Handbook of Secondary Fungal Metabolites, 3-Volume Set

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Handbook of Secondary Fungal Metabolites VOLUME I

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Handbook of Secondary Fungal Metabolites VOLUME

I

RICHARD J. COLE

Albany, Georgia

MILBRA A. SCHWEIKERT National Peanut Research Laboratory Dawson, Georgia

ACADEMIC PRESS An imprint of Elsevier Science Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

Cover images: Photography by Dr. Bruce Horn and design by Brian E. Cole. The cover is a collage of various fungi, some presented in pure culture and others in their natural forms, i.e. mushrooms, which are the easily recognizable fruiting structures of some fungi, superimposed on these fungi are the chemical structures of some representative secondary fungal metabolites. Academic Press Rapid Manuscript Reproduction This book is printed on acid-free paper. @ Copyright

92003, Elsevier Science (USA).

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://elsevier.com), by selecting "Customer Support" and then "Obtaining Permissions." A c a d e m i c Press An imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com A c a d e m i c Press 84 Theobald's Road, London WC1X 8RR, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2003103019 International International International International

Standard Standard Standard Standard

Book Book Book Book

Number: Number: Number: Number:

0-12-179460-1 0-12-179461-X 0-12-179462-8 0-12-179463-6

(Set) (Volume 1) (Volume 2) (Volume 3)

PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 8 7 6 5 4 3 2 1

Contents

Preface I ix Acknowledgments I xi

Indole Alkaloids / 1 Diketopiperazines / 145

I~l

ChaetoglobosinslCytochalasins /

I~1

Aflavinines and Related Indoles / 355

I ~ l Tryptoquivalines /

377

I(~ I Penitrems/Lolitrems / 411

245

vi

[~J

Contents

Paspaline and Related Metabolites / 441 J a n , h , t r e m s , ,83

] ~ I Miscellaneous Indole Metabolites /

493

I~~l Loline Alkaloids / 525 l~i

Aflatoxins / 545

i~l

Versicolorins / 571

I~ ~1 Sterigmatocystin and Related Metabolites I 595 l~ ~i Chokols ! 613 I~ ~] Enaminomycins and Related Metabolites /

629

l~ ~1 Boviquinones and Related Metabolites / 651 I~ ~1 Fusicoccins I 665

Contents

vii

I~ ~1 Altenuene and Related Metabolites / 695 I~ ~] Viridin and Related Metabolites / 715 I~(~

Cercosporin and Related Metabolites /

I~J

Cyathanes / 751

I~1

Alliacolide and Related Metabolites / 797

~~

Botrydial and Related Metabolites /

813

I~~1 Herbarin and Related Metabolites /

837

I~~[ Miscellaneous Metabolites I Secondary Metabofite Index I 989 Molecular Formula Index I 995 Molecular Weight Index I 999 Fungal/Plant Source Index I 1003

897

729

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Preface

The "Handbook of Secondary Fungal Metabolites" is presented in three volumes and is comprehensive to the extent that all major groups of secondary fungal metabolites are included. The format is similar to that presented in the "Handbook of Toxic Fungal Metabolites" with the major exception that actual spectra are not included; however, spectral data are included where available. Also included in these volumes are the methods used by the authors to isolate and purify metabolites. Another major difference is that the appropriate references are presented with each metabolite, negating the need to turn to the end of each group to find the appropriate references. Each volume contains four indexes: secondary metabolite index, molecular formula index, molecular weight index, and fungal/plant source index. In a few instances, plant sources are included when the metabolites are closely related to fungal metabolites or the source of precursors may be fungal; i.e., the baccharins, which are found in extracts from Baccharis megapotamica. These metabolites are closely related to the macrocyclic trichothecenes found in extracts of fungi such as Myrothecium spp. and Stachybotrys spp. Also, metabolites from the fungal symbiont of lichens are sometimes presented. To aid in the interpretation of NMR data, the numbering system presented in the literature is included for the major representative fungal metabolite and, at times, for several related metabolites. Fungal sources are given as reported in the original references. It is recognized that the taxonomy in several cases has been revised, perhaps more than once. It is beyond the scope of these volumes to deal with what is "currently accepted taxonomy" because this is a dynamic science that, in many cases, is as yet undefined. The "Handbook" has been divided into sections, and the placement of metabolites is based on chemical relationships. One section of each volume contains a miscellaneous section to accommodate metabolites difficult to place into one of the sections. The miscellaneous section of Volume III contains some metabolites related to those that appear in Volumes I and II. This occurred when related metabolites were discovered after Volumes I and II were completed.

ix

x

Preface

It is hoped that this compilation of data on secondary fungal metabolites will aid investigators in the identification of known or related fungal metabolites. Because fungal metabolites represent a wide diversity of chemical species, these volumes will be useful to scientists interested in correlations of structural features with various spectral and biological characteristics. The known biological activity of metabolites is presented, which may aid in future studies related to the identification of new uses for fungal metabolites. Richard J. Cole Milbra A. Schweikert

Acknowledgments

The authors thank the following investigators for their assistance in producing the "Handbook of Secondary Fungal Metabolites." Their contributions made this compilation of data on fungal metabolites possible. Wayne L. Bryden

James K. Porter

University of Queensland Gotton, Queensland 4343 Australia

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

Brian E. Cole

Images, Inc. Snellville, Georgia Horace G. Cutler

Department of Pharmacology Mercer University Atlanta, Georgia Jens C. Frisvad

Department of Biotechnology Technical University of Denmark DK-2800 Lyngby Denmark Bruce Horn

USDA-ARS National Panut Research Laboratory Dawson, Georgia

Ronaid T. Riley

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia Victor S. Sobolev

USDA-ARS National Peanut Research Laboratory Dawson, Georgia Bruce B. Jarvis

Department of Chemistry and Biochemistry University of Maryland College Park, Maryland

William Norred Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

xi

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Indole Alkaloids

3-(3-Indolyl)propane-1,2,3-triol 3-(3,3-Diindolyl)propane-1,2-diol 4-(3-Indolyl)butane-l,2,3-triol N-Methyl-4-dimethylallyltryptophan Lysergic Acid Ergine; Lysergic acid amide 8-Hydroxyergine Erginine; Isolysergic acid amide 8-Hydroxyerginine Lysergol Lysergene Lysergine Ergonovine; Ergometrine; Ergobasine Ergonovinine; Ergometrinine; Ergobasinine Agroclavine 6, 7-seco-Agroclavine Dihydroagroclavine Festuclavine Elymoclavine Elymoclavine-O-B-fructofuranoside Elymoclavine-O-B-fructofuranosyl-(2-1)-O-B-Dfructofuranoside Chanoclavine-I; Chanoclavine Isochanoclavine-I Chanoclavine-II N-Demethylchanoclavine-II; Norchanoclavine II Setoclavine Isosetoclavine Costaclavine Pyroclavine Molliclavine Penniclavine Cycloclavine Ergotamine Ergotaminine 8-Hydroxyergotamine Ergosine Ergosinine Ergostine Ergostinine Ergonine Ergovaline Ergoptine Ergocomine

2

1.

Indole Alkaloids

Ergocominine O- 12'-Methylergoeomine Ergocristine Ergocristinine Ergosecaline Ergosecalinine Ergobalansine Ergobalansinine a-Ergocryptine O- 12'-Methyl-a-ergocryptine 13-Ergocryptine 5'-epimer of 13-Ergocryptine 13-Ergocryptam 13,13-Ergoannam Ergobutine Ergobutyrine Rugulovasine A 8-Chlororugulovasine A Rugulovasine B 8-ChlororugulovasineB Fumigaclavine A; 9~-Acetoxy-6,8a-dimethylergoline Roquefortine A; IsofumigaclavineA; 9-Acetoxy-6,8-dimethylergoline Fumigaclavine B; 9-Hydroxy-6,8-dimethylergoline Roquefortine B; IsofumigaclavineB; 9-Hydroxy-6,8-dimethylergoline Fumigaclavine C; 2-Dimethylallyl-9-acetoxy-6,8-dimethylergoline

1. Indole Alkaloids

3

Common/Systematic Name 3-(3-Indolyl)propane- 1,2,3-triol Molecular Formula/Molecular Weight CllH13NO3; M W = 207.08954

cH2oH OH

Io.

General Characteristics Red-violet color reaction with p-dimethylaminocinnamaldehyde. Fungal Source Balansia epichlod. Isolation/Purification Purification was achieved by column chromatography on Porapak Q and preparative TLC on silica gel GF254[TLC developing systems were chloroform-methanol (80:20, v/v) and benzene-dimethylformamide (86.5:13.5)]. Biological Activity Toxic to fertile Leghorn chicken eggs: 23~g/egg = 80%; 68~g/egg = 100% mortality. Spectral Data UV:

~,~ff" 220(log e=4.95), 273(4.00), 280(4.02), and 289nm (3.95). IR:

(KBr) 1550, 1420, 1410, 1335, 1065, 1050, 740, and 780cm"1. Mass Spectrum: 207.08, 189.07, 188.06, 186.05, 172.07, 171.06, 170.05, 160.07, 159.06, 146.05, 145.05, 144.08, 144.04, 142.06, 130.06, 118.06, 117.05, 116.05, 103.05, 91.05, 90.04, and 89.03m/e. Reference J. K. Porter, C. W. Bacon, J. D. Robbins, D. S. Himmelsbach, and H. C. Higman; Indole Alkaloids from Balansia epichlo~ (Weese); J. Agric. Chem., Vol. 25, pp. 88-93 (1977).

4

1.

Indole Alkaloids

Common/Systematic Name 3-(3,3-Diindolyl)propane-l,2-diol Molecular Formula/Molecular Weight C19H18N202; ~

= 306.13683

CH20H I CHOH

~!LNH~ ~NH/L~ General Characteristics Red-violet color reaction with p-dimethylaminocinnamaldehyde. Fungal Source Balansia epichlo~. Isolation/Purification Purification was achieved by column chromatography on Porapak Q and preparative TLC on silica gel GF254 [TLC developing systems were chloroform-methanol (80:20, v/v) and benzene-dimethylformamide (86.5 13.5, v/v)]. Biological Activity Toxic to fertile Leghorn chicken eggs: 20l.tg/egg = 20%; 60lag/egg = 55% mortality; 99ktg/egg = 100% mortality. Spectral Data UV:

~

MeOH max

221(1og e=4.88), 275(3.97), 282(4.01), and 291nm (3.96).

IR:

(KBr) 1550, 1410, 1335, 1080, 1050, and 780cm1. Mass Spectrum: 306.1368(M+), 272.1326, 270.1145, 258.1132, 257.1049, 256.0993, 245.1069, 218.0958, 217.0887, 188.0671, 171.0675, 170.06, 160.07, 159.06, 144.04, 142.06, 130.06, 118.06, 117.05, 116.05, 103.05, 91.05, 90.04, and 89.03role. Reference J. K. Porter, C. W. Bacon, J. D. Robbins, D. S. Himmelsbach, and H. C. Higman; Indole Alkaloids from Balansia epichlo~ (Weese); J. Agric. Chem., Vol. 25, pp. 88-93(1977).

1. Indole Alkaloids

5

Common/Systematic Name 4-(3-Indolyl)butane- 1,2,3-triol Molecular Formula/Molecular Weight C12H15NO3, M W = 221.10519

OH

{~}LNHI ~ H CH20 H General Characteristics Red-violet color reaction with p-dimethylaminocinnamaldehyde. Fungal Source Balansia epichlos Isolation/Purification Purification was achieved by column chromatography on Porapak Q and preparative TLC on silica gel GF254[TLC developing systems were chloroform-methanol (80:20, v/v) and benzene-dimethylformamide (86.5:13.5, v/v)]. Biological Activity Toxic to fertile Leghorn chicken eggs: 57~g/egg = 53% mortality; 113l.tg/egg = 100% mortality. Spectral Data UV:

~

MeOH max

221(log e=4.65), 272(3.78), 279(3.8), and 288nm (3.73).

IR:

(KBr) 1550, 1410, 1340 1080, 1030, and 780cmq. Mass Spectrum: 221.10(M+), 203.09, 201.08, 189.07, 188.07, 186.05, 172.07, 171.06, 170.06, 160.07, 159.06, 146.05, 145.05, 144.08, 144.04, 142.06, 130.06, 118.06, 117.05, 116.05, 103.05, 91.04, 91.05, 90.04, and 89.08role. Reference J. K. Porter, C. W. Bacon, J. D. Robbins, D. S. Himmelsbach, and H. C. Higrnan; Indole Alkaloids from Balansia epichlo3 (Weese), J. Agile. Chem., Vol. 25, pp. 88-93(1977).

6

1.

Indole Alkaloids

Common/Systematic Name N-Methyl-4-dimethylallyltryptophan Molecular Formula/Molecular Weight C]THz2NzO2; MW' = 286.16813

Me Me

/

CO2H HMe

NH General Characteristics N-Methyl-4-dimethylallyltryptophan crystallized from methanol as needles; mp., 232~ Fungal Sourc.e

Clavicepsfusiformis.

Isolation/Purification

Clavicepsfusiformis was grown aerobically in submerged cultures in both shaken flasks and stirred fermenters. When alkaloid production began, anaerobic conditions were imposed and the cultures stood for three days. Clavine alkaloids were extracted with chloroform at alkaline pH and then the amphoteric metabolites extracted with n-butanol at neutral pH. The butanol extract, which contained considerable quantities of chanoclavines and other oxygenated clavine alkaloids, was chromatographed on silica gel with chloroform/methanol/ammonia as the eluant.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~, m=M~" 274, 280, and 295nm.

1. Indole Alkaloids

7

IR~

(KBr) 3580, 3250(broad) 1640, 1400, and 770cm1. 1H NMR: (CDaCOOD) inter alia 8.64(s, 6H), 7.64(s, 3H), 5.06(t, 1H, J=-7.0Hz), and 6.3-7.0ppm (complex, 4H).

Mass Spectrum: 286, 198, 156, 155, and 154m/e. The fragmentation under electron-impact was very similar to bis-seco-dehydrocyclopiazonic acid with allylic cleavage of the amino acid side chain giving the ion ofm/e 198, followed by cyclization to a series oftricyclic ions m/e 156, 155, and 154 with elimination of a C-3 unit. Cyclization of this type is only possible if the two side chains are located in the peri-position of the indole nucleus, Le. at positions 3 and 4. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or l:l, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4: l, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References K. D. Barrow and F. R. Quigley, Ergot Alkaloids HI : The Isolation of N-Methyl.4dimethylallyltryptophan from Clavicepsfusiformis; Tetrahedron Letters, pp. 4269-4270 (1975). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978).

J. K. Porter, Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

8

1.

Indole Alkaloids

Common/Systematic Name Lysergic acid Molecular Formula/Molecular Weight CI6H16N202; ~

= 268.12118

H,, COOH '~N--Me

NH General Characteristics Hexagonal scales, plates from water (associated with one or two moles water); mp., 240~ (dec.); [tt]D2~ +40 ~ (C=0.5, in pyridine); pKa=3.44/pI~,=7.68. Moderately soluble in pyridine; sparingly soluble in water and neutral organic solvents; soluble in NaOR NH4OH, Na2CO3, and HCI solutions; and slightly soluble in dilute H2SO4. Methyl ester derivative, thin leaflets from benzene; mp., 168 ~C. Fungal Source Sclerotia and saprophytic culture of Claviceps purpurea. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data IR:

See A. Hofmann, 1964. 13C NMR:

(CDCI3) (methyl lysergate) C-2, 118.2; C-3, 110.2; C-4, 26.9; C-5, 62.6; C-7, 54.6; C8, 41.8; C-9, 117.6; C-10, 136.0; C-11,127.6; C-12, 112.0; C-13, 122.9; C-14, 109.4; C-15, 133.7; C-16, 125.9; C-17, 172.4; Me, 51.9; and NMe, 43.4ppm.

1. Indole Alkaloids

9

Mass Spectrum: LREIMS: 268(M+, 100%), 224, 221,207, 192, 180, 167,and 154role. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

10

1.

Indole Alkaloids

Common/Systematic Name Ergine; Lysergic acid amide Molecular Formula/Molecular Weight CI6HI7N30; M ~ = 267.13716

0 II

H2N--C. ,H N--Me

\

H

General Characteristics Crystallized from acetone as massive colorless prisms; m.p. 196~ [a]D 20 q" 414 ~ [a]54612~+ 520 ~ (c--1.0, in CHCI3); pK = 6.2 (in 80% methylcellosolve); blue color with Keller's reagent. Fungal Source Ergot of Clavicepspurpurea and Paspalum distichum L. (also isolated from seeds of Rivea corymbosa (L.) and Ipomoea tricolor; Convolvulaceae). Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some ergopeptine alkaloids are used routinely in medical practice. Central American Indians used seeds ofRivea corymbosa and Ipomoea tricolor as a magic drug called "Ololiuqui". Spectral Data IR:

See A. Hofmann, 1964. UV:

UV spectrum identical to that of lysergic acid or isolysergic acid.

1. Indole Alkaloids

11

Mass Spectrum: LREIMS: 267(M+, 100%), 249, 224, 221,207, 192, 180, 167, and 154role. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

12

1.

Indole Alkaloids

Common/Systematic Name 8-Hydroxyergine Molecular Formula/Molecular Weight C16HITN302; MW = 283.13208

0II C~NH2 HO""'~~8 7~N_Me 13 14

I 3 d

2

Fungal Source

Clcn,iceps paspali (strain MG-6).

Isolation/Purification The strain C. paspali MG-6 was isolated from the grass Paspalum dilatatum in the vicinity of Rome. Alkaloids were separated by adsorption on bentonite (Flieger et al., 1989b). A crude alkaloid mixture was chromatographed on Kieselgel 60 F254, Merck preparative TLC plates and eluted with chloroform-isopropyl alcohol-ammonia (90:10:0.036, v/v/v); Rf values of 8-hydroxyergine and 8-hydroxyerginine were 0.50 and 0.91, respectively. Prepurified alkaloids were chromatographed on a Separon SGX C~s column (Tessek, Czechoslovakia) particle size 71.tm. The mobile phase consisted of (A) MeOH-H20-NH3 (90:10:0.036, v/v/v) and (B) MeOH-H20-NH3 (20:80:0.036, v/v/v). The column was equilibrated with 4% A in B and subsequently eluted with a linear gradient up to 54% A in B. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic comlSonents). The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medicine.

1.

Indole Alkaloids

13

Spectral Data 1H NMR: (CDaOD) H=2, 6.984(J2,4a=l.8Hz); H-4a, 2.749; H-4b, 3.569(J2,4b=>0,J~,4b-= 14.5Hz); H-5, 3.265(J4~,5=11.8Hz, J4b,5=5.9Hz); H-7a, 2.936(Js~m,=-I1.7Hz, Js~,9=l.0I-Iz); H=7b, 2.965; H-9, 6.358(J4b,9=>0, J5,9=2.1Hz); H=12, 7.193(Jla, la--7.4I-Iz, .J12,14=0.7I-Iz);H-13, 7.107(J~a,14=7.9Hz); H14, 7.231; and N-Me, 2.590ppm. 13C NMR: (CDaOD) C-2, 120.66; C-3, 110.52; C-4, 27.15; C-5, 64.20; C-6, 62.7 1; C-7, 73.84; C-8, 121.00; C-9, 139.91; C-10, 128.13; C-11,113.43; C-12, 123.93; C-13, 111.95; C-14, 136.02; C-15, 128.13; C-16, 177.92; and N-Me, 43.75ppm.

Mass Spectrum: EIMS: 283(M+, C16H17N302,61%), 266(C16H16N30, 27), 265(C16HIsN30, 37), 248(C16H12N20, 59), 240(CIA-II2N202, 86), 223(Cl,d-IiEN202, 86), 221(C~5HlaN2, 42), 206(C14HloN2, 19), 195(CIaH9NO, 36), 194(CIaHsNO, 32), 181(CI3HIIN, 26), 180(ClaHloN, 26), 167(C12HaN, 83), and 154(C~HsN, 100). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References M. Flieger, R. Linhartova, P. Sedmera, J. Zima, P. Sajdl, J. Stuchlik, and L. Cvak; New Alkaloids ofClavicepspaspali; J. Nat. Prod., Vol. 52, pp. 1003-1007 (1989a). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

14

1.

Indole Alkaloids

Common/Systematic Name Erginine; Isolysergic acid amide Molecular Formula/Molecular Weight CIrHITN30; MW = 267.13716

O II H2N--C,,

H. N--Me

NH General Characteristics Crystallized from methanol as solvated prisms; mp., 132-134~ 608 ~ (C=0.5, in pyridine); pK=6.1 (in 80% methylcellosolve).

[tt]D2~+ 480 ~ [tt]54612~+

Fungal Source Ergot and saprophytic culture of Clavicepspurpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some ergopeptine alkaloids are used routinely in medical practice. Central American Indians used seeds of Rivea corymbosa and Ipomoea tricolor as a magic drug called "Ololiuqui". Spectral Data _

IR:

See A. Hofmann, 1964.

1.

Indole Alkaloids

15

TLC Purification Silica gel plates developed with methylene chloride-isopropyi alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

16

1.

Indole Alkaloids

Common/Systematic Name 8-Hydroxyerginine Molecular Formula/Molecular Weight C]6H]7N302; MW = 283.13208 O II

C_--NH2 HOy8 "' 7~N--Me

Fungal Source Claviceps paspali MG-6. Epimers are not considered as naturally occurring but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification The strain C. paspali MG-6 was isolated from the grass Paspalum dilatatum in the vicinity of Rome. Alkaloids were separated by adsorption on bentonite (Flieger et al., 1989b). A crude alkaloid mixture was chromatographed on Kieselgel 60 F254,Merck preparative TLC plates, and eluted with chloroform-isopropyl alcohol-ammonia (90:10:0.036, v/v/v); Rf values of 8-hydroxyergine and 8-hydroxyerginine were 0.50 and 0.91, respectively. Prepurified alkaloids were chromatographed on a Separon SGX C18 column (Tessek, Czechoslovakia) of particle size 71~. The mobile phase consisted of (A) MeOH-H20-NH3 (90:10:0.036, v/v/v)and (B) MeOH-H20-NH3 (20:80:0.036, v/v/v). The column was equilibrated with 4% A in B and subsequently eluted with a linear gradient up to 54% A in B. Spectral Data ]H NMR: (CDsOD) H-2, 6.974(J2,4a=l.6Hz); H-4a, 2.649; H-4b, 3.606(J2,4b=>0, dna,4b=-14.6Hz); H-5, 3.150(J4a,5=l 1.5Hz, Jnb,5=5.9Hz); H-7a, 3.080(J7~,Tb=-I1.3Hz, J7a,9=l.5Hz); H-Tb, 2.626; H-9, 6.268(Jab,9=0.8Hz, Js,9=2.2Hz); H- 12, 7.118(J]2,13=7.0Hz, J]2,14=l.THz); H- 13, 7.090(Jx3,14=7.2Hz); H- 14, 7.219ppm; and N-Me, 2.614ppm.

1.

Indole Alkaloids

17

13CNMR: (CD3OD) C-2, 120.53; C-3, 110.55; C-4, 28.27; C-5, 64.11; C-6, 63.06; C-7, 71.87; C8, 124.30; C-9, 139.13; C-10, 128.04; C-11, 113.15; C-12, 123.96; C-13, 111.72; C14, 135.95; C-15, 127.99; C-16, 179.59; and N-Me, 43.39ppm. Mass Spectrum: EIMS: 283(M§ C16H17N302, 100), 266(C16H16N30, 14), 265(C16H15N30, 29), 248(C16H12N20, 35), 240(C14H12N202, 93), 223(C14H12N202, 31), 221(C15I-I13N2,42), 206(C14H1oN2, 12), 195(C13HgN,60), 194(CI3HsN, 61), 181(C13HllN, 50), 180(C13HloN, 20), 167(C12H9N,94), and 154(C1lI-lsN, 96%). Reference M. Flieger, R. Linhartova, P. Sedmera, J. Zima, P. Sajdl, J. Stuchlfk, and L. Cvak; New Alkaloids of Clawcepspaspali; J. Nat. Prod.; Vol. 52, pp. 1003-1007(1989a).

18

1.

Indole Alkaloids

Common/Systematic Name Lysergol Molecular Formula/Molecular Weight C16HIsN20, MW' = 254.14191

HOH2C. ,H

.~'~N--Me

General Characteristics Colorless prisms from ethanol; mp., 245~ (uncorr. decomp.); as plates and prisms; mp., 253-255~ (dec.); [a]D 18 +49 ~ (C=0.2, in pyridine); [a]D2~ +54 ~ [a]5~sl2~ +87 ~ (c=0.3, in pyridine). Gave a light purple and purplish blue colors with van Urk's and Allport-Cocking's reagents, respectively. Sublimes at high vacuum at 1800C; pK=6.6 (in 80% aqueous methylcellosolve). Soluble in 350 parts of boiling methanol or 100 parts boiling ethanol, sparingly soluble in chloroform or water. Fungal Source Saprophytic cultures of Elymus-type ergot fungus. Isolation/Purification Purified either by countercurrent distribution or by column chromatography using Hyflo Super Cell treated with a buffer solution (Mcllvaine). Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~, M.o. 225, 242, and 312nm. max

1. Indole Alkaloids

19

IR; See A. Hofmann, 1964. Mass Spectrum: LREIMS: 254(M+, 100%), 235, 223,221,207, 205, 193, 192, 180, 167, and 154role. References M. Abe, S. Yamatodani, T. Yamano, and M. Kusumoto; Isolation of Lysergol, Lysergene and Lysergine from the Saprophytic Cultures of Ergot Fungi; Agile. Biol. Chem. [Tokyo], Vol. 25, pp. 594-595(1961). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

20

1. Indole Alkaloids

Common/Systematic Name Lysergene Molecular Formula/Molecular Weight Cl6Hl6N2, M W = 236.13135

CH2 N--Me

NH General Characteristics Colorless needles or prisms from ethyl acetate; mp., 244~ (uncorr. decomp.); colorless needles or prisms from methanol, 247-249~ (dec.); [a]o TM +461 (c=0.2, in pyridine); [a]Dz~ +504 ~ (C=0.4, in pyridine). Sparingly soluble in most organic solvents, moderately soluble in chloroform or pyridine. Gave a yellowish-green color with both van Urk's and Allport-Cocking's reagents, respectively. Fungal Source Saprophytic cultures of Elymus-type ergot fungus. Isolation/Purification Purified either by countercurrent distribution or by column chromatography using Hyflo Super Cell treated with a buffer solution (Mcllvaine). Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UW:

m~H 243, 263, and 335nm. IR:

See A. Hofmann, 1964.

1.

Indole Alkaloids

21

References M. Abe, S. Yamatodani, T. Yamano, and M. Kusumoto; Isolation ofLysergol, Lysergene and Lysergine from the Saprophytic Cultures of Ergot Fungi; Agile. Biol. Chem. Vol., pp. 594-595(1961). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

22

1. Indole Alkaloids

Common/Systematic Name Lysergine Molecular Formula/Molecular Weight CI6HlsN2; M W = 238.14700

Me

,H

N--Me

'

Nit

General Characteristics Colorless prisms from ethyl acetate; mp., 275~ (uncorr. decomp.); prisms from methanol, ethanol, or ethyl acetate, 286-289~ (dec.); [a]DTM = +70 ~ (c=0.2, in pyridine), [a]D2~ +65 ~ (C=0.5, in pyridine); sparingly soluble in methanol, ethanol, and ethyl acetate. Fungal Source Ergot or saprophytic cultures ofAgropyrum sp. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~

Very similar to lysergic acid. IR~

See A. Hofmann, 1964. References Abe, S. Yamatodani, T. Yamano, and M. Kusumoto; Isolation ofLysergol, Lysergene and Lysergine from the Saprophytic Cultures of Ergot Fungi; Agric. Biol. Chem. [Tokyo], Vol. 25, pp. 594-595(1961).

1.

Indole Alkaloids

23

B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook 0fExperimental Pharmacology; Springer-Verlag, New York (1978) A. Hofmann; Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964.

24

1.

Indole Alkaloids

Common/Systematic Name Ergonovine; Ergometrine; Ergobasine Molecular Formul~olecu!ar Weight C19H23N302; M W = 325.17903

0 II C

CH20H I

NH~C ~H

...)___.

Hi,

e

N--Me

NH General Characteristics Tetrahedra from ethyl acetate; fine needles from benzene; tendency to form solvated crystals; mp., 162~ (nonsolvated, mp., 212~ dec.); [a]D2~+ 90~ in water); -16 ~ (c=l.0, in pyridine); [aid 2~ + 41~ [a]546~2~ + 60 ~ (c=l.0, in alcohol); pK = 6.8. Freely soluble in lower alcohols, ethyl acetate, and acetone; more soluble in water than other principal alkaloids of ergot; slightly soluble in chloroform. Fungal Source

Claviceps purpurea, Balansia epichlo~, B. henningsiana, and B. claviceps.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central neivous system effects (bulbomedullary and mesodiencephalic components). MLD (IV) in rabbits was 7.5mg/kg.

1.

Indole Alkaloids

25

Spectral Data IR~

See A. Hofmann, 1964. UV~

Identical to that of lysergic acid or isolysergic acid. ~3CNMR: (DMSO-d6) C-2, 119.1; C-3, 108.9; C-4, 26.8; C-5, 62.6; C-7, 55.5; C-8, 42.8; C-9, 120.1; C-10, 135.0; C-11, 127.4; C-12, 111.0; C-13, 122.4; C-14, 109.0; C-15, 133.7; C-16, 125.8; C-17, 171.2; Me, 17.4; NCH, 46.4; OCH2, 64.4; and N-Me, 43.4ppm. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1) followed by rechromatography in chloroform-methanol (9:1 or 4:1) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography: a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. chem., Vol. 39, pp. 1272-1276(1974). C. W. Bacon, J. K. Porter, and J. D. Robbins; Lysergic Acid Amide Derivatives from

Balansia epichlo3 and Balansia claviceps (Clavicipitaceae); J. Nat. Prod., 42:309-314 (1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Laboratory Production of Ergot Alkaloids by Species ofBalansia; J. Gen. Microbiol., 113: 119-126(1979).

26

1.

Indole Alkaloids

C. W. Bacon, J. K. Porter, and J. D. Robbins; Ergot Alkaloids Biosynthesis by Isolates of

Balansia epichlo~ and Balansia henningsiana; Can. J. Bot., 59:2534-2538(1981).

C. W. Bacon, J. K. Porter, J. D. Robbins, and D. Betowski; Ergot Alkaloids Identification in Clavicipitaceae Systemic Fungi of Pasture Grass; J. Agric. Food Chem., 29:653-657 (1981). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. W. A. Jacobs and L. C. Craig; On an Alkaloid from Ergot; Science, Vol. 82, pp. 16-17 (1935). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

27

Common/Systematic Name Ergonovinine; Ergometrinine; Ergobasinine Molecular Formula/Molecular Weight C19H23N302, ~

O II C

= 325.17903

CH2OH I NH~C ~H

H

e N--Me

"

NH

General Characteristics Forms large colorless prisms from acetone; mp., 196~ (dec.); [a]D2~+ 414 ~ [a]5~l 2~ + 520 ~ (c=l.0, in CHCI3); pK=6.2 (in 80% methylcellosolve); blue color with Keller's reagent. Fungal Source

Claviceps purpurea.

Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism and adrenergic blockage) and central nervous system effects (bulbomeduUary and mesodiencephalic components). MLD (IV) in rabbits was 7.5mg/kg.

28

1. Indole Alkaloids

Spectral Data IR-

See A. Hofmann, 1964. UV:

Identical to that of lysergic acid or isolysergic acid. 13C N M R :

(DMSO-d6) C-2, 119.0; C-3, 108.9; C-4, 26.9; C-5, 62.0; C-7, 54.0; C-8, ca. 42.2; C9, 119.0; C-10, 136.1; C-11,127.6; C-12, 111.0; C-13, 122.1; C-14, 109.8; C-15, 133.7; C-16, 125.7; C-17, 172.1; Me, 17.2; NCH, 46.2; OCH2, 64.3; and N-Me, 43.6ppm. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NHs (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References N. J. Bach, ,. H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. chem., Vol. 39, pp. 1272-1276(1974). C. W. Bacon, J. K. Porter, and J. D. Robbins; Lysergic Acid Amide Derivatives from Balansia epichlo3and Balansia claviceps (Clavicipitaceae); J. Nat. Prod., 42:309-314 (1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Laboratory Production of Ergot Alkaloids

1. Indole Alkaloids

29

by Species ofBalansia; J. Gen. Microbiol., 113:119-126(1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Ergot Alkaloids Biosynthesis by Isolates of

Balansia epichlo~and Balansia henningsiana; Can. J. Bot., 59: 2534-2538(1981).

C. W. Bacon, J. K. Porter, J. D. Robbins, and D. Betowski; Ergot Alkaloids Identification in Clavicipitaceae Systemic Fungi of Pasture Grass; J. Agile. Food Chem., 29:653-657 (1981). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolo~_; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. W. A. Jacobs and L. C. Craig; On an Alkaloid from Ergot; Science, Vol. 82, pp. 16-17 (1935). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

30

1. Indole Alkaloids

Common/Systematic Name Agroclavine Molecular Formula/Molecular Weight C16HIgN2; M~W = 238.14700 Me N--Me ll~

NH

General Characteristics Colorless needles from acetone; mp., 205-206~ sublimed under high vacuum between 110-130~ [tt]D2~-155 ~ (C=0.9 in CHCIs); [a]D2~ -182 ~ (C=0.5 in pyridine); pK=6.8 (in 80% aqueous methylcellosolve). Violet/blue color with Keller's reagent. Fungal Source First found from sclerotia and cultures ofAgropyrum semicostatum Nees and A. ciliare Fr. Also found in Pennisetum typhoideum. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~ )~0o.

225, 284 and 293nm (log e=4.47, 3.88 and 3.81, respectively).

IR~ See A. Hofmann, 1964. IH NMR: (pyridine-ds) H-6, 2.38(3H, s); H-7, 2.73(1H, d); H-4, 2.90(1H, ddd); H-7', 3.18(1H, d); H-4', 2.51(1H, ddd); H-5, 3.36(1H, dd); H-10, 3.89(1H, m); 7-CHs, 1.68(3H, s);

1.

Indole Alkaloids

31

H-9, 6.30(1H, m); aromatic-H, 7.1-7.4(4H, m); and H-l, 11.43ppm (1H, s). (Note: Possible incorrect assignment of the 44, 413, and 5 hydrogens). (CDCI3) H-4a, 2.78(dd, ,/=15, 12Hz); H-413, 3.3 l(dd, J=15, 4Hz); H-5, 2.52(ddd, J=12, 9.5 and 4Hz); H-7a, 3.24(d, J=17Hz); H-713, 2.93(dd, broad signal, J=17, 4Hz); H-9a and 13, 6.18(s, broad signal); H- 10, 3.74(dd, broad signal, J=9.5, 4Hz); H- 17, 1.77(s); and N-Me, 2.49ppm (s). ~3CNMR: (pyridine-ds) C-2, 118.3; C-3, 111.2; C-4, 26.4; C-5, 63.6; C-7, 60.2; C-8, 131.9; C-9, 119.4; C-10, 40.8; C-11,131.9; C-12, 112.0; C-13, 122.0; C-14, 108.4; C-15, 134.0; C-16, 126.6; C-17, 19.9; and N-Me, 40.2ppm. Mass Spectrum: LREIMS: 238(M+, 52%), 237(100), 167(17), and 154role (16). References M. Abe, T. Yamano, Y. Kozu, and M. Kusumoto; Isolation of Further Two Water-soluble Ergot Alkaloids; J. Age. Chem. Soc., Vol. 28, pp. 501-510(1954). N. J. Bach, H. E. Boaz, E. C. Cornfield, C-J Chang, H. G. Floss, E. W. Hegemon, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology, Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). A. Stoll, A. Brack, H. Kobel, A. Hofmann, and R. Brunner; Die Alkaloide eines Mutterkornpilzes von Pennisetum typhoideum rich. und deren Bildung in Saprophytischer Kultur; Helvetica Chimica Acta, Vol. 37, pp. 1815-1825(1954).

32

1. Indole Alkaloids

Common/SystematicName

6,7-seco-Agroelavine

Molevular F0rmula/Moleeular Weight C16H2oN2;MW = 240.16265

......

HMe

General Characteristics Crystals; mp., 126-129~ subl., gives a blue color with Allport and Cocking's reagent. Fungal Source v

Claviceps purpurea, strain AA-218, Balansia epichlo~ B. strangukms, and Epichlo~ trphina.

Isolation/Purification Purified by HPLC followed by PLC using 1% conc. ammonia, 5% Meg)H, and 94% chloroform. TLC using silica gel with same solvent system. Soeetral Data _

UV~

m,x~~a 225, 283, and 293nm.

(CHCI3): 3480(indole NH), 3320(aliphatic NH), 1605, and 1445cm"~(C=C). ~H NMR: (CDCI3): 1.85(s, 6H); 2.53(s, with hyperfme splitting, 3H); 2.4(br s, 1H); 2.6-3.5(m, 4H); 3.75-4.15(m, 1H); 5.0-5.3(d, 1H (C-10-H)); 6.6-7.3(m, 4H); and 8.1-8.5ppm (br s, 1H(indole)). Mass Spectrum: EIMS: 240, 225, 208, 197, 184, 168, and 155m/e. References C. HorweH and J. P. Verge; Isolation and Identification of 6,7-seco-Agroclavine l~om Claviceps trurpurea, Phytoehemistry, Vol. 18, p. 519 (1979).

1. Indole Alkaloids

J. K. Porter, C. W. Bacon, J. D. Robbins, and D. Betowski; Ergot Alkaloid Identification in Clavicipitaceae Systemic Fungi of Pasture Grasses; J. Agric. Food Chem., 29:653-657 (1981).

33

34

1.

Indole Alkaloids

Common/Systematic Name Dihydroagroclavine Molecular Formula/Molecular Weight C16H20N2; ~

= 240.16265

Me

J

"

~N--Me

NH

General Characteristics Crystals as long needles from toluene, benzene, ether, chloroform, ethyl acetate, acetone, methanol, ethanol or pyridine; mp., 242~ (dec.); [a]D2~ - 69 ~ [tt]5~ 2~ - 83 o (C=0.5, in CHCI3); [et]D~ - 111 ~ [~]54612~ - 129 ~ (c=0.5, in pyridine). Insoluble in toluene, benzene and ether; readily soluble in chloroform, ethyl acetate, acetone, methanol, ethanol and pyridine. Succinate derivative, C16H20N2. 0.5 C4H604, crystals as prisms from water; mp., 213~ (dec.); [t~]D~7 - 87 ~ (C=0.13, in pyridine). Fungal Source Ergot and saprophytic culture of Phalaris and Agropyrum sp. Spectral Data Mass Data: Found: C 79.94, H 8.34, N 11.64 (calcd. for CI6H20N2:C 79.95, H 8.39, N 11.66). Reference M:Abe and S. Yamatodani; Isolation of Further Two Water Soluble Ergot Alkaloids; J. Agr. Chem. Soc., Vol. 28, p. 501 (1954).

1. Indole Alkaloids

35

Common/Systematic Name Festuclavine Molecular Formula/Molecular Weight C16I-I20N2; MW Me

-~

240.16265

H

N--Me

NH General Characteristics Crystals (long needles) from methanol; mp., 238-2390C (dec.); 242-244~ (dec.); [a]u 15 -98 ~ (c=0.3, pyridine); [a]D 20 -70 ~ [a]546120 -83 ~ (c=0.5, in CHCI3); [a]D z0 -110 ~ [a]546120 -128~ pK=7.4 (in 80% aqueous methylcellosolve); positive for van Urk's reaction; insoluble in petroleum ether, sparingly soluble in ethyl acetate, moderately soluble in benzene and chloroform and readily soluble in acetone, methanol, ethanol, and pyridine. Fungal Source Penicillium chermesinum (PC 106-1), Agropyrum type ergot fungus parasitic on Agropyrum semicostatum, Trisetum bifidum Ohwi, Festuca rubra L., etc. growing in Japan. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV:

~

MeOH max

224(log e=4.54), 275(3.81), and 281nm (3.84).

IR;

See A. Hofmann, 1964.

36

1.

Indole Alkaloids

1H NMR: (CDCI3) H-4a, 2.68(dd, J=-15.0, 11.5Hz); H-4p, 3.39(dd, J=15.0, 4.5Hz); H-5, 2.10(ddd, J=l 1.5, 9.5, 4.5Hz); H-7tt, 2.95(d[broad], J=-I 1.0Hz); H-713, 1.87(t, J=l 1.0Hz); H-8, 2.01(ddd, J=-12, 11, 6.5Hz); H-9a, 2.63(dd, J=12.0, 3.5Hz); H-9[i, 1.08(q, J=12.0Hz); H-10, 2.97(ddd, J=-12.0, 9.5, 3.5Hz); H-17, 0.99(d, J=6.5Hz); and N-Me, 2.45ppm (s). 13C NMR: (CDC13) C-2, 117.7; C-3, 110.5; C-4, 26.6; C-5, 66.7; C-7, 65.0; C-8, 30.2; C-9, 36.2; C-10, 40.4; C-11, 132.7"; C-12, 112.0; C-13, 122.0; C-14, 108.3; C-15, 133.1; C-16, 125.9; C-17, 19.3; and N-Me, 42.7ppm.

* Assignment may be reversed. Mass Spectrum: LREIMS: 240(M+, 100%), 197, 182, 167, 154, and 144m/e. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Haganmn, and E. Wenkert; Nuclear Magnetic Resonanc~ Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook

of Experimental Pharmacology; Springer-Vedag, New York (1978). A. Hofinann; Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. S. Ohmomo, T. Sato, T. Utagawa, and M. Abe; Isolation of Festuelavine and Three New Indole Alkaloids, Roquefortine A, B and C from the Cultures ofPenicillium roqueforti; Agr. Biol. Chem., Vol. 39, pp. 1333-1334(1975).

1.

Indole Alkaloids

37

Common/Systematic Name Elymoclavine Molecular Formula/Molecular Weight C16HlgN20; M ' W = 254.14191

CH20H s LH 12 13

General Characteristics Crystallized as prisms from methanol; mp., 245-249~ [a]D 20 -152 ~ (c=0.9, in pyridine), [a]D2~-111 ~ (C=0.1, in EtOH); pK=6.7 (in 80% aqueous methylcellosolve); violet-blue color with Keller's reagent. Fungal Source Saprophytic culture of ergot fungus Claviceps sp. SD 58 (ATCC 26019), Pennisetum typhoideum sclerotia and saprophytic cultures, and Elymus mollis. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV:

~

MeOH max

227, 283, and 293nm (log e=4.31, 3.84, and 3.76, respectively).

See A. Hofmann, 1964. ~H NMR: (pyridine-ds) H-6, 2.41(3H, s); H-7, 3.05(IH, d); H-4, 2.92(IH, ddd); H-7', 3.61(IH,

d); H-4', 2.59(IH, ddd); H-5, 3.35(IH, dd); H-10, 3.94(IH, m); H-17, 4.36(21-I); 17-

38

1.

Indole Alkaloids

OH, 4.75(1H, s); H-9, 6.71(1H, m); aromatic-H, 7.1-7.4(4H, m); and H-l, 11.40ppm(1H, s). (CD3OD) H-2, 6.922; H-4a, 2.798; H-413, 3.357; H-5, 2.622; H7a, 3.026; H-713, 3.444; H-9, 6.464; H-10, 3.798; H-12, 6.967; H-13, 7.121; H-14, 7.191; H-17upfield, 4.106; H-17downfield, 4.141; and N-Me, 2.523ppm. ~3C NMR: (CD3OD) C-2, 119.09; C-3, 111.45; C-4, 27.17; C-5, 64.76; N(6)-Me, 41.15; C-7, 57.20; C-8, 134.37; C-9, 121.55; C-10, 40.98; C-11, 131.74; C-12, 112.83; C-13, 123.12; C-14, 109.60; C-15, 136.12; C-16, 126.88; and C-17, 65.06ppm. Mass Spectrum: LREIMS: 254(M+, 52%), 253(100), 237(23), 167(36), and 154m/e (30). References N. J. Bach, H. E. Boaz, E. C. Komfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolo~; Springer-Verlag, New York (1978). M. Flieger, N. F. Zelenkova, P. Sedmera, V. Kren, J. Novak, V. Rylko, P. Sajdl, and Z. l~eh~i6ek, Ergot Alkaloid Glycosides from Saprophytic Cultures of Claviceps, I. Elymoclavine Fructosides; J. Natural Products, Vol. 52, pp. 506-510(1989). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). A. Stoll, A. Brack, H. Kobel, A. Hofmann, and R. Brunner; Die Alkaloide eines Mutterkornpilzes von Pennisetum typhoideum rich. und deren Bildung in Saprophytischer Kultur; Helvetica Chimica Acta, Vol. 37, pp. 1815-1825(1954).

1.

Indole Alkaloids

39

Common/Systematic Name Elymoclavine-O-13-fructofuranoside Molecular Formula/Molecular Weight C22H28N206, MW = 416.19474

17 HOH2C~ O ~ 2,/O--0 H2 5'H~H, HHO H~~ CH2OH 6'

OH

H

8

,..

N--Me

//' NH

Fungal Source Saprophytic culture of ergot fungus Claviceps sp. SD 58 (ATCC 26019) and 88 EP; evidence suggested that this alkaloid formed from elymoclavine and the sucrose in the medium by the action of invertase present in the fungal mycelium. Isolation/Purification Alkaloids were separated from the culture broth (pH adjusted to 7.5 with concentrated NH3) by adsorption on bentonite (Lachema, Brno, Czechoslovakia) and desorbed with MeOH and the crude alkaloid solution was concentrated to a final volume of 10ml under low pressure conditions. The MeOH solution was loaded on a Separon SGX ClS column and eluted with MeOH-H20-concentrated NH3 (30:70:0.34, v/v/v). The column effluent was monitored by UV (288nm). The first alkaloid fraction contained a mixture of all elymoclavine fructosides. The mixture of elymoclavine fructosides was repeatedly loaded on Separon SGX Cl8 column and eluted with the above-mentioned mixture. A base line separation of all fructosides was reached. The Separon SGX C~8 column with the same mobile phase was also used for checking purity. Column effluent was monitored by UV at 224nm. Spectral Data IH NMR: (CD3OD) 6.937, H-2; 2.797, H-4tt; 3.379, H-413; 2.691, H-5; 3.174, H-7a; 3.631, H713; 6.553, H-9; 3.854, H-10; 6.950, H-12; 7.163, H-13; 7.143, H-14; 4.112, H-17u; 4.327, H-17d; 2.588, N-Me; 3.590, H-l'u; 3.717, H-I'd; 4.154, H-3'; 4.008, H-4'; 3.779, H-5'; 3.628, H-6'u; and 3.734ppm, H-6'd.

40

1.

Indole Alkaloids

13CNMR: (CD3OD) C-2, 119.98; C-3, 111.58; C-4, 27.56; C-5, 65.87; N(6)-Me, 41.02; C-7, 58.13; C-8, 134.63; C-9, 123.34; C-10, 41.65; C-11,131.91; C-12, 113.51; C-13, 123.74; C-14, 110.35; C-15, 135.61; C-16, 127.74; C-17, 65.01; C-I', 62.41; C-2', 105.79; C-3', 78.85; C-4', 77.26; C-5', 83.82; and C-6', 64.88ppm. Mass Spectrum: CIMS: (NH3) 417(33%), 416(22), 254(23), 253(30), 237(100), 236(85), 223(9), 207(6), 167(9), 154(6), and 127m/e (1). References M. Flieger, N. F. Zelenkova, P. Sedmera, V. Kren, J. Novak, V. Rylko, P. Sajdl, and Z. l~eh~i~ek; Ergot Alkaloid Glycosides from Saprophytic Cultures of Claviceps, I. Elymoclavine Fructosides; J. Natural Products, Vol. 52, pp. 506-510(1989). H. G. Floss, H. Gunter, U. Mothes, and I. Becker. Z.; Isolierung von Elymocalvin-O-13fruktosid aus Kulturen des Mutterkompilzes; Naturforsch,. Vol. 22b, pp. 399-402(1967).

1. Indole Alkaloids

41

Common/Systematic Name Elymoelavine-O-13-fruetofuranosyl-(2-,l)-O-~-D-ffuetofuranoside Molecular Formula/Molecular Weight C28H35N2Oll, ~ = 578.24756 17

I'"

OH

a"l

H

OH

Fungal Source Saprophytic culture of ergot fungus Claviceps sp. SD 58 (ATCC 26019) and 88 EP. Isolati0n/Purification Alkaloids were separated from the culture broth (pH adjusted to 7.5 with concentrated NH3) by adsorption on bentonite (Lachema, Bmo, Czechoslovakia) and desorbed with MeOH and the crude alkaloid solution concentrated to a final volume of 10ml under low pressure conditions. The MeOH solution was loaded on a Separon SGX Cls column and eluted with MeOH-H20-eoneentrated NH3 (30:70:0.34, v/v/v). The column effluent was monitored by UV (288nm). The first alkaloid fraction contained a mixture of all elymoclavine fruetosides. The mixture of elymoclavine fruetosides was repeatedly loaded on Separon SGX C18 column and eluted with the above-mentioned mixture. A base line separation of all fructosides was reached. The Separon SGX C~8 column with the same mobile phase was also used for checking purity. Column effluent was monitored by UV at 224nm. Spectral Data 1H NMR: (CI)3OD) 6.949, H-2; 2.841, H-4a; 3.404, H-4~; 2.797, H-5; 3.230, H-7a; 3.676, H7~; 6.557,1-I-9; 3.851, H-10; 6.966, H-12; 7.073, H-13; 7.151,1-1-14; 4.131, H-17u; 4.328, H-17d; 2.648, N-Me; 3.577, H-l'u; 3.649, H-l'd; 4.165, H-3'; 4.012, H..4'; N. D., H-5'; 3.627, H-6'u; and N. D., H-6'd.

42

1.

Indole Alkaloids

~3CNMR:

(CD3OD)C-2, 120.07; C-3, 111.30; C-4, 27.38; C-5, 65.82; N(6)-Me, 40.81; C-7,

58.02; C-8, 134.13; C-9, 123.62; C-10, 41.43; C-11,131.58; C-12, 113.58; C-13, 123.76; C-14, 110.44; C-15, 135.60; C-16, 127.67; C-17, 64.81; C-I', 62.54; C-2', 105.52; C-3', 79.21; C-4', 77.06; C-5', 83.80; C-6', 64.77; C-I", 62.85; C-2", 105.00; C-3", 80.01; C-4", 76.50; C-5", 83.67; and C-6", 63.91ppm. Mass Spectrum: CIMS: (NH3) 579(25%), 578(8), 416(25), 254(60), 253(45), 237(100), 236(93), 223(7), 207(3), 167(9), 154(10), and 127m/e (24). Reference M. Flieger, N. F. Zelenkova, P. Sedmera, V. I~en, J. Novfik, V. Rylko, P. Sajdi, and Z. l~ehfi~ek; Ergot Alkaloid Glycosides From Saprophytic Cultures of Claviceps, I. Elymoclavine Fructosides; J. Natural Products, Vol. 52, pp. 506-510(1989).

1. Indole Alkaloids

43

Common/Systematic Name Chanoclavine-I; Chanoclavine Molecular Formula/Molecular Weight CI6H20N20; ~ = 256.15756

CH20H Me

Me

General Characteristics Prisms and polyhedral crystals from acetone/methanol; mp., 220-222~ (dec.), [ a ] D 20 240~ [tt]54612~ 294 ~ (c=l.0, in pyridine); [tt]D2~- 205 ~ (C=0.75, in alcohol); pI~,=5.80; pK=8.2 (in aqueous methylcellosolve); violet-blue color with Keller's or van Urk's reagents. N-acetyl derivative crystallized as massive prisms; mp., 226-227 ~ C (dec.); [tt]D2~ - 180 ~ (C=0.5, in pyridine). Fungal Source Saprophytic culture of ergot fungus isolated from a tropical millet (Pennisetum typhoideum). Ergots of Elymus sp., Phragmites sp., Phalaris sp., Agropyrum sp., Balansia epichlo~ B. strangulans, B. claviceps, B. henningsiana, and Acremonium

coenophialum.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degreel biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ,~ McOH max

225, 284, and 293nm (log c=4,44, 3.82, and 3.76, respectively).

IR: 1600-1650cm "~ (characteristic of indole); see A. Hofmann, 1964.

44

1. Indole Alkaloids

Mass Spectrum: 256(M+), 237, 183(100%), 182, 167, 168, 154, and 155m/e. References W. Achlin, T. Fehr, and D. Arigoni; The Stereoehemistry of Chanoelavine-I and Isochanoielavine-I; Chemical Communications, pp. 799-800(1966). C. W. Bacon, J. K. Porter, and J. D. Robbins; Lysergic Acid Amide Derivatives from

Balansia epichlo~and Balansia claviceps (Clavicipitaceae); J. Nat. Prod., 42:309-314 (1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Laboratory Production of Ergot Alkaloids by Species of Balansia; J. Cren. Mierobiol., 113: 119-126(1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Ergot Alkaloids Biosynthesis by Isolates of

Balansia epichlo~ and Balansia henningsiana; Can. J. Bot., 59: 2534-2538(1981).

C. W. Bacon, J. K. Porter, J. D. Robbins, and D. Betowski; Ergot Alkaloids Identitication in Clavieipitaeeae Systemic Fungi of Pasture Grass; J. Agile. Food Chem., 29:653-657 (1981). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart 218 pp., 1964. A. Hofinam~ R. Brunner, H. Kobel, and A. Braek; Neue Alkaloide aus der saprophytischen Kultur des Mutterkornpilzes von Pennisetum typhoideum Rich.; Helvetica Chimica Aeta, Vol. XI, pp. 1358-1373(1957) P. C. Lyons, R. D. Plattner, and C. W. Bacon; Occurance of Peptide and Clavine Ergot Alkaloids in Tall Fescue Grass; Science, 232: 487-489(1986). D. Stauffacher and H. Tscherter; Isomere des Chanoclavins aus Clavicepspurpurea (Ft.) Tul.; Helvetica Chimica Acta, Vol. 47, pp. 2186-2194(1964).

1. Indole Alkaloids

45

Common/Systematic Name Isochanoclavine-I Molecular Formula/Molecular Weight C16H20N20; ~

= 272.15248

Me

~ - - - - C H20H Me

NH General Characteristics Crystals; mp., 190~ [a]D -208 ~ (in pyridine); rods from isopropanol; mp., 1810C,; [a]D2~ -216 ~ (C=0.5, in pyridine); blue color with van Urk's reagent; violet-blue with Keller's reagent. Fungal Source Saprophytic culture of ergot fungus, Claviceps purpurea (Fr.). Isolation/Purification The alkaloid was purified by aluminum oxide column chromatography (Act. III) eluted with chloroform/0.75% MeOH. The crude alkaloid fraction was crystallized from acetone followed by isopropanol to give rods, mp., 181 ~ sublimed under high vacuum at 170~ Spectral Data UV:

/~

MeOH max

222, 281,275(sh), and 291nm (log c=4.5, 3.89, 3.86, and 3.82, respectively).

IR.:

See Stauffacher and Tscherter, 1964. ~H NMR: See Stauffacher and Tscherter, 1964. References W. Achlin, T. Fehr, and D. Arigoni; The Stereochemistry of Chanoclavine-I and Isochanoclavine-I; Chemical Communications, pp. 799-800(1966).

46

1.

Indole Alkaloids

D. Stauffacher and H. Tscherter; Isomere des chanoclavins aus Claviceps purpurea (Fr.) Tul. Helvetica Chimica Acta, Vol. 47, pp. 2186-2194(1964).

1. Indole Alkaloids

47

Common/Systematic Name Chanoclavine-II Molecular Formula/Molecular Weight C16H20N20; MW = 256.15756 CH2OH Me Me

'~

NH

General Characteristics Prisms from acetone; mp., 174 ~ [a]D 20 -332 ~ (c--0.5, in pyridine); violet-blue color with Keller's and blue with van Urk's reagents. HCl.salt crystals from alcohol; mp., 247~ [a]D2~ -271 ~ (C=0.5, in 50% alcohol). N-Acetyl derivative, large crystalline prisms from methanol; mp., 203 ~ [a]D2~-455 ~ (C=0.54, in pyridine). Fungal Souree Saprophytic culture of ergot fungus, Clawcepspurpurea(FR.). Spectral Data UV:

~.Mm~H 222, 281, and 291nm (log c=4,50, 3.89, and 3.82, respectively); shoulders at ~,=x 275(Iog c=3.86), 245, and 289nm. IR:

(Nujol) N-aeetyl derivative: 1610em"l, (N-C)CH3. 1H NMR: N-acetyl derivative: 4.82(1H, dd, J=-10 and 4Hz, H-10); 5.53(1H, octet, J=4, 5, and 11Hz, H-5); and 2.67-3.61ppm (2H, AB part of an ABX system, J~=14Hz, J~c=l 1Hz, Jsx=SHz, H-4). N-aeetyl derivative, 4.28ppm (2H, s, allyl-CH2-O group). Reference D. Stauffacher and H. Tseherter, Isomere des Chanoclavins aus Clavicepspurpurea (Fr.) Tul.; Helvetica Chimica Acta, Vol. 47, pp. 2186-2194(1964).

48

1.

Indole Alkaloids

Common/Systematic Name N-Demethylchanoclavine-II; Norchanoclavine II Molecular Formula/Molecular Weight C15HIsN20; MW = 242.14191

CH2OH Me 2

NH General Characteristics Gray color, turning blue with Ehrlich's reagent. Fungal Source

Claviceps sp. (strain SD 58).

Isolation/Purification Culture filtrates were made alkaline to pH 11 with ammonia and extracted several times with chloroform or chloroform-isopropanol (3:1, v/v). The extracts were combined and evaporated to dryness in a vacuum. The residue was dissolved in 2% aqueous succinic acid, the solution washed 3 times with methylene chloride, made alkaline with ammonia to pH 11 and extracted with methylene chloride. This alkaloid extract was dried over anhydrous sodium sulfate, concentrated in a vacuum and left in the refrigerator overnight. The solution was filtered through a fine sintered glass funnel to remove the crystallized elymoclavine, which was washed with 3ml cold methylene chloride. The filtrate and washings were then passed throughan alumina column(Brockmann activity II-III) suspended in methylene chloride. The column was eluted with methylene chloride containing 2% methanol until no more isochanoclavine-I could be detected in the eluate. These fractions contained agroclavine, elymoclavine and isochanoclavine-I, chanoclavine-II and chanoclavine-I. The elution was continued with methylene chloride containing 10% methanol to give two more fractions. The first contained the chanoclavine-I, some chanoclavine-II and N-demethylchanoclavine-II and the following fraction contained mainly N-demethylchanoclavine-II. These two fractions were evaporated and streaked, respectively, on silica gel G plates. The plates were developed twice in acetone-ethyl acetate-N,N-dimethylfohnamide (5:5:1, v/v/v) system. The band containing N-demethylchanoclavine-II was scraped off and the alkaloid was eluted from the gel. This material was rechromatographed in chloroform-methanol (9:1, v/v) in an ammonia atmosphere to yield a chromatographically homogeneous material.

1.

Indole Alkaloids

49

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. Spectral Data

UV;

~bMmax~H 223,274, 283, jand 294nm. Mass Spectrum: HREIMS: 242.1430 (calcd for CIsHIsN20, 242.1419). The mass spectrum also showed strong peaks at 154, 156, 167, 169, 182, 194, 209, and 223m/e. The M + ion peak (242re~e) was the base peak (100%). Reference J. M. Cassady, C. I. Abou-chaar, and H. G. Floss; Ergot Alkaloids. Isolation of N-Demethylchanoclavine-II from Claviceps Strain SD 58 and the Role of Demethylchanoclavines in Ergoline Biosynthesis; Lloydia, Vol. 36, pp. 390-396(1973).

50

1.

Indole Alkaloids

Common/Systematic Name Setoclavine Molecular Formula/Molecular Weight C16HlsN20, lk4Vr = 254.14191 Me

OH N--Me

NH General Characteristics Prisms from methanol-acetone; m.p. 229-234~ (dec.); [a]Dz~+ 174~ [a]546120 + 232 ~ (c=l.1, in pyridine); [a]Dz~+ 165 ~ (C=0.3, in alcohol); pK=6.4 (in 80% aqueous methylcellosolve). Fungal Source Saprophytic culture of ergot fungus isolated from a tropical millet (Pennisetum typhoideum); Elymus mollis, Agropyrum semicostatum, Trisetum bifidum, and Festuca

rubra.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~ ~b MeOH max

243 and 313nm (log e=4.38 and 4.04).

IR:

See A. Hofmann, 1964. Mass Spectrum: 254(M+), 236, 235, 234, 219, 211, 196, 181,168, and 154m/e (100%).

1. Indole Alkaloids

51

References B. Berde and H. O. Scheld (eds.), Ergot Alkaloids and Related Compounds, In Handbook of Experimental Pharmacology, Springer-Verlag, New York (1978). A. Hofmann, Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp, 1964. A. Hofinann, R. Brunner, H. Kobel, and A. Brack, Neue Alkaloide aus der saprophytischen Kultur des Mutterkompilzes von Pennisetum typhoideum Rich., Helvetica Chimica Acta, Vol. XI, pp. 1358-1373(1957).

52

1. Indole Alkaloids

Common/Systematic Name Isosetoclavine Molecular Formula/Molecular Weight CI6HIsN20, MW = 254.14191 HO

Me

N--Me

NH General Characteristics Large polyhedral crystals from methanol; m.p. 234-237~ [a]u 2~+ 107 ~ [a]s~il 2~ + 147 ~ (c=0.5, in pyridine); [a]D2~+ 129 ~ (C=0.4, in alcohol); pK=5.9 (in 80% aqueous methylcellosolve). Soluble in 70 parts boiling methanol, 60 parts boiling acetone or 160 parts boiling chloroform. Responses to various color reactions were similar to setoclavine. Hydrochloride crystallized as rosettes from methanol diluted with acetone, did not melt at temperatures up to 300~ Fungal Source Saprophytic culture of ergot fungus isolated from a tropical millet (Pennisetum

typhoideum).

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~

~

MeOH max

242 and 317nm (log e=4.42 and 4.10).

IR~ See A. Hofmann, 1964.

1. Indole Alkaloids

53

References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmar~logy_; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. A. Hofmann, R. Brunner, H. Kobel, and A. Brack; Neue Alkaloide aus der saprophytischen Kultur des Mutterkompilzes yon Pennisetum typhoideum Rich.; Helvetica Chimiea Aeta, Vol. XI, pp. 1358-1373(1957).

54

1.

Indole Alkaloids

Common/Systematic Name Costaclavine Molecular Formula/Molecular Weight C16H2oN2, M W = 240.16265

Me H ~:---~/N--Me

NH General Characteristics Crystals (prisms) from ether-acetone, acetone, methanol or ethanol; mp., 182-184 ~ (dec.); [a]D2~+ 44 ~ [a]546~2~+ 59 ~ (c=0.2 in pyridine); positive for van Urk's reaction; insoluble in petroleum ether, sparingly soluble in ethyl acetate, moderately soluble in benzene and chloroform and readily soluble in acetone, methanol, ethanol, and pyridine. Fungal Source Penicillium chermesmum (PC 106-1), Agropyrum type ergot fungus parasitic on Agropyrum semicostatum Nees., Trisetum bifidum Ohwi, and Festuca rubra L., etc. growing in Japan. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Dat.a. UV:

~

MeOH max

225,275, 282, and 292nm.

IR:

See A. Hofmann, 1964.

1.

Indole Alkaloids

55

References M. Abe, S. Yamatodani, T. Yamono, and M. Kusumoto; Bull. Agr. Chem. Soc. (Japan), Vol. 20, pp. 59-60(1956). S. L. Agurell; Costaclavine from Penicillium chermesinum; Experientia, Vol. 20, pp. 2526(1964). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Vedag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

56

1.

Indole Alkaloids

Common/Systematic Name Pyroclavine Molecular Formula/Molecular Weight C16H20N2; M W -- 2 4 0 . 1 6 2 6 5

••/N--Me H

Me

NH

General Characteristics Crystals (needles) from ethyl acetate, methanol or benzene, mp., 204~ (uncorr.); [a]D2~ - 90 ~ [a]54~ 2~- 105 ~ (c=0.2 in pyridine); gave a deep blue color with Allport and Cockings's reagent and coned, sulfuric acid; insoluble in petroleum ether; moderately soluble in ethyl acetate and benzene; readily soluble in chloroform, acetone, methanol, ethanol and pyridine. Color reactions were identical to festuclavine. Fungal Source

Agropyrum-type ergot fungus parasitic on Agropyrum semicostatum, Trisetum bifidum, Festuca rubra L., etc. growing in Japan.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~

~

MeOH max

225, 275, 282, and 292nm.

IR~

See A. Hofmann, 1964.

1.

Indole Alkaloids

57

References M. Abe, S. Yamatodani, T. Yamono, and M. Kusumoto; Bull. Agr. Chem. Soc. (Japan), Vol. 20, pp 59-60(1956). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

58

1.

Indole Alkaloids

Common/Systematic Name Molliclavine Molecular Formula/Molecular Weight C16HIsN202; M W = 270.13683

CH2OH HO

N--Me

General Characteristics Crystallized as prisms from methanol or acetone; mp., 253~ (dec.); [a]D 17 "+"3 0 ~ [~]546117 -k- 42 ~ (c=0.2, in pyridine); green color with Keller's and van Urk's reagent. Soluble in methanol and acetone; sparingly soluble in benzene and chloroform; and very soluble in water. Fungal Source Sclerotia and saprophytic cultures ofElymus mollis. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~, M,o. 226, 287, and 294nm. max

IR:

See A. Hofmann, 1964. References Abe and S. Yamatodani; Isolation of Further Two Water Soluble Ergot Alkaloids; J. Agric. Chem. Soc. Japan, Vol. 28, p. 501 (1954).

1. Indole Alkaloids

59

A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. B. Berde and H. O. Seheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology, Springer-Verlag, New York (1978).

60

1.

Indole Alkaloids

Common/Systematic Name Penniclavine Molecular Formula/Molecular Weight CI6HIsN202;

MW

-

270.13683

HOH2C. ,OH .~-"~N--Me

~"

H

General Characteristics Crystals from methanol or acetone; mp., 222-225~ [a]D2~ +151 ~ [a]s~il2~ +201 o (c=0.5, in pyridine); pK=6.4 (in 80% aqueous methylcellosolve). Fungal Source Sclerotia and cultures of Pennisetum typhoideum. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomeduUary and mesodiencephalic components). Spectral Data UV; MeOH ~ max

240 and 315nm (log e=4.29 and 3.93, respectively).

IR~ See A. Hofmann, 1964. IH NMR: (pyridine-ds) H-6, 2.46(3H, s); H-7, 2.79(1H, d); H-4, 2.73(1H, ddd); H-7', 3.17(1H, dd); H-4', 3.20(1H, ddd); H-5, 3.53('1H, dd); 17-CH2, 4.00(2H, s); 17-OH, 5.26.2(2H); H-9, 6.85(1H, m); aromatic-H, 7.1-7.4(4H, m); and H-l, 11.43ppm (1H, s).

1. Indole Alkaloids

61

Mass Spectrum: LREIMS: 270(M+, 93%), 243(23), 234(72), 219(98), 208(60), 196(57), 192(43), 181(57), 167(32), and 154m/e (100). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook 0fExperimental Pharmacoloffy; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis o f Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). A. Stoll, A. Brack, H. Kobel, A. Hofmann, and R. Brunner; Die Alkaloide eines Mutterkornpilzes von Pennisetum typhoideum rich. und deren Bildung in Saprophytiseher Kultur; Helvetica Chimica Acta, Vol. 37, pp. 1815-1825(1954).

62

1. Indole Alkaloids

Common/Systematic Name Cycloclavine Molecular Formula/Molecular Weight C16HIsN2; MW = 238.14700 Me

~N--Me

9

NH

General Characteristics Colorless needles from methanol; mp 164-165 ~ C; [a]D 22 + 40 ~ (c=0.175, in pyridine); [a]54622 + 58~ in pyridine); gave a reddish purple color, turning to bluish-purple, with Allport-Cocking's reagent; an orange color with Dragendorff reagent. Sparingly soluble in benzene; moderately soluble in acetone, methanol, ethanol, chloroform, and ethyl and butyl acetate; readily soluble in pyridine; almost insoluble in cold water; readily soluble in dilute acids. It yielded no isomer on treatment with acid or alkali. Fungal Source

Aspergillusjaponicus.

Isolation/Purification Mycelia extracted with 0.1N H2504, combined with the culture filtrate, pH adjusted to 10 and the mixture repeatedly extracted with ethyl acetate. The alkaloid residue purified with silica gel column chromatography eluting with chloroform-methanol (955, v/v). Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~ EtOH max

227(1og 6=19.6), 276(3.66), 282(3.84), and 293nm (2.66).

1.

Indole Alkaloids

63

IR~ (KBr) 3400(indole NH) and 3150cm"~. IH NMR: (CDCI3) 0.45 and 1.62ppm (2H, AB system, cyclopropyl-CH2); 1.69ppm (3H, s, CCH3); 2.35ppm (3H, s, N-CH3); 2.4-3.3ppm (5H, m, -CH-N- or-CH-C=C-); 6.77.3ppm (4H, m, indole protons); and 8.2ppm (1H, s, indole NH).

Mass Data: 238(M+), 237(base peak), 167, and 154m/e; anal. found; C, 80.24; H, 7.72; N, 11.77; calcd, for Cl6HlsN2: C, 80.63; H, 7.61; N, 11.75%. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). S. Ohmomo, T. Sato, T. Utagawa, and M. Abe; Isolation of Festuclavine and Three New Indole Alkaloids, Roquefortine A, B and C from the Cultures of Pellicillium roqueforti; Agr. Biol. Chem., Vol. 39, pp. 1333-1334(1975).

64

1.

Indole Alkaloids

Common/Systematic Name Ergotamine Molecular Formula/Molecular Weight C33H35NsOs; MW = 581.26382

o

Me

II

N H .......... "~' " ~ ' O " ~ N :/

C

" I.... .

OHI

,\

o _- -1

'

/ --

NI . i I ==O

General Characteristics Elongated prisms from benzene; mp., 212-214 ~ (dee.); [a]D2~ 160", [a]5~sl2~ -192" (c--0.5, in CHCI3); [a]Dz~ -12.7 ~ [a]5~ 2~ -8.6 ~ (C----0.5,in pyridine). Becomes solventfree only after prolonged heating at high vacuum; darkens and decomposes on exposure to air, heat, and light. Very hygroscopic; pK = 5.6 (in 80% aqueous methyleellosolve); blue color with Keller's reagent. Soluble in 70 parts methanol, 150 parts acetone, 300 parts ethanol; freely soluble in chloroform, pyridine, and glacial acetic acid; slightly soluble in benzene; almost insoluble in water and petroleum ether. Fungal Source

Claviceps purpurea.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergie blockage), and central nervous system effects (bulbomedullary and mesodiencephalie components). LDs0 in rats 62mg/kg (IV).

1. Indole Alkaloids

65

Spectral Data (See A. Hofmann, 1964). ~H NMR: (CDCIa) H-I, 8.14(s); H-2, 6.91(m); H-4a, 2.79(m, ,/=14.2, 11.9Hz); H-4b, 3.32(,/=14.2, 5.0Hz); H-5, 3.73(m, 3=11.9, 5.0Hz); 6-N-Me, 2.61(s); H-7a, 2.96(`/=11.9, 3.9Hz); H-7b, 2.78(dd, 3--11.9, 3.4Hz); H-8b, 3.18(,/=-5.5, 3.9, 3.4Hz); H-9, 6.34(J=5.5Hz); 8-NH, 9.04(s); H-12-14, 7.0-7.5(m); H-2'Me, 1.51(s); H-5', 4.69; OH, 6.97(s); and 7'-NH, not obs. 13C NMR: (DMSO-d6) C-2, 119.4; C-3, 108.8; C-4, 26.6; C-5, 62.4; C-7, 55.1; C-8, 42.5; C-9, 118.3; C-10, 136.0; C-11, 127.1; C-12, 111.0; C-13, 122.2; C-14, 110.2; C-15, 133.8; C-16, 125.9; C-17, 174.3; and N-Me, 43.4ppm. TLC Data Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References N. J. Bach, H. E. Boa.z, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968).

66

1. Indole Alkaloids

J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). J. K. Porter and D. Betowski; Chemical-ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from Cenchrus echinatus (Sandbur Grass) Infected with Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

67

Common/Systematic Name Ergotaminine Molecular Formula/Molecular Weight C33H35NsOs; MW' = 579.24817

o

Me

II C

NH..

H -

OHI

.......... ,, N ,T,,,.O/~ --_ l 1~ i

o=1

'

/

J

o

NH

General Characteristics Crystallizes solvent-free as thin rhombic plates from methanol; m.p. 241-243 ~ (dec.); [tt]o 2~+ 369 ~ (c=0.5, in CHCI3); [tt]D2~+ 397 ~ (C=0.5, in pyridine). Soluble in about 1000 parts boiling ethanol, 1500 parts boiling methanol; fairly soluble in chloroform, pyridine, and glacial acetic acid; pK = 5.6 (in 80% aqueous methylcellosolve); blue color with Keller's reagent; does not form salts. Fungal Source

Claviceps purpurea.

Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and-~-uantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

68

1. Indole Alkaloids

Spectral Data IR:

(See A. Hofmann, 1964.)

~H NMR: (CDCI3) H-I, 8.00(s); H-2, 6.91(m); H-4a, 3.59(m, J=15.3, 4.9Hz); H-4b, 2.62(J=15.3, 10.4Hz); H-5, 3.23(m, J=10.4, 4.9Hz); 6-N-Me, 2.61(s); H-7a, 3.13(J=11.7, 1.7Hz); H-7b, 2.76(dd, J=l 1.7, 3.8Hz); H-8b, 3.07(J=6.3Hz); H-9, 6.25(J=6.3Hz); 8-NH, 9.83(s); H-Z'Me, 1.49(s); H-5', 4.61; OH, 6.94(s); and 7'-NH, not obs. ~3CNMR: (DMSO-d6) C-2, 119.7; C-3, 109.0; C-4, 26.9; C-5, 61.7; C-7, 53.0; C-8, 41.8; C-9, 118.1; C-10, 137.1; C-11,127.9; C-12, 111.4; C-13, 122.4; C-14, 110.3; C-15, 133.8; C-16, 126.1; C-17, 175.3; and N-Me, 42.5ppm. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from Cenchrus echinatus (Sandbu~Grass) Infected with Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

69

Common/Systematic Name 8-Hydroxyergotamine Molecular Formula/Molecular Weight C33HasNsO6, M W = 5 9 7 . 2 5 8 7 3 13'

M e ~, O H I

.o oo .,oj ,

I

9'

8'

NN General Characteristics Crystals from hot ethyl acetate and chloroform were obtained; mp., 197~ [tt]D2~ +14" (C=1%, in pyridine); [a]2546.12~+32.9 (c=1%, in pyridine). The compound has good solubility in dioxane, pyridine, and dimethyl sulfoxide;is less soluble in methanol and chloroform. It gave positive van Urk's and Keller's tests. Fungal Source Natural ergot (Claviceps purpurea). Isolation/Purification Ground ergot from the field culture containing mainly ergotamine was extracted with a mixture of ether and ethanol (1:1, v/v) at room temperature. The extracted alkaloids were treated with 1% tartaric acid. After neutralization by aqueous ammonia to pH 7.5, crude alkaloid mixture was obtained. The peptide alkaloids were then transformed to their levorotatory forms by reaction with 1N sulfuric acid in absolute ethanol and acetic acid. The bases were prepared from the mixture of crude crystalline sulfates by aqueous sodium hydrocarbonate and extracted with ether. Ergotamine was removed by crystallization. Mother liquors were concentrated in vacuo at 40~ giving the bases. Ergotamine, ergocristine and ergostine were transformed into their poorly soluble dextrorotatory forms by boiling eight hours in twenty-fold excess of methanol and their crystals were filtered off alter cooling. The residue in the mother liquors was subjected again to the same operation. It was further separated by chromatography on silica gel using chloroform-ethanol, 99:1, v/v. The new alkaloid was rechromatographed on silica gel using chloroform-benzene, 4:1, v/v. The product was crystallized from hot ethyl acetate and then from hot chloroform.

70

1. Indole Alkaloids

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

Spectral Data UV: 3,~2~

261(1oge=3.95) and 381nm (3.57).

13C NM~: (DMSO-d6) Peptide part- C-2', 86.8 s; C-3', 166.9 s; C-5', 57.2 d; C-6', 165.3 s; C-8', 46.8 t; C-9', 22.6 t; C-10', 26.8 t; C-1 l', 64.8 d; C-12', 103.7 s; C-13', 24.8 q; C-14', 39.4 d; C-15', 139.7 s; C-16', C-20', 130.7 d; C-17', C-19', 128.6 d; and C-18', 128.5ppm d. Ergoline part- C-2, 120.6 d; C-3, 109.5 s; C-4, 26.8 t; C-5, 62.4 d; C-7, 62.9 t; C-8, 72.7 s; C-9, 121.5 d; C-10, 139.0 s; C-1 l, 127.1 s; C-12, 112.3 d, C-13, 123.2 d; C-14, 111.5 d; C-15, 134.8 s; C-16, 127.1 s; C-17, 176.6 s; and N-CH3, 43.8ppm q. Mass Data: 314(30%, C17HlgN204), 283(13, CI6HI7N302), 265(C16HIsN30), 244(C14I-'I16N202), 240(21, C~4H~2N202), 196(10, C~3HIEN2), 167(22, C~2HgN), 154(36, C~IHsN), 153(78, CTH9N202), 125(53, C6HgN20), 120(7, CsH~oN), 91(50, C7H7), 70(100, C4HsN), and 43m/e (535, CzH30); elemental analyses found: 66.88% C, 6.0% H, 11.7% N; for C33H35N506 calcd 66.32% C, 5.90% H, 11.72% N. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds, In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Krajicek, B. Trtik, J. Sp~i~il,P. Sedmera, J. Vokoun, and Z. Reh~t~,ek; 8Hydroxyergotamine, a New Ergot Alkaloid; Czechoslov. Chem. Commun. Vol. 44, pp. 2255-2260(1979).

1. Indole Alkaloids

71

Common/Systematic Name Ergosine Molecular Formula/Molecular Weight C3oH37NsOs; MW' = 547.27947

H,,

o II C "'

Me la 17

NH .........

.~~a~'N'N--Me H

0----~ a

0

OH, 11'

l

N s..'.,~J==O

/ _

H

/Me

CH2CH \Me

"

Nit

General Characteristics Prisms from ethyl acetate; mp., 228~ (dec.); [a]D2~- 161 o (c=l.0 ' in CHCI3), [tt]D2~-8 ~ (C=I.0, in pyridine). Soluble in chloroform; fairly soluble in methanol and acetone; sparingly soluble in ethyl acetate and benzene; pK = 5.5 (in 80% aqueous methylcellosolve); blue color with Keller's reagent; does not form salts. Fungal Source

Claviceps purpurea.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

72

1.

Indole Alkaloids

Spectral Data IR:

(See A. Hofmann, 1964.) Mass Spectrum: CIMS: (isobutane) 268, 281, and 21 lm/e; LREIMS: 267,196, 154, 125, 86, 70 (100%), 44, and 43m/e. TLC Data Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or l:l, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4: l, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Seheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann, Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter, Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock, J. Animal Science, Vol. 73, pp. 871-880(1994). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and/L Leuchtmann, Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from C e n c ~ echinatus (Sandbur Grass) Infected with Bakmsia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1.

Indole Alkaloids

73

Common/Systematic Name Ergosinine Molecular Formula/Molecular Weight CaoHaTNsOs; MW = 547.27947

0n C H :- 17

Me OH, , 18 , , , ,/, 1~" ~ / . ~11' .,J NH......... O~N~s'i~"-O N--Me 3' ~ . Me

r YIH' "

H'CH2CH

\Me

General Characteristics Prisms from ethyl acetate, 90% alcohol, aqueous acetone or benzene; mp., 228~ (dec.); [a]D 20 + 420 ~ (c=l.0, in CHCI3), [a]546120 + 522 ~ (c=l.0, in CHCI3), [a]D 20 + 3 8 0 ~ (c=l.0, in acetone). Very readily soluble in chloroform; readily soluble in acetone; less soluble in ethyl acetate; very sparingly soluble in methanol; almost insoluble in water; pK = 5.5 (in 80% aqueous methylcellosolve); blue color with Keller's reagent; some amorphous salts have been prepared, but only with difficulty. Fungal Source Claviceps purpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and

74

1.

Indole Alkaloids

adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data IR~ (See A. Hofmann, 1964.) TLC Data Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group, and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

75

CommordSystematic Name Ergostine Molecular Formula/Molecular Weight C34H37NsOs; M W - 5 9 5 . 2 7 9 4 7

Me

,

H,,

o C

....

I CI-12 O H ,

L

,7

~,

'

NH............. , ~ 0 s

N--Me

3'

,

o.i-

Y

N T~-i N~s'jJ===O '-_

.--.

NH

General Characteristics Prisms from acetone or ethyl acetate; mp., 211-212~ (dec.); [a]D 2~ - 169 ~ [a]5,s~ 2~ 203~ in CHCI3); [ a i D 20 - 38 ~ [ a ] 5 4 6 1 2 0 - 39~ in pyridine). Moderately soluble in acetone; slightly soluble in chloroform and ethanol; sparingly soluble in benzene and ethyl acetate. Blue color with Keller's reagent changing to blue-green in approximately 15 seconds. Fungal Source

Claviceps purpurea strain D-3-18.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Soectral Data IR:

(See Hofmann, 1964.)

76

1.

Indole Alkaloids

Mass Spectrum: 328, 267, 244, 154, 153, 125, 91, 70(100%), and 57m/e. CIMS: (isobutane) 268(74%), 329(100), and 245m/e (47). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology_; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~ehfi~ek, J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). J. K. Porter and D. Betowski; Chemical-Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981).

1. Indole Alkaloids

77

Common/Systematic Name Ergostinine Molecular Formula/Molecular Weight C34I-I37N505; MW = 595.27947 Me I

CH2 OH,

o

II

C H : 17

18

NH ......... O===i3,

0

-

11'

Ns . . ~ = = = O

~~'~N--Me

"

NH

General Characteristics Prisms from methanol; mp., 215-216~ (dec.); [a]D 20 + 357 ~ [a]s4612~+ 4460(c=1.0, in CHCI3); [a]D2~ + 429 ~ [a]54612~+ 538~ in pyridine). Slightly soluble in chloroform and sparingly soluble in benzene. Blue color with Keller's reagent changing to blue-green in 2-3 minutes. Fungal Source

Claviceps purpurea strain D-3-18. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data IR.: (See Hofmann, 1964.)

78

1.

Indole Alkaloids

TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolo~r Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1.

Indole Alkaloids

79

Common/Systematic Name Ergonine Molecular Formula/Molecular Weight C30H37NsOs; MW = 547.27947 Me

H,,

I

0 C

CH20HI

/

NH

|l,

N, "

~

I

o

Me NH General Characteristics Crystals from EtOH/diisopropylether; mp., 206-207~ (synthetic ergonine; mp., 207208~ Fungal Source Claviceps purpurea. Isolation/Purification The mother liquor of the ergokryptine-ergocornine producing strain of Claviceps purpurea consisting of ergosine (90%) and ergonine (10%) was chromatographed on alumina (activity II) atter removing the ergosine as the di-p-toluyl-L-tartaric acid salt (twice). Elution with dichloromethane/0.3% methanol followed by crystallization from ethanol/diisopropylether yielded purified ergonine. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

80

1.

Indole Alkaloids

Spectral Data UV: ~

Dichlommethzne max

238(1og 6=4.31) and 307.5nm (3.95).

IR:

(dichloromethane) 3460, 1728, 1668(sh), and 1649cmq. 1H NMR:

(CDCI3) 9.05(1H, s, N1-H); 8.20(1H, s, CONH); 7.0-7.3(3 + 1H, m, aromatic H, OH); 6.90(1H, s, C2-H); 6.20-6.5(1H, m, C9-H); 4.40(1H, d, J=5Hz, C5'-H); 1.54(--16H, m), 2.6(3H, s, N6-CH3); 1.15(3 + 3H, d, J=THz, CH(CH3)2; and 0.91(3H, t, J=THz, CH2-CHs). Mass Spectrum: (Field desorption) Found 547re~e;calcd for C30H37N505 547m/e. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. Brunner, P. L. Stutz, H. Tschekter, and P. A. Stadler; Isolation of ergovaline, ergoptine, and ergonine, new alkaloids of the peptide type, from ergot sclerotia ; Can. J. Chem., Vol. 57, pp. 1638-1641(1979).

1. IndoleAlkaloids

81

Common/Systematic Name Ergovaline Molecular Formula/Molecular Weight C29H3sNsOs; MW = 533.26382

o

Me

II

C

OHI

/

N H ......

H,. . . .

0

~N--Me

~

~

0

H-

'--_ CHiMe

I

Me

NH General Characteristics Crystallized from ethyl acetate; mp., 207-208~ (synthetic ergovaline; mp., 207-208"C; [t~]D2~- 172 ~ (C=0.5, in CHCI3). Fungal Source Claviceps purpurea (strain No. 235), Epichlo~ typhina and Acremonium coenophialum infected fescue (Festuca arundinacea Schreb.). Isolation/Purification Sclerotia of Claviceps purpurea were ground and defatted by extraction with petroleum ether. The dark violet residue was extracted with a mixture of 70% acetone and 30% water containing 5% tartaric acid. The extracts were concentrated in vacuo at 50~ The acidic solution was rendered alkaline by addition of Na2CO3, extracted with ethyl acetate, washed with water, dried 0Nla2SO4), and evaporated at 50~ m vacuo. Chromatography of the crude extract on alumina, activity II followed by chromatography on DS-O Camag, activity I and crystallization from ethyl acetate gave purified ergovaline. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

82

1.

Indole Alkaloids

Spectral Data UV:

~,m~O~.....~e

238(1oge=4.3) and 307nm (3.95).

IR:

(dichloromethane) 3466, 1727, 1665(sh), and 1650cm~. 'H NMR: (DMSO) 10.8(1H, s, N1-H); 9.37(1H, s, CONH); 7.0-7.3(4H, m, aromatic H); 6.75(1H, d, J-2Hz, OH); 6.33(1H, s, C9-H); 4.31(1H, d, J=5Hz, C5'-H); 2.61(3H, s, N6-CH3); 1.7-3.9(~14H, m); 1.58(3H, s, C2'-CH3); and 1.08ppm (6H, d, J=7H~ CH(CH3)2). Mass Spectrum: HRIMS: found 533.2602m/e; calcd for C29H35NsO5 533.2638; CIMS (isobutane): 534(M § + 1, 8), 268(100), 267(74), and 197m/e (12%). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology_, Springer-Verlag, New York (1978). R. Brunner, P. L. Stutz, H. Tschekter, and P. A. Stadler; Isolation of Ergovaline, Ergoptine, and Ergonine, New Alkaloids of the Peptide Type, from Ergot Sclerotia; Can. J. Chem., Vol. 57, pp. 1638-1641(1979). A. Hofmann; Die Mutterkorn Alkaloide; Elke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter, C. W. Bacon, J. D. Robbins, and D. Betowski; Ergot Alkaloid Identification in Clavicipitaceae Systemic Fungi of Pasture Grass; J. Agric. Food Chem., Vol. 29, pp. 653-657(1981). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981). M. R. Siegel, G. C. M. Latch, L. B. Bush, F. F. Fannin, D. D. Rowan, B. A. Tapper, C. W. Bacon, and M. C. Johnson; Fungal Endophyte-infected Grasses: Alkaloid Accumulation and Aphid Response; J. Chem. Ecol., 16:3301-3315(1990).

1.

Indole A l k a l o i d s

83

Common/Systematic Name Ergoptine Molecular Formula/Molecular Weight CalH3aNsOs; MW = 561.29512 Me

I

o

c�89

"

C

NH .....

/

~

~N--Me

i"~l i |,,

~

H

~

I CH

M/ \ Me /

NH

General Characteristics Crystals from acetone/water (7:3), mp., 198-199~ (synthetic ergoptine; mp., 199200~ [a]D2~-188 ~ (C=0.8, in CHCI3). Fungal Source

Claviceps purpurea.

Isolation/Purification The mother liquor from ergokaTptine-ergocomine production with a total alkaloid content of about 80% (determined colorimetrically with van Urk's reagent) was chromatographed on alumina (activity II). Elution with chloroform/0.3% methanol led to a first fraction, which consisted primarily of ergokryptine and ergocomine. A second fraction was obtained by elution with chloroform/0.6% methanol which contained about 25% ergoptine. This fraction was further enriched by chromatography on alumina (activity H). The first fraction obtained on elution with chloroform/0.3% methanol contained kryptine and ergocornine. Elution with chloroform/0.6% methanol gave ergoptine (80% purity). Ergoptine was further purified by conversion into a salt with di-p-toluyl-L-tartaric acid. The product was crystallized and cleaved by partition between chloroform Na2COs giving an amorphous resin which was again converted into the salt with di-p-toluyl-L-tartaric acid. The salt obtained, mp 177-178 ~ was cleaved by partition in chloroform/2 N Na2COs and afforded ergoptine with a 95% purity. Further purification of the base by silica gel column chromatography followed by crystallization from acetone/water (7:3, v/v) yielded homogeneous ergoptine.

84

1.

Indole Alkaloids

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). v

Soeetral Data UV:

238(1og e=4.34) and 308nm (3.98). IR:

(dichloromethane) 3470, 3150-3300, 1732, 1671(sh), and 1653em"1. 1H M R : (CDCI3) 9.58(1H, s, N1-H); 8.17(1H, s, CONH); 7.1-7.3(3 + 1H, m, aromatic I-I, OH); 6.99(1H, s, C2-I-I); 6.34-6.5(1H, m, C9-H); 4.58(1H, t, J----6I-Iz,C5'-H); 2.8-4(-~9H, m, C4-H, C5-H, C7-H, C8-H, C8'-H, and C11'-H); 2.68(3H, s C6-CI-I3); 1.7-2.4(--9H, m, C2'-CH2, C5'-CH2, C9'-CH2, C10'-CH2 and side chain CI-I); and 1.9-2.2ppm (9H, side chain CH3). Mass Spectrum: HRIMS" Found 561.2875m/e; ceded for C31H39N~Os,561.2951. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92: 8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook ofExperim, entfl Pharmacoloffy, Springer-Verlag, New York (1978).

1.

Indole Alkaloids

85

R. Brunner, P. L. Stutz, H. Tschekter, and P. A. Stadler; Isolation of ergovaline, ergoptine, and ergonine, new alkaloids of the peptide type, from ergot sclerotia, Can. J. Chem., Vol. 57, pp. 1638-1641(1979). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

86

1.

Indole Alkaloids

Common/Systematic Name Ergocornine Molecular Formula/Molecular Weight C31H39NsOs" M W = 561.29512

M e \ /Me CH _OH

o II

C

,NH.......

HIIll,

0

~ N - - M e

Nil General Characteristics Solvated polyhedra from methanol; mp., 182-184~ (dec.); [a]D2~ -188", [a]s~l 2~ 224~ in CHCI3); [a]D2~ -111 ~ [tg]546120 -129 ~ (c=l.0, in pyridine); pK = 5.5 (in 80% methylcellosolve); blue color changing to green with Keller's reagent. Soluble in acetone, chloroform, and ethyl acetate; slightly soluble in ethyl and methyl alcohol; nearly insoluble in water. Fungal Source Sclerotia of Claviceps purpurea. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects

1.

Indole Alkaloids

87

(bulbomedullary and mesodiencephalic components). LDs0 IV in rabbits 1.17mg/kg.

Spectral Data IR:

(See A. Hofmann, 1964.) Mass Spectrum: CIMS: (isobutane) 562(M+ + 1, 14%), 268(100), 297(72), and 197m/e (11). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology_; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. H. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785-789 (1983). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agile. Food Chem., Vol. 29, pp. 651-653(1981).

88

1. Indole Alkaloids

Common/Systematic Name Ergocorninine Molecular Formula/Molecular Weight C31H39N505; M W "- 561.29512

M e \ /Me CH

o H

II C

NH ....

\

H

N--

_OH O

Me/`` Me

NH

General Characteristics Crystallizes solvent-free, prisms from alcohol; mp., 228~ (dec.); [tt]D2~+ 404 ~ [a]54612~ + 504 ~ (c=l.0, in CHCI3); [t~]D2~+ 488 ~ [a]546120 "~"624 ~ (c= 1.0, in pyridine); pK = 4.8 (in 80% methylcellosolve); blue color changing to green with Keller's reagent. Soluble in 15 parts boiling ethanol, 25 parts boiling methanol, 30 parts boiling benzene, 30 parts boiling ethyl acetate; freely soluble in acetone and chloroform; nearly insoluble in water. Does not seem to form salts. Fungal Source Sclerotia of Clavicepspurpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform ot methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification, and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser

1.

Indole Alkaloids

89

degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some ergopeptine alkaloids are used routinely in medicine. LDs0 IV in rabbits 1.17mg/kg. Spectral Data. IR; See A. Hofmann, 1964. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. H. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785-789 (1983). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981).

90

1. Indole Alkaloids

.Common/Systematic Name O- 12'-Methylergocomine Molecular Formula/Molecular Weight C32H41NsOs, ~ = 575.31077 Me

Me

,3[

,c\ H/

0 II

~7C

H,, .... 8

18

Me_O

I

]

I ~ 0 . ~ i_--~ 1 ~11' ~ j " ~ I_ " N " ~ I H I O~ 3 N ~' 6 0

NH ...............' - r 2 ' Me

16

e)2

Fungal Source

Clavicepspurpurea Strain 231 FI.

Isolation/Purification Crude alkaloids were obtained by extraction of fungal cultures with CH2CI2-MeOH (80:20, v/v) followed by precipitation with petroleum ether; the crude alkaloid extract was chromatographed by Extrelut (Merck) column chromatography eluted with CH2C12. The crude alkaloids thus obtained were chromatographed on a Silica gel 60 (Merck) column eluted with CH2CI2-MeOH (98:2, v/v); the column was monitored by UV and TLC. Final purification was by preparative TLC in a cold room in the dark. TLC was performed on Silica gel F254, plates (Merck) using the following solvent systems: CH2C12-isopropyl alcohol (92:8, v/v) or CH2CI2-MeOH (90:10, v/v). The alkaloids were detected by examination under UV light at 254 and 366 nm while some were sprayed with N,N-p-dimethylaminobenzaldehyde. The various bands were scraped off and eluted with CH2CI2-MeOH (1:1, v/v). Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater of lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Soectral Data IH NMR: (CDCI3) 8.07(1H, s, NH-1); 2.66 (3H, s, N-CH3), 6.40(1H, dd, J=-2.0, 5.9Hz, H-9); 9.17 (1H, CONH); 3.31 (3H, s, OCH3); and 4.51ppm (1H, d, J--4.1Hz, H-5').

1.

Indole Alkaloids

91

~3CNMR (CDC13) C-2, 119.0"; C-3, 110.8; C-4, 26.6; C-5, 59.4; N(6)-CH3, 44.1; C-7, 49.2; C-8, 41.3; C-9, (119.2); C-10, 138.8; C-11, 129.8; C-12, 112.0; C-13, 123.4; C-14, 110.0; C-15, 133.8; C-16, 126.2; 8-CONH, 173.9; C-2', 89.2; C-3', 164.7"*, C-5', 60.7; C-6', 168.7"*; C-8', 45.8; C-9', 22.1"**; C-10', 22.2***; C-11', 61.4; C-12' , 106.8; 12'-OCH3, 49.2; (2')-C, 35.7; (2')-CH-(CH3)2, 16.0, 16.7; (5')-C, 32.2; and (5')-C-C-(CH3)2, 19.5, 19.2ppm. *, **, and *** assignments may be reversed. Mass Spectrum: EIMS: 575(M+), 543,446, 418, 347, 319, 304, 221, 196, 154, and 70m/e (100%). Reference N. Crespi-Perellino, M. Ballabio, B. Gioia, and A. Minghetti; Two Unusual Ergopeptines Produced by a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 50, pp. 1065-1074(1987).

92

1. Indole Alkaloids

Common/Systematic Name Ergocristine Molecular F0rmula/Molecular,Weight C35H39N505; MW 609.29512 -

-

Me

\/

Me

CH

o II

C

�9N H ......

OH,

.

'0

Nit General Characteristics Orthorhombic crystals from benzene with 2 molecules benzene of crystallization; the solvent-free base melts at 155-157~ (dec.); [a]D 2~ -183 ~ [a]5,s] 2~-217 ~ (c=1.0, in CHCI3); [a]D2~ -108 ~ [a]~] 2~ -125 ~ (C=I.0, in pyridine); pg = 5.5 (in 80% methylcellosolve); blue color slowly changing to olive-green with Keller's reagent. Very soluble in ethyl and methyl alcohol, acetone, chloroform, and ethyl acetate; slightly soluble in ether; practically insoluble in water and petroleum ether. Fungal Source

Claviceps purpurea.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification, and analysis using a combination of co-chromatography using TLC and/or I-IPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

1.

Indole Alkaloids

93

Spectral Data See A. Hofmann, 1964. ~3CNMR: (CDCI3) C-2, 119.2; C-3, 110.6; C-4, 30.9; C-5, 59.3; 6-NMe, 40.9; C-7, 48.2, C-8, 44.3; C-9, 118.8; C-10, 138.9; C-II, 129.6; C-12, 111.9; C-13, 123.3; C-14, 110.2; C15, 133.8; C-16, 125.9; 17-CONH, 176.2; C-2', 89.9; C-Y, 165.7"; C-5', 56.8; C-6', 165.4"; C-8', 46.1; C-9' 21.7; C-10', 22.4; C-I 1', 64.3; and C-12', 103.7ppm. * Assignment may be reversed. Mass Spectrmn: LREIMS: 342(10OA), 267(11), 244(29), 154(19), 153(73), 125(81), 91(64), and 70m/e (100); CIMS: (isobutane) 268(100%), 343(55), and 245m/e (47). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70OAalkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. H. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785-

94

1.

Indole Alkaloids

789(1983). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

95

Common/Systematic Name Ergocristinine Molecular Formula/Molecular Weight C35H39NsOs; ~

= 609.29512 Me

CH

o

II C H, -

Me

\/

la

17

0

NH .........

0-"-33,

N--Me

OH,

'

-

I 11'

Ns , , , , , ~ ~ O

.

//__,

c

/

,\

/

General Characteristics Crystallizes solvent-free as long prisms from absolute alcohol solution; mp., 226~ (dec.); [aiD 20 -I- 366 ~ [a]546120 d- 460 ~ (c=0.7, in CHCI3); laiD 20 -t- 462 ~ [a]546120 -I- 582 ~ (c=l.0, in pyridine); pK = 5.0 (in 80% methylcellosolve); blue color slowly changing to olivegreen with Keller's reagent. Much less soluble than ergocristine; apparently does not form salts. Fungal Source

Clavicepspurpurea strain D-3-18. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base.

Isolation/Purification via TLC Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25)

96

1.

b) c) d) e)

Indole Alkaloids

Chloroform-methanol (90:10; 80:20; 90:10 in saturated NHs atmosphere). Chloroform-methanol-NH3 (94:5:1). Chloroform-ethylamine (90:10). Benzene-dimethylformamide (86.5:13.5).

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data IR~

See A. Hofmann, 1964. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimenta! Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

97

Common/Systematic Name Ergosecaline Molecular Formula/Molecular Weight C24H28N404; MW = 436.21106

?o Me

O II C

NH,,,......

i,,

~=0

Me z

O==~N/CH~CH N--Me

H

Me

NH Fungal Source Claviceps purpurea strain D-3-18. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medical practice. Spectral Data IR:

See Hofmann, 1964. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica usifig methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25).

98

1.

Indole Alkaloids

b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1.

Indole Alkaloids

99

Common/Systematic Name Ergosecalinine Molecular Formula/Molecular Weight_ C24H2gN404; M W -- 436.21106 Me

O

C H =

NH,,,,.....,.."~ ~ = 0 /Me O==a,,.N/CH--CH ~N--Me H Me

NH General Characteristics Prisms from ethyl acetate; mp., 217~ (dec.); [a], ~8+ 298 ~ [tt]546~18 + 375 ~ (c=l.0, in CHCI3); [aiD Is + 417 ~ [a]s46~Is + 512 ~ (C=I.0, in pyridine); blue color with AllportCocking's reagent. Readily soluble in acetone and methanol; moderately soluble in ethyl acetate; sparingly soluble in benzene and chloroform. Fungal Source Sclerotia and saprophytic culture of Claviceps purpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data IR:

See Hofmann, 1964.

100

1. Indole Alkaloids

TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharm.acolo~; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1.

Indole Alkaloids

101

Common/Systematic Name Ergobalansine Molecular Formula/Molecular Weight CEgH35NsOs; MW = 521.26382

0 17~

~3' Me Me 0H/ 0 - : 11,

18

H,....

\

o 6 N--Me

/

"--_

H - / - - ---CH2 CH M e / Me NH

General Characteristics Ergobalansine was obtained as a white solid. Fungal Source Cenchrus echinatus infected with Balansia obtecta and Cyperus virens infected with B. cyperi. Isolation/Purification Aerial parts of C echinatus infected with B. obtecta were ground in a Wiley mill fitted with a 1/4 inch screen and extracted with 95% EtOH for 24 h. The extraction process was repeated three additional times and combined EtOH extracts were evaporated to a volume of 6 liters on a rotary evaporator below 42~ An aliquot was evaporated to dryness and a portion of the concentrate was coated onto silica gel. The material was added to the top of a silica gel column packed in methylene chloride and the column was eluted with CH2C12, 5%, 10%, and 25% MeOH in CH2C12. Fractions were analyzed by TLC and recombined into fractions on the basis of similarity of TLC patterns. The remaining extract was processed by dividing into 6 portions and repeating the chromatographic procedure 6 additional times. Like materials were combined and all 8 fractions were examined for the presence of ergot-peptide alkaloids. An alkaloid-enriched fraction was prepared by solvent partitioning the entire sample in EtOAc and aqueous 2% tartaric acid. The aqueous layer was partitioned a second time with EtOAc. The aqueous layer was adjusted to pH 9 by slow addition of concentrated NI-hOH. The resulting basic solution was extracted with EtOAc, and the EtOAc extract yielded an alkaloidal material. Analysis of the alkaloid-enriched fractions by reversed-phase I-IPLC revealed two highly fluorescent

102

1.

Indole Alkaloids

compounds. Final purification of ergobalansine and ergobalansinine was achieved by preparative TLC. Purified ergobalansine and ergobalansinine readily epimerized to an approximate 1:1 equilibrium mixture on standing in solutions containing MeOH, particularly at elevated temperatures. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data IH NMR: (CDCI3) H-l, 7.99(s); H-2, 6.93(m); H-4a, 2.82(m, J=14.3, 12.2Hz); H-4b, 3.32(m, J=14.3, 5.0Hz); H-5, 3.79(m, J=12.2, 5.0Hz); 6-NMe, 2.64(s); H-7a, 2.98(dd, ,/=-12.0, 3.7Hz); H-7b, 2.88(dd, J=12.0, 2.8Hz); H-8a, 3.18(m, J=5.6, 3.7, 2.8Hz); H-9, 6.35(dd, J=5.6Hz); 8-NH, 9.26(s); H-12-14, 7.18(m); H-2'Me, 1.52(s); H-5', 4.52(dd); 5'-CH2, 1.94(m); 5'-CH2EH, 1.94(m); 5'-CH2CH(CH3)2, 1.05(d), 1.00(d); H-11', 3.56(m); 11'-CH3, 1.34(d); OH, 5.84(s); and 7'-NH, 6.78ppm(bs). ~3CNMR: (CDCI3) C-2, 119.0; C-3, 110.8; C-4, 22.1; C-5, 59.6; 6-NMe, 41.1; C-7, 48.9; C-8, 44.1; C-9, 118.4; C-10, 139.2; C-11,129.5; C-12, 112.0; C-13, 123.4; C-14, 110.0; C15, 133.8; C-16, 126.2; 17-CONH, 175.7; C-2', 85.4; C-3', 166.3"; C-5', 53.2; C-6', 169.1"; C-8', not obs.; C-9' not obs.; C-10', not obs.; C-11', 57.0; C-12', 102.7; T-Me, 24.6; 5'-CH2, 43.5; 5'-CH2CH, 24.7; 5'-(Me)2, 21.9, 22.8; and 11'-CH3, 14.7ppm. "Assignment may be reversed. Mass Data: EIMS: [M]§ 521(3%), 337(6), 267(100), 224(30), 221(52), 207(49), 196(21), 184(38), 180(29), 167(19), 154(22), 141(17), 128(54), 113(21), 44(48), and 43(59); CIMS: (isobutane) MH+ 522(6%), 504(2), 268(93), 255(100), and 185(12); negative ion CIMS: (isobutane) [M] 521(36), and 254(100). The MS-MS daughter spectrum of 254m/e (negative ion CIMS) was: 254(27), 211(7), 183(100), 126(2), 113(2), and 86(4). Found for [AH]§ 268.1422 (C16HlsN30 requires 268.1450); [BH] § 255.1362(C12H19N20; requires 255.1345). TLC Data Analytical and preparative TLC were carried out on Silica gel 60-F254plates (E. Merck) developed with chloroform-methanol (9:1, v/v).

1.

Indole Alkaloids

103

HPLC Data Preparative HPLC utilized a Rainin Dynamax-60/~ silica column with isocratic elution using methylene chloride-methanol (93:7, v/v). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected with Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

104

1. Indole Alkaloids

Common/Systematic Name Ergobalansinine Molecular Formula/Molecular Weight C28H35N505; MW = 521.26382

17~

13' Me

18

| O

Me OH=

:

11'

......

,~

~N--Me

12

//'-____ _

H-

---CH2

I

CH Me/

~Me

NH

General Characteristics Ergobalansinine was obtained as a white solid; ergobalansine and ergobalansinine readily epimerized to an approximate 1:1 equilibrium mixture on standing in solutions containing MeOH. Fungal Source Cenchrus echinatus infected with Balansia obtecta and Cyperus virens infected with B. cyperi. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification Aerial parts of C. echinatus infected with B. obtecta were ground in a Wiley mill fitted with a ~Ainch screen and extracted with 95% EtOH 24 h. The extraction process was repeated three additional times and combined EtOH extracts were evaporated to a volume of 6 liters, below 42 ~ on a rotary evaporator. An aliquot was evaporated to dryness and a portion of the concentrate was coated onto silica gel. The material was added to the top of a silica gel column packed in methylene chloride and the column was eluted with CH2C12, 5%, 10%, and 25% MeOH in CH2C12. Fractions were analyzed by TLC and recombined into fractions on the basis of similarity of TLC patterns. The remaining extract was processed by dividing into 6 portions and repeating the chromatographic procedure 6 additional times. Like materials were combined and all 8 fractions were examined for the presence of ergot-peptide alkaloids. An alkaloid-enriched fraction was prepared by solvent partitioning the entire sample in EtOAc and aqueous 2% tartaric acid. The aqueous layer was partitioned a second time with EtOAc. The aqueous layer

1. Indole Alkaloids

105

was partitioned a second time with EtOAc. The aqueous layer was adjusted to pH 9 by slow addition of concentrated Nt-hOH. The resulting basic solution was extracted with EtOAc, and the EtOAc extract yielded an alkaloidal material. Analysis of the alkaloid-enriched fractions by reversed-phase I-IPLC revealed two highly fluorescent compounds. Final purification of ergobalansine and ergobalansinine was achieved by preparative TLC. Purified ergobalansine and ergobalansinine readily epimerized to an approximate 1:1 equilibrium mixture on standing in solutions containing MeOR particularly at elevated temperatures. Spectral Data IH NMR: (CDCI3) H-I, 8.05(s); H-2, 6.90(m); H-4a, 3.59(m, J=14.4, 5.3Hz); H-4b, 2.64(m, J=14.4, 12.0Hz); H-5, 3.20(m, ,/=12.0, 5.3Hz); 6-NMe, 2.61(s); H-7a, 3.12(J=l 1.9Hz); H-To, 2.75(dd, J=l 1.9, 3.5Hz); H-8b, 3.06(m, J=6.3Hz); H-9, 6.49(dd, J=6.3Hz); 8-NI-I, 9.85(s); H-12-14, 7.15(m); H-2'Me, 1.50(s); H-5 ~ 4.42(dd); 5'-CH2, 1.90(m); 5'-CH2CH, 1.90(m); 5'-CH2CH(CH3)2, 0.96(d), 0.95(d); H11', 3.51(m); 11'-CH3, 1.32(d); OH, 6.01(s); and 7'-NH, 6.77ppm (brs). 13C N M R :

(CDCI3) C-2, 118.4; C-3, 110.1; C-4, 27.7; C-5, 62.6; 6-NMe, 43.6; C-7, 54.5; C-8, 43.0; C-9, 117.6; C-10, 137.5; C-11,127.5; C-12, 112.8; C-13, 123.3; C-14, 110.2; C15, 133.9; C-16, 126.2; 17-CONH, 176.0; C-2', 85.4; C-3', 165.7~ C-5', 53.0; C-6', 169.1*; C-8', not obs.; C-9' not obs.; C-10', not obs.; C-11', 56.9; C-12', 102.5; T-Me, 24.5; 5'-CH2, 43.4; 5'-CH2CH, 24.6; 5'-(Me)2, 22.0, 22.7; and 11'-CH3, 14.7ppm. ,

Assignments may be reversed.

Mass Data: EIMS: [M] § 521(3%), 337(4), 267(64), 221(43), 207(47), 196(25), 184(36), 180(34), 167(22), 154(33), 141(25), 128(96), 113(33), 44(89), and 43(100); CIMS: (isobutane) MIT 522(8%), 504(2), 268(97), 255(100), and 185(13); negative ion CIMS (isobutane) ([M] 42), and 254(100). Found for [AH]§ 268.1412 (Cl6HlsN30 requires 268.1450); [BH]§ 255.1362 (C~2H~9N20;requires 255.1345). TLC Data Analytical and preparative TLC were carried out on silica gel 60-F254plates (E. Merck) developed with chloroform-methanol (9:1). HPLC Data Preparative HPLC utilized a Rainin Dynamax-60A silica column with isocratic elution using methylene chloride-methanol (93:7, v/v).

106

1. Indole Alkaloids

References R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures orB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990). B. Berde and H. O. Scheld (eds.), Ergot Alkaloids and Related Compounds, In Handbook of Experimental Pharmacolowr Springer-Verlag, New York (1978). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock, J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

107

Common/Systematic Name ct-Ergocryptine Molecular Formula/Molecular Weight C32H41NsOs" M W = 575.31077

Me

0 17 II C

~/ /

\/

Me

13,CH OH r----q la L O . ~ . . ~ ' _J NH.............'1~' ~ i.!.I "?2'

1

II /

Me/

'

kMe

19'

Fungal Source Claviceps purpurea strain D-3-18. Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by HPLC on a semipreparative column: MicroPak NH2, particle size 101.tM; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 10btM; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

108

1. Indole Alkaloids

Spectral Data IR: See A. Hofinann, 1964. 1H NMR: (CDCh) NH(1), 8.37(s); H-2, 6.94(s); H-9, 6.37(dd, J--6.4, 1.SHz); N-CH3(6), 2.63(s); N-H(18), 9.86(s); H-5', 4.520, J=6.4Hz); C-2' side chain methyls, 1.03(d, 6.1Hz), 0.91(d, 6.1Hz); and C-5' side chain methyls, 1.02ppm (d, J=-7.3Hz).

~3CNMR: (CDCI3) C-2, 119.2; C-3, 110.6; C-4, 26.5; C-5, 64.5; C-7, 48.1; C-8, 40.9; C-9, 118.8; C-10, 139.2; C-11,129.6; C-12, 111.9; C-13, 123.3; C-14, 110.1; C-15, 133.9; C-16, 126.3; C-17, 176.3; N-Me, 44.3; C-2', 89.7; C-3', 165.8; C-5', 53.3; C-6', 166.2; C-8', 46.0; C-9' 21.6; C-10', 22.2; C-11', 59.3; C-12', 103.4; C-lY, 34.3; C-14', 15.3"; C-15', 16.9"; C-16', 43.5; C-17', 25.1; C-18', 22.1"; and C-19', 22.6"ppm. *, **Assignments may be reversed. Mass Spectrum: No M + observed in the mass spectrum; 308(C16I-I24N204,3%), 267(C~6I-I~7N30,22); 210(C~H18N202, 14), 209(C~H~TN202, 33), 154(CTH~oN202, 53), 125(C6H9N20, 8), 86(C~I12N, 6), 71(C4H70, 61), and 70role (C~J-IsN, 100). CIMS: (isobutane) Results suggested that major fragments for the cyclol alkaloids are due to cleavage of the bond joining the tricyclic peptide moiety with the lysergic acid amide portion of the molecule. (M+ + 1)+ was not observed in the CI spectrum; however, daughter ions 268(100%), 309 (30), and 211(3) were observed. Quadrupole MS/MS: (CI with isobutane) 576[(M+H)§ 35%], 268(AW, 100), 309(BH+, 64), and 211(CI-F, 7); Negative CIMS, 575(M', 65%), 266[(A-H)', 0.1], 308(B, 100), and 209[(C-H), 0.1 ]. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel clwomatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 7525). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere).

1.

Indole Alkaloids

109

c) Chloroform-methanol-NH3 (94"5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References M. Flieger, P. Sedmera, J. Vokoun, Z. l~eh~i~ek, I. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim, New Alkaloids from a Saprophytic Culture of Clavicepspurpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). A. Hofmann, Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. G. Yates, and J. K. Porter, Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785789(1983). J. K. Porter and D. Betowski, Chemical Ionization Mass Spectrometry of Ergot Cyclol

Alkaloids, J. Agric. Food Chem., Vol. 29, pp. 650-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from Cenchrus echina~s (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

110

1. Indole Alkaloids

Common/Systematic Name O- 12'-Methyl-ct-ergokryptine Molecular Formula/Molecular Weight C33H43NsOs; MW = 589.32642 Me \ / Me

! Y8

H,,

13,0H

,=

C

NH .............. 'B'

~

OMe

ho." ~

...

~ 0--'43'

6 N--Me

-

i

l

'

1, "N n

N5 ~ O

..J i

16'

/"--_ 16'

Fungal Source

Clavicepspurpurea strain 231 FI.

Isolation/Purification Crude alkaloids were obtained by extraction of fungal cultures with CH2CI2-MeOH (80:20) followed by precipitation with petroleum ether; the crude alkaloid extract was chromatographed by Extrelut (Merck) column chromatography eluted with CH2C12. The crude alkaloids thus obtained were chromatographed on a Silica gel 60 (Merck) column eluted with CH2CI2-MeOH (98:2, v/v); the column was monitored by UV and TLC. Final purification was by preparative TLC in a cold room in the dark. TLC was performed on Silica gel F254plates (Merck) using the following solvent systems: CH2Cl2-isopropyl alcohol (92:8, v/v) or CH2CI2-MeOH (90:10, v/v). The alkaloids were detected by examination under UV light at 254 and 366nm while some were sprayed with N,N-p-dimethylaminobenzaldehyde. The various bands were scraped off and eluted with CH2CI2-MeOH (1:1, v/v). Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

1.

Indole Alkaloids

111

Spectral Data IH NIV[R: (CDCI3) NH(1), 8.09(s); N(6)-CH3, 2.66; H-9, 6.41(J=1.8, 5.5Hz); (8)-CONH, 9.16; OCH3-12', 3.24; and H-5', 4.62ppm (,/=3.8, 10.8Hz). 13CNMR: (CDCI3) C-2, 119.0"; C-3, 110.3; C-4, 26.4; C-5, 59.5; N(6)-CH3, 44.6; C-7, 48.4; C8, 41.3; C-9, 119.2"; C-10, 138.8; C-11,129.8; C-12, 112.1; C-13, 123.5; C-14, 110.0; C-15, 133.8; C-16, 126.2; 8-CONH, 174.0; C-2', 89.2; C-3', 165.7"*; C-5', 53.1; C-6', 167.4"*; C-8', 46.1; C-9', 21.9"**; C-10', 22.0"**; C-11', 61.9; C-12', 106.4; 12'-OCH3, 48.8; (2')-C, 35.9; (2')-CH-(CH3)2, 16.0, 16.8; (5')-C, 42.2; (5')-C-C, 25.0; and (5')-CC-(CH3)2, 22.1, 21.4ppm.

*, **, and *** Assignments may be reversed. Mass Spectrum: EIMS: 589(M+), 557, 432, 347, 319, 304, 221,196, 154, and 70m/e (100%). Reference N. Crespi-Perellino, M. Ballabio, B. Gioia, and A. Minghetti; Two Unusual Ergopeptines Produced by a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 50, pp. 1065-1074(1987).

112

1.

Indole Alkaloids

Common/Systematic Name 13-Ergocryptine Molecular Formula/Molecular Weight C32H41NsOs; M'W = 575.31077 Me

_

,o C

H,,I

Y s /.

' /

\ /

Me

13'CH

,=

NH .............,~,

~

OH,

Lo.i. I-

0----43,

6 N--Me

"~

I,,.1..]r) I"

"~_I'N

J

,

I H 16,

Ns...~O /-

16'

Me/ CH2--Me

'~ '"

'"

General Characteristics Prisms from methanol solution (solvated with MeOH); mp., 210-212~ (dec.); [a]o 2~ 1910, [~]546120 - 228 ~ (c=l.0, in CHCI3); [~]D2~- 117 ~ [et]5~ 2~ - 138 ~ (C=I.0, in pyridine); pK = 5.5 (in 80% methylcellosolve); blue color slowly changing to olive-green with Keller's reagent. Fungal Source

Claviceps purpurea strain D-3-18.

Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by HPLC on a semipreparative column: MicroPak NH2, particle size 101.tm; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 101~m; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser

1.

Indole Alkaloids

113

degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). LDs0 IV in rabbits, 1.05mg/kg. Spectral Data IR:

See A. Hofmann, 1964. IH NMR: (CDCI3) NH(1), 8.21(s); H-2, 6.92(s); H-9, 6.35(dd, J-6.1, 1.4Hz); N-CH3(6), 2.63(s); N-H(18), 9.88(s); H-5', 4.49(d, J=2.5Hz); C-2' side chain methyls, 1.113(d, 6.1Hz), 0.87(d, 6.1Hz); and C-5' side chain methyls, 1.04(d, J=6.8Hz, and 0.94ppm (t, J=7.3Hz). 13C N M R :

(CDC13) C-2, 119.1; C-3, 110.7; C-4, 26.6; C-5, 64.0; C-7, 48.0; C-8, 40.9; C-9, 119.2; C-10, 139.2; C-11, 129.7; C-12, 112.0; C-13, 123.3; C-14, 110.1; C-15, 133.8; C-16, 126.3; 17-CONH, 176.3; N-Me, 44.3; C-2', 89.5; C-3', 164.6; C-5', 59.7; C-6', 166.6; C-8', 45.9; C-9' 21.3; C-10', 22.2; C-11', 59.2; C-12', 106.3; C-13', 34.3; C-14', 15.3"; C-15', 17.0~ C-16', 39.4; C-IT, 27.9; C-18', 12.6; and C-19', 16.6ppm. ,

Assignments may be reversed.

Mass Spectrum: No M + observed in the mass spectrum; 308(C16H24N204, 2%), 267(C16HI7N30, 47); 210(CllHlsN202, 8), 209(CI1HITN202, 22), 154(C7H10N202, 90), 125(C6I-I9N20, 17), 86(CsH12N, 10%), 71(C4H70, 44), and 70m/e (C4I-IsN, 100). CIMS (isobutane) Results suggested that major fragments for the cyclol alkaloids are due to cleavage of the bond joining the tricyclic peptide moiety with the lysergic acid amide portion of the molecule. (M+ + 1)+ was not observed in the CI spectrum; however, daughter ions 268(100%), 309(53), and 211(4) were observed. Quadrupole MS/MS: (CI with isobutane) 576[(M+H) +, 52%], 268(AW, 100), 309(BIT, 67), and 211(CIT, 8); Negative ElMS 575(M', 37%), 266[(A-H)', 0.1], 308(B-, 100), and 209[(CH), 0.2]. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates

114

1.

Indole Alkaloids

ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook 0.f Experimental Pharmacology; Springer-Verlag, New York (1978). M. FUieger, P. Sedmera, J. Vokoun, Z. l~ehd~,ek, J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. G. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785789(1983). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 650-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

115

Common/Systematic Name 5'-epimer of p-Ergocryptine Molecular Formula/Molecular Weight C32H41N5Os, ~ = 575.31077 Me

\/

Me

CH

O II

_OH

H

ill,,

H2CHC

H

NH Fungal Source

Claviceps purpurea strain D-3-18.

Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether, the resulting alkaloid mixture was chromatographed on a silica gel column that was eluted with CHCI3. The alkaloids were further purified by HPLC on a semi-preparative column: MicroPak NH2, particle size 10btm, mobile phase, EhO-EtOH (22:3, v/v), flow rate, 220ml/h, UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 1Own; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data ~H NMR:

116

1.

Indole Alkaloids

(CDCI3) NH(1), 8.47(s); H-2, 6.94(s); H-9, 6.36(dd, J=5.5, 1.3Hz); N-CH3(6), 2.65(s); N-H(18), 9.84(s); H-5', 4.53(d, J=3.1Hz); C-2' side chain methyls, 1.03(d, 6.1Hz), 0.89(d, 6.1Hz); and C-5' side chain methyls, 1.14(d, J--6.8Hz), 0.96ppm (t, J=-7.3Hz). ~3CNMR: (CDCI3) C-2, 119.2; C-3, 110.5; C-4, 26.7; C-5, 64.1; C-7, 48.2; C-8, 40.9; C-9, 118.9; C-10, 139.0; C-11, 129.6; C-12, 111.9; C-13, 123.2; C-14, 110.2; C-15, 133.9, C-16, 126.3; C-17, 176.1; N-Me, 44.3; C-2', 89.5; C-3', 165.0; C-5', 60.4; C-6', 167.1; C-8', 46.0; C-9' 21.7; C-10', 22.1; C-11', 59.3; C-12', 103.7; C-13', 34.3; C-14', 15.3"; C-15', 16.9"; C-16', 39.8; C-17', 26.3; C-18', 12.5; and C-19', 16.6ppm. * Assignment may be reversed. Mass Spectrum: No M + observed in the mass spectrum; 308(C16H24N204, 1%), 267(Ct6H17N30, 15); 210(CnHlsN202, 8), 209(C~IHl~N202, 22), 154(C~H~oN202,67), 125(C6HgN20, 12), 86(CsH12N, 8), 71 (C4H70, 47), and 70m/e (C4I-IsN, 100). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds, In Handbook 0.f Experimental Pharmacology; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~ehfi6ek, J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Clavicepspurpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-881(1994).

1. Indole Alkaloids

117

Common/Systematic Name 13-Ergocryptam Molecular Formula/Molecular Weight C32H41NsO4; M W = 559.15855

M e \ /Me 13' CH

o

...........

H,,,I 17 /~ -

,,Vc.o J.

I 0----~3, N--Me

I H I N ~ O /-

.,

Me

,

Nil Fungal Source

Claviceps purpurea strain D-3-18.

Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted, into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by I-IPLC on a semipreparative column: MicroPak NH2, particle size 101.tm; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 101.tm; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine.

118

1.

Indole Alkaloids

Spectral Data 1H ~ : (CDCI3) NH(1), 8.30(s); H-2, 6.92(s); H-9, 6.46(dd, ,/--4.3, 1.2Hz); N-CH3(6), 2.67(s); N-H(18), 8.22(d, J=9.2Hz); H-2', 5.83(dd, :=-9.2, 3.1Hz); H-5', 4.96(d, d=9.2Hz); HI I', 4.36(t, d=7.8Hz); C-2' side chain methyls, 0.75(d, 6.7Hz), 0.94(d, 6.7Hz); and C-5' side chain methyls, 0.98(d, J=6.7Hz), and 0.93ppm (t, ,/=7.3Hz). 13C NMPx: (CDC13) C-2, 118.9; C-3, 110.9; C-4, 29.6; C-5, 63.2; C-7, 50.6; C-8, 41.7; C-9, 119.2; C-10, 137.8; C-11,129.4; C-12, 111.8; C-13, 123.2; C-14, 109.9; C-15, 133.9; C-16, 126.3; C-17, 174.4; N-Me, 44.3; C-2', 58.1; C-3', 173.6; C-5', 60.0; C-6', 165.6; C-8', 45.5; C-9' 22.9; C-10', 23.3; C-11', 60.4; C-12', 170.0; C-13', 30.3; C-14', 16.1"; C-15', 20.1*; C-16', 37.2; C-17', 25.7; C-18', 10.9; and C-19', 15.Sppm. " Assignments may be reversed. Mass Spectrum: M + observed in the mass spectrum, 559(1%), 349(C21H23N302, 2), 321 (C2oH23N30, 4), 221(C~sH~3N2, 9), 154(CTH10N202, 100), 125(C6HgN20, 26), 86(CsH~2N, 13), and 70role (CaHsN, 87). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde arid H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~eh~i6ek,J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984).

1.

Indole Alkaloids

119

J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

120

1.

Indole Alkaloids

Common/Systematic Name 13,13-Ergoannam Molecular Formula/Molecular Weight C33H43NsO4; MW = 573.33151

M e \ /CH2Me 13, CH

oII

I

'"

3'

N H

,,

"

J

18 O . . ........... , , c .

c

O CH~CH2Me I

Me

NH

Fungal Source Claviceps purpurea strain D-3-18. Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by HPLC on a semipreparative column: MicroPak NH2, particle size 101,tm; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 101.tm; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine.

1.

Indole Alkaloids

121

Spectral Data 1H NMR: (CDCI3) NH(I), 8.00(s); H-2, 6.92(s); H-9, 6.46(dd, J=4.4, 1.5Hz); N-CH3(6), 2.67(s); N-H(18), 8.26(d, J=9.8Hz); H-2', 5.94(dd, J=9.8, 2.4Hz); H-5 ~ 4.94(d, J=9.3Hz); H11', 4.36(t, J=7.6Hz); C-2' side chain methyls, 0.70(d, 6.8Hz), 0.90(d, 7.3Hz); and C-5' side chain methyls, 1.06(d, J=6.8Hz); 0.92ppm (t, J=7.3Hz). 13C NMR: (CDCI3) C-2, 118.8; C-3, 111.1; C-4, 29.8; C-5, 63.3; C-7, 50.4; C-8, 41.7; C-9, 119.8; C-10, 137.9; C-11,129.7; C-12, 111.8; C-13, 123.3; C-14, 109.8 C-15, 133.9; C-16, 126.3; C-17, 174.7; N-Me, 44.4; C-2', 56.5; C-3', 173.6; C-5', 60.0; C-6', 165.6; C-8', 45.5; C-9' 22.9; C-10 ~ 23.1; C-11', 60.3; C-IT, 170.0; C-13', 36.8; C-14', 27.4; C-15', 11.7; C-16', 13.6; C-IT, 37.3; C-18', 25.7; C-19', 10.9, and C-20', 15.5ppm. Mass Spectrum: M* observed in the mass spectrum, 573(4%), 363(C22H25NaO2, 7), 335(C21H25N30, 12), 221(C~sH13N2, 21), 154(CTH~oN202,91), 125(C6H9N20, 22), 86(CsH~2N, 12), and 70m/e (CaHsN, 100). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolowy; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~eh~i~ek,J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

122

1.

Indole Alkaloids

Common/Systematic Name Ergobutine Molecular Formula/Molecular Weight C29HasNsOs; MW = 533.26382 Me I

13' CI-12 _OH I

o

I n--- J

,7~

H , , ~ ,~ 12

"J

1

NH ............ ,,,l~z" ' ~ l ' N / II_ H 16'

O---a3, s/N--Me

,

Ns,,,,,5,~O / ' ""-.1..6'

H-

---cN I Me

NH Fungal Source C l a v i c e p s p u r p u r e a strain 231 FI.

Isolation/Purification The whole broth culture was acidified to pH 2.5 with solid tartaric acid, homogenized, and centrifuged. The sediment was extracted twice with an aqueous solution of tartaric acid. The pooled supernatants were adjusted to pH 9-10 with 5N NaOH and extracted three times with chloroform-methanol (1:1, v/v). The combined extracts were dried over anhydrous Na2SO4 and concentrated in vacuo. The alkaloids were precipitated with petroleum ether, washed with the same solvent and dried m vacuo. The solid material was subjected to partial purification by dissolving it into 15ml of methanol and 15ml of an aqueous solution of 5% tartaric acid (w/v). The solution was brought to 600ml with water, and aliquots of 20ml were adsorbed onto 30 Extrelut (Merck) columns. Each column was percolated with chloroform, and the combined eluates were concentrated; the alkaloids were precipitated with petroleum ether, and the recovered material was washed and dried as above. Thin-layer chromatography was performed with silica gel 60 F254 (Merck). The crude extract was applied atter being dissolved in chloroform-methanol (1:1, v/v). Solvent systems were: chloroform-isopropanol (92:8, v/v); chloroform-methanol (90:10, v/v); and chloroform-methanol-cone, ammonia (90:10:1, v/v/v). The alkaloids were detected under UV light at 254 and 366nm and/or detected by spraying with N,N-p-dimethylaminobenzaldehyde. The alkaloids were scraped and eluted from the silica gel with chloroform-methanol (11 v/v).

1.

Indole Alkaloids

123

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~ MeOH max

310nm typical for lysergic acid derivatives.

Mass Spectrum: Field desorption mass spectrum 533(M+) and 267m/e (fragment ion for lysergamide); EIMS: 533(M+), 351,267, 181, 154, and 70role (100%). Reference M. L. Bianchi, N. C. Perellino, B. Gioia, and A. Minghetti; Production by Claviceps purpurea of Two New Peptide Ergot Alkaloids Belonging to a New Series Containing aAminobutyric Acid; J. Natural Products, Vol. 45, pp. 191-196(1982).

124

1.

Indole Alkaloids

Common/Systematic Name Ergobutyrine v

Molecular Formula/Molecular Weightht C3oH37NsOs, M W = 547.27947

Me

\/

Me

13' C H

o

II

la

17C

NH ............ ,,

O--L, '

'"

N--Me 12

I

OH f

O

-

11' 0

N '--. is' Me

NH

Fungal Source Claviceps purpurea strain 231 FI.

Isolation/Purification The whole broth culture was acidified to pH 2.5 with solid tartaric acid, homogenized, and centrifuged. The sediment was extracted twice with an aqueous solution of tartaric acid. The pooled supernatants were adjusted to pH 9-10 with 5 N NaOH and extracted three times with chloroform-methanol (1:1, v/v). The combined extracts were dried over anhydrous Na2SOa and concentrated in vacuo. The alkaloids were precipitated with petroleum ether, washed with the same solvent and dried m vacuo. The solid material was subjected to partial purification by dissolving it into 15 ml of methanol and 15 ml of an aqueous solution of 5% tartaric acid (w/v). The solution was brought to 600ml with water, and aliquots of 20ml were adsorbed onto 30 Extrelut (Merck) columns. Each column was percolated with chloroform, and the combined eluates were concentrated; the alkaloids were precipitated with petroleum ether, and the recovered material was washed and dried as above. Thin-layer chromatography was performed with silica gel 60 F254 (Merck). The,crude extract was applied after being dissolved in chloroform-methanol (1:1, v/v). Solvent systems were: chloroform-isopropanol (92:8, v/v); chloroform-methanol (90:10, v/v); and chloroform-methanol-conc, ammonia (90:10:1, v/v/v). The alkaloids were detected under UV light at 254 and 366nm and/or detected by spraying with N,N-p-dimethylarninobenzaldehyde. The alkaloids were scraped and eluted from the silica gel with chloroform-methanol (1:1, v/v).

1.

Indole Alkaloids

125

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV; ~MeOH max

310nm typical for lysergic acid derivatives.

Mass Spectrum: Field desorption mass spectrum 547(M +) and 267m/e (fragment ion for lysergamide); EIMS: 547(M+), 365, 280, 267, 181, 154, and 70role (100%). Reference M. L. Bianchi, N. C. Perellino, B. Gioia, and A. Minghetti; Production by Claviceps purpurea of Two New Peptide Ergot Alkaloids Belonging to a New Series Containing aAminobutyric Acid; J. Natural Products, Vol. 45, pp. 191-196(1982).

126

1. IndoleAlkaloids

C0mmon/Systematic Name Rugulovasine A Molecular Formula/Molecular Weight C16HI6N202; MW = 268.12118 16

Me~

~O

17

[[ .'5OI .,,,,,NHMe

8

General Characteristics Crystallizes as large, clear cubes from chloroform; rap., 138~ (dec.); [a]43622 -3.0 ~ (c=l.0, in pyridine); [~]D22 0.0. Rugulovasine A can be interconverted with its diastereomer, rugulovasine B upon warming in polar solvents. Fungal Source Penicillium islandicum, P. concavo-rugulosum, P. verruculosum, P. rubrum, and P. biforme. Isolation/Purification See 8-chlororugulovasine A. Biological Activity Rugulovasine A exhibited hypotensive action on chloralose-urethanized cats; the minimum effective dose was 0.2-0.5mg/kg. Spectral Data UV: ~,E~

224(e=23,400), 277(5,000), 288(6,000), and 295nm (6,000).

~3C NMR: C-2, 119.6 d; C-3, 109.8 s; C-4, 126.4 s*; C-5, 128.2 s*; C-6, 114.8 d; C-7, 122.8 d; C-8. 111.4 d; C-9, 133.9 s; C-10, 25.4 t; C-11, 63.7 d; C-12, 88.5 s; C-13, 150.6 d; C-14, 129.2 s; C-15, 174.3 s;'C-16, 10.8 q; and C-17, 3S.lppm q. * Assignments could be reversed.

1. Indole Alkaloids

127

TLC Data Silica gel PF254; Solvent: chloroform-acetone-methanol, 93:7:5, v/v/v; Re: 0.22. Detection: rose-colored spot after spraying with 50% ethanolic H2SO4 followed immediately by spraying with ninhydrin and heating at 100 ~ for 5-10 min.

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 537 (1981). R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. Singh, and D. Kim; Structures of rugulovasine-A and rugulovasine-B and 8-chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two toxic indole alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744 (1976).

128

1.

Indole Alkaloids

Common/Systematic Name 8-Chlororugulovasine A Molecular Formula/Molecular Weight C16HIsN202CI; MW = 302.08221 16

Me~

~,O 17

I "/

,NHMe

]: CI Fungal Source Penicillium islandicum. Isolation/Purification Rugulovasines A & B and chlororugulovasines A & B were extracted from fungal cultures with ethyl acetate in a Waring Blender after the pH was adjusted to 10.5 with aqueous sodium carbonate. The ethyl acetate fraction was separated from the aqueous fraction and dried over anhydrous sodium sulfate. After filtering, the crude extract was evaporated to dryness under vacuum and chromatographed on a silica gel column (70-230 mesh). The column was packed as a slurry in ethyl acetate and the extract was applied to the column in ethyl acetate solution. Toxins were eluted from the column by a linear gradient of ethyl acetate to acetone. 8-Chlororugulovasine A can be interconverted with its diastereomer, chlororugulovasine B upon warming in polar solvents. Biological Activity LDs0 in day old cockerels dosed orally, 75-125mg/kg. Spectral Dat___~a UV~

~

EtOH max

225(e=21,400), 280(sh)(4,600), 292(5,200), and 298nm (5,400).

1H NMR: H-l, 8.60; H-2, 7.00; H-6, 6.77(`/=7.6); H-7, 7.11(J=7.6); H-10, 3.30; H-11, 3.30; NH-1, 1.43; H-13, 7.35(./=1.5); H-16, 2.02(,/=1.5); and H-17, 2.43ppm.

1.

Indole Alkaloids

129

13C NMR: C-2, 120.6 d; C-3, 109.9 s; C-4, 124.8 s; C-5, 127.4 s; C-6, 116.0 d; C-7, 122.3 d; C-8, 116.5 s; C-9, 131.1 s*; C-10, 24.3 t; C-11, 63.6 d; C-12, 87.4 s; C-13, 150.2 d; C-14, 129.8 s*; C-15, 173.5 s; C-16, 10.8 q; and C-17, 34.8ppm q. * Assignments could be reversed. Mass Spectrum: HREIMS mass spectrum showed 302.0800m/e (M+). The presence of a chlorine atom was supported by the expected isotope ratios (relative intensity) 302.0800 = 86.5 ; 304.0778 = 25. Base peak was at 259.064 lm/e. TLC Data Silica gel PF254, chloroform-acetone-methanol, 93:7:5, v/v/v; Re: 0.27. Detection: rose-colored spot atter spraying with 50% ethanolic H2SO4 followed immediately by spraying with ninhydrin and heating at 100 ~ for 5-10 min. References R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two Toxic Indole Alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744(1976). R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. Singh, and D. Kim; Structures of Rugulovasine-A and Rugulovasine-B and 8-Chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 528 (1981).

130

1. Indole Alkaloids

Common/Systematic Name Rugulovasine B Molecular Formula/Molecular Weight C16H16N202; M W -- 268.12118 16

Me"-r~-~O II ~(~

%,.,,,~

17 NHMe

8

General Characteristics Colorless resinous oil; [a]43622 +1.4 ~ (c=l.0, in pyridine); [a]D 22 0.0. Fungal Source Penicillium islandicum, P. concavo-rugulosum, P. rubrum, and P. biforme. Isolation/Purification See 8-chlororugulovasine A. Biological Activity Rugulovasine B exhibited hypotensive action in experimental animals; in chloralose-urethanized cats, the minimum effective dose was 0.025-0.05mg/kg. Spectral Data UV:

~,mE~H

224(e=14,500), 277(4,800), 288(5,400), and 295nm (5,200).

1H NMR: H-I, 8.68; H-2, 7.00; H-6, 6.83; H-7, 7.2: H-8, 7.2; H-10, 3.25; H-11, 3.25; NH-1, 1.70~ H-13, 6.98; H-16, 2.02; and H-17, 2.43ppm. 13C NMR: C-2, 120.0 d; C-3, 108.4 s; C-4, 125.8 s*;C-5, 126.1 s*; C-6, 114.4 d; C-7, 122.7 d; C-8, 111.2 d; C-9, 133.9 s; C-10, 24.3 t; C-11, 63.6 d; C-12, 88.2 s; C-13, 150.9 d; C-14, 129.2 s; C-15, 174.0 s; C-16, 10.7 q; and C-17, 34.7ppm q. " Assignments could be reversed.

1.

Indole Alkaloids

131

TLC Data Silica gel PF254; solvent chloroform-acetone-methanol, 93:7:5, v/v/v; Re: 0.34. Detection: rose-colored spot after spraying with 50% ethanolic H2SO4 followed immediately by spraying with ninhydrin and heating at 100 ~ for 5-10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 540 (1981). R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. Singh, and D. Kim; Structures of Rugulovasine-A and Rugulovasine-B and 8-Chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two Toxic Indole Alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744 (1976).

132

1.

Indole Alkaloids

Common/Systematic Name 8-Chlororugulovasine B Molecular Formula/Molecular Weight CI6HlsN202CI; M W

302.08221

=

16

M e ~ II

O 17 ~l

NHMe

k. ,,,,O / 4

3

CI Fungal Source

Penicillium islandicum.

Isolation/Purification See 8-chlororugulovasine A Biological Activity LDs0 of chlororugulovasine B in preliminary studies showed it to be as toxic as chlororugulovasine A in day-old cockerels dosed orally (75-125mg/kg). Spectral Data UV: ~ mEtOH ax

224(e=22,400), 278(4,700), 288(5,100), and 295nm (5,400).

~H NMR: H-l, 8.89; H-2, 7.09; H-6, 6.73(,/--7.6); H-7, 7.05(J=7.6); H-10, 3.07; H-11, 3.07; NH-1, 1.61; H-13, 6.78(J=1.5); H-16, 2.01(J=1.5); and H-17, 2.44ppm. 13C NMR: C-2, 120.1 d; C-3, 110.6 s; C-4, 124.9 s; C-5, 127.5 s; C-6, 115.8 d; C-7, 122.0 d; C-8, 116.5 s; C-9, 130.9 s*; C-10, 25.1 t; C-11, 63.0 d; C-12, 87.8 s; C-13, 149.9 d; C-14, 129.3 s*; C-15, 173.8 s;.C-16, 10.7 q; and C-17, 34.9ppm q. * Assignments could be reversed.

1.

Indole Alkaloids

133

TLC Data Silica gel PF2s4; solvent: chloroform-acetone-methanol, 73:7:5, v/v/v; Re 0.44. Detection: rose-colored spot after spraying with 50% ethanolic H2SO4 followed immediately with ninhydrin spraying and heating at 100 ~ for 5-10 min. References R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. SingJt, and D. Kim; Structures of Rugulovasine-A and gugulovasine-B and 8-Chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two Toxic Indole Alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744 (1976). R.J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabo!ites; Academic Press, New York, p. 532 (1981).

134

1. Indole Alkaloids

Common/Systematic Name Fumigaclavine A 9-Acetoxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight CIgH22N202; M W = 298.16813 Me

0

H

H

'--

"~

Nil

General Characteristics Colorless needles; mp., 84-~176 (crystals from aqueous MeOH); [tt]5~s~ -56.7 ~ (pyridine); intense blue color with AUport and Cocking's reagent. Hydrochloride: prisms from EtOH; mp., 3040-305 ~ (dec.), [tt]~24622, -56.7 ~ (c=1.5, in MeOH) Fungal Source

Aspergillusfumigatus.

Isolation/Purification Extracted with chloroform, filtered, and dried over anhydrous sodium sulfate. The crude chloroform extract was concentrated under vacuum, redissolved in ethyl acetate, and partitioned three times between an aqueous solution adjusted to pH 2.0 with HCI. The acidic phases were combined, made basic (pH 10.0) with sodium carbonate, and partitioned three times with chloroform. The chloroform fractions were pooled and washed three times with distilled water. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting concentrate was chromatographed on a column containing neutral alumina that was deactivated to activity grade IV. The column was packed as a slurry in benzene, the sample was applied in benzene solution, and the column was eluted with benzene. This yielded fumagaclavines A and C in pure form after crystallization from methanol solution. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

1. Indole Alkaloids

135

Spectral Data UV: M,K)H ~ m~

225, 275, 282, and 293nm (e max notreported).

IR~

See Hofmann, 1964 ~H NMR: 1-NH, 7.94; H-2, 6.86; H-9, 5.70; H-12, 7.12; H-13, 6.76; H-14, 7.16; H-17, 2.47; H18, 1.35(d=7.0); and H-20, 1.90ppm. 13C NMR: C-2, 117.7 d; C-3, 111.6 s; C-4, 26.8 t; C-5, 61.9 d; C-7, 57.9 t; C-8 33.2 d; C-9, 71.6 d; C-10, 39.8 d; C-11, 129.9 s; C-12, 112.9 d; C-13, 123.2 d; C-14, 108.3 d; C-15, 133.6 s; C-16, 126.8 s; C-17, 43.6 q; C-18, 16.8 q; C-19, 170.8 s; and C-20, 21.2ppm q. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Re: 0.05. Detection: purple spot after spraying with 50% ethanolic H:SO4 and heating at 100~ for 5-10 min. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. J. Cole, J. W. Kirksey, J. W. Domer, D. M. Wilson, J. C. Johnson, Jr., A. N. Johnson, D. M. Bedel, J. P. Springer, K. K. Chexal, J. C. Clardy, and R. H. Cox; Mycotoxins Produced by Aspergillusfumigatus Species Isolated From Molded Silage; Agile. Food Chem., Vol. 25, pp. 826-830(1977). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. F. Spilsbury and S. Wilkinson; J. Chem. Soc. p. 2085 (1961).

136

1. Indole Alkaloids

Common/Systematic Name Roquefortine A; Isofumigaclavine A 9-Acetoxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight CIgH22N202; M W = 298.16831 Me H M e C O 0 .......

N--Me

NH

General Characteristics Crystals from benzene; mp., 190~176

[tg]D22 -54.1 ~

(c=0.67, in CHCI3).

Fungal Source Penicillium roqueforti. Biological Activity Roquefortine A exhibited a variety of weak pharmacological actions, such as muscle relaxant, antidepressant, local anesthetic, etc., in experimental animals. Its LDs0 in mice dosed IP was 340m~kg. Spectral Data UWz

~,mM~" 226(e=46,300), 277(sh)(6,000), 283(6,500), and 293nm (5,600). IH NMR: 1-NH, 10.76i H-2, 7.07; H-9, 5.06; H-12, 6.70(J=6.7); H-13, 7.07; H-14, 7.25(J=8.5); H-17, 2.40; H-18, 0.95(J=4.0); and H-20, 2.28ppm. 13C N M R :

C-2, 119.1 d; C-3, 110.7 s; C-4,28.4 t; C-5, 62.3 d; C-7, 55.4 t; C-8, 37.5 d; C-9, 79.6 d; C-10, 38.5 d; C-11, 131.5 s; C-12, 113.9 d; C-13, 122.8 d; C-14, 110.0 d; C-15, 134.2 s; C-16, 113.9 s; C-17, 45.9 q; C-18, 16.4 q; C-19, 171.5 s; and C-20, 22.2ppm q.

1.

Indole Alkaloids

137

TLC Data Silica gel F 1500/LS 254 (Schleicher and Schuell); solvent: chloroform-methanol-28% ammonium hydroxide, 85 15"1 v/v/v; Re 0.62. Detection: a mauve spot after spraying with 50% H2SO4 and heating at about 100~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic F unga.! Metabolites; Academic Press, New York, p. 549 (1981). P. M. Scott, J. Polonsky, and M. A. Merrien; Configuration of the 3,12- Double Bond of Roquefortine; J. Agrie. Food Chem. Vol. 27, p. 201 (1979).

138

1.

Indole Alkaloids

Common/Systematic Name Fumigaclavine B 9-Hydroxy-6,8-dimethylergoline Molecular Formula/Molecular Weight CI6H2oN20; M W -- 2 5 6 . 1 5 7 5 6

Me

H

General Characteristics Needles from aqueous methanol; 244-245~ (at 260~ the melt solidified and remelted at 265-267~ [a]546~22 -6.3 ~ (c=1.2, in MeOH); -113 ~ (c=0.6, in pyridine). Fungal Source

Aspergillusfumigatus and Rhizopus arrhizus.

Spectral Data UV~

225(e=30,900), 275(6,200), 282(6,600), and 293nm (5,200). IR~

(See A. Hofmann, 1964). ~H NMR: 1-NH, 8.04; H-2, 6.96; H-9, 4.54; H-11, 6.87; H-12, 7.18; H-13, 7.21; H-17, 2.43; and H- 18, 1.30ppm. 13C NMR: C-2, 117.6 d; C-3, 111.6 s; C-4, 26.2 t; C-5, 60.9 d; C-7, 57.4 t; C-8, 34.0 d; C-9, 69.0 d; C-10, 41.6 d; C-11, 129.5 s; C-12, 112.2 d; C-13, 123.0 d; C-14, 108.7 d; C-15, 133.5 s; C-16, 126.9 s; C-17, 43.4 q; and C-18, 16.9ppm q. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Re origin. Detection: purple spot after spraying with 50% ethanolic H2SO4 and heating at 100~ for 5-10 rain.

1. Indole Alkaloids

139

References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 553 (1981). R. J. Cole, J. W. Kirksey, J. W. Dorner, D. M. Wilson, J. C. Johnson, Jr., A. N. Johnson, D. M. Bedel, J. P. Springer, K. K. Chexal, J. C. Clardy, and R. H. Cox; Mycotoxins Produced by Aspergillusfumigatus Species Isolated from Molded Silage; Agric. Food Chem., Voi. 25, pp. 826-830(1977). A. Hofmann; Die Mutterkorn Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. J. F. Spilsbury and S. Wilkinson; J. Chem. Soc., p. 2085 (1961).

140

1.

Indole Alkaloids

Common/Systematic Name Roquefortine B; Isofumigaclavine B 9-Hydroxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight CI6H2oN20; MW = 256.15756 18

.o"N-Me Me

General Characteristics Melting point, 278~176 (dec.); colorless needles from methanol; mp., 222~ (760mm Hg); [a]D~s -147~ in pyridine). Fungal Source

Penicillium roqueforti.

Biological Activity The LD~0 of roquefortine B dosed IP to mice was 1000mg/kg. Spectral Data UV: ~mM~176 224(e=44,700), 275(6,200), 282(7,000), and 294nm(6,000); ~ , 225(e=25,100), 277(sh)(4,300), 283(4,600), and 293nm(3,800). TLC Data Silica gel F 1500FLS254 thin-layer sheets; chloroform-methanol-28% ammonium hydroxide, 85:15:1 v/v/v; Rf: 0.31. Detection: a mauve spot after spraying with 50% H2SO4 and heating at about 100~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 557 (1981).

1.

Indole Alkaloids

141

P. M. Scott, J. Polonsky, and M. A. Merrien; Configuration of the 3,12-Double Bond of Roquefortine; J. Agric. Food Chem. Vol. 27, p. 201 (1979).

142

1.

Indole Alkaloids

Common/Systematic Name Fumigaclavine C 2-Dimethylallyl-9-acetoxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight C23H3oN202; MW = 366.23073

Mecoo N-Mo

LL. General Characteristics Crystals from ethanol, acetone, or ethyl acetate (prisms), mp 191-193 ~ Needles from methanol; mp., 198~ [a]o 22 -90; [a]546122 -119 ~ (c=l.0, in CHCI3); [a]o 22 -132~ [~]546122 -160 ~ (C-'I.0, in pyridine). Fungal Source

Aspergillusfumigatus.

Isolation/Purification See fumigaclavine A. Biological Activity The LDs0 of fumigaclavine C dosed orally to day-old cockerels was 150mg/kg. Histopathology of surviving cockerels showed vacuolation of the hepatic parenchymal cells that may have been directly related to the toxic action of the toxin or due to anorexia. Spectral Data UV:

M.o. 229, 284, and 292nm (e max not reported); ~,.~M.o. 227(10,200), 283(11,000), and 292nm(9,500).

225(e=34,700),

IH ~ : 1-NH, 7.76; H-9, 5.67; H-12, 7.04; H-13, 6.72; H-14, 7.08; H-17, 2.46; H-18, 1.35; H-20, 1.90; H-22, 1.56; H-23, 1.56; H-24, 6.10; H-25a, 5.05; and H-25b, 5.20ppm.

1.

Indole Alkaloids

143

~3C NMR: C-2, 131.1 s; C-3, 106.2 s; C-4, 28.1 t; C-5, 61.6 d; C-7, 57.8 t; C-8, 33.1 d; C-9, 71.4 d; C-10, 39.4 d; C-11,129.1 s; C-12, 112.8 d; C-13, 122.2 d; C-14, 107.6 d; C-15, 136.6 s; C-16, 128.0 s; C-17, 43.5 q; C-18, 16.7 q; C-19, 170.8 s; C-20, 21.2 q, C-21, 39.1 s; C-22, 27.3 q; C-23, 27.4 q; C-24, 145.7 d; and C-25, 111.Sppm t. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Re: 0.14. Detection: blue spot aiter spraying with 50% ethanolic H2SO4 and heating at 100~ for 5-10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 560-561 (1981). R. J. Cole, J. W. Kirksey, J. W. Dorner, D. M. Wilson, J. C. Johnson, Jr., A. N. Johnson, D. M. Bedel, J. P. Springer, K. K. Chexal, J. C. Clardy, and R. H. Cox; Mycotoxins Produced by Aspergillusfumigatus Species Isolated from Molded Silage; Agile. Food Chem., Vol. 25, pp. 826-830(1977).

This Page Intentionally Left Blank

Diketopiperazines Roquefortine; Roquefortine C Aurantiamine Viridamine Okaramine A Okaramine B

2

cyclo-L-Phenylalanyl-L-alanine cyclo-L-Prolylglycine L-Propyldiketopiperazine B; L-Homoleucyl-D-proline-lactam L-Propyl-L-tyrosine Isoleucylisoleucyl anhydride Phomamide D-Valyl-L-tryptophan anhydride L-Alanyl-L-tryptophan anhydride

cyclo-(L-Isoleucyl-L-valine ) cyclo-(L-Alanyl-L-proline)

Neoxaline Oxaline Aszonalenin LL-$49013; Acetylaszonalenin Bipolaramide Austamide 12,13-Dihydroaustamide 12,13-Dihydro- 12-hydroxyaustamide Cycloechinulin 12,13-Dehydroprolyl-2-(l', l'-dimethylallyltryptophyl)diketopiperazine Alanyl-2-(1,1-dimethyl-2-propenyl)dehydrotryptophan anhydride 10,20-Dehydro [ 12,13-dehydroprolyl]-2-( 1', 1'-dimethylallyltryptophyl)diketopiperazine Deoxybrevianamide E Brevianamide C Brevianamide D Brevianamide F Eehinulin Preechinulin Neoechinulin Neoechinulin A Neoechinulin B; Cryptoechinulin C; E 10 Neoechinulin C; Cryptoechinulin A; E8 Neoechinulin D Neoechinulin E Isoechinulin A Isoechinulin B Isoechinulin C E-7 Cryptoechinulin G

145

146

2. Diketopiperazines

Cryptoechinulin B; Aurechinulin; E6 Cryptoechinulin D Fumitremorgin A Fumitremorgin B; Lanosulin Fumitremorgin C; SM-Q Verruculogen 15-Acetoxyverruculogen TR-2 Epiamauromine N-Methylepiamauromine Ditryptophenaline Exserohilone 9,10-Dihydroexserohilone Epoxyexserohilone

2. Diketopiperazines

147

Common/Systematic Name Roquefortine; Roquefortine C 1013-(Dimethyl-2-propenyl)-3-(imidazol-4-ylmethylene)-5 a, 1013,11,11atetrahydro-2H-pyrazino [ 1',2': 1,5]pyrrolo[2,3-b]indole- 1,4-(3H,6H)-dione Molecular Formula/Molecular Weight C22H23NsO2; M W = 389.18518 20

16

F,7 O General Characteristics Colorless needles of the solvate from methanol-water; mp., 195-200~ (dec.); [a]o 22 -703 ~ (c=1.0, in CHCI3). Fungal Source Penicillium roqueforti, P. notatum, P. oxaficum, P. commune, P. corymbiferum, P. expansum, and P. urticae. Isolation/Purification Fungal cultures were filtered and the mycelium macerated with acetone in a Waring blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v). Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, after crystallization from aqueous methanol, crystalline roquefortine. Biological Activity The LDs0 (IP, male mice) was 15-20mg/kg. Doses of 50-100mg/kg caused prostration and an atonic posture. The prostration was interrupted for a few seconds by opisthotonoid seizures which could be brought about by noise or a tap on the cage. Death followed within a few hours. At doses of 10mg/kg, convulsions were replaced by simple contractions and the state of prostration by equilibrium disorders during movement.

148

2. Diketopiperazines

Spectral Data UV~

EtOH

~max

209(e= 29,500), 240(16,200), and 328nm (27,000).

IR~

(CHCI3) 3430, 3380, 3190, 1685, 1665, and 1608cm"~. IH NMR: (CDC13) H-5a, 5.71; H-7-10, 6.58-7.30; H-11, 2.56; H-1 la, 4.00; H-12, 6.48; H-15, 7.73; H-17, 7.20; H-19, 6.12; H-20, 5.10; H-21, 1.06; and H-22, 1.17pm. ~3C NMR: (CDCI3) C-I, 166.2 s; C-3, 123.7 s; C-4, 159.8 s; C-5a, 78.9 d; C-6a, 151.9 s; C-7, 110.3 d; C-8, 129.8 d; C-9, 118.9 d; C-10, 125.6 d; C-10a, 129.4 s; C-10b, 62.1 d; C11,38.0 t; C-1 la, 59.4 d; C-12, 109.6 d; C-13, 125.6 d; C-15, 137.3 d; C-17, 134.5 d; C-18, 41.7 s; C-19, 144.9 d; C-20, 114.2 t; C-21, 23.0 q; and C-22, 23.3ppm q. Mass Spectrum: LREIMS: 389(M+), 320(base peak), 198, 192, 157, 130, and 108m/e. TLC Data TLC: silica gel F 1500/LS254; chloroform-methanol-28% ammonium hydroxide, 90:10:1 v/v/v; Rf: 0.46. Detection: a blue spot after spraying with 50% H2SO4 and heating at 110~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp.564 (1981). A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels, and W. E. Hull; Tremorgenic Mycotoxins from Penicillium crustosum: Isolation ofPenitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983). P. M. Scott; Roquefortine, In Mycotoxins-Production, Isolation, Separation, and Purification; V. Betina (ed.) Elsevier Science Publishers B.V., Amsterdam (1984). P. M. Scott, M. A. Merrien, and J. Polonsky; Roquefortine and Isofumigaclavine A, Metabolites from Penicillium roqueforti; Experientia, Vol. 32, pp. 140-141(1976). P. M. Scott, M. A. Merrien, and J. Polonsky; Roquefortine and Isofumigaclavine A, Metabolites from Penicillium roqueforti; Annales De La Nutrition et De L'Alimentation, Vol. 31, pp. 693-698(1977). P. M. Scott, J. Polonsky, and M. A. Merrien; Configuration of the 3, 12 Double Bond of Roquefortine; J. Agric, Food Chem. Vol. 27, p. 201 (1979).

2. Diketopiperazines

149

Common/Systematic Name Aurantiamine (Isomeric with viridamine) Molecular Formula/Molecular Weight C16H22N402; ~

= 302.17428

~

Me

"

H

1

12

NH

Me

0

General Characteristics Crystals from ethyl acetate; mp., 238-239~

[a]D23 -116 ~ (C=0.5, in MeOH).

Fungal Source Aurantiamine was produced by the majority of strains ofPenicillium aurantiogriseum var. aurantiogriseum and var. neoechinulatum. P. aurantiogriseum Diercks var. aurantiogriseum is the most common of all fungi in cereals and it is considered highly toxigenic. Isolation/Purification The fungal mat was extracted with CHCI3-MeOH (2:1, v/v). The extract was taken to dryness and the residue chromatographed on a silica gel column eluted with tolueneEtOAc (1:1 to 1:4, v/v). The fractions containing aurantiamine by TLC were pooled and taken to dryness, dissolved in hot EtOAc and filtered through activated charcoal. Crystals were formed on cooling and partial evaporation of the solvent. This procedure was repeated to give pure aurantiamine. Spectral Data UV:

~m~x (H20/MeCN with CF3CO2H) 230(40) and 320nm (100%). IH NIVIR:

(CDCI3) 1;2.16(exch., brs, H-12); 10.0(exch., brs, H-I); 7.57(brs, H-2); 6.96(s, H-6); 6.81(exch., brd, J=2Hz, H-9); 6.04[dd, 3--10.8 and 17.1Hz (X part of ABX pattern) H-17]; 5.15(2H, AB part of ABX pattern, CH2-18); 4.08(dd, ,/=2 and 3Hz, H-10); 2.47(dhept, J=3 and 7Hz, H-13); 1.49(6H, s, Me-19 and Me-20); and 10.9 and 0.95ppm (each 3H, d, J=7.0Hz, Me-14 and Me-15). 13C NMR: (CDCI3) C-2, 132.6 Dd; C-4, 132.1 Sdd; C-5, 136.7 Sm; C-6, 105.3 D; C-7, 123.5 Sd;

150

2. Diketopiperazines

C-8, 160.8 Sm; C-10, 61.1 Dm; C-11,165.1 Stm; C-13, 33.0 Dm; C-14, 18.6 Qm; C15, 15.8 Qm; C-16, 37.5 Sm; C-17, 144.6 Dm; C-18, 113.1 DDs; and C-19/20, 27.9 Qm. (CD3OD) C-2,133.0 Ds ; C-4,130.5 Sdd ; C-5, 137.0 Sm; C-6, 105.3 D; C-7, 122.5 S; C-8, 160.6 Sdd; C-10, 60.0 Dm; C-11,165.5 Sddd; C-13, 33.1 Dm; C-14, 16.8 Qm; C-15, 14.8 Qm; C-16, 36.8 Sm; C-17, 144.4 Dm; C-18, 110.9 DDs; and C19/20, 26.5 Qm. * Capital letters refer to directly bonded protons and small letters to long-range couplings. Mass Spectrum: EIMS: 302(M+, 50%), 287(15), 260(28), 231(35), 203(100), and 188m/e. Reference T. O. Larsen, J. C. Frisvad, and S. R. Jensen; Aurantiamine, a Diketopiperazine from Two Varieties ofPenicillium aurantiogriseum; Phytochemistry, Vol. 31, pp. 1613-1615 (1992).

2. Diketopiperazines

151

Common/Systematic Name Viridamine 2-(3',3'-DimethylaUyl)-6,7-didehydrocyclo-L-prolylhistdyl Molecular Formula/Molecular Weight C16H22N402; M W -" 302.17428

Me

,

HN---~

O

O

3N

Me General Characteristics A colorless glass;

[ a ] D 20 -

95,0 ~ (c=l.0, in CHCI3).

Fungal Source Penicillium viridicatum (strain no. CSIR 354). Isolation/Purification Viridamine was extracted from the dried moldy maize meal with 1:1 chloroform-methanol. An equal volume of water was added to the extract and the chloroform phase was collected. The aqueous phase was re-extracted with chloroform, the chloroform extracts were combined and the solvent was removed under reduced pressure. The residue was dissolved in a minimum of chloroform and diethyl ether was added. Precipitated material was removed by filtration and the diethyl ether-chloroform solution was repeatedly extracted with ice cold 2N hydrochloric acid. The hydrochloric acid extract was neutralized with sodium carbonate and extracted with chloroform. The chloroform was washed with 5N ammonia solution and water. The chloroform solution was dried over anhydrous Na2SO4 and evaporated under reduced pressure to yield a basic fraction which was chromatographed over alumina. The column was developed in chloroform-benzene (3:1, v/v) and chloroform. Elution with chloroform-methanol (95:5, v/v) gave viridamine as a colorless glass which could not be induced to crystallize. Spectral Data UV: MeOH

315(e=33,795), shii~ing to 300(28,280), and 338nm (39,610) on the addition of HCl and NaOH, resp.

~j'~max

152

2. Diketopiperazines

IR~ (CHCI3) 3460, 3420, 3140-3340, 1680, and 1645 cm"~. IH NMR: (CDI3) 11.88(s, 12-a); 10.1(s, l-H); 7.22(d, ,]=2.0nz, 9-n); 7.05(s, 5-n); 6.63(s, 6H); 5.32(t, J=7Hz, 17-H); 4.04(t, J=2 and 4Iq~ 10-H); 3.43(d, J=-7Hz, 16-1-12);2.35(m, J13,14and Jl3,1s=7Hz, Jl3,10=4Hz); 1.71 and 1.65(2xs, 19-and 20-H3); 1.05 and 0.94ppm (2xd, ,]14,13and Jls, 13=7Hz; 14- and 15-H3). (DMSO-d6) 12.14(s, 12-H); 11.56(s, I-H); 8.21(d, J=2Hz, 9-H); 7.31(s, 5-H); 6.45(s, 6-H); 5.33(t, J=7Hz, 17-H); 3.91(q, J=4 and 2Hz, 10-H); 3.41(d, J=7Hz, 16-1-12); 2.14(m, J13,14and Jls,13=7Hz, Jl3,1o=4Hz, 13-H); 1.68(s, 19-and 20-H3); 0.93 and 0.83ppm (2xd, J14,13and Jls, la=7Hz, 14- and 15-H3). Mass Spectrum: LREIMS: 302(49%), 287(4), 274(3), 259(3), 258(4), 231(11), 203(12), 176(13), 175(100), 174(5), 120(13), 119(24), 79(5), and 69m/e (5); HREIMS: found: M +, 302.1701; calc. for CI6H22N402, 302.1743. Reference C. W. Holzapfel and J. J. Marsh; Isolation and Structure of Viridamine, A New Nitrogenous Metabolite of Penicillium viridicatum; S. Afr. J. Chem., Vol. XXX, pp. 197204(1977).

2. Diketopiperazines

153

Common/Systematic Name Okaramine A Molecular Formula/Molecular Weight C32H32N403; MW' = 520.24744

11

.~,,.11,'

10' r ~

-~.

11b'/~~ ,1~

.

l

91

3"N'~_

General Characteristics Pale green needles from toluene; mp., 210-212~

[aid 30 +101 (c--0.09, in MeOH).

Fungal Source Penicillium simplicissimum (AK-40) -" P. brasilianum.

Isolation/Purification The acetone extract of okara (the insoluble residue of whole soybean) fermented with this fungal strain was concentrated and partitioned against ethyl acetate. Column chromatography of the ethyl acetate extract resulted in the isolation of okaramine A. Biological Activity Okaramine A caused about 90% of the test insects to die within 24hr at a dose of 30ppm in the diet when applied to the third instar larvae ofBombyx mori. Spectral Data UV:

~

MeOH

229(e=29,900), 255(21,100). 284(17,400), and 371 nm (19,400).

max

(KBr) 3460. 3300, 1610, 1480, 1375, and 742cm"~. ~H M R : Spectrum indicated the presence of the following partial structures: four tertiary methyls [1.52(3H, s), 1.65(3H, s), 1.69(3H s), 1.70(3H, s)];-CH2CH-[2.55(1H, dd,

154

2. Diketopiperazines

J=13.5, 9.6Hz); 3.1 l(1H, dd, J=13.5, 5.9); 4.54(1H, dd, J=9.6, 5.9)]; a vinyl [5.04(1H, dd,`/=l 1.0, 1.1); 5.17(1H, dd, J=17.8, 1.1); 6.11(1H, dd,`/=17.8, 11.0)]; an isolated methine [5.52(1H, s)], a cis substituted ene [5.60(1H, d, J=7.9), 5.87(1H, d, J=7.9)]; a 2,3-disubstituted indole [7.08-7.20(2H, m); 7.38(1H, dd, `/=7.2, 1.0); 7.2 1H, dd, J=6.5, 2.6); 10.64(1H, br, s)]; a 1,2-disubstituted benzene ring [6.75(1H, td, ,/=7.2, 1.0); 6.90(1H, dd, `/=8.4, 1.0); 7.05(1H, ddd, `/=8.4, 7.2, 1.5); 7.72(1H, dd, ,/=7.2, 1.5)]; an olefinic methine [7.68(1H, s)], and a hydroxyl [4.71(1H, s)]. Mass Spectrum: The molecular formula of okaramine A was established to be C32H32N403,by high-resolution mass spectral data (found, 520.2477; caled., 520.2457). Reference S. Murao, H. Ayashi, K. Takiuchi, and M. Arai; Okaramine A, a Novel Indole Alkaloid with Insecticidal Activity, from Penicillium simplicissimum AK-40; Agrie. Biol. Chem.,Vol. 52, pp. 885-886(1988).

2. Diketopiperazines

155

Common/Systematic Name Okaramine B Molecular Formula/Molecular Weight C33H34N4Os; M W = 5 6 6 . 2 5 2 9 2 14

,,,,!.0. 12,,,,!,!

N

4

O\ I~ OHm3OH 10'

11a'

Me

11

j3'N-"%_o

~ N

A General Characteristics Crystallized from methanol to afford okaramine B as pale yellow needles; mp., 295-298"C (dec.); [a]D2~+570 ~ (C=0.24, pyridine). Fungal Source Penicillium simplicissimum (AK-40) -, P. brasilianum.

Isolation/Purification The cultures were extracted with acetone, partitioned with ethyl acetate and chromatographed on a column containing Wakogel C-200 eluted with various concentrations of hexane in acetone. The active fractions eluted with 40 and 60% acetone-hexane. The active fractions were concentrated and chromatographed on alumina eluted with various concentrations of ethyl acetate-hexane. The 60% and 80% ethyl acetate fractions contained okaramine B which was crystallized from methanol as pale yellow needles. Biological Activity Insecticidal activity against third instar larvae of silkworm at 0.3ppm, 100% of the larvae were killed within 24hr. Spectral Data El"V:

~

MeOH max

233(e=27,000), 288 (12,900), and 375nm (17,900).

156

2. Diketopiperazines

IR:

(KBr) 3400, 3320, 1670, 1610, 1465, 1360, and 750cm-1. IH M R : (DMSO-d6) 8-3, 4.19 s; 8-4, 7.38 d (2'=7.8); H-5, 7.01 t (2"=7.8);8-6, 7.20 t (2"=7.8); H-7, 6.72 d (2"=7.8); H-11, 3.10 q (,/=7.3); H-12, 1.23 d (/=7.3); H-13, 1.65 s; H-14, 0.86 s; H-I', 7.39 s; H-4', 5.90 d (2'=8.3); H-5', 5.67 d (,/=8.3); H-7', 11.42 br s; H-8', 7.41 d (2"=4.4); H-9', 7.14 m; H-10' 7.13 m; H-11', 7.59 dd (2"=5.4, 2.8); H-13', 1.58 s; H-14', 1.66 s; 2-OH, 6.42 s; 3-OMe, 3.73 s; and 3a-OH, 5.26ppm s. 13C NMR: (DMSO-d6) C-2, 86.0; C-3, 82.7; C-3a, C-3b, 126.4; C-4, 125.2; C-5, 123.0; C-6, 129.4; C-7, 117.8; C-7a, 140.1; C-8a, 93.6; C-9, 164.1; C-10, 62.3; C-I 1, 40.9; C-12, 11.1; C-13, 25.2; C-14, 26.3; C-I', 112.1; C-2', 150.3; C-4', 139.5; C-5', 122.3; C-6', 36.2; 6a', 148.1; C-7a', 134.2; C-8', 113.8; C-9', 121.9; C-10', 120.8; C-11', 116.7, C-1 la', 129.8; C-1 lb', 104.6; C-12', 162.4; C-13', 28.1; C-14' 27.3; and 3-OMe, 60.1ppm.

Reference H. Hayashi, K. Takiuchi, S. Murao, and M. Arai; Okaramine B and Insecticidal Indole Alkaloid, Produced by Penicillium simplicissimum AK-40; Agrie. Biol. Chem., Vol. 52, pp. 2131-2133(1988).

2. Diketopiperazines

157

Common/Systematic Name cyclo-L-Phenylalanyl-L-alarfine Molecular Formula/Molecular Weight C12HI4N202; M W = 218.10553

O

"~

"Me

O General Characteristics Crystals from methanol; mp., 272-274~ + 63 ~(c=0.95, in acetic acid).

285~ (sealed tube); sublimed at 240~

[a]D~9

Fungal Source Entoloma haastii (fruiting bodies). Spectral Data UV: /~MeOH

max

204 and 285nm (weak).

IR:

(KBr) 3340, 3200, 1660, 1600, 1500, 750, 700, 1340, 1245, 1212, 1192, 1160, 970, 923, and 780cm"1. Mass Data: HREIMS: 318.1060m/e; found: C, 65.9, 66.1; H, 6.8, 6.9; N, 11.1, 12.1; calcd for CI2HI4N202: C, 66.0; H, 6.4; N, 12.9%. Reference E. P. White; 2,5-Dioxopiperazines from the Fungal Genera Entoloma and Fusarium; New Zealand J. of Science, Vol. 15, pp. 178-181(1972).

158

2. Diketopiperazines

Common/Systematic Name cyclo-L-Prolyl glycine Molecular Formu.la/M01ecul.arWeight CTHIoN202; ~

= 154.07423

O

O General Characteristics Crystals from ethanol-ethyl acetate; mp., 202-204~

[ a ] D 20 -

88~

in water).

Fungal Source Fusarium equiseti, and F. scirpi (CMI 112503). Isolation/Purification Cultures were extracted with chloroform, reduced to dryness, and material soluble in hot ether concentrated to yield heavy crystals. These were washed with acetone and recrystallized from ethanol-ethyl acetate. Spectral D.ata UV: ~MeOH

max

202nm

IR:

(Nujol) 3150, 3160, 3200, 1675, 1640, 1295, 1270, 1110, 1005, 900, 890, 790, and 775cm~. Mass Analysis: LREIMS: 154m/e; found: C, 54.3; H, 6.7; N, 18.2; calcd for c7nl0N202, C, 54.5; H, 6.5; N, 18.1%. Reference E. P. White; 2,5-Dioxopiperazines from the Fungal Genera Entoloma and Fusarium; New Zealand J. of Science, Vol. 15, pp. 178-181 (1972).

2. Diketopiperazines

159

Common/Systematic Name Prolyldiketopiperazine B; L-Homoleucyl-D-proline laetam Molecular Formula/Molecular Weight CI2H2oN202, M W = 224.15248

O

Me2CH(CH2)2~~L~ N 0 Fungal Source Sclerotia and saprophytic cultures of Clawceps sp. Spectral Data Mass Spectrum: LREIMS: 224(M+), 223, 195, 180, 153(100%), 99, 69, 43, and 28m/e. Reference S. Ohmomo and M. Abe; On a New Prolyldiketopiperazine Produced by Ergot Fungi; Nippon Nogei Kagaku Kaishi, Vol. 50, pp. 37-40(1976).

160

2. Diketopiperazines

Common/Systematic Name L-Propyl-L-tyrosine Molecular Formula/Molecular Weight CI4HI6N203; MW - 260.11609

NH./L'CH2~ OH General Characteristics Crystals from benzene (contained benzene of crystallization); mp., 127~ atter drying over P2Os for 24hrs. at 70~ mp., 154~ [a]D25 - 58.60(C=0.5, in MeOH). Fungal Source

Fusarium nivale Fn-2-B.

Isolation/Purification The metabolite was adsorbed onto activated carbon directly from the culture broth. The metabolite was eluted from the carbon with methanol, precipitated with the addition of chloroform, and purified with silica gel column chromatography by elution with chloroform-methanol (1 O:1, v/v) followed by crystallization from benzene solution. Spectral Data ~

MeOH max

205(e=9,620), 228(5,850), and 280nm (1,040).

IR:

(KBr) 3300, 3200, 3005, 2945, 2855, 1660, 1635, 1602, 1590, 1510, 1450, 1440, 1340, 1320, 1210, 1180, 1010, 870, and 805cm"i. 1H NMR: (CDCI3) 1.60; 2.30(2H, dd, J=72 and 11Hz); 2.02(2H, quintet); 3.32(2H, dd, J=43 and 15Hz); 3.70(2H); 4.32(1H); 4.68(1H, t); and 7.05ppm (4H, aromatic, dd, ,/=39 and 9Hz). Mass Data: Found: C, 71.27; H, 7.55; N, 8.27; calcd for CI4HI6N203oC6H6: C, 70.92, H, 6.39; N, 8.31; found: C, 65.68; H, 6.29; N, 9.37; calcd for C14I-I16N203: C, 64.74; H, 6.14; N, 10.75%.

2. Diketopiperazines

161

Reference T. Tatsuno, M. Sato, Y. Kubota, and H. Tsunoda; Recherches Toxicologiques des substances Metaboliques du Fusarium nivale. VIII. La Quatrieme Substance Metabolique de F. nivale; Chem. Pharm. Bull., Vol. 19, pp. 1498-1500(9171).

162

2.

Diketopiperazines

Common/Systematic Name Isoleucylisoleucyl anhydride Molecular Formula/Molecular Weight C 12H22N202; MW' = 226.16813

H2C/Me O NH

I

CH Me

Me~CH~NH~ O I Me"~CH2 Fungal Source

Ustilago cynodontis, a plant pathogenic microorganism isolated from Bermuda grass, and Beauveria bassiana.

Isolation/Purification Fungal cultures were extracted with dichloromethane after the removal of the mycelium by centrifugation. After evaporation of the dichloromethane, a small amount of ether was added to afford the white crystals of isoleucylisoleucyl anhydride. The metabolite sublimed at 253 ~ Spectral Data IR;

(Nujol) 3200 and 1660cm"! for N-H and amide carbonyl groups. Mass Data: 226(M+), 170(M + - C4I-I8), 113(M+ - C~t-I~7), 86, 69, and 57re~e; found: C, 63.29; H, 10.25; N, 12.32; calcd, for C~2Hz2N202: C, 63.68; H, 9.80; N, 12.38%. References J. F. Grove and M. Pople; Nitrogen-Containing Minor Metabolic Products of Beauveria bassiana; Phytochem., Vol. 20, pp. 815-816(1981). Y. Yamada, S. Sawada, and H. Okada; Production of Isoleucylisoleucyl Anhydride by Ustilago cynodontis; J. Ferment. Technol., Vol. 52, pp. 143-145(1974).

2. Diketopiperazines

163

Common/Systematic Name Phomamide Molecular Formula/Molecular Weight CITH22N204; M W = 318.15796

s

O'OH2

HN~0 CH20H

General Characteristics White prisms from ethyl acetate; mp., 213-215 ~

[a]D 20 - 7 6 ~

(MeOH).

Fungal Source

Phoma lingam.

Isolation/Purification The fungal cultures were extracted with ethyl acetate and submitted to silica gel column chromatography using a gradient of methanol in ethyl acetate as eluant; the more polar fraction was combined and a second chromatography eluting with chloroform-ethyl acetate-methanol (12:4:1, v/v) afforded a crude product (amorphous). The final purification was achieved on a Sephadex LH 20 column using methanol for elution while monitoring by TLC (Re 0.50; ethyl acetate-methanol, 4:1, v/v; Schleicher-Schiill F254 SiO2 films). The pure phomamide was obtained after crystallization from ethyl acetate as white prisms. Biological Activity Biological activity not reported, but the metabolite is related to the sirodesmin group of antibiotics. Spectral Data UV:

~

MeOH max

209(e=12,400), 229(13,500), and 277nm (1,450).

(KBr) 3500, 3320, 3200, 1680, 1650, 1620, 1585, 1515, and 825cm4. CD: (MeOH) 217(-10.4), 227(-0.2), and 275nm (+l).

164

2. Diketopiperazines

~H N M R : [(CD3)2SO] 1.69(3H, s, 17-H3); 1.73(3H, s, 16-H); 2.91(IH, m, A part of A~B~X~ system, ,/=4.5 and 14I-Iz,8-Ha); 2.91(IH, m, A part of A2B2X2 system, ,/=6 and I IHz, 7-Ha); 3.01(I H, dd, B part of A~BIX~ system,`/=6.I and 14Hz, 8-I-Ib);3.35(IH, m, B part of A2B2X2 system, J=3 and 11Hz, 7-Hb); 3.67 (I H, m, X part of A2B2X2 system, ,/=6 and 3Hz, 6-1-1);3.99(IH, m, X part of AzB~X~ system, ,/=4.5 and 6.IHz, 3-H); 4.48(2H, d, ,/=6.8Hz, 13-H2); 4.89(IH, t, exchangeable with D20, 7-OH); 5.41(IH, m, 14-H); 6.95(4H, m, aromatic);and 7.90 and 7.92ppm (2H, m, exchangeable with D20, I- and 4-NH). 13C NMR: (CD3)2SO 17.9(q, C-17); 25.3(q, C-16); 38.9(t, C-8); 55.6(d, C-3 or-6); 57.1(d, C-3 or-6); 63.1(t, C-7); 64.2(t, C-13); 114.3(d, C-11 and-11'); 120.1(d, C-1 and C-10'); 128.3(s, C-9); 130.9(d, C-14); 136.7(s, C-15); 157.2(s, C-12); 165.8(s, C-2 or-5); and 166.7ppm (s, C-2 or C-5, C-carbonyl atoms). Mass Data: 318(M+, 2%), 250(30), 144(95), 107(97), and 69m/e (100); found: C, 64.3; H, 7; N, 8.5; O, 20.2; calcd, for CiTH22N204: C, 64.1; H, 7; N, 8.8; O, 20.1%. Reference J-P Ferezou, A. Quesneau-Thierry, M. Barbier, A. Kollmann, and J-F Bousquet; Structure and Synthesis ofPhomamide, A New Piperazine-2,5-dione Related to the Sirodesmins, Isolated from the Culture Medium ofPhoma lingam Tode; J. C. S. Perkin I, pp. 113115(1980).

2. Diketopiperazines

165

Common/Systematic Name D-Valyl-L-tryptophan anhydride Molecular Formula/Molecular Weight C16H19N302; M W = 285.14773

~~ L .... ~I NH

.~ ,~ Me_/..

0"" NH

"CH Me

General Characteristics Rhombic crystals changed to long needles upon heating between 235-255"C; melted between 277-279~ [a]D +78 ~ (C=0.16, in acetic acid). Fungal Source Aspergillus chevalieri = Eurotium chevalieri. Isolation/Purification Isolated by silica gel column chromatography eluted with 1.5% methanol in chloroform. Partially purified fractions were combined and rechromatographed as above; D-valyl-Ltryptophan anhydride containing fractions crystallized as rhombic crystals. Spectral Data UV:

~

EtOH m~

290(~=5,800), 281(6,900), 274.5(6,500), and 220nm (43,400).

IR:

(KBr) 1660cm"~indicated amide functionality. ~H NMR: Spectrum revealed a six proton doublet of doublets at 0.85 and 0.88ppm (derived from proline) that was coupled to a single proton centered at 2.23ppm (m). A one proton doublet appeared at 3.23ppm. Mass Spectrum: LREIMS: 285 and 130m/e(lO0%). HREIMS indicated that 285 was CI6HIgN302 (M+), and 130m/e was CgI-IsN. Reference R. D. Stipanovic, H.W. Schroeder, and H. Hein, Jr.; Identification of D-Valyl-Ltryptophan Anhydride from Aspergillus chevalieri; Lloydia, Vol. 39, pp. 158-159(1976).

166

2. Diketopiperazines

Common/Systematic Name L-Alanyl-L-tryptophan anhydride Molecular Formula/Molecular Weight C1~-I15N302; MW = 257.11643 O

HNg"Me

I I/"Y )..NH O

General Characteristics Colorless needles; mp., 290-292~ (decomp.); [a]D 25 -F 36 ~ (c=0.5, in EtOH); positive to Ehrlich's reagent, negative to ninhydrin reagent. Treatment with 6N HCI afforded Lalanine and L-tryptophan. Fungal Source

Aspergillus chevalieri, IFO 4090.

Isolation/Purification The fungus was grown as a surface culture for 3 weeks at 24~ on potato extract medium. The culture filtrates were stirred with activated charcoal and the adsorbed metabolites were eluted with acetone. The acetone extracts were chromatographed on a column of silicic acid and eluted with benzene-acetone (4:1, v/v) which gave L-alanyl-2-(1,1-dimethylallyl)-L-tryptophan anhydride. Elution with benzene-acetone (2:1, v/v) gave L-alanyl-L-tryptophan anhydride. Spectral Data UV: Zm~x 220(e=38,000), 273(6,100), 280(6,300), and 290nm (5,800). IR:

3420(NH), 1675(C=O, amide), and 1665cm~ (C=O, amide). IH NMR: The spectrum revealed: a doublet at 0.48ppm (3H, doublet, J=7Hz) due to the alanyl methyl protons which showed coupling with a proton at 3.63ppm (1H, quartet of doublets, J=7 and 2Hz); two doublets of doublets at 3.05ppm (1H, doublet of doublets, ,/--15 and 5Hz) and 3.22ppm (1H, doublet of doublets, J=15 and 3Hz) assigned to the allylic methylene protons which showed coupling with a proton at 4.12ppm (1H, multiplet); a signal at 10.86ppm (IH, broad) assigned to the -NH group proton in the

2. Diketopiperazines

167

indole ring system. This proton coupled with a proton at 7.09ppm (1H, doublet, J=3Hz) because of a proton at position 2 in the indole ring system. Protons at 7.90ppm (1H, broad) and 7.95ppm (1H, broad) assigned to -NH groups in the dioxopiperazine ring system showed coupling with protons at 3.63 and 4.12ppm, respectively. These three protons at 7.90, 7.95, and 10.86ppm disappeared on treatment with deuterium oxide. Signals of the four aromatic protons were observed at 6.8-7.8ppm (4H, multiplet). Mass Data: LREIMS: 257(M+); found: C, 65.00; H, 5.79; N, 16.18; calcd, for cl4nlsN302: C, 65.35; H, 5.88; N, 16.33% Reference T. Hamasaki, K. Nagayama, and Y. Hatsuda; A New Metabolite, L-Alanyl-L-tryptophan Anhydride from Aspergillus chevalier/; Agr. Biol. Chem., Vol. 40, p. 2487(1976).

168

2. Diketopiperazines

Common/Systematic Name

cyclo-(L-Isoleucyl-L-valine)

Molecular Formula/Molecular Weight Clln2oN202; M W = 212.15248 Me

0

NH

M e ~ N H ~ O

.,i- Me "

Me General Characteristics Sublimed without melting at 250~

[a]D22 -400(c=0.908, in EtOH)

Fungal Source

Beauveria bassiana.

Isolation/Purification The metabolite was purified with silica gel column chromatography by elution with benzene-ethyl acetate (9:1, v/v) followed by sublimation at 250~ Spectral Data IR~

(Nujol) 3195, 3050, and 1642 cm"~for N-H and amide carbonyl groups. Mass Spectrum: LREIMS: 212(M+), 197(-CH3), 183(-C2H5), 170, 156(100%), 141, 127, 114, 113(100), 86, 85, 72, 69, and 57m/e. References S. Eriksen and I. S. Fagerson; Mass Spectra of Some Cyclic Dipeptides (2,5Diketopiperazines); J. Agile. Food Chem., Vol. 24, pp. 1242-1243(1976). J. F. Grove and M. Pople; Nitrogen-Containing Minor Metabolic Products of Beauveria bassiana; Phytochem., Vol. 20, pp. 815-816(1981).

2. Diketopiperazines

169

Common/Systematic Name cyclo-(L-Alanyl-L-proline) Molecular Formula/Molecular Weight CsHI2N202; ~

0

NH

= 168.08988

Me

General Characteristics Crystals from methanol; mp., 162-166~

[ a i D 22 -

85~

in EtOH)

Fungal Source Beauveria bassiana. Isolation/Purification The metabolite was purified with silica gel column chromatography by elution with benzene-ethyl acetate (9:1, v/v) followed by crystallization from methanol. Spectral Data IR:

(Nujol) 3280, and 1657cm"l for N-H and amide carbonyl groups. Mass Spectrum: HREIMS: 168.0904m/e (M+); CsHI2N202 requires 168.0898. Reference J. F. Grove and M. Pople; Nitrogen-Containing Minor Metabolic Products of Beauveria bassiana; Phytochem., Vol. 20, pp. 815-816(1981).

170

2. Diketopiperazines

Common/Systematic Name Neoxaline Molecular Formula/Molecular Weight C23H25NsO4; M W - 4 3 5 . 1 9 0 6 5 23

18

MeO

0

NH

General Characteristics Colorless needles from benzene; mp., 202~ (dec.); laiD 24 -16.3~(c=l.0, in CHCI3). Fungal Source Penicillium sp. Fg-234 and Aspergillusjaponicus Fg-551. Spectral Data UV:

~ 9

MeOH max

330(e=29,560) and 237nm (17,620).

IR:

3500(OH), 3425(CONH), 3200(NH, imidazole), 1710, and 1685cm~ (NC=O). CD: (c=0.0009, in EtOH) - 430(372nm) (negative maximum), + 1,200(339nm) (positive maximum), + 800(335.5nm) (negative maximum), + 1,200(328nm) (positive maximum), + 800(311nm) (negative maximum), + 4,800(290nm) (positive maximum), + 4,750(285nm) (negative maximum), + 8,970(267nm) (positive maximum), 11,200(245.5nm) (negative maximum), and + 5,600(223nm) (positive maximum). -

1H NMR: (acetone-d6) H-4, 7.58(dd, ,/=8.0, 2.014_z);H-5, 7.02(dt, ,/--2.0, 8.0Hz); H-6, 7.27(dt, ,/=2.0, 8.0Hz); H-7, 6.91(dd, J=2.0, 8.0Hz); H-8, 2.44(t, J=12.0Hz), 2.23(dd, ,/--12.0, 6.0Hz); H-9, 4.60(dd, J=12.0, 6.0Hz); n-15, 8.29(s); n-18, 7.79(s); H-20, 7.3 l(s); H22, 6.12(dd, J=18.0, 10.0Hz); H-23, 5.11(d, J=18Hz), ca. 4.97; 1-OMe, 3.73(s); 21OMe2, 1.3 l(s), 1.3 l(s); NH, 8.98(br s), 2.32(s); and OH, ca. 1.3 lppm.

2. Diketopiperazines

171

13C NMR:

C-2, 100.6(s); C-3, 53.3(s); C-3a, 128.2(s); C-4, 124.8(d); C-5, 123.6(d); C-6, 128.9(d); C-7, l ll.5(d); C-7a, 145.7(s); C-8, 40.8(t); C-9, 66.4(d); C-10, 171.65(s); C-12, 122.3(s); C-13, 165.6(s); C-15, 110.3(d); C-16, 125.8(s); C-18, 136.8(d); C-20, 134.4(d); C-21, 43.5(s); C-22, 144.5(d); C-23, 114.1(t); 1-OMe, 65.2(q); and 21-Me2, 24.7(q), 24.5ppm (q). Mass Data: HREIMS: 435.191m/e (M+) (calcd. 435.190); anal calcd, for C23H25N504;C, 63.43%; H, 5.79; N, 16.08; found: 63.23; H, 5.70; N, 15.90. Reference Y. Konda, M. Onda, A. Hirano, and S. Omura; Oxaline and Neoxaline, Chem. Pharm. Bull., Vol. 28, pp. 2987-2993(1980).

172

2. Diketopiperazines

Common/Systematic Name Oxaline Molecular Formula/Molecular Weight C24H25NsO4; M W = 4 4 7 . 1 9 0 6 5

22•,

OMe

L MeO

~0

~NH

General Characteristics Colorless prisms from acetone; rap., 230-232~

[aiD 22

-45 ~ (c=0.3, in MeOH).

Fungal Source Penicillium sp. Fg-234, P. oxalicum M-555, and Aspergillusjaponicus Fg-551.

Isolation/Purification The culture broth including mycelium was adjusted to pH 10.0 with aqueous ammonia and extracted with butyl acetate. The extract was concentrated in vacuo and the precipitate removed by filtration. The filtrate, which was dried over Na2SO4 and concentrated m vacuo, yielded pale brownish crystals which were recrystallized from acetone to yield colorless prisms. Biological Activity Biologically inactive. Spectral Data UV~

~

MeOH max

345(e=25,200) and 228nm (21,300).

IRz

(CHCI3) 3420(CONH), 3180(NH), 1710, and 1680cm q (NC=O). CD: (c=0.001, in MeOH) - 24,400(344nm) (negative maximum), + 38,200(273nm) (positive maximum), + 34,300(260nm) (negative maximum), + 42,00(247nm) (positive maximum), and - 98,300 (223nm) (negative maximum).

2. Diketopiperazines

173

IH NMR:

(CDCI3) H-4, 7.57(d, J=8.0Hz); H-5, 7.06(t, J=8.0Hz); H-6, 7.27(t, J=g.0Hz); H-7, 6.95(d, J=8.0Hz); H-S, 5.12(s); H-15, 8.32(s); H-18, 7.42(s); H-20, 7.02(s); H-22, 6.09(dd, ,/=18.0, 10.0Hz); H-23, 5.06(d, J=18Hz), 5.02(d, J=10.0Hz); 1-OMe, 3.70(s); 9-OMe, 3.62(s); 21-Me2, 1.29.(s), 1.25(s); NH, 12.76(s); and 9.59ppm (s). 13CNMR:

C-2, 101.7(s); C-3, 52.5(s); C-3a, 126.1(s); C-4, 124.7(d); C-5, 123.2(d); C-6, 128.5(d); C-7, l12.0(d); C-7a, 146.8(s); C-8, 106.9(d); C-9, 146.4(s); C-10, 157.5(s); C-12, 123.2(s); C-13, 166.3(s); C-15, 109.6(d); C-16, 126.1(s); C-IS, 136.4(d), C-20, 133.8(d); C-21, 42.5(s); C-22, 142.8(d); C-23, 113.9(t); 1-OMe, 65.2(q); 9-OMe, 55.7(q); and El-Me2, 24.1(q), 23.7ppm (q). Reference Y. Konda, M. Onda, A. Hirano, and S. Omura; Oxaline and Neoxaline, Chem. Pharm. Bull., Vol. 28, pp. 2987-2993(1980).

174

2. Diketopiperazines

Common/Systematic Name Aszonalenin Molecular Formula/Molecular Weight C23H23N302; MW = 3 73.17903 26

24 2 2 ~

25

General Characteristics Colorless needles from chloroform-methanol; mp., 244-247~ CHCI3); positive to Ehrlich's reagent.

[aiD 20 q-

53 ~ (c=1.31, in

Fungal Source Aspergillus zonatus IFO 8817. Isolation/Purification The fungus was stationarily cultured at 24 ~ for 21 days in malt extract medium. The acetone extract from the dry mycelial mats was chromatographed on a silica gel column eluted with benzene-acetone (19:1, v/v). After elution ofLL-S49013 , aszonalenin was obtained as a crude substance, which was rechromatographed over silica gel eluted with benzene-ethyl acetate (19:1, v/v) to give colorless crystals of aszonalenin. Biological Activity Application of aszonalenin at a concentration of 50~g/ml apparently induced the abnormal cleavage of sea urchin embryos. Spectral Data UV:

~

EtOH max

210(e=44,700), 233(sh, 25,100), and 290nm (5,050).

IR~ (KBr) 3400(NH), 1700(C-O, amide), 1640(C-O, amide), 1620(C=C), and 1578cmq (aromatic).

2. Diketopiperazines

175

~H NMR: (CDCI3) ~H NMR spectrum was very similar to that ofLL-S490g. A signal at 2.59ppm due to methyl protons of an acetyl group in the spectrum of LL-S49013 was not observed in the NMR spectrum of aszonalenin; an additional signal at ca. 7.0ppm assignable to -NH- was present in the spectrum of aszonalenin. Signals from a 1,1dimethyl-2-propenyl group were observed at 1.08, 1.16, 5.04, 5.07, and 6.10ppm; a signal at 8.77ppm was assigned to an amide proton; and signals between 6.55-7.92ppm were assigned to eight aromatic protons.

13C NMR: (CDCI3) Signals at 24.2(q) and 171.7(s) due to an acetyl group in spectrum of LL$49013 were not observed in this case. Signals from a 1,1-dimethyl-2-propenyl group were observed at 22.6, 22.9, 41.7, 114.3, and 144.1ppm; C-10, 33.6; C-11, 57.5; C-12, 170.2; C-16, 168.0; twelve signals between 119.3-141.8, aromatic carbons; 82.0, C-2; and 61.1 ppm, C-3. Mass Spectrum: LREIMS: 373(M+), 304(M + - 69, base peak), and 130m/e (due to indoline-3-methylene ion). Reference Y. Kimura, T. Hamasaki, and H. Nakajima; Structure of Aszonalenin, a New Metabolite ofAspergillus zonatus; Tetrahedron Letters, Vol. 23, pp. 225-228(1982).

176

2. Diketopiperazines

Common/Systematic Name LL-$49013; Acetylaszonalenin Molecular Formula/Molecular Weight C2sH25N3Oa; MW = 415.18959

4

O

General Characteristics Crystals from ethyl acetate-benzene; mp., 238-240~

[aiD 20 +

425 ~ (c=0.20, in MeOH).

Fungal Source

Aspergillus sp.

Isolation/Purification The whole mash from a fermentation was extracted with an equal volume of ethyl acetate at pH 5.0. The extract was concentrated to dryness and the residue was partitioned between methanol and heptane to remove fatty material. Evaporation to dryness of the methanol portion gave a crude residue. This residue was chromatographed over a silica gel column (acid washed) packed in methylene chloride. A gradient elution between 0.5% methanol-methylene chloride and 3% methanol-methylene chloride provided cladosporin after evaporation of the solvent and crystallization from ethyl acetate-benzene. Further elution, removal of the solvent, and crystallization from ethyl acetate gave LL-$49013. Spectral Data UV~ ~

MeOH max

210(e=61,000), 245(22,000), and 284nm (sh, 3,940).

IR~ (KBr) 3300(NH), 1689(C=O, amide), and 1647cmq (C=O, amide). IH NMR: (CDCI3) 1.02 and 1.21(3H, s); 2.60(3H, s); 2.46(q, J~=14, Jp~= 8.0Hz); 3.42(q, J~=14, JBx=8.5Hz); 3.90(t, J=8 Hz); 5.16(m, AB ofvinylidene); 5.92 (q, Jvo,~=18, Jcis=9.5Hz, X ofvinylidene); 6.00(1H, s); 6.83-8.17(8H, m); and 8.45ppm (1H, S).

2. Diketopiperazines

177

Mass Data: HREIMS: 415.18919 (calcd.for C25H25N303, 415.18959). Reference G. A. Ellestad, P. Mirando, and M. P. Kunstmann; Structure of the Metabolite LL-S49013 from an Unidentified Aspergillus species; J. Org. Chem., Voi. 38, pp. 4204-4205(1973).

178

2. Diketopiperazines

Common/Systematic Name Bipolaramide Molecular Formula/Molecular Weight ClsH14N204; MW' = 322.09536

6

4

0

OH

O2 OH

0

General Characteristics Crystallized from acetone as colorless needle-shaped crystals; mp., 296-297~ 210 ~ (C=1.0, in acetone).

[a]D -

Funsal Source

Bipolaris sorokiniana (IMI 115076); a toxigenic strain isolated in the Karroo from an indigenous weed Tribulis terrestris.

Spectral Data UV:

~,~"

218, 260, and 294nm (log e=4.65, 4.17, and 3.88, respectively).

IR:

(KBr) 1645(amide CO) and 1610cm"l. IH NMR:

(acetone-d6) 7.15(1H, t, J=7.5Hz); 6.87(1H, dd, J=7.5, 2.8Hz); 6.80(1H, dd, J=7.5, 2.8Hz); 5.53(1H, t, J=9.8Hz); 3.78(1H, dd, J=17.3, 9.8Hz); 3.51(1H, dd, J=17.3, 9.8Hz); and 11.40ppm (D20-exchangeable hydroxy group). ~3C NMR: [(CD3)zCO] 164.5(s); 145.1(s); 132.4(d); 127.5(d); 126.3(d); 116.4(d); 115.4(d); 61.4(d); and 30.3ppm (t). These data indicated the presence of three contiguous aromatic protons and of an ABX aliphatic system. The vicinal coupling constants (J=9.8Hz) observed for the ABX system are in accordance with the dihedral angular relationship obtained from X-ray crystallography: 149.8 and 31.6 ~ for H(3)-C(3)-C(4)H(4A) and H(3)-C(3)-C(4)-H(4B), respectively. Mass Spectrum: 322(M +) and 16 lm/e.

2. Diketopiperazines

179

Reference C. M. Maes, P. S. Steyn, P. H. van Rooyen, and C. J. Rabieb; The Structure of Bipolaramide, a Novel Dioxopiperazine from Bipolaris sorokiniana; J. Chem. Soc. Chem. Commun., pp. 350-351(1982).

180

2. Diketopiperazines

Common/Systematic Name Austamide Molecular Formula/Molecular Weight C21H21N303; M W -- 363.15829

O

:

O

19

General Characteristics A yellow amorphous compound or homogeneous powder;

l a i D 20 + 1 5 2 ~

(c=l, in EtOH).

Funsal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856). Isolation/Purification The dried Aspergillus ustus cultures were extracted with CHCI3-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material. This represented the main toxic component of the fungal culture. The latter in CHCI3 was extracted twice with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity III). Elution with mixtures of benzene and CHCI3 gave pure austamide and a mixture of austamide and 12,13-dehydroaustamide. Biological Activity Austamide was one of the active components in extracts ofA. ustus (cultured on maize) causing acute toxicosis in day old ducklings. Spectral Data UV~

~,~m~ H 234(r 256(117), 268 sh (1,096), 282(8,709), and 392nm (2,691) (unchanged upon addition of acid or base). IR:

(CHCI3) 3420, 1700, 1680, and 1650cm "l.

2. Diketopiperazines

181

CD: (in MeOH) 390(+2.0), 285(+1.8), and 234nm (20-25). ~3CNMR: 23.6, 26.1, 28.0, 41.9, 42.0, 45.5, 62.5, 70.7, 111.9, 118.7, 120.3, 120.4, 124.4, 125.4, 128.3, 132.2, 137.3, 154.7, 160.1, 161.1, and 200.8ppm.

TLC Data Silica gel; chloroform-methanol, 97:3 v/v; Re not reported. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp.484 (1981). P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

182

2. Diketopiperazines

Common/Systematic Name

12,13-Dihydroaustamide

Molecular Formula/Molecular Weight C21H23N303; MW = 365.17394

4

0

O~N~~2 13 ]17

T,,,, H

General Characteristics Crystallized from acetone; mp., 235-238~

[a]D 22 + 5 5 ~

(c=l.1, in CHCI3).

Fungal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856) and Penicillium italicum. Isolation/Purification The dried moldy maize culture was extracted with CHCI3-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material which represented the main toxic components of the fungal culture. The latter in CHCI3 was twice extracted with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity III). Elution with mixtures of benzene and CHCI3 gave a mixture of austamide and 12,13-dehydroaustamide. The latter metabolites were separated by repeated preparatory silica gel TLC developed in CHCI3-MeOH (97:3, v/v) yielding pure 12,13-dehydroaustamide. Biological Activity Toxic to ducklings. Spectral Data UV:

~

EtOH max

238(1og c=4.49), 256(sh)(4.13), and 390nm (3.52).

IR;

(CHCI3) 3420, 3335, 3,000, 1670, and 1620cm"l.

2. Diketopiperazines

183

~H NMR: The NMR spectrum of 12,13-dihydroaustamide when compared with that of austamide showed the absence of the olefinic triplet at 3.74z and the newly formed proton at C12 at 5.82z. Concurrently the peaks comprising the proline part became more complex. Mass Spectrum: HREIMS: 365.1750(M+, C21H23N303 requires 365.1739), 192.1268 (CIIHI6N20 requires 192.1262), and 70.0656m/e (C~HsN requires 70.0656). Reference P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

184

2. Diketopiperazines

Common/Systematic Name 12,13-Dihydro- 12-hydroxyaustamide 2S, 9S, 12R- 12,13-Dihydro- 12-hydroxyaustamide Molecular Formula/Molecular Weight C21H23N304; M W -- 381.16886

oxy

4

0

H,,,.I

"

I....'OH

General Characteristics Crystals from MeOH; mp., 164-165~ Fungal Source

Aspergillus ustus.

Isolation/Purification Isolated from A. ustus fermented corn and separated by chromatography on formamideimpregnated cellulose powder; further purified by extensive column chromatography and TLC on silica gel and aluminum oxide; crystallized from methanol. Biological Activity Toxic to ducklings. Spectral Data UV: ,~ McOH max

231,255, and 390nm (log e = 4.48, 4.04, and 3.47, respectively).

CD: ,~ MeOH max

420(0), 374(-2.0), 355(0), 341(+2.35), 3140, 305(-0.34), and 290 (0).

IR: (CHCI3) 3430-3350, 1670, and 1618cm"~. IH N]VIR: (CDCI3) Spectrum showed 2 singlets at 8.48(3H) and 9.16z (3H) which were assigned to the two geminal methyl groups; the cis-olefirfic protons appeared as an AB-pattern

2. Diketopiperazines

185

at 3.29 and 4.98Z(JAa=10Hz); protons at C-8 and C-9 appeared as an ABX system, HA being I-ls~q, 7.13z(q, JAa=15Hz, JBx=12Hz); a complex pattern centered around 6.361: was assigned to the methylene protons adjacent to the proline N-atom; the remaining 4 methylene protons which comprised the proline ring appeared as an unresolved multiplet between 7.6 and 8.1z; the proton at C-12 resonated between 5.80-5.90z; and the splitting pattern of the aromatic region was identical to that of dihydroaustamide. Mass Spectrum: HR IMS: 381.1685(M+, C21Hz3N304 requires 381.1688) and 363m/e for C~zH~4N202. Reference P. S. Steyn and R. Vleggaar; 12,13-Dihydro-12-hydroxyaustamide, A New Dioxopiperazine from Aspergillus ustus; Phytochemistry, Vol. 15, pp. 355-356(1976).

186

2. Diketopiperazines

Common/Systematic Name Cycloechinulin Molecular Formula/Molecular Weight C2oH21N303, MW = 352.15829 22 Me H N ~ .... ,H

4

r"

\17

MeO-/~~~8 NI~I /eMe 7 2o General Characteristics Cycloechinulin was isolated as a yellow solid; [~]D -23.3 ~ (c=0.06g/dl, in CHCIa). Fungal Source Sclerotia ofAspergillus ochraceus (NRRL 3519). Isolation/Purification Intact sclerotia of A. ochraceus were extracted at room temperature with hexane, then CHCI3. After removal of the solvent in vacuo, the hexane extract was subjected to reversed-phase HPLC [MeOH-H20 (9:1, v/v), 2ml/min} to give N-methylepiamauromine. The CHCI3 extract, after removal of the solvent in vacuo, was fractionated through a column of Sephadex LH-20 (25-1001,t) using CH2Cl2-hexane (1:1, v/v), then CH2CI2-MeOH (1:1, v/v) as eluents. The fractions obtained with CH2Cl2-hexane (1:1, v/v) were combined, concentrated, and purified by HPLC as above to give epiamauromine and N-methylepiamauromine. The fractions eluted with CH2CI2-MeOH (1:1, v/v) were likewise combined, concentrated, rechromatographed on Sephadex LH-20 using CHCI3-MeOH (2:1, v/v), and purified by reversed-phase HPLC to yield cycloechinulin. Biological Activity Caused moderate reduction in weight gain in assays against the lepidopteran crop pest Helicoverpa zea.

Spectral Data UV:

~

MeOH max

214(1og e=4.3), 228(4.2), 267(4.0), 300(4.1), and 377nm (3.9).

2. Diketopiperazines

187

IH N]VIR: (CDCI3) H-1,8.39, br s;H-4, 7.63, d(J=8.7);H-5, 6.84, dd(J=2.2, 8.7);H-7, 6.81, d(J=2.1); H-10, 7.57, s;H- 13, 6.37, br s; H-14, 4.13, dq(J=2.3, 6.9);H-17, 5.82, d(J=8.2); H-18, 5.94, d(J=8.2);H-20, 1.68, s;H-21, 1.67, s;H-22, 1.51, d(J=6.9); and H-23, 3.81ppm, s. 13CNMR: (CDCI3) C-2, 145.8; C-3, 105.7; C-4, 118.7; C-5, 110.9; C-6, 157.2; C-7, 94.8; C-8, 134.0; C-9, 124.4; C-10, 115.4; C-11,125.0; C-12, 165.6; C-14, 51.1; C-15, 167.2; C-17, 122.4; C-18, 139.7; C-19, 36.0; C-20, 27.2; C-21, 27.0; C-22, 18.4; and C-23, 55.8ppm. Mass Spectrum: EIMS: [M]§ 351(77), 336(100), 308(20), 296(26), 293(37), 280(93), 265(34), 252(46), 251(47), 237(94), 225(82), 222(31), and 197m/e (64); HRFABMS found 352.1671; calcd for C20H21N303+H 352.1661. HPLC Data Beckman Ultrasphere 51.tm(10mm x 25cm) C~8 reversed-phase column was used on all HPLC separations; HPLC retention time for cycloechinulin was 7.5 min. Reference F. S. de Guzman, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; New Diketopiperazine Metabolites from the Sclerotia of Aspergillus ochraceus; Journal of Natural Products, Vol. 55, pp. 931-939(1992).

188

2. Diketopiperazines

Common/Systematic Name 12,13-Dehydroprolyl-2-(l', l'-dimethylallyltryptophyl) diketopiperazine Molecular Formula/Molecular Weight C21H23N302; MW = 349.17903

4 ~ N _ \1o~/OST/ 7

20/19

General Characteristics Homogenous powder;

l a i D 22 - 3 8 ~

(c=1.3, in CHCI3).

Fungal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856) and Penicillium italicum. Isolation/Purification The dried moldy corn was extracted with CHCIa-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material which represented the main toxic components of the fungal culture. The latter in CHCI3 was twice extracted with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity III). Elution with benzene gave a mixture of compounds. The latter material was separated by silica gel TLC developed in CHCI3MeOH (98:2, v/v) yielding pure 10,20-dehydro[ 12,13-dehydropropyl]-2-(l', l'-dimethylallyltryptophyl)diketopiperazine. Elution of the foregoing A1203 column with CHCI3benzene (6:4, v/v) gave pure 12,13-dehydroprolyl-2-(l', 1'- dimethylallyltryptophyl)diketopiperazine as a homogenous powder. Biological Activity Toxic to ducklings. Spectral Data UV:

223(1og e=4.54), 268(sh)(4.03), 283(4.00), and 292nm (3.89).

2. Diketopiperazines

189

IR;

(CHCI3) 3482, 3460, 3380, 1670, and 1650cm"~. ~H NMR: The NMR spectra of deoxybrevianamide E and 12,13-dehydroprolyl-2-(1 ', 1'-dimethyl allyltryptophyl)diketopiperazine had many features in common, the most striking difference being due to protons of the proline ring. These protons appeared as an A2M2X pattern, represented by a triplet at 6.0~ (2H, J=-9Hz, C-15 CH2), a sextet at 7.311: (2H, ,/=3, 9, 9I-~ C-14 CH2), and a triplet at 3.94~ (1H, J-=-3I-~ C-13 CH). Mass Spectrum: HREIMS: 349.1720(M +, C21H23N302 requires 349.1708). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 453 (1981). P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

190

2. Diketopiperazines

Common/Systematic Name Alanyl-2-(1,1-dimethyl-2-propenyl)dehydrotryptophan anhydride Molecular Formula/Molecular Weight CwH21N302; MW = 323.16338 O

[~N

NHMe

General Characteristics Crystals; mp., 262-264 ~C. Fungal Source Aspergillus ruber -, Eurotium rubrum isolated from beet pulp.

Isolation/Purification Mycelia was extracted with ethyl acetate. The ethyl acetate-soluble/neutral fraction was chromatographed on a silica gel column using benzene-ethyl acetate. The benzene-40% ethyl acetate eluant gave two indole metabolites which were separated with preparative thin-layer chromatography developed with benzene-ethyl acetate (1:1, v/v). Biological Activity Growth retardant for silkworm larvae (Bombyx mori L.). Spectral Data UV: 1' MeOH ^ ....

224(E=31,600), 283 (8,300), 290(8,050), and 338nm (9,340).

IR;

(Nujol) 1675 and 1633cm"1. ~H NMR: (DMSO-dr) 11.03(IH, s, -NH-, indole nucleus), 8.57(1H, s, -NH-CO-), 8.3 I(1H, s,-NH-CO-), 7.44-7.00(4H, m), 6.92(1H, s), 6.10(1H, dd, J=9.9Hz and 17.8Hz -CH=CH2), 5.02(1H, dd, J=9.9Hz and 1.1Hz; 1H, dd, J=17.8Hz and 1.1Hz, -CH=CH2), 4.17(1H, q, J=7.0Hz, =CH-CH3), 1.48(6H, s, (CH3)2C=), and 1.38ppm (3H, d, J=7.0Hz, =CH-CH3).

2. Diketopiperazines

191

Mass Spectrum: 323.160 lm/e (M +) and 254(M + - C5H9); calcdfor C19H2~N302, 323.1631. Reference H. Nagasawa, A. Isogal, K. Ikeda, S. Sato, S. Murakoshi, A. Suzuki, and S. Tamura; Isolation and Structure Elucidation of a New Indole Metabolite from Aspergillus ruber; Agr. Biol. Chem., Vol. 39, pp. 1901-1902(1975).

192

2. Diketopiperazines

Co.mmon/Systematic Name 10,20-Dehydro [ 12,13-dehydroprolyl]-2-( 1', 1'-dimethylallyltryptophyl)diketopiperazine Molecular Formula/Molecular Weight C21H21N302;

MW

-- 3 4 9 . 1 6 3 3 8

13

O 8 4

I

7 Fungal Source v Aspergillus ustus (C.S.I.R. 1128, NRRL 5856) and Penicillium italicum. Isolation/Purification Dried moldy maize was extracted with CHCI3-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material which represented the main toxic components of the fungal culture. The latter in CHCI3 was twice extracted with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity HI). Elution with benzene gave a mixture of compounds. The latter material was separated by silica gel TLC developed in CHCI3MeOH (98:2, v/v) yielding pure 10,20-dehydro[ 12,13-dehydropropyl]-2-(l',l'-dimethylallyltryptophyl)diketopiperazine. Elution of the foregoing A1203 column with CHCl3benzene (6:4, v/v) gave pure 12,13-dehydroprolyl-2-(1 ', 1'-dimethylallyltryptophyl)diketopiperazine as a homogenous powder. Biologic~ Activity Toxic to ducklings. Spectral Data UV"

~ EtOH max

224(1og e=4.44), 272(sh)(3.93), 284(3.84), and 292nm (3.71).

2. Diketopiperazines

193

IR; (CHCI3) 3485(sharp peak), 3350(weak broad band), 3,000, 1675, and 1650cm "l. 1H The NMR spectrum showed the presence of an indole NH-proton 1.70% (s) and four contiguous aromatic protons 2.4-3.1% (m). The two 3-proton singlets at 8.39 and 8.66% were assigned to the gem-dimethyl group and the 2-proton singlet at 4.26% to the olefinic protons at positions 19 and 20. The three protons at C-8 and C-9 appeared as an ABX pattern as quartets: Hx at 5.751: (J=l, 6Hz, C9-H), HA at 6.40(J=1, 15Hz, CS-H, q), and lib at 6.54z (,/=6, 15Hz, C8 H,x.). The small coupling constant (1Hz) between C9-H and C8-I-I~ is consonant with a dihedral angle of close to 90 ~ while JBX 6Hz is consistent with a dihedral angle of close to 135"; the dihedral values are in agreement with those obtained from an inspection of a Dreiding model of 10,20-dehydro [ 12,13-dehydroprolyi]-2-( 1', 1'-dimethylallyl tryptophyl)diketopiperazine. A two-proton triplet at 6.421: was assigned to the protons at C15, a 2-proton sextet at 8.17~ (,/=3, 10, 10Hz) was assigned to the methylene protons at C~4, while the olefinic proton at C~3 resonated as a triplet at 4.58z (J=3Hz). Irradiation at the center of this latter triplet led to the collapse of the sextet to a triplet at 8.17% (J=-I 0Hz). Mass Spectrum: HREIMS: 347.161(M +, C21H21N302requires 347.162). Reference P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

194

2. Diketopiperazines

Common/Systematic Name Deoxybrevianamide E L-Prolyl-2-( 1', 1'-dimethylallyl)-L-tryptophyldiketopiperazine Molecular Formula/Molecular Weight C21H25N302; M W -- 351.19468 O

4

8 ~

N

General Characteristics Homogeneous powder from benzene;

is

[a]D 22

-59 ~

(c=1.2, in CHCI3).

Fungal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856) and Penicillium italicum. Biological Activity Toxic to ducklings. Spectral Data UV;

~

EtOH max

225(E=32,300), 275(sh)(7,000), 283(8,100), and 29 lnm (7,000).

IR;

(CHCI3) 3481, 3458, 3366, 1670, and 1665cml. lH N]V[R; Two exchangeable ringlets at 1.25 and 4.28ppm were assigned to NH protons. A multiplet at 2.48-3.05ppm was attributed to the four neighboring aromatic protons. A 6-proton singlet at 8.50 was due to the gem-dimethyl group while the three exocyclic olefinic protons appeared as an AA'X system at 3.90(1H, X part, dax 18.2, Jh~:9Hz, C19-H) and 4.92(2H, AA' part of AA'X system, J,x 18.2, JA~ 9Hz,-C=CH2). The protons at C-8 and C-9 appeared as an ABX system at 5.56(1H, X part of ABX system, Jgx 4.0, JBx 11.0Hz, C9-H, 6.25 and 6.83(2H, AB part of an ABX system, HA(6.25) JAB 15.5, JAX 4.0Hz, and HB(6.83) JAB 15.5, JBx 11.0Hz, 8 CH2). The triplet at 5.95ppm (J=7.0Hz) was assigned to the methine proton at position 12. The protons adjacent to the proline nitrogen resonated as an ill-defined triplet at 6.34ppm; the other four protons comprising the proline ring appeared as an unstructured multiplet between 7.6-8.2ppm.

2. Diketopiperazines

195

Mass Spectrum: Only one prominent fragment ion at 198role (C14HI6N) from cleavage of the 8,9-bond. TLC Data Silica gel, chloroform-acetone, 9:1 v/v, Rf: 0.75. Detection: blue spot atter spraying with 65% H2SO4 and heating at ca. 120~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 453 (1981). P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

196

2. Diketopiperazines

Common/Systematic Name Brevianamide C Molecular Formula/Molecular Weight C21H23N303; MW' = 365.17394

0 0 N

N

Me

General Characteristics Brevianamide C was obtained as an orange glass that could not be induced to crystallize. Fungal Source

Penicillium brevi-compactum.

Isolation/Purification The fungus was cultured on Czapek-Dox broth at 25 ~ Brevianamide C was isolated from the crude pigment mixture remaining after the crystallization ofbrevianamide A by repetitive preparative TLC using 5% methanol in chloroform or 1% ethanol in ether. The various metabolites in descending gf, brevianamide C, D, A, B, and F were scraped from the plates and eluted with methanol. Spectral Data UV~

~m~

234, 259, 277(in0, 300(in0, and 450rim.

IR~

(CHCI3) 3410, 3350, 1710, 1680, and 1615cm"l. ~H NMR: Refer to Birch and Russell (1972) for copy of spectrum. Mass Spectrum: HREIMS: 365.174Ira~e, M +, C21H23N303requires 365.1739; LREIMS" 365(13%), 322(15), 295(100), 177(5), 171(4), and 146m/e (3).

2. Diketopiperazines

Reference A. J. Birch, and R. A. Russell; Studies in Relation to Biosynthesis-XLIV Structural Elucidations of Brevianamides-B, -C, -D and-F; Tetrahedron, Vol. 28, pp. 2999-3008 (1972).

197

198

2. Diketopiperazines

Common/Systematic Name Brevianamide D Molecular Formula/Molecular Weight C21H23N303; M W -- 365.17394

General Characteristics Brevianamide D was obtained as a red glass that could not be induced to crystallize. Fungal Source Penicillium brevi-compactum. Isolation/Purification The fungus was cultured on Czapek-Dox broth at 25 ~ Brevianamide C was isolated from the crude pigment mixture remaining after the crystallization of brevianamide A by repetitive preparative TLC using 5% methanol in chloroform or 1% ethanol in ether. The various metabolites in descending Rf, brevianamide C, D, A, B, and F were scraped from the plates and eluted with methanol. Spectral Data UV~

~.,,~x 235, 264, 306, and 470nm. IR~

3440, 3200, 1710(sh), 1680, 1630, and 1615cmq. IH NMR: Refer to Birch and Russell (1972) for copy of spectrum. Mass Spectrum: HREIMS: 365.1738re~e, M +, C21H23N303requires 365.1739; LREIMS: 365(25%), 322(14), 295(100), 177(14), 171(6), 146(3), and 133role (7). Reference A. J. Birch, and R. A. Russell; Studies in Relation to Biosynthesis-XLIV Structural Elucidations ofBrevianamides-B, -C, -D and -F; Tetrahedron, Vol. 28, pp. 2999-3008 (1972).

2. Diketopiperazines

199

Common/Systematic Name Brevianamide F; L-Tryptophanyl-L-prolyl anhydride Molecular Formula/Molecular Weight CI6HITN302; M W = 283.13208

0 N

HN

General Characteristics Brevianamide F was obtained as white needles from ethanol; mp., 173-175~ with Ehrlich's reagent.

blue color

Fungal Source Penicillium brevi-compactum. Isolation/Purification The fungus was cultured on Czapek-Dox broth at 25 ~C. Brevianamide C was isolated from the crude pigment mixture remaining after the crystallization of brevianamide A by repetitive preparative TLC using 5% methanol in chloroform or 1% ethanol in ether. The various metabolites in descending Re, brevianamide C, D, A, B, and F were scraped from the plates and eluted with methanol. Spectral Data UV:

~,m~x 277(inf), 283, and 292nm. IR:

3280, 1670, 1650(weak), and 1640 cm"~(weak). ~H NMR: (DMSO-dr) 1.2-2. l(4H, methylene multiplet); 2.9-3.5(4H, =CCHAHa, N-CH2CH2); 4.05(1H, broad triplet, NHCHCO, J=7.0Hz); 4.30(1H, triplet, NCHCO, J=6Hz); 6.97.6(4H, rn, aromatic H); 7.98(1H, d, indole NHCH=C, J~rHJCh,=2Hz);7.66(1H, s, NH); and 10.8ppm (1H, b.s., indole NH): Mass Data: LREIMS: 282(9%), 154(8), 130(100), and 83role (9); found: C, 67.8; H, 6.1; N, 14.8 CI6H17N302 requires C, 67.7; H, 5.9; N, 14.7%.

200

2. Diketopiperazines

Reference A. J. Birch and R. A. Russell; Studies in Relation to Biosynthesis-XLIV Structural Elucidations of Brevianamides-B, -C, -D and-F; Tetrahedron, Vol. 28, pp. 2999-3008 (1972).

2. Diketopiperazines

201

Common/Systematic Name Echinulin 3{[2•(•••-Dimethy•al•y•)-5,7-bis(3•methy••2•buteny•)ind•••3-y•]methyl}•6•methy•-2•5piperazinedione Molecular Formula/Molecular Weight

C29H39N302;MW- 461.30423

~ ~

28 ~

~

4

10

~11

0

[[

General Characteristics Needles from butanol; mp., 242-243 ~ Soluble in glacial acetic acid, chloroform, pyridine, dioxane; slightly soluble in warm alcohol, butanol, benzene, ethyl ether, acetone, and carbon tetrachloride. Fungal Source Aspergillus amstelodami = Eurotium amstelodami, A. chevalieri = E. chevalieri, and A. echinulatus = E. echmulatum.

Spectral Data

UV: Z ~m~

230(39,810), 279(9,549), and 286nm (9,120).

13C NMR: C-2, 141.1 s; C-3, 104.5 s; C-4, 121.5 d; C-5, 130.4 s; C-6, 114.5 d; C-7, 124.4 s; C-8, 131.8 s; C-9, 130.4 s; C-10, 29.9 t; C-11, 55.2 d; C-12, 167.4 s; C-14, 50.4 d; C-15, 167.8 s; C-17, 20.2 q; C-18, 38.9 s; C-19, 27.9 q; C-20, 27.9 q; C-21, 145.9 d; C-22, 111.2 t; C-23, 30.4 t; C-24, 122.3 d; C-25, 130.4 s; C-26, 25.6 q; C-27, 17.7 q; C-28, 34.3 t; C-29, 123.2 d; C-30, 131.9 s; C-31, 25.6 q; and C-32, 17.7ppm q. TLC Data Silica gel HF2s4; A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Detection: not reported.

202

2. Diketopiperazines

Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 480 (1981).

2. Diketopiperazines

203

Common/Systematic Name Preechinulin (+)-L-Alanyl(1,1-dimethylallyl)tryptophan anhydride Molecular Formula/Molecular Weight C19Hx3N302, ~

= 325.17903

O 4

10 11 I [

General Characteristics Fine white needles from methylene chloride-benzene; mp., 294~176 (C=0.38, in acetic acid); [a]D2~ +22.3 o (in acetic acid).

[a]D 24

+50 ~

Fungal Source Aspergillus amstelodami = Eurotium amstelodami, A. chevalieri = E. chevalieri, and A. repens = E. repens.

Spectral Data UV: EtOH

225(e=32,500), 283(7,750), and 291nm (7,000).

IR:

(KCI) 3380, 3210, 1670, 1460, 993, and 910cmq. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 459 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983). ,

204

2. Diketopiperazines

Common/Systematic Name Neoechinulin Molecular Formula/Molecular Weight C23H25N303; M ' ~ = 391.18959 O 4

26

~ 25~y

~

10

11

NH

NH

~7

27 22

Fungal Source A s p e r g i l l u s a m s t e l o d a m i = E u r o # u m amstelodami.

Spectral Data UV:

~, ~,O=H 231(C=32,300), 287(13,100), and 420nm (9,700). ~H NMR: H-4,7.11; H-5,6.83; H-7,7.51; H-19,1.57; H-20,1.57; H-21, 6.10; H-22, 5.12; 5.11; H23, 3.42; H-24, 5.38; H-26, 1.75; H-27, 1.75; and NH, 9.59; 11.06; 12.07ppm. 13C N M R :

C-2, 131.1; C-3, 103.4; C-4, 116.3; C-5, 119.2; C-6, 124.0; C-7, 110.8; C-8, 134.6; C9, 123.1; C-10, 120.9; C-11, 145.7; C-12, 152.0; C-14, 157.0; C-15, 160.2; C-18, 38.9; C-19, 27.7; C-20, 27.7; C-21,145.1; C-22, 111.9; C-23, 33.9; C-24, 124.0; C25, 135.7; C-26, 25.4; and C-27, 17.6ppm. TLC Data Silica gel I-'~254. Solvent: A, ethyl acetate-hexane, 2:1 v/v; B, benzene-ethyl acetate, 1:1 v/v; C, benzene-ethyl acetate, 2:1 v/v. Rr: A, 0.75; B, 0.50; C, 0.30. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolit.e_s; Academic Press, New York, pp. 463 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

2.

Diketopiperazines

205

Common/Systematic Name Neoechinulin A Molecular Formula/Molecular Weight CI9H21N302; M W = 323.16338

O 4

10 11 [ [ 3

Nil

General Characteristics Ivory crystals from methanol; mp., 264-265~ Fungal Source A s p e r g i l l u s ruber = E u r o t i u m rubrum

and A.

amstelodami = E. amstelodami.

Spectral Data UV~

Z ~m"~

229(e=23,300), 286(11,700), 292(10,400), and 338nm (9,500).

IR~

(KBr) 3360, 2980, 1670, and 1630cm~. IH NMR: H-l, 8.30; H-4, 7.00-7.50; H-5, 7.00-7.50; H-6, 7.00-7.50; H-7, 7.00-7.50; H-10, 6.93; H-13, 8.30; H-14, 4.15; H-16, 10.96; H-17, 1.42; H-19, 1.50; H-20, 1.50; H-21, 6.12; H-23, 5.04; and H-24, 5.06ppm. ~3CNMR: C-2, 144.1; C-3, 103.3; C-4, 118.7; C-5, 120.8; C-6, 119.5; C-7, 110.2; C-8, 135.2; C9, 126.0; C-10, 111.6; C-11, 124.8; C-12, 159.7; C-14, 50.5; C-15, 166.3; C-17, 17.9; C-18, 40.2; C-19, 27.5; C-20, 27.5; C-21,145.2; and C-22, 111.6ppm. TLC Data Silica gel HF254; A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Re: A, 0.24, B, 0.17, C, 0.09. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New

206

2. Diketopiperazines

York, pp. 469 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

2.

Diketopiperazines

207

Common/Systematic Name Neoechinulin B; Cryptoechinulin C; E 10 Molecular Formula/Molecular Weight C19HI9N302; MW' = 321.14773 O 4

10 11 I I

3

NH

VNH~'8

N 5

17

N General Characteristics Yellow crystals from methanol; mp., 234-236~ Fungal Source Aspergillus amstelodami = Eurotium amstelodami.

Spectral Data UV: Z E~

228(e=28,100), 273(19,000), 284(18,100), and 374nm (10,400).

IR;

(KBr) 3360, 2980, 2925, 1680, and 1645cm~. ~H NMR: H-I, 8.36; H-4-7, 6.70-7.50; H-10, 6.85; H-13, 10.61; H-16, 10.86; H-17, 5.08, 5.34; H-19, 1.50; H-20, 1.50; H-21, 6.07; H-23, 5.07; and H-24, 5.09ppm. TLC Data Silica gel HF254;A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Re: A, 0.77; B, 0.46; C, 0.26. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 471 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

208

2. Diketopiperazines

Common/Systematic Name Neoechinulin C; E8; Cryptoechinulin A Molecular Formula/Molecular Weight C24H27N302; ]~[W' = 389.21033

O

4

10 11 I I 3

NH

2021~ 6 22

General Characteristics Optically inactive; yellow crystals from light petroleum-benzene, 1"1 v/v; mp., 205-207"C. Fungal Source Aspergillus amstelodami = Eurotium amstelodami.

Spectral Data UV:

231 (e=34,600), 275(22,300), 290(18,600), and 380nm (12,000). IR;

(KBr) 3350, 2970, 2925, 1680, and 1640cmq. IH NMR:

H-l, 8.66; H-4,5,7, 6.80-7.40; H-10, 7.01; H-13, 10.79; H-16, 10.96; H-17, 4.96, 5.27; H-19, 1.48; H-20, 1.48; H-21, 6.07; H-23, 5.06; H-24, 5.08; H-25, 3.40; H-26, 5.35; H-28, 1.76; and H-29, 1.76ppm. 13CNMR: C-2, 131.1; C-3, 102.8; C-4, 112.0; C-5, 118.5; C-6,124.0; C-7, 110.9; C-8, 134.4; C9, 124.0; C-10, 120.9; C-11, 142.2; C-12, 155.7; C-14, 134.8; C-15, 157.1; C-17, 100.0; C-18, 38.6; C-19, 27.5; C-20, 27.5; C-21, 144.1; C-22, 111.7; C-23, 33.9; C24, 124.0; C-25, 135.6; C-26, 25.4; and C-27, 17.6ppm. TLC Data Silica gel I-IF254;A, ethyl acetate-hexane, 2:1 v/v; B, benzene-ethyl acetate, 1"1 v/v; C, benzene-ethyl acetate, 2:1 v/v. Rf: A, 0.80; B, 0.53; C, 0.39. Detection: not reported.

2. Diketopiperazines

209

R.eferences R.. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 473 (1981). A. Dossena, R. Marchelli, and A. Pochini; Neoechinulin D, a New Isoprenylated Dehydrotryptophyl Metabolite from Aspergillus amstelodami; Experientia, Vol. 31, p. 1249 (1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 407 (1983).

210

2. Diketopiperazines

Common/Systematic Name Neoechinulin D Alanyl-2-(1,1-dimethyl-2-propenyl-6-isopentyl)-dehydrotryptophan anhydride Molecular Formula/Molecular Weight C24H29N302; MW = 391.22598 4

lO

O ]1 NH

20

2021~

L~

6 22

General Characteristics Ivory crystals from methanol; mp., 223-225 ~ Fungal Source Aspergillus amstelodami = Eurotium amstelodami

and A. rubrum = E. t u b e r

(Isolated from beet pulp). Isolation/Purification Neoechinulin D was isolated from the crude ethereal extract of the mycelium by silica gel 60 column chromatography eluted with hexane-ethyl acetate (1:1, v/v). Neoechinulin D rich fractions were combined, concentrated, and further purified by preparatory TLC followed by crystallization from petroleum ether-benzene as ivory crystals; recrystallized from methanol. Biological Activity Growth retardant for silkworm larvae (Bombyx mori L.). Spectral Data UV: ~

EtOH max

231(e=34,600), 265(11,400), 296(10,400), and 345nm (10,900).

IR:

(KBr) 3340, 2940, 1670, and 1630. IH NMR: H-l, 8.32; H-4,5,7, 6.95-7.40; H-10, 7.01; H-13, 10.38; H-14, 4.25; H-16, 10.62; H17, 1.53; H-19, 1.48; H-20, 1.48; H-21, 6.11; H-23, 5.06; H-24, 5.08; H-25, 3.40; H26, 5.33; H-28, 1.72; and H-29, 1.72ppm.

2. Diketopiperazines

211

Mass Spectrum: 391.2262m/e (M+); calcd for C24H29N302,391.2258. TLC Data Silica gel HF254;A: ethyl acetate-hexane, 21 v/v; B benzene-ethyl acetate, 1"1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Rf: A, 0.37; B, 0.23; C, 0.12. Detection not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 466 (1981). A. Dossena, R. Marchelli, and A. Pochini; Neoechinulin D, a New Isoprenylated Dehydrotryptophanyl Metabolite from Aspergillus amstelodami; Experientia, Vol. 31, p. 1249 (1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983). H. Nagasawa, A. Isogal, K. Ikeda, S. Sato, S. Murakoshi, A. Suzuki, and S. Tamura; Isolation and Structure Elucidation of a New Indole Metabolite from Aspergillus ruber; Agr. Biol. Chem., Vol. 39, pp. 1901-1902(1975).

212

2. Diketopiperazines

Common/Systematic Name_ Neoechinulin E Molecular Formula/Molecular Weight CIgHITN303; ~ = 323.12670 O 4

10

1,1

II

N

H

NH

8 N 2~

k~x

O 22

General Characteristics Orange-red crystals from methanol; mp., 275~ Funsal Source v

Aspergillus amstelodami = Eurotium amstelodami.

Spectral Data UV:

Z Em'~ 228(6=37,100), 281(9,100), and 410nm (8,100). IR:

(KBr) 3330, 3150, 3020, 2840, 1740, 1690, and 1600cmq. IH NMR: H-I, 8.64, H-4-7, 6.90-7.55, H-10, 7.40; H-13, 10.58; H-16, 10.58; H-19, 1.54; H-20, 1.54; H-21, 6.06; H-23, 5.06; and H-24, 5.20ppm. TLC Data Silica gel HF254; A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Rf: A: 0.51; B: 0.36; C: 0.17. Detection: visible light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 464 (1981). A. Dossena, R. Marchelli, and A. Pochini; Neoechinulin D, a New Isoprenylated Dehydrotryptophyl Metabolite from Aspergillus amstelodami; Experientia, Vol. 31, p. 1249(1975).

2. Diketopiperazines

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

213

214

2. Diketopiperazines

Common/Systematic Name Isoechinulin A Molecular Formula/Molecular Weight C24H29N302; M W ~---3 9 1 . 2 2 5 9 8

23

4

10

I

24

,,

3

Ir , T

NH "

17

7

20 2 1~_2 2 6

Fungal Source

Aspergillus ruber = Eurotium rubrum.

Biological Activity Isoechinulin A inhibited growth of silkworm larvae. Spectral Data UV: EtOH ~m~x

227(e=31,500), 289(8,900), and 34 lnm (9,400).

IR:

(KBr) 3260, 1675, and 1633cmq. ~H NMR: H-I, 10.21; H-4, 7.10; H-6, 6.99; H-7, 7.34; H-10, 7.15; H-13, 7.92; H-14, 4.28; H16, 7.53; H-17, 1.55; H-19, 1.55; H-20, 1.55; H-21, 6.16; H-23, 5.13; H-24, 5.13; H25, 3.40; H-26, 5.38; H-28, 1.71" and H-29, 1.71ppm. 13C N M R :

C-2, 143.9; C-3, 102.8; C-4, 123.2"; C-5, 132.9'*; C-6, 117.9; C-7, 111.1"**;C-8, 134.6; C-9, 126.4; C-10, 112.1"**"C-11,124.3; C-12, 160.3" C-14, 51.6; C-15,165.8; C-17, 20.9; C-18, 39.3; C-19, 27.4; C-20, 27.4; C-21, 144.1; C-22, 113.1; C-23, 34.6; C-24, 124.0"; C-25, 132.0"*; C-26, 25.8" and C-27, 17.9. *, **, *** Assignments may be reversed. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 475 (1981).

2. Diketopiperazines

W. B. Turner and D. C. Aldridge; Fungal Metabolites I.I; Academic Press, New York, New York, p. 407 (1983).

215

216

2. Diketopiperazines

Common/Systematic Name Isoechinulin B Molecular Formula/Molecular Weight C24H27N302; M3vV= 389.21033

4

24

10

0 H

I HN J~. NH ~ , 8 " ~ "C H2 ~21

0

22

Fungal Source Aspergillus ruber = Eurotium rubrum.

Spectral Data UV: ~, max MeOH

228(e=27,900), 272(18,700), 285(sh) (16,600), and 370nm (10,400).

IR:

(KBr) 3310, 1678, and 1643cm"l. IH NMR: (CDCI3) H-I, 10.23; H-4, 7.00-7.20; H-6, 6.96; H-7, 7.30; H-10, 7.00-7.20; H-13, 9.80; H-16, 8.08; H-17, 5.00-5.40; H-19, 1.54; H-20, 1.54; H-21, 6.13; H-23, 5.00-5.40; H-24, 5.00-5.40; H-25, 3.38; H-26, 5.00-5.40; H-28, 1.68; and H-29, 1.68ppm. ~3CNMR: C-2, 144.6; C-3, 102.7; C-4, 123.2"; C-5, 133.0"*; C-6, 118.0; C-7, 111.3***;C-8, 134.7; C-9, 126.0; C-10, 113.5"**;C-11,124.0; C-12, 158.5; C-14, 133.6; C-15, 156.1; C-17, 103.0; C-18, 39.2; C-19, 27.4; C-20, 27.4; C-21,144.2; C-23, 34.5; C-24, 124.0"; C-25, 132.0"*; C-26, 25.7; and C-27, 17.8ppm. 9 **

, ,

***

Assignmentsmay be interchanged.

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 476 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

2.

Diketopiperazines

217

Common/Systematic Name Isoechinulin C Molecular Formula/Molecular Weight C24H27N303; M W -- 405.20524

.~

~,~

4

10 3

-

~

i

~

0

/

12 NH

.,~1HN

~L,~ 7

"~-NH/~18 ys "~CH2 20 21~22 O

Fungal Source Aspergillus ruber = Eurotium rubrum.

Spectral Data UV:

Meott

229(e=26,700), 272(18,000),286 sh (15,800), and 371nm (9,700).

IR:

(KBr) 3250, 1676, and 1644cm"1. 1H NIVIR:

H-I, 10.21; H-4, 7.02; H-6, 7.02; H-7, 7.31; H-10, 7.07; H-13, 9.73; H-16, 8.12; H17, 5.00; 5.34; H-19, 1.56; H-20, 1.56; H-21, 6.11; H-23, 5.08; H-24, 5.05; H-25, 2.70-3.10; H-26, 2.70-3.10; H-28, 1.22; and H-29, 1.33ppm. ~3CNMR: C-2, 140.2; C-3, 104.5; C-4, 124.5; C-5, 131.2; C-6, 119.9; C-7, 112.8"; C-8, 136.4; C-9, 127.8; C-10, 112.9"; C-11,126.4; C-12, 158.3; C-14, 135.5; C-15, 157.0; C-17, 100.6; C-18, 40.6; C-19, 28.4; C-20, 28.4; C-21,146.2; C-22, 112.9~ C-23, 36.7; C24, 65.9; C-25, 59.4; C-26, 25.5; and C-27, 19.6ppm. "Assignments may be interchanged. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p.477 (1981). W. B. Turner and D. C. Aldri.dge; Fungal Metabolites II; Academic Press, New York, New York, p. 407 (1983).

218

2. Diketopiperazines

Common/Systematic Name E7 Molecular Formula/Molecular Weight C29H35N302; M ~ = 457.27293

O

H2cHN General Characteristics Crystals from ether; mp., 146-148 ~

Fungal Source Aspergillus amstelodami IFO 6667 = Eurotium amstelodami. Spectral Data IR~

(KBr) 1680, and 1640cm"~. Mass Spectrum: HREIMS: 457.2697(M+), 402, 388, 332, 294, and 69m/e. Reference S. Inoue, J. Murata, N. Takamatsu, H. Nagano, and Y. Kishi; Synthetic Studies on Echinulin and Related Natural Products. V. Isolation, Structure and Synthesis of Echinulin-Neoechinulin Type Alkaloids Isolated from Aspergillus amstelodami; Yakugaku Zasshi, Vol. 97, pp. 576-581(1977).

2. Diketopiperazines

219

Common/Systematic Name Cryptoechinulin G Molecular Formula/Molecular Weight C29H35N302; M W ' = 4 5 7 . 2 7 2 9 3

24

H 22

General Characteristics Amorphous solid, optically inactive. Fungal Source

Aspergillus ruber = Eurotium rubrum.

Spectral Data ~3CNMR*: C-2, 126.6; C-3, 101.3; C-4, 128.9; C-5, 130.3; C-6, 123.0; C-7, 109.5; C-8, 134.5; C9, 130.2; C-10, 114.6; C-11, 142.2; C-12, 156.6; C-14, 134.6; C-15, 156.1; C-17, 100.4; C-21,145.4; C-22, 111.1; C-24, 124.9; C-25, 130.2; C-29, 124.7; and C-30, 130.2. * Data for saturated carbons not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 468 (1981). G. Gatti; Molecular Structure of Cryptoechinulin G, an Isoprenylated Dehydrotryptophan Metabolite Isolated from Aspergillus ruber; Tetrahedron Lett., p. 2605-2606(1978). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

220

2. Diketopiperazines

Common/Systematic Name Cryptoechinulin B; Aurechinulin; E6 Molecular Formula/Molecular Weight C43H49N3Os; ~

= 687.36722

3'a

3'

aa a

~T/O H

HO.~ ~.~1~H 0 0 7' ae~ t~, b

S General Characteristics Crystals; mp., 188 ~C. Fungal Source Aspergillus amstelodami -, Eurotium amstelodami. Spectral Data 1H NMR: [(CD3)250] Similar to that of cryptoechinulin D except for the addition of two methyl resonances at 1.7ppm and a -CH2CH= pattern at 3.3 and 5.3ppm.

Mass Spectrum: LREIMS: 687, 618, 481,412, 344, 320, 298, 265, 243,206, and 69m/e. References R. Cardillo, C. Fuganti, D. Ghiringhelli, and P. Grasselli; New Minor Metabolites of Asperg#lus amstelodami; La Chimica E L'Industria, Vol. 57, pp. 678-679(1975). G. Gatti, R. Cardillo, C. Fuganti, and D. GhiringheUi; Structure Determination of Two Extractives from Aspergillus amstelodami by NMR Spectroscopy; J. C. S. Chem. Comm., pp. 435-436(1976).

2. Diketopiperazines

221

Common/Systematic Name Cryptoechinulin D Molecular Formula/Molecular Weight

C38H41N305;h/IW'= 619.30462 3'0~

3'a 3' ~ , , . . 0 H

HO./~ O ~/~"-' 7' (~HO �9 "

3 ~

....

"NHIg'

o

General Characteristics Crystals; mp., 198 ~C. Fungal Source Aspergillus amstelodami -, Eurotium amstelodami.

Spectral Data ~H NMR: [(CD3)2SO] Five protons that were exchangeable with D20 (11.8; 10.9; 9.2; 8.8, CONH; and 7.8ppm, CONH); 10.9(NH); 6.9-7.5ppm (4 aromatic protons); 1.4(3H); 6.97(3a-H); 6.7(7'H); 3.2 and 5.2ppm (-CH2CH=); 1.6ppm (3H); and 10.2ppm (CHO). 13C NM~:

[(CD3)250] 161.5 and 167.8ppm (two CONH groups); 36.8 (suggests the tt, ttdimethylallyl chain in position 2 of tryptophan); 144.4(C-3b) and 111.7ppm (C-3a); 17.7 and 25.6ppm (methyl resonances); 197.3ppm (CHO). References R. Cardillo, C. Fuganti, D. Ghiringhelli, and P. Grasselli; New Minor Metabolites of Aspergillus amstelodami, La Chimica E L'Industria, Vol. 57, pp. 678-679(1975). G. Gatti, R. Cardillo, C. Fuganti, and D. Ghiringhelli; Structure Determination of Two Extractives from Aspergillus amstelodami by NMR Spectroscopy; J. C. S. Chem. Comm., pp. 435-436(1976).

222

2. Diketopiperazines

Common/Systematic Name Fumitremorgin A Molecular Formula/Molecular Weight C32H41N307; M W - 5 7 9 . 2 9 4 4 5

0 0 ~ OH

~ 26

" 22

General Characteristics Colorless prisms from methanol; mp., 206-209~ [a]D 10 +61 o (acetone). Soluble in chloroform and ethyl acetate; slightly soluble in methanol and ethanol; unstable to prolonged exposure to light. Fungal Source

Aspergillus fumigatus and A. caespitosus.

Biological Activity Tremorgenic: lmg dosed IP to mice caused perceptible tremors; 5mg dosed IP to mice caused sustained tremors and 70% mortality. Spectral Data UV:

~

EtOH

226(6=31,700), 277(5,300), and 296nm (4,900).

1H NMR: H-4, 6.59; H-5, 6.82; H-7, 7.68; H-10, 5.50; 11 OH, 4.48; H-14, 3.63; H-15, 1.60-2.20; H-16, 1.60-2.20; H-17, 6.13; H-20, 5.06; H-21, 2.40; H-23, 0.99; H-24, 2.00; H-25, 6.62; H-26, 5.02; H-28, 1.71; H-29, 1.71; H-30, 3.84; H-31, 4.71; H-32, 5.60; H-34, 1.81; and H-35, 1.81ppm. TLC Data Silica gel G-HR; A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-formic acid,

2. Diketopiperazines

223

5:4:1, v/v/v; Rf: A, 0.30, B, 0.65. Detection: slate gray-blue spot in visible light or mustard-colored spot under UV light; develops immediately alter spraying with 50% ethanolic H2SO4. NOTE: The color response and Rf's ofverruculogen are identical to those of fumitremorgin A in the above conditions. References R. J. Cole and R. H. Cox; Handbook of Toxic Funga_! Metabolites; Academic Press, New York, pp. 357-359(1981). M. Yamazaki, H. Fujimoto, and T. Kawasaki; Tremorgenic Toxins from Aspergillus

fumigatus; Tetrahedron Lett., p. 1241 (1975).

224

2. Diketopiperazines

Common/Systematic Name Fumitremorgin B; Lanosulin Molecular Formula/Molecular Weight C27H33N3Os; M W -- 479.24202 OH 0 - OH

MeO

N H "'

II r~

0 - -

General Characteristics Colorless needles from methanol; mp., 211-212 oC. Fungal Source

Aspergillus fumigatus, A. caespitosus (NRRL 1929), Penicillium lanosum, and P. piscarium.

Isolation/Purification Cultures were comminuted in 70% aqueous acetone, filtered and extracted with chloroform, adsorbed on a silica gel column and eluted in sequence with benzene and 3% methanol/benzene. The tremorgenic fractions were further purified on a second silica gel column eluted with 1.5% acetone in chloroform. Verruculogen and fumitremorgen B were crystallized from benzene-ethanol solution and finally from methanol solution. Biological Activity Tremorgenic: Fumitremorgin B dosed at lmg/mouse (IP) caused perceptible tremors; at 5mg/mouse (IP), it caused sustained tremors and 70% mortality. Spectral Data UV:

~

MeOH max

228(e=36,400), 278(7,500), and 297nm (8,400).

IR:

(KBr) 3460, 3420, and 1665cm "~. ~H NMR: (CDCI3) H-4, 7.80(J=9.0); H-5, 6.73(J=9.0, 2.0); H-7, 6. 64(J=2. 0); H-10, 5.72;

2. Diketopiperazines

225

H-14, 3.80; H-15, 2.00; H-16, 2.00; H-17, 4.50; H-20, 5.95; H-21, 4.70; H-23, 1.67; H-24, 1.97; H-25, 4.50; H-26, 4.50; H-28, 1.60; H-29, 1.81; and H-30, 3.82ppm.

13C NMR: (CDCI3) C-2, 130.8 s; C-3, 104.2 s; C-4, 121.1 d; C-5, 104.0 d; C-6, 155.8 s; C-7, 93.7 d; C-8, 137.6 s; C-9, 120.3 s; C-10, 68.8 d; C-I 1, 82.8 d; C-12, 170.0 s; C-14, 48.9 t; C-15, 22.5 t; C-16, 28.9 t; C-17, 58.6 d; C-18, 165.8 s; C-20, 58.2 d; C-21, 122.7 d; C-22, 134.8 s; C-23, 25.5 q; C-24, 18.3 q; C-25, 45.1 t; C-26, 120.0 d; C-27, 134.4 s; C-28, 25.5 q; C-29, 18.3 q; and C-30, 55.6ppm q. References R. J. Cole and R. H. Cox; Handbook of Toxic Fung.a.l_Metabolites..; Academic Press, New York, pp. 360-363(1981). H. W. Schroeder, R. J. Cole, H. Hein, Jr., and J. W. Kirksey; Tremorgenic Mycotoxins from Aspergillus caespitosus; Applied Microbiology, pp. 857-858(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, K. Sasago, and K. Mikyaki; Tremorgenic Toxins from Aspergillus

fumigatus Fres.; J. Chem. Soc., Chem. Commun., p. 408 (1974).

226

2. Diketopiperazines

Common/Systematic Name Fumitremorgin C (SM-Q) Molecular Formula/Molecular Weight C22H25N303; M W -- 379.18959

O 10

MOO"

v

-NH

15

General Characteristics Crystals from ethyl acetate; mp., 125-130~ Fungal Source

Aspergillusfumigatus isolated from moldy silage suspected of causing disease in beef cattle.

Biological Activity Tremorgenic to day-old cockerels at levels down to 25mg/kg (oral). Spectral Data UV: ~.m,~M~O" 224, 272, and 294nm. IH N]V[R:

(CDCI3) I-NH, 8.32;H-4, 7.48(J=9.0);H-5, 6.86(/=9.0,2.0);H-7, 6.92(,/=2.0);H-17, 4.16; H-20, 4.98(J=9.0);H-21, (6.06)(J=9.0);H-23, 2.07;H-24, 1.72;and H-25, 3.91ppm. 13C N M R :

(CDCI3) C-2, 132.1 s; C-3, 106.2 s; C-4, 118.8 d; C-5, 109.4 d; C-6, 156.4 s; C-7, 95.2; C-8, 136.9 s; C-9, 120.7 s; C-10, 45.4 t; C-11, 51.0 d; C-12, 169.4 s; C-14, 45.4 t; C-15, 23.0 t; C-16, 28.6 t; C-17, 59.2 d; C-18, 165.6 s; C-20, 56.8 d; C-21,124.1 d; C-22, 133.9 s; C-23, 18.1 q; C-24, 25.7 q; and C-25, 55.7ppm, q TLC Data Silica gel G-H~ toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Rf: 0.55. Detection: bright orange spot develops immediately after spraying with 50% ethanolic H2SO4 and minimal heating.

2. Diketopiperazines

227

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 364-367(1981). R. J. Cole, J. W. Kirksey, J. W. Dorner, D. M. Wilson, J. Johnson, Jr., D. Bedell, J. P. Springer, K. K. Chexal, and J. Clardy; Ann. Nutr. Alim., Vol. 31, pp. 685-692 (1977).

228

2. Diketopiperazines

Common/Systematic Name Verruculogen Molecular Formula/Molecular Weight C27H33N307; M W = 511.23185

OH 0 .--- OH '

MeO~,....-.~..f.~N / ~

\~/

N

~

.i-" \ 2~ II

!

,s

0

General Characteristics Colorless plates from benzene-ethanol, 1 1, v/v; mp., 233-235~ (dec.); [a]D -27.7 ~ (in CHCI3). Soluble in benzene, ethyl acetate, and acetone; slightly soluble in ethanol; and very soluble in CHCI3. Verruculogen is unstable to prolonged exposure to light. Fungal Source. Penicillium verruculosum (ATCC 24640; NRRL 5881) --" P. brasilianum, P. paraherquei -, P. brasilianum, P. piscarium -, P. brasilianum, P. janthinellum, P. parilli --"P. graminicola, P. estinogenum --, P. brasilianum, Aspergillus caespitosus (NRKL 1929), and A. fumigatus.

Isolation/Purification Cultures were extracted in hot chloroform. Chloroform extracts were filtered, dried, evaporated to dryness in vacuo. The crude extract was chromatographed on a silica gel (Merck 70-325 mesh) column packed in n-hexane and eluted with n-hexane to remove non-polar triglycerides; the toxin was eluted with ethyl ether. The toxin was purified further on a silica gel column packed in toluene and eluted with toluene followed with a linear gradient from toluene to ethyl acetate. The tremorgen was further purified using a column packed with Florisil and eluted with 5% ethyl acetate in hexane. Active fractions were combined, concentrated m vacuo, and crystallized from benzene-ethanol solution (1:1, v/v). Biological Activity The EDs0 for tremor response in mice was 0.39mg/kg (IP); in day-old cockerels it was 0.33mg/kg (IP). Tremors are sustained at higher levels, intermittent at lower levels, and enhanced by enforced movement. Verruculogen administration produced a decrease in y-aminobutyric acid levels in mouse central nervous system; verruculogen-induced tremor is mediated by a loss of inhibitory GAB A function. The LDs0 of verruculogen to mice was 2.4mg/kg (IP) and 126.7mg/kg (oral); to day-old cockerels, 15.2mg/kg (IP) and 265.5mg/kg (oral).

2. Diketopiperazines

229

Spectral Data UV: ~, Em ~~

226(E=47,500), 277(11,000), and 295nm (9,750).

CD: AE (in EtOH solution): 290(+0.16) and 265nm (+0.56). 1H NIVIR:

H-4, 7.90(J=9.0); H-5, 6.80(,/=9.0,2.0);H-7, 6.60(,/=2.0);H-10, 5.65; 10-OH, 4.80, H-14, 3.65; H-15, 2.00; H-16, 2.00; H-17, 4.50; H-20, 6.05; H-21, 2.00; H-23, 1.02; H-24, 1.80;H-25, 6.70(J=8.0);H-26, 5.05; H-28, 1.80;H-29, 2.00; and H-30, 3.86ppm. ~3C NMR: C-2, 131.6 s; C-3, 105.7 s; C-4, 121.7 d; C-5, 109.3 d; C-6, 156.4 s; C-7, 93.9 d; C-8, 136.3 s; C-9, 121.1 s; C-10, 68.7 d; C-11, 82.6 s; C-12, 170.7 s; C-14, 51.3 t; C-15, 22.7 t; C-16, 29.1 t; C-17, 58.7 d; C-18, 166.2 s; C-20, 48.9 d; C-21, 45.3 t; C-22, 82.1 s; C-23, 27.1 q; C-24, 25.6 q; C-25, 85.8 q; C-26, 118.7 d; C-27, 142.9 s, C-28, 18.8 q; C-29, 24.2 q; and C-30, 55.7ppm q. TLC Data Silica gel G-HR; A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Re: A = 0.30, B = 0.65. Detection: slate gray-blue spot in visible light or mustard-colored spot under UV light; develops immediately atter spraying with 50% ethanolic H2SO4. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolit.e.S.; Academic Press, New York, pp. 368-373(1981). R. J. Cole, J. W. Kirksey, J. H. Moore, B. R. Blankenship, U. L. Diener, and N. D. Davis; Tremorgenic Toxin from Penicillium verruculosum; Applied Microbiology, pp. 248-256 (1972).

230

2. Diketopiperazines

Common/Systematic Name 15-Acetoxyverruculogen Molecular Formula/Molecular Weight C29H3sN3Og; ~

= 569.23733 OH 0 - OH

i@

N

'l" ,~1

o

o -

~Me

~ ~~~'o_o ._Z_ 0

General Characteristics Melting point, 217-218~ (dec.) Fungal Source P e n i c i l l i u m v e r r u c u l o s u m -, P. brasilianum.

Biological Activity Tremorgenic but activity less than that observed for comparable amount of verruculogen. Spectral Data UV:

~t-~M~~ 227 and 295nm (e max not reported). TLC Data Silica gel G-HR, toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Rf: 0.66. Detection: slate-gray spot develops immediately atter spraying with 50% ethanolic H2SO4. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 374-376(1981).

2. Diketopiperazines

231

Common/Systematic Name TR-2 Molecular Formula/Molecular Weight C22H27N306; M~V = 429.18999

OH

4 5

"

21

0

o ll -

14

~OH

General Characteristics Crystals from benzene-ethyl acetate, 95:5, v/v; mp., 150-152~ Fungal Source Penicillium verruculosum --, P. brasilianum, and Asperg#lus fumigatus Fres. Also,

obtained as one of two products from reductive cleavage of verruculogen. Biological Activity Perceptible tremors in day-old cockerels dosed orally at levels down to 12.5mg/kg. Spectral Data UV:

~

EtOH max

224(e = 37,400), 268(6,830), and 294nm (7,540).

~H NMR: (CDCI3) H-4, 7.60(d-9.0); H-5, 6.60(J=9.0, 3.0); H-7, 6.87; H-10, 5.52; 10-OH, 5.00; 11-OH, 4.20; H-14,3.54; H-15, 1.85; H-16, 1.85; H-17, 4.33; H-20, 5.37; H-21, 1.85; H-23, 1.05; H-24, 1.30; 22-OH, 5.94; and H-25, 3.71ppm. ~3C NMR: (CDC13) C-2, 131.8 s; C-3, 105.1 s; C-4, 121.2 d; C-5, 109.8 d; C-6, 156.6 s; C-7, 95.2; C-8, 137.2 s; C-9, 120.8 s; C-10, 68.8 d; C-11, 83.4 d; C-12, 171.4 s; C-14, 49.9 t; C-15, 22.7 t; C-16, 29.6 t; C-17, 58.9 d; C~ 166.2 s; C-20, 57.4 d; C-21, 45.3 t; C-22, 71.2 s; C-23, 31.9 q; C-24, 29.7 q; and C-25, 55.7ppm q. Mass Spectrum: 429 M + with prominent losses of-15(CH3), 18(OH), and 84m/e (C5H80).

232

2. Diketopiperazines

TLC Data Silica gel G-HR, toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Re: 0.46. Detection: light brown fluorescent spot after spraying with 50% ethanolic H2SO4 and heating at 100~ for 5 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 377-381(1981). R. J. Cole and J. W. Kirksey; The Mycotoxin Verruculogen: a 6-O-Methylindole; Agric. Food Chem., Vol. 21, pp. 927-929(1973).

2. Diketopiperazines

233

Common/Systematic Name Epiamauromine Molecular Formula/Molecular Weight C32H36N402; MW = 508.28383 5//Is

u 0

"2N

H HN,..j."'~.

~

''~"

0 General Characteristics Epiamauromine was isolated as a white solid; mp., 134"C; [a]D -50.0* (C=0.18g/dl, in CHCI3) and [a]D-29.1 ~ (c=0.46g/dl, CHCI3). Fungal Source Sclerotia ofAspergillus ochraceus (NRRL 3519). Isolation/Purification Intact sclerotia ofA. ochraceus were extracted at room temperature with hexane, then CHCI3. After removal of the solvent in vacuo, the hexane extract was subjected to reversed-phase HPLC [MeOH-H20 (9:1, v/v), 2ml/min] to give N-methylepiamauromine. The CHC13 extract, after removal of the solvent in vacuo, was fractionated through a column of Sephadex LH-20 (25-1001.tm) using CH2Cl2-hexane (1:1, v/v), then CH:CI2-MeOH (1:1, v/v) as eluents. The fractions obtained with CH2C12-hexane (1:1, v/v) were combined, concentrated, and purified by HPLC as above to give epiamauromine and N-methylepiamauromine. The fractions eluted with CH2CI2-MeOH (1:1, v/v) were likewise combined, concentrated, rechromatographed on Sephadex LH-20 using CHCIa-MeOH (2:1, v/v), and purified by reversed-phase HPLC to yield cycloechinulin. Biological Activity Caused moderate reduction in weight gain in assays against the lepidopteran crop pest Helieoverpa zea. Spectral Data UV;

~

Mr max

215(1og e=5.0), 243(5.0), and 300nm (4.6).

1H NMR: (CDCI3) H-1,5.36, br s; H-2, 5.30, s; H-4, 7.16(dd, J=-l.2, 7.8); H-5, 6.74(ddd, J=l.2,

234

2. Diketopiperazines

4.2, 7.2); H-6, 7.08(dt, J=l.2, 7.8); H-7, 6.56(ddd, ,/=0.6, 1.2, 7.8); H-10, 2.45(dd, ,/=9.6, 13.8); 2.75(dd, ,/=9.0, 13.3); H-11, 4.04(dt, ,/=1.8, 9.0); H-15, 5.86(dd, ,/=10.8, 17.4); H-16, 5.06(dd, ,/=1.2, 17.4); 5.09(dd, ,/=0.6, 10.8); H-17, 1.10, s; H-18, 0.93, s; H-I', 4.91, br s; H-2', 5.30, s; H-4', 7.12(dd, ,/=1.2, 7.2); H-5', 6.72(dd, J=l.2, 4.2, 7.2); H-6', 7.06(dt, ,/=1.2, 7.8); H-7', 6.52(dm, ,/=0.6, 1.2, 7.8); H-10', 2.50(dd, ./=6.0, 12.6); 2.35(dd, ./=10.8, 12.6); H-11', 3.90(ddd, ,/=1.8, 6.0, 11.4); H-15', 5.90(dd, ./=10.8, 17.4); H-16', 5.01(dd, ,/=1.2, 17.4); 5.07(dd, ,/=0.6, 10.8); H-17',1.05 s; and H-18', 0.95ppm s. ~3CNMR: (CDCI3) C-2, 79.3; C-3, 62.1; C-4, 125.7; C-5, 118.6; C-6, 128.3; C-7, 108.8; C-8, 148.4; C-9, 131.4; C-10, 36.0; C-11, 60.7; C-12, 166.0; C-14, 41.7; C-15, 143.3; C-16, 14.7; C-17, 22.9; C-18, 22.5; C-2', 77.6; C-3', 61.8; C-4', 125.0; C-5', 118.9; C-6', 128.8; C-7', 109.3; C-8', 150.0; C-9', 129.0; C-10', 35.2; C-11',62.0; C-12', 168.0; C-14', 40.8; C-15', 143.4; C-16', 114.4; C-17', 22.9; and C-18', 22.5ppm. Mass Spectrum: EIMS: [M]§ 508(0.03), 199(61), 158(20), 150(7), and 131m/e (100); HREIMS: found 508.2846; calcd for C32H36N402, 508.2838. HPLC Data Beckman Ultrasphere 5kt (10mm x 25cm) C~s reversed-phase column was used on all HPLC separations; HPLC retention time for epiamauromine was 12.7 min. Reference F. S. de Guzman, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; New Diketopiperazine Metabolites from the Sclerotia ofAspergillus ochraceus; Journal of Natural Products, Vol. 55, pp. 931-939(1992).

2.

Diketopiperazines

235

Common/Systematic Name N-Methylepiamauromine Molecular Formula/Molecular Weight C33H38N402; M W -- 522.29948 ~8

Me

Me

H

M

O

-

"

�9

N

H N

12,

N

,

Me

0 General Characteristics N-Methylepiamauromine was isolated as a white solid; [0g]D -29.1 ~ (c=0.46g/dl, in CHCI3). Fungal Source Sclerotia ofAspergillus ochraceus (NRRL 3519). Isolation/Purification Intact sclerotia of A. ochraceus were extracted at room temperature with hexane, then CHCI3. After removal of the solvent in vacuo, the hexane extract was subjected to reversed-phase HPLC [MeOH-H20 (9:1, v/v), 2ml/min] to give N-methylepiamauromine. The CHCI3 extract, alter removal of the solvent in vacuo, was fractionated through a column of Sephadex LH-20 (25-1001.tm) using CH2Cl2-hexane (1:1, v/v), then CH2CI2-MeOH (1:1, v/v) as eluents. The fractions obtained with CH2Cl2-hexane (1:1, v/v) were combined, concentrated, and purified by HPLC as above to give epiamauromine and N-methylepiamauromine. The fractions eluted with CH2CI2-MeOH (1:1, v/v) were likewise combined, concentrated, rechromatographed on Sephadex LH-20 using CHCI3-MeOH (2:1, v/v), and purified by reversed-phase HPLC to yield cycloechinulin. Biological Activity Caused moderate reduction in weight gain in assays against the lepidopteran crop pest Helieoverpa zea.

Spectral Data UV:

~

MeOH max

214(Iog e=4.4), 244(4.1), and 303nm (3.8).

IR:

(film on NaCI plate) 3380, "1665, and 1605cm"~.

236

2. Diketopiperazines

IH ~ : (CDCI3) H-I, 5.37, s; H-2, 5.32, S; H-4, 7.16(d, J=7.5); H-5, 6.73(dt, ,/=0.9, 7.5); H-6, 7.07, dd (`/=
Mass Spectrum: EIMS: [M] § 522(29), 453(59), 384(14), 255 (17), 184(14), 171(58), 157(17), and 144role (100); HREIMS: found 522.3010; calcd,for C33H38NaO2,522.2994. I-IPLC Data Beckman Ultrasphere 5~ (10mm x 25cm) Cl8 reversed-phase column was used on all HPLC separations; I-IPLC retention time for N-methylepiamauromine was 17.9 min. Reference F. S. de Guzman, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; New Diketopiperazine Metabolites from the Sclerotia ofAspergillus ochraceus; Journal of Natural Products, Vol. 55, pp. 931-939(1992).

2. Diketopiperazines

237

Common/Systematic Name Ditryptophenaline Molecular Formula/Molecular Weight C42H4oN604, ~ "- 692.31110

O

Cr .4VNMe O

~L N

026

y 0.Me

H o (3 General Characteristics Clear octahedral crystals from methylene chloride-methanol; mp., 204-205 ~ -330 ~ (c=0.52, in CH2CI2).

[a]D23s

Fungal Source

Aspergillusflavus (strains MIT-M25, 26 and 27).

Isolation/Purification Extracted with methylene chloride, precipitated with petroleum ether, and purified by column and preparative thin-layer chromatography. The Re of ditryptophenaline measured on 0.25mm silica gel plates developed with CHCI3:MeOH (20:1, v/v) was 0.80, and a dark green color was produced with van Urk's reagent. Biological Activity Possesses neither significant toxic nor antibiotic properties.

238

2. Diketopiperazines

Spectral Data UV~

~

EtOH max

244(e=15,250) and 303nm (6,200).

IR:

(CHCI3) 3415, 2995, 2930, 1662 br, 1611, 1498, 1480, 1470, 1455, 1401, 1355, 1323, 1270, 1162, 1151, 1135, 1078, 1052, 1027, 1020, 994, 974, 948, 906, 870, and 700cm"~. IH NMR*: (CDCI3) 1.59(t, I-H, J=12Hz), 2.05(d of d, I-H, J=12 and 5Hz), 3.03(s, 3-H), 3.21(d of d, l-H, J=14 and 5Hz), 3.55(d of d, l-H, J=14 and 4Hz), 3.65 (d of d, l-H, ,/=-12 and 5Hz), 1.25(br mult, l-H), 4.79(s, l-H, exchanges), 4.89(s, l-l-H), 6.5-6.9(m, 2-H), 6.5 - 7.3(m, 4-H), and 7.4-7.7ppm (m, 3-H). * Indicated a dimer. Mass Spectrum: The high resolution electron-impact mass spectrum gave a parent ion at 692.3095m/e (calcd for C42I-I40N604,692.3111). Reference J. P. Springer, G. Biichi, B. Kobbe, and A. L. Demain; The Structure of Ditryptophenaline-: A New Metabolite ofAspergillusflavus; Tet. Lett., pp. 2403-2406(1977).

2. Diketopiperazines

239

Common/Systematic Name Exserohilone Molecular Formula/Molecular Weight C2on22N206S2; M W = 450.09193

4

~

OH

O

..-'f~2A'"

MeS H 2 j

/2

HO

General Characteristics Colorless glass; [a]D 21

-247 ~

(c=0.32, in CHCI3).

Fungal Source Exserohilum holmii, a pathogenic fungus of the weedy plant Dactyloctenium aegyptium (crowfoot grass), which is a serious weed in all major tropical and semitropical agricultural areas of the world. Isolation/Purification The mycelium ofE. holmii was cultured on M-I-D culture broth shaken at 200 rpm for 2-3 weeks at 26 ~ under luminescence. Both the culture filtrate and the fluid obtained atter centrifugation of the homogenized mycelium were extracted with EtOAc, and the extract evaporated to dryness under reduced pressure and subjected to flash chromatography using (a) toluene-EtOAc (2:1, v/v) and (b) CHCI3-MeOH (70:1, v/v). Both exserohilone and 9,10-dihydroexserohilone were isolated by reversed phase HPLC (Merck, RP~8) with CH3CN-H20 (63:35, v/v) as the eluting solvent (lml/min). The retention time of both exserohilone and 9,10-dihydroexserohilone was 3.5 min in this HPLC system. Spectral Data IR:

(KBr) 3320, 2910, 1680, 1640, 1395, and 1260cm"~. ~H NMR: (CHCI3) 2.26(6H, s, SMe); 2.23(2H, overlapping, d, d, ,/--12.6, 13.6Hz, 1-1=-7,I-I,-14); 2.67(2H, d, d, ,/=4.7, 13.6Hz, 1-1=-7,1-1=-14);3.49(2H, d, d, ,/--4.7, 12.6Hz, H-7,, H-14,); 3.89(2H, d, d, d=8.7, 12.6Hz, (H-4,, H-11,); 4.70(2H, d, d, d, ,/--1.9, 1.9, 8.7Hz, H-4, H-11); 6.10(2H, s, OH); 6.1 l(2H, d, d, J=l.9, 9.1Hz, H-3, H-10); and 6.93ppm (2H, d, d, J=-1.9, 9.1Hz, H-2, H-9).

240

2. Diketopiperazines

13C NMR: (CDCI3) 15.1(q, SMe); 32.1(t, C-7, C-14); 46.3(d, C-7a, C-14,); 69.0(d, C-4,, C-11,); 72.4(s, C-6a, C-13a); 72.9(d, C-4, C-11); 128.6(d, C-2, C-9); 151.1(d C-3, C-10); 168.0(s, C-6, C-13); and 195.2ppm (s, C-l, C-8).

Mass Data: LREIMS: 450(2%), 405(24), 403(100), 375(27), 356(27), 355(28), 329(9), 328(18), 160(6), 133(8), 132(8), 110(5), and 95m/e (7); HREIMS: (M+; obsd 450.0919re~e; calcd for C20H22N206S2,450.0920) and C I 9 H I g N 2 0 6 S I ( M + - SMe; obsd 403.0963m/e; calcd 403.0964). TLC Data Re values on silica gel using CHCI3-MeOH (50:1, v/v) were 0.19 for exserohilone and 0.15 for 9,10-dihydroexserohilone. Reference K. Sugawara, F. Sugawara, G. A. Strobel, Y. Fu, H. Cun-Heng, and J. Clardy; Exserohilone: a Novel Phytotoxin Produced by Exserohilum holmii; J. Org. Chem., Vol. 50, pp. 5631-5633(1985).

2. Diketopiperazines

241

Common/Systematic Name 9,10-Dihydroexserohilone Molecular Formula/Molecular Weight C2oH24N20682; M~W - 450.09193

4

OH

O

1~ , 4 a 4 a / ~ N " ~ ~ "

_H /O

MeS ~) H - y HO General Characteristics [a]D 21 -110 ~ (c=0.33, in CHCI3). Fungal Source

Exserohilum holmii, a pathogenic fungus of the weedy plant Dactyloctenium aeDptium (crowfoot grass), which is a serious weed in all major tropical and semitropical agricultural areas of the world.

Isolation/Purification The mycelium of E. holmii was cultured on M-1-D culture broth shaken at 200 rpm for 2-3 weeks at 26 ~ under luminescence. Both the culture filtrate and the fluid obtained after centrifugation of the homogenized mycelium were extracted with EtOAc, and the extract evaporated to dryness under reduced pressure and subjected to flash chromatography using (a) toluene-EtOAc (2:1, v/v) and (b) CHCIrMeOH (70:1, v/v). Both exserohilone and 9,10-dihydroexserohilone were isolated by reversed phase HPLC (Merck, RP~s) with CH3CN-H20 (63:35, v/v) as the eluting solvent (lml/min). The retention time of both exserohilone and 9,10-dihydroexserohilone was 3.5 min in this HPLC system. Spectral Data IH NMR:

(CHCI3) 1.6(2H, m, H-10); 1.96(IH, m, H-7); 2.238(3H, s, SMe); 2.242(3H, s, SMe); 2.27(IH, d, d, ,/--12.0,13.7Hz, Ha-14); 2.51(3H, overlapping,m, H-7', H-9); 2.64(IH, d, d, J=4.7, 13.7I-Iz,I-I~-14),3.49(2H, m, H-7a, H-14a); 3.77(IH, d, d, ,/=8.6, 13.9Hz, H-I la);3.86(IH, d, d, J=8.6, 13.9Hz, H-4,); 4.27(II-I,d, d, J=ca. 6.0, 8.6Hz, H-II); 4.69(IH, d, d, d, ,/=ca.1.6, 2.0, 8.6Hz, H-4), 5.66(IH, s, OH), 6.09(IH, d, d, ,/=2.0, 10.1Hz, H-3); 6.12(IH, s, OH); and 6.92ppm (IH, d, d, ,/--1.6,I0.II-Iz,H-2).

242

2. Diketopiperazines

Mass Data: LREIMS: 452(1.8%, M+), 406(8), 405(33), M*- SMe, 359(9), 358(33, M* - 2 SMe), 357(100), 341(5), 339(7), 323(96), and 273role (13); HREIMS: (M +, obsd 452.1075m/e; calcd for C2oH2~N206S2452.1077) and CIgH21N206SI (M+ - SMe; obsd 405.1120m/e; calcd 405.1121). TLC Data Re values on silica gel using CHCI3-MeOH (50:1, v/v) were 0.19 for exserohilone and 0.15 for 9,10-dihydroexserohilone. Reference K. Sugawara, F. Sugawara, G. A. Strobel, Y. Fu, H. Cun-Heng, and J. Clardy; Exserohilone: a Novel Phytotoxin Produced by Exserohilum holmii, J. Org. Chem., Vol. 50, pp. 5631-5633(1985).

2. Diketopiperazines

243

Common/Systematic Name Epoxyexserohilone Molecular Formula/Molecular Weight C2oH22N206S2; MW' 450.09193 =

H

O

N ,.L.Me 10 HH O 20 19X ~ -~11,,0H MeS ~

~ , ~ , ~ ' ~H 0 H

General Characteristics Crystals from benzene-acetone; mp., 190-192~ Fungal Source

Nigrospora sphaerica.

Isolation/Purification Cultures were extracted with acetone, reduced in volume and reextracted with ethyl acetate. The crude extract was chromatographed on a silica gel column eluted with a polarity series: benzene, t-butylmethyl ether, ethyl acetate, acetone, and acetonitrile. The epoxyexserohilone-rich fractions (t-butylmethyl ether, ethyl acetate, and acetone) were combined, reduced in volume, and chromatographed on a silica gel column eluted with a linear gradient from benzene to acetone. Epoxyexserohilone crystallized from enriched fractions upon sitting for 72hr at room temperature. Spectral Data UV: ~,,~E~" End absorption. IR:

(KBr) 1384, 1068, 1022, 971,892, 878, 852, 834, 792, 725, 672, 659, and 645cm "~. 1H NMR: (acetone-d6) H-10, 5.90; H-11, 4.57(m, ,/=8.8, 5.7, 4.5, ca. 1.5Hz); H-12, 3.45(ddd, J=4.5, 3.4, 1.0Hz); H-13, 3.75(dt, 3.5, 0.4Hz); H-14, 4.3 l(sextet, 1.4Hz); OH, 3.71(d, 8.8Hz); H-8,,,, 3.00(ddt, 14.1, 2.2, 1.SHz); H-8~q, 2.91(dm, 14.1Hz); and CH3S, 2.15ppm (s).

244

2. Diketopiperazines

Mass Spectrum: 450(M+), 403.0964(100%)(M + - SCHa), 356, 337, and 160role. TLC Data Silica gel G, F254thin-layer plates developed in toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Re 0.10-0.16; detected as a lemon yellow-colored spot by spraying with anisaldehyde and heating to 100~ Reference H. G. Cutler, K. Hoogsteen, R. H. Littrell, and B. H. Arison; Epoxyexserohilone, a Novel Metabolite from Nigrospora sphaerica; Agric. Biol. Chem., Vol. 55, pp. 2037-2042 (1991).

Chaetoglobosins/Cytochalasins Chaetoglobsin A Chaetoglobosin B Chaetoglobosin C Chaetoglobosin D Chaetoglobosin E Chaetoglobosin F Chaetoglobosin G Chaetoglobosin J

19-O-AcetylchaetoglobosinA 19-O-AcetylchaetoglobosinB

19-O-Acetylchaetoglobosin D Chaetoglobosin K Chaetoglobosin L Chaetoglobosin M Cytochalasin A; Dehydrophomin Cytochalasin B; Phomin Cytochalasin C Cytochalasin D; Zygosporin A Cytochalasin F Cytochalasin L Cytochalasin M Zygosporin D Zygosporin E Zygosporin F; Cytochalasin D monoacetate Zygosporin G Cytochalasin H; Kodocytochalasin-1; Paspalin-P Cytochalasin U Deacetylcytochalasin H; Cytochalasin J; Kodocytochalasin-2; Paspalin-P2 Epoxycytochalasin H Deacetylepoxycytochalasin H; Epoxydeacetylcytochalasin H; Epoxycytochalasin J Engleromycin Pyrichalasin H Cytochalasin Npho Cytochalasin Opho Cytochalasin Ppho Cytochalasin Qpho Cytoehalasin Rpho' Cytochalasin S Cytochalasin Nhyp Cytochalasin Ohyp Cytochalasin Phyp Cytochalasin Qhyp 19,20-Epoxycytochalasin Q

245

246

3. Chaetoglobosins/Cytochalasins

19,20-Epoxydeacetylcytochalasin Q Cytochalasin RHYP Cytochalasin G Aspoehalasin A Aspoehalasin B Aspoehalasin C Aspoehalasin D Cytochalasin E Rosellichalasin Cytochalasin KASP Deoxaphomin Proxiphomin Protophomin Ascochalasin Cytochalasin KCHA

3. Chaetoglobosins/Cytochalasins

247

Common/Systematic Name Chaetoglobosin A (6R,7S, 16S,19R)-6,7-Epoxy- 10-(indol-3-yl)-19-hydroxy-16,18-dimethyl[13]cytochalasa13(E), 17(E),21(E)-triene- 1,20,23-trione Molecular Formula/Molecular Weight C32H36N2Os, M W = 5 2 8 . 2 6 2 4 2

Me,.,

Me, .... H

~ N H

0 , iiii

....'Me 0 Iq "0 Me

General Characteristics Crystals from dichloromethane; mp, 168-170~

[tt]D -270 ~ (in MeOH).

Fungal Source

Chaetomium globosum, C. cochfioides, C. mollipifium, C. rectum, and C. subaffine.

Isolation/Purification The culture filtrates of Chaetomium globosum were extracted with methylene chloride and the major metabolites, chaetoglobosin A and 19-O-acetylchaetoglobosin A separated by crystallization. Biological Activi'ty Cytotoxicity to HeLa cells, EDs0 between 3.2 and 101,tg/ml. Caused inhibition of cytoplasmic cleavage resulting in polynucleate cells. LDs0 in male mice dosed subcutaneously, 6.5mg/kg; in female mice, 17.8mg/kg. Spectral Data UV: i, MeOH max

223(e=40,700), 274(6,600), 282(6,600), and 291rim (5,300); 196(1og c=4.63), 220(4.62), 271(3.82), 280(3.82), and 290nm (3.72).

IR:

(KBr) 3600-3200, 1665, 1610, 1450, 1420, 1380, 1290, 1240, 1040, and 965cm"~.

248

3. Chaetoglobosins/Cytochalasins

1H NMR: H-2, 5.85; H-3, 3.81(J=3.0, 4.3, 7.5); H-4, 3.03(J=3.0, 5.0); H-5, 1.85(,/--5.0, 7.0); H-7, 2.78(J=5.0), H-8, 2.14(J=5.0, 9.5); H-10, 2.63(J=1.5, 7.5, 15); H-11, 1.24(J=7.0); H-12, 1.29; H-13, 6.05(J=9.5, 15); H-14, 5.20(J--4.0, 10.0, 15); H-15, 1.8-2.4; H-16, 2.42(J=6.5, 9.0); H-17, 5.57(J=9.0); H-19, 5.01; H-21, 7.72(J=16.5); H-22, 6.50(J=16.5); H-24, 1.00(J=6.5); H-25, 1.31; 19-OH, 3.84; H-I', 8.21; and H-2', 6.94; H-4', 7', 7.0-7.5. (400 MHZ, CDCI3): 0.99(d J=6.5, 3H); 1.25(d J=7.5 3H); 1.30(s, 3H); 1.32(d J=l, 3H); 1.85(m, 1H); 2.0-2.1(m, 1H); 2.13(dxd J=5 and 10, 1H); 2.2-2.3(m, 1H); 2.4-2.6(m, 1H); 2.64(dxd J=15, J=8, 1H); 2.79(d J=5, 1H); 2.96(dxd J=15, J=4, 1H); 3.03(dxd J=-3 and 5, 1H); 3.81(m, 1H); 3.87(d J---4, 1H; exchangeable with D20); 5.03(d J=4, 1H); 5.22(m, 1H); 5.59(dxd J=-l, 1H); 5.94(s, 1H; exchangeable with D20); 6.06(dxd J= 15 and 10, 1H); 6.48(d J= 17, 1H); 6.9-7.5(m, 5H); 7.73(d J= 17, 1H); and 8.25ppm (br. s, 1H; exchangeable with D20). ~3CNMR: (CDCI3) C-I, 173.2; C-3, 52.8; C-4, 46.8; C-5, 36.3; C-6, 58.0; C-7, 62.4; C-8, 48.8; C-9, 63.4; C-10, 33.7; C-11, 13.2; C-12, 19.7; C-13, 128.3; C-14, 133.1; C-15, 41.7; C-16, 32.0; C-17, 139.9; C-18, 132.3; C-19, 81.7; C-20, 201.4; C-21, 131.6; C-22, 136.1; C-23, 197.1; C-16CH3, 20.9; C-18CH3, 10.5; C-2', 121.7; C-3', 109.4; C-3a', 127.4; C-4', 118.4; C-5', 124.0; C-6', 119.3; C-7', 111.7; and C-74',136.6ppm. Mass Spectrum: 528(M+), 398, and 130m/e. TLC Data Silica gel G-H~ Solvent A: chloroform-acetone, 93:7, v/v; Solvent B: toluene-ethyl acetate-formic acid, 5:4: !, v/v/v. Re values of A: 0.05; B: 0.65. Detection: a light olivegray fluorescent spot under UV light alter spraying with 50% ethanolic H2SO4 and heating. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 304 (1981). A. Probst and C. Tamm; 19-O-Acetylchaetoglobosin B and 19-O-Acetylchaetoglobosin D, Two New Metabolites of Chaetomium globosum; Helvetica Chimica Acta, Vol. 64, pp. 2056-2064(1981). S. Nator and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991). S. Sekita, K. Yoshihira, S. Natoriand, and H. Kuwano; Structures of Chaetoglobosins A and B, Cytotoxic Metabolites of Chaetomium globosum; Tetrahedron Lett., p. 2109 (1973).

3. Chaetoglobosins/Cytochalasins

249

Common/Systematic Name Chaetoglobosin B (7S, 16S, 19R)- 10-(Indol-3-yl)-7,19-dihydroxy- 16,18-dimethyl[ 13]cytochalasa5(6), 13(E), 17(E),21 (E)-tetraene- 1,20,23-trione Molecular Formula/Molecular Weight C32Hs6N205;

MW

"- 5 2 8 . 2 6 2 4 2

Me

[ jj NH

-

Me,.... ~ O H H ...... HN

o

I

I

....'Me e

General Characteristics Crystals from benzene; mp., 186-187~

[a]D -176 ~ (in MeOH).

Fungal Source Chaetomium globosum, C. cochlioides, C. mollipifium, C. rectum, and C. subaffine. Biological Activity Cytotoxicity to HeLa cells, EDs0 between 3.2 and 101.tg/ml; caused inhibition of cytoplasmic cleavage, resulting in polynucleate cells. Spectral Data UV: Ef~

222(e=43,600), 274(7,900), 281(7,900), and 290nm (6,700).

IH NMR: H-4, 3.40; H-7, 3.53; H-8, 2.06; H-11,1.64; and H-12, 1.73ppm. The remainder of the protons are the same as in chaetoglobosin A. TLC Data Silica gel G-H~ Solvent: A: chloroform-acetone, 93:7 v/v; B: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v.Rf values for A: 0.05; B: 0.61. Detection: a dark olive-gray fluorescent spot under UV light after spraying with 50% ethanolic H2SO4 and heating.

250

3. Chaetoglobosins/Cytochalasins

References R. J. Cole and R. H. Cox; Handbo_okof Toxic Fungal Metabolites; Academic Press, New York, p. 308 (1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991). S. Sekita, K. Yoshihira, S. Natori, and H. Kuwano, Structures of Chaetoglobosins A and B, Cytotoxic Metabolites of Chaetomium globosum; Tetrahedron Lett., p. 2109 (1973).

3. Chaetoglobosins/Cytochalasins

251

Common/Systematic Name Chaetoglobosin C (6R,7S, 16S)-6,7-Epoxy- 10-(indol-3-yl)- 16,18-dimethyl[ 13]cytochalasa- 13(E), 17(E)diene- 1,19,20,23-tetraone Molecular Formula/Molecular Weight C32H36N205; M W -- 5 2 8 . 2 6 2 4 2

Me.,.

Me, ....

~'NH

H

0 ,,llll

General Characteristics Colorless leaflets from acetone; mp, 260-263 ~ colorless crystals; mp, 259-261 oC.

'Me

[t~]o -30 ~ (in MeOH); acetone gave

Fungal Source Chaetomium globosum (strain Lederle H-124), C cochlioides, C. mollipilium, C rectum, C. subaffine, and Penicillium auranaovirens. Isolation/Purification To extract the culture broth, an equal volume of ethanol was added, the mixture kept overnight at 4~ and the mycelium separated by filtration. The ethanol was then evaporated and the aqueous layer extracted with CH2C12. The organic layer was dried (Na2SO4) and evaporated to dryness. The brown residue was dissolved in benzene and kept 3 days at room temperature. The crystallized mixture of chaetoglobosin A and 19-0acetylchaetoglobosin A was separated by filtration and chromatographed on silica gel using increasing amounts of methanol in CH2C12. The benzene solution was evaporated and the residue chromatographed on silica gel in the same manner to yield 19-O-acetylchaetoglobosin A, ehaetoglobosin A, chaetoglobosin C, 19-O-acetylchaetoglobosin D, and 19-O-a.cetylchaetoglobosin B. Biological Activity Cytotoxicity to HeLa cells, EDs0 between 10 and 32ktg/ml. Oral LDs0 determination using day-old chicks was not successful due to insolubility of purified toxin; crude extracts were highly toxic.

252

3. Chaetoglobosins/Cytochalasins

Spectral Data UV:

~

EtOH max

222(e=36,300), 273(6,700), 281(6,700), and 291nm (5,700).

13C NMR: C-I, 173.8 s; C-3, 52.3 d; C-4, 48.4 d; C-5, 36.1 d; C-6, 56.6 s; C-7, 60.3 d; C-8, 48.4 d; C-9, 62.2 s; C-10, 39.5 t; C-11, 9.9 q; C-12, 19.3 q; C-13, 127.0 d; C-14, 133.1 d; C-15, 32.6 t; C-16, 37.0 d; C-17, 155.8 d; C-18, 130.9 s; C-19, 196.1 s; C-20, 205.2 s; C-21, 31.9 t; C-22, 31.9 t; C-23,208.1 s; C-24, 12.3 q; C-25, 19.1 q; C-2', 120.9 d; C-3', 108.0 s; C-4', 118.6 d; C-5', 125.1 d; C-6', 118.3 d; C-7', 111.3 d; C-8', 135.8 s; and C-9', 127.6ppm s. TLC Data Silica gel G-I-I~ Solvent A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Rf value of A: 0.13; B: 0.71. Detection: a slate gray fluorescent spot under UV light after spraying with 50% ethanolic H2SO4 and heating. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 313 (1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991). A. Probst and C. Tamm; 19-O-Acetylchaetoglobosin B and 19-O-Acetylchaetoglobosin D, Two New Metabolites of Chaetomium globosum; Helvetica Chimica Acta, Vol. 64, pp. 2056-2064(1981). J. P. Springer, J. Clardy, J. M. Wells, R. J. Cole, J. W. Kirksey, R. D. MacFarlane, and D. F. Torgerson; Tetrahedron Lett., p. 1355 (1976).

3. Chaetoglobosins/Cytochalasins

253

Common/Systematic Name Chaetoglobosin D (7S, 16S,19R)-7,19-Dihydroxy-10-(indol-3-yl)-16,18-dimethyl[13]cytochalasa6(12), 13(E), 17(E),21 (E)-tetraene- 1,20,23-trione Molecular Formula/Molecular Weight C32H36N205; M ~ r = 5 2 8 . 2 6 2 4 2

CH2 Me,....r / ~ O H H

~ N H

,lllll

'Me

General Characteristics Pale yellow prisms from chloroform solution; mp, 216~

[a]D -269 ~ (in MeOH).

Fungal Source Chaetomium globosum, C. cochfioides, C. mollipifium, C. rectum, and C. subaffine. Biological Activity Cytotoxicity to HeLa cells, EDs0 between 3.2 and 10~g/ml. St)corral ~ t a UV: ~,Em~ 221(e=43,600), 273(9,100), 281(9,100), and 290nm (7,500). IR:

(KBr) 3421, 3280, 1686, 1606, 972, 908, and 750cm~. 1H NMR: H-4, 3.30; H-5, 3.14; H-7, 4.59; H-8, 2.92; H-11, 1.23; H-12, 5.21, 5.66; H-13, 6.53; and H-14, 5.44. The remainder of the protons are the same as in chaetoglobosin A. Mass Spectrum: Molecular ion (M§ 528.26lm/e; base peak 130.037m/e (C9I-IsN). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 319 (1981).

254

3. Chaetoglobosins/Cytochalasins

S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, g. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338 (1991). S. Sekita, K. Yoshihira, S. Natori, and H. Kuwano, Structures of Chaetoglobosins A and B, Cytotoxic Metabolites of Chaetomium globosum; Tetrahedron Lett. p. 2109 (1973).

3. Chaetoglobosins/Cytochalasins

255

Common/Systematic Name Chaetoglobosin E (7S, 16S,20S)-7,20-Dihydroxy- 10-(indol-3-yl)- 16,18-dimethyl[ 13]eytochalasa5(6), 13(E), 17(E)-triene- 1,19,23-trione Molecular Formula/Molecular Weight C32H38N205, MW = 530.27807 Me

Me.OH H NH

,,llll

Me

General Characteristics Colorless needles from methanol; mp., 279-280~

[a]D + 158 ~ (in Met)H).

Fungal Source Chaetomium globosum, C. cochfioides, C. mollipifium, C. rectum, and C. subaffine. Biological Activity Cytotoxicity to HeLa calls, EDs0 between 3.2 and 101~g/ml. Spectral Data UV:

~m~

221(e=56,200), 275(7,000), 281(7,000), and 291nm (6,300).

IR:

(KBr) 3410, 1704, 1676, 1048, and 746cm ~. IH NMR: H-7, 4.56; 7-OH, 5.97; H-11, 1.49; H-12, 1.90; H-17, 6.34; H-20, 5.29; 20-OH, 6.10; H-21, 2.41; and H-22, 3.41ppm. The remainder of the protons are the same as in chaetoglobosin A. Mass Spectrum: 530.275m/e (M+); base peak 130.13 lm/e (CgI-IsN).

256

3. Chaetoglobosins/Cytochalasins

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 322 (1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991). S. Sekita, K. Yoshihira, S. Natori, and H. Kuwano; Structures of Chaetoglobosins A and B, Cytotoxic Metabolites of Chaetomium globosum; Tetrahedron Lett., p. 2109 (1973). S. Sekita, K. Yoshihira, S. Natori, and H. Kuwano; Structures of Chaetoglobosins C, D, E, and F, Cytotoxic indol-3-yl[ 13]cytochalasins from Chaetomium globosum; Tetrahedron Lett., p. 1351 (1976).

3. Chaetoglobosins/Cytochalasins

257

Common/Systematic Name Chaetoglobosin F (6R,7S, 16S,20S)-6,7-Epoxy-20-hydroxy- 10-(indol-3-yl)- 16,18-dimethyl[ 13]cytochalasa- 13(E), 17(E)-diene- 1,19,23-trione Molecular Formula/Molecular Weight C32H35N2Os; MW

=

530.27807

Me,,,

Me, ....

H

[

0 ,,|iii

~NH

i'Me

General Characteristics Colorless leaflets from benzene; mp., 177-178~

[a]D-69 ~ (in CHCI3).

Fungal Source Chaetomium globosum. Biological Activity Cytotoxicity to HeLa cells, EDs0 10~g/ml. Spectral Data UV:

Z Em~

222(C=47,800), 276(6,900), 283(6,700), and 292nm (6,000).

IR:

(KBr) 3346, 1676, 1618, 979, 879, and 740cm"l. 1H NMR:

H-5, 2.01; H-7, 3.12; H-11, 0.97; H-12, 1.25; H-17, 6.26; H-20, 5.14; H-21, 2.27; H-22, 2.95; and H-25, 1.93ppm. The remainder of the protons are the same as in chaetoglobosin A. Mass Spectrum: 530.275m/e (M+); base peak 130.107m/e (C9HaN).

258

3. Chaetoglobosins/Cytochalasins

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites Academic Press, New York, p. 325 (1981). S. Natori and I. Yahara; In Mycotoxins and Phyt0alexin..s, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991). S. Seldta, K. Yoshihira, S. Natori, and H. Kuwano; Structures of Chaetoglobosins C, D, E, and F, Cytotoxic Indoi-3-yl[ 13]cytochalasins from Chaetomium globosum; Tetrahedron Lett., p. 1351 (1976).

3. Chaetoglobosins/Cytochalasins

259

Common/Systematic Name Chaetoglobosin G (7S, 16S)-7-Hydroxy- 10-(indol-3-yl)-16,18-dimethyl[13]cytochalasa-5(6), 13(E), 17(E)triene- 1,19,20,23-tetraone Molecular Formula/Molecular Weight C32H36N2Os; MW = 528.26242 Me

M e ~ H

OH tlt111

@J~NH

Me

0

0

General Characteristics Colorless leaflets from methanol; mp, 251-253 ~

[a]D + 89 ~

Fungal Source

Chaetomium globosum.

Biological Activity Cytotoxicity to cultured HeLa cells at ca. 3pg/ml, forms multinucleated cells. Spectral Data UV:

Z~

222(e=32,300), 275(6,000), 282(6,000), and 291nm (5,300).

IR:

(KBr) 3455, 3300, 1713, 1693, 1646, 1623, 987, 948, and 741cmq. ~H NMR: Perhydroisoindolone protons same as in chaetoglobosin B and E. Thirteen-membered ring protons are the same as in chaetoglobosin C. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 328 (1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

260

3. Chaetoglobosins/Cytochalasins

S. Sekita, K. Yoshihira, S. Natori, and H. Kuwano; Chaetoglobosins G and J, Cytotoxic Indol-3-yl-[ 13]-Cytochalasins from Chaetomium globosum; Tetrahedron Lett., Vol. 32, p. 2771 (1977).

3. Chaetoglobosins/Cytochalasins

261

Common/Systematic Name Chaetoglobosin J (16S, 19R)-19-Hydroxy- 10-(indol-3-yl)-16,18-dimethyl[13]cytochalasa6(7), 13(E), 17(E),21(E)-tetraene- 1,20,23-trione Molecular Formula/Molecular Weight C32H36N204; MW = 512.26751 Me

H

Q

2

...... ,3

....'Me

H

0

~Me 01~ "OH

General Characteristics Pale yellow prisms from benzene; mp, 149-151~ Fungal Source

Chaetomium globosum.

Biological Activity Cytotoxicity to cultured HeLa cells at ca. 3~g/ml. Unlike other members of the group, chaetoglobosin J does not form multinucleated cells. Spectral Data UV:

~

EtOH max

224(c=47,800), 270(7,000), 280(7,000), and 290nm (6,000).

IR:

(KBr) 3412, 3273, 1683, 1639, 1612, 980, 975, 925, and 750cm"1. IH NMR: H-4, 3.12; H-5, 2.46; H-7, 5.30; H-8, 2.56; H-11, 1.42; and H-12, 1.80ppm. The remainder of the protons are the same as in chaetoglobosin A. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 329 (1981).

262

3. Chaetoglobosins/Cytochalasins

S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991). S. Sekita, K. Yoshihira, S. Natori, and H. Kuwano, Chaetoglobosins G and J, Cytotoxic Indol-3-yl-[ 13]-Cytochalasins from Chaetomiumglobosum; Tetrahedron Lett., Vol. 32, p. 2771 (1977).

3. Chaetoglobosins/Cytochalasins

263

Common/Systematic Name 19-O-Acetylchaetoglobosin A (6R, 7S, 16S, 19/?)- 19-Acetoxy- 10-(indol-3-yl)- 16,18-dimethyl[ 13]cytochalasa13(E), 17(E),21 (E)-triene- 1,20,23-trione Molecular Formula/Molecular Weight C34I-I35N206; M W = 570.27299

Me,,.

Me,,,

H Oo

0 '~176

~J~NH

....'Me Ofl

o~~

General Characteristics

19-O-Acetylchaetoglobosin A was crystallized from benzene to give a yellow powder; nap, 223-225~ [a]D2~-304 ~ (c=0.5, in CHCI3).

Fungal Source Chaetomium globosum (strain Lederle H-124). Isolation/Purification The culture filtrates of Chaetomium globosum were extracted with methylene chloride and the major metabolites chaetoglobosin A and 19-O-acetylchaetoglobosin A separated by crystallization. Biological Activity Biological activity not reported but probably typical activity of other related metabolites. Spectral Data UV:

~

EtOH max

196(1og c=4.65), 220(4.64), 271(3.83), 280(3.83), and 289nm (3.74).

IR:

(KBr): 3380, 1675, 1620, 1450, 1425, 1365, 1240, 1220, 1180, 1030, 970, and 960cmq. ~H NMR: 2N-H, 6.05, br s; H-3, 3.80, m; H-4, 2.93, dxd, J=3 and 5; H-5, 1.84, m; H-7, 2.80, d,

264

3. Chaetoglobosins/Cytochalasins

J=5; H-8, 2.14, dxd, J=5 and 10; H-10, 2.91, dxd, J=15, and 4 ; H-10, 2.67, dxd, 3=15 and 7.5; H-11, 1.22, d, J=7; H-12, 1.28, s; H-13, 6.08, dxd, ,/=15 and 10; H-14, 5.18, m; H-15, 2.3-2.3, m; 1.9-2.0, m; H-16, 2.4-2.6, m; H-17, 5.66, dxd, J=9.0 and 1; H-19, 5.90, s; H-21, 7.55, d, J=17; H-22, 6.61, d, J=17; 16-CH3, 0.99, d, 3=6.5; 18-CH3, 1.45, d, J=l; 19-O-acetyl, 2.17, s; I'-NH, 8.31, br, s; and 5H-indolyl, 7.0-7.5ppm. 13C NMR: [(CD3)2SO] C-l, 173.6; C-3, 52.8; C-4, 47.3; C-5, 36.4; C-6, 58.0; C-7, 62.3; C-8, 48.3; C-9, 63.4; C-10, 34.0; C-11, 13.3; C-12, 19.7; C-13, 128.3; C-14, 133.4; C-15, 41.3; C-16, 32.2; C-17, 142.5; C-18, 127.6; C-19, 83.4; C-20, 194.8; C-21, 133.4; C-22, 134.8; C-23, 196.9; C-16CH3, 20.7; C-18CH3, 11.5; acetyl-CH3, 20.7; Acetyl-CO, 170.1; C-2', 122.3; C-3', 110.1; C-3a', 127.3; C-4', 118.3; C-5', 123.7; C-6~ 119.9; C-7', 111.7; and C-7a', 136.5ppm. Mass Spectrum: 570(NC) and 527m/e. References A. Probst and C. Tamm; 19-O-Acetylchaetoglobosin B and 19-O-Acetylchaetoglobosin D, Two New Metabolites of Chaetomium globosum; Helvetica Chimica Acta, Vol. 64, pp. 2056-2064(1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

265

Common/Systematic Name 19-O-Acetylchaetoglobosin B (7S, 16S, 19R)- 19-Acetoxy- 10-(indol-3-yl)-7-hydroxy- 16,18-dimethyl[ 13]cytochalasa(6), 13(E), 17(E),21 (E)-tetraene- 1,20,23-trione Molecular Formula/Molecular Weight C34I-I3sN206; MW = 570.27299

'......r ~ H

OH 1111||

I

I

...... '

General Characteristics Gave yellow needles from benzene; mp., 154-157~

[a]D2~ -148 ~ (c--0.5, in CHCI3).

Fungal Source Chaetomium globosum (strain Lederle H- 124). Isolation/Purification The culture filtrates of Chaetomium globosum were extracted with methylene chloride and the major metabolites chaetoglobosin A and 19-O-acetylchaetoglobosin A separated by crystallization. The remaining extract was purified by column chromatography on silica gel. Chaetoglobosin C, 19-O-acetylchaetoglobosin B and D were enriched. Crystallization ofchaetoglobosin C led to a mixture of 19-O-acetylchaetoglobosin B and 19-O-acetylchaetoglobosin D which were separated and obtained pure by chromatography on silica gel 60 GF254using the chromatotron. Spectral Data

UV: ~

EtOH

max

221(1og e=4.64), 272(3.83), 279(3.83), and 289nm (3.74).

IR: (KBr) 3600, 3200, 1730, 1710, 1620, 1455, 1430, 1375, 1290, 1250, 1230, 1035, and 970cm"~. IH NMR: 2N-H, 5.84, br s; H-3, 3.55, m; H-4, 3.24, br s, J=
266

3. Chaetoglobosins/Cytochalasins

t, J=10 and 10; H-10, 2.86, dxd, J=14, 6; H-10, 2.71, dxd, J=14,8; H-11, 1.63, br s; H-12, 1.72, br, s; H-13, 6.18, dxd, d=16, 10; H-14, 5.28, m; H-15, 2.3-2.4, m 2.0-2.1, m; H-16, 2.4-2.6, m; H-17, 5.64, dxd, J=10,1; H-19, 5.87, s; H-21, 7.54, d, ,/--17; H-22, 6.73, d, J=17; 16-CH3, 1.02, d, ,/=6.5; 18-CH3, 1.51, d, J=l; 19-O-acetyl, 2.17, s; I'-NH, 8.20, br, s; 5H-indoyl, 7.0-7.5; and 7-OH, 1.88ppm, br, s. 13C NMR: C-l, 172.4; C-3, 57.8; C-4, 46.9; C-5, 125.2; C-6, 133.7; C-7, 67.7; C-8, 50.1; C-9, 60.8; C-10, 31.7; C-11, 14.4; C-12, 16.9; C-13, 127.2; C-14, 133.7; C-16, 32.0; C-17, 141.7; C-18, 127.2; C-19, 83.1; C-20, 194.0; C-21, 134.1; C-22, 135.2; C-23, 199.8; C-16CH3, 20.3; C-18CH3, 11.1; Acetyl-CH3, 20.6; Acetyl-CO, 169.2; C-2', 121.0; C-3', 109.9; C-3a' 126.1; C-4', 118.1; C-5', 123.5; C-6', 118.4; C-7', 111.4; and C-Ta', 136.2ppm. Mass Spectrum: 570(M+), 528, 185, 173, 130, 103, and 60role. References A. Probst and C. Tamm; 19-O-Acetylchaetoglobosin B and 19-O-Acetylchaetoglobosin D, Two New Metabolites of Chaetomium globosum; Helvetica Chimica Acta, Vol. 64, pp. 2056-2064(1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

267

Common/Systematic Name

19-O-Acetylchaetoglobosin D (7S, 16S, 19R)- 19-Acetoxy-7-hydroxy- 10-(indol-3-yi)- 16,18-dimethyl[ 13]cytochalasa6(12), 13(E), 17(E),21 (E)-tetraene- 1,20,23-trione

Molecular Formula/Molecular Weight C34H38N206; M3cV

=

570.27299

Me,,, Hle.r ~ O H L~

lOll

NH

....'Me o"

o Me

General Characteristics Gave yellow prisms from CHCI3; mp., 239-241 ~ ; [a]D2~ -176 ~ Fungal Source

Chaetomium globosum (strain Lederle H- 124).

Isolation/Purification The culture filtrates of Chaetomium globosum were extracted with methylene chloride and the major metabolites chaetoglobosin A and 19-O-acetylchaetoglobosin A separated by crystallization. The remaining extract was purified by column chromatography on silica gel. Chaetoglobosin C, 19-O-acetylchaetoglobosin B and D were enriched. Crystallization of chaetoglobosin C led to a mixture of 19-O-acetylchaetoglobosin B and 19-O-acetylchaetoglobosin D which were separated and obtained pure by chromatography on silica gel 60 GF254using the chromatotron. Spectral Data UV: EtOH ~, max

221(1og e=4.64), 272(3.83), 279(3.83), and 289nm (3.74).

IR:

(KBr) 3600-3200, 1730, 1700-1680, 1610, 1450, 1425, 1370, 1250, 1230, 1030, 970, and 900cmq. 1H NMR: 2N-H, 5.88, br s; H-3, 3.48, m; H-4, 2.96, br s, J=5 and 5; H-5, 2.84, m; H-7, 3.95, br

268

3. Chaetoglobosins/Cytochalasins

d, J=10; H-8, 2.33, t, J=10 and 10; H-10, 3.04, dxd, J=14, 3.56; H-10, 2.62, dxd, J=14, 9; H-11, 1.31, d, J=7; H-12, 5.41, br, s; H-13, 5.92, dxd, J=-lS, 10; H-14, 5.31, m; H-15, 2.3-2.4, m 2.0-2.1, m; H-16, 2.4-2.6, m; H-17, 5.70, dxd, J=9.5, 1; 1-1-19, 5.98, s; H-21, 7.88, d, J=17; H-22, 6.55, d, J=17; 16-CH3, 0.99, d, J=7.5; 18-CH3, 1.48, d, J=l; 19-O-acetyl, 2.16, s; I'-NH, 8.30, br, s; 5H-indolyl, 7.0-7.5; and 7-OH, 1.88ppm, br, s. 13C NMR:

[(CD3)2SO] C-l, 171.9; C-3, 52.0; C-4, 44.8; C-5, 31.3; C-6, 151.0; C-7, 70.1; C-8, 47.9; C-9, 61.3; C-10, 31.8; C-11, 13.4; C-12, 111.4; C-13, 128.3; C-14, 133.1; C-16, 32.3; C-17, 141.9; C-18, 127.4; C-19, 82.8; C-20, t94.2; C-21,133.3; C-22, 136.0; C-23, 198.9; C-.16CH3, 20.3; C-18CH3, 11.1; acetyl-CH3, 20.6; acetyl-CO, 169.3; C-2', 120.8; C-3', 109.4; C-3a' 126.4; C-4', ll8.0; C-5', 124.0; C-6', 118.4; C-7', 111.4; and C-7a', 136.2ppm. Mass Spectrum: 570(NV), 528, 185, 173, 130, 103, 60, and 43m/e. References A. Probst and C. Tamm; 19-O-Acetylchaetoglobosin B and 19-O-Acetylchaetoglobosin D, Two New Metabolites of Chaetomium globosum; Helvetica Chimica Acta, Vol. 64, pp. 2056-2064(1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

269

Common/Systematic Name Chaetoglobosin K (6R,7S, 16S, 19R)-6,7-Epoxy-5-ethyl- 19-hydroxy- 10-(indol-3-yl)- 10,16,18-trimethyl- 11nor[ 13] cytochalasa- 13(E), 17(E),21 (E)-triene- 1,20,23-trione Molecular Formula/Molecular Weight C34H40N2Os; MW = 556.29372 Me

U

NH

i'Me.

General Characteristics Rectangular trapezoids from acetone; mp., 264-266 oC. The uncorrected melting point was 264-266 ~C, though the crystals turned white at 60 ~ and most probably indicated the loss of acetone of crystallization. Fungal Source

Diplodia macrospora (ATCC accession No. 36896).

Isolation/Purification Extracted with ethyl acetate, dried over anhydrous sodium sulfate, reduced to a small volume under vacuum, then placed on a silica gel (70-230 mesh) column that had been slurry packed in benzene; followed by a stepwise elution with 1.0L each of benzene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was biologically active and was further fractionated on a silica gel (70-230 mesh) column packed in benzene and eluted with a linear gradient of benzene to acetone. The biologically active fractions were combined, reduced in volume and crystallized from acetone solution. Biological Activity - Significant growth inhibitor of wheat coleoptiles; activity down to 107 M. Studies indicated that chaetoglobosin K is biologically active as a plant growth regulator and is also toxic'to day-old chicks (LDs0 between 25 and 62.5mg/kg body weight). Spectral Data UV:

EtOH

~, max

219nm (E=44,600); also absorptions at 274, 282, and 290nm in concentrated

270

3. Chaetoglobosins/Cytochalasins

solutions. IR~ (KBr) 3340, 2970, 2930, 2880, 1725, 1685, 1655, 1615, 1455, 1427, 1375, 1250, 1156, 1105, 1048, 972, 875 and 742cm "~. IH NMR: H-2, 6.23; H-3, 3.89(J=4.4); H-4, 3.14; H-5, 1.90; H-7, 2.74(,/--5.3); H-8, 2.16; H-10, 3.14; H-10', 1.16(J=7.3); H-11, 1.51; H-11', 1.01(J=7.0); H-12, 1.29; H-13, 6.09(J=10.2, 15.6); H-14, 5.01; H-15, 2.16; H-16, 2.35; H-16', 1.12(,/=7.1); H-17, 5.56(J=8.4); H-18', 1.29; H-19, 4.99(,/=4.5); H-21, 7.65(,/--16.5); H-22, 6.49; H-I', 8.53; H-2', 6.90; H-4-7, 7.0-7.5ppm.

~3C NMR: C-I, 173.2 s; C-3, 56.7 d; C-4, 49.2 d; C-5, 36.3 d; C-6, 57.2 s; C-7, 61.6 d; C-8, 44.0 d; C-9, 63.9 s; C-10, 44.0 d; C-10', 10.6 q; C-11, 21.7 t; C-11', 13.8 q; C-12, 19.6 q; C-13, 133.2 d; C-14, 128.2 d; C-15, 41.9 t; C-16, 31.9 d; C-16', 12.6 q; C-17, 140.1 d; C-18, 125.8 s; C-18', 20.9 q; C-19, 81.6 d; C-20, 190.0 s; C-21,130.9 d; C-22, 136.4 d; C-23, 193.9 s; C-2', 122.3 d; C-3', 116.5 s; C-19', 132.2 s; C-4', 118.4 d; C-5', 119.7 d; C-6, 121.7 d; C-7', 111.5 d; and C-8', 136.4ppm s. TLC Data The Rf of chaetoglobosin K was 0.53-0.56 on silica gel 60 thin-layer plates developed with toluene/ethyl acetate/formic acid (5:4:1, v/v/v). It was observed as a dark spot under shortwave light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 330 (1981). H. G. Cutler, F. G. Crumley, R. H. Cox, R. J. Cole, J. W. Domer, J. P. Springer, F. M. Latterell, J. E. Thean and A. E. Rossi; Chaetoglobosin K: A New Plant Growth Inhibitor and Toxin from Diplodia macrospora; J. Agric. Food Chem., Vol. 28, pp. 139-142 (1980). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

3.

Chaetoglobosins/Cytochalasins

271

Common/Systematic Name Chaetoglobosin L (7S, 16S, 19R)-5-Ethyl-7,9-dihydroxy- 10-(indol-3-yl)ethyl-10,16,18-trimethyl- 11nor[ 13]cytochalasa-6(12), 13(E), 17(E),-21 (E)-tetraene- 1,20,23-tetraone Molecular Formula/Molecular Weight C3&'I40N2Os;MW = 556.29372 EL

Me

"--..

�9

CH2 OH

HN

Oo

~7

....'ME e

General Characteristics Yellow gum. Fungal Source Diplodia macrospora (strain ATTC No. 36,896). Isolation/Purification The microorganism was grown in standing culture on a medium containing 400g of shredded wheat, 150g of sucrose, 50g of mycological broth (Difco) and 20g of yeast extract (Difco) per 1000ml of H20. After incubation for 27 days at 27"C, the cultures were refluxed with CHCI3. The mixture was filtered and the extraction repeated 4 times. The combined extracts were dried (Na2SO4) and evaporated. The residue was washed with petroleum ether. The crude extract was purified on a silica gel column (increasing amounts of MeOH in CH2C12)yielding chaetoglobosin K and chaetoglobosin L. Spectral Data IR:

(film) 3400, 2930, 1720, 1640s, 1360, 1220, 970, 900, and 740cm"1.

~H NMR: (CDCI3) H-3, 3.55(m); H-4, 2.55(m); H-7, 3.90(d, J=10Hz); H-8, 2.39(m); H-10, 3.10(m); H-11, 1.57(m), 1.88(m); n-12, 5.29(s), 5.33(s); a-13, 5.99(dd); n-14, 5.37(m); n-15, 2.03(m), 2.28[m); H-16, 2.47(m); H-17, 6.61(d, J=9.0Hz); H-21, 6.63(d, J=18Hz); H-22, 7.94(d, J=l 8Hz); H-2', 6.90(d, J=lHz); H-4', 7.50(d, J=7Hz);

272

3. Chaetoglobosins/Cytochalasins

H-5', 7.10-7.22(m); H-6', 7.10-7.22(m); H-7', 7.37(d, J=7Hz); H-10' 1.10-1.15; H-11', 1.10-1.15; H-16', 1.00(d, J=7Hz); H-18', 1.32(s); OH-7, 2.00; OH-19, 3.90; NH-1, 5.61; and NH-I', 8.35ppm (s). ~3CNMR: (CDCi3) C-l, 172.6; C-3, 56.1; C-4", 39.8; C-5 ~ 41.0; C-6, 146.8; C-7, 71.1; C-8, 50.6; C-9, 62.2; C-10, 35.0; C-11, 21.4; C-12, 114.3; C-13, 128.5; C-14, 136.0; C-15, 42.0; C-16, 32.1; C-17, 140.3; C-18, 137.7; C-19, 81.7; C-20, 201.6; C-21"~ 131.5; C22~ 137.0; C-23, 197.7; C-2', 121.4; C-3', 117.7; C-4', 118.7; C-5', 122.5; C-6', 119.8; C-7', 111.6; C-10"", 12.9; C-11"", 12.1; C-16', 21.1; C-18', 10.7; C-3'a, 126.0; and C7'a, 136.6ppm. *, **and, *** Assignments may be reversed. Mass Spectrum: 556(M+), 423,412, 157, 144(100), 130, and 117role. References C. Spondlin and C. Tamm; Chaetoglobosin L, a New Metabolite of Diplodia macrospora; Helvetica Chimica Acta, Vol. 65, pp. 1543-1546(1982). C. Spondlin and C. Tamm; Chaetoglobosin M, a New Metabolite of a Mutant of Diplodia macrospora, Belonging to the Family of (1H-Indol-3-yl)-Substituted 10,11Diethyl- 10,11-dinorcytochalasins; Helvetica Chimica Acta, Vol. 71, pp. 1881-1884(1988).

3. Chaetoglobosins/Cytochalasins

273

Common/Systematic Name Chaetoglobosin M 6,7-Epoxy-5-ethyl-3-[ 1-(1H-indol-3-yl)ethyl]- 16,18-dimethyl- 10,1 l-dinor[ 13]cytochalasa- 13,17-diene- 1,19,21,23-tetraone Molecular Formula/Molecular Weight C34H4oN2Os; M W = 556.293 72

EL Me H'---"

CH2 //

Oo

lr

....IMe fl

OH

OH

General Characteristics A slightly yellow gum; [a]D 22 -18.3 ~ (c=0.92, in CH2C12). Fungal Source

Diplodia macrospora (strain DM 7).

Isolation/Purification The microorganism was grown in standing culture on a medium containing 400g of shredded wheat, 150g of sucrose, 50g of mycological broth (Difco) and 20g of yeast extract (Difco) per 1000ml of H20. After incubation for 27 days at 27~ the cultures were refluxed with CHC13. The mixture was filtered and the extraction repeated 4 times. The combined extracts were dried (Na2SO4) and evaporated. The residue was washed with petroleum ether. The crude extract was purified on a silica gel column (increasing amounts of MeOH in CH2C12) yielding chaetoglobosin K and chaetoglobosin L. The chaetoglobosin M containing fractions were further purified on a silica-gel column a second time with the same solvent system. Spectral Data UV~ ~ Methylenechloride max

230(1og e=4.09) and 290nm (3.60).

274

3. Chaetoglobosins/Cytochalasins

IR:

(film) 3350, 3030, 2970, 2930, 2880, and 1695cm"~. IH NMR: (CDCI3) H-3, 3.78(m); H-4, 1.5- 1.7; H-7, 2.65(d, J=7Hz); H-8, 2.28(m); H-10, 3.12(m); H-11, 1.5-1.7(m), 1.7-1.8(m); H-12, 1.22(s); H-13, 6.14(dd); H-14, 5.10(m); H-15, 1.88(m), 2.35(m); H-16, 2.14-2.4 or 2.6-2.8; H-17, 6.04(d, J=9.0Hz); H-21, 2.1-2.4; H-22, 2.6-2.8; H-2', 6.90(d, J=2Hz); H-4', 7.55(d, J=7Hz); H-5', 7.13-7.25; H6', 7.137.25; H-7', 7.35(d, J=7Hz); H-10', 1.30(d, J=7.0Hz); H-11', 0.97(d, J=7Hz); H16', 1.05(d, J=7Hz); H-18', 1.82(d, J=lHz); NH-1, 7.05(s); and NH-I', 8.55ppm (s). 13C NM~:

(CDCI3) C-I, 175.5; C-3, 57.8; C-4", 46.6; C-5", 44.4; C-6, 56.7; C-7, 60.6; C-8, 49.6; C-9, 63.5; C-10, 36.3; C-11, 21.5; C-12, 19.4; C-13, 126.7; C-14, 134.5; C-15, 40.2; C-16, 33.4; C-17, 156.5; C-18, 132.3; C-19, 196.8; C-20, 205.6; C-21"*, 32.6; C-22"*, 38.3; C-23,208.3; C-2', 122.5; C-3', 115.9; C-4', 118.5; C-5',122.5; C-6', 120.0; C-7', 111.6; C-10'~176176 16.2; C-11~176176 12.6; C-16', 19.4; C18', 10.3; C-3'a, 126.7; and C-7'a, 136.1ppm. *, "" and "" Assignmentsmay be reversed. Mass Spectrum: 556(M§ 538, 413,412, 394, 214, 179, 144(100%), 130, and 117m/e. Reference C. Spondlin and C. Tamm; Chaetoglobosin M, a New Metabolite of a Mutant of Diplodia macrospora, Belonging to the Family of (1H-Indol-3-yl)-Substituted 10,11Diethyl- 10,11-dinor-cytochalasins; Helvetica Chimica Acta, Vol. 71, pp. 18811884(1988).

3. Chaetoglobosins/Cytochalasins

275

Common/Systematic Name Cytochalasin A; Dehydrophomin (7S, 16R)-7-Hydroxy- 16-methyl- 10-phenyl-24-oxa[ 14]cytochalasa6(12), 13(E),21 (E)-triene- 1,20,23-trione Molecular Formula/Molecular Weight C29H35NOs; lk4"W= 477.25152 I

CH2 I

I

I

)-

Y

,......l / J ~

"

0

General Characteristics Crystals; mp., 193-195~

OH

0 [a]D2~ +83.7 ~ (C=I.0, in MeOH).

Fungal Source

Helminthosporium dematioideum, Phoma exigua var. exigua, and Ascochyta heteromorpha.

Biological Activity Cytotoxic to HeLa cells with an EDs0 of 3.21ag/ml. Inhibits movement (reversible) and cytoplasmic cleavage in cultured cells leading to multinucleated giant cells; at higher levels nuclear extrusion occurred. Acts selectively as a respiratory inhibitor in bacteria and fungi. Inhibited spore germination and hyphal tip growth in Gilbertellapersicaria. Cytochalasin A interfered with normal growth patterns and also affected the intracellular organization of Mucor mucedo. The presence of an a,[3-unsaturated carbonyl group in the macrolide moiety of cytochalasin A with appropriate bioisoteric placement were the requisite molecular features for its microbiological activity. Spectral Data 13C NMR: (CDCI3) C-l, 169.9 s; C-3, 52.8 d; C-4, 46.7 d; C-5, 30.9 d; C-6, 150.8 s; C-7, 68.1 d; C-8, 49.2 d; C-9, 83.5 s; C-10, 42.3 t; C-I 1, 13.5 q; C-12, 112.2 t; C-13, 128.1 d; C-14, 133.5 d; C-15, 32.4 t; C-16, 30.9 d; C-17, 30.6 t; C-18, 21.3 t; C-19, 40.8 t; C-20, 201.6 s; C-21,140.5 d; C-22, 127.9 d; C-23, 163.5 s; C-24, 20.2 q; C-I', 137.4 s; C-2', 6', 129.5 d; C-3', 5', 128.1 d; and C-4', 126.2ppm d.

276

3. Chaetoglobosins/Cytochalasins

TLC Data Adsorbent; silica gel G-HR; solvent A: chloroform-acetone, 93:7 (v/v); B: toluene-ethyl acetate-formic acid, 5:4:1 (v/v/v); Re value for A: 0.29, B: 0.68; detection: a blue fluorescent spot under UV light after spraying with 50% ethanolic HzSO4 and heating. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 276 (1981). E. K. Manavathu and D. D. S. Thomas; FEMS, Microbiol. Lett., Vol. 7, pp. 199-202 (1980). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991). W. Rothweiler and C. Tamm; Isolation and Structure ofPhomin; Experientia, Vol. 22, p. 750 (1966).

3. Chaetoglobosins/Cytochalasins

277

Common/Systematic Name Cytochalasin B; Phomin (7S, 16R,20R)-7,20-Dihydroxy- 16-methyl- 10-phenyl-24-oxa[ 14] cytochalasa6(12), 13(E),21 (E)-triene- 1,23-dione Molecular Formula/Molecular Weight C29H37NOs; M'W = 4 7 9 . 2 6 7 1 7 ,

12CH2

......I ~ O H

_-

HN~I--O'q~ O O HO H General Characteristics Crystals; mp., 221-223 ~ [0~]D 25 + 8 6 . 7 ~ (c=0.9, in MeOH); crystals from chloroform solution; mp., 218~ [tt]D25 +84 ~ (C=I.0, in EtOH). Fungal Source Helmmthosporium dematioideum, Hormiscium spp., Phoma exigua var. exigua, Curvularia lunata, Drechslera biseptata, Gnomonia erythrostoma, Hypomyces odoratus, and Ascochyta heteromorpha. Isolation/Purification Purified by methanol extraction, followed by reverse-phase C~a silica gel chromatography and elution with hexane-tetrahydrofuran (1:1, v/v). Final purification was by crystallization, followed by preparative TLC and recrystallization. Biological Activity Cytochalasin B inhibited cellular motility of moving L cells. Cytoplasmic cleavage was inhibited without interference with nuclear division, resulting in binudeate and multinucleate cells. The effect was reversible. Mammalian cells can be enucleated by treatment with cytochalasin B (10~g/ml) followed by centrifugation. Cytochalasin B has been reported to inhibit the following cellular functions: release of growth hormone; thyroid secretion; phagocytosis; platelet aggregation and clot retraction; and hexase transport. Cytotoxieity to HeLa cells, EDs0 between 1 and 2.5~g/ml. It was reported to inhibit certain gliding mycoplasmas and internalization of Herpes simplex virus type 1 (HSV)KOS strain by HEp-2 cells was reversibly inhibited by pretreatment with cytochalasins B and D.

278

3. Chaetoglobosins/Cytochalasins

Spectral Data UV:

~bEmtOaxH 220(e=44,668), 257(sh), 265(sh), and 269 (sh). 13C NMR: C-l, 170.6 s; C-3, 52.3 d; C-4, 46.5 d; C-5, 30.4 d; C-6, 150.9 s; C-7, 69.4 d; C-8, 47.5 d; C-9,83.3 s; C-10, 42.9 t; C-11, 13.2 q; C-12, 112.2 t; C-13, 128.4 d; C-14, 133.0 d; C-15, 34.8 t; C-16, 30.9 d; C-17, 34.2 t; C-18, 20.1 t; C-19, 41.6 t; C-20, 68.6 d; C-21,153.2 d, C-22, 118.1 d; C-23, 164.0 s; C-24, 20.2 q; C-I', 136.9 s; C-2', 6', 129.6 d; C-3', 5', 128.1 d; and C-4', 126.3ppm d. TLC Data Silica gel G-HR; solvent: A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Re value of A: 0.11 B: 0.60; detection: a blue fluorescent spot under UV light after spraying with 50% ethanolic H2SO4 and heating. HPLC Data Lichrosorb Si60 silica gel column, mobile phase was hexane-tetrahydrofuran and UV detection at 230nm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 281 (1981). M. A. Kolber, K. O. Broschat, and B. Landa-Gonzalez; Cytochalasin B induces cellular DNA fragmentation, FASEB J., Vol 4, pp. 3021-3027(1990). K. M. Lipski, J. D. McQuiggan, K. H. Loucy, and T. P. Fondy; Cytochalasin B: Preparation, analysis in tissue extracts and pharmacokinetics after intraperitoneal bolus administration in mice; Anal. Biochem., Vol. 161, pp. 332-340(1987). J. Maniloff and U. Chaudhuri; Gliding mycoplasms are inhibited by cytochalasin B and contain a polymerizable protein fraction; J. Supramol. Struct., Vol. 12, pp. 299-304 (1979). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991). A. Pribela, J. Tomko, and L. Dolejs; Chem. Zvesti Vol. 28, p. 710 (1974). K. Rosenthal, R. Perez, and C. Hodnichak; Inhibition of Herpes simplex type 1 virus penetration by cytochalasins B and D; J. Gen. Virol., Vo166, pp. 1601-1605(1985). W. Rothweiler and C. Tamm; Isolation and Structure of Phomin; Experientia, Vol. 22, p. 750 (1966).

3. Chaetoglobosins/Cytochalasins

279

C0mmon/Systematic Name Cytochalasin C (7S, 16S, 18R,21R)-21-Acetoxy-7,18-dihydroxy-16,18-dimethyl- 10-phenyl[ 11 ]cytochalasa-5(6), 13E, 19(E)-triene- 1,17-dione Molecular Formula/Molecular Weight

~

C3oH37NO6; M'W = 507.26209

.....y

OH

.....

/o. General Characteristics Crystals; mp, 260~ (dec.);

[a]D 25 - 1 4 . 7 ~

o (c=0.82, in dioxane).

Fungal Source

Metarrhizium anisopliae and Hypoxylon terricola.

Biological Activity Cytochalasin C showed essentially the same biological activity as cytochalasin B except it is about 10 times more potent in mammalian cell culture (inhibited movement and cytoplasmic cleavage); at higher doses caused nuclear extrusion which may lead to total enucleation. Spectral Data UV:

End absorption. 13C NMR: C-l, 173.9 s; C-3, 48.9 d; C-4, 59.6 d; C-5, 125.4 s; C-6, 133.0 s; C-7, 67.8 d; C-8, 48.5 d; C-9, 52.5 s; C-10, 43.6 t; C-11, 14.1 q; C-12, 16.4 q; C-13, 131.0 d; C-14, 131.5 d; C-15, 37.1 t; C-16, 40.4 d; C-17, 209.8 s; C-18, 77.5 s; C-19, 127.5 d; C-20, 131.7 d; C-21, 74.9 d; C-22, 19.2 q; C-23, 24.6 q; C-I', 137.6 s; C-2', 6', 129.2 d; C-3', 5', 128.4 d; C-4', 126.4 d; CH3C=O, 170.1 s; and CH3C=O, 20.5ppm q. TLC Data TLC: silica gel G-H~ solvent A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-

formic acid, 5:4" 1, v/v/v. Re A: 0.09; B" 0.55 detection: a dull yellow fluorescent spot under UV light atter spraying with 50% ethanolic H2SO4 and heating.

280

3. Chaetoglobosins/Cytochalasins

References D. C. Aldridge, J. J. Armstrong, R. N. Speake, and W. B. Turner; The Structures of Cytochalasins A and B; Chem. Commun. p. 1667 (1967). D. C. Aldridge and W. B. Turner; Structures of Cytochalasins C and D; J. Chem. Soc., p. 923 (1969). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 294 (1981). R. L. Edwards, D. J. Maitland, and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. CytochalasinN, O, P, Q, and R, New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

281

Common/Systematic Name Cytochalasin D; Zygosporin A (7S, 16S, 18R,21R)-21-Acetoxy-7,18-dihydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa-6(12), 13E, 19(E)-triene- 1,17-dione Molecular Formula/Molecular Weight C30H37NO6; M W "- 507.26209

CH2 , .....

~

~

.,|IIII

0

General Characteristics Needles from acetone-petroleum ether; mp, 267-271 ~

[a]D 25 -7.5 ~ (c=0.55,

in dioxane).

Fungal Source Metarrhizium anisopliae, Zygosporium mason#, Coriolus vernicipes, Hypoxylon terricola, and Engleromyces goetzii. Isolation/Purification The culture filtrate was extracted with ethyl acetate, washed with sodium carbonate and water and evaporated to dryness in vacuo. The residue gave cytochalasin D (zygosporin A) as colorless needles from acetone. Biological Activity Cytochalasin D showed similar activity to cytochalasin B, except it was about 10 times more potent in mammalian cells (inhibited movement and cytoplasmic cleavage); at higher doses causes nuclear extrusion which may lead to total enucleation. It was embryolethal with a tendency toward teratogenicity in chick embryos. It inhibited the growth of Bipolaris sorokiniana and Rhizoctonia solani and induced profuse hyphal branching; at high concentrations it induced swellings in the hyphal tips. It was reported to reversely inhibit internalization of Herpes simplex virus type 1 (HSV) KOS strain by HEp-2 cells. Spectral Data UV:

Meox

~,~

286nm (e=330)

282

3. Chaetoglobosins/Cytochalasins

IR: (KBr) 3404, 3240, 1742, 1703. 1693, 1233, 1049, 1009, 962, 907, 751, and 706 cm"~. 1H NMR: (CDC13) 9.07(d, J=6.8, Me), 8.82(d, J=6.3, Me), 8.51(s, MeC-OH), 7.76(OAc), 6.23(dm, J=10.4, OH), 4.95-3.76(8H), and 2.77ppm (5H, Ph).

13C NM~: C-l, 173.4 s; C-3, 52.4 d; C-4, 47.6 d; C-5, 31.5 d; C-6, 150.6 s; C-7, 70.0 d; C-8,46.0 d; C-9, 53.1 s; C-10,43.6 t; C-ll, 12.7 q; C-12, 111.3 t; C-13, 130.7 d; C-14, 131.6 d; C-15, 36.8 t; C-16, 40.2 d; C-17, 209.8 s; C-18, 77.4 s; C-19, 126.9 d; C-20, 132.0 d; C-21, 76.3 d; C-22, 19.1 q; C-23, 24.5 q; C-I', 136.9 s; C-2',6', 129.5 d; C-3',5', 128.1 d; C-4', 126.4 d; CHaC=O, 169.8 s; CHaC=O, 20.4ppm q. Mass Data: M + 507role (found: C, 71.1; H, 7.35, N, 2.5; C30H37NO6 requires C, 71.0; H, 7.35; N,

2.75%). TLC Data Silica gel G-HR; solvent: A: chloroform-acetone, 93:7 (v/v); B: toluene-ethyl acetateformic acid, 5:4:1 (v/v/v); Re: A: 0.06, B: 0.51; detection: a bright yellow fluorescent spot under UV light after spraying with 50% ethanolic HzSO4 and heating. References D. C. Aldridge, J. J. Armstrong, R. N. Speake, and W. B. Turner; The Structures of Cytochalasins A and B; Chem. Commun., p. 1667 (1967). D. C. Aldridge and W. B. Turner; Structures of Cytochalasins C and D; J. Chem. Soc., p. 923 (1969). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 299 (1981). R. L. Edwards, D. J. Maitland, and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. Cytochalasin N, O, P, Q, and R, New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. H. Gilani and C. Kreshal; Teratogenicity of cytochalasin D in chick embryos; J. Environ. Pathol. Toxicol. Oncol., Vol.6, pp 67-72(1986). H. Minato and M. Matsumoto; Studies on the Metabolites of Zygosporium masonii. Part I. Structure of Zygosporin A; J. Chem. Soc. (C), pp. 38-45(1970). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

K. Rosenthal, R. Perez, and C. Hodnichak; Inhibition of Herpes simplex type 1 penetration by cytochalasins B and D; J. Gen. Virol., Vol 66, pp. 1601-1605(1985).

283

284

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin F (6R,7S, 16R,20R)-6,7-Epoxy-20-hydroxy- 16-methyl- 10-phenyl-24-oxa[ 14]cytochalas13(E),21 (E)-diene- 1,23-dione Molecular Formula/Molecular Weight C29H37NO5; ~

= 479.26717

NOTE: There is some confusion in the literature as to the correct structure of cytochalasin F. The structure proposed here is based on X-ray analysis of cytochalasin E (B~chi et al., 1973).

Me,,,

Me,,, "

H"

0 .. IIii ~

"Me

0 HO

N

Fu.ngal Source Helminthosporium dematioideum. References D. C. Aldridge, B. F. Burrows, and W. B. Turner; The Structures of the Fungal Metabolites Cytochalasin E and F; J. C. S. Chem. Comm., pp. 148-149(1972). G. BiJchi, Y. Kitaura, S. Yuan, H. E. Wright, J. Clardy, A. L. Demain, T. Glinsukon, N. Hunt, and G. N. Wogan; Structure of Cytochalasin, a Toxic Metabolite of Aspergqllus clavatus; J. Am. Chem. Soc., Vol. 95, p. 5423-5425(1973). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 286 (1981). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

285

Common/Systematic Name Cytochalasin L (6R, 7S, 16S, 19/?)- 19-Acetoxy-6,7-epoxy- 16,18-dimethyl-10-phenyl-24-oxa[ 14] cytochalasa- 13(E), 17(E),21 (E)-triene- 1,20,23-trione Molecular Formula/Molecular Weight C32H37NOT;MW' = 547.25700

Me,,.

,0

'llll j ''Ii 0

e Me

0

OAc

General Characteristics Cytochalasin L was amorphous; [a]D 25 -165 o (in EtOH). Fungal Source Chalara microspora. Isolation/Purification Extracted with EtOAc and isolated using reversed-phase chromatography. Spectral Data UV~ No characteristic absorption observed.

Imz (KBr) 3400(OH)and 1720cm4 (broad, C=O). IH N-Ik,IR: (CDCI3) NH, 6.00; H-3, 3.71(J3.~o,=8.8,J3.~ob=4.8,J3.4=3.3Hz);H-4, 2.80(J4.s=4.5); H-5, 2.24(A.~=7.5); H-7, 2.67; H-8, 2.68(A.~3=I0.0);H-10a, 3.02(J~o,.~o~=13.3); H-10b, 2.89; 3H-I I, 1.03; 3H-12, 1.29;H-13, 6.17(J~344=I4.5);H-14, 5.40(J~445a=10.0,J~4.~sb=3.0);H-15a, 2.23(Jmqsb=I3.0); H-15b, 2.35; H-16, 2.6(J~6.~6Mo=7.0,J~647=7.5);H-17, 5.72; H-19, 5.70; H-21, 7.49(J2~.2~=16.0);H-22, 6.52; 16-CH3, 1.10; 18-CH3, 1.56;-OOCCH3, 2.19; and H-2', H-3', H-4', 7.1-7.4Hz.

286

3. Chaetoglobosins/Cytochalasins

13CNMR: (CDCI3) C-l, 171.1; C-3, 54.4; C-4, 49.3a; C-5, 33.0b; C-6, 57.5; C-7, 60.4; C-8, 48.5a; C-9, 84.1; C-10, 44.0; C-11, 13.5c; C-12, 20.0d; C-13, 130.0e; C-14, 134.7f; C-15, 41.2; C-16, 35.7b; C-17, 136.8f; C-18, 127.1e; C-19, 84.9; C-20, 193.59; C-21, 142.7; C-22, 126.5f; C-23, 164.0; 16-CH3, 11.5c; 18-CH3, 21.6d;-OOCCH3, 169.5; -OOCCH3 20.6d; C-I', 136.8; C-2', 129.2; C-3', 128.9; and C-4', 127.1ppm e. (a-f may be interchanged). Mass Spectrum: HRMS: 547.2535re~e, C32H37NO7;calcd 547.2570. References T. Fex; Structures of Cytochalasin K, L and M, Isolated From Chalara microspora; Tet. Lett., Vol. 22, pp. 2703-2706(1981). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

287

Common/Systematic Name Cytochalasin M (6R,7S, 16S,20S)-6,7-Epoxy-20-hydroxy-10-phenyl-24-oxa[14]cytochalasa13(E), 17(E)-diene-1,19,23-trione Molecular Formula/Molecular Weight C30H37NO6; M W -- 5 0 7 . 2 6 2 0 9

Me M e ....

.

.

.

.

HO~O GeneralCharacteristics

"Me

Cytochalasin M crystallized from methanol-water; mp., 161-162~

[a]D 25 q- 18 ~ (EtOH).

Fungal Source Chalara microspora. Isolation/Purification Extracted with EtOAc and isolated using reversed-phase chromatography. Spectral Data UV;

Abs.EIOH

~,m..

235nm (e=l 1,300).

IR: (KBr) 3380(OH), 1750, 1705(broad, C=O), and 1670cm~ (C=C). ~HNMR: (CDCI3) NH, 5.63; H-3, 3.63 (J3.~o,=10.0,J3.~Ob-4.0,J3.4-4.0Hz); H-4, 2.53(J4.5=4.0); H-5, 2.07(Js.~,=7.0); H-7, 2.70(JT.s=5.5); H-8, 2.49(Js.,3=9.5); H-10a, 3.17(Jlo,qob=13.5); H-10b, 2.88; 3H-11, 1.08; 3H-12, 1.32; H-13, 5.89,(J13.14=15.0), J13.15=1.5; H-14, 5.46(J14.15=10.0 and 3.5); 2H-15, 2.2-2.5; H-16, 2.85 (Jl6.16r~,=7.0, J~6.~7=9.5); H-17, 6.55(J~7.~sM==l.3);H20, 4.98(J2o.2~=4.0 and 6.0, J2O.OH=5.8);2H-21, 1.95; 2H-22, 2.45; 16-CH3, 1.13; 18-CH3, 1.88; 20-OH, 3.67; and H-2', H-3', H-4', 7.2-7.45Hz.

288

3. Chaetoglobosins/Cytochalasins

~3CNMR: (CDC13) C-I, 171.3; C-3, 54.5; C-4, 49.2a; C-5, 33.6c; C-6, 57.5; C-7, 59.7; C-8, 50.8b; C-9, 82.7; C-10, 43.0; C-11, 13.8d; C-12, 20.3e; C-13, 127.6f; C-14, 132.5f, C-15, 39.8; C-16, 35.9c; C-17, 150.2; C-18, 132.5; C-19, 203.2; C-20, 70.9; C-21, 30.5g; C-22, 33.1gf; C-23, 171.9a; 16-CHa, 12.0d; 18-CH3, 19.6e; and C-I', 137.8; C-2', 128.9; C-3', 128.9; and C-4', 126.9fppm. (a-g: may be interchanged). Mass Spectrum: HRMS: 507.2708re~e, C30H37NO6;calcd 507.2631. References T. Fex; Structures of Cytochalasin K, L and M, Isolated from Chalara microspora; Tet. Lett., Vol. 22, pp. 2703-2706(1981). S. Natori and I. Yahara; Cytochalasins: In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

289

Common/Systematic Name Zygosporin D (7S, 16S, 18R,21R)-7,18,21-Trihydroxy- 16,18-dimethyl-10-phenyl[11]cytochalasa6(12), 13(E), 19(E)-triene- 1,17-dione Molecular Formula/Molecular Weight C28H37NO4; M W -" 4 5 1 . 2 7 2 2 6

,.....

.~~.. '-

\ j

"

CH2

~

......

'

. ..... |

General Characteristics Colorless plates from methanol; mp 180-190~

[0~]D23 - 1 4 . 9 ~

(c=0.759, in dioxane).

Fungal Source Zygosporium masonii. Biological Activity Cytotoxicity to HeLa cells. Spectral Data IR:

(KBr) 3400, 1700, 1127, 1107, 1075, 1005, 968, and 910cmq. TLC Data Silica gel; Solvent: chloroform-methanol, 10:1, v/v; Rf=0.50. Detection: not reported References H. Minato and M. Matsumoto; Studies on the Metabolites of Zygosporium masonii. Part I. Structure of Zygosporin A; J. Chem. Soc., p. 38 (1970). S. Natori and I. Yahara; Cytochalasin; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

290

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Zygosporin E (7S, 16R, 18R)-21-Acetoxy-7-hydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa6(12), 13(E), 19(E)-triene- 1,17-dione Molecular Formula/Molecular Weight C3oH37NO5; M W = 491.26717

CH2 '.....i

N/ 0

.OH =.lllll

0

General Characteristics Colorless needles from acetone-n-hexane; mp 218-223.5~ dioxane).

[a]o 24 +6.20(c=0.971 in

Fungal Source Zygosporium masoni i. Biological Activity Cytotoxic to HeLa cells. Spectral Data IR:

(KBr)

3524, 3420, 1743, 1703, 1113, 1010, 973, and 912cm"~.

TLC Data Silica gel; solvent: ethyl acetate; Rf=0.48. Detection: not reported. References H. Minato and M. Matsumoto; Studies on the Metabolites of Zygosporium masonii. Part I. Structure of Zygosporin A; J. Chem. Soc., p. 38 (1970). S. Natori and I. Yahara; Cytochalasin; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

291

Common/Systematic Name Zygosporin F; Cytochalasin D monoacetate (7S, 16S, 18R,21R)-7,21-Diacetoxy- 18-hydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa-6(12), 13(E), 19(E)-triene- 1,17-dione Molecular Formula/Molecular Weight C32H39NO7; M W -" 549.27265

CH2 ,......~J.~OAc . . . . . .

General Characteristics Colorless prisms from diisopropyl ether; mp, 126-129"C; [a]D -12.0" (C----0.775,in dioxane). Fungal Source Zygosporium masonii. Biological Activity Cytotoxic to HeLa cells. Spectral Data IR:

(KBr)

3420, 1740, 1704, 1113, 1043, 1009, 965, and 910cm "1.

TLC Data Silica gel; solvent: toluene-methanol, 10:1, v/v; Rr

detection: not reported.

References H. Minato and M. Matsumoto; Studies on the Metabolites of Zygosporium masonii. Part I. Structure of Zygosporin A; J. Chem. Soc., p. 38 (1970). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991).

292

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Zygosporin G (16S, 18R,21R)-2 l-Acetoxy- 18-hydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa6(7), 13(E), 19(E)-triene- 1,17-dione Molecular Weight~olecular Formula C30H37NOs; MW = 491.26717

III~"E

H

-

.......

. OAc /%H"O General Characteristics Colorless prisms from diisopropyl ether; mp, 115-125~ dioxane).

[tt]D24 -82.0* (C=0.870, in

Fungal Source Zygosporium masoni i.

Biological Activity Cytotoxic to HeLa cells. Spectral Data IR:

(KBr) 3410, 1743, 1701, 1122, 1045, 1008, and 965cm1; (Nujol)3430, 3285, 1742, 1693, 1228, 1121, 1044, 1007, 965, 745, and 703cmq. TLC Data Silica gel; solvent: toluene-methanol, 10:1, v/v; Rr~0.35; detection: not reported. References H. Minato and M. Matsumoto; Studies on the Metabolites ofZygosporium masonii. Part I. Structure of Zygosporin A; J. Chem. Soc., p. 38 (1970). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

293

Common/Systematic Name Cytochalasin H; Kodocytochalasin- 1; Paspalin-P (7S, 16S, 18R,2 IR)-21-Acetoxy-7,18-dihydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa-6(12), 13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C3oH39NOs; MW' = 493.28282

CH2 HI]OH

II

'~/~

, J,,, HII,,,'H 0

M6

OH

General Characteristics Crystals from ethyl ether; mp, 255-257~ colorless needles from methanol; mp, 268-269~ [a]D25 +63 ~ (C=0.103, in MeOH). White needles from chloroform-ethyl ether; mp, 258-263 ~ [a]D -9.0 ~ (in CHCI3); +91.2 ~ (in MeOH). Isolation/Purification Cultures were extracted with chloroform-methanol, 2:1 (v/v) and chromatographed on silica gel 60 eluted with a linear gradient from hexane to ethyl ether. Cytochalasin H was crystallized from ethyl ether solution. Fungal Source

Phomopsis spp. and Phomopsispaspalli.

Biological Activity LDs0 12.5mg/kg dosed orally to day-old cockerels. A plant growth inhibitor; inhibited floral development in tobacco. Spectral Data UV:

End absorption and weak x-x* transition due to the aromatic ring. 13C NM~: C-l, 174.4 s; C-3, 57.8 d; C-4, 50.3 d; C-5, 32.9 d; C-6, 148.2 s; C-7, 69.8 d; C-8, 47.3 d; C-9, 51.9 s; C-IO, 45.6 t; C-1 l, 14.0 q; C-12, 113.8 t; C-13, 127.2 d; C-14, 138.3 d; C-15, 42.9 t; C-16, 31.2 d; C-17, 57.8 t; C-18, 74.2 s; C-19, 127.0 d; C-20,

294

3. Chaetoglobosins/Cytochalasins

138.5 d; C-21, 77.5 d; C-22, 26.5 q; C-23, 28.5 q; C-I', 137.4 s; C-2',6', 129.1 d; C-3',5', 128.9 d; C-4', 125.9 d; CH3C=0, 170.0 s; and CH3C=0, 20.8ppm q. TLC Data Silica gel G-HR. Solvent: A: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. B: chloroform-acetone, 93:7, v/v. Re value for A: 0.62, B: 0.13. Detection: 50% ethanolie HzSO4 sprayed alone or in combination with 3% ethanolic phosphomolybdic acid followed by heating for 5 min at 100 oc. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 289 (1981). R. J. Cole, J. M. Wells, R. H. Cox, and H. G. Cutler; Isolation and Biological Properties ofDeacetyleytochalasin H from Phomopsis sp.; J. Agile. Food Chem., Vol. 29, pp. 205-206(1981). S. Natori and I. Yahara; In Myeotoxins and Phytoalexins, R. P. Sharma, and D. K. Salunkhe (eds.), CRC Press, pp. 291-338(1991 ). S. A. Patwardhan, R. C. Pandey, S. Dev, and G. S. Pendse; Toxic Cytochalasins of

Phomopsispaspalli, a Pathogen of Kodo Millet; Phytoehemistry., Vol. 13, p. 1985 (1974).

3. Chaetoglobosins/Cytochalasins

295

Common/Systematic Name Cytochalasin U Molecular Formula/Molecular Weight C30H39NOs; MW = 493.28282

CH20H ,,,Me

(-0~ FungalSource

Mg Me

OH

Isolated from the fermentation broth ofPestalotia sp. (AB 1942R-114). The fungus was isolated from a partridge pea plant (Cassiafasciculata) collected in Missouri, USA.

Isolation/Purification The whole broth was centrifuged and the supernatant adsorbed in batch onto SP-207 (Mitsubishi Kasei Corp.). The resin was washed in a fritted glass filter with water, 50% methanol-water and methanol, successively. The active methanol eluate was concentrated to dryness under reduced pressure and the residue was triturated with ethyl acetate. The soluble material was purified on a C18 column using a methanol-water gradient. Final purification was achieved by HPLC using 5% isopropylhexane over silica (Waters I.tBondapak). Biological Activity In vitro immunosuppressant activity as measured by the ability to inhibit [3H]thymidine uptake in mixed BALB/c and C57BL 6 spleen cell suspensions. MLR ICs0 was 5.21~g/ml for cytochalasin U compared with 0.21.tg/ml for cytochalasin H. Spectral Data ]H NMR and 13CNMR: Both spectra were similar to that of cytochalasin H except at positions 6, 7 and 12. The 13 vinyl signal at 130.14ppm was that of a methine; its proton coupling partners (8 and 12) were indicative of its position on 7. The coupling patterns in the heteronuclear and homonuclear correlation experiments of the methylene signal at 63.16ppm in ~3C and 4.03ppm in the ]H were indicative of its position at 12. Chemical shifts were well suited for a methylene disubstituted.with an olefin and hydroxyl. Mass Spectrum: DCI-NH3 showed a (M + H) § peak at 494m/e and a high resolution peak match was

296

3. Chaetoglobosins/Cytochalasins

determined to be 494.2924 which corresponded to a formula of C30H40NOs (calcd

494.2906).

Reference N. S. Burres, U. Premachandran, P.E. Humphrey, M. Jackson, and R. H. Chen; a New Immunosuppressive Cytochalasin Isolated from a Pestalotia sp.; J. Antibiotics, Vol. 45, pp. 1367-1369(1992).

3. Chaetoglobosins/Cytochalasins

297

Common/Systematic Name Cytochalasin J; Deacetylcytochalasin H; Kodocytochalasin-2; Paspalin-P2 (7S, 16S, 18R,21R)-7,18,2 l-Trihydroxy- 16,18-dimethyl- 10-phenyl[ 11]eytochalasa6(12), 13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C28H37NO4; M ~ = 451.27226

llll.

..,llll

0

HO

--

General Characteristics Purified deacetylcytochalasin H formed large crystals from ethyl ether; mp, 274-276"C (partial decomposition); [a]D27 +47.8 ~ (C=0.43, in MeOH). Fungal Source

Phomopsis sp.

Isolation/Purification Extraction was identical to that of cytochalasin H. They eluted together from silica gel 60 columns eluted with ethyl ether. A second silica gel 60 column eluted with a linear gradient from n-hexane to ethyl ether effected separation of cytochalasin H from the more polar deacetylcytochalasin H. Biological Activity Biological data presented an LDs0 in day-old chickens of a single oral dose at 37.5mg/kg and plant growth inhibitory activity down to 10.5 M in wheat coleoptiles. Spectral Data UV~

Showed strong end absorption and a series of weak bands in the region of 240-280nm due to the x-x* transition of the monosubstituted aromatic ring. IR~

Conspicuous in the IR spectrum of deacetylcytochalasin H was the absence of ester carbonyl absorption (173 5cm'~), which was present in the IR spectrum of cytochalasin H.

298

3. Chaetoglobosins/Cytochalasins

13C NMR: C-l, 175.84 s; C-2(6.64); C-3, 53.14 d(3.20); C-4, 49.23 d(2.97); C-5, 32.69 d(2.70); C-6, 149.72 s; C-7, 70.05 d(3.94); C-8, 45.54 d(3.20); C-9, 53.24 s; C-10, 42.91 t(2.70,2.83); C-11, 13.72 q(0.87); C-12, 112.19 t(4.97,5.21); C-13, 128.02 d(5.76); C-14, 137.24 d(5.31); C-15, 45.00 t(1.80); C-16, 32.69 d(1.75); C-17, 53.77 t(1.75); C-18, 73.42 s; C-19, 136.37 d(5.83); C-20, 130.65 d(5.90); C-21, 75.71 d(4.52); C-22, 26.39 q(1.01); C-23, 28.19 q(1.28); C-I', 137.53 d; C-2', 6', 129.24 d(7.23); C-3',C-5', 128.24(7.23); and C-4', 126.38ppm d(7.23). Mass Spectrum: The HRP spectrum of deacetylcytochalasin H gave a molecular ion peak at 451.2676 (calcd for C:sH3~NOa, 451.2722). In addition to the molecular ion peak, the LRP mass spectrum showed major peaks at m/e 415, 361,342, 324, 270, 120, and 91(base peak). TLC Data It was visualized on TLC as a rose-colored spot at It4=0.44 compared to 0.53 for cytochalasin H. References R. J. Cole, J. M. Wells, R. H. Cox, and H. G. Cutler; Isolation and Biological Properties ofDeacetylcytochalasin H from Phomopsis sp.; J. Agric. Food Chem., pp. 205-206 (1981). S. Natori and I. Yahara; In Mycotoxins and Phytoalexins, R. P. Sharma, and D. K.

Salunkhe (eds.), CRC Press, pp. 291-338(1991). S. A. Patwardhan, R. C. Pandey, S. Dev, and S. E. Pendse; Toxic Cytochalasins of

Phomopsispaspalli, a Pathogen of Kodo Millet; Phytochem., Vol. 13, p. 1985 (1974).

3. Chaetoglobosins/Cytochalasins

299

Common/Systematic Name Epoxycytochalasin H (6R, 7S, 16S, 18R,21R)-21-Acetoxy-6,7-epoxy- 18-hydroxy- 16,18-dimethyl- 10-phenyl[ 11 ]cytochalasa-6(12), 13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C30H39NO4; M W = 493.28282

,,lllll

0

HO

-

General Characteristics Purified epoxycytochalasin H formed crystals from ethyl ether; mp., 128-130~ -84.68 ~ (c=0.33, in CHCI3).

[a]D 30

Fungal Source

Phomopsis sojae.

Isolation/Purification The crude chloroform extract was fractionated on a silica gel 60 column packed as a slurry in benzene and eluted with 3 column volumes each of benzene, ethyl ether, ethyl acetate, acetone, and methanol. The ethyl ether and ethyl acetate fractions were highly toxic, and TLC analyses suggested the same toxins were involved. These two fractions were combined, concentrated under vacuum at 60~ and added to a second silica gel 60 column packed in ethyl ether. The column was eluted with a linear gradient from ethyl ether to ethyl acetate. The fractions associated with acute toxicity to day-old chickens were combined and reduced to dryness under vacuum at 60~ The combined fractions were applied to a C~s reverse phase column packed in acetonitrile and reequilibrated with 10% acetonitrile-water. The column was eluted with a linear gradient from 10% acetonitrile-water to 100% acetonitrile. Toxicity was associated with two distinct compounds: one combined fraction yielded epoxycytochalasin H and the other yielded epoxydeacetylcytochalasin H. Biological Activity Acutely toxic to day-old chicks and showed plant growth inhibition in wheat coleoptile assay.

300

3. Chaetoglobosins/Cytochalasins

Spectral Data UV: Showed strong end absorption and a series of weak bands in the region of 240-280nm. IR:

1735cm"~ (carbonyl ester). 13C NMR: C-l, 175.3 s; C-2 (6.14); C-3, 54.2 d; C-4, 50.7 d; C-5, 36.7 d; C-6, 57.1 s; C-7, 62.9 d; C-8, 45.2 d; C-9, 53.6 s; C-10, 42.9 t; C-11, 12.6 q(0.83, J=7.0); C-12, 19.7 q(1.33); C-13, 127.1 d(5.86, J=9.6, 16.2); C-14, 138.2 d(5.28, J=-5.5, 16.2); C-15, 45.9 t; C-16, 30.7 t; C-17, 53.7 t; C-18, 74.2 s; C-19, 127.7 d(5.48, J=2.2, 16.6); C-20, 135.2 d(5.77, J=2.2, 16.6); C-21, 76.1 d(5.67, J=-2.2); C-22, 26.4 q(1.04); C-23, 28.2 q(1.19); C-I', 136.9 s; C-2', C-6', 129.2 d(7.17, m); C-3',5',128.9 d(7.26, m); C-4', 125.4 d(7.26, m); CH3CO, 170.2 s, and CH3CO, 20.8ppm q(2.20).

Mass Spectrum: The LRP electron-impact mass spectra of epoxycytochalasin H gave ions at nominal mass m/e 493(M+), 475(M+ -H20), 433(M+ -H20- C2H20), 415(M + -2H20 - C2H20), 402, 343, 324, 270, 240, 120, and 91(base). The HRP electron-impact mass spectrum gave a molecular ion peak at 493.2748 (calcd for C30HagNOs, 493.2828). References R. J. Cole, D. M. Wilson, J. L. Harper, R. H. Cox, T. W. Cochran, H. G. Cutler, and D. K. Bell; Isolation and Identification of Two New [ 11]Cytochalasins from Phomopsis sojae; J. Agric. Food Chem., Vol. 30, pp. 301-304(1982). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

301

Common/Systematic Name Epoxydeacetylcytochalasin H; Deacetylepoxycytochalasin H; Epoxycytochalasin J (6R,7S, 16S, 18R,21R)-6,7-Epoxy- 18,21-dihydroxy- 16,18-dimethyl- 10-phenyl[ 11 ]cytochalasa-6(12), 13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C28HaTNO4; M W = 451.27226

,ooolll

0

HO

-

Fungal Source

Phomopsis sojae.

Isolation/Purification The crude chloroform extract was fractionated on a silica gel 60 column packed as a slurry in benzene and eluted with 3 column volumes each of benzene, ethyl ether, ethyl acetate, acetone, and methanol. The ethyl ether and ethyl acetate fractions were highly toxic, and TLC analyses suggested the same toxins were involved. These two fractions were combined, concentrated under vacuum at 60 oC, and added to a second silica gel 60 column packed in ethyl ether. The column was eluted with a linear gradient from ethyl ether to ethyl acetate. The fractions associated with acute toxicity to day-old chickens were combined and reduced to dryness under vacuum at 60 ~C. The combined fractions were applied to a C18 reversed-phase column packed in acetonitrile and reequilibrated with 10% acetonitrile-water. The column was eluted with a linear gradient from 10% acetonitrile-water to 100% acetonitrile. Toxicity was associated with two distinct compounds: one combined fraction yielded epoxycytochalasin H and the other yielded epoxydeacetylcytochalasin H. Biological Activity Acutely toxic to day-old chicks and showed plant growth inhibition in wheat coleoptile assay. Spectral Data UV:

Showed strong end absorption and a series of weak bands in the region of 240-280nm.

302

3. Chaetoglobosins/Cytochalasins

IR: The distinguishing feature in the IR spectrum of epoxydeacetylcytochalasin H was the absence of carbonyl ester absorption (1735cm~) which was present in the IR spectrum of epoxycytochalasin H. ~3C NMR: C-I, 176.4; C-2 (5.88); C-3, 54.3 d; C-4, 51.1 d; C-5, 36.6 d; C-6, 57.2 s; C-7, 63.2 d; C-8, 43.9 d; C-9, 54.7 s; C-10, 42.8 t; C-11, 12.8 q(1.05, ,/--7.0); C-12, 19.9 q(1.35); C-13, 128.1 d(5.85, J=10.1, 16.2); C-14, 136.8 d(5.22, ,/=6.1, 16.2); C-15, 46.2 t; C-16, 30.7 t; C-17, 53.7 t; C-18, 74.5 s; C-19, 134.8 d(5.72, J=-2.2, 16.9); C-20, 130.4 d(5.95, ,/--2.2, 16.9); C-21, 75.3 d(4.23, ,/--2.2); C-22, 26.4 q(1.03, ,/=5.4); C-23, 28.2 q(1.22); C-I', 137.1 s; C-2',6', 129.0 d(7.17 m); C-3', 5', 128.7 d(7.25, m); and C-4', 126.9ppm d(7.25, m). Mass Spectrum: The LRP spectrum of epoxydeacetylcytochalasin H gave a molecular ion at 451 with additional peaks at 433(M + - H20), 415(M + - 2H20), 342, 210, 240, 120, and 91(base peak). The HRP spectrum gave a molecular ion at 451.2682 (calcd for C28H37NO4= 451.2722). References R. J. Cole, D. M. Wilson, J. L. Harper, R. H. Cox, T. W. Cochran, H. G. Cutler, and D. K. Bell; Isolation and Identification of Two New [ 11]Cytochalasins from Phomopsis sojae; J. Agric. Food Chem., Vol. 30, pp. 302-304(1982). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3.

Chaetoglobosins/Cytochalasins

303

Common/Systematic Name Engleromycin (7S, 16S, 18S,21S)-7,18,21-Trihydroxy- 19R,20S-epoxy-16,18-dimethyl- 10-phenyl[ 11]cytochalasa-6(12), 13(E)-diene- 1,17-dione Molecular Formula/Molecular Weight C2sH35NO6; ~

= 481.24644

OH2

H

Me, ....

U I

II

I

H o.

I.~II I.~H ,, /

_-H

~3

'~ .... ,Me

General Characteristics Colorless needles from ethanol-water; mp, 226-228~

[a]D25 +64 ~ (in EtOH).

Fungal Source Engleromyces goe tzei. Soectral Data _

IH NMR: (deuteropyridine) N-2, 8.66; H-3, 3.6; H-4, 2.7-2.9; H-5, 3.2; H-7, 4.42(,/--10.7); H-8, 3.5(,/=10.8); H-10, 2.7-2.9; H-11, 0.93(,/--6.7); H-12, 5.27 or 5.08(d=1.4); H-13, 6.59(,/--15.5); H-14, 5.96(J=9.9, 5.6); H-15, 2.7-2.9 or 2.02(3"=12.6, 2.5); H-16, 3.2(3"=2.5); H-19, 3.99(3"=2.1); H-20, 4.27(3"=2.1, 0.8); H-21, 4.29(,/--0.8,4.2); C-16Me, 1.07(3"=6.8); C-18Me, 1.67; C-21OH, 7.14; C-7OH, 5.2; and C-18OH, 5.6ppm. 13CNMR: (deuteropyridine) C-I, 177; C-3, 54.24; C-4, 52.07; C-5, 33.61; C-6, 152.33; C-7, 71.61; C-8, 46.58; C-9, 54.80; C-10, 45.59; C-11, 13.86; C-12, 111.78; C-13, 131.79; C-14, 133.10; C-15, 38.47; C-16, 42.67; C-17, 217; C-18, 77.72; C-19, 60.90; C-20, 56.38; C-21, 74.93; C-I', 138.69; C-2', C-6',130.08; C-3', C-5', 128.83; C-4', 126.89; C-16Me, 19.17; and C-18Me, 23.08ppm. References E. J. Pedersen, P. Larsen, and P. M. Boll; Engleromycin, a new cytochalasin from Engleromyces goetzei Hennings; Tet. Lett., Vol. 21, pp. 5079-5082(1980). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

304

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Pyrichalasin H (7S, 16S, 18R,21R)-21-Acetoxy-7,18-dihydroxy- 16,18-dimethyl- 10-p-methoxyphenyl[ 11]cytochalasa-6(12), 13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C31H41NO6, MW

=

523.29339

CH2 H

.,, H

OH

M J~,, F H

Me, .... ~"

._

II OAc z ~ / \ 0 M6 OH General Characteristics Crystallized from ethyl acetate to afford colorless plates; mp 207-209"C; [tt]n23 -18.4" (C=1.05, in CHCI3). Fungal Source

Pyricularia grisea.

Isolation/Purification The filtered culture broth was passed through a column of XAD-2, the column was washed with methanol. The methanol washings were combined and concentrated to an aqueous suspension which was extracted with ethyl acetate. The ethyl acetate extract was chromatographed on a silica gel column with ethyl acetate-hexane. The fractions containing pyrichalasin H as indicated by TLC (silica gel 60 F254), aluminum sheet (Merck), chloroform-methanol, 10:1 (v:v); Rf=0.47 were combined, crystallized from ethyl acetate and recrystallized to give colorless plates. Biological Activity Phytotoxic. Spectral Data UV:

~, ~~ Exhibited intense absorption maxima at 203(e=22,000) and 224nm(17,000), with inflections at 276(2,000) and 283nm (1,640). IR:

(CHCI3) bands at 3590, 3540, and 3410cm4 (OH and NH); 1735(ester C=O); 1690(amide C=O); 1610, 1580, and 1512(benzene ring); and 965cm"l (trans double bond).

3. Chaetoglobosins/Cytochalasins

305

1H NMR: H-2, 5.56 s; H-3, 3.21 m; H-4, 2.1 l(dd, ,/=4, 5); H-5, 2.77 m; H-7, 3.83(d, 3=10); H-8, 2.94(dd, J=10, 10); H-10, 2.59(dd, J=-14, 10); 2.80(dd, J=14, 5); H-11, 0.99(d, J=7); H-12, 5.11 br s; 5.35, br s: H-13, 5.74(dd, ,/--15, 10); H-14, 5.40 m; H-15, 2.04 m; 1.80 m; H-16, 1.79 m; H-17, 1.88(dd, 3=14,3); 1.57(,/=14, 2); H-19, 5.87(dd, J=15, 2); H-20, (dd, J=15, 2); H-21, 5.51(d, J=2); H-22, 1.05(d, J=7); H-23, 1.35 s; H-2',H-6', 7.06(d, J=8); H-3', H-5', 6.85(d, 3--8); Ac, 2.24 s; OMe, 3.79ppm s. :3C NMR: C-l, 174.18; C-3, 53.85; C-4, 50.31; C-5, 32.84; C-6, 148.01; C-7, 69.73; C-8, 47.19; C-9, 51.77; C-10, 44.70; C-11, 14.08; C-12, 113.99; C-13, 127.15; C-14, 138.64; C-15, 42.74; C-16, 28.43; C-17, 53.69; C-18, 74.28; C-19, 125.98; C-20, 138.08; C-21, 77.47; C-22, 26.42; C-23, 31.14; C-I', 129.37; C-2', C-6', 129.99; C- 3',C-5', 114.32; C-4', 158.68; CH3COO, 170.06; CH3COO, 20.82; and OMe, 55.29ppm. Mass Data: ELMS: spectrum of pyrichalasin H showed the following prominent ions: 523(2, M+), 506(2, M + -17, OH), 505(2, M+ -18, H20), 464(4, -59, OAt), 463(3,-60, AcOI-I), 446(9, -77 (AcOH + OH or AcO + H20)), 445(11, -78 (AcOH + HzO)), 402(12, - 121(MeOC6Iq_4CH2), 385( 1, - 138 (MeOC6I-I4-CH2+ OH)), 384( 5, - 1390VIeOCd-I4CHz + H20)), 343(3, -180 (MeOCd-hCH2 + OAc)), 342(12, -181 (MeOCd-I4CHz + AcOH)), 325(9, -198(MeOCd-hCH2 + AcOH + OH)), 324(33, -199 (MeOCd-I4CH2 + AcOH + H20)), 306(8, -217(MeOC6I-hCH2 + 2H20 + OAc)), and 121m/e (100, MeOC6I-I4CH2+ ion); found: C 70.86; H 7.95: N 2.47; calcd for C3:I-I4~NO~,C. 71.10; H. 7 89; N 2.67. References M. Nukina; Pyrichalasin H, a New Phytotoxic Metabolite Belonging to the Cytochalasins from Pyricularia grisea (Cooke) Saccardo; Agric. Biol. Chem., Vol. 51, pp. 2625-2628(1987). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

306

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin NpHo (7S, 16S, 18R,21R)-21-Acetoxy-7,18-dihydroxy- 16,18-dimethyl- 10-phenyl[ 11 ]cytochalasa-5,13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C3oH39NOs; m w = 493.28282

OH

,,Ollll

0

HO -

General Characteristics Colorless powder; mp., 253-254~

[a]o +85.4 ~ (in MeOH).

Fungal Source

Phomopsis sp.

Isolation/Purification Extracted with dichloromethane and chromatographed on silica gel with hexane-acetone as elution solvent. Final purification by HPLC using Nucleosil 50-5 with hexane-acetone as mobile phase. Biological Activity Cytotoxic (binds specifically to actin). Spectral Data UV: ZmUff" 208rim (e=19,800).

(KBr) 3400, 2725, 1690, 1470, 1235, 1150, 960, and 700cmq.

IH NMR: (DMSO-d6) H-2, 8.15(s);H-3, 3.17(ddd,,/=-4.3,I0.5);H-4, 2.27(m);H-7, 3.60(m); H-8, 2.36(dd,J=10, I0);H-10, 2.96(dd, 12.8,4.3);2.72(dd, 12.8, 10.5);H-I I, 1.51(s);H-12, 0.87(s);H-21, 5.66(dd, 1.8,2.0);and 21-Ac, 2.25ppm (s).

3. Chaetoglobosins/Cytochalasins

307

13CN M ~ : (DMSO-d6) C-I, 174.5(s);C-3, 49.1(d);C-4, 60.0(d);C-5, 125.5(s);C-6, 133.2(s); C-7, 68.3(d);C-8, 48.5(d);C-9, 51.4(s);C-10, 43.3(0; C-I I, 14.4(q);C-12, 16.5(q); C-21, 75.1(d);and 21Ac, 20.4(q), 170.4ppm (s). References S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991). T. Tomioka, Y. Izawa, K. Koyama, and S. Natori; Three New 10-Phenyl[ 11] cytochalasins, Cytochalasins N, O, and P from Phomopsissp.; Chem. Pharm. Bull., Vol. 35, pp. 902-905(1987).

308

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin Opno (7S, 16S, 18R,21R)-7,18,21-Trihydroxy- 16,18-dimethyl-10-phenyl[11]cytochalasa5,13(E), 19(E)-trien- 1-one Molecular Formula/Molecular Weight C28H37NO4; M W = 451.27226

OH

.|11111

0

HO

General Characteristics Colorless needles; mp., 187-188~

--

[a]D +59.6 ~ (in MeOH).

Fungal Source

Phomopsis sp.

Isolation/Purification Extracted with dichloromethane and chromatographed on silica gel with hexane-acetone as elution solvent. Final purification by HPLC using Nucleosil 50-5 with hexane-acetone as mobile phase. Biological Activity Cytotoxic (b inds specifically to actin). Spectral Data UV:

~M~O. 206rim(e=20,800) max

IR:

(KBr) 3425, 1670, 1250, 1140, 960, and 700cm"~. IH NMR: (DMSO-d6) H-2, 7.83(s); H-3, 3.12(ddd, J=5.0, 9.8); H-4, 2.85(m); H-7, 3.57(m); H-8, 2.36(dd, J=10.1, 10.1); H-10, 2.92(dd, 12.9, 5.0); 2.70(dd, 12.9, 9.8); H-11, 1.51 (s); H- 12, 0.95(s); and H-21, 5.27ppm (s).

3. Chaetoglobosins/Cytochalasins

309

13C NMR: (DMSO-d6) C-I, 176.6(s); C-3, 48.5(d); C-4, 59.8(d); C-5, 126.4(s); C-6, 132.4(s), C-7, 68.6(d); C-8, 48.0(d); C-9, 53.1(s); C-10, 43.4(t); C-11, 14.3(q); C-12, 16.6((1); and 73.4ppm (d).

References S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991). T. Tomioka, Y. Izawa, K. Koyama, and S. Natori; Three New 10-Phenyl[ 11] cytochalasins, Cytochalasins N, O, and P from Phomopsissp.; Chem. Pharm. Bull., Vol. 35, pp. 902-905(1987).

310

3.

Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin PPao (6S,7S, 16S, 18R,21R)-21-Acetoxy-6,7, ! 8-trihydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa- 13(E), 19(E)-dien- 1-one Molecular Formula/Molecular Weight C3oH4]NO6; M W = 5 1 1 . 2 9 3 3 9

~k---OH

,

......~ , ~ ~ O H

.,lllll

0

HO

-

General Characteristics Crystals from chloroform; mp., 117-118~ [a] -116 ~ (in MeOH). Fungal Source

Phomopsis sp.

Isolation/Purification Extracted with dichloromethane and chromatographed on silica gel with hexane-acetone as elution solvent. Final purification by HPLC using Nucleosil 50-5 with hexane-acetone as mobile phase. Biological Activity Cytotoxic (binds specifically to actin). Spectral Data UV: Mr ~.max

208rim(e=l 5,200).

IR:

(KBr) 3425, 2920, 1680, 1370, 1230, 960, and 700cm"]. IH NMR: (DMSO-d6) H-2, 7.92(s); H-3, 4.02(ddd, J=4.6, 5.4, 5.0); H-4, 1.75(dd, 5.0, 5.1); H-5, 1.63(dq, 5.1, 7.0); H-7, 3.27(dd, 5.9, 11.4); H-8, 2.46(dd, J=l 1.4, 10.2); H-10, 2.57(dd, 13.8, 5.4); 2.78(dd, 13.8, 4.6); H-11, 0.81(d, 7.0); H-12, 0.98(s), H-21, 4.77(dd, 2.4, 2.1); and H-21Ac, 2.01(s).

3. Chaetoglobosins/Cytochalasins

311

13C NMR: (DMSO-d6) C-l, 174.3(s); C-3, 52.6(d); C-4, 48.8(d); C-5, 37.8(d); C-6, 75.0(d); C-7, 75.5(d); C-8, 45.4(d); C-9, 52.9(s); C-10, 42.9(0; C-11, 12.8(c0; C-12, 22.4(q); C-21, 77.6; and C-21Ac, 20.5(q), 169.2(s).

References T. Tomioka, Y. Izawa, K. Koyama, and S. Natori; Three New 10-Phenyl[ 11] cytochalasins, Cytochalasins N, O, and P from Phomopsissp.; Chem. Pharm. Bull., Vol. 35, pp. 902-905(1987). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

312

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin QPHO (6S,7S, 16S, 18R,21R)-6,7,18,21-Tetrahydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa13(E), 19(E)-dien- 1-one Molecular Formula/Molecular Weight C2sHagNOs MW = 469.28282 N, ,,OH ' ......( ~ , , , O H

.....

,

HO General Characteristics Colorless powder; mp, 158-159~ (hexane-acetone); [a]D23 -47.80. Fungal Source

Phomopsissp.

Isolation/Purification The mold was incubated in stationary culture on sterilized wheat at 26~ for 20 days. The moldy wheat was extracted three times with methylene chloride for 24 hrs at room temperature. The extract was chromatographed over silica gel using a gradient system of hexane-acetone as the developing solvents to afford several fractions (1, hexane-acetone, 5:1; 2, 3:1; 3, 2:1; 4, 1:1; and 5, 1:2, v/v). Fraction 5 was chromatographed on columns containing silica gel and eluted with chloroform-methanol (15" 1, v/v) followed by hexane-acetone (5:3, v/v) and subjected to HPLC on a Nucleosil 50-5 column with hexane-acetone (5:4, v/v) as the first mobile phase followed by hexane-acetone (5:3, v/v) to give cytochalasins R, S, and Q respectively. Biological Activity Biological activity typically that of other cytochalasins which is based on an interaction with the protein actin. Spectral Data UV: ~,,~M~O. 209rim (e=48,750). IR: (KBr) 3400, 2910, 1680, 1455, 1370, and 962cmq.

3. Chaetoglobosins/Cytochalasins

313

~H NMR: (DMSO-d6) H-2, 7.65 l(s); H-3, 4.039(ddd, J3,4=5Hz, d3,,o,=3.8I-Iz,J3,,Obb=5.7Hz); H-4, 2.068(dd, J4,s=5.0Hz); H-5, 1.785(dq, J5,~=7.1Hz); H-7, 3.285(dd, JT,s=12.0I-Iz); H-8, ca. 2.50(Js.~3=10.1Hz); H-10a, 2.528(dd); H-10b, 2.853(dd); H-11, 0.932(d); H-12a, 0.969(s); H-13, 5.323(dd, Jl3,14=15.0nz); H-14, 4.881(ddd, d~4,~s,=4.2Hz, J~4,~sb=l1.0Hz); H-15a, 1.536(ddd); H-15b, 1.850(ddd); H-16, ca. 1.68; H-17a, 1.325(dd); H-17b, 1.625(dd); H-19, 5.51 l(dd, Jlg,20=16.8Hz, Jlg,21=2.3Hz); H-20, 5.883(dd, J20m=2.2Hz); H-21, 3.313(ddd); 16-CH3, 0.929(d); 18-CH3, 1.100(s); H-2', H-6', 7.176(d); H-3', H-5', 7.276(dd); H-4', 7.184(dd); 21-OH, 4.751(d, J2~mon=6.6Hz); 6-OH, 4.151 (s); 7-OH, 3.746(d, JT,7orr=4.7I-Iz);and 18-OR 4.142ppm

(s).

13C NMR.:

(DMSO-d6) C-l, 175.5(s); C-3, 52.4(d); C-4, 48.7(d); C-S, 37.9(d); C-6, 75.2(s); C-7, 75.4(d); C-8, 43.4(d); C-9, 54.3(s); C-10, 43.0(0; C-11, 12.6(q); C-12, 22.2(q); C-13, 127.6(d); C-14, 135.5(d); C-15, 42.9(t); C-16, 28.1(d); C-17, 53.2(t); C-18, 72.4(s); C-19, 135.5(d); C-20, 131.8(d); C-21, 75.5(d); C-22, 26.2(q); C-23, 32.4(q); C-I', 137.4(s); C-2', C-6', 130.1(d); C-3', C-5', 127.8(d); and C-4', 126.0ppm (d). Mass Spectrum: 469.2808(M+, calcd for C28H39NO5,469.2826), 451,433, 417, 360, 324, 120, and 9 lm/e.

References Y. Izawa, T. Hirose, T. Shimizu (nee Tomioka), K. Koyama, and S. Natori; Six New 10-Phenyl[ 11]Cytochalasins, Cytochalasins N-S from Phomopsis sp.; Tetrahedron, Vol. 45, pp. 2323-2335(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

314

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin RpHO (6R,7S, 16S, 18R,21R)-6,7,18,21-Tetrahydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa13(E), 19(E)-dien- 1-one Molecular Formula/Molecular Weight C2sHaaNOs; MW = 469.28282

OH ',.....~ O H I

_

o OH

_

..... '

INO

General Characteristics Colorless powder; mp, 106-107 ~ (hexane-acetone); [{g]D23

-46.0

~

Fungal Source

Phomopsis sp.

Isolation/Purification The mold was incubated in stationary culture on sterilized wheat at 26~ for 20 days. The moldy wheat was extracted three times with methylene chloride for 24 hrs at room temperature. The extract was chromatographed over silica gel using a gradient system of hexane-acetone as the developing solvents to afford several fractions: (1, hexane-acetone, 5:1; 2, 3:1; 3, 2:1; 4, 1:1; and 5, 1:2, v/v). Fraction 5 was chromatographed on columns containing silica gel and eluted with chloroform-methanol (15:1, v/v) followed by hexane-acetone (5:3, v/v) and subjected to HPLC on a Nucleosil 50-5 column with hexane-acetone (5:4, v/v) as the first mobile phase followed by hexane-acetone (5:3, v/v) to give cytochalasins R, S, and Q respectively. Biological Activity Biological activity typically that of other cytochalasins which is based on an interaction with the protein actin. Spectral Dat.a.. UV~

~, M~H 206nm (6=23,200). max

IR:

(KBr) 3400, 2910, 1680, 1455, 1370, and 962cm"1.

3. Chaetoglobosins/Cytochalasins

315

IH NMR: (DMSO-d6) H-2, 7.814(s);H-3, 3.425(m, J3,4=5.0Hz);H-4, 2.245(dd, J4,s=5.0Hz); H-5, 1.985(dq,Js,~=7.2Hz); H-7, 2.905(d,J7,8=I1.8Hz); H-8, 2.735(Js,~3=9.9Hz);H10a, ca. 2.66; H-If, O.681(d); 12a, 1.007(s);H-13, 5.372(dd, J~3,~4=I5.1Hz);H-14, 4.922(ddd, J~4,15,=4.II-Iz,J~4,~sb=l1.0Hz); H-15a, 1.536(ddd);H-15b, 1.848(ddd); H-16, ca. 1.68;H-17a, 1.340(dd);H-17b, 1.617(dd);H-19, 5.585(dd, J~o,2o=16.51-Iz, Jz9,2~=2.2Hz);H-20, 5.825(dd,J2o,2~=1.9Hz);H-21, 3.480(dd); 16-CH3, 0.930(d); 18CH3, 1.120(s);H-2', H-6', ca. 7.21 ;H-3', H-5', 7.290(dd); and H-4', ca. 7.21ppm. 13C NMR: (DMSO-d6) C-I, 176.0(s); C-3, 52.4(d); C-4, 47.3(d); C-5, 38.2(d); C-6, 71.5(s); C-7, 72.5(d); C-8, 41.5(d); C-9, 54.2(s); C-10, 44.2(t); C-I 1, 12.7(q); C-12, 24.5(q); C-13, 127.9(d); C-14, 134.2(d); C-15, 43.0(t); C-16, 27.9(d); C-17, 53.4(t); C-18, 72.4(s); C-19, 136.0(d); C-20, 131.3(d); C-21, 75.2(d); C-22, 26.2(q); C-23, 31.9(q); C-l', 137.1(s); C-2', C-6', 129.9(d); C-3', C-5', 127.9(d); and C-4', 126.2ppm (d).

Mass Spectrum: 469.2808(M +, calcd for C28H39NOs,469.2826), 451,433, 415, 360, 324, 120, and 9 lm/e.

References Y. Izawa, T. Hirose, T. Shimizu (nee Tomioka), K. Koyama, and S. Natori; Six New 10-Phenyl[11]cytochalasins, Cytochalasins N-S from Phomopsis sp.; Tetrahedron, Vol. 45, pp. 2323-2335(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

316

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin S (5S, 6S, 7S, 16S, 18R,21R)-5,7,18,21-Tetrahydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa- 13(E), 10(E)-dien- 1-one Molecular Formula/Molecular Weight C2sHa9NO5; MW = 469.28282

HO,,I~..,r~OH ,(

,

.

..... , HO

General Characteristics Colorless powder; mp., 149-151 ~ (hexane-acetone); [a]D23 -62.9 ~ Fungal Source

Phomopsis sp.

Isolation/Purification The mold was incubated in stationary culture on sterilized wheat at 26 ~ for 20 days. The moldy wheat was extracted three times with methylene chloride for 24 hrs at room temperature. The extract was chromatographed over silica gel using a gradient system of hexane-acetone as the developing solvent to afford several fractions (1, hexane-acetone, 5:1; 2, 3:1; 3, 2:1; 4, 1:1; and 5, 1:2, v/v). Fraction 5 was chromatographed on columns containing silica gel and eluted with chloroform-methanol (15:1, v/v) followed by hexane-acetone (5:3, v/v) and subjected to HPLC on a Nucleosil 50-5 column with hexane-acetone (5:4, v/v) as the first mobile phase followed by hexane-acetone (5:3, v/v) to give cytochalasins R, S, and Q respectively. Biological Activity Biological activity typically that of other cytochalasins (inhibition of a variety of cellular movements) which is based on an interaction with the protein actin. Spectral Data U-V: Z M~.

208nm (6=13,370).

IR: (KBr) 3350, 2900, 1680, 1445, 1362, and 960cm"l.

3. Chaetoglobosins/Cytochalasins

317

IH NMR: (DMSO-d6) H-2, 7.833(s); H-3, 3.557(ddd, ,13,4=4.7H~J3,~0,=5.2H~ ,JlO,,b=13.8Hz); H-4, 2.078(d); H-6, ca. 1.53(J6,~2=7.3Hz);H-7, 2.851(m, dT,s=l 1.2Hz); H-8, 3.043(dd); H-10a, 2.650(dd); H-10b, ca. 2.90; H-11, 1.130(d); H-12a, 1.030(d); H-13, 5.393(dd, d~3,t4=16.6Hz); H-14, 4.889(ddd, dt4,~5,=3.9Hz, d~4,~sb=l1.2Hz); H-15a, ca. 1.53; H-15b, 1.866(ddd); H-16, 1.720(m); H-17a, 1.330(dd); H-17b, 1.592(dd); H-19, 5.578(dd); H-20, 5.666(dd); H-21, 3.243(m); 16-CH3, 0.920(d); 18-CH3, 1.102 (s); H2', H-6', ca. 7.21; H-3', H-5', 7.290(dd); H-4', ca. 7.21; 21-OH, 5.414(d); and 7-OH, 3.877ppm (d, dT,OH=5.1Hz). ~SCNMR: (DMSO-d6) C-I, 174.8(s); C-3, 52.7(d); C-4, 55.5(d); C-5, 72.1(s)*; C-6, 49.0(d); C-7, 72.2(d); C-8, 45.1(d); C-9, 54.7(s); C-10, 42.0(0; C-11, 25.3(q); C-12, 16.9(q); C-13, 127.8(d); C-14, 135.5(d); C-15, 43.0(t); C-16, 28.0(d); C-17, 53.5(0; C-18, 72.3(s)*; C-19, 136.5(d); C-20, 129.8(d); C-21, 74.2(d); C-22, 26.2(q); C-23, 31.9(q); C-I', 136.7(s); C-2', C-6', 130.2(d); C-3', C-5', 127.9(d); and C-4', 126.3ppm (d). * Assignments may be reversed. Mass Spectrum: 469.2792(M+, calcd for C2sHsgNOs,469.2826), 451,433,415, 378, 360, 324, 306, 288, 270, 242, 216, 120, and 91role. References Y. Izawa, T. Hirose, T. Shimizu (nee Tomioka), K. Koyama, and S. Natori; Six New 10-Phenyl[ 11]cytochalasins, Cytochalasins N-S from Phomopsis sp.; Tetrahedron, Vol. 45, pp. 2323-2335(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoa!exins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

318

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin NHVp (7S, 16S, 18/2,21R, 13E, 19E)-21-Acetoxy-5,6-epoxy-7,18-dihydroxy- 16,18dimethyl-10-phenyl[11]cytochalasa- 13(E), 19(E)-diene- 1,17-dione Molecular Formula/Molecular Weight Cs0H37NOT;MW = 523.25700

O.

Me

,,,Me 00Ac

Me OH 0

General Characteristics Hair-like needles from methanol; mp., 272~

[a]D23 -4 o (c=0.5 ' in MeOH).

Fungal Source Hypoxylon terricola (habitat restricted to dead and decaying coniferous needles; only found in one location in the U.S.A. and in the Atlantic Pyrenees). Isolation/Purification The mycelia were extracted with chloroform. Evaporation yielded a solid that was chromatographed using Merck Kieselgel PF. The sample was applied to the column in chloroform-methanol (95:5, v/v) and eluted with the same solvent to yield cytochalasin C and a mixture of cytochalasins C, D, and N which were further purified by flash chromatography using chloroform-butanol (97:3, v/v) as the eluting solvent. Biological Activity Marked cytostatic effects on mammalian cells in tissue culture; target protein was actin. Spectral Data IR:

(KBr) 3410, 1742, 1705, and 1699cm~. 1H N1V[R: (CsDsN) 2-NH, 9.55(s); 3=H, 4.05(ddd, J=7.0, 7.0. 2.0); 4=H, 2.88(d, ,/=2.0); 7=H, 4.15(dd, J=10.5, 6.0); 8-H, 3.81(dd, J=10.5, 10.5); 10-Ha, Fib, 3.16(d, 7.0); 11-I-I, 1.59(s); 12-H, 1.25(s); 13-H, 6.61(dd, J=15.5, 10.5); 14-H, 5.47(ddd, d=15.5, 10.5, 4.5); 15-Ha, 1.95(ddd, J=ll.5, 5.5, 1.0); 15-Hb, 2.58-2.75(m); 16-H, 2.58-2.75(m); 19=H, 5.65(dd, ,/=16.0, 2.5); 20=H, 6.76(dd, J=16.0, 2.5); 21-H, 6.22(dd, J=2.5, 2.5);

3. Chaetoglobosins/Cytochalasins

319

22-H, 1.02(d, J=6.5); 23-H, 1.56(s); 21-OAc, 2.38(s); 7-OH, 6.06(d, J=6.0); 18-OH, 6.02(s, br); and Ar-5H, 7.29-7.38ppm (m). ~3C NMR: (CH3) 14.83, 19.40, 20.16, 20.74, and 24.64ppm; (CH2) 38.70, and 45.43ppm; (CH) 42.42, 44.50, 49.02, 56.76, 70.15, 76.72, 127.04, 128.23, 129.03 x 2, 129.95 x 2, 131.18, 133.41, and 134.05ppm; and (C) 55.57, 63.42, 65.67, 78.45, 138.39, 170.73, 174.89, and 210.90ppm. Mass Data: EIMS: 523m/e; found: C, 69.1; H, 7.2; N, 2.6, C30H37NO7requires C, 68.8; H, 7.1: N, 2.7%. References R. L. Edwards, D. J. Maitland, and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. Cytochalasin N, O, P, Q, and 1L New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

320

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin On~ (6R or 6S,7S,16S, 18R,21R)-21-Acetoxy-6,7,18-trihydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa- 13(E), 19(E)-diene- 1,17-dione Molecular Formula/Molecular Weight C30H39NOT; ~

= 525.27265

OH

Me

'It

.olllJ

General Characteristics Needles from toluene-methanol; mp., 258-265~

[a]D23 -39.3 o (C=I.0 ' in MeOH).

Fungal Source Hypoxylon terricola (habitat restricted to dead and decaying coniferous needles; only found in one location in the U.S.A. and in the Atlantic Pyrenees). Isolation/Purification The mycelium was extracted with chloroform; evaporation yielded a solid that was chromatographed using Merck Kieselgel PF. The sample was applied to the column in chloroform-methanol (95:5, v/v) and eluted with the same solvent to give two fractions, one containing cytochalasins C, D, and N and the other containing eytoehalasin O. Biological Activity Marked cytostatic effects on mammalian cells in tissue culture; target protein was actin. Spectral Data IR:

(KBr) 3410, 1741, 1603, and 1691cm"l. 1H NIVIR: (CsDsN) 2-NH, 9.25(s), 3-H, 3.98(ddd, J=7.6, 5.6.4.6); 4-H, 2.43(dd, J=-5.3, 4.6); 5-H, 2.6-2.8(m); 7-H, 3.76(d, d=l 1.5); 8-H, 3.55(dd, J=-10.0, 11.5); 10-Ha, 2.95(dd, 13.5, 5.2); 10-Hb, 2.84(dd, J=13.5, 7.6); 1 l-H3, 1.14(d, J=6.3); 12-H3, 1.44(s); 13-H, 6.32(dd, J=15.3, 10.0), 14-1-1,5.43(ddd, J=15.3, 10.7, 4.7); 15-Ha, 1.97(dd, J=12.3, 4.4); 15-Hb, 2.60-2.80(m); 16-H, 2.60-2.80(m); 19-H, 5.64(dd, J=-15.9, 2.5); 20-H, 6.98(dd, ,/=15.9, 2.5); 2 I-H, 5.94(dd, J=2.5, 2.5); 22-H, 1.04(d, J=6.6); 23-H, 1.56(s), 21-OAt, 2.31(s), 6-OH, 5.37(s, br), 7-OH, 6.08(s, br); 18-OH, 5.99(s, br); and kr-5H, 7.22-7.34ppm (m).

3. Chaetoglobosins/Cytochalasins

321

~3CNMR: (CH3) 13.55, 19.44, 20.53, 24.68, and 25.30ppm; (CH2) 38.82 and 46.60ppm; (CH) 39.85, 42.59, 44.73, 49.97, 54. 18, 73.42, 78.77, 127.07, 127.46, 129.04 x 2, 130.09 x 2, 131.05, 133.49, and 134.59ppm; and (C) 55.52, 72.56, 78.37, 138.43, 170.36, 175.66, and 211.39ppm. Mass Data: EIMS: 525m/e; found: C, 68.7; H, 7.6; N, 2.6, C30H39NO7requires C, 68.5; H, 7.4: N, 2.7%. References R. L. Edwards, D. J. Maitland and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. Cytochalasin N, O, P, Q, and It, New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; g. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

322

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin PnYP (6S or 6R,7S, 16S, 18R,21R)-2 l-Acetoxy-6,7,18-trihydroxy- 16,18-dimethyl- l 0-phenyl[ 11]cytochalasa- 13(E), 19(E)-diene- 1,17-dione Molecular Formula/Molecular Weight C30Hs9NOT; MW = 525.27265 OH Me "

j

Me OH "1111

,11111

I~n

General Characteristics Needles from acetone-light petroleum; mp., 169-173 oc;

[ a ] D 23 -3

5.8 o

(C,=

1.0, in MeOH).

Fungal Source

Hypoxylon terricola (habitat restricted to dead and decaying coniferous needles; only found in one location in the U.S.A. and in the Atlantic Pyrenees).

Isolation/Purification The mycelia were extracted with chloroform. Evaporation yielded a solid that was chromatographed using Merck Kieselgel PF. The sample was applied to the column in chloroform-methanol (95:5, v/v) and eluted with the same solvent to give several fractions; one fraction was a viscous yellow gum. The gum was triturated with ethyl acetate, filtered and afforded a mixture containing cytochalasins C, O, and N. Evaporation of the filtrate and crystallization of the residue from acetone-light petroleum gave cytochalasin P as needles. B.iological Activity Marked cytostatic effects on mammalian cells in tissue culture; target protein was actin. S..pectral Data IR:

(KBr) 3425, 1747, 1700, and 1697cm"l. 1H NMR: (CsDsN) 2NH, 9.10(s); 3-H, 4.79(ddd, J=8.0, 4.5, 4.0); 4-H, 2.3-2.41(m); S-H, 2.3-2.41(m); 7-H, 4.29(dd, J=l 1.0, 3.0); 8-H, 3.26(dd, J=l 1.0, 10.3); 10-Ha,

3. Chaetoglobosins/Cytochalasins

323

2.60-2.85(m); 10-Hb, 3.12(dd, J=13.7, 3.4); l l-H, 1.39(d, J=6.9); 12-H, 1.72(s), 13-H, 6.33(dd, J=15.0, 9.8); 14-H, 5.47(ddd, J=15.0, 10.6, 4.4); 15-Ha, 2.01(dd, J=12.5, 4.4); 15-Hb, 2.60-2.85(m); 16-H, 2.60-2.85(m); 19-H, 5.67(dd, J=15.9, 2.4); 20-H, 7.06(dd, J=15.9, 2.8); 21-H, 5.94(dd, J=2.6, 2.6); 22-H, 1.06(d, J=6.8); 23-H, 1.60(s); 21-OAt, 2.43(s); 6-OH, 4.90(under HOD signal); 7-OH, 5.80(d, J=3.0); 18-OH, 6.01(s); and Ar-5H, 7.16-7.34ppm (m). 13C NMR: (CH3) 13.42, 19.43, 20.63, 23.30 and 24.73ppm; (CH2) 38.73 and 46.26; (CH) 39.22, 42.72, 47.08, 51.82, 54.62, 76.81, 79.10, 126.67, 127.17, 128.80 x 2, 129.86 x 2, 131.06, 133.66, and 134.98ppm; and (C) 55.72, 76.08, 78.32, 39.04, 170.08, 175.59, and 211.56ppm.

Mass Data: EIMS: 525re~e;found: C, 68.6; H, 7.5; N, 2.7, C30H39NO7requires C, 68.6; H, 7.4: N, 2.7%. References R. L. Edwards, D. J. Maitland and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. Cytochalasin N, O, P, Q, and R, New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

324

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin QHYP (6R,7S, 16S,18R,21R)-21-Acetoxy-6,7-epoxy- 18-hydroxy- 16,18-dimethyl- 10-phenyl[ 11]-cytochalasa- 13(E), 19(E)-diene- 1,17-dione Molecular Formula/Molecular Weight C3oH37NO6; M W -- 5 0 7 . 2 6 2 0 9

Me -

~|;i

21

0

I

16 ,, ,,Me

General Characteristics Fine, silky, acetone solvated needles from acetone-light petroleum; mp., 145-147 ~C; [tt]D23 (solvate) -94.5 ~ (c=l.0, in CHCI3). Fungal Source

Hypoxylon terricola (habitat restricted to dead and decaying coniferous needles; only found in one location in the U.S.A. and in the Atlantic Pyrenees).

Isolation/Purification The mycelia were extracted with chloroform. Evaporation yielded a solid that was chromatographed using Merck Kieselgel PF. The sample was applied to the column in chloroform-methanol (95:5, v/v) and eluted with the same solvent to give several fractions; one fraction was a viscous yellow gum which was dissolved in ethyl acetate, filtered alter 24 hours to yield a mixture containing cytochalasins C, D, O, and N. The mother liquor was evaporated and the gum was redissolved in ethyl acetate, filtered after 24 hours to give large rhombs comprising mainly cytochalasin R. The viscous filtrate containing the bulk of the cytochalasin Q was evaporated and the resulting gum dissolved in a mixture of benzene-ethyl acetate-acetic acid (50:49:1, v/v) and applied to a silica gel column eluted with the same solvent mixture; evaporation gave two fractions, one a gummy yellow foam which yielded fine, silky acetone-solvated needles of cytochalasin Q. Biological Activity Marked cytostatic effects on mammalian cells in tissue culture; target protein was actin. Spectral Data IR;

(KBr) 3440, 1745, and 17.02cm"l.

3. Chaetoglobosins/Cytochalasins

325

1H NMR: (CsD5N) 2NH, 9.50(s); 3-H, 3.92(dd, ,/=6.6, 7.9); 4-H, 2.45(dd, ,/--5.7, 1.8); 5-I-I, 1.90-2.05(dq, ,/=7.3, 5.7); 7-H, 3.06 (d, ,/=5.8); 8-H, 2.97(dd, ,/=9.9, 5.7); 10-Ha, 2.84(dd, ,/=13.2, 7.9); 10-Hb, 3.09(dd, ,/=13.2, 6.6); 1l-H3, 0.74(d, ,/=7.3); 12-I-I3, 1.21(s); 13=H, 6.46(dd, ,/=15.6, 10.1); 14-H, 5.46(ddd, `/=15.6, 10.4, 5.2); 15=Ha, 1.90-2.05(m); 15-Hb, 2.64-2.77(m); 16-H, 2.64-2.77(m); 19-H, 5.62(dd, ,/=15.8, 2.4); 20=H, 6.85(dd, J=15.8, 2.4); 21-H, 6.24(dd, ,/=2.2, 2.4); 22=H, 1.07(d, `/=6.2); 23-H, 1.57(s); 21-OAt, 2.38(s); 18-OH, 6.05-6.15(s, br); and Ar-SH, 7.20-7.40ppm (m). 13CNMR: (CH3) 12.58, 19.39, 19.63, 20.58, and 24.69ppm; (CH2) 38.30, and 46.19ppm; (CI-I) 37.36, 42.49, 46.03, 50.54, 54.49, 63.16, 76.64, 127.05, 128.60, 129.00 x 2, 130.10 x 2, 131.94, 132.14, and 133.03ppm; and (C) 56.11, 78.26, 138.26, 170.66, 175.47, and 210.88ppm.

Mass Data: ELMS: 507re~e;found: C, 70.1; H, 7.3; N, 2.7, C30H37NO6; requires C, 70.1; H, 7.7: N, 2.5%. References R. L. Edwards, D. J. Maitland and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. Cytochalasin N, O, P, Q, and 1L New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

326

3.

Chaetoglobosins/Cytochalasins

Common/Systematic N.am_e 19,20-Epoxycytochalasin Q (13E)-21 -Acetoxy-6, 7,19,20-diepoxy- 18-hydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa- 13-ene- 1,17-dione Molecular Formula/Molecular Weight C3oH37NO7; M W = 5 2 3 . 2 5 7 0 0

12 11 Me\ ~0 Me,....~

~

10

H~b

l_

I

]

4

14

..... f " ~ ~

o

22 ....,Me

"o 23

General Characteristics Crystals; mp., 266-268~

[a]D 23

-73 ~ (c=l.1, in CHCI3).

Funsal Source Fruit bodies ofXylaria obovata, a wood inhabiting fungus. Isolation/Purification The fungus was cultured on rice; the rice cultures were extracted with chloroform. Purification was achieved by silica gel column chromatography by elution with a hexaneacetone gradient followed by preparative TLC. Biological Activity The cytochalasin was lethal to brine shrimp (LCs0 2.51~g/ml) and cytotoxic to HL-60 cells at 11zg/ml. It also inhibited mammalian cell growth with high potency as demonstrated in the Vero monkey cell growth inhibition assay (ICs0 0.46l.tg/ml). Spectral Data

IR: (KBr) 1220, 1370, 1450, 1690, 1740, 2970, and 3440cm 4. 1H N]VIR:

(CsDsN) NH, 9.58(s);3-H, 3.92(brt,d=7.0; 4-H, 2.52(dd,d=5.8, 2.0);5-H, 1.89(dq, d=7.2, 5.8);7-H, 3.02(d,J=5.6); 8-H, 2.75(m); 10-H,, 2.87(dd,J=13.2, 8.0): 10-I-Ib, 3.08(dd, J=13.2, 6.0); I I-H, 0.63(d,d=7.2); 12-H, 1.17(s);13-H, 6.64(dd,J=15.2, 10.0), 14-H, 5.83(sept,J=15.6, I0.0,6.0); 15-I-I,,2.00(m); 15-I-Ib,2.75(m), 16-H,

3. Chaetoglobosins/Cytochalasins

327

3.24(m); 19-H, 3.72(d, J=2.0); 20-H, 4.30(dd, J=8.0, 2.0); 21-H, 6.18(br s); 22-H, 1.07 (d, J=6.8); 23-H, 1.64(s); OAc, 2.18(s); U-2', n-6', 7.40(d, ,/=-6.7); H-3', n-5', 7.34(t, ,/=7.8); and H-4 ~ 7.23ppm (d, J= 6.7Hz).

13CNMR.: (CsDsN) C-I, 175.4(s);C-3, 54.9(d);C-4, 51.2(d);C-5, 37.3(d);C-6, 57.4(s);C-7, 63.1(d); C-8, 45.7(d);C-9, 55.6(s);C-10, 46.1(t);C-I I, 12.4(c0;C-12, 19.6(q);C-13, 132.5(d);C-14, 131.6(d);C-15, 37.9(0; C-16, 42.2(d);C-17, 216.3(s);C-18, 77.4(s); C-19, 60.8(d); C-20, 53.9(d); C-21, 73.7(d); C-22, 19.2(q); C-23, 22.7(q); C-1 ~ 138.2(s); C-2',6', 130.2(d); C-3',5', 128.9(d); C-4', 127.1(d); CH3CO, 20.4(q); and CHACO, 170.Sppm (s). Mass Spectrum: HREIMS: 523.2648(M~, 8%, calcd for C3oH37NO7,523.2569), 495(M+ -CO, 17), 480, 464, 432, 338, and 91m/e (100), Reference E. Dagne, A. A. Leslie Gunatilaka, S. Asmellash, D. Abate, D. G. I. Kingston, G. A. Hofmann, and R. K. Johnson; Two New Cytotoxic Cytochalasins from Xylaria obovata; Tetrahedron, Vol. 50, pp. 5615-5620(1994).

328

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name 19,20-Epoxydeacetylcytochalasin Q (13E)-6, 7,19,20-Diepoxy- 18,21 -dihydroxy- 16,18-dimethyl- 10-phenyl[ 11]eytochalasa- 13ene- 1,17-dione Molecular Formula/Molecular Weight C2sI-I35NO6;MW = 481.24644 12

~ I

' II

11 ~ ~O h',,,.r~ ~I 10 I_

14

.. ~ " ~

\ 2

-~ 0%~ 19

~ 16 ) ...... ,

23

General Characteristics Crystals; mp., 119-121 ~ C. Fungal Source Fruit bodies of Xylaria obovata, a wood inhabiting fungus. Isolation/Purification The fungus was cultured on rice; the rice cultures were extracted with chloroform. Purification was achieved by silica gel column chromatography by elution with a hexaneacetone gradient followed by preparative TLC. Biological Activity The cytochalasin was lethal to brine shrimp (LCs0 2.51ag/ml) and cytotoxic to HL-60 cells at l~g/ml. It also inhibited mammalian cell growth with high potency as demonstrated in the Vero monkey cell growth inhibition assay (IC50 1.91ag/ml) Spectral Data IR:

(KBr) 1370, 1450, 1685, 2970, and 3400cm "1. IH NMR: (CsDsN) NH, 9.25(s); 3-H, 3.91(br t, J=7.0; 4-H, 2.10(m); 5-H, 1.96(m); 7-H, 3.09(d, J=5.6); 8-H, 2.75(m); 10-H,, 2.85(dd, J=13.2, 7.0); 10-Hb, 3.06 (dd, J= 13.2, 5.8); llH, 0.82(d, J=7.2); 12-H, 1.20(s); 13-H, 6.66(dd, J=15.4, 9.6); 14-H, 5.85(sept, J=15.6, 10.0, 4.0); 15-H,, 1.97(m); 15-Hb, 2.75(m); 16-H, 3.21(m); 19-H, 3.98(d, J=2.0); 20-H, 4.27(t, J=l.6); 21-H, 4.97(br s); 22-H, 1.06(d, J=6.4Hz); 23-H, 1.67(s); and H-4', 7.20ppm (m).

3. Chaetoglobosins/Cytochalasins

329

13C NMP~: (CsDsN) C-I, 177.2(s); C-3, 54.6(d); C-4, 52.8(d); C-5, 37.5 (d); C-6, 57.6(s); C-7, 63.6(d); C-8, 44.7(d); C-9, 56.7(s); C-10, 46.2(t); C-I 1, 12.7(q); C-12, 19.8(q); C-13, 132.9(d); C-14, 131.1(d); C-15, 38.1(t); C-16, 42.2(d); C-17, 216.8(s); C-18, 77.7(s); C-19, 60.8(d); C-20, 56.1(d); C-21, 73.3(d); C-22, 19.2(q); C-23, 22.8(q); C-I', 138.3(s); C-2', C-6', 130.1(d); C-3', C-5', 128.9(d); and C-4~ 126.1ppm (d).

Mass Spectrum: HR IMS: 481.2464(NV, 5%, calcd for C28H35NO6,481.2467), 390(M+ - 91, 100%), and 338m/e (96). Reference E. Dagne, A. A. Leslie Gunatilaka, S. Asmellash. D. Abate, D. G. I. Kingston, G. A. Hofmann, and R. K. Johnson; Two New Cytotoxic Cytochalasins from Xylaria obovata; Tetrahedron, Vol. 50, pp. 5615-5620(1994).

330

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin Rm,p 21-Acetoxy-6,7,13,14-diepoxy- 18-hydroxy- 16,18-dimethyl- 10-phenyl[ 11]cytochalasa19(E)-ene- 1,17-dione Molecular Formula/Molecular Weight C30H37NO7; M W -" 523.25700 Me O Me O Me

General Characteristics Glistening solvated rods from methyl cyanide; mp., 159-167~ [a]oz3 (ethyl acetate solvate) -73.1 ~ (c=l.0, in CHCI3); [a]D23 MeCN solvate - 49.3 ~ (c=l.0, in MeCN). Fungal Source Hypoxylon terricola (habitat restricted to dead and decaying coniferous needles; only found in one location in the U.S.A. and in the Atlantic Pyrenees). Isolation/Purification The mycelia were extracted with chloroform. Evaporation fielded a solid that was chromatographed using Merck Kieselgel PF. The sample was applied to the column in chloroform-methanol (95:5, v/v) and eluted with the same solvent to give several fractions; one fraction was a viscous yellow gum which was dissolved in ethyl acetate, filtered after 24 hours to yield a mixture containing cytochalasins C, D, O, and N. The mother liquor was evaporated, the gum was redissolved in ethyl acetate and filtered after 24 hours to give large rhombs comprising mainly cytochalasin R. Biological Activity Marked cytostatic effects on mammalian cells in tissue culture, target protein was actin. Spectral Data

(KBr) 3460, 3315, 1750, 1712, and 1700cm"1. ~H NMR: (CsDsN) 2NH, 9.70(s); 3-H, 3.96(ddd, J=7.0, 7.1, 2.0); 4=H, 2.40(dd, ,/-5.7, 2.0); 5=H, 1.85(dq, J-7.3, 5.7); 7-H, 3.39(d, J--5.7); 8-H, 1.98(dd, J--8.6, 5.7); 10=Ha,

3. Chaetoglobosins/Cytochalasins

331

2.84(dd, J=13.2, 7.7); 10-Hb, 3.11(dd, J=13.2, 6.4); 1l-H3, 0.71(d, 3"=7.3); 12-H3, 1.20(s); 13-H, 4.37(dd, J=8.6, 2.0); 14-H, 3.0-3.12(m); 15-Ha, 1.95-2.09(m); 15-Hb, 2.12(dd, J=12.4, 1.5); 16-H, 3.0-3.12(m); 19-H, 6.22(dd, J=12.5, 2.6); 20-H, 7.18(dd, 3'=12.5, 2.6); 2 I-H, 6.25(s); 22-H, 1.03(d, J=6.8); 23-H, 1.62(s); 21-OAc, 2.30(s); 18-OH, 6.54(s); and Ar-5H, 7.20-7.38ppm (m). 13C NMR: (CH3) 12.50, 19.55, 20.39, 20.48, and 24.77ppm; (CH2) 38.79, and 46.16ppm; (CH) 37.45, 38.23, 44.78, 50.79, 54.75, 59.16, 61.79, 62.20, 76.16, 127.11, 129.03 x2, 130.10 x 2, 130.56, and 132.22ppm; and (C) 55.20, 56.11, 78.76, 138.15, 170.61, 175.45, and 213.21ppm.

Mass Data: EIMS: 523m/e; found: C, 68.1; H, 7.1; N, 5.0, C30H37NO7; requires C, 68.1; H, 7.1: N, 5.0%. References R. L. Edwards, D. J. Maitland, and A. J. S. Whalley; Metabolites of the Higher Fungi. Part 24. Cytochalasin N, O, P, Q, and R, New Cytochalasins from the Fungus Hypoxylon terricola Mill.; J. Chem. Soc. Perkin Trans. I, pp. 57-65(1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

332

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Cytochalasin G (6R,7S, 16S)-6,7-Epoxy- 10-(indol-3-yl)- 16-methyl[ 11]cytochalasa- 13(E)-ene- 1,18,21trione Molecular Formula/Molecular Weight C29H34N204; M W -" 474.25186

Me, 0 Me,,, ~ ; ~

OLN..

H i,

''llll

.,Me ....

0

0

General Characteristics Needles from methanol; mp, 255-257~ l a i D 24 - 9 9 ~ (c=0.35, in MeOH). Colorless crystals; mp, 254-256~ [00D2~ -100 ~ (C=0.3, in MeOH). Fumzal Source

Nigrosabulum spp. and Pseudeurotium zonatum.

Isolation/Purification Extracted twice with ethyl acetate, dried over anhydrous Na2SO4 and evaporated to dryness. The dark brown extract was chromatographed on silica gel using increasing amounts of methanol in CH2C12. Cytochalasin G eluted in the fractions containing 1.25% methanol. Final purification was by crystallization from acetone/hexane. Spectral Data UV:

maxMr 222(e=3,250) ' and 283nm (535); ,~m , axMeOH 198(1oge=3.95), 219(4.24), 274(3.75), 281(3.77), and 29Ohm (3.72). m:

(Nujol) 3360, 3200, 1716, 1678, 1112, 978, 878, and 740cmq; (KBr) 3360, 1710, 1675, 1610, 1450, 1420, 1380, 1355, 1290, 1230, 1100, 1080, 1050, and 970cmq. ~H NMR:

[(CD3)2SO] 0.88(d,J=8 3H); 0.91(d,J=8, 3 H); 1.15(s,31-I);1.56(m, 2H); 1.79(dxd, J=6 and 10, IH); 1.79(m, IH); 2.59(d,J=6, 21-I);2.69(d,J=6, IH); 2.69(m, 2H); 3.13(m, IH); 3.72(m, II-I);4.78(m, IH); 6.07(dxd,J=6, 2H); 2.69(d,,/=6, If-I); 3.13(m, IH); 3.72(m, IH); 4.78(m, IH); 6.07(dxd,J=10 and 15 If-I);7.0-7.5(m, 5H); 8.32(s, IH, exchangeable with DzO); and 10.89ppm (s, IH, exchangeable with DzO).

3. Chaetoglobosins/Cytochalasins

333

13C NMR: (CDCI3) C-l, 173.2 s; C-3, 51.7 d; C-4, 49.1 d; C-5, 35.6 d; C-6, 56.9 s; C-7, 60.7 d; C-8, 47.4 d; C-9, 64.1 s; C-10, 42.1 t; C-11, 12.1 q; C-12, 22.4 q; C-13, 128.4 d; C-14, 132.8 d; C-15, 32.4 t; C-16, 27.4 d; C-17, 36.2 t; C-18, 206.5 s; C-19, 46.1 t; C-20, 46.2 t; C-21,207.4 s; C-22, 19.0 q; C-2', 120.7 d; C-3', 108.3 s; C-4', 118.4 d; C-5',124.9 d; C-6', 118.4 d; C-7', 111.0 d; C-8', 136.0 s; and C-9 ~ 127.6ppm s. References A. F. Cameron, A. Freer, B. Hesp, and C. J Strawson; Isolation and Crystal and Molecular Structure of Cytochalasin G, an [ 11]cytochalasin Containing an Indole Group; Chem. Sot., Perkin Trans 2, p. 1741 (1974). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 272 (1981). A. Probst and C. Tamm; 19-O-Acetylchaetoglobosin B and 19-O-Acetylchaetoglobosin D, Two New Metabolites of Chaetomium globosum; Helvetica Chimica Acta, Vol. 64, pp. 2056-2064(1981). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

334

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Aspochalasin A 10-Isopropyl- 14-methyl[ 11]cytochalasa-6,13-diene- 1,17,18,21-tetrone Molecular Formula/Molecular Weight C24I-I33NO4;MW = 399.24096 Me

Me",,,,.r~

Me

Me Me

0

General Characteristics Bright yellow amorphous powder; [tt]t)25 -20 ~ (c=0.27, in CHCI3). Fungal Source Aspergillus microcysticus. Spectral Data UV;

EtOH ~ max

265(log 6=2.61) and 410nm (log e=l.09).

IR~

(KBr) 1710 and 1690cm~. 13C N M R :

(25 MHZ, CDCI3): 13.4(qa), 14.7(qa), 19.9(qa), 20.9(qa), 23.7(qa), 24.9(d), 31.2(2t), 35.4(d), 36.5(t), 39.0(t), 43.6 (d), 49.0(t), 51.2(d), 52.0(d), 66.7(s), 124.9(2d), 135.4(s), 140.1(s), 176.2(s), 197.3(s), 202.3(s), and 208.7ppm (s). Mass Spectrum: 399(M+, C24HaaNO4, 19%), 314(18), 222(100), 194(49), and 177m/e (30). References W. yon Keller-Schierlein, and E. Kupfer; Stoffwechselprodukte yon Mikroorganismen: Uber die Aspochalasine A, B, C und D; Helvetica Chimica Acta, Vol. 62, Fasc. 5, pp. 1501-1524(1979). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

335

Common/Systematic Name Aspochalasin B (17R)- 17-Hydroxy- 10-isopropyl- 14-methyl[ 11]cytochalasa-6,13,19-triene- 1,18,21-trione Molecular Formula/Molecular Weight C24I-I33NO4;MW = 399.24096 Me

Me,,,,,,]~ Me Me 0

// 0

"OH

General Characteristics Pale yellow amorphous powder; [a]D 25 =118 ~ (c=1.37, in CHCI3). 17-O-Acetyl derivative: amorphous powder; [tt]Da5 -127 ~ (c=l.00, in CHCI3). Fungal Source Aspergillus microcysticus. Biological Activity Antibiotic. Spectral Data UV:

x max E~H

225nm (log e=3.99).

IR:

(CHCI3) 3425, 3200 br, 1740, 1690, and 1620cm"~. 1H N]VIR: (cDc13) 0.88(d, ,/=6, 3H); 0.90(d, J=6, 3H); 1.22(br, s, 3H); 1.24(d, J=6, 3H); 1.25-1.7(m, 2H); 1.80(d, J=l, 3H); 1.85-2.05(br, 2H); 2.08(s, 3H); 2.15-3.25(br, 7I~; 5.3-5.55(m, H-C(7) and H-C(17)); 6.37(br, d, J-11, H-C(13); 6.34 (d J-16, 1H); 6.58(br, s, HN); and 8.38ppm (d, dr=16, 1H). 13C NMR:

25 MHZ, (CDC13) 13.8(qa), 15.4(qa), 20.2(qa), 23.8(qa), 25.0(qa), 32.3(t), 34.8(d), 39.8(t), 41.5(d), 47.4(d), 48.3(0, 52.0(d), 69.2(s), 74.5(d), 124.5(d), 126.4(2 d), 136.0(s), 138.6(d) 141.5(s), 173.6(s), 195.3(s), and 204.9ppm (s).

336

3. Chaetoglobosins/Cytochalasins

Mass Data: 399(M+, 98%), 381(33), 371(16), 356(11), 342(15), 320(11), 222(36), 220(100), 57(35), 55(81), 43(86), and 41role (48). C2,H33NO4(399.58), found C, 72.15; H, 8.33; N 3.51% required C 71.96; H 8.38 N 3.35%. References W. von Keller-Schierlein, and E. Kupfer; Stoffwechselprodukte von Mikroorganismen: Ober die Aspochalasine A, B, C und D; Helvetica Chimica Acta, Vol. 62, Fasc. 5, pp. 1501-1524(1979). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

337

Common/Systematic Name Aspochalasin C (17S, 18R)-17,18-Dihydroxy- 10-isopropyl- 14-methyl[ 11]cytochalasa-6,13,19triene-1,21-dione (isomeric with aspochalasin D) Molecular Formula/Molecular Weight C24H35NO4; MW' = 401.25661 Me

Me,,,,,..~

Me

Me

O

/ OH

"OH

General Characteristics Colorless, amorphous powder; [a]D25 -86 ~ (C=1.37, in CHCI3). 17,18-O-Diacetyl derivative: Colorless crystals from benzene-hexane (3:1); rap., 113-114~ (dec.); [a]n -38 ~ (c=1.30, in CHC13).

Fungal Source

Aspergillus microcysticus.

Spectral Data UV:

~.

max

240nm (log e = 3.80).

IR:

17,18-O-Diacetyl derivative: (CHCI3) 3420, 3180 br, 1735, 1690, and 1630cm"l. 1H NMR:

17,18-O-Diacetylaspochalasin C: (CDCI3): 0.95(d J=6, 6H); 1.05-2.65(m, 8 I-I); 1.25(d d=6, 3 H); 1.49(d J=l, 3 H); 1.80(br, s, 3 H); 2.05(s, 3 H); 2.10(s, 3H); 2.85(br, d 3'--11, 1 H); 2.9-3.3(m, 2 H); 4.82(m, H-C(17)); 5.30[t, d=-8, H-C(18)]; 5.40(br, s 1 H); 6.02[br, dJ=l 1, H-C(13)]; 6.18[dxd dl=16, ,]2=8, H-C(19)]; 7.25(br, s H ~ ; and 7.45ppm [d J= 16, H-C(20)]. Mass Spectrum: 401(M+, C24H35NO4, 10~ 383(8), 365(4), 344(1), 326(2), 262(37), 205(11), 121(100), 57(18), and 55m/e (50).

338

3. Chaetoglobosins/Cytochalasins

References W. von Keller-Schierlein and E. Kupfer; Stoffwechselprodukte von Mikroorganismen: Ober die Aspochalasine A, B, C und D; Helvetica Chimica Acta, Vol. 62, Fasc. 5, pp. 1501-1524(1979). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

339

Common/Systematic Name Aspochalasin D (17S, 18S)-17,18-Dihydroxy- 10-isopropyl- 14-methyl[11]cytochalasa-6,13,19triene- 1,21-dione Diastereomer of aspoehalasin C (different configuration at C(I 7) or C(18). Molecular Formula/Molecular Weight C24I-I35NO4; M"W = 401.25661 Me

Me'"',,.i~ Me Me 0

/ OH

"OH

General Characteristics Colorless needles; mp, 148 oC; [a]D 25 -81 o (c= 1.43, in EtOH). 17,18-O-Diacetylaspochalasin D: Amorphous colorless powder; [aiD25 -43 ~ (C=1.05, in CHC13). Fungal Source

Aspergillus microcysticus.

Spectral Data UV~

~,m~xE~" 248nm (log e = 3.92). 1H NMR:

(CDC13) 17,18-O-diacetyl derivative: 0.88(d, J=6, 3H); 0.90(d, J=6, 3H); 1.1-1.8(m, 4H); 1.26(d, J=7, 3H); 1.33(br, s, 3H); 1.80(br s, 3H); 1.9-3.3(m, 7H); 2.04(s. 3H); 2.13(s, 3H); 4.79[br, H-C(17)]; 5.43[br, H-C(18)]; 5,72[br, H-C(7)]; 6.10[br, d, J=12, H-C(13)]; 6.20[dxd J1=17, J2=5, H-C(19)]; 7.1 l(br, s, HN); and 7.35ppm [dxd J1= 17, J2=1.5, H-C(20)]. 13C NMR:

(CDCI3) 17,18-O-diacetyl derivative: 13.5(qa), 15.6(qa), 19.9(qa), 20.9(2qa), 21.2(qa), 23.6(qa), 24.7(d), 28.0(t), 35. l(d), 38.7(0, 43.5(d), 48.5(t), 48.9(d), 51.4(d), 68.4(s), 73.8(d), 78.6(d), 125.2(2d), 130.8(d), 136.3(d), 136.3(s), 140.3(s), 169.5(2s), 174.8(s), and 196.7ppm (s). Mass Data: 401(M+, 2%), 383(2), 365(1), 326(2), 316(16), 262(37), 220(3), 178(24), 139(55),

340

3. Chaetoglobosins/Cytochalasins

124(69), 121(61), 97(99), 73(80), 69(98), 60(86), 57(69), 55(80), 43(100), and 41m/e (96); C24HasNO4(401.60); found C, 71.79; H, 8.79; N, 3.49%; required C, 71.65; H, 8.84; N, 3.44%. References W. yon Keller-Schierlein and E. Kupfer; Stoffwechselprodukte yon Mikroorganismen: Ober die Aspochalasine A, B, C und D; Helvetica Chimica Acta, Vol. 62, Fast. 5, pp. 1501-1524(1979). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexin.s.; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

341

Common/Systematic Name Cytochalasin E (6R,7S, 16S, 18R)-6,7-Epoxy- 18-hydroxy- 16,18-dimethyl- l 0-phenyl-21,23-dioxa[ 13]cytochalasa- 13(E), 19(E)-diene- 1,17,22-trione Molecular Formula/Molecular Weight C28H33NO7; MW' = 495.22570

_

Me,.,

Me, ....~

(,

O

_Me

oH;;

o

0

General Characteristics Crystals from acetone-hexane; mp, 206-208~ (dec.) [a]D25 -25.6 ~ (in MeOH); mp, 210~ (from acetone-n-hexane). Soluble in dichloromethane, chloroform, acetone, and methanol; sparingly soluble in water. It is unstable in chloroform solution and under acidic conditions. Fungal Source

Rosellinia necatrix and Aspergillus clavatus.

Biological Activity LDs0 in rats; oral 9. l mg/kg, IP 2.6mg/kg. Death was due to circulatory collapse caused by massive extravaseular effusion of plasma. The LDs0 values for a single IP dose of cytochalasin E were 1-day-old rats, 0.98mg/kg; adolescent rats, 2.60mg/kg; mice, 4.60mg/kg; and guinea pigs, 0.5-1.5mg/kg. In adolescent rats dosed orally, LDs0 was 1.3mg/kg. Histopathologic examination showed congestive degenerative changes, necrosis of liver, kidney, spleen, pancreas, and small intestine, brain edema, pulmonary hemorrhages, and injury to vascular walls. Cytochalasin E rarely produced nuclear extrusion typical of other cytochalasins; however, it produced a unique halo around the nuclei. Spectral Data, UV:

Weak absorptions between 240 and 300nm due in part to the n-n* transition of the monosubstituted aromatic ring. IR:

(CHC13) 3410, 2970, 1762, 1720, and 1662cm"].

342

3. Chaetoglobosins/Cytochalasins

CD: (MeOH) Ae325 O, Ae294 -2.6, Ae259 -0.58, Ae229 -5.14, and Ae218 0. ~3C NMR: C-I, 168.9 s; C-3, 52.1 d; C-4, 45.6 d; C-5, 35.5 d; C-6, 56.1 s; C-7, 58.8 d; C-8, 45.9 d; C-9, 86.9 s; C-10, 42.6 t; C-11, 12.4 q; C-12, 19.8 q; C-13, 127.6 d; C-14, 131.4 d; C-15, 38.5 t; C-16, 39.6 d; C-17, 211.4 s; C-18, 76.5 s; C-19, 121.0 d; C-20, 140.5 d; C-22, 148.8 s; C-24, 19.1 q; C-25, 24.8 q; C-I', 136.1 s; C-2', C-6', 130.1 d; C-3', C-5', 127.9 d; and C-4', 126.3ppm d. Mass Data: 495m/e (M+); found; C, 67.8; H, 6.85; N, 3.0%. C2gH33NOT;requires C, 67.86; H, 6.71; N, 2.83%. TLC Data Adsorbent: silica gel GF254; Solvent, acetone-hexane, 2:3, v/v; Rf, 0.25-0.30. Adsorbent: silica gel G-HR; Solvent, A: chloroform-acetone, 93:7, v/v. B: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Re, A: 0.10; B: 0.60. Detection: a dark purple fluorescent spot under UV light after spraying with 50% ethanolic H2SO4 and heating. References G. Bfichi, Y. Kitaura, S. Yuan, H. E. Wright, J. Clardy, A. L. Demain, T. Glinsukon, N. Hunt, and G. N. Wogan; Structure of Cytochalasin E, a Toxic Metabolite of Aspergillus clavatus; J. Am. Chem. Soc., Vol. 95, p. 5423-5425(1973). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p.266 (1981). T. Glinsukon, R. C. Shank, G. N. Wogan, and P. M. Newberne; Acute and Subacute Toxicity of Cytochalasin E in Rats; Toxicol Appl. Pharmacol., Vol. 32, p. 135-139 (1975). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoa!exins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991). P. S. Steyn, F. R. van Heerden, and C. J. Rabie; Cytochalasins E and K, Toxic Metabolites from Aspergillus clavatus; J. Chem. Soc. (London), Perkin Trans I, pp. 541-544(1982).

3. Chaetoglobosins/Cytochalasins

343

Common/Systematic Name Rosellichalasin 6,7-Epoxy- 10-phenyl-5,6,16,18-tetramethyl-22-oxa[ 12]cyclochalas- 13,19-diene- 17,21dione Molecular Formula/Molecular Weight C28H33NOs, M W = 463.23 587

Me,.. Me,,,

,o

(

~

"11

"I

O

DH ]

,L,

A~

~176 0

~e

.....~,,~'x,...-h -

0

,,)-=o

Me

General Characteristics Colorless needles from n-hexane-acetone; mp, 121-123 ~ MeOH).

[tt]D2~-2.7 ~ (C=I.0, in

Fungal Source Rosellinia necatrix, a plant pathogenic fungus that causes white root rot to several economically important crops. Isolation/Purification Mycelial mats were extracted with acetone and purified by a silica gel column with a solvent system of benzene-acetone. Rosellichalasin eluted with benzene-acetone (9:1, v/v) and was crystallized as colorless needles from n-hexane-acetone. Biological Activity Physiological effects in mammalian cells, microorganisms and plants. Spectral Data WW:

Z ~H

245nm (6=12,500).

IR:

(KBr) 1665 and 1640cm"l. ~H ~ : (CDCI3) l l-H, 1.00(d, J=7.3 Hz); 22-H, 1.13(d, J=6.8Hz); 12-H, 1.23(s); 23-H, 1.83(br, s); 5-I-I, 2.23(dq, J=5.4, 7.3 Hz); 15-H, 2.03(m), 2.24(m); 16-H, 3.27(m); 10-H, 2.85(21-1, d, J=5.4); 4-H, 2.72(dd, J=2.0, 5.4 Hz); 8-I-I, 2.86(dd, J=5.4, 10.3

344

3. ChaetoglobosinslCytochalasins

Hz); 3-H, 3.73(m); 7-H, 2.67(d, J=5.4 Hz); 19-H, 6.35(br, dd, J=6.8, 11.0 Hz); 13-H, 5.83(ddd, 1.5, 10.3, 15.1 Hz); 3', 5'-H, 7.28(m); 2',6'-H, 7.28(m); 14-1-1,5.44(ddd, 3.9, 11.2, 15.1 Hz); 20-H, 2.98(dd, 6.8, 12.1 Hz), 3.23(dd, 11.0, 12.1 Hz); and NH, 6.69ppm (br). 13C NMR: (CDCI3) C-11, 12.4(q); C-22, 17.4(q); C-12, 19.6(q); C-23, 12.8(q); C-5, 36.0(d), C-15, 36.6(0; C-16, 39.8(d); C-10, 44.1(d); C-4, 48.8(d); C-8, 47.0(d); C-3, 53.9(d); C-6, 57.2(s); C-7, 60.3(d); C-18, 143.1(s); C-9, 84.7(s); C-19, 135.1(d); C-4', 125.7(d); C-13, 127.1(d), C-3',5', 128.8(dd); C-2',6', 129.6(dd); C-14, 131.8(d); C-I', 136.6(s); C-20, 39.8(t); C-21, 171.8(s); C-l, 170.1(s); and C-17, 205.4ppm (s).

Mass Spectrum: ELMS: 463(M+), 372(M+- 91), 190(CI1H~2NO2+), 120(CsH~oN+),and 91m/e (base peak). References Y. Kimura, H. Nakajima, and T. Hamasaki; Structure of Rosellichalasin, a New Metabolite Produced by Rosellinia necatrix; Agric. Biol. Chem., Vol. 53, pp. 1699-1701 (1989). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

345

Common/Systematic Name Cytochalasin K~sp (7S, 16S, 18R)-7,8-Dihydroxy- 16,18-dimethyl- 10-phenyl-21,23-dioxa[ 13]cytochalasa5,13(E), 19(E)-triene- 1,17,22-trione The same trivial name was given to two different cytochalasms nearly simultaneously (see cytoehalasin K from Chalara microspora). Molecular Formula/Molecular Weight C28H33NO7, MW' -- 4 9 5 . 2 2 5 7 0

OH

Oh

' ~ 0 II

0

II

0

4'

I""~ I,.I

General Characteristics Cytochalasin K crystallized from hexane-acetone as white crystals; mp, 246-248 ~ Fungal Source Aspergillus clavatus (MRC 1181). The isolate was obtained from a sample of industrial sorghum malt. Isolation/Purification The culture material was extracted with chloroform-methanol (1:1, v:v). The crude extract was partitioned between 90% methanol and hexane, and the methanol extract was evaporated to dryness. The residue was partitioned between chloroform and water and the chloroform extract evaporated to dryness to give a toxic gum. This toxic material was purified by chromatography on silica gel using benzene-acetone (4:1, v:v) to remove lipids. The remaining material was purified by column chromatography on silica gel under pressure (150 kPa) using chloroform-methanol (95:5, v:v) as eluant to give cytochalasins E and K. Biological Activity Acutely toxic to ducklings and rats. Spectral Data UV: b MeOH max

263sh, 257, and 252nm sh (log e=3.44, 3.64, and 3.58).

346

3. Chaetoglobosins/Cytochalasins

IR~

(KBr) 3440, 2920, 1765, 1720, and 1662cmq. ~HNMR: (CDCIa) 7.2-7.04(m, Ph), 6.57(d, J=13 Hz, H-20), 6.21-6.05(m, H-13), 5.70(s, NH), 5.67(d, J=13Hz, n-19), 5.47-5.20(m, n-14), 4.39(s, OH), 3.87-3.76(m, H-4, H-7), 3.38(t, J=SHz, H-a), 3.0-2.50(m, H-8, n-10, H-15a, n-16), 2.08(m, H-15b), 1.61(s, Me), 1.47(s, Me), 1.44(s, Me), and 1.10ppm (d, ,/--6 Hz, 16-Me). Mass Data: 495role (M+);found: C, 67.2; H, 6.45; N, 2.65%. C2~LI33NO7;requires C, 67.86; H, 6.71; N, 2.83%. References S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991). P. S. Steyn, F. R. van Heerden, and C. J. Rabie; Cytochalasins E and K, Toxic Metabolites from Aspergillus clavatus; J. Chem. Soc. (London), Perkin Trans. I, pp. 541-544(1982).

3. Chaetoglobosins/Cytochalasins

347

Common/Systematic Name Deoxaphomin (7S, 16R,2OR)-7,20-Dihydroxy-5,16-dimethyl- 10-phenyl[ 13]cytochalasa6(12), 13(E),21 (E)-triene- 1,23-dione Molecular F ormul _.aJMolecular Weight C29H37NO4; M W -- 4 6 3 . 2 7 2 2 6

H CH2 OH

General Characteristics Amorphous solid; [~]D -50.8 ~ (C=0.178, in CHC13). Fungal Source Phoma exigua var. exigua and Ascochyta heteromorpha. Biological Activity Biological activity comparable to phomin: cytostatic activity (in vitro) of P-185 mastoeyte cells, EDs0 0.29mg/liter; in target cell destruction test treatments of 10, 1, and 0.1 t~g/liter gave inhibition of 100, 76, and 9%, respectively. Spectral Data UV:

Zm~" 217(e=14,100) and 234nm (8,700). (CH2C12) 3590, 3540, 3410, 3060, 3020, 1710, 1670, 1622, 1600, 1490, and 1020cm"l. ]H NMR:

H-2, 6.36; H-3, 2.81; H-4, 3.15; H-5, 3.44; H-7, 3.97; H-8, 2.50; H-10, 2.60; H-I I, 0.94; H-12, 5.09, 5.29; H-13, 6.18; H-14, 5.40; H-15-19, 1.0-2.0;H-20, 4.20; H-21, 6.77; H-22, 7.20; H-24, 0.98; H-2'-6',7.20; and OH, 3.10, 2.35ppm. LREIMS: 463(8%), 445(15), 372(30), 354(20), 174(18), and 91role (100).

References M. Binder and Ch. Tamm; Deoxaphomin, des erst [ 13]-Cytochalasin, ein m6glicher

348

3. Chaetoglobosins/Cytochalasins

biogenetischer VorL/iufer der 24-oxa-[14]-cytochalasane; Helv. Chim. Acta, Vol. 56, p. 966 (1973). R. Capasso, A. Evidente, G. Ramdazzo, A. Ritieni, A. Bottalico, M. Vurro, and A.Logrieco; Isolation of Cytochalasins A and B from Ascochyta heteromorpha; J. Nat., Prod., Vol. 50, p. 989 (1987). R. Capasso, A. Evidente, A. Ritiewi, G. Ramdazzo, M. Vurro, and A. Bottalico; Ascochalasin, a New Cytochalasin from Ascochytaheteromorpha;J. Nat., Prod., Vol. 51, p. 567-571(1988). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexin..s.; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

349

Common/Systematic Name Proxiphomin (16R)- 16-Methyl- 10-phenyl[ 13]cytochalasa-6(7), 13(E),21 (E)-triene- 1,23-dione Molecular Formula/Molecular Weight C29H37NO2; M W -- 431.28243

!

""l|ll

General Characteristics Amorphous; [a]D 24 -140 ~ (c=0.156, in CHCI3). Fungal Source Phoma exigua var. exigua. Spectral Data UV~

~

EIOH max

206(e=22,900), 217(12,300), and 243nm (10,700).

IR~

(CC14) 3420, 3200, 3080, 3060, 3020, 1700, 1670, 1610, and 1490em"l. 1H N M R :

H-2, 5.84; H-3, 3.24; H-4, 3.24; H-7, 5.44; H-10, 2.52, 2.85; H-I 1, 1.22; H-12, 1.80; H-13, 6.30; H-14, 5.24; H-15-18, 1.0-3.0; H-21, 6.80; H-22, 7.26; H-24, 0.95; and H-2'-6', 7.26ppm. Proxiphomin[ 5,6,16-T rimethyl- 10-phenyl[13]eytoehalasa-6(7), 13,21trien- 1,23-dione]. References M. Binder and Ch. Tamm; Proxiphomin und Protophomin, zwei neue eytochalasane; Helv. Chim. Acta, Vol. 56, p. 2387 (1973). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

350

3. Chaetoglobosins/Cytochalasins

Common/Systematic Name Protophomin (16R)- 16-Methyl- 10-phenyl[ 13]cytochalasa-5 (6), 13(E),21 (E)-triene- 1,7,23-trione Molecular Formula/Molecular Weight C29I-I35NO3; M W = 447.27734

O H

9 Ioo,,. 13 16 .

General Characteristics Crystals from acetone; mp, 252-256~

11111

[a]D 24 -112 ~ (c--0.42, in CHCI3).

Fungal Source

Phoma spp.

Soectral Data UV~

~

EtOH max

207(14,100) and 245nm (13,200).

IRz

(KBr) 3600-3200, 1700, 1680, 1660, 1610, and 1490cm~. ~H NMR: H-2, 6.53; H-3, 3.50; H-4, 3.71; H-8, 3.13; H-10, 2.53, 2.70; H-11, 1.53; H-12, 1.70; H-13, 6.24; H-14, 5.09; H-15-19, 1.0-3.0; H-21, 7.07; H-22, 6.73; H-24, 0.84; and H-2'-6', 7.24ppm. References M. Binder and Ch. Tamm; Proxiphomin und Protophomin, zwei neue cytochalasane; Helv. Chim. Acta, Vol. 56, p. 2387 (1973). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

351

Common/Systematic Name Ascochalasin (7S, 16R,20R)-7,20-Dihydroxy- 16-methyl- 10-phenyl[ 13]cytochalasa-6(12), 13(E)-diene1,23-dione Molecular Formula/Molecular Weight C29H39N04, M-W = 4 6 5 . 2 8 7 9 1 12

,,

CH2

HUH

Mel ...."~ O " H -

0

Me

0

General Characteristics Ascochalasin was an amorphous solid. Fungal Source

Ascochyta heteromorpha was isolated in 1985 from oleander (Nerium oleander) grown in a nursery near Bari, and deposited in the fungus collection of the Istituto Tossine e Micotossine da Parassiti Vegetali del CNR, Bari, Italy.

Isolation/Purification The culture filtrates were lyophilized, resuspended in distilled H20 and extracted with chloroform. The organic extracts were combined, dried (Na2SO4) and evaporated under reduced pressure. The residue was chromatographed on a SiO~ column eluted with chloroform- isopropyl alcohol (9:1, v/v). The first fraction yielded crude cytochalasin A; the successive eluate comained small amounts of cytochalasin A, cytochalasin B and a mixture of products. This mixture was further purified on SiO2 column eluted with chloroform- isopropyl alcohol (93:7, v/v). Crude cytochalasin B was obtained and a fraction consisting of small amounts of cytochalasin B and a mixture of two products with lower Re than B. The latter was further purified on SiO2 plates with chloroform-isopropyl alcohol (93:7, v/v) affording crude cytochalasin B and a crude mixture of two products. The mixture was purified on reversed-phase plates (CH3CN-H~O, 6:4, v/v), giving pure ascochalasin and deoxaphomin. .Spectral Data UV:

3.~, <220nm.

352

3. Chaetoglobosins/Cytochalasins

IR~

3580-3500, 3400, 1715, and 1605cm"1. IH NMR: H-3, 3.30(ddd, J3,4=3.3Hz, J3a0=5.5Hz, J3.w=3.3Hz); H-4, 3.14(dd, J4,5=5.9Hz); H-5, 2.88(dq, Js, ll=6.6Hz); H-7, 3.94(d, JT,s=10.3Hz); H-8, 2.48(dd, Js,13=9.6Hz); H-10, 2.74(dd, J10,10~13.6Hz); H-10', 2.50(dd); 3H-11, 1.06(d; H-12, 5.32, brs); H-12', 5.12(brs); H-13, 6.20(ddd, J~3,~4=15.4Hz,J~3,~5=l.8Hz); H-14, 5.42(ddd, J~4,~5=9.6Hz, J~4,lS~3.3Hz); H-15, 2.15(ddd, J~5,~5~-12.3Hz);2H-17, 0.85-2.00; H-20, 3.30(m); H21, 0.85-2.00; H-22, 0.85-2.00; Me-C-16, 0.94; and H-3',5', 7.20ppm. 13C NMR: C-l, 164.1, s; C-3, 53.2, d; C-4, 45.8, d; C-5, 31.9, d; C-6, 143.9, s; C-7, 70.6, d; C-8, 50.7, d; C-9, 65.7, s; C-10, 44.3, t; C-I 1, 13.6, q; C-12, 114.1, t; C-13, 127.4, d; C-14, 137.4, d; C-15, 29.6, d; C-16, 33.8, d; C-17,34.7, t; C-18, 25.2, t; C-19, 41.5, t; C-20, 70.3, d; C-21,39.2, t; C-22, 29.6, t; C-23, 191.6, s; 16Me, 22.2, q; C-I', 129.3, s; C-2', 129.1, d; C-3', 128.9, d; C-4', 127.0, d; C-5', 128.9, d; and C-6', 129.1ppm, d. Mass Spectrum: HREIMS: 465.2873 (M+); calcd for C29H39NO4(465.2869). Other significant peaks were detected at 463.2715 (C29HaTNO4), likely formed by loss of H2 in the source from the molecular ion (465.2873); 372.2191 (C22H3oNO4), and 354.2075(C~H28NO4) originated from the ion at 463.2715 by loss of a benzyl group and 1-120, respectively; the ion at m/z 270.1496(CITH20NO2) arose from the fragment at 354.2075 by losses of 1-120 and a cyclopentadienyl moiety. This fragmentation pattern was coincident with that of deoxaphomin except for the loss of two hydrogens. References R. Capasso, A. Evidente, A. Ritieni, G. Randazzo, M. Vurro, and A. Bottalico; Ascochalasin, A New Cytochalasin from Ascochyta heteromorpha; J. Nat. Prod., Vol. 51, pp. 567-571(1988). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoale.xins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

3. Chaetoglobosins/Cytochalasins

353

Common/Systematic Name Cytochalasin K c ~ (6R,7S, 16S, 19S)- 19-Acetoxy-6,7-epoxy- 16,18-dimethyl- 10-phenyl[ 13 ]cytochalasa13(E), 17(E),21 (E)-triene- 1,20,23-trione The same trivial name was given to two different cytochalasms nearly simultaneously. Molecular Formula/Molecular Weight C32H37NO6, M W = 531.26209

Me,,.

0

i,

I

....'Me "Me 0

OAc

General Characteristics Cytochalasin K was amorphous; [t~]D25 - 177 ~ (EtOH). Fungal Source Chalara microspora. Isolation/Purification Extracted with EtOAc and isolated using reversed-phase chromatography. Biological Activity Biological activity not reported but assumed to be similar to the other cytoehalasins. Spectral Data I.J-V:

/~^~. E~o. max

232nm (log e=l 1,300).

IR: (KBr) 3380(OH), 1750, and 1700cm 1 (broad, C=O). IH NMR:

(CDr NI-I,5.85;H-3, 3.66(J3.,o:5.5,J3.lOb=8.0,J3-~=2.SI-Iz);H-4, 2.99(A.5=5.5); H-5, 1.83(J~.,:6.5);H-7, 2.83(J7_~=5.0);H-S, 2.21(J~.:I0.0); H-10a, 2.71(J10a.10b=13.5);H-10b, 2.53; 3H-I I, 1.03;3H-12, 1.28;H-13, 6.16(J13.14=15.5); H-14, 5.27(J14_lSa=10.2,Jl4.1Sb=3.5);H-15a, 2.07(J1sa.lSb=13.5),H-15b, 2.32; H-16,

354

3. Chaetoglobosins/Cytochalasins

2.5(,/~6.16M==7.3,J16.~7=9.0);H-17, 5.73; H-19, 5.94; H-21, 7.66(,/21.22=15.5);H-22, 6.71; 16CH3, 1.09; 18CH3, 1.50; -OOCCH3, 2.18; and H-2', H-3 ~and H-4', 7.1-7.4Hz. ~3CNMR: (CDCI3) C-I, 172.9; C-3, 53.7; C-4, 48.0a; C-5, 32.3b; C-6, 58.0; C-7, 62.2; C-8, 46.4a; C-9, 63.0; C-10, 44.3; C-11, 13.1c; C-12, 19.8d; C-13, 128.1e; C-14, 134.1f, C-15, 41.2; C-16, 36.2b; C-17, 133.6f; C-18, 128.1e; C-19, 83.3; C-20, 194.59; C-21, 142.4; C-22, 134.9f, C-23, 196.99; 16-CH3, 11.5c; 18-CH3, 20.8d;-OOCCH3, 169.8; -OOCCH3, 20.8d; C-I', 136.2; C-2', 129.4; C-3', 128.9; and C-4', 127.1ppm e. (a-f: May be interchanged.). Mass Spectrum: HR S: 531.2660m/e, C32H37NO6;requires 531.2621. References T. Fex; Structures of Cytochalasin K, L and M, Isolated from Chalara microspora; Tet. Lett., Vol. 22, pp. 2703-2706(1981). S. Natori and I. Yahara; Cytochalasins; In Mycotoxins and Phytoalexins; R. P. Sharma, and D. K. Salunkhe (eds.); CRC Press, pp. 291-338(1991).

Aflavinines and Related Indoles

Aflavinine Dihydroxyaflavinine(20,25-Dihydroxyaflavinine) 20-Hydroxyaflavinine 24,25-Dehydro-10,11-dihydro-20-hydroxyaflavinine 10,11-Dihydro-11,12-dihydro-20-hydroxyaflavinine 14-epi- 14-Hydroxy-10,23-dihydro-24,25-dehydroaflavinine 10,23-Dihydro-24,25-dehydroaflavinine 10,23-Dihydro-24,25-dehydro-21-oxoaflavinine Aflavazole

355

This Page Intentionally Left Blank

4. Aflavinines and Related Indoles

357

Common/Systematic Name Aflavinine Molecular Formula/Molecular Weight C28H39NO; M W = 4 0 5 . 3 0 3 1 7 28

-..

14 12~

17

27 , , ' " " ~

"'OH

Me General Characteristics Aflavinine was obtained as colorless needles from EtOAc; mp., 102-104~ (c=0.98, in CHCI3).

[a]D

+24.9 ~

Fungal Source Aspergillus flavus and A. tubingensis. Isolation/Purification Extracted with CHCI3; chromatographed on silica gel with CHCl3-cyclohexane (1:9, v/v) as eluant. Biological Activity Antiinsectan activity. Spectral Data UV: k MeOH max

291(e=2710), 283(3010), and 225nm (18,700).

1H NMR: (CDCI3) H-I, 8.02(br, s); H-2, 6.88(d, J=2.2); H-5, 7.42(br d, J=7.8); H-6, 7.08(rid, J=7.1, 7.8); H-7, 7.17(dd, J=7.1, 8.1); H-8, 7.36(br, d, J=8.1); H-11, 2.43(br, d, J=5.1); H-12, 1.96(m); H-13, 1.14(m), 1.64(m); H-14, 1.18(m), 1.55(m); H-16, 2.03(m); H-17, 1.23(m), 1.69(m); H-18, 1.75(m), 2.06(m); H-19, 4.46(br, s); H-21, 1.73(m), 1.85(m); H-22, 2.10(m), 2.22(m); H-24, (septet, J= 7.0); H-25, 0.82(d, J=7.1); H-26, 0.96(d, J=6.8); H-27, 1.08(d, J=7.1); H-28, 0.75(d, J=6.8); and H-29, 0.98ppm (s).

358

4.

Aflavinines and Related Indoles

13C NMR: (CDC13) C-2, 121.2; C-3, 118.6; C-4, 127.6; C-5, 119.6; C-6, 118.6; C-7, 121.8; C-8, 111.0; C-9, 135.9; C-10, 125.5; C-11, 43.7; C-12, 31.3; C-13, 25.7; C-14, 27.6; C-15, 38.5; C-16, 29.1; C-17,25.4; C-18, 21.8; C-19, 71.1; C-20, 42.4; C-21, 30.1; C-22, 20.5; C-23, 141.0; C-24, 31.0; C-25, 21.9; C-26, 20.8; C-27, 18.1; C-28, 15.7; and C-29, 18.1ppm.

References R. T. Gallagher, T. McCabe, K. Hirotsu, and J. Clardy; Aflavinine, a Novel Indole-mevalonate Metabolite from Tremorgen-producingAspergillusflavus Species; Tet. Lett., Vol. 21, pp. 243-246(1980). M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Three New Aflavinines from The Sclerotia ofAspergillus tubingensis; Tetrahedron, Vol. 45, pp. 4961-4968(1989).

4.

Aflavinines and Related Indoles

359

Common/Systematic Name Dihydroxyaflavinine; 20,25-Dihydroxyaflavinine Molecular Formula/Molecular Weight C25H39NO3; M W = 4 3 7 . 2 9 2 9 9

OH

27

20

28 t," 29 ""'"

NH

H

25

General Characteristics Crystals suitable for X-ray diffraction experiments formed from methanol with symmetry P2~ with a = 9.448; colorless plates from ethyl acetate; mp., 254-256"C; [a]D +22.9* (C=0.50, in MeOH). Fungal Source

Aspergillusflavus (NRRL 3251) and A. flavus (NRRL 6541).

Isolation/Purification The cultures were extracted with hot chloroform; the crude chloroform extract was chromatographed on a silica gel column eluted with benzene, ethyl ether, ethyl acetate, and acetone. Dihydroxyaflavinine crystallized from the ethyl acetate fraction as colorless plates. Hexane and CHCI3 extracts of milled sclerotia were subjected to reversed phase flash chromatography followed by reversed-phase HPLC [5-ktm C]8 column, MeOH-H20 (70:30, v/v), 2.0mL/min, monitored at 215nm] to afford 20,25-dihydroxyaflavinine, 20-hydroxyaflavinine, 24,25-dehydro- 10,11-dihydro-20-hydroxyaflavinine, and 10,11-dihydro- 11,12-dehydro-20-hydroxyaflavinine. Retention time for 20,25-dihydroxyaflavinine, 14.4min. Biological Activity Antiirisectan but not tremorgenic. Antifeeding activity against Carpophilus hemiptaerus. Spectral Data UV;

~, M~, 225(r 282-284(7,400), and 290nm (6,000), was typical of a 3-substituted indole chromophore.

360

4.

Aflavinines and Related Indoles

IR:

The IR spectrum showed absorptions indicating the presence of OH and indole groups (3250, 3450, and 1035cm~), gem-dimethyl groups (doublet 1370, 1380cm't), and four adjacent aromatic hydrogens (745cm1). ~H NMR: The 100-MHZ ~H NMR spectrum showed three doublets (3"=7Hz) at 0.83, 0.97, and 1.15ppm and a singlet at 1.22ppm for the four methyl groups. In addition to multiplets at 1.2-2.7 and 3.2-4.0ppm, other peaks were observed at 4.3(br s), 6.7-7.2(indole ring protons), and 8.9ppm(N-H). H-2, 6.91(s); H-5, 7.44(br d, 3'=7.8); H-6, 7.03(dd, ,/=7.8, 6.4); H-7, 7.14(dd, ,/=8.0, 6.4); H-8, 7.40(br d, 3"=8.0); H-12, 2.34(m); 2.16(m); n-13, 2.02(m); 1.75(m); H-15, 4.54(br s); n-16, 2.1 l(m); 1.78(m); n-17, 1.86(m); 1.09(m); n-18, 2.09(m); n-20, 3.94(dd, 3"=10.0, 2.0); H-21, 1.91(m); 1.42(dd, 3'=12, 2); H-22, 2.21(m); H-23, 2.56(br d, J=6.0); H-24, 2.61(br q, 3'=7.0); H-25, 3.51(dd, 3'=11, 7); H-26, 0.81(d, J=7.0); H-27, 0.95(d, ,/=7.0); H-28, 1.19(s); and H-29, 1.15ppm (d, 3"=8.0). 13C NM~:

The ~3CNMR spectrum showed 10 peaks for the sp2 carbons at 137.0(s), 135.8(s), 127.8(s), 126.9(s), 122.1(d), 120.6(d), 118.8(d), 118.3(d), 116.5(s), and 111. lppm (d). The hydroxy-bearing carbons appear at 70.5(d), 68.9(d), and 65.7ppm (t). The remainder of the aliphatic carbons have absorptions at 44.2(s), 43.1 (s), 38.9, 35.1 (t), 31.1(d), 30.3(t), 29.6(d), 27.3(t), 21.8(t), 21.5(t), 19.4(q), 19.2(q), 15.4(q), and 13.1ppm (q). (CD3OD): 138.5(s), 137.7(s), 129.9(s), 128.4(s), 123.2(d), 122.2(d), 120.0(d), 119.6(d), 118.0(s), 112.2(d), 72.6(d), 71.0(d), 67.3(t), 49.9(d), 45.9(s), 44.7(s), 40.4(d), 35.9(t), 32.5(d), 31.3(t), 31.2(d), 28.6(t), 23.1(t), 22.8(t), 20.1(q), 19.6(q), 15.6(q), and 13.6ppm (q). Mass Spectrum: The high-resolution mass spectrum gave a molecular ion peak at 437.2909m/e corresponding to a molecular formula of C28H39NO3(calcd 437.29297m/e). In addition to the molecular ion peak in the low-resolution mass spectrum, peaks were observed at 419(M-H20), 401(M-2H20), 338, and 130m/e. EIMS: 437(M +, rel. int. 12.5%), 419(3.5), 328(3.0), 302(5.4), 288(5.4), 260(18), 234(31), 218(44), 194(47), 180(45), 167(49), 154(30), 144(40), 130(100), and 117(85); HREIMS: obsd 437.2931; calcd for C2sH39NO3 437.2936. TLC Data Purified dihydroxyaflavinine was a reddish-purple spot on silica gel 60 F254at Rf=0.44 [toluene-ethyl acetate-formic acid (5:4:1, v/v/v)].

4.

Aflavinines and Related Indoles

361

References R. J. Cole, J. W. Domer, J. P. Springer, and R. H. Cox; Indole Metabolites from a Strain ofAspergillusflavus; J. Agric. & Food Chem., Vol. 29, pp. 293-295(1981). J. B. Gloer, M. R. TePaske, J. S. Sima, D. T. Wicklow, and P. F. Dowd; Antiinsectan Aflavinine Derivatives from the Sclerotia ofAspergillusflavus; J. Organ. Chem., Vol. 53., pp. 5457-5460(1988).

362

4.

Aflavinines and Related Indoles

Common/Systematic Name 20-Hydroxyaflavinine Molecular Formula/Molecular Weight C28H39NO2; M W = 4 2 1 . 2 9 8 0 8

OH

27

20

29'..... '~,~ ~'"'OH 25

General Characteristics Melting point 174-176~ dec; [a]D +23.8 ~ (c=0.56, in MeOH). Fungal Source

Aspergillusflavus (NRRL 6541).

Isolation/Purification Hexane and CHCI3 extracts of milled sclerotia were subjected to reversed-phase flash chromatography followed by reversed-phase HPLC [ 5-I,tm C18 column, MeOH-H20 (70:30, v/v), 2.0mL/min, monitored at 215nm] to afford 20,25-dihydroxyaflavinine, 20-hydroxyaflavinine, 24,25-dehydro- 10,11-dihydro-20-hydroxyaflavinine, and 10,11-dihydro-11,12-dehydro-20-hydroxyaflavinine. Retention time for 20-hydroxyaflavinine, 34.2min. Biological Activity Antifeeding activity against Carpophilushemiptaerus. Spectral Data UV: M,~. 224(e=14,920), 283(2,760), and 290nm (2,520). :H NMR:

H-2, 6.89(s); H-5, 7.44(br d, J=7.8); H-6, 7.08(dd, J=7.8, 6.3); H-7, 7.18(dd, J=8.3, 6.3); H-8, 7.36(br d, J=8.3); H-12, 2.3 l(m); 2.21(m); H-13, 1.99(m); 1.67(m); H-15, 4.44(br s), H-16, 2.04(m); 1.63(m); H-17, 1.79(m); 1.1 l(m); H-18, 2.12(m), H-Z0, 3.99(dd, J=12.8, 2.7); H-Z1, 1.95(m); 1.46(m); H-22, 2.10(m), H-23, 2.47(br d,

4.

Aflavinines and Related Indoles

363

J=5.5); H-24, 2.61(septet, J=6.8); H-25, 0.82(d, J=6.8); H-26, 0.96(d, J---6.8); H-27, 0.99(d, J=6.6); H-28, 1.24(s); and H-29, 1.16ppm (d, ,/=7.6).

13CNMR.: (CD3OD) 141.8(s),137.7(s),128.5(s),127.2(s),122.8(d),122.1(d),120.0(d), 119.5(d),112.2(d),112.0(s),72.6(d),71.0(d),49.8(d),46.0(s),44.7(s),36.0(t), 32.5(d), 32.0(d), 31.5(t), 31.2(d), 28.6(t), 23.2(t), 22.1(t), 21.5(q), 21.1(q), 19.9(q), 19.6(q), and 13.6ppm (q). Mass Spectrum: EIMS: 421(M +, 20), 403(2.0), 378(3.1), 360(3.1), 342(4.3), 288(4.4), 234(12), 220(26), 206(22), 194(35), 180(28), 168(62), 154(25), 144(26), 130(100), and 117m/e(50); HR IMS: obsd 421.2981, calcd for C25H39NO2,421.2980. Reference J. B. Gloer, M. R. TePaske, J. S. Sima, D. T. Wicklow, and P. F. Dowd; Antiinsectan Aflavinine Derivatives from the Sclerotia ofAspergillusflavus; J. Organ. Chem., Vol. 53, pp. 5457-5460(1988).

364

4.

A f l a v i n i n e s and R e l a t e d I n d o l e s

Common/Systematic Name 24,25-Dehydro- 10,11-dihydro-20-hydroxyaflavinine Molecular Formula/Molecular Weight C28H39NO2;

MW

= 421.29808

OH

27 .%

20

"

17

15 29 I"'"'

NH

H

25

General Characteristics Melting point 259-262~ dec; [(t]D+0.9 ~ (C=0.34, in MeOH). Fungal Source

Aspergillusflavus (NRRL 6541).

Isolation/Purification Hexane and CHCI3 extracts of milled sclerotia were subjected to reversed-phase flash chromatography followed by reversed-phase HPLC [5-ktm C~g column, MeOH-H20 (70:30, v/v), 2.0mL/min, monitored at 215nm] to afford 20,25-dihydroxyaflavinine, 20-hydroxyaflavinine, 24,25-dehydro- 10,11-dihydro-20-hydroxyaflavinine and 10,11-dihydro- 11,12-dehydro-20-hydroxyaflavinine. Retention time for 24,25-dehydro- 10,11-dihydro-20-hydroxyaflavinine, 25.8 min. Biological Activity Antifeeding activity against Carpophilushemiptaerus. Spectral Data UV: McOH ~ max

227(~=26,260), 284(6,060), and 291nm (5,720).

~HNMR: H-2, 7.06(s); H-5, 7.46(br d, J-7.8); H-6, 6.98(dd, J=7.8, 6.3); H-7, 7.06(dd, ,/-8.1, 6.3); H-8, 7.30(br d, J=8.1); H-10, 3.66(dd, J-13, 6); H-11, 3.22(ddd, ,]-13, 12, 6); H-12, 1.95(m); 1.65(m); H-13, 2.17(m); 1.78(m); H-15, 4.64(br s); n-16, 1.96(m); 1.17(m); H-17, 1.85(m); 1.27(m); H-18, 2.23(m); H-20, 3.92(dd, ,]--12.9, 2.7); H-21,

4.

Aflavinines and Related Indoles

365

1.95(ddd, 3--13, 12, 5.5); 1.21(m); H-22, 1.55(m); H-23, 2.67(dd, 3--5, 6); H-25, 4.81(br s); H-26, 1.50(br s); H-27, 1.03,(d, 3=6.6); H-28, 1.28(s); and H-29, 1.32ppm (d, J-7.3). 13C NMP~:

(CD3OD) 151.7(s), 137.7(s), 128.6(s), 124.6(d), 122.0(d), 119.3(d), 118.7(d), 116.2(s), 112.3(d), 111.6(t), 72.6(d), 69.1(d), 47.4(s), 45.5(s), 44.8(d), 39.5(d), 38.9(t), 35.7(d), 32.6(d), 32.3(d), 31.1(t), 29.2(t), 28.5(t), 25.8(t), 23.1(q), 19.7(q), 18.4(q), and 13.8ppm (q). Mass Spectrum: EIMS: 421(M+; 23), 406(1.5), 403(1.7), 388(1.0), 330(4.3), 302(2.4), 288(1.2), 248(2.0), 220(4.0), 210(10), 196(28), 184(14), 168(28), 143(13), 130(100), and 117m/e (16); HREIMS: obsd 421.2975; calcd for C25H39NO2,421.2980. Reference J. B. Gloer, M. R. TePaske, J. S. Sima, D. T. Wicklow, and P. F. Dowd; Antiinsectan Aflavinine Derivatives from the Sclerotia ofAspergillusflavus; J. Organ. Chem., Vol. 53, pp. 5457-5460(1988).

366

4.

Aflavinines and Related Indoles

Common/Systematic Name 10,11-Dihydro- 11,12-dehydro-20-hydroxyaflavinine Molecular Formula/Molecular Weight C28H39NO2;MW = 421.29808

~H

ON _27 20

29 =''''''

-

17

N. aZ 25

General Characteristics Melting point 276-278~C dec; [~7']D +1.7~ (c-0.10, in MeOH). Fungal Source

Aspergillusflavus (NRRL 6541).

Isolation/Purification Hexane and CHCI3 extracts of milled sclerotia were subjected to reversed-phase flash chromatography followed by reversed-phase HPLC [5-:m Cls column, MeOH-H20 (70:30, v/v), 2.0mL/min, monitored at 215nm] to afford 20,25-dihydroxyaflavinine, 20-hydroxyaflavinine, 24,25-dehydro-10,11-dihydro-20-hydroxyaflavinine, and 10,11-dihydro-11,12-dehydro-20-hydroxyaflavinine. Retention time for 10,11-dihydro- 11,12-dehydro-20-hydroxyaflavinine, 35.5 min. Biological Activity Antifeeding activity against Carpophilus hemiptaerus. Spectral Data UV-

MeOH

8~

224(,=20,030), 251 (3,600), and 285nm (3,380).

1H NMR: H-2, 7.00(s); H-5, 7.46(br d, J=7.8); H-6, 7.02(dd, ,/=7.8, 6.1); H-7, 7.09(dd, J=8.0, 6.1); H-8, 7.36(br d, J=8.0); H-10, 4.17(d, J=5); H-12, 5.66(br d, J=5.6); H-13, 2.63(dd, J= 13.5, 5.6); 2.29(m); H- 15, 4.29(br s); H- 16, 2.19(m); 1.80(m); H- 17, 1.81(m); 1.26(m); H-18, 2.22(m); H-20, 3.95(dd, J=-12.8, 3.0); H-21, 1.78(m); 1.32(m); H-22, 1.49(m); H-23, 2.53(dd, J=5.3, 5); H-24, 2.71 (septet, J=6.8); H-25, 1.10(d, J=6.8); H-26, 0.72(d, J=6.8); H-27, 1.02,(d, J=-6.6); H-28, 1.24(s); and H-29, 1.14ppm (d, J=7.1).

4.

Aflavinines and Related Indoles

367

13CNMR: (CD3OD) 144.7(s), 137.5(s), 129.5(s),124.5(d), 122.2(d), 121.6(d), 120.0(s),119.6(d), 118.3(d), 112.3(d), 73.1(d), 70.7(d), 46.1(s), 44.9(s), 40.1(d), 37.9(t), 37.1(d), 31.5(d), 31.4(d), 30.9(t), 30.2(d), 29.0(0, 28.7(t), 23.4(q), 21. l(q), 19.7(q), 17.9(q), and 13.6ppm (q). Mass Spectrum: ELMS: 421(M+, 2.8), 403(0.6), 388(0.8), 342(1.6), 274(3.1), 260(4.4), 246(7.7), 232(8.9), 220(14), 210(24), 196(30), 180(32), 168(100), 143(32), 130(95), and 117m/e (81); HREIMS: obsd 421.2990; calcd for C25H39NO2,421.2980. Reference J. B. Gloer, M. R. TePaske, J. S. Sima, D. T. Wicklow, and P. F. Dowd; Antiinsectan Aflavinine Derivatives from the Sclerotia ofAspergillusflavus; J. Organ. Chem., Vol. 53, pp. 5457-5460(1988).

368

4.

Aflavinines and Related Indoles

Common/Systematic Name 14-epi- 14-Hydroxy- 10,23-dihydro-24,25-dehydroaflavinine Molecular Formula/Molecular Weight

C28H39NO2;MW = 421.29808

OH~8

14-

27 I " ' " ' ~ ~

-

~7

'''iON

102~H2 2

~J~NH~j7 General Characteristics Melting point, 79-82~

25

[a]D -5.6 ~ (c=1.06, in CHCI3).

Fungal Source

Aspergillus tubingensis (NRRL 4700), A. flavus, and A. parasiticus.

Isolation/Purification Sclerotia were ground to a powder with a mortar and pestle and extracted with hexane. Filtration and evaporation of solvent afforded a yellow oil. Reversed-phase HPLC of the hexane extract using an Altex reversed-phase semipreparative column (C]8-5l.tm particle size; UV detection at 215nm; 20ml/min; 90:10, v/v, MeOH-H20) afforded pure 14-epi- 14-hydroxy- 10,23-dihydro-24,25-dehydroaflavinine along with fractions containing 10,23-dihydro-24,25-dehydroaflavinine and 10,23-dihydro-24,25-dehydro-21-oxoaflavinine. 10,23-Dihydro-24,25-dehydroaflavinine was isolated by reversed-phase HPLC using a solvent composition of [MeOH-H20 (85:15, v/v); 2.5ml/min], and 10,23-dihydro24,25-dehydro-21-oxoaflavinine was isolated by further chromatography on a 10-~m Hamilton PRP-I column (100% MeOH, v/v, 1.0ml/min). Biological Activity No antiinsectan activity; mild antibacterial activity against Bacillus subtilis at 1001,tg/disk. Spectral Data

UV: ~

MeOH max

292(e=2,100), 284 (2,290), and 226nm (16,400).

4. Aflavinines and Related Indoles

369

1H NMR: H-I, 8.04(br, s); H-2, 7.08(br s); H-5, 7.52(br, d, J=7.6); H-6, 7.1 l(dd, J=7.6, 6.8); H-7, 7.16(dd, J=8.1, 6.8); H-8, 7.32(d, J=8.1); H-10(dd, J=5.9, 12.9); H-11, 2.73(dd, J=5.9, 5.8); H-12, 1.42(m); H-13, 0.85(m, 1.68, m); H-14, 4.740r, s); H-16, 2.18(m); H-17, 1.19(m, 1.68, m); H-18, 1.38(m, 1.78, m); H-19, 4.73(br, m); H-21, 1.74(m, 2.15, m); H-22, 1.62(m, 2.31, m); H-23, 3.38(ddd, J=4.6, 13, 13); H-25, 4.77, 4.860ar, s); H-26, 1.44(s); H-27, 1.29(d, J=5.8); H-28, 0.78(d, J=5.8); and H-29, 1.02ppm (s). 13CNMR: C-2, 123.4; C-3, 115.9; C-4, 127.5; C-5, 118.3; C-6, 119.0; C-7, 121.6; C-8, 111.1; C-9, 135.7; C-10, 34.8; C-11, 38.0; C-12, 29.3; C-13, 29.9; C-14, 71.7; C-15, 39.3; C-16, 32.2; C-17, 28.5; C-18, 31.4; C-19, 68.8; C-20, 46.9; C-21, 24.9; C-22, 30.9; C-23, 39.5; C-24, 150.0; C-25, 111.4; C-26, 18.1; C-27, 18.1; C-28, 15.9; and C-29, 17.9ppm. Mass Spectrum: 421(M+40%), 388(0.3), 334(7.2), 306(6.9), 243(3.3), 226(5.1), 196(14), 182(45), 168(43), 159(16), 146(18), 130(100), and 117m/e (14); HREIMS: obs. 421.2962; calcd, for C2sH39NO2421.2980. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Three New Aflavinines From The Sclerotia ofAspergillus tubingensis; Tetrahedron, Vol. 45, pp. 4961-4968 (1989).

370

4.

Aflavinines and Related Indoles

Common/Systematic Name l 0~23-Dihydro-24,25-dehydroaflavinine Molecular Formula/Molecular Weight C2sHagNO; MW = 405.30317 28 14

''

27

I'"""

(.~NH~ 2 ~ H 8

General Characteristics Melting point 192-194~

'",l H 25

[~]D -1.2 ~ (C=0.5, in CHCI3).

Fungal Source

Aspergillus tubingensis (NRRL 4700), A. flavus and A. parasiticus.

Isolation/Purification Sclerotia were ground to a powder with a mortar and pestle and extracted with hexane. Filtration and evaporation of solvent afforded a yellow oil. Reversed-phase HPLC of the hexane extract using an Altex reversed phase semipreparative column [C~r5l.tm particle size; UV detection at 215nm; 20ml/min; MeOH-H20 (90:10, v/v)] afforded pure 14-epi- 14-hydroxy- 10,23-dihydro-24,25-dehydroaflavinine along with fractions containing 10,23-dihydro-24,25-dehydroaflavinine and 10, 23-dihydro-24,25-dehydro-21oxoaflavinine. 10,23-Dihydro-24,25-dehydroaflavinine was isolated by reversed-phase HPLC using a solvent composition of [MeOH-H20 (85:15, v/v); 2.5ml/min] and 10,23-dihydro-24,25-dehydro-21-oxoaflavinine was isolated by further chromatography on a 10-I,tm Hamilton PRP-I column (100% MeOH 1.0ml/min). Biological Activity No antiinsectan activity; mild antibacterial activity against Bacillus subtilis at 100l.tg/disk. Spectral Data UV: ~,,~.

291 (e--4,100), 284(4,400), and 226nm (31,600).

IH N/VIR: H-l, 7.89, br, s; H-2, 7.02, d, J=2.2; H-5, 7.52, br, d, J=7.8; H-6,7.09, dd, ,/=6.8, 7.8; H-7, 7.16, dd, J=6.8, 8.1; H-8, 7.32, d, J=8.1 H-10, 3.68, dd, J=5.1, 12.9; H-11, 2.62,

4.

Aflavinines and Related Indoles

371

m; H-12, 1.45, m; H-13, 1.16, m, 1.54, m; H-14, 0.86, m, 1.63, m; H-16, 2.18, m; H-17, 1.36, m, 1.75, m; H-18, 1.86, m, 2.21, m; H-19, 4.84, br, s; H-21, 1.88, m, 2.04, m; H-22, 1.75, m, 1.84, m; H-23, 3.19, m; H-25, 4.64, 4.78, br, s; H-26, 1.51, s; H-27, 1.24, d, J=7.1; H-28, 0.77, d, J=6.8; and H-29, 0.97ppm, s. 13C NMR: C-2, 122.8; C-3, 116.7; C-4, 127.4; C-5, 118.4; C-6, 119.0; C-7, 121.6; C-8, 111.1; C-9, 136.0; C-10, 34.6; C-11, 43.6; C-12, 29.8; C-13, 27.9; C-14, 25.3; C-15, 39.3; C-16, 31.4; C-17, 28.2; C-18, 28.9; C-19, 69.0; C-20, 44.0; C-21, 24.6; C-22, 30.1; C-23, 38.7; C-24, 150.3; C-25, 111.0; C-26, 18.4; C-27, 21.9; C-28, 15.8; and C-29, 18.4. Mass Spectrum: 405(M + 22%), 390(1.3), 372(2.6), 330(2.4), 236(1.8), 210(7.8), 196(20), 184(14), 182(14), 168(15), 156(27), 143(17), 130(100), and l l5m/e (11); obs. 405.3044; caled. for C2sH39NO, 405.3032. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Three New Aflavinines From The Sclerotia ofAspergillus tubingensis; Tetrahedron, Vol. 45, pp. 4961-4968 (1989).

372

4.

Aflavinines and Related Indoles

Common/Systematic Name 10,23-Dihydro-24,25-dehydro-21-oxoaflavinine Molecular Formula/Molecular Weig.h_t. C2sHaTNO2; MW = 419.28243 28

14

15

\

'

17

"OH 27 le"""

25

General Characteristics Melting point 138-141~

[a]D -13.5 ~ (c=0.72, in CHCI3).

Fungal Source Aspergillus tubingensis (NRRL 4700), A. flavus, and A. parasiticus. Isolation/Purification Sclerotia were ground to a powder with a mortar and pestle and extracted with hexane. Filtration and evaporation of solvent afforded a yellow oil. Reversed-phase HPLC of the hexane extract using an Altex reversed-phase semipreparative column [C~r51~m particle size; UV detection at 215nm; 20ml/min; MeOH-H20 (90:10, v/v)] afforded pure 14-epi- 14-hydroxy- 10,23-dihydro-24,25-dehydroaflavinine along with fractions containing 10,23-dihydro-24,25-dehydroaflavinine and 10,23-dihydro-24,25-dehydro-21-oxoaflavinine. 10,23-Dihydro-24,25-dehydroafiavinine was isolated by reversed-phase HPLC using a solvent composition of [MeOH-H20 (85:15, v/v); 2.5ml/min] and 10,23-dihydro24,25-dehydro-21-oxo-aflavinine was isolated by further chromatography on a 10-I.tm Hamilton PRP-1 column (100% MeOH 1.0ml/min). Biological Activity Caused a 68% weight reduction in Hefiothis zea fed at a rate of 125ppm for 1 week; caused a 38% reduction in feeding rate by Carpophilus hemipterus; and exhibited mild antibacterial activity against Bacillus subtilis at 100~g/disk. Spectral Data UV:

~,~"

291(c=1,750), 283(2,100), 273(1,400), 267(1,200), and 224nm (18,200).

4.

Aflavinines and Related Indoles

373

IH NMR: H-I, 8.00, br, s; H-2, 7.03, br, s; H-5, 7.55, d, `/=7.8; H-6,7.13, dd, ,/=6.8, 7.8; H-7, 7.19, dd, ,/=6.8, 7.6; H-8, 7.36, d, ,/=7.6; H-10, 4.05, dd, ,/=5.4, 13.2; H-11, 3.00, dd, ,/=5.5, 4.6; H-12, 1.47, m; H-13, 1.54, m, 2.15, m; H-14, 0.93, m, 1.21, m; H-16, 2.08, m; H-17, 1.73, m, 1.96, m; H-18, 1.41, m, 2.32, m; H-19, 5.19, br, s; H-22, 2.42, dd, ,/=6.8, 14.4, 3.08, dd, ,/=12. 5, 14.4; H-23, 3.67, ddd,,/=13, 12.5, 6.8; H-25, 4.68, 4.81, br, s; H-26, 1.60, br, s; H-27, 1.03, d, ,/=6.1; H-28, 0.75, d, ,/=6.8; and H-29, 1.38ppm, S.

13C NMR: C-2, 122.5; C-3, 114.9; C-4, 127.2; C-5, 118.0; C-6, 119.4; C-7, 122.0; C-8, 112.4; C-9, 136.1; C-10, 34.0;'C-11, 43.9; C-12, 29.6; C-13, 29.6; C-14, 28.3; C-15, 38.4; C-16, 31.3; C-17, 24.7; C-18, 30.2; C-19, 72.7; C-20, 58.1; C-21,214.2; C-22, 44.9; C-23, 47.3; C-24, 146.8; C-25, 111.4; C-26, 18.2; C-27, 18.0; C-28, 14.7; and C-29, 18.1ppm. Mass Spectrum: 419(M+ 15%), 401(3.7), 322(2.9), 302(2.7), 265(3.8), 248(3.7), 242(5.3), 232(5.2), 225(7.9), 217(5.2), 208(4.8), 196(28), 182(82), 168(60), 157(70), 146(27), 130(100), and 117role (17); obs. 419.2839; calcd, for C28H37NO2, 419.2824. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Three New Aflavinines From The Sclerotia ofAspergillus tubingensis; Tetrahedron, Vol. 45, pp. 4961-4968 (1989).

374

4. Aflavinines and Related Indoles

Common/Systematic Name Aflavazole Molecular Formula/Molecular Weight C28H35NO2; M W = 417.26678

OH L

..

13 1iiiI Ill"

H

jill II'

I0 [

General Characteristics A light yellow crystalline solid; mp., 156-160oC dec; [a]D +2.8 o (c=0.35, in MeOH) Fungal Source

Aspergillus flavus (NRRL 13462) and A. parasiticus.

Isolation/Purification Ground A. flavus sclerotia were exhaustively extracted with hexane, followed by chloroform and the chloroform extract was subjected to flash chromatography on a reversed phase column using a step gradient from 50 to 70% MeOH-H20; aflavazole eluted at 60% MeOH-H20. Separation of the resulting fraction by reversed phase HPLC; MeOH-H20 (70:30, v/v) at 2.0mL/min afforded pure aflavazole; HPLC retention time under the above conditions, 25.3min. Biological Activity Aflavazole displayed significant antifeedant activity against the fungivorous beetle

Carpophilus hemipterus.

Spectral Data UV:

~,mU~" 341(e=1600), 327(1,300), 297(7,600), 263(7,500), 243(17,300), and 219rim (15,300). IH NMR: (CDC13) H-5, 7.95(br, d, J=-8.0); H-6, 7.14(br, dd, J=8.0, 7.1); H-7, 7.1227(br, dd, J=7.8, 7.1); H-8, 7.39(br d, J=7.8); H-11, 4.18(br, d, d=-7.1); H-12, 2.86(m); H-13,x, 2.38(m); H-13~q, 1.68(br, dd, J=12.9, 2.2); H-14, 4.22(dd, J=12.9, 3.4); H-16,

4.

Aflavinines and Related Indoles

375

2.60(m); H-17~,, 1.26(m); H-17~q, 1.78(m); H-18,x, 1.37(br, dd, J=12.7, 1.9); H-18,q, 1.84(m); n-19, 3.84(br, d, J=2.9); H-21, 2.15(m); 2.75(br, dd, J=111 7.1; H-22, 2.89(br, dd, J=17.6, 7.1); 2.39(m); H-25, 7.12(br, s); H-26, 2.35(br, s); H-27, 0.64(d, J=7.6); H-28, 1.19(d, ,/=6.8); and H-29, 1.33ppm (s). lac NMR: (CDCI3) C-2, 140.29; C-3, 119.57: C-4, 123.88: C-5, 122.33; C-6, 119.64; C-7,125.09; C-8, 111.67; C-9, 141.53; C-10, 135.8; C-11, 40.05; C-12, 32.79; C-13, 36.43; C-14, 72.48; C-15, 44.94; C-16, 32.60; C-17, 28.72; C-18, 25.22; C-19, 71.47; C-20, 45.23; C-21, 23.05; C-22, 30.77; C-23, 126.40; C-24, 135.13; C-25, 110.25; C-26, 20.68; C-27, 19.34; C-28, 13.70; and C-29, 19.83. Mass Spectrum: EIMS: 417(rel int 100%), 399(37), 381(13), 316(40), 284(8), 272(13), 258(32), 254(16), 244(36), 231(42), 217(62), 204(29), 194(47), 182(20), 168(21), and 130m/e (60); HREIMS: obsd 417.2694, calcd for C2d-I35NO2,417.2669. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Aflavazole: A New Antiinsectan Carbazole Metabolite from the Sclerotia ofAspergillusflavus; J. Organ. Chem., Vol. 55, pp. 5299-5303(1990).

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Tryptoquivalines Tryptoquivaline; Tryptoquivaline A; Tryptoquivaline C; FTC Nortryptoquivalone; Tryptoquivalone; Tryptoquivaline B Nortryptoquivaline; Tryptoquivaline D; Norisotryptoquivaline; FTD Deoxytryptoquivaline Deoxynortryptoquivaline Tryptoquivaline E; FTE Tryptoquivaline F Tryptoquivaline G; FTG Tryptoquivaline H Tryptoquivaline I Tryptoquivaline J; FTJ Tryptoquivaline L Tryptoquivaline M Tryptoquivaline N; Deoxynortryptoquivalone 2 7-epi-Tryptoquivaline 27-epi-Nortryptoquivaline

377

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5. Tryptoquivalines

379

Common/Systematic Name Tryptoquivaline; Tryptoquivaline A; 'Tryptoquivaline C'; FTC Molecular Formula/Molecular Weight C29H3oN407; M W = 546.21145

o

0

HI

I

II

12

General Characteristics Colorless prisms from dichloromethane-hexane; mp., 155-157~ in CHCI3). Fungal Source Aspergillus clavatus (N

[a]D 25 +

130 ~ (c--0.22,

5890) and A. fumigatus.

Isolation/Purification See Bfichi et al., 1977. Biological Activity Tryptoquivaline (dosed IP, up to 501~1DMSO solution) induced persistent tremors in rats. Spectral Data UV:

~,~m~" 228(e=42,200), 232sh (40,200), 252sh (19,500), 268(10,900), 279(9,500), 307(3,700), and 319nm (3,000).

CD: ~

95% EIOH max

258(-5.3), 282(0), 305(2.2), and 327nm (0).

'H NMR: (CDCI3) H-2, 5.00;H-5-8, 7.12-7.90;H-11, 1.50;H-12, 1.52;H-13a, 3.10(J=I0);H13b, 3.15(,/=10);H-14, 5.70(J=I0);H-18, 8.22(J=8);H-19-21, 7.12-7.90;H-24, 5.61(J=9); H-25, 2.63;H-26, 1.03;H-27, 1.17;CH3CO, 2.19; and NOH, 3.63, 4.04ppm.

380

5. Tryptoquivalines

Mass Spectrum: HREIMS: 546.21554 (100%). TLC Data Silica gel GF254, benzene-acetone, 9:1 v/v; Rf not reported. Detection: not reported. References G. Biichi, K. C. Luk, B. Kobbe, and J. M. Townsend; Four New Mycotoxins of Aspergillus clavatus Related to Tryptoquivaline; J. Org. Chem., Vol. 42, pp. 244-246 (1977). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 414-417(1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 434 (1983).

5. Tryptoquivalines

381

Common/Systematic Name Nortryptoquivalone; 'Tryptoquivalone'; 'Tryptoquivaline B' Molecular Formula/Molecular Weight C26II24N406; MW

= 488.16958

O"~ N 21 o _HI I II '

3,..... ,!

~'5 i~'~ ~.,,,,H

0

f~,,,H 0 12 General Characteristics Colorless prisms from dichloromethane-hexane; mp., 208~176 (c=0.30, in CHCI3).

[a]D 25 q- 255 ~

Fungal Source

Aspergillus clavatus (NRRL 5890).

Biological Activity Nortryptoquivalone dosed IP (up to 501.tl DMSO) induced persistent tremors in rats. Spectral Data UV:

Z ~m~ " 228(sh) (E = 31,1300), 232(31,800), 253sh (16,900), 278(7,600), 289(6,500), 304(5,400), and 316nm sh (4,500).

CD: ~

95%EtOH max

256(-3.8), 273(0), 298(3.6), and 342nm (0).

IH NIVIR: (CDCI3) H-2, 5.24; H-5-8, 7.1 I-7.74;H-I I, 1.53;H-12, 1.53;H-13a, 3.06(J=I0); H13b, 3.06(J=I0); H-14, 5.65(J=I0); H-18, 8.20; H-19-21, 7.1 I-7.74;H-24, 5.52(J=9); H-25, 2.54; H-26, 1.04(/=7);H-27, 1.20(/=7);and CH3CO, 2.16ppm.

382

5. Tryptoquivalines

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 418 (1981). W. B. Turner and D. C. Aldridge, Fungal Metabolites II, Academic Press, New York, New York, p. 434 (1983).

5. Tryptoquivalines

383

Common/Systematic Name Nortryptoquivaline; 'Tryptoquivaline D'; Norisotryptoquivaline'; FTD Molecular Formula/Molecular Weight C2sH25N407; M W = 532.19580

"-42/ q HI

I

II

.O

O General Characteristics Colorless prisms from dichloromethane-hexane; mp., 256-258"C; [a]D2s 170 ~ (c--0.64, in CHCI3). Fungal Source Aspergillus clavatus (NRRL 5890) and A. fumigatus. Biological Activity Tremorgenic activity not reported in detail; however, metabolite is related to the reported tremorgens, tryptoquivaline and nortryptoquivalone. Spectral Data ~,.

~,~t~ 228(e=43,600), 233 sh (42,000), 254 sh (18,700), 267 sh (11,900), 279(10,200), 306(4,500), and 319nm (3,500). CD: 95% EtOH ~ max

254(-5.4), 287(0), 307(2.1), and 325 (0).

IR:

3490, 2980, 2940, 2880, 1790, 1728, 1670, 1610, 1485, 1471, and 1410cm"1. 13C NMR: (CDCI3) H-2, 5.10; H-5-8, 7.01-7.79; H-11, 4.28(]=-7); H-12, 1.58(J=7), H-13a,

384

5. Tryptoquivalines

2.94(`/=10,13); H-lab, 3.18(J=10,13); H-14, 5.65(J=10); H-18, 8.12; H-19-21, 7.01-7.79; H-24, 5.54(J=9); H-25, 2.57; H-26, 1.02(J=7); H-27, 1.16(,/=7); and CHACO, 2.16ppm. Mass Spectrum: HREIMS: 532.19525(100%). References G. Bfichi, K. C. Luk, B. Kobbe, and J. M. Townsend; Four New Mycotoxins of Aspergillus clavatus Related to Tryptoquivaline; J. Org. Chem., Vol. 42, pp. 244-246 (1977). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p.422 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 434 (1983).

5. Tryptoquivalines

385

Common/Systematic Name Deoxytryptoquivaline Molecular Formula/Molecular Weight C29H3oN406; M3cV= 530.21653

q HI

0

I

o

II

12

General Characteristics White needles from dichloromethane-hexane; mp., 150-152"C; [a]D25 +56.8* (C----0.78,in CHCI3). Fungal Source Aspergillus clavatus. Biological Activity Details of tremorgenic activity not reported; however, metabolite is chemically related to known tremorgens, tryptoquivaline and nortryptoquivalone. Spectral Data UV: ~,Em~H 227(e=44,500),232(sh) (41,900), 252(sh) (18,500), 267(sh) (12,000), 278(sh) (10,300), 304(3,300), and 318nm (sh) (2,700). IR:

(CHCI3) 3360, 3310, 2980, 2935, 2875, 1790, 1720, 1676, 1604, 1483, and 1469cm"~. 1H NMR: (CDCI3) H-2, 5.24; H-5-8, 7.11-7.74; H-11, 1.53; H-12, 1.53; H-13a, 3.06(J=10); H-13b, 3.06(,]--10); H-14, 5.65(,/--10); H-18, 8.20; H-19-21, 7.11-7.74; H-24, 5.52(J=9); H-25, 2.54; H-26, 1.04(,/=7); H-27, 1.20(,/=7); and CH3CO, 2.16ppm.

386

5. Tryptoquivalines

Mass Spectrum: HREIMS: 530.21527(100%). References G. Biichi, K. C. Luk, B. Kobbe, and J. M. Townsend; Four New Mycotoxins of Aspergillus clavatus Related to Tryptoquivaline; J. Org. Chem., Vol. 42, pp. 244-246 (1977). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 426 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 434 (1983).

5. Tryptoquivalines

387

Common/Systematic Name Deoxynortryptoquivaline Molecular Formula/Molecular Weight C2sH28N406; MW = 516.20088

~N~,,H 0 General Characteristics Colorless prisms from diethyl ether; mp., 158-160~

[a]D 25 69.5 ~

(c=0.82, in CHCI3).

Fungal Source Aspergillus clavatus. Biological Activity Details of tremorgenic activity not reported; however, the metabolite is related to the reported tremorgens, tryptoquivaline and nortryptoquivalone. Spectral Data UV: Z ~mt~ 228(e=43,900), 233(sh) (40,100), 254(sh) (15,600), 268(11,700), 278(sh) (10,500), 305(4,100), and 317nm (3,300). IR:

(CHCI3) 3360, 2975, 2935, 2880, 1790, 1724, 1676, 1607, and 1483cm"~. 1H N]VIR: (CDCI3) H-2, 5.22; H-5-8, 7.02-7.74; H-11, 4.12(J=7); H-12, 1.55(,/--7); H-13a, 2.86(,/--10, 13); H-lab, 3.07(,/--10, 13); H-14, 5.65(J=10); H-18, 8.14; H-19-21, 7.02-7.74; H-24, 5.55(J=9), H-25, 2.58; H-26, 1.00(J=7); H-27, 1.15(J=7); and CHACO, 2.16ppm.

388

5. Tryptoquivalines

Mass Spectrum: HR IMS: 516.20420(100%). References G. Bfichi, K. C. Luk, B. Kobbe, and J. M. Townsend; Four New Mycotoxins of Aspergillus clavatus Related to Tryptoquivaline; J. Org. Chem., Vol. 42, pp. 244-246 (1977). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 434 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p.435 (1983).

5. Tryptoquivalines

389

Common/Systematic Name Tryptoquivaline E; FTE Molecular Formula/Molecular Weight C22HIsN40s MW = 418.12772

,~N

21

N

~

0

,,,H 12

General Characteristics Colorless feathers from acetone; mp., --257~ (dec.);

[a]D 15"5 + 2 5 7 ~

(c=0.009, in CHCIs).

Fungal Source Aspergillus fumigatus and A. clavatus. Biological Activity Related to tremorgen, tryptoquivaline; however, not demonstrated to be tremorgenic. Spectral Data UV:

Z~" 225.5(e=32,300), 232(sh) (29,800), 254(sh) (16,000), 265.5(sh) (11,700), 275.5(sh) (8,400), 291(sh) (3,500), 303(3,000), and 315nm (2,600). IR:

(KBr) 3430, 1780, 1740, 1732, 1677, 1658, and 1614cm"~. ]H NMR: (pyridine) H-2, 5.42; H-5-8, 6.83-7.80; H-I I, 4.26(J-7); H-12, 1.63(./=7); H-13a, 3.38(J=10, 13); H-13b, 3.56(J=10, 13); H-14, 6.48(J=10); H-18, 8.17; H-19-21, 6.83-7.80; H-23, 8.59; and N-OH, 10.42ppm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981).

390

5. Tryptoquivalines

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazmki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

391

Common/Systematic Name Tryptoquivaline F Molecular Formula/Molecular Weight C22HIsN404; M'W = 402.13281

~N

21

O H O~,,, ' ....,N

General Characteristics Colorless fine needles from methanol; mp.,-277~ (dec.); [a]D~5s -109~ (C=0.006, in CHCIa). Fungal Source

Aspergillusfumigatus.

Biological Activity Reportedly related to tremorgen tryptoquivaline; however, not demonstrated to be tremorgenic. Spectral Data UV: ~,~" 226(e=33,400),232(sh) (31,200), 255(sh) (12,400), 265(sh) (11,101), 276(sh) (8,100), 290(sh) (3,100), 303(2,800), and 315nm (2,200). IR:

(KBr) 3365, 1775, 1725, 1664, and 160cm"~. 1H NMR: (CDOla) H-2, 5.65; H-5-8, 7.12-8.07; H-11, 4.4(J=7); H-12, 1.63(./=7); H-13a, 3.19(J=9); H-lab; 3.33(,/--9,13); n-14, 5.40(,/--9); n-18, 8.21(,/=7); H-19-21, 7.12-8.07; H-23 and CHACO,2.15ppm.

392

5. Tryptoquivalines

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazmki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

393

Common/Systematic Name Tryptoquivaline G; FTG M_olecular Formula/Mol.ecular Weight C23H2oN4Os; M W - 4 3 2 . 1 4 3 3 7

~

0

,,,Me 12

General Characteristics Colorless prisms from acetone; mp., 240-241.5~ (dec.); [a]D 11 +215 ~ (c=0.011, in acetone). F.ungal Source

Aspergillusfumigatus.

Biological Activity Reportedly related to tremorgen tryptoquivaline; however, not demonstrated to be tremorgenic. Spectral Data

UV: )~mM~H 226(e=34,300), 232(sh) (31,700), 253(sh) (17,400), 265(sh) (11,800), 275(sh) (8,200), 291(sh) (3,700), 302(3,000), and 315nm (2,500). JR;

(KBr) 3470, 1778, 1738, 1662, and 1610cm~. IH N-IV[R:

(pyridine) H-2, 5.30;H-5-8, 6.96-7.96;H-I I, 4.26(J=7);H-12, 1.50, 1.62;H-13a, 3.43(J=I0, 14);H-13b, 3.64;H-14, 6.56(J=I0);H-18, 8.23 H-19-21, 6.96-7.96; H-23, 8.66; and N- OH, 10.61ppm.

394

5. Tryptoquivalines

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 438 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. I 11117(1978).

5. Tryptoquivalines

395

Common/Systematic Name Tryptoquivaline H Molecular Formula/Molecular Weight C22HIsN4Os; MW = 418.12772

N,.,g,j

0 ...... I H

0

General Characteristics Colorless fine needles from methanol; mp., -~2740C (dec.); [tt]D11 -155 ~ (C=0.021, in acetone) Fungal Source_

Aspergillusfumigatus.

Biological Activity Related to the reported tremorgen tryptoquivaline; however, not demonstrated to be tremorgenic. Spectral Dat_a UV:

Z~" 226(e=33,100), 232(sh) (30,900), 255(sh) (16,600), 266(s:1) (11,300), 276(sh) (8,500), 291(sh) (3,600), 303(3,100), and 315nm (2,500). IR:

(KBr) 3430, 1780, 1742, 1734, 1667, and 1609cm~. IH NMR: (pyridine) H-2, 5.25; H-5-8, 6.68-7.88; H-11, 3.98(J=8); H-12, 1.45(J=8); H-13a, 2.96(J=10,13); H-13b, 3.60(d=10, 13); H-14, 5.76(,/=10); H-18, 8.11; H-19-21, 6.68-7.88; H-23, 8.38; and N-OH, 10.6ppm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981).

396

5. Tryptoquivalines

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

397

Common/Systematic Name Tryptoquivaline I Molecular Formula/Molecular Weight C27H26N406; MW = 502.18523

0" ~

0

N

21

12

General Characteristics Colorless leaflets from methylene-methanol; mp., 232-235.5~ (dec.); [a]D~4 +239* (C=0.16, in CHCI3). Fungal Source

Aspergillusfumigatus.

Biological Activity Related to the reported tremorgen tryptoquivaline; however, not demonstrated to be tremorgenic. Spectral Data UV:

/~

MeOH max

235(~=31,700), 250(sh) (21,400), 292(9,600), and 321nm (sh) (6,100).

IR:

(KBr) 3480, 1780, 1732, 1710, 1675, and 1609cm"~. 1H NMR: (CDCI3) H-2, 4.99; H-5-8, 7.00-7.94; H-12, 1.49; H-13a, 3.06(J=10, 14); H-13b, 3.39(J=10, 14); H-14, 5.47(J=10); H-18, 8.24(,/--7); H-19-21, 7.00-7.94; H-25, 4.07(,/--7); H-26, 1.22; H-27, 1.28; and NOH, 7.01ppm.

398

5. Tryptoquivalines

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

399

Common/Systematic Nam.e Tryptoquivaline J, FTJ Molecular Formula/Molecular Weight C22HIsN404; MW = 402.13281 ~N

~

,

,

21

H

General Characteristics Colorless fine needles from acetone-methanol; mp., 254-258~ in acetone).

[a]D14 +135~ (C=0.024,

FunRal Source v

Aspergillusfumigatus.

Biological Activity Related to the reported tremorgen tryptoquivaline; however, not demonstrated to be tremorgenic. Spectral Data UV;

XM~" 225.5(e=41,100), 23 l(sh) (38,000), 253(sh)(16,200), 264(sh) (12,800), 275(sh) (9,700), 290(sh) (4,200), 302(3,900), and 3 lOnm (3,1oo).

IR: (KBr) 3375, 1780, 1713 1670, and 1710cmq. IH N]VIR: (DMSO) H-Z, 5.41(J=6);H-5-8, 7.20-8.02;H-I 1, 3.87(J=-7);H-I2, 1.41(J=7);H-13a, 3.08; H-13b, 3.10;H-14, 6.01(,/=10);H-18, 8.17;H-19-21, 7.20-8.02;H-23, 8.49; and NH, 3.76ppm.

400

5. Tryptoquivalines

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

401

Common/Systematic Name Tryptoquivaline L Molecular Formula/Molecular Weight C23H2oN4Os; M W = 432.14337

H ~N~ C)~'~..'N ..~i~~f~J

General Characteristics Colorless leaflets from acetone; mp., 265-268~ sulfoxide).

[a]D 23 - 2 2 9 ~

(c=0.03, in dimethyl

Fungal Source

Aspergillusfumigatus.

Spectral Data UV:

3,U~" m~ 216(sh) (e=33,900), 226(33,000), 231(sh) (30,800), 252(sh) (17,400), 264(sh) (12,300), 274(sh) (8,400), 290(sh) (3,700), 302(3,000), and 315nm (2,400). IR:

(KBr) 3230, 1784, 1749, 1670, 1616, 1485, 1260, and 1200cm"~. 1n NMR: (DMSO) H-2, 5.21" H-5-8, 7.24-8.00; H-12, 1.26, 1.36; H-13, 3.03, 3.43; H-14, 5.57; H-18, 8.23; H-19-21, 7.24-8.00; H-23, 8.53; and NOH, 8.74ppm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981). W. B. Turner and D. C. Aldridge; Fungal Meta.bolites II; Academic Press, New York, New York, p. 435 (1983).

402

5. Tryptoquivalines

M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

403

Common/Systematic Nam_e. Tryptoquivaline M Molecular Formula/Molecular Weight C2sH2sN4OT; M W = 532.19580

q HI

0

I

O

II

12

General Characteristics Colorless plates from methanol-water; mp., 157-164~

[a]D 24 - 1 5 4 ~

(c=0.50, in CHCI3).

Fungal Source

Aspergillusfumigatus.

Spectral Data UV;

XM~. 228(e=32,700),232(30,700), 255(15,400), 278(9,300), 305(2,900), and 317nm (2,700). IR:

(KBr) 3400, 1788, 1725, 1678, 1600, 1480, 1464, and 1210cmq. ~HNMR: H-2, 5.22; H-5-8, 7.12-7.88; H-11, 4.15(J=7); H-12, 1.55(,/=7); H-13, 2.68, 3.65(J=11,10); H-14, 5.87(J=10); H-18, 8.24(,/=-8); H-19-21, 7.12-7.88; H-24, 5.54(J=10); H-25, 2.74; H-26, 0.92(,/=6); H-27, 1.04(,/=-6); CH3CO, 2.16; and OH, 6.86ppm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981).

404

5. Tryptoquivalines

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J; Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

5. Tryptoquivalines

405

Common/Systematic Name Tryptoquivaline N; Deoxynortryptoquivalone Molecular Formula/Molecular Weight C26I-I24N405; M W = 4 7 2 . 1 7 4 6 7

0" ~

~

~ N ~ 0

N

21

,,H 12

General Characteristics Colorless needles from methanol; mp., 193-197~ sulfoxide).

[a]D 24 +

127 ~ (c=0.66, in dimethyl

Fungal Source Aspergillus fumigatus and A. clavatus. Spectral Data UV;

ZmM~" 232(c=32,300), 251(sh) (17,900), 291(9,000), and 320nm (sh) (6,100). IR~

(KBr) 3360, 1780, 1722, 1705, 1680, 1607, 1481, and 1250cm"~. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 438 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II, Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, H. Fujimoto, and E. Okyama; Structure Determination of Six Fungal Metabolites, Tryptoquivaline E, F, G, H, I, and J, Chem. Pharm. Bull., Vol. 26, pp. 111117(1978).

406

5. Tryptoquivalines

Common/Systematic Name

27-epi-Tryptoquivaline

Molecular Formula/Molecular Weight C29H30N407; MW = 546.21145

M e - - C - - O , ......K,,~...~N~

o

'

2

H I

H

I,... o Me

General Characteristics Colorless needles from MeOH; mp., 225-227~

[tt]D25 -138 ~ (C=0.020, in CHCI3).

Fungal Source

Corynascus setosus (IFM 4648).

Isolation/Purification

Corynascus setosus was cultivated on sterilized rice at 25 ~ for 17 days. The moldy rice was extracted two times with ethyl acetate with shaking at room temperature for 2 hours, to give a crude extract which was partitioned with n-hexane-MeOH (1:1, v/v). The defatted layer was then partitioned with ethyl acetate-H20 (1:1, v/v) and the ethyl acetate layer was chromatographed on a silica gel column with n-hexane-ethyl acetate (2:1, v/v), (1:1, v/v), and (1:2, v/v), ethyl acetate and MeOH two times to give fractions I, II, III, and IV. Fraction III was further chromatographed on silica gel columns with n-hexaneethyl acetate and CHCI3-MeOH repeatedly to afford helvolic acid, helvolinic acid, setosusin, and 2-(1-oxo-2-hydroxyethyl)furan. Fraction I was chromatographed on silica gel columns with n-hexane-ethyl acetate repeatedly and on an octadecyl silica gel (ODS) column with acetonitrile-HzO to furnish 27-epi-tryptoquivaline and 27-epi-nortryptoquivaline. Fraction II was also chromatographed in a similar manner to afford fiscalin B, previously identified from Neosartoryafischeri.

Biological Activity

27-epi-Tryptoquivaline and 27-epi-nortryptoquivaline caused a weak tremor when administered at 50mg/kg, i.p. The tremor appeared at ca. 30 and 120 minutes atter injection and continued for ca. 120 and 30 minutes, respectively.

5. Tryptoquivalines

407

Spectral Data UV: ~,mM~a

209(sh, log e=4.67), 289(sh, 3.83), 306(sh, 3.54), and 318nm (sh, 3.44).

CD: (Ac) 0(325), + 1.8(305), + 3.6(281), 0(260), and - 1.9nm (253) (c= 0.011, in EtOH). IR:

(CHCI3) 3500, 3000, 1790. 1735, 1675, and 1610cm~. ~H NMR: (CDCIs) 5.05(1H, s, H-2); 41(1H, br d, J=7.6Hz, H-5); 7.28(1H, td, 3--7.6, 1.1Hz, H= 6); 7.48(1H, td, J=7.6, 1.1Hz, H-7); 7.64(1H, br d, J=7.6Hz); 5.88(1H, t-like, J=10.0Hz, H-12); 3.12(1H, dd, 3=13.5, 9.7Hz, H-13); 3.14 (1H, dd, 3--13.5, 10.1Hz, H-13); 6.99(1H, s, 16-OH); 8.25(1H, dd, ,/=7.2, 1.3Hz, H-20); 7.55(1H, td, J=7.2, 1.2Hz, H-21); 7.84(1H, td, J=7.2, 1.3Hz, H-22); 7.79(1H, br d, d=7.2Hz); 5.65(1H, d, 3--10.1, H-27); 2.69(1H, m, H-28); 1.06(3H, d, J=6.8Hz); 1.17(3H, d, J=6.8Hz); 2.22(3H, s, H-33); 1.49(3H, s, H-34); and 1.50ppm (3H, s, H-35). 13C NMR:

(CDCI3) 87.6, C-2(d); 83.9, C-3(s); 133.7, C-4(s); 123.7, C-5(d); 125.6, C-6(d); 131.9, C-7(d); 116.1, C-8(d); 138.1, C-9(s); 169.4, C-1 l(s); 54.7, C-12(d); 34.6, C13(t); 169.4, C-14(s); 71.4, C-15(s); 161.7, C-18(s); 120.3, C-19(s); 126.8, C-20(d); 128.2, C-21(d); 135.5, C-22(d); 128.0, C-23(d); 146.5, C-24(s); 151.5, C-26(s); 78.9, C-27(d); 31.8, C-28(d); 18.9, C-29(q); 18.9, C-30(q); 170.6, C-32(s); 20.8, C-33(q); 16.9, C-34(q); and 22.8ppm (q). Mass Spectrum: HKFAB-MS: 547.2183m/e (C29H31N407 requires 547.2193 [(M+H)+]). Reference H. Fujimoto, E. Negishi, K. Yamaguchi, N. Nishi, and M. Yamazaki; Isolation of New Tremorgenic Metabolites from an Ascomycete, Corynascussetosus; Chem. Pharm. Bull.; Vol. 44, pp. 1843-1848(1996).

408

5. Tryptoquivalines

Common/Systematic Name 2 7-epi-N ortryptoquivaline Molecular Formula/Molecular Weight C2sH28N4OT; MW = 532.19580

Me.~ 28/Me

27~/H~N~ Me--C--O--~,,,, O H '"| [I

~N~ I "

I'~

~~L~N/~N/OH O/J~

I'M"2''H

General Characteristics Colorless needles from MeOH, mp., 236-238~

[a]o ~s + 196" (c=0.292, in CHCI3).

Fungal Source Corynascus setosus (IFM 4648). Isolation/Purification Corynascus setosus was cultivated on sterilized rice at 25 ~ for 17 days. The moldy rice was extracted two times with ethyl acetate with shaking at room temperature for 2 hours, to give a crude extract which was partitioned with n-hexane-MeOH (1:1, v/v). The defatted layer was then partitioned with ethyl acetate-H20 (1:1, v/v) and ethyl acetate layer was chromatographed on a silica gel column with n-hexane-ethyl acetate (2:1, v/v), (1:1, v/v), and (1:2, v/v), ethyl acetate and MeOH two times to give fractions I, II, III, and IV. Fraction III was further chromatographed on silica gel columns with n-hexaneethyl acetate and CHCI3-MeOH repeatedly to afford helvolic acid, helvolinic acid, setosusin, and 2-(1-oxo-2-hydroxyethyl)furan. Fraction I was chromatographed on silica gel columns with n-hexane-ethyl acetate repeatedly and on an octadecyl silica gel (ODS) column with acetonitrile-H20 to furnish 27-epi-tryptoquivaline and 27-epi-nortryptoquivaline. Fraction II was also chromatographed in a similar manner to afford fiscalin B, previously identified from Neosartoryafischeri. Biological Activity 27-epi-Tryptoquivaline and 27-epi-nortryptoquivaline caused a weak tremor when administered at 50mg/kg, i.p. The tremor appeared at ca. 30 and 120 minutes after injection and continued for ca. 120 and 30 minutes, respectively.

5. Tryptoquivalines

409

Spectral Data UV;

~

MeOH max

209(sh, log e = 4.64), 290(sh, 3.80), 307(sh, 3.53), and 318nm (sh, 3.43).

CD: (AE) 0(325), + 1.9(305), + 4.1(278), 0(259), and - 2.6(252nm) (c= 0.011, in EtOH). IR~

(CHCI3) 3500, 3000, 1780. 1720, 1660, and 1600cm"1. IH NMR: (CDCI3) 5.23 (1H, s, H-2); 7.41(1H, br d, J=7.6Hz, H-5); 7.28(1H, td, J=7.6, 1.1Hz, H-6); 7.49(1H, td, J=7.6, 1.1Hz, H-7); 7.63(1H, br d, J=7.6Hz); 5.87(1H, t-like, J=10.1Hz, U-12); 3.09(1H, dd, J=13.3.9.5Hz, n-13); 3.15(1H, dd, J=13.3.10.7Hz, H-13); 4.35(1H, q, J=7.2Hz, n-15); 7.16(1H, s, 16-OH), 8.22(1H, dd, J=8.4, 1.5Hz, H-20); 7.55(1H, td, J=8.4, 1.3Hz, n-21); 7.84(1H, td, J=8.4, 1.5I-~ H-22); 7.79(IH, br d, d=8.4Hz); 5.66(1H, d, J=9.8, H-27); 2.68(1H, m, H-28); 0.98(3H, d, J=6.6Hz); 1.16(3H, d, J=6.6Hz); 2.21(3H, s, H-33); and 1.59ppm (3H, d, J=7.2Hz, H-35). 13C N M R :

(CDCl3) 89.9, C-2(d); 83.9, C-3(s); 133.7, C-4(s); 125.7, C-5(d); 125.7, C-6(d); 131.9, C-7(d); 116.1, C-8(d); 137.8, C-9(s); 169.3, C-1 l(s); 54.6, C-12(d), 34.1, C13(t); 169.2, C-14(s); 67.9, C-15(s); 161.8, C-18(s); 120.3, C-19(s); 126.7, C-20(d); 128.3, C-21(d); 135.5, C-22(d); 128.0, C-23(d); 146.5, C-24(s); 151.5, C-26(s), 79.0, C-27(d); 31.8, C-28(d); 19.0, C-29(q); 18.9, C-30(q); 170.5, C-32(s), 20.8, C-33(q); and ll.0ppm (q). Mass Spectrum: HRFAB-MS: 533.2023m/e (C2sH29NaO7 requires 533.2036 [(M+H)+]). Reference H. Fujimoto, E. Negishi, K. Yamaguchi, N. Nishi, and M. Yamazaki; Isolation of New Tremorgenic Metabolites form an Ascomycete, Corynascus setosus; Chem. Pharm. Bull.; Vol. 44, pp. 1843-1848(1996).

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Penitrems/Lolitrems Penitrem A Penitrem B Secopenitrem B 10-Oxo- 11,33-dihydropenitrem B Penitrem C Penitrem D Penitrem E Penitrem F Pennigritrem PC-M4 Lolitrem B Lolitrem E (Lolitrem C) Lolitriol

6

411

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6. Penitrems/Lolitrems

413

Common/Systematic Name Penitrem A Molecular Formula/Molecular Weight C37H44NO6CI; MW = 633.28572

H Me .......~,,,,Me 16. 0 H

'"

,e,O.o.T.i, i 0 k ' V ~~" : ~ ~-~TMe

General Characteristics White amorphous solid. Fungal Source Penicillium crustosum.

Isolation/Purification P. crustosum cultures were filtered and the mycelium macerated with acetone in a Waring

blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v) to give the mixture of penitrems. Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, atter crystallization from aqueous methanol, roquefortine, mp., 196-200 ~C. The mixture of penitrems was separated and purified by column chromatography on silica gel using benzene-acetone (85:15, v/v) to yield the six penitrems in the following order of descending Re values: penitrem F, B, A, E, C, and D. Biological Activity Highly tremorgenic and acutely toxic. Spectral Data UV:

~

MeOH max

233 and 295nm (e=37,000 and 11,600).

414

6.

Penitrems/Lolitrems

IR: (KBr) 3400, 2930, 1710, and 1650cmq. CD: 325, 0; 315, 0.91; 300, 0; 255, 0; 236, -14.09; and 210nm, 0. ~H NMR:

[(CD3)2CO] The intensities of four of the signals (10.03, s; 4.16, s; 3.40, d, J=7.5Hz; and 3.32ppm, s) decreased upon addition of deuterium oxide to the sample as well as through saturation transfer upon irradiation of the water resonance at 2.84ppm. The signal at 10.03ppm was assigned to the NH proton of the indole moiety whereas the remaining signals were attributed to hydroxy protons. The one-proton aromatic singlet at 7.24ppm was assigned to 7-H; five three-proton signals at 1.75, 1.71, 1.40, 1.22, and 1.07ppm were assigned to tertiary methyl groups. The signal at 1.71ppm showed broadening, which was resolved in the resolution enhanced spectrum, due to long-range couplings. The remainder of the ~H-spectrum exhibited extensive fine structure (see de Jesus, et al., 1983). ~3CNMR: [(CD3)2CO] C-2, 154.36; C-3,120.64; C-4, 133.29; C-5, 125.80; C-6, 124.56; C-7,111.86; C-8, 121.99; C-9, 139.3; C-10, 35.06; C-11,149.48; C-12,47.01; C-13, 24.67; C-14, 52.71; C-15, 81.01; C-16, 76.09; C-18,72.44; C-19, 58.79; C-20, 18.56; C-21, 30.59; C-22, 78.24; C-23, 66.11; C-24, 61.92; C-25, 66.31; C-26, 74.67; C-28, 71.99; C-29, 28.89; C-30, 26.91; C-31, 43.55; C-32, 50.08; C-33, 107.10; C-34, 20.32; C-35, 31.06; C-36, 19.70; C-37, 143.27; C-38, 111.64, C-39, 18.98; and C-40, 21.35ppm. Mass Spectrum: HREIMS showed M§ at 633.2859, C37H44NO6CIrequires 633.2857. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels, and W. E. Hull, Tremorgenic Mycotoxins from Penicillium crustosum: Isolation of Penitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983).

6. Penitrems/Lolitrems

415

Common/Systematic Name Penitrem B Molecular Formula/Molecular Weight C37H45NOs; MW' = 583.32977 H

H,,

r,~l

Me l.,,,,Me

~47'"~'~'~,-,

6 ~,,"'~NI-:I

Ir

....

'H

40 ~ ~ ~ M e

A

38

General Characteristics White amorphous solid. Fungal Source Penicillium crustosum.

Isolation/Purification P. crustosum cultures were filtered and the mycelium macerated with acetone in a Waring

blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v) to give the mixture of penitrems. Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, alter crystallization from aqueous methanol, roquefortine; mp., 196-200 ~C. The mixture of penitrems was separated and purified by column chromatography on silica gel using benzene-acetone (85:15, v/v) to yield the six penitrems in the following order of descending Rf values: penitrem F, B, A, E, C, and D. Biological Activity Highly tremorgenic and acutely toxic. Spectral Data UV: 220 and 286nm (6=31,900 and 11,900, respectively).

416

6.

Penitrems/Lolitrems

IR: (KBr) 3400, 2930, 1700, and 1650cmq. CD: 305, 0; 285, -2.56; 252, -0.60; and 236nm, -9.50. IH NMR: The ~HNMR of penitrem B showed that the structure differed from penitrem A in that both the chlorine atom and the C-15 hydroxy group are replaced by hydrogen atoms. The chemical shit, s and coupling constants of tings A-F are similar to those of penitrem D. ~3CNMR: C-2, 152.97; C-3, 119.40; C-4, 128.84; C-5, 128.13; C-6, 120.99; C-7, 110.26; C-8, 123.19; C-9, 139.34; C-10, 38.75; C-11,150.22; C-12, 35.04; C-13, 26.69; C-14, 52.32; C-15, 39.35; C-16, 75.47; C-18, 72.10; C-19, 59.12; C-20, 18.61; C-21, 30.58; C-22, 78.26; C-23, 66.16; C-24, 61.95; C-25, 66.33; C-26, 74.68; C-28, 72.03; C-29, 28.92; C-30, 26.85; C-31, 43.60; C-32, 49.68; C-33, 105.86; C-34, 18.61; C-35, 28.80; C-36, 19.71; C-37, 143.29; C-38, 111.64, C-39, 18.92; and C-40, 21.24ppm. Mass Spectrum: HREIMS: found, M+, 583.3302; C37H45NO5requires 583.3297. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels and W. E. Hull; Tremorgenic Mycotoxins from Penicillium crustosum: Isolation of Penitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983).

6.

Penitrems/Lolitrems

417

Common/Systematic Name Secopenitrem B Molecular Formula/Molecular Weight C37I-I47NOs;MW = 585.34542

/ ""I'I

,^ _~

20

.., . _ ,

o

General Characteristics Orange-brown solid; mp., 185-189~ dec; laiD-5.2 ~ (c=0.005g/ml, in CHCI3). Fungal Source Sclerotia ofAspergillus sulphureus (NRRL 4077). Isolation/Purification Powdered sclerotia ofA. sulphureus were sequentially extracted with pentane and CH2CI2 using a Soxhlet apparatus. A portion of the total CH2C12extract was fractionated by silica gel column chromatography. A stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3 was employed resulting in the elution and collection of a distinct red band at 4% MeOH. This active fraction was further separated by reversed-phase HPLC (MeOH-H20, 89:11, v/v) to yield sulpinines A, B, C, and secopenitrem B (HPLC retention time, 8.5 min) and penitrem B. Biological Activity Exhibited potent activity against the lepidopteran crop pest Helicoverpa zea. Spectral Data ~H NMR: (CDCI3) H-6, 6.88(d, d=8.1); H-7, 7.07(d, J=8.1); H-10, 3.90(br d, J-=15.1), 3.40(d, ,/=15.3); H-12, 3.15(dd, J=8.9, 8.8); H-13, 2.33(m), 1.85(m); H-14, 2.48(m); H-15, 4.08(dd, J=8.5, 8.7); H-18, 2.99(dd, J=13.0, 6.1), 2.47(m); H-19, 2.75(m); H-20, 1.89 (m), 1.62 (m); H-21, 1.53(m), 1.50(m); H-24, 3.64(d, 3=2.3); H-25, 4.03(br s); H-26, 4.10(br s); H-28, 4.30(dr, ,/=9, 9); H-29, 2.36(m), 1.93(m); H-30, 2.67(ddd, J=13.6, 13.6, 5.1), 1.33(m); H-33, 4.83(br s), 4.72(br s); H3-34, 1.16(s); H3-35, 1.07(s); H3-36, 1.72(br s); H-38, 5.16(br s), 5.02(br s); H3-39, 1.163(s); and H3-40, 1.175ppm

(s).

418

6.

Penitrems/Lolitrems

13C NMR: (CDCI3) C-2, 152.0; C-3, 116.3; C-4, 131.3; C-5, 129.7; C-6, 121.1; C-7, 109.3; C-8, 123.4; C-9, 138.9, C-10, 37.6; C-11,149.2; C-12, 36.5; C-13, 25.8; C-14, 52.3; C-15, 37.0, C-16, 71.4; C-18, 29.4; C-19, 49.9; C-20, 20.5; C-21, 30.2; C-22, 78.0; C-23, 65.8; C-24, 61.8; C-25, 64.7; C-26, 73.6; C-28, 71.5; C-29, 27.7; C-30, 27.4; C-31, 42.4; C-32, 50.2; C-33, 108.3; C-34, 26.5; C-35, 27.3; C-36, 19.5; C-37, 141.1; C-38, 112.3; C-39, 18.6; and C-40, 15.6ppm. Mass Spectrum: EIMS: 585(M+, 4), 667(11), 552(17), 531(6), 516(7), 499(84), 484(40), 437(33), 422(35), 369(32), 354(26), 314(70), 301(31), 246(100), 233(37), and 194m/e (38); HRFABMS: obsd 586.3589, calcd for C37I-h7NO5+ H, 586.3532. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Sulpinines A-C and Secopenitrem B: New Antiinsectan Metabolites from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 2066-2071(1992).

6.

Penitrems/Lolitrems

419

Common/Systematic Name 10-Oxo- 11,33-dihydropenitrem B Molecular Formula/Molecular Weight C37H45NO6;/VIW = 599.32469 13

3 3 .....

~.,,,,

118

o

38

General Characteristics Obtained as a light yellow solid, [a]D -78.6 ~ (c=0.2 g/dL). Fungal Source Sclerotia of Aspergillus sulphureus (NRRL 4077). Isolation/Purification Sclerotia ofA. sulphureus (NRRL 4077) were ground with a Tecator mill and sequentially extracted with pentane and CH2C12 using a Soxhlet apparatus. A portion of the total CH2C!2 extract was fractionated by silica gel column chromatography. A stepwise gradient from 0-10% (v/v) MeOH in CHCI3 was employed, resulting in the elution of a distinct red band at 4% MeOH. The fraction that yielded 10-oxo-I 1,33-dihydropenitrem B eluted immediately after this distinct band. This fraction was separated further by reversed-phase HPLC (92:8(v/v), MeOH-H20; Beckman Ultrasphere ODS column, 5~m particles, 250x10mm, 2.5 mL/min) to give 10-oxo-11,33-dihydropenitrem B as a light yellow solid (HPLC retention time, 8.9 min). Biological Activity Exhibited potent activity in dietary assays against the corn earworm, Helicoverpa zea and was also moderately effective as a feeding deterrent against the dried fruit beetle,

Carpophilus hemipterus.

Spectral Data UV:

~

Mr max

261(e=28,900) and 289nm (7,310).

420

6.

Penitrems/Lolitrems

IR~

(neat) 3532, 3342, 1660, 1457, 1231, 1038, 934, and 757cm"~. ~HNMR: (CD3OD) H-6, 7.38(d, ,/=8.5Hz); H-7, 7.18(d, ,/=8.5Hz); H-I I, 2.81(br q, J=6.41-Iz); H-12, 2.27(m); H-13a, 2.18(m); H-13b, 1.91(m); H-14, 2.77(m); H-15, 3.83(dd, ./--9.2, 9.21-Iz); H-18, 4.89(d, `/=8.2Hz); H-19, 2.65(m); H-20~, 1.93(m); H-20~q, 1.77(m); H21~, 1.47(ddd, ./=13.4, 12.7, 4.0Hz); H-21~q, 1.74(m); H-24, 3.49(br, J=81-Iz), H-25, 4.04(br, s); H-26, 4.03(br, s); H-28, 4.29(dd, :=-9.2, 8.6Hz); H-29~, 2.10(m); H-29~, 2.29(m); H-30,~, 2.61(ddd, ./=13.8, 13.3, 5.2Hz); H-30~q, 1.62(dd, ./=13.0, 6. IHz); H= 33, 1.14(d, J=6.4Hz); H-34, 1.55(s); H-35, 1.20(s); H-36, 1.71(br, s); H-38a, 5.090ar, s); H-38b, 4.91(br, s); H-39, 1.23(s); and H-40, 1.42ppm (s). 13CNMR.:

(CD3OD) C-2, 155.1; C-3, 120.8; C-4, 136.5; C-5, 125.8; C-6, 118.9; C-7, 111.4; C8, 143.4; C-9, 122.7; C-10, 203.8; C-11, 46.6; C-12, 34.4; C-13, 28.7; C-14, 50.5; C15, 35.7; C-16, 77.3; C-18, 73.2; C-19, 59.9; C-20, 18.8; C-21, 30.5; C-22, 78.5; C23, 66.7; C-24, 62.2; C-25, 66.5; C-26, 75.1; C-27, 72.7; C-28, 29.0; C-30, 27.0; C31, 44.0; C-32, 50.9; C-33, 12.7; C-34, 19.0; C-35, 28.7; C-36, 19.8; C-37, 143.2; C38, 111.9; C-39, 19.0; and C-40, 21.2ppm. Mass Spectrum: EIMS: 599(M+, 26%), 493(15), 469(26), 400(9), 265(12), 264(65), 134(26), 133(23), 131(23), 130(100), and 119m/e (54); HREIMS: obsd 599.3244re~e, calcd for C37H45NO6, 599.3247.

Reference J. A. Laakso, J. B. Gloer, D.T. Wicklow, and P. F. Dowd; A New Penitrem Analog with Antiinsectan Activity from the Sclerotia of Aspergillus sulphureus; J. Agric. Food Chern, Vol. 41, pp. 973-975(1993).

6. Penitrems/Lolitrems

421

~ommon/Systematic Name Penitrem C Molecular Formula/Molecular Weight C37H44NO4CI, M'W = 601.29589

H

Me

Me

ethYlS, M e lI"'H' H,,,19H~ :

~,OH A

38

General Characteristics White amorphous solid. Fungal Source Penicillium crustosum.

Isolation/Purification P. crustosum cultures were filtered and the mycelium macerated with acetone in a Waring

blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v) to give the mixture of penitrems. Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, after crystallization from aqueous methanol, roquefortine, m.p. 196-200~ The mixture of penitrems was separated and purified by column chromatography on silica gel using benzene-acetone (85:15, v/v) to yield the six penitrems in the following order of descending Re values: penitrem F, B, A, E, C, and D. Biological Activity Highly tremorgenic and acutely toxic.

422

6.

Penitrems/Lolitrems

Spectral Data UV:

ImMff" 232 and 292nm (e=38,200 and 12,500).

IR: (KBr) 3400, 2930, 1700, and 1650cm"~. CD: 310, 0; 299,-2.11; 260,-0.40; 235,-13.85; 220,-10.46; and 21 lnm,-14.27. ~H NMR: Refer to the reference below for detailed IH NMR data. 13C NMR: [(CD3)2CO] C-2, 154.38; C-3, 119.33; C-4, 130.73; C-5, 125.73; C-6, 125.28; C-7, 110.56; C-8, 122.33; C-9, 138.75; C-10, 35.63; C-1 l, 148.94; C-12,34.86; C-13, 26.61; C-14, 52.37; C-15, 39.63; C-16, 75.49; C-18, 72.03; C-19, 58.89; C-20, 19.06; C-21, 35.00; C-22, 77.45; C-23, 148.33; C-24, 119.70; C-25, 64.27; C-26, 74.36; C-28, 80.38; C-29, 29.19; C-30, 27.71; C-31, 43.67; C-32, 49.91; C-33, 107.35; C-34, 18.64; C-35, 28.77; C-36, 19.97; C-37, 143.90; C-38, 110.78, C-39, 20.10; and C-40, 21.15ppm. Mass Spectrum: HREIMS showed M~ at 601.2956, C37H44NO4CIrequires 601.2958. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vieggaar, P. L. Wessels and W. E. Hull; Tremorgenic Mycotoxins from Penicillium crustosum: Isolation ofPenitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983).

6.

Penitrems/Lolitrems

423

Common/Systematic Name Penitrem D Molecular Formula/Molecular Weight C37H45NO4; M 3 ~ = 5 6 7 . 3 3 4 8 6

H

H,,,

Me

r'.,,.V ~.,,,,Me 147""~'~

38

General Characteristics Penitrem D crystallized from benzene as white needles; mp., >300~ (decomp.). Fungal Source Penicillium crustosum.

Isolation/Purification P. crustosum cultures were filtered and the mycelium macerated with acetone in a Waring

blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v) to give the mixture of penitrems. Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, after crystallization from aqueous methanol, roquefortine; mp., 196-200 ~C. The mixture of penitrems was separated and purified by column chromatography on silica gel using benzene-acetone (85:15, v/v) to yield the six penitrems in the following order of descending Rf values: penitrem F, B, A, E, C, and D. Biological Activity Highly tremorgenic and acutely toxic.

424

6.

Penitrems/Lolitrems

Spectral Data UV~ ~, MeOH max

220 and 286nm (e=34,300 and 11,200).

CD: 310, 0; 282,-1.95; 255,-0.60; 232,-12.85; 223,-9.83; 209, -18.89; and 199nm, 0. IR~

(KBr) 3400, 1700, and 1650cm"~. IH NMR: Refer to de Jesus et al., 1983. ~3CNMR: [(CD3)2CO] C-2, 153.49; C-3, 119.16; C-4, 128.80; C-5, 128.09; C-6, 120.91; C-7, 110.22; C-8, 123.18; C-9, 139.27 C-10, 38.75; C-I 1,150.23; C-12, 35.04; C-13, 26.70; C-14, 52.36; C-15, 39.37; C-16, 75.44; C-18, 72.17; C-19, 58.87; C-20, 19.11; C-21, 35.08; C-22, 77.48; C-23, 148.44; C-24, 119.59; C-25, 64.28; C-26, 74.40; C-28, 80.39; C-29, 29.24; C-30, 27.68; C-31, 43.71; C-32, 49.77; C-33, 105.83; C-34, 18.64; C-35, 28.81; C-36, 19.98; C-37, 143.92; C-38, 110.75, C-39, 20.11; and C-40, 21.32ppm. Mass Spectrum: HREIMS showed M+ at 567.3339; C37H45NO4requires 567.3348. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels, and W. E. Hull; Tremorgenic Mycotoxins from Penicillium crustosum: Isolation of Penitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983).

6. Penitrems/Lolitrems

425

Common/Systematic Name Penitrem E Molecular Formula/Molecular Weight C37I"I4sNO6; ~

= 599.32469

H Me ~',,,.I L, ,Me 147"'''1'~
%,H H

....

.27 ~

OH

.,Me

General Characteristics White amorphous solid. Fungal Source Penicillium crustosum.

Isolation/Purification P. crustosum cultures were filtered and the mycelium macerated with acetone in a Waring

blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v) to give the mixture of penitrems. Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude r0quefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, atier crystallization from aqueous methanol, roquefortine, m.p. 196-200~ The mixture of penitrems was separated and purified by column chromatography on silica gel using benzene-acetone (85:15, v/v) to yield the six penitrems in the following order of descending Rf values: penitrem F, B, A, E, C, and D. Biological Activity Highly tremorgenic and acutely toxic.

426

6.

Penitrems/Lolitrems

Spectral Data UV:

MeOH 220 and 286nm (c=34,200 and 11,200). ~ max

IR:

(KBr) 3400, 2930, 1700, and 1650cm"~. CD: 325, 0; 317, 0.58; 300, 0; 250, 0; 234, -11.28; and 210nm, 0. 1H N M R :

The only major differences between the IH NMR spectra of penitrem A and E are observed for those protons in close proximity to the chlorine atom. Two ortho-substituted protons were evident from the magnitude of the coupling constant (8.3Hz) observed for resonances at 6.70 and 7.09ppm. The chemical shift difference for the C-10 protons (10-Ha and 10-I-Ib)was 0.57ppm for penitrem A but -0.26ppm in the case of penitrem E (see de Jesus, et al., 1983 for fine structure in IH NMR spectrum). 13C N M R :

[(CD3)2CO]C-2, 153.48; C-3, 120.38; C-4, 131.52; C-5, 128.16; C-6, 120.34; C-7, 111.63; C-8, 24.27; C-14, C-22, 78.28; 28.94; C-30, 31.10; C-36,

122.75; C-9, 140.16; C-10, 38.11; C-11,150.91; C-12, 47.41; C-13, 52.78; C-15, 81.08; C-16, 76.09; C-18, 72.52; C-19, 58.86; C-20, 18.63; C-23, 66.17; C-24, 61.95; C-25, 66.34; C-26, 74.69; C-28, 72.05; C-29, 26.89; C-31, 43.58; C-32, 49.92; C-33, 105.47; C-34, 20.28; C-35, 19.70; C-37, 143.31; C-38, 111.62, C-39, 18.99; and C-40, 21.53ppm.

Mass Spectrum: HREIMS showed M + at 599.3252; C37H45NO6, requires 599.3246. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels, and W. E. Hull; Tremorgenic Mycotoxilas from Penicillium crustosum: Isolation of Penitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983).

6. Penitrems/Lolitrems

427

Common/Systematic Name Penitrem F Molecular Formula/Molecular Weight C37I-I44NO5C1, M W = 617.29080

H Me 34 r,~ I l,,Me

,,./1~

......' ~ _

16 (,)

_ . .

6

c ' ~ N H

1

~e~o/._~~7-

.~.. OH i '"H

Me

38

General Characteristics White amorphous solid. Fungal Source Penicillium crustosum.

Isolation/Purification P. crustosum cultures were filtered and the mycelium macerated with acetone in a Waring

blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v) to give the mixture of penitrems. Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, after crystallization from aqueous methanol, roquefortine, m.p. 196-200~ The mixture of penitrems was separated and purified by column chromatography on silica gel using benzene-acetone (85:15, v/v) to yield the six penitrems in the following order of descending Rf values: penitrem F, B, A, E, C, and D. Biological Activity Highly tremorgenic and acutely toxic.

428

6.

Penitrems/Lolitrems

Spectral Data UV~ 232 and 292nm (e=35,500 and 11,30, respectively). IR~ (KBr) 3400, 2930, 1700, and 1650cm"~. CD: 315, 0; 290, -2.50; 255, 0; 220, - 10.10, and 200nm, 0. IH NMR; In the IH ~ spectrum of penitrem F, a new proton signal was observed at 3.87ppm. The presence of additional (H,H) couplings between the cyclobutane protons as well as the changes in their chemical shit't values show that the C-15 hydroxyl group in penitrem A is replaced by a hydrogen atom in F. The effect of replacing the C-15 hydroxyl group with a hydrogen atom is especially pronounced for 18-H and the C-34 methyl protons owing to a decrease in steric hindrance as all three of these groups are located below the plane of the eight-membered ring (refer to de Jesus, et al., 1983 below for detailed IH NMR data). ~3C NMR: C-2, 153.86; C-3, 119.56; C-4, 130.76; C-5, 125.76; C-6, 125.36; C-7, 110.60; C-8, 122.33; C-9, 138.81 C-10, 35.63; C-11,148.92; C-12, 34.86; C-13, 26.61; C-14, 52.34; C-15, 39.54; C-16, 75.51; C-18,71.97; C-19, 59.13; C-20, 18.55; C-21, 30.49; C-22, 78.23; C-23, 66.10; C-24, 61.93; C-25, 66.31; C-26, 74.67; C-28, 71.97; C-29, 28.88; C-30, 26.89; C-31, 43.55; C-32, 49.82; C-33, 107.36; C-34, 18.62; C-35, 28.75; C-36, 19.70; C-37, 143.26; C-38, 111.65, C-39, 18.91; and C-40, 21.06ppm. Mass Spectrum: HR IMS: found, M+, 617.2908; C37H44NOsClrequires 617.2907. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels and W. E. Hull; Tremorgenic Mycotoxins from Penicillium crustosum: Isolation of Penitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983).

6. Penitrems/Lolitrems

429

Common/Systematic Name Pennigritrem Molecular Formula/Molecular Weight C37H44NO6CI, M3~ = 633.28572

HbMe Me Ha,, I H ~Hb .... ~ \ H,....~ 0 Hb .Ha Ha~~,T;"x~.,, I,H,,,,.[H ~- Hb. Ha,,,,.~o ~ ' O E , / / q ~ f ~ I ,,,Ha Hb 3~ "QH H H b c ' ~ 7 J ~ ~ ~0:r ~ ~ _":I HH~ /- A H i~le''Me HbI Fla 38 -

General Characteristics Pennigritrem was isolated as a white amorphous solid. Fungal Source

Penicillium nigricans (IM1228669).

Isolation/Purification Extracted with acetone, evaporated to dryness and the residue dissolved in acetonechloroform (1:1, v/v); silica gel 60 (Merck 230-400 mesh) was added and the mixture evaporated and fractionated by flash chromatography using a silica gel column eluted with chloroform-acetone (19:1, v/v). Final purification was with preparative HPLC using Dynamax C~s reversed-phase chromatography with MeOH-water (3:1, v/v) at 1lml/min. and detection at 233nm. Biological Activity Tremorgenic; albino mice (male) administered a colloidal suspension (4.6mg/300 mm3, ethanol-dimethyl sulfoxide-water, 1:1:1, v/v/v) produced tremor within 0.5hr. This represents a marked reduction in tremorgenic activity when compared to penitrem A (50~tg/200mm3). Spectral Data UV:

~.,~ 235 and 295nm (e=35,700 and 10,300). ~H NMR: 1N-H, 10.0; H-7, 7.24; H-10a, 3.63, H-10b, 3.26; H-12, 2.96; H-13a, 2.41, H-13b,

430

6.

Penitrems/Lolitrems

2.24; H-14, 2.45; OH-15, 4.19; H-16 CH3, 1.05, 1.74; H-18,4.90; H-19, 2.60; H-20a, 1.94, H-20b, 1.79; H-21a, 2.03, H-21b, 1.62; OH-22, 3.48; H-24, 3.35; H-25, 4.60; (1H, d=4.17Hz); H-26, 3.87(1H, J=4.17Hz); H-28, 3.50(1H, J=4.21Hz and 7.18Hz); H-29a, 2.66(1H, d=4.21Hz); H-29b, 1.93(1H, d=7.18Hz); H-30a, 2.53; H-30b, 1.74; H-31 CH3, 1.35; H-32 CH3, 1.43; H-33a, 5.01; and H-37, 2X CH3, 1.40, and 1.20ppm. ~3C NMR: C-2, 154.11 s; C-3, 120.47 s; C-4, 133.26 s; C-5, 125.76 s; C-6, 124.50 s; C-7, 111.78 d; C-8, 121.97 s; C-9, 139.66 s; C-10, 35.02 t; C-11,149.42 s; C-12, 46.90 d; C-13, 24.63 t; C-14, 52.63 d; C-15, 80.85 s; C-16, 76.04 s; C-18, 72.59 d; C-19, 58.39 d; C-20, 18.78 t; C-21, 30.65 t; C-22, 77.36 s; C-23, 67.15 s; C-24, 56.52 d; C-25, 70.45 d; C-26, 75.56 s; C-28, 73.79 d; C-29, 27.53 t; C-30, 26.70 t; C-31, 42.89 s; C-32, 50.35 s; C-33, 107.08 t; C-34, 20.26 q; C-35, 31.01 q; C-36, 22.96 q; C-37, 85.93 s; C-38, 21.87 t, q; C-39, 25.71 q; and C-40, 21.46ppm q. Reference J. Penn, J. R. Biddle, P. G. Mantle, J. N. Bilton, and R. N. Sheppard; Pennigritrem, a Naturally-Occurring Penitrem A Analogue with Novel Cyclisation in the Diterpenoid Moiety; J. Chem. Soc., Perkin Trans I, pp. 23-26(1991).

6.

Penitrems/Lolitrems

431

Common/Systematic Name PC-M4 Molecular F.ormula/Molecular Weight C37H49NO4; M W --- 571.36616 OH H

1"

9 |1,

iIH

7c

7a

7

H

6

11

OH 12

2'

General Characteristics Crystalline powder, mp., 269-272~ (dec.). Fungal Source Penicillium crustosum (strain NHL 6491).

Isolation/Purification Penicillium crustosum was cultivated at 25 ~ for 14 days using Czapek-Dox medium

supplemented with 0.2% yeast extract. The mycelium was extracted with CH2C12at room temperature. The residue obtained by evaporation of the extract was ehromatographed on silica gel with C6I-I6-Me2CO (5:1, v/v) followed by repeated purification by LPLC [C6H6-Me2CO (20:1, v/v) or C6Hla-EtOAc-MeOH (12:6:1, v/v/v to obtain PC-M4 and with C6H6-Me2CO (3:1, v/v)] followed by purification by HPLC using the solvent system CrH6-EtOAc (5:2, v/v) to give PC-M5. Biological Activity Biological activity not presented but based on structure PC-M4 is probably tremorgenic. Spectral Data UV: MeOH

~, max

233(log c=4.09) and 283nm (3.71).

IR:

(KBr) 3600 and 3400cm~ (NH, OH).

432

6.

Penitrems/Lolitrem$

CD: (c=4.06x105, MeOH) +1.6(214), -2.0(240), -0.6(271), and -0.Snm (292). ~HNMR: [(CD3)2SO] 0.96(6H, s, Me-13C, Me-l"); 1.21(3H, s, Me-13b); 1.23(3H, s, Me-l"); 1.25(3H, s, Me-10); 1.27(3H, s, Me-10); 1.55(1H, brd, J=l 1.5 Hz, H-5a); 1.62(1H, brd, J=12.2 Hz, H-6B); 1.67-1.88(5H, m, H-6a, H-14B, H-15B, H2-9); 1.72(3H, brs, Me-l'); 1.93(2H, m, H-5B, H-8); 2.05(1H, m, H-15a); 2.29(1H, dd, J=13.1, 10.5 Hz, H-7a); 2.46(1H, ddd, ,/=12.8, 12.8, 5.8 Hz, H-14a); 2.70(2H, m, H-6a, H-9a); 2.84(1H, dd, J=13.1, 5.8 Hz, H-7B); 3.70(1H, brd, J=9.0Hz, H-7d); 3.71(1H, brs, H-2); 3.84(1H, brd, d=4.7Hz, H-3); 4.50(1H, br dd, d=9.2, 7.9Hz, H-15a); 4.85(1H, br s, H-2'); 5.01(1H, brs, H-2'); 5.58(1H, br d, J=4.7Hz, H-4); 6.74(1H, d, J=8.2Hz, H-11); 7.12(1H, d, J=8.2Hz, H-12); and 10.53ppm [1H, br s, H-13 (NH)]. laC NMR: C-2, 79.1; C-3, 62.6; C-4, 117.7; C-4a, 146.7; C-4b, 75.7; C-5, 33.2; C-6, 21.1; C-6a, 49.3; C-7, 28.3; C-7a, 113.7; C-7b, 121.4; C-7c, 133.7; C-7d, 44.2; C-8, 48.6; C-9, 23.4; C-9a, 49.0; C-10, 44.8; Me-10, 21.7, 32.1; C-10a, 141.8; C-11,113.9; C-12, 110.6; C-12a, 139.7; C-13a, 153.0; C-13b, 49.9; Me-13b, 16.2; C-13, 42.1; Me-13c, 19.2; C-14, 26.3; C-15, 27.9; C-15a, 72.9; C-I', 142.9; C-2', 100.0; C-3'(Me), 19.6; C-I", 70.5; and C-Me-l", 27.1, 27.Sppm. Mass Spectrum: HRMS: 571.3662(M+, 17); (calcd for C37I-I49NO4,571.3662), 485(100), and 470role (35). Reference T. Yamaguchi, K. Nozawa, T. Hosoe, S. Nakajima, and K. Kawai; Indoloditerpenes Related to Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Phytochemistry, Vol. 32, pp. 1177-1181(1993).

6. Penitrems/Lolitrems

433

Common/Systematic Name Lolitrem B Molecular Formula/Molecular Weight C42H55NO7; MW = 685.39785 v

Me Me

M

O'

~

42

~ ~ 1 ~H 2 1

..N.T,/ " ' h""J'~~ : Me

HH

0,,! _

.

.,

Me.

.

43

~,Me

H ll,iO

...o.j .... /.i2

"-

H He/\M e General Characteristics Crystals; mp., 303-304 ~ Fungal Source Acremonium lolii, the endophyte of ryegrass (Lolium perenne L.).

Isolation/Purification The lolitrems were extracted from ground endophyte-infected perennial ryegrass seed, containing lolitrem B, by Soxhlet extraction with petroleum spirit (35-60"C) for 11 hours. After concentration in vacuo, the filtered petroleum extract was partitioned against ethanol-water (4:1, v/v). The alcoholic extract was diluted by addition of water, extracted with dichloromethane, the extract was dried (MgSO4), and the solvent was removed in vacuo. The resulting brown oil contained lolitrem B. This oil was dissolved in petroleum spirit, extracted with acetonitrile-water (13:7, v/v), and the petroleum fraction was discarded. The aqueous acetonitrile extract was washed with petroleum spirit, diluted by addition of water, and extracted with dichloromethane. The extract was dried (MgSO4) and the solvent was removed in vacuo to give a brown oil containing lolitrem B. This oil was applied in the minimum volume of dichloromethane to a flash column, packed as a slurry in petroleum spirit. The column was eluted first with petroleum spirit, then with petroleum spirit-dichloromethane (1:1, v/v), dichloromethane, and then with a stepwise gradient of acetonitrile-dichloromethane (1:19, 1:9, 3:17, 1:4, 2:3, and 3:2, v/v). Fractions containing lolitrem B (by HPLC) were concentrated in vacuo to give a brown oil containing lolitrem B. This material was purified by flash chromatography using acetonitrile-dichloromethane (3:47, then with 2:23, v/v) to afford a yellow solid containing lolitrem B. The solid was further purified by flash chromatography using acetonitrile-dichloromethane first at 3:97, and then at 3:47, v/v. Fractions containing

434

6.

Penitrems/Lolitrems

lolitrem B were concentrated in vacuo to give a pale yellow solid containing 62% of lolitrem B. Crystallization from acetonitrile-dichloromethane gave a colorless solid containing lolitrem B and E. The mixture was reacted with acetic anhydride-pyridine (1:1, v/v) and 4-(N,N-dimethylamino)-pyridine for 24 hours to acetylate lolitrem E. The mixture was dissolved in dichloromethane, washed with water, dried (MgSO4), and eluted from a flash column with acetonitrile-dichloromethane (3:17, v/v). The residue was crystallized from acetonitrile-dichloromethane to give lolitrem B. The mother liquor from the first crystallization afforded a second crop of colorless crystals containing lolitrem B. From the resulting mother liquor was obtained a third crop, as a pale yellow solid, containing lolitrem B along with lolitrem F. Recrystallization of this material gave a cream colored solid containing lolitrem B. The lolitrem B from the first, second, and third crops was then combined, recrystallized to give lolitrem B as a colorless solid. Biological Activity A major agent involved in the etiology of ryegrass staggers, a nervous disorder of sheep, cattle, horses, and deer grazing perennial ryegrass (Lolium perenne L.) pastures infected with Acremonium lolii. The disorder is characterized by severe incoordination and hypersensitivity to external stimuli and is of considerable importance to agriculture in New Zealand and Australia, with occasional outbreaks also being reported in other countries. Spectral Data UV: ~H 290(e=6,700) and 267nm (26,800) in the UV spectrum oflolitrem B suggested the presence of a 2,3-disubstituted indole nucleus with a carbonyl group. IR: (KCI) 3530, 3480, and 3315cm"1, assigned to OH and NH groups and a carbonyl absorption band at 1664cm ~. 1H NMR: (CDCI3) A feature of the 500.13 MHZ IH NMR spectrum of lolitrem B was the threeproton signals at 1.717(d, J=l.3Hz), 1.71 l(d, J=l.3Hz), 1.515, 1.370, 1.301, 1.276, 1.276, 1.266, 1.240, and 1.134ppm assigned to the protons often tertiary methyl groups. The remainder of the resonances exhibited extensive fine structure. First-order analyses of these multiplets yielded the values of the proton chemical shifts. 1-NH, 8.313; H-5a, 1.367; H-5b, 2.698; H-6a, 1.749; H-6b, 2.253; H-7, 4.306; H-9, 3.550; H-12, 5.519; H-14, 3.903; H-15, 3.602; H-18a, 1.564; H-18b, 1.423; H-19a, 1.928; H-19b, 1.627; H-20, 2.84; H-21a, 2.617; H-21b, 2.920; H-25a, 3.425; H-25b, 2.961; H-26, 2.660; H-30, 2.763; H-33, 7.831; H-34, 7.194; and H-40, 5.281ppm. ~3C NMR: (CDCI3) C-2, 152.77(s); C-3, 50.69(s); C-4, 42.36(s); C-5, 27.38(t); C-6, 27.98(t); C7, 71.45(d); C-9, 71.21(d); C-10, 74.70(s); C-12, 92.66(d); C-14, 71.1 l(d); C-15,

6.

Penitrems/Lolitrems

435

61.13(d); C-16, 67.73(s); C-17,78.04(s); C-18, 30.26(t); C-19, 20.49(t); C-20, 50.08(d); C-21, 29.16(t); C-22, 118.58(s); C-23, 125.96(s); C-24, 123.89(s); C-25, 28.28(t); C-26, 49.87(d); C-27, 79.26(s); C-29, 79.93(s); C-30, 59.93(d); C-31, 196.51(s); C-32, 136.97(s); C-33, 120.23(d); C-34, 110.42(d); C-35, 142.00(d); C-36, 15.91(q)'; C-37, 16.59(q)'; C-38, 18.87(q)~ C-39, 25.01(q)~ C-40, 121.96(d); C-41, 139.54(s); C-42, 25.65(q); C-43, 18.63(q); C-44, 25.10(q)~ C-45, 28.28(q)'; C-46, 29.3 l(q)~ and C-47, 30.63ppm (q)'. May be interchanged. Mass Spectrum: Molecular ion at m/e 685.3974 with an abundant ion at m/e 348 arising through cleavage of the C-3, C-4 and C-18, C-19 bonds with transfer of a hydrogen atom to the indole-containing fragment. A similar diagnostic fragmentation was observed for the related tremorgens aflatrem, paspalinine, and penitrem A. I-IPLC Data Zorbax Silica, particle size 5-6~m; solvent, dichloromethane-acetonitrile (8020, v/v), 1.8ml/min.; fluorescence detection, excitation at 268nm, emission at 440nm; and UV detection at 268nm. References R. M. Ede, C. O. Miles, L. P. Meagher, S. C. Munday, and A. L. Wilkins; Relative Stereochemistry of the A/B Rings of the Tremorgenic Mycotoxin Lolitrem B; J. Agile. Food Chem., Vol. 42, pp. 231-233(1994). R. T. Gallagher, A. D. Hawkes, P. S. Steyn, and R. Vleggaar; Tremorgenic Neurotoxins from Perennial Ryegrass Causing Ryegrass Staggers Disorder of Livestock: Structure Elucidation of Lolitrem B; J. Chem. Soc. Chem. Commun., pp. 614-616(1984). C. O. Miles, S. C. Munday, A. L. Wilkins, R. M. Ede, and N. R. Towers; Large Scale Isolation of Lolitrem B and Structure Determination of Lolitrem E; J. Agile. Food Chem., Vol. 42, pp. 1488-1492(1994).

436

6.

Penitrems/Lolitrems

Common/Systematic Name Lolitrem E; Lolitrem C Molecular Formula/Molecular Weight C42H57NOT; MW = 687.41350 40

Me

38

O-~/___Me

0

o-

-T ' ~ / ~ ' 2 4 NH/2

16

2.'6

= -""

~e

,,

o

13 = , , ~

,,,,OH 10 ]' 41

29

28

47

e

46

General Characteristics A colorless solid which decomposed without melting above 150 ~C. Fungal Source Acremonium lofiL the endophyte of ryegrass (Lolium perenne L.). Isolation/Purification Lolitrem E was isolated from fractions enriched in minor lolitrems obtained during the purification of lolitrem B. These were subjected to repeated flash chromatography, using acetonitrile-dichloromethane. Eluted fractions were monitored by HPLC, and fractions enriched in lolitrem E were pooled for further purification. After 4 flash chromatography purifications, the crude lolitrem E was dissolved in the minimum of dichloromethane and an equal volume of petroleum spirit was added, precipitating most of the lolitrem E; the bulk of the other lolitrems remained in the mother liquor. The precipitate was treated with dilute acidic ethanol to convert contaminating lolitrems into lolitriol (lolitrem E is stable to acid hydrolysis). The residue was then treated briefly with NaBH4 to reduce contaminating ketosterols to diols. Lolitrem E was then easily separated from the more polar lolitriol and dihydroxysterols by flash chromatography to give a colorless solid. Biological Activity Chemically related to the agents involved in the etiology of ryegrass staggers, a nervous disorder of sheep, cattle, horses, and deer grazing perennial ryegrass (Lolium perenne L.) pastures infected with Acremonium lolii. Lolitrem E was not tremorgenic in mice.

Spectral Data

6.

Penitrems/Lolitrems

437

Soeetral Data _

UV:

Qualitatively, the UV absorbance spectrum was identical to that reported by Gallagher (1985) for lolitrem B; ~,~H 290 and 267nm.

et al.

'H NMR: (CsDsN) 1.34(3H, s, H-40); 1.38(3H, s, H-29); 1.40(3H, s, H-28); 1.45(3H, s, H-26); 1.49(3H, s, H-39); 1.54(3H, s, H-38); 1.58(3H, s, H-47); 1.59(3H, s, H-46); 1.69(3H, s, H-37); 1.75(3H, s, H-25); 2.95(1H, d, d=14. lI-Iz, H-31); 3.59(1H, dd, J=15.6, 4.1Hz, H-36); 3.93(IH, d, J=9.6Hz, H-9); 3.96(1H, br s, H-11); 4.06(2H, br d, J=7.2Hz, H-43); 4.36(1H, d, d=-8.6Hz, H-10); 4.63(1H, t, d=8.5Hz, H-7); 5.36(1H, t, J=6.6Hz, H-44); 5.65(1H, m, 10-OH); and 6.37ppm (1H, s, 13-OH). ~3CNMR: (CsDsN) C-2, 154.7; C-3, 51.5; C-4, 43.1; C-5, 26.9; C-6, 28.7; C-7, 72.1; C-9, 76.2; C-10, 67.8; C-I 1, 64.5; C-12, 69.3; C-13, 77.6; C-14, 30.1; C-15, 21.2; C-16, 50.7; C-17, 29.8; C-18, 118.5; C-19, 126.3; C-20, 124.9; C-21,137.4; C-22, 119.8; C-23, 111.0; C-24, 143.5; C-25, 16.5; C-26, 18.7; C-27, 78.7; C-28, 23.9; C-29, 20.5; C-30, 196.5; C-31, 60.5; C-32, 79.7; C-34, 79.3; C-35, 50.5; C-36, 28.7; C-37, 31.0; C-38, 25.5; C-39, 29.6; C-40, 25.1; C-43, 58.9; C-44, 122.5; C-45, 135.9; C-46, 25.4; and C47, 17.7ppm. Mass Spectrum: EIMS: 688(21%), 687.4101(M+, 687.4135 for C42H57NOT,34), 619(18), 604(18), 586(14), 472(19), 471(30), 468(15), 457(16), 456(30), 349(29), 348(100), 336(15), 335(34), 278(14), and 277m/e (18). HPLC Data Zorbax Silica, particle size 5-6l.tm; solvent, dichloromethane-acetonitrile (80:20, v/v), 1.8ml/min.; fluorescence detection, excitation at 268nm, emission at 440nm; and UV detection at 268nm. Reference C. O. Miles, S. C. Munday, A. L. Wilkins, R. M. Ede, and N. R. Towers; Large Scale Isolation ofLolitrem B and Structure Determination of Lolitrem E; J. Agrie. Food Chem., Vol. 42, pp. 1488-1492(1994).

438

6.

Penitrems / Lolitrems

Common/Systematic Name Lolitriol Molecular Formula/Molecular Weight C37I-I49NO7; M W --- 619.35090

J ao

12o

17

15

28

29

General Characteristics A pale yellow solid; mp., 322-325~ Fungal Source Acremonium lo#i, endophyte on perennial ryegrass.

Isolation/Purification Extracted with dichloromethane, dried over Na2SO4; removal of solvent in vacuo afforded a colorless solid. This was purified by flash chromatography using methanol-chloroform, 6:94, v/v, to afford lolitriol as a pale yellow solid. Biological Activity Nontremorgenic to mice. Spectral Data UV:

,~, maxAeet~

263(1oge=4.72) and 290nm (sh).

IH NMR: (CsDsN) H-5, 2.97; H-6, 2.34; H-7, 4.60; H-9, 3.91; H-14, 1.66; H-15, 2.25; H-17, 2.76; H-22, 8.26; H-23, 7.49; H-25, 1.69; H-26, 1.42; H-28, 1.50; H-29, 1.52; H-31, 2.94; H-35, 2.78; H-36, 3.05; H-37, 1.69; H-38, 1.54; H-39, 1.50, and H-40, 1.36ppm. 13C NMR: (CsDsN) C-2, 154.6; C-3, 50.5" C-4, 42.8' C-5, 27.1 C-6, 29.0; C-7, 72.1" C-9, 77.5; C-10, 68.4; C-1 l, 64.7; C-12, 69.5 C-13, 77.3; C-14, 30.2; C-15" 21.4; C-16, 50.9;

6.

Penitrems / Lolitrems

439

C-17, 29.7; C-18, 118.2; C-19, 125.9; C-20, 124.6; C-21, 137.4; C-22, 120.0; C-23, 111.2; C-24, 143.3; C-25, 16.4; C-26, 18.6; C-27, 72.5; C-28,.24.7; C-29, 28.3; C-30, 196.4; C-31, 60.6; C-32, 79.6; C-34, 79.2; C-35, 50.6; C-36, 28.8; C-37, 30.9; C-38, 25.4; C-39, 29.5; and C-40, 25.0ppm. Mass Spectrum: HREIMS: 619.3528m/e (1Vr, 619.3496 for C37I-hgNOT,43%), 604(41), 471(55), 456(44), 348(100), and 335(44). Reference C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday, and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992).

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Paspaline and Related Metabolites Paspaline Paspalicine Paspilinine Paxilline a-Paxitriol 13-Paxitriol 3-O-Acetoxypaxilline l'-O-Acetylpaxilline Aflatrem (a,a-Dimethylallylpaspalinine) 13-Aflatrem Paspalitrem A (3-Methyl-2-butenylpaspalinine) Paspalitrem B (3-Methyl-3-hydroxy- 1-butenylpaspalinine) Paspalitrem C Sulpinine A Sulpinine B Sulpinine C PC-M4 PC-MS PC-M5' PC-M6

441

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7. Paspaline and Related Metabolites

443

Common/Systematic Name Paspaline Molecular Formula/Molecular Weight C28H39NO2; MW = 421.29808 4

H 3

NH

29

_

6

25

General Characteristics Crystals from methanol; mp., 254~

sublimed sample mp., 264~

[a]D-23 (c=0.36).

Fungal Source

Clavicepspaspali.

Biological Activity Nontremorgenic in mice dosed IP up to 500mg/kg; chemically related to paspalitrem group tremorgens. Spectral Data UV:

/~,max 228-231(C=25,118), 282(7,943), and 291nm (6,309). 13C NMR: C-2, 150.7 s; C-3, 118.3 s; C-4, 118.3 d; C-5, 119.4 d; C-6, 120.4 d; C-7, 111.3 d; C-8, 139.9 s; C-9, 125.1 s; C-10, 52.9 s; C-11, 42.2 s; C-12, 21.9 t; C-13, 24.6 t; C-14, 84.7 d; C-15, 85.7 d; C-16, 37.7 t; C-17, 25.2 t; C-18, 36.4 s; C-19, 46.4 d; C-20, 21.9 t; C-21, 27.5 t; C-22, 48.7 d; C-23, 33.8 t; C-24,71.9 s; C-25, 33.7 qt; C-26, 26.1 qt; C-27, 19.9 q; C-28, 14.5 q; and C-29, 12.7ppm q. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Rf: 0.76. Detection: dull fluorescent spot atter spraying with 50% ethanolic H2SO4 and heating at 100~ for 5 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 390-393(1981).

444

7.

Paspaline and Related Metabolites

J. P. Springer and J. Clardy; Paspaline and Paspalicine, Two Indole-Mevalonate Metabolites from Clavicepspm7~li; Tet. Lett., Vol. 21, pp. 231-234(1980).

7. Paspalineand RelatedMetabolites

445

Common/Systematic Name Paspalicine Molecular Formula/Molecular Weight C27H31NO3, M W = 417.23039 4

H

3

6~ N H

0

" I General Characteristics Large needles from acetone; mp., 240~ (dec.); [a]D +173 ~ (c=0.5); crystals from methanol; mp., 230~ Fungal Source

Clavicepspaspali.

Biological Activity Nontremorgenic relative of paspalinine. No clinical signs in mice dosed (IP) up to 500mg/kg. Spectral Data UV:

~.~x

231(e=47,863), 250(17,782), and 275nm (12,589).

CD: Ae +11.8 13C NMR:

C-2, 149.4 s; C-3, 118.4 s; C-4, 118.6 d; C-5, 119.9 d; C-6, 120.8 d; C-7, 111.6 d; C-8, 140.2 s; C-9, 125.3 s; C-10, 51.5 s; C-11, 39.9 s; C-12, 2.6 t; C-13, 27.7 t; C-14, 104.4 s; C-15, 88.4 s; C-16, 197.6 s; C-17, 118.4 d; C-18, 171.8 s; C-19, 37.5 d; C-20, 28.6 t; C-21, 29.4 t; C-22, 48.9 d; C-23, 32.1 t; C-24, 78.2 s; C-25, 23.8 q; C-26, 29.0 q; C-27, 23.2 q; and C-28, 14.9ppm q. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Rf, 0.85. Detection: dark spot with fluorescent halo under UV light after spraying with 50% ethanolic HzSO4 and heating at 100 ~C for 5 min.

446

7.

Paspaline and Related Metabolites

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 394-397(1981). J. P. Springer and J. Clardy; Paspaline and Paspalicine, Two Indole-Mevalonate Metabolites from Clavicepspaspali; Tet. Lett., Vol. 21, pp. 231-234(1980).

7.

Paspaline and Related Metabolites

447

Common/Systematic Name Paspalinine Molecular Formula/Molecular Weight C27H31NO4; l v r w = 4 3 3 . 2 2 5 3 1

4

6

H

NH

13 (~) -

I"

I

Fungal Source

Claviceps paspali and Aspergillusflavus sclerotia.

Isolation/Purification Extracted with chloroform. The crude extract chromatographed on a silica gel column. Tremorgens eluted with ethyl ether and ethyl acetate. The active fractions were chromatographed on a silica gel column eluted with a linear gradient from benzene to ethyl ether followed by a second gradient from ethyl ether to ethyl acetate. Aflatrem and paspalanine were isolated in the first gradient while dihydroxyaflavinine was isolated during the second gradient elution. Biological Activity EDs0 for tremor > 14mg/kg IP in mice. Other clinical signs: uncoordinated movement, diarrhea, rough hair coat and hypersensitivity to sound stimuli. Gross clinical signs very similar to those produced by paxilline. Spectral Data UV: ~,~"

232(e=39,800) and 275nm (10,500).

13C N M R :

C-2, 152.1 s; C-3, 117.1 s; C-4, 118.4 d; C-5, 119.5 d; C-6, 120.3 d; C-7, 111.5 d, C-8, 139.9 s; C-9, 125.2; C-10, 51.5 s; C-11, 39.9 s; C-12, 21.1 t; C-13, 26.3 t; C-14, 104.5 s, C-15, 87.9 s; C-16, 197.2 s; C-17, 117.5 d; C-18, 169.9 s, C-19, 77.4 s, C-20, 27.4 t; C-21, 28.2 t; C-22, 48.6 d; C-23, 32.8 t; C-24, 78.6 s; C-25, 23.5 q; C-26, 28.8 q; C-27, 23.0 q; and C-28, 16.3ppm q.

448

7.

Paspaline and Related Metabolites

TLC Data Silica gel G-HR; chloroform-acetone, 93:7, v/v; Re: 0.52. Detection: blue spot after spraying with 50% ethanolic HzSO4 and heating at 100~ for 5 min; fluorescent under UV light. References 1L J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 398-401 ( 1981). R. J. Cole, J. W. Domer, J. P. Springer, and R. H. Cox, Indole Metabolites from a Strain

ofAspergillusflavus; J. Agric. Food Chem., Vol. 29, pp. 293-295(1981).

R. T. Gallagher, J. Finer, J. Clardy, A. Leutwiler, F. Weibel, W. Acklin, and D. Arigoni; Paspalanine, A Tremorgenic Metabolite from Clavicepspaspali Stevens et Hall, Tet. Lett., Vol. 21, pp. 235-238(1980).

7. Paspaline and Related Metabolites

449

Common/Systematic Name Paxilline Molecular Formula/Molecular Weight C27H33NO4, MW = 43 5.24096 4

H

I

21 iOH

I -OH General Characteristics Clear cubes from acetone; mp., 252~ (paxilline crystallized with 1 molecule of acetone H-bonded to the hydrogen of the indole nitrogen). Fungal Sour.ce

Penicillium paxilli, Emericella desertorum, E. foreolata, E. striata, and Acremonium lolii, an endophyte of perennial ryegrass (Lolium perenne L.).

Isolation/Purification The tremorgen was extracted with hot chloroform and the crude extract was chromatographed on a silica gel column; the tremorgen eluted with diethyl ether. The ether fraction was chromatographed on a silica gel column eluted with a linear gradient from toluene to ethyl acetate. Paxilline was crystallized from acetone solution. Biological Activity In day-old cockerels dosed orally: at 25mg/kg (lmg/cockerel) tremors were intermittent; at 100mg/kg (4mg/cockerel) tremors were more sustained. Mortality was observed at higher dosage levels. In mice dosed intraperitoneally, intermittent tremors were observed at levels down to 35mg/kg. Tremors were enhanced when the animal tried to move or was forced to move. Mice were noticeably hypersensitive to sound stimuli. No mortality was observed in mice dosed up to 227mg/kg. Mice continued to feed or attempt to feed. Suspected as one of the tremorgens responsible for the clinical signs of ryegrass staggers. Spectral Data UV: ~b McOH max

230(E=41,500) and 281nm (8,000).

IR: (KBr) 3420, 1650, 1355, 1365, and 740cm "~.

450

7.

Paspaline and Related Metabolites

CD: AE 335(+ 1.2) and 300nm (+ 3.3). 13C NMR: C-2, 152.1 s; C-3, 114.5 s; C-4, 117.2 d; C-5, 118.4 d; C-6, 118.8 d; C-7, 111.4 d; C-8, 139.3 s; C-9, 124.1 s; C-10, 50.0 s; C-11, 39.9 s; C-12, 20.6 t; C-13, 25.8 t; C-14, 72.2 d; C-15, 83.1 d; C-16, 196.9 s; C-17, 117.9 d; C-18, 169.9 s; C-19, 75.4 s; C-20, 26.7 t; C-21, 28.1 t; C-22, 49.1 d; C-23, 32.4 t; C-24, 70.8 s; C-25, 25.7 qt; C-26, 25.8 q; C-27, 18.6 q; and C-28, 16.2ppm q. Mass Data: 435.2422(M+), 420.2165, 378.2033, 377.1998, 363.1779, and 362.1753role. (Anal. found: C, 75.29; H, 7.68; N, 3.12; calcd for C27H33NO4; C, 74.48; H, 7.58; N, 3.21; O, 14.70). TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Rf: 0.75. Detection: A dark spot with 50% ethanolic H2SO4 spray; a greenish-gray spot with 3% ethanolic phosphomolybdic acid. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 386-389(1981). R. J. Cole, J. W. Kirksey, and J. M. Wells; The Structure of Paxilline, a Tremorgenic Metabolite of Penicillium paxilli Bainier; Can. J. Microbiol., Vol. 20, pp. 1159-1162(1974). C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday, and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992). J. P. Springer, J. Clardy, J. M. Wells, R. J. Cole, and J. W. Kirksey; The Structure of Paxilline, a Tremorgenic Metabolite ofPenicillium paxilli Bainier; Tet. Lett. pp. 2531-2534(1975).

7. Paspaline and Related Metabolites

451

Common/Systematic Name a-Paxitriol Molecular Formula/Molecular Weight C27H35NO4; M W = 437.25661 17

27.,OH

O" ,,,,,,-,~

General Characteristics Colorless solid that decomposed without melting at 250-3000C. Fungal Source A c r e m o n i u m lolii

endophyte on perennial ryegrass.

Isolation/Purification Extracted with dichloromethane and dried over Na2SO4 overnight at 4~ in the dark. Removal of solvent in vacuo afforded a pale yellow oil. Addition of dichloromethane resulted initially in dissolution of the product, followed by precipitation. Atter standing at 4~ for 30 min, solvent was removed by decantation. The off-white crystals were washed with fresh solvent to afford pure 13-paxitriol. The washings were purified by flash chromatography (acetone-dichloromethane, 3:17, v/v) to give pure a-paxitriol (the less mobile diastereoisomer). Biological Activity Nontremorgenic to mice at 100mg/kg; caused lethargy and rough hair coats, and normal activities such as walking, rearing and preening were greatly reduced for several hours. Spectral Data UV:

X^~,a~o

23 l(log e=4.53) and 281nm (3.91).

~H NMR: (CDCla-DMSO) H-5, 2.57; H-6, 2.01; H-7, 4.60; H-9, 3.03; H-1 l, 5.35; H-14, 1.69; H-15, 2.06; H-17, 2.36; H-20, 7.28; H-21, 6.92; H-22, 6.91" H-23, 7.27; H-25, 1.29; H-26, 0.95" H-28, 1.22; and H-29, 1.26ppm. 13C NMR:

(CDCI3-DMSO) C-2, 153.5; C-3, 50.5; C-4, 42.5; C-5, 26.8; C-6, 28.3; C-7, 73.3;

452

7. Paspaline and Related Metabolites

C-9, 81.7; C-10, 64.5; C-11,120.2; C-12, 144.5; C-13, 75.8; C-14, 33.5; C-15, 21.2; C-16, 49.7; C-17, 27.1; C-18, 115.1; C-19, 124.7; C-20, 117.6; C-21, 118.4; C-22, 119.1; C-23, 111.7; C-24, 139.9; C-25, 16.3; C-26, 19.5; C-27, 72.4, C-28, 24.2; and C-29, 27.8ppm. Mass Spectrum: 437.2587(M+, 437.2557 for C27HasNO4,55%), 422(47), 419(42), 404(28), 343(15), 328(18), 208(9), 182(100), 169(54), and 230m/e (56). Reference C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday, and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992).

7.

Paspaline and Related Metabolites

453

Common/Systematic Name 13-Paxitriol Molecular Formula/Molecular Weight C27H35NO4;1VIW= 437.25661 17

(

20

OH 2 ~ ,,,,"

General Characteristics Colorless solid that decomposed without melting at 250-300~ Fungal Source A cremonium lolii

endophyte on perennial ryegrass.

Isolation/Purification Extracted with dichloromethane and dried over Na2SO4 overnight at 4 ~ in the dark. Removal of solvent in vacuo afforded a pale yellow oil. Addition of dichloromethane resulted initially in dissolution of the product, followed by precipitation. ARer standing at 4 ~ for 30 min, solvent was removed by decantation. The off-white crystals were then washed with fresh solvent to afford pure [3-paxitriol. The washings were purified by flash chromatography (acetone-dichloromethane, 3:17, v/v) to give pure tt-paxitriol (the less mobile diastereoisomer). Biological Activity Nontremorgenic to mice at 100mg/kg; caused lethargy and rough hair coats, and normal activities such as walking, rearing and preening were greatly reduced for several hours. Spectral Data UV: ~ Acetonitfil 9 max

23 l(log e=4.58) and 280nm (3.94).

~H NMR: (CsDsN) H-5, 2.61; H-6, 2.19; H-7, 4.61; H-9, 3.06; H-10, 4.19; H-11, 5.64; H-14, 1.69; H-15, 2.03; H-17, 2.39; H-20, 7.33; H-21, 6.96; H-22, 6.94; H-23, 7.32; H-25, 1.33; H-26, 1.04; H-28, 1.29; and H-29, 1.30ppm. IaC NMR: (CsDsN) C-2, 153.4; C-3, 50.5; C-4, 42.4; C-5, 26.8; C-6, 28.3; C-7, 74.1; C-9, 80.4;

454

7.

Paspaline and Related Metabolites

C-10, 63.3; C-11, 117.5; C-12, 147.9; C-13, 76.2; C-14, 33.7; C-15, 21.2; C-16, 49.8; C-17, 27.1; C-18, 115.2; C-19, 124.7; C-20, 117.6; C-21, 118.5; C-22, 119.2; C-23, 111.8; C-24, 139.9; C-25,16.3; C-26, 19.6; C-27, 72.0; C-28, 26.7; and C-29, 27.1ppm. Mass Spectrum: 437.2558(M § 437.2557 for C27H35NO4,55%), 422(52), 419(15), 343(18), 328(18), 208(17), 182(100), 69(52), and 130m/e (57). Reference C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992).

7.

Paspaline and Related Metabolites

455

Common/Systematic Name 3-O-Acetoxypaxilline Molecular Formula/Molecular Weight C29H37NOs; ~

8

11

= 479.26717

H

NH

6

i''~ 13

,4 R

H ['OH

General Characteristics Colorless crystalline powder; mp., 162-164 ~C. Fungal Source Penicillium crustosum (strain NHL 6491) isolated from mycelia of contaminated bread

~ i c h had been intended for school lunches in Shinagawa-ku, Tokyo, Japan in 1983. Isolat~ion P. crustosum was cultivated at 25 ~ for 14 days containing Czapek-Dox medium

supplemented with 0.2% yeast extract. The mycelium was extracted with CH2C12; the residue obtained by evaporation of the extract and chromatography on silica gel with benzene-acetone (100:1, v/v) gave ergosterol; with benzene-acetone (50:1, v/v) followed by purification by LPLC using benzene gave penitrems F and B; with benzene-acetone (20:1, v/v) followed by purification by LPLC using benzene-acetone (100:1, v/v) gave penitrems A and E; with benzene-acetone (10:1, v/v) followed by further purification by LPLC [benzene-acetone (50:1, v/v)] gave penitrem D; with benzene acetone (5:1, v/v) followed by repeated purification by LPLC using benzene-acetone (20:1, v/v) and/or hexane-AcOEt-EtOH gave cyclopenin and 3-O-acetoxypaxilline; and with benzene-acetone (3:1, v/v) followed by purification by HPLC using benzene/AcOEt (5:2, v/v) gave dihydrodehydroxypaxilline. Biological Activity Probable biosynthetic precursor for the penitrems and probably tremorgenic; however, tremorgenic activity was not reported. Spectral Data UV:

~

MeOH max

229(1og e=4.74) and 279nm (4.05).

IR:

(KBr) 3500, 3460, 3400(NH, OH), and 1720cm1 (OAc).

456

7. Paspaline and Related Metabolites

CD: (c=4.55 x 105, MeOH) [0] 2~ -61,000(232), +4,900(286), and +5,000nm (294). ~H NMR: [(CD3)2CO] 1.00(3H, s, 12C-Me), 1.20(3H, 5, 2'-Me), 1.26(3H, s, 3'-Me). 1.36(3H, s, 12b-Me), 1.70(3H, m), 1.82(1H, dddd, J=13.5, 13.5, 8.5, 5.2), 1.95(1H, ddd, ,/--13.5, 13.5, 4.5), 1.99(3H, s, 3-OCOMe), 2.10(1H, m), 2.39(1H, dd, J=13.0, 10.8, 7B-H), 2.67(1H, dd, ,/=13.0, 6.3, 7a-H), 2.69(1H, ddd, ,]--13.3, 13.3, 4.8), 2.85(1H, m), 3.32(1H, d, J=l.8, 2-H), 3.48(1H, s, OH), 3.49(1H, s, OH), 4.65(1H, br dd, J=10.5, 7.6, 14a-I-I), 5.20(1H, ddd, J=5.8, 1.8, 1.8, 3-H), 5.69(1H, dd, J=5.8, 1.8, 4-I-I), 6.94(2H, m, 9-H, 10-H), 7.28(1H, m, 8-H or 1l-H), 7.32(1H, m, 11-H or 8-H), a~nd 9.85ppm [1H, br s, 12-H (NH)]. 13C NMP~:

[(CD3)2CO]C-2, 82.56; C-3, 66.01; C-4, 115.21; C-4a, 151.29; C-4b, 77.52; C-5, 34.71; C-6, 22.0; C-6a, 50.67; C-7, 30.56; C-7a, 116.71; C-7b, 126.11; C-8, 118.66; C-9, 119.54; C-10, 120.36; C-11,112.45; C-1 la, 141.23; C-12a, 153.84; C-12b, 51.61; 12b-Me, 16.73; C-12c, 43.53; 12-C-Me, 19.93; C-13, 27.87; C-14, 29.13; C-14a, 75.06; C-I', 71.58; C-2', 25.99; C-3', 27.95; MeCOO, 21.28; and MeCOO, 170.69ppm. Mass Spectrum: El-MS" 479.2669(M*, 479.2670 for C29H37NOs,63), 464.2433[(M-Me)+, 464.2433 for C28H34NOs, 100], 182(87), and 130m/e (68). Reference T. Hosoe, K. Nozawa, S. Udagawa, S. Nakajima, and K. Kawai; Structures of New Indoloditerpenes, Possible Biosynthetic Precursors of the Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Chem. Pharm. Bull., Vol. 38, pp. 3473-3475 (1990).

7. Paspaline and Related Metabolites

457

Common/Systematic Name 1'-O-Acetylpaxilline Molecular Formula/Molecular Weight C29H35NOs; M W = 477.25152

8

7

H

6

,,

~

~4 R -

R

I~OAc !

3'

General Characteristics Colorless needles from ethyl acetate; mp., 262-265~ (sublim.). Fungal Source Emericella striata, strain 80-NE-22. Isolation/Purification The dried mycelia were extracted with acetone at room temperature. The acetone extract was chromatographed on silica gel with benzene-acetone followed by column chromatography with benzene-ethyl acetate (5:1, v:v). It was further purified by repeated LPLC [hexane-ethyl acetate (10:1, v:v)] to yield l'-O-acetylpaxilline. Biological Activity Tremorgenic. Spectral Data UV: ~EtOH max

23 l(log E=4.62). 280(3.97), and 290nm (sh) (3.92).

IR:

3480, 3400(OH, NH), 1728(OAc), and 1680cm~ (-CO-). CD: e

-80400(244), -1090(268sh), +8260(296), and +3430nm (338).

1H NMR: (CDCI3) 1.02(3H, s, Me); 1.32(3H, s, Me); 1.43(3H, s, Me); 1.50(1H, m); 1.61(1H, s, OH), 1.65(3H, s, Me); 1.55-2.08(6H, m); 2.04(3H, s, OAc); 2.27(1H, m), 2.44(1H, dd J=13.1, 10.9Hz), 2.73(1H, dd J=13.1, 6.1Hz); 2.80(1H, m); 4.77(1H, brt, J=10.0Hz,

458

7.

Paspaline and Related Metabolites

CH2-CH-O-); 4.79(1H, d J=l.8Hz, (=CH-O-)); 5.78(1H, d J=I.8Hz(=C=CH); 7.07(2H, m); 7.29(1H, m); 7.43(1H, m); and 7.83ppm (1H, brs. NH). 13CNMR: [CDCI3 plus trace (CD3)280] C-2, 80.43; C-3, 195.31; C-4, 120.07; C-4a, 166.21; C-4b, 77.37; C-5, 28.05; C-6, 28.37; C-6a, 49.54; C-7, 34.23; C-7a, 117.49; C-7b, 125.15; C-8, 118.52; C-9, 119.73; C-10, 120.64; C-I 1, 111.52; C-1 la, 139.81; C-12a, 151.79; C-12b, 50.85; C-12c, 43.14; C-13, 20.94; C-14, 27.22; C-14a, 72.94; C-I', 81.89; C-2', 22.84; C-3', 23.86; 12b-Me, 16.21 12c-Me, 19.73" O-CO-Me, 22.31" and O-CO-Me, 170.92ppm. Mass Data: El-MS: 477(100% M~), 462(75 [M-Me]), 417(22 [M+-AcOH], 402(25[M+-AcOH-Me]), 384(20 [M-AcOH-Me-H20]), 182(65), and 130(45); anal calcd for C29H35NOs;C, 72.93: H, 7.39: N, 2.93; found: C, 73.07: H, 7.42: N, 2.81. Reference K. Nozawa, Y. Horie, S. Udagawa, K. Kawai, and M. Yamazaki; Isolation of a New Tremorgenic Indoloditerpene, l'-O-Acetylpaxilline, from Emericella striata and Distribution of Paxilline in Emericella spp.; Chem. Pharm. Bull., Vol. 37, pp. 1387-1389 (1989).

7. Paspaline and Related Metabolites

459

Common/Systematic Name Aflatrem; a, a-Dimethylallylpaspalinine Molecular Formula/Molecular Weight C32H39NO4; M W = 5 0 1 . 2 8 7 9 1

29~ ~

H

30

12~~00~24 General Characteristics Colorless needles from ethyl ether-acetone; mp., 222-224 ~ Fungal Source

Aspergillus flavus.

Isolation/Purification Fungal cultures were extracted with chloroform and the crude extract was chromatographed on a silica gel column. Tremorgens eluted with ethyl ether and ethyl acetate. The active fractions were combined and chromatographed on a silica gel column eluted with a linear gradient from benzene to ethyl ether followed by a second gradient from ethyl ether to ethyl acetate. Aflatrem and paspalanine were isolated in the first gradient. Biological Activity Tremorgenic when dosed IP or orally to mice, guinea pigs, and rats. A dose of lmg of partially purified toxin to mice via stomach tube initially resulted in inactivity but animals responded to auditory and tactile stimuli. Tremors became marked when animals attempted to move. Trembling became more pronounced in 1-2 hr after dosing and continued for several hours. Marked improvement was usually evident the day after treatment with the toxin. Spectral Data UV:

MeOH

231(e=27,750), 282(9,050), and 292nm (sh) (7,850).

460

7. Paspaline and Related Metabolites

13C NMR: C-2, 151.8 s; C-3, 115.1 s; C-4, 140.6 s; C-5, 115.8 d; C-6, 118.9 d; C-7, 110.6 d; C-8, 139.3 s; C-9, 123.3 s; C-10, 50.5 s; C-11, 39.4 s; C-12, 21.0 t; C-13, 26.4 t; C-14, 104.5 s; C-15, 87.6 d; C-16, 197.0 s; C-17, 116.9 d; C-18, 170.0 s; C-19, 77.6 s; C-20, 28.2 t; C-21, 32.6 t; C-22, 48.0 d; C-23, 33.8 t; C-24, 78.0 s; C-25, 28.8 q; C-26, 22.9 q; C-27, 22.9 q; C-28, 16.0 q; C-29, 111.1 t; C-30, 140.6 d; C-31, 41.3 s; C-32, 29.3 q, and C-33, 29.5ppm q. TLC Data Silica gel G-HR; chloroform-acetone, 93:7 (v/v); Re: 0.65. Detection: blue spot after spraying with H2SO4 followed by 1% dimethyl aminobenzaldehyde (ethanolic) and heating at 100 ~C for 1-2 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 410-413(1981). R. J. Cole, J. W. Dorner, J. P. Springer, and R. H. Cox; Indole Metabolites from a Strain ofAspergiilusflavus; J. Agric. Food Chem., Vol. 29, pp. 293-295(1981). R. T. Gallagher and B. J. Wilson; Aflatrem, the tremorgenic mycotoxin from Aspergillus

flavus; Tet. Lett., Vol. 21, pp. 239-240(1980).

W. B. Turner and D. C. Aldfidge; Fungal Metabolites II, Academic Press, New York, New York, pp. 298-300(1983).

7. Paspaline and Related Metabolites

461

Common/Systematic Name 13-Aflatrem Molecular Formula/Molecular Weight C32H39NO4, ~

= 501.28791

23

21

19

17

General Characteristics 13-Aflatrem was obtained as a yellow crystalline solid with the following properties: mp., 188-190~ [a]D +77.9 ~ (C--0.011, in CHCI3). Fungal Source

Aspergillusflavus (NRRL 13462) sclerotia, A. parasiticus (NRRL 6433, 13539), and A. subolivaceus (NRRL 4998).

Isolation/Purification

A. flavus (NRRL 13462) sclerotia were subjected to exhaustive extraction with hexane, and this extract was directly subjected to preparative reversed-phase HPLC [81.tm C~8 column, 2.14x25cm, MeOH:H20 (90:10, v/v) at 8.4ml/min] to afford the following major components: paspalinine (retention time, 17 min), afiatrem (26 min), 13-aflatrem (32 min), nominine (34 min), aflavinine (38.5 min), and paspaline (66 min).

Biological Activity Exhibited significant activity against Helicoverpa zea, causing a 57% reduction in weight gain relative to controls when incorporated into a standard diet at a concentration of 100ppm. Spectral Data UV: Ms ~ max

233(e=18,500), 264(6,300), and 296nm (2,700).

IR:

(CH2CI2) 3572, 3468, 3060, 2971, 2937, 1690, 1614, 1455, 1275, and 894cmq. ~H NMR: (CDCI3) H-l, 7.73, s; H-5, 7.43, d, J=1.5; H-7, 7.13, dd, J=l.5, 8.8; H-8, 7.27, d,

462

7. Paspaline and Related Metabolites

,/--8.8; H-12, 2.26, m, 2.69, m; H-13, 1.75, m, 1.82, m; H-15, 4.32, s; H-17, 5.86, s; H-20, 2.73, m, 2.83, m; H-21, 2.45, dd, ,]--12.5, 10.3, 2.73, m; H-22, 2.83, m; H-23, 1.92, m, 1.97, m; H-25, 1.14, s; H-26, 1.42, s; H-27, 1.19, s; H-28, 1.23, s; H-30, 6.12, dd, J=10.5, 17.3; H-31, 5.05, br d, J=10.5, 5.09, br d, J=17.3; and H-32/33, 1.48ppm, br s. ~3CNMR: (CDCI3) C-2, 152.45; C-3, 117.22; C-4, 124.94; C-5, 115.32; C-6, 140.11; C-7, 119.54; C-8, 111.04; C-9, 138.14; C-10, 51.45; C-11, 39.86; C-12, 26.93; C-13, 21.14; C-14, 104.36; C-15, 87.96; C-16, 197.28; C-17, 117.58; C-18, 169.84; C-19, 77.61; C-20, 28.25; C-21, 27.58; C-22, 48.55; C-23, 33.82; C-24, 78.71; C-25, 23.07; C-26, 28.76; C-27, 23.55; C-28, 16.27; C-29, 41.05; C-30, 149.01; C-31, 109.87; and C-32/33, 28.83, 28.76ppm. Mass Spectrum: 501(M+, 20), 4.86(21), 483(18), 468(18), 443(10), 428(16), 425(31), 410(48), 387(17), 372(4), 344(5), 250(31), 237(37), 222(23), 198(35), 182(50), 168(37), and 130m/e (100); HREIMS: found 501.2877; calcd for C32H39NO4, 501.2881. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Aflavarin and [3-Aflatrem: New Anti-Insectan Metabolites From The Sclerotia ofAspergillusflavus; J. Natural Products, Vol. 55, pp. 1080-1086(1992).

7. Paspaline and Related Metabolites

463

Common/Systematic Name Paspalitrem A; 3-Methyl-2-butenylpaspalinine Molecular Formula/Molecular Weight C32H39NO4; M W - 5 0 1 . 2 8 7 9 1

23

H

21

Fungal Source

Claviceps paspali sclerotia (collected from Paspalum dilatatum infected plants) and an endophytic Phomopsis sp. (MF5670) isolated from the living bark of Cavendishia pubescens, a shrub or small tree of dry or exposed slopes and thickets in upland regions of northwest South America. This isolate was from Colombia. A potential ecological role of this metabolite in regard to endophytism of the woody host is possible.

Isolation/Purification Fungal sclerotia were extracted with chloroform; the chloroform extract was partitioned between hexane and 80% aqueous methanol. The aqueous methanol extract was chromatographed on a silica gel column eluted with a linear gradient from benzene to ethyl ether. The active fractions were chromatographed on a silica gel column eluted with a linear gradient from benzene to 10% ethyl ether/benzene. Biological Activity EDs0 in mice dosed IP was <14.0mg/kg. Clinical signs were intermittent tremors, diarrhea, rough hair coat, and hypersensitivity to sound stimuli. Gross clinical signs very similar to those produced by paxilline. Spectral Data UV:

/~, maxMeOH 234 and 278nm (e max not reported). ~H NMR: (CDCI3) 1.18, 6H, s; 1.35, 3H, s; 1.42, 3H, s; 1.77, 6H, s; 1.8-2.9, llH, m; 3.62, 21-1, m; 4.29, 1H, s; 5.38, 1H, m; 5.73, 1H, s; and 6.8-8.0ppm, 5H, m. 13C NMR: C-2, 151.2 s; C-3, 116.7 s; C-4, 117.6 d; C-5, 127.9 s; C-6, 120.9 d; C-7, 109.4 d, C-8,

464

7.

Paspaline and Related Metabolites

139.8 s, C-9, 124.6 s, C-10, 51.2 s; C-11, 39.9 s, C-12, 21.1 t, C-13, 27.0 t, C-14, 104.4 s; C-15, 88.0 s, C-16, 197.3 s, C-17, 117.6 d; C-18, 169.8 s; C-19, 77.6 s, C-20, 28.3 t, C-21, 29.4 t, C-22, 48.7 d, C-23, 33.8 t, C-24, 78.7 s, C-25, 23.6 q, C-26, 28.9 q; C-27, 23.1 q, C-28, 16.3 q, C-29, 32.0 t; C-30, 123.7 d, C-31, 133.0 s, C-32, 18.0 q, and C-33, 25.8ppm q. Mass Spectrum: 501.2885m/e (M +) (requires 501.2878) TLC Data Silica gel G-H~ chloroform-acetone, 93:7, v/v, Rr~.60. Detection: blue spot after spraying with 50% ethanolic H2SO4 and heating at 100~ for 5 min, fluorescent under UV

light. References G. F. Bills, R. A. Giscobbe, S. H. Lee, F. Pelaez, and J. S. Tkacz, Tremorgenic mycotoxins, paspalitrem A and C, from a tropical Phomopsis; Mycol. Res., Vol. 96, pp.

977-983(1992).

R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, Ne~' York, p. 402 (1981). R. J. Cole, J. W. Domer, J. A. Lansden, R. H. Cox, C. Papr B. Cunfer, S. S. Nicholson, and D. M. Bedell; Paspalum Staggers: Isolation and Identification of Tremorgenic Metabolites from Sclerotia of Clavicepspaspali; Agric. FooA Chem., Vol. 25, pp. 1197-1201(1977).

7. Paspaline and Related Metabolites

465

Common/Systematic Name Paspalitrem B, 3-Methyl-3-hydroxy- 1-butenylpaspalinine Molecular Formula/Mo!eeular Weight C32H39NOs; M~V = 517.28282

.

.

.

.

.

o

.

2

a

~

General Characteristics Paspalitrem B readily formed a methylated derivative in methanol solution and acidic conditions. The methylation occurred with the tertiary hydroxyl group located on the 3-methyl-3-hydroxy-l-butenyl moiety. Fungal Source

Clavicepspaspali sclerotia.

Isolation/Purification See paspalitrem A. Biological Activity EDs0 for tremorgenic activity was < 14mg/kg in mice dosed IP; other clinical signs were diarrhea, rough hair coat, and hypersensitivity to sound stimuli. Gross clinical signs very similar to those produced by paxilline. Spectral Data UV:

~

MeOH max

227, 248, 305, and 336nm (e max not reported).

~H NMR: (CDC13) 1.21, 3H, s; 1.25, 3H, s; 1.41, 3H, s; 1.47, 3H, s; 1.50, 6H, s; 1.90-2.77, IIH, m; 4.32, IH, s; 5.81, 1H, s; 6.42, 1H, d, J=16.0Hz; 7.0-7.30, 4H, m; and 7.76ppm, 1H, s N-H. 13C NMR: (CDCI3) C-2, 152.3 s, C-3, 116.4 s;,C-4, 116.0 d; C-5, 128.6 s; C-6, 120.9 d; C-7, 110.5 d, C-8, 140.3 s; C-9, 124.2 s; C-10, 51.2 s; C-11, 39.9 d; C-12, 21.1 t; C-13, 27.0 t; C-14, 104.4 s; C-15, 87.9 s; C-16, 197.3 s; C-17, 117.6 d; C-18, 169.7 s; C-19,

466

7.

Paspaline and Related Metabolites

s; C-20, 28.3 t; C-21, 29.9 t; C-22, 48.6; C-23, 33.8 t; C- 24, 78.7 s; C-25, 23.6 q; C-26, 28.8 q; C-27, 23.0 q; C-28, 16.3 q; C-29, 124.7 d; C-30, 137.5 d; C-31, 71.2 s; C-32, 29.9 q; and C-33, 29.9ppm q. TLC Data Chloroform-acetone, 93:7 (v/v); Rf: 0.20. Detection: spray with 50% ethanolic H2SO4 and heat at 100~ for 5 min. Blue-green spot atier spraying; turns blue atter heating; fluorescent under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 406-409 (1981). R. J. Cole, J. W. Dorner, J. A. Lansden, R. H. Cox, C. Pape, B. Cunfer, S. S. Nicholson, and D. M. Bedell; Paspalum Staggers: Isolation and Identification of Tremorgenic Metabolites from Sclerotia of Clavicepspaspali; Agric. Food Chem., Vol. 25, pp. 1197-1201(1977).

7. Paspalineand Related Metabolites

467

Common/Systematic Name Paspalitrem C Molecular Formula/Molecular Weight C32H39NO4, MW = 501.28791

I 23 H 21

6~ N H

_

0

0

124

Fungal Source Claviceps paspali sclerotia and an endophytic Phomopsis sp. (MF5670) isolated from the living bark of Cavendishia pubescens, a shrub or small tree of dry or exposed slopes and thickets in upland regions of northwest South America. This isolate was from Colombia. A potential ecological role of this metabolite in regard to endophytism of the woody host is possible. Isolation/Purification Fungal cultures were extracted three times with methyl ethyl ketone which provided a brown oily residue upon the removal of solvent. Atter adding methylene chloride to the residue, the mixture was sonicated and filtered through sintered glass. The filter cake was washed twice with portions of fresh methylene chloride. The filtrates were pooled, and the solvent was removed in vacuo. The material from methylene chloride was then subjected to silica gel flash chromatography (Silica Gel 60, particle size 0.040-0.063mm, 230-400 mesh ASTM; EM Science). The column was eluted stepwise with methylene chloride-methanol; the effluent was monitored at 240nm. Analysis of fractions by analytical reversed phase HPLC (Whatman Partisil 10 ODS-3, 4-6mm x 25cm; 80% aqueous methanol, isocratic, l ml per min, ambient temperature) revealed the presence of the major components (paspalitrems A and C). The fractions rich in the major component were recovered and subsequently subjected to preparative reversed-phase HPLC (Whatman Partisil 10 ODS-3; 70% aqueous methanol, isocratic, 10ml/min, ambient temperature) to provide the major component (paspalitrem C) and a minor component (paspalitrem A). The retention times for the two compounds by the analytical HPLC procedure were 20.1 and 21.8 minutes, respectively. Biological Activity Tremorgenic in vertebrates and toxic in some insect species.

468

7.

Paspaline and Related Metabolites

Spectral Data UV: M~on max

236 and 282nm.

IH NMR:

(CD2C12) 7.83(H-1, IH, s); 7.13(H-7, 1H, dd, d-8.1, 0.6Hz); 6.96(H-6, 1H, dd, d=8.1, 7.2I-Iz); 6.81(H-5, 1H, dd, J=7.2, 0.6Hz); 5.83(H-17, 1H, d, J=l.2Hz); 5.39(H-30, 1H, tq, J=7.2, 1.5Hz); 4.30(H-15, 1H, d, J=l.5Hz); 3.59(H-29, 2H, d, ,/=7.2); 2.84(H-22, 1H, m); 2.80, 2.07(H-21, 2H, m); 2.76, 2.07(H-20, 2H, m); 2.03(H-13, 2H, m); 1.93(H-23, 2H, m); 1.81(H-12, 2H, m); 1.77(H-33, 3H, s); 1.75(H-32, 3H, s); 1.43(H-26, 3H, s); 1.38(H-28, 3H, s); 1.24(H-27, 3H, s); and 1.17ppm (H-25, 3H, s). 13C NMR:

(CD2C12) 197.4(C=16, s); 169.9(C=18, s), 151.7(C=2, s), 140.3(C-8, s); 133.3(C-31, s), 132.0(C-4, s); 124.9(C-9, s); 124.2(C-30, d); 121.1(C-6, d); 119.2(C-5, d); 118.1(C=17, d); 116.9(C=3, s); 109.6(C=7, d); 104.7(C=14, s); 88.4(C=15, d); 79.0(C-24, s); 78.0(C-19, s); 51.6(C-10, s); 49.1(C-22, d); 40.2(C-11, s); 34.2(C-23, t), 32.4(C-29, t), 29.7(C-21, t); 29.0(C-26, q); 28.7(C-20, t); 27.3(C=13, t); 25.8(C=32, q); 23.8(C-27, q); 23.2(C-25, q); 21.4(C-12, t); 18.0(C-33, q); and 16.3ppm (C-28, q). Mass Spectrum: HREIMS: 501.2894m/e (M+); calcd. 501.2894. References G. F. Bills, R. A. Giscobbe, S. H. Lee, F. Pelaez, and J. S. Tkacz; Tremorgenic mycotoxins, paspalitrem A and C, from a tropical Phomopsis; Mycol. Res., Vol. 96, pp. 977-983(1992). J. W. Dorner, R. J. Cole, R. H. Cox, and B. M. Cunfer; Paspalitrem C, a New Metabolite from Sclerotia of Clavicepspaspali; J. Agric. and Food Science, Vol. 32, pp. 1069-1071 (1984).

7. Paspaline and Related Metabolites

469

Common/Systematic Name Sulpinine A Molecular Formula/Molecular Weight C32I-I41NO4; MW = 503.30356 4

33

34

~

21

A

~

27

26

General Characteristics Sulpinine A was isolated as a yellow-orange solid with the following properties: mp, 149-152~ dec; [a]D -29.6 ~ (C=0.005, in CHCI3). Fungal Source Sclerotia ofAspergillus sulphureus (NRKL 4077). Isolation/Purification Powdered sclerotia of A. sulphureus were sequentially extracted with pentane and CH2C12 using a Soxhlet apparatus. A portion of the total CHzCI2 extract was fractionated by silica gel column chromatography. A stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3 was employed resulting in the elution and collection of a distinct red band at 4% MeOH. This active fraction was further separated by reversed phase HPLC (MeOH-H20, 89:11, v/v) to yield sulpinines A (HPLC retention time 16.0 min), B, and C and secopenitrern B and penitrem B. Biological Activity Exhibited potent activity against the lepidopteran crop pest Helicoverpa zea. Spectral Data UV: ~,m=M"OH 235(22,600) and 283nm (5,600). IR;

3473, 2933, 2873, 1457, 1371, 1043, 911, and 756cm "~. ~H NMR: (CD3OD) H-4, 7.20(d, J=8.3); H-5, 6.95(dd, J=8.3, 1.7); H-7, 7.28(d, J=l.3); H-12, 1.63(m), 2.60(m); H-13=q, 2.09(m); H-13,x, 2.25(m); H-14~,, 4.28(br dd, J=9.0, 8.8);

470

7.

Paspaline and Related Metabolites

H-16,x, 4.04(br s); H-17~q, 4.03(br d, ,/=3.1); H-18~, 3.47(br d, J=2.0); H-21, 1.37(m), 1.60(m); H-22oq, 1.49(dm, J=10.0); H-22~x, 1.89(dddd, J=-12, 12, 12, 3); H-23,~, 2.69(m); H-24, 2.32(m), 2.56(m); H-26, 4.91(br s), 5.10(br s); H-27, 1.70(br s); H-28, 1.14(s); H-29, 1.26(s); H-31, 6.07(dd,`/=17.5, 10.6); H-32, 4.97(dd, J=10.6, 1.5), 5.07(dd, ,/=17.5, 1.5); H-33, 1.41(s); and H-34, 1.41ppm (s). 13CNMR: (CD3OD) C-2, 153.9; C-3, 116.7; C-4, 118.1; C-5, 118.6; C-6, 142.0; C-7, 110.0; C-8, 141.2; C-9, 124.4; C-10, 52.0; C-11, 43.8; C-12, 27.4; C-13, 29.1; C-14, 72.1; C-16, 75.1; C-17, 66.5; C-18, 66.3; C-19, 66.8; C-20, 78.8; C-21, 27.2; C-22, 21.9; C-23, 51.5; C-24, 30.5; C-25, 143.2; C-26, 111.9; C-27, 19.8; C-28, 19.1; C-29, 16.5; C-30, 42.1; C-31,150.5; C-32, 110.2; C-33, 29.3; and C-34, 29.3ppm. Mass Spectrum: EIMS: 503(M+, 32), 488(43), 467(12), 452(13), 434(10), 397(10), 373(24), 358(29), 250(100), 222(35), 198(33), 182(72), and 167m/e (21); HREIMS: obsd 503.3039; calcd for C32H4~NO4,503.3036. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Sulpines A-C and Secopenitrem B: New Antiinsectan Metabolites from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 2066-2071(1992).

7. Paspaline and Related Metabolites

471

.Common/Systematic Name Sulpinine B Molecular FormulaJ~olecular Weight C32H41NO3; M W = 487.30864 24

26

General Characteristics Sulpinine B was an orange oil; [tt]o -43.2 ~ (C=0.003, in CHCi3). Fungal Source Sclerotia of Aspergillus sulphureus (NRRL 4077). Isolation/Purification Powdered sclerotia ofA. sulphureus were sequentially extracted with pentane and CH2C12 using a Soxhlet apparatus. A portion of the total CH2CI2extract was fractionated by silica gel column chromatography. A stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3 was employed resulting in the elution and collection of a distinct red band at 4% MeOH. This active fraction was further separated by reversed phase HPLC (MeOH-H20, 89:11, v/v) to yield sulpinines A, B (HPLC retention time, 13.8 min), C and secopenitrem B, and penitrem B. Biological Activity Exhibited potent activity against the lepidopteran crop pest Helicoverpa zea. Spectral Data IH NMR: (CD3OD) H-I, 7.72(br s); H-4, 7.34(d, ./=8.3); H-5, 7.07(dd, ,/=8.3, 1.5); H-7, 7.28(d, ,/=0.8); H-12, 2.68(dm, `/=6.0), 1.59(m); H-13, 2.23(m), 1.84(m); H-14, 4.61(br dd, `/=9.1, 8.8); H-16, 3.86(br s); H-17, 3.96(br d, ,/=5.6); H-18, 5.82(br d, `/=4.7); H-21, 1.71(m), 1.34(m); H-22, 2.06(m), 2.02(m); H-23, 2.80(m); H-24, 2.65(dm, J=4.4), 2.39(dd, J=12.9, 12.9); H-26, 5.20(br s), 5.03(br s); H-27, 1.79(br s); H-28, 1.10(s); H-29, 1.28(s); H-31, 6.05(dd, J=17.4, 10.6); H-32, 5.06(dd,`/=17.3, 1.2), 4.99 (dd, ./=10.6, 1.2); H-33, 1.41(s); and H-34, 1.41ppm (s). ~ac NMR: (CD3OD) C-2, 152.2; C-3, 116.9; C-4, 117.9; C-5, 118.5; C-6, 141.2; C-7, 108.9; C-8, 139.8; C-9, 123.1; C-10, 50.7; C-11, 42.9; C-12, 28.2; C-13, 29.3; C-14, 73.7;

472

7. Paspaline and Related Metabolites

C-16, 79.1; C-17, 62.8; C-18, 118.7; C-19, 148.1; C-20, 77.7; C-21, 27.3; C-22, 21.1; C-23, 49.7; C-24, 34.7; C-25, 141.6; C-26, 111.7; C-27, 19.8; C-28, 20.2; C-29, 16.2; C-30, 41.1; C-31,148.8; C-32, 110.0; C-33, 28.6; and C-34, 28.6ppm. Mass Spectrum: ELMS: 487(M+ 21), 472(16), 469(10), 451(26), 436(11), 250(100), 198(34), 182(71), 167(23), 129(45), 115(28), and 105(21); HRELMS: obsd 487.3104; calcd for C32H41NO3,487.3086. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Sulpines A-C and Secopenitrem B: New Antiinsectan Metabolites from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 2066-2071(1992).

7. Paspaline and Related Metabolites

473

Common/Systematic Name Sulpinine C Molecular Formula/Molecular Weight C32H41N06; MW = 535.29339 4

O

%____~4

26

General Characteristics Yellow oil; [a]o

+17.1~ (C=0.003, in CHCI3).

Fungal Source Sclerotia ofAspergillus sulphureus (NRRL 4077). Isolation/Purification Powdered sclerotia ofA. sulphureus were sequentially extracted with pentane and CH2C12 using a Soxhlet apparatus. A portion of the total CH2C12extract was fractionated by silica gel column chromatography. A stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3 was employed resulting in the elution and collection of a distinct red band at 4% MeOH. This active fraction was further separated by reversed phase HPLC (89:11 (v/v), MeOHH20) to yield sulpinines A, B, C (HPLC retention time, 6.0 rain), and secopenitrem B and penitrem B. Biological Activity Exhibited potent activity against the lepidopteran crop pest Helicoverpa zea. Soectral Data _

UV:

~

MeOH max

229(e=25,600) and 262nm (18,200).

IR:

3483, 2965, 2872, 1685, 1647, 1603, 1391, and 756cm"1. 1H N]VIR: (CD3OD) H-I, 7.13(br s); H-4, 7.62(d, ,/--8.1); H-5, 7.32(dd, J=8.1, 1.8); H-7, 6.99(d, ,/--1.7); H-12,x, 2.41(m); H-12~, 1.89(dm, d=l 1.4); H-13=,, 1.70(m); H-13.q, 2.25(m); H-14,x, 4.19(dd, J=9.3, 9.0); H-16~x, 4.04(br s); H-17~, 3.96(br d, ,/--1.9);

474

7.

Paspaline and Related Metabolites

H-18, 3.47(br d, J=2.3); H-20(OH), 1.45(br s); H-21~, 1.21(m), H-21,, 1.34(m); H-22,x, 1.76(dd m, d--13.4, 3.4); H-22oq, 1.34(m); n-23,x, 2.99(m); H-24a, 3.05(dd, d=17.8, 4.4); H-24b, 2.40(dd, J=17.8, 4.0); H-26a, 5.13(br s); H-26b, 5.00(d, ,I=1.6); H-27, 1.68(br s); H-28, 0.99(s); H-29, 1.55(s); H-31, 5.93(dd, d=17.4, 10.6); H-32a, 5.09(dd, J=10.6, 0.9); H-32b, 5.06(dd, d-17.4, 0.9); H-33, 1.37(s); and H-34, 1.37ppm

(s).

13C NMR: (CD3OD) C-2, 176.6; C-3, 202.4; C-4, 129.4; C-5, 125.3; C-6, 154.5; C-7, 125.0; C-8, 136.8; C-9, 131.6; C-10, 57.5; C-11, 43.4; C-12, 24.1; C-13, 27.3; C-14, 71.1; C-16, 73.4; C-17, 64.6; C-18, 61.8; C-19, 65.5; C-20, 77.1; C-21, 28.1; C-22, 24.4; C-23, 36.1; C-24, 47.7; C-25, 140.9; C-26, 112.5; C-27, 19.4; C-28, 18.7; C-29, 16.5; C-30, 41.3; C-31,146.4; C-32, 112.1; C-33, 28.0; and C-34, 28.0ppm.

Mass Spectrum: EIMS: 535(M+, 13%), 254(3), 242(3), 228(3), 214(11), 200(4), 188(100), 172(5), 145(9), 130(9), 115(5), and 105m/e (6); HREIMS: obsd 535.2945; calcd for C3zI-I41NO6,535.2934. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Sulpines A-C and Secopenitrem B: New Antiinsectan Metabolites from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 2066-2071(1992).

7. Paspaline and Related Metabolites

475

Common/Systematic Name PC-M4 Molecular Formula/Molecular Weight C37H49NO4; MW = 571.36616

|| | | | H

r

71)

11

7a

7 H 6

5 ~

O

H

2' General Characteristics Crystalline powder, mp., 269-272~ (dec.). Fungal Source

Penicillium crustosum (strain NHL 6491).

Isolation/Purification_

Penicillium crustosum was cultivated at 25 ~ for 14 days using Czapek-Dox medium

supplemented with 0.2% yeast extract. The mycelium was extracted with CH2C12 at room temperature. The residue obtained by evaporation of the extract was chromatographed on silica gel with C6I-I6-Me2CO (5:1, v/v) followed by repeated purification by LPLC [C6I-I6-Me2CO (20:1, v/v) or C6H14-EtOAc-MeOH [12:6:1, v/v/v to obtain PC-M4 and with C6H6-Me2CO (3:1, v/v)] followed by purification by HPLC using the solvent system C6I-I6-EtOAc (5:2, v/v) to give PC-M5. Biological Activity Biological activity not presented but based on structure PC-M4 is probably tremorgenie. Spectral Data UV~ ;~Mm~" 233(Iog e=4.09) and 283nm (3.71).

(KBr) 3600 and 3400em "1 (NH, OH).

476

7.

Paspaline and Related Metabolites

CD: (c=4.06x105, MeOH) +1.6(214), -2.0(240), -0.6(271), and -0.5nm (292). 1H NMR: [(CD3)2SO] 0.96(6H, s, Me-13C, Me-l"); 1.21(3H, s, Me-13b); 1.23(3I-I, s, Me-l"); 1.25(3H, s, Me-10); 1.27(3H, s, Me-10); 1.55(1H, brd, J=l 1.5 Hz, H-5a); 1.62(1H, brd, J=12.2 Hz, H=6B); 1.67-1.88(5H, m, H-6a, H-14B, H-15B, H2=9); 1.72(3H, brs, Me-l'); 1.93(2H, m, H-5B, H=8); 2.05(1H, m, H-15a); 2.29(1H, dd, J=13.1, 10.5 I-~ H-7a); 2.46(1H, ddd, J=12.8, 12.8, 5.8 Hz, H-14a); 2.70(2H, m, H-6a, H-9a); 2.84(1H, dd, J=13.1, 5.8 Hz, H=7B); 3.70(1H, brd, d=9.0I-~ H-7d); 3.71(1H, brs, H=2); 3.84(1H, brd, J=4.7Hz, H-3); 4.50(1H, br dd, J--9.2, 7.gHz, H-15a); 4.85(1H, br s, H-2'); 5.01(1H, brs, H-2'); 5.58(1H, br d, J=4.7Hz, H-4); 6.74(1H, d, J=8.2Hz, H-11); 7.12(1H, d, J=8.2Hz, H-12); and 10.53ppm [lI-I, br s, H-13 (NH)]. 13CNMR: C-2, 79.1; C-3, 62.6; C-4, 117.7; C-4a, 146.7; C-4b, 75.7; C-5, 33.2; C-6, 21.1; C-6a, 49.3; C-7, 28.3; C-7a, 113.7; C-7b, 121.4; C-7c, 133.7; C-7d, 44.2; C-8, 48.6; C-9, 23.4; C-9a, 49.0; C-10, 44.8; Me-10, 21.7, 32.1; C-10a, 141.8; C-11, 113.9; C-12, 110.6; C-12a, 139.7; C-13a, 153.0; C-13b, 49.9; Me-13b, 16.2; C-13, 42.1; Me-13c, 19.2; C-14, 26.3; C-15, 27.9; C-15a, 72.9; C-I', 142.9; C-2', 100.0; C-3'(Me), 19.6; C-I", 70.5; and C-Me-I", 27.1, 27.8ppm. Mass Spectrum: HRMS: 571.3662(M*, 17); (calcd for C37H49NO4,571.3662), 485(100), and 470m/e (35). Reference T. Yamaguchi, K. Nozawa, T. Hosoe, S. Nakajima, and K. Kawai; Indoloditerpenes Related to Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Phytochemistry, Vol. 32, pp. 117%1181(1993).

7. Paspaline and Related Metabolites

477

Common/Systematic Name PC-M5 Molecular Formula/Molecular Weight C32I-I41NO4; ~

"- 5 0 3 . 3 0 3 5 6

OH

T

]

7 H

s

,4A-

ii 2'

General Characteristics Pale yellow needles from ethyl acetate; rap., 218-220~ (dec.). Fungal Source Penicillium crustosum

(strain NHL 6491).

Isolation/Purification Penicillium crustosum

was cultivated at 25 ~ for 14 days using Czapek-Dox medium supplemented with 0.2% yeast extract. The mycelium was extracted with CH2CI2 at room temp. The residue obtained by evaporation of the extract was chromatographed on silica gel with C6I-I~-Me2CO(5:1, v/v) followed by repeated purification by LPLC [C6I-I6-Me2CO (20:1, v/v) or C6I-I14-EtOAc-MeOH (12:6:1, v/v/v)] to obtain PC-M4 and with C6H6-Me2CO (3:1, v/v) followed by purification by HPLC using the solvent system C6I-I6-EtOAc (5:2, v/v) to give PC-MS.

Biolggical Activity Biological activity not presented but based on the structure of PC-M4 it is probably tremorgenic. Spectral Data UV: MeOH max

226sh (log e=4.30), 246(4.22), 305(3.74), and 325nm (3.74).

IR:

(KBr) 3525 and 3400cm" (NH, OH).

478

7.

Paspaline and Related Metabolites

CD: (C=4.49x10 5, MeOH)-1.3(242), +0.4(259), +0.6(291), and +l.0nm (334). 1H NMR: [(CD3)2SO] 1.04(3H, s, Me-12C); 1.38(3H, s, Me-12b); 1.41(6H, s, Me-3" x 2); 1.69(2H, m, H-5, H-6); 1.75(1H, m, H-13B); 1.77(3H, br s, Me-l'); 1.87(2H, m, H-5, H-14); 2.06(1H, ddd, J=12.7, 12.7, 3.SHz, H-13); 2.06(1H, m, H-6); 2.16(1H, m, H-14); 2.60(1H, ddd, J=13.5, 12.7, 4.6Hz, H-13a); 2.68(1H, dd, J=13.3, 4.6Hz, H-7B); 2.90(2H, m, H-6a, H-7a); 3.15(1H, d, J=9.3Hz, OH-3); 3.40(1H, br s, OH); 3.72(1H, br s, OH); 3.80(1H, br s, H-2); 3.909(1H, ddd, J=9.3,5.8, 1.8Hz, H-3); 4.62(1H, br dd, J=10.2, 8.2Hz, H-14a); 4.87(1H, qd, J-1.4, 1.4Hz, H-2'); 5.09(1H, br s, H-2'); 5.76(1H, dd, J=5.8, 1.8Hz, H-4); 6.42(1H, d, J=15.9Hz, H-2"); 6.92(1H, dd, ,/=8.0, 7.7Hz, H-10); 7.10(1H, d, J=15.9Hz, H-I"); 7.14(1H, d, J=7.7Hz, H-9), 7.16(1H, d, J=8.0Hz, H-II); and 9.87ppm [1H, br s, H-12 (NH)].

13CNMR,: C-2, 77.8; C-3, 65.3; C-4, 114.5;C-4a, 150.6;C-4b, 77.8; C-5, 34.7; C-6, 22.5; C-6a, 49.5; C-7, 28.7, C-7a, 116.7;C-7b, 122.0;C-7c, 134.4;C-Td, 44.4; C-8, 49.2, C-9, 24.0, C-9a, 49.6; C-10, 45.6; Me-10, 21.9, 32.3;C-10a, 143.7;C-I I, 115.6; C-12, 110.7; C-12a, 139.7;C-13a, 152.4;C-13b, 50.5;Me-13b, 16.3,Me-13c, 20.1; C-14, 28.3; C-15, 28.1; C-15a, 73.4; C-I', 141.1;C-2', III.8;C-3'(Me), 19.6,C-l", 72.9; C-Me-I", 26.6, 28.0ppm, MeCOO, 21.1; and MeCOO, 170.9ppm. Mass Spectrum: EIMS: 485([M-H20] § 100), 470([M-H20] § 34), 400(16), 382(28), and 248m/e (28). Reference T. Yamaguchi, K. Nozawa, T. Hosoe, S. Nakajima, and K. Kawai; Indoloditerpenes Related to Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Phytochemistry, Vol. 32, pp. 1177-1181(1993).

7. Paspaline and Related Metabolites

479

Common/Systematic Name PC-M5' Molecular Formula/Molecular Weight C29H37NOs; M W = 479.26717

H

-

1 3 _=.~.

l-oN

General Characteristics Colorless crystalline powder; mp, 162-164 ~C. Fungal Source Penicillium crustosum, strain NHL 6491, isolated from contaminated bread which had

been intended for school lunches in Shinagawa-ku, Tokyo. Isolation/Purification Penicillium crustosum was cultivated at 25~ for 14 days on Czapek-Dox medium

supplemented with 0.2% yeast extract. The fresh mycelium was extracted with CH2C12at room temperature. The residue obtained by evaporation of the extract was chromatographed on silica gel with benzene-acetone (50:1, v/v) followed by purification by LPLC using benzene to obtain penitrems F and B, with benzene-acetone (20:1, v/v) followed by purification by LPLC using the solvent system of benzene-acetone (100:1, v/v) to give penitrems A and E, with benzene-acetone (10:1, v/v) followed by further purification hy LPLC [benzene-acetone (50:1, v/v)] to obtain penitrem D, with benzeneacetone (5:1, v/v) followed by repeated purification by LPLC [benzene-acetone (20:1, v/v) and/or hexane-AcOEt-EtOH] to give cyclopenin and PC-M5', with benzene-acetone (3:1, v/v) followed by purification by HPLC using the solvent system ofbenzene-AcOEt (5:2, v/v) to give PC-M6. Spectral Data UV: ~,m~,M~~ 229(Iog 6=4.74) and 279nm (4.05).

IR: 3500, 3460, 3400(NH, OH), and 1720cm~ (AcO). 1H NMR: [(CD3)2CO] 1.00(3H, s, 12c-Me); 1.00(3H, s, 2'-Me); 1.26(3H, s, 3-Me); 1.36(3H, s,

480

7.

Paspaline and Related Metabolites

12b-Me); 1.70(3H, m); 1.82(1H, dddd, J=13,5, 13.5, 8.5, 5.2Hz); 1.95(1H, ddd, J=13.5, 13.5, 4.5Hz); 1.99(3H, s, 3-OCOMe); 2.10(1H, m); 2.39(1H, dd, ,/--13.0, 10.8Hz, 7a-H); 2.67(1H, dd, J=13.0, 6.3Hz, 7a-H); 2.69(1H, ddd, J=13.3.13.3, 4.8Hz); 2.85(1H, m); 3.32(1H, d, J=l.8Hz, 2-H); 3.48(1H, s, OH); 3.49(1H, s, OH); 4.65(1H, br dd, J=10.5, 7.6Hz, 14a-H); 5.20(1H, ddd, J=5.8, 1.8, 1.8Hz, 3-H); 5.69(1H, dd, ,/=5.8, 1.8Hz, 4-H); 6.94(2H, m, 9-H, 10-H); 7.28(1H, m, 8-H or 1l-H); 7.32(1H, m, 11-H or 8-H); and 9.85ppm (1H, br s, 12-H (NH)). ~3CNMR: [(CD3)2CO] C-2, 82.56; C-3, 66.01; C-4, 115.21; C-4a, 151.29; C-4b, 77.52; C-5, 34.71; C-6, 22.01; C-6a, 50.67; C-7, 30.56; C-7a, 116.71; C-7b, 126.11; C-8, 118.66; C-9, 119.54; C-10, 120.36; C-11,112.45; C-1 la, 141.23; C-12a, 153.84; C-12b, 51:61; C-12b-Me, 16.73; C-12c, 43.53; C-12c-Me, 19.93; C-13, 27.87; C-14, 29.13; C-14a, 75.06; C-I', 71.58; C-2', 25.99; C-3', 27.95; C-MeCOO, 21.28; and MeCOO; 170.69ppm. Mass Spectrum: HREIMS: 479.2669m/e (M+), C29H37NO5 requires 479.2670. Reference T. Hosoe, K. Nozawa, S. Udagawa, S. Nakajima, and K. Kawai; Structures of New Indoloditerpenes, Possible Biosynthetic Precursors of the Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Chem. Pharm. Bull. Vol. 38, pp. 3473-3475 (1990).

7. Paspaline and Related Metabolites

481

Common/Systematic Nam.e PC-M6 Molecular Formula/Molecular Weight C27H35NO3; M W - 421.26169 H O

NH

OH

_

1 3

--

I:1

H

General Characteristics Colorless crystalline powder; mp. (dec.), 260-263~ Fungal Source

Penicillium crustosum, strain NHL 6491, isolated from contaminated bread which had

been intended for school lunches in Shinagawa-ku, Tokyo. Isolation/Purification

Penicillium crustosum was cultivated at 25~ for 14 days on Czapek-Dox medium

supplemented with 0.2% yeast extract. The fresh mycelium was extracted with CH2C12at room temperature. The residue obtained by evaporation of the extract was chromatographed on silica gel with benzene-acetone (50:1, v/v) followed by purification by LPLC using benzene to obtain penitrems F and B, with benzene-acetone (20:1, v/v) followed by purification by LPLC using the solvent system of benzene-acetone (100:1, v/v) to give penitrems A and E, with benzene-acetone (10:1, v/v) followed by further purification by LPLC [benzene-acetone (50:1, v/v)] to obtain penitrem D, with benzeneacetone (5:1, v/v) followed by repeated purification by LPLC [benzene-acetone (20:1, v/v) and/or hexane-AcOEt-EtOH] to give cyclopenin and PC-M5', with benzene-acetone (3:1, v/v) followed by purification by HPLC using the solvent system ofbenzene-AcOEt (5:2, v/v) to give PC-M6. Spectral Data

UV~

~

MeOH max

229(log 6=4.64) and 280nm (3.98).

IR~

(KBr) 3560, 3420, and 3270cm"1 (NH, OH).

482

7. Paspaline and Related Metabolites

1H NMR: [(CD3)2SO] 0.88(3H, s, 12b-Me); 0.98(3H, s, 12c-Me); 1.17(3H, s, 2'-Me); 1.20(3H, s, 3'-Me); 1.46(1H, ddd, d-11.5, 11.5, 4.6Hz); 1.54(1H, br d, d=12.THz); 1.61(IH, m); 1.71(2H, m); 1.80(1H, ddd, J=l 1.5, 11.5, 3.5Hz); 1.84(1H, ddd, J=-12.7, 12.7, 3.5Hz); 2.03(2H, m); 2.19(1H, br d, J=l 1.5Hz); 2.3 I(1H, dd, J=12.7, 10.4I-~ 7a-H); 2.60(1H, dd, J=12.7, 6.9Hz, 7b-H); 2.70(1H, m); 3.00(1H, d,J =- 1.8Hz, 2-H); 3.92(1I-I, br dd, J= 10.2, 7.2Hz, 14a-H); 4.04(1H, dd, J=6.0, 1.8Hz, 3-H); 5.45(1I-I, d, J---6.0H~ 4-H); 6.90(1H, br t, J=7.0Hz, 9-H or 10-H); 6.94(1H, br t, J=7.0Hz, 10-H or 9-H); 7.27(2I-I, dd, J=7.0, 1.2Hz, 8-H, 1l-H); and 10.70ppm (1H, br s, NH). 13CNMR:

[(CD3)2CO] C-2, 80.91; C-3, 62.48; C-4, 117.54; C-4a, 144.11; C-4b, 40.50; C-5, 31.32; C-6, 24.17; C-6a, 48.94; C-7, 29.15; C-7a, 115.61; C-7b, 124.32; C-8, 118.34; C-9, 119.13; C-10, 119.27; C-11, 111.75; C-1 la, 139.99; C-12a, 150.73; C-12b, 49.62; C-12b-Me, 14.50; C-12c, 40.37; C-12c-Me, 15.41; C-13, 26.15; C-14, 26.87; C-14a, 76.13; C-I', 71.35; C-2', 26.46; and C-3', 27.24ppm. Mass Spectrum: HREIMS: 421.2616(M+), C27H35NO3 requires 421.2616, 406m/e (M+-Me). Reference T. Hosoe, K. Nozawa, S. Udagawa, S. Nakajima, and K. Kawai; Structures of New Indoloditerpenes, Possible Biosynthetic Precursors of the Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Chem. Pharm. Bull., Vol. 38, pp. 3473-3475 (1990).

Janthitrems Janthitrem B Janthitrem E Janthitrem F (10-O-Aeetyljanthitrem E) Janthitrem G

483

This Page Intentionally Left Blank

8.

Janthitrems

485

Common/Systematic Name Janthitrem B Molecular Formula/Molecular Weight C37H47NOs; IVIW' = 5 8 5 . 3 4 5 4 2

e'

-

/

-'r-"

Me 4O

15 14

20

Me H ~ 7 . . ~ ~ 9 ~ -

H

i

HQ

37

'~

\\

I, '

I '

'

~

10

OH

35

General Characteristics Obtained as a pale-yellow glass. Gray-green color response to Erhlich's reagent on TLC. Fungal Source Penicillium janthinellum. Isolation/Purification Purified janthitrem B was obtained as a pale-yellow glass from column chromatography on Mallinckrodt Silic AR CC-7 silica gel following repeated preparatory thin-layer chromatography. Flash chromatography was performed on silica gel (E Merck 9385) using toluene-acetone (17:3, v:v) as the eluent to further purify decomposed samples of janthitrem B from storage. Biological Activity Tremorgenic in mouse bioassay. I.P. injection of 200~g of toxin in propylene glycol solution elicited a characteristic tremor response accompanied by incoordination and hypersensitivity to sound and touch. Spectral Data UV"

m,~M'~ 228(E=15,420), 258(25,170), 265(27,300), and 329nm (16,600); ~,,~M'~ Fluorescence emission: 385nm. IH NMR: [ ( C D 3 ) 2 C O ] H-5, 2.60, 1.58; H-6, 2.04, 1.79; H-7, 4.58; H-9, 3.77; H-10, 3.91; H-11, 5.69; H-14, 1.58, 1.70; H-15, 2.04, 1.57; H-16, 2.68; H-17, 2.35, 2.65; H-20, 7.374; H-22, 4.90; H-23, 2.65; H-27, 5.97; H-30, 7.366; H-32, 1.32; H-33, 0.89; H-35, 4.86, 5.07; H-36, 1.76; H-37, 1.41; H-38, 1.09; H-39, 1.25; H-40, 1.29; 10-OH, 3.03(d, J=9.3 Hz); 13-OH, 3.33(s, br); 22-OH, 4.27(d, J=7.6 Hz); and NH, 9.81ppm (s br).

486

8. Janthitrems

13C NMR:

(CD3)2CO C-2, 156.0; C-3, 51.8; C-4, 43.5; C-5, 28.1; C-6, 29.2; C-7, 74.3; C-9, 80.5; C-10, 64.2; C-11, 119.5; C-12, 148.5; C-13, 77.5; C-14, 34.7; C-15, 22.1; C-16, 50.5; C-17, 27.9; C-18, 116.9; C-19, 127.8; C-20, 114.1; C-21,140.1; C-22, 76.4; C-23, 60.3; C-24, 74.3; C-26, 72.7; C-27, 120.2; C-28, 137.2; C-29, 131.6; C-30, 103.6; C-31,142.2; C-32, 16.7; C-33, 20.0; C-34, 143.9; C-35, 110.8; C-36, 20.1; C-37, 30.6; C-38, 23.7; C-39, 30.4; and C-40, 32.5ppm. Mass Spectrum: ELMS: 585.3422m/e (NV, 585.3442 for C37H47NOs), 568(35), 567(M~ - H20, 71), 553(29), 552(NV - H20- CH3, 81), 550(34), 549(69), 535(30), 534(78), 533(21), 532(34), 531(45), 529(24), 525(40), 524(97), 517(25), 516(48), 514(25), 507(42), 506(100), 504(21), 490(22), 489(35), 488(75), 486(44), 464(21), 436 (29), and 330(36). TLC Data Thin-layer chromatography was performed on silica gel plates (E. Merck 55543) using toluene-acetone (3:2, v:v) as the eluent. Janthitrems were visualized by their fluorescence when irradiated at 366nm. Reference A. L. Wilkins, C. O Miles, R. M. Ede, R. T. Gallagher, and S. C. Munday; Structure Elucidation of Janthitrem B, a Tremorgenic Metabolite of Penicilliumjanthinellum, and Relative Configuration of the A and B Rings of Janthitrems B, E, and F; J. Agric. Food Chem., Vol. 40, pp. 1307-1309(1992).

8.

Janthitrems

487

Common/Systematic Name Janthitrem E Molecular Formula/Molecular Weight C37H49NO6; MW = 603.35599 H 15

Me' ......

Me 40

Me

OH

35

General Characteristics Colorless amorphous solid. Fungal Source

Penicillium janthinellum (TDD4).

Isolation/Purification The cultures of Penicilliumjanthinellum were filtered and the mycelium macerated with acetone in a Waring blender. The acetone solution was evaporated and the aqueous residue partitioned between ethyl acetate and water. The residue of the ethyl acetate solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between ethyl acetate and water. The crude extract obtained from the ethyl acetate solution was separated by column chromatography on silica gel using benzene-acetone (70:30, v/v) as eluant to yield the three janthitrems in order of descending Re values: janthitrem G, F, and E. Biological Activity Tremorgenic. Spectral Data UV:

~

MeOH

228(e=17,700), 258sh (27,300), 265(30,000), and 330nm (17,000).

IR~

(KBr) 3400, 2900, and 1700cm"1.

488

8.

Janthitrems

~3C NMR: (acetone-d6) C-2, 155.92 s; C-3, 51.75 s; C-4, 43.39 s; C-5, 27.85 t; C-6, 29.18 t; C-7, 75.03 d; C-9, 81.82 d; C-10, 64.48 d; C-11, 119.15 d; C-12, 148.56 s; C-13, 77.49 s; C-14, 34.51 t; C-15, 22.02 t; C-16, 50.33 d; C-17, 28.04 t; C-18, 116.85 s; C- 19, 127.77 s: C-20, 113.99 d; C-21, 139.92 s; C-22, 76.45 d; C-23, 60.32 d; C-24, 74.26 s; C-26, 72.78 s; C-27, 120.13 d; C-28, 137.11 s; C-29, 131.51 s; C-30, 103.73 d; C-31, 142.11 s; C-32, 16.58 q; C-33, 20.20 q; C-34, 72.65 s; C-35, 27.28 q; C-36, 27.21 q; C-37, 30.43 q; C-38, 23.76 q; C-39, 30.63 q; and C-40, 32.51ppm q. Mass Spectrum: Found: M +, 603.355; C37H49NO6requires 603.356. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, and R. Vleggaar; Structure Elucidation of the Janthitrems, Novel Tremorgenic Mycotoxins from Penicilliumjanthinellum; J. Chem. Soc. Perkin Trans. I, pp. 697-701 (1984).

8. Janthitrems

489

Common/Systematic Name Janthitrem F; 10-O-Acetyljanthitrem E Molecular Formula/Molecular Weight C39Hs1NOT; ~

HO

37

3s

= 645.36655

Me

H 15 20

~

)-..~.,~"~a~.~x

o,26 //

~, ...... Me,,,,,,~~Me27

1

4

I Me[

....

4O

11

,...X,L, Me

-~ A u

M~eeOH 35

General Characteristics Colorless amorphous solid. Fungal Source

Penicillium janthinellum (TDD4).

Isolation/Purification The cultures of Penicilliumjanthinellum were filtered and the mycelium macerated with acetone in a Waring blender. The acetone solution was evaporated and the aqueous residue partitioned between ethyl acetate and water. The residue of the ethyl acetate solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between ethyl acetate and water. The crude extract obtained from the ethyl acetate solution was separated by column chromatography on silica gel using benzene-acetone (70:30, v:v) as eluant to yield the three janthitrems in order of descending Rf values: janthitrem G, F, and E. Biological Activity Tremorgenic. Spectral Data UV: ~ MAD1-1 228(e=14,200), 258sh (20,500), 265(22,400), and 330nm (12,200). IR"

(KBr) 3400, 2900, and 1720cm "~. ~3C NMR: (acetone-d6) C-2, 155.64 s; C-3, 51.72 s; C-4, 43.37 s; C-5, 27.70 t; C-6, 29.14 t, C-7,

490

8. Janthitrems

75.03 d; C-9, 82.44 d; C-10, 66.00 d; C-11, 115.14 d; C-12, 151.26 s; C-13, 77.42 S; C-14, 34.50 t; C-15, 21.88 t; C-16, 50.27 d; C-17, 27.85 t; C-18, 116.84 s; C-19, 127.67 s:. C-20, 114.08 d; C-21,139.74 s; C-22, 76.35 d; C-23, 60.10 d; C-24, 74.22 s; C-26, 72.70 s; C-27, 120.13 d; C-28, 136.85 s; C-29, 131.57 s; C-30, 103.36 d; C-31, 142.02 s; C-32, 16.60 q; C-33, 19.87 q; C-34, 71.61 s; C-35, 27.81 q; C-36, 25.95 q; C-37, 30.25 q; C-38, 23.68 q; C-39, 30.61 q; and C-40, 32.40 q; COCH3, 170.86; and COCH3, 21.37ppm. Mass Spectrum: Found: 645.367, M+; C39Hs~NO7 requires 645.366. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, and R. Vleggaar; Structure Elucidation of the Janthitrems, Novel Tremorgenic Mycotoxins from Penicilliumjanthinellum; J. Chem Soc. Perkin Trans. I, pp. 697-701 (1984).

9.

Miscellaneous Indole Metabolites

491

Common/Systematic Name Janthitrem G Molecular Formula/Molecular Weight C39Hs1NO6; ~ = 629.37164 I

37

as

Me

Me' ......

B

20

r

~

Me 4O

H is

I i_el

14

~

Me

OH

35

General Characteristics Colorless amorphous solid. Fungal Source

Penicillium janthmellum (TDD4).

Isolation/Purification The cultures of Penicilliumjanthinellum were filtered and the mycelium macerated with acetone in a Waring blender. The acetone solution was evaporated and the aqueous residue partitioned between ethyl acetate and water. The residue of the ethyl acetate solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between ethyl acetate and water. The crude extract obtained from the ethyl acetate solution was separated by column chromatography on silica gel using benzene-acetone (70:30, v:v) as eluant to yield the three janthitrems in order of descending Re values: janthitrem G, F, and E. Biological Activity Tremorgenic. Spectral Data UV:

~

MeOH

228(6=23,000), 258sh (15,300), 263(15,700), and 331nm (10,080).

IR:

(KBr) 3400, 2900, and 1720cm l.

492

9.

Miscellaneous Indole Metabolites

~3C NMR: (acetone-d6) C-2, 155.38 s; C-3, 51.70 s; C-4, 43.45 s; C-5, 27.74 t; C-6, 29.15 t; C-7, 75.06 d; C-9, 82.47 d; C-10, 66.08 d; C-11,115.16 d; C-12, 151.28 s; C-13, 77.47 s; C-14, 34.56 t; C-15, 21.92 t; C-16, 50.39 d; C-17, 27.87 t; C-18, 116.46 s; C-19, 127.60 s: C-20, 114.11 d; C-21, 136.69 s; C-22, 33.53 d; C-23, 49.79 d; C-24, 74.74 s; C-26, 72.94 s; C-27, 119.53 d; C-28, 140.79 s; C-29, 133.31 s; C-30, 103.96 d; C-31, 141.17 s; C-32, 16.60 q; C-33, 19.86 q; C-34, 71.62 s; C-35, 27.83 q; C-36, 25.94 q; C-37, 29.82 q; C-38, 22.53 q; C-39, 30.49 q; and C-40, 32.30 q; COCH3, 170.69; and COCH3, 21.33ppm. Mass Spectrum: Found: 629.372, M+; C39Hs~NO6 requires 629.372. Reference A. E. de Jesus, P. S. Steyn, F. R. van Heerden, and R. Vleggaar; Structure Elucidation of the Janthitrems, Novel Tremorgenic Mycotoxins from Penicilliumjanthinellum; J. Chem Soc. Perkin Trans. I, pp. 697-701 (1984).

Miscellaneous Indole Metabolites 2,4-Dimethylindole 4-Hydroxymethyl-2-methylindole 4-Methoxymethyl-2-methylindole Marcfortine A Marcfortine B Marcfortine C Paraherquamide Nominine Aspernomine Tubingensin A Tubingensin B Radarin A Radarin B Radarin C Radarin D

S

493

This Page Intentionally Left Blank

9.

Miscellaneous Indole Metabolites

495

Common/Systematic Name 2,4-Dimethylindole Molecular Formula/Molecular Weight Cl0HllN; MW = 145.0891 9

Me

41

7

8

General Characteristics 2,4-Dimethylindole was obtained as a yellow oil. Fungal Source Fruit bodies of Tricholoma sciodes and T. virgatum. Isolation/Purification Fruit bodies of Tricholoma sciodes were collected in the vicinity of Lund, Sweden. The fruit bodies were ground in a meat grinder, and the mush was extracted with ethyl acetate. The organic phase was separated, dried with Na2SO4 and the solvent was evaporated. The fresh fruit bodies of T. virgatum were frozen at -20 ~C, rapidly broken with a hammer and extracted at -20~ with ethyl acetate. The organic phase was dried with MgSO4 and filtered through a short A1203 column to remove free fatty acids. Final purification from crude extracts was achieved by silica gel column and radial chromatography. Spectral Data UV:

Z Em~" 223(C=19,200), 268 (4,400), 271 (4,400), 273(4,300), and 278nm (2,900). IR: (KBr) 3380, 2920, 1690, 1610, 1160, and 770cm "1. ~H NMR: (CDCIa) H-l, 7.82(br s); n-3, 6.24(m); H-5, 6.88(d, J=7.2Hz); n-6, 7.03(dd, ,/=7.0, 8.0Hz); H-7, 7.13(d, J=8.1Hz); H-8, 2.45(s); and H-9, 2.52ppm (s). 13C N M ~ :

(CDCI3) C-2, 134.3(s); C-3, 98.9(d); C-3a, 128.9(s); C-4, 129.1(s); C-5, 119.8(d); C6, 121.0(d); C-7, 107.8(d); C-7a, 135.7(s); C-8, 13.7(q); and C-9, 18.8ppm (q).

496

9.

Miscellaneous Indole Metabolites

Mass Spectrum: HREIMS: 145.0892m/e (M+, 67%, CloHttN), requires 145.0891, 144(100), and 130role (15). Reference L. Garlaschelli, Z. Pang, O. Sterner, and G. Vidari; New Indole Derivatives from the Fruit Bodies of Tricholoma Sciodes and T. Virgatum; Tetrahedron, Vol. 50, pp. 3571-3574 (1994).

9.

Miscellaneous Indole Metabolites

497

Common/Systematic Name 4-Hydroxymethyl-2-methylindole Molecular Formula/Molecular Weight C]0HI1NO; MW = 161.08406 9

CH20H

7

8

General Characteristics 4-Hydroxymethyl-2-methylindole was obtained as a yellow oil. Fungal Source Fruit bodies of Tricholoma sciodes and T. virgatum. Isolation/Purification Fruit bodies of Tricholoma sciodes were collected in the vicinity of Lund, Sweden. The fruit bodies were ground in a meat grinder, and the mush was extracted with ethyl acetate. The organic phase was separated, dried with Na2SO4 and the solvent was evaporated. The fresh fruit bodies of T. virgatum were frozen at -20 oC, rapidly broken with a hammer and extracted at -20~ with ethyl acetate. The organic phase was dried with MgSO4 and filtered through a short A1203 column to remove free fatty acids. Final purification from crude extracts was achieved by silica gel column and radial chromatography. Spectral Data UV: E~o~ 222(e=25,400) and 279nm (6,100). IR:

(KBr) 3380, 2920, 1620, 1570, 1450, 1420, 1400, 1360, 1300, 1200, 1170, 1100, and 780cm ~. 1H NMR: (CDCI3) H-l, 7.91(br s); H-3, 6.34(m); H-5, 7.04(d, J=7.1Hz); H-6; 7.08(dd, ,/=7.0, 8.0Hz); H-7, 7.24(d, J=7.8Hz); H-8, 2.45(d, J=0.6Hz); H-9, 4.72(s); and OCH3, 3.41 ppm (s). 13C N M ~ :

(CDC|3) C-2, 135.2(s); C-3, 98.9(d); C-3a, 128.0(s); C-4, 128.8(s); C-5, 119.3(d); C6, 120.7(d); C-7, 110.0(d); C-7a, 136.2(s); C-8, 13.7(q); C-9, 64.1(t); and OCH3, 58.0ppm (q).

498

9.

Miscellaneous Indole Metabolites

Mass Spectrum: HREIMS: 161.0842m/e (M +, 100%, C~oHI~NO),requires 161.0840, 144(71), 132(58), 117(23), 101(33), 84(50), and 59m/e (63). Reference L. Gadaschelli, Z. Pang, O. Sterner, and G. Vidari; New Indole Derivatives from the Fruit Bodies of Tricholoma Sciodes and T. Virgatum; Tetrahedron, Vol. 50, pp. 3571-3574 (1994).

9.

Miscellaneous Indole Metabolites

499

Common/Systematic Name 4-Methoxymethyl-2-methylindole Molecular Formula/Molecular Weight

CH2OMe 4~Me

CllHI3NO; M W = 175.09971 9

7

8

General Characteristics 4-Methoxymethyl-2-methylindole was obtained as a yellow oil. Fungal Source Fruit bodies of Tricholoma sciodes and T. virgatum. Isolation/Purification Fruit bodies of Tricholoma sciodes were collected in the vicinity of Lund, Sweden. The fruit bodies were ground in a meat grinder, and the mush was extracted with ethyl acetate. The organic phase was separated, dried with Na2SO4 and the solvent was evaporated. The fresh fruit bodies of T. virgatum were frozen at -20 oC, rapidly broken with a hammer and extracted at -20~ with ethyl acetate. The organic phase was dried with MgSO4 and filtered through a short A1203 column to remove free fatty acids. Final purification from crude extracts was achieved by silica gel column and radial chromatography. Spectral Data UV:

~,Em~" 219(e=22,500), 266 (4,700), and 274nm (5,600). IR:

(KBr) 3360, 2920, 1440, 1420, 1390, 1340, 1290, 1220, 1060, 1000, and 780cmq. 1H M R :

(CDCI3) H-l, 8.18(br s); H-3, 6.32(m); H-5, 7.08(d, J=7.0Hz); H-6, 7.12(dd, ,/=7.0, 7.0Hz); H-7, 7.21(d, J=7.0Hz); H-8, 2.38(s); and H-9, 4.90ppm (s). 13C~

~

(CDCI3) C-2, 135.6(s); C-3, 98.4(d); C-3a, 127.4(s); C-4, 131.4(s); C-5, 118.3(d); C6, 120.9(d); C-7, 110.2(d); C-7a, 136.3(s); C-8, 13.7(q); and C-9, 64.1ppm (t).

500

9.

Miscellaneous Indole Metabolites

Mass Spectrum: HREIMS: 175.0999(M +, 38%, ClIHI3NO), requires 175.0997, 159(10), 144(100), 132(17), and 130m/e (21). Reference L. Garlaschelli, Z. Pang, O. Sterner, and G. Vidari; New Indole Derivatives from the Fruit Bodies of Tricholoma Sciodes and T. Virgatum; Tetrahedron, Vol. 50, pp. 3571-3574 (1994).

9. Miscellaneous Indole Metabolites

501

Common/Systematic Name Marcfortine A Molecular Formula/Molecular Weight C28H35N304; MW = 477.26276

22

J

20 . . . f "

...'

M e ~ , , 26

Me""

24

General Characteristics Crystals; mp., 242-244~

[~]D 22 -5.3 ~ (c= 1.14, in CHC13).

Fungal Source

Penicillium roqueforti.

Isolation/Purification Marcfortine A was purified by chromatography of the alkaloidal extract obtained from lyophilized mycelium. Spectral Data UV" Lm~x 226(e =30,080) and 268nm sh (4,625). IR: (CHC13) 3400(NH), 1700, and 1630cm-I (C=O). IH NMR: (CDCI3) The spectrum revealed an aromatic ring which has two contiguous protons (H-4 and H-5) giving rise to an AB quartet (doublets at 6.79 and 6.6 lppm, Jga=8.1Hz); a widely separated olefinic AB quartet (doublet at 6.38ppm and doublet at 4.92, Jga=7.SHz), assigned to H-24 and H-25, respectively; signals due to four methyl groups [0.84, 1.13 and 1.45ppm (6H)] due to the two isoprene units; and two ofthe three nitrogen atoms were exchangeable with D20 (8.7ppm) and one was an N-CH3 (3.12ppm) group.

502

9.

Miscellaneous Indole Metabolites

13C NMR: (CDC13) C-2, 183.3, s; C-3, 60.6, s; C-4, 120.2, d; C-5, 114.9", d; C-6, 146.2, s; C-7, 132.8"*, s; C-8, 135.4"*, s; C-9, 124.9, s; C-10, 37.0, t; C-11, 63.1, s; C-12, 61.5, t; C13, 64.2, s; C-14, 31.7, t; C-15, 20.7, t; C-16, 25.9, t; C-17, 54.5, t; C-18, 173.9, s; C19, 31.7, t; C-20, 52.9, d; C-21, 46.5, s; C-24, 139.2, d; C-25, 117.2", d; C-26, 79.8, s; N-ME, 26.4, q; 21-Me, 20.7, q, 25.8"**, q; and 26-Me, 29.9ppm, q. , ,

Assignments may be reversed.

Mass Spectrum: HREIMS: 477.2602(M +) and a base peak at 418.2248m/e for C26H3oN203 (M+ MeNHCHO). Reference J. Polonsky, M-A. Merrien, T. Prange, C. Pascard, and S. Moreau; Isolation and Structure (X-Ray analysis) of Marcfortine A, a New Alkaloid from Penicillium roqueforti; J. C. S. Chem. Commun., pp. 601-602(1980).

9.

Miscellaneous Indole Metabolites

503

Common/Systematic Name Marcfortine B Molecular Formula/Molecular Weight C27H33N304; M3~ = 463.24711

22

20 12e,,,,,

...

23 M e ~ ' " ' " ' " b 8 Me,,,,.v_._21 -1 "NH ~ "~0

24

General Characteristics Crystals from ethyl acetate; mp., 178-180~

[0g]D22 -67.7 ~ (c= 1.77, in CHCI3).

Fungal Source Penicillium roqueforti strain B26. Isolation/Purification Marcfortine B was purified by chromatography of the alkaloidal extract obtained from lyophilized mycelium. Spectral Data UV:

)~m,x 229nm (e=20,755) IH NMR: (CDCI3) H-4, 6.66(d, J=8. lHz); H-5, 6.79(d); H-24, 6.39(d, d=7.5Hz); H-25, 4.91(d); n-12, 3.71(,/=11.2Hz), 2.48(br d); H-17, 2.70(dd, J=l 1.2Hz), 2.48(br, d); 26-Me, 1.45, 1.43; and 21-Me, 1.08, 0.81ppm.

13C NMR: (CDCI3) C-2, 183.1, s; C-3, 61.4, s; C-4, 120.4, d; C-5, 115.0", d; C-6, 146.1, s; C-7, 132.6 *~ s; C-8, 135.4 ~ s; C-9, 124.9, s; C-10, 39.9, t; C-11, 61.4, s; C-12, 61.4, t; C13, 63.1, s; C-14, 31.1, t; C-15, 20.9, t; C-16, 25.9, t; C-17, 54.6, t; C-18, 177.1, s; C19, 31.8, t; C-20, 54.2, d; C-21, 46.7, s; C-24, 139.1, d; C-25, 117.8 ~ d; C-26, 79.8, s; 21-Me, 23.9, q, 25.8 ~176176 q; and 26-Me, 30.1ppm, q.

504

9. Miscellaneous Indole Metabolites

, ,

Assignments may be reversed.

Mass Spectrum: t-[REIMS: 463.2479m/e (M +) for C27H33N304. References J. Polonsky, M-A. Merrien, T. Prange, C. Pascard, and S. Moreau; Isolation and Structure (X-Ray analysis) of Marcfortine A, a New Alkaloid from Penicillium roqueforti; J. C. S. Chem. Commun., pp. 601-602(1980). T. Prange, M.-A. Billion, M. Vuilhorgne, C. Pascard, J. Polonsky, and S. Moreau; Structures ofMarcfortine B and C, Alkaloids from Penicillium roqueforti; Tet. Lett., Vol. 22, pp. 1977-1980(1981).

9.

Miscellaneous Indole Metabolites

505

Common/Systematic Name Marcfortine C Molecular Formula/Molecular Weight C27H33N303; M'W = 447.25219

22 .e

12jR1~3

Mg 28

General Characteristics Colorless prisms from ethyl acetate; mp., 264-2670C; [aiD 22 -64.4 ~ (c=l.1, in CHCIs). Fungal Source Penicillium roqueforti strain B26. Isolation/Purification Marcfortine C was purified by chromatography of the alkaloidal extract obtained from lyophilized mycelium. Spectral Data UV:

~,,=x 246nm (e=l 1,490). IH NMR: (CDCI3) H-4, 6.86(d, J=8.1Hz); H-5, 6.42(d); H-24, 6.44(d, J=10Hz); H-25, 5.82(d); H-12, 3.68(d, J=l 1.2Hz), 2.45(br d); H-20,3.08(t, J=10Hz); H-17, 2.67(dd, J=l 1.2Hz); 26-Me, 1.45, 1.43; and 21-Me, 1.08, 0.81ppm. 13C NMR: (CDCI3) C-2, 185.5, s, C-3, 61.0, s; C-4, 124.4, d; C-5, 109.4, d; C-6, 153.1, s, C-7, 105.6, s; C-8, 137.8, s; C-9, 121.2, s; C-10, 39.6, t; C-11, 61.2, s; C-12, 61.4, t, C-13, 62.6, s; C-14, 30.8, t; C-15, 20.7, t; C-16, 25.7, t; C-17, 54.5, t, C-18, 177.2, s; C-19, 31.5, t, C-20, 54.3, d; C-21, 46.3, s; C-24, 116.3, d; C-25, 131.4, d; C-26, 76.8, s, 26Me, 27.8, 27.8; and 21-Me, 23.7, q, 25.8ppm, q.

506

9.

Miscellaneous Indole Metabolites

Mass Spectrum: LREIMS: 447m/e (M +) for C27H33N303. References J. Polonsky, M-A. Merrien, T. Prange, C. Pascard, and S. Moreau; Isolation and Structure (X-Ray analysis) of Marcfortine A, a New Alkaloid from Penicillium roqueforti; J. C. S. Chem. Commun., pp. 601-602(1980). T. Prange, M.-A. Billion, M. Vuilhorgne, C. Pascard, J. Polonsky, and S. Moreau; Structures of Marcfortine B and C, Alkaloids from Penicillium roqueforti; Tet. Lea., Vol. 22, pp. 1977-1980(1981).

9.

Miscellaneous Indole Metabolites

507

Common/Systematic Name Paraherquamide Molecular Formula/Molecular Weight C2sH35N3Os; MW = 493.25767 Me

O

NH

General Characteristics Colorless prisms from ethyl acetate; mp., 244-247~ (dec.); [a]o 22 -28~ MeOH); positive color on Dragendorfs test.

in

Fungal Source Penicillium paraherquei. Biological Activity Reported to be toxic but details not presented. Spectral Data UV:

226(e=32,400), 260(6,100), and 290nrn (1,600). IR:

(KBr) 3510, 3430, 3245, 1714, and 1650cm"~. IH NMR~

(CDCl3) 0.86(3H, s); 1.10(3H, s); 1.45(6H, s); 1.65(3H, s); 1.85(1H, d, J=15Hz); 1.77-2.40(5H, m); 2.55(1H, d, J=l 1Hz); 2.58(1H, s, disappeared with D20); 2.67(1H, d, J=15Hz); 2.93-3.25(2H, m); 3.03(3H, s); 3.58(1H, d, J=l 1Hz); 4.87(1H, d, J=SHz); 6.30(1H, d, J=8Hz); 6.64(1H, d, J=8Hz); 6.78(1H, d, J=8Hz); and 8.33ppm (1H, s, disappeared with D20). Mass Spectrum: LREIMS: 493m/e (M+).

508

9. Miscellaneous Indole Metabolites

Reference M. Yamazaki, E. Okuyama, M. Kobayashi, and H. Inoue; The Structure of Paraherquamide, A Toxic Metabolite from Penicillium paraherquei; Tet. Lett., Vol. 22, pp. 135-136(1981).

9.

Miscellaneous Indole Metabolites

509

Common/Systematic Name Nominine Molecular Formula/Molecular Weight C2sH39NO; MW = 405.30317 29 -

13

28

-__ .

:"

5

: 21~ 10

:

General Characteristics Melting point, 54-55~

:

OH

[a]D +23.6 ~ (c=0.85, in MeOH).

Fungal Source Sclerotia ofAspergillus nomius (NRRL 1313 7). A. nomius is closely related to the aflatoxin-producing fungi, A. flavus and A. parasiticus, both of which selectively allocate antiinsectan aflavinine derivatives to their sclerotia. Isolation/Purification Sclerotia of A. nomius were ground with a mortar and pestle and triturated repeatedly with hexane. The combined hexane extracts were filtered and evaporated to afford a fight yellow oil which was subjected to reversed-phase semipreparative HPLC [51.tm, Cls column; 90:10 (v/v) MeOH-H20 at 2.0mL/min] to afford nominine as an off-white powder. Biological Activity Nominine exhibited potent activity against the widespread crop pest Hefiothis zea, causing 40% mortality and a 97% reduction in weight relative to controls when incorporated into a standard test diet at 100ppm dry weight. Spectral Data ~H NMR Data: (CDCI3) H-l, 7.88(br, s); H-2, 6.93(br s); H-5, 7.59(br d, J= 7.8Hz); H-6, 7.1 l(dd, ,/=7.1, 7.8); H-7, 7.17(dd, J=7.1, 8.1); H-8, 7.32(br d, J=8.1); H-10, 3.04(br d, J=15.4Hz; 3.10(dd, J=9.0, 15.4Hz); H-11, 3.22(br d, J=9.0Hz; H-13, 2.1 l(ddd, J=3.2,

510

9.

Miscellaneous Indole Metabolites

3.5, 12.7Hz); 2.21(m); H-14, 1.51(m); 1.68(m); H-16, 2.45(m); H-17, 1.33(m); 1.70(m); H-18, 1.64(m); 1.92(m); H-19, 4.52(br s); H-21, 1.36(m); 1.75(m); H-22, 2.21(m); H-23, 5.17(br t, J=7.1Hz); H-25, 1.67(br s); H-26, 1.71(br s); H-27, 4.81(br s); 4.92(br s); H-28, 0.82(d, J=6.6Hz); and H-29, l l.01ppm (s). ~3CNMR: (CDCI3) C-2, 121.2(d); C-3, 117. l(s); C-4,127.7(s); C-5, 118.5(d); C-6, 119.1(d); C-7, 121.8(d); C-8, 111.0(d); C-9, 136.0(s); C-10, 21.4(t); C-11, 45.9(d); C-12, 148.9(s); C-13, 33.5(t); C-14, 34.4(0; C-15, 40.9(s); C-16, 31.1(d); C-17, 25.4(0; C-18, 25.8(0; C-19, 70.0(d); C-20, 47.9(s); C-21, 29.2(t); C-22, 23.9(t); C-23, 125.8(d); C-24, 131.2(s); C-25, 17.8(q); C-26, 25.8(q); C-27, 107.7(t); C-28, 16.6(q); and C-29, 18.7ppm (q). Mass Spectrum: EIMS: (70 eV) 405(M~; rel intensity 57), 387(100), 318(15), 304(52), 302(70), 288(17), 248(15), 232(15), 196(42), 180(40), 168(28), and 156(16); HREIMS obsd 405.3035; calcd for C2sH39NO405.3031. Reference J. B. Gloer, B. L. Rinderknecht, D. T. Wicklow, and P. F. Dowd; Nominine: A New Insecticidal Indole Diterpene from the Sclerotia ofAspergillus nomius; J. Org. Chem., Vol. 54, pp. 2530-2532(1989).

9.

M i s c e l l a n e o u s Indole Metabolites

511

Common/Systematic Name Aspernomine Molecular Formula/Molecular Weight C28Ha9NO2; ~

28

29

Ill~

= 421.29808

'! 16 ~

5

a

26

H

General Characteristics [tt]D +225 ~ (C=0.12 g/dL; MeOH). Fungal Source Sclerotial metabolite from Aspergillus nomius (NRRL 6552). The strain ofA. nomius was originally isolated from a pine sawfly (Diprion similis). Isolation/Purification Sclerotia of A. nomius were ground with a mortar and pestle and then extracted with n-pentane. Concentration of the resulting n-pentane extract afforded a yellow-orange oil. A portion of this extract was subjected to silica gel chromatography using a hexane-ethyl acetate gradient. Aspernomine was obtained as fine white needles from 90% hexane-ethyl acetate. Aspernomine was also purified by reversed-phase HPLC separation of the pentane extract, eluting slightly before nominine with a retention time of 20.9 min, using 90:10(v/v) MeOH-H20 on a Beckman Ultrasphere 5ktm Cls column at 2.0 mL/min. Biological Activity Exhibited moderate activity against the corn earworm (Helicoverpa zea); 100ppm in a standard diet resulted in a 35% reduction in weight gain of test insects relative to controls. Exhibited cytotoxicity toward three human solid tumor cell lines. EDs0 values were 3.09, 4.93, and 3.08ktg/ml (A-549 lung carcinoma, MCF-7 breast adenocarcinoma and HT-29 colon adenocarcinoma). Spectral Data El-V;

~

Ma3H

333(e=1,150), 302(2,510), 244(5,760), and 222nm (5,460).

512

9.

Miscellaneous Indole Metabolites

IR:

(neat) 3500, 3360, 2970, 2930, 1698, 1606, 1490, and 750cm~. ~H NMR: (CDCI3) H-I, 4.35(br s); H-2, 3.75(br s); H-5, 7.43(br d, J=7.2Hz); H-6, (ddd, J=l.1, 7.2, 7.2); H-7, 7.04(ddd, J=l.1, 7.8, 7.8Hz); H-8, 6.51(dd, J=l.1, 7.8Hz); H-1 la, 2.08(dd, J=9.3, 17.6); H-1 lb, 2.49(d, J-17.6); H-12, 2.64(br d, J=9.3Hz); H-13~, 1.28(m); H-13ax, 2.58(ddd, J=4.1, 14.5, 14.5Hz); H-14=q, 1.39(m); H14~, 1.68(m); H-16, 2.40(m); H-17a, 1.34(m); 17b, 1.73(m); H-18~, 1.53(br dd, J=3.1, 14.0Hz); H-18=q, 1.82(m); H-19, 4.01(br s); H-21a, 1.63(m); H-21b, 1.97(m); H-22a, 1.98(m); H-22b, 2.18(m); H-23, 5.01(br t); H-25, 1.60(s); H-26, 1.68(s); H-27a, 1.79(br d, J=14.0); H-27b, 2.93(br dd, J=3.7, 14.0Hz); H-28, 0.93(d, J=7.7Hz); and H-29, 1.09(s). 13C NM~:

(CDCI3) C-2, 56.97(d); C-3, 36.30(s), C-4, 130.70(s); C-5, 125.59(d), C=6, 118.37(d); C=7, 127.58(d); C-8, 114.65(d); C=9, 142.70(s); C-10, 209.20(s); C1 I-a, 37.33(t), C-12, 47.25(d), C-13, 30.62(t); C-14, 28.95(t); C=15, 40.09(s); C=16, 31.33(d); C=17, 24.97(0; C-18,29.92(t); C=19, 69.84(d); C=20, 46.38(s); C-21, 30.20(t), C-22, 23.88(0; C-23, 125.59(d), C-24, 131.72(s); C-25, 17.91(q); C-26, 25.57(q); C-27, 34.63(t), C=28, 15.92(q); and C-29, 18.84(q). Mass Spectrum: ELMS: (70 eV) 421(M +, rel intensity 4), 184(7), 156(100), 143(37), 130(28); HREIMS obsd 421.3024; calcd for CzsH39NO2,421.2981. Reference G. M. Staub, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Aspernomine: A Cytotoxic Antiinsectan Metabolite with a Novel Ring System from the Sclerotia of Aspergillus nomius; J. Amer. Chem. Soc., Vol. 114, pp. 1015-1017(1992).

9.

Miscellaneous Indole Metabolites

513

Common/Systematic Name Tubingensin A Molecular Formula/Molecular Weight C2sH35NO; MW = 401.27186 25

L\ 15

5

3

12

28

8

General Characteristics A light yellow solid from MeOH-H20; mp., 95-98~

[a]D 13.6 ~ (c=l.0, in CHCI3).

Fungal Source

Aspergillus tubingensis (NRRL 4700), a member of the Aspergillus niger taxonomic group.

Isolation/Purification Sclerotia of A. tubingensis were ground with a mortar and pestle and triturated repeatedly with hexane. The combined hexane extracts were filtered and evaporated to afford a yellow oil. This residue was subjected to crude preliminary separation by reversed-phase semipreparative HPLC [5-I.tm C~8 column; 250x10mm; MeOH-H20 (90:10, v/v) at 2.0mL/mi~ HPLC retention time 17.7min] [MeOH-HzO (90:10, v/v)]. Fractions containing tubingensin A were rechromatographed on the same column at MeOH:H20 (85:15, v/v) to afford tubingensin A as a light yellow solid. Biological Activity Tubingensin A exhibited activity against the widespread crop pest Heliothis zea and displayed in vitro antiviral activity against Herpes simplex virus type 1. Spectral I~ata ~.~,M~. 340(e=480), 326(480), 302(6,780), 262(6,930), 239(18,200), and 218nm (:14,900).

514

9.

Miscellaneous Indole Metabolites

~H NMR: (CDC13) H-l, 7.81(br s); H-5, 7.98(br d, J=7.8), H-6, 7.18(dd, J=3.9, 7.6, 7.8); H-7, 7.34(m); H-8, 7.34(m); H-10, 7.92(s); H-13, 2.99(ddd, J=7.3, 12.9, 17.6, I-I,x);2.88(br, dd, J=6.6, 17.6, I-~); H-14, 1.52(m); 2.01(m); H-16, 1.74(m); H-17, 1.17(m); 1.70(m); H-18, 1.66(m); 2.05(m); H-19, 4.99(br, s); H-21, 1.71(m); 2.08(m); H-22, 1.76(m); 2.06(m); H-23, 5.03(dd, J=6.6, 5.9); H-25, 1.43(br, s); H-26, 1.58(br, s); H-27, 7.1 l(s), H-28, 0.85(d, J=5.6); and H-29, 1.21ppm (s). ~SCNMR: (CDCI3) C-2, 137.84(s); C-3, 121.32(s); C-4, 123.78(s); C-5, 119.77(d); C-6, 119.13(d), C-7, 125.27(d), C-8, 110.41(d), C-9, 139.99(s), C-10, 118.45(d), C-11, 132.43(s); C-12, 135.08(s); C-13, 27.06(0; C-14, 29.42(t); C-15, 38.76(s), C-16, 32.58(d); C-17, 25.36(0; C-18, 29.61(0; C-19, 71.35(d), C-20, 47.23(s), C-21, 34.91(0; C-22, 23.10(0; C-23, 125.00(d), C-24, 131.48(s), C-25, 17.62(q); C-26, 25.62(q), C-27, 110.65(d), C-28, 0.85(q), and C-29, 18.35ppm (s). Mass Spectrum: EIMS: 401(M+, 38%), 318(100), 300(52), 260(11), 246(40), 234(21), 232(25), 220(28), 206(51), 180(32), 146(10), and 130m/e(5); HREIMS: obsd 401.2698; caled for C2oHssNO401.2720. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Tubingensin A: An Antiviral Carbazole Alkaloid from the Sclerotia ofAspergillus tubingensis; J. Org. Chem., Vol. 54, pp. 4743-4746(1989).

9.

Miscellaneous Indole Metabolites

515

Common/Systematic Name Tubingensin B Molecular Formula/Molecular Weight C28H35NO; ~ = 401.27186 28

29 14

)16

13 .,

25 8

Fungal Source Aspergillus tubingensis (NRRL 4700). Isolation/Purification Sclerotia extracted with hexane and fractionated by fig HPLC (see Tubingensis A). Biological Activity Exhibited antiinsectan activity against Heliothis zea and antiviral activity against Herpes simplex. Spectral Data UV:

Characteristic of a carbazole moiety. 'H NMR: (CDCI3) H-I, 7.86(br s); H-5, 7.98(br, d, 0=7.8); H-6, 7.15(ddd, J=l.5, 7.3, 7.8); H-7, 7.32(ddd, J=l.l, 6.6, 7.3); H-8, 7.30(dd, J=1.5, 6.6); H-10, 8.05(br s); H-13=q, 1.40(m); H-13~, 1.82(ddd, d=4.4, 12.7, 14.1); H-14~q, 1.24(m); H-14~, 0.17(ddd, d=5.0, 13.4, 14.1); H-16, 1.22(m); H-17, 1.31(m), 1.72(m); H-18~q, 2.1 l(ddd, J=3.2, 3.4, 10.7); H-18,x, 2.68m; H-19, 4.36 brt); H-21, 1.76(m); 2.59 (br dd, J=3.6, 10.3); H-22, 1.44(m); 1.66(m); H-24, 2.41(qq, J=6.6, 6.8); H-25, 1.00(d, ,/=6.6); H-26, 1.09(d, J=6.8); H-27, 7.32(br s); H-28, 0.70(d, J=6.8); and H-29, 1.15ppm (s). ~3CNMR: (CDCI3) C-2, 137.95; C-3, 119.8; C-4, 123.65; C-5, 119.7; C-6, 119.19; C-7, 125.08; C-8, 110.8; C-9, 139.62; C-10, 116.83; C-1 l, 134.80; C-12, 142.80; C-13, 38.36;

516

9.

Miscellaneous Indole Metabolites

C-14, 38.08; C-15, 40.67; C-16, 38.79; C-17, 25.65; C-18, 33.67; C-19, 76.65; C-20, 47.63; C-21, 27.70; C-22, 25.63; C-23, 42.47; C-24, 35.05; C-25, 19.45; C-26, 17.06; C-27, 107.39; C-28, 16.89; and C-29, 14.08ppm. Mass Spectrum: ELMS: 401(M+; rel. int. 91%), 383(1.0), 358(15), 340(4), 330(1.7), 314(1.4), 300(1.6), 288(36), 260(31), 246(49), 230(34), 218(100), 204(16), 191(5), 180(11), 167(5), 144(7), and 130role (28); HREIMS, obs. 401.2733; calcd, for C2sH3sNO, 401.2720 Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; The Structure of Tubingensin B: A Cytotoxic Carbazole Alkaloid from the Sclerotia of Aspergillus tubingensis; Tet. Lett., Vol. 30, pp. 5965-5968(1989).

9. Miscellaneous Indole Metabolites

517

Common/Systematic Name Radarin A Molecular Formula/Molecular Weight C28H39NO2; MW -- 421.29808

HO/ ~

-~NH~

27

29O".,,.L19J 30 ~'"

y

0 General Characteristics Radarin A was isolated as a pink oil; [a]D +11.1 o (c=0.005 ' in CHCI3). Fungal Source Sclerotia of Aspergillus sulphureus (NRRL 4077). Isolation/Purification Ground sclerotia ofA. sulphureus (NRRL 4077) were exhaustively extracted using a Soxhlet apparatus, first with pentane and then with methylene chloride. The methylene chloride extract was subjected to silica gel column chromatography, using a stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3. Fractions of similar composition as determined by TLC were pooled. The resulting active fractions were separated further by reversed-phase HPLC (51.tm-Beckman Ultrasphere ODS column; 250x10mm; with detection at 215nm; flow rate 2.0mL/min) using various MeOH-H20 mixtures to yield pure radarins A-D. Radarin A: HPLC retention time, 25.5 min (MeOH-H20, 88:12, v/v). Biological Activity Exhibited significant biological activity against the corn earworm Helicoverpa zea, as well as cytotoxicity in assays against three human solid tumor cell lines. Spectral Data UV: ~,~H

210(e=6,222), 226(6,985), and 290nm (1,512).

IR: 3405, 2925, 2956, 1700, 1628, 1452, and 757cm"~.

518

9.

Miscellaneous Indole Metabolites

CD: (MeOH) 290(-78.6), 233(-31.9), and 216nm (-34.3). 1H NMR: (acetone-d6) H-2, 6.89(br s); H-4, 7.36(d, ,/=8.6); H-5, 6.63(dd, ,/=8.6, 2.2); H-7, 6.78(d, J=2.1); H-10a, 2.74(d, J=15.1); H-10b, 2.65(d, J=15.1); H-12,~, 1.57(m); H-13,x, 1.39(m); H-13~, 1.25(m); H-la,x, 0.71(m); H-14~, 1.58(m); H-16,x, 1.28(m); H-17~,, 1.83(m); H-17~, 1.54(m); H-18,x, 2.22(m); H-18~, 2.13(m); H-20~, 2.10(m); H-22,x, 1.20(m); H-22,~, 1.72(ddd, J=12.9, 3.3, 3.2); H-23,x, 1.50(m); H-23m, 1.96(m); H-24,,,, 1.10(dd, J=l 1.9,1.9); H-25, 0.82(s); H-26, 1.02(d, ,/=-6.7); H-27, 0.86(s); H-28, 0.74(d, J=6.7); and H-29, 0.68ppm (s). 13C NMI~:

(acetone-d6) C-2, 123.2; C-3, 112.2; C-4, 120.2; C-5, 109.8; C-6, 153.9; C-7, 97.4; C-8, 138.1; C-9, 124.1; C-10, 34.5; C-11, 41.3; C-12, 36.8; C-13, 28.1; C-14, 40.2; C-15, 38.7; C-16, 59.3; C-17, 22.8; C-18, 41.7; C-19, 211.4; C-20, 58.3; C-21, 42.1; C-22, 41.0; C-23, 19.7; C-24, 51.1; C-25, 18.1; C-26, 17.6; C-27, 16.8; C-28, 7.2; and C-29, 15.0ppm. Mass Spectrum: EIMS: 421(M§ 4.3%), 189(4.2), 175(2.6), 161(3.7), 146(100), 123(6.6), 121(9.3), 119(5.8), 109(8.0), 107(9.0), and 105m/e (7.0); HREIMS: obsd 421.2996; calcd for C2sH39NO2, 421.2981. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Radarins A-D: New Antiinsectan and Cytotoxic Indole Diterpenoids from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 138-141(1992).

9.

Miscellaneous Indole Metabolites

519

Common/Systematic Name Radarin B Molecular Formula/Molecular Weight C28H41NO2; M'W = 423.31373 4

H

27

29 IIIl**

28 =o"'"

OH General Characteristics Radarin B was a yellow solid; mp., 115-118~ (dec); [a]D +39.4 ~ (c=0.003, in CHCI3). Fungal Source Sclerotia of Aspergillus sulphureus (NRRL 4077). Isolation/Purification Ground sclerotia of A. sulphureus (NRRL 4077) were exhaustively extracted using a Soxhlet apparatus, first with pentane and then with methylene chloride. The methylene chloride extract was subjected to silica gel column chromatography, using a stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3. Fractions of similar composition as determined by TLC were pooled. The resulting active fractions were separated further by reversed-phase HPLC (51am-Beckman Ultrasphere ODS column; 250x10mm; with detection at 215nm; flow rate 2.0mL/min) using various MeOH-H20 mixtures to yield pure radarins A-D. Radarin B: I-IPLC retention time, 22.2 min (MeOH:HzO, 86:14, v/v). Biological Activity Exhibited biological activity against the corn earworm Helicoverpa zea, as well as cytotoxicity in assays against three human solid tumor cell lines. Spectral Data IR:

3408, 2927, 2856, 1628, 1453, 1384, and 757cm"~. CD: (MeOH) 228nm (-64.2).

520

9.

Miscellaneous Indole Metabolites

~H NMR: (acetone-d6) H-2, 6.88(br s); H-4, 7.38(d, J=8.5); H-5, 6.61(dd, J=8.6, 2.2); H-7, 6.80(d, J=2.0); H-10a, 2.75(d, J=15.2); H-10b, 2.66(d, J=15.1); H-12,~, 1.52(m); H-13,~, 1.38(m); H-13,a, 1.21(m); H-14,~, 0.58(dm, J=3.9); n-14~, 1.58(m); H-16,~, 0.64(dd, J=l 1.7, 1.9); H-17,x, 1.50(m); H-17,a, 1.30(m); H-18~, 1.40(m); H-18,~, 1.76(m); H-19~, 3.62(m);H-20~,, 1.07(m); H-22,x, 0.95(m); H-22m, 1.76(m); H-23~, 1.45(m); H-23~, 1.89(dm, J=13.5); H-24,x, 1.04(m); H-25, 0.83(s); H-26, 1.04(d, J=7.5); H-27, 0.89(s); H-28, 0.92(d, J=7.2); and H-29, 1.02ppm (s). 13CNMR: (acetone-d6) C-2, 123.0; C-3, 112.2; C-4, 120.2; C-5, 109.7; C-6, 153.8; C-7, 97.3; C-8, 138.1; C-9, 124.2; C-10, 34.3; C-11, 41.3; C-12, 36.7; C-13, 28.2; C-14, 40.2; C-15, 38.3; C-16, 61.9; C-17, 16.6; C-18, 36.5; C-19, 71.8; C-20, 50.3; C-21, 38.4; C-22, 41.7; C-23, 19.1; C-24, 51.3; C-25, 17.9; C-26, 17.7; C-27, 17.1; C-28, 12.2; and C-29, 17.0ppm. Mass Spectrum: EIMS: 423(M+, 1.3%), 405(3.8), 243(7.7), 163(11), 147(100), 121(19), 107(22), 91(28), and 81m/e (22); HRFABMS: obsd 424.3245; calcd for C2d-I4~NO2+ H, 424.3215. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Radarins A-D: New Antiinsectan and Cytotoxic Indole Diterpenoids from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 138-141(1992).

9.

Miscellaneous Indole Metabolites

521

Common/Systematic Name Radarin C Molecular Formula/Molecular Weight C2sHagNO; MW = 405.30317 _-'3 4

--:

"

".',,,.L,, ,) 28

="

~]~

O General Characteristics Radarin C was a yellow oil; [a]D +6.7 ~ (c=0.002, in CHCI3). Fungal Source Sclerotia of Aspergillus sulphureus (NRRL 4077). Isolation/Purification Ground sclerotia ofA. sulphureus (NRRL 4077) were exhaustively extracted using a Soxhlet apparatus, first with pentane and then with methylene chloride. The methylene chloride extract was subjected to silica gel column chromatography, using a stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3. Fractions of similar composition as determined by TLC were pooled. The resulting active fractions were separated further by reversed-phase HPLC (5~m-Beckman Ultrasphere ODS column; 250x10mm; with detection at 215nm; flow rate 2.0mL/min) using various MeOH-H20 mixtures to yield radarins A-D. Radarin C: I-IPLC retention time, 40 min (MeOH-H20, 90:10, v/v). Biological Activity Exhibited biological activity against the corn earworm Helicoverpa zea, as well as cytotoxicity in assays against three human solid tumor cell lines. Spectral Data IR:

3410, 2925, 2857, 1707, 1456, 1388, and 741cm"~. CD: (MeOH) 298(-58.7), 229(-36.7), and 212nm (+68.4).

522

9.

Miscellaneous Indole Metabolites

~HNMR: (acetone-d6) H=2, 7.13(br s); H-4, 7.61(br d, J=8.0); H-5, 7.00(ddd, J=8.0, 7.1, 1.0); H-6, 7.07(ddd, ,/=8.0, 7.0, 1.0); H-7, 7.38(br d, ,/-8.0); H-10a, 2.83(d, J=lS.0); H-10b, 2.76(d, J=15.2); H-12=,, 1.60(m); H-13~, 1.42(m); H-13~, 1.30(m); H-14=,, 0.73(m); H-14~, 1.62(m); H-16~, 1.36(m); H-17~, 1.87(m); H-17~, 1.56(m); H-18~, 2.32(m); H-18~, 2.25(m); H-20=,, 1.07(m); H-22=,, 0.95(m); H-22~, 1.76(m); H-23=,, 1.45(m); H=23~, 1.89(dm, J=13.5); H=24~, 1.04(m); H-25, 0.83(s); H-26, 1.04(d, J=7.5); H-27, 0.89(s); H-28, 0.92(d, J=7.2); and H-29, 0.72ppm (s). ~3CNMR: (acetone-d6) C-2, 125.0; C-3, 112.2; C-4, 119.8; C-5, 109.3; C-6, 121.7; C-7, 112.1; C-8, 137.0; C-9, 130.1; C-10, 34.3; C-11, 41.3; C-12, 36.8; C-13, 28.1; C-14, 40.2; C-15, 38.4; C-16, 59.3; C-17, 22.8; C-18, 41.7; C-19, 211.5; C-20, 58.2; C-21, 42.1; C-22, 41.0; C-23, 19.7; C-24, 51.2; C-25, 18.1; C-26, 17.6; C-27, 16.7; C-28, 7.1; and C-29, 15.0ppm. Mass Spectrum: EIMS: 405(M+, 2.0%), 137(2.2), 130(100), 121(3.2), 111(3.3), 109(3.2), and 107m/e (3.5); HR IMS; obsd 405.3008; calcd for C2sH39NO,405.3032. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd, Radarins A-D: New Antiinsectan and Cytotoxic Indole Diterpenoids from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 138-141(1992).

9.

Miscellaneous Indole Metabolites

523

Common/Systematic Name Radarin D Molecular Formula/Molecular Weight C28H41NO; MW = 407.31882 _ _: . 4

3

"

"

27

II1'1 ~ 28 ll~'"'

OH General Characteristics Radarin D was a yellow oil; [{X]D +31.8 ~ (c=0.003, in CHCI3). Fungal Source Sclerotia of Aspergillus sulphureus (NRRL 4077). Isolation/Purification Ground sclerotia ofA. sulphureus (NRRL 4077) were exhaustively extracted using a Soxhlet apparatus, first with pentane and then with methylene chloride. The methylene chloride extract was subjected to silica gel column chromatography, using a stepwise gradient from 0 to 10% (v/v) MeOH in CHCI3. Fractions of similar composition as determined by TLC were pooled. The resulting active fractions were separated further by reversed-phase HPLC (51.tm-Beckman Ultrasphere ODS column; 250x10mm; with detection at 215nm; flow rate 2.0mL/min) using various MeOH-H20 mixtures to yield radarins A-D. Radarin D: HPLC retention time, 45 min (MeOH-H20, 90:10, v/v). Biological Activity Exhibited biological activity against the corn earworm Helicoverpa zea, as well as cytotoxicity in assays against three human solid tumor cell lines. Spectral Data IR:

3414, 2924, 2856, 1456, 1384, and 742cm ~. CD: (MeOH) 228(-58.0), 298(-58.7), 229(-36.7), and 212nm (+68.4).

524

9.

Miscellaneous Indole Metabolites

1H NMR: (acetone-d6) H-2, 7.08(br s); H-4, 7.60(br d, J=8.0); H-5, 7.05(ddd, ,/=7.0, 7.0, 1.0); H-6, 7.05(ddd, J=7.0, 7.0, 1.0); H-7, 7.36(d, J=8.1); H-10a, 2.81(d, d-15.2); H-10b, 2.72(d, J=15.1); H-12ax, 1.57(m); H-13,x, 1.38(m); H-13~, 1.21(m); H-14,~, 0.58(ddd, d-13.0, 13.0, 3.8); H-14~, 1.60(m); H-16,x, 0.62(dd, J=12.0, 1.8); H-17,~, 1.50(m); H-17~, 1.30(m); H-18~, 1.40(m); H-18~, 1.78(m); H-19~, 3.61(dm, d-2.6); H- 20,~, 1.06(m); H-22,~, 0.95(ddd, J=12.8, 12.8, 3.7); H-22~, 1.76(m); H-23=,, 1.45(m); H-23~, 1.89(dm, J=16.6); H-24,x, 1.03(dm, J-1.8); H-25, 0.86(s); H-26, 1.05(d, d-6.5); H-27, 0.89(s); H-28, 0.90(d, J=7.1); and H-29, 1.02ppm (s). ~SCNMR: (acetone-d6) C-2, 124.8; C-3, 112.3; C-4, 119.8; C-5, 119.2; C-6, 121.6; C-7, 112.0; C-8, 136.9; C-9, 130.2; C-10, 34.2; C-11, 41.4; C-12, 36.8; C-13, 28.2; C-14, 40.3; C-15, 38.3; C-16, 62.0; C-17, 16.7; C-18, 36.5; C-19, 71.7; C-20, 50.3; C-21, 38.4; C-22, 41.8; C-23, 19.1; C-24, 51.4; C-25, 17.9; C-26, 17.7; C-27, 17.1; C-28, 12.2; and C-29, 17.1ppm. Mass Spectrum: EIMS: 407(M§ 0.5%), 389(1.7), 163(4.3), 149(5.5), 131(100), 121(6.9), 111(7.1), 109(5.1), and 107m/e (6.9). HREIMS: obsd 407.3176; calcd for C28I-h~NO,407.3188. Reference J. A. Laakso, J. B. Gloer, D. T. Wicklow, and P. F. Dowd, Radarins A-D: New Antiinsectan and Cytotoxic Indole Diterpenoids from the Sclerotia of Aspergillus sulphureus; J. Org. Chem., Vol. 57, pp. 138-141(1992).

Loline Alkaloids Loline; Festuline N-Formylloline N-Acetylloline N-Methylloline Norloline N-Formylnorloline N-Acetylnorloline Peramine Festucine

525

This Page Intentionally Left Blank

10. Loline Alkaloids

527

Common/Systematic Name Loline; Festucine Molecular Formula/Molecular Weight CsH14N20; M W = 154.11061 O

,' ~, H

Me

,' HH_\ _N--H 'e -. t f

e

Hb,, .... H

....

H

Hb H6 General Characteristics Colorless, viscous oil. Fungal Source Present in caryopses of tall fescue (Festuca arundinacea) infected with the endophyte Acremonium coenophialum. Also found in Lolium temulentum, L. cuneatum, and

A denocarpus decorticans.

Isolation/Purification Air dried caryopses were pulverized and extracted with ligroin (Soxhlet method) for 12h followed by cold methanol. The filtrate was evaporated to dryness. The viscous brown residue was suspended in 2.5% HCI and was kept in the refrigerator overnight. Atter filtration, the aqueous solution was extracted with Et20 and CHzCI2, respectively. The aqueous solution was evaporated to dryness and a small quantity of methanol was used to suspend the residue; the solution was adjusted to pH 8-10 with 30% NaOH and a crude alkaloid mixture was obtained alter column chromatography on alumina. Further purification of loline was accomplished with gas chromatography of the crude alkaloid mixture(column length 2m, diameter 3 mm; 3% SE 30 on Chromosorb PN AW; column temperature 110~ injection temperature 150~ flow rate was 50ml Nz/min, retention time = 8.5). Loline was obtained as a colorless oil. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue; however, in tests for central nervous system activity lolineoHC1 was ineffective in displacing prazosin, serotonin, QNB, bungarotoxin, and flunitrazepam from binding sites.

528

10.

Loline Alkaloids

Spectral Dat.a IR."

(CHCI3) 2793,2715, 1476, 1432, 1367, 1311, 1293, 1251, 1218, 1134, 1090, 1044, 1025, 991, and 956cm~. 1H NMR:

(CDCI3) H-I, 3.03(dd,Jl,2=1.6Hz);H-2, 3.71(dd,J2,a,=<2.0Hz);H-3a, 3.10(dd,

,]3a,3b=l1.6Hz); H-3b, 2.10(d); H-5a, 2.62(ddd, Js~,sb=12.3Hz, Jsa,6a=9.SI-tz, Js~,6b=7.3Hz, JSb,6==3.8Hz,JSb,6b=8.3Hz);H-5b, 2.77(ddd); H-6a, 1.65(dddd, J6,,6b=14.3Hz, J6,,7=4.3Hz); H-6b, 1.74(ddd); H-7, 4.10(dd, ,/7,s=l.9Hz); H-8, 2.84(dd, Js.l = 1.8Hz); and N-Me, 2.19ppm (s). (1)20) H-I, 4.23(dd, J~,2=<2.0Hz); H-2, 4.79(dd, J2a,=l.0Hz); H-3a, 4.15(dd, J3,,3b=13.9Hz); H-3b, 3.55(d); H-5a, 3.73(ddd, Js,,Sb=12.SHz, Js,,6,=8.2I-Iz, Js,,6b=7.7Hz, JSb,6,=5.0Hz, JSb,6b=9.6Hz);H-5b, 3.73(ddd); H-6a, 2.28(dddd, J6=,6b=14.6Hz, J6=,v=4.8Hz); H-6b, 2.37(ddd); H-7, 4.72(dd, JT,S=2.2Hz); H-8, 4.79(dd, JS,l =l.9Hz); NH, 4.76(bm); and N-Me, 2.79ppm (s). 13C NMR:

(CHCI3) C-I, 67.8(d); C-2, 73.4(d); C-3, 60.6(t); C-5, 54.0(0; C-6, 33.5(0; C-7, 81.0(d); C-8, 69.0(d); and N-Me, 34.7ppm (q). (D20) C-I, 65.9(d); C-2, 73.9(d); C-3, 64.2(t); C-5, 58.1(0; C-6, 31.6(0; C-7, 83.2(d); C-8, 72.2(d); and N-Me, 36.5ppm (q). Mass Spectrum: EIMS: 154(M§ 3.5), 123(12), 110(34), 95(31), and 82m/e (100%). References G. Dannhardt and L. Steindl; Alkaloids ofLo#um temulentum; Isolation, Identification and Pharmacological Activity; Planta Medica, pp. 212-214(1985). R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989). R. G. Powell and R. J. Petroski; The Loline Group of Pyrrolizidine Alkaloids; In S. W. Pelletier (Ed.); The Alkaloids: Chemical and Biological Perspectives; Vol. 8, p. 320, Springer-Verlag, New York (1992). S. G. Yates and H. L. Tookey, Festucine; An Alkaloid from Tall Fescue (Festuca arundinacea Schreb.): Chemistry of the Functional Groups; Aust. J. Chem., Vol. 18, pp. 53-60(1965).

10. Loline Alkaloids

529

Common/Systematic Name N-Formylloline Molecular Formula/Molecular Weight C9H14N202, M'W --- 182.10553 O

,' ~,

0

�9t

Me I

H ,,' HH,+N--C HO

H,. b ,,,,. / ' aT ' \~ H "' ~ a

t

Hb HI:3 General Characteristics Clear, viscous oil. Fungal Source Present in caryopses of tall fescue (Festuca arundinacea) infected with the endophyte Acremonium coenophialum; also found in Lolium cuneatum. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue. Studies have shown a positive association between endophyte infection, loline alkaloid production and resistance to certain insect pests. N-Formylloline was shown to be toxic to the large milkweed bug (Oncopeltus fasciatus) (EDs0 2~g). Spectral Data IR; (CHCI3) 2937, 2880, 1671, 1473, 1386, 1353, 1084, 1050, 1024, and 962cm"l. IH NMR: (CDCI3) H-I, 3.68(dd, J~,2=l.5Hz); H-2, 4.05(dd, J2,a,=<2.0Hz); H-3a, 3.09(dd, J3a,3b=l 1.9Hz); H-3b, 2.33(d); H-5a, 2.83(ddd, Js,,Sb=13.1Hz, Js,,6,=9.3Hz, Jsa,6b=7.1Hz, Jsb,6,=4.4Hz, Jsb,6b=7.SHz); H-Sb, 2.9 l(ddd); H-6a, 1.80(dddd, J6,,6b=14.3Hz, J6,,7=4.3Hz); H-6b, 1.93(ddd); H-7, 4.34(dd, JT,8=l.8Hz); H-8, 3.30(dd, Js,~ =2.0Hz); N-Me, 2.76(s); and HC=O, 7.25ppm (s). lac NMR: (CHCI3) C-l, 65.3(d); C-2, 73.9(d); C-3, 60.4(t); C-5, 54.4(t); C-6, 32.8(t); C-7, 81.8(d); C-8, 67.4(d); N-Me, 33.4(q); and HC=O, 162.1ppm (d).

530

10.

Loline Alkaloids

Mass Spectrum: EIMS: 154(M + - 28, 11.0), 123(9), 110(9), 95(24) and 82m/e (100%); GC-MS: 182(1), 154(15), 139(1), 123(10), 111(11), 110(12), 96(9), 95(25), 83(17), 82(100), 80(21), 69(16), 55(16), 42(45), and 41m/e (22%). References R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989). S. G. Yates, J.C. Fenster, and R. J. Bartelt; Assay of Tall Fescue Seed Extracts, Fractions and Alkaloids Using the Large Milkweed Bug; J. Agric. Food Chem. Vol. 37, pp. 354-357 (1989). S. G. Yates and H. L. Tookey; Festucine, and Alkaloid from Tall Fescue (Fesmca arundinacea Schreb.): Chemistry of the Functional Groups; Aust. J. Chem., Vol. 18, pp. 53-60(1965).

10.

Loline Alkaloids

531

Common/Systematic Name N-Acetylloline Molecular Formula/Molecular Weight CloHI6N202; MW = 196.12118

t

i

O ,' ~i.

i

MI ~ I

H_ ,' H H_', ,,N--Ac al,,

Ha" Hb

-- "Ha H6

General Characteristics Clear, viscous oil. Fungal Source Present in caryopses of tall fescue (Festuca arundinacea) infected with the endophyte Acremonium coenophialum; also found in Lolium cuneatum. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue. Studies have shown a positive association between endophyte infection, loline alkaloid production, and resistance to certain insect pests. Spectral Data IR:

(CHCI3) 2937, 2878, 1652, 1472, 1400, 1348, 1023, and 957cmq.

IH NM]~: (CDCI3) H-I, 4.05(dd,J~,2=2.7Hz);H-2, 4.38(dd,J~,3,=<2.0Hz);H-3a, 3.03(dd, J3~,3b=11.7Hz); H-3b, 2.26(d);H-5a, 2.84(ddd,Js,,sb=12.8Hz,Js,,6,=9.IHz, JSa,6b=7.4Hz,Jsb,6a=4.2Hz,JSb,6b=8.4I-Iz); H-5b, 2.93(ddd);H-6a, 1.81(dddd, J6~,6b=14.2Hz,J6a,7=4.31-'Iz); H-6b, 1.92(ddd);H-7, 4.29(dd,J7,s=<2.0Hz);H-8, 3.10(dd,Js,~=<2.0Hz); and N-Me, 3.04(s);MeC=O, 1.96ppm (s).

13CNMI~: (CHCI3) C-l, 64.1(d); C-2, 73.4(d); C-3, 61.0(0; C-5, 54.7(0, C-6, 33.0(t), C-7, 80.6(d); C-8, 67.8(d); N-Me, 33.9(q); MeC=O, 171.5(s); and MeC=0, 22.3ppm (q).

532

10.

Loline Alkaloids

Mass Spectrum: EIMS: 196(M+, 2.1), 167(5), 153(8), 123(23), 95(43), 82(100), and 42m/e (42%). Reference R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989).

10. Loline Alkaloids

533

Common/Systematic Name N-Methylloline Molecular Formula/Molecular Weight CgI-Ii6N20; MW = 168.12626 O , ' x, Me

H ,'

HH,tN--Me

Hb,, .... ~

H

"'

H

Hb H6 General Characteristics N-Methylloline-HCI recrystallized from EtOH; mp., 212-216~ Fungal Source Present in endophyte-infected Lolium cuneatum. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue. Studies have shown a positive association between endophyte infection, loline alkaloid production, and resistance to certain insect pests. Spectral Data IR~

(CHCIa) 2942, 2875,2818, 2768, 1600, 1466, 1367, 1314, 1272, 1184, 1094, 1042, and 960cml. IH NMR:

(CDCI3) H-l, 2.53(dd, J~,2=l.2Hz); H-2, 3.82(dd, J2,a,=l.2Hz); H-3a, 3.36(dd, daa,ab=10.9Hz); H-3b, 2.18(d); H-5a, 2.78(ddd, Jsa,Sb=12.8Hz,Js,,6,=9.3Hz, Js~,6b=7.2Hz, dSb,6,=4.2Hz,Jsb,6b=8.4Hz); H-5b, 2.89(ddd); H-6a, 1.75(dddd, d6,,6b=14.0Hz, d6,,v=4.4Hz); H-6b, 1.86(ddd); H-7, 4.25(dd, dv,8=l.8Hz); n-8, 3.01(dd, ds,~=l.3Hz); NMe, 2.12(s); and N-Me, 2.12ppm (s). 13C NMR:

(CHCI3) C-l, 74. l(d); C-2, 74.2(d); C-3, 61.2(0; C-5, 54.3(0; C-6, 33.40(0; C-7, 82.0(d); C-8, 69.2(d), NMe, 44.4(q); and N-Me, 44.4ppm (q).

534

10.

Loline Alkaloids

Mass Spectrum: ELMS: 168(M+, 1.7), 123(42), 95(68), and 82m/e (100%). Reference R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989).

10.

Loline Alkaloids

535

Common/Systematic Name Norloline Molecular Formula/Molecular Weight CTH~2N20; MW = 140.09496

i

i

0 e~ Il

H. ,,' H H',s.NI-12 i,,

Ha~ i Hb

H

- "Ha H6

General Characteristics Clear viscous oil; very soluble in water and very volatile. Fungal Source Present in endophyte-infected Lolium cuneatum. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall rescue. Studies have shown a positive association between endophyte infection, loline alkaloid production, and resistance to certain insect pests. Spectral Data IR;

(CHC13) 3376, 3293, 3181, 2964, 2937, 2876, 1616, 1473, 1293, 1249, 1216, 1174, 1041, 1000, 998, and 974cm "~. ~H NMR: (CDCI3) H-I, 3.48(dd, J~,2=<2Hz); H-2, 3.72(dd, J2,3.=l.6Hz); H-3a, 3.38(dd, J3.,3b=l 1.7Hz); H-3b, 2.29(d); H-5a, 2.80(ddd, Js.,sb=12.8Hz, Js.,s.=9.3Hz, Js.,6b=7.5Hz, Jsb,6.=3.8Hz, Jsb,6b=8.3Hz); H-5b, 2.98(ddd); H-6a, 1.84(dddd, J6~,6b=14.3Hz, J6,,7=4.4Hz); H-6b, 1.94(ddd); H-7, 4.29(dd, JT,s=l.9Hz); and H-8, 2.92ppm(dd, Js,~= 1.8Hz). 13C N M R :

(CHCI3) C-I, 60.5(d); C-E, 76.2(d); c-a, 60.8(0; C-5, 54.5(0; C-6, 34.1(t), C-7, 81.7(d); and C-8, 71.9ppm (d).

536

10.

Loline Alkaloids

Mass Spectrum: EIMS: 140(M+, 4.3), 123(24), 111(15), 97(22), 95(15), 82(100), and 69m/e (26%). Reference R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. PoweU; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989).

10.

Loline Alkaloids

537

Common/Systematic Name N-Formylnorloline Molecular Formula/Molecular Weight CsH12N202, M W = 168.08988

O eeee ~lI

H. ,,' HI'I-',,,,N--CHO Hb,, .... ~ , Hb H6 General Characteristics Clear, viscous oil. Fungal Source Present in endophyte-infected Lolium cuneatum. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue. Studies have shown a positive association between endophyte infection, loline alkaloid production, and resistance to certain insect pests. Soectral Data IR,:

(CHCI3) 3190, 2972, 2880, 2790, 1681, 1600, 1541, 1388, 1339, 1247, 1105, and 1007cm"~. IH N]V[P.~:

(CDCI3) H-I, 4.52(dd,d~,2=2.IHz); H-2, 4.29(dd,J2,3,=<2.0Hz),H-3a, 3.43(dd, J3a,3b=12.0Hz);H-3b, 2.53(d);H-5a, 3.04(ddd,Js,,sb=12.9Hz,Js,,6,=9.3I-Iz, Jsa,6b=7.5Hz,Jsb,6,=4.0Hz,Jsb,6b=8.6Hz);H-b, 3.17(ddd);H-6a, 2.07(dddd, J6,,6b=14.3Hz,J6,,7=3.9Hz);H-6b, 2.18(ddd);H-7, 4.50(dd,JT,8=2.2Hz);H-8, 3.32(dd, Js,~=<2.0Hz),and NH, 7.39ppm (bm).

13C ]~-M~2

(CHCI3) C-l, 55.8(d); C-2, 73.6(d); C-3, 60.8(0; C-5, 53.7(0; C-6, 31.7(0; C-7, 80.0(d); C-8, 69.7(d); and HE=O, 161.8ppm (d).

538

10.

Loline Alkaloids

Mass Spectrum: ELMS: 168(M+, 0.3), 140(3), 123(5), 95(17), 82(100), and 69m/e (26%). Reference R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989).

10.

Loline Alkaloids

539

Common/Systematic Name N-Acetylnorloline Molecular Formula/Mol .ecular Weight C9H14N202; M3cV = 182.10553

O i i

% l %

H ,,' H H_,sNH--Ac S

Hb,, ....- H

~

"

H

Hb HI5 General Characteristics Clear, viscous oil. Fungal Source Present in caryopses of tall fescue (Festuca arundinacea) infected with the endophyte Acremonium coenophialum; also found in Lolium cuneatum. Biological Activity The loline alkaloids are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue. Studies have shown a positive association between endophyte infection, Ioline alkaloid production, and resistance to certain insect pests. Spectral Data IR:

(CHCI3) 3284, 2878, 1673, 1542, 1473, 1436, 1374, 1294, 1106, and 962cmq. ~H NMR:

(CDCI3) H-I, 4.41(dd,Jz,2=2.5Hz);H-2, 4.17(dd,J2,3~=l.0Hz);H-3a, 3.29(dd, J3a,3b=l 1.8Hz); H-3b, 2.42(d); H-5a, 2.90(ddd, Jsa,sb=12.8Hz, Js=,6==9.3Hz,

Js,,6b=7.4Hz, Jsb,6,=3.6Hz, Jsb,6b=8.3Hz); H-5b, 3.10(ddd); H-6a, 1.98(dddd, J6,,6b=14.4Hz, J6,,7=4.3Hz); H-6b, 2.08(ddd); H-7, 4.44(dd, JT,8=l.9Hz); H-8, 3.09(dd, Js, l=l.6Hz); and NH, 6.21 and MeC=O, 1.97ppm (s). 13C N M R :

(CHCI3) C-I, 57.6(d); C-2, 73.8(d); C-3, 60.9(t); C-5, 54.6(0; C-6, 33.9(0; C-7, 80.9(d); C-8, 69.6; MeC=O, 170.2(s); and MeC=O, 23.1ppm (q).

540

10.

Loline Alkaloids

Mass Spectrum: EIMS: 153(M+ - 29, 2.6), 139(1), 123(7), 95(21), 82(100), and 69m/e (34%). Reference R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell; Isolation, Semi-synthesis, and NMR Spectral Studies of Loline Alkaloids; J. Natural Products, Vol. 52, pp. 810-817 (1989).

10.

Loline Alkaloids

541

Common/Systematic Name Peramine 3-(3'-Guanidinopropyl)-2-methyl- 1,2-dihydropyrrolo[ 1,2-a] pyrazin- 1-one Molecular Formula/Molecular Weight C12HITNsO; MW = 247.14331 8

1'2

7

15

Me/N~~J 4

NH

General Characteristics Peramine gave a positive reaction with the Sakaguchi reagent and no reaction with ninhydrin. Crystalline peramine diacetate had mp. 141-142 oC. Fungal Source

Acremonium loliae infected perennial ryegrass (Lolium perenne).

Isolation/Purification Peramine was obtained as its acetate salt after ethanol extraction ofA. loliae-infected perennial ryegrass, solvent partitioning and repeated chromatography. Biological Activity Perennial ryegrass infected with the endophyte Acremonium loliae (Latch, Christensen and Samuels) is resistant to the Argentine stem weevil (Listronotus bonariensis Kusehel) as well as to some five other diverse insect species. Resistance against stem weevil is considered to be due to the presence of feeding deterrents which reduce the population of egg-laying adult insects on endophyte-infected plants. Peramine has been identified as the principal feeding deterrent. Spectral Data UV: ~, mMH ~ 237 and 285nm were similar to those reported for phakellin and its derivatives which contain a pyrrole ring with a carbonyl group at the 2 position. IR;

(20% MeOH-CHCI3) 3100, 1670, 1615, and 1580cm~ assigned to NH and amide groups.

542

10.

Loline Alkaloids

1H N]VIR~ (CDCI3) diacetate derivative: ~H NMR showed the two methyl ringlets (2.133 and 2.189ppm) and the two NH singlets of the acetylated guanidino moiety, -NH-C(=NAc)NHAc. Connectivities were established by couplings and nuclear Overhauser effect enhancements. The methylene propyl protons showed vicinal couplings (J1,2~7.5, J2,3~-~THz). The C-3' protons showed a 6Hz coupling to the higher-field guanidino NH singlet and the C-I' protons showed both an allylic coupling (J=0.8Hz) and an nuclear Overhauser effect (7% enhancement) with the Cis-olefirfic proton H-4. This proton, in turn, showed a long range coupling (d=-0.7Hz) to H-8 and an nuclear Overhauser effect enhancement (13%) to H-6 of the pyrrole moiety. The three aromatic protons (H-6, -7, -8) showed couplings (,/6,7=2.56, J6,8=1.52, dT,s=3.98Hz) and nuclear Overhauser effect enhancements characteristic of a 1,2-disubstituted pyrrole. Nuclear Overhauser effect enhancements (-3%) were observed between the geminally related heterocyclic amide N-Me and C-1' methylene protons. 13C NMR: (CDC13) 25.1(CH3CONH); 27.6(C-2'), 27.9(C-1'), 28.7(CH3CON=), 29.0[N(2)-Me], 39.7(C-3'); 106.1(C-4); 109.8(C-7); 112.3(C-6); 117.5(C-8); 123.1(C-3); 127.3(C-8a); 155.7(guanidino); 157.1(C-I); 172.8(CH3CONH); and 186.1ppm (CH3CON=).

Reference D. D. Rowan, M. B. Hunt, and D. L. Gaynor; Peramine, a Novel Insect Feeding Deterrent from Ryegrass Infected with the Endophyte Acremonium loliae; J. Chem. Soc., Chem. Commun., pp. 935-936(1986).

10. Loline Alkaloids

543

Common/Systematic Name Festucine Molecular Formula/Molecular Weight CsH14N20, MW = 154.11061

H ~H '~-N~Me

0,. !

General Characteristics Free base was a clear volatile oil; the dihydrochloride salt formed as orthorhombic needles which melted with decomposition at 237-242~ [tt]D25 +4.6 ~ (C=4.365 in water); pK, values were 8.25 and 2.5-3.0. Festucine is a positional isomer ofloline. Fungal Source Found contaminating tall fescue presumed to be of fungal origin by an endophyte Acremonium sp. Isolation/Purification Alkaloids in tall fescue were extracted with 80% ethanol and then partitioned into chloroform. The hydrochloride salt was separated by paper chromatography (n-butanolacetic acid-water, 10:1:3, v/v; Rf 0.12) and recrystallized from absolute ethanol as the dihydrochloride salt. Biological Activity Festucine did not contract isolated guinea pig uterus or intestine. It killed mice when administered intravenously at 400mg/kg, but produced no apparent effects when administered orally at 1000mg/kg. Spectral Data UV:

No significant absorption between 220-400nm. ~H NMR: (CDCI3) 7.531: (N-CH3); all values were in the range 5.57-8.151:; dihydrochloride salt showed N-CH3 at 6.591: and all values between 4.51-7.29z. Reference S.9 G. Yates and H. L. Tookey; Festucine, an Alkaloid from Tall Fescue (Festuca arundinacea Schreb.): Chemistry of the Functional Groups; Aust. J. Chem. Vol. 18, pp. 53-60(1965).

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Aflatoxins Aflatoxin B Aflatoxin B2 Aflatoxin G~ Aflatoxin G2 Aflatoxin M~ Aflatoxin M2 Aflatoxin P1 Aflatoxin D1 Parasiticol (Aflatoxin B3) Aflatoxin Q1 Aflatoxicol A (Ro) Aflatoxicol B Aflatoxin B2~ Aflatoxin G2~ Aflatoxicol O-ethyl ether A Aflatoxicol O-ethyl ether B

545

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11. Aflatoxins

547

Common/Systematic Name Aflatoxin B~ Molecular Formula/Molecular Weight C17H1206; M W = 312.06339

O H

O

O

General Characteristics Crystals from chloroform; mp., 268~176 dimethylformamide).

(dec.); [a]o -480 ~ (c=0.1, in

Fungal Source

Aspergillus flavus, A. nomius, and A. parasiticus.

Biological Activity The toxic effects of aflatoxin B~ vary with species, age, sex, and general nutrition. The primary organ affected is the liver with some changes occurring in other organs. The induction of liver lesions, liver carcinoma, and bile duct proliferation by aklatoxin B~ has been demonstrated in several species of animals. Some species, such as the trout, duckling, rat and pig, are very susceptible to aflatoxicosis, whereas others, such as sheep and cattle, are more tolerant. In Fischer rats and rainbow trout, it was the most potent hepatocarcinogen known. The LDs0 in 1-day-old rats dosed orally was 0.56mg/kg, in weanling rats, 5.5mg/kg (male), 7.4mg/kg (female); in 100gm male rats, 7.2mg&g; in 150gm female rats, 17.9mg/kg. The LDs0 in Macaque monkeys dosed orally was 7.8mg/kg. Spectral Data UW:

~,m.,x 223(e=25,600), 265(13,400), and 362nm (21,800). Fluorescence emission, 425nm. IH NMR: H-4, 2.56; H-5, 3.34; H-9, 6.38; H-13, 6.75(J=7.0); H-14, 4.72(J=7.0, 3.0); H-15, 5.42(J=3.0, 3.0); H-16, 6.40; and H-17, 3.93ppm.

548

11. Aflatoxins

13C NMR: C-l, 154.7 s; C-2, 117.0 s; C-3,200.6 s; C-4, 35.0 t; C-5, 29.0 t; C-6, 176.5 s; C-7, 103.7 s; C-8, 161.0 s; C-9, 90.6 d; C-10, 165.3 s; C-I 1,107.5 s; C-12, 152.5 s; C-13, 113.2 s; C-14, 47.8 d; C-15, 102.3 d; C-16, 144.8 d; and C-17, 56.4ppm q. TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Rf, 0.33; detected as bright blue fluorescence under UV light. I-IPLC Data Reverse phase Nova-Pak C~s (Waters) column, water-MeOH, 40:60 (v/v), 60mg/L KBr, 100~I/L nitric acid. Post column derivatization with bromine (also iodine may be used). Immunoassay Numerous immunoassays are commercially available for both qualitative and quantitative analysis. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 15-19(1981). J. W. Dorner and R. J. Cole; Rapid Determination of Aflatoxins in Raw Peanuts by Liquid Chromatography with Post column Iodination and Modified Minicolumn Cleanup; J. Assoc. Off. Anal. Chem., Vol. 71, pp. 43-47(1988).

11.

Aflatoxins

549

Common/Systematic Name Aflatoxin B2 Molecular Formula/Molecular Weight C:7H]406; MW = 314.07904

0 H

0

0

Lo .o< General Characteristics Crystals from chloroform-pentane; mp., 287-289~ (dec.); [a]D -430* (C=I, in CHCI3). Crystals from chloroform; mp., 305~ (dec.); [a]D -490 ~ (C=I.0, in CHCI3). Fungal Source

Aspergillus flavus, A. nomius, and A. parasiticus.

Biological Activity The toxic properties of aflatoxin B2 were similar to those for aflatoxin B:; however, potency was markedly reduced compared to aflatoxin B:. A dosage in ducklings of 50ktg of aflatoxin B2 was required to produce the same degree of bile duct proliferation produced by 3.9~g of aflatoxin B]. Spectral Data ug:

~

MeOH max

220(e=20,500), 265(12,700), and 363nm (24,000).

:3C NMR: C-I, 155.8 s; C-2, 115.6 s; C-3,200.0 s; C-4, 34.6 t; C-5, 28.5 t; C-6, 176.6 s; C-7, 105.5 s; C-8, 160.8 s; C-9, 90.0 d; C-10, 165.8 s; C-11, 105.9 s; C-12, 152.0 s; C-13, 113.3 d; C-14, 42.8 d; C-15, 30.6 t; C-16, 66.9 t; and C-17, 56.6ppm q. TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Rf, 0.31; detection as bright blue fluorescence under UV light. HPLC Data Reverse phase Nova-Pak C:8 (Waters) column, water-MeOR 40:60(v/v), 60mg/L KBr, 1001.d/L nitric acid. Postcolumn derivatization with bromine (also iodine may be used). Fluorescence detection-excitation at 365nm, emission at 418nm.

550

11.

Aflatoxins

Immunoassay Numerous immunoassays are commercially available for both qualitative and quantitative analysis. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 34-37(1981). J. W. Dorner and R. J. Cole; Rapid Determination of Atlatoxins in Raw Peanuts by Liquid Chromatography with Postcolumn Iodination and Modified Minicolumn Cleanup; J. Assoc. Off. Anal. Chem., Vol. 71, pp. 43-47(1988).

11.

Aflatoxins

551

Common/Systematic Name Aflatoxin G1 Molecular Formula/Molecular Weight C17H1207; M W ' = 3 2 8 . 0 5 8 3 0

0 0

0 0

"0-"~0,'~ ~OMe H General Characteristics Crystals from chloroform-methanol; mp., 244- 246~ (dec.); Crystals from acetone; mp., 257-259~ [a]D -556 ~ (c=l, in CHCI3). Fungal Source

Aspergillus flavus, A. nomius, and A. parasiticus.

Biological Activity The toxicity of aflatoxin G~ was similar to aflatoxin Bl; acute toxicity was less than B~ but greater than B2. The LDs0 in ducklings was 39.2~g/duckling using dimethylformamide as carrier; 45.7~g/duckling (0.785mg/kg) in another trial using dimethylformamide. The LDs0 in the rat was twice that of aflatoxin B I. The lesions induced by G~ in ducklings are the same as from BI; in the rat, the zone in the affected liver lobule was the same as in B~ but the consistent pattern seen with B~ was absent. Spectral Data UV;

~

EtOH max

243(e=11,500), 257(9,900), 264 (10,000), and 362nm (16,100).

IH NMR: H-4, 4.41; H-5, 3.46; H-9, 6.43; H-13, 6.80(J=7.1); H-14, 4.75(,/--7.1, 2.5); H-15, 5.45(,/--2.5, 2.5); H-16, 6.47(J=2.5); and H-17, 3.95ppm. 13C N M R :

C-l, 154.8 s; C-2, 113.2 s; C-3, 159.9 s; C-4, 64.3 t; C-5, 28.8 t; C-6, 161.1 s; C-7, 106.9 s; C-8, 161.0 s; C-9, 91.0 d; C-10, 164.6 s; C-11,107.5 s; C-12, 151.7 s; C-13, 113.2 d; C-14, 47.7 d; C-15, 102.3 d; C-16, 144.8 d; and C-17, 56.4ppm q.

552

11.

Aflatoxins

TLC Data Silica gel G-I-IR (chloroform-acetone, 93:7, v/v) R~, 0.29; detected as blue-green fluorescence under UV light. HPLC Data Reverse phase Nova-Pak C~s (Waters) column, water:MeOH, 40:60(v/v), 60mg~ KBr, 100~tl/L nitric acid. Postcolumn derivatization with bromine (also iodine may be used). Fluorescence detection excitation at 365nm, emission at 418nm. Immunoassay Numerous immunoassays are commercially available for both qualitative and quantitative analysis. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 25-28(1981). J. W. Dorner and R. J. Cole; Rapid Determination of Aflatoxins in Raw Peanuts by Liquid Chromatography with Postcolumn Iodination and Modified Minicolumn Cleanup; J. Assoc. Off. Anal. Chem., Vol. 71, pp. 43-47(1988).

11.

Aflatoxins

553

Common/Systematic Name Aflatoxin 02 Molecular Formula/Molecular Weight C17H1407; M W ~-- 330.07395

0

0

O

O

'~ O...T...O..~ .,.""~ OMe General Characteristics Crystals from ethyl acetate; mp., 230~

[a]D -454 ~ (C=I.0, in CHCI3).

Fungal Source Aspergillus flavus, A. nomius, and A. parasiticus. Biological Activity Aflatoxin G2 was the least acutely toxic of the 4 major naturally occurring aflatoxins. The LDs0 in ducklings was 172.5lag/duckling using dimethylformamide as carrier in a 6-day test. Spectral Data UV:

Xmax 217(e=28,000), 245(12,900), 265(11,200), and 365nm (19,300). 1H NMR: H-4, 3.46; H-5, 2.30; H-9, 6.34; H-13, 6.47; H-14, 3.60; H-15, 4.16; H-16, 4.42; and H- 17, 3.94ppm. 13C NMR: C-l, 154.9 s, C-2, 113.7 s, C-3, 160.0 s, C-4, 64.3 t, C-5, 28.9 t, C-6, 161.3 s, C-7, 102.1 s, C-8, 161.0 s, C-9, 90.3 d, C-10, 166.1 s, C-11,106.6 s, C-12, 152.2 s, C-13, 113.7 d, C-14, 43.9 d, C-15, 31.4 t, C-16, 67.7 t, and C-17, 56.4ppm q. TLC Data Silica gel GoHR (chloroform-acetone, 93:7, v/v), Rf: 0.27, detected as blue-green fluorescence under UV light.

554

11.

Aflatoxins

HPLC Data Reverse phase Nova-Pak C~8 (Waters) column, water-MeOH, 40:60(v/v), 60mg/L KBr, 1001.tl/L nitric acid. Postcolumn derivatization with bromine (also iodine may be used). Fluorescence detection excitation at 365nm, emission at 418nm. Immunoassay Numerous immunoassays are commercially available for both qualitative and quantitative analysis. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 45-48(1981). J. W. Dorner and R. J. Cole; Rapid Determination of Afiatoxins in Raw Peanuts by Liquid Chromatography with Postcolumn lodination and Modified Minicolumn Cleanup; J. Assoc. Off. Anal. Chem., Vol. 71, p. 43-47(1988).

11.

Aflatoxins

555

Common/Systematic Name Aflatoxin MI Molecular Formula/Molecular Weight C 1 7 H 1 2 0 7 ; M3~V = 3 2 8 . 0 5 8 3 0

0 1

OH

0

O ~

.o .o Ao o

'

General Characteristics Crystals from methanol in dimethylformamide; mp., 299~ (dec); [tt]D-280 ~ (C=0.1, in dimethylformamide). Fungal Source

Aspergillus flavus (NRRL 3251), and A. parasiticus. Note: Aflatoxin M~ is excreted in the milk and urine of animals ingesting aflatoxin B l" The conversion of B1 to Ml occurs in the liver.

Isolation/Purification Pull milk-water (1:1, v/v) through C18 cartridge using slight vacuum; wash with waterCH3CHOH (95:5, v/v). Place silica gel cleanup column beneath C18 cartridge; wash with ether. Elute aflatoxin MI from column using CH2Cl2-isopropyl alcohol (95:5, v/v); dry gently under nitrogen using heat. Derivative by adding 2001,ti hexane and 200~1 trifluoroacetic acid (TFA); mix and let stand 10 min. at 40~ evaporate to dryness; add 2ml water-CH3CN (75:25, v/v); mix. Biological Activity Aflatoxin MI is a hydroxylated metabolite of aflatoxin B~, secreted in milk of mammals receiving aflatoxin B~. It is potentially hepatocarcinogenic. The LDs0 value for aflatoxin M~ in day old ducklings was 16.61.tg/duckling compared to 12~g/duckling for aflatoxin BI determined simultaneously. Pathology and clinical signs similar to aflatoxin B~. Ducklings dosed with aflatoxin MI showed liver lesions indistinguishable from those induced by aflatoxin BI; however, M1 also induced a renal tubular necrosis not seen with B~. In rainbow trout and rats, aflatoxin M~ was slightly less effective than B~ in inducing hepatic carcinoma. Spectral Data UV:

~

EtOH max

226(e=23,100), 265(11,600), and 357nm (19,000).

556

11.

Aflatoxins

TLC Data Kieselgel G (chloroform-methanol, 97:3, v/v); Rf: 0.34; detection as blue fluorescence under long wave UV light. Adsorbosil-1, isopropyl alcohol-acetone-chloroform, 5:10:85, v/v/v; Rf, 0.46; detection as blue fluorescence under long wave UV light. HPLC Data Column: C18 bonded silica gel, 0.4 x 25cm, 5ktm, mobile phase: water-isopropyl alcoholCHaCN (80:12:8, v/v/v); detection: fluorescence at 365nm excitation and >400nm emission. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 29-33(1981). K. Helrich, Ed.; AOAC Official Methods of Analysis, 15th Edition, Vol. II; AOAC, Inc., Arlington, VA, pp. 1199-1205(1990) C. H. Holzapfel, P. S. Steyn, and I. F. H. Purchase; Isolation and Structure of Aflatoxins, M~ and M2; Tet. Lett. p. 2799 (1966).

11.

Aflatoxins

557

Common/Systematic Name Aflatoxin M2 Molecular Formula/Molecular Weight C17H1407; MW = 330.07395 O

O

OH 0~~"~

o,l,o,(Yo,

General Characteristics Crystals from methanol-chloroform; mp., 293 ~ (dec.). Fungal Source

Aspergillus flavus and A. parasiticus.

Biological Activity Aflatoxin M2 was excreted in the urine and milk of animals ingesting mixed aflatoxins. The LDs0 value for aflatoxin M2 was 621.tg/day old duckling compared to 121.tg for aflatoxin B~ in the same assay done simultaneously. Spectral Data UV:

~.~x 221(e=20,000), 264(10,900), and 357nm (21,000). IR:

(CHCI3) 3350, 1760, and 1690cm "l. TLC Data Adsorbosil- 1 silica gel, isopropyl alcohol-acetone-chloroform (5:10:85, v/v/v); Rf, 0.42; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 49-50(1981). C. W. Holzapfel, P. S. Steyn, and I. F. H. Purchase; Isolation and Structure of Aflatoxins, M~ and Mz; Tet. Lett., p. 2799 (1966).

558

11.

Aflatoxins

Common/Systematic Name Atlatoxin Pl Molecular Formula/Molecular Weight C16H1oO6; ~

= 298.04774

0

0

0

General Characteristics Pale yellow needles from methanol-benzene-hexane; mp., >320~ in MeOH).

[a]D2~ -574 ~ (C=0.08,

Source Aflatoxin PI was the principal urinary metabolite of aflatoxin B1 in rhesus monkeys. It occurred in the urine in unconjugated (3%), sulfate (10%), and glucuronide (50%) forms. Biological Activity In a mouse bioassay using IP injection, aflatoxin P l showed considerably less toxicity than aflatoxin B1. At a dosage of 100mg/kg, no mortalities were observed; at 150mg/kg there were 2 mortalities in 15 animals; and at 200mg/kg, no mortalities occurred. Atlatoxin B l in the same assay had an LDs0 of 9.5mg/kg. Spectral Data UV:

~..... 226(e=20,400), 267(11,200), 342(14,900), 362(15,400) and 425nm (2,500). References G. Buchi, D. Spitzner, S. Paglialunga, and G. N. Wogan; Life Science, Vol. 13, p. 1145 (1973). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 3-5 (1981). J. Dalezios, G. N. Wogan, and S. M. Weinreb; Science, Vol. 171, p. 584 (1971).

11.

Aflatoxins

559

Common/Systematic Name Aflatoxin D~ Molecular Formula/Molecular Weight C16H1405; M W = 286.08412

0 OH

General Characteristics Sublimed without melting at 2200-290~ in sealed tube crystals decomposed without melting at 255~ Acetate derivative; mp., 155-157~ Fungal Source A major product formed from reacting aflatoxin B~ with ammonium hydroxide at 100~ under pressure. Biological Activity Specific toxicity data not known. It has been reported that the nonfluorescent compounds formed in alkaline solutions are strongly toxic (mixture included aflatoxin D~). Spectral Data UV:

~,~H 227(~=20,792) and 324nm (17,074) [corrected from 227(15,920), and 324nm (12,440)].

IH NMR: H-2, 6.24; H-4, 2.40; H-5, 3.10; H-8, 6.25; H-12, 6.76; H-13, 4.70; H-14, 5.40; H-15, 6.63; and H-16, 3.75ppm. 13C NMR: C-l, 208.6 s; C-2, 131.5 d; C-3, 170.9 s; C-4, 31.9 t; C-5, 34.2 t; C-6, 106.1 s*; C-7, 159.5 s; C-8, 86.6 d; C-9, 158.6 s; C-10, 106.7 s*; C-11,151.3 s, C-12, 111.5 d; C-13, 47.6 d; C-14, 103.1 d; C-15, 144.1 d; and C-16, 55.8ppm q. * Assignment may be reversed.

560

11.

Aflatoxins

TLC Data Silica gel: chloroform-acetone, 95:5, v/v; Re, 0.31. Detection: turns gray-brown after spraying with 10% FeCI3; orange-yellow with 2,4-dinitrophenylhydrazine. Acetate Re, 0.78. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, pp. 6-9(1981). J. B. Stanley, L. S. Lee, A. F. Cucullu, and I. V. deGruy; J. Agric. Food Chem., Vol. 23, p. 447 (1975).

11.

Aflatoxins

561

Common/Systematic Name Parasiticol; Aflatoxin B3 Molecular Formula/Molecular Weight C16H1406; M W = 302.07904

O O

II I

I

CH20H

'~OJ~O~/'~,OMe General Characteristics Crystals from chloroform; mp., 217~ Fungal Source

Aspergillusflavus, A. parasiticus. Parasiticol appears to be more prominent in older cultures ofA. flavus and A. parasiticus. It was suggested to be the first step in the biological degradation of aflatoxin G~ and was reported as an intermediate in the biodegradation of aflatoxin G~ by Rhizopus spp.

Biological Activity Parasiticol had the same acute toxicity to ducklings as aflatoxin B1, but it had little tendency to cause biliary hyperplasia. It was only 1/100 as toxic as BI in chick embryo studies. Spectral Data UV;

ZmM~H 229(sh) (C=10,000), 253(7,300), 262(7,550), and 326nm (9,350); ~,mx 225(sh) (12,600), 253(6,800), 262(7,400), and 325nm (9,700). 1H NMR: H-2, 5.94; H-6, 6.62; H-10, 6.85 (,/--7.0); H-11, 4.72(,/=7.0, 3.0); H-12, 5.36 (,/--3.0, 3.0); H-13, 6.66; H-14, 3.00; H-15, 3.61; H-16, 3.85; and OH, 4.59ppm. 13C NMR: C-l, 158.6 s*; C-2, 110.8 d; C-3, 154.7 s; C-4, 103.5 s; C-5, 158.8 s*; C-6, 91.0 d; C-7, 160.6 s; C-8, 106.8 s; C-9, 150.7 s; C-10, 112.6 d; C-11, 47.2 d; C-12, 102.0 d; C-13, 145.1 d; C-14, 41.1 t; C-15, 59.9 t; and C-16, 56.4ppm q. * Assignment may be reversed.

562

11.

Aflatoxins

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites.; Academic Press, New York, pp. 10-14(1981). R. J. Cole and J. W. Kirksey; J. Agric. Food Chem., Vol. 19, p. 222 (1971).

11.

Aflatoxins

563

Common/Systematic Name Aflatoxin Q~ Molecular Formula/Molecular Weight C17Hn207; ~ = 328.05830

0

0

0 o.

General Characteristics Colorless needles from methanol-chloroform or hot acetonitrile; mp., 295 ~ (dec.); crystals from chloroform-hexane; mp., 266~ (dec.). Fungal Source Atlatoxin Q~ was a major metabolite of aflatoxin B~ metabolism in monkey, rat, and human liver preparations (in vitro). Biological Activity Aflatoxin Q~ was approximately 18 times less toxic than aflatoxin B~ by the air cell route in the chicken embryo test. No mutagenic activity was detected by the bacterial mutagenesis test, using Salmonella typhimurium TA 1538 with or without microsomal activities. Spectral Data UV: ~MeOH max

366(E=17,500), 267(11,450), and 223nm (19,030); ~, max E~H 365(18,800), 266(11,700), 242(sh) (10,000), and 224nm (20,500). TLC Data Adsorbosil- 1 silica gel, chloroform- acetone-n-hexane, 88:15:20, v/v/v, Rf, 0.23. Detection: intense yellow-green fluorescence under UV light. Silica gel, chloroform-methanol, 20:1, v/v, Rf: 0.25. Detection: yellow fluorescence in 350nm UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 20-24(1981). M. S. Masri, W. F. Hadden, R. E. Lundin, and D. P. H. Hsieh; J. Agric. Food Chem., Vol. 22, p. 512 (1974).

564

11.

Aflatoxins

Common/Systematic Name Aflatoxicol A (Ro) Molecular Formula/Molecular Weight C17H1406; M W "- 3 1 4 . 0 7 9 0 4

0

OH

O

General Characteristics Crystals from benzene-n-hexane; mp., 224-226~ Fungal Source Aflatoxicol A (Ro) is produced from the m vitro incubation of aflatoxin B~ with submitochondrial liver fractions from several animal species. It was also produced from biological reduction of aflatoxin BI by Tetrahymenapyriformis, Dactylium dendroides, Rhizopus spp., and other microorganisms. It was reported to occur together with a diastereoisomer (aflatoxicol B). Biological Activity Reportedly 18 times less toxic than aflatoxin B~ in the duckling biliary hyperplasia assay. Spectral Data UV~

~ mEtOH 332(e = 14,100), 261 (10,800), and 254nm (6,790). Fluorescence emission, ax 425nm.

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re, 0.30; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 38-40(1981 ). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

11.

Aflatoxins

565

Common/Systematic Name Aflatoxicol B Molecular Formula/Molecular Weight C17H1406; MW = 314.07904 0

OH _

0

General Characteristics Crystals from benzene-n-hexane, decomposed over broad range, starting at 233 ~ Fungal Source Aflatoxicol B was obtained from biological reduction of aflatoxin B~ by Rhizopus spp., Dactylium dendroides, Tetrahymenapyriformis, and other microorganisms. Spectral Data UV:

Z~

325(E=14,100), 261(10,800), and 254nm (6,790).

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re, 0.26; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 41-44(1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

566

11.

Aflatoxins

Common/Systematic Name Aflatoxin B2, Molecular Formula/Molecular Weight C17H1407; M W "- 3 3 0 . 0 7 3 9 5

O

O

H General Characteristics Melting point, 240~ (dec.). Fungal Source

Aspergillusflavus and A. parasiticus. Also biotransformation product of aflatoxin B~ by the liver of some animals and forms spontaneously in acidic conditions.

Biological Activity Aflatoxin B2, was more than 200 times less toxic than aflatoxin BI as measured by the initiation of bile duct proliferation in the standard duckling assay. No acute toxicity was noted in Khaki Cambell ducklings (day-old) at levels up to 12001.tg/duckling. Spectral Data UV:

228(e=17,600), 256(10,300), and 363nm (20,400). ~3C NMR: C-I, 153.9 s; C-2, 117.4 s; C-3, 200.0 s; C-4, 34.6 t; C-5, 28.5 t; C-6, 176.7 s; C-7, 106.9 s; C-8, 160.8 s; C-9, 90.5 d; C-10, 165.3 s; C-11,108.6 s; C-12, 152.0 s; C-13, 113.6 d; C-14, 41.3 d; C-15, 37.2 t; C-16, 99.7 d; and C-17, 56.6ppm q. TLC Data Kieselgel G (chloroform-methanol, 98:2, v/v); Rf, 0.13; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 51-54(1981). J. G. Heathcote and J. R. Hibbert; Biochem. Soc. Trans. Vol. 2, p. 301 (1974).

11.

Aflatoxins

567

Common/Systematic Name Aflatoxin G2a Molecular Formula/Molecular Weight Cl7Hl4Os, MW

= 346.06887

O

H

O

.o o o

O

I

O

H

General Characteristics Melting point, 190~ (dec.). Fungal Source

Aspergillusflavus and A. parasiticus. Also biotransformation product of aflatoxin B~ by the liver of some animals and forms spontaneously in acidic conditions.

Biological Activity There were no significant differences in growth and no characteristic liver lesions in day-old Khaki Cambell ducklings dosed up to 1600~g/duckling (LDs0 of aflatoxin BI in same assay was 18.21.tg/duckling). Spectral Data UV:

~b max

MeOH

223(e=18,600), 242(10,100), 262(8,700), and 365nm (18,000).

13C NMR: C-I, 153.9 s; C-2, 111.0 s; C-3, 159.6 s; C-4, 64.1 t; C-5, 28.4 t; C-6, 161.9 s; C-7, 105.3 s; C-8, 161.6 s; C-9, 91.1 d; C-10, 164.9 s; C-11,108.6 s; C-12, 151.6 s; C-13, 113.7 d; C-14, 41.9 d; C-15, 41.9 t; C-16, 91.1 d; and C-17, 56.7ppm q. TLC Data Kieselgel G (chloroform-methanol, 98:2, v/v); Re, 0.10; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 55-58(1981). J. G. Heathcote and J. R. Hibbert; Biochem. Soc. Trans. Vol. 2, p. 301 (1974).

568

11.

Aflatoxins

Common/Systematic Name Aflatoxicol O-ethyl ether A Molecular Formula/Molecular Weight C19H1806; MW = 342.11034 0

OCH2Me

0

General Characteristics Colorless crystals from ethanol solution; mp., 198-200 ~ Fungal Source Produced spontaneously from aflatoxicol A during silica gel column chromatography using chloroform (ethanol preservative) as the eluting solvent. Spectral Da.ta UV: ~,Em~H 332(C=14,200), 261(9,660), and 255nm (8,830). TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Rr, 0.81; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 59-61(1981). R. J. Cole, J. W. Kirksey, and B. R. Blankenship; J. Agric. Food Chem., Vol. 20, p. 11 O0 (1972).

11. Aflatoxins

569

C.ommon/Systematic N.ame Aflatoxicol O-ethyl ether B Molecular Formula/M01ecular Weight ClgHlsO6; MW = 342.11034

O o

I"-

OCH2Me

General Characteristics Colorless crystals from ethanol solution; mp., 194-196 ~C. Fungal Source Produced spontaneously from aflatoxicol B during silica gel column chromatography using chloroform (ethanol preservative) as the eluting solvent. Soectral Data _ UV: ~ , EtOH max

331(e=15,750), 261(12,280), and 255nm (11,170).

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re, 0.73; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handboo.k of Toxic Fungal Met abolites; Academic Press, New York, pp. 62-64(1981). R. J. Cole, J. W. Kirksey, and B. R. Blankenship; J. Agric. Food Chem., Vol. 20, p. 1100 (1972).

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Versicolorins Versicolorin A Versicolorin B Versicolorin C Averufin Norsolorinic acid Versiconal hemiacetal acetate Versiconol acetate Versiconol Nidurufin Dimethylnidurufin Aversin O-Methylaversin; Tri-O-methylversicolorin B Versicolorone Hydroxyversicolorone

571

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12. Versicolorins

573

Common/Systematic Name Versicolorin A Molecular Formula/Molecular Weight C1ffli007; MW = 338.04265

OH

Logo2

0

OH

0

General Characteristics Orange-yellow needles from chloroform-methanol; mp., 303-306~ [ a i D 18 _ 354 ~ (c=0.75, in dioxane). Trimethyl ether; mp., 241 ~ Versicolorin A was soluble in acetone, dioxane, ethyl acetate, ether, and ethanol; sparingly soluble in chloroform and benzene; insoluble in water and aqueous bicarbonate. Versicolorin A is a biosynthetic precursor of the aflatoxins. Fungal source Aspergillus versicolor and A. parasiticus (mutant strain 1- 11-105 wk- 1). Isolation/Purification The mycelium was harvested after 4-7 days growth on a low salts medium. The pigment was obtained by chloroform-methanol (1:1, v/v) extraction of the mycelium in a Soxhlet apparatus, followed by solvent partition with hexane-90% methanol. The 90~ methanol layer contained versieolorin A and was separated by chromatography on silica gel H under pressure (1 kg/cm2). The column was developed with chloroform-methanol (97:3, v/v). Crystallization from chloroform-methanol gave pure versicolorin A. Spectral Data UV: ~ EtOH max

222(c=1og 4.45), 255(4.13), 267(4.26), 290(4.40), 326(3.83), and 450nm

(3.85). IR:

(KBr) 3340, 1679, 1625, and 1610cm ~. 13C N M R :

(CDC13) C-l, 158.6, s; C-2, 119.7, s; C-3, 165.1, s; C-4, 100.9, d, C-5, 108.6, d, C-6, 164.8, s, C-7, 107.6, d, C-8, 163.8, s, C-9, 188.4, s, C-10, 180.1, s; C-11, 43.3, t, C-12, 30.0, t; ,C-13, 66.9, d; C-14, 112.9, d, C-la, 110.4, s; C-4a, 134.2, s, C-5a,

574

12. Versicolorins

134.8, s; and C-8a, 107.9ppm, s.

TLC Data Silica gel (benzene-acetic acid. 95:5, v/v); Re: 0.23; detected as yellow spot in visible fight. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 102 (1981). T. Hamasaki, Y. Hatsuda, N. Terashima, and M. Renbutsu; Studies on the Metabolites of Aspergillus versicolor (Vuillemin) Tiraboschi Part V. Isolation and Structures of Three New Metabolites, Versicoloilns A, B, and C; Agile. Biol. Chem., Vol. 31, pp. 11-17 (1967). G. P. Gorst-Allmart, P. S. Steyn, P. L. Wessels, and D. B. Scott; Carbon-13 Nuclear ~ n e t i c Resonance Assignments and Biosynthesis of Versicolorin A in AspergiUus parasitieus; J. Chem. Soc., Perkin Trans. 1, pp. 961-964(1978).

12. Versicolorins

575

Common/Systematic Name Versicolorin B MolecuLar Formula/Molecular Weight C18H1207; ~

= 340.05830

OH

0

OH

A spergi llus versicolor.

Spectral Data UV:

I.,~, 223(c=23,900), 255(13,500), 266(19,500), 291(24,000), 324(12,800), and 450nm (8,700). TLC Data Silica gel (benzene-acetic acid, 95:5, v/v); Re, 0.23; detected as yellow spot in visible light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 99 (1981). G. P. Gorst-Allman, P. S. Steyn, P. L. Wesseis, and D. B. Scott; J. Chem. Soc., Perkin

Trans. 1, p. 961 (1978).

576

12. Versicolorins

Common/Systematic Name Versicolorin C Molecular Formula/Molecular Weight C15H1207; ~

= 340.05830

OH

0

OH

General Characteristics Orange-red needles from acetone; mp., 310~ [a]D 25 (c--0.44, in dioxane); trimethyl ether; mp., 216~ Versicolorin C was soluble in acetone, dioxane, ethyl acetate, ether, and ethanol; sparingly soluble in chloroform and benzene; insoluble in water and aqueous bicarbonate. Versicolorin C is a biosynthetic precursor of the aflatoxins. Fungal Source

Aspergillus versicolor.

Spectral Data UV:

Z ~2~ 223(r 450nm (10,700).

255(15,800), 267(20,400), 292(28,800), 326(10,000), and

13C NMR: C-l, 158.6 s; C-2, 119.7 s; C-3, 165.1 s; C-4, 100.9 d; C-5, 108.6 d; C-6, 164.8 s; C-7, 107.6 d; C-8, 163.8 s; C-9, 188.4 s; C-10, 180.1 s; C-11, 43.3 t; C-12, 30.0 t; C-13, 66.9 d; C-14, 112.9 d; C-la, 110.4 s; C-4a, 134.2 s; C-5a, 134.8 s; and C-Sa, 107.9ppm s. TLC Data Silica gel (benzene-acetic acid. 95:5, v/v); Rr, 0.23; detected as yellow spot in visible light. References R. J. Coleand R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 102 (1981). T. Hamasaki, Y. Hatsuda, N. Terashima, and M. Renbutsu; Agric. Biol. Chem., Vol. 31, p. 11 (1967).

12. Versicolorins

577

Common/Systematic Name Averufin Molecular Formula/Molecular Weight C20H1607; 1VIW'= 368.08960

OH

O

I

OH 8

OH O General Characteristics Bright orange-red laths from acetone; mp., 280-282* (dec.); [aiD 23 > 1* (c--0.30, in EtOH); crystals from acetone; mp., 283-289 ~ (dec.); tri-O-acetyl derivative, yellow needles from ethanol; mp., 297-311 *C; [tt]D22 -15 ~ (Sg/liter in chloroform). Averufm is unstable toward alkali; stable toward acid. It is a biosynthetic precursor of aflatoxin Bl.

Fungal Source Aspergillus versicolor, A. parasiticus (mutant, ATCC 15517), and A. ustus. Spectral Data UV:

~,~~ 223(c=33,000), 256(sh)(16,500), 265(18,500), 286(sh)(24,800), 294(30,800), 319(12,500), and 454nm (10,500); tri-O-acetyl derivative: X~.~," 244(e=18,300), 248(18,300), 281 (45,000), 282(45,000), 335(4,900), and 37ohm (4,250). IH NMR: H-4, 6.98; H-5, 7.08(/=2.5); H-7, 6.56(,/--2.5); H-11, 5.26; H-16, 1.58; and oI-rs, 11.30, 12.04, 12.42ppm. 13C NM~: C-l, 158.0 s; C-2, 115.8 s; C-3, 159.7 s; C-4, 107.0 d; C-5, 108.8 d; C-6, 165.1 s; C-7, 107.9 d; C-8, 164.1 s; C-9, 188.6 s; C-10, 180.5 s; C-1 l, 65.9 d; C-12, 26.8 t; C-13, 15.3 t; C-14, 35.1 t; C-15, 101.1 s; C-16, 27.3 q; C-la, 108.3 s; C-4a, 132.9 s; C-8a, 134.7 s; and C-5a, 108.5ppm s. TLC Data Silica gel (Eastman 6060, chloroform-acetone-acetic acid, 97:2:1, v/v/v); Re, 0.5; detected as a red spot in visible light.

578

12. Versicolorins

References R. J. Cole and R. H. Cox; _Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 107 (1981). J. A. Donkersloot and R. I. Mateles; Biochim. Biophys. Acta., Vol. 86, p. 418 (1964).

12. Versicolorins

579

Common/Systematic Name Norsolorinic acid 2-n-Hexanoyl- 1,3,6, 8-tet rahydroxyanthraquino ne Molecular Formula/Molecular Weight C2oH1807; M~W = 370.10525

OH

0

OH

0

O General Characteristics Red prisms from acetone; mp., 256-257~ (dec.); formed tetramethyl ether; rap.,131132~ Norsolorinic acid was sparingly soluble in acetone, ethanol, and methanol; it was insoluble in water, hexane, ether, and sodium bicarbonate; it dissolved in sodium carbonate and sodium hydroxide to give a purple color. Norsolorinic acid is a precursor in atlatoxin biosynthesis. Fungal Source Aspergillus versicolor, A. parasiticus (mutant, NRRL, A-27,996), and the lichen Sorlorina crocea (L.) Ach. Spectral Data UV~

ZEm~ 235(e=24,500), 269(16,900), 284(18,600), 297(19,900), 314(22,900), and 465nm (7,760); ~,Em~ ~ 234(e=23,667), 265(16,650), 283(17,352), 297(sh) (19,872), 313(23,763), and 465nm (7,336). IH N-IV[R:

(CDCI3) H-4, 7.22;H-5, 7.13(/=2.5);H-7, 6.61(J=2.5);H-12, 2.81;H-13, 1.60;HI4, 1.30;H- 15, 1.30;and H- 16, 0.88ppm. 13C NMR:

(CDCI3) C-I, 161.0, s; C-2, 121.1,s; C-3, 163.0, s; C-4, 108.7, d; C-5, 108.0, d; C-6, 165.0, s; C-7, 107.8, d; C-8, 164.1, s; C-9, 188.0, s; C-10, 181.1, s; C-11,202.6, s; C12, 43.5, t; C-13, 22.6, t; C-14, 30.6, t; C-15, 21.8, t; C-16, 13.7, q; C-la, 108.9, s; C4a, 134.7, s; C-5a, 134.7, s; C-5a, 134.5, s; and C-8a, 108.0ppm s. TLC Data Adsorbosil-l; A: chloroform-acetone-n-hexane, 85:15:20 v/v/v. B: ethyl acetate-2propanol-water, 10:2:1 v/v/v; Re: A, 0.69; B, 1.0. Detected as orange-red spot in normal light.

580

12. Versicolorins

References R. J. Cole and R. H. Cox; Handbook. of Toxic Fungal Metabolites; Academic Press, New York, p. 102 (1981). G. P. Gorst-Allman, P. S. Steyn, P. L. Wessels, and D. B. Scott; Carbon-13 Nuclear Magnetic Resonance Assignments and Biosynthesis of Versicolorin A in Aspergillus parasiticus; J. Chem. Soc., Perkin Trans. 1, pp. 961-964 (1978).

T. Hamasaki, Y. Hatsuda, N. Terashima, and M. Renbutsu; Studies on the Metabolites of

Aspergillus versicolor (Vuillemin) Tiraboschi Part V. Isolation and Structures of Three New Metabolites, Versicolorins A, B, and C; Agric. Biol. Chem., Vol. 31, pp. 11-17 (1967).

12. Versicolorins

581

Common/Systematic Name Versiconal hemiacetal acetate 2,3-Tetrafurano- 1,6,8-trihydroxy- 15-ethyl acetate Molecular Formula/Molecular Weight C20H1609; MW' = 400.07943

OH O OH 7 11 13 1 2 I HO

15~..

O II L;H2OCMe

O~OH O

OH

0

It

0

o

CH2OCMe

0 General Characteristics Orange needles from chloroform-acetone; mp., 216-220~ from acetone; mp., 234-236~ In polar solvents, such as dimethyl sulfoxide, versiconal hemiacetal acetate existed as an equilibrium mixture of isomers; in acetone solution, the angular hemiacetal form was absent. Fungal Source Versiconal hemiacetal acetate accumulated in cultures of Aspergillusflavus and A. parasiticus treated with the insecticide dichlorvos, an inhibitor of aflatoxin biosynthesis. It was an intermediate in the biosynthesis of the aflatoxins. Spectral Data UV:

225(E=23,800), 267(14,000), 298(23,000), 323(11,300), and 480nm. ~3C NMR: C- 1, 159.1 s; C-2, 120.5 s; C-3, 164.2 s; C-4, 103; C-5, 108.8 d; C-6, 165.1 s; C-7, 107.0 d; C-8, 164.2 s; C-9, 188.9 s; C-10, 180.9 s; C-11, 134.7 s; C-12, 107.9 s; C-13,

582

12. Versicolorins

110.0 s; C-14, 134.7 s; C-15, 43 d; C-16, 113.0 d; C-17, 28.9 t; C-18, 61.7 t; C-19, 170.1 s; and C-20, 20.5ppm q. Mass Spectrum: Electron-impact mass spectral analysis showed 382.0688m/e due to NV-H20 (C20H1408); chemical-ionization showed distinct 40 lm/e due to M~ + IF. TLC Data Silicar TLC-7G silica gel; A: toluene-ethyl acetate, 27:12, v/v; B: chloroform-acetone, 85:15, v/v; Re: A, 0.32; B, 0.33. Detected as orange-red spot in normal light. References R. J. Cole and R. H. Cox; H.andbook of Toxic Fungal Metabolites; Academic Press, New York, p. 115 (1981). T. Hamasaki, M. Renbutsu, and Y. Harsuda; Agric. Biol. Chem., Vol. 31, p. 1513 (1967).

12.

Versicolorins

583

Common/Systematic Name Versiconol acetate Molecular Formula/Molecular Weight C19H1609; MW = 388.079432

OH 0 II

L

HO" V

L

OH

11

2

11 1cH2OH2

~L ~

y 0

"~a\OH

0

H/~CH2OCMe

General Characteristics Isolated as a glass. Fungal Source A mutant ofAspergillus parasiticus. Spectral Data UV:

(8,500).

225(E=30,900), 265(sh) (15,800), 294(25,700), 315(12,800), and 453nm

IR:

(KBr) 1700(sh), 1620(broad), and 1600cm"l. 1H NMR: H-4, 7.37; H-5, 7.23(,]--2.3); H-7, 6.61(,/=2.3); H-11, 3.83; H-12, NR; H-13, 2.17; H-14, 4.03; H-16, 2.00; 1-OH, 13.02; and 8-OH, 12.31ppm. 13CNMR: (DMSO, CDCI3, 1:1)C-l, 163.0; C-la, 108.1; C-2, 121.4; C-3, 163.3; C-4, 108.6; C-4a, 132.2; C-5, 108.6; C-5a, 134.6; C-6, 164.8; C-7, 107.9; C-8, 164.2; C-8a, 108.6; C-9, 188.8; C-10, 181.0; C-11, 62.9; C-12, 35.0; C-13, 27.8; C-14, 62.9; C-15, 169.8; and C- 16, 20.8ppm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 120 (1981). P. S. Steyn, R. Vleggaar, L. Wessels, R. J. Cole, and D. Scott; J. Chem. Soc., Perkin Trans. I, p. 451 (1979).

584

12. Versicolorins

Common/Systematic Name Versiconol Molecular Formula/Molecular Weight C18H1608; MW' -" 360.08452

OH

HO

0

OH

L

s

11

~C H-~CI--12OH

~3"OH H 0

General Characteristics Crystals from acetone; mp., 257-259~ -3 5.8 ~ (C=0.35, in dioxane).

Orange-red needles; mp., 2650C (dec.); [a]D25

Fungal Source A mutant ofAspergillus parasiticus. Spectral Data UV:

~i,M.=~" 224(e=30,100), 266(14,100), 294(23,900), 315(12,300), and 455nm (7,400); g ~~~ 224(e=44,600), 255(22,300), 265(22,300), 295(33,800), 322(13,800), and 460rim (10,400). 1H NMR;

(DMSO) H-4, 7.18; H-5, 7.23(,/=2.3); H-7, 6.54(,/=2.3); H-11, 3.0-4.0; H-12, 3.0-4.0; H-13, 1.95; H-14, 3.0-4.0; 1-OH, 12.79; and 8-OH, 12.15ppm. ~3CNMR: (DMSO, CDCI3, 1:1)C-I, 162.9; C-la; 108.1; C-2, 122.9; C-3, 163.1; C-4, 108.9; C-4a, 132.1; C-5, 108.6; C-5a, 134.7; C-6, 164.7; C-7, 107.9; C-8, 164.2; C-8a, 108.6; C-9, 188.8; C-10, 181.0; C-11, 63.2; C-12, 34.9; C-13, 32.5; and C-14, 60.1ppm. Mass Spectrum: Electron-impact mass spectral analysis shows 342.0739m/e due to M* -H20. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 121 (1981). P. S. Steyn, R. Vleggaar, L. Wesseis, R. J. Cole, and D. Scott; J. Chem. Soc., Perkin

12. Versicolorins

Trans. I, p. 451 (1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

585

586

12. Versicolorins

Common/Systematic Name Nidurufin Molecular Formula/Molecular Weight C20H1608; M W -- 384.08452

OH

OH

O

OH

OH O General Characteristics Crystals from chloroform-methanol; mp., 188 ~C. Fungal Source Aspergillus nidulans. Spectral Data UV:

3,~m~ 223(e=33,500), 253(15 300), 264(19,000), 291(30,400), 319(10,700), and 450nm (10,000) IR:

(KBr) 1673 and 1725cm"1. CD: Ac 230(-10.4), 240(0), 256(sh) (1.9), 260(2.84), 270(0), 280(0.95), 288(0), 296(o7.6), 308(0), 325(2.8), 348(0), 365(-0.76), 380(-0.57), 400(0), 444(4.17), and 480nm (0.76). References P. J. Aucamp and C. W. Holzapfel; J. S. Afr. Chem. Inst., Vol. 23, p. 40 (1970). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 122 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

12. Versicolorins

587

Common/Systematic Name Dimethylnidurufin Molecular Formula/Molecular Weight C22H2008; MW = 412.11582 OH

Me

OH

0

0

OMe

5

OMe

.General Characteristics Crystals from acetone-hexane; rap., 211-213 ~C; [a]D25 -77 ~ (c=0.15, in CHCI3). Fungal Source

Aspergillus versicolor.

Spectral Data UV:

~E~

224(e=48,200), 251(19,000), 288(30,900), 314(8,540), and 444nm (8,790).

IR: (KBr) 3500, 3420, 2940, 1680, 1625, 1600, 1560, 1490, 1460, 1400, 1330, 1300, 1250, 1220, 1170, 1068, 1050, 1000, 970, 890, and 850cm~. IH ]N:~: (CDCI3) H-4, 7.28; H-5, 7.46(J=2.5); H-7, 6.86(J=2.5); H-11, 5.30(J=2.0), H-12, 4.16 m; H-13, 14, 1.6-2.5 m; H-16, 1.64; 12-OH, 1.60; OCH3, 4.00, 4.16ppm. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 123 (1981). D. G. I. Kingston, P. N. Chen, and J. R. Vercellotti; Phytochemistry, Vol. 15, p. 1037

(1976). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

588

12. Versicolorins

Common/Systematic Name Aversin Molecular Formula/Molecular Weight C20H1607; 1VIW = 368.08960 OH

0

OMe

~OMe General Characteristics Slender golden needles from acetone solution; mp., 217~ CHCI3).

[a]D 20 -222* (c=0.248, in

Fungal Source

Aspergillus versicolor.

Spectral Data UV:

~.~ff" 224(e=36,700), 251(13,400), 285(33,600), 313(8,900), 363(4,960), and 440nm (7,830); ~,s~" 240(e=21,300), 263(34,800), 307(13,800), 355(5,040), 498(6,890), and 501nm (6,890). IR:

(KBr) 3470, 3090, 2970, 2935, 2880, 2840, 1725, 1665, 1628, 1595, 1558, 1479, 1436, 1423, 1375, 1354, 1329, 1298, 1243, 1210, 1174, 1160, 1118, 1104, 1084, 1076, 1044, 990, 953,911,864, 821,798, 771,758, and 747cm "1. References E. Bullock, D. Kirkaldy, J. C. Roberts, and J. G. Underwood; J. Chem. Soc., p. 829 (1963). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 125 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

12. Versicolorins

589

Common/Systematic Name O-Methylaversin; Tri-O-methylversicolorin B Molecular Formula/Molecular Weight C21H1807; M W = 382.10525

OMe 0

t.oAo , X

OMe

~OMe

0

General Characteristics Fine golden-yellow needles from chloroform-methanol; mp., 216-217" C; [a]D 127" (c=0.1804, in CHCI3); golden-yellow crystals from methanol; mp., 212-213 ~ sublimed sample, mp., 229-230 ~ 25

-

Fungal Source

Aspergillus versicolor,

Spectral Data UV:

~

EtOH max

222(e=33,800), 285(39.200), 348(4,700), and 407nm (4,200).

IR:

(KBr) 3460, 3100, 3089, 3053, 3019, 3004, 2986, 2947, 2937, 2880, 2845, 2664, 1673, 1667, 1602, 1572, 1510, 1463, 1440, 1431, 1419, 1357, 1349, 1332, 1323, 1307, 1281, 1255, 1238, 1209, 1201, 1188, 1178, 1163, 1114, 1103, 1085, 1060, 1035, 990, 958, 942, 925, 914, 897, 871,850, 841,836, 787, 778, and 753cm"~. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 126 (1981). G. M. Holmwood and J. C. Roberts; J. Chem. Soc., p. 3899 (1971). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

590

12. Versicolorins

Common/Systematic Name Versicolorone Molecular Formula/Molecular Weight C20H1607, MW' = 368.08960

OH

0

OH

0

0 General Characteristics Recrystallized from acetone-water; mp., 208-210 ~C. Probable intermediate in the biosynthesis of the aflatoxins. Fungal Source Aspergillus parasiticus [Mutant WE-47 (hvn- 1)]. Derived from ATCC 15517. Isolation/Purification Purification by preparative TLC (40% acetone-hexane) and Chromatotron (50% ethyl acetate-hexane).The product was recrystallized five times from acetone-water and dried under vacuum. Spectral Data UV:

~,~" 222(c=26,700), 252(13,300), 264(16,500), 298(17,700), 318(26,400), 468(6,000), and 483nm (6,400). IR:

(CHCI3) 3020, 1700, 1600, 1185, 800, and 670cm"l. ~H NMR: (DMSO-dr) 1.94(2H, m, H-3'), 2.06(3H, 8, H-6'), 2.20(1H, dt, ,/--7.4, 17.6, H-4'), 2.34(1-H, dt, ,/=7.8, 17.6Hz, H-4'), 3.38(1H, m, H-2'), 3.68(1H, dd, ,/=6.6, 10.0Hz, H-I'), 3.75(1H, dd, ,/=7.6, 10.0Hz, H-I'), 6.57(1H, d, ,/=2.6Hz, H-7), 7.09(1H d, J=2.6Hz, H-5), 7.19(1H, s, H-4), 11.21(1H, brs, 6-OH), 12.18(1H, s, 8-OH); and 12.82ppm (1 H, s, 1-OH). Mass Spectrum: 368(28), 325(6), 310(100), 297(19), and 285m/e (6); HRMS: obsd 368.0905, C20H1707 requires 368.0896.

12. Versicolorins

591

Reference C. A. Townsend, K. A. Plavcan, K. Pal, S. W. Brobst, M. S. Irish, E. W. Ely, Jr., and J. W. Bennett; Hydroxyversicolorone: Isolation and Characterization of a Potential Intermediate in Aflatoxin Biosynthesis; J. Org. Chem., Vol. 53, pp. 2472-2477(1988).

592

12. Versicolorins

Common/Systematic Name Hydroxyversicolorone Molecular Formula/Molecular weight C2oH1608; M W = 384.08452

OH

0

OH

0

2' ,,,,L,,. L

"o. 0

o.

IL

0 General Characteristics Obtained as an orange powder from acetone; mp., 247-249~ CHCI3).

[a]o 25 0 ~ (C=0.006, in

Fungal Source

Aspergillus parasiticus [(Mutant WE-47 (hvn- 1)]. Derived from ATCC 15517. Probable intermediate in the biosynthesis of the aflatoxins.

Isolation/Purification The mycelia were filtered, washed well with water, and pulverized in a blender with acetone. The residual cells were extracted with acetone until colorless. The solvent was removed, and the material was extracted with pentane. The residue was preadsorbed and chromatographed on silica gel eluted with chloroform-methanol (97:3, v/v). The fraction containing mainly hydroxyversicolorone was recrystallized several times from ethyl acetate-hexane to afford pure hydroxyversicolorone. Spectral Data UV:

Z ~m~" 223(e=24,000), 265(20,300), 293(19,500), 316(22,600), 476(5,300), and 528nm (4,000).

12. Versicolorins

593

IR:

(CHCI3) 3390, 1705, 1600(s) 1440, 1320, and 970cm~. 1H NIV[R:

(DMSO-d6) 1.68(1-H, m, H-3'), 1.94(1-H, m H-3'), 2.06(3-H, 8, H-6'), 2.56(2-H, t, J=6.6Hz, H-4'), 3.09(1=H, dt, J=l.5Hz, H-2'), 5.89(1-H, br dd, J=5.9, 1.5Hz, H-I'), 6.56(1/zH, d, J=2.2Hz, *H-7), 6.58Q/zH, d, J=2.2Hz, H-7), 7.06(1/zI-I, d, J=2.2Hz, *H-5), 7.10Q/zH, d, J=2.2 Hz, H-5), 7.11Q/zH, s, H-4), 7.26(1/21-1,s, *H-4), 7.73 (<1 H, br d, J=5.9Hz, I'-OH), 11.09(3/2H, br s, 3 (OH)), 12.20Q/zH, br s, 13.8Hz, OH), 12.53Q/zH, br s, 17.1Hz, OH), 13.38Q/zH, s, 17.1Hz, OH); and 13.38ppm (1/zl-I, s, 17.1Hz, OH). * Asterisk denotes angular isomer 13C NMR:

(DMSO-d6) (Linear form) C-5', 207.7; C-9, 189.0; C-10, 180.8; C-6(164.9, 164.2, 164.1,164.0); C-3(163.6); C-I, 159.1; C-14(135.2, 134.7, 134.5, 134.4); C-2, 121.5; C-4, 108.7; C-13, 110.5; C-5, 108.7; C-12, 108.4; C-7, 107.9; C-I', 102.4; C-2', 46.2; C-6', 29.8; C-3', 23.9; and C-4' 23.9ppm; (angular form) C-9, 184.7; C- 10, 181.5; C-6(164.9, 164.2, 164.1,164.0); C-3, 160.8; C-I, 159.0; C-14(135.2, 134.7, 134.5, 134.4); C-2, 122.7; C-4, 108.8; C-13, 109.4; C-5, 107.1; C-I', 107.7; and C-2', 45.4ppm. Mass Spectrum: CIMS: (NH3) 385m/e ( M + +1, 16), 369(100); HRFABMS: obsd 285.0927m/e OVI+ 1)+, C20HlTOsrequires 384.0923. EIMS: 384(2), 382(22), 368(28), 367(60), 366(100), 347(21), 340(26), 339(32), 324(97), 313(34), 312(48), 311(92), 310(86), 309(62), 299(46), 298(76), 297(86), 296(78), 285(46), 270(26), 269(22) 245 (20), 155(28), 70(35), and 61m/e (47). Reference C. A. Townsend, K. A. Plavcan, K. Pal, S. W. Brobst, M. S. Irish, E. W. Ely, Jr., and J. W. Bennett; Hydroxyversicolorone: Isolation and Characterization of a Potential Intermediate in Aflatoxin Biosynthesis; J. Org. Chem., Vol. 53, pp. 2472-2477(1988).

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Sterigmatocystin and Related Metabolites

Sterigmatocystin Dihydrosterigmatocystin O-Methylsterigmatocystin Dihydro-O-methylsterigmatocystin Aspertoxin 5-Methoxysterigmatocystin Dihydrodemethylsterigmatocystin Dimethoxysterigmatocystin Sterigmatin

595

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13. Sterigmatocystin and Related Metabolites

597

Common/Systematic Name Sterigmatocystin 3a, 12c-Dihydro-8 -hydroxy-6-methoxyfuro[3',2':4, 5]furo[ 3,2-c]xanthen-7-one Molecular Formula/Molecular Weight C18H1206; MW = 324.06339

o ~0

0

e

General Characteristics Pale yellow crystals; mp., 246~ (dec.) of a sublimed sample. Sterigmatocystin was relatively insoluble in most solvents tested; solubility was best in chloroform (7138 mg/100 ml) and pyridine (1815 mg/100 ml); [tt]o2~ -398 ~ (c=l.0, in CHCI3). Fungal Source Aspergillus versicolor, A. amstelodami = Erotium amstelodami, A. chevalieri = E. chevalierL A. ruber = E. rubrum, A. unguis, A. multicolor, A. nidulans, Bipolaris sorokiniana, A. aurantio-brunnel, A. quadrilineatus, A. ustus Bainier, A. variecolor, Chaetomium thielavioideum, and Farrowia sp. Also an intermediate in biosynthesis of aflatoxins by A. parasiticus and A. flavus.

Biological Activity The LDs0 values for sterigmatocystin in albino rats was 166mg/kg (male rats, per os, dimethylformamide solvent): 60mg/kg (male rats, IP, dimethylformamide solvent); 120mg/kg (female rats, per os, wheat germ oil carrier); 65mg/kg (male rats, IP, wheat germ oil carrier). The LDs0 in male monkeys was 32mg/kg (dosed IP, with DMSO as carrier). Acute toxicity: caused liver and kidney damage and renal necrosis in rats; it was cirrhogenic; caused hepatic damage in primates. In monkeys, oral administration (20mg/kg) every 14 days for several months caused chronic hepatitis and hyperplasia. It has been hypothesized that sterigmatocystin may be implicated in the etiology of chronic liver disease in humans in Africa. Spectral Data UV:

~

EtOH max

208(c=19,000), 235(24,500), 249(27,500), and 329nm (13,100).

598

13. Sterigmatocystin and Related Metabolites

IH NIVIR: H-I, 6.48(,/=2.0, 2.5); H-2, 5.42(J=2.5); H-3, 4.75(,/=2.0, 7.25); H-4, 6.79(/=7.25); H-5, 6.35; H-8, 6.78(`/=8.25, 1.0); H-9, 7.45(,/=8.25); H-10, 6.70(,/=8.25, 1.0); and H- 18, 3.96ppm.

13C NMR.: C-l, 145.1 d; C-2, 105.7 d; C-3, 47.9 d; C-4, 113.1 d: C-5, 90.4 d; C-6, 163.0 s; C-7, 154.7 s; C-8, 106.4 d; C-9, 135.4 d; C-10, 111.0 d; C-I 1,162.1 s; C-12, 108.8 s, C-13, 180.9 s; C-14, 105.7 s; C-15, 153.7 s; C-16, 106.4 s; C-17, 164.3 s; and C-18, 56.6ppm q. TLC Data Silica gel (chloroform-methanol, 98:2, v:v); Rf: 0.5; detection as orange-red fluorescent spot under UV light; it exhibited a light yellow fluorescence, if sprayed with acetic acid. GLC Data Chromosorb W, Liquid phase: 1.5% SE-30, 220~ to cholestane).

relative retention time 1.21 (relative

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 68-69(1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

13. Sterigmatocystin and Related Metabolites

599

Common/Systematic Name Dihydrosterigmatocystin; 1,2-Dihydro-6-methoxy-7-hydroxydifuroxanthone Molecular Formula/Molecular Weight C18H1406; M W = 326.07904

oS Lo. (o7.o

I

o

e

General Characteristics Pale yellow needles; mp., 230~ (dec.); [a]D 20 -311.7 ~ (c=0.85, in CHCI3). Yellow plates from ethanol (sublimed sample); mp., 229-230~ Fungal Source

Aspergillus versicolor.

Biological Activity Dihydrosterigmatocystin markedly inhibited mitosis of primary kidney epithelial cells of Cercopithecus aethiops but had negligible effects on nucleolar morphology. It also markedly inhibited the incorporation of [~3H]thymidine and [3H]uridine, which indicated impaired DNA and RNA synthesis. Spectral Data UW:

~.~=," 233(6=27,600), 247(32,200), and 325nm (16,600); ~.n=,, 208(e=20,400), 232(26,300), 247(30,900), and 325nm (16,300). ~H NMR: (CDCl3) H-l, 4.10; H-2, 2.23; H-3, 3.62(J=5.5); H-4, 6.41(,]=5.5); H-5, 6.25; H=8, 6.72(J=8.0, 1.0); H-9, 7.39(J=8.0); H-10, 6.64(J=8.0, 1.0); H=18, 3.90; and 7-OH, 13.26ppm. 13C NMR: (CDCI3) C-l, 67.6 t; C-2, 31.4 t; C-3, 44.2 d; C-4, 113.1 d; C-5, 89.6 d; C-6, 163.1 s; C-7, 154.6 s; C-8, 105.5 d; C-9, 135.2 d; C-10, 110.7 d; C-11,161.9 s; C-12, 108.7 s; C-13, 180.8 s; C-14, 105.1 s; C-15, 154.6 s; C-16, 106.7 s; C-17, 165.7 s; and C-18, 56.6ppm q.

600

13. Sterigmatocystin and Related Metabolites

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re: 0.83; detected as red-orange fluorescent spot under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 73-74(1981). Y. Hatsuda, T. Hamasaki, M. Ishida, K. Matsui, and S. Hara; Agric. Biol. Chem., Vol. 36, p. 521 (1972). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 631 (1983).

13. Sterigmatocystin and Related Metabolites

601

Common/Systematic Name O-Methylsterigmatocystin; 6,7-Dimethoxydifuroxanthone Molecular Formula/Molecular Weight C19H1406; MW = 338.07904

O.~~OM e

,Lo os

General Characteristics Colorless prisms from ethanol; mp., 274 ~ (synthetic compound); crystals from methanol and chloroform-heptane; mp., 265~ (dec.); faint yellow, slender rods from methanol; mp., 265-267~ Funsal Source

Aspergillus flavus, A. parasiticus, Chaetomium thielavioideum, and Farrowia sp.

Spectral Data UV:

~,maxm'~ 236(e=40,700) and 310nm (16,500).

IH N-MR: (CDCI3) H-I, 6.48(J=2.5.2.0);H-2, 5.43(J=2.5);H-3, 4.76(J=7.1,2.0);H-4, 6.78(d=7.1); H-5, 6.36; H-7, 6.92(/=8.3, 1.0); H-8, 7.48(/=8.3); H-9, 6.74(,/--8.3, 1.0); H-18, 3.95; and H-19, 3.91ppm. ~3C NMR: (CDCI3) C-I, 145.0 d; C-2, 102.6 d; C-3, 48.1 d; C-4, 112.9 d; C-5, 90.3 d; C-6, 162.8 s; C-7. 156.5 s; C-8, 106.2 d; C-9, 133.4 d; C-10, 108.9 d; C-11, 160.4 s; C-12, 106.2 s; C-13, 174.6 s; C-14, 105.6 s; C-15, 152.9 s; C-16, 106.2 s; C-17, 162.7 s; C-18, 56.3 q; and C-19, 56.3ppm q. TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Rr, 0.38; detected as blue fluorescent spot under UV light; turns to yellow fluorescent spot after spraying with 50% ethanolic H2SO4.

602

13. Sterigmatocystin and Related Metabolites

References R. J. Cole and R. H. Cox; _Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 77 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983). S. Udagawa, T. Muroi, H. Kurata, S. Sekita, K. Yoshihira, S. Natori, and M. Umeda; Can. J. Microbiol., Vol. 25, p. 170 (1979).

13. Sterigmatocystinand Related Metabolites

603

Common/Systematic Name Dihydro-O-methylsterigmatocystin; 1,2-Dihydro-6-methoxy-7-hydroxydifuroxanthone Molecular Formula/Molecular Weight C19H1606; MW "- 340.09469

O,~~OM

LO.~O~01~0Me

e

General Characteristics Colorless rods from methanol; mp., 282-283 ~ chloroform-methanol; mp., 281-282~

colorless needles from

Fungal Source

Aspergillus flavus and A. parasiticus.

Biological Activity The toxicity of dihydro-O-methylsterigmatocystin, as evaluated in primary cell culture (primary kidney epithelial cells of Cercopithecus aethiops) showed negligible effects on mitosis and nucleolar morphology; also negligible effects on the incorporation of [3H]thymidine and [3H]uridine, indicating no inhibition on DNA and RNA synthesis. Spectral Data UV: ~,mMff" 238 and 313nm (e max not reported); ~.m~x 203(26,300), 237(38,900), and 311 nm (17,300). IH ~ :

H-I, 4.20; H-2, 2.34; H-3, 3.72; H-4, 6.48(J=6.0); H-5, 6.34; H-8, 6.97(J=8.0, 1.0); H-9, 7.52 (J=8.0); H-10, 6.78(J=8.0, 1.0); H-18, 4.00; and H-19, 3.96ppm. 13C NMR:

C-1, 67.5 t; C-2, 31.4 t; C-3, 44.3 d; C-4, 112.8 d; C-5, 89.5 d; C-6, 162.7 s; C-7, 156.3 s; C-8, 106.0 d; C-9, 133.1 d; C-10, 108.7 d; C-11, 160.3 s; C-12, 106.0 s; C-13, 156.3 s; C-14, 104.2 s; C-15, 153.2 s; C-16, 106.0 s; C-17, 164.1 s; C-18, 56.3 q; and C- 19, 56.3ppm q.

604

13. Sterigmatocystin and Related Metabolites

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v:v); Rf, 0.32; detected as blue fluorescent spot under UV light changing to yellow fluorescent after spraying with 50% ethanolic H2SO4. References W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 81-82(1981). R. J. Cole, J. W. Kirksey, and H. W. Schroeder; Tet. Lett., p. 3109 (1970).

13. Sterigmatocystin and Related Metabolites

605

Common/Systematic Name Aspertoxin; 3-Hydroxy-6,7-dimethoxydifuroxanthone Molecular Formula/Molecular Weight C19H1407; M W

=

354.07395

OH

it r

OMe

~

~0../"'~0~/,/~..OMe

General Characteristics Crystals from dioxane/H20; mp., 240-280~ (dec.); from dimethylformamide; mp., 3 25-3 27 oC; [tt]D27 - 140 ~ (C=0.015, in dimethylformamide). Aspertoxin is insoluble in most organic solvents. Fungal Source

Aspergillus flavus.

Biological Activity In developing chicken embryo, injections of 2.0ktg/egg (yolk or air sac) killed 100% of the embryos; while 0.7t.tg/egg killed 50% of the embryos. Microscopically, the embryos showed beak malformations, generalized edema, loss of muscle tone and hemorrhage from the umbilical vessels. Spectral Data UV:

~

MeOH max

241(e=33,900) and 310nm (12,100).

1H NMR: H-I, 6.72; H-2, 5.76; H-4, 6.38; H-5, 6.56; H-8, 7.05; H-9, 7.61; H-10, 6.95; H-18, 3.93; and H-19, 3.93ppm. TLC Data Silica gel (chloroform-acetic acid, 9:1 v:v); Rf=0.55-0.60; detected as blue fluorescent spot under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 85 (1981).

606

13. Sterigmatocystin and Related Metabolites

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

13.. Sterigmatocystin and Related Metabolites

607

Common/Systematic Name 5-Methoxysterigmatocystin Molecular Formula/Molecular Weight C20H1607, ~ l W = 368.08960

General Characteristics Pale yellow needles from sublimed sample; mp., 223~ (dec.); [a]o 2~ -360* (c=0.238, in CHCI3).

Fungal Source

Aspergillus versicolor.

Spectral Data UV;

)~~2~" 232(E=24,100), 248(26,800), 279(11,200), and 33 lnm (12,100). IH N]%,IR: Similar to sterigmatocystin except for two coupled aromatic protons at 6.71 and 7.18ppm. IR;

(KBr) 3447, 3383, 3121, 3096, 3016, 2971, 2927, 2853, 1662, 1634, 1618, 1595, 1559, 1498, 1462, 1444, 1419, 1398, 1360, 1345, 1339, 1302, 1285, 1262, 1248, 1233, 1223, 1199, 1187, 1168, 1148, 1140, 1099, 1078, 1060, 1042, 1017, 979, 960, 930, 881,853, 825, 788, 767, 744, 737, and 724cm"~. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 89 (1981). W. B. Turner and D. C. Aldndge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

608

13. Sterigmatocystin and Related Metabolites

Common/Systematic Name Dihydrodemethylsterigmatocystin Molecular Formula/Molecular Weight C17H1206; MW = 312.06339

OH

o

General Characteristics Pale yellow needles from acetone; mp., 202~ (dec.); [a]D 25 - 3 7 6 . 6 ~ (c=0.90, in CHCI3); green ferric reaction in ethanol. Fungal Source Aspergillus versicolor. Spectral Data UV:

233(e=26,800), 250(33,100), 259(30,300), and 335nm (19,400). IR:

(KBr) 1659, and 1625cm"l. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 91 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

13. SterigmatocystinandRelatedMetabolites

609

Common/Systematic Name Dimethoxysterigmatocystin; 6,9,10-Trimethoxy-7-hydroxydifuroxanthone Molecular Formula/Molecular Weight C20H16Os; MW = 384.08452

MeO~

OMe

2 -o.

II /

I" IY '~

~ O J ' ~ O ~~"~,~"~,OM e General Characteristics Pale yellow needles from acetone; mp., 253-254~ dihydro derivative; mp., 241-242"C: acetate derivative; mp., 188-189~ monomethyl ether derivative; mp., 257-258~ Fungal Source Aspergillus versicolor. Spectral Data UV:

~

EtOH max

233(e=27,200), 275(sh) (7,700), 284(34,000), and 330nm (19,200).

IR:

(KBr) 3125, 1655, 1635, and 1610cm~. 1H NMR: (CDCI3) H-I, 6.64(,]=2, 2.5); H-2, 5.41(J=2, 2.5); H-3, 4.80(,/--2, 2.5, 7); H-4, 6.75(J=7); H-5*, 6.28; H-8*, 6.32; 7-OR 13.20; OCH3, 3.91, 3.94, and 3.98ppm. * Assignments may be reversed. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 92 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

610

13. Sterigmatocystin and Related Metabolites

Common/Systematic Name Sterigrnatin Molecular Formula/Molecular Weight C17H1006; M~W = 310.04774

OH

0

OH

H General Characteristics Pale yellow needles from acetone; mp., 253 ~ green ferric reaction in ethanol.

[aiD 20 -377 ~ (c=0.50, in CHCI3); gave a

Fungal Source

Aspergillus versicolor.

Isolation/Purification Mycelium extracted with acetone and extract chromatographed on silica gel column chromatography. Recrystallized from acetone to give pale yellow needles. Spectral Data UV: )t~ EtOH max

225(e=23,400), 250(33,800), 259(23,800), 268(20,000), and 324nm (16,900).

IR:

(KBr) 1670, 1640, and 1610cm"1. 1H NMR:

(CDCI3) 11.85 and 12.1 lppm (2 x OH protons, s); 6.44(1H, s); 6.76(1H, dd, J=l and 8.5Hz); 6.85(1H, dd, J=l and 8.5 Hz); 7.55(1H, t, J=8.5Hz); 4.68(1H, dt, J=2.5 and 7Hz); 5.44(1H, t, J=2.5Hz); 6.46(1H, t, J=2.5Hz); and 6.77ppm (1H, d, J=7.0Hz, dihydro furan ring system). Mass Spectrum: EIMS: 3 lOm/e (M +) References K. Fukuyama, T. Tsukihara, Y. Katsube, T. Hanasaki, Y. Hatsuda, N. Tanaka, T. Ashida, and M. Kakudo; The Crystal and Molecular Structure of Sterigmatin, A Metabolite of Aspergillus versicolor; Bull. Chem. Soc. Japan, Vol. 48, pp. 1639-1640(1975).

13. Sterigmatocystin and Related Metabolites

T. Hamasaki, K. Matsui, K. Isono, and Y. Hatsuda; A New Metabolite from Aspergillus

versicolor; Agric. Biol. Chem. Vol. 37, pp. 1769-1770(1973).

611

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Chokols Chokol A Chokol B Chokol C Chokol D Chokol E Chokol F Chokol G

613

This Page Intentionally Left Blank

14.

Chokols

615

Common/Systematic Name Chokol A 4-(3-Hydroxy-2,3-dimethylcyclopentyl)penta-4-en- 1-ol Molecular Formula/Molecular Weight C12H2202; MW = 198.16198

/

HOH2C General Characteristics Colorless syrup; [{I]D24 -45.5 ~ (C=0.4, in MeOH). Fungal Source

Epichlo~ typhinaon Phleumpratense.

Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The ethyl acetate extract was chromatographed on a silica gel (CHCI3-MeOH, 95:5, v/v) followed by several chromatographies on silica gel (n-hexane-EtOAc gradient system), Sephadex LHo20 (CHCIrMeOH, 1:1, v/v) and silica gel (twice, CHCI3-MeOH, 95:5, v/v) and cyclohexane-EtOAc, 1:1, v/v). Final purification was achieved by HPLC (IzBondapak C is; H20-CH3CN, 4:1, v/v). Biological Activity Moderate fungitoxic activity (TLC plate bioassay using Cladosporium herbarum); minimal qnhibition was about 25~g/spot. Spectral Data IR:

(film) 3350, 3050, 2920, 1730, 1640, 1440, 1380, 1150, 1050, and 880crn].

~H NMR: (CDCI3) 4.79(IH, s,H-14a); 4.78(IH, d, J=l.5I-Iz,H-14b), 3.67(21-I,t,J=6.6, H-10); 2.39(IH, ddd, s~--11.3,9.2, 9.2Hz, H-6); 2.06(2H, br. t,J=7.8H~ H-8); 1.98(II-I,m, H-5a); 1.76(2H, t,d-=7.81-1z,H-4); 1.71-1.78(2I-I,rn,H-9); 1.56(II-I,dq, J=11.3, 6.8Hz, H-2); 1.42(1H, m, H-5b); 1.28(3H, s, H-13); and 0.87ppm (3H, d, J----6.8Hz, H-l). 13C NMR: (CHCIa) 10.7(C-1), 47.6(C-2), 80.3(C-3), 39.9(C-4), 28.8(C- 5), 51.7(C-6), 151.3(C-7), 30.2(C-8), 31.1(C-9), 62.7(C-10), 26.5(C-13) and 108.2ppm (C-14).

616

14. Chokols

Mass Spectrum: FI-MS: 198role (M+, 31%), and 181(M+-OH, 100); El-MS: 198.1628(M+, C~2H2202, calcd 198.1620, 0.4%), 180.1502(M+-H20, C~2H200;calcd. 180.1503, 0.2), 136(36), 121(34), 71(43), and 43role (100). Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxie Sesquiterpenoids, Chokols A-G, from Stromata of Epichlo~ typhina and the Absolute Configuration of Chokol E; Agile. Biol. Chem., Vol. 53, pp. 789-796(1989).

14.

Chokols

617

Common/Systematic Name Chokol B 6-(3-Hydroxy-2,3-dimethylcyelopentyl)-2-methylhepta- 1,6-dien-3-ol Molecular Formula/Molecular Weight CasH2602; MW = 238.19328 14

....2 ~ a , . , , OH

15

~0 12

10

H

1

Ii13

General Characteristics Colorless syrup. Fungal Source Epichlo~ typhina on Phleum pratense. v

Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The hexane extract was chromatographed on a silica gel (CHCI3-MeOH, 95:5, v/v) followed by several chromatographies on silica gel (CHCI3-MeOH and benzene-EtOAc gradient system), Sephadex LH-20 (CHCI3-MeOR 1:1, v/v), and silica gel (cyelohexane-EtOAc, 75:25, v/v). Biological Activity Inhibited the growth of Cladosporium herbarum at the amount of more than 5ixg/spot (TLC plate bioassay). Soectral Data IR:

(film) 3370, 3080, 2920, 1720, 1640, 1445, 1380, 1080, and 880cm"1. ]H NMR: (CDCI3) 4.95(1H, br. s, H-12a); 4.85(1H, br. s, H-12b); 4.79(1H, d, J=l.0Hz, H-14a); 4.7g(1H, d, J=-l.0Hz, H-14b); 4.09(1H, dd, J=6.8, 5.9Hz, H-10); 2.39(1H, ddd, J=l 1.2, 9.3, 8.8Hz, H-6); 2.09(1H, dddd, ,/=9.3, 6.4, 5.9 1.0Hz, H-8a); 1.92-2.02(2H, m, H-5a and 8b); 1.77(2H, t, J=7.8Hz, n-4); 1.74(3H, s, H-15); 1.63-1.78(2H, rn, H-9); 1.56(1H, dq, J=l 1.2, 6.8Hz, n-2); 1.44(1H, rn, n-5b); 1.28(3H, s, H-13); and 0.87ppm (3H, d, J=6.8Hz, H-l).

618

14.

Chokols

~3CNMR: (CHCI3) 10.7(C-1), 47.7(C-2), 80.3(C-3), 40.0(C-4), 28.8(C-5), 51.9(C-6), 151.5(C-7), 33.5"(C-8), 30.0'(C-9), 75.8(C-10), 147.6(C-11), 110.0(C-12), 26.6(C-13), 108.2(C-14), and 17.6ppm (C-15). * Assignments may be exchanged. Mass Spectrum: FD-MS: 239m/e (MI-F); El-MS: 220.1834(M+-H20, C15H240;calcd 220.1827), 205(M+-Me-H20), 202(M+-2H20, 4.0%), and 43m/e (base peak). Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxic Sesquiterpenoids, Chokols A-G, from Stromata of Epichlog typhina and the Absolute Configuration of Chokol E; Agric. Biol. Chem., Vol. 53, pp. 789-796(1989).

14.

Chokols

619

Common/Systematic Name Chokol C 6-(3-Hydroxy-2,3-dimethylcyclopentyl)-2-methylhepta-2(E),6-dien- 1-ol Molecular Formula/Molecular Weight C15H2602; M W = 2 3 8 . 1 9 3 2 8 14

HOH2C ~ \~/~o

/

~ 1 "

....." 13

,

12

General Characteristics Colorless needles; mp., 51.0-53.0~

[a]D 22

-32.0 ~ (c=0.25, in EtOH).

Fungal Source Epichlo~ typhina on Phleum pratense. Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The hexane extract was chromatographed on silica gel (CHC13-MeOH, 95:5, v/v) followed by several chromatographies on silica gel (CHCI3-MeOH and benzene-EtOAc gradient system), Sephadex LH20 (CHCI3-MeOH, 1:1, v/v) and silica gel (cyclohexane-EtOAc, 75:25, v/v). Final purification was achieved by HPLC (~Bondapak C~g;H20-CH3CN, 7:3, v/v). Biological Activity Inhibited the growth of Cladosporium herbarum at the amount of more than 5l.tg/spot (TLC plate bioassay). Soectral Data _

IR~

(KBr) 3340, 3080, 2970, 1650, 1370, 1230, 1170, 1020, and 890em"~. ~H NMR: (CDCI3) 5.43(IH, tq, J=7.0, 1.3Hz, H-10); 4.79(IH, s, H-14a); 4.76(IH, d, J=l.5Hz, H-14b); 4.00(21-1, s, H-12); 2.39(1H, ddd, J=l 1.3, 9.4, 9.0Hz, H-6); 2.20(2H, dt, ,/=7.0, 7.3Hz, H-9); 2.05(2H, dt, J=3.7, 7.3Hz, H-8); 1.96(1H, dddd, ,/=13.3, 9.4, 8.2, 7.6Hz, H-5a); 1.76(2H, dd, J=-8.2, 7.6Hz, H-4); 1.68 (3H, s, H-15); 1.55(11-1,dq, J=l 1.3, 7.0Hz, H-2); 1.42(1H, dddd, J=13.3, 9.0, 8.2, 7.6Hz, H-Sb); 1.28(3H, s, H-13); and 0.87ppm (3H, d, J=7.0Hz, H-I).

620

14.

Chokols

13C NMR:

(CHCI3) 10.7(C-1), 47.6(C-2), 80.3(C-3), 40.0(C-4), 28.7(C-5), 51.8(C-6), 151.4(C-7), 33.6(C-8), 26.4(C-9), 125.9(C- 10), 134.9(C-11), 68.8(C-12), 26.6(C-13), 108.2(C- 14) and 13.8ppm (C- 15). Mass Spectrum: FD-MS: 238m/e (M +, 68%); El-MS: 238(M§ 0.2%), 220.1864(M+-H20, C~5H240, calcd. 220.1827, 2.0), 202(20), 187(25), 159(18), 133(20), 121(37), 107(61), 95(59), 93(42), 79(41), and 43m/e (100). Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxic Sesquiterpenoids, Chokols A-G, from Stromata of Epichlog typhina and the Absolute Configuration of Chokol E; Agrie. Biol. Chem., Vol. 53, pp. 789-796(1989).

14. Chokols

621

Common/Systematic Name Chokol D 6-(3-Hydroxy-2,3-dimethylcyclopentyl)-2-methylhepta-2,6-dien- 1-ol Molecular Formula/Molecular Weight C15H2602, ~

= 238.19328 14

15

7' ...z ~ a , . . , O H

H O H 2 C X ~ / o "1 ~

/

"' "

13

,

12

General Characteristics Colorless syrup; [a]D22 -44.4 ~ (C=0.054, in EtOH). Fungal Source Epichlo~ typhina on Phleum pratense. Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The hexane extract was chromatographed on a silica gel (CHCI3-MeOH, 95:5, v/v) followed by several chromatographies on silica gel (CHCI3-MeOH and benzene-EtOAc gradient system), Sephadex LH20 (CHCI3-MeOR 1:1, v/v) and silica gel (cyclohexane-EtOAc, 75:25, v/v). Final purification was achieved by HPLC (ktBondapak C~s; H20-CH3CN, 7:3, v/v). Biological Activity Inhibited the growth of Cladosporium herbarum at the amount of more than 51.tg/spot (TLC plate bioassay). Spectral Data IR:

(film) 3370, 3080,2970, 1730, 1645, 1450, 1375, 1200, 1100, 1005, 920, and 885cm1. ~H NMR: (CDC13) 5.31(1H, t, J=7.1Hz, H-10); 4.79(1H, s, H-14a); 4.75(1H, d, J=l.5Hz, H-14b); 4.12(2H, s, H-15), 2.38(1H, ddd, J=l 1.2, 9.0, 8.3Hz, H-6), 2.22(2H, dt, J-7.1, 7.3Hz, H-9); 2.01(2H, tit, J=3.4, 7.3Hz, H-8); 1.96(1H, dddd, J=13.2, 9.0, 7.8, 7.8Hz, H-5a); 1.80(3H, d, J=-l.0Hz, H-12); 1.76(2H, t, J=-7.8Hz, H-4); 1.55(1H, dq, J=l 1.2, 6.6Hz, H-2); 1.41(11-1,dddd, J=13.2, 8.3, 7.8 7.8Hz, H-5b); 1.28(3H, s, H-13); and 0.87ppm (3H, d, J=6.6Hz, H-l).

622

14.

Chokols

13C NM~:

(CHCI3) 10.7(C-1), 47.6(C-2), 80.3(C-3), 40.0(C-4), 28.7(C-5), 51.8(C-6), 151.4(C-7), 34.1(C-8), 26.3(C-9), 128.2(C- 10), 134.6(C-11), 21.3(C-12), 26.6(C-13), 108.3(C-14), and 61.7ppm (C-15). Mass Spectrum: FD-MS: 239(MH+, 54%), 238(M§ 55), and 220m/e (M+-H20,. 100); EI-MS: 238(M~, 0.1%), 220.1795(M+-H20, C~5H240,calcd 220.1827, 1.3), 202(9.0), 187(13), 162 (11), 161(10), 133(15), 121(20), 107(35), 95(27), and 43m/e (100). Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxic Sesquiterpenoids, Chokols A-G, from Stromata of Epichlo~ typhina and the Absolute Configuration of Chokol E; Agric. Biol. Chem., Vol. 53, pp. 789-796(1989).

14.

Chokols

623

Common/Systematic Name Chokol E (3R)•6-[(•R•2S•3R)•3•Hydr•xy•2•3•dimethy••yc••penty•]-2-methy•hepta-6-ene-2•3-di•• Molecular Formula/Molecular Weight C15H2803; M W -- 2 5 6 . 2 0 3 8 4 14

HO) 12

,3 OH

General Characteristics Colorless syrup; [tt]o2~ -15.8 ~ (c=0.67, in EtOH). Fungal Source

Epichlog typhina on Phleum pratense.

Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The ethyl acetate extract was chromatographed on silica gel (CHCI3-MeOH, 95:5, v/v) followed by silica gel (CHCl3-acetone gradient system), Sephadex LH-20 (CHCIrMeOH, 1:1, v/v), and silica gel (twice, eyclohexane-EtOAc, 3:7, v/v, and benzene-EtOAc, 3:7, v/v) chromatographies. Biological Activity Relatively weak fungitoxic activity (TLC plate bioassay using Cladosporium herbarum); minimum amount of inhibition was about 501~g/spot. Spectral Data IR: (film) 3400, 3080, 2970, 1730, 1650, 1460, 1380, 1160, and 1080cm~. CD: (CHC13, Eu(fod)3) 305(-3.8 Ac), 290(0), and 285nm (+1.5). 1H NMR: (CDCI3) 4.82(1H, s, H-14a); 4.78(1H, s, H-14b); 3.40(1H, br. d, J=10.7Hz, H-10); 2.41(1H, ddd, J=l 1.7, 8.8, 8.8Hz, H-6); 2.28(1H, ddd, J=15.1, 9.5, 4.9Hz, H-8a); 2.06(1H, ddd, J=15.1, 10.0, 6.4Hz, H-8b); 1.98(1H, dddd, J=13.2, 8.8, 7.8 7.8Hz, H-

624

14.

Chokols

5a); 1.77(2H, t, J=7.8Hz, H-4); 1.67(1H, dddd, J=13.3, 9.5, 6.4, 2.0Hz, H-9a); 1.55(1H, dq, J=l 1.7, 6.8Hz, H-2); 1.48(1H, dddd, J=13.3, 10.7, 10.0, 4.9Hz, H-9b); 1.45(1H, dddd, J=13.2, 8.8, 7.8, 7.8Hz, H-5b), 1.29(3H, s, H-13); 1.23(3H, s, H-12); 1.18(3H, s, H-15); and 0.88ppm (3H, d, J=6.8Hz, H-I). 13C NMR.: (CHCI3) 10.7(C-1), 47.8(C-2), 80.3(C-3), 39.9(C-4), 28.8(C-5), 51.9(C-6), 151.7(C-7), 31.1(C-8), 30.0(C-9), 78.4(C- 10), 73.1(C-11), 26.5"(C-12), 26.5(C-13), 108.2(C- 14), and 23.3*ppm (C- 15).

* Assignments may be exchanged. Mass Spectrum: FD-MS" 257role (MH+); El-MS" 256.2083(M*, C~5H2803,calcd 256.2038, 0.3%), 238(M+-H20, 3.0), 220(M+-2H20, 4.0), 205(2.9), 202(2.1), 180(5.2), 161(7.5), 136(34), 121(52), 107(30), 95(49), 81(28), 71(40), 59(100), and 43m/e (74). Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxic Sesquiterpenoids, Chokols A-G, from Stromata of Epichlo~ typhina and the Absolute Configuration of Chokol E; Agric. Biol. Chem., Vol. 53, pp. 789-796(1989).

14.

Chokols

625

Common/Systematic Name Chokol F Molecular Formula/Molecular Weight C14H2403; M W -- 2 4 0 . 1 7 2 5 4

AcO---/ General Characteristics Colorless syrup; [a]D 24 -48.8 ~ (c=0.32, in EtOH). Fungal Source Epichlod Ophina on Phleumpratense. Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The ethyl acetate extract was chromatographed on silica gel (n-hexane-EtOAc gradient system), Sephadex LH20 (CHCI3-MeOH, 1:1, v/v), silica gel (twice, CHCl3-acetone, 99:1, v/v) and cyclohexane-EtOAc, 9:1, v/v) and silica gel (preparative TLC, n-hexane-EtOAc, 7:3, v/v). Final purification was achieved by HPLC (~-Porasil, n-hexane-EtOH, 9:1, v/v). Spectral Data IR:

(film) 3470, 3070, 2950, 2925, 1745, 1640, 1445, 1370, 1245, 1040, 920, and 885cm-1. 1H NMR: (CDC13) 4.81(1H, s, H-14a); 4.76(1H, d, J=l.0Hz, H-14b); 4.08(2H, t, J=6.6Hz, H-10); 2.38(1H, ddd, J=l 1.2, 9.3, 9.3Hz, H-6); 2.05(3H, s. H-At); 2.02-2.07(2H, m, H-8), 1.98(1H, dddd, J=12.2, 9.3, 7.8, 7.8Hz, H-5a); 1.78-1.83(2H, m, H-9); 1.76(21-1, t, J=7.8Hz, H-4); 1.55(1H, dq, J=l 1.2, 6.8Hz, H-2); 1.41(1H, dddd, J--12.2, 9.3, 7.8, 7.8Hz, H-5b); 1.29(3H, s, H-13), and 0.87ppm (3H, d, J--6.8Hz, H-l). Mass Spectrum: El-MS: 222.1621(M+-H20, C14I-I2202;calcd 222.1619, 5.1%), 201(2.5), 180(3.1), 162(8.7), 155(13), 147(16), 122(20), 121(21), 109(26), 107(24), 95(33), 93(31), 81(33), 67(34), 55(41) and 43m/e (100).

626

14.

Chokols

Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxic Sesquiterpenoids, Chokols A-G, from Stromata of Epichlog typhina and the Absolute Configuration of Chokol E; Agric. Biol. Chem., Vol. 53, pp. 789-796(1989).

14.

Chokols

627

Common/Systematic Name Chokol G 3-(3-Hydroxy-2,3-dimethylcyclopentyl)but-3-en- 1-ol Molecular Formula/Molecular Weight CllH2oO2; M~W = 184.14633

H0 89 General Characteristics Colorless syrup; [a]D24 -43.3 o (C=0.24, in MeOH). Fungal Source

Epichlo6 typhina on Phleum pratense.

Isolation/Purification Timothy chokes were extracted with 70% ethanol, partitioned between n-hexane and water and subsequently between ethyl acetate and water. The ethyl acetate extract was chromatographed on silica gel (CHCI3-MeOH, 95:5, v/v) followed by several chromatographies on silica gel (n-hexane-EtOAc gradient system), Sephadex LH20 (CHCI3-MeOR 1:1, v/v) and silica gel (twice, CHC13-MeOH, 95:5, v/v and cyclohexane-EtOAc, 1:1, v/v). Final purification was achieved by HPLC (IzBondapak C~s; H20-CH3CN, 4:1, v/v). Biological Activity Relatively weak fungitoxic activity (TLC plate bioassay using Cladosporium herbarum); minimal inhibition was about 501~g/spot. Spectral Data IR:

(film) 3370, 3070, 2950, 1720, 1640, 1440, 1370, 1040, 920, and 880cm "1. 1H NMR: (CDC13) 4.91(1H, s, H-14a); 4.84(1H, dd, J=2.6, 1.5Hz, H-14b), 3.75(2H, t, J-6.6Hz, H-9), 2.40(1H, ddd, J-I 1.4, 9.0, 9.0Hz, H-6); 2.28(2H, t, J=6.6Hz, H-8), 2.00(IH, dddd, J=13.9, 9.0, 7.7, 7.7Hz, H-5a); 1.77 (2H, t, J=7.7Hz, H-4); 1.56(1H, dq, J=l 1.4, 6.6Hz, H-2); 1.43(1H, dddd, J=13.9, 9.0, 7.7, 7.7Hz, H-5b); 1.29(3H, s, H-13); and 0.88ppm (3H, d, J=6.6Hz, H-l). .

13C NMR:

(CHO3) 10.6(C-1),47.6(C-2), S0.2(C-3), 39.9(C-4), 2S.7(C-5), 51.7(C-6), 148.1(C-?), 37.1(C-8), 60.9(C-9), 26.6(C-13), and 108.gppm (C-14).

628

14.

Chokols

Mass Spectrum: FIMS" 184(M+, 16%), 167(M+-OH, 100), and 166m/e (M+- H20, 75); EIMS: 166.1349(M+-H20, C1~H~80,ealed 166.1358, 19%), 151(12), 148(9), 135(40), 121(33), 107(32), 105(34), 95(54), and 43m/e (100). Reference H. Koshino, S. Togiya, S. Terada, T. Yoshihara, S. Sakamura, T. Shimanuki, T. Sato, and A. Tajimi; New Fungitoxie Sesquiterpenoids, Chokols A-G, from Stromata of Epichlo~ typhina and the Absolute Configuration of Chokol E; Agric. Biol. Chem., Vol. 53, pp. 789-796(1989).

Enaminomycins and Related Metabolites Enaminomycin A (Antiphenicol) Enaminomycin B Enaminomyein C Antibiotic G7063-2 Epoformin (Desoxyepoxydon) (:t:)-Epoxydon Acetylepoformin (+)-Epoxydon monoacetate (+)-Desoxyepiepoxydon (• [(+)-Isoepoxydon] Coriloxin

629

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15. Enaminomycins and Related Metabolites

631

Common/Systematic Name Enaminomycin A; Antiphenicol 2-Amino-3-carboxy- 1,4-benzoquinone 5,6-epoxide; 4-Amino-2,5-dioxo-7oxabicyclo[4.1.0]hept-3-ene-3-carboxylic acid Molecular Formula/Molecular Weight CTHsNOs; MW = 183.01677 O

l cOOH

0 General Characteristics Enaminomycin A (antiphenicol) is an acidic, lipophilic, white amorphous powder; mp., 105~ (dec.); [a]o 2~ -20.9 ~ (c--0.83, in MeOH). Crystallized from ethyl ~ t e to give yellow needles, mp 153-155~ (dec.); pK~ 6.1 and 9.7. Antiphenicol was positive to 2,4dinitrophenylhydrazine. It decolorized aqueous potassium permanganate solution and becomes brown with ninhydrin, orange-red with sodium nitroprusside and grayish violet with isatine, respectively. It is soluble in water, methanol, ethanol, acetone and ethyl acetate and insoluble in benzene and chloroform. Fungal Source Streptomycesfulvoviolaceus No.851 and S. baarnensis No. 13120. Isolation/Purification The cultured broth was filtered and the filtrate was adjusted to pH 6 and extracted with ethyl acetate. The extract was concentrated and applied to a column of silicic acid. Active fractions eluted with ethyl acetate were collected and concentrated to remove the solvent. The concentrate was applied onto a silica gel column. The active substance was eluted with benzene-ethyl acetate (95:5, v/v). The active fractions were concentrated and yielded a yellowish powder. Antiphenicol was recrystallized from ethyl acetate as yellow needles. Biological Activity Showed noticeable antagonistic action against chloramphenicol in Escherichia coll. Antiphenicol antagonized the activities of antibiotics such as chloramphenicol, thiamphenicol, lineomycin and erythromycin, all of which have effects on the ribosome 50S subunit of E. coll. The enaminomycins are active against Gram-positive and Gramnegative bacteria as well as against some species of fungi. Enaminomycin A is also active against L1210 mouse leukemia cells m vitro.

632

15. Enaminomycins and Related Metabolites

Spectral Data UV: MeOH ~ max 244(e=9,150), 292 (15,200), and 349nm (660). Im:

(KBr) 3350, 3250, 2730, 1700, 1590, 1500, 1440, 1370, 1250, 1050, 990, 880, 750, and 570cmq. CD: (MeOH) [0]25, +25,600(250), +27,900(253), +65,800(273), -79,200(300), and -900(250). ~H NMR: (acetone-d6) 4.02(1H, d, J=lHz); 4.22(1H, d, J=4Hz); 8.6(1H, broad); 10.0(IH, broad); and 13.2ppm (1H, broad). 13C N M R :

(acetone-d6) 53.2(d, J=52.4Hz); 55.4(d, J=52.7Hz); 96.4(s); 155(s); 168.7(s); 187.4(s); and 192.Sppm (s). Mass Spectrum: HREIMS: 183.0146m/e (M+); calcd, for C7HsNO5 183.0167. References Y. Imagawa, S. Shima, A. Hirota, and H. Sakai; Isolation and Structure Elucidation of Antiphenicol, An Antagonist of Chloramphenicoi; Agrie. Biol. Chem., Vol. 43, pp. 24372440(1979). Y. Itoh, T. Haneishi, M. Arai, T. Hata, K. Aiba, and C. Tamura, New Antibiotics, Enaminomycins, B and C. III. The Structures of Enaminomycins A, B and C, J. Antibiot., Vol. 31, pp. 838-846(1978).

15. Enaminomycins and Related Metabolites

633

Common/Systematic Name Enaminomycin B 2-Ox o-4-amino- 5-hydroxy-5-acetonyl-7-oxabicyclo [4.1.0 ]hept-3-ene- 3-carboxylic acid Molecular Formula/Molecular Weight C10H]INO6, M W = 241.05864

~

y-C02 H

O~NI-12 HO CH2OOMe General Characteristics Enaminomycin B is an acidic, lipophilic, colorless crystalline substance; mp., 160~ (dee.), [a]D2~ + 60.1 o (C=0.99, in MeOH). Fungal Source Streptomyces fulvoviolaceus No.851 and S. baarnensis No. 13120. Biological Activity The enaminomycins are active against Gram-positive and Gram-negative bacteria as well as against some species of fungi. Spectral Data UV:

~mM~" 244(e=315) and 293nm (580). IR:

(KBr) 3500-2500, 1720, 1670, and 1650cm1. CD: (MeOH) 296(-80400) and 269nm (+94400).

1H NMR: (acetone-d6) The NMR spectrum exhibited a methyl singlet at 2.20, a methylene singlet at 3.15 and a hydroxy singlet at 5.22, in addition to two epoxide protons at 3.60(J=4.5Hz) and 4.10ppm (J=4.5Hz). Mass Spectrum: 241m/e (M+).

634

15.

Enaminomycins and Related Metabolites

Reference Y. Itoh, T. Haneishi, M. Arai, T. Hata, K. Aiba, and C. Tamura; New Antibiotics, Enaminomycins A, B and C. HI. The Structures of Enaminomycins A, B and C; J. Antibiot., Vol. 31, pp. 838-846(1978).

15. Enaminomycins and Related Metabolites

635

Common/Systematic Name Enaminomycin C 2-Oxo-4-amino-5-hydroxy-7-oxabicyclo[4.1.0]hept-3-ene-3-carboxylic acid Molecular Formula/Molecular Weight CTHTNOs; MW = 185.03242

~

CO2H

O'HO~~HI NH2 General Characteristics Enaminomycin C is an acidic, lipophilic, colorless crystalline substance; mp., 173 ~ (dee.); [a]D2~ +31.4 o (C=0"14, in MeOH). Treatment of enaminomyein C with diazomethane in methanol gave a biologically active substance, which showed the same Re value as enaminomycin A on silica gel TLC. Fungal Source

Streptomyces baarnensis No. 13120.

Biological Activity The enaminomyeins are active against Gram-positive and Gram-negative bacteria as well as against some species of fungi. Spectral Data UV: ~t, MeOH max

245(e=436) and 290nm (930).

IR:

3500-2500, 1680, and 1605cm "~. CD: (MeOH) 294(-78000) and 268nm (+64200). IH NMR: (DMSO-d~) The NMR spectrum exhibited two protons at 3.63(J=4.5Hz) and 3.82(J=4.5Hz) corresponding to epoxide protons, a proton giving a broad double doublet at 4.81(J=8.5, 2.0Hz) and an exchangeable proton giving doublet at 6.56ppm (J=8.5Hz). One of the epoxide protons was coupled to a proton (4.81ppm) on the carbon linked to the hydroxy group.

636

15. Enaminomycins and Related Metabolites

Mass Spectrum: 1 8 5 m / e (M+) .

Reference Y. Iyoh, T. Haneishi, M. Arai, T. Hata, K. Aiba, and C. Tamura, New Antibiotics, Enarninomycin A, B and C. III.The Structures of Enaminomycin A, B and C; J. of Antibiot., Vol. 31, pp. 838-846(1978).

15. Enaminomycins and Related Metabolites

637

Common/Systematic Name Antibiotic G7063-2 4- Amino-7-oxabicyclo[4.1.0 ]hept- 3 -ene-2, 5-dione- 3 -carboxamide Molecular Formula/Molecular Weight C7H6N204;/VIW = 1 8 2 . 0 3 2 7 6

O

O~~i cONI-12 ( "NI-12 0 General Characteristics G7063-2 is a yellow crystalline antibiotic (decomposes > 180~ soluble in water, methanol, dimethyl sulfoxide, and acetone and slightly soluble in chloroform. It turns red with the loss of antibiotic activity in sodium carbonate solution at pH 10. Fungal Source Streptomyces sp. (NCIB- 11306). Isolation/Purification The fungal broth was adjusted to pH 7 with sulfuric acid and centrifuged to separate aqueous solution from mycelium. The aqueous solution was extracted with 3 one-third volumes of ethyl acetate and the combined ethyl acetate extracts were evaporated to dryness. The residue was extracted with n-butanol-methanol-water (4:1:2, v/v/v), filtered to remove inactive solid and the extract fractionated on a column of Sephadex LH20 in the same solvent mixture. Fractions active against both Staphylococcus aureus and Escherichia coli were separated from fractions active only against Staphylococcus aureus. The fractions with activity against both organisms were combined and evaporated to dryness. The residue was extracted with ethyl acetate and filtered. The filtrate was evaporated to 30ml and applied to a column of Sephadex LH20 packed in chloroform-ethyl acetate (1:2, v/v). Elution was with the same solvent and active fractions were combined, evaporated to 70ml and kept at -20 ~C. After 3 days yellow crystals were collected, washed with cold ethyl acetate and dried under reduced pressure to give pure G7063-2. Biological Activity G7063-2 had moderate activity against a wide range of Gram-positive and Gram-negative bacteria; weak to moderate activity against fungi; and an LDs0 IP to mice was about 17mg/kg body weight.

638

15.

Enaminomycins and Related Metabolites

Spectral Data UV:

maxW~' r 248(e=430), 292(740), and 358nm (72); ~ m=~HC, 358nm (75); Z~ N'~ 249sh (870) and 260nm (1010).

249(450), 292(690), and

IR:

(Nujol mull) showed peaks at 3440 and 3300 (-NH2) and at 1720, 1630, and 1555cm"~ [NH2CO-C(CO-R)=C-COR]. 1H NMR: (acetone-d6) Spectrum of G7063-2 showed signals centered at 5.921: (d, 4 I ~ 1H) and 6.13(d, 4Hz, 1H) for vicinally coupled epoxide protons, at 1:1.40 and 3.421: (2H) for amide protons and at 0.601: for amine protons (2H). 13C NMR: (DMSO-d6) Showed seven carbon resonances at 52.5; 55.0; 97.6; 154.7; 169.4; 188.5; and 188.8ppm. Mass Data: The mass spectrum showed a base peak-molecular ion at m/e 182; an accurate mass measurement for this peak gave a value of 182.0326 indicating a molecular formula of C7I--I6N204. Fragmentation peaks at 137 and 109m/e corresponded with the loss of CH3NO and C2H3NO2 moieties; elemental analysis gave: C, 46.2; H, 3.4; N, 15.2; C7I-~N204 requires C, 46.2; H, 3.3; N, 15.4%. TLC Data Samples were applied to either layers of cellulose containing fluorescent indicator or to layers of Kieselgel 60F254(E. Merck, Darmstadt, Germany). Development was with butanol-methanol-water (4:1:2, v/v/v) for cellulose plates and with ethyl acetate for silica plates. After development at 24 ~ sheets were air-dried, examined under UV light (254 and 356nm). Rf values were 0.80 and 0.67, respectively. Reference M. Noble, D. Noble, and R. B. Sykes; G7063-2, A New Nitrogen-containing Antibiotic of The Epoxydon Group, Isolated from the Fermentation Broth of A Species of Streptomyces; J. Antibiot., Vol. 30, pp. 455-459(1977).

15. Enaminomycins and Related Metabolites

639

Common/Systematic Name Epoformin; Desoxyepoxydon 2,3-Epoxy-4-hydroxy-6-methyl-5-cyclohexen- l-one Molecular Formula/Molecular Weight CTHsO3; MW = 140.04734 0 Me 0 OH General Characteristics Colorless needles; mp., 75-77"C; [a]D 22 +114.3 ~ (c=l.0, in EtOH); recrystallized from benzene (mp., 89-9 I~ Fungal Source

Penicillium claviforme and Penicillium patulum (NRRL 2159A) = P. griseofulvum.

Isolation/Purification Culture filtrate was extracted with ethyl acetate, dried and subjected to preparative TLC: silica gel GF254, chloroform-EtOH (8:2, v/v), Rf 0.46. Biological Activity Weak antimicrobial activity; strong cytotoxicity against PS-cells; and LDs0 in mice, ca. 100 mg/kg (IV). Spectral Data UV: ~,~" max

240nm (c=5540); ~ M,o. max

242nm (6=4700).

[R: (CHCI3) 3550(OH), 1680(conjugated C=O), 1650(-C=C-),1230, and 880cm q (epoxide); (KBr) 3410, 2950, and 1655cm q. ORD: 365 = +114~ 335 = 0~ 312.5 =-70~ 290 = 0~ 260 = +263~ and 250 =0 ~ IH NMR: (CDCI3) 6.25(1H, m); 4.63(1H, m, ,/=-3.0, 2.5, 2.0Hz); 3.80(1H, dt, J=4.0, 3.0, 2.5Hz); 3.47(1H, d, J=4.0Hz); 1.83(3H, t, ,1=2.0, 2.0); and 3.07ppm (broad singlet, exchangeable with D20).

640

15. Enaminomycins and Related Metabolites

Mass Data: Calcd C7H803; C, 59.99; H, 5.75 found C, 59.88, 59.86; H, 5.70, 5.70. References A. I. Scott, L. Zamir, G. T. Phillips, and M. Yalpani; The Biosynthesis of Patulin; Bioorganic Chemistry, Vol. 2, pp. 1124-1139(1973). I. Yamamoto, E. Mizuta, T. Henmi, T. Yamano, and S. Yamatodani; Epoformin, A New Antibiotic Produced by Penicillium claviforme Bainier; J. Takeda Res. Lab. Vol..32, pp. 532-538(1973).

15. Enaminomycins and Related Metabolites

641

Common/Systematic Name (• Molecular Formula/Molecular Weight C7H804; MW = 156.04226

HOH2C~o 0

OH General Characteristics Oil; [a]D 23 91.9~ (c=0.123, in EtOH); [a]D 24 +105 ~ (c=0.98, in EtOH). Fungal Source Penicillium claviforme, P. patulum = P. griseovulvum, Ascochyta chrysanthemi, Mycosphaerella ligulicola, and Phoma chrysanthemi. Isolation/Purification Cultures were extracted with ethyl acetate, dried over sodium sulfate and evaporated to dryness. The crude extract was chromatographed by preparative TLC on sifica gel, containing 1% KH2PO4 using hexane-ethyl acetate (1:1, v/v) and chloroform-methanol, 19:1 (v/v) as eluants to give purified (+)-epoxydon and its monoacetate. Biological Activity Phytotoxic; cytotoxic PS-cells, 3.1; LDs0 in mice (IV), 110-219mg/kg body wt. Spectral Data UV: maxE~n 238nm (e=4900). IH ~ : (DMSO-d~) 3.42(1H, d, J=5.0Hz, H-2); 3.76(1H, m, H-3); 4.03(2H, -CH2OH); 4.70(1H, m, H-4); 4.97(1H, OH); 5.78(1H, d, J=5.0I-~ OH); and 6.42ppm (1H, m, H-

5). Mass Data: Anal calcd C7H804; C, 53.83; H, 5.16; Mol. Wt., 156.13; found: C, 53.96, 53.57; H, 5,18, 5.04; Mol. Wt. 151 (V.P.O.).

642

15. Enaminomycins and Related Metabolites

References G. Assante, L. Camarda, L. Merlini, and G. Nasini; Secondary Metabolites from Mycosphaerella ligulicola; Phytochem., Vol. 20, pp. 1955-1957(1981). I. Yamamoto, E. Mizuta, T. Henmi, T. Yamano, and S. Yamatodani; Epoformin, A New Antibiotic Produced by Penicillium claviforme Bainier; J. Takeda Res. Lab., Vol., 32, pp. 532-538(1973).

15. Enaminomycins and Related Metabolites

643

Common/Systematic Name Acetylepoformin Molecular Formula/Molecular Weight C9Hlo04; MW = 182.05791

"e' o OCOMe General Characteristics Crystals; mp., 62-63~ [IZ]D22 +113.4 ~ (c=l.0, in EtOH). Fungal Source

Penicillium claviforme, Ascochyta chrysanthemL Mycosphaerella ligulicola, and Phoma chrysanthemi.

Spectral Data UV:

/~ EtOi-I

max

237nm (e=6,680).

]H NMR: (CDCI3) 6.15, CS-H(1H, m, J=2.5, 2.5, 1.SHz); 3.85, C3-H (double triplet, J=4.0, 2.5, 2.SHz); 3.48, C2-H(1H, d, J--4.0); 1.83(3H, t, J=l.5, 2.0Hz); and 5.74ppm, C4-H(1H, m, J=2.0, 2.5, 2.SHz). Mass Data: Anal calcd C9H]004; C, 59.36; H, 5.49; found: C, 58.90; H, 5.04. Reference I. Yamamoto, E. Mizuta, T. Henmi, T. Yamano, and S. Yamatodani; Epoformin, A New Antibiotic Produced by Penicillium clm~iforme Bainier; J. Takeda Res. Lab., Vol. 32, pp. 532-538(1973).

644

15. Enaminomycins and Related Metabolites

Common/Systematic Nam.e (• monoacetate Molecular Formula/Molecular Weight C9H1005; 1V[W= 198.05282

O AcOH2C

i'::,,o

General Characteristics Needles; mp., 60-62~ [a]D23 +90.4 ~ (C=0.125, in EtOH); [aID24 + 105~ (C=0.98, in EtOH). Fungal Source

Ascochyta chrysanthemi, Mycosphaerella ligulicola (CBS 367 67), and Phoma chrysanthemL

Isolation/Purification Cultures were extracted with ethyl acetate, dried over sodium sulfate and evaporated to dryness. The crude extract was chromatographed by preparative TLC on silica gel, containing 1% KH2PO4 using hexane-ethyl acetate (1:1, v/v) and chloroform-methanol, 19:1 (v/v) as eluants to give purified (+)-epoxydon and its monoacetate. Biological Activity Phytotoxic. Spectral Data IR:

(Nujol) 3430(OH), 1725(acetate C=O), and 1670cm"l (conjugated CO). IH NMR:

(CDCI3) 2.07(3H, s, COMe); 3.15(1H, Br, s, OH); 3.52(1H, d, J=-3I-~ H-2); 3.85(1H, m, H-3); 4.72(3H, s, -CH2-O- and H-4); and 6.53ppm (1H, m, H-5). Mass Data: 198(M+), 166, 156, 138, and 127m/e. Reference G. Assante, L. Camarda, L. Merlini, and G. Nasini; Secondary Metabolites from Mycosphaerella ligulicola; Phytochem., Vol. 20, pp. 1955-1957(1981).

15. Enaminomycins and Related Metabolites

645

Common/Systematic Name (• Molecular Formula/Molecular Weight C7H803; M W -~ 140.04734

O Me

O

General Characteristics An amorphous solid; [a]D +221 ~ (C=0.83, in EtOH). Fungal Source Isolated from the culture filtrate of an unidentified fungus, which was separated from the diseased leaf of crepe-myrtle (Lagerstroemia indica L.). Isolation/Purification The culture filtrate was extracted with ethyl acetate and fractionated into the neutral, acidic and basic fractions. The neutral fraction showed marked inhibition against germination of lettuce seeds, Lactuca sativa L. Purification was monitored using the lettuce seed bioassay. The neutral fraction was applied onto a silica gel column eluted stepwise with a mixture of benzene and ethyl acetate. The effluent containing 10% ethyl acetate-benzene was subjected to preparative TLC on silica gel GF2s4 developed with benzene-ethyl acetate (1:1, v/v) and the extract corresponding to Rf 0.40 gave desoxyepiepoxydon as an amorphous solid. The fraction eluted with 70% ethyl acetate-benzene from the silica gel column was further fractionated with preparative TLC on silica gel GF254 developed with benzene-ethyl acetate (1:3, v/v) and the extract (Rf 0.52) yielded (+)-epiepoxydon as an amorphous solid. Biological Activity In the bioassay using lettuce seeds, it completely inhibited the germination at a concentration of 50ppm. Spectral Data UV: ~, m=~~" 237nm (e=4,440); ~.~"§ 32(sh) (6,670), 262(3,440), and 324nm (sh) (870). UV absorption in ethanol was irreversibly changed by addition of alkaline solution, suggesting that desoxyepiepoxydon was labile in an alkaline conditions.

646

15. Enaminomycins and Related Metabolites

IR;

(film) 3440(OH), 1668(C=O), 1282, 1042(C-O), 830, and 727cm1. CD: (c=0.01, in dioxane) [O]25(nm): 0(385), +6,300(340), + 140(282), and + 11,600(254). 1H NMR: (CDCI3) 6.40(IH, m, -C=CH-); 4.61(1H, broad s, -CHOH); 3.73(1H, m); 3.46(1H, d, J=4Hz); 2.20(1H, broad s, -OH); and 1.82ppm (3H, s, -C=C-CH3). The NMR spectrum was quite similar to those of epoformin and desoxyepoxydon. However, slight differences were observed in the chemical shifts of signals due to -C=CHand -CHOH. From these facts, it was concluded that desoxyepiepoxydon has the structural feature similar to epoformin and that the difference may lie in the relative configuration of hydroxyl group at C-4 and oxyran ring.

Mass Spectrum: HR IMS: 140.0448(M +, 140.0472, calcd for C7HsO3), 112, 111, 97, 83, 71, and 69role.

Reference H. Nagasawa, A. Suzuki, and S. Tamura; Isolation and Structure of (+)-Desoxyepiepoxydon and (+)-Epiepoxydon, Phytotoxic Fungal Metabolites; Agile. Biol. Chem., Vol. 42, I:P. 1~ 33-1304(1978).

15. Enaminomycins and Related Metabolites

647

Common/Systematic Name (• ( •)-Isoepoxydon Molecular Formula/Molecular Weight C7H804; MW = 156.04226

O ~ cH2OH OH General Characteristics An amorphous solid;

[a]D 24 + 1 9 4 ~

(c=1.57, in EtOH).

Fungal Source Isolated from the culture filtrate of an unidentified fungus, which was separated from the diseased leaf of crepe-myrtle (Lagerstroemia indica L.). Isolation/Purification The culture filtrate was extracted with ethyl acetate and fractionated into the neutral, acidic and basic fractions. Only the neutral fraction showed the marked inhibition against the germination of lettuce seeds, Lactuca sativa U Purification was monitored using the lettuce seed bioassay. The neutral fraction was applied onto a silica gel column eluted stepwise with the mixture of benzene and ethyl acetate. The effluent with benzene containing 10% ethyl acetate was subjected to preparative TLC on silica gel GFz54 plates developed with benzene-ethyl acetate (1:1, v/v) and the extract corresponding to Re 0.40 gave (+)-desoxyepiepoxydon as an amorphous solid. The fraction eluted with benzene containing 70% ethyl acetate from the silica gel column was further fractionated with PTLC on silica gel GF254 plate developed with benzene-ethyl acetate (1:3, v/v) and the extract (Re 0.52) yielded (• as an amorphous solid. Biological Activity In the bioassay using lettuce seeds, it showed 30% inhibition at a concentration of 300ppm. Soectral Data IR:

(film) 3340(OH), 1675(C=O), 1397, 1240, 1113, 1031(C-O), 1012(C-O), and 825cm ~. CD (c=0.01, dioxane) [a]2~(nm): 0(390), +7,800(341), +470(282), and + 13,300(256).

648

15. Enaminomycins and Related Metabolites

IH N1VIR: (CDCI3) 6.65(1H, m), 4.80(1H, broad s, -OH), 4.62(1H, broad s), 4.18(2H, AB quartet, J=17Hz, -CHzOH), 4.05(1H, broad s, -OH), 3.75(1H, m), and 3.38ppm (1H, d, J=4Hz). The ~H NMR spectrum was identical with that of authentic (•

Mass Spectrum: 156m/e (M+, CTHs04). Short communication. Reference H. Nagasawa, A. Suzuki, and S. Tamara; Isolation and Structure of (+)-Desoxyepiepoxydon and (• Phytotoxic Fungal Metabolites; Agric. Biol. Chem., Vol. 42, pp. 1303-1304(1978).

15.

Enaminomycins and Related Metabolites

Common/Systematic Name Coriloxin 5,6-Epoxy-4-hydroxy-3-methoxy-5-methyl-2-cyclohexen- 1-one Molecular Formula/Molecular Weight CaHsO4; MW = 168.04226

MeO~Me 0

0 FungalSource

Coriolus vernicipes.

Reference Nissan Chem. Ind, Co. Ltd., Japanese Patent J5 5089 274.

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Boviquinones and Related Metabolites Boviquinone-3 Bovinone Methylenediboviquinone-3,3 Diboviquinone-3,4 Diboviquinone-4,4 Amitenone (Methylenediboviquinone-4,4)

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16. Boviquinones and Related Metabolites

653

Common/Systematic Name Boviquinone-3 2, 5-Dihydroxy-3-farnesyl- 1,4-benzoquinone Molecular Formula/Molecular Weight C21H2804; M W = 344.19876

O

HO" T O

~(CH2CH=CCH2)3H

General Characteristics Boviquinone-3 crystallized from acetic acid as yellow needles turning orange in air; mp., 90-92~ Fungal Source

Gomphidius rutilus, Chroogomphus helveticus, and C. rutilus.

Isolation/Purification Freshly collected fruiting bodies of G. rutilus were sliced and steeped in cold ethanol for 2 hr. The bright red solution was decanted and the mushrooms were extracted overnight with fresh alcohol. Treatment of the combined extracts with an excess of aqueous lead acetate solution yielded the blue-green lead salt which was filtered off and washed twice with water and then twice with methanol. Methanolic hydrogen chloride was added dropwise to the stirred suspension of the lead salt in methanol. The mixture was filtered and the filtrate was evaporated at 35 ~ to yield a red gum. Boviquinone-3 was crystallized from acetic acid. Spectral Data UV: ~, EtOH max

288nm (log e=4.27).

IR:

(KBr) 3290, 2960, 2915, 2850, 1615, and 1335cm'~; (CHCI3) 3360, 2920, 1645, and 1370cm"l. 1H N M R : (CC14) 2.9(1H); 4.77-5.18(3H); 6.79(d, J=7Hz, 2H); 7.74(61-1); 7.77(6H); 8.02(8H); 8.33(6H); and 8.42z (6H).

654

16. Boviquinones and Related Metabolites

Mass Data: LREIMS: 314m/e (M+); Leucotetraacetate (C29H3008): 514, 472, 430, 388, 346, 320, 294, 277, 252, 239, 154, 81, and 69m/e (100%); found: C, 73.4; H, 8.4; C21H2804 requires C, 73.3; H, 8.1%. Reference P. C. Beaumont and R. L. Edwards; Constituents of The Higher Fungi. Part XI. Boviquinone-3,(2,5-Dihydroxy-3-farnesyl- 1,4-benzoquinone), Diboviquinone-3,4, Methylenediboviquinone-3,3, and Xerocomic Acid from Gomphidius rutilus Fr. and Diboviquinone-4,4 from Boletus (Suillus) bovinus (Linn ex Fr.) Kuntze; J. Chem. Soc. (C), pp. 2582-2585(1971).

16. Boviquinones and Related Metabolites

655

Common/Systematic Name Bovinone 2, 5-Dihydroxy-3-geranylgeranyl- 1,4-benzoquinone Molecular Formula/Molecular Weight C26H3604; M W "- 412.26136 O

HO" T O

~(CH2CH=CCH2)4H

General Characteristics Crystallized from acetic acid as yellow needles which turned orange when dried in air. It was readily soluble in organic solvents but insoluble in water. The quinonoid nature of bovinone was indicated by the reversible reduction and oxidation of its aqueous alcoholic solution and dithionite and air; yellow needles from glacial acetic acid; mp., 84-85~ Fungal Source Boletus (Suillus) bovinus. Isolation/Purification Bovinone was best obtained by acid decomposition of its insoluble olive-green lead salt. The freshly collected sporophores ofBoletus bovinus were sliced and steeped in cold ethanol for 1 hr. The bright red solution was decanted and the mushrooms were extracted overnight with fresh, cold alcohol. The combined extracts were treated with an excess of aqueous lead acetate solution; the dark olive-green precipitate was filtered off and washed twice with water and twice with methanol. A suspension of the lead salt in methanol was treated dropwise with 5% methanolic hydrogen chloride until it was completely decomposed. During the decomposition, the mixture was filtered periodically and the red filtrates were evaporated at 35 ~ under reduced pressure until a red solid precipitated; this was recrystallized from glacial acetic acid as yellow needles. Spectral Data UV:

~,Em ~t~

287nm (log 6=4.31).

IR~

(KBr) 3300, 2959, 2909, 2848, 1610, and 858cm~; (CHCIs) 3360, 2960, 2919, 2853,1640, 1360, and 850em "l.

656

16. Boviquinones and Related Metabolites

Mass Data: LREIMS: 414(M + 2), 413(M + 1), 412(M+), 343(M - 69), 275(M- 69-68), 207(M 9-2 x 68), 153, 154, and 155m/e (benzylium ions), 81, 69(base), 55, 41, and 28m/e; HR IMS: 412.2617role (M+), requires 412.2614; found: C, 75.5; H 3.4; calcd for C26H3604, C 75.7; H 8.7%. Reference P. C. Beaumont, and R. L. Edwards; Constituents of The Higher Fungi. Part XI. Boviquinone-3, (2,5-dihydroxy-3-farnesyl- 1,4-benzoquinone), Diboviquinone-3,4Methylenediboviquinone-3,3, and Xerocomic Acid from Gomphidius rutilus Fr. and Diboviquinone-4,4 from Boletus (Suillus) bovinus (Linn ex Fr.) Kuntze; J. Chem. Soc. (C), pp. 2582-2585(1971).

16. Boviquinones and Related Metabolites

657

Common/Systematic Name Methylenediboviquinone-3,3 Molecular Formula/Molecular Weight C43H5608; MW' = 700.39752 Me ,

0

0

H(CH2CH=CHCH2)3

H H

HO" y 0

~

Me '

(CH2CH=CCH2)3H y

"OH

0

General Characteristics Recrystallization from acetic acid gave yellow fatty needles; mp., 197-201 ~ Fungal Source Gomphidius rutilus. Isolation/Purification Freshly collected fruiting bodies of G. rutilus were sliced and steeped in cold ethanol for 2 hr. The bright red solution was decanted and the mushrooms were extracted overnight with fresh alcohol. Treatment of the combined extracts with an excess of aqueous lead acetate solution yielded the blue-green lead salt which was filtered off and washed twice with water and then twice with methanol. Methanolic hydrogen chloride was added dropwise to the stirred suspension of the lead salt in methanol. The mixture was filtered and the filtrate was evaporated at 35 ~ to yield a red gum. The gum was dissolved in acetic acid and set aside for three days. Filtration and recrystallization of the yellow residue six times from acetic acid gave methylenediboviquinone-3,3 as yellow fatty needles. Spectral Data UV: ~, m~x~~~ 288nm (log e=4.40); ~,~EO"+N'O" 327nm (log e=4.56). Im: (KBr) 3305, 2965, 2923, 2850, 1620, and 1048cm"~. IH NMR: (CDCI3) 2.25-2.48(4H); 4.68-5.05(6H); 6.41(2H); 6.86(d, .]=7Hz, 4H); 7.99(16H); 8.27(6H); 8.30(6H); 8.40(12H); (CCl4) 2.9(1H); 4.77-5.18(3H); 6.79(d, J=7Hz, 2H); 7.74(6H); 7.77(6H); 8.02(8H); 8.33(6H); and 8.421: (6H).

658

16. Boviquinones and Related Metabolites

Mass Data: 700(M+), 546, 358, and 344m/e (M § boviquinone-3); found: C, 73.6; H, 8.25; C43H5608requires C, 73.7; H, 8.0%. Reference P. C. Beaumont, and R. L. Edwards; Constituents of The Higher Fungi. Part XI. Boviquinone-3, (2, 5-d ihydroxy-3-farnesyl-1,4-benzoquinone), Diboviquinone-3,4Methylene-diboviquinone-3,3, and Xerocomic Acid from Gomphidius rutilus Fr. and Diboviquinone-4,4 from Boletus (Suillus) bovinus (Linn ex Fr.) Kuntze; J. Chem. Soc. (C), pp. 2582-2585(1971).

16. Boviquinones and Related Metabolites

659

Common/Systematic Name Diboviquinone-3,4 Molecular Formula/Molecular Weight C47I-I6208; M W "- 754.44447

Me

'

O

H(CH2CH=CHCH2)3

O

OH HO

.o- 1 0

Me

'

(CH2CH=CCH2)4H

y-o. 0

General Characteristics Clusters of small orange-yellow fatty needles from acetic acid; mp., 137-139 ~C. Fungal Source

Gomphidius rutilus.

Isolation/Purification Freshly collected fruiting bodies of G. rutilus were sliced and steeped in cold ethanol for 2 hr. The bright red solution was decanted and the mushrooms were extracted overnight with fresh alcohol. Treatment of the combined extracts with an excess of aqueous lead acetate solution yielded the blue-green lead salt which was filtered off and washed twice with water and then twice with methanol. Methanolic hydrogen chloride was added dropwise to the stirred suspension of the lead salt in methanol. The mixture was filtered and the filtrate was evaporated at 35 ~ to yield a red gum; boviquinone-3,4 was recrystallized five times from acetic acid. Spectral Data UV: ,/~ m~ EtOH

288nm (log 6=4.46); ~,max ~H+N,OH

326nm (log 6=4.63).

IR:

(KBr) 3305, 2955, 2923, 2850, 1620, and 1050cm q. IH ~ :

(CDCI3) 2.03-2.40(4H);4.67-5.13(7H);6.88(d,J=7Hz, 4I-I);8.02(20H), 8.27(6I-I), 8.32(6H); 8.41(15H); (CCl4) 2.9(1H); 4.77-5.18(3H); 6.79(d, J=7Hz, 2H); 7.74(6H); 7.77(6H); 8.02(8H); 8.33(6H); and 8.42z (6H). Mass Data: LREIMS: 684, 616, 548, 529, 461,412(M +, boviquinone-4), and 344m/e (M + Boviquinone-3); found: C, 74.5; H, 8.15. C47H62Osrequires C, 74.8; H, 8.2%.

660

16. Boviquinones and Related Metabolites

Reference P. C. Beaumont and R. L. Edwards; Constituents of The Higher Fungi. Part XI. Boviquinone-3, (2, 5- d ihydroxy-3-farnesyl- 1,4-benzoquinone), Diboviquinone-3,4Methylenediboviquinone-3,3, and Xerocomic Acid from Gomphidius rutilus Fr. and Diboviquinone-4,4 from Boletus (Suillus) bovinus (Linn ex Fr.) Kuntze; J. Chem. Soc.(C), pp. 2582-2585(1971).

16. Boviquinones and Related Metabolites

661

Common/Systematic Name Diboviquinone-4,4 Molecular Formula/Molecular Weight Cs2HT00$; M'W = 822.50707

0

Me

0

H(CH2CH-CHCH2)4

OH

HOy

H

(CH2CH=CCH2)4H

F 0

Me

'OH

0

General Characteristics Recrystallization from acetic acid gave small orange-red needles; mp., 132.5-134 ~C. Fungal Source Boletus (Suillus) bovirrus and Gomphidius rutilus. Isolation/Purification Freshly collected fruiting bodies ofB. bovinus were sliced and steeped in cold ethanol for 2 hr. The extract was treated with an excess of aqueous lead acetate solution yielded the blue-green lead salt which was filtered off and washed twice with water and then twice with methanol. Methanolic hydrogen chloride was added dropwise to the stirred suspension of the lead salt in methanol. The mixture was filtered and the filtrate was evaporated to yield an orange solid. The solid was recrystallized from acetic acid to give small orange-red needles. Spectral Data UV:

~, ~" max

287nm (log e=4.53); ~, E~H+N,O. 253inf and 325nm. max

IR:

(KBr) 3320, 2970, 2920, 2860, 1620, and 1320cm'~; (CHCI3) 3340, 2960, 2920, 2850, 1640, and 1355cm ~.

IH ~ :

(CDCI3)4.69-5.12(8H);6.86(d,J=71-Iz,4H);7.98(24H);8.28(6I-I);8.38(6I-I);and 8.681:(18H). Mass Data: LREIMS" 684, 616, 529, and 412m/e (M +, boviquinone-4); found: C, 76.4; H, 8.50, Cs2HT008 requires C, 76.0; H, 8.5%.

662

16. Boviquinones and Related Metabolites

Reference P. C. Beaumont, and R. L. Edwards; Constituents of'The Higher Fungi. Part XI. Boviquinone-3, (2,5-dihydroxy-3-farnesyl- 1,4-benzoquinone), Diboviquinone-3,4 Methylenediboviquinone-3,3, and Xerocomic Acid from Gomphidius rutilus Fr. and Diboviquinone-4,4 from Boletus (Suillus) bovinus (Linn ex Fr.) Kuntze; J. Chem. Soc. (C), pp. 2582-2585(1971).

16. Boviquinones and Related Metabolites

663

Common/Systematic Name Amitenone; Methylenebis(2,5-dihydroxy-4-all-trans-gerangeranyl-3,6-benzoquinone); Methylenediboviquinone-4,4 Molecular Formula/Molecular Weight C53H7208; ] ~ W = 836.52272

Me ,

0

0

H(CH2CH=CHCH2)4

H

H01 ~

H

~

0

Me '

(CH2CH=CCH2)4H

~I~

"OH

0

General Characteristics Yellow orange crystals; mp., 187-188oC. Fungal Source Amitake mushroom and Suillus bovinus. Isolation/Purification Extracted with acetone; purification not described. Spectral Data UV~ /~EtOH max

288(log c=4.45); ~,maxE~H+NaOH323nm.

IR~

(KBr) 3290(quinone), 1300, 1050, 840, 770, 690; (in CHCI3), 3358(OH), 1646(quinone) deuterium substituted (in CHCI3), 2490(OD). 1H NMR: (CDCI3) 2.42(4H, s; exchangeable by D20; Q-OH), 4.85-5.05(8H); 6.48(2H, s); 6.93(4H, d); 7.85-8.15(24H); 8.30(6H, s); 8.35(6H, s); and 8.43z (18H, s). Mass Data: EIMS: 426.279, 412.258, 343,275, 207, 81, 69(base), 55, 41, and 28re~e; calcd for C53H7208; C, 76.04% H, 8.67%; found C, 75.84%, H, 8.44%. Reference K. Minami, K. Asawa, and M. Sawada; The Structure of Amitenone; Tet. Lett., No. 49, pp. 5067-5070(1968).

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Fusicoccins Fusicoccin; Fusicoccin A Allofusicoccin Monodeacetylfusicoccin; 3'-Deacetylfusicoccin; F/IV Dideacetylfusicoccin; F/VII Isofusicoccin; F/Ill 12-O-Acetyldideacetylfusicoccin 12-O-Acetylfusicoccin 12-O-Acetylisofusicoccin 16-O-Demethyl-19-deoxydideacetyl-3-epifusicoccin; F-V/1 19-Deacetylallofusicoccin; F-V/2 19-Deacetylisofusieoccin; F-VI 19-Deacetylfusicoccin; F-VIII 19-Deoxydideacetylfusicoccin; Fusicoccin J 19-Deoxy-3a-hydroxydideacetylfusicoccin; F-V/3

665

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17.

Fusicoccins

667

Common/Systematic Name Fusicoccin; Fusicoccin A Molecular Formula/Molecular Weight C36H56012; ~ = 680.37718

OH AcO~~rJC

Me I H20--CIMe--C H--C H2

HO' ....t " ~ .- 0 HO - -~-CH2OAc

bH CH2OMe General Characteristics Crystallization from acetone-light petroleum (bp., 40-60 ~C) afforded fusicoccin as colorless prisms; mp., 152-154 ~ containing approximately a 0.5 molar equivalent of acetone. A_Rer24 hr. at 50 ~C/0.2mm, the acetone of solvation was completely removed to give fusicoccin; mp., 151-153~ [a]D3~ +70.5 (C=0.8, in CHCI3). Fusicoccin aglycone; mp., 161-162~ Fungal Source

Fusicoccum amygdali, the fungus responsible for a common wilting disease of peach and almond trees in southern Europe.

Isolation/Purification The original purification of fusicoccin was as follows: the phytotoxic metabolite was exhaustively extracted with butyl acetate and evaporated to dryness; the residue was taken up in CHCI3 and the insoluble fraction discarded; the residue from the CHCI3 extract was taken up in methanol (insoluble fraction discarded); the residue from the methanol extract was washed with hexane to remove lipid impurities and taken up in ethyl ether (insoluble fraction discarded); and the residue from the ether extract was dissolved in about 10 times the volume of chloroform containing 3% acetone and passed through a Florisil column on which the active material was firmly adsorbed. The column was washed with about three column volumes of the same solvent. Fractional elution was effected with chloroform containing 6-10% acetone. The fractionation was followed at first by biological assay and later by thin-layer chromatography with chloroform-isopropanol (92:8), using a sulfuric acid spray.

668

17.

Fusicoccins

Spectral Data

(Nujol) 3400(OH), and 1745cmq (C=O).

IH NMR: 4.24(dd, 1H, J=10, 17Hz); 6.71(s, 3H); 7.89(s, 3H); 7.96(s, 3H); 8.77(6H, s); 8.82(s, 3H); 8.89(d, 3H, J=6.5Hz); and 9.16ppm (d, 3H, J=7.0I-Iz). Mass Data: LREIMS: 680m/e (M+), C36Hs6012molecular wt., 680; minor ion at 722m/e presumed due to thermally induced transacctylation; Found: C, 63.55; H, 8.25, C36H56012 requires C, 63.5; H, 8.3%. References A. Ballio, M. Brufani, C. G. Casinovi, S. Cerrini, W. Fedeli, R. PeUicciari, B. Santurbano, and A. Vaciago; The Structure of Fusicoccin A; Experientia, Vol. 24, pp. 631-635(1968). A. Ballio, E. B. Chain, P. De Leo, B. F. Erlanger, M. Mauri, and A. Tonolo; Fusicoccin: a New Wilting Toxin Produced by Fusicoccum amygdali Del.; Nature, Vol. 203, p. 297 (1964). K. D. Barrow, D. H. R. Barton, E. Cha, C. Conlay, T. C. Smale, R. Thomas, and E. S. Waight; Fusicoccin. Part 1. The Nature of the Substituent Groups; J. Chem. Soc.(C), pp. 1259-1264(1971). K. D. Barrow, D. H. R. Barton, E. Chain, U. F. W. Ohnsorge, and R. Thomas, Fusicoccin. Part II. The Constitution of Fusicoccin, pp. 1265-1274( 1971 ).

17.

Fusicoccins

669

Common/Systematic Name Allofusicoccin Molecular Formula/Molecular Weight C36I-I56012; M W = 6 8 0 . 3 7 7 1 8

OH

Me

-:

I

HO~CH20--CI --CH=CH2 I.~ / Me AcO' ....~ / 0 19 _CH2OAc HO H q~) H

OH CH2OMe General Characteristics Allofusicoccin was obtained as a partially crystalline substance by precipitation with fight petroleum from acetone solution; mp., 83-86~ [a]o 25 + 35 ~ (C=1.26). Fungal Source Culture filtrates ofFusicoccum amygdali. AUofusicoccin formed (during the production phase) when fusicoccin was incubated at room temperature at the same pH as the culture brew. Therefore it might arise nonenzymatically from fusicoccin during the course of the fermentation. Isolation/Purification Allofusicoccin was obtained by chromatographic fractionation of the residue left in ethyl acetate after crystallization of fusicoccin. In particular, repeated fractionations on silica gel columns (Kieselgel S-H~ Machery and Nagel) yielded chromatographically pure allofusicoccin, which was eventually obtained as a partially crystalline substance by precipitation with light petroleum (bp., 30-50~ from an acetone solution. Biological Activity Phytotoxic. Spectral Data IR:

The IR spectrum in the region 850-3700cm ~ was superimposable upon that of fusicoccin, whereas it was different in the range 500-850cm q.

670

17.

lH

Fusicoccins

NMR: (CDCI3) The NMR spectrum clearly indicated the same features observed in fusicoccin and isofusicoccin, namely a vinyl on a quaternary carbon, an olefinic proton on a trisubstituted double bond, 1 O-Me, 20-Ac, 2 secondary and 3 tertiary C-Me groups. Unambiguous assignment of the acetylated position for each isomer was attained through NMR- and NMDR-spectra. Allofusicoccin dissolved in acetone-d6 showed a dd centered at 4.6ppm (1 H) with splittings (3.5 and 10Hz) consistent with l'(eq)2'(ax) and 2' (ax)- 3'(ax) couplings, as expected for an tt-glucopyranoside esterified at C-2'. The dd collapsed to a d centered at 4.65ppm (J=10Hz) on irradiation of the d at 4.99ppm (J=3.6Hz, anomeric proton) and to a d centered at 4.70ppm (J=3.5Hz) on irradiation at 3.96ppm (which therefore must correspond to the chemical shitt of CH-3'). Thus, allofusicoccin has a structure differing from fusicoccin by the location of one acetoxy group on C-2' instead of C-3'.

Mass Spectrum: The mass spectrum was also very similar to that fusicoccin, showing the molecular ion at 680role and characteristic ions at 408(aglycone), 205(monoacetylglucosyl), 69(C5H9+), and 43m/e (CH3CO+). Reference A. Ballio, C.G. Casinovi, M. Framondino, G. Grandolini, F. Menichini, G. Randazzo, and C. Rossi; The Structures of Isofusicoccin and Allofusicoccin; Experientia, Vol. 15, pp. 126-127(1972).

17. Fusicoccins

671

Common/Systematic Name Monodeacetylfusicoccin; F/IV Molecular Formula/Molecular Weight C34H54Oll; M W --- 6 3 8 . 3 6 6 6 1

OH HO~CH20--C--C I-

Ho' .... HO

/ :

-

Me I

H=CH2

Me

- CH2OAc

-7

bH CH2OMe General Characteristics [a]D 25 +1 8.5 (c=0.12, in EtOH). Fungal Source Produced in submerged cultures of the phytopathogenic fungus Fusicoccum amygdali. Also, formed when dilute solutions of fusicoccin are incubated at room temperature in buffer having the same pH (about 7.0) as the culture brew during the production phase; therefore, probably arises nonenzymatically from fusicoccin during the course of the fermentation. Isolation/Purification The brown oily residue leg in ethyl acetate after crystallization of fusicoccin was dissolved in chloroform and fractionated on a Florisil column by extended r with chloroform. Each fraction was tested by thin-layer chromatography (silica gel G, 8% 2-propanol in chloroform; sulfuric acid spray). After fusicoccin (F/I), which emerged first, a very small amount of F/II appeared, followed by isofusicoccin (F/III) and monodeacetylfusicoccin (F/IV), and finally by a mixture ofF/V, F/VI and dideacetylfusicoccin (F/VII), separated from isofusicoccin and dideacetylfusicoccin. Appropriate fractions were pooled; those containing mixtures of isofusicoccin and monodeacetylfusicoccin were further fractionated by chromatography under the same conditions described above, whereas dideacetylfusicoccin was obtained pure after repeated chromatography on silica gel (Machery and Nagel S-H~ 50% benzene in acetone). By these procedures monodeacetylfusicoccin was obtained chromatographically pure.

672

17.

Fusicoccins

Biological Activity While isofusicoccin is nearly as phytotoxic as fusicoccin in the assay with tomato plants, monodeacetylfusicoccin and dideacetylfusicoccin are respectively 12 and 100 times less active. Spectral Data UV: ~,mx <215nm. IR:

The IR spectrum was very similar to that of fusicoccin. ~H NMR: The NMR spectrum indicated the occurrence of the same groups observed in fusicoccin, with the only difference that a single O-Ac group was present. This was corroborated by the observation that monodeacetylfusicoccin, contrary to fusicoccin, was oxidizable with periodate. NMR spectra also yielded evidence that the O-Ac group cannot be located on either of the secondary alcoholic functions of the aglycone moiety. Mass Spectrum: That a single O-Ac was present was confirmed by the mass spectrum which showed the molecular ion peak at 638m/e. Furthermore, the aglycone peak at 408m/e was still present, thus indicating that the missing acetyl group was on the glucosyl moiety. Reference A. Ballio, C. G. Casinovi, G. Randazzo, and C. Rossi; Characterization of By-products of Fusicoccin in Culture Filtrates ofFusicoccum amygdali Del.; Experientia, Vol. 26, pp. 349-351(1970).

17.

Fusicoccins

673

Common/Systematic Name Dideacetylfusicoccin; F/VII Molecular Formula/Molecular Weight Ca2Hs201o; MW 596.35605 =

OH Me --I HO~y~~,CH20--?--C I/ Me

H=C H2

HO'....I ~ . . 0 ,9 HO = CH2OH ,

L---

bn CH2OMe General Characteristics [a]D 25 +9 (c=0.90, in EtOH). Fungal Source Produced in submerged cultures of the phytopathogenic fungus Fusicoccum amygdali. Also formed when dilute solutions of fusicoccin were incubated at room temperature in buffer having the same pH (about 7.0) as the culture brew during the production phase, therefore probably arises nonenzymatically from fusicoccin in the course of the fermentation. Isolation/Purification The brown oily residue left in ethyl acetate after crystallization of fusicoccin was dissolved in chloroform and fractionated on a Florisil column by extended washing with chloroform. Each fraction was tested by thin layer chromatography (silica gel G, 8% 2-propanol in chloroform, sulfuric acid spray). After fusicoccin (F/I), which emerged first, a very small amount of F/II appeared, followed by isofusicoccin (F/III) and monodeacetylfusicoccin (F/IV), and finally by a mixture ofF/V, F/VI, and dideacetylfusicoccin (F/~I), separated from isofusicoccin and dideacetylfusicoccin. Appropriate fractions were pooled; those containing mixtures of isofusicoccin and monodeacetylfusicoccin were further fractionated by chromatography under the same conditions described. Dideacetylfusicoccin was obtained pure after repeated chromatography on silica gel (Maehery and Nagel S-HIL 50% benzene in acetone). By these procedures dideacetylfusicoccin was obtained chromatographically pure.

674

17.

Fusicoccins

Biological Activity While isofusicoccin was nearly as phytotoxic as fusicoccin in the assay with tomato plants, monodeacetylfusicoccin and dideacetylfusicoccin were respectively 12 and 100 times less active. Spectral Data UV: ~,,~x <215nm. Ig:

The IR spectrum showed the absence of O-Ac groups. IH NMR: The NMR spectrum indicated similar features observed in the spectrum of fusicoccin and confirmed the absence of O-Ac groups. Mass Spectrum: The mass spectrum showed the molecular ion peak at 596role and strong signals at 366role (deacetylaglycone) and 69role (C5H9+). Reference A. Ballio, C. G. Casinovi, G. Randazzo, and C. Rossi; Characterization of By-products of Fusicoccin in Culture Filtrates of Fusicoccum amygdali Del., Experientia, Vol. 26, pp. 349-351(1970).

17.

Fusicoccins

675

Common/Systematic Name Isofusicoccin; F/III Molecular Formula/Molecular Weight C36H56012; M W = 680.37718

OAc .--HO~~rjCH20--CI

.~

u

-

.,."

,O

Me I - - C H - - C H2

- CH2OAc

--, ""

I-I

CH2OMe

General Characteristics [aiD 25 +11 (c=0.15, in EtOH). Fungal Source Isofusicoccin was produced in submerged cultures of the phytopathogenic fungus Fusicoccum amygdali. It also formed when dilute solutions of fusicoccin were incubated at room temperature in buffer having the same pH (about 7.0) as the culture brew during the production phase; therefore, it probably arises nonenzymatically from fusicoccin during the course of the fermentation. Isolation/Purification The brown oily residue lef~ in ethyl acetate after crystallization of fusicoccin was dissolved in chloroform and fractionated on a Florisil column by extended washing with chloroform. Each fraction was tested by thin-layer chromatography (silica gel G, 8% 2-propanol in chloroform, sulfuric acid spray). After fusicoccin (F/I), which emerged first, a very small amount of F/II appeared, followed by isofusicoccin (F/Ill) and monodeacetylfusicocein (F/IV), and finally by a mixture ofF/V, F/VI, and dideacetylfusicoccin (F/VII), separated from isofusicoccin and dideacetylfusicoccin. Appropriate fractions were pooled; those containing mixtures of isofusicoccin and monodeacetylfusicoccin were further fractionated by chromatography under the same conditions described above. Dideacetylfusicoccin was obtained pure after repeated chromatography on silica gel (Machery and Nagel S-H~ 50% benzene in acetone). By these procedures isofusicoccin was obtained chromatographically pure.

676

17.

Fusicoccins

Biological Activity Isofusicoccin was a highly active phytotoxic metabolite produced in submerged cultures of the phytopathogenic fungus Fusicoccum amygdali. Isofusicoccin was nearly as phytotoxic as fusicoccin in the assay with tomato plants. Spectral Data UV:

~.m~x <215nm. IR;

The IR spectrum in the region 850-3700cm j was superimposable upon that of fusicoccin, whereas it was quite different in the region 720-850cm "~. ~H NMR: The NMR spectrum clearly indicated the same features observed in fusicoccin, namely a vinyl on a quaternary carbon, an olefinic proton on a trisubstituted double bond, an O-Me, two O-Ac, 2 secondary and 3 tertiary C-Me groups. Mass Spectrum: The mass spectrum was also very similar to that of fusicoccin, showing the molecular ion peak at 680m/e and strong signals at 408(aglycone), 205(monoacetylglucosyl), 69(C5H9+), and 43m/e (CH3CO+). Reference A. Ballio, C. G. Casinovi, G. Randazzo, and C. Rossi; Characterization of By-products of Fusicoccin in Culture Filtrates ofFusicoccum amygdali Del.; Experientia, Vol. 26, pp. 349-351(1970).

17. Fusicoccins

677

Common/Systematic Name 12-O-Acetyldideacetylfusicoccin; F-V/4 Molecular Formula/Molecular Weight C341-154011; M W = 6 3 8 . 3 6 6 6 1

OH Me : I H O ~ ~ r l C H 2 0 - - C I --CH:CH2 HO'....I~/_. 0 19 HO

.,,O

Me

-.___CH; O H

CH2OMe General Characteristics Crystals from acetone; mp., 186-188~

[a]D25 +11.5 (C=0.49, in CHCI3).

Fungal Source

Fusicoccum amygdali.

Isolation/Purification Purified by extensive chromatographic fractionations on silica gel columns (Kieselgel SMacherey and Nagel) of the ethyl acetate mother liquor from the crystallization of fusicoccin. Biological Activity Phytotoxic. Spectral Data 1H NMR: (acetone-a~) Spectrum showed the signal expected for the CH-OAc at 4.91 ppm as a m partially overlapping the AB part of the ABX system due to CH2-CH- and clearly affected on irradiation at 2.33ppm. This value corresponded to the center of the AB system (2 out of 8 lines were covered by the signals of acetone) of an ABX system which can be attributed to C(CH3)2=C(CH3)-CH2-CH(OAc)-C(CH3)3. This was further supported in the spectrum of the dihydro derivative; (pyridine-ds) which showed separate signals for the anomeric proton (5.44pprn, d, J=3.5Hz, collapsed to an s upon

678

17.

Fusicoccins

irradiation at 4.05ppm) and for the acetylated function (IH, 5.27ppm, dd, collapsed to a d, J=5.0Hz upon irradiation at 2.88Hz; the latter two frequencies each corresponded to the center of a four line system, which gave rise to a d, J= 16.0Hz, upon irradiation at 5.27ppm). Mass Spectrum: 638(NV), 408(acetate on aglycone), 366(aglycone), and 205role (monoacetylglucosyl). Reference A. Ballio, C. G. Casinovi, M. Framondino, G. Grandolini, G. Randazzo, and C. Rossi; The Structures of Three Isomers of Monodeacetylfusicoccin; Experientia, Vol. 28, pp. 11501151(1972).

17.

Fusicoccins

679

Common/Systematic Name 12-O-Acetylfusicoccin Molecular Formula/Molecular Weight C38HssO13; MW = 722.38774

OH AcOuSTiC

Me I

H20--CI --C H--C H2 Me

HO' ....i ~ / . . 0 ,9 -- --_ CH2OAc HO .-'O- " - - ~ H

bAc

CH2OMe General Characteristics 12-O-Acetylfusicoccin crystallized from ethyl ether-light petroleum (40-70~ 83 ~ and [tt]D2s +30.0 ~ (C=0.9, in CHCI3).

mp., 82-

Fungal Source

Fusicoccum amygdafi.

Isolation/Purification Isolated by repeated column and thin-layer chromatography on silica gel of fractions obtained by chromatographic fractionation of the ethyl acetate mother liquor after crystallization of fusicoccin. It was crystallized from ethyl ether-light petroleum. Biological Activity Phytotoxic. Spectral Data IH NMR: NMR spectra (CDCIs) demonstrated besides other features characteristic of fusicoccin and related compounds, the presence of 30-Ac groups. Both 12-O-acetylfusicoccin and 12-O-acetylisofusicoccin contained O-Ac groups on C-12 and C-19 of the aglycone moiety; in the former the third O-Ac was located on C-2' while in the latter it was located on C-4' on the sugar moieties. Since 12-O-acetylfusicoecin was not oxidized by periodate it was located on C-2' in contrast to 12-O-acetylisofusicoccin.

680

17.

Fusicoccins

Mass Spectrum: Mass spectra showed 12-O-acetylfusicoccin to be isomeric with 12-O-acetylisofusicoccin with a molecular ion at. 722(M +) for each, resulting in a molecular formula of C3d-IssO13 for each metabolite; each with daughter ions at 405 and 205role which corresponded to the 20-Ac groups in the aglycone and 1 in the sugar moiety. Reference A. Ballio, C. G. Casinovi, G. Grandolini, G. Randazzo, C. Rossi, and M. Sorrentino; 12-O-Acetylfusicoccin and 12-O-Acetylisofusicoccin, Two New Minor Metabolites of Fusicoccum amygdali Del.; Experientia, Vol. 30, pp. 1108-1109(1974).

17. Fusicoccins

681

Common/Systematic Name 12-O-Acetylisofusicoccin Molecular Formula/Molecular Weight C3sI-IssOl3; MW = 722.38774

OAc HO~y/~rjC

Me

I H20--?--C H=C H2 Me

HO'....I~.. 0 ,9 - = CH2OAc HO ,,,O -"--~H

bAc CH2OMe General Characteristics ]2-O-Acetylisofusicoccin crystallized from ethyl ether-light petroleum (40-70~ 59~ and

[a]D 25 + 3 5 . 1 ~

mp.,

(c=1.4, in CHCI3).

Fungal Source

Fusicoccum amygdali.

Isolation/Purification Isolated by repeated column and thin-layer chromatography on silica gel of fractions obtained by chromatographic fractionation of the ethyl acetate mother liquor at~er crystallization of fusicoccin. It was crystallized from ethyl ether-light petroleum. Biological Activity Phytotoxic. Spectral Data IH NMR: NMR spectra (CDCI3) demonstrated, besides other features characteristic of fusicoccin and related compounds, the presence of 30-Ac groups. Both 12-O-acetylfusicoccin and 12-O-acetylisofusicoccin contained O-Ac groups on C-12 and C-19 of the aglycone moiety; they differed in the position of the O-Ac on the sugar moiety. NMR data indicated that the third O-Ac group in 12-O-acetylisofusicoccin was located on C4'. Irradiation around 3.90ppm (a value compatible with CH-3' and CH-5') resulted in a triplet centered around 4.72ppm collapsing to a broad singlet; multiplicity and splitting are compatible with values found for CH(4')OAc in isofusicoccin.

682

17.

Fusicoccins

Mass Spectrum: Mass spectra showed it to be isomeric with 12-O-acetylfusicoccin with a molecular ion at 722(M +) for a molecular formula of C3sH~sO~3; with daughter ions at 405 and 205role which corresponded to the 20-Ac groups in the aglycone and 1 in the sugar moiety. Reference A. Ballio, C. G. Casinovi, G. Grandolini, G. Randazzo, C. Rossi, and M. Sorrentino; 12-O-Acetylfusicoccin and 12-O-Acetylisofusicoccin, Two New Minor Metabolites of Fusicoccum amygdali Dei.; Experientia, Vol. 30, pp. 1108-1109(1974).

17.

Fusicoccins

683

Common/Systematic Name 16-O-Demethyl- 19-deoxydideacetyl-3-epifusicoccin; F-V/1 Molecular Formula/Molecular Weight C31H1509; MW = 531.07161

OH Me I HO~CH20--C--C

H--CH2

Me

6

HO

---H

H

CH2OH General Characteristics

16-O-Demethyl-19-deoxydideacetyl-3-epifusicoccinwas obtained as white crystals from acetone; mp., 129-130~ [a]D2s -10.2 ~ (C=1.05, in EtOH). Aglycone was crystallized from ethyl acetate as white crystals; mp., 194-197~ [a]D25 - 94 ~ (C=I.0, in EtOH).

Fungal Source The phytopathogenic fungus Fusicoccum amygdali. Isolation/Purification Partially purified fraction further purified by chromatography on silica gel (benzene followed by benzene-acetone, 8:2, v/v). It was finally purified by crystallization as white crystals from acetone. Aglycone was isolated after silica gel chromatography (benzene with increasing amounts of methanol) as white crystals from ethyl acetate. Biological Activity Phytotoxic. Spectral Data UV: ~, E~on <22Ohm. max

Mass Spectrum: 566(M+), 336(aglycone, C20H3204), and 69m/e (C5H9). Aglycone, 336role (h/f).

684

17.

Fusicoccins

Reference A. Ballio, C. G. Casinovi, G. Grandolini, M. Pomponi, G. Randazzo, and C. Rossi; XIX/16-O-Demethyl- 19-deoxydideacetyl-3-epifusicoccin; Gazzetta Chimica Italiana, Vol. 105, pp. 647-650(1975).

17. Fusicoccins

685

Common/Systematic Name 19-Deacetylallofusicoccin; F-V/2 Molecular Formula/Molecular Weight C34H54011; M W -- 638.36661

OH Me = I HO~CH20--?--CH=CH2 Me

AcO' ....L , , y ~ 0 19 - - --_ CH2OH HO .--'O -'--- H

CH2OMe

General Characteristics Crystals; mp., 87-90~ [a]D25 +15.0 (C=0.65, in CHC13). Fungal Source Fusicoccum amygdali. Isolation/Purification Purified by extensive chromatographic fractionations on silica gel columns (Kieselgel SH1L Machery and Nagel) of the ethyl acetate mother liquor from the crystallization of fusicoccin. Biological Activity Phytotoxic. Spectral Data IH N]k,[R:

(acetone-d~) Spectrum showed dd(IH) centered at 4.70ppm with (eq)(ax)and (ax)(ax) couplings (J----4and 9Hz), which collapsed to a d on irradiationat 4.91ppm (II-I,d, J=4Hz; anomeric proton, partiallyoverlappingthe A B part of the A B X system due to CH2=CH-). The dd can be attributedto the CH(2') OAc of 19-deacetylfusicoccin.

Mass Spectrum: LREIMS: 638(NF), 366(aglycone),and 205m/e (monoacetylglucosyl).

686

17.

Fusicoccins

Reference A. Ballio, C. G. Casinovi, M. Framondino, G. Grandolini, G. Randazzo, and C. Rossi; The Structures of Three Isomers ofMonodeacetylfusicoccin; Experientia, Vol. 28, pp. 11501151(1972).

17.

Fusicoccins

687

Common/Systematic Name

19-Deacetylisofusicoccin; F-VI

Molecular Formula/Molecular Weight C34Hs4011; MW = 638.36661

OAc Me --_ I H O ~ ~ , . , ~ C H20--CI--C H=C H2 HO'....~ 0 HO 6

Me 19

- _CH2OH

bH CH2OMe General Characteristics Crystals from ethyl acetate; mp., 211-213~

[a]D25 +8.0 (C=0.50, in CHC13).

Fungal Source

Fusicoccum amygdafi.

Isolation/Purification Purified by extensive chromatographic fractionations on silica gel columns (Kieselgel SH ~ Machery and Nagel) of the ethyl acetate mother liquor from the crystallization of fusicoccin. Biological Activity Phytotoxic. Spectral Data 1H NMR: (CDCI3) Spectrum of dihydro derivative showed the anomeric proton, partially overlapping the AB part of the ABX proton as a d (d=3.5Hz) at 5.12ppm, coupled to a proton centered at 3.69ppm (overlapped by other resonances), a frequency which does not affect the t (1I-I, J=9.0Hz) resonating at 4.86ppm; the latter collapses to a broad s on irradiation around 3.86pprn, a value compatible with CH-3' and CH-5'.

688

17.

Fusicoccins

Mass Spectrum: 638(M+), 366 (aglycone), and 205m/e(monoacetylglucosyl). Reference A. Ballio, C. G. Casinovi, M. Framondino, G. Grandolini, G. Randazzo, and C. Rossi; The Structures of Three Isomers ofMonodeacetylfusicoccin; Experientia, Vol. 28, pp. 11501151(1972).

17.

Fusicoccins

689

Common/Systematic Name ] 9-Deacetylfusicoccin; F-VIII Molecular Formula/Molecular Weight C34I-I54011; M W = 6 3 8 . 3 6 6 6 ]

OH = AcO~~y~C

Me I H20--?--C H=C H2 Me

HO'....

HO

0

,,

~. - CH2OH .,,,O - - H

b. CH2OMe General Characteristics Crystals from cyclohexane-ethyl acetate; mp., ] 12-1 ]4~ CHCI3); not oxidizable with periodate.

[a]D25 +36.6 (C----0.42,in

Fungal Source Fusicoccum amygdafi; originally reported as a product of alkaline isomerization of either 19-deacetylisofusicoccin or dideacetylfusicoccin. Isolation/Purification Purified by extensive chromatographic ffactionations on silica gel columns (Kieselgel SH ~ Machery and Nagel) of the ethyl acetate mother liquor from the crystallization of fusicoccin. Biological Activity Phytotoxic. Spectral Data 1H NMR: NMR and NMDR spectra yielded the same type information about the position of the acetoxy group as with allofusicoccin and isofusicoccin. Mass Spectrum: 638(M+), 366(deacetylaglycone), 205(monoacetylglucosyl), 69(CsHf), and 43m/e (CHACO+).

690

17.

Fusicoccins

References A. Ballio, C. G. Casinovi, M. Framondino, G. Grandolini, F. Menichini, G. Randazzo, and C. Rossi; The Structures of Isofusicoccin and Allofusicoccin; Experientia, Vol. 28, pp. 126-127(1972). A. Ballio, C. G. Casinovi, G. Grandolini, G. Randazzo, C. Rossi, and M. Sorrentino; 12O-Acetylfusicoccin and 12-O-Acetylisofusicoccin, Two New Minor Metabolites of Fusicoccum amygdali Del.; Experientia, Vol. 30, pp. 1108-1109(1974).

17. Fusicoccins

691

Common/Systematic Name 19-Deoxydideacetylfusicoccin; Fusicoccin J Molecular Formula/Molecular Weight C32H5209; M W -" 5 8 0 . 3 6 1 1 3

OH HO~/~rIC

Me I H20--CI --C H=C H2 Me

H o ' .... HO

: 9 "

:

z

~""

H

bH CH2OMe General Characteristics Crystals from ethyl acetate-light petroleum ether (30-50~ mp., 91~ [et]Dz5 +21 (C=0.3, in CHCI3). Aglycone: C21H3404(M+ 350); amorphous solid; [a]Dz5 +4 (C=I.0, in CHCI3). Fungal Source Fusicoccum amygdali. Isolation/Purification

19-Deoxydideacetylfusicoccin was purified by silica gel chromatography followed by treatment with 0.1N NaOH for thirty minutes, extraction with 1-butanol and chromatography on a silica gel column. 19-Deoxydideacetylfusicoccin was eluted from the column with chloroform-methanol (8:2, v/v) and crystallized from ethyl acetate-light petroleum ether. Aglycone prepared by treatment of periodate reaction product with alkali; isolated by silica gel column chromatography. Aglycone eluted with chloroform-2propanol, 95:5 (v/v) as an amorphous solid.

Biological Activity Phytotoxic. Spectral Data UV: ,;L~'~ max

<220nm.

692

17.

Fusicoccins

IR~

(CHCI3) 3500(OH), 1630(olefinic), and 920cm"~(vinyl). ~H NMR: (CDCl3-pyridine-ds) Spectrum very similar to dideacetylfusicoccin but with signals for 3 secondary C-methyl groups; 2 being part of an isopropyl group whose CH resonated at 3.22ppm. Mass Spectrum: LREIMS: 580(M+), 512(M+- 68), 350(aglycone), and 69role (C5H9+). Reference A. Ballio, C. G. Casinovi, V. D'Alessio, G. Grandolini, G. Randazzo, and C. Rossi; 19Deoxydideacetylfusicoccin, a Minor Metabolite of Fusicoccum amygdali Del.; Experientia, Vol. 30, pp. 844-845(1974).

17.

Fusicoccins

693

Common/Systematic Name 19-Deoxy-3 a-hydroxydideacetylfusicoccin; F-W3 Molecular Formula/Molecular Weight C32H5201o, MW

=

596.35605

OH

Me

-

I

HO~L,y~C H20--?--CH=C1-12 Me

HO' ....L...j.. 0 HO

."

:

- --" -'- H

OH ""OH

CH2OMe General Characteristics Obtained as white crystals; mp., 137-138~ (from AcOEt); [tt]o25 +22.0 (c=0.26, in CHCI3). Fungal Source Fusicoccum amygdali. Isolation/Purification 19-Deoxy-3tt-dideacetylfusicoccin was purified by repetitive silica gel column chromatography. Acid hydrolysis yielded approximately one mole of D-glucose (glucose oxidase).

Biological Activity Phytotoxic. Spectral Data UV:

~,E~H max

<220nm.

IR:

(CHCI3) 3500(OH), 1630(olefinic), and 920cm"l (vinyl).

694

17.

Fusicoccins

IH ~ : (CsDsN) Spectrum was similar to that of 19-deoxy-3tt-dideacetylfusicoccin with the following differences: proton H-C (1) appeared as a doublet (d=2Hz) instead of a triplet; in the region where the protons H-C (3) and H-C (6) resonate, only one proton rather than two was present; the C(16)H2 group gave signals typical of an AB system instead of an ABX system. 13C NMR: (CsDsN) Spectrum very similar to that of deoxydideacetylfusicoccin except for C(2), C(3), and C(4), which were shifted to higher frequencies (4.2, 33.8, and 4.4ppm, respectively). Mass Spectrum: Whereas in the electron impact mass spectrum the highest detectable peak was at m/e=578 (C32H500, by high-resolution measurements), the spectrum obtained by the field ion-desorption technique contained a peak at m/e=596 thus suggesting that the molecular formula of 19-deoxy-3a-hydroxydideacetylfusicoccin is C32H52010. Furthermore, the electron impact mass spectrum showed losses of CH40 (confirmed by metastable peaks) and C5H8, as well as the signal expected for the aglycone (C21H3405, m/e=366); the base peak was at m/e=69 (C5H9+). Reference A. Ballio, C. G. Casinovi, G. Grandolini, M. Marta, and G. Randazzo; XXIX/ 19-Deoxy-3tt-hydroxydideacetylfusicoccin; Gazzetta Chimica Italiana, Vol. 105, pp. 1325-1328(1975).

Altenuene and Related Metabolites (+)-Altenuene (+)-Dehydroaltenusin Botrallin Alternariol Monomethyl Ether Alternariol Altenuisol (Altertenuol) Altenusin Dehydroaltenusin Altenuic Acid I (+)-Altenuic Acid II Altenuic Acid III

695

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18.

Altenuene and Related Metabolites

697

Common/Systematic Name (• 2',3',4',5'-Tetrahydro-3,3',4'-trihydroxy-5-methoxy-6'-methyldibenzo[a]pyrone Molecular Formula/Molecular Weight C15H1606; ~

= 292.09469

M,vOO. 0

:

HC)

\

OMe

General Characteristics The metabolite crystallized from acetone-hexane mixtures as colorless needles that melted sharply at 190-191 ~ (corr.); produced an intense purple color with addition of ethanolie FeCI3. Altenuene dissolved readily in acetone, 1M NaOH, methanol, pyridine, and tetrahydrofuran. It was slightly soluble in diethyl ether and chloroform and insoluble in benzene, hexane, 1M HCI, saturated aqueous NaI-ICO3, and water. Fungal Source The isolate ofAlternaria tenuis (NRRL L5872; 5873) was obtained from naturally infected tobacco in 1965. Isolation/Purification The rice culture was extracted with acetone-water (7:3, v/v) and allowed to stand for 15-20 min. The slurry was filtered, evaporated to dryness, and repeatedly extracted with hot tetrahydrofuran. The altenuene was purified from the crude tetrahydrofiaran extract by column chromatography on silica gel G. Biological Activity The compound showed cytotoxic activity against five bacteria tested by the paper disc agar plate method: Bacillus mycoides (ATTC 6462), B. subtilis (ATTC 6633), Neisseria gonorrhoeae (ATTC 23050), Sarcmia lutea (ATTC 9341), and Staphylococcus aureus (ATTC 6538P). Spectral Data UV:

~,9mS~E~" 319(6,600), 278(10,000), and 240nm (30,000). IR:

(KBr) 1635cm q.

698

18.

Altenuene and Related Metabolites

IH N]VIR: C-4 H, 6.66 or 6.42, ./(4,6)=2.5; C-6 H, 6.42 or 6.66, ,/(6,4)=2.5; C-2~-I, 6.27, J(2',3')=2.5; C-3 H, 4.10, J(3',2')=2.5;(3,4)=5.5; C-4'H, 3.86, J(4',3')=5.5, J(4',5'a)=8.5, J(4',5b')=4.0; C-Sa, 1.97, J(Sa',5b')=l 5, `/(5a',4)'=8.5; C-Sb, 2.40, J(5b',5a')=15, J(5b',4')=4.0; C-3 OH, 11.39; C-3' OH, 2.98; C-4' OH, 2.98; C-50CH3, 3.90; and C-6'CH3, 1.52ppm. 13CNMR:

C-4, 100.4(d);C-6, I02.4(d);C-7, 165.7(s);C-8, 55.5(q);C-2', 81.0(s);C-3', 40.2(t); C-4', 69.6(d); C-5', 72.4(d);C-6', 132.5(d);C-7', 27.8(d); 130.9(s);139.1(s); 143.6(s); 165.7(s);and 168.7ppm (s). Mass Spectrum: High resolution mass spectrometry indicated a parent ion with mol. wt. 292.0930, with a molecular formula of C16H1606. TLC Data Silica gel G; (A) benzene-ethanol, 95:5, v/v; (B) chloroform-methanol, 95:5, v/v; Rf (A) 0.14, (B) 0.13; detected as blue fluorescent spot under UV light. GLC Data: Solid support: Gas Chrom Q; liquid phase OV-17 (3%); column temp, 100~176 at 8~ rise; injector temp, 3000C; detector temp, 300~ retention time, about 24 min. References R. J. Cole and R. H. Cox; Hand.book of Toxic Fungal Metabolites; Academic Press, New York, pp. 628-634(1981). R. W. Pero, R. G. Owens, S. W. Dale, and D. Harvan; Isolation and Identification of a New Toxin, Altenuene, from the Fungus Alternaria tenuis; Biochimica et Biophysica Acta, 230, 170-179(1971).

18.

Altenuene and Related Metabolites

699

Common/Systematic Name (_+)-Dehydroaltenusin Molecular Formula/Molecular Weight C15H1206; MW' = 288.06339

General Characteristics Dehydroaltenusin crystallized from ethanol as yellow needles; mp., 189-190~ (deeomp.); also crystallized from acetone as plates; mp., 190-193 ~ dehydroaltenusin is only slightly soluble in water. Aqueous NaHCOa readily extracted the compound from ethyl acetate solution but dissolves the solid only slowly; the bicarbonate solution is colored yellow. In aqueous NaOH the color is yellow-green, soon changing to yellow. An ethanolic solution gives an intense brown color (with FeCI3), identical with that produced by the action of excess FeCIa on altenusin; the color is essentially unchanged by the addition of water. An ethanolic solution yields a red precipitate with Brady's reagent. Fungal Source

Alternaria tenuis and other Alternaria spp.

Isolation/Purification Dehydroaltenusin was initially isolated by column chromatography of the ether extracts by a gradient elution technique. Subsequently, it was extracted with chloroform, redissolved in acetone and left standing for several days after which dehydroaltenusin crystallized out as plates (mp., 190-193 ~C). Spectral Data UV; ~..... 217(1og e=4.53), 249(4.10), and 300nm (3.88). IR;

(KCI Disc) 3380, 1655, 1645sh, 1625, 1580, 1275, 1230, 1210, 1165, 1080, 1032, 975, 865, 800, and 750cm1. IH NMR: (acetone-d6) Three-proton singlets at 1.76 and 4.00ppm; one-proton singlets at 6.23 and 6.85ppm; one-proton doublets (J=2.5Hz) at 6.68 and 7.05; and two exchangeable protons at 8.00(broad) and 11.32ppm (sharp).

700

18.

Altenuene and Related Metabolites

Mass Data: LREIMS: 288role (M+); (Found: C, 62.4, 62.3; H, 4.4, 4.5; OMe, 11.1, 11.1. C15H1206 requires C, 62.5; H, 4.2; OMe, 10.8 %). References R. G. Coombe, J. J. Jacobs, and T. R. Watson; Metabolites of Some Alternaria Species. The Structures of Altenusin and Dehydroaltenusin; Aust. J. Chem., Vol. 23, pp. 23432351(1970) D. Rogers and D. J. Williams; The crystal structure of (+)-dehydroaltenusin; Chem. Commun., pp. 390-393(1971). T. Rosett, R. H. Sankhala, C. E. Stickings, M. E. U. Taylor, and R. Thomas; Studies in the Biochemistry of Micro-organisms; Biochem. J., Voi 67, pp. 390-399(1957).

18.

Altenuene and Related Metabolites

701

Common/Systematic Name Botrallin Molecular Formula/Molecular Weight C16H1407; ~

= 318.07395

MeO

H

J %. General Characteristics Yellow prisms from ethanol; mp., 165-185~ (decomp. ). The compound showed no sharp melting point. At about 165~ decomposition started, at 185~ the crystals were fully transformed into a dark brown tar. Botrallin was unstable in alkaline solutions. In dilute ammonia a red color was observed which rapidly changed through brown into yellow. The compound dissolved in aqueous sodium bicarbonate with a violet-red color. This color was more stable. It took several hours before the color changed to brown. An alcoholic solution ofbotrallin gave a violet-brown color with ferric chloride. Reduction of an ethyl acetate solution by shaking with aqueous sodium dithionite led to a slightly yellow solution which did not give a color with aqueous sodium bicarbonate. Evaporation of this solution exposed to the air yielded a residue which again gave the color reaction with alkali. The supposed quinonoid character of the compound was confirmed by a positive Craven test 2. The reddish solution in alcoholic ammonia turned to an intense green upon addition of a drop of ethyl cyanoacetate. Fungal Source

Botrytis allii, strain Walker, obtained from the Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands

Isolation/Purification Botrallin was extracted from fungal cultures with ethyl acetate. The residue obtained on evaporating the ethyl acetate was extracted with boiling benzene. Evaporation of the benzene yielded a solid residue which was recrystallized as yellow prisms from ethanol. Biological Activity Botrallin in a concentration of 100ppm had no inhibitory effect on the growth of the fungi

Botrytis allii, Penicillium italicum, Cladosporium cucumerinum, and Aspergillus niger and the bacteria Escherichia coli and Bacillus subtilis. Since Botrytis allii is a parasite of onions, it was tested to see if botrallin had a toxic effect on onion scales; however, no effect was observed.

702

18. Altenuene and Related Metabolites

Spectral Data UV:

~

gtOH max

238(1og e=4.36), 273(4.20), and 37Onto (3.95).

IR:

(KBr) 1656, 1629, and 1580cm "1. 1H NMR: The NMR spectrum of botrallin, taken in CDCIs, led to the conclusion that the compound was a benzoquinone with three substituents. One of these substituents was a triply substituted phenyl group. Taking into consideration the formula C16H1407the following assignments were made: quinone Me at 1.77ppm (singlet, 3 H); OMe groups at 3.80 and 3.90ppm (singlets, each 3 H); quinone proton at 6.14ppm (singlet, 1 H); two meta-coupled aromatic protons at 6.54 and 7.34ppm (doublets J=2.4Hz, each 1 H); OH at 7.23ppm (singlet, 1 H, exchangeable with D20); and COOH at 11.36ppm (singlet, 1 H, exchangeable with D20). Mass Data: HR IMS: 318.07321m/e (NV)(calcd, 318.07394); found C 60.4%; H 4.6%; O 34.8%; calcd for C16H1407, C 60.38; H 4.43; O 35.19. References K. Kameda, H. Aoki, M. Namimi, and J. C. Overeem; An Alternative Structure For Botrallin A Metabolite ofBotrytis allii;, Tetrahedron Letter, No. 1, pp. 103-106 (1974). J. C. Overeem and A. Van Dijkman; Botrallin, A Novel Quinone Produced By Botrytis

allii; Recueil, Vol. 87, pp. 940-944(1968).

18. Altenuene and Related Metabolites

703

Common/Systematic Name Altemariol monomethyl ether 3,4'-Dihydroxy-5-methoxy-6'-methyldibenzo[a]pyrone Molecular Formula/Molecular Weight C15H1205; MW = 272.06847

. ?_~_0 /OH \

Me

\

OMe

General Characteristics Crystals from ethanol; mp., 266-267~ (dec.); sublimed without decomposition at 180-200~ in high vacuum; [a]D2~ 0 ~ (C=0.8, in EtOH). Fungal Source Alternaria tenuis (NRRL 5831), A. dauci, and A. cucumerina. Alternariol monomethyl ether and alternariol have also been found in tobacco, grain sorghum, and pecans. Isolation/Purification Alternariol and alternariol monomethyl ether were extracted with methanol-water (80:20, v/v). The crude extract was first separated on columns containing Baker 3405 silica gel. These columns were packed with 3% acetone in chloroform and developed with the same solvent system. An easily visualized component fluorescing blue under long-wavelength UV was recognized during the monitoring of fractions of the first separations of the crude extract. A compound with similar fluorescent characteristics but with a lower Rf was also observed, but appeared to be present in smaller amounts. These two compounds were further purified by a consecutive series of column separations on columns containing E. M. Laboratories silica gel 60 (particle size less than 0.063mm) developed with 1.75% methanol in benzene. The extracts were concentrated and yielded high purity crystals of alternariol and alternariol monomethyl ether, respectively. Biological Activity Alternariol monomethyl ether showed cytotoxic activity to bacteria and mammalian cells. Inhibition to Bacillus mycoides was observed at 500~tg/disc; the IDs0 to HeLa cells was 8-141~g/ml. In mice dosed IP (DMSO cartier) at 400mg/kg, 1 of 10 mice died; mice were often sedated within a few minutes of dosing, and the eyes of the mice were dull; there were occasional stomach spasms and periodic panting.

704

18. Altenuene and Related Metabolites

Spectral Data UV;

3.,~, 335-342(broad), 301,290, 257, and 230nm (e max not reported). IH NMR: H-4, 6.29; H-6, 7.12; H-8, 3.84; H-3', 7.60; H-5', 6.60; and H-8', 2.68ppm. ~3C NMR: C-2, 98.7 s; C-4, 98.5 d; C-6, 103.4 d; C-7, 165.0 s; C-8, 55.4 q; C-3', 101.9 d; C-5', 117.5 d; C-6', 137.8 s; C-8', 25.4 q; 137.9 s; 146.7 s; 152.6 s; 158.2 s; 164.4 s; and 166.0ppm s. Mass Spectrum: LREIMS showed a molecular ion at nominal mass 272re~e;HREIMS showed a molecular ion at 272.0699m/e; calcd for C~5H~205is 272.0684. TLC Data Silica gel G, (A) benzene-ethanol, 95:5, v/v ; (B) chloroform-methanol, 95:5, v/v. Re (A) 0.39; 03) 0.54. Detection: blue fluorescent spot under UV light. GLC Data Solid support: Gas Chrom Q; Liquid phase: OV-17 (3%); Column temp: 100-250~ at 8 ~ min rise; detector temp: 300"C; injector temp: 3000C; retention time: approximately 31 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 615-627(1981). H. W. Schroeder and R. J. Cole; Natural Occurrence of Alternariol in Discolored Pecans; Agile. Food Chem.; Vol. 25, pp. 204-206(1977).

18. Altenuene and Related Metabolites

705

Common/Systematic Name Alternariol 3,4',5-Trihydroxy-6'-methyl-dibenzo[a]pyrone Molecular Formula/Molecular Weight C1~-I1005; MW = 258.05282

,,

/

0-~

\

O

\

/ \

Me

OH

OH

General Characteristics Crystals from ethanol; mp., 350~ (dec.). Sublimed without decomposition at 250~ in a high vacuum. [a]D2~ 0 ~ (C=0.8, in EtOH). Triacetate from ethanol; mp., 167-169~ Fungal Source Alternaria tenuis (NRRL 5255; 5872; 5873), A. dauci, and A. cucumerina. Alternariol and altemariol monomethyl ether have been found in tobacco, grain sorghum, and peemas. Isolation/Purification Altemariol and altemariol monomethyl ether were extracted with methanol-water, 80:20. The crude extract was first separated on columns containing Baker 3405 silica gel. These columns were packed with 3% acetone in chloroform and developed with the same solvent system. An easily visualized component fluorescing blue under long-wavelength UV was recognized during the monitoring of fractions of the first separations of the crude extract. A compound with similar fluorescent characteristics but with a lower Rf was also observed, but appeared to be present in smaller amounts. These two compounds were further purified by a consecutive series of column separations on columns containing E. M. Laboratories silica gel 60 (particle size less than 0.063mm) developed with 1.75% methanol in benzene. The extracts were concentrated and yielded high purity crystals of alternariol monomethyl ether and alternariol, respectively. Biological Activity Altemariol completely suppresses growth of Staphylococcus aureus at concentration of 1:40,000; E. coli at concentration of 1:20,000. The IDs0 to HeLa cells was 61~g/ml. In mice dosed IP (DMSO carrier) at 200mg/kg, 3 of 10 mice died; mice were often sedated within a few minutes of dosing; the eyes of mice recovering were dull; there were occasional stomach spasms and periodic panting.

706

18.

Altenuene and Related Metabolites

Spectral Data WW~

Z ~m~

218, 258(C=38,000), 302, and 330nm.

IR~

(KBr) 3460(OH), 3100(intramolecular hydrogen-bonded OH), 1672(lactone shit~ed downfield due to H-bonding with OH), 1590, 1430(phenyl nucleus), 1365(CH3), 1200(phenolic OH), and 850em "1 (isolated H on aromatic ring). 1H NMR: H-4, 6.30(J=2.0); H-6, 7.14(,/=2.8); H-3', 7.65; H-5', 6.58; and H-8', 2.66ppm. 13C NMR: C-2, 97.6 s; C-4, 100.9 d; C-6, 104.2 d; C-7, 165.0 s; C-3', 101.7 d; C-5', 117.3 d; C-6', 137.8 s; C-8', 25.4 q; 128.6 s; 138.2 s; 152.6 s; 158.0 s; 164.3 s; and 165.0ppm s. Mass Spectrum: LREIMS showed molecular ion at nominal mass 258m/e. TLC Data Silica gel G; A: benzene-ethanol, 95:5 v/v; B: chloroform-methanol, 95:5 v/v; Rf: A: 0.21, B: 0.19. Detection: blue fluorescent spot under UV light. GLC Data Gas Chrom Q; Liquid phase OV-17 (3%); Column temp. 100-250~ at 8 ~ min rise; Injector temp. 300~ Detector temp. 300~ and retention time, about 30 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 615 (1981). H. W. Sehroeder and R. J. Cole; Natural Occurrence of Alternariol in Discolored Pecans; Agrie. Food Chem., Vol. 25, pp. 204-206(1977).

18. AltenueneandRelatedMetabolites

707

Common/Systematic Name Altenuisol; Altertenuol 3,5,5'-Trihydroxy-4'-methoxydibenzo[a]pyrone Molecular Formula/Molecular Weight C14H1006; M W = 274.04774

O

M e O ~ / HO

OH \

OH

General Characteristics Altenuisol crystallized from hot acetic acid as slender needles; mp., 277-282~ It was soluble in acetone, ethanol, and ethyl acetate; only slightly soluble in chloroform and diethyi ether; and insoluble in benzene and hexane. Fungal Source

A lternaria tenuis.

Isolation/Purification Altenuisol was extracted with 70% acetone, chromatographed on silica gel G, eluted with increasing tetrahydrofuran in benzene, and further purified on silica gel G using ethyl acetate as eluant. Biological Activity The IDs0 dose with HeLa cells was 81~g/ml and zones of inhibition were noted with Bacillus mycoides from 5-2001,tg/assay disc. Spectral Data UV;

)LE~m~x" 216, 256, 278nm sh (e=9,000, 11,000, and 3,500, respectively). IR~

(KBr) 3380, 3280, 1653, 1620, 1600, 1560, 1530, 1510, 1440, 1370, 1275, 1200, 1180, 1170, 1160, 1095, 1035, 985, 915, 857, 830, 815, and 780cm"~. ~H NMR: Tetrahydrofuran-ds: singlet 7.38, doublet, 6.92, singlet 6.70, doublet 6.47, and three methoxy protons at 3.89ppm; chemical shitt at 11.62ppm H-bonded OH group.

708

18.

Altenuene and Related Metabolites

Mass Spectrum: 274.0495m/e (M+); calcd for C~4H~oO6,274.0476.

TLC Data Altenuisol had an Rf of 0.33 in 20% tetrahydrofuran in benzene using silica gel G and fluorescence detection under both short and long wave ultraviolet light. Reference R. W. Pero, D. Harvan, and M. C. Blois; Isolation of the Toxin Altenuisol From the Fungus, Alternaria tenuis Auct.; Tet. Lett., pp. 945-948(1973).

18. Altenuene and Related Metabolites

709

Common/Systematic Name Altenusin Molecular Formula/Molecular Weight C15H1406; MW = 290.07904

\

Me

\

OMe

General Characteristics Altenusin crystallized from CHCI3 in colorless prisms. After drying at 100 ~ C under reduced pressure, it melted at 202-203 o C with effervescence to a yellow liquid; undried crystals melted at about 95 ~ C, resolidified, and remelted at the higher temperature. Altenusin can also be crystallized from benzene or water. It was readily soluble in ether, ethanol and methanol; it also dissolved in aqueous NaHCO3. Its ethanolic solution, treated with ethanolic FeCI3, gave a pale-gray color, ttmaing to deep brown with excess. Altenusin does not reduce Fehling's solution in the cold, though the solution turns green; on boiling reduction takes place. Tetramethylaltenusin was a pale-brown gum that recrystallized several times from methanol to gave colorless prisms, mp., 116-117 ~ Fungal Source Alternaria tenuis, A. citri, A. dauci, A. alternata, Corynespora smithii. Isolation/Purification Continuous ethyl ether extraction followed by crystallization from chloroform yielded crystalline material that melted at 201.5-203"~C. Spectral Data UV-

~max 217, 247sh, and 290nm. IR: (KC1 disc) 3350(broad), 1635, 1607, 1520, 1460, 1440, 1365, 1255, 1230, 1205, 1185, 1075, 1040, 980, 875, 860, and 800cm "l. IH NMR: (DMSO-d6) Three-proton singlets at 1.88 and 3.77ppm; one-proton doublet (J=-2.5Hz) at 6.15; one proton singlet at 6.85ppm and two-proton singlet at 6.46ppm; three exchangeable protons at 8.60ppm (very broad) and one exchangeable in region 56ppm.

710

18.

Altenuene and Related Metabolites

Mass Data:

290m/e (M+); found, after drying at 100~ in a high vacuum: C, 62.1; H, 5.2; OMe,

10.4; C-Me, 5.2. C~5H1406required C, 62.1; H, 4.9; OMe, 10.7; C-Me, 6.2 %. References R. G. Coombe, J. J. Jacobs, and T. R. Watson; Metabolites of Some Alternaria Species. The Structures of Altenusin and Dehydroaltenesin; Aust. J. Chem., Vol. 23, pp. 23432351(1970). T. Rosett, R. H. Sankhala, C. E. Stickings, M. E. U. Taylor, and R. Thomas; Studies in the Biochemistry of Micro-organisms; Biochem. J., Vol. 67, pp. 390-399(1957).

18.

Altenuene and Related Metabolites

711

Common/Systematic Name Dehydroaltenusin Molecular Formula/Molecular Weight C15H1206; ~

Me

= 288.06339

0

0

OH

Me

General Characteristics Yellow plate crystals from acetone; mp., 190-193~ (dec.). Fungal Source

Alternaria tenuis, A. dauci, A. cucumerina, and A. alternata.

Spectral Data UV:

~,~"

217(e=33,800), 249(12,600), and 30Ohm (7,500).

IRz

(KBr) 3380, 1655, 1645(sh), 1625, 1580, 1275, 1230, 1210, 1165, 1080, 1032, 975, 856, 800, and 750cm"l. IH NMR: (acetone-d6) H-4, 6.68(J=2.5); H-6, 7.05(J=2.5); H-8, 4.00; H-3', 6.23; H-6', 6.85; H7', 1.76; OH, 8.00; and 11.32ppm. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 634 (1981).

712

18. Altenuene and Related Metabolites

Commo0/Systematic N.ame Altenuic acid I Molecular Formula/Molecular Weight C15H1408, IV[V~ = 322.06687 O

'o o General Characteristics Altenuic acid I crystallized from water or ethanol as colorless needles; mp., 183-184~ with effervescence; if heated further, it reset at about 200~ and remelted at 224-230~ with further decomposition. The compound retained solvent even when dried to constant weight at 100 ~ in high vacuum. To obtain solvent-free material, it was necessary to dry for 8 hr. at 120~ in a high vacuum. It dissolved on heating in methanol, ethanol, dioxane, acetic acid and water, but was not very soluble in the cold. An ethanolic solution gave a pale purple-brown color on addition of ethanolic FeCI3, essentially unchanged on addition of water. Its aqueous solution gave no precipitate with Brady's reagent. Altenuic acid I, II, and III are readily interconverted under basic conditions. Fungal Source A lternaria tenuis.

Isolation/Purification Isolated by adsorption on charcoal, followed by elution with ethanol. The isolated crystalline compound was a colorless acidic substance. Reference T. Rosett, R. H. Sankhala, C. E. Stickings, M. E. U. Taylor, and R. Thomas, Metabolites ofAlternaria tenuis Auct., Culture Filtrate Products, Vol. 67, pp. 390-399(1957).

18. Altenuene and Related Metabolites

713

Comm0n/Systematic Name (+_)-Altenuic acid II Molecular Formula/Molecular Weight C15H1408, M'W" = 322.06887

0

O

0

General Characteristics Altenuic acid II crystallized from aqueous dioxane in small colorless rectangular plates; mp., 245-246~ (decomp.) (uncorr.). It also crystallized from aq. acetic acid. Altenuic acid II was very sparingly soluble in water and the commoner organic solvents. A solution in dioxane/ethanol (1:1, v/v) gave a pale-brown ferric color, essentially unchanged on addition of water. Readily interconvertible with its isomers altenuie I and III under basic conditions. Fungal Source Alternaria tenuis.

Isolation/Purification Isolated by adsorption on charcoal, followed by elution with ethanol, which gave a colorless acidic compound called altenuic acid II. References T. Rosett, R. H. Sankhala, C. E. Stickings M. E. U. Taylor, and R. Thomas; Metabolites ofAlternaria tenuis Auct.; Culture Filtrate Products, Biochem. J., Vol 67, pp. 390-399 (1957). D. J. Williams; The Crystalline Structure of (+)-Altenuic Acid II; Tet. Lett., pp. 639-640 (1973).

714

18.

Altenuene and Related Metabolites

Common/Systematic Name Altenuic acid III Molecular Formula/Molecular Weight C15H1408; MW = 322.06687

0

OH

0 HO2C.~J

OMe

General Characteristics Altenuic acid III crystallized from aq. methanol or acetic acid in colorless prisms. The melting point was variable; crystals from aqueous methanol usually melted around 185~ with effervescence followed by resetting, complete about 195~ a further melt occurred between 215 and 235~ with effervescence. Crystals from acetic acid melted first in the range 198-202~ with effervescence, reset immediately and remelted at about 225~ with effervescence. Altenuic acid III was readily soluble in ethanol and methanol, sparingly soluble in ether, and less soluble in chloroform, benzene or light petroleum. An ethanolic solution gave a pale-wine ferric color, deepening to an intense purple on addition of water. Readily interconvertible with its isomers altenuic I and II under basic conditions. Forms a colorless dimethyl derivative; mp., 143.5-144.5~ Fungal Source A lternaria terruis.

Isolation/Purification Mixtures of altenuic acids I and III were difficult to separate. Separation on a preparative scale was achieved by means of counter-current distribution using ethyl acetate-water and extraction from ethyl acetate containing fractions after acidification. Reference T. Rosett, R. H. Sankhala, C. E. Stickings, M. E. U. Taylor, and R. Thomas; Metabolites ofAlternaria tenuis Auct.; Culture Filtrate Products, Biochem. J., Vol 67, pp. 390-399 (1957)

Viridin and Related Metabolites Viridin Desmethoxyviridin Viridiol Desmethoxyviridiol Wortmannin

11-Desaeetoxywortmannin

715

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19. Viridin and Related Metabolites

717

Common/Systematic Name Viridin 1 [3-Hydroxy-213-methoxy-18-norandrosta-5,8,11,13-tetraeno[6,5,4-bc]furan-3,7,17-trione Molecular Formula/Molecular Weight C20H1606; ~

= 352.09467

OH

16

MeO

O"

~

y -oy

0

19

General Characteristics Prisms from benzene; rap., 245~ (dec.); needles from acetone; rap., 222-224~ plates from methanol; rap., 1400C; hemimethanolate, 242~ (dec.); [a]D~9 -224~ acetyl derivative: microcrystalline solid; rap., 121-126~ [a]D17 -176~ soluble in water, and chloroform; sparingly soluble in carbon disulfide and carbon tetrachloride. Fungal Source

Gliocladium virens and Trichoderma viride.

Biological Activity Viridin possesses strong antifungal activity; minimum concentration preventing germination ofBotrytis allii conidia, 0.0051.tg/ml. Spectral Data UV:

~,m~x 242(e=30,900) and 300nm (16,500). IR:

(Nujol) 3390, 3145, 1692, 1675, 1622, 1587, 1532, 1126, 1090, 1070, 1032, 1019, 1005, 983, and 970cmq; (hemimethanolate) 3450, 3105, 1709, 1675, 1622, 1587, 1532, 1120, 1085, 1064, 1015, 1001, 982, and 970cmq. ~HNMR: H-l, 4.45(,]--5); H-2, 3.95; H-11, 8.10(,]=8); H-12, 8.80; H-15, 2.90; H-16, 3.80; H18, 1.73; H-19, 8.45; and H-20, 3.75ppm. Mass Data: C, 68.18%; H, 4.58%; O, 27.24%.

718

19. Viridin and Related Metabolites

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p.747 (1981). J. F. Grove, P. McClosky, and J. S. Moffatt; The Structure of Viridin; Chem. Commun., pp. 343-344(1965).

19. Viridin and Related Metabolites

719

Common/Systematic Name Desmethoxyviridin Molecular Formula/Molecular Weight C19H1405; MW = 322.08412

0 OH

16

H

is

0

0 19

General Characteristics Melting point variable depending on solvent of crystallization; two ranges were 145-160~ (dec.) or 220-240~ (dec.); crystals from acetone; rap., 230-2400C.

Fungal Source

Nodulisporium hinnuleum (ATCC 24911), an unidentified fungus (ACC 3199), and Apiospora camptospora.

Isolation/Purification Extracted with hot CHCI3. The CHCI3 extract was evaporated to dryness and the residue applied to a silica gel column packed in a slurry of hexane and eluted sequentially with nhexane, Et20, CHCI3, EtOAc, Me2CO, and MeOH. Biological activity, as measured by acute toxicity to orally dosed day-old cockerels, was found in the EtOAc fraction, which was further purified on a second silica gel column eluted with a linear gradient from CHCI3 to EtOAc. The nonpolar fraction contained desmethoxyviridin. Biological Activity Desmethoxyviridin was toxic to day-old cockerels, but the level of toxicity was not determined. Spectral Data UV: Xm~x 243(e=27,000) and 303nm (11,600).

13C NMR: C-l, 70.8 d; C-2,46.5 t; C-3, 189.9 s; C-4, 122.9 s; C-5, 144.3 s; C-6, 145.6 s; C-7, 172.5 s; C-8, 156.9 s; C-9, 136.3 s; C-10, 41.5 s; C-11,126.2 d; C-12, 128.4 d; C-13, 156.2 s; C-14, 129.6 s; C-15, 28.1 t; C-16, 35.7 t; C-17, 205.4 s; C-18, 25.2 q; and C19, 149.3ppm d.

720

19. Viridin and Related Metabolites

IH NMR: H-I, 4.33(J=6.0); H-2, 2.72(J=6.0); H-11, 7.83(J=8.5); H-12, 8.58(J=8.5); H-15, 2.75; H-16, 3.58; H-18, 1.58; H-19, 8.75; and 1-OH, 6.12ppm. TLC Data Adsorbent: silica gel G-HR; solvent: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Re 0.57. Detection: orange spot atter spraying with 50% H2SO4 and heating for 5 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites.; Academic Press, New York, pp. 739-742(1981). R. J. Cole, J. W. Kirksey, J. P. Springer, J. Clasrdy, H. G. Cutler, and K. H. Garren; Desmethoxyviridiol, A New Toxin from Nodulisporiumhinnuleum;Phytochemistry, Voi. 14, pp. 1429-1432 (1975).

19. Viridin and Related Metabolites

721

Common/Systematic Name Viridiol Molecular Formula/Molecular Weight C2oHIsO6; M W = 354.11034

O 12

OH MeO

.o- y - y i' 19

0

General Characteristics Crystals; mp., 198-201 ~ (dec.). Fungal Source Trichoderma viride (NRRL 1828) and Gfiocladium virens (ACC 213). Spectral Data UV~

~.m,,, 250(1oge=29,500) and 317nm (11,700).

(Nujol) 1680 and 1695cm'1; (CHCI3) 1673 and 1712cm"l. 13C N M R :

C-l, 71.7 d; C-2, 81.7 d; C-3, 61.6 d; C-4, 121.9 s; C-5, 142.2 s; C-6, 142.2 s; C-7, 173.1 s; C-8, 158.4 s; C-9, 136.7 s; C-10, 42.2 s; C-11,127.0 d; C-12, 127.0 d: C-13, 157.6 s; C-14, 127.0 S; C-15, 28.4 t; C-16, 36.4 t; C-17, 206.2 s; C-18, 30.5 q; C-19, 145.3 d; and C-20, 60.5ppm q. IH NIVIR: H-I, 4.32; H-2, 3.60; H-3, 5.12; H-11, 7.93(J=8.0Hz); H-12, 8.25(J=8.0Hz); H-15, 2.71; H-16, 3.64; H-18, 1.74; H-19, 7.77; and H-20, 3.75ppm. Mass Spectrum: LREIMS: 354m/e (M +) with losses of 18, 33, 46, and 74m/e mass units. TLC Data Adsorbent, silica gel G; solvent, chloroform-methanol, 9:1, v/v; Re 0.36; detection, green fluorescence under UV light; red-brown atter AgNO3 spray.

722

19. Viridin and Related Metabolites

References R. J. Cole and R. H. Cox; H.andboo_k of Toxic Fungal Metabolites; Academic Press, New York, pp. 748-752(1981). J. S. Moffatt, J. D. Bu'Lock, and T. H. Yuen; Viridiol, A Steroid-like Product from

Trichoderma viride; Chem. Commun., p. 839 (1969).

19.

Viridin and Related Metabolites

723

Common/Systematic Name Desmethoxyviridiol Molecular Formula/Molecular Weight C19H1605; MW = 324.09977 O OH

16 15

HO

O 19

General Characteristics Crystals from ethyl acetate; mp., 155-157~ soluble in ethyl ether, chloroform, ethyl acetate, and acetone; moderately soluble in toluene, methanol, and ethanol; insoluble in hexane and water. Fungal Source

Nodulisporium hinnuleum (ATCC 24911) and an unidentified fungus (ACC 3199).

Isolation/Purification Extracted with hot CHCI3. The CHCI3 extract was evaporated to dryness and the residue applied to a silica gel column packed in a slurry of hexane and eluted sequentially with nhexane, Et20, CHCI3. EtOAc, Me:CO, and MeOH. Biological activity, as measured by acute toxicity to orally dosed day-old cockerels, was found in the EtOAc fraction,which was further purified on a second silica gel column eluted with a linear gradient from CHCI3 to EtOAc. The polar fraction was crystallized from EtOAc at room temperature. Biological Activity Desmethoxyviridiol had an oral LDs0 of 4.2mg/kg in day-old cockerels. It also produced plant-growth regulating and phytotoxic effects in plant assay systems. Spectral Data UV:

)Eto. 251(c=30,000) and 322nm (16,000); ~E~. 249(c=23,500 ) and 319nm (11,400). max

max

Iac NMR: (CDCI3) C-1, 70.3 d; C-2, 41.8 t; C-3, 59.4 d; C-4, 129.6 s; C-5, 143.9 s; C-6, 143.9

724

19. Viridin and Related Metabolites

s; C-7, 172.2 s; C-8, 157.4 s; C-9, 135.7 s; C-10, 41.8 s; C-11, 125.5 d; C-12, 127.6 d; C-13, 154.4 s; C-14, 129.6 s; C-15, 27.9 t; C-16, 35.6 t; C-17, 205.7 s; C-18, 27.4 q; and C- 19, 146.0ppm d. ~H NMR: H-I, 4.00; H-2, 2.12; H-3, 4.78(,/-6.0); H-11, 7.76(,/-8.0); H-12, 8.56(,/--8.0); H-15, 2.60; H-16, 3.55; H-18, 1.50; H-19, 7.97; and OH's, 5.46, 5.70ppm. TLC Data Adsorbent; silica gel G-I-~ solvent; toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Re: 0.66; detection: gray-blue spot after spraying with 50% ethanolic H2SO4 and heating at 110~ for 5 min. Reference R. J. Cole, J. W. Kirksey, J. P. Springer, J. Clardy, H.G. Cutler, and K. H. Garren; Desmethoxyviridiol, A New Toxin from Nodulisporium hinnuleum; Phytochemistry, Vol. 14, pp. 1429-1432(1975).

19. Viridin and Related Metabolites

725

Common/Systematic Name Wortmannin Molecular Formula/Molecular Weight C23H2408; M W = 428.14712

O ,o

0

-

-.yo -o

0

19

General Characteristics Crystallized from methanol or benzene to give needles; mp., 238-239~ [a]D 26 + 8 9 ~ (c=l. 1, in CHCI3); the mono-2,4-dinitrophenylhydrazone crystallized from acetic acid as yellow needles; mp., 266-268 oC. Fungal Source

Penicillium wortmannii and Myrothecium roridum.

Spectral Data UV:

~.m~, 257 and 292nm (e=l 1,770 and 7,700). IR;

(KBr) 3112, 1751, 1732, 1684, 1656, and 1547cm1; (CHC13)3150, 1750, 1683, 1653, and 1553cm"1. ~H NMR: (CDC13) H-l, 5.25; H-2, 6.52, 7.02; H-11, 3.90; H-12, 7.30, 8.0; H-14, 7.15; H-20, 1.78; H-18, 9.06; H-19, 8.29; 2-OMe, 6.87; and 11-AOAc, 7.911:(15 protons at 6.80 and 7.80z). Coupling constants: J1,2=2.0; J1,2'=7.0; ,/2,2'=11.0; J11,12=8.0; J11,12'=8.0; J12,12'=12.5; J11,14=3.0; J14,15=6.0 and J14,15=12.2Hz. The four three-proton singlets were assigned to the 13-methyl (9.06), the 10 methyl (8.29), the acetoxy (7.91) and the methoxy (6.87) protons. The one proton singlet at 1.78z was of similar chemical shift to the furanoid proton in the acid and was assigned to the 20proton. An ABX-system at 7.02, 6.52, and 5.25 with JAB 11, JAX 2, and JBX 6Hz was assigned to the protons at C-2 and C-1. A double triplet at 3.9ppm was assigned to the C-11 proton, which showed an 8Hz coupling to each of the C-12 protons at 7.3 and 8.41: (J12A,12B, 12.5Hz). In addition, the 1l proton showed a homoallylic

726

19. Viridin and Related Metabolites

coupling of 3Hz to the C-14 methine proton at 7.151:. The 15-proton signals were located at 6.8 and 7.8z and those of the 16-protons at 7.4 and 7.91:. Mass Data: ELMS: 428.145(M +, 0.1%), 413(8.0), 385(19), 368(46.0), 354(68), 323(94), 294(32), 268(55), 266(100), 239(41), 238(62), 223(40), 45(37),44(45), and 43role (55); found a~er crystallization from methanol and drying in v a c u o at 100~ for 15 h; C, 63.5, 64.1, 63.9; H, 5.6, 5.9, 5.9~ Found after crystallization from benzene and drying in v a c u o at 100~ for 6 h: C, 64.8, 65.0, 65.4, 64.3; I-I, 5.7, 5.7, 5.6, 5.9%; found atter sublimation: C, 63.8, 63.6; H, 5.8, 5.8%; C23H2408 requires C, 64.5; H, 5.6%. References J. MacMillan, A. E. Vanstone, and S. K. Yeboah; Fungal Products. Part III. Structure of Wortmannin and Some Hydrolysis Products; J. Chem. Soc. Perkin I, pp. 2898-2903 (1972). J. MacMillan, T. J. Simpson, and S. K. Yeboah; Absolute Stereochemistry of the Fungal Product, Wortmannin; J.Chem. Soc. Chem Commun., p. 1063 (1972). T. J. Petcher, H. -P. Weber, and Z. Kis; Crystal Structure and Absolute Configuration of Wortmannin and ofWortmannin p-Bromobenzoate; J. Chem. Soc., Chem. Commun., pp. 1061-1062(1972).

19. Viridin and Related Metabolites

727

Common/Systema.tic Name 11-Desacetoxywortmannin Molecular Formula/Molecular Weight C21H2206; MW = 3 70.14164 0

MeOH2C..~ "

~

16 15

19

General Characteristics Crystallized from methylene chloride-ethyl ether; mp., 178-180~ (c=0.967, in CHCI3).

[a]o 2~ + 104 ~

Fungal Sourc.e

Penicillium funiculosum , P. wortmannii, and Aspergillusjanus.

Biological Activity Antiinflammatory metabolite. Spectral Data UV:

~,Cm~~176 258.5(Iog C=4.08) and 293.5nm (3.86). IR;

(CH2C12) 3140, 1755, 1745, 1680, 1650, and 1510cm"~.

~H NMR: (CDCI3) 0.90 s (3H), 1.71 s (3H), 3.11 s (3H), 3.15 m (2H), 4.82 t(d=5 Hz; 1H), and 8.221: s (1H). Mass Data: LREIMS: 370(100%), 325(85), 312(48), 297(43), 296(48), 281(43), 268(33), and 240role (67); HR IMS: C2~H2206, 370.40Ira~e; obsd, C, 68.1; H, 6.0; O, 25.9~ required C, 67.9; H, 5.8; O, 26.3%. Reference von W. Haefliger and D. Hauser; Isolierung und Strukturaufklarung von 11-Desacetoxywortmannin; Helvetica Chimica Acta, Vol. 56, pp. 2901-2903(1973).

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Cercosporin and Related Metabolites Cercosporin Amphieereosporin Protocercosporin Hypocrellin 2'-Acetylcercosporin 2',2"-Diacetylcercosporin 2',2"-Dibenzoylcereosporin Neosporin (Neocercosporin) Elsinochrome D Pleichrome

729

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20. Cercosporin and Related Metabolites

731

Common/Systematic Name Cercosporin 1,12-(2-Hydroxypropyl)-2,11-dimethoxy-4,9-dihydroxy-6,7-methylenedioxyperylene-3,10quinone Molecular Formula/Molecular Weight C29H2601o; MW 534.15260 " -

OH 0 6

4

0

,,,,,,,,70M~H ~

<0 7

M

e

~ M e ~I~oMOH OH 0

General Characteristics The pigment melted at 241 *C without sublimation or decomposition. Cercospodn was optically active and showed a rotation of [tQ2~ +470* which changed on heating in a high boiling solvent, such as toluene, or fusing the crystals at the melting point to a value of-152"C. Fungal Source Produced by the mold Cercosporina kilcuchii responsible for the disease called purple speck disease of Japanese soy beans; Cercospora hay& the causal organism of brown spot of banana fruit. There is widespread distribution of cercosporin producing ability in the genus Cercospora. Assante et al. (1977) showed that 24 of 61 species tested produced cercosporin. These include C. ariminiensis, C. bertoreae, C. beticola, C. bizzozeriana, C. canescena, C. carotae, C. chenopodii, C. cistinearum, C. cladosporioides, C. diazu, C. dulcamarae, C. erysimi, C. kikuchii, C. malvacearum, C. malvicola, C. medicaginis, C. nicotianae, C. oryzae, C. personata, C. plantaginis, C. setariae, C. unamunoL and C. violae. Isolation/Purification The pigment was purified by silicic acid column chromatography (acid treated Florisil, 100/200 mesh), packed in an acetone slurry and eluted with chloroform, or it could be obtained by extraction with ethyl ether followed by crystallization from ether to give dark red crystals. Biological Activity Phytotoxic.

732

20. Cercosporin and Related Metabolites

Spectral Data UV:

~~"

max

223,260, 271 275, and 470nm.

IR:

(KBr) 3400m, 2940m, 1619vs, 1585vs, 1554vs, 1455m, 1428m, 1395w, 1348w, 1315m, 1268s, 1223m, l170s, 1145m, ll13m, 1075m, 1055m, 1017m, 978w, 938w, 921w, and 860cm"~m; bands in the 1700-1550cm"~region (CHCI3), 1614vs; 1583s, and 1551cm'~; (tetrahydrofuran) 1615, 1585, and 1651cm"~. IH NMR: The extreme sharpness of the per/-hydroxy, the aromatic, the methylenedioxy, the methoxy, and the side-chain hydroxy protons (2.94, 4.24, 5.79, and 8.42ppm), integrating in the ratios 2:2:2:2:6:2, is consistent with a symmetrical arrangement of the substituents on the parent ring system. A methyl doublet(3=6Hz) and an ABX-type multiplet, both representing six protons, appeared at 9.37 and 6.7ppm, respectively, and represent the side-chain protons. The assignment of the methoxy groups at C-2 and C-11, rather than at C-5 and C-8, is based upon the position of their NMR resonance, which is comparable to the corresponding groups in the spectrum of elsinochrome A. Moreover, the methoxy resonance failed to undergo an appreciable solvent shift (CDCI3, C6D6), which is compatible with the position of the methoxy groups on the parent ring system. The position of the side-chains at C-1 and C-12 is evident from the results of zinc dust distillation which yielded a hydrocarbon with an electronic absorption spectrum closely resembling that of benzo[ghi]perylene.

Mass Spectrum: Major ions in the mass spectrum were 534.1466(M+), 516.1380, 503.1359, 490.1230, 475.1030, 471.1103, 459.1093, 445.0880, 431.0777, 415.0854, 401,387, 371,360, 344, 330, and 313role. TLC Data Supelcosil 12A, benzene-MeOH, 1:1, v/v, Rf 0.70; Chromatogram Sheet, chloroformMeOH, 18:1, v/v, Re 0.87. References G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Masini; Screening of the Genus Cercospora for Secondary Metabolites; Phytochemistry, Vol. 16, pp. 243-247(1977). R. J. J. Ch. Lousberg, U. Weiss, C. A. Salemink, A. Arnone, L. Metlini, and G. Nasini; The Structure of Cercosporin, a Naturally Occurring Quinone; Chem. Comm., pp. 14631464(1971). R. O. Mumma, F. L. Lukezic, and M. G. Kelly; Cercosporin from Cercosporahayii; Phytochemistry, Vol. 12, pp. 917-922(1973).

20. Cercosporin and Related Metabolites

733

Common/Systematic Name Amphicercosporin 4, 8-Dihydroxy- 1,12-(2-hydroxypropyl)-2,11-dimethoxy-6,7-(methylenedioxy)perylene3,10-quinone Molecular Formula/Molecular Weight C29H26Olo; MW = 534.15260

OH

0

~'O~Me HO.~'~,~oMOeH .0"

~

Y

v

"Me

General Characteristics Reddish purple needles; mp., 195-196~C. Fungal Source Cercospora kilcuchii. Spectral Data _

UV:

~

EtOH max

260(1og ~=4.42), 315-325(3.48), 475(4.23), and 565nm (3.77).

IR:

(Kbr) Showed characteristic absorptions at 1705 and 1630cmq (nonchelated and chelated carbonyl, respectively). IH NMR:

(CDCI3) showed signals at 1.05(6H, d, J=7.2Hz, 2Me); 1.50(2H, s, 2OH); 2.80(2H, dd, J=7.2 and 12Hz; 3.45(2H, m); 3.74(2H, m); 4.28(3H, s, MeO); 4.30(3H, s, MeO); 5.82(2H, s); 7.10(H, s, Ph-H); 7.12(1H, s, Ph-H); 11.80(1H, s, free Ph-OH); and 14.60ppm (H, s, chelated Ph-OH). Reference S. Matsueda, K. Takagaki, M. Shimoyama, T. Imaizumi, and M. Koreeda; Structure of Amphicercosporin and Protocercosporin; Chem. Ind. (London), p. 58 (1982).

734

20.

Cercosporin and Related Metabolites

Common Name/Systematic Name Protocercosporin Molecular Formula/Molecular Weight C27H2009; MW = 488.11073

OH

~O ~ OH

u~

0

~

,,. 'Me

---~'-'C H2 O

General Characteristics Orange orthorhombic crystals; mp., 260~ (sub.); protocercosporin is chemically stable and not photosensitive. Fungal Source Cercospora kikuchii. Spectral Data UV:

~.,~x 237(1og e=4.06), 266(4.06), 298sh (3.64), 340-360(3.55), and 413nm (3.91) showed the extended quinone skeleton. IR:

The infrared spectrum (KBr) of protocercosporin showed absorption at 1625cmq (chelated carbonyl). 1H NMR:

(CDCI3) Showed signals at 1.55(3H, d, J=6Hz, C-15 linked Me); 2.00 and 2.55(1H, dd, J=3, 13, 5Hz and 1H, dd, J=10, 13.5Hz, respectively, C-14 CH2); 3.45 and 4.25(1H, d, J=17Hz and 1H, d, J=17Hz, respectively, C-13 CH2); 4.40(1H, m, C-15 linked H); 3.48(3H, s, C-2 linked OMe); 4.16(3H, s, C-11 linked OMe); 5.75(2H, dd, ./=6, 12Hz, -O-CH2-O-); 12.00(1H, s, C-4 linked OH); and 13.45ppm (1H, s, C-9 linked OH). Reference S. Matsueda, K. Takagaki, M. Shimoyama, T. Imaizumi, and M. Koreeda; Chem. Ind. (London), p. 58 1982.

20. Cercosporin and Related Metabolites

735

Common/Systematic Name Hypocrellin Molecular Formula/Molecular Weight C3oH2601o; MW = 546.15260

OH

0

5

2,~"OMe 1

MeO

13

MeO7 ~ ~ ~

Me

ON Me ~OMe

ON

0

General Characteristics Dark red crystals from acetone; mp., 209-210~ Fungal Source Hypocrella bambusae. Biological Actiyity Photodynamic activity toward microorganisms. Spectral Data UV:

~,~" 215(1ogc=4.78), 268(4.60), 283(4.59), 342(3.50), 367(3.40), 465(4.48), 539(4.04), 581(4.13), 608nm (3.80); ~N~. 217(4.56), 251(4.22), 285(4.30), 473(3.19), 581(4.13) and 608rim (4.15). Im;

(KBr) 3500(OH), 1720(=C-O), 1620, 1610, 1574, and 1525cmq (aromatic ring). IH N]V[R:

(CDCI3) H-5, 6.52(s); H-S, 6.56(s); H-13, 3.53(I-L~), 2.64(HB, J=12Hz); H-15, 3.74(s); H-16, 1.71(s); H-18, 1.90(s); 2-OCH3, 4.12(s); 6-OCH3, 4.07(s); 7-OCH3, 4.08(s); 11-OCH3, 4.07; 1.68; 15.08; and 1509ppm (3 X D20 exchangeable protons). laC NMR: C-l, 133.2; C-la, 127.7; C-2, 150.9; C-3, 181.9; C-3a, 106.7; C-3b, 167.5; C-4, 179.7; C-5, 102.4; C-6, 172.0; C-6a, 124.8; C-7, 171.0; C-7a, 124.8; C-7b, 167.5; C-8, 102.4;

736

20. Cercosporin and Related Metabolites

C-9, 180.1; C-9a, 106.7; C-10, 179.7; C-11,150.7; C-12, 134.0; C-12a, 127.7; C-13, 42; C-14, 78.8; C-15, 61.7; C-16, 27.0; C-17, 207.5; C-18, 30.1; C-2-OCH3, 62.1; C6-OCH3, 56.6; C-7-OCH3, 60.9 and C-11-OCH3, 56.5ppm. Mass Spectrum: EIMS: 546(M+), 528, 515, 503,497, 489, 471,457, 446, 429, and 415role. Reference C. Wei-shin, C. Yuan-teng, W. Xiand-yi, E. Friedrich, H. Puff, and E. Breitmaier; Die Struktur des Hypocrellins und seines Photooxidationsproduktes Peroxyhypocrellin; Liebigs Ann. Chem., pp. 1880-1885(1981).

20. Cercosporin and Related Metabolites

737

Common/Systematic Name 2'-Acetylcercosporin Molecular Formula/Molecular Weight C31H28011, M W = 576.16316

6

<0 0 7

OH 0 ~. /OMe ~ ~,Ac ~ v 2' "Me ~Me 4

~

H

OH

0

General Characteristics 2'-Acetylcercosporin crystals; mp., 133-134 oC. Fungal Source Cercospora setariae. Isolation/Purification Cercospora setariae cultures were extracted with EtOAc and the crude extract was evaporated to dryness. 2'-Acetylcercosporin was isolated from the crude extract by preparative TLC with benzene-ethyl ether-formic acid (50:50:1, v/v/v). Biological Activity Phytotoxic. Spectral Data UV:

~, E~ 253,291,299sh, 480, 590sh, and 640nm (e=15,500, 23,000, 23,300, 14,100, 5,000, and 6,200, respectively). IR.:

(Nujol) 3450(OH), 1740(acetate), and 1620cm"l (conjugated C=O). CD: (in EtOH, c=0.9 x 10.2 g/100ml) 240, 297, 352, and 410nm (A~ +23.6, -42.2, +1.92, and -8.00).

738

20. Cercosporin and Related Metabolites

IH NMR: (CDCI3): 0.56 and 0.64(d, 2 Methyls); 1.66(Acetate); 2.80-3.80(m, -CHz-O and 2CH2-CH-O); 4.24 and 4.22ppm (O-Methoxyl groups); 4.64(m,-CH2-CH-OAc); 5.76(s, O-CH2-O); 7.04(s, 2 aromatic H's); 14.82 and 14.76ppm (2 chelated OH's). Mass Spectrum: EIMS: 576role (M+) . Reference G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Nasini; Screening of The Genus Cercospora for Secondary Metabolites, Phytochemistry, Vol. 16, pp. 243-247(1977).

20. Cercosporin and Related Metabolites

739

Common/Systematic Name 2',2"-Diacetylcercosporin Molecular Formula/Molecular Weight C33H3oO12; M W = 618.17373

OH

0 ~OMe

0 <0

/

V

2' "Me

~Me

7

OH

0

General Characteristics 2',2"-Diacetylcercosporin was obtained as an orange-red powder; mp., 80-82~ Fungal Source Cercospora setariae. Isolation/Purification Cercospora setariae cultures were extracted with EtOAc and the crude extract was evaporated to dryness. 2',2"-Diacetylcercosporin was isolated from the crude extract by preparative TLC with benzene-ethyl ether-formic acid (50:50:1, v/v/v). Biological Activity Phytotoxic. Spectral Data UV:

~Om~" 290, 297sh, 485, 600, and 640nm (e=32,700, 32,400, 21,800, 8,600, and 12,000, respectively). IR:

(Nujol) 1740(acetate) and 1620cm"l (conjugated C=O). CD: (in EtOH, c=2.1 x 10.2 g/100ml) 246, 297, 320, 352, and 410nm (Ae +23.5, -43.5, -7.95, +2.94, and -7.35).

740

20. Cercosporin and Related Metabolites

1H NMR: (CDC13) 0.55(d, 2 methyls, J=6Hz); 1.70(2 acetates); 3.73 and 3.0ppm (m, 2-CH2CH-O); 4.28(20-methoxy groups); 4.68(m, 2-CH2-CH-OAc); 5.8(s, O-CH2-O); 7.04(s, 2 aromatic H's); and 14.76ppm (2 chelated OH's). Mass Data: EIMS: 618m/e (M+); found: C, 63.17; H, 4.50; C33H3oO~2 requires C, 64.07; H, 4.89%). Reference G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Nasini; Screening of The Genus Cercospora for Secondary Metabolites, Phytochemistry, Vol. 16, pp. 243-247

20. Cercosporin and Related Metabolites

741

Common/Systematic Name 2',2"-Dibenzoylcercosporin Molecular Formula/Molecular Weight C43H34012; M W

" - "

742.20503

OH 0 L~~/OMe

;LL,oL OH

()COPh

0

General Characteristics 2',2"-Dibenzoylcercosporin was obtained as a red solid; mp., 120-123 ~C. Fungal Source

Cercospora setariae.

Isolation/Purification

Cercospora setariae cultures were extracted with EtOAc and the crude extract was

evaporated to dryness. 2',2"-Dibenzoylcercosporin was isolated from the crude extract by preparative TLC with benzene-ethyl ether-formic acid (50:50:1, v/v/v). Biological Activity Phytotoxic. Spectral Data UV:

~ EtOH 224, 270, 475, and 565nm (e=36,700, 16,300, 11,800, and 4,300 respectively). max

IR:

(Nujol) 1715(OCOPh) and 1620cm "l (conjugated C=O). CD: (in EtOH, c= 1.23 x 102 g/100ml) 240, 297, 318, and 410nm (Ae +21, -24.2, -6.95, and -3.62).

742

20. Cercosporin and Related Metabolites

~HNMR: (CDCI3): 0.83(d, 2 methyl's); 3.40-3.86(m, 2-CH2-CH-O); 4.32(2 methoxy groups); 5.0-5.2(m, 2 -CH2-CH-OCOPh); 5.62(s, O-CH2-O); 6.80(s, 2 aromatic ITs); 7.0-7.40(10 aromatic H's); and 14.70ppm (2 chelated OH's). Mass Data: EIMS" 742m/e (M+); found: C, 68.76; H, 4.60, C43H34012requires: C, 69.6: H, 4.62. Reference G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Nasini; Screening of The Genus Cercospora for Secondary Metabolites, Phytochemistry, Vol. 16, pp. 243-247(1977).

20. Cercosporin and Related Metabolites

743

Common/Systematic Name 2'-Acetyl-2"-benzoylcercosporin Molecular Formula/Molecular Weight C38H32012, M]vV = 680.18938

OH

O

.O e

o- "T Y

< O ~ M e ~-. ')..,o'r 6COPh - f "oMe

OH

0

General Characteristics 2'-Acetyl-2"-benzoylcercosporin was obtained as red crystals; mp., 153-155 ~C. Fungal Sourc.e Cercospora setariae. Isolation/Purification Cercospora setariae cultures were extracted with EtOAc and the crude extract was evaporated to dryness. 2'-Acetyl-2"-benzoylcercosporin was isolated from the crude extract by preparative TLC with benzene-ethyl ether-formic acid (50:50:1, v/v/v). Biological Acti~ty Phytotoxic. Spectral Data UV;

~,~2~" 223,273, 380sh, 480, 570 and 620nm (e=41,500, 24,400, 4,500, 18,700, 5,850, and 4,000 respectively). IR: (Nujol) 1740(acetate), 1720(OCOPh), and 1620cm"~(conjugated C=O). CD: (in EtOH, c=3.21 x 102 g/100ml): 240, 298, 320, 360, and 410nm (Ae +16.2, -20.0, 4.86, +0.42, and -3.82).

744

20. Cercosporin and Related Metabolites

1H NMR: (CDCI3) 0.53 and 0.82ppm (d, methyl); 1.61(acetate); 2.92-3.88(m, 2-CI-I2-CH-O); 1.28 and 4.30ppm (methoxy); 4.65(m, CH2-CH-OAc); 5.07(m, CH2-CH-OCOPh); 5.70(d, O-CH2-O, J = 7.0Hz); 6.85 and 7.06ppm (s, 2 aromatic protons); 7.167.50(C6H6); 14.66 and 14.80ppm (2/chelated OH's). Mass Data: EIMS: 680m/e (M+); found: C, 67.00; H, 4.74, C38H32012requires: C, 67.05: H, 4.75. Reference G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Nasini; Screening of The Genus Cercospora for Secondary Metabolites, Phytochemistry, Vol. 16, pp. 243-247(1977).

20. CercosporinandRelatedMetabolites

745

Common/Systematic Name Neosporin; Neocerosporin 5,8-Dihydroxy- 1,12-(2-hydroxypropyl)-2,1 l-dimethoxy-6,7-(methylenedioxy) perylene-3,10-quinone Molecular Formula and Molecular Weight C29H2601o; MW = 534.15260

O

4

< 0 ~

0

v z "Me ~ M e

OH" "~" ~ ~ O M ! H 9 0 General Characteristics The novel fungal pigment; mp., 237 ~C, was isolated together with cercosporin; reddish violet orthorhombic crystals. Fungal Source Cercosporina kikuchii. Spectral Data UV:

Spectrum showed the skeleton of neosporin was like that of cercosporin; ~,mM~H 222(e=49,000), 270(31,500), 475(23,500), and 562nm (8500). IR:

(KBr) Showed absorption at 1710cml (non-chelated carbonyl), which was the characteristic different feature from that of cercosporin. IH NMR: The IH NMR spectrum (CDCI3) of neosporin showed signals at 0.90(6H, d, J=6H2, 2Me); 1.40(2H, s, 2OH); 2.9(2H, dd, J=6H2 and 12); 4.15(6H, s, 2MeO); 5.65(2H, s, O-CH2-O-); 6.95(2H, s, 2Ph-H); and 11.2ppm (2H, s, 2Ph-OH). Mass Spectrum: The mass spectrum showed the main fragments as follows: 534(M+ - C~-I26010), 516(M +- H20), 502(M+- CH3OH), 490(M+- C2I-hO), 475(M +- C3H70), 458(M +C3HsO2), 43 l(base peak, M + -C5H1102_),415(M + - C5H~O3), and 401(M + - C6H~303).

746

20. Cercosporin and Related Metabolites

Reference S. Matsueda; Structure of Neosporin; Chem. Ind., pp. 233-234(1978).

20.

Cercosporin and Related Metabolites

747

Common/Systematic Name Elsinochrome D Molecular Formula/Molecular Weight C27H15OT(OCH3)3; MW = 544.13695 v

OH

0 4

MeO

~

I .

MeO

~/,Jx,0 OH OH

o---/

General Characteristics Elsinochrome D is a monoether of a peri-dihydroxyquinone. Recrystallized from nhexane-chloroform (8:1, v/v), the orange crystals of elsinochrome D were dried for 48 hr. at 70~ and 0.1mm; mp., 159-161 ~ soluble in most organic solvents, slightly soluble in hot hexane, and very slightly soluble in water; and the solution in aqueous alkali is brownish-green. The brownish-green solution of elsinochrome D in weak alkali turned pink on addition of a few drops of 1N sodium dithionite. When zinc was added to the deep orange solution in acetic acid, the color changed quickly to yellow with green fluorescence. In both cases, the original color was rapidly restored on aeration. Fungal Source Elsinoe annonae.

Isolation/Purification The filtered mycelium was extracted several times with ethyl acetate, the combined extracts were evaporated in vacuo, and the residue was taken up in chloroform, washed several times with aqueous sodium hydrogen carbonate, dried (MgSO4) and evaporated to dryness. The residue was chromatographed over calcium hydrogen phosphate with chloroform-ethyl acetate (1:1, v/v) as eluant. The fraction containing mainly elsinochrome D was evaporated in vacuo and redissolved in methanol. A white precipitate (amorphous) was filtered off, and the orange solution was evaporated. The residue was taken up in carbon tetrachloride and chromatographed first over calcium hydrogen phosphate, with chloroform as eluant, and subsequently over calcium hydrogen phosphate (activated), with chloroform-acetone-ethanol (131.2:8.7:2, v/v/v) as eluant. The fraction which contained mainly elsinochrome D was evaporated and the residue was recrystallized several times consecutively from n-pentane-chloroform (5:1, v/v), n-hexane-methanol (10:1, v/v), and n-hexane-chloroform (8:1, v/v).

748

20. Cercosporin and Related Metabolites

Soectral Data UV:

~,~m"~ 224, 253, 267, 348, 463, 526, and 560nm (log c 4.70, 4.50, 4.46, 3.66, 4.28, 4.05, and 3.15). IR:

(KBr)

3400, 1634, 1620, 1600, 1460, 1360, 1145, 1090, and 925cm"~.

1H NMR: 8.91(d) and 8.83(d) (each CH-CH3); 8.00(OH); 6.17; 6.06; and 5.80(OMe); 6.15(m) and 6.07(m) (CH-CH-OH); 3.90 and 3.77ppm (quinonoid H); 3.67 and 3.73(each 1H, d, J=0.5Hz, O-CH2-O); and 6.00ppm (OH). The positions of the signals at 6.15 and' 6.071: was determined from a spectrum of elsinochrome D in [2H]chloroform[2I-I6]benzene, and may not correspond exactly with the positions of these multiplets in the spectrum of elsinochrome D in pure CDCI3. Mass Data: Found: C, 65.7; H, 5.1-OMe, 15.5%; MW, 546.1530 C27HIsOT(OMe)3requires C, 65.9; H, 4.8; OMe, 17.0%; M, 546.1523. Reference R. J. J. Ch. Lousberg, C. A. Salemink, and U. Weiss; Pigments of Elsinoe Species. Part V. The Structure of Elsinochrome D; J. Chem Soc., pp. 2159-2162(1970).

20. Cercosporin and Related Metabolites

749

Common/Systematic Name Phleichrome 1,12-Bis(2-hydroxypropyl)-2,6,7,11 -tetramethoxy-4,9-dihydroxyperylene-3,10-quinone Molecular Formula/Molecular Weight C3oH3o01o; M W -- 5 5 0 . 1 8 3 9 0

OH

MeO

0

,~

Me

OH 0 General Characteristics Obtained as a red pigmented powder. The color in alkaline solution changed from red to green. Fungal Source

Cladosporium phlei, which causes a leaf spot disease of timothy (Phleum pratense). It forms a characteristic eye spot of light grayish-fawn centers with purplish margins on the plant leaves.

Isolation/Purification The culture filtrates were concentrated m vacuo and then extracted with ethyl ether. The extract was dried on Na2SO4, concentrated to dryness, and then chromatographed on a polyamide column with water-methanol (1:1, v/v). Its homogeneity was confirmed by TLC on silica gel with 2% oxalic acid solution; Rf 0.56 (reference compound, elsinochrome A, 0.59) in acetone and 0.14 (0.50) in benzene-ethyl acetate (1:1, v/v). Biological Activity_ Phytotoxic. Spectral Data UV: X,~ 226(sh) (e=52,600), 260(sh) (32,200), 274(33,900), 474(22,800), 540(sh) (11,400), and 584nm (11,800).

750

20. Cercosporin and Related Metabolites IR:

Absorption bands at 1610(extended quinone system), 1110 (secondary alcohol), and 839cm "1 (hexasubstituted phenyl). ORD: (c=0.28 x 10-3 g/ml in MeOH) [O] (nm); -29,400(600), -23,520(589), +13,720(490), -47,040(380), +3,920(330), and +9,800(304). ~H NMR: (acetone-d6) assignments as follows: CH3- at 0.35(d, J=SHz); -CH2- and HC- at 2.73.2(m) and 3.3-3.9(m); CH30- at 4.21(s) and 4.23(s); and Ar-H at 6.80 and Ar-OH at 15.1-16.3ppm (br). Mass Spectrum: (Tetraacetate) 718m/e. Reference T. Yoshihara, T. Shimanuki, T. Araki, and S. Sakamura; A New Phytotoxic Compound Produced by Cladosporiumphlei; Agr. Biol. Chem., Vol. 39, pp. 1683-1684(1975).

Cyathanes Cyathin A3 Cyathin A4 Cyathin B2 Cyathin B3 Cyathin C3 (1,2-Dehydrocyathin B3) Cyathin C5 Allocyathin B2 Allocyathin B3 Neoallocyathin A4 Cyafrin A4 Cyaffin B4 Cyafrin A5 Allocyafrin B4 Cyathatriol 11-O-Acetylcyathatriol 15-O-Acetylcyathatriol 11,15-0,O-Diacetylcyathatriol Sarcodonin A Sarcodonin G Striatin A Striatin B Striatin C

751

This Page Intentionally Left Blank

21. Cyathanes

753

Common/Systematic Name Cyathin A3 Molecular Formula/Molecular Weight C2oH3oO3; 1VIW' = 318.21950

1 16

.,,i

HO

....

15 CH2OH

OH

CH2OH

General Characteristics In the solid state cyathin A3 existed and crystallized in the internal hemiketal form. Acetylation gave the diacetyl derivative of the hydroxyketone form, whereas treatment with methanolic HCI gave an internal ketal. Fungal Source Metabolite of the bird's nest fungus Cyathus helenae and C. africanus. Isolation/Purification The original isolation procedure was based mainly on a combination of column and thin-layer chromatography on the ethyl acetate extract obtained from the culture broth. A material with Rf 0.4 (silica gel G, chloroform-methanol, 9:1, v/v) which appeared chromatographically pure was obtained and called cyathin A3. However, careful inspection of the NMR spectrum of the acetyl derivative indicated that this material consisted of two components: allocyathin B3 and its dihydro derivative cyathin A3. The metabolites were separated and purified using TLC. The plates were developed with Skellysolve B-acetone 2:3, sprayed with a 0.2% solution of 2',7'-dichlorofluorescein in ethanol, and viewed under longwave UV light to locate the zones containing allocyathin B3 and cyathin A3. The zones were scraped off and eluted with ether to obtain, after evaporation, allocyathin B3 and cyathin A3. Cyathin A3 was crystallized after trituration with acetone and further purified by recrystallization from benzene or benzene-cyclohexane mixtures. Biological Activity The metabolite possessed antibiotic activity.

754

21. Cyathanes

Spectral Data UV:

~,'~=~ 323(e=130), and shoulder at 225nm (8,700) (assigned to n-g* and rt-n* transitions of the a,13-unsaturated carbonyl system). IRi

Solid phase IR spectrum lacked absorption in the 1750-1650cm q region. In solution an IR maximum at 1670cmq (medium intensity) suggested that in solution cyathin As existed as an equilibrium mixture of the tautomeric forms. CD: Maximum at 330nm (Ae +0.73). IH NMR: O,O-Diacetylcyathin As derivative: H-l, H-7, and H-8, 1.9-1.1(u), H-2, 2.3(u); H-5b, 2.78(dd, J=lO and 2.5I-Iz); H-10a, 2.05(ddd, ,/=-14, 10 and 7Hz); H-10b, 2.53(ddd, ,/--14,6 and 2.5Hz); H-1 lb, 5.36(ddddd, J=7,6, 0.5, 0.5 and 0.5Hz); H-13a or b, 6.13(ddd, ,/=1.5, 0.5 and 0.5 Hz); H-15a, 4.75(ddd, ,/=14, 1.5 and 0.5Hz); H-15b, 4.63(ddd, 14,1.5 and 0.SHz); H-16 and H-17, 1.15 and 1.06(s, s); H-18, 2.84(qq, ,/=-7 and 7Hz); and H-19, H-20, 1.00 and 0.99ppm (d, d, J--'7 and 7Hz). Reference W. A. Ayer and H. Taube; Metabolites of Cyathus helenae. A New Class of Diterpenoids; Can. J. Chem., Vol. 51, pp. 3842-3854(1973).

21. Cyathanes

755

Common/Systematic Name Cyathin A4 Molecular Formula/Molecular Weight C20H3004; MW = 334.21441

..,""

HO

0

CH20H

General Characteristics Evaporative distillation at 220 oC/0.2mm gave a clear oil; cyathin A4 was further characterized as its crystalline tri-p-bromobenzoate; crystallization from methylene chloride-methanol gave tri-p-bromobenzoylcyathin A4; mp., 188-189~ Fungal Source Cyathus helenae strain 1500-102. Isolation/Purification The active constituents of the culture broth were extracted with ethyl acetate. Examination of the crude extract (referred to as cyathin) by TLC revealed the presence of at least eight components. A solution of the crude cyathin complex was analyzed by preparative TLC, developed with benzene-dioxane-acetic acid (100:25:1, v/v/v). Under UV, bands were detected at Rf 0.54 (cyathin C5), 0.51 (2,4,5-trihydroxybenzaldehyde), 0.27 (cyathin A3), and 0.06 (cyathin A4). Cyathin A4 was isolated by subjecting crude cyathin from which most of the trihydroxybenzaldehyde has been removed (by treatment with chloroform) to preparative TLC using benzene-acetone-acetic acid (75:25:1, v/v/v) as the developing system; cyathin A4 Rf 0.10 gave a brown oil, which on further chromatography gave cyathin A4 as a white foam. Tri-p-bromobenzoate: purified by preparative TLC, developed with benzene-acetone (49:1, v/v). Biological Activity A paper disc-agar plate method for the assay of antibiotic substances was used. Grampositive as well as gram-negative bacteria were sensitive to cyathin, but the former were the more sensitive. All species of Micrococcus tested proved to be sensitive. Staphylococcus aureus 'penicillin-sensitive' as well as 'penicillin-resistant' strains were found to be sensitive. However, E. coli, Serratia sp., and Proteus mirabilis showed little sensitivity. Among pathogenic forms, only Pneumococcus multocida, Corynebacterium diphtheriae, Clostridium welchii, Diplococcus pneumoniae, Streptococcus pyogenes,

756

21. Cyathanes

Neisseria meningitidis, and Haemophilusparainfluenzae were sensitive. None of these pathogenic forms, however, exhibited large zones of inhibition. On the other hand, Agrobacterium tumefaciens (the cause of crown gall disease of many horticultural plants) was found to be the most sensitive of all the organisms screened. All fungi tested against cyathin were sensitive to some degree. Most of the sensitive fungi belong to the class Deuteromycetes. The most striking examples of fungi sensitive to cyathin included species of Aspergillus, Penicillium, Fusarium, Trichoderma, Chaetomium, and Gliocladium. However, none of the Phycomycetes proved to be sensitive to cyathin. The same was true of yeasts. Of the six 'dermatophytes' tested, all showed some degree of sensitivity to cyathin. The most striking examples were Microsporum canis, Trichophyton rubrum, and T. terrestre. Spectral Data IR: (CHCI3) 3600, 3400, 1700(w), 1650(m), and 1375cm l. Tri-p-bromobenzoate: (CHCI3) 1715 1680, 1580, and 1480cm "1.

IH NMR: (CDCI3) 4.02(1H, narrow m); 5.6(1H, m); 5.79(2H, narrow m); 6.0(3H, disappeared on DzO exchange); 6.62(2H, m); 9.02(3H, s); 9.04(3H, s); and 9.07ppm (3H, d, splitting 6Hz). Tri-p-bromobenzoate (CDCI3) 2.0-2.5(12H, aromatic protons); 3.70(1H, narrow m); 4.22(1H, t); 5.04(2H, m); 5.80(2H, AB part of ABX system); 6.72(1FI, q); 7.0-8.0 (several multiplets); 8.0-8.7(complex); 8.84(3H, s); 8.96(3H, d, splitting 7I-Iz); and 9.02ppm (3H, s). Mass Data: HREIMS: 334.2146re~e; anal. calcd for C20H3004 334.2144; C, 71.82, H, 9.04; found: C, 72.06, H, 8.53. Tri-p-bromobenzoate: anal. calcd for C41H39OTBr3:C, 55.72; H, 4.45; O, 12.67; Br, 27.16; found: C, 56.00; H, 4.49; O, 12.72; Br, 27.25. Reference A. D. Allbutt, W. A. Ayer, H. J. Brodie, B. N. Johri, and H. Taube; Cyathin, a new antibiotic complex produced by Cyathus helenae; Can. J. Microbiol., Vol. 17, pp. 1401-1407(1971).

21. Cyathanes

757

Common/Systematic Name Cyathin B2 Molecular Formula/Molecular Weight C20H2502, M'W = 300.20893 17 1 16

18 5

CHO General Characteristics Cyathin B2 was obtained as a viscous syrup. Fungal Source Cyathus earlei is a tropical or subtropical species of bird's nest fungus which is known to occur in Cuba, Puerto Rico, Mexico, and Hawaii. Isolation/Purification The mycelium was extracted with ethyl acetate aider 65 days growth on Brodie medium which gave six compounds belonging to the "cyathin" group of diterpenoids. The ethyl acetate extract was washed with water, dried over sodium sulfate, and concentrated to a viscous brown oil. The crude extract was subjected to silica gel column chromatography eluted with SkeUysolve B-benzene to give cyathin B2. Biological Activity Moderate antibiotic activity against Staphylococcus aureus. Spectral Data UV;

~,~"

228nm (6=6,100).

IR:

(CC14) 2710, 1710, 1698, and 1645cm"l. IH NIVIR:

(CCIa) 0.99(s, 3H); 1.02(d, J=6Hz, 6H); 1.10(s, 3H); 3.32(d, J=14Hz, H on C-13); 3.64(dd, J=14 and 1Hz, H on C-13); 6.60(m, C-11, H); and 9.38ppm (s, 1H).

758

21. Cyathanes

Mass Spectrum: HREIMS: 300.2092m/e (M+); exact mass calcd for C20H2802 300.2090. Reference W. A. Ayer and S. P. Lee, Metabolites of bird's nest fungi. Part II. Diterpenoid metabolites of Cyathus earlei Lloyd; Can. J. Chem., Vol. 57, pp. 3332-3337(1979).

21.

Cyathanes

759

Common/Systematic Name Cyathin B3 Molecular Formula/Molecular Weight C2oH2803, M W -- 3 1 6 . 2 0 3 8 4 17

19

8

-

HO

CHO

General Characteristics Colorless needles from ether-pentane; mp., 130-132 ~C. Isolation/Purification The ethyl acetate soluble metabolites of Cyathus helenae were chromatographed on a column containing silicic acid containing electronic phosphor using chloroform (0.75% EtOH) as eluent. The progress of the separation was monitored with a short wavelength UV lamp. Crystals of the mixture were applied to TLC plates coated with silica gel-10% AgNO3 and developed with Skellysolve B-acetone (3:2, v/v). Two overlapping bands were detected by UV. These were removed from the plates in 3 fractions; the top and bottom fractions were rechromatographed on silica gel. In this way, it was possible to obtain cyathin B3 only slightly contaminated with cyathin C3 (as judged by NMR and MS). Cyathin B3 methyl ketal was purified from cyathin C3 methyl ketal by preparative TLC using silica gel - 10% AgNO3 (solvent system, Skellysolve B-acetone (3:1, v/v), Re values 0.58 cyathin B3 methyl ketal and 0.51 cyathin C3 methyl ketal). Neither could be induced to crystallize. Biological Activity The fungus Cyathus helenae grown on liquid culture produced a mixture of substances showing antibiotic activity, including cyathin B3. Spectral Data Optical Rotation: [a]ff 4 -105"C (c= 0.1, in isooctane). UV:

~.~,o,~

233nm (e 5,300)

760

21. Cyathanes

CD: (c= O.1, in isooctane) AE346-0.97 and AE235-2.78. IR; (CC14) 2850, 2720, 1692(s), and 1625cm1. ~H NMR: (CC14) 9.88(s, H-15); 7.00(s, H-13); 4.97(br s, H-11); 3.26(s, methoxyl); 2.93(septet, H-18, J=7Hz); 2.3-1. l(complex); 0.98(d, J=7Hz, one isopropyl methyl), 0.94(s, 3H, H-16 or -17); 0.90(s, 3H, H-17 or -16); and 0.89ppm (d, J=7Hz, one isopropyl methyl). Mass Spectrum: LREIMS: 330(24%), 315(14), 301(20), 299(23), 255(21), 227(23), 204(21), 199(21), 189(100), 175(31), 161(33), 133(35), 119(47), 105(63), 91(65), 55(67), and 41m/e (86); exact mass measurement 330.2184 (calcd for C2~H3oO3,330.2195). Appropriate parent peak at role 330 and no M+ -2 peak. Reference William A. Ayer and Leslie L. Carstens; Diterpenoid Metabolites of Cyathus helenae, Cyathin B~ and Cyathin C3, Can. J. Chem., Vol. 51, pp. 3157-3160(1973).

21. Cyathanes

761

Common/Systematic Name Cyathin C3; 1,2-Dehydrocyathin Ba Molecular Formula/Molecular Weight C2oH2603; M W -- 314.18819 17 1

8

7

2

..,,"

2

0

15

19

-

HO

CliO

General Characteristics of Mixture (Cyathin C3 and Cyathin B3) Colorless needles from ether-pentane; mp., 131-133~ Fungal Source The bird's nest fungus Cyathus helenae. Isolation/Purification Ethyl acetate soluble metabolites of Cyathus helenae were chromatographed on a column containing silicic acid containing electronic phosphor using chloroform (0.75% EtOH) as eluent. The progress of the separation was monitored with a short wavelength UV lamp. Crystals of the mixture were applied to TLC plates coated with silica gel-10% AgNO3 and developed with SkeUysolve B-acetone (3:2, v/v). Two overlapping bands were detected by UV. These were removed from the plates in 3 fractions; the top and bottom fractions were rechromatographed on silica gel. In this way, it was possible to obtain cyathin B3 only slightly contaminated with cyathin C3 (as judged by NMR and MS). Biological Activity The fungus Cyathus helenae grown on liquid culture produced a mixture of substances showing antibiotic activity, including cyathin C3. Spectral Data (Cyathin C3 and Cyathin B3 mixture) UV:

Z ~ "=~ 233nm (c=6,800). IR:

(CCI4) 3585, 3350(br), 3050(w), 2810, 2710, 1692(v.s.), and 1625cm"~ (w).

762

21. Cyathanes

IH N]k,IR:

(CDCI3) 9.91(s),9.61(s),7.01(s),6.6-6.2(complex),and 5.01ppm (m), methyl region complex. Mass Spectrum: 314(11), 301(22), 299(5), 273(11), 255(12), 204(23), 203(23), 201(21), 189(58), 187(21), 175(14), 173(10), 161(28), 159(17), 119(37), 105(39), 91(46), 55(57), and 41m/e (100%). Cyathin C3 methyl ketal: The spectroscopic properties are similar to those of cyathin B3 except for those attributable to the cyclopentadiene moiety. Optical Rotation: [eC]D24 -89~ (c=0.043, in isooctane). UV;

~~e

236(e=7,400) and 257nm (sh) (4,300).

IR;

(CCh) Characteristic olefinic C-H stretching bands appeared at 3050, 2850, 2810, 2720, 1693(s), and 1625cm"1. CD: (c=0.043, in isooctane) AE356-1.23, AE25s-4.31, and Ae2a5 -9.23. ~H NMR: (CC14) 9.92(s, H-15); 7.03(s, H-13); 6.20 and 6.10(AB quartet, H-1 and-2, J=5.SHz); 5.05(m, H-11); 3.29(s, methoxyl); 2.95(septet, H-18, J=7I-Iz); 2.5-21(complex, 2H); 1.8-1.1 (complex); 1.09 and 1.03(two nonequivalent isopropyl methyls, J's=7Hz); 0.88(s, 3H, H- 16 or - 17); and 0.77ppm (s, 3H, H- 17 or H- 16). The olefinic protons at C-1 and C-2 appear as an AB quartet (J=5.5Hz) centered at 6.15ppm. Mass Spectrum: 328(39), 313(13), 299(25), 295(21), 253(25), 225(20), 202(27), 187(100), 173(28), 159(23), 145(32), 133(20), 119(24), 105(22), 101(45), 91(25), 75(77), 55(16), and 4 lm/e (29); exact mass measurement 328.2042 (caicd for C2~H2sO3,328.2039). Reference William A. Ayer and Leslie L. Carstens; Diterpenoid Metabolites of Cyathus helenae, Cyathin B3 and Cyathin C3; Can. J. Chem., vol. 51, pp. 3157-3160(1973).

21. Cyathanes

763

Common/Systematic Name Cyathin C5 Molecular Formula/Molecular Weight C2oH2605, M W ' -" 3 4 6 . 1 7 8 0 2

..,"

OH

CliO General Characteristics Colorless crystals from ethyl ether-pentane; mp., 225-226 ~ Fungal Source

Cyathus helenae.

Isolation/Purification The active constituents of the culture broth were extracted with ethyl acetate. Examination of the crude extract (referred to as cyathin) by TLC revealed the presence of at least eight components. A solution of the crude cyathin complex was analyzed by preparative TLC, developed with benzene-dioxane-acetic acid (100:25:1, v/v). Under UV, bands were detected at 1%0.54 (cyathin C5), 0.51 (2,4,5-trihydroxybenzaldehyde), 0.27 (cyathin A3), and 0.06 (cyathin A4). The material with 1%0.54 was eluted from the adsorbent with ethermethanol (10:1, v/v) to give a brown oil. Further purification by preparative TLC yielded a material which crystallized from ether-pentane to give colorless crystals of cyathin Cs. Biological Activity Gram-positive as well as gram-negative bacteria were sensitive to cyathin, but the former were more sensitive. All species of Micrococcus tested proved to be sensitive. Staphylococcus aureus 'penicillin-sensitive' as well as 'penicillin-resistant' strains were found to be sensitive. However, E. coli, Serratia sp., and Proteus mirabilis showed little sensitivity. Among pathogenic forms, only Pneumococcus multocida, Corynebacterium

diphtheriae, Clostridium welchiL Diplococcus pneumoniae, Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus parainfluenzae were sensitive. None of these pathogenic forms, however, exhibited large zones of inhibition. On the other hand,

Agrobacterium tumefaciens (the cause of crown gall disease of many horticultural plants) was found to be the most sensitive of all the organisms screened. All fungi tested against cyathin were sensitive to some degree. Most of the sensitive fungi belonged to the class Deuteromycetes. The most striking examples of fungi sensitive to cyathin included species

of Aspergillus, Penicillium, Fusarium, Trichoderma, Chaetomium, and Gliocladium.

764

21. Cyathanes

However, none of the Phycomycetes proved to be sensitive to cyathin. The same was true of yeasts. Of the six 'dermatophytes' tested, all showed some degree of sensitivity to cyathin. The most striking examples were Microsporum canis, Trichophyton rubrum, and

T. terrestre.

Spectral Data

IR: (Nujol) 3400, 3200, 1695, 1675, 1235, and 1220cm"i. 1H NMR:

(acetone-d6) 0.1(IH, s);5.16(2H, m); 5.9-6.6(5-7H,m); 8.92(3H, d, splitting7Hz); 9.05(3H, s);and 9.15ppm (3H, s). Mass Data: HREIMS: 346.1774m/e; anal calcd for C20H2605,346.1780: C, 69.34; H, 7.57; O, 23.09; found: C, 68.85; H, 7.66; O, 23.47; (Tri-p-bromobenzoate) anal calcd for C41H3907Br3: C, 55.72; H, 4.45; O, 12.67; Br, 27.16; found: C, 56.00; H, 4.49; O,12.72; Br, 27.25. Reference A. D. Allbutt, W. A. Ayer, H. J. Brodie, B. N. Johri, and H. Taube; Cyathin, a new antibiotic complex produced by Cyathus helenae; Can. J. Microbiol., Vol. 17, pp. 1401-1407(1971).

21. Cyathanes

765

Common/Systematic Name Allocyathin B2 Molecular Formula/Molecular Weight C2oHzsO2; MW = 300.20893 17 1

g 8

..-'" OH

2

i|l H 19 20

11

CHO 15

General Characteristics Allocyathin B2 was obtained as a yellow syrup which showed a single spot on TLC; [a]o 25 + 87.1 ~ (c=0.95, in CHaOH). Fungal Source Cyathus earlei is a tropical or subtropical species of bird's nest fungus which is known to occur in Cuba, Puerto Rico, Mexico, and Hawaii. Isolation/Purification The mycelium was extracted with ethyl acetate after sixty-five days growth on Brodie medium which gave a crude extract containing six compounds belonging to the "cyathin" group of diterpenoids. The ethyl acetate extract was washed with water, dried over sodium sulfate, and concentrated to a viscous brown oil. The crude extract was subjected to column chromatography over silica gel by elution with benzene which afforded allocyathin B2. Biological Activity Moderate antibiotic activity against Staphylococcus aureus. Spectral Data UV:

~,"m~=~

266(e=5,150) and 325nm (12,100).

IR:

(CC14) 3600, 2720, 1675, and 1570cm "l.

~H NMR: (eel4) 0.96 and 1.05(each 3H, d, J=6Hz); 0.97(s, 3H); 1.01(s, 3H); 2.80(heptet, J=6Hz, H on C-18); 3.09(dd, ,/=18 and 6Hz, 1H on C-13); 3.63(dd, J's 6 and 1Hz, H

766

21. Cyathanes

on C-14); 5.84(d, J=8Hz, H on C-10); 6.68(dd, J=8 and 1Hz, H on C-11); and 9.37ppm (s, 1H). Mass Data: HREIMS: 300.2092; exact mass calcd, for C20H250 2 300.20893; anal. calcd, for C20H2sO2: C 80.02, H 9.40; found: C 80.04, H 9.70. Reference W. A. Ayer and S. P. Lee; Metabolites of bird's nest fungi. Part II. Diterpenoid metabolites ofCyathus earlei Lloyd; Can. J. Chem., Vol. 57, pp. 3332-3336(1979).

21. Cyathanes

767

Common/Systematj.c Name Allocyathin B3 Molecular Formula/Molecular Weight C20H2803, M3cV = 316.20384

1 16 ..,,

-

H5

0

\15

CH2OH

CH2OH

General Characteristics In the solid state allocyathin B3 existed in the hydroxyketone form. Acetylation gave the diacetyl derivative of the hydroxyketone form, whereas treatment with methanolic HCI gave an internal ketal. Allocyathin B3 could only be crystallized after it had been subjected to an acetylation-deacetylation procedure, gecrystallization from Skellysolve B-benzene; mp., 143-144~ Fungal Source The bird's nest fungus, Cyathus helenae and C. africanus. Isolation/Purification The original isolation procedure was based on a combination of column and TLC of the ethyl acetate extract of the culture broth. A material with Re 0.4 which appeared chromatographically pure was obtained and designated cyathin As. Careful inspection of the NMR spectrum of the acetyl derivative indicated that this material consisted of two components, allocyathin B3 and its dihydro derivative cyathin A3. Chromatography on silica gel impregnated with 10% silver nitrate separated the two components (solvent Skellysolve B-acetone, 2:3, v/v, Re 0.51). Biological Activity Possessed antibiotic activity. Spectral Data UV:

The UV spectrum has a somewhat unsymmetrical maximum at 235nm (MeOH), e=5,000 resulting from two overlapping chromophores. Acetylated allocyathin B3: The UV spectrum (isooctane) showed maxima at 325(e=140), 254(4800), and 225nm (9,000).

768

21. Cyathanes

IR:

Spectrum in chloroform displayed hydroxyl absorption and a broad band of medium intensity at 1650cm"~. This band was assigned to an a,13-unsaturated carbonyl group and it appeared in the same position in the solid-phase spectrum but was much stronger and sharper; other absorptions were 3580 and 3400cm~. Acetyl derivative: 1745, 1685, and 1655(sh) cm~ attributed to an a,13-unsaturated carbonyl system. CD: Spectrum displayed a positive maximum at 327nm with a dichroic absorption + 1.6, assigned to an a,13-unsaturated carbonyl group. IH NMR:

The NMR spectrum had one readily analyzable feature: an AB quartet centered at 6.26ppm, J=5Hz; the remainder of the spectrum was poorly defined (tautomerism?) and of limited value in structural studies. Allocyathin B3 diacetyl derivative (CDCI3): H- 1, 6.34, d, J=5.5Hz; H-2, 6.31, d, J=5.5Hz; H-13, 6.15, ddd, 1.5, 1.5, 0.5Hz; H-11, 5.39, ddd, 6.5, 6.5, 0.5Hz; H-15a, 4.75, dd, J=14.5 and 1.5Hz; H-15b, 4.68, dd, ,/=14.5 and 1.5Hz; H-18, 2.96, qq, ,/--7.0 and 7.0; H-5, 2.87, qq, J=10.5 and 3.0Hz; H-10a, 2.56, ddd, ,/=-14, 6.5, and 3.0; H10b, 2.27, ddd, ,/=14, 10.5, and 6.5Hz; H-7, H-8, 1.9-1.1, u; H-16, H-17, 1.04, 1.00, s, s; H-19, H-20, 1.12, 1.10, d, d, ,/--7 and 7Hz; C-11 or C-15 OAC, 2.12 or 2.05ppm, s, S.

Mass Spectrum: Composition C20H2803 was determined by high resolution mass spectrometry; calcd, 316.2039; found, 316.2031. TLC Data Re 0.4 using chloroform-methanol, 9:1 (v/v), on 10% silver nitrate silica gel. Reference W. A. Ayer and H. Taube; Metabolites of Cyathus helenae: A New Class of Diterpenoids; Cand. J. Chem., Vol. 51, pp. 3842-3854(1973).

21. Cyathanes

769

Common/Systematic Name Neoallocyathin A4 Molecular Formula/Molecular Weight C2oH3oO4, ~

= 334.21441

""

H()

0

CH20H

General Characteristics Neoallocyathin ,M acetonide, C23H3404; mp., 144.5-146 ~C (from ether-pentane). Fungal Source Cyathus he lenae. Isolation/Purification (Neoallocyathin A4 acetonide) The crude metabolites of C. helenae were chromatographed over silicic acid using 1.5% MeOH in CHCI3 as eluant. All fractions showing peaks at 334m/e were combined and subjected to TLC on silica gel G (E. Merck) (containing 5% added inorganic phosphor) using acetone-Skellysolve B (7:3, v/v) as the solvent system. The band centered at Re 0.66 was removed and eluted with ether to give a clear oil. The latter material was again subjected to TLC with silica gel G impregnated with 10% AgNO3 developed with acetone-Skellysolve B (7:3, v/v). After drying, the plate was sprayed lightly with 2',7'-dichlorofluorescein and observed under long wave UV light. Neoallocyathin A4 acetonide appeared at Re 0.49. Spectral Data (Acetonide) UV"

/~MeOH

max

232(6=12,000) and 322nm (100).

IR~

(CHCI3) 1685(sh) and 1635cm'~; (neat) 3400 br, and 1642cm"l br. CD: (c=0.001 M in MeOH) E300 -0.18 and e270 -0.23.

770

21. Cyathanes

IH Nh/IR: (CDCl3) 1.01(s, 3H, C-16, CH3); 1.06(d, 3H, J=7Hz) and 1.10(d, 3H, J=-7ilz, isopropyl methyls); 1.24(s, 3H, C-17 CH3); 4.0-4.5(m, 3H, C-l 1 and C-15 H's); and 6.03ppm (bs, 1H, C-13 H): (acetonide)(CDCIa) 1.03(s, 3H, C-16, CHa); 1.08(apparent t, 6H, isopropyl CHa's); 1.24(s, 3H, C-17, CH3); 1.40, 1.50(each 3H singlets, acetonide methyls); 4.15-4.35(m, 3H, C-15 and C-11 H's); and 7.25ppm (bs, 1H, C-13 H). Mass Spectrum: HR IMS: 334.2138; exact mass calcd for C2oH3oO4334.2144: NeoaUocyathin A4 acetonide 374.7452re~e; exact mass calcd for C23H3404 374.2457; LREIMS: 374 (M+, 45%), 359(11), 189(26), 149(21), 79(25), 61(30), and 43m/e (100). TLC Data (Neoallocyathin A4 acetonide) Subjected to TLC on silica gel G impregnated with 10% AgNO3 using acetone-Skellysolve B (7:3, v/v). Atter drying, the plate was sprayed lightly with 2',7'-dichlorofluorescein and observed under long wave UV light. It appeared at Re 0.49. Reference W. A. Ayer, L. M. Browne, J. R. Mercer, D. R. Taylor, and D. E. Ward; Metabolites of bird's nest fungi. Part 8. Some minor metabolites of Cyathus helenae and some correlations among the cyathins; Can. J. Chem., Vol. 56, pp. 717-721(1978).

21. Cyathanes

771

Common/Systematic Name Cyafrin A4 Molecular Formula/Molecular Weight C2oH3o04; M W -- 334.21441

HO

HO

CH20H

General Characteristics Cyafrin A4 crystallized from methanol-benzene as colorless needles; mp., 200-201 ~C; [a]D25 -125 ~ (C=0.4, in MeOH). Fungal Source

Cyathus africanus (Nidulariaceae).

Isolation/Purification The crude extract was chromatographed over silicic acid (MN-Kieselgel) using chloroform-methanol (19:1, v/v) as eluant. Fractions were monitored by TLC (solvent system B). Fractions 101-110 contained a mixture of cyathin A3 and allocyathin B3 which was separated into its components by preparative TLC over 10% AgNOa-silica gel (solvent system A). Fractions 123-130 contained cyafrin B4 and allocyafrin B4. Separation by preparative TLC (solvent system D) gave cyafrin B4 (Re 0.6) and aUocyafrin B4 (Re 0.5). Fractions 228-254 yielded crude cyafrin A4 which was purified by recrystallization from acetone. Fractions 255-276 gave a mixture of cyafrin A4 and cyafrin As, which was separated by preparative TLC (solvent system C triple elution). Cyafrin A5 was purified by recrystallization from acetone-benzene. Preparative TLC was carried out on 0.75mm layers of silica gel G (W. Merck, Darmstadt) containing 1% electronic phosphor (General Electric, Cleveland). The solvent systems used were: A, Skellysolve B-acetone (3:1, v/v), B, benzene-acetone-acetic acid (75:25:1, v/v); C, chloroform-acetonitrile (3:2, v/v); and D, benzene-methanol-acetic acid (18:2:1, v/v). Spectral Data

UV:

~,~x 230nm (sh) (e=1500).

772

21. Cyathanes

IR; (KBr) 3300(br) and no carbonyl. ~H NMR: (pyridine) Indicated a mixture of hydroxyketone and hemiacetal forms. Mass Spectrum:

334.2152m/e (M*); exact mass calcd for C20H3oO4, 334.2144.

Reference W. A. Ayer, T. Yoshida, and D. M. J. Van Schie; Metabolites of bird's nest fungi. Part 9. Diterpenoid metabolites of Cyathus africanus Brodie; Can. J. Chem., Vol. 56, pp. 21132120(1978).

21. Cyathanes

773

Common/Systematic Name Cyafrin B4 Molecular Formula/Molecular Weight C2oH2804; M W = 332.19876

O

.'"

HO

...~

CH2OH

O

OH

CH2OH

General Characteristics Cyafrin B4 was obtained as a colorless syrup; [a]D25 -156.5 ~ (C=0.34, in CHCIs). Fungal Source

Cyathus africanus.

Isolation/Purification The crude extract was chromatographed over silicic acid (MN-Kieselgel) using chloroform-methanol (19:1, v/v) as eluant. Fractions were monitored by TLC (solvent system B). Fractions 101-110 contained a mixture of cyathin A3 and allocyathin B3 which was separated into its components by preparative TLC over 10% AgNO3-silica gel (solvent system A). Fractions 123-130 contained cyafrin B4 and allocyafrin B4. Separation by preparative TLC (solvent system D) gave cyafrin B4 (Re 0.6) and allocyafrin B4 (Re 0.5). Fractions 228-254 yielded crude cyafrin A4 which was purified by recrystallization from acetone. Fractions 255-276 gave a mixture of cyafrin A4 and cyafrin As, which was separated by preparative TLC (solvent system C triple elution). Cyafrin A5 was purified by recrystallization from acetone-benzene. Preparative TLC was carried out on 0.75mm layers of silica gel G (W. Merck, Darmstadt) containing 1% electronic phosphor (General Electric, Cleveland). The solvent systems used were: A, Skellysolve B-acetone (3:1, v/v), B, benzene-acetone-acetic acid (75:25:1, v/v); C, chloroform-acetonitrile (3:2, v/v); and D, benzene-methanol-acetic acid (18:2:1, v/v). Spec.tral Data UV:

~,~x 241rim (e=l 1,000). IR:

(CHCI3) 3630, 3420, 1695, and 1623cm "l.

774

21. Cyathanes

1H NMR: Poorly resolved (tautomeric equilibrium), Mass Spectrum:

332.1983m/e (M+); exact mass calcd for CzoH2sO4, 332.1988.

Reference W. A. Ayer, T. Yoshida, and D. M. J. Van Schie; Metabolites of bird's nest fungi. Part 9. Diterpenoid metabolites of Cyathus africanus Brodie; Can. J. Chem., Vol. 56, pp. 21132120(1978).

21. Cyathanes

775

Common/Systematic Name Cyafrin A5 Molecular Formula/Molecular Weight C2oH3oO5, M W = 350.20932

HO

.,,"

OH

CH2OH General Characteristics Cyafrin As, was obtained as colorless needles from acetone-benzene; mp., 208-209"C; [a]o 25 -77.9 ~ (c=0.154, in MeOH). Fungal Source

Cyathus africanus.

Isolation/Purification The crude extract was chromatographed over silicic acid (MN-Kieselgel) using chloroform-methanol (19:1, v/v) as eluant. Fractions were monitored by TLC (solvent system B). Fractions 101-110 contained a mixture of cyathin A3 and allocyathin B3 which was separated into its components by preparative TLC over 10% AgNO3-silica gel (solvent system A). Fractions 123-130 contained cyafrin B4 and allocyafrin B4. Separation by preparative TLC (solvent system D) gave cyafrin B4 (Rf 0.6) and allocyafrin B4 (Rf 0.5). Fractions 228-254 yielded crude cyafrin A4 which was purified by recrystallization from acetone. Fractions 255-276 gave a mixture of cyafrin A4 and cyafrin As, which was separated by preparative TLC (solvent system C triple elution). Cyafiin A5 was purified by recrystallization from acetone-benzene. Preparative TLC was carried out on 0.75mm layers of silica gel G (W. Merck, Darmstadt) containing 1% electronic phosphor (General Electric, Cleveland). The solvent systems used were: A, Skellysolve B-acetone (3:1, v/v), B, benzene-acetone-acetic acid (75:25:1, v/v); C, chloroform-acetonitrile (3:2, v/v); and D, benzene-methanol-acetic acid (182:1, v/v). ..Spectral Data

(film) 3350(br) and 1703cm"~ (w).

776

21.

Cyathanes

IH NMR: (CsDsN + D20) 0.88 and 0.99ppm (each 3H, d, d=7Hz, isopropyl methyls); 1.34(s, 3H); 1.36(s, 3H); 4.18(s, 1H); 4.20 and 4.30(dd, J=18Hz); 4.22(t, 1H, d=8Hz); and 4.75ppm (m, 1H). 13C N M ~ :

(CD3OD) 12.3; 17.0; 21.2; 22.5(CH3); 28.5; 29.6; 35.6; 37.1; 58.8(CH2); 26.4; 40.8; 56.9; 74.0; 82.4(CI-1); 43.7; 49.6; 63.9; 164.3(C); 136.5; and 138.0ppm (C=C). Mass Spectrum: HREIMS 350.2093m/e (M+); exact mass calcd, for C20H3005 , 350.2092. Reference W. A. Ayer, T. Yoshida, and D. M. J. Van Schie; Metabolites of bird's nest fungi. Part 9. Diterpenoid metabolites of Cyathus africanus Brodie; Can. J. Chem., Vol. 56, pp. 21132120(1978).

21. Cyathanes

777

Common/Systematic Name Allocyafrin B4 Molecular Formula/Molecular Weight C20H2804, ~

= 332.19876

o �9 -"

H()

o

CI-120H

General Characteristics Allocyafrin B4 was obtained as a colorless syrup; [a]D25 -157.8 ~ (C----0.27,in CHCI3). Fungal Source

Cyathus africanus.

Isolation/Purification The crude extract was chromatographed over silicic acid (MN-Kieselgel) using chloroform-methanol (19:1, v/v) as eluant. Fractions were monitored by TLC (solvent system B). Fractions 101-110 contained a mixture of cyathin A3 and allocyathin B3 which was separated into its components by preparative TLC over 10% AgNO3-silica gel (solvent system A). Fractions 123-130 contained cyafrin B4 and allocyafrin B4. Separation by preparative TLC (solvent system D) gave cyafiin B4 (Rf 0.6) and allocyafrin B4 (Re 0.5). Fractions 228-254 yielded crude cyafrin A4 which was purified by reerystallization from acetone. Fractions 255-276 gave a mixture of cyafrin A4 and cyafi~ As, which was separated by preparative TLC (solvent system C triple elution). Cyafiin A5 was purified by recrystallization from acetone-benzene. Preparative TLC was carried out on 0.75 mm layers of silica gel G (W. Merck, Darmstadt) containing 1% electronic phosphor (General Electric, Cleveland). The solvent systems used were: A, Skellysolve B-acetone (3:1, v.v); B, benzene-acetone-acetic acid (75:25:1); C, chloroform-acetonitrile (3:2); and D, benzene-methanol-acetic acid (18:2:1). Spectral Data

UV:

~,,=x 239nm (e=16,400).

778

21. Cyathanes

IR; (CHCI3) 3600, 3440, 1700, 1680 (sh), and 1600cm1. 1H ~ : (CDCI3) 1.08(s, 3H); 1.12(s, 3H); 1.22 and 1.26(each 3H, d, J=7Hz); 4.42(rn, 3H); 5.98(bs, 1H); and 6.04ppm (bs, 1H).

Mass Spectrum: HREIMS: 332.1985m/e (M+); exact mass calcd for C20H2804, 332.1988. Reference W. A. Ayer, T. Yoshida, and D. M. J. Van Schie; Metabolites of bird's nest fungi. Part 9. Diterpenoid metabolites of Cyathus africanus Brodie; Can. J. Chem., Vol. 56, pp. 21132120(1978).

21. Cyathanes

779

Common/Systematic Name Cyathatriol Molecular Formula/Molecular Weigh_t. C2oH3203; M W = 320.23515 17 1 16

OH

18

HO

5

CI--120H

General Characteristics Cyathatriol was obtained as a white powder which crystallized from acetone; mp., 172-173~ Fungal Source Cyathus earlei is a tropical or subtropical species of bird's nest fungus which is known to occur in Cuba, Puerto Rico, Mexico, and Hawaii. Isolation/Purification The ethyl acetate extract of fungal mycelium after sixty-five days growth on Brodie medium gave six compounds belonging to the "cyathin" group of diterpenoids. The ethyl acetate extract was washed with water, dried over sodium sulfate, and concentrated to a viscous brown oil. The crude extract was subjected to silica gel column ehrornatography eluted with ether-benzene (2:3, v/v) which gave cyathatriol. Biological Activity Moderate antibiotic activity against Staphylococcus aureus. Spectral Data IR:

(film) 3300cm "l. 1H ~ : (acetone-d~) 0.80(s, 3H); 0".94(d, J=6Hz, 3H); 0.95(d, J=6Hz, 3H); 1.07(s, 3H); 3.00(m, H's on C-5 and C-18); 3.76(d, J=6Hz, H on C-14); 4.12(s, 2H on C-15), 4.85(ddd, J=8, 4, and 1Hz, H on C-11); and 5.87ppm (dd, J=6 and 1Hz, H on C-13).

780

21. Cyathanes

Mass Data: HREIMS: 320.2336re~e;exact mass calcd for C2oH320 3 320.2351; anal. ealcd for C2oH3203: C 74.94, H, 10.07; found: C 74.67, H 10.14%. Reference W. A. Ayer and S. P. Lee; Metabolites of bird's nest fungi. Part II. Diterpenoid metabolites ofCyathus earlei Lloyd; Can. J. Chem., Vol. 57, pp. 3332-3337(1979).

21. Cyathanes

781

Common/Systematic Name 11-O-Acetylcyathatriol Molecular Formula/Molecular Weight C22H3404; MW = 362.24571 17 1 16

OH 18

AcO

5

CI--120H

General Characteristics 11-O-Acetylcyathatriol crystallized from ether; mp., 165-166~ Fungal Source Cyathus earlei is a tropical or subtropical species of bird's nest fungus which is known to occur in Cuba, Puerto Rico, Mexico, and Hawaii. Isolation/Purification The ethyl acetate extract of the fungal mycelium after 65 days growth on Brodie medium gave six compounds belonging to the "cyathin" group of diterpenoids. The ethyl acetate extract was washed with water, dried over sodium sulfate, and concentrated to a viscous brown oil. The crude extract was subjected to silica gel column chromatography eluted with ether-benzene (1:4, v/v) to give 11-O-acetylcyathatriol. Biological Activity Moderate antibiotic activity against Staphylococcus aureus. Spectral Data_ IR:

(CC14): 3500 and 1710cmq. 1H N]VIR: (CDC13) 0.80(s, 3H); 0.97(d, J=6Hz, 6H); 1.10(s, 3H); 2.10(s, 3H); 3.02(br d, J=10Hz, H on C-5); 3.90(d, d=6Hz, H on C-14); 4.06(s, 2H on C-15); 5.86(dd, J=6 and 1Hz, H on C-13); and 5.95ppm (m, H on C-11). Mass Data: HREIMS: found: 362.2496re~e; exact mass calcd for C22H3404, 362.2457; anal calcd for C22H3404: C 72.87, H 9.45; found: C 72.51, H 9.54.

782

21. Cyathanes

Reference W. A. Ayer and S. P. Lee; Metabolites of bird's nest fungi. Part II. Diterpenoid metabolites ofCyathus earlei Lloyd; Can. J. Chem. Vol. 57, pp. 3332-3337(1979).

21. Cyathanes

783

Common/Svstematic Name 15-O-Acetylcyathatriol Molecular Formula/Molecular Weight C22H3404; M W -- 362.24571 17 1 16

OH

18 5

H0

CH2OAc

General Characteristics

15-O-Acetylcyathatriol was obtained as a viscous oil.

Fungal Source Cyathus earlei is a tropical or subtropical species of bird's nest fungus which is known to occur in Cuba, Puerto Rico, Mexico, and Hawaii. Isolation/Purification The ethyl acetate extract of the fungal mycelium gave six compounds belonging to the "cyathin" group of diterpenoids. The ethyl acetate extract was washed with water, dried over sodium sulfate, and concentrated to a viscous brown oil. The crude extract was subjected to silica gel column chromatography eluted with ether-benzene (1:9, v/v) which gave 15-O-acetylcyathatriol. Biological Activity Moderate antibiotic against Staphylococcus aureus. Svectral Data IR:

(CCh) 3600, 3500, and 1745em~. 1H N M R :

(CC14) 0.75(s, 3H); 0.98(d, J=6Hz, 6H); 1.07(s, 3H); 2.05(s, 3H); 3.00(m, 2H, H's on C-5 and C-18); 3.82(d, J=6Hz, H on C-14); 4.60(s, 2H on C-15); 4.70(m, H on C-11); and 5.65ppm (d, J=6Hz, H on C-13). Mass Data: HREIMS: 362.2479re~e; exact mass calcd for C22H3404 362.2457; anal c~lcd for C22H3404: C 72.87, H 9.45; found: C 72.58, H 9.48.

784

21. Cyathanes

Reference W. A. Ayer and S. P. Lee; Metabolites of bird's nest fungi. Part II. Diterpenoid metabolites of Cyathus earlei Lloyd; Can. J. Chem., Vol. 57, pp. 3332-3337(1979).

21. Cyathanes

785

Common/Systematic Name 11,15-O, O-Diacetylcyathatriol Molecular Formula/Molecular Weight C24H3605; M W = 4 0 4 . 2 5 6 2 7 17 1 16

OH

18 5

AcO

CH2OAc

General Characteristics 11,15-0, O-Diacetylcyathatriol crystallized from Skellysolve B-ether; mp., 123-124 ~ Fungal Source Cyathus earlei is a tropical or subtropical species of bird's nest fungus which is known to occur in Cuba, Puerto Rico, Mexico, and Hawaii. Isolation/Purification The ethyl acetate extract of the mycelium after sixty-five days growth on Brodie medium gave six compounds belonging to the "cyathin" group of diterpenoids. The ethyl acetate extract was washed with water, dried over sodium sulfate, and concentrated to a viscous brown oil. The crude extract was subjected to silica gel column chromatography; elution with ether-benzene (1:9, v/v) gave 15-O-acetylcyathatriol followed by 11,15-0, Odiacetylcyathatriol. Biological Activity Moderate antibiotic activity against Staphylococcus aureus. Spectral Data IR:

(CC14) 3600, 3500, and 1745cm"l. ~H NMR: (CC14) 0.70(s, 3H); 0.95(d,.d=6Hz, 6H); 1.10(s, 3H); 1.98(s, 3H); 2.00(s, 3H); 2.90(heptet, d=6Hz, H on C-18); 3.00(dd, J=10 and 1Hz, H on C-5); 3.70(d, J=6Hz, 1H); 4.34(dd, d=14 and 1Hz, H on C-15); 4.54(dd, d=14 and 1Hz, H on C-15); 5.82(dd, ,/=6 and 1Hz, H on C-13); and 5.85ppm (m, H on C-11).

786

21. Cyathanes

Mass Data: The highest peak in the mass spectrum corresponded to the molecular ion-H20; exact mass calcd for C2,I-I3404 (M+-H20): 386.2457; found: 386.2454m/e; anal calcd for C2,I-I3605; C 71.25, H 8.96; found: C 70.96, H 9.07(%). Reference W. A. Ayer and S. P. Lee; Metabolites of bird's nest fungi. Part II. Diterpenoid metabolites of Cyathus earlei; Can. J. Chem., Vol. 57, pp. 3332-3337(1979).

21. Cyathanes

787

Common/S.ystematic Name Sarcodonin A Molecular Formula/M.olecular Weight C2oH2803, M W = 316.20384

..,," ,,H Me

,H

(~H20H ~

H CHO

General Characteristics Yellow syrup; [a]D2t +917 ~ (C=0.1, in CHCIa); intense bitterness; threshold value was 3.1 10 9 "6M. Gave 2,4-dinitrophenylhydrazone, a red amorphous solid; mp., 220-222"C. Fungal Source

Sarcodon scabrosus.

Isolation/Purification Dried fruiting bodies of the fungus were extracted with methylene chloride. The neutral fraction was chromatographed on silica gel repeatedly (methanol-chloroform or benzene-ethyl acetate). Spectral Data UV"

~m,x 338nm (e=46,000). IR:

(KBr) 3400, 2950-2850, 2700, 1670, 1620, 1580, 1440, 1420, 1380, 1170, and 1040cm"1. ~H NMR: (CDCI3) 9.45(1H, s); 6.83(1H, dd, J=8.2, 2.3Hz), 6.19(1H, d, J=8.2Hz); 3.73(1H, d, J-5.8Hz); 3.58(2H, d, J-7.2Hz); 3.15(1H, dd, J=18.2, 5.8Hz); 2.95(1H, sextet, J=7.2Hz); 2.53(1H, d, J=18.4Hz); 2.52(1H, m); 2.41(2H, m); 1.72(4H, m), 1.35(1H, dt, J=13.9, 3.3Hz); 1.03(3H, s); 0.96(3H, s); and 0.95ppm (3H, d, J=7.2Hz). 13C NMR:

(CDCI3) 194.3, 154.2, 146.5, 144.5, 141.1,138.1, 119.8, 74.0, 66.4, 49.6, 48.3, 38.3, 36.4, 35.2, 33.6, 29.4, 29.3, 26.5, 24.3, and 15.9ppm.

788

21. Cyathanes

Mass Spectrum: HRMS" 316.2073m/e (M ~, calcd, for C2oH2803, 316.2037); EIMS: 316(M+, 100), 300(10), 298(15), 285(85), 211 (48), and 9 lm/e (88). Reference H. Shibata, T. Tokunaga, D. Karasawa, A. Hirota, M. Nakayama, H. Nozaki, and T. Tada; Isolation and Characterization of New Bitter Diterpenoids from the Fungus Sarcodon scabrosus; Agric. Biol. Chem., Vol. 53, pp. 3373-3375(1989).

21. Cyathanes

789

.Common/Systematic Name Sarcodonin G Molecular Formula/Molecular Weight C2oH2803; M W -- 316.20384

.,.""

O

llllll

Clio General Characteristics Colorless prisms; mp., 158-161 ~ threshold value, 3.1 10 9 .5 M.

[(~]D 21

-64.9 ~ (c=0.2, CHCI3). Intense bitterness;

Fungal Sourc.e

Sarcodon scabrosus.

Isolation/Purification Dried fruiting bodies of the fungus were extracted with methylene chloride. The neutral fraction was chromatographed on silica gel repeatedly (methanol-chloroform or benzene-ethyl acetate). Spectral Data UV~

~,~x 220nm. IR~

(KBr) 3400, 2900-2850, 2700, 1705, 1660, 1640, 1450, 1420, 1380, 1280, 1090, and 1050cm~.

~H NMR: (CDCI3) 9.3 I(1H, s); 6.71(1H, m); 3.72(1H, br, d, J=13.6Hz); 3.45(2H, d, J=7.3Hz); 3.42(1H, d, J=13.9Hz); 3.34(1H, d, J=12.0Hz); 3.16(1H, dd, ,/--13.4 and 6.4Hz); 3.02(1H, sextet, J=7.3Hz); 2.74(1H, m); 2.31(3H, m); 1.94(1H, dt, J=13.3 and 5.3Hz); 1.59(4H, m); 1.24(1H, dt, J=13.2 and 3.9Hz); 1.12(3H, s); 1.01(3H, s); and 0.96ppm (3H, d, J=7.3Hz). 13C NMR~ (CDCI3) 210.6, 192.4, 153.5, 141.1,135.9, 135.8, 65.8, 55.3, 49.8, 39.5, 37.7, 35.5, 35.2, 34.1, 32.7, 32.1, 28.6, 24.8, 15.6, and 12.7ppm.

790

21. Cyathanes

Mass Spectrum: HRMS: 316.2059m/e (M+, calcd, for C20H2803316.2038); EIMS: 316(M +, 55), 300(58), 284(35), 266(38), 255(36), 174(85), 121(98), 91role (100%). Reference H. Shibata, T. Tokunaga, D. Karasawa, A. Hirota, M. Nakayama, H. Nozaki, and T. Tada; Isolation and Characterization of New Bitter Diterpenoids from the Fungus Sarcodon scabrosus; Agric. Biol. Chem., Vol. 53, pp. 3373-3375(1989).

21. Cyathanes

791

Common/Syst .ematic Name Striatin A Molecular Formula/Molecular Weight C2sH40Os; MW = 504.27232 17

......'

20

,;"--k

O,

u

H

'

'sj~--O"bH "OAc

MeO General Characteristics Colorless crystals from MeOH; mp., 144-145~ they were soluble in methanol, ethanol, ethyl acetate, and chloroform, and were poorly soluble in water and cyclohexane. Striatin A crystallized with one molecule of methanol, forming a system of hydrogen bonds with the angular OH groups at C(2') and C(3'). The striatins give positive reactions with KMnO4, and a modified Kagi-Miescher reagent. Upon standing in solution the striatins undergo rapid transformation into several new compounds, some of which show antibiotic activity. All striatins were very sensitive to water in the presence of traces of acid resulting in typical changes which were in accord with the formation of ketoaldehydes. Fungal Source

Cyathus striatus.

Isolation/Purification The mycelium was extracted with methanol-acetone (2:1, v/v). The combined extracts were evaporated to dryness, the residue dissolved in methanol, and applied to a Sephadex LH-20 column and eluted with the same solvent. The fractions containing the striatins were pooled, concentrated to 1/10 of the original volume, and kept at 4~ After 2-3 days the crystals of striatin A were removed, and the mother liquid evaporated to dryness. The residue containing the striatins B and C was dissolved in chloroform and applied to a column with silica gel (Mallinckrodt). Striatins B and C were eluted with chloroform-ethanol (99:1, v/v) and chloroform-ethanol-methanol (97:1:2, v/v/v) and crystallized from chloroform (striatin B) and ethanol (striatin C). Biological Activity The striatins are highly active against fungi imperfecti and a variety of gram-positive bacteria, as well as against some gram-negative bacteria.

792

21. Cyathanes

Soectral Data UV:

The UV spectra of the striatins in ethanol showed no absorption maxima up to 220nm. 1H NMR: (CDCI3) The 5'-1-12and 4'-H formed an ABX-pattem (3.49, 3.93, and 4.98; J= 11, 9, and 5Hz); r-H and 15-H singlets (5.00 and 5.25); 14-H a doublet (4.19, J=10Hz); and 1 l-H, 13-H, and 10-1-12multiplets (6.04, 3.04, and 2.50). In striatin B an additional hydroxy group was attached to C(10). This followed from the NMR spectrum in which 10-H appeared at 4.84 as a doublet due to 8 Hz coupling to 11-H. The pseudoaxial orientation of this OH-group was deduced from the low field shift of 14-H (0.42), 18H(0.49), and 16-Me (0.23) induced by the nearby oxygen atom. As a consequence the dihedral angle between 5-H and 10-H became 90 ~ and no vicinal coupling was observed. Striatin C differed from B in lacking the acetyl group (2.15) which led to increased shielding of 4'-H (3.98). Mass Spectrum: 472m/e (M" -32) with prominent ion at M+-47 and a characteristic fragment ion at 203m/e (C15H23). TLC Data Merck silica gel plates: Benzene-methanol-ethyl acetate, 17:2:1, v/v/v; striatin ARt, 0.51; B, 0.39; and C, 0.24. cyclohexane-ethyl acetate, 1:1, v/v; R~, A, 0.38; B, 0.30; and C, 0.16. Benzene-acetone-acetic acid, 70:30:1, v/v/v; Re, A, 0.55; B, 0.47; and C, 0.26. References T. Anke, F. Oberwinkler, W. Steglich, and G. Hofle; The Striatins - New Antibiotics from the Basidiomycete Cyathus striatus (Huds. ex Pers.) Willd.; J. Antibiot., Vol. 30, pp. 221-225(1977). H. Hecht, G. Hofle, W. Steglich, T. Anke, and F. Oberwinlder; Striatin A, B, and C: Novel Diterpenoid Antibiotics from Cyathus striatus; X-Ray Crystal Structure of Striatin A; J. Chem. Sot., Chem. Commun., pp. 665-666(1978).

21. Cyathanes

793

Common/Systematic Name Striatin B Molecular Formula/Molecular Weight C28I-I4009; MW = 520.26723

~ ] 6 ot III16 L,;,,~..~iv H,~

'

H

l~176 0

HO 2'1,

19

3' 4'

MeO

iiH

Ac

General Characteristics Colorless crystals from MeOH; mp., 143-144~ soluble in methanol, ethanol, ethyl acetate, and chloroform, and poorly soluble in water and cyclohexane. Striatin B gave positive reactions with KMnO+, and a modified Kagi-Miescher reagent. Upon standing in solution it rapidly transformed into several new compounds, some of which showed antibiotic activity. All striatins were very sensitive to water in the presence of traces of acid resulting in typical changes which were in accord with the formation of ketoaldehydes. Fungal Source

Cyathus striatus.

Isolation/Purification The mycelium was extracted with methanol-acetone (2:1, v/v). The combined extracts were evaporated to dryness; the residue was dissolved in methanol, applied to a Sephadex LH-20 column, and eluted with the same solvent. The fractions containing the striatins were pooled, concentrated to 1/10 of the original volume, and kept at 4~ Atter 2-3 days the crystals of striatin A were removed, and the mother liquid evaporated to dryness. The residue containing the striatins B and C was dissolved in chloroform and applied to a column with silica gel (MaUinckrodt). Striatins B and C were eluted with chloroformethanol (99:1, v/v) and chloroform-ethanol-methanol (97:1:2, v/v/v) and crystallized from chloroform (striatin B) and ethanol (striatin C). Biological Activity The striatins are highly active against fungi imperfecti and a variety of gram-positive bacteria, as well as against some gram-negative bacteria.

794

21. Cyathanes

Spectral Data UV: The UV spectra of the striatins in ethanol showed no absorption maxima up to 220nm. IH NMR: (CDCI3) The 5'-H2 and 4'-H formed an ABX-pattern (3.49, 3.93, and 4.98; J=l 1, 9, and 5Hz); I'-H and 15-H singlets (5.00 and 5.25); 14-H a doublet (4.19, J=10Hz); and 1 l-H, 13-H, and 10-1-12multiplets (6.04, 3.04, and 2.50). In striatin B an additional hydroxy group was attached to C(10). This followed from the NMR spectrum in which 10-H appeared at 4.84 as a doublet due to 8Hz coupling to 11-H. The pseudoaxial orientation of this OH-group was deduced from the low field shitt of 14-H (0.42), 18H (0.49), and 16-Me (0.23) induced by the nearby oxygen atom. As a consequence the dihedral angle between 5-H and 10-H became 90 o, and no vicinal coupling was observed. Mass Spectrum: 488role (M +-32) with prominent ion at M+-47 and a characteristic fragment ion at 203m/e (C15H23). TLC Data Merck silica gel plates: Benzene-methanol-ethyl acetate, 17:2:1, v/v/v; striatin A: Rf, 0.51; B, 0.39; and C, 0.24. Cyclohexane-ethyl acetate, 1:1, v/v; Rf, A, 0.38; B, 0.30; and C, 0.16. Benzene-acetone-acetic acid, 70:30:1, v/v/v; Rf, A, 0.55; B, 0.47; and C, 0.26. References T. Anke, F. Oberwinkler, W. Steglich, and G. Hofle; The Striatins - New Antibiotics from the Basidiomycete Cyathus striatus (Huds. ex Pers.) Willd.; J. Antibiot., Vol. 30, pp. 221-225(1977). H. Hecht, G. Hofle, W. Steglich, T. Anke; and F. Oberwinkler; Striatin A, B, and C: Novel Diterpenoid Antibiotics from Cyathus striatus; X-Ray Crystal Structure of Striatin A; J. Chem. Soc., Chem. Commun., pp. 665-666(1978).

21. Cyathanes

795

Common/Systematic Name Striatin C Molecular Formula/Molecular Weight C26H3808, M W = 4 7 8 . 2 5 6 6 7 17

16

O,

Hf 19

HO

II1'

H

27,

0

3' 4'

2O

MeO

,,H

H

General Characteristics Colorless crystals from MeOH; mp., 144-145~ soluble in methanol, ethanol, ethyl acetate, and chloroform, and poorly soluble in water and cyclohexane. The antibiotic gave positive reactions with KMnO4, and a modified Kagi-Miescher reagent. Upon standing in solution the striatins undergo rapid transformation into several new compounds, some of which show antibiotic activity. All striatins were very sensitive to water in the presence of traces of acid resulting in typical changes which were in accord with the formation of ketoaldehydes. Fungal Source

Cyathus striatus.

Isolation/Purification The mycelium was extracted with methanol-acetone (2:1, v/v). The combined extracts were evaporated to dryness, the residue dissolved in methanol, applied to a Sephadex LH-20 column, and eluted with the same solvent. The fractions containing the striatins were pooled, concentrated to 1/10 of the original volume, and kept at 4~ Atter 2-3 days the crystals of striatin A were removed, and the mother liquid evaporated to dryness. The residue containing the striatins B and C was dissolved in chloroform and applied to a column with silica gel (Mallinckrodt). Striatins B and C was eluted with chloroformethanol (99:1, v/v) and chloroform-ethanol-methanol (97:1:2, v/v/v) and crystallized from chloroform (striatin B) and ethanol (striatin C). Biological Activity The striatins are highly active against fungi imperfecti and a variety of gram-positive bacteria, as well as against some gram-negative bacteria.

796

21. Cyathanes

Spectral Data UV: The UV spectra of the striatins in ethanol showed no absorption maxima up to 220rim. ~H NMR: (CDCI3) The 5'-1-12and 4'-H formed an ABX-pattern (3.49, 3.93, and 4.98; J=l 1, 9, and 5Hz); r-H and 15-H singlets (5.00 and 5.25); 14-H a doublet (4.19, J=10Hz); and 1 I-H, 13-H, and 10-1-12multiplets (6.04, 3.04, and 2.50). In striatin B an additional hydroxy group was attached to C(10). This followed from the NMR spectrum in which 10-H appeared at 4.84 as a doublet due to 8Hz coupling to 11-H. The pseudoaxial orientation of this OH-group was deduced from the low field shift of 14-H (0.42), 18H(0.49), and 16-Me (0.23) induced by the nearby oxygen atom. As a consequence the dihedral angle between 5-H and 10-H became 90 ~ and no vicinal coupling was observed. Striatin C differs from striatin B in lacking the acetyl group (2.15ppm) which leads to increased shielding of 4'-H (3.98ppm). Mass Spectrum: 446m/e (M +-32) with prominent ion at M+-47 and a characteristic fragment ion at 203m/e (C15H23). TLC Data Merck silica gel plates: Benzene-methanol-ethyl acetate, 17:2:1, v/v/v; striatin A: Re, 0.51; B, 0.39; and C, 0.24. Cyclohexane-ethyl acetate, 1:1, v/v; Re, A, 0.38; B, 0.30; and C, 0.16. Benzene-acetone-acetic acid, 70:30:1, v/v/v; Rr A, 0.55; B, 0.47; and C, 0.26. References T. Anke, F. Oberwinkler, W. Steglich, and G. Hofle; The Striatins - New Antibiotics from the Basidiomycete Cyathus striatus (Huds. ex Pers.) Willd.; J. Antibiot., Vol. 30, pp. 221-225(1977). H. Hecht, G. Hofle, W. Steglich, T. Anke; and F. Oberwinkler; Striatin A, B, and C: Novel Diterpenoid Antibiotics from Cyathus striatus; X-Ray Crystal Structure of Stfiatin A; J. Chem. Soc., Chem. Commun., pp. 665-666(1978).

Alliacolide and Related Metabolites

22

Alliacolide Alliacide Alliacol A (Alliacolide II) Alliacol B (12-Hydroxydehydroalliacolide) 11-Hydroxyalliacolide 12-Hydroxyalliacolide 12-Noralliacolide

797

This Page Intentionally Left Blank

22.

Alliacolide and Related Metabolites

799

Common/Systematic Name Alliacolide Molecular Formula/Molecular Weight C 15H2204; M W -- 266.15181

y

10 _-- s o

|l o

Me" 2 "CO 12

I"1

13

General Characteristics Crystals from ethyl acetate-light petroleum; mp., 192-194~ (corr.) (sealed capillary) (193~ [a]D2~ - 35 ~ (589), -62~ and -80 ~ (365nm) (c= 0.1, in CHCI3); [a]D - 33 ~ (C= 0.8, in CHCI3). Fungal Source

Marasmius alliaceus.

Isolation/Purification Isolated from the ether extract of the culture broth by a combination of sublimation (the compound sublimes readily), silica gel chromatography, and crystallization (ethyl acetate-light petroleum). Spectral Data UV;

End absorption. IR;

~ c~oo,~.cuo,~ 3590, 3450, and 1785cm"l. IH N]V[R:

(CDCI3) H-I, 1.85; H-6, 1.29, 1.96(J6,6~14Hz); H-8, 3.22; H-10, 1.14(d, d=7.0Hz); H-11, 2.69(q, J=7.0Hz); H-12, 1.18; and H-14 and H-15, 1.12ppm. 13C NMR: (CDCI3) C-l, 31.4; C-2, 25.3; C-3, 28.2; C-4, 77.2; C-5, 92.6; C-6, 40.8; C-7, 38.5; C-8, 68.2; C-9, 68.6; C-10, 24.3; C-11, 45.0; C-12, 7.6; C-13, 176.1; C-14, 23.9*; and C- 15, 17.4ppm*. *Assignments may be reversed.

800

22. Alliacolide and Related Metabolites

Mass Spectrum: HREIMS: 266.1518role (M+); LREIMS 266(M+, 25%), 238(15), 211(11), 210(11), 193(100), 182(46), 151(26), 142(42), and 125role (39). References I. A. FarreU, T. G. Halsall, and V. Thaller; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus; J. C. S. Perkin Trans I, pp. 1790-1793(1981). T. J. King, I. W. Farrell, T. G. Halsall, and V. Thaller; A New Bicyclic Sesquiterpene Epoxy-lactone with a Novel Carbon Skeleton from Cultures of the Fungus Marasmius alliaceus; J.C.S. Chem. Comm., pp. 727-728(1977).

22.

Alliacolide and Related Metabolites

801

Common/Systematic Name Alliacide Molecular Formula/Molecular Weight C15H2203; M W = 250.15689 lO

b I

CO

12

13

General Characteristics Needles from light petroleum; mp., 168-171 ~C. Fungal Source Marasmius alliaceus, a wood rotting fungus. Isolation/Purification Extracted from the culture broth with ethyl acetate and purified by chromatography on silica gel with chloroform-ethyl acetate-acetic acid, 40:10:1, v/v/v as eluent. Rechromatographed on silica gel column chromatography using 10% ethyl acetatepetroleum ether; final purification was by crystallization from petroleum ether as needles. Spectral Data IR:

(Nujol) 1770cm"l ~H NMR: (CDCi3) H-8, 3.25; H-10, 1.30(d, J=7.0Hz); H-12, 1.50; and H-14, H-15, 1.18ppm 13C NMR: (CDCI3) C-l, 31.6; C-2, 26.7; C-3, 19.7; C-4, 31.6; C-5, 90.6; C-6, 46.8; C-7, 38.6; C-8, 69.5; C-9, 67.9; C-10, 24.3; C-1 l, 42.5; C-12, 9.6; C-13, 178.0; C-14, 23.8; and C- 15, 17.0ppm. Mass Data: HREIMS: 250.1571(M + ; found C, 72.1; H, 8.7%); C15H2203 requires 250.1569; C, 72; H, 8.9%; LREIMS: 250(M+, 27%), 235(25), 222(12), 195(50), 194(36), 193(21), 182(36), 180(34), 179(32), 177(69), 165(100), 125(55), and 97nm (63).

802

22.

Alliacolide and Related Metabolites

References T. Anke, W. H. Watson, B.M. Giannetti, and W. Steglich; New Antibiotics from the BasidiomyceteMarasmius alliaceus; Planta Med., Vol. 39, p. 194 (1980). I. A. Farrell, T. G. Halsall, and V. Thaller; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus; J. C. S. Perkin Trans I, pp. 1790-1793(1981).

22.

Alliacolide and Related Metabolites

803

.Common/Systematic Name Alliacol A; Alliacolide II Molecular Formula/Molecular Weight C15H2oO4; M W

-" 2 6 4 . 1 3 6 1 6

10

8 I

HO'~I

0

H2C" ""C I 12

"

13

I't20 General Characteristics Crystallized from ethyl acetate-light petroleum as needles; mp., 156-1580C; [tt]D 10 ~ (C=0.95, in CHCI3). Fungal Source Marasmius alliaceus, a wood rotting fungus. Isolation/Purification Isolated from the ether extract of the culture broth by a combination of sublimation (the compound sublimes readily at 140~ 0.3mm Hg), silica gel chromatography, and crystallization (ethyl acetate-light petroleum). Biological Activity Both alliacol A and B showed weak antibacterial and antifungal activity; the growth of sensitive bacteria and fungi were inhibited by concentrations of 25-50~tg/m! Both strongly inhibited DNA synthesis in cells of the ascitic form of Ehrlich carcinoma at 5btg/ml whereas RNA synthesis was somewhat less affected. No inhibition of protein synthesis was observed at concentrations up to 20btg/ml. Spectral Data UV:

End absorption. IR: ~ caiboa teaachlofide max

3580, 3450, 1775, 1680, 1125, and 900cm ~.

804

22.

Alliacolide and Related Metabolites

IH NIV[R: (CDCI3) H-6, 1.30, 2.00(,/6,6~14Hz);H-8, 3.20; H-10, 1.20(d,J=7.0I-Iz);H-12, 5.9, 6.35; and H-14 and H-15, 1.20ppm. 13C NMR: (CDCI3) C-l, 31.5; C-2, 26.3; C-3, 38.6; C-4, 76.5; C-5, 95.1; C-6, 41.7; C-7, 39.2; C8, 67.1; C-9, 69.6; C-10, 24.4; C-11, 142.9; C-12, 124.7; C-13, 168.8; C-14, 24.0; and C-15, 19.4ppm. Mass Spectrum: LREIMS: 264(M+, 30%), 249(29), 247(20), 236(45), 193(70), 151(75), and 83m/e (100). References T. Anke, W. H. Watson, B.M. Giannetti, and W. Steglich; New Antibiotics from the Basidiomycete Marasmius alliaceus; Planta Med., Vol. 39, p. 194 (1980). I. A. Farrell, T. G. Halsall, and V. Thaller; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus; J. C. S. Perkin Trails I, pp. 1790-1793 (1981).

22.

Alliacolide and Related Metabolites

805

Common/Systematic Name Alliacol B; 12-Hydroxydehydroalliacolide Molecular Formula/Molecular Weight C15H2004; ~

-- 2 6 4 . 1 3 6 1 6

10

12 Iill 0

~~1

HOH2C

CO 13

Fungal Source

Marasmius alliaceus, a wood rotting fungus.

Isolation/Purification Extracted from the culture broth with ethyl acetate and purified by chromatography on silica gel with chloroform-ethyl acetate-acetic acid, 40:10:1 (v/v/v) as eluent. Final purification was by preparative TLC, using chloroform-methanol, 20:1 (v/v). Biological Activity Both alliacol A and B showed weak antibacterial and antifungal activity; the growth of sensitive bacteria and fungi was inhibited by concentrations of 25-501.tg/ml. Both strongly inhibited DNA synthesis in cells of the ascitic form of Ehrlich carcinoma at 51ag,/ml whereas RNA synthesis was somewhat less affected. No inhibition of protein synthesis was observed at concentrations up to 201,tg/ml. Spectral Data IR:

(Nujol) 3520, 3440, 1735, and 1665cm"1. ~H NMR: (CDCI3) H-6, 1.20, 1.87(J6,6~14Hz); H-8, 3.25; H-10, 1.00(d, J=7.0Hz); H-12, 4.43; and H-14 and H-15, 1.20ppm. 13CNMR: (CDCla) C-l, 31.4; C-2, 23.1; C-3, 35.3, C-4,170.4; C-5, 94.7; C-6, 43.8; C-7, 41.4; C-8, 67.8; C-9, 68.2; C-10, 24.2; C-11,127.0; C-13, 173.6, C-14, 23.1; and C-15, 19.2ppm.

806

22.

Alliacolide and Related Metabolites

References T. Anke, W. H. Watson, B.M. Giannetti, and W. Steglich; New Antibiotics from the Basidiomycete Marasmius alliaceus; Planta Med., Vol. 39, p. 194 (1980). I. A. Farrell, T. G. HalsaU, and V. Thaller; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus; J. C. S. Perkin Trans I, pp. 1790-1793(1981).

22.

Alliacolide and Related Metabolites

807

Common/Systematic Name 11-Hydroxyalliacolide Molecular Formula/Molecular Weight C15H2205; M W = 282.14672

HO'""111 b 12 : 13 OH

General Characteristics Crystallized from ethyl acetate in polymorphic form; mp., 199-201.5~ and 228-229"C; [tt]D -8.6 ~ (C=0.56, in CHCI3). Fungal Source Marasmius alliaceus, a wood rotting fungus. Isolation/Purification Isolated from the ether extract of the culture broth by a combination of silica gel column chromatography, preparative thin-layer chromatography (chloroform-methanol, 20:1, v/v), and crystallization from ethyl acetate solution. Biological Activity Chemically related to alliacol A and B; both showed weak antibacterial and antifungal activity; both strongly inhibited DNA synthesis in cells of the ascitic form of Ehrlich carcinoma; RNA synthesis was somewhat less affected; and no inhibition of protein synthesis was observed with either metabolite. Spectral Data UV: End absorption. IR;

(Nujol) 3470, 3350, and 1755cmq. 1H NIV[R: (CDCI3) H-6, 1.50, 2.00(J6,n~14Hz); H-8, 3.22; H-10, 1.18(d, J=7.0Hz); H-12, 1.42; and H-14 and H-15, 1.18ppm

808

22.

Alliacolide and Related Metabolites

13CNMR: (CDCI3) C-l, 31.5; C-2, 25.3; C-3, 28.9; C-4, 76.6; C-5, 94.1; C-6, 42.6; C-7, 38.6; C-8, 67.3; C-9, 68.6; C-10, 24.2; C-11, 74.4; C-12, 17.4"; C-13, 174.9; C-14, 24.1; and C-15, 18.0*ppm. * Assignments may be interchanged. Mass Spectrum: LREIMS: 282(M+, 10%), 211(16), 205(7), 193(100), 179(18), 165(20), 151(26), 147(15), and 143m/e (35). Reference I. A. Farrell, T. G. HalsaU, and V. ThaUer; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus; J. C. S. Perkin Trans I, pp. 1790-1793(1981).

22.

Alliacolide and Related Metabolites

809

Common/Systematic Name 12-Hydroxyalliacolide Molecular Formula/Molecular Weight C15H2205; MW = 282.14672 10

O. -

o,~

8 I

HO"" 111

HOH2C~cIo 12 H 13 General Characteristics Crystallized from ethyl acetate-light petroleum as needles; mp., 191-193 ~ (c=0.47, in CHCI3).

[a]D -32.0"

Fungal Source Marasmius alliaceus, a wood rotting fungus. Isolation/Purification Isolated from the ether extract of the culture broth by a combination of silica gel column chromatography, preparative thin-layer chromatography (chloroform-methanol, 20:1, v/v), sublimation, and crystallization from ethyl acetate solution. Biological Activity Chemically related to alliacol A and B; both showed weak antibacterial and antifungal activity; both strongly inhibited DNA synthesis in cells of the ascitic form of Ehrlich carcinoma; RNA synthesis was somewhat less affected; and no inhibition of protein synthesis was observed with either metabolite. Spectral Data IR:

(Nujol)

3470 and 1745cm"1.

1H NIVJR:

(CDCI3) H-l, 1.83; H-6, 1.29, 1.98(d6,6,=14Hz);H-8, 3.23; H-10, 1.20(d, J=7.0Hz); H-11, 2.91; H-12, 4.05; and H-14 and H-15, 1.12ppm ~3CNMR: (CDCI3) C-l, 31.4; C-2, 25.5; C-3, 28.9; C-4, 77.0; C-5, 93.1; C-6, 40.8; C-7, 38.6;

810

22.

Alliacolide and Related Metabolites

C-8, 68.1; C-9, 68.3; C-10, 24.3; C-11, 51.7; C-12, 57.6; C-13, 174.5; C-14, 23.9; and

C-15, 17.5ppm.

Mass Spectrum: LREIMS: 282(M +, 1%), 267(5), 249(5), 211(20), and 193role (100). Reference I. A. Farrell, T. G. Halsall, and V. Thaller; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus; J. C. S. Perkin Trans I, pp. 1790-1793(1981).

22.

Alliacolide and Related Metabolites

811

Common/Systematic Name 12-Noralliacolide

Molecular Formula/Molecular Weight C14H2oO4; M W = 2 5 2 . 1 3 6 1 6 lO

HO' ....Ill

0

~(~/ 13 II 0

General Characteristics Crystallized as needles; mp., 198 ~C. Fungal Source Marasmius alliaceus, a wood rotting fungus. Isolation/Purification Isolated from the ether extract of the culture broth by a combination of silica gel column chromatography, preparative thin-layer chromatography (chloroform-methanol, 20:1), and crystallization. Biological Activity Chemically related to alliacol A and B; both showed weak antibacterial and antifungal activity; both strongly inhibited DNA synthesis in cells of the ascitic form of Ehrlich carcinoma; RNA synthesis was somewhat less affected; and no inhibition of protein synthesis was observed with either metabolite. Spectral Data IRz

(Nujol) 3410 and 1755cm"l. IH NMR: ([2H]pyridine) H-6, 1.47, 2.37(J6,6~14Hz); H-8, 3.25; H-10, 1.18(d, J=7.0Hz); H-11, 2.75, 3.15(d, J=17Hz); H-14, 1.13; and H-15, 1.09ppm ]3C NMR: ([2H]pyridine) C-I, 32.4; C-2, 27.0; C-3, 32.4; C-4, 75.2; C-5, 96.3; C-6, 42.1; C-7, 39.1; C-8, 67.1; C-9, 69.1; C-10, 24.9; C-11, 44.4; C-13, 174.2; C-14, 24.2; and C-15, 19.2ppm.

812

22.

Alliacolide and Related Metabolites

Mass Spectrum: LREIMS: 252.136(M+), 237(11%), 210(10), 193(25), 182(37), 167(18), 151(19), 142(46), 140(42), 125(23), 113(25), 109(18), 97(31), and 83m/e (100). Reference I. A. Farrell, T. G. Halsall and V. Thaller; Structures of Some New Sesquiterpenoid Metabolites ofMarasmius alliaceus, J. C. S. Perkin Trans I, pp. 1790-1793(1981).

Botrydial and Related Metabolites Botrydial O-Methyldihydrobotrydial Deacetyl-O-methyldihydrobotrydialone Dihydrobotrydial Botryaloic Acid Botryaloic Acid Acetate Botryoloic Acid Norbotryal Acetate Botrydienal Dehydrobotrydienal Deacetyldihydrotrydial

813

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23.

Botrydial and Related Metabolites

815

Common/Systematic Name Botrydial Molecular Formula/Molecular Weight C17H2605; M W -- 3 1 0 . 1 7 8 0 2

CliO

CliO ,,,|

OAc

General Characteristics Crystals from petroleum ether; mp., 108-110~ [aiD 20 +34 ~ (c=0.14, in CHCI3). Fungal Source Botrytis cmerea. Biological Activity Antibiotic with activity against fungi and gram-positive bacteria. Spectral Data CD: (dioxane) + 1.2(308) and - I.I(220nm). IR:

(CC14) 3465, 1735(OAc), 1717(CH=O-), and 1240cmq (OAc). ~H N M R : (CDCI3) 0.96(d,J=6.0Hz, CH-CH3); I.I0; 1.29; 1.350e, s, 3 CH3); 2.05(s, AcO); 5.08(X part of ABCX system, JAx=4.5, JBx=Jcx=l 1.5Hz, 4H); 9.62(s, CH=O); and

9.80ppm (d, J=2.5Hz, CH-CH=O). Mass Spectrum: 310(M+, 2%), 250(M+- OAc, 14), 204(C~4H2oO§ 87), 180(CllH~602+, 32), 179(C1~H1502+, 33), 175(C~3H~9+, 100), 119(C9H11§ 55); and 112m/e (CTH120+, 30). TLC Data: Re 0.3 with Kieselgel-G, PF2s4using petroleum ether-acetone, 10:3, v/v as solvent.

816

23.

Botrydial and Related Metabolites

Reference H. -W. Fehlhaber, R. Geipel, H. -J. Mercker, R. Tschesche, and K. Weimar; Botrydial, ein Sesquiterpen-Antibiotikum aus der N~ihrl6sung des Pilzes Botrytis cmerea; Chem. Ber., Vol. 107, pp. 1720-1730(1974).

23. Botrydial and Related Metabolites

817

Common/Systematic Name O-Methyldihydrobotrydial Molecular Formula/Molecular Weight C1sI-I3oOs;M3V = 326.20932

MeO%.....O-

i o."1 ,,.,

OAc General Characteristics Crystals from n-hexane; mp., 93~

[{I]D20 -t-85 (c=l.0, in MeOH).

FunRal Source Botrytis squamosa. Isolation/Purification The fungus was statically cultured in an onion extract medium. The culture filtrate was adsorbed onto active charcoal at pH 2.0 with HC1 and subsequently eluted with acetone. After evaporating the solvent, the residual brown syrup was purified by silica gel column chromatography, eluting with benzene. The eluate gave O-methyldihydrobotrydial which was recrystallized from n-hexane. Biological Activity Biological activity was examined using lettuce seedlings. O-Methyldihydrobotrydial inhibited the hypocotyl elongation by approximately 70% at a dose of 10mg/L. However, it accelerated the root growth in proportion to concentrations from 3mg/L to 100mg/L. At a dose of 100mg/L, the root growth was increased by 70% over the control seedlings. On the other hand,deacetyl-O-methyldihydrobotrydialone inhibited the hypocotyl elongation by 50%, even at a dose of 300 mg/L, and root growth was hardly influenced by deacetylO-methyldihydrobotrydialone at concentrations from 1mg/L to 1000mg/L. Spectral Data

(KBr) 3520, 1740, 1250, 1120, 1080, and 1050cm "~.

~H NMR: H-I, 1.60 d(J=IIHz); H-2, 1.83 qddd(J=2.5, 5.5, II.0, I 1.0),H-3, l.II ddd(J=9.8. 11.0, 11.0Hz), 2.05 ddd(J=2.5, 4.5, 11.0Hz);H-4, 5.09 ddd(J=4.5.9.8.9.81-1z);H-5,

818

23.

Botrydial and Related Metabolites

1.92 d(J=9.8Hz); H-7, 1.05 d(J=10.5Hz), 1.86 d(J=10.5Hz); H-10, 4.82 s; H-11, 0.93 d(J=5.5Hz); H-12, 1.30 s; H-13, 1.10 s; H-14, 1.25 s; H-15, 3.22 d(J=9.5Hz), 3.96 d(J=9.5Hz); 4 CH3COO-, 2.03 s; and 10 CH30-, 3.37ppm s. 13C NMR: C-I, 54.8 d; C-2, 28.6 d; C-3, 40.0 t; C-4, 72.8 d; C-5, 59.5 d; C-6, 38.9 s; C-7, 50.4 t; C-8, 45.5 s; 82.6 s; C-10, 98.7 s; C-11, 20.1 q; C-12, 35.6 q; C-13, 27.3 q; C-14, 25.3 q; C-15, 67.5 t; 4 CH3COO-, 21.5 q; 4 CH3COO-, 170.5 s; and CHaO-, 55.0 q. Mass Data: EIMS: 308role (M +-18); found: C, 66.19; H, 9.81; calcd, for ClsH30Os: C, 66.23; H, 9.26%. Reference Y. Kimura, H. Fujioka, H. Nakajima, T. Hamasaki, and A. Isogai; Isolation and Structures of O-Methyldihydrobotrydial and Deacetyl-O-methyldihydrobotrydialone Produced by Botrytis squamosa; Agric. Biol. Chem., Vol. 52 (7), pp. 1845-1847(1988).

23.

Botrydial and Related Metabolites

819

Common/Systematic Name Deacetyl-O-methyldihydrobotrydialone Molecular Formula/Molecular Weight C16H2605; MW -- 298.17802

MeO~,/O

0

OH General Characteristics Crystals from methanol; mp., 236-237~ [aiD 20 +106 (c=0.49, in MeOH). Fungal Source Botrytis squamosa. Isolation/Purification The fungus was statically cultured in an onion extract medium. The culture filtrate was adsorbed onto active charcoal at pH 2.0 with HCI and subsequently eluted with acetone. After evaporating the solvent, the residual brown syrup was purified by silica gel column chromatography, eluting with benzene-acetone (49:1, v/v). The metabolite was applied to a Sephadex LH20 column and eluted with methanol. Final purification was by preparative TLC (silica gel I--IF254;benzene-ethyl acetate, 6:4, v/v) followed by recrystallization from methanol. Biological Activity See O-methyldihydrobotrydial. Spectral Data IR:

(KBr) 3450, 1765, 1285, and 1200cm~. 1H NMR: H-I, 1.71 dd(J=5.0, 12.0Hz); H-2, 1.61 qddd(J-3.0, 6.0, 12.0, 12.0Hz); H-3, 1.22 ddd(J=12. 0, 12.0, 12.0Hz), 1.90 ddd(J=3.0, 4.5, 12.0Hz); H-4, 3.80 ddd(J-4.5.10.5, 12.0Hz); H-5, 1.65 d(J=10.5Hz); H-7, 1.50 d(J=13.0Hz), 2.46 d(J=13.0Hz); H-10, 5.19 d(J=5.0Hz); H-11, 0.99 d(J=6.0Hz); H-12, 1.29 s; H-13, 1.25 s; H-14, 1.33; and 10-CH30-, 3.58ppm s.

820

23.

Botrydial and Related Metabolites

13C NM~: C-l, 55.6 d; C-2, 32.6 d; C-3, 43.1 t; C-4, 69.5 d; C-5, 64.55 d; C-6, 40.0 s; C-7, 49.0 t; C-8, 53.8 s; C-9, 84.4 s; C-10, 108.0 d; C-11, 20.4 q; C-12, 36.3 q; C-13, 27.3 q; C14, 20.4 q; C-15, 173.9 s; and 10 CH30-, 57.0ppm q. Mass Data: EIMS: 280(M + -18), 266, 252, 238, 222, 195, and 180m/e; CIMS: 299(M + +1); found: C, 62.96; H, 8.70; ealed, for C~6Hz605 + 1/2 H20: C, 62.54; H, 8.79%. Reference Y. Kimura, H. Fujioka, H. Nakajima, T. Hamasaki, and A. Isogai; Isolation and Structures of O-MethyldihydrobotrydiaI and Deaeetyl-O-methyldihydrobotrydialone Produced by Botrytis squamosa; Agrie. Biol. Chem., Vol. 52 (7), pp. 1845-1847(1988).

23.

Botrydial and Related Metabolites

821

Common/Systematic Name Dihydrobotrydial Molecular Formula/Molecular Weight C17H2805; MW = 312.19367

HO,,,,/O.

! o."I ......,

OAc

General Characteristics Crystals from petroleum ether; mp., 158-160~

[~]D 22 +

56~ (c=0.94, in petroleum ether).

Fungal Source Botrytis cinerea.

Biological Activity Antibiotic with activity against fungi and gram-positive bacteria. Spectral Data IR:

(CC14) 3595, 3535(OH), 1730, and 1235cm~ (OAc). 1H NMR:

(CDCI3) 0.98(d, J=6.0Hz, CH-CH3); 1.14(s, CH3); 1.28(s, 2CH3); 2.03(s, AcO), 3.25, and 4.20(AB-system, JAB=10Hz, CH20); 5.12(X part of ABCX system, Jhx=4.5, JBx=Jcx = 11.5Hz, 4H); and 5.36ppm (s, 1OH). 13C NM~:

(CDCI3) C-11, 20.0(q), 21.4(q); C-14, 25.4(q); C-13, 27.2(q); C-2, 28.6(d); C-12, 35.9(q); C-6, 38.8(s); C-3, 39.9(t); C-8, 45.4(s); C-7, 50.4(0; C-l, 55.0(d); C-5, 59.8(d); C-15, 67.4(t); C-4, 72.7(d); C-9, 84.4(s); and C-10, 92.2(d), 170.3ppm (s). Mass Spectrum: 294(M+ - H20, 26%), 234(M§ - H20 - HOAc, 50), 175(C13H~9§ 77), and 96m/e (100). TLC Data Re 0.3 5 with Kieselgel-G, PF254using petroleum ether-acetone, 10:3, v/v as solvent.

822

23.

Botrydial and Related Metabolites

References H. -W. Fehlhaber, R. Geipel, H. -J. Mercker, R. Tschesche, and K. Weimar; Botrydial, ein Sesquiterpen-Antibiotikum aus der N~ihrl6sung des Pilzes Botrytis cinerea; Chem. Ber., Vol. 107, pp. 1720-1730(1974). Y. Kimura, H. Fujioka, H. Nakajima, T. Hamasaki, M. Irie, K. Fukuyama, and A. Isogai; Isolation, X-Ray Structure and Biological Activity of Deacetyldihydrobotrydial Produced by Botrytis squamosa; Agric. Biol. Chem., Vol. 50, pp. 2123-2125(1986).

23.

Botrydial and Related Metabolites

823

Common/Systematic Name Botryaloic acid (Isolated as methyl botryaloate) Molecular Formula/Molecular Weight C15H24Os; MW = 284.16237 CliO

C02H .,|'

13

OH

12

General Characteristics Methyl botryaloate: Crystallized from ether as needles; mp., 115-117~ (C=0.43, in CHCI3).

[tt]D +67 ~

Fungal Source Botrytis cinerea. Isolation/Purification The culture broth was saturated with sodium chloride and acidified to pH 2 with hydrochloric acid. The culture broth was extracted with ethyl acetate and the extracts were shaken with aqueous sodium hydrogen carbonate; the sodium hydrogen carbonate extracts were acidified to pH 2 with 6N hydrochloric acid and extracted with ethyl acetate. These extracts were dried and the solvent evaporated to give an oil which was dissolved in ether and treated with an excess of diazomethane. The solvent and excess of reagent were evaporated to give an oil which was chromatographed on a dry column of silica (Merck). Elution with 20% ethyl acetate-light petroleum gave methyl acetylbotryaloate which crystallized from ether as needles. Further elution with 25-30% ethyl acetate-light petroleum gave methyl botryaloate which crystallized from ether as needles. Fractions eluting with 40% ethyl acetate-light petroleum were rechromatographed on preparative TLC using chloroform-ethyl acetate-acetic acid (40:10:1, v/v) to yield methyl botryoloate. Spectral Data (Methyl botryaloate) IR:

(Nujol mull) 3460, 3360, 2750, 1730, and 1710 cm4. 1H NMR: (CDCI3) 0.90(3H, d, J=SHz); 1.33(9H, s); 3.68(3H, s, OMe); 3.95(1H, sextet, 14.5, 11, and 11 Hz); and 9.86ppm (1H, d, J=3Hz).

824

23. Botrydial and Related Metabolites

~3CNMR: (CDCI3) C-I, 68.5; C-2, 28.5; C-3, 43.5; C-4, 70.0; C-5, 66.3; C-6, 38.6; C-7, 54.9; C8, 55.1; C-9, 88.2; C-10, 204.1; C-11, 20.7; C-12, 27.8; C-13, 36.2; C-14, 20.7; C-15, 179.0; and OMe, 52.5ppm. Mass Data: Found: C, 64.35; H, 8.8; C16H2605requires C, 64.4; H, 8.8%. Reference A. P. W. Bradshaw and J. R. Hanson; Three New Sesquiterpenoid Metabolites of Botry~s cinerea; J. Chem. Soc., Perkin Trans I, pp. 741-743(1980).

23. Botrydial and Related Metabolites

825

Common/Systematic Name Botryaloic acid acetate (Isolated and purified as methyl acetyl botryaloate) Molecular Formula/Molecular Weight C17H2606; M W - 3 2 6 . 1 7 2 9 4 10

15

CHO CO2H 11

14

13

OAc

12

General Characteristics Methyl acetyl botryaloate: crystallized from ether as needles; mp., 106-108~ (C=0.46, in CHCI3).

[r

+810

Fungal Source. Botrytis cmerea.

Isolation/Purification The culture broth was saturated with sodium chloride and acidified to pH 2 with hydrochloric acid. The culture broth was extracted with ethyl acetate and the extracts were shaken with aqueous sodium hydrogen carbonate; the sodium hydrogen carbonate extracts were acidified to pH 2 with 6N hydrochloric acid and extracted with ethyl acetate. These extracts were dried and the solvent evaporated to give an oil which was dissolved in ether and treated with an excess of diazomethane. The solvent and excess of reagent were evaporated to give an oil which was chromatographed on a dry column of silica (Merck). Elution with 20% ethyl acetate-light petroleum gave methyl aeetyl botryaloate which crystallized from ether as needles. Further elution with 25-30% ethyl acetate-light petroleum gave methyl botryaloate which crystallized from ether as needles. Fractions eluting with 40% ethyl acetate-light petroleum were rechromatographed on preparative TLC using chloroform-ethyl acetate-acetic acid (40:10:1, v/v/v) to yield methyl botryoloate. Spectral Data (Methyl acetyl botryaloate) IR:

(Nujol mull) 3445, 2760, 2720, 1740, and 1710cm"1.

:H NMR: (CDCI3) 0.86(3H, d, J=5Hz); 1.06, 1.30, 1.36, 2.03, and 3.70(each 3H, s); 5.06(1H, sextet J=4.5, 11, and 11Hz); and 9.86ppm (1H, d, J=-3Hz).

826

23.

Botrydial and Related Metabolites

~3CNMR: (CDCI3) C-I, 68.3; C-2, 28.2; C-3, 38.6; C-4, 72.9; C-5, 61.7; C-6, 38.6; C-7, 55.0; C8, 55.0; C-9, 88.0; C-10, 203.5; C-11, 20.9*; C-12, 27.5; C-13, 35.7; C-14, 20.5*; C15, 178.9; OMe, 52.6; and OAc, 21.4, 170.3ppm. * Assignments may be interchanged. Mass Data: Found: C, 63.5; H, 8.3. C18H2806requires C, 63.5; H, 8.3%. Reference A. P. W. Bradshaw and J. R. Hanson; Three New Sesquiterpenoid Metabolites of Botrytis cinerea; J. Chem. Soc., Perkin Trans I, pp. 741-743(1980).

23.

Botrydial and Related Metabolites

827

Common/Systematic Name Botryoloic acid Molecular Formula/Molecular Weight ClvH2sO6; M W -- 3 2 8 . 1 8 8 5 9 10

15

CO2H CH2OH OHI. ....., 14

13

OAc

12

General Characteristics Methyl botryoloate: Crystallized from methanol as plates; mp., 123-125~ (c=0.24, in CHC13).

[O~]D

+34 ~

Fungal Source Botrytis cinerea. Isolation/Purification The culture broth was saturated with sodium chloride and acidified to pH 2 with hydrochloric acid. The culture broth was extracted with ethyl acetate and the extracts were shaken with aqueous sodium hydrogen carbonate; the sodium hydrogen carbonate extracts were acidified to pH 2 with 6N hydrochloric acid and extracted with ethyl acetate. These extracts were dried and the solvent evaporated to give an oil which was dissolved in ether and treated with an excess of diazomethane. The solvent and excess of reagent were evaporated to give an oil which was chromatographed on a dry column of silica (Merck). Elution with 20% ethyl acetate-light petroleum gave methyl acetyl botryaloate which crystallized from ether as needles. Further elution with 25-30% ethyl acetate-light petroleum gave methyl botryaloate which crystallized from ether as needles. Fractions eluting with 40% ethyl acetate-light petroleum were rechromatographed on preparative TLC using chloroform-ethyl acetate-acetic acid (40:10:1, v/v/v) to yield methyl botryoloate. Spectral Data (Methyl botryoloate) IR~

(Nujol mull) 3560, 3480(sh), 3230(br), 1725, and 1705cm"~. IH NMR: (CDC13) 0.92(3H, d, J=6Hz); 1.04, 1.10, 1.31, and 2.08ppm (each 3H, s); 3.28 and 3.60ppm (each 1H, d, J=13Hz); 3.80(3H, s); and 5.10ppm (IH, sextet, J=4, 11, and 11Hz).

828

23.

Botrydial and Related Metabolites

Mass Data: Found: C, 63.5; H, 8.3. CIsH280605H20 requires C, 63.5; H, 8.3%. Reference A. P. W. Bradshaw and J. R. Hanson; Three New Sesquiterpenoid Metabolites of Botrytis cmerea; J. Chem. Sot., Perkin Trans I, pp. 741-743(1980).

23. Botrydial and Related Metabolites

829

Common/Systematic Name Norbotryal acetate Molecular Formula/Molecular Weight C16H2403; m w

-- 2 6 4 . 1 7 2 5 4

O--CH 9

,~ll

OAc General Characteristics An unstable oil; [I~]D20

+102 ~

(c=0.2, in CHCI3): bp., 84~

Fungal Source Botrytis cmerea (GCRI strain 216). Isolation/Purification The fungal broth was extracted with EtOAc and separated into acidic and neutral fractions with aqueous NaHCO3. The neutral fraction was chromatographed on silica gel, deactivated with 12% water. Elution with 15% EtOAc-petrol gave a fraction which was further purified by TLC on silica gel in EtOAc-petrol (1:4, v/v) to afford norbotryal acetate as an unstable oil. The material had Rf 0.53 in the above system and gave a pink coloration with a MeOH-H2SO4 spray. Spectral Data UV:

~,m~x 253nm (e=10,300). IR:

2740, 1735, 1670, 1625, and 1235cm1. IH NMR: (CDCI3) H-2, 2.85(m); H-3, 1.38 and 2.14 =(m); H-4, 4.75(octet, ,/--3, 9, 10Hz); H-5, 2.54(q, J=2 and 9Hz); H-7, 1.8(m); H-8, 3.2(m); H-10, 10.0(s); H-11, 1.12(d, J=6Hz); H-12, 1.25(s); H-13, 0.75(s); H-14, 1.28(d, J=7Hz); and Me, 2.00ppm (s). ~3CNMR: (CDCI3) C-l, 135.8; C-2, 32.5; C-3, 37.5; C-4, 70.2; C-5, 56.0; C-6, 40.9; C-7, 49.3; C-8, 29.1; C-9, 169.0; C-10, 190.4; C-11, 21.9; C-12, 28.3; C-13, 20.6; C-14, 24.2; C=O, 170.1; and C-Me, 21.3ppm.

830

23.

Botrydial and Related Metabolites

Mass Spectrum: LREIMS: 264(M+), 204(base peak), 189, metastable 175.1 (for 204-189), 161, 119, 105, 43, and 41role. HREIMS: 204.15148m/e; Cl4H2oO§ (M + - MeCO2H) requires 204.151407. Reference O. Cuevas and J. R. Hanson; Norbotryal Acetate, A Nor-Sesquiterpenoid Aldehyde from Botrytis cinerea; Phytochemistry, Vol. 16, pp. 1061-1062(1977).

23.

Botrydial and Related Metabolites

831

Common/Systematic Name Botrydienal Molecular Formula/Mole.cular Weight C15H2002; MW = 232.14633

CliO CliO ,, II II

[',,, General Characteristics Colorless needles; mp., 69-70~

[IX]D30 + 190 ~ (c=0.18, in n-hexane).

Fungal Source

Botryotinia squamosa (IFO 9432).

Isolation/Purification The mycelia ofB. squamosa and agar medium were macerated with acetone. The acetone extract evaporated to an aqueous residue, which was extracted with ethyl acetate at pH 9. Purification was monitored by a turnip seedling bioassay. The phytotoxin was purified by column chromatography on silica gel followed by preparative TLC (silica gel) and HPLC (Develosil Cs), affording botrydienal as the active principle of the cultured material. Two related metabolites, dehydrobotrydienal and deacetyldihydrobotrydial, also were isolated from the same cultured material ofB. squamosa. Biological Activity Phytotoxic to turnip seedlings. The respective 50% inhibitory concentrations (IC50) of botrydienal, dehydrobotrydienal and deacetyldihydrobotrydial were 10, 40, and 10001.tg/ml. Botrydienal and dehydrobotrydienal also showed anti-bacterial activity, the respective MICs against Escherichia coli B 110 being 10 and 20txg/ml, and those against Staphylococcus aureus IFO 12732 50 and 50~g/ml. Spectral Data

UV: ~

EtOH max

245(sh, 6=2,670) and 324nm (6,940).

IR:

(KBr) 1720, 1660, and 1635cm "~.

832

23. Botrydial and Related Metabolites

IH N]V[R:

(CDCI3) 0.92(d,J2~l=7.1Hz, 11-CH3); 1.06, 1.25, 1.54(s,12-, 13-and 14-CH3); 1.63, 2.24(d,Jg~,=I3.9Hz, 7-CH2); 2.17, 2-48(ddd,J2,3,=8.I,J,,4=3-2,J2,3a=2.0,J3B,4=6.I and Jg=m=l8Hz, 3a-, 3b-H); 2.98(m, 2-H); 5.87(ddd,,/2,4=0.5,J3,4=3.2andJ3B,4=6.1Hz, 4-H); and 9-67, 9.74ppm (s, 15- and 10-CHO). 13C NMR: (CDCI3) 18.3; 25.3; 28.7; 30.5(q, Ell3 x 4); 30.5; 50.4(t, CH2 x 2); 24.2(d, CH); 40.4; 56.3(s, quaternary C x 2); 123.5(d,-CH=); 135.7; 149.2; 154.9(s,-C=x 3); 189.5; and 199.5(s, CHO x 2).

Mass Spectrum: HREIMS: 232.1479m/e (M+); calcd, for C15H2oO2,232.1469. Reference T. Kimata, M. Natsume, and S. Marumo; Botrydienal, A New Phytotoxin, and its Related Metabolites, Dehydrobotrydienal and Deacetyldihydrobotrydial Produced by Botryotinia squamosa; Tetrahedron Letters, Vol. 26, pp. 2097-2100(1985).

23. Botrydial and Related Metabolites

833

Common/Systematic Name Dehydrobotrydienal Molecular Formula/Molecular Weight C]5H2002; MW = 230.13063 CHO CHO g

.

I

III

"IIii

General Characteristics Colorless oil; [tt]D3~ -22 ~ (C=0.14, in n-hexane). Fungal Source Botryotinia squamosa (IFO 9432). Isolation/Purification See botrydienal. Biological Activity See botrydienal. Spectral Data UV:

~m~

260(sh c=6,390) and 315nm (2,080).

IR:

(KBr) 1725, 1685, and 1585cm"1. IH NMR:

(CDCI3) 1.32, 1.33, 1.55, and 2.68ppm (4 X CH3; positioned on C-6(2), C-8, and C-2, respectively; 9.60 and 10.40ppm (2 X CHO); 7.22(J=-7.8Hz on C-3); 7.32(J=7.8, 15Hz on C-4); and 1.86, 2.21ppm (2H on C-7, J=13.SHz). 13C NMR:

(CDCI3) 19.2; 22.1; 30.5; 31.4(q, CH3 x 4); 51.3(t, CH2); 42.4; 57.9(s, quatemary-C x 2); 128.3; 132.2(d,-CH= x 2); 129.7; 140.8; 141.2; 153.1(s,-C=x 4); 191.3; and 200.Sppm (d, CHO x 2). Reference T. Kimata, M. Natsume, and S. Marumo; Botrydienal, A New Phytotoxin, and its Related Metabolites, Dehydrobotrydienal and Deacetyldihydrobotrydial Produced by Botryotinia squamosa; Tetrahedron Letters, Vol. 26, pp. 2097-2100(1985).

834

23. Botrydial and Related Metabolites

Common/Sy.stematic Na.me Deacetyldihydrobotrydial Molecular Formula/Molecular Weight ClsH2604; MW = 270.18311

HO~,.,,,,,O. 7

.,i I

H'~H .. 1" OH

General Characteristics Colorless prisms; mp., 163-165~

[I~]D25 + 2 2 . 4 ~

(c=1.2, in MeOH).

Fungal Sourc_e Botryotinia squamosa (IFO 9432). Isolation/Purification See botrydienal. Biological Activity See botrydienal. Spectral Data Im:

(KBr) 3460, 3370, and 1000cmq. IH NMR: The presence of three OH groups was shown by D20 exchangeable signals at 5.60, 5.82, and 8.31 ppm. 13C N M R :

In the ~3CNMR spectrum measured with a coaxial dual tube of CD3OD/CDaOR the upfield shifted carbons at 70.3, 84.7, and 93.3ppm were assigned to the three respective OH-bearing carbons at 4-C, 9-C, and 10-C. The two carbons vicinal to the OH-bearing carbons at 1-C and 5-C (57.3 and 64.9ppm or vice versa) showed large upfield values (0.136 and 0.141, respectively), clearly indicative that they are sandwiched between the two OH-beating carbons; 20.6, 26.0, 36.5, and 27.gppm (4 methyl groups); 46.0, 51.8, and 68.4ppm (3 CH2 groups); 39.8 and 46.5ppm (2 quaternary carbons); and 57.3, 30.1, 70.3, 64.9, and 93.3ppm (5 methine carbons).

23.

Botrydial and Related Metabolites

835

References T. Kimata, M. Natsume, and S. Marumo; Botrydienal, A New Phytotoxin, and its Related Metabolites, Dehydrobotrydienal and Deacetyldihydrobotrydial Produced by Botryotinia squamosa; Tetrahedron Letters, Vol. 26, pp. 2097-2100(1985). M. Irie, K. Fukuyama, and A. Isogai; Isolation, X-Ray Structure and Biological Activity of Deacetylhydrobotrydiol Produced by Botrytis squamosa; Agric. Biol. Chem.; Vol. 50, pp. 2123-2125(1986).

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Herbarin and Related Metabolites

Herbarin Dehydroherbarin O-Methylherbarin Anhydrofusarubin 8-O-Methylfusarubin O-Ethylfusarubin Dihydrofusarubin Dihydrofusarubin (S-form) Hydroxydihydrofusarubin O-Ethyldihydrofusarubin 8-O-Methylbostrycoidin Fusarubin (Oxyjavanicin) Fusarubin Methyl Ether Javanicin Norjavanicin 8-O-Methyljavanicin 8-O-Methylsolaniol Marticin Isomarticin 5,8-Dihydroxy-6-methoxy-3-methyl-2-aza-9,10-anthracenedione

26

rel-(3R,4aR)-5,10-Dioxo-3,4,4a,5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9trihydroxy-1H-naphtho[2,3-c]pyran

rel-(3R,4aR, 10aS)-5,10-Dioxo-3,4,4a,5,10,10a-hexahydro-7-methoxy-3-methyl3,6,9-trihydroxy-1H-naphtho[2,3-c]pyran rel-(3R,4aR, 10aR)-5,10-Dioxo-3,4,4a,5,10,10a-hexahydro-3,7-dimethoxy-3-

methyl-6,9-dihydroxy-1H-naphtho[2,3-c]pyran Anhydrofusarubin Lactone 1,5,10-Trihydroxy-7-methoxy-3-methyl-1H-naphtho[2,3-c]pyran-6,9-dione 5,10-Dihydroxy-1,7-dimethoxy-3-methyl-1H-naphtho[2,3-c]pyran-6,9-dione 2,3-Dihydro-5,8-dihydroxy-6-methoxy-2-hydroxymethyl_3_(2_hydroxypropyl)_ 1,4-naphthalenedione 2,3-Dihydro-5-hydroxy-4-hydroxymethyl-8-methoxynaphtho[1,2-b]furan-6,9dione 5,8-Dihydroxy-2-methoxy-6-hydroxymethyl-7-(2-hydroxypropyl)-1,4naphthalenedione 2,5,8-Trihydroxy-6-methoxy-3-(2-oxypropyl)-1,4-naphthoquinone Nectriafurone 8-O-Methyl ether nectriafurone

837

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24.

Herbarin and Related Metabolites

839

Common/Systematic Name Herbarin Molecular Formula/Molecular Weight v

C16H1606; M W "- 304.09469

O

MeO~Me OMe0 General Characteristics Herbarin crystallized from methanol as long, bright yellow needles; mp., 192-193 ~C. It was insoluble in aqueous sodium hydroxide and gave no color with ethanolic ferric chloride. In concentrated sulfuric acid a blood red color developed which on standing turned brown. Fungal Source

Torula herbarum, a dermataceous fungus regularly associated with dry leaves and twigs ofFelia microphylla.

Isolation/Purification Fungal cultures were extracted (pH 4.5-5.0, unacidified) with ethyl acetate, washed twice with water and reduced in volume. A highly pigmented crystalline material separated after standing at -5~ overnight. Herbarin was purified by column chromatography or by preparative thin-layer chromatography and crystallized from methanol solution as long, bright yellow needles. Biological Activity Herbarin showed weak antibacterial and antifungal activity. The pigment mixture containing herbarin also showed antiamoebic activity against Entamoeba histolytica at a concentration of 201,tg/ml. Spectral Data UV:

l Em'~ 415, 266, and 216nm (log e=3.48, 4.18, and 4.83, respectively); intl. 350, 285, and 237nm (log e=3.15, 3.94, and 3.91, respectively). IR:

(KBr) 3325(OH), 1678, 1650(C=O), 1607, 1572, and 1567cm~ (C=C).

840

24.

Herbarin and Related Metabolites

CD: Optical activity; in ethanolic solution herbarin showed negligible rotation at D line, but ORD showed a negative Cotton effect at 345nm. IH NMR: (pyridine-ds) 2.61(1H, d, 3--2.5); 3.18(1H, d, ,/=2.5); 4.9-5.0(2H, m); 5.4(1H, broad singlet); 6.17(3H, s); 6.21(3H, s); 6.86(1H, m,,/=18); 7.33(1H, m, ,/=18Hz); and 8.26ppm (3H, s). Mass Data: Molecular weight determination by osmometric method gave 299. HREIMS showed 304.0905(M +) C12H1606 requires 304.0947; 286.0846, M ~ - H20; 276.1028, M + - CO ; and 271.0643, M + -H20,-CH3. Anal. C, 62.9; H, 5.15; calcd for Ct6H~606, C, 63.15; H, 5.26. Reference M. V. Kadkol, K. S. Gopalkrishnan, and N. Narasimhachari; Isolation and Characterization of Naphthaquinone Pigments from Torula herbarum (Pers.) Herbarin and Dehydroherbarin; J. Antibiot., Vol. 24, pp. 245-248(1971).

24.

Herbarin and Related Metabolites

841

Common/Systematic Name Dehydroherbarin Molecular Formula/Molecular Weight C16H1405; MW = 286.08412

MeO,~~~~~Me O

OMe 0 General Characteristics Dehydroherbarin crystallized from methanol as bright red glistening needles; mp., 186188~ It was soluble in ethyl acetate, chloroform, diethyl ether, and insoluble in water; was insoluble in aqueous sodium hydroxide, and did not give color with ethanolic ferric chloride. Funsal Source Torula herbarum, a dermataceous fungus regularly associated with dry leaves and twigs of Felia microphylla. Isolation/Purification Fungal cultures were extracted (pH 4.5-5.0, unacidified) with ethyl acetate, washed twice with water and reduced in volume. Dehydroherbarin was separated by silica gel column chromatography and crystallized from methanol. Biological Activity Dehydroherbarin showed weak antibacterial and antifungal activity. The pigment mixture containing dehydroherbarin also showed antiamoebic activity against Entamoeba histolytica at a concentration of 201,tg/ml. Spectral Data UV; ~E:~ 485, 400, 335, 272, 250, and 217nm (log c=3.15, 3.15, 3.28, 3.85, 3.85, and 4.12, respectively). IR;

(KBr) 1675, 1667(C=O), 1632, 1610, 1600, and 1565cm~ (C=C). ~H NMR: (CDCIa) 2.75(1H, d, J=2.5); 3.30(1H, d, ,/=2.5); 4.18(1H, d, J=l.0Hz); 4.88(2H, s); 6.05(3H, s); 6.07(3H s); and 8.02ppm (3H, s).

842

24.

Herbarin and Related Metabolites

Mass Data: HREIMS showed M + 286.082 I(C16H14Os, 286.0841), M + -CH3, 271.0561, and M + CO, 258.0888m/e. Anal: C, 67.3; H, 5.0; calcd for C16H1405, C, 67.51; H, 4.90. Reference M. V. Kadkol, K. S. Gopalkrishnan, and N. Narasimhachari; Isolation and Characterization of Naphthaquinone Pigments from Torula herbarum (Pers.) Herbarin and Dehydroherbarin; J. Antibiot., Vol. 24, pp. 245-248(1971).

24.

Herbarin and Related Metabolites

843

Common/Systematic Name O-Methylherbarin Molecular Formula/Molecular Weight C17H1806; MW =- 318.11034

O

OMe

0

General Characteristics O-Methylherbarin was crystallized from ethyl acetate as yellow needles and prisms which melted at 188-190 ~ C . Fungal Source Torula herbarum, a dermataceous fungus regularly associated with dry leaves and twigs of Felia microphylla. Isolation/Purification Purified by preparative thin-layer chromatography and crystallized from ethyl acetate as yellow needles and prisms. Spectral Data UV:

Eto.

218, 267, 285, and 415nm.

IR:

(CHCI3) Spectrum very similar to herbarin except no hydroxyl absorption observed. 1H NMR.:

(CDCI3) 6.06 and 6.08(aromatic OCH3); 6.72 (OCH3); 8.52 (CH3), 2.73 and 3.29 (aromatic protons); H-9eq, 5.25, H-9ax, 5.65; H-12cq, - 7.2; and H-12,x-7.55z. Mass Spectrum: 318(M+), 303,286, 271,244(base peak), and 243m/e. Reference M. V. Kadkol, K. S. Gopalkrishnan, and N. Narasimhachari; Isolation and Characterization of Naphthaquinone Pigments from Torula herbarum (Pers.) Herbarin and Dehydroherbarin; J. Antibiot., Vol. 24, pp. 245-248(1971).

844

24. Herbarinand Related Metabolites

Common/Systematic Name Anhydrofusarubin Molecular Formula/Molecular Weight C15H1206; M ' W = 2 8 8 . 0 6 3 3 9

0

OH

~~~~"~Me

MeO 0

OH

General Characteristics Crystallized from benzene-methylene chloride; mp., 197-198 ~C uncorr. Fungal Source Fusarium solani isolated from diseased citrus roots.

Isolation/Purification Culture filtrates were extracted three times with ethyl acetate; the combined extracts were dried with anhydrous sodium sulfate and reduced in volume. The extract was evaporated to dryness, dissolved in acetone, and fractionated by preparative TLC on silica gel HF254 (type 60) plates. Plates were developed with benzene-acetone (85:15, v/v). Seven bands were collected and eluted from the gel with acetone; any remaining colored material was eluted with ethanol. Both acetone and ethanol eluates were diluted and assayed for toxicity in a root growth test. Compounds possessing toxic activity were further purified by TLC with hexane-acetone (75:25, v/v) or hexane-chloroform-ether (1:1:1, v/v/v). Anhydrofusarubin was crystallized from benzene-methylene chloride. Biological Activity Phytotoxin that disrupts plant metabolism by inhibiting anaerobic and oxidative decarboxylation reactions. Suspected to be involved in root rot diseases of herbaceous plants and citrus blight, a complex of symptoms also called young tree decline. Anhydrofusarubin was effective in reducing root growth of germinating rough lemon seeds. SDectral Data _ UV: maxEtOH

207, 234, 292, 544, and 578nm.

IH NMR: (CDCI3) 2.04(3H, s, CH3); 3.94(3H, s, CH3-O); 5.24(2H, s, CH2); 6.02(1H, s, CH); 6.15(1H, s, H-6); 12.70(s, OH); and 13.05ppm (s, OH).

24.

Herbarin and Related Metabolites

845

Mass Spectrum: 288, 272, 244, and 217m/e. TLC Data A, C61-16-nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexane-HOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, Cd-I~-EtOAc-isoPrOH-HOAc (145:50:5:1, v/v/v); E, Cd-I6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6H6-Me2CO (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, Cd-I6-EtOAciso-PrOH-HOAc (16:3:1:0.1, v/v/v). Rf for anhydrofusarubin: A=0.54; B=0.57; C=0.37, and D=0.61. Systems E-H were used for isolation by preparative TLC. References R. A. Baker, J. H. Tatum, and S. Nemec, Jr.; Toxin Production by Fusarium solani from Fibrous Roots of Blight-Diseased Citrus; Physiology and Biochemistry, Vol. 71, pp. 951953(1981). J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

846

24. HerbarinandRelatedMetabolites

Common/Systematic Name 8-O-Methylfusarubin Molecular Formula/Molecular Weight C16H1607; M W "- 320.08960

OH

0

M e O ~ M e

OMe 0

General Characteristics Crystals from methanol; mp., 138-139~ Fungal Source

Fusarium moniliforme (strain MRC 602). The toxicogenic strain ofF. moniliforme (MRC 602) was isolated from moldy ears of corn in the Transkei, S.A.

Isolation/Purification Metabolite was extracted with chloroform and partitioned between hexane-90% aqueous methanol; aqueous methanol fraction concentrated and partitioned between chloroformwater. The crude extract was chromatographed on formamide-impregnated cellulose powder eluted with benzene-CH3COOEt (1:1, v/v); following that, Merck silica type H column chromatography eluted with chloroform-methanol (95:5, v/v) gave 8-0methylfusarubin. Spectral Data UV: ~,max 226, 282.5, 484, 510, and 550nm (sh) (log E=4.48, 4.05, 3.83, 3.80, and 3.49, respectively). IR:

(CHCIs) 1600cmq. IH NM]~:

[CDCI3-(CD3)2SO, 1:1] H-l, 4.66, ABX/, Jab--18Hz; H-4, 2.5, 2.78, Jgx=Jax=2.7Hz; H-7, 6.85; H-15, 1.56; OCH3, 4.01; OCH3, 4.05; and OH, 13.16ppm. 13C NMR~

[CDCIa-(CD3)2SO, l:l] C-l, 57.7; C-3, 93.1; C-4, 31.7; C-5, 147.6; C-6, 155.0; C-7, 103.0; C-8, 155.3; C-9, 179.0; C-10, 188.7; C-11, 113.7; C-12, 108.9; C-13, 144.6; C14, 136.8; C-15, 28.3; 6-OCH3, 56.1; and 8-OCH3, 56.1.

24.

Herbarin and Related Metabolites

847

Mass Data: 8-O-Methylfusarubin failed to give a molecular ion in its mass spectrum upon electron ionization. The compound was converted into a monoacetate which exhibited the appropriate molecular ion in its mass spectrum at 362.099m/e. Found: C, 56.88; H, 5.39% C16HI6OT.H20requires C, 56.80; H, 5.33%. Reference P. S. Steyn, P. L. Wessels, and W. F. O. Marasas; Pigments from Fusarium moniliforme Sheldon: Structure and ~3C Nuclear Magnetic Resonance Assignments of an Azaanthraquinone and Three Naphthoquinones; Tetrahedron, Vol. 35, pp. 1551-1555 (1979).

848

24.

Herbarin and Related Metabolites

Common/Systematic Name O-Ethylfusarubin Molecular Formula/Molecular Weight ClTHlgOT; MW = 334.10525 OH

0

OH

0

General Characteristics Reversibly decolorized by sodium hydrosulfite. Readily converted to fusarubin. .Fungal Source Fusarium solani.

Isolation/Purification Extracted from acidified fungal broth with chloroform. Separated by column chromatography using Mallinckrodt CC-4, 100-200 mesh and elution with chloroform and increasing concentrations of ethyl acetate. Biological Activity Antibiotic activity. Spectral Data UV:

Xmax 301,472, 485(sh), 498, 522(small), and 535nm. IR: 1600, 1580 sh, 1210 s, 1170 s, 1150 s, 1100 s, 1080 s, 1055 s, 970, 950, 840, 810 s, 665, 650, 580, 540, 510, 445, 410, 390cm l. IH NMR: (CDCI3) 4.0(OCH3); 12.7 and 13.0(phenolic OH); 6.2(aromatic H); 1.3(CH3); 3.7, q; 1.3 t, J=5.0 (OEt); and 4.7ppm br, d (CH2-C). Reference N. N. Gerber and M. S. Ammar; New Antibiotic Pigments Related to Fusarubin from Fusarium solani (Mart.) SACC., II. Structure Elucidations; J. Antibiot., Vol.32, pp. 685688(1979).

24.

Herbarin and Related Metabolites

849

Common/Systematic Name Dihydrofusarubin rel-(3R,4aR, 10aR)-5,10-Dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9trihydroxy- 1H-naphtho[2,3-c]pyran Molecular Formula/Molecular Weight C15H1607, M~V = 308.08960 OH

O

H

' OH

2

" O,

O

General Characteristics Reddish-orange needles from CH2CI2; mp., 153-154~ acetone).

[a]D 20 +

145.4 ~ (c=0.31, in

Fungal Source

Fusarium solani.

Isolation/Purification Cultures ofF. solani grown on a maltose ammonium tartrate- salt medium were harvested at 2 days when the pH had dropped to 3 from an initial value of 5. An ethyl acetate extract of the filtrate was taken to dryness and the residue chromatographed on silicic acid. Elution with a 5:1 (v/v), benzene-ethyl acetate mixture gave dihydrofusarubin; rel-( 3R, 4aR, 10aR)- 5,10-dio xo-3,4,4a, 5,10,10a-hexahydro- 7-methoxy-3-methyl-3,6, 9trihydroxy-1H-naphtho[2,3-c]pyran. Decreasing the benzene-ethyl acetate ratio to 3:1, v/v, gave rel-(3R,4aR, 10aS)-5,10-dioxo-3,4,4a,5,10,10a-hexahydro-7-methoxy-3methyl-3,6,9-trihydroxy-lH-naphtho[2,3-c]pyran. Biological Activity Antibiotic. Spectral Data UV; ~ 9~. 213,243,273, 300, and 391nm (log 6=4.17, 4.30, 3.87, 3.71, and 3.94, respectively). IH NMR: The signals for the methyl and methoxy groups of (3R,4aR, 10aR)-5,10dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9-trihydroxy- 1H-

850

24.

Herbarin and Related Metabolites

naphtho[2,3-c]pyran and (3R,4aR, 10aS)-5,10-dioxo-3,4,4a,5,10,10a-hexahydro-7methoxy-3-methyl-3,6,9-trihydroxy- 1H-naphtho[2,3-c]pyran were readily assigned. Resonances for the ketal and intra-molecularly hydrogen-bonded phenolic hydroxyl groups were temperature dependent and removed by exchange with D20. H-8 was deshielded relative to its counterpart fusarubin (6.36ppm) and was, therefore, present in a phenolic rather than a quinone ring. From the chemical shitts and spin-spin coupling constants the ketal ring systems giving rise to the coupled six-spin systems had the conformations shown, with H-4a and H-10a antiperiplanar in the R form and synclinal in the S form. Conformational free energy calculations indicated that the hydroxyl group at C-3 in both diastereoisomers were axially orientated, assuming a methyl group has a stronger 1,3-diaxial interaction with hydrogen than a hydroxyl group and the 'anomeric effect' favors the epimer with an axial hydroxyl group. Mass Spectrum: 308.0905m/e (M+); calcd for C15H1607:308.08961. Reference I. Kurobane, L. C. Vining, A. G. Mclnnes, and D. G. Smith; Diastereoisomeric 44,10aDihydrofusarubins: True Metabolites ofFusarium solani; Can. J. Chem., Vol. 56, pp. 1593-1594(1978).

24.

Herbarin and Related Metabolites

851

Common/Systematic Name Dihydrofusarubin (S form) rel-(3R,4aR, 10aS)-5,10-Dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9trihydroxy- 1H-naphtho [2,3-c] pyran Molecular Formula/Molecular Weight C15H1607; ~ = 308.08960

OH

0

7~, MeO ~ ' ~ . OH

H

2

HOH 0

H

General Characteristics Pale yellow needles from methanol; mp., 117-118~

[a]D2~ +23.3 ~ (C=0.31, in acetone).

Fungal Source

Fusarium solani.

Isolation/Purification Cultures ofF. solani grown on a maltose ammonium tartrate-salt medium were harvested at 2 days when the pH had dropped to 3 from an initial value of 5. An ethyl acetate extract of the filtrate was taken to dryness and the residue chromatographed on silicic acid. Elution with a 5:1 (v/v) benzene-ethyl acetate mixture gave dihydrofusarubin, rel-(3R,4aR, 10aR)-5,10-dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9trihydroxy-1H-naphtho[2,3-c]pyran. Decreasing the benzene-ethyl acetate ratio to 3:1, v/v, gave rel-(3R,4aR, 10aS)-5,10-dioxo-3,4,4a,5,10,10a-hexahydro-7-methoxy-3methyl- 3,6,9-trihydroxy- 1H- naphtho [2,3 -c] pyran. Biological Activity Antibiotic Spectral Data UV: Xgm~'~ 213, 243, 273, 300, and 391nm (log c = 4.15, 4.31, 3.89, 3.70 and 3.96, respectively). IH NMR: The signals for the methyl and methoxy groups of rel-(3R,4aR, 10aR)-5,10dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9-trihydroxy- IHnaphtho[2,3-c]pyran and rel-(3R,4aR, 10aS)-5,10-dioxo-3,4,4a,5,10,10a-hexahydro-7-

852

24.

Herbarin and Related Metabolites

methoxy-3-methyl-3,6,9-trihydroxy- 1H-naphtho[2,3-c]pyran were readily assigned. Resonances for the ketal and intramolecularly hydrogen-bonded phenolic hydroxyl groups were temperature dependent and removed by exchange with 2H20. H-8 was deshielded relative to its counterpart fusarubin (6.36ppm) and was, therefore, present in a phenolic rather than a quinone ring. From the chemical shills and spin-spin coupling constants the ketal ring systems giving rise to the coupled six-spin systems had the conformations shown, with H-4a and H-10a antiperiplanar in the R form and synclinal in the S form. Conformational free energy calculations indicated that the hydroxyl group at C-3 in both diastereoisomers were axially orientated, assuming a methyl group has a stronger 1,3-diaxial interaction with hydrogen than a hydroxyl group and the 'anomeric effect' favors the epimer with an axial hydroxyl group. Mass Spectrum: M + peak too small for mass measurement, M - 18 (also observed in the mass spectrum of R form) at 290.0784m/e; calcd for C~5H1406,290.07904. Reference I. Kurobane, L. C. Vining, A. G. Mclnnes, and D. G. Smith; Diastereoisomeric 44,10aDihydrofusarubins: True Metabolites ofFusarium solani; Can. J. Chem., Vol. 56, pp. 1593-1594(1978).

24.

Herbarin and Related Metabolites

853

Common/Systematic Name Hydroxydihydrofusarubin Molecular Formula/Molecular Weight C15H1608; ~

= 324.08452

MeO'~~~Me OH

0

OH

0

General Characteristics A yellow pigment reversibly decolorized by sodium hydrosulfite; readily converted to fusarubin; exhibited a strong bluish fluorescence. Fungal Source Fusarium solani Isolation/Purification Extracted from acidified fungal broth with chloroform. Separated by column chromatography using Mallinckrodt CC-4, 100-200 mesh and elution with chloroform and chloroform with increasing concentrations of ethyl acetate. Biological Activity Antibiotic activity. Spectral Data UV~

Xmax 272, 300(weak), and 388nm. IR~

3600w, 1730w, 1635s, 1465-1480, 1440, 1410, 1310, 1270, 1170, and 1005cm"~. IH ~ :

(CDC13) 4.0(OCH3); 12.1 and 12.2(phenolic OH); 6.75(aromatic H); 1.5(CH3); 4.2 dd; 1.57 m; and 2.3ppm m. Reference N. N. Gerber and M. S. Ammar; New Antibiotic Pigments Related to Fusarubin from Fusarium solani, II. Structure Elucidations; J. Antibiot., Vol.32, pp. 685-688(1979).

854

24.

Herbarin and Related Metabolites

Common/Systematic Name O-Ethyldihydrofusarubin (Diastereoisomeric-3-O-ethyl ether derivatives ofdihydrofusarubin, R and S forms) Molecular Formula/Molecular Weight C17H2007; M W -- 3 3 6 . 1 2 0 9

OH

0

OH

0

General Characteristics Yellow pigments that form during crystallization of the parent diastereoisomers from ethanol. The possibility that they are laboratory artifacts cannot be excluded. .Fungal Source Fusarium solani (cholesterol-metabolizing strain PP 96); probably form non-enzymatically in the culture broth or during purification. Biological Activity Antibiotic activity. Spectral Data (Spectral characteristics corresponded closely those of dihydrofurarubins) Mass Spectrum: CIMS with methane as carrier gas gave five peaks: 377(1.5%, M + 41), 365(23, M + ), 337(100, M § +1), 319(19, M + +1 - H20), and 291role (93, M ~ +1 - EtOH). References N. N. Gerber and M. S. Ammar; New Antibiotic Pigments Related to Fusarubin from Fusarium solani (Mart.) SACC., II. Structure Elucidations; J. Antibiot., Vol. 32, pp. 685688(1979). I. Kurobane, L. C. Vining, A. G. Mclnnes, and N. N. Gerber; Metabolites of Fusarium solani Related to Dihydrofusarubin; J. Antibiot., Vol. 33, pp. 1376-1379(1980).

24. Herbarinand RelatedMetabolites

855

Common/Systematic Name 8-O-Methylbostrycoidin 6,8-Dimethoxy-5-hydroxy-methyl-2-azaanthraquinone Molecular Formula/Molecular Weight C16H13NOs, ~

= 299.07937

OH

0

MeO.~~~~Me

OMe 0 General Characteristics 8-O-Methylbostrycoidin was the major pigment from cultures of this strain ofF. moniliforme (MRC 601). Crystals from chloroform-methanol; mp., 215-216 oC. Treatment of 8-O-methylbostrycoidin with Zn +2 in acetic acid led to a change in the color from dark-red to light pink, however, upon removal of the Zn by filtration, the material was readily oxidized to starting material which indicated that the metabolite was a quinone. 8-O-Methylbostrycoidin formed blue alkali-metal salts and exhibited strong chelating properties with e.g. Ca +2 and Mg +2. Acetylation of gave a fluorescent lightyellow monoacetate; mp., 254-246 ~ Fungal Source

Fusarium moniliforme (strain MRC 602). The toxicogenic strain ofF. moniliforme (MRC 602) was isolated from moldy ears of maize in the Transkei, S.A.

Isolation/Purification Metabolite was extracted with chloroform and partitioned between carbon tetrachloridewater. The crude extract was chromatographed on the following systems: (1) formamideimpregnated cellulose powder eluted with benzene-CH3COOEt followed by (2) Merck silica type H column chromatography eluted with chloroform-methanol (96:4, v/v) and (3) preparative TLC using silica gel Hzs4 (chloroform-methanol; 96:4, v/v). Rf for 8-0methylbostrycoidin was 0.51. Spectral Data UV:

(log e) 247.5(4.50), 318(3.92), and 480nm (3.83); ~^~ir 227(4.32), 262(4.20), 310(3.88), and 510nm (3.73); ~,~M~o. 259(4.44), 306(3.82), and 546nm.

/~,mM ~H

856

24.

Herbarin and Related Metabolites

IR:

(CHCI3) 1641, 1591, 1311, and 1265cm"1. IH NMR: (CDCI3) C-l, 1H, 9.44; C-4, 1H, 7.85; C-7, 1H, 6.86; C-15, 1H, 2.76; OCHa X 2= 4.05; and OH, 13.19ppm. Mass Data:

299.080m/e, calcd 299.083; found: C 63.75, H 4.39, N 4.63%; requires C 64.2, H 4.35, N 4.68%.

Reference P. S. Steyn, P. L. Wessels, and W. F. O. Marasas; Pigments from Fusarium moniliforme Sheldon: Structure and ~3CNuclear Magnetic Resonance Assignments of an Azaanthraquinone and Three Naphthoquinones; Tetrahedron, Vol. 35, pp. 1551-1555 (1979).

24.

Herbarin and Related Metabolites

857

Common/Systematic Name Fusarubin; Oxyjavanicin Molecular Formula/Molecular Weight C15H1407; MW = 306.07395

0

OH 0 OH

MeO

0

OH

General Characteristics Red prisms from benzene; mp., 196-198~ (uncorrected); charring about 200~ but mp. 218 ~ Soluble in glacial acetic acid, tetrahydrofuran, acetone, dioxane, pyridine; slightly soluble in cold chloroform, cold alcohol, ether; practically insoluble in carbon disulfide, cyclohexane, cold benzene. Dissolves in dilute NaOH with a violet color; practically insoluble in bicarbonate solution. Fungal Source Fusarium solani isolated from diseased citrus roots, and F. javanicum. Isolation/Purification Culture filtrates were extracted three times with ethyl acetate; the combined extracts were dried with anhydrous sodium sulfate and reduced in volume. The extract was evaporated to dryness, dissolved in acetone, and fractionated by preparative thin-layer chromatography (TLC) on silica gel HF254 (type 60) plates. Plates were developed with benzene-acetone (85:15, v/v). Seven bands were collected and eluted from the gel with acetone; any remaining colored material was eluted with ethanol. Both acetone and ethanol eluates were diluted and assayed for toxicity in a root growth test. Compounds possessing toxic activity were further purified by TLC with hexane-acetone (75:25, v/v) or hexanechloroform-ether (1:1:1, v/v/v). Fusarubin was crystallized from benzene. Biological Activity Phytotoxin that disrupts plant metabolism by inhibiting anaerobic and oxidative decarboxylation reactions. Suspected to be involved in root rot diseases of herbaceous plants and citrus blight, a complex of symptoms also called young tree decline. Fusarubin was effective in reducing root growth of germinating rough lemon seeds. Spectral Data UV: Ether

m~

535 and 499nm.

858

24.

Herbarin and Related Metabolites

~H NMR: (CDCI3) 1.68(3H, s, CH3), 2.27(d, J=2, OH, disappears on addition of D20), 2.72(1H, dd, J=18, d=2, CH2), 3.05(1H, d, d=lS, CH2), 3.94(3H, s, CH3-O), 4.90(2H, s,-CH2O-), 6.18 (1H, s, n-6), 12.69(s, OH), and 12.95ppm (s, OH). Mass Data: 306, 288, 273,259, and 246m/e; C 58.82%, H 4.61%, O 36.57%. TLC Data Solvent system A, C6H6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-MezCO-HOAc (15:5:0.3, v/v/v); D, Cd-I6-EtOAciso-PrOH-HOAc (145:50:5:1, v/v/v); E. Cd-I6-hexane-MezCO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6I-I6-Me2CO, (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, C61-16EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for fusarubin: A=0.23; B=0.29; C=0.23, and D=0.39. Systems E-H were used for isolation (preparative TLC). References R. A. Baker, J. H. Tatum, and S. Nemec, Jr.; Toxin Production by Fusarium solani From Fibrous Roots of Blight-Diseased Citrus; Physiology and Biochemistry, Vol. 71, pp. 951953(1981). J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24. Herbarin and Related Metabolites

859

Common/Systematic Name Fusarubin methyl ether Molecular Formula/Molecular Weight C16H1607; MW = 320.08960 0

OH

MeO

OMe

0

OH

General Characteristics Red crystals from EtOAc; mp., 188-190 ~ (lit. 190 ~C). Fungal Source

Fusarium solani isolated from diseased citrus roots.

Isolation/Purification Culture filtrates were extracted with ethyl acetate; the combined extracts were dried with anhydrous sodium sulfate and reduced in volume. The extract was evaporated to dryness, dissolved in acetone, and fractionated by preparative thin-layer chromatography (TLC) on silica gel HF254 (type 60) plates. Plates were developed with benzene-acetone (85:15, v/v). Seven bands were collected and eluted from the gel with acetone; any remaining colored material was eluted with ethanol. Both acetone and ethanol eluates were diluted assayed for toxicity in a root growth test. Compounds possessing toxic activity were further purified by TLC with hexane-acetone (75:25, v/v) or hexane-chloroform-ether (1:1:1, v/v/v). The methyl ether of fusarubin was crystallized from EtOAc. Biological Activity Suspected to be phytotoxic and may be involved in the etiology of citrus decline. Spectral Data WW:

~

EtOH max

228, 306, 475, 499, and 534nm.

~H NMR: (CDCI3) 1.55(3H, s, CH3); 2.66(dt, J=18.0, 4eq and 2.0Hz); 3.02(dd, J=18.0 (4ax) and 1.5Hz); 3.33(OMEO); 3.94(s, 7-OMe); 4.65(dt, J=18.0 (leq) and 2.7Hz); 4.88(dd, J=18.0 (lax) and 1.5Hz); 6.18(H-8); 12.69(OH); and 12.95ppm (OH).

860

24.

Herbarin and Related Metabolites

Mass Spectrum: 320m/e (M+). TLC Data Solvent system A, C6H6 nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3); D, C6I-l~-EtOAc-isoPrOH-HOAc (145:50: 5:1, v/v/v); E, C6I-I6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6H6-Me2CO (17:3, v/v); G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, Cd-I~-EtOAciso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for methyl ether of fusarubin: A=0.43; B=0.50; C=0.36, and D=0.55. Systems E-H were used for isolation (preparative TLC). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

861

Common/Systematic Name Javanicin 2-Acetonyl-3-methyl-7-methoxynaphthazarin Molecular Formula/Molecular Weight C15H1406 ; M ' ~ = 2 9 0 . 0 7 9 0 4

0

OH ~fMe

MeO

0

0

~/"~'~CH2--~;--Me OH

General Characteristics Crystallized from benzene; mp., 207-208~ uncorr. (lit. 207-208~ Fungal Source Fusarium solani isolated from diseased citrus roots.

Isolation/Purification Culture filtrates were extracted three times with ethyl acetate;the combined extracts were dried with anhydrous sodium sulfate and reduced in volume. The extract was evaporated to dryness, dissolved in acetone, and fractionated by preparative TLC on silica gel HF254 (type 60) plates. Plates were developed with benzene-acetone (85:15, v/v). Seven bands were collected and eluted from the gel with acetone; any remaining colored material was eluted with ethanol. Both acetone and ethanol eluates were diluted and assayed for toxicity in a root growth test. Compounds possessing toxic activity were further purified by TLC with hexane-acetone (75:25, v/v) or hexane-chloroform-ether (1" 1"1, v/v/v). Javanicin was crystallized from benzene. Biological Activity Phytotoxin that disrupts plant metabolism by inhibiting anaerobic and oxidative decarboxylation reactions. Suspected to be involved in root rot diseases of herbaceous plants and citrus blight, a complex of symptoms also called young tree decline. Javanicin was effective in reducing root growth of germinating rough lemon seeds. Spectral Data UV: ~,~"

227, 305, 478, 504, and 541.

IH NMR: (CDCI3) 2.22(3H, s, CH3), 2.28(2H, s, CH2), 3.88(2H, s, CH2), 3.91(3H, s, CH3), 6.18 (1H, s, H-6), 12.82(s, OH), and 13.21ppm (s, OH).

862

24.

Herbarin and Related Metabolites

Mass Spectrum: 290, 248, 230, 219, 205, and 43role. TLC Data Solvent system A, C6H6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, C6H6-EtOAciso-PrOH-HOAc (145:50:5:1, v/v/v); E. C6H6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6H6-Me2CO (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v), H, C6I-I6EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for javanicin A=0.36; B=0.45; C=0.30, and D=0.51. Systems E-H were used for isolation (preparative TLC). References R. A. Baker, J. H. Tatum, and S. Nemec, Jr.; Toxin Production by Fusarium solani from Fibrous Roots of Blight-Diseased Citrus; Physiology and Biochemistry, Vol. 71, pp. 951953(1981). J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

863

Common/Systematic Name Norjavanicin 2-Acetonyl-7-methoxynaphthazarin Molecular Formula/Molecular Weight C14I-I1206; MW = 276.06339

0

OH 0 II C H2--C--Me

MeO

0

OH

General Characteristics Red needles from CH2CIz-MeOH; mp., 195-200~ Fungal Source Fusarium solani isolated from diseased citrus roots. Isolation/Purification Culture filtrates were extracted with ethyl acetate; the combined extracts were dried with anhydrous sodium sulfate and reduced in volume. The extract was evaporated to dryness, dissolved in acetone, and fractionated by preparative thin-layer chromatography (TLC) on silica gel I-IF254(type 60) plates. The extract was developed in solvent system F and norjavanicin was isolated as a minor component and crystallized from methylene chloridemethanol to give red crystals. Biological Activity Phytotoxic. Suspected to be involved in root rot diseases of herbaceous plants and citrus blight, a complex of symptoms also called young tree decline. Spectral Data UV/Visible: ~,~H 225, 299, 477, 503, and 540nm.

~H NMR: (CDCI3): 2.31, 3H, s; 3.81, 2H, s; 7.18, H-10a; 3.93, 7-OMe; 6.18, H-8; 12.55, OH; and 12.59ppm, OH. Mass Spectrum: 276role (M+).

864

24.

Herbarin and Related Metabolites

TLC Data Solvent system A, C6I-I6-nitromethane-HOAc(75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, C~I~-EtOAciso-PrOH-HOAc (145:50:5:1, v/v/v); E, C6H6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6H6-Me2CO (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, C6I-I~EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Rf for norjavanicin: A=0.33; B=0.35; C=0.28, and D=0.41. Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

865

Common/Systematic Name 8-O-Methyljavanicin Molecular Formula / Mole.cular Weight C16H1606; ]VI3vV= 3 0 4 . 0 9 4 6 7

OH

0

MeO~Me T

Y

OMe0

-Me

General Characteristics Recrystallized from acetone to yield red needles; mp., 196-198~ 8-O-methyljavanicin was positive to ferric chloride solution and 2,4-dinitrophenylhydrazine solution. F.ungal Source Fusarium moniliforme and F. solani isolated from rice. Isolation/Purification The culture broth was filtered and extracted twice with ethyl acetate at pH 6.5. The combined extracts were concentrated under reduced pressure below 40 ~ to give a dark brown residue. The residue was dissolved in acetone and mixed with acid-washed Celite. After drying under reduced pressure, the impregnated Celite was charged onto a SilicAR CC-4 column which was eluted with ethyl acetate. Atter evaporation, the solid was subjected to SilicAR CC-4 column chromatography eluted with benzene and ethyl acetate; solid obtained from above 20% and 30% ethyl acetate fractions was applied onto a LH20 column eluted with methanol. After evaporation of the solvent, the residue was applied onto a SilicAR CC-4 column. The active substance eluted with benzene-acetone (49:1, v/v). Fractions with the biological activity were combined and evaporated to dryness to afford red crystals. Biological Activity A growth inhibitor on lettuce seedlings and on the root growth of lettuce seedlings (subjected to the growth assay of lettuce seedlings according to the method of Frankland and Wareing). In the bioassay using lettuce seeds at a concentration of 500mg/liter showed a weak inhibitory effect on germination when observed atter 24 hr. However, the activity at doses of 1-500mg/liter revealed approximately the same extent as that of controls after 48 hr. Suppression of hypocotyl elongation of lettuce seedlings was observed at a concentration of 10mg/liter, although at the same concentration it did not inhibit the root growth; at a concentration of 500mg/liter it inhibited the root growth with accompanying necrosis. Biological activities to rice seedlings were examined according to the method of Murakami at doses of 1-1000mg/liter; it did not inhibit the growth of the second leaf sheath of rice.

866

24.

Herbarin and Related Metabolites

Soectral Data UV:

~,~x 283(e=31,300), 486(18,600), 510(sh, 16,900), and 550nm (sh, 7,300); ~M,~. 226, 282.5, 482, 510, and 550nm (sh) (log e=4.56, 4.04, 3.80, 3.75, and 3.80). IR:

(KBr) 3300-3400(OH), 1715(C=O), 1635(C=O), 1620, 1595, 1480, 1435, 1395, 1315, 1280, 1260, 1220, 1080, 1035, 943,900, and 830cmq (indicated the presence of naphthoquinone moiety). IH NMR:

(CDCI3) Exhibited signals at 2.10(3H, s, CH3); 2.29(3H, s, CHa); 3.74(2H, s, CH2); 3.96(3H, s, OCH3); 3.98(3H, s, OCH3); 6.72(1H, s, ArH); and 13.0ppm (IH, s, OH). 13CNMR:

(CDCI3) C-l, 13.9; C-3,203.1; C-4, 41.1; C-5, 147.9; C-6, 155.1; C-7, 102.9; C-8, 155.6; C-9, 181.2; C-10, 189.4; C-I 1, 114.3; C-12, 110.6; C-13, 150.0; C-14, 137.8; C-15, 30.2; 6-OCH3, 56.4; and 8-OCHa, 57.0ppm. References Y. Kimura, T. Hamasaki, and H. Nakajima; Isolation, Identification and Biological Activities of 8-O-Methyljavanicin Produced by Fusarium solani; Agric. Biol. Chem., Vol. 45, pp. 2653-2654(1981). P. S. Steyn, P. L. Wessels, and W. F. O. Marasas; Pigments from Fusarium moniliforme Sheldon: Structure and ~3CNuclear Magnetic Resonance Assignments of an Azaanthraquinone and Three Naphthoquinones; Tetrahedron, Vol. 35, pp. 1551-1555 (1979).

24.

Herbarin and Related Metabolites

867

Common/Systematic Name 8-O-Methylsolaniol Molecular Formula/Molecular Weight C16H1806; 1VI'W= 306.11034

OH 0 M e O ~ M e

L IA. L TOMe "f0

,-o.

General Characteristics Crystals from methanol; mp., 152-154~C. Fungal Source

Fusarium moniliforme (strain MRC 602); the toxicogenic strain ofF. moniliforme isolated from moldy ears of maize in the Transkei, S.A.

Isolation/Purification Metabolite was extracted with chloroform and partitioned between hexane-90% aqueous methanol; aqueous methanol fraction concentrated and partitioned between chloroformwater; the crude extract was chromatographed on formamide-impregnated cellulose powder eluted with benzene-CHaCOOEt (1:1, v/v) followed by Merck silica type H column chromatography eluted with chloroform-methanol (98:2, v/v) which gave an 8-0methylsolanion-containing fraction. This was separated by chromatography on Merck precoated TLC plates Si gel F254,thickness 2mm, developed in CHCI3-MeOH (94:6, v/v). Spectral Data UV:

~,~8 226, 285, 476, and 510nm (sh) (log 6=4.49, 4.025, 3.82, and 3.69, respectively).

(CHCI3) 1628, 1470, 1435, and 1272cm"~. ~H NMR:

(CDCI3) H-I, 2.12;H-3, 4.18;H-4, 2.62,2.82;H-7, 6.49;H-15, 1.36(d,3=7I-Iz); OCH3, 3.87;OCH3, 3.92;OH, 3.41(d);and OH, 13.22ppm

~3CM R : (CDCIa) C-l, 13.9; C-3, 67.2, C-4, 36.7; C-5, 147.4; C-6, 154.7; C-7, 101.8, C-8,

868

24.

Herbarin and Related Metabolites

155.0; C-9, 181.3; C-10, 190.5; C-11, 114.1; C-12, 109.9; C-13, 149.6; C-14, 141.4; C-15, 23.9; 6-OCH3, 56.2; and 8-OCH3, 56.5ppm. Mass Data: Found: 306.11 lm/e; C16H1806requires 306.110. Reference P. S. Steyn, P. L. Wessels, and W. F. O. Marasas; Pigments from Fusarium moniliforme Sheldon: Structure and ~3CNuclear Magnetic Resonance Assignments of an Azaanthraquinone and Three Naphthoquinones; Tetrahedron, Voi. 35, pp. 1551-1555 (1979).

24. Herbarin and Related Metabolites

869

Common/Systematic Name Marticin Molecular Formula/Molecular Weight C15H1609; M W = 376.07943

o

.o

A A ..A ,o y "~ ~. "Me 6 H6H'

MeO"

General Characteristics Red crystals from benzene; 180-182 ~ C . Fungal Source Fusarium solani isolated from diseased citrus roots. Isolation/Purification Culture filtrates were extracted with ethyl acetate; the extract was dried with anhydrous sodium sulfate and reduced in volume. The extract was fractionated by preparative TLC on silica gel HF254(type 60) plates. Marticin was crystallized from benzene. Biological Activity Phytotoxic. Spectral Data UV;

;~Em~H 229, 304, 474, 501, and 536nm. IH NMR: (CDCI3) 1.68(s, 3-Me); 2.73(d, J=19.0Hz, H-4eq); 3.12(d, J=19.0Hz, H-4ax); 2.10(m, H-1 leq); 2.86(m, H-1 lax); 5.42(d, H-12ax, 9.0Hz (H-1 lax); 3.94(7-OMe); 4.58(m, H leq); 6.18(H-8); 12.53(OH); and 12.93ppm (OH).

Mass Spectrum: 3 76role (M+). TLC Data Solvent system A, C6H6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, Cd-I6-EtOAc-

870

24.

Herbarin and Related Metabolites

iso-PrOH-HOAc (145:50:5:1, v/v/v); E. C6H6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6H6-Me2CO (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, C6I-I6EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for marticin: A=0.12; B=0.13; C=0.10, and D=0.17. Systems E-H were used for isolation (preparative TLC). Systems E-H were used for isolation (preparative TLC). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

871

Common/Systematic Name Isomarticin Molecular Formula/Molecular Weight C18H1609; MW = 376.07943

o

.o "COOH

General Characteristics Red crystals from methanol; mp., 160-163~ Fungal Source Fusarium solani isolated from diseased citrus roots. Isolation/Purification Culture filtrates were extracted with ethyl acetate; the extract was dried with anhydrous sodium sulfate and reduced in volume. The extract was fractionated by preparative TLC on silica gel HF254 (type 60) plates. Isomarticin was crystallized from methanol. Biological Activity Phytotoxic. Spectral Data UV/Visible: 3,~m t~ 229, 304, 474, 501, and 536nm. IH NMR:

(CDCI3) 1.70(s, 3-Me); 3.0(d, J=19.0Hz, H-4eq); 3.13(d, ,/--19.0 Hz, H=4ax); 2.04(m, H-1 leq); 2.30(3-OH); 5.58(d, H-1 lax); 4.3 l(dd, J=12.0Hz, 1leq, 3.0Hz, 1lax); 6.20(H-8); 12.51(OH); and 12.91ppm (OH). Mass Spectrum: 3 76m/e (M+). TLC Data Solvent system A, C6I-I6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, C6I-I6-EtOAc-

872

24.

Herbarin and Related Metabolites

iso-PrOH-HOAc (145:50:5:1, v/v/v); E, C6H6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6H6-Me2CO (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, Cd-16EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for isomarticin: A=0.11; B=0.12; C=0.09; and D=0.15. Systems E-H were used for isolation (preparative TLC). References S. Nemec, R. A. Baker, and J. H. Tatum; Toxicity of Dihydrofusarubin and Isomarticin from Fusarium solani to Citrus Seedlings; Soil Biol. Biochem., Vol. 20, pp. 493-499 (1988). J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24. Herbarinand Related Metabolites

873

Common/Systematic Name 5,8-Dihydroxy-6-methoxy-3-methyl-2-aza-9,10-anthracenedione Molecular Formula/Molecular Weight C15H11NOs;MW = 285.06372 0

OH

~~"~Me

MeO 0

OH

General Characteristics Red crystals from benzene/methylene chloride; mp., 231-235 oc. F.ungal Source Fusarium solani isolated from diseased citrus roots.

Isolation/Purification Culture filtrates were extracted with ethyl acetate; the extract was dried with anhydrous sodium sulfate and reduced in volume. The extract was fractionated by preparative TLC on silica gel HF254(type 60) plates. 5,8-Dihydroxy-6-methoxy-3-methyl-2-aza-9,10anthracenedione was crystallized from benzene/methylene chloride. Biological Activity Suspected phytotoxin. Spectral Data UV:

)E~H max

253 322, 476, 500, and 530nm.

1H NMR: (CDCI3) 2.81(s, 3-Me), 7.96(H-4); 4.02(7-OMe); 9.50(H-1); 6.75(H-8); 13.10(O1-1); and 13.50ppm (OH). Mass Spectrum: 2 8 5m/e (M§ TLC Data Solvent system A, C6H6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCla-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v), C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v), D, C6H6-EtOAciso-PrOH-HOAc (145:50:5" 1, v/v/v); E, C6I%-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1,

874

24.

Herbarin and Related Metabolites

v/v/v); F, C6H6-Me2CO (17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, CnI-I~EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for 5,8-dihydroxy-6-methoxy-3-methyl-2aza-9,10-anthracenedione: A=0.31; B=0.38; C=0.35; and D=0.43. Systems E-H were used for isolation (preparative TLC). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

875

Common/Systematic Name rel-(3R,4aR)-5,10-Dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9trihydroxy- 1H-naphtho[2,3-c]pyran Molecular Formula/Molecular Weight C15H1607, M3~ = 308.08960

OH

0

.~ MeO

H

Fi 0 Me OH

II fl I OH 0 H

General Characteristics Pale red crystals from ethyl acetate or methylene chloride; 153-154~C. Fungal Source Fusarium solani isolated from diseased citrus roots. Isolation/Purification Culture filtrates were extracted with ethyl acetate; the extract was dried with anhydrous sodium sulfate and reduced in volume. The extract was fractionated by preparative TLC on silica gel HF254(type 60) plates, rel-(3R,4aR)-5,10-Dioxo-3,4,4a,5,10,10a-hexahydro7-methoxy-3-methyl-3,6,9-trihydroxy-1H-naphtho[2,3-c]pyran was crystallized from ethyl acetate or methylene chloride. Biological Activity Suspected phytotoxin. Spectral Data UW:

~,E~

214, 245, 277, 304, and 394nm.

IH NMR: (CDCI3) 1.54(s, 3-Me); 1.69(dd, J-13.5Hz, 4eq, l l.0Hz, 4a); 2.42(dd, 13.5Hz, 4ax, 3.5Hz, 4a); 2.97(m, 13.5Hz, 4a, 11.0Hz, lax, 5.5Hz, leq); 2.02(d, 2.4Hz); 3.43(m, J=13.5Hz, 10a, 11.0Hz, 4ax, 3.5Hz, 4eq); 3.97(7-OMe); 4.22(m, H-lax); 4.22(H-leq); 6.68(H-8); 12.05(OH); and 12.22ppm (OH). Mass Spectrum: 308m/e (M+).

876

24.

Herbarin and Related Metabolites

TLC Data Solvent system A, C6H6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, C6Hn-EtOAciso-PrOH-HOAc (145:50:5:1, v/v/v); E, C6H6-hexane-Me2CO (9:9:2, v/v/v) or (3"6:1, v/v/v); F, C6I-I6-Me2CO(17:3, v/v); G, hexane-Me2CO-HOAc (15:5:0.1, v/v/v); H, C6I-I6EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for rel-(3R,4aR)-5,10dioxo-3,4, 4a, 5,10,10a-hexahydro-7-methoxy- 3-methyl-3,6, 9-trihydroxy- 1H-naphtho [2,3c]pyran: A=0.21; B=0.27; C=0.21; and D=0.38. Systems E-H were used for isolation (preparative TLC). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

877

Common/Systematic Name rel-(3R,4aR, 10aS)-5,10-Dioxo-3,4,4a, 5,10,10a-hexahydro-7-methoxy-3-methyl-3,6,9trihydroxy- 1H-naphtho[2,3-c]pyran Molecular Formula/Molecular Weight C15H1607; M ~ r = 308.08960

OH

0

H

_ROMe MeO

OH

II H I OH 0 H

General Characteristics Yellow crystals from methanol; mp., 126-129~ Fungal Source

Fusarium solani isolated from diseased citrus roots.

Isolation/Purification Culture filtrates were extracted with ethyl acetate; the extract was dried with anhydrous sodium sulfate and reduced in volume. The extract was fractionated by preparative TLC on silica gel HF2s4 (type 60) plates, rel-(3R,4aR, 10aS)-5,10-Dioxo-3,4,4a,5,10,10ahexahydro-7-methoxy-3-methyl-3,6,9-trihydroxy- 1H-naphtho[2,3-c]pyran was crystallized from methanol. Biological Activity Suspected phytotoxin. Spectral Data UV:

~,maxE~" 212, 245, 277, 304, and 394nm. IH N M R :

(CDCI3) 1.47(s, 3-Me); 1.63(t, J=13.0Hz, 4eq); 2.04(dd, J=13.0Hz, 4ax, J=4.0Hz, 4a); 2.99(m, H-10a); 2.02(3-OH); 3.62(m, H-4a); 4.06(dd, J=l 1.5Hz, leq, J=3.0Hz, 10a); 4.61(d, J=l 1.5Hz, lax); 6.75(H-8); 12.37(OH); and 12.69ppm (OH). Mass Spectrum:

308m/e (IVY).

878

24.

Herbarin and Related Metabolites

TLC Data Solvent system A, C6H6-nitromethane-HOAc (75:25:2, v/v/v); B, CHCIs-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, C6I-I~-EtOAciso-PrOH-HOAc (145:50:5:1, v/v/v); E, C6I%-hexane-Me2CO (9:9:2, v/v/v)or (3:6:1, v/v/v); F, C6H6-Me2CO(17:3, v/v): G, hexane-MezCO-HOAc (15:5:0.1, v/v/v); H, C6I~EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Rt for rel-(3R,4aR, 10aS)-5,10dioxo-3,4,4a, 5,10,10a-hexahydro-7-metho xy-3-methyl-3,6,9-t rihydroxy- 1H-naphtho [2,3c]pyran: A=0.18; B=0.21; C=0.17; and D=0.33. Systems E-H were used for isolation (preparative TLC). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24. Herbarinand RelatedMetabolites

879

Common/Systematic Name rel-(3R,4aR, 10aR)-5,10-Dioxo-3,4,4a,5,10,10a-hexahydro-3,7-dimethoxy-3-methyl-6,9dihydroxy- 1H-naphtho[2,3-c]pyran Molecular Formula/Molecular Weight C16H1807; M W -- 3 2 2 . 1 0 5 2 5

OH

0

H

MeO,/[~~

H i

OH

II H / O H

Me OMe

General Characteristics Yellow crystals from methanol; mp., 176-180~ Fungal Source

Fusarium solani isolated from diseased citrus roots.

Isolation/Purification Culture filtrates were extracted with ethyl acetate; the extract was dried with anhydrous sodium sulfate and reduced in volume. The extract was fractionated by preparative TLC on silica gel HF254 (type 60) plates, rel-(3R,4aR, lOaR)-5,10-dioxo-3,4,4a,5,10,lOahexahydro-3,7-dimethoxy-3-methyl-6,9-dihydroxy-1H-naphtho [2,3 -c] pyran was crystallized from methanol. Biological Activity Suspected phytotoxin. Spectral Data UV:

~,rm~n 210, 245, 277, 304, and 394nm.

~HNMR: (CDCI3) 1.47(s,3-Me), 1.69(dd, 13.5Hz, 4eq, I 1.0I-Iz,4a), 2.42(dd, 13.5Hz, 4ax, 3.5Hz, 4a); 2.97(m, 13.5Hz, 4a, I 1.0Hz, lax, 5.51-Iz,leq); 3.24(3-OMe); 3.43(m, 13.5Hz, 10a, I 1.0I-Iz,4ax, 3.51-Iz,4eq); 3.98(7-OMe); 3.88(dd, 12.0I-Iz,leq, I 1.0Hz, 10a); and 4.22ppm (dd, 12.0Hz, la• 5.5Hz, 10a). Mass Spectrum:

322role (M+).

880

24.

Herbarin and Related Metabolites

TLC Data Solvent system A, C6H6 nitromethane-HOAc (75:25:2, v/v/v); B, CHCl3-EtOAc-hexaneHOAc (10:5:5:0.3, v/v/v); C, hexane-Me2CO-HOAc (15:5:0.3, v/v/v); D, C6I-I6-EtOAciso-PrOH-HOAc (145:50:5:1, v/v/v); E, C6H6-hexane-Me2CO (9:9:2, v/v/v) or (3:6:1, v/v/v); F, C6I-I6-Me2CO(17:3, v/v): G, hexane-Me2CO-HOAc (15:5:0.1); H, C6H6EtOAc-iso-PrOH-HOAc (16:3:1:0.1, v/v/v). Re for rel-(3R,4aR, 10aR)-5,10dioxo-3,4,4a,5,10,10a-hexahydro-3,7-dimethoxy-3-methyl-6,9-dihydroxy- 1Hnaphtho[2,3-c]pyran: A=0.43; B=0.57; C=0.35; and D=0.63. Systems E-H were used for isolation (preparative TLC). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

881

Common/Systematic Name Anhydrofusarubin lactone 5,10-Dihydroxy-7-methoxy-3-methyl- 1H-naphtho[2,3-c] pyran- 1,6,9-trione Molecular Formula/Molecular Weight C15H1oO7, M~V = 302.04265

0

OH

0

I)~

0

MeO

Me

0

OH

H

General Characteristics Dark crystals from CHCI3; mp., 300 ~C. Fungal Source

Fusarium solani, a common soil fungus in citrus groves which can be readily isolated from

vascular tissue of fibrous roots from citrus trees affected with blight. First isolated from

Nectria haematococca.

Isolation/Purification Column chromatography using Kieselgel 60; column was deactivated with HOAc and H20, washed with Me2CO and then CHCI3; and then eluted with CHCI3, CHCI3-Me2CO, 2, 5, 10, and 15%. Biological Activity Suspected phytotoxin. Spectral Data UV~

;~mM~" 255(Iog C=4.25), 312(3.55), 330 inf(3.46), 508 inf(3.72), 536(3.77), 576(3.72), and 612nm (3.49). IR:

(KBr) 1745, 1650sh, 1610, 1582, 1532, 1470, 1435, 1400, 1375sh, 1350, 1300, 1275sh, 1260, 1230, 1190, 1175, 1155, 1135, 1115, 1060, 1025w, 980, 955, 935w, 875, 865sh, 845sh, 800, 780, 725, and 710cm1. IH NMR: (CDCI3) 2.38(3H, s, Me-3), 3.97(3H, s, MeO-7); 6.30(1H, s, H-S), 6.84(1H, s, H-4), 12.75(1H, s, OH-5); 14.28(1H, s, OH-10); addition of D20 removed the OH signals at 12.75 and 14.75ppm.

882

24. Herbarin and Related Metabolites

Mass Spectrum: 302(M § 100%) and 287m/e (14.5). TLC Data Solvent system (A) C6H6-MeNO2-HOAc(7525:2, v/v/v), (B) C6H6-Me2CO-HOAc (40:10:1, v/v/v), (C)CHCI3-HOAc (200:1, v/v), (D)CHCIa-CHaNO2 (9:1, v/v). TLC plates were silica gel GF (analytical) and silica gel HF 60 (preparative). Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

883

Common/Systematic Name 1,5,10-Trihydroxy-7-methoxy-3-methyl- 1H-naphtho[2,3-c] pyran-6,9-dione Molecular Formula/Molecular Weight C15H1207; M3~ = 304.05830

0

0

HO HO H

OH

General Characteristics Deep purple crystals from acetone; mp., 245~ on a preheated block. Fungal Source Fusarium solani, a common soil fungus in citrus groves which can be readily isolated from vascular tissue of fibrous roots from citrus trees affected with blight. Isolation/Purification Column chromatography using Kieselgel 60; column was deactivated with HOAc and H20, washed with Me2CO and then CHCI3; and then eluted with CHCI3, CHCI3-Me2CO, 2, 5, 10, and 15%. Biological Activity Suspected phytotoxin. Spectral Data UV:

ZmM~" 228(1oge=4.06), 263(4.05), 278sh (4.01), 343(3.23), 509(3.80), 535(3.87), and 572nm (3.71). Im:

(KBr) 3425s, 1600, 1580, 1440, 1400, 1385sh, 1280sh, 1250, 1225sh, l190w, 1155, 1110w, 1070, 1040, 990, 955, 890, 860br, and 805cmq. 1H NMR: (CDCI3) 2.18(3H, s, Me-3); 3.94(3H, s, MeO-7); 6.26(1H, s, H-8); 6.45(1H, s, H-4); 6.73(1H, s, H-I); 12.85(1H, s, OH-5); 13.25(1H, s, H-10); addition of D20 removed the OH signals at 12.85 and 13.25ppm. (CD3)2SO 2.04(3H, s, Me-3); 3.84(3H, s, MeO-7); 6.10(1H, s, H-8); 6.20(1H, s, H-4); 6.55(1H, d, J=6, H-I); 6.82(1H, d, ,]--6, OH-I); 12.76(1H, s, OH-5); 13.22(1H,

884

24.

Herbarin and Related Metabolites

s, OH-10). Addition of DzO removed the OH signals at 6.82, 12.76, 13.22, and the doublet at 6.55ppm appeared as a singlet at 6.63ppm. Mass Spectrum: LREIMS: 304(M +, 31.4%), 289(11), and 261(100); HR IMS: requires 304.0581, found 304.0590. Reference J. H. Tatum and R. A. Baker; Naphthoquinones Produced by Fusarium solani Isolated from Citrus; Phytochemistry, Vol. 22, pp. 543-547(1983).

24.

Herbarin and Related Metabolites

885

Common/Systematic Name 5,10-Dihydroxy- 1,7-dimethoxy-3-methyl-1H-naphtho[2,3 -c] pyran-6,9-dione Molecular Formula/Molecular Weight C16H1407; M W - 318.07395

MeOL ~' 0 Me 0 OH General Characteristics Purple crystals from benzene; mp., 190 ~ on a preheated block. Fungal Source Fusarium solani, a common soil fungus in citrus groves which can be readily isolated from vascular tissue of fibrous roots from citrus trees affected with blight. Isolation/Purification Column chromatography using Kieselgel 60; column was deactivated with HOAc and H20, washed with Me2CO and then CHCI3; and then eluted with CHCI3, CHCIa-Me2CO, 2, 5, 10, and 15%. Biological Activity Suspected phytotoxin. Spectral Data UV:

ZmM~" 228(Iog E=4.01), 263(3.98), 278sh (3.96), 342(3.15), 507(3.68), 535(3.75), and 572nm (3.63). Im:

(KBr) 1605, 1585, 1430, 1380, 1310w, 1250, 1220, 1185, 1150, 1080, 1050, 1015, 955, 885w, 865w, 815, and 790cm~ sh. 1H NMR: (CDCl3) 2.15(3H, s, Me-3); 3.62(3H, s, MeO-1); 3.93(3H, s, MeO-7); 6.24(1H, s); 6.25(1H, s); 6.29(1H, s); 12.80(1H, s, OH-5); and 13.24ppm (1H, s, OH~ addition of D20 removed signals at 12.80 and 13.24ppm.

886

24.

Herbarin and Related Metabolites

Mass Spectrum: LREIMS: 318(M+, 34.7%), 287(88.9), and 286role (100): HREIMS: Cv,I-Ii407 requires 318.0738; found: 318.0764m/e. TLC Data Solvent system (A) CsI-I6-MeNO2-HOAc (75:25:2, v/v/v), (B) Cd-I6-Me2CO-HOAc (40:10:1, v/v/v), (C) CHCI3-HOAc (200:1, v/v), (D) CHCI3-CH3NO2 (9:1, v/v). TLC plates were silica gel GF (analytical) and silica gel HF 60 (preparative). Reference J. H. Tatum, R. A. Baker, and R. E. Berry; Metabolites ofFusarium solani, Phytochemistry, Vol. 28, pp. 283-284(1989).

24.

Herbarin and Related Metabolites

887

Common/Systematic Name 2,3-Dihydro-5,8-dihydroxy-6-methoxy-2-hydroxymethyl-3-(2-hydroxypropyl)-l,4naphthalenedione Molecular Formula/Molecular Weight C15H1507; M W ' = 3 1 0 . 1 0 5 2 5

OH

0

OH

O

General Characteristics Light red crystals from EtOAc; mp., 135 ~ on preheated block. Fungal Source Fusarium solani, a common soil fungus in citrus groves which can be readily isolated from vascular tissue of fibrous roots from citrus trees affected with blight. Isolation/Purification Column chromatography using Kieselgel 60; column was deactivated with HOAc and 1-120, washed with Me2CO and then CHCI3; and then eluted with CHCIa, CHCIa-Me2CO, 2, 5, 10, and 15%. Biological Activity Suspected phytotoxin. Spectral Data UV/Visible: ~,mU~" 213, 244, 277, 303 sh, 391, and 400nm (sh) (log 6=3.98, 4.21, 3.83, 3.64, 3.88, and 3.83 respectively). IR:

(KBr) 3300, 1610, 1570 sh, 1470, 1430, 1400, 1380, 1315, 1290, 1270, 1220, 1205, 1175, 1125, 1095, 1065, 1040, 1020, 1000 sh, 990, 925, 900, 870 w, 825, and 805cm"1

(sh). 1H N~/[R:

(CDCI3) 1.22(3I-I,d, J=6Hz, Me-12); 1.79(IH, m, H-10), 1.96(IH, m, H-10), 2.42(IH, s, OH); 2.90(IH, m, H-2); 3.28(II-I,m, H-3), 3.86(2H, m); 3.95(3H, s, MeO-6), 4.24(IH, dd, J=l 1.2,2.51-1z);6.70(IH, s,H-7); 12.22(IH, s, OH-5), and

888

24.

Herbarin and Related Metabolites

12.58ppm (1H, s, OH-8); addition of D20 removed the OH signals at 2.42, 12.22, and 12.58ppm. When 2,3-dihydro-5,8-dihydroxy-6-methoxy-2-hydroxymethyl-3-(2-hydroxypropyl)1,4-naphthalenedione was treated with D20 the hydroxyls at 12.58, 12.22, and 2.42ppm disappeared. This treatment also caused the H-1 distorted dd at 4.24ppm to become a sharp dd, J=l 1.2, 2.5Hz and the 2H multiplet at 3.86ppm became rounded with a projecting dd out the top (J=l 1.2, 4.5Hz) which indicated a CH2 coupled to a hydroxyl. Decoupling at 4.24ppm changed the 2H multiplet at 3.86ppm to a multiplet and the 1H multiplet at 2.90ppm to a multiplet which showed that they were coupled. Deeoupling at 3.86ppm changed the dd at 4.24ppm to a broad sing,let, the IH multiplet at 2.90 became a dd (J=6.2, 2.5Hz), the 1H multiplet at 1.96 became add (J=l 5, 7.5Hz), the 1H multiplet at 1.79 became a broad d (J= 15Hz) and the 3H doublet at 1.26ppm became a singlet. The C-9 carbon was CH2-OH at 4.24ppm, 3.86ppm and was split by a single hydrogen at 2.90ppm on C-2 and distorted by the hydroxyl. The other H at 3.86ppm was coupled to CH2 at 1.96 and 1.79ppm and a methyl at 1.26ppm. Decoupling of the 1H multiplet at 3.28ppm changed the multiplet at 2.90ppm to a dd (J=4.5, 2.5Hz), the multiplet at 1.96ppm became a broad doublet (J=l 5Hz) and the multiplet at 1.79ppm became add (J=15, 10Hz). Decoupling of the 1H multiplet at 2.90ppm caused the dd at 4.22ppm to become a doublet (J= 11.2Hz) and the multiplet at 3.86ppm to show a distorted doublet (J= 11.2Hz). The C-10 carbon was CH2 at 1.96, 1.79ppm which was attached to the C-3 position and was split by the C-3 hydrogen at 3.28ppm and the C-11 hydrogen at 3.86ppm. The C-12 was a methyl that was split by the C-11 hydrogen. The H-2 and H-3 hydrogens were shown to be coupled and established their relative positions. The small coupling constant of 6.2Hz indicated they were in axial-equatorial position. The methoxy resonance at 3.95ppm was assigned to the 6 position. The lone unsplit hydrogen at 6.70ppm was at the C-7 position on the aromatic portion of the ring. Mass Spectrum: LREIMS: 292role (M + - 18); CIMS: (isobutane) 31 lm/e (M++ 1); ClsHlsO7 requires 310.1051; found: 310.1046role. TLC Data System (A) CHC13-nitromethane-MeOH-HOAc (170:20:6: I, v/v/v/v); (B) CHCI3-CH2CI2HOAc-MeOH (140:50:4:4, v/v/v/v). TLC plates were silica gel GF (analytical) and silica gel HF 60 (preparative). Reference J. H. Tatum, R. A. Baker, and R. E. Berry; Metabolites ofFusarium solani; Phytochemistry, Vol. 24, pp. 3019-3021 (1985).

24. Herbarin and Related Metabolites

889

Common/Systematic Name 2,3-Dihydro-5-hydroxy-4-hydroxymethyl-8-methoxynaphtho[l,2-b]furan-6,9-dione Molecular Formula/Molecular Weight C15H1406; MW' = 290.07904

O

Me

1

2

MeO,

~"C O

H2OH

OH

General Characteristics Red crystals from CH2Cl2-hexane; mp., 202-203 ~C. Fungal Source Fusarium solani, a common soil fungus in citrus groves which can be readily isolated from vascular tissue of fibrous roots from citrus trees affected with blight. Isolation/Purification Column chromatography using Kieselgel 60; column was deactivated with HOAc and H20, washed with Me2CO and then CHCI3; and then eluted with CHCI3, CHCI3-Me2CO, 2, 5, 10, and 15%. Biological Activity Suspected phytotoxin. Spectral Data UV:

ImM~" 223,297, 485, 502, and 538nm (log e=4.06, 3.62, 3.50, 3.47, and 3.17, respectively).

l-R:

(KBr) 3490, 1660, 1625, 1585, 1420, 1400, 1390sh, 1355, 1315, 1220, 1160, 1075, 1040, 1015, 955, 915, 885, 855, 830, 815, and 780cm~. IH NMR: (CDCI3) 1.59(3H, d, J=6Hz, 12-Me); 2.67(1H, t, J=6Hz, CH2 OH); 2.92(1H, dd, ,/-17, 6.5Hz, H-3); 3.46(1H, dd, J=17, 9Hz, H-3); 3.90(3H, s, MeO-8); 4.74(2H, d, J=6Hz, H-4); 5.21(1H, m, H-2), 6.07(1H, s, H-7); and 13.60ppm (1H, s, C-5); addition of D20 removed the OH signals at 2.67 and 13.60ppm. The NMR spectrum had an

890

24.

Herbarin and Related Metabolites

eight line multiplet with 1H at (5.21 ppm). When the spectrum was decoupled at 5.21ppm and the 1H dd at 3.46ppm (J=17.9 Hz) became a d (J=17Hz), the 1H dd at 2.92ppm (J= 17, 6.5Hz) became a doublet (J= 17Hz) and the 3H doublet at 1.59ppm became a singlet. Thus, a single hydrogen was split by a CH2 and a methyl. Decoupling the 2H doublet at 4.8ppm (J=6Hz) caused the triplet at 2.67ppm (J=6Hz) to become a singlet. A CH2 was split by a single H. Decoupling the 3H doublet at 1.6ppm caused the multiplet at 5.21 ppm to become add (J=9, 6.SHz). When D20 was added the singlet at 13.60ppm and the triplet at 2.67ppm disappeared and the 2H doublet at 4.74ppm became a singlet. This indicated a methyl C-H attached to CH2 and an isolated CH2 attached to a hydroxyl. A single unsplit hydrogen at 6.07ppm was on a quinone ring at C-7 while a singlet 3H at 3.90ppm indicated a methoxy at the C-8. Mass Spectrum: LREIMS: 290(M+); C~5H~406 requires 290.0789; found: 290.0795m/e. TLC Data System (A) CHCl3-nitromethane-MeOH-HOAc (170:20:6:1, v/v/v/v); (B) CHCIa-CH~_CI2HOAc-MeOH (140:50:4:4, v/v/v/v). TLC plates were silica gel GF (analytical) and silica gel HF 60 (preparative). Reference J. H. Tatum, R. A. Baker, and R. E. Berry; Metabolites of Fusarium solani; Phytochemistry, Vol. 24, pp. 3019-3021(1985).

24.

Herbarin and Related Metabolites

891

Common/Systematic Name 5,8-Dihydroxy-2-methoxy-6-hydroxymethyl-7-(2-hydroxypropyl)-l,4-naphthalenedione Molecular Formula/Molecular Weight C15H1607; ~

MeO

= 308.08960

0

OH 8L~ lO ? H 12 B~ ' ~ C H2-C H--Me

O

OH

General Characteristics Red crystals from EtOAc; mp., 206-212~ (dec). Fungal Source Fusarium solani, a common soil fungus in citrus groves which can be readily isolated from vascular tissue of fibrous roots from citrus trees affected with blight. Isolation/Purification Column chromatography using Kieselgel 60; column was deactivated with HOAc and H20, washed with Me2CO and then CHCI3; and then eluted with CHCI3, CHCIa-Me2CO, 2, 5, 10, and 15%. Biological Activity Suspected phytotoxin. Spectral Data UV:

~,~m"~ 227, 306, 452 sh, 482, 509, and 546nm (log c=4.38, 3.87, 3.56, 3.76, 3.80, and 3.59, respectively). IR:

(KBr) 3330, 1615, 1550, 1450, 1420, 1390, 1295, 1265, 1245, 1220, 1205, l180w, 1150, 1115, 1105, 1075, 1025, 1010, 995 w, 940 w, 855, 825, and 815cmq (sh). 1H NMR: (CDCI3) 1.39(3H, d, J=6Hz, Me-12); 2.86(1H, dd, J=13.5, 10Hz, H-10); 3.13(1H, dd, J=13.5, 3Hz, I~I-10); 3.95(3H, s, MeO-2); 4.16(1H, m, H-11); 4.63(1H, d, J=12Hz, H-9); 4.97(1H, d, J=12Hz, H-9); 6.20(1H, s, H-3); 12.86(1H, s, OH-5); and 13.35ppm (1H, s, OH-8); addition of D20 removed the OH signals at 12.86 and 13.35, the other OH's were not observed.

892

24.

Herbarin and Related Metabolites

Mass Spectrum: LREIMS: 308(M+); C15Hi607requires 308.0895; found: 308.0862m/e. TLC Data System (A) CHCl3-nitromethane-MeOH-HOAc (170:20:6:1, v/v/v/v); (13) CHCI3-CH2CI2HOAc-MeOH (140:50:4:4, v/v/v/v). TLC plates were silica gel GF (analytical) and silica gel HF 60 (preparative). Reference J. H. Tatum, R. A. Baker, and R. E. Berry; Metabolites ofFusarium solani; Phytochemistry, Vol. 24, pp. 3019-3021(1985).

24.

Herbarin and Related Metabolites

893

Common/Systematic Name 2,5,8-Trihydroxy-6-methoxy-3-(2-oxopropyl)- 1,4-naphthoquinone Molecular Formula/Molecular Weight C14H1207; M W "- 292.05830

OH

0 OH

MeO

CH2- 0 OH

H--Me

0

General Characteristics Red crystals from CHCI3; mp., 231-234 ~C. Fungal Source Fusarium solani. Isolation/Purification Isolated by preparative TLC using (A) C6H6-Me2CO-HOAc (160:40:1, v/v/v), 03) chloroform or (C) benzene-nitromethane (3:1, v/v). Biological Activity Strongly phytotoxic to tomatoes and peas. Spectral Data UV:

~,~ffH 212, 238, 261,320,470, 490 and 520nm (log c=4.22.4.14, 407, 3.88, 3.84, 3.82, and 3.66, respectively). IR:

3300, 1710, 1630, 1600, 1570, 1480, 1430w, 1405, 1360, 1320, 1280, 1210, l190w, 1085, 1045, 1025, 975, 885, 840w, 820sh, 810, and 760cm"~. Mass Spectrum: LREIMS: 292(M~); C~4H~207requires 292.0581; found: 292.0609m/e. Reference J. H. Tatum, R. A. Baker, and R. E. Berry; Naphthoquinones and Derivatives from Fusarium; Phytochemistry, Vol. 26, pp. 795-798(1987).

894

24.

Herbarin and Related Metabolites

Common/Systematic Name Nectriafurone 5,8-Dihydroxy-4,9-dione-3-(2-hydroxyethyl)-7-methoxynaphtho[2,3-c]furan Molecular Formula/Molecular Weight C15H1207; 1VIVr = 3 0 4 . 0 5 8 3 0

OH

0 o

OH

0

C--OH \ Me

General Characteristics Yellow-brown crystals from MeOH; mp., 222-225 ~C. Fungal Source Fusarium oxysporum and Nectria haematococca. Isolation/Purification Extracted with Me2CO and the aqueous mixture obtained by vacuum concentration reextracted (x4) with EtOAc. The EtOAc residue was redissolved in CHCi3 and applied to a Sephadex LH20 column packed in CHCI3-MeOH (49:1, v/v) and the pigments eluted with CHCI3-MeOH (49:1, v/v). The first pigments to be eluted gave a dark violet fraction containing anhydrofusarubin as the main product. Then a red fraction was obtained which was shown to be a mixture ofjavanicin, norjavanicin and anhydrojavanicin. Nectriafurone was isolated in a yellow fraction. It showed a strong fluorescence in UV which distinguished this pigment from the preceding ones. Next, anhydrofusarubin lactone was eluted as a purple fraction followed by fusarubin. Biological Activity Suspected phytotoxin; tested for antibiotic activity in a broth microdilution assay against Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhi, Serratia marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Klebsiella pneumoniae. None of the organisms were inhibited at 128~g/ml. Sp.ectral Data UV: Ef~ 242, 258, 324, 444, a,ld 468nm (log e-4.26, 4.23, 3.84, 4.07, and 3.97, respectively).

24.

Herbarin and Related Metabolites

895

IR: (KBr) 3420, 3100, 1605, 1560, 1450, 1425, 1410, 1360w, 1300, 1250, 1215, 1175, 1160, 1110, 1007(sh), 1005, 975, 950, 900w, 865, 850, 820w, and 810cmq. ~H NMR: (CDCI3) 1.64(3H, d, J=7Hz, Me); 3.99(3H, s, MeO=6); 4.83(1H, d, J=7I--Iz, OH), 5.19(1H, m, 51, H); 6.70(1H, s, H-7); 8.08(1H, s, H-I); 13.07(1H, s, OH-5) and 13.39ppm (1H, s, OH-8). Mass Spectrum: LREIMS: 304m/e (M"). References D. Parisot, M. Devys, J. P. Ferezou, and M. Barbier; Pigments from Neetria haematococca: Anhydrofusarubin Lactone and Nectriafurone; Phytochemistry, Vol. 22, pp. 1301-1303(1983). J. H. Tatum, R. A. Baker, and R. E. Berry; Naphthoquinones and Derivatives from Fusarium; Phytochemistry, Vol. 26, pp. 795-798(1987).

896

24. Herbarin and Related Metabolites

Common/Systematic Name 8-O-Methyl ether nectriafurone 5-Hydroxy-4, 9-dione-3-(2-hydroxyethyl)-7,8-dimethoxynaphtho[2,3-c] furan Molecular Formula/Molecular Weight C16H1407; ~ = 318.07395

MeO

OMe

0

OH

0

"

C--OH \ Me

General Characteristics Yellow-brown needles from MeOH; mp., 214-222 ~ (Dec.). Fungal Source Fusarium oxysporum. Biological Activity Suspected phytotoxin. Spectral Data UV:

EtOH ~ max

236, 255(sh), 322, and 443nm (log e=4.37, 4.21, 3.90, and 4.02, respectively).

IR: (KBr) 3430, 3120, 1645, 1625, 1600, 1540, 1470, 1435, 1375, 1345, 1295(sh), 1265(sh), 1245, 1215, 1165, 1120(sh), 1110, 1070, 1035, 1005w, 955, 895, 855, 820, 810, and 765cm1. IH NMR: (CDCI3) 1.64(3H, d, ,/=7Hz, Me); 4.00(3H, s, MeO-6); 4.03(3H, s, MeO-6); 4.03(3H, s, MeO-8); 4.78(1H, d, J=7Hz, OH); 5.19(1H, m, 51, H); 6.83(1H, s, 8-7); 7.99(1H, s, H-l); and 13.30ppm (1H, s, OH-5). Mass Spectrum: LREIMS: 304m/e (M+). Reference J. H. Tatum, R. A. Baker, and R. E. Berry; Naphthoquinones and Derivatives from Fusarium; Phytochemistry, Vol. 26, pp. 795-798(1987).

Miscellaneous Metabolites Alternaric acid Ampullicin Isoampullicin Acremoauxin A 2-(3,4-Dihydroxyhepta- 1,5-dienyl)-6-hydroxybenzyl alcohol Achaetolide Achaetolidone 8-(10,11-Dihydroxyhexa- 12-enyl)-3-hydroxydihydroisbenzylfuran 8-(10,11-Dihydroxyhexa- 12-enyl)-3-hydroxydihydroisbenzylfuran (isomeric) 6-( 1-Ethoxyethyl)-5-hydroxy-2,7-dimethoxy- 1,4-naphthoquinone 6-( 1-Hydroxyethyl)-5-hydroxy-2,7-dimethoxy- 1,4-naphthoquinone 6-Ethyl- 1-acetonyl-1,5-dihydroxy-2,7-dimethoxy-1,4-naphthoquinone Pyriculariol Sydonol (3-Hydroxy-4-(1-hydroxy- 1,5-dimethylhexyl)benzyl alcohol) Homotrichione p-Toluquinone (Toluquinol) Aszonapyrone A Pyrenochaetic acid A Pyrenochaetic acid B Pyrenochaetic acid C Candidusin A Candidusin B Ankalactone Alterporriol D Alterporriol E Paecilospirone O-Acetylbenzeneamidinocarboxylic acid Helicascolide A Helicascolide B Arthrosporone Anhydroarthrosporone Arthrosporol Aurantiein A Auranticin B Shiraiachrome A Shiraiachrome B Shiraiachrome C Cavoxin Cavoxone Cavoxinine Cavoxinone Armillyl everninate Arnamiol trans-Resoreylide 897

898

25.

CisoResorcylide Cochliohydroquinone A (Alboleersin) Cochlioquinone A (Luteoleersin) Cochlioquinone B Stemphone

Miscellaneous Metabolites

25. Miscellaneous Metabolites

899

Common/Systematic Name Alternaric acid 12-(6, 6-Dihydro-4-hydroxy-6-methyl-2-ox opyran-3-yl)-4,6-dihydroxy-3-methyl-9methylene- 12-oxododec-6-ene-6-carboxylic acid Molecular Formula/Molecular Weight C21HaoOs; MW = 410.19407 Me

OH

OH

CH2

O

OH

Et ~ C H--C H--C--C H ~ C H--C H2~C--C H2~C H2~ I

CO2H

0 "/ "(

Me

General Characteristics Alternaric acid, mp.,138~ is a highly crystalline, colorless, optically inactive, unsaturated dibasic acid; crystallized from benzene in needles, mp. 135 ~ Specimens dried at room temperature gave high values for carbon due presumably to traces of adsorbed benzene, drying at 100~ caused discoloration so that the agreement between the observed and the calculated analytical figures was perhaps fortuitous. Fungal Source Certain strains of Alternaria solani. Isolation/Purification The crude yellow solid (mp. 122-128~ obtained by chloroform extraction of culture filtrates, previously adjusted to pH 3.6, was taken up in hot benzene and a little amorphous insoluble material rejected. The solid which separated on cooling was twice recrystallized from dry benzene, giving a colorless product, mp. 132~ The bright yellow benzene mother-liquors were bulked and concentrated to yield slightly less pure material, mp. 130-132~ The first fraction was dissolved in saturated sodium hydrogen carbonate solution and extracted with ether (removing only a trace of gum); alternaric acid was recovered by acidification of the bicarbonate solution with concentrated hydrochloric acid, dried in vacuo at room temperature, and crystallized twice from aqueous methanol. It formed colorless, thin, rectangular plates, mp. 135-136 oC of the monohydrate. Alternaric acid was crystallized from absolute methanol or ethanol and formed elongated prisms, mp. 138~ Biological Activity A substance with remarkably specific antifungal activity but low antibacterial properties. It was shown to inhibit germination of fungal spores but it also caused a characteristic stunting of the germ tubes. Extreme specificity has been noted; some fungi, e.g., Myrothecium verrucaria, are extremely sensitive, germination being inhibited by low concentrations of alternaric acid; others, e.g.,Fusarium caeruleum, are very resistant, high concentrations having no noticeable effect; still others, e.g.,B, allii, show stunted germ

900

25.

Miscellaneous Metabolites

tubes at low concentrations, though much higher concentrations fail to prevent germination. Alternaric acid is markedly phytotoxic and it is possible that it is, at least in part, responsible for plant diseases caused by A. solani. Spectral Data UV:

maxEtOH 274 and 210nm (log e=4.04 and 4.07). IR~ The infrared spectra were obtained with Nujol "mulls". The infrared spectrum of alternaric acid crystallized from methanol or from carbon tetrachloride showed bands at 1560, 1338, 1292, 1270, ca. 1225, 1215, 1175, 1149,1117, 1069, 1000, 975, 955, 932, ca. 910, 900, 820, 800, and 760cm "~ in addition to 1710 and 1732cm ~. Mass Data: Found atter drying in vacuo over phosphoric oxide at 20~ C, 61.9, 62.0, 62.7, 62.4; H, 7.5, 7.3, 7.4, 7.3. At 100~ C, 61.4; H, 7.7: required C, 61.4; H, 7.4; 3C-Me, 11.1; 4C-Me, 14.8%; equiv. (dibasic acid), 205; M, 410. References J. R. Bartels-Keith; Alternaric Acid. Part III. Structure; J. Chem. Soc., pp. 1662-1665 (1960). P.'W. Brian, P. J. Curtis, H. G. Hemming, E. G. Jefferys, C. H. Unwin, and J. M. Wright; Alternaric Acid; A Biologically Active Product ofAlternaria solani; its Production, Isolation and Antifungal Properties; J. Gen. Microbiol, Voi. 5, pp. 619-632(1951). J. F. Grove; Alternaric Acid. Part 1. Purification and Characterization; J. Chem. Soc.,pp. 4056-4059(1952).

25.

Miscellaneous Metabolites

901

Common/Systematic Name Ampullicin Molecular Formula/Molecular Weight ClsHITNO3, MW = 259.12084 O 141[

8

13

~

1

Me 12

General Characteristics

Colorless needles from n-hexane-benzene-acetone; mp., 197-198 ~C; [a]D20 -32 ~ (C=0.5, in MeOH). Fungal Source

Ampulliferma-like fungus sp. No. 27, obtained from a dead pine tree (Pmus thunbergii).

Isolation/Purification Culture filtrate was treated at pH 2.0 with active charcoal, which was successively extracted with acetone. The extract was concentrated, the residue was chromatographed on silica gel (benzene-acetone, 9:1, v/v). Crystallized from n-hexane-benzene-acetone. Biological Activity Accelerated the root growth of lettuce seedlings by 200% at 300 and 30mg/L. Spectral Data UV: ~H

max

281nm (e=14,700).

IR: (KBr) 3400, 2950, 2875, 1739(y-lactone), 1715, 1690, 1660, 1390, 1310, 1210, 910, 888, and 819cm "~.

IH N]VIR: (CDCI3) 1.75(IH,d, J=10.3I-Iz,H-I); 2.40(IH, ddd, J=I0.3, 4.9,4.9,H-I); 2.77(IH, d, J=4.9, H2); 2.03(2H, m, H-4); 1.96(2H, m, H-5); 2.77(IH, br, d, J=4.9, H-6); 5.53(IH, br, s,H-8); 6.73(IH, br, d, J=l.5, H-10); 1.99(3H, d, J=I.5, H-12); 1.53(IH, s,H-13); and 8.67ppm (IH, br, s,NH, D20 exch.).

902

25.

Miscellaneous Metabolites

13C NMR: (CDCI3) 23.8(t, C-I), 52.7(d, C-2), 88.0(s, C-3), 29.7(t, C-4), 23.0(t, C-5), 46.7(d, C6), 56.0(s, C-7), 109.4(d, C-8), 136.3(s, C-9), 128.5(d, C-10), 141.1(s, C-I 1), 10.9(q, C-12), 24.9(q, C-13), 176.1(s, C-14), and 171.9ppm (s, C-15). Mass Data: 259(NC), 218, 204, 187, 134, 91, and 72m/e; found: C, 67.27; H, 6.39; N, 5.45; calcd for CIsHITNO3 + 1/2 H20: C, 67.15; H, 6.76; N, 5.22%. Reference Y. Kimura, H. Nakajima, T. Hamasaki, T. Matsumoto, Y. Matsuda, and A. Tsuneda; Ampullicin and Isoampullicin, New Metabolites from an Ampulliferina-like Fungus sp. No.27; Agric. Biol. Chem., Vol. 54, pp. 813-814(1990).

25.

Miscellaneous Metabolites

903

Common/Systematic Name Isoampullicin Molecular Formula/Molecular Weight C15HI7NO3, MW = 259.12084

O

Me

I 2,,//~

.I.. 1

xl,~_sMe 0

12

General Characteristics Colorless crystals; mp., 202-203~

[a]D 20 +110 ~ (c=0.5, in MeOH).

Fungal Source

Ampulliferina-like fungus sp. No. 27, obtained from a dead pine tree (Pinus thunbergii).

Isolation/Purification Culture filtrate was treated at pH 2.0 with active charcoal, which was successively extracted with acetone. The extract was concentrated, the residue was chromatographed on silica gel, benzene-acetone, 19:1, v/v. Biological Activity Accelerated the root growth of lettuce seedlings by 200% at 300 and 30mg/L. Spectral Data UV:

~,maxE~" 284nm (e=20,500). IR:

(KBr) 3200, 2950, 1759, 1700, 1680, 1660, 1618, 1205, 1170, 1080, 1020, 910, 890, and 760cm "~. ~H NMR: (CDCI3) d, J-4.9, 5.16(1H, s, H-13);

1.76(H1, d, J-10.8Hz, H-I); 2.40(1H, ddd, J=10.8, 4.9, 4.9, H-I), 2.79(H1, H-2); 2.02(2H, m, H-4); 1.96(2H, m, H-5); 2.79(1H, br, d, J=4.9, H-6); br, s, H-8); 6.62(1H, br, d, J=l.5, H-10); 1.96(1H, d, J-1.5, H-12), 1.52(1H, and 8.47ppm (br, s, NH).

13C NMR.:

(CDCI3) 23.8(t,C-I), 51.0(d,C-2), 88.7(s,C-3),29.6(t,C-4),23.2(t,C-5),45.80, C-

904

25.

Miscellaneous Metabolites

6), 56.1(s, C-7), 107.2(d, C-8), 133.9(s, C-9), 133.3(d, C-!O), 139.9(s, C-11), lO.4(q, C-12), 24.7(q, C-13), 176.4(s, C-14), and 173.0ppm (s, C-15). Mass Data: 259(M§ 218, 204, 187, 134, 91, and 72re~e;found: C, 69.48; H, 6.61; N, 5.40; ealed for C15HITNO3: C, 69.59; H, 6.71; N, 5.41%. Reference Y. Kimura, H. Nakajima, T. Hamasaki, T. Matsumoto, Y. Matsuda, and A. Tsuneda; Ampullicin and Isoampullicin, New Metabolites from an Ampulliferina-like Fungus sp. No. 27; Agile. Biol. Chem., Vol. 54, pp. 813-814(1990).

25.

Miscellaneous Metabolites

905

Common/Systematic Name Acremoauxin A Molecular Weight/Molecular Formula CI6H21NO6; ~ = 323.13689 H OH

HOH2

II

H e

-

~IH

General Characteristics Colorless needles from EtOH-EtOAc; mp., 142-144~ MeOH).

l a i D 19 + 5 3 . 6 ~

(c=0.35, in

Fungal Source Acremonium roseum

(I 4267).

Isolation/Purification Culture filtrate (pH 5.6) was saturated with NaCI and extracted with ethyl acetate. The extract was chromatographed on silica gel (ethyl acetate-acetone (3:1, v/v), containing 1% AcOH), crystallized from CHCI3-EtOH (5:1, v/v) and recrystallized from EtOH-EtOAc. Biological Activity Strongly inhibited the growth of Chinese cabbage seedlings at concentrations higher than 2.0ppm. Showed high activity in a rice lamina inclination test for auxins and brassinosteroids at concentrations higher than 5.0ppm. Spectral Data UV:

~

MeOH max

288(e=6000), 278 (7000), and 225nm (9600).

IR:

(KBr) 3430, 3260, 1730, 1450, 1330, 1228, 1169, 1088, 1037, 879, and 746cm~. ~H NMR: (MeOH-d4) 7.59(4', md, J-7.9Hz); 7.32(7', md, J-8.2Hz); 7.15(2', s); 7.08(6', ddd, J=8.2, 7.0, 0.9Hz); 7.00(5', ddd, J-7.9, 7.0, 0.8Hz); 4.17(1", dd, J-11.1, 6.6Hz); 4.13(1", dd, J-11.1, 6.6Hz); 4.05 (2, q, J-7.2Hz); 4.03(2", ddd, J-6.6, 6.6, 1.5Hz); 3.72(5", dd, J-11.1, 3.3Hz); 3.64(4", ddd, J=8.5, 6.2, 3.3Hz); 3.54(5", dd, J-11.1, 6.2Hz); 3.40(3", dd, ,/-8.5, 1.5Hz); and 1.58ppm (3, d, J-7.2Hz).

906

25.

Miscellaneous Metabolites

Mass Spectrum: HRMS: 305.1265m/e (M+-H20, calcd for CI6H~gNOs,305.1263); EIMS: 323(M*, 3%), 305(7), 287(1), 231(1), 189(23), 171(2), 144(100), and ll5m/e (10); FDMS: 669(2M+Na, 32%), 646(2M, 18), 346(M+Na, 4), and 323m/e (IV[*, 100). Reference T. Sassa, N. Yoshida, and E. Haruki; Isolation and Structural Elucidation of Acremoauxin A, a Novel Auxin Derivative Produced by Acremonium roseum; Agric. Biol. Chem., Vol. 53, pp. 3105-3107(1989).

25.

Miscellaneous Metabolites

907

Common/Systematic Name 2-(3,4-Dihydroxyhepta- 1,5-dienyl)-6-hydroxybenzyl alcohol Molecular Formula/Molecular Weight C]4I-I]sO4;MW = 250.12051 1

CH2OH

OH 14

General Characteristics Gummy substance; [a]40020 q-100 ~ (in EtOH). Fungal Source Pyricularia oryzae. Isolation/Purification Extracted with ethyl acetate. Repeated chromatography on silica gel. Biological Activity Promoted root elongation of rice seedlings at 250ppm. Spectral Data UV: ~ EtOH max

219(e=29500), 254(9400), and 293nm (3300).

IR: (CHCI3) 3600-3510(OH groups) and 1600cm"~(aromatic group). 1H NMR: (CD3COCD3) 1.67(3H, d, J=6.0Hz, H-14); 4.02(1H, q, J=12.5, 6.5Hz, H-10); 4.20(1H, q, J=12.5, 6.0Hz, H-11); 4.84(2H, s, H-I); 5.75(1H, o, J=15.0, 6.0Hz, H13); 6.10 (1H, q, J=15.0, 6.0Hz, H-12); 6.15(1H, q, J=16.0, 6.5Hz, H-9); 6.95(1H, d, J=16.0Hz, H-8); 6.75, 6.90(2H, 2d, J=7.0Hz, H-4, H-6); and 7.05ppm (1H, t, J=7.0, 7.0Hz, n-5). Reference S. Iwasaki, H. Muro, K. Sasaki, S. Nozoe, S. Okuda, and Z. Sato; Isolation of Phytotoxic Substances Produced by Pyricularia o~zae Cavara; Tetrahedron Lett., No. 37, pp. 3537-3542(1973).

908

25.

Miscellaneous Metabolites

Common/Systematic Name Achaetolide Molecular Formula/Molecular Weight C16H2805, M ~

"- 3 0 0 . 1 9 3 6 7

OH HO

OH

(CH2)6--Me General Characteristics Melting point, 120-122~ C . Fungal Source A chaetomium cristalliferum. Biological Activity Transpiration amplifier on barley shoots. Reference B. Bodo, L. Molho, D. Davoust, and D. Molho; Isolation and Structure Elucidation of Achaetolide and Achaetolidone, Two New Macrolides from a Fungus, 12th IUPAC Int. Symp. Chem. Nat. Prod., Tenerife, 1980, Abstr. B98.

25.

Miscellaneous Metabolites

909

Common/Systematic Name Achaetolidone Molecular Formula/Molecular Weight C16H2806; M W = 316.18859 0 HO

OH

/OH

(CH2)6--Me General Characteristics Melting point, 150-152~ C . Fungal Source A chaetomium cristalliferum. Reference B. Bodo, L. Molho, D. Davoust, and D. Molho; Isolation and Structure Elucidation of Achaetolide and Achaetolidone, Two New Macrolides from a Fungus; 12th IUPAC Int. Symp. Chem. Nat. Prod., Tenerife, 1980, Abstr. B98.

910

25.

Miscellaneous Metabolites

Common/Systematic Name 8-(10,11-Dihydroxyhexa- 12-enyl)-3-hydroxydihydroisobenzylfuran Molecular Formula/Molecular Weight C14H1804; M W -- 250.12051

OH

General Characteristics Melting point, 189~ [a]40026 +61.1 o 0EtOH); stereoisomeric to 8-(10,11-dihydroxyhexa- 12-enyi)-3-hydroxydihydroisobenzylfuran at C-8. Fungal Source. Pyricularia oryzae (Ken 53-55). Isolation/Purification Extracted with ethyl acetate; purified by repeated chromatography on silica gel. Biological Activity Promoted root elongation of rice seedlings at 250ppm. v

Spectral Data UV:

k

EtCH ,max

270(e=2600) and 278nm (2370).

IR:

(CHCI3) 3400-2800, 1605, 1470, 1380, 1290, and 1100-950cmq. IH NMR: (CD3OD) 1.73(3H, d, J=6.0Hz, n-14); 1.80(2H, q, J=6.0, 4.0Hz, H-9); 3.7-4.1(2H, m, H-10, H-11)*; 5.02(2H, br, s, H-I); 5.42(1H, t, J=6.0Hz, H-8); 5.52(1H, q, ,/=15.0, 6.0Hz, H-12); 5.75(1H, o, J=15.0, 6.0Hz, H-13); 6.68(2H, 2d, J=8.0Hz, H-4, H-6); and 7.12ppm (1H, t, J=8.0Hz, H-5). * t~-Glycol moiety should possess an erythro configuration. Reference S. Iwasaki, H. Muro, K. Sasaki, S. Nozoe, S. Okuda, and Z. Sato; Isolation of Phytotoxic Substances Produced by Pyricularia oryzae Cavara; Tetrahedron Lett., No. 37, pp. 3537-3542(1973).

25.

Miscellaneous Metabolites

911

Common/Systematic Name 8-(10,11-Dihydroxyhexa- 12-enyl)-3-hydroxydihydroisobenzylfuran Molecular Formula/Molecular Weight C14H1804; MW = 250.12051 I

.o

"~

0

H

OH I 13 OH

General Characteristics Gummy substance; [tt]4o026 -4.8 ~ (EtOH); stereoisomeric to 8-(10,11-dihydroxyhexa- 12-enyl)-3-hydroxydihydroisobenzylfuran at C8. Fungal Source Pyricularia oryzae. Isolation/Purification Extracted with ethyl acetate. Repeated chromatography on silica gel. Biological Activity Promoted root elongation of rice seedlings at 250ppm. Spectral Data UV:

~, Em~ 270(6=2600) and 278nm (2370). IR:

(CHCI3) 3400-2800, 1605, 1470, 1380, 1290, and 1100-950cm "]. 1H NMR:

(CD3OD) 1.70(3H, d, J=5.0Hz, h-14); 1.77(1H, H-9)*, 1.97(1H, s, J=13.0, 4.0, 4.0Hz, ); 3.7(2H, m, H-10, H-11)**; 4.97(1H, d, J=12.5Hz, H-I), 5.10(1H, d, J=12.5Hz); 5.20-5.50(1H, difused q, H-8); 5.55(1H, q, J=15.0, 5.0Hz, H-12); 5.77 (1H, o, J=15.0, 5.0Hz, H-13); 6.67(1H, d, J=8.0Hz, H-4, H-6), 6.90(1H, d, J=8.0Hz); and 7.10ppm (1H, t, J=8.0Hz, H-5). * Splittings of the signal were not observable because of the overlapping of C 14-H signal. ** a-glycol moiety should possess an erythro configuration.

912

25.

Miscellaneous Metabolites

Reference S. Iwasaki, H. Muro, K. Sasaki, S. Nozoe, S. Okuda, and Z. Sato; Isolation of Phytotoxic Substances Produced by Pyricularia oryzae Cavara; Tetrahedron Lett., No. 37, pp. 3537-3542(1973).

25. Miscellaneous Metabolites

913

Common/Systematic Name 6-(1-Ethoxyethyl)-5-hydroxy-2,7-dimethoxy-l,4-naphthoquinone Molecular Formula/Molecular Weight C16H1806; ~ = 306.11034 O

II MeO~~~C

Me--CH T I OCH2Me OH

,Me

1]

0

General Characteristics Orange needles from chloroform-hexane; mp., 136-137~ Fungal Source

Guignardia laricina GM-7.

Isolation/Purification Filtrated mycelia were extracted with acetone. After removal of acetone, the aqueous concentrate was extracted with ethyl acetate followed by chromatography on silica gel (CHCI3; CHCI3-EtOAc, 9:1, v/v; CHCI3-MeOH, 9:1, v/v). Crystallized from chloroform-hexane. Biological Activity Inhibited the growth of hypocotyls and roots of lettuce seeds 100% at 100ppm; more than 95% at 50ppm. Spectral Data UV: ~mM~H 221(Iog e=4.08), 234(sh, 4.01), 259(sh, 3.78), 264(3.79), and 310nm (3.64). IR: (KBr) 1675, 1630, 1590, 1300, and 1100cmq. 1H NMR: (CDCI3, TMS) 12.76(s, 1H, peri-OH); 7.23(s, 1H, ArH); 6.03(s, 1H, OH); 5.19(q, IH, J=6.84Hz); 3.99, 3.91(both s, 3H, -OCH3); 3.43, 3.34(both dq, 1H, J=9.52, 6.84Hz); 1.61(d, 3H, J-6.84Hz); and 1.18ppm (t, 3H, J-6.84Hz).

914

25.

Miscellaneous Metabolites

Mass Spectrum: 306.110 lm/e (M+); calcd for CI6HIgO6,306.1101.

Reference N. Otomo, H. Sato, and S. Sakamura; Novel Phytotoxin Produced by the Causal Fungus of the Shoot Blight of Larches; Agric. Biol. Chem., Vol. 46, pp. 861-863(1982).

25.

Miscellaneous Metabolites

915

Common/Systematic Name 6-(1-Hydroxyethyl)-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone Molecular Formula/Molecular Weight C14H1406; M W

-- 2 7 8 . 0 7 9 0 4

Me_ec~T ,yOMe O

OH

0

OH

General Characteristics Orange needles from chloroform-hexane; mp., 201-204 ~

Optically inactive.

Fungal Source Guignardia laricina GM-7 and Hendersonula toruloidea. Isolation/Purification Filtrated mycelia were extracted with acetone. After removal of acetone, the aqueous concentrate was extracted with ethyl acetate followed by chromatography on silica gel (CHC13, CHC13-EtOAc, 9:1, v/v; CHC13-MeOH, 9:1, v/v). Crystallized from chloroform-hexane. Biological Activity Inhibited the growth of hypocotyls and roots of lettuce seeds 100% at 50ppm, more than 95% at 10ppm and more than 55% at 5ppm. Antimicrobial activity (MIC) against Candida albicans, Staphylococcus aureus and Bacillus subtilis was 20, 2, and 1ppm respectively. Not active at 50ppm against other gram-negative rods: Escherichia coli, Salmonella typhosa, Shigella sonnei and Klebsiella pneumoniae. Spectral Data UV:

~

MeOH max

221(1og e=4.14), 23 l(sh, 4.07), 258(sh, 3.88), 263(3.89), and 310nm (3.72).

IR:

(KBr) 1680, 1630, 1585, 1300, and 1100cm~. ~H NMR: (CDCl3, TMS) 12.85(s, 1H, peri-OH); 7.25(s, 1H, ArH); 6.03(s, IH, OH); 5.34(dq, 1H, J=l 1.72, 6.84Hz); 4.00, 3.91(both s, 3H,-OCH3); 3.74(d, 1H, J=l 1.72Hz, D20 exch.); and 1.56ppm (d, 3H, J=6.84Hz).

916

25.

Miscellaneous Metabolites

Mass Spectrum: 278.078m/e (M+); calcd for C14H1406,278.0789.

Reference N. Otomo, H. Sato, and S. Sakamura; Novel Phytotoxin Produced by the Causal Fungus of the Shoot Blight of Larches; Agric. Biol. Chem., Vol. 46, pp. 861-863(1982).

25.

Miscellaneous Metabolites

917

Common/Systematic Name. 6-Ethyl- 1-acetonyl- 1,5-dihydroxy-2,7-dimethoxy-4-naphthoquinone Molecular Formula/Molecular Weight C17H2oO6;MW = 320.12599

MeO~OMe HO

MeH2C"

T OH

CH2COMe

O

General Characteristics Pale yellow needles from chloroform-hexane; mp., 133 ~

Optically inactive.

Fungal Source

Guignardia laricina GM-7.

Isolation/Purification Filtrated mycelia were extracted with acetone. After removal of acetone, the aqueous concentrate was extracted with ethyl acetate followed by chromatography on silica gel (CHCI3; CHCIa-EtOAc, 9:1, v/v, CHCI3-MeOH, 9:1, v/v). Crystallized from chloroform-hexane. Spectral Data UV: ~,~'m ~H ~

256(10ge=4.45), 265(sh, 4.41), and 336nm (4.25).

IR;

(KBr) 1725(sh), 1710, 1640, and 1590cmq. ~H NMR: (CDCI3, TMS) 13.02(s, 1H, peri-OH); 6.80(s, IH, ArH); 5.61(s, 1H, QH); 4.68(s, 1H, D20 exch.); 3.95, 3.88(both s, 3H, -OCH3); 2.98(s, 2H, acetonylmethylene); 2.70(q, 2H, J=7.4Hz); 2.1 l(s, 3H, acetonylmethyl); and 1.10ppm (t, 3H, d=7.4Hz). Mass Spectrum: 320.1207m/e (M+); calcd for C17H2o06, 320.1258. Reference N. Otomo, H. Sato, and S. Sakamura; Novel Phytotoxin Produced by the Causal Fungus of the Shoot Blight of Larches; Agric. Biol. Chem., Vol. 46, pp. 861-863(1982).

918

25.

Miscellaneous Metabolites

Common/Systematic Name Pyriculariol Molecular Formula/Molecular Weight C14H1604; M W

-" 2 4 8 . 1 0 4 8 6

(or its enantiomer)

HO.

~

CHO

,H

Me

OH

General Characteristics Pale yellow needles from CHCI3-CCI4; mp., 105.5-106.5~ CHCI3).

[0g]D 24 -

3.4 ~ (c=l.0, in

Fungal Sou.rce Pyricularia oryzae. Isolation/Purification Filtered broth extracted with ethyl acetate, followed by chromatography on silica gel (EtOH-CHCI3, 2:98, v/v). The substance then was purified on Sephadex LH20 (acetone) and crystallized from CHCI3-CCI4. Biological Activity At 300ppm the length of root and 2nd leaf sheath of rice seedlings (Kiyonishiki) were 17 and 30% resp. When 5~tg of the substance was applied into a pin hole of rice leaf, a dark necrotic spot developed within 24hr. Spectral Dat.a UV: ~EtOH

251(e=26800), 292(16600), and 366nm (8700); 3,m~a'INN'~ 309(13800), and 406nm (9200). max

246(e=35000),

l-R:

(CHCI3) 3650, 3560, 3400(OH), 1640(chelated Ar-CHO), 1600, 1565(phenyl), and 990cm q (conjugated trans double bond). ~H NMR: (CDCI3) 1.19(3H, d, d=6.5Hz, H-14); 3.94(1H, dq, J=3.5, 6Hz, H-13); 4.22(1H, dd, J=3.5, 6Hz, H-12); 1.8(2H, br., C12- and C13-OH, D20 exch.); 5.94(1H, dd, J=14.5,

25.

Miscellaneous Metabolites

6Hz, H-11); 6.4-6.7(2H, m, H-9 and H-10); 7.08(1H, d, J=14.5Hz, H-8); 6.88, 6.97(each 1H, d, J=8Hz, H-4 and H-6); 7.45(1H, t, J=8Hz, H-5); 10.28(1H, s, H-l); and 11.84ppm (1 H, s, C3-OH). Mass Spectrum: 248.1072(19%, calcd for C~4H~6Oa,248.1048; M~), 204.0822(83, C12H1203),and 203.0732m/e (22, CI2HIlO3). Reference M. Nukina, T. Sassa, M. Ikeda, T. Umezawa, and H. Tasaki; Pyriculariol, a New Phytotoxic Metabolite ofPyricularia oryzae Cavara; Agric. Biol. Chem., Vol. 45, pp. 2161-2162(1981).

919

920

25. Miscellaneous Metabolites

Common/Systematic Name Sydonol 3-Hydroxy-4-(1 -hydroxy- 1,5-dimethylhexyl)benzyl alcohol Molecular Formula/Molecular Weight C]sH2403; MW = 252.17254 HO HO

Me

HOH2C~ I le

General Characteristics Colorless needles from ethyl acetate-hexane; mp., 65-67~ MeOI-I).

[tt]D2~ +7.2* (C=I.0, in

Fungal Source Aspergillus sp. Isolation/Purification Culture filtrate (pH 4.8) was extracted with ethyl acetate and chromatographed on silica gel (ethyl acetate-hexane, 1:4, v/v) followed by purification on Sephadex LH2o (acetone) and crystallization from ethyl acetate-hexane. Biological Activity Inhibited colonial growth and successively induced to form irregularly swollen hyphae of a phytopathogenic fungus Cochliobolus lunata (IFO 6299) at 501.tg/disc. Spectral Data UV: .=M'~ 221(e=6700), and 280nm (2100); ~,~M'~176 243, and 295nm. IR:

(CHCI3) 3320, 1630, 1575, and 1510cm"l. 1H Nlk,IR: 0.80(6H, d, J=6Hz, isopropyl); 4.46(2H, s, hydroxymethyl attached to benzene ring); 1.55(3H, s, tertiary methyl); 6.94(1H, d, d=8Hz), 6.68(1H, dd, ,/=-8, 2Hz), 6.65(1H, d, d=2Hz) (ortho- and meta-coupled three benzenoid protons); 9.49(1H, br, s), and 1.9ppm (2H, br.) (three hydroxyl protons, one of which is phenolic).

25.

Miscellaneous Metabolites

921

13C NMR:

(acetone-d6) 157.1(s), 143.1(s), 129.8(s), 126.9(d), 117.7(d), 115.8(d), 78.3(s), 64.2(t), 44.0(t), 40.0(t), 29.5(q), 28.5(d), 22.8(t), and 22.4ppm (q). Mass Spectrum: 252.1721(26%, M+; calcd for C15H2403, 252.1725), 234(41, M*- H20), 167(77, M*C6HI3), and 164m/e (100, M* - C5H120). Reference M. Nukina, Y. Sato, M. Ikeda, and T. Sassa; Sydonol, a New Fungal Morphogenic Substance Produced by an Unidentified Aspergillus sp.; Agric. Biol. Chem., Vol. 45, pp. 789-790(1981).

922

25.

Miscellaneous Metabolites

Common/Systematic Name Homotrichione Molecular Formula/Molecular Weight C19HlsOs; MW = 374.10017 OH

0

'~~'~~OH 0

0

0

General Characteristics Orange anthraquinone pigment. Fungal Source Metatrichia vesparium. Biological Activity Antibiotic activity. References L. Kopanski, H. Besl, and W. Steglich; Unpublished results. W. Steglich; Biologically Active Compounds From Higher Fungi; Pure and Applied Chemistry, Vol. 53, pp. 1233-1240(1981).

25. MiscellaneousMetabolites Common/Systematic Name p-Toluquinone ~

Toluquinol

Molecular Formula/Molecular Weight C7I-I602; MW = 122.03678 ~ 0

Me

923

C7HgO2,MW = 124.05243 OH

~.,

.-~

0

7

Me

OH

General Characteristics p-Toluquinone formed crystals from hexane; mp., 66-67~ toluquinol formed crystals from benzene; mp., 123-124~ (sublimes at 112~ Toluquinol yielded p-toluquinone on oxidation with silver carbonate/celite. Fungal Source The hypocrealean fungus, Nectria erubescens, Penicillium lanosum, P. patulum = P griseofulvum, and P. urticae. Isolation/Purification Cultures extracted with ethyl acetate, evaporated to dryness under vacuum, and ptoluquinone and toluquinol purified by silica gel column chromatography. The quinone was eluted with ethyl acetate-Skellysolve B (3:1); while toluquinol eluted with ethyl acetate. The quinone crystallized from hexane and the hydroquinone crystallized from benzene. Spectral Data UV: Z HmY~~176 294nm (e=3100). IR:

(Quinone) 1660(broad) and 1605cm"l. IH NMR: (Quinone) 2.05(3H, d J=l.5Hz); 6.63(1H, m); and 6.73ppm (2H, broad). Reference S. T. Carey and M. S. R. Nair; Metabolites of Pyrenomycetes X: Isolation of pToluquinone and Toluquinol from Nectria erubescens; J. Natural Products (Lloydia), Vol. 42, p. 231 (1974).

924

25.

Miscellaneous Metabolites

Common/Systematic Name Aszonapyrone A Molecular Formula/Molecular Weight C28H4oO5;MW = 456.28757 5'

MeC02MA,~~vJMe

Me

0

General Characteristics Colorless needles from CHCI3-MeOH; mp., 242-244~

[a]D2~ +33* (c=1.06, in CHCI3)

Fungal Source

Aspergillus zonatus (IFO 8817).

Isolation/Purification Dry mycelial mats were extracted with acetone and twice chromatographed on silica gel (benzene-acetone, 19:1, v/v and benzene-ethyl acetate, 9:1, v/v). The substance was crystallized from CHCI3-MeOH. Biological Activity Antimicrobial; showed an MIC of 6.31.tg/ml against Staphylococcus aureus 209P and S. aureus res. Soectral Data UV:

3,max'~ 210(e = 16900) and 290nm (7900). Im:

(KBr) 3400, 3090, 2940, 2850, 1728, 1665, 1640, 1580, 1445, 1410, 1380, 1255, and 893cmq. IH NIV[R"

(pyridine-ds) 1.10(IH,dd, J=2, 12Hz); 1.33(2H,m); 1.51(2H,m); 1.62(IH, dd, J=4,

12Hz); 1.69(1H, m); 1.93(3H, s); 2.05(1H, m); 2.08(3H, s); 2.09(1H, m); 2.39(1H, m); 2.88(1H, d, J=13Hz); 3.12(1H, d, J=12Hz); 3.18(1H, dd, J=12, 13Hz); 4.64(1H, dd, J=4, 12Hz); 5.02(1H, s); 5.57(1H, s); and 6.09ppm (1H, s).

25.

Miscellaneous Metabolites

925

13C NMR:

15.5, 16.5, 16.7, 19.1(t), 19.3(q, C-17), 20.0(C-15), 21.0(q), 23.9(t, C-2), 24.2(C-11), 28.0(q, C-18), 37.8(s, C-4), 38.1(s, C-10), 38.4(C-12), 38.6(t, C-I), 40.6(t, C-8), 55.3(d, C-5), 55.4(C-9), 60.0(C-14), 80.8(d, C-3), 100.8(d, C-5'), 103.2(s, C-3'), 107.0(t, C-16), 149.7(s, C-13), 159.4(s, C-6'), 165.8(s, C-4'), 165.9(s, C-2'), and 170.4ppm (s). Mass Spectrum: EIMS: 456(M+), 396, 317, 257, 192, 139, 135, 121, and 107m/e. TLC Data Detection: under UV light. Reference Y.Kimura, T.Hamasaki, A.lsogai, and H.Nakajima; Structure of Aszonapyrone A, a New Metabolite Produced by Aspergillus zonatus; Agric. Biol. Chem., Vol. 46, pp. 1963-1965 (1982).

926

25.

Miscellaneous Metabolites

Common/Systematic Name Pyrenochaetic acid A; 4-Crotonoyl-3-methoxy-5-methylbenzoic acid Molecular Formula/M.olecular Weight C13H1404; MW = 234.08921

OMe 0 H02C1~

Me

General Characteristics Needles from chloroform-n-hexane; mp., 176-179~ Fungal Source

Pyrenochae ta terrestris.

Isolation/Purification Culture filtrate was extracted with ether. The extract was chromatographed on silica gel (benzene-ethyl acetate). The substance was crystallized from chloroform-n-hexane. Biological Activity Inhibited the root growth of onion seedlings 100% at 250ppm, and inhibited that of rice seedlings 100% at 500ppm. Stimulated the root elongation of lettuce seedlings at concentrations of not more than 100ppm and inhibited that at higher concentrations. Spectral Data UV:

~

MeOH max

240(sh, e=8900) and 303nm (1700).

IR:

(KBr) 2900, 1680, 1657, 1580, 880, and 765cm"]. 1H NMR:

(CDCI3, TMS) 1.88(3H, d, J=5.4Hz); 2.18(3H, s); 3.73(3H, s); 6.13(1H, d, J-16Hz); 6.35(1H, dq, ,]-5.4, 16Hz); 7.45(1H, br, s); 7.57(1H, br, s); and 9.08ppm (1H, br, s). Mass Spectrum: HRMS: 234.0894m/e (M~), calcd for C13H1404, 234.0892. 234(M~, 219(base peak, M%CH3), 205(M+-C2Hs), 193(M~-C3Hs), 175, 160, and 77m/e.

25.

Miscellaneous Metabolites

TLC Data Silica gel; benzene-EtOAc (1:1, v:v); Re: 0.35; detection by spraying with anisaldehyde reagent. Reference H. Sato, K. Konoma, and S. Sakamura; Three New Phytotoxins Produced by Pyrenochaeta terrestris: Pyrenochaetic Acids A, B and C; Agrie. Biol. Chem., Vol. 45, pp. 1675-1679(1981).

927

928

25. Miscellaneous Metabolites

Common/Systematic Name Pyrenochaetic acid B; 4-(3-Hydroxybutyroyl)-3-methoxy-5-methylbenzoic acid Molecular Formula/Molecular Weight C13H1605; MW = 252.09977

OMe 0 H02C1<

OH

Me

General Characteristics Needles from chloroform-n-hexane; mp., 139 ~C. Fungal Source Pyrenochaeta terrestris. Isolation/Purification Culture filtrate was extracted with ether. The extract was chromatographed on silica gel (benzene-ethyl acetate). The substance was crystallized from chloroform-n-hexane. Biological Activity Stimulated the root elongation of lettuce seedlings at concentrations from 100 to 500ppm, the roots lengthening two times relative to the control at 500ppm. Spectral Data IR: (KBr) 3400, 2960, 1695, 1580, 885, and 770cm "~. ~H NMR: (CDCI3, TMS) 1.26(3H, d, J=6Hz); 2.18(3H, s); 2.28(3H, s); 2.88(1H, d, J---4Hz) 2.93(1H, s); 3.88(3H, s); 4.42(1H, m); 7.47(1H, br, s); and 7.56ppm (1H, br, s). Mass Spectrum: HRMS: 252.1030m/e (M+); calcd for C~3H~605, 252.0998. TLC Data Silica gel; benzene-EtOAc (1:1, v:v); Rf: 0.08; detection by spraying with anisaldehyde reagent. Reference H. Sato, K. Konoma, and S. Sakamura; Three New Phytotoxins Produced by Pyrenochaeta terrestris: Pyrenochaetic Acids A, B and C; Agfic. Biol. Chem., Vol. 45, pp. 1675-1679(1981).

25.

Miscellaneous Metabolites

929

Common/Systematic Name Pyrenochaetic acid C; 4-Butyroyl-3-methoxy-5-methylbenzoic acid Molecular Formula/Molecular Weight C13H1604; M3cV= 236.10486

OMe

0

13 2 ~ / 4 ~ 1 2 ~ e H02C 6

Me

General Characteristics Plates from chloroform-n-hexane; mp., 148-152~ Fungal Source Pyrenochaeta terrestris. Isolation/Purification Culture filtrate was extracted with ether. The extract was chromatographed on silica gel (benzene-ethyl acetate). The substance was crystallized from chloroform-n-hexane. Biological Activity Inhibited the root growth of onion seedlings 100% at 500ppm, and inhibited that of rice seedlings 54% at 500ppm. Stimulated the root elongation of lettuce seedlings at concentrations of not more than 100ppm and inhibited that at higher concentrations. Spectral Data UV:

~mU~" 249(C=8500) and 303nm (3100). IR:

(KBr) 2950, 1690, 1570, 880, and 765cm"~. IH NMR:

(CDCI3, TMS) 1.04(3H, t, J=7Hz); 1.81(2H, sextet, J=7Hz); 2.35(3H, s); 2.81(2H, t, J=7Hz); 3.94(3H, s); 7.44(1H, br. s); 7.56(1H, br. s); and 9.70ppm (1H, br. s). ~3CNMR: (CDCI3, TMS) 135.6(s, C-I), 109.4(d, C-2), 156.1(s, C-3), 130.3(s, C-4), 136.5(s, C5), 125.4(d, C-6), 207.2(s, C-7), 46.2(t, C-8), 16.5(t, C-9), 13.3(q, C-10), 55.5(q, C11), 18.5(q, C-12) and 171.7ppm (s, C-13).

930

25.

Miscellaneous Metabolites

Mass Spectrum: HRMS: 236.1047role (h/Y); calcd for C13H1604,236.1047; 236(M-~), 193m/e (base peak, M+-C3HT). TLC Data Silica gel; benzene-EtOAc (1:1, v:v); Re: 0.39; detection by spraying with anisaldehyde reagent. Reference H. Sato, K. Konoma, and S. Sakamura; Three New Phytotoxins Produced by Pyrenochaeta terrestris: Pyrenochaetic Acids A, B and C; Agric. Biol. Chem., Vol. 45, pp. 1675-1679(1981).

25.

Miscellaneous Metabolites

931

Common/Systematic Name Candidusin A 2,2'-Epoxy-3',6'-dimethoxy- 1,1',4', 1"-terphenyl-4,4", 5-triol Molecular Formula/Molecular Weight C20H1606; ~ "-" 352.09469 v

OMe

OH

5

HO

OH MeO

O~ -

3

General Characteristics Colorless rods from benzene-EtOH; mp., 123 ~ Fungal Source.

Aspergillus candidus.

Isolation/Purification Culture filtrate was adjusted to pH 3 and extracted with ethyl acetate. The extract was chromatographed on silica gel (toluene-acetone). The substance was crystallized from benzene-EtOH. Biological Activity Cytotoxic on the sea urchin (Hemicentrotus pulcherrimus) embryos totally blocked the initial cleavage at 1.0 x 10-4 M, when added five minutes after fertilization. At the concentration of 5.0 x 10-5 M, partial fragmentation of the unequal-sized blastomeres was observed in the molura stage. At the concentrations of 5.0 x 10.5 - 1.0 x 10-4 M the substance did not inhibit the furrow formation when it was added before anaphase. The substance is expected to have the same inhibitory activity toward DNA and RNA synthesis as does candidusin B. Antibacterial activity against Bacillus subtilis (15mm-diam. inhibitory zone around 8mm-diam. paper disc containing 1.35 x 10-4 M (501.tg/ml) of the compound). Spectral Data UV;

~

EtOH max

216(e=32100), 280(17300), 296(15900), and 335nm (2310).

Im; (KBr) 3240, 1600, 1469, 1438, 1377, 1330, 1230, 1107, 1059, 981,912, and 8 2 8 c m "1.

IH NMR: (CDCI3, TMS) 3.83(3H, s,);4.04(3H, s),6.76(IH, s);6.92(2H, d, J=SHz), 7.12(IH, s);7.48(2H, d, J=8Hz); and 7.54ppm (IH, s).

932

25.

Miscellaneous Metabolites

Mass Spectrum: HRMS: 352.0947m/e (M§ calcd for C20H1606,352.0947. TLC Data Silica gel; toluene-acetone (7:3, v/v); Rf: 0.38; detection by spraying with FeCIrHCI and vanillin-H2SO4. Reference A. Kobayashi, A. Takemura, K. Koshimizu, H. Nagano, and K. Kawazu; Candidusin A and B: Newp-Terphenyls with Cytotoxic Effects on Sea Urchin Embryos; Agric. Biol. Chem., Vol. 46, pp. 585-589(1982).

25. Miscellaneous Metabolites

933

Common/Systematic Name Candidusin B 2,2'-Epoxy-3',6'-dimethoxy- 1,1 ',4', 1"-t erphenyl-3 ",4,4", 5-tetrol Molecular Formula/Molecular Weight C20H1607, ~

= 368.08960

HO

OMe

/

MeO

OH

~0 ~

General Characteristics Colorless plates from benzene-EtOH; mp., 259-260~ Fungal Source

A spergi llus candidus.

Isolation/Purification Culture filtrate was adjusted to pH 3 and extracted with ethyl acetate. The extract was chromatographed on silica gel (toluene-acetone). The substance was crystallized from benzene-EtOH. Biological Activity Cytotoxic to sea urchin (Hemicentrotus pulcherrimus) embryos; totally blocked the initial cleavage at 5.0 x 10-5 M, when added five minutes after fertilization. At the concentration of 3.0 x 10-5 M, partial fragmentation of the unequal-sized blastomeres was observed in the molura stage. Treatment of the embryos in the gastrula stage with 10ktg/ml (2.7 x 10.5 M) of the substance almost completely suppressed 3H-thymidine uptake into DNA. 3H-uridine uptake into RNA was also inhibited by 86% at 2.76x10 "5 M. At concentrations of 5.0 x 10-5 - 1.0 x 10-4 M the substance did not inhibit the furrow formation when it was added before anaphase. Antibacterial activity against Bacillus subtilis [ 16- mm-diam, inhibitory zone around 8- mm-diam, paper disc containing 1.35 x 10-4 M (50~g/ml) of the compound]. Spectral Data UV: ~t, EtOH max

221(e=37,600), 281(16,700), 299(16,900), and 333nm (21,900).

IR:

(KBr) 3260, 1600, 1469, 1322, 1221, 1120, 1092, 1060, 986, and 811cm "~.

934

25.

Miscellaneous Metabolites

~H NMR: (CDCI3, TMS) 3.81(3H, s,); 4.02(3H, s); 6.75(1H, s); 6.89(1H, d, d, J=-8, 0.5Hz); 7.03(1H, d, d, J=8, 2Hz); 7.13(1H, s); 7.17(1H, d, d, J=2, 0.SHz); and 7.56ppm (1H, s). Mass Spectrum: HR S: 368.0848m/e (M+); calcd for C20H1607,368.0886. TLC Data Silica gel (toluene-acetone, 7:3); Rt: 0.28; detection by spraying with FeCI3-HCI and vanillin-H2SO4. Reference A. Kobayashi, A. Takemura, K. Koshimizu, H. Nagano, and K. Kawazu; Candidusin A and B: New p-Terphenyls with Cytotoxic Effects on Sea Urchin Embryos; Agric. Biol. Chem., Vol. 46, pp. 585-589(1982).

25.

Miscellaneous Metabolites

935

Common/Systematic Name Ankalactone Molecular Formula/Molecular Weight C18H2606; M W -" 338.17294 9

1

0

8

-

15

6 .__

"",%

0 General Characteristics Colorless crystals from chloroform; mp., 133.0-134.0~ Fungal Source

Monascus anka.

Isolation/Purification Culture filtrate was extracted with ethanol. The extract was repeatedly chromatographed on silica gel (chloroform-ethyl acetate, 4:1, v/v). The substance was crystallized from chloroform. Biological Activity Showed a gross inhibitory effect against Escherichia coli and Bacillus subtilis at a concentration of 5mg/disk (inhibition zone diameters 10.0 and 10.2mm; paper disk, diameter 8.0mm). Spectral Data UV~

~, ~m ~H

270.8nm (e=l 7,000).

IR:

(KBr) 3590(sh, hydroxyl) and 174 lcm 1 (ct,13-unsaturated y-lactone). IH N]VIR:

(CDCl3) 5.79(d, d=l.8Hz, H-l); 5.59(d, J=l.8Hz, H-3); 6.50(s, H-7); 2.17(m, H-9); 1.42(m, H-10); 0.87(t, J=7.0Hz, H-15); 1.08(s, n-16); 3.87(s, n-18); 2.87(1H, br, s, exch. with D20); and 4.17ppm (1H, s, exch. with D20). 13C NMR:

(CDCI3) 111.55(d,C-I), 173.63(s,C-2),83.43(d,C-3), 79.70(s,C-4), 83.00(s,C-5),

936

25.

Miscellaneous Metabolites

152.77(s, C-6), 118.53(d, C-7), 161.31(s, C-8), 31.66(t, C-9), 27.67(t, C-10), 29.00(t, C-11), 29.22(t, C-12), 31.69(t, C-13), 22.57(t, C-14), 14.03(q, C-15), 15.59(q, C-16), 172.78(s, C17) and 54.08ppm (q, C-18). Mass Spectrum: HR-EIMS: 338.1749m/e (C1aH2606,/2.0mmu); CI-MS: 339m/e ((M+lf). X-ray: Triclinic crystals with a=17.038 (2), b=8.038 (2), c=6.997 (2) A, a=107.72 (3), 13=99.64 (2), y=82.07 (2) ~ Centrosymmetric space group P 1; one molecule of the substance per asymmetric unit (pc=1.26g cm'3), lntramolecular contact: bond between O(4)-H and 0(5) [1.94 (3) A]; the molecules in the crystal were linked by an intermolecular hydrogen bond O(3)-H---O(1) [ 1.90 (3) A]. Reference H. Nozaki, S. Date, H. Kondo, H. Kiyohara, D. Takaoka, T. Tada, and M. Nakayama; Ankalaetone, a New a,[3-Unsaturated y-Lactone from Monascus anka; Agile. Biol. Chem., Vol. 55, pp. 899-900(1991).

25. Miscellaneous Metabolites

937

Common/Systematic Name Alterporriol D Molecular Formula/Molecular Weight C32H30016; ]VIW = 670.15339

7~ I Me O ~

OH O I ^11

OH .!

I1~,.11 M , O 0 0

5' MeO

OH .

OH

4~,. /IVie x]~

,

OH

.....,OH I H

xl~,,,,O H

la' ~ 1 1 '""OH I

O

OH

General Characteristics Orange amorphous solid; mp., 285~ (dec.);

[a]D 20 - 8 0 0 ~

(c=0.01, in EtOH).

Fungal Source

Alternaria porri (IFO, strain No. 9762).

Isolation/Purification Culture liquid was extracted with ethyl acetate. The aqueous liquid was concentrated to 1/10th in volume, activated carbon (ca 1%) was added. The substances adsorbed to the carbon were eluted with hot acetone followed by preparative TLC on silica gel (chloroform-acetone-formic acid, 200:100:1, v/v/v). Final purification of the substance was achieved by reversed phase chromatography on ODS-C~s adsorbent (water-methanol, 9:1, v/v) and HPLC (water-methanol, 4:1, v/v). Spectral.Data .UV:

Z~f~

225(e=1.456106), 270(4.176105), and 440nm (3.556102).

IR:

(KBr) 3600-3000(OH), 1660(shoulder, free C=O), 1640(chelated C=O), 1600(phenyl), 1210(At-OH), 1160(tert-OH), and 1080cm-1 (sec-OH).

938

25.

Miscellaneous Metabolites

1H NMR: (THF..ds, TMS) 4.74(d, J=6.5Hz, H-l); 4.32(d, J=6.5Hz, H-2); 4.26(H-4); 6.77(H-7); 1.33(C3-Me); 3.71(C6-OMe); 4.74(d, J=6.5Hz, H-I'); 4.32(d, J=6.5Hz, H-2'), 4.26(H4'); 6.77(H-7'); 1.33(C3'-Me); and 3.71ppm (C6'-OMe). 13C NMR: (THF-ds, TMS) 22.2(Me-3), 56.7(OMe-6), 68.2(CH-4), 68.4(C-1), 73.7(C-2), 104.1(C-7), 72.8(C3, quat. C), 109.3(C-9a), 121.4(C-5), 129.8(C-10a), 142.7(C-4a), 144.5(C-la), 163.5(C-8a), 163.6(C-6), 184.0(C-10), and 188.8ppm (C-9). Mass Data: FDMS: 693[(M+Na)§ l 1], 671[(M+H)§ 46.2], 670(M+, 100), 634[(M+-2H20, 54.4], and 598m/e (M+-4H20, 16.9); found: C, 54.93; H, 4.40; calcd for C32H3oO16"2H20: C, 54.39; H, 4.56%. Reference R. Suemitsu, Y. Sakurai, K. Nakachi, I. Miyoshi, M. Kubota, and K. Ohnishi; Alterporriol D and E, Modified Bianthraquinones from Alternaria porri (Ellis) Ciferri; Agfic. Biol. Chem., Vol. 53, pp. 1301-1304(1989).

25.

Miscellaneous Metabolites

939

Common/Systematic Name Alterporriol E Molecular Formula]Molecular Weight C32H30016; M W = 670.15339

OH

O

OH

0

OH

~OH

9

7

,

I

0

OH

~:L . .

,L, ~

~

y

OH

-.O

....,o~

OH

General Characteristics Orange amorphous solid; mp., 285~ (dec.);

[tg]D 20 - 6 9 5 ~

(c=0.01, in EtOH).

Fungal Source

Alternaria porri (IFO, strain No. 9762).

Isolation/Purification Culture liquid was extracted with ethyl acetate. The aqueous liquid was concentrated to 1/10th in volume, activated carbon (ca 1%) was added. The substances adsorbed to the carbon were eluted with hot acetone followed by preparative TLC on silica gel (chloroform-acetone-formic acid, 200:100:1, v/v/v). Final purification of the substance was achieved by reversed phase chromatography on ODS-C~8 adsorbent (water-methanol, 9:1, v/v) and HPLC (water-methanol, 4:1, v/v). Spectral Data UV:

/~

EtOH max

225(c=8.326105), 275(3.556105), and 440nm (1.026102).

IR:

(KBr) 3600-3000(OH), 1660(shoulder, free C=O), 1640(chelated C=O), 1595(phenyl), 1210(Ar-OH), 1160(tert-OH), and 1080cm"~ (sec-OH).

940

25.

Miscellaneous Metabolites

IH NMR: (TI-IF-ds, TMS) 4.75(d, J=6.4Hz, H=I); 4.35(d, J=6.4Hz, H-2); 4.26(H=4), 6.77(H7); 1.36(C3-Me); 3.67(C6=OMe); 4.75(d, J=6.4Hz, H-I'); 4.35(d, J=6.4Hz, H-2'), 4.26(H-4'); 6.77(H-7'); 1.36(C3'-Me); and 3.67ppm (C6'-OMe). 13CNMR.: (THF-ds, TMS) 22.2(Me-3), 56.8(OMe=6), 68.3(CH-4), 68.3(C=1), 73.8(C-2), 103.9(C=7), 72.9(C3, quat. C), 109.3(C-9a), 122.5(C-5), 128.9(C-10a), 142.8(C-4a), 143.5(C=1a), 163.9(C=8a), 164.7(C=6), 183.8(C=10), and 188.8ppm (C-9).

Mass Data: FDMS: 634[(M+-2H20, 7.5], 598(M+-4H20, 100), and 582m/e (598+2H-H20, 79.2); found: C, 52.26; H, 5.32; calcd for C32H30OI6.4H20: C, 51.75; H, 5.16 %. Reference R. Suemitsu, Y. Sakurai, K. Nakachi, I. Miyoshi, M. Kubota, and K. Ohnishi; Alterporriol D and E, Modified Bianthraquinones from Alternaria porri (Ellis) Ciferri; Agile. Biol. Chem., Vol. 53, pp. 1301-1304(1989).

25.

Miscellaneous Metabolites

941

Common/Systematic Name Paecilospirone Molecular Formula/Molecular Weight ClaHI4Os; MW = 250.08412 HO

0

, ....

7

1'

~ 3'

General Characteristics Colorless needles from n-hexane-ethyl acetate; mp., 128~ MeOH).

[a]Dz3 -322.7 ~ (C=0.022, in

Fungal Source Paecilomyces sp. Isolation/Purification Culture filtrate was extracted with ethyl acetate. The neutral fraction was chromatographed on silica gel (benzene-acetone, 95:5, v/v). The substance was crystallized from n-hexane-ethyl acetate. Biological Activity MIC of the substance against Bacillus subtilis was 51ag/ml at 25~ At 37~ it did not show any antimicrobial activity, probably due to the rapid destruction of the substance (labile at 37 ~C). Spectral Data

UV: ~,mU~" 211(e=9800) and 237nm (sh,7360). IR;

(KBr) 3430, 3380 (sh), 1800 (sh), 1770, 1695, 1675, 1150, 1110, 1095, and 870cm"~. IH NMR: (CD3EOCD3) 5.37(1H, ddd, J=6, 2.5, 2.5Hz, H-4); 3.99(1H, m, H-6), 6.73(1H, dd, J=10, 2.5Hz, H-7); 6.13(1H, dd, J=10, 3Hz, H-8); 4.37(1H, d, s, H-10); 5.53(1H, ddq, J=15.5, 7.5, 1Hz, H-I'); 5.66(1H, ddq, J=15.5, 6.5, 1Hz, H-2'); 1.62(3H, dd, J=6.5, 1Hz, H-3'); 4.59(1H, dd, ,/=-2.5, 2.5Hz) and 4.68(1H, dd, ,/=2.5, 2.5Hz, C3-CH2); 5.80(1H, d, ,/-6, C4-OH); and 4.66ppm (IH, d, ,/=-3, CI0-OH).

942

25.

Miscellaneous Metabolites

~3CNMR:

(CD3COCD3) 172.5(s,C-I), 159.6(s,C-3),70.0(d,C-4),62.7(s,C-5), 41.5(d,C-6), 149.9(d,C-7), 127.4(d,C-8), 197.7(s,C-9),73.1(d,C-10), 128.2(d,C-I'),132.5(d, C2'), 18.6(q, C-3') and 87.9ppm (t, CH2-3).

Mass Data: EIMS: 250(M+, 1.9), 232(M+-18, 2.6), 222(M+-28, 2.0), 108(100), and 80m/e (96); found: C, 62.44; H, 5.59%; calcd for C13H1405: C, 62.39; H, 5.64%. Reference A.Hirota, M. Nakagawa, and H. Hirota; Structure ofPaecilospirone, a New Antibiotic from Paecilomyces; Agric. Biol. Chem., Vol. 55, pp. 1187-1188(1991).

25.

Miscellaneous Metabolites

943

Common/Systematic Name O-Acetylbenzeneamidinocarboxylic acid Molecular Formula/Molecular Weight C10Hl0N203; M W = 206.06914

~

?

NH II

8

N--C--CO2H ~Me I 7

0

General Characteristics Colorless needle crystals; mp., 175-175.5 ~ Low solubility in hexane, benzene, chloroform, and ether; readily soluble in pyridine, DMSO and dioxane, pK=.in 50% dioxane: 10.65 and < 2. Fungal Source Gibberella saubinetii (IAM 8049).

Isolation/Purification Culture filtrate and mycelium were separately extracted with ethyl acetate followed by repeated chromatography on silicic acid and silica gel (benzene-ethyl acetate, 6:4, v/v). Biological Activity Selective cytotoxicity in vitro using SV40-C3H-2K and C3H-2K from the first day. Caused formation of a large number of rounded and detached cells on the third day at concentrations of 62.5 and 2000~g/ml in SV40-C3H-2K and C3H-2K respectively. Showed a slight antitumor activity against Ehrlich carcinoma in vivo (a 30% inhibition of tumor growth in ddy mice at dose levels of 30mg/kg three times). Spectral Data UV: E~

207(e=7800), 242(sh, 4000), 295(sh, 2700), and 306nm (2850).

I_R:

(KBr)

3390, 3200, 1720, 1680, 1580, 1510, 1380, 1350, 1240, and 745cm "~.

lI-t NMR: (DMSO-d6) 12.65(br. s.,-COOH); 8.58(dd, J=7.8, 1.2Hz, =CH-); 8.30(br. s.,=C-NH); 7.84(dd, J=7.9, 1.6Hz,=CH-); 7.76(br. s.,-C=NH); 7.56(ddd, J=7.6, 7.6, 1.3, =CH); 7.19(ddd, J-7.6, 7.6, 1.5, =CH); and 2.43ppm (s,-CH3).

944

25.

Miscellaneous Metabolites

13C NMR: 196.3(s, C-9), 170.2(s, C-8), 158.6(s, C-7), 138.0(s, C-l), 132.2(d, C-5), 128.7(d, C3), 123.4(d, C-4), 120.7(s, C-2), 119.8(d, C-6), and 24.1ppm (q, C-10).

Mass Spectrum: CIMS: 207m/e (M++H). Reference M. Munekata, H. Seto, and G. Tamura; Isolation of O-Acetylbenzeneamidinocarboxylic Acid, a New Metabolite of Gibberella saubinetii; Agric. Biol. Chem., Vol. 46, pp. 1711-1713(1982).

25.

Miscellaneous Metabolites

945

Common/Systematic Name Helicascolide A Molecular Formula/Molecular Weight C12H2oO3; M W = 2 1 2 . 1 4 1 2 4

O

HO'....~

a

r.

I 9

General Characteristics White crystalline solid; mp., 97-98~

[tg]D -25.0 ~ (c=1.4, in CHCI3, 31 ~

Fungal Source

Helicascus kanaloanus (ATCC 18591).

Isolation/Purification Filtered culture broth was extracted with EtOAc followed by chromatography on Sephadex LH~0 (eluted with CH2Cl2-hexane, 4:1, v/v). Early fractions were chromatographed on silica gel (gradient of 0-10% EtOAc in hexane) and reversed-phase HPLC (Cls). Spectral Data IR;

(CHCI3) 3636, 3458(br), 3020, 2982, 1717, 1460, 1398, and 1147cml. CD: (MeOH) [0]221.5 = +3680. IH NMR: (CDCI3) 3.53(dd, ,/=3.7, 1.7Hz, H-3); 2.08(d, J=3.7Hz, C3-OH); 2.28(ddq, ,J=l 1.2, 6.8, 1.7Hz, H-4); 4.66(d, J=l 1.2Hz, H-5); 5.54(br. q, J=5.6Hz, H-7); 1.62(br. d, J=5.6Hz, H-8); 1.60(br. s, H-9); 0.89(d, J=6.8Hz, H-10); 1.28(s, H-I 1); and 1.32ppm (s, n-12). 13C N M R :

(CDCIs) 177.2(C-1), 44.1(C-2), 77.3(C-3), 31.2(C-4), 87.8(C-5), 131.5(C-6), 126.5(C7), 13.2(C-8), 10.3(C-9), 13.8(C-10), 26.5(C-11), and 22.6ppm (C-12).

946

25.

Miscellaneous Metabolites

Mass Spectrum: HREIMS: 212.1418m/e (M+); calcd for C12H2003,212.1412; EIMS: 212(M +, 2.5), 197(0.9), 195(1.8), 156(11), 154(6.1), 142(13), 128(19), 125(4.3), 113(5.2), and 96role (100). HPLC Data Retention time 10.1 rain; 5-I.tm C~s column, 250 x 10 ram, MeOH-H20 (70:30, v/v), 2.0 ml/min, 215nm. Reference G. K. Poch and J. B. Gloer; Helicascolides A and B: New Lactones from the Marine Fungus Helicascus kanaloanus; J. Nat. Prod., Vol. 52, No. 2, pp.257-260(1989).

25.

Miscellaneous Metabolites

947

Common/Systematic Name Helicascolide B Molecular Formula/Molecular Weight C12H2oO3; MW -- 212.14124

O lli,'

H

a 9

General Characteristics White crystalline solid; mp., 61-62~

laiD -27.6 ~ (c=0.4, in CHCI3, 31 *C).

Fungal Source

Helicascus kanaloanus (ATCC 18591).

Isolation/Purification Filtered culture broth was extracted with EtOAc followed by chromatography on Sephadex LH20 (eluted with CH2Cl2-hexane, 4:1, v/v). Early fractions were chromatographed on silica gel (gradient of 0-10% EtOAc in hexane) and reversed phase HPLC (Cls). Spectral Data IR:

(CHCI3) 3626, 3450(br), 3018, 2981, 2939, 1724, 1470, 1384, and 1266cm"l. CD" (MeOH) [O]221.5 = +3330. IH N]V[R:

(CDCI3) 3.43(dd, J=10.6, 5.5Hz, H-3); 1.80(d, J=5.5Hz, OH-3); 2.06(m, H-4); 4.12(d, J-11.0Hz, H-5); 5.53(br. q, J=6.8Hz, H-7); 1.64(br. d, J=6.8Hz, H-8); 1.61(hr. s, H-9); 0.93(d, J=6.4Hz, H-10); 1.26(s, H-11); and 1.36ppm (s, H-12). 13C N M R :

(CDC13) 177.0(C-1), 44.5(C-2), 77.4(C-3), 33.8(C-4), 89.0(C-5), 131.4(C-6), 127.0(C-7), 13.3(C-8), 10.4(C-9), 13.5(C-10), 23.8(C-11), and 20.6ppm (C-12). Mass Spectrum: HREIMS: 212.1416m/e (M+); calcd for C12H2003,212.1412; EIMS 212(M+, 1.5), 197(0.5), 194(0.3), 128(8.5), 125(2.0), 113(2.2), 96(48), 85(100), and 70m/e (91).

948

25.

Miscellaneous Metabolites

HPLC Data Retention time 9.1 min; 5-I.tm Cls column, 250 x 10 ram, MeOH-H20 (70:30, v/v), 2.0 ml/min, 215nm. Reference G. K. Poch and J. B. Gloer; Helicascolides A and B New Lactones from the Marine Fungus Helicascus kanaloanus; J. Nat. Prod., Vol. 52, No. 2, pp. 257-260(1989).

25. MiscellaneousMetabolites

949

Common/Systematic Name Arthrosporone Molecular Formula/Molecular Weight C15H2403; MW = 252.17254 Me

M e m o Me" ~

~ OH

__1 OH

General Characteristics Colorless needles from Skellysolve B/EhO; mp., 139-141 ~ CHC13).

[a]D -140.8 ~ (c=0.9, in

Fungal Source Arthroconidial fungus (UAMH 4262), isolated as a contaminant on a culture of Ceratocystis ulmi. Believed to be a haploid basidiomycete; has not been fully identified. Isolation/Purification Dried mycelium and culture filtrate were separately extracted with Et20 followed by EtOAc. Combined EtzO-soluble extracts were dissolved in EtOAc and separated into neutral, strong acidic and weak acidic compounds by standard acid-base extraction. The neutral extract was chromatographed on silica gel (eluted with CH2CI2-MeCN, 3:2, v/v). The substance was crystallized from Skellysolve B/Et20. Biological Activity Weak antifungal activity against several Ceratocystis species. Spectral Data UV:

,~ McOH

max

280nm (e=650).

FT-IR: (CHCI3') 3440, 2951, 2866, 1731, 1381, 1360, 1275, 1189, and 1015cm"l.

IH NMR: (CDCI3, TMS) 2.56(dd,J=8, 121-Iz,H-I); 2.57(q,J=7Hz, H-3); 2.69(dd,J=l, 20Hz, H-5); 2.20(d,J=201-1z,H-5); 2.40(d,J=16I-Iz,H-7); 2.21(d,J=16 I-Iz,H-7); 1.96(d, J=14Hz, H-9); 1.79(dd,./=2.5,14Hz, H-9); 1.70(t,J=12Hz, H-I I); 1.59(ddd,J=2.5, 8, 12Hz, H-I I); 1.16(s,H-12); 1.08(s,H-13); 0.84(s,H-14), and 1.02ppm (d,J=7Hz, H-15). (pyridine-ds,TMS) 2.77(dd,J=9.5, 12Hz, H-I); 2.92(dq,J=I.7I-Iz,H-3);

950

25.

Miscellaneous Metabolites

2.55(d, d=20Hz, H-5); 2.47(dd, d=l, 20Hz, H-5); 2.75(d, d=l 6Hz, H-7); 2.46(d, J=16Hz, H-7); 2.14(d, d=14Hz, H-9); 2.06(dd, J=3, 14Hz, H-9); 1.97(t, J=-12Hz, H-11); 1.47(ddd, 3'=3, 9.5, 12Hz, H-11); 1.24(s, H-12); 0.96(s, H-13); 0.94(s, H-14); 1.00(d, 3"=7Hz, H-15); and 5.60ppm (s, OH). 13CNMR: (CDCI3, TMS) 216.4(s, C-4), 91.0(s, C-6), 87.0(s, C-8), 60.6(d, C-3), 58.9(t, C-5), 56.9(t, C-7), 55.5(d, C-I), 54.6(s, C-2), 49.9(t, C-9), 44.7(t, C-11), 40.2(s, C-10), 29.6(q), 26.9(q), 11.3(q), and 8.3ppm (q).

Mass Spectrum: HREIMS: 252.1723(36); calcd, for C~5H2403, 252.1722; 235(38), 234(28), 219(13), 216(2), 206(12), 192(89), 191(24), 177(22), 163(21), 125(100), 102(25), 95(33), and 83m/e (40); CIMS: (NH3): 270([M+NH4]§ 100) and 252m/e (NC, 40). TLC Data Silica gel 60, F254;Me2CO-C6H6, 3:2 (v/v); Rf: 0.5 relative to the Rf of cholesterol (was assigned the value of 1.00). Detected under UV light (~,254 or ~.350nm); spraying with H2SO4-vanillin reagent or phosphomolybdic acid-H2SO4-ceric sulfate reagent, followed by charring. Reference E. Amouzou, W. A. Ayer, and L. M. Browne; Antifungal Sesquiterpenoids from an Arthroconidial Fungus; J. Nat. Prod., Vol. 52, No. 5, pp. 1042-1054(1989).

25.

Miscellaneous Metabolites

951

Common/Systematic Name Anhydroarthrosporone Molecular Formula/Molecular Weight C15H2202; M W = 234.16198 Me

M e m o Me \~' ~,. ~" "

/

V

OH General Characteristics Crystals from Skellysolve B/Et20; mp., 118-119~

[a]D -140.8 ~ (c=0.9, in CHCIs).

Fungal Source Arthroconidial fungus (UAMH 4262), isolated as a contaminant on a culture of Ceratocystis ulmi. Believed to be a haploid basidiomycete; has not been fully identified. Isolation/Purification Dried mycelium and culture filtrate were separately extracted with Et20 followed by EtOAc. Combined Et20-soluble extracts were dissolved in EtOAc and separated into neutral, strong acidic and weak acidic compounds by standard acid-base extraction. The neutral extract was chromatographed on silica gel (eluted with CH2CI2-MeCN, 3:2, v/v). The substance was crystallized from Skellysolve B/Et20. Biological Activity Weak antifungai activity against several Ceratocystis species. Spectral Data UV: ~,~"

230nm (e=13700).

FT-IR: (CHC13) 3461, 1693, 1632, 1466, 1448, 1372, 1364, and 872cm "~. IH NMR: (CDCI3, TMS) 2.38(dd, J-9, 11Hz, H-I); 2.32(q, J=7Hz, H-3); 5.85(d,J=l.3 Hz, H-5), 2.79(d, J=16Hz, H-7); 2.72(dd, J=l, 16Hz, H-7), 1.87(dd, J=2, 14Hz, H-9); 1.68 (d, J=14Hz, H-9);. 1.72(ddd, ,/=2, 9, 13Hz, H-11); 1.47(dd, J=l 1, 13Hz, H-11); 1.20(s, H-12), 1.13(s, H-13); 0.94(s, H-14); 1.1 l(d, J=7Hz, n-15) and 1.63ppm (s, OH). (pyridine-ds, TMS) 2.63(t, J=10Hz, H-l); 2.41(q, J=7.5Hz, H-3), 5.92(d, J=2Hz, H-5); 2.96(d, J=15Hz, H-7); 2.80(dd, ,/=2, 15Hz, H-7); 2.08(dd, ,]--2, 14I-Iz,

952

25.

Miscellaneous Metabolites

H-9), 1.68(d, J=14Hz, H-9); 1.65(ddd, ,]=2, 9, 13Hz, H-11), 1.45(dd, J=-10, 13Hz, H-11), 1.40(s, H-12); 1.10 (s, n-13); 0.86(s, H-14); 1.14(d, J=7.5Hz, H-15) and 6.21 ppm (s, OH). 13C NMR:

(CDCI3, TMS) 211.6(s, C-4), 177.0(s, C-6), 122.9(d, C-5), 92.7(s, C=8), 63.4(d, C-3), 57.7(d, C=I), 55.9(t, C-7), 53.4(s, C-2), 44.0(t, C-9), 43.3(s, C=10), 41.8(t, C=I 1), 30.2(q), 28.2(q), 21.8(q, C-14), and 9.5ppm (q, C-15). Mass Spectrum: HREIMS: 234.1617(63); calcd for C~5H2202, 234.1616, 219(32), 216(34), 201(23), 173(23), 123(78), 122(100), 111(10), 95(13), and 55aVe (20). CIMS (NHa): 252([M+NH4] +, 2), 235([M+H] +, 100), and 234m/e (20). TLC Data Silica gel 60, F254;Me2CO-Cd-I6, 3:2 (v/v); Rf: 0.74 relative to the 1%of cholesterol (was assigned the value of 1.00). Detected under UV light (~.254 or ~.350nm); spraying with H2SO4-vanillin reagent or phosphomolybdic acid-H2SO4-ceric sulfate reagent, followed by charring. Reference E. Amouzou, W. A. Ayer, and L. M. Browne; Antifungal Sesquiterpenoids from an Arthroconidial Fungus; J. Nat. Prod., Vol. 52, No. 5, pp. 1042-1054(1989).

25. MiscellaneousMetabolites

953

Common/Systematic Name Arthrosporol Molecular Formula/Molecular Weight C15H2603; MW = 254.18819

M

e

~

~

Me

H

Me" \9 /' -\6 / 'OH OH

OH

General Characteristics Crystals from Et20; mp., 163-164~ MeOH).

[et]D -29 ~ (C=2.0, in CHCI3), -62.1 ~ (c=l.0, in

Fungal Source Arthroconidial fungus (UAMH 4262), isolated as a contaminant on a culture of Ceratocystis ulmi. Believed to be ~ haploid basidiomycete; has not been fully identified. Isolation/Purification Dried mycelium and culture filtrate were separately extracted with Et20 followed by EtOAc. Combined EhO-soluble extracts were dissolved in EtOAc and separated into neutral, strong acidic and weak acidic compounds by standard acid-base extraction. The neutral extract was chromatographed on silica gel (eluted with CH2CI2-MeCN, 3:2, v/v). The substance was crystallized from Et20. Biological Activity Weak antifungal activity against several Ceratocystis species. Spectral Data FT-IR: (CHCIa) 3376, 2951, 2933, 2456, 1380, 1372, 1088, and 1012cm"l. 1H NMR: (CDCI3, TMS) 2.40(dd, ,/-8, 12Hz, H-!); 2.01(m, Ho3); 3.94(dt, ,/=5, 9Hz, H-4); 2.01(m, H-5); 2.32(d, J=15Hz, H-7); 2.22(d, J=16Hz, H-7); 1.86(d, J=13I-Iz, H-9); 1.70(dd, ,/=2, 13Hz, H-9); 1.54(t, J=12I-Iz, H-11); 1.47(ddd, ,/--2, 8, 12Hz, H-11); 1.1 l(s, H-12); 1.04(s, H-13); 0.76(s, H-14); 1.02(d, J=7Hz, H-15); 1.77(s, OH) and 1.64ppm (s, OH). ~3CNMR: (CDCIa, TMS) 91.0(s, C-6 or C-8), 90.3(s, C-8 or C-6), 76.8(d, C-4), 60.9(d, C-3 or

954

25.

Miscellaneous Metabolites

C-I), 58.5(t, C-5 or C-7), 58.2(t, C-7 or C-5), 56.3(s, C-2), 52.1(d, C-1 or C-3), 49.0(t, C-11 or C-9), 45.3(t, C-9 or C-11), 40.0(s, C-10), 30.7(q), 28.6(q), 12.9(q), and 12.Sppm (q). Mass Spectrum: HREIMS: 236.1778([M-H20] +, 11, calcd, for C~5H2402, 236.1776), 218(100), 203(31), 190(8), 182(38), 174(33), 150(54), 139(32), 135(33), 127(32), 125(16), 123, 121(15), 110(25), 109(38), 107(34), 95(31), 83(54), 69(38), and 55m/e (65). CIMS (NH3): 272([M+NH4] +, 100), 254(18), and 237role (15). TLC Data Silica gel 60, F254;Me2CO-C6I-I6, 3:2 (v/v); Re: 0.39 relative to the Re of cholesterol (was assigned the value of 1.00). Detected under UV light (),254 or )~350nm); spraying with H2SO4-vanillin reagent or phosphomolybdic acid-H2SO4-ceric sulfate reagent, followed by charring. Reference E. Amouzou, W. A. Ayer, and L. M. Browne; Antifungal Sesquiterpenoids from an Arthroconidial Fungus; J. Nat. Prod., Vol. 52, No. 5, pp. 1042-1054(1989).

25.

Miscellaneous Metabolites

955

Common/Systematic Name Auranticin A Molecular Formula/Molecular Weight C24H2408; M W = 440.14712

C02H Me~J 0

~~0 Me~ CH2OH/

Me

7

z

General Characteristics White, crystalline solid; mp., 264~ (dec). Funual Source Preussia aurantiaca (ATCC 14745).

Isolation/Purification Filtered broth was extracted with EtOAc-Me2CO (9:1, v/v). The extract was evaporated to afford a yellow oil, which was triturated with Me2CO and the residual insoluble white solid was chromatographed on silica gel (gradient of 0-50% MeOH in CHCI3; the substance was eluted at 30% MeOH). Biological Activity Antimicrobial activity (50ktg/disk) against most of the Bacillus subtilis and Staphylococcus aureus strains. Spectral Data UV:'

maxM~~ 284nm (6=23823). IR:

(neat) 3387, 3335(br, COOH), 2962, 1721, 1708 (br), 1640, 1616, and 1565cm"l. 1H N]k,IR: 0)MSO-d6) 6.65(s, H-2); 6.55(s, H-7); 4.58(s, C4-CH2-O); 3.77(s, C8-OMe); 2.10(s, C9-Me); 5.55(qq, J=6.8, 1.4Hz, H-2'); 1.78(d, J=6.8H~ H-3'); 2.05(s br., H-4');

956

25.

Miscellaneous Metabolites

5.60(d, J=l.3Hz, H-2"); and 2.25ppm (s br., H-4"). 13C NMR: (DMSO-d6) 148.24(C-1), 112.77(C-2), 161.01(C-3)*, 118.65(C-4), 161.78(C-4a)*, 142.29(C-5a), 135.83(C-6), 107.67(C-7), 154.19(C-8), 116.30(C-9), 142.75(C-9a), 162.87(C-11), 110.13(C-1 la), 52.27(C-4-CH20), 55.95(C8-OMe), 8.18(C-9-Me), 133.15(C-1'), 125.74(C-2'), 17.60(C-3'), 13.72(C-4'), 154.66(C-1"), 119.48(C-2"), 166.77(C-3"), and 19.94ppm (C-4"). * Assignments may be interchanged. Mass Spectrum: FABMS (glycerol): 441([M+H] +, 36%), 423(49), 405(21), 377(19), 361(14), 349(9.5), 240(25), 217(20), 203(23), 189(48), 150(100), and 131m/e (29). HRFABMS: 441.1527m/e ([M+H]+); calcd for C24I-I24Os+H, 441.1529role. HPLC Data Retention time 13.4 min; 5~m C~s column, 250 x 10 mm, MeOH-H20 (70:30, v/v), 2.0ml/min, 215nm. Reference G. K. Poch and J. B. Gloer; Auranticins A and B: Two New Depsidones from a Mangrove Isolate of the Fungus Preussia aurantiaca; J. Nat. Prod., Vol. 1, pp. 213-217(1991).

25. Miscellaneous Metabolites

957

CQrnmon/Systematic Name Auranticin B Molecular Formula/Molecular Weight C24I-I2208; M W

4-

= 438.13147

C02H

Me~ 0 HO/

y "O--~6 ~ O M e CHO _~ Me

'~ 2'

7

General Characteristics Orange oil. Fungal Source Preussia aurantiaca (ATCC 14745).

Isolation/Purification Filtered broth was extracted with EtOAc-Me2CO (9:1, v/v). The extract was evaporated to afford a yellow oil, which was triturated with Me2CO and the Me2CO soluble portion was chromatographed on silica gel (gradient of 0-25% MeOH in CHCI3). Late fractions were chromatographed on Sephadex LH-20 (eluted with CH2Cl2-he~ane, 4:1, v/v). Biological &ctivity Antimicrobial activity (50~g/disk) against most of the Bacillus subtilis and Staphylococcus aureus strains. Spectral Data UV: b MeOH max IR:

350(e=5,957) and 266rma (18,578).

1130era

(neat) 3264(br, COOH), 2915, 2851, 1734, 1717, 1653, 1616, 1416, 1265, and "].

958

25.

Miscellaneous Metabolites

1HN M R : (DMSO-d6) 6.83(s, H-2); 6.60(s, H-7); 9.85(s, CHO-4); 3.78 s, OMe-8); 2.1 l(s, Me9); 5.47(qq, J=6.8, 0.8Hz, H-2'); 1.78(d, J=6.8Hz, H-3'); 2.05(s br., H-4'); 5.65(d, Jr=1.3Hz, H-2"); and 2.23ppm (s br, H-4").

13CN M R : (DMSO-d6) 153.20(C-I),I14.88(C-2),163.68(C-3)*, III.89(C-4), 165.04(C-4a)*, 141.31(C-5a), 135.85(C-6),I07.88(C-7),154.38(C-8),I16.82(C-9), 142.27(C-9a), 161.53(C-11), 110.83(C-1 la), 191.62(CHO-4), 56.03(OMe-8), 8.82(Me-9), 132.18(C-1'), 126.19(C-2'), 17.90(C-3'), 13.52(C-4'), 154.58(C-1"), 120.38(C-2"), 166.41(C-3"), and 19.39ppm (C-4"). * Assignments may be interchanged. Mass Spectrum: EIMS: 438(M+, 3.7%), 410(0.9), 393(31), 377(3.2), 365(7.2), 323(2.5), 307(7.2), 241(1.0), 217(24), 203(6.2), 191(53), 129(39), and ll5m/e (18). HPLC Data Retention time, 8.4 min (broad peak); 51,tmC18column, 250 x 10mm, MeOH-HzO (70:30, v/v), 2.0ml/min, 215nm. Reference G. K. Poch and J. B. Gloer; Auranticins A and B: Two New Depsidones from a Mangrove Isolate of the Fungus Preussia aurantiaca; J. Nat. Prod., Vol. 54, No. 1, pp. 213-217 (1991).

25. Miscellaneous Metabolites

959

Common/Systematic Name Shiraiachrome A Molecular Formula/Molecular Weight C3oH2601o, MW -- 546.15260

o/H,,,O 4

MeO~

I:

I ['

I~.i OMe Ha 13 "'.~. Me

b,,

MeO

O~H,,.'O General Characteristics Deep red crystals; mp., 247-250~ Fungal Source

Shiraia bambusicola (Chinese bamboo fungus).

Isolation/Purification The mycelium was extracted with Me2CO, the solvent was evaporated and the extract was crystallized from Me2CO to give deep red crystals. The crystalline substance was chromatographed on Sephadex LH20 (eluted with CHCI3-MeOR 1:1, v/v). The pigment fraction was chromatographed on Cls column (eluted with MeOH-H20, 85:15, v/v) to yield the substance. Spectral Data UV: ZmM~" 580(Iog C=4.09), 540(4.07), 465(4.41), 338(3.51), 285(4.48), 265(4.56), and 215nm (4.74). CD: (MeOH) positive Cotton effects at 588, 540, and 350nm; negative Cotton effects at 450, 283, and 255nm. IR: (KBr) 3415, 2939, 1700, 1608, 1529, 1451, 1285, 1215, 1160, 997, 968, 835, 668, 589, and 532cml.

960

25.

Miscellaneous Metabolites

1H NMR: (CHCI3, TMS) 6.56(s, H=5); 6.57(s, H-8); 3.68(d, J=14Hz, H=13a); 2.35(d, J-14Hz, H-13b); 3.74(s, H-15); 15.98(s, 4=OH); 16.10(s, 9-OH); 4.19(s, 2=OMe); 4.08(s, 6-OMe); 4.08(s, 7-OMe); 4.28(s, 11-OMe); 1.79(s, 14-Me); and 1.84ppm (s, 15=Ac). 13CNMR: (CDCI3, TMS) 24.8, 28.5, 42.5, 56.4, 56.4, 61.0, 61.8, 64.1, 78.6, 101.6, 102.0, 106.6, 106.9, 124.3, 124.3, 127.7, 127.7, 131.1, 134.9, 149.0, 151.6, 167.1,167.5, 171.5, 171.5, 179.1, 179.4, 179.5, 179.9, and 206.Sppm. Mass Spectrum: 546(1.9%), 528(46), 513(15), 479(16), 485(23), and 455m/e (13). HRMS: 546.1535m/e; calcd for C3oH2601o,546.1526 HPLC Data Retention time, 6.1 min; Cls column, 150 x 5.7mm, MeOH-H20 (85:15, v/v). Reference H. Wu, X. -F. Lao, Q. -W. Wang, R. -R. Lu, C. Shen, F. Zhang, M. Liu, and L. Jia; The Shiraiachromes: Novel Fungal Perylenequinone Pigments from Shiraia bambusicola; J. Nat. Prod., Vol. 52, No. 5, pp. 948-951(1989).

25.

Miscellaneous Metabolites

961

Common/Systematic Name Shiraiachrome B Molecular Formula/Molecular Weight C3oH26010; M W -- 5 4 6 . 1 5 2 6 0

o/H,,,O

Meo 4

MeO

OMe b,,Ha

13

"'.~. Me

I~ O~H,,..O

General Characteristics Deep red crystals; mp., 247-248~ Fungal Source

Shiraia bambusicola (Chinese bamboo fungus).

Isolation/Purification The mycelium was extracted with Me2CO, the solvent was evaporated and the extract was crystallized from Me2CO to give deep red crystals. The crystalline substance was chromatographed on Sephadex LH20 (eluted with CHCIa-MeOH, 1:1, v/v). The pigment fraction was chromatographed on C~s column (eluted with MeOH-H20, 85:15, v/v) to yield the substance. Spectral Data UV;

~,mM~" 580(1oge=4.12), 540(4.09), 465(4.42), 340(3.75), 283(4.50), 267(4.55), and 215nm (4.74). IR;

(KBr) 3423, 2939, 1823, 1702, 1609, 1528, 1451, 1217, 1161, 1091, 1050, 997, 971, 950, and 835cm~. CD: (MeOH) Opposite to CD of hypocrellin.

�9 962

25.

Miscellaneous

Metabolites

1H NMR: (CDCI3, TMS) 6.55(s, H-5); 6.57(s, H-8); 3.52(d, d=12Hz, H-13a); 2.64(d, J=12Hz, H-13b); 3.47(s, H-15); 15.95(s, 4-OH); 15.99(s, 9-OH), 4.08(s, 2-OMe); 4.07(s, 6-OMe); 4.07(s, 7-OMe); 4.12 (s, 11-OMe); 1.71(s, 14-Me); and 1.90ppm (s, 15-Ac). Mass Spectrum: EIMS: 546(M§ 11%), 528(10), 513(4), 497(4), 485(15), 471(3), 457(7), and 445m/e (8); HRMS: 546.1535m/e; calcd for C30H26010,546.1526. HPLC Data Retention time, 6.8 min; C~8 column, 150 x 5.7mm, MeOH-H20 (85:15, v/v). Reference H. Wu, X. -F. Lao, Q. -W. Wang, R. -R. Lu, C. Shen, F. Zhang, M. Liu, and L. Jia; The Shiraiachromes: Novel Fungal Perylenequinone Pigments from Shiraia bambusicola; J. Nat. Prod., Vol. 52, No. 5, pp. 948-951(1989).

25. Miscellaneous Metabolites

963

Common/Systematic Name Shiraiachrome C Molecular Formula/Molecular Weight C30H2409; MW

"- 5 2 8 . 1 4 2 0 3

o/H.,,O 4 13

MeO~

I

.OMe 13

MeO O~H,,,.O

Me

o2:

General Characteristics Deep red crystals; mp., 278-280~ Fungal Source

Shiraia bambusicola (Chinese bamboo fungus).

Isolation/Purification The mycelium was extracted with Me2CO, the solvent was evaporated and the extract was crystallized from Me2CO to give deep red crystals. The crystalline substance was chromatographed on Sephadex LH20 (eluted with CHCI3-MeOH, 1:1, v/v). The pigment fraction was chromatographed on Cl8 column (eluted with MeOH-H20, 85:15, v/v) to yield the substance. Spectral Data UV: ,~ MeOH max

587(1og 6=4.09), 550(4.24), 461(4.57), and 334nm (4.09).

IR~ (KBr) 3397, 1689, 1609, and 1523cm"~. 1H NM]~: (CDCI3, TMS) 6.43.(s, H-5); 6.44(s, H-8); 4.05(d, d-11.5Hz, H-13a); 3.22(d, d=l 1.5Hz, H-13b); 16.03(s, 4-OH); 16.05(s, 9-OH); 4.09(s, 2-OMe), 4.05(s, 6-OMe); 4.05(s, 7-OMe); 4.15(s, 11-OMe); 1.84(s, 14-Me); and 2.38ppm (s, 15-Ac).

964

25.

Miscellaneous Metabolites

13C NMR: (CDCI3, TMS) 20.55, 29.33, 34.64, 56.41 x 2, 60.97, 61.18, 102.90 x 2, 107.23, 108.42, 121.75, 123.05, 123.81 x 2, 133.99, 124.32 x 2, 144.61,146.56, 149.38, 163.55, 164.87, 167.90, 168.12, 185.46 x 4, and 200.08ppm. Mass Spectrum: EIMS: 528(M § 11%), 513(17), 497(15), 485(19), and 455m/e (7). HR S: 528.1409m/e; calcd for C30H2409,528.1420. I-IPLC Data Retention time, 8 min; C~8 column, 150 x 5.7mm, MeOH-H20 (85"15, v/v). Reference H. Wu, X.-F. Lao, Q. -W. Wang, R. -R. Lu, C. Shen, F. Zhang, M. Liu, and L. Jia, The Shiraiachromes: Novel Fungal Perylenequinone Pigments from Shiraia bambusicola; J. Nat. Prod., Vol. 52, No. 5, pp. 948-951(1989).

25. Miscellaneous Metabolites

965

Common/Systematic Name. Cavoxin 2-Hydroxy-3-(1-oxo-2E,4E-octadienyl)-4-methoxy-6-hydroxymethyl benzoic acid Molecular Formula/Molecular Weight C]7H2006; M W = 3 2 0 . 1 2 5 9 9 16

OMe 0 41 JJ 9 s ~ C = C 17~ I~ HOH2C" " ~ "OH

,o

"C---Ck 15 CH2CH2Me

C02H 7

General Characteristics Pale yellow needles from EtOAc-petroleum ether; mp., 125-128 ~C. Showed positive test with FeCI3. Gave cavoxone with quantitative yield by treatment with 6N HCI at reflux. Fungal Source P h o m a cava

(CBS 535.66).

Isolation/Purification Lyophilized solid residue of culture filtrate was dissolved in H20 (1/9 of the initial filtrate volume) and extracted with CHCI3 followed by chromatography on Sephadex LH20 (eluted with CHCIa-isoPrOH, 9:1, v/v). The substance was crystallized from EtOAc-petroleum ether (bp., 40-70~ Biological Activity Phytotoxic when supplied to tomato cuttings, caused a vascular browning and rapid wilting of leaflets. Spectral Data UV:

~,~

325(1oge=4.19) and 286nm (4.38).

IR:

(CHCI3) 3540, 3000, 1745, 1640, 1620, and 1590cm"~. IH NMR: (CDCI3-CD3OD, 2:1; TMS) 6.47(s, H-5); 6.61(d,`/=15.4Hz, H-9); 7.21(dd,`/=15.4, 9.2Hz, H-10); 6.32(dd, J=9.2, 15.1Hz, H-11); 6.20(dt, J=15.1, 6.6Hz, H-12); 2.17(td, ./=6.6, 14Hz, 2H-13); 1.46(tq, ./=14Hz, 2H-14); 0.91(t, ./=7.0Hz, 3H-15); 3.56(s, 2H- 17); and 3.89ppm (s, OMe).

966

25.

Miscellaneous Metabolites

13C NMR: (CDCI3-CD3OD, 2:1; TMS) 125.3(s, C-I), 145.8(s, C-2), 133.1(s, C-3), 150.1(s, C4), 106.6(d, C-5), 121.0(s, C-6), 174.3(s, C-7), 197.3(s, C-8), 129.6(d, C-9), 146.4(d, C-10), 129.7(d, C-11), 147.3(d, C-12), 35.6(t, C-13), 22.2(t, C-14), 13.8(q, C-15), 56.2(q, C- 16), and 40.1 ppm (t, C- 17).

Mass Spectrum: HRMS: 320.1263(M§ calcd for C17H2006, 320.1260; 277.0718(C14I-I1306); 233.0829(C~3H~304); 224.0317(C~0H806); 206.0246(C~0H605, base peak), and 196.0375m/e (C9HsOs). MS: 320(M+, 23%), 302(4), 278(9), 277(87), 224(21), 206(100), 150(23), and 121m/e (11). TLC Data Kieselgel 60 F254;detected under UV light or by spraying with H2SO4-phosphomolybdic acid reagent followed by heating. Reference A. Evidente, G. Randazzo, N. S. Iacobellis, and A. Bottalico; Structure of Cavoxine, a New Phytotoxin from P h o m a cava and Cavoxone, its Related Chroman-4-one; J. Nat. Prod., Vol. 48, No. 6, pp.916-923(1985)

25.

Miscellaneous Metabolites

967

Common/Systematic Name Cavoxone 2-(1E-Pentenyl)-5-methoxy-7-hydroxymethyl-8-carboxylic acid chroman-4-one Molecular Formula/Molecular Weight C17H2oO6;M~W= 320.12599 14 OMe

HOH2C-

a'~

-I CO2H

0

U

" ~C=C

16

"CH2CH2Me 13

General Characteristics White needles from EtOAc; mp., 210-213 oC. Formed from cavoxin with quantitative yield by treatment with 6N HC1 at reflux. Probably formed from cavoxin and may be considered as an artifact. Fungal Source Phoma cava

(CBS 535.66).

Isolation/Purification Lyophilized solid residue of culture filtrate was dissolved in 1-120(1/9 of the initial filtrate volume) and extracted with CHCI3 followed by chromatography on Sephadex LH20 (eluted with CHCI3-isoPrOH, 9:1, v/v). The substance was crystallized from EtOAc. Biological Activity Inactive to tomato cuttings. Spectral Data UV: ~,Em~

288(Iog e=3.87) and 242nm (3.95).

IR:

(CHCI3) 3540, 2950, 1750, 1710, 1675, and 1610cm1. 1H ~ : (CDCI3-CD3OD, 2:1; TMS) 4.94(ddd, d=10.7, 4.0, 10Hz, H-2); 2.80(dd, d=10.7, 16.7Hz, 2H-3); 2.69(dd, J=q,,.0, 16.7Hz, 2H-3); 6.46(s, H-6); 5.72(dd, ,/=15.4, 10Hz, H,9); 5.88(dt, J = 1 5 . ~ H-10); 2.07(td, J=6.6, 12Hz, 2H-11); 1.44(tq, d=12Hz, 2H-12); 0.92(t, J=7.0Hz, 3H- 13); 3.96(d, J=-16.9Hz, 2H- 15); 3.88(d, J= 16.9Hz, 214-15); and 3.94ppm (s, OMe).

968

25.

Miscellaneous Metabolites

13C NMR:

(CDCI3-CD3OD, 2:1; TIVIS) 78.8(d, C-l), 41.3(t, C-2), 193.9(s, C-3), 134.0(s, C-4), 152.4(s, C-5), 109.7(d, C-6), 114.4(s, C-7), 128.7(s, C-8), 151.3(s, C-9), 127.7(d, C10), 136.3(d, C-11), 34.6(t, C-12), 22.3(t, C-13), 13.7(q, C-14), 56.4(q, C-15), 44.3(t, C-16), and 174.7ppm (s, C-17). Mass Spectrum: HRMS: 320.1237(M§ calcd for C17H2006,320.1260; 224(C~0I-IsO6);206(C~0H605, base peak), and 196m/e (C9HsOs); MS: 320(M+, 31%), 278(22), 277(85), 224(28), 206(100), 196(20), 150(31), and 121m/e (27). TLC Data Kieselgel 60 F254;Detected under UV light or by spraying with H2SO4-phosphomolybdic acid reagent followed by heating. Reference A. Evidente, G. Randazzo, N. S. Iacobellis, and A. Bottalico; Structure of Cavoxin, a New Phytotoxin from Phoma cava and Cavoxone, its Related Chroman-4-one; J. Nat. Prod., Vol. 48, No. 6, pp. 916-923(1985).

25. Miscellaneous Metabolites

969

Common/Systematic Name Cavoxinine Molecular Formula/Molecular Weight C17H2oO5; MW = 304.13107

4

7 1~, HO2CH2C" ~

OMe

I

O I

~,

9

10

"C=C 1

15 "CH2CH2Me

General Characteristics Pale yellow oil resistant to crystallization; showed positive test with FeCI3. Fungal Source P h o m a cava

(CBS 535.66).

Isolation/Purification Lyophilized solid residue of culture filtrate was dissolved in H20 (1/9 of the initial filtrate volume) and extracted with CHCI3 followed by chromatography on Sephadex LH20 (eluted with CHCI3-isoPrOH, 9:1), and repeated preparative TLC on silica gel (CHCI3-isoPrOH, 9:1, v/v, and Cd-I6-Me2CO, 6:4, v/v, as eluents). Spectral Data. UV:

~

EtOH max

315(log e=3.92), and 280nm (4.06).

IR;

(CHCI3) 3520, 3000, 1740, 1710, 1670, 1610, and 1575cmq. 1H NIV[R:

(TMS) 6.42(d, J=2.6Hz, H-I)*; 6.40(d, d=2.6Hz, H-5)*; 6.59(d, d=l 1Hz, H-9); 7.3 l(ddd, d=l 1, 12, 2.9Hz, H-10); 6.27(dd, J=12 Hz, H-11); 6.22(ddt, ,/=15.1, 2.9Hz, H-12); 2.17(td, J=6.3, 14Hz, 2H-13); 1.46(tq, J=14Hz, 2H-14); 0.92(t, J=7.3Hz, 3H-15); 3.84(d, J=16.9Hz, 2H-17); 3.76(d, J=16.9Hz, 2H-17); and 3.80ppm (s, OMe). * Assignments may be reversed. 13C NMR,:

(TMS) 100.6(d,C-I), 163.1(s,C-2), 135.7(s,C-3), 163.7(s,C-4), II 1.2(d,C-5), 117.1(s,C-6), 174.5(s,C-7), 195.3(s,C-8), 127.9(d,C-9), 145.2(d,C-10), 129.1(d,CI I), 147.2(d,C-12), 35.2(t,C-13), 21.8(t,C-14), 13.5(q,C-15), 55.4(q,C-16), and 40.6ppm (t,C 17).

970

25.

Miscellaneous Metabolites

Mass Spectrum: HRMS: 304.1185(M+); calcd, for C~7H2oO5,304.1311; 261.0795(C~4HIaOs); 190.0227(C10H604); 180.0437(C9HSO4); and 164.0507m/e (CgHsO3). MS: 304(M+, 18%), 286(9), 262(9), 261 (100), 243(27), 217(64), 208(5), 190(36), 180(40), 165(40), and 164m/e (50). TLC Data Kieselgel 60 F254;mobile phase: CHCl3-isoPrOH (9:1, v/v) and C6H6-Me2CO (6:4, v/v); detected under UV light or by spraying with H2SO4-phosphomolybdic acid reagent followed by heating. Reference A. Evidente; Cavoxinine, a Minor Metabolite from Phoma cava, and Cavoxinone, its Corresponding Chroman-4-one; J. Nat. Prod., Vol. 50, No. 2, pp. 173-177(1987).

25.

Miscellaneous Metabolites

971

Common/Systematic Name Cavoxinone (Probably formed from the open precursor cavoxinine and may be considered as an artifact.) Molecular Formula/Molecular Weight CITH20Os; ~

= 304.13107

OMe O 3

HOOCH2C

---C

13

\CH2CH2Me

General Characteristics Colorless oil resistant to crystallization. Fungal Source P h o m a cava

(CBS 535.66).

Isolation/Purification Lyophilized solid residue of culture filtrate was dissolved in H20 (1/9 of the initial filtrate volume) and extracted with CHCI3 followed by chromatography on Sephadex LH20 (eluted with CHCI3-isoPrOH, 9:1, v/v), and repeated preparative TLC on silica gel (CHCI3-isoPrOH, 9:1, v/v, and Cd-I6-Me2CO, 6:4, v/v, as eluents). Soectral Data UV:

/~

EtOH max

309(1og c=3.00), 275(3.42), and 280nm (sh).

IR:

(CHCI3) 3000, 1740, 1710, 1670, 1605, and 1575cm"l. IH NMR: (TMS) 4.87(ddd, J=l 1.7, 10, 3.68Hz, H-2); 2.81(dd, J=l 1.7, 16.8Hz, 2H-3); 2.69(dd, J=3.68, 16.8Hz, 2H-3); 6.40(d, J=2.2Hz, H-6)*; 6.47(d, J=2.2Hz, H-8)*; 5.64(dd, J=10, 15.4Hz, H-9); 5.87(dt, J=15.4Hz, H-10); 2.08(td, J=6.6, 12Hz, 2H-11); 1.44(tq, J=12Hz, 2H-12); 0.92(t, J=7.3Hz, 3H-13); 3.93(s, 2H-15); and 3.82ppm (s, OMe). * Assignments may be reversed.

972

25.

Miscellaneous Metabolites

Mass Spectrum: HRMS: 304.1294m/e (M+); calcd for C17H2oO5,304.1311; 190.0292(C1oH604); 180.0414(C9HSO4) ;and 164.0465m/e (CgHgO3). MS: 304(M +, 12%), 286(20), 262(15), 261(100), 243(45), 217(70), 190(28), 180(40), 165(35), and 164m/e (55). TLC Data Kieselgel 60 F254,mobile phase: CHCI3-isoPrOH (9:1, v/v) and Cd-In-Me2CO (6:4, v/v); detected under UV light or by spraying with H2SO4-phosphomolybdic acid reagent followed by heating. Reference A. Evidente; Cavoxinine, a Minor Metabolite from Phoma cava, and Cavoxinone, its Corresponding Chroman-4-one; J. Nat. Prod., Vol. 50, No. 2, pp. 173-177(1987).

25.

Miscellaneous Metabolites

973

Common/Systematic Name Armillyl everninate Molecular Formula/Molecular Weight C24H3206; M W -- 416.21989 1

OH

HOH2C

15

""% 14

o,. o 8'

,111

OH

OMe

General Characteristics Needles from n-hexane-EtOAc; mp., 86-87~

[(I]D25 -66 ~

(c=0.79, in MeOH).

Fungal Source

Armillaria mellea (CPC 111.29).

Isolation/Purification The mycelium was extracted with MeOH, the solvent was evaporated and partitioned between CHCI3-MeOH-H20 (13:7:4, v/v/v). The CHCla layer was evaporated and chromatographed on Sephadex LH20 (MeOH). Silica gel chromatography (CHCI3-MeOH, 99:1, v/v) of the nonpolar fractions yielded a mixture of two compounds (armillyl everninate and arnamiol), which were separated on silica gel (n-hexane-EtOAc-MeOH, 40:15:1, v/v/v). The substance was crystallized from n-hexane-EtOAc. Spectral Data UV:

~

MeOH max

21 l(log e=4.33), 261(4.04), and 298nm (3.78).

IR:

(KBr) 3360 and 1640cm4. IH NMR: (CDCI3) 4.30, 4.38(dd, ,/=13.0Hz, H-I); 4.21(dd, ,/=8.8, 2.9I-Iz, H-3); 5.9(ddd, ,/=6.6,

974

25.

Miscellaneous Metabolites

8.5, 2.9Hz, H-5); 1.96(dd, d=6.6, 11.7Hz, H-6a); 2.62(dd, J=8.5, 11.7Hz, H-6b); 2.32.5(m, H-9,13); 1.18(dd, d=l 1.7, 2.0Hz, H-10a); 1.83(dd, J=l 1.7, 5.9Hz, H-10b); 1.32(dd, ,/=12.5, 9.5Hz, H-12a); 1.45(dd, d=12.5, 7.3Hz, H-12b); 1.1 l(s, CH3-8); 1.07(s, CH3-14), 0.98(s, CH3-15); 6.28(d, d=2.2Hz, H-4'), 6.33(d, J=2.2Hz, H-6'); 2.53(s, CH3-8'), and 3.80ppm (s, OCH3). 13C NMR:

(CDCI3) 58.8(t, C-l), 142.5(s, C-2), 74.4(d, C-3), 133.2(s, C-4), 69.9(d, C-5), 40.7(t, C-6), 39.9(s, C-7), 20.9(q, C-8), 47.2(d, C-9), 46.4(t, C-10), 38.6(s, C-11), 46.0(t, C12), 49.8(d, C-13), 29.3(q, C-14), 26.8(q, C-15), 170.7(s, C-I'), 104.7(s, C-2'), 163.8(s, C3'), 98.6(d, C-4'), 165.4(s, C-5'), 111.0(d, C-6'), 142.9(s, C-7'), 24.2(q, C8'), and 55.1ppm (q, OCH3). Mass Data:

[MH]+ 417m/e; EIMS: 398(2%), 380(1), 234(6), 217(28), 201(6), 182(35), 173(5), and 165m/e (100); found: C, 69.29; H, 7.96; calcd for C24I-I3206;C, 69.17; H, 7.74%.

FABMS:

Reference D. M. X. Donnelly, D. J. Coveney, N. Fukuda, and J. Polonsky; New Sesquiterpene Aryl Esters fromArmillaria mellea; J. Nat. Prod., Vol. 49, No. 1, pp. 111-116(1986).

25. Miscellaneous Metabolites

975

Common/Systematic Name. Arnamiol Molecular Formula/Molecular Weight C24I-I3106C1;MW = 450.18092

HOH2C~ 1~ I

OH

""% 14

5/,~/',,,,,, 0~,

0 OH

8'

OI ~

6' ",~, OMe

General Characteristics Needles from n-hexane-EtOAc; rap., 132-134~

[~]D 25 -91 o

(c=0.73, in MeOH).

Fungal Source

Armillaria mellea (CPC 111.29).

Isolation/Purification The mycelium was extracted with MeOH, the solvent was evaporated and partitioned between CHCI3-MeOH-H20 (13:7:4, v/v/v). The CHCi3 layer was evaporated and chromatographed on Sephadex LH20 (MeOH). Silica gel chromatography (CHCI3-MeOH, 99:1, v/v) of the nonpolar fractions yielded a mixture of two compounds (armillyi everninate and arnamiol), which were separated on silica gel (n-hexane-EtOAc-MeOH, 40:15:1, v/v/v). The substance was crystallized from n-hexane-EtOAc. SpectralData UV:

~

MeOH max

217(1og e=4.36), 259(4.16), and 298nm (3.93).

IR:

(KBr) 3360 and 1640cm4.

976

25.

Miscellaneous Metabolites

~H NMR: (CDCI3) 4.31, 4.38(dd, J=12.1Hz, H-I); 4.22(dd, J=8.2, 2.6Hz, H-3); 6.01(ddd, ,/=7.3, 7.3, 2.6Hz, H-5); 1.98(dd, ,/--7.3, I 1.7Hz, H-6a); 2.64(dd, ,/=7.3, I 1.711z, H-6b); 2.3-2.5(m, H-9,13); 1.18(dd, J=l 1.7, 2. IHz, H-10a); 1.82(dd, d=-I 1.7, 6.1Hz, H-10b); 1.36(dd, ,/=13.0, 10.2Hz, H-12a); 1.43(dd, J=13.0, 7.3Hz, H-12b); 1.10(s, CH3-8); 1.07 (s, CH3-14); 0.98(s, CH3-15); 6.41(s, H-6'); 2.65(s, CH3-8'); and 3.90ppm (S, OCH3). 13C NMR: (CDCI3) 59.0(t, C-I), 142.5(s, C-2), 74.6(d, C-3), 133.8(s, C-4), 70.8(d, C-5), 40.9(t, C-6), 40.1(s, C-7), 21.2(q, C-8), 47.4(d, C-9), 46.6(t, C-10), 39.0(s, C-I 1), 46.2(t, C12), 50.1(d, C-13), 29.5(q, C-14), 27.0 (q, C-15), 170.4(s, C-l'), 106.3(s, C-2'), 159.7(s, C3'), 98.5(d, C-4'), 163.0(s, C-5'), 115.7(s, C-6'), 139.7(s, C-7'), 19.8(q, C-8'), and 56.3ppm (q, OCH3).

Mass Data:

[M-H]" 449m/e; EIMS: 432(2%), 388(1), 234(8), 217(28), 216(68), 201(33), 199(100), 187(10), and 172m/e (25); found: C, 63.91; H, 6.77; CI, 8.19; caled for C24I-I3106C1:C, 63.92; H, 6.93%; CI, 7.86%.

FABMS:

Reference D. M. X. Donnelly, D. J. Coveney, N. Fukuda, and J. Polonsky; New Sesquiterpene Aryl Esters from Armillaria mellea; J. Nat. Prod., Vol. 49, No. 1, pp. 111-116(1986).

25.

Miscellaneous Metabolites

977

Common/Systematic Name

trans-Resorcylide

Molecular Formula/Molecular Weight Cld-IlsOs; MW = 290.11542 OH

HO~

0

,H 0

""

General Characteristics Colorless solid; [a]D +78 ~ (MeOH); photosensitive (in the MeOH solution, it slowly converted into the sole product cis-resorcylide) Fungal Source

Penicillium sp.

Isolation/Purification The culture filtrate was extracted with EtOAc and readily separated by chromatography on silica gel (eluted with benzene-acetone, 10:2, v/v). Biological Activity Inhibited the root elongation of Chinese cabbage, lettuce and rice in the seedling test for a week at 100ppm. Spectral Data UV':

/~

MeOH

260(sh., ~=5100) and 293nm (2800).

IR:

(CHCI3) 3240, 1708, 1645, and 1623cm"1. IH Nlk,iR:

(acetone-tin, TMS) 6.95(dt, J=l 7, 7Hz,-CH=CH-CO-); 6.47, 6.38(ABq, J=2H~ ArH); 5.95(d, J=17Hz,-CH=CH-CO-); 5.5-4.9(m,-CH-OCO-); 4.32, 3.62(ABq, J=13Hz, ArCH2-); 1.32(d, J=6Hz, -CH3); and 9.5-8.5ppm (2H, br., OH). ~SCNMR: (CDCI3, TMS) a,13-unsaturated ketone: 131.0 (Ca) and 150.3ppm (CI3).

978

25.

Miscellaneous Metabolites

Mass Spectrum: 290m/e (M+). TLC Data Kieselgel GF254(benzene-acetone, 10:3, v/v); Re: 0.5. Reference H. Oyama, T. Sassa, and M. Ikeda; Structures of New Plant Growth Inhibitors, trans- and cis-Resorcylide; Agric. Biol. Chem., Vol. 42, pp. 2407-2409(1978).

25.

Miscellaneous Metabolites

979

Common/Systematic Name

cis-Resorcylide

Molecular Formula/Molecular Weight C16H1805; M3~ = 290.11542

HO ......,,i0

General Characteristics Melting point 215-216~ purple).

~ H

[a]D +5.0 ~ (MeOH). Gave positive test with FeCI3 (dark

Fungal Source

Penicillium sp.

Isolation/Purification The culture filtrate was extracted with EtOAc and readily separated by chromatography on silica gel (eluted with benzene-acetone, 10:2, v/v). Biological Activity Less active than trans-resorcylide in the seedling tests. Spectral Data UV:

~,~oo. 268(e=13,000) and 306nm (7,100). IR:

(KBr) 3360, 1676, 1646, and 1608cm~. ~H NMR: (acetone-d6, TMS) 6.55(d, J=12Hz,-CH=CH-CO-); 6.38, 6.28(ABq, J=2Hz, ArH); 5.79(ddd, J=12, 9, 6Hz,-CH=CH-CO-); 5.4-4.8(m,-CH-OCO-); 4.63, 3.67(ABq, J=18Hz, ARCH2-); 1.29(d, J=6Hz, -CH3); 12.0(1H, s, OH); and 9.2ppm (1H, br., OH). 13C NMR: (CDCI3, TMS) a,13-unsaturated ketone: 132.5(Ca), and 138.Sppm (C13).

980

25.

Miscellaneous Metabolites

TLC Data Kieselgel GF254 (benzene-acetone, 10:3, v/v); Rf: 0.65. Reference H. Oyama, T. Sassa, and M. Ikeda; Structures of New Plant Growth Inhibitors, trans- and cis-Resorcylide; Agric. Biol. Chem., Vol. 42, pp. 2407-2409(1978).

25.

Miscellaneous Metabolites

981

Common/Systematic Name Cochliohydroquinone A; Alboleersin Molecular Formula/Molecular Weight C30H46Og; M W -- 5 3 4 . 3 1 9 2 7

Aco, .... F'.

HO\ ~ r

HO 1

.....,H

i'

zO

4 2

H

6

General Characteristics Repeated crystallization from benzene yielded cochliohydroquinone A as plates; mp., 210-212~ (lit., 215~ [tt]D23 +98 ~ (C=0.06). Fungal Source The plant pathogenic fungus Helmmthosporium leersii -. Drechslera oryzae has been found as a parasite on leaf blades of Leersia virginica, and Cochliobolus miyabeanus = Drechslera oryzae, a parasitic mold of rice. Isolation/Purification Extracted with n-hexane and separated by fractional crystallization. Crystallized from hexane and hexane-diethyl ether. Spectral Data UW: ~max

295nm (6=3,300); ~.max M~o.

270(9,140) and 388nm (1,060).

IR:

3400(OH), and 1700cm ~ (C=O); (CHCI3) 3575, 3515, 1740, 1673, 1645, and 1 6 2 0 c m "l. INMR: (CDCI3) 3.75(1H, s), 4.94(1H, dd, J-4.2 and 8.5Hz), 4.98(1H, s), 8.2(3H, s), 8.75(3H, s), 8.82(6H, s), 8.83(3H, d, J=6.5Hz), 8.95(3H, s), 9.09(3H, d, J=6.5Hz), and 9.1 lppm (3H, t, J=7.0Hz),

982

25.

Miscellaneous Metabolites

Mass Data: 534(M+), 516, 501,456, 441(100%), and 387re~e; found: M+-1-120, 516.3036. C30I-I4407, requires 516.3087). The hydroquinone showed a very weak molecular ion at role 534, but a much stronger ion at m/e 516(M+ -H20) suitable for mass measurement; the formula of this ion was C30H4407, and therefore that of the hydroquinone is C3oH46Og. References K. D. Barrow and W. S. Murphy; The Structures of Alboleersin and Luteoleersin; the Identity of Luteoleersin with Cochlioquinone A; J. C. S. Perkin I, pp. 2837-2839(1972). J. Beljak, O. Gribanovski-Sassu, B. M. Ranzi, and A. Scala; Production of Two New Quinones, Cochlioquinone A and B from Cochliobolus miyabeanus (Helminthosporium oryzae Breda de Haan); Ann. Ist. Super. Sanith, Vol. 7, pp. 14-22(1971).

25.

Miscellaneous Metabolites

983

_Common/Systematic Names Cochlioquinone A; Luteoleersin Molecular Formula/Molecular Weight C3oH4408; M W = 532.30362

'"7'. 2 4

1

O 7

H

General Characteristics Luteoleersin (mp., 126-135 ~ was repeatedly crystallized from ethyl acetate-light petroleum (bp., 60-80 ~C), and finally from light petroleum alone, yielding cochlioquinone A as yellow needles; mp., 132-136~ (1it.,130-132~ [a]D23 +158~ (c=0.09). Funga! Source

Helminthosporium leersii = Drechslera leersii is widely distributed in America and has been found as a parasite on the leaf blades ofLeersia virginica (Homolocenchrus virginicus), and Cochliobolus miyabeanus = Drechslera oryzae a parasitic mold of flee.

Spectral Data UV:

~,m~x 390(900), 280(11,000), and 245nm(3300). IR:

1720(C=O) and 3350cm" (OH). 'H NMR: (CDCI3) 3.54(1H, s), 5.05(1H, dd, 3=4.6 and 7.2Hz), 5.15(1H, d, 3=10.5Hz), 6.65-7(3H, m), 8.08(3H, s), 8.68(3H, s), 8.82(6H, s), 8.85(3H, d, 3=6.5Hz), 8.98(3H, s), 9.1 l(3H, d, ,J=6.5Hz), and 9.12ppm (3H, t, 3=6.5Hz). The signal at 3.54(1H, s) was for the single olefinic proton, and a double doublet at 5.05(3=4.6 and 7.2Hz) is assigned to a =CH-CH(OAc)-CH- system. The allylic proton at C-11 appeared as a doublet at 5.15ppm (J= 10.5Hz) and is disposed trans diaxially to the proton at C-9.

984

25.

Miscellaneous Metabolites

Mass Data: 532(M+), 516, 514, 501,499, 473,456, 441(100%), and 386m/e; found: M + 532.2981" calcd for C30H4408"M, 532.3036. TLC Data Silica gel using chloroform-ethyl acetate (7:3, v:v); R~0.47. Reference K. D. Barrow and W. S. Murphy; The Structures of Alboleersin and Luteoleersin; the Identity of Luteoleersin with Cochlioquinone A; J. C. S. Perkin I, pp. 2837-2839(1972).

25.

Miscellaneous Metabolites

985

Common/Systematic Name Cochlioquinone B Molecular Formula/Molecular Weight C28H4006; M W -- 4 7 2 . 2 8 2 4 9

o:T,. O'

H

~

....,H

i~i 6

General Characteristics Crystals from diethyl ether; mp., 168-169~ from diethyl ether;

[a]D 20 + 1 5 3 ~

Fungal Source

Cochfiobolus miyabeanus, a parasitic mold of flee.

Isolation/Purification Extracted with n-hexane and separated by fractional crystallization followed by chromatography using Florisil and benzene-chloroform as eluent. Crystallized as long needles from diethyl ether. (Note: The structure of cochlioqumone B was established and confirmed from chemical, spectroscopic evidence and from X-ray crystallographic studies of a para-iodobenzene-sulphonyl derivative. Spectral Data UV:

~,mM~" 260(e=10,150), 398(892), and 303nm (1,990). IR:

(Nujol) 3520, 1708, 1673, 1650, 1630, and 1610cm ~.

986

25.

Miscellaneous Metabolites

TLC Data Silica gel using chloroform-ethyl acetate (7:3, v/v); Rf=0.56. References K. D. Barrow and W. S. Murphy; The Structures of Alboleersin and Luteoleersin; the Identity of Luteoleersin with Cochlioquinone A, J. C. S. Perkin I, pp. 2837-2839(1972). J. Beljak, O. Gribanovski-Sassu, B. M. Ranzi, and A. Scala; Production of Two New Quinones, Cochlioquinone A and B from Cochliobolus miyabeanus (Helminthosporium oryzae Breda de Haan); Ann. Ist. Super. SanitY, Voi. 7, pp. 14-22(1971). J. g. Carruthers, S. Cerrini, W. Fedeli, C. G. Casinovi, C. Galeffi, A. M. Torracca Vaccaro, and A. Scala; Structures of Cochlioquinones A and B, New Metabolites of Cochliobolus miyabeanus: Chemical and X-ray Crystallographic Determination; Chem. Commun., pp. 164-166(1971).

25.

Miscellaneous Metabolites

987

Common/Systematic Name Stemphone Molecular Formula/Molecular Weight C3on420g; M W "- 5 3 0 . 2 8 7 9 7

0

0

OH

7

3

2//OH

\

32

General Characteristics Crystallized as needles from hexane; mp., 160.5-161.5~ [~]D 29 +146 ~ (c-l, in EtOH). Insoluble in water, 2N HCI and NH4OH; slightly soluble in hexane; soluble in acetone, chloroform, ethanol, and benzene. F .ungal Source

Stemphylium sarcinaeforme is an important pathogen of red clover (Trifolium pratense) and is also reported to cause leaf spot disease in grain. The fungus is commonly used as a test organism for screening fungicides.

Isolation/Purification The cultures were extracted with ethyl acetate. The extract was dried, filtered and evaporated.The residue was dissolved in a small volume of chloroform and added to a column of Merck silica gel (70-325 mesh ASTM). After an initial wash of the column with benzene-chloroform (1:2, then 1:3, v/v), a yellow band was eluted by chloroform. Evaporation of this fraction yielded a yellow oil which crystallized on titration with n-hexane. Biological Activity Stemphone was toxic to 10-day-old chick embryos; 100~g injected into the yolk sac gave 33% mortality (control 5%) within 24 hours. Stemphone also killed zebrafish larvae (average LCs0 at 24 hours was 1.6~g/ml), and inhibited growth of Bacillus megaterium and Sarcina lutea at levels of 10~tg per cup in a cylinder plate assay. Spectral Data UV:

X~" 267(e=9,890) and 390nm (e=l,030), and maximum at 294nm (e= 4,500) after the addition of one drop O.1M sodium borohydride solution.

988

25.

Miscellaneous Metabolites

IR:

(carbon tetrachloride) bands at 3578, 3510, 1735, 1673, 1645, 1620, and 1596cmq that were due to hydroxyl, carbonyl, and olefinic groups. 13C NM~: (CDCI3) 188.8(C-1); 133.0(dd, C-2); 148.7(C-3); 181.5(C-4); 151.6(C-5); 119.2(C-6); 51.0(C-8); 132.1(C-24); 125.3(dq, C-25); and 169.9ppm (CH3CO).

Mass Data: M + 530; found: C, 67.89, 68.06; H, 8.11, 8.05; O, 24.17%; C30I-I4208required: C, 67.90; H, 7.98; O, 24.12%. References C. S. Huber; The Structure of Stemphone, a Yellow Fungal Metabolite; Acta Cryst., B31, pp. 108-113(1975). C. Huber, W. A. Court, J. P. Devlin, and O. E. Edwards; Stemphone: A New Type of Natural Quinone; Tetrahedron Letters, pp. 2545-2548(1974). P. M. Scott and J. W. Lawrence; Stemphone, A Biologically Active Yellow Pigment Produced by Stemphylium sarcinaeforme (Cav.) Wiltshire; Can. J. Microbiol., Vol. 14, pp. 1015-1016(1969).

Secondary Metabolite Index Secondary metabolite 9~-acetoxy-6,8t~dimethylergoline 3-O-Acetoxypaxilline 15-Acetoxyverruculogen Acetylaszonalenin O-Acetylbenzeneamidinocarboxylic acid 2'-Acetyl-2"benzoylcercosporin 2'-Acetylcercosporin 19-O-Acetylchaetoglobosin A. 19-O-Acetylchaetoglobosin B 19-O-Acetylchaetoglobosin D 11-O-Acetylcyathatriol 15-O-Acetylcyathatriol 12-O-Acetyldideacetylfusicoccin Acetylepoformin 12-O-Acetylfusicoccin 12-O-Acetylisofusicoccin 10-O-Acetyljanthitrem E N-Acetylleline N-Acetylnorloline l'-O-Acetylpaxilline Achaetolide Achaetolidone Acremoauxin A Aflatoxicol A (Ro) Aflatoxicol B Aflatoxicol O-ethyl ether A Aflatoxicol O-ethyl ether B Aflatoxin B1

Page

134, 136 455 230 176

943 743 737 263 265 267 781 783 677 643 679 681 489 531 539 457 908 909 91)5 564 565 568 569 547

Secondary metabolite

Page

Secondary metabolite

Page

Aflatoxin B2 Aflatoxin BEa Aflatoxin B3 Aflatoxin D1 Aflatoxin G1 Aflatoxin G2 Aflatoxin G2a Aflatoxin M1 Aflatoxin M2 Aflatoxin P~ Aflatoxin Q~ Aflatrem ~l-Aflatrem Aflavazole Aflavinine Agroclavine 6,7-seco-agroclavine Alanyl-2-(1,1-dimethyl2-propenyl) dehydrotryptophan anhydride

549 566 561 559 551 553 567 555 557 558 563 459 461 374 357 30 32

705

proline) t-Alanyl-e-tryptophan anhydride Alboleersin Alliacide Alliacol A Alliacol B 'Alliacolide Alliacolide II Allocyafrin B4 Allocyathin BE Allocyathin B3 Allofusicoccin Altenuene Altenuic acid I Altenuic acid II Altenuic acid III Altenuisol Altenusin Alternaric acid

169

Altemariol Alternariol monomethyl ether Alterporriol D Alterporriol E Altertenuol Amitenone Amphicercosporin Ampullicin Anhydroarthrosporone Anhydrofusarubin Anhydrofusarubin lactone Ankalactone Antibiotic G7063-2 Antiphenicol Armillyl everninate Amamiol Arthrosporol Arthrosporone Ascochalasin Aspernomine Aspertoxin Aspochalasin A Aspochalasin B Aspochalasin C Aspochalasin D Aszonalenin Aszonapyrone A Aurantiamine Auranticin A Auranticin B Aurechinulin Austamide Aversin Averufin Bipolaramide Botrallin Botryaloic acid Botryaloic acid acetate

178 701 823 825

cyclo-(L-Alanyl-L-

190

166 981 801 803 805 799 803 777 765 767 669 697 712 713 714 707 709 899

703 937 939 707 663 733 901 951 844 881 935 637 631 973 975 953 949 351 511 605 334 335 337 339 174 924 149 955 957 220 180 588 577

989

990

Secondary Metabolite Index

Secondary metabolite

Page

Secondary metabolite

Page

Secondary metabolite

Botrydial Botrydienal Botryoloic acid Bovinone Boviquinone-3 Brevianamide C Brevianamide D Brevianamide F

815 831 827 655 653 196 198 199 931 933 965 969 971 967 731 247 249 251 253 255 257 259 261 269 271 273 43 43 47

207 221 219 771 775 773 779 753 755 757 759 761 763 62 186 275 277 279 281

Deacetylepoxycytochalasin H

Candidusin A Candidusin B Cavoxin Cavoxinine Cavoxinone Cavoxone Cercosporin Chaetoglobosin A Chaetoglobosin B Chaetoglobosin C Chaetoglobosin D Chaetoglobosin E Chaetoglobosin F Chaetoglobosin G Chaetoglobosin J Chaetoglobosin K Chaetoglobosin L Chaetoglobosin M Chanoclavine-I Chanoclavine Chanoclavine-II 8-Chlororugulovasine A 8-Chlororugulovasine B Chokol A Chokol B Chokol C Chokol D Chokol E Chokol F Chokol G Cochliohydroquinone A Cochlioquinone A Cochlioquinone B Coriloxin Costaclavine Cryptoechinulin A Cryptoechinulin B

Cryptoechinulin C Cryptoechinulin D Cryptoechinulin G Cyafrin A4 Cyafrin A5 Cyafrin B4 Cyathatriol Cyathin A3 Cyathin A4 Cyathin B2

128

Cyathin B3 Cyathin C3 Cyathin C5 Cycloclavine Cycloechinulin Cytochalasin A Cytochalasin B Cytochalasin C Cytochalasin D Cytochalasin D monoacetate Cytochalasin E Cytochalasin F Cytochalasin G Cytochalasin H Cytochalasin J Cytochalasin KASP Cytochalasin KCHA Cytochalasin L Cytochalasin M Cytochalasin NHyp Cytochalasin NpHO Cytochalasin Ony P

132 615 617 619 621 623 625 627 981 983 985 649 54 208 220

Cytochalasin OpHo Cytochalasin PHYP Cytochalasin PPHO

Cytochalasin QHYP Cytochalasin QPHO Cytochalasin RHyp Cytochalasin RpHo Cytochalasin S Cytochalasin U 19-Deacetylallofusicoccin Deacetylcytochalasin H Deacetyldihydrobotrydial

291 341 284 332 293 297 345 353 285 287 318 306 320 308 322 310 324 312 330 314 316 295

685 297 834

19-Deacetylfusicoccin

19-Deacetylisofusicoccin Deacetyl-Omethyldihydrobotrydialone Dehydroaltenusin Dehydroaltenusin Dehydrobotrydienal

1,2-Dehydrocyathin B3 10,20-Dehydro[ 12,13dehydroprolyl]-2(l',l-dimethylallyltryptoallyltryptophyl)diketopiperazine 24,25-Dehydro-10,11dihydro-20hydroxyaflavinine Dehydroherbarin Dehydrophomin 12,13-Dehydroprolyl-2(l',l'-dimethylallyltryptophyl)diketopiperazine N-Demethylchanoclavine II 16-O-Demethyl-19deoxydideacetyl-3epifusicoccin Deoxaphomin Deoxybrevianamide E 19-Deoxydideacetylfusicoccin 19-Deoxy-30thydroxydideacetylfusicoccin Deoxynortryptoquivaline Deoxynotryptoquivalone Deoxytryptoquivaline 11-Desacetoxywortmannin Desmethoxyviridin Desmethoxyviridiol Desoxyepiepoxydon

Page

301 689 687

819 699 711 833 761

192

364 841 275

188 48

683 347 194 691

6e 387 405 385 727 719 723 645

Secondary Metabolite Index

991

Secondary metabolite

Page

Secondary l~etabolite

Desoxyepoxydon 2',2"-Diacetylcercosporin 11,15-O, O-Diacetylcyathatriol 2',2"-Dibenzoylcercosporin Diboviquinone-3,4 Diboviquinone-4,4 Dideacetylfusicoccin Dihydroagroclavine 12,13-Dihydroaustamide Dihydrobotrydial 10,23-Dihydro-24,25dehydroafiavinine Dihydrodemethylsterigmatocystin 10,23-Dihydro-24,25dehydro-21oxoaflavinine 10,11-Dihydro-ll,12dehydro-20hydroxyaflavinine 2,3-Dihydro-5,8dihydroxy-6methoxy-2hydroxymethyl-3(2-hydroxypropyl)1,4-naphthalenedione 9,10-Dihydroexserohilone Dihydrofusarubin Dihydrofusarubin (s form) 12,13-Dihydro-12hydroxyaustamide 2,3-Dihydro-5-hydroxy4-hydroxymethyl-8methoxynaphtho [1,2-b]furan-6,9dione Dihydro-O-methylsterigmatocystin Dihydrosterigmatocystin 20,25-Dihydroxyaflavinine

639

5,10-Dihydroxy-l,7dimethoxy-3methyl-lHnaphtho[2,3-c] pyran-6,9-dione 2,5-Dihydroxy-3farnesyl-l,4benzoquinone 2-(3,4-Dihydroxyhepta1,5-dienyl)-6hydroxybenzylalcohol 8-(10,11Dihydroxyhexa-12enyl)-3-hydroxydihydroisobenzylfuran

739 785 741 659 661 673 34 182 821 370 608 372 366

887 241 849 851

184

5,8-Dihydroxy-2methoxy-6hydroxymethyl-7(2-hydroxypropyl)1,4-naphthalenedione 5,8-Dihydroxy-6methoxy-3-methyl2-aza-9,10anthracenedione 5,10-Dihydroxy-7methoxy-3-methyl1H-naphtho[2,3-c] pyran-l,6,9-trione 3-(3,3-Diindolyl) propane-l,2-diol Dimethoxysterigmatocystin (~,(~-Dimethylallylpaspalinine 2,4-Dimethylindole Dimethylnidurufin

rel-(3R,4aR,lOaR)-

889 603 599 359

5,10-Dioxo3,4,4a,5,10,10ahexahydro-3,7dimethoxy-3methyl-6,9dihydroxy-lHnaphtho[2,3-c] pyran

Page

Secondary metabolite

Page

rel-(3R,4aR)-5,10Dioxo-3,4,4a,5,10, 10a-hexahydro-7methoxy-3-methyl3,6,9-trihydroxy1H-naphtho[2,3-c] pyran

875

5,10-Dioxo-3,4,4a, 5,10,10ahexahydro-7methoxy-3-methyl3,6,9-trihydroxy1H-naphtho[2,3-c] pyran

849

5,10-Dioxo-3,4,4a, 5,10,10ahexahydro-7methoxy-3-methyl3,6,9-trihydroxy1H-naphtho[2,3-c] pyran

rel-(3R,4aR,lOaS)-

877

891

873

5,10-Dioxo-3,4,4a, 5,10,10ahexahydro-7methoxy-3-methyl3,6,9-trihydroxy1H-naphtho[2,3-c] pyran Ditryptophenaline

851 237

885 653

907

rel-(3R,4aR,lOaR)-

rel-(3R,4aR,lOaS)910, 911

881 4 609 459 495 587

879

E6 E7 E8 El0 Echinulin Elsinochrome D Elymoclavine Elymoclavine-O-13fructofuranoside Elymoclavine-O-13fructofuranosyl(2---)I)-O-~-Dfructofuranoside Enaminomycin A Enaminomycin B Enaminomycin C

220 218 208 207 201 747 37 39

41 631 633 635

992

Secondary metabolite

Secondary Metabolite Index

Page

Engleromycin 303 Epiamauromine 233 Epiepoxydon 647 Epoformin 639 Epoxycytochalasin H 299 Epoxycytochalasin J 301 19,20-Epoxycytochalasin Q 326 Epoxydeacetylcytochalasin H 301 19,20-Epoxydeacetylcytochalasin Q 328 Epoxydon 641 Epoxydon monoacetate 644 Epoxyexserohilone 243 Ergine 10 Erginine 14 13,13-Ergoannam 120 Ergobalansine 101 Ergobalansinine 104 Ergobasine 24 Ergobasinine 27 Ergobutine 122 Ergobutyrine 124 Ergocornine 86 Ergocorninine 88 Ergocristine 92 Ergocristinine 95 13-Ergocryptam 117 0~-Ergocryptine 107 I]-Ergocryptine 112 [~-Ergocryptine-5'epimer 115 Ergometrine 24 Ergometrinine 27 Ergonine 79 Ergonovine 24 Ergonovinine 27 Ergoptine 83 Ergosecaline 97 Ergosecalinine 99 Ergosine 71 Ergosinine 73 Ergostine 75 Ergostinine 77 Ergotamine 64 Ergotaminine 67 Ergovaline 81

Secondary metabolite 6-(1-Ethoxyethyl)-5hydroxy-2,7dimethoxy-l,4naphthoquinone 6-Ethyl-l-acetonyl-l,5dihydroxy-2,7dimethoxy-4naphthoquinone O-Ethyldihydrofusarubins O-Ethylfusarubin Exserohilone F-V/1 F-V/2 F-V/3 F-V/4 F/Ill F/IV F-VI F/VII F-VIII Festucine Festuclavine N-Formylloline N-Formylnorloline FTC FTD FTE FTG FFJ Fumigaclavine A Fumigaclavine B Fumigaclavine C Fumitremorgin A Fumitremorgin B Fumitremorgin C (SM-Q) Fusarubin Fusarubin methyl ether Fusicoccin Fusicoccin A Fusicoccin J Helicascolide A Helicascolide B Herbarin L-Homoleucyl-Dproline-lactam

Page

913

917 854 848 239 683 685 694 671 675 671 687 673 689 543 35 529 537 379 383 389 393 399 134 138 142 222 224 226 857 859 667 667 691 945 947 839 159

Secondary metabolite

Page

Homotrichione 20-Hydroxyaflavinine

922 362 807 809

11-Hydroxyalliacolide 12-Hydroxyalliacolide

12-Hydroxydehydroalliacolide 14-epi-14-Hydroxy- 10, 23-dihydro-24,25dehydroaflavinine Hydroxydihydrofusarubin 9-Hydroxy-6,8dimethylergoline 9-Hydroxy-6,8dimethylergoline 8-Hydroxyergine 8-Hydroxyerginine 8-Hydroxyergotamine 6-(1-Hydroxyethyl)-5hydroxy-2,7dimethoxy-l,4naphthoquinone 3-Hydroxy-4-(1hydroxy-l,5dimethylhexyl) benzyl alcohol 4-Hydroxymethyl-2methylindole Hydroxyversicolorone Hypocrellin 4-( 3- Indolyl) butane1,2,3-triol 3-(3-Indolyl)propane1,2,3-triol Isoampullicin Isochanoclavine-I Isoechinulin A Isoechinulin B Isoechinulin C Isoepoxydon Isofumigaclavine A Isofumigaclavine B lsofusicoccin Isoleucylisoleucyl anhydride

cyclo-( L-Isoleucyl-Lvaline)

805

368 853 138 140 12 16 69

915

920 497 592 735

5 3 903 45 214 216 217 647 136 140 675 162 168

Secondary Metabolite Index

Secondary metabolite

Page

Isolysergic acid amide Isomarticin Isosetoclavine

14 871 52

Janthitrem Janthitrem Janthitrem Janthitrem Javanicin

485 487 489 491 861

B E F G

Kodocytochalasin- 1 Kodocytochalasin-2

293 297

Lanosulin LL-S490~ Loline Lolitrem B Lolitrem C Lolitrem E Lolitriol Luteoleersin Lysergene Lysergic acid Lysergic acid amide Lysergine Lysergol

224 176 527 433 436 436 438 983 20 8 10 22 18

Marcfortine A Marcfortine B Marcfortine C Marticin 4-Methoxymethyl-2methylindole 5-Methoxysterigmatocystin O-Methylaversin 8-O-Methylbostrycoidin 3-Methyl-2-butenylpaspalinine O-Methyldihydrobotrydial N-Methyl-4-dimethylallyltryptophan Methylenediboviquinone-3,3 Methylenediboviquinone-4,4

501 503 505 869

993

Secondary metabolite N-Methylepiamauromine O-12'-Methylergocornine O-12'-Methyl-txergocryptine 8-O-Methyl ether nectriafurone 8-O-Methylfusarubin O-Methylherbarin 3-Methyl-3-hydroxy-1butenylpaspalinine 8-O-Methyljavanicin N-Methylloline 8-O-Methylsolaniol O-Methylsterigmatocystin Molliclavine Monodeacetylfusicoccin

Page

Secondary metabolite

Page

235

10-Oxo-11,33-dihydropenitrem B Oxyjavanicin

419 857

90 110 896 846 843 465 865 533 867 601 58 671

Nectriafurone Neoallocyathin An Neocerosporin Neoechinulin Neoechinulin A Neoechinulin B Neoechinulin C Neoechinulin D Neoechinulin E Neosporin Neoxaline Nidurufin Nominine 12-Noralliacolide Norbotryal acetate Norchanoclavine II Norisotryptoquivaline Norjavanicin Norloline Norsolorinic acid Nortryptoquivaline

817

27-epi-Nortrypto-

894 769 745 204 205 207 208 210 212 745 170 586 509 811 829 48 383 863 535 579 383

6

quivaline Nortryptoquivalone

408 381

Okaramine A Okaramine B Oxaline

153 155 172

499 607 589 855 463

657 663

Paecilospirone Paraherquamide Parasiticol Paspalicine Paspaline Paspalinine Paspalin-P Paspalin-P2 Paspalitrem A Paspalitrem B Paspalitrem C Paxilline cz-Paxitriol ~-Paxitriol PC-M4 PC-M5 PC-M5" PC-M6 Penitrem A Penitrem B Penitrem C Penitrem D Penitrem E Penitrem F Penniclavine Pennigritrem Peramine

cyclo-L-PhenylalanylL-alanine Phleichrome Phomamide Phomin Preechinulin Prolyldiketopiperazine B

cyclo-L-Prolylglycine L-Propyl-L-tyrosine Protocercosporin Protophomin Proxiphomin Pyrenochaetic acid A Pyrenochaetic acid B Pyrenochaetic acid C Pyrichalasin H

941 507 561 445 443 447 293 297 463 465 467 449 451 453 431, 475 477 479 481 413 415 421 423 425 427 60 429 541 157 749 163 277 203 159 158 160 734 350 349 926 928 929 304

994

Secondary Metabolite Index

Secondary metabolite

Page

Secondary metabolite

Page

Secondary metabolite

Page

Pyriculariol Pyroclavine

918 56

Radarin A Radarin B Radarin C Radarin D c/s-Resorcylide trans- R esorcyli de Roquefortine Roquefortine A Roquefortine B Roquefortine C Rosellichalasin Rugulovasine A Rugulovasine B

517 519 521 523 979 977 147 136 140 147 343 126 130

Sulpinine A Sulpinine B Sulpinine C Sydonol

469 471 473 920 923 923 231

397 399 401 403 405 381 513 515

Sarcodonin A Sarcodonin G Secopenitrem B Setoclavine Shiraiachrome A Shiraiachrome B Shiraiachrome C SM-Q Stemphone Sterigmatin Sterigmatocystin Striatin A Striatin B Striatin C

787 789 417 50 959 961 963 226 987 610 597 791 793 795

Toluquinol p-Toluquinone TR-2 1,5,10-Trihydroxy-7methoxy-3-methyl1H-naphtho[2,3-c] pyran-6,9-dione 2,5,8-Trihydroxy-6methoxy-3-(2oxopropyl)-l,4naphthoquinone Tri-O-methylversicolorin B L-Tryptophanyl-Lprolyl anhydride Tryptoquivaline B Tryptoquivaline C Tryptoquivaline D Tryptoquivaline 27-epi-Tryptoquivaline Tryptoquivaline A Tryptoquivaline E Tryptoquivaline F Tryptoquivaline G Tryptoquivaline H

Tryptoquivaline I Tryptoquivaline J Tryptoquivaline L Tryptoquivaline M Tryptoquivaline N Tryptoquivalone Tubingensin A Tubingensin B

883

893 589 199 381 379 383 379 406 379 389 391 393 395

D-Valyl-L-tryptophan anhydride Verruculogen Versicolorin A Versicolorin B Versicolorin C Versicolorone Versiconal hemiacetal acetate Versiconol Versiconol acetate Viridamine Viridin Viridiol

165 228 573 575 576 590 581 584 583 151 717 721

Wortmannin

725

Zygosporin A Zygosporin D Zygosporin E Zygosporin F Zygosporin G

281 289 290 291 292

Molecular Formula Index Molecular formula

Page

C7

C7HsNO5 C7H602 C7H6N204 C7H7NO5 C7H803 C7H804 C7HloN202 C7H12N20

631 923 637 635 639, 645 641,647 158

535

C8

C8H804 C8HnN202

C8H14N20 C~ C9HloO4 C9HloO5

C9H14N202 CgH16N20 Clo ClonloN203 CloHllN CloHllNO CloHllNO6 CloH16N202 Cll C11H13NO CllH13NO3 CllH2oO2 C11HEoN202 C12 C12H14N202 C12H15NO3 C12H17NsO C12H2oO3 CI2H2oN202

C12H2202 C12HEEN202 C13 C13H1404 C13H1405 C13H1604

649 169, 537 527,543 643 644 157, 529, 539

Molecular formula

Page

C13H1605 El4 C14HloO5

928 705

C14HloO6

707

C14H1207 C14H1206 C14H1406 ClaH15N302 C14H1604 ClaH16N203 C14H1804

893 863 915 166 918

C14H2oO4 C14H2403 C15 C15HloO7 C15HllNO5 C15H1205

533

C15H1206

943 495 497 633 531

C15H1207 C15H1406

499 3 627 168

C15H1606 C15H1607

157 5 541 945,947 159 615 162 926 941 929

C15H1407 C15H1408

C15H1608 CI5H17NO3 C15H1807 C15HlsN20 C15H2oO2 C15H2oO4 C15H2202 C15H2203 C15H2204 C15H2205 C15H2403

160 907, 910, 911 811

625 881 873 703 699, 711, 844 883,894 709, 861, 889 857 712,713, 714 697 849, 851, 875, 877, 891 853 901,903 887 48, 50, 52 831,833 803, 805 951 801 799 807, 809 920, 949

Molecular formula

C15H2405 C15H2602 C15H2603 C15H2604 C15H2803 C16 C16HloO6 C16H13NO5 C16H1405 C16H1406 C16I-I1407 C16H15N202C1 C16H1606 C16H1607 C16H16N2 C16H16N202 C16I-I17N302 C16H17N30 C16H1805 C16H1806 C16H1807 C16HlsN2 CI6H18N20 C16H18N202 C16H19N302 C16H2oN2

C16H2oN20 C16H21NO6 C16H22N402 C1682403 C16I-I2605 C16H2805

Page 823 617,619, 621 953 834 623 558 855 559,841 561 701,885, 896 128,132 839, 865 846, 859 20 8,10,14 12,16, 199 10,12, 14,16, 199 977, 979 867, 913 879 22, 30, 62 18, 37 58, 60 165 32, 34, 35, 54, 56 43, 45, 47,138, 140 905 149,151 829 819 908

995

996

Molecular Formula Index

Molecular formula

Page

Molecular formula

C16H2806 C17 C17H1006 C17H1206 C17H1207

909

C2o C2oH1606 C2oH1607

C17H1406 C17H1407 C17H1408 C17H1806 C17H1807 C17H2oO5 C17H2oO6 C17H2oO7 C17H22N202 C17H22N204 C17H2605 C17H2606 C17H2805 C17H2806 C18 C18H1007 C18H1206 C18H1207 C18H1406 C18H14N204 C18H1608 C18H1609 C18H17N303 C18H22N202 C18H2606 C18H3oO5 C19 C19H1405 C19H1406 C19H1407 C19H1605 C19H1606 C19H1609 C19H1806 C19H1808 C19H18N202 C19H19N302 C19H21N302 C19H23N302

610 547,608 551,555, 563 549,564, 565 553,557, 566 567 843 848 969,971 917,965, 967 854 6 163 815 825 821 827 573 597 575,576 599 178 584 869,871 212 134,136 935 817 719 601 605 723 603 583 568,569 922 4 207 190,205 24,27, 203

C2oH1608 C2oH1609 C2oH1806 C20H1807 C2oH21N303 C2oHE2N206S2 C2oH24N20652 C16H16N202 C20H2605 C2oH2802 C2oH2803 CzoH2804 C2oH3oO3 C2oH3oO4

C2oH3oO5 C2oH3203 C21 C21H1807 CzlHE1N302

C21H21N303 C-~1H2206 C21H23N302 C21H23N303 C21H23N304 C21H25N302 C21H2804 C21H3oO8 C22 C22HlsN404 C22H18N405

C22H2oO8 C22H23NsO2 C22H25N303 C22H27N306 C22H28N206 C22H3404 C23 C23H2oN405 C23HE3N302 C23H2408

Page

717,931 577,588, 590,607, 933 586,592, 609 581 721 579 186 239,243 241 761 763 757,765 759,767, 787,789 773,777 753 755,769, 771 775 779 589 192 180 727 188 182, 196, 198 184 194 653 899

391,399 389,395 587 147 226 231 39 781,783 393,401 174 725

Molecular formula

Page

C23H25N303 C23H25NsO4 C23H3oN202 C24 C24H2208 C24H2408 C24H25NsO4 C24H27N302 C24H27N303 C24828N404 C24H29N302 C24H3106C1 C24H3206 C24H33NO4

204 170

C24835NO4 C24H3605 C25 C25H25N303 C26 C26H24N405 C26H24N406 C26H3604 C26H3808 C27 C27H1507 (OCH3)3 C27H2oO9 C27HE6N406 C27H31NO3 C27H31NO4 C27H33NO4 C27H33N303 C27H33N304 C27H33N305 C27H33N307 C27H35NO3 C27H35NO4 C28 C28H28N406

C28H28N407 C28H33NO5 C28H33NO7 C28H35NO C28H35NO2 C28H35N304 C28H35N305 C28H35NsO5 C28H35NO6 C28H37NO2

142

957 955 172 208, 216

217 97, 99 210, 214

975 973 334, 335 337, 339

785 176 405 381 655 795 747 734 397 445 447 449 505 503 224 228 481 451,453 387 383, 403, 408 343 341,345 513,515 374 501 507 101,104 303, 328 372

Molecular Formula Index

Molecular formula

C~H37NO4 C28H38N2011 C28H39NO C28H39NO2

C28H39NO3 C28H39NO5 C28H4oO5

C28H4oO6 C28H4oO8 C28H4oO9 C28H41NO C28H41NO2 C29 C29H26Olo C29H3oN406 C29H3oN407 C29H34N204 C29H35NO3 C29H35N302 C29H35NO5 C29H35N505 C29HasN309 C29Ha7NO2

C29H37NO4 C29H37NO5 C29H39NO4 C29H39N302 C3o C3oH2409 C3oH26Olo

C3oH3oOlo C3oH37NO5 C3oH37NO6 C3oH37NO7 C3oH37N505

Page 289,297, 301,308 41 357,370, 509,521 362,364, 366,368, 443,511, 517 359 312,314, 316 924 985 791 793 523 519 731,733, 745 385 379,406 332 350 218,219 275,457 81,122 230 349 347 277,284, 455,479 351 201 963 735,959, 961 749 290,292 279,281, 287,324 318,326, 330 71,73, 79,124

997

Molecular formula

C3oH39NO4 C3oH39NO5 C3oH39NO7 C3oH41NO6 C3oH4208 C30H4408 C3oH4608 C31 C31H1509 C31H28Oll C31H39NsO5 C31H41NO6 C32

C32H3oO16 C32H32N403 C32H36N204 C32H36N205 C32H36N402 C32H37NO6 C32H37NO7 C32H38N205 C32H39NO4 C32H39NO5 C32H39NO7 C32H41NO3 C32H41NO4 C32H41NO6 C32H41N307 C32H41NsO4 C32H41N505 C32H5209 C32H52Olo C33 C33H3oO12 C33H34N405 C33H35NsO5 C33H35NsO6 C33H38N402 C33H43N504 C33H43NsO5

Page

Molecular formula

299 293,295, 306 320,322 310 987 983 981

C34 C34H37N505 C34H38N206

683 737 83, 86, 88 304

C35 C35H39NsO5 C36 C36H56012

937,939 153 261 247,249, 251,253, 259 233 353 285 255,257 459,461, 463,467 465 291 471 469,477 473 222 117 90,107, 112,115 691 673,694 739 155 64,67 69 235 120 110

C34H4oN205 C~Hs4Oll

C37 C37H44NO4CI C37H,uNOsCI C37H44NO6Cl C37H45NO4 C37H45NO5 C37H45NO6 C37H47NO5 C37H49NO4 C37H49NO6 C37H49NO7

C38 C38H32012 C3sH41N305 C38H58013

C39 C39H51NO6 C39Hs1NO7 C42 C42H4oN604 C42H55NO7 C42H57NO7 C43 C43H34012 C43H49N305 C43H5608 C47 C47H6208 C~2 C52H7oO8 C53 C53H7208

Page 75,77 263,265, 267 269,271, 273 671,677, 685,687, 689 92, 95 667, 669, 675 421 427 413,429 423 415 419,425 417,485 431,475 487 438 743 221 679,681 491 489 237 433 436 741 220 657 659 661 663

This Page Intentionally Left Blank

Molecular Weight Index Molecular weight 122.03678 140.04734 140.09496 145.08910 154.07423 154.11061 156.04226 161.08406 168.04226 168.08988 168.12626 175.09971 182.03276 182.05791 182.10553 183.01677 184.14633 185.03242 196.12118 198.05282 198.16198 206.06914 207.08954 212.14124 212.15248 218.10553 221.10519 224.15248 226.16813 230.13063 232.14633 234.08921 234.16198 236.10486 236.13135 238.14700 238.19328 240.16265 240.17254 241.05864

Page

Molecular weight

Page

923 639, 64 535 495 158 527, 543 641,647 497 649 169, 537 533 499 637 643 529,539 631 627 635 531 644 615 943 3 945,947 168 157 5 159 162 833 831 926 951 929 20 22, 30, 62 617, 619, 621 32, 34, 35, 54, 56 625 633

242.14191 247.14331 248.10486 250.08412 250.12051 250.15689 252.09977 252.13616 252.17254 254.14191 254.18819 256.15756 256.20384 257.11643 258.05282 259.12084 260.11609 264.13616 264.17254 266.15181 267.13716 268.12118 270.13683 270.18311 272.06847 272.15248 274.04774 276.06339 278.07904 282.14672 283.13208 284.16237 285.06372 285.14773 286.08412 286.16813 288.06339 290.07904 290.11542 292.05830

48 541 918 941 907,910,911 801 928 811 920,949 18,37,50,52 953 43,47,138,140 623 166 705 901,903 160 803,805 829 799 10,14 8,126,130 58,60 834 703 45 707 863 915 807,809 12,16,199 823 873 165 559,841 6 699,711,844 709,861,889 977,979 893

Molecular weight

Page

292.09469 298.04774 298.16813 298.17802 299.07937 300.19367 300.20893 302.04265 302.07904 302.08221 302.17428 304.05830 304.09467 304.13107 306.07395 306.11034 306.13683 308.08960

697 558 134,136 819 855 908 757,765 881 561 128,132 149,151 883,894 839,865 969,971 857 867,913 4 849,851,875,877, 891 310.04774 610 310.10525 887 310.17802 815 312.06339 547,608 312.19367 821 314.07904 549,564,565 314.18819 761 316.18859 909 316.20384 759,767,787,789 318.07395 701,885,896 318.11034 843 318.15796 163 318.21950 753 320.08960 846,859 320.12599 917,965,967 320.23515 779 321.14773 207 322.06687 712,713,714 322.08412 , 719 322.09536 178 322.10525 879

999

1000

Molecular weight 323.12670 323.13689 323.16338 324.06339 324.08452 324.09977 325.17903 326.07904 326.17294 326.20932 328.05830 328.18859 330.07395 332.19876 334.10525 334.21441 336.12090 338.04265 338.07904 338.17294 340.05830 340.09469 342.11034 344.19876 346.06887 346.17802 349.16338 349.17903 350.20932 351.19468 352.09467 352.15829 354.07395 354.11034 360.08452 362.24571 363.15829 365.17394 366.23073 368.08960 370.10525 370.14164 373.17903 374.10017 376.07943 379.18959

Molecular Weight Index

Page 212 905 190, 205 597 853 723 24, 27, 203 599 825 817 551,555,563 827 553, 557, 566 773, 777 848 755, 769, 771 854 573 601 935 575,576 603 568, 569 653 567 763 192 188 775 194 717, 931 186 605 721 584 781,783 180 182, 196, 198 142 577,588, 590, 607, 933 579 727 174 922 869, 871 226

Molecular weight 381.16886 382.10525 384.08452 388.07943 389.18518 389.21033 391.18959 391.22598 399.24096 400.07943 401.25661 401.27186 402.13281 404.25627 405.20524 405.30317 407.31882 410.19407 412.11582 412.26136 415.18959 416.19474 416.21989 417.23039 417.26678 418.12772 419.28243 421.26169 421.29808 423.31373 428.14712 429.18999 431.28243 432.14337 433.22531 435.24096 436.21106 437.25566 437.29299 438.13147 440.14712 447.19065 447.25219 447.27734 450.09193 450.18092

Page 184 589 568, 592, 609 583 147 208, 216 204 210,214 334, 335 581 337,339 513,515 399 391 217 357, 370, 509, 521 523 899 587 655 176 39 973 445 374 389, 395 372 481 362, 364,366, 368, 443,511,517 519 725 231 349 393,401 447 449 97, 99 451,453 359 957 955 172 505 350 239,241,243 975

Molecular weight 451.27226 453.19065 456.28757 457.27293 461.30423 463.24711 463.23587 463.27226 465.28791 469.28282 472.17467 472.28249 474.25186 477.25152 477.26276 478.25667 479.26717 481.24644 487.30864 488.11073 488.16958 491.26717 493.25767 493.28282 495.22570 501.28791 502.18523 503.30356 504.27232 507.26209 508.28383 511.23185 511.29339 512.26751 516.20088 517.28282 520.24744 520.26723 521.26382 522.29948 523.25700 523.29339 525.27265 528.14203 528.26242 530.21653

Page 289,297,301,308 170 924 218,219 201 503 343 347 351 312,314,316 405 985 332 275,457 501 795 277,284,455,479 303,328 471 734 381 290,292 507 293,295,299,306 341,345 459,461,463,467 397 469,477 791 279,281,287,324 233 228 310 261 387 465 153 793 101,104 235 318,326,330 304 320,322 963 247,249,251,353, 259 385

Molecular Weight Index

Molecular weight 530.27807 530.28797 531.07161 531.26209 532.19580 532.30362 533.26382 534.15260 534.31927 535.29339 544.13695 546.15260 546.21145 547.25700 547.27947 549.27265 550.18390 556.29372 559.15855 561.29512 566.25292 567.33486

1001

Page

Molecular weight

Page

255,257 987 683 353 383,403,408 983 81,122 731,733,745 981 473 747 735,961 379,406 285 71,73,79,124 291 749 269,271,273 117 83,86,88 155 423

569.23733 570.27299 571.36616 573.33151 575.31077 576.16316 578.24756 579.24817 579.29445 580.36113 581.26382 583.32977 585.34542 589.32642 595.27947 596.35605 597.25873 599.32469 601.29589 603.35599 609.29512 617.29080

230 263,265,267 431,475 120 90, 107, 112, 115 737 41 67,224 222 691 64 415 417,485 110 75,77 673,694 69 419,425 421 487 92,95 427

Molecular weight 618.17373 619.30462 619.35090 629.37164 633.28572 638.36661 645.36655 670.15339 680.18938 680.37718 685.39785 687.36722 687.41350 692.31110 700.39752 722.38774 742.20503 754.44447 822.50707 836.52272

Page 739 221 438 491 413,429 671,677, 685,687, 689 489 937, 939 743 667, 669, 675 433 220 436 237 657 679, 681 741 659 661 663

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Fungal/Plant Source Index A

Achaetomium cristalliferum, 908, 909 Acremonium coenophialum, 43, 81,527, 529, 533,539,543 Acremonium lolii, 433,436, 438, 449, 451,453, 541 Acremonium roseum, 905 Adenocarpus decorticans, 433,527 Agropyrum ciliate, 30 Agropyrum semicostatum, 30, 35, 50, 54,56 Agropyrum sp., 22, 34, 43 Agropyrum type ergot fungus, 35, 43, 54, 56 Alternaria alternata, 699, 709, 711 Alternaria citri, 709 Alternaria cucumerina, 703,705, 711 Alternaria dauci, 703,705,709, 711 Alternaria porri, 937, 939 Alternaria solani, 899 Alternaria sp., 699 Alternaria tenuis, 697,699, 703,705,707,709, 711,712,713 Ampulliferina-like fungus, 901,903 Apiospora camptospora, 719 Armillaria mellea, 973,975 Ascochyta chrysanthemi, 641,643,644 Ascochyta heteromorpha, 275,277,351 Aspergillus amstelodami, 201,203,204, 205, 207, 208, 210, 212, 218, 220, 221,597 Aspergillus aurantio-brunnel, 597 Aspergillus caespitosus, 222, 224, 228 Aspergillus candidus, 931,933 AspergiUus chevalieri, 165,166, 201,203, 579 Aspergillus clavatus, 341,345,379, 381,383, 385,387,389, 405 Aspergillus echinulatus, 201 AspergiUus flavus, 237, 357,359, 362, 364, 366, 368, 372, 374, 447, 459, 461,547, 549, 551, 553, 555,557, 561,566, 567, 601,603, 605 Aspergillus fumigatus, 134, 138, 142, 222, 224, 226, 228, 231,379, 383,389, 391,393, 395, 397, 399, 401,403,405 Aspergillus janus, 727

Aspergillus japonicus, 62, 170, 172 Aspergillus microcysticus, 334, 335,337, 339 AspergiUus multicolor, 597 Aspergillus nidulans, 586, 597 Aspergillus nomius, 509, 511,547, 549, 551, 553 Aspergillus ochraceus, 186, 233,235 Aspergillus parasiticus, 368, 372, 461,547, 549, 551,553, 555,557,561,566, 567, 573, 577, 579, 581,583, 584, 590, 592, 601,603 Aspergillus quadrilineatus, 597 Aspergillus repens, 203 Aspergillus rubrum, 190, 205,210, 214, 216, 217,219,579 Aspergillus sp., 176, 920 Aspergillus subolivaceus, 461 Aspergillus sulphureus, 417, 419, 469, 471,473, 517,519, 521,523 AspergiUus tubingesis, 357, 368, 370, 372, 513, 515,370 Aspergillus unguis, 597 Aspergillus ustus, 180, 182, 184, 188, 192, 194, 577, 597 Aspergillus variecolor, 597 Aspergillus versicolor, 573, 575,576, 577, 579, 587,588, 589, 597, 599, 607, 608, 609, 610 Aspergillus zonatus, 174, 9?5 B Balansia claviceps, 24, 43 Balansia cyperi, 101,104 Balansia epichloe, 3, 4, 5, 24, 32, 43 Balansia henningsiana, 24, 43 Balansia obtecta, 101,104 Balansia strangulans, 32, 43 Beauveria bassiana, 162, 168, 169 Bipolaris sorokiniana, 178, 597 Boletus (Suillus) bovinus, 655, 661 Botryotinia squamosa, 833,834 Botrytis aUii, 701 Botrytis cinerea, 815,821,823,825, 827, 829 Botrytis squamosa, 817, 819, 831

1003

1004

C Cercospora ariminiensis, 731 Cercospora bertoreae, 731 Cercospora beticola, 731 Cercospora bizzozeriana, 731 Cercospora canescena, 731 Cercospora carotae, 731 Cercospora chenopodii, 731 Cercospora cistinearum, 731 Cercospora cladosporioides, 731 Cercospora diazu, 731 Cercospora dulcamarae, 731 Cercospora erysimi, 731 Cercospora hayii, 731 Cercospora kikuchii, 731,733, 734, 745 Cercospora malvacearum, 731 Cercospora malvicola, 731 Cercospora medicaginis, 731 Cercospora nicotianae, 731 Cercospora oryzae, 731 Cercospora personata, 731 Cercospora plantaginis, 731 Cercospora setariae, 731,737, 739, 741,743 Cercospora unamunoi, 731 Cercospora violae, 731 Chaetomium cochliodes, 247, 249, 251,253, 255 Chaetomium globosum, 247,249, 251,253, 255,257, 259, 261,263, 265, 267 Chaetomium mollipilium, 247,249, 251,253, 255 Chaetomium rectum, 247,249, 251,253, 255 Chaetomium subaffine, 247, 249, 251,253, 255 Chaetomium thielavioideum, 597, 601 Chalara microspora, 285,287, 353 Chroogomphus helveticus, 653 Chroogomphus rutilus, 653 Cladosporium phlei, 749 Claviceps fusiformis, 6 Claviceps paspali, 12, 16, 443, 445, 447, 463, 465,467 Claviceps purpurea, 8, 10, 14, 24, 27, 32, 45, 47, 64, 67, 69, 73, 75, 77, 79, 81, 83, 86, 88, 90, 92, 95, 97, 99, 107, 110, 112, 115, 117, 120, 122, 124 Claviceps sp., 37, 39, 41, 48, 159 Cochliobolus miyabeanus, 981,983, 985 Coriolus vernicipes, 281,649 Corynascus setosus, 406, 408 Corynespora smithii, 709 Curvularia lunata, 277

Fungal/Plant Source Index

Cyathus africanus, 753, 767, 771,773, 775,777 Cyathus earlei, 757, 765,779, 781,783,785 Cyathus helenae, 753, 755,759, 761,763, 767, 769 Cyathus striatus, 791,793, 795 D

Diplodia macrospora, 269, 271,273 Drechslera biseptata, 277 Drechslera leersii, 983 Drechslera oryzae, 981,983 E

Elsinoe annonae, 747 Elymus mollis, 37, 50, 58 Elymus sp., 43 Elymus-type ergot fungus, 18, 20 Emericella desertorum, 449 Emericella foreolata, 449 Emericella striata, 449, 457 Engleromyces goetzei, 281,303 Entoloma haastii, 157 Epichloe typhina, 32, 81,615,617, 619, 621, 623,625,627 Erotium amstelodami, 201,203, 204, 205,207, 208, 210, 212, 218, 220, 221,587 Erotium chevalieri, 165,201,203, 597 Erotium echinulatum, 201 Erotium repens, 203 Erotium rubrum, 190, 205,214, 216, 217, 219, 597 Exserohilum holmii, 239, 241 F

Farrowia sp., 597, 601 Festuca rubra, 35, 50, 54, 56 Fusartum equiseti, 158 Fusartum javanicum, 857 Fusarium moniliforme, 846, 855, 865, 867 Fusarlum nivale, 160 Fusarlum oxysporum, 894, 896 Fusartum scirpi, 158 Fusarium solani, 844, 848, 849, 851,853, 855, 857, 859, 861,863,865, 869, 871,873,875, 877, 879, 881,883, 885, 887, 889, 891,893 Fusicoccum amygdali, 667, 669, 671,673, 675,677, 679, 681,683, 685,687, 689, 691, 693

Fungal/Plant Source Index

G Gibberella saubinetti, 943 Gliocladium virens, 717,721 Gnomonia erythrostoma, 279 Gomphidius rutilus, 653,657, 659, 661 Guignardia laricina, 913,915,917 H

Helicascus kanaloanus, 945,947 Helminthosporium dematioideum, 275,277,284 Helminthosporium leersi, 981,983 Hendersonula toruloidea, 915 Hormiscium spp., 213 Hypocrella bambusae, 735 Hypomyces odoratus, 277 Hypozylon terricola, 279, 281,318, 320, 322, 324, 330

Ipomoea tricolor, 10

I

L Lolium cuneatum, 527,529, 531,533,535,537, 539 Lolium temulentum, 527 M

Marasmius aUiaceus, 799, 801,803, 805, 807, 809, 811 Metarrhizium anisopliae, 279, 281 Metatrichia vesparium, 922 Monascus anka, 935 Mycosphaerella ligulicola, 641,643,644 Myrothecium roridum, 725 N

Nectria erubescens, 923 Nectria haematococca, 894, 881 Nigrosabulum spp., 332 Nigrospora sphaerica, 243 Nodulisporium hinnuleum, 719, 723 P

Paecilomyces sp., 941 Paspalum distichum, 10

1005

Penicillium aurantiogriseum var. neoechinulatum, 149 Penicillium aurantiovirens, 251 Penicillium biforme, 126, 130 Penicillium brasilianum, 153, 155,228, 230, 231 Penicillium brevi-compactum, 196, 198, 199 Penicillium chermesinum, 35, 54 Penicillium claviforme, 639, 641,643 Penicillium commune, 147 Penicillium concavo-rugulosum, 126, 130 Penicillium corymbiferum, 147 Penicillium crustosum, 413, 415,421,423, 425, 427,431,455,475,477,479, 481 Penicillium estinogenum, 228 Penicillium expansum, 147 Penicillium funiculosum, 727 Penicillium graminicola, 228 Penicillium griseofulvum, 639, 641,923 Penicillium islandicum, 126, 128, 130, 132 Penicillium italicum, 182, 188, 192, 194 Penicillium janthinellum, 228, 485,487, 489, 491 Penicillium lanosum, 224, 923 PeniciUium nigricans, 429 Penicillium notatum, 147 Penicillium oxalicum, 147, 172 Penicillium paraherquei, 228, 507 Penicillium patulum, 639, 641,923 Penicillium paxilli, 228, 449 Penicillium piscarium, 224, 228 Penicillium roqueforti, 136, 140, 147, 501,503, 5O5 Penicillium rubrum, 126, 130 Penicillium simplicissimum, 153, 155 Penicillium sp., 170, 172, 977, 979 Penicillium urticae, 147, 923 Penicillium verruculosum, 126, 228, 230, 231 Penicillium viridicatum, 151 Penicillium wortmannii, 725,727 Pennisetum typhoideum, 37, 52, 160 Pestulotia sp., 295 Phalaris sp., 34, 43 Phoma cava, 965,967, 969, 971 Phoma chrysanthemi, 641,643, 644 Phoma exigua, 275,277, 347, 349 Phoma lingam, 163 Phoma sp., 350 Phomopsis paspalli, 293 Phomopsis sojae, 299, 301

1006

Fungal/Plant Source Index

Phomopsis sp., 293,297, 306, 308, 310, 312, 314,316 Phragmites sp., 43 Preussia aurantiaca, 955, 957 Pseudeurotium zonatum, 332 Pyrenochaeta terrestris, 926, 928, 929 Pyricularia grisea, 304 Pyricularia oryzae, 907,910, 911,920 R

Rhizopus arrhizus, 138 Rivea corymbosa, 10 Rosellinia necatrix, 341,343 S

Sarcodon scabrosus, 787, 789 Shiraia bambusicola, 959, 961,963 Sorlorina crocea (Lichen), 579 Stemphylium sarcinaeforme, 987 Streptomyces baarnensis, 631,633,635 Streptomyces fulvoviolaceus, 631,633

Streptomyces sp., 637 Suillus bovinus, 663 T Torula herbarum, 839, 841,843 Trichoderma viride, 717, 721 Tricholoma sciodes, 495, 497, 499 Tricholoma virgatum, 495, 497, 499 Trisetum bifidum, 35, 50, 54, 56 U

Ustilago cynodontis, 162 X

Xylaria obovata, 326, 328

Z

Zygosporium masonii, 281,289, 290, 291,292

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Handbookof Secondary Fungal Metabolites V O L U M E II

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Handbook of Secondary Fungal Metabolites VOLUME

II

RICHARD J. COLE

Albany, Georgia

MILBRA A. SCHWEIKERT

National Peanut Research Laboratory Dawson, Georgia

ACADEMIC PRESS An imprintof ElsevierScience Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

Cover images: Photograph by Dr. Bruce Horn and design by Brian E. Cole. The cover is a collage of various fungi, some presented in pure culture and others in their natural forms, i.e. mshrooms, which are the easily recognizable fruiting structures of some fungi, superimposed on these fungi are the chemical structures of some representative secondary fungal metabolites. Academic Press Rapid Manuscript Reproduction

This book is printed on acid-free paper. Copyright

92003, Elsevier Science ( U S A ) .

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://elsevier.com), by selecting "Customer Support" and then "Obtaining Permissions." A c a d e m i c Press An imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com A c a d e m i c Press 84 Theobald's Road, London WC1X 8RR, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2003103019 International International International International

Standard Standard Standard Standard

Book Book Book Book

Number: Number: Number: Number:

0-12-179460-1 0-12-179461-X 0-12-179462-8 0-12-179463-6

(Set) (Volume 1) (Volume 2) (Volume 3)

PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 8 7 6 5 4 3 2 1

Contents

Preface I ix Acknowledgments I xi

I ~ I C27 Sterols / 1 I~1

C28 Sterols / 25

I~1

C29 Sterols / 91

I~1

C3o Sterols / 127

I ~ I C31Sterols/ I~1

173

C32 Sterols / 211

vi

Contents

I~1

Miscellaneous Sterols /

I~1

Antheridiolsand Oogoniols /

I~1

Ganoderic Acids /

223

249

271

I~ �9 Ganoderiols / 351 q~l

GanolucidicAcids / 379

I~ ~1 Lucidenic Acids / I~ ~1 Lucidones /

423

I~ ~1 Azasterols /

431

[~+!

Fasc,cu,o,s,

391

447

Fusidanes and Protostanes /

461

LL-Z1271 Antibioticsand Related Metabolites /

493

Contents

vii

I~ ~1 LL-Z1272 Antibioticsand Related Metabolites / 505 I~ ~1 Helminthosporols/

527

]~�9 Hebevinosidesand Hebelomic Acids / 543 I~1

Siccanochromenesand Grifolins / 587

~~

Fusarochromanones/

I~1

Aphidicolins / 619

609

Neovasinins / 629 i~1

Koninginins/ 641

~(~

Curvularins /

~1

ArcyroxepinA and Related Bisindoles /

I~1

Arugosins / 685

653

675

viii

Contents

Hericenes and Hericenones / 697

Miscellaneous Metabolites / 715 Secondary Metabofite Index / 801 Molecular Formula Index / 809 Molecular Weight Index / 813 Fungal/Plant Source Index I 815

Preface

The "Handbook of Secondary Fungal Metabolites" is presented in three volumes and is comprehensive to the extent that all major groups of secondary fungal metabolites are included. The format is similar to that presented in the "Handbook of Toxic Fungal Metabolites" with the major exception that actual spectra are not included; however, spectral data are included where available. Also included in these volumes are the methods used by the authors to isolate and purify metabolites. Another major difference is that the appropriate references are presented with each metabolite, negating the need to turn to the end of each group to find the appropriate references. Each volume contains four indexes: secondary metabolite index, molecular formula index, molecular weight index, and fungal/plant source index. In a few instances, plant sources are included when the metabolites are closely related to fungal metabolites or the source of precursors may be fungal; i.e., the baccharins, which are found in extracts from Baccharis megapotamica. These metabolites are closely related to the macrocyclic trichothecenes found in extracts of fungi such as Myrothecium spp. and Stachybotrys spp. Also, metabolites from the fungal symbiont of lichens are sometimes presented. To aid in the interpretation of NMR data, the numbering system presented in the literature is included for the major representative fungal metabolite and, at times, for several related metabolites. Fungal sources are given as reported in the original references. It is recognized that the taxonomy in several cases has been revised, perhaps more than once. It is beyond the scope of these volumes to deal with what is "currently accepted taxonomy" because this is a dynamic science that, in many cases, is as yet undefined. The "Handbook" has been divided into sections, and the placement of metabolites is based on chemical relationships. One section of each volume contains a miscellaneous section to accommodate metabolites difficult to place into one of the sections. The miscellaneous section of Volume III contains some metabolites related to those that appear in Volumes I and II. This occurred when related metabolites were discovered after Volumes I and II were completed.

ix

x

Preface

It is hoped that this compilation of data on secondary fungal metabolites will aid investigators in the identification of known or related fungal metabolites. Because fungal metabolites represent a wide diversity of chemical species, these volumes will be useful to scientists interested in correlations of structural features with various spectral and biological characteristics. The known biological activity of metabolites is presented, which may aid in future studies related to the identification of new uses for fungal metabolites. Richard J. Cole Milbra A. Schweikert

Acknowledgments

The authors thank the following investigators for their assistance in producing the "Handbook of Secondary Fungal Metabolites." Their contributions made this compilation of data on fungal metabolites possible. Wayne L. Bryden

James K. Porter

University of Queensland Gotton, Queensland 4343 Australia

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

Brian E. Cole

Images, Inc. Snellville, Georgia Horace G. Cutler

Department of Pharmacology Mercer University Atlanta, Georgia ,lens C. Frisvad

Department of Biotechnology Technical University of Denmark DK-2800 Lyngby Denmark Bruce Horn

USDA-ARS National Panut Research Laboratory Dawson, Georgia

Ronaid T. Riley

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia Victor S. Sobolev USDA-ARS National Peanut Research Laboratory Dawson, Georgia Bruce B. Jarvis

Department of Chemistry and Biochemistry University of Maryland College Park, Maryland

William Norred

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

xi

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C27 Sterols Cholest-5-en-313-ol (Cholesterol) Cholesta-5,24-dien-313-ol (Desmosterol) Cholest-7-en-313-ol (Lathosterol) Cholesta-5,22-dien-313-ol (2-Dehydrocholesterol) Cholest-8-en-313-ol Cholesta-5,7-dien-313-ol (7-Dehydrocholesterol) Cholesta-7,24-dien-313-01 Cholesta-8,24-dien-313-ol (Zymosterol) Cholesta-5,7,24-trien-313-ol Cholesta-5,8,24-trien-313-01 Cholesta-7,22,24-trien-313-01 Cholesta-8,22,24-trien-313-01 Cholesta-5,7,22,24-tetraen-313-01 Cholesta- 5,8,22,24-tetraen-313-ol

This Page Intentionally Left Blank

1.

C27 Sterols

3

Common[Systematic Name Cholesterol; Cholesterin Cholest-5-en-3 I~-ol Molecular Formula/Molecular Weight C27H460; M W -" 3 8 6 . 3 5 4 8 7

HO General Characteristics Monohydrate, pearly leaflets or plates from dilute alcohol; becomes anhydrous at 70-80~ When anhydrous, mp. 148.5~ Sublimes as orthorhombic needles. [a]D2~ -31.5 o (C=2' in ether); [trio 2~ -39.5 o (c=2 ' in CHC13). Practically insoluble in water (about 0.2mg/100ml H20). Slightly soluble in alcohol (1.29%, w/w at 20~ more soluble in hot alcohol (100 g of saturated 96% alcoholic solution contains 28g at 80~ One gram dissolves in 2.8ml ether, in 4.5ml chloroform, in 1.5ml pyridine. Also soluble in benzene, petroleum ether, oils, and fats. Soluble in aqueous solutions of bile salts: Gives intense red color with rosaniline in chloroform solution. Methyl ether, crystals from acetone; mp., 84~ ; [a]D2~-45.8~ (c=1.2, in CHCI3). Acetate, needles from acetone; mp., 115-116~ [t~]D2~ -47.4 ~ (C=2, in CHCI3). Benzoate; mp., 145.5~ (the melt becomes clear at 180~ and [t~]D25 -13.7 ~ (c=0.9, in CHCI3). Fungal Source

Acaulospora laevis, Achlya bisexualis, Allomyces macrogynus, Allomyces spp., Amoebidium parasiticum, Aspergillus oryzae, Blastocladia ramosa, Colletotrichum dematium, Dipsacomyces acummosporus, Glomus caledonius, G. mosseae, Gnomonia leptostyla, Leptosphaeria typhae, Linderina pennispora, Monoblepharella sp., Nectria galligena, Phycomyces blakesleeanus, Rhizophlyctis rosea, Smittium sp., and Ustilago maydis.

Isolation/Purification Extracted with chloroform-methanol (3:1, v/v); the extract was saponified with ethanolic KOH and the non-saponifiable fraction extracted with n-hexane. The non-saponifiable fraction was fractionated on alumina (activity III). The nonpolar components were eluted with hexane, while diethyl ether eluted the sterol fraction. The free sterols were acetylated and separated on silica gel impregnated with silver nitrate. Cholesteryl acetate was identified using GLC.

4

1.

027 Sterols

Also, see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids, W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York NY, 341 pp. (1989). Spectral Data UV~

End absorption. 13C NMR: (CDCI3) C-l, 37.2; C-2, 31.5; C-3, 71.4; C-4, 42.2; C-5, 140.5; C-6, 121.3; C-7, 31.8; C-8, 31.8; C-9, 50.0; C-10, 36.4; C-11, 21.0; C-12, 39.7; C-13, 42.2; C-14, 56.6; C15,24.2; C-16, 28.2; C-17, 56.1; C-18, 11.8; C-19, 19.2; C-20, 35.7; C-21, 18.7; C22, 36.1; C-23, 23.8; C-24, 39.4; C-25, 27.9; C-26, 22.5; and C-27, 22.7ppm. Mass Data:

M + 386.64m/e, also 343,301,275, 247, 260, and 273re~e; Acetate derivative, 428, 413,368(100), 353(28), 301,299, 315, 273,255, and 213role. C, 83.37%; H, 11.99%;

O, 4.14%. References W. D. Nes, and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner, and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

1.

027 Sterols

5

Common/Systematic Name Desmosterol; 24-Dehydrocholesterol Cholesta-5,24-dien-313-01 Molecular Formul.a/Molecular Weight C27H440; MW = 384.33922

HO General Characteristics Platelets from methanol; mp., 121.5~ also reported as 119-119.5 ~ C; [ a ] D (c=l, in CHC13).

27 -

41.0 ~

Funsal Source Smittium culisetae, S. culicis, S. simuliL S. mucronatum, S. spp., and Aplanopsis spp. v

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data 13C NMR: (CDCI3) C-15, 24.4; C-16, 28.3; C-17, 56.2; C-18, 12.0; C-20, 36.2; C-21, 18.7; C-22, 35.7; C-23, 24.8; C-24, 125.5; C-25, 130.9; C-26, 17.7; and C-27, 25.7ppm (most other chemical shills were omitted because these chemical shills were consistent with those of cholesterol). Mass Spectrum: Acetate: 426(1W), 411,366(100), 356, 351(14), 315, 313, 296(4), 281,273, 259, 255(8), 253(36), 229, 213(16), and m/e. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989).

6

1.

C27 Sterols

W. B. Turner and D. C. Aldridge; Fungal Metabolites I.!; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

1.

027 Sterols

7

Common/Systematic Name Lathosterol Cholest-7-en-313-ol Molecular Formula/Molecular Weight C27H460; M W -- 3 8 6 . 3 5 4 8 7

H Fungal Source Blastocladia ramosa, Nectria galligena, and Dictyuchus monosporus. Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data UV~

End absorption. 13C NMR: (CDCI3) C-l, 37.1; C-2, 31.3; C-3, 70.7; C-4, 37.8; C-5, 40.2; C-6, 29.6; C-7, 117.2; C-8, 139.3; C-9, 49.4; C-10, 34.1; C-11, 21.5; C-12, 39.5; C-13, 43.2; C-14, 54.9; C15, 22.9; C-16, 27.9; C-17, 56.1; C-18, 11.8; C-19, 12.9; C-20, 36.1; C-21, 18.8; C-22, 36.1; C-23, 23.9; C-24, 39.4; C-25, 27.9; C-26, 22.5; and C-27, 22.7ppm. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

8

1.

C27 Sterols

Common/Systematic Name 22-Dehydrocholesterol Cholesta-5,22-dien-313-o1 Molecular Formula/Molecular Weight C27H440; MW

-- 3 8 4 . 3 3 9 2 2

Fungal Source v Physarum polycephalum, and Rhizophlyctis rosea. Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data UV~

End absorption. 13C NMR: (CDCI3) (22E-dehydrocholesterol) C-15, 24.3; C-16, 28.6; C-17, 56.0; C-18, 12.1; C20, 40.1; C-21, 20.9; C-22, 138.1; C-23, 126.2; C-24, 42.0; C-25, 28.6; C-26, 22.3; and C-27, 22.3ppm; (22Z-dehydrocholesterol) C-15, 24.3; C-16, 28.1; C-17, 56.3; C18, 12.2; C-20, 34.3; C-21, 20.7; C-22, 137.2; C-23, 125.5; C-24, 36.8; C-25, 28.8; C26, 22.4; and C-27, 22.6ppm. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

1.

C27 Sterols

9

Common/Systematic Name Cholest-8(9)-en-313-01 Molecular Formula/Molecular Weight C27H460; MW = 386.35487 IIIiii I

H Fungal Source B l a s t o c l a d i a ramosa.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Soectral Data UV:

End absorption. 13C NMR: (CDC13) C-l, 35.1; C-2, 31.5; C-3, 70.9; C-4, 38.2; C-5, 40.7; C-6, 25.4; C-7, 27.1; C-8, 128.0; C-9, 134.8; C-10, 35.6; C-11, 22.7; C-12, 36.9; C-13, 42.0; C-14, 51.8; C15, 23.9; C-16, 28.7; C-17, 54.8; C-18, 11.2; C-19, 17.8; C-20, 36.2; C-21, 18.7; C22, 36.1; C-23, 23.7; C-24, 39.4; C-25, 27.9; C-26, 22.4; and C-27, 22.7ppm. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

10

1.

C27 Sterols

Common/Systematic Name 7-Dehydrocholesterol; Provitamin D3 Cholesta-5,7-dien-313-01 Formula/Molecular Weight C27H440; M W = 384.33922

General Characteristics Hydrated plates from ethyl ether-methanol; mp., 150-151 ~ (anhydrous); [a]D 20---113.6 ~ (c=l.0, in CHC13); [a]D2~ - 127.1 o (in benzene). Insoluble in water; soluble in usual organic solvents; gives pink to blue color with antimony trichloride, blue with chloral hydrate and red with 90% trichloroacetic acid. Acetate, crystals from methanol; mp., 129-130~ [a]D2~= -85.3 ~ (C=1.2, in benzene). Fungal Source Lactarius sp., Nectria galligena, Russula aeruginosa, and R. decolorans. Isolation/Purification 7-Dehydrocholesterol was isolated from the neutral lipid fraction by column chromatography on neutral alumina (grade IV) using 10%, 20%, 50% benzene/n-hexane and benzene. The sterol containing fraction was analyzed by silica gel-G TLC (rhodamineimpregnated plates, 0.1%) or acetylated with pyridine-acetic anhydride and purified by argentation TLC chromatography (20% AgNO3-impregnated plates). Also see R. C. Heupel: Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.); Academic Press Inc., New York, NY, pp. 1-27(1989). Biolo~cal Activity Converted to vitamin D3 by UV irradiation. Spectral Data UV:

M~on 260, 271,281.5, and 293.5nm. tntx

1.

C27Sterols

11

Mass Spectrum: LREIMS: 384(NY), 369(M § - methyl), 366(M § - H20), 299(M § - 1-120 - C5H7), and 271role ( M + - side chain). References B. Dehorter, M. F. Brocquet, L. Lacoste, J. Alais, A. Lablache-Combier, A. Maquestiau, Y. van Harverbeke, R. Flammang, and H. Mispreuve; Phytochemistry, Vol. 19, pp. 23112316(1980). R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.); Academic Press Inc., New York, NY, pp. 1-27(1989). L. G. Jayko, T. I. Baker, R. D. Stubblefield, and R. F. Anderson; Nutrition and Metabolic Products o f Lactarius species; Canad. J. Microbiol., Vol. 8, pp. 361-371(1962). A. M. Pierce, A. M. Unrau, A. C. Oehlschlager, and R. A. Woods; Azasterol Inhibitors in Yeast. Inhibition of the A24 -Sterol Methyltransferase and the 24-Methylene Sterol A24(2s)Reductase in Sterol Mutants o f Saccharomyces cerevisiae; Canad. J. Biochem., Vol. 57, pp. 201-208(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, N.Y., p. 307(1983).

12

1.

C27 Sterols

Common/Systematic Name Cholesta- 7,24-dien-313-ol Molecular Formula/Molecular Weight C27H440; MW --- 384.33922

Fungal Source

Saccharomyces cerevisiae.

Isolation/Purification Sterols from the fungal extract were purified by preparative TLC (plates were coated with silica gel GF254impregnated with 45% silver nitrate and 0.25% rhodamine 6G and developed with cyclohexane-ethyl acetate, 1:1, v/v) of their acetate derivatives. The free sterol was purified via the benzoate. Also see Heupel, R. C.; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.); Academic Press Inc., New York, NY, 341 pp, (1989). Soectral Data _ 1H NMR: (CDC13) 0.58(s, 3H, 18-H3); 0.84(s, 3H, 19-H3); 0.97(d, 3H, J20,2a=6.0Hz, 21-H); 1.64 and 1.72(2s, 6H, 26-H3 and 27-H3); 3.68(m, 1H, H-3); and 5.16ppm (m, 2H, H-7,24). Mass Spectrum: LREIMS: 384m/e (M +, 31%) (calcd for C27H440, 384), 369(M § CH3, 29), 330(17), 300(17), and 27 lm/e (100) (M + - side chain); acetate, 422role (M +) calcd for C29H4202

.

References L. Avruch, S. Fischer, H. Pierce, Jr., and A. C. Oehlschlager; The Induced Biosynthesis of 7-Dehydrocholesterol in Yeast: Potential Sources of New Provitamin D3 Analogs; Can. J. Biochem., Vol. 54, pp. 657-665(1976). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp.(1989).

1.

C27 Sterols

13

W. B. Turner and D.C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research; Vol. 23, pp. 115-167(1989).

14

1.

C27Sterols

Common/Systematic Name Zymosterol; Cholesta-8,24-dien-313-ol Formula/Molecular Weight C27H440; M W = 384.33922

HO General Characteristics Plates from chloroform-methanol and acetone-methanol; mp., 110-113~ [IX]D25 "q- 53.7 ~ (c=1.55, in CHC13); benzoate, prisms from chloroform-methanol; mp., 126-128~ [Ct]D27 = + 44.8 ~ (C=1.3, in CHC13). Fungal Source Saccharomyces cerevisiae. Isolation/Purification Sterol residues with most of the ergosterol removed were chromatographed by column chromatography on neutral alumina using benzene and various combinations of benzene-nhexane, benzene-acetone and benzene-methanol. The sterol was finally purified as the benzoate by silica gel GF254 TLC or by argentation TLC chromatography (20% AgNO3 impregnated plates) and fractional crystallization. Also see R. C. Heupel: Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.); Academic Press Inc., New York, NY, pp. 1-27(1989). Spectral Data UV: ~k,MeOH

max End absorption.

IR:

(KBr) 890 and 1640cm "1. ~H NMR: (CDC13) 9.00 and 9.3 8T (singlet methyl chemical shifts); 4.92x (24-H).

1.

C27 Sterols

15

Mass Spectrum: Acetate, LREIMS: 426(M § 100%), 411(M + - methyl, 41), 366(M +- ROH, 19), 351 (M +- Me - ROH), and 313role (M ~-- side chain - 2H). References D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenoids. Part VI. The Sterols of Yeast; J. Chem. Sot., Perkin Trans. I, pp. 513-522(1972). R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.) Academic Press Inc., New York, NY, pp. 1-27(1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, N.Y., p. 308 (1983).

16

1.

C27 Sterols

Common/Systematic Nam.e Cholesta-5,7,24-trien-313-01 Molecular Formula/Molecular Weight C27H420; MW = 382.32357 ff

General Characteristics Needles from methanol; mp., 103.5-105~ [~]D - 118.9 ~ (c=0.35, in CHC13) mp., 102102.5~ [a]D 9 - 111.0~ benzoate, plates from chloroform-methanol; mp., 131-134~ [~t]D- 55.0 ~ (c=0.39, in CHCI3). Fungal Source Saccharomyces cerevisiae. Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol removed in vacuo, and ether soluble material converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254 TLC followed by silver nitrate-impregnated TLC. Spectral Data (Benzoate) UV: ~Lmax 229(g = 18,200), 252sh (7,400), 262sh (10,800), 272(15,100), 282(15,300), and 294nm (8,200). Mass Data: Found: C, 84.0; H, 9.4; calcd for C34H4602" C, 83.9; H, 9.5%. References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc. Perkin Trans I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of terpenes and steroids. Part X. The Sterols of Some Yeast

1.

C27 Sterols

17

Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc. Perkin Trans I, pp. 88-92 (1975).

18

1.

C27 Sterols

Common/Systematic Name Cholesta-5,8,24-trien-313-01 Molecular Formula2Molecular Weight C27I-I420; M W -- 382.32357

Fungal Source Saccharomyces cerevisiae. Isolation/Purification The nonsaponifiable fraction was acetylated and the sterol was purified by preparative argentation TLC (silica gel GF254 coated with 25% AgNOa) of the acetate derivative using benzene as mobile phase. 313-Acetoxycholesta-5,8,24(28)-triene eluted together with a small amount of 313-acetoxycholesta-5,8,22,24(28)-tetraene from a band at Re 0.31-0.38. The fraction was further purified with three consecutive developments in benzene-hexane 3:2, 1:2, and 1:5 (v/v); two major bands were obtained at Rf 0.17-0.26 and 0.26-0.30. After further purification of both bands by TLC (silica gel GF2s4, eluted with benzene), the upper band yielded 313-acetoxycholesta-5,8,24-triene and the lower band yielded 313-acetoxycholesta-5,8,22,24-tetraene. Spectral Data 1H N~/[R:

(CDCI3) (acetate)0.66(3H, s,H3-18); 0.95(3H, d, Jao,2~=6.OHz,H-21; 1.09(3h,d, H-21); 1.21(3H, s, H-19); 2.08(3H, s, CHACO-); 4.64(1H, m, H-3); 4.82(2H, m, H-28); 5.41(1H, m, H-6); 5.51(1H, dd, J2o,22=8.5Hz); and 5.92ppm (1H, d, J22,23= 15.3Hz). Mass Spectrum: Acetate, LREIMS: 436(M +, 5.9%), 376(70.5), 361(100), 253(25.3), 251(23.2), 211(12.2), 157(31.2), 143(24.8), 123(17.2), and 81m/e (23.0). Reference A. M. Pierce, A. M. Unrau, A. C. Oehlschlager, and R. A. Woods; Azasterol Inhibitors in Yeast. Inhibition of the A24-Sterol Methyltransferase and the 24-Methylene Sterol A292s) Reductase in Sterol Mutants of Saccharomyces cerevisiae; Can. J. Biochem., Vol. 57, pp. 201-208(1979).

1.

C27 Sterols

19

Common/Systematic Name Cholesta-7,22,24(28)-trien-313-ol Molecular Formula/Molecular Weight C27H420; MW = 382.32357

HO Fungal Source

Saccharomyces cerevisiae.

Isolation/Purification See R. C. Heupel: Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids, W.D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, pp. 1-27(1989). References A. M. Pierce, A. M. Unrau, A. C. Oehlschlager, and R. A. Woods; Azasterol Inhibitors in Yeast. Inhibition of the A24-Sterol Methyltransferase and the 24-Methylene Sterol A2~28)Reductase in Sterol Mutants of Saccharomyces cerevisiae, Can. J. Biochem., Vol. 57, pp. 201-208(1979). R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids, W. D. Nes and E. J. Parish (eds.) Academic Press Inc., New York, NY, pp. 1-27(1989).

20

1.

C27 Sterols

Common/Systematic Name Cholesta-8,22,24-trien-313-ol Molecular Formula/Molecular Weight C27H420; MW = 382.32357

HO Fungal Source

Saccharomyces cerevisiae.

Isolation/Purification See R. C. Heupel: Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, pp. 1-27(1989). References R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.) Academic Press Inc., New York, NY, pp. 1-27(1989). A. M. Pierce, A. M. Unrau, A. C. Oehlschlager, and R. A. Woods; Azasterol Inhibitors in Yeast. Inhibition of the A24-Sterol Methyltransferase and the 24-Methylene Sterol A24(28)Reductase in Sterol Mutants of Saccharomyces cerevisiae; Can. J. Biochem., Vol. 57, pp. 201-208(1979).

1.

C27 Sterols

21

Common/Systematic Name Cholesta-5,7,22,24-tetraen-3 g-ol Molecular Formula/Molecular Weight C27H400; MW = 380.30792

HO General Characteristics Crystals from methanol; mp., 139-141 ~ (141-1430C); acetate, crystals from chloroformmethanol; mp., 137-13 8 oc (sealed, evacuated capillary). Fungal Source Saccharomyces cerevisiae. Isolation/Purification Sterols from the fungal extract were purified by preparative TLC (plates were coated with silica gel GFz54impregnated with 45% silver nitrate and 0.25% rhodamine 6G and developed with cyclohexane-ethyl acetate, 1:1) of their acetate derivatives. The free sterol was purified via the benzoate. Also see Heupel, R. C.; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.); Academic Press Inc., New York, NY, 341 pp, (1989). Spectral Data UV: ~. EtOHmax 232sh(e = 27,170), 238(29,200), 247sh (21,900), 261(9,900), 271(12,800), 281(13,000), and 293nm (7,600); (acetate) 233sh (e = 28,400), 239(30,200), 247sh (23,000), 261(10.100), 271(13,100), 282(13,400), and 293nm (7,680). ~H NMR: (CDCI3) 0.71(s, 3H, 18-H3); 1.01(s, 3H, 19-H3); 1.13(d, J2o,zl=6.5Hz, 21-H); 1.81(s, 6H, 26-H3 and 27-H3); 3.65(m, 1H, H-3); 5.19-5.89(m, 4H, H-6, 7,22,24); and 6.19ppm (q, f122,23=13.0I-Iz,f123,24=10.0Hz, 1H); (Acetate) 0.72(s, 3H, 18-H3); 1.02(s, 3H, 19-H3); 1.12(d, ff2o,21=6.5Hz, 21-H); 1.82(s, 6H, 26-H3 and 27-H3); 2.10(s, 3H, CH3CO-); 4.72(m, 1H, H-3); 5.40(AB, J6,~=5.5Hz, H-7); 5.44(dd, f122,23=14.5Hz, J21,22=8.2Hz, H-22); 5.60(1H, H-6); 5.78(d, m, 1H, J23,24=10.5Hz, J24,26,27=lnz, H-24); and 6.18ppm (dd, 1H, H-24).

22

1.

C27 Sterols

Mass Spectrum: LREIMS: 380m/e (12%) (calcd for C27H400,380), 347(M + - 1-120 - CH3, 11), and 109m/e (100) (NC - side chain); acetate, 422role (M+) calcd for C29H4202. References L. Avruch, S. Fischer, H. Pierce, Jr., and A. C. Oehlschlager; The Induced Biosynthesis of 7-Dehydrocholesterol in Yeast: Potential Sources of New Provitamin D3 Analogs; Can. J. Biochem., Vol. 54, pp. 657-665(1976). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp.(1989). W. B. Turner and D.C. Aldridge; Fungal Metabolite.s. !I; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research; Vol. 23, pp. 115-167(1989).

1.

C27 Sterols

23

Common/Systematic Name Cholesta- 5,8,22,24-tetraen-313-ol Molecular Formula/Molecular Weight C27H4oO; M W -- 3 8 0 . 3 0 7 9 2

HO Fungal Source Saccharomyces cerevisiae. Isolation/Purification The nonsaponifiable fraction was acetylated and the sterol was purified by preparative argentation TLC (silica gel GF254 coated with 25% AgNO3) of the acetate derivative using benzene as mobile phase. 313-Acetoxycholesta-5,8,24(28)-triene eluted together with a small amount of 3 [3-acetoxyergosta-5,8,22,24(28)-tetraene and 313-acetoxy-cholesta5,8,22,24(28)-tetraene from a band at Re 0.31-0.38. The fraction was further purified with three consecutive developments in benzene:hexane 3:2, 1:2, and 1:5 (v/v); two major bands were obtained at Rf 0.17-0.26 and 0.26-0.30. After further purification of both bands by TLC (silica gel GF254,benzene), the upper band yielded 313-acetoxycholesta5,8,24(28)-triene and the lower band yielded 313-acetoxycholesta-5,8,22,24(28)-tetraene. Spectral Data (Acetate) UV:

H .... max

232(sh), 238, and 247nm.

~H NMR: (CDC13) 0.69(3H, s, H3-18); 1.06(3H, d, J2om=6.3Hz, H-21); 1.21(3H, s, H-19); 1.75(6H, s, 26-CH3 and 27-CH3); 2.05(3H, s, CH3-CO-); 4.61 (1H, m, H-3); 5.32(1H, dd, J2o,22=9.2Hz, H-22); 5.41(1H, m, H-6); 5.69(1H, d, J23,24=12.0Hz, H-24); and 6.07ppm (1H, d, J22,23=4.5Hz, H-23). Mass Spectrum: LREIMS: 422(M*, 5.7%), 362(34), 347(55.3), 253(26.7), 251(16.1), and 109m/e (100).

24

1.

C27 Sterols

Reference A. M. Pierce, A. M. Unrau, A. C. Oehlschlager, and R. A. Woods; Azasterol Inhibitors in Yeast. Inhibition of the A24-Sterol Methyltransferase and the 24-Methylene Sterol A24~28). Reductase in Sterol Mutants of Saccharomyces cerevisiae; Can. J. Biochem., Vol. 57, pp. 201-208(1979).

C2s Sterols (24R) 24-Methylcholest-5-en-313-ol(Campesterol) (24R) 24-Methylcholesta-5,22-dien-313-ol(Brassicasterol, Ergosta-5,22-dien-ol) (24R) 24-Methyleholestan-313-ol(Campestanol) (24S) 24-Methylcholesta-313-ol(Ergostanol) (24S) 24-Methylcholest-8-en-313-ol(Ergost-8-en-313-ol) 24S) 24-Methylcholesta-5,8-dien-313-ol(Ergosta-5,8-dien-313-ol) 24-Methylenecholesterol (Ergosta-5,24(28)-dien-313-ol) Ergo sta-8,24(28)-dien-313-ol(Fecosterol) (24S) 24-Methylcholest-7-en-313-ol(Fungisterol, Ergost-7-en-313-ol) (24S) 24-Methylcholesta-5,7,14-trien-313-ol (Ergosta-5,7,14-trien-313-ol) Ergosta-5,7,22-trien-313-ol(Ergosterol) Ergosta-5,7-dien-313-01 Ergosta- 7,22-dien-313-ol(5,6-Dihydroergosterol) Ergosta-7,24(28)-dien-313-ol(Episterol) Ergosta- 5,7,24(28)-trien-313-ol Ergosta-8,14,22-trien-313-ol (24S)24-Methylcholest-5-en-313-ol(Ergost-5-en-313-ol) (24S)24-Methylcholesta-5,8,22-trien-313-ol(Lichesterol) Ergosta- 5,7-dien-313-ol (22,23-Dihydroergosterol) (24S) 24-Methylcholesta-7,16-dien-313-ol Ergosta-4,6,8(14),22-tetraen-3-one (24S) 24-Methylcholesta-8,14-dien-313-ol(Ignosterol) (24S) 24-Methylcholesta-8,22-dien-313-ol(Ergosta-8,22-dien-313-ol) Ergosta-7,22,24(28)-trien-313-01 Ergo sta-8,14,24(28)-trien-313-ol Ergosta-8,22,24-trien-313-ol (24S)24-Methylcholesta-5,7,9,(11),22-tetraen-313-ol(Ergosta-5,7,9(11),22tetraen-313-ol) Ergosta- 5,8,22,24-tetraen-3[3-ol Ergosta-4,7,22-trien-3-one Ergo sta-7,22-diene-313,5Gt-diol Ergosta-8,24-diene-313,6a-diol Portensterol (Hydroxy-iso-ergosterol) Pyrocalciferol Ergosta- 5,7,22,24(28)-tetraen-313-ol Ergosta-7,22-dien-3-one Ergosta-7,22-dien-Sa,613,713-triol(Cerevisterol) 14Qt-Methylcholest-7-en-313-ol 14a-Methylcholest-8-en-313-ol 140t-Methylzymosterol 4Qt-Methylzymosterol

2

25

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2.

C28 Sterols

27

_Common/Systematic Name Campesterol; 24a-Methylcholesterol (24R)24-Methylcholest-5-en-313-01 Molecular Formula/Molecular Weight C28H480; MW

-- 4 0 0 . 3 7 0 5 2 _ ?

HO General Characteristics Crystals from acetone; mp., 157-158~ [ ~ ] D 23 - 3 3 ~ (22.5mg, in 5ml CHCI3). Acetate: C30H5002, crystals from alcohol; mp., 137-138~ [t~]D23 -35 ~ (28.8mg, in lml CHCI3). Fungal Source

Allomyces macrogynus, Fusarium sporotrichioides, Glomus mosseae, Hyphochytrium catenoides, ~izidiomyces apophysatus, Stachybotrys alternans, Spizellomyces punctatum, Monoblepharella sp., Acaulospora laevis, and Physarum polycephalum.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data UV: End absorption. 13C NMR: (CDCI3) C-15, 24.4; C-16, 28.3; C-17, 56.2; C-18, 11.9; C-20, 36.0; C-21, 18.8; C22, 33.8; C-23, 30.4; C-24, 38.9; C-25, 32.5; C-26, 20.3; C-27, 18.3; and C-28, 15.4ppm. (Skeletal carbon chemical shitis essentially similar to cholesterol). Mass Data: 400(M+), 385, 382, 367, 315, 289, 274, 273,261,255, 246, 229, 231, and 213re~e; C 83.93%. H 12.08%, O 3.99%.

28

2.

C28 Sterols

References M. Lenfant, M. F. Lecompte, and G. Farrugi; Identification des St6rols de Physarum polycephalum; Phytochemistry, Vol. 9, pp. 2529-2535(1970). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2.

C28 Sterols

29

Common/Systematic Name Brassicasterol (24R)24-Methylcholesta-5,22-dien-313-o1 Molecular Formula/Molecular Weight C2sH460; MW = 398.35487 |e,,

.o.i Fungal Source

Aspergillus oryzae, Cladonia gonecha, Stereocaulon tomentosum, Trichophyton rubrum, Tuber melanosporum, T. brumale, Terfezia sp., Protomyces sp., Gnomonia leptostyla, Clavicepspurpurea, and C. fusiformis. Widespread in the genera Protomyces and Taphrina.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data 13C N M R :

(CDCI3) C-15, 24.4; C-16, 28.6; C-17, 56.1; C-18, 12.1; C-20, 40.2; C-21, 21.1; C-22, 135.8; C-23, 131.8; C-24, 42.9; C-25, 33.2; C-26, 20.0; C-27, 19.7; and C-28, 17.7ppm. Mass Spectrum: Acetate derivative; 440(M+), 425, 397, 380(100), 365, 337, 315, 313,273,255(47), 253, and 213m/e (13). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983).

30

2.

C28Sterols

J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2. C28Sterols

31

Common/Systematic Name Campestanol (24R)-24-Methylcholestan-313-oi Molecular Formula/Molecular Weight C28H500; MW = 402.38617

HO~

H

Fungal Source

Physarumpolycephalum.

Isolation/Purification See R. C. Heupel: Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, pp. 1-27(1989). Soectral Data UV: End absorption. Mass Spectrum: LREIMS: (acetate) 444(M +, 35%), 429(M + - methyl, 7), 384(NY - ROH, 34), 369(M +- Me - ROH, 23), 317(M § side chain), 275(NY- side chain -42, 28), 257(M + side chain- ROH, 16), and 215role (M§ side chain - ROH-42). References M. Lefant, M. F. Lecompte, and G. Farugia; Identification Des St&ols de Physarum polycephalum; Phytochemistry, Vol. 9, pp. 2529(1970). W. B. Turner and D. C. Aldridge; Fungal Metabolites .II; Academic Press Inc., New York, New York, p. 308(1983).

32

2.

C28 Sterols

Common/Systematic Name Ergostanol (24S)24-Methylcholestan- 3 [3-ol Molecular Formula/Molecular Weight CzsHs00; MW = 402.38617

HOr

H

General Characteristics Crystals; mp., 144-145~ [tg]D20 +15.9 ~ (c=1.8, in CHCI3); acetate: C30H5202; mp., 145~ [tt]D2~+6 ~ (C=1.8, in CHCI3); benzoate: C35H5402; mp., 163-165~ Fungal Source

Physarum polycephalum and P. flavicomum.

Isolation/Purification See R. C. Heupel: Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.); Academic Press Inc., New York, NY, pp. 1-27(1989). References E. Bullock and C. J. Dawson; Sterol Content of the Myxomycetes Physarum polycephalum and P. flavicomum; J. Lipid Res., Vol. 17, p. 565(1976). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press Inc., New York, New York, 631 pp.(1983).

2. C2a Sterols

33

Common/Systematic Name (24S)24-Methylcholest-8-en-3 [3-ol; Ergost-8-en-3 [3-ol Molecular Formula/Molecular Weight C2sHasO; MW = 400.37052

H O ~ _

H

General Characteristics Benzoate, plates from chloroform-methanol; mp., 152-155~ [a]D + 33.8 ~ (C=0.60, in CHCI3) (mp., 147-148~ [a]D + 34~ Fungal Source Candida albicans, C. utilis, Torulopsis glabrata, and Saccharomyces cerevisiae. Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether-soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254TLC followed by silver nitrate-impregnated TLC. Spectral Data ~H NMR: (CDCI3) 0.63(3H, s, 13[3-Me); 1.01(3H, s, 10[3-Me); and 4.9ppm (1H, m, 3a-H). Mass Data: Benzoate: LREIMS: 504(M+, 100%), 489, 382, 377, 367, 335, 289, 255, 229, and 213m/e; found: C, 83.3%; H, 10.4; calcd for C35H5202; C, 83.3; H, 10.4. References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc.,Trans Perkin I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc.,Perkin Trans I, pp. 88-92(1975).

34

2.

C28 Sterols

Common/Systematic Name (24S)24-Methylcholesta-5,8-dien-313-ol; Ergosta-5,8-dien-313-ol Molecular Formula/Molecular Weight C2sH460; MW = 398.35487

General Characteristics Plates from acetone-methanol; mp., 119-122~ [ a ] D = 0.40 ~(c=0.2, in CHCI3); benzoate, plates from acetone-methanol; mp., 136-139~ [tt]D + 0.5 ~ (calcd + 2.0 ~ )(c=0.25, in CHCI3). Fungal Source Saccharomyces cerevisiae. Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether-soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254TLC followed by silver nitrate-impregnated TLC. Spectral Data (Benzoate) UV: Xmax 230nm (e = 10,630) IH N]VIR: (CDCI3) 0.67(3H, s, 1313methyl); 1.25(3H, s, 1013methyl); 4.90(IH, m, 3(~-H); and 5.45ppm (IH, m, 6-H).

Mass Data: LREIMS: 502(M+), 380(100%), 365, 253, and 251re~e; found: C, 83.8%; H, 10.0; calcd for C35H5oO2;free alcohol, 398(M+), 383,380, 365(100%), and 271m/e (M+side chain); found: C, 84.0; H, 11.5; calcd for C35H5oO2;C, 84.3; H, 11.6.

2.

C28 Sterols

35

Reference D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc.,Perkin Trans I, pp. 88-92 (1975).

36

2.

C28 Sterols

Common/Systematic Name 24-Methylene cholesterol 24-Methyicholesta-5,24(28)-dien-313-01 Molecular Formula/Molecular Weight C2sI-h60; MW = 398.35487 |

HO General Characteristics Acetate crystallized from ethanol as plates; mp., 131-132~ Fungal Source

Achlya bisexualis, Glomus caledonius, Saprolegniaferax, S. megasperma, Leptolegnia caudata, Pythiopsis cymosa, and Rhizophydium sphaerotheca.

Isolation/Purification The mycelium from Achlya bisexualis was air dried, saponified with ethanolic KOH, and the lipids were extracted with hexane. The non-saponifiable material was chromatographed on alumina (activity III) and squalane and other hydrocarbons eluted with light petrol. Further elution with diethyl ether gave a sterol fraction which was subsequently acetylated and separated by preparative TLC on silica gel-impregnated with silver nitrate. Also, see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV:

End absorption. 13C R :

(CDCI3) (Acetate derivative) C-15, 24.3; C-16, 28.2; C-17, 56.0; C-18, 11.9; C-20, 35.8; C-21, 18.7; C-22, 34.7; C-23, 31.0; C-24, 156.7; C-25, 33.8; C-26, 21.9; C-27, 22.0; and C-28, 105.9ppm. (Skeletal carbon chemical shit, s essentially similar to cholesterol).

2.

C28 Sterols

37

TLC Data TLC was carried out on silica gel G plates impregnated with AgNO3 and rhodamine 6G. Plates were developed with benzene. Re for 24-methylenecholesteryl acetate was 0.470.51. GC Data GC was performed using 1% QF-1.24-Methylenecholesteryl acetate had relative retention time of 1.28 (relative to cholesteryl acetate). References M. Lenfant, M. F. Lecompte, and G. Farrugia; Identification des St~,rols de Physarum polycephalum; Phytochemistry, Vol. 9, pp. 2529-2535(1970). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, pp. 341 (1989). C. R. Popplestone and A. M. Uurau; Major Sterols

ofAchlya bisexualis; Phytochemistry,

Vol. 12, pp. 1131-1133(1973). W. B. Turner and D.C. Aldridge; Fungal Metabolite.s II; Academic Press Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi; Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

38

2.

C28 Sterols

Common/Systematic Name Fecosterol; Ergosta-8,24(28)-dien-313-ol 24-Methylcholesta- 8,24(28)-dien-313-ol Molecular Formula/Molecular Weight C2sH460; MW = 398.35482

HO

R

General Characteristics Plates from chloroform-methanol; mp., 126-130~ [~]D 24 q- 44.9 ~ (c=0.64, in CHCI3); benzoate: plates from acetone-methanol; mp., 127-128~ mp., 127-128~ + 43.2 (c=0.40) (mp., 127-129~ [tX]D+ 39.9~ acetate: plates from acetone-methanol; mp., 136-138~ [t~]D + 34.7 ~ Fungal Source

Botrytis cinerea, Candida albicans, C. utilis, Lobaria pulmonaria, L. scobiculata, Neurospora crassa, Pichia sp., Saccharomyces carlsbergensis, S. cerevisiae, Torulopsis glabrata, and Usnea longissima.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data UV: End absorption. IR:

(KBr) 3600, 1640, and 890cm ~. Benzoate: 1710, 1640, and 890cm~. Acetate: 1710, 1640, and 890cm"~. IH NMR: (CDCI3) 5.35(2H, m, 28-H); 6.4(1H, m, 3tt-H); 8.99(3H, d, J=7.0Hz, 26- and 27-Me); 9.05(3H, s, 10-Me);.and 9.391: (3H, s, 13-Me); benzoate: 5.1(1H, m, 3a-H); 5.3(2H,

2. C28 Sterols

39

m, 28-H); 9.00(6H, d, J=8Hz, 26- and 27-Me); 9.02(3H, s, 10-Me); 9.38(3H, s, 13Me); and 2.0 and 2.5z (5H, m, aromatic-H). Acetate: 5.31(1H, m, 28-H); 5.35(1H, m, 3a-H); 8.0(3H, s, Ac); 8.98(6H, d, J=7.0Hz, 26- and 27-Me); 9.03(3H, s, 10-Me); and 9.38~ (3H, s, 13-Me). Mass Spectrum: (Acetate derivative) 440(M+, 100%), 425,380(64), 365, 356(7), 341,315(10), 313(49), 296(3), 281,273(32), 255(31), 253(30), and 213m/e (100). References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc.,Perkin Trans I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc.,Perkin Trans I, pp. 88-92(1975). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II, Academic Press, New York, New York, 314 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

40

2.

C28 Sterols

Common/Systematic Name Fungisterol; Ergost-7-en-313-ol (24S)24-Methylcholest-7-en-313-01 Molecular Formula/Molecular Weight C2sII4sO;MW = 400.37052

R HO

H

Fungal Source

Agaricus bisporus, Armillaria mellea, Boletus luridus, Candida albicans, C. utilis, C. tropicalis, Coriolus pargamenus, C. versicolor, Cronartium fusiforme, Cryptoderma citrinum, Fomitopsis pinicola, Gloeophyllum sepiarium, Glomus mosseae, Grifola frondosa, Hymenomycetes sp., Lenzites trabea, Linderina pennispora, Neurospora crassa, Penicillium claviforme, Pholiota aegerita, Phycomyces blakesleeanus, Puccinia graminis var. triticil, P. striiformis, Rhizopus arrhizus, Russula senecis, Saccharomyces cerevisiae, Torulopsis glabrata, Ustilago maydis, and U. nuda. The only C2s sterol detected in rusts and many Basidiomycetes.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). _Spectral Data UV: End absorption. IH NIV[R:

(CDCI3) 0.53;0.80(each 3H, s,H3-18, -19);0.78(3H, d, J=5.2Hz); 0.83(3H, d, J=5.51--Iz);0.86(3H, d, J=5.5I-Iz);0.92(3H, d, d=6. II-Iz);3.60(IH, m, H-3); and 5.18ppm (IH, m, H-7).

13CNMR.: (CDCI3) I 1.8(q,C-18); 13.0(q,C-19); 15.4(q,C-28); 17.6(q,C-27), 20.5(q,C-26); 19.0(q, C-21); 21.5(t, C-11); 22.9(t, C-15); 27.9(t, C-16); 29.6(t, C-6); 29.6(t, C-23);

2. C28 Sterols

41

30.7(t, C-2); 31.5(d, C-25); 33.6(t, C-22); 34.2(s, C-10); 36.6(d, C-20); 37.1(t, C-I); 38.0(t, C-4); 39.0(d, C-24); 39.5(t, C-12); 40.2(d, C-5); 43.3(s, C-13); 49.4(d, C-9); 55.1(d, C-14); 56.0(d, C-17); 71.0(d, C-37); 117.4(d, C-7); and 139.6ppm (s, C-8). Mass Spectrum: Acetate derivative: 442(M +, 72%), 427(M + - Me, 15), 382(M + - AcOH, 36), 353(8), 315(11), 288(8), 273(16), 255(100), 229(26), and 213role (67); benzoate: 504(M+), 489, 382, 377, and 255m/e. References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids, Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc.,Perkin Trans I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids, Part X. The Sterols of Some Yeasts Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc.,Perkin Trans I, pp. 88-92(1975). H. Fujimoto, K. Maeda, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum III. Isolation and Structures of Three New Glycosides, Hebevinosides XII, XIII and XIV and Productivity of the Hebevinosides at Three Growth Stages of the Mushroom; Chem. Pharm. Bull., Vol. 39, pp. 1958-1961(1991). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 312 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi; Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

42

2.

C28 Sterols

Common/Systematic Name ( 24S)24-M ethylcholest a- 5,7,14-trien-313-ol Molecular Formula/Molecular Weight C28I-I440; M W "- 3 9 6 . 3 3 9 2 2 "5

Fungal Source

Rhizopus arrhizus.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2.

C28 Sterols

43

Common/Systematic Name Ergosterol Ergosta-5,7,22-trien-313-ol Molecular Formula/Molecular Weight C 2 8 H 4 4 0 ; M W "- 396.33922

General Characteristics Small hydrated plates from alcohol; hydrated needles from ether. The best crystallized form contains 1.5 moles ofH20; mp., 168~ Complete removal of water is almost impossible and results in an amorphous mass; melting range 166-183 ~ [tt]Dz~ -13 5 ~ (C=1.2, in CHC13 calcd as anhydrous). Practically insoluble in water. One gram dissolves in 660ml alcohol, in 45 ml boiling alcohol, in 70ml ether, in 39ml boiling ether, in 3 lml chloroform. Precipitated by digitonin. Affected by light and air; turns yellow. Oxygen forms peroxides and hydrogen may form polyhydro compounds. Acetate, C30H46Oz; mp., 179~ clear at 181~ Benzoate, C3sHasO2; plates or needles; mp., 169-171~ [a]D23 -71 o (C= 1.1, in CHC13). Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Fungal Source The major fungal sterol in many fungal species. The major sterol of most species of the family Mucoraceae, most Ascomycetes, and with the exception of rusts, most Basidiomycetes.

Agaricus campestris, Alternaria alternata, A. brassicicola, A. kikuchiana, A. tenuis, Amoebidium parasiticum, Amelaria mellea, Aspergillus niger, Boletus luridus, B. spp., Candida albicans, C. guilliermondii, C. utilis, Candida sp., Cantharellus cibarius, Corioluspargamenus, C. sangamenus, C. versicolor, Cryptoderma citrinum, Cyathus helenae, Fomes allardii, F. annosus, Fomitopsis cytisina, F. pinicola, F. pubertatis, Fusarium sporotrichioides, Ganoderma applanatum, Geotrichum flavo-brunneum, Gloeophyllum sepiarium, Glomus mosseae, Gnomonia leptostyla, Grifola frondosa, G. umbellata, Hemispora stellata, Leccinum aurantiacum, Leccinum spp., Lentinus lepideus,

44

2.

C28 Sterols

Lenzites trabea, Leptosphaeria typhae, Lobaria pulmonaria, L. scobiculata, Microporus flabelliformis, Monilinia fructigena, Mucor pusillus, Nectria galligena, Neurospora crassa, Penicillium claviforme, Phellinus gilvus, P. igniarius, P. robustus, Pholiota aegerita, Phycomyces blakesleeanus, Pichia sp., Polyporus cretaceous, P. dryadeus, Poria cocos, Rhizopus arrhizus, Saccharomyces cerevisiae, Scleroderma aurantium, Sclerotinia fructicola, Smittium sp., Spicaria elegans, Suillus bovinus, S. variegatus, Torulopsis glabrata, Trametes feei, T. lilacino-gilva, Usnea longissima, Ustilago maydis, U. nuda, and Xanthoria parietina. Biological Activity When irradiated with UV light, ergosterol develops powerful vitamin DI (q.v.) activity. Spectral Data UV: ~, EtOH 262, 271 282, and 293nm. max

13CN M R : (CDCI3) C-l, 38.5; C-2, 32.1; C-3, 70.5; C-4, 40.9; C-5, 139.8; C-6, 119.7; C-7, 116.4; C-8, 141.3; C-9, 46.3; C-10, 37.1; C-I I, 21.1; C-12, 39.2; C-13, 42.9; C-14, 54.6; C-15, 23.0; C-16, 28.3; C-17, 55.9; C-18, 12.1; C-19, 16.3; C-20, 40.4; C-21, 21.2; C-22, 135.6; C-23, 132.1; C-24, 42.9; C-25, 33.1; C-26, 22.0; C-27, 19.7; and C28, 17.6ppm. Mass Spectrum: 396(M +, 72%), 381(7), 378, 363(100), 271,253,229, 227, 211,158, and 143m/e. Acetate derivative; 438(M +, 9%), 423, 378(100), 363, 313,253, 211, and 143role. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2. C28 Sterols

45

Common/Systematic Name (24S)24-Methylcholesta-5,7-dien-3 [3-ol; Ergosta-5,7-dien-313-ol Molecular Formula/Molecular Weight C2si-I460; MW = 398.35487

HO General Characteristics Crystals from chloroform-methanol; mp., 148-150~ [a]D - 73.6 ~ (C=0.41); acetate, crystals from acetone-methanol; mp., 153-155~ [a]D- 87.6 ~ (C=0.45) (mp., 157-158~ [a]D- 74.8~ Fungal Source Found in several Hymenomycetes species. Agaricus bisporus, Amelaria mellea, Aspergillusparasiticus, Boletus luridus, B. spp., Candida albicans, C. utilis, Coriolus

pargamenus, C. sangamenus, C. versicolor, Daedalea quercina, Leccinum aurantiacum, L. spp., Microporus flabelliformis, Mucor pusillus, Pholiota aegerita, Phycomyces blakesleeanus, Pichia sp., Piptoporus betulinus, Polyporus paragamenus, Saccharomyces cerevisiae, S. carlsbergensis, Schizophyllum commune, Suillus bovinus, S. variegatus, Torulopsis glabrata, Tremella fuciformis, and Ustilago maydis.

Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254 TLC followed by silver nitrate-impregnated TLC. Spectral Data UV:

(acetate) ~.=~ 262(e = 8,400), 272(12,400), 282(13,600), and 294nm (7,400). Mass Data:

(benzoate) Found: C, 84.1; H, 9.6; calcd for C35H4gO2; C, 83.9; H, 9.7%.

46

2.

C28 Sterols

References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc., Trans Perkin I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc., Perkin Trans I, pp. 88-92(1975).

2. C28 Sterols

47

Common/Systematic Name 5,6-Dihydroergosterol; Ergosta-7,22-dien-313-ol 24-Methylcholesta-7, 22-dien-313-ol Molecular Formula/Molecular Weight C28H460; M W -- 398.35487

HO

Fungal Source

Acremonium coenophialum, Amelaria mellea, Aspergillus fennelliae, A. oryzae, Boletus luridus, B. spp., Candida albicans, C. utilis, C. tropicalis, Candida sp., Coriolus pargamenus, C. versicolor, Cryptoderma citrinum, Daedalea quercina, Flammulina velutipes, Fomes annosus, F. fomentarius, Fomitopsis cytisina, Gloeophyllum sepiarium, Leccinum aurantiacum, Leccinum spp., Hymenomycetes sp., Leptosphaeria typhae, Neurospora crassa, Phellinus gilvus, Pholiota aegerita, Phycomyces blakesleeanus, Piptoporus betulinus, Rhizopus arrhizus, Saccharomyces cerevisiae, Suillus bovinus, S. variegatus, Torulopsis glabrata, and Xanthoria parietina. Many Basidiomycetes.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV;

End absorption. IH NMR: (CDCI3) 0.54.0.80(each 3H, s, 1-/3-18, -19); 0.78(3H, d, J=6.7Hz); 0.82(3H, d, J=6.7Hz); 0.91(3H, d, J=7.0Hz); 1.02(3H, d, J=6.7Hz); 3.60(1H, m, H-3); and 5.18ppm (3H, m, H-7, -22, -2). 13C NMPx:

(CDCI3) 12.1(q, C-18); 13.0(q, C-19); 17.6(q, C-28); 19.6(q, C-26); 19.9(q, C-27); 21.1(q, C-E1); 21.5(t, C-11); 22.9(t, C-15); 28.1(t, C-16); 29.6(t, C-6); 31.5(t, C-2);

48

2.

C28 Sterols

33.1(d, C-25); 34.2(s, C-IO); 37.1(t, C-l); 38.0(t, C-4); 39.4(t, C-12); 40.2(d, C-5); 40.4(d, C-2); 42.8(d, C-24); 43.3(s, C-13); 49.4(d, C-9); 55.0(d, C-14); 56.0(d, C-17); 71.O(d, C-3); 117.4(d, C-7); 131.9(d, C-23); 135.6(d, C-22); and 139.6ppm (s, C-8). Mass Spectrum: EIMS: (acetate derivative)440(M +, 60%), 425(15), 397, 380(9), 365(9), 337, 315, 313(100), 273(12), 255(81), 253(15), and 213m/e (23). TLC Data Rf 0.33 with silica gel plates developed with benzene-diethyl ether (9:1, v/v). I-IPLC Data Relative retention time was 0.86 (relative to cholesterol) performed with C~s reversed phase column eluted with 4% aqueous methanol at ambient temperature with a flow rate of 1.6ml/min. GLC Data Relative retention time was 1.26 relative to cholesterol using 3% SE-30 at 245~ References H. Fujimoto, K. Maeda, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum III. Isolation and Structures of Three New Glycosides, Hebevinosides XII, XIII and XIV and Productivity of the Hebevinosides at Three Growth Stages of the Mushroom; Chem. Pharm. Bull., Vol. 39, pp. 1958-1961 (1991). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungai Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2.

C28 Sterols

49

Common/Systematic Name Episterol Ergosta-7,24( 28)-alien-313-ol Molecular Formula/Molecular Weight C 2 8 H 4 6 0 ; M W -" 398.35487

H O ~

H

Fungal Source Amanita caesaria, Coprinus atramentarius, Flammulina velutipes, Hygrocybe punicea, Lampteromycesjaponicus, Lentinus edodes, Leucopaxillus giganteus, Russula foetens, R. nigricans, R. senecis, Stachybotrys atra, Dictyuchus monosporus, Agaricus campestris, Aspergillus oryzae IFO 4290 (occurs as acylsterol with linoleic acid), Candida albicans, C. utilis, C. tropicalis, Inocybe macrosperma, Leptosphaeria typhae, Lobaria pulmonaria, L. scobiculata, Nectria galligena, Neurospora crassa, Phycomyces blakesleeanus, Saccharomyces cerevisiae, Torulopsis glabrata, Usnea longissima, and Xanthoria parie tina. Isolation/Purification The non-saponifiable lipid extract was purified by TLC on silica gel G developed with ethanol-chloroform (1:99, v/v) to give crude sterol fractions. These were acetylated, purified on TLC with silica gel G developed with cyclohexane-benzene (1:1, v/v) and analyzed by gas chromatography and gas chromatography-mass spectroscopy using 1% SE-30. Further purification was achieved with 10% AgNO3-silica gel G TLC developed with cyclohexane-benzene (1:1, v/v) or alumina column chromatography (neutral grade III) using increasing amounts of ethyl ether in petroleum ether. See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes, and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV: End absorption.

50

2.

C28 Sterols

Mass Spectrum: LREIMS: (Acylsterol with linoleic acid) 660(M +, 12.5%), 578, 563, 535(M + - side chain, 15.2), 533,381(M + - RCOOH, 100), 380(27.3), 297, 255, 253, 228, and 213m/e. References J. R. Lenton, L. J. Goad, and T. W. Goodwin; Sterols of the Mycobiont and Phycobiont Isolated From The Lichen Xanthoria parietina; Phytochemistry, Vol~ 13, pp. 22492253(1973). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). M. Ohnishi and Y. Fujino; Principal Molecular Species of Acylsterols in Aspergillus oryzae; Agric. Biol. Chem., Vol. 44, pp. 681-683(1980). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2.

C28 Sterols

51

Common/Systematic Name 24-Methylcholesta- 5,7,24(28)-t rienol Ergo sta-5,7,24(28)-trien-313-ol Molecular Formula/Molecular Weight C2sI-LuO; MW = 396.33922

.o.2 Fungal Source

Candida albicans, C. utilis, Phycomyces blakesleeanus, Leptosphaeria typhae, Saccharomyces cerevisiae, and S. carlsbergensis.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV: Typical 5,7-diene absorption. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

52

2.

C28 Sterols

Common/Systematic Name (24S)24-Methylcholesta-8,14,22-trien-313-ol; Ergosta-8,14,22-trien-313-ol Molecular Formula/Molecular Weight C25I--I440; M W -- 396.33922

HOe'S= H General Characteristics Benzoate, plates from chloroform-methanol; mp., 140-142~

[tt]D- 30.3 ~ (C=0.89).

Fungal Source

Saccharomyces cerevisiae.

Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether-soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254TLC followed by silver nitrate-impregnated TLC. Spectral Data (Benzoate) UV:

~,m~x 230(E = 26,200) and 252nm sh (22,000). Mass Data: Found: C, 84.1; H, 9.6; calcd for C35H4802;C, 83.9; H, 9.7%. References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and their Relationship to the Biosynthesis of Ergosterol; J. Chem. So% Trans Perkin I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc., Trans Perkin I, pp. 88-92(1975).

2.

C28 Sterols

53

Common/Systematic Name Ergost-5-en-313-ol (24S)24-Methylcholest-5-en-313-o1 Molecular Formula/Molecular Weight C2sH4sO; MW = 400.3 7052

HO Fungal Source

Physarum polycephalum, P. flavicomum, Hyphochytrium catenoides, Trichophyton mentagrophytes, and Fusarium culmorum.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV: End absorption. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

54

2.

C28 Sterols

Common/Systematic Name Lichesterol (24S)24-Methylcholesta-5,8,22-trien-313-ol; Ergosta-5,8,22-trien-313-ol Molecular Formula/Molecular Weight C28H440; MW = 396.33922

.o.I General Characteristics Acetate, plates from acetone-methanol; mp., 138-140~ [~]D" 40.2 ~ (c=0.39); benzoate, needles from chloroform-methanol; mp., 153-155~ [t~]D- 6.1 ~ (C=0.46). Fungal Source Candida albicans, Flammulina velutipes, Leptosphaeria typhae, Lobaria pulmonaria, L. scobiculata, Nectria galligena, Neurospora crassa, Saccharomyces cerevisiae, Usnea longissima, and Xanthoria parietina. Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether-soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF2s4 TLC followed by silver nitrate-impregnated TLC. Also, see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data Mass Spectrum: Acetate derivative, LREIMS: 438(M+, 7%), 423,378(70), 363(100), 313,253(48), 251, 211(18), and 337m/e (10). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983).

2.

C28 Sterols

55

J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

56

2.

C28 Sterols

Common/Systematic Name 22,23-Dihydroergosterol; Ergosta-5,7-dien-313-ol Molecular Fo.rmul~olecular Weight C28H460; MW = 398.35487 IIIItl .

HO.J Fungal Source Found in several Hymenomycetes species. Agaricus bisporis, Amelaria mellea, Aspergillus parasiticus, Boletus luridus, B. spp., Candida albicans, C. utilis, Coriolus

pargamenus, C. sangamenus, C. versicolor, Daedalea quercina, Leccinum aurantiacum, L. spp., Microporus flabelliformis, Mucor pusillus, Pholiota aegerita, Phycomyces blakesleeanus, Pichia sp., Piptoporus betulinus, Polyporus paragamenus, Saccharomyces cerevisiae, S. carlsbergensis, Schizophyllum commune, Suillus bovinus, S. variegatus, Torulopsis glabrata, Tremella fiwiformis, and Ustilago maydis.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV:

Typical 5,7-diene system (see ergosterol). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabo!ites II.; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

2.

C28 Sterols

57

Common/Systematic Name (24S)24-Methyl- 5tt-cholesta-7,16-dien-313-ol Molecular Formula/Molecular Weight C 2 8 H 4 6 0 ; M W -" 398.35487

H, H General Characteristics Crystals from methanol; mp., 166-169.5 ~C; acetate; mp., 168-170 ~ Fungal Source Ganoderma applanatum = Fomes applanatus. Isolation/Purification The ground fruit bodies of G. applanatum were extracted with methanol, evaporated to dryness and chromatographed on aluminum oxide (activity II) followed by chromatography on silica gel impregnated with silver nitrate using a gradient from hexane to ethyl acetate (2:1, v/v). The final product was crystallized from methanol. Spectral Data ~H NMR: (CDCl3) 0.76(s, 19-CH3); 0.56(s, 18-CH3); and 5.08-5.2ppm (m, =CH). Mass Spectrum: LREIMS: 398(M +) and 269m/e (base peak). References L. I. Strigina, Yu. N. Elkin, and G. B. Elyakov; Steroid Metabolites of Ganoderma applanatum Basidiomycete; Phytochemistry, Vol. 10, pp. 2361-2365(1971). W. B. Turner and D.C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, New York, 631 pp. (1983).

58

2.

C28 Sterols

Common/Systematic Name Ergosta-4,6, 8(14),22-tetraen-3-one Molecular Formula/Molecular Weight C25H400; MW -- 392.30792

General Characteristics Crystals; mp., 114-115 ~C; highly fluorescent. Fungal Source Alternaria alternata, Aspergillus flavus, A. fumigatus, A. ochraceus, Candida utilis, Fomes officinalis, F. applanatus, Fusarium moniliforme, Lampteromycesjaponicus, Penicillium citrinum, P. patulum = P. griseofidvum, P. rubrum, and P. islandicum. Isolation/Purification Fungal cultures were extracted twice with methanol, saponified with ethanolic KOH, and extracted with n-hexane. The n-hexane extracts were reduced in volume, applied to a silica gel 60 column eluted in succession with hexane, ethyl acetate-hexane (5:95, v/v), and ethyl acetate-hexane (10:90, v/v). Ergosta-4,6,8(14),22-tetraen-3-one purification monitored with long-wave UV light was eluted in the 5% ethyl acetate-hexane fraction and recrystallized from petroleum ether. Soectral Data UV: /~ EtOH max

351(1og e 25,800), 282(5,800), and 239nm (4,100).

IR:

(KBr) 2940, 2860, 1650, 1562, 1445, 1347, 1320, 1265, 1217, 1191, 964, 946, 872, and 758cm "~. 13C NMR: (CDCI3) 16.64; 17.62; 18.97; 19.44; 19.64; 19.96; 21.21; 25.34; 27.65; 33.05; 33.91; 34.09; 35.59; 36.70; 39.19; 42.82; 43.93; 44.31; 55.66; 122.83; 124.29; 132.35; 133.72; 155.73; 163.97; and 198.97ppm.

2.

C28 Sterols

59

Mass Spectrum: LREIMS: 392(M § 21%), 268(100), 267(55), 242(5), and 214role (22). TLC Data Brinkman SIL G-HR-25 precoated plates were activated for 1 hr at 110 ~ C; solvent systems were chloroform-acetone (88:12, v/v) (Rf 0.87) and benzene-acetone (90:10, v/v) (Rf 0.68). The metabolite was detected as a blue fluorescent spot with UV light. HPLC Data SIL-X-I column, mobile phase was isooctane-ethyl acetate (92.5:7.5, v/v) at 2.5ml/min flow rate; detection at 350nm and elution at 2.5min. References R. G. Cooks, R. D. Daftary, and Y. Pomeranz; Changes in Mold-Damaged Wheat Flours Stored at Various Temperatures; J. Agric. Food Chem., Vol. 18, pp. 620-623(1970). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, New York, p. 341 (1989). L. M. Seitz and J. V. Paukstelis; Metabolites ofAlternaria alternata: Ergosterol and Ergosta-4,6,8(14),22-tetraen-3-one; J. Agric. Food Chem., Vol. 25, pp. 838-841(1977). W. B. Turner and D.C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, New York, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi; Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

60

2.

C28 Sterols

Common/Systematic Name Ergosta-8(9), 14-dien-313-ol; Ignosterol (24S)24-Methylcholesta-8(9), 14-alien-313-ol Molecular Formula/Molecular Weight C28H460; MW = 398.35487

.o.i General Characteristics Crystals from acetone-MeOH; mp., 133-134~

[a]D + 11 ~ (C=1%, in CHCI3).

Fungal Source Saccharomyces cerevisiae strain 3701B and Chlorella ellipsoidea. Isolation/Purification Ignosterol was isolated from the neutral lipid fraction by column chromatography using Woelm grade II alumina and eluting with increasing amounts of ethyl ether in n-hexane. The sterol fraction was acetylated and further chromatographed by argentation chromatography eluting with increasing amounts of ethyl ether in n-hexane. Soectral Data _

UV~ ~, M~.m~ 25 lnm (e = 18,000). IR;

(KBr) 3050 and 795cm"~. IH NMR: The C-18 and C-19 methyl resonances appeared at 0.82 and 0.99ppm, respectively, which is in good agreement with the calculated shitts of 0.83 and 0.94ppm and compares favorably to those of ergosta-8,14,22-trien-313-yl benzoate (0.85 and 0.94ppm). A broad one-proton absorption appeared at 5.33ppm for the olefinic hydrogen of the 8, 14 conjugation.

2.

C28 Sterols

61

Mass Spectrum: LREIMS: 398(M+), 383,365, and 371m/e. GC Data (Acetate derivative, relative retention time (RRT) relative to cholesteryl acetate) QF-1, RRT 1.24; Hi-Eff-8-BP, 1.44; PMPE, 1.38; and SE-30, 1.32. References L. G. Dickson, G. W. Patterson, C. F. Cohen, and S. R. Dutky; Two Novel Sterols from Inhibited Chlorella ellipsoidea; Phytochem., Vol. 11, pp. 3473-3477(1972). P. R. Hays, L. W. Parks, H. D. Pierce, Jr., and A. C. Oehlschlager; Accumulation of Ergosta-8,14-dien-313-ol by Saccharomyces cerevisiae Cultured with an Azasterol Antimycotic Agent; Lipids, Vol. 12, pp. 666-668(1977). P. R. Hays, W. D. Neal, and L. W. Parks; Physiological Effects of an Antimycotic Azasterol on Cultures of Saccharomyces cerevisiae; Antimicrobial Agents and Chemotherapy, Vol. 12, pp. 185-191(1977).

62

2.

C28 Sterols

Common/Systematic Name (24S)24-Methylcholesta-8,22-dien-313-ol; Ergosta-8(9),22-dien-313-ol Molecular Form_ula/Molecular Weight C2sH460; MW = 398.35487

HO

General Characteristics Free alcohol, lathes from acetone; rap., 165-168~ [a]D + 27.6 ~ (C=0.51, in CHCI3) (mp., 166-169~ [et]D + 30~ Benzoate: plates from chloroform-methanol; mp., 152-153~ [tt]D + 26.9 ~ (C=0.42, in CHCI3) (mp., 147-148~ [t~]D+ 34~ Fungal Source Candida albicans, C. utilis, Neurospora crassa, Saccharomyces cerevisiae, and Torulopsis glabrata. Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol removed m vacuo, and ether soluble material converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254TLC followed by silver nitrate-impregnated TLC. The yeast cells were saponified by the alkaline pyrogaUol procedure as modified for large volumes. The nonsaponifiable fraction was extracted into hexane and recovered by evaporating the solvent under nitrogen. The sterols were dissolved in ethanol, precipitated with digitonin, recovered by dimethyl sulfide cleavage and recrystallization from ethanol. The partially purified sterols were then separated by thin-layer chromatography on AgNO3-silica gel G. To obtain large quantities of the sterol for detailed analyses by various spectroscopic procedures, the sterols were recrystallized three times from alcohol atter digitonin precipitation. The recovered product was streaked onto plates of silica gel G (1.0mm), and the plates were developed in benzene-ethyl acetate (5:1, v/v). The sterol band was scraped from the plates and eluted with CHCI3. Spectral Data IH NMR: (CDCI3) The spectrum showed the presence of two olefinic hydrogens (broadened triplet) at 5.14ppm and a sirigle hydrogen on a carbon bearing oxygen as a broadened

2.

C28 Sterols

63

multiplet at 3.56ppm. In addition, there were clearly six distinct methyl resonances, with singlets at 0.54(C-18) and 0.79ppm (C-19). There were doublet methyls due to C21, C-26, C-27, and C-28 at 0.81(J=7), 0.82(J=7), 0.90(J=6), and 1.00ppm (d=6Hz). In particular, the nonidentity of the C-26 and C-27 methyl groups required the presence of an asymmetric center at C-24. Significant parts of the spectrum were similar to that of ergosterol. In particular, ergosterol showed its 22-23 olefinic hydrogens as a broad ended triplet at 5.15ppm and four secondary C-methyl groups as doublets centered at 0.82, 0.83, 0.90, and 1.01ppm. This clearly defined the nature of the side chain as being identical with that of ergosterol. Mass Data: Benzoate: Found: C, 83.6%; H, 10.1; calcd for C35H5202;C, 83.6; H, 10.0. The mass spectrum of the free alcohol showed a molecular ion at 398m/e (C2sH460) with significant fragment peaks at 383m/e (M+ - CH3), 380(M + - H20), 365(M+ - CH3 and -H20), 273(M + - side chain), and 255(M +- side chain and - H20). References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc., Trans Perkin I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc., Trans Perkin I, pp. 88-92(1975). L. W. Parks, F. T. Bond, E. D. Thompson, and P. R. Starr; AS(9)'22-Ergostadiene-313-ol, an Ergosterol Precursor Accumulated in Wild-type and Mutants of Yeast; J. Lipid Res., Vol. 13, pp. 311-316(1972).

64

2.

C28 Sterols

Common/Systematic Name (24S)24-Methylcholesta-7,22,24(28)-trien-313-ol; Ergosta-7,22,24-trien-313-ol Molecular Formula/Molecular Weight C28H440; MW = 396.33922

HO

H

General Characteristics Crystals from acetone-methanol; 119-121~ [a]D 25 + 36.5 ~ (c=0.85, in CHCI3). Benzoate, crystals from chloroform-methanol; mp., 139-141~ [a]D26 + 13.5~ (C=1.56, in CHCI3). Acetate, plates from chloroform-methanol; 133-136~ [a]D24 + 6.4 ~ (C=0.85, in CHCI3). Fungal Source

Saccharomyces cerevisiae, S. carlsbergensis, Candida albicans, C. utilis, and Leptosphaeria typhae.

Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether soluble material was chromatographed by alumina column chromatography, eluting with various combinations of benzene, acetone, light petroleum ether or methanol. Alternatively, they were converted into benzoate derivatives and separated with preparative silica gel GF254 TLC followed by silver nitrate-impregnated TLC. Spectral Data UW: ~.m,x 232(e = 18,600), 226sh (18,300), and 240nm sh (12,800); benzoate, ~m~x 232nm (e = 33,800); acetate, ~.n~x 227sh (e = 22,100), 232(23,200), and 240nm sh (15,700). IR:

(KBr) 3400, 1640, 1600, and 890cm~; benzoate, 1710, 1640, 1600, and 890cm~; acetate, 1710, 1640, 1600, and 890cm"1.

2.

C28 Sterols

65

1H N-MR:

(CDCI3) 4.12(IH, d, J=16Hz, H-23); 4.50(IH, dd, J=8.0, 16Hz, H-22); 4.90(IH, m, H-7); 5.25(2H, m, H-28); 6.46(IH, m, H-3a); 7.52(IH, septet,J=7.0Hz, H-25); 8.98(6H, d, J=7.0Hz, 26- and 27- CH3); 9.23(3H, s, 10-Me); and 9.46z (3H, s, 13Me). Mass Data: Found: C, 84.5%; H, 11.0; calcd for C2sl-~O; C, 84.8; H, 11.2; benzoate, found: C, 83.7; H, 9.4; calcd for C3sHasO2; C, 83.9; H, 9.7; acetate, LREIMS: 438(M+), 423, 395, 378, 363, 354, 315, and 313m/e (100%); found: C, 82.0%; H, 10.6; calcd for C30H4602;C, 82.1; H, 10.6. References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J. Chem.Soc., Perkin Trans I, pp. 1326-1333(1974). D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenes. Part VI. The Sterols of Yeast; J. Chem. Soc.,Perkin Trans I, pp. 513-522(1972).

66

2.

C28 Sterols

.Commo_n/SystematicName (24S)24-Methylcholesta-8,14,24(28)-trien-313-ol; Ergosta-8,14,24-trien-313-ol Molecular Formula/Molecular Weight CzsI-I440; MW = 396.33922

_

H O ~ _

H

General Characteristics Benzoate, plates from chloroform-methanol; mp., 148-151~ [a]D- 4.1 ~ (c=0.38). Fungal Source. Saccharomyces cerevisiae. Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether-soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF254TLC followed by silver nitrate-impregnated TLC. Spectral Data (Benzoate) UV:

~.max 230(e = 25,800) and 251nm sh (21,400). Mass Data: LREIMS: 500(M+, 100%), 485, 457, 441,378, 375, 373,363,335, 279, 271,253, 251,239, 238, and 237m/e; found: C, 83.8; H, 9.5" calcd for C35H4sO2; C, 83.9%; H, 9.7. References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J.Chem. Soc., Trans Perkin I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Soc., Trans Perkin I, pp. 88-92 (1975).

2.

C28 Sterols

67

Common/Systematic Name Ergosta-8,22,24(28)-trien-313-ol Molecular Formula/Molecular Weight C28H440; M W -- 396.33922

Fungal Source Saccharomyces cerevisiae (SM36 mutant strain).

Isolation/Purification Yeast cells were refluxed in 10% methanolic KOH, methanol was removed in vacuo, and ether-soluble material was converted into benzoate derivatives. The benzoates were separated with preparative silica gel GF/s4 TLC followed by silver nitrate-impregnated TLC. Spectral Data 1H NMR: (CDCI3) 0.65(s, 1313-methyl); 1.02(s, 1013-methyl); and 1.04ppm (9H, d, 20-methyl and 25-methyls). Mass Spectrum: Benzoate, LREIMS: 500m/e (M+). References D. H. R. Barton, J. E. T. Corrie, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part IX. The Sterols of Some Mutant Yeasts and Their Relationship to the Biosynthesis of Ergosterol; J. Chem. Soc., Trans Perkin I, pp. 1326-1333(1974). D. H. R. Barton, A. A. L. Gunatilaka, T. R. Jarman, D. A. Widdowson, M. Bard, and R. A. Woods; Biosynthesis of Terpenes and Steroids. Part X. The Sterols of Some Yeast Mutants Doubly Defective in Ergosterol Biosynthesis; J. Chem. Sot., Trans Perkin I, pp. 88-92(1975).

68

2.

C28 Sterols

Common/Systematic Name Ergosta-5,7,9(11),22-tetraen-313-ol Molec.ular Formula/Molecular Weight C28H420; M W -- 394.32357

I

Fungal Source

Mucor rouxii, Saccharomyces cerevisiae, and Gibberella fufikuroi.

Isolation/Purification Purification was by TLC (silica gel GF), followed by repeated crystallization to give pure ergo sta- 5,7,9,22-tetraen- 313-ol. SpectralData UV:

~m~x 23 l(e = 21,000), 271(11,700), 282(12,000), and 293nm (6,600).

IR:

(Nujol mull) 3350, 1610, 1060, 1040, 980, and 900cm"~. 1H NMR: 9.37(3H, s, !3-Me); 9.08(3H, s, 10-Me); 8.95(9H, d, J=6.5Hz, 21-, 26-, 25-Me); 6.42(1H, m, 3a-H); and 5.23-5.21ppm (2H, m, 28-1-I). Mass Data: Benzoate, Anal. calcd for (C35I-I4602);C, 84.29%; H, 93.0; found: C, 83.26; H, 9.15. TLC Data Re 0.33 with silica gel plates developed with benzene-diethyl ether (9:1, v/v). HPLC Data Relative retention time was 0.56 (relative to cholesterol) performed with C~8 reversed phase column eluted with 4% aqu.eous methanol at ambient temperature with a flow rate of 1.6ml/min.

2. C2s Sterols

69

GC Data Relative retention time was 1.11 relative to cholesterol using 3% SE-30 at 245~ References L. Atherton, J. M. Duncan, and S. Safe; Isolation and Biosynthesis of Ergosta5,7,9(11),22-tetraen-313-ol fromMucor rouxii; J. Chem. Soc., Chem. Commun., pp. 882883(1972). D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenes. Part VI. The Sterols of Yeast; J. Chem. Sot., Perkin Trans I, pp. 513-522(1972). W. D. Nes, S. Xu, and W. F. Haddon; Evidence for Similarities and Differences in t he Biosynthesis ofFungal Sterols; Steroids, Vol. 53, pp. 533-558(1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, 310 pp. (1983).

70

2. C28 Sterols

Common/Systematic Name Ergo sta-5,8,22,24(28 )-tetraen-313-ol Molecular Formula/Molecular Weight C2sH420; MW = 394.32357

General Characteristics Acetate, crystals from chloroform-methanol; mp., 104-106 ~ Fungal Source

Saccharomyces cerevisiae.

Isolation/Purification The sterol was purified by .t)reparative argentation TLC (silica gel coated with AgNO3) of the acetate derivative using benzene as mobile phase. 313-Acetoxyergosta-5,8,22,24(28)tetraene was eluted from a band at Re 0.33 and recrystallized from chloroform-methanol. Spectral Data UWz

~, nox~e (acetate) 225sh(c=25,000), 231(26,200), and 239.5nm sh (16,700). max

~H NMR: (CDCI3) (acetate) 0.70(3H, s, H3-18); 1.09(6H, d, J=7.0Hz, H3-26 and H3-27); 1.09(3H, d, H-21); 1.21(3H, s, H-19); 2.08(3H, s, CH3CO-); 4.64(1H, m, H-3); 4.82(2H, m, H-28); 5.41(1H, m, H-6); 5.51(1H, dd, J20,22=8.5Hz); and 5.92ppm (1H, d, J12.23=15.3Hz). Mass Spectrum: LREIMS: (acetate) 436(M +, 5.9%), 376(70.5), 361(100), 253(25.3), 251(23.2), 211(12.2), 157(31.2), 143(24.8), 123(17.2), and 8Ira~e(23.0).

2. C28 Sterols

71

Reference A. M. Pierce, A. M. Unrau, A. C. Oehlschlager; and R. A. Woods; Azasterol Inhibitors in Yeast. Inhibition of the A24-Sterol Methyltransferase and the 24-Methylene Sterol A 2 4 ( 2 8 ) Reductase in Sterol Mutants of Saccharomyces cerevisiae; Can. J. Biochem., Vol. 57, 112-116(1979).

72

2.

C28 Sterols

Comm.0n/Systematic Name Ergosterone Ergosta-4,7,22-trien-3-one ..Common[Systematic Name C28H420; MW = 394.32357

General Characteristics Crystals from acetone-light petroleum; 134~ [tt]o -12 ~ (c=4.5, in CHC13). Funsal Sour.ce v

F o m e s annosus.

Isolation/Purification Sporophores were extracted with light petroleum and evaporated to dryness to give a yellow wax. The extract was chromatographed on a column of neutral alumina with increasing concentrations of chloroform in benzene. The benzene-chloroform (1:1, v/v) eluant contained ergosterol and ergosta-4,7,22-trien-3-one which were separated by preparative TLC using chloroform as developing solvent. Spectral Data UV;

~,m~, 240nm (log e = 4.11). IR:

(KBr) 1680cm~. Mass Data: Found: C, 84.8; H, 10.5; calcd, for C2sH420: C, 85.2; H, 10.7%. References F. Johnson, G. T. Newbold, and F. S. Spring; Steroids. Part XIII. The Conversion of Ergosterol into Progesterone; J. Chem. Soc. (C), pp. 1302-1306(1954). H. D. Munro and O. C. Musgrave; Extractives from Sporophores of Some F o m e s Species; J. Chem. Soc. (C), pp. 685-688(1971).

2. C28 Sterols

73

Common/Systematic Name Ergosta-7,22-diene-313,5t~-diol .Common/Systematic Name C28H4602; MW = 414.34978

H

-

Fungal Source

Saccharomyces cerevisiae.

Biological Activity Shown to be an intermediate in the biosynthesis of ergosterol. Reference M. Fryberg, A. C. Oehlschlager, and A. M. Unrau; Formation of A 5tt-Hydroxy Sterol by Saccharomyces cerevisiae; Biochem. Biophys. Res. Commun., Vol. 51, pp. 219222(1973).

74

2.

C28 Sterols

Common/Systematic Name Ergosta-8,24(28)-diene-313,6tt-diol Common/Systematic Name C28I-I4602;MW = 414.34978

.o.i

i

OH

Fungal Source Saccharomyces cerevisiae (mutant erg 2). Isolation/Purification The non-saponifiable fraction was acetylated and separated by preparative TLC on silica gel 254, AgNO3 plates developed in benzene. Ergosta-8,24(28)-diene-313,6a-diol was finally purified from preparative TLC by eluting the band at Rr 0.14. Biological Activity Shown to be an intermediate in the biosynthesis of ergosterol. Spectral Data ~H NMR: Acetate: (CDCI3) 0.61(H-18); 1.08(H-19); 0.97(J=7.5Hz, H-21); 1.05(J=7.0Hz, H-26, 27); 2.05, 2.07(COOCH3); ca. 4.65(H-3tQ; ca. 3.90(H-613); and 4.65ppm (J=5.0Hz, H-

28).

13C NMR:

Acetate (CDCI3) 34.8(C-1); 27.0(C-2); 72.7(C-3); 28.1(C-4); 44.3(C-5); 70.2(C-6); 33.9(C-7); 125.9(C-8); 134.5(C-9); 37.4(C-10); 22.4(C-11); 36.4(C-12); 41.8(C-13); 51.2(C-14); 23.4(C-15); 28.4(C-16); 54.4(C-17); 11.0(C-18); 18.4(C-19); 35.9(C-20); 18.4(C-21); 34.4(C-22); 30.8(C-23); 156.5(C-24); 33.5(C-25); 21.6(C-26); 21.6(C27); 105.3(C-28); 20.9, 21.2(COOCH3); 170.2; and 170.6ppm (COOCH3). Mass Spectrum: HREIMS: Acetate 498.3716re~e; C28H4602, requires 498.3709. CIMS: 498m/e.

2.

C28 Sterols

Reference A. M. Pierce, H. D. Pierce, Jr., A. M. Unrau, and A. C. Oehlschlager; Identification of Ergosta-8,24-diene-313,6a-diol in a As to A7 Sterol Isomerase-Blocked Mutant; Lipids, Vol. 14, pp. 876-879(1979).

75

76

2.

C28 Sterols

Common/Systematic Name Portensterol; Hydroxyisoergosterol Molecular Formula/Molecular Weight C28H4402; M W = 4 1 2 . 3 3 4 1 3

HO

HO ~

~"

General Characteristics Colorless irregular prisms from ethanol; 248-250~ 255~ in capillary tube; [a]D 20 - 53 ~ (c=0.010, in CHCIa/MeOH). Acetate: C32I-I4sO4;mp., 160~ in capillary tube; [tt]D2~ - 119 ~ (C=0.010, in CHCI3). Portensterol was very soluble in methanol and ethanol; soluble in chloroform, acetone, and benzene; sparingly soluble in ethyl ether; and insoluble in petroleum ether. Fungal Source

Clitocybe nebularis, Rhodopaxillus nudus, and Tricholomaportentosum.

Isolation/Purification Fruit bodies of Clitocybe nebularis, Rhodopaxillus nudus, and Tricholomaportentosum were extracted with benzene, evaporated to dryness, redissolved in minimal benzene and chromatographed on a neutral alumina (15% water) column. The column was eluted with a gradient from benzene to ethyl ether. Rechromatography of the impure metabolite with neutral alumina (25% water) eluted with a gradient from ethyl ether to 10% ethanol/ethyl ether followed by recrystallization from ethanol yielded pure portensterol. Spectral Data Mass Data: Found: C, 77.42; H, 10.61; O, 12.22%; calcd, for C28H4402*H20;C, 78.09; H, 10.77; O, 11.15%. GC Data Column: 10% UCW 98 coated onto Chromosorb GAW; flame-ionization detector at 290~ column temperature 260~ isothermal; injector temperature 240~ retention time acetate, 7.2 min, TMS 10.3 min.

2.

C28 Sterols

Reference F. Regerat and H. Pourrat; Isolement d'un nouveau st6rol, le portenst&ol, des carpophores de Tricholoma portenosum, Rhodopaxillus nudus, et Clitocybe nebularis; Annales Pharmaceutiques Franqaises, Vol. 34, pp. 323-328(1976).

77

78

2.

C28 Sterols

Common/Systematic Name Pyrocalciferol Molecular Formula/Molecular Weight C2gH440; M W = 396.33922

/

HO General Characteristics Mycelium extracted with petroleum ether, removal of extracting solvent followed by crystallization from ethanol yielded pure pyrocalciferol. Crystals from ethanol; 93-95~ [tx]o25 + 502~ positive Liebermann-Burchard test. Fungal Source Penicillium notatum and Trichoderma koningii. Isolation/Purification Mycelium extracted with petroleum ether; removal of extracting solvent followed by crystallization from ethanol yielded pure pyrocalciferol. Spectral Data UV:

maxM~~ 295(e = 3111), 285(4375), 271(4733), and 263nm (4368). IR:

(Nujol) 3436(OH), 3322, 2873, 1736, 1724, 1643, 1618, 1471, 1390, 1370, 1242, 1163, 1073, 1059, 1042, 975, 883, 806, and 727cm~. 1H NMR: (CDCI3) 9.35 and 9.22(two singlets for angular methyl groups at carbons 10 and 13); 9.1(12H, d, gem-dimethyls and two C-methyls at C-25); 5.75(OH at C-3, disappeared on deuteration); 4.78(2H, s, two protons in ring B at C-6 and -7); and 7.5-8.81: (23H, broad multiplet for remaining protons of the ring and side chain).

2.

C28 Sterols

79

Mass Data: LREIMS: 396(M § intense), 378(- H20), 363(-CH3), 337, 231,211,199, 197, and 147m/e; found: C, 85.04; H, 10.95; O, 4.11%; calcd, for C2si-I440; C, 84.79; H, 11.18; O, 4.O3% Reference A. Kamal, R. Akhtar, and A. A. Qureshi; Studies in The Biochemistry of Microorganisms; Pakistan J. Sci., Ind. Res., Vol. 14, pp. 71-78(1971).

80

2.

C28 Sterols

Common/Systematic Name Ergo sta- 5,7,22,24( 28)-tetraen- 3 g-ol Molecu!.ar Formula/Molecular Weight C2sH420; MW = 394.32357

HO Fungal Source

Aspergillus oryzae, Candida albicans, C. utilis, Saccharomyces cerevisiae, and Gibberella fujikuroi.

Soectral Data UV~

Xm~x 232 and 282nm. TLC Data Rf 0.33 with silica gel plates developed with benzene-diethyl ether (9:1, v/v). HPLC Data Relative retention time was 0.67 (relative to cholesterol) performed with C~8 reversed phase column eluted with 4% aqueous methanol at ambient temperature with a flow rate of 1.6ml/min. GC Data Relative retention time was 1.30 relative to cholesterol using 3% SE-30 at 245~

References D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenes. Part VI. The Sterols of Yeast; J. Chem. Sot., Perkin Trans I, pp. 513-522(1972). M. Fryberg, A. C. Oehlschlager, and A. M. Urau; Sterol Biosynthesis in Antibiotic Sensitive and Resistant Candida; Archiv. Biochem. and Biophys., Vol. 173, pp. 171177(1975). W. D. Nes, S. Xu, and W. F. Haddon; Evidence for Similarities and Differences in The Biosynthesis ofFungal Sterols; Steroids, Vol. 53, pp. 533-558(1989).

2.

C28 Sterols

81

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 316(1983).

82

2.

C28 Sterols

Common/Systematic Name (24S)24-Methylcholesta-7,22-dien-3-one; Ergosta-7,22-dien-3-one Molecular Formula/Molecular Weight C28I-I440; MW = 396.33922

O

General Characteristics Needles from acetone; mp., 182-183.5~ 185~ [a]D + 1.0~

[0~]D22 -I- 1.63~ (c=4.90, in CHCI3) (mp., 183-

Fungal Source Ganoderma applanatum = Fomes applanatus, Fomes fomentarius, F. annosus, and F. pini. Isolation/Purification Sporophores were collected from the trunks of dead hairy birch trees in the Forest of Birse, near Aboyne, and chopped into small pieces. The air-dried fungus was exhaustively extracted with light petroleum followed by evaporation to yield a yellow wax. The light petroleum extract was evaporated to dryness, redissolved in ethyl ether, washed with aqueous sodium hydrogen carbonate, followed by acidification. The ethereal solution was evaporated to dryness, redissolved in benzene and chromatographed through a column of neutral alumina which was eluted with light petroleum-benzene (1:1, v/v), (1:2, v/v), benzene, benzene-chloroform (3:4, v/v), chloroform, chloroform-methanol (4:1, v/v), and methanol. The benzene fraction was crystallized from acetone to yield ergosta-7,22-dien3-one as needles. Spectral Data IR~

(KBr) 1717cm "1 (six membered ring C=O). Mass Data: HREIMS: 396.3392(M +, C28H440) and 244.1828m/e (C17H240); found: C, 84.75; H, 11.3; calcd for C2gH440: C, 84.8; H, 11.2%.

2.

028 Sterols

TLC Data Rf 0.41 using Kieselgel plates developed with petroleum ether-benzene-ethyl acetate, 85:10:5 (v/v/v). References H. R. Arthur, T. G. Halsall, and R. D. Smith; The Chemistry of Triterpenes and Related Compounds. Part XXXIII. The Isolation of Ergosta-7,22-dien-3-one from Fomes fomentarius; Vol. 29, pp. 2603-2605(1958). H. D. Munro and O. C. Musgrave; Extractives from Sporophores of Some Fomes Species; J. Chem. Soc. (C), pp. 685-688(1971). H. Ripperger and H. Budikieqicz; Steroide aus Ganoderma applanatum; Phytochem., Vol. 14, pp. 2297-2298(1975).

83

84

2.

C28 Sterols

Common/Systematic Name Cerevisterol Ergosta-7,22-diene- 5tt, 613,7[3-triol Molecular Formula/Molecular Weight C28I-~O3; MW = 430.34470

OH General Characteristics Crystals; mp., 252-255~

[a]D- 80.5 ~ (pyridine); diacetate; mp., 167-171~

[tt]D- 147~

Fungal Source Acremonium luzulae, Boletaceae (family), Cantharellus cibarius, Fomes allardiL Fusarium moniliforme, Polyporus dryadeus, and Russula sardonia. Isolation/Purification Cultures extracted with butanol and the residue was purified by silica gel column chromatography using benzene-ethyl acetate as eluant. Spectral Data Mass Spectrum: LREIMS: 412(M + -18), 394, 376, 361,269, 251, and 69m/e. References D. Andina, M. De Bernardi, A. Del Vecchio, G. Fronza, G. Mellerio, G. Vidari, and Po Vita-Finzi; Sesquiterpenes from Russula sardonia; Phytochemistry, Vol. 19, pp. 9397(1980). P. Ceccherelli, R. Fringuelli, and G. F. Madruzza; Cerevisterol and Ergosterol Peroxide from Acremonium luzulae; Phytochemistry, Vol. 14, p. 1434(1975). Yu. P. Cherotchenko and A. N. Shivrina; Cerevisterol in Fungi of the Family Boletaceae; Dokl. Akad. Naukssr., Vol. 212, pp. 486-487(1973).

2.

C28 Sterols

85

E. P. Serebryakov, A. V. Simolin, V. F. Kuchrov, and B. V. Roskkynov; New Metabolites ofFusarium moniliforme Sheld.; Tetrahedron, Vol. 26, pp. 5215-5223(1970). W. B. Turner and D. C. Aldridge; Eungal Metaboljtes II; Academic Press, New York, 316 pp. (1983).

86

2.

C28 Sterols

Common/Systematic Name 14ct-Methylcholest-7-en-313-ol Molecular Formula/Molecular Weight C28H4sO; MW = 400.37052

Fungal Source Blastocladia ramosa.

Isolation/Purification The neutral lipid was fractionated on Woelm neutral grade II alumina, the sterols were isolated by digitonin precipitation, and final purification was by argentation chromatography. Spectral Data Mass Spectrum: 14a-Methylcholest-7-en-313-yl acetate; LREIMS: 442(M+), 427, 381,329, 287, 273, 255, 245, 227, 201, 161, 147, 133, 107, 95, 81, 69, 55, and 43m/e (100%). GLC Data Analysis of free sterols was with 3% SE-30, 1% QF-1 and 3% OV-17; sterol acetates were with SE-30 and QF-1. Reference M. A. Southall, J. J. Motta, and G. W. Patterson; Identification and Phylogenetic Implications of Fatty Acids and Sterols in Three Genera of Aquatic Phycomycetes; Amer. J. Botany, Vol. 62, pp. 246-252(1972),

2.

C28 Sterols

87

Common/Systematic Name 14a-Methylcholest-8-en-313-ol Molecular Formula~.. olecular Weight C28Ha80; MW = 400.37052

HO

Funsal Source v

Blastocladia ramosa.

Isolation/Purification The neutral lipid was fractionated on Woelm neutral grade II alumina, the sterols were isolated by digitonin precipitation, and final purification was by argentation chromatography. Soectral Data Mass Spectrum: 14a-Methylcholest-8-en-313-yl acetate; LREIMS: 442(M+), 427, 382, 371,368, 353, 329, 287, 273,255, 245, 229, 227, 213,201, 187, 173, 161, 145, 119, 107, 95, 69, and 43m/e (100%). GLC Data Analysis of free sterols was with 3% SE-30, 1% QF-1, and 3% OV-17; sterol acetates were with SE-30 and QF-1. Reference M. A. Southall, J. J. Motta, and G. W. Patterson; Identification and Phylogenetic Implications of Fatty Acids and Sterols in Three Genera of Aquatic Phycomycetes; Amer. J. Botany, Vol. 62, pp. 246-252(1972).

88

2.

C28 Sterols

Common/Systematic Name 14a-Methylzymosterol Molecular Formula/Molecular Weight C2sH460; MW = 398.35487

HO General Characteristics Crystals from methanol; mp., 103-104.5~ Fungal Source

Saccharomyces cerevisiae (25-Aza-inhibited cultures) and Candida albicans (mutant).

Isolation/Purification The yeast cells were saponified, free sterols separated by TLC followed by recrystallization from methanol. Sterol acetates separated by argentation TLC chromatography. Spectral Data 1H NMR: (CDCI3) 0.72(3H, s, C-18 H); 0.89(3H, s, C-32 H); 0.90(3H, d, J20,2~=6.0Hz, C-21 H); 0.95(3H, s, C-19 H); 1.60 and 1.68(2 X 3H, 2 X s, C-26 and C-27 H); 3.57(1H, m, C3 H); and 5.08ppm (1H, m, C-24 H). Mass Spectrum: LREIMS: 398(M+, 71.9%), 383(100), 365, 313,271,245, 231,219, 213,201, 187, 161,159, 149, 147, 145, 137, 135, 133, 131,123, 121,119, 109, 107, 105, 95, 93, 91, 83, 81, 79, 69, 67, 55, 43, and 4 lm/e. TLC Data TLC was conducted with silica gel HF-254 + 366 (Merck type 60) containing rhodamine 6G developed in methylcyclohexane-ethyl acetate (3:1, v/v). Sterol acetates were separated with argentation TLC chromatography (25% AgNO3) developed with diethyl ether.

2. C28 Sterols

GC Data Analysis of TMS derivatives was with OV-101 at 245~ 245~ and 3% SilicAR-10C on Gas Chrom-Q at 220~

89

acetates with OV-101 at

Reference A.M. Pierce, R. B. Mueller, A. M. Unrau, and A. C. Oehlschlager; Metabolism of A24Sterols by Yeast Mutants Blocked in Removal of the C-14 Methyl Group; Can. J. Biochem., Vol. 56, pp. 794-800(1978).

90

2.

C28 Sterols

Common/Systematic Name 4tt-Methylzymosterol Formula/Molecular Weight C28H460; MW = 398.35487

HO H General Characteristics (Acetate) Plates from acetone; mp., 143-145.5~ +61 ~

[tt]D25 + 65.4 ~ (mp., 138-139~

[a]D

Fungal Source

Saccharomyces cerevisiae, S. carlsbergensis, Candida albicans, Candida sp., C. utilis, Hemispora stellata, Pichia sp., and Torulopsis glabrata.

Isolation/Purification Yeast sterol residues were absorbed on an alumina grade V column and eluted with different ratios of benzene-petroleum ether; 4a-methylzymosterol eluted in the benzenepetroleum ether, 2:3 (v/v), fraction. The partially purified fraction was chromatographed again on alumina grade V. The 4tt-methylzymosterol was acetylated and final purification was accomplished by fractional crystallization; triangulation from acetone-methanol. References D. H. R. Barton, D. M. Harrison, G. P. Moss, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenoids. Part II. Role of 24-Methylene Derivatives in the Biosynthesis of Steroids and Terpenoids; J. Chem. Soc. (C), pp. 775-785(1970). D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenoids. Part VI. The Sterols of Yeast; J. Chem. Soc., Perkin Trans I, pp. 513-522(1972).

C29 Sterols

(24R) 24-Ethylcholesta-4,6,8(14),22-tetraen-3-one (24S) 24-Ethylcholest-5-en-313-ol(Sitosterol) (24R) 24-Ethylcholes-5-en-313-ol(Clionasterol) (24R) 24-Ethylcholesta-5,22-dien-313-ol(Stigmasterol) (24R) 24-Ethyleholesta-5,24(28)-dien-313-ol(Fucosterol) (24R) 24-Ethylcholesta-5,7,24-trien-313-ol(7-Dehydrofucosterol) (24R) 24-Ethylcholest-22-en-313-ol Stigmasta-7(Z),24(28)-dien-313-01 (24S) 24-Ethylcholest-7-en-313-ol(Stigmast-7-en-313-ol) (24S) 24-Ethyleholesta-5,7-dien-313-ol(Stigmasta-5,7-dien-313-ol) (24S) 24-Ethylcholesta-5,22-dien-313-ol(Poriferasterol) (24S) 24-Ethylcholestan-313-ol(Stigmastanol) 4a, 14a-Dimethylcholesta-8,24-dien-313-ol 14-Demethyllanosterol

14-Methylfecosterol 4,4-Dimethylcholesta-8,14,24-trien-313-ol Lanosta-7,9(11),24-trien-313-oi Senexonol Senexadione Oxidosenexone

91

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3.

C29 Sterols

93

Common/Systematic Name (24R)24-Et hylcholest a-4, 6, 8(14), 22-t et raen-3-one Molecular Formula/Molecular Weight C29H420; M W -- 4 0 6 . 3 2 3 5 7

J

O General Characteristics Crystals from petroleum ether; rap., 107-109.5 ~ (mixed with Czs analog); highly fluorescent. Fungal Sourc.e Moldy flour; most abundant molds were A3pergillus niger, A. candidus, A. versicolor, and A. flavus. Isolation/Purification Fungal cultures were extracted twice with methanol, saponified with ethanolic KOH, and extracted with n-hexane. The n-hexane extracts were reduced in volume, applied to a silica gel 60 column eluted in succession with hexane, ethyl acetate-hexane (5:95, v/v), and ethyl acetate-hexane(10:90). (24R)24-Ethylcholesta-4,6,8(14),22-tetraen-3-one purification was monitored with long-wave UV light and was eluted in the 5% ethyl acetate-hexane fraction; crystallization was from petroleum ether. Spectral Data UV:

/~ EtOH max

351(1og e = 25,800), 282(5,800), and 239nm (4,100).

IR;

(KBr) 2940, 2860, 1650, 1562, 1445, 1347, 1320, 1265, 1217, 1191,964, 946, 872, and 758cm 1. Mass Spectrum: LREIMS: 406(M § 6%), 268(100), 253(17), 240(7), and 214m/e (22).

94

3.

C29 Sterols

References R. G. Cooks, R. D. Daftary, and Y. Pomeranz; Changes in Mold-Damaged Wheat Flours Stored at Various Temperatures; J. Agric. Food Chem., Vol. 18, pp. 620-623(1970). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press, Inc. New York, NY, pp. 341 (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, p. 631 (1983). J. D. Weete; Structure and Function of Sterols in Fungi; Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

95

.Common/Systematic Name Sitosterol; 13-Sitosterol; Stigmast_5.en.313.ol; Cupreol; Rhamnol; tt-Dihydrofucosterol; aPhytosterol; Cinchol; Quebrachol; Sitosterin; 22,23-Dihydrostigmasterol (24S)24-Ethylcholest-5-en-313-ol Molecular Formula/Molecular Weight C29H500; ~ = 414.38617 =

NO General Characteristics Plates from alcohol; rap., 140~ [t~]D25 -37 ~ (C=2.0, in CHCI3); precipitated by digitonin. Acetate, C3~H5202;mp., 127-128~ [Ct]D25 -41 ~ (C= 2.0, in CHCI3). Fungal Source Allomyces macrogynus, Aspergillus oryzae, Atta sexdens rubropilosa, Candida sp., C. utilis, Coriolus versicolor, Fusarium sporotrichioides, Glomus mosseae, Hypochytrium catenoides, Physarum polycephalum, Rhizophlyctis rosea, and Stachybotrys alternans. Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Soectral Data _

UV:

End absorption. 1H NMR: H-l, 1.15, 1.89; H-2, 1.85, 1.56; H-3, 3.53; H-4, 2.28; H-6, 5.36 (br d, J=5Hz); H-7, 1.53; H-8, 1.93; H-9, 0.98; H-11, 1.51; H-12, 1.19, 2.03; H-14, 1.00; H-15, 1.13, 1.61; H-16, 1.35, 1.86; H-17, 1.10; H-18, 0.68 (s) ; H-19, 1.009 (s) ; H-20, 1.37; H-21, 0.922 (d, J=7Hz); H-22, 1.04, 1.35; H-23, 1.20; H-24, 0.97; H-25, 1.70; H-26(Pro-S, 0.836 (d, J=7Hz); H-27(Pro-R), 0.814 (J=7Hz); H-28, 1.27 and H-29, 0.846ppm (t, J=7Hz).

96

3.

C29 Sterols

13CNMR: C-l, 37.2; C-2, 31.6; C-3, 71.8; C-4, 42.5; C-5, 14; C-6, 121.7; C-7, 31.8; C-8, 31.9; C-9, 50.1; C-10, 36.5; C-11, 21.1; C-12, 39.7; C-13, 42.3; C-14, 56.7; C-15, 24.3; C16, 28.2; C-17, 56.0; C-18, 11.9; C-19, 19.4; C-20, 36.1; C-21, 18.8; C-22, 33.9; C23, 26.0; C-24, 45.8; C-25, 29.1; C-26(S), 19.8; C-27(R),19.1; C-28, 23.0 and C-29, 12.0ppm. Mass Data: 414(M*, 100%), 399(45), 396(54), 381(42), 329(50), 285,273(54), 255(60), 231(58), and 213m/e (74). Acetate derivative; 456(M§ 441,396(100%), 381,329, 327, 315, 313,273,255(45), and 213m/e (29); C 83.99%, H 12.15%, O 3.86%. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

97

.Common/Systematic Name Clionasterol; 22-Dihydroporiferasterol (24R)24-Ethylcholest-5-en-313-ol Molecular Formula/Molecular Weight C29H500; MW = 414.38617

General Characteristics Monohydrate plates from alcohol when dry; mp., 147-148~ [t~]Dz~-43 ~ (C=1.9, in CHCI3). Acetate, C3~H5202;mp., 143-144~ [t~]D2~-45.3 ~ (C=1.9, in CHC13). Fungal Source Physarum polycephalum and P. flavicomum. Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV: End absorption. 13C NMR: C-15, 24.3; C-16, 28.2; C-17, 56.0; C-18, 11.9; C-20, 36.3; C-21, 18.8; C-22, 33.9; C23, 26.4; C-24, 46.1; C-25, 29.0; C-26(S), 19.6; C-27(R), 19.0; C-28, 23.0; and C-29, 12.3ppm. Mass Spectrum: 414(M*, 100%), 399(45), 396(54), 381(42), 329(50), 285, 273(54), 255(60), 231(58), and 213m/e (74%). Acetate derivative: 456(M*), 441,396(100%), 381,329, 327, 315, 313,273, 255(45), and 213m/e (29).

98

3.

C29 Sterols

References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

99

.Common/Systematic Name Stigmasterol; Stigmasta-5,22-dien-313-ol (24R)24-Ethylcholesta-5,22-dien-313-ol Molecular Formula/Molecular Weight C29H480; MW = 412.37052

HO General Characteristics Monohydrate; crystals from alcohol, when dry; mp., 170~ [tg]D 22 -51 o (c=2 ' in CHCI3). Insoluble in water; soluble in the usual organic solvents. Acetate derivative, C31H5002; mp., 144~ [tt]D2~ -55.6 ~ (C=2, in CHCI3). Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Fungal Source Physarum polycephalum, Debaromyces hansenii, Fusarium sporotrichioides, Glomus mosseae, Hypochytrium catenoides, Pullularia pullulans, and Stachybotrys alternans. Spectral Data UV: End absorption. 13C NMR: C-15, 24.2; C-16, 29.0; C-17, 56.1; C-18, 12.1; C-20, 40.5; C-21, 21.1; C-22, 138.4; C-23, 129.3; C-24, 51.3; C-25, 31.9; C-26, 21.3; C-27, 19.0; C-28, 25.4; and C-29, 12.3ppm. (Assignments for skeletal carbon signals were almost consistent with cholesterol) Mass Spectrum: (Acetate derivative)454(M*), 411,439, 394(100), 379, 351,315, 313, 273,255(95), 253, and 213m/e.

100

3.

C29 Sterols

References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press, Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

101

Common/Systematic Name Fucosterol (24R)24-Ethylcholesta-5,24(28)-dien-313-ol Molecular Formula/Molecular Weight C 2 9 H 4 8 0 ; M W -" 412.37052

HO

i

General Characteristics Crystals from methanol; mp., 124~ [a]D2~ -38.4 ~ (CHC13). Soluble in most organic solvents. Acetate derivative: crystals; mp., 120-121 ~ [a]Dz~ -45 ~ (CHC13). Fungal Source

Fucus vesiculosus (predominant sterol in the brown marine algae, Phaeophyceae), Achyla bisexualis, Aplanopsis terrestris, Apodachlya brachynema, A. minima, Apodachlyella completa, Leptolegnia caudata, Pythiopsis cymosa, Saprolegniaferax, and S. megasperma.

Isolation/Purification The mycelium from Achlya bisexualis was air dried, saponified with ethanolic KOH, and the lipids were extracted with hexane. The non-saponifiable material was chromatographed on alumina (activity III), and squalane and other hydrocarbons were eluted with light petrol. Further elution with diethyl ether gave a sterol fraction which was subsequently acetylated and separated by preparative TLC on silica gel impregnated with silver nitrate. Also, see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV: End absorption.

~3CNMR: (CDCI3) C-15, 24.3; C-16, 28.2; C-17, 55.7; C-18, 11.9; C-20, 36.4; C-21, 18.7; C-22, 35.2; C-23, 25.7; C-24, 146.7; C-25, 34.8; C-26, 22.2; C-27, 115.4; and C-28, 13.1.

102

3.

C29 Sterols

Mass Data: Acetate derivative; 454(M+), 439, 394,379, 356, 341,315, 313,296(100%), 281,273, 255, 253, and 213m/e. C, 84.40%, H, 11.72%. O, 3.88%. TLC Data TLC was carried out on silica gel G plates impregnated with AgNO3and rhodamine 6G. Plates were developed with benzene. Rf fucosteryl acetate, 0.60-0.70. GC Data GC was performed using 1% QF-1 Fucosteryl acetate had relative retention time of 1.51 (relative to cholesteryl acetate). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). C. R. Popplestone and A. M. Unrau; Major Sterols ofAchlya bisexua#s; Phytochemistry,

Vol. 12, pp. 1131-1133(1973). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

103

Common/Systematic Name 7-Dehydrofucosterol (24R)24-Ethylcholesta-5,7,24(28)-trien-313-ol Molecular Formula/Molecular Weight C29H460; MW = 410.35487

J

General Characteristics Colorless crystals from methanol; mp., 127-130~ (129-131 ~ Fungal Source A chlya bisexualis .

Isolation/Purification The mycelium from Achlya bisexualis was air dried, saponified with ethanolic KOH, and the lipids were extracted with hexane to give a yellow oil. The non-saponifiable material was chromatographed on alumina (activity III), and squalene and other hydrocarbons were eluted with light petrol. Further elution with diethyl ether gave a sterol fraction which was subsequently acetylated and separated by preparative TLC on silica gel impregnated with silver nitrate. Also, See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, New York, 341pp.(1989). Spectral Data UV: Typical 5,7-diene system (see ergosterol). Mass Spectrum: Acetate derivative: 392(M § - MeCO2H), corresponding to a molecular weight of 452, 297, 253, 143, 157, and 158m/e.

104

3.

C29 Sterols

TLC Data TLC was carried out on silica gel G plates impregnated with AgNO3 and rhodamine 6G. Plates were developed with 10% ethyl acetate-benzene. Re (7-dehydrofucosteryl acetate) 0.19-0.33. GC Data GC was performed using 1% QF-1. Fucosteryl acetate had relative retention time of 1.67 (relative to cholesteryl acetate). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press, Inc., New York, New York, 34 l pp. (1989). C. R. Popplestone and A. M. Unrau; Major Sterols ofAchlya bisexualis; Phytochemistry, Vol. 12, pp. 1131-1133(1973). W. B. Turner and D.C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, New York, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research; Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

105

Common/Systematic Name 24-Ethylcholesta-22-enol; A22-Stigmasta-313-ol (24R)24-Ethylcholest-22-en-313-o1 Molecular Formula/Molecular Weight C29H500; ~ = 414.38617

H General Characteristics Anisotropic needles from ethanol" rap., 156-157~ from ether-methanol; mp., 156~

[[X]D +5.2 ~ Acetate, glistening plates

Fungal Source

Dictyostelium discoideum.

Isolation/Purification The neutral alcoholic lipids were chromatographed on neutral alumina (activity grade III); eluted increasing amounts of benzene in petroleum ether. 24-Ethylcholest-22-enol was isolated and crystallized (from ethanol solution) from the 25% and 50% benzene in petroleum ether fractions. Also see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Biological Activity Demonstrated weak acrasin-type activity (aggregation of individual slime mold cells into multicellular units) as measured by the Shaffer bioassay. Spectral Data UV:

End absorption.

106

3.

C29 Sterols

IR;

(CS2) strong absorption maxima at 3.42 and 3.50~ (C-H stretching), 9.641.t (C-O stretching of OH) and 10.29~ (trans-disubstituted double bond); weak absorption maxima at 8.56, 8.82, 9.31, 10.10, 10.44, and 10.711.t which was correlated with a 31~hydroxy A/B trans-sterol. Mass Data: LREIMS: (acetate derivative) 456(M +, 27%), 413(M+ - C3H7), 441(M + - CH3), 396(M +- ROH, 13), 381(M + - CH3- ROH), 353(M+- C3H7- ROH), 317(23), 315(32), 275, 257(100), 255(50), and 215m/e (29); found: C, 83.98%; H, 12.09%. References E. Hettmann, B. E. Wright, and G. U. Liddel; The Isolation of Stigmast-22-en-3It-ol from Dictyostelium discoideum; Archives of Biochemistry and Biophysics, Vol. 91, pp. 266270(1960). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

029 Sterols

107

Common/Systematic Name Stigmasta-7(Z),24(28)-dien-313-01 Molecular Formula/Molecular Weight C29H480; M W = 410.35487

H Fungal Source

Aspergillus oryzae, Cronartiumfusiforme, Dictyuchus monosporus, Melampsora lini, and Uromycesphaseoli (bean rust spores).

Isolation/Purification Spores were broken and extracted with chloroform-methanol, 2:1, v/v. The concentrated extract was chromatographed by preparative TLC using silica gel with a solvent of ethyl ether-heptane-acetic acid (70:30:1, v/v/v). The sterols were eluted from the TLC adsorbent with chloroform and rechromatographed (preparative TLC) with a solvent of 0.5% acetic acid in chloroform. The sterols were acetylated and chromatographed by argentation TLC using 0.5% acetic acid in chloroform as developing solvent. Final purification was by recrystallization from acetone solution. See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV~

End absorption. 1H NMR: The resonances were 0.535(s, CH3-18); 0.81(s, CH3-19); 0.93(broad doublet, J=7Hz, CH3-21); 0.97(doublet, J=7Hz, CH3-26 and CH3-27); 1.58(doublet, J=7Hz, CH3-29); 2.01(singlet, CH3-CO); 2.82(septet, J=7Hz, CH-25); 4.72(broad, CH-3); and 5.18ppm (cal)(broad,CH-7 and CH-28).

108

3.

C29 Sterols

Mass Spectrum: The mass spectrum of the sterol acetate showed a molecular ion at 454 m/e (36%); strong peaks were observed at 439(3.6, M + - CH3), 394(1.4, M +- acetate), 379(3.1, M +- CHs and acetate), 356(46.7, M +- C7H~4of side chain), 341(5.6, M +- C7H14 and CH3), 313(100, M+-side ehain-2H), 296(5.7, 356 - acetate), 288(7.9, M +- side chain and Cls, 17), 281(4.9, 296-CHs), 255 (12.4, M § side chain and acetate), and 213role (18.6%, M + - side chain - 42, and -acetate). References H. K Lin and H. W. Knoche; Origin of Sterols in Uredospores of Uromyces phaseoli; Phytochemistry, Vol. 13, pp. 1795-1799(1974). H. K Lin, R. J. Langenbach, and H. W. Knoche; Sterols of Uramycesphaseofi Uredospores; Phytochemistry, Vol. 11, pp. 2319-2322(1972). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press, Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

109

Common/Systematic Name Stigmast-7-en-313-ol (24S)24-Ethylcholest-7-en-313-01 Molecular Formula/Molecular Weight C29H500; M W -- 414.38617

HO' Fungal Source

Aspergillus oryzae, Cronartium fusiforme, Linderina pennispora, Melampsora linL Puccinia graminis var. triticL P. striiformis, Uromycesphaseoli, and Ustilago nuda.

Isolation/Purification Spores were broken and extracted with chloroform-methanol, 2:1, v/v. The concentrated extract was chromatographed by preparative TLC using silica gel with a solvent of ethyl ether-heptane-acetic acid (70:30:1, v/v/v). The sterols were eluted from the TLC adsorbent with chloroform and rechromatographed (preparative TLC) with a solvent of 0.5% acetic acid in chloroform. The sterols were acetylated and chromatographed by argentation TLC using 0.5% acetic acid in chloroform as developing solvent. Final purification was by recrystallization from acetone solution. See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV:

End absorption. Mass Spectrum: (Acetate derivative) LREIMS: 456(M +, 81%), 441(18), 396(21), 381(13), 315(11), 273(13), 255(84), 229(27), 213(40), and 288m/e (9); 456(M § 26.7), peaks at 441(26, M +- CH3), 396(52, M § - acetate), 381(27.4, M + - acetate and CH3), 315(17, M + - side chain and 2H), 288(22.3, M + - side chain and C16,17), 273(14, M § - side chain and 42), 255(100, M + - side chain and acetate), and 213m/e (65.6%, M * - side chain -42 and -acetate).

110

3.

C29 Sterols

References H-K Lin, R. J. Langenbach, and H. W. Knoche; Sterols of Uromycesphaseoli Uredospores; Phytochemistry, Vol. 11, pp. 2319-2322(1972). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

111

Common/Systematic Name St igmast a- 5,7-dien- 3 ~-ol (24S)24-Ethylcholesta-5,7-dien-313-01 Molecular Formula/Molecular Weight C29H480; M W -" 4 1 2 . 3 7 0 5 2

HO Funzal Source Cronartium fusiforme and Melampsora lini. Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). S.pectral Data UV: Typical 5,7-diene chromophore (i.e. 7-dehydrocholesterol or ergosterol). Mass Spectrum: (Acetate derivative) LREIMS 454(M § 4%), 394(100), 379(40), 339, 313, 271, 253(37), 227(43), 211(17), 158(40), and 143m/e (55). References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989)~ W. B. Turner and D. C. Aldridge; Fungal Me.tabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

112

3.

C29 Sterols

Common/Systematic Name Poriferasterol; Stigmasta-5,22-dien-313-ol (24S)24-Ethylchole.sta-5,22-dien-313-01 Molecular Formula/Molecular Weight C29H480; M W = 412.37052

HO Fungal Source

Physarumflavicomum and P. polycephalum.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV~

End absorption. 13C NMR: (CDCI3) C-15, 24.4; C-16, 28.8; C-17, 56.1; C-18, 12.1; C-20, 40.5; C-21, 21.0; C-22, 138.3; C-23, 129.4; C-24, 51.3; C-25, 31.9; C-26(S), 19.0; C-27, 21.2; C-28, 25.4; and C-29, 12.4ppm. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aid.ridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

3.

C29 Sterols

113

Common/Systematic Name Stigmastanol; Dihydro-13-sitosterol; [3-Sitostanol; Fucostanol (24S)24-Ethylchol est~in-313-ol Molecular Formula/Molecular Weizht C29H520; MW = 416.40182

HO General Characteristics Monohydrate: crystals; mp., 138-139~ When dry mp. 144-145~ [0~]D20 + 2 5 in CHCI3); Acetate: C31H5402;mp., 137-138~ [t~]D2~ + 14 ~ (C=1.8, in CHCI3).

~

(c=l.1,

Fungal Source

Dictyoste#um discoideum and Physarumpolycephalum.

Isolation/Purification See R. C. Heupel: Isolation and Primary'Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, pp. 1-27(1989). Spectral Data Mass Data: C 83.58%, H 12.58%, and O 3.84%. Reference W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press Inc., New York, New York, p. 337(1983).

114

3.

C29 Sterols

Common/Systematic Name 4t~, 14a-Dimethylcholesta-8,24-dien-313-ol; 4tt, 14a-Dimethylzymosterol Molecular Formula/M01ecular Weight C29H480; M W -- 412.3 7052

Me

HO

H

: H Me

Fungal Source Saccharomyces cerevisiae (strains D-587-4B, Nys 1, and Erg2) and Candida albicans. Isolation/Purification The yeast cells were saponified and sterols (as acetate derivatives) separated by TLC on AgNO3-impregnated silica gel. Further purification of free sterols was achieved by liquid chromatography. Spectral Data ~H NMR: (CDCI3) TMS derivative: 0.71(s, 13-CH3); 0.88(s, 14-CH3); 0.93(20-CH3); 0.96(s, 10CH3); 0.99(d, J=6Hz, 4a-CH3); 1.60 and 1.68ppm (geminal vinyl methyl groups at C26 and C-27); 5.12(a vinyl proton at C-24); and 3.42ppm (a methyl group at C-4, d, J=6Hz which collapsed to a singlet at 3.42ppm when proton at C-4 was irradiated at 4.85ppm). With Eu(dpm)3, had 2.08(s, 10-CH3, -1.12); and 3.42(d, J=6Hz, 4a-methyl, -2.43); and 4.84ppm (H-4). Mass Spectrum: LREIMS: 412(NV'), 397, 379 and 245m/e; trimethylsilyl ether showed M + at nominal mass of 484m/e. TLC Data Rf 0.56 using silica gel G TLC plates developed with benzene-ethyl acetate (4 1, v/v). HPLC Data Rf 2.07 minutes using ktBondapak C~s column with a solvent system consisting of tetrahydrofuran-acetonitrile-water, 5:5:2 v/v/v, and a flow rate of 0.5ml/min.

3.

C29 Sterols

115

GLC Data Relative retention time of trimethylsilyl ether relative to squalene was 4.78 minutes using 1% neopentyl glycol succinate on Chromosorb W. References P. J. Trocha, S. J. Jasne, and D. B Sprinson; Novel Sterols in Ergosterol Deficient Yeast Mutants; Biochemical and Biophysical Research Communications, Vol.59, pp. 666671(1974). P. J. Trocha, S. J. Jasne, and D. B Sprinson; Yeast Mutants Blocked in Removing the Methyl Group of Lanosterol at C-14. Separation of Sterols by High-Pressure Liquid Chromatography; Biochemistry, Vol. 16, pp. 4721-4722(1977).

116

3.

C29 Sterols

Common/Systematic Name 14-Demethyllanosterol; 4,4-Dimethylzymosterol Molecular Formula/Molecular Weight C29H480; M W - 412.37052

HO

-

H Me

General Characteristics Colorless needles from methanol; mp., 136-138.5~ [a]D 23 q- 40.4 ~ (c=l.0, in CHC13); positive Tortelli-Jaffe and Liebermann-Burchard tests; acetate; mp., 128-132~ [~]Dz3 + 41.8 ~ Fungal Source

Saccharomyces cerevisiae, Candida albicans, C. sp., C. utilis, and Torulopsis glabrata.

Isolation/Purification Isolated from the lipid fraction by preparative TLC on Kieselgel G using hexane-benzene (1:4, v/v) as developing solvent followed by crystallization from methanol. Spectral Data IR:

3470(0H), 1680(C=C), and 1630cm1 (CH=C). 1H NMR:

(CCla) 8.45(3H); 8.40(26-, 27-CH3); 8.05(CH2-7); 6.92(CH-3); and 5.05z (CH-24). Mass Data: HREIMS: C29H480 n20 9 (421.7); found: C, 82.97; H, 11.40; calcd for C29H4gO; C, 82.6%; H, 11.71. References D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenoids. Part VI. The Sterols of Yeast; J. Chem. Soc., Perkin Trans I, pp. 513-522(1972). H. Morimoto, I. Imada, T. Murata, and N. Matsumoto; Uber die Sterine von Candida utilis; Liebigs. Ann. Chem., Vol. 78, pp. 230-240(1967).

3.

C29Sterols

117

Common/Systematic Name

14a-Methylergosta-8,24-dien-313-ol;

14-Methylfecosterol

Molecular Formula/Molecular Weight C29H4sO; MW = 412.37052

Fungal Source

Saccharomyces cerevisiae (strains D-587-4B, Nys 1, and Erg 2) and Ustilago maydis.

Isolation/Purification The yeast cells were saponified and sterols (as acetate derivatives) separated by AgNO3impregnated silica gel TLC. Further purification of free sterols was achieved by liquid chromatography (see below). Spectral Data 1H NM~: (CDCI3) TMS derivative, 0.72(3H, s, H-13); 0.92(9H, d, J - 5-6Hz, H=20 and CC(CH3)2); 0.99(3H, s, H=14; 1.06(3H, s, H-10); 4.68 and 4.73ppm (br s, C=CH2). With Eu(dpm) 3, 2.57(H-10, =1.65); other methyl group protons were shifted only about 0.2ppm. Mass Spectrum: LREIMS: 412(M +), 397(base peak), 379, 369, 313,299, 245, and 23 lm/e; trimethylsilyl ether showed M + at nominal mass of 484role. TLC Data Rf 0.47 using silica gel G TLC plates developed with benzene-ethyl acetate (4:1, v/v). HPLC Data Retention time 1.94 minutes using I.tBondapak ClS column with a solvent system consisting of tetrahydrofuran-acetonitrile-water, 5:5:2 (v/v/v), and a flow rate of 0.5ml/min. GLC Data Relative retention time of trimethylsilyl ether relative to squalene was 4.00 minutes using 1% neopentyl glycol succinate on Chromosorb W.

118

3.

C29 Sterols

References N. N. Ragsdale; Specific Effects of Triafimol on Sterol Biosynthesis in Ustilago maydis; Biochimica et Biophysiea Acta, Vol. 380, pp. 81-96(1975). P. J. Trocha, S. J. Jasne, and D. B Sprinson; Novel Sterols in Ergosterol Deficient Yeast Mutants; Biochemical and Biophysical Research Communications, Vol.59, pp. 666671(1974). P. J. Trocha, S. J. Jasne, and D. B Sprinson; Yeast Mutants Blocked in Removing the Methyl Group of Lanosterol at C-14. Separation of Sterols by High-Pressure Liquid Chromatography, Biochemistry, Vol. 16, pp. 4721-4722(1977).

3.

C29 Sterols

119

Common/Systematic Name 4,4-Dimethylcholesta-8,14,24-trien-313-ol Molecular Formula/Molecular Weight C 2 9 H 4 6 0 ; M W -" 410.3 5487

HO

General Characteristics Purified as 313-acetoxy derivative; crystals from methanol; mp., 126-128.5~ Fungal Source Saccharomyces cerevisiae cultures treated with 23-azacholesterol. Isolation/Purification The sterol mixture from the non-saponifiable fraction of yeast cultures grown in the presence of 23-azacholesterol was separated by preparative TLC using silica gel H F 2 5 4 containing 0.2% rhodamine 6G developed with methylcyclohexane-ethyl acetate, 3:1. The 4,4-dimethylcholesta-8,14,24-trien-3 D-ol containing fraction was acetylated and further purified by preparative TLC using silica gel HFz54 impregnated with 25% silver nitrate and 0.2% rhodamine 6G developed with benzene. The 4,4-dimethylcholesta-8,14,24-trien-313ol containing fraction was chromatographed on silica gel HF254 developed with benzene followed by recrystallization from methanol. Spectral Data UV;

313-acetoxy derivative:

~, Hmax ....

249nm (e=19,600).

IH ~ : 1.04(10[3-methyl); 0.81 ( 13 [3-methyl) and (acetate) 1.06 and 0.81 ppm (1013-methyl and 1313-methyl). Mass Spectrum: LREIMS: 410m/e (M+); acetate: 452role (M~).

120

3.

C29 Sterols

GLC Data 3% Silar-10C on Gas Chrom Q; retention time relative to cholestanol acetate; 4,4dimethylcholesta-8,14,24-trien-3 [3-yl acetate relative retention time 1.92; OV- 101 capillary relative retention time 1.11. References H. D. Pierce, Jr., A. M. Pierce, R. Srinivasan, A. M. Unrau, and A. C. Oehlschlager; Azasterol Inhibitors in Yeast; Biochimica et Biophysica Acta, Vol. 529, pp. 429437(1978). W. B. Turner and D. C. Aldridge; Fungal Metabolites II.; Academic Press, New York, pp. 327(1983).

3.

C29 Sterols

121

Common/Systematic Name Lanosta-7,9(11),24-triene-313,21-diol Molecular Formula/Molecular Weight C29H4602; M W "- 426.34978

HOH2 ......

HO General Characteristics Colorless needles from ethanol; 194-197~

[0QD25 +

72~ (c=3%, in CHC13).

FunRal Source v

Polyporus pinicola and Trichoderma koningii.

Isolation/Purification Mycelium was extracted with petroleum ether, extraction solvent removed, followed by crystallization from ethanol to yield pure pyrocalciferol. The mother liquor was subjected to preparative TLC (solvent system petroleum ether-ethyl ether, 4: Iv/v; Rf 0.50) followed by crystallization oflanosta-7,9(11),24-triene-313,21-diol from ethanol. Soectral Data UV.

~"

325(E = 31,152) and 275nm (20,157).

IR:

(Nujol) 3448(OI-1), 2941, 2857, 1645, 1604, 1471(CH=CH), and 1371crn1. 1H NMR:

(CDC13) 8.40(15H, s, 5 x CH3 groups); 6.68(8H, m, 4 x CH2 groups); 5.15(3H, m, 3 x CH groups); and 4.581~. Mass Data: LREIMS: 440(M+, intense), 425, 422, 408, 394, 368, 346, 339, 338, 313, 312, 311, 285, 280, 264, 255, 231,222, 213,207, 199, 185, 171, 129, and llOm/e; found: C, 81.59; H, 11.35; O, 7.45%; calcd, for C30H4sO2;C, 81.76; H, 10.98; O, 7.26%

122

3.

C29 Sterols

Reference A. Kamal, R. Akhxar, and A. A. Qureshi; Studies in The Biochemistry of Microorganisms; Pakistan J. Sci., Ind. Res., Vol. 14, pp. 71-78(1971).

3.

C29 Sterols

123

Common/Systematic Name Senexonol 6a,22-Dihydroxy-4a, 14~-dimethylcholesta-8,24-dien-3-one Molecular Formula/Molecular Weight C29H4603; MW

-/

-- 4 4 2 . 3 4 4 7 0

0H

General Characteristics Crystals; mp., 118-119~ [~]D + 78 ~ (in CHC13); positive Liebermann-Burchard and tetranitromethane color reactions. Fungal Source The wood-rotting fungus Fomes senex. Spec.tra! Data Mass Spectrum: LREIMS: 426(M +) and 69m/e (base peak). References A. K. Batta and S. Rangaswami; Structure of Senexonol, a Novel Tetracyclic Nortriterpene from Fomes senex; Indian J. Chem., Vol. 7, pp. 1063-1064(1969). A. K. Batta and S. Rangaswami; New Tetracyclic Triterpenes from F o m e s senex: Senexonol, Senexdione, Oxidosenexone and Senexdiolic Acid; Current Science, Vol. 39, pp. 416-417(1970). S. Rangaswami; New Tetracyclic Triterpenes o f F o m e s senex; J. Indian Chem. Soc., Vol. L-11, pp. 179-184(1975).

124

3.

C29 Sterols

Common/Systematic Name Senexadione 4tt, 14a-Dimethylcholesta-8,24-diene-3,22-dione Molecular Formula/Molecular Weight C29H4402; MW = 424.33413 O J

i H

~-

General Characteristics Crystals; mp., 108-110~ [a]D + 52 ~ (in CHCI3); positive Liebermann-Burchard and tetranitromethane color reactions. Fungal Source The wood-rotting fungus Fomes senex. Spectral Data Mass Spectrum: LREIMS: 424m/e (M+). References A. K. Batta and S. Rangaswami; Structure of Senexonol, a Novel Tetracyclic Nortriterpene from Fomes senex; Indian J. Chem., Vol. 7, pp. 1063-1064(1969). A. K. Batta and S. Rangaswami; New Tetracyclic Triterpenes from F o m e s senex: Senexonol, Senexdione, Oxidosenexone and Senexdiolic Acid; Current Science, Vol. 39, pp. 416-417(1970). S. Rangaswami; New Tetracyclic Triterpenes o f Fomes senex; J. Indian Chem. Sot., Vol. L-11, pp. 179-184(1975).

3.

C29 Sterols

125

Common/Systematic Name Oxidosenexone Molecular Formula/Molecular Weight C29H4602; M W -~ 4 2 6 . 3 4 9 7 8

H ~Funsal Source The wood-rotting fungus Fomes senex. References A. K. Batta and S. Rangaswami; Structure of Senexonol, a Novel Tetracyclic Nortriterpene from Fomes senex; Indian J. Chem., Vol. 7, pp. 1063-1064(1969). A. K. Batta and S. Rangaswami; New Tetracyclic Triterpenes from F o m e s senex: Senexonol, Senexdione, Oxidosenexone, and Senexdiolic Acid; Current Science, Vol. 39, pp. 416-417(1970). S. Rangaswami; New Tetracyclic Triterpenes o f F o m e s senex; J. Indian Chem. Soc., Vol. L-11, pp. 179-184(1975).

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C3o Sterols Lanosterol Lanoast-8-en-313-ol Lanoasta-8,23 -di en- 313,25-d io 1 La n o asta-8,2 4-di e n- 313,23-d io 1 Lanoasta-8,23-dien-3 a,22,25-triol 313-Hydroxylanosta-7,9(11),24-trien-21-oic acid 313,15Qt-Dihydroxylanosta-8,24-diene-21,26-dioic acid Trametenolic acid 15a-Hydroxytrametenolic acid Lanost-8-ene- 313,22-dio1 Obtusifoliol Parkeol Obliquol (inotodiol) Deacetylechinodone Deacetyl-3-epiechinodol Deacetoxy-3-epiechinodol Deacetoxyechinodol Deacetoxyechinodone Pinicolic acid A Zeorin Glochidone Glochidonol Betulin Cyathic acid Friedelin Alnusenone Officinalic acid Senexdiolic acid

127

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4.

030Sterols

129

Common/Systematic N.ame Lanosterol; Lanosta-8,24-dien-313-ol Molecular Formula/Molecular Weight C30H500; MW = 426.38617

HO General Characteristics Crystals; mp., 138-140~ 65.5 ~ (c=1.12, in CHC13).

[(~]D20 q-

62.0 ~ (CHC13). Acetate; mp., 131.5-133~

[~]D 20 +

Fungal Source

Agaricus campestris, Allomyces macrogynus, Alternaria kikuchiana, Aspergillus oryzae, Blastocladiella emersoniL Blastocladia ramosa, Candida albicans, C utilis, Catenaria anguillulae, Chytridium confervae, Coriolus heteromorphus, C. pargamenus, Cryptoderma citrinum, Fomitopsis pinicola, Grifola frondosa, Microporus labelliformis, Mucor pusillus, Neurospora crassa, Phycomyces blakesleeanus, Physarum flavicomum, P. polycephalum, Pichia sp., Saccharomyces cerevisiae, Torulopsis glabrata, Hyphochytrium catenoides, Hymenomycetes spp., Rhizidiomyces apophysatus, Rhizophlyctis rosea, Spizellomyces punctatum, and Uromyces phaseoli.

Isolation/Purification See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data UV~

End absorption. Mass Spectrum: LREIMS: (acetate derivative) 468(M+, 65%), 454(100), 393(74), 383(5), 341(4), 325(5), 311(5), 301(6), 289(5), 271(5), 255(6), 243(8), 241(9), 229(9), 227(10), and

215m/e (10).

130

4.

030 Sterols

References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, 631 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol 23, pp. 115-167(1989). J. D. Weete, M. S. Fuller, M. Q. Huang, and S. Gandhi; Fatty Acids and Sterols of Selected Hyphochytriomycetes and Chytridiomycetes; Experimental Mycology, Vol. 13, pp. 183-195(1989).

4. C3o Sterols

131

Common/Systematic Name Lanost-8-en-313-ol; 24-Dihydrolanosterol Molecular Formula/Molecular Weight C30H520; MW = 428.40182

HO General Characteristics Crystals from methanol; mp., 144.5-145.5~

[~]D + 65.7 ~ (c=1.6, in CHCI3).

FunRal Source Saccharomyces cerevisiae, Fomes fomentarius, F. annosus, and F. pini. v

Isolation/Purification Lanost-8-en-313-ol was purified from yeast sterol residues using column chromatography [alumina-Brockmann activity grade III, TLC using silica gel GF254 (Merck), and silica gel impregnated with silver nitrate (20%) with a solvent system consisting of benzene-light petroleum or ethyl acetate-benzene (20:80, v/v]. Spectral Data IR~

(KBr) 3380 and 1630cm "1. Reference H. D. Munro and O. C. Musgrave; Extractives from Sporophores of Some Fomes Species; J. Chem. Soc. (C), pp. 685-688(1971).

132

4.

C30Sterols

Common/Systematic Name Lanosta-8,23-diene-313,25-diol Molecular Formula/Molecular Weight C30H5002; M W -- 442.38108 IIIIiii '

H

HO

General Characteristics Crystals; mp., 189-190~

[0lID 21 q-

60.3.

Fungal Source Peridia of Scleroderma aurantium. Isolation/Purification Fresh peridia were extracted with acetone, concentrated, and partitioned between 60% aqueous ethanol and benzene. Purification was achieved by repeated silicic acid column chromatography. Also, see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Spectral Data IR:

(KBr) 3380, 1635, and 1090cm"1 IH NMR:

(CDCI3) 1.00(s), H-30; 0.81(s), H-30; 0.98(s), H-19; 0.70(s), H-18; 0.87(s), H-32; 0.89(d, J21,20= 6Hz, H-21; 1.31(s), H-26; 1.31(s), H-27; 3.23 (m,J- 15Hz, H-3); 5.59(complex unresolved multiplet, H-23); 5.59(complex unresolved multiplet, H-24); and 1.57ppm, OH. Mass Spectrum: LREIMS: 442(M+), 427(C29H4702), 424(C3oH480), 409(C29H450), 391(C29H450), 327(C23H350), 309(C~H33), and 255role (C19H27).

4.

C30Sterols

133

References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; F.u.ngal Metabolites II; Academic Press, Inc., New York, NY, 319 pp. (1983). J. Vrko~,, M. Bud6~insk~ and L. Dolejg; Constituents of the Basidiomycete Scleroderma

aurantium; Phytochemistry, Vol. 15, pp. 1782-1784(1976).

J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989). J. D. Weete, M. S. Fuller, M. Q. Huang, and S. Gandhi; Fatty Acids and Sterols of Selected Hyphochytriomycetes and Chytridiomycetes; Experimental Mycology, Vol. 13, pp. 183-195(1989).

134

4.

C30Sterols

.Common/Systematic Name Lanosta-8,24-diene- 313,23-diol Molecular Formula/Molecular Weight C30H5002, M W = 442.38108

HO General Characteristics Crystals; mp., 189-190~

[0[,]D 21 "+" 37.4 ~ (in

CHC13).

Fungal Source S c l e r o d e r m a aurantium.

Isolation/Purification Fresh peridia of S c l e r o d e r m a a u r a n t i u m were homogenized in acetone and the concentrated extract was distributed between 60% aqueous ethanol and benzene. The benzene layer was concentrated and the free acids were converted to methyl esters. The methyl ester mixture was repeatedly chromatographed on silica gel (15% water) columns using a gradient from benzene to benzene-acetone (9:1, v/v) to obtain purified metabolites. Spectral Data IR~

kbR

y m~ 3400, 1635, and 1090cm ~. ~H NMR: (CDCI3) 1.00(s, H-30); 0.81(s, H-31), 0.98(s, H-19); 0.72(s, H-18), 0.87(s, H-32); 0.98(d, J21,20= 6Hz, H-21); 1.67(broadened signals with J26,E4=JE7,E4=l.3Hz, H-26); 1.68(broadened signals with J26,E4=J27,E4=l.3Hz, H-27); 3.23 (broadened signals with J26,E4=J27,24=l3nz, H-3); 4.46(dt, J2~.22= 3.0 and 9.0Hz, H-23); 5.20(broad doublet, ,]24,23 = 9.0Hz, J24,E6=J24,27=13Hz), and 1.55ppm (OH). Mass Spectrum: LREIMS: 442(M+), 427(C29H4702 ), 424(C3oH4sO), 409(C29H450), 391(C29H43), 327(C23H350), 309(C23H33), and 255role (C19H27).

4.

C30Sterols

135

Reference J. Vrko6, M. Bud6~inslc~ and L. Dolej~; Constituents of the Basidiomycete Scleroderma aurantium; Phytoehemistry, Vol. 15, pp. 1782-1784(1976).

136

4.

C30Sterols

Common/Systematic Name (22R)-Lanosta-8,23-diene-3 a,22,25-triol; (22R)-3 ct,22,25-Trihydroxylanosta-8,23-diene (Isolated and identified as diacetate, (22R)-25-Hydroxylanosta-8,23-diene-3tz,22-diacetate) Molecular Formula/Molecular Weight C30H5003, ~ = 458.37600 (Diacetate: C34I-I5405; MW = 542.39713) OH .. H

HO ......

General Characteristics Diacetate: crystals from methanol; mp., 190-192~ Fungal Source Fruiting bodies ofPisolithus

[0t]D + 6 ~ (C=1.5, in CHCls).

w

tinctorius.

Isolation/Purification Dried fruit bodies were extracted with methanol, which was divided into benzene soluble and insoluble fractions. The benzene soluble fraction was chromatographed on a silica gel flash column to give five fractions. Fraction 3 was acetylated and further fractionated with silica gel column chromatography using hexane-ethyl acetate (7:3, v/v) to yield purified (22R)-25-hydroxylanosta-8,23-diene-3a,22-diacetate). Spectral Data 1H NMR: (CDC13) (Diacetate) 0.72 (3H, s); 0.87(6H, s); 0.92 (3H, s); 0.94 (31-1, d, J= 6Hz); 1.00 (3H, s); 1.32(6H, s); 2.05 (3H, s); 2.06 (3H, s); 4.57(1H, d, J= 7Hz); 5.25 (1H, dd, J l = 7.5Hz, J2 = 3.7Hz); 5.56(1H, d, Jt = 15Hz, J2 = 7.5Hz); and 5.79ppm (1H, d, J= 15Hz). Mass Spectrum: 542[M +, C34H5405]§ (44%), 482[M-AcOH] § (5), 467[M-AcOH-Me] § (32), 449[M-AcOH-Me-H20] + (42), 407[M-2AcOH-Me] § (26), 389[M-2AcOH-Me-H20] § (26), 356[M-CloH~803] § (11), 341 [M-CloH~sO3] § (11), and 3 2 5 m / e [M-C~oHIsOa-AcOH] § (14).

4.

C30Sterols

137

Reference A. M. Lobo, M. de Abreu, S. Prabhakar, R. Jones, H. S. Rzepa, and D. J. Williams, Triterpenoids of the Fungus Pisolithus tinctorius, Phytochem., Vol. 27, pp. 3569-3572 (1988).

138

030 Sterols

4.

Common/Systematic Name 313-Hydroxylanosta-7,9 (11),24-trien-21-oic acid Molecular Formula/Molecular Weight C30H4603; M W -- 454.34470 HOOC,,

H O ....

General Characteristics Methyl ester; colorless needles from methanol; mp., 142-142.5~ CHC13).

[a]D 24 +

42 ~(c=l.0, in

Fungal Source Sclerotia o f Poria cocos. Isolation/Purification The powdered sclerotia were extracted with ethyl ether, the extract was concentrated, and crude pachymic acid was separated out. The ethyl ether mother liquor was evaporated to dryness, washed with hexane, methylated with diazomethane, and the methyl esters were chromatographed on a alumina column (Wako) eluted with benzene-ether (95:5, v/v). The acid was recrystallized from methanol as colorless needles. Biological Activity "Fuling" is the naturally occurring sclerotium o f Poria cocos and has been frequently prescribed in Chinese medicine as a diuretic and used for palpitations. Spectral Data UV:

/~ EtOH (Methyl Ester) 237(log c = 4.08), 244 (4.15), and 252nm (3.97).

IR:

(KBr) 3310(OH), 1725(C=O), and 810cm~ (>C=C-). 1H NMR: (CDC13) 9.42(3H, s); 9.13(6H, s); 9.05(3H, s); 9.02(3H, s); 8.43(3H, s); 8.33(3H, s); 6.78(1H, m); 6.36(3H, s); 4.94(1H, m); 4.72(1H, m); and 4.53z (1H, m).

4.

C30Sterols Mass Data: Anal. calcd, for

139

C31H4803 " C,

79.43; H, 10.32; found: C, 79.26%; H, 10.11%.

Reference A. Kanematsu and S. Natori; Triterpenoids of Hoelen (ruling), Sclerotia of Poria c o c o s Wolf. II. 3[B-Hydroxylanosta-7,9(11),24-trien-21-oic Acid; Chem. Pharm.Bull., Vol. 18, pp. "/79-783(1970).

140

4. C3oSterols

Common/Systematic Name 313,15(t-Dihydroxylanosta-8,24-diene-21,26-dioic acid Molecular Formula/Molecular Weight C30H4606; MW-- 502.32944

HOOC..... dOOH

~. HO ....,,,,

-

"OH

General Characteristics Dimethyl derivative, needles from methanol, mp 149-153~ [~]D q- 59 ~ (c=l.0, in CHC 13); diacetate derivative, needles from methanol, mp 172-178 ~ Fungal Source Tramee tes odorata.

Isolation/Purification The finely ground fungus was extracted with cold methanol for three weeks at room temperature. The extract when concentrated under reduced pressure gave a waxy gel which was extracted with ethyl ether, filtered, and separated into acidic and neutral fractions. The acidic fraction was methylated and chromatographed on deactivated alumina eluted with benzene. The dimethyl 313,15a-dihydroxylanosta-8,24-diene-21,26dioate containing fraction was further purified by preparative TLC using benzenemethanol, 9:1 (v/v) followed by crystallization from methanol to yield needles of dimethyl 313,15Qt-dihydroxylanosta-8,24-diene-21,26-dioate. Spectral Data (Dimethyl ester) UV:

m~M~' 222 nm (e = 17,000). IR:

(KBr) 3550, 3385 (OH), 1735 (COzMe), 1702 (0t,13-unsaturated CO2Me), 1653 (C=C), 1060 (OH) and 755cm1 (trisubstituted C=C).

4.

C30Sterols

141

1H NMR: (CDC13) 0.77, 0.80, 0.93, 0.96, and 0.99ppm (tertiary methyls); 1.63 (2OH's exchanged with D20), 1.80 (Me2C=); 3.20 (1H, m, H-3 proton); 3.65, 3.72 (2 X 3H, 2 x CO2Me); 4.20 (1H, m, H-15 proton); and 6.65ppm (1H, m, H-24 proton). Diacetate derivative; 0.80, 0.87, 0.87, 0.98, and 0.98ppm (tertiary methyls); 1.8(MeC=); 2.04(6H, 2 x OAt); 3.66, 3.83(2 x CO2Me); 4.45(1H, m, H-3 proton); 4.96(1H, m, HI 5 proton); and 6.65 ppm (1H, m, H-24 proton). Mass Data:

HREIMS 530.3601m/e (M§ C32H5006 requires 530.3607. Found: C, 71.8; H, 9.25;

Calcd. for C32H5006: C, 72.4; H, 9.5%. References W. B. Turner and D. C. Aldfidge; Fungal Metabolites !I; Academic Press, New York, p.320 (1983). R. C. Cambie, R. N. Duve and J. C. Parnell, Constituents of Trametes odorata, New Zealand Journal of Science, Vol. 15, pp. 200-208 (1972).

142

4.

C30Sterols

Common/Systematic Name Trametenolic acid 313-Hydroxylanosta-8,24-dien-21-oic acid Molecular Formula/Molecular Weight C30H4sO3; MW = 456.36035 Iw|,,,

HO General Characteristics Colorless needles from methanol; mp., 252-258~ [~]D + 45 ~ (in CHC13), acetate derivative, needles from methanol; mp., 145-146 o C; [a]D2~+ 68 o (C=0.75, in CHC13). Fungal Source

Daedalea trabea, Fomes senex, Gloephyllum abietinum, G. odoratum, G. sepiarium, G. striatum, G. trabeum, Heterobasidion tasmanica, Laetiporus sulphureus, Len#nus lepideus, Lenzites trabea, Phellinus gilvus, Spongiporus appendiculatus, Trametes odorata, and Veluticeps angularis.

Isolation/Purification The fruiting bodies ofPhellinus gilvus collected from the Changa Manga Forest near Lahore (Specimen No. 6376 deposited at herbarium PCSIR Peshawar) were extracted with EtOH at room temperature for 24 days. The brown extract was concentrated under reduced pressure and chromatographed on silica gel eluted with C6I-I6followed by crystallization from methanol solution to give a crystalline mixture. Further elution with CHCI3 and crystallization with MeOH gave colorless needles of pinicolic acid. Elution with Me2CO-CHC13 (5:95, v/v) and crystallization from MeOH gave colorless needles of trametanolic acid. Spectral Data UV:

~, max236, 243, and 252nm. IR:

methyl ester (CHC13) 3600, 3425, and 1725cml.

4.

C30Sterols

143

1H NMR: (CDC13) 0.72, 0.83, 0.88, 0.98, and 0.99ppm (tertiary methyls); 1.58, 1.68(Me2C=); 3.65(CO2Me); and 5.08ppm (m, C-24 proton). Acetate derivative; 0.72, 0.87, 0.87, 0.87, and 0.98ppm (tertiary methyls); 1.58, 1.68(Me2C=); 2.03(OAc); 3.65(CO2Me); and 5.10ppm. Mass Data: Peaks at 44 lm/e = M+-I 5(M+-CH3), 423role = M+-I 5,-18(M+-CH3-H20); Acetate derivative; found: C, 77.0%; H, 10.2; calcd, for C33H5204: C, 77.3%; H, 10.2. TLC Data Silica gel plates with sodium carbonate and a solvent of benzene-acetone, 9:11, v/v) gave an Rf of .0.42. References S. Ahmad, G. Hussain, and S. Razaq; Triterpenoids ofPhellinus gilvus; Phytochemistry, Vol. 15, p. 2000(1976). R. C. Cambie and J. C. Parnell; Chemistry of Fungi. Part VII Constituents of Heterobasidion tasmanica; New Zealand J. Sci., Vol. 14, pp. 292-298(1991). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 321(1983). V. R. Villanueva; Triterpenes et Sterols de Lenzites trabea; Phytochemistry, Vol. 10, pp. 427-4300970).

144

4. C3o Sterols

Common/Systematic Name 15Qt-Hydroxytrametenolic acid 313,15a-Dihydroxylanosta-8,24-dien-21-oic acid Molecular Formula/Molecular Weight C3oH4804; MW-- 472.35526

HOO

H i,,,

~OH HO General Characteristics Methyl ester, needles from methanol, mp. 180-182~ Diacetate, needles from methanol, mp. 104-106 o C;

[(t]D + 64 ~ (c=1.2, in CHC13)

Fungal Source Daedalea trabea, Lenzites trabea, and Trameetes odorata. Isolation/Purification The mycelium was defatted with light petroleum and extracted with ethyl ether. The acidic fraction was separated from the neutral fraction and methylated with diazomethane. The methylated fraction was eluted on an alumina column with benzene-ether (19:1,v/v). 15aHydroxytrametenolic acid was finally purified as the methyl ester on a silica gel column impregnated with 12.5% silver nitrate eluted with benzene-ether (9:1,v/v) followed by crystallization from methanol. Spectral Data IR:

Methyl ester, (KCI) 3448, 1724 and 1259cm1. Mass Spectrum: LREIMS peaks at M +-15(M+-CH3), M +-15,- 18(M+-EH3-H20), NV- 15,-36(NV-CH32H20). (Methyl ester) 486.3703m/e (M +) calc. for C31H5004 486.3709. Found: C, 76.5; H, 10.6%; C31HsoOa requires C, 76.5; H, 10.4%;

4.

C30Sterols

145

TLC Data Silica gel with sodium carbonate (solvent, benzene-acetone, 9:11,v/v); Re = 0.54. Reference V. R. Villanueva, Triterpenes et Sterols de Lenzites Trabea; Phytochemistry, vol. 10, pp. 427-430 (1970). W. Lawrie, J. McLean and J. Watson, A New Triterpenoid Acid from Lenzites trabea, J. Chem. Soc. (C), pp. 1776-1779 (1967).

146

4.

C30Sterols

Common/Systematic Name Obtusifoliol Molecular Formula/Molecular Weight C30H500; M W = 426.38617

HO -S.

Fungal Source Candida albicans, Monilinia fructigena, Mucor pusillus, Saccharomyces cerevl'siae (strains D-587-4B, Nys 1, and Erg 2), and Ustilago maydis. Isolation/Purification The yeast cells were saponified and sterols (as acetate derivatives) separated by AgNO3impregnated silica gel TLC. Further purification of free sterols was achieved by liquid chromatography. See R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp.(1989). Spectral Data UV:

End absorption. IH NMR: (CDCI3) TMS derivative: 0.72(s, 13-CH3); 0.93(d, J=7Hz, 20-CH3 and C-C(CH3)2); 0.99(s, 14-CH3); 0.99(d, J=6Hz, 4a-methyl); 1.06(s, 10-CH3); 4.67 and 4.72ppm (br s, C=CH2). With Eu(dpm)3, 1.98(s, 10-CH3,-0.92); 3.23(d, J=6.0Hz, 4a-methyl,- 2.24); and 4.60ppm (H-4). Upon irradiation at 4.60, the doublet of 4a-methyl collapsed to a singlet at 3.23ppm.

4.

C30Sterols

147

13C NMR: (CDCla) C-l, 34.9; C-2, 31.1; C-3, 76.4; C-4, 39.2; C-5, 47.0; C-6, 20.7; C-7, 28.2; C-8, 135.5"; C-9, 134.5"; C-10, 36.3; C-11, 21.7; C-12, 25.5; C-13, 44.5; C-14, 49.8; C-15, 31.2; C-16, 31.0; C-17, 50.3; C-18, 15.7; C-19, 18.7"*; C-20, 36.4; C-21, 18.2"*; C-22, 34.9; C-23, 30.7; C-24, 156.8; C-25, 33.8; C-26, 21.8"**; C-27, 22.0***; C-28, 105.9; C-30, 15.0; and C-32, 24.4ppm. *, **, *** Assignments may be interchangeable. Mass Spectrum: 426role (M+); (trimethylsilyl ether) 498m/e; acetate derivative, 468(M+, 6%), 453(M+ - 15, 4), 408(M+- ROH, 100), 393(81), 385, 369, 343,341,324(6), 309, 301, 283(31), 281,269, and 24 lm/e (15). TLC Data Re 0.57 using Silica gel G TLC plates developed with benzene-ethyl acetate (4:1, v/v). HPLC Data Retention time 1.96 minutes using I,tBondapak C18 column with a solvent system consisting of tetrahydrofuran-acetonitrile-water, 5:5:2 (v/v/v), and a flow rate of 0.5ml/min. GC Data Relative retention time of trimethylsilyl ether relative to squalene was 3.13 minutes using 1% neopentyl glycol succinate on Chromosorb W. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, pp. 341 (1989). N. N. Ragsdale; Specific Effects of Triarimol on Sterol Biosynthesis in Ustilago maydis; Biochimica et Biophysica Aeta, Vol. 380, pp. 81-96(1975). P. J. Trocha, S. J. Jasne and D. B Sprinson, Novel Sterols in Ergosterol Deficient Yeast Mutants, Biochem.Biophys.Res.Commun., Vol. 59, pp. 666-671 (1974). P. J. Trocha, S. J. Jasne, and D. B Sprinson; Yeast Mutants Blocked in Removing the Methyl Group of Lanosterol at C-14. Separation of Sterols by High-Pressure Liquid Chromatography; Biochemistry, Vol. 16, pp. 4721-4722(1977). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 327 pp. (1983). J. D. Weete; Structure and Function of Sterols in Fungi; Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

148

4.

C30Sterols

Common/Systematic Name Parkeol Molecular Formula/Molecular Weight C30H500, M3cV= 426.38617

H O ......

.

--:

General Characteristics Parkeol was obtained as needles from chloroform-methanol; mp., 159.5-160~ [0t,]D + 78.4 ~ (c=1.49, in CHCI3). Parkeyl benzoate gave needles from chloroform-methanol; mp., 200-203~ (200.5-201 ~ [a]D + 93 .4 ~ (C=0.57, in CHCI3)([a]D + 96.4 ~ ). Parkeyl acetate was obtained as plates; mp., 159.5-160~ [a]D + 90 ~ (c=1.82, in CHC13). Fungal Source

Saccharomyces cerevisiae.

Isolation/Purification Parkeol was purified from sterol residues of yeast using column chromatography (alumina Brockmann activity grade III), TLC using silica gel GF254 (Merck), and silica gel impregnated with 20% silver nitrate with a solvent system consisting of benzene-light petroleum or ethyl acetate-benzene (20:80, v/v). Spectral Data 1H N:VIR: (CDCl3)(benzoate) 4.80(1H, m, H-11); 4.96(1H, t, J=6Hz, H-24); 5.36(1H, m, H-3Qt); 8.36 (3H, s, Me-26 or 27); 8.44 (3H, s, Me-27 or 26); 8.91(3H, s, Me-10); 8.98 (3H, s, Me-4Qt); 9.09 (3H, s, Me-413); 9.27(3H, s, Me-14); and 9.35x (3H, s, Me-13). Reference D. H. R. Barton, U. M. Kempe, and D. A. Widdowson; Investigations on the Biosynthesis of Steroids and Terpenoids. Part VI. Sterols of Yeast; J. Chem. Sot., Perkin Trans I, pp. 513-522(1972).

4.

C30Sterols

149

Common/Systematic Name Obliquol; Inotodiol Molecular Formula/Molecular Weight C30H5002; M W = 442.38108

OH

HO

General Characteristics Needles from acetone; mp., 188-190~ [0[,]D20 q- 61 ~ (c=1.19, in CHC13); diacetate, lamina from ethanol-water; mp., 162-164~ [Qt]D2~+ 39 ~ (C=1.39, in CHC13); dibenzoate, needles from acetone-ether; mp., 179-181~ [Ct]D2~+ 75.3 ~ (C=1.13, in CHC13). The Liebermann-Burchard test gave a pale yellow color turning green, then orange. The Salkowski test gave a rust-colored ring, tetranitromethane gave a yellow color and the Rosenheim test was negative. Fungal Source Poria obliqua = lnonotus obliquus.

Isolation/Purification The dried, powdered, sterile conks were extracted with hexane and the hexane extract concentrated to a residue. The residue was heated with ethyl ether and filtered. The ethereal filtrate was extracted with 1% sodium hydroxide which produced a flocculent insoluble material. The flocculent material was suspended in water, acidified, and extracted with ether. The residue was recrystallized from acetone and chromatographed on alumina to yield obliquol. Spectral Data UV:

~,~ 244(8 = 270) and 252nm (250). IR:

3350, 1410, 1048, and 1030cm "1.

150

4.

C30Sterols

Mass Data: Anal. calcd for C3oH5oO2: C, 81.39; H, 11.38 found: C, 81.72; H, 11.48%. References L. B. Kier; Triterpenes ofPoria 471474(1961).

obliqua; J. Pharmaceutical Sciences, Vol. 50, pp.

L. B. Kier and W. S. Brey, Jr.; Structural Studies on The Triterpene Obliquol; J. Pharmaceutical Sciences, Vol. 52, pp. 465-468(1963).

4.

C30Sterols

151

Common/Systematic Name Deacetylechinodone Molecular Formula/Molecular Weight C30H4603; MW

--- 4 5 4 . 3 4 4 7 0

OH

iiiiii1|~ " ,|,,llll~r/i /

0 General Characteristics Colorless needles from methanol; mp., 186-187~

[Ct]D2~ + 54.2 ~ (C=I.0, in CHC13).

Funsal Source v Fruit bodies of Echinodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification Fruit bodies of Echinodontium tsugicola, collected at Marunuma, Gumma Prefecture, Japan, in October 1969, were air-dried, crushed, and extracted twice with ethyl ether at room temperature. The combined extracts were treated with hot benzene to remove an orange-yellow amorphous powder. The benzene extract was chromatographed on a column of alumina (Woelm neutral) and eluted successively with (1) benzene, (2) benzene-ether (99:1, v/v), (3) benzene-ether (98.5" 1.5, v/v), (4) ether, and (5) MeOH. Fraction (3) was rechromatographed under similar conditions and each fraction was examined by TLC and GLC. The fractions eluted with benzene that contained deacetoxyechinodone and echinodone were combined and further separated by preparative thin-layer chromatography (silica gel HFz54). Echinodone and deacetoxyechinodone were recrystallized from MeOH to give colorless needles. The next fraction containing a diterpene ester was recrystallized from MeOH to colorless prisms. The fractions eluted with benzene-ether (98:2, v/v)containing deacetoxy-3-echinodol and deacetoxyechinodol were combined and further purified by preparative thin-layer chromatography. Deacetoxy-3-epiechinodol was recrystallized from MeOH, while deacetoxyechinodol was obtained as colorless needles from MeOH. The benzene-ether (95:5, v/v) fractions were collected and again separated by preparative thin-layer chromatography into three __ components" 3-epiechinodol, deacetylechinodol, and deacetyl-3-epiechinodol.

152

4.

C30Sterols

Spectral Data UV~

,~Et~ 278nm (log e 1.8). IR~

(KBr) 3440 and 1710cm"1. 1H NMR: (CDC13) 3.75 (m, H-16); 2.96 (2.96 (t, J=9.0Hz, H-22); 3.95 (t, J=9.0Hz, H-23); 5.18 (bd, J=9.0Hz, H-24); 0.72; (3H, H-18); 1.12 (H-19); H-21, (3H) obscured by other peaks; 1.77(bs, H-26); 1.05 (H-30, -31); and 1.07ppm

(H-32). Mass Data: HREIMS: 454.342m/e (M*); calcd for C30I-h603 454.345; LREIMS: 71(8%), 297(47), 313(100), 439(8), and 454m/e (NV, 17). Reference A. Kanematsu and S. Natori; Triterpenoids of Echmodontium tsugicola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

4.

C30Sterols

153

Common/Systematic Name Deacetyl-3-epiechinodol Molecular Formula/Molecular Weight C30H4803; MW --- 456.36035 OH

. H

i ,,,"

General Characteristics Colorless needles from methanol; mp., 232-233 ~

[~]D 17 -k- 45 o

(c-l.0, in CHC13).

Fungal Source Fruit bodies of Echlnodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification (See deacetylechinodone). Spectral Data IR: (KBr) 3480cm1. 1H Nlk/IR: (CDC13) 3.42 (m, broadened triplet of about 4Hz, H-3); 3.75 (m, H-16); 2.92 (t, J=9.0Hz, H-22); 3.90 (t, d=9.0Hz, H-23); 5.14 (bd, J=8.0Hz, H-24); 0.69 (3H, H-18); 0.97 (3H, H-19); obscured by other peaks (3H, H-26, -27); 1.75 (bs); 0.97 (3H, H-30) 0.88 (H-31); and 1.03ppm (3H, H-32). Mass Data: Anal calcd for C30H4803: C, 78.9; H, 10.6; found: C, 78.6%; H, 10.5. Reference A. Kanematsu and S. Natori; Triterpenoids ofEchmodontium tsugicola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

154

4.

C30Sterols

Common/Svstematic Name Deacetoxy-3-epiechinodol Molecular Formula/Molecular Weight C3oH4802; M W -- 4 4 0 . 3 6 5 4 3

011111'|,,,,1'1111~

H

HO"" General Characteristics Colorless needles from methanol; mp., 194-195 ~

[a]D 19 + 3 2 . 5 o

(c=l.0, in CHC13).

Fungal Source Fruit bodies of Echinodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification (See deacetylechinodone). Spectral Data IR:

(KBr) 3450cmq. 1H NMR: (CDCI3) 3.39 (m, broadened triplet of about 4Hz, H-3); 3.63(m, H-16); 4.12(bt, J=9.0Hz, H-23), 5.12(bd, J=9.0Hz, H-24); 0.68 (3H, H-18); 1.00 (3H, H-19), 0.95(3H, d, J=6.0Hz, H-21); 1.73(3H each, bs, H-26, -27); 1.02 (3h,H, H-30); 0.91 (3H, H-31); and 1.07ppm (3H, H-32). Mass Data: HREIMS: 440.366role (M*), calcd for C3ot-I4sO2440.365; LREIMS: 109(75%), 273(4), 299(7), 315(4), 425(100), and 440m/e (M*, 75); anal calcd, for C30H4sO2:C, 81.8; H, 11.0; found: C, 81.3%; H, 10.9. Reference A. Kanematsu and S. Natori; Triterpenoids ofEchinodontium tsugqcola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

4.

C30Sterols

155

Common/Systematic Name Deacetoxyechinodol Molecular Formula/Molecular Weight C30H4sO2; MW = 440.36543

1......; ......Y HIIIlll H

.,"

General Characteristics Colorless needles from methanol; mp., 194-195~

[(I]D Is + 5 8 . 3 ~

(c=0.96, in CHC13).

Fungal Source Fruit bodies of Echinodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification (See deacetylechinodone).. Spectral Data IR:

(KBr) 3450cm1. 1H NMR: (CDC13) 3.20(m, H-3); 3.60(m, H-16); 4.10(bt, J=9.0Hz, H-23); 5.12(bd, J=9.0Hz, H-24); 0.68 (an, H-18); 1.03 (an, H-19); 0.91 (3H, d, J=6.0Hz, n-21); 1.72(3H each, bs, H-26, -27); 1.03 (3H, H-a0); 0.82(3H, n-31); and 1.06ppm (3H, H-32). Mass Data: HREIMS: 440.366m/e (1W), calcd for C30HasO2440.365; LREIMS: 109(33%), 273(4), 299(7), 315(2), 425(100), and 440m/e (M+, 73); anal calcd, for C30I-hsO2: C, 81.8; H, 11.0; found: C, 81.3; H, 10.9%. Reference A. Kanematsu and S. Natori; Triterpenoids of Echinodontium tsugicola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

156

4.

C30Sterols

Common/Systematic Name Deacetoxyechinodone Molecular Formula/Molecular Weight C30H4602; M W -- 4 3 8 . 3 4 9 7 8 IIIIIi1,.

..11IIII

Y

0 General Characteristics Colorless needles from methanol; mp., 182-183~ [0t]o24+ 84.1o (c=0.82. in CHC13). Fungal Source Fruit bodies of Echinodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification (See deacetylechinodone). Spectral Data IR:

(KBr) 1710cmq. 1H NMR: (CDCI3) 3.60(m, H-16); 4.12(bt, J=9.0Hz, H-23); 5.15(bd, J=9.0Hz, H-24); 0.70 (3H, H-18); 1.18 (3H, H-19); 0.92(3H, d, J=6.0Hz, H-21); 1.72(3H each, bs H-26, -27); 1.11 (3H each, H-30, -31); and 1.13ppm (3H, H-32). Mass Data: HREIMS: 438.346m/e (M+), calcd for C3oH4602438.350; LREIMS: 109(100%), 271(5), 297(12), 313(3), 423(49), and 438m/e (M+, 40); anal calcd, for C3oH4602: C, 81.8; H, 11.0; found: C, 81.3; H, 10.9%. Reference A. Kanematsu and S. Natori; Triterpenoids of Echinodontium tsugicola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

4.

C30Sterols

157

Common/Systematic Name Pinicolic acid A _Molecular Formula/Molecular Weight C30H4603; M W -- 4 5 4 . 3 4 4 7 0

HO0

0 General Characteristics Colorless needles ofpinicolic acid from methanol; mp., 198-202~ methyl ester; mp., 120-121~ [Ct]D2~+ 69 ~ (C=0.27, in CHC13).

[0t]D q- 65 ~ (in

CHC13);

Fungal Source Phellinus gilvus, Fomes senex, and Heterobasidion tasmanica. Isolation/Purification The fruiting bodies ofPhellinus gilvus collected from the Changa Manga Forest near Lahore (Specimen No. 6376 deposited at herbarium PCSIR Peshawar) were extracted with EtOH at room temperature for 24 days. The brown extract when concentrated under reduced pressure was chromatographer on silica gel eluted with benzene and crystallized from methanol solution to give a crystalline mixture. Further elution with CHC13 and crystallization with MeOH gave colorless needles of pinicolic acid. Spectral Data IR:

7max 1700 and 1730cm"1. 1H NMR: (CDCI3) methyl ester, 0.76; 0.89; 1.07; 1.09, 1.09(tertiary methyls); 1.58; 1.67(Me2C=); 3.66(CO2Me); and 5.66ppm (m, C-24 proton)., Mass Data: LREIMS: 454m/e (M). Methyl ester: found; C, 79.1%; H, 10.3; O, 10.6, calcd for C31H4sO3; C, 79.4%; H, 10.3; O, 10.2.

158

4.

C30Sterols

References S. Ahmad, G. Hussain, and S. Razaq; Triterpenoids ofPhellinus gilvus; Phytochemistry, Vol. 15, p. 2000(1976). R. C. Cambie and J. C. Parnell; Chemistry of Fungi. Part VII. Constituents of

Heterobasidion tasmanica; New Zealand J. Sci., Vol. 14, pp. 292-298(1991).

W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p.321 (1983).

4.

030 Sterols

159

Common/Systematic Name Zeorin Molecular Formula/Molecular Weight C30H5202; MW = 444.39673

~'OH

OH General Characteristics Crystals from chloroform; mp., 222-224~ [~]D 24 q- 73.41 o (c=2-3%, in CHCI3). Acetate; crystals from chloroform-methanol; mp., 225-230~ [t~]D + 78 ~ (C=0.5, in CHC13). Benzoate; crystals from benzene-acetone; mp., 236-246~ [t~]D + 73 ~ (C=0.5; in CHCI3). _Fungal Source Found in Cyathus helenae, plants from the genus Glochidion, and the lichen, Nephroma

arcticum.

Isolation/Purification Mycelium was extracted with ethyl acetate; the ethyl acetate was washed with water, dried, and concentrated to give a brown oil. The crude extract was chromatographed on a column containing silica gel eluted with methylene chloride. Zeorin was crystallized from chloroform solution. Spectral Data UV~

Xm~ 228nm (log e=4.00). IR~

(CHC13) 1660 and 885cml. Mass Data: Acetate: Found: C, 79; H, 11.05. C32H5403 requires C, 78.95; H, 11.2%; benzoate: Found: C, 80.7; H, 9.95. C37H5603 requires C, 80.95; H, 10.3%.

160

4.

C30Sterols

References W. A. Ayer, L. M. Browne, J. R. Mercer, D. R. Taylor, and D. Edward; Metabolites of Bird's Nest Fungi. Part 8. Some Minor Metabolites of Cyathus helenae and Some Correlations Among the Cyanthins; Can. J. Chem., Vol. 56, pp. 717-721(1978). D. H. R. Barton and T. Bruun; Triterpenoids. Part VI. Some Observations on the Constitution ofZeorin; J. Chem. Soc. (London), pp. 1683-1690(1952). D. H. R. Barton, P. De Mayo, and J. C. On'; Triterpenoids. Part XXIV. Further Investigations on the Constitution ofZeorin; J. Chem. Soc. (London), pp. 22392248(1958). A. K. Ganguly, T. R. Govindachari, P. A. Modhamed, A. D. Rahimtulla, and N. Viswanathan; Chemical Constituents of Glochidion hohenackeri; Tetrahedron, Vol. 22, pp. 1513-1519(1966). I. Yosioka, T. Nakanishi, and I. Kitagawa; The Chemical Proof of Hoptane Skeleton of Zeorin; Chem. Pharm. Bull., Vol. 15, pp. 353-355(1967).

4.

C30Sterols

161

Common/Systematic Name Glochidone Molecular Formula/Molecular Weight C30H460; M W = 422.3 5487

CH2 H

0

General Characteristics Crystals; mp., 158-160 oC; needles from chloroform-methanol; mp., 164-165 oC; [a]D24 + 73.41 o (c=2_3%, in CHC13). Funsal Source

Cyathus helenae and in plants from the genus Glochidion.

Isolation/Purification The mycelium was extracted with ethyl acetate; the ethyl acetate was washed with water, dried, and concentrated to give a brown oil. The crude extract was chromatographed on a column containing silica gel eluted with methylene chloride to yield purified glochidone. Soectral Data UV:

~,m~ 228nm (log e=4.00). IR:

(CHC13) 1660 and 885cm-1. Mass Data: Found: C, 84.72; H, 10.65. C30H460 requires C, 85.24; H, 10.97%. References W. A. Ayer, L. M. Browne, J. R. Mercer, D. R. Taylor, and D. E. Ward; Metabolites of Bird's Nest Fungi. Part 8. Some Minor Metabolites of Cyathus helenae and Some Correlations Among the Cyanthins; Can. J. Chem., Vol. 56, pp. 717-721 (1978). A. K. Ganguly, T. R. Govindachari, P. A. Modhamed, A. D. Rahimtulla, and N. Viswanathan; Chemical Constituents of Glochidion hohenackeri, Tetrahedron, Vol. 22, pp. 1513-1519(1966).

162

4.

C30Sterols

Common/Systematic Name Glochidonol Molecular Formula/Molecular Weight C301-I4802; M W -" 440.36543

CH2

.o

General Characteristics Amorphous solid from light petroleum; mp., 228 ~C; acetate derivative, needles from methanol; mp., 194-195~ [a]D + 49.4 ~ (in CHC13). Readily converts to glochidone. Fungal Source

Cyathus helenae, also in plants from the genus Glochidion.

Isolation/Purification The mycelium was extracted with ethyl acetate; the ethyl acetate was washed with water, dried, and concentrated to give a brown oil. The crude extract was chromatographed on a column containing silica gel eluted with methylene chloride. An amorphous solid was obtained from light petroleum. Spectral Data UV:

Xm~x End absorption. IR:

(Nujol mull) 3430(OH), 3080, 1725(C=O), 1650, and 880cm "]. Mass Data: Acetate derivative: Found: C, 80.00; H, 10.2. C32H5003 requires C, 79.6; H, 10.40%. TLC Data Silica gel G developed with chloroform, Re 0.20.

4.

C30Sterols

163

References W. A. Ayer, L. M. Browne, J. R. Mercer, D. R. Taylor, and D. E. Ward, Metabolites of Bird's Nest Fungi. Part 8. Some Minor Metabolites of Cyathus helenae and Some Correlations Among the Cyanthins; Can. J. Chem., Vol. 56, pp. 717-721(1978). W. H. Hui and M. L. Fung; An Examination of the Euphorbiaceae ofHong Kong. Part VI. Isolation and Structure of Glochidonol, a New Triterpene Ketol from Glochidion wrightii; J. Chem. Soc. (C), pp. 1710-1712(1969).

164

4.

C30Sterols

Common/Systematic Name Betulin (lup-20(29)-ene-313,28-diol) Molecular Formula/Molecular Weight C30Hs002; MW = 442.38108

H20H

HO"

,~H v

General Characteristics Needles from methanol; mp., 250-251 ~ derivative; mp., 216-217~

[a]D 20 +

16~ (c=0.3, in EtOH); diacetate

Fungal Source The wood-rotting fungus, Polyporuspmicola. Betulin is an important constituent of the bark of birch trees and it has been suggested that the compound may be translocated from the bark directly into the fungus (in this case Fomesfomentarius). Although Polyporus pinicola is usually found on pines, betulin may arise in a similar manner or from small pieces of bark adhering to the fungus. Soectral Data IR~

(Nujol) 3279, 1658, 1265, and 915cmq. 1H NMR: (CDC13) 0.78(3H, s, 24-CH3); 0.85(3H, s, 25-CH3); 0.99(6H, s, 23-, 27-CH3); 1.04(3H, s, 26-CH3); 1.40(2H, s, D20 exchangeable); 1.70(3H, s, 29-CH3); 3.25(1H, m, 3-H); 3.43; 3.75(2H, 28-CH2); and 4.63, 4.73ppm (2H, 30-CH3). Mass Data: LREIMS: 442(M+, 91%), 411(82), 424(14), 234(44), 220(30), 203(62), 205(10), 207(85), and 189m/e (94.5). Diacetate, found: C, 81.0; H, 11.35%; calcd for C30Hs002: C, 81.4; H, 11.4%; found: C, 77.4; H, 10.3%; calcd for C34Hs404 C, 77.5 H, 10.3%.

4.

030 Sterols

165

References R. C. Cambie; Chemistry of Fungi, 12. Betulin from Polyporuspinicola Fr.; New Zealand J. Science, Vol. 21, pp. 565-567(1978). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 330(1983).

166

4.

C30 Sterols

Common/Systematic Name Cyathic acid Molecular Formula/Molecular Weight C30I-I4804; M W -- 4 7 2 . 3 5 5 2 6

H OH

HO

General Characteristics Crystals from chloroform; mp., 307-309~ (after sublimation at 280~ O,Odiacetylcyathic acid: clear white needles from methanol; mp., 311-312~ sublimed at 280~ Fungal Source Cyathus helenae Isolation/Purification Cyathic acid was purified as O, O-diacetylcyathic acid. The mycelium was extracted with methanol, concentrated to dryness, tritiated with methanol, concentrated, and chromatographed on Sephadex LH20 eluted with methanol. The first fractions contained glochidonol followed by glochidone and finally O, O-diacetylcyathic acid. Spectral Data UV:

~. M~on 210nm (e = 100). max IR:

(Thin film) 3400, 2960, and 1685cm"1" O, O-diacetylcyathic acid: (CHCI3) 3500-3100, 3080, 2900, 1741, 1685, 1250, and 1240cmq. Mass Data: LREIMS: 472(M+, 65%), 457(21), 445(19), 429(34), 219(14), 218(67), 203(77), 189(74), 175(50), 135(83), 121(100), 107(96), and 95m/e (94). O,O-Oiacetylcyathic acid: 556(M+, 36%), 496(9), 436(30), 219(34), 218(100), 203(79), 189(75), 175(43), 161(33), 149(35), 147(47), 135(91), and 121m/e (86); found: C, 72.39; H, 9.46. C32H5206requires C, 72.35; H, 9.41%.

4.

C30 Sterols

167

References W. A. Ayer, L. M. Browne, J. R. Mercer, D. R. Taylor, and D. Edward, Metabolites of Bird's Nest Fungi. Part 8. Some Minor Metabolites of Cyathus helenae and Some Correlations Among the Cyanthins; Can. J. Chem., Vol. 56, pp. 717-721(1978). R. J. Flanagan, Ph.D. Thesis, University of Alberta, Edmonton, Canada, (1978).

168

4.

C30Sterols

Common/Systematic Name Friedelin Molecular Formula/Molecular Weight C30H500; M W = 4 2 6 . 3 8 6 1 7

General Characteristics Crystals from light petroleum; mp., 255-257~ (262-263~ CHC13) ([a]D - 21~

[tt]D - 25 ~ (C=1.2, in

Fungal Source

Ganoderma applanatum.

Isolation/Purification Crushed, dried pieces of the fungus were extracted with ethanol. The extract was evaporated to dryness and the residue extracted with boiling light petroleum, filtered, and evaporated to dryness to give an oil. The oil was chromatographed on a silica gel column eluted with light petroleum followed by light petroleum-benzene, 9:1 (v/v). Spectral Data IR~ (CHC13) 1712cm1 (C=O). Mass Spectrum: LREIMS: 426(M +, 4%, C30H500), 411(3, C29H470), 341(2, C25H41), 302(5, C21H340), 273(12, C~9H290 ), 205(13), and 69m/e (100). References J. Protiva, H. Skorkovsk~i, J. Urban, and A. Vystreil; Triterpenes and Steroids from Ganoderma applanatum; Collection Czechoslov. Commun., Vol. 45, pp. 27102713(1980) W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 331 (1983).

4.

030 Sterols

169

Common/Systematic Name Alnusenone; Friedoolean-5-en-3-one Molecular Formula/Molecular Weight C30H480; M W = 424.37052

O~ ____ General Characteristics Crystals from light petroleum; mp., 230-233~ (247~

[t~]D + 24 ~ (C=0.05, in CHC13).

Fungal Source Ganoderma applanatum. Isolation/Purification Crushed, dried pieces of the fungus were extracted with ethanol. The extract was evaporated to dryness and the residue extracted with boiling light petroleum, filtered, and evaporated to dryness to give an oil. The oil was chromatographed on a silica gel column eluted with light petroleum followed by light petroleum-benzene, 9:1 (v/v). _Spectral Data IR:

(CHC13) 1712cm1 (C=O). Mass Spectrum: LREIMS: 424(M +, 12%, C30H480), 409(6, C29H450), 274(55, C20H3a), 259(37, C19H31), 205(36), and 69m/e (100). References J. Protiva, H. Skorkovsk~i, J. Urban, and A. Vystr~il; Triterpenes and Steroids from Ganoderma applanatum; Collection Czechoslov. Commun., Vol. 45, pp. 27102713(1980). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 331 (1983).

170

4.

030 Sterols

Common/Systematic Name Officinalic acid Molecular Formula/Molecular Weight C30H4406; M W --- 500.31379 I

H~l,

0

(

0 O "

.

COOH

General Characteristics Crystals; mp., 272~ [~]D -60 ~ (c=0.5, in dioxane). Monomethyl ester; rap., 236~ -54 ~ (c=0.15, in dioxane).

[a]D

Fungal Source Fomes officinalis, a wood-rotting fungus found on the trunks of living or dead coniferous trees in the Pacific Northwest United States, Canada and in Europe. Spe.ctral Data IR:

3210, 1730, and 1709 cml; monomethyl ester, no OH and 1736cm~. 1H NMR: (CDC13) 2.05.3; 172.6, and 168.5ppm (3 carbonyls). Monomethyl ester: C31I--I4606, 105.3ppm (ketal carbon); six methyl resonances at 0.82, 0.84, 0.85, 0.89, 1.04, and 1.29ppm (all singlets); 2.84(1H, d, J=13Hz); 3.24(2H, s); and 2.49ppm (2H, s). References W. W. Epstein, F. W. Sweat, G. VanLear, F. M. Lovell, and E. J. Gabe; Structure and Stereochemistry of Officinalic Acid, A Novel Triterpene from Fomes of-ficinalis; J. Am. Chem. Soc., Vol. 101, pp. 2748-2750(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 331 (1983).

4.

030 Sterols

171

Common/Systematic Name Senexdiolic acid Molecular Formula/Molecular Weight C3oH4804; MW

= 472.35526

OH

HO

OH

General Characteristics Positive Liebermann-Burchard and tetranitromethane tests. FunRal Source v The wood-rotting fungus F o m e s senex. Spectral Data Mass Spectrum: LREIMS: 440(M+ - 32) and 69m/e (base peak). References A. K. Batta and S. Rangaswami; Structure of Senexonol, a Novel Tetracyclic Nortriterpene from F o m e s senex; Indian J. Chem., Vol. 7, pp. 1063-1064(1969). A. K. Batta and S. Rangaswami; New Tetracyclic Triterpenes from F o m e s senex: Senexonol, Senexdione, Oxidosenexone, and Senexdiolic Acid; Current Science, Vol. 39, pp. 416-417(1970). S. Rangaswami; New Tetracyclic Triterpenes o f F o m e s senex; J. Indian Chem. Soc., Vol. L-11, pp. 179-184(1975).

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C3~ Sterols Dehydroeburicoic acid ketone 3-Ketodehydrosulfurenic acid Eburicodiol Eburical 24-Methyl-24,25-dihydrolanosterol Stowardolic acid Stowardonic acid Eburicoic acid Tumulosic acid Polyporenic acid C 6~t-Hydroxypolyporenic acid C Polyporenic acid D Dehydroeburicoic acid Dehydrotumulosic acid 24-Methylene-24,25-dihydrolanosterol (Eburicol) Pisolactone 3-Oxopisolactone (22S, 24R) 24-Methyllanost-8-en-22,28-epoxy-313,28~t-diol (22S, 24S) 24-Methyllanost-8-en-22,28-epoxy-313,2813-diol 24-Methylenelanosterol (22S) Lanosta-8,24 (28)-diene-313,22-diol

173

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5.

C31 Sterols

175

Common/Systematic Name Dehydroeburicoic acid ketone Molecular Formula/Molecul.ar Weight C31I--I4603, M W --- 4 6 6 . 3 4 4 7 0

HO0

0

General Characteristics Crystals of methyl ester from methanol; mp., 157-159~ [~]D 25 q- 30.2 ~ (c=0.86, in CHC13). Ethyl ester; mp., 240-242~ [a]D24 + 29~ (C=1.2, in pyridine); 2,4-dinitrophenylhydrazone; mp., 144-145~ Fungal Source

Fomes officinalis.

Spectral Data UV:

252(log e = 3.79), 243(3.93), and 236nm (3.90). IR~

(KBr) 3450, 1718, 1640, and 893cml Mass Spectrum: LREIMS: 466role (M+). Reference K. E. Schulte, G. Wicker, and H. Fachmann; Zur Struktur Der "Agaricolsaure", Tet. Lett., pp. 4823-4825(1967).

176

5.

C31 Sterols

Common/Systematic Name 3-Ketodehydrosulfurenic acid (Isolated and identified as methyl-3-ketodehydrosulfurenate) Molecular Formula/Molecular Weight v

C31H4604, M W = 482.33961

HOOC

H 0

General Characteristics Methyl-3-ketoacetyldehydrosulfurenate: Long slender needles from ethanolic KOH; mp., 173-175~ [0t]D24 + 67.5 ~ (C=2.0, in CHCI3). Fungal Source Fomes officinalis.

Isolation/Purification The chloroform extract from the fungus was separated into neutral, strong and weak acids. The weak acid fraction was reduced in vacuo, taken up in methanol, and allowed to stand for several days. The precipitate was removed and the methanol mother liquor removed, reduced in vacuo, and acetylated. The resulting weak acids were methylated, and chromatographed on Florisil to yield methyl-3-ketoacetyldehydrosulfurenate which was allowed to stand overnight in 0.1N ethanolic KOH to yield long slender needles. Spectral Data (Methyl ester) UV~

)q~ 236(log e = 4.10), 2.43(4.23), and 252nm (4.00). IR~

(KBr) 3540, 1725, 1700, 1650, 1060, and 890cm -1. 1H NMR: (CDC13) several methyl resonances in the region of 8.8-9.5(21H); 6.32(3H); 5.32(1H); 5.25(1H); 4.48(1H); and 4.101(1H).

5.

C31 Sterols Mass Data: LREIMS: 496m/e (M+).

Reference C. G. Anderson, W. W. Epstein, and G. Van Lear; Minor Triterpenoids of Fomes officinalis; Phytochemistry, Vol. 11, pp. 2847-2852(1972).

177

178

5.

C31 Sterols

Common/Systematic Nam_e Eburicodiol 24-Methylenelanost- 8-ene-313,21 -diol Molecular Formula/Molecular Weight C31H5202; M~V = 456.39673

HOH2

HO General Characteristics Needles from methanol; mp., 211-212~ [a]D 28 + 57~ 132-133~ [aiD2s + 46~ in CHCIs).

in CHC13); diacetate; mp.,

Fungal Source

Fomes officinafis.

Isolation/Purification The non-saponifiable lipids were chromatographed on Florex. The benzene fractions were combined, concentrated, and rechromatographed on Florex. The benzene-ether (1:1, v/v) eluents were concentrated and rechromatographed on Florex; the ether fraction was crystallized from methanol to yield purified eburicodiol as needles. Spectral Data Mass Spectrum: LREIMS: 456m/e OW). Reference C. G. Anderson and W. W. Epstein; Metabolic Intermediates in the Biological Oxidation of Lanosterol to Eburicoic Acid; Phytochemistry, Vol. 10, pp. 2713-2717(1971).

5.

C31 Sterols

179

Common/Systematic Name Eburical 24-Methylenelano st - 8-en- 313-ol-21=al Molecular Formula/Molecular Weight C31H5002; M W = 4 5 4 . 3 8 1 0 8

OH

I

HO General Characteristics Needles from methanol; mp., 177-179~ [~]D 26 -[- 57 ~ (c=1.25, in CHC13); acetate, small plates from methanol; mp., 154-155~ [a]D24 + 57 ~ (C=1.4, in CHC13). Fungal Source

Fomes officinalis.

Isolation/Purification The neutral extract was defatted and chromatographed on Florex. The benzene fraction was rechromatographed on Florisil; the eburical eluted with benzene-ethyl acetate (95:5, v/v) from the benzene-ethyl acetate (8:2, v/v) fraction chromatographed on a second Florisil column. Spectral Data

(CC14) 2850sh, 274 l(w), and 1725 cml; acetate: 2849sh (w), 2740(w), and 1725cm"1

(s).

~H NMR: (CDCI3) I proton singlet at 9.55ppm (CHO); (CC14) acetate, broad I proton singlet at 9.58ppm (CHO). Mass Spectrum: LREIMS: 454role (M+); acetate 496role (M+).

180

5.

C31 Sterols

Reference C. G. Anderson and W. W. Epstein; Metabolic Intermediates in the Biological Oxidation of Lanosterol to Eburicoic Acid; Phytochemistry, Vol. 10, pp. 2713-2717(1971).

5.

C31 Sterols

181

Common/Systematic Name 24-Methyl-24,25-dihydrolanosterol Molecular Formula/Molecular Weight C31H540; ~

= 442.41747

HO Fungal Source Candida albicans, C. utilis, and Neurospora crassa. Isolation/Purification The lyophilized mycelium was extracted with acetone; benzene-acetone (1:1, v/v); ethyl acetate-absolute methanol (1:1, v/v) and methanol. The fatty acids were removed by partition and the organic phase was concentrated and chromatographed on silica gel G TLC plates using hexane-diethyl ether-methanol (80:20:2, v/v/v) as solvent to separate free sterols from sterol esters. The sterols and sterol esters were eluted from TLC plates with ethyl ether, the sterol esters saponified, and both free sterols and free sterols from saponification rechromatographed as above. The sterols were analyzed by GLC using OV101 and Silar 10C on Gas Chrom Q at 220~ References M. Fryberg, A. C. Oehlschlager, and A. M. Unrau; Sterol Biosynthesis in Antibiotic Sensitive and Resistant Candida; Archives of Biochem. and Biophys., Vol. 173, pp. 171177(1975). A. M. Pierce, H. D. Pierce, Jr., A. M. Unrau, and A. C. Oehlschiager; Lipid Composition and Polyene Antibiotic Resistance of Candida albicans Mutants; Can. J. Biochem., Vol. 56, pp. 135-142(1978). A. M. Pierce, H. D. Pierce, Jr., A. M. Unrau, A. C. Oehlschlager, R. E. Subden, and R. L. Renaud; The Biosynthesis of the Free Sterols and Sterol Esters of Neurospora crassa; Can. J. Biochem. Vol. 57, pp. 112-116(1979). R. L. Renaud, R. E. Subden, A. M. Pierce, and A. C. Oehlschlager; Sterol Composition of Neurospora crassa, Lipids, Vol. 13; pp. 56-58(1978).

182

5.

C31 Sterols

Common/Systematic Name Stowardolic acid 3a-Hydroxy-24~-methyl-23-oxolanosta-8,25-dienoic acid Molecular Formula/Molecular Weight C31H4804; M W = 484.35526

H

H

-=

H 0"" General Characteristics Needles from acetone-light petroleum; mp., 172-181~ Fungal Source

Trametes stowardii.

Isolation/Purification Fungal cultures were extracted with 95% ethanol, evaporated to dryness, and redissolved in ethyl ether. The ether soluble fraction was partitioned between 2N HC1 and 5% NaHCO3. Acidification of the bicarbonate solution and extraction with ether gave a crude product that was chromatographed on a silicic acid column followed by crystallization from acetone-light petroleum to yield stowardolic acid. Spectral Data UV:

max210nm (e = 10,400). IR:

(Nujol null) 2500-3600, 1670-1740, 1065, and 960cmq. IH NMR: (CDC13) 3.45(J=6.OHz, H-3); 3.7(J=7.0Hz, H-24); 5.90(J=3.0Hz, H-26); 6.2(2.5Hz, H-26); 1.35(3H, d, J=7.0Hz, H-31); and 5.1ppm (2H, OH, CO2H). Mass Spectrum: HREIMS: 484.355m/e (M+); C31H4804 requires 484.355.

5.

C31 Sterols

Reference H. T. Cheung, J. C. F. Seeto, and T. R. Watson; Triterpene Metabolite of Trametes stawardii; Aust. J. Chem., Vol. 26, pp. 609-618(1973).

183

184

5.

C31 Sterols

Common/Systematic Name Stowardonic acid Molecular Formula/Molecular Weight C31H4604; M W -- 482.33961

0'~. . . . .

H

General Characteristics Crystals from ethyl acetate; mp., 180-184~ Fungal Source Trametes stowardii.

Isolation/Purification Fungal cultures were extracted with 95% ethanol, evaporated to dryness, and redissolved in ethyl ether. The ether soluble fraction was partitioned between 2N HC1 and 5% NaHCO3. Acidification of the bicarbonate solution and extraction with ether gave a crude product that was chromatographed on a silicic acid column followed by crystallization from ethyl acetate to yield stowardonic acid. Spectral Data UV:

~, max 210nm (e = 11,100). IR:

(Nujol null) 2500-3600, 1730, 1680, and 940cm 1. 1H NMR: (CDC13) 3.7(q, J=7.0Hz, H-24); 5.95(J=2.5Hz, H-26); 6.2(1.5Hz, H-26); 1.35(3H, d, J=7.0Hz, H-31); and 9.0ppm (1H, CO2H). Mass Spectrum: HREIMS: 482.34 lm/e (M§ C31H4604 requires 482.340.

5.

C31 Sterols

Reference H. T. Cheung, J. C. F. Seeto, and T. R. Watson; Triterpene Metabolite of Trametes stowardii; Aust. J. Chem., Vol. 26, pp. 609-618(1973).

185

186

5.

C31 Sterols

Common/Systematic Name Eburicoic acid Molecular Formula/Molecular Weight C31H5003; M W = 470.37600

CH2 HO0

HO General Characteristics Needles from ethanol; mp., 289-290~ (sublime); [a]D2~+ 35 ~ (c=l.0, in pyridine); acetate; mp., 256-258~ Fungal Source

Daedalea trahea, Fomes officinalis, Gloephyllum ahietinum, G. sepiarium, G. striatum, G. trabeum, Laetiporus sulphureus, Lentinus lepideus, Lenzites trabea, Polyporus cretaceous, Trametes lilacino-gilva, and Spongiporus appendiculatus.

Spectral Data UV;

max 236, 243, and 252nm. IR~

(KBr) 3538, 3422(OH), 2940, 2870, 2838, 1720, and 1702cm"1. Mass Spectrum:

LREIMS: 470(M+), 456, 455, 441,437, 423,409, and 395m/e.

TLC Data Silica gel with sodium carbonate (solvent, benzene-acetone, 9:11)gave Rf= 0.52. References L. H. Briggs, R. C. Cambie, I. C. Dean, S. H. Dromgoole, B. J. Fergus, W. B Ingra~ K. G. Lewis, C. W. Small, R. Thomas and D. A. Walker; 10. Metabolites of Some Fungi, New Zealand Journal of Science, Vol. 18, pp. 565-576(1975)

5.

C31 Sterols

187

K. E. Schulte, G. Rucker, and H. Fachmann, Zur Struktur Der "Agaricolsaure", Tet. Lett., pp. 4823-4825(1967). V. R. Villanueva; Triterpenes et Sterols de Lenzites trabea; Phytochemistry, Vol. 1O, pp. 427-430(1970).

188

5.

C31 Sterols

Common/Systematic Name Tumulosic acid Molecular Formula/Molecular Weight C31H5004; MW = 486.37091

CH2 HOO

HO

,,,..-

General Characteristics Methyl ester, needles from aqueous methanol; mp., 163-164~

[r

+ 25 ~

Fungal Source

Daedalea dickinsii, Melanoporia cajanderi, M. juniperina, M. nigra, M. purpuracea, M. rosea, Spongiporus appendiculatus, Trametes feel, T. lilacmo gilva, and T. dickinsii.

Isolation/Purification (purified as methyl ester) The fungus was extracted with ethyl ether, methylated, chromatographed on alumina, and eluted with benzene (3 different fractions) and benzene-chloroform, 1:1 (v/v). The later benzene fraction yielded a 1:1, v/v, mixture of methyl tumulosate and methyl dehydrotumulosate. Methyl tumulosate was obtained by hydrolysis of methyl-3-Omethyltumulosate followed by precipitation with water and recrystallization from aqueous methanol. Biological Activity Antibiotic. Spectral Data (Methyl ester) UV~

max243nm (broad, a = 500). IR~

(CHC13) 3540, 3400, 725, 1645, and 890cm l (C=CH2). 1H NMR: (CDC13) 4.7(2H, m, vinylic protons); 4.10(1H, m, H-16); 3.7(3H, s, CO2Me); and

5.

C31 Sterols

189

3.2ppm(1H, q aider D20, ,]=4.0, 10Hz, H-3); and 1.07ppm (OH). Mass Data: Found: C, 74.2; H, 10.3; calcd for C32H5204 9H20; C, 74.1; H, 10.5. References J. T. Pinhey, B. J. Ralph. J. J. H. Simes, and M. Wootton; Extractives of Fungi 1. The Constituents of Trametes lilacino-gilva; Aust. J. Chem., Vol. 23, pp. 2141-2146(1970). J. T. Pinhey, B. J. Ralph, J. J. H. Simes, and M. Wootton; Extractives of Fungi II. The Constituents of Trametesfeei, 6~t-Hydroxypolyporenic Acid C; Aust. J. Chem., Vol. 24, pp. 609-619(1971).

190

5.

C31 Sterols

Common/Systematic Name Polyporenic acid C Molecular Formula/Molecular Weight C3]I-I4604; MW = 482.33961

CH2

r "r General Characteristics Methyl ester derivative: crystals from aqueous methanol; mp., 198-199~

[~]D + 10~

Fungal Source Daedalea dickinsii, D. tanakae, Fomitopsis pmicola, Melanoporia cajanderi, M. juniperina, M. nigra, M. purpuracea, M. rosea, Piptoporus betulinus, Poria cocos, Trametes feel, and T. dickinsii. Isolation/Purification The fungus was extracted with ethyl ether, methylated, chromatographed on alumina, and eluted with benzene (3 different fractions) and benzene-chloroform, 1:1 (v/v). The first benzene fraction was crystallized from methanol solution, saponified, remethylated, and purified by alumina column chromatography and recrystallization from aqueous methanol to give the purified methyl ester of polyporenic acid C. Biological Activity Antibiotic. Spectral Data (Methyl ester) UV:

~,,~x 236(e = 14,500), 243(16,600), and 252nm (11,100). IR:

(CHC13) 3600, 3480(OH), 1725(CO2Me), 1705(cyclohexanone), 1645, and 890cm ~ (C=CH2); (Nujol) 3400, 1730, 1685, 1645, and 890cm ].

5.

C31 Sterols

191

1H NMR: (CDC13) 5.5(2H, m, vinylic protons at C-7 and C-11); 4.77 and 4.72(2H, m, C=CH2); 4.1(1H, m, CHOH); 3.7(3H, s, CO2Me); and 1.9ppm (OH). Mass Data: LREIMS: 496(M§ calcd, for C32H4604496; Found: C, 77.3; H, 9.7; calcd for C32H4604; C, 77.4; H, 9. 7. Reference J. T. Pinhey, B. J. Ralph, J. J. H. Simes, and M. Wootton; Extractives of Fungi II. The Constituents of Trametesfeei, 6a-Hydroxypolyporenic Acid C; Aust. J. Chem., Vol. 24, pp. 609-619(1971).

192

5.

C31 Sterols

Common/Systematic Name 6a-Hydroxypolyporenic acid C Molecular Formula/Molecular Weight C31H4sOs; MW = 500.35017

CH2

F H

OH

General Characteristics Methyl ester derivative: crystals from aqueous acetone; mp., 222-223~

[(l,]D + 99 ~

Fungal Source Trametes feel. Isolation/Purification The fungus was extracted with ethyl ether, methylated, chromatographed on alumina, eluted with benzene (3 different fractions) and benzene-chloroform, 1:1 (v/v). The benzene-chloroform fraction was purified by preparative TLC (silica gel HF254; chloroform-methanol, 50:1, v/v). The more non-polar component was saponified and purified by preparative TLC (silica gel HF254; chloroform), recrystallized from aqueous acetone to give purified the methyl ester of 6a-hydroxypolyporenic acid C Biological Activity Antibiotic. Spectral Data (Methyl ester) UV:

max 237(E = 12,600), 243(16,600), and 252nm (9,900). IR:

(CHC13) 3600, 3440(OH), 1730(CO2Me), 1705(cyclohexanone), 1645, and 890cmq (C=CH2); (Nujol) 3540, 3450, 1735, 1675, 1645, and 890cm~. :H NMR:

5.

C31 Sterols

193

(CDC13) 5.5(2H, m, vinylic protons at C-7 and C-11); 4.78 and 4.70(2H, m, C=CH2); 4.4(1H, d after deuteration, J=10Hz, C-60H); 4.1(1H, m, C-16HOH); 3.7(3H, s, CO2Me); and 1.3-0.63ppm (7 X Me). Mass Data: HREIMS: 512.3509m/e (M+); calcd, for C32H4805 512.3502; found: C, 74.7; H, 9.5; calcd for C32HasOs, C, 75.0%; H, 9.4%. Reference J. T. Pinhey, B. J. Ralph, J. J. H. Simes, and M. Wootton; Extractives of Fungi II. The Constituents of Trametesfeei, 6Qt-Hydroxypolyporenic Acid C; Aust. J. Chem., Vol. 24, pp. 609-619(1971).

194

5.

Common/Systematic Name Polyporenic acid D 3r

C31 Sterols

5tz-ergosta-8,24( 28)-dien-26-oic acid

Molecular Formula/Molecular Weight C31H5003; M~W = 470.37600

CH2 COOH

HO

/

General Characteristics A white crystalline solid from methanol; mp., 268-270~ a white solid; mp., 235~ [a]D + 23.2 ~

[0[]D + 40 ~ (in pyridine); acetate,

Fungal Source

Polyporus officinalis.

Isolation/Purification Soxhlet extraction of the powdered fungus with n-hexane gave an extract which deposited a white crystalline material on chilling; recrystallization from methanol gave a white crystalline solid. Spectral Data IR:

3525(OH), 2700-2500, 1710, 1698 (C=O), 1640(-C=CH2), and 898cm1. 1H NMR:

(CsDsN) 0.90-1.02(6 methyls); 1.23(d, J=2Hz, H-27); 4.81(2H, d, H-28); and 3.27ppm (1H, t, H-3). Mass Spectrum: LREIMS: 470(M+), 468, 455, 437, 409, 354, 279, 273,247, 234, 220, 219, 211,210, 203,202, 201, 186, 185, 144, 116 and 115role.

5.

C31 Sterols

195

Reference R. K. Thappa. S. G. Agarwal, K. L. Dhar, and C. K. Atal; A New Triterpenic Acid from the Wood Rotting Fungi; Phytochemistry, Vol. 20, pp. 1746-1747(1981).

196

5.

031 Sterols

Common/Systematic Name Dehydroeburicoic acid Molecular Formula/Molecular Weight C31HasO3; MW = 468.36035

CH2 /

COOH

I

HO ......

____---

General Characteristics Crystals; mp., 240-242~

[0t]D21 + 28 ~ (C=0.8). Methyl ester; 159-160~

[a]D21 + 29.5 ~

(c=2.5). Fungal Source

Daedalea trabea and Fomes officinalis.

Spectral.Data Mass Spectrum: LREIMS: 468m/e (M+). References L. H. Briggs, R. C. Cambie, I. C. Dean, S. H. Dromgoole, B. J. Fergus, W. B Ingram, K. G. Lewis, C. W. Small, R. Thomas, and D. A. Walker; 10. Metabolites of Some Fungi; New Zealand Journal of Science, Vol. 18, pp. 565-576(1975). K. E. Schulte, G. Rocker, and H. Fachmann; Zur Struktur Der "Agaricolsaure", Tet. Lett. pp. 4823-4825 (1967).

5.

031 Sterols

197

Common/Systematic Name Dehydrotumulosic acid Molecular Formula/Molecular Weight C31H4804; M W -- 4 8 4 . 3 5 5 2 6

HO0

H,, H

General Characteristics Crystals from methanol; mp., 135-137~ Fungal Source Daedalea dickinsii, Melanoporiajuniperina, M. rosea, Trametes dickinsii, T. feel, and T. lilacino gilva. Spectral Data (All spectral data on methylate) UV~

Ef~ 236(log e = 3.91), 244(3.92), and 252nm (3.73). IR~

(KBr) 3440, 1715, 1648, and 890cm1. 1H NMR: (CDCI3) 3.66(3H, s); 3.22(1H, m); 4.06(1H, m); 4.65(1H, m); and ca 5.30ppm (1H, m). Reference A. Yokoyama and S. Natori; Triterpenoids of Lanostane Group from Fruit Bodies of Nine Basidiomycetous Species; Chem. Pharm. Bull., Vol. 22, pp. 877-883(1974).

198

5.

031 Sterols

Common/Systematic Name Eburicol 24-Methylene-24,25-dihydrolanosterol Molecular Formula/Molecular Weight C31H520, MW = 440.40182

HO~:

General Characteristics A clear yellow color with Liebermann-Burchard reagent, indicating the presence of a 14amethyl group; crystals; mp., 150-151 ~ Fungal Source Phycomyces blakesleeanus, Candida albicans, C. utifis, Neurospora crassa, and Agaricus campestris. Isolation/Purification The fungi were extracted twice with ethyl ether-ethanol (1:1), filtered, diluted with water and extracted with ethyl ether-petrol and saponified. The crude extract was chromatographed on alumina (Brockmann Grade III). The 4,4-dimethyl sterol fraction was acetylated, chromatographed by preparative TLC on 10% AgNO3-silica plates developed with 40% benzene-hexane to give purified eburicol. Spectral Data UV:

End absorption. IR: (KBr) 887 and 1640cm"l. ~H M R : (CDC13) (acetate derivative) Signals at 9.31 (C-18 protons); 9.12(4a, 48 and 14amethyl protons; 9.02(C-19 protons); 9.00(C-26 protons); 8.95(C-27 protons); 7.98(3BO-acetyl protons); 5.3 5(C-28 protons); and 5.50z (3a- proton).

5.

C31 Sterols

199

Mass Spectrum: (acetate derivative) 482(M+), 467(M § - Me), 422(M + - AcOH), and 407m/e (M + [AcOH + Me]). Reference G. Goulston, E. Ian Mercer, and L. J. Goad; The Identification of 24-Methylene-24,25dihydrolanosterol and Other Possible Ergosterol Precursors in Phycomyces blakesleeanus and Agaricus campestris; Phytochemistry, Vol. 14, pp. 457-462(1975).

200

5.

C31 Sterols

Common/Systematic Name Pisolactone (22S)24-Methyllanost-8-en-22,28-epoxy-313-ol-28-one Molecular Formula/Molecular Weight C31H5003; MW = 470.37600

o

~r~ ~ 2B

19

:t2

NO :to

31

General Characteristics Colorless prisms from methanol; mp., 279-280~ Monoacetate, crystals from methanol; mp., 280~

60~ (c=l.0, in CHC13). [a]D2s + 53~ in CHC13).

[ ~ ] D 21 +

Fungal Source Spores ofPisolithus tinctorius. Isolation/Purification Spores were extracted with ethyl ether and purified by chromatography. Also see Heupel, R.C.; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.); Academic Press Inc., New York, NY, 341 pp, (1989). Spectral Data IR:

(KBr) 3410, and 1745cm1; acetate derivative: 1746, and 1725cm]. ~H NMR: Four singlets; 0.72(3H), 0.97(6H), 0.81(3H), and 0.88ppm (3H) were attributed to methyl groups at C-18, C-19, C-30, C-31, and C-32, respectively. References A. M. Lobo, M. de Abreu, S. Prabhakar, and L. S. Godinho; Pisolactone, A Novel Triterpenoid Isolated From The Fungus Pisolithus tinctorius; Tetrahedron Letters, Vol.

5. C31 Sterols

201

24, pp. 2205-2208(1983). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, NY, 341 pp.(1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York,; NY, p. 319 (1983).

202

5.

C31 Sterols

Common/Systematic Name 3-Oxopisolactone Molecular Formula/Molecular Weight C31H4803; M W -- 4 6 8 . 3 6 0 3 5

o

,~O

O General Characteristics White crystalline solid; mp., 248-250~

[01,]D +79 ~ (c=1.4, in CHC13).

Fungal Source Fruiting bodies of Pisolithus tinctorius. Isolation/Purification Powdered, dried fruiting bodies were extracted by percolation with Et20 followed by MeOH. Removal of the Et20 and of the MeOH gave brown oils which were chromatographed separately on a column of silica gel. The Et20 extract was purified on a chromatographic column eluted with hexane-EtOAc (6:4, v/v). Three fractions were collected (F-1, F-2 and F-3) by order of the compounds eluted and analyzed by TLC (silica gel GF254, hexane-EtOAc, 7:3, v/v). Purification of Fraction F-1 by flash column chromatography (silica gel, hexane-EtOAc, 9:1, v/v) gave a white crystalline solid of 3-oxopisolactone. Spectral Data Im:

(KBr) 1705, and 1740cm1 (C=O). 1H N M R : (CDC13) 0.71(3H, s); 0.87(3H, s); 0.89(3H, d, J=7Hz); 0.91(3H, d, J=7Hz); 0.98(3H, d, J=7Hz); 1.02(3H, s); 1.07(3H, s); 1.08(3H, s); 2.47(1H, m); 2.50(2H, m); and 4.35ppm (1H, m).

5. C31 Sterols

203

Mass Spectrum: LREIMS: 468m/e [M+, C31H4803]+ (20%), 453 [M-Me] + (35), 435[M-Me-H20] + (10), 313[M-CgH~502] + (5), 298[M-C9H~502-Me] + (15). Reference A. M. Lobo, M. de Abreu, S. Prabhakar, R. Jones, H. S. Rzepa, and D. J. Williams; Tnterpenoids of The Fungus Pisolithus tinctorius; Phytochem., Vol. 27, pp. 3569-3574(1988).

204

5.

031 Sterols

Common/Systematic Name (22S,2 4R )-2 4-M ethyllanost-8-en-2 2,2 8-epoxy-3 f~,28a-diol Molecular Formula/Molecular Weight C31H5203, MW = 472.39165 0

~ ......OH

it,,,,.. ..,,,,|1[~

3

HO General Characteristics Colorless amorphous powder. Fungal Source Fruiting bodies of Pisolithus tinctorius (proposed to be non-enzymatically formed from pisolactone through its enolic form at position 28). Isolation/Purification Dried fruit bodies were extracted with methanol, which was divided into benzene soluble and insoluble fractions. The benzene soluble fraction was chromatographed on a silica gel flash column to give five fractions. Fraction 4 was further fractionated with silica gel flash column chromatography using benzene-ethyl acetate (8:1, v/v) followed by a medium pressure liquid chromatography (silica gel) eluting with benzene-ethyl acetate (7:1, v/v) to afford 24-methyllanosta-8,24(28)-diene-313,22~-diol and pisolactone. Fraction five was fractionated using silica gel flash column chromatography and medium pressure liquid chromatography using ODS with methanol as eluting solvent to give pisolactone, (22S,24R)-24-methyllanost-8-en-22,28-epoxy-3~,28a-diol, (22S,24R)-24Methyllanost-8-en-22,28-epoxy-313,2813-diol, and 24-methyllanosta-8,24(28)-diene313,22~-diol. Biological Activity (22S,24R)-24-Methyllanost-8-en-22,28-epoxy-313,28a-diol suppressed proliferation of mouse spleen lymphocytes stimulated with the mitogens, concanavalin A and lipopolysaccharide. IDs0 calculated to be 2.3ktg/ml against the Con A-induced proliferation and 1.91xg/mlagainst the LPS-induced proliferation of mouse spleen lymphocytes.

5. 031 Sterols

205

Spectral Data 1H NMR:

1.23(m, H-I); 1.74(m); 1.55(m, H-2); 1.68(m); 3.24(dd, J=l 1.6, 4.5Hz, H-3, H-3); 1.05(dd, J=12.5, 2.0Hz, H-5); 1.52(m, H-6); 1.68(m); 2.04(2H, m, H-7); 2.02(2H, m, H-11); 1.21(m, H-12); 1.75(m); 130(m, H-15), 1.80(m); 138(m, H-16); 2.03(m); 1.82(m, H-17); 0.70(3H, s, H-18); 0.98(3H, s, H-19); 1.46(m, H-20); 0.91(3H, d, J=6.4Hz, H-21); 4.28(ddd, J=10.9, 5.1, 2.0Hz, H-22); 1.42(m, H-23); 1.84(m); 1.78(m, H-24); 1.60(m, H-25); 0.91(3H, d, Jr= 6.4Hz, H-26); 0.99(3H, d, J=6.8Hz, H-27); 5.17(d, J=3.4Hz, H-28); 0.81(3H, s, H-29); 1.00(3H, s, H-30); and 0.90ppm (3H, s, H-31). 13CNMR:

C-l, 35.58(0; C-2, 27.85(t); C-3, 78.99(d); C-4, 38.89(s); C-5, 50.40(d); C-6, 18.27(t); C-7, 26.49(0; C-8, 34.35(s); C-9, 34.47(s); C-10, 37.02(s); C-11, 21.02(t); C-12, 30.92(t); C-13, 44.63(s); C-14, 49.72(s); C-15, 30.97(0; C-16, 27.87(0; C-17, 47.88(d); C-18, 15.55(q); C-19, 19.16(q); C-20, 39.05(d); C-21, 12.98(q); C-22, 80.52(d); C-23, 33.40(t); C-24, 54.63 (d); C-25, 30.58(d); C-26, 20.76(q); C-27, 21.41(q); C-28, 102.21(d); C-29, 15.43(q); C-30, 27.97(q); and C-31, 24.40ppm (q). Mass Spectrum: HRFAB-MS: 472.3918m/e (M+); calcd for C31H5203,472.3916. Reference H. Fujimoto, M. Nakayama, Y. Nakayama, and M. Yamazaki; Isolation and Characterization of Immunosuppressive Components of Three Mushrooms, Pisolithus tinctorius, Microporus flabelliformis and Lenzites betulina; Chem. Pharm. Bull., Vol. 42, pp. 694-697(1994).

206

5.

C31 Sterols

Common/Systematic Name (22S,24S)-24-Methyllanost-8-en-22,28-epoxy-313,2813-diol Molecular Formula/Molecular Weight C31H5203, MW" = 4 7 2 . 3 9 1 6 5

O

.I

OH

HO General Characteristics Colorless amorphous powder. Fungal Source Fruiting bodies ofPisolithus tinctorius (proposed to be non-enzymatically formed from pisolactone through its enolic form at position 28). Isolation/Purification See (22S,24R)-24-Methyllanost-8-en-22,28-epoxy-313,28~t-diol isolation. Biological Activity Suppressed proliferation of mouse spleen lymphocytes stimulated with the mitogens, concanavalin A and lipopolysaccharide. IDs0 calculated to be 2.31xg/ml against the Con Ainduced proliferation and 1.9pg/ml against the LPS-induced proliferation of mouse spleen lymphocytes. Spectral Data IH NMR: H-I, 1.23(rn, H-l), 1.74(m); 1.55(m, H-2), 1.68(m); 3.24(dd, J-11.6, 4.5Hz, H-3), 1.05(dd, J=12.5, 2.0Hz, n-5), 1.52(m, n-6); 1.68(m); 1.04(21, m, H-7); 2.02(21, m, n-11); 1.21(m, n-12), 1.75(m); 1.70(m, n-15), 1.80(m); 1.54(m, n-16); 2.01(m), 1.86(m, H-17); 0.70(31, s, ); n-18), 0.98(31, s, H-19), 1.57(m, n-20); 0.93(31, d, J=6.4Hz, H-21); 4.13(ddd, J-10.9, 5.1, 2.0Hz, H-22); 1.22(m, H-23); 1.76(m); 1.72(m, H-24); 1.67(m, H-25); 0.99(31, d, J=6.8Hz, H-26); 0.95(d, J=6.8Hz, H-27); 5.27(d, J=3.9Hz, H-28); 0.81(s, H-29); 1.00(s, H-30), and 0.90ppm (3H, s, H-31).

5.

C31 Sterols 13CNMR: C-I, 35.58(t); C-2, 27.85(t); C-3, 78.99(d); C-4, 38.89(s); C-5, 50.40(d); C-6, 18.27(t), C-7, 26.49(t), C-8, 34.35(s); C-9, 34.47(s); C-10, 37.02(s); C-11, 21.02(t); C-12, 30.93(t); C-13, 44.61(s); C-14, 49.77(s), C-15, 30.89(t); C-16, 27.92(t); C-17, 48.00(d); C-18, 15.58(q); C-19, 19.16(q); C-20, 39.35(d); C-21, 13.01(q); C-22, 83.01(d); C-23, 31.29(t); C-24, 52.94(d); C-25, 27.81(-d); C-26, 21.48(q); C-27, 22.06(q); C-28, 97.77(d); C-29, 15.43(q); C-30, 27.97(q); and C-31, 24.36ppm (q). Mass Spectrum: HRFAB-MS: 472.3918m/e (M+); calcd for C31H5203,472.3916.

Reference H. Fujimoto, M. Nakayama, Y. Nakayama, and M. Yamazaki; Isolation and Characterization of Immunosuppressive Components of Three Mushrooms, Pisolithus tinctorius, Microporus flabelliformis and Lenzites betulina; Chem. Pharm. Bull., Vol. 42, pp. 694-697(1994).

207

208

5.

C31 Sterols

_Common/Systematic Name 24-Methylenelanosterol 24-Methylene-5~t-lanost-8-en-313-01 Molecular Formula/Molecular Weight C31H520; MW = 440.40182

HO

Isolation/Purification The unsaponifiable material from P i c h i a sp. was separated into five fractions by preparative TLC (Wakogel B-10 coated plates developed with hexane-ether, 7:3, v/v). Preparative argentation (10% silver nitrate coated onto Wakogel B-10 plates, developed with hexane-benzene, 3:2, v/v) TLC of the acetylated 4,4-dimethyl fraction produced lanosterol and 24-methylenelanosterol. Fungal Source P i c h i a sp. (an alkane-utilizing yeast).

Spectral Data Mass Spectrum: LREIMS: 440(M +, 6%), 425(M +- CH3, 17), 407(M +- CHs- 1-120, 11), 356(M + C6H12, 2), 341(M + - C6H12 - CHs, 4), 323(M +- C6H12 - CHs- H20, 6), 315(M +- C9H17, 3), 297(M + - C9H~7 - H20, 3), 273(M + - C9H~7 - C3H6, 5), 259[M + - C9H17 - Call6- CH2 (14a-methyl - H), 11 ], and 24 l m / e ( M ~ C 9 H 1 7 - Call6 - CH2 - 1-120, 11); weak ions at 438(M + - H) and 423role ( M + - CH3) indicative of a As bond. -

GC Data OV-17 coated onto Gas Chrom-Z; column temperature 256~ detector 280~ cartier gas N2 at 50ml/min. Relative retention time relative to 13-sitosterol for 24- methylenelanosterol wasl. 18 and the acetate derivative was 1.43. Reference T. M. Jeong, T. Itoh, T. Tamura, and T. Matsumoto; Sterols from a Species o f P i c h i a , A n-Alkane-utilizing Yeast; Steroids, Vol. 25, pp. 741-751(1975).

5. C31

Sterols

209

Common/Systematic Name Lanosta-8,24(28)-diene-313,22-diol; (22S)-Lanosta-8,24(28)-diene-313,22-diol Molecular Formula/Molecular Weight C31H5202; M W = 456.39673

H0~.....,,,, General Characteristics Crystals from methanol; mp., 161-165~

[a]D + 29 ~ (C=0.16, in CDC13).

Fungal Source Fruiting bodies of Pisolithus tinctorius. Isolation/Purification Powdered, dried fruiting bodies were extracted by percolation with Et20 followed by MeOH. Removal of the Et20 and MeOH gave brown oils which were chromatographed separately on a column of silica gel. The Et20 extract was purified on a chromatographic column eluted with hexane-EtOAc (6:4,v/v). Three fractions were collected (F-1, F-2 and F-3 by order of the compounds eluted) and analyzed by TLC (silica gel GF254, hexane-EtOAC, 7:3, v/v). A portion of fraction two was acetylated and purified by silica gel column chromatography (hexane-ethyl acetate, 9:1, v/v) to yield the diacetate of lanosta-8,24(28)diene-3f3,22-diol, which was recovered after saponification and recrystallization from methanol. Spectral Data IR:

(KBr) 3450 and 890cm"1. 1H N~/IR: (CDC13) 0.70(3H, s); 0.81(3H, s); 0.91(3H, s), 0.92(3H, d, ,/--6 Hz); 0.98(3H, s); 1.01(3H, d, ,/=6 Hz); 1.04(3H, s); 1.06(3H, d, J=6Hz); 3.23(1H, m); 3.80(1H, m); 4.78(1H, s): and 4.88ppm (1H, s).

210

5.

C31 Sterols

13C NMR: (CDC13) 153.9; 135.0; 134.8; 128.6; 78.3; and 71.0ppm. Mass Spectrum: LREIMS: 456[M +, C31H5202]+(26%), 441 [M-Me] § (7), 438 [Me-H20] § (1), 423[M-Me-H20] § (9), 372[M-C6H12] § (16), 357[M-C6H12-Me] § (100), 339[M-C6HIE-Me-H20] § (45), 314[M-C9H~80] § (10), and 281m/e [M-CaHlsO-H20] § (18). Reference A. M. Lobo, M. de Abreu, S. Prabhakar, R. Jones, H. S. Rzepa, and D. J. Williams; Triterpenoids of The Fungus Pisolithus tinctorius; Phytochem., Vol. 27, pp. 3569-3574(1988).

C32 Sterols Echinodol 3-Epiechinodol Echinodone (22R) 3Qt,25-Dihydroxylanosta-8,23-diene-22-acetate 1213-Acetoxy-4,4-dimethyl-24-methylene-5~t-cholesta-8,14-diene-313,11ct-diol

8

211

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6.

C32 Sterols

213

Common/Systematic Name Echinodol Molecular Formula/Molecular Weight C32H5004; M W -- 4 9 8 . 3 7 0 9 1

OAc

I... ~~,~

.0

~

HO General Characteristics Crystals; mp., 236-238~

[0[,]D25 +48~ diacetate; mp., 270-271~

[~]D 25 +51 ~

Fungal Source Echmodontium tinctorium, a wood-rotting fungus commonly found on conifers, particularly in the Northwest. Isolation/Purification The dried fungus was extracted with petroleum ether, followed by column chromatography on alumina and repeated recrystallizations. Biological Activity The fungus was used as a bactericidal agem by the Wasco Indians, and tumor-inhibiting activity of crude material was indicated in preliminary tests. Spectral Data 1H NMR: (CDC 13) H-3, 3.20(m); H- 16, 3.74(m); H-22, 4.51 (t, J 9Hz); H-23, 4.10(t, J=9Hz); H-24, 5.16(allylically broadened doublet); 18-CHs, 0.69; 19-CH3, 1.00; 21-CH3, 0.90(d, J=7.0Hz); 26, 27-CH3, 1.69(d, J=l.SHz); 30-CH3, 0.82; 31-CH3, 1.00; and 32CH3, 1.05ppm. Reference F. H. Bond, D. S. Fullerton, L. A. Sciuchetti, and P. Catalfomo; The Isolation and Structure ofEchinodol, a Triterpene Acetate; J. Am. Chem. Sot., Vol 88, pp. 3882-3883 (1966).

214

6.

C32 Sterols

Common/Systematic Name 3-Epiechinodol Molecular Formula/Molecular Weight C32H5oO4; M W = 498.37091

OCOMe

Illlll%o

H

,,1.1111~1~

Y

"--

HO General Characteristics Colorless needles from methanol; mp., 230-23 I~ CHC13).

[~]D 19 +

45 ~ (c=l.0, in

Fungal Source Fruit bodies ofEchinodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification Fruit bodies ofEchinodontium tsugicola, collected at Marunuma, Gumma Prefecture, Japan, in October 1969, were air-dried, crushed, and extracted twice with ether at room temperature. The combined extracts were treated with hot benzene to remove an orange-yellow amorphous powder. The benzene extract was chromatographed on a column of alumina (Woelm neutral) and eluted successively with (1) benzene, (2) benzene-ether (99:1, v/v), (3) benzene-ether (98.5:1.5, v/v), (4) ethyl ether, and (5) MeOH. Fraction (3) was rechromatographed under similar conditions and each fraction was examined by TLC and GLC. The fractions eluted with benzene that contained deacetoxyechinodone and echinodone were combined and further separated by preparative thin-layer chromatography (silica gel I-IF254). Echinodone and deacetoxyechinodone were recrystallized from MeOH to give colorless needles. The next fraction containing a diterpene ester was recrystallized from MeOH to colorless prisms. The fractions eluted with benzene-ether (98:2, v/v) containing deacetoxy-3-echinodol and deacetoxyechinodol were combined and further purified by preparative thin-layer chromatography. Deacetoxy-3-epiechinodol was recrystaUized from MeOH, while deacetoxyechinodol was obtained as colorless needles from MeOH. The benzene-ether (95:5, v/v) fractions were collected and again separated by preparative thin-layer chromatography into

6.

C32 Sterols three components 3-epiechinodol, deacetylechinodol and deacetyl-3-epiechinodol.

Spectral Data IR~ (KBr) 3480, 1750, and 1228cm"1. 1H NMR: (CDC13) 3.42(m, broadened triplet of about 4Hz, H-3); 3.79(m, H-16); 4.52(t, J=9.0Hz, H-22); 4.03(t, J=9.0Hz, H-23), 5.12(bd, J=9.0Hz, H-24); 0.70 (3H, H-18); 0.98 (3H, H-19); 0.91(3H, d, J=6.0Hz, H-21); 1.70(3H each, bs, H-26, -27); 0.98 (3H, H-30); 0.87; 1.05 (H-32); and 1.98ppm (3H, -OCOCH3.) Mass Data: Anal calcd for C3zH5004: C, 77.1; H, 10.1; found: C, 76.8; H, 10.0%. Reference A. Kanematsu and S. Natori; Triterpenoids ofEchinodontium tsugicola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

215

216

6.

C32 Sterols

Common/Systematic Name Echinodone Molecular Formula/Molecular Weight C32HagO4= 496.35526 OAc i|1||,..

...,,11'.N~

V

General Characteristics Colorless needles from methanol; mp., 225-227~ (227-229~ CHCI3).

[~]D24 + 67~ (c=l.0, in

Fungal Source Fruit bodies ofEchmodontium tsugicola. The fungus is indigenous to Japan and saprophytic to Tsuga diversifolia. Isolation/Purification See 3-epiechinodol for isolation. Spectral Data UV~

~, m,~Et~ 278nm (log e = 1.8). IR~

(KBr) 1748, 1705, and 1225cm~. IH N]VIR: (CDC13 3.20(m, H-3); 3.72(m, H-16); 4.52(t, J=9.0Hz, H-22); 4.03(t, J=9.0Hz, H-23); 5.12(bd, J=9.0Hz, H-24); 0.68 (CH3-18); 0.98 (CH3-19); 0.90 (d, J=6.0Hz, CH3-21); 1.69(6H, bs, CH3-26,-27); 0.80 (6H, CH3-30,-31); 0.98; 1.03(CH3-32); and 1.98ppm (3H, OCOCH3,H-22). Mass Spectrum: HREIMS: 496.346m/e (M), calcd for C32H4804 496.352; LREIMS: 71(10%), 297(55), 313(8), 421(16), 436(100), 481(2), and 496role (M +, 3).

6.

C32 Sterols

217

Reference A. Kanematsu and S. Natori; Triterpenoids ofEchinodontium tsugicola; Chem. Pharm. Bull., Vol. 20, pp. 1993-1999(1972).

218

6.

C32 Sterols

Common/Systematic Name (22R)25-Hydroxylanosta-8,23-dien-3 ~t-ol-22-monoacetate; (22R)3Gt,25-Dihydroxylanosta-8,23-diene-22-acetate Molecular Formula/Molecular Weight C32H5204; ~

= 500.38656

OAc

H

HO~....% General Characteristics Crystals from methanol; mp., 187-190~ Fungal Source Fruiting bodies ofPisolithus tinctorius. Isolation/Purification Dried fruit bodies were extracted with methanol, which was divided into benzene soluble and insoluble fractions. The benzene soluble fraction was chromatographed on a silica gel flash column using hexane-ethyl acetate (7:3, v/v) to yield purified (22R)-3a,25dihydroxylanosta-8,23-diene-22-acetate. Spectral Data IR~

(KBr) 3370(OH) and 1720cm~ (C=O). 1H NMR: (CDC13) 0.70(3H, s); 0.82(3H, s); 0.87(3H, s); 0.94(6H, d, J=6Hz); 0.97(3H, s); 0.98(3H, s); 1.32(6H, s); 2.05(3H, s); 3.42(1H, J~,2=7Hz); 5.30(1H, dd, Jl=7.5Hz, J2=3.7Hz); 5.65(1H, dd, Jl=14.7Hz, J2=7.5Hz); and 5.86ppm (1H, d, J=14.7Hz). Mass Spectrum: 500[M +, C32H5204]§ (10%), 485[M-Me] § 467[M-H20-Me] + (2), 440[M-AcOH] + (26), 425[M-AcOH-Me] + (30), 407[M-AcOH-Me-H20] + (40), 389[M-AcOH-Me-2H20] + (4), 325[M-CsH~303-H20] § (14), and 314m/e [M-CsH~303]§ (8).

6.

C32 Sterols

219

Reference A. M. Lobo, M. de Abreu, S. Prabhakar, R. Jones, H. S. Rzepa, and D. J. Williams; Triterpenoids of The Fungus Pisolithus tinctorius; Phytochem., Vol. 27, pp. 3569-3574 (1988).

220

6.

C32 Sterols

Common/Systematic Name

1213-Acetoxy-4,4-dimethyl-24-methylene-5a-cholesta-8,14-diene-313,11 a-diol

Molecular Formula/Molecular Weight C32H5oO4; M W "-- 4 9 8 . 3 7 0 9 1

AcO

HO

. ~

- lh

General Characteristics Crystallized from acetone-hexane as needles; mp., 180-181 ~ CHC13).

[tt] 25 + 32.7 ~ (c=0.44, in

Fungal Source Fusarium sporotrichioides 921

Isolation/Purification Fungal cultures were extracted with ethyl alcohol, evaporated to dryness, diluted with water, and partitioned with light petroleum, diethyl ether, ethyl acetate, chloroform, and methylene chloride. The light petroleum and diethyl ether fractions were combined and treated with diazomethane. The mixture was concentrated in vacuo and the residue diluted with light petroleum ether. White needles of T-2 toxin formed and were removed by filtration. 313-Acetoxy-4,4-dimethyl-24-methylene-5a-cholesta-8,14-diene-3 D,11 a-diol was isolated from the mother liquor by chromatography on a Florisil column eluted with light petroleum and light petroleum-ethyl acetate followed by crystallization from acetonehexane. Spectral Data IR:

(KBr) 3450, 1712, 1260, 1060, and 880cm".

'H NMR: (CDCI3) 2.07(s,12-OAc); 3.33(m, H-3); 4.3(s,H-I I);4.75(2H, d, J=SHz, H-28); 5.03(s,H-12); and 5.67ppm (s,H-15).

6.

C32 Sterols

221

13CNMR: (CDCI3) C-l, 34.36; C-2, 27.38; C-3, 79.16; C-4, 39.04; C-5, 50.15; C-6, 18.07; C-7, 26.94; C-8, 125.85; C-9 138.39; C-10, 37.17; C-11, 68.55; C-12, 79.02; C-13, 46.45; C-14, 146.69; C-15, 128.82; C-16, 35.17; C-17,48.97; C-18, 16.68; C-19, 22.05; C20, 33.25; C-21, 18.07; C-22, 33.25; C-23, 30.89; C-24, 156.38; C-25, 33.81; C-26, 21.85; C-27, 21.99; C-28, 105.99; C-29, 15.51; C-30, 28.19; CH3CO, 170.73; and CH3CO, 21.26ppm. Mass Spectrum:

LREIMS: 498(M+), 438(M+ - 60), and 423m/e (M +- 60- 15).

Reference B. Yagen, P. Horn, A. Z. Joffe, and R. H. Cox; Isolation and Structural Elucidation of a Novel Sterol Metabolite ofFusarium sporotrichioides 921; J. Chem. Sot., Perkin Trans I, pp. 2914-2917(1980).

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Miscellaneous Sterols Pachymic acid (313-Acetoxy-16~t-hydroxylanosta-8,24(28)-dien-21-oic acid) 3-O-Acetyltumulosic acid Carbomethoxyacetylquercinic acid Carboxyacetylquercinic acid 1213-Hydroxycarboxyacetylquercinic acid 1213-Hydroxycarbomethoxyacetylquercinic acid Carboxyacetylstowardolic acid 23,24,25,2 6,2 7-Pentano rlano st-8-ene- 313,22-dio1 24E-Ethylidene-5~t-lanosta-8-ene-313-acetate 22~-Acetoxy-313,23~-dihydroxy-24( 28)Z-ethyli denelanost-8-ene 22(-Acetoxy-313,23~-dihydroxy-24(28)-methylenelanost-8-ene Terretonin 313,613-Dihydroxy-4,4,14a-trimethyl- 5Gt-pregn-8-en-20-one 3Gt-Hydroxy-4,4,14~t-trimethyl-5a-pregn-8-en-20-one 3a-Hydroxy-513-cholest-11-en-24-oic acid

223

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7.

Miscellaneous Sterols

225

Common/Systematic Name Pachymic acid 3 D-Acetoxy- 16tt-hydroxylanosta-8,24(28)-dien-21-oic acid Molecular Formula/Molecular Weight C33H5205; MW = 528.38148

MeCO0 General Characteristics Methyl ester: crystals; mp., 185-187~

[{g]D 14 +

41.5 ~

Fungal Source Sclerotia o f Poria cocos. Isolation/Purification The powdered sclerotia were extracted with ethyl ether and the extract was concentrated, from which crude pachymic acid separated out. The crude acid was methylated with diazomethane followed by final purification on an alumina column. Biological Activity "Fuling" is the naturally occurring sclerotium o f Poria cocos and has been frequently prescribed in Chinese medicine as a diuretic and for palpitations. Spectral Data (Methyl ester) UV~

/~

M~)H

234(e= 2,500), 243(2,950), and 25 lnm (2,000).

1H NMR: (CDC13) 0.58, 0.72; 0.87; 0.96; 1.06; and 1.13ppm.

226

7.

Miscellaneous Sterols

References A. Kanematsu and S. Natori; Triterpenoids of Hoelen (ruling), Sclerotia o f P o r i a cocos Wolf II. 313-Hydroxylanosta-7,9(11),24-trien-21-oic Acid; Chem. Pharm Bull., Vol. 18, pp. 779-783(1970). J. Valisolalao, L. Bang. J-P. Beck, and G. Ourisson; Etude Chimique et Biochimique de Drogues Chinoises. V. Cytotoxicite de Triterpenes de P o r i a cocos (Polyporacee et de Substances Analogues); Bull. Soc. Chim. Fr., pp. 473-477(1980).

7.

Miscellaneous Sterols

227

_Common/Systematic Name 3-O-Acetyltumulosic acid Molecular Formula/Molecular Weight C33H5205; MW = 528.38148

HO0

AcO General Characteristics Methyl ester, crystals from aqueous methanol; mp., 184-186~

[0~]D + 41 o

Fungal Source Trametes feei, T. lilacino gilva, and Poria cocos. Isolation/Purification The fungus was extracted with ethyl ether, methylated, chromatographed on alumina, eluted with benzene (3 different fractions), and benzene-chloroform, 1:1 (v/v). The first benzene fraction in methanol and dioxane together with sodium borohydride was stirred at room temperature for 2 hours. Water was added and the resultant precipitate was chromatographed on alumina using benzene and recrystallized from aqueous methanol to give the purified methyl ester of 3-O-acetyltumulosic. Biological Activity Antibiotic Spectral Data (Methyl ester) UV~

max243nm (broad, e = 500). IR~

(CHC13) 3480(OH), 1715(unresolved CO2Me, OAc), 1260(OAc), 1645, and 887cm 1 (C=CH2).

228

7.

Miscellaneous Sterols

1H NMR: (CDC13) 4.7(2H, m, vinylic protons); 4.50(1H, q, J=4.0, 10Hz, CHOAc); 4. I(1H, m, CHOH); 3.7(3H, s, CO2Me); 2.04(3H, s, OCOMe); and 1.82ppm (OH). Mass Data: LREIMS: 542m/e (M); calcd for C34H5405,542; found: C, 75.2; H, 9.9%; calcd for C34Hs405; C, 75.2; H, 10.0%. References J. T. Pinhey, B. J. Ralph, J. J. H. Simes, and M. Wootton; A New Source of Tumulosic Acid and 3-O-acetate tumulosic Acid; Aust. J. Chem., Vol. 23, pp. 2141-2146(1970). J. T. Pinhey, B. J. Ralph, J. J. H. Simes, and M. Wootton; Extractives of Fungi I1. The Constituents of Trametesfeei, 6tx-Hydroxypolyporenic Acid C; Aust. J. Chem., Vol. 24, pp. 609-619(1971).

7.

Miscellaneous Sterols

229

Common/Systematic Name Carbomethoxyacetylquercinic acid Molecular Formula/Molecular Weight C35H5407, MW = 586.38696

H

MeOCOCH2CO0 ...... General Characteristics Crystals; mp., 165.5-167~

[~t]D + 0.7 ~

Fungal Source

Trametes dickinsii (Japanese name Hoorokutake).

Spectral Data 1H NMR: (Methylated derivative) 6.59(s, 2H); 6.72(s, 3H); and 6.33x (s, 3H). Reference H. Inouye and K. Tokura; Absolute Configuration of Carboxyacetylquercinic Acid, Chemical Correlation of Carboxyacetylquercinic Acid with Lanosterol; Z. Naturforsch., Teil B, Vol. 25, pp. 1194-1195(1970).

230

7.

Miscellaneous Sterols

Common/Systematic Name Carboxyacetylquercinic acid Molecular Formula/Molecular Weight C34H5207; MW = 572.37130 _

H

HOCOCH2CO0 ......

General Characteristics Crystals; mp., 221-224~

[~]D +

8.80.

Fungal Source Trametes dickinsii (Japanese name Hoorokutake) and Daedalea quercina. Spectral Data 1H NMR: (Methylated derivative) 6.59(s, 2H); 6.72(s, 3H); and 6.33x (s, 3H). References H. Inouye and K. Tokura; Absolute Configuration of Carboxyacetylquercinic Acid, Chemical Correlation of Carboxyacetylquercinic Acid with Lanosterol; Z. Naturforsch., Teil B, Vol. 25, pp. 1194-1195(1970). S. Natori, A. Yokoyama (nee Kanematsu), and K. Aoshima; Distribution of Tetracyclic Triterpenoids of Lanostane Group in Pore Fungi (Basidiomycetes). In Chemistry in Botanical Classifications, G. Bendz, J. Santesson, eds., Nobel Foundation, Stockholm, Academic Press, New York, pp. 173-179(1974).

7.

Miscellaneous Sterols

231

Common/Systematic Name 1213-Hydroxycarboxyacetylquercinic acid Molecular Formula/Molecular Weight C34H5208; MW = 588.36622 .

OH

t.. HO2CCH2CO0 ......

Fungal Source Melanoporia rosea and M. nigra. v

Isolation/Purification Fruit bodies were extracted with ethyl ether, methylated, and purified by preparative TLC using silica gel H or HF254and developing solvents of hexane-ethyl acetate (7:3, v/v) and chloroform-methanol (9:1, v/v); detection was by spraying 20% vanillin-phosphoric acid and heating. Spectral Data ~H NMR: (CDC13) 0.74(3H, s, H-18); 1.00(3H, s, H-19); 0.92(6H, H-30, -18); 0.88(3H, s, H32); 1.04(3H, d, J=6Hz, H-27); 1.12(3H, d, J=6Hz, H-28); 4.66(1H, m); 3.61(3H, s, OCH3); and 3.38ppm (2H, s, OCH2-). C-21 methyl appeared at 0.9-1.0ppm overlapping with other methyl signals. References S. Natori, A. Yokoyama (nee Kanematsu), and K. Aoshima; Distribution of Tetracyclic Triterpenoids of Lanostane Group in Pore Fungi (Basidiomycetes). In Chemistry in Botanical Classifications, G. Bendz, J. Santesson, eds., Nobel Foundation, Stockholm, Academic Press, New York, pp. 173-179(1974). W. B. Turner and D. C. Aldridge; Fungal Metabo_lites II; Academic Press, New York, p. 323 (1983). A. Yokoyama and S. Natori; Triterpenoids of Lanostane Group from Fruit Bodies of Nine Basidiomycetous Species; Chem. Pharm. Bull., Vol. 22, pp. 877-883(1974).

232

7.

Miscellaneous Sterols

A. Yokoyama, S. Natori, and K. Aoshima; Distribution of Tetracyclic Triterpenoids of Lanostane Group and Sterols in The Higher Fungi Especially of the Polyporaceae and Related Families; Phytochemistry, Vol. 14, pp. 487-497(1975).

7.

Miscellaneous Sterols

233

Common/Systematic Name 1213-Hydroxycarbomethoxyacetylquercinic acid Molecular Formula/Molecular Weight C35H54Os; MW = 602.38187

L. 6 OH ........

coo.

MeO2CCH2COO ....... General Characteristics (Methyl ester) Crystals from methanol; mp., 116-117 ~ [~]D 21 + 4.66 o (c=0.97, in CHC13); oily monoacetate; [a]D21 + 5.26~ diol monomethyl ester; [a]D21 + 19.0 ~ Fungal Source Me lanoporia rosea.

Isolation/Purification Fruit bodies were extracted with ethyl ether, methylated, and purified by preparative TLC using silica gel H o r I--~254 and developing solvents ofhexane-ethyl acetate (7:3, v/v) and chloroform-methanol (9:1, v/v); detection was by spraying 20% vanillin-phosphoric acid and heating. Spectral Data IR:

(methyl ester) 3420, 1750, 1735, 1708, and 1700cm"1. 1H ~:

(CDCI3) 0.72(3H, s, H-18); 1.01(3H, s, H-19); 0.92(6H, H-30, -18); 0.87(3H, s, H32); 1.03(3H, d, d--6Hz, H-27); I.12(3H, d, d=6Hz, H-28); 4.68(IH, m); 4. I0(IH, m); 3.62(3H, s, OCH3); and 3.38ppm (2H, s, OCH2-). C-21 methyl appeared at 0.9-1.0ppm overlapping with other methyl signals.

234

7.

Miscellaneous Sterols

Mass Spectrum: (methyl ester) HREIMS: 616.409(M § calcd for C36H5608:616.398), 5 9 8 ( M + - H 2 0 ) , 583(M + - CH3- H20), 498(M +- C4H604), 483(M +- CH3- C4H604), 465(M +- CH3 H20 - C4H604), 325(M § - CH3- Cr - C8H~403), and 307m/e (M § - CH3-1-120 C4I-I604 - C8H1403).

References W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p.323 (1983). A. Yokoyama and S. Natori; Triterpenoids of Lanostane Group from Fruit Bodies of Nine

Basidiomycetous Species; Chem. Pharm. Bull., Vol. 22, pp. 877-883(1974).

7.

Miscellaneous Sterols

235

Common/Systematic Name Carboxyacetylstowardolic acid 3a_Carboxyacetoxy_24~-methyl-23-oxolanosta-8,25(26)-dien-27-oic acid Molecular Formula/Molecular Weight C34H5007; M W -- 5 7 0 . 3 5 5 6 5

COOH

HO2CCH2OCO General Characteristics Crystals from aqueous methanol; mp., 184-186~ Fungal Source Trametes stowardii. Isolation/Purification Fungal cultures were extracted with 95% ethanol, evaporated to dryness, and redissolved in ethyl ether. The ether soluble fraction was partitioned between 2N HC1 and 5% NaHCO3. Acidification of the bicarbonate solution and extraction with ether gave a crude product that was chromatographed on a silicic acid column. Spectral Data UV: ~, max

210nm (e = 11,800)

IR: (Nujol mull) 1765, 1740, 1725, 1715, and 1695cm1. 1H N M R : (CDC13) 4.75(J=6.0Hz, H-3); 3.7(q, J=7.0Hz, H-24); 5.95(3.0Hz, H-26); 6.2(2.5Hz, H-26); 1.35(3H, d, J=7.0Hz, H-31); 10.0(2H, OH, CO2H); and 3.45ppm (2H, COCH2CO). Reference H. T. Cheung, J. C. F. Seeto, and T. R. Watson; Triterpene Metabolite of Trametes stowardii; Aust. J. Chem., Vol. 26, pp. 609-618(1973).

236

7.

Miscellaneous Sterols

Common/Systematic Nam.e 23,24,25,26,27-P entanorlanost-8-ene- 3 J3,22-diol Molecular Formula/Molecular Weight C25H4202; M W -- 3 7 4 . 3 1 8 4 8

....... CH20H

NO General Characteristics Prisms or needles from methanol; mp., 206-207~ atter a transition at 90~ for loss of solvent; diacetate; mp., 175-177~ Fungal Source

Verticillium lecanii (entomopathogenic fungus).

Isolation/Purification The dried mycelium was extracted with petrol followed by ethyl acetate. The extracts in benzene were chromatographed on a silica gel column eluted from benzene to ethyl ether. Crystallization of the benzene-ethyl ether fraction from methanol yielded the purified 23,24,25,26,27-pentanodanost-8-ene-313,22-diol. Soectral Data UV: End absorption. IR~

(KBr) 3380 and 1622(w) cm~. IH NMR: 0.70; 0.79; 0.86; 0.96; 0.98(3H, all s); 1.01(3H, d, J=6.0Hz); 1.1-2.0(19H); 2.0(20H, br); 3.21(dd, J=l 1.5Hz, H-3); 3.34; and 3.63ppm (AB, J=10.5, 6.5, 2.0Hz, H-22).

7.

Miscellaneous Sterols

237

13C NMP~: C-I, 35.6(2); C-2, 27.6" (2); C-3, 79.0(1); C-4, 38.9(0); C-5, 50.4(1); C-6, 18.3(2); C-7, 27.9" (2); C-8, 134.3"* (0); C-9, 134.6"* (0); C-10, 37.0(0); C-11, 21.0(2); C-12, 26.5(2); C-13, 44.7(0); C-14, 49.6(0); C-15, 30.9"** (2); C-16, 31.0 *~"(2); C-17, 46.81; C-18, 15.9 3 ; C-19, 19.1 3 ; C-20, 39.5 1 ; C-21, 16.7 3 ; C-22, 68.2 2; C-28, 24.3(3); C-29, 28.0(3); and C-30, 15.4ppm*'* (3).

, ,

Assignments may be reversed.

Mass Data: HR IMS: 374.3185m/e (M*, C25I-I4202requires 374.3185); found: C, 79.6; H, 11.4; calcd C, 80.1; H, 11.3%. Reference J. F. Grove; 23,24,25,26,27-Pentanodanost-8-ene-313,22-diol from Verticillium lecanii; Phytochemistry, Vol. 23, pp. 1721-1723(1984).

238

7.

Miscellaneous Sterols

Common/Systematic Name E-24-Ethylidene-5a-lanost-8-ene-313-acetate Molecular Formula/Molecular Weight C34H5602; M W -- 4 9 6 . 4 2 8 0 3

AcO

General Characteristics Crystals; mp., 135-138~ Fungal Source M u c o r rouxii isolated from sheep rumen contents.

Isolation/Purification The freeze-dried mycelium o f M u c o r rouxii was extracted with chloroform-methanol (2:1, v/v) and purified by argentation TLC chromatography. Also, see Heupel, R.C.; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.); Academic Press Inc., New York, New York, 341 pp. (1989). Spectral Data UV~ End absorption. 1H NMR: (CDC13) 0.69(3H, H-18); 0.87(9H, H-4Qt, -413, -14); 0.95(3H, H-21); 1.00(3H, H=19); 0.97(3H, H-26,-27); 1.58(3H, H-29); 2.05(3H, 313-O-COCH3); 2.19(1H, H-25); 4.50(1H, H-3a); and 5.19ppm (1H, H-28); free sterol 0.60(3H, s, H-18); 0.81 and 0.87(6H, s, H-4ot and -4[3); 0.96-1.02(15H, m, H-19, -14, -21, -26, and -27); 1.57(3H, d, J=6.0Hz, H-29); 2.29(1H, heptet, J=6.0Hz, H-25); 3.24(1H, q, d=5.0Hz, H-3~); and 5.29ppm (1H, q, J=6.0Hz, H-28).

7.

Miscellaneous Sterols

239

Mass Spectrum: LREIMS: 496, 481, and 42 lm/e; free sterol, HREIMS: 454.4169role (M+); calcd for C32H540, 454.4174. References W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc., New York, New York, 341 pp. (1989). S. Safe and L. M. Safe; The Isolation and Synthesis of E-24-Ethylidene-5a-lanost-8-en-313 Acetate; Canadian J. Chem., Vol. 53, pp. 3247-3249(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

240

7.

Miscellaneous Sterols

Common/Systematic Name 22~-Acetoxy-313,23~-dihydroxy-24( 28 )Z-ethylidenelanost-8-ene Molecular Formula/Molecular Weight C34H5604; MW

-

-

528.41786

AcO , / " I1|

Ho

-

General Characteristics Free alcohol (C32H5403): mp., 192-196~ [(I,]D20 + 39~ (c=l.1, in CHC13); triacetate: mp., 160-165~ [~t]D2~ + 30 ~ (C=0.9 in CHC13); triol: mp., 193-195~ [a]D2~ + 38 ~ (c=l. 1, in CHC13). Fungal Source

Pisofithus tinctorius.

Isolation/Purification Purified by extraction with dichloromethane and chromatographic purification of the crude extract. Spectral Data IR:

(KBr) 1735cml. 1H NMR: (CDC13) Free alcohol: 0.745; 0.819; 0.921; 0.990; and 1.005 attributed to C-18, C-30, C-31, C-32, and C-19 tertiary methyl groups (each 31-1,s); 0.936(3H, d, J=6.0Hz) assigned to secondary methyl group at C-21; 1.078(3H, d, J=6.0Hz), 1.152(3H, d, J=6.0Hz); and 2.833(1H, d, J=6.0Hz) assigned to the isopropyl group attached to C24; 1.470(3H, d, J=-6.0Hz); and 5.636(1H, q, J= 6.0Hz) suggested for CH3-CH=CR2 group; 3.225(1H, m, H-3a); 3.984(1H, d, J=9.0Hz); and 3.627(1H, d, J=9.0Hz) attributed to carbons bearing secondary OH groups. Reference A. M. Lobo, P. Macedo de Abreu, S. Prabhakar, and L. S. Godinho; Novel Triterpenoids from The Fungus Pisolithus tinctorius; Tetrahedron Letters, Vol. 26, pp. 2589-2592(1985).

7.

Miscellaneous Sterols

241

Common/Systematic Name 22~-Acetoxy-313,23~-dihydroxy-24-methylenelanost'8-ene .Molecular Formul.a/Molecular Weight C33H5404; M W - 5 1 4 . 4 0 2 2 1

AcO

,,

HO

General Characteristics Triol; mp., 193-195~

[~]D 20 +

38~ (c=l.1, in CHC13).

Funsal Source v

Pisolithus tinctorius.

Isolation/Purification Purified by extraction with dichloromethane and chromatographic purification (preparative TLC) of the crude extract. Soectral Data IR~

(KBr) 1735cm"1. 1H NMR: (CDC13) Spectrum similar to 22~-acetoxy-313,23~-dihydroxy-24(28)Z-ethylidenelanost-8-ene except for substitution on side chain. Reference A. M. Lobo, P. Macedo de Abreu, S. Prabhakar, and L. S. Godinho; Novel Triterpenoids from The Fungus Pisolithus tinctorius; Tetrahedron Letters, Vol. 26, pp. 2589-2592 (1985).

242

7.

Miscellaneous Sterols

Common/Systematic Name Terretonin Molecular Formula/Molecular Weight C26H3209; M'W --- 4 8 8 . 2 0 4 6 3

H2C

Me

0 Me

Me 0

~ 0 Me" Me OH

......I0~

O--Me

0

Fungal Source Aspergillus terreus. Isolation/Purification Fungal cultures were extracted with chloroform, filtered, dried over anhydrous sodium sulfate, and reduced in volume. The crude extract was chromatographed on a Florisil column eluted with a gradient from benzene to ethyl acetate. The biologically active fractions were combined, reduced in volume, and chromatographed on a Woelm neutral grade IV alumina column eluted with a gradient from benzene to ethyl acetate. The active fractions were combined, reduced in volume, and allowed to sit at 5~ until crystals formed. Biological Activity The metabolite had an LDs0 in day-old cockerels of between 250-375mg/kg body weight. Spectral Data UV~

maxM~H276nm (e = 7,960). Im~

(KBr) 3470, 3340, 2940, 1764, 1748, 1729, 1708, 1685, 1641, 1452, 1372, 1307, 1265, 1252, 1171, 1150, 1118, 1018, 999, and 919cm"1. CD: (c-0.54, in MeOH) [01221 - 9200 ~ [0]275 -20,900 ~ [0]305 -3400 ~ [01317 - 3,700 ~ and [0]329- 2,700.

7.

Miscellaneous Sterols

243

1H NMR: (CDCI3) 1.22; 1.45; 1.48 x 2; 1.73; 1.94(s, 3H x 6, H-20 ABC, H-21 ABC, H-24 ABC, H-26 ABC, H-28 ABC, H-30 ABC); 1.80(cm, 1H, H-1A eq); 1.80(s, ex, 1H, H-25); 2.28(bd, J=14Hz, 1H, H-11 A or B); 2.38(ddd, J=14, 11,8Hz, 1H, H-1Bax); 2.54(ddd, J=19, 11, 8Hz, 1H, H-2Bax); 2.73(bdd, J=19, 8Hz, 1H, H-2Aeq); 2.99(bd, J=14Hz, 1H, H-11 A or B); 3.55(s, 1H, H-14); 3.81(s, 3H, H-34 ABC); 5.11, 5.49(bs, 1H x 2, H-27 AB); and 6.16ppm (s, ex, 1H, H-22).

13C NMR: (CDC13) C-l, 32.7a; C-2, 34.4a; C-3,214.5; C-4, 52.2; C-5, 130.0b; C-6, 139.8b; C-7, 197.3; C-8, 43.2; C-9, 77.5; C-10, 47.7c; C-11, 28.1; C-12, 139.1; C-13, 49.1c; C-14, 43.9; C-15, 168.2; C-17, 85.2; C-18, 201.8; C-20, 21.3d; C-21, 24.0d; C-24, 21.3d; C-26, 19.4~; C-27, 115.2; C-28, 18.6a; C-30, 23.5d; C-31,168.2; and C-34, 53.4ppm. a, b, e, d Assignmentsmay be reversed.

Mass Data: HREIMS: 488.2066(M +, calcd for C26H3209, 488.2046), 473.1844(-CHs), 470.1959 (-H/O), 357.1702(- C5H704), and 330.1850m/e (- C6H605); Found C, 64.07%; H, 6.80; and O by difference 29.13. Required for C26H3209: C, 63.9%; H, 6.6; 0,29.5. TLC Data Appeared as a green spot at Rf 0.3 using silica gel GH-R plates developed with chloroform-acetone, 93:7, v/v and detected with 50% ethanolic sulfuric acid spray followed by heating at 125~ for 5 min. Reference J. P. Springer, J. W. Domer, R. J. Cole, and R. H. Cox; Terretonin, A Toxic Compound from Aspergillus terreus; J. Organic Chemistry, Vol. 44, pp. 4852-4854(1979).

244

7.

Miscellaneous Sterols

Common/Systematic Name 313,613-Dihydroxy-4,4,14a-trimethyl-5 a-pregn-8-en-20-one Molecular Formula/Molecular Weight C24H3803; M W = 3 7 4 . 2 8 2 1 0

z

HO OH General Characteristics Prisms from isopropyl ether; mp., 188-189~ [{X]D24 + 20 ~ (c= 1.0, in CHC13); diacetate: crystals from methanol; mp., 258-260~ [a]D + 24 ~ (C=0.24, in CHC13). Fungal Source F o m e s officinalis

Isolation/Purification See Epstein and G. van Lear; J. Org. Chem., Vol. 31 p. 3434(1966). Spectral Data IR~

(CC14) 3350 and 1685cm1; diacetate: 1739, 1718, and 1247cm~. ORD: [a]600 + 54 ~ [a]ss9 + 54~ [a]313 + 895 ~ [a]268 - 574 ~ and [a] 247 0~ (c=0.14, in dioxane). 1H NMR: (CDC13) 9.28, 9.14, 9.05, 9.01, and 7.80z (methyl resonances in the ratio of 1 1:2:1:1); diacetate; 7.99z (6H) methyl resonances. Mass Data: LREIMS: 374m/e (M+); anal calcd for C24H380 3" C, 76.96; H, 10.23%; Found: C, 76.66; H, 10.24%; diacetate, 398role ( M ~ - 60). Reference C. G. Anderson, W. W. Epstein, and G. Van Lear; Minor Triterpenoids o f F o m e s officinalis; Phytochemistry, Vol. 11, pp. 2847-2852(1972).

7.

Miscellaneous Sterols

245

Common/Systematic Name 3 a-Hydroxy-4,4,14 a-trimethyl- 5 a-pregn-8-en-20-one Molecular Formula/Molecular Weight Cz4H3sO2; NYW = 358.28718 M

HC/ General Characteristics Crystals from methanol; mp., 230-232~ [a]D + 109~ (C=0.18, in hexane); acetate, crystals from methanol; mp., 250-252~ [a]D + 48 ~ (C=0.05, in CHC13). _Fungal Source

Fomes officinalis

Isolation/Purification Finely ground fungus was extracted with petroleum ether, concentrated, and redissolved in methanol. The methanol solution was allowed to sit for several days; the precipitate was removed and the mother liquor was separated into neutral, basic and acidic components. The neutral materials were recrystallized from methanol to give ergosterol. The mother liquor was concentrated and treated with complexing material (urea) to remove fatty substances, which were discarded. The neutral material was isolated, saponified, and noncomplexing materials chromatographed on Florisil. The less polar fractions were rechromatographed on Florisil; 3a-hydroxy-4,4,14a-trimethyl-5a-pregn-8-en-20-one eluted from the column with ether-benzene. Final purification was by recrystallization from methanol. Spectral Data UV~

~, EtO. 235(1og e=3.08), 243(3 11), and 250nm (2.90). IR:

3350 and 1695cm1; diacetate, 1739, 1718 and 1247cm "1. ORD: [a]625 -'1- 90 ~ [a]ss9 + 109 ~ [a]312 + 2370 ~ [0~]267- 2170 ~ and [ a ] 247 - 1950 ~ (c--0.18, in dioxane).

246

7.

Miscellaneous Sterols

IH NMR: (CDC13) 9.27, 9.12, 9.02, and 7.92z (methyl resonances in the ratio of 1:1:3:1). Mass Data: LREIMS: 358role

(M+).

TLC Data Silica gel G plates developed with acetone-chloroform (1:9, v/v). Reference W. W. Epstein and G. Van Lear; Metabolites ofFomes 31, pp. 3434-3435(1966).

officinalis;

J. Org. Chem., Vol.

7.

Miscellaneous Sterols

247

Common/Systematic Name 3tt-Hydroxy-5[3-cholest- 11-en-24-oic acid .Molecular Formula/Molecular Weight C24H3803; M W -- 374.28210 COOH

HO,, ~

H

General Characteristics Crystallized from benzene solution to give plates; mp., 166~ [~]D 19 + 25.9 ~ (c=2.2, in CHC13). Others 164-166~ 165-165.5~ and 168-169~ Acetoxy derivative: crystals from methanol; mp., 126-127~ Fungal Source Curvularia

spp.

Spectral Data IR:

(KBr) 3420(OH), 2660 (H-bonded OH), and 1729cm1 (carboxylic C=O). 1H N M R :

(CDC13) 3.91, 4.57, and ca. 7.72z (AMXm, J A i l 0 , Jgx=2, J~c=3Hz, -CH=CH CH=); 4.26br (1H, s, =CHOH); ca. 6.35(1H, J1,2 ca. 20Hz, axial =CHOH); and 9.12 and 9.28z (each 3H, s, CH3 at C-10 and C-13); acetoxy derivative: 3.90, 4.58 and ca. 7.70z (AMXm, JA~10, Jgx=2, JMx=3Hz, -CH=CH-CH=); ca. 5.26(1H, J1,2 ca. 20Hz, axial =CHOAc); 7.99(3H, s, CH3CO2R); and 9.12 and 9.281: (each 3H, s, CH3 at C-10 and C-13). Mass Data: LREIMS: 374(M § 9%), 356(100), 341(35), 255(45), 228(400), and 2 1 3 m / e (25); acetoxy derivative: 416(M*, 0.2%), 356(66), 341(29), 255(71), 228(63), and 213role (39). Found: C, 76.8; H, 10.3%; C24H3sO3requires C, 76.95; H, 10.25%; acetoxy derivative HREIMS: 3 5 6 . 2 7 1 7 m / e , C24H3602 requires 356.2715. Reference H. D. Munro, O. C.Musgrave, and A. B. Turner; 3~-Hydroxy-5[3-chol-11-en-24-oic Acid, a New Fungal Metabolite; J. Chem. Soc., Perkin Trans. I, pp. 1597-1598(1974).

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Antheridiols and Oogoniols Antheridiol 23-Deoxyantheridiol Oogoniol Oogoniol-1 Oogoniol-2 Oogoniol-3 15-Keto-oogoniol-2 24(28)-Dehydro-oogoniol 24(28)-Dehydro-oogoniol-1 24(28)-Dehydro-oogoniol-2 24(28)-Dehydro-oogoniol-3

249

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8. Antheridiols and Oogoniols

251

Common/Systematic Name Antheridiol Molecular Formula/Molecular Weight C29H4205; M W -" 470.30322 HO,,,

H

Tu /

1

General Characteristics Crystals; rap., 250-255~ (dec.) Fungal Source A chlya bisexualis. Isolation/Purification The fungal mycelium was removed by filtration. The filtrate was adjusted to pH 4.00 and the hormone activity was extracted with methylene chloride. Concentration of the extract produced a brown gum that was chromatographed on silica gel with ethyl acetate as eluant. Biological activity eluted late in the column elution. Final purification was achieved by preparative TLC of the biologically active fraction on silica gel H with chloroformmethanol (10:1, v/v) as solvent. Antheridiol formed crystals in the yellow gum eluted from preparative TLC which were removed by dissolving the impurities away from the crystals with methanol-ethyl ether. Biological Activity Sex hormone secreted by the female that induces formation of antheridial hyphae on the male. Spectral Data UV:

l=ax 222nm (log e 4.22). IR:

3390, 1742, and 1672cm "1.

252

8. Antheridiols and Oogoniols

1H NMR: (CDCI3-MeOD, 3:1, v/v) 0.70(18-H); 1.17 and 1.22(pair of d, J=7Hz, isopropyl CH3); 1.20(19-H); 4.95(d, J=8Hz, 23-H); 5.69(6-H); and 5.77ppm (24'-H). Mass Spectrum: LREIMS: 470(NV), 344, 326, 298, 287, 269, and 25 lm/e. References G. P. Arsenault, K. Biemann, A. W. Barksdale, and T. C. McMorris; The structure of Antheridiol, a Sex Hormone inAchlya bisexualis; J. Am. Chem. Soc., Vol. 90, pp. 56355636 (1968). J. A. Edwards, J. S. Mills, J. Sundeen, and J. H. Fried; The Synthesis of the Fungal Sex Hormone Antheridiol; J. Am. Chem. Soc., Vol. 91, pp. 1248-1249(1969). J. A. Edwards, J. Sundeen, W. Salmond, T. Iwadare, and J. H. Fried; A New Synthetic Route to the Fungal Sex Hormone Antheridiol and the Determination of its Absolute Stereochemistry; Tetrahedron Letters, pp. 791-794(1972). T. C. McMorris and A. W. Barksdale; Isolation of a Sex Hormone from the Water Mold

Achlya bisexualis; Nature, Vol. 215, pp. 320-321(1967).

8. Antheridiols and Oogoniols

253

.Common/Systematic Name 23-Deoxyantheridiol Molecular Formula/Molecular Weight C29H4204; M W -- 4 5 4 . 3 0 8 3 1

HO' General Characteristics Crystals; mp., 265-270~ Fungal Source

Achlya ambisexualis (No. 734) and A. bisexualis (No. 369).

Isolation/Purification Liquid cultures of the mold were extracted with methylene chloride and the concentrated extract shaken briefly with dilute sodium hydroxide solution to remove fatty acid material. The solvent was removed and the residue chromatographed on silica gel eluted with ethyl acetate-petroleum (1:1, v/v). Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. Deoxyantheridiol possessed an activity of about 1/100th that of antheridiol but may have been due to the contamination with small amounts of antheridiol.

254

8. Antheridiols and Oogoniols

Spectral Data UW:

~

EtOH max

230nm (e = 17,900); treatment with acid changed the spectrum to ~.,~, 212 and 278nm.

IR; KBr V max

3470, 1706, 1672, and 1633cm "1.

CD: [ 0]400 +0 ~ [ ~3s, -30 ~ [ fl]3so -65 ~ [ fl]376 -77 ~ [ 0]370 +0 ~ [/7]360 +330 ~ [ fl]35o + 1160 ~ [ 0]340 +2170 ~ [/7]330 +3030 ~ [ 0]320 +2870 ~ [ ~3~o +2110 ~ [/7]300 +1040 ~ [fl]2s9 +0 ~ [fl]2so -2180~ -6300~ [0]260 -14,710 ~ [/7]250 -24,740 ~ [ 0]240 -22,400 ~ and [ 0]23o -14,140 ~ ~H M R : (CDCI3) 0.71(18-H); 1.02(1H, d, J=6.0Hz, 21-H); 1.10(3H, d, J=7.0Hz, isopropyl CH3); 1.19(1H, 19-H); 3.3-3.8(1H, m, 3a-H); 4.3, 4.4(1H, pair of broad triplets, 22H); 5.69(6H); and 5.75ppm (1H, lactone olefinic H). Mass Spectrum: LREIMS: 454(M +, 28%), 436(23), 316(41), 245(100), 192(33), 161(30), and 139m/e

(45).

Reference D. M. Green, J. A. Edwards, A. W. Barksdale, and T. C. McMorris; The Isolation and Structure of 23-Deoxyantheridiol and the Synthesis of its C-22 Epimer; Tetrahedron, Vol. 27, pp. 1199-1203(1971).

8. Antheridiols and Oogoniols

255

Common/Systematic Name Oogoniol Molecular Formula/Molecular Weight C29H4805 ; M W --- 4 7 6 . 3 5 0 1 7

HOH2C~ IIIll,,..

HO,, OH HO Fungal Source

Achlya heterosexualis and A. bisexualis.

Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. References T. C. McMorris, R. Seshadri, G. R. Weihe, G. P. Arsenault, and A. W. Barksdale; Structures of Oogoniol-1, -2, and -3, Steroidal Sex Hormones of the Water Mold, Achlya; J. Amer. Chem. Soc., Vol. 97, pp. 2544-2545(1975). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R313,11t~,1513,29-Tetrahydroxystigmast-5-en-7-one and Identification of 24(28)Dehydrooogoniols as Hormones in Achlya; J. Amer. Chem. Soc., Vol. 101, pp. 30663071(1979).

256

8. Antheridiols and Oogoniols

Common/Systematic Name Oogoniol-1 Molecular Formula/Molecular Weight C33H5406; 1VIW" - 546.39204

HOH2C~ i11111 '

HO,, Me

~CHCO0

OH

Fungal Source

A chlya heterosexualis.

Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1"1, v/v) to give oogoniol-1. Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. Spectral Data UV:

,~ EtOH 235nm (e = 13,500). IR: Chloroform

3625, 3525, 1732, and 1662cm"~.

8. Antheridiols and Oogoniols

257

IH N]V[R: (CDCI3) 0.84(t,J=71-Iz,29-H); 0.950, J=6I-Iz,27-H); 0.98(18-H); 1.01(d,J=7.0Hz, 2 I-H, probably due to the presence of the dehydro derivativesalso present in the sample); 1.16(d,J=7Hz, isobutyrate-CH3);1.36(19-H); 3.65(m, 26-H), 4.16(m, I II~-H, OH); 4.71(m, 3~H, 15a-H); and 5.82ppm (s, 6-H). Mass Spectrum: LREIMS: 458(100), 301(5), 299(21), 283(30), and 161m/e (62). HREIMS: 546.3908m/e (calcd for C33H5406, 546.3920). References T. C. McMorris, R. Seshadri, G. R. Weihe, G. P. Arsenault, and A. W. Barksdale; Structures of Oogoniol- 1, -2, and -3, Steroidal Sex Hormones of the Water Mold, Achlya; J. Amer. Chem. Soc., Vol. 97, pp. 2544-2545(1975). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R3[3,11 t~,1513,29-Tetrahydroxystigmast-5-en-7-one) and Identification of 24(28)Dehydrooogoniols as Hormones in Achlya; J. Amer. Chem. Soc., Vol. 101, pp. 30663071(1979).

258

8. Antheridiols and Oogoniols

Common/Systematic Name Oogoniol-2 Molecular Formula/Molecular Weight C32H5206; M W = 532.37639

HOH2C~

HO,, OH MeCH2COO General Characteristics Crystals from ethyl acetate-petroleum ether; mp., 161-163 ~C. Fungal Source

Achlya heterosexualis.

Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1:1, v/v), to give oogoniol-2. Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. Spectral Data UV:

~,

EtOH max

235nm (e

=

14,300).

8. Antheridiols and Oogoniols

259

IR: Chloroform max

3625, 3540, 1735, and 1660cm~.

1H N]VIR: (CDC13) 0.84(t, J=7Hz, 29-H); 0.95(d, J=7 Hz, 27-H); 0.98(18-H), 1.05(d, J=7.0Hz, 21-H); 1.10(t, J=6 Hz, propionate-CH3); 1.36(19-H); 2.32(q, J=6.0Hz, propionateCH2); 3.65(m, 26-H); 4.16(m, II[3-H, OH); 4.74(m, 3a-H, 15a-H); and 5.82ppm (s, 6H). Mass Spectrum: LREIMS: 532(M+, 9), 458(100), 440(27), 301(7), 299(20), 283(28) and 161role (57); HREIMS: 532.3762role (calcd for C32H5206, 532.3764). References T. C. McMorris, R. Seshadri, G. R. Weihe, G. P. Arsenault, and A. W. Barksdale; Structures of Oogoniol- 1, -2, and -3, Steroidal Sex Hormones of the Water Mold, Achlya; J. Amer. Chem. Soc., Vol. 97, pp. 2544-2545(1975). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R313,11a, 1513,29-Tetrahydroxystigmast-5-en-7-one) and Identification of 24(28)Dehydrooogoniols as Hormones in Achlya; J. Amer. Chem. Soc., Vol. 101, pp. 30663071(1979).

260

8. Antheridiols and Oogoniols

Common/Systematic Name Oogoniol-3 Molecular Formula/Molecular Weight C31H5006, M'W = 518.36074

~

J

~

~

HOH2C~

0

OH "OH

MeCO0

Fungal Source Achlya heterosexualis. Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1:1, v/v), to give oogoniol-3. Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. Spectral Data IR: Cl'dorofom~

V~

3620, 1735, and 1660cm"~.

IH NMR: (CDCI3) 0.84(t, J=7Hz, 29-H); 0.95(d, Jr- 6Hz, 27-H); 0.98(18-H); 1.03(d, J=7.0Hz, 21-H); 1.36(19-H); 2.04(acetate); 3.66(m, 26-H); 4.14(m, ll[3-n, OH); 4.72(m, art-H, 15tt-H); and 5.82ppm (s, 6-H).

8. Antheridiols and Oogoniols

Mass Spectrum: HREIMS: 518.3588m/e

261

(M+).

References T. C. McMorris, R. Seshadfi, G. R. Weihe, G. P. Arsenault, and A. W. Barksdale; Structures of Oogoniol-1, -2, and -3, Steroidal Sex Hormones of the Water Mold, Achlya; J. Amer. Chem. Soc., Vol. 97, pp. 2544-2545(1975). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R313,11a, 1513,29-Tetrahydroxystigmast-5-en-7-one) and Identification of 24(28)Dehydrooogoniols as Hormones inAchlya; J. Amer. Chem. Sot., Vol. 101, pp. 30663071(1979).

262

8. Antheridiolsand Oogoniols

Common/Systematic Name 15-Keto-oogoniol-2 Molecular Formula/Molecular Weight C32H5006; ~ -" 530.36074

HOH2C~

HO,,

MeCH2COO Fungal Source

A chlya heterosexualis.

Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1:1, v/v) followed by crystallization from methanol. The dehydro and 15-keto derivatives occurred as minor metabolites together with parent metabolites and final purification could only be achieved with HPLC (I.t-Bondapak Cls column eluted with a mobile phase of 70% methanol in water and a flow rate of 2.6ml/min at 2000psi). Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. References T. C. McMorris; Sex Hormones of the Aquatic Fungus Achlya; Lipids, Vol. 13, pp. 716722(1978).

8. Antheridiols and Oogoniols

263

M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R-313,11~, 1513,29-Tetrahydroxystigmast-5-en-7-one) and Identification of 24(28)-Dehydrooogoniols as Hormones inAchlya; J. Amer. Chem. Soc., Vol. 101, pp. 3066-3071(1979).

264

8. Antheridiols and Oogoniols

Common/Systematic Name 24(28)-Dehydro-oogoniol Molecular Formula/Molecular Weight

i,,,,,%,~

C29H4605; M W -- 4 7 4 . 3 3 4 5 2

H04,~I~~~0

-OH

Funsal Source

A chlya heterosexualis.

Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1:1, v/v) followed by crystallization from methanol. The dehydro derivatives occurred as minor metabolites together with parent metabolites and final purification could only be achieved with HPLC (~t-Bondapak Cls column eluted with a mobile phase of 70% methanol in water and a flow rate of 2.6ml/min at 2000psi). Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. 24(28)Dehydrooogoniol was active at 50~tg/ml (induced oogonial initials). References T. C. McMorris; Sex Hormones of the Aquatic Fungus Achlya; Lipids, Vol. 13, pp. 716722(1978). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R-313,1let, 1513,29-Tetrahydroxystigmast-5-en-7-one)and Identification of 24(28)-Dehydrooogoniols as Hormones inAchlya; J. Amer. Chem. Sot., Vol. 101, pp. 3066-3071(1979).

8. Antheridiols and Oogoniols

265

Common/Systematic Name 24(28)-Dehydro-oogoniol- 1 11 tt, 1513,29-Trihydroxy-5,24(28)(E)-dien-7-one-313-isobutyrate Molecular Formula/Molecular Weight C33H5206; M W "- 544.37639

Me

iii,,,,,~

"'"~~ 0

~CHCO0

"OH OH

M~~

Fungal Source

Achlya heterosexualis.

Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1:1, v/v) followed by crystallization from methanol. The dehydro derivatives occurred as minor metabolites together with parent metabolites, and final purification could only be achieved with HPLC (I.t-Bondapak C~s column eluted with a mobile phase of 70% methanol in water and a flow rate of 2.6ml/min at 2000psi). 24(28)-Dehydro-oogoniol-1 eluted at 11.9 minutes. Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. 24(28)-Dehydrooogoniol-1 was active at 501.tg/ml (induced oogonial initials).

266

8. Antheridiols and Oogoniols

Spectral Data

IH NIVIR: (CDCI3) 0.84(t,J=7Hz, 29-H); 0.94(d,J=7Hz, 27-H); 0.98(3-H, s); 1.01(6-H, d, J=7.0Hz, isobutyrateCH3, C-26, and C-27 methyls); 1.36(3-H, s); 3.65(broad peak, H29, the methylene protons on C-29 sometimes appear together with the C-I 113 protons at about 4.1ppm); 4.13(m, 1 I13-H);4.69(m, 3a-H, 15a-H, 15a-H); and 5.83ppm (s). Mass Spectrum: LREIMS: 456(M*-88, 100), 440(31), 425(9), 422(11), 407(6), 301(6), 299(3), 283(37), 265(14), 245(9), 227(11), and 161m/e (54). Reference M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R-313,1 ltt, 1513,29-Tetrahydroxystigmast-5-en-7-one) and Identification of 24(28)-Dehydrooogoniols as Hormones inAchlya; J. Amer. Chem. Soc., Vol. 101, pp. 3066-3071(1979).

8. Antheridiols and Oogoniols

267

Common/Systematic Name 24(28)-Dehydro-oogoniol-2 Molecular Formula/Molecular Weight C32H5006; M W -- 530.36074

[ll~l,I, HO,, OH MeCH2CO0

Fungal Source A chlya heterosexualis. Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether (1:1) followed by crystallization from methanol. The dehydro derivatives occurred as minor metabolites together with parent metabolites, and final purification could only be achieved with HPLC (I.t-Bondapak C18 column eluted with a mobile phase of 70% methanol in water and a flow rate of 2.6ml/min at 2000psi). Biological Activity Sexual reproduction in Achlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. 24(28)-Dehydrooogoniol-2 was active at 50[xg/ml (induced oogonial initials). Spectral Data ]H NMR: (CDC13) 0.84(t, J-7Hz, 29-H); 0.94(d, J=7Hz, 27-H); 0.98(3-H,s); 1.01(6-H, d, J-7.0Hz, isobutyrate CH3, C-26 and C-27 methyls); 1.36 (3-H, s); 3.65(broad peak, H29, the methylene protons on C-29 sometimes appear together with the C-11 [3 protons at about 4.1ppm); 4.13(m, ll[3-H); 4.69(m, 3a-H, 15~-H); and 5.83ppm (s).

268

8. Antheridiols and Oogoniols

Mass Spectrum: LREIMS: 456(M+- 88, 100), 440(31), 425(9), 422(11), 407(6), 301(6), 299(3), 283(37), 265(14), 245(9), 227(11), and 161role(54). References T. C. McMorris; Sex Hormones of the Aquatic FungusAchlya; Lipids, Vol. 13, pp. 716722(1978). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R-313,1 la, 1513,29-Tetrahydroxystigmast-5-en-7-one) and Identification of 24(28)-Dehydrooogoniols as Hormones inAchlya; J. Amer. Chem. Soc., Vol. 101, pp. 3066-3071(1979).

8. Antheridiols and Oogoniols

269

Common/Systematic Name 24(28)-Dehydro-oogoniol-3 Molecular_Formula/Molecular Weight C31H4806; M W -- 5 1 6 . 3 4 5 0 9

tlll,l,i, ~ MeCO0~.,.~,~~~ 0

-OH

Fungal Source A chlya heterosexualis. Isolation/Purification Crude active crystalline fractions from chromatography of methylene chloride extracts of the culture liquid were further purified by multiple development preparative thin-layer chromatography with ethyl acetate-petroleum ether ( 1:1, v/v) followed by crystallization from methanol. The dehydro derivatives occurred as minor metabolites together with parent metabolites, and final purification could only be achieved with HPLC (l,t-Bondapak C]s column eluted with a mobile phase of 70% methanol in water and a flow rate of 2.6 ml/min at 2000psi). Biological Activity Sexual reproduction in A chlya is mediated by the steroid hormones antheridiol and the oogoniols. Sexual reproduction is initiated by the vegetative female mycelium which secretes the first hormone, antheridiol (hormone A). In response to antheridiol, male hyphae react by putting out numerous thin lateral branches which secrete another hormone, the oogoniol (hormone B). These in turn induce the development of spherical oogonial initials on female hyphae. Oogonial initials are believed to secrete relatively large amounts of antheridiol (hormone A), which directs the growth of the antheridial hyphae to the developing oogonium, leading to conjugation of the sex organs. Spectral Data ]H NMR: (CDC13) The dehydro derivatives of the oogoniols can be distinguished in the NMR from their parent oogoniols by the presence of a strong chemical shift at 1.01ppm (9H, d, J=7.0Hz, isobutyrate CH3, C-26, and C-27 methyls) and the methylene protons on

270

8. Antheridiols and Oogoniols

References T. C. McMorris; Sex Hormones of the Aquatic FungusAchlya; Lipids, Vol. 13, pp. 716722(1978). M. W. Preus and T. C. McMorris; The Configuration at C-24 in Oogoniol (24R-313,1 la, 1513,29-Tetrahydroxystigmast-5-en-7-one)and Identification of 24(28)-Dehydrooogoniols as Hormones inAchlya; J. Amer. Chem. Soc., Vol. 101, pp. 3066-3071(1979).

Ganoderic Acids Ganoderic acid A Ganoderic acid B Ganoderic acid C1 Ganoderic acid C2 Ganoderic acid D Ganoderic acid E Ganoderic acid F Ganoderic acid G Ganoderic acid H Ganoderic acid I Ganoderic acid J Ganoderic acid K Ganoderic acid L Compound B8 Compound B9 Ganoderic acid Ma Ganoderic acid Mb Ganoderic acid Me Ganoderic acid Md Ganoderic acid Me Ganoderic acid Mf Ganoderic acid Mg Ganoderic acid Mh Ganoderic acid Mi Ganoderic acid Mj Ganoderic acid Mk Ganoderic acid M Ganoderic acid N Ganoderic acid O Ganoderic acid R Ganoderic acid S Ganoderic acid T Ganoderic acid U Ganoderic acid V Ganoderic acid W Ganoderic acid X Ganoderic acid Y Ganoderic acid Z Ganodermic acid R Ganodermic acid S

271

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9. Ganoderic Acids

273

Common/Systematic Name Ganoderic acid A 713,15a-Dihydroxy-3,11,23-trioxo-5 tt-lanost-8-en-26-oie acid Molecular Formula/Molecular Weight C3oH4407; M W -- 5 1 6 . 3 0 8 7 0 2O

General Characteristics An amorphous powder; [a]D 27 + 153.8 ~ (c = 0.156, in CHCI3). Funsal Source Isolated from the dried fruiting bodies (mushrooms) of Ganoderma lucidum (Polyporaeeae). Isolation/Purification The dried chipped epidermis of Ganoderma lucidum was extracted twice with CHCI3. The combined extracts were evaporated at 40-45 ~ under reduced pressure to about 1/10 of the original volume, and the resulting solution was extracted with sat. aq. NaHCO3 solution. The combined extracts were acidified with 6N HC1 (pH 3-4) at 0 ~C. The precipitate was extracted with CHC13, dried (Na2SO4) and the solvent removed under reduced pressure, leaving crude material. A part of the crude material was chromatographed on silica gel. Elution with CHCI3:MeOH, 98:2 (v/v), gave a bitter substance as an amorphous powder (ganoderic acid A). Further elution with CHCI3:MeOI-I, 95:5 (v/v) gave a slightly bitter component as an amorphous powder (ganoderic acid B). Biolo~cal Activity The mushrooms have long been used as a home remedy in China and Japan. Spectral Data IR;

(CHC13) 3300, 2600-2400, 1700, 1650, 1250, 1100, and 1000em"1.

274

9.

Ganoderic Acids

~HN M R : (CDCI3) H-I~, 2.85; H-la, 1.42(ddd, J=14, 8, and 8Hz); H-5a, 1.67(dd, J=12.5, 1.5Hz); H-6a, 1.70(ddd, J-12.5, 12.5, 9.7Hz); H-6~, 2.06(ddd, J--12.5, 7.6, 1.5Hz); H-7a, 4.62(dd, J=9.7, 7.6Hz); H-15~, 4.97(dd, J-9.2, 7.8Hz); H-16, 1.81; 18CH3, 0.98(s); 19CH3, 1.26(s); 21CH3, 0.90(d, J=6.2Hz); H-22, 2.41(dd, J=17.6, 3.2Hz); H22, 2.30(dd, J=16.7, 8.6Hz); H-24, 2.85(dd, J=18.0, 8.6Hz); H-24, 2.50(dd, J=18.0, 4.9Hz); H-25, 2.97(qadd, J-7.3, 8.6, 4.9Hz); 26CH3, 1.23(d, J=7.3Hz); 30CH3, 1.10(s); 31CH3, 1.29(s); COOH(DMSO), 12.05(br, s); and OH(DMSO), 5.32 and 4.50ppm. Iac NMR: (CDCI3) C-I, 35.7; C-2, 34.4; C-3,208.4; C-4, 47.0; C-5, 49.2; C-6, 29.2; C-7, 69.1; C-8, 159.6; C-9, 140.6; C-10, 46.8; C-11,200.0; C-12, 51.9; C-13, 38.2; C-14, 54.2; C-15, 72.6; C-16, 36.2; C-17, 48.3; C-18, 17.4; C-19, 19.6; C-20, 32.8; C-21, 19.4; C22, 49.8; C-23, 217.3; C-24, 46.7; C-25, 34.8; C-26, 27.4; C-27, 180.1; C-30, 17.0; C31, 20.8; and C-32, 19.8. Mass Spectrum: 516.3013(M+, 50%; calcd for C30H~O7, 516.3087), 498(21), 139(52), and l l5m/e (57). Reference T. Kubota, Y. Asaka, I. Miura, and H. Moil; Structures of Ganoderic Acid A and B, Two New Lanostane Type Bitter Triterpenes from Ganoderma lucidum (FR.) Karst.; Helvetica Chimica Acta, Vol. 65, pp. 611-619(1982).

9. Ganoderic Acids

275

Common/Systematic Name Ganoderic acid B Molecular Formula/Molecular Weight C3oH4407; M W -- 5 1 6 . 3 0 8 7 0

O

H ...... HO

COOH

H

General Characteristics Ganoderic acid B was crystallized from EtOAc-benzene as pale yellow needles; mp., 2122130C; [tZ]o14 +138 ~ (c=0.2, in MeOH). Acetate derivative; pale yellow prisms from ethyl acetate; mp., 214-215~ Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was acidified (pH 3-4) with 6N HCI, and the resulting precipitate was applied to silica gel column chromatography using a solvent system of chloroform-methanol. The eluate was rechromatographed on the silica gel column to give two strongly bitter compounds (lucidenic acid A and ganoderic acid C) and three slightly bitter compounds (lucidenic acid B, lucidenic acid C, and ganoderic acid B). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UVI

/~. EtoH 254nm (E=10,740). max

9.

276

Ganoderic Acids

IR;

(KBr) 3420, 3200-2400(br), 2950, 2875, 1720, 1640, 1450, 1360, 1240, 1160, and 1025cm"l. CD:

/~ MeOH max

293(- 3.8), 279(0), 254(+ 17.5), 228(0), and 212nm (-8.6).

(CDCI3) 4.80(IH, dd, J=8.9 and 8.9Hz); 3.19(IH, dd, J=I0.8 and 5.4Hz); 2.78(IH, d, J=I6.8Hz); 2.69(IH, d, J=16.8Hz); 1.34(3H, s); 1.22(3H, d, J=5.5Hz); 1.21(3H s); 1.03(3H, s); 1.00(3H, s);0.99(3H, d, J=5.8Hz); and 0.85ppm (3H, s). 13C N M ~ :

(CDCI3) 217.3(s); 207.5(s); 197.7(s); 180.2(s); 156.7(s); 142.6(s); 78.2(d); 66.8(d); 28.1 (q); 24.3(q); 19.5(q); 18.3(q); 17.3(q); 16.9(q); and 15.4ppm (q). Mass Spectrum: 516.3110(M+, C30I-~O7, 18%), 498(4), 488(19), 376(11), 358(32), 331(27), 149(15), 139(19), 105(27), 69(36), and 43m/e (100). References T. Kubota, Y. Asaka, I. Miura, and H. Moil; Structures of Ganoderic Acid A and B, Two New Lanostane Type Bitter Triterpenes from Ganoderma lucidum (FR.) Karst.; Helvetica Chimiea Acta, Vol. 65, pp. 611-619(1982). T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agile. Biol. Chem., Vol. 48, pp. 2905-2907(1984). T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agile. Biol. Chem., Vol. 49, pp. 1793-1798(1985).

9. Ganoderic Acids

277

Common/Systematic Name Ganoderic acid C1 713-Hydroxy-3,11,15,23-tetraoxo-5a-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight

C3oH4207; MW -- 514.29305 O

~

oo"

14

15

[~0

o---

General Characteristics Methyl ester: colorless prisms from ethanol; mp., 171-173~ CHC13).

[a]D + 168 ~ (c=l.0, in

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The dried powdered surface of the gills of the fruiting bodies of Ganoderma lucidum was extracted with ethyl ether. The extract was concentrated and partitioned between ethyl ether and water. The acidic fraction was obtained, methylated with diazomethane, and chromatographed on a silica gel column eluted with different ethyl acetate-benzene mixtures to give 13 fractions. Most fractions were further purified by silica gel column chromatography eluted with various mixtures of ethyl acetate-benzene or various mixtures of acetone-chloroform Fractions 3 and 4 were combined and recrystaUized from ethanol to give methyl ganoderate C 1. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) 1H NIk4R: (CDCI3) 1.47(1H, dt, J=13.5, 8.5Hz, H-la); 2.95(1H, ddd, J=13.5, 7.5, 5.50Hz, H113); 2.46(1H, ddd, J=16.0, 8.0, 5.5Hz, H-2a); 2.54(1H, ddd, J=16.0, 8.5, 7.5Hz, H213); 1.56(1H, dd, J=13.0, 1.5Hz, n-5); 2.11(1H, ddd, d=13.0, 8.0, 1.5Hz, n-6a); 1.67(1H, td, J=13.0, 9.2Hz, H-613); 4.87 (1n, ddd, J=9.2, 8.0, 4.5Hz, n-7a); 2.77(1H,

278

9.

Ganoderic Acids

br d, J=17.5Hz, H-12a); 2.72(1H, d, J=17.5Hz, H-1213); ca. 2.07(1H, H-16); 2.68(1H, H-16); ca. 2.13(1H, m, H-17); 2.16(1H, m, H-20); 2.37(2H, d, J=5.0Hz, H2-22); 2.44(1H, dd, J-17.5, 5.0Hz, H-24); 2.85(1H, dd, ,/-17.5, 8.8Hz, H-24); 2.96(1H, dqd, J=9.0, 7.0, 5.0Hz, H-25); 1.03(3H, s, H3-18); 1.26(3H, s, H3-19); 1.00(an, d, J=6.0Hz, H3-21); 1.19(3H, d, J=7.0Hz, H3-27); 1.13(3H, s, H3-30); 1.1 l(3H, s, H331); 1.34(3H, s, H3-32); 4.055(1H, d, J=4.5Hz, OH); and 3.69ppm (3H, s, COOCH3). 13CNMR: (CDCI3) 35.5, t, C-l; 34.1, t, C-2; 217.3, s, C-3; 46.7, s, C-4; 48.7, d, C-5; 27.6, t, C6; 66.1, d, C-7; 157.7, s, C-8; 141.0, s, C-9; 38.1, s, C-10; 197.4, s, C-11; 50.0, t, C12; 44.8, s, C-13; 59.2, s, C-14; 216.3, s, C-15; 40.8, t, C-16; 45.6, d, C-17; 17.5, q, C-18; 18.0, q, C-19; 31.8, d, C-20; 19.5, q, C-21; 48.9, t, C-22; 207.6, s, C-23; 46.6, t, C-24; 34.4, d, C-25; 175.9, s, C-26; 16.9, q, C-27; 26.8, q, C-30; 20.6, q, C-31; 24.5, q, C-32; and 51.7ppm q, CH30-. Mass Spectrum: HREIMS: 532.3395(M+, calcd for C31H4sO7 532.3400), 514, 496, 392, 388, 230, 171, 144, 139, and 129role. Reference T. Kikuchi, S. Kanomi (nee Matsuda), S. Kadota, Y. Murai, K. Tsubono, and Z. Ogita; Constituents of the Fungus Ganoderma lucidum (Fr.) Karst. I. Structures of Ganoderic Acids C2, E, I, and K, Lucidenic Acid F and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 3695-3712(1986).

9.

Ganoderic Acids

279

Common/Systematic Name Ganoderic acid C2 313,713,15a-Trihydroxy- 11,23-dioxo-5a-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C30I-I4607; MW = 518.32435 �9

lhh,,. ~

C

OOH

O H !

.....OH

HO

General Characteristics Methyl ester: colorless prisms from ethyl ether; mp., 199-2020C; [a]D + 98 ~ (c=l.0, in CHCI3). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The dried powdered surface of the gills of the fruiting bodies of Ganoderma lucidum was extracted with ethyl ether. The extract was concentrated and partitioned between ethyl ether and water. The acidic fraction was obtained, methylated with diazomethane, and chromatographed on a silica gel column eluted with different ethyl acetate-benzene mixtures to give 13 fractions. Most fractions were further purified by silica gel column chromatography eluted with various mixtures of ethyl acetate-benzene or various mixtures of acetone-chloroform Fraction 13 was subjected to preparative TLC (acetonechloroform, 1:9); the more mobile band contained methyl ganoderate C2, methyl ganoderate K and compound B9. Methyl ganoderate K was crystallized as yellow prisms from ethyl ether. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV: /~ MeOH 253nm (log e = 4.01). max

280

9.

Ganoderic Acids

IR: (KBr) 3450, 1720, 1710, 1700, and 1650cm"1. 1H ~ : (CDC13) ca. 0.93(1H, H-ltt); 2.74(1H, dt, J=13.5, 4.00Hz, H-l[3); ca. 1.60(11--1,H2a); ca. 1.66(1H, H-213); 3.20(1H, dd, J=10.8, 5.8Hz, H-3); 0.92(1H, dd, J=13.0, 1.0Hz, H-5); 2.14(1H, ddd, J=12.5, 7.5, 1.0Hz, H-6tt); 1.59(1H, td, J=12.5, 10.5Hz, H-6~); 4.54(1H, dd, J=10.3, 7.5Hz, H-7t~); 2.76(1H, br d, J=15.5Hz, H-12a); 2.46(1H, d, J=15.5Hz, H-1213); 4.74(1H, br t, J=7.5Hz, H-15); ca. 1.78(2H, H2-16); ca. 1.80(1H, H-17); 2.02(1H, m, H-20); 2.24(1H, dd, J=16.5, 9.5Hz, H-22); 2.40(1H, dd, ,/--16.5, 3.0Hz, H-22); 2.46(1H, dd, ,]--16.5, 3.0Hz, H-24); 2.83(1H, dd, J=17.8, 9.0Hz, H-24); 2.94(1H, dqd, J=9.0, 7.0, 5.0Hz, H-25); 0.96(3H, s, H3-18); 1.25(3H, s, H3-19); 0.88(3H, d, J=6.5Hz, H3-21); 1.18(3H, d, J=7.5Hz, H3-27); 1.02(3H, s, H330); 0.84(3H, s, H3-31); 1.24(3H, s, H3-32); and 3.68ppm (3H, s, COOCH3). Mass Spectrum: HREIMS: 532.3395(NV, calcd for C31H4807 532.3400), 514, 496, 392, 388, 230, 171, 144, 139, and 129role. Reference T. Kikuchi, S. Kanomi (nee Matsuda), S. Kadota, Y. Murai, K. Tsubono, and Z. Ogita; Constituents of the Fungus Ganoderma lucidum fir.) Karst. I. Structures of Ganoderic Acids C2, E, I, and K, Lucidenic Acid F and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 3695-3712(1986).

9.

Ganoderic Acids

281

Common/Systematic Name Ganoderic acid D 713,1213-Dihydroxy-3,11,15,23-tetraoxo-5 ~-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C3oH4208; M W : 530.28797

OH H

"OH

General Characteristics Ganoderic acid D was obtained as colorless needles from EtOAc; mp., 201-203~ + 185 (c=0.1, in EtOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with chloroform and chromatographed on a silica gel column using various concentrations of methanol in chloroform. The bitter principles were further purified with a second silica gel column eluted in a like manner. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy, also bitter tasting. The intensity of bitterness was evaluated to be lucidenic acid D, ganoderic acid C, lucidone A, lucidenic acid A, ganoderic acid B, lucidenic acid B, lucidenic acid C, and lucidenic acid E. Lucidenic acid A, ganoderic acid C, lucidenic acid D, and lucidone A had intense bitterness, and their threshold values were determined to 2.0 x 106M, 5 x 10SM, 5 x 10l~ and 1 x 106M, respectively, when the organoleptic test was carried out by an ascending series of concentrations in a 10% ethanol solution. Spectral Data UV:

~,o. max

252nm

(c

-

8800).

282

9.

Ganoderic Acids

IR;

3410, 3400-2500(br), 1730, 1720, and 1660cm"~. 1H NMR: (CsDsN) 5.16(1H, dd, ,/--8.4 and 8.4Hz); 4.69(1H, s); 1.48(3H, s); 1.45(3H, s); 1.40(3H, d. d=6.7Hz); 1.36(3H, d, J=7.3Hz); 1.21(3H, s); 1.16(3H, s); and 1.10ppm (3H, s). The ~H NMR data clearly reflect a pyridine-induced solvent shift and the signals at 5.16 and 4.69 showed an upfield shift in CDCI3 to 4.83 and 4.34, respectively. These IH and ~3C NMR spectra were very similar to those of ganoderic acid C, but the ~3C NMR signal at 79.2 and the ~H NMR signal at 4.69(1H, s) showed the presence ofa hydroxy group at C(12) by analogy with the case oflucidenic acids A and B. When the ~H NMR spectrum was measured in CDCI3, the signal of H-C(12) was observed at 4.38 for lucidenic acid B, which has a fl-hydroxy group at C(12), and 3.83 for lucidenic acid E, which has an tt-hydroxy group at C(12). On the basis of this finding, the configuration of the hydroxyl group at C(12) for ganoderic acid D was assigned as 13, because the signal of H-C(12) was observed at 4.34 in CDCI3. Thus, the structure of ganoderic acid D was established to be 713,12[3-dihydroxy-3,11,15,23-tetraoxo-5a-lanost-8-en-26-oic acid. 13C NMR: (C~DsN) (number of bonded H): 216.6(0), 215.1(0), 208.9(0), 200.9(0) 178.1(0), 159.7(0) 140.8(0), 79.2(1), and 65.7ppm (1).

Mass Spectrum: The molecular formula, C30I"I420$, was based on HRMS (M +, 530.2875; calcd for C30I-h2Os, 530.2881). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids from Ganoderma lucidum and Their Bitterness; Agric. Biol. Chem., Vol. 49, pp. 1547-1549(1985).

9.

Ganoderic Acids

283

Common/Systematic Name Ganoderic acid E

3,7,11,15,23-Pentaoxo-5a-lanost-8-en-26-oic acid

Molecular Formula/Molecular Weight C3oH4oO7; M W -" 5 1 2 . 2 7 7 4 0

o O-/~

v

o. "O

General Characteristics Methyl ester: Yellow needles; mp., 206-208~

[aiD q-

167~ (in CHCI3).

Fungal Source Isolated from Ganoderma lucidum. .Spectral Data

(Methyl ester)

~H NMR: (CDCI3) 0.89(3H, s, 18-CH3); 1.29(3H, s, 19-CH3); 0.99(3H, d, J=6.0Hz, 21-CH3); 1.18(3H, d, J=7.0Hz, 27-CH3); 1.12(3H, s, 30-CH3); 1.15(3H, s, 31-CH3); 1.66(3H, s, 32-CH3); and 3.70ppm (3H, s, COOCH3). 13C NMR: (CDCI3) 37.3, C-l, t; 34.7, C-2, t; 217.2, C-3, s; 43.9, C-4, s; 50.9, C-5, d; 33.8, C-6, t; 199.3, C-7, s; 149.7, C-8, s; 146.8, C-9, s; 39.4, C-10, s; 199.3, C-11, s; 48.9, C-12 ~ t; 47.0, C-13, s ; 57.2, C-14, s; 206.8, C-15, s; 39.8, C-16, t; 44.5, C-17, d; 16.1, C-18, q; 18.6, C-19, q; 32.0, C-20, d; 19.8, C-21, q; 49.1, C-22 ~ t; 207.6, C-23, s; 46.7, C24, t; 34.6, C-25, d; 176.1, C-26, s; 17.1, C-27,q; 26.6, C-30, q; 20.9, C-31**, q; 20.3, C-32 ~ q; and 59.9ppm, CH30-, q. *, ** Assignments may be reversed. Reference T. Kikuchi, S. Matsuda, S. Kadota, Y. Murai, and Z. Ogita; Ganoderic Acid D, E, F, and H and Lucidenic Acid D, E, and F, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2624-2627(1985).

284

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid F 3,7,11,15,23-Pentaoxo- 12-acetoxy-5a-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C32I-I4209; MW = 570.28288

oAc

O~" ~

v

~-O

General Characteristics Methyl ester, C33H4409;amorphous; [~]D -I- 111 o (in CHC13). Fungal Source Isolated from Ganoderma lucidum. Isolation/Purification The acidic fraction from the ether extract of the gills of dried fruit bodies of Ganoderma lucidum was methylated with diazomethane and the crude product was separated repeatedly by a combination of silica gel chromatography and preparative thin-layer chromatography to give pure triterpenoids. Spectral Data

(Methyl ester)

UV~ ~,max 252nm (log e = 3.91). IR~ 1745, 1735, 1710, and 1700cm~.

~H NMR: (CDCI3) 0.85(3H, s, 18-CH3); 1.33(3H, s, 19-CH3), 0.99(3H, d, J=6.0Hz, 21-CH3); 1.18(3H, d, J=7.0Hz, 27-CH3); 1.12(3H, s, 30-CH3); 1.14(3H, s, 31-CH3); 1.80(3H, s, 32-CH3); 3.68(3H, s, COOCH3); 2.26(OAc); and 5.68ppm (1H, s, 12-H). 13C NMR: (CDCI3) 37.5, C-l, t; 34.1, C-2, t; 214.8, C-3, s; 46.9, C-4, s; 51.0, C-5, d; 33.7, C-6, t; 198.7, C-7, s; 149.9, C-8, s; 146.1, C-9, s; 39.3, C-10, s; 194.1, C-11, s; 79.0, C-12,

9.

Ganoderic Acids

285

d; 47.7, C-13, s ; 58.6, C-14, s; 205.4, C-15, s; 37.8, C-16, t; 44.5, C-17, d; 12.1, C18, q; 18.7, C-19, q; 29.4, C-20, d; 21.6, C-21, q; 48.5, C-22, t; 207.4, C-23, s; 46.7, C-24, t; 34.7, C-25, d; 176.0, C-26, s; 17.1, C-27,q; 27.6, C-30, q; 20.8, C-31~ q; 20.4, C-32 ~ q; 51.9, CH30-, q; 20.9, COCH3", s; and 170.2ppm (COCH3, s). * Assignments may be reversed. Reference T. Kikuchi, S. Matsuda, S. Kadota, Y. Murai, and Z. Ogita, Ganoderic Acid D, E, F, and H and Lucidenic Acid D, E, and F, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2624-2627(1985).

286

9.

Ganoderic

Acids

Common/Systematic Name Ganoderic acid G Molecular Formula/Molecular Weight C30I'~Os; MW = 532.30362 21 HO l n ' ~ ~ ~ n ' ~ ~ r

" 1 27 H

- 32 H O 31 "

""~ 3o

General Characteristics Isolated and identified as methyl ester. Colorless prisms; mp., 134-135~ CHCI3).

[a]D -k- 64 ~ (in

Fungal Source

Ganoderma lucidum.

Spectral Data

(Methyl ester)

UV:

)t~x 252.5nm (log e = 4.01) IR:

(KBr) 3450, 1730, 1720, and 1660cm "1. 1H NMR:

(CDCI3) 0.88(3H, s, 18-CH3); 1.32(3H, s, 19-CH3); 1.19(3H, d, J=7.0Hz, 27-CH3); 1.04(3H, s, 30-CH3); 0.80(3H, s, 31-CH3); 1.43(3H, s, 32-CH3); 3.68(3H, s, COOCH3); 3.22(1H, dd, J=10.0, 6.0Hz, 3-s 4.80(1H, td, J=8.5, 4.5Hz, 7-H); 4.38(1H, d, J=2.5Hz, 12-H); 4.43(1H, d, J=4.5Hz, 7-OH); and 4.03ppm (1H, d, J=2.5Hz, 12-OH). 13C NMR: (CDCI3) C-l, 34.6, t; C-2, 27.6, t; C-3, 78.3, d; C-4, 38.6, s; C-5, 49.2, d; C-6, 26.9, t, C-7, 66.2, d; C-8, 157.4, s; C-9, 141.9, s; C-10, 38.4, s; C-11,199.3, s; C-12, 77.9, d; C-13, 51.9, s; C-14, 60.3, s; C-15,216.8, s; C-16, 38.4, t; C-17, 45.8, d; C-18, 12.0, q; C-19, 18.8, q; C-20, 28.7, d; C-21, 21.4, q; C-22, 48.4, t; C-23,208.1, s; C-24, 46.4, t; C-25, 34.6, d; C-26, 176.1, s; C-27, 17.1, q; C-30, 28.1, q; C-31, 15.4, q; C-32, 23.1, q; and OCH3, 51.9ppm.

9. Ganoderic Acids

287

Mass Spectrum: LREIMS: 546(M+), 375, 306, 139, and 129m/e. Reference T. Kikuchi, S. Matsuda, Y. Murai, and Z. Ogita; Ganoderic Acid G and I and Ganolucidic Acid A and B, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2628-2631(1985).

288

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid H 313-Hydroxy-7,11,15,23-tetraoxo- 1213-acetoxy-5a-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C32H4409; M W = 572.29853

OAc'~,~,,n,,,"~hl ~

_

HO"

~..%v

,coo

-o ~'0

General Characteristics Methyl ester, C33H4609; yellow needles; mp., 155-156~

laiD d- 55 ~ (in CHC13).

Fungal Source

Ganoderma lucidum.

Isolation/Purification The acidic fraction from the ether extract of the gills of dried fruit bodies of Ganoderma lucidum was methylated with diazomethane, and the crude product was separated repeatedly by a combination of silica gel chromatography and preparative thin-layer chromatography to give pure triterpenoids. Spectral Data (Methyl ester) UV:

~.max 256nm (log e=3.87). 1H NMR: (CDCI3) 0.88(3H, s, 18-CH3); 1.33(3H, s, 19-CH3); 0.98(3H, d, J=6.0Hz, 21-CH3), 1.18(3H, d, J=7.0Hz, 27-CH3); 1.03(3H, s, 30-CH3); 0.82(3H, s, 31-CH3); 1.73(3H, s, 32-CH3); 3.68(3H, s, COOCH3); 2.26(OAc); 3.27(1H, dd, J=10.0, 6.0Hz, 3-H); and 5.63ppm (1H, s, 12-H). 13C NMR: (CDCI3) 36.6, C-l, t; 27.3, C-2, t; 77.3, C-3, d; 40.4, C-4, s; 51.4, C-5, d; 33.2, C-6, t; 198.7, C-7, s; 151.6, C-8, s; 145.7, C-9, s; 39.1, C-10, s, 193.9, C-11, s, 79.1, C-12, d, 47.9, C-13, s; 58.4, C-14, s; 205.5, C-15, s; 37.8, C-16, t; 44.7, C-17, d, 12.1, C-18, q; 17.9, C-19, q; 29.3, C-20, d; 21.6, C-21, q; 48.4, C-22, t; 207.4, C-23, s; 46.6, C24, t; 34.6, C-25, d; 176.0, C-26, s; 17.0, C-27,q; 27.9, C-30, q; 15.5, C-31, q, 21.2, C-32 ~ q; 51.8, CH30-, q; 20.8, COCH3~ s; and 170.1ppm (COCH3, s). * Assignments may be reversed.

9.

Ganoderic Acids

289

Reference T. Kikuchi, S. Matsuda, S. Kadota, Y. Murai, and Z. Ogita; Ganoderic Acid D, E, F, and H and Lucidenic Acid D, E, and F, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2624-2627(1985).

290

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid I Molecular Formula/Molecular Weight C30H4408; MW = 532.30362 21

OH

18~~.~..,.,.~-

, g ~ ~ ]

HO" ~'xg" v 31"" "% 30

t3

27

COOH

"OH

General Characteristics Isolated and identified as methyl ester. Colorless prisms; mp., 279-281~ CHCI3).

[a]D + 132~ (in

Fungal Source

Ganoderma lucidum.

Spectral Data (Methyl Ester) UV:

~m,x 254.5nm (log e = 3.86). IR:

(KBr) 3450, 1730, 1710, and 1660cm"l. IH NMR:

(CDCI3) 1.14(3H, s, 18-CH3); 1.21(3H, s, 19-CH3); 1.40(3H, d, J=6.0Hz, 21-CH3); 1.19(3H, d, J=7.0Hz, 27-CH3); 1.03(3H, s, 30-CH3); 0.86(3H, s, 31-CH3); 1.35(3H, s, 32-CH3); 3.71(3H, s, COOCH3); 3.22(1H, dd, J-10.0, 6.0Hz, 3-H); 4.80(1H, td, J=8.5, 4.5Hz, 7-H); and 4.12ppm (1H, d, J=4.5Hz, 7-OH). 13CNMR: (CDCI3) C-l, 34.9, t; C-2, 27.8, t; C-3, 78.4, d; C-4, 38.9, s; C-5, 49.2, d; C-6, 26.7, t; C-7, 66.9, d; C-8, 156.6, s; C-9, 142.3, s; C-10, 38.7, s; C-11,197.8, s; C-12, 50.7, t; C-13, 45.7, s; C-14, 59.7, s; C-15, 217.7, s; C-16, 36.1, t; C-17, 49.3, d; C-18, 19.0, q; C-19, 18.4, q; C-20, 73.0, s; C-21, 26.7, q; C-22, 52.7, t; C-23,210.4, s; C-24, 47.7, t; C-25, 34.5, d, C-26, 175.9, s; C-27, 17.0, q; C-30, 28.2, q; C-31, 15.5, q; C-32, 24.8, q; and OCHa, 52.0ppm.

9.

Ganoderic Acids

291

Reference T. Kikuchi, S. Matsuda, Y. Murai, and Z. Ogita; Ganoderic Acid G and I and Ganolucidic Acid A and B, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2628-2631(1985).

292

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid J Molecular Formula/Molecular Weight C30I--I4207;MW --- 514.29305 Methyl ester: C3~I-/4407;MW = 528.30870

H O~~i

......OH

General Characteristics Methyl ester: Obtained as a pale yellow syrup; [tl]D23 + 174 ~ (C=0.1, in MeOH). Fungal Source Isolated from the dried fruiting bodies (mushrooms) of Ganoderma lucidum. Isolation/Purification The dried chipped epidermis of Ganoderma lucidum was extracted with CHCI3. The combined extracts were evaporated at 40-45 ~ under reduced pressure to about 1/10 of the original volume, and the resulting solution was extracted with saturated aqueous NaHCO3 solution. The combined extracts were acidified with 6N HCI (pH 3-4) at 0 ~C. The precipitate was extracted with CHCI3, dried (Na2SO4) and the solvent removed under reduced pressure, leaving crude material. The acidic material was chromatographed on silica gel column chromatography [Fr. 1-11, CHCI3-MeOH, 98:2 (v/v); Fr. 12, CHC13-MeOH, 9:1 (v/v), Fr. 13, MeOI-I]. Fractions 7-9 were combined and rechromatographed on silica gel to give a mixture of ganoderic acid J and A, which was methylated with diazomethane and separated by further silica gel column chromatography and preparative TLC. Methyl ganoderate J was obtained as a pale yellow syrup. Fraction 12 was chromatographed on a Lobal column (RPs, Merck) and the second fraction was methylated with diazomethane. The methylated products were subjected to silica gel column chromatography and methyl ganolucidate C was obtained as colorless needles. Biological Activity A bitter principle; the mushrooms of Ganoderma lucidum (Fr.) Karst. (Polyporaceae), have long been used as a home remedy in China and Japan.

9.

Ganoderic Acids

293

Spectral Data UV: k MeOH max

270nm (7,500) (methyl ester).

IR:

3480, 1735, 1710, and 1670cm-~ (methyl ester). 1H NMR: (methyl ester)(CDCls) 0.87(3H, d, J= 6.4Hz); 0.91(3H, s); 1.12(3H, s); 1.14(3H, s); 1.18 =(3H, d, J=6.4Hz); 1.19(3H, s), 1.27(3H, s); 3.68(3H, s, COOCH3); 4.29(1H, ddd, J=7.8, 7.8 and 1.5Hz); 4.45(1H, d, d=l.5Hz). The 1H NMR data were very similar to those of ganoderic acid A, but the signal due to the carbinol methine proton was only observed at 4.29ppm (1513-H). This indicated the presence of a carbonyl group at C(7) of methyl ganoderate J instead of the 7-hydroxyl group for ganoderic acid A.

(Free acid)(CDCls) 0.87(1H, d, J=6.2 Hz); 0.90(3H, s); 1.12(3H, s); 1.14(3H, s); 1.18(3H, s), 1.22(3H, d, J=7.0Hz), 1.27(3H, s), 1.29ppm (1H, dd, J=7.2 and 7.2Hz). Mass Spectrum: Methyl ganoderate J showed a molecular ion peak at 528.3084m/e (calcd for C31H4407, 528.3088); ganoderic acid J (M+, 514.2968, calcd. 514.2932). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganoderic Acid J and Ganolucidic Acid C from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 49, pp. 3637-3638(1985).

294

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid K 313,15t~-Dihydroxy-7,11,23-trioxo-5t~-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight Cs0H44OT; MW = 516.30870 C OOH 0 H

OH HO General Characteristics Methyl ester: pale yellow prisms from ethyl ether; mp., 166-167~ CHC13).

[0~]D + 165 ~ (c=0.3, in

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The dried powdered surface of the gills of the fruiting bodies of Ganoderma lucidum was extracted with ethyl ether. The extract was concentrated and partitioned between ethyl ether and water. The acidic fraction was obtained, methylated with diazomethane, and chromatographed on a silica gel column eluted with different ethyl acetate-benzene mixtures to give 13 fractions. Most fractions were further purified by silica gel column chromatography eluted with various mixtures of ethyl acetate-benzene or various mixtures of acetone-chloroform Fraction 13 was subjected to preparative TLC (acetonechloroform, 1:9, v/v) the more mobile band contained methyl ganoderate C2, methyl ganoderate K, and compound B9. Methyl ganoderate K was crystallized as yellow prisms from ethyl ether. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV: ,/~,MeOH

255nm (log e = 3.43).

9.

Ganoderic Acids

295

IR:

(KBr) 3450, 1730, 1715, and 1665cm"~. ~H N M R : (CDCI3) 1.28(1H, td, J=13.0, 4.50Hz, H-la); 2.84(1H, dt, J=13.5, 3.50Hz, H-l~3); 1.77(1H, dq, J=13.0, 4.5Hz, H-2t~); 1.69(1H, tdd, J=13.0, 11.5, 3.SHz, H-213); 3.28(1H, br dd, J=l 1.0, 5.0Hz, H-3); 1.54(1H, dd, J=12.5, 4.5Hz, H-5); 2.54(1H, dd, J=16.0, 4.SHz, H-6tt); 2.59(1H, dd, J=16.0, 13.0Hz, H-613); 2.83(1H, d, J=17.0Hz, H12t~); 2.54(1H, d, J=17.0Hz, H-1213); 4.335(1H, td, J=7.5, 1.8Hz, H-15); 1.84(2H, dlike, H2-16); ca. 1.85(1H, H-17); 2.01(1H, m, H-20); 2.245(1H, dd, J=16.5, 9.5Hz, H22); 2.41(1H, dd, J=16.6, 3.0Hz, H-22); 2.455(1H, dd, J=17.5, 5.0Hz, H-24); 2.823(1H, dd, J=17.5, 8.SHz, H-24); 2.945(1H, dqd, J=8.5, 7.5Hz, H-25); 0.89(3H, s, H3-18); 1.29(3H, s, H3-19); 0.87(3H, d, J=6.5Hz, H3-21); 1.18(3H, d, J=7.0Hz, H327); 1.03(3H, s, H3-30); 0.89(3H, s, H3-31); 1.14(3H, s, H3-32); 4.485(OH); and 3.67ppm (3H, s, COOCH3). ~ac NMR: (CDC13) 34.3, t, C-1; 27.7, t, C-2; 77.5, d, C-3, 40.2, s, C-4; 49.8, d, C-5; 36~5, t, C-6; 205.3, s, C-7, 154.6, s, C-8; 149.8, s, C-9; 38.9, s, C-10, 201.3, s, C-11; 52.3, t, C-12; 48.0, s, C-13; 52.8, s, C-14; 72.1, d, C-15; 36.3, t, C-16; 48.2, d, C-17; 17.4, q, C-18; 17.6, q, C-19; 32.4, d, C-20; 19.5, q, C-21; 49.5, t, C-22; 208.2, s, C-23; 46.8, t, C-24, 34.7, d, C-25; 176.1, s, C-26; 17.1, q, C-27; 27.8, q, C-30; 15.4, q, C-31; 20.3, q, C32; and 51.9ppm, q, CH30-. Mass Spectrum: HREIMS: 530.3252(M +, calcd for C31H4607 530.3244), 499, 368, 341,303, 209, 171, 144, 139, and 129role. Reference T. Kikuchi, S. Kanomi (nee Matsuda), S. Kadota, Y. Murai, K. Tsubono, and Z. Ogita; Constituents of the Fngus Ganoderma lucidum (Fr.) Karst. I. Structures of Ganoderic Acids C2, E, I, and K, Lucidenic Acid F and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 3695-3712(1986).

296

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid L 313,713,15tt,20-Tetrahydroxy- 11,23-dioxo-5tt-lanost-8-en-26-oic acid (Isolated and identified as the methyl ester.) Molecular Formula/Molecular Weight C30I-/46Os; MW = 534.31927 OH 0

H /~

HO

......OH

OH

General Characteristics Methyl ganoderate L: colorless prisms; mp., 228-230~

[a]D 23 +

66 ~ (c=0.2, in MeOH).

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was separated into thirteen fractions; fraction 12 was subjected to Lobar column (RPs, Merck) chromatography and the second fraction was treated with diazomethane. The resulting product was rechromatographed on silica gel and the Lobar (RPs) column to give methyl ganoderate L, lucidone C, and methyl lucidenate G. The fourth fraction in the chromatography of Fr. 12 was purified on a silica gel column, preparative TLC and HPLC to give ganolucidic acid D. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Monomethyl derivative) UV:

3,M~~ max

256nm (E=6750).

9.

Ganoderic Acids

297

IR:

(KBr) 3430, 1720, and 1655cm"1. :H NMR: (CsDsN) 5.46(1H, dd, J=9.2 and 7.3Hz); 5.00(1H, overlapped); 3.52(1H, dd, J=10.6 and 5.1Hz); 1.64(3H, s); 1.59(3H, s); 1.57(3H, s), 1.54(3H, s); 1.29(3H, s); 1.15(3H, d, J-6.6Hz); and 1.11ppm (3H, s). 13CNMR:

(CsDsN) (number of bonded H): 209.4(0); 200.2(0); 176.2(0); 160.3(0); 141.7(0); 77.6(1); 73.8(0); 72.4(1); and 69.5ppm (1). Mass Spectrum: The FD-MS showed 548(M+, 5.3%), 404(100), and 144m/e (33.4); ElMS did not give a molecular ion peak. Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganolucidic Acid D, Ganoderic Acid L, Lucidone C, and Lucidenic Acid G, from the Fungus Ganoderma lucidum; Agrie. Biol. Chem., Vol. 50, pp. 809-811 (1986).

298

9.

Ganoderic Acids

Common/Systematic Name Compound B8 7tt, 15a-Dihydroxy-3,11,23-trioxo-5tt-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C3oH4407; M W "- 5 1 6 . 3 0 8 7 0

o

"•/•ir/•],/COOH ......OH

General Characteristics Methyl ester: colorless prisms from ether; mp., 158-163~ CHCI3).

[a]D q-

128~ (c=0.5, in

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The dried powdered surface of the gills of the fruiting bodies of Ganoderma lucidum was extracted with ethyl ether. The extract was concentrated and partitioned between ethyl ether and water. The acidic fraction was obtained, methylated with diazomethane, and chromatographed on a silica gel column eluted with different ethyl acetate-benzene mixtures to give 13 fractions. Most fractions were further purified by silica gel column chromatography eluted with various mixtures of ethyl acetate-benzene or various mixtures of acetone-chloroform Fractions 10-12 were combined, repeatedly chromatographed by preparative TLC (acetone-chloroform, 15:85, v/v), and recrystallized from ethyl ether to give compound B8 (7tt, 15a-dihydroxy-3,11,23-trioxo-5a-lanost-8-en-26-oic acid). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV: ~, EtOH max

254.5nm (log e = 3.49).

9.

Ganoderic Acids

299

IR;

(thin film) 3400, 1730, 1710, and 1650cm1. 1H NMR: (CDCI3) 1.71(1H, dr, J-15.0, 8.0Hz, H-la); 2.97(1H, ddd, J=14.0, 8.5, 5.50Hz, H113); 2.45(1H, ddd, J=15.5, 9.0, 7.0Hz, H-2a); 2.61(1H, ddd, ,/-15.5, 9.5, 6.0Hz, H213); 2.09(1H, dd, J=12.5, 3.0Hz, U-5); ca. 1.72(1H, m, H-6a); ca. 1.78(1H, m, H-613); 4.58(1H, dd, J--5.0, 1.SHz, H-713); 2.75(1H, br d, J=lS.0Hz, H-12a); 2.42(1H, d, J=17.7Hz, H-1213); 4.605(1H, dd, J-9.0, 6.0Hz, H-15); ca. 1.82(1H, H-16); 1.95(1H, H-16); ca. 1.95(1H, H-17); 2.00(1H, m, H-20); 2.27(1H, dd, J=16.3, 8.5Hz, H-22), 2.38(1H, dd, 3=16.0, 3.0Hz, H-22); 2.46(1H, dd, 3"=17.3, 5.0Hz, H-24); 2.84(1H, dd, 3"=17.5, 8.5Hz, H-24); 2.95(1H, m, H-25); 0.89(3H, s, H3-18); 1.03(3H, s, H3-19); 0.86(3H, d, 3"=5.8Hz, H3-21); 1.18(3H, d, 3"=7.0Hz, H3-27); 1.16(3H, s, H3-30); 1.08(3H, s, H3-31); 1.30(3H, s, H3-32); and 3.68ppm (3H, s, COOCH3). 13C NMR:

(CDC13) 34.8, t, C-I; 34.1, t, C-2; 217.8, s, C-3; 47.2, s, C-4; 45.2, d, C-5; 27.9, t, C6; 66.7, d, C-7; 159.3, s, C-8; 140.0, s, C-9; 38.0, s, C-10; 199.1, s, C-11; 51.8, t, C12; 46.4, s, C-13; 53.4, s, C-14; 72.4, d, C-15; 37.8, t, C-16; 49.0, d, C-17; 17.5, q, C18; 17.5, q, C-19; 32.5, d, C-20; 19.3, q, C-21; 49.6, t, C-22; 208.3, s, C-23; 46.9, t, C-24; 34.7, d, C-25; 176.2, s, C-26; 17.1, q, C-27; 27.6, q, C-30; 20.5, q, C-31; 21.1, q, C-32; and 51.9ppm, q, CH30-. Mass Spectrum: HR IMS: 530.3293(M +, calcd for C31H4607 530.3244), 392, 364, 230, 171, 144, 139, and 129role. Reference T. Kikuchi, S. Kanomi (nee Matsuda), S. Kadota, Y. Murai, K. Tsubono, and Z. Ogita; Constituents of the Fungus Ganoderma lucidum (Fr.) Karst. I. Structures of Ganoderir Acids C2, E, I, and K, Lueidenir Acid F and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 3695-3712(1986).

300

9.

Ganoderic Acids

Common/Systematic Name Compound B9 313,7a, 15a-Trihydroxy- 11,23-dioxo-5a-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C3oH4607; M W -- 518.32435

........I j . - ~ . ~ ~ C O O H 0 H...... HO v

~

/

.-.,,,,

v

....O H

General Characteristics Amorphous powder. Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The dried powdered surface of the gills of the fruiting bodies of Ganoderma lucidum was extracted with ethyl ether. The extract was concentrated and partitioned between ethyl ether and water. The acidic fraction was obtained, methylated with diazomethane, and chromatographed on a silica gel column eluted with different ethyl acetate-benzene mixtures to give 13 fractions. Most fractions were further purified by silica gel column chromatography eluted with various mixtures of ethyl acetate-benzene or various mixtures of acetone-chloroform Fraction 13 was repeatedly chromatographed by preparative TLC (acetone-chloroform, 1:9, v/v) to give compound B9 (313,7a, 15a-trihydroxy-11,23-dioxo5 a-lanost-8-en-26-oic acid). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV:

~,maxE~'" 255nm (Iog e = 3.43) IR:

(thin film) 3450, 1730, 1715, and 1650cmq.

9.

Ganoderic Acids

301

IH NMR:

(CDCI3) 1.17(1H, td, J=13.5, 4.5Hz, H-la); 3.00(1H, dt, J=14.0, 3.5Hz, H-113); ca. 1.65(1H, m, H-2a); 1.72(1H, br d-like, H-213); 3.3 I(1H, dd, J=l 1.5, 5.0Hz, H-3); 1.28(1H, dd, J=13.0, 2.0Hz, H-5); 1.83(1H, br d, `/=14.0Hz, H-6a); 1.73(1H, td, J=13.5, 5.0Hz, H-6~3); 4.56(1H, br d, J=5.0Hz, H-713); 2.75(1H, br d, `/=17.5Hz, H12a); 2.39(1H, d, J=17.7Hz, H-1213); 4.57(1H, dd, J=9.0, 5.5Hz, H-15); ca. 1.78(1H, H-16); 1.94(1H, n-16); ca. 1.92(1H, H-17); 2.00(1H, m, H-20); 2.25(1H, dd, ,/=16.2, 9.0Hz, H-22); 2.38(1H, dd, ,/=16.2, 3.0Hz, H-22); 2.46(1H, dd, ,/=17.5, 5.0Hz, H-24); 2.83(1H, dd, ,/=17.5, 8.5Hz, H-24); 2.94(1H, dqd, ,/=9.0, 7.5, 5.5Hz, H-25); 0.87(3H, s, 1-13-18); 1.04(3H, s, 1-13-19);0.85(3H, d, ,/=6.0Hz, 1-13-21); 1.18(3H, d, ,/=7.0Hz, 1-1327); 1.06(3H, s, 1-13-30);0.84(31-1, s, H3-31); 1.27(3H, s, 1-13-32);and 3.68ppm (3H, s, COOCH3). 13CNMR: (CDCI3) 34.2, C-l; 27.3, C-2; 78.5, C-3; 39.1, C-4; 47.7, C-5; 28.0, C-6; 68.0, C-7; 158.8, C-8; 141.6, C-9; 38.6, C-10; 199.4, C-11; 52.2, C-12; 46.1, C-13; 53.5, C-14; 72.3, C-15; 37.8, C-16; 49.0, C-17; 17.3, C-18; 17.3, C-19; 32.5, C-20; 19.3, C-21; 49.6, C-22; 208.3, C-23; 46.9, C-24; 34.7, C-25; 176.4, C-26; 17.1, C-27; 28.2, C-30; 15.8, C-31; 21.1, C-32; and 51.9ppm CH30-. Mass Spectrum: HREIMS: 532.3413(M § calcd for C31H4807 532.3400), 514, 496, 364, 230, 171, 144, 139, and 129m/e. Reference T. Kikuchi, S. Kanomi (nee Matsuda), S. Kadota, Y. Murai, K. Tsubono, and Z. Ogita; Constituents of the Fungus Ganoderma lucidum (Fr.) Karst. I. Structures of Ganoderic Acids C2, E, I, and K, Lucidenic Acid F and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 3695-3712(1986).

302

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Ma 3 a, 7 a-Diacetoxy- 15a-hydroxy- 5 a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C3,I-I5207; MW = 572.37130

COOH

i_ A c O ...... . . . . .

.....OH

OAc

General Characteristics Colorless syrup; [a]D 24 -16 ~ (c=0.3, in MeOH). Fungal .Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"). Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated on a silica gel column into seven fractions (Fr. 1-7). Fraction 5 was successively rechromatographed on a silica gel column, preparative TLC, and/or reversed-phase LC to afford ganoderic acid Ma and eight other related compounds. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data UV: M~H 21Ohm (19,300). max

IR:

(thin film) 3200-2500, 1715, and 1640cmq (sh). IH N]V[R: (CDCI3) The spectrum showed the presence of five tertiary methyl groups 0.67(3H, s), 0.82(3H, s), 0.91(3H, s), 1.00(3H, s), 1.09(3H, s); one secondary methyl group 0.91 (3H, d, J=6.2); one olefinic methyl group 1.84(3H, d, J=l.1), 6.87(1H, tq, ,I=7.5, 1.1);

9.

Ganoderic Acids

303

two secondary acetoxyl groups 2.04(3H, s), 2.10(3H, s), 4.69(1H, t, J=2.6; 313-H), 5.43(1H, br. s; 713-H); and one secondary hydroxyl group 4.26ppm (1H, dd, J=9.5, 5.5, 1513-H). 13CNM~: (CDCI3) C-l, 30.1; C-2, 23.3; C-3, 77.3; C-4, 36.2; C-5, 40.5; C-6, 26.1; C-7, 70.0; C-8, 131.1; C-9, 145.1; C-10, 38.3; C-11, 21.4; C-12, 31.2; C-13, 45.8; C-14, 52.1; C15, 72.4; C-16, 39.7; C-17, 48.8; C-18, 16.3; C-19, 17.7; C-20, 36.7; C-21, 18.4; C22, 34.7; C-23, 25.8; C-24, 145.2; C-25, 126.8; C-26, 172.4; C-27, 12.0; C-28, 27.2; C-29, 22.7; C-30, 17.9; OCOCH3, 171.9, 170.8; OCOCH3, 21.9ppm. Mass Spectrum: FD-MS: 572m/e (M+); HREIMS: 554.3629m/e (M +-1-/20, C34I--I5006, calcd 554.3609). Reference T. Nishitoba, H. Sato, S. Shirasu, and S. Sakamura; Novel Triterpenoids from the Mycelial Mat at the Previous Stage of Fruiting of Ganoderma lucidum; Agrie. Biol. Chem., Vol. 51, pp. 619-622(1987).

304

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Mb 3a, 15a,22-Triacetoxy-7a-hydroxy-5a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C36H5209; M W = 630.37678 OAc

COOH

.... l

.,,o.

General Characteristics Colorless syrup; [0~]D27 -4 ~ (c=0.2, in MeOH). Fungal Source Isolated from mycelium of Ganoderma lucidum CSaegusa"). Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated on a silica gel column into seven fractions (Fr. 1-7). Fraction 5 was successively rechromatographed on a silica gel column, preparative TLC and/or reversed-phase LC to afford ganoderic acid Mb and eight other related compounds. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data UV: ~, MeOHmax 21 Ohm (22,300). IR:

(thin film) 3450, 3200-2500, 1720, and 1640cm1 (sh). 1H N~/[R:

(CDCI3) 4.11(IH, br, s, 713-H);4.69(IH, t,J=2, 313-H); 5.03(IH, t,J=7.0, 22-I-I); 5.15ppm (IH, dd, J=9.5, 5.5, 15~-H).

9.

Ganoderic Acids

305

~3CNMR: (CDC13) C-I, 30.3; C-2, 23.3; C-3, 77.5; C-4, 36.3; C-5, 40.0; C-6, 27.4; C-7, 66.5; C-8, 133.8; C-9, 141.8; C-10, 38.4; C-11, 20.7; C-12, 31.2; C-13, 45.3; C-14, 51.3; C15, 76.0; C-16, 36.3; C-17, 45.9; C-18, 16.3; C-19, 17.5; C-20, 39.9; C-21, 12.7; C22, 74.4; C-23, 31.9; C-24, 138.9; C-25, 129.5; C-26, 172.0; C-27, 12.3; C-28, 27.4; C-29, 22.0; C-30, 20.2; OCOCH3, 170.9, 170.6, 170.5; and OCOCH3, 21.4, 21.2, 21.0ppm. The ~H and 13CNMR data resembled those of ganoderic acid W and showed the presence of an additional acetoxyl group at C-22, analogous to ganoderic acids R, S, and T. Mass Spectrum: FD-MS: 630m/e (M§ HREIMS: 612.3636m/e (M ~ -1-120, C36H5208, calcd 612.3664). Reference T. Nishitoba, H. Sato, S. Shirasu, and S. Sakamura; Novel Triterpenoids from the Mycelial Mat at the Previous Stage of Fruiting of Ganoderma lucidum; Agric. Biol. Chem., Vol. 51, pp. 619-622(1987).

306

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Mc 3 a,7a,22-Triacetoxy- 15a-hydroxy-5a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C36H5409; ]ViW'= 630.37678

OAc COOH

-

.....OH

AcO. . . . . . . . . . OAc

General Characteristics Colorless syrup; [tt]D 27 -23" (c=0.2, in MeOH). Fungal Source Isolated from mycelium of Ganoderma lucidum CSaegusa"). Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated on a silica gel column into seven fractions (Fr. 1-7). Fraction 5 was successively rechromatographed on a silica gel column, preparative TLC and/or reversed-phase LC to afford ganoderic acid Me and eight other related compounds. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data UV: ~, M~H 210nm (17,000). max

IR:

(thin film) 3450, 3200-2500, 1720, and 1640cm"l (sh). IH N1V[R: (CDCI3) The ~H and ~3C NMR data (see below) were very similar to those of ganodermic acid Mb, but indicated that an acetyl group was attached to the 7a-

9.

Ganoderic Acids

307

hydroxyl group in ganoderic acid Mc instead of to the 15t~-hydroxylgroup in ganoderic acid Mb. 13CNMR; (CDC13) C-l, 30.1; C-2, 23.3; C-3, 77.3; C-4, 36.2; C-5, 40.5; C-6, 26.0; C-7, 69.9; C-8, 131.1; C-9, 145.0; C-10, 38.3; C-11, 21.2; C-12, 31.2; C-13, 45.6; C-14, 52.1; C15, 72.2; C-16, 39.3; C-17, 45.5; C-18, 16.2; C-19, 17.7; C-20, 39.5; C-21, 12.9; C22, 74.6; C-23, 31.7; C-24, 139.1; C-25, 129.3; C-26, 171.7; C-27, 12.3; C-28, 27.2; C-29, 21.9; C-30, 18.0; OCOCH3, 171.9, 170.8, 170.5; and OCOCH3, 21.7, 21.4, 21.0ppm. Mass

Spectrum: FD-MS: 630m/e (M+); HREIMS: 612.3609m/e (M +-H20), C36H5208,calcd 612.3464.

Reference T. Nishitoba, H. Sato, S. Shirasu, and S. Sakamura; Novel Triterpenoids from the Mycelial Mat at the Previous Stage of Fruiting of Ganoderma lucidum; Agric. Biol. Chem., Vol. 51, pp. 619-622(1987).

308

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Md 3 a,22-Diacetoxy-7a-methoxy-5a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C35H5407, M'W' = 586.38695 OAc COOH

AcO. . . . . . . . . .

OMe

General Characteristics Colorless needles; mp., 180-182~

[tt]D 24 -20 ~ (c=0.33, in MeOH).

Funsal Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"). Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated on a silica gel column into seven fractions (Fr. 1-7). Fraction 5 was successively rechromatographed on a silica gel column, preparative TLC and/or reversed-phase LC to afford ganoderic acid Md and eight other related compounds. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data_ UV:

~, M~OH~x21Ohm (17,600). IR.:

(thin film) 3400-2500, 1720, 1680, and 1640cm "~. ~H NMR: (CDCI3) 3.29(3H, s); 3.61(IH, br, d, ,/=7.9, 713-H); 4.69(IH, br, s, 383-H), 5.11ppm (IH, t, J=7.0, 22-H). The signals at 4.69 and 5.11ppm indicated the presence of 3a-

9.

Ganoderic Acids

309

and 22-acetoxyl groups, and the signals at 3.29 and 3.6 l ppm indicated the presence of a 7a-methoxy group, which were confirmed by conversion into ganoderic acid R on treatment with H2SO4. 13C NMR: (CDCI3) C-I, 30.2; C-2, 23.4; C-3, 77.6; C-4, 36.4; C-5, 40.4; C-6, 22.4; C-7, 76.5; C-8, 134.3; C-9, 141.3; C-10, 38.1; C-11, 21.0; C-12, 31.1; C-13, 45.0; C-14, 50.0; C15, 30.2; C-16, 27.9; C-17, 47.2; C-18, 15.9; C-19, 17.5; C-20, 39.8; C-21, 12.9; C22, 74.8; C-23, 31.9; C-24, 139.6; C-25, 129.1; C-26, 172.1; C-27, 12.3; C-28, 27.3; C-29, 22.2; C-30, 25.6; OCOCH3, 171.1; and OCOCH3, 21.6, 21.0ppm. Mass Spectrum: HREIMS: 586.3996m/e (M+), C35H5407,calcd 586.3871. Reference T. Nishitoba, H. Sato, S. Shirasu, and S. Sakamura; Novel Triterpenoids from the Mycelial Mat at the Previous Stage of Fruiting of Ganoderma lucidum; Agric. Biol. Chem., Vol. 51, pp. 619-622(1987).

310

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Me 3a, 15 a-Diacetoxy-5 a-lanosta-7,9( 11),24E-trien-26-oic acid Molecular Formula/Molecular Weight C34H5006; M W -- 5 5 4 . 3 6 0 7 4

COOH

i

......O A c

A c O .....

General Characteristics Colorless syrup; [a]D24 + 53 ~ (C= 0.26, in MeOH). Fungal Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"). Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated on a silica gel column into seven fractions (Fr. 1-7). Fraction 5 was successively rechromatographed on a silica gel column, preparative TLC and/or reversed-phase LC to afford ganoderic acid Me and eight other related compounds. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data UV: /~, MeOH max

220(c=17,400), 235(16,400), 244(16,200), and 252nm (10,900).

IR:

(thin film) 3200-2500, 1720, 1680 (sh) and 1640cm "~ (sh). IH NMR: (CDCI3) 4.68(1H, br, s, 313-H); 5.08(1H, dd, ,/=9.3, 4.9, 1513-H); 5.34(1H, d, ,/--5.9, 1 l-H); and 5.48ppm (1H, br, s, 7-H). The ~H M R data were identical with that of 3O-acetyl methyl ganoderate X, except for the loss of the methyl ester signal.

9.

Ganoderic Acids

311

13CNMR: (CDC13) C-l, 30.9; C-2, 23.2; C-3, 78.1; C-4, 36.6; C-5, 44.0; C-6, 22.9; C-7, 121.2; C-8, 140.3; C-9, 145.9; C-10, 37.4; C-11, 115.7; C-12, 38.0; C-13, 44.2; C-14, 51.5; C-15, 77.5; C-16, 37.0; C-17, 48.9; C-18, 16.0; C-19, 22.7*; C-20, 36.0; C-21, 18.5; C-22, 34.7; C-23, 26.0; C-24, 144.9; C-25, 126.8; C-26, 172.0; C-27, 12.1; C-28, 27.8; C-29, 22.5*; C-30, 18.2; OCOCH3, 171.2, 170.8; and OCOCH3, 21.4, 21.3ppm. * Assignments may be reversed. Mass Spectrum: HREIMS: 554.3624m/e (M+); C34H5006,calcd 554.3609. Reference T. Nishitoba, H. Sato, S. Shirasu, and S. Sakamura; Novel Triterpenoids from the Myeelial Mat at the Previous Stage of Fruiting of Ganoderma lucidum; Agile. Biol. Chem., Vol. 51, pp. 619-622(1987).

312

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Mf 3 a-Acetoxy- 15 a-hydroxy-5 a-lanosta-7,9(11),24E-trien-26-oic acid Molecular Formula/Molecular Weight C32H4805; MW

"- 5 1 2 . 3 5 0 1 7

COOH

i

.....OH

A c O .....

General Characteristics Colorless syrup; [a]D24 -{"42 ~ (C=0.20, in CHC13). Fungal Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"). Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCla layer was concentrated and separated on a silica gel column into seven fractions (Fr. 1-7). Fraction 5 was successively rechromatographed on a silica gel column, preparative TLC and/or reversed-phase LC to afford ganoderic acid Mf and eight other related compounds. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data UV: Z M~oH 219(e=27,900), 236(26,200), 244(26,200), and 253nm (16,900). max IR:

(thin film) 3400, 3200-2500, 1720, 1680, and 1640cm ~. 1H NMR: (CDCI3) 4.29(1H, dd, J-9.5, 5.1, 15~3-H); 4.68(1H, t, ,/--2.3, 3~3-H); 5.32(1H, d, J=6.2, 1l-H); 5.86(1H, br, d, ,/=3.3, 7-H). The IH and 13C NMR data closely resembled those of ganoderic acid Me, but showed that ganoderic acid Mflost the acetyl group bonded to the 15a-hydroxyl group in ganoderic acid Me.

9.

Ganoderic Acids

313

13CNMR: (CDCI3) C-l, 30.7; C-2, 23.2; C-3, 78.1; C-4, 36.6; C-5, 44.1; C-6, 22.8; C-7, 121.3; C-8, 140.8; C-9, 146.2; C-10, 37.4; C-11, 115.7; C-12, 38.5; C-13, 44.5; C-14, 52.1; C-15, 74.7; C-16, 40.1; C-17, 48.9; C-18, 16.0; C-19, 22.5*; C-20, 36.0; C-21, 18.3; C-22, 34.8; C-23, 25.9; C-24, 145.2; C-25, 126.8; C-26, 172.1; C-27, 12.1; C-28, 27.8; C-29, 22.7*; C-30, 17.2; OCOCH3, 170.8; and OCOCH3, 21.3ppm. * Assignments may be reversed. Mass Spectrum: HREIMS: 512.3531m/e (M+); C32H4805,calcd. 512.3503. Reference T. Nishitoba, H. Sato, S. Shirasu, and S. Sakamura; Novel Triterpenoids from the Mycelial Mat at the Previous Stage of Fruiting of Ganoderma lucidum; Agile. Biol. Chem., Vol. 51, pp. 619-622(1987).

314

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Mg 3 a,22-Diacetoxy- 15 a-hydroxy-7a-methoxy-5a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C35H54Os; M W = 6 0 2 . 3 8 1 8 7

OAc

......

24

26

COOH

o.

OAc 29

28

General Characteristics An amorphous powder; mp., 126-129~

[ a ] D 23 -

23 ~ (c=0.2, in MeOH).

Fungal Source Mycelial mat of Ganoderma lucidum. Isolation/Purification The mycelial mat of G. lucidum was extracted with EtOR reduced in volume, partitioned between water and chloroform, and the chloroform removed under vacuum. The residue was subjected to silica gel column chromatography eluted with a gradient from benzene to ethyl acetate to yield seven fractions. Fraction six was chromatographed twice more using chloroform-methanol, 19:1 (v/v) and benzene-ethyl acetate, 1:1 to yield six more fractions (6a-6 0. Fraction 6d was applied to preparative TLC developed with benzene-ethyl acetate, 1:1 (v/v) and chloroform-methanol, 19:1 (v/v) to yield purified ganoderic acid Mg. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

~, EtOH 210(e = 18,100). max

IR:

(KBr) 3430, 3300-2500(br), 1720,1680(sh), and 1640cm l.

9. Ganoderic Acids

315

~HNMR:

(CDCI3) 6.81(IH, tq,,/=7.3,l.IHz, H-24); 5.01(IH, td,,/=7.0,1.4Hz, H-22), 4.69(IH, t,J=2.9Hz, H-3~); 4.34(IH, t,J=7. IHz, 15~-H); 4.02(IH, br d, J=3.31-Iz,H713);3.33(3H, s);2.57(IH, ddd, ,:=14.5,7.3,7.0Hz, H-23); 2.08(3H, s);2.05(3H, s); 1.87(3H, d, J=I. IHz, H3-27); 1.04(3H, s,H3=30); 0.98(3H, s,H3=28); 0.96(3H, d, J=6.2Hz, H3=21); 0.95(3H, s,H3-29); 0.92(3H, s, 143-19);and 0.65ppm (3H, s,H3=18). 13C NMR: (CDCI3) 30.3, C-1; 23.2, C-2; 77.3, C-3; 36.5, C-4; 40.1, C-5; 21.3, C-6; 76.3, C-7; 132.8, C-8; 143.6, C-9; 38.4, C-I0; 20.9, C-II; 31.4, C-12; 46.2, C-13; 52.8, C-14; 72.0, C-15; 37.3, C-16, 46.4, C-17; 16.6,C-18; 17.2,C-19; 39.3, C-20; 13.0, C-21; 74.9, C-22; 31.9, C-23; 139.1,C-24; 129.3,C-25; 172.0, C-26; 12.3,C-27; 27.3, C28; 22.0, C-29; 18.5,C-30; 170.9, 170.4,CH3OC-; 21.5, 21.0, CH3OC; and 54.4ppm CH30-7. Mass Spectrum: HREIMS: 602.3835(M+); C35H5408,calcd 602.3820; 4.5%, 570(-CH3OH, 5.3), 495, 405, 175, and 43role (base peak). Reference T. Nishitoba, H. Sato, and S. Sakamura; Novel Mycelial Components, Ganoderic Acid Mg, Mh, Mi, Mj, and Mk from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 51, pp. 1149-1153(1987).

316

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Mh 3 tt,22-Diacetoxy-7tt, 15 a-dihydroxy-5 a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C34H520$; M W -" 5 8 8 . 3 6 6 2 2

OAc

.

26

,COOH

.,,

AcO ..... 29

24

"OH 28

General Characteristics A colorless syrup; [tt]D23 + 20 (C=0.2, in MeOH). Fungal Source Mycelial mat of Ganoderma lucidum. Isolation/Purification The mycelial mat of G. lucidum was extracted with EtOH, reduced in volume, partitioned between water and chloroform, and the chloroform removed under vacuum. The residue was subjected to silica gel column chromatography eluted with a gradient from benzene to ethyl acetate to yield seven fractions. Fraction six was chromatographed twice more using chloroform-methanol (19:1, v/v) and benzene-ethyl acetate (1:1, v/v) to yield six more fractions (6a-6 0. Fractions 6e and 6f were combined and chromatographed on a silica gel column (chloroform-methanol, 9:1, v/v), reverse-phase LC (RP~8, methanol-water, 17:3, v/v), and preparative TLC (chloroform-methanol, 24:1, v/v) to yield purified ganoderic acid Mh. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

~, EtoH max

210rim (e = 17,900).

9.

Ganoderic Acids

317

IR:

(KBr) 3400, 3200-2500(br), 1720,1680(sh), and 1640cm~. ~HNMR: (CDCI3) 6.79(1H, tq, J=7.3, 1.0, 24-H), 5.03(1H, t, J=7.0Hz, H-22); 4.69(1H, br s, H-3~); 4.42(1H, t, J=7.0Hz, 15~-H); 4.39(1H, br s, H-7~); 2.56(1H, ddd, ,/--14.4, 7.3, 7.0Hz, 23-H); 2.37(1H, ddd, J=14.4, 7.3, 7.0Hz, 23-H); 2.08(3H, s); 2.06(3H, s); 1.86(3H, s, H3-27); 1.08(3H, s, H3-30); 0.96(3H3, s, H3-28); 0.96(3H, d, J-6.2Hz, 1-1321); 0.93(3H, s, H3-29); 0.92(3H, s, H3-19); and 0.66ppm (3H, s, H3-18). ~3CNMR: (CDC13) 30.3, C-l; 23.3, C-2; 77.5, C-3; 36.4, C-4; 40.5, C-5; 28.1, C-6; 66.8, C-7; 134.5, C-8; 141.7, C-9; 38.1, C-10; 20.6, C-11; 31.7, C-12; 45.7, C-13; 52.4, C-14; 72.4, C-15; 38.1, C-16; 46.4, C-17; 16.4, C-18; 17.3, C-19; 39.5, C-20; 12.9, C-21; 74.7, C-22; 31.9, C-23; 139.1, C-24; 129.3, C-25; 171.4, C-26; 12.4, C-27; 27.4, C28; 21.9, C-29; 19.2, C-30; 170.9, 170.6, CH3OC-; and 21.5, 21.0ppm CH3OC. Mass Spectrum: FD-MS: 588(M+, 30%) and 570m/e ( M ~ - H20, 100%); HREIMS: 570.3524(M + 1-120, C3oH5oO7,calcd. 570.3558, 19%), 495, 435, and 4 3 m / e (base peak). Reference T. Nishitoba, H. Sato, and S. Sakamura; Novel Mycelial Components, Ganoderic Acid Mg, Mh, Mi, Mj, and Mk from the Fungus G a n o d e r m a lucidum; Agric. Biol. Chem., Vol. 51, pp. 1149-1153(1987).

318

9. Ganoderic Acids

_Common/Systematic Name Ganoderic acid Mi 3 a-Acetoxy-7a-methoxy-5a-lanosta-8,24E-dien-26-oic acid _Molecular Formula/Molecular Weight C33H5206; 1V[W' = 544.37639 24

AcO ..... / , ~ ~ 29

v

26

COOH

....OMe

28

General Characteristics A colorless syrup; [a]D23 - 11~ (C=0.2, in MeOH). Funsal Source Mycelial mat of Ganoderma lucidum. Isolation/Purification The mycelial mat of G. lucidum was extracted with EtOH, reduced in volume, partitioned between water and chloroform, and the chloroform removed under vacuum. The residue was subjected to silica gel column chromatography eluted with a gradient from benzene to ethyl acetate to yield seven fractions. Fraction six was chromatographed twice more using chloroform-methanol (19:1, v/v) and benzene-ethyl acetate (1:1, v/v) to yield six more fractions (6a-6f). Fraction 6b was applied to preparative TLC (benzene-ethyl acetate, 1:1, v/v; chloroform-methanol, 19:1, v/v) to yield purified ganoderic acid Mi. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

~, ,~Et~

210rim (e = 19,900).

IR:

(KBr) 3430, 3200-2500(br), 1720, 1680(sh), and 1640cm ~.

9.

Ganoderic Acids

319

1H ~ : (CDCI3) 6.88(IH, tq, J=7.5, 1.1Hz, H-24); 4.27(IH, t, J=2.6Hz, H-313); 4.34(IH, t, J=7.7Hz, 1513-H);4.03(1H, br d, J=3.3Hz, H-713); 3.33(3H, s); 2.08(3H, s); 1.84(3H, d, J= 1.1Hz, H3-27); 1.05(3H, s, 1-13-30);0.98(31-13, s, 1-13-28);0.95(3H, s, H3-29); 0.92(3H, s, 1-13-19);0.91(3H, d, J=6.6Hz, 1-13-21);and 0.65ppm (3H, s, 1-13-18). Mass Spectrum: HREIMS: 544.3739(M+, C33H5206, calcd. 544.3766, 4.2%), 512(M+ -MeOH, 6.0), 437(46), 347(53), 175(49), 95(62), and 43m/e (base peak). Reference T. Nishitoba, H. Sato, and S. Sakamura; Novel Mycelial Components, Ganoderic Acid Mg, Mh, Mi, Mj, and Mk from the Fungus Ganoderma lucidum, Agrie. Biol. Chem., Vol. 51, pp. 1149-1153(1987).

320

9. Ganoderic Acids

Common/Systematic Name Ganoderic acid Mj 22-Acetoxy-3 a-hydroxy-7a-methoxy-5 tt-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C33H5206; MW' = 544.37639 OAc

HO ..... ~ ~ 29

v

24

26

COOH

....OMe

28

General Characteristics A colorless s y r u p ;

[a]D 23 - 8 ~ ( C - 0 . 0 5 ,

in MeOH).

Fungal Source Mycelial mat of Ganoderma lucidum. Isolation/Purification The mycelial mat of G. lucidum was extracted with EtOH, reduced in volume, partitioned between water and chloroform, and the chloroform removed under vacuum. The residue was subjected to silica gel column chromatography eluted with a gradient from benzene to ethyl acetate to yield seven fractions. Fraction six was chromatographed twice more using chloroform-methanol (19:1, v/v) and benzene-ethyl acetate (1:1, v/v) to yield six more fractions (6a-6f). Fraction 6b was applied to preparative TLC (benzene-ethyl acetate, 1:1, v/v; chloroform-methanol, 19:1, v/v) to yield purified ganoderic acid Mi and Mj. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

EtOH 210nm (e = 22,000). max

IR:

(KBr) 3440, 3200-2500(br), 1720, 1680, and 1640cm "~.

9.

Ganoderic Acids

321

1HI~V[R:

(CDCI3) 6.81(1H, tq, J=7.3, 1.1Hz, H-24); 5.10(1H, td, J=6.9, 1.1Hz, H-22); 3.43(1H, t, J=2.9Hz, H-3~3);3.59(1H, br d, J=3.5Hz, H-7~); 3.27(3H, s); 2.57(1H, ddd, d=14.7, 7.3, 6.9, H-23); 2.37(1H, ddd, J=14.7, 7.3, 6.9Hz, H-23); 2.05(3H, s); 1.87(3H, d, J=l.lHz, H3-27); 1.02(3H3, s, H3-28); 1.00(3H, s, H3-29); 0.98(3H, d, J=6.6Hz, H3-21); 0.96(3H, s, H3-30); 0.90(3H, s, H3-19); and 0.61ppm (3H, s, H3-18). Mass Spectrum: HREIMS: 544.3745(M+, C33H5206, calcd. 544.3766, 3.4%), 529(7.9), 404(24), 262(11), 205(14), 173(18), 95(48), 55(38), and 43m/e (base peak). Reference T. Nishitoba, H. Sato, and S. Sakamura; Novel Mycelial Components, Ganoderic Acid Mg, Mh, Mi, Mj, and Mk from the Fungus Ganoderma lucidum, Agric. Biol. Chem., Vol. 51, pp. 1149-1153(1987).

322

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Mk 3 a,22-Diacetoxy- 15 a-hydroxy-5 a-lanosta-7,9(11),24E-dien-26-oic acid Molecular Formula/Molecular Weight C34H5007; M W --" 5 7 0 . 3 5 5 6 5

OAc

24

26

COOH

-

AcO'"

,,.

29

"OH

30

28

General Characteristics A colorless syrup; [a]D23 + 23 ~ (C=0.2, in MeOH). Fungal Source Mycelial mat of Ganoderma lucidum. Isolation/Purification The mycelial mat of G. lucidum was extracted with EtOH, reduced in volume, partitioned between water and chloroform, and the chloroform removed under vacuum. The residue was subjected to silica gel column chromatography eluted with a gradient from benzene to ethyl acetate to yield seven fractions. Fraction six was chromatographed twice more using chloroform-methanol (19:1, v/v) and benzene-ethyl acetate (1:1, v/v) to yield six more fractions (6a-6f). Evaporation of fraction 6c afforded ganoderic acid Mk. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

~, Eton max

218nm (e = 11,700).

IR:

(KBr) 3450, 3300-2500(br), 1720, 1680(sh), and 1640cm ~.

9.

Ganoderic Acids

323

IH N]VIR: (CDCla) 6.86(1H, td, J=7.3, 1.1Hz, 24-H); 5.88(1H, br d, d=S.lHz, H-7); 5.31(1H, d, J=6.2Hz, H-11); 5.04(1H, t, J=6.8Hz, H-22); 4.86(1H, br s, H-313); 4.27(1H, dd, J=9.3, 4.9Hz, 15~-H); 2.57(1H, ddd, J=14.7, 7.3, 6.8Hz, 23-H); 2.07(3H, s); 2.05(3H, s); 1.87(3H, s, H3-27); 1.00(an, s, H3-28); 0.99(3H, s, H3-29); 0.97(3H, s, H3-30); 0.97(3H, d, J-6.6Hz, H3-21); 0.88(3H, s, Ha-19); and 0.62ppm (3H, s, 1"t3-18). 13CNMR: (CDCI3) 30.6, C-l; 23.1, C-2; 78.1, C-3; 36.5, C-4; 44.0, C-5; 22.8, C-6; 121.6, C-7; 140.5, C-8; 146.2, C-9; 37.3, C-10; 115.3, C-11; 38.4, C-12; 44.3, C-13; 52.1, C-14; 74.5, C-15; 39.6, C-16; 45.5, C-17; 15.8, C-18; 22.7~ C-19; 39.3, C-20; 12.8, C-21; 74.7, C-22; 31.7, C-23; 139.1, C-24; 129.4, C-25; 172.2, C-26; 12.3, C-27; 27.8, C28; 22.5", C-29; 17.3, C-30; 170.8, 170.7, CH3OC-; and 21.3, 21.0ppm CH3OC. Assignments may be reversed. Mass Spectrum: HREIMS: 570.3549(M~, C34H5oO7,calcd 570.3557, 0.9%), 510(2.8), 466(3.2), 428(8.3), 353(9.5), 95(20), 60(60), and 43m/e (base peak). Reference T. Nishitoba, H. Sato, and S. Sakamura; Novel Mycelial Components, Ganoderic Acid Mg, Mh, Mi, Mj, and Mk from the Fungus Ganoderma lucidum, Agric. Biol. Chem., Vol. 51, pp. 1149-1153(1987).

324

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid M 713,12a-Dihydroxy-3,11,15,23-tetraoxo-5t~-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C30I-I4208; M W -- 530.28797

29

28

General Characteristics Methyl ester: needles from methanol; mp., 206-210~ Funsal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol ( 9:1, v/v; fraction 12), and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using preparative TLC (benzene-ethyl acetate, 1:1 or 2:1, v/v), silica gel column chromatography (chloroform-methanol, 99:1, v/v), or reversed-phase LC (methanol-water, 7:3, v/v, Lobar RP~8). These separations gave purified methyl ganoderate M, N, O, H, E, methyl lucidenate K, L, and E2. Biological Activi.ty Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ester)

UV~

~, MeOH max

258nm (r

9.

Ganoderic Acids

325

CD: ~, MeOH max

A292 -1.0, A572 +7.3, A23o0, and A218-2.8.

IR~

(KBr) 3450, 2900, 1710, and 1660cm"l. 1H NMR:

(CDC13) 1.82(1H, ddd, ,/--13.6, 8.7, 8.3Hz, H-la); 2.89(1H, ddd, J=13.6, 7.8, 5.9Hz, H-l[3); 2.50(1H, ddd, J=15.1, 8.3, 7.8Hz, H-2a); 2.54(1H, ddd, J=15.1, 8.7, 5.9Hz, H2[3); 1.74(1H, dd, J=13.7, 1.0Hz, H-Sa); 2.11(1H, ddd, J=13.2, 7.3, 1.0Hz, H-6a); 1.65(1H, ddd, J=13.7, 13.2, 9.8Hz, H-613);4.85(1H, dd, J=9.8, 7.3Hz, H-7a); 3.78(1H, s, H-1213), 2.73(1H, dd, J=22.5, 8.3Hz, H-16a); 2.11(1H, dd, J--22.5, 12.7Hz, H-1613), 2.73(1H, m, H-17); 1.03(3H, s, H3-18); 1.15(3H, s, H3-19); 2.18(1H, m, H-20); 1.09(3H, d, J=6.8Hz, H3-21); 2.41(1H, dd, J=16.1, 8.3Hz, H-22), 2.37(1H, dd, J=6.1, 4.4Hz, H-22); 2.87(1H, dd, J=17.6, 8.3Hz, H-24); 2.47(1H, dd, J=17.6, 5.4Hz, H-24); 2.96(1H, m, H-25); 1.19(3H, d, J=7.3Hz, H3-27); 1.10(3H, s, H3-28); 1.14(3H, s, H3-29); 1.32(3H, s, H3-30); and 3.69ppm (3H, s, COOCH3). Mass Spectrum: HREIMS: 544.3058(M § C3~H4408,calcd 544.3037, 3%), 526(16), 355(6), 304(100), 139(23), 129(31), 69(25), and 59m/e (37). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

326

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid N 7[3,20-Dihydroxy-3,11,15,23-tetraoxo-5et-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C30H42Og; M W -- 530.28798 OH

O~ 12%~[~

29

13

(~OOH

28

General Characteristics Methyl ester: prisms from ethyl acetate-cyclohexane; mp., 164-167~

[tg]D 24 -k-

153 ~

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12), and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol, 19:1 to give several fractions. Some of the fractions were methylated and further fractionated using preparative TLC (benzene-ethyl acetate, 1:1 or 2:1, v/v), silica gel column chromatography (chloroform-methanol, 99:1, v/v), or reversed-phase LC (methanol-water, 7:3, v/v, Lobar RP~8).These separations gave purified methyl ganoderate M, N, O, H, E, methyl lucidenate K, L and E2. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV:

~. M,-oH 254nm (e=7,180). max

9.

Ganoderic Acids

327

IR:

(KBr) 3450, 2950, 1720, 1700, and 1650cmq. CD:

MeOH /~ max

A292 -2.5, Asso+0, A255 +18.1, A229 0, and A215 -7.3.

]H NMR: (CDCI3) 1.47(IH, ddd, J=13.6, 8.5,8.5Hz, H-le); 2.95(IH, m, H-I~); 2.50(IH, m, H-2a); 2.50(IH, m, H-213),1.55(IH, dd, J=12.6, 1.1Hz, H-5a), 2.12(IH, ddd, J=12.6, 7.7, I.IHz, H-6a); 1.68(IH, ddd, J=12.6, 12.6,9.0I-Iz,.1-1-613); 4.84(IH, dd, J=9.0, 7.7Hz, H-7a); 2.77(1H, d, J=17.2Hz, H-12a); 2.85(1H, d, J=17.2Hz, H-1213); 2.86(1H, dd, J=19.4, 10.3Hz, n-16tt); 2.45(1H, m, H-1613); 2.25(1H, dd, J=10.0, 9.0Hz, n-17); 1.17(3H, s, H3-18); 1.26(3H, s, H3-19); 1.41(3H, s, H3-21), 2.66(1H, d, J=16.5Hz, H-22); 2.49(1H, d, J=16.5Hz, H-22); 2.95(1H, m, H-24); 2.45(1H, m, H24); 2.95(1H, m, H-25); 1.20(3H, d, J=7.0Hz, H3-27); 1.1 l(3H, s, H3-28), 1.12(3H, s, H3-29); 1.34(3H, s, H3-30); and 3.69ppm (3H, s, COOCH3). 13C NMR:

(CDCI3) 35.7, C-l; 34.3, C-2; 217.8, C-3; 45.3, C-4; 49.4, C-5; 27.7, C-6; 66.3, C-7; 157.6, C-8; 141.0, C-9; 38.3, C-10; 197.6, C-11; 50.5, C-12; 46.8, C-13; 59.78, C-14; 216.8, C-15; 36.3, C-16; 49.0, C-17; 19.3, C-18; 18.1, C-19; 72.9, C-20; 26.7, C-21; 52.7, C-22; 210.4, C-23; 47.7, C-24; 34.5, C-25; 175.9, C-26; 17.0, C-27; 27.0, C-28; 20.8, C-29; 25.1, C-30; and 52.0ppm, CH3OOC-. Mass Spectrum: HREIMS: 544.3057(M+, C3]H4408, calcd 544.3037, 0.06%), 400(2), 372(5), 129(7), 112(18), 87(21), 59(210), and 43m/e (100). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lueidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

328

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid O 20-Hydroxy-3,7,11,15,23-pentaoxo-5 tt-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight C30I-h0Os; MW = 528.27232 OH 0

29

12

o.

28

General Characteristics Methyl ester: pale yellow needles from ethyl ether-hexane; mp., 168-17 I~ Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12), and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using preparative TLC (benzene-ethyl acetate, 1:1 or 2:1, v/v), silica gel column chromatography (chloroform-methanol, 99:1, v/v), or reversed-phase LC (methanol-water, 7:3, v/v, Lobar RP~s). These separations gave purified methyl ganoderate M, N, O, H, E, methyl lucidenate K, L, and E2. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

(Methyl ester)

MeOH

Z max

252nm (c=6,610).

9.

Ganoderic Acids

329

IR:

(KBr) 3475, 2950, 1740, and 1700cm"1. CD: /~ MeOH max

A330 0, A305 -5.0, A290 0, A277 +5.1, A259 0, A259 0, A252 -1.5, A243 0, and A212 0.

1H NMR: (CDCI3) 1.79(1H, ddd, ,/=14.1, 10.4, 5.9Hz, H-la); 3.00(1H, m, H-l[3); 2.46(1H, ddd, J=15.6, 8.8, 5.9Hz, H-2(x); 2.66(1H, ddd, J=15.6, 10.4, 5.9Hz, H-213); 2.48(1H, dd, J=14.7, 2.4Hz, H-Sa); 2.53(1H, dd, J=13.4, 2.4Hz, H-6(x); 2.76(1H, dd, J=14.7, 13.2Hz, H-613); 3.25(1H, d, J=16.6Hz, H-12(x); 3.12(1H, d, J=16.6Hz, H-1213); 3.00(1H, m, H-16a); 3.00(1H, m, H-1613); 2.92(1H, dd, J=8.3, 8.3Hz, H-17); 1.38(3H, s, H3-18); 1.32(3H, s, H3-19); 1.67(3H, s, H3-21); 2.85(1H, d, J=14.7Hz, H-22); 2.85(1H, d, J=14.7Hz, H-22); 3.00(1H, m, H-24); 2.70(1H, dd, J=17.0, 4.0Hz, H-24); 3.00(1H, m, H-25); 1.16(3H, d, J=7.aHz, H3-27); 1.06(3H, s, H3-28); 1.1 l(3H, s, H329); 1.92(3H, s, H3-30); and 3.62ppm (3H, s, COOCH3). Mass Spectrum: FD-MS: 542(M+, 100%), 398(43), 355(20), 187(90), and 144m/e (31); LREIMS: 398(C24H3005+, 6%), 355(4), 300(42), 215(23), 87(15), 59(15), and 43m/e (100). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

330

9. Ganoderic Acids

Common/Systematic Name Ganoderic acid R (22S,24E)-3 a,22-Diacetoxy-5 tt-lanosta-7,9(11),24-trien-26-oic acid Molecular Formula/Molecular Weight C34H5006; M W -- 5 5 4 . 3 6 0 7 4

OAc

lh,,,,.

COOH

_

AcO.....

,

General Characteristics Colorless needles; mp., 201-202~

[a]D

+8.7~ (c=0.092, in CHCI3).

Fungal Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"), called Reishi in Japan. Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated by repeated chromatography on silica gel column and/or reversed-phase LC to afford purified ganoderic acids, 1L S, and T. Biological Activity Strongly antihepatotoxic in galactose-induced cytotoxicity using primary cultured rat hepatocytes. Spectral Data UV:

~

MeOH max

224nm (log c = 4.0), 234(4.0), 242(4.0), and 250nm (3.8).

IR:

(thin film) 3400(OH), 2870(CH), 1720(COO), and 1675cm~ (COO). ~H NMR: (CDCI3) The IH NMR spectrum was very similar to that of ganoderic acid T except for the loss of the methine proton signal at 5.03ppm (15a-H) and loss of one acetoxyl methyl proton signal.

9.

Ganoderic Acids

331

~3CNMR: (CDCI3) C-I, 30.5; C-2, 23.0; C-3, 78.0; C-4, 36.4; C-5, 44.0; C-6, 27.5; C-7, 120.2; C-8, 142.2; C-9, 145.8; C-10, 37.1; C-11, 115.5; C-12, 37.6; C-13, 43.6; C-14, 50.3; C-15, 31.3; C-16, 22.8; C-17, 47.3; and C-18, 15.4ppm. The 13CNMR spectrum was also closely similar to that of ganoderic acid T except for the appearance of a methylene carbon signal (31.3ppm) in ganoderic acid R instead of the methine carbon signal (77.4ppm) in T. Mass Data: Anal. calcd for C34H5006:C, 73.61; H, 9.09%; found C, 73.77; H, 9.13%. Reference M. Hirotani, C. Ino, T. Furuya, and M. Shirob; Ganoderic Acids T, S, and R, New Triterpenoids in the Cultured Mycelia of Ganoderma lucidum; Chem. Pharm. Bull., Vol. 34, pp. 2282-2285(1986).

332

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid S (22S, 24E)-22-Acetoxy-3a-hydroxy-5a-lanosta-7,9(11),24-trien-26-oic acid Molecular Formula/Molecular Weight C34I-I4sO5; MW = 512.35017

OAc IIIiii,"

,COOH

HO"" General Characteristics Ganoderic acid S crystallized as needles; mp., 194-196~ in CHC13).

[a]D + 19.8~ (c= 0.085,

Fungal Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"), called Reishi in Japan. Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with ethanol. The extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated by repeated chromatography on silica gel columns and/or reversed-phase LC to afford purified ganoderic acids R, S, and T. Biological Activity Strongly antihepatotoxic in galactose-induced cytotoxicity using primary cultured rat hepatocytes. Spectral Data UV:

~ EtOH

231(log c = 4.2), 240(4.2), and 248nm (4.0).

IR:

(Thin film) 3410(OH), 1720(COO), and 1675cm~ (COO).

9.

Ganoderic Acids

1H NMR: (CDC13) The proton NMR spectrum of ganoderic acid S showed a methine proton signal at 5.1 lppm (ddd, J=6.8, 6.0, 1.7Hz, H-22) and a proton signal at 3.45ppm (dd, J=l.4, 1.4ppm, H-313) which was absent in the proton spectra of T andR. 13CNMR: (CDCI3) 29.8, C-l; 25.5, C-2; 76.0, C-3; 37.1", C-4; 43.1, C-5; 27.5, C-6; 120.3, C-7; 142.2, C-8; 145.9, C-9; 37.3*, C-10; 115.5, C-11; 37.6, C-12; 43.6, C-13; 50.3, C-14; 31.2, C-15; 22.9, C-16; 47.3, C-17; 15.4, C-18; 22.7, C-19; 39.3, C-20; 12.6, C-21; 74.6, C-22; 31.7, C-23; 139.5, C-24; 129.0, C25; 172.2, C-26; 12.2, C-27; 28.1, C-30; 22.5*, C-31; 25.7, C-32; 21.0, acetyl methyl; and 170.6ppm, C=O. * Assignments may be reversed. Mass Data: Anal. Calcd. For C32I-hsOs: C, 74.96; H, 9.44%; found C, 74.93; I-I, 9.55%. Reference M. Hirotani, C. Ino, T. Furuya, and M. Shirob; Ganoderic Acids T, S, and R, New Tripenoids in the Cultured Mycelia of Ganoderma lucidum; Chem. Pharrn. Bull., Vol. 34, pp. 2282-2285(1986).

333

334

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid T (22S,24E)3 a, 15a,22-Triacetoxy-5 a-lanosta-7,9(11),24-trien-26-oic acid Molecular Formula/Molecular Weight C36H5208; M W -" 612.36622 OAc COOH

II111% '

"OAc A c O .....

General Characteristics Colorless needles; mp., 200-202~

[a]D +23 ~ (C=0.13, in CHCI3).

Fungal Source Isolated from mycelium of Ganoderma lucidum ("Saegusa"), called Reishi in Japan. Isolation/Purification The mycelial mat grown on a solid medium was harvested and then extracted with EtOH. The extract was partitioned between CHCI3 and water. The CHCI3 layer was concentrated and separated by repeated chromatography on silica gel column and/or reversed-phase LC to afford purified ganoderic acids, R, S, and T. Biological Activity Possibly cytotoxic to hepatoma cells or antihepatotoxic. Spectral Data UV:

~, maxM~H 216rim (log E = 4.0), 225(4.00), 234(4.0), and 242nm (4.0). IR:

(thin film) 3420, 2940, 1720, and 1240cm"1. IH NMR: (CDCI3) The spectrum showed the presence of five tertiary methyl (0.66, 0.88, 0.98, 0.99, and 1.03ppm), one allyl methyl (1.86ppm) and one secondary methyl (0.97ppm, d, J=6.7Hz) signals. Also observed were signals due to three secondary acetoxyl

9.

Ganoderic Acids

335

groups 2.05(3H, s), 2.07(3H, s), 2.08(3H, s), 4.68(1H, dd, J=3.0, 3.0 H-3), 5.08(H, dd, J=10.0, 5.1Hz, H-15), and 5.03ppm (H, ddd, J=7.1, 7.0, 1.4Hz, H-22) and three olefinic proton signals 5.32ppm (1H, d, J=6.4Hz, H-11), 5.48(1H, bs, H-7), 6.78(1H, dd, J=7.5, 7.SHz, H-24). These spectral data suggested that ganoderic acid T was a lanostane-type triterpenoid having a heteroannular diene moiety and three acetoxyl groups. 13CNMR: (CDCI3) C-l, 30.8; C-2, 23.3; C-3, 78.2; C-4, 36.8; C-5, 44.1; C-6, 22.7; C-7, 121.6; C-8, 140.2; C-9, 146.2; C-10, 37.5; C-11, 115.5; C-12, 38.1; C-13, 44.1; C-14, 51.6; C-15, 77.4; C-16, 36.7; C-17, 45.6; and C-18, 15.9ppm. Reference M. Hirotani, C. Ino, T. Furuya, and M. Shirob; Ganoderic Acids T, S, and R, New Triterpenoids in the Cultured Mycelia of Ganoderma lucidum; Chem. Pharm. Bull., Vol. 34, pp. 2282-2285(1986).

336

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid U Molecular Formula/Molecular Weight C3oH4804; M W

-- 4 7 2 . 3 5 5 2 6

COOH

11,,,,,.

HO ....

.

0H

General Characteristics Crystals; mp., 166-169~

[tg]D 22 q-

35 ~ (c=3.5, in EtOH).

FunRal Source v Isolated from mycelium of Ganoderma lucidum. Biological Activity. In vitro cytotoxicity on hepatoma cells. Soectral Data IR~

(CHCI3 or CCI4) 3610, 3550-3300, 1690, and 1645cm"l. 1H NMR: (CDCI3) 0.62(3H, s); 0.89(3H, s); 0.92(3H, d, J=6.0Hz); 0.97(3H, s); 1.01(3H, s); 1.04(3H, s); 1.84(3H, s); 3.48(1H, t, J=2.5Hz); 4.16(1H, m); and 6.91ppm (1n, t, J=7.0Hz) 13C NMR: (CDCI3) Methyl ester derivative: 29.7, C-I; 28.8, C-2; 75.8, C-3; 37.2, C-4; 39.0, C-5; 25.6, C-6; 67.3, C-7; 135.9, C-8; 141.4, C-9; 38.3, C-10; 20.9, C-11; 29.9, C-12; 45.1, C-13; 49.7, C-14; 31.1, C-15; 28.2, C-16; 50.62, C-17; 16.1, C-18; 17.3, C-19; 36.5, C-20; 18.4, C-21; 34.9, C-22; 25.9, C-23; 145.4, C-24; 126.8, C-25; 172.7, C-26; 12.0, C-27; 27.9, C-30; 22.3, C-31; 26.3, C-32; and 51.7ppm -COECH. Mass Spectrum: LREIMS" 472(NV), 454(M + - H20), 526(M +- AcOH), 508(NV - AcOH-H20), 493(M + - AcOn- CH3- n20), 451(M + - AcOn - CH3), 433(NV - 2AcOn - CH3- n20), 293(M + - 2AcOH - H20 - C 9 H 1 5 0 2 ) , and 239m/e.

9. Ganoderic Acids

337

TLC Data A rose-violet colored spot at Rf 0.55 of methyl ester using silica gel Merck HE254with methylene chloride-MeOH, 19:1 as developing solvent and spraying with 50% sulfuric acid and heating. Reference J. O. Toth, B. Luu, J. P. Beck, and G. Ourisson; Triterpenes Cytotoxiwues de Ganoderma lucidum. Structures des acides Ganoderiques U-Z; J. Chem. Res.(M), pp. 27222787(1983).

338

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid V Molecular Formula/Molecular Weight C32H4806; MW = 528.34509

~ ~.~ yi

COOH .......

OAc

General Characteristics Isolated as methyl ester (C33H5006), white amorphous powder; CHCI3). Fungal Source Isolated from

laiD 20 q-

83 ~ (c=0.92, in

Ganoderma lucidum.

Biological Activity Cytotoxic Spectral Data (Methyl ester) IR:

(CHCI3 or CCh) 3600, 1713, and 1649cm"~. 1H NIVIR:

(CDCI3) 0.72(3H, s); 0.92(3H, d, J=6.0Hz); 1.05(3H, s); 1.07(3H, s); 1.13(3H, s); 1.16(3H, s); 1.82(3H, s); 3.73(3H, s); 5.10(1H, dd, J=9.0, 5.0Hz); and 6.72ppm (1n, t, J=7.0Hz) ~ac NMR: (CDC13) 35.2, C-l; 34.3, C-2; 217.3, C-3; 46.7, C-4; 44.8, C-5; absent, C-6; 66.2, C7; 134.7, C-8; 140.0, C-9; 38.1, C-10; 20.8, C-11; 28.4, C-12; 45.3, C-13; 51.1, C-14; 76.6, C-15; 36.5, C-16; 49.32, C-17; 16.6, C-18; 17.3, C-19; 36.2, C-20; 18.2, C-21; 34.8, C-22; 25.7, C-23; 142.5, C-24; 127.4, C-25; 168.7, C-26; 12.4, C-27; 26.5, C-30; 21.2, C-31; 20.1, C-32; 51.7,-CO2CH; 21.2, CH3COO-; and 170.6ppm CH3COO-. Mass Spectrum: LREIMS: NV absent, 524(M~ -H20), 493,464(M~ - AcOH-H20), 449(M + - hcOHCH3- H20), 335, and 309role ( M + - AcOH - C9H1502).

9.

Ganoderic Acids

339

TLC Data Re 0.35 of methyl ester using silica gel Merck I-IF254 using ethyl ether-hexane, 1:1 as developing solvent. Appeared as yellow then chestnut-violet spot atter spraying with 50% sulfuric acid and heating. Reference J. O. Toth, B. Luu, J. P. Beck, and G. Ourisson; Triterpenes Cytotoxiwues de Ganoderma lucidum. Structures des Acides Ganoderiques U-Z; J. Chem. Res. (M), pp. 27222787(1983).

340

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid W Molecular Formula/Molecular Weight C34H5207; M W -- 572.37130

C OOH

_. i AcO

.....

�9 ....,,,

...... O A c

OH

General Characteristics Amorphous solid; mp., 114-117~ methyl ester: [a]D21 + 4~ (C=0.5, in EtOH). Fungal Source Isolated from mycelium of Ganoderma lucidum. Biological Activity. Cytotoxic. Spectral Data UV:

~.m~x 215nm (log e=4.1). IR:

(CHCI3 or CCI4) 3600, 3000-2500, 1743,1690, and 1645cm~; 3600, 1742, 1728-1718, and 1650cm"~ IH NMR: (CDCI3) 0.71(3H, s); 0.93(9H, m); 0.97(3H, s); 1.20(3H, s); 2.07(3H, s); 2.09(3H, s); 1.84(3H, s); 4.12(1H, m); 4.69(1H, t, J=2.5Hz); 5.09(1H, dd, J=9.0, 5.0Hz); and 6.86(1H, t, J=7.0Hz); methyl ester: 0.71(3H, s); 0.93(9H, m); 0.97(3H, s); 1.12(3H, s); 2.07(3H, s); 2.09(3H, s); 4.1 l(1n, m); 4.69(1H, t, J=2.5Hz); 5.14(1H, dd, J=9.0, 5.0Hz); and 6.72ppm (1H, t, J=7.0Hz). 13C NMR: (CDCI3) Methyl ester derivative: 30.2, C-l; 25.6, C-2; 77.4, C-3; 36.3, C-4; 40.0, C-5; 23.3, C-6; 66.4, C-7; 133.8, C-8; 141.7, C-9; 38.4, C-10; 20.7, C-11; 27.3, C-12; 45.4, C-13; 51.1, C-14; 76.5, C-15; 36.5, C-16; 49.2, C-17; 16.5, C-18; 17.4, C-19; 36.2, C-

9.

Ganoderic Acids

341

20; 18.2, C-21; 34.7, C-22; 25.6, C-23; 142.6, C-24; 127.4, C-25; 168.7, C-26; 12.4, C-27; 27.4, C-30; 21.9, C-31; 20.2, C-32; 51.7-CO2CH3; 21.4, CH3COO-; 171.9, CH3COO- ; 21.2, CH3COO; and 170.5ppm CH3COO. Mass Spectrum: LREIMS: Methyl ester: M+(absent), 568(M+ -H20), 526(M+ - AcOH), 508(M ~ AcOH-H20), 493(M +- AcOH- CH3- H20), 451(M+- AcOH- CH3), 433(M +2AcOH- CH3- H20), 293(M + - 2AcOH- 1-120 - C9H1502), and 239m/e. TLC Data Rf 0.43 of methyl ester using silica gel impregnated with silver nitrate with ethyl etherhexane, 4:1 (v/v) as developing solvent. Reference J. O. Toth, B. Luu, J. P. Beck, and G. Ourisson; Triterpenes Cytotoxiwues de Ganoderma lucidum. Structures des acides Ganoderiques U-Z; J. Chem. Res. (M), pp. 27222787(1983).

342

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid X Molecular Formula/Molecular Weight C32H4805; M W = 512.35017 l,,,,,,

i

NO .....

COOH

.....OAc

.",%

General Characteristics Isolated as methyl ester (C33H5005), crystals; mp., 161-163~ [a]D 23 + 76~ (c=3.0, in CHC13). Fungal Source Isolated from Ganoderma lucidum. Biological Activity Cytotoxic. Spectral Data (Methyl ester only) UV:

220(1og e = 4.1), 233(4.0), 242(4.0), and 251nm (3.8). Im:

(CHCI3 or CC14) 3608, 3000-2500, 1710, and 1648cm1. 1H NMR: (CDCI3) 0.66(3H, s); 0.92(3H, d, J=6.0Hz); 0.93(3H, s); 0.99(6H, s), 1.02(3H, s), 1.82(3H, s); 2.08(3H, s); 3.73(3H, s); 3.44(1H, t, J=2.5Hz); 5.08(1H, dd, J=9.0, 5.0Hz); 5.35(1H, m); 5.48(1H, m); and 6.73ppm (1H, t, J=7.0Hz). 13C N M R :

(CDCI3) 30.0, C-l; 25.7, C-2; 76.0, C-3; 37.1, C-4; 43.0, C-5; 23.0, C-6; 121.3, C-7, 140.3, C-8; 146.1, C-9; 37.4, C-10; 115.6, C-11; 38.0, C-12; 44.2, C-13; 51.5, C-14, 77.5, C-15; absent, C-16; 48.9, C-17; 16.0, C-18; 22.8, C-19; 35.9, C-20; 18.5, C-21; 34.8, C-22; 25.7, C-23; 142.7, C-24; 127.4, C-25; 168.7, C-26; 12.4, C-27, 28.2, C-30; 22.7, C-31; 18.2, C-32; 51.7,-CO2_C_CH; 21.5, CH3COO-; and 171.2ppm CHaCOO-.

9.

Ganoderic Acids

343

Mass Spectrum: HREIMS: 5 2 6 . 3 6 6 1 m / e (M+), calcd, for (C33H5oO5) 526.3658; LREIMS: 526(M+), 508(M +- HzO), 493(M +- CH3- H20), 466(M +- AcOH), 451(M +- AcOH- CH3), 448(M + - AcOH - H20), 433(M + - CH3-1-120 - AcOH), 337, 311(M + - AcOH CgHlsO2), and 293role ( M + - AcOH- H/O - CgH15Oz). TLC Data Re 0.32 of methyl ester using silica gel Merck HF254using ethyl ether-hexane, 1:1 (v/v) as developing solvent. Appeared as yellow turning to maroon-violet spot after spraying with 50% sulfuric acid and heating. Reference J. O. Toth, B. Luu, J. P. Beck, and G. Ourisson; Triterpenes Cytotoxiwues de G a n o d e r m a l u c i d u m . Structures des acides Ganoderiques U-Z; J. Chem. Res. (M), pp. 27222787(1983).

344

9.

Ganoderic Acids

Common/Systematic Name Ganoderic acid Y 313-Hydroxy-5 a-lanosta-7,9(11),24-trien-26-oic acid Molecular Formula/Molecular Weight C3oH4603; M W "-- 4 5 4 . 3 4 4 7 0

COOH

.oJ, General Characteristics Crystals from ethyl ether-ethanol, mp., 203-206~ [a]D 25 + 5 4 ~ (c= 0.8, in ethanol); Methyl ester; mp., 139-141~ [a]D26 +53 ~ (C= 0.6, in chloroform). Fungal Source Isolated from mycelium of Ganoderma lucidum. Biological Activity. Cytotoxic. Spectral Data IR: (CHCI3 or CC14) 3600, 3440, 2700-2200, 1690, and 1647cm1. 1H NMR:

(CDCI3) 5.33 (IH, m); 5.47 (IH); and 8.88ppm (IH, t,J= 7.0Hz). Methyl ester;3.73 (3H, s); 5.31 (1H, m); 5.48 (11-1, m), and 6.72ppm (1H, t , J = 7.0Hz).

Mass Spectrum: LREIMS: 454(M+), 439(M + - CH3), and 42 lm/e (M + - Ell3 - H20 ). Methyl ester; 468 (M+), 453 (M~- CH3), 435(M + - CH3- H20), and 302m/e.

Reference

J. O. Toth, B. Luu, J. P. Beck, and G. Ourisson; Triterpenes Cytotoxiwues de Ganoderma lucidum. Structures des acides Ganoderiques U-Z; J. Chem. Res. (M), pp. 2722-2787 (1983).

9.

Ganoderic Acids

345

Common/Systematic Name Ganoderic acid Z Molecular Formula/Molecular Weight C3oH4803; M W -- 456.3603 5 ..

COOH

General Characteristics Crystals; mp., 137-140~ [a]D 24 + 59~(c=0.13, in CHCla); methyl ester; mp., 123-125~ [tZ]D22 + 56~ (C=0.7, in EtOH). Funsal Source Isolated from mycelium of Ganoderma lucidum. Biological Activity Cytotoxic. Spectral Data IR:

(CHC13 or CCI4) 3600, 3470, 2700-2300, 1685, and 1645cm"~. 1H NMR:

(CDCI3) 6.90(1H, t, J=7.0Hz); methyl ester: 0.69(3H, s); 0.81(3H, s); 0.88(3H, s); 0.92(3H, d, J=6Hz); 1.00(3H, s); 1.84(3H, s); 3.24(1H, m); 3.73(3H, s); and 6.76ppm (1H, t, J=7.0Hz). 13C NMR:

(CDC13) 313-acetylmethylester derivative: 35.3, C-l; 24.2, C-2; 80.9, C-3; 37.9, C-4; 50.4, C-5; 18.2, C-6; 26.4, C-7; 134.4, C-8; 134.4, C-9; 37.0, C-10; 21.0, C-11; 30.8, C-12; 44.6, C-13; 49.9, C-14; 31.0, C-15; 28.2, C-16; 50.6, C-17; 15.8, C-18; 18.6, C19; 36.4, C-20; 19.2, C-21; 35.0, C-22; 25.7, C-23; 143.2, C-24; 127.2, C-25; 168.8, C-26; 12.3, C-27; 28.0, C-30; 16.6, C-31; 24.3, C-32; 51.6 -CO2CH3; 21.3, CH3COO-; and 171.0ppm CHaCOO-. Mass Spectrum: LREIMS: Methyl ester: 470(M+), 455(M-CH3), 442, 437(M-CH3-H20), 427, and 409m/e.

346

9.

Ganoderic Acids

TLC Data Rf 0.28 of methyl ester using silica gel impregnated with silver nitrate with ethyl etherhexane, 1:4 (v/v) as developing solvent. Reference J. O. Toth, B. Luu, J. P. Beck, and G. Ourisson; Triterpenes Cytotoxiwues de Ganoderma lucidum. Structures des acides Ganoderiques U-Z; J. Chem. Res. (M), pp. 27222787(1983).

9.

Ganoderic Acids

347

Common/Systematic Name Ganodermic Acid R Lanosta-7,9(11),24-triene-3 t~,15t~-diacetoxy-26-oic acid Molecular Formula/Molecular Weight C34H5006; MW

-- 5 5 4 . 3 6 0 7 4

COOH

H AcO

-

~DAc

\

General Characteristics Amorphous powder from n-hexane-CHCls; mp., 126-129~ Fungal Source

Ganoderma lucidum (strains TP-1 and AT- 4).

Isolation/Purification Dried, ground mycelia were extracted with MeOH; the extract was partitioned between nhexane and H20. The aqueous layer was re-extracted with EtOAc and chromatographed on silica gel (gradient from MeOH to CHC13) followed by reversed phase (Cls) HPLC (90% aqueous MeOH). Pure substance was obtained through repeated recrystallization from n-hexane-CHC13. Spectral Data UV~ ,~ MeOH max

210(log e = 4.08), 235(4.14), 243(4.16), and 251nm (3.97).

IR:

(CHC13) 3025, 2945, 1715, 1680, and 1240cml. CD: (c=0.0001, in MeOH)

[0]230 -- q-

17,200.

ORD:

(c=0.0001, in MeOH) 20,700.

[(I)]248 + 8 2 0 0 , [(D] 223 -

7700,

[(i)]211

-11,600, and [(I)]201

-

348

9.

Ganoderic Acids

IH NMR: (CDCI3) 4.64(1H, bs, H-3); 5.45(1H, m, H-7); 5.30(1H, dd, J=5.7Hz, H-11), 5.04(1H, dd, J=4.5, 10Hz, H-15); 0.63(3H, s, H-18); 0.95(3H, s, H-19); 0.88(3H, d, J=6.3Hz, n-21), 6.83(1H, m, H-24); 1.79(3H, s, H-27); 0.85(3H, s, H-29); 0.94(3H, s, H-a0), 2.01(3H, s, AcCH3) and 2.05ppm (3H, s, AcCH3). 13C NMR:

(CDC13) 30.51(t, C-I); 23.03(t, C-2); 77.98(d, C-3); 36.42(s, C-4); 43.82(d, C-5); 22.70(t, C-6); 121.04(d, C-7); 140.09(s, C-8); 145.40(s, C-9); 37.22(s, C-10); 115.52(d, C-11); 37.87(t, C-12); 44.04(s, C-13); 51.30(s, C-14); 77.26(d, C-15), 36.89(t, C-16); 48.76(d, C-17); 15.85(q, C-18); 22.52(q, C-19), 35.85(d, C-20); 18.08(q, C-21); 34.54(t, C-22); 25.82(t, C-23); 144.99(d, C-24), 126.72(s, C-25); 172.92(s, C-26); 11.84(q, C-27); 18.31(q, C-28); 27.65(q, C-29); 22.32(q, C-30); 21.15(q, COOCH3); 21.26(q, COOCH3); 170.65(s, AcCO); and 171.02ppm (AcCO). Mass Spectrum: 554(M~, C34I-I5oO6,100%), 494(M§- CH3COOH, 40), 479(M+-CH3COOH-CH3, 25), 434(M+- 2CH3COOR 33), 419(M+- 2CH3COOH-CH3, 90), 353(M+ -CH3COOHC1~-I13Ozside chain, 12), 299(D-ring cleavage-CH3, 14), 293(M+- 2CH3COOHClsH1302 side chain, 19), and 239role (299- CH3COOR 44). Reference M. S. Shiao, L. J. Lin, S. F. Yen, and C. S. Chou, Two New Triterpenes of the Fungus Ganoderma lucMum; J. Nat. Prod., Vol. 50, No. 5, pp. 886-890(1987).

9.

Ganoderic Acids

349

Common/Systematic Name Ganodermic Acid S Lanosta-7,9(11),24-triene-313,15a-diacetoxy-26-oic acid Molecular Formula/Molecular Weight C34I-I5006, M W -- 5 5 4 . 3 6 0 7 4

COOH

H,,

i

''OAc

Ac General Characteristics Colorless needles from n-hexane-CHC13; mp., 123-124~ Fungal Source

Ganoderma lucidum (strains TP-1 and AT-4).

Isolation/Purification Dried, ground mycelia were extracted with MeOH; the extract was partitioned between hexane and H20. The aqueous layer was re-extracted with EtOAc and chromatographed on silica gel (gradient from MeOH to CHC13) followed by reversed phase (Cls) HPLC (90% aqueous MeOH). Pure substance was obtained through repeated recrystallization from n-hexane-CHC13. Spectral Data UV~

~"

211(log e = 4.17), 235(4.21), 243(4.22), and 251nm (4.04).

IR~

(CHC13) 3030, 2950, 1720, 1695, and 1245cm"1. CD: (c=0.0001, in MeOH) [0]230 = +13,700. ORD: (c=0.0001, in MeOH) [~]2o4 - 4400.

[(I)]248 q-

7500, [(I)]225

-

2700,

[(I)]217 -

10800, [(I)]209

-

400, and

350

9.

Ganoderic Acids

1H NMR: (CDCI3) 4.49(1H, dd, J=4.5, 11Hz, H-3); 5.44(1H, m, H-7); 5.29(1H, dd, J=5.7Hz, n-11); 5.04(1H, dd, J=4.5, 10Hz, H-15); 0.62(3H, s, H-18); 0.97(3H, s, H-19); 0.88(3H, d, J=6.5Hz, H-21); 6.84(1H, m, H-24); 1.80(3H, s, H-27); 0.86(3H, s, H29); 0.92(3H, s, H-30); 2.03(3H, s, COOCH3); and 2.06ppm (3H, s, COOCH3). 13C NMR: (CDC13) 35.26(t, C-l); 24.06(t, C-2); 80.60(d, C-3); 37.43(s, C-4); 48.83(d, C-5); 22.69(t, C-6); 120.93(d, C-7); 140.01(s, C-8); 145.52(s, C-9); 37.16(s, C-10); 116.00(d, C-11); 37.86(t, C-12); 43.92(s, C-13); 51.19(s, C-14); 77.15(d, C-15); 36.84(t, C-16); 48.67(d, C-17); 15.82(q, C-18); 22.69(q, C-19), 35.79(d, C-20); 18.03(q, C-21); 34.49(t, C-22); 25.78(t, C-23); 144.90(d, C-24); 126.72(s, C-25); 173.01(s, C-26); 11.78(q, C-27); 18.19(q, C-28); 27.97(q, C-29); 16.79(q, C-30); 21.06(q, COOCH3); 21.18(q, COOCH3); 170.83(s, AcCO); and 171.04ppm (AcCO). Mass Spectrum: 554(M*, C34H5oO6,95%), 494(M*- CH3COOH, 35), 479(M* - CH3COOH-CH3, 25), 434(M§ 2CH3COOH, 23), 419(M § - 2CH3COOH-CH3, 100), 353(M§ CH3COOHC18H1302 side chain, 36), 299(D-ring cleavage-CH3, 70), 293(NV- 2CH3COOHC18H1302 side chain, 34), and 239role (299- CH3COOH, 62). Reference M. S. Shiao, L. J. Lin, S. F. Yen, and C. S. Chou; Two New Triterpenes of the Fungus Ganoderma lucidum; J. Nat. Prod., Vol. 50, No. 5, pp. 886-890(1987).

Ganoderiols Ganoderiol A Ganoderiol B Ganoderiol C Ganoderiol D Ganoderiol E Ganoderiol F Ganoderiol G Ganoderiol H Ganoderiol I Ganodermanondiol Ganodermanontriol Ganodermenonol Ganodermadiol Ganodermatriol

351

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10.

Ganoderiols

353

Common/Systematic Name Ganoderiol A 5 a-Lanosta-7, 9( 11)-diene-313,24,25,26-tetraol Molecular Formula/Molecular Weight C3oH5oO4, MW = 474.37091

OH ........~ ~ . ~ ~ C H 2 0 H

NO H Io'''

.

i -.

-"

General Characteristics Colorless needles from chloroform; mp., 232-234~

[a]D 23 +20 (c=0.1, in EtOH).

Fungal Source Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with EtOH. The extract was concentrated and partitioned between CHC13 and water. The CHCI3 layer was concentrated and extracted with sodium bicarbonate solution. The CHCI3 layer was washed with water, dried with anhydrous sodium sulfate and evaporated to dryness. The neutral fraction was subjected to chromatography on a silica gel column and eluted successively with MeOH-CHCI3 (2:98, 1:9, 1:1, v/v) with monitoring by TLC. Eluates were separated into three fractions. Fraction 1 was rechromatographed on a silica gel column eluted with CHCI3-EtOAc (9:1, v/v) to yield five fractions. Fraction 4 from this column was subjected repeatedly to silica gel chromatography and eluted with CHCI3acetone (9:1, v/v) to yield six fractions. After evaporation of the solvent, Fraction 3 of the latter column on recrystallization from CHC13 yielded ganoderiol A as colorless needles. Fraction 2 of the latter column was rechromatographed on a silica gel column eluted with CHCI3-MeOH (9:1, v/v) to yield six fractions. Further purification of Fraction 4 of this column was achieved by a Lobar column RPls (MeOH-H20, 95 + 5, v/v), and ganodermanontriol (colorless needles) and ganodermatriol (colorless plates) were isolated. Fraction 2 of the original column was rechromatographed on a silica gel column and eluted with CHC13-MeOH (95:5, v/v) to yield seven fractions. Fractions 4 and 5 of the latter column were combined and rechromatographed on a silica gel column eluted with CHCI3-EtOAc; six fractions were collected. After evaporation of the solvent, Fraction 4 of the latter column gave ganoderiol A, which was recrystallized from CHCI3 to yield

354

10.

Ganoderiols

colorless needles. Fraction 5 of the latter column was rechromatographed on a Lobar column RP-8 (MeOH-H20, 9:1, v/v). Further purification by preparative TLC with CHCI3-MeOH (9:1, v/v) gave ganoderiol B as an amorphous powder. Spectral Data UV~ ~ EtOH

max

237(E= 8058), 244(9362), and 253nm (6518).

IR:

(KBr) 3350, 2950, 2900, 2850, 1430, 1360, and 1060cm~. ~H NMR: (CDCI3) 5.48(1H, m, 7-H); 5.32(1H, d, J=5.8Hz, 1l-H); 3.83, 3.47(1H each, d, J=10.6Hz, 26-H); 3.46(1H, t, J=12.1Hz, 24-H); 3.25(1H, dd, J=5.0 and 10.5Hz, 3-Ha); 1.1 l(3H, s, 27-H); 1.01(3H, s, 19-H); 0.98(3H, s, 28-H); 0.92(3H, d, J=6.2Hz, 2 I-H); 0.88(6H, s, 29 and 30-H); and 0.57ppm (3H, s, 18-H). Mass Spectrum: 474(M+, C3oH5oO4, 100%). 459(11), 456(13), 398(22), 311(39), 271(83), 69(72), 55(91), and 43m/e (95). Reference H. Sato, T. Nishitoba, S. Shirasu, K. Oda, and S. Sakamura; Ganoderiol A and B, New Triterpenoids from the Fungus Ganoderma lucidum (Reishi); Agric. Biol. Chem., Vol. 50, pp. 2887-2890(1986).

10.

Ganoderiols

355

Common/Systematic Name Ganoderiol B 15t~,26,27-Trihydroxy-5a-lanosta-7, 9(11),24-trien-3-one Molecular Formula/Molecular Weight C30H4604; M W "- 470.33961

......./V~,T/CH20H

~

~--H

CH2OH

"OH

General Characteristics Amorphous powder. Fungal Source

Ganoderma lucidum.

Isolation/Purification (See ganoderiol A). Spectral Data UV: EtOH

237(6 = 4740), 245(5400), and 253nm (2850).

IH N M R :

(CDC13) 5.89(18, d, J=6.2Hz, 7-H); 5.54(1H, t, J= 7.3Hz, 24-H); 5.38(1H, d, J=6.6Hz, 1l-H); 4.33(2H, s, 27-H); 4.29(1H, dd, J=5.5 and 9.9Hz, 15HB); 4.33(2H, s, 26-H); 2.77(1H, ddd, J=5.4, 14.4 and 14.4Hz, 2-H); 1.19(3H, s, 19-H); 1.13(3H, s, 28-H); 1.09(3H, s, 29-H); 0.93(3H, s, 30-H); 0.9(3H, d, J=6.6Hz, 21-H); and 0.64ppm

(3H, s, 18-H).

Mass Spectrum: HREIMS: 470.3394(M+, C30H4604, 17%), 352(100), 55(59), and 43m/e (55). Reference H. Sato, T. Nishitoba, S. Shirasu, K. Oda, and S. Sakamura; Ganoderiol A and B, New Triterpenoids from the Fungus Ganoderma lucidum (Reishi); Agric. Biol. Chem., Vol. 50, pp. 2887-2890(1986).

356

10.

Ganoderiols

Common/Systematic Name Ganoderiol C Molecular Formula/Molecular Weight C32H5405; MW-- 518.39713 OH

OH H

O

"--_ H

.....OEt

General Characteristics (Diacetyl derivative) Colorless syrup; [a]D23 + 54 ~ (C=0.1, in EtOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II, and Fr. III) using C~s Sep-Pak "(MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-C~s column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, v/v, 0.Sml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C and 24,26-di-Oacetylganoderiol D, respectively. Ganoderiol F was crystallized (CHCI3-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254 and RPls F254 S) ofFr. P alter methylation with CH2N2. Ganoderiols G, H, and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; also bitter tasting. Spectral Data (Diacetyl derivative) IR:

(KBr, film) 3460, 2960, 2870, 1735, and 1705cm "~.

10.

Ganoderiols

357

1H N]V[R: (CDCI3) 4.90(1H, dd, J=10.3, 2.6Hz, 24-H); 4.14(1H, d, J=l 1.4Hz, 26-H); 3.91(1H, d, J=l 1.4Hz, 26-H); 3.77(1H, br, s, 713-H); 3.61(1H, dq, J=8.8, 7.0Hz, 7a-OC__H_2H CHa); 3.35(1H, dq, J=8.8, 7.0Hz, 7a-OCH C2_C__H~H_1.21(3H, ); dd, J=7.0, 7.0Hz, 7a-OCH2C__H_a); 1.21(3H, s, 27-H); 1.11(3H, s, 28-H); 1.08(3H, s, 29-H); 1.06(3H, s, 19 or 30-H); 1.05(3H, s, 19 or 30-H), 0.90(3H, d, J-6.6Hz, 21-H); 0.62(3H, s, 18-H); 2.1 l(3H, s, OCOCH3); and 2.07ppm (3H, s, OCOCH3). 13C NMR: (CDCI3) C-1, 35.2, t, C-2, 34.4, t, C-3,217.6, s; C-4,46.8, s; C-5, 45.0, s; C-6, 24.2, t; C-7, 76.4, d; C-8, 135.5, s; C-9, 139.2, s, C-10, 37.8, s; C-11, 21.1, t; C-12, 30.3, t; C13, 45.0, s; C-14, 50.0, s; C-15, 28.1, t; C-16, 31.1, t; C-17, 50.4, d; C-18, 16.2, q*; C19, 17.4, q; C-20, 36.7, d; C-21, 18.7, q; C-22, 32.7, t; C-23, 26.0, t; C-24, 74.8, d, C25, 73.3, s; C-26, 68.5, t; C-27, 20.2, q; C-28, 25.3, q; C-29, 21.4, q; C-30, 26.3, q; 7a-OCH2CH3, 64.0, t; 7a-OCH2CH3, 16.0, q'; OCOCH3, 171.1, s, 170.6, s; and OCOCH3, 21.0ppm, q, 20.8, q. * Assignment may be reversed. Mass Spectrum: M +, 602.4191(C36I-I5807, 2%), 587(5), 556(M+ - C2HsOH, 8%), 496(8), 039(46), and 43m/e (100). Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agile. Biol. Chem., Vol. 52, pp. 367-372(1988).

358

10.

Ganoderiols

Common/Systematic Name Ganoderiol D Molecular Formula/Molecular Weight C3oH4805; M W -" 488.35017 OH

~

O

CH20H H

General Characteristics (Diacetyl derivative) Colorless syrup; [a]D 21 + 8 ~ (C=0.1, in MeOH). F ungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II and Fr. III) using C18 Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-C~8 column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, v/v, 0.8ml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C and 24,26-di-Oacetylganoderiol D, respectively. Ganoderiol F was crystallized (CHL,13-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254and RP18 F254 S) ofFr. P after methylation with CH2N2. Ganoderiols G, H, and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; they are also bitter tasting. Spectral Data

(Diacetyl derivative)

~M eOH max 254nrn (9730).

UV"

10.

Ganoderiols

359

IR:

(KBr, film) 3440, 2950, 2860, 1730, 1705, 1655, and 1575cm"~. ~H N M R : (diacetate) (CDC13) 4.90(1H, dd, J=9.9, 2.6Hz, 24-H); 4.13(1H, d, J=l 1.4Hz, 26-H); 3.90(1H, d, J=l 1.4Hz, 26-H); 2.70(1H, ddd, J=15.8, 12.1, 7.0Hz, 213-H); 2.54(1H, dd, J=16.0, 14.5Hz, 613-H); 2.46(1H, ddd, J=15.8, 5 9, 3.7Hz, 2a-H); 2.33(1H, dd, J=16.0, 3.3Hz, 6a-H); 2.14(1H, dd, J=14.5, 3.3Hz, 5a-H); 1.33(3H, s: 30-H); 1.20(3H, s, 27-H); 1.12(3H, s, 28-H); 1.10(3H, s, 29-H); 0.93(3H, s, 19-H); 0.92(3H, d, J=5.9Hz, 2 l-H); and 0.67ppm (3H, s, 18-H). 13C NMR:

(flee ganoderiol) (CDC13) C-l, 35.4, t; C-2, 34.4, t; C-3,214.6, s; C-4, 47.3, s; C-5, 49.1, s, C-6, 37.2, t; C-7, 198.1, d; C-8, 139.6, s, C-9, 162.8, s; C-10, 39.5, s; C-11, 23.9, t; C-12, 30.2, t, C-13, 45.0, s, C-14, 47.8, s, C-15, 28.7, t; C-16, 31.9, t; C-17, 50.5, d; C-18, 16.0, q, C-19, 17.9, q, C-20, 36.6, d, C-21, 18.9, q, C-22, 33.6, t; C-23, 28.8, t; C-24, 79.1, d; C-25, 73.9, s; C-26, 67.7, t; C-27, 20.9, q; C-28, 25.4, q, C-29, 21.4, q; and C-30, 26.3ppm, q. Mass Spectrum: M +, 572.3639(C34H5207, 6%), 557(6), 339(15), 327(13), 69(65), 57(87), and 43m/e

(loo).

Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 52, pp. 367-372(1988).

360

10.

Ganoderiols

Common/Systematic Name Ganoderiol E Molecular Formula/Molecular Weight C3014_4804;MW = 472.35526

~v,/~~CH2

OH 2OH

HOv ~.____~v "O General Characteristics (Triacetyl derivative) Isolated as 3,26,27-tri-O-acetylganoderiol E; colorless syrup; [aid 21 + 18~ (C=0.1, in MeOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II and Fr. III) using C~s Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-C~s column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, v/v, 0.Sml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C, 24,26-di-Oacetylganoderiol D and 3,26,27-tri-acetyl ganoderiol E, respectively. Ganoderiol F was crystallized (CHCI3-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254and RPI8 F254 S) ofFr. P after methylation with CH2N2. Ganoderiols G, H, and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; also bitter tasting. Spectral Data (Triacetyl derivative) UV: M~oH 255nm (9160). max

10.

Ganoderiols

361

IR:

(KBr film) 3000, 2940, 2860, 1720, 1650, and 1570cm"~. IH NMR:

(CDCI3) 5.77(1H, t, J=7.3Hz, 24-H), 4.66(2H, s, 27-H); 1.56(7H, s, 26-H); 1.51(1H, dd, J=l 1.4, 4.4Hz, 3-H); 7.46(1H, dd, J=16.0, 12.0Hz, 613-H); 2.38(1H, dd, J=16.0, 5.0Hz, 6a-H); 1.19(3H, s, 30-H); 0.95(3H, s, 19-H); 0.93(3H, d, J=5.9Hz, 21-H); 0.91(3H, s, 28-H); 0.89(3H, s, 29-H); 0.65(3H, s, 18-H); and 2.06ppm (9H s, OCOCH3 x 3). 13CNMPx:

(CDCIa) C-l, 34.6, t; C-2, 23.7, t*; C-3, 79.6, s; C-4, 37.8, s; C-5, 49.0, s; C-6, 35.8, t; C-7, 198.6, s; C-8, 139.0, s; C-9, 164.6, s; C-10, 39.7, s; C-1 l, 23.9 ~ t; C-12, 30.2, t; C-13, 45.0, s; C-14, 47.8, s; C-15, 28.8, t; C-16, 32.0, t; C-17, 49.9, d; C-18, 15.8, q; C-19, 16.4, q; C-20, 36.2, d; C-21, 18.5, q*; C-22, 36.5, t; C-23, 24.7, t; C-24, 137.2, d; C-25, 128.8, s; C-26, 66.8, t; C-27, 59.9, q; C-28, 27.4, q; C-29, 21.2, q; C-30, 25.0, q; OCOCH3, 170.0, s, 170.8, s and 170.9 s; and OCO__CH3,18.7, q', 21.0, q and 20.9ppm. * Assignments may be reversed. Mass Spectrum: M +, 598.3836(C36H5407, 13%), 583(11), 538(15), 463(28), 369(23), 121(42), and 43role (100). Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agrie. Biol. Chem., Vol. 52, pp. 367-372(1988).

362

10.

Ganoderiols

Common/Systematic Name Ganoderiol F Molecular Formula/Molecular Weight

~

C3oH4603, M W = 454.34470

CH2OH

H2OH

--H

o

General Characteristics Pale yellow needles from chloroform-methanol; mp., 116-120~ MeOH).

[tt]D21 + 42 ~ (C=0.1, in

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions fir. I, Fr. II and Fr. III) using C]s Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-C]s column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, v/v, 0.8ml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C and 24,26-di-Oacetylganoderiol D, respectively. Ganoderiol F was crystallized (CHCI3-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254and RP~s F254 S) ofFr. P after methylation with CH2N2. Ganoderiols G, H, and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; also bitter tasting. Spectral Data UV: ,~ MeOH max

237(e=16,600), 245(19,200), and 253nm (12,500).

10.

Ganoderiols

363

IR:

(KBr film) 3350, 2900, 2850, and 1690cm"1. 1H NMR: (CDCI3) 5.56(1H, t, J=7.3Hz, 24-H); 5.51(1H, d, J=6.9Hz, 7-1-1); 5.39(1H, d, J=5.9Hz, 1l-H); 4.33(2n, s, 27-H); 4.22(2H, s, 26-H); 2.78(1H, ddd, J=14.7, 14.7, 5.5Hz, 213-H); 1.20(3H, s, 19-H); 1.13(3H, s, 28-H); 1.09(3H, s, 29-H), 0.92(3H, d, J=6.6Hz, 21-H); 0.88(3H, s, 30-H); and 0.59ppm (3H, s, 18-H). 13CNMR: (CDC13) C-l, 36.1, t; C-2, 34.9, t; C-3,216.8, s; C-4, 47.5, s; C-5, 50.8, s; C-6, 23.7, t; C-7, 120.0, d; C-8, 142.9, s; C-9, 144.6, s; C-10, 37.3, s; C-11, 117.3, d; C-12, 37.9, t; C-13, 43.8, s; C-14, 50.4, s; C-15, 27.9, t; C-16, 31.5, t; C-17, 50.9, d; C-18, 15.8, q ; C-19, 22.5, q; C-20, 36.1, d; C-21, 18.4, q; C-22, 36.7, t; C-23, 24.4, t; C-24, 131.7, d; C-25, 136.8, s; C-26, 67.7, t; C-27, 60.2, t; C-28, 25.5 ~ q; C-29, 21.3, q; and C-30, 25.4*ppm, q.

Assignments may be reversed. Mass Spectrum: M +, 454.3430(C30I-h603, 4%), 436(6), 309(100), 69(24), 55(40), and 41m/e (31). Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 52, pp. 367-372(1988).

364

10.

Ganoderiols

Common/Systematic Name Ganoderiol G Molecular Formula/Molecular Weight C31H5205; MW = 504.38140 OH

General Characteristics A colorless syrup; [a]D24 + 34 ~ (C=0.1, in MeOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II and Fr. III) using C18 Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-Cls column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, v/v, 0.8ml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C and 24,26-di-Oacetylganoderiol D, respectively. Ganoderiol F was crystallized (CHCI3-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254 and RPls F254 S) of Fr. P after methylation with CH2N2. Ganoderiols G, H and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; they are also bitter tasting. Spectral Data IR:

(KBr film) 3380, 2920, 2870, and 1700cm "1.

10.

Ganoderiois

365

~H NMR: (CDC13) 3.84(1H, d, J=l 1.4Hz, 26-H); 3.67(1H, br, s, 713-H); 3.19(1H, d, J=l 1.0Hz, 26-H); 3.47(1H, t, J=8.0Hz, 24-I-I); 3.32(3H, s, 7a-OCH3); 1.26(3H, s, 27-H); 1.12(3H, s, 28-H); 1.09(3H, s, 29-H); 1.05(3H, s, 30-H); 1.05(3H, s, 19-H); 0.90(3H, d, J=8.8I-Iz, 21-I-I); and 0.64ppm (3H, s, 18-H). Mass Spectrum: M +, 504.3860(C3~H5205, 22%), 489(43), 472(17), 457(19), 366(64), 95(55), 69(62), 55(78), and 43m/e (100). Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agile. Biol. Chem., Vol. 52, pp. 367-372(1988).

366

10.

Ganoderiols

Common/Systematic Name Ganoderiol H Molecular Formula/Molecular Weight C3oH5oO5; M W = 490.36583 OH

~

HO

O

cH2OH H

-\

General Characteristics Colorless needles; mp., 200-201.5~

[a]D 24 -b 22 ~ (c=0.1, in MeOH).

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II and Fr. III) using C18 Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-Cl8 column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, v/v, 0.8ml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C and 24,26-di-Oacetylganoderiol D, respectively. Ganoderiol F was crystallized (CHC13-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254 and RP18 F254 S) ofFr. P after methylation with CH2N2. Ganoderiols G, H, and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; they are also bitter tasting. Spectral Data UV:

~, M~Hr~,x 255rim (9170).

10. Ganoderiols

367

IR~

(KBr film) 3380, 2950, 2860, and 1640cm"1. 'H NMR: (CDCI3) 3.82(1H, d, J=l 1.4Hz, 26-H); 3.46(1H, d, J=l 1.4Hz, 26-H); 3.43(1H, dd, J=10.0, 1.5Hz, 24-H); 3.27(1H, dd, J=l 1.2, 4.9Hz, 3t~-H); 2.46(1H, dd, J=16.5, 11.7Hz, 6a-H); 2.38(1H, dd, J=16.5, 6.2Hz, 6et-H); 1.17(3H, s, 30-H); 1.11(an, s, 27-H); 1.00(3H, s, 28-H); 0.93(3H, d, J=7.7Hz, 21-H); 0.92(3H, s, 19-H); 0.88(3H, s, 29-H); and 0.66ppm (3H, s, 18-H). 13C NMR: (CDC13) C-l, 35.2, t; C-2, 28.9, t*; C-3, 77.1, s; C-4, 40.1, s; C-5, 49.6, s; C-6, 37.2, t; C-7, 198.7, s; C-8, 138.9, s; C-9, 164.9, s; C-10, 39.5, s; C-11, 23.8, t; C-12, 30.5, t; C13, 45.2, s; C-14, 48.2, s; C-15, 28.3, t; C-16, 32.7, t; C-17, 50.6, d; C-18, 16.1, q; C19, 18.3, q; C-20, 37.3, d; C-21, 19.3, q; C-22, 34.5, t; C-23, 29.1, t*; C-24, 77.1, d; C25, 74.8 s; C-26, 69.3, t; C-27, 20.1, q; C-28, 27.9, q; C-29, 16.0, q; C-30, 25.2ppm, qo Assignments may be reversed. Mass Spectrum: M +, 490.3625(C3oH~oO5, 30%), 475(47), 415(46), 381(35), 329(35), 121(74), 69(59), 55(68) and 43m/e (100). Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 52, pp. 367-372(1988).

368

10.

Ganoderiols

Common/Systematic Name Ganoderiol I Molecular Formula/Molecular Weight C31Hs0Os; MW = 502.36583

~ f~v~r/CH20H

./~.I/~~

"

'-..

CH20H

"OH

.....

General Characteristics Colorless syrup; [a]D22 + 53 (c=0" 1, in MeOH).

o

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II and Fr. III) using C~s Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-Cls column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, 0.Sml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Fractions D and K were acetylated (Ac20/pyridine) and subjected to silica gel column chromatography to give 24,26-di-O-acetylganoderiol C and 24,26-di-Oacetylganoderiol D, respectively. Ganoderiol F was crystallized (CHCI3-MeOH) from Fr. K. Methyl ganolucidate D was obtained by preparative TLC (Kieselgel 60 F254and RP~s F254 S) ofFr. P after methylation with CH2N2. Ganoderiols G, H, and I were obtained by repetitive column chromatography on silica gel followed by preparative TLC and recrystallizations. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; also bitter tasting. Spectral Data I]?c

(KBr film) 3380, 2920, 2870, and 1700cm"~.

~H NIVIR:

10. Ganoderiols

369

(CDCI3) 5.55(1I-I, t, J=7.3Hz, 24-H); 4.35(1/-1, t, d=S.SHz, 1513-H); 4.33(2I-1, s, 27-I-I); 4.22(2H, s, 26-H); 1.06(1H, br s, 713-H); 3.33(3H, s, 7tt-OCHa); 2.58(1H, ddd, J=15.9, 10.6, 7.3Hz, 213-H); 2.46(1H, dad, J-~5.9, 7.3, 3.SHz, 2tt-H); 1.13(3H, s, 28-1-I); 1.10(3H, s, 29- or 30-1-1); 1.08(3H, s, 29- or 30-H); 1.02(3H, s, 19-1-1);0.89(3H, d, J=6.6Hz, 21-H); and 0.66ppm (3H, s, 18-I-I). Mass Spectrum: M +, 502.3675(C3~H~oO~, 3%), 470(7), 452(2), 349(59), 325(45), 175(66), 69(60), 55(99), and 43m/e (I00). Reference T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 52, pp. 367-372(1988).

370

10.

Ganoderiols

Common/Systematic Name Ganodermanondiol 24(S),25-Dihydroxy-5 et-lanosta-7,9(11)-dien-3-one Molecular Formula/Molecular Weight Cs0I-I4sOs; MW = 456.36035 OH OH

0 General Characteristics Colorless needles from MeOH; mp., 182-183~ [(X]D 23 + 45.8 ~ (c=0.5 ' in CHCI3). Showed a positive Liebermann-Burchard reaction. Fungal Source Ganoderma lucidum. Isolation/Purification The fresh fruiting bodies were extracted with MeOH and the extract was partitioned between petroleum ether and MeOH-H20 (90:10, v/v). The 90% MeOH extract was chromatographed on silica gel (0-100% gradient MeOH in CHC13). The CHCI3 eluate was rechromatographed on silica gel using a gradient of EtOAc in hexane; the substance was eluted at 20% EtOAc). Spectral Data UV:

)~ ~ "

235(1og e = 4.00), 243(4.06), and 25 lnm (3.89).

IR:

(KBr) 3390,2960,2930, 1710, 1460, 1370, 1165, 1110, 1080, and 810cm 1. 1H NMR: (CDC13) 2.77(1H, m, H-2); 5.40(1H, dd, J=2.0, 6.0Hz, H-11); 5.52(1H, dd, J=2.0, 6.0Hz, H-7); 0.58(3H, s, H-18); 1.07(3H, s, H-29); 0.91(3H, d, J=6.4Hz, H-21); 3.30(1H, dd, J=3.0, 8.0Hz, H-24); 1.15(3H, s, H-26); 1.18(3H, s, H-19); 0.86(3H, s, H-28), 1.1 l(3H, s, H-30); and 1.21ppm (3H, s, H-27).

10.

Ganoderiols

371

13CNMR: (CDC13) 36.63(t, C-I); 34.87(t, C-2); 216.92(s, C-3); 47.50(s, c-a); 50.70(d, C-5); 23.67(t, C-6); 119.91(d, C-7); 142.81(s, C-8); 144.46(s, C-9); 37.17(s, C-10); 117.23(d, C-11); 37.79(t, C-12); 43.75(s, C-13); 50.3r(s, c-14); 31.45(t, C-15); 27.85(t, C-16); 50.94(d, C-17); 15.71(q, C-18); 22.06(q, C-19); 36.53(d, C-20); 18.62(q, C-21); 33.47(t, C-22); 28.71(t, C-23); 79.58(d, C-24); 73.26(s, C-25); 23.18(q, C-26); 26.57(q, C-27); 25.45(q, C-28); 25.32(q, C-29); and 22.46ppm (q, C-

30). Mass Spectrum: HREIMS: 456.3610role (M+); calcd for C30H48033,456.3603. LREIMS: 456(M+, 7%), 438(M+-H20, 6), 424(M%OH-Me, 3), 309(7), 269(9), 69(11), 55(14), and 32m/e (100). References M. Arisawa, A. Fujita, T. Hayashi, M. Shimizu, and N. Morita; Revision of IH- and 13CNMR Assignments of Lanostanoids from Ganoderma lucidum by 2D-NMR Studies; J. Nat. Prod., Vol. 51, pp. 54-59(1988). A. Fujita, M. Arisawa, M. Saga, T. Hayashi, and N. Morita; Two New Lanostanoids~from Ganoderma lucidum; J. Nat. Prod., Vol. 49, pp. 1122-1125(1986).

10. Ganoderiols

372

Common/Systemic Name Ganodermanontriol 24(S),25,26-Trihydroxy-5a-lanosta-7,9(11)-dien-3-one Molecular Formula/Molecular Weight C30H4804; M W ----472.35526

OH

General Characteristics Colorless needles from MeOH; mp., 161-162~ [~]D 23 Showed a positive Liebermann-Burchard reaction.

+

35.70 ~ (c--1.0, in CHC13).

Fungal Source

Ganoderma lucidum.

Isolation/Purification The fresh fruiting bodies were extracted with MeOH and the extract was partitioned between petroleum ether and MeOH-H20 (90:10). The 90% MeOH extract was chromatographed on silica gel (0-100% gradient MeOH in CHC13). The 1% MeOHCHCI3 eluate was repeatedly chromatographed on silica gel using CHC13-EtOAc-Me2CO (14:1:1, v/v/v) and hexane-EtOAc-Me2CO (6:1:1, v/v/v) to give ganodermanontriol. Spectral Data UV~

~, ~ "

236 (log e = 4.06), 243(4.11), and 25 lnm (3.95).

IR~

(KBr) 3400,2960,2925,2875, 1700, 1460, 1445, 1370, 1110, 1040, 1000, and 810cm q. 1H NMR: (CDCI3) 2.80(11, m, H-2), 5.39(11, dd, J=l.5, 5.0Hz, 1H-11), 5,51(11, dd, J=2.0, 6.1Hz, H-7), 0.58(31, s, H-18), 1.09(31, s, H-29), 0.92(31, d, J=6.1Hz, H-21), 3.48(11, m, H-24); 3.48, 3.84(each, 1H, d, J=l 1.3Hz, 2H-26); 1.21(31, s, H-27);

10.

Ganoderiols

373

13C NMR: (CDC13) 36.63(t, C-l), 34.87(t, C-2); 216.92(s, C-3), 47.50(s, C-4), 50.70(d, C-5), 23.67(t, C-6), 119.91 (d, C-7); 142.81 (s, C-8); 144.46(s, C-9); 37.17(s, C-10); 117.23(d, C-11), 37.79(t, C-12); 43.75(s, C-13), 50.31(s, C-14), 31.45(t, C-15), 27.85(t, C-16), 50.94(d, C-17), 15.71(q, C-18); 22.06(q, C-19); 36.53(d, C-20), 18.62(q, C-21); 33.47(t, C-22), 28.71(t, C-23); 79.58(d, C-24); 73.26(s, C-25); 23.18(q, C-26), 26.57(q, C-27); 25.45(q, C-28), 25.32(q, C-29), and 22.46ppm (q,

c-3o). Mass Spectrum: HREIMS: 456.3610m/e (M+); calcd for C30H48033, 456.3603. LREIMS: 456(M+, 7%), 438(M+-H20, 6), 424(M+-OH-Me, 3), 309(7), 269(9), 69(11), 55(14), and 32m/e

(10o). References M. Arisawa, A. Fujita, T. Hayashi, M. Shimizu, and N. Morita; Revision of 1Hand 13C-NMRAssignments of Lanostanoids from Ganoderma lucidum by 2D-NMR Studies; J. Nat. Prod., Vol. 51, pp. 54-59(1988). A. Fujita, M. Arisawa, M. Saga, T. Hayashi, and N. Mofita; Two New Lanostanoids from Ganoderma lucidum; J. Nat. Prod., Vol. 49, pp. 1122-1125(1986).

374

10.

Ganoderiols

Common/Systematic Name Ganodermenonol 26-Hydroxy-5 tt-lanosta-7,9(1 l),24-trien-3-one Molecular Formula/Molecular Weight Ca0I-I4602; MW = 438.34978 ,,.

CH20H

General Characteristics Colorless needles from methanol; mp., 109-111 ~C; positive Liebermann-Burchard reaction; [tt]D23 +38.96 ~ (C=I.0, in CHCI3). Fungal Source The dried fruiting bodies of Ganoderma lucidum. Isolation/Purification The fruiting bodies of G. lucidum were cut into small pieces and extracted three times with MeOH at room temperature for 3 days. The MeOH extract was partitioned between H20 and CHCIa and the CHCI3 fraction was partitioned between petroleum ether and MeOH-H20 (90:10). The 90% MeOH extract was chromatographed on a silica gel column by stepwise elution with CHCI3, 1% MeOH/CHCI3, 5% MeOH/CHC13, 10% MeOH/CHCI3 and MeOH. The CHCI3 elution was rechromatographed on a silica gel column by stepwise elution with an EtOAc/hexane solvent system to give ganodermenonol from 5% EtOAc/hexane and ganodermadiol from 10% EtOAc/hexane. The 1% eluate was repeatedly separated by silica gel column chromatography (CHCI3-EtOAc-MeECO, 14:l:l, v/v/v, and hexane-EtOAc-Me2CO, 6:1:1, v/v/v) to afford a mixture of colorless crystals. The mixture was treated overnight with Ac20 and pyridine at room temperature and was separated by TLC to give ganodermatriol acetate. Biological Activity The dried fruiting bodies of G. lucidum has been prescribed in Chinese medicine as a tonic and sedative drug and have been used in the management of hepatopathy, hypertension, arthritis, neuasthenia, bronchitis, etc. _Spectral Data UV: /~ MeOH

236(log e = 4.17), 243(4.23), and 251nm(4.06).

10.

Ganoderiols

375

Im:

(KBr) 3425, 2930, 2880, 1700, 1450, 1375, 1110, 1000, and 815cmq. 1H N]V[R:

(CDCl3) 0.59(s, 18-CH3); 1.20(s, 19-CH3); 0.92(d, J=6.2Hz, 21-CH3); 0.88(s, 28-CH3); 1.09(s, 29-CH3); 1.13(s, 30-CH3); 4.00(s, 26-2H); 1.67(s, 27-CH3); 2.78, 2.35(2-CH2); 5.51(dd, J=6.3Hz, 7-H); 5.41(m, 1l-H); and 5.41ppm (m, 24-H). 13C NMR:

(CDCI3) C-I, 36.59 (t); C-2, 34.82 (t); C-3, 16.95 (s); C-4, 47.43 (s); C-5, 50.66 (d); C-6, 23.62 (t); C-7, 119.83 (d); C-8, 142.81 (s); C-9, 144.47 (s); C-10, 37.17 (s); C-11, 117.24 (d); C-12, 37.76 (t); C-13, 43.68 (s); C-14, 50.27 (s); C-15, 31.44 (t); C-16, 27.86(0; C-17, 50.86 (d); C-18, 15.65 (4); C-19, 22.40 (q); C-20, 36.01 (d); C-21, 18.36 (q); C-22, 35.87 (t); C-23, 24.50 (t); C-24, 126.86 (d); C-25, 134.34 (s); C-26, 69.01 (t); C-27, 13.62 (q); C-28, 25.38 (q); C-29, 25.28 (q); and C-30, 22.43ppm (q). Mass Spectrum: ELMS: 438(M+, 80%), 423(M +- Me, 14), 420(M+- H20, 10), 405(M+- Me- 1-120, 12), 309(M+-side chain-2H, 100), 269(M+ - side chain- 42, 50), 244(16), 199(16), 185(20), 171(30), 157(40), 145(34), 133(365), 119(36), 109(38), 95(44), 81(54), 69(64), and 55m/e (100); anal calcd for C30H4602438.3498; found 438.3507. References M. Arisawa, A. Fujita, T. Hayashi, M. Shimizu, N. Morita, T. Kikuchi, S. Kakota, and Y. Tezuka; Revision of IH and ~3C-NMRAssignments of Lanostanoids from Ganoderma lucidum by 2D-NMR Studies; J. Natural Products, Vol. 51, pp. 54-59(1988). M. Arisawa, A. Fujita, M.Saga, H. Fukumura, T. Hayashi, M. Shimizu, and N. Morita; Three New Lanostanoids from Ganoderma lucidum; J. Natural Products, Vol. 49, pp. 621-625(1986).

376

10.

Ganoderiols

Common/Systematic Name Ganodermadiol 5 t~-Lanosta-7,9(11),24-triene-3 [3,26-diol Molecular Formula/Molecular Weight C3oi-I4802; M W = 4 4 0 . 3 6 5 4 3 ~,,,,,

CH2OH

HO H

I1,'

General Characteristics Colorless needles from methanol; mp., 168-170~ reaction; [tt]o 23 + 53.0 ~ (c=l.0, in CHC13).

positive Liebermann-Burchard

Fungal Source The dried fruiting bodies of Ganoderma lucidum. Isolation/Purification The fruiting bodies of G. lucidum were cut into small pieces and extracted three times with MeOH at room temperature for 3 days. The MeOH extract was partitioned between H20 and CHC13 and the CHC13 fraction was partitioned between petroleum ether and MeOH-H20 (90:10). The 90% MeOH extract was chromatographed on a silica gel column by stepwise elution with CHCI3, 1% MeOH-CHC13, 5% MeOH-CHC13, 10% MeOH-CHC13, and MeOH. The CHC13 elution was rechromatographed on a silica gel column by stepwise elution with an EtOAc-hexane solvent system to give ganodermenonol from 5% EtOAc-hexane and ganodermadiol from 10% EtOAc-hexane. The 1% eluate was repeatedly separated by silica gel column chromatography (CHC13EtOAc-Me2CO, 14:1:1, v/v/v, and hexane-EtOAc-Me2CO, 6:1:1, v/v/v) to afford a mixture of colorless crystals. The mixture was treated overnight with Ac20 and pyridine at room temperature and was separated by TLC to give ganodermatriol acetate. Biological Activity The dried fruiting bodies of G. lucidum have been prescribed in Chinese medicine as a tonic and sedative drug and have been used in the management of hepatopathy, hypertension, arthritis, neuasthenia, bronchitis, etc. Spectral Data UV:

/~

MoOH

236(log e = 4.08), 243(4.12), and 25 lnm (3.95).

10.

Ganoderiols

377

IR; (KBr) 3340, 2930, 1440, 1430, 1370, 1070, 1040, and 1010cm"1. IH N]VIR: (CDCI3) 0.57(s, 18-CH3); 0.98(s, 19-CH3); 0.92(d, J=6.3Hz, 21-CH3); 0.88(s, 28CH3); 1.00(s, 29-CH3); 0.88(s, 30-CH3); 4.00(s, 26-2H); 1.67(s, 27-CH3); 3.25(d, J=5.4, 10.1Hz); 5.47(m, 7-H); 5.34(m, 1l-H); and 5.42ppm (m, 24-H). 13CN M R : (CDCI3) C-l, 35.76 (t); C-2, 27.82 (t); C-3, 78.98 (d); C-4, 38.73 (s); C-5, 49.15 (d); C-6, 23.13 (t); C-7, 120.25 (d); C-8, 142.69 (s); C-9, 145.93 (s); C-10, 37.36 (s); C11, 116.26 (d); C-12,-37.85 (t); C-13, 43.84 (s); C-14, 50.35 (s); C-15, 31.53 (t); C16, 27.95(0; C-17, 50.94 (d); C-18, 15.71 (4); C-19, 22.79 (q); C-20, 36.12 (d); C-21, 18.44 (q); C-22, 35.98 (t); C-23, 24.97 (t); C-24, 126.99 (d); C-25, 134.33 (s); C-26, 69.11 (t); C-27, 13.65 (q); C-28, 26.60 (q); C-29, 28.17 (q); and C-30, 15.80ppm (q).

Mass Spectrum: ELMS: 440(M+, 100%), 425(M+-Me, 12), 422(M+-H20, 8), 407(M+-Me-H20, 12), 31 l(M+-side chain, 50), 271(M+-side chain-40, 44), 253(30), 171(28), 157(32), 145(34), 119(38), 107(40), 95(48), 81 (54), 69(70), 55(98), 109(38), 95(44), 81(54), 69(64), and 55m/e (100); anal calcd for C3oH4sO2 440.3654; found 440.3612. References M. Arisawa, A. Fujita, T. Hayashi, M. Shimizu, N. Morita, T. Kikuchi, S. Kakota, and Y. Tezuka; Revision of ~H and ~3C-NMR Assignments of Lanostanoids from Ganoderma lucidum by 2D-NMR Studies; J. Natural Products, Vol. 51, pp. 54-59(1988). M. Arisawa, A. Fujita, M.Saga, H. Fukumura, T. Hayashi, M. Shimizu, and N. Morita; Three New Lanostanoids from Ganoderma lucidum; Journal of Natural Products, Vol. 49, pp. 621-625(1986).

378

10.

Ganoderiols

Common/Systematic Name Ganodermatriol Molecular Formula/Molecular Weight C30H4803; M W -- 4 5 6 . 3 6 0 3 5 21

26

24

,8......f~-~~~CH20H 2

8

3

30

29

Fungal Source Fresh fruiting body of the fungus Ganoderma lucidum. Spectral Data (Acetate derivative) 1H NMR: (CDCI3) 18-H3, 0.56(s); 19-H2, 1.01(s), 0.92(d, d=7.4Hz); 28-H3, 0.87(s); 29-H3, 0.89(s); 30-H3, 0.96(s); 26-H2, 4.57(s, 2H); 27-H2, 4.66(s, 2H); 3a-n, 4.50(s); 7-n, 5.46(m); 1 l-H, 5.33(m); 24-H, 5.78(dd, ,/=7.3, 7.4Hz); and 3 X OAc, 2.06(3H), 2.07ppm (6H). 13C N M R :

(CDCI3) C-I, 35.40 (t); C-2, 24.33 (t); C-3, 80.82 (d); C-4, 37.22 (s); C-5, 49.27 (d); C-6, 22.90 (t); C-7, 120.01 (d); C-8, 142.63 (s); C-9, 145.64 (s); C-10, 37.82 (s); Ell, 116.50 (d); C-12, 37.95 (t); C-13, 43.81 (s); C-14, 50.32 (s); C-15, 31.50 (t); C16, 27.91 (t); C-17, 50.83 (d); C-18, 15.69 (q); C-19, 22.87 (q); C-20, 36.07 (d); C-21, 18.41 (q); C-22, 35.78 (t); C-23, 24.81 (t); C-24, 137.83 (d); C-25, 128.64 (s); C-26, 66.83 (t); C-27, 59.84 (t); C-28, 25.56 (q); C-29, 28.10 (q); and C-30, 16.97ppm (q). Reference M. Arisawa, A. Fujita, T. Hayashi, M. Shimizu, N. Morita, T. Kikuchi, S. Kadota, and Y. Tezuka; Revision of 1H- and ]3C-NMR Assignments of Lanostanoids from Ganoderma lucidum by 2D-NMR Studies; J. Nat. Products, Vol. 51, pp. 54-59(1988).

Ganolucidic Acids Ganolucidie acid A Ganolucidic acid B Ganolucidic acid C Ganolucidic acid D Ganolucidic acid E

379

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11.

Ganolucidic Acids

381

Common/Systematic Name Ganolucidic acid A Molecular Formula/Molecular Weight C3oH4406; M W = 5 0 0 . 3 1 3 7 9

0 Ot__..__._.L~H

H

.

"

-:

OH ,,.

General Characteristics Isolated and identified as methyl ester. Colorless needles; mp., 192-194~ CHCI3).

[a]o + 188~ (in

Fungal Source

Ganoderma lucidum.

Spectral Data (Methyl ester) UV:

~.m~x 256.5nm (log e = 4.00). IR~

(KBr) 3450, 1730, 1710, and 1645cm"~. 1H NMR: (CDCI3) 0.91(3H, s, 18-CH3); 1.12(3H, s, 19-CH3); 1.18(3H, d, J=7.0Hz, 27-CH3); 1.08(3H, s, 30-CH3); 1.12(3H, s, 31-CH3); 1.18(3H, s, 32-CH3); 3.70(3H, s, COOCH3); and 4.41ppm (1H, dd, J=9.0, 5.5Hz, 15-H). 13C NMR: (CDC13) C-l, 35.1, t; C-2, 34.2, t; C-3,217.7, s; C-4, 47.0 ~ s; C-5, 51.7, d; C-6, 18.7, t; C-7, 29.6, t; C-8, 163.2, s; C-9, 138.6, s; C-10, 37.1, s; C-11, 198.1, s; C-12, 51.7, t; C-13, 46.8 ~ s; C-14, 53.6, s; C-15, 72.9, d; C-16, 38.6, t; C-17, 48.7, d; C-18, 17.2, q; C-19, 18.8~ q; C-20, 32.6, d; C-21, 19.4, q; C-22, 49.6, t; C-23,208.3, s; C-24, 46.8, t; C-25, 34.7, d; C-26, 176.2, s; C-27, 17.1, q; C-30, 27.8, q; C-31, 20.6, q; C-32, 19.0 ~176 q; and OCH3, 51.gppm. *, ** Assignments may be reversed.

382

11.

Ganolucidic Acids

Mass Spectrum: LREIMS: 514(M+), 417, 371,287, 171, 139, and 129m/e. Reference T. Kikuchi, S. Matsuda, Y. Murai, and Z. Ogita; Ganoderic Acid G and I and Ganolucidic Acid A and B, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2628-2631(1985).

11.

Ganolucidic Acids

383

Common/Systematic Name Ganolucidic acid B Molecular Formula/Molecular Weight CsoH4606; MW = 502.32944

0

COOH .

-

H

H,, HO

General Characteristics Isolated and identified as methyl ester. Colorless needles; mp., 167-169~ CHC13).

[a]D + 114~ (in

Fungal Source

Ganoderma lucidum.

Spectral Data (Methyl ester) UV:

~.max 256.5nm (log e = 4.00). IR:

(KBr) 3450, 1730, 1710, and 1645cm "1. 1H NMR: (CDCI3) 0.89(3H, s, 18-CH3); 1.1 l(3H, s, 19-CH3); 1.18(3H, d, J=7.0Hz, 27-CH3); 1.01(3H, s, 30-CH3); 0.82(3H, s, 31-CH3); 1.16(3H, s, 32-CH3); 3.68(3H, s, OOCH3); 3.25(1H, dd, J=10.0, 6.0Hz, 3-H); and 4.39ppm (1H, dd, J=9.0, 5.5Hz, 15-H).

13CNMR: (CDC13) C-l, 34.4, t; C-2, 28.0, t; C-3, 78.7, d; C-4, 39.0, s; C-5, 51.8, d; C-6, 17.4, t; C-7, 30.4, t; C-8, 162.9, s; C-9, 140.0, s; C-10, 37.8, s; C-11, 198.3, s; C-12, 52.1, t; C-13, 47.2, s; C-14, 53.5, s; C-15, 73.0, d; C-16, 38.7, t; C-17, 48.7, d; C-18, 17.1, q; C-19, 18.8", q; C-20, 32.5, d; C-21, 19.4, q; C-22, 49.7, t; C-23,208.3, s; C-24, 46.8, t; C-25, 34.6, d; C-26, 176.1, s; C-27, 17.1, q; C-30, 28.3, q; C-31, 15.7, q; C-32, 19.0 ~ q; and OCH3, 51.9ppm. Assignments may be reversed.

384

11.

Ganolucidic Acids

Mass Spectrum: LREIMS: 516(M+), 373,289, 171, 139, and 129m/e.

Reference T. Kikuchi, S. Matsuda, Y. Murai, and Z. Ogita; Ganoderic Acid G and I and Ganolucidic Acid A and B, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2628-2631(1985).

11.

Ganolucidic Acids

385

Common/Systematic Name Ganolucidic acid C 313,15a,29-Trihydroxy- 11,23-dioxo-5a-lanost-8-en-26-oic acid Molecular Formula/Molecular Weight _ C30H4607;

MW

Methyl ester:

= 518.32435

C31H4gOT; M W --

532.34000

H ......O H

2

General Characteristics Methyl ester: Crystallized from methanol as colorless needles; mp., 230-23 I~ 124 ~ (c=0.1, in EtOH).

[~]D 25 -F

FunRal Source v Isolated from the dried fruiting bodies (mushrooms) of Ganoderma lucidum (Fr.) Karst. (Polyporaceae). Isolation/Purification The dried chipped epidermis of Ganoderma lucidum was extracted twice with CHCIa. The combined extracts were evaporated at 40-45 ~ under reduced pressure to about 1/10 of the original volume and the resulting solution was extracted with sat. aq. NaHCO3 solution. The combined extracts were acidified with 6N HC1 (pH 3-4) at 0 ~ The precipitate was extracted with CHC13, dried (Na2SO4) and the solvent removed under reduced pressure, leaving crude material. The acidic material was chromatographed on silica gel column chromatography (Fr. 1-11, CHC13-MeOH = 98:2, v/v; Fr. 12, CHCI3-MeOH = 9:1, v/v, Fr. 13, MeOH). Fractions 7-9 were combined and rechromatographed on silica gel to give a mixture of ganoderic acid J and A, which was methylated with diazomethane and separated by further silica gel column chromatography and preparative TLC. Methyl ganoderate J was obtained as a pale yellow syrup. Fraction 12 was chromatographed on a Lobal column (RPs, Merck) and the second fraction was methylated with diazomethane. The methylated products were subjected to silica gel column chromatography and methyl ganolucidate C was obtained as colorless needles. Biological Actifity A bitter principle; the mushrooms of Ganoderma lucidum (Fr.) Karst. (Polyporaceae) have long been used as a home remedy in China and Japan.

386

11.

Ganolucidic Acids

Spectral Data (Methyl ester) UV~

Eton 258nm (7,930). max IR:

(KBr) 3400, 1720 and 1620cm"~. ~H N M R : (CsDsN) 0.92(3H, d, J=5.9Hz); 0.96(3H, s); 1.19(3H, d, ,]--7.3 Hz); 1.36(3H, s); 1.48(3H, s), 1.56(3H, s), 3.70(1H, dd, J=12.0 and 4.6Hz); 3.74(1H, J=l 1.2 Hz); 4.59(1H, d, J=ll.2Hz); and 1.63ppm (1H, dd, J=9.3 and 5.9Hz). (Free acid) (CsDsN) 0.96(3H, s); 0.97(3H, d, 3'--5.9 Hz); 1.36(3H, d, J=6.9 Hz); 1.37(3H, s); 1.48(3H, s); 1.56(3H, s); 3.71(1H, dd, J=l 1.1 and 4.4Hz); 3.75(1H, d, J=10.8Hz); 4.60(1H, d, J=10.8Hz); and 4.64ppm (1H, dd, J=9.2 and 5.6Hz). 13C NMR:

(CsDsN)(number of bonded H): 208.6(0); 198.2(0); 176.2(0); 164.5(0); 139.7(0); 79.8(1); 72.0(1); 64.4(2); 51.6(3); 23.8(3); 20.0(3); 19.7(3); 19.4(3); 17.2(3); and 17. lppm (3). Mass Spectrum: Methyl ganolucidate C showed a molecular ion peak at 532.3408m/e (calcd. for Cs=I-hsO7, 532.3401); ganolucidic acid C (M+, 518.3268, calcd. 518.3245). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganodeilc Acid J and Ganolucidic Acid C, from the Fungus Ganoderma lucidum; Agile. Biol. Chem., Vol. 49, pp. 3637-3638(1985).

11. GanolucidicAcids

387

Common/Systematic Name Ganolucidic acid D 15a,23-Dihydroxy-3,11-dioxo-5a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C30H4406; M W = 500.313 79 COOH

0 .

,.

~_

"OH

General Characteristics Ganolucidic acid D was a crystalline solid; [tt]o 23 + 192 ~ (c=0.1, in EtOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was separated into thirteen fractions; Fraction 12 was subjected to Lobar column (RPs, Merck) chromatography and the second fraction was treated with diazomethane. The resulting product was rechromatographed on silica gel and the Lobar (RPs) column to give methyl ganoderate L, lucidone C, and methyl lucidenate G. The fourth fraction in the chromatography of Fr. 12 was purified on a silica gel column, preparative TLC and I-IPLC to give ganolucidic acid D. .Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Monomethyl derivative) UV;

~, mEtoH 257nm (e=7800). ax IR:

(KBr) 3400, 1700, and 1640cmq.

388

11.

Ganolucidic Acids

1H N ~ :

(CsDsN) 7.17(IH, dq, ,/--8.8and 1.5Hz); 4.95(IH, overlapped);4.62(IH, ddd, ,/--8.8, 5.9, and 5.4Hz); 2.01(3H, d, J=l.5Hz); 1.47(3H, s), 1.26(3H, s), 1.13(3H, s); 1.12(3H, s); 1.09(3H, d, J=6.8Hz); and 0.95ppm (3H, s). 13C ~ :

(CsDsN) (number of bonded H): 217.1(0); 198.3(0); 168.6(0); 165.5(0); 146.2(I); 138.2(0); 127.3(0),72.0(I);and 66.Tppm (I). These data indicatedthat methyl ganolucidate D was differentfrom methyl ganolucidateA in the side chain moiety, having a double bond between C-24 and C-25 and a hydroxyl group at C-23. In the ~H N M R spectrum of methyl ganolucidateD, the signaldue to H-24 resonated at 6.59ppm in CDCI3, which resembled those of tiglicacid and ganoderic acidsU-Z, so the configurationof the double bond between C-24 and C-25 was assigned as E. From these observations,the structureof ganolucidicacid D was concluded to be 15a,23dihydroxy-3,I l-dioxo-5a-lanosta-8,24E-dien-26-oicacid. Mass Spectrum: HREIMS: M + 500.3135; C30H4406. Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganolucidic Acid D, Ganoderic Acid L, Lucidone C, and Lucidenie Acid G, from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 50, pp. 809-811(1986).

11.

Ganolucidic Acids

389

Common/Systematic Name Ganolucidic acid E 15tt-Hydroxy-3,11-dioxo-5a-lanosta-8,24E-dien-26-oic acid Molecular Formula/Molecular Weight C30I-I4405; MW = 484.31887

H

O

i

"oH

General Characteristics Methyl ganolucidate E was a colorless syrup; [a]D24 "k-154 (C=0.1, in MeOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification The neutral fraction obtained from the EtOH extract of G. lucidum (a mixture of two strains) was separated into three fractions (Fr. I, Fr. II and Fr. III) using C~8 Sep-Pak (MeOH; Waters Assoc.) and reversed-phase HPLC (Resolve-C~8 column, Waters Assoc.), 2% AcOH-CH3CN (2.7:1, 0.8ml/min., detector set at 254nm). Fraction I was further separated by silica gel and Lobar column chromatographies into several fractions (Frs. D, K, M, and P). Methyl ganolucidate E was obtained by preparative TLC (precoated Kieselgel 60 F254 and RP~8 F254) ofFr. P alter methylation with diazomethane. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ganolucidate)

UV; /~ EtOH max

219(e = 12,100) and 257nm (7,080).

IR;

(KBr) 3450, 2940, 2920, 2870, 1700, 1635, and 1570cm"~.

390

11.

Ganolucidic Acids

IH NMR: (CDCI3) 6.73(1H, tq, J=7.5,1.5Hz, 24-H); 4.39(1H, dd, J=9.2, 5.7 Hz, 1513-H); 3.01(1H, ddd, J=13.9, 8.2, 5.6 Hz, l13-H); 2.72(1H, d, J=17.2 Hz, 12a-H); 2.45(1H, d, J=16.9 Hz, 1213-H); 1.83(3H, s, 27-H); 1.18(3H, s, 30-H); 1.13(3H, s, 19- or 28-H); 1.12(3H, s, 19- or 28-H); 1.08(3H, s, 29-H); 0.89(3H, s, 18-H); 0.89(3H, d, J=6.2, 21H); and 3.74ppm (3H, s, COOCH3). Mass Spectrum: HR IMS: 498.3335(M+, C3~I-I4605),470(18%), 401(100), 287(69), 95(34), 69(28), 55(36), and 4 lm/e (29). References T. Nishitoba, K. Oda, H. Sato, and S. Sakamura; Novel Triterpenoids from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 52, pp. 367-372(1988). T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganolucidic Acid D, Ganoderic Acid L, Lucidone C, and Lucidenic Acid G, from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 50, pp. 809-811 (1986).

Lucidenic Acids Lucidenic acid A Lucidenic acid B Lucidenic acid C Lucidenic acid D 1 Lucidenic acid D2 Lucidenic acid E1 Lucidenic acid E2 Lucidenic acid F Lucidenic acid G Lucidenic acid H Lucidenic acid I Lucidenic acid J Lucidenic acid K Lucidenic acid L Lucidenic acid M

391

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12.

Lucidenic Acids

393

Common/Systematic Name Lucidenic acid A 3,11,15-Trioxo-713-hydroxy-5a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3806; M'W = 458.26684

~

COOH

0

0 " f "'rf-.& v

/-_n

"OH

General Characteristics Crystallized from EtOAc as colorless needles; mp., 194-195~ CHC13).

[a]D24 +173 o (C=0.2, in

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was acidified (pH 3-4) with 6N HCI, and the resulting precipitate was applied to silica gel column chromatography using a solvent system of chloroform-methanol. The eluate was rechromatographed on the silica gel column to give two strongly bitter compounds (lucidenic acid A and ganoderic acid C) and three slightly bitter compounds (lucidenic acid B, lucidenic acid C, and ganoderic acid B). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV:

~, Eton 254nm (e=7,680). max

IR:

(KBr) 3440, 3300-2500(br), 2975, 2875, 1720, 1700, 1680, 1380, 1270, 1170, 1060, and 750cm "~.

394

12.

Lucidenic Acids

CD: (MeOH) 295(- 1.9), 282(0), 255(+ 15.0), 227(0), and 212nm (-7.2). ~HNMR: (CDC13) 4.86(1H, dd, J=9.5 and 7.7Hz, 7a-H); 2.94(1H, ddd, J=13.6, 7.7 and 5.5Hz, l[3-H); 2.81(1H, dd, J=19.6 and 8.2Hz, 16a-H); 2.78(1H, d, J=17.2Hz, 12a-H); 2.75(1H, d, J=17.2 Hz, 1213-H);2.52(1H, 2-H); 2.45(2H, 2-H and 23-H); 2.34(1H, ddd, J=16.4.8.2 and 8.2Hz, 23-H); 2.17(1H, dd, J=19.6 and 9.9Hz, 1613-H);2.12(1H, ddd, J=13.0, 7.7 and 1.2 Hz, 6a-H); 2.01(1H, ddd, J=9.9, 9.0 and 8.2Hz, 17-H); 1.80(1H, m, 22-H); 1.68(1H, ddd, J=13.0, 13.0 and 9.5Hz, 613-H); 1.60(1H, m, 20-H); 1.58(1H, dd, J=13.0 and 1.2Hz, 5a-H); 1.48(1H, ddd, J=13.6, 8.3 and 8.3Hz, la-H); 1.37(1H, 22-H); 1.35(3H, s, 27-H); 1.26(3H, s, 19-H); 1.14(3H, s, 25-H); 1.1 l(3H, s, 26-H); 1.01(3H, s, 18-H) and 0.99ppm (3H, d, J=6.6Hz, 21-H). Mass Spectrum: 458.2698(M+, C27H3806,4%), 430(2), 329(10), 320(5), 55(36), 44(100), 43(36), and 40m/e (71). References T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agile. Biol. Chem., Vol. 48, pp. 2905-2907(1984). T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agile. Biol. Chem., Vol. 49, pp. 1793-1798(1985).

12.

Lucidenic Acids

395

Common/Systematic Name Lucidenic acid B 3,11,15-Trioxo-7[3,12[3-dihydroxy-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3807; M W - 4 7 4 . 2 6 1 7 5

HO ~ C O O H 0

o

"OH

General Characteristics Crystallized from EtOAc as colorless needles; mp., 179-181~ MeOH).

[a]D24 + 168.9 ~ (C=0.22, in

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was acidified (pH 3-4) with 6N HC1, and the resulting precipitate was applied to silica gel column chromatography using a solvent system of chloroform-methanol. The eluate was rechromatographed on the silica gel column to give two strongly bitter compounds (lucidenic acid A and ganoderic acid C) and three slightly bitter compounds (lucidenic acid B, lucidenic acid C, and ganoderic acid B). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV~

,~ EtOH 252nm (e=6,010). max

396

12.

Lucidenic Acids

CD: (MeOH) 290(- 7.0), 277(0), 248(+ 25.6), 224(0), and 208nm (-12.1). IR; (KBr) 3380, 3200-2400(br), 2950, 2925, 2860, 1730, 1700, 1660, 1380, 1270, 1160, and 1050cmq. IH ~ :

(CDCI3) 4.83(IH, dd, J=8.7 and 8.7Hz, 7a-H); 4.38(IH, s, 12a-H), 2.79(IH, ddd, J=13.7, 7.0 and 4.21-Iz,ll3-I-I);2.68(IH, dd, J=19.5 and 8.5Hz, 16or-I-I);2.64(IH, ddd, J=15.6, I 1.0 and 7.0Hz, 2~-H), 2.45(2H, 17-H and 23-I-I);2.40(IH, 23-H); 2.40(IH, ddd, J=15.6, 6.6 and 4.2Hz, 2or-I-I);2.32(IH, dd, J=19.5 and 10.21-1z,1613-H); 2.18(IH, ddd, J=13.7. 8.7 and 1.8Hz, 6a-H); 1.96(IH, m, 20-H), 1.86(IH, m, 22-I-I); 1.79(IH, ddd, J=13.7, 13.7 and 8.7Hz, 6~-H); 1.48(IH, dd, J=13.7 and 1.8Hz, 5a-H); 1.44(3H, s, 27-H); 1.43(3H, s, 19-H); 1.35(IH, ddd, J=13.7, II.0 and 6.6Hz, la-H); 1.30(1H, m, 22-H); 1.15(3H, d, J=6.7Hz, 21-H); 1.13(3H, s, 25-H); 1.13(3H, s, 26-H); and 0.83ppm (3H, s, 18-H). 13CNMR: (CDC13) C-l, 35.1(0; C-2, 34.3(t); C-3,215.7(s); C-4, 46.9(s); C-5, 49.4(d); C-6, 27.7(0; C-7, 65.8(d); C-8, 158.1(s); C-9, 140.1(s); C-10, 37.8(s); C-11, 199.6(s), C12, 78.3(0; C-13,451.6(s); C-14, 60.2(s); C-15, 217.1(s); C-16, 37.6(t), C-17, 46.6(d); C-18, 12.1(q); C-19, 18.3(q); C-20, 31.5(d); C-21, 20.5(q); C-22, 29.5(0; C23, 31.8(0; C-24, 178.7(s); C-25, 26.3(q); C-26, 21.2(q); and C-27, 23.3ppm (q).

The ~H and 13CNMR data oflucidenic acid B were very similar to those oflucidenic acid A, but lucidenic acid B showed the new signals at 4.38ppm (singlet) in the ~H NMR and at 78.3ppm (doublet) in the 13C NMR, and lacked the signals of the methylene protons at C(12) in the ~H NMR. This indicated the presence of a secondary hydroxy group at C(12) in lucidenic acid B. Mass Spectrum: 474.2579(M +, C27H3sO7, 4%), 456(3), 304(100), 55(9), 44(96), 43(10), and 40m/e (14). References T. Nishitoba, H. Sato, T.Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agric. Biol. Chem., Vol. 48, pp. 2905-2907(1984). T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agric. Biol. Chem., Vol. 49, pp. 1793-1798(1985).

12.

Lucidenic Acids

397

Common/Systematic Name Lucidenic acid C 11,15-Dioxo-3 [3,713,12[3-trihydroxy-5a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27I-I4007; MW = 476.27740

HO - . ~ C O O H 0

H..o ~ o H :-H " General Characteristics Crystallized from CHCl3-hexane as pale yellow needles; mp., 199-200~ (c=0.2, in MeOH).

[a]D 24 q-140 ~

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was acidified (pH 3-4) with 6N HC1, and the resulting precipitate was applied to silica gel column chromatography using a solvent system of chloroform-methanol. The eluate was rechromatographed on the silica gel column to give two strongly bitter compounds (lucidenic acid A and ganoderic acid C) and three slightly bitter compounds (lucidenic acid B, lucidenic acid C, and ganoderic acid B). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Soectral Data UV:

i moll 254nm (e=5,960). max

398

12.

Lucidenic Acids

CD: (MeOH) 290(- 5.8), 276(0), 248(+ 15.4), 224(0), and 207nm (-7.8). IR: (KBr) 3350, 3200-2400(br), 2950, 2850, 1720, 1675, 1260, 1090, 1040, and 1020cmq. IH NMR: (CDCI3) The NMR spectrum of lucidenic acid C was very similar to that of lucidenic acid B, but it showed three signals at 3.21(double doublet J=6.4, 10.1Hz), 4. 73 (singlet) and 4.78ppm (double doublet, J=8.7, 8.7Hz) due to methine groups beating the hydroxyl groups. This provided support for a secondary hydroxyl group at C-3 in lucidenic acid C, instead of a carbonyl group. 13C NMR:

(CDCI3) C-I, 33.8(0; C-2, 23.8(0; C-3, 79.0(d);C-4, 37.7(s);C-5, 49.1(d);C-6, 25.0(t); C-7, 68.8(d); C-8, 154.4(s); C-9, 146.0(s); C-10, 38.2(s); C-11,192.0(s); C-12, 80.2(t); C-13, 50.2(s); C-14, 58.2(s); C-15, 210.5(s); C-16, 37.9(t); C-17, 46.4(d); C-18, 13.4(q); C-19, 18.2(q); C-20, 31.8(d); C-21, 20.3(q); C-22, 29.6(t); C-23, 31.7(t); C-24, 178.3(s); C-25, 28. l(q); C-26, 16.5(q); and C-27, 25.5(q). Mass Spectrum: 476.2753(M +, Cz7I-I4007,2%), 458(2), 307(14), 306(100), 55(17), 44(12), and 43m/e (16). References T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agric. Biol. Chem., Vol. 48, pp. 2905-2907(1984). T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura; New Bitter C27 and C30 Terpenoids from the Fungus Ganoderma lucidum (Reishi); Agric. Biol. Chem., Vol. 49, pp. 1793-1798(1985).

12. Lucidenic Acids

399

Common/Systematic Name Lucidenic acid D 1 3,7,11,15-Pentaoxo-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3407; M W "- 470.23045 '

COOH

0

0

0

General Characteristics Crystallized from EtOAc-cyclohexane as pale yellow needles; mp., 229-23 I~ 84 ~ (C=0.1, in EtOH).

[t~]D22 +

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with chloroform and chromatographed on a silica gel column using various concentrations of methanol in chloroform. Lucidenic acid D 1 and lucidone A were further purified with a second silica gel column eluted in a like manner. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; also bitter tasting. The intensity of bitterness was evaluated to be lucidenic acid D, ganoderic acid C, lucidone A, lucidenic acid A, ganoderic acid B, lucidenic acid B, lucidenic acid C, and lucidenic acid E. Lucidenic acid A, ganoderic acid C, lucidenic acid D, and lucidone A had intense bitterness and their threshold values were determined to 2.0 x 106M, 5 x 10SM, 5 x 10t~ and 1 x 106M, respectively, when the organoleptic test was carried out by an ascending series of concentrations in a 10% ethanol solution. Spectral Data UV: ~ EtOH max

224(sh, e=4,160) and 266nm (5,170).

400

12.

Lucidenic Acids

IR:

3400-2400(br), 2960, 2875, 1755(sh), 1730, and 1685cm "~. ~H NMR: (CDC13) The ~H NMR spectrum showed the presence of six methyl groups; 1.56(s), 1.36(s), 1.17(s), 1.16(s), 1.14(s), and 0.91ppm (d, J=6.1Hz), but did not show any signal due to the methine group bearing the hydroxy group, which was observed in the case of lucidenic acids A, B, and C. These data led to the structure of lucidenic acid D being a pentaketo acid. 13C N M ~ :

(CDCI3) (number of bonded H); 214.4(0),204.2(0),198.2(0).197.2(0),192.8(0), 178.4(0), 150.0(0), 149.6(0), 61.0(0), 59.0(0), 51.0(1), 47.0(0). 39.3(0). 39.1(2). 39.0(1), 37.3(2), 35.5(1), 33.6(2), 33.5(2), 30.9(2), 30.1(2), 27.5(3), 23.3(3), 20.3(3), 18.6(3), 17.8(3), and 12.5ppm (3). Mass Spectrum: Field-desorption mass spectrum gave a molecular ion peak at 470m/e. Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids from Ganoderma lucidum and Their Bitterness; Agric. Biol. Chem., Vol. 49, pp. 1547-1549(1985).

12.

Lucidenic Acids

401

_Common/Systematic Name Lucidenic acid D2 3,7,11,15-Tetraoxo-12-acetoxy-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C29H3808; ]~[Vl/-- 514.25667

",~COOH

AcO

0

0-~ ~ / .....,, v

~'0

General Characteristics Isolated and identified as methyl ester. Yellow amorphous powder; [a]D + 136~ (C=I.0, in CHCIa). Fungal Source

Ganoderma lucidum.

Isolation/Purification The acidic fraction from the ether extract of the gills of dried fruit bodies of Ganoderma lucidum was methylated with diazomethane. The crude product was separated repeatedly by a combination of silica gel chromatography and preparative thin-layer chromatography to give pure triterpenoids. Spectral Data (Methyl ester) UV: ~m~x 252.5nm(log e=3.96). IR:

(KBr) 1745, 1735(sh), 1700, 1680, and 1220cm~. 1H N1VIR: (CDCI3) 173(1H, ddd, J-14.5, 10.0, 6.5Hz, H-la); 2.77(1H, ddd, J-14.0, 8.5, 6.0Hz, H-113); 2.425(1H, ddd, J-15.5, 8.0, 5.5Hz, H-2a); 2.61(ddd, J=15.5, 9.5, 6.0Hz, H2[3); 2.3 l(1n, dd, J=15.0, 2.5Hz, H-5); 2.49(1H, dd, J=15.0, 2.5Hz, H-6a); 2.73(1H, dd, J=14.8, 14.0Hz, H-6~); 5.67(1H, s, n-12a); 2.83(1H, dd, J=18.5, 10.0Hz, H-16a); 2.08(1H, dd, J=18.5, 8.0Hz, n-16~); 2.48(1H, dt, J=10.0, 8.0Hz, n-17); 1.64(1H, m, H-20); 1.30(1H, dtd, J=13.5, 8.5, 5.0Hz, H-22); 1.84(1H, dtd, J=13.5, 8.5, 2.5Hz, H22); 2.28(1H, dt, J=16.0, 8.5Hz, H-23); 2.41(1H, ddd, J=16.0, 8.5, 5.5Hz, H-23);

402

12.

Lucidenic Acids

0.86(3H, s, 18-CH3); 1.33(3H, s, 19-CH3); 1.00(3H, d, J-6.8Hz, 21-CH3); 1.14(3H, s, 30-CH3); 1.12 (3H, s, 31-CH3); 1.81(3H, s, 32-CH3); and 3.68ppm (3H, s, COOC__H_aH_). 13C NMR: (CDCI3) C-l, 34.0, t; C-2, 33.8, t; C-3,214.8, s; C-4, 46.9, s; C-5, 51.0, d; C-6, 37.4, t; C-7, 198.5~ s; C-8, 149.7, s; C-9, 146.2, s; C-10, 39.3, s; C-11, 194.1*, s; C-12, 79.1, d; C-13, 47.6, s; C-14, 58.6, s; C-15, 205.8, s; C-16, 37.4, t; C-17, 45.2, d; C-18, 12.0, q; C-19, 18.6, q; C-20, 33.0, d; C-21, 20.1, q; C-22, 30.1, t; C-23, 31.6, t; C-24, 173.6, s; C-30, 27.6, q; C-31, 20.45, q; C-32, 20.8, q; OCH3, 51.6, q; OCOCH3, 20.8, q; and OCOCH3, 170.0ppm, s. Assignments may be reversed. Mass Data: LREIMS: 528(M+, 14%), 486(M+- CH2CO-, 22), 468(27), 413(5), 371(11), 353(8), 302(100), 227(30), 185(84), 153(32), 167(95), 167(95), 135(22), 115(9), and 55m/e (49); HR IMS: 528.2722role (calcd for C30H4008 528.2722); anal. calcd for C30H4008 ~ 1/21-120C, 67.03; H, 7.68; found, C, 67.20; H, 7.47. References T. Kikuchi, S. Kanomi (nee Matsuda), Y. Murai, S. Kadota, K. Tsubono, and Z. Ogita; Constituents of the Fungus Ganoderma lucidum fir.) Karst. II. Structures of Ganoderic Acids F, G, and H, Lucidenic Acids D2 and E2, and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 4018-4029(1985). T. Kikuchi, S. Matsuda, S. Kadota, Y. Murai, and Z. Ogita; Ganoderic Acid D, E, F, and H and Lucidenic Acid D, E, and F, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull. Vol. 33, pp. 2624-2627(1985).

12.

Lucidenic Acids

403

Common/Systematic Name Lucidenic acid E1 7~, 12a-Dihydroxy-3,11,15-trioxo-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3807; M W = 474.26175

HO_ .,.y....,~,..,fc OOH 0

i

0

General Characteristics Lucidenic acid E was crystallized from EtOAc as colorless plates; mp., 216-219~ + 229 ~ (c=0.2, in MeOH).

[a]D 20

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with chloroform and chromatographed on a silica gel column using various concentrations of methanol in chloroform. The bitter principles were further purified with a second silica gel column eluted in a like manner. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy; also bitter tasting. The intensity of bitterness was evaluated to be lucidenic acid D, ganoderic acid C, lucidone A, lucidenic acid A, ganoderic acid B, lucidenic acid B, lucidenic acid C, and lucidenic acid E. Lucidenic acid A, ganoderic acid C, lucidenic acid D, and lucidone A had intense bitterness and their threshold values were determined to 2.0 x 106M, 5 x 10SM, 5 x 10~~ and 1 x 106M, respectively, when the organoleptic test was carried out by an ascending series of concentrations in a 10% ethanol solution. Spectral Data UV:

~, MeOH 258(e = 6,640). max

404

12.

Lucidenic Acids

IR: (KBr) 3450, 3400-2400(br), 2950, 2880(sh), 1725, 1705, and 1640cmq.

(CDCI3) 4.87(1H, dd, ,/--9.5 and 7.3Hz); 3.83(1H, s); 1.34(3H, s), 1.17(3H, s); 1.14(3H, s); 1.10(an, s); 1.08(3H, d, J=6.7Hz); and 0.98ppm (an, s). 13CN-MR: (CDCl3)(number of bonded H) 218.8(0), 216.6(0), 201.I(0), 178.3(0), 160.7(0), 139.2(0), 78.9(I), 66.2(I), The ~3C and ~H N M R data oflucidenic acid D were very similarto those oflucidenic acid B; however, the signalof H-C(12) was observed at 3.83(IH, s), in contrastto 4.38ppm (IH, s) for lucidenicacid B.

Mass Spectrum: C27H3807by HR-MS (M+, 474.2624; calcd for C27HasOT,474.2618). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids from Ganoderma lucidum and Their Bitterness; Agric. Biol. Chem., Vol. 49, pp. 1547-1549(1985).

12.

Lucidenic Acids

405

Common/Systematic Name Lucidenic acid E2 Molecular Formula/Molecular Weight C29H4008; MW = 516.27232

AcO ~ . / , ' ~ C O O H 0 H HO" ~ Y

v

~'0

General Characteristics Isolated and identified as methyl ester. Yellow needles from ether; mp., 141-144~ [a]D + 86~ (c=0.5, in CHC13). Fungal Source

Ganoderma lucidum.

Spectral Data (Methyl ester) UV:

Zmax 256nm (log c = 3.75). IR:

(KBr) 3400, 1745, 1735(sh), 1720(sh), 1690, 1680(sh), and 1220cm~0 ~H NMR: (CDCI3) 1.18(IH, td, J=13, 4.5Hz, H-I~); 2.73(IH, dt, ,/=13.5, 3.51-Iz, H-I13); 1.72(2H, m, H-2a and 13);3.26(IH, dd, J=l 1.0, 5.0Hz, H-3); 1.56(IH, dd, 0=14.2, 2.5Hz, H-5); 2.58(IH, dd, J=14.0, 2.5Hz, H-6e); 267(IH, t, d=14.0Hz, H-613); 5.62(IH, s, H-12a); 2.79(IH, dd, J=18.2, 9.5Hz, H-16a); 2.08(IH, dd, J=18.2, 8. II-Iz, H-1613); 2.46(IH, dt, J=9.0, 8.0I-Iz, H-17); 1.66(IH, m, H-20); 1.29(IH, dtd, d=14.0, 8.5, 5.0I-Iz, H-22); 1.84(IH, dtd, ,/=13.5, 8.0, 2.5I-Iz, H-22); 2.27(IH, dt, J=16.0, 8.0Hz, H-23); 2.40(IH, ddd, J=16.0, 8.8, 5.5Hz, H-23); 0.83(3H, s, 18-CH3); 1.33(3H, s, 19-CH3); 1.00(3H, d, J=6.7Hz, 21-CH3); 1.03(3H, s, 30-CH3); 0.89(3H, s, 31-CH3); 1.73(3H, s, 32-CH3); 3.67(3H, s, COOCH3), and 2.22ppm (3H, CHACO.). 13C NMR:

(CDCI3) C-l, 33.3, t; C-2, 27.4, t; C-3, 77.5, d; C-4, 40.5, s; C-5, 51.4, d; C-6, 36.7, t; C-7, 198.8~ s; C-8, 151.6, s; C-9, 146.0, s; C-10, 39.2, s; C-11, 194.1~ s; C-12, 79.4, d; C-13, 48.0, s; C-14, 58.5, s; C-15, 206.0, s; C-16, 37.6, t; C-17, 45.5, d; C-18, 12.1,

406

12.

Lucidenic Acids

q; C-19, 18.0, q; C-20, 33.0, d; C-21, 20.2, q; C-22, 30.2, t; C-23, 31.8, t; C-24, 173.7, s; C-30, 27.9, q; C-31, 15.6, q; C-32, 21.4, q; OCH3, 51.6, q; OCOCH3, 20.9, q; and OCOCH3, 170.1ppm, s. Assignments may be reversed. Mass Data: LREIMS: 530(M+, 5%), 488(M+- CH2CO-, 2), 470(10), 415(2), 373(5), 355(2), 304(100), 227(10), 185(23), 167(30), 153(10), 135(8), 115(3), and 55role (10); HR IMS: 530.2879role (calcd for C30I-h208, 530.2879); anal. calcd for C30I-h2Os 9 1/21-120: C, 66.78; H, 8.03%; found; C, 66.98; H, 7.94%. Reference T. Kikuchi, S. Kanomi (nee Matsuda), Y. Murai, S. Kadota, K. Tsubono, and Z. Ogita; Constituents of the Fungus Ganoderma lucidum (Fr.) Karst. II. Structures of Ganoderic Acids F, G, and H, Lucidenic Acids D2 and E2, and Related Compounds; Chem. Pharm. Bull., Vol. 34, pp. 4018-4029(1985).

12.

Lucidenic Acids

407

Common/Systematic Name Lucidenic acid F 3,7,11,15-Tetraoxo-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3606; M W = 456.25119 COOH

0

General Characteristics Yellow needles; mp., 208-21 l~

0

[a]D + 195 ~ (in CHCI3).

Fungal Source Isolated from Ganoderma lucidum. Isolation/Purification The acidic fraction from the ether extract of the gills of dried fruit bodies of Ganoderma lucidum was methylated with diazomethane and the crude product was separated repeatedly by a combination of silica gel chromatography and preparative thin-layer chromatography to give pure triterpenoids. Spectral Data (Methyl ester)

(CDCI3) 0.85(3H, s, 18-CH3); 1.27 (3H, s, 19-CH3); 0.96 (3H, d, J=6.0Hz, 21-CH3); 1.11 (3H, s, 30-CH3); 1.14 (3H, s, 31-CH3); 1.65H, s, 32-CH3); and 3.68ppm (3H, s COOCn3) 13C NMR: (CDC13) 37.3, C-l, t; 34.6, C-2, t; 215.3, C-3, s; 43.9, C-4, s; 51.0, C-5, d; 33.9, C-6, t; 199.5, C-7, s; 149.7, C-8, s; 146.9, C-9, s; 39.4, C-10, s; 194.4, C-11, s; 49.0, C-12, t; 47.0, C-13, s ; 57.2, C-14, s; 207.3, C-15, s; 39.9, C-16, t; 45.2, C-17, d; 16.1, C-18, q; 18.6, C-19, q; 35.4, C-20, d; 18.3, C-21, q; 30.8, C-22, t; 31.0, C-23, t; 173.8, C-24, s; 27.7, C-30, q; 20.9, C-31" q; 20.3, C-32", q; and 51.7ppm, CH30-, q. * Assignments may be reversed.

408

12.

Lucidenic Acids

Reference T. Kikuchi, S. Matsuda, S. Kadota, Y. Murai, and Z. Ogita; G-anoderic Acid D, E, F, and H and Lucidenic Acid D, E, and F, New Triterpenoids from Ganoderma lucidum; Chem. Pharm. Bull., Vol. 33, pp. 2624-2627(1985).

12.

Lucidenic Acids

409

Common/Systematic Name Lucidenic acid G 3,11-Dioxo-413,713,15 a-trihydroxy-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H4007; M W "- 476.27740

0

O HOH2C

~

COOH

-

General Characteristics Colorless syrup; [a]D 21 -1- 127~ (c=0.2, in MeOH). .Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was separated into thirteen fractions; Fraction 12 was subjected to Lobar column (RPs, Merck) chromatography and the second fraction was treated with diazomethane. The resulting product was rechromatographed on silica gel and the Lobar (RPs) column to give methyl ganoderate L, lucidone C, and methyl lucidenate G. The fourth fraction in the chromatography of Fr. 12 was purified on a silica gel column, preparative TLC and HPLC to give ganolucidic acid D. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV:

uooH 254nm (e=8040). max

410

12.

Lucidenic Acids

IR: (KBr) 3400, 1730(sh), 1700, and 1660cmq.

~H NMR: (CsDsN) 5.24(IH, dd, ,/--9.3and 7.1 I-Iz);4.99(IH, dd, ,/=9.9and 7.3I-Iz);4.37(IH, d, J=l 1.0Hz); 3.99(1H, d, J=l 1.0Hz); 1.72(3H, s); 1.52(3H, s); 1.51(3H, s); 1.07(3H, s); and 0.81ppm (3H, d, J=5.9Hz). 13C N M ~ :

(CsDsN) 214.4(0); 199.8(0);174.1(0); 161.3(0); 140.6(0);72.2(I);69.2(I);and 65. lppm (2). Mass Spectrum: HREIMS: 490.2905m/e (M+). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganolucidic Acid D, Ganoderic Acid L, Lucidone C and Lueidenie Acid G from the Fungus Ganoderma lucidum; Agile. Biol. Chem., Vol. 50, pp. 809-811 (1986).

12. Lucidenic Acids

411

Common/Systematic Name Lucidenic acid H

11,15-Dioxo-313,713-dihydroxy-5tz-lanost-8-en-24-oic acid

Molecular Formula/Molecular Weight C27H4007; M W -- 476.27740

COOH

0

HO

0

-CH20H

General Characteristics Methyl ester: pale yellow prisms from ethyl acetate-cyclohexane; mp., 190-192~ + 136~ (c=0.2, in MeOH).

[aiD 23

Funsal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12)and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using silica gel column chromatography (chloroform-methanol, 19:1, v/v; chloroform-acetone, 2:1, v/v; benzene-ethyl acetate, 7:1, v/v) and reversed-phase LC (methanol-water, 7:3, v/v, Lobar RP~s) to yield methyl lucidenate H, I, and J. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ester)

UV;

u~on 255nm (e = 7,550). max

412

12.

Lucidenic Acids

(thin film) 3400, 2925, 1720, and 1655cm"~. CD:

~l, MeOH nlax

A293 - 3.3, A28o0, A25s + 14.9, A23o 0, and A21o - 8.3.

~H NMR: (CDC13) 1.29(1H, ddd, ,]=13.6, 12.5, 6.9Hz, H-I~); 3.27(1H, ddd, ,I=13.6, 3.5, 3.5Hz, I-1-113);2.05(1H, m, H-2a); 2.05(1H, m, H-213);4.30(1H, dd, ,1=11.7, 5.5Hz, H-3r 1.91(1H, d, ,1=12.7Hz, H-5e0; 2.58(1H, dd, d=12.1, 7.5Hz, H-6a); 1.95(1H, m, H-613); 5.25(1H, dd, J=7.5, 7.5Hz, H-7~); 2.95(1H, dq, J=16.8, 1.0Hz, H-12a); 2.81(1H, d, `1=16.8Hz, H-1213); 2.77(1H, dd, ,1=19.2, 8.1Hz, H-16a); 2.21(1H, dd, ,/--19.2, 9.9Hz, H-1613); 2.00(1H, m, H-17); 1.10(3H, s, H3-18); 1.54(3H, s, H3-19); 1.50(1H, m, H20); 0.86(3H, d, J=6.2Hz, H3-21); 1.80(1H, m, H-22), 1.30(1H, m, H-22); 2.43(1H, ddd, J=15.0, 9.5, 5.5Hz, H-23); 2.30(1H, ddd, J=15.0, 8.8, 7.0Hz, H-23); 4.24(1H, d, J=10.4Hz, H-25); 3.78(1H, d, ,1=10.4Hz, H-25); 1.1 l(3H, s, H3-26); 1.30(3H, s, H327); and 3.65ppm (3H, s, COOCH3). 13C NMR.:

(CDCI3) 35.4, C-l; 28.3, C-2; 72.0, C-3; 43.0, C-4; 42.5, C-5; 27.8, C-6; 66.7, C-7; 158.8, C-8; 142.9, C-9; 39.2, C-10; 198.3, C-11; 51.1, C-12; 45.6, C-13; 59.2, C-14; 216.8, C-15; 41.4, C-16; 46.2, C-17; 17.8, C-18; 19.2, C-19; 35.4, C-20; 18.1, C-21; 31.0, C-22; 31.0, C-23; 174.0, C-24; 66.6, C-25; 13.2, C-26; 24.8, C-27; and 51.4ppm CH3OOC-. Mass Spectrum: HREIMS: 490.2932(M +, C28I-I4207,38%; calcd 490.2932), 472(22), 462(30), 347(100), 334(63), 322(36), 107(61), 95(35), 81(36), 69(43), 55(66), and 43m/e (88). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

12.

Lucidenic Acids

413

.Common/Systematic Name Lucidenic acid I 7,11,15-Trioxo-313-hydroxy-5a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3807; M W = 474.26175

~ 0

HO

COOH

0

-CH2OH

General Characteristics Methyl ester: pale yellow syrup; [a]D23 -I- 118~ (C'-0.2, in MeOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12) and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using silica gel column chromatography (chloroform-methanol, 19:1, v/v; chloroform-acetone, 2:1, v/v; benzene-ethyl acetate, 7:1, v/v) and reversed-phase LC (methanol-water, 7:3, v/v, Lobar RP18) to yield methyl lucidenate H, I, and J. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data (Methyl ester) UV: /~ MeOH max

262nm (e = 6,830).

414

12.

CD: ~

MeOH max

Lucidenic Acids

A333 0, A3o5- 4.9, A292 0, A275 + 8.3, A24s0, A231 + 0.2, A225 0, and A2o7 -4.4.

(thin film) 3400, 2925, 1735, and 1675cm"~. 1H N]k,IR: (CDCI3) 1.45(1H, m, H-la); 3.10(1H, ddd, J=13.6, 3.5, 3.5Hz, H-113); 2.05(1H, m, H-2a); 2.05(1H, m, H-213); 4.29(1H, dd, J=l 1.2, 5.3Hz, H-3a); 2.65(1H, dd, J=13.6, 2.6Hz, H-5et); 2.92(1H, dd, J=13.9, 2.6Hz, H-6a); 2.80(1H, dd, J=13.9, 13.6Hz, H613); 3.09(1H, d, J=15.3Hz, H-12a); 2.81(1H, d, J=15.3Hz, H-1213); 2.78(1H, m, n16a); 2.10(1H, m, H-1613); 2.05(1H, m, H-17); 0.93(3H, s, H3-18); 1.47(3H, s, H3-19); 1.45(1H, m, H-20); 0.86(3H, d, J=6.6Hz, H3-21); 1.80(1H, m, H-22); 1.40(1H, m, H22); 2.45(1H, ddd, J=15.1, 9.3, 5.2Hz, H-23); 2.31(1H, ddd, J=15.1, 8.8, 7.0Hz, H23); 4.16(1H, d, J=10.SHz, H-25); 3.61(1H, d, J=10.8Hz, H-25); 1.05(3H, s, H3-26); 1.63(3H, s, H3-27); and 3.65ppm (3H, s, COOCH3). ~3CNMR: (CDC13) 36.7, C-l; 27.9, C-2; 70.7, C-3; 43.6, C-4; 44.5, C-5; 34.1, C-6; 200.4, C-7; 152.2, C-8; 147.4, C-9; 40.8, C-10; 199.8, C-11; 49.9, C-12; 44.5, C-13; 57.6, C-14; 208.0, C-15; 40.5, C-16; 45.5, C-17; 16.4, C-18; 18.6, C-19; 35.5, C-20; 18.2, C-21; 31.0, C-22; 31.1, C-23; 174.0, C-24; 65.1, C-25; 13.1, C-26; 21.6, C-27; and 51.4ppm CH3OOC-. Mass Spectrum: HREIMS: 488.2796(M +, C28FL,oO7,30%; calcd. 490.2775), 470(12), 265(20), 141(33), 109(42), 95(36), 81(33), 69(40), 55(73), and 44m/e (100). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

12.

Lucidenic Acids

415

Common/Systematic Name Lucidenic acid J 3,11,15-Trioxo-313,1213-dihydroxy-5 a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H3808; ~ - 490.25667

O

HO

""-1/"',-/"COOH

HO~ ~_ -CH2OH General Characteristics Methyl ester: pale yellow syrup; [a]D23 + 78 ~ (C=0.1, in MeOH). Funsal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12) and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using silica gel column chromatography (chloroform-methanol, 19:1, v/v; chloroform-acetone, 2:1, v/v; benzeneethyl acetate, 7:1, v/v) and reverse-phase LC (methanol-water, 7:3, v/v, Lobar RPls) to yield methyl lucidenate H, I, and J. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ester)

UV: ~, mM~oa 255nm (e = 6,400). ax

416

12.

Lucidenic Acids

CD: ~ MeOH max

A346 0, A3os - 6.0, A292 0, A279 + and A228 + 5.7.

6.0, A262 0, A255 - 1.2, A24s 0, A215 0,

IR:

(thin film) 3430, 2930, 1740, and 1680cm"1. IH NMR:

(CDC13) 1.35(1H, m, H-la); 2.97(1H, ddd, J=13.6, 3.5, 3.5Hz, H-1[3); 2.05(1H, m, H-2a); 2.05(1H, m, H-213); 4.27(1H, dd, J=l 1.5, 5.2Hz, H-3a), 2.65(1H, dd, ,/=13.6, 2.6Hz, H-5a); 2.93(1H, dd, J=13.6, 2.6Hz, H-6a), 2.83(1H, dd, J=13.6, 13.6Hz, H6[3); 4.92(1H, s, n-12a); 2.87(1H, dd, J=17.6, 9.2Hz, n-16a); 2.26(1H, dd, ,/=17.6, 8.1Hz, H-1613); 2.70(1H, ddd, J=8.0, 8.0, 8.0Hz, n-17); 0.98(3H, s, H3-18); 1.52(3H, s, H3-19); 1.95(1H, m, n-20), 1.28(3H, d, J=6.6Hz, H3-21), 1.95(1H, m, H-22), 1.41(1H, m, H-22), 2.48(1H, ddd, J=16.2, 9.2, 5.8Hz, H-23); 2.37(1H, ddd, J=16.2, 8.7, 6.9Hz, H-23); 4.16(1H, d, J=10.6Hz, H-25), 3.61(1H, d, J=10.6Hz, H-25); 1.04(3H, s, H3-26); 1.78(3H, s, H3-27); and 3.65ppm (3H, s, COOCH3). Mass Spectrum: HREIMS: 504.2746(M+, C28H4oO8,51%; calcd. 504.2724), 458(25), 371(65), 353(33), 155(39), 115(42), 95(34), 55(73), and 44m/e (100). Reference T. Nishitoba, H. Sato, and S. Sakamura, Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

12.

Lucidenic Acids

417

Common/Systematic Name Lucidenic acid K 3,7,11,15-Tetraoxo- 12a-hydroxy-5 a-lanost-8-en-24-oic acid Molecular Formula/M01ecular Weight C27H3607; M W - 472.24610

H 0

COOH :

----i

~-0

General Characteristics Methyl ester: pale yellow syrup. Fungal Source Fruiting'bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12) and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using preparative TLC (benzene-ethyl acetate, 1:1 or 2:1, v/v), silica gel column chromatography (chloroform-methanol, 99:1, v/v), or reversed-phase LC (methanol-water, 7:3, v/v, Lobar RPls).These separations gave purified methyl ganoderate M, N, O, H, E, methyl lucidenate K, L, and E2. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ester)

UV;

~, M~ori 258nm (e = 5,440). max

418

12.

Lucidenic Acids

CD:

~ MeOH A337 0, Aaos-4.1, A291 0, A272 + 4.4, A233+ 3.4, and A21s 0.

IR:

(thin film) 3450, 2920, 1735, 1700, and 1680cm"~. IH NMR: (CDCI3) 2.08(1H, m, H-le0; 2.78(1H, ddd, J=14.2, 8.3, 5.8Hz, H-1Ia); 2.55(1H, ddd, J=15.6, 8.3, 7.8Hz, n-2et); 2.61(1H, ddd, ,/=-15.6, 8.8, 5.8Hz, n-EIa); 2.39(1H, dd, J=14.6, 3.4Hz, H-5tt); 2.54(1H, dd, `/=15.1, 3.4Hz, H-6tt); 2.68(1H, dd, J=15.1, 14.6Hz, H-613); 3.94(1H, s, n-lEIa); 2.84(1H, dd, ./=18.6, 9.3Hz, H-16a); 2.07(1H, dd, `/=18.6, 9.aHz, H-16~); 2.07(1H, ddd, ,/=9.3, 9.3, 9.3Hz, H-17); 0.84(3H, s, H318); 1.23(3H, s, H3-19); 1.55(1H, m, n-20); 1.06(3H, d, `/=6.8Hz, Ha-E1); 1.82(1H, m, H-22); 1.43(1H, m, H-22); 2.43(1H, ddd, ,/=16.1, 9.3, 5.4Hz, H-23); 2.30(1H, ddd, 3=16.1, 8.8, 7.3Hz, H-23); 1.12(3H, s, H3-25); 1.15(3H, s, H3-26); 1.58(3H, s, H3-27); and 3.68ppm (3H, s, COOCH3). Mass Spectrum: HREIMS: 486.2601(M+, C25H3807,63%, calcd 486.2618), 458(19), 371(39), 353(22), 302(100), 287(35), 55(55), and 43role (57). Reference T. Nishitoba, H. Sato, and S. Sakamura, Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

12.

Lucidenic Acids

419

Comm0n/Systematic Name Lucidenic acid L 7,11,15-Trioxo-313,1213-dihydroxy-5a-lanost-8-en-24-oic

acid

Molecular Formula/Molecular Weight C27H3807; M W -- 474.26175 HO

" - - . , r . . ~ ~ cOOH

o

o

Ho General Characteristics Methyl ester: a pale yellow syrup. Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12) and methanol (fraction 13). Fraction 5 was rechromatographed 3 times on a silica gel column eluted with chloroform-methanol (19:1, v/v) to give several fractions. Some of the fractions were methylated and further fractionated using preparative TLC (benzene-ethyl acetate, 1:1 or 2:1, v/v), silica gel column chromatography (chloroform-methanol, 99:1, v/v), or reversed-phase LC (methanol-water, 7:3, v/v, Lobar RP~8). These separations gave purified methyl ganoderate M, N, O, H, E, methyl lucidenate K, L, and E2. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ester)

UV:

~M~OH max

255nm (e = 6,930) 9

420

12.

Lucidenic Acids

CD:

,~ MeOH max

A345 0, A307 - 7.1, A291 0, A278 + 6.2, A264 0, A256 - 2.1, A246 0, A227 + 8.3, and A213 0.

IR:

(thin film) 3450, 2920, 1740, and 1680cmq. ~H NMR: (CDCI3) 1.14(IH, ddd, ,/=13.6,13.6,5.8Hz, H-la); 2.76(IH, ddd, J=13.6, 3.7, 3.5Hz, H-tO); 1.75(IH, m, H-2a); 1.75(IH, m, H-2~));3.27(IH, dd, 0'=9.9,6.2Hz, H-3a); 1.55(IH, dd, ,/=14.3,2.6Hz, H-5a); 2.58(IH, dd, d=14.3, 2.6Hz, H-6a); 2.70(IH, dd, ,/=14.3, 14.3Hz, H-613); 4.51(1H, s, H-12a); 2.74(1H, dd, J=18.0, 9.9Hz, H-16a); 2.06(1H, dd, J=18.0, 8.1Hz, H-1613); 2.55(1H, ddd, ,/=9.9, 8.1, 8.3Hz, H-17); 0.64(3H, s, H3-18); 1.37(3H, s, H3-19); 1.80(1H, m, H-20); 1.11(3H, d, J=7.0Hz, H321); 1.80(1H, m, H-22); 1.30(1H, m, H-22); 2.41(1H, ddd, J=15.8, 9.2, 5.5Hz, H-23); 2.27(1H, ddd, J=15.8, 8.8, 7.0Hz, H-23); 1.04(3H, s, H3-25); 0.90(3H, s, H3-26); 1.69(3H, s, H3-27); and 3.66ppm (3H, s, COOCH3). Mass Spectrum: HREIMS: 488.2766(M+, C2sH4oO7, 10%; calcd 488.2775), 304(100), 191(21), 129(35), 95(21), 69(26), 55(43), and 43m/e (78). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

12.

Lucidenic

Acids

421

Common/Systematic Name Lucidenic acid M 11-Oxo-313,7a, 15a-trihydroxy-5a-lanost-8-en-24-oic acid Molecular Formula/Molecular Weight C27H4206; M W -" 462.29814

COOH 0 _

HO

.....OH

.....OH

General Characteristics Methyl ester: a syrup. Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of Ganoderma lucidum were extracted with ethanol. The extract was concentrated and partitioned between chloroform and water. The acidic fraction was obtained and chromatographed on a silica gel column eluted with chloroformmethanol (49:1, v/v; fractions 1-11), chloroform-methanol (9:1, v/v; fraction 12) and methanol (fraction 13). Fraction 12 was subjected to reversed-phase LC (methanol-water, 13:7, v/v, Lobar RPls) and separated into four fractions (12a-12d). Methyl ganoderate M was isolated from the methylated fraction 12d after a combination of silica gel column chromatography (chloroform-methanol, 9:1), preparative TLC (chloroform-acetone, 2:1, v/v) and reversed-phase HPLC (methanol-water, 3:2, v/v, ktBondapak Cls). Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data

(Methyl ester)

UV:

~, MooH 255nm (e = 7,900). max

422

12.

Lucidenic Acids

~H NMR: (CDCI3) 3.00(1H, ddd, J=13.2, 3.5, 3.5Hz, H-l[3); 3.32(1H, dd, J=l 1.1, 4.7Hz, H3a); 4.55(1H, m, H-613); 2.76(1H, d, J=17.6Hz, n-12a); 2.40(1H, d, J=17.6Hz, H1213); 4.58(1H, dd, J-19.9, 5.1Hz, n-15[~); 0.84(3H, s, H3-18); 1.06(3H, s, H3-19); 0.85(3H, d, J=5.8Hz, H3-21); 1.05(3H, s, H3-25); 0.84(3H, s, H3-26); 1.28(3H, s, H327); and 3.67ppm (3H, s, COOCH3). Mass Spectrum: HR IMS: 476.3105(M +, C28H4406, 100%; calcd 476.3139), 458(37), 336(56), 236(53), 69(58), 55(92), and 43m/e (96). Reference T. Nishitoba, H. Sato, and S. Sakamura; Triterpenoids from the Fungus Ganoderma lucidum; Phytochemistry, Vol. 26, pp. 1777-1784(1987).

Lucidones Lucidone A Lucidone B Lucidone C

423

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13.

Lucidones

425

Common/Systematic Name Lucidone A Molecular Formula/Molecular Weight C24H3405; M W -- 4 0 2 . 2 4 0 6 2

0

0 HO'~~ _

0

General Characteristics Lucidone A was isolated as a pale yellow amorphous powder; mp., 280.3~ (c=0.2, in CHC13).

[(g]D24 +210

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with chloroform and chromatographed on a silica gel column using various concentrations of methanol in chloroform. The bitter principles were further purified with a second silica gel column eluted in a like manner. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy, also bitter tasting. Spectral Data UV:

~, max EtOH 256nm (e=l 1,320). IR:

(KBr) 3450, 2960, 2925, 1720(sh), 1715, and 1660cm"1. 1H NMR: (CDC13) 4.82(1H, dd, J=9.1 and 8.5 Hz); 3.21(1H, dd, J--ll.0 and 5.2Hz); 3.02(1H, dq, J=16.2 and 0.7 Hz); 2.77(1H, d, J=16.2); 2.22(3H, s); 1.41(3H, s); 1.22(3H, s); 1.04(3H, s); 0.88(3H, s); and 0.85ppm (3H, s).

426

13.

Lucidones

13CN M R : (CDCI3) 215.8 (s); 204.8 (s); 196.3 (s); 156.2 (s); 142.6 (s); 78.1 (d); 66.6 (d); 31.2 (q); 28.1 (q), 24.5 (q); 19.0 (q); 18.3 (q); and 15.3ppm (q). These data closely resemble those of ganoderic acids and lucidenic acids, although lucidone A differs from them in having twenty-four carbons. The signals at 2.22(3H, s) in the IH NMR spectrum and 31.2 (q) in the ~3C NMR spectrum indicated the presence of an acetyl group at C(17) instead of the side chains of ganoderic acids and lucidenic acids. Mass Spectrum: The molecular formula was assigned as C2&I3405 based on HRMS (M +, 402.2428; ealed for C2,I-I34Os, 402.2407). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids from Ganoderma lucidum and Their Bitterness; Agile. Biol. Chem., Vol. 49, pp. 1547-1549(1985).

13.

Lucidones

427

Common/Systematic Name Lucidone B 7[3-Hydroxy-4,4,14a-trimethyl-3,11,15,20-tetraoxo-5 a-pregn-8-ene Molecular Formula/Molecular Weight C24H3205; M W "- 4 0 0 . 2 2 4 9 7

0 0

0

H

General Characteristics Lucidone B was crystallized from EtOAc as colorless prisms; mp., 270-271~ +278 ~ (c=0.2, in CHC13).

[a]D 22

Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with chloroform and chromatographed on a silica gel column using various concentrations of methanol in chloroform. The bitter principles were further purified with a second silica gel column eluted in a like manner. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy, also bitter tasting. The intensity of bitterness was evaluated to be lucidenic acid D, ganoderic acid C, lucidone A, lucidenic acid A, ganoderic acid B, lucidenic acid B, lucidenic acid C, and lucidenic acid E. Lucidenic acid A, ganoderic acid C, lucidenic acid D, and lucidone A had intense bitterness and their threshold values were determined to be 2.0 x 106M, 5 x 10SM, 5 x 10I~ and 1 x 106M, respectively, when the organoleptic test was carried out by an ascending series of concentrations in a 10% ethanol solution. Soectral Data UV;

~. MoOH 255nm (e=7,900). max

428

13.

Lucidones

IR~

(KBr) 3500, 2960, 1730, 1705, and 1660em"~. IH NMR and 13CNMR: The 1H and 13CNMR spectra oflucidone B were very similar to those oflueidone A, but the 1H-NMR data for lueidone B did not show any signal around 3.21 (H-3) of lueidone A. The 13C-NMR data also did not show any signal around 78.1(C-3) of lueidone A], but gave a new signal at 216.2. These facts indicated the presence of a carbonyl group at C-3 instead of the hydroxy group. Thus, lueidone B was confirmed to be 713-hydroxy-4,4,14tx-trimethyl-3,11,15,20-tetraoxo-50t-pregn-8-ene. Mass Spectrum: It had a molecular formula C24H3205from HRMS data (M +, 400.2267; caled for C24I-I3205, 400.2251). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids from Ganoderma lucidum and Their Bitterness; Agrie. Biol. Chem., Vol. 49, pp. 1547-1549(1985).

13.

Lucidones

429

Common/Systematic Name Lucidone C 313,713,15a-Trihydroxy-4,4,14a-trimethyl- 11,20-dioxo-5 a-preg-8-ene Molecular Formula/Molecular Weight C24H3605; M W -- 404.25627

O

"OH HO

H

General Characteristics Colorless syrup; [a]D24 -1- 145~ (C=0.2, in MeOH). Fungal Source Fruiting bodies of Ganoderma lucidum. Isolation/Purification Dried chipped fruiting bodies of G. lucidum were extracted with ethanol; the extract was partitioned between chloroform and water. The chloroform layer was concentrated and separated into its acidic and neutral fractions. The acidic fraction was separated into thirteen fractions. Fraction 12 was subjected to Lobar column (RP8, Merck) chromatography and the second fraction was treated with diazomethane. The resulting product was rechromatographed on silica gel and the Lobar (RPs) column to give methyl ganoderate L, lucidone C, and methyl lucidenate G. The fourth fraction in the chromatography of Ft. 12 was purified on a silica gel column, preparative TLC and HPLC to give ganolucidic acid D. Biological Activity Fruiting bodies of G. lucidum produce some physiological effects when used as a home remedy. Spectral Data UV: ,~ MeOH max

255nm (e=7680).

IR:

(KBr) 3500(sh), 3430, 1700, and 1660cm1.

430

13.

Lucidones

1H NMR: (CsDsN) 5.34(IH, dd, J=9.5 and 7.:3 Hz); 4.98(IH, dd, J=9.5 and 7.3I-Iz); 3.51(IH, dd, J=I0.8 and 5.3Hz); 2.11(3H, s); 1.59(3H, s); 1.51(3H, s), 1.29(3H, s); I.II(3H, s), and 1.07ppm (3H, s). 13C NMR: (CsDsN) 207.6(0); 198.9(0); 159.9(0); 141.9(0); 77.5(1); 72.4(1); and 69.4ppm (1).

Mass Spectrum: HR IMS: 404.2527m/e (NC). Reference T. Nishitoba, H. Sato, and S. Sakamura; New Terpenoids, Ganolucidic Acid D, Ganoderic Acid L, Lucidone C, and Lucidenic Acid G from the Fungus Ganoderma lucidum; Agric. Biol. Chem., Vol. 50, pp. 809-811 (1986).

Azasterols Antibiotic Antibiotic Antibiotic Antibiotic Antibiotic Antibiotic Antibiotic

A25822A A25822B A25822D A25822L A25822M A25822N A25822H

431

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14.

Azasterols

433

Common/Systematic Name Antibiotic A25822A 4,4-Dimethyl-313-hydroxy-24-methylene- 14a-aza-D-homo- 5 a-cholesta- 8( 9), 14( 14a)diene Molecular Formula/Molecular Weight C30I--I49NO; M W

HO

.

= 439.38142

-

General Characteristics Crystals; mp., 147~ [tg]D 25 - 72 ~ (c=1.15, in MeOH). pKa, = 8.0 [66% DMF]. Dihydrodiacetate derivative: crystals; mp., 130-132~ Fungal Source

Geotrichumflavo-brunneum (NRRL 3862).

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated, adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate (extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H, and L. Extract A was dissolved in acetone (50ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with n-hexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The later fractions containing antibiotic A25822D were dissolved in chloroform and chromatographed on a silica gel column eluted with a linear gradient from chloroform to chloroform-40% methanol. The antibiotic A25822D containing fractions were combined and concentrated to yield the antibiotic as a dry foam of amorphous material that could not be crystallized. Biological Activity Broad spectrum antifungal activity.

434

14.

Azasterols

.Spectral Data UV~

,~ EtOHmax239nm (6=12,600). IR;

(CHCI3) 3584(OH) and 1621 cml. Mass Spectrum: HREIMS: 439.3776m/e (M+). References J. W. Chamberlin, M. O. Chaney, S. Chen, P. V. Demarco, N. D. Jones, and J. L. Occolowitz; Structure of Antibiotic A25822 B, A Novel Nitrogen-containing C2s-Sterol With Antifungal Properties; J. Antibiotics, Vol. 27, pp. 992-993(1974). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 229(1983).

14.

Azasterols

435

Common/Systematic Name Antibiotic A25822B 3 ~-Hydroxy-24-methylene- 14a-aza-D-homo-5a-cholesta-8(9), 14(14a)-diene Molecular Formula/Molecular Weight C28H45NO, MW = 411.35012 II ~ " '

NO"

~_-

H

General Characteristics Crystals; mp., 115-118 ~ C; [ a ] D 25 - 20 ~ (c=0.775, in MeOH). pKa. = 8.4 [66% DMF]. Dihydro-diacetate derivative: crystals; mp., 130-132 ~C. Fungal Source

Geotrichumflavo-brunneum (NRRL 3862)

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated, adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate (extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H, and L. Extract A was dissolved in acetone (50ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with n-hexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The later fractions containing antibiotic A25822D were dissolved in chloroform and chromatographed on a silica gel column eluted with a linear gradient from chloroform to chloroform-40% methanol. The antibiotic A25822D containing fractions were combined and concentrated to yield the antibiotic as a dry foam of amorphous material that could not be crystallized. Biological Activity Broad spectrum antifungal activity, primarily against Candida and Trichophyton.

436

14.

Azasterols

Soectral Data UV:

~

EtOH max

238nm (e=12,300); shift to 277nm (e=12,400) in acidic solution.

IR:

(CHCI3) 3571 and 1618cm "1. Mass Spectrum:

HREIMS" 411.3497role (M+).

References L. D. Boeck, M. M. Hoehn, J. E. Westhead, R. K. Wolter, and D. N. Thomas, New Azasteroidal Antifungal Antibiotics from Geotrichumflavo-brunneum. I. Discovery and Fermentation Studies; J. Antibiotics, Vol. 28, pp. 95-101(1975). J. W. Chamberlin, M. O. Chaney, S. Chen, P. V. Demarco, N. D. Jones, and J. L. Occolowitz; Structure of Antibiotic A25822 B, A Novel Nitrogen-containing C2rSterol with Antifungal Properties; J. Antibiotics; Vol. 27, pp. 992-993(1974). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 229 (1983).

14.

Azasterols

437

Common/Systematic Name Antibiotic A25822D 313, 15-Dihydroxy-24-methylene- 14a-aza-D-homo-5a-cholesta-8(9), 14(14a)-diene Molecular Formula/Molecular Weight C25I-I45NO2, M W = 427.34503

H H, HO

A

General Characteristics An amorphous material that could not be crystallized; [a]D25 + 39 ~ (C=0.722, in MeOH). pKa 7.7 (66% DMF). Fungal Source

Geotrichumflavo-brunneum.

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated, adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate (extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H, and L. Extract A was dissolved in acetone (50ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with n-hexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The later fractions containing antibiotic A25822D were dissolved in chloroform and chromatographed on a silica gel column eluted with a linear gradient from chloroform to chloroform-40% methanol. The antibiotic A25822D containing fractions were combined and concentrated to yield the antibiotic as a dry foam of amorphous material that could not be crystallized. Biological Activity Antibiotic activity against a broad spectrum of pathogenic fungi, including Candida spp. and Trichophyton spp.

438

14.

Azasterols

Spectral Data UV;

~

EtOH max

235nm (neutral and basic pH, e = 10,400); 270nm (acidic pH, e = 8,600).

IR~

(CHCI3) 3600(OH), 1640(-C=C -, -C=N-), and 890cm "1 (exocyclic methylene). Mass Data: LREIMS: 427(M+). Found: C, 78.40; H, 10.28; N; 3.17, O, 8.06%; calcd for C2sI-hsNO2: C, 78.40; H, 10.54; N, 3.28" O, 7.49%. TLC Data Silica gel plates developed with diethyl ether-ethanol (31, v/v); Rf = 0.27. GC Data 3% OV-17 coated onto Chromosorb W; column temperature 275~ flash heater 285~ flow rates, hydrogen 47ml/min, helium 75ml/min, and air 350ml/min; relative retention time of 8.8 (relative to cholesterol). References K. H. Michel, R. L. Hamill, S. H. Larsen, and R. H. Williams; New Azasteroidal Antifungal Antibiotics from Geotrichumflavo-brunneum;J. of Antibiotics, Vol. 28, pp. 102-111(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, pp. 329 and 332(1983).

14.

Azasterols

439

Common/Systematic Name Antibiotic A25822L 313-Hydroxy-24-methylene- 14a-aza-D-homo-Sa-cholesta-8(9), 14(14a)-dien- 15-one Molecular Formula/Molecular Weight C25I-I43NO2, M W = 4 2 5 . 4 2 9 3 8

H General Characteristics An amorphous powder that could not be crystallized; [a]D25 + 75 ~ (C=0.072, in MeOH). pK, 4.75 (66% DMF). Fungal Source

Geotrichumflavo-brunneum.

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated, adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate (extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H and L. Extract A was dissolved in acetone (50 ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with nhexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The later fractions containing antibiotic A25822D were dissolved in chloroform and chromatographed on a silica gel column eluted with a linear gradient from chloroform to chloroform-40% methanol. The antibiotic A25822L containing fractions were dissolved in a minimal amount of chloroform and chromatographed on a Woelm activity III column eluted with chloroform and the A25822L containing fractions concentrated to yield the antibiotic as an amorphous powder.

440

14.

Azasterols

Biological Activity Antibiotic activity against a broad spectrum of pathogenic fungi, including Candida spp. and Trichophyton spp. Spectral Data UV~

~, EtoH 262nm (neutral and basic pH, e = 10,200); 278nm (acidic pH, e = 10,800). max IR~

(CHCI3) 3620(OH), 1670-1685(amide), and 890cmq (exocyclic methylene). ORD" [a]36025 -270 ~ MeOH).

[~]27225 +7550 ~

[tt]23525-3500 ~ and

[a]20625 +16,700 ~ (c=0.072, in

Mass Data: LREIMS: 425(M+); found: C, 78.66; H, 9.74; N; 3.06, O, 8.37%; calcd for C2sH43NO2: C, 79.01; H, 10.18; N, 3.29; O, 7.52%. TLC Data Silica gel plates developed with diethyl ether-ethanol (3:1, v/v); Re = 0.67. GC Data 3% OV-17 coated onto Chromosorb W; column temperature 275~ flash heater 285~ flow rates, hydrogen 47ml/min, helium 75ml/min, and air 350ml/min; relative retention time of 8.4 (relative to cholesterol). References K. H. Michel, R. L. Hamill, S. H. Larsen, and R. H. Williams; New Azasteroidal Antifungal Antibiotics from Geotrichumflavo-brunneum; J. of Antibiotics, Vol. 28, pp. 102-111(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, ppo 329 and 332(1983).

14.

Azasterols

441

Common/Systematic Name Antibiotic A25822M 313-Acetoxy-24-methylene- 14a-aza-D-homo-5a-cholesta-8(9), 14(14a)-diene Molecular Formula/Molecular Weight C3oH47NO2; M W -- 4 5 3 . 3 6 0 6 8

H

AcO

General Characteristics A yellowish amorphous powder;

[ ~ ] D 25 -

15 ~ (c=0.021, in MeOH). pKa 7.9 (66% DMF).

Funsal Source

Geotrichumflavo-brunneum.

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated, adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate (extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H, and L. Extract A was dissolved in acetone (50ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with n-hexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The antibiotic A25822M and A25822N containing fraction was dissolved in acetone and placed at -20~ that formed a white crystalline precipitate of A25822N. The filtrate was reduced to an oily residue which was dissolved in a minimal amount of chloroform and chromatographed on a column containing Woelm basic alumina deactivated with 6% water and eluted with chloroform. The antibiotic A25822M containing fraction was dissolved in benzene and lyophilized to give a yellowish amorphous powder that could not be crystallized. Biological Activity Antibiotic activity against a broad spectrum of pathogenic fungi, including Candida spp. and Trichophyton spp.

442

14.

Azasterols

Spectral Data UV: ~, mEtOH ax

239nm (neutral and basic pH, e = 13,900); 278nm (acidic pH, e = 14,200).

IR:

(CHCI3) 1730(carbonyl), 1625(-C=C-, -C=N-), and 890cm 4 (exocyclic methylene). Mass Data: LREIMS: 453(M+); found: C, 79.57; H, 10.26; N; 2.92, O, 7.21%; calcd for C30I-LtTNO2: C, 79.42; H, 10.44; N, 3.09; O, 7.05%. TLC Data Silica gel plates developed with diethyl ether-ethanol (3:1, v/v); Re = 0.42. GLC Data 3% OV-17 coated onto Chromosorb W; column temperature 275~ flash heater 285~ flow rates, hydrogen 47ml/min, helium 75ml/min, and air 350ml/min; relative retention time of 9.2 (relative to cholesterol). References K. H. Michel, R. L. Hamill, S. H. Larsen, and R. H. Williams; New Azasteroidal Antifungal Antibiotics from Geotrichumflavo-brunneum; J. of Antibiotics, Vol. 28, pp. 102-111(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, pp. 329 and 332(1983).

14.

Azasterols

443

Common/Systematic Name Antibiotic A25822N 3-Keto-24-methylene-14a-aza-D-homo-5a-cholesta-8(9),14(14a)-diene Molecular Formula/Molecular Weight C28H43NO; MW' = 409.33447

General Characteristics A white crystalline precipitate; mp., 165~ (66% DMF).

[a]D2S -14 ~ (C=0.05, in MeOH). pK, 7.45

Fungal Source

Geotrichumflavo-brunneum.

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated, adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate(extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H, and L. Extract A was dissolved in acetone (50ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with n-hexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The antibiotic A25822M and A25822N containing fraction was dissolved in acetone and placed at -20~ that formed a white crystalline precipitate of A25822N. The filtrate was reduced to an oily residue which was dissolved in a minimal amount of chloroform and chromatographed on a column containing Woelm basic alumina deactivated with 6% water and eluted with chloroform. The antibiotic A25822M containing fraction was dissolved in benzene and lyophilized to give a yellowish amorphous powder that could not be crystallized.

444

14.

Azasterols

Biological Activity Antibiotic activity against a broad spectrum of pathogenic fungi, including Candida spp. and Trichophyton spp. Spectral Data UV;

~

EtOH max

239nm (neutral and basic pH, e = 13,900); 278nm (acidic pH, e = 14,100).

IR:

(CHCI3) 1715(carbonyl), 1620(-C=C -, -C=N-), and 890cm"~ (exocyclic methylene). Mass Data: LREIMS: 409(M+); found: C, 81.82; H, 10.61; N; 3.53, O, 3.91%; calcd for C2~q43NO C, 82.16; H, 10.52; N, 3.43" O, 3.89%. TLC Data Silica gel plates developed with diethyl ether-ethanol (3:1, v/v); Re = 0.40. GLC Data 3% OV-17 coated onto Chromosorb W; column temperature 275~ flash heater 285~ flow rates, hydrogen 47ml/min, helium 75ml/min, and air 350ml/min; relative retention time of 7.1 (relative to cholesterol). References K. H. Michel, R. L. Hamill, S. H. Larsen, and R. H. Williams; New Azasteroidal Antifungal Antibiotics from Geotrichumflavo-brunneum; J. of Antibiotics, Vol. 28, pp. 102-111(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, pp. 329 and 332 (1983).

14.

Azasterols

445

Common/Systematic Name Antibiotic A25822H (isomeric with Antibiotic A25822L) Molecular Formula/Molecular Weight C2sH43NOz; MW = 425.32938

Hll HO

General Characteristics An amorphous powder that could not be crystallized; [t~]D25 + 15 ~ (C=0.147, in MeOH). pK, 6.9 (66% DMF). Fungal Source

Geotrichumflavo-brunneum.

Isolation/Purification The mycelium of Geotrichumflavo-brunneum was extracted with methanol, concentrated,. adjusted to pH 10.0, and extracted with ethyl acetate. The organic phase was concentrated, partitioned between 2.5N aqueous sodium hydroxide and ethyl ether, washed with water, and extracted with 0.25N sulfuric acid. The aqueous acidic phase was adjusted to pH 10.0 with sodium hydroxide and extracted with n-hexane (extract A) followed by ethyl acetate (extract B). Extract A contained antibiotic A25822A, B, M, and N. Extract B contained antibiotics A25822D, H, and L. Extract A was dissolved in acetone (50ml) and n-hexane (150ml) and the clear solution was adjusted to one liter with n-hexane. Antibiotic A25822A crystallized in small white needles. The filtrate was combined with extract B and reduced in Volume to give extract C. Extract C was dissolved in a minimal volume of ethyl acetate-n-hexane-water (80:16:4, v/v/v) and chromatographed on a column of basic alumina eluted with the same solvent combination. The fractions containing antibiotic A25822B and H were dissolved in chloroform and chromatographed on a Woelm basic alumina column eluted with chloroform. The antibiotic A25822H containing fractions were dissolved in a minimal amount of acetone and chromatographed on preparative silica gel F-254 plates developed with diethyl etherethanol (3:1) followed by silica gel column chromatography eluted with ethyl acetate to yield the antibiotic as a white amorphous powder. Biological Activity Antibiotic activity against a broad spectrum of pathogenic fungi, including Candida spp. and Trichophyton spp.

446

14.

Azasterols

Spectral Data UV;

~

EtOH max

235nm (neutral and basic pH, e = 10,000); 270nm (acidic pH, e = 8,700).

IR;

(CHCI3) 3550(OH), 1625(-C=C -, -C=N-), and 890cm "1 (exocyclic methylene). Mass Data: LREIMS: 443(M + H20, 9 425m/e + H20); found: C, 74.50; H, 10.15; N; 3.54, O, 11.36%; calcd for C2ffI43NO2 + H20" C, 75.84; H, 10.20; N, 3.16; O, 10.80%. TLC Data Silica gel plates developed with diethyl ether-ethanol (31, v/v); Re = 0.54. GLC Data 3% OV-17 coated onto Chromosorb W; column temperature 275~ flash heater 285~ flow rates, hydrogen 47ml/min, helium 75ml/min, and air 350ml/min; relative retention time of 8.4 (relative to cholesterol). References K. H. Michel, R. L. Hamill, S. H. Larsen, and R. H. Williams; New Azasteroidal Antifungal Antibiotics from Geotrichumflavo-brunneum; J. of Antibiotics, Vol. 28, pp. 102-111(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, pp. 329 and 332 (1983).

Fasciculols Fasciculol A Fasciculol B Fasciculol C Fasciculol C depsipeptide E Fasciculol C depsipeptide F Fasciculol B depsipeptide

447

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15.

Fasciculols

449

Common/Systematic Name Fasciculol A 2a,313,24(R),25-T etrahydroxylanost-8-ene Molecular Formula/Molecular Weight C30H5204;/V[V~ = 476.38656 OH ~H

General Characteristics Crystals; mp., 209~ [a]D21 +50.7 ~ (c=l.0, in MeOH). Triacetate 177~ [a]D2~ +7.7 (C=I.0, in MeOH).

(C36H5807);mp.,

Fungal Source Fruit bodies of Neamatolomafasciculare. Isolation/Purification Fruit bodies were extracted with ethyl acetate and the neutral fraction was applied to a silica gel column (chloroform-ethanol, 100:4, v/v), followed by preparative TLC with Kieselgel GF254 plates and chloroform-ethanol, 10:1 (v/v) as developing solvent. Also see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Biological Activity Inhibited the growth of Chinese cabbage seedlings. Spectral Data UV:

~, Moon 207(6 = 5,700). max

450

15.

Fasciculols

IR;

(KBr) 3380cm ~ (no carbonyl bands); triacetate 3560 and 1736cm~ ~H NMR: (pyridine-ds) 0.77; 0.90; 1.08; 1.11; 1.22; (each 3H, s); 0.93; (3H, d, J=6Hz); 1.46(6H, s); 3.30(1H, d, J=10Hz); 3.62(1H, br s); and 3.96ppm (1H, td, J=10, 10, 4.5Hz). Triacetate: (CDCI3) eight methyl signals in the region from 0.72 to 1.20ppm; three acetyl methyl signals at 1.99, 2.01, and 2.10; acetoxy methine protons at 5.14(1H, td, J=10, 10, 4.5ppm); 4.72(1H, d, J=10Hz) and 5.0-4.7ppm (1H, m, overlapping with other signals).. Mass Spectrum: LREIMS: 476(M+), 331,289, 215, 187, 173, and 145m/e. TLC Data Kieselgel GF2s4 plates were developed with chloroform-ethanol, 10:1 (v/v). Fasciculol A had an Re of 0.85 and was detected by spraying plates with 0.5% vanillin in concentrated H2SO4, followed by heating at 120 ~ References M. Ikeda, Y. Sato, M. Izawa, T. Sassa, and Y. Miura; Isolation and Structure of Fasciculol A, a New Plant Growth Inhibitor from Neamatolomafasciculare; Agric. Biol. Chem., Vol. 41, pp. 1539-1541(1977). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc. New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, p. 319 (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

15.

Fasciculols

451

Common/Systematic Name Fasciculol B 2et,313,12a,24(R),25-Pentahydroxylanost-8-ene Molecular Formula/Molecular Weight C3oH5205; MW = 492.38148 OH lh

OH ---

......

H

HOII, HO

General Characteristics Crystals; mp., 235~ [a]D21 +76.5 ~ (c=l.0, in MeOH). Positive Liebermann-Burchard reaction and tetranitromethane test. Triacetate (C36H5608);mp., 191 ~C. Fungal Source Fruit bodies of Neamatolomafasciculare. Isolation/Purification Fruit bodies were extracted with ethyl acetate and the neutral fraction was applied to a silica gel column (chloroform-ethanol, 100:7, v/v). Further purification and separation of fasciculol B from fasciculol D was achieved by silica gel column chromatography by elution with ethyl acetate-ethanol (100: 4, v/v) for fasciculol B and (100:13, v/v) for fasciculol D. Final purification was by preparative TLC with Kieselgel GF254 plates and chloroform-ethanol, 20:3 (v/v) as developing solvent. Also see R. C. Heupel; Isolation and Primary Characterization of Sterols, In Analysis of Sterols and Other Biologically Significant Steroids; W. D. Nes and E. J. Parish (eds.), Academic Press Inc., New York, NY, 341 pp. (1989). Biological Activity Inhibited the growth of Chinese cabbage seedlings. Spectral Data UV: ~. MCOHmax 207(e = 6,000).

452

15.

Fasciculols

IR~ (KBr) 3400cm q (no carbonyl bands); triacetate 3560, and 1736cmq IH ~ :

(pyridine-ds)0.78; 1.13; 1.16; 1.26; 1.43;(each 3H, s); 1.39;(3H, d, J=6I-Iz);1.48(6H, s); 3.34(IH, d, J=101-Iz);3.74 (IH, br s);4.09(IH, td,J=10, 10, 4.5Hz); and 4.22ppm (IH, d, J=6.0Hz). Triacetate:(CDCI3) Three acetoxy methine protons at 4.72(IH, d, J=10Hz), 5.14(IH, td,J=10, 10, 4.5Hz), and 5.0-4.7ppm (IH, m, overlappingwith other signals). 13CNMR: (pyridine-ds) C-l, 44.7 t; C-2, 68.9 d, C-3, 83.5 d; C-4, 39.7 s*; C-5, 51.0 d; C-6, 18.7 t; C-7, 28.3 t; C-8, 134.9 s; C-9, 133.6 s; C-10, 38.3 s*; C-11, 26.7 t; C-12, 72.0 d; C-13, 49.9 s; C-14, 49.9 s; C-15, 32.6 t**; C-16, 29.1 t**; C-17, 43.4 d; C-18, 16.7 q***; C-19, 20.4 q; C-20, 36.8 d; C-21, 18.0 q; C-22, 34.7 t****; C-23, 34.2 t; C-24, 79.1 d; C-25, 72.7 s; C-26, 26.1 q*****; C-27, 25.8 q*****; C-28, 25.2 q*****; C29, 29.1 q*****; and C-30, 17.4ppm q**** *, **, ***, ****, ***** Assignments may be interchanged. Mass Spectrum: LREIMS: 492(M+), 187, and 145role. TLC Data Kieselgel GF2s4 plates were developed with chloroform-ethanol, 20:3 (v/v). Fasciculol B had an Re of 0.3 (dark green spot) and fasciculol D was at Re 0.7 (dark green spot). Detection was by spraying plates with 0.5% vanillin in concentrated H2SO4, followed by heating at 120 ~C. References M. De Bernardi, G. MeUerio, G. Vidari, P. Vita-Finzi, G. Fronza, M. Kocbr, and J. St. Pyrek; Fungai Metabolites. IX. Triterpenes from Neamatoloma sublateritium; J. Natural Products, Vol. 44, pp. 351-356(1981). M. Ikeda, Y. Sato, M. Izawa, T. Sassa, and Y. Miura; Isolation and Structure of Fasciculol A, a New Plant Growth Inhibitor from Neamatolomafasciculare; Agile. Biol. Chem., Vol. 41, pp. 1539-1541(1977). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc. New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, p. 319 (1983). J. D. Weete; Structure and Function of Sterols in Fungi, Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

15.

Fasciculols

453

Common/Systematic Name Fasciculol C 2a,313,12a,21,24(R),25-Hexahydroxylanost-8-ene Molecular Formula/Molecular Weight C3oH5206; M W -- 508.37639

OH HOH2C_ OH ! I ~~~'OH

HO S

HO-

General Characteristics Crystals; mp., 244~

[a]D 2] + 67.5~ triacetate (C36H5608), mp., 96-97~

Fungal Source Fruit bodies of Neamatolomafasciculare. Isolation/Purification Fruit bodies were extracted with ethyl acetate and the neutral fraction was applied to a silica gel column (chloroform-ethanol, 100:7, v/v). Further purification and separation was achieved by silica gel column chromatography by elution with ethyl acetate-ethanol (100:4, v/v) and (100:13, v/v). Final purification was by preparative TLC with Kieselgel GF254 plates and chloroform-ethanol, 20:3 (v/v) as developing solvent; detected at Re 0.2 as a dark purple spot by spraying with 0.5% vanillin in concentrated sulfuric acid and heating at 120 ~C. Biological Activity Inhibited the growth of Chinese cabbage seedlings. Spectral Data UV:

MeOH 207(E = 6,000). max

IR:

(KBr) 3400cm 1 (no carbonyl bands); tetraacetate 3400 and 1730cm].

454

15.

Fasciculols

IHNMR (pyridine-ds) 0.72; 1.10; 1.13; 1.26; 1.37(each 3H, s); 1.48(6H, s); 3.34(1H, d, J=10Hz); and 3.6-4.4ppm (5H, m). ~3C NMR: (pyridine-ds) C-I, 44.4 t; C-2, 68.9 d; C-3, 83.5 d; C-4, 39.6 s'; C-5, 51.0 d; C-6, 18.7 t; C-7, 28.3 t'~ C-8, 134.7 s; C-9, 133.9 s; C-10, 38.4 s'; C-11, 26.8 t; C-12, 73.1 d; C-13, 50.6 s'"; C-14, 50.2 s'"; C-15, 32.3 t"; C-16, 29.7 t'*; C-17, 38.6 d; C-18, 17.1 q ; C-19, 20.3 q; C-20, 44.0 d; C-21, 61.2 q; C-22, 28.1 t; C-23, 33.1 t; C-24, 78.9 t; C-25, 72.6 d; C-26, 26.0 q ..... C-27, 9 25.7 q ..... ; C-28, 24.1 q ..... ; C-29, 29.0 q ..... ; and C-30, 17.4ppm q .... ~176176

. . . .

Assignments may be interchanged.

Mass Spectrum: LREIMS: 508(M+), 347, 305, 203, and 161role. TLC Data Kieselgel GF2s4 plates were developed with chloroform-ethanol, 20:3 (v/v). Fasciculol C had an Re of 0.2 and appeared as a dark purple spot after spraying the TLC plate with 0.5% vanillin in concentrated sulfuric acid and heating at 120~ References M. De Bernardi, G. Mellerio, G. Vidari, P. Vita-Finzi, G. Fronza, M. Kocbr, and J. St. Pyrek; Fungal Metabolites. IX. Triterpenes from Neamatoloma sublateritium; J. Natural Products, Vol. 44, pp. 351-356(1981). M. Ikeda, G. Niwa, K. Tohyama, T. Sassa, and Y. Miura; Structures of Fasciculol C and Its Depsipeptides, New Biologically Active Substances From Neamatolomafasciculare; Agile. Biol. Chem., Vol. 41, pp. 1803-1805(1977). W. B. Turner and D.C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, New York, p. 319 (1983).

15. Fasciculols

455

Common/Systematic Name Fasciculol C depsipeptide E Molecular Formula/Molecular Weight. C39H65NOll; MW = 723.45576

HOH2C%..... ~ H.,~H / HC~ ~ v "~0 H O HO OH oHO"~ . ~ .A ~ II I II H2-/-C H2-C MeO-C-CH2-N-C-C /

_

Me

General Characteristics Crystals; mp., 105-106~

[a]D 21 +30.6 ~ Positive ninhydrin test.

Fungal Source Fruit bodies of Neamatolomafasciculare. Isolation/Purification Fruit bodies were extracted with ethyl acetate and the neutral fraction was applied to a silica gel column (chloroform-ethanol, 100:7, v/v). Further purification and separation was achieved by silica gel column chromatography by elution with ethyl acetate-ethanol (100:4, v/v) and (100:13, v/v). Final purification was by preparative TLC with Kieselgel GF254 plates and chloroform-ethanol, 20:3, v/v, as developing solvent; detected at Re 0.2 as a dark purple spot by spraying with 0.5% vanillin in concentrated sulfuric acid and heating at 120~ Biological Activity. Inhibited the growth of Chinese cabbage seedlings. Spectral Data UV: EtOH max

208(e

=

5,000).

IR:

(KBr) 3600-3300, 1730, 1655, and 1530cm"1. IH NMR: (CDCI3) (acetonide) 0.59(3H, s); 0.87(6H, s); 1.06(9H, s); 1.22(3H, s); 1.3 l(3H, s); 1.39(6H, s); 2.65(2H, br d, J=l-2Hz); 2.73(2H, s); 3.72(3H, s); 4.00(2H, d, J=5Hz); 4.57(1H, d, J=10Hz); and 7.51ppm (1H, t, J=5Hz).

456

15.

Fasciculols

TLC Data Kieselgel GF254plates were developed with chloroform-ethanol, 20:3 (v/v). Fasciculol C depsipeptides had an Rf of 0.25 (dark purple spot) and Rf 0.5 (dark purple spot). Detection was by spraying plates with 0.5% vanillin in concentrated H2SO4, followed by heating at 120 ~C. Reference M. Ikeda, G. Niwa, K. Tohyama, T. Sassa, and Y. Miura; Structures ofFasciculol C and Its Depsipeptides, New Biologically Active Substances From Neamatolomafasciculare, Agric. Biol. Chem., Vol. 41, pp. 1803-1805(1977).

15. Fasciculols

457

Common/Systematic Name Fasciculol C depsipeptide F Molecular Formula/Molecular Weight C39I-I65NOll; M W - 7 2 3 . 4 5 5 7 6

HOH2C-____. A OH "

OH ~,.

/

0 H0 OH 0 II I II H2_/_C H 2 II- C - O . ~ MeO-C-C H2-N-C-C Me

HO" ~ _ General Characteristics Crystals; mp., 102-103~

[a]D 2] +9.9 ~ Positive ninhydrin test.

Fungal Source Fruit bodies of Neamatolomafasciculare. Isolation/Purification Fruit bodies were extracted with ethyl acetate and the neutral fraction was applied to a silica gel column and eluted with chloroform-ethanol, 100:7 (v/v). Further purification and separation was achieved by silica gel column chromatography by elution with ethyl acetate-ethanol (100: 4, v/v) and (100:13, v/v). Final purification was by preparative TLC with Kieselgel GF2s4 plates and chloroform-ethanol, 20:3 (v/v) as developing solvent; detected at Rf 0.5 as a dark purple spot by spraying with 0.5% vanillin in concentrated sulfuric acid and heating at 120~ Biological Activity Inhibited the growth of Chinese cabbage seedlings. Spectral Data UW:

EtOHmax 208(e-= 5,000). IR:

(KBr) 3600-3300, 1730, 1655, and 1530cm"].

458

15.

Fasciculols

1H ~:

(CDCI3) (acetonide) 0.59(3H, s);0.90(3H, s); 1.09(12H, s); 1.25(3H, s); 1.32(3H, s); 1.40(6H,s);2.65(2H, br d, J=12Hz); 2.73(2H,s);3.24(IH, d, J=IOHz); 4.02(2H, d, J=51-1z);5.00(II-I,td,J=I0, 4.5Hz); and 7.40ppm (IH, t,J=5Hz). TLC Data Kieselgel GF2s4plates were developed with chloroform-ethanol, 20:3 (v/v). Fasciculol C depsipeptide F had an Re of 0.5 (dark purple spot). Detection was by spraying plates with 0.5% vanillin in concentrated H2SO4, followed by heating at 120~ Reference M. Ikeda, G. Niwa, K. Tohyama, T. Sassa, and Y. Miura; Structures ofFasciculol C and Its Depsipeptides, New Biologically Active Substances from Neamatolomafasciculare; Agric. Biol. Chem., Vol. 41, pp. 1803-1805(1977).

15. Fasciculols

459

Common/Systematic Name Fasciculol B depsipeptide Molecular Formula/Molecular Weight C39I-I6sNO10; ~

= 707.46085

OH OH O II

HO I II

OH I

H

O II

MeO-C-CH2-N-C-CH2-C-CH 2 - C - O ~

Me

General Characteristics Crystals; mp., 95-97~

~

_

HO

[tg]D 21 + 1 4 . 1 ~

(c=l.0, in MeOH). Positive ninhydrin test.

Fungal Source Fruit bodies of Neamatolomafasciculare. Isolation/Purification Fruit bodies were extracted with ethyl acetate and the neutral fraction was applied to a silica gel column (chloroform-ethanol, 100:7, v/v). Further purification and separation of fasciculol B from fasciculol D was achieved by silica gel column chromatography by elution with ethyl acetate-ethanol (100:4, v/v) for fasciculol B and (100:13, v/v) for fasciculol D. Final purification was by preparative TLC with Kieselgel GF254plates and chloroform-ethanol, 20:3 (v/v) as developing solvent. Biological Activity Inhibited the growth of Chinese cabbage seedlings and showed weak antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae. Spectral Data UV:

~, maxEt~ 208(e = 3,900). IR:

(KBr) 3600-3400, 1655, and 1530cm"~.

~H NMR: (CDCI3) nine methyl groups in the region 0.62-1.42; a methoxy group at 3.78; a

460

15.

Fasciculols

secondary methylene amino group at 4.07(2H, d, J=5.0Hz); 7.32(1H, t, J=5.0Hz, disappeared on addition of D20); an acyloxy methine proton at 5.14(1H, td, J= 10, 10, 4.5Hz); and a hydroxy methine proton at 3.24ppm (1H, d, dr--10Hz). Six protons in the region of 2.4-4.2 were unassigned. TLC Data Kieselgel GF254 plates were developed with chloroform-ethanol, 20:3, v/v. Fasciculol B had an Re of 0.3 (dark green spot) and fasciculol D was at Rf 0.7 (dark green spot). Detection was by spraying plates with 0.5% vanillin in concentrated H2SO4, followed by heating at 120 ~C. References M. Ikeda, Y. Sato, M. Izawa, T. Sassa, and Y. Miura; Isolation and Structure of Fasciculol A, a New Plant Growth Inhibitor from Neamatolomafasciculare;Agric. Biol. Chem., Vol. 41, pp. 1539-1541(1977). W. D. Nes and E. J. Parish (eds.); Analysis of Sterols and Other Biologically Significant Steroids; Academic Press Inc. New York, NY, 341 pp. (1989). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, Inc., New York, NY, p. 319 (1983). J. D. Weete; Structure and Function of Sterols in Fungi; Advances in Lipid Research, Vol. 23, pp. 115-167(1989).

Fusidanes and Protostanes Virdominic acid A Virdominic acid B Virdominic acid C Cephalosporin P 1 3-Ketofusidic acid 11-Ketofusidic acid 3-Epifusidic acid 11-Epifusidic acid 9,11-Anhydrofusidic acid 9,11-Anhydro- 12-hydroxyfusidic acid 9,11- Anhydro-%t, 110t-epoxyfusidic acid 7,8-Dehydropseudofusidic acid Fusilactidic acid Fusidic acid Helvolic acid

461

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16.

Fusidanes and Protostanes

463

Common/Systematic Name Viridominic acid A Molecular Formula/Molecular Weight C33H4809; M W -- 5 8 8 . 3 2 9 8 3

OOH OAc

HO'"' .... v

v

---

~OH

6Ac

General Characteristics Crystals; mp., 151-155~ [a]D22 + 49 ~ (C=0.1, in MeOH); diketo derivative; mp., 124derivative; mp., 119-121 ~ monoethyl ester; [a]D25 +23 ~ (C=0.1, in 127.5 ~C, dihydrool 9 MeOH). Fungal Source

Cladosporium sp. (501-7y).

Isolation/Purification The culture filtrate was acidified to pH 3 and extracted with ethyl acetate. The extract was washed with water, concentrated and extracted with saturated NaHCO3 solution. The aqueous layer was acidified, extracted with ethyl acetate, dried over anhydrous sodium sulfate and evaporated to a viscous residue. The chloroform soluble fraction was chromatographed on a silicic acid column eluted with benzene and benzene-ethanol (25:1, v/v). Biologically active fractions from the benzene-ethanol eluate were chromatographed on a silicic acid column eluted with benzene-ethanol (100:1, 50:1, and 25:1, v/v). Final purification was by repetitive silica gel column chromatography eluted with chloroform and chloroform-ethanol (50:1, v/v). Biological Activity Chlorosis-inducing activity in higher plants (six day old seedlings of Chinese cabbage Brassica campestris L. var. pekinensis; seven day old seedlings of barnyard grass Panicum crus-galli L. var. frumentaceum).

464

16.

Fusidanes and Protostanes

Spectral Data UV:

~, EtOH 220(sh, e = 6,500) and 290nm (103); monoethyl ester: (r max

~t, EtOH max

217nm

(KBr) 3420(OH), 2660(H-bonded OH), and 1729cm q (carboxylic C=O); monoethyl ester: 3500, 1710(br), 1245, 1020, and 900cm q. ~H NMR:

(CDCI3) Methyl ester, 3.70(brs,H-3); 4.48(d,J=I II-Iz,H-6); 3.50(s,H-7); 3.28(s,H9); 5.84(d,J=8.0Hz, H-16), 5.05(brt,J=8.0Hz, H-24); 0.92(d,J=7.0Hz, secondary methyl); 1.03, 1.28,and 1.47(tertiarymethyls), 1.60, 1.67ppm (vinylmethyls);and 1.95, 2.03ppm (each 3H, s, acetoxy groups).

Mass Spectrum: LREIMS" methyl ester, 533m/e (M+-69). References H. Kaise, K. Munakata, and T. Sassa; Structures of Viridominic Acids A and B, New Chlorosis-inducing Metabolites of a Fungus; Tet. Lett., pp. 3789-3782(1972). H. Kaise, Y. Ogawa, T. Sassa and K. Munakata, Studies on the Chlorosis-Inducing Substances Produced by a Fungus. Part I. Isolation and Biological Activities of Viridominic Acids A, B, C and Cephalosporin P~; Agr. Biol. Chem., Vol. 36, No. 1, pp. 120-124(1972).

16.

Fusidanes and Protostanes

465

Common/Systematic Name Viridominic acid B Molecular Formula/Molecular Weight C33H4sO10; MW = 604.32475

HO

OOMe OAc

HO'"'" v

v

"OH

6Ac _

General Characteristics Amorphous; [a]D 17 + 64 ~ (c=0.37, in MeOH); diketo acetate derivative; mp., 236-240~ Fungal Source

Cladosporium sp. (501-7y).

Isolation/Purification The culture filtrate was acidified to pH 3 and extracted with ethyl acetate. The extract was washed with water, concentrated and extracted with saturated NaHCO3 solution. The aqueous layer was acidified, extracted with ethyl acetate, dried over anhydrous sodium sulfate and evaporated to a viscous residue. The chloroform soluble fraction was chromatographed on a silicic acid column eluted with benzene and benzene-ethanol (25:1, v/v). Biologically active fractions from the benzene-ethanol eluate were chromatographed on a silicic acid column eluted with benzene-ethanol (100:1, 50:1, and 25:1, v/v). Final purification was by repetitive silica gel column chromatography eluted with chloroform and chloroform-ethanol (50:1, v/v). Biological Activity Chlorosis-inducing activity in higher plants (six day old seedlings of Chinese cabbage Brassica campestris L. var. pekinensis; seven day old seedlings of barnyard grass Panicum crus-galli L. var. frumentaceum).

466

16.

Fusidanes and Protostanes

Spectral Data UV:

~.toH 213nm (E=8,000). max

IR;

(KBr) 3580, 3400, 1728(br.), 1250, 1120, and 930cm "~. IH NMR: (CDCI3) Methyl ester, 3.73(br s, H-3); 4.45(d, J=10Hz, H-6); 3.50(s, H-7); 3.39(s, H9); 4.28(d, J=8.0Hz, H-12); 5.73(d, J=8.0Hz, H-16); 5.12(br t, J=7.5Hz, H-24); 0.92(d, J=6.5Hz, secondary methyl); 0.90, 1.32, and 1.56(tertiary methyls); 1.58, 1.65ppm (vinyl methyls); 1.58 and 1.65(each 3H, s), 5.14(br t), isopentenyl group; and 1.98, 2.03ppm (each 3H, s, acetoxy groups). Mass Spectrum: LREIMS: methyl ester, 594role (M+-69). TLC Data Silicic acid plates developed with chloroform-ethanol (8" 1, v/v), Re 0.45. References H. Kaise, K. Munakata, and T. Sassa; Structures of Viridominic Acids A and B, New Chlorosis-inducing Metabolites of a Fungus; Tet. Lett., pp. 3789-3782(1972). H. Kaise, Y. Ogawa, T. Sassa, and K. Munakata; Studies on the Chlorosis-inducing Substances Produced by a Fungus. Part I. Isolation and Biological Activities of Viridominic Acids A, B, C and Cephalosporin P~; Agr. Biol. Chem., Vol. 36, No. 1, pp. 120-124(1972).

16.

Fusidanes and Protostanes

467

Common/Systematic Name Viridominic acid C Molecular Formula/Molecular Weight C33H5009; MW' = 590.34548

OOH ;

-

OAc

6Ac N

General Characteristics Crystals; mp., 168-171~ [a]D 25 198~ lactone; mp., 248-250~

+38 ~

(c=0.1, in MeOH); diacetate derivative; mp., 196-

Fungal Source

Cladosporium sp. (501-7y).

Isolation/Purification The culture filtrate was acidified to pH 3 and extracted with ethyl acetate. The extract was washed with water, concentrated and extracted with saturated NaHCO3 solution. The aqueous layer was acidified, extracted with ethyl acetate, dried over anhydrous sodium sulfate and evaporated to a viscous residue. The chloroform soluble fraction was chromatographed on a silicic acid column eluted with benzene and benzene-ethanol (25:1, v/v). Biologically active fractions from the benzene-ethanol eluate were chromatographed on a silicic acid column eluted with benzene-ethanol (100:1, 50:1, and 25:1, v/v). Final purification was by repetitive silica gel column chromatography eluted with chloroform and chloroform-ethanol (50:1, v/v). Biological Activity Chlorosis-inducing activity in higher plants (six day old seedlings of Chinese cabbage Brassica campestris L. var. pekinensis; seven day old seedlings of barnyard grass Panicum crus-galli L. var. frumentaceum).

468

16.

Fusidanes and Protostanes

Spectral Data UV"

~.EtoH 22Onto(e=8,100). max

IR:

(KBr) 3450, 1715(br), 1265, and 915cm"~. IH NMR: (CDCI3/DMSO-d6) 3.51(br s, H-3), 4.51(d, J=12Hz, H-6); 3.30(s, H-7); 3.70(m, HI 1); 5.74 (d, J=7.5Hz, H-16); 5.05(br t, J-7.5Hz, H-24); 0.77(d, J=6.5Hz, secondary methyl); 0.99, 1.07 and, 1.16(tertiary methyls); 1.57, 1.64ppm (vinyl methyls); 3.56; methyl ester derivative (3H, s, methyl ester); 4.83(1H, d, J=5.0Hz); 4.34, D20 exchangeable = sec. OH); (1H, d, J=7.0Hz, D20 exchangeable, sec. OH); 3.14ppm (1H, d, J=3.5Hz, D20 exchangeable, see. OH). Mass Spectrum: LREIMS: methyl ester, 535m/e ( M ~ -69). TLC Data Silicic acid plates developed with chloroform-ethanol (8:1, v/v), Re 0.35. References H. Kaise, K. Munakata, and T. Sassa; Structures of Viridominic Acids A and B, New Chlorosis-inducing Metabolites of A Fungus; Tet. Lett., pp. 3789-3782(1972). H. Kaise, K. Munakata, and T. Sassa; Structure of Viridominic Acid C, A New Steroidal Metabolite of a Fungus Having Chlorosis-inducing Activity; Tet. Lett., pp. 199202(1972). H. Kaise, Y. Ogawa, T. Sassa, and K. Munakata, Studies on the Chlorosis-Inducing Substances Produced by a Fungus. Part I. Isolation and Biological Activities of Viridominic Acids A, B, C and Cephalosporin P1; Agr. Biol. Chem., Vol. 36, No. 1, pp. 120-124(1972). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 332(1983).

16.

Fusidanes and Protostanes

469

Common/Systematic Name Cephalosporin Pl Molecular Formula/Molecular Weight C33H5oO8; MW = 574.35057

OOH OAc

NO........ i

: i

General Characteristics Colorless powder; monomethyl ester: crystals from acetone-hexane; mp., 99-101.5 ~C; [tt]D + 25 ~ (C=0.5, in MeOH). Fungal Source

Cladosporium sp. (501-7),) and C. acremonium.

Isolation/Purification The culture filtrate was acidified to pH 3 and extracted with ethyl acetate. The extract was washed with water, concentrated and extracted with saturated NaHCO3 solution. The aqueous layer was acidified, extracted with ethyl acetate, dried over anhydrous sodium sulfate and evaporated to a viscous residue. The chloroform soluble portion of the residue was chromatographed on a silicic acid column eluted with benzene and benzene-ethanol (25:1, v/v). Biologically active fractions from the benzene-ethanol eluate were chromatographed on a silicic acid column eluted with benzene-ethanol (100:1, 50:1, and 25:1, v/v). Final purification was by repetitive silica gel column chromatography eluted with chloroform and chloroform-ethanol (50:1, v/v). Biological Activity Antibiotic and chlorosis-inducing activity in higher plants (six day old seedlings of Chinese cabbage Brassica campestris L. var. pekinensis; seven day old seedlings of barnyard grass Panicum crus-galli L. var. frumentaceum).

470

16.

Fusidanes and Protostanes

Spectral Data UV;

Monomethyl ester, ~, ~.toH 218nm (e = 8,300); monodesacetylcephalosporin PI; 1 Eto~ 20nm (e = 7,000). max

IR:

Monodesacetylcephalosporin P~; (KBr)3400, 2920, 1700, 1440, 1370, and 1260cm 1. IH NMR: (CDCI3/DMSO-d~) 0.80(3H, d, J=6.5Hz, secondary methyl); 0.99, 1.09, and 1.09ppm (3 X tertiary methyls); 1.52 and 1.60ppm (vinyl methyls); 3.52(br s, H-3); 4.50(d, J=10Hz, H-6); 3.38(s, H-7); 5.67(d, J=8.0Hz, H-16); and 5.00ppm (br t, J=7.5Hz, H24). TLC Data Silicic acid plates developed with chloroform-ethanol (8" 1, v/v), Re 0.50. References T. S. Chou, E. J. Eisenbraun, and R. T. Rapala; The Chemistry of Cephalosporin Pl, Tet. Lett., pp. 409-414(1967). T. G. Halsall, E. R. H. Jones, G. Lowe, and C. E. Newall; Cephalosporin P l; Chem. Commun., pp. 685-687(1966). H. Kaise, K. Munakata, and T. Sassa; Structure of Viridominic Acid C, A New Steroidal Metabolite of a Fungus Having Chlorosis-inducing Activity; Tet. Lett., pp. 199-202 (1972). H. Kaise, Y. Ogawa, T. Sassa, and K. Munakata; Studies on the Chlorosis-Inducing Substances Produced by a Fungus. Part I. Isolation and Biological Activities of Viridominic Acids A, B, C and Cephalosporin P1; Agr. Biol. Chem., Vol. 36, No. 1, pp. 120-124(1972). P. Oxley; Cephalosporin Pl and Helvolic Acid; Chem. Commun., pp. 729-730(1966). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 332(1983).

16.

Fusidanes and Protostanes

471

Common/Systematic Name 3-Ketofusidic acid Molecular Formula/Molecular Weight C31I-I4606; MW 514.32944 -

"

OOH

H HO.....9 ~ ~ O A c

General Characteristics Crystals from ethyl ether, dimorphic forms; mp., 177-178~ and 191-192~ 23 ~ methyl ester: crystals from ether-light petroleum; mp., 125-126~

[a]D 2~ -I-

Fungal Source Fusidium coccineum.

Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate after collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, after seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The acetone filtrate was evaporated to dryness and the residue dissolved in ethanol, followed by acidification to pH 3. Addition of ether completed the precipitation of dicyclohexamine hydrochloride. The ether filtrate was washed with water, dried and evaporated to yield a solid mixture of acidic metabolites. This mixture was fractionated on a column containing silicic acid eluted with ether-light petroleum ether-acetic acid (60:40:0.5, v/v/v). An additional fractionation with a silicic acid column eluted with ether-light petroleum ether-acetic acid (50:50:0.5, v/v/v) gave a mixture which yielded 3-ketofusidic acid after crystallization from ether solution.

472

16.

Fusidanes and Protostanes

Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coll. Spectral Data UV: ~, EtOH max

204(c = 10,500) and 225nm (infl e = 7,500); methyl ester: ~, Eto8 max 204(e = 10,200) and 225nm (c = 8,900).

IR:

(KBr) 3550(hydroxyl), 1730, 1255(acetoxy), and 1700cm "~(carboxyl); methyl ester: 3540, 1720, 1700, and 1250cm "~. IH NMR:

(CDCIa) methyl ester: 4.42(1H, m, 1 la-H); 3.05(1H, m, 13a-H); 5.92(1H, d, J=7.58.5H~ 16a-H); 5.14(1H, m, H-24); 1.63 and 1.68(26- and 27-methyls); 0.97, 1.18, and 1.33(18-, 19- and 32-tertiary methyls); 1.99(16-acetate group); and 3.68ppm (21methoxy group). Mass Data: Anal. calcd for C31I-I~O6: C, 72.34; H, 9.01; found: C, 72.22; H, 8.95%; methyl ester, anal. calcd for C32I-I4sO6"C, 72.69; H, 9.15; found" C, 72.75; H, 9.17%. TLC Data Silica gel (Merck HF254) plates developed with (A) ether-acetic acid (100:0.5, v/v), (n) ether-dichloromethane-acetic acid (50:50:0.5, v/v/v), (C)dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20:80:2.5:10, v/v/v/v). Re = 0.42, 0.22, 0.35, and 0.60, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was crimson changing to blueviolet. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal, and W. D. Ollis, Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16. Fusidanes and Protostanes

473

Common/Systematic Name 1 l-Ketofusidic acid Molecular Formula/Molecular Weight C31H4606; M W = 514.32944

:OA~ OOH

HO .......

;

General Characteristics Crystals from ethyl ether; mp., 196-197~ ether-light petroleum; mp., 160-162 ~

[a]D 20 q- 550; methyl ester: crystals from

Fungal Source Fusidium coccineum. Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate atter collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and aider seeding and standing, and the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The acetone filtrate was evaporated to dryness and the residue dissolved in ethanol, followed by acidification to pH 3. Addition of ether completed the precipitation of dicyclohexamine hydrochloride. The ether filtrate was washed with water, dried and evaporated to yield a solid mixture of acidic metabolites. This mixture was fractionated on a column containing silicic acid eluted with ether-light petroleum ether-acetic acid (60:40:0.5, v/v/v). An additional fractionation with a silicic acid column eluted with ether-light petroleum ether-acetic acid (50:50:0.5, v/v/v) gave a mixture which yielded 11-ketofusidic acid after crystallization from ether solution.

474

16.

Fusidanes and Protostanes

Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coll. Spectral Data UV:

~,EtOH 204(e = 10,900) and 225nm (infi e = 6,950); methyl ester: 204(e=10,300) and 225nm (e=7,700). max

~,EtOH max

IR:

(KBr) 3450, 1710, 1260, and 1260cmq; methyl ester: 3560, 1720, 1250, and 1720cmq. ~H NMR: (CDCI3) Methyl ester:3.79(IH, m, 3~3-H);2.62(9~3-H);2.80(3H, m, 12a-, 1213-and 13a-I-I);5.93(IH, d, J=7.5-8.5Hz, 16a-H); 5.12(IH, m, H-24); 0.92(3H, d, ,/=7.58.51-1z,30-secondary methyl); 1.62 and 1.68(26- and 27-methyls); 1.03, 1.18, and I.18(18, 19 and 32-tertiarymethyls);2.01(16-acetategroup); and 3.68ppm (21-methoxy group). Mass Data: HREIMS: methyl ester: 528.3436m/e (M+); anal. calcd for C31H4606:C, 72.34; H, 9.01; found: C, 72.19; H, 8.92%; methyl ester, anal. calcd for C32H4sO6: C, 72.69; H, 9.15; found: C, 72.66; H, 9.04%. TLC Data Silica gel (Merck HF254) plates developed with (A) ether-acetic acid (100:0.5, v/v), (B) ether-dichloromethane-acetic acid (50:50:0.5, v/v/v), (C) dichloromethane-methanol-acetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20:80:2.5:10, v/v/v/v). Re = 0.68, 0.54, 0.50, and 0.73, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was orange changing to brown. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal, and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16. Fusidanesand Protostanes

475

Common/Systematic Name 3-Epifusidic acid Molecular Formula/Molecular Weight C31H4806; M W - 5 1 6 . 3 4 5 0 9

OOH H

HO.....9 _ _~OAc H, NO

:

General Characteristics Colorless crystals from chloroform; rap., 211 ~ [tg]D 20 + 10 ~ (in pyridine); methyl ester: crystals from ether-light petroleum; mp., 142-143 oC. Fungal Source Fusidium coccineum. Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate atter collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, atter seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The acetone filtrate was evaporated to dryness and the derived acids were fractionated on a column containing silicic acid eluted with cyclohexane-chloroform-methanol-acetic acid (20:80:2.5:0.5, v/v/v/v). 3Epifusidic acid separated on standing in etheral solution and was recrystallized from chloroform. Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coli.

476

16.

Fusidanes and Protostanes

Spectral Data V

"

/~

EtOH max

204(e = 9,800) and 225nm (7,350); methyl ester: ~, EtOH 204(e = 10,000) max and 225nm (infl e = 8,400).

(KBr) 3560, 1715, 1690, and 1260cmq" methyl ester: 3510, 3410, 1740, 1240, and 1700cmq . ~H NMR:

(CDCI3) methyl ester:3.05(IH, m, 3a-H); 4.37(IH, m, II~-H); 3.05(IH, m, 13a-H); 5.87(IH, d, J=7.5-8.5Hz, 16a-I-I);5.10(IH, m, H-24); 1.61 and 1.67(26-and 27methyls);0.93, 1.02,and 1.33(18-,19-and 32-tertiarymethyls); 1.97(16-acetate group); and 3.65ppm (21-methoxy group). Mass Data: Anal. calcd for C3~H4sO6: C, 72.06; H, 9.36%; found: C, 71.89; H, 9.48%; methyl ester: anal. calcd for C32Hs006: C, 72.41; H, 9.50%; found: C, 72.32; H, 9.48%. TLC Data Silica gel (Merck HF254) plates developed with (A) ether-acetic acid (100:0.5, v/v), 03) ether-dichloromethane-acetic acid (50: 50: 0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20"80:2.5:10, v/v/v/v). Re= 0.45, 0.35, 0.34, and 0.58, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was grayish changing to red. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal, and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16. Fusidanesand Protostanes

477

Common/Systematic Name 11-Epifusidic acid Molecular Formula/Molecular Weight C31H4sO6; MW = 516.34509

OOH H HO :

~OAc.

H HO.......

; ~

General Characteristics Colorless crystals from acetone; mp., 202-203 ~C; cyclohexamine salt; mp., 172-173 ~C. Fungal Source Fusidium coccineum. Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate after collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, alter seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The mixture of dicyclohexylamine salts was acidified and extracted with ether. 11-Epifusidic acid was purified from the acidic fraction by chromatography on silicic acid eluted with ether-dichloromethane-acetic acid, 25:75:0.5 (v/v/v) followed by crystallization from acetone. Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coll.

478

16.

Fusidanes and Protostanes

Spectral Data UV:

~.to. 204(E = 9,750) and 225nm (infl 7,050); methyl ester: ~, Eton 204(e = 10,000) and 225nm (infl e = 8,200). max

max

IR:

(KBr) 3600, 3400, 1710, and 1270cm'1; methyl ester: 3460, 3440, 1740, 1700, and 1230cm~ . 1H N]V[R.:

(CDCI3) Methyl ester: 3.80(1H, m, 3~3-H); 3.88(1H, m, 1la-H); 5.87(1H, d, J=7.58.5Hz, 16a-H); 5.10(1H, m, H-24); 5.13(1H, m 24-H); 0.92(1H, d, X component of ABX system JAx 6.0-6.5Hz and JBx=0Hz, 30-secondary methyl); 1.62 and 1.68(26and 27-methyls); 0.99, 1.12, and 1.17(18-, 19- and 32-tertiary methyls); 1.98(16acetate group); and 3.67ppm (21-methoxy group). Mass Data: Anal. calcd, for C31I'-I4gO6 91/2I--I20, C, 70.85; H, 9.40%; found" C, 70.68; H, 9.32%; methyl ester: anal. calcd, for C32H5006: C, 72.41; H, 9.50%; found: C, 72.30; H, 9.46%. TLC Data Silica gel (Merck HF254)plates developed with (A) ether-acetic acid (100:0.5, v/v), (B) ether-dichloromethane-acetic acid (50:50:0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20:80:2.5:10, v/v/v/v). Rf= 0.58, 0.44, 0.40, and 0.64, respectively. Detected by spraying plates with concentrated H2SO4 and heating; color was red. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal, and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16.

Fusidanes and Protostanes

479

Common/Systematic Name 9,11-Anhydrofusidic acid Molecular Formula/Molecular Weight C31H4605; M W "-- 4 9 8 . 3 3 4 5 2

~ H O ......

OOH OAc

F-I _

General Characteristics Crystals from ether-light petroleum; mp., 135-137~ methyl ester: an amorphous solid, dicyclohexylamine salt; mp., 163-164 ~ [a]D2~ -34 ~ Fungal Source Fusidium coccineum Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate after collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, after seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The mixture of dicyclohexylamine salts was acidified and extracted with ether. 9,11-Anhydrofusidic acid was purified from the acidic fraction by chromatography on silicic acid eluted with ether-dichloromethaneacetic acid, 25:75:0.5 (v/v/v) followed by crystallization of its dicyclohexylamine salt from early fractions from the silicic acid column. Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coli.

480

16.

Fusidanes and Protostanes

Spectral Data UV:

/~

EtOH msx

204(e = 15,400) and 225nm (infl 8,250).

IR:

(KBr) 3440, 1740, 1690, 1625, and 1250cmq. 1H NMR:

(CDCI3) methyl ester: 3.73(1H, m, 313-H); 5.47(1H, t, J=3.SHz, 11-olefinic H); 5.95(1H, d, J=7.5-8.5Hz, 16a-H); 5.12(1H, m, H-24); 1.60 and 1.68(26- and 27methyls); 0.88, 0.98, and 1.18(18-, 19- and 32-tertiary methyls); 1.97(16-acetate group); and 3.65ppm (21-methoxy group). Mass Data: Anal. calcd for C3~I-h6Os:C, 74.66; H, 9.30%; found: C, 74.53; H, 9.25%; methyl ester: anal. calcd for C32I-I4sO6:C, 74.96; H, 9.44%; found: C, 74.55; H, 9.32%. TLC Data Silica gel (Merck I-IF254)plates developed with (A) ether-acetic acid (100:0.5, v/v), (B) ether-dichloromethane-acetic acid (50: 50:0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20:80:2.5:10, v/v/v/v). Re = 0.71, 0.53, 0.48, and 0.75, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was crimson changing to blueviolet. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal, and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431 (1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16. Fusidanesand Protostanes

481

_Common/Systematic Name 9,11-Anhydro- 12-hydroxyfusidic acid Molecular Formula/Molecular Weight C31I-I4606;

MW

-" 5 1 4 . 3 2 9 4 4

HO

OOH H _

,,~OAc

HO........~ H . General Characteristics An amorphous solid; [a]D2~ + 32~ methyl ester: an amorphous solid. Funsal Source v Fusidium coccineum. Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate after collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, after seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The mixture of dicyclohexylamine salts was acidified and extracted with ether. 9,11-Anhydro-12-hydrofusidic acid was purified by chromatography on silicic acid eluted with dichloromethane-ethyl acetateacetic acid, 70:30:0.5 (v/v/v) followed by preparative TLC (ether-acetic acid, 100:0.5, v/v) of the more polar column fractions. _Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coll.

482

16.

Fusidanes and Protostanes

Spectral Data UW: ~ EtOH max

205(e = 15,700) and 225nm (infl 6,300).

IR:

(KBr) 3450, 1720, and 1250cm1; (CHCI3) 3640, 3600, 3020, 1725, 1700, and 1240cm"~. ~H NMR: (CDCI3) methyl ester: 3.75(1H, m, 313-H); 5.53(1H, m, olefinic 7-H); 4.37(2H, dd, 12a- and 1213-H); 2.67(1H, d, J=8.0Hz, 13a-H); 5.89(1H, d, J=7.5-8.5Hz, 16a-H); 5.18(1H, m, H-24); 1.60 and 1.69(C-26 and C-27 methyls); 0.90, 1.02, and 1.23(18-, 19-, and 32-tertiary methyls); 1.98(16-acetate group); and 3.68ppm (21-methoxy group). Mass Data: Methyl ester: anal. calcd for C31H4606 n20: 9 C, 71.12; H, 9.05%; found: C, 71.35; H, 9.10%; methyl ester: HREIMS: 510.3360role (M§ - H20, C32I-h605); metastable transition detected (M+, 528 -. role 510). TLC Data Silica gel (Merck HF254)plates developed with (A) ether-acetic acid (100:0.5, v/v), (B) ether-dichloromethane-acetic acid (50:50:0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20:80:2.5"10, v/v/v/v). Re = 0.43, 0.27, 0.30, and 0.50, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was brown-violet changing to blue-violet. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal; and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16.

Fusidanes and Protostanes

483

Common/Systematic Name 9,11-Anhydro-9a, l 1a-epoxyfusidic acid Molecular Formula/Molecular Weight CslH4606; MW = 514.32944

OOH

H

.~]jOAc

..

HO

......

_

General Characteristics An amorphous solid; salt; mp., 135-138~

[ a ] D 2~ -

40~ methyl ester: an amorphous solid, dicyclohexylamine

Funsal Source

Fusidium coccineum.

Isolation/Purification Following the usual procedure for the isolation of fusidic acid (see Godtfredsen, et al., 1979), the benzene filtrate after collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, alter seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The mixture of dicyclohexylamine salts was acidified and extracted with ether. 9,11-Anhydro-9a, 11 a-epoxyfusidic acid was purified from the mother liquor of the acidic fraction by chromatography on silicic acid eluted with ether-light petroleum-acetic acid, 60:40:0.5 (v/v/v). The 9,11-anhydro9a, 11 a-epoxyfusidic acid containing fraction was converted to its dicyclohexylamine salt and chromatographed on silicic acid eluted with ether-light petroleum-acetic acid, 50:50:0.5 (v/v/v) to give the purified salt.

484

16.

Fusidanes and Protostanes

Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coll. Spectral Data UV;

~ EtOH raax

204(c = 10,900) and 225nm (infl 8,000).

IR:

(KBr) 3450, 1730, 1710, and 1240cm "~. 1H NMR:

(CDCIa) Methyl ester: 3.72(1H, m, 313-H); 3.17(1H, m, 1l13-H); 5.95(1H, d, J=7.5 8.5I-~ 16tt-H); 5.13(1H, m, H-24); 0.97(3H, d, X component of an ABX system, JAX=6.0-6.SHz, 30-secondary methyl); 1.62 and 1.70(26- and 27-methyls); 0.97, 1.07, and 1.12(18-, 19-, and 32-tertiary methyls); 1.98(16-acetate group); and 3.68ppm (21methoxy group). Mass Data: Anal. calcd, for C31H4606:C, 71.12; H, 9.05; found: C, 71.17; H, 9.14%; methyl ester: HREIMS: 468.3250role ( M +- HOAc, C30H4404); metastable transition detected (M +, 528 -. role 468). TLC Data Silica gel (Merck I--IF254)plates developed with (A) ether-acetic acid (100:0.5, v/v), 03) ether-dichloromethane-acetic acid (50:50:0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20"80:2.5:10, v/v/v/v). Re = 0.62, 0.49, 0.45, and 0.68, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was brown-violet changing to blue-violet. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal, and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites I.I.;Academic Press, New York, p. 333(1983).

16.

Fusidanes and Protostanes

485

_Common/Systematic Name 7, 8-Dehydropseudofusidic acid Molecular Formula/Molecular Weight C31I-I4606; MW = 514.32944

OOH

HO

.~OAc

HO........ ; General Characteristics An amorphous solid;

[ ~ ] D 20 --

33 o; methyl ester: an amorphous solid.

Fungal Source Fusidium coccineum. Isolation/Purification Following the usual procedure for the isolation of fusidic acid, the benzene filtrate after the collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, after seeding and standing, thel precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The mixture of dicyclohexylamine salts was acidified and extracted with ether. 7,8-Dehydrofusidic acid was purified by chromatography on silicic acid eluted with dichloromethane-ethyl acetate-acetic acid, 70:30:0.5 (v/v/v), followed by preparative TLC (ether-acetic acid, 100:0.5, v/v) of the more polar column fractions. Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coli. Spectral Data UV:

~

EtOH max

204(e=15,300) and 225nm (infl 8,200).

486

16.

Fusidanes and Protostanes

IR:

(KBr) 3450, 1720, and 1255cm"~. 1H NMR: (CDCI3) methyl ester: 3.73(1H, m, 313-H); 5.35(1H, m, olefinic 7-H); 4.48(1H, m, 11[~-H); 5.95(1H, d, J=7.5-8.5Hz, 16tt-H); 5.17(1H, m, H-24); 0.95(3H, d, X component of an ABX system, JAx=6.0-6.5Hz, ,/Bx=0, 30-secondary methyl); 1.62 and 1.70(26- and 27-methyls); 0.96, 1.06, and 1.25(18-, 19- and 32-tertiary methyls); 1.98(16-acetate group); and 3.68ppm (21-methoxy group). Mass Data: Anal. calcd, for C31I--I4606 9H20" C, 69.89; H, 9.08%; found: C, 70.17; H, 9.00%; methyl ester: HREIMS" 468.3250m/e (NV - HOAc, C30I-I4404);metastable transition detected (NV, 528 -- role 468). TLC Data Silica gel (Merck HF254)plates developed with (A) ether-acetic acid (100:0.5, v/v), (B) ether-dichloromethane-acetic acid (50:50:0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyclohexane-chloroform-methanol-acetic acid (20:80:2.5:10, v/v/v/v). Rf= 0.52, 0.38, 0.37, and 0.60, respectively. Detected by spraying plates with concentrated H2SO4 and heating; initial color was brown-violet changing to blue-violet. References W. O. Godtffedsen, N. Rastrup-Andersen, S. Vangedal; and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431 (1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16.

Fusidanes and Protostanes

487

Common/Systematic Name Fusilactidic acid Molecular Formula/Mo.lecular Weight C31H4607; M W ----530.32435 25

24

OOH 0

-

OAc

..

NO ......

i _

General Characteristics Crystals from ether; mp., 192-193~

[ a ] D 2~ -

14~ methyl ester: mp., 192-193~

Fungal Source Fusidium coccineum. Isolation/Purification Following the usual procedure for the isolation of fusidic acid, the benzene filtrate alter the collection of the fusidic acid-benzene solvate was evaporated yielding an oil. This oil was dissolved in acetone, diethanolamine was added and, alter seeding and standing, the precipitate of crystalline diethanolamine salts was collected and washed with cold acetone. The mixture of crystalline diethanolamine salts was suspended in ethanol and acidified to pH 3. Benzene and water were added; the benzene layer was concentrated to give a fusidic acid-benzene solvate. Evaporation of the benzene filtrate and solution of the residue in acetone and addition of dicyclohexylamine gave a mixture of crystalline dicyclohexylamine salts. The mixture of dicyclohexylamine salts was acidified and extracted with ether. Fusilactidic acid was purified from the mother liquor of the acidic fraction by chromatography on silicic acid eluted With ether-light petroleum-acetic acid, 60:40:0.5 (v/v/v). The fusilactidic acid containing fraction was crystallized from ether on standing. Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coli.

488

16.

Fusidanes and Protostanes

Spectral Data UV: ~

EtOH max

208(c=9,900) and 225nm (infl 6,800); methyl ester: X max EtOH 205(C=10,800) and 225nm (intl 7,450).

IR:

(KBr) 3460, 3540, 1735, 1720, 1680, and 1240cm~ ; methyl ester: 3560, 1735, 1725, 1705, and 1250cm~. 1H N M R : (CDCI3) methyl ester, 3.70(1H, m, 313-H); 2.83(1H, 913-H); 4.82 and 4.47(2H, m, 12a- and 1213-H); 2.97(1H, m, 13a-H); 5.83(1H, d, J=7.5-8.5Hz, 16a-H); 5.09(1H, m, H-24); 0.98(3H, d, X component of an ABX system, JAX=6.0-6.5Hz; JBx = 0); 1.62 and 1.69(C-26 and C-27 methyls); 1.13, 1.13 and 1.18(18-, 19-and 32-tertiary methyls); 1.99(16-acetate group); and 3.67ppm (21-methoxy group). Mass Data: Anal. calcd for C31I-I4607:C, 70.16%; H, 8.74; found: C, 70.16; H, 8.73%; methyl ester: anal. calcd for C31I-I4sO7:C, 70.56; H, 8.88%; found: C, 70.45; H, 8.97%; HR IMS: 544.3391m/e; 3-O-acetate, 586.3503role. TLC Data Silica gel (Merck HF254) plates developed with (A) ether-acetic acid (100:0.5, v/v), (B) ether-dichloromethane-acetic acid (50: 50:0.5, v/v/v), (C) dichloromethane-methanolacetic acid (95:5:0.5, v/v/v), and (D) cyciohexane-chloroform-methanol-acetic acid (20:80:2.5:10, v/v/v/v). Rf= 0.32, 0.35, 0.41, and 0.61, respectively. Detected by spraying plates with concentrated H2SO4 and heating; color was yellow. References W. O. Godtfredsen, N. Rastrup-Andersen, S. Vangedal and W. D. Ollis; Metabolites of Fusidium coccineum; Tetrahedron, Vol. 35, pp. 2419-2431(1979). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 333(1983).

16.

Fusidanes and Protostanes

489

Common/Systematic Name Fusidic acid Molecular Formula/Molecular Weight C31I-I4sO6; MW = 516.34509

H OOH HO,,,H

HO"

-

~OCOMe

t

'H

-

General Characteristics Colorless needles from benzene; mp., 133.5-135~ .F.ungal Source

Isaria kogane (a mushroom parasitic mainly on larvae and imagos of beetles), Acremonium fusidioides, Calcarisporium arbuscula, Cephalosporium acremonium, and Gabarnaudia tholispora.

Isolation/Purification The culture filtrate was extracted with ethyl acetate, evaporated to dryness and chromatographed on silica gel twice. The biologically active fractions were combined and fusidic acid was crystallized from benzene. Biological Activity Antibiotic activity against Gram-positive bacteria and, in particular, a strain of Staphylococcus aureus; no activity against the Gram-negative bacterium, Escherichia coll. Soectral Data UV:

,~ EtOH 220rim (log E = 3.86). max

IR:

(KBr) 3400(hydroxyl), 1725, 1250(acetoxy), and 1715cm "~(carboxyl).

490

16.

Fusidanes and Protostanes

IH NMR: (CDCI3) 0.90, 0.96, and 1.36ppm (3 methyl singlets, C-18, C-19 and C-32); 0.91(3H, d, J=6Hz, C-30-methyl); 1.58, 1.66ppm (3H, d, d=4.0Hz, C-26 and C-27 methyls); 1.94ppm (3H, s, C-16-OCOCH3); 3.73(1H, m, C-3-H); 4.30(1H, m, C-11-H); -~5.1(1H, m, C-24-H); and 5.84ppm (1H, d, ,/--9.0 Hz, C-16-H). Mass Data: LREIMS" 516(M+), 456, 438, 420, 405, 351,283,232, 189, 95, 93, 69, 60, 45, 43, and 41m/e; anal. calcd for C3~H4806 ~ 1/2H20" C, 69.94; H, 9.40%; found: C, 70.12; H, 9.34%. References H. Hikino, Y. Asada, S. Arihara, and T. Takemoto; Fusidic Acid, a Steroidal Antibiotic from lsaria kogane; Chem. Pharm. Bull., Vol. 20, pp. 1067-1069(1972). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 334(1983).

16.

Fusidanes and Protostanes

491

Common/Systematic Name Helvolic acid; Fumigacin Molecular Formula/Molecular Weight C33H4408; M W "-" 5 6 8 . 3 0 3 6 2

OOo" ~ - / J L , ~ .,~OCMe

~

7y 2829

1,7

0~~_~

=-

I

I 2J..Meo

FI II

0

..........,..H..

'UUMe

3031

General Characteristics Crystals from aqueous methanol; mp., 208-212~

[aiD 25 -

121 ~ (in CHCI3).

Fungal Source

Aspergillusfumigatus, Cephalosporium caerulens, and Emericellopsis terricola.

Biological Activity Antibiotic activity towards bacteria; Gram-positive bacteria are more sensitive than Gramnegative bacteria. Early mammalian toxicity reports for helvolic acid were in error due to contamination by gliotoxin. Spectral Data UV: ,~, EtOH

23 lnm (e = 17,400).

max

13C N M R :

(CDCI3) 13.0; 17.7; 17.9; 18.2; 20.4; 20.6; 23.9; 25.6; 25.9; 27.4; 28.2; 28.5; 30.1" 40.4; 40.7; 41.8; 46.5; 47.2; 49.4; 52.6; 73.4; 73.7; 122.6; 127.6; 130.2; 132.6; 147.4; 156.8; 168.5; 169.8; 200.9; and 208.3ppm. Mass Spectrum: LREIMS: The largest detectable ion in the mass spectrum was 508(due to M + - 60), 490, 479, 473,465, 439(100), 430, 415, 387, 343, and 283m/e.

492

16.

Fusidanes and Protostanes

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 806-809(1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 324-328(1983).

LL-Z1271 Antibiotics and Related Metabolites

LL-Z1271a LL-Z127113 LL-Z12717 Wentilaetone A Wentilaetone B

493

This Page Intentionally Left Blank

17.

LL-Z1271 Antibiotics and Related Metabolites

495

Common/Systematic Name LL-Z1271tt Molecular Formula/Molecular Weight C17H2oO5, MW = 304.13107 O

11~ 0

o;

o

General Characteristics Recrystallization from either acetone-hexane or methylene chloride-hexane gave crystals with mp. 214-215~ [t~]D25 -203 ~ (C=0.29, in MeOH). Fungal Source Acrostalagmus sp.

Isolation/Purification The culture was adjusted to pH 3.0 with hydrochloric acid and extracted with ethyl acetate. Concentration of the extract in vacuo followed by drying over sodium sulfate and the addition of hexane caused essentially pure LL-Z 1271 t~ to crystallize. Final purification was by recrystaUization from either acetone-hexane or methylene chloride-hexane. Biological Activity Antifungal activity. Spectral Data UV; ~, MooH max 257nm (e=13,500). IR; (KBr) 1775(y-lactone) and 1730cm"~(6-1actone). 1H NMR: (CDC13) 1.11 and 1.33(s, CH3's at C-10 and C-4); 1.93(d, H=5, J5,6-5.3 Hz); 3.70(s, OMe); 5.07(t, H-6); 5.76(2H's, H=I 1 and -14); and 6.53ppm (m, H-7); (benzene-d6) 1.08 and 1.16(s, CH3's); 3.60(s, OMe); 4.73(t, H-6); 5.52(t, H-14); 5.70(d, H-11); and 6.43ppm (m, H-7).

496

17. LL-Z1271 Antibioticsand Related Metabolites

Mass Spectrum: EIMS: 304m/e (M +, Cl~H20Os). Reference G. A. Ellestad, R. H. Evans, Jr., M. P. Kunstma~ J. E. Lancaster, and G. O. Morton, Structure and Chemistry of Antibiotic LL-Z1271 a, an Antifungal Carbon-17 Terpene; J. Am. Chem. Soc., Vol. 92, pp. 5483-5489(1970).

17.

LL-Z1271 Antibiotics and Related Metabolites.

497

Common/Systematic Name LL-Z1271~ Molecular Formula/Molecular Weight C1~H24Os; MW = 296.16237 .COOH

H -

COOH

General Characteristics Crystals; mp., 200~ (dec); [a]D +22.5 (in MeOH). Fungal Source

Acrostalagmus sp.

Spectral Data IR: (KBr) 3500(hydroxyl), 2750, 1710(carboxyl), 1650, and 900cm"l (exocyclic methylene). 1H NMR: (CDCI3) The NMR spectrum showed signals for two tertiary C-methyl groups at 0.53 and 1.15ppm and characteristic broadened doublet for an exocyclic methylene group at 4.44 and 5.08ppm. Also present was a broad one-proton signal at 3.75 assigned to an axial carbinol proton. Mass Spectrum: EIMS" 296(M+), 167, 139, and 121m/e. The latter three were the result of cleavage of the allylic bonds at C-6 and C-9. The base peak was at 123m/e. Reference G. A. Ellestad, R. H. Evans, Jr., and M. P. Kunstmann; LL-Z127113, and Additional C~6 Terpenoid Metabolite from an Acrostalagmus Species; Tetrahedron Letters, pp. 497500(1971).

498

17.

LL-Z1271 Antibiotics and Related Metabolites

Common/Systematic Name LL-Z 12717 Molecular Formula/Molecular Weight C16HlsOs, ~ = 290.11542

1,/k o

General Characteristics Crystals from ether; mp., 241-247~

[a]D 25 - 2 5 6 ~

(c=0.43, in MeOH).

Fungal Source Acrostalagmus sp.

Isolation/Purification The methanol extracts of the mycelium pads were combined and concentrated m vacuo to an aqueous phase, which was extracted twice with one-half volumes of chloroform. The chloroform layer was separated, dried over sodium sulfate and concentrated to an oily residue. Silica gel chromatography (slurry packed in methylene chloride) by elution with methylene chloride and ether-methylene chloride (1:4, v/v) (monitored at 255nm) gave two bands. The antibiotic was crystallized from both bands from ethyl ether solution. Biological Activity Antifungal activity. Spectral Data UV;

~, ~oH max

257nm (E=15,400).

IR~

(KBr) 1780(y-lactone) and 1680cm"1 (6-1actol, occurs at 1720cm"1 in CHC13). ~H NMR: (CDCI3 with 2 drops of DMSO4) 1.16 and 1.33ppm (s, CH3's at C-10 and C-4); 1.95(d, H-5, ds,6=5.0Hz); 5.07(t, H-6); 5.77(d, H-11, JT,11=2.0Hz); 6.08(broadened s, H-14); and 6.46ppm (m, H-6).

17. LL-Z1271 Antibioticsand Related Metabolites

499

Mass Data: EIMS: 290m/e (M§ C16H1805); anal. calcd for C16H1805, C, 66.19; H, 6.25%; found: C, 66.46; H, 6.31%. Reference G. A. Ellestad, R. H. Evans, Jr., M. P. Kunstmann, J. E. Lancaster and G. O. Morton; Structure and Chemistry of Antibiotic LL-Z1271tt, an Antifungal Carbon-17 Terpene; J. Am. Chem. Soc., Vol. 92, pp. 5483-5489(1970).

500

17. LL-Z1271Antibioticsand Related Metabolites

Common/Systematic Name Wentilactone A Molecular Formula/Molecular Weight C16H1606; M W -- 3 0 4 . 0 9 4 6 9

O

0

HO

[

OH

,,,,,""

General Characteristics Crystalline cubes from methanol-ethyl acetate (1:1); mp., 258~ Soluble in water and acetone, sparingly soluble in methanol and insoluble in hexane, benzene, ethyl ether, chloroform, and ethyl acetate. Fungal Source

Aspergillus wentii (NRRL 6435).

Isolation/Purification Fungal cultures were extracted with water saturated chloroform, dried over sodium sulfate and concentrated to near dryness. The crude extract was fractionated using a series of silica gel column chromatography systems (elution with a linear gradient from benzene to ethyl acetate) followed by a final purification using Florisil column chromatography (elution with a linear gradient from benzene to ethyl acetate) to separate wentilactone A from B. Purification was monitored with a one-day-old chick bioassay. Biological Activity Wentilactone A had an LDs0 of 7mg/kg in day-old chicks; it was phytotoxic showing growth inhibition of 81, 81, and 48% in wheat coleoptiles at 103, 10-4 and 10-5 M concentrations, respectively. Spectral Data UV: /~ MeOH max

259nm (1.34 X 104 M ~ cm"~) (indicates diene lactone system).

17.

LL-Z1271 Antibiotics and Related Metabolites

501

IR; (KBr) 3500, 1780, and 1710cm"l.

CD: (MeOH) 259nm =-8.23 X 10a deg cm2/dmol. 1H NMR: (DMSO-d6) H-I, 3.63(J=4.4Hz); n-2, 3.38(,/=4.4, 6.1Hz); H-3, 4.30(J=6.1Hz); n-5, 2.22(J=5.6Hz); H-6, 5.09(J=5.6, 4.4Hz); H-7, 6.36(,/= 4.4, 1.5Hz); H-11, 6.22(J=l.5Hz); H-14, 5.04; H-18, 1.10; and H-20, 1.38ppm.

13C NMPx: (DMSO-d6) C-l, 50.20; C-2, 54.69; C-3, 66.78; C-4, 48.21; C-5, 47.72; C-6, 69.49; C7, 121.82; C-8, 131.15; C-9, 156.45; C-10, 35.57; C-11, lll.91; C-12, 162.95; C-14, 68.81; C-18, 18.65; C-19, 175.84; and C-20, 24.85ppm. Mass Spectrum: HREIMS: 304.0914m/e (M+, consistent with a molecular formula of C16H1606; calculated mass 304.0946). TLC Data Re 0.18 appeared as a brown quenching spot under long-wave UV light atter spraying with Ehrlich's reagent and heating (silica gel GH-R, solvent system, chloroform-acetonemethanol, 17:2:1, v/v/v). Reference J. W. Dorner, R. J. Cole, J. P. Springer, R. H. Cox, H. G. Cutler, and D. T. Wicklow; Isolation and Identification of Two New Biologically Active Norditerpene Dilactones from Aspergillus wentii; Phytochemistry, Vol. 19, pp. 1157-1161(1980).

502

17.

LL-Z1271 Antibiotics and Related Metabolites

Common/Systematic Name Wentilactone B Molecular Formula/Molecular Weight C16HlsOs; MW' = 290.11542 O

I HO

o

.

General Characteristics Crystalline cubes from methanol-ethyl acetate (1:1); mp., 260~ Soluble in water and acetone, sparingly soluble in methanol and insoluble in hexane, benzene, ethyl ether, chloroform, and ethyl acetate. Fungal Source

Aspergillus wentii (NRRL 6435).

Isolation/Purification Fungal cultures were extracted with water saturated chloroform, dried over sodium sulfate and concentrated to near dryness. The crude extract was fractionated using a series of silica gel column chromatography systems (elution with a linear gradient from benzene to ethyl acetate) followed by a final purification using Florisil column chromatography (elution with a linear gradient from benzene to ethyl acetate) to separate wentilactone A from B. Purification was monitored with a one-day-old chick bioassay. Biological Activity Wentilactone B was not toxic to day-old chicks dosed up to 250mg/kg; it was less phytotoxic than A showing growth inhibition of 100 and 48% in wheat coleoptiles at 10s and 10.4 M concentrations respectively. Spectral Data UV~

maxM~n 259nm (1.40 X 10a M "1 cm"1) (indicates diene lactone system). IR~

(KBr) 3500, 1780, and 1710cm1.

17.

LL-Z1271 Antibiotics and Related Metabolites

503

CD: (MeOH) 260nm =-8.23 X 103 deg cm2/dmol. IH NMR: (DMSO-d6) H-I, 1.3-2.3; H-2, 1.3-2.3; H-3, 4.98; H-5, 2.07(J=5.4Hz); H-6, 5.19(J=5.4, 4.4Hz); H-7, 6.35(J=4.4, 1.7Hz); H-11, 5.74(J=l.7Hz); H-14, 4.98; H-18, 1.11; and H-20, 1.30ppm. 13C NMR: (DMSO-d6) C-I, 39.63; C-2, 36.33; C-3, 63.22; C-4, 42.24; C-5, 45.53; C-6, 71.30; C7, 121.64; C-8, 131.38; C-9, 158.17; C-10, 35.56; C-11, 111.85; C-12, 163.16; C-14, 69.32; C-18, 23.15; C-19, 180.70; and C-20, 27.37ppm. Mass Spectrum: HREIMS: 290.1166m/e (M+, consistent with a molecular formula of C16H1805; calculated mass 290.1153). TLC Data Rf 0.12 appeared as a yellow fluorescent spot under long-wave UV light alter spraying with Ehrlich's reagent and heating (silica gel GH-R, solvent system, chloroform-acetonemethanol, 17:2:1, v/v/v). Reference J. W. Dorner, R. J. Cole, J. P. Springer, R. H. Cox, H. G. Cutler, and D. T. Wicklow; Isolation and Identification of Two New Biologically Active Norditerpene Dilactones from Aspergilluswentii; Phytochemistry, Vol. 19, pp. 1157-1161(1980).

LL-Z1272 Antibiotics and Related Metabolites LL-Z1272Qt LL-Z127213 LL-Z12728 LL-Z1272e LL-Z1272Cj LL-Z 12727 (Ascochlorin) Ascofuranone Ascofuranol Chloronectrin Deacetylchloronectrin

505

18.

LL-Z1272 Antibioticsand Related Metabolites

507

Common/Systematic Name Antibiotic LL-Z 1272a Molecular Weight/Molecular Formula C23H3103C1; MW = 390.19617 CI

Fungal Source Unknown Fusarium species designated LL-Z1272. Isolation/Purification Fungal culture extracted with ethyl acetate, chromatographed on a silica gel column, eluted with CH2C12, followed by a second silica gel column eluted with a gradient of CH2Cl2-ethyl ether, 1"1 (v/v). Final purification was by countercurrent distribution using a MeOH-heptane system. General Characteristics Crystallization from MeOH-isooctane and then MeOH-water; mp., 72.5-73 o Biological Activity This compound inhibited the growth of the protozoan, Tetrahymenapyriformis. Soectral Data _

UV:

~,m~x 228, 293, and 345nm (c = 11,150, 10,400, and 7800, respectively). IR:

Spectrum is characterized by strong carbonyl absorption around 1630cm~ in accord with a 2,4-dihydroxybenzaldehyde system. 1H NMR: 759(C-2, chelated OH); 389(C-4, non-chelated OH); 605(C-1, aldehyde H); 153(C-6, Ar-CH3); 203(C-1', AR-CH2); 315(C-2',-CH=); and 108Hz (C-3', CH3). The non-aromatic portions which covered the residual C~sHz5portion of the molecule

508

18.

LL-Z1272 Antibiotics and Related Metabolites

suggested the presence of a famesyl moiety. The NMR spectrum exhibited an aldehydic proton signal at 600-607Hz and the chelated phenolic hydrogen signal at 757-761Hz. The remaining para phenolic hydroxyl signal was at 389-405Hz by exchange with CD3OD. The 3-proton singlet at 146-155Hz was assigned to the aromatic Me group which is considerably deshielded by the adjacent carbonyl. Mass Data: Found: C, 70.73; H, 8.05; CI, 9.65%; C23H3~O3C1requires: C, 70.66; H, 7.99; CI, 9.07%. Reference G. A. Ellestad, R. H. Evans, Jr., and M. P. Kunstmann; Some New Terpenoid Metabolites from an Unidentified Fusarium Species; Tetrahedron, Vol. 25, pp. 1323-1334(1969).

18.

LL-Z1272 Antibiotics and Related Metabolites

509

Common/Systematic Name Antibiotic LL-Z 127213 Molecular Weight~olecular Formula C23H3203; M W --- 356.23515

Fungal Source Unknown Fusarium species designated LL-Z 1272. Isolation/Purification Fungal culture extracted with ethyl acetate, chromatographed on a silica gel column eluted with CH2C12, followed by a second silica gel column eluted with a gradient of CH2Cl2-ethyl ether, 1:1 (v/v). Final purification was by countercurrent distribution using a MeOH-heptane system. General Characteristics A semicrystalline material was recrystallized several times from MeOH-water; mp., 97.5~ Biological Activity This compound inhibited the growth of the protozoan, Tetrahymenapyriformis. Spectral Data UV:

)tmax 223,233 sh, 297 and 340nm sh (6=15,100, 11,500, 16,000, and 3900, respectively). IR:

Spectrum was characterized by strong carbonyl absorption around 1630cm"a in accord with a 2,4-dihydroxybenzaldehyde system. ~H NMR: 759(C-2, chelated OH); 396(C-4, non-chelated OH); 600(C-1, aldehyde H); 148(C-6, AR-CH); 372(C-5, AR-H); 203(C-1', AR-CH2); 316(C-2',-CH=); and 108ppm (C-3',

510

18.

LL-Z1272 Antibiotics and Related Metabolites

CH3). The non-aromatic portions which cover the residual C15H25portion of the molecule suggested the presence of a farnesyl moiety. The NMR spectrum exhibited an aldehydic proton signal at 600-607Hz and the chelated phenolic hydrogen signal at 757-761Hz. The remaining para phenolic hydroxyl signal was observed at 389-405Hz by exchange with CD3OD. The 3-proton singlet at 146-155Hz was assigned to the aromatic Me group which was considerably deshielded by the adjacent carbonyl. Mass Data: Found: C, 77.08; H, 8.96%. C23H3203requires: C, 77.49; H, 9.05%. Reference G. A. EUestad, R. H. Evans, Jr., and M. P. Kunstmann; Some New Terpenoid Metabolites from an Unidentified Fusarium Species; Tetrahedron, Vol. 25, pp. 1323-1334(1969).

18.

LL-Z1272 Antibiotics and Related Metabolites

511

Common/Systematic Name Antibiotic LL-Z 12726 Molecular Weight/Molecular Formula C23H3104C1; MW -- 406.19109

.O.f Me CI

I~' ~"2 ~ C H O H

0 Fungal Source Unknown Fusarium species designated LL-Z 1272. Isolation/Purification Fungal culture extracted with ethyl acetate, chromatographed on a silica gel column, eluted with CH2C12, followed by a second silica gel column, eluted with a gradient of CH2Cl2-ethyl ether, 1:1 (v:v). Final purification was by countercurrent distribution using a MeOH-heptane system. General Characteristics Fractional crystallization from acetone-hexane; mp., 129.5-130.5~ MeOH).

[tt]D25 +6 ~ (C=I.0, in

Biological Activity This compound inhibited the growth of the protozoan, Tetrahymenapyriformis. Spectral Data UV:

~,max 231,293, and 346nm (e = 23,000, 12,000, and 9150, respectively). Im:

Spectrum is characterized by strong carbonyl absorption around 1630cm"1 in accord with a 2,4-dihydroxybenzaldehyde system. IH NMR: 757(C-2, chelated OH); 399(C-4, non-chelated OH); 604(C-1, aldehyde H); 155(C-6,

512

18.

LL-Z1272 Antibiotics and Related Metabolites

AR-CH3); 203(C-1', AR-CH2); 315(C-2',-CH=); 108(C-3' CH3); 33(C-6' CH3); 52, 53Hz (C-7' and C-11' CH3). The non-aromatic portions which cover the residual C~5H25 portion of the molecule suggested the presence of a farnesyl moiety. Spectrum exhibited an aldehydic proton signal at 600-607Hz and the chelated phenolic hydrogen signal at 757-761Hz. The remaining para phenolic hydroxyl signal was observed at 389-405Hz by exchange with CD3OD. The 3-proton sing,let at 146-155Hz was assigned to the aromatic Me group which is considerably deshielded by the adjacent carbonyl. Mass Data: Found: C, 67.93, H, .7.81; C1, 9.08%; C~H3~O4C1 requires: C, 67.89, H, 7.68; CI, 8.71 %. Reference G. A. Ellestad, R. H. Evans, Jr., and M. P. Kunstmann; Some New Terpenoid Metabolites from an Unidentified Fusarium Species; Tetrahedron. Vol. 25, pp 1323-1334(1969).

18. LL-Z1272 Antibiotics and Related Metabolites

513

Common/Systematic Name Antibiotic LL-Z1272e Molecular Weight/Molecular Formula C23H3204; MW -- 372.23006 HO,

Me

H 0

Fungal Source Unknown Fusarium species designated LL-Z 1272. Isolation/Purification Fungal culture extracted with ethyl acetate, chromatographed on a silica gel column, eluted with CH2C12, followed by a second silica gel column, eluted with a gradient of CH2Cl2-ethyl ether, 1:1 (v/v). Final purification was by countercurrent distribution using a MeOH-heptane system. General Characteristics Crystallization from AcOEt-hexane; mp., 171.5-172.5 ~

[a]D 25 + 6 ~

(c=0.93, in MeOH).

Biological Activity This compound inhibited the growth of the protozoan, Tetrahymenapyriformis. Spectral Data UV: )~,~x 223, 233 sh, and 340nm sh (c = 15,600, 11,500, 16,500, and 3700, respectively). IR: Spectrum was characterized by strong carbonyl absorption around 1630cm"~in accord with a 2,4-dihydroxybenzaldehyde system.

IH N~R: 758(C-2, chelatedOH), 396(C-4, non-chelatedOH); 600(C-1, aldehydeI-I);146(C-6, AR-CH3); 376(C-5, ARH); 196(C-1', ARCH2); 315(C-2',-CH=), 108(C-3' CH3); 32(C-6' CHa); 51, 51Hz (C-7' and C-11' CH3). The non-aromatic portions which cover

514

18.

LL-Z1272 Antibioticsand Related Metabolites

the residual C15H25 portion of the molecule suggested the presence of a farnesyl moiety. Spectrum exhibited an aldehydic proton signal at 600-607Hz and the chelated phenolic hydrogen signal at 757-761Hz. The remaining para phenolic hydroxyl signal was observed at 389-405Hz by exchange with CD3OD. The 3-proton singlet at 146-155Hz was assigned to the aromatic Me group which is considerably deshielded by the adjacent carbonyl. Mass Data: Exhibited a molecular ion at m/e of 3 72.228 in accord with the molecular formula C23H3204; found: C, 73.15; H, 8.62%; C23H3204 requires: C, 74.16; H, 8.66%. Reference G. A. Ellestad, R. H. Evans, Jr., and M. P. Kunstmann; Some New Terpenoid Metabolites from an Unidentified Fusarium Species; Tetrahedron, Vol. 25, pp. 1323-1334(1969).

18.

LL-Z1272 Antibiotics and Related Metabolites

515

Common/Systematic Name Antibiotic LL-Z 1272~ Molecular Weight/Molecular Formula C25H3106C1, ~ = 462.18092 Cl

HO~Me 8,

OAc r" ~

H

"CliO

0 Fungal Source Unknown Fusarium species designated LL-Z 1272. Isolation/Purification Fungal culture extracted with ethyl acetate, chromatographed on a silica gel column eluted with CH2C12, followed by a second silica gel column eluted with a gradient of CH2Cl2-ethyl ether, 1:1 (v/v). Final purification was by countercurrent distribution using a MeOH-heptane system. General Characteristics SemicrystaUine solid from acetone-hexane; mp., 156.5-157.0~ MeOH).

[a]D2~ -1.5 ~ (c=l.0, in

Biological Activity This compound inhibited the growth of the protozoan, Tetrahymenapyriformis. Spectral Data UV:

X,~x 229, 293, and 347nm (e = 39,800, 11,700, and 9600, respectively). IR:

Spectrum was characterized by strong carbonyl absorption around 1630cm"1in accord with a 2,4-dihydroxybenzaldehyde system. 1H NMR: 761 (C-2, chelated OH); 400(C-4, non-chelated OH); 607(C- 1, aldehyde H); 155(C-6,

516

18.

LL-Z1272 Antibiotics and Related Metabolites

AR-CHa); 212(C-1', ARCH2); 334(C-2',-CH=); 116(C-3' CH3); 322(C-7' CH3); 355(C-4'-C-5'); 44(C-6' CH3);, 53, 53Hz (C-7' and C-11' CH3). The non-aromatic portions which cover the residual C~5H25portion of the molecule suggested the presence of a famesyl moiety. Spectrum exhibited an aldehydic proton signal at 600-607Hz and the chelated phenolic hydrogen signal at 757-761Hz. The remaining para phenolic hydroxyl signal was observed at 389-405Hz by exchange with CDaOD. The 3-proton singlet at 146-155Hz was assigned to the aromatic Me group which is considerably deshielded by the adjacent carbonyl. Mass Data: Found: C, 65.01; H, 7.03; C1, 8.02%; C25H3~O6C1requires: C, 64.86; H, 6.75; CI, 7.66%. Reference G. A. Ellestad, R. H. Evans, Jr., and M. P. Kunstmann; Some New Terpenoid Metabolites from an Unidentified Fusarium Species; Tetrahedron, Vol. 25, pp. 1323-1334(1969).

18.

LL-Z1272 Antibiotics and Related Metabolites

517

Common/Systematic Name Ascochlorin; LL-Z 1272y 2,4-Dihydroxy-3-substituted-5-chloroorcylaldehyde Molecular Formula/Molecular Weight C23H3oO4C1, M W = 404.17544

.O Me CI

~~CHO

General Characteristics Ascochlorin appeared as pale yellow crystalline needles when extract was concentrated in vacuo. Recrystallization from acetone-hexane; mp., 161.0-163.0 ~C. Analytical sample had mp., 172-173~ [r -1.5 ~ (C=I.0, in MeOH). Fungal Source Ascochyta viciae, Nectria coccinea, N. lucida, Acremonium luzulae, Cylindrocladium iliciola, and Fusarium species.

Isolation/Purification The fungus was grown in glucose, peptone, and ammonium chloride and the mycelia were collected by filtration. Several spots positive to FeCI3 were seen on a thin-layer chromatogram of the mycelial acetone extract using n-hexane-acetone (4:1, v/v) as the solvent. The mother liquor aider crystallization was concentrated in vacuo and the residue was chromatographed on a column packed with silica gel. The column was eluted with n-hexane-acetone (9:1, v/v). Further eluted with n-hexane-acetone (4:1, v/v). Each fraction was spotted on a silica gel thin-layer plate and the plate was developed with n-hexane-acetone (4:1, v/v). The fractions containing substances positive to FeC13 were collected and concentrated in vacuo. The following substances were eluted successfully and obtained as crystalline forms; LL-Z 11272/5, LL-Z 1272e, ascochlorin, ascofuranone, ascofuranol, and 4'-hydroxy-5'-hydroascochlorin. Biological Activity Antibiotic against viruses and fungi in vitro. Some of these prenyl phenols show hypolipidemic activity in both normolipidemic and hyperlipidemic rats.

518

18.

LL-Z1272 Antibiotics and Related Metabolites

Spectral Data UV:

~.~,x 250(e = 29,600), 292(e = 10,600), and 350nm (e = 11,000). IR:

IR spectrum showed a strong chelated carbonyl band at 1630cm4, probably due to the 2,4-dihydroxybenzaldehyde system. ~H NMR: (CDCI3) 10.14(1H, s0 C-l-aldehyde); 12.72(1H, s, C-2-chelated OH); 6.57(1H, s, C-4-OH); 2.57(3H, s, C-6-methyl); 3.50(2H, d, C-I'); 5.50(1H, t, C-2'); 1.92(3H, s, C3 '-methyl); 5.91(1H, d, C-4'); 5.32(1H, d, C-5'); 0.70(3H, s, C-6'-methyl); 0.81(3H, d, C-7'-methyl); and 0.83ppm (3H, d, C-11-methyl). References G. A. Ellestad, R. H. Evans, Jr., and M. P. Kunstmann; Some New Terpenoid Metabolites from an Unidentified Fusarium Species; Tetrahedron, Vol. 25, pp. 1323-1334(1969). Y. Nawata, K. Ando, G. Tamura, K. Arima, and Y. Iitaka; The Molecular Structure of Ascochlorin; J. Antibiotics, Vol. 22, pp. 511-512(1969). H. Sasaki, T. Hosokawa, Y. Nawata, and K. Ando; Isolation and Structure of Ascochlorin and Its Analogs; Agr. Biol. Chem., (8), pp. 1463-1466(1974).

18.

LL-Z1272 Antibiotics and Related Metabolites

519

Common/Systematic Name Ascofuranone Molecular Formula/Molecular Weight C23H2905C1, M W = 420.17035 CliO

General Characteristics Colorless needles from n-hexane-acetone (4" 1); mp., 84-85~ [aiD 21 -50 ~ (C=I, in MeOH). Gave intense purple-brown color with FeC13. Insoluble in water, slightly soluble in n-hexane, readily soluble in most organic solvents. Fungal Source Ascochyta viciae (mutant strain No. 34). Isolation/Purification Culture filtrate was extracted with chloroform-methanol. The extract was chromatographed on silica gel (n-hexane-acetone, 95:5, v/v). The substance was crystallized from n-hexane-acetone, 4:1 (v/v). Biological Activity Less toxic to mice and rats than ascochlorin; effectively lowered serum lipid levels. Spectral Data UV: /~ MeOH max

228@=20300), 290(15000), and 350nm (8200); gave 57nm bathochromic shift of principal ET band under alkaline condition.

m: (KBr) 3300(hydroxyl), 1735(carbonyl), and 1630cm"1 (carbonyl). IH NMR: (CDCI3, TMS) 10.12(s, C1-Aldehyde); 12.68(s, C2-Chelated OH); 6.55(s, C4-OH); 2.50(s, C6-Methyl); 3.38(d, 41'-H); 5.21(t, C2'-H); 1.63(s, C3'-Methyl); 2.07(s, C4',5'-

520

18.

LL-Z1272 Antibioticsand Related Metabolites

H); 5.50(m, C6'-H); 1.79(s, C7'-Methyl); 4.50 (t, C8'-H); 2.39(d, C9'-H); 1.20 and 1.26ppm (2s, C 1l'-Dimethyl). Mass Data: 420(M +, 12%), 334(30), 330(6), 267(8), 253(87), 239(25), 221(12), 199(100), 167(13), and 153m/e (23); found: C, 65.24; H, 6.93; CI, 8.26%; calcd for C23H2905C1, C, 65.62; H, 6.94; CI, 8.43%. TLC Data Silica gel; n-hexane-acetone (4:1); Rf=0.42. Detection: spraying with sulfuric acid and heating at 110 ~ for 5 minutes. Reference H. Sasaki, T. Hosokawa, M. Sawada, and K. Ando; Isolation and Structure of Ascofuranone and Ascofuranol, Antibiotics with Hypolipidemic Activity; J. Antibiotics, Vol. 26, No. 11, pp. 676-680(1973).

18.

LL-Z1272 Antibiotics and Related Metabolites

521

Common/Systematic Name Ascofuranol Molecular Formula/Molecular Weight Cz3H31OsCl; M W = 422.18600

CliO

General Characteristics Colorless needles from n-hexane-acetone, 4:1; mp., 75~ [a]D 21 - 7 ~ (c=l, in MeOH); gave positive reaction with FeCIa; the solubility was nearly the same as that of ascofuranone. Fungal Source Ascochyta viciae (mutant strain No. 34) Isolation/Purification Culture filtrate was extracted with chloroform-methanol. The extract was chromatographed on silica gel (n-hexane-acetone, 4:1, v/v). The substance was crystallized from n-hexane-acetone, 4:1 (v/v). Spectral Data UV;

~, MOOHmax228(e=15800), 290(8700), and 352nm (13900); gave 57nm bathochromic shill of principal ET band at alkaline condition. IR;

(KBr) 3300(hydroxyl), 1630(carbonyl), and 1090cm "1(secondary alcohol).

IH NMR: (CDCI3, TMS) 10.1l(s,CI-aldehyde), 12.66(s,C2- chelatedOH); 6.98(s,C4-OH), 2.58(s, C6-methyl); 3.37(s, CI'- H); 5.18(t, C2'-H), 1.60(s, C3'-methyl), 2.06(s, C4', 5'-H); 5.48(m, C6'-H); 1.78(s, C7'-methyl); 4.30 (t, C8 ~H); 2.35 (d, C9'-H); 1.20 and 1.26(2s, C1 l'-dimethyl); 2.38(s, C10'-OH); and 3.95(t, C10'-H).

522

18.

LL-Z1272 Antibiotics and Related Metabolites

Mass Data: 422(M+, 11%), 253(26), 239(19), 223(9), 199(100), 183(9), 169(24), 155(21), 151(31), and 13role (636); found: C, 65.31; H, 7.35; CI, 8.42%. TLC Data Silica gel; n-hexane-acetone (4:1); Rf=0.23; detection: spraying with sulfuric acid and heating at 110 ~C for 5 minutes. Reference H. Sasaki, T. Hosokawa, M. Sawada, and K. Ando; Isolation and Structure of Ascofuranone and Ascofuranol, Antibiotics with Hypolipidemic Activity; J. Antibiot., Vol. 26, No. 11, pp. 676-680(1973).

18.

LL-Z1272 Antibiotics and Related Metabolites

523

Common/Systematic Name Chloronectrin Molecular Formula/Molecular Weight C25H3306C1, MW = 464.19657 CHO

0-/ " ~ 1 ~

"OAc

General Characteristics A gum. Fungal Source Nectria coccinea (CMI 120337C).

Isolation/Purification Mycelium extracted with chloroform, evaporated to dryness, triturated with petroleum ether to give a solid. The solid was chromatographed on a silica gel column; chloronectrin was eluted with benzene-chloroform (1:1, v/v). This fraction was subjected to repeated TLC using Merck GF silica gel to yield chloronectrin as a gum. Biological Activity Antibiotic. Spectral Data IR:

(CHCI3) 3515, 1728, 1712, and 1633cmq. 1H NMR: (CDCI3) -2.44(bonded OH);-0.I I(CHO); ca 3.5 br (OH); 4.44(t, J=8.0Hz, CH2CH=); 4.64(dd, J=4.0 and 8.0Hz, CHOAc); 6.62(d, J=8.0Hz), CH2CH=); 7.43(ARCH3); 7.99(OCOCH3); 8.21(d, J=2.0Hz, CH=CCH3); 9.06(d, J=7.0Hz, CHCH3); 9.22(d, J=7.0Hz, CHCH3), and 9.48z (CCH3).

524

18.

LL-Z1272 Antibioticsand Related Metabolites

Mass Spectrum: EIMS: 464.1979(M+), C25H3306C1requires 464.1965. Reference D. C. Aldridge, A Borrow, R. G. Foster, M. S. Large, H. Spencer, and W. B. Turner; Metabolites ofNectria coccinea; J. Chem. Sot., Perkin Trans I, pp. 2136-2141 (1972).

18.

LL-Z1272 Antibiotics and Related Metabolites

525

Common/Systematic Name Deacetylchloronectrin 4'-Hydroxy-5-hydroascochlorin Molecular Formula/Molecular Weight C23H3105C1; M W = 422.18600

CliO

~/"cl

" lv

-OH

General Characteristics 4'-Hydroxy-5-hydroascochlorin was obtained as pale yellow needles. RecrystaUized from n-hexane-acetone (4:1); formed colorless crystalline needles; mp., 138 ~ A deacetylation product of chloronectin. Funsal Source Ascochyta viciae. Biological Activity Some of these prenyl phenols show hypolipidemic activity in both normolipidemic and hyperlipidemic rats. Isolation/Purification See Ascochlorin. Spectral Data UV:

Absorption maxima, 228(e 16,400), 293(e 9400), and 356nm (e 13,500). IR:

(I~r) 3400(OH), 1710(C=O), and 1630(chelated carbonyl) ~H NMR: (CDCI3) 10.12(1H, s, Cl-aldehyde); 12.68(1H, s, C2-chelated OH); 6.49(1H, s, C4=OH); 2.60(3H, s, C6=methyl); 3.39(2H, d, CI'); 5.Sl(1H, t, C2'); 1.83(3H, s,

526

18.

LL-Z1272 Antibioticsand Related Metabolites

C3'-methyl); 4.22(1H, d, d, C4'); 1.53(1H, d, 1.60 1H, d, C5'); 0.55(3H, s, C6'-methyl); 0.82(3H, d, C7'-methyl); and 0.96ppm (3H, d, C11-methyl). Mass Data: The base peak, m/e 139, was derived from the trimethylcyclohexanone moiety. Prominent chlorine-containing peaks, m/e 265 and 251, are formed by fission of either C-4'-5' or 5'-6' followed by rearrangement with accompanying loss ofH20. A chlorine-containing peak at m/e 199 was a tropylium ion common to ascochlorin and its analogs. Analysis: calculated for C23H3105C1, C 65.32, H 7.33, C1 8.40%: found C 65.39, H 7.41, CI 8.36%. Reference H. Sasaki, T. Hosokawa, Y. Nawata, and K. Ando; Isolation and Structure of Ascochlorin and Its Analogs; Agr. Biol. Chem., (8), pp. 1463-1466(1974).

Helminthosporois Helminthosporol Prehelminthosporolactone Prehelminthosporol Dihydroprehelminthosporol Helminthosporal acid Helminthosporic acid Helminthosporal

527

This Page Intentionally Left Blank

19. Helminthosporols

529

Common/Systematic Name Helminthosporol Molecular Formula/Molecular Weight C15H2402; MW = 236.17763

L General Characteristics Recrystallization from n-hexane gave colorless needles melting at 98~ [aiD 23 -28.7 ~ (c=1.93, in CHC13). It was soluble in most organic solvents such as benzene, chloroform, ethyl acetate, ether, acetone, and alcohols, but almost insoluble in water. Fungal Source Culture broth ofHelminthosporium sativum, reclassified as Bipolaris sorokiniana which is known to cause seedling blight, foot and root rot, head blight, and leaf spot of cereals and grasses. Isolation/Purification The cultured broth was filtered and the filtrate treated with charcoal. The charcoal and the mycelia were separated and extracted respectively with acetone. The neutral fraction from these acetone extracts was purified by silicic acid column chromatography using benzene, benzene-2% ethyl acetate and benzene-7% ethyl acetate. Helminthosporol was obtained from the latter fraction and crystallized from n-hexane. The fungus was cultured on liquid CZ8 medium. The cultures were stirred at 350rpm for two days at 30~ and aerated at 10,000cm3/min. The mycelia were separated from the culture filtrate by gravity filtration. The filtrate was extracted with EtOAc in a separatory funnel and concentrated under reduced pressure. The crude extract was suspended in a 9:1 (v/v) mixture of H20-MeOH and the resulting aqueous solution extracted three times with hexane, three times with EtOAc and once with n-BuOH (2:1, v/v). Drying with anhydrous Na2SO4 and evaporation yielded low, medium, and high polarity fractions, respectively. Leaf-spot assay of the three extracts indicated that the strongest biological activity was located in the low polarity fraction. The toxic, low polarity fraction was purified by preparative TLC C6I-I6-Me2CO (80:20, v/v; Re 0.47), CH2CI2-Me2CO(90:10, v/v ,Re 0.50) which yielded highly purified helminthosporol. Biological Activity Plant growth regulator; promoted shoot growth of rice seedlings.

530

19. Helminthosporols

Spectral Data UV:

~ EtOH 267nm (c - 9,700) max

IR:

(Nujol) 3440, 1645, 1610, and 1025cml; (CHCI3) 3500, 2740, 1665, and 1620cm"1. IH NMR: (CDCI3) 10.03(1H, s, H-14); 3.63(1H, dd, ./=11,5, 5.5Hz, H-13); 3.32(1H, dd, 3"--11, 8Hz, n-13); 2.00(3H, s, n-12); 1.66(1H, dd, ./=8, 5Hz, H-8); 1.05(3H, d, ./=6Hz, H-10 or H-l 1); 1.02(3H, s, H-15); and 0.75ppm (3H, d, ./=5Hz, H-10 or H-11). Mass Data: Found: C, 76.22; H, 10.14%; MW 227 (Rast); calcd for C15H2402; C, 76.22; H, 10.24%; MW 236. References L. M. Pefia-Rodriguez, N. A. Armingeon, and W. S. Chilton; Toxins from Weed Pathogens, I. Phytotoxins from A Bipolaris Pathogen of Johnson Grass; J. Natural Products, Vol. 51, pp. 821-828(1988). S. Tamura, A. Sakurai, K. Kainuma, and M. Takai; Isolation of Helminthosporol as a Natural Plant Growth-Regulator and Its Chemical Structure; Agric. Biol. Chem., Vol. 29, pp. 216-221(1965).

19. Helminthosporols

531

Common/Systematic Name Prehelminthosporolactone Molecular Formula/Molecular Weight C15H2202; MW = 234.16198

Fungal Source

Bipolaris sp. strain 36 (ATTC 64838), a fungal pathogen of Johnson grass [Sorghum hatepense (L.) Pers].

General Characteristics Colorless needles Isolation/Purification Purification of the crude fungal mixture was accomplished by flash column chromatography using Merck silica gel 60 and stepwise elution with n-hexane-Et20 (90:10 to 30:70, v/v). Biological Activity Phytotoxic, produced lesions on Johnson grass and sorghum resembling those observed in the fungal disease in the field; a reddish brown center surrounded by a black circle at the edge of the drop containing the phytotoxin, with an outer, chlorotic zone extending beyond the drop. Reference L. M. Pefia-Rodriguez and W. S. Chilton; Victoxinine and Prehelminthosporolactone, Two Minor Phytotoxic Metabolites Produced by Bipolaris sp., A Pathogen of Johnson Grass; J. Natural Products, Vol. 52, pp. 899-901(1989).

532

19. Helminthosporols

Common/Systematic Name Prehelminthosporol Molecular Formula/Molecular Weight

~

C15H2402; M W -- 2 3 6 . 1 7 7 6 3 9

HO.~

3

Is

H" \0 General Characteristics A colorless oil. Prehelminthosporol occurs as a mixture of epimers at the hemiacetal carbon. It is hydrophobic, has low water solubility, and is unstable to heat. Fungal Source

Bipolaris sp., a pathogen of Johnson grass (Sorghum halepense L.); Helminthosporium sativum (reclassified as Bipolaris sorokiniana, which is known to cause seedling blight, foot and root rot, head blight and leaf spot of cereals and grasses); Cochliobolus sativus and C. setariae, which are the perfect stages ofBipolaris sorokiniana and B. setariae.

Isolation/Purification The fungus was cultured on liquid CZ8 medium. The cultures were stirred at 350RPM for two days at 30~ and aerated at 10,000 cm3/min. The mycelia were separated from the culture filtrate by gravity filtration. The filtrate was extracted with EtOAc in a separatory funnel and concentrated under reduced pressure. The crude extract was suspended in a 9:1 (v/v) mixture of H20-MeOH and the resulting aqueous solution extracted three times with hexane, three times with EtOAc and once with n-BuOH (2:1, v/v). Drying with anhydrous NazSO4 and evaporation yielded low, medium, and high polarity fractions, respectively. Leaf-spot assay of the three extracts indicated that the strongest biological activity was located in the low polarity fraction. The toxic, low polarity fraction was purified by flash column chromatography by stepwise elution with Cd-I6-Me2CO mixtures (95:5 to 70:30, v/v) which yielded a highly active colorless oil identified as prehelminthosporol. Biological Activity Very phytotoxic to sorghum and Johnson grass at 25~g/51.tl droplet. Toxin interacts with host membranes (plasmalemma and tonoplast) resulting in cell death and leakage of metabolites; induces both chlorosis and necrosis in plant tissues.

19. Helminthosporols

533

Spectral Data

IR: (thin film on KBr) 3404, 3067, and 1655cmq. 1H NMR: (CDCI3) major epimer 4.94ppm*(1H, d, J=2.7Hz, H-14); 4.90(1H, s, H-12); 4.81(1H, s, H-12); 3.99(1H, dd, J=l 1.5, 2Hz, H-13); 3.57(1H, dd, J=l 1.5, 2Hz, H-13); 3.18(1H, brs, OH); 2.69(1H, br s, H-6); 2.52(1H, br s, H-5); 1.16(3H, s, H-15); 0.94(3H, d, J=6Hz, H-10 or H-11); and 0.88ppm (3H, d, J=6Hz, H-10 or H-11); minor isomer 4.84(1H, s, H-12), 4.73(1H, s, H-12); 4.67"(1H, br s, H-14); 3.93(1H, dd, J=l 1.5, 1.SHz, H-13); 3.77(1H, dd, J=l 1.5, 2Hz, H-13); 2.73(1H, brs, OH); 2.61(1H, br s, H-6); 2.09(1H, br s, H-5); 1.19(3H, s, H-15); 0.97(3H, d, J=6Hz, H-10 or H-11); and 0.87ppm (3H, d, J=6Hz, H-10 or H-11). Carlson et al., 1991 have assigned a different shift and multiplicity for H-14 (H-14 gave a doublet (J=12Hz) at 4.66ppm for the major epimer and an unresolved doublet at 4.95ppm for the minor isomer).

13C NM~: (CDC13) epimeric mixture 157.83(s, C-7); 157.25(s, C-7'); 101.92(t, C-12), 101.07(t, C-12'); 97.09(d, C-14); 96.00(d, C-14'); 67.64(t, C-13); 64.71(t, C-13'); 51.64(d, C-5, C-6 or C-8); 50.48(d, C-5, C-6 or C-8 and C-5', C-6' or C-8'); 48.87(d, C-5', C-6' or C8'); 46.54(d, C-5, C-6 or C-8); 4.78(s, C-I); 44.53(d, C-5',C-6' or C-8'); 43.60(s, C-I'); 41.95(t, C-2 and C-2'); 36.96(d, C-4 and C-4'); 30.73(d, C-9 and C-9'); 25.79(t, C-3 and C-3'); 20.91(q, C-10, C-11 or C-15 and C-10', C-11' or C-15'); 28.81(q, C-10, C11 or C-15); 20.16(q, C-10', C-11' or C-15'); and 18.88ppm (q, C-10, C-11 or C-15 and C-10',C-11' or C-15'). Mass Spectrum: HR S: [M] + calcd for C~5H2402, 236.1776, found 236.1796m/e; CIMS: (isobutane) 237(37%), 219(100), and 191role (77). Plasma spray mass spectroscopy 237(M + + 1) and 219role (loss of water). TLC Data Rf: 0.70(C6H6-Me2CO; 80:20, v/v), 0.58(C6H6-Et20; 60:40, v/v), and 0.52(petroleum ether-Me2CO; 85:15, v/v). HPLC Data Normal phase column was Nucleosil 50-5, silicic acid (Machery-Nagel, Duren, FRG). The mobile phase was hexane/ethyl acetate (80:20, v/v) at a flow rate of 3ml/min. Reverse phased column was Kromasil 100-5 (C~s) with a mobile phase of acetonitrile/water (70:30, v/v) at a flow rate of 1 ml/min.

534

19. Helminthosporols

References H. Carlson, P. Nilsson, H. B. Jansson, and G. Odham; Characterization and Determination ofPrehelminthosporol, a Toxin from the Plant Pathogenic Fungus Bipolaris sorokiniana, Using Liquid Chromatography/Mass spectrometry; J. Microbiol. Methods, Vol. 13, pp. 259-269(1991). L. M. Pefia-Rodriguez, N. A. Armingeon, and W. S. Chilton; Toxins from Weed Pathogens, I. Phytotoxins from A Bipolaris Pathogen of Johnson Grass; J. Natural Products, Vol. 51, pp. 821-828(1988).

19. Helminthosporols

535

Common/Systematic Name Dihydroprehelminthosporol Molecular Formula/Molecular Weight C15H2602; m w = 2 3 8 . 1 9 3 2 8

12

HOH2C

CH2OH

Fungal Source Bipolaris sp., a pathogen of Johnson grass (Sorghum halepense L.); Helminthosporium sativum (reclassified as Bipolaris sorokiniana which is known to cause seedling blight, foot and root rot, head blight and leaf spot of cereals and grasses); Cochliobolus sativus and C. setariae, which are the perfect stages ofBipolaris sorokiniana and B. setariae. Isolation/Purification The fungus was cultured on liquid CZ8 medium. The cultures were stirred at 350RPM for two days at 30~ and aerated at 10,000 cm3/min. The mycelia were separated from the culture filtrate by gravity filtration. The filtrate was extracted with EtOAc in a separatory funnel and concentrated under reduced pressure. The crude extract was suspended in a 9:1 (v/v) mixture of H20-MeOH and the resulting aqueous solution extracted three times with hexane, three times with EtOAc and once with noBuOH (2:1, v/v). Drying with anhydrous Na2SO4 and evaporation yielded low, medium, and high polarity fractions, respectively. Leaf-spot assay of the three extracts indicated that the strongest biological activity was located in the low polarity fraction. The toxic, low polarity fraction was purified by preparative TLC [C6I-Ls-Et2CO(30:70, v/v; Re ,0.43)] which yielded highly purified dihydroprehelminthosporol. Biological Activity Very phytotoxic to sorghum and Johnson grass at 12.51~g/51~1droplet. Soectral Data IR:

(thin film on KBr) 3335, 3076, and 1647cm"1. 1H N]V[R(CDCI3) 4.90(1H, d, J=3Hz, H-12); 4.79(1H, d, J=3Hz, H-12); 3.68(1H, dd, J=ll, 6Hz, n-14); 3.66(1H, dd, J=l 1, 6.5Hz, n-13); 3.46(1H, dd, J=l 1, 9Hz, H-14);

536

19. Helminthosporols

3.75(1H, dd, J=l 1, 9Hz, H-13); 2.52(1H, br t, J=8Hz, H-6); 2.37(1H, s, H-5); 1.57(1H, br t, ,/=8 Hz, n-8); 0.99(3H, s, n-15); 0.96(3H, d, J=7Hz, n-10 or H-11); and 0.84ppm (3H, d, J=7Hz, H-10 or H-11).

13C NMR: (CDCI3) 158.69(s, C-7); 104.50(t, C-12); 66.44(t, C-13 or C-14); 63.11(t, C-13 or C-14); 57.39(d, C-5, C-6 or C-8); 49.27(d, C-5, C-6 or C-8); 47.28(s, C-l); 45.67(d, C-5, C-6 or C-8); 42.72(t, C-2); 37.71(d, C-4); 30.61(d, C-9); 24.97(t, C-3); 21.34(q, C-10, C-11 or C-15); 20.66(q, C-10, C-11 or C-15); and 20.00ppm (q, C-10, C-11 or C-15). Mass Spectrum: HRMS: [M] § calcd for C~5H2602, 238.1933; found 238.1928re~e; CIMS: (isobutane) 239(16%), 221(100), 208(16), 203(78), and 191m/e (86). Reference L. M. Pefia-Rodriguez, N. A. Armingeon, and W. S. Chilton; Toxins from Weed Pathogens, I. Phytotoxins from A Bipolaris Pathogen of Johnson Grass; J. Natural Products, Vol. 51, pp. 821-828(1988).

19. Helminthosporols

537

Common/Systematic Name Helminthosporal acid Molecular Formula/Molecular Weight C15H2203; MW

--

250.15689

9

15 14

COOH 13

Fungal Source

Bipolaris sp., a pathogen of Johnson grass (Sorghum halepense L.) and Helminthosporium sativum (reclassified as Bipolaris sorokiniana which is known to cause seedling blight, foot and root rot, head blight and leaf spot of cereals and grasses.).

Isolation/Purification The fungus was cultured on liquid CZ8 medium. The cultures were stirred at 350RPM for two days at 30~ and aerated at 10,000 cma/min. The mycelia were separated from the culture filtrate by gravity filtration. The filtrate was extracted with EtOAc in a separatory funnel and concentrated under reduced pressure. The crude extract was suspended in a 9:1 (v/v) mixture Of HEO-MeOH and the resulting aqueous solution extracted three times with hexane, three times with EtOAc and once with n-BuOH (2:1, v/v). Drying with anhydrous Na2SO4 and evaporation yielded low, medium, and high polarity fractions, respectively. Leaf-spot assay of the three extracts indicated that the strongest biological activity was located in the low polarity fraction. The toxic, low polarity fraction was purified by preparative TLC (C6H6-Et2CO, 70:30, v/v; Rf, 0.62) which yielded highly purified helminthosporal acid. Spectral Data UV: MeOH

max

267nm.

IR:

(thin film on KBr) 1702, 1670, and 1631cm"~.

~H N-MR: (CDCI3) 9.99(1H, s, H-14); 3.39(1H, s, H-S); 2.35(1H, s, H-8); 2.02 (3H, s, H-12); and 1.17ppm (3H, s, H-15).

538

19. Helminthosporols

Mass Spectrum: HRMS [M] § calcd for C15H2203,250.1542; found 250.1563re~e; CIMS: (isobutane) 251 (100%) and 233role (1). Reference L. M. Pefia-Rodriguez, N. A. Armingeon, and W. S. Chilton; Toxins from Weed Pathogens, I. Phytotoxins from A Bipolaris Pathogen of Johnson Grass; J. Natural Products, Vol. 51, pp. 821-828(1988).

19. Helminthosporols

539

Common/Systematic Name Helminthosporic acid Molecular Formula/Molecular Weight C15H2403; MW' - 252.17254 9

..._.

HOOC

I

-15

~4 CH2OH 13

Fungal Source

Bipolaris sp., a pathogen of Johnson grass (Sorghum halepense L.), Helminthosporium sativum (reclassified as Bipolaris sorokiniana which is known to cause seedling blight, foot and root rot, head blight and leaf spot of cereals and grasses.).

Isolation/Purification The fungus was cultured on liquid CZ8 medium. The cultures were stirred at 350RPM for two days at 30~ and aerated at 10,000 cm3/min. The mycelia were separated from the culture filtrate by gravity filtration. The filtrate was extracted with EtOAc in a separatory funnel and concentrated under reduced pressure. The crude extract was suspended in a 9:1 (v/v) mixture of H20-MeOH and the resulting aqueous solution extracted three times with hexane, three times with EtOAc and once with n-BuOH (2:1, v/v). Drying with anhydrous Na2SO4 and evaporation yielded low, medium, and high polarity fractions, respectively. Leaf-spot assay of the three extracts indicated that the strongest biological activity was located in the low polarity fraction. The toxic, low polarity fraction was purified by preparative TLC (C6I-I~-Me2CO, 80:20, v/v; Rf 0.24) which yielded highly purified helminthosporic acid. Spectral Data IR;

(thin film on KBr) 3343, 1670, and 1623cm"~. 1H NMR: (CDCI3) 3.66(1H, dd, J=11, 4Hz, H-13); 3.39(1H, dd, J=11, 9Hz, H-13); 3.15(1H, s, H-5); 2.00(3H, s, n-12); 1.63(1H, dd, J=9, 4Hz, H-8); 1.02(3H, d, J=6Hz, H-10 or H-11); 0.96(3H, s, H-15); and 0.77ppm (3H, br s, H-10 or H-11) Mass Spectrum: HRMS" [M] + calcd for C15H2403, 252.1725; found 252.1725re~e; CIMS: (isobutane) 253(5%) and 235m/e (100).

540

19. Helminthosporols

Reference L. M. Pefia-Rodriguez, N. A. Armingeon, and W. S. Chilton; Toxins from Weed Pathogens, I. Phytotoxins from A Bipolaris Pathogen of Johnson Grass; J. Natural Products, Vol. 51, pp. 821-828(1988).

19. Helminthosporols

541

Common/Systematic Name Helminthosporal Molecular Formula/Molecular Weight C15H2202; MW = 234.16198

.•L,,

CliO CHO

General Characteristics Crystallized twice from light petroleum to give helminthosporal; mp., 56-59~ (C= 1.18, in CHCI3). Unstable to air, even at low temperature.

[a]D -19 ~

Fungal Source Helminthosporium sativum, a fungus which produces a seedling blight, foot and root rot, head blight, and leaf spot of cereals and grasses. The disease is widespread in North America and elsewhere and in particular affects wheat and barley crops. Isolation/Purification The culture filtrate was adjusted to pH 2 and activated charcoal was added and stirred. The charcoal with the absorbed toxin was separated from the liquid in a Tolhurst centrifuge and suspended in ethanol. After filtering on a Buchner funnel, the charcoal cake was suspended in chloroform, stirred, and filtered. This process was repeated three times. The chloroform extracts were combined and the solvent removed in vacuo to yield a crude oil extract. The oil was heated under nitrogen at 120~ for 12 hours. During this time small amounts of water, ethanol, and chloroform were lost, leaving a residue which was dissolved in light petroleum (bp, 60-80 ~C) and applied to a column of alumina (Merck, acid-washed). Elution with the same solvent gave a trace of oil. Elution with ether gave, after evaporation, a brown oil. Distillation of this oil (bp, 115-120/0.015mm) afforded a yellow oil which slowly set. A portion was crystallized twice from light petroleum to give helminthosporal. Biological Activity Phytotoxic, produces seedling blight, foot and root rot, and leaf spot of cereals in North America. The action of the fungus in producing disease is largely dependent on the action of the toxin. Spectral Data UV: ~. mEtOH 266nm (c=l 1,000). ax

542

19. Helminthosporols IR;

(CC14) 1715, 1685, and 1618cmq. Mass Data: Found: C, 76.72; H, 9.27; C-Me, 12.56%; MW. (isopiestic, av.) 232, (Rast, av.) 247; calcd for C15H2202: C, 76.88; H, 9.46; C-Me, (2), 12.83%; MW 234. References P. De Mayo. E. Y. Spencer, and R. W. White; Helminthosporal, The Toxin from Helminthosporium sativum: Isolation and Characterization; Canadian Journal of Chemistry, Vol.39, pp. 1608-1612(1961). P. De Mayo. E. Y. Spencer, and R. W. White; The Constitution ofHelminthosporal; J. Am. Chem. Soc., Vol. 84, pp. 494-495(1962). P. De Mayo. E. Y. Spencer, and R. W. White; IV. The Structure and Stereochemistry of Helminthosporal; Canadian Journal of Chemistry, Vol.41, pp. 2996-3004(1963).

Hebevinosides and Hebelomic Acids Hebevinoside I Hebevinoside II Hebevinoside III Hebevinoside IV Hebevinoside V Hebevinoside VI Hebevinoside VII Hebevinoside VIII Hebevinoside IX Hebevinoside X Hebevinoside XI Hebevinoside XII Hebevinoside XIII Hebevinoside XIV Hebelomic acid A (HS-A) Hebelomic acid B Hebelomic acid E Hebelomic acid F HS-B HS-C

543

This Page Intentionally Left Blank

20.

Hebevinosides and Hebelomic Acids

545

Common/Systematic Name Hebevinoside I 313,1613-Dihydroxy-713-methoxycucurbita-5,24-diene-3-O-13-D-xylopyranoside- 16-0-(6-0acetyl)-13-D-glucopyranoside Molecular Formula/Molecular Weight C44H72013; M W -- 808.49729

H2CH=CMe2

,,.

H H O

o~O~

Me

AcOH2COI

HO" ~ OH

OH H OH

General Characteristics Amorphous; dihydrohexaacetate: colorless needles from methanol; m.p. 176-177 ~ [a]D22 + 16. ~ (C=0.51, in MeOH). Fungal Source

Hebeloma vinosophyllum (a toxic mushroom).

Isolation/Purification The n-butanol soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with C6I-I6-acetone and on Sephadex LH-20 with CHCI3-MeOH had afforded several hebevinosides. Spectral Data UV:

~. A0oto~i~o End absorption. max

IR:

(KBr) 3400, 1735(ester), 1635, 1075, and 1040cm"1.

546

20.

Hebevinosides and Hebelomic Acids

IHNMR (CDCI3) 0.88(3H, d, J=6.3Hz); 0.89(6H, d, J=6.6Hz); 1.99, 2.01, 2.0, 2.04, 2.06(each 3H, s, Ac x 5); and 2.02ppm (6H, s, Ac x 2). ~3CNMR: (CsDsN) C-3, 87.3; C-5, 148.0; C-6, 119.7; C-7, 77.9; C-16, 82.3; C-24, 126.9; C25, 130.1; OMe, 56.3; xyl-1, 107.3; xyl-2, 74.8; xyl-3, 78.5; xyl-4, 71.1; xyl-5, 66.8; Glu-1, 106.6; glu-2, 75.1; glu-3, 78.0; glu-4, 71.7; glu-5, 75.5; glu-6, 64.7; COCH3, 20.8; and COCH3, 170.6ppm. Mass Spectrum: (FD-MS of dihydrohexaacetate). 1062m/e (M~). Reference H. Fujimoto, K. Suzuki, H. Hagiwara, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. I. Isolation and Structures of Hebevinosides I, II, III, IV and V, Chem. Pharm. Bull., Vol. 34, pp. 88-89(1986).

20.

Hebevinosides and Hebelomic Acids

547

Common/Systematic Name Hebevinoside II 313,713, 16[3-Trihydroxycucurbita-5,24-diene-3-O- 13-D-xylopyranoside- 16-O-(6-O-acetyl)13-D-glucopyranoside Molecular Formula/Molecular Weight C4sH72014; MW = 836.49221

H

O~

O

"'

........ C~H2~H2CH=CMe2

AcOH2C O I

AcO"--'7 OH

H

OH OH

General Characteristics Amorphous; [a]D 20 + 3 0 ~ (c=0.20, in acetone). Fungal Source

Hebeloma vinosophyllum (a toxic mushroom).

Isolation/Purification The n-butanol soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with C6H6-acetone and on Sephadex LH-20 with CHCI3-MeOH had afforded several hebevinosides. Spectral Data UV: Aeetonitrile max

End

absorption.

IR:

(KBr) 3400, 1735(ester), 1640, 1075, and 1035cm1.

548

20.

Hebevinosides and Hebelomic Acids

IH~: (CDCI3) 0.88, 1.12, 1.17, 1.38, 1.61(each3H, s); 1.04(3H, d, J=6.8Hz, H3-21); 1.70, 1.73(each 3H, s,H3-26 and 1-13-27);1.96,2.04(each 3H, s, Ac); 4.42(IH, brd, J=5. IHz, H-7); 4.67, 4.82(each IH, d, J=7.7Hz, H-x3,1-1and H-glc-1); 5.53(IH, brt,J=7.7Hz, H-24); and 6.12ppm (IH, d, J=5.1Hz, H-6); acetate(CDCI3) 1.99,2.01, 2.04, 2.10(each 3H, s, Ac X 4); 2.00, 2.02ppm (each 6H, s, Ac X 4). 13CNMR: (CsDsN) C-3, 87.4; C-5, 146.2; C-6, 122.5; C-7, 67.5; C-16, 82.2; C-24, 126.8; C-25, 130.0; xyl-1, 107.3; xyl-2, 74.9; xyl-3, 74.9; xyl-4, 73.1; xyl-5, 63.1; glu-1, 106.5; glu-2, 75.2; glu-3, 78.3; glu-4, 71.7; glu-5, 75.4; glu-6, 64.7; COCH3, 20.8, 20.8; and COCH3, 170.5, 170.7ppm. Reference H. Fujimoto, K. Suzuki, H. Hagiwara, and M. Yamazaki; New Toxic Metabolites from Mushroom, Hebeloma vinosophyllum. I. Isolation and Structure of Hebevinosides I, II, III, IV, and V; Chem. Pharm. Bull., Vol. 34, pp. 88-89(1986).

20.

Hebevinosides and Hebelomic Acids

549

Common/Systematic Name Hebevinoside III 313,713,1613-Trihydroxycucurbita-5,24-diene-3-O- [3-D-xylopyranoside- 16-O-(6-O-acetyl)[3-D-glucopyranoside Molecular Formula/Molecular Weight C43H70013; M W -- 794.48164

H2CH=CMe2

IIIi,,,.

O o~O~ HO

OH AcOH2C IO~HH ~

~

OH

HO OH

General Characteristics Amorphous. Funsal Source

Hebeloma vinosophyllum, a toxic mushroom.

Isolation/Purification The n-butanol soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with CrI-Ir-acetone and on Sephadex LH20 with CHCI3-MeOH had afforded several hebevinosides. Spectral Data UV: ~, Acetonitrile max

End

absorption.

IR:

(KBr) 3400, 1735 (ester), 1640, 1080, and 1040cm"1.

550

20.

Hebevinosides and Hebelomic Acids

1H ~ : (CDCI3) 0.88, 1.12, 1.18, 1.36, 1.61(each 3H, s); 1.04(3H, d, J=6.0Hz, H3-21); 1.69, 1.72(each 3H, s, H3-26 and H3-27); 2.05(3H, s, Ac); 4.41(IH, brd, J=5.6I-Iz, H-7); 4.67, 4.8(each IH, d, J=7.7Hz, H-xyl-I and H-glc-l); 5.52(IH, brt, d=6.0I-Iz, H-24) and 6.12ppm (IH, d, J=5.6Hz, H-6). 13CNMR: (CsDsb0 C-3, 87.4; C-5, 146.3; C-6, 122.4; C-7, 67.5; C-16, 82.2; C-24, 126.8; C25, 129.9; xyl-1, 107.7; xyl-2, 75.1; xyl-3, 78.4; xyl-4, 71.1; xyl-5, 66.8; glu-1, 106.6; glu-2, 75.0; glu-3, 78.6; glu-4, 71.9; glu-5, 75.6; glu-6, 64.8; COCH3, 20.8; and COCH3, 170.Sppm.

Reference H. Fujimoto, K. Suzuki, H. Hagiwara, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. I. Isolation and Structures of Hebevinosides I, II, III, IV and V, Chem. Pharm. Bull., Vol. 34, pp. 88-89(1986).

20.

Hebevinosides and Hebelomic Acids

551

Common/Systematic Name Hebevinoside IV 313,1613-Dihydroxy-713-methoxycucurbita-5,24-diene-3-O-13-D-xylopyranoside Molecular Formula/Molecular Weight C36I--I6007 ; M W -" 6 0 4 . 4 3 3 9 0

. . .,,,.. . . CH2CH2CH=CMe2 OH

0v . ~

,,,,,-- \

"r

"OMe

HO--- I OH General Characteristics Colorless needles from methanol; mp., 172-173 ~ positive to both the Liebermann-Burchard and Molisch reactions. On catalytic hydrogenation afforded a dihydro derivative and on acetylation, a tetraacetate. Catalytic hydrogenation of acetate gave dihydrotetraacetate; mp., 122-125~ [a]D17 +64 ~ (C=0.081, in MeOH). Tetraacetate; mp., 129-130~ Fungal Source

Hebeloma vinosophyllum (a toxic mushroom).

Isolation/Purification The n-butanol soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with C6I-I6-acetone and on Sephadex LH20 with CHC13-MeOH had afforded several hebevinosides. Spectral Data UV: /~, Aeetonitrile E n d max

absorption.

IR:

(KBr) 3410, 1638(ester), 1075, and 1038cm1.

552

20.

Hebevinosides and Hebelomic Acids

IH ~ : (CDCI3) 0.69,0.97, 1.00, 1.12, 1.22(each3H, s);0.99(3H, d, J=8.1Hz, H3-21); 1.62, 1.70(each 3H, s,H3-26 and H3-27); 3.34(3H, s, OCH3); 3.42(IH, brd,J=5.6Hz, H-7); 3.98(IH, dd, 0"I=I1.8,J2=4.5Hz, H-3); 4.36(IH, d, J=6.0Hz, H-xyl- I);4.44(IH, dd, JI=7.3, J2=13.7Hz, H-16); 5.19(IH, brt, J=7.TI-Iz,H-24); and 5.74ppm (IH, d, J=5.6Hz, H-6). dihydro derivative(CDCI3) 0.88(6H, d, J=7Hz); 0.97ppm (3H, d, J=SHz). acetatederivative(CDCI3) 2.03(6H, s, Ac • 2); 2.04, and 2.05ppm (each 3H, s, Ac x 2). 13C NMR: (CsDsN) C-3, 87.4; C-5, 147.9; C-6, 119.3; C-7, 77.8; C-16, 71.1; C-24, 126.2; C25, 130.5; OMe, 56.2; xyl-1,107.8; xyl-2, 74.9; xyl-3, 78.5; xyl-4, 71.2; and xyl-5, 67.1ppm. Mass Spectrum: EIMS: 572role (19%, M + - CH3OH); FDMS: (acetate derivative) 772m/e (M+). Reference H. Fujimoto, K. Suzuki, H. Hagiwara, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. I. Isolation and Structures of Hebevinosides I, II, III, IV and V, Chem. Pharm. Bull., Vol. 34, pp. 88-89(1986).

20.

Hebevinosides and Hebelomic Acids

553

Common/Systematic Name Hebevinoside V 313,1613-Dihydroxy-713-methoxycucurbita-5,24-diene-3-O-(4-O-acetyl)-13-D-xylopyranoside- 16-O-(6-O-acetyl)-13-D-glucopyranoside Molecular Formula/Molecular Weight C46H74014; MW = 850.50786

..... CH2~H2CH=CMe2 ',..

H O co. c

AcO~

OH

H

~ I

OH OH

General Characteristics Obtained as colorless needles from methanol; mp., 165-166~ CHCI3).

[a]D 20 +83 ~ (era0. 053, in

Fungal Source

Hebeloma vinosophyllum (a toxic mushroom).

Isolation/Purification The n-butanol soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with C6H6-acetone and on Sephadex LH20 with CHCI3-MeOH had afforded several hebevinosides. Spectral Data UV:

Ao=~m~o

max

End absorption.

IR:

(KBr) 3420, 1743, 1725, 1640, 1087, and 1032cm"1.

554

20.

Hebevinosides and Hebelomic Acids

1H NMR:

(CDCI3) 0.68, 0.97, 1.01, 1.12, 1.22(each 3H, s); 0.96(3H, d, J=5.6Hz, H3-21); 1.62, 1.69(each 3H, s, 1-I3-26and H3-27); 2.10(6H, s, hc x 2); 3.34(3H, s, OMe); 3.42(1H, br d, J=6.2Hz, H-7); 4.07(1H, dd, Jl=12.0, ,/2=4.3 Hz, H-glc-6); 4.16(1H, dd, partly overlapped with other signals, I-~-glc. 6); 4.42(2H, d, J=6.4Hz, H-xyl-I and H-glc-l); 4.81(1H, dt, J 1=J2=7.7, J3=5.0Hz, H-xyl-4); 5.14(1H, br t, Jl=J2=6.9Hz, H=24); and 5.75ppm (1H, d, J=6.2Hz, n-6). 13C M R : (CsDsN) C-3, 87.4; C-5, 147.8; C-6, 119.5; C-7, 77.8; C-16, 82.2; C-24, 126.9; C25, 130.0; OMe, 56.3; xyl-1, 107.5; xyl-2, 75.0; xyl-3, 78.4; xyl-4, 73.1; xyl-5, 63.2; glu-1, 106.8; glu-2, 75.0; glu-3, 78.3; glu-4, 71.7; glu-5, 75.4; glu-6, 64.5; COCH3, 20.9, 20.9; and COCH3, 170.5, 170.6ppm. Reference H. Fujimoto, K. Suzuki, H. Hagiwara, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vmosophyllum. I. Isolation and Structures of Hebevinosides I, II, III, IV and V, Chem. Pharm. Bull., Vol. 34, pp. 88-89(1986).

20.

Hebevinosides and Hebelomic Acids

555

Common/Systematic Name Hebevinoside VI 6-3-O-[3-D-Xylopyranoside- 16-O-13-D-glucopyranoside Molecular Formula/Molecular Weight C41H6sO12; MW = 752.47108

lllli,,,

H

0

0

HO "=-=I OH

General Characteristics Amorphous; [tt]D2a + 56~

H

OH

o' OH

in pyridine).

Fungal Source

He beloma vinosophyllum.

Isolation/Purification Dried fruit bodies obtained by cultivation ofH. vinosophyllum were cut into fine pieces and shaken in n-hexane at room temperature. The defatted fruit bodies were extracted with acetone whose pH was kept at 7 by occasional addition of small amounts of pyridine at room temperature to afford the acetone extract. The acetone extract was separated by centrifugal TLC with CHCl3-acetone (1:1, v/v), CHCl3-acetone-tetrahydrofuran (THF) (1:1:0.1), (1:1:0.2), (1:1:0.5, v/v), and THF. A mixture of the latter half of the fraction eluted with CHCl3-acetone (1:1, v/v) and the first half of the fraction eluted with CHC13-acetone-THF (1:1:0.1, v/v/v) was further chromatographed on a Sephadex LH20 column with CHC13 and CHCI3-THF (20:1, v:v) to afford hebevinoside VIII. A mixture of the latter half of the fraction eluted with CHC13-acetone-THF (1:1:0.1, v/v/v) and the fraction eluted with CHCl3-acetone-THF (1:1:0.2, v/v/v) was further chromatographed on a Sephadex LH20 column with CHC13-THF (10:1, v/v) to afford hebevinoside II, hebevinoside VII, and hebevinoside IX. A mixture of the fraction eluted with CHCl3-acetone-THF (1:1:0.5, v/v/v) and the first half of the fraction eluted with THF was further chromatographed on a Sephadex LH20 column with CHCI3-THF (10:1, v/v) and THF to afford hebevinosides III and VI.

556

20.

Hebevinosides and Hebelomic Acids

Biological Activity IP administration of 100mg/kg to mice was LD~oo ; mice died atter paralysis; LDs0 was 66mg/kg (IP dose). Spectral Data UV~

End absorption. IR~

(KBr) 3380, 1635, 1075, and 1035cm"1. IH NMR:

(CsDsN) 0.78; 1.12; 1.17; 1.35; 1.63(3H each, s); 1.04(3H, d, J=6.4Hz, H-21); 1.68, 1.72(3H each, s, H-26 and -27); 4.27(1H, d, J=4.8Hz, H-7); 4.74, 4.81(1H each, d, J=7.9Hz, H-glc-1 and H-xyl-1); 5.52(1H, t,J=7.1Hz, H-24); and 6.03ppm (1H, d, J=4.8Hz, H-6). 13C NMR: (CsDsN) C-3, 87.4; C-5, 146.2; C-6, 122.6; C-7, 67.3; C-16, 81.9; C-24, 126.8; C-25, 130.0; xyl-1,107.6; xyl-2, 75.0; xyl-3, 78.5; xyl-4, 71.1; xyl-5, 67.1; glu-1,106.6; glut, 75.7; glu-3, 78.6; glu-4, 72.1; glu-5, 78.1; and glu-6, 63.2ppm. Reference H. Fujimoto, H. Hagiwara, K. Suzuki, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. II. Isolation and Structures ofHebevinosides VI, VII, VIII, IX, X, and XI; Chem. Pharm. Bull., Vol. 35, pp. 2254-2269(1987).

20.

Hebevinosides and Hebelomic Acids

557

Common/Systematic Name Hebevinoside VII 6-3-O-13-D-Xylopyranoside- 16-O-(4,6-di-O-acetyl)-13-D-glucopyranoside Molecular Formula/Molecular Weight C43H70013; m w = 794.48164

H_

H

O

H

e~176

HO" " I OH General Characteristics Amorphous; [tt]D 2~ + 8 2

~ '

OH H

~

OH

(c=0.22, in acetone).

Fungal Source He be loma vinosophyllum. Isolation/Purification Dried fruit bodies obtained by cultivation of H. vinosophyllum were cut into fine pieces and shaken in n-hexane at room temperature. The defatted fruit bodies were extracted with acetone whose pH was kept at 7 by occasional addition of small amounts of pyridine at room temperature to afford the acetone extract. The acetone extract was separated by centrifugal TLC with CHCl3-acetone (1:1, v/v), CHCl3-acetone-tetrahydrofuran (THF) (1:1:0.1), (1:1:0.2), (1:1:0.5, v/v/v), and THF. A mixture of the latter half of the fraction eluted with CHCl3-acetone (1:1, v/v) and the first half of the fraction eluted with CHCl3-acetone-THF (1:1:0.1, v/v/v) was further chromatographed on a Sephadex LH20 column with CHC13 and CHC13 -THF (20:1, v/v) to afford hebevinoside VIII. A mixture of the latter half of the fraction eluted with CHC13-acetone-THF (1:1:0.1, v/v/v) and the fraction eluted with CHCl3-acetone-THF (1:1:0.2, v/v/v) was further chromatographed on a Sephadex LH20 column with CHCI3-THF (10:1, v/v) to afford hebevinoside II, hebevinoside VII and hebevinoside IX. A mixture of the fraction eluted with CHC13-acetone-THF (1:1:0.5, v/v/v) and the first half of the fraction eluted with THF was further chromatographed on a Sephadex LH20 colurfin with CHCI3-THF (10:1, v/v) and THF to afford hebevinosides III and VI.

558

20.

Hebevinosides and Hebelomic Acids

Spectral Data WW: L Acetomtrilr E n d max

absorption.

IR:

(KBr) 3450, 1735(ester), 1630, 1070, and 1035cm1. ~H NMR: (CsDsN) 0.86; 1.08; 1.19; 1.38; 1.63(each 3H, s); 1.03(3H, d, J=6.0Hz, 21-1-13); 1.71, 1.75(each 3H, s, 26 and 271-I3); 2.03, 2.09(each 3H, s AC); 4.43(1H, partly overlapped with other signals, H-7); 4.66(1H, d, J=7.7Hz, H-glc-1); 4.82(1H, d, J=7.7Hz, H-xyl1); 5.51(1H, t, J=9.8Hz, H-24); and 6.12ppm (1H, d, J=4.9Hz, H-6). 13C NMR: (CsDsN) C-3, 87.4; C-5, 146.4; C-6, 122.5; C-7, 67.4; C-16, 82.5; C-24, 126.9; C-25, 130.1; xyl-1, 107.6; xyl-2, 75.1; xyl-3, 78.5; xyl-4, 71.1; xyl-5, 67.1; glu-1, 106.5; glu-2, 75.6~ glu-3, 75.5*; glu-4, 72.4; glu-5, 72.4; glu-6, 63.6; COCH3, 20.8, 20.9; COCH3, 170.5 and 170.7ppm. * Assignments may be reversed. Mass Spectrum: LREIMS 818(M + - H20), 686(18.5%, M - CsHloOs[xyl.]), 668(10.2%, NY - H20, CsHloOs[xyl.]), and 653m/e (21.3%, M + - H20 - CsHloO5 - CH3). Reference H. Fujimoto, H. Hagiwara, K. Suzuki, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. II. Isolation and Structures ofHebevinosides VI, VII, VIII, IX, X, and XI; Chem. Pharm. Bull., Vol. 35, pp. 2254-2269(1987).

20.

Hebevinosides and Hebelomic Acids

559

Common/Systematic Name Hebevinoside VIII 6-3-O-(4-O-Acetyl)- [3-D-xylopyranoside- 16-0-(4, 6-di-O-acetyl)- 13-D-glucopyranoside Molecular Formula/Molecular Weight C47H74015; MW = 878.50277

H

O

MeOCO----] OH

OH

MeOCOH2Co~H (~ MeOCO"-"r OH

General Characteristics Amorphous; [t~]D2~= + 36.5 ~ (C=0.52, in CHC13). _Fungal Source

He beloma vinosophyllum.

Isolation/Purification Dried fruit bodies obtained by cultivation of H. vinosophyllum were cut into fine pieces and shaken in n-hexane at room temperature. The defatted fruit bodies were extracted with acetone whose pH was kept at 7 by occasional addition of small amounts of pyridine at room temperature to afford the acetone extract. The acetone extract was separated by centrifugal TLC with CHC13-acetone (1:1, v/v), CHC13-acetone-tetrahydrofuran (THF) (1:1:0.1), (1:1:0.2), (1:1:0.5, v/v), and THF. A mixture of the latter half of the fraction eluted with CHC13-acetone (1:1, v/v) and the first half of the fraction eluted with CHC13-acetone-THF (1:1:0.1, v/v/v) was further chromatographed on a Sephadex LH20 column with CHC13 and CHC13-THF (20:1, v/v) to afford hebevinoside VIII. A mixture of the latter half of the fraction eluted with CHCl3-acetone-THF (1:1:0.1, v/v/v) and the fraction eluted with CHC13-acetone-THF (1:1:0.2, v/v/v) was further chromatographed on a Sephadex LH20 column with CHC13-THF (10:1, v/v) to afford hebevinoside II, hebevinoside VII, and hebevinoside IX. A mixture of the fraction eluted with CHCl3-acetone-THF (1:1:0.5, v/v/v) and the first half of the fraction eluted with THF was further chromatographed on a Sephadex LH20 column with CHCI3-THF (10:1, v/v) and THF to afford hebevinosides III and VI.

560

20.

Hebevinosides and Hebelomic Acids

Spectral Data UV:

~, A ~ t ~ max

End absorption.

IR:

(KBr) 3420, 1740(ester), 1640, 1075, and 1025cm "~. 1H NMR: (CDCI3) 0.66; 1.00; 1.08; 1.22; 1.26(each 3H, s); 0.95(3H, d, J=6.6Hz, 21-H3); 1.61, 1.68(each 3H, s, 26 and 27H3); 2.10, 2.12, 2.12(each 3H, s, Ac); 3.29(1H, dd, Jl=l 1.7, J2=8.1Hz, H-xyl-5a); 3.55(1H, m, H-glc-5); 3.62(1H, t, J=9.5Hz, H-xyl-3); 3.70(1H, t, J=8.1Hz, H-xyl-3); 3.93(1H, d, J=5.5Hz, H-7); 3.95(1H, dd, partly overlapped with the signal at 3.93ppm, H-xyl-5b); 4.07(1H, dd, J1=12.1, J2=4.8Hz, H-glc-6a); 4.16(1H, dd, partly overlapped with other signals, H-glc-6b); 4.22(1H, d, J=5.6Hz, H-glc-l); 4.40(1H, d, J=5.9Hz, H-xyl-1); 4.81(1H, dt, J~=J2=8.1, J3=5.1Hz, H-xyl-4); 4.91(1H, t, J=9.5Hz, H-glc-4); 5.14(1H, t, J=6.8Hz, H-24); and 5.74ppm (1H, d, J=5.5Hz, H-6). 13C NMR: (CsDsN) C-3, 87.4; C-5, 146.3; C-6, 122.6; C-7, 67.5; C-16, 82.5; C-24, 126.9; C-25, 130.1; xyl-1,107.4; xyl-2, 75.2; xyl-3, 74.9; xyl-4, 73.1; xyl-5, 63.2; glu-1, 106.5; glu2, 75.6"; glu-3, 75.5"; glu-4, 72.4; glu-5, 72.4; glu-6, 63.6; COCH3, 20.8, 20:9, 20.9; COCH3, 170.5, 170.6, and 170.7ppm. Assignments may be reversed. Mass Spectrum: LREIMS 860(47.1%, M + - H20), 686(27.4%, M - CTn~206[xyl.]), 668(83.9%, M + H20 - C7H~206 [xyl-1]), and 653m/e (36.5%, M + - H20 - C-tH~206 - CH3). Reference H. Fujimoto, H. Hagiwara, K. Suzuki, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. II. Isolation and Structures of Hebevinosides VI, VII, VIII, IX, X, and XI; Chem. Pharm. Bull., Vol. 35, pp. 2254-2269(1987).

20.

Hebevinosides and Hebelomic Acids

561

Common/Systematic Name Hebevinoside IX 6-3-O-p-D-Xylopyranoside Molecular Formula/Molecular Weight C35HssO7; MW = 590.41825

I|llll,,

H

0

Ho

"-7 OH

General Characteristics Amorphous. Funsal Source

He beloma vinosophyllum.

Isolation/Purification Dried fruit bodies obtained by cultivation ofH. vinosophyllum were cut into fine pieces and shaken in n-hexane at room temperature. The defatted fruit bodies were extracted with acetone whose pH was kept at 7 by occasional addition of a small amounts of pyridine at room temperature to afford the acetone extract. The acetone extract was separated by centrifugal TLC with CHC13-acetone (1:1, v/v), CHCl3-acetone-tetrahydrofuran (THF) (1:1:0.1), (1:1:0.2), (1:1:0.5, v/v), and THF only. A mixture of the latter half of the fraction eluted with CHCl3-acetone (1:1, v/v) and the first half of the fraction eluted with CHCl3-acetone-THF (1:1:0.1, v/v/v) was further chromatographed on a Sephadex LH20 column with CHCI3 and CHC13-THF (20:1, v/v) to afford hebevinoside VIII. A mixture of the latter half of the fraction eluted with CHC13-acetone-THF (1:1:0.1, v/v/v) and the fraction eluted with CHC13-acetone-THF (1:1:0.2, v/v/v) was further chromatographed on a Sephadex LH20 column with CHCI3-THF (10:1, v/v) to afford hebevinoside II, hebevinoside VII, and hebevinoside IX. A mixture of the fraction eluted with CHC13-acetone-THF (1:1:0.5, v/v/v) and the first half of the fraction eluted with THF was further chromatographed on a Sephadex LH20 column with CHC13-THF (10:1, v/v) and THF to afford hebevinosides III and VI.

562

20.

Hebevinosides and Hebelomic Acids

Spectral Data

UV: ~. A~=~,

End absorption.

IH NIk,IR: (CDCI3) 0.61; 0.93; 0.97; 1.02; 1.15(each 3H, s);0.90(3H, d, J=6.2Hz, H3-21); 1.54, 1.62(each 3H, s,H3-26 and 1-13-27);3.89(IH, d, J=4.9Hz, H-7); 4.33(IH, d, J=5.91-Iz, H-x3,1-1);5.10(IH, t,J=6.2Hz, H-24); and 5.68ppm (IH, d, J=4.9Hz, H-6). Reference H. Fujimoto, H. Hagiwara, K. Suzuki, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. II. Isolation and Structures ofHebevinosides VI, VII, VIII, IX, X, and XI; Chem. Pharm. Bull., Vol. 35, pp. 2254-2269(1987).

20.

Hebevinosides and Hebelomic Acids

563

Common/Systematic Name Hebevinoside X 7-3-O- 13-D-Xylopyranoside- 16-O-13-D-glucopyranoside Molecular Formula/Molecular Weight C42H7o012; M W -- 7 6 6 . 4 8 6 7 3

,,. H

H2CH=CMe2

H

O

Me

HOH2C OI HO - ' - ] OH

OH H OH

General Characteristics Amorphous; [ a ] D 20

+63 o

(c=0.49, in MeOH).

Fungal Source

Hebeloma vinosophyllum; however, it was shown to be an artifact produced during purification of other natural hebevinosides.

Isolation/Purification The n-butanol-soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with C6I-I6-acetone and on Sephadex LH:0 with CHCI3-MeOH had afforded several previously reported hebevinosides. A residual chromatographic fraction was further chromatographed on a column of silica gel with Cd-I6-acetone (5:1) and (2:1, v/v) to afford hebevinoside XI; C6I-I6-acetone (1:1, v/v) afforded hebevinoside X. Spectral Data UV:

~, M~oH max

End absorption.

564

20.

Hebevinosides and Hebelomic Acids

IR:

(KBr) 3400, 1740(ester), 1075, and 1035cmq. IH N-MR: (CDCI3) 0.74; 1.10; 1.16; 1.24; 1.63(each 3H, s); 1.08(3H, d, J=7.3Hz, 21-1-13);1.69, 1.73(each 3H, s, 26 and 27H3); 3.21(3H, s, OMe); 3.40(IH, d, J=5.3Hz, H-7); 4.79(2H, d, J=7.6Hz, H-glc-1 and H-xyl-1);5.55(IH, t,J=6.8Hz, H-24); and 5.88ppm (1H, d, J=5.3Hz, H-6). 13C NMR: (CsDsN) C-3, 87.4; C-5, 147.7; C-6, 119.4; C-7, 77.6; C-16, 82.1; C-24, 126.8; C-25, 130.0; OMe, 56.2; xyl-l, 107.7; xyl-2, 74.9; xyl-3, 78.5; xyl-4, 71.1; xyl-5, 67.0; glu-1, 106.8; glu-2, 75.6; glu-3, 78.7; glu-4, 72.0; glu-5, 78.2; and glu-6, 63.2ppm. Reference H. Fujimoto, H. Hagiwara, K. Suzuki, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. II. Isolation and Structures of Hebevinosides VI, VII, VIII, IX, X, and XI; Chem. Pharm. Bull., Vol. 35, pp. 2254-2269(1987).

20.

Hebevinosides and Hebelomic Acids

565

Common/Systematic Name Hebevinoside XI 7-3-O- [3-D-Xylopyranoside- 16-0-(4,6-di-O-acetyl)-13-D-glucopyrano side Molecular Formula/Molecular Weight C44I-I72013; MW = 808.49729

Ih,,,..

0"

~

v

"OMe

HO ~ ' ~

OH OH

H OH

General Characteristics Amorphous; [tt]D20 +65 o (C=0"17, in MeOH). Fungal Source

Hebeloma vinosophyllum; however, it was shown to be an artifact produced during purification of other natural hebevinosides.

Isolation/Purification The n-butanol-soluble fraction was isolated from the 90% MeOH extract of dried fruit bodies of cultivated H. vinosophyllum. Repeated column chromatography of the n-BuOH-soluble fraction on silica gel with C6H6-acetone and on Sephadex LH20 with CHCI3-MeOH had afforded several previously reported hebevinosides. A residual chromatographic fraction was further chromatographed on a column of silica gel with CnH6-acetone (5:1) and (2:1, v/v) to afford hebevinoside XI; CnI-Ir-acetone (1:1, v/v) afforded hebevinoside X. Spectral Data UV:

~, M~H max

End absorption.

IR:

(KBr) 3450, 1747(ester), 1645, 1085 and 1040cm"1.

566

20.

Hebevinosides and Hebelomic Acids

IH NMR: (CDCI3) 0.68; 0.97; 1.01; 1.12; 1.21(each 3H, s); 0.96(3H, d, J=5.0Hz, 21-H3); 1.69, 1.60(each 3H, s, 26 and 27H3); 2.07, 2.12(3H, s, Ac); 3.33(1H, d, J=5.3Hz, H-7); 4.79(2H, d, J=7.6Hz, H-glc-1 and H-xyl-1); 5.55(1H, t, J=6.8Hz, H-24); and 5.88ppm (1H, d, J-5.3Hz, H-6). 13C NMR: (CsDsN) C-3, 87.4; C-5, 147.7; C-6, 119.4; C-7, 77.6; C-16, 82.1; C-24, 126.8; C-25, 130.0; OMe, 56.2; xyl-I, 107.7; xyl-2, 74.9; xyl-3, 78.5; xyl-4, 71.1; xyl-5, 67.0; glu-1, 106.8; glu-2, 75.6; glu-3, 78.7; glu-4, 72.0; glu-5, 78.2; and glu-6, 63.2ppm. Reference H. Fujimoto, H. Hagiwara, K. Suzuki, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum. II. Isolation and Structures of Hebevinosides VI, VII, VIII, IX, X, and XI; Chem. Pharm. Bull., Vol. 35, pp. 2254-2269(1987).

20.

Hebevinosides and Hebelomic Acids

567

Common/Systematic Name Hebevinoside XII

16-3-O-fi-D-Xylopyranoside-16-O-(4-O-acetyl)-fJ-D-glucopuranoside

Molecular Formula/Molecular Weight C45H74Oli; ~ = 790.52311 Ill

U

H _

O

OH

ii III

HOH

OH

OH

General Characteristics Amorphous solid;

[tg]D 22 + 6 0 ~

(c=0.36, in pyridine).

Fungal Source

Hebeloma vinosophyllum (poisonous mushroom).

Isolation/Purification Dried mycelia with primordia at growth stage II were cut into fine pieces and extracted 4 times with acetone at room temperature for 12h. The combined acetone extracts were divided with n-hexane into hexane soluble and hexane insoluble fractions. The hexane insoluble fraction was successively chromatographed on an ODS column in the HPLC system with 20% aqueous acetone (flow rate: 4.0ml/min) and an ODS column in the HPLC system with 75% aqueous CH3CN (flow rate: 2.0ml/min). Biological Activity Neurotoxic. Spectral Data UV:

~, A~tom~l= max

End absorption.

568

20.

Hebevinosides and Hebelomic Acids

IR;

(KBr) 3400, 1730, 1640, 1075, and 1035cm"l. IH NMR: (CsDsN) 0.78; 1.11; 1.20; 1.38; 1.67(each 3H, s); 1.05(3H, d, J=6.4Hz, 1-I3-21); 1.73; 1.77(each 3H, s, 1-13-26,-27); 2.06(3H, s, Ac); 4.36 (1H, d, J=4.9Hz, H-7); 4.75 (1H, d, J=7.6Hz, H-glc-1); 4.84 (1H, d, J=7.6Hz, H-xyl-1); 5.56 (IH, t, J=7.7Hz, H-24); 5.65 (1H, t, J=9.6Hz, H-glc-4); and 6.04ppm (1H, d, J=4.9Hz, H-6). 13CNMR: (CsDsN) C-3, 87.5; C,5, 146.2; C-6, 122.6; C-7, 67.3; C-16, 82.1; C-24, 126.9; C-25, 130.0; xylose-1, 107.7; xylose-2, xylose-3, 78.5; xylose-4, 71.1; xylose-5, 67.1; glucose-l, 106.5; glucose-2, 75.7~ glucose-3, 75.9*; glucose-4, 73.1; glucose-5, 75.9~ glucose-6, 62.5; COC__H3,21.1; and COCH3, 170.6ppm. * Assignments may be interchanged. Reference H. Fujimoto, K. Maeda, and M. Yamazaki, New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum III. Isolation and Structures of Three New Glycosides, Hebevinosides XII, XIII and XIV and Productivity of the Hebevinosides at Three Growth Stages of the Mushroom; Chem. Pharm. Bull., Vol. 39, pp. 1958-1961(1991).

20.

Hebevinosides and Hebelomic Acids

569

Common/Systematic Name Hebevinoside XIII 16-3-0-(3,4-Di-O-acetyl)- 13-D-xylopyranoside- 16-0-(4, 6-di-O-acetyl)- 13-Dglucopyranoside Molecular Formula/Molecular Weight C51H8oO14; M W = 916.55481 I|h,,.

U O MeOCO

L

" H

MeO MeC00 H0~C - - ' 7 MeOCO~ OH

OH General Characteristics Amorphous solid; [~]D22 +35 ~ (C=0.42, in CHC13). Fungal Source Hebeloma vinosophyllum (poisonous mushroom). Isolation/Purification Dried mycelia with primordia at growth stage II were cut into fine pieces and extracted 4 times with acetone at room temperature for 12h. The combined acetone extracts were divided with n-hexane into hexane soluble and hexane insoluble fractions. The hexane insoluble fraction was successively chromatographed on an ODS column in the HPLC system with 20% aqueous acetone (flow rate: 4.0ml/min) and an ODS column in the HPLC system with 75% aqueous CH3CN (flow rate: 2.0ml/min). Biological Activity Neurotoxic. Spectral Data UV:

~, Aoo~o,t,~o End absorption. max

570

20.

Hebevinosides and Hebelomic Acids

IR:

(KBr) 3430, 1750, 1645, 1070, and 1040cm"~. 1H NMR: (CDCI3) 0.66; 1.00; 1.05; 1.08; 1.21(each 3H, s); 0.95(3H, d, J=6.6Hz, 1-/3-21); 1.61; 1.68(each 3H, s, 1-13-26,-27); 2.02; 2.08; 2.11; 2.12(each 3H, s, Ac); 3.31(1H, dd, J~=l 1.6, J2=8.7 Hz, H-xyl-Sa); 3.64(1H, m, H-glc-5); 3.67(1H, t-like, H-glc-3); 4.06(1H, d, J=5.2Hz, H-7); 4.08(1H, dd, Jl=l 1.8, J2=5.0 Hz, H-glc-6a); 4.22(1H, dd, H-glc-6b); 4.45(1H, d, J=7.6Hz, H-glc-1); 4.51(1H, d, J=6.7Hz, H-xyl-1); 4.90(1H, t, J=7.8Hz, H-glc-4); 5.00(1H, brt, J=8.7Hz, H-xyl-4); 5.08(1H, t, J=9.3Hz, H-xyl-3); 5.13(1H, t, J=8.5Hz, H-24); and 5.61ppm (1H, d, J=5.2Hz, H-6). 13C NMR: (CsD~N) C-3, 87.6; C-5, 146.1; C-6, 122.7; C-7, 67.4; C-16, 82.5; C-24, 126.9; C-25, 130.0; xylose-1, 106.9; xylose-2,72.1; xylose-3, 75.4; xylose-4, 70.6; xylose-5, 62.8; glucose-l, 106.5; glucose-2, 75.6; glucose-3, 75.4; glucose-4, 72.4; glucose-5, 72.4; glucose-6, 63.5; COCH3, 20.6, 20.8, 20.9, 20.9; and COCH3, 170.2, 170.4, 170.4, 170.6ppm. Reference H. Fujimoto, K. Maeda, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum III. Isolation and Structures of Three New Glycosides, Hebevinosides XII, XIII and XIV and Productivity of the Hebevinosides at Three Growth Stages of the Mushroom; Chem. Pharm. Bull., Vol. 39, pp. 1958-1961(1991).

20.

Hebevinosides and Hebelomic Acids

571

Common/Systematic Name Hebevinoside XIV 16-3-O-(3,4-Di-O-acetyl)- 13-D-xylopyranoside- 16-O-(6-O-acetyl)- 13-D-glucopyranoside Molecular Formula/Molecular Weight C49H78013; ~ -" 874.54424 Illll,,,.

H

O

O

MoO MeOCO~

MeOCOH2~_~ .0

OH

OH

General Characteristics Amorphous solid; [tt]D24 + 15 (C=0.40, in CHCI3). Fungal Source

Hebeloma vinosophyllum (poisonous mushroom).

Isolation/Purification Dried mycelia with primordia at growth stage II were cut into fine pieces and extracted 4 times with acetone at room temperature for 12h. The combined acetone extracts were divided with n-hexane into hexane soluble and hexane insoluble fractions. The hexane insoluble fraction was successively chromatographed on an ODS column in the HPLC system with 20% aqueous acetone (flow rate: 4.0ml/min) and an ODS column in the HPLC system with 75% aqueous CH3CN (flow rate: 2.0ml/min). Biological Activity Neurotoxic. Spectral Data UV: /~ Acetoni~le max

End

absorption.

572

20.

Hebevinosides and Hebelomic Acids

IR;

(KBr) 3450, 1740, 1635, 1065, and 1030cm"~. IH NMR: (CDCI3) 0.66; 1.00; 1.04; 1.07; 1.21(each 3H, s); 0.94(3H, d, J=6.4Hz, H3-21); 1.61; 1.68(each 3H, s, H3-26, -27); 2.03; 2.09; 2.13(each 3H, s, Ac); 3.3 I(1H, dd, J~=12.2, J2=9.0Hz, H-xyl-5a); 3.52(1H, m, H-glc-5); 3.94(1H, d, J=4.8Hz, H-7); 4.03(1H, dd, Jl=l 1.6, J2=5.2Hz, H-glc-6a); 4.16(1H, dd, H-glc-6b); 4.18(1H, d, J=7.6Hz, H-glc-1); 4.34(1H, d, J=7.3Hz, n-xyl-1); 4.89(1H, dt, ,]~=J2=9.0Hz, Ja=5.2Hz, H-xyl-4); 5.09(1H, t, J=9.0Hz, H-xyl-3); 5.13(1H, t, J=7.3Hz, H-24); and 5.71ppm (1H, d, J=4.8Hz, n-6). 13C NMR:

(CsDsN) C-3, 87.5; C-5, 146.1; C-6, 122.7; C-7, 67.5; C-16, 82.2; C-24, 126.8; C-25, 130.0; xylose-1, 106.8; xylose-2, 72.2; xylose-3, 75.5; xylose-4, 70.6; xylose-5, 62.7; glucose-l, 106.5; glucose-2, 75.9; glucose-3, 78.3; glucose-4, 71.8; glucose-5, 75.5; glucose-6, 64.7; COCH3, 20.6, 20.8, 20.9; and COCH3, 170.2, 170.4, 170.8ppm. Reference H. Fujimoto, K. Maeda, and M. Yamazaki; New Toxic Metabolites from a Mushroom, Hebeloma vinosophyllum III. Isolation and Structures of Three New Glycosides, Hebevinosides XII, XIII and XIV and Productivity of the Hebevinosides at Three Growth Stages of the Mushroom; Chem. Pharm. Bull., Vol. 39, pp. 1958-1961(1991).

20.

Hebevinosides and Hebelomic Acids

573

Common/Systematic Name Hebelomic acid A 313-Acetyl-2a-(3'-hydroxy-3'-methyl)glutarylcrustulinol Molecular Formula/Molecular Weight C38I--I6o011; M W -" 6 9 2 . 4 1 3 5 6

6

Me I 3'

HO~Ov~ OH ~.21~ 24~-12 ---- ~ ~

0

II HOOC--CH2--C--C H2--C-- ,,,. s'

.o.

H

-~

,,c o .

General Characteristics A colorless powder; mp., 201-202~

[a]D17 -7 9 ~ (c=l.0, in MeOH).

Fungal Source

Hebeloma spoliatum, H. crustuliniforme, and H. sinapizans.

Isolation/Purification Dried fruit-bodies were cut into small pieces and extracted for 3 hours with MeOH at room temperature. The methanolic extract was divided with 60% (v/v) aqueous MeOH-acetone (3:5, v/v) into a supernatant and a precipitate. The supernatant was chromatographed on a silica gel column eluted with CHCI3-MeOH (100:1), (100:1), (50:1), (15:1) and (5:1, v/v) to afford five fractions. Fraction IV was subjected to MPLC with n-hexane-acetone (2:1) and (1:1, v/v) at a flow rate of 12ml/min and HPLC with MeOH-H20 (40:1, v/v) at a flow rate of 4.5 ml/min successively, to afford HS-A, which was treated with n-hexane-acetone to give HS-A in a pure state. Fraction III was subjected to HPLC with CH3CN-acetone (3:1, v/v) at a flow rate of 2.Sml/min and MPLC with CHCI3-MeOH (70:1, v/v) to afford HS-B and HS-C. Biological Activity Toxic to mice; IP dose of 100mg/kg produced death atter paralysis of the limbs. It appeared to exhibit a paraverine-like relaxation of smooth muscle. Spectral Data UV:

~ MeOH max

End absorption.

574

20.

Hebevinosides and Hebelomic Acids

IR~

3425(0-H), 2930(C-H), 1735(C=O), and 1375cm1 (C-O). 1H NMR: (CDCI3) 0.61; 0.91; 0.94; 1.05; 1.11; 1.15; 1.18; 1.37(3H each, s, H-18,-30,-29,-19, -26, -27, -28, -6'); 2.06(3H, s, CH3CO); 2.62, 2.66(1H each, AB-type d, J=14.8Hz); 2.65, 2.71(each 1H, AB-type d, J=l 5.5Hz, H-2', -4'); 3.72(1H, dd, J~=l 1.6, J2=2.0Hz, H-24); 3.87(1H, d, J=8.2Hz, H-12); 4.79(1H, d, J=10.3Hz, H-3); 5.18(1H, td, Jl=10.3, J2=5.0Hz, H-2); and 5.46ppm (1H, brs, H-21). 13C NMR: (CsDsN) i2-1, 40.9; C-2, 70.3; C-3, 80.2; C-4, 39.5; C-5, 50.2; C-6, 18.3; C-8, 135.6; C-9, 132.9; C-10, 38.2; C-12, 72.7; C-13, 50.5; C-14, 50.0; C-17, 39.9; C-18, 17.9; C-19, 19.9; C-20, 44.0; C-21, 93.3; C-24, 75.0, C-25, 71.2; C-26, 26.1; C-27, 26.8; C-28, 24.2; C-29, 28.3; C-30, 17.2; C-I', 171.3; C-2', 46.5; C-3', 69.9; C-4', 46.6; C-5', 174.8; C-6', 28.4; COCH3, 170.8; and COCH3, 21.1ppm. Mass Data: FAB-MS positive ion 715m/e [(M - Na)§ positive ion (plus KI) 73 lm/e [(M + K)§ negative ion 691role [(M-H)]. Anal. calcd for C3sH60Ol1: C, 65.87; H, 8.73%; found: C, 65.45; H, 8.67%. Reference L. Garlaschelli, G. Vidari, M. Virtuani, P. Vita-Finzi, and G. Mellerio; The Structures of New Lanostane Triterpenes from the Fruiting Bodies of Hebeloma senescens; J. Nat. Prod., Vol. 58, pp. 992-1002(1995). H. Fujimoto, Y. Takano, and M. Yamaazaki; Isolation, Identification and Pharmacological Studies on Three Toxic Metabolites from a Mushroom, Hebeloma spoliatum; Chem. Pharm. Bull., Vol. 40, pp. 869-872(1992).

20. Hebevinosides and Hebelomic Acids

575

Common/Systematic Name Hebelomic acid B Molecular Formula/Molecular Weight C4oi--I62012; M W

-- 7 3 4 . 4 2 4 1 3

HO

O H

OH

AcO Me I

O

II HOOC--CH2--C--CH2--C--O,,, I

OH

.... 2

AcO" 2'.,,,, "

General Characteristics Amorphous powder; mp., 160-163~

[a]D 21 "4- 27.7 ~ (c=0.7,

in CHCI3).

Fungal Source Hebeloma senescens, an inedible mushroom.

Isolation/Purification The more polar fractions from silica gel column chromatography containing metabolites showing a free carboxylic group (yellow spots with bromocresol green solution on TLC plates) were further purified. Concentration of fraction 14 by rotary evaporation gave a white precipitate, which was crystallized from acetonitrile and found to be identical to hebelomic acid A. Concentration of fraction 12 gave a precipitate consisting of a 1:1 (v/v) mixture of hebelomic acids A and B, which were then separated from one another. Fraction 11 was further separated by silica gel column chromatography, using a methylene chloride-acetone-acetic acid gradient, to provide, in order of elution, hebelomic acids D and C, along with additional quantities of hebelomic acid B and hebelomic acid A. Fraction 10 was further separated by silica gel column chromatography with methylene chloride-acetone-acetic acid (75:25:2.5 and 70:30:2.5, v/v/v) to afford fractions A, B, C, D, E, F, G, and H. Fractions were monitored by silica gel TLC developed with methylene chloride-acetone-acetic acid (73:24.5:2.5, v/v/v). Crystallization of fraction B from acetone/hexane afforded hebelomic acid F. Fractions A, F, and H contained almost pure hebelomic acids D, B, and A, respectively. Fractions C, D, and E were pooled and separated on different silica gel columns, eluted with hexane-acetone-acetic acid (48.7:48.7:2.5), benzene-acetone-methanol (85:10:5), benzene-methanol-acetic acid (93:5:2), benzene-methylene chloride-methanol-acetic acid (26:59:3:2, v/v/v/v), to afford hebelomic acid E.

576

20.

Hebevinosides and Hebelomic Acids

Biological Activity Showed moderate antibacterial activity in the Kirby-Bauer test against Bacillus subtilis and Staphylococcus oxford, but no activity against Escherichia coli or Candida albicans. The LDs0 value in the brine shrimp (Artemia salina) lethality assay was 3861.tg/ml. Spectral Data IR:

(KBr) 3450(OH), 1725(C=O), 1375, 1235, 1073, 1030, 1000, 950, and 897cm1. IH NMR: (CDCI3) 0.75;0.91;0.94; 1.11; 1.12; 1.15; 1.37(24H overall, 7xs, H3-18,-30,-29, -19, -28, -26, -27, -6'); 2.04(3H, s, MeCOO-12); 2.06(3H, s, MeCOO-3); 2.52; 2.69 (1H each, Abq, J=16.0Hz, H2-2' or H2-4'); 2.64, 2.69(1H each, Abq, J=15.5Hz, H2-4' . or H2-2'); 3.69(1H, dd, Jl=l 1.5Hz, J2=2.0Hz, H-24); 4.78(1H, d, J2,3=10.0Hz, H-3); 4.93(1H, br d, J=7.3Hz, H-12); 5.18(1H, td, J2,3=J2,~,=l1.0Hz, J2,~p=4.SHz, H-2); and 5.28ppm (1H, br s, H-21). (CsDsN) 0.75; 0.95; 1.02; 1.31; 1.34; 1.74(24H overall, 6 x s, H3-18, -30, -29, -19, -28, -26, -27, -6'); 2.10; 2.16(3H each, 2 X s, 2 X MeCOO-); 3.1 l(2H, ABq, 1-12-2or H2-4 ); 3.18(2H, ABq, H2-4 or H2-2 ); 4.20(1H, dd, Jl=l 1.5 Hz, J2=2.0Hz, H-24); 5.08(1H, d, J2,3=10.0Hz, H-3); 5.18(1H, br d, d=7.SHz, H-12); 5.51(1H, td, J2p,3==dlr 1.0Hz, J2.~p=4.5Hz, H-2); and 5.70ppm (1H, br s, H-21). 13C NMR: (CDCI3) C-I, 40.9(t); C-2, 69.8(d); C-3, 80.1(d); C-4, 39.5(s); C-5, 50.1(d); C-6, 18.2(0; C-7, 27.0~ C-8, 135.6(s); C-9, 131.8(s); C-10, 38.1(s); C-11, 26.6"(t); C-12, 73.8(d); C-13, 49.7(s); C-14, 48.2(s); C-15, 32.2(t); C-16, 31.4(0; C-17, 38.9(d); C18, 17.6"'(q); C-19, 19.9(q); C-20, 44.2(d); C-21, 93.6(d); C-22, 26.2(t); C-23, 23.6(0; C-24, 74.5(d); C-25, 71.3(s); C-26, 26.2""(q); C-27, 26.6""(q); C-28, 25.4(q); C-29, 28.3(q); C-30, 17.1"'(q); C-I', 170.8(s); C-2', 46.4 .... (t); C-3', 69.8(s); C-4', 46.3 .... (t); C-5', 174.6(s); C-6', 28.3(q); COMe, 171.2(s), 170.5(s); and COMe, 21.1(q), 21.7ppm (q).

, ,

,

Assignmentsmay be reversed.

Mass Data: CIMS: (NH3) 734(M++ NH4-H20), 674(734 - HOAc), and 632m/e (M ++ NH4- 2 X HOAc); DCIMS: (NH3) 752(M+ + NH3) and 734m/e (M ~ + NH4-H20). EIMS: 656(86%) (M+- H20- HOAc), 641role (656- Me), 641(77), 554(25), 514(20), 512(46), 497 (24), 479 (32), 436(21), 435(43), 419(28), 376(23), 353(22), 295(30), 277(23), 253(31), 251(25), 237(23), 226(20), 211(29), 208(33), 197(20), 181(100), 173(25), 171(30), 168(60), 159(33), 157(29), 145(44), 133(35), 124(42), 123(38), 121(30), 109(24), 107(33), 95(41), 93(21), 81(21), and 43m/e (29); anal. calcd, for C40H62012: C, 65.37; H, 8.50%; found: C, 65.57, H, 8.38%. TLC Data Silica gel GF254 plates developed with acetone-methylene chloride-acetic acid (24:73.5:2.5, v/v/v), Rf= 0.22.

20. Hebevinosides and Hebelomic Acids

577

Reference L. Garlaschelli, G. Vidari, M. Virtuani, P. Vita-Finzi, and G. Mellerio; The Structures of New Lanostane Triterpenes from the Fruiting Bodies ofHebeloma senescens; J. Nat. Prod., Vol. 58, pp. 992-1002(1995).

578

20.

Hebevinosides and Hebelomic Acids

Common/Systematic Name Hebelomic acid E Molecular Formula/Molecular Weight C3si-I60010; MW = 676.41865

HO

oH

b~

HOOC--C H2--C--CH2--C--O ...../ OH

~

T-

~"

AcO" 2"" , General Characteristics Amorphous powder; mp., 170-172~

[ a ] f I - 9.6 ~ (c=0.6, in CH2C12).

Fungal Source Hebeloma senescens, an inedible mushroom.

Isolation/Purification The more polar fractions from silica gel column chromatography containing metabolites showing a free carboxylic group (yellow spots with bromocresol green solution on TLC plates) were further purified. Concentration of fraction 14 by rotary evaporation gave a white precipitate, which was crystallized from acetonitrile and found to be identical to hebelomic acid A. Concentration of fraction 12 gave a precipitate consisting of a 1:1 (v/v) mixture of hebelomic acids A and B, which were then separated from one another. Fraction 11 was further separated by silica gel column chromatography, using a methylene chloride-acetone-acetic acid gradient, to provide, in order of elution, hebelomic acids D and C, along with additional quantities of hebelomic acid B and hebelomic acid A. Fraction 10 was further separated by silica gel column chromatography with methylene chloride-acetone-acetic acid (75:25:2.5 and 70:30:2.5, v/v/v) to afford fractions A, B, C, D, E, F, G, and H. Fractions were monitored by silica gel TLC developed with methylene chloride-acetone-acetic acid (73:24.5:2.5, v/v/v). Crystallization of fraction B from acetone/hexane afforded hebelomic acid F. Fractions A, F, and H contained almost pure hebelomic acids D, B, and A, respectively. Fractions C, D, and E were pooled and separated on different silica gel columns, eluted with hexane-acetone-acetic acid (48.7:48.7:2.5), benzene-acetone-methanol (85:10:5), benzene-methanol-acetic acid (93:5:2), benzene-methylene chloride-methanol-acetic acid (26:59:3:2, v/v/v), to afford hebelomic acid E.

20.

Hebevinosides and Hebelomic Acids

579

Biological Activity Showed moderate antibacterial activity in the Kirby-Bauer test against Bacillus subtilis and Staphylococcus oxford, but no activity against Escherichia coli or Candida albicans. Spectral Data IR:

(KBr) 3424(OH), 2948, 1742(C=O), 1456, 1376, 1237, 1094, 1076, 1032, 951,902, 803, 752, and 730cmq. 1H NMR: (CDC13) 0.72; 0.88; 0.90; 0.94; 1.13; 1.15; 1.18; 1.36(3H each, 8 X s, H3-18, -30, -29, -19, -28,-26, -27, -6'); 2.10(3H, s, MeCOO-); 2.40-2.80(4H, m, H2-2' and H2-4'); 3.78(1H, br d, J=l 1.0Hz, H-24); 4.77(1H, d, d=10.0Hz, H-3); 5.17(1H, br t, d=l 1.0Hz, H-2); and 5.38ppm (1H, br s, H-21). 13C

NM~:

(CDCI3) C-I, 41.1(t);C-2, 71.1(d);C-3, 80.3(d);C-4, 39.4(s);C-5, 49.9(d);C-6, 18.0(t); C-7, 27.0(0; C-8, 135.0(s); C-9, 133.2(s), C-10, 38.1(s); C-11, 21.3~ C-12, 26.2~ C-13, 44.1(s); C-14, 49.7(s); C-15, 30.6**(t); C-16, 30.5"'(t); C-17, 45.0***(d); C-18, 16.6(q); C-19, 20.1(q); C-20, 42.8***(d);C-21, 93.0(d); C-22, 26.0~ C-23, 23.2(0; C-24, 74.2(d); C-25, 72.2(s); C-26, 26.4 .... (q); C-27, 24.6 .... (q); C-28, 23.8(q); C-29, 27.2(q); C-30, 17.5(q); C-I', 171.5(s); C-2 ~ 45.1(0; C-3', 69.9(s); C-4', 45.1(0; C-5', 174.7(s); C-6~ 28.2(q); COMe, 171.3(s); and COMe, 21.1ppm (q). ~ ~

, ,

,~

,

~176176

Assignments may be reversed.

Mass Data: CIMS: (NH3+ NH4CI) 676(M++ NH4-H20), 658(676 - H20), and 574m/e (M ++ NH4 - 2 X HOAc); DCIMS: (NH3) 694(M§ + NH4§ and 676m/e ( M ~+ NH4-H20); anal. calcd, for C3sH60010: C, 67.43; H, 8.93%; found: C, 67.66, H, 8.76%. TLC Data Silica gel GF254 plates developed with acetone-methylene chloride-acetic acid (24:73.5:2.5, v/v/v), Rf= 0.29. Spots were visualized under UV light or by spraying plates with 0.5% vanillin solution in sulfuric acid-ethanol (4:1, v/v) and heating at 120~ for 1 rain. or with 0.04% bromocresol green solution in water in the cold. Reference L. Garlaschelli, G. Vidari, M. Virtuani, P. Vita-Finzi, and G. Mellerio; The Structures of New Lanostane Triterpenes from the Fruiting Bodies ofHebeloma senescens; J. Nat. Prod., Vol. 58, pp. 992-1002(1995).

580

20.

Hebevinosides and Hebelomic Acids

Common/Systematic Name Hebelomic acid F Molecular Formula/Molecular Weight C4oH62Oll;

MW

-- 7 1 8 . 4 2 9 2 1

ACOvO O

Me I

II

HOOC-CH2--C-CH2--C-O.~ I

~ =

OH AcO General Characteristics Amorphous powder;

~

H

[(I,]D 21 -

..

32.5 ~ (c=0.8, in methylene chloride).

Fungal Source H e b e l o m a senescens, an

inedible mushroom.

Isolation/Purification The more polar fractions from silica gel column chromatography containing metabolites showing a free carboxylic group (yellow spots with bromocresol green solution on TLC plates) were further purified. Concentration of fraction 14 by rotary evaporation gave a white precipitate, which was crystallized from acetonitrile and found to be identical to hebelomic acid A. Concentration of fraction 12 gave a precipitate consisting of a 1:1 (v/v) mixture of hebelomic acids A and B, which were then separated from one another. Fraction 11 was further separated by silica gel column chromatography, using a methylene chloride-acetone-acetic acid gradient, to provide, in order of elution, hebelomic acids D and C, along with additional quantities of hebelomic acid B and hebelomic acid A. Fraction 10 was further separated by silica gel column chromatography with methylene chloride-acetone-acetic acid (75:25:2.5 and 70:30:2.5, v/v/v) to afford fractions A, B, C, D, E, F, G, and H. Fractions were monitored by silica gel TLC developed with methylene chloride-acetone-acetic acid (73:24.5:2.5, v/v/v). Crystallization of fraction B from acetone/hexane afforded hebelomic acid F. Fractions A, F, and H contained almost pure hebelomic acids D, B, and A, respectively. Fractions C, D, and E were pooled and separated on different silica gel columns, eluted with hexane-acetone-acetic acid 9 (48.7: 48.7:2.5), benzene-acetone-methanol (85:10: 5), benzene-methanol-acetic acid (93:5:2), benzene-methylene chloride-methanol-acetic acid (26: 59:3:2, v/v/v), to afford hebelomic acid E.

20.

Hebevinosides and Hebelomic Acids

581

Biological Activity Showed moderate antibacterial activity in the Kirby-Bauer test against Bacillus subtilis and Staphylococcus oxford, but no activity against Escherichia coli or Candida albicans. The LDs0 value in the brine shrimp (Anemia saliva) lethality assay was 19.71.tg/ml. Spectral Data IR:

(KBr) 3464(OH), 2953, 1743(C=O), 1455, 1373, 1337, 1238, 1162, 1118, 1078, 1036, 1012, 959, 941,928, 910, and 866cm 1. 1H NMR: (CDC13) 0.73; 0.86; 0.92; 0.95; 1.12; 1.13; 1.15; 1.38(3H each, 8 x s, H3-18, -30, -29, -19, -28, -26, -27, -6'); 2.07, 2.10(3H each, 2 x s, 2 x MeCOO-); 2.54, 2.64(2H, ABq, J=15Hz, 1-12-2'and HZ-4'); 2.61, 2.72(2H, ABq, J=15.0Hz, H2-4' or H2-2'); 3.48(1H, br d, J=15.0Hz, H-24); 4.78(1H, d, J=10Hz, H-3); 5.17(1H, td, J213,3~JE~1~=4.2Hz,H2); and 6.10ppm (1H, br s, H-21 ).

13CNMR: (CDCI3) C-I, 41.1(t); C-2, 71.2(d); C-3, 80.3(d); C-4, 39.3(s); C-5, 50.0(d); C-6, 18.0(0; C-7, 27.1(t); C-8, 134.7(s); C-9, 133.4(s); C-10, 38.1(s); C-11, 21.2(0; C-12, 26.2*(0; C-13, 43.9(s); C-14, 49.7(s); C-15, 30.6**(0; C-16, 29.7**(0; C-17, 45.0"**(d); C-18, 16.5(q); C-19, 20.1(q), C-20, 41.2"**(d); C-21, 93.0(d); C-22, 25.3*(0; C-23, 23.8(0; C-24, 76.6(d); C-25, 71.6(s); C-26, 25.6 .... (q); C-27, 24.4 .... (q); C-28, 23.8(q); C-29, 27.1(q); C-30, 17.5(q); C-l', 171.4(s); C-2', 45.0(0; C-3', 69.6(s); C-4', 44.9(0; C-5', 174.0(s); C-6', 28.2(q); COMe, 171.2(s), 170.1(s); and COMe, 21.0(q), 21.1 ppm (q). , ,

,

Assignments may be reversed.

Mass Data: CIMS: (NH3) 676(M + + NH4-HOAc), 658, 614(676 - H20), and 574m/e (M ++ NH4HMGA); DCIMS: (NH3) 676(M + + NH4+ + HOAc). EIMS: 658(M+ - HOAc)(2%), 556(M+- HMGA) (20), 541(556- Me) + (13), 496(556- HOAc) (10), 481(496- Me) + (3), 421(481- HOAc) (20), 294(29), 168(100), 125(20), 119(16), 107(21), 95(20), 69(19), and 43role (68); Anal. calcd for C40H62Ol1: C, 66.83; H, 8.69%; found: C, 66.71, H, 8.82%. TLC Data Silica gel GF254plates developed with acetone-methylene chloride-acetic acid (24:73.5:2.5, v/v/v), Rf = 0.47. Spots were visualized under UV light or by spraying plates with 0.5% vanillin solution in sulfuric acid-ethanol (4:1, v/v) and heating at 120~ for 1 min. or with 0.04% bromocresol green solution in water in the cold.

582

20.

Hebevinosides and Hebelomic Acids

Reference L. Garlaschelli, G. Vidari, M. Virtuani, P. Vita-Finzi, and G. Mellerio; The Structures of New Lanostane Triterpenes from the Fruiting Bodies ofHebeloma senescens; J. Nat. Prod., Vol. 58, pp. 992-1002(1995).

20.

Hebevinosides and Hebelomic Acids

583

Common/Systematic Name HS-B 313,21-Diacetyl-2-(3'-hydroxy-3'-methyl)glutarylcrustulinol Molecular Formula/Molecular Weight C4oH62012; M W -- 7 3 4 . 4 2 4 1 3

H OH 6

s'

Me

I

12-

a'

O

[I

OH

~

AcO"

2"',.

General Characteristics A colorless amorphous solid; [aiD 25 -21.4 ~ (c=0.70, in MeOH). Fungal Source

Hebeloma spoliatum.

Isolation/Purification Dried fruit bodies were cut into small pieces and extracted for 3 hours with MeOH at room temperature. The methanolic extract was divided with 60% (v/v) aqueous MeOH-acetone (3:5, v/v) into a supernatant and a precipitate. The supernatant was chromatographed on a silica gel column eluted with CHCI3-MeOH (100:1), (100:1), (50:1), (15:1) and (5:1, v/v) to afford five fractions. Fraction IV was subjected to MPLC with n-hexane-acetone (2:1) and (1:1, v/v) at a flow rate of 12ml/min and HPLC with MeOH-H20 (40:1, v/v) at a flow rate of 4.5 ml/min successively, to afford HS-A, which was treated with n-hexane-acetone to give HS-A in a pure state. Fraction III was subjected to HPLC with CH3CN-acetone (3:1, v/v) at a flow rate of 2.5ml/min and MPLC with CHC13-MeOH (70:1, v/v) to afford HS-B and HS-C. Biological Activity Toxic to mice; IP dose of 100mg/kg produced death after paralysis of the limbs. It appeared to exhibit a paraverine-like relaxation of smooth muscle. Spectral Data UV: ~, M~o, End absorption.

584

20.

Hebevinosides and Hebelomic Acids

IR.:

3450(O-H), 2950(C-H), 1740(C=O), and 1370cm~ (C-O). IH NMR: (CDaCOCD3) 0.70; 0.91; 0.94; 1.07; 1.07; 1.14; 1.16; 1.35(each 3H, s. H3-18,-30, -29, -19, -26, -27, -28, -6'); 2.02, 2.03(each 3H, s, CHACO); 2.61, 2.64(each 1H, AB-type d, J=15.1Hz); 2.65, 2.70(each 1H, AB-type d, J=15.7Hz) (H2-2', -4'); 3.51(1H, dd, J~=10.8, J2=l.9Hz, H-24); 3.77(1H, d, J=7.2Hz, n-12); 4.76(1H, d, J=10.8Hz, H-3); 5.16(1H, td, J~=10.8, JE=4.4Hz, H-2); and 6.14ppm (1H, br s, H-21). 13C NMR:

(CsDsN) C-I, 41.2; C-2, 70.3; C-3, 80.1; C-4, 39.5; C-5, 50.1; C-6, 18.3; C-8, 135.6; C-9, 132.3; C-10, 38.1; C-12, 71.4; C-13, 49.8; C-14, 49.4; C-17, 38.4; C-18, 17.6; C19, 19.9; C-20, 42.4; C-21, 93.0; C-24, 77.2, C-25, 70.9; C-26, 25.6; C-27, 26.9; C28, 25.7; C-29, 28.3; C-30, 17.3; C-I', 170.2; C-2', 46.5; C-3', 69.9; C-4', 46.4; C-5', 174.6; C-6', 28.4; COCHa, 170.8; and COCHa, 21.0ppm. Mass Data: FAB-MS positive ion 757role [(M + Na)+]; positive ion (plus KI) 773role [(M + K)+]; negative ion 733role [(M - H)]; HRFAB-MS positive ion(plus KI) calcd for C40I-~20~2 [(M + K) § 773.3879; found: 773.7859; negative ion calcd for C4olq~20~2[(M - H)] 733.4163role. Reference H. Fujimoto, Y. Takano, and M. Yamaazaki; Isolation, Identification and Pharmacological Studies on Three Toxic Metabolites from a Mushroom, Hebeloma spoliatum; Chem. Pharm. Bull., Vol. 40, pp. 869-872(1992).

20.

Hebevinosides and Hebelomic Acids

585

Common/Systematic Name HS-C 313-Acetyl-2-(3'-hydroxy-3-methyl)glutarylanhydrocrustulinol Molecular Formula/Molecular Weight C38H58010; M W

= 674.40300

21

O

....... "

OH 5,

Me I 3,

~176

o.

0

II

AcO" ~ .

General Characteristics A colorless powder; [a]D 25 -3.1 ~ (c=0.38, in MeOH). Fungal Source

He be loma spo liatum.

Isolation/Purification Dried fruit bodies were cut into small pieces and extracted for 3 hours with MeOH at room temperature. The methanolic extract was divided with 60% (v/v) aqueous MeOH-acetone (3:5, v/v) into a supematant and a precipitate. The supernatant was chromatographed on a silica gel column eluted with CHC13-MeOH (100:1), (100:1), (50:1), (15:1) and (5:1, v/v) to afford five fractions. Fraction IV was subjected to MPLC with n-hexane-acetone (2:1) and (1:1, v/v) at a flow rate of 12ml/min and HPLC with MeOH-H20 (40:1, v/v) at a flow rate of 4.5 ml/min successively, to afford HS-A, which was treated with n-hexane-acetone to give HS-A in a pure state. Fraction III was subjected to HPLC with CH3CN-acetone (3:1, v/v) at a flow rate of 2.5ml/min and MPLC with CHCI3-MeOH (70:1, v/v) to afford HS-B and HS-C. Biological Activity Toxic to mice; IP dose of 100mg/kg produced death after paralysis of the limbs. It appeared to exhibit a paraverine-like relaxation of smooth muscle Spectral Data UV:

~. MoOH End absorption. max

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Siccanochromenes and Grifolins Siccanochromene A Siccanochromene B Siccanochromene C Sicr E Siccanin Grifolin Grifolin Monomethyl Ether Grifolin Dimethyl Ether Neogrifolin Neogrifolin Dimethyl'Ether Grifolin- 1-acetoxy-3-methyl Ether Scutigeral

587

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21.

Siccanochromenes and Grifolins

589

Common/Systematic Name Siccanochromene A

HO~Me

Molecular Formula/Molecular Weight C22H3oO2; M W = 3 2 6 . 2 2 4 5 8

General Characteristics Noncrystalline solid;

[~]D 9 -I-"68.8 ~ (EtOH).

Fungal Source Helminthosporium siccans, the plant pathogenic fungus which is parasitic on rye-grass, Lo#um multifolium Lam. Isolation/Purification The mycelia were separated from the fermented broth by filtration, extracted with hot acetone, and most of the acetone evaporated in vacuo and combined with the filtrated broth. The combined filtrates were extracted with ether or EtOAc, dried (Na2SO4) and evaporated to dryness. The crude metabolites were directly or a~er hydrolysis (in 5% KOH-EtOH at room temperature overnight) fractionated on silica gel columns and/or Florisil columns repeatedly. Changing eluting solvents from 0-100% ether in benzene on silica gel columns, siccanochromene A and H, siccanin, siccanochromene B, C, D, E, F, and G were eluted out successively; each fraction was further purified by changing solvent systems (to 0-100% MeOH in CHCI3 and/or packing material to Florisil) until no impurity was detected by TLC, GC, NMR, and/or MS. The purity was also confirmed on acetylation. Biological Activity Some chromenes exhibit antibacterial activity against Staphylococcus aureus. Spectral Data UV:

/~

EtOH

229.5(e=25,700), 278.5(9,320), and 286.5nm (8,700).

21.

590

Siccanochromenesand Grifolins

IR: (CHC13) 1630 and 1580cm"1. 1H NMR: The NMR spectrum of siccanochromene A indicated the presence of a geminal dimethyl group (0.84 and 0.92, 6H), a methyl attached to an oxygen atom (1.26, 3H), an aromatic methyl group (2.17, 3H), an exocyclic methylene group 4.71, 4.48, 2H, d, J=2 cps), protons on a phenyl-conjugated disubstituted double bond (AB pattern at 6.55, 5.31, 2H, J=l 1 cps) and two aromatic protons (6.10, 6.96, 2H, J=l.5 cps); C14Methyl, C15-methyl, 0.92, 0.82 (6H, s); C12-methyl, 1.26 (3H, s); C7'-methyl, 2.17 (3H, s); C13-H, 4.48, 4.71(2H, J=2.0Hz); C10-H, 5.3 l(2H, JAa=l 1Hz); C1 I-H, 6.55; and C3'-H, C5'-H, 5.96, 6.10ppm (2H, s).

Mass Spectrum: 326(M+), 311(M + -150, 247(M + -79), and 175m/e (base peak). References S. Nozoe and K. T. Suzuki; The Isolation and Structure of Siccanochromenes; Tetrahedron, Vol. 27, pp. 6063-6071 (1971). S. Nozoe, K. T. Suzuki, and S. Okuda; The Isolation and the Structure of Siccanochromenes; Tet. Lett., pp. 3643-3646(1968).

21. Siccanochromenes andGrifolins

591

Common/Systematic Name Siccanochromene B Molecular Formula/Molecular Weight C22H3003; M W = 342.21950

HO~,Me

(

I

O

General Characteristics Noncrystalline solid; [a]Ds5 +121 ~ (EtOH). Fungal Source Helminthosporium siccans, the plant pathogenic fungus which is parasitic on rye-grass, Lolium multifolium. Isolation/Purification The mycelia were separated from the fermented broth by filtration, extracted with hot acetone, and most of the acetone evaporated in vacuo and combined with the filtrated broth. The combined filtrates were extracted with ether or EtOAc, dried (Na2SO4) and evaporated to dryness. The crude metabolites were directly or after hydrolysis (in 5% KOH-EtOH at room temp. overnight) fractionated on silica gel columns and/or Florisil columns repeatedly. Changing eluting solvents from 0-100% ether in benzene on silica gel columns, siccanochromene A and H, siccanin, siccanochromene B, C, D, E, F, and G were eluted out successively; each fraction was further purified by changing solvent systems (0-100% MeOH in CHC13 and/or packing material to Florisil) until no impurity was detected by TLC, GC, NMR, and/or MS. The purity was also confirmed on acetylation. Biological Activity Some chromenes exhibit antibacterial activity against Staphylococcus aureus. v

Soectral Data UV: /~EtOH

229.5(e=25,700), 278.5(9,320), and 286.5nm (8,700).

592

21.

Siccanochromenes and Grifolins

IH NMR: The NMR spectrum of siccanochromene B showed singlets at 2.36 and 2.53(2H, AB pattern, J=5.0Hz) due to the methylene protons of the oxirane ring instead of the exocyclic methylene of siccanochromene A. Additional chemical shifts were at 0.82; 1.04(geminal dimethyl, 6H); 1.29(methyl attached to an oxygen atom, 3H); 2.16(aromatic methyl, 3H); 6.55; 5.33(protons on a disubstituted double bond, AB pattern, 2H, J=l 1Hz); 5.97; 6.08(aromatic protons, 2H, d=l.5Hz); C14-methyl, C15methyl, 1.04, 0.82(6H, s); C12-methyl, 1.28(3H, s); C7'-methyl, 2.16(3H, s); C13-H, 2.36, 2.53 (2H, AB, J=-5.0Hz); C10-H, 5.33(2H, JAa=I 1Hz); C1 I-H, 6.55; and C3'-H, C5'-H, 5.97, and 6.08ppm (2H, s). Mass Spectrum: 342(M+), 327(M + -15), 324(M + - 18), 309(M+ -33), and 175m/e (base peak). References S. Nozoe and K. T. Suzuki; The Isolation and Structure of Siccanochromenes; Tetrahedron, Vol. 27, pp. 6063-6071 (1971). S. Nozoe, K. T. Suzuki, and S. Okuda; The Isolation and the Structure of Siccanochromenes; Tet. Lett., pp. 3643-3646(1968).

21.

Siccanochromenes and Grifolins

593

Common/Systematic Name Siccanochromene C

.OMe

Molecular Formula/Molecular Weight C22H3003; MW = 342.21950

HOH2C

II

0I

General Characteristics Noncrystalline solid. Fungal Source Helmmthosporium siccans, the plant pathogenic fungus which is parasitic on rye-grass, Lolium multifolium Lam. Isolation/Purification See siccanochromene A. Biological Activity Some chromenes exhibit antibacterial activity against Staphylococcus aureus. Spectral Data UV: Essentially the same as siccanochromene A. IR: Similar to siccanochromene A. 1H NMR: The NMR spectrum indicated the presence of an unsaturated primary alcohol (3.95, 2H, s) which changed to an AB type quartet at 4.29 and 4.43, d=12Hz, upon acetylation and an olefinic proton (5.54,1H, multiplet); C 14-methyl, C 15-methyl, 0.91, 0.82(6H, s); C12-methyl, 1.27(3H, s); C7'-methyl, 2.09(3H, s); C13-H, 3.92(2H, s); C10-H, 5.28(2H, JAB=I 1Hz); C1 l-H, 6.54; C3'-H, C5'-H, 5.97, 6.06(2H, s); and C4-H, 5.54ppm (1H, m).

594

21.

Siccanochromenes and Grifolins

Mass Spectrum:

ELMS:

342role(M+).

Reference S. Nozoe and K. T. Suzuki; The Isolation and Structure of Siccanochromenes; Tetrahedron, Vol. 27, pp. 6063-6071 (1971).

21. Siccanochromenes andGrifolins

595

Common/Systematic Name Siccanochromene E Molecular Formula/Molecular Weight C22H3003; MW -- 342.21950

HO

Me

HOH2C' i r ~

General Characteristics Crystals; mp., 191-192~

[tg]D 20 -86 ~ (EtOH).

Fungal Source Helminthosporium siccans Drechsler, a parasitic fungus of rye grass (Lothium multifolium Lam.). Isolation/Purification Siccanochromene E was isolated from the non-saponifiable fraction of the fermentation broth ofH. siccans after repeated silica gel or Florisil column chromatography. Biological Activity Possible antifungal and antibacterial activity. Soectral Data _ UV: ~

EtOH max

234(e=26,000), 287(13,900), and 293nm(14,100).

IR: (CHCI3) 1628 and 1580cm "1.

IH NMR: (CDCI3) 0.94, 1.01, 1.39, and 2.18(4 X methyl groups); 3.80(2H, AB quartet, J= 11Hz); 6.21 and 6.25(2H, s, aromatic H's), and 6.59ppm (1H, s, olefinic proton). Mass Spectrum: EIMS: 342(M+), 327(M + -CH3), 311(M § -31), and 175m/e (base peak).

596

21.

Siccanochromenesand Grifolins

Reference K. Hirai, K. T. Suzuki, and S. Nozoe; The Structure and the Chemistry of Siccanin and Related Compounds; Tetrahedron, Vol. 27, pp. 6057-6061(1971).

21. Siccanochromenes and Grifolins

597

Common/Systematic Name Siccanin Molecular Formula/Molecular Weight C22H3oO3, ~

= 342.21450

HO~~/!Me

?

General Characteristics Crystals; mp., 138~ [tg]D 16 -150~ (c=7.75, in CHCI3). Fungal Source Helminthosporium siccans Drechsler, the plant pathogenic fungus which is parasitic on rye-grass, Lolium multifolium Lam. Biological Activity Siccanin exhibits inhibitory activity against a variety of fungi, especially strong activity against Trichophyton interdigitale and T. asteroide at 0.1~g/ml. Spectral Data UV: ~ EtOH max

278(sh) and 285nm (e=l,800).

IR:

(CHCI3) 3500, 1633, and 1575cm"1. 1H NMR:

(CDC13) The NMR spectrum showed the signals at 0.80, 0.84(6H, gem-dimethyls), 1.25(-OCCH3), 2.20(aromatic methyl), 6.15 and 6.30(aromatic protons), 3.46, 4.24(2H, AB quartet, -CH20-), 5.16(1H, d, benzylic methine), 1.94(methine), and 6.57ppm (-OH).

Mass Spectrum: 342role (M+). Reference K. Hirai, S. Nozoe, K. Tsuda, Y.Iitaka, K. Ishibashi, and M. Shirasaka; The Structure of Siccanin; Tet. Lett., pp. 2177-2179(1967).

598

21. SiccanochromenesandGrifolins

Common/Systematic Name Grifolin

E,E-5-Methyl-(3,7,11-trimethyl-2, 6,10-dodecatrienyl)- 1,3-benzenediol

Molecular Formula/Molecular Weight C22H3202; M W = 328.24023

HO~Me

General Characteristics Colorless needles from petroleum ether; mp., 43~ Fungal Source.

Grifola confluens (shiromaitake), Albatrellus confluens, A. ovinus = Polyporus ovinus, an

edible mushroom. Isolation/Purification Fresh Albatrellus ovinus mushrooms were extracted with acetone, concentrated and partitioned between 60% aqueous EtOH and benzene. The benzene layer was dried and repeatedly chromatographed on silica gel using petrol-benzene (3:1, v/v), benzene and benzene-ethyl ether (9:1, v/v). Grifolin was recrystallized from petroleum ether to give colorless needles. Biological Activity Antibiotic activity. Spectral Data UW:

275(1og e = 2.97) and 281nm (2.96). IR:

(KBr) 3430, 1631, 1590, 1515, and 1043cm'~; (CC14) 3620, 3460, 2920, 1635, 1585,1450, 1170, and 1044cm"~. ~H NMR: (CDCI3) I-H, 6.24(s); 5-H, 6.24(s); 8-H, 2.21 (s);l'-H, 3.38(d); 2'-H, 5.20(m);

21.

Siccanochromenes and Grifolins

599

4',5',8',9'-H, 1.92, 2.10(m); 6', 10'-H, 5.08(m); 12'-H, 1.67(s); 13'-H, 1.58(s); 14'-H, 1.59(s); and 15'-H, 1.81ppm (s). Mass Spectrum: EIMS: 328(M +, 3%), 201(18), 191(21), 175(100), 137(100), and 69m/e (74). References H. Besl, G. H6fle, B. Jendrny, E. J~igers, and W. Steglich; Farnesylphenole aus Albatrellus-Arten (Basidiomycetes); Chem. Ber., Vol. 110, pp. 3370-3376(1977). T. Goto, H. Kakisawa and Y. Hirata, The Structure of Gfifolin, an Antibiotic from a Basidiomycete, Tetrahedron, Vol. 19, pp. 2079-2083(1963). J. Vrko6, M. Bude~insky, and L. Dolej?,; Phenolic Meroterpenoids from the Basidiomycete Albatrellus ovinus; Phytochemistry, Vol. 16, pp. 1409-1411 (1977).

600

21.

Siccanochromenes and Grifolins

Common/Systematic Name Grifolin monomethyl ether E,E- 5-Methyl-(3,7,11 -trimethyl-2, 6,10-dodecatrienyl)- 1-hydroxy-3-methoxybenzene Molecular Formula/Molecular Weight C23H3402; MW = 342.25588

MeC)~Me OH

Fungal Source

Albatrellus ovinus and Polyporus ovinus, an edible mushroom.

Isolation/Purification Fresh Albatrellus ovinus mushrooms were extracted with acetone, concentrated and partitioned between 60% aqueous EtOH and benzene. The benzene layer was dried and repeatedly chromatographed on silica gel using petrol-benzene (3:1, v/v), benzene and benzene-ethyl ether (9:1, v/v). Purity of chromatographic fractions was monitored by silica gel TLC (benzene-ethyl ether, 9:1, v/v) (Rf=0.85) and GLC [Gas Chrom-Q coated with 3% QF-1 (220~ and 3% OV-17(240~ Biological Activity Antibiotic activity Spectral Data Im:

(KBr) 3450, 1615, 1585, and 1510cmq. ~H NMR: (CDCI3) 3.77, 3H; 2.26, Ar-methyl; 6.30, Ar-H(2H); 3.37 Ar-CH2, 1.58(6H); 1.67(3H); 4.95-5.35(3H, -CH=); and 2.03ppm, (-CH2-CH2-). Mass Spectrum: EIMS: 342(M+, 7%), 327(3), 299(3), 286(5), 273(1), 260(1), 231(3), 229(5), 217(2), 205(21), 204(3), 203(6), 191(12), 190(7), 189(11), 177(5), 163(5), 151(100), 121(15), 69(28), 43(4), and 41m/e (43).

21.

Siccanochromenes and Grifolins

601

Reference J. Vrko6, M. Bude~insky, and L. Dolej?,; Phenolic Meroterpenoids from the Basidiomycete Albatrellus ovinus; Phytochemistry, Vol. 16, pp. 1409-1411(1977).

602

21.

Siccanochromenes and Grifolins

Common/Systematic Name Grifolin dimethyl ether E,E- 5-Methyl-2-(3,7,11 -trimethyl-2, 6,10-dodecatrienyl)- 1,3-dimethoxybenzene Molecular Formula/Molecular Weight C24H3602; M W -- 3 5 6 . 2 7 1 5 3

OMe

"OMe

Fungal Source

Albatrellus ovinus and Polyporus ovmus, an edible mushroom.

Isolation/Purification Fresh Albatrellus ovinus mushrooms were extracted with acetone, concentrated and partitioned between 60% aqueous EtOH and benzene. The benzene layer was dried and repeatedly chromatographed on silica gel using petrol-benzene (3:1, v/v), benzene and benzene-ethyl ether (9:1, v/v). Purity of chromatographic fractions was monitored by silica gel TLC (benzene-ethyl ether, 9:1, v/v)(R~0.85) and GLC (Gas Chrom-Q coated with 3% QF-1 (220 ~ C) and 3% OV-17 (240~ Biological Activity Antibiotic activity. Spectral Data IR:

(KBr) 1608, 1566, 1492, and 1118cm"~. IH NMR: (CDCI3) 3.78, 3H; 3.78, 3H; 2.32, Ar-methyl; 6.36, Ar-H; 3.32 Ar-CH2; 1.58(6H); 1.68(3H); 4.95-5.30(3H, -CH=); and 1.99ppm (3H, -CH2-EH2-). Mass Spectrum: EIMS: 356(M +, 13%), 219(46), 165(100), 151(8), 150(8), and 69m/e (17). Reference J. Vrko~, M. Bude~insky, and L. Dolej~,; Phenolic Meroterpenoids from the Basidiomycete Albatrellus ovinus; Phytochemistry, Vol. 16, pp. 1409-1411 (1977).

21. Siccanochromenes and Grifolins

603

Common/Systematic Name Neogrifolin E,E-5-Methyl-4-(3,7,11-trimethyl-2, 6,10-dodecatrienyl)- 1,3-benzenediol Molecular Formula/Molecular Weight C22H3202; M W = 328.24023

Me

,OH

General Characteristics Crystals; mp., 43-45~ Fungal Source Grifola confluens (shiromaitake), Albatrellus confluens, A. ovinus, and Polyporus ovinus, an edible mushroom. Isolation/Purification Fresh Albatrellus ovinus mushrooms were extracted with acetone, concentrated and partitioned between 60% aqueous EtOH and benzene. The benzene layer was dried and repeatedly chromatographed on silica gel using petrol-benzene (3:1, v/v), benzene and benzene-ethyl ether (9:1, v/v). Purity of chromatographic fractions was monitored by silica gel TLC (benzene-ethyl ether, 9:1, v/v) (Rf=0.85) and GLC [Gas Chrom-Q coated with 3% QF-1 (220~ and 3% OV-17 (240~ Biological Activity Antibiotic activity. Spectral Data UV: /~ Aeetomtrile max

280(log E=3.54) and 227nm (3.83).

IR:

(KBr) 3400, 1600, 1516, 1496, and lll0cmq; (CHC13) 3600, 3550-3150, 3030, 3005, 2975, 2925, 2860, 1720, 1610, 1590, 1485, 1455, 1435, 1370, 1325, 1220, 1130, 1040, 975, and 885cm q.

604

21.

Siccanochromenes and Grifolins

1H NMR: (CDC13) 2.22, At-methyl; 6.19(1H),6.24(IH), At-H; 3.29 Ar-CH2; 1.58(6H); 1.67(3H); 5.10(3H,-CH=); and 2.03ppm, (-CH2-CH2-). 13CNMR: (CDCI3) C-l, 109.9; C-2, 154.2; C-3, 101.1; C-4, 155.3; C-5, 118.1; C-6, 138.6; C-8, 20.0; C-I', 26.7; C-2', 124.4; C-3', 131.3; C-4', 25.9; C-5', 17.7, C-6', 16.2; and C-7', 16.0ppm. Mass Spectrum: EIMS: 328.2407(M+, 20%), 191(42), 177(10), 175(27), and 137m/e (100). References H. Besl, G. H6fle, B. Jendrny, E. J/igers, and W. Steglich; Famesylphenole aus Albatrellus-Arten (Basidiomycetes); Chem. Ber., Vol. 110, pp. 3370-3376(1977). J. Vrko~, M. Bude~insky, and L. Dolej~,; Phenolic Meroterpenoids from the Basidiomycete

Albatrellus ovinus; Phytochemistry, Vol. 16, pp. 1409-1411(1977).

21. Siccanochromenesand Grifolins

605

Common/Systematic Name Neogrifolin dimethyl ether E,E-5-Methyl-4-(3,7,11-trimethyl-2, 6,10-dodecatrienyl)- 1,3-dimethoxybenzene Molecular Formula/Molecular Weight C24H3602; M W -- 3 5 6 . 2 7 1 5 3

OMe MeO< Fungal Source Grifola confluens (shiromaitake), Albatrellus confluens, A. ovinus, and Polyporus ovinus, an edible mushroom. Isolation/Purification Fresh Albatrellus ovinus mushrooms were extracted with acetone, concentrated and partitioned between 60% aqueous EtOH and benzene. The benzene layer was dried and repeatedly chromatographed on silica gel using petrol-benzene (3:1, v/v), benzene and benzene-ethyl ether (9:1, v/v). Purity of chromatographic fractions was monitored by silica gel TLC (benzene-ethyl ether, 9:1, v/v) (Rf=0.85) and GLC .[Gas Chrom-Q coated with 3% QF-1 (220~ and 3% OV-17 (240~ Biological Activity Antibiotic activity. Spectral Data. m;

(KBr) 1608, 1586, 1492, and 1118cmq.

~H NMR: (CDCI3) 3.77(OCH3), 3.77(OCH3); 2.25,At-methyl;6.32(2H), A_r-H;3.28 Ar-CH2, 1.58(6H); 1.67(3H);5.07(3H,-CH=); and 1.99ppm, (-CH2-CH2-). Mass Spectrum: Identical with that of grifolin dimethyl ether except for the intensity of some peaks. Reference J. Vrko6, M. Bude~insky, and L. Dolej~,; Phenolic Meroterpenoids from the Basidiomycete Albatrellus ovinus; Phytochemistry, Vol. 16, pp. 1409-1411 (1977).

606

21.

Siccanochromenes and Grifolins

Common/Systematic Name Grifolin- 1-acetoxy-3 -methyl ether

E,E-5-Methyl-(3 ,7,11-trimethyl-2, 6,10-dodecatrienyl)- 1-acetoxy-3-methoxybenzene

Molecular Formula/Molecular Weight C25H3603; M W -" 384.26645

OCOMo /

J

/

'

OMe

General Characteristics Liquid. Fungal Source

Albatrellus ovinus= Polyporus ovinus, an edible mushroom.

Isolation/Purification Fresh Albatrellus ovinus mushrooms were extracted with acetone, concentrated and partitioned between 60% aqueous EtOH and benzene. The benzene layer was dried and repeatedly chromatographed on silica gel using petrol-benzene (3:1, v/v), benzene and benzene-ethyl ether (9:1, v/v). Grifolin-1-acetoxy-3-methyl ether was obtained as a liquid. Biological Activity Antibiotic activity. Spectral Data IR.:

(KBr) 1768, 1595, 1485, and 1212cm4. IH ~ :

(CDCI3) 2.26, CHaC=O-; 3.77(OCH3); 2.25, At-methyl; 6.47(2H), 6.51 At-H; 3.32 Ar-CH2, 1.58(6H), 1.68(31-I); 5.07(3H, -CH=); and 2.00ppm (-CH2-CH2-). Mass Spectrum: EIMS: 384(M+, 8%), 342(8), 273(3), 260(8), 248(11), 247(5), 246(5), 231(5), 218(6), 217(10), 205(100), 204(6), 193(33), 191, 175(10), 163(7), 151(95), 137(13), 121(23), 109(7), 107(8), 95(8), 93(6), 91(7), 81(19), 69(61), 67(8), 57(2), 55(9), 53(5), 43(14), and 41m/e (47). Reference J. Vrko(3, M. Bude~insky, and L. Dolej~,; Phenolic Meroterpenoids from the Basidiomycete Albatrellus ovinus; Phytochemistry, Vol. 16, pp. 1409-1411(1977).

21.

Siccanochromenes and Grifolins

607

Common/Systematic Name Scutigeral Molecular Formula/Molecular Weight

Me.OH

C23H3204; M W = 372.23006 CHO

,4'

f,,'s~,"

"OH

15' 12'

13'

General Characteristics Crystals from n-hexane; mp., 8 I~ Fungal Source Albatrellus ovinus = Polyporus ovinus

and Albatrellus

subrubescens

(edible mushrooms).

Biological Activity Antibiotic activity. Spectral Data UV~

~, MeOH m~ 310nm (log e=3 .74); ~" MeOH+r~.on max 425 and 295nm (3.33 and 3.64 respectively). IR:

(KBr) 3500-3200, 3440, 2970, 1230, 1195, 1150, 1120, 1090, 1075, 915, 890, 840, and 715cm"~. 1H NMR: (CDCI3) 7-H, 10.17(s); 8-H, 2.43(s); I'-H, 3.35(d); 2'-H, 5.07(m); 4'-, 5'-, 8'-, 9'-H, 1.95, 2.04(m); 6', 10'-n, 5.07(m); 12'-n, 1.67(s); 13'-H, 1.58(s); 14'-n, 1.58(s); and 15'-H, 1.77ppm (s). 13C NMR:

(CDCI3) C-l, 113.1; C-2, 149.8; C-3, 128.6; C-4, 150.2; C-5, 120.1; C-6, 133.1; C-7, 194.3; C-8, 12.9; C-I', 24.5; C-2', 122.0; C-3', 135.1; C-4', 39.8; C-5', 26.6; C-6', 124.1; C-7', 135.7; and C-8', 39.8ppm.

608

21.

Siccanochromenes and Grifolins

Mass Spectrum: ELMS: 372.2319(M~, 18%), 236(11), 235(11), 221(13), 219(24), 191(12), 182(11), 181(100), 153(11), 152(20), 151(24), 136(14), and 125m/e (12). Reference H. Besl, G. H6fle, B. Jendmy, E. J/igers, and W. Steglich; Farnesylphenole aus Albatrellus-Arten (Basidiomycetes); Chem. Ber., Vol. 110, pp. 3370-3376(1977).

Fusarochromanones

Fusarochromanone Diacetylfusarochromanone C-3'-N-Acetylfusarochromanone Formylfusarochromanone

609

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22. Fusarochromanones

611

Common/Systematic Name Fusarochromanone Molecular Formula/Molecular Weight ClsH2oN204; MW = 292.14231

0II NH2 O H2 HOCH2~CH--CH2--C~M

e Me

General Characteristics Crystals from methylene chloride-hexane; mp., 132-134~ Fungal Source Fusarium equiseti (Corda) Sacc. (Previously identified as F. roseum graminearum)

collected from overwintered oats in Fairbanks, Alaska. Isolation/Purification Five hundred grams of crude culture material was extracted 5 times with CHCI3-MeOHNH4OH (90:10:1, v/v/v). The extracts were pooled and concentrated in vacuo at 40~ to give an oily residue which was dissolved in 50mL methanol in 200mL of distilled water. This mixture was further concentrated to approximately 150mL of solution and applied to an XAD-2 column rinsed with 1L of distilled water, and eluted with 1L of 90% aqueous methanol. The methanol fraction was concentrated in vacuo at 40 ~ and reconstituted in 50ml CHCI3-MeOH (9:1, v/v). This solution was mixed with 50g of anhydrous sodium sulfate and the mixture was dried at room temperature. The dry yellow powder was then packed on the top of a column containing 150g of Florisil and eluted with 2L of CHC13MeOH (9:1, v/v) followed by 2L of CHC13-MeOH-NH4OH (90:10:1, v/v/v). Fusarochromanone was detected by irradiating the column with long-wave UV (365nm) light since the toxin produced an intense blue fluorescence under the UV light. The fractions containing fusarochromanone were concentrated (40 ~C) and subsequently purified by TLC using CHC13-MeOH-NHaOH (80:20:2, v/v/v). Fusarochromanone was crystallized from methylene chloride-hexane. Biological Activity Caused tibial dyschondroplasia, a bone malformation in poultry which is characterized by the presence of a cone of cartilage which extends distally from the proximal tibiotarsal physis; also causes low hatchability of fertile eggs. Spectral Data UV:

~

MeOH max

248(e = 21,800), 277(8,800), and 383nm (11,700).

612

22.

Fusarochromanones

IR;

(KBr) 3373, 3267, 3160(NH2, OH), 1655(CO), and 1562cmq (CO,-H bonded). 1H NMR: (CDCI3) 2.7, H-3; 7.86, H-7; 6.08, H-8; 3.03, 3.88, H-2'; 3.50, H-3'; 3.60, H-4'; 1.26, CMe2; and 9.46, 9.60ppm, ArNH2. ~3CNMR: (CDCI3) 79.32, C-2, 48.96, C-3; 193.70, C-4; 154.71, C-5, 111.71, C-6; 140.16, C-7; 104.10, C-8, 165.99, C-9, 104.10, C-10, 198.80, C-I', 42.44, C-2', 49.71, C-3', 65.82, C-4', 26.28ppm, C-11 and C-12. Mass Spectrum: LREIMS yielded an ion at nominal mass of 292. Fast atom bombardment mass spectrometry indicated a protonated species at 293m/e, which was peak matched to 293.1524 to fit a composition ClsH20N204 within 7.8ppm. The FAB also showed an ion 558.2960role which fitted to within 6.3ppm for a composition of C30H41N4Os, presumably due to a dimer. Reference S. V. Pathre, W. B. Gleason, Y. Lee, and C. J. Mirocha; The Structure of Fusarochromanone: New Mycotoxin from Fusarium roseum, "Graminearum"; Can. J. Chem., Vol. 64, pp. 1308-1311(1986).

22.

Fusarochromanones

613

Common/Systematic Name Diacetylfusarochromanone 2,2-Dimethyl-5-amino-6-(3'-N-acetyl-4'-O-acetylbutyryl)-4-chromone Molecular Formula/Molecular Weight CIgH24N206, ~ = 376.16344

NHAc O

NH2 0

Me

General Characteristics Obtained as needle-shaped colorless crystals in Me2CO. Fungal Source Fusarium equiseti (previously identified as Fusarium roseum graminearum) was isolated from an oat kernel collected in Fairbanks, Alaska and deposited as Alaska 2-2. Isolation/Purification The F. equiseti isolate was grown on autoclaved rice with 60% water for 4 weeks at room temperature. The dried, powdered rice culture was extracted with 90% aqueous MeOH. The extract was concentrated under reduced pressure, defatted with petroleum ether, and partitioned with CH2C12. The CH2C12 layer was concentrated and fractionated on a Florisil column. Elution was with CHC13-MeOH (19:1, v/v) followed by CHCI3-MeOH (9:1, v/v). Fractions containing 2,2-dimethyl-5-amino-6-(3'-N-formyl-4'-O-hydroxylbutyryl)-4-chromone by TLC were combined and further purified by preparative TLC in CHCI3-MeOH (9:1, v/v); combined fractions containing 2,2-dimethyl-5-amino-6-(3'-Nacetyl-4'-O-acetylbutyryl)-4-chromone were purified by TLC in EtOAc-Me2CO (9:1, v/v). The TLC spots and bands exhibited blue fluorescence under long wavelength UV. Biological Activity Chemically related to fusarochromanone (which caused tibia dyschondroplasia, a common bone disease in chickens), and reduced hatchability in fertile chicken eggs. Spectral Data UV: ~, MooH 278, 248, and 214nm. max

IR: (film) 3395, 3285, 2998, 2932, 2856, 1734, 1662, 1653, 1595, 1560, 1498, 1466, 1375, 1319, 1278, 991, and 893cm"~.

614

22.

Fusarochromanones

~H NMR: (CDC13) 1.44(6H, s, H-11 and H-12); 1.97(3H, s, NHAc); 2.04(3H, s, OAc); 2.69(2H, s, H-3); 3.01(1H, dd, J=6.2, 16Hz, Ha-2'); 3.25(1H, dd, J=5.7, 16Hz, Hb-2'); 4.14(1H, dd, J=5.3, 11Hz, Ha-4'); 4.32(1H, dd, J-6.1, 11Hz, Hb-4'); 4.48( 1H, m, H-3'); 6.02(1H, d, J--8.9Hz, H-8); 6.39(1H, d, J=8.3Hz, NHAc); 7.82(1H, d, J-8.9Hz, H-7); and 9.34-9.60ppm (2H, b, ArNH). Mass Spectrum: ElMS yielded an M + at m/e 376. FABMS yielded an [M + H] § at m/e 377 and [M + Na] § at role 399, and negative CIMS yielded an [M] at role 376. The HREIMS indicated a molecular ion at role 376.1661 (calcd 376.1634 for C19H24N206). Reference W. Xie, C. J. Mirocha, and Y. Wen; Formyi Fusarochromanone and Diacetyl Fusarochromanone, Two New Metabolites ofFusarium equiseti; J. Nat. Prod., Vol. 54, pp. 1165-1167(1991).

22.

Fusarochromanones

615

Common/Systematic Name C-3'-N-Acetylfusarochromanone Molecular Formula/Molecular Weight C17H22N2Os; M W - 3 3 4 . 1 5 2 8 7

NHAc O

NH2 0

Me General Characteristics Yellow-green, needle-shaped crystals from acetone-petroleum ether. Fungal Source Fusarium equiseti (previously identified as F. roseum graminearum) collected from

overwintered oats in Fairbanks, Alaska. Isolation/Purification Dried rice culture material was extracted with 90% aqueous methanol three times, filtered and concentrated in vacuo. The filtrate was defatted by partitioning with petroleum ether. The aqueous methanol fraction was partitioned three times with dichloromethane, evaporated to dryness in vacuo, and redissolved in a small volume of chloroform. This extract was applied to a Florisil column eluted with chloroform-methanol (9:1, v/v). Fractions were monitored by TLC (chloroform-methanol, 4:1 v/v). Fractions containing mainly C-3'-N-acetylfusarochromanone were combined, evaporated to dryness, redissolved in acetone; petroleum ether was added to the solution dropwise until the solution became cloudy. The test tube was placed in a freezer overnight. The mother liquor was pipetted out and the yellow-green, needle-shaped crystals were vacuum dried and collected. Biological Activity Chemically related to fusarochromanone, a metabolite that caused tibial dyschondroplasia and reduced hatchability of fertile eggs. Spectral Data ~H NMR: (CDCI3) 2.68(2H, s, H-3); 7.92(1H, d, J=9.1Hz, H-7); 6.06(1H, d, J=4.1Hz, H-8); 3.06, 3.28(2H, m, H-2'); 4.4(1H, d, J=7.4Hz,, H-3'); 3.70(2H, m, n-4'); 1.49(6H, s, CMe2); 9.43,950(2H, b, ArNH2); 1.89(3H, NAc); and 6.50ppm (1H, d, J=7.5Hz, NHAc).

616

22.

Fusarochromanones

13C NMR: (CDCI3) 79.52(C-2, s), 48.95(C-3, t); 193.81(C-4, s); 154.97(C-5, s); 111.48(C-6, s); 140.62(C-7, d); 164.55(C-8,d); 166.39(C-9, s); 104.55(C10, s); 26.53(C-11, C-12, q, 26.50, q); 198.82(C-1 ~ s);39.58( C-2', t); 49.70(C-3', d); 64.27(C-4', t); 23.48(CH3C=O, q); and 170.73ppm (CHaC-O, s). Mass Spectrum: Spectrum displayed a base peak at 218m/e and molecular ion peak at 334m/e. The molecular ion of 334 was confirmed by the EI and CI mass spectra of the TMS derivative obtained from GC-MS. The EI spectrum yielded a molecular ion of 406m/e, which was 334 plus a TMS (72) group. The CI spectrum in which ion 407(M + + 41) were found confirmed the molecular ion of the TMS derivative as 406m/e. HPLC Data Reversed-phase laBondpak C~8 column, mobile phase, methanol-water-acetic acid (120:20:1, v/v/v) eluted at lml/minute. Fluorescence detection at 450 (emission) and 384 (excitation). References S. V. Pathre, W. B. Gleason, Y. Lee, and C. J. Mirocha; The Structure of fusarochromanone: NewMycotoxin from Fusarium roseum, "Graminearum"; Can. J. Chem., Vol. 64, pp. 1308-1311(1986). W. Xie, C. J. Mirocha, R. J. Pawlosky, Y. Wen, and X. Xu; Biosynthesis of Fusarochromanone and Its Monoacetyl Derivative by Fusarium equiseti; Appl. Environ. Microbiol., Vol. 55, pp. 794-797(1989).

22.

Fusarochromanones

617

Common/Systematic Name Formylfusarochromanone 2,2-Dimethyl-5-amino-6-(3'-N-formyl-4'-O-hydroxylbutyryl)-4-chromone Molecular Formula/Molecular Weight CI6H2oOsN2; MW = 320.13722

NHCHO I O

NH2 O

Me

General Characteristics Obtained as a yellow oil. Fungal Source Fusarium equiseti (previously identified as Fusarium roseum graminearum) was isolated from an oat kernel collected in Fairbanks, Alaska and deposited as Alaska 2-2. Isolation/Purification The F. equiseti isolate was grown on autoclaved rice with 60% water for 4 weeks at room temperature. The dried, powdered rice culture was extracted with 90% aqueous MeOH. The extract was concentrated under reduced pressure, defatted with petroleum ether, and partitioned with CH2C12. The CH2C12 layer was concentrated and fractionated on a Florisil column. Elution was with CHCI3-MeOH (19:1, v/v) followed by CHC13-MeOH (9:1, v/v). Fractions containing 2,2-dimethyl-5-amino-6-(3'-N-formyl-4'-O-hydroxylbutyryl)-4-chromone by TLC were combined and further purified by preparative TLC in CHCI3-MeOH (9:1, v/v); combined fractions containing 2,2-dimethyl-5-amino-6-(3'-Nformyl-4'-O-acetylbutyryl)-4-chromone were purified by TLC in EtOAc-Me2CO (9:1, v/v). The TLC spots and bands exhibited blue fluorescence under long wavelength UV. Biological Activity Chemically related to fusarochromanone (which caused tibia dyschondroplasia, a common bone disease in chickens), and reduced hatchability in fertile chicken eggs. Spectral Data UV~ ~, M~OH 278, 248, and 214nm. max

IR~ (film) 3399, 3287, 2932, 2864, 1724, 1682, 1657, 1599, 1568, 1560, 1496, 1466, 1388, 1373, 1277, 1174, 1159, 1109, and 893cm~.

618

22.

Fusarochromanones

IH NIVIR:

(CDCI3) 1.45(6H, s,H-I I and H-12); 2.70(2H, s,H-3); 3.12(IH, dd, ,/=5.9, 16.31-Iz, Ha-2'); 3.34(IH, dd, ,/=6.4,16.3Hz, Hb-2'); 3.79(2H, d, J=4.21-Iz,H-4');4.26-4.39(IH, m, H-3'); 6.08(IH, d, J=9. IHz, H-8); 6.57(2H, d, ,/=6.0Hz, NH-3'); 7.89(IH, d, J=9. IHz, H-7); 8.14(II-I,d, ,/=l.6Hz, CHO); and 9.50ppm (2H, s, ArNH). Mass Spectrum: ELMS: 320(M +) and at 302m/e (M+-H20) indicating the presence of an -OH group. Acetylation yielded a monoacetate with a molecular ion at 362role (M+). FABMS yielded an [M + H] § at 321 and {M + Na} § at 343role ; negative CLMS yielded M- at role 320role (M+). HR IMS: calcd 320.1372 for a molecular formula of C16H20OsN2, which was compatible with 8 double bond equivalents. Reference W. Xie, C. J. Mirocha, and Y. Wen; Formyl Fusarochromanone and Diacetyl Fusarochromanone, Two New Metabolites ofFusarium equiseti; J. Nat. Prod., Vol. 54, pp. 1165-1167(1991).

Aphidicolins Aphidicolin 3-Deoxyaphidicolin Aphidicolin 17-monoacetate Aphidicolin 3,18-orthoacetate

619

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23. Aphidicolins

621

Common/Systematic Name Aphidicolin Aphidicolane-3 a, 16,17,18-tetraol Molecular Formula/Molecular Weight C2oH3404; M W -- 3 3 8 . 2 4 5 7 1

HO

,,CH20H H

HO ...... HOH2C

General Characteristics Needles from ethyl acetate; mp., 227-233~

[a]D27 +12 ~ (C=I.0, in MeOH).

Fungal Source

Cephalosporium aphidicola Petch, Nigrospora sphaerica, and Harziella entomophilla.

Isolation/Purification The culture medium was filtered, adjusted to pH 6.6 and extracted with chloroform. The extract was concentrated and aphidicolin crystallized over time; it was recrystallized from ethyl acetate to give needles. Biological Activity Aphidicolin reduced the mitotic rate of mouse "L" cells growing in tissue culture; inhibited the growth of Herpes simplex type 1 both in cultures of human embryonic lung cells and in the rabbit cornea; and showed marked inhibitory activity in lettuce and rice seedlings (root growth). Spectral Data IR: (Nujol) 3520, 3440, 3360, 1077, 1047, and 1027cm"~. 1H NNIR: (pyddine-d~) 0.76(3H, s); 0.98(3H, s); 1.2-3.0(ca 20H, br m); 3.70(3H, br); 5.60(1H, br); 5.84(1H, br); and 6.08ppm (1H, br).

622

23. Aphidicolins

13C NMR: (CD3CO2D) C-1 or 7, 27.4(t); C-3, 76.9(d); C-4, 41.2(s); C-5, 41.7(d); C-7 or-1, 27.1(t); C-8, 34.6(d); C-9, 50.0(s); C-10, 40.6(s); C-12, 41.0(d); C-15, 28.3(0; C-16, 77.3(s); C-17, 68.1(t); C-18, 17.8(qt); C-19, 71.5(t); C-20, 15.5(qt); C-2, 33.5(t); C-6, 32.1(t); C-11, 27.6(t); C-13, 25.3(t); and C-14, 23.9ppm (t). Mass Spectrum: ELMS: 307m/e OVf - CH2OH) References K. M. Brundret, W. Dalziel, B. Hesp, J. A. J. Jarvis, and S. Neidle; X-Ray Crystallographic Determination of the Structure of the Antibiotic Aphidicolin: a Tetracyclic Diterpenoid Containing a New Ring system; J. C. S. Chem. Commun., pp. 1027-1028(1972). W. Dalziel, B. Hesp, K. M. Stevenson, and J. A. J. Jarvis; The Structure and Absolute Configuration of the Antibiotic Aphidicolin: a Tetracyclic Diterpenoid Containing a New Ring System; J. Chem. Soc., Perkin Trans. I, pp. 2841-2851(1973). K. Kawada, Y. Kimura, K. Katagiri, A. Suzuki, and S. Tamura, Isolation of Aphidicolin as a Root Growth Inhibitor from Harziella entomophilla, Agric. Biol. Chem., Vol. 42, pp. 1611-1612(1978). A. N. Starratt and S. R. Loschiavo; The Production of Aphidicolin by Nigrospora sphaerica; Canad. J. Microbiol., Vol. 20, pp. 416-417(1974).

23. Aphidicolins

623

Common/Systematic Name 3-Deoxyaphidicolin Molecular Formula/Molecular Weight C2oH3403; M W -- 3 2 2 . 2 5 0 8 0

OH ......CH20H ~

.~H

HOH2C General Characteristics Crystals; mp., 138.0-140.5~

[aiD 27 +22.6 ~ (c=0.98, in EtOH).

Fungal Source Phoma betae Fr., which causes a leaf spot disease on sugar beet.

Isolation/Purification The culture broth was separated by filtration into mycelium and filtrate, and the mycelium was extracted with acetone. To the extracts from the mycelium was added water and the mixture was again extracted with ether to give brown extracts. The residual aqueous layer was further extracted with ethyl acetate and the extracts were combined with the ether extracts. After removal of the solvent, the dark brown residue was chromatographed on a silica gel column which was eluted with chloroform-methanol (9:1, v/v) to give active fractions for a bioassay using lettuce seedlings. The active fractions which inhibited the root elongation of lettuce seedlings were further separated by repeated chromatography. The aphidicolin derivatives appeared as pinkish spots on TLC after anisaldehyde reagent spray and gave in the order of elution aphidicolin 3,18-orthoacetate, 3-deoxyaphidicolin, aphidicolin 17-monoacetate and aphidicolin. Biological Activity Aphidicolin 3,18-orthoacetate, 3-deoxyaphidicolin, aphidicolin 17-monoacetate, and aphidicolin inhibited the root growth of lettuce seedlings by 54.5, 50.9, 58.1 and 74.3%, respectively. Spectral Data IR:

(KBr) 3360, 2930, 2850, 1440, and 1030cm4; diacetate: 3470, 2930, 1730, 1230, and 1030cm4.

624

23. Aphidicolins

~H NMR: (pyridine-ds)0.88(3H, s); 1.03(3H, s);3.2(IH, dd, ,:=10.8,3.4Hz); 3.68(IH, dd, J=I0.8, 3.4Hz); 3.72(IH, d, J=10.81-1z);3.81(IH, d, J=10.8 Hz). Diacetate(CDCI3) 0.87(3H, s);0.93(IH, dd, ,/=13.3,5.9I-Iz);0.98(3H, s);2.07(3H, s);2.10(3H, s); 3.63(IH, d, J=I0.81-1z);3.82(IH, d, J=I0.8I-Iz);3.95(IH, d, J=l 1.51-Iz);and 3.99(IH, d, J=l 1.5Hz). Mass Spectrum: 3-Deoxyaphidicolin has the molecular formula C20H3403from the high resolution mass spectrum, 291.2319(M +-CH~OH, C19H3102, calcd 291.2321) and the field desorption mass spectrum 322(M § and 304(M § -1-120). Reference K. Hayashi, M. Hashimoto, S. Sakamura, and R. Sakai; 3-Deoxyaphidicolin and Aphidicolin Analogues as Phytotoxins from Phoma betae; Agric. Biol. Chem., Vol. 48, pp. 1687-1689(1984).

23. Aphidicolins

625

Common/Systematic Name Aphidicolin 17-monoacetate Molecular Formula/Molecular Weight C22H3605,/VIW = 380.25627 OH

......CH2OAc H

HO ....... HOH2

fi

General Characteristics Crystals; mp., 196.5-199.0~

193.5-196~

[a]D 27 q-

5.0 ~

Fungal Source

Phoma betae Fr., which causes a leaf spot disease on sugar beet.

Isolation/Purification See 3-deoxyaphidicolin. Biological Activity Aphidicolin 3,18-orthoacetate, 3-deoxyaphidicolin, aphidicolin 17-monoacetate, and aphidicolin inhibited the root growth of lettuce seedlings by 54.5, 50.9, 58.1, and 74.3%, respectively. Spectral Data IR:

(KBr) 3300 and 1750cm"1. ~H NMR:

(pyridine-ds) 0.79(3H, s); 1.03(3H, s); 1.95(3H, s);3.65(IH, br, d, J=IOHz); 3.82(IH, dd, ,/=10.7,4.31-1z);3.92(IH, br, s);and 4.29ppm (2H, s).

Mass Spectrum: Aphidicolin 17-monoacetate had the molecular formula C22H3605from the high resolution mass spectrum, 362.2451 (M+ -H20, C22H3404; calcd 362.2454).

626

23. Aphidicolins

Reference K. Hayashi, M. Hashimoto, S. Sakamura, and R. Sakai; 3-Deoxyaphidicolin and Aphidicolin Analogues as Phytotoxins from Phoma betae; Agric. Biol. Chem., Vol. 48, pp. 1687-1689(1984).

23. Aphidicolins

627

Common/Systematic Name Aphidicolin 3,18-orthoacetate Molecular Formula/Molecular Weight C22H3605; M ~ r = 3 8 0 . 2 5 6 2 7

OH ......CH20H H

General Characteristics Oily compound; [a]D25 -5.4 ~ (C=1.6, in CHC13). Fungal Source Phoma betae Fr., which causes a leaf spot disease on sugar beet.

Isolation/Purification See 3-deoxyaphidicolin. Biological Activity Aphidicolin 3,18-orthoacetate, 3-deoxyaphidicolin, aphidicolin 17-monoacetate, and aphidicolin inhibited the root growth of lettuce seedlings by 54.5, 50.9, 58.1, and 74.3%, respectively. Spectral Data IR:

(neat) 3400cm q. ~H NMR: (CDC13) 0.73(3H, s); 0.99(3H, s); 1.41(3H, s); 3.23(1H, d, J=12.2Hz); 3.37(1H, d, J-10.7Hz); 3.40(1H, d, J=10.7Hz); 3.62(1H, d, J=12.2Hz); and 3.62-3.65ppm (1H, m). Mass Spectrum: C22H3605 from HRMS 363.2572 (M + - H20 + IT, C22H3504, calcd. 363.2536), and

FD-MS 380~f).

628

23. Aphidicolins

Reference K. Hayashi, M. Hashimoto, S. Sakamura, and R. Sakai; 3-Deoxyaphidicolin and Aphidicolin Analogues as Phytotoxins from Phoma betae; Agric. Biol. Chem., Vol. 48, pp. 1687-1689(1984).

Neovasinins Neovasinin Neovasinone Neovasipyridone A Neovasipyridone B Neovasipyridone C

629

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24. Neovasinins

631

Common/Systematic Name Neovasinin Molecular Formula/Molecular Weight C17H2405; M ' W = 3 0 8 . 1 6 2 3 7

13

OH

0-I 1"-(,HO-

Me Me 15

16

Me

General Characteristics Colorless plates; mp., 204-206~

17

monoacetyl derivative; mp., 127-129~

Fungal Source

Neocosmospora vasinfecta (strain NHL 2298), a pathogen that causes root- and fruit-rot and seedling damping off in the Malvaceae, Leguminosae, Piperaceae, Cucurbitacea, etc., including pepper, peanuts, soybeans, beans, coconuts, and Albizziac.

Isolation/Purification Fungal culture filtrate was treated with active charcoal at pH 2, extracted with acetone and the extract concentrated. The concentrated extract was chromatographed on a silica gel column eluting with benzene and acetone. Neovasinone eluted with 5% acetone in benzene; neovasinin eluted with 10% acetone in benzene. Neovasinin was further purified by Sephadex LH20 column chromatography eluting with methanol. It was crystallized from n-hexane-ethyl acetate solution as colorless plates. Biological Activity The metabolite inhibited root growth of lettuce seedings 50% at 300mg/liter. Spectral Data UV: /~ EtOH max

212(6 = 36,800), 240 (3,900), and 289nm (11,300).

IR:

(KBr) 3560, 3120, 1670, 1590, 1240, and lll0cm "l. 1H NMR: (DMSO-d6) 3.70(s, H-7); 5.07(dd, ,/=9.3, 1.2Hz, H-9); 2.33(m, H-10); 1.25(m, H-11); 0.84(t, J=7.3Hz, H-12); 1.85(s, H-13); 4.29, 4.55(Abq, J=15.1Hz, H-14); 1.22(s, H15); 1.70(d, J=l.2Hz, H-16); 0.91(d, J=6.3Hz, H-17); 5.29(br, s, 3-OH); and 3.40ppm (br, s, 6-OH).

632

24.

Neovasinins

13C NMR(DMSO-d6) 164.3(C-1, s); 98.9(C-2, s); 161.8(C-3, s); 107.6(C-4, s); 155.7(C-5, s); 68.2(C-6, s); 87.7(C-7, d); 131.1(C-8, s); 136.2(C-9, d); 33.2(C-10, d); 29.9(C-11, t); 11.9(C-12, q); 9.1(C-13, q); 61.7(C-14, d); 20.6(C-15, q); 13.3(C-16, q); and 20.5ppm

(c-17, q). Mass Spectrum: LREIMS: 308(M +, 6%), 182(80), 139(100), 126(15), and l llm/e (15). Reference H. Nakajima, K. Nishimura, T. Hamasaka, Y. Kimura, and S. Udagawa; Structure of Neovasinin, A New Metabolite Produced by the Fungus, Neocosmospora vasinfecta E.F. Smith, and Its Biological Activity to Lettuce Seedlings; Agric. Biol. Chem., Vol. 51, pp. 2831-2833(1987).

24. Neovasinins

633

Common/Systematic Name Neovasinone Molecular Formula/Molecular Weight C17H2206; ~ --" 322.14164 O/H

Me,~ 0 "~ "(

O

0 H HO" Me Me

Me

Me

General Characteristics Colorless plates from ethyl acetate-n-hexane; mp., 193-195~ MeOH).

[a]o 2S - 90 ~ (c=0.20, in

Fungal Source

Neocosmospora vasinfecta var africana.

Isolation/Purification The culture filtrate was treated at pH 2.0 with active charcoal and the active charcoal was extracted with acetone. The extract was concentrated and the residue chromatographed on a silica gel column with 5% acetone in benzene. The combined neovasinone fractions were further purified by silica gel column chromatography using 30% ethyl acetate in n-hexane followed by crystallization from ethyl acetate-n-hexane to afford colorless plates. Biological Activity Plant growth regulator activity; the administration of 10 and 100mg/L of neovasinone accelerated the root elongation of lettuce seedlings. Spectral Data UV; )~ MeOH max

223(~=19,000), 271(9900), and 300nm (2400).

IR:

(KBr) 3450, 1700, 1680, 1600, and 1460cmq. IH NMR: (CDCIa) 4.68(s, 1H, H-7); 5.32(dd, 1H, J=9.8, 1.3 Hz, H-9); 2.43(dddd, 1H, ,/--9.9, 7.5, 6.8, 7.2Hz, H-10); 1.35(m, 2H); 0.89(t, J=7.7Hz, 3H, H-12); 1.94(s, 3H, H-13); 1.60(s, 3H, H-15); 1.87(d, J=l.3Hz, 3H, H-16); and 1.00ppm (d, J=7.2 Hz, 3H, H-17).

634

24.

Neovasinins

13CN M R : (CDCI3) 162.8(Sq),C-I; 101.4(Sdq),C-2; 162.3(Sqd),C-3; 98.1(Sd), C-4; 168.8(Sbr), C-5; 69.4(Sbr,q), C-6; 90.54(Dm), C-7; 127.3(Sm), C-8; 142.2(Dm), C-9; 34.2(Dm), C-10; 30.0(Tin),C-I I; 12.1(Qdt),C-12; 8.3(Qs),C-13; 167.1(Sbr s),C14; 20.5(Qbr s),C-15; 13.2(Qdd), C-16; and 201ppm (Qm), C-17. Mass Data: LREIMS: 322role (M+); found C, 63.18; H, 6.81%; calcd for C17H2206;C, 63.34; H, 6.88%. Reference H. Nakajima, K. Nishimura, T. Hamasaki, Y. Kimura, T. Dyokota, and S. Udagawa; Structure ofNeovasinone, A New tt-Pyrone Plant Growth Regulator Produced by the Fungus, Neocosmospora vasinfecta E.F. Smith; Agric. Biol. Chem., Vol.51, pp. 12211224(1987).

24.

Neovasinins

635

Common/Systematic Name Neovasipyridone A Molecular Formula/Molecular Weight C21H35NO3; MW = 349.26169 O

Me~N/CH2CH(I~e

)CH21~le

HO 12

13

General Characteristics An oil; [a]D2~+ 366 ~ (C=I.0, in EtOH). Fungal Source

Neocosmospora vasinfecta (NHL 2298), a pathogen which causes root and fruit rot and seedling damping-off in the Malvaceae, Leguminosae, Piperaceae, and Cucurbitaceae.

Isolation/Purification See Neovasipyridone B for isolation. Spectral Data UV~

)~ ~

202 (log e = 3.79), 262 (4.10), and 322nm (4.14).

IR: (thin film)3398,2964,2930,2874,2858, 1647, 1580, 1462, 1375, 1350, 1315,1267, 1241, 1172, and 1114cm1. 1H NMR: (CDCI3) 3.82(s, H-2); 8.21(s, H-6); 5.12(dq, J-9.5, 0.9Hz, H-8); 2.28(m, H-9); 1.10-1.40(m, H-10); 0.83(t, J=7.6Hz, H-11); 1.43(d, J-0.9Hz, H-12); 0.93(d, J=6.8Hz, H-13); 1.38(s, H- 14); 2.86(dq, J= 17.1,7.2Hz, H- 16); 2.98(dq, J= 17.1, 7.2Hz, H- 16); 1.10(t, J=7.2Hz, H- 17); 3.10(dd, J=13.5, 8.6Hz, H-18); 3.23(dd, J-13.5, 8.6Hz, H-18); 1.80(m, H-19); 1.25(m, H-20); 0.95(t, J=7.2Hz, H-21); and 0.98ppm (d, J=6.3Hz, H-22).

636

24.

Neovasinins

13CNMR: (CDCI3) 76.7, C-2; 71.1, C-3; 191.7, C-4; 104.9, C-5; 158.8, C-6; 127.6, C-7; 140.1, C-8, 34.0, C-9; 30.1, C-10; 11.8, C-11; 13.1, C-12; 20.6, C-13; 28.8, C-14; 197.6, C-15; 34.9, C-16; 8.5, C-17; 61.2, C-18; 33.7, C-19; 26.4, C-20; 10.8, C-21; and 16.8ppm, C-22. Mass Spectrum: FABMS: 350[M+H] § (100%), 332(10), 306(14), 236(30) and 196m/e (12); exact mass calcd for C2~H36NO3349.26169; found 349.2655role. Reference H. Nakajima, K. Shimomum, T. Furumoto, and T. Hamasaki; Neovasipyridones A, B and C: Metabolites Related to Neovasinin, A Phytotoxin of the Fungus, Neocosmospora vasinfecta; Phytochemistry, Vol. 40, pp. 1643-1647(1995).

24.

Neovasinins

637

Common/Systematic Name Neovasipyridone B Molecular FormulaJMolecular Weight C21H35NO3; MW = 349.26169 17

O nn

M e ~

18

21, 22

N/CH2CH2CH(Me)2

O-'~r~..,,K,,n,.,y / y "Me HO ~4 M e M e 12

13

General Characteristics An oil; [(/,]D 20 + 392 ~ (c-l.0, in EtOH). Fungal Source

Neocosmosporavasinfecta(NHI, 2298),a pathogen which causes root and fruit rot and seedling damping-off in the Malvaceae, Leguminosae, Piperaceae, and Cucurbitaceae.

Isolation/Purification The dried mycelial mats were soaked in acetone overnight atter which the acetone extract was obtained by filtration. The acetone extract was evaporated to dryness, the residue dissolved in NaHCO3 and treated with ethyl acetate. The ethyl acetate was placed over Na2SO4 and evaporated to dryness. The residue was partitioned with MeOH-H20 (9:1, v/v) and n-hexane. The n-hexane layer was extracted with MeOH-H20 (9:1, v/v). The combined MeOH solutions were evaporated to dryness and the residue was subjected to silica gel column chromatography. The column was eluted successively with each of 0%, 2%, 5%, 10% and 20% acetone in n-hexane. Fractions 1-3 eluted with 10% acetone in n-hexane contained the neovasipyridones. The first fraction was purified by Sephadex LH20 column chromatography with MeOH as the solvent. Each 7ml of eluate constituted one fraction and fractions 41-44 were combined and evaporated to dryness. The residue was further purified by preparatory TLC with a solvent system of acetone-chloroform (5:95, v/v) to afford neovasipyridone A. The second and third fractions were combined and evaporated to dryness. The residue was subjected to Sephadex LH20 column chromatography with MeOH as the solvent. Each 10ml of eluate constituted one fraction, and fractions 56-61 were combined and evaporated to dryness. The residue contained neovasipyridones A, B, and C. Removal of neovasipyridone A from the mixture was achieved by preparatory TLC with ethyl acetate-benzene (7:93, v/v) (quadruple development), giving neovasipyridone A as a constituent of a higher Re band on TLC. The lower Rf band contained neovasipyridones B and C. Neovasipyridone B was separated from C by repeated HPLC [column: DAISOPAK SP-120-5-ODS-A, solvent: MeOH-H20 (9:1, v/v), flow rate: 1.0ml min"1, detector: 220nm), giving neovasipyridones B (Rt, 28.0min) and C (Rt, 33.0mini.

638

24.

Neovasinins

Spectral Data UV; ~ EtOH max

200(log e = 3.71), 263(4.15), and 322nm (4.19).

IR;

(thin film) 3362, 2960, 2874, 1638, 1574, 1460, 1392, 1377, 1334, 1292, 1257, 1201, 1156, 1116, 1083, and 1029cm"~. IH N]V[R:

(CDCI3) 3.80(s,H-2); 8.25(s,H-6); 5.12(dq,J=10.8, 1.2Hz, H-8); 2.25(m, H-9); I.I01.42(m, H-10), 0.82 (t,J=7.5Hz, H-I I), 1.41(d,J=I.2Hz, H-12); 0.91(d,Jr= 6.8Hz, H13), 1.34(s,H-14), 2.84(dq,J=16.5, 7.5 I-Iz,H-16), 2.97(dq,J=16.5, 7.5Hz, H-16), 1.09(t, d=7.5Hz, H-17); 3.29(dd, .I=13.5, 7.5Hz, H-18); 3.39(dd, .1=13.5, 7.0Hz, H-18); 1.60(m, H-19); 1.60(m, H-20); 0.95(t, .1--6.5Hz, H-21); and 0.96ppm (d, .1=6.5Hz, H22). 13C N M R :

(CDCI3) 76.1, C-2; 70.9, C-3; 191.6, C-4; 104.8, C-5; 158.7, C-6; 127.7, C-7; 140.0, C8, 34.0, C-9, 30.1, C-10, 11.8, C-11; 13.0, C-12, 20.6, C-13; 28.6, C-14; 197.6, C-15; 34.8, C-16; 8.6, C-17; 53.6, C-18; 37.6, C-19; 25.7, C-20, 22.0, C-21; and 22.5ppm, C22. Mass Spectrum: FABMS: 350[M+H]+(100%), 306 (15), 196 (10),154 (39), 149(27), 136 (33), and 107m/e (15); exact mass calcd for C21H36NO3350.2695; found 350.2666m/e. Reference H. Nakajima, K. Shimomum, T. Furumoto, and T. Hamasaki; Neovasipyridones A, B and C: Metabolites Related to Neovasinin, A Phytotoxin of the Fungus, Neocosmospora vasinfecta; Phytochemistry, Vol. 40, pp. 1643-1647(1995).

24. Neovasinins

639

Common/Systematic Name. Neovasipyridone C Molecular Formula/Molecular Weight w C20H33NO3; MW = 335.24604 17

O II

6

18

20,21

Me~~~.~N~CH2CH(Me)2

0"/_~.__ ~ y "Me HO" Me Me Me 14

12

13

General Characteristics An oil; [tt]D2~ + 383 ~ (C= 1.0, in EtOH). Funsal Source

- N e o c o s m o s p o r a v a s i n f e c t a ( N H L 2298), a pathogen which causes root and fruit rot and

seedling damping-off in the Malvaceae, Leguminosae, Piperaceae and Cucurbitaceae. Isolation/Purification The dried mycelial mats were soaked in acetone overnight after which the acetone extract was obtained by filtration. The acetone extract was evaporated to dryness, the residue dissolved in NaHCO3 and treated with ethyl acetate. The ethyl acetate was placed over Na2SO4 and evaporated to dryness. The residue was partitioned with MeOH-H20 (9:1, v/v) and n-hexane. The n-hexane layer was extracted with MeOH-HzO (9:1). The combined MeOH solutions were evaporated to dryness and the residue was subjected to silica gel column chromatography. The column was eluted successively with each of 0%, 2%, 5%, 10% and 20% acetone in n-hexane. Fractions 1-3 eluted with 10% acetone in n-hexane contained the neovasipyridones. The first fraction was purified by Sephadex LH20 column chromatography with MeOH as the solvent. Each 7ml of eluate constituted one fraction, and fractions 41-44 were combined and evaporated to dryness. The residue was further purified by preparatory TLC with a solvent system of acetone-chloroform (5:95, v/v) to afford neovasipyridone A. The second and third fractions were combined and evaporated to dryness. The residue was subjected to Sephadex LH20 column chromatography with MeOH as the solvent. Each 10ml of eluate constituted one fraction, and fractions 56-61 were combined and evaporated to dryness. The residue contained neovasipyridones A, B and C. Removal of neovasipyridone A from the mixture was achieved by preparatory TLC with ethyl acetate-benzene (7:93, v/v) (quadruple development), giving neovasipyridone A as a constituent of a higher Rf band on TLC. The lower Re band contained neovasipyridones B and C. Neovasipyridone B was separated from C by repeated HPLC [column: DAISOPAK SP-120-5-ODS-A, solvent: MeOH-HzO (9:1, v/v), flow rate: 1.0 ml min"1, detector: 220 nm, giving neovasipyridones B (Rt, 28.0 min) and C (Rt, 33.0 min)].

640

24.

Neovasinins

Spectral Data UV;

203 (log E = 3.98), 262 (4.11), and 323nm (4.14). (thin film)3406, 2967,2926,2874, 1639, 1579, 1460, 1369, 1315, 1261, 1157, and 1113cm~. IH NMR:

(CDCl3) 3.80(s, H-2); 8.20(s, H-6); 5.13(dq, J=9.5, 1.0Hz, H-8); 2.28(m, H-9); 1.071.40 (m, H-10); 0.Sl(t, J=7.5Hz, H-l 1); 1.41(d, J=l.0Hz, H-12); 0.91(d, J=6.8Hz, H13); 1.37(s, H-14); 2.84(dq, J=17.0, 7.0 Hz, H-16); 2.97(dq, J=17.0, 7.0Hz, H-16); 1.08(t, J=7.0Hz, H-17); 2.94 (dd, J=14.0, 9.0Hz, H-18); 3.28(dd, J=14.0, 5.2Hz, H18); 2.03(m, H-19); 0.97(d, J=6.SHz, H-20); and 0.98ppm (d, Jr-- 6.8Hz, H-21). ~3CNMR: (CDCI3) 76.3, C-2; 71.0, C-3; 191.7, C-4; 104.8, C-5; 158.1, C-6; 127.5, C-7; 140.1, C8; 34.0, C-9; 30.1, C-10; 11.8, C-11; 13.1, C-12; 20.6, C-13; 28.9, C-14; 197.6, C-15; 34.9, C-16; 8.5, C-17; 62.7, C-18; 27.2, C-19; 19.8, C-20; and 20.1ppm, C-21. Mass Spectrum: ElMS 335 (M § 55%), 292 (100), 278 (23), 222 (15), 182 (41), and 125m/e (19); exact mass calcd for C2oH33NO3335.2461; found 335.2488role. Reference H. Nakajima, K. Shimomum, T. Furumoto, and T. Hamasaki; Neovasipyridones A, B and C: Metabolites Related to Neovasinin, A Phytotoxin of the Fungus, Neocosmospora vasinfecta; Phytochemistry, Vol. 40, pp. 1643-1647 (1995).

Koninginins Koninginin A Koninginin B Koninginin C Koninginin D Koninginin E

641

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25. Koninginins

643

Common/Systematic Name Koninginin A Molecular Formula/Molecular Weight C16H2sO4; M W = 2 8 4 . 1 9 8 7 6

HO

H O~ 0

H ;Me

H General Characteristics Crystallized from isooctane; mp., 80-84~

[tt]D -22 ~ (C=0.7, in CHC13).

.Fungal Source

Trichoderma koningii was isolated from Diffenbachia sp. (ATCC 46314) and T. harzianum was isolated from wheat roots.

Isolation/Purification Fungal cultures were extracted with acetone, reduced to an aqueous phase under vacuum, and partitioned twice with ethyl acetate. The ethyl acetate extract was chromatographed on a silica gel column eluted with benzene, ethyl ether, ethyl acetate, acetone, and acetonitrile. The ethyl ether and ethyl acetate fractions were combined, reduced in volume and further purified by silica gel column chromatography eluted with a linear gradient from benzene to acetone. The koninginin A-containing fractions were further purified with a silica gel Cls reversed-phase column eluted with different proportions of acetonitrilewater. The koninginin A containing fractions were combined, frozen and the supematant liquid removed. The supematant fluid was reduced in volume and chromatographed on a column containing hydrated silica gel eluted with benzene-ethyl acetate (5:4, v/v). Final purification was performed on a silica gel-silver nitrate column eluted with methylene chloride-acetone (9:1, v/v) followed by crystallization as fine needles. Biological Activity The metabolite inhibited the growth of etiolated wheat coleoptiles at 10-3 M. It was not active against several bacteria and fungi. Spectral Data UV~

E~n

End absorption.

644

25.

Koninginins

IR~ (KBr) 3500, 3460, 2950, 2925, 2855, 1450, 1410, 1340, 1255, 1242, 1216, 1202, 1175, 1132, 1102, 1080, 1068, 1052, 1042, 1002, 955, 938, 915, 855, 820, 770, 722, 665, and 640cm"~. 13C N M R : (CDCI3) 72.66(C-1, d); 30.85(C-2, t); 25.38(C-3, t); 69.78(C-4, d); 109.20(C-5, s); 41.43(C-6, d); 20.60(C-7, t); 27.26(C-8, t); 79.00(C-9, d); 79.30(C-10, d); 35.12(C11, t); 25.54(C-12, t); 29.08(C-13, t); 31.69(C-14, t); 25.53(C-15, t); and 14.02ppm (C-16, q).

1H NMR: 0.88(3H, J=6.6Hz, H-a); 1.30(8H, m, H-b's); 1.50(2H, J=-6.7Hz, H-c); 1.57(3H, J=3.2, 7.2Hz, H-d); 1.72(1H, J=6.3, 13.9Hz, H-e); 1.85(2H, J=3.6, 11.6, H-f); 1.96(1H, J=6.8, 12.THz, H-g); 2.11(1H, J=4.7, 9.5Hz, H-h); 2.26(1H, J=3.6, 6.1, 13.1, H-i); 3.61(1H, J=5.4, 11.6Hz, H-j); 3.89(1H, J=2.5Hz, H-k); 4.03(1H, J=6.6Hz, H-I); and 4.32ppm (1H, J=2.1Hz, H-m). TLC Data Silica gel 60 thin-layer plates developed with toluene-ethyl acetate-formic acid (5:4:1, v/v/v), Rf = 0.56-0.60; detected as a salmon-pink colored spot when treated with anisaldehyde followed by heating. References F. Almassi, E. L. Ghisalberti, M. J. Narbey, and K. Sivasithamparam; New Antibiotics from Strains of Trichoderma harzianum; J. Natural Products, Vol. 54, pp. 396-402(1991). H. G. Cutler, D. S. Himmelsbach, R. F. Arrendale, P. D. Cole, and R. H. Cox; Koninginin A: A Novel Plant Growth Regulator from Trichoderma koningii; Agric. Biol. Chem., Vol. 53, pp. 2605-2611(1989).

25. Koninginins

645

Common/Systematic Name Koninginin B Molecular Formula/Molecular Weight C16H2604; M W : 282.18311 OH

OH 16

3

O General Characteristics Colorless crystalline solid; mp., 72-74 ~ Fungal Source

Trichoderma koningii was isolated from Diffenbachia sp. (ATCC 46314).

Isolation/Purification Fungal cultures were extracted with acetone, reduced to an aqueous phase under vacuum, and partitioned twice with ethyl acetate. The ethyl acetate extract was chromatographed on a silica gel column eluted with benzene, ethyl ether, ethyl acetate, acetone, and acetonitrile. The ethyl ether and ethyl acetate fractions were combined, reduced in volume and further purified by silica gel column chromatography eluted with a linear gradient from benzene to acetone. The koninginin B fractions were reduced in volume and chromatographed on a column containing hydrated silica gel eluted with benzene-ethyl acetate (5:4, v/v). Biologically active fractions were pooled and chromatographed on Cls reversed-phase silica gel with acetonitrile-water (1:1, v/v). Final purification was performed on a silica gel-silver nitrate column eluted with methylene chloride-acetone (9:1, v/v) to yield koninginin B. Biological Activity The metabolite inhibited the growth of etiolated wheat coleoptiles at 10-3 M. It was marginally active against gram-positive bacteria and inactive against gram-negative bacteria. Spectral Data UV:

~, Etori

262nm (log c = 4.24).

646

25. Koninginins

IR~

(KBr) 3450, 2960, 2930, 2860, 1650, 1612, 1453, 1402, 1381, 1287, 1242, 1196, 1152, 1078, 1048, 981,860, 728, and 692cm1. ~H NMR: (CDC13) 2.50(J~2b=- 17Hz, H-2a); 2.56(J~3a = 14.4, Jza,3b=2.3,J2b,3a--5.1,JEb,3b=5.2Hz, n-2b); 1.79(Ja~,ab=12.9Hz, n-3a); 2.34(Ja~a=-12.1, Jab,4=4.8Hz, H-3a); 4.05(H-4); 2.09(J7~Tb=- 15.9Hz, H-7a); 2.57(JT~Sa= 11.6, J7~Sb=6.1,J7b,Sa= 13.2, J7b,Sb=2.1Hz, H-7b); 1.68(JS~Sb=-13.0Hz, H-8a); 1.96(J8~,9=5.2,JSb,9=l.9Hz, H-8b); 3.80(J9,~o=11.2Hz); 3.68(JlO,lj=5.9Hz, n-10); 1.550-1-11); 1.30, 1.55(H-12); 1.30(I4-13); 1.30(H-14); 1.30(J15,16=6.8Hz, H-15); and 0.89ppm (H-16). 13C NMR:

(CDCI3) 198.10(C-1); 2714(C-2); 20.20(C-3); 71.02(C-4); 171.58(C-5); 109.9(C-6); 17.70(C-7); 22.59(C-8); 80.80(C-9); 72.89(C-10); 32.76(C-11); 25.44(C-12); 29.25(C-13); 31.75(C-14); 22.59(C-15); and 14.06ppm (C-16). Reference H. G. Cutler, D. S. Himmelsbach, B. Yagen, R. F. Arrendale, J. M. Jacyno, P. D. Cole, and R. H. Cox; Koninginin B: A Biologically Active Congener ofKoninginin A from Trichoderma koningii; J. Agile. Food Chem., Vol. 39, pp. 977-980(1991).

25.

Koninginins

647

Common/Systematic Name Koninginin C Molecular Formula/Molecular Weight C16H2804; M W =- 284.19876

O

Me

HO General Characteristics Fine white crystals from isooctane; mp., 70-72 oC. Fungal Source

Trichoderma koningii was isolated from Diffenbachia sp. (ATCC 46314) and T. harzianum was isolated from wheat roots.

Isolation/Purification Fungal cultures were extracted with acetone, reduced to an aqueous phase under vacuum, and partitioned twice with ethyl acetate. The ethyl acetate extract was chromatographed on a silica gel 60 column eluted with benzene, ethyl ether, ethyl acetate, acetone, and acetonitrile. The koninginin C containing fractions were further purified with a silica gel Cls reversed-phase column eluted with different proportions of acetonitrile-water; hydrated silica gel eluted with benzene-ethyl acetate (5:4, v/v). The koninginin C was crystallized as fine white crystals from isooctane. Biological Activity The metabolite inhibited the growth of etiolated wheat coleoptiles at 103 M. Spectral Data UW~

~, E~n

End absorption.

13C NMR:

(CDCl3) 25.15(C-1, t), 76.89(C-2, d); 29.01(C-3, t); 69.30(C-4, d); 108.47(C-5, s); 36.15(C-6, d); 20.42(C-7, t); 27.07(C-8, t); 78.92(C-9, d); 79.17(C-10, d); 35.65(C11, t); 23.32(C-12, t); 29.17(C-13, t); 31.71(C-14, t); 22.54(C-15, t); and 14.04ppm (C-16, q). Mass Spectrum: HREIMS: 284.198929m/e (M+); calcd for C16H2804,284.198760.

648

25. Koninginins

TLC Data Silica gel 60 thin-layer plates developed with toluene-ethyl acetate-formic acid (5:4:1, v/v/v); Rf, 0.56-0.60; detected as a salmon-pink colored spot when treated with anisaldehyde followed by heating. Reference S. R. Parker, H. G. Cutler, and P. R. Schreiner; A Biologically Active Natural Product from Trichoderma koningqi; Bioscience, Biotech., Biochem., Vol. 59, pp. 1126-1127 (1995).

25.

Koninginins

649

Common/Systematic Name Koninginin D 4,8-Dihydroxy-2-(1 -hydroxyheptyl)-3,4,5,6, 7,8-hexahydro-2H- 1-benzopyran- 5-one Molecular Formula/Molecular Weight C16H2605, ]VIW= 298.17802 OH

OH

O

OH

General Characteristics Crystallized from CHCl3/pentane as solvated needles which collapsed to a powdery solid on recovery. The triol had mp of 122-123~ [a]D + 166.9 ~ (C=0.3, in CHC13). Fungal Source

Trichoderma koningii (IMI 308477) isolated from soil.

Isolation/Purification The culture broth was extracted with ethyl acetate, dried over anhydrous sodium sulfate and evaporated to dryness. Column chromatography on neutral alumina (activity I) using ethyl acetate as eluting solvent gave 4,8-dihydroxy-2-(1-hydroxyheptyl)-3,4,5,6,7,8hexahydro-2H-1-benzopyran-5-one which was crystallized from chloroform-pentane. Biological Activity The metabolite inhibited the growth of the take all fungus, Gaeumannomyces graminis, and inhibited the growth of several other soil fungi. Spectral Data UV~ ~, EtCH

260rim (log e = 3.9).

IR:

(CHC13) 3600, 3380, 1655, and 1620crn"1. 1H NMR: (CDC13) 2.38(J2~,2b=l6.9Hz, J~3a=6.3, J~3b=4.9, H-2a); 2.64(J2b,3a=9.4, J2b,3b=5.7Hz, n-2b); 1.6(J3~,3b=14,J3~,4=4.7, n-3a); 2. 2(J3b,4=4.7, J4,7=l.0Hz, n-3b); 5.81(d7,s~3.5, JT,sb=2.1Hz), 1.75(ds~,sb=l5, Jsa,9=3.5Hz, H-Sa), 1.99(dab,9=2.2, H-8b); 4.15(d9,1o=3.9I-Iz); 5.08(Jlo, l~=6.7Hz); 1.3, 1.6; 1.25, 1.25, 1.25, 1.25; and 0.9ppm (J15,16=6.5Hz).

650

25.

Koninginins

13C NMR: (CDCI3) 194.8(C-1); 31.6(C-2); 26.4(C-3);.66.5(C-4); 170.4(C-5); 112.1(C-6); 59.6(C-7); 29.1(C-8), 74.2(C-9); 72.9(C-10); 29.9(C-11); 25.1(C-12); 29.2(C-13), 32.8(C-14); 22.5(C-15); and 14.0pp (C-16). Mass 9 Spectrum: LREIMS: 298(M+,3%), 280(5), 262(4), 244(8), 236(6), 209(13), 191(7), 181(12), 170(18), and 165role (39); HREIMS: 298.1759re~e, C16H2605 requires 298.1780. TLC Data Silica gel thin-layer plates developed with ethyl acetate; Rf, 0.13; detected under UV light. Reference R. W. Dunlop, A. Simon, K. Sivasithamparam, and E. L. Ghisalberti; An Antibiotic From Trichoderma koningii Active Against Soilborne Plant Pathogens; J. Natural Products, Vol. 52, pp. 67-74(1989).

25. Koninginins

651

Common/Systematic Name Koninginin E Molecular Formula/Molecular Weight C16H2604; M W --- 282.18311

OH

H

OH

General Characteristics Off-white amorphous solid from isooctane. Fungal Source

Trichoderma koningii isolated from Diffenbachia sp. (ATCC 46314).

Isolation/Purification Following incubation, cultures were extracted with acetone. The extract was filtered under vacuum and the filtrate reduced to a non-volatile, aqueous volume. The aqueous volume was extracted three times with an equal volume of ethyl acetate and the combined extract washed with an equal volume of distilled water. Atter drying over anhydrous sodium sulfate, the ethyl acetate extract was reduced to an oily residue and thoroughly azeotroped with benzene to remove any remaining water. The residue was mixed 1:1 (w/w) with Celite and used to fill a 10ml stainless steel extraction thimble. The contents were made compact using a blunt ended glass rod and the sealed thimble placed in the extraction chamber of an SFX 2-10 extraction module. A ten minute static extraction was followed by a dynamic extraction, at constant pressure, with supercritical carbon dioxide. SFE fractions were dissolved in acetone and analyzed directly by GC-MS. Koninginin E was observed in the methanol amended fractions. The combined koninginin E containing SFE fractions were reduced under nitrogen and applied to a C18 column prepared in 1:1 (v/v) acetonitrile-water. The column was eluted stepwise with each of 1:1 (v/v) followed by 3:1 (v/v) acetonitrile-water. Final purification was achieved by precipitation from isooctane solution. Biological Activity The metabolite significantly inhibited (60%) the growth of etiolated wheat coleoptiles at 10-3 M.

652

25. Koninginins

Spectral Data UV~

~

EtOH max

262nm.

IR:

(KBr) 3450, 3366, 2955, 2925, 2856, 1650, 1620, 1592, 1402, 1379, 1295, 1250, and 1082cm1. 13C N M R :

(CDC13) 197.6(C-1); 33.3(C-2); 29.0(C-3); 65.9(C-4); 169.3(C-5); 111.4(C-6); 17.6(C-7); 22.8(C-8);-81.3(C-9); 73.4(C-10); 32.7(C-11); 25.1(C-12); 29.3(C-13); 31.8(C-14); 22.6(C-15); and 14.1ppm (C-16). TLC Data Silica gel 60 thin-layer plates developed with toluene-ethyl acetate-formic acid (5"4:1, v/v/v); Re, 0.28-0.30; detected by spraying with acidified anisaldehyde and heating. References E. L. Ghisalberti, and C. Y. Rowland; Antifungal Metabolites from Trichoderma harzianum; J. Nat. Prod., Vol. 56, pp. 1799-1804(1993). S. R. P~rker, H. G. Cutler, and P. R. Schreiner; Koninginin E: Isolation of a Biologically Active Natural Product from Trichoderma koningii; Bioscience Biotech Biochem., Vol. 59, pp. 1747-1749(1995).

Curvularins Curvularin 6-Chlorodehydrocurvularin 11-Acetyldehydrocurvularin 1113-Hydroxycurvularin

trans-Dehydrocurvularin cis-Dehydroeurvularin 11~t-Hydroxylcurvularin 12-Oxocurvularin Methoxycurvularin Citreofuran

653

This Page Intentionally Left Blank

26.

Curvularins

655

Common/Systematic Name Curvularin Molecular Formula/Molecular Weight C17H2205; MW = 306.14672

0•0•

HO~ OH

16

0

General Characteristics Curvularin separated as plates from benzene containing a small amount of methanol; mp., 206-206.5~ [a]DIs -36.3 ~ (C=3.8, in ethanol). Curvularin was soluble in ethanol, methanol, dioxane, acetone, pyridine, and concentrated sulfuric acid, moderately soluble in acetic acid and ether, and sparingly soluble in benzene, light petroleum, chloroform, and water. It gave yellow solutions with aqueous ammonia, sodium carbonate, and sodium hydroxide, from which it was recovered unchanged by acidification. Alkaline solutions of curvularin darken rapidly in air, eventually becoming purple. This sensitivity to oxygen in the presence of alkali is also demonstrated by the rapid decomposition of curvularin at its m.p. when heated in open soda-glass capillaries. Funsal Source Curvularia sp. (No. F334 in the Nobel Division culture collection; Commonwealth Mycological Institute Cat. No. I.M.I. 52,980), Drechslera sp. (tentatively identified as D. rostrata, IMI 356287), Cercospora spp., Penicillium citreo-viride B (hybrid strain derived from IFO 6200 and 4692), P. gilmanii, Alternaria cinerariae, and Penicillium sp. 511 (isolated from soil). Isolation/Purification The culture filtrate of Curvularia sp. was decanted from the mycelium and extracted with ethyl ether which,~ after removal of the solvent, gave crude curvularin as colorless or pale yellow-brown crystals. The crude curvularin was recrystallized from hot benzene containing a small amount of methanol to give plates. Penicillium sp. 511 was grown on potato-sucrose-agar for thirty days. The slant cultures were soaked in acetone for one week at room temperature. An aqueous concentrate atter evaporating acetone was partitioned against EtOAc. The EtOAc extract was chromatographed on a silica gel column (Wakogel C-100). The column was eluted with 20, 30, 40, and 50% EtOAc/benzene. Purification of curvularin, 8-dehydrocurvularin, 8-hydroxycurvularin, and 8-methoxycurvularin was guided by a sea urchin egg assay.

656

26.

Curvularins

Biological Activity Non-specific phytotoxic action, caused stem collapse and vascular necrosis of cut zinnia (Zinnia elegans Jacq.) seedlings at concentrations down to 0.1 and 0.33mg/ml respectively within 24hr; caused necrosis of cuttings of Canada thistle (Cirsium arvense L.) Scop. within 16 hours; cytotoxic against sea urchin (Hemicentrotuspuicherrimus) embryogenesis where it induced barbell-shaped mitotic spindles. It was shown to act on components of the mitotic apparatus and to effectively inhibit cell division. Spectral Data

UV: /~EtOH 223, 272, and 304.5nm (E = 11,300, 6350, and 5100, respectively) and an max

inflection at 229nm (e = 10,150), unaffected by the addition of hydrochloric acid and 0.005N aqueous sodium hydroxide at 233 and 35 lnm (e = 10,200 and 9300) and inflections at 258 and 328nm (r = 7,250 and 8,100).

(KBr) 3300, 1706, 1665, 1615, 1608, 1160, 1050, and 841cm q. ~H NMR:

(acetone-d6) 6.37(IH, d, J=2.21-1z);6.33(IH, d, J=2.2Hz); 4.90(IH, m); 3.76(IH, d, J=151-1z);3.69(IH, d, J=151-1z);3.09(IH, m); 2.76(IH, m); 1.2-~1.8(8H,m); and 1.10ppm (3H, d, J=6.3Hz).

Mass Spectrum: HREIMS: 292.1309m/e M +, (C~rH20Os, calcd 292.1309). References R. G. Coombe, J. J. Jacobs, and T. R. Watson; Constituents of Some Curvularia Species; Aust. J. Chem., Vol. 21, pp. 783-788(1968). A. Kobayoshi, T. Hino, S. Yata, T. J. Itoh, H. Sato, and K. Kawazi; Unique Spindle Poisons, Curvularin and Its Derivatives, Isolated from Penicillium Species; Agric. Biol. Chem., Vol. 52, pp. 3119-3123(1988). S. Lai, Y. Shizuri, S. Uamamura, K. Kawai, Y. Terada, and H. Furukawa; Novel Curvularin-Type Metabolites of a Hybrid Strain Me 0005 Derived from Penicillium citreo-viride B. IFO 6200 and 4692; Tet. Lea., Vol. 70, pp. 2241-2244(1989). O. C. Musgrave; Curvularin. Part I. Isolation and Partial Characterization of a Metabolic Product from a New Species of Curvularia; J. Chem. Soc., pp. 4301-4305(1956). H. Raistrick and F. A. H. Rice; 2,3-Dihydro-3,6-dihydroxy-2-methyl-4-pyrone and Curvularin from Penicillium gilmanii; J. Chem. Soc. (C), pp. 3069-3070(1971). D. J. Robeson and G. A. Strobel; a,13-Dehydrocurvularin and Curvularin from Alternaria cinerariae; Z. Naturforsch., Vol. 36, pp. 1081-1083(1981).

26. Curvularins

657

Common/Systematic Name 6-Chlorodehydroeurvularin E-6-Chloro- 10,11-dehydrocurvularin Molecular Formula/Molecular Weight C16I-I1705C1; MW = 324.07645 H

-----

OH

0

General Characteristics Crystals from CH2CI2-EtOAc as needles; mp., 189-190~

[a]D-51~ (c=0.2, in EtOH).

Fungal Source Drechslera species (Deuteromycotina) (tentatively classified as D. rostrata) was isolated from soil in which wheat seedlings were growing (culture deposited with the Commonwealth Mycological Institute; IMI 356287). Drechslera sp. are an important group of fungal plant pathogens which have been responsible for devastating disease epidemics, particularly on flee, corn, and sorghum. Isolation/Purification The fungal culture was grown on Czapek-Dox medium. The liquid medium of a 3-monthold culture of Drechslera sp, after filtration from the mycelial mat, was extracted repeatedly with EtOAc. The combined organic layer was dried over Na2SO4 and evaporated under reduced pressure to give a residue. The mycelial mat was extracted with MeOH and the extract was washed with EtOAc to give a residue. TLC analysis (Si gel; CH2CI2-EtOAc, 1:1, v/v) of both extracts showed them to be similar and to contain mainly two components. Radial plate chromatography of the EtOAc-soluble portion of the mycelial extract using gradient elution (CH2C12to EtOAc) gave two fractions. Fraction 1 appeared to be mainly linoleic acid. Fraction 2 was purified by column chromatography on silica gel to give a crystalline sample of curvularin. The fungal culture was also grown on potato-dextrose broth. A 3-month-old culture ofDrechslera sp. grown on this medium was similarly processed to give two residues: EtOAc extract and EtOAc-soluble portion of mycelial extract. TLC analysis (Si gel; CH2C12-EtOAc, 1:1, v/v) of both fractions showed them to be similar. Radial plate chromatography of the EtOAc extract and gradient elution (CH2CI2-EtOAc, 1"1, v/v, to EtOAc) yielded a fraction, corresponding to the major component, 6-chlorodehydrocurvularin, as a solid.

658

26.

Curvularins

_Biological Activity Biological activity of 6-chlorodehydrocurvularin is not known but the close chemical relationship to the phytotoxic metabolite curvularin suggests it may also be phytotoxic. _Spectral Data 1H NMR: (CDCI3) 6.68(1H, ddd, J=16.0, 3.5, 3.5Hz, H-11); 6.66(1H, br d, J=16.0Hz, H-10); 6.48(1H, br s, H-4); 4.85(1H, ddq, ,/--2.0, 8.0, 6.5Hz, H-15); 4.02(1H, br d, J=17.8Hz, H-2a); 3.54(1H, br d, J=17.8, H-2b); and 1.25(3H, d, J=6.5Hz, H-16). The following assignments were made with the aid of decoupling techniques: 2.5 and 2.35, H-12; 1.99 and 1.69, H-13; 1.89 and 1.65ppm, H-14; (500MHz, acetone-d6) 6.77(1H, d, J-15.4, H-11); 6.62(1H, ddd, J=15.4, 8.9, 4.9Hz, H-10); 6.57(1H, br s, H-4); 4.74(1H, m, H-15); 4.05(1H, br d, J=17.6Hz, H-2a); 3.62(1H, br d, J=17.6, H-2b); and 1.17ppm (3H, d, J=6.4Hz, H-16). 13C NMR; (CDC13) C-l, 170.9; C-2, 43.7; C-3, 130.9; C-4, 112.5; C-5, 156.1; C-6, 107.5; C-7, 160.8; C-8, 114.9; C-9, 198.3; C-10, 135.9; C-11, 149.7; C-12, 32.8; C-13, 24.1; C-14, 34.0; C-15, 72.8; and C-15 CH3, 20.0ppm.

Mass Spectrum: EIMS: 326(15), 324(36) (M+), 239(29), 237(9), 231(6), 229(14), 226(16), 225(11), 224(24), 223(18), 213(12), 211(37), 210(31), 209(32), 203(12), 201(37), 186(15), 184(46), 160(24), 115(26), 81(60), and 55m/e (100). Reference E. L. Ghisalberti, and C. Y. Rowland; 6-Chlorodehydrocurvularin, A New Metabolite from a Drechslera sp.; Journal of Natural Products (Lloydia), Vol. 56, pp. 2175-2177 (1993).

26. Curvularins

659

Common/Systematic Name 11-Acetyldehydrocurvularin Molecular Formula/Molecular Weight ClsH2006; MW = 332.12599

"~0"~ Me

OAc

0

General Characteristics Crystals; mp., 105-110~

[~]D20 -31.7 ~ (c=0.4, in MeOH).

Funsal Source

Cercospora scirpicola (CBS 104.40).

Biological Activity Phytotoxic. Soectral Data UV~

222, 232sh, 275, and 308nm (e = 10,200, 8,800, 5,800, and 4,900, respectively). ~H NMR: (acetone-d6/DMSO-d6) 1.10(d, 3H, d, J=6Hz); 1.2-2.1(6 aliphatic protons); 2.09(Ac), 3.74(2H, q, Ar-CHz-CO); 4.94(1H, m, O-C___HH-Me);and 6.26-6.48ppm (12 aromatic and 2 vinylic protons). Reference G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Nasini; Screening of the Genus Cercospora for Secondary Metabolites; Phytochemistry, Vol. 16, pp. 243-247(1977)o

660

26.

Curvularins

Common/Systematic Name 1113-Hydroxycurvularin Molecular Formula/Molecular _Weight C16H2oO6; M W

= 308.12599

O

O

HO

OH

O

H

OH

General Characteristics 13-Hydroxycurvularin was obtained as a colorless solid; mp.; 138-140~ (c=0.19, in EtOH).

[ a i D 24 -

10.9 ~

Fungal Source Penicillium citreo-viride B (hybrid strain derived from IFO 6200 and 4692), Alternaria tomato, and Penicillium sp. 511 (isolated from soil). Isolation/Purification Penicillium sp. 511 was grown on potato-sucrose-agar for thirty days. The slant cultures were soaked in acetone for one week at room temperature. Following evaporation of acetone the resulting aqueous concentrate was partitioned against EtOAc. The EtOAc extract was chromatographed on a silica gel column (Wakogel C-100). The column was eluted with 20, 30, 40, and 50% EtOAc/benzene. Purification of curvularin, 8-dehydrocurvularin, 8hydroxycurvularin, and 8-methoxycurvularin was guided by a sea urchin egg assay. Biological Activity Highly phytotoxic. Marked sporulation-suppressing activity against Alternaria tomato. Cytotoxic against sea urchin (Hemicentrotus pulcherrimus) embryogenesis where it caused miniature spindles. It was shown to act on components of the mitotic apparatus and to effectively inhibit cell division. Spectral Data UV:

/~

EtOH max

223,275, and 306nm (log e = 3.98, 3.71, and 3.66, respectively).

IR:

(KBr) 3300br, 1700, 1605, and 1585cm ~.

26.

Curvularins

661

~H NMR: (CD3OD) 1.16(3H, d, J=5.9Hz); 1.32(2H, m); 1.48(2H, m); 1.62(2H, m); 2.96(1H, dd, 3--9.0, 13.7Hz); 3.62(1H, dd, ,/--5.1, 13.7Hz); 3.67(1H, d, J=15.6Hz); 3.98(1H, d, J=15.6Hz); 4.08(1H, m); 4.87(1H, m); 6.24(1H, d, J=2.2Hz; and 6.28ppm (1H, d, Y=2.2Hz). Mass Spectrum: HREIMS: 308.1223Ore~e, M +, (C~6H2006); prominent M + - 18 ion for loss of H20. References S. Hyeon, A. Ozaki, A. Suzuki, and S. Tamura; Isolation of a,13-Dehydrocurvularin and 13-Hydroxycurvularin from Alternaria tomato as Sporulation Suppressing Factors; Agric. Biol. Chem., Vol. 40, pp. 1663-1664(1976). A. Kobayoshi, T. Hino, S. Yata, T. J. Itoh, H. Sato, and K. Kawazi, Unique Spindle Poisons, Curvularin and Its Derivatives, Isolated from Penicillium Species; Agric. Biol. Chem., Vol. 52, pp. 3119-3123(1988). S. Lai, Y. Shizuri, S. Uamamura, K. Kawai, Y. Terada, and H. Furukawa; Novel Curvularin-Type Metabolites of a Hybrid Strain Me 0005 Derived from Penicillium citreo-viride B. IFO 6200 and 4692; Tet. Lett., Vol. 70, pp. 2241-2244(1989).

662

26. Curvularins

Common/Systematic Name a,13-Dehydrocurvularin; trans-Dehydrocurvularin; 10,11-Dehydrocurvularin Molecular Formula/Molecular Weight C16H1805; MW = 290.11542 16

HO. 0~~.~.0.~ 5 12

OH

0

General Characteristics Pale yellow crystals from benzene; mp., 223-224~ -82.4 ~ (c=0.9, in EtOH).

[a]D -82.4 ~ (c=0.4, in MeOH); [a]D

Fungal Source Curvularia scirpicola, Penicillium sp. 511 (isolated from soil), Alternaria cinerariae (syn. Alternaria senecionis) a pathogen of the ornamental Cinerariae (ATCC 11784), and A lternaria tomato. Isolation/Purification The fungus was cultured in shaking flasks containing a medium composed of 1% potato starch (Difco) and 2% sucrose at 25~ for 6 days in darkness. The culture filtrate was extracted with ethyl acetate at pH 6.4. After evaporation of the solvent in vacuo, the residue was applied to a silica gel column (Wacogel C-100), and the column was eluted with benzene and benzene-ethyl acetate, successively. Marked sporulation-suppressing activity was observed in eluates with benzene containing 10% and 50% ethyl acetate. After evaporation of the solvent from 10% ethyl acetate eluate, the residue was recrystallized from benzene to give an active principle as pale yellow crystals. Biological Activity, Exhibited antifungal activity against Cladosporium herbarium; highly phytotoxic. Marked sporulation-suppressing activity against A. tomato. Spectral Data UV~

227(c = 14,700), 299(5,710), and 330nm (5,180). IR~

(KBr) 3428, 3312, 1712, and 1636cm ~.

26.

Curvularins

663

IH NMR: 1.21, 4-Me; 1.70, 1.90, 6-1-12;2.37, 2.43, 7-1-I2; 1.67, 1.90, 5-1-12;3.62, 4.07, 1-1-12; 4.70, 4-H; 6.30, 12-H; 6.34, 14-H; 6.74, 9-H; and 6.54ppm, 8-H. ~3CNMR: 20.23(C-4Me); 24.90(C-6); 33.15(C-7); 34.83(C-5); 43.59(C-1); 73.09(C-4); 103.16(C-12); 113.90(C-14); 116.21(C-16); 133.11(C-9); 139.63(C-15); 150.35(C-8); 163.51(C-13); 165.92(C-11); 172.47(C-2); and 198.11ppm (C-10). Mass Spectrum: HR IMS: 290.1177role M+, calcd 290.1157. References K. Arai, B. J. Rawlings, Y. Yoshizawa, and J. C. Vederas; Biosyntheses of Antibiotic A26771 B by Penicillium turbatum and Dehydrocurvularin by Alternaria cinerariae: Comparison of Stereoehemistry of Polyketide and Fatty Acid Enoyl Thiol Ester Reduetases; J. Am. Chem. Soe., Vol. 111, pp. 3391-3399(1989). G. Assante, R. Locci, L. Camarda, L. Merlini, and G. Nasini; Screening of the Genus Cercospora for Secondary Metabolites; Phytochemistry, Vol. 16, pp. 243-247(1977). S. Hyeon, A. Ozaki, A. Suzuki, and S. Tamura; Isolation of a,13-Dehydrocurvulailn and 13-Hydroxyeurvulailn from Alternaria tomato as Sporulation Suppressing Factors; Agile. Biol. Chem., Vol. 40, pp. 1663-1664(1976). D. J. Robeson, and G. A. Strobel; a,13-Dehydrocurvularin and Curvularin from Alternaria cinerariae; Z. Naturforseh., Teil C, Vol. 36, pp. 1081-1083(1981).

664

26.

Curvularins

Common/Systematic Name

cis-Dehydrocurvularin

Molecular Formula/Molecular Weight

Cl6HlsOs; ]VIVr= 290. I 1542 16

.o,

OH

0

General Characteristics

cis-Dehydrocurvularin was obtained as an amorphous powder; [tt]D22 +7.3 ~ (C=0.781, in

EtOH). Fungal Source

Penicillium citreo-viride B (IFO 6200 and 4692).

Isolation/Purification Polished rice was inoculated with a suspension of mycelium of the hybrid strain ME 0005, incubated stationarily at 25 ~ for 23 days, and extracted with acetone and then with EtOAc. The combined extracts were partitioned between EtOAc and water. The EtOAc extract was chromatographed on silica gel using a gradient solvent of MeOH-CHCI3 (150%). The dark brown oil eluted with CHCI3-MeOH (100:3-4, v/v) was separated by repeated preparative TLC (Kieselgel PF2s4) using hexane-EtOAc (5:1, v/v), hexaneEtOAc (2:1, v/v), benzene-acetone (5:1, v/v), chloroform-MeOH (15:1, v/v), chloroformacetone (2:1, v/v), and then chloroform-MeOH (10:1, v/v) to give cis-dehydrocurvularin and citreofuran. Spectral Data UV: ~, ~.toi~ 210, 220, 272, 256, and 287nm (log e = 4.21, 4.20, 3.91 and 3.74, respectively). max

IR:

(KBr) 3300br, 1705, 1620, and 1590cm~. IH N]VIR:

(CD3OD) 1.23(3H, d, J=6.4Hz); 1.77(IH, m); 2.03(IH, m); 2.66(IH, ddd, ,/=2.9, I I.5, 15.0Hz); 2.84(IH, ddd, ,/=2.9,5.9, 15I-Iz);3.10(IH, d, J=14.4Hz); 3.20(IH, d, J=14.4Hz); 5.16(IH, m); 6.09(IH, d, J=3.2FIz);6.21(IH, d, J=3.2Hz); 6.30(1H, d, ,/=2.4Hz); and 6.34ppm (IH, d, J=2.4Hz).

26.

Curvularins

665

~3C NMR: (CD3OD) 21.2(q); 26.5(t); 37.2(0; 42.5(t); 73.9(d); 102.5(d); 107.5(d); l ll.0(d); l ll.8(s); 112.2(d); 139.7(s); 148.7 (s); 156.0(s); 157.3(s); 159.5(s); and 174.1ppm (s). Mass Spectrum: HREIMS: 290.116 lm/e (M +, C16H1805). Reference S. Lai, Y. Shizuri, S. Uamamura, K. Kawai, Y. Terada, and H. Furukawa; Novel Curvularin-Type Metabolites of a Hybrid Strain Me 0005 Derived from Penicillium citreo-viride B IFO 6200 and 4692; Tet. Lett., Vol. 70, pp. 2241-2244(1989).

666

26.

Curvularins

Common/Systematic Name 11 tt-Hydroxycurvularin Molecular Formula/Molecular Weight C16H~006; MW = 308.12599

oyo OH

16

6 HO H

General Characteristics et-Hydroxycurvularin; mp., 150-152~ [~]D26 -29.4 ~ (c=0.33, in EtOH). FunRal Source v

Penicillium citreo-viride B (hybrid strain derived from IFO 6200 and 4692).

Isolation/Purification Polished rice was inoculated with a suspension of mycelium of the hybrid strain ME 0005, incubated stationarily at 25 ~ for 23 days, and extracted with acetone and then with EtOAc. The combined extracts were partitioned between EtOAc and water. The EtOAc extract was chromatographed on silica gel using a gradient solvent of MeOH-CHCI3 (150%). The dark brown oil eluted with CHC13-MeOH (100:9-10, v/v) was separated by repeated preparative TLC (Kieselgel PF254)using CHCI3-MeOH (10:1, v/v) then hexane-methylene chloride-EtOAc-MeOH (3:3:3:1, v/v/v/v) to afford a pale yellow solid, which was further separated by preparative HPLC (Develosil ODS-5) using CH3CN-H20 then MeOH-H20 (1:1, v/v) to afford a-hydroxycurvularin. Spe.ctral Data UV: /~EtOH max

222, 274, and 307nm (log e = 3.98, 3.78, and 3.69, respectively).

IR:

(KBr) 3300br, 1700, 1605, and 1585cm"l. ~H NMR: (CD3OD) 1.13(3H, d, J=6.3Hz); 1.45(4H, m); 1.61(1H, m); 1.68(1H, m); 3.23(2H, d, J=6.8Hz); 3.59(1H, d, J=15.6Hz); 3.97(1H, d, J=15.6Hz); 4.09(1H, m); 4.98(1H, m); 6.21(1H, d, J=2.2Hz); and 6.28ppm (1H, d, J=2.2Hz).

26.

Curvularins

Mass Spectrum: HREIMS: 308.1242m/e M +, (C16H2006); prominent M + - 18 for loss of H20. Reference S. Lai, Y. Shizuri, S. Uamamura, K. Kawai, Y. Terada, and H. Furukawa; Novel Curvularin-Type Metabolites of a Hybrid Strain Me 0005 Derived from Penicillium citreo-viride B. IFO 6200 and 4692; Tet. Lett., Vol. 70, pp. 2241-2244(1989).

667

668

26.

Curvularins

Common/Systematic Name 12-Oxocurvularin Molecular Formula/Molecular Weight C16H1806; ~

= 306.11034

0

0

HO

OH

0

General Characteristics 12-Oxocurvularin was an amorphous powder; [aiD 29 -43.5 ~ (c=0.47, in EtOH). Fungal Source Penicillium citreo-viride B (hybrid strain derived from IFO 6200 and 4692)

Isolation/Purification Polished rice was inoculated with a suspension of mycelium of the hybrid strain ME 0005, incubated stationarily at 25 ~ for 23 days, and extracted with acetone and then with EtOAc. The combined extracts were partitioned between EtOAc and water. The EtOAc extract was chromatographed on silica gel using a gradient solvent of MeOH-CHC13 (150%). The dark brown oil eluted with CHCI3-MeOH (100:5-6, v/v) was separated by repeated preparative TLC (Kieselgel PF254) using CHC13-MeOH (10:1, v/v), hexaneacetone (1:1, v/v), chloroform-MeOH (15:1, v/v), hexane-acetone (2:10, v/v), hexaneEtOAc-MeOH (7:2:1,v/v/v), then benzene-MeOH (4:1, v/v) to give 12-oxocurvularin. Spectral Data UV: ~ EtOH max

222, 272, and 299nm (log e = 3.99, 3.69, and 3.60, respectively).

(KBr) 3300br, 1710, 1608, and 1590cm q. ~H NMR:

(CD3OD) 1.13(3H, d, J=6.4Hz); 1.72(IH,m); 2.04(IH, m); 2.25(IH, ddd, J=2.9, 7.8, 16.6Hz); 2.66(IH, m); 2.73(II-I,ddd, J=2.3, 9.8, 16.6Hz);3.01(2H, m); 3.42(IH, m); 3.54(IH, d, J=14.91-Iz);3.64(IH, d, J=14.9Hz); 4.94(IH, m); 6.20(IH, d, :=2.4Hz); and 6.28ppm (IH, d, J=2.4Hz).

26.

Curvularins

669

13CNMR: (CD3OD) 19.8(q); 30.5(t); 38.3(t); 38.8(t); 39.7(t); 40.4(t); 71.8(d); 102.4(d); l ll.4(d); 120.6(s); 137.1(s); 158.7(s); 161.2 (s); 172.4(s); 208.3(s); and 212.5ppm (s). Mass Spectrum: HREIMS: 306.1082m/e M +, (C~6HlsO6). Reference S. Lai, Y. Shizuri, S. Uamamura, K. Kawai, Y. Terada and H. Furukawa, Novel Curvularin-Type Metabolites of a Hybrid Strain Me 0005 Derived from Penicillium citreo-viride B. IFO 6200 and 4692; Tet. Lett., Vol. 70, pp. 2241-2244(1989).

670

26.

Curvularins

Common/Systematic Name Methoxycurvularin Molecular Formula/Molecular Weight C17H2206; M W = 322.14164

OH

0

OMe

General Characteristics Methoxycurvularin was obtained as a colorless oil from 50% EtOAc eluate; [a]D 20 -13.9 ~ (c=3.3, in EtOH). Fungal Source

Penicillium sp. 511 (isolated from soil).

Isolation/Purification Penicillium sp. 511 was grown on potato-sucrose-agar for thirty days. The slant cultures were soaked in acetone for one week at room temperature. ARer evaporation of acetone the resulting aqueous concentrate was partitioned against EtOAc. The EtOAc extract was chromatographed on a silica gel column (Wakogel C-100). The column was eluted with 20, 30, 40 and 50% EtOAc/benzene. Purification of curvularin, 8-dehydrocurvularin, 8hydroxycurvularin and 8-methoxycurvularin was guided by a sea urchin egg assay. Biological Activity Cytotoxic against sea urchin (Hemicentrotus pulcherrimus) embryogenesis. It was shown to act on components of the mitotic apparatus and to effectively inhibit cell division. Spectral Data UV:

223,237, 275, and 308nm (e = 9,800, 8,200, 6,100, and 15,400, respectively).

/X, EtOH max

l-R:

(KBr) 3300br, 1710, 1615, 1595, and 1460cm "~. IH NMR: (CDCl3) 6.10(1H, d, J=3Hz); 6.00(1H, d, J=3Hz); 4.70(1H, m); 4.04-3.60(1H, m);

26.

Curvularins

3.92(1H, d, J=16Hz); 3.62(1H, d, J=16Hz); 3.26(3H, s); 3.20(1H, dd, J=3, 15Hz); 2.94(1H, dd, J=2.5, 15Hz); 1.44(6H, m); and 1.08ppm (3H, d, J=6.5Hz). 13CNMR: (CD3OD) 203.7, s; 171.3, s; 160.4, s; 137.1, s; 136.1, s; 111.5, d; 101.7, d; 77.0, d; 74.3, d; 56.1, q; 40.0, t; 33.8, t; 32.1, t; 23.3, t; 21.3, t; and 18.9ppm, q. Mass Spectrum: EIMS: 322(M+, 6%), 290(100), 203(57), 177(38), 167(39), 150(59), 121(35), and 69m/e (67). Reference A. Kobayoshi, T. Hino, S. Yata, T. J. Itoh, H. Sato, and K. Kawazi; Unique Spindle Poisons, Curvularin and Its Derivatives, Isolated from Penicillium Species; Agric. Biol. Chem., Vol. 52, pp. 3119-3123(1988).

671

672

26.

Curvularins

Common/Systematic Name Citreofuran Molecular Formula/Molecular Weight C16H1605; MW = 288.09977

oyo.:

HO 0

General Characteristics Melting point, 203-205~

[a]D 27 4"

112~ (c=0.18, in EtOH).

Fungal Source Penicillium citreo-viride B (hybrid strain derived from IFO 6200 and 4692). Isolation/Purification Polished rice was inoculated with a suspension of mycelium of the hybrid strain ME 0005, incubated stationarily at 25 ~ for 23 days, and extracted with acetone and then with EtOAc. The combined extracts were partitioned between EtOAc and water. The EtOAc extract was chromatographed on silica gel using a gradient solvent of MeOH-CHCI3 (150%). The dark brown oil eluted with CHCI3-MeOH (100:3-4, v/v) was separated by repeated preparative TLC (Kieselgel PF254) using hexane-EtOAc (5 1, v/v), hexaneEtOAc (2:1, v/v), benzene-acetone (5 1, v/v), chloroform-MeOH (15 1, v/v), chloroformacetone (2:1, v/v), and then chloroform-MeOH (10" 1, v/v) to give cis-dehydrocurvularin and citreofuran. Spectral Data UV~

~, EtoH 210, 220, 272, 256, and 287nm (log e = 4.21, 4.20, 3.91, and 3.74, respectively). max

IR:

(KBr) 3300br, 1705, 1620, and 1590 cm"l. IH ~ :

(CDaOD) 1.23(3H,d, J=6.4Hz); 1.77(II-I,m); 2.03(II-I,m); 2.66 (IH, ddd, J=2.9, 11.5, 15.0Hz); 2.84(1H, ddd, ,/=2.9, 5.9, 15Hz); 3.10(1H, d, J=14.4Hz); 3.20(1H, d, J=14.4 Hz); 5.16(1H, m); 6.09(1H, d, J=3.2Hz); 6.21(1H, d, J=3.2Hz); 6.30(1H, d, J=2.4Hz); and 6.34ppm (1H, d, J=2.4Hz).

26.

Curvularins

673

13CNM~: (CD3OD) 21.2(q); 26.5(t); 37.2(t); 42.5(0; 73.9(d); 102.5(d); 107.5(d); 111.0(d); l ll.8(s); 112.2(d); 139.7(s); 148.7 (s); 156.0(s); 157.3(s); 159.5(s); and 174.1ppm (s). Mass Spectrum HREIMS: 288.10150m/e (M+, C16H1605). Reference S. Lai, Y. Shizuri, S. Uamamura, K. Kawai, Y. Terada, and H. Furukawa; Novel Curvularin-Type Metabolites of a Hybrid Strain Me 0005 Derived from Penicillium citreo-viride B. IFO 6200 and 4692, Tet. Lett., Vol. 70, pp. 2241-2244(1989).

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Arcyroxepin A and Related Bisindoles Arcyroxepin A Arcyriaflavin B Arcyriaflavin C Arcyriarubin B Arcyriarubin C

675

This Page Intentionally Left Blank

27.

Arcyroxepin A and Related Bisindoles

677

Common/Systematic Name Arcyroxepin A Molecular Formula/Molecular Weight v

C2oHIIN303; M W = 341.08004

H

o-f H

N

H

General Characteristics Red crystals; mp., 268-270~ Fungal Sou.rce

Arcyria denudata (fruiting bodies).

Isolation/Purification Extracted with methanol, separated by chromatography on Sephadex LH-20 (eluent methanol). Biological Activity In the platelet diffusion test, arcyroxepin A exhibited medium inhibiting action against Bac;llus brevis and B. subtilis. Spectral I?~t___ga UV" -#

MeOH max

471(1og e=3.68), 362(3.51), 283(3.83), 273(3.82), and 226nm (4.44).

IR~

(KBr) 3420 br, s; 1760 w; 1710, ss; 1625, w; 1510, m; 735cm"~ s. 1H NMPx:

(CDaCOCD3, TMS) 7.0-7.40(m); 9.82(br. s, 1,1'-NH); 111.22ppm (very br. s,

10-NH).

~3C NMR: (CD3OD, TMS) 92"(C-3);I12.4(CH); II6.1(CH); 123";124.5";125.9(CH); 133"; 143.2(C-7a);153.2"(C-2);and 177.0ppm (C-9). * Signals are broadened by coalescence; assignment of the signals of coupled carbon by

678

27.

Arcyroxepin A and Related Bisindoles

selective decoupling and increment calculation. TLC Data Silica gel F254,benzene-ethyl formate-formic acid (10:5:3, v/v/v); Rf=0.42. Reversible color change to violet over NH3 vapor. Reference W. Steglich, B. Steffan, L. Kopanski, and G. Eckhardt; Indole Pigments from the Bodies of the Slime Mold Arcyria denudata; Angew. Chem. Int. Ed. Engl., Vol. 19, pp. 459-460(1980).

27.

Arcyroxepin A and Related Bisindoles

679

Common/Systematic Name Arcyriaflavin B Molecular Formula/Molecular Weight C20HllN303; M W "- 341.08004 H

o=("3=o H

H

General Characteristics Pale yellow crystals; mp., 350 ~C. Fungal Source

Arcyria denudata (fruiting bodies).

Isolation/Purification Extracted with methanol, separated by chromatography on Sephadex LH-20 (eluent methanol). Spectral Data UV: /~ M 9 max

414(1og e=3.19), 323(4.11), 280(3.75), 271(3.70), and 229nm (4.07).

IR:

(KBr) 3420 br, s; 2955 s; 2880 m; 1750 w; 1705 s; and 1620cm"~. br, m. 1H NMR: (CD3COCD3, TMS) 6.93(dd, J=8.6 + 2.1Hz, 5-H); 7.16(d, J-2.1Hz 7-H), 7.24-7.80(m, 5',6',7'-H); 8.96(d, J=8.6Hz, 4-H); 9.15 (broadened d, J=7.9Hz, 4'-H); 9.64(br. s, 10-NH); and 11.65ppm (br. s, 1, I'-NH). TLC Data Silica gel F254,benzene-ethyl formate-formic acid (10:5:3, v/v/v); Rf=0.33. Detection: strong bright yellow fluorescence at 366nm. Reference W. Steglich, B. Steffan, L. Kopanski, and G. Eckhardt; Indole Pigments from the Bodies of the Slime Mold Arcyria denudata; Angew. Chem. Int. Ed. Engl., Vol. 19, pp. 459-460(1980).

680

27.

Arcyroxepin A and Related Bisindoles

Common/Systematic Name Arcyriaflavin C Molecular Formula/Molecular Weight C2oHllN304; MW = 357.07496

H

HornH/ ~N~OH H

General Characteristics Pale yellow crystals; mp., 350~ Fungal Source

Arcyria denudata (fruiting bodies).

Isolation/Purification Extracted with methanol, separated by chromatography on Sephadex LH-20 (eluent methanol). Spectral Data UV:

/~

MeOH max

422(1og e = 3.34), 330.5(4.29), 318(sh, 4.09), 280(3.70), 270(3.66), 255(sh, 3.77), and 229nm (4.14).

IR:

(KBr) 3420 br, s; 2960 s; 2880 m; 1740 w; 1710 s; and 1635cmq br, s. 1H N M R :

(CD3COCD3, TMS) 6.89(dd,,/=8.4+ 2.2Hz, 5,5'-H);7.13(d,J=2.2Hz, 7,7'-H); 8.93(d, J=8.41-Iz,4,4'-I-I);9.56(br. s, 10-NH); 11.92ppm (br. s, I,I'-NH). TLC Data Silica gel F254; benzene-ethyl formate-formic acid (10:5:3, v/v/v); Rf=0.21; detection: strong bright yellow fluorescence at 366nm. Reference W. Steglich, B. Steffan, L. Kopanski, and G. Eckhardt; Indole Pigments from the Bodies of the Slime Mold Arcyria denudata; Angew. Chem. Int. Ed. Engl., Vol. 19, pp. 459-460(1980).

27.

Arcyroxepin A and Related Bisindoles

681

Common/Systematic Name Arcyriarubin B Molecular Formula/Molecular Weight C 2 0 H I 3 N 3 0 3 ; M W -- 343.09569 H

o=(3=o H

H

General Characteristics Red crystals; mp., 154-155 ~C. Fungal Source

Arcyria denudata (fruiting bodies).

Isolation/Purification Extracted with methanol, separated by chromatography on Sephadex LH-20 (eluent methanol). Biological Activity In the platelet diffusion test, arcyriarubin B exhibited medium inhibiting action against Bacillus brevis and B. subtilis. Spectral Data UV:

/~

MeOH max

465(1og e=3.77), 392(sh, 3.57), and 281nm (3.93).

(KBr) 3440 br, s; 2960 m; 1760 m; 1715 s; 1630 w; 1538cmq s. ~H NMR: (CD3COCD3, TMS) 6.24(dd, 3'-8.7 + 2.1Hz, 5-H), 6.72(d, J=8.7Hz, 4-H), 6.85 (d, J=2.4Hz, 7-H); 6.72-7.05 (m, 5', 6', 7'-H), 7.42("d", J=8Hz, 4'H); 7.73 (d, J=2.7Hz, 2-H), 7.82(d, J-2.9Hz, 2'-H), 9.63(br. s, 10-NH); 10.52(br. s, I'-NH), 10.78ppm (br. s, 1-NH). ~3CNMR:

(CD3OD, TMS) 97.7(C-7),I07.7(C-3/3');II0.8(C-5),I12.3(C-7');120.7(CH); 120.9(C-3a), 122.7(CH); 123.0(CH); 123.2(C4), 127.3(C-3a'); 128.9(C-2/8'),

682

27.

Arcyroxepin A and Related Bisindoles

129.7(C-2'); 129.9(C-8); 137.8(C-7a'); 138.9(C-7a); 154.5(C-6); 175.3ppm (C-9/9'). TLC Data Silica gel F254;benzene-ethyl formate-formic acid (10:5:3, v/v/v); Re=0.33. Reference W. Steglich, B. Steffan, L. Kopanski, and G. Eckhardt; Indole Pigments from the Bodies of the Slime Mold Arcyria denudata; Angew. Chem. Int. Ed. Engl., Vol. 19, pp. 459-460(1980).

27.

Arcyroxepin A and Related Bisindoles

683

Common/Systematic Name Arcyriarubin C Molecular Formula/Molecular Weight C2oHI3N304, ~ = 359.09061 H

H

H

General Characteristics Red crystals; mp., 205-206~ Fungal Source

Arcyria denudata (fruiting bodies).

Isolation/Purification Extracted with methanol, separated by chromatography on Sephadex LH-20 (eluent methanol). Biological Activity In the platelet diffusion test, arcyriarubin C exhibited medium inhibiting action against Bacillus brevis and B. subtilis. Spectral Data UV;

~

MeOH max

474(1og r = 3.76) and 283nm (3.93).

IPx;

(KBr) 3430 br, s; 1755 w; 1705 s; 1625 m; 1535cm"1 s.

~H MR: (CD3COCD3, TMS) 6.27(dd,J=8.4 + 2.2Hz, 5,5'-H);6.79(d,J=8.4Hz,-4,4'-H), 6.85(d,J=2.2Hz, 7,7'-H);7.66(d,J=2.5Hz, 2,2'-H);9.51(br.s, 10-NH); 10.42ppm (br. s, I,I'-H). 13C

NMPx:

(CDaOD, TMS) 97.7(C-7); 107.8(C-3); 110.9(C-5); 121.0(C-3a); 123.3(C-4); 128.7(J=190Hz, C-2); 129.2(C-8); 138.8(C-7a); 154.4(C-6); and 175.3ppm (C-9).

684

27.

Arcyroxepin A and Related Bisindoles

TLC Data Silica gel F254;benzene-ethyl formate-formic acid (10:5:3); Rf=0.21. Reference W. Steglich, B. Steffan, L. Kopanski, and G. Eckhardt; Indole Pigments from the Bodies of the Slime Mold Arcyria denudata; Angew. Chem. Int. Ed. Engl., Vol. 19, pp. 459-460(1980).

Arugosins Arugosin A Arugosin B Arugosin C Arugosin D 25-O-Methylarugosin A

685

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28. Arugosins

687

Common/Systematic Name Arugosin A Molecular Formula/Molecular Weight C25H2806, ~ 424.18859 =

~

OH Me~

0

OH

Me CH2CH=C/

O Me

Me

Me

General Characteristics Optically inactive yellow oil. Fungal Source Aspergillus rugulosus (I.M.I. strain 84338) Isolation/Purification A. rugulosus was grown for 15 days at 25 ~ on a low-nitrogen medium in stationary culture. The mycelium, alter separation from the culture fluid, was macerated in a blender with distilled water and extracted with ether. The extract was concentrated in vacuo to yield a dark brown viscous residue. Portions of the residue were distributed equally between the first six tubes of a 200-tube automatic counter-current distribution apparatus and subjected to 200 transfers with light petroleum-methanol-water (8:10:1, v/v/v). Tubes 50-80 yielded impure arugosin A as a yellow syrup, which was subjected to preparative thin-layer chromatography on Kieselgel G plates (light petroleum-ethyl acetate, 72:28, v/v). A light yellow band at Re 0.75 was collected and the metabolite eluted with ethyl acetate. The metabolite was obtained as a viscous yellow oil alter concentration m vacuo. Spectral Data UV: ~ EtOH max

227, 280, and 365nm (log e 4.32, 3.97, 3.86, 4.01, and 395, respectively);

~, maxEtOn'N'on 241, 272, 384, and 427nm (log e 4.58, 4.27, 3.94, and 3.93 respectively). IR:

(CHC13) 3395(OH) and 1735cm ] (C=O).

688

28. Arugosins

1H NMR: (CDC13) ArOH,-3.37(1H, s); ArOH,-1.09(1H, s); ArH, 2.76(1H, d, J=8.0Hz); ArH, 3.19(1H, s); ArCH(OH)-O, 3.46(1H, d, d=5.0Hz); ArH, 3.56(1H, d, J=8.0Hz); =CHCH2-O-, 4.48(1H, m); ArCH2-CH=, 4.73(1H, m); ArCH(OH)-O-, 5.0(1H, d, d=5.0Hz); O-CH2-CH, 5.66(2H, rn, ,/--4.0, 9.0Hz); ARCH3,6.74(2H, d, d=8.0Hz); ARCH3, 7.68(3H, s); and (CH3)2C=, 8.25z (6H, d, J=7.0Hz). 13C NMR: (CDC13) C-I, 194.4; C-3, 6, 8, 12, = 160.1, 155.4, 150.6, 143.6; C-21,139.0; C-20, 137.4(d); C-15, 135.8; C-16, 130.8; C-7, 128.5; C-5, 121.9; C-10, 120.7; C-2 or 13, 118.9; C-4, 118.8(d); C-2 or 13, 117.3; C-11,112.5; C-9, 110.0(d); C-25, 92.4; C-19, 70.8(t); and C-14, 17, 18, 22, 23, 24, = 30.2, 27.3, 25.4, 17.3, 17.4, 16.5ppm (m). Mass Spectrum: HREIMS: 424.1886m/e (M§ C25H2806requires 424.1886. References J. A. Ballantine, V. Ferrito, C. H. HassaU, and M. L. Jenkins; The Biosynthesis of Phenols. Part XXIV. Arugosin C, a Metabolite of a Mutant Strain ofAspergillus rugulosus; J. Chem. Soc., Perkin I, pp. 1825-1830(1973). J. A. Ballentine, D. J. Francis, C. H. Hassall, and J. L. C. Wright; The Biosynthesis of Phenols. Part XXI. The Molecular Structure of Arugosin, a Metabolite of a Wild-type Strain ofAspergillus rugulosus; J. Chem. Soc. (C), pp. 1175-1182(1970). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

28. Arugosins

689

Common/Systematic Name Arugosin B Molecular Formula/Molecular Weight C25H2806; M W = 4 2 4 . 1 8 8 5 9

OH

Me

0

OH

2.,

O..v~ Me

Me

CH2-CH=C[

Me

General Characteristics Optically inactive yellow oil. FunRal Source Aspergillus rugulosus (I.M.I. strain 84338) Isolation/Purification A. rugulosus was grown for 15 days at 25 ~ on a low-nitrogen medium in stationary culture. The mycelium, after separation from the culture fluid, was macerated in a blender with distilled water and extracted with ether. The extract was concentrated in vacuo to yield a dark brown viscous residue. Portions of the residue were distributed equally between the first six tubes of a 200-tube automatic counter-current distribution apparatus and subjected to 200 transfers with light petroleum-methanol-water (8:10:1, v/v/v). Tubes 50-80 yielded impure arugosin B as a yellow syrup, which was subjected to preparative thin-layer chromatography on Kieselgel G plates (light petroleum-ethyl acetate, 72:28, v/v). A light yellow band at Re 0.60 was collected and the metabolite eluted with ethyl acetate. The metabolite was obtained as a viscous yellow oil after concentration m vacuo. Spectral Data UV:

~, E~r~,x 227, 280, and 365nm (log e 4.32, 3.97, 3.86, 4.01, and 395, respectively; /~ EtOH-NaOH

m~

241, 272, 384, and 427nm (log e = 4.58, 4.27, 3.94, and 3.93).

IR:

(CHC13) 3395(OH) and 1735cm 1 (C=O).

690

28. Arugosins

1H NMR: (CDCI3) ArOH,-2.78(1H, s); ArOH,-0.87(1H, s); ArH, 2.76(1H, d, J=8.0Hz); ArH, 3.19(1H, s); ArCH(OH)-O, 3.46(1H, d, J=5.0Hz); ArH, 3.56(1H, d, J=8.0Hz); =CHCH2-O-, 4.48(1H, m); ArCH2-CH=, 4.73(1H, m); ArCH(OH)-O-, 5.0(1H, d, J=5.0Hz); O-CH2-CH, 5.66(2H, m, ,]=4.0, 9.0Hz); ARCH3,6.74(2H, d, J=8.0Hz); ARCH3, 7.68(3H, s); and (CH3)2C=, 8.25z (6H, d, J=7.0Hz). 13C NMR: (CDCI3) C-l, 194.4; C-3, 6, 8, 12, = 159.4, 154.8, 149.9, 143.5; C-21,138.8; C-20, 137.4(d); C-15, 135.2; C-16, 130.6; C-5, 121.0; C-10, 121.1; C-2 or, 13, 118.9; C-4, 118.8(d); C-2 or 13, 116.3; C-11, 116.0; C-9, 108.4(d); C-25, 91.6; C-19, 70.1(t); and C-14, 17, 18, 22, 23, 24, = 28.2, 27.3, 25.0, 17.3, 17.4, 16.5ppm (m). Mass Spectrum: HREIMS: 424.1886role (M+); C25H2sO6requires 424.1886. References J. A. Ballantine, V. Ferrito, C. H. Hassall, and M. L. Jenkins; The Biosynthesis of Phenols. Part XXIV. Arugosin C, a Metabolite of a Mutant Strain of Aspergillus rugulosus; J. Chem. Soc., Perkin I,, pp. 1825-1830(1973). J. A. Ballentine, D. J. Francis, C. H. Hassall, and J. L. C. Wright; The Biosynthesis of Phenols. Part XXI. The Molecular Structure of Arugosin, a Metabolite of a Wild-type Strain ofAspergillus rugulosus; J. Chem. Soc. (C), pp. 1175-1182(1970). W. B. Turner and D. C. Aldridge; Fungal Metabolites H; Academic Press, New York, New York, 631 pp. (1983).

28. Arugosins

691

Common/Systematic Name Arugosin C 1,12a-Dihydro-6,8-dihydroxy- 1-(1-hydroxy- 1-methylethyl)-4-methyl-9-(3-methylbut-2enyl[ 1]benzopyrano[4,5-bc][ 1]benzoxepin-7(2H)-one Molecular Formula/Molecular Weight C25H2806; M~V -- 424.18859

OH 0

OH 1 5 ~

Me"5"~ ,,,.,"~0"8"~ 019"Y~OH General Characteristics Obtained as a golden yellow oil. Funsal Source A mutant strain ofAspergillus rugulosus (A.R.M. 325). v

Isolation/Purification A mutant strain ofA. rugulosus was grown for 14 days at 25 ~ on a low-nitrogen medium in stationary culture. The mycelium, atter separation from the culture fluid, was macerated in a blender with distilled water and extracted with ether. The extract was concentrated in vacuo to yield a dark brown viscous residue. Portions of the residue were distributed equally between the first six tubes of a 200-tube automatic counter-current distribution apparatus and subjected to 200 transfers with light petroleum-methanol-water (10:10:1, v/v/v). Tubes 50-80 yielded impure arugosin C as a yellow syrup, which was subjected to preparative TLC on Kieselgel G plates. A light yellow band at Rf 0.55 was collected and the metabolite eluted with ethyl acetate. The metabolite was further purified by gel filtration through Sephadex LH20 in methanol to yield pure arugosin C. Soectral Data UV: ~ EtOH rallx

227, 271,293,309, and 407nm (log e 4.34, 3.94, 3.97, 4.01, and 395, respectively).

IR:

(CHCI3) 3600(OH), 3440br (OH), and 1620cm~ (C=O).

692

28. Arugosins

CD: (MeOR c= 8.4) 409, 307, 262, and 223nm (mol. ellipticity-12,640, +12,060, + 10,200, and +44,220). ORD: (c=8.4, in MeOH) 446, 371,333,295, 274 and 236nm (molecular rotation -4,950, -16,800, -25,500, -9,100, +10,900, and +23,600~ ~H NMR: (CDCI3) ArOU, -3.85(s); ArOH, -0.69(s); ArH, 2.76(d, d=8.0Hz); ArH, 3.23(s); ArH, 3.64(d, J=8.0Hz); ArCH2-CH=, 4.72(m); ArCHO, 4.96(d, J=4.0Hz); O-CH2-CH, 5.76(m, d=4.0, 9.0Hz); ARCH?.,6.72(d, J=8.0Hz); O-CH2-CH-CI-I, 7.67(m, d=4.0, 9.0Hz); ARCH3, 7.78(s); (CH3)2C=, 8.28(d, J= 7.0Hz); C-OH, 8.44(s); (CH3)2C-O, 8.70 (s); and (CH3)2C-O, 8.75z (s). ~3C NMR: (CDC13) C-I, 194.0; C-3, 6,8, 12, = 160.8, 156.6, 153.3, 143.0; C-15, 135.3; C-7, 134.2; C-16, 130.7; C-5, 122.1; C-10, 120.1; C-2 or, 13, 118.7; C-4, 118.2(d); C-2 or 13, 117.8; C-11, 110.8; C-9, 107.2(d); C-25, 73.1; C-21, 69.8; C-19, 64.2(0; C-20, 48.7(d); and C-14, 17, 18, 22, 23, 24, = 28.3, 27.7, 27.3, 25.3, 17.4, 16.1ppm. Mass Spectrum: HREIMS: 424.1886m/e (M+) C25H2806requires 424.1886; LREIMS: 424(31%), 366(10), 349(7), 293(11), 205(14), 203(17), 189(54), 188 (100), 163(12), 162(58), 161 (21), 149(25), and 59m/e (26). References J. A. Ballantine, V. Ferrito, C. H. Hassall, and M. L. Jenkins; The Biosynthesis of Phenols. Part XXIV. Arugosin C, a Metabolite of a Mutant Strain ofAspergillus rugulosus; J. Chem. Soc., Perkin I, pp. 1825-1830(1973). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

28. Arugosins

693

Common/Systematic Name Arugosin D 1,12a-Dihydro-6,8-dihydroxy-l-(1-hydroxy-l-methylethyl)-4-methyl-9-(2-hydroxy3-methylbut-3-enyl) [ 1]benzopyrano[4, 5-bc] [ 1]benzoxepin-7(2H)-one Molecular Formula/Molecular Weight C25H2807; M W = 440.18350 22

Me

21/~"OH

0" Me

~

"Me

25

23

OH Me I

I

H2-CH--C=CH2 OH

0

OH

General Characteristics Isolated as a viscous yellow oil; Re 0.40 [ether-benzene (1:1)]; [a]D -31.2 ~ (c=0.95, in CHC13). Fungal Source

Aspergi llus variecolor

Isolation/Purification The cultures of the individual strains of A. variecolor were extracted with light petroleum. The isolations of the mycelial metabolites from the methanol-soluble fractions of the light petroleum extracts were carried out as for shamixanthone and tajixanthone. Arugosin D was isolated from the mycelium by preparative TLC on silica gel GF (Merck). Spectral Data 1H NMR: (CDC13) I-OH, -0.60 or -3.88; 3-H, 2.70; 5-H, 3.20; 14-CH2, ca. 7.10; 15-H, ca. 5.65; 17 and 18-CH3, 8.20(5.00, 5.16); 19-CH2, ca. 5.70; 20-H, ca. 7.58; 23-CH2, 24-CH3, 8.26, 8.31; 24-CH3, 7.80; 25-CH, 4.90; 4-H, 3.58; 11-OH, -3.88 or-0.601:. Mass Spectrum: EIMS: 440.182(M +) C25H2807 requires 440.182. References K. K. Chexal, J. S. E. Holker, and T. J. Simpson; The Biosynthesis ofFungal Metabolites. Part VI. Structures and Biosynthesis of Some Minor Metabolites from Variant Strains of Aspergillus variecolor; J. C. S. Perkin I, pp. 549-554(1975).

694

28. Arugosins

J. S. E. Holker, R. D. Lapper, and T. J. Simpson; The Biosynthesis ofFungal Metabolites. Part IV Tajixanthone: 13C NMR Spectrum and Feedings with [ 1-13C]- and [2-~3C]Acetate; J. C. S. Perkin I, 2135-2142(1974).

28. Arugosins

695

Common/Systematic Name 25-O-Methylarugosin A 1,1•-Dihydr•xy-6-meth•xy-8-methy•-2-(3-methy•but-2-eny•)-7-(3-methy•but-2-eny••xy)dibenz[b, e]oxepin- 11(6H)-one Molecular Formula/Molecular Weight C26H3oO6; M W --- 4 3 8 . 2 0 4 2 4 22

Me

~9 j " / "Me 0/

OMe 17

/

Me

H=C 16 OH

0

OH

\

Me 18

General Characteristics Isolated as a viscous yellow oil; Re 0.50 (benzene). Fungal Source Aspergillus variecolor Isolation/Purification The cultures of the individual strains of A. variecolor were extracted with light petroleum. The isolations of the mycelial metabolites from the methanol-soluble fractions of the light petroleum extracts were carried out as for shamixanthone and tajixanthone. 25-0Methylarugosin A was isolated from the mycelium by preparative TLC on silica gel GF (Merck). Spectral Data 1H NMR: (CDC13) I-OH, -0.83 or-2.43; 3-H, 2.71; 5-H, 3.14; 14-CH2, 6.65; 15-H, 4.68; 17 and 18-CH3, 8.12, 8.18; 19-CH2, 5.67, 5.74; 20-H, 4.48; 23-CH2, 24-CH3, 8.26, 8.31; 24CH3, 7.66; 25-CH, 3.63; 4-H, 3.50; 1I-OH, -2.43 or-0.83; and 25-OMe, 5.45z. Mass Spectrum: EIMS: 438.207(M+) C26H3006requires 438.204.

696

28. Arugosins

References K. K. Chexal, J. S. E. Holker, and T. J. Simpson; The Biosynthesis ofFungal Metabolites. Part VI. Structures and Biosynthesis of Some Minor Metabolites from Variant Strains of Aspergillus variecolor; J. C. S. Perkin I, pp. 549-554(1975). J. S. E. Holker, R. D. Lapper, and T. J. Simpson; The Biosynthesis ofFungal Metabolites. Part IV Tajixanthone: 13C NMR Spectrum and Feedings with [ 1-13C]- and [2-13C]Acetate; J. C. S. Perkin I, 2135-2412(1974).

Hericenes and Hericenones Hericene A Hericene B Hericene C Hericenone A Hericenone B Hericenone E Hericenone F Hericenone G

697

This Page Intentionally Left Blank

29.

Hericenes and Hericenones

699

Common/Systematic Name Hericene A Palmitic acid ester of 4(3',7'-dimethyl-2',6'-octadienyl)-2-formyl-3-hydroxyl-5methoxylbenzyl alcohol Molecular Formula/Molecular Weight C35H5605; 1VIW': 556.41278 9'

10'

/

/

~

OH

8

CHO MeO 9

O--CO(CH2)14Me 6

7

General Characteristics Hericene A was isolated as an oil. Fungal Source Hericium erinaceum (strain CBS 233.87)

Isolation/Purification Fungal cultures were extracted with EtOAc containing 1% MeOH and the extracts were evaporated to give a mixture of crude metabolites. The mixture was chromatographed on a silica gel column using hexane-EtOAc (4:1, v/v) to yield hericenes A-C, fatty acids (palmitic, linoleic, and oleic acids); the residual material was then eluted with EtOAc containing MeOH (1%) to give erinapyrone C. Further purification of the above mixture of compounds by preparative TLC (RP18 plates Me2CO-H20, 8:1, v/v) and HPLC with a Si-60 column [250 X 50mm, 6ml/min, hexane-EtOAc (98:2)] with detection at 298nm gave hericenes A-C in a 5:3:2 ratio. Spectral Data IR:

(thin film) 3450, 1730, 1630, and 1570cm"1. 1H NMR: (CDCI3) H-6, 6.52(s); H-7, 5.32(s), H-8, 10.10(s); H-9, 3.91(s); 3-OH, 12.37(s), H-I', 33.4(br d, J=7.0Hz); n-2', 5.17(br t, J=7.0Hz); U-4', 1.97(m); n-5', 2.03 (m); H-6', 5.05(br t, J=6.8Hz); H-8', 1.63(br s); H-9', 1.60(br s), H-10', 1.77(br s), H-2", 2.33(t, J=7.5Hz); H-3", 1.60(m); and H-16", 0.88ppm (t, J=6.5Hz).

700

29.

Hericenes and Hericenones

13C NMR: (CDC13) C-l, 138.41; C-2, 112.87; C-3, 162.88; C-4, 118.06; C-5, 163.47; C-6, 105.60; C-7, 62.96; C-8, 193.09; C-9, 55.90; C-I', 21.35; C-2', 121.19; C-3', 131.23"; C-4', 39.77; C-5', 26.67; C-6', 124.36; C-7', 135.75"; C-8', 25.67; C-9', 17.65; C-10', 16. 10; C-I", 173.21; C-2", 34.23; and C-16", 14.14ppm. Assignments may be reversed. Mass Spectrum: EllS: 556(M§ 300, and 257re~e; CIMS: 557(M+ + 1) Reference A. Amone, R. Cardillo, G. Nasini, and O. Vajna De Pava; Secondary Mold Metabolites: Part 46. Hericenes A-C and Erinapyrone C, New Metabolites Produced by the Fungus Hericium erinaceum; J. Nat. Prod., Vol. 57, pp. 602-606(1994).

29.

Hericenes and Hericenones

701

Common/Systematic Name Hericene B Oleic acid ester of 4(3',7'-dimethyl-2',6'-octadienyl)-2-formyl-3-hydroxyl-5methoxylbenzyl alcohol Molecular Formula/Molecular Weight C37HssOs; MW = 582.42843 9'

~

1(~

/

8'

/

6'

4'

OH 2'

II

8

CHO I

Meo~O--CO( 9

6

7

1"

18"

c H2)7CH=CH(CH2)7Me

General Characteristics Hericene B was isolated as an oil. Fungal Source Hericium erinaceum (strain CBS 233.87).

Isolation/Purification Fungal cultures were extracted with EtOAc containing 1% MeOH and the extracts were evaporated to give a mixture of crude metabolites. The mixture was chromatographed on a silica gel column using hexane-EtOAc (4:1, v/v) to yield hericenes A-C, fatty acids (palmitic, linoleic, and oleic acids); the residual material was then eluted with EtOAc containing MeOH (1%) to give erinapyrone C. Further purification of the above mixture of compounds by preparative TLC (RPls plates Me2CO-H20, 8:1, v/v) and HPLC with a Si-60 column [250 X 50mm, 6ml/min, hexane-EtOAc (98:2, v/v)] with detection at 298nm gave hericenes A-C in a 5:3:2 ratio. Spectral Data 1H NMR: (CDC13) The data of the alcoholic portion of hericene B paralleled those of hericene A; the vinylic protons of the oleic portion resonated at 5.34ppm, while the remaining protons resonated between 2.4 and 0.8ppm. 13C NMR: (CDC13) The data of the alcoholic portion of hericene B paralleled those of hericene A; the vinylic carbons of the oleic portion resonated at 130.02 and 129.71ppm, while the remaining carbons resonated between 32 and 14ppm. Mass Spectrum: EIMS: 582(M+), 300, and 283m/e.

702

29.

Hericenes and Hericenones

Reference A. Amone, R. Cardillo, G. Nasini, and O. Vajna De Pava, Secondary Mold Metabolites: Part 46. Heficenes A-C and Efinapyrone C, New Metabolites Produced by The Fungus Hericium erinaceum, J. Nat. Prod., Vol. 57, pp. 602-606(1994).

29.

Hericenes and Hericenones

703

Common/Systematic Name Hericene C Steric acid ester of 4(3',7'-dimethyl-2',6'-octadienyl)-2-formyl-3-hydroxyl-5methoxylbenzyl alcohol Molecular Formula/Molecular Weight C37H6005; M W "-- 5 8 4 . 4 4 4 0 8

9'

10'

/

J

~

OH

8

CHO MeO 9

O~CO(CH2)16Me 6

7

General Characteristics Hericene C was isolated as an oil. Fungal Source Hericium erinaceum (strain CBS 233.87)

Isolation/Purification Fungal cultures were extracted with EtOAc containing 1% MeOH and the extracts were evaporated to give a mixture of crude metabolites. The mixture was chromatographed on a silica gel column using hexane-EtOAc (4:1, v/v) to yield hericenes A-C, fatty acids (palmitic, linoleic, and oleic acids); the residual material was then eluted with EtOAc containing MeOH (1%) to give erinapyrone C. Further purification of the above mixture of compounds by preparative TLC (RPls plates Me2CO-H20, 8:1, v/v) and HPLC with a Si-60 column [250 X 50mm, 6ml/min, hexane-EtOAc (98:2, v/v)] with detection at 298nm gave hericenes A-C in a 5:3:2 ratio. Spectral Data ~H NMR: (CDC13) The data of the alcoholic portion ofhericene B and C paralleled those of hericene A; the vinylic protons of the oleic portion of B resonated at 5.34ppm, while the remaining protons of B and C resonated between 2.4 and 0.8ppm. 13C M R : (CDC13) The data of the alcoholic portion of hericene B and C paralleled those of hericene A; the vinylic carbons of the oleic portion of B resonated at 130.02 and 129.71 ppm, while the remaining carbons of hericenes A and C resonated between 32 and 14ppm.

704

29.

Hericenes and Hericenones

Mass Spectrum: EIMS: 584(M+), 300, and 285m/e. Reference A. Arnone, R. Cardillo, G. Nasini, and O. Vajna De Pava; Secondary Mold Metabolites: Part 46. Hericenes A-C and Erinapyrone C, New Metabolites Produced by the Fungus Hericium erinaceum; J. Nat. Prod., Vol. 57, pp. 602-606(1994).

29.

Hericenes and Hericenones

705

Common/Systematic Name Hericenone A Molecular Formula/Molecular Weight C19H2205; M W -" 330.14672 a

"

0

.

OH

MeO"

0

" ~ " "~ -"3'0

General Characteristics Colorless crystals from chloroform; mp., 100-102~ reagent.

reacted with Folin-Ciocalteu

Fungal Source

Hericium erinaceum, an edible mushroom.

Isolation/Purification Fresh fruiting bodies ofH. erinaceum were extracted with acetone and the extract concentrated and fractionated by solvent partitions (chloroform then ethyl acetate). Repeated silicic acid column chromatography followed by recrystallization of the chloroform extract gave purified hericenone A as colorless crystals. Biological Activity Cytotoxic to HeLa cells; the minimum concentration completely inhibiting growth of HeLa cells was 100~g/ml. Spectral Data IR:

1760, 1660 and 3300-2600cm "1. 1H N ~ :

(CDC13) 5.25(s, H-3); 6.97(s, H-4); 3.59(d, J=6.41Hz, H-I'); 5.30(t, J=6.41Hz, H-2'); 3.18(s, H-4'); 6.09(s, H-6'); 1.91(s, H-8'); 1.81(s, 3'-CH3); 2.17(s, 7'- CH3); and 3.89ppm (s, OCH3). lSc NMR: (CDCIs) 199.08(C-5'); 171.86(C-1); 159.18(C-5 or C-7); 157.48(C-7 or C-5); 150.61(C-7'); 133.63(C-3a); 128.22(C-3'); 125.80(C-7a or 25; 125.02(C-2' or 7a); 123.05(C-6'); 121.35(C-6); 98.45(C-4); 68.31(C-3), 56.21(OCH3); 54.40(C-4'); 27.82(C-8'); 23.32(C-15; 21.06(C-7'-CHs); and 17.18ppm (C-3'-CH3).

706

29.

Hericenes and Hericenones

Mass Spectrum: FAB-MS exhibited MH § ion at 33 lm/e; HREIMS: 330.1494m/e (M +, C19H2205). Reference H. Kawagishi, M. Ando, and T. Mizuno; Hericenone A and B as Cytotoxic Principles from The Mushroom Hericium erinaceum; Tetrahedron Letters, Vol. 31, pp. 373376(1990).

29.

Hericenes andHericenones

707

Common/Systematic Name Hericenone B Molecular Formula/Molecular Weight C27H31NO4; ~ 0

8

'

= 433.22531 ~

OH

~

M

e

O

~

0

Nc

H2CH2~

General Characteristics Colorless crystals from chloroform, mp. 136-138~ Fungal Source Hericium erinaceum, an edible mushroom.

Isolation/Purification Fresh fruiting bodies ofH. erinaceum were extracted with acetone and the extract concentrated and fractionated by solvent partitions(chloroform then ethyl acetate). Repeated silicic acid column chromatography followed by recrystallization of the chloroform extract gave purified hericenone B as colorless crystals. Biological Activity Cytotoxic to HeLa cells; the minimum concentration completely inhibiting growth of HeLa cells was 6.1 ~g/ml. Spectral Data IR: 1680cm 1

1H NMR: (CDC13) 4.20(s, H-3); 6.96(s, H-4); 3.56(d, J=6.74Hz, H-I'); 5.30(t, J=6.74Hz, H-2'); 3.14(s, H-4'); 6.08(s, H-6'); 1.88(s, H-8'); 1.81(s, 3'-CH3); 2.16(s, 7'- CH3); 3.84(t, J=7.33Hz, H-I'); 2.97(t, J=7.33Hz, H-2"); 7.20, 7.26(m, H-2'" -6'"); and 3.84ppm (s, OCH3). 13C NMR:

(CDC13) 198.98(C-5'); 168.94(C-1); 158.48(C-5 or C-7); 156.81(C-7 or C-5); 150.70(C-7'); 138.76(C-1'"); 132.85(C-3a or 4'"); 132.20(C-4'" or C-3a); 128.70(C-3'); 128.61(C-2'" or C-6'"); 126.51(C-2'); 123.01(C-6'); 122.00(C-7a); 118.48 (C-6); 97.69(C-4); 56.21(OCH3); 54.65(C-4'); 48.28 (C-3); 44.21(C-1"); 34.90(C-2"); 27.76(C-8'); 23.09(C-1'); 20.94(C-7'-CH3); and 17.00ppm (C-3'-CH3).

708

29.

Hericenes and Hericenones

Mass Spectrum: FAB-MS: 434(MH+) and 456m/e (M+Na+); HREIMS: 433.2243re~e; LREIMS: 433(M+, 12.9%), 342(64.6), 205(3.3), 137(4.5), 105(15.5), 83(100), and 55m/e (36.3). Reference H. Kawagishi, M. Ando, and T. Mizuno; Hericenone A and B as Cytotoxic Principles from The Mushroom Hericium erinaceum; Tetrahedron Letters, Vol. 31, pp. 373376(1990).

29.

Hericenes and Hericenones

709

Common/Systematic Name Hericenone E 4-(3', 7'-Dimethyl-5'-oxo-2', 6'-octadienyl)-2-formyl-3-hydroxy-5-methoxybenzyl linoleate Molecular Formula/Molecular Weight C37H5406; M W -- 5 9 4 . 3 9 2 0 4

8

i

'

0

~

C

.

H

I'

O

OH

MeO~ - [/ ~ ~ O - c . ( C H 2 ) 7 0II

9"

~--/

/~

12"

\--/

_(C H2)4M"e

General Characteristics Colorless oil. Fungal Source Hericium erinaceum, an edible mushroom.

Isolation/Purification Fresh fruiting bodies ofH. erinaceum were extracted with acetone, and the extract was concentrated and fractionated by solvent partitions (chloroform then ethyl acetate). Repeated silicic acid column chromatography followed by HPLC with an ODS column using methanol-water(95:5, v/v) as mobile phase gave purified hericenone E as a colorless oil. Biological Activity Hericenone E was non-cytotoxic to HeLa cells; however, it exhibited stimulating activity to the synthesis of nerve growth factor in vitro. In the presence of 331.tg/ml, mouse astroglial cells secreted 13.9pg/ml NGF into culture medium. Spectral Data IR: 1660, 1720, and 1730cm"1. 1H NMR: (CDC13) 6.53(s, H-6); 5.32(s, H-I-CH2); 0.1 l(s, CHO); 12.38(s, OH); 3.91(s, OCH3); 3.40(d, J-7.33Hz, H-I'); 5.32(t, J=7.33Hz, H-2'); 3.01(s, H-4'); 6.09(s, H-6'); 1.84(s, H-8'); 1.78(s, H-7'-CH3); 2.12(s, H-7'-CH3); fatty acid, 2.33(dd, J=7.70, 7.32Hz, H2"); 1.61(m, H-3"); 0.88(t, J=6.96Hz, terminal CH3); and 1.25ppm (m, H-7", 15").

710

29.

Hericenes and Hericenones

13C NMR: (CDCI3) 199.53(C-5'); 1.93.11(CHO); 173.19 (C-I"); 163.47(C-5); 162.93(C-3); 155.45(C-7); 138.68(C-1); 130.34(C-3'); 126.25(C-2'); 122.81(C-6'); 117.29(C-4); 112.88(C-2); 105 55(C-6); 62.90(C-1-CH2); 55.93(OCH3); 55.56(C-4'); 34.23(C-2"); 27.67(C-8'); 21.61(C-1'), 20.67(C-7'-CH3); 16.40(C-Y-CH3); 14.32(C-16"); 31.93; 29.70; 29.68; 29.66; 29.65; 29.44; 29.36; 29.23; 29.12; 24.88; and 22.70ppm (C-3"15").

Mass Spectrum: FAB-MS: 595m/e MIT; HR-FAB-MS: 595.4016m/e for C37H5406. Reference H. Kawagishi, M. Ando, H. Sakamoto, S. Yoshida, F. Ojima, Y. Ishiguro, N. Ukai, and S. Furukawa; Hericenones C, D, and E, Stimulators of Nerve Growth Factor (NGF) Synthesis, from the Mushroom Hericium erinaceum, Tetrahedron Letters, Vol. 32, pp. 4561-4564(1991).

29.

Hericenes and H e r i c e n o n e s

711

_Common/Systematic Name Hericenone F 8-Formyl-5-methoxy-2-methyl-2-(4'-methyl-2'-oxo-3'-pentenyl-)-7-chromanylmethyl palmitate Molecular Formula/Molecular Weight C35H5406; M W -- 5 7 0 . 3 9 2 0 4 5'

Z

2

10

CHO

Meo~O~c~(c

II 0

H2)14Me

General Characteristics Pale yellow oil. Fungal Source Hericium erinaceum, an edible mushroom.

Isolation/Purification Fresh fruiting bodies ofH. erinaceum were extracted with acetone, and the extract was concentrated and fractionated by solvent partitions between chloroform and water, then ethyl acetate. The chloroform layer was concentrated and chromatographed on a silicic acid column eluted with benzene and chloroform-acetone mixtures (9:1, 7:3, and 2:3, v/v) followed by repeated silica gel column chromatography, preparative TLC (chloroformacetone, 49:1) and HPLC with an ODS column using methanol-water, 19:1, v/v as mobile phase gave purified hericenone F as a pale yellow oil. Biological Activity Hericenone F exhibited stimulating activity to the synthesis of nerve growth factor in quiescent mouse astroglial cells. Spectral Data IR:

1740, 1670, and 1570cm1. 1H NMR: (CDC13) 2.01(ddd,,J-13.94, 6.61, 6.61, H-3)and 1.91(13.94, 6.60, 6.60, H-3); 2.64(dd, J=6.61, 6.60, H-4); 6.55(s, H-6); 10.41(s, CHO); 1.44(s, 2-CH3); 5.51(s, 7CH2); 3.88(s, OCH3); 2.82(d, J=13.94Hz, H-I'), 2.66(d, J=13.94Hz, H-I'); 6.06(s, H3'); 2.14(s, 4'-CH3); 1.86(s, H-5'); fatty acid, 2.41(t, J=7.33Hz, H-2"); 1.69(m, H-3");

712

29.

Hericenes and Hericenones

0.88(t, J=6.60Hz, terminal CH3), and 1.25ppm (H- 11', -8", - 14", -7", - 15", -9", - 10", 12",-13", others).

13CNMP~: (CDCI3) 76.9(C-2); 30.1(C-3); 16.5(C-4), 109.2(C-4a), 161.9(C-5), 100.8(C-6); 139.7(C-7), 115.8C-8); 158.3(C-8a); 24.5(C-2 methyl); 52.5(C-1'), 197.9(C-2'); 125.0(C-3'); 156.4(C-C-4'); 20.8(C-4' methyl); 27.8(C-5'); 190.4(CHO), 55.6(OCH3); 64.5(C-7'-CH2); fatty acid, 173.3(C- 1"); 34.5(C-2"), 25.1 (C-3"): 14.1(terminal methyl); and 31.9; 29.7; 29.7; 29.6; 29.5; 29.4; 29.3; 29.3; 22.7ppm (others). Mass Data: FAB-MS: 571m/e MIT; HR-FAB-MS: 571.3981m/e calcd for C35H5406 571.3999 Reference H. Kawagishi, M. Ando, K. Shinba, H. Sakamoto, S. Yoshida, F. Ojima, Y. Ishiguro, N. Ukai, and S. Furukawa; Chromans, Hericenones F, G and H, from the Mushroom Hericium erinaceum; Phytochemistry, Vol. 32, pp. 175-178(1993).

29. Hericenes and Hericenones

713

Common/Systematic Name Hericenone G 8-Formyl-5-methoxy-2-methyl-2-(4'-methyl-2'-oxo-3'-pentenyl)-7-chromanylmethylsterate Molecular Formula/Molecular Weight C37H5806; M W = 598.42334 5'

2'

2

4

. 8

CHO

MeO~~/~O~c__(C

II 0

H2)16Me

General Characteristics Crystallized as yellow plates from chloroform-ethanol; mp., 56-58~ Fungal Source Hericium erinaceum, an edible mushroom.

Isolation/Purification Fresh fruiting bodies ofH. erinaceurn were extracted with acetone, and the extract was concentrated and fractionated by solvent partitions between chloroform and water, then ethyl acetate. The chloroform layer was concentrated and chromatographed on a silicic acid column eluted with benzene and chloroform-acetone mixtures (9:1, 7:3, and 2:3, v/v) followed by repeated silica gel column chromatography, preparative TLC (chloroformacetone, 49:1, v/v) and HPLC with an ODS column using methanol-water, 19:1, as mobile phase gave purified hericenone G as a yellow plate from chloroform-ethano! solution. Biological Activity Hericenone G exhibited stimulating activity to the synthesis of nerve growth factor in quiescent mouse astroglial cells. Spectral Data IR:

1740, 1680, and 1570cm"1. 1H N ~ : (CDC13) 2.00(ddd, J=13.92, 6.60, 6.60, H-3)and 1.90(J=13.94, 6.60, 6.60, H-3); 2.63(dd, J=6.96, 6.60, H-4); 6.45(s, H-6); 10.41(s, CHO), 1.44(s, 2-CHs); 5.51(s, 7CH2); 3.88(s, OCHs); 2.82(d, J=14.29Hz, H-I'), 2.66(d, J=14.29Hz, H-I'); 6.06(s, H-

714

29.

Hericenes and Hericenones

3'); 2.14(s, 4'-CH3); 1.86(s, H-5'); fatty acid, 2.41(t, J=7.33Hz, H-2"); 1.69(m, H-3"); 0.88(t, J=6.59Hz, terminal CH3); and 1.25ppm (H-11',-8",-14",-7",-15",-9",-10",12", - 13", others). ~3CNMR: (CDC13) 76.9(C-2); 30.1(C-3); 16.5(C-4); 109.2(C-4a); 161.9(C-5); 100.8(C-6); 139.7(C-7); 115.8(C-8); 158.3(C-8a); 24.5(C-2 methyl); 52.5(C-1'); 197.9(C-2'); 125.0(C-3'); 156.4(C-C-4'); 20.8(C-4' methyl); 27.8(C-5'); 190.4(CHO); 55.6(OCH3); 64.5(C-7'-CH2); fatty acid, 173.3(C- 1"); 34.5(C-2"); 25.1(C-3"): 14.1(terminal methyl); and 31.9; 29.7; 29.7; 29.7; 29.6; 29.5; 29.4; 29.3; 29.3; 22.7ppm (others). Mass Data: FAB-MS: 599m/e MIT; HR-FAB-MS: 599.4299m/e calcd for C37H5906,599.4312. Reference H. Kawagishi, M. Ando, K. Shinba, H. Sakamoto, S. Yoshida, F. Ojima, Y. Ishiguro, N. Ukai, and S. Furukawa; Chromans, Hericenones F, G, and H from the Mushroom Hericium erinaceum; Phytochemistry, Vol. 32, pp. 175-178(1993).

Miscellaneous Metabolites Variecoxanthone A Variecoxanthone B Variecoxanthone C Nodicl: ~,xanthone T-Decalactone 5-Decanolide Jasmine lactone; cis-7-Decen-5-olide 5-Hydroxy-3-methoxy-6-oxo-2-decanoic acid 8-1actone 6-( 1-Hydroxypentyl)-4-methoxypyran-2-one Pestalotin; LL-P880a LL-P88013; (6S, l'S,2'R)-4-Methoxy-5,6-dihydro-6-(l',2'-dihydroxypentyl)-2H-pyranone LL-P880T; 4-Methoxy-6-(l',2'-dihydroxypentenyl)-2H-pyran-2-one 3,5-Dihydroxydecanoic acid 8-1actone Trichoacorenol; ( 1S,4S, 5S,7R)- 1-Isopropyl-4,8-dimethylspiro [4.5]dec-8-en-7-ol Aszonapyrone A Oxysporone Ascladiol Anhydrosepedonin Sepedonin Alterporriol B LL-$49113 LL-S491T cis-Sativenediol trans-Sativenediol; Isosativenediol Isosativenetriol Patulin; Claviformin; Clavitin; Clavicin; Expansion; Penicidin; Mycoin; Leucopin; Tercinin Isopatulin Acrostalidic acid Acrostalic acid Pimara-8(9), 15-diene Isoacrostalidic acid Deoxypatulinic acid; 2,3-Dihydro-4-pyrone-5-acetic acid Dihydropenicillic acid (-)-Longifolene Victoxinine Antibiotic A26771B IFO 6635 Aversion Factor Penicillic acid; 3-Methoxy-5-methyl-4-oxo-2,5-hexadienoic acid 5,5-Dimethyl-9-(2-hydroxyethyl)-8-hydroxymethyl-1-methylbicyclo[4.2.1]non-7-ene Cynodontin; 1,4,5,8-Tetrahydroxy-2-methylanthraquinone; 2,5,7-trihydroxyemodin Cyclonerodiol Cyperine Erinapyrone A; (2S)-2,3-Dihydro-6-hydroxymethyl-2-methyl-4H-pyran-4-one Erinapyrone B; (2R)-2,3-Dihydro-2-hydroxymethyl-6-methyl-4H-pyran-4-one 715

716

30.

Miscellaneous Metabolites

Erinapyrone C Asperfl avin; (+)-3,4-Dihydro-3,6,9-trihydroxy-8-methoxy-3-methylanthracen-1(2H)-one Diplodiol; Diplosporin 5-Deoxydiplosporin Betulin (Lup-20(29)-ene-313,28-dioi)

30.

Miscellaneous Metabolites

717

Common/Systematic Name Variecoxanthone A

Molecular Formula/Molecular Weight C20H2005; M W -- 340.13107

General Characteristics Variecoxanthone A gave pale yellow needles from methanol; mp., 135 oc. Fungal Source

Aspergillus variecolor (strain CBS 135-55)

Isolation/Purification

Aspergillus variecolor was grown in static culture for 15 days at 25 oC. The dried mycelium was ground and continuously extracted with light petroleum (bp., 60-80 ~C). The resulting semi-solid was triturated with warm methanol and the solution evaporated to give a yellow solid which was fractionated by preparative TLC (toluene-ether, 97:3, v/v). Variecoxanthone C was eluted from the band at Re 0.3 and gave yellow needles from methanol; variecoxanthone A was eluted from the band at Re 0.5 and gave pale yellow needles from methanol; and variecoxanthone B was eluted from the band of Rf 0.55 and afforded yellow prisms from methanol.

Soectral Data UV~

~

EtOH

374(e = 5,500), 292(11,100), and 260nm (26,000).

IR:

(CHC13) 3500-3100 and 1643cm"1. ~H M R : (CDC13) -2.64, 1-OH; 3.22, 2-CH; 2.42, 3-CH; 3.12, 4-CH; 2.71, 5-CH; 5.58, 19CH2; 4.42, 20-H; 8.20, 22-CH3; 8.29, 23-CH3; 7.56, 24-CH3; 4.95, 25-CH2; and 5.60z 25-OH.

718

30.

Miscellaneous Metabolites

Mass Data: EIMS: 340.130(M+), 272(35), 271(48), 254(100), and 242m/e (13%), C2oH2oO5 requires 340.131; found: C, 70.5; H, 5.9%; requires C, 70.6; H, 5.9%. Reference K. K. Chexal, J. S. E. Holker, T. J. Simpson, and K. Young; Part V. Structure of Variecoxanthones A, B and C, Metabolites ofAspergillus variecolor Conversion of Variecoxanthone A into (• J. C. S. Perkin I, pp. 543548(1975).

30. MiscellaneousMetabolites

719

Common/Systematic Name Variecoxanthone B; Emericellin Molecular Formula/Molecular Weight C25H2805; M W -- 408.19367 17

M

~

~

3Me

. ~ CH20H Me~ 21 "Me General Characteristics Variecoxanthone B afforded yellow prisms from methanol; mp., 113-115~ crystals from ethanol as yellow prisms; mp., 110-112~ [tt]D O~ gave a dark green color with ferric chloride. Fungal Source Aspergillus variecolor (strain CBS 135.55), A. nidulans, A. rugulosus, and A. quadrilineatus. Isolation/Purification See Variecoxanthone A. Spectral Data UV:

~, E~n

353(e = 26,000), 258(28,400), 275(30,400), 294(28,400), and 380nm 15,400) (see Chexal, et al., 1975);

,~ EtOH

~'~ 234(24,400), 245(29,500), 256(29,800), 287(11,900), and 383nm (8,800) (characteristic of xanthone) (see Ishida, et al., 1975). IR:

(CHC13) 3520-3100 and 1640cm"1. IH NMR: (CDC13) -2.86, 1-OH; 3.23, 2-CH; 2.59, 3-CH; 2.82, 5-CH; 6.66, 14-CH2; 4.71, 15CH; 8.26, 17-CH3; 8.26, 18-CH3; 5.60, 19-CH2; 4.43, 20-H; 8.20, 22-CH3; 8.29, 23CH3; 7.59, 24-CH3; 4.98, 25-CH2; and 5.601:, 25-OH.

720

30.

Miscellaneous Metabolites

The NMR spectrum showed two signals at 4.35ppm (1H, triplet, J=8Hz) and 12.50ppm (1H, singlet) disappeared by addition of deuterium oxide and assigned to the primary hydroxy and hydrogen bonded phenolic hydroxy protons. The signal at 7.28ppm (1H, singlet),which showed weak long range coupling with the aromatic methyl proton (2.45ppm, 3H, singlet),was assigned to the proton at the position 5 in xanthone nucleus. The signals at 6.68ppm (1H, doublet, J=8.5Hz) and 7.40ppm (1H, doublet, J=8.5Hz) were assigned to the protons at the position 2 and 3, or 4 and 3. The signals at 1.75-1.80ppm (3H x 4) were assigned to the protons of the four olefinic methyl groups. The signals at 4.44ppm (2H, doublet, J=7Hz) and 5.62ppm (1H, triplet, J=7Hz) were assigned to the methylene proton attached to the oxygen-bearing carbon and olefinic proton, respectively. These data indicated the presence of an O-isoprenyl group. The signals at 3.47ppm (2H, doublet, J=7Hz) and 5.31 ppm (1H, triplet, J=7Hz) were assigned to the methylene and olefinic protons of the C-isoprenyl group. Mass Data: ELMS: 408.195(M +, 5%), 340(37), 339(3500), and 322role (925), C25H2sO5requires 408.194; found: C, 73.4; H, 7.0%; requires C, 73.5; H, 6.9%. References K. K. Chexal, J. S. E. Holker, T. J. Simpson, and K. Young; Part V. Structure of Variecoxanthones A, B and C, Metabolites ofAspergillus variecolor Conversion of Variecoxanthone A into (+)- De-C-Prenylepishamixanthone; J. C. S. Perkin I, pp. 543548(1975). M. Ishida, T. Hamasaki, Y. Hatsuda, K. Furuyama, T. Tsukihara, and Y. Katsube; Emericellin, a New Metabolite from Aspergillus nidulans; Agr. Biol. Chem., Vol. 39, pp. 291-292(1975).

30.

Miscellaneous Metabolites

721

Common/Systematic Name Variecoxanthone C Molecular Formula/Molecular Weight C25H2806; M W -- 4 2 4 . 1 8 8 5 9

H~,,, H2C]4 ~ e Me ,~_O___ 17

M

o

.~L CH2OH

6.

Me" 2~"Me General Characteristics Variecoxanthone C gave yellow needles from methanol; mp., 118-120~ Fungal Source Aspergillus variecolor (strain CBS 135.55) Isolation/Purification See variecoxanthone A. Spectral Data UV:

E~H~ 252(e=30,700), 258(24,700), 274(30,000), 294(27,800), and 376nm (15,000). IR:

(CHC13) 3500-3100 and 1641cm4. 1H NMR: (CDC13) -2.62, 1-OH; 3.23, 2-CH; 2.50, 3-CH; 2.69, 5-CH; 6.97, 14-CH2; 6.97, 15CH; 8.56, 17-CH3; 8.67, 18-CH3; 5.58, 19-CH2; 4.43, 20-H; 8.20, 22-CH3, 8.29, 23CH3; 7.55, 24-CH3; 4.94, 25-CH2; and 5.601: 25-OH.

722

30.

Miscellaneous Metabolites

Mass Spectrum: EIMS: 424.188(M+, 4%), 356(95), 355(96), and 338m/e (100); C25H2806 requires 424.188. Reference K. K. Chexal, J. S. E.Holker, T. J. Simpson, and K. Young; Part V. Structure of Variecoxanthones A, B and C, Metabolites of Aspergillus variecolor Conversion of Variecoxanthone A into (+)-De-C-prenylepishamixanthone; J. C. S. Perkin I, pp. 543548(1975).

30. Miscellaneous Metabolites

723

Common/Systematic Name Norlichexanthone 3,6, 8-Trihydroxy- 1-methylxanthone Molecular Formula/Molecular Weight C14Hlo05; M W = 258.05282

HO~OH Me

0

OH

General Characteristics Crystals from aqueous acetone; mp., 272-274 oC. Gives blue-green color with FeCl3. Fungal Source Penicillium patulum, a mutant strain. Isolation/Purification The filtered broth was acidified with mineral acid (pH 2), extracted with ethyl acetate, and evaporated to dryness. The residue was redissolved in methanol and evaporated onto silica gel. This was packed on top of additional silica gel and the substance was eluted with chloroform-ethyl acetate (17:3, v/v) and chloroform-ethyl acetate-ethanol (17:3:1, v/v/v). Biological Activity Antibacterial (MIC vs Clostridium welchii = 25ppm) Spectral Data UV: ~ EtOH max

241(e=36500), 311nm (22500).

IR:

3500, 3050, 1650, 1620, 1500, 830, 812, and 760cm1. 1H NMR: 3.4(OH); 3.4(2H, m, ArH), 3.85(AB q, J=2Hz, ArH); and 7.24z (Ar-CH3). References D. Broadbent, R. P. Mabelis, and H. Spencer; 3,6,8-Trihydroxy-l-methylxanthone - An Antibacterial Metabolite from Penicillium patulum; Phytochemistry, Vol. 14, pp. 2082-2083(1975). J. Santesson; Chemical Studies on Lichens. 16. The Xanthones ofLecanora straminea. I. Arthothelin and Thiophanic Acid; Ark. Kemi, Vol. 30, pp. 449-454(1969).

724

30.

Miscellaneous Metabolites

Common/Systematic Name y-Decalactone Molecular Formula/Molecular Weight C1oH1802, MW' = 170.13068

General Characteristics A flavor component of deteriorated milk fat, butter, y-irradiated milk fat, raspberry oil, peach, strawberry, apricot, orange, carrot seed oil, and bilberry. Fungal Source

Sporobolomyces odorus.

Isolation/Purification The culture broth was extracted with an equal volume of ethyl ether. The ether extract was washed with 2% sodium carbonate solution and dehydrated with anhydrous sodium sulfate. The extract was reduced in volume by fractional distillation and finally a stream of nitrogen gas. y-Decalactone was isolated in pure form by preparative gas chromatography (10% PEG-20M as the liquid phase on Celite 545 AW DMCS at 190~ Spectral Data IR~

(Thin film) 2910, 2880, 1770(y-lactone ring), 1458, 1178, and 720cm "1. Mass Spectrum: LREIMS: 170(M+), 152, 134, 128, 100, 85(base peak), 55, 43, and 41m/e. GLC Data Retention time 7.2 and 4.0min. on Celite 545AW DMCS coated with 10% PEG-20M; carrier gas N2 at 23ml/min; column temperature 180~ injection port temperature 250~ and on 1.5% OV-17 on Chromosorb W; carrier gas N2 at 24ml/min; column temperature 140 oC, respectively. Reference S. Tahara, K. Fujiwara, H. Ishizaka, J. Mizutani, and Y. Obata; y-Decalactone, One of the Constituents of Volatiles in Cultured Broth of Sporobolomyces odorus; Agric. Biol. Chem., Vol. 36, pp. 2585-2587(1972).

30.

Miscellaneous Metabolites

725

Common/Systematic Name 5-Decanolide Molecular Formula/Molecular Weight C10H1802, ~ = 170.13068

General Characteristics 5-Decanolide occurs widely in the flavor components of milk and fruits. Fungal Source Sporobolomyces odorus AHU 3246. Isolation/Purification The neutral fraction was obtained and fractionated on a silica gel column packed in isopentane and eluted with ether-isopentane (1:3, v/v) followed by ether-isopentane (1:1, v/v). The tS-lactone fractions were further purified by preparative GLC (10% PEG-20M column, column temperature 183 ~ injection port 270~ N2 carrier at 22.5ml/min., retention time 4.72 min.). Spectral Data Mass Spectrum: LREIMS: 170(M+), 152, 141, 134, 114, 99(base peak), 71, 55, and 41m/e. Reference S. Tahara and J. Mizutani; 6-Lactones Produced by Sporobolomyces odorus; Agile. Biol. Chem., Vol. 39, pp. 281-282(1975).

726

30.

Miscellaneous Metabolites

Common/Systematic Name Jasmine lactone cis-7-Decen-5-olide Molecular Formula/Molecular Weight C10H1602; M W --- 168.11503

0 Me j General Characteristics Jasmine lactone was originally isolated from the oil and wax ofJasmmum grandiflorum. Fungal Source Sporobolomyces odorus AHU 3246 Isolation/Purification The neutral fraction was obtained and fractionated on a silica gel column packed in isopentane and eluted with ether-isopentane (1:3, v/v) followed by ether-isopentane (1:1, v/v). The 6-1actone fractions were further purified by preparative GLC (10% PEG-20M column, column temperature 183 ~ injection port 270~ N2 carrier at 22.5ml/min., retention time 4.72 min.). Spectral Data Mass Spectrum: LREIMS: 168(NF), 150, 139, 108, 99, 81, 71(base peak), 55, and 41m/e. Reference S. Tahara and J. Mizutani; 6-Lactones Produced by Sporobolomyces odorus; Agric. Biol. Chem., Vol. 39, pp. 281-282(1975).

30.

Miscellaneous Metabolites

727

Common/Systematic Name 5-Hydroxy-3-methoxy-6-oxo-2-decanoic acid 8-1actone Molecular Formula/Molecular Weight CllH1604; ~

"- 2 1 2 . 1 0 4 8 6

OMe

~ O O General Characteristics Crystallized from ether-cyclohexane as colorless needles; mp., 83-84 ~ (c=0.266, in MeOH).

[~]D 22 - 4 . 4 ~

Fungal Source Penicillium

sp. and an unidentified fungus.

Isolation/Purification Filtrates from fungal liquid shake cultures were extracted thoroughly with chloroform. After removal of the solvent in vacuo TLC analysis of the brown residue revealed two major components. The mixture was chromatographed on a column of silica gel (Davidson Grade 923) and eluted with chloroform-methylene chloride which afforded 5-hydroxy-3methoxy-6-oxo-2-decanoic acid-8-1actone slightly contaminated by other products. The second major metabolite, again containing small amounts of impurities, was eluted with chloroform. The less polar compound was purified by preparative TLC on silica gel plates, using the solvent system benzene-n-hexane-acetone (10:9:1, v/v/v). 5-Hydroxy-3methoxy-6-oxo-2-decanoic acid-8-1actone crystallized from ether-cyclohexane as colorless needles. Soectral Data UV~ ~,m~x 233nm(c = 10,600) IR~ (KBr) 1710, 1620, 1248, and 1230cm"1. 1H NMR:

(CDC13) 0.92(3H, t, J=7Hz); 1.34(2H, sextet, slightly broadened, J=7Hz); 1.59(2H, quintet, slightly broadened, J=7Hz); 2.75(4H, m, simplified by irradiation at both 1.59 and 4.80ppm); 3.78(3H, s); 4.80(1H, dd, JAx + JBx =14Hz); and 5.17ppm (1H, s).

728

30.

Miscellaneous Metabolites

Mass Data: LREIMS: 212(M), 127(base peak, M + - 85, - C5H90), and 85role (C5H90+); anal. calcd, for CllH1604: C, 62.25; H, 7.60; O, 30.15; found: C, 62.44; H, 7.32; O, 30.20. Reference G.M. Strunz, C. J. Heissner, M. Kakushima, and M. A. Stillwell; Metabolites of an Unidentified Fungus: A New 5,6-Dihydro-2-pyrone Related to Pestalotin; Can. J. Chem., Vol. 52, pp. 825-826(1974).

30.

Miscellaneous Metabolites

729

Common/Systematic Name 6-(1-Hydroxypentyl)-4-methoxypyran-2-one Molecular Formula/Molecular Weight CllH1604, M W -- 212.10486 OMo

Me--C H2--C H2--C H2--C--'-~~.O.,,.~-.. O OH General Characteristics Oil; [~]D 23 + 78.5 ~ (c=l.0, in MeOH); monoacetate: an oil; [0~]D23 MeOH).

+

85.3 ~ (c=0.19, in

Fungal Source Penicillium sp.

Isolation/Purification The culture filtrate was extracted with ethyl acetate at pH 2, washed with sodium bicarbonate and water, and evaporated to dryness. The residue was chromatographed on a silica gel column eluted with a gradient from benzene to ethyl acetate. The 30% ethyl acetate fraction was concentrated and further purified using preparative silica gel TLC plates developed with benzene-ethyl acetate (1:1, v/v). Spectral Data UV: /~ EtOH max

227(sh, E=3,650) and 289nm (9,850).

IR~

(thin film) 3460, 1720, 1700, 1650, 1570, 1410, and 1255cm1; monoacetate: (thin film) 1730, 1650, 1565, 1405, 1245, 1225, 1035, and 1020cm"1. IH NMR: (CDCI3) 0.89(3H, t, J=7.0Hz); 1.33(4H, m), 1.75(2H, m), 2.95(1H, br), 3.72(3H, s), 4.30(1H, m); 5.33(1H, d, J=2.0Hz); and 6.02ppm (1H, d, J=2.0Hz); monoacetate: 0.87(3H, t, J=7.0Hz); 1.25(4H, m), 1.80(2H, m), 3.72(3H, s), 5.32(2H, d and t, J=7.0 and 2.0Hz); and 5.82ppm (1H, d, J=2.0Hz). Mass Spectrum: Acetate: 212(M+), 125m/e (base peak, C6H503+), LREIMS: 254(M+), 211 (M§ CH3CO), and 125role (base peak).

730

30.

Miscellaneous Metabolites

TLC Data Silica gel G254plates developed with benzene-ethyl acetate (1:1, v/v); Rf 0.34. Reference Y. Kimura, W. J. McGahren, A. Suzuki, and S. Tamura; Structure of a New Fungal Pyrone, from an Unidentified Penicillium sp.; Agile. Biol. Chem., Vol. 42, pp. 16251626(1978).

30.

Miscellaneous Metabolites

731

Common/Systematic Name Pestalotin; LL-P880a Molecular Formula/Molecular Weight CllH1804, M W = 214.12051 OMe 4

1'

0

General Characteristics Crystallization from n-hexane-benzene gave pestalotin as colorless plates melting at 88.088.5~ [a]D-86.2 ~ (C=0.14, in MeOH); acetate, a colorless oil. Pestalotin was soluble in most organic solvents such as benzene, chloroform, ethyl acetate, ether, acetone, and alcohols, but sparingly in n-hexane. Fungal Source

Pestalotia cryptomeriaecola, a pathogen on Japanese cedar (Cryptomeriajaponica) and Penicillium sp.

Isolation/Purification After addition of an equal volume of acetone, the culture broth was filtered to remove mycelia and was evaporated under reduced pressure. The residual aqueous solution was extracted repeatedly with ethyl acetate at pH 3. The combined extracts were washed successively with 5 % sodium bicarbonate solution and water. The ethyl acetate was dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. The residue was dissolved in benzene and applied to a column of silicic acid eluted stepwise with 100 ml each of benzene and benzene containing 10, 20, 30, 40, and 50% ethyl acetate, respectively. The eluate with benzene containing 30% ethyl acetate afforded crude pestalotin, which was subjected to preparative TLC using silica gel GF254, and benzene-ethyl acetate (1:1, v/v) as the developing solvent. The zone containing pestalotin was extracted with ethyl acetate. The extract was evaporated to dryness under reduced pressure to yield pestalotin. Final purification of pestalotin was by recrystallization from nhexane-benzene. Biological Activity Biological activity as a gibberellin synergist. No effect observed when applied alone on rice seedlings at dosages of 30-300mg/liter. However, when applied in combination with gibberellin A3, pestalotin enhanced the stimulative effect of gibberellin A3 on shoot growth.

732

30.

Miscellaneous Metabolites

Spectral Data UV: 95%EtOH

233nm

(e=12,200)

IR:

(Nujol) 3480, 1710, 1635, 1255, 1090, and 1025cm"1. CD: (0.82mg/10cc MeOH) AE243 -7.90 IH NMR:

(CDCI3) 0.91(3H, t, J=6.3 Hz); 1.30~1.60(6H, broad); 2.22(1H, broad); 2.25(1H, q, J=4.0, 17.0Hz); 2.79(1H, 3~--2.0, 12.7, 17.0Hz); 3.62(1H, broad); 3.75(3H, s); 4.28(1H, m, J=4.0, 4.0, 12.7 Hz); and 5.14ppm (1H, d, J=2.0Hz). Mass Spectrum: LREIMS: 214(M*), 196, 183, 181,170, 167, 157, 153, 139, and 127role (base peak). TLC Data Re 0.35 on silica gel using benzene-ethyl acetate (1" 1, v/v) as developing solvent. References G. A. Ellestad, W. J. McGahren, and M. P. Kunstmann; Structure of a New Fungal Lactone, LL-P8804, from an Unidentified Penicillium sp.; J. Org. Chem., Vol. 37, pp. 2045-2047(1972). Y. Kimura, K. Katagiri, T. Inoue, and S. Tamura; Isolation and Biological Activity of Pestalotin, A Gibberellin Synergist from Pestalotia cryptomeriaecola; Agric. Biol. Chem., Vol. 35, pp. 1313-1314(1971). Y. Kimura, K. Katagiri, and S. Tamura; Structure ofPestalotin, A New Metabolite from Pestalotia cryptomeriaecola; Tet. Lett., pp. 3137-3140(1971). Y. Kimura and S. Tarnura; Isolation and Structure ofPestalotin, A Gibberellin Synergist from Pestalotia cryptomeriaecola; Agric. Biol. Chem., vol. 36, pp. 1925-1930(1972).

30.

Miscellaneous Metabolites

733

Common/Systematic Name LL-PS80[3 (6S, 1'S,2'R)-4-Methoxy- 5,6-dihydro-6-( 1',2'-dihydroxypentyl)-2H-pyran-2-one Molecular Formula/Molecular Weight CllH/805, MW' = 230.11542 OMe

HO

H -

H

~

~

General Characteristics White crystals from ethyl aeetate-hexane; mp., 135.5-136~ MeOH).

[a]D25 -59.8 ~ (C= 0.96, in

Fungal Source

Penicillium sp.

Isolation/Purification The fungal cultures were extracted with ethyl acetate. The extract was concentrated to a dark red solution, which, on being allowed to remain in the cold room for 2 days, yielded pure LL-P88013. Spectral Data UV:

~, M~on 234nm (e =13,800) IR:

(KBr) 1710 and 1624crnq. CD: AE246 -12.5, Ae22o +6.6, shoulder at Ae21o + 4.2(c=0.236, in MeOH). ~H NMR: (CDCI3) 0. 93 (-CH2CH3, t); 1.50((CH2)2CH3, m); 2.36(Hc, dd, Jcb=l 7.5, J~=12.5Hz); 2.87(Hb, qq, Jbd=4Hz); 3.47(I-I~, dd, Jof=3.8, J~=4.2Hz); 3.78(Ha, m, hidden by OCH3 signal); 3.80(OCH3, s), 4.51(Ha, tt); and 5.16ppm (Ha, d, Jac ~ 2Hz).

734

30.

Miscellaneous Metabolites

Mass Data:

Anal. calcd, for CI~HlsOs: C, 57.38; H, 7.88; found: C, 57.02; I-I, 7.76. Reference W. G. McGahren, G. A. Ellestad, G. O. Morton, M. P. Kunstmatm, and P. Mullen; A New Fungal Lactone. LL-P88013, and A New Pyrone, LL-P880y, from Penicillium sp.; J. Org. Chem., Vol. 38, pp. 3542-3544(1973).

30.

Miscellaneous Metabolites

735

_Common/Systematic Name LL-P880 T 4-Methoxy-6-(l',2'-dihydroxypentenyl)-2H-pyran-2-one Molecular Formula/Molecular Weight CllH1605; M'W = 2 2 8 . 0 9 9 7 7

OMe

HO H

I

General Characteristics Colorless needles from ethyl acetate; mp., 116-118~

[a]D 20 -31 o (c=l.0 ' in MeOH).

Funsal Source v

Penicillium citreo-viride and Penicillium sp.

Isolation/Purification The fungus was cultured statically in a malt extract medium containing 3% peptone at 24 ~ for 21 days. The culture broth was filtered and the filtrate was extracted with ethyl acetate at pH 2.0. The combined extracts were concentrated under reduced pressure below 40~ to give a dark brown residue which was dissolved in acetone and mixed with acid-washed Celite. ARer drying under reduced pressure, the impregnated Celite was charged onto a SilicAR CC-4 column and eluted with benzene-acetone mixtures, the content of acetone in the mixture being increased stepwise. The 5% and 10% acetone fractions were concentrated and the residual solid was recrystallized from ethyl acetate to afford colorless plates. The pale yellow solid obtained from the 15% and 20% acetone fractions was subjected to a silica gel column, which was eluted with benzene-ethyl acetate mixtures, the content of ethyl acetate being increased stepwise. The eluates with benzene containing more than 35% ethyl acetate fractions were evaporated to dryness to afford pale brown solids, which were recrystallized from ethyl acetate to give LL-P880T as colorless needles. Biological Activity Acted as a gibberellin synergist using dwarf rice seeds (Oryzae sativa). LL-P880y alone at dosages of 1.0 -~ 300rag/liter did not affect the growth of rice seedlings. When applied together with GA3 at ling/liter, it enhanced the stimulative effect of GA3 on the elongation of the second leaf sheath.

736

30.

Miscellaneous Metabolites

Soectral Data UV~

~, M~on 225(sh, e=3,500) and 280nm (8,400). IR~

(KBr) 3300, 3100, 2690, 1735, 1660, 1580, and 1265cm"1. 1H NMR: (pyridine-ds) The signals at 5.50(1H, d, J=2.0Hz) and 6.58ppm (1H, d, J=2.0Hz) were due to olefinic protons and at 3.51ppm (3H, s) due to -OCH3. The signals at 4.35(1H, m) and 4.62ppm (1H, d, J=3.0Hz) were assigned to C-2' and -1' protons, respectively. The presence of an n-propyl group was evident by the signals at 0.86(31-1, s) and 1.75ppm (-CHz-CH2-, m). Mass Spectrum: LREIMS: 229(M+ + 1), 211,185, 155, 125, 110, 99, 83, 69m/e (base peak). TLC Data Silica gel thin-layer plates developed with benzene-ethyl acetate (3:7v/v), Rf=0.21. References Y. Kimura, T. Hamasaki, and H. Nakajima; Stereochemistry and Biological Activities of LI,-P880y, A Pestalotin Analogue, Produced by Penicillium citreo-viride; Agric. Biol. Chem., Vol. 50, pp. 1649-1650(1986).

30.

Miscellaneous Metabolites

737

Common/Systematic Name 3,5-Dihydroxydecanoic acid 6-1actone Molecular Formula/Molecular Weight C10H1803; M ~ r = 1 8 6 . 1 2 5 5 9

OH

Me(CH2)4~0~0 General Characteristics Isolated as an oil; [t~]D25 + 24.7 ~ (C=I 1.7, in CHC13). Funual Source Cephalosporium recifei (NRRL 5161). v

Isolation/Purification Fungal cultures were extracted with hexane. The remaining hexane insoluble material was fractionated by silicic acid column chromatography (Bio Sil A), and eluted with ethyl ether-hexane (50:50, v/v) to yield 3,5-dihydroxydecanoic acid 8-1actone as an oil. Spectral Data IR~

(Neet) 3430 and 1720-1730cm 1. 1H NMR: (CDC13) 5.2-5.4(CH at junction oflactonic oxygen); 5.66(CH-OH); 6.94(OH); 7.36(doublet, CH2 adjacent to carbonyl); and 9.12z (C-CH3). Mass Data: LREIMS: Highest detectable mass was at 168m/e (M + - H20), 97(-H20 from 115m/e, 100%) with fragment ions at 115m/e (- hydrocarbon chain at C-5), and 97m/e (- H20 from 115m/e; anal. calcd for C10H1803: C, 64.49; H, 9.74; found: C, 64.6; H, 9.54. Reference R. F. Vesonder, F. H. Stodola, and W. K. Rohwedder; Formation of the 6-Lactone of 3,5Dihydroxydecanoic Acid by the Fungus Cephalosporium recifei; Can. J. Bioehem., Vol. 50, pp. 363-365(1972).

738

30.

Miscellaneous Metabolites

Common/Systematic Name Trichoacorenol ( 1S,4S,5S, 7R)- 1-Isopropyl-4,8-dimethylspiro [4.5 ]dec-8-en- 7-oi Molecular Formula/Molecular Weight C15H260; MW = 222.19837 13

12 11

i

14

~)H

General Characteristics Colorless amorphous solid; [0~]D 5.2 ~ (c=0.12, in CHC13). Fungal Source Trichoderma koningii. Isolation/Purification The culture broth was extracted with butanol and the butanol extract subjected to silica gel column chromatography with a chloroform-methanol gradient (100:0 - 50:50) to give eight fractions. Fraction 2 was further purified by silica gel column chromatography with chloroform to give pure trichoacorenol. Spectral Data IR: (CHCI3) 3600cm -! IH NMR: (acetone-d6) 0.84(3H, d, J=7.0Hz, 14-H3); 0.86; 0.93(each 3H, d, J-6.5Hz, 12-H3, 13-H3); 1.09(1 H, m, 3-H); 1.27(1 H, m, 1-H); 1.29(1 H, dd, Jr- 14.0, 10.0Hz, 6-H); 1.46(1 H, tdd, dr=11.5, 10.0, 4.0Hz, 2-H); 1.58(1 H, ddq, J= 10.0, 9.0, 6.5Hz, 4-H); 1.70(1H, m, 3-H); 1.70(1H, m, 1 I-H); 1.72(1H, m, 6-H); 1.73(3H, brs, 15-H3); 1.74(1H, m, 2-H); 1.80(1H, br d, J-9.5Hz, 10-H); 2.10(1H, ddq, J=9.5, 5.5, 2.5Hz, 10H); 3.51(1H, d, J=6.5Hz, 7-OH); 4.22(1H, br s, J=19.5Hz, 7-H); and 5.39ppm (1H, m, 9-H). 13C NMR: (acetone-d6) 15.0(q, C-14); 90.0(q, C-15); 23.8, 24.3(each q, C-12, C-13); 27.0(t, C2); 30.1(t, C-3); 31.5(d, C-11); 33.4(t, C-6); 36.7(t, C-10); 46.2(s, C-5); 48.0(d, C-4); 61.2(d, C-I); 68.6(d, C-7); 124.6(d, C-9); and 138.6ppm (s, C-8).

30.

Miscellaneous Metabolites

739

Mass Spectrum: LREIMS: 222(M +, 100), 207(M +- CH3, 7), 204(M § 1-I20, 4), 179(29), 166(20), 161(21), 151(70), and 138m/e (61). HREIMS: found 299.197Ore~e; calcd for C15H260,222.1984. Reference Q. Huang, Y. Tezuka, Y. Hatanaka, T. Kikuchi, A. Nishi, and K. Tubaki; Studies on Metabolites of Mycoparasitic Fungi. III. New Sesquiterpene Alcohol from Trichoderma koningii; Chem. Pharm. Bull., Vol. 43, pp. 1035-1038(1995).

740

30.

Miscellaneous Metabolites

Common/Systematic Name Aszonapyrone A Molecular Formula/Molecular Weight CasH4oOs; MW = 456.28757

H2 ~

Z?Me

294' 19

12

1 1 1

22

~ 1, 2 ~ L . . . j 9

16

28

2s O~

j.

r

T

# 17

_-18

General Characteristics (Monomethyl ether) Crystals from benzene-acetone; mp., 276-278~ Fungal Source Mycelial mats of Aspergillus zonatus IFO 8817. Biological Activity Antibacterial activity. Spectral Data (monomethyl ether of OH on carbon 23) UV:

~. E,oH 298nm (e=8,600) IR:

(KBr) 1740, 1700, 1690(sh), 1650, 1560, 1460, 1440, 1240, and 910cm"1. 1H N]VIR: (CDCI3) 0.76(3H, s); 0.84(9H, s); 1.00---2.80(17H, m); 2.04(3H, s); 2.23(3H, s); 3.92(3H, s); 4.54(II-I, dd, J=4.12I-Iz); 4.72(IH, s); 4.88(IH, s); and 6.06ppm (IH, s). ~SC NMR: (CDCIs) 15.2(q); 16.4(q); 16.5(q); 19.0(0; 19.3(q); 20.1(q); 21.1(q); 23.7(0; 23.9(t); 28.1(q), 37.7(s); 38.0(s); 38.4(0; 38.5(0; 40.3(s), 40.3(t); 54.2(d); 55.6(d); 56.0(q); 60.1(d); 81.1(d); 95.1(d); 105.9(s); 106.2(0; 149.0(s); 160.7(s); 165.1(s); 165.5(s); and 170.6ppm (s). Mass Spectrum: 470(M+), 455, 411,395, 154, and 153m/e.

30.

Miscellaneous Metabolites

References Y. Katsube, Y.Kimura, T. Hamasaki, H. Nakajima, and A. Isogai; Structure of Aszonapyrone A Monomethyl Ether-1, a Derivative of Aszonapyrone A, Produced by Aspergillus zonatus; Agile. Biol. Chem.. Vol. 49,. pp. 551-553(1985). Y. Kimura, T. Hamasaki, A.Isogai, and H. Nakajima; Structure of Aszonapyrone A, a New Metabolite Produced by Aspergillus zonatus; Agile. Biol. Chem., Vol. 46, p. 1963(1982).

741

742

30.

Miscellaneous Metabolites

Common/Systematic Name Oxysporone Molecular Formula/Molecular Weight C7H804, M ' W = 1 5 6 . 0 4 2 2 6

OH

0 H

General Characteristics [tt]D25 +39.5 ~ (C= 0.9%, MeOH); gave a monoacetate derivative. Fungal Source Fusarium oxysporum IMI 211881 and Pestalotia longiseta PL8501 (IFO 32172) which causes tea gray blight, the most important disease of tea plantations in Japan. Isolation/Purification Separated on silica gel using ethyl acetate-petrol (2:3, v/v). Biolo~cal Activity Phytotoxin; caused leaf necrosis of cv. Yabukita at ca. 15ppm. Agent responsible for the antibiotic activity of earthworm casts used in some parts of Nigeria to treat chronic dysentery. Spectral Data _ UV:

~,m~ 215(e = 1277) and 284nm (417). IR:

(CHC13) 3600(OH), 1806(y-alkoxy-y-lactone), and 1654(C=C) cm]. ]H NMR: 6.37(1H, J--6Hz, H-2); 5.06(1H, ddd, J=6, 5.5, 1.0Hz, H-3 coupled to H-2, H-4 and one of the high field protons possibly H-5); 5.82(11-1, d, J=4.5Hz, H-6); 4.16(1H, dd, J=-5.5 and 2.0Hz, H-4); and four high field protons between 62 and 3, one of which was OH (exch. with D20). 13C NMR:

175.3(C-8), 143.5(C-2), 100.1(C-3), 96.0(C-6), 60.0(C-4), 41.8(C-7), and 29.5ppm. (C-5); (CDCI3) 174.5(C-8), 144.1(C-2), 99.8(C-3), 95.8(C-6), 60.3(C-4), 41.8(C-5), and 29.4ppm (C-7).

30.

Miscellaneous Metabolites

743

Mass Spectrum: EIMS: 156(M+, 11%), 128(12); 114(21); 110(19); 84(100), 73(73), and 55m/e (79). HREIMS: 156.042m/e; calcd. For CTHsO4, 156.042. TLC Data Silica gel; ethyl acetate-acetone (6:4, v/v); Rf=0.53. References E. K. Adesogan, and B. I. Alo; Oxysporone, A New Metabolite from Fusarium oxysporum; Phytochemistry, Vol. 18, pp. 1886-1887(1979). T. Nagata, and Y. Ando; Oxysporone, a Phytotoxin Isolated from the Tea Gray Bright Fungus Pestalotia longiseta; Agile. Biol. Chem., Vol. 53, p. 2811(1989).

744

30.

Miscellaneous Metabolites

Common/Systematic Name Ascladiol Molecular Formula/Molecular Weight C7H804; ~

HOH2C

= 156.04226

CH2OH

General Characteristics Crystals from chloroform-acetone; mp., 65-66~

very hygroscopic.

Fungal Source

Aspergillus clavatus (WF-38-11) isolated from wheat flour.

Isolation/Purification Liquid culture was shaken with activated charcoal, which was centrifuged and extracted with acetone-water (4:1, v/v) followed by concentration and extraction with ether. The ether extract was chromatographed on silica gel using chloroform-acetone (50:2, v/v). The substance was crystallized from chloroform-acetone. Biological Activity Acute toxicity (I.P.) was about 1/4 times as strong as that ofpatulin. Spectral Data UV: ~, EtOH marx

271nm

IR:

(KBr) 3300(associated OH), 1750(5-membered lactone-ring), and 1735cm 1 (5-membered lactone-ring). 1H NMR: (CD3COCD3) 6.29(1H, q); 5.87(1H, m); 4.74(4H, broad s); and 4.30ppm (2H). Mass Data: Found: C, 53.01, 53.40; H, 5.41, 5.29; calcd, for C7nsO4'H20: 53.23; H, 5.23. Reference T. Suzuki, M. Takeda, and H. Tanabe; A New Mycotoxin Produced by Aspergillus clavatus; Chem. Pharm. Bull., Vol. 19, pp. 1786-1788(1971).

30.

Miscellaneous Metabolites

745

Common/Systematic Name Anhydrosepedonin Molecular Formula/Molecular Weight CllH1004; ~ =

0

206.05791

OH

Me

General Characteristics Glistening, yellow needles; mp., 205 ~ (decomp.); sublimed unchanged in high vacuum at 170~ Optically inactive. Insoluble in light petroleum (bp., 40-60~ Sparingly soluble in acetone, ethanol, methanol, ethyl acetate, dioxane, chloroform, and benzene. Dissolved slowly in aqueous NaHCO3 but readily in aqueous NaOH, and also in cone. HC1 from which it was precipitated unchanged on dilution with water. Gave a strong brown color with FeC13 solution. Anhydrosepedonin probably formed spontaneously in the culture filtrate, and during the isolation process, by chemical dehydration of sepedonin.

Funsal Source

Sepedonium chrysospermum isolated from Boletus luridus.

Isolation/Purification Culture filtrate was acidified with HC1 and extracted with ether. The extract was evaporated to dryness, and the resulting gum was triturated with acetone, giving an orange solid (mp., 190-197~ which was crystallized from absolute ethanol or glacial acetic acid. Soectral Data UV;

~, m, xEtOU 249(r = 15750), 286(29400), 302(inflection, 18700), 354(inflection, 8270), 375(7250), and 386nm (8130). IR;

(Nujol) 1660, 1607, 1465, 1430, 1370, 1340, 1310, 1230, 1170, 1140, 1105, 885, 835, and 725cm "1. 1H NMR: (2N NaDH in D20) 6.58(1H oftropolone ring); 6.20(1H oftropolone ring at C-3);

746

30.

Miscellaneous Metabolites

IH NMR: (2N NaDH in D20) 6.58(1H oftropolone ring); 6.20(1H oftropolone ring at C-3); 5.49(1H, olefinic); 5.00(2H, methylene); and 1.87ppm (3H, methyl). Mass Data: Mass peaks higher than the expected molecular-ion peak at 206, probably due to the existence of oligomers in the solid state. After hydrogenation of the substance a mass spectrum showed expected molecular-ion peak at 206m/e;found: C, 64.0, 64.2; I-I, 5.52, 5.59; O, by direct estimation, 31.2, OCH3, nil; CCH3, 5.5; active H atoms in pyridine, 0.86%; titration equivalent to phenolphthalein, 204. CllH1004 required: C, 64.1; H, 4.89; O, 31.0; 1-CCH3, 7.3; 2 active H atoms, 0.98%, equivalent titrating as a monobasic acid, 206. Reference P. V. Divekar, H. Raistrick, T. A. Dobson, and L. C. Vining; Studies in the Biochemistry of Microorganisms. Part 117. Sepedonin, a Tropolone Metabolite of Sepedonium chrysospermum Fries; Can. J. Chem., Vol. 43, pp. 1835-1848(1965).

30. MiscellaneousMetabolites

747

Common/Systematic Name Sepedonin Molecular Formula/Molecular Weight v

CllH1205; ~[W = 224.06847

OH

HO/~

Me OH

General Characteristics Pale-yellow prisms from ethyl acetate; mp., 190-205~ (decomp.); [~]D 26 0 ~ (c=0.2, in EtOH). Drying at 80 ~ caused decomposition. Insoluble in petroleum ether, carbon tetrachloride, benzene, and dilute NaHCO3. Poorly soluble in chloroform, ethyl acetate, acetone, and water. Fairly soluble in ethanol, pyridine, dioxane, dilute Na2CO3 or NaOH solution, also in NHC1 giving a straw-colored solution which rapidly turned bright yellow. Gave a yellow solution in concentrated H2SO4 and deep-green color with FeC13 solution. Fungal Source

Sepedonium chrysospermum isolated from Boletus luridus.

Isolation/Purification The broth was acidified with HC1 and extracted with ethyl acetate. The extract was crystallized after removal of the solvent and recrystallized several times from ethyl acetate to yield sepedonin. Spectral Data UW:

~ EtOH

253(6 = 36800) and 327nm (6400).

IR:

(KBr) 3265, 2920, 2850, 1656(sh), 1600, 1573, 1525, 1462, 1378, 944, 870, 796, and 705cm "1.

Mass Data: Found in a sample dried in high vacuum at room temp.: C, 58.58; H, 5.63; OC2H5, 3.14%.

748

30.

Miscellaneous Metabolites

Reference P. V. Divekar, H. Raistrick, T. A. Dobson, and L. C. Vining; Studies in the Biochemistry of Microorganisms. Part 117. Sepedonin, a Tropolone Metabolite of Sepedonium chrysospermumFries; Can. J. Chem., Vol. 43, pp. 1835-1848(1965).

30.

Miscellaneous Metabolites

749

Common/Systematic Name Alterporriol B 2,2••Dimeth•xy•4•6,4'•5••6'•7',8'•heptahydr•xy•7,7••dimethy•-(5'S•6'R•7•S•8'R)•5'•6••7',8•• tetrahydro- 1, l'-bianthraquinone Molecular Formula/Molecular Weight C32H26013; M W -- 618.13734

OH

0 OH

MeO

Me

0 O MeO

OH

Me

/,,,OH ......OH OH

O

General Characteristics Melting point >300~

i

OH [a]D 25 -310 ~ (c=0.05, in EtOH).

Fungal Source

Alternariaporri, a causal fungal agent of black spot disease of stone leek.

Isolation/Purification The coloring matter obtained from an ethyl acetate soluble part of the culture liquid and myeelium was separated into 5% sodium bicarbonate soluble and insoluble parts and the latter was further separated into 5% sodium carbonate soluble and insoluble parts. Repeated silieie acid chromatography (Merck Kiesel Gel 60) of the 5% sodium carbonate soluble part with a solvent system of methanol-chloroform-formic acid (5:95:2, v/v/v) and preparative TLC (Merck Kiesel Gel GF254)with a solvent system of chloroform-acetone-formic acid (200:100:1, v/v/v) gave a dark-red pigment named alterporriol B. Spectral Data IR:

(KBr) 3000-3700(broad OH), 1670, 1650(non-chelated C=O), 1640(chelated C=O), 1210(phenolic OH), 1160(tert-OH), and 1110ppm (see-OH).

750

30.

Miscellaneous Metabolites

1H NMR: (da-acetic acid) 4.40(1H, s, H-8'); 4.01(1H, d, Js,,6,=7.8Hz, H-6); 4.91(1H, d, Js.,6~7.8Hz, H-5'), 6.81(1H, s, H-4'); 6.81(1H, s, H-3'); 1.39(3H, s, C-7'-CH3); 3.71(3H, s, C-2'-OCH3); 7.55(1H, s, H-8); 7.60(1H, s, H-5); 6.82(1H, s, H-3); 6.82(1H, s, H-I); 2.26(3H, s, C-7-CH3); and 3.72ppm (31-1, s, C-2-OCH3). Mass Spectrum: The field desorption mass spectrum of alterporriol B showed only a characteristic fragment ion role 582 (C32H220~). However, the molecular secondary ion mass spectrum showed fragment ions at 641 (M + +Na) and 619(M§ +H). The high resolution mass spectrum (In Beam El) gave peaks at role 582.1161 (C32Hz20~1, M + - 21-120), 566.1204(C32H22010, 582 +2H -H20), 551.1026(C31H19010), 535.1000(C31H2909, 566 -OCH3), 284.0695(C~6H~205), 255.0646(C~5H~104, 284 -CHO), and 241.094 lm/e

(C14H904). Reference R. Suemitsu, T. Sano, M. Yamamoto, Y. Anmoto, F. Morimatsu, and T. Nabeshima; Structural Elucidation of Alterporriol B, a Novel Metabolic Pigment Produced by Alternariaporri (Ellis)Ciferri; Agric. Biol. Chem., Vol. 48, pp. 2611-2613(1984).

30.

Miscellaneous Metabolites

751

Common/Systematic Name LL-S49113 Molecular Formula/Molecular Weight C2oH2605; MW" = 346.17802

General Characteristics Melting point, 180-185~

[a]D +112.4 ~ (MeOH).

Fungal Source

Aspergillus chevalieri (Lederle culture $491).

Biological Activity Antibacterial activity against certain gram-positive organisms. Strong antiprotozoal activity against Tetrahymenapyriformis. Spectral Data UV~

uooH 241nm (c=5850); ~ UoOH§ acid; a-diketone chromophore). nlax

263nm (c=4600) (reversible on addition of

IR~

(KBr) 3500(-OH), 1755(y-lactone), 1710(ketone), and 1620cm"1 (=bond). CD: Negative Cotton effect at 245nm with a Ac=3.7. 1H NMR:

(CDC13) 5.02-5.65(2H, 8-line pattern); 5.84(1H, 4-line system); 1.09(3H, s, 3C-CH3); 1.20(6H, s, 3C-CH3); and 1.90 and 5.25ppm (two 1H exchangeable, sharp s, two tertiary OH). Reference G. A. Ellestad, M. P. Kunstmann, P. Mirando, and G. O. Morton; Structures of Fungal Diterpene Antibiotics LL-$49113 and y; J. Amer. Chem. Sot., Vol. 94, pp. 6206-6208(1972).

752

30.- Miscellaneous Metabolites

Common/Systematic Name LL-S491y Molecular Formula/Molecular Weight C20H2805;~ = 348.19367

il

General Characteristics Melting point, 190-195~

[a]D +63.3 ~ (MeOH).

Fungal Source Aspergillus chevalieri (Lederle culture $491).

Biological Activity Antiviral activity against Herpes simplex. Strong antiprotozoal activity against Te tr a hy m e na p y r if o rm i s.

Spectral Data. UV~ End absorption IR~ (KBr) 1745cm-1 (y-lactol)

CD: Positive Cotton effect at 209nm with a Ae=8.3. 1H NMR: (CDCI3) 5.83(1, d, JT,14=2.0Hz, H-14); and 4.30ppm (H-7). Reference G. A. Ellestad, M. P. Kunstmmm, P. Mirando, and G. O. Morton; Structures of Fungal Diterpene Antibiotics LL-$49113 and y; I. Amer. Chem. Soc., Vol. 94, pp. 6206-6208 (1972).

30.

Miscellaneous Metabolites

753

Common/Systematic Name cis-Sativenediol Molecular Formula/Molecular Weight C15H2402; M W -" 2 3 8 . 1 9 3 2 8

HO , ~ \ ~ HO/\H H General Characteristics Oil; [a]D - 119 ~ (C=0.94, in CHCI3). Fungal Source Cochliobolus setariae IFO 6635 and Helminthosporium sativum. Isolation/Purification Both species of fungi were cultured by shake culture on malt-dextrose medium. The culture broth was extracted with ethyl acetate, cis- and trans-sativenediol were isolated from the crude extract by silicic acid column chromatography and elution with 3% ethyl acetate in n-hexane. Final purification of these diols from each other was accomplished by treating the mixture with 2,2-dimethoxypropane and p-TsOH. Pure trans-sativenediol and an acetonide of the cis-diol were isolated from the reaction mixture, and the latter compound was subsequently hydrolyzed liberating cis-sativenediol in pure form. Biological Activity A plant growth promoter with gibberellin-like activity that promoted elongation of rice seedings; at concentrations of 3, 30, and 300~g/ml; cis-sativenediol promoted elongation at 77%, 176%, and 110% of control seedlings, respectively. Spectral Data IR:

(CC14) 3670 and 3360cm 1 (OH groups). 1H NMR: (CDCI3) Two 3H doublets at 0.89, and 0.95ppm, J=7Hz, for an isopropyl group; a tertiary methyl (3H singlet at 1.08ppm); an exocyclic methylene (two 1H doublets at 4.66 and 4.98ppm); two vicinal hydroxyl groups (two 1H doublets at 3.68 and 4.08ppm, J=6Hz) which were decoupled to each other in double resonance

754

30.

Miscellaneous Metabolites

experiments and shitted downfield (4.72 and 5.05ppm, respectively); acetylation to the diacetate resulted in two CH3COO groups at 2.05ppm. These data were indicative of a dihydroxy derivative of sativene. Mass Spectrum: 236role (M+); diacetate derivative, 320role (M+). Reference

M. Nukina, H. Hattori, and S. Marumo; cis-Sativenediol, a Plant Growth Promotor, Produced by Fungi; J. Am. Chem. Sot., Vol. 97, pp. 2542-2543(1975).

30.

Miscellaneous Metabolites

755

Common/Systematic Name trans-Sativenediol; Isosativenediol Molecular Formula/Molecular Weight C15H2402; M W -- 2 3 6 . 1 7 7 6 3

H

I OH

General Characteristics Colorless needles; mp., 176~

[a]D 27 - 3 . 4 ~

(c=1.4, in CHC13).

Fungal Source Cochliobolus setariae IFO 6635, Helminthosporium sativum (reclassified as Bipolaris sorokiniana, which is known to cause seedling blight, foot and root rot, head and leaf spot of cereals and grasses), and H. victoriae. Isolation/Purification Fungi were cultured by shake culture on malt-dextrose medium. The culture broth was extracted with ethyl acetate, cis- and trans-sativenediol were isolated from the crude extract by silicic acid column chromatography and elution with 3% ethyl acetate in n-hexane. Final purification of these diols from each other was accomplished by treating the mixture with 2,2-dimethoxypropane and p-TsOH. Pure trans-sativenediol and an acetonide of the cis-diol were isolated from the reaction mixture, and the latter compound was subsequently hydrolyzed liberating cis-sativenediol in pure form. Biological Activity A plant growth inhibitor that inhibited elongation of rice seedlings only slightly; at concentrations of 3, 30, and 300ktg/ml trans-sativenediol inhibited elongation 8%, 15%, and 16% of control seedlings, respectively. Spectral Data 1H NM]~: (CDCI3) Two 3H doublets at 0.95 and 1.05ppm, J=6.5Hz, for an isopropyl group; a tertiary methyl (3H singlet at 1.00ppm); an exocyclic methylene (two 1H doublets at 4.75 and 5.05ppm); two vicinal hydroxyl groups (two 1H of a slightly splitting broad singlet at 3.68 and 4.45ppm, J=<2Hz) which were deeoupled from each other by double resonance experiments and shitied downfield to 4.70 and 4.92ppm, respectively); acetylation to the diacetate resulted in two CH3COO groups at 1.99, and 2.07ppm.

756

30.

Miscellaneous Metabolites

Mass Spectrum: 218m/e (M + - H20), diacetate derivative, 320m/e (M+).

References F. Dora and D. Arigoni; Zwei neue Sesquiterpene mit Isosativangerust aus Helminthosporium sativum: Derivatives of lsosativene from Helminthosporium sativum; Experientia, Vol. 31, pp. 753-754(1975). M. Nukina, H. Hattori, and S. Marumo; cis-Sativenediol, a Plant Growth Promotor, Produced by Fungi; J. Am. Chem. Soc., Vol. 97, pp. 2542-2543(1975).

30.

Miscellaneous Metabolites

757

Common/Systematic Name Isosativenetriol Molecular Formula/Molecular Weight C15H2403; MW = 252.17254

HO

HO

General Characteristics Crystals; mp., 206~

[t~]D25 +11 o (EtOH).

Fungal Source

Helminthosporium sativum.

Biological Activity Phytotoxin. Reference F. Dora and D. Arigoni; Zwei neue Sesquiterpene mit Isosativangerust aus Helminthosporium sativum: Derivatives of lsosativene from Helminthosporium sativum; Experientia, Vol. 31, pp. 753-754(1975).

758

30.

Miscellaneous Metabolites

Common/Systematic Name Patulin; Claviformin; Clavitin; Clavicin; Expansion; Penicidin; Mycoin; Leucopin; Tercinin 4-Hydroxy-4H-furo[3,2-c]pyran-2(6H)-one Molecular Formula/Molecular Weight C7I-I604; MW = 154.02661

"0"

"OH

General Characteristics Crystals from benzene; mp., 110-111 ~ [a]D 0 ~ Patulin 9 is soluble in water, alcohols, acetone, ethyl acetate, chloroform; sparingly soluble in ethyl ether, benzene; insoluble in petroleum ether. It is somewhat unstable in polar solvents such as water and methanol and loses its biological potency in alkaline conditions. Acetyl derivative, prisms from methanol, mp., 118-120 ~C. Phenylhydrazone, orange plates, mp., 152-153 ~C. Oxime, crystals from water, mp., 152-153 ~ (dec.). Isopatulin, prisms from ethyl acetate, mp., 88-900C. Fungal Source.

Penicillium expansum (P. leucopus), P. patulum Bainier (P. urticae, P. griseofulvum), P. claviforme, P. equinum (19. terrestre), P. novae-zeelandiae, P. lapidosum, P. granulatum (P. divergens), P. lanosum, P. melinfi, P. cyclopium, P. cyaneofulvum, P. roqueforh, Aspergillus clavatus, A. giganteus, A. terreus, and Byssochlamys nivea (Gymnoascus spp.).

Biological Activity Patulin was originally isolated because of its antibiotic properties against gram-positive and gram-negative bacteria. However, it was soon found to be too toxic for use as an antimicrobial agent in humans. The LDs0 of patulin in White Leghorn cockerels was 170mg/kg. Sublethal doses produced hemorrhaging in the digestive tract. LDs0 (IP) in mice was 5mg/kg. Patulin has been reported to be carcinogenic and a potent mitotic toxin in addition to being toxic to many protozoa, fungi, mammals, plants, HeLa cells, and viruses. Patulin has been found contaminating apple juice and has been implicated in the death of cattle. Spectral Data UV~

~.m~x 276nm (e =16,600)

30.

Miscellaneous Metabolites

759

1H NMR: 6.96(H-3); 5.90(H-4); 4.32(H-Sa); 4.66(H-Sb); 5.90(H-6); and 5.94ppm (4-OH). ~3CNMR: 168.5(C-2, s); 109.8(C-3, d); 88.4(c-4, d); 59.1(C-5, t); 107.9(C-6, d); 151.5(C-7, s); and 146.1ppm (C-8, s). TLC Data Adsorbent: silica gel G-HR; Solvent: toluene-ethyl acetate-formic acid, 5:4:1 v/v/v, Rf=0.58; Detection: brown spot alter spraying with 50% ethanolic H2SO4 and heating at 100~ for 5 min. GC Data Solid support: Gas Chrom-Q, Liquid phase: 10% DC-200, Column temp: 175~ Detector temp, 190 ~C, Injector temp, 190 ~C. Relative retention time (relative to 13-BHC and analyzed as TMS derivative): 0.56. Solid support: Gas Chrom-Q, 100-120 mesh; Liquid phases: 3% Dexsil 300, 3% OV-17 and 3% OV-25. Reference R. J. Cole, and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 937(1981).

760

30.

Miscellaneous Metabolites

Common/Systematic Name Isopatulin Molecular Formula/Molecular Weight C7H604; M W -" 154.02661

09 HO

"~~

General Characteristics Prisms from ethyl acetate; mp., 88-90~ Fungal Source Penicillium urticae

(2159k, cells).

Isolation/Purification Acrylamide gel with fungi cells embedded in it was acidified and extracted with ethyl acetate. The extract was chromatographed on preparative TLC plates (chloroform-ethyl acetate-ethyl ether, 5:2:1, v/v/v). Isopatulin crystallized from ethyl acetate. Spectral Data UV~

~. M~oH 269nm (e=12800) IR~

(Nujol) 3400, 1780, 1755, 1675, 1610, 1440, 1400, 1280, 1225, 1175, 1110, 1070, 1040, 1000, 980, 900, 845, 820, and 795cmq. CD: Optically inactive. ~H NMR: (CD3COCD3) 3.08(s, 5-OH); 4.74(dd, Ja_b-16.9, Jb.7=l.2Hz, 1-Hb); 5.04(dd, Ja.b=16.9, J~.7=2.2Hz, 1-Ha; shifts of Ha and Hb may be interchanged); 5.72(m, 5-H); and 6.01 ppm (m, 7-H). 13C NMR:

(CD3COCD3) 58.2(t, l-C); 90.4(d, 5-C); 109.0, 110.8(each d, for 4 and 7-C); 150.2(s, 2-C); 153.4(s, 3-C); and 169.6ppm (s, 8-C).

30.

Miscellaneous Metabolites

761

Mass Data: 154(M+), 136, 126, 112, 97, 67, and 39m/e; found: C, 54.79; H, 4.15; calcd, for C7H604: C, 54.55; H, 3.92. TLC Data Chloroform-ethyl acetate-ethyl ether (5:2:1, v/v/v); Rf=0.30. Detection: UV light. Reference J. Sekiguchi, G. M. Gaucher, and Y. Yamada; Biosynthesis of Patulin in Penicillium urticae: Identification of Isopatulin as a New Intermediate; Tetrahedron Lett., No. 1, pp. 41-42(1970).

762

30.

CommnnlSvstematic j

Miscellaneous Metabolites

Name

Acrostalidic acid MoleclJlar Fnrmlda/Molecldzr Weight C16H2204; M W = 278.15181 O

H

9

n

14

"

COOH

General Characteristics Recrystallization from ethyl acetate gave crystals; mp., 210-211 ~C. Funozl s

Acrostalagmus sp. (NRRL 3481). L~nlatinn/Pnrific:~d nn

The fermented mash was adjusted to pH 2 with 2N hydrochloric acid. The mash was filtered through Celite and the filter pad was washed with water. The filtrate was extracted with ethyl acetate, the organic layer was successively washed with water and saturated salt solution, dried (Na2SO4), and evaporated under reduced pressure to give an oily residue. This was washed with n-hexane, and the crude product was dissolved in ethyl acetate and precipitated with benzene. The supematant was evaporated to give an oil. Sufficient acetone was added to dissolve the oil and silica gel was added to the solution. The solvent was removed under reduced pressure and the dry silica gel was placed on a silica gel column slurry packed in benzene. The column was eluted successively with benzene, benzene containing increasing amounts of chloroform, chloroform, and chloroform containing increasing amounts of ethyl acetate. From fractions eluted by chloroform-ethyl acetate, LL-Z 1271 cx, LL-Z 127113, and LL-Z 1271 ~i were isolated, respectively. Further column chromatography of fractions eluted by chloroform-ethyl acetate, (6:1 - 3:1) was carried out on silica gel by the usual method. The column was eluted successively with chloroform and chloroform containing an increasing proportion of ethyl acetate crude; acrostalidic acid was obtained from fractions eluted by chloroform-ethyl acetate (25:1). Recrystallization from ethyl acetate gave crystals of acrostalidic acid. Riala~cal Activi~ Related to the antifungal metabolite LL-Z 1271 cx. Therefore, may also be antifungal.

30.

Miscellaneous Metabolites

763

Sneetral Data IR; (KBr) 3030cm~ (olefinie); (CHC13) 1720 and 1690cm1. ~HNMR: (CDC13); 2.03(H-5); 2.33(H-8); 1.57(H-9); 2.23(dd, J=18 and 12.5Hz, H=1113); 2.62(dd, J=18 and 5Hz, H-1 lcx); 3.87(dd, J=l 1.5 and 10.5Hz, H-14cx); 4.45(dd, J=10.5 and 5Hz, H-1413); and 10.1ppm (COzH). ~3CNMR: (CDC13) 54.7; 46.1; 43.1; 37.0; 35.5; 35.2(2 C); 30.6; 28.0; 18.9; and 10.7ppm. Mass Spectrum: 278(M+), 263, 260, 233(intense), 217, 173, 145, and 105m/e (base peak); high resolution 278.150(M+; C16H2204requires M, 278.152, 263.126(C15H1904,M-CH3), 233.153(C15H2102, M-COzH), 217.116(C~4H1702), 173.132(C~4H~7), 145.102(C~H13); and 105.070m/e (C8H9). Reference

M. Sato and H. Kakisawa; Structures of Three New C16 Terpenoids from an Acrostalagmus Fungus; J. Chem. Sot., Trans Perkin I, pp. 2407-2413(1976).

764

30.

Miscellaneous Metabolites

_Common/Systematic Name Acrostalic acid ._Molecular Formula/Molecular Weight C16H2404; M W -- 2 8 0 . 1 6 7 4 6

_COOH

COOH General Characteristics Recrystallization from ethyl acetate gave crystals; mp., 219-220 oC. Fungal Source

Acrostalagmus sp. (NRRL - 3481).

Isolation/Purification The fermented mash was adjusted to pH 2 with 2N .hydrochloric acid. The mash was filtered through Celite and the filter pad was washed with water. The filtrate was extracted with ethyl acetate and the organic layer was successively washed with water and saturated salt solution, dried (Na2SO4), and evaporated under reduced pressure to give an oily residue. This was washed with n-hexane, and the crude product was dissolved in ethyl acetate and precipitated with benzene. The supernatant was evaporated to give an oil. Sufficient acetone was added to dissolve the oil and silica gel was added to the solution. The solvent was removed under reduced pressure and the dry silica gel was placed on a silica gel column slurry packed in benzene. The column was eluted successively with benzene, benzene containing increasing amounts of chloroform, chloroform, and chloroform containing increasing amounts of ethyl acetate. From fractions eluted by chloroform-ethyl acetate (12.5:1,v/v/v) crude acrostalic acid was obtained. It was recrystallized from ethyl acetate to give crystals. Biological Activity Related to the antifungal metabolite LL-Z1271 a. Therefore it may also be antifungal. Spectral Data

(KBr) 3080, 1645, and 890cm q (=CH2). IH NMR:

((CD3)2CO) 0.66 and 1.23ppm (s, I0- and 4-methyls).

30.

Miscellaneous Metabolites

765

Mass Spectrum: 280(M+), 262, 234, and 109re~e; high resolution 280.169m/e (M+); C16H2404requires M, 280.167, 262.150(C16H2203, M-H20), 234.159(C~5H2202, M-HCO2H), and 109.107m/e (Csi-I13). Reference M. Sato and H. Kakisawa; Structures of Three New C16Terpenoids from an AcrostalagmusFungus; J. Chem. Soc., Trans Perkin I, pp. 2407-2413(1976).

766

30.

Miscellaneous Metabolites

Common/Systematic Name Pimara-8(9), 15-diene Molecular Formula/Molecular Weight C2oH32; M W = 272.25040

Fungal Source Trichothecium roseum, Cephalosporium aphidicola, and Nigrospora sphaerica. Isolation/Purification Mycelium was extracted with acetone, acetone extract was concentrated in vacuo and extracted with ethyl acetate. The extract was dried, evaporated, and the residue adsorbed onto silica gel and chromatographed on alumina. Elution with light petroleum gave a mixture of hydrocarbons which was analyzed by GLC on carbowax. Spectral Data Mass Spectrum: ELMS: 272(M+), 257(base peak), 215, 201,189, 187, 175, 161,157, 147, 133, 121, 119, 109, 107, and 105m/e. References M. R. Adams and J. D. Buq_,oek; Biosynthesis of the Diterpene Antibiotic, Aphidieolin, by Radioisotope and 13C-Nuclear Magnetic Resonance Methods; J. Chem. Soc. Chem. Commun., pp. 989-991(1975). B. Dockerill and J. R. Hanson; Studies in Terpenoid Biosynthesis. Part 19. Formation of Pimara-8(9),15-diene by Trichothecium roseum; J. Chem. Soc., Perkin Trans I, pp. 324327(1977).

30.

Miscellaneous Metabolites

767

Common/Systematic Name Isoacrostalidic acid Molecular Formula/Molecular Weight C15H2004, m w = 2 6 4 . 1 3 6 1 6

O

COOH General Characteristics Recrystallization from ethyl acetate gave crystals; mp., 204-206 ~C. Fungal Source

Acrostalagmus sp. (NRRL -

3481).

Isolation/Purification The fermented mash was adjusted to pH 2 with 2N hydrochloric acid. The mash was filtered through Celite and the filter pad was washed with water. The filtrate was extracted with ethyl acetate and the organic layer was successively washed with water and saturated salt solution, dried (Na2SO4), and evaporated under reduced pressure to give an oily residue. This was washed with n-hexane, and the crude product was dissolved in ethyl acetate and precipitated with benzene. The supernatant was evaporated to give an oil. Sufficient acetone was added to dissolve the oil and silica gel was added to the solution. The solvent was removedunder reduced pressure and the dry silica gel was placed on a silica gel column slurry packed in benzene. The column was eluted successively with benzene, benzene containing increasing amounts of chloroform, chloroform, and chloroform containing increasing amounts of ethyl acetate. Fractions eluted by chloroform-ethyl acetate (50:1,v/v) followed by recrystallization from ethyl acetate giving needles of isoacrostalidic acid. Bioloceal Activity Related to the antifungal metabolite LL-Z1271 a. Therefore it may also be antifungal. So_ectral Data IR~

(KBr) 3050crn ~ (olefinic); (CHC13) 1755cm~.

768

30.

Miscellaneous Metabolites

Mass Spectrum: 278m/e (M+); high resolution 278.152m/e (M*, C16I-I2204requires M), 278.152, 263.132(C15H1904, M-CH3), 232.146(C15H2002), 217.120(C~4H~702), 189.127, and 109.107role (CgH13). Reference M. Sato and H. Kakisawa; Structures of Three New C16 Terpenoids from an Acrostalagmus Fungus; J. Chem. Sot., Trans Perkin I, pp. 2407-2413(1976).

30.

Miscellaneous Metabolites

769

Common/Systematic Name Deoxypatulinic acid 2,3-Dihydro-4-pyrone-5-acetic acid Molecular Formula/Molecular Weight CTHsO4; MW = 156.04226

0

C02H

General Characteristics Crystals; mp., 115-115.5~ (with effervescence).

soluble in water (to give an acidic pH) and aqueous NanCO3

Fungal Source Penicillium patulum (19. urticae), strain No. 562 from mixed feed. Isolation/Purification Extracted with ethyl acetate and chromatographed on silica gel. The substance was eluted with benzene-ethyl acetate (85:15 to 75:25, v/v); crystallized from isopropyl ether-n-hexane and toluene, followed by sublimation. Biological Activity Had no inhibitory effect (46mg/ml) on Bacillus megaterium, B. subtilis, B. cereus, Staphylococcus aureus, S. epidermidis, Sarcina lutea, Micrococcus flavus, and Saccharomyces cerevisiae. Spectral Data UV:

~, E~a

267nm (e=7960)

IR:

(CHCI3) 3510(OH), 3000-2400(COOH), 1718(COOH dimer), 1672(conjugated C-O), and 1621cml (C-C). 1H NMR: (CDC13) 1.53(1H, s, exchanged with D20); 2.63(1H, s, COC=CHO), 5.48(2H, t, J=7Hz, CH20), 6.83(2H, s, COCH2C=C); 7.34(21-1, t, J=THz, COH2), each triplet collapsed to a singlet on irradiation at the frequency of the other triplet.

770

30. Miscellaneous Metabolites

Mass Data: 156(M+), 112(M-CO2), 83, 60[CH2=E(OH)2+], 55, 39, and 27re~e;found: C, 53.94; H, 5.16; calcd, for C7HsO4: C, 53.85, H, 5.16%). TLC Data Silica gel F254,toluene-ethyl acetate-formic acid (6:3:1, v/v/v); Rf= 0.22. Detection: dark spot under short-wavelength UV. Reference P. M. Scott, B. Kennedy, and W. van Walbeek, Desoxypatulinic Acid from a Patulin-Producing Strain of Penicillium patulum; Experientia, Vol. 28, p. 1252(1972).

30. Miscellaneous Metabolites

771

Common/Systematic Name Dihydropenicillic acid Molecular Formula/Molecular Weight C8H1204, MW = 172.07356

Meo.'OLo MeO~

Me

General Characteristics Colorless needles from ethyl acetate-hexane; mp., 84 ~C. Hydrogenation with 5% palladium on charcoal gave penicillic acid. Fungal Source Unidentified fungus. Isolation/Purification Fungal culture acidified to pH 3.0 and extracted with ethyl acetate. The ethyl acetate extracts were concentrated and extracted several times with sodium bicarbonate solution. Concentration of the ethyl acetate solution produced crystals which were recrystallized from ethyl acetate-hexane to give colorless needles. SDectral Data _

UV:

Quite similar to that of peniciUic acid. IR:

Quite similar to that of penicillic acid. 1H ~ :

0.88(3H, d, J=7.0Hz); 1.06(1H, d, J=7.0Hz); 2.18(1H, septet, J=7.0Hz); 3.90(3H, s, methoxy); and 5.02ppm (1H, vinyl proton). Reference T. Sassa, S. Hayakari, M. Ikeda, and Y. Miura; Plant Growth Inhibitors Produced by Fungi: Part I. Isolation and Identification of Penieillic Acid and Dihydropenieillie Acid; Agric Biol. Chem., Vol. 35, pp. 2130-2131 (1971).

772

30.

Miscellaneous Metabolites

Common/Systematic Name (-)-Longifolene Molecular Formula/Molecular Weight C 15H24, M W = 2 0 4 . 1 8 7 8 0

Fungal Source Helminthosporium sativum, H. victoriae, and H. setariae. References F. Dora and D. Argigoni; Ein Bicyclischer Abkommling von (-)-Longifolen aus Helmmthosporium sativum und H. victoriae; Experientia, Vol. 30, pp. 851-852(1974). F. Dora and D. Argigoni; Sesquiterpenoide Metaboliten aus Helmmthosporium-Arten: Struktur und Biosynthese; Chemica, Vol. 29, pp. 24-25(1975).

30.

Miscellaneous Metabolites

773

Common/Systematic Name Victoxinine Molecular Formula/Molecular Weight C17H29NO, M W = 2 6 3 . 2 2 4 9 1

,•9

5

1 15 ,~

N~

CH20H ~7

Funsal v Source Bipolaris sp. strain 36 (ATTC 64838), a fungal pathogen of Johnson Grass (sorghum halepense (L.) Pers.), Helmmthosporium victoriae, the causal agent of Victoria blight of oats. Vietoxinine has been reported from culture filtrates ofHelminthosporium sativum (reclassified as Bipolaris sorokiniana). Isolation/Purificatio.n Purification of the crude fungal mixture was accomplished by flash column chromatography using Merck silica gel 60 and stepwise elution with n-hexane-Et20 (90:10 to 30:70, v/v). Biological Activity Phytotoxic, inhibited root growth of H. victoriae susceptible and resistant oat plants at 75pg/ml. Vietoxinine is a component of victorin, the host-specific toxin ofH. victoriae. Spectral. Data IR:

(KBr) 3464, 3063 and 1655cm"~.

~H NMR: (CDCI3) 4.75(IH, s, H-12a); 4.59(IH, s, H-12b); 3.50(2H, m, H-17); 2.99(IH, dd, 3.0, 10.9Hz, H-13a), 2.81(1H, dd, 4.7, 9.3Hz, H-14a), 2.58(1H, br m, H-6), 2.52(1H, ddd, 4.7, 8 0, 12.6Hz, H-16a), 2.41(1H, dt, 4.4, 12.5Hz, H-16b); 2.34(1H, dd, 1.6, 10.9Hz, n-13b); 2.23(11-1, d, 9.4Hz, n-14b); 1.82(1H, br s, H-5); 1.42(1H, m, H-8); 1.35(1H, rn, H-9), 1.13(3H, s, H-15), 0.90(3H, d, 6.6Hz, H-10), 0.86(3H, d, 6.6, H-11). Js,s, J6,8, ,]6,14 all -" < 1Hz; J6,14a=4.7Hz, Js,13a=3.0nz, Js,13b=1.6I--Iz, J:3~,b=10.9Hz; J:4~b=9.3Hz; J16~b=4.7Hz, J16~l-n,=8.0Hz,J16b,17a-4.4Hz, and J16b,17b=4.4Hz.

774

30.

Miscellaneous Metabolites

Mass Spectrum: CIMS: (CH4) 264(M+, 100%), and 232role (39); HRMS: (parent ion not observed) {M- CH2OH}+; calcd for C~6H22N232.2065, found 232.2066. References F. Dorn and D. Arigoni; Structure of Vietoxinine; J. C. S. Chem. Commun., pp. 13421343(1972). L. M. Pefia-Rodriguez and W. S. Chilton; Victoxinine and Prehelminthosporolactone,

Two Minor Phytoxic Metabolites Produced by Bipolaris sp., A Pathogen of Johnson Grass; J. Natural Products, Vol. 52, pp. 899-901(1989).

R. B. Pringle; Compaative Biochemistry of the Phytopathogenic Fungus Helmmthosporium. XVI. The Production of Victoxinine by H. sativum and H. victoriae; Can. J. Biochem., Vol. 54, pp. 783-787(1976). R. B. Pringle and A. C. Braun; Isolation of Victoxinine from Cultures of Helmmthosporium victoriae; Phytopathology, Vol. 50, pp. 324-325(1960).

30.

Miscellaneous Metabolites

775

Common/Systematic Name Antibiotic A26771B Molecular Formula/Molecular Weight C20H3007~ M W -- 3 8 2 . 1 9 9 1 5

O~O.

N

O

~

16

.,,,,Me

o

0 General Characteristics Crystals from ethyl acetate-hexane; mp., 121-123~ [a]D -13.1o (c=0.65, in MeOH); [a]D -14 ~ (C=0.13, in MeOH). Fungal Source Penicillium turbatum (ATCC 28797).

Isolation/Purification The fungal mycelium was filtered, dried, and extracted (3X) with ethyl acetate. The combined extracts were dried (Na2SO4), concentrated m vacuo, and chromatographed on preparative TLC plates with ethyl acetate-chloroform (2:1, v/v) to give a lipid fraction and crude antibiotic A26771B. The latter was recrystallized from ethyl acetate-hexane to give pure A2677 lB. Biological Activity Antibiotic Spectral Data IR:

(KBr) 3420, 1748, 1713, and 1701cm "l. ~H ~ : (CDCI3) 1.13-1.60(15H, m); 1.29(3H, d, J=6.5Hz); 1.68(1H, m); 1.89(2H, m), 2.72(4H, m); 5.13(1H, m); 5.31(1H, br t, J=5.5Hz); 6.73(1H, d, J=15.5Hz); and 7.22ppm (1H, d, J=15.5Hz).

776

30.

Miscellaneous Metabolites

13CNMR: (CDCI3) C-16, 19.54; C-7, 22.08; C-13, 23.33; C-9, 26.75; C-10, 26.91; C-8, 27.05; C-12, 27.66; C-11, 27.77; C-2', 28.11; C-3', 28.41; C-6, 28.72; C-14, 34.34; C-15, 72.57; C-5, 77.32; C-2, 132.78; C-3, 135.59; C-I, 165.28; C-I', 171.85; C-4', 177.03; and C-4, 195.95ppm Mass Spectrum:

CIMS: (NHs) 400(M+ + NH4+) and 38 lm/e OVI§ H+).

Reference K. Arai, B. J. Rawlings, Y. Yoshizawa, and J. C. Vederas; Biosyntheses of Antibiotic A26771B by Penicillium turbatum and Dehydrocurvularin by Alternaria cinerariae: Comparison of Stereochemistry of Polyketide and Fatty Acid Enoyl Thiol Ester Reductases; J. Am. Chem. Soc., Vol. 111, pp. 3391-3399(1989).

30.

Miscellaneous Metabolites

777

Common/Systematic Name IFO 663 5 Aversion Factor Molecular Formula/Molecular WeiRht C30H4604; MW = 470.33961 v

General Characteristics A labile oily substance that yields one molecule of prehelminthosporol and one molecule of prehelminthosporal on standing. Fungal Source

Cochliobolus setariae strain IFO 663 5.

Isolation/Purification Isolated by silicic acid column chromatography eluted with ethyl acetate in n-hexane. Biological Activity Antibiotic that specifically causes inhibition of different strains of the same fungal species. Spectral Data IR~

(Acetate derivative) (CCI4) 1760 (acetyl C=O) 1H NMR: (CDC13)(acetate derivative) 5.8 l ppm (acetyl carbon as a broad singlet that sharpened upon irradiation at a frequency of 1.59 ppm, which was assigned to the vicinal methine proton); and 2.02ppm (acetyl methyl). Mass Spectrum: (Acetate derivative) 512(M+), 23 5(45%), 219(100), and 277role (3 5). Reference M. Nukina and S. Marumo; Aversion Factors, Antibiotics among Different Strains of a Fungal Species: Aversion Factors of Cochliobolus setariae; Agile. Biol. Chem., Vol. 40, pp. 2121-2123(1976).

30. Miscellaneous Metabolites

778

Common/Systematic Name Penicillic acid 3-Methoxy-5-methyl-4-oxo-2,5-hexadienoic acid Molecular Formula/Molecular Weigh_t C8H1004; MW = 170.05791

00Me II I H2C:C--C--C-'C--CO2H I Me

MeO~

H~

Me--C II CH2

General Characteristics Crystals from petroleum ether; mp., 86-87~ from H20, 58-64~ [ a ] D -" 0 ~ Methyl ester, colorless prisms; mp., 35 oc. Penicillic acid can occur as keto or lactone form. Fungal Source Penicillium fividum, P. puberulum, P. griseum, P. simplicissimum, P. cyclopium, P. thomii, P. roqueforti (P. suavolens), P. martensii, P. fenelliae, P. aurantio-virens, P. janthinellum, P. viridicatum, P. palitans, P. baar ense, P. madriti, P. lilacinum, P. canescens, P. chrysogenurn, P. olivino-viride, Aspergillus ochraceus, A. sulphureus, A. melleus (,4. quercinus), A. sclerotiorum, A. alliaceus, A. ostianus and Paecilomyces ehrlichii. Biological Activity Subcutaneous injection of penicillic acid in mice at ca. 200-300mg/kg was lethal. The LDs0 to mice dosed SC was 110mg/kg; IV was 250mg/kg. The LDs0 for rabbits dosed SC was 100-200mg/kg. Penicillic acid has been shown to have antibacterial (mostly gram-negative), antiviral, antitumor, and cytotoxic effects. Spectral Data UV:

~. M,oH 221rim (c - 10,500) max

1H NMR: 5.09(H-2); 1.76(H-6); 5.18(H-7a); 5.44(H-7b); and 3.90ppm (H-8). 13C NMR:

169.5(C-1, s); 89.6(C-2, d); 178.9(C-3, s), 102.1(C-4, s); 139.8(C-5, s); 16.9(C-6, q); 114.0(C-7, t); and 59.6ppm (C-8, q).

30.

Miscellaneous Metabolites

779

TLC Data Adsorbent: Kieselgel G; Solvent: benzene-methanol-acetic acid, 18:1"1, v/v/v. Rf=0.25 (patulin has same Re in this system). Detection: fluoresces under UV light after exposure to NH3; yellow fluorescence with NH3 and phenylhydrazine. GLC Data Soliu support: Gas Chrom-Q liquid phase: 10% DC-200; Column temp: 175~ Detector temp: 190~ Injector temp: 190~ Relative retention time (relative to 13-BHC and were analyzed as TMS derivatives): 0.34; relative retention time for patulin in same system: 0.56. Solid support: Gas Chrom-Q, 100-102 mesh liquid phase: 3% Dexsil 300 3% OV-17 and 3% OV-25. Reference R. J. Cole, and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 520-524(1981).

780

30.

Miscellaneous Metabolites

Common/Systematic Name 5,5-Dimethyl-9-(2-hydroxyethyl)-8-hydroxymethyl-1-methylbicyclo[4.2.1]non-7-ene Molecular Formula/Molecular Weight C15H2602, MW = 238.19328

HOH2C

2OH

General Characteristics Crystals; mp., 117~

[ a ] D 25 --

+3 o (in CHC13), diacetate derivative, [ a ] D 25

--

+ 9~

Fungal Sou..rce

Helmmthosporium sativum and H. victoriae.

Isolation/Purification Extracted from cultures with ethyl ether followed by silica gel column chromatography. Spectral Data IH N]VIR:

(CDC13) Signals for three methyl groups at 0.94(6H); 1.04(3H); 5.71(an olefinic proton); 3.67(m, 2H); 4.08(2H); and two D20 exchangeable protons at 1.7ppm. Mass Data: Calcd. for C15H2602:

C,

75.58%, H, 11.00%; found: C, 75.52%, H, 11.00%

Reference F. Dora and D. Arigoni; Ein Bicyclischer Abkommling von (-)-Longifolen aus Helmmthosporium sativum und H. victoriae; Experientia, Vol. 30, pp. 851-852(1974).

30.

Miscellaneous Metabolites

781

Common/Systematic Name Cynodontin 1,4,5,8-Tetrahydroxy-2-methylanthraquinone; 2,5,7-trihydroxyemodin Molecular Formula/Molecular Weight C15H1006; M~V = 2 8 6 . 0 4 7 7 4

OH

O

OH Me

OH

O

OH

General Characteristics Brown-bronze crystals (no melting point reported) from ethyl acetate. Fungal Source

Curvularia lunata NRRL 2380, C. geniculata, C. ramosa, C. trifolii, Drechslera halodes, D. spicifera, D. sorokiniana, D. dematioidea, and D. setariae.

Isolation/Purification Extracted and defatted with ethyl acetate; recrystallized from ethyl acetate. Spectral Data UV:

~

EtOH

243,297, 474sh, 484, 506sh, 517, 542, 554, and 603nm.

Mass Spectrum: HREIMS: 286.04945m/e (M+); calcd, for C15H1006, 286.04773. Reference G. W. van Eijk, and H. J. Roeymans; Cynodontin, The Tetrahydroxyanthraquinone of Curvularia and Drechslera spp.; Experientia, Vol. 10, pp. 1283-1284(1977).

782

30. Miscellaneous Metabolites

Common/Systematic Name Cyclonerodiol Molecular Formula/Molecular Weight C15H2802, M W = 240.20893

OH

OH General Characteristics I,iquid. Fungal Source

Gibberella fujikuroi, Trichoderma koningii isolated from Diffenbachia sp. (ATTC 46314), Trichothecium sp. (Z. roseum), and Fusarium culmorum.

Isolation/Purification Fungal cultures were extracted with acetone, the acetone reduced to an aqueous phase under vaeuurn, and partitioned twice with ethyl acetate. The ethyl acetate extract was chromatographed on a silica gel column eluted with benzene, ethyl ether, ethyl acetate, acetone and acetonitrile. The ethyl ether and ethyl acetate fractions were combined, reduced in volume, and further purified by silica gel column chromatography eluted with a linear gradient from benzene to acetone. The koninginin B fractions were reduced in volume and chromatographed on a column containing hydrated silica gel eluted with benzene-ethyl acetate (5:4, v/v). Biologically active fractions were pooled and chromatographed on C ls reverse phase silica gel with acetonitrile-water (1:1, v/v). Final purification was performed on a silica gel-silver nitrate column eluted with methylene chloride-acetone (9:1, v/v) to yield koninginin B and cyclonerodiol. Cyclonerodiol was separated from koninginin B on silica gel, eluting with ethyl acetate-benzene (9:1, v/v). Biological Activity The metabolite inhibited the growth of etiolated wheat coleoptiles 60% at 103M but did not visibly affect them at 10"4 or -10~V[. Spectral Data UV~

No UV absorption above 210nm.

30.

Miscellaneous Metabolites

783

IR:

(KBr) 3460(broad OH), 2965, 2025, 1446, 1375, 1285, 1200, 1143, 1110, 1025, 983, 916, 882, and 830crn1. ~H NMR: (Acetone-d6) 0.99(d, J=6.8Hz, H-l), 1.65-1.40(H-2); 1.79(m, H-4); 1.65-1.40(H-5), 1.65-1.40(H-6), 1.43(t, J=8.5Hz, H-8); 2.03(m, n-9); 5.10(br t, J=7.2Hz); 1.63"(s, H12); 1.17*(s, H-13); 1.10*(s, H-14); 1.55"(s, H-15); and 2.88ppm (br s, OH). Assignments may be reversed.

~SC NMR: (Acetone-d6) 15.2(C-1); 44.8(C-2); 80.5(C-3); 41.6(C-4); 24.9(C-5); 55.1(C-6); 74.0(C-7), 41.4(H-8); 23.3(C-9), 125.9(C-10), 131.0(C-11), 25.7(C-12), 25.2(C-13), 26.4(C-14); and 17.5ppm (C-15). TLC Data Silica gel 60 thin-layer plates were developed with toluene-ethyl acetate-formic acid (5:4:1, v/v/v), Rf = 0.59-0.64; detected as a bright pink spot when sprayed with acidified anisaldehyde followed by heating. References H. G. Cutler, J. M. Jacyno, R. S. Phillips, R. L. VonTersch, P. D. Cole, and N. Montemurro; Cyclonerodiol from a Novel Source, Trichoderma koningii: Plant Growth Regulatory Activity; Agile. Biol. Chem., Vol. 55, pp. 243-244(1991). S. Nozoe, M. Goi, and N. Morisaki; Structure of Cyclonerodiol; Tet. Lett., pp. 12931296(1970).

784

30.

Miscellaneous Metabolites

Common/Systematic Name Cyperine _Molecular Formula/Molecular Weight C15H1604; MW' = 2 6 0 . 1 0 4 8 6

OH

OH Fungal Source

Ascochyta cypericola, a new pathogen of purple nut sedge isolated in India near the center of the origin of the weed. Purple nut sedge (Cyperus rotundus L.) is one of the world's most notorious and ubiquitous weeds, reported throughout Africa, India, southern Asia, Australia, South and Central America, and southern United States.

Isolation/Purification Three-week-old liquid cultures ofAscochyta cypericola were extracted with three equal volumes of ethyl acetate. This crude extract, which induced lesion formation on purple nut sedge, was purified by repeated bioassay-guided size exclusion chromatography (Sephadex LH-20). It yielded the extremely phytotoxic metabolite cyperine. Biolo~cal Activity Cyperine is an extremely active phytotoxin with modest host-selectivity within the genus Cyperus. Cyperus rotundus, the source ofA. cypericola, exhibited the greatest sensitivity to cyperine among the species of Cyperus tested. The fungus was isolated from surfacesterilized diseased leaves of C. rotundus. Soectral Data IR~

A strong, broad IR absorption at 3400cm q suggested a phenolic moiety, and the absence of signals between 2000 and 1620cmq argued against carbonyl functionality. ~H NMR: The five ~H NMR signals between 6.0 and 6.4ppm indicated the presence of five aromatic methines. The signals at 6.35 and 6.29ppm were meta-coupled to each other (J=2.7Hz), as were the three higher field signals(6.22, 6.12, and 6.02, J=2.1Hz). Methyl singlets at 2.03 and 2.18 indicated the presence of two aromatic methyl groups; a methyl singlet at 3.72ppm indicated a methoxyl substituent. There was benzylic coupling between the methyl singlet at 2.18 and the aromatic protons at 6.12 and

30.

Miscellaneous Metabolites

785

6.22ppm, and between the methyl singlet at 2.03 and the aromatic protons at 6.29 and 6.34ppm. Irradiation at 3.72ppm resulted in enhancement of the two aromatic protons at 6.34 and 6.29 (3.5 and 4.2%, respectively), which had been assigned to the tetrasubstituted benzene ring. Irradiation of the methyl singlet at 2.10 resulted in enhancement (4.1%) of the aromatic proton at 6.34ppm. Mass Spectrum: HREIMS established the molecular formula of C15H1604,indicating a compound with eight sites ofunsaturation. Reference A. Steerle, R. Upadhyay and G. Strobel; Cyperine, A Phytotoxin Produced by Ascochyta cypericola, A Fungal Pathogen of Cyperusrotundus;Phytochemistry, Vol. 30, pp. 21912192(1991).

786

30.

Miscellaneous Metabolites

Common/Systematic Name Erinapyrone A (2S)-2,3-Dihydro-6-hydroxymethyl-2-methyl-4H-pyran-4-one Molecular Formula/Molecular Weight C7H1003; MXV = 1 4 2 . 0 6 2 9 9

O

6

HOH2

2 1

Me

General Characteristics A colorless oil. Fungal Source Hericium erinaceum, an edible mushroom.

Isolation/Purification Culture filtrate from H. erinaceum was fractionated by solvent partition between water and ethyl acetate. Repeated silica gel column chromatography followed by HPLC with an ODS column gave purified erinapyrones A and B as colorless oils. Biological Activity Erinapyrone A exhibited cytotoxicity against HeLa cells; LD100was 0.88mM. Soectral Data 1608, 1655, and 3392cm1. CD: Ae - 1.14 at 316nm (EtOH). 1H NMR: (CDC13) 1.48(d, J=6.23Hz, H-methyl); 2.36(dd, J=16.85, 4.76, H-3eq); 2.40(dd, J=16.85, 12.07, H3ax); 4.18(d, J=15.93, H-6CH2); 4.23(d, J=15.93Hz, H-6CH2); 4.57(ddq, J-12.07, 4.76, 6.23, H-2); and 5.61ppm (s, H-5); Monoacetate: 1.40(d, J--6.59Hz, 2-methyl); 2.07(s, acetate); 2.37(dd, J=16.85, 4.13Hz, H-3eq); 2.42(dd, J=16.85, 13.92Hz, H-3ax); and 4.51ppm (ddq, J=13.92, 4.13, 6.59Hz, H-2);

13C NMR: (CDCI3) 20.4(C-2 methyl);43.1 (C-3); 61.9 (C-2); 76.1 (C-6-CH2); 102.4 (C-5); 174.9 (C-6);and 192.8ppm (C-4).

30.

Miscellaneous Metabolites

Mass Spectrum: HREIMS: 142.0615m/e for C7H1003. Reference H. Kawagishi, M. R. Shirai, H. Sakamoto, S. Yoshida, F. Ojima, and Y. Ishiguro, Erinapyrones A and B from the Cultured Mycelia of Hericium erinaceum; Chemistry Letters, (Japan)pp. 2475-2476(1992).

787

788

30.

Miscellaneous Metabolites

Common/Systematic Name Erinapyrone B (2R)-2,3-Dihydro-2-hydroxymethyl-6-methyl-4H-pyran-4-one Molecular Formula/Molecular Weight C7H[003; MW = 142.06299 O

6

Me

2 1

' CH20H

General Characteristics A colorless oil. Fungal Source Hericium erinaceum, an

edible mushroom.

Isolation/Purification Culture filtrate from H. erinaceum was fractionated by solvent partition between water and ethyl acetate. Repeated silica gel column chromatography followed by HPLC with an ODS column gave purified erinapyrones Aand B a s c o ~ ~ ~ f Biological Activity Erinapyrone B exhibited cytotoxicity against HeLa cells; LD~00was 1.76mM. Soectral Data _ IR:

1606, 1655, and 3394cmq. CD: Ae -0.56 at 315nm (EtOH). 1H NMR: (CDCI3) 2.01(s, H-6-methyl); 2.30(dd, J=16.85, 3.66, H-3eq); 2.61(dd, J-16.85, 14.29, H-3ax); 3.76(dd, J=12.46, 5.50, H-2-CH2); 3.87(dd, J=12.46, 3.30Hz, H2CH2); 4.46(dddd, J=14.29, 3.66, 3.30, 5.50Hz, H-2); and 5.32ppm (s, H-5); 13C NMPx:

(CDCI3) 20.9(C-6 methyl), 36.7(C-3), 63.7(C-2-CH2), 79.5(C-2), 104.9(C-5), 174.2(C-6), and 192.6ppm (C-4).

30.

Miscellaneous Metabolites

Mass Spectrum: HREIMS: 142.0659m/e for C7H1003. Reference H. Kawagishi, M. R. Shirai, H. Sakamoto, S. Yoslfida, F. Ojima, and Y. Ishiguro; Erinapyrones A and B from the Cultured Mycelia ofHericium erinaceum; Chemistry Letters, (Japan) pp. 2475-2476(1992).

789

790

30.

Miscellaneous Metabolites

Common/Systematic Name Erinapyrone C Molecular Formula/Molecular Weight C8Hlo05; M W = 186.05282 9

Me

0

HO OH

6 1

7

General Characteristics Erinapyrone C was isolated as a solid; mp., 72~

[a]D 25 + 141 o (c=l.0, in MeOH).

Fungal Source Hericium erinaceum (strain CBS 233.87).

Isolation/Purification Fungal cultures were extracted with EtOAc containing 1% MeOH and the extracts were evaporated to give a mixture of crude metabolites. The crude mixture was chromatographed on a silica gel column using hexane-EtOAc (4:1, v/v) to yield hericenes A-C, fatty acids (palmitic, linoleic, and oleic acids); the residual material was then eluted with EtOAc containing MeOH (1%) to give erinapyrone C. Spectral Data UV:

,~ EtorI

280(e = 9,700)

1H NMR: (CDCI3 - DMSO-d~) H-3, 5.72(t, ,/=1.2 Hz); H-6, 5.74(s); H-7a, 4.16(ddd, ,/=16.7, 6.1, and 1.2Hz); H-To, 4.07(ddd, ,/=16.7, 6.1 and 1.2Hz); H-8, 4.41(dq, ,/=6.1 and 6.4Hz); H-9, 1.28(q, J=6.4Hz); 7-OR 5.400, J=6.1Hz); and 8-O1-I, 4.67ppm (d, J=6.1Hz). 13C NMR:

(CDCI3 - DMSO-d6) C-2, 170.61(s); C-3, 101.60(d); C-4, 189.47(s); C-5, 64.90(s); C6, 80.98(d); C-7, 59.980); C-8, 60.68(0; and C-9, 18.17ppm (q).

30.

Miscellaneous Metabolites

791

Mass Data: ELMS: 186(M+), 168, and 155re~e; anal. found: C, 51.6; H, 5.4: calcd for C8I-IloO5 C, 51.3; H, 5.7. Reference A. Arnone, R. Cardillo, G. Nasini, and O. Vajna De Pava; Secondary Mold Metabolites: Part 46. Hericenes A-C and Erinapyrone C, New Metabolites Produced by The Fungus Hericium erinaceum; J. Nat. Prod., Vol. 57, pp. 602-606(1994).

792

30. Miscellaneous Metabolites

Common/Systematic Name Asperfl avin (+)-3,4-Dihydro-3,6,9-trihydroxy- 8-methoxy-3-methylanthracen- 1(2H)-one Molecular Formula/Molecular Weight

HO' O;

C16H1605; ~

= 288.09977

OMe

OH

0

General Characteristics Fractional crystallization from ethyl acetate gave citrine prisms; mp., 225-230~ resetting to needles which decomposed at ca. 280~ without melting; [a]Dz~+ 4 ~ (C=0.3, in MeOH). It was insoluble in sodium hydrogen carbonate, but dilute solutions in sodium carbonate had an intense yellowish green fluorescence. In 2N sodium hydroxide it gave a yellow solution which rapidly became orange. Dilute solutions in organic solvents showed a green fluorescence. It gave an intense dark green color with iron chloride in ethanol. Sublimation at 160~ and 10-1 mmHg gave a solid which crystallized from ethyl acetate in needles (decomp. 290~ without melting). Asperflavin was unstable in light and air. Solutions in organic solvents slowly became dark green, while thin layers of silica impregnated with asperflavin rapidly changed color from citrine to green. Fungal Source An entomogenous strain of Aspergillus flavus (probably A. nomius), A. repens, and Leucopaxi llus tricolor. Isolation/Purification Extracted with chloroform and purified by fractional crystallization from ethyl acetate solution atter separation of 5'-hydroxyasperentin. Spectral Data UV:

/~

MeOH

230, 269, 317, 335, and 392nm (log e =4.13, 4.51, 3.64, 3.40, and 3.91).

IR:

(Nujol) 3490, 3350, 1632, and 1582cmq. ]H NMR: (CDsOD) 3.25(10-H); 3.50, 3.57(5-, 7-H); 6.08(OMe); 7.00, 7.22(2-, 4-1-12); 8.64(3-Me); and 3.821: (OH). [(CDs)2CO] 3.22(10-H); 3.38, 3.50(5-, 7-H); 6.10(OMe); 6.94, 7.18(2-, 4-H/); 8.62(3-Me);-5.0, 3.721: (OH).

30.

Miscellaneous Metabolites

793

Mass Data: LREIMS: 288m/e; an abundant resonance-stabilized rearrangement ion at 230m/e (M +

- C3H60); anal. found C, 66.4; H, 5.75; C16H1605requires C, 66.7; H, 5.6. Reference J. F. Grove; New Metabolic Products of Aspergillusflavus. Part II. Asperflavin, Anhydroasperflavin, and 5,7-Dihydroxy-4-methylphthalide; J. C. S. Perkin I, pp. 24062411(1972).

794

30.

Miscellaneous Metabolites

Common/Systematic Name Diplodiol; Diplosporin trans- 6-Ethyl- 5-hydroxy-3 -hydroxymethyl- 5,6,7, 8-tetrahydrochromone; (5 S, 6X)-6-Ethyl5-hydroxy-3-hydroxymethyl- 5,6,7,8-tetrahydrobenzo[ b ]pyran-4-one Molecular Formula/Molecular Weight C12H1604; M W - 2 2 4 . 1 0 4 8 6

OH O / H 11 MeH2C~~~/cH2OH

13

General Characteristics Fine white crystals from benzene-acetone; mp., 47-48~ Colorless crystals from benzenehexane; mp., 83-84~ [tt]D2~ + 49.7 ~ (C=I.01, in MeOH). Fungal Source Diplodia macrospora (ATTC 36896), a fungal pathogen of corn, causing ear- and stalkrot in tropical and subtropical areas of the world and under humid conditions a severe leaf spot. Isolation/Purification Fungal cultures were extracted with acetone, reduced to an aqueous phase, and partitioned between ethyl acetate. The ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, reduced in volume, and chromatographed on a silica gel column eluted with a series of solvents; benzene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further fractionated on a silica gel column eluted with a linear gradient from benzene to acetone. The diplodiol containing fractions were combined, reduced in volume, and diplodiol was crystallized as fine white needles. Biological Activity Toxic to day-old chicks dosed orally, LDs0 88mg/kg; not biologically active in the Avena coleoptile assay. Spectral Data UV:

/~

EtOH

217(log e = 3.90) and 251nm (3.90); Z~x 214(log e = 4.04), 252(4.08), and 256nm (4.07).

IR~

(KBr) 3400(broad, OH), 2960(methyl), 2930(methylene), 2870(methylene), 1653(strong, y-pyrone), 1585(y-pyrone), 1450, 1315, 1235, 1180 1125, and 1020cm 1.

30.

Miscellaneous Metabolites

795

~H NMR: (CDCI3) 7.79(H-2); 4.59(H-5); 1.40(H-6); 2.05, 1.75(H-7); 2.60(H-8); 4.46(H-I I); 1.67(H-12); and 0.98ppm (H-13). ~3CNMR: (CDCI3) 152.05(C-2); 123.43(C-3); 180.29(C-4); 68.48(C-5); 41.22(C-6); 24.17(C-7); 22.96(C-8); 165.91(C-9); 126.80(C-10); 57.73(C-11); 26.73(C-12); and 11.28ppm (C13) (Cutler et al); 152.0 (C-2); 127.0 (C-3); 179.9 (C-4);68.2 (C-5); 41.1 (C-6); 22.8 (C-7); 26.3 (C-8); 165.6 (C-9); 123.3 (C-10); 23.9 (C-11), 10.8 (C-12); and 57.2ppm (C- 13) (Chalmers et al). Mass Spectrum: HREIMS: 224.1051 (M*, calculated mass for C12Hi604,224.1048), 208(M+ - O), 206(M+- 1-120), 193(M+- CH2OH), and 188role (M ~- 21420). TLC Data Rf of diplodiol was 0.33-0.36 on silica gel-60 TLC plates using toluene-ethyl acetateformic acid (5:4:1, v/v/v) as developing solvent. References A. A. Chalmers, C. P. Gorst-Allman, P. S. Steyn, R. Vleggaar, W. F. O. Marasas, and N. P. J. Kriek; The Structure ofDiplosporin; S. Aft. J. Chem., Vol. 31, p. 111(1978). H. G. Cutler, F. G. Crumley, R. H. Cox, R. J. Cole, J. W. Dorner, F. M. Latterell, and A. E. Rossi; Diplodiol: A New Toxin from Diplodia macrospora; Agric. Food Chem., Vol. 28, pp. 135-138(1980).

796

30. Miscellaneous Metabolites

Common/Systematic Name 5-Deoxydiplosporin Molecular Formula/Molecular Weight C 12H1603; MW = 208.10994 la

HOH2C

0

11~

General Characteristics Isolated as an oil; [tZ]D2~ + 55 ~ (C=I.1, in MeOH). Fungal Source Diplodia macrospora (ATTC 36896), a fungal pathogen of com, causing ear and stalk rot and under humid conditions a severe leaf spot. Isolation/Purification Fungal culture filtrates were extracted with chloroform, dried over anhydrous sodium sulfate, filtered, reduced in volume, and chromatographed on a silica gel column under pressure and eluted with chloroform-acetone-hexane (7:2:1, v/v/v). 5-Deoxydiplosporin was obtained as an oil from the first fractions to elute from the column. Spectral Data UV:

/~ EtOH

219(1og c = 3.72) and 258nm (3.73).

IR:

(KBr) 3400, 1660, 1610, 1590, and 1450cm1. 1H NMR: (CDCI3) 7.79(H-2), 4.59(H-5); 1.40H-6); 2.05, 1.75(H-7); 2.60(H-8); 4.46(H-11); 1.67(H-12); and 0.98ppm (H-13). 13C NMR:

151.30(d, C-2), 125.8(s, C-3), 179.3(s, C-4), 27.4*(t, C-5), 34.6(d, C-6), 26.6~ C-7), 27.4(t, C-8), 164.4(s, C-9), 122.1(s, C-10); 28.4(t, C-11), 11.8(q, C-12), and 58.4ppm (t, C-13). (CDCI3)

Assignments may be reversed.

30.

Miscellaneous Metabolites

Mass Spectrum: HREIMS: 208.1104m/e (M+, calculated mass for C12H1603, 208.1099). Reference A. A. Chalmers, C. P. Gorst-Allman, P. S. Steyn, and R. Vleggaar; Biosynthesis of Diplosporin by Diplodia macrospora. Ring Formation Involving Methionine-derived Carbon Atoms; J. Chem. Soc. (Perkin Trans I), pp. 1481-1484(1979).

797

798

30.

Miscellaneous Metabolites

C_omrnon/Systematic Name Betulin (Lup-20(29)-ene-313,28-diol) Molecular Formula/Molecular Weight C30H5002; MW' = 442.38108

H2OH

HO General Characteristics Needles from methanol; mp., 250-251 ~ derivative; mp., 216-217~

[a]D 20 +

16 ~ (c=0.3, in EtOH); diacetate

Fungal Source The wood-rotting fungus, Polyporuspmicola. Betulin is an important constituent of the bark of birch trees and it has been suggested that the compound may be translocated from the bark directly into the fungus (in this case Fomesfomentarius). Although Polyporus pmicola is usually found on pines, betulin may arise in a similar manner or from small pieces of bark adhering to the fungus. S_pectral Data IR.~

(Nujol) 3279, 1658, 1265, and 915cm"~. 1H NMR: (CDCI3) 0.78(3H, s, 24-CH3); 0.85(3H, s, 25-CH3); 0.99(6H, s, 23-, 27-CH3); 1.04(3H, s, 26-CH3); 1.40(2H, s, D20 exchangeable); 1.70(3H, s, 29-CH3); 3.25(1H, m, 3-H); 3.43; 3.75(2H, 28-CH2); and 4.63, 4.73ppm (2H, 30-CH3). Mass Data: LREIMS: 442(M +, 91%), 411(82), 424(14), 234(44), 220(30), 203(62), 205(10), 207(85), and 189m/e (94.5). Diacetate, found: C, 81.0; H, 11.35%; calcd for C3oH5oO2: C, 81.4; H, 11.4%; found: C, 77.4; H, 10.3%; calcd for C34H~404 C, 77.5; H, 10.3%.

30.

Miscellaneous Metabolites

799

References R. C. Cambie; Chemistry of Fungi, 12. Betulin from Polyporuspinicola Fr.; New Zealand J. Science, Vol. 21, pp. 565-567(1978). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, p. 330 (1983).

This Page Intentionally Left Blank

Secondary Metabolite Index Secondary metabolite 22~-Acetoxy-3[~,23~dihydroxy-24(28)Zethylidenelanost-8ene 22~-Acetoxy-313,23~dihydroxy-24methylenelanost8-ene 121~-Acetoxy-4,4dimethyl-24methylene-5o~cholesta-8,14-diene3[~,lla-diol 313-Acetoxy-160thydroxylanosta-8, 24(28)-dien-21-oic acid 30t-Acetoxy-150thydroxy-5a-lanosta7,9(ll),24E-trien26-oic acid (22S, 24E)-22Acetoxy-3othydroxy-5alanosta-7,9(ll),24trien-26-oic acid 22-Acetoxy-3othydroxy-7amethoxy-5alanosta-8,24E-dien26-oic acid 3a-Acetoxy-7~methoxy-5~lanosta-8,24E-dien26-oic acid 313-Acetoxy-24methylene-14~-azao-homo-5o~cholesta-8(9), 14(14a)-diene ll-Acetyldehydrocurvularin

Page

240

241

220

225

312

332

320

318

441 659

Secondary metabolite C-3'-N-Acetylfusarochromanone 31~-Acetyl-2-(3'hydroxy-3-methyl) glutarylanhydrocrustulinol 3l~-Acetyl-2~-(3'hydroxy-3'methyl)glutarylcrustulinol 3-O-Acetyltumulosic acid 6-3-O-(4-O-Acetyl)[~-o-xylopyranoside16-O-(4,6-di-Oacetyl)-13-oglucopyranoside Acrostalic acid Acrostalidic acid Alnusenone Anhydroasperflavin 9,11-Anhydro-9a,1 lotepoxyfusidic acid 9,11-Anhydofusidic acid 9,11-Anhydro-12hydroxyfusidic acid Anhydrosepedonin Antheridiol Antibiotic A25822A Antibiotic A25822B Antibiotic A25822D Antibiotic A25822H Antibiotic A25822L Antibiotic A25822M Antibiotic A25822N Antibiotic A26771B Antibiotic LL-Z1272a Antibiotic LL-Z12721~ Antibiotic LL-Z1272~5 Antibiotic LL-Z1272e

Page 615

585

573 227

559 764 762 169 223 483 479

481 745 251 433 435 437 445 439 441 443 775 507 509 511 513

Secondary metabolite

Page

Antibiotic LL-Z1272~ Aphidicolane-3c~,l 6, 17,18-tetraol Aphidicolin Aphidicolin-17monoacetate Aphidicolin-3,18orthoacetate Arcyriaflavin B Arcyriaflavin C Arcyriarubin B Arcyriarubin C Arcyroxepin A Arugosin A Arugosin B Arugosin C Arugosin D Ascladiol Ascochlorin Ascofuranol Ascofuranone Asperflavin Aszonapyrone A

515

Betulin Bostrycin Brassicasterol

164 798 29

Campestanol Campesterol Carbomethoxyacetylquercinic acid 3a-Carboxyacetoxy24~-methyl-23oxolanosta-8,25 (26)-dien-27-oic acid Carboxyacetylquercinic acid Carboxyacetylstowardolic acid

31 27

621 621 625 627 679 680 681 683 677 687 689 691 693 744 517 521 519 792 740

229

235 230 235

801

802

Secondary Metabolite Index

Secondary metabolite

Page

Secondary metabolite

Page

Secondary metabolite

Cephalosporin P1 Cerevisterol 6-Chlorodehydrocurvularin E-6-Chloro-10,11dehydrocurvularin Chloronectrin Cholesta-5-en-313-ol Cholesta-7-en-313-ol Cholesta-8(9)-en313-ol Cholesta-5,7-dien313-ol Cholesta-5,22-dien313-ol Cholesta-5,24-dien313-ol Cholesta-7,24-dien313-ol Cholesta-8,24-dien313-ol Cholesta-5,7,24-trien313-ol Cholesta-5,8,24-trien315-ol Cholesta-7,22,24(28)trien-3~-ol Cholesta-8,22,24-trien313-ol Cholesta-5,7,22,24tetraen-3[~-ol Cholesta-5,8,22,24tetraen-313-ol Cholesterin Cholesterol Cinchol Citreofuran Clavicin Claviformin Clavitin Clionasterol Compound B8 Compound B9 Cupreol Curvularin Cyathic acid Cyclonerodiol Cynodontin Cyperine

469 84

Deacetoxyechinodol Deacetoxyechinodone Deacetoxy-3epiechinodol Deacetylchloronectrin Deacetylechinodone Deacetyl-3epiechinodol ),-Decalactone 5-Decanolide 7-Dehydrocholesterol 22-Dehydrocholesterol 24-Dehydrocholesterol 0t,13-Dehydrocurvularin

155 156

3t~,22-Diacetoxy-15cthydroxy-7txmethoxy-5ctlanosta-8,24(E)dien-26-oic acid (22S, 24E)-3o~,22Diacetoxy-5t~lanosta-7,9(11),24trien-26-oic acid 3t~,15o~-Diacetoxy-5t~lanosta-7,9(ll),24Etrien-26-oic acid 30t,22-Diacetoxy-7txmethoxy-5ctlanosta-8,24E-dien26-oic acid Diacetylfusarochromanone 313,21-Diacetyl-2-(3'hydroxy-3'-methyl) glutarylcrustulinol 16-3-O-(3,4-Di-Oacetyl)-13-oxylopyranoside16-O-(4,6-di-Oacetyl)-13-Dglucopyranoside 16-3-O-(3,4-Di-Oacetyl)-13-Dxylopyranoside-16O-(6-O-acetyl)-13-oglucopyranoside 5,6-Dihydroergosterol 22,23-Dihydroergosterol 0~-Dihydrofucosterol (2R)-2,3-Dihydro-2hydroxymethyl-6methyl-4H-pyran-4one (2S)-2,3-Dihydro-6hydroxymethyl-2methyl-4H-pyran-4one 24-Dihydrolanosterol Dihydropenicillic acid 22-Dihydroporiferasterol Dihydroprehelminthosporol

657 657 523 3 7 9 10 8

cis-Dehydrocurvularin trans-

5

Dehydrocurvularin 10,11Dehydrocurvularin Dehydroeburicoic acid Dehydroeburicoic acid ketone 7-Dehydrofucosterol 24(28)Dehydrooogoniol 24(28)Dehydrooogoniol-1 24(28)Dehydrooogoniol-2 24(28)Dehydrooogoniol-3 7,8-Dehydropseudofusidic acid Dehydrotumulosic acid 14-Demethyllanosterol 23-Deoxyantheridiol 3-Deoxyaphidicolin 5-Deoxydiplosporin Deoxypatulinic acid Desmosterol 3c~,22-Diacetoxy-7a, 15a-dihydroxy-50~lanosta-8,24E-dien26-oic acid 3ot,7a-Diacetoxy-15o~hydroxy-5o~lanosta-8,24E-dien26-oic acid

12 14 16 18 19 20 21 23 3 3 95 672 758 758 758 97 298 300 95 655 166 782 781 784

154 525 151 153 724 725 10 8 5 662 664 662 662 196 175 103 264 265 267 269 485 197 116 253 623 796 769 5

316

302

Page

314

330

310

308 613

583

569

571 47 56 95

788

786 131 771 97 535

Secondary Metabolite Index

Secondary metabolite 2,3-Dihydro-4-pyrone5-acetic acid Dihydro-l]-sitosterol 22,23Dihydrostigmasterol (+)-3,4-Dihydro-3,6,9trihydroxy-8methoxy-3methylanthracen-l(2H)-one 3,5-Dihydroxydecanoic acid 8-1actone 313,15~-dihydroxy-8,24dien-21-oic acid 6t~,22-Dihydroxy-4ct, 14t~-dimethylcholesta8,24-dien-3-one 15ot,23-Dihydroxy-3,11dioxo-5ot-lanosta-8, 24E-dien-26-oic acid 4,8-Dihydroxy-2-(1hydroxyheptyl)3,4,5,6,7,8hexahydro-2H-1benzopyran-5-one

Page 769 113 95

792 737 144

123

387

649

(22R)3~,25Dihydroxylanosta -8,23-dien-22-acetate 218 313,5ot-Dihydroxylanosta8,24-dien-21,26dioic acid 140 24(S),25-Dihydroxy5a-lanosta-7,9(ll)dien-3-one 370 3[~,161~-Dihydroxy-713methoxycucurbita5,24-dien-3-O-l]-Dxylopyranoside 551 3~,15-Dihydroxy-24methylene-14a-azao-homo-5ct-cholesta8(9),14(1400-diene 437 2,4-Dihydroxy-3substituted-5chloroorcylaldehyde 517 713,121~-Dihydroxy3,11,15,23-tetraoxo5a-lanost-8-en26-oic acid 281,324

803

Secondary metabolite 31~,613-Dihydroxy-4,4, 14o~-trimethyl-5o~pregn-8-en-20-one 713,12t~-Dihydroxy-3, ll,15-trioxo-5ctcholesta-8-en-24oic acid 7ct,15ot-Dihydro~y-3, 11,23-trioxo-50~lanost-8-en-26-oic acid 713,15ct-Dihydroxy-3, ll,23-trioxo-50~lanost-8-en-26-oic acid 313,15ct-Dihydroxy-7, 11,23-trioxo-5ctlanost-8-en-26-oic acid 4ct,14o~Dimethylcholesta8,24-dien-3,22dione 4ct,14aDimethylcholesta8,24-dien-31]-ol 4,4-Dimethylcholesta8,14,24-trien-313-ol 5,5-Dimethyl-9-(2hydroxyethyl)-8hydroxymethyl-1methyl-bicyclo [4,2,1]non-7-ene 4,4-Dimethyl-31]hydroxy-24methylene-14ct-azaD-homo-5ot-cholesta8(9),14(14t~)-diene 4,4Dimethylzymosterol 4ct,14ctDimethylzymosterol ll,15-Dioxo-313,713dihydroxy-5ot-lanost8-en-24-oic acid 3,11-Dioxo-413,7~,15ottrihydroxy-5ctlanost-8-en-24-oic acid

Page

Secondary metabolite

Page

Diplodiol Diplosporin

794 794

Eburical Eburicodiol Eburicoic acid Eburicol Echinodol Echinodone Emericellin 3-Epiechinodol 3-Epifusidic acid ll-Epifusidic acid Episterol Ergost-5-en-3~-ol Ergost-7-en-313-ol Ergost-8-en-3[3-ol Ergosta-8,24(28)-dien313,6t~-diol Ergosta-5,7-dien313-ol Ergosta-5,8-dien-3~-ol Ergosta-7,22-dien313-ol Ergosta-7,24(28)-dien313-ol Ergosta-7,22-dien-3one Ergosta-7,22-dien-313, 5t~-diol Ergosta-8(9),14-dien313-ol Ergosta-8(9),22-dien31]-ol Ergosta-8,24( 28)-dien313-ol Ergosta-7,22-dien-5ct, 613,713-triol Ergostanol Ergosta-5,7,9(ll),22tetraen-31]-ol Ergosta-5,7,22,24(28)tetraen-313-ol Ergosta-5,8,22,24( 28)tetraen-31]-ol Ergosta-4,6,8(14),22tetraen-3-one Ergosta-4,7,22-trien-3one

179 178 186 198 213 216 719 214 475 477 49 53 40 33

244

403

298

273

294

124

114 119

780

433 116 114

411

409

74 45, 56 34 47 49 82 73 60 62 38 84 32 68 80 70 58 72

804

Secondary metabolite

Secondary Metabolite Index

Page

Ergosta-5,7,22-trien313-ol 43 Ergosta-5,7,24(28)trien-313-ol 51 Ergosta-5,8,22-trien315-ol 54 Ergosta-7,22,24-trien313-ol 64 Ergosta-8,14,22-trien3[~-ol 52 Ergosta-8,14,24-trien31]-ol 66 Ergosta-8,22,24(28)trien-313-ol 67 Ergosterol 43 Ergosterone 72 Erinapyrone A 786 Erinapyrone B 788 Erinapyrone C 790 (24S)24Ethylcholestan31~-ol 113 (24R)24-Ethylcholest5-en-313-ol 97 (24S)24-Ethylcholest5-en-313-ol 95 (24S)24-Ethylcholesta7-en-313-ol 109 (24R)24Ethylcholesta-22en-313-ol 105 (24S)24Ethylcholesta5,22-dien-313-ol 111, 112 (24S)24Ethylcholesta5,7-dien-313-ol 109 (24R)24-Ethylcholesta5,22-dien-313-ol 99 (24R)24-Ethylcholesta5,24(28)-dien-3[~-ol 101 24-Ethylcholesta-22enol 105 (24R)24-Ethylcholesta4,6,8(14),22tetraen-3-one 93 (24R)24-Ethylcholesta5,7,24(28)-trien313-ol 103

Secondary metabolite (5S,6X)-6-Ethyl-5hydroxy-3hydroxymethyl5,6,7,8tetrahydrobenzo [b]pyran-4-one

trans-6-Ethyl-5-

Page

794

hydroxy-3hydroxymethyl5,6,7,8tetrahydrochromone 794 E-24-Ethylidene-5c~lanost-8-en-313 acetate 238 Expansion 758 Fasciculol A Fasciculol B Fasciculol B depsipeptide Fasciculol C Fasciculol C depsipeptide E Fasciculol C depsipeptide F Fecosterol Formylfusarochromanone Friedelin Friedoolean-5-en-3one Fucostanol Fucosterol Fumigacin Fungisterol Fusarochromanone Fusidic acid Fusilactidic acid

169 113 101 491 40 611 489 487

Ganoderic Ganoderic Ganoderic Ganoderic Ganoderic Ganoderic Ganoderic Ganoderic Ganoderic Ganoderic

273 275 277 279 281 283 284 286 288 290

acid acid acid acid acid acid acid acid acid acid

A B C1 C2 D E F G H I

449 451 459 453 455 457 38 617 168

Secondary metabolite

Page

Ganoderic acid J Ganoderic acid K Ganoderic acid L Ganoderic acid Ma Ganoderic acid Mb Ganoderic acid Mc Ganoderic acid Md Ganoderic acid Me Ganoderic acid Mf Ganoderic acid Mg Ganoderic acid Mh Ganoderic acid Mi Ganoderic acid Mj Ganoderic acid Mk Ganoderic acid M Ganoderic acid N Ganoderic acid O Ganoderic acid R Ganoderic acid S Ganoderic acid T Ganoderic acid U Ganoderic acid V Ganoderic acid W Ganoderic acid X Ganoderic acid Y Ganoderic acid Z Ganoderiol A Ganoderiol B Ganoderiol C Ganoderiol D Ganoderiol E Ganoderiol F Ganoderiol G Ganoderiol H Ganoderiol I Ganodermadiol Ganodermanondiol Ganodermanontriol Ganodermatriol Ganodermenonol Ganodermic Acid R Ganodermic Acid S Ganolucidic acid A Ganolucidic acid B Ganolucidic acid C Ganolucidic acid D Ganolucidic acid E Glochidone Glochidonol

292 294 296 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330 332 334 336 338 340 342 344 345 353 355 356 358 360 362 364 366 368 376 370 372 378 374 347 349 381 383 385 387 389 161 162

Secondary Metabolite Index

805

Secondary metabolite

Page

Secondary metabolite

Grifolin Grifolin-l-acetoxy-3methyl ether Grifolin dimethyl ether Grifolin monomethyl ether

598

3a-Hydroxy-513cholest-11-en-24oic acid llaHydroxycurvularin

Hebelomic acid A Hebelomic acid B Hebelomic acid E Hebelomic acid F Hebevinoside I Hebevinoside II Hebevinoside III Hebevinoside IV Hebevinoside V Hebevinoside VI Hebevinoside VII Hebevinoside VIII Hebevinoside IX Hebevinoside X Hebevinoside XI Hebevinoside XII Hebevinoside XIII Hebevinoside XIV Helminthosporal Helminthosporal acid Helminthosporic acid Helminthosporol Helvolic acid Hericene A Hericene B Hericene C Hericenone A Hericenone B Hericenone E Hericenone F Hericenone G 2a,3~,12a,21,24(R),25Hexahydroxylanost8-ene HS-B HS-C 121~-Hydroxycarbomethoxyacetylquercinic acid 121~-Hydroxycarboxyacetylquercinic acid

573 575 578 58O 545 547 549 551 553 555 557 559 561 563 565 567 569 571 541 537 539 529 491 699 701 703 705 707 709 711 713

606 602 600

453 583 585

233 231

Page

247 666

Hydroxycurvularin 15a-Hydroxy-3,11dioxo-5a-lanosta8,24E-dien-26-oic acid 4-Hydroxy-4H-furo

660

one 4'-Hydroxy-5hydroascochlorin Hydroxyisoergosterol (22R)25Hydroxylanosta8,23-dien-3a-ol-22monoacetate 3~-Hydroxylanosta-8, 24-dien-21-oic acid 31~-Hydroxy-5alanosta-7,9(ll),24trien-26-oic acid 313-Hydroxylanosta7,9(ll),24-trien-21oic acid 26-Hydroxy-5alanosta-7,9(ll), 24-trien-3-one 5-Hydroxy-3methoxy-6-oxo2-decanoic acid ~-lactone 31~-Hydroxy-24methylene-14aaza-o-homo-5acholesta-8(9), 14(14a)-diene 3a-Hydroxy-24{methyl-23oxolanosta-8,25dienoic acid 6-(1-Hydroxypentyl)-4methoxypyran-2one

758

389

[3,2-c]pyran-2(6H)525 76

Secondary metabolite 6a-Hydroxypolyporenic acid C 313-Hydroxy-7,11,15,23tetraoxo-121~acetoxy-5a-lanost8-en-26-oic acid 15o~-Hydroxytrametenolic acid 3a-Hydroxy-4,4,14trimethyl-5~xergosta-8,24(28)dien-26-oic acid 3a-Hydroxy-4,4,14atrimethyl-5a-pregn8-en-20-one 713-Hydroxy-4,4,14cxtrimethyl-3,11,15,20tetraoxo-5a-pregn8-ene

Page

192

288 144

194

245

427

IFO 6635 Aversion Factor Ignosterol Inotodiol Isoacrostalidic acid Isopatulin

344

(1S,4S,5S,7R)-I-

777 60 149 767 760

138

Isopropyl-4,8dimethyl-spiro [4,5]-dec-8-en-7-ol Isosativenediol Isosativenetriol

738 755 757

Jasmine lactone

726

218

142

374

727

435

182

729

3-Ketodehydrosulfurenic acid 3-Ketofusidic acid ll-Ketofusidic acid 3-Keto-24-methylene14~-aza-o-homo5a-cholesta-8(9), 14(14a)-diene 15-Keto-oogoniol-2 Koninginin A Koninginin B Koninginin C Koninginin D Koninginin E

176 471 473

443 262 643 645 647 649 651

806

Secondary metabolite Lanosta-8,23-diene313,25-diol 5~-Lanosta-7,9(ll)diene-313,24,25,26tetraol (22R)-Lanosta-8,23diene-3~,22,25-triol Lanosta-8,24-diene315-ol Lanosta-8,24-diene313,23-diol Lanosta-8,24(28)diene-313,22-diol Lanost-8-en-313-ol Lanosta-7,9(11 ),24triene-3ot,15adiacetoxy-26-oic acid Lanosta-7,9(11),24triene-313,15t~diacetoxy-26-oic acid Lanosta-7,9(ll),24triene-313,21-diol 5ct-Lanosta-7,9(ll),24triene-3~,26-diol Lanosterol Lathosterol Leucopin Lichesterol LL-P880ct LL-P8801~ LL-P8807 LL-S491~ LL-S491y LL-Z1271t~ LL-Z127113 LL-Z1271y LL-Z1272y (-)-Longifolene Lucidenic acid A Lucidenic acid B Lucidenic acid C Lucidenic acid D1 Lucidenic acid D2 Lucidenic acid E1 Lucidenic acid E2 Lucidenic acid F Lucidenic acid G

Secondary Metabolite Index

Page 132

353 136 129 134 209 131

347

349 121 376 129 7 758 54 731 733 735 751 752 495 497 498 517 772 393 395 397 399 401 403 405 407 409

Secondary metabolite

Page

Secondary metabolite

Lucidenic acid H Lucidenic acid I Lucidenic acid J Lucidenic acid K Lucidenic acid L Lucidenic acid M Lucidone A Lucidone B Lucidone C lup-20(29)-ene-313,28diol

411 413 415 417 419 421 425 427 429

Methoxycurvularin

670

24-Methylcholesta8,24(28)-dien-313-ol 38 (24S)24Methylcholesta5,7,14-trien-3~-ol 42 (24S)24Methylcholesta5,8,22-trien-313-ol 54 24-Methylcholesta5,7,24(28)-trienol 51 (24S)24Methylcholesta7,22,24(28)-trien31~-ol 64 (24S)24Methylcholesta8,14,22-trien-313-ol 52 (24S)24Methylcholesta8,14,24(28)-trien3~1-ol 66 (24R)24Methylcholestan31]-ol 31 (24S)24Methylcholestan31]-ol 32 (24R)Z4Methylcholest-5en-313-ol 27 (24S)24Methylcholest-5en-3~-ol 53 140t-Methylcholest-7en-313-ol 86 (24S)24Methylcholest-7en-3~-ol 40 (24S)24-Methylcholest-8en-313-ol 33

(6S, I'S,2"R)-4-

Methoxy-5,6dihydro-6-(l',2'dihydroxypentyl)2H-pyran-2-one 4-Methoxy-6-(l',2'dihydroxypentenyl)2H-pyran-2-one 3-Methoxy-5-methyl4-oxo-2,5hexadienoic acid 25-O-Methylarugosin A (24S)24Methylcholesta-5,7dien-3~-ol (24S)24-Methylcholesta5,8-dien-313-ol (24R)24Methylcholesta5,22-dien-313-ol 24-Methylcholesta5,24(28)-dien-313-ol (24S)24-Methyl-5~cholesta-7,16-dien313-ol 24-Methylcholesta7,22-dien-313-ol (24S)24-Methylcholesta7,22-dien-3-one (24S)24Methylcholesta8(9),14-dien-3~-ol (24S)24Methylcholesta8,22-dien-3~-ol

164

733

735

778 695

45 34

29 36

57 47 82

60

62

Page

14ct-Methylcholest-8-

en-313-ol 240~-Methylcholesterol 24-Methyl-24,25dihydrolanosterol 24-Methylene cholesterol 24-Methylene-24,25dihydrolanosterol

87 27 181 36 198

Secondary Metabolite Index

Secondary metabolite 24-Methylenelanost-8en-3[~-ol-21-al 24-Methylenelanosterol 24-Methylene-5alanost-8-en31~-ol 14a-Methylergosta-8, 24-dien-3[~-ol 14-Methylfecosterol (22S,24S)-24Methyllanost-8-en22,28-epoxy-31], 28[~-diol (22S,24R)-24Methyllanost-8-en22,28-epoxy-3[~, 28a-diol (22S)24-Methyllanost8-en-22,28-epoxy313-ol-28-one E,E-5-Methyl-(3,7,11trimethyl-2,6,10dodecatrienyl)-lacetoxy-3methoxybenzene E,E-5-Methyl-(3,7,11trimethyl-2,6,10dodecatrienyl)1,3-benzenediol E,E-5-Methyl-4-(3,7, ll-trimethyl-2,6,10dodecatrienyl)-l,3benzenediol E,E-5-Methyl-2-(3,7, 11-trimethyl-2,6,10dodecatrienyl)-l,3dimethoxybenzene E,E-5-Methyl-4-(3,7, 11-trimethyl-2,6,10dodecatrienyl)-l,3dimethoxybenzene E,E-5-Methyl-(3,7,11trimethyl-2,6,10dodecatrienyl)-lhydroxy-3methoxybenzene 4a-Methylzymosterol 14ot-Methylzymosterol Mycoin

Page 179 208

208 117 117

206

204

200

606

598

603

602

605

600 90 88 758

807

Secondary metabolite

Page

Secondary metabolite

Page

Neogrifolin Neogrifolin dimethyl ether Neovasinin Neovasinone Neovasipyridone A Ne0vasipyridone B Neovasipyridone C Norlichexanthone

603 605 631 633 635 637 639 723

Polyporenic acid D Poriferasterol Portensterol Prehelminthosporol Prehelminthosporolactone Provitamin D3 Pyrocalciferol

194 112 76 532 531 10 78

Quebrachol

95

Obliquol Obtusifoliol Officinalic acid Oogoniol Oogoniol-1 Oogonial-2 Oogoniol-3 Oxidosenexone 12-Oxocurvularin 3-Oxopisolactone Oxysporone

149 146 170 255 256 258 260 125 668 202 742

Rhamnol

95

cis-Sativ enediol trans-Sa tivenediol

Pachymic acid Parkeol Patulin Penicidin Penicillic acid 2ot,313,12o~,24(R),25Pentahydroxylanost8-ene 23,24,25,26,27Pentanorlanost-8ene-313,22-diol 3,7,11,15,23-Pentaoxo12-acetoxy-5otlanost-8-en-26-oic acid 3,7,11,12,15-Pentaoxo5c~-lanost-8-en-24oic acid 3,7,11,15,23-Pentaoxo5o~-lanost-8-en-26oic acid Pestalotin c~-Phytosterol Pimara-8(9),15-diene Pinicolic acid A Pisolactone Polyporenic acid C

225 148 758 758 778

753 755 607 124 171 123 747 597 589 591 593 595 113 95 95 95 95 109

451

236

284

399

283 731 95 766 157 200 190

Scutigeral Senexadione Senexdiolic acid Senexonol Sepedonin Siccanin Siccanochromene A Siccanochromene B Siccanochromene C Siccanochromene E [~-Sitostanol Sitosterin Sitosterol I~-Sitosterol Stigmast-5-en-3a-ol Stigmast-7-en-313-ol Stigmasta-5,7-dien31~-ol Stigmasta-5,22-dien313-ol Stigmasta-7 (Z)-24( 28)dien-3[~-ol Stigmastanol Stigmasterol Stowardolic acid Stowardonic acid Tercinin Terretonin 3ot,7~,15ot, 20Tetrahydroxy-ll,23dioxo-5a-lanost-8en-26-oic acid

111, 112 99 107 113 99 182 184 758 242

296

808

Secondary metabolite

Secondary Metabolite Index

Page

2~,313,24(R),25Tetrahydroxylanost8-ene 449 1,4,5,8-Tetrahydroxy-2methylanthraquinone 781 3,7,11,15-Tetraoxo-12acetoxy-5a-cholesta8-en-24-oic acid 401 3,7,11,15-Tetraoxo-12o~hydroxy-5o~-lanost8-en-24-oic acid 417 3,7,11,15-Tetraoxo-5alanost-8-en-24-oic acid 407 Trametenolic acid 142 3a,70t,22-Triacetoxy15ot-hydroxy-5alanosta-8,24E-dien26-oic acid 306 3ot,15ot,22-Triacetoxy7a-hydroxy-5alanosta-8,24E-dien26-oic acid 304 (22S, 24E)-3a,15at,22Triacetoxy-5alanosta-7,9(11),24trien-26-oic acid 334 Trichoacorenol 738 11a,1513,29-Trihydroxy5,24(28)(E)-dien-Yone-31]-isobutyrate 265 313,15~,29-Trihydroxy11,23-dioxo-5at-lanost8-en-26-oic acid 385 313,7a,15a-Trihydroxy11,23-dioxo-5c~-lanost8-en-26-oic acid 300

Secondary metabolite

Page

313,7~,15a-Trihydroxy11,23-dioxo-5ctlanost-8-en-26-oic acid 2,5,7Trihydroxyemodin (22R)-3a,e2,25Trihydroxylanosta8,23-diene 24(S),25,26Trihydroxy-5txlanosta-7,9(11)dien-3-one 15tx,26,27-Trihydroxy50~-lanosta-7,9(11), 24-trien-3-one 3,6,8-Trihydroxy-1methylxanthone 3~,7~,15tx-Trihydroxy4,4,14a-trimethyl11,20-dioxo-5txpreg-8-ene 3,11,15-Trioxo-3~,12~dihydroxy-5txlanost-8-en-24oic acid 7,11,15-Trioxo-3~,12[~dihydroxy-5ctlanost-8-en-24-oic acid 7,11,15-Trioxo-313hydroxy-5~lanost-8-en-24oic acid Tumulosic acid

413 188

Variecoxanthone A Variecoxanthone B

717 719

279

Secondary metabolite

Page

Variecoxanthone C Victoxinine Viridominic acid A Viridominic acid B Viridominic acid C

721 773 463 465 467

Wentilactone A Wentilactone B

500 502

781

136

372

355 723

6-3-O-p-oXylopyranoside 16-3-O-13-0Xylopyranoside-16O-(4-O-acetyl)-i3-oglucopuranoside 6-3-O-13-oXylopyranoside- 16O-(4,6-di-O-acetyl)-

561

567

~=D= 429

glucopyranoside 7-3-0-15-oXylopyranoside-16O-(4,6-di-O-acetyl)-

557

~-D= 415

419

glucopyranoside 6-3-O-13-oXylopyranoside-16O-~-oglucopyranoside 7-3-O-13-oXylopyranoside-16-

565

555

O-~-Dglucopyranoside Zeorin Zymosterol

563 159 14

Molecular Formula Index Molecular formula

C7 C7H604 C7H804 C7HloO3

Page

758, 760 742, 744, 769 786, 788

C8

C8HloO4 C8HloO5 C8H1204 Clo CloH1602 CloH1802 CloH1803 Cn CllHloO4 CllH1205 CllH1604 CllH1605 CnH1804 CNH1805 C12 C12H1603 C12H1604 C14 C14HloO5 C15 C15HloO6 C15H1604 C15H2oN204 C15H2oO4 C15H2202 C15H2203 C15H24 C15H2402 C15H2403 C15H260 C15H2602 C15H2802 C16 C16H1605 C16H1606

778 790 771 726 724, 725 937 745 747 727,729 735 731 733 796 794 723

Molecular formula

C16H1705C1 C16H1805 C16HlsO6 C16H2oN205 C16H2o06 C16H2204 C16H2404 C16H2405 C16H2604 C16H2605 C16H2804 C17 C17H2oO5 C17Hz2N205 C17H2205 C17H2206 C17H2405 C17Hz9NO C18 C18H2oO6 C19 C19H2205 C19H24N206 C2o C2oHuN303

781 784 611 767 531,541 537 772 529,532, 753,755 539,757 738 535,780 782

C2oH11N304 C2oH13N303 C2oH13N304 C2oH2oO5 C2oH2605 C2oH2805 C2oH3oO7 C2oH32 C2oH33NO3 C2oH3403 C2oH3404 C21 C21H35NO3

672, 792 500

C22 C22H3o02

Page 657 498, 502, 662, 664 668 617 660, 666 762 764 497 645, 651 649 643, 647 495 615 655 633,670 631 773 659

Molecular formula

C22H3oO3 C22H3202 C22H3605 C23 C23H2905C1 C23H3oO4C1 C23H3105C1 C23H3103C1 C23H3104C1 C23H3203 C23H3204 C23H3402 C24 C24H3205 C24H3405 C24H3602 C24H3605 C24H3802 C24H3803 C25 C25H2805

705 613 677 679, 680 681 683 717 751 752 775 766 639 623 621 635, 637 589

C25H2806 C25H2sO7 C25H3106C1 C25H3306C1 C25H3603 C25H4202 C26 C26H3oO6 C26H3209 C27 C27H31N40 C27H3407 C27H3606 C27H3607 C27H3806 C27H3807 C27H3808 C27H4oO

Page 591,593, 595,597 598, 603 625,627 519 517 521,525 507 511 509 513,607 600 427 425 602,605 429 245 244,247 719 687,689, 691,721 693 515 523 606 236 695 242 707 399 407 417 393 395,403, 413,419 415 21,23

809

810

Molecular formula

C27H4007 C27H420 C27H4206 C27H440 C27H460 C28 C28H4oO C28H4oOs

C28I--142O C28H43NO C28H43NO2 C~H440

C28H4402 C2sH45NO

C28I'-I45NO2 C28H460

C~H4602

C28H4603 C~H~80

C~HsoO

C29 C29H3808 C291-14oO8 C29H420 C29H4204 C29H4205 C29H4402 C29H460 C29H4602 C29H4603 C29H4605

Molecular Formula Index

Page 397, 409, 411 16,18, 19, 20 421 5,8,10, 12,14 3,7,9 58 740 68,70, 72,80 443 439,445 42,43, 51,52, 54,64, 66,67, 78,82 76 435 437 29,34, 36,38, 45,47, 49,56, 57,60, 62,88, 90 73,74 84 27,33, 40,53, 86,87 31,32 401 405 93 253 251 124 103, 119 121, 125 123 264

Molecular formula

C29848O

C29H4805 C29H5oO C29H520 C3o C3oH4oO7 C3oH4oO8

C3oH4207 C3oH4208 C3oH4405

C3oH4406 C3oH4407 C3oH4408 C3oH460 C3oH4602

C3oH4603 C3oH4604 C3oH4606 C3oi-'I4607 C3oH4608 C3oi"I47NO2 C30I~O C3oH4802

C3oH4803

C3oH4804

C3oH4805 C3on49NO C3oH5oO C3oH5oO2 C3oH5oO3 C3oH5oO4

Page

Molecular formula

Page

99,101, 107,111, 112,114, 116,117 255 95,97, 105,109 113

C3oH5oO5 C3oH520 C3oH5202 C3oH5204 C3oH5205 C3oH5206 C31 C31H4603 C31H4604

366 131 159 449 451 453

283 328 277,292 281,324, 326 389 170,381, 387 273,275, 294,298 286,290 161 156,374 138,151, 157,344, 362 355,777 140,383 279,300, 385 296 441 169 154,155, 162,376 142,153, 345,370, 378 144,166, 171,336, 360,372 358 433 129,146, 148,168 132,134, 149,164, 798 136 353

C3~H4605

C31H4606 C31H4607 C31H4803 C31H4804 C31I'I4805

C31H4806 C31H5oO2 C31H5oO3 C31H5oO4 C31H5oO5 C31H5oO6 C31H520 C31H5202 C31H5203 C31H5205 C31H540 C32 C32H26013

C32H4209 C32H4409 C32H4804 C32H4805 C32H4806 C32H5oO4 C32H5oO6 C32H5204 C32H5206 C32H5405 C33 C33H4408 C33I--I4809 C33H48Olo C33H5oO8

175 176, 184, 190 479 471,473, 481,483, 485 487 196, 202 182, 197 192 269, 475, 477, 489 179 186, 194, 200 188 368 260 198, 208 178, 209 204, 206 364 181 749 248 288 216 312,342 338 213,214, 220 262,267 218 258 356 491 463 465 469

Molecular Formula Index

Molecular formula

C33H5009 C33H5205 C33H5206 C33H5404 C33H5406 C34 C34H4805 C34H5oO6 C34H5oO7 C34H5207 C34H5208 C34H5602 C34H5604 C35 C35H5406 C35H5407 C35H5408 C35H5605 C35H5807

811

Page

Molecular formula

467 225,227 265,318, 320 241 256

C36 C36H5208 C36H5209 C36H5409 C36H6007 C37 C37H5406 C37H5805 C37H5806 C37H6oO5

709 701 713 703

C38 C38H58Oao C38H6oOlo C38H6oOll

585 578 573

C39 C39H65NOlo C39H65NOll

459 455,457

332 310,330, 347,349 235,322 230,302, 340 231,316 238 240 711 229, 308 233,314 699 561

Page

334 304 306 551

C40

C4oH62Oll C4oH62012 C41 C41H68012

580 575,583 555

Molecular formula

C42 C42H7oO12 C43 C43H70013 C44 C44H72013 C45 C45H72014 C45H74Oll C46 C46H74014 C47 C47H74015 C49 C49H78013

Page

563 549,557 546, 565 547 567 553 559 571

C51

C51H80014

569

This Page Intentionally Left Blank

Molecular Weight Index Molecular weight 142.06299 154.02661 156.04226 168.11503 170.05791 170.13068 172.07356 186.05282 186.12559 204.18780 206.05791 208.10994 212.10486 214.12051 222.19837 224.06847 224.10486 228.09977 230.11542 234.16198 236.17763 238.19328 240.20893 250.15689 252.17254 258.05282 260.10486 263.22491 264.13616 272.25040 278.15181 280.16746 282.18311 284.19876 286.04774 288.09977 290.11542 292.14231 296.16237 298.17802

Page

Molecular weight

Page

786,788 758,760 742,744,769 726 778 724,725 771 790 737 772 745 796 727,729 731 738 747 794 735 733 531,541 529,532,755 535,753,780 782 537 539,757 723 784 773 767 766 762 764 645,651 643,647 781 672,792 498,502,662,664 611 497 649

304.09469 304.13107 306.11034 306.14672 308.12599 308.16237 320.13722 322.14164 322.25080 324.07645 326.22458 328.24023 330.14672 332.12599 334.15287 335.24604 338.24571 340.13107 341.08004 342.21950 342.25588 343.09569 346.17802 348.19367 349.26169 356.23515 356.27153 357.07496 358.28718 359.09061 372.23006 374.28210 374.31848 376.16344 380.25627 380.30792 382.19915 382.32357 384.26645 384.33922

5OO 495 668 655 660,666 631 617 633,670 623 657 589,598 603 7O5 659 615 639 621 717 677,679 591,593,595,597 6O0 681 751 752 635,637 509 602,605 68O 245 683 513,607 244,247 236 613 625,627 21,23 775 16,18,19,20 606 5,8,10,12,14

Molecular weight 386.35487 390.19617 392.30792 394.32357 396.33922

398.35487

400.22497 400.37052 402.24062 402.38617 404.17544 404.25627 406.19109 406.32357 408.19367 409.33447 410.35487 414.34978 414.38617 416.40182 420.17035 422.18600 422.35487 424.18859 424.33413 424.37052 425.32938 425.42938 426.34978 426.38617 427.34503 428.40182 430.34470 433.22531 438.20424

Page 3,7,9 507 58 68,70,72,80 42,43,51,52,54, 64,66,67,78, 82 29,34,36,38,45, 47,49,56,57,60, 62,88,90 427 27,33,40,53,86, 87 425 31,32 517 429 511 93 719 443 103,107,119 73,74 95,97,105,109 113 519 521,525 161 687,689,691,721 124 169 445 439 121,125 129,146,148,168 437 131 84 707 695

813

814

Molecular weight 438.34978 439.38142 440.18350 440.36543 440.40182 442.38108 442.34470 442.41747 444.39673 453.36068 454.30831 454.34470 454.38108 456.25119 456.28757 456.36035 456.39673 458.26684 458.37600 462.18092 462.29814 464.19657 466.34470 468.36035 470.23045 470.30322 470.33961 470.37600 472.24610 472.35526 472.39165 474.26175 474.33452 474.37091 476.27740 476.35017 476.38656 482.33961 484.31887

Molecular Weight Index

Page 156,374 433 693 154, 155, 162, 376 198, 208 132, 134, 149, 164, 798 123 181 159 441 253 138, 151,157, 344, 362 179 407 740 142, 153, 345, 370, 378 178, 209 393 136 515 421 523 175 196, 202 399 251 355, 777 186, 194, 200 417 144, 166, 171,336, 360, 372 204, 206 395,403,413,419 264 353 397, 409, 411 255 449 176, 184, 190 389

Molecular weight 484.35526 486.37091 488.20463 488.35017 490.25667 490.36583 492.38148 496.35526 496.42803 498.33452 498.37091 500.31379 500.35017 500.38656 502.32944 502.36583 504.38140 508.37639 512.27740 512.35017 514.25667 514.29305 514.32944 514.40221 516.27232 516.30870 516.34509 518.32435 518.36074 518.39713 528.27232 528.34509 528.38148 528.41766 530.28797 530.32435 530.36074 532.30362 532.37639 534.31927 544.37639 546.39204

Page 182,197 188 242 358 415 366 451 216 238 479 213,214,220 170,381,387 192 218 140,383 368 364 453 283 312,332,342 401 277,292 471,473,481,483, 485 241 405 273,275,294,298 269,475,477,489 279,300,385 260 356 328 338 225,227 240 281,324,326 487 262,267 286,290 258 296 265,318,320 256

Molecular weight

Page

554.36074 556.41278 568.30362 570.28288 570.35565 570.39204 572.29853 572.37130 574.35057 582.42843 584.44408 586.38695 588.32983 588.36622 590.34548 590.41825 594.39204 598.42334 602.38187 604.32475 604.43390 612.36622 618.13734 630.37678 674.40300 676.41865 692.41356 707.46085 718.42921 723.45576 734.42413 752.47108 766.48673 790.52311 794.48164 808.49729 836.49221 850.50786 874.54424 878.50277 916.55481

310,330,347,320 699 491 284 235,322 711 288 230,302,340 469 701 703 229,308 463 231,316 467 561 709 713 233,314 465 551 334 749 304,306 585 578 573 459 580 455,457 575,583 555 563 567 549,557 545,565 547 553 571 559 569

Fungal/Plant Source Index A

Acaulospora laevis, 3, 27 Achlya ambisexualis, 253,255 A. bisexualis, 3, 36, 101,103,251,253,255 A. heterosexualis, 256, 258, 260, 262, 264, 265, 267, 269 Acremonium coenophialum, 47 A. fusidioides, 489 A. luzulae, 84, 517 Acrostalagmus sp., 495,497, 498, 763,764 Agaricus bisporis, 40, 45, 56 A. campestris, 43, 49, 129, 198 Albatrellus confluens, 598, 603,605 A. ovinus, 598, 600, 602, 603,605, 606, 607 A. subrubescens, 607 Albizziac, 631 Allomyces macrogynus, 3, 27, 95, 129 Allomyces spp., 3 Alternaria alternata, 43, 58 A. brassicicola, 43 A. cinerariae, 622 A. cucumerina, 655 A. kikuchiana, 43, 129 A. porri, 749 A. scirpicola, 662 A. senecionis, 662 A. tenuis, 43 A. tomato, 660, 662 Amanita caesaria, 49 Amelaria mellea, 43, 44, 45, 47, 56 Amoebidium parasiticum, 3, 43 Aplanopsis spp., 5 A. terrestris, 101 Apodachlya brachynema, 101 A. minima, 101 Apodachlyella completa, 101 Arcyria denudata, 677, 679, 680, 681,683 Armillaria mellea, 40 Ascochyta cypericola, 784 A. viciae, 517, 519, 521,525 Aspergillus alliaceus, 778 A. candidus, 93 A. chevalieri, 751,752 A. clavatus, 744, 758

A. fennelliae, 47 A. flavus, 58, 93,792 A. fumigatus, 58, 491 A. giganteus, 758 A. melleus, 778 A. nidulans, 719 A. niger, 43, 93 A. nomius, 792 A. ochraceus, 58, 778 A. oryzae, 3, 29, 47, 49, 80, 95, 107 A. ostianus, 778 A. parasiticus, 45, 56 A. quadrilineatus, 719 A. quercinus, 778 A. repens, 792 A. rugulosus, 687, 689, 691,719 A. sclerotiorum, 778 A. sulphureus, 778 A. terreus, 242, 758 A. variecolor, 693,695,717, 719, 721 A. versicolor, 93 A. wentii, 500, 502 A. zonatus, 740 Atta sexdens rubropilosa, 95

B

Bipolaris sorokiniana, 529, 532, 535, 537, 539, 755 Bipolaris sp., 531,532, 535,537, 539, 773 Blastocladia ramosa, 3, 7, 9, 86, 87, 129 Blastocladiella emersonii, 129 Boletus luridus, 40, 43, 45, 47, 56, 745,747 Boletus spp., 43, 45, 47, 56 Botrytis cinerea, 38 Byssochlamys nivea, 758

C Calcarisporium arbuscula, 489 Candida albicans, 33, 38, 40, 43, 45, 47, 49, 51, 54, 56, 62, 64, 80, 90, 114, 116, 129, 146, 181,198

815

816

Secondary Metabolite Index

C. guilliermondii, 43 Candida sp., 43, 47, 90, 95 C. tropicalis, 40, 47, 49 C. utilis, 33, 38, 40, 43, 45, 47, 49, 51, 56, 58, 62, 64, 80, 90, 95, 116, 129, 181,198 Cantharellus cibarius, 43, 84 Catenaria anguillulae, 129 Cephalosporium acremonium, 489 C. aphidicola, 621,766 C. caerulens, 491 C. recifei, 737 Cercospora scirpicola, 659 C. spp., 655 Chlorella ellipsoidea, 60 Chytridium confervae, 129 Cladonia gonecha, 29 Cladosporium acremonium, 469 Cladosporium sp., 463,465,467, 469 Claviceps fusiformis, 29 C. purpurea, 29 Clitocybe nebularis, 76 Cochliobolus sativus, 532, 535 C. setariae strain IFO 6635, 532, 535,753,755, 777 Colletotrichum dematium, 3 Coprinus atramentarius, 49 Coriolus heteromorphus, 129 C. pargamenus, 40, 43, 45, 47, 56, 129 C. sangamenus, 43, 45, 56 C. versicolor, 40, 43, 45, 47, 56, 95 Cronartium fusiforme, 40, 107, 109, 111 Cryptoderma citrinum, 40, 43, 47, 129 Curvularia geniculata, 781 C. lunata, 781 C. ramose, 781 C. spp., 247, 655 C. trifolii, 781 Cyathus helenae, 43, 159, 161,162, 166 Cylindrocladium iliciola, 517 D

Daedalea dickinsii, 188, 190, 197 D. quercina, 45, 47, 56 D. tanakae, 190 D. trabea, 142, 144, 186, 196 Debaromyces hansenii, 99 Dictyostelium discoideum, 105,113 Dictyuchus monosporus, 49 Diplodia macrospora, 107, 794, 796 Dipsacomyces acuminosporus, 3

Drechslera dematioidea, 781 D. halodes, 781 D. setariae, 781 D. sorokiniana, 781 Drechslera sp., 655,657 D. spicifera, 781 E

Echinodontium tinctorium, 213 E. tsugicola, 151,153, 154, 155, 156, 214, 216 Emericellopsis terricola, 491 F

Flammulina velutipes, 47, 49, 54 Fomes allardii, 43, 84 E annosus, 43, 47, 72, 82, 131 E applanatus, 58, 82 E fomentarius, 47, 82, 131 E officinalis, 58, 170, 175, 176, 178, 179 E pini, 82, 131 E senex, 123, 124, 125, 142, 157, 171 Fomitopsis cytisina, 43, 47 E pinicola, 40, 43, 190 E pubertatis, 43 Fucus vesicudosus (brown marine algae), 101 Fusarium culmorum, 53,782 E equiseti, 611,613,615, 617 E moniliforme, 50, 84 E oxysporum, 742 Fusarium sp., 507, 509, 511,513,515, 517 E sporotrichioides, 27, 43, 95, 99, 220 E coccineum, 471,473,475,477, 479, 481,483, 485,487 G Gabarnaudia tholispora, 489 Ganoderma app~natum, 43, 57, 82, 168, 169 G. lucidum, 273, 277, 279, 281,283,284, 286, 288,290,292,294,296,298,300,302,304, 306,308,310,312,314,316,318,320,322, 324,326,328,330,332,334,336,338,340, 342,344,345,347,349,353,355,356,358, 360,362,364,366,368,370,372,374,376, 378,381,383,385,387,389,393,395,397, 399,401,403,405,407,409,411,413,415, 417,419,421,425,427,429

Fungal/Plant Source Index

Geotrichum flavo-brunneum, 43,433, 435,437, 439, 441,443,445 GibbereUa fujikuroi, 68, 80, 782 Gloeophyllum abietinum, 142, 186 G. odoratum, 142 G. sepiarium, 40, 43, 47, 142, 186 G. striatum, 142, 186 G. trabeum, 142, 186 Glomus caledonius, 3, 36 G. mosseae, 3, 27, 40, 43, 95, 99 Gnomonia leptostyla, 3, 29, 43 Grifola confluens, 589, 603, 605 G. frondosa, 40, 43, 129 G. umbellate, 43 Gymnoascus spp., 758

H Harziella entomophilla, 621 Hebeloma crustuliniforme, 573 H. senescens, 575,578, 581 H. sinapizans, 573 H. spoliatum, 573, 583, 585 H. vinosophyllum, 545,547, 549, 551,553, 555,557, 559, 561,563,565, 567, 569, 571 Helminthosporium sativum, 529, 532, 535, 537, 541,753,755, 757, 772, 773, 780 H. setariae, 772 H. siccans, 589, 591,593, 595,597 H. victoriae, 755,772, 773, 780 Hemispora stellata, 43, 90 Hericium erinaceum, 699, 701,703,705,707, 709, 711,713,786, 788, 790 Heterobasidion tasmanica, 142, 157 Hygrocybe punicea, 49 Hymenomycetes sp., 40, 47, 129 Hyphochytrium catenoides, 27, 53, 95, 99, 129

817

Lentinus edodes, 49 L. lepideus, 43, 142, 186 Lenzites trabea, 40, 44, 142, 144, 186 Leptolegnia caudata, 36 Leptosphaeria typhae, 3, 44, 47, 49, 51, 64 Leucopaxillus giganteus, 49 L. tricolor, 792 Linderina pennispora, 3, 40, 109 Lobaria pulmonaria, 38, 44, 49, 54 L. scobiculata, 38, 44, 49, 54 M

Melampsora lini, 107, 109, 111 Melanoporia cajanderi, 188, 190 M. juniperina, 188, 190, 197 M. nigra, 188, 190, 231 M. purpuracea, 188, 190 M. rosea, 188, 190, 197,231,233 Microporus flabelliformis, 44, 45, 56 M. labelliformis, 129 Monilinia fructigena, 44, 146 Monoblepharella sp., 3, 27 Mucor pusillus, 44, 45, 56, 129, 146 M. rouxii, 68, 238 N

Neamatoloma fasciculare, 449, 451,453,455, 457, 459 Nectria coccinea, 517, 523 N. galligena, 3, 5, 7, 10, 44, 49, 54 Neocosmospora vasinfecta, 631,633,635,637, 639 Nephroma arcticum, 159 Neurospora crassa, 38, 40, 44, 47, 49, 54, 62, 129, 181,198 Nigrospora sphaerica, 621,766 P

I

Inocybe macrosperma, 49 L Lactarius sp., 10 Laetiporus sulphureus, 142, 186 Lampteromyces japonicus, 49, 58 Leccinum aurantiacum, 43, 45, 47, 56 Leccinum spp., 43, 45, 47, 56

Paecilomyces ehrlichii, 778 Penicillium aurantio-virens, 778 P. baarnense, 778 P. canescens, 778 P. chrysogenurn, 778 P. citreo-viride, 655,660, 664, 666, 668, 672, 735 P. citrinum, 58 P. claviforme, 40, 44, 758 P. cyaneofulvum, 758

818

P. cyclopium, 758, 778 P divergens, 758 P equinum, 758 P. expansum, 758 P fenelliae, 778 P gilmanii, 655 P granulatum, 758 P. griseofulvum, 58, 758 P griseum, 778 P. islandicum, 58 P janthinellum, 778 P. lanosum, 758 P lapidosum, 758 P leucopus, 758 P lilacinum, 778 P lividum, 778 P. madriti, 778 P. martensii, 778 P. melinfi, 758 P. notatum, 78 P. novae-zeelandiae, 758 P. olivino-viride, 778 P. palitans, 778 P. patulum, 58, 723, 758, 769 P. puberulum, 778 P. roqueforti, 758, 778 P. rubrum, 58 P. simplicissimum, 778 Penicillium sp., 655, 660, 662, 670, 727 P. suavolens, 778 P. terrestre, 758 P. thomii, 778 P. turbatum, 775 P. urticae, 758, 760 P viridicatum, 778 Pestalotia cryptomeriaecola, 731 P. longiseta, 742 Phellinus gilvus, 44, 47, 142, 157 P igniarius, 44 P robustus, 44 Pholiota aegerita, 40, 44, 45, 47, 56 Phoma betae, 623, 625, 627 Phycomyces blakesleeanus, 3, 40, 44, 45, 47, 49, 51, 56, 129, 198 Physarum flavicomum, 32, 53, 97, 99, 112, 129 P polycephalum, 8, 27, 31, 32, 53, 95, 97, 112, 113, 129 Pichia sp., 38, 44, 45, 56, 90, 129, 208 Piptoporus betulinus, 45, 47, 56, 190 Pisolithus tinctorius, 136, 200, 202, 204, 206, 209, 218, 240, 241

Fungal/Plant Source Index

Polyporus cretaceous, 44, 186 P dryadeus, 44, 84 P. officinalis, 194 P ovinus, 598, 600, 602, 603, 605, 606, 607 P. paragamenus, 45, 56 P pinicola, 121,164, 798 Poria cocos, 44, 138, 190, 225,227 P. obliqua = Inonotus obliquus, 150 Protomyces sp., 29 Puccinia graminis var. triticil, 40, 109 P striiformis, 40, 109 Pullularia pullulans, 99 Pythiopsis cymosa, 36, 101

R

Rhizidiomyces apophysatus, 27, 129 Rhizophlyctis rosea, 3, 8, 95, 129 Rhizophydium sphaerotheca, 36 Rhizopus arrhizus, 40, 42, 44, 47 Rhodopaxillus nudus, 76 Russula aeruginosa, 10 R. decolorans, 10 R. foetens, 49 R. nigricans, 49 R. sardonia, 84 R. senecis, 40, 49

S

Saccharomyces carlsbergensis, 38, 45, 51, 56, 90, 64 S. cerevisiae, 12, 14, 16, 18, 19, 20, 21, 23, 33, 34, 38, 40, 44, 45, 47, 49, 51, 52, 54, 56, 60, 62, 64, 67, 68, 70, 73, 74, 80, 88, 90, 114, 116, 117, 119, 129, 131,146, 148 Saprolegnia ferax, 36, 101 S. megasperma, 36, 101 Schizophyllum commune, 45, 56 Scleroderma aurantium, 44, 132, 134 Sclerotinia fructicola, 44 Sepedonium chrysospermum, 745,747 Smittium culicis, 5 S. culisetae, 5 S. mucronatum, 5 S. simulii, 5 Smittium sp., 3, 5, 44 Spicaria elegans, 44 Spizellomyces punctatum, 27, 129 Spongiporus appendiculatus, 142, 186, 188

Fungal/Plant Source Index

819

Sporobolomyces odorus, 724, 725,726 Stachybotrys alternans, 99 S. atra, 27, 95 Stereocaulon tomentosum, 29 Suillus bovines, 44, 45, 47, 56 S. variegates, 44, 45, 47, 56

Trichophyton mentagrophytes, 53 T. rubrum, 29 Trichothecium roseum, 766, 782 Trichothecium sp., 782 Tuber brumale, 29 T. melanosporum, 29

T Terfezia sp., 29 Torulopsis glabrata, 33, 38, 40, 44, 45, 47, 49, 56, 62, 90, 116, 129 Trametes dickinsii, 188, 190, 197, 229, 230 T. feei, 44, 188, 190, 192, 197, 227 T. lilacino-gilva, 44, 186, 188, 197, 227 T. odorata, 140, 142, 144 T. stowardii, 182, 184, 235 Tremella fuciformis, 45, 56 Trichoderma harzianum, 643, 647 T. koningii, 78, 121,643,645,647,649, 651, 738, 782 Tricholoma portentosum, 76

U Uromyces phaseoli, 107, 109, 129 Usnea longissima, 38, 44, 49, 54 Ustilago maydis, 3, 40, 44, 45, 56, 117, 146 U. nuda, 40, 44, 109

V Veluticeps angularis, 142 Verticillium lecanii, 236 X

Xanthoria parietina, 44, 47, 49, 54

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Handbook of Secondary Fungal Metabolites V O L U M E III

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Handbook of Secondary Fungal Metabolites VOLUME

III

RICHARD J. COLE

Albany, Georgia

BRUCE B. JARVIS

Department of Chemistry and Biochemistry University of Maryland �9 College Park, Maryland

MILBRA A. SCHWEIKERT

National Peanut Research Laboratory Dawson, Georgia

ACADEMIC PRESS An imprint of Elsevier Science Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

Cover images: Photography by Dr. Bruce Horn and design by Brian E. Cole. The cover is a collage of various fungi, some presented in pure culture and others in their natural forms, i.e. mushrooms, which are the easily recognizable fruiting structures of some fungi, superimposed on these fungi are the chemical structures of some representative secondary fungal metabolites. Academic Press Rapid Manuscript Reproduction This book is printed on acid-free paper. @ Copyright

92003, Elsevier Science (USA).

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://elsevier.com), by selecting "Customer Support" and then "Obtaining Permissions."

Academic Press

An imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press 84 Theobald's Road, London WC1X 8RR, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2003103019 International International International International

Standard Standard Standard Standard

Book Book Book Book

Number: Number: Number: Number:

0-12-179460-1 0-12-179461-X 0-12-179462-8 0-12-179463-6

(Set) (Volume 1) (Volume 2) (Volume 3)

PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 8 7 6 5 4 3 2 1

Contents

Preface I ix Acknowledgments I xi

I ~ I Atranones / 1 Austalides / 17 I~1

Altertoxins / 43

I~[

Cercophorins / 51

~1 Fiscalins / 59 [(~1 Palmarumycins /

67

vi

Contents

Phomosines /

107

I ~ J Fumiquinazolines ! 115 ~i

Ganomastenols / 131

L~~1 Memnobotrins / 141 1~~ I Tsugicolines / 151 I~ ~1 Radicinins / 161 I~ ~l Stachybotrylactone and Related Metabolites ! 173 I~ ~1 Trichothecenes and Related Metabolites / 199 l~l

ModifiedTrichothecenes / 325

[~ ~1 Macrocyclic Trichothecenes and Related Metabolites / 349 I~ ~1 Miotoxins / 467

oo

Contents

wl

Roritoxins / 487

h|

Myrotoxins and Mytoxins / 497

Baccharinoids / 517

Fumonisins, AAL Toxins, and Related Metabolites I 561

Ochratoxins and Related Metabolites / 613

Miscellaneous Metabolites / 625 Secondary Metabolite Index I 661 Molecular Formula Index I 667 Molecular Weight Index I 669 Fungal/Plant Source Index I 671

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Preface

The "Handbook of Secondary Fungal Metabolites" is presented in three volumes and is comprehensive to the extent that all major groups of secondary fungal metabolites are included. The format is similar to that presented in the "Handbook of Toxic Fungal Metabolites" with the major exception that actual spectra are not included; however, spectral data are included where available. Also included in these volumes are the methods used by the authors to isolate and purify metabolites. Another major difference is that the appropriate references are presented with each metabolite, negating the need to turn to the end of each group to find the appropriate references. Each volume contains four indexes: secondary metabolite index, molecular formula index, molecular weight index, and fungal/plant source index. In a few instances, plant sources are included when the metabolites are closely related to fungal metabolites or the source of precursors may be fungal; i.e., the baccharins, which are found in extracts from Baccharis megapotarnica. These metabolites are closely related to the macrocyclic trichothecenes found in extracts of fungi such as Myrothecium spp. and Stachybotrys spp. Also, metabolites from the fungal symbiont of lichens are sometimes presented. To aid in the interpretation of N M R data, the numbering system presented in the literature is included for the major representative fungal metabolite and, at times, for several related metabolites. Fungal sources are given as reported in the original references. It is recognized that the taxonomy in several cases has been revised, perhaps more than once. It is beyond the scope of these volumes to deal with what is "currently accepted taxonomy" because this is a dynamic science that, in many cases, is as yet undefined. The "Handbook" has been divided into sections, and the placement of metabolites is based on chemical relationships. One section of each volume contains a miscellaneous section to accommodate metabolites difficult to place into one of the sections. The miscellaneous section of Volume III contains some metabolites related to those that appear in Volumes I and II. This occurred when related metabolites were discovered after Volumes I and II were completed.

ix

x

Preface

It is hoped that this compilation of data on secondary fungal metabolites will aid investigators in the identification of known or related fungal metabolites. Because fungal metabolites represent a wide diversity of chemical species, these volumes will be useful to scientists interested in correlations of structural features with various spectral and biological characteristics. The known biological activity of metabolites is presented, which may aid in future studies related to the identification of new uses for fungal metabolites. Richard J. Cole Bruce B. Jarv& Milbra A. Schweikert

Acknowledgments

The authors thank the following investigators for their assistance in producing the "Handbook of Secondary Fungal Metabolites." Their contributions made this compilation of data on fungal metabolites possible. Wayne L. Bryden

William Norred

University of Queensland Gotton, Queensland 4343 Australia

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

Brian E. Cole

Images, Inc. Snellville, Georgia Horace G. Curler

Department of Pharmacology Mercer University Atlanta, Georgia Jens C. Frisvad

Department of Biotechnology Technical University of Denmark DK-2800 Lyngby Denmark Bruce Horn

James K. Porter

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia Ronald T. Riley

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia Victor S. Sobolev

USDA-ARS National Peanut Research Laboratory Dawson, Georgia

USDA-ARS National Panut Research Laboratory Dawson, Georgia

xi

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Atranones Atranone A Atranone B Atranone C Atranone D Atranone E Atranone F Atranone G

This Page Intentionally Left Blank

1.

Atranones

3

Common/Systematic Name Atranone A Molecular Formula/Molecular Weight C24I-I3206; M W --" 416.21989 General Characteristics Crystals; mp., 179-184~ Me

Me 8

x.,o., M .

Me 16

[~]D 20 + 2 9 ~ (c =

Me

0.86 in CHC13).

Me

~s

Fungal Source

Stachybotrys chartarum (S. atra) S-11.

Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. chartarum, S- 11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give 11.5g of a black gum. The majority of the extract was chromatographed over PEI silica, eluting with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 2 (eluted with 100% methylene chloride) was applied to a 2mm Chromatotron plate and eluted with ethyl acetate-hexane-methanol (40:60: 5, v/v/v) to give atranone B. A later fraction from the plate was found to contain a 2:1 mixture of atranones A and C. These were resolved by semipreparative HPLC using the following conditions: reversed phase Cls column (Phenomenex, 10 x 250mm); solvent methanolwater (isocratic; 2:5, v/v); flow 3.5ml/min; detection UV 250nm; retention time for atranone A and C was 35.8 and 40.9 min, respectively. Spectral Data UV:

,,=M'~ 224nm (e = 10,500). IR:

(CHCI3) 3684, 3598, 3036, 1789, 1706, 1522, 1424, and 1212em"1.

4

1.

Atranones

1H NMR: (benzene-d~) 0.71(3H, d, d=6.7Hz, H-19); 0.77(3H, d, J=-6.8Hz, H-20); 0.80(3H, s, H-15) 1.27(1H, ddd, J-2.7, 12.5, and 14.0Hz, H-2B); 1.40(3H, s, H-23), 1.46(1I-I, m, H-10B) 1.48(3H, br s, H-17); 1.54(1H, m, H-3B); 1.67(3H, d, J=I.5H~ H-16); 1.81(1H, m, H-3A); 1.82(1H, m, H-10A); 1.98(1H, dt, ,/=5.2 and 4.0Hz, H-2A); 2.08 (1H, qq, J=6.7 and 6.8Hz, H-18); 2.47(1H, br dd, J=2.5 and 7.9Hz, H-11); 2.67(1H, d, d=l 1.1Hz, H-21); 2.91(1H, ddq, ,/-1.5, 4.1 and 11.1Hz, H-6); 5.16(1H, d, J=4.1Hz, H-7); 5.23(1H, br dd, J=4. land 12.0Hz, H-9); and 5.82ppm (1H, d, d=2.5Hz, H-13). 13CNMR: (benzene-d6) 84.7, C-l; 39.2, C-2; 26.8, C-3; 111.4, C-4; 145.7, C-5; 47.8, C-6; 80.7, C-7; 134.3, C-8; 128.9, C-9; 23.4, C-10; 44.0, C-11; 166.8, C-12; 114.7, C-13; 163.5, C-14; 20.8, C-15; 14.9, C-16; 17.6, C-17; 29.5, C-18; 21.8, C-19; 22.8, C-20; 57.5, C-21; 105.1, C-22; 25.6, C-23; and 170.3ppm C-24. References B. B. Jarvis, Y.-W. Lee, S. N. Yatawara, and C. S. C0mezoglu; Trichothecenes Produced by Stachybotrys atra from Eastern Europe; Appl. Environ. Microbiol., Vol. 51, pp. 915-918(1986). S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis; Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lett., Vol. 40, pp. 2725-2728(1999). S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis; Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra; Phytochemistry, Vol. 55, pp. 663-673 (2000).

1. Atranones

5

Common/Systematic Name Atranone B Molecular Formula/Molecular Weight C25H3407; M W = 446.23045

Me

Me

0.7 ~

0

0"-

HO'""'~ "~t
2,

~

9

Me,

H

y

Me

112 OMe

~/-'0" Me 15 16

Me

General Characteristics Crystals; mp., 213-218~

"0

[et]D2~+25 ~ (C=1.50 in CHC13).

Fungal Source

Stachybotrys chartarum (S. atra) S-11.

Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. chartarum, S- 11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give 11.5g of a black gum. The majority of the extract was chromatographed over PEI silica, eluting with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 2 (eluted with 100% methylene chloride) was applied to a 2mm Chromatotron plate and eluted with ethyl acetate-hexane-methanol (40:60: 5, v/v/v) to give atranone B. A later fraction from the plate was found to contain a 2:1 mixture of atranones A and C. These were resolved by semipreparative HPLC using the following conditions: reversed phase ClS column (Phenomenex, 10 x 250mm); solvent methanolwater (isocratic, 2:5, v/v); flow 3.5ml/min; detection UV 250nm; retention time for atranone A and C was 35.8 and 40.9 min, respectively. Spectral Data UV:

2, maxMO ' H 23 lnm (e = 10,800). IR:

(CHC13) 3684, 3599, 3018, 1790, 1704, 1522, 1424, 1212 and 1136cm"]. 1H NMI~: (benzene-d6) 0.86(3H, s, H-15); 1.10(3H, d, J=7.0Hz, H-20); 1.22(3H, d, J=6.9Hz,

6

1.

Atranones

H-19); 1.22(1H, m, H-2B); 1.33(3H, s, H-23); 1.44(1H, m, H-10B); 1.46(3H, br s, H-17); 1.50(1H, br dt, d=2.3 and 14Hz, H-3B); 1.64(3H, br s, H-16); 1.75(1H, dt, d=5.0 and 14.5Hz, H-3A); 1.82(1H, m, H-10A); 1.94(1H, dt, 5.0 and 14.5Hz, H-2A); 2.19(1H, qq, J=6.9 and 7.0Hz, H-18); 2.58(1H, br d, J=8.1Hz, H-11); 2.64(1H, d, J=l 1.2Hz, H-21); 2.90(1H, ddq, d=2.0, 4.0, and 11.2Hz, H-6); 3.63(3H, s, OMe); 5.14(1H, d,J=4.0Hz, H-7); and 5.73ppm (1H, br dd, J=4.1 and 12.0Hz, H-9). 13CNMR: (benzene-d6) 84.0, C-I; 39.2, C-2; 26.5, C-3; 111.3, C-4; 145.7, C-5; 47.8, C-6; 80.9, C-7; 134.3, C-8; 128.9, C-9; 24.4, C-10; 43.4, C-11; 143.6, C-12; 142.5, C-13; 160.1, C-14; 20.5, C-15; 14.8, C-16; 17.6, C-17; 29.1, C-18; 20.1, C-19; 20.4, C-20; 57.4, C-21; 105.0, C-22; 25.6, C-23; 170.1, C-24; and 58.9ppm, OMe. References B. B. Jarvis, Y.-W. Lee, S. N. Yatawara, and C. S. COmezoglu; Trichothecenes Produced by Stachybotrys atra from Eastern Europe; Appl. Environ. Microbiol., Vol. 51, pp. 915-918(1986). S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis; Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lett., Vol. 40, pp. 2725-2728(1999). S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis; Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra; Phytochemistry, Vol. 55, pp. 663-673 (2000).

1. Atranones

7

Common/Systematic Name Atranone C Molecular Formula/Molecular Weight C24H3206; M W -- 416.21989

Me

Me

Me

Me

HOH.... 0 0

Me

General Characteristics Crystals; mp., 204-208~

Me

0

[a]D 20 +

0

74 ~ (c= 0.44 in CHC13).

Fungal Source

Stachybotrys chartarum (S. atra) S-11.

Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. chartarum, S-11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give 11.5g of a black gum. The majority of the extract was chromatographed over PEI silica, eluting with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 2 (eluted with 100% methylene chloride) was applied to a 2 mm Chromatotron plate and eluted with ethyl acetate-hexane-methanol (40:60: 5, v/v/v) to give atranone B. A later fraction from the plate was found to contain a 2:1 mixture of atranones A and C. These were resolved by semipreparative HPLC using the following conditions: reversed phase Cls column (Phenomenex, 10 x 250mm); solvent methanolwater (isocratic; 2:5, v/v); flow 3.5ml/min; detection UV 250nm; retention time for atranone A and C was 35.8 and 40.9 min, respectively. Spectral Data UV:

Z maxM~O" End absorption only. IR~

(CHCI3) 3607, 2970, 2935, 1791, 1716, 1521, 1475, 1386, 1297 and 1136cm"l. ~H NMR: (benzene-d6) 0.52(3H, d, J=6.6Hz, H-19); 0.68(3H, d, J=6.8Hz, H-20), 0.92(3H, s,H-15); 1.38(3H, s, H-23); 1.39(1H, m, H-2B); 1.53(3H, br s, H-17); 1.61(1H, dt, J -

8

1.

Atranones

2.7 and 13.3Hz, H-3B); 1.63(3H, br s, H-16); 1.95(1H, m, H-3A); 1.98(1H, dt, ,/-4.8 and 14.3Hz, H-2A); 2.38(1H, m, H-10B); 2.51(1H, br d,J=20.6Hz, H-13B); 2.53(1H, m, H-18); 2.56(1H, dd, J=12.0 and 14.8Hz, H-10A); 2.61(1H, d, J=l 1.4Hz, H-21); 2.87(1H, d, J=20.6Hz, H-13A); 2.92(1H, br ddq, J=2, 4.6, and 11.4Hz, H-6); 5.18(11-1, d, J=4.6Hz, H-7); and 5.40ppm (1H, dd, ,/=6.3 and 12.0Hz, H-9). 13CNMR: (benzene-d6) 86.6, C-l; 39.1, C-2; 27.2, C-3; 111.3, C-4; 145.4, C-5; 47.8, C-6; 80.9, C-7; 133.3, C-8; 128.9, C-9; 25.6, C-10; 129.5, C-11; 135.5, C-12, 29.3, C-13; 168.9, C-14; 28.2, C-15; 14.7, C-16; 17.8, C-17; 29.7, C-18; 19.6, C-19; 20.0, C-20; 57.4, C-21; 105.1, C-22; 25.6, C-23; and 170.4ppm C-24. References B. B. Jarvis, Y.-W. Lee, S. N. Yatawara, and C. S. COmezoglu; Trichothecenes Produced by Stachybotrys atra from Eastern Europe; Appl. Environ. Microbiol., Vol. 51, pp. 915-918(1986). S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis; Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lett., Vol. 40, pp. 2725-2728(1999). S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis; Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra; Phytochemistry, Vol. 55, pp. 663-673 (2000).

1. Atranones

9

Common/Systematic Name Atranone D Molecular Formula/Molecular Weight C24H3404; M W -- 3 8 6 . 2 4 5 7 1

Me. H

HO~

Me

M e ~ ..Me

H H ~

Me

H T

Me ~

General Characteristics Clear film; [~]D 20 + 21 o (c=0.70 in CHC13). Fungal Source Stachybotrys chartarum (S. atra) S-11. Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. chartarum, S- 11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give a black gum. The majority of the extract was chromatographed over PEI silica, elution with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 1 (eluted with hexane to 80% methylene chloride-hexane) was applied to a 2mm Chromatotron plate (eluting with 10% ethyl acetate in methylene chloride) to yield atranones D and E. Spectral Data UV: ~, MaOH

max 23 lnm (e= 14,800).

IR:

(CHC13) 3684, 3607, 3015, 2971, 2935, 1780, 1693, 1522, 1424, and 1213cm"1. 1H NMR: (CDC13) 1.01(3H, br s, H-15), 1.14(3H, d, J=6.9Hz, H-20), 1.16(3H, d, J=6.7Hz, H-19); 1.27(1H, m, H-2B), 1.30(1H, m, H-3B), 1.32(3H, d, J=6.4Hz, H-23), 1.69(1H, m, H-10B); 1.72(3H, br s, H-17), 1.80(3H, s, H-16), 1.84(1H, m, H-3A), 2.22(1H, dddd, d=3.0, 5.9, 12.4, and 12.4Hz, H-9B), 2.24(1H, m, H-10A), 2.31(1H, m, H-2A), 2.42(1H, m, H-9A), 2.62(1H, qq, J--6.7 and 6.9Hz, H-18); 2.70(1H, dq, J-1.0 and 12.2Hz, H-11), 3.03(1H, dd, J=5.2 and 11.5Hz, H-21), 4.07(1H, dq, J=5.2 and 6.4Hz,

10

1.

Atranones

H-22); 4.15(1H, br dd, J= 10.4 and 11.5Hz, H-6), 5.51(1H, br d, Jr=10.4Hz, H-7); and 5.83ppm (1H, d, d=l.0 Hz, H-13). 13CNMR: (CDCI3) 52.0, C-l; 35.7, C-2; 27.0, C-3; 114.5, C-4; 144.9, C-5; 38.1, C-6; 126.7, C-7; 137.3, C-8; 36.3, C-9; 24.0, C-10; 43.7, C-11; 188.0, C-12; 124.3, C-13; 212.0, C-14; 24.0, C-15; 15.8, C-16; 16.6, C-17; 29.1, C-18; 21.2, C-19; 22.1, C-20; 50.5, C-21; 66.8, C-22; 20.9, C-23; and 175.3ppm C-24. References S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis; Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lett., Vol. 40, pp. 2725-2728(1999). S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis; Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra; Phytochemistry, Vol. 55, pp. 663-673 (2000).

1. Atranones

11

Common/Systematic Name Atranone E Molecular Formula/Molecular Weight C24I--I3404; M W = 3 8 6 . 2 4 5 7 1

Me. H

Me

M e ~ ..Me

HO ~Ho~.~.; 7~ 8 H~L Me

Me ~5

General Characteristics Clear film; [a]D2~-- 16~(C=0.75 in CHC13). Fungal Source

Stachybotrys chartarum (S. atra) S-11.

Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. chartarum, S- 11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give a black gum. The majority of the extract was chromatographed over PEI silica, eluting with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 1 (eluted with hexane to 80% methylene chloride-hexane) was applied to a 2mm Chromatotron plate (eluting with 10% ethyl acetate in methylene chloride) to yield atranones D and E. Spectral Data UV: maxM~' 226nm (e= 12,600). IR:

(CHCI3) 3688, 3608, 3518, 2970, 2937, 2874, 1773, 1692, 1606, 1459, 1386, 1374, 1277, 1133, and 1068cm"~. 1H NMR: (CDC13) 1.10(3H, s, H-15); 1.14(3H, d, J=6.8Hz, H-20); 1.16(3H, d, J=6.7Hz, H-19); 1.21(1H, m, H-3B); 1.30(3H, d, J=6.4Hz, H-23); 1.35(1H, m, H-ZB); 1.69(1H, m, H-10B); 1.71(3H, d, J=2Hz, H-17); 1.80(3H, s, H-16); 1.99(1H, ddd, J=2.1, 12.2, andl2.8Hz, H-3A); 2.23(1H, m, H-9B); 2.24(1H, ddd, J=4.5, 12.9, and 13.1Hz, H-10A); 2.34(1H, ddd, J=5.5, 12.8, and 13.4Hz, H-2A); 2.40(1H, m, H-9A); 2.50(1H,

12

1.

Atranones

dd, ,/=6.3 and 8.9Hz, H-21); 2.62(1H, qq, ,/=6.7 and 6.8Hz, H-18); 2.70(1H, br d, J=12.9Hz, H-11); 3.82(1H, br dd, J=8.9 and 9.5Hz, H-6); 4.00(1H, dq, J=6.3 and 6.4Hz, H-22); 5.44(1H, d, J= 9.5Hz, H-7); and 5.82ppm (1H, br s, H-13). 13CNMR: (CDC13) 52.0, C-I; 35.8, C-2; 27.0, C-3; 115.6, C-4; 143.6, C-5; 39.6, C-6; 129.6, C-7; 135.0, C-8; 36.4, C-9; 23.9, C-10, 43.6, C-11; 188.0, C-12; 124.2, C-13; 212.0, C-14; 24.2, C-15; 15.5, C-16; 17.0, C-17; 29.1, C-18; 21.2, C-19; 22.1, C-20; 52.9, C-21; 67.5, C-22; 20.8, C-23; and 175.3ppm C-24. References S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis, Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lett., Vol. 40, pp. 2725-2728(1999). S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis; Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra, Phytochemistry, Vol. 55, pp. 663-673 (2000).

1.

Atranones

13

Common/Systematic Name Atranone F Molecular Formula/Molecular Weight C24H3207; M W = 4 3 2 . 2 1 4 8 0 17

IV~e

Mek

Me.

0

o

Me

.

Me

Me

o

General Characteristics Pale yellow film; [(~]D20 Jr 24 ~ (c=0.70 in CHC13). Fungal Source

Stachybotrys chartarum (S. atra) S-11.

Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. chartarum, S- 11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give a black gum. The majority of the extract was chromatographed over PEI silica, eluting with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 3 (eluted with 1-20% methanol-methylene chloride) was loaded onto a 2mm Chromatotron plate and eluted with ethyl acetate-hexane-methanol (gradient from 1:9:0, v/v/v to 70:30:1, v/v/v) to give atranones F and G. Spectral Data UV:

)~ m~M~~ 237nm (e = 4,170). IR:

(CHCI3) 3369(br), 2969, 2934, 1807, 1743, 1712, 1691, 1382, 1230, 1196, 1030, and 991cm1. 1H NMR: (CDC13) 1.06(38, d, J--7.0Hz, 8-20); 1.18(3H, d, J=6.6Hz, 8-19); 1.31(3H, s, H-15); 1.75(38, s, H-16); 1.77(38, br s, 8-17); 1.87(1H, m, H-10B); 2.00(1H, m, n-2a); 2.02(3H, s, 8-23); 2.14(18, m, n-3a); 2.34(18, m, H-aA); 2.62(1H, qq, J=6.6 and 6.7Hz, n-18); 2.71(18, AB, J=18.1Hz, n-21a); 2.74(18, m, H-10A); 3.24(18,

14

1.

Atranones

dd, J=l 1.0 and 11.4Hz, H-2A); 3.27(1H, dd, J=2.2 and 9.2 Hz, H-11); 3.43(1H, AB, J=18.1Hz, H-21A); 5.79(11-1, s, H-13); 5.83(1H, brt, J=8.4Hz, H-9); and 6.08ppm (1H, d, Jr=1Hz, H-7). 13C NMR: (CDCI3) 86.4, C-l; 37.4, C-2; 23.6, C-3; 117.0, C-4; 149.5, C-5; 76.5, C-6; 75.2, C-7; 132.6, C-8; 134.0, C-9; 25.7, C-10; 43.9, C-11; 169.1, C-12; 112.6, C-13; 165.0, C-14; 21.8, C-15; 16.9, C-16; 18.9, C-17; 31.0, C-18; 20.6, C-19, 23.7, C-20; 41.0, C-21; 169.7, C-22; 20.7, C-23; and 170.9ppm C-24. References S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis; Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lett., Vol. 40, pp. 2725-2728(1999).

S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis, Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra; Phytoehemistry, Vol. 55, pp. 663-673 (2000).

1.

Atranones

15

Common/Systematic Name Atranone G Molecular Formula/Molecular Weight C25H3408; M W = 4 6 2 . 2 2 5 3 7 17

o.o o Me

Me

\

2"

Me.

Me

Me

--o ~s

General Characteristics Pale yellow film; [~]D 20 + 28~ (c=0.20 in CHC13). Funsal Source

Stachybotrys chartarum (S. atra) S-11.

Isolation/Purification After four weeks of growth, a rice culture of Stachybotrys atra (S. charta~m, S- 11) was extracted with methanol-chloroform and, following solvent removal, the crude extract triturated with hexane to give a black gum. The majority of the extract was chromatographed over PEI silica, eluting with increasing proportions of methylene chloride in hexane, then increasing amounts of methanol in methylene chloride to give four fractions. Fraction 3 (eluted with 1-20% methanol-methylene chloride) was loaded onto a 2mm Chromatotron plate and eluted with ethyl acetate-hexane-methanol (gradient from 1:9:0, v/v/v to 70:30:1, v/v/v) to give atranones F and G. Spectral Data UV:

~, MO ,m . ax237nm (e= 4,170). IR:

(CHC13) 3390, 2963, 2930, 1808, 1743, 1718, 1374, 1260, 1233, 1092, and 1023cm1. 1H N]k,IR: (CDCh) 1.21(3H, d, J-6.9Hz, H-19); 1.31(3H, d, J=6.9Hz, H-20); 1.36(3H, s, H-15); 1.73(3H, s, H-16); 1.76(31-1,br s, H-17); 1.87(1H, dd, J=8.0 and 15.5Hz, H-10B); 1.97(1I-I, m, H-2B); 2.01(3H, s, H-23); 2.10(1H, dd, J=5.1 and 11.8Hz, H-3B); 2.24(1H, ddd, J=8.3, 11.8 and 14.0Hz, H-3A); 2.49(1H, br qq, J-6.9 and 6.9Hz, H-18) 2.70(1H, m, H-10A); 2.74(1H, AB, J=18.0Hz, H-21B); 3.22(1H, dd,

16

1.

Atranones

J=12.7 and 13.3Hz, H-2A); 3.26(1H, br d, J=9.0Hz, H-11); 3.45(1H, AB, J=18.0Hz, H-21A) 3.67(3H, s, OMe); 5.80(1H, dd, ,/--8.0 and 8.1Hz, H-9) and 6.05ppm (11t, s, H-7). 13C NMR: (CDC13) 85.7, C-l; 37.2, C-2; 23.5, C-3; 116.5, C-4; 149.7, C-5; 76.1, C-6; 75.4, C-7; 132.6, C-8; 134.3, C-9; 26.1, C-10; 44.5, C-11; 147.1, C-12; 141.0, C-13; 161.1, C-14; 21.3, C-15; 16.5, C-16, 20.6, C-17, 30.3, C-18; 20.3, C-19; 20.8, C-20, 40.9, C-21; 169.7, C-22; 20.6, C-23; 171.0, C-24, and 59.4ppm OMe.

References S. F. Hinkley, J. Jiang, E. P. Mazzola, and B. B. Jarvis; Atranones: Novel Diterpenoids from the Toxigenic Mold Stachybotrys atra; Tetrahedron Lea., Vol. 40, pp. 2725-2728(1999). S. F. Hinkley, E, P. Mazzola, J. C. Fettinger, Y.-F. Lam, and B. B. Jarvis, Atranones A-G: A Unique Series of Metabolites from the Toxigenic Mold Stachybotrys atra; Phytochemistry, Vol. 55, pp. 663-673 (2000).

Austalides Austalide A Austalide B Austalide C Austalide D Austalide E Austalide F Austalide G Austalide H Austalide I Austalide J Austalide K Austalide L

17

This Page Intentionally Left Blank

2.

Austalides

19

Common/Systematic Name Austalide A Molecular Formula/Molecular Weight C2si-I3609; MW = 516.23 593 23

Me

24

34 33 Me / MeOCO.. ~ . . . ~ O ~ 7 i~..~9j~ 25

�9

13[

Me ~ M e '60

"'

.u-l.'-"

"1"11

1

T"

iT-

] \

~3J 0

I.

A.^

O

MeO 28

General Characteristics Crystals from chloroform-methanol; mp., 212-214~

[(Z]D24 -84.4 ~ (c=l.00, in CHCI3).

Isolation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 25"C. Cultures were dried at 450C for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform andwater, yielding toxic material in the organic layer. This material was fraetionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction A was highly lipidic in nature and was dissolved in aqueous methanol (90%) and repeatedly extracted with n-hexane. The resulting polar material was subjected to column chromatography on silica gel 60H with chloroform as eluant, to give, in order of elution, austoeystin A, austalide A, and 7-ehloro2-(3-furyl)-l,3,8-trimethoxyxanthone. Fungal Source Aspergillus ustus (isolate MRC 1163).

Biolo~calActivity Reportedtobe toxicto ducklings.

20

2.

Austalides

Spectral Data UV:

~

MeOH

222(~ = 35,400) and 267nm (17,140).

IR"

(KBr) 1740emq. 1H NMR: (CDCI3) 0.970(3H, s, H-27); 1.213(3H, s, Me); 1.318(3H, s, Me); 1.569(3H, s, Me); 1.822-1.946(4H, rn, H-18 and H-19); 2.030(3H, s, H-23); 2.530(1H, dd, J=16.1 and 2.2Hz, H-12); 2.136(1H, dd, d=16.1, and 4.4Hz, H-12); 2.398(1H, d, J=-8.2Hz, H-21), 5.067(1H, dd, J=4.4 and 2.2Hz, H-13); 2.827(1H, dd, dr-- 18.7 and 8.2Hz, I-k-22); 2.930 (1H, d, J=-18.7Hz, Ha-22); 3.417 (3H, s, H-28); 4.087 (3H, s, H-29); and 5.070ppm (2H, s, H-I). 13CNMR.: 10.365, q, C-23; 17.92, t, C-22; 17.95, q, C-27; 21.01, q; 27.39, q; 28.45, q; 25.81, q; 30.28, t; 30.56, t; 35.86, d, C-21; 40.35, s, C-20; 38.00, t, C-12; 48.69, q, C-28; 61.78, q, C-29; 68.04, t, C-I; 70.84, d, C-13; 75.48, s, C-11; 84.41, s; 85.33, s; 107.27, s; 113.79, s; 115.57, s; 119.24, s; 145.53, s; 155.23, s; 157.79, s; and 169.13ppm,s, C-3. Mass Data: HREIMS: 516.236re~e; found: C, 55.05; H, 5.85%; C2gI-I3609.CHCI3requires 516.236; C, 54.77; H, 5.86%. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuelear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985).

2.

Austalides

21

Common/Systematic Name Austalide B Molecular Formula/Molecular Weight C 2 6 H 3 4 0 8 , M W -" 474.22537

24

HO..... 13ri"~~11 0 25Me ....L Me,,L ~ . 26 M e

23

Me

\~~j0~~21" ~ ~ 17y

~..J~3 2

ONe 29

-'~

0

MeO 28

General Characteristics Crystals from benzene-n-hexane; mp., 243-245~

[~]D 24 -46.2 ~ (c=l.00, in CHC13).

Isolation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 25~ Cultures were dried at 45~ for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction B was purified by silica gel column chromatography with chloroform as eluant to give, in order of elution, 4,6-0, Obisdemethylaustocystin A, 4-O-demethylaustocystin A, 6-O-demethylaustocystin A, 8deoxy-6-O-methylversicolorin A, and a mixture. The mixture was subjected to column chromatography on silica gel with n-hexane-ethyl acetate (1:1, v/v) as eluant to give austocystin H, austalide A, austalide B, and austalide C. Fungal Source Aspergillus ustus (isolate MRC 1163).

22

2.

Austalides

Biological Activity Reported to be toxic to ducklings. Spectral Data UV~

~

MeOH max

223(e = 28,700) and 269nm (16,800).

IR~

(KBr) 1740cm"1. 1H NMR: (CDCls) 0.969(3 H, s, H-27); 1.249(3H, s, Me); 1.462(3H, s, Me); 1.631(3H, s, Me); 1.689-1.935(4H, m, H-18 and -19); 2.022(3H, s, H-23); 2.322(2H, d, J=3.3Hz, H-12); 2.379(1H, d, J=7.7Hz, H-21); 2.491(1H, d, J=8.5, OH-13); 2.85(1H, dd, J=18.7 and 7.7Hz, Hb-22); 2.925(1H, d, J=l 8.7Hz, Ha-22); 3.397(3H, s, H-28); 4.098(1H, dd, J= 8.5 and 3.3Hz, H-13); 4.122(3H, s, H-29); and 5.104ppm (2H, s, H-l). 13CNMR: (CDCI3) 10.65, q, C-23; 18.53, t, C-22; 19.23, q, C-27; 26.18, q; 27.14, q; 28.84, q; 30.4, t; 31.54, t; 36.68, d, C-21; 39.74, s, C-20; 41.32, t, C-12; 48.75, q, C-28; 62.01, q, C-29; 68.15, t, C-I; 69.91, d, C-13; 78.29, s, C-11; 84.85, s; 86.35, s; 108.22, s; 114.26, s; 116.57, s; 118.95; 145.64, s; 155.43, s; 157.13, s; and 169.07ppm, s, C-3. Mass Data: LREIMS: 474re~e; found: C, 65.65; H, 7.2%; C26H3408requires 474; C, 65.81 H, 7.22%. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites of Aspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985).

2. Austalides

23

Common/Systematic Name Austalide C Molecular Formula/Molecular Weight C 3 0 H 3 5 O l l ; M W --- 574.24141 34

33

MeOCO..

23 Me

24

I

Me

~I/0~7~,.~9

2. 16 0 -u

u-~. "i" ~i"

~x

Ld ~ A... OMe -s

0 "

MeO 28

General Characteristics A white amorphous solid; [{~]D24

-99.0 ~

(c=l.00, in CHCI3).

Isolation/Purification See austalide B. Fungal Source

Aspergillus ustus (isolate MRC 1163).

Biological Activity Reported to be toxic to ducklings. Spectral Data UV:

J,~,xM~~ 221(e = 26,900) and 265nm (13,800). IR:

(KBr) 1740cm1. :H NMR: (CDCI3) 1.023(3H, s, H-27); 1.232(3H, s, Me); 1.348(3H, s, Me), 1.705(3H; s, Me); 1.951(1H, d, J=15.0Hz, I-Ib-18); 1.966(3H, s, H-34), 2.034(3H, s, Me); 2.043(3H, s, H-32); 2.147(1H, dd, J=16.0 and 4.2I-~ I-Ib-13); 2.202(1H, d, J=8.5 Hz, H-21); 2.302(1H, dd, J=15.0 and 6.1Hz, I-I~-18);2.600(1H, dd, J=16.0 and 2.1Hz, H-12); 2.979(1H, dd, J=19.0 and 8.5Hz, I-Ib-22), 3.266(1H, d, J=19.0Hz, I-I~-22); 3.41(3H, s, H-28); 4.142(3H, s, H-29); 5.077(2H, s, H-l); 5.147(1H, dd, J=4.2 and 2.1I-~ H-13); and 5.506ppm (1H, d, J=6. IHz, H-19).

24

2.

Austalides

13C NMR: (CDC13) 10.38, q, C-23; 12.94, q, C-27; 19.35, t, C-22; 21.02, q, C-34; 21.06, q; 25.44, q; 27.63, q; 29.37, q; 37.12, t; 37.61, d, C-21; 37.8, t; 45.50, s, C-20; 48.79, q, C-28; 62.04, q, C-29; 68.05, t, C-I; 70.72, d; 70.95 d; 75.47, s, C-11; 85.25, s; 85.33, s; 107.76, s; 113.67, s; 115.34, s; 117.83, s, C-17; 145.61, s; 155.50, s; 157.36, s; 169.04, s; 169.22, s, C-33; and 170.06ppm, s, C-31. Mass Data: HREIMS: 574.242m/e; found: C, 62.45; H, 6.5%; C30H38Oll requires 574.241; C, 62.7; H, 6.6%. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345- 356 (1985).

2. Austalides

25

Common/Systematic Name Austalide D Molecular Formula/Molecular Weight C28I'-I36010; M W "- 532.23085 23

Me

24

Me HO,, ~ , , ~ . , , ~ 0 ~ 9 i'3r

25

28Me ----

. . . . "~~~i

":'

I"11

..~

& r"

,T

OMe ....OCOM32e OMe

' \

o

,o

MeO 28

General Characteristics Crystals from acetone; mp., 259-261 ~

[OQD24 -73.4 ~ (c=l.00, in CHCIs).

Isolation~urification

Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21

days at 25~ Cultures were dried at 45~ for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction C was purified by silica gel column chromatography; elution with n-hexane-ethyl acetate (3:2, v/v) gave,in order of elution, 8deoxy-6-O-methylversicolorin A, austocystins A, B, and H, and austalides D and E. Funsal Source

Aspergillus ustus (isolate MRC 1163).

Biological Activity Reported to be toxic to ducklings.

26

2.

Austalides

Spectral Data UV~ ~mMffH 222(e = 32,700) and 268nm (17,700).

IR: (KBr) 1745cm~. 1H NMR: (CDC13) 0.964(3H, s, H-27); 1.258(3H, s, Me); 1.488(3H, s, Me); 1.771(3H, s, Me); 2.274(1H, dd, J=15.0 and 6.1Hz, H-18); 1.935(1H, d, J=15.0Hz, H-18); 2.019(3H, s, H-23); 2.399(1H, dd, J=15.8 and 2.2Hz, H-12); 2.320(1H, dd, J=15.8 and 4.2Hz, H12); 2.176(1H, d, J=8.5Hz, H-21), 4.184(1H, m, J= 4.2 and 2.2Hz, H- 13); 2.630(1H, d, J=8.0Hz, OH-13); 2.975 (1H, dd, J-18.9 and 8.5Hz, Hb-22); 3.229(1H, d , J = 18.9 Hz, Ha-22); 3.378(3H, s, H-28); 4.119(3H, s, H-29); 2.041(3H, s, H-32 and 5.087ppm (2H, s, H-l). 13C NMR: 10.61, q, C-23; 19.73, t, C-22; 14.07, q, C-27; 21.13, q; 27.35, q; 29.65, q; 25.73, q; 37.34, t; 38.49, d, C-21; 44.98, s, C-20; 40.84, t, C-12; 48.17, q, C-28; 62.16, q, C-29; 68.10, t, C-I; 69.92, d, C-13; 78.23, s, C-11; 86.10, s, C-14, 85.69, s, C-15; 71.07, d, C-19; 108.63, s; 114.10, s; 116.26, s; 117.47, s; 145.76, s; 155.61, s; 156.58, s; 170.27, s; and 168.93ppm, s, C-3.

Mass Data: LREIMS: 532m/e; found: C, 63.15; H, 6.8%; C28H3601orequires 532; C, 63.14; H, 6.1%. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids, J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985).

2.

Austalides

27

Common/Systematic Name Austalide E Molecular Formula/Molecular Weight C28I-I36010; M W -" 532.23085 34

24

3a

Me

23

MeOCO,,~ , , J ~ O ~ ~ 9 2s i'gr -' :11 M~ ~LMe~ ~r-"

26Me

'~S'JO~, jH21V 17~~,//19 ....OH

MeO 28

Me '

.:x ,r ,\ L3/O

~~OMe~0 '~'~

General Characteristics Crystals from acetone; mp., 262-264~

[a]D 24 - 7 3 . 4 ~

(c=l.00, in CHCI3).

Isolation/Purification See austalide D. Fungal Source

Aspergillus ustus (isolate MRC 1163).

Biological Activity Reported to be toxic to ducklings. Spectral Data UV:

~

MeOH max

222(e= 34,600) and 266nm (19,600).

IR:

(KBr) 1745em"1.

:H NMR: (CDCI3) 1.162(3H, s, H-27); 1.200(3H, s, Me); 1.357(3H, s, Me); 1.690(3H, s, Me); 1.976(3H, s, H-34), 2.043(1H, d, J=14.6Hz, 18-I-Ib),2.058(3H, s, Me); 2.108(1H, d, J=- 9.1Hz, H-21); 2.142 (1H, dd, J=16.0 and 3.5, I-tb-12), 2.355(1H, dd, J=14.6 and 5.0Hz, H-18); 2.558(1H, br d, J=16.0Hz, I-I~-12);2.873(1H, dd, J=18.8 and 9.1Hz, I-Ib22); 3.009(1H, d, J=-lS.8H~ Ha-22); 3.410(3H, s, H-28), 4.020(1H, m, H-19); 4.105(3H, s, H-29); 5.092(2H, s, H-I); and 5.115ppm (1H, rn, H-13).

28

2.

Austalides

13C NMR: (CDC13) 10.38, q, C-23; 13.34, q, C-27; 19.10, t, C-22; 21.09, q, C-34; 25.89, q; 27.63, q; 29.20, q; 37.61, t; 37.75, d, C-21; 39.03, t, C-18; 46.29, s, C-20; 49.17, q, C28; 62.00, q, C-29; 68.08, t, C-l; 70.76, d, C-19; 70.82, d, C-13; 75.53 s, C-11; 85.78, s; 86.03, s;.107.33, s; 113.66, s; 115.10, s; 118.76, s, C-17; 145.63, s; 155.33, s; 157.57, s; 169.13, s; and 169.36, s, C-33.

Mass Data: LREIMS: 532re~e; found: C, 63.05; FI, 6.85%; C2sH36010 requires 532; C, 63.14; H, 6.18%. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites of Aspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985).

2. Austalides

29

Common/Systematic Name Austalide F Molecular Formula/Molecular Weight C~H3409; MW = 490.22028 23

Me

24

Me HO,, i , ~ L ~ o ~ 7 ~ ,

=Me 16

0

ieJ.

i'3r "' Tll

., ,

o ..:~ '\

&

IJ )o

r--

,r

OMe

0

MeO 28

General Characteristics Crystals from acetone; mp., 261-263~

[tt]D24 -57.7 ~ (c=l.00, in CHCIa).

Isolation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 25*C. Cultures were dried at 450C for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to dayold ducklings. Fraction C was purified by silica gel column chromatography; elution with n-hexane-ethyl acetate (3:2, v/v) gave,in order of elution, 8-deoxy-6-O-methylversicolorin A, austocystins A, B, and H, and austalides D and E. Subsequent elution of the column with n-hexane-ethyl acetate (3:7, v/v) gave austalide F. Fungal Source Aspergillus ustus (isolate MRC 1163). Biological Activity Reported to be toxic to ducklings.

30

2.

Austalides

Spectral Data UV~

~

MeOH max

220(e = 30,900) and 264nm (16,800).

IR~

(KBr) 1740cm~. 1H NMR: (CDCI3) 1.144(3H, s, H-27); 1.240(3H, s, Me); 1.491(3H, s, Me); 1.764(3H, s, Me); 2.034(3H, s, Me); 2.067-2.096(2H, m, H-18); 2.313-2422(3H, m); 2.710(1H, d, J=8.7Hz, OH); 2.895(1H, dd, J=18.8 and 8.3Hz, Hb-22); 2.988(1H, d, J=18.8 Hz, Hb22); 3.051(1H, d, J=10.1Hz, OH); 3.393(3H, s, H-28); 3.922(1H, m, H-19); 4.126(3H, s, H-29); 4.131(1H, m, H-13); and 5.110ppm (2H, s, H-l). 13C N M R :

(CDC13) 10.54, q, C-23; 14.32, q, C-27; 19.47, t, C-22; 26.02, q; 27.23, q; 29.36, q; 38.14, t, C-18; 39.06, d, C-21; 40.78, t, C-12; 45.60, s, C-20; 49.09, q, C-28; 62.05, q, C-29; 68.07, t, C-I; 69.72, d, C-13; 71.11, d, C-19; 78.31, s, C-11; 86.i2, s; 86.93, s; 108.16, s; 114.08, s; 116.02, s; 118.40, s, C-17; 145.72, s; 155.35, s; 156.71, s; and 168.96ppm s, C-3. Mass Data: LREIMS: 49Ore~e;found: C, 63.65; H, 6.9%; 6.99%.

C26H3409

requires 490; C, 63.66; H,

References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985).

2.

Austalides

31

Common/Systematic Name Austalide G Molecular Formula/Molecular Weight C28H3809; MW 518.25158 -

-

Me Me

MeOCO,,, , / - , . ~ 0 ~

I

HO"

Me I

H ~ H

~...t k

II 3o

I

OMe '6

MeO.~ 0 General Characteristics Isolated as a colorless glass; [a]D24 -100.2 ~ (C=I.00, in CHC13). Isolation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 25~ Cultures were dried at 45~ for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction C was purified by silica gel column chromatography; elution with n-hexane-ethyl acetate (3:2, v/v) gave, in order of elution, 8deoxy-6-O-methylversicolorin A, austocystins A, B, and H, and austalides D and E. Subsequent elution of the column with n-hexane-ethyl acetate (3:7, v/v) gave austalides F, G, and I. Fungal Source Aspergillus ustus (isolate MRC 1163). Biological Activity Reported to be toxic to ducklings.

32

2.

Austalides

Spectral Data UV:

Zm~" 2213(e=26,500) and 267nm (14,100). IR: (KBr) 1720-1770cm q. 1H ~ : (CDC13) 1.022(3H, s, H-27); 1.171(3H, s, Me); 1.380(3H, s, Me); 1.593(1H, s br, OH); 1.626(1H, d, J=8.2Hz, H-21), 1.697(1H, d, J=2.2Hz, H-14), 1.767(1H, dd, J-16.0 and 4.2Hz, Hb-12), 1.823(1H, ddd, J--15.0, 11.5 and 60Hz, Hb-19); 1.967(3H, s, Me); 1.968(3H, s, Me); 2.029(3H, s, Me), 2.250(1H, ddd, J=15.3, 11.2, and 6.0Hz, Hb-18); 2.417(1H, ddd, J--15.0, 11.2, and 3.6 Hz, Ha-19); 2.538(1H, ddd, J--15.3, 11.5, and 3.6 Hz I-L-18); 2.718 (1H, dd, J=18.6 and 8.2Hz, Hb-22); 2.624(1H, dd, ,/=16.0 and 2.3Hz, H-12); 3.004(1H, d, J=18.6Hz, Ha-22); 3.673(3H, s, H-28); 4.11 l(3H, s, H-29); 5.088(2H, s, H-I); and 5.391ppm (1H, ddd, J-4.2, 2.3, and 2.2Hz, H-13).

13C NMR: (CDC13) 10.53, q, C-23; 17.24 t, C-22; 20.66, q, C-27; 21.54, q, C-34; 27.81, q; 29.21, t, C-19; 29.80, q; 34.26, q; 34.50, t, C-18; 41.01, d, C-21; 41.30, s, C-20, 42.01, t, C-12; 51.67, d, C-14; 51.67, q, C-28; 62.04, q, C-29; 68.15, t, C-I; 75.15, s; 71.03, d, C-13; 75.56, s; 107.38, s; 113.79, s; 115.36, s; 145.58, s; 155.46, s; 158.10, s; 169.27, s; 170.29, s; and 174.47ppm, s, C-17. Mass Data: HREIMS: 518.25 lm/e (M) +, C28H3809 requires 518.252. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaxtr, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 3. Structure Elucidation of Austalides G-L; J. Chem. Soc. Perkin Trans. I, pp. 363367(1985).

2. Austalides

33

Common/Systematic Name Austalide H Molecular Formu!aiMolecular Weight C26H3608; MW = 476.24102

Me

Me

HO,,, , ~ 0 ~

Me_ I Uel

HC) H ) H MeO,,~

.,.,~.........X

I-

II ;o

OMe

'O

0 ~ n e r a l Characteristics Isolated as a colorless glass; [a]D 24 -19.5 ~ (c=l.00, in CHCIa). !solation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 25"C. Cultures were dried at 45"C for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction D was purified by silica-gel column chromatography with chloroform-methanol (98:2, v/v) as eluant to give, in order of elution, 8-deoxy-6-O-methylversieolorin A, austocystin D, and a mixture. This mixture was subjected to column chromatography on silica gel 60H with n-hexane-ethyl acetate (1:1, v/v) as eluant to afford austalide F, G, and H. Fungal Source Aspergillus ustus (isolate MRC 1163). Biolo~eal Activity Reported to be toxic to ducklings.

34

2.

Austalides

Spectral Data

UV~ 221 (e=20,400) and 267nm (9,600).

IR~ (KBr) 1740cm1. 1H NMR: (CDC13) 0.962(3, s, H-27); 1.244(3H, s, Me), 1.322(1H, d, J-2.5Hz, H-14); 1.431(3H, s, Me); 1.437(3H, s, Me); 1.655(1H, d, J=7.9 Hz, H-21); 1.799(1H, dd, J=15.3 and 3.7Hz, Hb-12); 1.885-1.998(1H, m); 2.022(3H, s, H-23); 2.187-2.273(2H, m); 2291-2.389(1H m); 2.504(1H, dd, J= 15.3 and 3.1Hz Ha- 12); 2.771 (1H, dd, J=18.7 and 7.9Hz, Hb-22); 3.071(1H, J=18.7Hz, Ha-22); 3.683(3H, s, H-28); 4.128(3H, s, H-29), 4.629(1H, m, H-13); and 5.100ppm (2h, s, H-l). 13C NMR: (CDC13) 10.76, q, C-23; 18.49, t, C-22; 21.48, q, C-27; 27.44, q; 29.62, t; 31.67, q; 33.49, q; 35.00, t; 40.41, d, C-21; 40.96, s, C-20; 43.99, t, C-12; 49.43, d, C-14; 51.82, q, C-28; 62.17, q, C-29; 68.15, t, C-I; 69.96, d, C-13; 75.61, s; 78.60, s; 108.51, s; 114.09, s; 116.00, s; 145.75, s; 155.51, s; 156.50, s; 160.0, s, C-3; and 173.87ppm s, C- 17. Mass Data: HREIMS: 476.240m/e (M) +, C26H3608 requires 476.241. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. technique to the Structure elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 3. Structure Elucidation of Austalides G-L; J. Chem. Soc. Perkin Trans. I, pp. 363367(1985).

2.

Austalides

35

Common/Systematic Name Austalide I Molecular Formula/Molecular Weight C27H3408, ~ "- 486.22537 Me

Me

MeOCO,,, ,,...,~~0,.~ ~ . ~ Me '"l Me ] l- II (~~,~

H H

;o

OMe ~0

0 General Characteristics Crystals; mp., 236-238~

[a]D24 -132.6 ~ (c=l.00, in CHCI3).

Isolation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 25~ Cultures were dried at 45~ for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1"1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction D was purified by silica-gel column chromatography with chloroform-methanol (98:2, v/v) as eluant to give, in order of elution, 8-deoxy-6-O-methylversicolorin A, austocystin D, and a mixture. This mixture was subjected to column chromatography on silica gel 60H with n-hexane-ethyl acetate (1:1, v/v) as eluant to afford austalide F, G, and H. Subsequent elution of the column with n-hexane-ethyl acetate (3:7, v/v) gave austalides F, (3, I, and J. Fungal Source Aspergillus ustus (isolate MRC 1163). Biological Activity Reported to be toxic to ducklings.

36

2.

Austalides

Spectral Data UV:

maxM~~ 221 (e=35,600) and 266nm (17,000). IR~

(KBr) 1720-1750cm 1. 1H NMR:

(CDC13) 1.065(3H, s, H-27); 1.205(3H, s, Me); 1.486(3H, s, Me); 1.501(3H, s, Me); 1.571(1H, d, J=8.1Hz, H-21); 1.829(1H, m); 1.862(1H, d, J=3.9 Hz, H-14); 1.989(3H, s, Me); 2.005(3H, s, Me); 2.085-2.135(1H, m); 2.677-2.726(2H, m); 2.783-2.848(2H, m); 2.844(1H, dd, J=18.8 and 8.1Hz, Hb-22); 3.079(1H, d, J=18.8Hz, H-22); 4.127(3H, s, H-29); 5.103(2H, s, H-I); and 5.407ppm (1H, m, H-13). lac NMR: (CDCI3) 10.23, q, C-23; 15.84, q, C-27; 18.10, t, C-22; 21.23, q, C-34; 26.48, q; 27.43, q; 31.24, t, C-19; 33.89, q; 36.59, t, C-18; 40.86, s, C-20; 41.63, t, C-12; 45.94, d, C-21; 55.35, d, C-14; 61.86, q, C-29; 67.99, t, C-I; 69.92, d, C-13; 74.96, s, C-11; 85.12, s, C-15; 107.30, s; 113.74, s; 114.82, s; 145.56, s; 155.14, s; 157.58, s; 168.97, s; 169.76, s; and 174.40ppm s, C-17. Mass Data: HR IMS: 486.225m/e (M) § C27H3408 requires 486.225; found: C, 66.4%; H, 6.8 requires C, 66.65%; H, 7.04. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 3. Structure Elucidation of Austalides G-L; J. Chem. Soc. Perkin Trans. I, pp. 363367(1985).

2. Austalides

37

Common/Systematic Name Austalide J Molecular Formula/Molecular Weight C25H3207; i W

"-- 4 4 4 . 2 1 4 8 0

Me

H,,,,

'"IMel I ~~_~ H 0

Me

Me

x

II ;o ~0 OMe

General Characteristics Crystals from methanol; mp., 284-286~

laiD 24 -42.1 o (c=l.00, in CHCI3).

Isolation/Purification Aspergillus ustus was grown in bulk on wet, sterilized, whole yellow maize kernels for 21 days at 250C. Cultures were dried at 45~ for 24 hours and milled to a fine meal. The resulting material was acutely toxic to day-old ducklings. The dried, milled, moldy maize was extracted with chloroform-methanol (1:1, v/v) for 48 hours. The solvent was removed under reduced pressure and the resulting, toxic extract was partitioned between aqueous methanol (90%) and n-hexane. The methanol solution was evaporated and the residual material partitioned between chloroform and water, yielding toxic material in the organic layer. This material was fractionated on a formamide impregnated cellulose powder column which was eluted with solvent of increased polarity from n-hexane, n-hexanebenzene, benzene, to benzene-ethyl acetate (1:1, v/v). Fractions (100ml) were collected and combined on the basis of their chromatographic behavior on thin-layer chromatography affording five fractions (A-E). Each of these fractions was partitioned between chloroform and water to remove any traces of formamide. All five fractions were acutely toxic to day-old ducklings. Fraction D was purified by silica-gel column chromatography with chloroform-methanol (98:2, v/v) as eluant to give, in order of elution, 8-deoxy-6-O-methylversicolorin A, austocystin D, and a mixture. This mixture was subjected to column chromatography on silica gel 60H with n-hexane-ethyl acetate (1:1, v/v) as eluant to afford austalide F, G, and H. Subsequent elution of the column with n-hexane-ethyl acetate (3:7, v/v) gave austalides F, (3, I, and J. Funaal Source Aspergillus ustus (isolate MRC 1163). Biological A~ivity Reported to be toxic to ducklings.

38

2.

Austalides

Spectral Data

UV; ~,~H

221(~=31,900) and 267nm (16,200).

IR; (KBr) 1720, 1750, and 1760cm"~. 1H N M R : (CDCI3) 0.852(31-I,s, H-27); 1.190(3H, s, Me); 1.365(3I-I,s, Me); 1.385(3H, s, Me); 1.719-1.748(1H, m); 1.826-1.900(1H, m); 1.925-1.965(2H, m); 2.020 3H, s, H-23); 2.035-2.176(2H, m); 2.417-2.517(2H, m); 2.620-2.683(1H, m); 2.799(1H, dd, J=18.6 and 8.0Hz, Hb-22); 2.909(1H, d, Jr=18.6 Hz, Ha- 22), 4.091 (3I-I, s, H-29); and 5.090ppm (2H, s, H- 1). 13CNMR: (CDCI3) 10.57, q, C23; 17.94, t, C-22; 20.75, q, C-27; 25.22, t; 26.90, q; 27.14, t; 29.13, t, C-19; 29.13, q; 30.12, q; 32.98, t; 37.43, d, C-21; 39.73, s, C-20; 61.93, q, C-29; 68.18, t, C-I; 75.85, s, C-11; 79.58, s, C-15; 91.33, s, C-14; 107.38, s; 114.38, s; 115.26, s; 145.63, s; 155.18, s; 158.22, s; 169.27, s; and 173.38ppm s, C-17. Mass Data: HREIMS: 444.214m/e (M)+, C25H3207requires 444.213; found: C, 67.75%; H, 7.35: requires C, 67.55%; H, 7.26. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuelear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; Ji Chem. Soc. Perkin Trans. I, pp. 345-356(1985). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 3. Structure Elucidation of Austalides G-L; J. Chem. Soc. Perkin Trans. I, pp. 363367(1985).

2.

Austalides

39

Common/Systematic Name Austalide K Molecular Formula/Molecular Weight C25H3205; M W = 4 1 2 . 2 2 4 9 7

H, .....

0-/

v

Me

Me

MeO

General Characteristics Isolated as a white glass; [{X]D24 -75.9 ~ (c=l.00, in CHC13). Isolation/Purification Aspergillus ustuswas cultured on malt extract medium at 25~ in static culture. After 14 days the cultures were filtered and the mycelium macerated with acetone in a Waling blender. The acetone mixture was filtered, evaporated, and the resulting brown gum was partitioned between aqueous methanol (90%) and n-hexane. Evaporation of the methanol solution gave a residue which was partitioned between chloroform and water. The organic layer was concentrated and percolated through an aluminum oxide column with chloroform-methanol (95:5, v/v). The combined eluants were evaporated to give a lightyellow gum. This material was subjected to column chromatography on silica gel with nhexane-ethyl acetate (1:1, v/v) as eluant. Fractions were evaluated by TLC (n-hexaneethyl acetate, 1:1, v/v) and appropriate fractions combined to give three fractions: Re 0.48, 0.31, and 0.13. The latter fraction crystallized from n-hexane-acetone to give austalide J. The material with Rr 0.48 was purified on a silica gel column. Elution with chloroformmethanol (99:1, v/v) gave austalide A and austalide K. Fungal Source Aspergillus ustus (isolate MRC 1163). Biological Activity Reported to be toxic to ducklings. Spectral Data or:

~.mMffH

222(e=18,900) and 267nm (11,000).

40

2.

Austalides

IR:

(KBr) 1700 and 1745cm1. 1H N M R : (CDC13) 0.697(3H, s, H-27); 0.999(3H, s, Me); 1.090(3H, s, Me); 1.165(3H, s, Me); 1.458-1.518(4H, m, Hb-13, H-14, Hb-19, and H-21); 1.615(1H, ddd, J--13.9, 13.9, and 4.2Hz, H-12); 1.785(1H, dddd, J--13.9, 13.9, 13.9, and 3.0Hz, H-13); 2.018(3H, s, H23); 2.072(1H, ddd, J--13.3, 6.9, and 3.8Hz, H- 19); 2.260(1H, ddd J=13.9, 3.0, and 3.0Hz, H-12); 2.365(1H, ddd, J=16.1, 6.5, and 3.8Hz, Hb-18); 2.485(1H, ddd, J=16.1, 11.6, and 6.9Hz, H-18); 2.790(1H, dd, J=18.5 and 8.0 Hz, Hb-22); 2.910(1H, d, J--18.5 Hz, H-22); 4.078(3H, s, H-29); and 5.087ppm (2H, s, H-I). 13C NMR: (CDCI3) 10.51, q, C-23; 14.07, q, C-27; 18.15, t, C-22; 21.53, q; 26.97; 26.61, q; 29.54, t; 33.91, t, C- 18; 37.49, s, C-20; 38.24, t; 39.60, t, C-19; 46.95, d, C-21; 47.09, s, C-15; 54.07, d, C-14; 61.75, q, C-29; 68.07, t, C- 1; 76.20, s, C-11; 107.18, s; 114.29, s; 115.14, s; 145.38, s; 155.23, s; 158.45, s; 169.17, s, C-3; and 216.23ppm s, C-17. Mass Data: HREIMS: 412.224m/e (M) +, C25H3205 requires 412.225; found: C, 72.6%; H, 7.9; requires C, 72.79%; H, 7.82. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 3. Structure Elucidation of Austalides G-L; J. Chem. Soc. Perkin Trans. I, pp. 363367(1985).

2.

Austalides

41

Common/Systematic Name Austalide L Molecular Formula/Molecular Weight C25H3206; M W = 428.21989

Me

Me

0 t

v

General Characteristics Crystals; mp., 207-208~

MeO

"o

[~]D 24 -71.0 ~ (c=l.00, in CHCh).

Isolation~urification Aspergillus ustus was cultured on malt extract medium at 250C in static culture. After 14 days the cultures were filtered and the mycelium macerated with acetone in a Waring blender. The acetone mixture was filtered, evaporated, and the resulting brown gum was partitioned between aqueous methanol (90%) and n-hexane. Evaporation of the methanol solution gave a residue which was partitioned between chloroform and water. The organic layer was concentrated and percolated through an aluminum oxide column with chloroform-methanol (95:5, v/v). The combined eluants were evaporated to give a lightyellow gum. This material was subjected to column chromatography on silica gel with nhexane-ethyl acetate (1:1, v/v) as eluant. Fractions were evaluated by TLC (n-hexaneethyl acetate, 1:1, v/v) and appropriate fractions combined to give three fractions: Rf 0.48, 0.31, and 0.13. The material associated with Rf 0.31 was purified on a silica gel column eluted with chloroform-methanol (99:1, v/v) to give purified austalides D and L. Fungal Source Aspergillus ustus (isolate MRC 1163). Biological Activity Reported to be toxic to ducklings. Spectral Data UV:

~,maxM~~ 223(e=31,200) and 269nm (16,600). IR:

(KBr) 1700 and 1745cm "1.

42

2.

Austalides

1H NMR: (CDC13) 0.780(3H, s, H-27); 1.092(3H, s, Me); 1.128(3H, s, Me); 1.167(3H, s, Me); 1.467(1H, ddd, d=13.4, 4.0, and 4.0 Hz, Hb-12); 1.751(1H, ddd, d=12.9, 8.0 and 4.0Hz, Hb-19); 1.979-2.038(2H, m, Hb-13 and H-19); 2.015(3H, s, H-23); 2.075(1H, ddd, J=13.4, 13.4, and 4.0Hz, H~-13); 2.155(1H, ddd, J--13.4, 13.4, and 4.0Hz, H-12); 2.254(1H, dd, J--7.0 and 1.9Hz, H-21); 2.463(1H, ddd, J=15.6, 8.0, and 8.0Hz, Hb-18); 2.549(1H, ddd, J-- 15.6, 8.0, and 4.0Hz, Ha-18); 2.740-2.831 (2H, m, H22); 4.061(3H, s, H-29); and 5.078ppm (2H, s, H-l).

13C NMR: (CDC13) 10.61, q, C-23; 18.04, t, C-22; 18.28, q, C-27; 21.64, q; 23.59, q; 24.19, t; 26.81, q; 33.16, t, C-19; 33.39, t; 33.79, t, C-18; 40.73, d, C-21; 41.23, s, C-20; 52.62, s, C-15; 61.83, q, C-29; 68.18, t, C-I; 76.14, s, C-11; 79.53, s, C-14; 107.23, s; 114.40, s; 115.84, s; 145.41, s; 155.29, s; 158.59, s; 169.35, s, C-3 and 216.40ppm, s, C-17. Mass Data: HR IMS: 428.219m/e (M) § C25H3206requires 428.220; found: C, 69.8%; H, 7.4; requires C, 70.07%; H, 7.53. References R. M. Horak, P. S. Steyn, P. H. Van Rooyen, R. Vleggaar, and C. J. Rabie; Structures of the Austalides A-E, Five Novel Toxic Metabolites from Aspergillus ustus; J. C. S. Chem. Comm., pp. 1265-1267(1981). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 1. Application of the Heteronuclear Selective Population Inversion (SPI) N.M.R. Technique to the Structure Elucidation of the Austalides A-F, Novel Ortho Ester Meroterpenoids; J. Chem. Soc. Perkin Trans. I, pp. 345-356(1985). R. M. Horak, P. S. Steyn, and R. Vleggaar; Metabolites ofAspergillus ustus. Part 3. Structure Elucidation of Austalides G-L; J. Chem. Soc. Perkin Trans. I, pp. 363367(1985).

Altertoxins Altertoxin I Altertoxin II Altertoxin III

43

This Page Intentionally Left Blank

3.

Altertoxins

45

Common/Systematic Name Altertoxin I; Dihydroalterperylenol Molecular Formula/Molecular Weight C20H1606, MW' = 352.09469 1

2

3

11 12 12a12b~4 O-/ v

,OH

0

"OH

General Characteristics Crystals from ehloroform:hexane; m.p., > 180~ (dec.); [a]D + 484 ~ (c=0.002, in CHCh); bright yellow fluorescence under UV light. Fungal Source Alternaria tenuis (strain 42, deposited with the Division of Microbiology, Food and Drug Administration, Washington D.C.), A. tenuis, and A. mali. Isolation/Purification Moldy rice was broken up with a spatula, blended with 1L CHCI3 and filtered. The filter cake was re-blended with CHCI3 and filtered. The combined filtrates were concentrated using a rotary evaporator. The concentrated extracts were added to a silica gel column (E. Merck silica gel 60, 0.06 - 0.2mm) and eluted with 3L CH2C12 to obtain altertoxin III, then 3L CHCI3 for altertoxin II, and finally with 3L CHCI3-MeOH (98:2) to obtain altertoxin I. Crystallization from CHCl3:hexane yielded purified altertoxins. Biological Activity The IDs0 of altertoxin I to HeLa cells was 20~tg/ml; toxicity to Bacillus mycoides was 250~tg/dise on agar plates. In mice dosed IP (DMSO carrier), an LDso was between 100-200mg/kg; at 200mg/kg, an LDl00 was observed. Spectral Data UV; MeOH max

215(6=25,500), 256(34,600), 285(16,300), 296sh (13,300), and 356nm

(6,000). IR(KBr) 3450, 1641, 1598, 1489, 1465, 1368, 1337, 1234, 1170, 1062, 951, and 829cm 1.

46

3.

Altertoxins

CD: +391, -351, +313(Ae +19.7, -8.8, +7.3) (c=2 x 10.4 in CHCI3). 1H NMR: (CDCI3) H-l, 7.87"(d, J=-8.8Hz); H-2, 7.01(d, J=8.SHz); H-5eq, 2.65(dt, J=15.0Hz); H-5ax, 3.17(m); H-6ax, 2.43(dt, J=13.0, 3.0Hz); H-6eq, 3.17(m); H-6b, 3.09(d, J=9.0Hz); H-7, 4.78(ddd, J=-12.0, 9.0, 5.0Hz); H-Sax, 2.94(dd, J=16.0, 12.0Hz); H8eq, 3.07(dd, J=16.0, 5.0Hz); H-11, 6.92(d, J=-8.SHz); H-12, 7.82"(d, J=-8.8Hz); H-3OH, 12.7; and H-10-OH, 12.4ppm. * Assignments may be interchanged. ~3CNMR: (CDCI3) C-l, 132.7(d); C-2, 119.5(d); C-3, 162.3"; C-3a, 116.9"; C-4, 205; C-5, 34.0*(0; C-6, 34.5*(0; C-6a, 69.2; C-6b, 51.9(d); C-7, 66. l(d); C-8, 47.7(0; C-9, 202; C-9a,, 117.4"; C-9b, 139.1; C-10, 162.0"; C-11, 117.5(d); C-12, 132.4(d); C-12a, 124.1; C-12b, 122.7; and C-12c, 135.5ppm. * Assignments may be interchanged. Mass Spectrum: HREIMS: 352.0948(M+, calcd for C20H1606~352.0946), 334(M+ - 1-120), 316(M+ 2H20), 314(M+ - 21-120 - 2H), 291(M+ -1-120- C2H30), 290(M+- Czi-I602), and 275m/e (M+ -2H20 - C9-IO). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 635 (1981). M. E. Stack, E. P. Mazzola, S. W. Page, and A. E. Pohland; Mutagenic Perylenequinone Metabolites ofAlternaria alternata: Altertoxins I, II, and III; J. Nat. Prod., Vol. 49, pp. 866-871(1986).

3. Altertoxins

47

Common/Systematic Nam,.,,r Altertoxin II Molecular Formula/Molecular Weight v

C20H1406, M W = 3 5 0 . 0 7 9 0 4

1

2

3 OH

~2 12b~O

o--" H General Characteristics Crystals from ehloroform:hexane; m.p., 245-250~ (dec.); [a]D + 636 ~ (C=0.001, in CHCI3); bright yellow fluorescence under UV light. Fungal Source Altemaria tenuis (strain 42, deposited with the Division of Microbiology, Food and Drug Administration, Washington D.C.), A. tenuis, and A. marl. Biological Activity The IDs0 of altertoxin II to HeLa cells was 0.51ag/ml; inhibition of Bacillus mycoides was observed at 250lag/disc on agar plates. In mice dosed IP (DMSO carrier), an LDs0 was between 100-200mg/kg; at 200mg/kg, an LD10o was observed. Isolation/Purification Moldy rice was broken up with a spatula, blended with 1L CHC13 and filtered. The filter cake was re-blended with CHC13 and filtered. The combined filtrates were concentrated using a rotary evaporator. The concentrated extracts were added to a silica gel column (E. Merck silica gel 60, 0.06 - 0.2mm) and eluted with 3L CH2C12 to obtain altertoxin HI, then 3L CHCI3 for altertoxin II, and finally with 3L CHCI3-MeOH (98:2) to obtain altertoxin I. Crystallization from CHCl3:hexane yielded purified altertoxins. Biological Activity The IDso of altertoxin I to HeLa cells was 201ag/ml; toxicity to Bacillus mycoides was 250lag/disc on agar plates. In mice dosed IP (DMSO carrier), an LDs0 was between 100-200mg/kg; at 200mg/kg, an LDloo was observed.

48

3.

Altertoxins

Spectral Data UV~ Z ~ " 215(e=27,700), 258(31,700), 286sh (17,000), 297sh (13,500), and 358nm (5,300). IR~ (KBr) 3462, 1643, 1598, 1485, 1384, 1342, 1321, 1296sh, 1223, 1184, 1138, 1103, 1095, 1061, 1041,952, 833, and 774cm ~.

CD: 395, 327(Ae +21.0, 8.8) (c=2 x 10.4 in CHCI3). 1H NMR: (CDC13) H-l, 7.91"(d, J=8.8Hz), H-2, 7.12**(d,d=8.8Hz), H-5~q, 2.83(ddd, J=17.0, 3.0, 2.0Hz), H-5ax, 3.26(ddd, J=17, 14, 4Hz), H-6~x, 2.41(dt, J=14.0, 3.0Hz), H-6eq, 2.89(ddd, J=14, 4, 2.0Hz), H-6b, 3.55(d, J=0.5Hz), H-7, 4.23(d, J=3.0Hz), H-8ax, 3.71(dd, d=3.0, 0.5Hz), H-11, 7.07"*(d, J=8.8Hz), H-12, 7.86"(d, d=8.8Hz), H-3-OH, 12.7, and H-10-OH, 12.1ppm.

*, **, *** Assignments may be interchanged. 13C NMR: (CDCI3) C-l, 133.0(d), C-2, 119.9(d), C-3, 163.3", C-3a, 113.5", C-4, 204.1 C-5, 32.1"**(0, C-6, 33.3***(0, C-6a, 68.3, C-6b, 45. l(d), C-7, 55.7(d), C-8, 52.8(0 , C-9, 196.6, C-9a, 114.6"*, C-9b, 138.8, C-10, 162.6", C-11, 118.0(d), C-12, 132.6(d), C12a, 124.0, C-12b, 122.4, and C-12c, 133.5ppm.

*, ** Assignments may be interchanged. Mass Spectrum: HREIMS 350.0784(M+, calcd for C20H1406, 350.0790), 332(M+ - H20), 321(M+ CHO), 305(M+ - CHO2), and 29 lm/e (M + -2CHO2 - H). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 635 (1981). M. E. Stack, E. P. Mazzola, S. W. Page, and A. E. Pohland, Mutagenic Perylenequinone Metabolites ofAlternaria alternata: Altertoxins I, II, and III; J. Nat. Prod., Vol. 49, pp. 866-871(1986).

3.

Altertoxins

49

Common/Systematic Name Altertoxin III Molecular Formula/Molecular Weight C2oH1206, M W -- 348.06339

H H.....~ 0 11~ O H

N General Characteristics Crystals from chloroform:hexane, m.p., 175-230~ (subl.); [a]D + 845 ~ (~--4 x 10-4, in CHCI3). Fungal Source Alternaria tenuis (strain 42, deposited with the Division of Microbiology, Food and Drug Administration, Washington D.C.). Isolation/~fification Moldy rice was broken up with a spatula, blended with 1L CHCI3 and filtered. The filter cake was re-blended with CHCh and filtered. The combined filtrates were concentrated using a rotary evaporator. The concentrated extracts were added to a silica gel column (E. Merck silica gel 60, 0.06 - 0.2mm) and eluted with 3L CH2C12 to obtain altertoxin IN, then 3L CHCI3 for altertoxin II, and finally with 3L CHCI3-MeOH (98:2) to obtain altertoxin I. Crystallization from CHCl3:hexane yielded purified altertoxins. Biological Activity The IDso of altertoxin I to HeLa cells was 20~tg/ml; toxicity to Bacillus mycoides was 250~tg/dise on agar plates. In mice dosed IP (DMSO carder), an LDso was between 100-200mg/kg; at 200mg/kg, an LD]oo was observed. Spectral Data

~max

210(6=19,500), 265(14,500), and 352nm (5,200).

IR"

(KBr) 3452, 1650, 1620, 1420, 1473, 1440, 1382, 1372, 1249, 1234, 1198, 1045,

50

3.

Altertoxins

1003, 806, 746, and 715cm~. CD: 344nm (Ae +39.2) (c=2 x 10"5 in CHCI3). 1H NMR: (CDCI3) H-l, 4.60(d, J=-3.4Hz); H-2, 3.86(dd, J=-3.4, 1.0Hz); H-5eq, 6.90(d, J=8.6Hz); H-6, 7.60(d, J=g.6Hz); H-6b, 4.20(br s); H-7, 4.60(d, J=3.4Hz); H-8ax, 3.86(dd, J=3.4, 1.0Hz); H-11, 6.90(d, J=8.6Hz); H-12, 7.60(d, J=8.6Hz); H-12b, 4.2(br s); H-3-OH, 11.5; and H-10-OH, 11.5ppm. ~3CNMR: (CDCI3) C-l, 56.0(d); C-2, 53.6(d); C-3, 196.8; C-3a, 112.3; C-4, 159.6; C-5, 114.5(d); C-6, 132.1(dt); C-6a, 128.8; C-6b, 37.5(d); C-7, 56.0(d); C-8, 53.6(d); C-9, 196.8; C-9a, 112.3; C-9b, 143.0; C-10, 159.6; C-11, 114.5(d); C-12, 132.l(d); C-12a, 128.8; C-12b, 37.5; and C-12c, 143.0ppm. Mass Spectrum: HREIMS: 348.0633(M+, ca.lcd for C20H1206,348.0634), 3 4 6 ~ - 2H), 331(M+ OH), 329(M+- 2H20- H), 319(M+ CHO), 314(M+- 2OH), 291(M+- CHO-CO), 263, and 250m/e. Reference M. E. Stack, E. P. Mazzola, S. W. Page, and A. E. Pohland; Mutagenic Perylenequinone Metabolites ofAlternaria alternata: Altertoxins I, II, and III; J. Nat. Prod., Vol. 49, pp. 866-871(1986).

Cercophorins Cercophorin A Cercophorin B Cercophorin C

51

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4. Cercophorins

53

Common/Systematic Name Cercophorin A Molecular Formula/Molecular Weight C20H1609; l ~ W -- 400.07943

2 1 " ~ / ' ~ ~ ~ ~--~ OH 2 I 7 o

0

OH

HO

~ ' ~ ~8~OMe

OMe 2O

General Characteristics White solid; rap., 208-210~ Fungal Source

Cercophora areolata (JS-166 = UAMH 7495) isolated from porcupine dung.

Isolation/Purification The filtered broth was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to yield a dark red oil. The extract was subjected to silica gel column chromatography with a step gradient from 0 to 40% (v/v) MeOH in CHC13, and 8mL fractions were collected. Similar fractions (as determined by TLC) were pooled and bioassayed. Active fractions were further purified by Sephadex LH20 column chromatography eluting with hexane-toluene-MeOH (3:1:1, v/v/v), followed by reversedphase semipreparative HPLC using a gradient from 20 to 70% MeOH in 0.01M NHnOAc over 20 min and then 70-100% MeOH in 0.01M NH4OAc over 2 min. HPLC fractions were concentrated m vacuo to afford 4-acetyl-8-hydroxy-6-methoxy-5-ethylisocoumarin, cercophorin B, and cercophorin C. The dried mycelial material was extracted with ethyl acetate, followed by filtration and concentration of the solvent to yield a dark red solid. This extract was processed by silica gel and Sephadex LH20 column chromatography as described above and then by preparative HPLC using a gradient from 70 to 100% MeOH in 1-120 over 45 min to afford decarboxycitrinone, cercophorin A, and roridin E. Biological Activity Cercophorins A-C inhibited the growth of the early successional coprophilous fungi Sordariafimicola (NRRL 6459) and Ascobolusfurfuraceus (NRRL 6460) at 200lag/disk in centerpoint inoculation disk assays. Decarboxycitrinone caused a 100% reduction in radial growth against both fungi, while 4-acetyl-8-hydroxy-6-methoxy-5-methylisocou-

54

4. Cercophorins

marin and cercophorins A, B, and C caused 46, 34, 10, and 51% growth reduction against S. fimicola, respectively. Roridin E caused a 100% reduction in growth of S. fimicola and A. furfuraceus and an inhibition zone of 29mm against Candida albicans (ATCC 14053) at the same concentration. In standard disk assays, cercophorin A was the most potent of these metabolites against Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 29213), affording zones of inhibition of 26 and 16mm, respectively, at 200pg/disk. Cercophorins A, B, and C showed only limited cytotoxicity in the NCI tumor cell line bioassay panel, displaying average GIs0 values of 9.4, 26.2, and 20.8pg/mL, respectively. Spectral Data UV;

~

MeOH max

253 (e=8,400), 318 (6,900), and 348nm (4,900).

IR~

(KBr) 3586, 2950, 2830 1671, and 1241cm"l. 1H NMR: (CDC13) 6.15(1H, s, H=4); 6.34(1H, s, H-5); 12.52(1H, s, OH=O; 12.17(1H, s, OH8); 7.00(1H, d, J=2.3Hz, H-14); 6.60(1H, d, J-2.3Hz, H-16); 7.42(1H, br s, OH-17); 3.57(3H, s, OMe); 3.85(3H, s, OMe); and 2.23ppm (3H, s, CH3-21). I3C NMR: (CDC13) 166.6, C-I; 157.0, C-3; 104.4, C-4; 103.4, C-5; 168.5, C-6; 110.5, C-7; 165.1, C-8; 98.1, C-9; 143.3, C-10; 199.1, C-11; 120.8, C-12; 132.6, C-13; 108.7, C14; 162.1, C-15; 105.5, C-16; 156.7, C-17; 166.4, C-18; 52.3, C-19; 55.7, C-20; and 19.6ppm, C-21. Mass Spectrum: FABMS (thioglycerol): 401 ([M + H] § 20), 369(15), 341 (30), 219(100), and 209role (86); EIMS: 400(M § 2), 351(18), 341(100), 219(36), and 209m/e (64); HRFABMS: 401.0867m/e (M + H) § calcd for (C2oH1609 + H) § 401.0873. HPLC Data Retention time, 26.5min (84:16, v/v; MeOH-H20). Reference A. C. Whyte, J. B. Gloer, J. A. Scott and D. Malloch; Cercophorins A-C Novel Antifungal and Cytotoxic Metabolites from the Coprophilous Fungus Cercophora areolata; J. Nat. Prod., Vol. 59, pp. 765-769(1996).

4. Cercophorins

55

Common/Systematic Name Cercophorin B Molecular Formula/Molecular Weight C2oH16Olo; M W = 4 1 6 . 0 7 4 3 5

2

I 0

7 OH

0

17 MeO 2O

OH 15

OMe 19

General Characteristics Yellow solid; mp., 99-101~

Fungal Source

Cercophora areolata (JS-166 = UAMH 7495) isolated from porcupine dung.

Isolation/Purification The filtered broth was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to yield a dark red oil. The extract was subjected to silica gel column chromatography with a step gradient from 0 to 40% (v/v) MeOH in CHCI3, and 8ml fractions were collected. Similar fractions (as determined by TLC) were pooled and bioassayed. Active fractions were further purified by Sephadex LH20 column chromatography eluting with hexane-toluene-MeOH (3:1:1, v/v/v), followed by reversed-phase semipreparative HPLC using a gradient from 20 to 70% MeOH in 0.01M NH4OAc over 20 min and then 70-100% MeOH in 0.01M NH4OAc over 2 min. HPLC fractions were concentrated m vacuo to afford 4-acetyl-8-hydroxy-6-methoxy-5-methylisocoumarin, cercophorin B, and cercophorin C. The dried mycelial material was extracted with ethyl acetate, followed by filtration and concentration of the solvent to yield a dark red solid. This extract was processed by silica gel and Sephadex LH20 column chromatography as described above and then by preparative HPLC using a gradient from 70 to 100% MeOH in H20 over 45 min to afford decarboxycitrinone, cercophorin A, and roridin E. Biological Activity Cercophorins A-C inhibited the growth of the early successional coprophilous fungi Sordariafimicola (NRRL 6459) and Ascobolusfurfuraceus (NRRL 6460) at 200~tg/disk in centerpoint inoculation disk assays. Decarboxycitrinone caused a 100% reduction in radial growth against both fungi, while 4-acetyl-8-hydroxy-6-methoxy-5-methylisocou-

56

4. Cercophorins

matin and cercophotins A, B, and C caused 46, 34, 10, and 51% growth reduction against S. fimicola, respectively. Roridin E caused a 100% reduction in growth of S. fimicola and A. furfuraceus and an inhibition zone of 29mm against Candida albicans (ATCC 14053) at the same concentration. In standard disk assays, cercophorin A was the most potent of these metabolites against Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 29213), affording zones of inhibition of 26 and 16mm, respectively, at 2001Jg/disk. Cercophorins A, B, and C showed only limited cytotoxicity in the NCI tumor cell line bioassay panel, displaying average GIs0 values of 9.4, 26.2, and 20.81Jg/mL, respectively. Spectral Data UV:

ZmM~H 252(e=9,400) and 333nm(6,500). IR:

(KBr) 2987, 2855 1720sh, 1688, and 1619cm"l. 1H NMR: (CDCI3) 6.26(1H, s, H-4); 6.17(1H, s, H-5); 11.73(1H, br s, OH-6); 11.60(1H, s, OH8); 10.38(1H, s, OH-13), 7.30(1H, d, d=2.9Hz, H-15); 7.11(1H, d, d=2.9Hz, H-17); 3.98(3H, s, OMe-19); 3.81(3H, s, OMe-20); and 2.21ppm (3H, s, CH3-21). 13CNMR: (CDCI3) 166.6~ C-I; 156.7, C-3; 104.9, C-4; 101.7, C-5; 162.3, C-6; 112.1, C-7; 161.1, C-8; 101.6, C-9; 141.3, C-10; 165.1", C-11; 143.0, C-12; 148.8, C-13; 114.8, C-14; 111.1, C-15; 152.8, C-16, 117.1, C-17; 170.4, C-18, 53.3, C-19; 56.3, C-20; and 19.3ppm, C-21. * Assignments may be reversed. Mass Spectrum: FABMS (glycerol): 439([M + Na] § 75), 417([M + H] § 28), 399(60), 299(74), 219(68), and 172m/e (100), EIMS: 416(M § 2), 372(29), 340(29), 267(13), and 236m/e (10); HRFABMS: 439.0660m/e (M + Na) § calcd for (C2oH~sOlo+ Na)§ 416.0743. HPLC Data Retention time, 26.7min (MeOH). Reference A. C. Whyte, J. B. Gloer, J. A. Scott and D. Malloch; Cercophorins A-C: Novel Antifungal and Cytotoxic Metabolites from the Coprophilous Fungus Cercophora areolata; J. Nat. Prod., Vol. 59, pp. 765-769(1996).

4. Cercophorins

57

Common/Systematic Name Cercophorin C Molecular Formula/Molecular Weight C20H16010; ~ "- 416.07435

21",,,.~/~"~~ ~ ~ 6 0 H 2 I 7 0

OH

0

0

is I OMe 2O

General Characteristics Yellow solid; mp., 102-105~ Fungal Source

Cercophora areolata (JS-166 = UAMH 7495) isolated from porcupine dung.

Isolation/Purification The filtered broth was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to yield a dark red oil. The extract was subjected to silica gel column chromatography with a step gradient from 0 to 40% (v/v) MeOH in CHC13, and 8ml fractions were collected. Similar fractions (as determined by TLC) were pooled and bioassayed. Active fractions were further purified by Sephadex LH20 column chromatography eluting with hexane-toluene-MeOH (3:1:1, v/v/v), followed by reversed-phase semipreparative HPLC using a gradient from 20 to 70% MeOH in 0.01M NHaOAc over 20 min and then 70-100% MeOH in 0.01M NH4OAc over 2 min. HPLC fractions were concentrated in vacuo to afford 4-acetyl-8-hydroxy-6-methoxy-5-ethylisocoumarin, cercophorin B, and cercophorin C. The dried mycelial material was extracted with ethyl acetate, followed by filtration and concentration of the solvent to yield a dark red solid. This extract was processed by silica gel and Sephadex LH20 column chromatography as described above and then by preparative HPLC using a gradient from 70 to 100% MeOH in H20 over 45 min to afford decarboxycitrinone, cercophorin A, and roridin E. Biological Activity Cercophorins A-C inhibited the growth of the early successional coprophilous fungi Sordariafimicola (NRRL 6459) and Ascobolusfurfuraceus (NRRL 6460) at 200~tg/disk in centerpoint inoculation disk assays. Decarboxycitrinone caused a 100% reduction in radial growth against both fungi, while 4-acetyl-8-hydroxy-6-methoxy-5-methylisocou-

58

4. Cercophorins

matin and cercophotins A, B, and C caused 46, 34, 10, and 51% growth reduction against S. fimicola, respectively. Roridin E caused a 100% reduction in growth of S. fimicola and A. furfuraceus and an inhibition zone of 29mm against Candida albicans (ATCC 14053) at the same concentration. In standard disk assays, cercophorin A was the most potent of these metabolites against Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 29213), affording zones of inhibition of 26 and 16mm, respectively, at 200pg/disk. Cercophorins A, B, and C showed only limited cytotoxicity in the NCI tumor cell line bioassay panel, displaying average GIs0 values of 9.4, 26.2, and 20.8pg/mL, respectively. Spectral Data UV~

MeOH j~ max

247(E=9,100) and 327nm (4,200).

IR~

(KBr) 2950, 1726, 1689, 1620, 1354, and 1197cm"~. 1H NMR: (CDCI3) 6.40(1H, s, H-4); 6.17(1H, s, H-5); 11.70(1H, br s, OH-8); 6.98(1H, d, J=3.0Hz, H-14); 6.82(1H, d, J=3.0Hz, H-16); 3.71(3H, s, OMe-19); 3.84(3H, s, OMe20); and 2.21ppm (3H, s, CH3-21). 13C NMR: (CDC13) 166.5", C-l, 157.0, C-3; 105.0, C-4; 101.3, C-5, 163.4, C-6; 111.1, C-7; 161.5, C-8; 101.5, C-9; 141.7, C-10; 165.7", C-11; 135.0, C-12; 126.4, C-13; 107.7, C-14; 158.7, C-15; 108.1, C-16; 152.4, C-17; 165.9, C-18; 52.7, C-19; 56.1, C-20, and 19.3ppm, C-21. * Assignments may be reversed. Mass Spectrum: FABMS (glycerol): 439([M + Na] § 12), 417([M + H] § 3), 399(21), 379(12), 355(36), 219(39), and 116m/e (100), ELMS: 372([M+ - CO2], 100), 340(91), 312(24), 297(14), and 269role (14); HRFABMS: 439.0661role (M + Na) § calcd for (C2oH16Olo + Na) § 416.0743. HPLC Data Retention time, 24.9min (MeOH). Reference A. C. Whyte, J. B. Gloer, J. A. Scott and D. Malloch; Cercophorins A-C: Novel Antifungal and Cytotoxic Metabolites from the Coprophilous Fungus Cercophora areolata; J. Nat. Prod., Vol. 59, pp. 765-769(1996).

Fiscalins Fiscalin A Fiscalin B Fiscalin C

59

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5. Fiscalins

61

Common/Systematic Name Fiscalin A Molecular Formula/Molecular Weight C26I--I27N504; M W "- 4 7 3 . 2 0 6 3 0 lc 10

11

N>-

la

H. 1-

~,~,lOa~N.~ ~ 2 r~ .Y ~ l a "NH

0

~ -OH8, H , ~ r HN3= \ j l ~j

General Characteristics Fisealin A was recrystallized from dichloromethane-methanol (1:1, v/v) to provide colorless orthorhombic crystals; mp., 242--243*C; [t~]D -169* (C=0.5, in MeOH). Isolation/Purification The fermentation was carried out at 26~ with shaking at 220rpm. Fiscalin production and cell growth peaked after 6 days. The majority of fiscalins was associated with the cell mass. Harvested cells were extracted with ethyl acetate to yield a brown residue. After redissolving in a mixture of methanol and dichloromethane (1:1, v/v), the sample was subjected to flash silica gel column chromatography with stepwise elution using chloroform, chloroform-methanol (1:1, v/v), and methanol. Bioactive eluents were collected from the chloroform-methanol fraction, and dried in vacuo to yield a white powder. After rinsing with methanol, the white solids were redissolved in a mixture of diehloromethane-methanol (1:9, v/v) and then subjected to semi-preparative I-IPLC using a YMC reverse phase column (C18) and isocratic elution with 86% methanol and 14% water at lml/minute. Eluents were collected to yield fiscalin A, B, and C. Fungal Source Neosartoryafischeri isolated from a plant rhizosphere collected near the We Fung Chi Cascade region of Taiwan. Anamorph stage is Aspergillusfischerianus. Biological Activity Inhibited the binding of radiolabeled substance P ligand to the human neurokinin (NK-1) receptor, with Ki value of 5711M.

62

5.

Fiscalins

Soeetral Data _

UV:

~,M~oH 226, 278, and 306nm. max CD: ~,~x 228(-62.1) and 307nm (-9.5Ae). IR:

(KBr) 3378-3279(NH)and 1689em~ (amide). 1H N - ~ : (CDC13 + CD3OD or DMSO-d~) 4.41(1H, H-I); 8.43(1H, NH); 5.31(1H, H-4); 7.99(1H, H-7); 7.26(1H, H-8); 7.55(1H, H-9); 7.43(1H, H-10); 0.94(3H, H-la); 2.85(1H, H-lb); 0.68(3H, H-le); 2.51, 2.68(2h, H-4a); 3.14(11-1,NH-I'); 3.47(1H, H-2'); 0.82(3H, H-2a'); 7.17(1H, H-5'); 6.88(1H, H-6'); 7.07(1H, H-7'); 7.14(1H, H8'); and 4.93ppm (1H, H-9a'). 13CNMR: (CDCI3) 60.4, C1; 172.4, C-3; 54.4, C-4; 163.9, C-6; 122.2, C-6a; 128.4, C-7; 129.1, C-8; 136.8, C-9; 129.2, C-10; 149.4, C-10a; 152.2, C-11a; 20.0, C-la; 31.2, C-lb; 16.5, C-lc; 40.1, C-4a; 62.9, C-2'; 18.0, C-2a'; 177.0, C-3'; 139.1, C-4a'; 117.3, C-3'; 127.5, C-6'; 132.2, C-7'; 126.4, C-8'; 139.2, C-8a'; 76.3, C-9'; and 84.4ppm C-9a'. Mass Spectrum: HREIMS: 473.2032re~e;calculated, 473.2063. Reference S-M. Wong, L. L. Musza, G. C. Kydd, R. Kullnig, A. M. Gillum, and R. Cooper; Fiscalins: New Substance P Inhibitors Produced by The Fungus Neosartorya fischeri, Taxonomy, Fermentation, Structures, and Biological Properties; The Journal of Antibiotics, Vol. 46, pp. 545-553(1993).

5.

Fiscalins

63

Common/Systematic Name Fiscalin B Molecular Formula/Molecular Weight C23H22N402, ~ = 386.17428 H

-

General Characteristics Fiscalin B was obtained as a white amorphous solid. Isolatiort~urification See fiscalin A. Fungal Source

Neosartoryafischeri isolated from a plant rhizosphere collected near the We Fung Chi Cascade region of Taiwan. Anamorph stage is Aspergillusfischerianus.

Biological Activity Inhibited the binding of radiolabeled substance P ligand to the human neurokinin (NK-1) receptor, with Ki value of 157~M. Spectral Data UV: UV suggested the presence of quinazolone and indole chromophores. CD: ~,~x 228(-62.1) and 307nm (-9.5AE). 1H NIV[R: (CDCI3) 2.70(1H, H-l), 5.97(1H, NH); 5.63(1H, H-4); 8.33(1H, H-7), 7.49(1H, H-8); 7.74(1H, H-9); 7.53(1H, H-10); 0.61(3H, H-la); 2.60(1FL n-lb); 0.62(3H, H-lc); 3.69, 3.59(2h, n-4a); 8.21(1H, H-4'); 7.38(1H, n-5'); 6.88(1H, n-6'); 7.08(1H, H-7'); 7.25(1H, n-8'); and 6.57ppm (1H, n-9a').

64

5.

Fiscalins

13CNMR: (CDC13) 58.4, C1,170.2, C-3, 57.1, C-4, 161.6, C-6, 120.7, C-6a, 127.4, C-7, 127.7, C-8, 135.3, C-9, 127.7, C-10, 147.7, C-10a, 151.0, C-11a, 19.0, C-la, 29.8, C-lb, 14.9, C-lc; 27.6, C-4a; 127.8, C-4a'; 119.2, C-3', 120.5, C-6'; 123.1, C-7'; 111.6, C-8'; 136.6, C-8a', 109.7, C-9', and 124.2ppm C-9a'. Mass Spectrum: 387m/e (M + H) + and H R I M S of 386.1743m/e suggesting a molecular formula of C23H2zN4Oz, with two major fragments 257.1158 (C1A-I15N302)and 130.0649m/e

(Cd-hN). Reference S-M. Wong, L. L. Musza, G. C. Kydd, R. Kullnig, A. M. GiUum, and R. Cooper; Fiscalins: New Substance P Inlfibitors Produced by The Fungus Neosartoryafischeri, Taxonomy, Fermentation, Structures, and Biological Properties; The Journal of Antibiotics, Vol. 46, pp. 545-553(1993).

5.

Fiscalins

65

Common/Systematic Name Fiscalin C Molecular Formula/Molecular Weight C27H29NsO4; M W -- 4 8 7 . 2 2 1 9 5

H

-

0 General Characteristics Fiscalin C was obtained as a white amorphous solid. Isolation/Purification See fiscalin A. Fungal Source

Neosartoryafischeri isolated from a plant rhizosphere collected near the We Fung Chi Cascade region of Taiwan. Anamorph stage is Aspergillusfischeriatms.

Biological Activity Inhibited the binding of radiolabeled substance P ligand to the human neurokinin (NK-1) receptor, with Ki value of 68~M. Spectral Data UV;

I.W suggested the presence of quinazolone and indole chromophores. CD" ~.m,~ 228(-44.6) and 307rim (-5.0At).

66

5.

Fiscalins

1HNI~: (CDCI3) 4.56(1H, H-l); 6.12(1H, NH), 5.82(1H, H-4); 8.30(1H, H-7), 7.51(1H, H8); 7.79(1H, H-9), 7.68(1H, n-10); 0.95(3H, H-la); 3.16(1H, U-lb); 1.18(3H, H-lc), 2.47, 2.69(2H, n-4a); 1.39(3H, n-3a'); 1.49(1H, n-4'), 7.51(1H, H-5'), 7.09(1H, n-6'); 7.32(1H, n-7'); 7.32(1H, n-8'), and 5.25ppm (1H, n-9a'). 13CNMR: (CDCI3) 48.6, C-1,170.8, C-3, 51.8, C-4, 161.3, C-6, 120.9, C-6a, 127.7, C-7, 128.0, C-8; 135.5, C-9; 128.1, C-10; 147.5, C-10a, 149.8, C-1 la; 19.8, C-la; 29.0, Clb; 15.6, C-lc; 40.0, C-4a; 65.4, C-2'; 25.4, C-2a', 26.4, C-2b', 175.9, C-Y, 138.0, C4a', 116.3, C-5', 125.8, C-6', 130.8, C-7', 124.7, C-8', 138.5, C-8a', 74.5, C-9', and 78.9ppm C-9a'. Mass Spectrum: HREIMS: 488.2318m/e (M + H) +. Reference S-M. Wong, L. L. Musza, G. C. Kydd, R. KuUnig, A. M. Gillum, and R. Cooper, Fiscalins: New Substance P Inhibitors Produced by The Fungus Neosartoryafischeri, Taxonomy, Fermentation, Structures, and Biological Properties; The Journal of Antibiotics, Vol. 46, pp. 545-553(1993).

Palmarumycins

6

Palmarumycin CP1 Palmarumycin CP2 Palmarumycin CP3 Palmarumycin CP4 Palmarumycin C1 Palmarumycin C2 Palmarumycin C3 Palmarumycin C4 Palmarumycin C5 Palmarumycin C6 Palmarumycin C7 Palmarumycin C8 Palmarumycin Clo Palmarumycin Cll Palmarumycin C12 Palmarumycin C13 Palmarumycin C14 Palmarumycin C15 Palmarumycin C16

67

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6. Palmarumycins

69

Common/Systematic Name Palmarumycin CP1 5-Hydroxyspiro [naphthalene- 1(4H),2'-naphtho [ 1,8-de] [ 1,3 ]dioxin]-4-one Molecular Formula/Molecular Weight C2oH1204; M~V = 316.07356 0

d

OH

4'

General Characteristics Light yellow crystals from methylene chloride-petroleum ether; mp., 170~ (dec.); optically inactive. Isolation/Purification The palmarumycins were extracted with chloroform and CP1 and CP3 were isolated in pure form using preparative thin-layer chromatography [silica gel on aluminum foil (Merck)]. Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP1 showed no biological activity against the test fungus Cladosporium cucumerinum. Spectral Data UV~ ~,mM~y'~~ (3,731).

232(10g e = 4,633), 288(3,961), 296(3,966), 330(3,785), and 362nm

IR~ (KBr) 3456(OH), 3057, 1662(C=O), 1611, 1455, 1410, 1379, 1340, 1268(Ar-O), 1236, 1112, 1075, 942, and 744cm "1.

70

6. Palmarumycins

1H NMR: (CDCI3) 12.16(1OH, s); 7.66[1H, pseudo-t (dd), J6,7=8.2Hz, J7,8=7.9Hz, H-7]; 7.58(2H, d, J3,4,,(5,,6,)=8.3Hz, 4'-H and 5'H); 7.4712H, pseudo-t (dd), J3',4;(5"6')- 8.4Hz, Jz3,c6,,7,)=7.5Hz,H-3' and H-6']; 7.46(1H, dd, J6,s=lHz, H-8); 7.14(1H, dd, J6,7=8.4Hz, J6,8=lHz, H-6); 7.02(1H, d, J2,3=10.4Hz, H-2); 6.98(2H, d, J2,,3,(6,,7,)=7.5Hz, H2' and H7'); and 6.36ppm (1H, d, J2,3=10.6Hz, H-3). 13CNMR: (CDC13) 188.8(C-4, s); 161.9(C-5, s); 147.2(C-1', C-8', s); 139.7(C-2, d); 138.8(C-8a, s); 136.6(C-7, d); 134.1(C-4a', s); 129.7(C-3, d); 127.6(C-3', C-6', d); 121.4(C-4', C-5', d); 119.7(C-8, d); 119.4(C-6, d); 113.9(C-4a, s); 113.0(C-8a', s); 109.9(C-2', C-7', d); and 93.0ppm (C- 1, s).

Mass Data: 316(M§ 100%), 288(24, M§ - CO), 287(31, M+ - CO), 209(27), 169(40), and 167m/e (32); HREIMS: obsd 316.0736m/e; calcd for C20H1204, 316.0736; anal C =75.5, H=3.68; calcd C=75.94, H=3.82. Reference K. Krohn, A. Michel, U. Florke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CP1 -CP4 from Coniothyriumpalmarum: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1093-1097(1994).

6. Palmarumycins

71

Common/Systematic Name Palmarumycin CP2 5-Hydroxy-2,3-dihydrospiro [naphthalene- 1(4H),2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-4-one _Molecular Formula/Molecular Weight C20H1404; M W = 318.08921 0

O

OH

ii I1'

General Characteristics Crystals; mp., 170~ (dec.); optically inactive. Isolation/Purification The palmarumycins were extracted with ethyl acetate and CP2 was isolated in pure form using repeated column chromatography on silica gel using toluene as eluant. Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP1 showed no biological activity against the test fungus Cladosporium cucumerinum. However, it showed weak activity against Ustilago violacea and Eurotium repens. Spectral Data UV: Z mM~=~176 ~ 285(Iog e = 3.81), 297(4.12), 300(4.11), 315(3.98), and 329nm (3.99). IR: ~

(KBr) 3438(OH), 3056, 1640(C=O), 1609, 1586, 1412, 1381, 1348, 1331, 1237, 1117, 1107, 1074, 920, 885, 819, 804, 795, 758, and 743cm"1.

72

6. Palmarumycins

1H NMR: (CDCI3) 12.43(1OH, s, H-7); 7.62[1H, pseudo-t (dd), J6,7= 8.2Hz, J=7,s7.9Hz, H-7]; 7.54(2H, dd, ffY,4"(5;6')-" 8.3HZ, ff2,,4,(5,,7,)= 0.6Hz, H-4' and H-5'); 7.4612H, pseudo-t (dd), J2,3,(6,,7~=7.3Hz, ff3',4'(5',6~-- 8.3Hg, H-3' and H-6']; 7.45 (1H, dd, Jz, s= 7.6Hz, J6,s = 1.0Hz, H-8); 7.10 (1H, dd, J6,7= 8.3Hz, d6,s=l.OHz, H-6); 6.97(2H, dd, J2,3,(6,z,)=7.3Hz, J2,4,o,,7~ = 0.6Hz, H-2' and H-7' ); 2.85(2H, t, Jz~= 6.5Hz, H-3); and 2.49ppm (2H, t, Jz3=6.5Hz, H-2). 13C NUll: (CDC13) 203.3(C-4, s); 162.5(r s); 147.5(C-1', C-8', s); 140.9(C-8a, s); 137.2(C-7, d); 134.3(C-4a', s); 127.6(C-3', C-6', d); 120.9(C-4', C-5', d); 119.6(C-8, d); 116.7(C-6, d); 115.5(C-4a, s); 109.5(C-2', d); 113.4(C-8a', s); 98.4(C-1, s); 34.1(C-3, t) and 29.5ppm (C-2, t).

Mass Data: ELMS: 318(M§ 100%), 301(M+-OH, 44), 159(36), and 132m/e (26); HR IMS: obsd 318.089m/e; calcd for C20H1404, 318.0892; anal C= 75.21, H= 4.23; calcd C=75.46, H=4.43. Reference K. Krohn, A. Michel, U. Florke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CP1 CP4 from C o n i o t h y r i u m p a l m a r u m : Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1093-1097(1994). -

6. Palmarumycins

73

Common/Systematic Name Palmarumycin CP3 2S*,4 aS*, 8S*, 8aS*-2,3,8,8a-T etrahydro spiro [2,8-epoxynaphthalene- 1(4H),2'naphtho [ 1,8-de ][ 1,3 ] dioxin]-4,5 (4aH)-dione Molecular Formula/Molecular Weight C20H1405~, ~

0

II1''

= 334.08412

0

~.

General Characteristics Colorless prisms from methylene chloride-petroleum ether; mp., 190~ (dec.); - 108.8 ~ (c=l.0, in CDCls).

[ ~ ] D 20 -"

Isolation/Purification The palmarumycins were extracted 3 times with ethyl acetate. The combined extracts were dried (MgSO4), filtered, and concentrated under reduced pressure. The crude extract was subjected to preparative liquid chromatography four successive times using ethyl acetatemethylene chloride (1:1, v/v); diethyl ether-methylene chloride (5:15, v/v); diethyl ethermethylene chloride (1:10, v/v), and diethyl ether-methylene chloride (2:98, v/v). Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP3 showed biological activity against a variety of bacteria and fungi. Spectral Data ugz Chloroform

1~ (4.06). max

290(log e=4.06), 299(4.34), 307(4.27), 313(4.29), 3.27(4.22), and 381nm

74

6. Palmarumycins

IR~

(KBo 3079, 2982, 2955, 2901, 1728(C=O), 1688(C=O), 1607, 1584, 1414, 1381, 1275(Ar-O), 1252, 1230, 1169, 1153, 1124, 1055, 928, 833, and 770cm1. 1H N1V[R:

(CDCI3) 7.57(1H, d, Jy,6~-8.3Hz, H-5'); 7.56(1H, d, Js,4~-8.3Hz, H-4'); 7.47[1H, pseudo-t (dd), Jzs~7.6Hz, Js,,4,-=8.3Hz,H-3']; 7.45[ 1H, pseudo-t (dd), Jy,6~8.3Hz, J6,z,~-7.8Hz, H-6']; 7.07(1H, dd, Jz, s~7.5Hz, Jz,4~0.6Hz, H-2'; 6.99(1H, dd, J6,7=10.1Hz, Jzs-=5.1Hz, H-7); 6.95(1H, dd, J6,7~lO.1Hz, Jzs-=5.1Hz, H-7); 6.14(1H, d, J6,7=10.1Hz, H-6); 4.97[1H, pseudo-t (dd), Jzs=4.8Hz, Js,sa=4.8Hz, H-8]; 4.55(1H, m, H-2); 3.65(1H, dJ4a,sa=5.4Hz, H-4a); 3.18[1H, pseudo-t (dd), J4a,sa=4.2Hz, Js,s,,=4.9Hz, Jzs~=I.4Hz, H-8a]; 3.04[1H, d, (AB)dd, Js=,s~q=18.7Hz,Jzs~q=2.4Hz, Js~q,4,~=O.7Hz,H-3eq]; and 2.84ppm [ 1H, d (AB)dd, Js,=,s~q=l8.7Hz, Jz3==2.9Hz, J~,4~=l. 1Hz, H-3ax]

13C NMR:

(CDC13) C-4, 200(s); C-5, 189.9(s), C-8', 146.7(s); C-l', 146.5(s); C-7, 143.1(d); C4a', 134.5(s), C-6, 1131.4(d); C-3', 127.7(d); C-6', 127.5(d); C-5', 122.0(d); C-4', 121.5(d); C-8a', 113.9(s); C-2', 110.1(d); C-7', 109.5(d); C-I, 105.6(s); C-2, 76.3(d), C-8, 70. l(d); C-4a, 58.1(d); C-3, 45.6(0; and C-8a, 42.4ppm (d). Mass Data: 334(M+, 100%), 305(8), 263(31), 235(30), 218(18), 212(15), 199(15), 171(14), 115(39), 114(24), 91(18), and 65m/e (24); anal, C=72.05, H=4.31; calcd C=71.85, H=4.22. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CI-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

75

Common/Systematic Name Palmarumycin CP4 (8R *,8 aS*)-4, 8-Dihydroxy-6, 7,8,8a-tetrahydro spiro [naphthalene- 1( 5H),2'-naphtho [ 1,8de][ 1,3 ]dioxin]-5-one Molecular Formula/Molecular Weight C20H1605, M W = 336.09977

OH

0

11III

General Characteristics Colorless prisms from methylene chloride; mp., 186~ (dec.); mp., 193~ (diethyl ether); [Ct]D2~= + 495 ~ (C=0.2, in CH2C12). Isolation/Purification The palmarumycins were extracted 3 times with ethyl acetate. The combined extracts were dried (MgSO4), filtered, and concentrated under reduced pressure. The crude extract was subjected to preparative liquid chromatography four successive times using ethyl acetatemethylene chloride (1:1, v/v); diethyl ether-methylene chloride (5:15, v/v); diethyl ethermethylene chloride (1:1, v/v); and diethyl ether-methylene chloride (2:98, v/v). The polar fractions of the chromatography were further separated by PLC (Chromatotron, 2mm, CH2C12-CH3OH; 99:1, v/v) to afford pure palmarumycin CP4. Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP3 showed biological activity against a variety of bacteria and fungi. Spectral Data UV:

~, Cm~~176176 283(1og e=3.62), 295(4.03), 301(4.07), 304(4.07), 314(4.10), and 328nm (4.08).

76

6.

Palmarumycins

IP-~

(KBr) 3551(OH), 3063, 2932, 2905, 1638, 1611, 1580, 1412, 1375, 1323, 1252, 1252, 1213, 1103, 1082, 1065, 1036, 959, 930, 919, 826, and 762cm"1. 1H N1VIR:

(CDC13) 15.5(1H, s, OH); 7.55(1H, dd, ds,6~8.2Hz, Jy,7~-O.6Hz,H-5'); 7.54(1H, d, J3,4~8.2Hz, H-4'); 7.48(1H, t(dd), dz,3~7.4Hz, d3,4~8.3Hz, H-Y); 7.4[ 1H, pseudo-t (dd), Js,6,~-8.2Hz,J6,7,=7.7Hz, H-6']; 7.04(1H, dd, Jz,3~-8.2Hz, Jz,4,~O.6Hz, H-2']; 6.91(1H, d, J6,,7~7.5Hz, H-7'); 6.44(1H, d, Jz3=10.3Hz, H-2); 6.13(1H, d, dz3=10.3Hz, H-3); 4.86[1H, pseudo-t (dd), dT~,8=2.3Hz,ds,oH=2.3Hz, H- 8]; 3.77(2H, d, Js,~,sa=2.3Hz, H-8a); 3.4(1H, s, OH); 2.98(1H, ddd, J6~, 6eq=19.1Hz, J6~,TAX=12.4Hz,d6,~,6~q=6.8Hz,H-6ax); 2.41 (1H, dd, J6~, 6~q=l9.1Hz, J~q,7~=5.5Hz, H-6eq); 2.25(1H, m, H-7eq); and 1.82ppm (1H, m, H-7ax).

13CNMR:

(CDCI3) C-4, 181.3(s); C-5, 188.4(s); C-8', 146.5(s); C-I', 145.9(s); C-2, 137.3(d); C4a', 134.2(s); C-3, 130.6(d); C-3', 127.7(d); C-6', 127.4(d); C-5', 121.3(d); C-4', 121.7(d); C-8a', 113.6(s); C-2', 110.1(d); C-7', 110.1(d); C-l, 99.3(s); C-8, 63.4(d); C8a, 46.0(d); C-6, 27.6(d); and C-7, 27.1ppm (t). Mass Data: 336(M § 100%), 318(94, M+ - n20), 289(15, 318 - CHO), 279(76, 318 - C3H3), 160(24), 159(76, 1,8-dihydroxynaphthalene-H), 144(19), 131(50), and 115re~e; HREI]~S: C20H1605,obsd 336.0997re~e; calcd, 336.0998; anal, C, 71.26, H, 4.61; calcd, C, 71.42, H, 4.79. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

77

Common/Systematic Name Palmarumycin C1 3-Chloro-5-hydroxyspiro[naphthalene- 1(4H),2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-4-one Molecular Formula/Molecular Weight C20HllCIO4; ~

Cl

0

OH

4

~

1111

,

~ 6'

6

7

8

8,(. r

= 350.03459

' 4'

General Characteristics Slightly yellow needles; mp., 280~ (dec.). Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV: Chloroform ~, max

285(log 6=3.52), 296(3.96), 301(4.05), 314(3.81), and 329nm (3.71).

IR:

(KBr) 3438(OH), 3202, 3067, 1734, 1718, 1580, 1704, 1653(C=O), 1628, 1611, 1458, 1412, 1381, 1271(Ar-O), 1231, 1167, 1070, 939, 903, 845, 826, 802, and 756cm -1.

78

6. Palmarumycins

~H NMR: (CDCI3) I 1.84(IH, s, OH); 7.67[IH, pseudo-t (dd), J6,z=8.4I-Iz, dT,s=7.9Hz, H- 7]; 7.59(2H, d, J3,,4,a,,69=8.3Hz, H-4', H-5'); 7.47121-1,pseudo-t (dd), d2;3,(a;79=7.61-1z, Js,,4,(5,,6.)=8.3Hz,H-3', H-6']; 7.45(IH, dd, JT,~7.9Hz, J6,8=l.01-Iz, H-8); 7.16(IH, (dd), J6,7=8.41-Iz, d6,s=l.OHz, H-6); 7.16(2H, s, H-2); and 6.98ppm (2H, d, d2,3,(6,7,)=7.61-Iz, H-2', H-7'). 13CNMP-~:

(CDC13) C-4, 181.7(s); C-5, 161.9(s), C-8', 146.5(s); C-I', 146.5(s); C-8a, 137.8(s); C-7, 137.1(d); C-2, 135.5(d); C-4a', 113.0(s); C-3, 133.2(d); C-Y, 127.5(d); C-6', 127.5(d); C-4a, 113.0(s); C-8a', 112.4(s), C-2', 109.9(d); C-7', 109.9(d); and C- 1, 93.3ppm (s). Mass Spectrum: LREIMS: 352(M+, 35%), 350(100), 315(32, M+- CI), 287(20), 259(13), 116(29), 96(42), 71(29), and 56role (53); HREIMS: C2oHl1C104,obsd 350.0345m/e, calcd 350.0346. Reference K. Krohn, A.Michel, U. FlSrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1C~6 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

79

Common/Systematic Name Palmarumycin C 2 (2S*, 3R *)- 5-Hydroxyspiro [2,3-epoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de] [ 1,3 ]dioxin]4(3H)-one Molecular Formula/Molecular Weight C20H1205; M'W = 332.0647

0

OH

General Characteristics Crystallized in long yellow needles; mp., 228~ (dec.); [ ~ ] D with 2,4-dinitrophenylhydrazine and bromocresol green.

20 - - -

341 ~ Positive reaction

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin Cz. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV:

c~o~oro,. 295(log 6-=4.11), 300(4.13), 314(4.08), and 328nm (4.10).

80

6. Palmarumycins

IR:

(KBr) 3432(OH), 3052, 1651(C=O), 1611, 1355, 1410, 1381, 1333, 1269(Ar-O), 1240, 1179, 1115, 1105, 1065, 1016, 970, 918, 874, 818, 506, 758, 752, 739, and 506cml. :H NMR:

(CDCla) 11.26(1H, s, OH); 7.6411H, pseudo-t (dd), J6,z=8.2Hz, Jzs-=7.9Hz, H- 7]; 7.59(1H, dd, J3,4~8.3Hz, Jz,4~0.7Hz, H-4'); 7.56(1H, d, Jy, D~8.3Hz, H-5'); 7.52[1H, pseudo-t (dd), J2,,3~7.SHz, Jy,4~-8.3Hz, H-3']; 7.4511H, pseudo-t (dd), Jy,6~8.3Hz, JD,,7~7.4Hz, n-6']; 7.45(1H, dd, Jzs=7.9Hz, JD,8=l.OHz,H-8); 7.16(1H, (dd), JD,7=8.4Hz,J6,,r=l.OHz,n-6); 7.44(1H, dd, J7,,s~7.9Hz, J6,,s~l.OHz, H- 8); 7.18(1H, dd, Jz,3~7.4Hz, Jz,4~0.7Hz,H-2'); 7.13(1H, dd, JD,7=8.4Hz,J6,s=l.0Hz, H-6); 6.92(1H, dd, J6,,7~7.4Hz, Jy,7~0.6Hz, H-7'); 4.08(1H, d, Jz3=4.0Hz, H-3); and 3.68ppm (2H, d, Jz3=4.0Hz, n-2).

13C NMR:

(CDC13) C-4, 196.5(s), C-5, 161.9(s); C-8', 146.9(s); C-I', 146.7(s); C-8a, 136.9(s); C-7, 137.7(d); C-2, C-3, 53.2(d); C-4a', 134.2(s); C-3', 127.8(d); C-6', 127.6(d); C-4a, 112.2(s); C-8a', 112.8(s); C-2', 109.3(d); C-7', 109.3(d); C-l, 96.0(s); C-4', 121.4(d); C-5', 121.3(d); C-6, 120.1(d); and C-8, 119.1ppm (d). Mass Data: LREIMS: 332(NV, 100%), 287(20), 246(19), 232(50), 204(30), 176(20), 173(33), 159(22), 154(20), 145(20), 130(20), and 43m/e (55); HREIMS: C20H1205,obsd 332.0660m/e; calcd 332.0685; calcd, C=72.29, H=3.64; found C=72.16, H=3.59. Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz, Palmarumycins C1-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

81

Common/Systematic Name Palmarumycin C3 (2S*,3R*)-5,8-Dihydroxyspiro [2,3-epoxynaphthalene- 1(2/~,2'-naphtho [ 1,8de][1,3 ] dioxin]-4(3H)-one Molecular Formula/Molecular Weight C2oH1206, MW

0

-- 3 4 8 . 0 6 3 3 9

OH

I

General Characteristics Crystallized as greenish needles; mp., 220~

[0~]D 20 --

-300 ~

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins Ca and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Column chromatography was continued with a gradient of methylene chloride-methanol (0-10%). The second fraction was further purified by column chromatography with petroleum ethermethylene chloride (1:1, v/v) as eluant to afford additional palmarumycin C2 and palmarumycin C3. Funsal Source v An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV:

X c~o~ofo~m296(1og e=4.14), 299(4.14), 312(4.01), 327(3.91), and 380nm (3.91).

82

6. Palmarumycins IR:

(KBr) 3499(OH), 3060, 1651(C=O), 1613, 1595, 1476, 1410, 1375, 1335, 1264(ArO), 1237, 1223, 1175, 1098, 1067, 1026, 954, 893, 876, 818, 797, 764, and 754cm1 1H NMR: (CDC13) 11.48(1H, s, OH); 7.67(1H, d, J3,4~8.4Hz, H-4'); 7.61(1H, d, Jy,o~8.2Hz, H5'); 7.54[ 1H, pseudo-t (dd), Jz.3~7.7Hz, J3,4~8.3Hz, H-3']; 7.49[ 1H, pseudo-t (dd), Jy,o~-8.2Hz, J6, 7~-7.7Hz, H-6']; 7.35(1H, s, OH); 7.29(1H, d, Jo,7=9.2Hz, H-7); 7.25(1H, d, Jz,3~7.5Hz, H-2'); 7.11(1H, d, Jo.7=9.3Hz,H-6); 7.03(1H, d, Jo,7~-7.6Hz, H-7'); 4.03(1H, d, Jz3=4.0Hz, H-3); and 3.63ppm (1H, d, Jz3=4.0Hz, H-Z). 13CNMR: (CDC13) C-4, 195.5(s); C-5, 157.0(s), C-8, 149.1(s); C-8', 146.3(s); C-l', 144.9(s); C4a', 134.0(s); C-7, 129.6(d); C-6', 127.8(d); C-3', 127.5(d); C-4', 122.7(d); C-6, 122.3,(d); C-5', 121.6(d); C-8a, 115.9(s); C-8a', 115.9(s); C-4a, 111.0(s); C-2', 111.0(d); C-7', 110.2(d); C-l, 98.8(s); C-2, 53.0 (d); and C-3, 52.5ppm (d). Mass Data: LREIMS: 348(M§ 100%), 303(11), 189(9), 171(9), 125(11), 111(17), 97(22), 85(20), 83(22), 71(27), and 57m/e (35); calcd, C=68.97, H=3.47, found C=68.75, H=3.43. Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6.

Palmarumycins

83

Common/Systematic Name Palmarumycin C4 (4aRS, 8aRS)3-Chlorospiro [4a, 8a-epoxynaphthalene- 1(4H),2'-naphtho [ 1,8de][1,3 ]dioxin]-4,5,8-trione Molecular Formula/Molecular Weight C20H9C106; M~W = 380.00877

O

O

CI

(,Z) ~ General Characteristics An orange amorphous material; [a]D2~-285 ~ (C=I.0, in CH2C12). Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins Ca and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV~

c~o~oro,, 285(log c=3.52), 296(3.96), 301(4.05), 314(3.81), and 329nm (3.71).

84

6. Palmarumycins

IR~

(KBr) 3438(OH), 3202, 3067, 1734, 1718, 1580, 1704, 1653(C=O), 1628, 1611, 1458, 1412, 1381, 1271(Ar-O), 1231, 1167, 1070, 939, 903, 845, 826, 802, and 756cm1. 1H NMR:

(CDC13) 7.62(1H, d, J3,4~8.3Hz, H-4'); 7.60(1H, d, Jy,D~8.2Hz, H-5'), 7.52[ 1H, pseudo-t (dd), J2,3~7.6Hz, J3,4~8.2Hz, H-3']; 7.45[ 1H, pseudo-t (dd), Jy, D~8.2Hz, J6,z~7.6Hz, H-6']; 7.12(1H, dd, J2,3~7.SHz, J2,4~-O.5Hz,H-2'), 6.91(1H, d, J6,z~7.6Hz, H-7'); 6.75(s, H-2); 6.72(1H, d, J6,7=10.8Hz, H-7); and 6.71ppm (1H, d, J6,7=10.8Hz, H-6).

13C NMR:

(CDCI3) C-4, 178.6(s); C-5, 183.3(s); C-8, 184.4(s); C-8', 184.5(s); C-I', 145.0(s); C4a', 134.5(s); C-7, C-6, 136.8(d); C-6', 127.8(d); C-3', 128.0(d); C-4', 122.2 (d); C-5', 122.2 (d); C-8a, 55.3(s); C-8a', 112.6(s); C-4a, 62.1(s); C-2', 110.1(d); C-7', lO9.8(d); C-I, 95.2(s); C-2, 133.6(d); and C-3, 132.2ppm (s). Mass Spectrum: LREIMS: 382(36), 380(M§ 100), 352(M§ CO, 5), 317(4), 289(10), 230(12), 195(42), and 114m/e (28); HREIMS: C2oH9CIO6,found 380.O087m/e; calcd, 380.0088. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CI-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

85

Common/Systematic Name Palmarumycin C5 (4R *)-3,4-Dihydro-4-methoxyspiro [2,3-epoxynaphthalene- 1,(2H)2'-naphtho [ l, 8de][1,3 ]dioxin]-5,8-dione Molecular Formula/Molecular Weight C21H1605; ~

- 348.09977

OMe 0

I ,

11III

General Characteristics The compound slowly decomposed on exposure to air. Isolotion/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Column chromatography was continued with a gradient of methylene chloride-methanol (0-10%). The second fraction was further purified by column chromatography with petroleum ethermethylene chloride (1:1, v/v) as eluant to afford additional palmarumycin C2 and palmarumycin C3. The third fraction was subjected to low-pressure column chromatography with methylene chloride as eluant to afford additional palmarumycin C3 and palmarumycin C5. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo.

86

6. Palmarumycins

Spectral Data UV;

l, Chl m~176176286(1og e=3.35), 299(4.02), 313(4.20), 317(4.16), and 329nm (4.04). IR;

(KBr) 3050, 2930, 1665(C=O), 1608, 1583, 1414, 1381, 1354, 1333, 1271(Ar-O), 1237, 1183, 1119, 1084, 1047, 1034, 959, 926, 851,818, and 750cmq 1H ~ :

(CDCI3) 7.50(1H, dd, Js,4~-8.5Hz, J2,4~0.8Hz, H-4'); 7.49(1H, dd, Jy,6~8.3Hz, Jy,7~0.8Hz, H-5'); 7.42[ 1H, pseudo-t (dd), Jz,3~7.4Hz, Js,4~8.3Hz, H-3']; 7.41[ 1H, pseudo-t (dd), Jy,6~8.4Hz, J6,7~7.4Hz, H-6']; 6.79(1H, d, J6,7=10.2Hz, H- 7); 6.89(1H, dd, J2,s~7.3Hz, J2,4~0.9Hz, H-2'); 6.86(1H, d, J6,7=10.3Hz, H-6); 6.87(1H, dd, J6,7~-7.4Hz, Jy,7~0.8Hz, H-7'); 4.44[ 1H, pseudo-t (dd), J3eq,,,=3.0Hz, J3,~,t=3.0I-Iz, H-4]; 3.47(3H, s, OCH3, H-9); 2.22(2H, m, H-2ax, H-2eq); 2.04(1H, dq, J3eq,s== 14.6Hz, Jseq,4eq=3.0Hz, H-3eq]; and 1.87ppm (1H, dddd, JS~q,.~==14.6Hz, J~=,3=--11.3Hz, J~q,3=,~5 .9Hz, J3,=,4=3 .0Hz, H-3ax). 13C NMR:

(CDCI3) C-4, 68.4(d); C-5, C-8, 186.5(s); C-8', 147.6(s); C-l', 146.3(s); C-4a', 134.2(s), C-7, 137.8(d), C-6', 127.2(d), C-3', 127.4(d); C-4', 120.7(d), C-6, 135.3(d); C-5', 120.4(d); C-Sa, 142.3(s); C-8a', 112.9(s); C-4a, 138.6(s); C-2', 109.2(d); C-7', 109.18(d); C-I, 98.7(s); C-2, 22.4(0; C-9, 58.3(q); and C-3, 26.6ppm (t). Mass Spectrum: LREIMS: 350(10%), 349(14), 348(M+, 56), 318(18), 317(24), 316(M§ CH3OH, 100), 299(10), 287(19), 271(10), 159(11), 131(14), 115(16), 114(14), 71(15), 57(13), and 43m/e (16); HREIMS: C21H1605,found 348.0997re~e, calcd 348.0998. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CI-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

87

Common/Systematic Name Palmarumycin C6 4,7-Dihydroxyspiro [ 1H-inden- 1, 2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-3 (2/O-one Molecular Formula/Molecular Weight C19H12Os; MW = 320.06847

0

OH

General Characteristics Obtained as a microcrystalline powder that slowly turned dark upon contact with air; mp., 191-192~ (dec.). Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of soluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ethermethylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20 mg of palmarumycin C3 was isolated. From the second fraction 22mg of palmarumycin C6 was crystallized (petroleum ether). From fraction four and five liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and Cs could be isolated. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C10, 30mg of palmarumycin Cll, and 198mg of palmarumycin Cn. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

88

6.

Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV: ~Cm~~176176286(1og e=3.47), 296(3.76), 300(3.74), 314(3.55), 346(3.52), and 329nm (3.57). IR: (KBr) 3468(OH), 3376(OH), 3064, 2926, 1638(C=O), 1609, 1584, 1561, 1509, 1476, 1412, 1377, 1264(Ar-O), 1235, 1181, 1092, 1021, 938, 909, 874, 820, 793, 762, and 681 cm~. 1H NMR: (DMSO-d6) 9.83(1H, OH); 9.56(1H, OH); 7.61(2H, d, J3,,4yr,6~=8.1Hz, H-4', H- 5'); 7.5012H, pseudo-t (dd), J2;3y6,,7,~=7.6Hz, J3,,4ys:6~=8.2Hz, H-3', H-6']; 7.14(1H, d, Js,6=8.8Hz, H-6); 7.00(2H, d, ff2,3y6,,79=7.5Hz,J2,4~O.9Hz, H-2', H-7'); 6.96(1H, d, Js,6~8.8Hz, H-5); and 2.76ppm (2H, s, H-2). 13CNMR: (DMSO-d6) C-4, 148.1(s); C-5, 120.4(d); C-8', C-I', 148.26(s); C-4a', 134.0(s); C- 7, 147.5(s); C-6', C-3', 127.6(d); C-4', C-5', 120.8(d); C-6, 125.7(d); C-3, 196.9(s), C-8a', 113.5(s); C-3a, 123.1(s); C-2', C-7', 109.6(d); C-l, 103.5(s); C-2, 49.1(0; and C-7a, 131.8ppm (s).

Mass Spectrum: LREIMS: 320(M § 100%), 303(M+ - OH, 55), 160(10), and 115m/e (11); LRCIMS: (NH3, positive ion) 338(M ++ NH4, 55%), 321(M + + H, 100), 320(M+, 22), 236(21), and 161m/e (30); HREIMS: C19H1205,found 320.0684m/e, calcd 320.0685. Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1- C~6 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

89

Common/Systematic Name Palmarumycin C7 (4aRS, 8aRS)-3-Chloro-5-hydroxyspiro [4a, 8a-epoxynaphthalene- 1(4H),2'-naphtho [ 1,8de][ 1,3]-dioxin]-4,8(5H)-dione Molecular Formula/Molecular Weight C2oH1]C106, MW = 382.02442 0 Cl

OH :

General Characteristics Obtained as the major component of a mixture with palmarumycin C8. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether/methylene chloride (1:1, v/v) as eluant.The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. From the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether). From fractions four and five liquid chromatography (Chromatotron, 99.5% methylene chloride- 0.5% methanol) an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8 could be isolated. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride- 0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C~0, 30mg of palmarumycin CI~, and 198mg ofpalmarumycin C12. An additional 246mg ofpalmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

90

6. Palmarumycins

Fungal Source An unidentified Coniothyrium sp.(intemal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data IH NMR: (CDC13) 7.58(2H, m, H-4', H-5'); 7.50[ 1H, pseudo-t (dd), J2,3~7.5Hz, J3,4=8.2Hz, H3']; 7.45[1H, pseudo-t (dd), J6,7=7.5Hz, Jy.6~8.2Hz, H-6']; 7.11(1H, d, J2,3=7.5Hz, H2'), 6.95(1H, d, J6,,7=7.5t-Iz, H-7'), 6.85(1H, s, H-2), 6.78(1H, d, J6,z=10.6Hz, J5,6 = 5.1Hz, H-6); 6.16(1H, d, J6,7 = 10.6Hz, J5,7= 0.9Hz, H-7); and 5.32ppm (1H, m, H-5). 13C NMR: (CDC13) C-8, 184.8(s); C-4, 184.3(s); C-8', 145.1(s); C-I', 144.4(s); C-6, 140.8(d); C2, 135.9(d); C-4a', 134.1(s); C-3, 131.3(s); C-3', 127.9(d); C-7, 127.6(d); C-6', 127.5(d); C-4', 121.7(d); C-5', 121.6(d); C-4a', 112.3(s); C-2', 110.4(d); C- 7', 109.5(d); C-l, 95.0 (s); C-4a, 63.2(s); C-5, 61. l(d), and C-Sa, 60.4ppm (s). Reference K. Krohn, A. Michel, U. FlOrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp." Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

91

Common/Systematic Name Palmarumycin C8 (4aRS, 8aRS)-3-Chloro-6,7-dihydro-5-hydroxyspiro [4a,8a-epoxynaphthalene- 1(4H),2'nap htho [ 1,8-de ][ 1,3]-dioxin ]-4, 8(5H)-dione Molecular Formula/Molecular Weight C20H13C106; MW = 384.04007 0

OH

C

General Characteristics Obtained as the minor component of a mixture with palmarumycin C7. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. From liquid chromatography of the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether). From liquid chromatography of fractions four and five (Chromatotron, 99.5% methylene chloride-0.5% methanol) an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8 were isolated. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C10, 30mg of palmarumycin Cll, and 198mg of palmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

92

6. Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activit3 against growth of garden cress. Spectral Data 1H NMR: (CDC13) 7.58 0(2H, m, H-4', H-5'); 7.50 [1H, pseudo-t(dd), J2,,3,=7.5Hz, ,]3,4,- 8.2Hz, H-3']; 7.43 [1H, pseudo-t(dd), J6,,7,-7.5Hz, Js,,6,-8.3Hz, H-6']; 7.11(1H, d, J2',s' =7.5Hz, n-2'); 6.93(1H, d, JD,7,-7.6Hz, H-7'); 6.78(1H, s, H-E); 5.12(1H, s, H-5); 2.81(1H, ddd, J7ax,7eq=18.6Hz,J6~,7ax=l 1.4Hz, J6eq,7ax=3.6Hz, H-7ax); 2.5 I(1H, td, J7ax,7eq-18.6Hz, J6ax,7eq(6eq,7eq)=3.6Hz,H-7eq); and 2.09ppm (2H, m, H-6ax, H-6eq). 13C NMR: (CDC13) C-8, 194.4(s); C-4, 185.3(s); C-8', 145.1(s); C-I', 144.5(s); C-6, 23.3(0; C-2, 135.4(d); C-4a', 134.1(s); C-3, 131.4(s); C-3', 127.6(d); C-7, 32.7(0; C-6', 127.4(d); C4', 121.6(d); C-5', 121.55(d); C-8a', 112.4(s); C-2', 110.4(d); C- 7', 109.5(d); C-I, 95.5(s); C-4a, 62.9(s); C-5, 61.4(d); and C-Sa, 61.3ppm (s). Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C~6 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

93

Common/Systematic Name Palmarumycin C10

(2S*,3S*,4R*,4aR *,8aS)-3 ,4-Dihydro-4-hydroxyspiro[2,3 :4a,8a-diepoxynaphthalene1,(2H) 2'-naphtho[ 1,8-de][ 1,3 ]-dioxin]-5,8-dione

Molecular Formula/Molecular Weight C20H1207; MW = 364.05830 OH

0

O

CL General Characteristics A yellow solid; mp., 236~ (dec.); [a]D 2~=-48.2 ~ Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin Cs was isolated; from the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether).Liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) of fractions four and five produced an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg ofpalmarumycin C10, 30mg ofpalmarumycin Cll, and 198mg of palmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

94

6.

Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data UV~

EmtOaxH/10%MethyleneChl~293(log e =4.19), 299(4.31), 307(4.22), 313(4.24), and 327nm (4.16). IR: (KBr) 3480(OH), 1692(C=O), 1609, 1414, 1381, 1275(Ar-O), 1111, 1042, 951,818, and 754cm1. 1H NMR: (DMSO--d6) 7.66(1H, dd, Jy4~-8.3nz, H-4'); 7.65(1H, d, Js:6,=8.3Hz, H-5'); 7.53(2H, m, Jy,6,=8.3Hz, H-3', H-4'); 7.13(1H, d, J2,,.~,=7.5Hz, H-2'); 7.03(1H, d, J6, 7,= 7.5Hz, H-7'); [1H, pseudo-t(dd), Js,,6,=8.2Hz,J6.,7,=7.6Hz, H-6']; 6.77(1H, d, J6,7= 10.7Hz, H6); 6.66(1H, d, J6.7=lO.7Hz, H-7); 5.02(1H, d, J3.4=3.0nz, H-4); 3.46(11-1, d, J2,3 =4.1Hz, H-2); and 3.40ppm (1H, d,[pseudo-t(dd), J2.3~3,.0=3.6Hz, H-3). 13C NMR:

(DMSO-d6) C-8, 190.1(s); C-5, 187.3(s); C-8', 145.2(s); C-I', 144.9(s); C-7, 136.4(d); C-6, 135.7, (d); C-4a', 133.8(s); C-3', 127.9(d); C-6', 127.8(d); C-4', C-5', 121.0(d); C8a', 111.50(s); C-2', 109.2(d); C-7', 109.0(d); C-l, 94.5(s); C-4a, 65.4(s); C- 8a, 63.5(s); C-4, 59.4(d); C-2, 53.2(d); and C-3, 55.2ppm (d). Mass Data: LREIMS: 366(M++ 2H, 22%), 365(M*+ H, 100), 364(M§ 86), 211(6), 171(8), 159(8), 115(14), and 114m/e (18); HREIMS: C20H1207calcd. 364.058303; found 364.058305; calcd. C=65.92, H=3.32, found C=65.85, H=3.40. Reference K. Krohn, A. Michel, U. FlOrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6.

Palmarumycins

95

Common/Systematic Name Palmarumycin all (2S*,3R*,4S*)-3,4-Dihydrospiro [2,3-epoxynaphthalene- 1,(2H) 2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-4,5-diol Molecular Formula/Molecular Weight C20H1405; ~ = 334.08412 OH

OH

General Characteristics Crystallized as colorless needles; mp., 207-208~

[Q~]D20"- -153 ~

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. From the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether). Liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) of fractions four and five provided an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and Cs. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920 mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C10, 30mg of palmarumycin Cll, and 198mg of palmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

96

6. Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data UV: ~,~e,,c,.o,d~ 293(log e=4.05), 296(4.09), 300(4.10), 314(3.83), and 327nm (3.62). IR: (KBr) 3540(OH), 3070(OH), 3000, 2900, 1619, 1590, 1467, 1412, 1381, 1327, 1232, 1180, 1130, 1111, 1072, 1028, 968, 872, 822, and 798cm"1. 1H N1V[R: (CDC13) 8.28(1H, s, OH), 7.57(1H, dd, J3,.4~8.1Hz, J2,,4~-0.9Hz, H-4'), 7.55(1H, d, Jy6 ,= 8.1Hz, H-5'); 7.51[1H, pseudo-t(dd), Jz3,=7.4Hz, Jy4,=8.3Hz, H-3']; 7.44[1H, pseudo-t(dd), Jy,6,=8.2nz, J6,,7,-_7.6Hz,H-6']; 7.37(1H, d, [pseudo-t(dd), J7.8,=7.9Hz, Ja,7=7.5Hz, H-7); 7.14(1H, dd, J2,3,=7.3Hz, J2,.4,=O.9Hz, H-2'); 7.04(1H, dd, J6,7=7.0Hz, J6,s=2.4Hz, H-6); 6.92(1H, d, J6, 7,=7.4Hz, H-7'); 3.74(1H, dd, Jz3=4.4Hz, J~,4=2.7Hz, H-3), 3.ppm(1H, d, Jz3=4.Hz, H-2); 7.41(1H, dd, J7,s=8.0Hz, J6,s=2.3Hz); 5.44(1H, dd, J4.on=l 1.3Hz, J3,4=2.7Hz, H-4); and 3.15ppm (1H, d, J4,OH= 11.3Hz, OH). 13C NMR: (CDCl3) C-4, 66.7(d), C-5, 156.5(s); C-8, 119.3(d); C-8', 147.3(s); C-l', 147.2(s); C-4a', 134.1(s), C-7, 130.6(d), C-6', 127.4(d), C-3', 127.7(d), C-4', 121.3(d), C-6, 118.9(d), C-5', 120.96(d); C-8a, 132.0(s); C-4a, 118.5(s); C-2', 109.9(d); C-7', 109.0(d); C-l, 96.7(s); C-2, 54. l(d); and C-3, 52.8ppm (d).

Mass Data: LREIMS: 335(M ++ H, 27%), 334(M § 8), 285(17), 209(70), 169(100), and 152m/e (72); HREIMS: C2oH~405calcd. 334.0841 found 334.084 lm/e; calcd. C= 71.85, H, 4.22; found C, 71.57, H, 4.07. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp. Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

97

Common/Systematic Name Palmarumycin C~2 (2S*,3S*,4S*)-3,4-Dihydrospiro[2,3: 4a,8a-epoxynaphthalene- 1,(2/-/) 2'-naphtho[ 1,8de][1,3 ]~-diordn]-4,5 , 8-triol Molecular Formula/Molecular Weight C20H1406; ~

= 350.07904

OH

OH

I

General Characteristics Colorless prisms with poor solubility in methylene chloride; mp., 207-208~ (dec.); [a]o 2~ = _179.6 o. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. Palmarumycin C6 crystallized (petroleum ether) from the second fraction. Liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) of fractions four and five provided an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C~0, 30mg ofpalmarumycin Cll, and 198mg ofpalmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

98

6. Palmarumycins

Fungal Source An unidemified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data UV: ~,

1096Methylene Chloride max

288(log e=3.64), 292(3.97), 299(4.10), 302(4.10), 313(3.99), and

327nm (3.46). IR:

(KBr) 3476(OH), 1609, 1586, 1474, 1445, 1377, 1304, 1265(Ar-O), 1190, 1183, 1069, 1061, 1049, 1030, 947, 909, 870, 835, 820, 793,760, and 739cm1. 1H NMR: (CDCI3/CD3OD) 7.54(1H, d, J3,4~8.4Hz, H-4'), 7.49(1H, d, Jy,o,=8.4Hz, H-5'), 7.43[ 1H, pseudo-t(dd), J2,~,=7.5Hz, J3,r H-3']; 7.39[ 1H, pseudo-t(dd), Jy,6,=8.4Hz, d6,,v'=7.5Hz, H-6']; 7.08(1H, d, d2,~,=7.5Hz, H-2'); 6.92(1H, d, ,I6,7, =7.5Hz, H-7'), 6.87(1H, d, Jo,7=8.9Hz, H-7); 6.82(1H, d, J6,7=8.9Hz, H-6); 5.29 (1H, d, J3,4=2.3Hz, H-4); 3.67(1H, d, J2,3=4.4Hz, H-2); and 3.57ppm (1H, dd, J2,r~4.3Hz, ,]3,4=2.7Hz, H-3). ~3CNMR: (CDCla/CDaOD) C-8, 149.6(s); C-5, 149.3(s); C-8', 147.0(s); C-I', 145.7(s); C-4a', 134.0(s), C-3', 127.5(d); C-6', 127.4(d); C-4', 122.4 (d); C5', 121.2(d); C-4a, 120.0(s); C-7, 119.9(d), C-6, 118.9(d); C-8a, 115.4(s), C-8a', 113.0(s); C-2', 110.6(d), C-7', 109.8(d); C-l, 99.4(s); C-4, 65.5(d), C-2, 51.5(d); and C-3, 54.0ppm (d). Mass Data: LREIMS: 351(M++ H, 59%), 350(M+, 90), 332(NC- H20, 100), 316(21), 304(332CO, 34), 303(332 - CHO, 34), 287(20), 275(16), 159(41), 131(23), 115(36), and 114m/e (33), HREIMS: C20H1406calcd. 350.0790, found 350.0790m/e. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C~-C16from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

99

Common/Systematic Name Palmarumycin C13; Diepoxide rl (2S*,3 S*,4R*,4aR *,5R*,8aS*)-3,4-Dihydro-4, 5-dihydroxyspiro [2,3:4a, 8adiepoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de] [ 1,3 ] -dioxin]-8(5H)-one Molecular Formula/Molecular Weight C20H1607; MW -- 366.07395

OH

OH

General Characteristics Isolated as a 1:1 mixture with palmarumycin C14. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins C13 and C14 as a 1:1 mixture, along with palmarumycins C15, and C16. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria, fungi and biological activity against growth of garden cress.

100

6. Palmarumycins

Spectral Data ~HNMR: (CD3OD) 7.57(2H, m, J~,4,o,6,)=8.4Hz, H-4', H-5'); 7.50[ 1H, pseudo-t(dd), J2,3,=7.4Hz, J3,4,=8.4Hz, H-3']; 7.46[ 1H, pseudo-t(dd), Jy,o,=8.4Hz, Jo,7,=7.5Hz, H6']; 7.04(1H, dd, Jz,3,=7.4Hz, Jz,4,= 1.0Hz,H-2'); 6.94(1H, dd, Je,7~-7.5Hz, Jy,7,=0.6Hz, H-7'); 5.91(1H, dd, Jo,7=lO.6Hz, Js.7=O.9Hz, H-7); 6.74(1H, dd, Jo,7=lO.6Hz, Js,6=4.9Hz, H-6); 5.11(1H, m, H-4); 4.82(1H, dd, Js,o=4.9Hz, J5,7= 0.9Hz, H-5); and 3.45ppm (2H, m, H-2, H-3). 13C NMR:

(CD3OD) C-8, 190.1(s); C-5, 62.8(d); C-8', 147.5(s); C-I', 145.2(s); C-4a', 136.0(s); C-3', 129.0(d); C-6', 128.8(d); C-4', 122.2(d); C-5', 122.1(d); C-4a, 72.3(s); C- 7, 127.4(d); C-6, 145.1(d); C-8a, 65.4(s); C-8a', 113.7(s); C-2', 110.5(d); C-7', 110.0(d); C-l, 96.8(s); C-4, 62.5(d); C-2, 54.9(d); and C-3, 57.0ppm (d). Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumyeins

101

Common/Systematic Name Palmarumycin C~4; Diepoxine

(2S*,3S*,4R*,4aR *,5R *,8aS*)-3 ,4,6,7-Tetrahydro-4,5-dihydroxyspiro[2,3 :4a, 8a_

diepoxynaphthalene- 1(2/-/), 2'-naphtho[ 1,8-de] [ 1,3 ]-dioxin]-8(5H)-one Molecular Formula/Molecular Weight C20H1607, ~

OH

= 368.08960

!

OH

General Characteristics Isolated as a 1:1 mixture with palmarumycin C13. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins Ca and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2 mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins C~3 and C14 as a 1:1 mixture, along with palmarumycins C]5, and C16.

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress.

102

6. Palmarumycins

Spectral Data ~HNMR: (CD3OD) 7.56(2H, m, Jy,4,(y69=8.4Hz, H-4', H-5'); 7.49[ 1H, pseudo-t(dd), J2,,3~7.5Hz, Jy,4,=8.4Hz, H-3']; 7.45 [ 1H, pseudo-t(dd), Js,,6,=8.4I-Iz, J6,,7,=7.5I-'Iz, H6']; 7.03(1H, dd, J2,,3,=7.5Hz, J2,,4,=l.OHz, H-2'); 6.95(1H, dd, J6,,7~7.5Hz, Jy,7,=0.6Hz, H-7'); 4.87(1H, dd, J3,4=2.5Hz, H-4); 4.71(1H, t, Js,6ax(5,6eq)=3.0nz,H-5), 3.45(2H, m, H-2, H-3); 2.57(1H, ddd, J7~x,7eq=18.1Hz,J6~,Tax--12.4Hz, J7eq,7~x=6.5Hz, H-7ax); 2.32(1H, ddd, J7a~,7eq=l8.1Hz, J6ax,7eq=5.6I--Iz,J6eq,7eq=2.4Hz, H-7eq); 2.04(1H, dddd, J6ax,6eq = 14.2Hz, J6~,7~-12.4Hz, J6ax,Teq=5.6nz, Js,6ax=3.0nz, H-6ax); and 1.83ppm (1H, dddd, J6ax,6eq= l 4.2Hz, J6eq,7ax=6. 5Hz, Js,6eq --3 .0 n z , J6eq,Teq=2.4Hz, H-6eq). 13C NMR: (CD3OD) C-8, 200.2(s); C-8', 147.5(s); C-I', 147.2(s); C-4a', 136.0(s); C-3', 129.0(d), C-6', 128.8(d), C-4', 122.2,(d); C-5', 122.1(d), C-4a, 71.3(s), C-7, 33.9(0; C-6, 25.7(0; C-8a, 64.1(s); C-8a', 113.7(s); C-2', 1lO.5(d); C-7', 1lO.O(d); C-l, 96.8(s); C-4, 63.2(d); C-2, 55.0(d); C-5, 64. l(d); and C-3, 56.9ppm (d).

Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C~-C16from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

103

Common/Systematic Name Palmarumycin C15 (2S*,3S* 4R* 4aR*,5R* 8RS, 8aS*)-3,4,5,8-Tetrahydrospiro[2,3:4a,8adiepoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de ] [ 1,3 ]-dioxin]-4, 5,8-tri ol Molecular Formula/Molecular Weight C20H1607; M~V = 367.08178

OH

OH

General Characteristics Isolated as a white powder; mp., 150~ (dec.); [a]D2~ -18.1 ~ Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins Cls and C14 as a 1:1 mixture, along with palmarumycins C15, and C16.

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activit3 against growth of garden cress.

104

6. Palmarumycins

Spectral Data UV: ~, ~.,.Ch,o,d. 287(1og e = 3.79), 297(4.07), 312(3.71), and 327nm (3.44). IR: (KBr) 3426(OH), 2361, 1611, 1414, 1381, 1273(Ar-O), 1136, 1088, 1047, 1032, 941,822, and 758cm1. 1H NNtR: (CDC13/CD3OD) 7.51(1H, d, J3,~8.2Hz, H-4'); 7.50(1H, d, Js,,6~8.3Hz, H-5'); 7.42(2H, m, H-3', H-6'); 7.11(1H, d, Jz,3,=7.3Hz, H-2'); 6.96(1H, d, J6.,7.=7.4Hz, H7'); 5.75(1H, ddd, J6,7=lO.SHz, J s,6=4.SHz, ff6,s=l.7Hz, H-6); 5.60(1H, dd, ,]6,7= 10.5Hz, JT,s-2.60Hz, H-7); 5.18(1H, s, H-8); 4.93(1H, d, J3,~2.8Hz, H-4); 4.61(1H, d, J4,5=4.5Hz, H-5); 3.48(1H, d, Jz3=4.3Hz, H-2); and 3.42ppm(1H, dd, Jz3=4.3Hz, J3,r=2.SHz, H-3). 13CNMR: (CDC13/CD3OD) C-8, 63.0(d); C-5, 61.0(d); C-8', 145.8(s); C-l', 145.1(s), C-4a', 134.0(s), C-3', 127.7(d), C-6', 127.3(d), C-4', 121.5(d); C-5', 121.3(d), C-4a, 68.3(s); C-7, 126.9(d); C-6, 125.6(d); C-8a, 66.2(s); C-8a', 112.7(s); C-2', 110.3(d); C-7', 109.4(d); C-l, 96.6(s); C-4, 61.4(d); C-2, 53.2(d), and C-3, 55.8ppm (d).

Mass Data: LREIMS: 368(M § 100%), 160(21), 125(26), 111(38), 97(51), 82(48), 71(55), and 57role (68); HREIMS C2oH1607calcd. 368.0896; found 368.089m/e; calcd. C,65.22, H, 4.42; found C, 65.14, H, 4.42. Reference K. Krohn, A. Michel, U. Flfrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1C16 from Coniothyrium sp." Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

105

Common/Systematic Name Palmarumycin C 16 (2S*, 3S*,4R*,4aR *, 5R *, 8RS, 8 aS*)-3,4,5,6, 7, 8-Hexahydro spiro [2,3: 4a, 8adiepoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de ][ 1,3] -dio~n]-4, 5,8-triol Molecular Formula/Molecular Weight C20H1807; ~

OH

= 369.09743

OH

General Characteristics Crystals; mp., 187-188~ (dec.);

[I~]D 20 -'-

-43.3 ~

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins C13 and C14 as a 1:1 mixture, along with palmarumycins C15 and C16.

Fungal Source An unidentified Coniothyriumsp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activit2 against growth of garden cress.

106

6. Palmarumycins

Spectral Data UV: L ~.o.c,,o,d, 287 (log e=3.60), 298 (3.81), 307 (3.66), 313 (3.68), and 327nm (3.60). IR:

(KBr) 3426(OH), 2938, 2361, 1611, 1412, 1381, 1273(Ar-O), 1090, 1047, 1034, 1005, 965, 939, 820, and 758cm1. :H ~ :

(CD3OD) 7.59(1H, dd, J~,,4~8.SHz, J~,,4~l.lHz, H-4'); 7.58(1H, d, Js,,6~8.SHz, H-5'); 7.52 [1H, pseudo-t(dd), J2,,3,=7.4Hz, J2,,4,=8.SHz, H-3']; 7.49[1H, pseudo-t(dd), Js,a,=8.5Hz, J6,,7.=7.5Hz, H-6']; 7.14(1H, dd, J2,~,=7.3Hz, J2,.4,=l.OHz, H-2'); 7.04(1H, dd, J6,z,--7.5Hz, ds,,7,=0.7Hz, H-7'); 4.82(1H, d, d3.4=2.8Hz, H-4); 4.68(1H, m, H-8); 4.47(1H, m, H-5); 3.45(1H, d, Jz3=4.3Hz, H-2); 3.41(1H, dd, Jz3=4.3Hz, J3,4=2.8Hz, H-3); 1.94(2H, m, J=l 1.4Hz, H-6eq, H-7eq); and 1.48ppm (2H, m, J= 11.4Hz, H-6ax, H-7ax).

13CNMR: (CDCI3/CD3OD) C-8, 63.4(d), C-5, 64.3(d); C-8', 147.9(s); C-I', 147.6(s); C-4a', 135.9(s); C-3', 129.1(d); C-6', 128.9(d); C-4', C-5', 122.4(d), C-4a, 69.3(s); C-7, 26.0(0; C-6, 24.0(0; C-8a, 68.0(s); C-8a', 114.3(s), C-2', 111.4(d); C-7', 110.5(d); C-I, 98.4(s); C-4, 63.2(d); C-2, 54.0(d), and C-3, 57.2ppm (d). Mass Data: LREIMS 370(M§ 100%), 352(11), 213(16), 172(20), 160(72), 159(20), 139(31), 121(27), 116(26), 99(38), 86(60), and 56m/e (37); HR IMS: C20H1807calcd. 370.1052; found 370.1052re~e; calcd. C, 64.86, H, 4.90; found C, 64.31, H, 4.76. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp. Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

Phomosines Phomosine A Phomosine B Phomosine C

107

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7. Phomosines

109

Common/Systematic Name Phomosine A Molecular Formula/Molecular Weight C18H1807; ~

= 346.10525

H

OH

0

Me 5//.,. i ~ 0 HO

OH

~'~J "Me "~, OMe

Me

General Characteristics Long, pale yellow needles from dichloromethane-petroleum ether; mp., 203-204~ Isolation/Purification Culture medium from Phomopsis sp. was homogenized with a Waring blender, diluted with water and then extracted four times with ethyl acetate. The organic extracts were dried (Na2SO4), filtered, and evaporated m vacuo. The main compounds were detected in the residue on TLC at Rf values: phomosine A: Rf=0.71; phomosine B: Rf=0.53; phomosine C: Rf=0.48, 5% methanol-methylene chloride detected by UV irradiation at 254nm. Column chromatography on silica gel (methylene chloride-20% ethyl ether) gave four fractions. The second fraction gave material that was further purified by crystallization from dichloromethane-petroleum ether to afford phomosine A. The third fraction was further separated by Chromatotron chromatography (methylene chloridepetroleum ether; 1:1, v/v) into four fractions. The second fraction was purified by thinlayer chromatography (5% methanol-methylene chloride) and crystallization from dichloromethane-petroleum ether to afford additional phomosine A. From the fourth fraction, phomosine B and phomosine C were isolated by the same procedure. Fungal Source Phomopsis sp. Biological Activity All three metabolites showed moderate fungicidal and antibacterial activity against four fungal and two bacterial (one Gram-positive and one Gram-negative) test organisms. Sp,,ectral Data UV:

~mM~y~=~176176232(1og C = 5.23) and 269nm (4.52).

110

7.

Phomosines

IR~

(KBr) 3320(OH), 2955(CH3), 1653(CO), 1637(CO), 1607, 1582, 1437, 1331, 1302, 1211, 1200, 1096, and 808cm~ 1H NMR: (CDCI3) 2.10(3H, s, H-3a); 2.16(3H, s, H-5'a); 2.28(3H, s, H-2a); 3.92(3 H, s, H-lb); 5.80(1H, s, H-6'); 6.36(1H, s, H-4'), 8.92(1H, OH); 10.43(1H, s, H-2'a); 11.87(1H, s, OH), and 11.92ppm (1H, s, OH). 13C NMR: (CDC13) 8.4, C-Sa, (q); 15.3, C-2a (q); 22.5, C-5'a (q); 52.3, C-lb (q); 104.3, C-6' (q); 104.9, C-1 (s); 109.2, C-2' (s); 111.2, C-5 (s); 111.6, C-4' (d); 131.6, 133.0, C-2, C-3 (2s); 150.9, C-5' (s); 153.8, C-4 (s); 161.0, C-3' (s); 161.1, C-6 (s); 164.1, C-I' (s); 172.9, C-la (s); and 194.3ppm C-2a (s). Mass Data: 346(M+, 100%), 314(98), 286(54), 258(26), 165(32), and 151m/e (53); HREIMS: (ClsHIsO7): 346.1050; calcd. 346.1052; found C=62.29, H=5.44; calcd. C=62.41, H=5.24. Reference K. Krohn, A. Michel, E. R6mer, U. F16rke, H.-J. Aust, S. Draeger, B. Schulz, and V. Wray, Biologically Active Metabolites from Fungi 6, Phomosines A-C, Three New Biaryl Ethers from Phomopsis sp.; Natural Product Letters, Vol. 6, pp. 309-314(1995).

7.

Phomosines

111

Common/Systematic Name Phomosine B Molecular FormulafMolecular Weight C19H2207; 1VIW = 362.13655

OH

HO"

"~ 0-"

CH2OMe

"Me "OMe

, Me

General Characteristics Colorless powder from dichloromethane-petroleum ether; mp., 204-206~ Isolation/Purification Culture medium from Phomopsis sp. was homogenized with a Waring blender, diluted with water and then extracted four times with ethyl acetate. The organic extracts were dried (Na2SO4), filtered, and evaporated m vacuo. The main compounds were detected in the residue on TLC at Rf values: phomosine A: Rf=0.71; phomosine B: Rf=0.53; phomosine C: Rf=0.48, 5% methanol-methylene chloride detected by UV irradiation at 254nm. Column chromatography on silica gel (methylene chloride-20% ethyl ether) gave four fractions. The second fraction gave material that was further purified by crystallization from dichloromethane-petroleum ether to afford phomosine A. The third fraction was further separated by Chromatotron chromatography (methylene chloridepetroleum ether; 1:1, v/v) into four fractions. The second fraction was purified by thinlayer chromatography (5% methanol-methylene chloride) and crystallization from dichloromethane-petroleum ether to afford additional phomosine A. From the fourth fraction, phomosine B and phomosine C were isolated by the same procedure. Fungal Source Phomopsis sp. Biological Activity All three metabolites showed moderate fungicidal and antibacterial activity against four fungal and two bacterial (one Gram-positive and one Gram-negative) test organisms. Phomosines B and C also inhibited the algal organism, Chlorellafusca.

112

7.

Phomosines

Spectral Data UV:

~,mM~y~'~176

232(1og e = 4.51), 267(4.25), and 312nm (3.74).

IR: (KBr) 3413(OH), 2953(CH3), 1647, 1622, 1599, 1456, 1422, 1337, 1300, 1285, 1223, 1088, 1047,and 934cm 1. 1H N-MR:

(CDC13) 2.08(3H, s, H-3a), 2.11(3H, s, 5'a-H); 2.44(3H, s, H-6a), 3.47(3H, s, H2'b); 3.97(3H, s, H-lb); 4.78(2H, s, H-2'a); 5.94(1H, s, H-6'a); 6.46(1H, s, H-4'); 8.69(1H, s, OH), 9.10(1H, s, OH); and 11.94ppm (1H, s, OH). lSc NMR:

(CDCI3) 8.3, C-3a (q); 15.8, C-6a (q); 21.5, C-5'a (q), 52.4, C-lb (q); 58.5, C-2'b (q); 64.6, C-2'a (t), 104.3, C-1 (s); 107.1, C-6' (d); 111.1, C-3 (s), 111.5, C-2' (s); 111.5, C-4' (d); 132.3, 135.5, C-2, C 5 (2s); 140.9, C-5' (s), 154.7, C-4 (s), 157.2, C-3' (s), 158.5, C-I' (s); 161.1, C-2 (s); and 173.4ppm, C-la (s). Mass Spectrum: 362(M +, 20%), 330(62), 298(100), 283(26), and 270m/e (18); HREIMS: (C19H2207) 362.136Ore~e; calcd, 362.1365. Reference K. Krohn, A. Michel, E. R0mer, U. FlOrke, H.-J. Aust, S. Draeger, B. Schulz, and V. Wray; Biologically Active Metabolites from Fungi 6; Phomosines A-C, Three New Biaryl Ethers from Phomopsis sp.; Natural Product Letters, Vol. 6, pp. 309-314(1995).

7.

Phomosines

113

Common/Systematic Name Phomosine C Molecular Formula/Molecular Weight C17H1607; M W -- 3 3 2 . 0 8 9 6 0

o.

.yo

HO-~~ I~Me 1 ~L~s,I 0~ ~OMe Me General Characteristics White powder from dichloromethane-petroleum ether; mp., 192-193~ Isolation/Purification Culture medium from Phomopsis sp. was homogenized with a Waring blender, diluted with water and then extracted four times with ethyl acetate. The organic extracts were dried (Na2SO4), filtered, and evaporated in vacuo. The main compounds were detected in the residue on TLC at Re values: phomosine A, Rf=0.71; phomosine B, Rf=0.53; phomosine C, Rf=0.48, 5% methanol-methylene chloride detected by UV irradiation at 254nm. Column chromatography on silica gel (methylene chloride-methylene chloride20% ethyl ether) gave four fractions. The second fraction gave material that was further purified by crystallization from dichloromethane-petroleum ether to afford phomosine A. The third fraction was further separated by Chromatotron chromatography (methylene chloride-petroleum ether; 1:1, v/v) into four fractions. The second fraction was purified by thin-layer chromatography (5% methanol-methylene chloride) and crystallization from dichloromethane-petroleum ether to afford additional phomosine A. From the fourth fraction, phomosine B and phomosine C were isolated by the same procedure. Fungal Source Phomopsis sp. Biological Activity All three metabolites showed moderate fungicidal and antibacterial activity against four fungal and two bacterial (one Gram-positive and one Gram-negative) test organisms. Phomosines B and C also inhibited the algal organism, Chlorellafusca.

114

7.

Phomosines

Spectral Data UV:

~,~yl~oC,o,~ 232(log e = 4.43), 263(4.00), and 273nm (3.93). IR:

(KBr) 1420(OH), 3195, 2955, 1647, 1623, 1431, 1329, 1285, 1254, 1198, and 1074cm1. 1H NMR:

(CDC13) 2.21(3H, s, H-5'a), 2.38(3H, s, H-2a); 3.96(3 H, s, H-lb), 5.93(1H, s, H-6'); 6.42(1H, s, H-4'); 6.46(1H, s, H-5), 9.15(1H, OH); 10.50(1H, s, H-Z'a); 11.43(1H, s, OH); and 11.95ppm (1H, s, OH). 13C NMR:

(CDC13) 15.4, C-2a (q); 22.5, C-5'a (q); 52.5, C-lb (q); 103.1, C-5 (d); 105.0, C-1 (s); 105.0, C-6' (d), 109.6, C-2' (s), 111.7, C-4' (d), 130.7, 135.8, C-2, C-3 (2s), 151.4, C-5' (s), 156.4, C-4 (s), 161.8, C-3' (s); 162.8, C-6 (s); 164.3, C-l' (s); 172.5, C-la (s), and 194.Sppm, C-2a (s). Mass spectrum: 332(M +, 100%), 300(71), and 272m/e (36); HREIMS: (C]7H]607): 332.0890; calcd, 332.0896. Reference K. Krohn, A. Michel, E. ROmer, U. F1Orke, H.-J. Aust, S. Draeger, B. Schulz, and V. Wray; Biologically Active Metabolites from Fungi 6; Phomosines A-C, Three New Biaryl Ethers from Phomopsis sp. Natural Product Letters, Vol. 6, pp. 309-314(1995).

Fumiquinazolines Fumiquinazoline A Fumiquinazoline B Fumiquinazoline C Fumiquinazoline D Fumiquinazoline E Fumiquinazoline F Fumiquinazoline G

115

This Page Intentionally Left Blank

8. Fumiquinazolines

117

Common/Systematic Name Fumiquinazoline A Molecular Formula/Molecular Weight C24H23NsO4; MW' = 445.17500 16

d."'~.c,I~NH 7

-

H_

H,"';4N~, "0

II

o . ,,,,

.,Me

H "ItOH

H

179

ll,,, 20

N

\

N

~

//

\xj

General Characteristics Fumiquinazoline A was obtained as a pale yellow powder; mp., 178-182~ (c=0.47, in CHCI3).

[(I]D-214.5 ~

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH2o using methanol as the eluant. The third fraction was ehromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C alter purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillusfumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells.

118

8.

Fumiquinazolines

Spectral Data UV"

~,M~n 208(1og e = 4.58), 226(4.29), 233(4.44), 256(4.17), 265(4.13), 277(4.01), 305(3.54), and 327nm (3.44). CD: (c=1.47 x 10-5 mol/dm3 in ethanol) 230(Ae -11.82), 245(0), 251(+2.11), 261(0), 278(-3.53), 284(-2.99), 290(-2.65), 297(-2.31), 305(-2.45), 312(-1.90), 316(- 0.20), and 334nm (0). IR:

3349(OH, NH), 1680(CON), and 1608em1 (Ar-C-C). 1H NMR: (CDCI3) 66.1(1H, dd, J-0.9, 0.3Hz, NH-2); 4.88(1H, qd, J=7.1, 0.3Hz, H-3); 7.67(1H, dd, J=8.2, 1.0Hz, H-7); 7.75(1H, ddd, J-8.2, 7.0, 1.0Hz, H-8); 7.49(1H, ddd, J=7.9, 7.0, 1.0Hz, H-9); 8.23(1H, dd, J=7.9, 1.0Hz, H-10); 5.97(1H, ddd, ,/=10.9, 6.0, 0.9Hz, H-14); 2.28(1H, dd, J-13.7, 6.0Hz, H-15A); 2.51(1H, dd, J=13.7, 10.9Hz, H-15B); 1.79(1H, d, J=7.1Hz, H-16); 5.49(1H, s, H=18); 2.79(1H, br s, NH19); 4.22(1H, q, J=6.THz, H=20); 7.52(1H, dd, J=7.5, 1.0Hz, H-24); 7.31(1H, td, J=7.5, 1.0Hz, H-25); 7.16(1H, ddd, J=7.5, 6.8, 1.0Hz, H-26); 7.61(1H, dd, J=6.8, 1.0Hz, H-27); 1.35(3H, d, J=6.7Hz, H-29); and 4.89ppm (1H, s, OH). 13C NMR:

(CDCI3) 172.36(C-1, q); 49.15(C-3, t); 150.78(C-4, q); 146.87(C-6, q); 127.57(C-7, t); 134.80(C-8, t); 127.45(C-9, t); 126.77(C-10, t); 120.18(C-11, q); 160.48(C-12, q); 52.98(C-14, t); 36.72(C-15, s); 16.75(C-16, p); 80.20(C-17, q); 86.28(C-18, t); 59.01(C-20, t); 170.53(C-21, q); 136.17(C-23, q); 115.01(C- 24, t); 129.76(C-25, t); 125.58(C-26, t); 124.85(C-27, t); 138.59(C-28, q); and 18.63ppm(C-29, p). Mass Spectrum: EIMS: 445(M+, 5%), 428(M+- OH, 36), 229(13), 228(4), 217(100), 199(20), and 146m/e (22); HREIMS: 445.1769re~e;C24Hz3NsO4,requires 445.1750. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

119

Common/Systematic Name Fumiquinazoline B Molecular Formula/Molecular Weight C24H23NsO4; M W = 445.17500

0 General Characteristics Fumiquinazoline B was obtained as a pale yellow powder; mp., 174-176~ (C=0.38, in CHCIa).

[a]D-196.6 ~

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by I-IPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fishPseudolabrus

japonicus.

Biological ,Activity Exhibited moderate cytotoxicity against cultured P388 cells.

120

8. Fumiquinazolines

Spectral Data UV:

~M~, 206(log 6 = 4.77), 225(4.69), 232(4.65), 256(4.38), 266(4.32), 277(4.24), 305(3.79), and 317rim (3.68). CD: (c=2.22 x 10.5 mol/dm-3 in ethanol) 227(Ae -11.04), 246(0), 252(+2.32), 259(0), 280(-7.36), 287(-5.31), 297(-4.36), 305(-4.77), 318(-3.13), and 329nm (0). IR:

3350(OH, NH), 1673(CON), and 1604cm1 (Ar-C-C). 1H M R : (CDCI3) 7.34(1H, dd, J=4.9, 0.9Hz, NH-2); 4.72(1H, qd, J=7.2, 4.9Hz, H-3), 7.56(1H, dd, J=8.0, 1.0Hz, n-7); 7.73(1H, ddd, J=8.0, 7.0, 1.0Hz, H-8); 7.45(1H, ddd, J=7.8, 7.0, 1.0Hz, H-9), 8.19(1H, dd, J=7.8, 1.0Hz, H-10), 5.79(1H, ddd, J=l 1.2, 4.8, 0.9Hz, H-14); 2.48(1H, dd, J=13.3, 4.8Hz, H-15A); 2.61(1H, dd, J=13.3, ll.2Hz, H-15B); 1.83(1H, d, J=7.2Hz, H-16); 5.42(1H, br s, H-18); 2.75(1H, br s, NH-19); 4.14(1H, q, J=6.7Hz, n-20); 7.51(1H, dd, J=7.5, 1.0Hz, H-24); 7.30(1H, td, J=7.5, 1.0Hz, H-25); 7.17(1H, td, J=7.5, 1.0Hz, H-26); 7.61(1H, dd, J=7.5, 1.0Hz, H-27); 1.29(3H, d, J=6.7Hz, H-29); and 5.47ppm (1H, s, OH). 13C NMR:

(CDC13) 170.69(C-1, q), 52.73(C-3, t), 150.72(C-4, q), 147.00(C-6, q), 126.88(C-7, t), 134.97(C-8, t), 127.27(C-9, t); 126.88(C-10, t), 120.01(C-11, q), 160.30(C-12, q); 52.01(C-14, t); 38.97(C-15, s), 24.88(C-16, p), 80.20(C-17, q), 80.43(C-18, t), 59.07(C-20, t), 170.56(C-21, q), 136.56(C-23, q), 114.86(C-24, t), 129.73(C-25, t), 125.50(C-26, t), 125.02(C-27, t), 138.62(C-28, q), and 18.14ppm (C-29, p). Mass Spectrum: EIMS: 445(M+, 5%), 229(10), 228(2), 217(100), 199(6), and 146m/e (15); HREIMS: 445.174 lm/e; C2aH23NsO4,requires 445.1750. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish, J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

121

Common/Systematic Name Fumiquinazoline C Molecular Formula/Molecular Weight C24H21NsO4; M W = 443.15935

0 General Characteristics Fumiquinazoline C was obtained as colorless prisms, 244-246~ in CHC13).

[a]D -193.7 ~ (C=0.31,

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Acti~ty Exhibited moderate cytotoxicity against cultured P388 cells.

122

8.

Fumiquinazolines

Spectral Data UV:

~M~, 207(1og e = 4.56), 225(4.48), 260(4.06), 271(4.02), 282(3.98), 304(3.61), and 317nm (3.50). CD: (c = 4.02 x 10-5 mol/dm3 in ethanol) 226 (Ae -15.84), 238(0), 243(+5.28), 247(+ 3.77), 253(+ 4.90), 269(0), 303(-13.58), 310(-10.18), 314(-10.56), and 328nm (0). IR;

3343, 3257(NH), 1715(CON), and 1611cm-1 (Ar-C-C). 1H NMR:

(CDCI3) 8.04(1H, br s, NH-2); 7.78(1H, dd, J=7.4, 1.7Hz, H-7); 7.81(1H, ddd, J-7.4, 6.3, 1.THz, H-8); 7.60(1H, ddd, J=7.4, 6.3, 1.THz, H-9); 8.35(1H, dd, J=7.4, 1.7Hz, H-10); 5.72(1H, dd, J=10.9, 6.0Hz, H-14); 2.14(1H, dd, J-13.7, 6.0Hz, H15A); 2.98(1H, dd, J=13.7, 10.9Hz, H=15B); 2.06(1H, s, H-16); 5.34(1H, d, J=6.9Hz, H=18), 1.04(1H, dd, J=6.9, 6.THz, NH-19); 3.71(1H, qd, J--6.9, 6.THz, H- 20); 7.45(1H, dd, J-7.4, 1.0Hz, H=24); 7.32(1H, td, ,/=7.4, 1.0Hz, H=25); 7.19(1H, td, J=7.4, 1.0Hz, H-26); 7.37(1H, dd, J-7.4, 1.0Hz, H-27); and 1.06ppm (3H, d, J=6.9Hz, H-29). 13C NMR.:

(CDCI3) 171.02(C-I, q), 84.16(C-3, q); 150.39(C=4, q), 146.32(C-6, q), 128.45(C-7, t), 134.91(C-8, t), 128.56(C-9, t), 126.9S(C-10, t), 121.34(C-11, q); 159.53(C-12, q), 51.39(C-14, t), 31.36(C-15, s), 24.43(C-16, p), 87.07(C-17, q), 87.07(C-18, t), 58.61(C-20, t), 170.90(C-21, q), 135.73(C-23, q), 115.46(C-24, t), 130.23(C-25, t), 126.17(C-26, t), 124.88(C-27, t), 138.41(C-28, q), and 18.71ppm (C-29, p). Mass Spectrum: HREIMS: 443.159 lm/e; C24H21NsO4,requires 443.1594. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish, J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

123

Common/Systematic Name Fumiquinazoline D Molecular Formula/Molecular Weight C24H21N504, M ~ = 443.1593 5

O General Characteristics Fumiquinazoline D was obtained as colorless prisms from acetone, mp., 214-216~ + 68.9 ~ (C=0.27, in CHC13).

[a]D

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells.

124

8. Fumiquinazolines

Spectral Data UV~

ZM~" 205(1og e = 4.30), 225(4.26), 232(4.24), 254(3.91), 265(3.84), 276(3.77), 304(3.43), and 316nm (3.54). CD: (c = 4.33 x 10.5 mol/dm3 in ethanol) 232(Ae -1.75), 235(0), 247(+6.12), 258(0), 268(- 4.90), 275(- 5.25), 294(- 2.45), 303(-2.80), 310(-2.10), 315(-2.10), and 326nm

(o). IR~

3418(OH, NH), 1703(CON), and 1611em1 (At-C-C). ~H NMR: (CDCI3) 9.16(1H, br s, NH-2); 7.66(1H, dd, J=8.3, 1.2Hz, H-7); 7.75(1H, ddd, J=8.3, 6.8, 1.2Hz, H-8); 7.50(1H, ddd, J=7.9, 6.8, 1.2Hz, H-9); 8.19(1H, dd, J=7.9, 1.2Hz, n-10), 5.65(1H, d, J=10.3Hz, n-14); 2.27(1H, d, J=15.1Hz, H-15A); 3.38(1H, dd, ,/=15.1, 10.3Hz, H-15B); 2.02(1H, s, n-16); 5.52(1H, d, J=l.3Hz, U-18); 3.96(1H, qd, J=6.5, 1.3Hz, n-20); 7.41(1H, dd, J=7.4, 1.0Hz, H-24); 7.23(1H, td, J=7.4, 1.0Hz, H-25); 7.05(1H, td, ,/=7.4, 1.0Hz, H-26); 7.44(1H, dd, J=7.4, 1.0Hz, H-27); 1.08(3H, d, J=6.5Hz, H-29); and 5.27ppm (1H, br s, OH). 13C NMR:

(CDCI3) 172.58(C-1, q), 70.84(C-3, q), 152.14(C-4, q), 146.34(C-6, q), 127.79(C-7, t); 134.83(C-8, t); 127.68(C-9, t), 126.85(C-10, t), 120.35(C-11, q), 160.86(C-12, q); 52.76(C-14, t); 43.44(C-15, s), 18.77(C-16, p); 84.13(C-17, q); 85.59(C-18, t), 59.11(C-20, t), 171.41(C-21, q), 137.60(C-23, q), 115.43(C-24, t), 130.07(C-25, t), 125.77(C-26, t), 124.31(C-27, t); 137.38(C-28, q); and 17.41ppm (C-29, p). Mass Spectrum: HR IMS: 443.1588re~e; C24I"I21N504,requires 443.1594. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

125

Common/Systematic Name Fumiquinazoline E Molecular Formula/Molecular Weight CzaH25NsOs; MW = 475.18557

o ,,

HN--V

O General Characteristics Fumiquinazoline E was obtained as a pale yellow powder, mp., 168-172~ (C=0.18, in CHC13).

[tt]D-143.3 ~

Isolation/Purification / The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water;7:3,v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v).

Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

126

8.

Fumiquinazolines

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells. Spectral Data UV:

ZM~" 210 (log e = 4.54), 226 (4.48), 233 (4.44), 256 (4.16), 278 (4.06), 304 (3.62), and 317nm (3.52). CD: (c = 4.33 x 10-5 mol/dm3 in ethanol) 219(Ae -2.69), 221(-2.34), 233(-7.73), 245(0), 251(+ 1.70), 259(0), 278(- 3.98), 290(- 2.87), 296(-2.34), 312(-2.58), 310(-2.05), 314(-1.99), and 329nm (0). IR:

3427(OH, NH), 1685(CON), and 1608cma (Ar-C-C). 1H NMR:

(CDC13) 7.57(1H, br d, NH-2); 7.74(1H, dd, J=7.7, 1.5Hz, H-7); 7.79(1H, ddd, J=8.2, 7.7, 1.5Hz, H-8); 7.53(1H, ddd, J=8.2, 6.8, 1.5Hz, H-9), 8.25(1H, dd, J=6.8, 1.5Hz, H-10); 5.93(1H, ddd, J=8.9, 5.2, 1.0Hz, H-14), 2.34(1H, dd, J=14.4, 5.2Hz, H-15A), 2.80(1H, dd, J=14.4, 8.9Hz, H-15B); 1.97(1H, s, H-16); 5.45(1H, s, H-18), 4.16(1H, q, J=6.6Hz, H-20), 7.56(1H, dd, J=7.2, 0.8Hz, H-24); 7.33(1H, ddd, J=8.0, 7.2, 1.0Hz, H-25); 7.16(1H, ddd, J=8.0, 7.2, 0.8Hz, H-26); 7.57(1H, dd, J=7.2, 1.0Hz, H-27); 1.33(3H, d, J=6.6I-Iz, H-29); 3.33(3H, s, OMe); and 4.55ppm (1H, s, OH-17). 13C NMR:

(CDC13) 172.70(C-1, q); 84.83(C-3, q), 148.17(C-4, q), 146.18(C-6, q), 127.96(C-7, t), 134.82(C-8, t); 127.96(C-9, t); 126.95(C-10, t); 120.60(C-11, q); 161.01(C-12, q); 53.38(C-14, t); 38.91(C-15, s); 20.88(C-16, p), 80.11(C-17, q), 86.29(C-18, t); 59.20(C-20, t); 171.28(C-21, q); 136.78(C-23, q); 115.09(C-24, t); 129.74(C-25, t), 125.16(C-26, t); 124.69(C-27, t); 138.61(C-28, q); 17.85(C-29 p); and 50.80ppm (COMe, p). Mass Spectrum: FAB: 476(MH +, 11%), 442(8), 228(18), 217(38), 199(34), 154(100), and 136m/e

(90). Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

127

Common/Systematic Name Fumiquinazoline F Molecular Formula/Molecular Weight C21HlaN402, MW = 358.14298

N

General Characteristics Fumiquinazoline F was obtained as a pale yellow powder, mp., 88-90~ (c= 1.36, in CHCI3).

[a]D -411.2 ~

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene ehioride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by I-IPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells.

128

8.

Fumiquinazolines

Spectral Data UV;

~,Mm ~H ~x 207(1og e = 4.71), 219(4.73), 270(4.13), 277(4.13), 289(3.99), 306(3.78), and 320nm (3.66). IR;

3264(NH), 1679(CON), and 1610cmq (At-C-C). IH NMR: (CDCI3) 6.60(1H, br s, NH-2); 3.14(1H, q, J=6.8, 0.3Hz, H-3); 7.60(1H, dd, J=7.8, 1.0Hz, H-7); 7.77(1H, td, ,/=7.8, 1.8Hz, H-8); 7.53(1H, td, ,/=7.8, 1.0Hz, H-9); 8.37(1H, dd, J=7.8, 1.8Hz, H-10); 5.68(1H, dd, J=5.2, 3.6Hz, H-14); 3.64(1H, dd, 3'=15.0, 5.2Hz, H-15A); 3.71(1H, dd, 3'=15.0, 3.6Hz, H-15B); 1.37(1H, d, J=6.8Hz, H16); 6.71(1H, d, J=2.5Hz, H-18); 8.26(1H, br s, NH-19); 7.30(1H, dd, 3'=8.0, 0.8Hz, H-21); 7.13(1H, td, J=8.0, 0.8Hz, H-22); 6.92(1H, td, 3'=8.0, 0.8Hz, H-23); and 7.40ppm (1H, dd, J=8.0, 0.8Hz, H-25). X3CNMR: (CDCI3) 169.33(C-1, q); 49.18(C-3, t); 151.68(C-4, q); 147.08(C-6, q); 127.30(C-7, t); 134.70(C-8, t); 127.12(C-9, t); 126.85(C-10, t); 120.24(C-11, q); 160.82(C-12, q); 57.53(C-14, t); 27.04(C-15, s); 19.08(C-16, p); 109.39(C-17, q); 123.55(C-18, t); 135.98(C-20, q); 111.22(C-21, t); 122.57(C-22, t); 120.01(C-23, t); 118.48(C-24, t); and 127.30ppm (C-25, q). Mass Spectrum: EIMS: 358(M +, 3%), 229(4), 228(1), and 130m/e (100); HRElMS: C21HlsN402, requires 358.1430.

358.1436re~e;

Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata, Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

129

Common/Systematic Name Fumiquinazoline G Molecular Formula/Molecular Weight C21HIsN402; MW = 358.14298

N

General Characteristics Fumiquinazoline G was obtained as a pale yellow powder; mp., 119-121~ - 462.8 ~ (C=0.61, in CHCI3).

[a]D =

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was ehromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells. w

Ganomastenols

g

Ganomastenol A Ganomastenol B Ganomastenol C Ganomastenol D

131

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9.

Ganomastenols

133

Common/Systematic Name Ganomastenol A

rel-3a,8,9a-Trihydroxycadina-4,10(15)-diene

Molecular Formula/Molecular Weight C15H2403; M~cV = 2 5 2 . 1 7 2 5 4 15

H

,

|,

~

R

-

13"i~14

i

General Characteristics Isolated as a white powder. Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. After 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2 M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHCI3 and the CHCI3 fraction was dried and evaporated to dryness. The combined CHC13 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane (1:1, v/v; 500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v, 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v, 940ml); and fraction 6 with ethyl acetate-n-hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane, (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source

Ganoderma mastoporum.

Spectral Data IR:

(KBr) 3425ernq (OH).

134

9.

Ganomastenols

1HN:VIR: (CDC13) 1.89(1H, ddd, J=12.0, 11.0, 2.5Hz, H-I), 1.48(1H, ddd, J=12.0, 12.0, 10.0Hz, H-2~); 2.24(1H, ddd, J=12.0, 6.0, 2.5Hz, H-2a); 4.16(1H, dd, J=10.0, 6.0Hz, H-313); 5.61(1H, dd, J=l.0, 1.0Hz, H-5); 1.62(1H, ddd, J=l 1.0, 11.0, 1.0Hz, H-6); 1.24(1H, ddd, J=l 1.0, 11.0, 1.0Hz, H-713); 3.12(1H, dd, J=l 1.0, 10.0Hz, H-8a); 3.80(1H, ddd, J=10.0, 1.5, 1.5Hz, H-913); 1.76(1H, br s, H-11); 2.27(1H, qqd, J=7.0, 7.0, 1.0Hz, H-12); 1.00(3H, d, J=7.0Hz); 1.09(3H, d, J=7.0Hz, H-14); 4.76 91H, dd, J=3.0, 1.SHz, H-15a); and 5.09ppm (1I-I, dd, J=3.0, 1.SHz, H-15b). 13CNMR: (CDCI3) 43.1, C-l; 37.2, C-2; 71.6, C-3; 139.4, C-4; 127.3, C-5; 44.9, C-6, 52.7, C7; 79.0, C-8; 79.1, C-9; 152.2, C-10; 20.1, C-11; 28.0, C-12; 19.9, C-13; 22.1, C-14 and 103.2ppm, C- 15. Mass Spectrum: FAB-MS: 275m/e (M + Na)+; HREIMS: 234.1625m/e (M - H20) +, C15H2403,requires 252.1725; EIMS: 234(97%), 191(49), and 173m/e (100). Reference M. Hirotani, C. Ino, A. Hatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

9.

Ganomastenols

135

Common/Systematic Name Ganomastenol B rel- 313,8[3,9a-Trihydroxycadina-4,10( 15)-diene Molecular Formula/Molecular Weight C15H2403; MW" = 252.17254

HO

.,

~,,-

: v FI _

H

0

~DH

General Characteristics Isolated as needles; mp., 139-140~ Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. After 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHC13 and the CHC13 fraction was dried and evaporated to dryness. The combined CHCI3 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane (1:1, v/v; 500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v; 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v; 940ml); and fraction 6 with ethyl acetate-n-hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source Ganoderma mastoporum. Spectral Data IR:

(KBr) 3320(OH) and 2880cm ~.

136

9.

Ganomastenols

1H NMR:

(CDC13) 2.05(1H, ddd, J-11.0, 11.0, 2.0Hz, H-I); 1.63(1H, ddd, J-13.0, 11.0, 4.0Hz, H-213);2.00(1H, ddd, ./=13.0, 2.0, 2.0Hz, H-2a); 3.99(1H, dd, ./- 4.0, 2.0Hz, H-3a); 5.72(1H, dd, ./--1.0, 1.0I-Iz, H-3[~); 1.48(1H, ddd, ./=11.0, 11.0, 1.0I-lz, H-6); 1.3 I(1H, ddd, ./--11.0, 11.0, 1.0Hz, H-713); 3.14(1H, dd, J--11.0, 10.0Hz, H-8a); 3.85(1H, ddd,./-- 10.0, 1.5, 1.5Hz, H-9[~); 1.81(1H, br s, H-11); 2.31(1H, qqd, J=7.0, 7.0, 1.0Hz, H-12); 1.02(3H, d, `/-7.0Hz, H-13); 1.09(3H, d, J-7.0Hz, H- 14); 4.73 (1H, rid,`/=3.0, 1.5Hz, H-15a); and 5.08ppm (1H, rid,`/-3.0, 1.5Hz, H-15b). 13CNMR: (CDC13) 37.7, C-l; 36.2, C-2; 68.7, C-3; 137.1, C-4; 128.6, C-5; 44.9, C-6; 55.2, C7; 79.2, C-8; 79.2, C-9; 152.8, C-10; 21.8, C-11; 27.7, C-12; 19.9, C-13; 22.0, C-14 and 103.1ppm C-15. Mass Spectrum: FAB-MS: 275m/e (M + Na)+; HREIMS: 234.1614m/e (M - H20) +, C15H2403requires 252.1725; EIMS: 234(10%), 216(25), 173(70), and 145m/e (100). Reference M. Hirotani, C. Ino, A. Hatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

9.

Ganomastenols

137

Common/Systematic Name Ganomastenol C

rel-313,813,9tt-Trihydroxycadina-10(15)-ene

Molecular Formul .a/Molecular Weight C15H2603; M W -- 2 5 4 . 1 8 8 1 9

HO

H v - v R :

"OH

General Characteristics Isolated as needles; mp., 155.5-157~ Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. After 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2 M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na/SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHC13 and the CHC13 fraction was dried and evaporated to dryness. The combined CHC13 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane (1:1, v/v; 500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v; 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v; 940ml); and fraction 6 with ethyl acetate-n-hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source

Ganoderma mastoporum.

Spectral Data IR:

(KBr) 3360(OH) and 2890cm1

138

9.

Ganomastenols

1HNIV[R:

(CDCI3) 2.03(1H, ddd, J=13.0, 11.0, 2.0Hz, H-l); 1.54(1H, ddd, J=13.0, 11.0, 2.5Hz, H-213); 1.95(1H, ddd, J=13.0, 4.0, 2.0Hz, H-2a); 3.85(1H, dd, J= 4.0, 2.5Hz, n-3a); 1.47(1H, m, H-4); 1.13(1H, ddd, J=13.0, 12.0, 12Hz, H-5); 1.67(1H, ddd, J=13.0, 3.5, 3.0Hz, H-5); 0.93(1H, dddd, J=13.0, 12.0, 11.0, 3.0Hz, H-6); 1.26(1H, ddd, J=12.0, 11.0, 1.5Hz, H-713); 3.07(1H, dd, J-11.0, 9.0Hz, H-Sa); 3.79(1H, ddd, J= 9.0, 1.5, 1.5Hz, H-913); 0.96(1H, d, Jr--7.0Hz, H-11); 2.24(1H, qqd, J-7.0, 7.0, 1.0Hz, H-12); 0.98(3H, d, J=7.0Hz); 1.04(3H, d, J=7.0Hz, H-14); 4.70(1H, dd, J=2.5, 1.5Hz, H-15a); and 5.06ppm (1H, dd, J=2.5, 1.5Hz, H-15b). 13CNMR: (CDC13) 38.7, C-l; 37.8, C-2; 71.1, C-3; 37.6, C-4; 35.4, C-5; 45.2, C-6; 53.8, C-7; 78.9, C-8; 79.2, C-9; 153.3, C-10; 19.3, C-11; 27.7, C-12; 20.3, C-13; 21.6, C-14 and 103.0ppm C- 15. Mass Spectrum: FAB-MS: 277m/e (M + Na)+; HREIMS: 236.1778m/e (M- H20)§ C~sH2603requires 254.1881; EIMS: 236(38%), 218(40), 175(100), and 91m/e (28). Reference M. Hirotani, C. Ino, A. qatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

9.

Ganomastenols

139

Common/Systematic Name Ganomastenol D

rel-8[~,9a-Dihydroxy-4-hydroxymethylcadina-4,10(15)-diene

Molecular Formula/Molecular Weight C15H2403; M W = 2 5 2 . 1 7 2 5 4

.,

H

HOH2C" "~" ~ ~..~ ~)H

General Characteristics Isolated as a white powder; mp., 155.5-157~ Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. A_fter 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2 M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHCI3 and the CHC13 fraction was dried and evaporated to dryness. The combined CHC13 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane, 1: lv/v (500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v; 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v; 940ml); and fraction 6 with ethyl acetate-n- hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source

Ganoderma mastoporum.

.Spectral Data IR:

(KBr) 3360(OH) and 2890cm ~.

140

9.

Ganomastenols

1H NMR: (CDC13) 2.03(1H, ddd, J=13.0, 11.0, 2.0Hz, H-I); 1.54(1H, ddd, J=13.0, 11.0, 2.5Hz, H-213); 1.95(1H, ddd, J=13.0, 4.0, 2.0Hz, H-2a); 3.85(1H, dd, J = 4.0, 2.5Hz, H-3a); 1.47(1H, m, H-4); 1.13(1H, ddd, J=13.0, 12.0, 12Hz, H-5); 1.67(1H, ddd, J=13.0, 3.5, 3.0Hz, H-5); 0.93(1H, dddd, J=13.0, 12.0, 11.0, 3.0Hz, H-6); 1.26(1H, ddd, J=12.0, 11.0, 1.5Hz, H-713); 3.07(1H, dd, J=l 1.0, 9.0Hz, H-8a); 3.79(1H, ddd, J=9.0, 1.5, 1.5Hz, H-913); 0.96(1H, d, J=7.0Hz, H-11); 2.24(1H, qqd, J=7.0, 7.0, 1.0Hz, H-12); 0.98(3H, d, J=7.0Hz); 1.04(3H, d, J=7.0Hz, H-14); 4.70(1H, dd, J=2.5, 1.5Hz, H-15a); and 5.06ppm (1H, dd, J=2.5, 1.5Hz, H-15b). 13CNMR: (CDC13) 38.7, C-l; 37.8, C-2; 71.1, C-3; 37.6, C-4; 35.4, C-5; 45.2, C-6; 53.8, C- 7; 78.9, C-8; 79.2, C-9; 153.3, C-10; 19.3, C-11; 27.7, C-12; 20.3, C-13; 21.6, C-14 and 103.0ppm, C- 15. Mass Spectrum: FAB-MS: 277m/e (M + Na)§ HREIMS: 236.1778m/e (M- H20) § C15H2403requires 252.1725; ELMS: 236(38%), 218(40), 175(100), and 91m/e (28). Reference M. Hirotani, C. Ino, A. Hatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

Memnobotrins Memnobotrin A Memnobotrin B Memnoconone Memnoconol

141

This Page Intentionally Left Blank

10.

Memnobotrins

143

Common/Systematic Name Memnobotrin A Molecular Formula/Molecular Weight C25H33NOs; MW = 427.23587 O NH

~~

""II I I 12

3

AcO

\n 14

13

General Characteristics Crystalline solid; m.p., 240-250~ (dec.);

[ a ] D 20 --

+ 9.6 ~ (C=I. 14, in MeOH).

Fungal Source Memnoniella echinata (JS6308). Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Spectral Data UV:

~.~"

217.0 (e= 26,200), 257.5 (5,600), and 300nm (2,300).

IR:

(CHC13) 3596, 3455, 3316,2931,2854, 1716, 1692, 1612, 1458, 1368, 1136, and 1071cm 1

144

10.

Memnobotrins

1H NNtIR:

[(CD3)3CO)] 0.71(3H, s, H-15); 0.84(3FL s, n-13); 0.89(3H, s, n-14); 1.13(1H, m, H-5), 1.19(3H, s, H-12); 1.20(1H, m, H-1B); 1.55(1H, m, H-6B); 157 (1n, m, H-9), 1.61(2H, m, H-2); 1.70(2H, m, H-6A, H-7B); 1.89(1H, dt, J=3.4, 13.2Hz, H-1A), 1.98(3H, s, Ac), 2.16(1H, br, d, J= 9.60Hz, H-7A), 2.71(1H, ABX, J=8.2, 18.8Hz, H-11B); 2.87(1H, AB, J=18.SHz, H-1A); 4.21(2H, br AB, d=16.9Hz, H-8'); 4.46(1H, br dd, J=6.3, 9.3Hz, H-3); and 6.80ppm (1H, s, H-3'). 13C NMR:

[(CD3)3CO] 38.4, C-1, 24.1, C-2; 80.9, C-3; 38.3, C-4; 54.8, C-5; 18.5, C-6, 40.8, C-7, 76.3, C-8, 49.0, C-9; 38.3, C-10; 18.9, C-11, 27.2, C-12; 17.2, C-13, 28.7, C-14; 14.6, C-15; 114.7, C-I', 156.3, C-2', 100.6, C-3', 132.3, C-4'; 123.6, C-5'; 151.3, C-6'; 171.8, C-7'; 43.0 C-8'; 21.0, Ac-CH3; and 170.8ppm Ac-CO. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis; Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata; J. Antibiotics, Vol. 52, pp. 988-997 (1999).

10.

Memnobotrins

145

Comm0n/Systematic Name Memnobotrin B Molecular Formula/Molecular Weight C27H37NO6; M W -- 4 7 1 . 2 6 2 0 9

O NCH2CH2OH 15

Aco

\ 14

13

General Characteristics Cream colored powder; m.p., 186-192~

[0~]D20"- + 1 1 . 7

~ (c=1.25, in MeOH).

Fungal Source Memnoniella echinata (JS6308).

Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Biological Activity Cytotoxic. Spectral Data UV:

MeOH max

218.5 (c = 37,200), 259.0 (10,600), and 302nm (3,300).

146

10.

Memnobotrins

IR:

(CHC13) 3598, 3323, 2933,2855, 1717, 1669, 1616, 1458, 1368, 1317, 1135, and 1071cm1. ~HNMR: [(CD3)3CO] 0.69(3H, s, H-15); 0.82(3H, s, H-13); 0.87(3H, s, H-14); 1.13(1H, br d, J=9.8Hz, H-5); 1.17(3H, s, H-12); 1.20(1H, m, H-1B); 1.57(1H, m, H-9); 1.61(2H, m, H-2); 1.63(2H, m, H-6); 1.70(1H, m, 7B); 1.90(1H, dt, J=3.4, 13.3Hz, H-1A); 1.96(3H, s, Ac); 2.16(1H, br, dd, J=2.0, 10.4Hz, H-TA); 2.71(1H, ABX, J=8.1, 18.9Hz, H-11B); 2.87(1H, AB, J=18.9Hz, H-1A); 3.65(2H, t, J=5.4Hz, H-9'); 3.77(2H, t, J=5.4Hz, H-10'); 4.36(2H, br AB, J=l 7.0Hz, H-8'); 4.46(1H, br dd, J=6.7, 9.8Hz, H-3); and 6.81ppm (1H, s, H-3'). 13CNMR: [(CD3)3CO] 38.4, C-l; 24.1, C-2; 80.9, C-3; 38.3, C-4; 54.7, C-5; 18.5, C-6; 40.8, C-7; 76.4, C-8; 49.0, C-9; 38.7, C-10; 18.8, C-11; 27.2, C-12; 17.1, C-13; 28.6, C-14; 14.6, C-15; 114.4, C-I'; 156.2, C-2'; 100.6, C-3'; 132.5, C-4'; 121.6, C-5'; 151.0, C-6'; 169.6, C-7', 49.2 C-8'; 46.0, C-9', 61.2, C-10'; 21.0, Ac-CH3; and 170.8ppm At-CO. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis; Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata.; J. Antibiotics, Vol. 52, pp. 988-997 (1999).

10.

Memnobotrins

147

Common/Systematic Name Memnoconone Molecular Formula/Molecular Weight C23H3005; M W -- 386.20932

Ii~ "I

13'

0

7

, I

-

HO.o,i,~~ x,~O

14'

I

15'

O

oH

General Characteristics Light oil. Fungal Source Memnoniella echinata (JS6308).

Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Biological Activity Cytotoxic. Spectral Data UV: , ~ MeOH max

219.0(c = 38,000), 261.0(14,600), and 289nm (2,900).

IR:

(CHCI3) 3587, 3435, 3934, 2972, 1761, 1639, 1612, 1462, 1353, 1384, 1355, 1316, and 1124cm"1.

148

10.

Memnobotrins

1H NMR:

[(CD3)3CO] 1.1 l(6H, s, H-12', H-13'); 1.26(1H, dddd, J=4.4, 10.0, 10.1, and 13.6Hz, H-9'A); 1.55(3H, s, H-15'); 1.58(1H, dddd, J=l.6, 6.8, 9.9, and 13.6Hz, H-9'B); 1.76(3H, s, H-14'); 1.95(2H, br t, J=7.SHz, H-4'); 2.02(2H, m, H-5'); 2.10(2H, br t, J=7.5Hz, H-8'A); 2.49 (2H, m, H-9'); 2.60(1H, hept, J=7.0Hz, H-11'); 3.36 (2H, br d, J= 7.1Hz, H-I'); 5.07(1H, dt, J=l.1, 6.SHz, H-6'); 5.22(2H, s, H-3); 5.25(1H, dt, J=l. 1, 6.8Hz, H-2'); and 6.62ppm (1H, s, H-7). 13CNMR:

[(CD3)3CO] 173.0, C-l; 70.7, C-3; 103.7, 3a; 163.8, C-4; 114.2, C-5; 155.7, C-6; 101.5, C-7; 147.5, C-Ta; 22.1, C-I'; 123.0, C-2'; 135.4, C-3'; 40.3, C-4'; 27.1, C-5'; 125.0, C-6'; 134.9, C-7'; 34.1, C-8'; 39.5, C-9'; 213.7, C-10'; 41.0, C-11'; 18.4, C-12'; 18.4, C-13'; 16.2, C-14'; and 16.1ppm, C-15'. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis; Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata.; J. Antibiotics, Vol. 52, pp. 988-997 (1999).

10.

Memnobotrins

149

Common/Systematic Name Memnoconol Molecular Formula/Molecular Weight C23H3206; M W = 404.21989

O

OH OHI_~H 13'

-

I

14'

,

HO~6.,~ "1/ . "]~ \0

I

lo.

1~'

General Characteristics Light brown powder; m.p., 60-63~

[tZ]D2~= -14.0 ~ (C=1.08, in MeOH).

Fungal Source Memnoniella echinata (JS6308). Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Biological Activity Cytotoxic. Spectral Data UV:

)tmM~" 219.0(e = 28,700), 261.5(11,100), and 290nm (3,000). IR: (CHCI3) 3587, 3440, 3005,2978,2932, 1728, 1637, 1612, 1451, 1353, 1316, 1196, and 1161 cm1.

150

10.

Memnobotrins

1H~ : [(CD3)3CO] 1.1 l(6H, s, H-12', H-13'); 1.26(1H, dddd, J=4.4, 10.0, 10.1, and 13.6Hz, H-9'B); 1.55(3H, s, H-15'); 1.58(1H, dddd, J--1.6, 6.8, 9.9, and 13.6Hz, H-9'A); 1.76(3H, s, H-14'); 1.87(1H, m, H-8'B); 1.91(2H, br t, J=7.4Hz, H-4'); 2.02(2H, m, H-5'); 2.15(1H, br ddd, ,/=4.4, 9.9, and 13.7Hz, H-8'A); 3.18(1H, dd, J=l.6, 10.1Hz, H-10'); 3.30(2H, d, J=6.9Hz, H-I'); 5.07(1H, br t, J=6.SHz, H-6'); 5.15(2H, s, H-3); 5.20(1H, br t, J=6.9Hz, H-2'); and 6.52ppm (1H, s, H-7). 13C NMR: [(CD3)3CO] 172.9, C-l, 70.6, C-3; 103.6, 3a; 163.8, C-4; 115.6, C-5; 155.7, C-6; 101.5, C-7; 147.4, C-7a; 22.1, C-I'; 123.0, C-2'; 135.4, C-3'; 40.3, C-4'; 27.0, C-5'; 124.6, C-6'; 135.7, C-7'; 37.4, C-8'; 30.6, C-9'; 78.4, C-10'; 72.9, C-11'; 25.7, C-12'; 24.9, C-13'; 16.2, C-14'; and 16.1ppm, C-15'. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis, Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata.; J. Antibiotics, Vol. 52, pp. 988-997(1999).

Tsugicolines Tsugicoline A Tsugicoline B Tsugicoline C Tsugicoline D

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11. Tsugicolines

153

Common/Systematic Name Tsugicoline A Molecular Formula/Molecular Weight C 15H2204; M W -- 266.15181

H,.

OH

H

H O H 2 C ~

15

O '''~ %58

",14

H OH Fungal Source

Laurilia tsugicola = Echinodontium tsugicola (CBS 248.51, Centraal Bureau voor

Schimmel Cultures, Baarn). General Characteristics White crystals from methylene chloride-hexane; mp., 168-170~ [a]o - 156 ~ (c=0.15, in MeOH) ; triacetate, an oil; [a]D - 43.2 ~ (C=0.1, in CHC13); acetonide, white crystals from chloroform; mp., 146-148~ Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Then the extracts were dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether; the residue was composed of pure tsugicoline A; the mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B and D; Re, 0.10, 0.30 for tsugicoline A, respectively. Spectral Data

UV; ;~

260nm (c=8,500).

IR; (KBr) 3370(OH) and 1730crn1 (earbonyl).

154

11. Tsugicolines

1H NMR: (acetone-d6) 4.46(1H, J~,lb=16Hz, J~a=l.4Hz, H-la); 4.42(1H, Jlb,a=l.5Hz, H-lb); 4.23(1H, Ja,la=8.7Hz, H-a); 4.36(1H, n-6); 1.00(3H, n-8); 2.54(1H, Jg,lO~=10.4Hz, Ja,lo~=7.9Hz, J9,~3=11.5Hz, H-9); 1.5 I(1H, Jwa,~ol3=12.4Hz,Jw~,ls=0.8Hz, H-10a); 1.55(1H, Jlo13,1213=l.8Hz,H-10I]); 1.30(1H, JiE~A2fl=12.2Hz,J1Ea,]a=10.4Hz, J12~,ls=0.8Hz, H-12a); 1.86(1H, J121~,13=7.3Hz,H-1213); 2.32(1H, H-13); 1.13(3H, H14); 1.01(3H, n-15); 3.82(1H, OH-I); 4.57(1H, OH-3); and 5.16ppm (1H, OH-6); (CDC13) 4.57(1H, H-la); 4.57(1H, n-lb); 4.24(1H, n-3); 4.39(1H, H-6); 1.02(3H, n-8); 2.51(1H, n-9); 1.48(1H, U-10a); 1.62(1H, n-1013); 1.23(1H, H-lEa); 1.88(1H, H-1213); 2.33(1H, n-13); 1.15(3H, U-14); 1.00(3H, H-15); 1.55"(1H, OH-l); 2.15"(1H, OH-3); and 2.95ppm* (1H, OH-6). * Assignments may be interchanged. 13CNMR: 61.45, t, C-l; 153.02, s, C-2; 74.93, d, C-3; 143.98, s, C-4; 200.15, s, C-5; 90.33, d, C-6; 42.70, s, C-7; 14.66, q, C-8; 47.02, d, C-9; 42.50, t, C-10; 40.89, s, C-11; 47.51, t, C-12; 50.90, d, C-13; 29.79, q, C-14; and 27.23ppm, q, C-15. Mass Data: LREIMS: 266role (M+); CIMS: (isobutane) 267(MH+), 249(MH+ - H20, base peak), 23 I(MH+ - 2H20), and 203role (231 - 28), found: C, 67.5, H, 8.3. C15H2204requires C, 67.64; H, 8.33%. Reference A. Amone, U. Brambilla, G. Nasini, and O. Vajna de Pava; Isolation and Structure Elucidation of Tsugicolines A-D, Novel ProtoiUudane Sesquiterpenes from Laurilia tsugicola; Tetrahedron; Vol. 51, pp. 13357-13364(1995).

11. Tsugicolines

155

Common/Systematic Name Tsugicoline B Molecular Formula/Molecular Weight C15H2103; MW = 250.15689 OH

HOH

H �9. ,,,%

O ~

\H

Fungal Source Laurilia tsugicola = Echinodontium tsugicola (CBS 248.51; Centraal Bureau voor

Schimmel Cultures, Baam). General Characteristics Obtained as an oil; [a]D - 71.5 ~(C=4.5, in CHC13). Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Then the extractswere dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether; the residue was composed of pure tsugicoline A; the mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B and D; Rf, 0.50; 0.30 for tsugicoline B, respectively. Spectral Data UV:

Xm~x 254nm (e=5,100). IR:

(chloroform) 1730cm"1 (conjugated carbonyl). 1H NMR:

(CDCI3) 4.54(1H, H-la); 4.50(1H, Jlb,3-1.9Hz, H-lb); 4.24(1H, J3,~3-9.1Hz, H-3); 2.82(1H, H-6); 1.17(3H, H-8); 2.48(1H, J9,~o~-10.2Hz, Jo,lop-8.3I-Iz, J9,13=11.9Hz, H9); 1.46(1H, J~o~,~op=12.6I-~J~o~,lS=0.8H~ H-10a); 1.56(1H, J~op,~2p=l.8Hz, H-1013); 1.22(1H, J12a,12p-12.3Hz, J12~,13=11.3Hz, Jl2~,lS=0.8Hz, H-12a); 1.85(1H, J12p,~3=7.2Hz, H-12~); 2.44(1H, H-13); 1.14(3H, H-14); 0.99(3H, H-15); 3.00"(3H, OH-l); and 3.80ppm* (3H, OH-3). * Assignments may be interchanged.

156

11. Tsugicolines

13C NM~: 61.17, t, C-l; 149.99, s, C-2; 74.51, d, C-3; 148.67, s, C-4; 197.77, s, C-5; 60.31, t, C6; 36.35, s, C-7; 20.41, q, C-8; 46.40, d, C-9; 41.27, t, C-10; 40.81, s, C-11; 46.56, t, C-12; 52.45, d, C-13; 29.46, q, C-14; and 26.82ppm, q, C-15. Mass Data: ELMS: (isobutane) 251(MI-F, 100%), 233(MIT - H20, 95), 215(38), 205(30), 203(22), 187(28), and 173role (25); found: C, 71.6, H, 8.7; C~5H2203 requires C, 71.97; H, 8.86%. Reference A. Arnone, U. Brambilla, G. Nasini, and O. Vajna de Pava; Isolation and Structure Elucidation of T sugicolines A-D, Novel Protoilludane Sesquiterpenes from Laurilia tsugicola; Tetrahedron, Vol. 51, pp. 13357-13364(1995).

11. Tsugicolines

157

Common/Systematic Name Tsugicoline C Molecular Formula/Molecular Weight C15H2404; M W "-" 2 6 8 . 1 6 7 4 6

OH HOH2

H

HO"~,~. - 171 H I L : H OH

Fungal Source Laurilia tsugicola = Echinodontium tsugicola (CBS 248.51 Centraal Bureau voor

Schimmel Cultures, Baarn). General Characteristics Obtained as white crystals from methylene ehloride-hexane; mp., 74-760C; [a]D - 39.6* (c=3.00, in CHCI3). Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Then the extracts were dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether. The residue was composed of pure tsugicoline A. The mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B, C, and D; Re, 0.50; 0.30 for tsugicoline C. Spectral Data UV: ~.mx 208nm (e=5,600). IR:

(chloroform) 3400cm1 (OH). 1H N]~IR: (CDCla) 4.28(1H, Jla,lb=13.4Hz, Jl~a=l.0Hz, H-la); 4.26(1H, Jlb,a=l.2Hz, H-lb); 4.15(1H, d3,13=8.5Hz, H-a); 3.70(1H, n-6); 1.04(3H, n-8); 2.30(1H, Jg,lo~=10.2Hz, Jg,lO~=8.2Hz, Jg,la=12.0Hz, H-9); 1.35(1H, Jloa,lO~=12.6Hz, Jloa,15=0.8Hz, H-10a);

158

11. Tsugicolines

1.39(1H, Jlop,~2p=l.6Hz, H-10~); 1.17(1H, d~2~,~2p=12.6Hz, J12=,13=10.2Hz, J~2~,~5=0.8Hz,n-12a); 1.78(1I-I,J~2p,~a=7.2Hz,H-1213); 2.25(1H, H-13); 1.08(3H, H- 14); 0.97(3H, H- 15); 3.97"(1H, OH- 1); 4.50"(1H, 3-OH); and 3.95ppm* (1H, 6OH). * Assignments may be interchanged. 13CNMR: 59.437, t, C-l; 139.59, s, C-2; 74.16, d, C-3; 141.03, s, C-4; 70.90, d, C-5; 76.92, d, C-6; 51.03, s, C-7; 15.481, q, (~-8; 46.59, d, C-9; 41.82, t, C-10; 40.50, s, C-11; 47.70, t, C-12; 51.58, d, C-13; 29.87, q, C-14; and 27.35ppm, q, C-15. Mass Data: CIMS: (isobutane) 269(MH+, 100%) and 233m/e (MIT - 2H20, base peak). Reference A. Amone, U. Brambilla, G. Nasini, and O. Vajna de Pava, Isolation and Structure Elucidation of Tsugicolines A-D, Novel Protoilludane Sesquiterpenes from Laurilia tsugicola; Tetrahedron, Vol. 51, pp. 13357-13364(1995).

11. Tsugicolines

159

Common/Systematic Name Tsugieoline D Molecular Formul.dMolecular Weight C15H2003; M W = 248.14124 H

H ~

'".,,

H OH Fungal Source

l_xmrilia tsugicola = Echinodontium tsugicola (CBS 248.51; Centraal Bureau voor

Schimmel Cultures, Baarn). General Characteristics Obtained as a white solid; mp., 107-110~

[0~]D +

161.8~(c=0.1, in CHCh).

Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Thenthe extracts were dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether; the residue was composed of pure tsugicoline A; the mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B and D; Re, 0.70, 0.30 for tsugicoline D, respectively. Soe~ral Data UV" ~,~ 215(e=6,0,50) and 240nm (e=4,800). IR:

(KBr) 3400(OH), 1780, and 1680em1 (earbonyl). 1H ~ . (CDCI3) 9.47(1H, H-la), 6.77(1H, Ja,13=2.0Hz, H-3); 4.71(1H, H-6); 1.13(3H, H-8); 2.64(1H, J9,1oa=12.7Hz, J9,1Ol~=6.9Hz,J9,1a=7.4Hz, H-9); 1.07(1H, J1oa,1o13=12.6Hz, Jloa,15=~ 0Hz, n-10a), 1.59(1H, J1o1~,12~=~ 0Hz, n-1013); 1.72(1H, J12,~12~=la.5Hz, J12a,la=2.3Hz, J12~,lS=-q3Hz,n-12a); 2.10(1H, J121~,13=9.2Hz,n-12~); 3.16(1H, n-13); 1.08(3H, H-14); 1.01(3H, H-15); and 5.14ppm (1H, OH-6).

160

11. Tsugicolines

13CNMR: 193.97, t, C-l; 134.54, s, C-2; 154.21, d, C-3; 54.55, d, C-4; 203.02, s, C-5; 85.83, d, C-6; 38.38, s, C-7; 18.82, q, C-8; 43.02, d, C-9; 37.61, t, C-10; 44.40, s, C-11; 37.61, t, C-12; 47.26, d, C-13; 32.00, q, C-14; and 31.61pprn, q, C-15. Mass Data: CIMS: (isobutane) 249(MH+, 80%), 231(MH+ - H20, 100), 220(20), and 203m/e (25); found: C, 72.2, H, 8.0; C15H2003requires C, 72.55; H, 8.12%. Reference A. Arnone, U. Brambilla, G. Nasini, and O. Vajna de Pava; Isolation and Structure Elucidation of Tsugicolines A-D, Novel Protoilludane Sesquiterpenes from Laurilia tsugicola; Tetrahedron, Vol. 51, pp. 13357-13364(1995).

Radicinins Radicinin Radicinin Diastereomer (2S*, 3R*) Radicinol Diasteromer (2S*,3S*,4S*)

2S,3 S, 4S-epi-Radicino l

Radicinol

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12.

Radicinins

163

Common/Systematic Name Radicinin; Stemphylone Molecular Formula/Molecular Weight C12H1205; MW = 236.06847

SO,~~~dOH O

11

0

8

O

12

General Characteristics Needles from methanol; mp., 238-240~ (dec,),[t~]D2~-236 ~ (c--0.1, in CHCI3). Isolation/Purification The fungus was grown without shaking at 24~ for 21 days in the dark in liquid medium made up of glucose (3.0g/L), peptone (3.0g/L) and the extract from 50g~ of malt and water. The culture filtrate was acidified to pH 2.0 with HC1, after which the metabolites in the culture filtrate were extracted with ethyl acetate (3x). The ethyl acetate extracts were dried over Na2SO4 and concentrated. The residue was applied to a silica gel column and the column washed with 1500ml of 10% acetone in n-hexane, then developed successively with 750ml each 20, 30, and 40% acetone in n-hexane. Each 150ml eluate constituted one fraction. Fractions 10 and 11 were combined and evaporated. Recrystallization of the residue from methanol afforded radicinin as needles. The mother liquor was purified by Sephadex LH20 column chromatography. The column was washed with 500ml of methanol and fractions consisting of 7ml eluates were collected. Fractions 33-48 were combined and evaporated to dryness. Recrystallization of the residue from methanol afforded radicinin as needles. The diastereoisomer of radicinin (2S*,3R*) (Rf=0.38) in the mother liquor was separated from the mixture of the diastereoisomer of radicinol (2S*,3S*,4S*) and epiradicinol (2S*,3S*,4R*) bypreparative TLC [acetone-benzene (2:8, v/v) Rf=0.26]. The crude radicinin diastereoisomer was further purified with preparative TLC (ethyl acetaten-hexane; 7:3, v/v) which gave the radicinin diastereoisomer as needles [ethyl acetate-nhexane (7:3, v/v) Rf=0.63]. The diastereoisomer of radieinol (2S*,3S*,4S*) and epiradicinol (2S*,3S*,4R*) and radicinol epoxide were purified after preparative TLC (ethyl acetate-n-hexane; 7:3, v/v) of the mixture. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jobi L), Alternaria radicina, A. chrysanthemL A. helianthL Cochliobolus lunata, and Sternphylium radicinum.

Biological Activity In a leaf spot assay of Job's tears, radicinin caused necrotic lesions at 0.31zg/leaf but not active at 0. ll,tg/leaf.

164

12.

Radicinins

Spectral Data UV:

X~ff2 203(1oge = 4.00), 221(4.14), 270(3.66), 280(3.61), and 341nm (4.15). IR:

(KBr) 3462, 3098, 3034, 2990, 2924, 2918, 1760, 1659, 1605, 1522, 1456, 1435, 1379, 1325, 1228, 1172, and 1056cm~. ~HN M R : (CDCIa) 4.36(1H, dq, J=12.4, 6.3Hz, H-2); 3.98(1H, d, J=12.4Hz, H=3); 5.84(1H, s, H-8); 6.03(1H, dq, J=15.5, 1.8Hz, H-9); 6.95(1H, dq, ,/=15.5, 7.0Hz, H-10); 1.95(3H, dq, J=7.0, 1.8Hz, H-11); and 1.64ppm (3H, d, J=6.3Hz, H-12). 13CNMR: (CDCI3) C-2, 80.0; C-3, 72.0; C-4, 188.6; C4a, 97.9; C-5, 156.7; C-7, 164.3; C-8, 98.0; C-Sa, 176.3; C-9, 122.6; C-10, 141.0; C-11, 18.8; and C-12, 18.1ppm. Mass Spectrum: CIMS (isobutane): 236m/e (M)'; 236.0682m/e exact mass calcd for C~2H1205, 236.0684. Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe; Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears; Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

165

Common/Systematic Name Radicinin Diastereomer (2S*, 3R*) Molecular Formula/Molecular Weight C12H1205, ~

-- 236.06847

0

O ~

0

,,,OH

General Characteristics Colorless needles from methanol; mp., 202-220~ EtOH).

(dee,); [et]D2~ = -105 ~ (C=0.25, in

Isolation/Purification See radicinin for isolation/purification. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jobi L).

Biological Activity In a leaf spot assay of Job's tears, radicinin diastereomer caused necrotic lesions at 1.0~g/leaf but was not active at 0.3~g/leaf. Spectral Data UV: Z Emt~ 203(1og e = 4.00), 220(4.14), 269(3.67), 280(3.64), and 338nm (4.14). IR: (KBr) 3420, 2928, 2858, 1742, 1655, 1605, 1531, 1458, 1438, 1381 1263, 1170, 1118, and 1044cm1. 1H

NMR:

(CDCI3) 5.07(1H, dq, J-- 6.0Hz, 6.5Hz, H-2), 4.52(1H, d, J=6.0Hz, H-3); 5.83(1H, s, H-8), 6.03(1H, dq, J=15.3Hz, 1.3Hz, H-9), 6.96(1H, dq, J=15.3Hz, 7.0Hz, H-10), 1.96(3H, dd, J=7.0Hz, 1.3Hz, H-11), and 1.35ppm (3H, d, J=6.5Hz, H-12). 13C N M R :

(CDCI3) C-2, 78.9; C-3, 70.1; C-4, 188.0; C-4a, 97.6; C-5, 156.7; C-7, 164.5; C-8, 98.4; C-8a, 174.9; C-9, 122.6; C-10, 141.0; C-11, 18.8; and C-12, 12.1ppm. Mass Spectrum: CIMS (isobutane): 236role(M); 236.0682role exact mass calcd for C~2H1205, 236.0684

166

12.

Radicinins

Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe; Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears; Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

167

Common/Systematic Name Radicinol diasteromer (2S*,3S*,4S*) Molecular Formula/Molecular Weight C12H1405; M~W -- 238.08412

0

O~

OH

,,OH

General Characteristics An oil; [a]D2~-19 ~ (C----0.85,in EtOH). Isolation/Purification See radicinin for isolation/purification. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jobi L).

Biological Activity In a leaf spot assay of Job's tears, radicinol diasteromer caused no necrotic lesions up to 10~g/leaf. Spectral Data UV:

%Em ~~

224 (log e = 4.45), 260 (3.51), 271 (3.56), and 315nm (3.95).

IR:

(KBr) 3408, 2988, 2942, 1690, 1618, 15742, 1437, 1383, 1315, 1278, 1214, 1160, 1141, 1071, and 1025cm1. 1H NMR:

(CDC13) 4.37(1H, dq, J=l.1, 6.3Hz, H-2); 3.88(1H, br, H-3); 4.60(1H, br, H-4OH); 5.79(1H, s, H-8); 5.97(1H, dq, J=15.7, 1.5Hz, H-9); 6.72(1H, dq, J=15.7, 6.8I-Iz, H10); 1.91(3H, dd, J=6.8, 1.5Hz, H-11); and 1.50ppm (3H, d, J=6.3Hz, H-12). 13C NMR:

(CDC13) C-2, 72.4; C-3, 69,1; C-4, 63.2; C4a, 99.5; C-5, 165.4; C-7, 158.5; C-8, 99.3; C-8a, 166.6; C-9, 122.6; C-10, 135.2; C-11, 18.4; and C-12, 15.8ppm. Mass Spectrum: EIMS: 238(M+, 18%), 181(100), 152(7), 137(8), and lllm/e (26); 238.0845m/e exact mass calcd for C12H1405,238.0841.

168

12.

Radicinins

Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe; Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears; Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

169

Common/Systematic Name

2S,3S, 4S-epi-Radicinol

Molecular Formula/Molecular Weight C12H1406, M W = 254.07904

0

O ~ A

O--

Me"/>

/

"~

L

OH

"0"

,,OH "'Me

General Characteristics An oil; [0~]D20 -92 ~ (c= 1.0, in EtOH). Isolation/Purification See radicinin for isolation/purification. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jo bi L).

Biological Activity In a leaf spot assay of Job's tears, epi-radicinol caused no necrotic lesions up to 10~tg/leaf. Spectral Data UV: Era'~ 215 (log e = 4.34) and 288nm (4.31). IR: (Thin film) 3376, 3000, 2930, 1698, 1653, 1580, 1444, 1383, 1212, 1139, 1071 and 1033cm1. 1H N M R :

(CDC13) 4.37(1H, brq, J=6.7Hz, H-2), 3.84(1H, br, H-3); 4.56(1H, d, J=2.4Hz, H-4); 6.05(1H, s, H-8), 3.33(1H, d, J=l.9Hz, H-9), 3.21(1H, dq, J=l.9Hz, 5.4Hz, H-10), 1.42(3H, d, J=5.40Hz, H-11); and 1.49ppm (3H, d, J=6.7Hz, H-12). 13C N M R :

(CDC13) C-2, 72.6; C-3, 69,1; C-4, 63.3; C-4a, 100.4; C-5, 164.8; C-7, 160.4; C-8, 99.7; C-8a, 165.9; C-9, 55.1; C-10, 57.3; C-11, 17.3; and C-12, 15.7ppm. Mass Spectrum: ELMS: 254(M+, 28%), 197(100), 182(10), 153(79), and 139m/e (13);254.0795m/e exact mass calcd for C~2H1406,254.0790.

170

12.

Radicinins

Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe, Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears, Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

171

Common/Systematic Name Radicinol Molecular Formula/Molecular Weight C12H1405; MW" = 2 3 8 . 0 8 4 1 2 III ii1,.

HO

OH

0

General Characteristics Colorless viscous oil; [a]D 31 -175 ~ (c--1.02, in CHCI3). Isolation/Purification A mixture of radicinol and radicinin was obtained from the ethyl acetate extract of the fungal culture aider column chromatography using 60% ethyl acetate in n-hexane. The mixture was purified using Sephadex LH20 with acetone as eluanl. Fungal Source Cochliobolus lunata (IFO 6288). Spectral Data UV~ ,~ EtOH max

226(~ =39,300), 262(3,100), 271 (3,200), and 318nm (10,800).

IR~

(CHC13) 3425(OH) and 1680cmq (a-pyrone). 1H N M R :

(CDC13) 6.72(1H, dq, J=7.0, 16.0Hz, H-10); 3.76(1H, dd, ./=6.5, 8.0Hz, H-3); 4.75(2H, bs, 2 OH's); 5.85(1H, s, H-8); 6.02(1H, dq, ./=16.0, 2.0Hz, H-9); 1.92(3H, dd, J=7.0, 2.0Hz, H-11); 4.70(1H, d, J=6.5Hz, H-4); 4.23(1H, dq, ./=8.0, 6.6Hz, H-2); and 1.52ppm (3H, d, J=6.5Hz, H-12). 13C NMR: (CDCla) C-2, 76.8; C-3, 72.5; C-4, 68.0; C-4a, 100.6; C-5, 165.4; C-7, 158.9; C-8, 99.1; C-8a, 164.5; C-9, 122.7; C-10, 135.7; C-11, 18.4; and C-12, 17.0ppm. Mass Spectrum: HREIMS: 238.082re~e; exact mass calcd for C12H1405, 238.084.

172

12.

Radicinins

Reference M. Nukina and S. Marumo, Radicinol, A New Metabolite of Cochliobolus lunata, and Absolute Stereochemistry of Radicinin, Tetrahedron Letters, pp. 3271-3271 (1977).

Stachybotrylactone and Related Metabolites Stachybotrylactone K-76 Stachybotrylactone Acetate 2a-Hydroxystaehybotrylactone 2~t-Acetoxystachybotrylactone Acetate Stachybotrylactam Stachybotrylactam Acetate 2~t-Acetoxystachybotrylactone Acetate Stachybotramide Stachybotrydial Stachybotrin A Stachybotrin B

173

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13. Stachybotrylactone and Related Metabolites

175

Common/Systematic Name Stachybotrylactone Molecular Formula/Molecular Weight C23H3005; M W -- 3 8 6 . 2 0 9 3 2

O HO I

"

r

11

,,,.J 12

HO' .....

General Characteristics White or pale yellow crystalline solid; mp., 280~ (dec.); [0~]D -6.4 ~ (c=0.09, in acetone). Isolation/Purification Rice cultures of Stachybotrys chartarum (Egypt 1) were air dried, ground, and extracted with methanol followed by extraction with chloroform. The extracts were combined and evaporated to dryness. The crude extract was dissolved in methanol and filtered through a pad of ClS silica gel followed by elution with methanol to give a black gum alter removal of solvent. The gum was dissolved in methylene chloride and chromatographed on a PEI silica gel column eluted with methylene chloride-hexane (4:1, v/v), methylene chloride, and 10% methanol in methylene chloride. A combination of preparative HPLC and TLC gave purified satratoxin H, S-isosatratoxin H, stachybotrylactone, stachybotrylactone acetate, 2a-acetoxystachybotrylactone acetate and stachybotrylactam. Fungal Source

Stachybotrys chartarum and S. cylindrospora. Note: Ayer and Miao (1993) believed stachybotrylactone to be an artifact formed from stachybotrydial.

Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV: ~ Aoetonitrile max

218(c=28,000), 268(5,3000), and 309nm (2,600).

176

13. Stachybotrylactone and Related Metabolites

IR~

(CHsC1) 3600-3100, 2939, 1742, 1623, 1469, 1389, 1356, 1332, 954, 943,772, and 737cmq. 1H NMR:

(CDCls) 1.90(1H, H-la); 1.06 (1H, H-113); 1.60 (1H, H-2~); 1.44(1H, H-213); 3.32(1H, br s, H-313); 2.17(1H, dd, J=l 1.6, 2.7Hz, H-5~); 1.46(1H, H-6a); 1.55(1H, H-613); 1.60 (1H, H-7a); 1.45(1H, H-713); 1.86(1H, H-813); 3.26(1H, d, J=17.3Hz, HI la, proton on the side of C-10); 2.90(1H, d, J=17.3Hz, H-1 lb, proton on the side of C-8); 0.72(3H, d, J=6.5Hz, H-12a); 0.97(3H, s, H-141~); 0.86(3H, s, H-1413); 1.06(3H, s, H-1513); 6.76(1H, s, H-3'); 5.23(1H, d, J=14.7Hz, H-8'a); and 5.17ppm (1H, d, J=14.7Hz, H-8"o). ~SCNMR: (acetone-d6) 24.91, t, C-I; 21.60, t, C-2; 75.21, d, C-3; 38.29, s, C-4; 40.65, d, C-5; 25.91, t, C-6; 31.81, t, C-7; 37.78, d, C-8; 100.00, s, C-9; 43.09, s, C-10; 32.53, t, C11; 15.86, q, C-12; 28.94, q, C-13; 22.80, q, C-14; 16.36, q, C-15; 120.72, s, C-I'; 156.65, s, C-2'; 103.00, d, C-3'; 119.2, s, C-4'; 128.07, s, C-5'; 155.47, s, C-6'; 171.29, s, C-7'; and 67.84ppm, t, C-8'. Mass Spectrum: HREIMS: 386.2095(C23H3005,53%), 368.1977(C23H2sO4, 11), 353.1753(C22H2504, 9), 244.0730(C14H1204, 9), 230.0579(ClsH1004, 100), 217.049(C~2H904, 18), 207.1747(C14H230, 38), and 189.1642m/e (C~,I-I2~,35). References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Canad. J. Chem., Vol. 71, pp. 487-493(1993). B. B. Jarvis, J. Salemme, and A. Morais; Stachybotrys Toxins 1. Natural toxins; Vol. 3, pp. 10-16 (1995).

13.

Stachybotrylactone and Related Metabolites

177

Common/Systematic Name K-76 Molecular Formula/Molecular Weight C23H3oO6; MW = 402.20424

HO 1 . ~ - , , , . / . C HO

H ~

r'lU,

....

' .... '

.... , 1 2

General Characteristics Crystals; mp., 176 o; [tt]D20 _ 48 ~ (in MeOH). Fungal Source Stachybotrys complementi (ATCC 20511). Biological Activity Strongly inhibited the complement C5 step at ca. 10pg/ml in vitro and preliminary experiments indicated that it improved the symptoms of experimental glomerulonephritis. Spectral Data UV:

~

EtOH max

246(e=16,500), 307(6,700) and 359nm (5,400).

IR:

(KBr) 1600, 1585, 1385, and 880cm~. 1H NNIR:

(C6HsN) 0.82(3H, d, J=5.0Hz, H-12); 0.93, 1.04, and 1.27(3H each, s); 3.72(1H, d, J=2.0Hz, H-3); 4.30(1H, J=20.4Hz, H-2); 7.41(1H, s, H-3'); and 10.75, 11.02ppm (1H each, s, CHO). X-Ray: Structure confirmed by X-ray analysis of the p-bromobenzenesulfonate. References E. J. Corey and J. Das; Total Synthesis of the Complement Inhibitor K-76 in Racemic Form. Structural Assignment to "1(-76 monocarboxylic Acid"; J. Am. Chem. Sot., Vol. 104, pp. 5551-5553(1982).

178

13.

Stachybotrylactone and Related Metabolites

H. Kaise, M. Shinohara, W. Smiyazaki, T. Izawa, Y. Nakano, M. Sugawara, K. Sugiura, and K. Sasaki; Structure of K-76, A Complement Inhibitor Produced by Stachybotrys complementi nov. sp. K-76; J. C. S. Chem. Comm,pp. 726-727(1979).

13.

Stachybotrylactone and Related Metabolites

179

Common/Systematic Name Stachybotrylactone acetate Molecular Formula/Molecular Weight C25H3206; M W -- 428.21989

O

�9 /__
Aoo

.......

14~

7 ; ........' 12 13

General Characteristics Crystals; mp., 105-106~

[~]D -12 ~ (c=0.5, in MeOH).

Isolation/Purification Rice cultures of Stachybotrys chartarum (Egypt 1) were air dried, ground, and extracted with methanol followed by extraction with chloroform. The extracts were combined and evaporated to dryness. The crude extract was dissolved in methanol and filtered through a pad of C~s silica gel followed by elution with methanol to give a black gum after removal of solvent. The gum was dissolved in methylene chloride and chromatographed on a PEI silica gel column eluted with methylene chloride-hexane (4:1, v/v), methylene chloride, and 10% methanol in methylene chloride. A combination of preparative HPLC and TLC gave purified satratoxin H, S-isosatratoxin H, stachybotrylactone, stachybotrylactone acetate, 2~-acetoxystachybotrylactone acetate and stachybotrylactam. F ungal Source

Stachybotrys chartarum.

Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV:

maxA~~ 218, 265, and 306nm. IR:

(CHaC1) 3280(OH), 1754(C=0), and 1724crn1 (C = 0).

180

13. Stachybotrylactone and Related Metabolites

1H NMR: (CDCI3) 0.74(3H, d, J=6.4Hz, H-12); 0.90(3H, s, H-13); 0.91(3H, s, H-14); 0.98(3H, s, H-15); 1.4-1.9(9H, m, H-I, 2, 6, 7, 8); 2.02(3H, s, acetate); 2.05(1H, m, H-5); 2.90 and 3.30(1H each, AB, J=17.4, H-11); 4.60(1H, bs, H-3); 5.13 and 5.27(1H each, AB, J= 14.5, H-8'); and 6.90ppm (1H, s, H-3'). 13C NMR: 25.0, C-I; 20.7, C-2; 77.8, C-3; 37.0, C-4; 41.0, C-5; 22.3, C-6; 30.9, C-7; 36.8, C-8; 99.6, C-9; 42.2, C-10; 31.6, C-11; 15.5, C-12, 27.8, C-13; 21.3, C-14; 16.0, C-15; 118.9, C-I'; 153.9, C-2'; 103.3, C-3'; 120.0, C-4'; 127.5, C-5'; 155.7, C-6'; 170.4, C-7'; 67.6, C-8'; 21.9, and 170.5ppm (acetates). Mass Spectrum: CIMS (CH4): 429(7%), 369(12), 299(12), 281(19), and 69m/e (100); HREIMS: 428.2195m/e for C25H3206;calcd, 428.2199. Reference B. B. Jarvis, J. Salemme and A. Morals; Stachybotrys Toxins. 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

13.

Stachybotrylactone and Related Metabolites

181

Common/Systematic Name 2tt-Hydroxystachybotrylactone Molecular Formula/Molecular Weight C23H3006; ~

= 402.20424

O 2'

0

Te

--

HO.......(3 ;kl/~,,,,,() 14@" : ~ \

8'8'

/'//

13

General Characteristics Amorphous solidi [tt]D - 40 ~ (C=I.0, in MeOH). Fungal Source Stachybotrys chartarum. Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV:

Ao~om~em,x 218, 265, and 306nm. IR:

(CH3C1) 3296(OH) and 1750cm~ (C = O). ~H NMR: (CDC13) 0.67(3H, d, J=6.2Hz, H-10); 0.83(3H, s, H-14); 0.97(3H, s, H-13); 1.01(3H, s, H-15); 1.3-1.8(7H, m, H-I, 6, 7, 8); 2.05(1H, m, H-5); 2.85 and 3.2(1H each, AB, J=17.3Hz, H-11); 3.40(1H, bs, H-3); 4.02(1H, bd, J=l 1Hz, H-2); 5.10 and 5.24(1H each, AB, J=14.9Hz, H-8'); and 6.78ppm (1H, s, H-3'). 13C NMR: 30.9, C-l; 66.7, C-2; 78.7, C-3; 39.2, C-4; 38.3, C-5; 20.6, C-6; 30.9, C-7; 36.8, C-8; 98.9, C-9; 43.4, C-10; 33.0, C-11; 15.4, C-12; 28.4, C-13; 21.9, C-14; 16.9, C-15; 119.0, C-I'; 153.9, C-2'; 103.3, C-3'; 120.1, C-4'; 127.1, C-5'; 155.4, C-6'; 172.0, C-7'; and 68.1 pprn, C-8'.

182

13. Stachybotrylactone and Related Metabolites

Mass Spectrum: CIMS (El-h): 403(15%), 299(8), 257(2), 173(100), and 145m/e (43); HREIMS: 402.2047m/e for C23H3oO6;calcd, 402.2042. Reference B. B. Jarvis, J. Salemme and A. Morals; Stachybotrys Toxins. 1 Natural Toxins, Vol. 3, pp. 10-16(1995).

13. Stachybotrylactone and Related Metabolites

183

Common/Systematic Name 2tt-Acetoxystachybotrylactone acetate Molecular Formula/Molecular Weight C27H3408; M W = 4 8 6 . 2 2 5 3 7

O

AcQ \.

z

,s % / ~ . , ) dl T~

0

8'8'

A o O .......

14 ~

7//~........I 12 13

General Characteristics Amorphous solid; [tt]D -15 ~ (c=l.0, in MeOH). Fungal Source Stachybotrys chartarum. Isolation~urification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2C12-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH2C12) were combined to give A; fractions 85-87 (3% MeOH/CH2C12) were combined to give B; fractions 88-89 (3% MeOH/CH2C12) and 90-99 (4% MeOH/CH2C12) were combined to give C; fractions 100-103 (5% MeOH/CH2C12) and fractions 104-110 (6% MeOH/CH2C12) were combined to give D; fractions 111-112 (6% MeOH-CH2C12) were combined to give E; fractions 113-115 (10% MeOH-CH2C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction A was subjected to high speed countercurrent distribution [Vo = 850ml, MeOH (3)-1-120 (2)-CC14 (3.5)-CH2C12 (0.5)hexane (1), organic mobile phase at 3.2ml/min] to give 10 fractions (F1-F10). Fraction F5 was subjected to preparative TLC (Chromatotron, 2mm plate, 20% hexane in EtOAc) to give pure 2a-acetoxystachybotrylactone acetate. Biological Activity Cytotoxic and immunosuppressive.

184

13.

Stachybotrylactone and Related Metabolites

Spectral Data UV:

~,Am~~

218, 265, and 306nm.

IR:

(CH3C1) 3280(OH)and 1744cm1 (C = O). 1H NMR:

(CDC13) 0.73(3 H, d, J=6.4, H-12); 0.93(3H, s, H-14); 1.00(3H, s, H-13); 1.10( 3H, s, H-15); 1.3-1.8(7H, m, H-I, 6, 7, 8); 2.05(1H, m, H-5); 1.90 and 2.05(3H each, s, acetates); 2.95 and 3.35(1H each, AB, J=17.4Hz, H-11); 3.50(1H, bs, H-3); 4.95(1H, bd, J=10Hz, H-2); 5.18 and 5.37(1H each, AB, J=14.5Hz, H-8'); and 6.92ppm (1H, s, H-3'). 13CNMR: (CDC13) 30.7, C-l; 68.3, C-2; 77.0, C-3; 38.2, C-4; 40.6, C-5; 20.4, C-6; 30.6, C-7; 36.8, C-8; 99.0, C-9; 43.6, C-10; 32.0, C-11; 15.5, C-12; 27.7, C-13; 21.6, C-14; 16.9, C-15; 118.8, C-I'; 153.9, C-2', 103.6, C-3'; 119.9, C-4'; 127.6, C-5'; 155.4, C-6'; 171.6, C-7'; 67.8, C-8'; 21.1, 20.9, 170.4, and 170.5 ppm, acetates. Mass Spectrum: ELMS: 486(M+, 18%), 384(100), 351(3), 279(7), and 230m/e (21); HREIMS: 486.2250m/e for CzvH34Os;calcd, 486.2254. Reference B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1" Natural Toxins, Vol. 3, pp. 10-16(1995).

13.

Stachybotrylactone and Related Metabolites

185

Common/Systematic Name Stachybotrylactam Molecular Formula/Molecular Weight C23H31NO4; ~ "- 385.22531 O

HOrN H .o

....

~

14,"

-

~\

_, /2 " '

12

13

General Characteristics Amorphous solid. Fungal Source Stachybotrys chartarum. Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2CI2-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH:C12) were combined to give A; fractions 85-87 (3% MeOH-CH2C12) were combined to give B; fractions 88-89 (3% MeOH-CH:C1:) and 90-99 (4% MeOH/CH2C1:) were combined to give C; fractions 100-103 (5% MeOH-CH2C12) and fractions 104-110 (6% MeOH/CH2C12) were combined to give D; fractions 111-112 (6% MeOH/CHzC12) were combined to give E; fractions 113-115 (10% MeOH-CH:C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction D was subjected to reversedphase MPLC (Rainin, 18g, 81~, C-18, 4ml/min) with increasing MeOH, starting with 70% MeOH in H20, to give several fractions one of which upon reversed-phase HPLC (semipreparative Cls, 70% MeOH in 1-120) gave pure stachybotrylactam. Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV~

XA~~

216, 257, and 298nm.

186

13.

Stachybotrylactone and Related Metabolites

(CH3CI) 3450(NH), 3290(OH), and 1690cm1 (C=O). 1H NMR: (CDCls) 0.73(3H, d, J=6.0Hz, H-12), 0.87(3H, s, H-14), 0.98(3H, s, H-13), 1.03(3H, s, H-15), 1.5-1.9(9H, m, H-l, -2, -6, -7, -8), 2.20(1H, m, H-5), 2.83 and 3.18(1H each, AB, J=17.2Hz, H-11); 3.36(1H, bs, H-3), 4.25 and 4.35(1H each, AB, J=16.5Hz, H-8'), 6.80(1H, s, H-3'), and 8.0ppm (1H, bs, NH). 13CNMR: (CDC13) 24.9, C-l; 21.7, C-2; 75.5, C-3; 38.3, C-4; 40.6, C-5; 25.9, C-6; 31.9, C-7; 37.7, C-8; 99.0, C-9; 43.0, C-10; 32.6, C-11; 16.0, C-12; 28.6, C-13; 22.8, C-14; 16.4, C-15; 118.0, C-I'; 153.9, C-2'; 102.3, C-3'; 116.0, C-4'; 135.0, C-5'; 154.7, C-6'; 172.1, C-7'; and 43.3ppm, C-8'.

Mass Spectrum: EIMS: 386(8%), 385(Nf, 23), 279(10), 230(18), 179(15), and 149m/e (100), HREIMS: 385.2215m/e for C23H31NO4,calcd, 385.2253. Reference B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

13. Stachybotrylactone and Related Metabolites

187

Common/Systematic Name Stachybotrylactam acetate Molecular Formula/Molecular Weight C25H33NOs, ~ = 427.23587 O

HOz~/NH AcO

.......~

5 ~

''''0

14 13 General Characteristics Amorphous solid. Fungal Source

Stachybotrys chartarum.

Isolation/Purification A shake culture of Stachybotrys complementi (ATCC 20511) yielded a crude extract from which were isolated stachybotrylactam, stachybotrylactam acetate, and 2t~-hydroxystachybotrylactam acetate by a series of chromatographic steps similar to those described for stachybotrylactam. Biological Activity Cytotoxie and immunosuppressive. Spectral Data UV~

Aeetomtrilemax216, 257, and 298nm.

IR~ (CH3C1) 3450(NH), 3248(OH), 1730(C=O), and 1696cm~ (lactam). 1H NMR: (CDC13) 0.70(3H, d, J=6.0Hz, H-12); 0.89(3H, s, H-14); 0.97(3H, s, H-13); 1.23(3H, s, H-15); 1.4-1.9(9H, m, H-I, -2, -6, -7, -8); 1.99(3H, s, acetate); 2.00(1H, m, H-5); 2.84 and 3.19(1H each, AB, d=17.0Hz, H-11); 4.22 and 4.38(1H each AB, J=l 5.0Hz, H-8'); 4.58(1H, s, H-6); 6.78(1H, bs, NH); and 6.90ppm (1H, s, H-3').

188

13.

Stachybotrylactone and Related Metabolites

13C NMR: (CDC13) 24.9, C-I; 20.8, C-2; 77.9, C-3; 36.8, C-4; 40.9, C-5; 22.3, C-6; 30.8, C-7; 37.0, C-8; 98.6, C-9; 42.0, C-10; 31.8, C-11; 15.5, C-12; 27.8, C-13; 21.2, C-14; 16.0, C-15; 117.8, C-I'; 153.3, C-2'; 102.3, C-3'; 115.3, C-4'; 135.0, C-5'; 156.4, C-6'; 172.4, C-7'; 43.0, C-8'; 21.7, acetate; and 170.4ppm, acetate. Mass Spectrum: HREIMS: 427.2350m/e for C25Ha3NOs; calcd 427.2359. Reference B. B. Jarvis, J. Salemme and A. Morais, Stachybotrys Toxins. 1" Natural Toxins, Vol. 3, pp. 10-16(1995).

13. Stachybotrylactone and Related Metabolites

189

Common/Systematic Name 2a-Acetoxystachybotrylactam acetate Molecular Formula/Molecular Weight C27H35NO7;M ~ = 485.24135 O AcO -~-

~5 ~ N H ~

AcO....... (3[, F ' ~oP. 1,~'""06' - ~1 ,

14 ~

~-8'

_

7 / ........, 12 13

General Characteristics Amorphous solidi [0~]D -29 ~ (c=0.1, in MeOH). Fungal Source

Stachybotrys chartarum (MRC 1422).

Isolation/Purification A rice culture of S. chartarum yielded 3.2g of crude extract. Verrucarin J, roridin E, and satratoxins F, G, and H were isolated from this extract by a series of chromatographic procedures (see isolation of stachylbotrylactone acetate). Also isolated was 13mg of stachybotrylactone and 25mg of 2t~-acetoxystachybotrylactam acetate. Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV; ~,Am~r~~ 216, 257, and 298nm. IR~ (CHaC1) 3450(NH), 3280(OH), 1744(C=O, acetate), and 1696cm"1 (C=O, lactam). 1H NMR: (CDC13) 0.71(3H, d, J-6.4Hz, H-12); 0.93(3H, s, H-14); 1.02(3H, s, H-13); 1.10(3H s, H-15); 1.4-1.8(7H, m, H-I, -6, -7, -8); 2.05(1H, m H-5); 1.90 and 2.07(3H each, s, acetates); 2.90 and 3.24(1H each, AB, J=17.0Hz, H-11); 4.25 and 4.42(1H each AB, J=16.5Hz, H-8'); 4.95(1H, bs, H-3); 5.21(1H, bd, J=l 1Hz, H-2); 7.01(1H, s, H-3'); and 8.0ppm (1H, bs, NH).

190

13.

Stachybotrylactone and Related Metabolites

13C NMR: (CDCI3) 30.5, C-l; 68.2, C-2; 76.7, C-3; 38.1, C-4; 40.5, C-5; 20.4, C-6; 30.7, C-7; 36.7, C-8; 98.1, C-9; 43.5, C-10; 32.0, C-11; 15.4, C-12; 27.7, C-13; 20.9, C-14; 16.9, C-15; 117.6, C-I'; 153.4, C-2'; 102.5, C-3'; 115.3, C-4'; 133.0, C-5'; 156.1, C-6'; 172.0, C-7'; 43.5, C-8'; 21.0, 21.6; and 170.3, 170.6ppm, acetates.

Mass Spectrum: EIMS: 487(7%), 486(20), 485(60), 383(62), 279(14), and 229m/e (24); HREIMS: 485.2404m/e for C27H35NO7;calcd, 485.2414). Reference B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1" Natural Toxins, Vol. 3, pp. 10-16(1995).

13.

Stachybotrylactone and Related Metabolites

191

Common/Systematic Name Stachybotramide Molecular Formula/Molecular Weight C25H35NOs; M W = 429.25152 0

is ~NCH2CH2OH HO....~ , , , O

il

14- ~ 1 4 \

,/

[~

8'

....... , 1 2

13

General Characteristics Crystals; mp., 196-199~

[0~]D -16 ~ (c= 0.1, in MeOH).

Fungal Source

Stachybotrys chartarum and S. cylindrospora.

Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2C12-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH2C12) were combined to give A; fractions 85-87 (3% MeOH-CH2C12) were combined to give B; fractions 88-89 (3% MeOH-CH2C12) and 90-99 (4% MeOH-CH2C12) were combined to give C; fractions 100-103 (5% MeOH-CH2C12) and fractions 104-110 (6% MeOHCH2C12) were combined to give D; fractions 111-112 (6% MeOH-CH2C12) were combined to give E; fractions 113-115 (10% MeOH-CH2C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction F was filtered through a short PEI silica gel column which was eluted with increasing concentrations of MeOH in CH2C12 to give two fractions. The first fraction was crystallized from EtOAc-hexane to give stachybotramide. Acetylation of staehybotramide gave the triacetate derivative, which was identical to the compound reported by Ayer and Miao [ 1993]. Biological Activity Cytotoxic and immunosuppressive.

192

13.

Stachybotrylactone and Related Metabolites

Spectral Data UV~

~,A~on~o 216, 257, and 300nm. max IR;

(CH3C1) 3270(OH), and 1695cm1 (C=O, lactam). 1H M R :

(pyridin-ds) 0.84(3H, d, J-5.5Hz, H-12); 0.91(3H, s, H-14); 0.98(3H, s, H-13); 1.22(3H s, H-15); 1.20-1.90(9H, m, H-I, -2, -6, -7, -8); 2.05(1H, m, H-5); 3.10 and 3.52(1H each, d, J=l 7.0Hz, H-11); 3.60(1H, br s, H-2); 3.65(2H, m, H-9'); 3.90 (2H, m, H-10'); 4.07 and 4.35 (1Heach, AB, J=16.8Hz, H-8'); and 7.35ppm (1H, s, H-3'). 13C NMR:

(pyridin-ds) 25.7, C-I; 24.5, C-2; 74.6, C-3; 38.0, C-4; 40.2, C-5; 21.2, C-6; 31.4, C7; 37.2, C-8; 98.5, C-9; 42.5, C-10; 32.6, C-11; 15.7, C-12; 28.9, C-13; 22.5, C-14; 16.0, C-15; 117.4, C-I'; 155.2, C-2'; 101.6, C-3'; 113.0, C-4'; 135.0, C-5'; 156.7, C-6'; 168.8, C-7'; 48.3, C-8'; 45.7, C-9'; and 60.3ppm, C-10'. Mass Spectrum: HREIMS: 4292534m/e for C27H35NO7,calcd, 429.2515. References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993). B. B. Jarvis, J. Salemme and A. Morals; Stachybotrys Toxins. 1 Natural Toxins, Vol. 3, pp. 10-16(1995).

13. Stachybotrylactone and Related Metabolites

193

Common/Systematic Name Stachybotrydial Molecular Formula/Molecular Weight Cz3H30Os, ~

= 386.20932

HO,..v/~y,,...GHO

General Characteristics Crystals; mp., 266~ (dec.). Fungal Source

Stachybotrys chartarum and S. cylindrospora.

Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2C12-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH2C12) were combined to give A; fractions 85-87 (3% MeOH-CH2C12) were combined to give B; fractions 88-89 (3% MeOH-CH2C12) and 90-99 (4% MeOH-CH2C12) were combined to give C; fractions 100-103 (5% MeOH-CH2CI2) and fractions 104-110 (6% MeOHCH2C12) were combined to give D; fractions 111-112 (6% MeOH-CH2C12) were combined to give E; fractions 113-115 (10% MeOH/CH2C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction B was subjected to CCC (Vc = 850ml, MeOH (2)-H20(2)-EtOAc(2)-hexane(3), organic mobile phase at 3.2ml/min) to give a major fraction which was subjected to preparative TLC (Chromatotron, 4mm plate, increasing EtOAc in hexane) to yield a semisolid material. This material was subjected to silica gel HPLC (semipreparative column, 2.5% MeOH in CH2C12 [40%]-hexane [60%], 4ml/min, 0.2g/injection to give stachybotrylactone and stachybotrydial). _ _ Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV:

~on~e

232, 254, 248, and 360nm.

194

13.

Stachybotrylactone and Related Metabolites

IR~

(CHaC1) 3450(OH)and 1690cm1 (C=O). 1H NMR:

(CDC13) 0.72(3H, d, J=6.4Hz, H-12), 0.85(3H, s, H-14), 0.98(3H, s, H-13), 0.98(3H, s, H-15); 1.5-2.0(9H, m, H-l, -2, -6, -7, -8); 2.05(1H, dd, J-13 and 3Hz); 2.79 and 3.15(1H each, AB, J=17.4Hz, H-11); 3.39(1H, bs, H-3), 6.87(1H, s, H-3'), 10.34(1H, s, H-7'); and 10.57ppm ( 1H, s, H-8'). 13C NMR: (CDC13) 24.6, C-I, 24.0, C-2, 75.7, C-3; 37.5, C-4; 40.0, C-5; 20.8, C-6; 30.3, C-7; 36.9, C-8; 100.6, C-9; 42.1, C-10; 30.9, C-11; 15.4, C-12; 28.2, C-13; 22.2, C-14; 16.0, C-15; 111.0, C-I'; 158.0, C-2'; 109.3, C-3'; 137.9, C-4'; 119.3, C-5'; 167.5, C-6'; 188.7, C-7'; and 193.4ppm, C-8'. Mass Spectrum: HREIMS: 386.4878m/efor C25H3005; calcd, 386.4882. References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993). B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

13.

Stachybotrylactone and Related Metabolites

195

Common/Systematic Name Stachybotrin A Molecular Formula/Molecular Weight C23H31NOs; M"W = 401.22022

25 OH 22.

i

23

C H20H 24

7

0,,,~

OH

12 13

H/N--~O General Characteristics Isolated as a yellow oil; [tt]D + 8.8 ~ (C=0.612, in MeOH). Fungal Source

Stachybotrys sp. (ATCC 90017) collected from brackish water in Florida.

Isolation/Purification The culture filtrate was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to afford a red oil. The extract was dissolved in acetone and divided into four equal portions. Each portion was subjected to preparative TLC on silica gel GF plates developed twice with 9:1, v/v, chloroform-ethanol. Major bands observed under illumination (254nm) were collected and extracted with methanol. Bands 2 (Rf 0.18) and 4 (Rf 0.36) were further purified with the same TLC conditions to afford stachybotrin A (a yellow oil) and stachybotrin B (as a white solid). Biological Activity Stachybotrins A and B each showed activity in disk assays against Bacillus subtilis (ATCC 6051) at 10~g/disk (8- and 10-mm zones, respectively). They also inhibited the radial growth of the filamentous fungi Ascobolusfurfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459) by 50% in centerpoint inoculation assays at 20 and 10ktg/disk, respectively. Disk assays for activity against a strain of Candida albicans (ATCC 14053) were negative at 1001.tg/disk. Stachybotrin A was also evaluated for cytotoxicity against three human solid tumor cell lines (nonsmall carcinoma A-549, breast adenocarcinoma MCF-7, and colon adenocarcinoma HT-29), but afforded only mild activity (EDs0 values 20-30~g/ml). Spectral Data UV:

~,mM~" 220(e=17,000), 254(C=6,400) and 302nm (c=2,900).

196

13. Stachybotrylactone and Related Metabolites

IR:

(neat) 3262,2918, 1684, 1611, 1462, 1358, 1049, 1025 and 1006cmq. 1H NMR:

(CDC13) 6.74(1H, s, H-4); 2.65(1H, dd, J=17.6, 7.2Hz, H-7); 2.97(1H, dd, J=17.6, 5.5Hz, H-7); 3.88(1H, dd, J=7.2, 5.SHz, H-8); 4.24(2H, br s, H-13); 1.69(2H, m, H14); 2.27 (2H, m, H-15); 5.30(1H, dd, J=7.4, 7.4Hz, H-16); 2.08(2H, m, H-18); 2.07(2H, m, H-19); 5.07(1H, dd, J-1.4, 1.4Hz, H-20); 1.63(3H, s, H-22); 1.55(3H, s, H-23); 4.08(1H, d, J=12.2Hz, H-24); 4.05(1H, d, J=12.2Hz, H-24); and 1.26ppm (3H, s, H-25). 13CNMR: (CDC13) 174.3, C-2; 132.5, C-3; 101.1, C-4; 158.4, C-5; 113.6, C-6; 27.7, C-7; 68.4, C-8; 80.1, C-9; 150.1, C-11; 123.8, C-12, 44.2, C-13; 38.9, C-14; 22.1, C-15; 128.8, C-16; 139.7, C-17; 36.0, C-18; 27.9, C19; 125.4, C-20; 132.2, C-21; 25.5, C-22; 17.7, C-23; 59.9, C-24; and 18.7ppm, C-25. Mass Spectrum: EIMS: 401(M+, 24%), 383(10), 332(7), 314(4), 248(12), 232(26), 216(18), 190(23), 178(57), 147(19), 121(21), 93(24), and 69m/e (base peak); HREIMS: obsd for C23H31NOs, 401.2189, calcd, 401.2202. HPLC Data Beckman C18 Ultrasphere column; 250 x 4.6 mm; 90:10, v/v, MeOH-water, 1.0ml/min;tR 2.95 min. Reference X. Xu, F. S. de Guzman, and J. B. Gloer; Stachybotrins A and B: Novel Bioactive Metabolites from a Brackish Water Isolate of the Fungus Stachybotrys sp.; J. Org. Chem., Vol. 57, pp. 6700-6703(1992).

13.

Stachybotrylactone and Related Metabolites

197

Common/Systematic Name Stachybotrin B Molecular Formula/Molecular Weight C23H31NO4; MW" = 385.22531 r5 OH

o H/N General Characteristics Isolated as a white solid; mp., 178-180~

o. O [~]D q- 39.1 ~ (c=0.11, in MeOH).

Fungal Source

Stachybotrys sp. (ATCC 90017) collected from brackish water in Florida.

Isolation/Purification The culture filtrate was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to afford a red oil. The extract was dissolved in acetone and divided into four equal portions. Each portion was subjected to preparative TLC on silica gel GF plates developed twice with 9:1, v/v, chloroform-ethanol. Major bands observed under illumination (254nm) were collected and extracted with methanol. Bands 2 (Re 0.18) and 4 (Rf 0.36) were further purified with the same TLC conditions to afford stachybotrin A (a yellow oil) and stachybotrin B (as a white solid). Biological Activity Stachybotrins A and B each showed activity in disk assays against Bacillus subtilis (ATCC 6051) at 10~g/disk (8- and 10-mm zones, respectively). They also inhibited the radial growth of the filamentous fungi Ascobolusfurfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459) by 50% in centerpoint inoculation assays at 20 and 101,tg/disk, respectively. Disk assays for activity against a strain of Candida albicans (ATCC 14053) were negative at 100~g/disk. Stachybotrin A was also evaluated for cytotoxicity against three human solid tumor cell lines (nonsmall carcinoma A-549, breast adenocarcinoma MCF-7, and colon adenocarcinoma HT-29), but afforded only mild activity (EDs0 values 20-30~g/ml).

198

13.

Stachybotrylactone and Related Metabolites

Spectral Data IR~

(neat) 3293, 2919, 1653, 1608, 1457, 1350, and 1080cm1. 1H NMR.:

(CDCls) 6.74(1H, s, H-4); 2.64(1H, dd, J=18.0, 6.0Hz, H-7); 2.98(1H, dd, J=18.0, 6.0Hz, H-7), 3.87(1H, dd, J=6.0, 6.0Hz, H-8), 4.23(1, d, J=18.0Hz, H-13); 4.20(1H, d, d=18.0Hz, H-13); 1.67(2H, m, H-14); 2.18(2H, m, H-15); 5.14(1H, m, H-16); 1.95(2H, m, H-18); 2.02(2H, m, H-19); 5.05(1H, m, H-20); 1.63(3H, s, H-22); 1.55(3H, s, H-23), 1.57(3H, s, H-24); and 1.27ppm (3H, s, H-25). 13C NMR: (CDCI3) 174.2, C-2; 132.5, C-3; 100.8, C-4; 158.0, C-5; 113.5, C-6; 27.8, C-7; 68.4, C-8; 80.2, C-9; 150.2, C-11; 124.1, C-12, 44.2, C-13; 38.5, C-14; 22.6, C-15; 125.5, C-16; 136.2, C-17; 40.8, C-18; 27.8, C19; 125.3, C-20; 132.2, C-21; 25.8, C-22; 17.7, C-23; 15.9, C-24; and 18.8ppm, C-25. Mass Spectrum: EIMS: 385(M+, 36%), 311(22), 232(13), 216(16), 190(23), 178(38), 177(30), 147(9), 123(13), 109(15), 95(16), 81(25), and 69m/e (base peak); HREIMS: obsd for C23H31NO4, 385.2270m/e; calcd, 385.2254. HPLC Data Beckman Cls Ultrasphere column; 250 x 4.6 mm; 90:10, v/v, MeOH-water; 1.0ml/min; tR 4.05 min. Reference X. Xu, F. S. de Guzman, and J. B. Gloer; Stachybotrins A and B: Novel Bioactive Metabolites from a Brackish Water Isolate of the Fungus Stachybotrys sp.; J. Org. Chem., Vol. 57, pp. 6700-6703(1992).

Trichothecenes and Related Metabolites Culmorin Trichothecene 813-Hydroxytrichothecene Trichodermin Isotrichodermin 7-Hydroxyisotrichodermin 8a-Hydroxyisotrichodermin 7,8-Dihydroxyisotrichodermin NT- 1 Toxin 8-Ketoisotrichodermin Calonectrin 8-Ketocalonectrin 15-Deacetylcalonectrin 3-Deacetylcalonectrin 7a -Hydroxycalonectrin 8a -Hydroxycalonectrin 7a,8a -Dihydroxycalonectrin 4-Acetoxyscirpenediol 15-Acetoxyscirpenetriol 4,15-Diacetoxyscirpenol Scirpenetriol 7a-Hydroxydiacetoxyscirpenol 7a,8a-Dihydroxydiacetoxyscirpenol 3,15-Diacetoxyscirpenetriol 3,4,15-Triacetoxyscirpenetriol T-2 Tetraol T-2 Toxin HT-2 Toxin Acetyl T-2 Toxin 4-Propanyl HT-2 Toxin 3'-Hydroxy T-2 Toxin 3'-Hydroxy HT-2 Toxin 3'-Hydroxy T-2 Triol Neosolaniol Isoneosolaniol (Acuminatum) 8-Acetoxyneosolaniol 8-Propanylneosolaniol 8-Butyrylneosolaniol 8-Isobutyrylneosolaniol 15-Deacetylneosolaniol (NT-2 Toxin) 4-Deacetylneosolaniol Sporotrichiol 413,8a,15-Triacetoxy-3a,7a-dihydroxy- 12,13-epoxytrichothec-9-ene

199

200

14. Trichothecenes and Related Metabolites

3-Acetyl-4-deoxynivalenol Trichodermol 7a-Hydroxytrichodermol Verrucarol 4,15-Deacetylverrucarol Crotocin (Antibiotic T) Crotocol Trichothecolone Trichothecin Trichothecinol A Trichothecinol B Trichothecinol C Trichodermone Nivalenol 4-Deoxynivalenol (Rd Toxin, Vomitoxin) 3-Acetyl-4-deoxynivalenol 413,15-Diacetoxynivalenol 413,15-Diacetyl-7-deoxynivalenol

15-Acetyl-4-deoxynivalenol

3,15-Diaeetyl-4-deoxynivalenol Fusarenon-X (Nivalenol Monoacetate)

14. Trichothecenes and Related Metabolites

201

Common/Systematic Name Culmorin Molecular Formula/Molecular Weight C15H2602; MW

-- 2 3 8 . 1 9 3 2 8 4

3

1

HO/

14

7

OH

General Characteristics Colorless needles from tetrahydrofuran-cyclohexane; mp., 178-179~ (c=0.60, in CHC13).

[ ~ ] D 24

-

15~

Fungal Source Fusarium roseum (ATCC 28114), F. culmorum, F. graminearum, and F. crookwellense.

Isolation/Purification Fusarium roseum cultures were harvested, and the broth was extracted as described by Greenhalgh et al. (1984). Some of the oil from the methanol phase after it had been

partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A alter removal of 3-acetyldeoxynivaienol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). Culmorin was isolated from mixture A3 by elution with 50% ethyl acetate in hexane followed by recrystallization from tetrahydrofuran-cyclohexane.

202

14. Trichothecenes and Related Metabolites

Spectral Data 1H ~ : (CDC13) 0.70(3H, H-13), 0.86(3H, H-12), 0.90(3H, H-14), 1.02(3H, H-15), 1.32.1(6H, H-3, H-4, H-5, m), 1.64(2H, J1o,l~=6.8Hz, H-10), 1.74(1H, J7,s=5.0Hz, H-7), 1.90(1H, Y1,11=4.5Hz, H-l), 3.86(1H, J2,s=8.0Hz, H-8), and 4.35ppm (1H, dlo,~1=6.8, Yll,l=4.5Hz, H-11). Mass Spectrum: HR IMS: 238.1922m/e (M+); calcd for C15H2602,238.1922. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. Apsimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984). D. R. Lauren, A. Ashley, B. A. BlaekweU, R. Greenhalgh, J. D. Miller, and G. A. Neish, Tilchothecenes Produced by Fusarium crookwellense DAOM 193611, J. Agile. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes

and Related Metabolites

203

Common/Systematic Name Trichothecene 12,13-Epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2202; M W = 234.16198 ~o H

~ 6 ~ 0 . ~

~

I-

"1

H

13

2 3 I .~

~,~"u

i~le ~ ~ 15

4

14

General Characteristics Crystals from aqueous methanol; mp., 54-57~ Fungal Source Trichothecium roseum.

TLC Data Preparative TLC using silica gel developed with benzene-ethyl acetate (10:2, v/v). References Y. Fujimoto, S. Yokuna, T. Nakamura, T. Morikawa, and T. Tatsuno; Total Synthesis of 12,13-Epoxytrichothec-9-ene; Tetrahedron Letters, pp. 2523-2526(1974). N. Masouka and T. Kawikawa; A Synthesis of 12,13-Epoxytrichothec-9-ene; Tetrahedron Letters, pp. 1691-1694(1976).

204

14. Trichothecenes and Related Metabolites

Common/Systematic Name 8[3-Hydroxytrichothecene 813-Hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2203; MW = 250.15689

~o H

H

[-8 ';l

H O f ~

~2 14

Fungal Source

Fusarium sporotrichioides (MC-72083).

Spectral Data 1H NMR:

(CDCI3) 3.65(1H, d, J=3.9Hz, H-2); 1.91(1H, m, H-3); 1.57(1H, dd, J=13.0, 7.0Hz, H-3); 2.78(1H, dd, J=13.0, 7.0Hz, H-4); 1.68(1H, dd, J=13.6, 7.0Hz, H-4); 2.03(1H, dd, J=12.8, 5.5Hz, H-7); 1.15(1H, dd, J=12.5, 9.3Hz, H-7); 4.12(1H, m, H-8); 5.27(1H, m, H-10); 4.66(1H, m, H-11); 3.37(1H, d, J=4.1Hz, H-13); 3.06(1H, d, J=4.1Hz, H-13); 0.96(3H, s, CH3-14); 0.96(3H, s, CH3-15); and 1.77ppm (3H, s, CH316). 13CNMR: (CDC13) 81.6, C-2; 30.2, C-3; 36.8, C-4; 46.8, C-5; 44.8, C-6; 39.9, C-7; 69.2, C-8; 136.2, C-9; 128.9, C-10; 71.6, C-11; 69.1, C-12; 49.3, C-13; 20.4, C-14; 13.5, C-15; and 18.2ppm, C- 16. Mass Spectrum: HREIMS: 250.150m/e (M+); calcd for C15H2203, 250.156. References D. G. Corley, G. E. Rottinghaus, and M. S. Tempesta; Novel Trichothecenes from Fusarium sporotrichioides; Tetrahedron Letters, Vol. 27, pp. 427-430(1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

205

Common/Systematic Name Trichodermin 413-Acetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2404; ~

-- 2 9 2 . 1 6 7 4 6

lo H

H

I~le J s 14

'~A

C

General Characteristics Synthetic trichodermin; mp., 58-60~ natural trichodermin crystallized from pentane; mp., 45-46~ [a]D2~ - 11.0~ (C=I.0, in CHC13). Insoluble in water; soluble in all common organic solvents. Fungal Source

Trichoderma lignorum, T. viride, and Stachybotrys cylindrospora, a fungal pathogen of aspen (Populus tremuloides) that has been found to be strongly antagonistic to the bluestain fungus, Ceratocystiopsis crassivaginata.

Biological Activity A potent inhibitor of protein synthesis in mammalian cells. Trichodermin inhibited the elongation and/or termination steps in protein synthesis. In vitro, it was a potent inhibitor of the peptidyl transferase activity required for elongation and/or termination. LDs0 in mice dosed SC was 500-1000mg/kg. Trichodermin is also a potent antifungal antibiotic. Spectral Data UV:

205nm (e=2,400). I_R:

(KBr) 1730, 1682, 1245, 1225, and 1085cm1; (CHC13) 2970, 1735, 1374, 1244, 1220, 1081, 1031,996, and 970cm1. 1H NMR: (CDC13) 3.80, H-2; 1.9-2.5, H-3; 5.60, H-4; 1.9-2.5, H-7; 1.9-2.5, H-8; 5.4, H-10; 3.7, H-11; 2.80, ttH-13; 3.10, 13H-13; 0.95, H-14; 0.75, H-15; 1.70, H-16; and 2.05ppm, CH3COO-.

206

14. Trichothecenes and Related Metabolites

13C NMR: 79.1 d, C-2; 36.7 t, C-3; 75.0 d, C-4; 48.9 s, C-5; 40.4 s, C-6; 24.5 t, C-7; 28.0 t, C-8; 140.1 s, C-9; 118.6 d, C-10; 70.5 d, C-11; 65.5 s, C-12; 47.8 t, C-13; 5.8 q, C-14; 16.0 q, C-15; 23.3 q, C-16; 21.1 q, CH3COO-; and 170.8ppm, s, CH3COO. Mass Spectrum: HREIMS: 292.1676(C17H2404, Amm~=0.2, 100), 277.1441(C16H2104, 78), and 217.1230m/e (C14H1702, 10%). TLC Data A. Adsorbent, silica gel G; solvent, chloroform-methanol, 98:2, v/v; Re, 0.67; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent, silica gel G; solvent: benzene-tetrahydrofuran, 85:15, v/v; Rf, 0.51; detection, H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 162 (1981). W.O. Godtfredsen and S. Vangedal; Trichodermin, A N e w Antibiotic, Related to

Trichothecin; Proc. Chem. Sot., Vol. 19, pp. 188-189(1964). W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993).

14. Trichothecenes and Related Metabolites

207

Common/Systematic Name Isotrichodermin 3 a-Acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2404; MW

= 292.16746

lo H: c~ H�9

16

-.-

~"

13

~el~ ~ 15

2

H I

3

"'OAc

iI

4

14

General Characteristics Colorless crystals; mp, 97-97.5~

[a]D 24 d- 9.5 ~ (c=0.09,

in CHC13).

Fungal Source Fusarium culmorum, F. roseum, F. graminearum, and F. crookwellense.

Isolation/Purification Fusarium roseum culture broth was extracted with methylene chloride alter being treated

with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A alter removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselge160). Isotrichodermin was isolated from mixture A2 by elution with 20% ethyl acetate in hexane. Spectral Data 1H N]VIR:

(CDCI3) 0.74(3H, s, H-15); 0.80(3H, s, H-14); 1.0-2.0(6H, m, H-7, H-4, H-8);

2.08

14. Trichothecenes and Related Metabolites

1.71(3H, br s, H-16); 2.1 l(3H, s, Ac-CH3); 2.84, 3.07(2H, Jab=4.0Hz, H-13); 3.73(1H, d, JE,a=4.6Hz, H-2); 3.96(1H, d, JlO,ll=5.5Hz, H-11); 5.15(1H, dt, J24=10.0, JE,a=4.6Hz, H-3); and 5.44ppm (1H, dq, J1o,ll=5.5Hz, Jlo,16=l.5Hz, H-10). 13C NMR: (CDC13) 78.2, C-2; 71.7, C-3; 38.6, C-4; 45.3, C-5; 40.2, C-6; 24.4, C-7; 28.2, C-8; 139.5, C-9; 119.4, C-10; 71.5, C-11; 65.2, C-12; 48.4, C-13; 11.0, C-14; 15.9, C-15; 23.1, C-16; 20.9, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 293.174m/e (M+ + 1); calcd for C17H2504,293.168. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes and Related Metabolites

209

Common/Systematic Name 7-Hydroxyisotrichodermin 7a-Hydroxy-3a-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405; M'W -- 308.16237

1 6 ~ ~ 0 ~lo2 H

3,

, "4 -

" '

15

OH

14

General Characteristics Crystalline solid from ether-petroleum ethers; mp., 154-155.5oC; in CHC13).

[ a ] D 22 -

10.6 ~ (c=0.095,

Fungal Source Fusarium culmorum, F. roseum (ATCC 28114), and F. crookwellense.

Isolation/Purification Fusarium roseum culture broth was extracted with methylene chloride after being treated

with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil which was subjected to medium pressure liquid chromatography on a LiChroprep Si 60 column.The column was eluted with the following solvents: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. A metabolite eluting in the 10% ethyl acetate fractions was further purified by elution initially with chloroform and then 2% methanol-chloroform. The metabolite was recrystallized from ether-petroleum ether to give a crystalline solid of 7-hydroxyisotrichodermin. Spectral Data IH ~ : (CDC13) 0.82(3H, s, CH3-15); 1.04(3H, s, CH3-14); 1.09(1H, 7-OH, J=6.3Hz); 1.70(3 H, br s, CH3-16); 2.10(3H, s, Ac-CH3); 1.9, 2.2(2H, dd, J=17.2, 10.0; 17.2, 6.0Hz, H-8); 2.07, 2.10(2H, m, 2H-4); 3.08, 3.13(2H, d, J=4.4, 4.4Hz, H-13); 3.76(1H, d, J2,3=4.5Hz, H-2); 4.10(1H, d, J11,1o=5.4Hz, H-11); 4.46(1H, JT,s=5.9, 10.1, J7,1o=6.3Hz); 5.16(1H, ddd, J=9.8, 5.2, 4.5Hz, H-3); and 5.40ppm (1H, br d, J=5.4Hz, H-10).

210

14. Trichothecenes and Related Metabolites

13C NMR: (CDC13) 79.0, C-2; 74.1, C-3; 39.5, C-4; 44.6, C-5; 46.2, C-6; 74.7, C-7; 41.4, C-8; 138.0, C-9; 119.6, C-10; 69.0, C-11; 64.9, C-12; 47.7, C-13; 10.2, C-14; 15.0, C-15; 22.4, C-16; 20.9, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 308.163m/e (M~); calcd for C17I-I2405, 308.162. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 34, pp. 115-118(1986). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes and Related Metabolites

211

Common/Systematic Name 8tt-Hydroxyisotrichodermin 8t~-Hydroxy-3t~-acetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405,

16

~

= 308.16237

10 H U

2

HO'"[~~~~I~" ' 191e~ ~

3

o

....

15

14

General Characteristics Crystals from ethyl acetate-hexane; mp., 153-155.5~

[tg]D 21 -

18.2~ (c=0.11, in CHC13).

Fungal Source Fusarium culmorum, F. roseum (ATCC 28114), and F. crookwellense. Isolation/Purification Fusarium roseum culture broth was extracted with methylene chloride after being treated with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil which was subjected to medium pressure liquid chromatography on a LiChroprep Si 60 column. The column was eluted with the following solvents: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. An oil eluting in the 10% ethyl acetate fractions was further purified with a Chromatotron eluted with chloroform followed by 5% methanol in chloroform. 8-Hydroxyisotrichodermin and 8hydroxy-3tt-acetoxy-12,13-epoxytrichothec-9-ene were purified by preparative TLC with 7% methanol in chloroform. Spectral Data 1H NMR: (CDC13) 3.72(1H, d,J=4.6Hz, H-2); 5.16(1H, ddd, J=9.8, 5.5, 4.6Hz, H-3); 2.09(2H, m, J=9.8, 5.5Hz, H-4); 1.70(1H, dd, J=14.3, 1.4Hz, H-7); 2.21(1H, dd, J=14.3, 5.7Hz, H-7); 4.10(1H, br d, J=5.7Hz, H-8); 5.59(1H, d, J=5.7Hz, H-10); 4.04(1H, d, J=5.7Hz, H-11); 2.87(1H, d, J=4.1Hz, H-13); 3.06(1H, d, J=4.1Hz, H-13), 0.77(3H, s, CHs-14); 0.98(3H, s, CH3-15); 1.86(3H, br s, CH3-16); and 2.1 lppm (3H, s, CH3COO-).

212

14. Trichothecenes and Related Metabolites

13C NMR: (CDC13) 78.1, C-2; 71.5, C-3; 38.1, C-4; 45.4, C-5; 39.6, C-6; 33.6, C-7; 68.0, C-8; 138.8, C-9; 122.1, C-10; 71.3, C-11; 65.2, C-12; 48.5, C-13; 11.0, C-14; 19.0, C-15; 20.4, C-16; 20.9, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 308.166m/e (Mr); ealcd for C17H2405,308.162. References R. Greenhalgh, R.M. Meier, B. A. Blaekwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agrie. Food Chem., Vol. 34, pp. 115-118(1986). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichotheeenes Produced by Fusarium crookwellense DAOM 193611; J. Agrie. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes and Related Metabolites

213

Common/Systematic Name 7,8-Dihydroxyisotrichodermin 7,8-Dihydroxy-3 t~-acetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2406; MW : 324.15729 16

10 8

H 6

OH

13~

,,~

I

....OAc

14

General Characteristics White solidi mp., 80-84 ~ Fungal Source Fusarium crookwellense.

Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed on a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase to give pure 7,8-dihydroxyisotrichodermin. Spectral Data 1H NIV[R:

(CDC13) 3.76(1H, d, J=4.SHz, H-2); 5.17(1H, m, J=8.8, 6.9, 4.5Hz, H-3); 2.05(2H, m, Jt~=6.9, 7.8Hz, J][3=8.8, 7.8Hz, H-4); 4.41(1H, dd, J=9.7, 5.5Hz, H-7); 3.98(1H, bt, J=6.0Hz, H-8); 5.59(1H, dq, J=5.9, 1.6Hz, H-10); 4.16(1H, d, J=5.9Hz, H-11); 3.10(1H, d, J=4.3Hz, H-13); 3.17(1H, d, J=4.3Hz, H-13); 1.06(3H, s, CH3-14); 0.89(3H, s, CH3-15); 1.87(3H, br s, CH3-16); and 2.10ppm (3H, s, CH3COO-). 13C NMR: 78.9 C-2; 71.5, C-3; 40.6, C-4; 46.4, C-5; 44.2, C-6; 73.8, C-7; 71.1, C-8; 138.8, C-9; 122.6, C-10; 69.8, C-11; 65.0, C-12; 47.8, C-13; 13.2, C-14; 14.7, C-15; 20.3, C-16; 20.9, CH3COO-; and 170.6ppm, C-O. Mass Spectrum: LREIMS: 324(NV, 27%), 165(24), 148(47), 123(49), 135(61), 95(51), 107(75), and 100m/e (100).

214

14. Trichothecenes and Related Metabolites

Reference D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agile. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes and

Related Metabolites

215

Common/Systematic Name NT- 1 Toxin 413,8a-Diacetoxy-3 a, 15-dihydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608; MW = 382.16277 lo

..

H

H

1~)~O-,~

H

2 3 i .... 'OH

H

'2"0 -

l

- 14 CH2OH 15 General Characteristics

'I ,,H OAc

Crystals from benzene-n-hexane; mp., 172.5-173.5~ Fungal Source

Fusarium sporotrichioides (strain M- 1-1), F. tricinctum, F. equiseti, and F. acuminatum.

Isolation/Purification Extracted by charcoal absorption and elution with methanol. Extraction of methanolchloroform soluble materials produced a pale yellow powder. The powder was chromatographed on a silica gel column eluted with n-hexane, n-hexane-acetone, 12:5, 2:1, 1:1, 1:2 (v/v), acetone and methanol followed by a second silica gel column eluted with benzene-acetone (3:2) and n-hexane-acetone (1:1, v/v). NT-1 was crystallized with benzene-n-hexane. Biological Activity Inhibited uptake of [14C]leucine in protein synthesis with rabbit reticulocytes; IDs0 at concentrations of 0.23 and 0.251.tg/ml by NT-1 and NT-2, respectively. These activities were similar to that of neosolaniol (0.25~g/ml) in the same assay. LDs0in rats dosed IP was 1.2mg/Kg. Spectral Data Im: (KBr) 3450, 2940, 1720, and 1230cm"1. 1H NMP< (CDCl3) 3.66(H-2); 4.24(H-3), 5.40(H-4); m.96(H-7cx);2.34(H-7~); 5.36(H-8); 5.82(H-10); 4.27(H-11); 2.80(H-13a); 3.04(H-13~); 0.84(H-]4); 3.60(H-15a); 3.91(H-1513); 1.75(H-16); and 2.09, 2.14ppm (3H each, CHACO-).

216

14. Trichothecenes and Related Metabolites

Mass Data: LREIMS: 382m/e (M+); found: C, 59.69; H, 6.82; O, 33.49%; calcd for C19H2608 ; C, 59.65; H, 6.85; O, 33.50%. TLC Data Adsorbent: silica gel; solvent: ethyl acetate-petrol (3:1, v/v); Re, 0.4; detection: sky-blue fluorescence in UV after spraying withp-anisaldehyde and heating. References K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology, Vol. 42, pp. 541-543(1981). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico, and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Trichothecene; J. Agric. Food Chem., Vol. 37, pp. 1348-1351(1989).

14. Trichothecenes and Related Metabolites

217

Common/Systematic Name 8-Ketoisotrichodermin 8-Keto-3 tt-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2205; l~trW = 306.14672

~o H

~6~o.,J

H

2

3.,,"OAc

,,,o I

J: 15 14

General Characteristics White solidi mp., 122-124~ Fungal Source

Fusarium crookwellense.

Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed by a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase. Spectral Data 1H ~ :

(CDCI3) 3.83(1H, d, J=4.6Hz, H-2); 5.22(1H, m, ,/--4.6, 10.9, 4.6Hz, H-3); 2.03(2H, dd, Jtt=14.8, 10.9Hz, JI3=14.8, 4.6Hz, H-4); 2.26(1H, dd, J=15.3, 1.1Hz, H-7); 2.87(1H, d, J=15.3Hz, H-7); 6.51(1H, dd, J=5.7, 1.5Hz, H-10); 4.32(1H, d, J=5.7Hz, H-11); 2.86(1H, d, J=3.9Hz, n-13); 3.10(1H, d, J=3.9Hz, n-13); 0.75(3H, s, CH3-14); 0.92(3H, d, d=l. 1Hz,CH3-15); 1.81(3H, d, J=l.5Hz, CH3-16); and 2.14ppm (3H, s, cn3coo-). 13CNMR: (CDC13) 78.5, C-2; 71.4, C-3; 42.1, C-4; 45.4, C-5; 43.4, C-6; 37.8, C-7; 189.0, C-8; 137.7, C-9; 137.6, C-10; 71.6, C-11; 57.5, C-12; 48.1, C-13; 10.6, C-14; 18.3, C-15; 15.3, C-16; 20.8, CH3COO-; and 172.5ppm, C=O. Mass Spectrum: LREIMS: 306(M+, 3%), 216(27), 253(35), 138(92), 123(100), 107(42), and 79m/e (49).

218

14. Trichothecenes and Related Metabolites

Reference D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611" J. Agric. Food Chem., Vol. 35, pp. 884-889(1987).

14.

T r i c h o t h e c e n e s and Related M e t a b o l i t e s

219

Common/Systematic Name Calonectrin 3 t~, 15-Diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C 19H2606; MW -- 350.17294 16

1o H

H

s

r. 14

H

~i'''H H

CH2OAc 15

General Characteristics Prisms from ethyl ether-petroleum ether; mp., 83-85~

[ ~ ] D 27 -

14.6 ~ (c=l.0, in CHCI3).

Fungal Source Fusarium culmorum, F. roseum (ATCC 28114), and Calonectria nivalis.

Isolation/Purification. Fusarium roseum culture broth was extracted with methylene chloride atter being treated

with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase atter it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil which was subjected to medium pressure liquid chromatography on a LiChroprep Si 60 column. The column was eluted with the following solvents: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. The fractions collected (651) were reduced to 52 samples by combining appropriate fractions. Calonectrin was isolated from an oil eluted in fraction 13 and purified by column chromatography on silica gel eluted with 5% methanol in chloroform. Spectral Data l-R:

(KBr) 1745, 1725, 1240, and 965cm "1. 1H N:V[R: (CDCI3) 3.82(2H, d, J=12.2Hz, ttCH2-15); 4.06(2H, d, J=12.2Hz, 13CH2-15); 0.81(3 H, s, CH3-14); 1.78-2.19(2H, m, H-7); 1.70(3 H, br s, CH3-16); 2.01(3H, s, Ac-CH3);

220

14. Trichothecenes and Related Metabolites

1.7-2.19(2H, m, H-8); 2.1-2.19(2H, m, H-4); 2.84, 3.08(2H each, d, J=4.0, 4.0Hz, 2H13t~ and 2H-1313, respectively); 3.74(1H, d, J=4.6Hz, H-2); 4.00(1H, d, J=5.6Hz, HI 1; 5.15(1H, ddd, J=9.6, 7.4, 4.6Hz, H-3); 2.01(3H, s, CH3COO); 2.08(3H, s, CH3COO); and 5.45ppm (1 H, dq, J=5.4, 1.3Hz, H-10). 13CNMR: (CDC13) 78.0, C-2; 71.2, C-3; 39.35, C-4; 45.3, C-5; 42.9; C-6; 20.9, C-7; 28.1, C-8; 140.3, C-9; 118.9, C-10; 68.0, C-11; 64.9, C-12; 48.4, C-13; 12.0, C-14; 63.5, C-15; 23.1, C-16; 20.9, 21.0, CH3COO-; and 170.6, 170.7ppm, C=O. References R. Greenhalgh, D. Lavandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites of Fusarium culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R.M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J.W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114), Part 2; J. Agile. Food Chem., Vol. 34, pp. 115-118(1986). D. R. Lauren, A. Ashley, B. A. BlackweU, R. Greenhalgh, J. D. Miller, and G. A. Neish; Triehothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agile. Food Chem., Vol. 35, pp. 884-889(1987). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 192 (1981).

14. Trichothecenes and Related Metabolites

221

.Common/Systematic Name 8-Ketocalonectrin 3tz, 15-Diacetoxy-12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2407; M W -- 3 6 4 . 1 5 2 2 0

16

lo

H

_

~'i''O .... 'OAc -

C)O~2

:

14

i

4

~H2OAc 15

General Characteristics Isolated as an oil. Fungal Source

Fusarium culmorum.

Isolation/Purification Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was dissolved and chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2% methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 8-ketocalonectrin were combined and obtained pure by preparative TLC. Spectral Data ~H NMR: (CDC13) 0.81(3H, s, CH3-14), 1.82(3H, d, J16,1o=l.6Hz, CH3-16), 1.96(3H, s, CH315Ac); 2.14(3H, s, CHa-3Ac); 1.95-2.23(2H, m, H-4); 2.51, 2.85(2H, dd, JAB=16.0Hz, J7~,ls=l.5Hz, n-7); 2.88, 3.12(2H, d, JAB=3.8Hz, n-13); 3.84(2H, d, JE,a=4.6Hz, n-2); 4.07(2H, m, H-15); 4.44(1H, d, Jl1,~o=5.1Hz, H-11); 5.23(1H, ddd, da,E=4.6Hz, d3,4-5.5, 10.4Hz, H-3); and 6.55ppm (1H, dd, JLO,11=5.1Hz,Jll,16=l.6Hz, H-10).

13CNMR: (CDC13) 78.1, C-2, 71.2, C-3; 38.5, C-4; 45.0, C-5; 47.5, C-6; 38.4, C-7; 198.7, C-8, 138.3, C-9; 137.5, C-10; 68.2, C-11; 65.3, C-12; 48.3, C-13; 11.3, C-14; 64.2, C-15; 15.4, C-16, 20.9, CHaCOO-, and 170.5ppm, C=O.

222

14. Trichothecenes and Related Metabolites

Mass Spectrum: LREIMS: 364(11%, M§ 262 (18), 203, (13), 109 (19); 121, and 43m/e (100). Reference R. Greenhalgh, D. Lavandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production andCharacterization of Deoxynivalenol and Other Secondary Metabolites of Fusarium culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986).

14.

Trichothecenes and Related Metabolites

223

Common/Systematic Name 15-Deacetylcalonectrin 3 tt-Acetoxy- 15-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405; M W = 3 0 8 . 1 6 2 3 7

~6

lo H

H

H

- O...]2 [.,3,,,OAc :

l~

l'~,'''H

-

14

H

~H2OH

15

General Characteristics Colorless prisms from ethyl acetate-hexane, mp., 181-183~ [~]D 26 q- 10.2 ~ (c=0.8, in CHC13); prisms from ethyl ether-light petroleum; mp., 184-186~ Fungal Source Fusarium culmorum, F. roseum, F. graminearum, and Calonectria nivalis.

Isolation/Purification Fusariurn roseum was cultured, harvested, and the broth extracted as described by Greenhalgh et al. (1984). Some of the oil from the methanol phase alter it had been

partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750mL of(A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A alter removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). Isotrichodermin was isolated from mixture A2 by elution with 20% ethyl acetate in hexane. Changing the solvent to 20% ethyl acetate in hexane gave pure 15deacetylcalonectrin.

224

14. Trichothecenes and Related Metabolites

Spectral Data 1H NMR: (CDCI3) 3.72(1H, d, J2,3=4.68Hz, H-2); 5.13(1H, ddd, J=10, 5.0, 4.6Hz, H-3); 1.732.30(2H, m, H-4); 1.73-2.30(2H, m, H-7); 1.73-2.30(2H, m, H-8); 5.46(1H, dd, J=5.3, 1.5Hz, H-10); 3.96(1H, d, J=5.3Hz, H-11); 2.84, 3.07(1H each, d, J=4.0Hz, H-13); 0.90(3H, s, CH3-14); 3.47(1H, d, J=l 1.6Hz, H-15); 3.66(1H, ddd, J=l 1.6, 4.5, 3.2Hz, H-15); 1.71(3H, d, J=l.5Hz, H-16); and 2.09ppm (3H, s, CH3COO-). 13CNMR: (CDC13) 78.2, C-2; 71.5, C-3; 39.4, C-4; 45.3, C-5; 44.2, C-6; 20.8, C-7; 28.5, C-8; 140.5, C-9; 119.5, C-10; 68.3, C-11; 65.2, C-12; 48.5, C-13; 12.3, C-14; 62.8, C-15; 23.1, C-16; 20.8, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 309.167m/e

( M ++

1); calcd for C17H2505, 309.162.

References R. Greenhalgh, D. Levandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites o f F u s a r i u m culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites o f F u s a r i u m roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984).

14. Trichothecenes and Related Metabolites

225

Common/Systematic Name 3-Deacetylcalonectrin 3 a-Hydroxy- 15-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405; M ' W -- 3 0 8 . 1 6 2 3 7

16

lo

H

,OH

,, i'" = 14

(~H2OAc 15 Fungal Source Fusarium graminearum and F. culmorum. Isolation/Purification Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2% methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 3-deacetylcalonectrin were combined and further purified by preparative TLC using 10% acetone in chloroform to yield pure 3-deacetylcalonectrin (Re, 0.62). Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of

226

14. Trichothecenes and Related Metabolites

animals and man. Spectral Data 1H NMR: (CDC13) 0.79(3H, s, CH3-14); 1.70(3H, s, CH3-16); 2.02(3H, s, CH3-15Ac); 1.92.3(6H, unresolved m, H-4, -7, -8); 2.82, 3.06(2H, d, JA,B=4.0Hz, H-13); 3.48(1H, d, J2,3=4.6Hz, H-2); 3.82, 4.06(2H, d, JA,B=12.2Hz, H-15); 4.13(1H, d, Jll,10=5.4Hz, H11); 4.43(1H, ddd, J3,2=4.6, J3,4=ca 6, 9.5Hz, H-3); and 5.48ppm (1H, dd, Jlo,ll=5.4Hz, J10,16=l.4Hz, H-10). 13CNMR: (CDCI3) 68.4, C-2; 69.1, C-3; 42.4, C-4; 45.9, C-5; 45.9; C-6; 21.2, C-7; 28.4, C-8; 140.1, C-9; 119.3, C-10; 80.0, C-11; 65.6, C-12; 48.4, C-13; 12.3, C-14; 62.8, C-15; 23.2, C-16; 21.0, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: LREIMS: 308(M+), 106(70%), 248(80), 123(82); 220(82), and 91m/e (100). Reference R. Greenhalgh, D. Lavandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites of Fusarium culmorum (CIVIl 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986).

14. Trichothecenes and Related Metabolites

227

Common/Systematic Name 7a-Hydroxycalonectrin 3a, 15-Diacetoxy-7a-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; M W -- 3 6 6 . 1 6 7 8 5

lo

H

1 6 " ~ O ~

2

13 :

HO

3..,"OAc

I ,,O

~5

4

- 14 ~H2OAc

15

General Characteristics Crystals from ethyl acetate-hexane; mp., 174-175~

[a]D 23

- 14.3 o (c=0" 14, in CHC13).

Fungal Source Fusarium roseum.

Isolation/Purification Fusarium roseum Greenhalgh et al.

was cultured, harvested, and the broth was extracted as described by (1984). Some of the oil from the methanol phase atter it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750mL of (A) methylene chloride, (B) 0.5 methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A aider removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC (75% ethyl acetate in chloroform; Re, 0.50); fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60) eluted with chloroform followed by 5% methanol in chloroform. Final purification of 7-hydroxycalonectrin was by preparative TLC using ethyl acetate as developing solvent.

Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range

228

14. Trichothecenes and Related Metabolites

of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and man. Spectral Data 1H NMR: (CDC13) 3.78(1H, d, J2,3=4.4Hz, H-2); 5.17(1H, ddd, J=10, 8.0, 4.5Hz, H-3); 1.972.18(2H, m, H-4); 4.55(1 H, dd, J=5.9, 11.2Hz, H-7); 1.4 I(1H, d, J= 11.2Hz, OH-7); 1.90-2.0(2H, m, H-8), 2.41(dd, J=17.6, 5.9Hz H-8); 5.38(1H, dd, J=5.4, 1.3Hz, H10); 4.11(1H, d, J=5.4Hz, H-11); 3.09, 3.23(1H each, d, J=4.4Hz, H-13); 1.14(3H, s, CH3.14); 4.05(1H, d, J=12.2Hz, H-15); 4.3 I(1H, m, H-15); 1.72(3H, br s, CHs-16); and 2.08, 2.09ppm (each 3H, s, CH3COO-). 13C NMR: (CDC13) 78.9, C-2; 71.1, C-3; 39.6, C-4; 46.1, C-5; 46.4, C-6; 71.0, C-7; 41.7, C-8; 139.4, C-9; 119.0, C-10; 70.0, C-11; 64.3, C-12; 47.8, C-13; 15.9, C-14; 64.4, C-15; 22.4, C-16; 21.0 and 20.8, CH3COO-; and 170.0 and 170.1ppm, C=O. Mass Spectrum: HREIMS: 306.148m/e (M+ - AcOH); calcd for C17H2205, 306.147. TLC Data Silica gel 60 F-254 plates developed with 75% ethyl acetate in chloroform; Rf, 0.50. References R. Greenhalgh, D. Levandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites ofFusarium culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984).

14. Trichothecenes and Related Metabolites

229

Common/Systematic Name 8 t~-Hydroxycalonectrin 3et, 15-Diacetoxy-8t~-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; M W

~6

~o H -

= 366.16785

0

2

HO""~~2 __.

3r

,OAc

4

14

(~H2OAc 15

General Characteristics Crystals; mp., 168-170~

[0~]D 21 -

6.67 ~ (c=0.15, in CHC13).

Fungal Source Fusarium roseum, F. culmorum, and F. graminearum.

Isolation/Purification Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2%methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 8-hydroxycalonectrin were combined and the mixture was separated by preparative TLC (developed with 1% ethanol in ethyl ether; Rf, 0.27) to yield pure 8hydroxycalonectrin. Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on

230

14. Trichothecenes and Related Metabolites

polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The triehothecenes have been strongly implicated in natural intoxications of animals and man. Spectral Data 1H NMR: (CDC13) 3.73(1H, d, J2,3=4.6Hz, H-2); 5.15(1H, ddd, J=10, 5.1, 5.0Hz, H-3); 2.232.28(2H, m, H-4); 1.95(1H, d, J=14.6Hz, H-7), 2.19(1H, dd, J=14.6, 10.8Hz, H-7); 4.08-4.18(2H, m, H-8), 5.58(dd, J=5.7, 1.9Hz H-10); 4.08-4.18(1H, m, H-11); 2.86, 3.08(1H each, d, J=4.0Hz, H-13); 0.83(3H, s, CH3-14); 4.08-4.18(2H, m, H-15); 1.85(3H, d,J=O.6Hz, CH3-16); and 2.03, 2.1 lppm (each 3H, s, CH3COO-). 13CNMR: (CDC13) 77.8, C-2; 71.1, C-3; 39.1, C-4; 45.4, C-5; 42.3, C-6; 30.0, C-7; 67.8, C-8; 139.5, C-9; 121.5, C-10; 67.0, C-11; 64.9, C-12; 48.6, C-13; 11.9, C-14; 65.0, C-15; 20.5, C-16; 20.9 and 21.8, CH3COO-; and 170.3 and 170.4ppm, C=O. Mass Spectrum: HREIMS: 306.145m/e (M§ AcOH); calcd for C17H2205,306.147. TLC Data Silica gel 60 F254plates developed with 75% ethyl acetate in chloroform; Rf, 0.37. References R. Greenhalgh, D. Levandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites ofFusarium culmorum (CMI 14764, HLX 1503); J. Agile. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agile. Food Chem., Vol. 32, pp. 1261-1264(1984).

14.

T r i c h o t h e c e n e s and Related M e t a b o l i t e s

231

Common/Systematic Name 7 tt, 8 t~-Dihydroxycalonectrin 3 tt, 15-Diacetoxy-7 tt, 8 tt-dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608; ~ 16

m,~,., IVl~-

= 382.16277

10

n

A

2

13

H

,UAC A

~

A

3 .,~

i II

e (2H2OAc 15

General Characteristics Needles from ethyl ether; mp., 190-192~

[t~]D26 + 7.2 ~ (C=0.07, in CHC13).

Fungal Source Fusarium roseum (ATCC 28114), F. culmorum, and F. graminearum.

Isolation/Purification Fusarium roseum was cultured, harvested, and the broth was extracted as described by Greenhalgh et al. (1984). Some of the oil from the methanol phase aider it had been

partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750mL of (A) methylene chloride, (B) 0.5 methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A aider removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC (75% ethyl acetate in chloroform; Re, 0.31); fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). 3a,15-diacetoxy-7a,813dihydroxy-12,13-epoxytrichothec-9-ene was isolated from mixture A3 by elution with 50% ethyl acetate in hexane followed by recrystallization from ethyl ether.

232

14. Trichothecenes and Related Metabolites

Spectral Data 1HNMR:

(CDCI3) 1.12(3H,H-14); 1.86(3H, H-16, Jxo,16=l.0Hz); 2.02(3H, AC-CH3); 2.10(3H, AC-CH3); 2.13, 2.34(2H, H-4, ,/4,3=11.1, 4.6Hz); 2.71(1H, 8-OH, Js,ou=8.0Hz); 3.0(1H, 7-OH, JT,on=9.7Hz); 3.09, 3.20(2H, H-13, Jab=4.3Hz); 3.77(1H, H-Z, J2,3=4.4Hz); 3.99(1H, H-8, JT,s=5.8, Js,on=7.5Hz); 4.14, 4.44(2H, H-15, Jab=lZ.3Hz); 4.32(1H, H-11, J11,lO=5.8Hz); 4.50(1H, H-7, JT,on=9.6Hz,JT,s=5.4Hz); 5.15(1H, H-3, J3,2=4.4Hz, J3,4=4.5, 11.1Hz); and 5.58ppm (1H, H-10, Jlo,ll=5.8Hz, Jlo,a6=0.9Hz). 13CNMR: (CDC13) 78.5, C-2; 70.4, C-3; 41.3, C-4; 46.0, C-5; 46.2, C-6; 70.2, C-7; 71.0, C-8; 139.3, C-9; 122.1, C-10; 69.7, C-11; 64.6, C-12; 47.8, C-13; 15.0, C-14; 63.3, C-15; 21.1, C-16; 20.9, 20.3, CH3COO-; and 170.4, 170.0ppm, C=O. Mass Spectrum: HREIMS: 383.179role

( M ++

1); calcd for C19H27Os,383.171.

References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Part 2. Minor Metabolites o f F u s a r i u m r o s e u m (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 115-118(1984). R. Greenhalgh, R. M. Meier, B. A. Blaekwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites o f F u s a r i u m r o s e u m (ATCC 28114); J. Agrie. Food Chem., Vol. 32, pp. 1261-1264(1984). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from F u s a r i u m Fungi; In Myeotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

233

Common/Systematic Name 4-Acetoxyscirpenediol 413-Acetyl-3 tt, 15-dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2406; M W = 324.15729

1o _H "T"7 11"~ 8

~

6 13

H I

~ ~ ' 2 ~s _- Me 14

H

I..

~ '"OH

,,O 4

....,H OAc

CH2OH 15

General Characteristics Amorphous solid from benzene-hexane; mp., 100-110 oC. Fungal Source Fusarium roseum (NRRL 1181), F. sulphureum, and F. sambucinum (NRRL 13495). Isolation/Purification The mycelium was extracted with ethyl acetate, followed by silica gel column chromatography eluted with benzene-acetone (2:1, v/v), acetone, and methanol. 4-Acetoxyscirpenediol was purified by preparative TLC using silica gel G plates developed with hexane-acetone (1:1, v/v). Biological Activity 4-Acetoxyscirpenediol caused dermal necrosis in white rats. The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and man.

234

14. Trichothecenes and Related Metabolites

Spectral Data IR~ 3450, 2950, 1720, 1435, 1375, 1240, 1165, 1110, 1080, 1050, and 960cm1. 1H NMR: (CDC13) 3.68(1H, d,J=4.8Hz, H-2); 4.26(1H, dd, J-3.1, 4.1Hz, H-3); 5.53(1H, d, J-3.1Hz, H-3); 1.7-2.2(4H, m, H-7 and H-8); 5.58(1H, br d, J=5.0Hz, H-10); 4.20(1H, br d, J-5.0Hz, H-11); 2.78(1H, d, J=4.0Hz, H-13); 3.06(1H, d, J-4.0Hz, H13); 0.86(3H, s, CH3-14); 3.64, 3.69(2H, d, J=12.0Hz, CH2-15); 1.74(3H,br s, CH316); and 2.170ppm (3H, s, CH3-Ae).

Mass Data: LREIMS: 306(M+ - H20), 278, 234, 219, and 43re~e; TMS ether: 468(M+); anal. calcd for C17H2406: C, 62.95; H, 7.46%; found: C, 60.93; H, 6.65%. References K. Ishii, S. V. Pathre, and C. J. Mirocha; Two New Trichothecenes Produced by Fusarium roseum; J. Agile. Food Chem., Vol. 26, pp. 649-653(1978). K. E. Richardson, G. E. Toney, C. A. Haney, and P. B. Hamilton; Occurrence of Seirpentriol and Its Seven Acetylated Derivatives in Culture Extracts of Fusarium sambucinum NRRL 13495; J. Food Protect., Vol. 52, pp. 871-876(1989). D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, and M. S. Tempesta; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blaekwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Myeotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14.

T r i c h o t h e c e n e s and Related M e t a b o l i t e s

235

Common/Systematic Name 15-Acetoxyscirpenetriol 15-Acetoxy-3 ~,4[3-dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2406; MW = 324.15729

',,o ',',',,OH

~o H

H

-

15

=

Me

-

14

H

'H

4 OH

_

CH2OAc 15

General Characteristics Crystals from isooctane-ethyl acetate; mp., 172-173~ Fungal Source F u s a r i u m r o s e u m and F. sambucinum.

Isolation/Purification The dried cultures were moistened with water to a moisture content of 30% and extracted with ethyl acetate. The extract was concentrated to a gum and redissolved in acetonitrile and partitioned against petroleum ether; the petroleum ether layer was discarded. The acetonitrile layer was concentrated and then chromatographed on a column of silica gel. The components were eluted off the column with ethyl acetate-nhexane (1:3 to 1:7, v/v), ethyl acetate-methanol (5:1, v/v), and methanol. Each 100ml fraction was tested for toxicity by topical application to the shaved skin of the white rat. Fractions 4 through 12 were toxic and were pooled and rechromatographed on a column of silica gel using chloroform-methanol (98:2, v/v) as the eluting solvent. The fractions from this column were tested for toxicity and the one found toxic was purified by preparative TLC on silica gel G, developed in chloroform-methanol (98: ~, v/v) and made visible by charring with concentrated H2SO4. The monoacetoxyscirpenol was crystallized from methylene chloride-petroleum ether. Two recrystallizations from isooctane-ethyl acetate gave an analytical sample. Biological Activity Clinical signs in birds ingesting the toxin were bilateral inflammation of the beak area and gastrointestinal hemorrhaging. Application to shaved skin of rat resulted in hyperkeratosis and petechial hemorrhaging. The LDs0 in 20-day-old white female weanling rats dosed SC was 0.752mg/kg. A single 20kg female pig injected IV with 20mg monoacetoxyscirpenol (lmg/kg) exhibited emesis within 0.5 hr, lethargy, a staggered gait, and death within 13 hr.

236

14. Trichothecenes and Related Metabolites

Spectral Data IR:

(KBr) 3400,1715, and 1250 cm "1. 1H NMR: (CDCI3) 3.60(1H, d, J=4.6Hz, H-2); 4.21(1H, dd, J--2.7, 4.6Hz, H-3); 4.26(1H, br d, J=2.7Hz, H-4); 1.76(1H, dd, J=l 1.3, 4.8Hz, H-7); 2.01(1H, m, H-7); 1.98-2.00(2H, m, H-8); 5.46(1H, d, J=5.4Hz, H-10); 3.92(1H, br d, J=5 4Hz, H-11); 2.73(1H, d, J=3.9Hz, n-13); 3.01(1H, d, J=3.9Hz, n-13); 0.79(3H, s, CH3-14); 3.87(1H, d, J=12.3Hz, H-15); 4.18(1H, d, J=12.3Hz, H-15); 1.70(3H, br s, CH3-16); and 2.04ppm (s, CHaCOO-). 13C NMR: (CDCI3) 78.9, C-2; 80.0, C-3; 81.9, C-4; 49.2, C-5; 43.5, C-6; 21.1, C-7; 28.0, C-8; 140.7, C-9; 118.4, C-10; 68.0, C-11; 64.7, C-12; 46.9, C-13; 6.9, C-14; 63.6, C-15; 23.2, C-16; 21.3, CH3COO-; and 170.8ppm, CH3COO-. Mass Data: 264(36), 205(100), 191(9), 189(11), 187(30), and 177role (24); anal. calcd for C17H2406: C, 62.96; H, 7.41%; found: C, 63.42; H, 7.61%. TLC Data Adsorbent: silica gel G; Solvent: chloroform-methanol (90:10, v/v); Re, 0.37. Detection: H2SO4 or p-anisaldehyde spraying and heating at 110~for 10 min. Purple to brown color with H2SO4; purple color with p-anisaldehyde. GLC Data Liquid phase: 3% OV-1 on Gas Chrom Q (100-120 mesh); TMS, 9-10 min.; retention time: trifluoroaeetate, 5-6 min. (Derivative for GLC: trimethylsilyl ether or trifluoroacetate). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 168(1981). S. V. Pathre, C. J. Mirocha, C. M. Christensen, and J. Behrens; Monoacetoxyscirpenol. A New Mycotoxin Produced by Fusarium roseum Gibbosum; J. Agile. Food Chem., Vol. 24, pp. 97-103(1976). K. E. Richardson, G. E. Toney, C. A. Haney, and P. B. Hamilton; Occurrence of Scirpentriol and Its Seven Acetylated Derivatives in Culture Extracts of Fusarium sambucinum N R R L 13495; J. Food Protection, Vol. 52, pp.871-876(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257(1994).

14. Trichothecenes and Related Metabolites

237

Common/Systematic Name 4,15-Diacetoxyscirpenol; Anguidine 4~, 15-Diacetyl-3 t~-hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; M W -- 3 6 6 . 1 6 7 8 5

16

lo

H

H

~O.,,J2 13-

~ ~ ' 2 --

I

H

31. "OH

,,0 !" . ,,H

14

OAc

CH2OAc 15 General Characteristics Crystals from ether; mp., 162-164~ Benzoate: prisms from ethanol; mp., 178~ [t~]D24 - 27 ~ (C=1.28). Fungal Source

Fusarium roseum (NRRL 1181), F. sambucinum (NRRL 13495) = Gibberella pulicaris, F. tricinctum, F. equiseti, F. lateritium, F. graminearum, F. semitectum, F. sulphureum, F. diversisporum, F. scirpL and Gibberella intricans.

Isolation/Purification The mycelium was extracted with ethyl acetate, followed by silica gel column chromatography eluted with benzene-acetone (2:1, v/v), acetone, and methanol. 4,15Diacetoxyscirpenol was purified by repetitive preparative TLC using silica gel G plates developed with hexane-acetone (1:1, v/v). Biological Activity The LDs0 in mice dosed IP was 23.0mg/kg; LDs0 in chicken embryo test was 0.091,tg per egg. IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.03~g/ml; cell-free was 5.01.tg/ml; tumor cells were 0.30Ftg/ml; rat liver (cell-free) was >501.tg/ml. Dermal toxicity to rabbit, mouse, and guinea pig. Diacetoxyscirpenol was also highly phytotoxic. Spectral Data 1H NMR: (CDCI3) 3.65(1H, d, J=4.9Hz, H-2); 4.10(1H, dd, ,/--2.9, 4.9Hz, H-3); 5.1 l(1H, d, J=2.9Hz, H-4); 2.00(1H, d, J=l 1Hz, H-7); 1.71(1H, d, J=l 1.0Hz, U-7); 1.93(2H, m, H-8); 5.50(1H, d, J=5.3Hz, H-10); 4.02(1H, d, J=5.3Hz, H-11); 3.00(1H, d, J=4.0nz, n-13); 2.74(1H, d, J= 4.0Hz, n-13); 0.79(3H, s, CH3-14); 4.10, 3.90(2H, d, J=12.3Hz, CH2-15); 1.68(3H, s, CH3-16); and 2.00, 2.09ppm (3H, s, CHa-Ac).

238

14. Trichothecenes and Related Metabolites

13C NMR: 79.0, d, C-2; 76.3, d, C-3; 83.0, d, C-4; 49.1, s, C-5; 44.2, s, C-6; 21.1, t, C-7; 27.9, t, C-8; 139.4, s, C-9; 119.3, d, C-10; 67.4, d, C-11; 64.8, s, C-12; 46.6, t, C-13; 6.8, q, C-14; 63.4, t, C-15; 23.2, q, C-16; 21.1, q, and 21.1, q, CH3C = O; 170.3, s, and 170.3, s, CH3C__= O. Mass Data: LREIMS (TMS derivative): 439(M § + 1), 438(M+), 324, 379(base peak), 319, 301, 289, and 229m/e. TLC Data Adsorbent: Kieselgel G; solvent: chloroform-methanol, 97:3 (v/v); Rf, 0.55; detection: H2SO4 spraying and heating at 110~ for 5 min. Adsorbent: alumina; solvent: chloroformmethanol, 98:2 (v/v); Re, 0.86; detection: H2SO4 spraying and heating at 110~ for 5 min. GLC Data Support: Shimalite W; liquid phase: OV-17 (1.5%); retention time: 5.95; relative retention time: (relative to nivalenol) 1.67. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 171-172(1981). K. E. Richardson, G. E. Toney, C. A. Haney and P. B. Hamilton; Occurrence of Scirpentriol and Its Seven Acetylated Derivatives in Culture Extracts of Fusarium sambucinum NRRL 13495; J. Food Protect., Vol. 52, pp. 871-876(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp.59-257 (1994).

14. Trichothecenes and Related Metabolites

239

Common/Systematic Name Scirpenetriol 3 tt,4 fl, 15-Trihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2205; MW" = 282.14672 ~o

H

H

- ~ -

14

H

'~

,,H OH

CH2OH 15

General Characteristics Prisms from ethyl acetate; mp., 193~

[tg]D 23 0 ~

(c=1.27, in acetone).

Fungal Source Fusarium roseum and F. equiseti.

Isolation/Purification The dried cultures were rehydrated to a moisture content of 30% and extracted with ethyl acetate. The extract was concentrated to a gum and redissolved in acetonitrile and partitioned against petroleum ether; the petroleum ether layer was discarded. The acetonitrile layer was concentrated and then chromatographed on a column of silica gel. The components were eluted off the column with ethyl acetate-n-hexane (1:3 to 1:7, v/v), ethyl acetate-methanol (5:1, v/v), and methanol. Each 100ml fraction was tested for toxicity by topical application to the shaved skin of the white rat. Fractions 4 through 12 were toxic and were pooled and rechromatographed on a column of silica gel using chloroform-methanol (98:2, v/v) as the eluting solvent. Scirpenetriol was purified by preparative TLC on silica gel G, developed in chloroform-methanol (98:5, v/v) and made visible by charring with concentrated H2SO4. Biological Activity LDs0 in rats dosed IP was 0.8 lmg/kg (single dose). Spectral Data UV~

End absorption.

(Nujol) 3480, 3455, 3405, 1676, and 830cm 1.

240

14. Trichothecenes and Related Metabolites

1H NMR: 3.78, H-2; 4.08, H-3; 4.12, H-4; NR, H-7; NR, H-8; NR, H-10; NR, H-11; 2.70, H13a; 3.05, H-1313; 0.90, H-14; 3.59, H-15; and 1.72ppm, H-16. References P. W. Brian, A. W. Dawkins, J. F. Grove, H. G. Hemming, D. Lowe, and G. L. F. Norris; Phytotoxic Compounds Produced by Fusarium equiseti; J. Exp. Botany, Vol. 12, pp. 116123(1966). R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, New

York, p. 160 (1981). S. V. Pathre, C. J. Mirocha, C. M. Christensen, and J. Behrens; Monoacetoxyscirpenol. A New Mycotoxin Produced by Fusarium roseum Gibbosum; J. Agric. Food Chem., Vol. 24, p. 97(1976)

14. Trichothecenes and Related Metabolites

241

Common/Systematic Name 7tt-Hydroxydiacetoxyscirpenol 3a,7 tt-Dihydroxy-4~3,15t~-diacetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608; MW

=

382.16277

~o H

H

~ > ~ , , , ~111I_.:- H131, ost. I in, "'I''OHAc (~H2OAc 15

General Characteristics Needles from chloroform-hexane; mp., 201-203~ tetraacetoxy derivative, needles from benzene-n-hexane; mp., 147-149~ Fungal Source

Fusarium lateritium and Fusarium

spp. (K-5036).

Isolation/Purification The culture filtrate was mixed with activated charcoal, the charcoal washed with water and the materials adsorbed to the charcoal eluted with methanol. The methanol extract was concentrated and chloroform added which resulted in a precipitate. The chloroformmethanol was filtered and the filtrate evaporated to dryness. The residue was extracted with hot acetone, the acetone soluble materials were chromatographed on a silica gel column eluted with benzene-acetone 5:2, 2:1, 11 (v/v), acetone and methanol. The fractions were monitored by TLC (Kieselgel G) and the fractions containing 7ahydroxydiacetoxyscirpenol were combined and chromatographed on a silica gel column eluted with n-hexane-acetone, 1:1 (v/v); the metabolite at Rf0.5 was crystallized from chloroform-n hexane to give needles of 7a-hydroxydiacetoxyscirpenol. Biological Activity The LDs0 to mice dosed IP was about 3.5mg/kg; IDs0 of protein synthesis in rabbit reticulocytes was 0.31.tg/ml. Spectral Data UV:

End absorption. IR:

(KBr) 3500, 2950, 1720, 1380, 1280, and 1060cm.1.

242

14. Trichothecenes and Related Metabolites

1H NMR: (CDC13) 3.70(H-2); 4.25(H-3); 5.20(H-4); 4.62(H-7); 2.00(H-8a); 2.43(H-813); 5.50(H-10); 4.25(H-11); 3.07(H-13a); 3.17(H-1313); 1.13(H-14); 4.24(H-15a); 4.42(H-1513); 1.75(H-16); and 2.07, 2.15ppm (C_H_3H CO-). Mass Data: LREIMS: 364(M + - 18), 322(M + - 60), 304, and 263m/e; found: C, 59.25; H, 6.78%; C19H2608 requires: C, 59.65; H, 6.85%. TLC Data Adsorbent: silica gel; solvent: acetone-hexane (1:1, v/v); Rf, 0.5; detection: spraying with 20% H2SO4 and heating at 110~ References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 190(1981). K. Ishii; Two New Trichothecenes Produced by Fusarium sp.; Phytochemistry, Vol. 14, pp. 2469-2471(1975).

14. Trichothecenes and Related Metabolites

243

Common/Systematic Name 7a,8a-Dihydroxydiacetoxyscirpenol 3 a,7a,8 a-Trihydroxy-4[3,15 ~-diacetoxy- 12,13-epoxytrichothec-9-ene M.Qlecular Formula/Molecular Weight C19H2609, ~

-- 3 9 8 . 1 5 7 6 8

~o H

16

-

HO"

v

O

H

q

2

... -,~

H~) i ~

3

,.., o .

I

~J 0Ac

~-,H2OAc 15

General Characteristics Needles from ethanol-ether-n-hexane; mp., 167-169~ Fungal Source Fusarium lateritium, and F. spp. (K-5036). Isolation/Purification The culture filtrate was mixed with activated charcoal, the charcoal washed with water and the materials adsorbed to the charcoal eluted with methanol. The methanol extract was concentrated; chloroform was added which resulted in a precipitate. The chloroformmethanol was filtered and the filtrate evaporated to dryness. The residue was extracted with hot acetone, the acetone soluble materials were chromatographed on a silica gel column eluted with benzene-acetone 5:2, 2:1, 1:1 (v/v), acetone and methanol. The fractions were monitored by TLC (Kieselgel G) and the fractions containing 7t~hydroxydiacetoxyscirpenol were combined and chromatographed on a silica gel column eluted with n-hexane-acetone (1:1, v/v); the metabolite at Rf 0.38 was crystallized from EtOH-ether-n hexane to give needles of 7a,8ec-dihydroxydiacetoxyscirpenol. Biological Activity The LDs0 to mice dosed IP was about 6.0mg/kg; IDs0 of protein synthesis in rabbit reticulocytes was 0.61.tg/ml. Spectral Data UV:

End absorption. IR:

(KBr) 3500, 2950, 1750, 1390, 1260, and 1050cml.

244

14. Trichothecenes and Related Metabolites

1H NMR: (CDC13) 3.70(H-2); 4.20(H-3), 5.45(H-4), 4.01(H-7), 4.50(H-8); 5.65(H-10); 4.50(H-11), 3.05(H-13a); 3.15(H-1313); 1.10(H-14); 4.29(H-15a); 4.50(H-1513); 1.85(H-16); and 2.02, 2.13ppm (CH_H_3_CO-). TLC Data Adsorbent, silica gel; solvent, acetone-n-hexane (1:1, v/v); Rf, 0.38; detection, spraying with 20% H2SO4and heating at 110~ References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press' New York, p. 191 (1981). K. Ishii; Two New Trichothecenes Produced by Fusarium sp.; Phytochemistry, Vol. 14, pp. 2469-2471(1975).

14. Trichothecenes and Related Metabolites

245

Common/Systematic Name 3,15-Diacetoxyscirpenetriol 3t~, 15-Diacetyl-413-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; MW = 366.16785

16

~o H

H

"-0

2 i

o

_ :

is

4

.... ,o

c

~OH

14

~_,H2OAc 15

General Characteristics Colorless glass. Fungal Source Fusarium sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered,and the solid residue was re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum, and the nonpolar materials were removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone (4:1, 2:1, 1:1, v/v), acetone, and acetone-methanol (1:1, v/v). The toluene-acetone (4:1, v/v) fraction was further purified by flash chromatography eluting with toluene-acetone (4:1, v/v) This resulted in purified 3,15-diacetoxyscirpenol Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein

246

14. Trichothecenes and Related Metabolites

synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and man. Spectral Data UV: XAm~ 199nm(e=8,200, ~ to r~* transition). IR~ (film) 3403 and 1731cm~ (C=O). 1H N]VIR: (CDC13) 3.81(1H, d, J=4.9Hz, H-2); 4.85(1H, dd, J=2.9, 4.9Hz, H-3); 4.31(1H, d, J=2.9Hz, H-4); 1.7-2.1; 5.47(1H, b d, J=5.5Hz, H-10); 3.75(1H, b d, J=5.5Hz, H-11); 2.79(1H, H-13); 3.05(1H, d, J=4.0Hz, H-13); 0.87(3H, s, CH3-14); 3.90(1H, CH215); 4.13(1H, d, J=12.3Hz, CH2-15); 1.73(3H, b s, CH3-16); and 2.05, 2.20ppm (3H each, s, CH3-Ac).

13C NMR: 77.6, C-2; 83.5, C-3; 79.1, C-4; 48.9, C-5; 43.6, C-6; 20.9, C-7; 28.0, C-8; 141.0, C9; 118.2, C-10; 68.1, C-11; 64.0, C-12; 46.8, C-13; 6.7,C-14; 63.5, C-15; 23.2, C-16; 21.0, and 21.2, CH3C = O; not observed due to small sample size, CH3C = O. Mass Data: HR IMS: 307.154m/e; calcd for C17H2305 (M + - OAc); 307.174; LREIMS: 307(M + OAc, 5%), 205(68), 124(base peak), 105(97), and 81m/e (55). Reference D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989).

14. Trichothecenes and Related Metabolites

247

Common/Systematic Name 3,4,15-Triacetoxyscirpenetriol 3~,413,15-Triacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C21H2808; MW = 408.17842

- ~

10 H

16

O'~2l,,O3[....,OAc H

.- 5 ?

~'2 4- -=OAc

(~H2OAc 15 --

14

H

FI

General Characteristics Colorless crystals from benzene-n-hexane; mp., 124-126~ Fungal Source Fusarium sulphureum (MRC-514). Isolation/Purification Fungal cultures were extracted with chloroform-methanol (1:1, v/v), concentrated, and partitioned between 90% methanol-n-hexane. The aqueous methanol solution was concentrated and partitioned between chloroform and water. The chloroform solution was dried, filtered, evaporated to dryness, and chromatographed on a silica gel H column eluted with chloroform-methanol (19:1, v/v). The first fraction was filtered through a short alumina column (activity II-III) using chloroform; the yellow oil obtained was recrystallized from benzene-n-hexane to give colorless crystals of triacetoxyscirpenol. Spectral Data IH NMR: (CDC13) 0.76(3H, s, C-14 Me); 1.71(3H, s, C-16 Me); 2.04(3H, s, C-15 OAc); 2.09(3H, s, C-30Ac); 2.12(3H, s, C-40Ac); 2.77; 3.05(1H,each d, J~3,~4=4Hz C-13 H); 3.84(1H, d, ,/2,3=5 Hz, C-2 H); 3.99(d, 1H, d, Jlo, l~=5Hz, C-11 H); 4.03 and 4.25(11H, each d, Jls,ls=12Hz, C-15 H); 5.17(1H, dd, J3,a=3.5Hz, J2,3=5Hz, C-3 H); 5.46(1H, br d, Jlo, lt=5Hz, C-10 H); and 5.74ppm (1H, d, J3,4-3.5Hz, C-4 H). Mass Data: Found: C, 61.89; H, 6.68%; calcd for C21H2808"C, 61.75- H, 6.91%.

248

14. Trichothecenes and Related Metabolites

References R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 201(1981). P. S. Steyn, R. Vleggaar, C. J. Rabie, N. P. J. Kriek, and J. S. Harington, Trichothecene Mycotoxins from Fusarium sulphureum, Phytochemistry, Vol. 17, pp. 949-951(1978).

14.

Trichothecenes

and Related Metabolites

249

Common/Systematic Name T-2 Tetraol 3a,413,8a, 15-Tetrahydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2206; M W = 298.14164 ~o H I-

1~ OH

H I n o4 ~%1 .... , v

"1 13 ~1

Is - 1, CH2OH

i

.... , O H

4~ H

15

Fungal Source

Fusarium poae and F. sporotrichioides.

Spectral Data UV:

End absorption. IR:

(KBr) 3400, 2930, 1625, 1450, and 1380cm1. 1H NMR: (CDC13) 3.60(1H, d, J=4.8Hz, H-2); 4.07(1H, dd, J=4.8, 3.3Hz, H-3); 4.12(1H, d, J=3.3Hz, H-4); 1.97(1H, dr, J=14.4, 1.6Hz, ttH-7); 2.16(1H, dd, J=14.4, 5.3Hz, I]H7); 5.52(1H, dq, J=5.7, 1.5Hz, H-10); 3.80(1H, d, J=5.7Hz, H-11); 2.89(1H, d, J=4.1Hz, H-13); 2.76(1H, d, J=4.1Hz, H-13); 0.84(3H, s, CH3-14); 3.72(1H, d, J=2.4Hz, H-15); 3.40(1H, d, J=12.4Hz, H-15); and 1.82ppm (3H, s, CH3-16). 13C NMR: 78.5, C-2; 79.1, C-3; 80.0, C-4; 48.0, C-5; 45.1, C-6; 28.5, C-7; 64.8, C-8; 138.1, C-9; 121.6, C-10; 67.6, C-11; 64.3, C-12; 45.1, C-13; 6.3, C-14; 61.0, C-15; and 19.9ppm, C-16. (CDC13) 80.6, C-2; 80.8, C-3; 82.3, C-4; 50.4, C-5; 45.9, C-6; 29.9, C-7; 67.0, C-8; 141, C-9; 122.6, C-10; 70.0, C-11; 65.8, C-12; 47.2, C-13; 7.3, C-14; 63.1, C-15 and 20.7ppm, C- 16. TLC Data Adsorbent: silica gel G or Hi solvent: ethanol-ethyl acetate-acetone, 1:4:4, v/v/v; Re, 0.52; detection: H2SO4 spraying and heating at 150~ for 5 min.

250

14. Trichothecenes and Related Metabolites

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 162 (1981). R. A. Ellison and F. N. Kotsonis; Carbon-13 Nuclear Magnetic Resonance Assignments in the Trichothecene Mycotoxins; J. Org. Chem., Vol. 41, p. 576(1976). E. B. Smalley and F. M. Strong; Toxic Trichothecenes. In Mycotoxins; I. F. H. Purchase, ed.; pp. 199-228, Elsevier Scientific Pub. Co., New York (1974). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14.

T r i c h o t h e c e n e s and Related M e t a b o l i t e s

251

Common/Systematic Name T-2 Toxin 3 et-Hydroxy-413,15-diacetoxy-8tt- [3-methylbutyryloxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C24H3409; M W = 4 6 6 . 2 2 0 2 8

16~ Me

18

O_

~CHCH2~O

Me/ 21

H

H

042

"

~~

:

,4

(~H2OAc

,iO lH ,~

OAc

15

General Characteristics White needles from benzene-Skellysolve B; mp., 151-152~ [~]D 26 "at- 15~ (c=2.58, in EtOH). Acetate derivative: amorphous solid from ether-pentane; [a]D3~ +27 ~ (C=0.9, in 95% EtOH). Fungal Source Fusarium tricinctum, F. culmorum (F. roseum), F. solanL F. poae, F. sporotrichioides, and Trichoderma lignorum. Biological Activity Acute LDs0 in rats and swine dosed orally was 4mg/kg; LDs0 in mice dosed IP was 3.04mg/kg; LDs0 in chicks dosed orally was 1.84mg/kg; IDso of rabbit reticulocytes (whole cell) was 0.031,tg/ml; IDs0 of rabbit reticulocytes (cell-free) was 0.151.tg/ml. T-2 toxin inhibited the initiation step of protein synthesis on polyribosomes. It had emetic properties when administered orally, IP or IV. Minimum SC dosage to induce vomiting in ducklings was 0. l mg/kg; in cat, 0.1-0.2mg/kg. Major gross clinical signs in cats from T-2 toxin were emesis, vomiting, diarrhea, anorexia, ataxia of the hind legs, discharge from the eyes, and ejection of hemorrhagic fluid. Consecutive administration at sublethal dosages caused a marked decrease in white cells. Necropsy showed extensive cellular damage in the bone marrow, intestine, spleen, and lymph nodes. Also evident were meningeal hemorrhage of the brain, bleeding in the lungs, and vacuolic degeneration of the renal tubes. T-2 caused dermal necrosis when applied to the surface of the skin. It caused primary oral lesions in animals ingesting it. Spectral Data UV~

End absorption.

252

14. Trichothecenes and Related Metabolites

1H NMR: (CDC13) 3.68(1H, d, J=4.9Hz, H-2); 4.13(1H, ddd, d=4.9, 2.8, 2.8Hz, H-3); 3.18(1H, d, J=2.8Hz, 3-OH); 5.24(1H, d, J=2.8Hz, H-4); 2.38(1H, dd, d=15.1, 5.8Hz, all-7); 1.87(1H, dd, J=15.3, 1.5Hz, [3H-7); 5.27(1H, d, J=5.8Hz, H-8); 5.72(1H, dt, J=5.8, 1.2Hz, H-10); 4.33(1H, d, J=5.8Hz, H-11); 3.04(1H, d, J=3.9Hz, H-13); 2.78(1H, d, J=3.9Hz, H-13); 0.79(3H, s, CH3-14); 4.04(1H, d, J=12.6Hz, all-15); 4.27(1H, d, J=12.6Hz, 13H-15); 1.72(3H, br s, CH3-16); 2.10(2H, m, H-18); 2.01(1H, m, H-19); 0.94, 0.93(3H each, d, J=6.5Hz, 20 and 21 CH3); and 2.01, 2.12ppm (3H each, s, CH3COO-). 13C NMR: 78.7, C-2; 78.2, C-3; 84.3, C-4; 48.3, C-5; 42.9, C-6; 27.7, C-7, 68.0, C-8; 136.3, C-9; 123.8, C-10; 67.3, C-11; 64.2, C-12; 47.0, C-13; 6.6, C-14; 64.5, C-15; 20.1, C-16; 172.7, C-17; 43.4, C-18; 25.6, C-19; 22.2, C-20 and C-21; 20.8(3H, CH3COO-); and 170.2, 170.5ppm, (3H each, CH3COO-).

TLC Data A. Adsorbent: silica gel G; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.40; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.88; detection: H2SO4 spraying and heating at 110~ for 5 minutes. GLC Data (TMS derivatives) Support: Shimalite W; liquid phase: OV-17 (1.5 %); retention time 8.55; relative retention time: (relative to nivalenol) 2.40. References J. R. Bamberg and F. M. Strong; Mycotoxins of the Trichothecene Family Produced by Fusarium tricinctum and Trichoderma lignorum; Phytochemistry, Vol. 8, pp. 24052410(1969). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 185-186(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

253

Common/Systematic Name HT-2 Toxin 3a,413-Dihydroxy-15-acetoxy-8a- [3-methylbutyryloxy]-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C22H32Og; MW

= 424.20972

H

H .... , O H

Me. 20

18

0 ~

U

Me/CHCH2CO 21

~

_

~5

- ,, ~_,H2OAcO

4~)H

15

General Characteristics Pale yellow oil. Fungal Source

Fusarium sporotrichioides, F. poae, and F. culmorum.

Biological Activity LDs0 dosed IP to mice was 9.0mg/kg; LDs0 in chicken embryo assay was 0.5~g per egg. IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.03~g/ml. It inhibited the initiation step in protein synthesis. It also caused dermal necrosis similar to that of T-2. Spectral Data IR:

3400, 2950, 1720, 1635, and 1240cm1. 1H NMR: (CDC13) 3.60(1H, d, J=4.9Hz, H-2); 4.22(1H, m, H-3); 4.13(1H, br d, J=3.0Hz, H4); 2.00(1H, dr, J=15.0, 1.5Hz, all-7); 2.32(1H, dd, J=lS.0, 5.7Hz, 13H-7); 5.25(1H, br d, J=5.1Hz, H-8); 5.73(1H, br d, J=6.0Hz, H-10); 4.36(1H, br d, J=6.0Hz, H-11); 2.74(1H, d, J=4.0Hz, H-13); 3.00(1H, d, J=4.0Hz, H-13), 0.75(3H, s, CH3-14); 3.97(1H, d, J=12.5Hz, all-15); 4.29(1H, d, J=12.5Hz, ~H-15); 1.71(3H, s, CH3-16); 2.05-2.10(1H, m, H-19); 0.93, 0.92(3H each, d, J=6.4Hz, 20- and 21-CH3); and 2.01ppm (3H, s, CH3COO-). lSC NMR: 78.7, C-2; 80.6, C-3; 81.6, C-4; 48.9, C-5; 42.5, C-6; 27.6, C-7; 68.1, C-8; 136.4, C-9; 123.7, C-10; 67.3, C-11; 64.7, C-12; 46.9, C-13; 6.9, C-14; 64.5, C-15; 20.3, C-16; 172.7, C-17; 43.6, C-18; 25.7, C-19; 22.4, C-20 and C-21; 21.1(3H, C__H3COO-), and

254

14. Trichothecenes and Related Metabolites

170.4ppm, (3H, CH3COO-). Mass Spectrum: LREIMS: 424(M+), 322, 249, 203, 121,105, 85, and 57m/e (base peak). TLC Data A. Adsorbent: silica gel G; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.09; detection: H2SO4 spraying and heating at 110~ for 5 min. B. Adsorbent: alumina; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.32; detection: H2SO4 spraying and heating at 110~ for 5 min. References J. R. Bamberg and F. M. Strong; Mycotoxins of the Trichothecene Family Produced by Fusarium tricinctum and Trichoderma lignorum; Phytochemistry, Vol. 8, pp. 2405-2410 (1969). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 181-182(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. T r i c h o t h e c e n e s and Related M e t a b o l i t e s

255

Common/Systematic Name Acetyl T-2 Toxin 3 a,4~, 15-Triacetoxy-8a-[3-methylbutyloxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C26H36010; M W --- 5 0 8 . 2 3 0 8 5

lo

~

H -

H O..J~ ,

[~ 20 Me

0

II\CHCH2CO Me/ ,8

-

21

~1 ~

" ~ 1 .... ,v ~ ,

l

--~4 CH2OAc

OAc

15

General Characteristics An oil. Fungal Source

Fusarium poae (NRRL 3287).

Biological Activity Acetyl T-2 toxin was much less toxic than T-2 toxin in pigeons. It caused emesis at higher dosages (18.2mg/kg oral). No deaths occurred at tiffs dosage level. Spectral Dat.a UV:

End absorption. 1H NMR: (CDC13) 3.78, H-2; 5.07, H-3; 5.85, H-4; 1.85-2.1, H-7; 5.20, H-8; 5.65, H-10; 4.14, H-11; 2.75, all-13; 2.95, ~H-13; 0.65, H-14; 4.00, all-15; 4.27, 13H-15; 1.66, H16;1.93, H-18; 2.0, H-19; 0.96, H-20; 0.96, H-21; and 2.03ppm, (3H, CH3-COO-). 13CNMR: 76.2, C-2; 78.7, C-3; 78.2, C-4; 47.7, C-5; 42.0, C-6; 26.7, C-7; 67.2, C-8; 135.0, C-9; 124.0, C-10; 66.3, C-11; 63.6, C-12; 41.4, C-13; 6.1, C-14; 63.6, C-15; 19.4, C-16; 172.2, C-17; 42.4, C-18; 25.3, C-19; 21.7, C-20; 21.7, C-21; 170.6, CH3CO; and 20.3ppm, CH3CO. TLC Data Adsorbent: BrinkmanF254 silica gel; solvent: ethyl acetate-ethanol, 6:1, v/v; Rf, 0.74; detection: a quenching spot under UV light.

256

14. Trichothecenes and Related Metabolites

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 185-186(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

257

Common/Systematic Name 4-Propanyl HT-2 413-Propanyl-3a-hydroxy- 15-acetoxy-8a-[3-methylbutyryloxy]-12,13-epoxytrichothec-9ene Molecular Formula/Molecular Weight C25H3609; M W = 480.23 593

H .-

4

Me.

a" d'

,"

I-

.[

111

6 1

H v"J i

13

"~i~,, I

"~CHCH2COO'....- , , , G / i ~ / 1 2

Me/ 5"

~ ~e

-=

.... O H

,-,

ii u

1'

4

2'

3'

--~'OCOCH2Me

14

~;H2OAc 15

General Characteristics Isolated as white needles; mp., 141-142~ Fungal Source

Fusarium sporotrichioides (MC-72083) and F. sambucmum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil column chromatography eluted with benzene-hexane (2:1, v/v), methylene chloride, and chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil was further purified by normal phase preparative HPLC using benzene-acetone followed by preparative RPTLC to yield several trichothecenes including 4-propanyl HT-2 toxin. Spectral Data IR:

(film) 3430(OH), 1741, and 1730cm] (ester). 1H NMR:

(CDCla) 6.24(1H, t, J=-l.5Hz, H-2); 3.10(1H, d, ,/--18.2, H-4), 2.03(1H, d, J=18.2Hz, H-4); 1.95(1H, dd, ,/=15.0, 4.6Hz, n-7); 1.79(1H, d, J=15.0Hz, H-7); 1.45(2H, m, J=4.6Hz, H-8); 5.05(1H, br s, H-10); 4.11(1H, br s, H-11); 4.55(1H, d, d=17.3Hz, H 13); 4.34(1H, d, J=17.3Hz, n-13); 1.24(3H, s, CH3-14); 0.84(3H, s, CHa-15); and 1.61ppm (3H, br s, CH3-16). 13CNMR: (CDCI3) 129.5, C-2; 208.4, C-3; 50.4, C-4; 53.1, C-5; 41.9, C-6; 29.9, C-7; 27.4, C8, 135.7, C-9; 125.0, C-10; 71.3, C-11; 188.0, C-12; 61.0, C-13; 22.4, C-14; 13.1, C-

258

14. Trichothecenes and Related Metabolites

15; and 22.5ppm C-16. Mass Spectrum: CIMS: 470.236m/e (h/V); calcd for C25H3609,470.235. References D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene Mycotoxins of Fusarium sporotrichioides (MC- 72083); Tetrahedron Letters, pp. 41334136(1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

259

Common/Systematic Name 3'-Hydroxy T-2 toxin 3~-Hydroxy-413,15-diacetoxy-8a-[3-hydroxy-3-methylbutyryloxy]- 12,13-epoxytrichothec9-ene Molecular Formula/Molecular Weight C24I-I3401o;,M W -- 4 8 2 . 2 1 5 2 0 10

H

16~0.,j~--2 o _g "

o, ,,,' ,'

HO

5'

I A

-

H

I

3...., O H

14

(~�89 15

General Characteristics An oil. Fungal Source Fusarium sporotrichioides (MC-72083 and DAOM 165006). Isolation/Purification Fungal material was extracted with ethyl ether, chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Fraction five was further purified, first by activated LiChroprep Si-60 eluted with methylene chloride containing increasing amounts of ethyl acetate followed by preparative HPLC using 9% isopropanol-hexane to yield 3'-hydroxy T-2 toxin as an oil. Spectral Data UV:

End absorption. 1H NMR: (CDC13) 3.67(1H, d, J=4.9Hz, H-2), 4.14(1H, dd, J=4.9, 2.9Hz, H-3), 5.27(1H, d, J=2.9Hz, H-4), 2.40(1H, dd, J=lh.0, 5.2Hz, ~H-7); 1.90(1H, br d, J=lh.0Hz, ~H-7); 5.26(1H, d, J=5.3Hz, H-8); 5.79(1H, br d, J=5.9Hz, H-10); 3.04(1H, d, J=3.9Hz, H13); 2.78(1H, d, J=3.9Hz, H-13); 0.78(3H, s, CH3-t4); 4.04(1H, d, J=2.6Hz, all-15);

260

14. Trichothecenes and Related Metabolites

4.26(1H, d, J=12.6Hz, [3H-15); 1.73(3H, br s, CH3-16); 2.42(2H, s, 2'-CH2); 1.27(3H, s, 4' and 5'-CH3); and 2.02, 2.12ppm (3H each, s, CH3COO-). 13C NMR: 78.8, C-2; 78.4, C-3; 84.5, C-4; 48.4, C-5; 42.9, C-6; 27.7, C-7; 68.5, C-8; 135.8, C-9; 124.3, C-10; 67.2, C-11; 64.2, C-12; 47.1, C-13; 6.7, C-14; 64.6, C-15; 20.2, C-16; 172.8, C-I'; 46.6, C-2'; 69.1, C-3'; 29.0, 29.5, C-4', C-5'; 20.9, 21.0(3H each,_ CH3COO-); and 170.4, 172.6ppm, (3H each, CH3COO-).

Mass Spectrum: CIMS: (methane, positive ion) (TFA derivative) 601(M+ + 1), 561,541,501,451, 403,401(base peak), 341,311,281,215, 207, 151, and ll5m/e. EIMS: 185(48%), 180(45), 105(57), 121(100), and 59m/e (92). TLC Data Silica gel. Solvent system A: Benzene-acetone (12:7, v/v), Rf0.48; solvent system B: toluene-ethyl acetate-formic acid (6:3:1, v/v/v); Re, 0.19. Solvent system C: chloroformmethanol (9:1, v/v); Re, 0.59. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agrie. Food Chem., Vol. 36, pp.216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Myeotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Bottalico, and J. Chelkowski; Trichothecene Mycotoxins Produced by Fusarium sporotrichioides Strain P-11; Mycotoxin Research, Vol. 1, pp. 310(1985). T. Yoshizawa, T. Sakamoto, Y. Ayano, and C. J. Miroeha; 3'-Hydroxy T-2 and 3' Hydroxy HT-2 Toxins: New Metabolites ofT-2 Toxin, A Trichothecene Myeotoxin, In Animals; Agrie. Biol. Chem., Vol. 46, pp. 2613-2615(1982).

14. Trichothecenes and Related Metabolites

261

Common/Systematic Name 3'-Hydroxy HT-2 toxin 3 t~,413-Dihydroxy-15-acetoxy-8 t~-[3-hydroxy-3-methylbutyryloxy]- 12,13-epoxytrichothee9-ene Molecular Formula/Molecular Weight C22H3209; M W -- 440.20463

,~

,0 _H

H ....... o .

,'_~

.... 1'

"

"

1 ~

I

I

';1

CN2OAc 15

Fungal Source Fusarium acuminatum misidentified as F. heterosporum. The F. acuminatum was isolated from Claviceps paspali honey dew on Paspalum distichum. Isolation/Purification Fungal cultures were extracted with chloroform; the crude chloroform extract was chromatographed on a column containing silica gel eluted with toluene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further purified on a silica gel column eluted with a linear gradient from ethyl acetate to acetone. The 3'-hydroxy HT-2 toxin-containing fractions were combined, reduced in volume and the toxin was precipitated from solution. Biological Activity The LDs0 value for day-old chicks dosed orally was 8.5mg/kg; the toxin was inhibitory in the wheat coleoptile assay down to 10-5 M; EDs0 values for dermal toxicity on back skin of rabbits was >1.28~g compared to < 0.16~g for T-2 toxin in the same assay. Spectral Data UV~

End absorption. 1H NMR: (CDCI3) 3.28(1H, J=4.6Hz, H-2); 4.02(1H, H-3); 4.02(1H, H-4); 2.09(2H, H-7); 5.18(1H, n-8); 5.65(1H, J=5.EHz, n-10); 4.10(1n, n-11); 2.66(1H, J=4.0Hz, n-13); 2.88(1H, J=4.0Hz, n-13); 0.65(3H, s, CH3-14); 3.84(1H, J=12.0Hz, H-15); 4.10(1H, J=12.0Hz, H-15); 1.67(3H, s, CH3-16); 2.31(2H, s, 2'-CH2); 1.19(3H each, s, 4', 5'CH3); and 1.99ppm (3H, s, CHaCOO-). 13C N M R [

262

14. Trichothecenes and Related Metabolites

78.43, d, C-2; 78.87, d, C-3; 79.45, d, C-4; 47.95, s, C-5, 41.90, s, C-6; 26.40, t, C-7; 67.58, d, C-8; 134.43, s, C-9; 124.19, d, C-10; 66.22, d, C-11; 64.28, s, C-12; 45.44, t, C-13; 6.93, q, C-14; 63.85, t, C-15; 19.81, q, C-16; 169.89, s, C-I'; 47.86, t, C-2'; 68.05, s, C-3'; 29.17, 29.37, C-4', C-5'; 20.83, s, CH3COO-, and 169.89ppm, s, CH3COO-. Mass Spectrum: CIMS: 441(M § + 1), 423,405, 381,363,263,203, and 121m/e. EIMS: 381,203, and 121m/e. Reference R. J. Cole, J. W. Dorner, R. H. Cox, B. M. Cunfer, H. G. Cutler, and B. P. Stuart; The Isolation and Identification of Several Trichothecene Mycotoxins from Fusarium heterosporum; J. Nat. Prod., Vol. 44, pp. 321-330(1981).

14. Trichothecenes and Related Metabolites

263

Common/Systematic Name 3'-Hydroxy T-2 triol 3 tt,4~, 15-Trihydroxy-8tt-[3-hydroxy-3-methylbutyryloxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C2oH3oO8; M W -- 3 9 8 . 1 9 4 0 7

,~

,0 _H

O

Is

'

....

H

sl ~ 1

!

.,l~J I

I

(~H2OH 15

Fungal Source Fusarium acuminatum misidentified as F. heterosporum. The F. acuminatum was isolated from Clavicepspaspali honey dew on Paspalum distichum. Isolation/Purification Fungal cultures were extracted with chloroform; the crude chloroform extract was chromatographed on a column containing silica gel eluted with toluene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further purified on a silica gel column eluted with a linear gradient from ethyl acetate to acetone. The 3'-hydroxy T-2 triol-containing fractions were combined, reduced in volume and final purification was on a C~s reversed-phase column eluted with a linear gradient from 10-50% acetonitrile-water. Biological Activity The toxin was inhibitory in the wheat coleoptile assay down to 10-5 M; EDs0 values for dermal toxicity on back skin of rabbits was >l.281,tg compared to < 0.16~g for T-2 toxin in the same assay: Spectral Data UV:

End absorption.

IH N]VIR: (CDCI3) 3.21(IH, d, J=4.8Hz, H-2); 3.84(II--I,J=4.8, 2.9Hz, H-3), 4.42(IH, J=2.9Hz, H-4); 2.05(2H, H-7); 5.15(IH, J=5.81--Iz,H-8); 5.63(IH, J=5.4Hz, H-10); 4.05(IH, J=5.4Hz, H-11), 2.83(1H, J=4.2Hz, H-13), 2.61, 2.83(2H, J=4.2Hz, H-13), 0.68(3H, s, CH3-14); 4.24(1H, J=12.0Hz, H-15); 3.64(1H, J=12.0Hz, H-15); 1.66(3H, s, CH316); 2.40(2H, s, 2'-CH2); and 1.20ppm (3H, s, 4', 5'-CH3).

13C N M R :

264

14. Trichothecenes and Related Metabolites

78.49, d, C-2; 79.18, d, C-3; 79.18, d, C-4; 48.06, s, C-5; 43.37, s, C-6; 26.66, t, C-7; 68.12, d, C-8; 133.93, s, C-9; 124.76, d, C-IO; 66.45, d, C-11; 64.75, s, C-12; 45.58, t, C-13; 6.92, q, C-14; 61.37, t, C-15; 19.78, q, C-16; 170.11, s, C-I'; 47.81, t, C-2'; 67.91, s, C-3'; 29.08, q, C-4', and 29.64ppm, q, C-5' Reference R. J. Cole, J. W. Domer, R. H. Cox. B. M. Cunfer, H. G. Cutler, and B. P. Stuart; The Isolation and Identification of Several Trichothecene Mycotoxins from Fusarium heterosporum; J. Nat. Prod., Vol. 44, pp. 321-330(1981).

14. Trichothecenes and Related Metabolites

265

Common/Systematic Name Neosolaniol; Solaniol 413,15tt-Diacetoxy-3 t~,8a-dihydroxy- 12,13-epoxytrich9thee-9-ene Molecular Formula/Molecular Weight C19H2608;

MW

~o

I I~

o.

-" 3 8 2 . 1 6 2 7 7

H

H

~I 13 Iic~ ~I ~ I ...." "

i l

"

_--

14

..... ,OH

Ac

CH2OAc 15

General Characteristics Crystals from ethyl acetate-n-hexane; mp., 170-172~ F_ungal Source

Fusarium culmorum, F. solanL F. poae, F. sporotrichioides, F. lateritium, F. equiseti, and F. averaceum.

Biological Activity LDs0 to mice dosed IP was 14.5mg/kg; LDs0 in chicken embryo test was 5.01,tg per egg. IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.25~g/ml; rat liver (cellflee) was 20~g/ml. Clinical signs in animals receiving a fatal dose were marked cellular degeneration and karyorrhexis in actively dividing cells of thymus, lymph nodes, spleen, bone marrow, intestine, and testes reflecting the so-called "radiomimetic" property. Minimum dose for skin-irritant toxicity to rabbits was 1.0~g. Spectral Data 1H NMR: (CDC13) 3.66(1H, d, d-4.9Hz, H=2); 4.14(1H, dd, J-4.9, 2.9Hz, H-3); 5.25(1H, d, J=2.9Hz, H-4); 1.89(1H, dd, J=14.5, 1.4Hz, H-7); 2.33(1H, dd, J-14.5, 5.7Hz, H-7); 4.10(1H, d, d=5.7Hz, H-8); 5.65(1H, d, d-5.9Hz, H-10); 4.25(1H, d, J=5.9Hz, H-11); 2.78(1H, d, J=4.0Hz, H-13); 3.04(1H, d, J=4.0Hz, H-13); 0.83(3H, s, CH3-14); 4.29(1H, d, J=12.6Hz, H-15); 4.21(1H, d, J-12.6Hz, H-15); 1.82(3H, s, CH3-16); and 2.02, 2.13ppm (3H each, s, CH3COO-). 13C NMR: 78.7, C-2; 78.4, C-3; 84.7, C-4; 48.7, C-5; 43.4, C-6; 30.4, C-7; 66.7, C-8; 139.6, C-9; 121.1, C-10; 67.7, C-11; 64.4, C-12; 47.2, C-13; 6.7, C-14; 64.8, C-15; 19.9, C-16; 21.4 and 21.0 (3H each, CH3COO-); 169.8 and 172.0ppm (3H each, CH3COO-).

266

14. Trichothecenes and Related Metabolites

TLC Data Adsorbent: Kieselgel G; solvent: ethyl acetate-n-hexane, 3:1 (v/v); Rf, 0.15; detection: H2SO4 spraying and heating at 110~ for 5 min. GLC Data Solid Support: Shimalite W; liquid phase: OV-17 (1.5%); retention time: 7.50 (relative to nivalenol). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 177(1981). M. Mesilaakso, M. Moilanen, and E. Rahkamaa; 1H and 13CNMR Analysis of Some Trichothecenes; Arch. Environ. Contam. Toxicol., Vol. 18, pp. 365-373(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

267

Common/Systematic Name Isoneosolaniol; Acuminatum; 8a,15-Diacetoxy-T-2 tetraol 8a, 15-Diacetoxy-3 a,413-dihydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608, ~

"- 3 8 2 . 1 6 2 7 7

H

~ O . , J

H

18 1:1

6Ac

i A5

2

3 ,

,,o l'''~ '~H

CH2OAc 15

Fungal Source Fusarium acuminatum, F. tricinctum, F. compactum, and F. equiseti. Isolation/Purification Extraction was performed with MeOH- 1% aqueous NaC1 (55:45, v/v); after filtration, the residue was extracted with the MeOH-NaCI solution. The filtrate was concentrated, defatted with n-hexane and then extracted with methylene chloride. The methylene chloride extract was dried over anhydrous Na2SO4 and concentrated to dryness. The residue was reconstituted with MeOH and chromatographed on a silica gel 60 column eluted sequentially with CH2C12, CH2C12-MeOH (95:5, v/v), CH2C12-MeOH (90:10, v/v), CH2C12-MeOH (80:20, v/v), and MeOH. Eighteen fractions were collected and tested by TLC for trichothecenes. The compounds of major interest that were positive for trichotheeenes accumulated in fractions F6 and F7. Fraction F6 contained acuminatin which was further purified by preparative HPLC using an RP18 column with MeOH-H20 (40:60, v/v) as mobile phase. Biological Activity Highly toxic to brine shrimp larvae (Artemia salina) and inhibited the growth of tomato seedlings. Spectral Data 1H NMR: (CDC13) 3.64(1H, d, J=4.9Hz, H-2); 4.17(1H, dd, J=4.9, 3.0Hz, H-3); 4.94(1H, d, J=3.0Hz, H-4); 2.06(1H, dr, J=14.3, 1.6Hz, all-7); 2.19(1H, dd, J=14.3, 5.2Hz, 13H7); 4.12(1H, m, H-8); 5.65(1H, dq, J=5.8, 1.5Hz, H-10); 3.88(1H, d,J=5.8Hz, H-11); 2.79(1H, d, J=4.0Hz, H-13); 3.02(1H, d, J=4.0Hz, H-13); 0.89(3H, s, CH3-14); 3.48(1H, d, J-12.6Hz, H-15); 3.76(1H, d,J-12.6Hz, H-15); 1.85(3H, br s, CH3-16); and 2.13 ppm (3H, s, CH3COO-).

268

14. Trichothecenes and Related Metabolites

13CNMR: 78.7, C-2; 78.3, C-3; 84.9, C-4; 49.1, C-5; 44.9, C-6; 27.9, C-7; 66.8, C-8; 139.0, C-9; 122.0, C-10; 68.5, C-11; 64.4, C-12; 47.0, C-13; 7.0, C-14; 62.2, C-15; 20.59, C-16; 21.0, CH3COO-; and 172.8ppm CH3COO-. References K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology; Vol. 42, pp. 541-543(1981 ). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico, and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Triehothecene; J. Agrie. Food Chem., Vol. 37, pp. 1348-1351(1989).

14.

Trichothecenes and Related Metabolites

269

Common/Systematic Name 8-Acetoxyneosolaniol 413,8a, 15-Triacetoxy-3 tz-hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C21H2sO9; M W = 4 2 4 . 1 7 3 3 3

~o H H ~ 6 ~ 0 . . J 2

I-

"1 -

:

13

I

i

1.

3 ,r~

.... ,OH

AC

CH2OAc 15

Fungal Source

Fusarium acuminatum, F. tricinctum, F. compactum, and F. roseum.

Isolation/Purification Extraction was performed with MeOH- 1% aqueous NaC1 (55:45); after filtration, the residue was extracted with the MeOH-NaC1 solution. The filtrate was concentrated, defatted with n-hexane, and extracted with methylene chloride. The methylene chloride extract was dried over anhydrous NazSO4 and concentrated to dryness. The residue was reconstituted with MeOH and chromatographed on a silica gel 60 column eluted sequentially with CHuC12, CH2CIz-MeOH (95:5, v/v), CH/Clz-MeOH (90:10, v/v), CH2C12-MeOH (80:20, v/v), and MeOH. Eighteen fractions were collected and tested by TLC for trichothecenes. The compounds of major interest that were positive for trichothecenes accumulated in fractions F6 and F7. Fraction F6 contained 8acetoxyneosolaniol which was further purified by preparative HPLC using an RPI8 column with MeOH-H20 (40:60, v/v) as mobile phase. Biological Activity Highly toxic to brine shrimp larvae (Artemia salina) and inhibited the growth of tomato seedlings. LDs0 in day-old cockerels dosed via crop intubation was 0.789mg/kg; LD50 of T-2 toxin (dosed simultaneously) was 1.84mg/kg. Also, showed inhibition in wheat coleoptile assay down to 106 M. It showed a linear, rather than a curvilinear, response. Spectral Data 1H NMR: (CDC13) 3.67(1H, d, J=4.9Hz, H-2); 4.12(1H, dd, J=4.9, 2.9Hz, H-3); 5.21(1H, d, J=2.9Hz, H-4); 1.97(1H, d, J=15.1Hz, tzH-7); 2.35(1H, dd, J=15.1, 5.7Hz, [3H-7); 5.24(1H, d, J=5.7Hz, H-8); 5.79(1H, d, J=5.9Hz, H-10); 4.28(1H, d, J=5.9Hz, H-11); 2.77(1H, d, J=4.0Hz, H-13); 3.03(1H, d, J=4.0Hz, H-13); 0.80(3H, s, CH3-14);

270

14. Trichothecenes and Related Metabolites

4.05(1H, d, J=12.5Hz, H-15); 4.26(1H, d, J=12.5Hz, H-15); 1.73(3H, s, CH3-16); and 2.00, 2.01, 2.12ppm (3H, s, CH3COO-). ~3CNMR: 78.7, C-2; 78.4, C-3; 84.6, C-4; 48.6, C-5; 43.0, C-6; 27.4, C-7; 68.5, C-8; 136.2, C-9; 123.8, C-10; 67.4, C-11; 64.3, C-12; 47.2, C-13; 7.0, C-14; 64.4, C-15; 20.3, C-16; 21.0, 21.1, 21.1(3H each, CH3COO-); and 172.6, 170.5, 170.0ppm (3H each, CH3CO0-). Mass Data: Positive CIMS (TFA derivative), 521(8%), 461 (10), 401 (100), and 341m/e (10). TLC Data A. Benzene-acetone, 12:7 (v/v); Re, 0.6. B: Chloroform-methanol, 9:1 (v/v); Rf, 0.85. C: Toluene-ethyl acetate-formic acid, 6:3:1 (v/v/v); Re, 0.28. GLC Data (TFA derivative) DB5 capillary column, with covalently bonded liquid phase; injector temperature, 275~ detector temperature, 300~ and temperature programming from 150-280~ at 10~ followed by 5 min. at 280~ retention time, 10.51 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 178(1981). K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from

Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology, Vol.

42, pp. 541-543(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Trichothecene; J. Agile. Food Chem., Vol. 37, pp. 1348-1351(1989).

14.

Trichothecenes

and Related Metabolites

271

Common/Systematic Name 8-Propionylneosolaniol 8 t~-Propanyl-3 et-hydroxy-413,15-diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C22H3009; M W -- 4 3 8 . 1 8 8 9 8

~0 H H ",,,,,,,,~",,,,~/.-- O,,,~2

16

H 18

I-. 1ol

~ O II -

MeCH2CO

'

~

:

~5

_ 14

3......OH

,,o / 4

c

r189 15

General Characteristics Crystals; mp., 182-183~ Fungal Source

Fusarium sporotrichioides (MC-72083 and DAOM 165006).

Isolation/Purification Fungal material was extracted with ethyl ether, chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Further purification was achieved by preparative HPLC using various isocratic solvent systems of 2-propanol-hexane. Fraction three was further purified by preparative HPLC using 5% isopropanol-hexane followed by 3% isopropanol-hexane to yield crystalline 8-propionylneosolaniol in pure form. Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukoeytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on

272

14. Trichothecenes and Related Metabolites

polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans. Spectral Data 1H NMR: (CDC13) 3.68(J2,3=4.9Hz, H-2); 4.12(J3,2=4.9, J3,4=2.8Hz, H-3); 5.27(J4,3=2.8Hz, H4); 1.93(JAB=15.1Hz, H-70; 2.38(JT,s=5.8Hz, H-7); 5.27(Js,7=5.SHz, H-8); 5.80(J10,11-6.0nz, J10,16-l.2Hz, H-10); 4.33(J11,10=6.0Hz, H-11); 2.78(JAB=3.9Hz, H-13); 3.04(H13); 0.79(H-14); 4.03(JAB=12.6Hz, H-15); 4.27(H-15); 1.72(J16,10=l.2Hz, H-16); 2.28(2H, q, J2,,3.=7.5Hz, H-2'); 1.13(1H, t, Jy,2,=7.5Hz, H-3'); 2.01; and 2.13ppm (CH3Ac). 13C NMR: 78.7, C-2; 78.2, C-3; 84.3, C-4; 48.3, C-5; 42.9, C-6; 27.7, C-7; 68.0, C-8; 136.3, C-9; 123.8, C-10; 67.3, C-11; 64.2, C-12; 47.0, C-13; 6.6, C-14; 64.5, C-15; 20.1, C-16; 174.1, C-17; 27.6, C-18; 8.9, C-19; 20.8, 20.1(3H, CH3COO-); and 170.2, 170.5ppm (3H each, CH3COO-).

Mass Spectrum: EIMS: (lacked a significant M +) 185(35%), 180(61), 121(89), 105(48), and 57role (100). GLC Data DB-5 fused capillary column (20m x 0.32mm i.d., 0.25l.tm film); temperature programmed from 140-260~ at 15~ using helium as carrier gas. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agric. Food Chem., Vol. 36, pp. 216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; Plant Research Centre, Agriculture Canada, Ottawa, Ontario, K1A 0C6.

14. Trichothecenesand Related Metabolites

273

Common/Systematic Name 8-Butyrylneosolaniol 8 t~-Butyryloxy-3 t~-hydroxy-413,15-diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C23H3209, ~

= 452.20463

~o H

H

16~O-,~2

] ....,OH

o 11=113" I,,O H-~;O......k~'v>~'~:l'2 M CH C i i '\OAc -

14

(3�89 15

General Characteristics Isolated as an oil. Fungal Source

Fusarium sporotrichioides (MC-72083 and DAOM 165006).

Isolation/Purification Fungal material was extracted with ethyl ether and chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Further purification was achieved by preparative HPLC using various isocratic solvent systems of 2-propanol-hexane. Fraction three was further purified by preparative HPLC using 5% isopropanol-hexane followed by 3% isopropanol-hexane. Pure 8-butyrylneosolaniol was obtained as an oil. Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free

274

14. Trichothecenes and Related Metabolites

systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans. Spectral Data 1H NMR: (CDC13) 3.68(,J2,3=4.9Hz,H-2); 4.13(J3,2=4.9Hz, J3,4=2.9Hz, H-3); 5.27(.A,3=2.9Hz, H-4); 1.91(J~=15.9Hz, H-7); 2.39(JT,S=5.3Hz, H-7); 5.27(,Js,7=5.5Hz, H-8); 5.79(Jlo,1~= 4.9Hz, H-10); 4.30(,J~1,lo=4.9Hz, H-11); 2.78(J,~=3.9Hz, H-13); 3.04(H13); 0.79(H-14); 4.03(,L~=12.5Hz, H-15); 4.27(H-15); 1.72(J~6,1o=l.2Hz, H-16); 2.33(2H, q, Jz,r=7.4Hz, H-2'); 1.64(Jz,3,=,J3,,a,=7.4Hz, H-3'); 0.93(J4,,3~-7.4Hz, H-4'); 2.01; and 2.13ppm (CH3-Ac). Mass Spectrum: EIMS: (lacked a significant M+) 185(26%), 180(45), 121(100), 105(48), and 71m/e

(80).

GLC Data DB-5 fused capillary column (20m x 0.32mm i.d., 0.251.tm film); temperature programmed from 140-260~ at 15~ using helium as cartier gas. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agric. Food Chem., Vol. 36, pp. 216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

275

Common/Systematic Name 8-Isobutyrylneosolaniol 3 t~-Hydroxy-413, 15-diacetoxy-8 tt-[(2-methylpropionyl)oxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C23H3209; MW

16

Me\

-- 4 5 2 . 2 0 4 6 3

~o H -~?',~2 _[8

CHCOO~'"'~

Me /

H

3.... ,OH

61 '" ~1., ;''0 4L~ 12

i ~5 7. = 14

OAc

6�89 15

Fungal Source

Fusarium sporotrichioides (MC-72083 and DAOM 165006).

Isolation/Purification Fungal material was extracted with ethyl ether, chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Further purification was achieved by preparative HPLC using various isocratic solvent systems of 2-propanol-hexane. Fraction three was further purified by preparative HPLC using 5% isopropanol-hexane followed by 3% isopropanol-hexane. Pure 8-isobutyrylneosolaniol was obtained atter double development preparative TLC using hexane-acetone-formic acid (65:33:2, v/v/v; Rf 0.42). Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukoeytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans.

276

14. Trichothecenes and Related Metabolites

_Spectral Data 1H NMR: (CDCI3) 3.69(J2,3=4.9Hz, H-2); 4.14(J3,2=4.9Hz, J3,4=2.9Hz, H-3); 5.35(J4,3=2.9Hz, H-4); 1.83(JAB=15.0Hz, H-7); 2.40(H-7); 5.26(Js,7=5.6Hz, H-8); 5.80(Jx0,~=5.4Hz, H10); 4.38(Jlx,10=5.4Hz, H-11); 2.78(JAB=3.9Hz, H-13); 3.05(H-13); 0.79(H-14); 4.02(JAB=lZ.6Hz, H-15); 4.3 l(H-15); 1.73(J16,~0=l.2Hz, H-16); 2.45(2H, q, J2,,3~J2,,4~=7.4Hz, H-2'); 1.16(J2,,3~7.0Hz, H-3'); 1.16(J2,,4'=7.0Hz, H-4'); 2.01; and 2.13ppm (CH3-Ac). Mass Spectrum: EIMS: (lacked a significant M +) 195(38%), 191(62), 180(60), 121(100), 105(57), and 71m/e (98). GLC Data DB-5 fused capillary column (20m x 0.32mm i.d., 0.2511m film); temperature programmed from 140-260~ at 15~ using helium as carrier gas. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agrie. Food Chem., Vol. 36, pp. 216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

277

Common/Systematic Name 15-Deacetylneosolaniol; NT-2 Toxin; 4-Acetyl-T-2 tetraol 4~-Acetoxy-3a,8a, 15-trihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2407; MW

=

~o H I-

H

"1 13

-

340.15220

~ .c~ l .... ,OH

14

AC

(~H2OH 15

General Characteristics Needles from ethyl acetate-n-hexane; mp., 172-173~ FunRal Source Fusarium sporotrichioides (strain M-1-1), F. tricinctum, F. compactum, and F. averaceum. Isolation/Purification Extracted by charcoal absorption and elution with methanol. Extraction of methanolchloroform soluble materials produced a pale yellow powder. The powder was chromatographed on a silica gel column eluted with n-hexane, n-hexane-acetone (12: 5, 2:1, 1:1, 1:2, v/v), acetone and methanol followed by a second silica gel column eluted with benzene-acetone (3:2, v/v), and n-hexane-acetone (1:1, v/v). NT-2 was crystallized with ethyl acetate-n-hexane. Biological Activity Inhibited uptake of [14C]leucine in protein synthesis with rabbit reticulocytes; IDs0 at concentrations of 0.23 and 0.251,tg/ml by NT-1 and NT-2, respectively. These activities were similar to that of neosolaniol (0.25tzg/ml) in the same assay. Spectral Data IR:

(KBr) 3380, 2960, 1720, 1370, 1240, and 1025cm"1. ]H NMR: (CDCI3) 3.64(1H, d, J=4.9Hz, H-2); 4.17(1H, dd, J=4.9, 3.0Hz, H-3); 4.94(1H, d, J=3.0Hz, H-4); 2.06(1H, dt, J=14.3, 1.6Hz, all-7); 2.19(1H, dd, J=14.3, 5.2Hz, [~H7); 4.12(1H, m, H-8); 5.65(1H, dq, J=5.8, 1.5Hz, H-10); 3.88(1H, d, J=5.8Hz, H-11);

278

14. Trichothecenes and Related Metabolites

2.79(1H, d, J=4.0Hz, H-13); 3.02(1H, d, J=4.0Hz, H-13); 0.89(3H,s, CH3-14); 3.48(1H, d,J=12.6Hz, H-15); 3.76(1H, d, J=12.6Hz, H-15); 1.85(3H, br s, CH3-16); and 2.13 ppm (3H, s, CH3COO-). 13C NMR: 78.7, C-2; 78.3, C-3; 84.9, C-4; 49.1, C-5; 44.9, C-6; 27.9, C-7; 66.8, C-8; 139.0, C-9; 122.0, C-10; 68.5, C-11; 64.4, C-12; 47.0, C-13; 7.0, C-14; 62.2, C-15; 20.59, C-16; 21.0, CH3COO-; and 172.8ppm, 3H, CH3COO-. Mass Data: LREIMS: 340m/e (M+); found: C, 60.04; H, 7.10; O, 32.86%; calcd for C17H2407;C, 59.97; H, 7.11; O, 32.92%. References K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology, Vol. 42, pp. 541-543(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Trichothecene; J. Agric. Food Chem., Vol. 37, pp. 1348-1351(1989).

14. Trichothecenes and Related Metabolites

279

Common/Systematic Name 4-Deacetylneosolaniol 3a,4~,8a-Trihydroxy- 15-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2407; M W = 3 4 0 . 1 5 2 2 0

H H oJ2 ~r,~,,O

"

,o

] "OH ......

-" ~' OH (~H2OAc 15

Fungal Source Fusarium acuminatum misidentified as F. heterosporum. The F. acuminatum was isolated from Claviceps paspali honey dew on Paspalum distichum. Isolation/Purification Fungal cultures were extracted with chloroform; the crude chloroform extract was chromatographed on a column containing silica gel eluted with toluene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further purified on a silica gel column eluted with a linear gradient from ethyl acetate to acetone. The 15-acetoxy T-2 triol-containing fractions were combined, reduced in volume and final purification was on a Cls reversed-phase column eluted with a linear gradient from 50% acetonitrile to water. Biological Activity The toxin was inhibitory in the wheat coleoptile assay down to 10"3 M; EDs0 values for dermal toxicity on back skin of rabbits was > 1.28~g compared to < 0.161,tg for T-2 toxin in the same assay. The LDs0 value for day-old chicks dosed orally was > 10mg/kg. Spectral Data UV:

End absorption. :H NMR: (CDCI3) 3.24(1H, J=4.9Hz, n-2); 3.89(1H, H-3); 4.06(1H, H-4); 2.06(2H, n-7); 5.19(1H, H-8); 5.40(1H, H-10), 4.03(1H, H-I 1); 2.84(1H, J=4.1Hz, H-13); 2.61(1H, J=4.1Hz, H-13); 0.67(3H, s, CHa-14); 3.89, 4.03(2H, H-15); 1.72(3H, s, CH3-16); and 1.97ppm (3H, s, CHaCOO).

~3C NMR:

280

14. Trichothecenes and Related Metabolites

(CDC13) 78.48, d, C-2; 79.01, d, C-3; 79.49, d, C-4; 47.86, s, C-5; 42.57, s, C-6; 30.09, t, C-7; 64.82, d, C-8; 138.93, s, C-9; 120.38, d, C-10; 66.65, d, C-11; 64.61, s, C-12; 45.58, t, C-13; 6.78, q, C-14; 64.61, t, C-15; 20.34, q, C-16; 20.82, q, CH3COO) and 169,60ppm, s, CH3COO. Mass Spectrum: EIMS: 340(M+), 322, 203, 175, and 121m/e (base peak). Reference R. J. Cole, J. W. Domer, R. H. Cox, B. M. Cunfer, H. G. Cutler, and B. P. Stuart; The Isolation and Identification of Several Trichothecene Mycotoxins from Fusarium heterosporum; J. Nat. Prod., Vol. 44, pp. 321-330(1981).

14. Trichothecenes and Related Metabolites

281

Common/Systematic Name Sporotrichiol Molecular Formula/Molecular Weight C20H3006;MW = 366.20424

~o H 16

-

O

2

.,,,,," ~ ~ ' 2

Me2CHCH2COO

!

-_.

~5

,,OH 'O

14

(~H2OH 15

General Characteristics Isolated as an oil. Fungal Source Fusarium sporotrichioides (MC-72083). Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil column chromatography eluted with benzene-hexane (2:1, v/v), methylene chloride, and chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil was further purified by normal phase preparative HPLC using benzene-acetone followed by preparative RPTLC to yield several trichothecenes including sporol and sporotrichiol. Spectral Data IR: (thin film) 3422(OH) and 1726cm1 (ester). 1HN:V[R: (CDCI3) 3.49(1H, d, H-2), 4.49(1H, m, H-3); 2.05-2.25(2H, m, H-4); 1.96(1H, d, n-7), 2.30(1H, dd, n-7), 5.50(1H, d, n-8), 5.79(1H, d, n-10), 4.17(1H~ d, n-11), 2.57(1H, d, J=4.1Hz, H-13); 3.06(1H, d, d=4.1Hz, H-13), 0.87(3H, s, CH3-14); 3.53(1H, d, J=12.7Hz, n-15), 3.70(1H, d, J=12.7Hz, H-15), 1.75(3H, s, CH3-16); 2.21(2H, m, H-18); 2.05-2.30(1H, m, H-19); and 0.95ppm (3H each, d, J=7.0Hz, CH3-20 and -21).

13CNMR: (CDCI3) 79.6 C-2; 69.1, C-3, 42.1, C-4, 45.8, C-5, 43.3, C-6; 26.8, C-7, 68.4, C-8, 135.7, C-9; 125.4, C-10, 68.1, C-11, 65.1, C-12; 48.5, C-13; 12.44, C-14; 63.1, C-15; 20.4, C-16; 171.9, C-17; 43.7, C-18, 25.7, C-19; and 22.3ppm, C-20 and C-21.

282

14. Trichothecenes and Related Metabolites

Mass Spectrum: HREIMS: 366.204m/e; calcd for C20H3006,366.206. References D. G. Corley, G. E. Rottinghaus, and M. S. Tempesta; Novel Trichothecenes from Fusarium sporotrichioides; Tetrahedron Letters, Vol. 27, pp. 427-430(1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

283

Common/Systematic Name 413,8a, 15-Triacetoxy-3 t~,7a-dihydroxy-12,13-epoxytriehothec-9-ene Molecular Formula/Molecular Weight C21H28Olo;

~6.

~

M 3 v V -- 4 4 0 . 1 6 8 2 5

~o H

o

.

H

J

-

2

5

Ac0 ~)H 141 C;H2OAc

3

OAc

15

General Characteristics Crystals; mp., 185-188~ Fungal Source Fusarium equiseti. Biological Activity LDs0 in rats dosed IP was 1.2mg/kg. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 200(1981).

284

14. Trichothecenes and Related Metabolites

Common/Systematic Name 3 a-Acetoxy- 15-hydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2206, M W = 322.14164

,,,OAc

.... .

;

o I" 4

14

~H20H 15

General Characteristics Colorless needles; mp., 202-203~

[a]D 26 q- 41.7 ~ (c=0.40,

in CHCI3).

Fungal Source Fusarium roseum

(ATCC 28114).

Isolation/Purification Fusarium roseum Greenhalgh et al.

cultures were harvested and the broth was extracted as described by (1984). Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A after removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). 3 a-Acetoxy- 15-hydroxy- 12,13-epoxytrichothec-9-en-8-one was isolated from mixture A2 by elution with 20% ethyl acetate in hexane followed by rechromatography using the same solvent.

Spectral Data 1H NMR: (CDC13) 0.83(3H, H-14); 1.80(3H, H-16,J16,~o=l.4Hz); 2.12(3H, AC-CH3); 2.40, 2.17(2H, H-4, Jab=12.8Hz, J4,3=4.4Hz); 2.86, 2.42(2H, H-7, J~=16.8Hz); 2.86,

14. Trichothecenes and Related Metabolites

285

3.09(2H, H-13, Jab=3.9Hz); 3.64(2H, H-15); 3.81(1H, H-2, Jz,3=4.6Hz); 4.52(1H, H11, J11,10=5.7Hz), 5.18(1H, H-3, J3,z=4.6Hz, J3,4=lO.2, 4.4Hz); and 6.55 ppm (1H, HI0, J10,11=5.7Hz, J10,a6=l.4Hz). 13C NMR: (CDCI3) 78.1, C-2; 71.2, C-3; 38.5, C-4; 45.0, C-5; 47.5, C-6; 38.3, C-7; 198.7, C-8; 138.3, C-9; 137.7, C-10; 68.2, C-11; 65.3, C-12; 48.3, C-13; 11.2, C-14; 64.1, C-15; 15.3, C- 16; 20.9, CH3COO-; and 170.5ppm, C=O. Mass Spectrum: HREIMS: 323.149m/e (M ++ 1); calcd for C17H2306,323.150. References R. Greenhalgh, R.M. Meier, B. A. Blaekwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agile. Food Chem., Vol. 32, pp. 1261-1264(1984). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Triehothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agile. Food Chem., Vol. 35, pp. 884-889(1987).

286

14. Trichothecenes and Related Metabolites

Common/Systematic Name 3-Acetyl-4-deoxynivalenol 3 a-Acetoxy-Ta, 15-dihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2207; ~

= 338.13655

1 6 ~ 02q 1H 0

HO

H

"" " c 3.,",uP,

: 14 ~H2OH

15

General Characteristics Colorless needles from ethyl ether-n-pentane; mp.,185.5-1860C; [a]D 20 + 43.0* (c=1.3, in EtOH); [a]D25 + 40.5~ tetracetate; mp., 168-1700C. Isolation/Purification Fungal mycelium was extracted with methylene chloride, concentrated and the residue dissolved in methanol. The methanol phase was extracted with petroleum ether and evaporated to dryness. The residue was chromatographed on a silica gel column eluted with chloroform-ethanol (50:1, v/v) and then chloroform-ethanol (400:1, v/v). 3-Acetyl-4deoxynivalenol was recrystallized from hot ethyl ether. Fungal Source Fusarium culmorum (HLX 1503 ATCC 28114), F. graminearum (HLX 1506), and F. roseum. Biological Activity LDs0 in male ddS strain of mice dosed IP was 76.7mg/kg; females, 49.9mg/kg; ducklings, 37mg/kg. Minimum SC dosage to induce vomiting in ducklings was 10.0 mg/kg; dogs, S.C., 0.2mg/kg. Inhibited Tetrahymena pyriformis, 29.0~g/ml. Spectral Data UV:

~, E~. max

219nm (6=5,900).

IR:

(KBr) 3480, 3400, 1720, and 1680cml.

14. Trichothecenes and Related Metabolites

287

Spectral Data UV:

~,.=~~ 220nm (c=7,100). IR:

(Nujol) (needles) 3500, 3420, 1740, 1683, and 1668cm1; (needles or plates) 3480 br, 1750, 1690, 1672, and 1660cm"1. 1H NMR: (CDC13) 6.10(1H, d, J=4.5Hz, H-2); 4.72(1H, m, H-3); ca. 7.75(2H, m, H-4); 5.12(1H, d, J=l.5Hz, H-7); 3.36(1H, dd, J=5.5, 1.5Hz, H-10); 5.29(1H, d, J=5.5Hz, H-11); 6.84(2H, s, H-13); 8.81(1H, s, H-14); 6.14(2H, d, J=l.5Hz, H-15); 8.12(3H, s, H-16); 7.90(3H, OAt); and 6.15, 8.0ppm (1H each, OH). Mass Data: HREIMS: 338.1360, C17H2207requires 338.1365; found: C, 60.4; H, 7.0%; C17H2207 requires C, 60.35; H, 6.55%. Reference M. M. Blight and J. F. Grove; New Metabolic Products of Fusarium culmorum: Toxic Trichothec-9-en-8-ones and 2-Acetylquinazolin-4(3H)-one; J. C. S. Perkin I, pp. 16911693(1974).

288

14. Trichothecenes and Related Metabolites

Common/Systematic Name Trichodermol 4 I]-Hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2203; MW = 250.15689 ~o H

~~

~.;-

0 ~H

~

,~,,'-" 15

14

H

General Characteristics Needle crystals from light petroleum; mp., 116-119~ crystals from ether-n-hexane; mp., 117.5~176 [a] D2~ - 33.5~ in CHC13). Fungal Source Myrothecium roridum, Trichoderma polysporum, T. sporulosum, and Hypocrea austrograndis. Spectral Data UV~

End absorption. 1H NMR: (CDC13) 3.51, H-2; 1.7-2.5, H-3; 4.3, H-4; 1.7-2.5, H-7; 1.7-2.5, H-8; 5.41, H-10; 3.81, H-11; 2.95, H-12; 0.80, H-14; 0.85, H-15; and 1.70ppm, H-16. 13C NMR: (CDCh) 78.8, C-2; 40.2, C-3; 74.0, C-4; 49.2, C-5; 39.8, C-6; 24.5, C-7; 28.0, C-8; 140.1, C-9; 118.8, C-10; 70.4, C-11; 65.8, C-12; 47.6, C-13; 6.2, C-14; 15.8, C-15; and 23.2ppm, C- 16. Mass Data: HREIMS: 250.1569(C~5H2203),235.1335(C14H~903), 207.1384(C~3H~902), and 188.1200m/e (C13H160). TLC Data A. Adsorbent: silica gel G. Solvent: chloroform-methanol, 98:2, v/v; Re, 0.36; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina. Solvent: chloroform-methanol, 98:2, v/v; Rf, 0.86; detection: H2SO4 spraying and heating for 5 minutes.

14. Trichothecenes and Related Metabolites

289

References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993). J. R. Hanson, T. Marten, and M. Sivems; Studies in Terpenoid Biosynthesis. Part XII. Carbon-13 Nuclear Magnetic Resonance Spectra of the Trichothecenes and the Biosynthesis of Trichothecolone from [2-13C]Mevalonic Acid; J. Chem. Soc. Perkin I, pp. 1033-1036(1974).

290

14. Trichothecenes and Related Metabolites

Common/Systematic Name 7 t~-Hydroxytrichodermol 413,7a-Dihydroxy- 12,13-epoxytrichothec-9-ene Molecular formulamolecular Weight C15H2204; MW = 266.15181 lo

H

H

~I~

~

H

.......H

~1 '~ ~-;~.,,,,v ~

-IA ~e ~

H~)

~

"H

'H

15

General Characteristics Amorphous colorless solidi [a]D = +2.4* (C=0.76, in MeOH). Plant Source Myrothecium roridum.

Isolation/Purification An aqueous shake culture ofMyrothecium roridum (M4582) as filtered through Whatman # 1 filter paper, and the filtrate was extracted three times with ethyl acetate. The mycelia remaining from filtration were soaked in methanol overnight and filtered. The methanol filtrate was concentrated in vacuo until only an aqueous phase remained which was extracted three times with ethyl acetate. The ethyl acetate extracts were pooled and concentrated m vacuo to yield 4g of gum. The gum was subjected to filtration chromatography (50g of silica gel) with increasing amounts of ethyl acetate in hexane to yield five fractions: 1 (800mg, eluted with hexane), 2 (200mg, eluted with 10% ethyl acetate in hexane), 3 (400mg, eluted with 30% ethyl acetate in hexane), 4 (500mg, eluted with 50 to 70% ethyl acetate in hexane), and 5 (1.5g, eluted with ethyl acetate). Fractions 3 and 4 were subjected to purification on a Chromatotron (2-mm silica gel plates, ethyl acetate-hexane) to yield, after recrystallization (ethyl acetate-hexane), 25mg of trichodermol and 50 mg of 7a-hydroxytrichodermol. 7a-Hydroxytrichodermol also was isolated from rice culture: ca. 200mg/kg of flee. Spectral Data ~H NMR: (CDC13) 0.86(3H, s, H-14); 1.09(3H, s, H-15); 1.69(3H, s, H-16); 1.8 to 2.1(2H, m, H-3~ and H-8~); 2.24(1H, dd, J= 12.6 and 6.0Hz, H-8a); 3.06 and 3.1 0(1H each, AB, J=4.3 Hz, H-13); 3.61(1H, d, J-5.5Hz, H-11); 3.82(1H, d, J-5.0Hz, H-2); 4.26(1H, dd, J=7.6 and 4.0Hz, H-4); 4.46(1H, dd, J=12.0 and 6.0Hz, H-7); and 5.36ppm (1H, br d, J=5Hz, H- 10).

14. Trichothecenes and Related Metabolites

Mass Spectrum:

HREIMS: 266.1526m/e found for C15H2204;calcd 266.1512. Reference B. B. Jarvis, Y.-W. Lee, C. S. Yatawara, D. B. Mazzocchi, J. L. Flippen-Anderson, R. Gilardi, and C. George; 7a-Hydroxytrichodermol, A New Trichothecene from Myrothecium roridum; Appl. Environ. Microbiol., Vol. 50, pp. 225-228(1985).

291

292

14. Trichothecenes and Related Metabolites

Common/Systematic Name Verrucarol 413,15-Dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2204; MW - - 266.15181 10

[8

H -

H

61,3.,~

....

iu

. 14

OH20H 15

General Characteristics Crystals from ether-methylene chloride; mp., 158-159~ fine needles from acetone-ether; mp., 155-156~ [a]D22 -39 ~ (C= 1.069, in CHC13). Di-O-acetylverrucarol, colorless needles from acetone-ether-petroleum ether; mp., 148-150~ [a]D25 -17 ~ (C=1.22, in CHC13). Di-O-benzoylverrucarol, crystals from ether-petroleum ether; mp., 151-152~ [a]D24 - 64 ~ (c=l. 130, in acetone). Dihydroverrucarol, crystals from acetone-etherpetroleum ether; mp., 149-151~ [a]D25 - 6 ~ (C=I.101, in CHCI3). Fungal Source Formed as one of the products of alkaline hydrolysis of verrucarin A which is produced by Myrothecium vemwaria. Spectral Data UV~ EtOH

~. max 195nm (e=7,900). IR;

(CH2C12) 3610, 3570, 1675, 1380, 1335, 1079, 1045, 965, and 820cm"1. 1H NMR: 3.70, H-2; 1.7-2.5, H-3; 4.7, H-4; 1.7-2.5, H-7; 1.7-2.5, H-8; 5.45, H-10; 3.80, H-11; 2.95, H-12; 0.92, H-14; 3.70, H-15; and 1.72ppm, H-16. 13C NMR: 78.5, C-2; 39.5, C-3; 74.0, C-4; 48.7, C-5; 43.7, C-6; 20.9, C-7; 28.0, C-8; 140.4, C-9; 118.7, C-10; 66.4, C-11; 65.6, C-12; 47.4, C-13; 6.8, C-14; 62.1, C-15; and 22.9ppm, C-16.

14. Trichothecenes and Related Metabolites

293

TLC Data A. Adsorbent: silica gel G. Solvent: chloroform-methanol (98:2, v/v); Rf, 0.06; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina. Solvent: chloroform-methanol (98:2, v/v); Rf, 0.34; detection: H2SO4 spraying and heating at 110~ for 5 minutes. References J. Gutzwiller and C. Tamm; Uber die Verrucarine und Roridine. Struktur von Verrucarol; Helv. Chim. Acta, Vol. 46, pp. 1786-1790(1963). J. Gutzwiller and C. Tamm; Uber die Struktur von Verrucarin A, Helv. Claim. Acta, Vol. 48, pp. 157-176(1965).

294

14. Trichothecenes and Related Metabolites

Common/Systematic Name 4,15-Diacetylverrucarol 4[3,15-Diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2606; M W -- 350.17294 H 61

-

H

,~.,,,

,

14

CH2OAc

AC

15

General Characteristics White prisms from benzene-methanol; mp., 147-148~ [0~]D 25 - 14~ (c=l.01, in MeOH). Readily soluble in most organic solvents such as methanol, acetone, chloroform, ether, and benzene; sparingly soluble in hexane and petroleum ether. Fungal Source

Myrothecium spp.

Biological Activity Antifungal activity against Trichophytonasteroides (EDso, O.1ktg/ml) and T. interdigitale (EDs0, 10~g/ml). Spectral Data 1H NMR: (CDCI3) 3.80(H-2); NR(H-3); 3.24(H-4); NR(H-7); NR(H-8); 5.39(H-10); 3.75(H-11); 2.79(H-13e0; 3.09(H-1313); 0.79(H-14); 4.05(H-15a); 4.15(H-1513); and 1.70ppm (H- 16). 13C NMR: (CDC13) 79.0, C-2; 36.7, C-3; 75.4, C-4; 48.9, C-5; 43.4, C-6; 21.4, C-7; 28.1, C-8; 138.7, C-9; 119.7, C-10; 66.8, C-11; 65.2, C-12; 47.3, C-13; 6.8, C-14; 63.7, C-15; 23.0, C-16; 20.2; 2 each CH3CO; and 169.6, 169ppm CH3CO. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 189 (1981). M. Okuchi, M. Itoh, Y. Kaneko, and S. Doi; A New Antifungal Substance Produced by

Myrothecium; J. Agric. Biol. Chem. Jpn., Vol. 32, pp. 394(1968).

14. Trichothecenes and Related Metabolites

295

Common/Systematic Name Crotocin; Antibiotic T Molecular Formula/Molecular Weight C19H2405; M W "- 3 3 2 . 1 6 2 3 7

~ 0

~,D ;s

CCH=:CHMe

General Characteristics Colorless prisms from methanol; mp., 126-128~ 118~ [a]D25 + 7.17 ~ (C=3.98, in CHC13).

needles from ether-benzene; mp., 116-

Fungal Source

Cephalosporium crotocmigenum and Trichothecium roseum.

Biological Activity LDs0 of crotocin in mice dosed IV was 700mg/kg; fungistatic; negligible antitumor activity. Spectral Data UV:

Xmx 211(e=21,800) and 209nm. IR:

1710 and 1640cm~. 13C NMR: (CDCI3) 74.3, C-2; 36.2, C-3; 78.5, C-4; 47.7, C-5; 41.3, C-6; 58.1, C-7; 50.4, C-8; 137.2, C-9; 122.9, C-10; 69.4, C-11; 65.9, C-12; 46.8, C-13; 6.2, C-14; 15.9, C-15; 21.0, C-16; 166.4, C-17; 120.2, C-18; 146.5, C-19; and 14.8ppm, C-20. TLC Data Adsorbent, silica gel; solvent, ethanol-ethyl acetate-acetone, 1:4:4, v/v/v; Re, 0.67; detection: 50% H2SO4 spraying and heating at 110~ for 5 minutes. References B. A. Achilladelis and J. R. Hanson; Minor Terpenoids of Trichothecium roseum; Phytochemistry, Vol. 8, p. 765 (1969).

296

14. Trichothecenes and Related Metabolites

R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 195 (1981).

J. Gyimesi and A. Melera, On the Structure of Crotocin, an Antifungal Antibiotic, Tetrahedron Letters, p. 1665 (1967).

14. Trichothecenes and Related Metabolites

297

_Common/Systematic Name Crotocol 4 [3-Hydroxy-7, 8,12,13-diepoxytrichothec-9-ene Molecular Formula/Molecular Weight C 1 5 H 2 0 0 4 ; M W -- 264.13616

~ "

o

--

I s

General Characteristics Crystals; mp., 154~

4

[ ~ ] D 20 -

6.4 ~ (c=2.01, in CHC13); acetate derivative; mp., 128~

Fungal Source Formed upon mild alkaline hydrolysis of crotocin. Spectral Data UV~

Zm~, 210nm (e=4,830). TLC Data Adsorbent: silica gel; solvent, ethanol-ethyl acetate-acetone, 1:4:4, v/v/v; Rf, 0.50; detection: 50% H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 196 (1981). J. Gyimesi and A. Melera; On the Structure of Crotocin an Antifungal Antibotic; Tetrahedron Letters, p. 1665 (1967).

298

14. Trichothecenes and Related Metabolites

Common/Systematic Name Trichothecolone 4[3-Hydroxy-12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C15H2004; ~ = 264.13616 10

H

-

15

H

~5

'""~

14

4"%OH

General Characteristics Colorless needles from benzene-light petroleum; mp., 183-184~ [a]D 19"5 + 22.5 ~(c=l.0, in CHC13); [et]Ds + 17.0~ (C=I.0, in EtOH). Acetyl derivative, prisms from methanol; mp., 148-149~ 2,4-dinitrophenylhydrazone, deep orange rods from chloroform-ethanol; mp., 261-262~ Fungal Source F u s a r i u m r o s e u m and Trichothecium roseum.

Also, hydrolysis product of trichothecin.

Spectral Data UV:

)t, mLxMeOH226nm (e=8,000); acetate derivative: ~ maxM~O" 227nm (e=8,000). IR~

3560 and 1680cml. 1H NMR:

(CDC13) 3.90(1H, H-2); 1.93(1H, H-3); 2.43(1H, H-3); 4.35(1H, H-4); 6.49(1H, H10); 3.94(1H, H-11); 2.81, 3.12(2H, H-13); 1.00(3H, CH3-15); and 1.80ppm (3H, s, CH3-16). 13C NMR: 70.2, C-2; 40.2, C-3; 79.6, C-4; 49.5, C-5; 43.6, C-6; 42.3, C-7; 199.4, C-8; 138.5, C9; 137.7, C-10; 73.1, C-11; 66.0, C-12; 47.3, C-13; 6.2, C-14; 15.6, CH3-15; and 18.5ppm, CH3-16. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 222 (1981).

14. Trichothecenes and Related Metabolites

299

M. E. Savard and B. A. Blackwell; A. Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

300

14. Trichothecenes and Related Metabolites

Common/Systematic Name Trichothecin 4 I]-Crotonoyloxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2405; ~ ~6

lo

IJ~

= 332.16237 H

H

~1 61 is

13

I

,n

"~1 ....,v

14

General Characteristics Crystals; mp., 118~

i'OAd --

II 0

- - C 2'H = C \

Me 4'

[a]D 10 Jr 44 ~ (c=l.0, in CHCI3).

Fungal Source Fusarium roseum.

Biological Activity LDs0 in mice dosed IV was-~300mg/kg; IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.151xg/ml. Inhibited the elongation or termination steps in protein synthesis on polyribosomes. Spectral Data UV: /~, maxMeOH 215nm (c = 19,000).

1H ~ : 3.95(1H, H-2); 2.0-2.5(2H, H-3); 5.70(1H, H-4); 2.0-2.5(2H, H-7); 6.50(1H, H-10); 3.95(1H, n-11); 2.80(1H, n-laa), 3.10(1H, n-13b); 1.05(3H, n-14); 0.80(3H, n=15); 1.80(3H, n-16); 5.80(1H, n-2'); 6.30(1H, n-3'); and 2.10ppm (an, n-4'). 13C NMR: (CDCI3) 73.3, d, C-2; 36.9, t, C-3, 79.5, d, C-4; 49.0, s, C-5; 43.7, s, C-6, 42.1, t, C7; 198.5, s, C-8, 138.1, s, C-9, 137.0, d, C-10, 1,70.1, d, C-11, 65.4, s, C-12; 47.4, t, C-13, 5.7, q, C-14; 15.4, q, C-15, 18.5, q, C-16; 166.1, s, C-1', 120.3, d, C-2'; 145.9, d, C-3'; and 15.4ppm, q, C-4'.

14. Trichothecenes and Related Metabolites

301

TLC Data A. Adsorbent, silica gel G; solvent, chloroform-methanol, 98:2, v/v; Rf, 0.66; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent, silica gel G; solvent, benzene-tetrahydrofuran, 85:15, v/v; Rf, 0.53; detection, H2SO4 spraying and heating at 110~ for 5 minutes. GLC Data Support, Shimalite W; liquid phase, OV-17 (1.5%); retention time: 8.55min.; relative retention time (relative to nivalenol): 2.40 minutes. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 217(1981).

302

14.

Trichothecenes and Related Metabolites

Common/Systematic Name Trichothecinol A Molecular Formula/Molecular Weight C19H2406, ~ 16

"- 3 4 8 . 1 5 7 2 9

lo H

H TM

0r I~lOl 5

is ~4

'

i

.... O H

'1

~'

2'

3',,H

O--C--CH=C,, II Me 0 4'

General Characteristics [a]D 18 "4-81.5 ~ (c=0.70, in MeOH). Fungal Source Trichothecium roseum (TMI-32358 Supplied from the Tottori Mycological Institute; Tottori, Japan). Biological Activity Trichothecinol A was quite a potent antitumor promoter on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in Raji cells in comparison with t-carotene. Spectral Data UV: MeOH max

218nm (E=1.4 x 104).

IR: (CHC13) 3450, 1720, and 1680 c m "l. 1H NMR: (CDCI3) 3.78(d, d=5.0Hz, H-2); 4.29(ddd, d=2.6, 3.0, 5.0Hz, H-30); 4.99(d, J=3.0Hz, H-4); 2.3 l(dd, J=l.6, 15.2Hz, H-7a); 2.95(dd, ,/=1.2, 15.2Hz, H-713); 6.595(dq, `/=1.4, 5.8Hz, H-10); 4.41(dd, J=0.8, 5.8Hz, H-11); 2.81(d, `/=3.9Hz, H-13 pro-R); 3.08(d, ,/=3.9Hz, H-13 pro-S); 0.77(s, 5-Me); 1.05(d, ,/=l.2Hz, 6-Me); 1.84(dd, ,/=0.8, 1.4Hz, 9-Me); 3.50(d, ,/=2.6Hz, 3-OH); 5.88(dq, ,/=1.8, 11.5Hz, H-2'); 6.45(dq,,/=7.3, 11.5Hz, H-3'); and 2.17ppm (dd,,/=l.8, 7.3Hz, 3'-Me). 13C N M R :

(CDCI3) 79.30(d, C-2), 78.77(d, C-3), 83.17(d, C-4), 48.87(s, C-5), 44.39(s, C-6); 42.03(t, C-7); 198.46(s, C-8), 137.77(s, C-9), 137.24(d, C-10); 70.97(d, C-11); 64.50(s, C-12), 46.63(t, C-13), 5.90(q, C-14), 18.40(q, C-15); 15.33(q, C-16),

14. Trichothecenes and Related Metabolites

303

167.81(s, C-I'), 119.89(d, C-2'); 147.05(d, C-3'); and 15.57ppm (q, C-4'). Mass Spectrum: HR-CIMS: 349.1656re~e; MH+; calcd mass, 349.1651. Reference A. Iida, K. Konishi, H. Kubo, K. Tomioka, H. Tokuda, and H. Nishino; Trichothecinols A, B, C, Potent anti-Tumor Promoting Sesquiterpenoids from the Fungus Trichothecium roseum; Tetrahedron Letters, Vol. 37, pp-9219-9220.

304

14. Trichothecenes and Related Metabolites

Common/Systematic Name Trichothecinol B Molecular Formula/Molecular Weight C19H2605; ~

= 334.17802

IH6~%31 o H

I~lel14 15

H

s

4~0

'

2'

3'/H

C--CH--C,, II

O

Me 4'

General Characteristics [tt]D19 - 14.1 o (C=0.10, in MeOH). Fungal Source

Trichothecium roseum (TMI-32358 Supplied from the Tottori Mycological Institute;

Tottori, Japan). Biological Activity Trichothecinol B was quite a potent antitumor promoter on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in Raji cells in comparison with [}-carotene. Spectral Data UV: ~M~. 210nm (e-l.5 x 104). I-R:

(neat) 3450 and 1720cm "1. 1H NMR:

(CDC13) 2.58 (dd, J=7.8, 15.5Hz, H-3a); 2.04 (ddd, J=3.6, 5.2, 15.5Hz, H-313); 4.15 (d, J=5.8Hz, H-313). Compare spectrum with trichothecinol A. 13C NMR:

(CDCI3) 36.91 (t, C-3) and 67.83ppm (d, C-8). Compare spectrum with trichothecinol A. Mass Spectrum: HR-CIMS: 335.1859re~e; MI-F, calcd mass, 335.1858.

14. Trichothecenes and Related Metabolites

305

Reference A. Iida, K. Konishi, H. Kubo, K. Tomioka, H. Tokuda, and H. Nishino; Trichothecinols A, B, C, Potent Anti-Tumor Promoting Sesquiterpenoids from the Fungus Trichothecium roseum; Tetrahedron Letters, Vol. 37, pp-9219-9220, 1996.

306

14.

Trichothecenes

and R e l a t e d M e t a b o l i t e s

Common/Systematic Name Trichothecinol C Molecular Formula/Molecular Weight C 19H2606; ~

-" 3 5 0 . 1 7 2 9 4

~o H

H

HO,,'~k~"O ! ~el~ '6 ~ 0 . . . . . ]

1~

2

1,

3.,,,OH

'

--

d

II 0

2'

--CH=C\

3' /

H Me 4'

General Characteristics [a]DTM +28.8 ~ (c=0.2, in MeOH). Fungal Source Trichothecium roseum

(TMI-32358 Supplied from the Tottori Mycological Institute;

Tottori, Japan). Biological Activity Trichothecinol C was quite a potent anti-tumor promoter on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in Raji cells in comparison with l-carotene. Spectral Data UV: ~M~.max 211nm(e = 1.2x 104). IR:

(CHC13) 3420 and 1705cm"1. 1H NMR: (CDC13) 4.22(ddd, ./-2.7, 3.0, 4.9I-Iz, H-3[~); and 4.13ppm (d, J-4.9Hz, H-813). Compare spectrum with trichothecinol A. 13C N M R :

(CDC13) 78.81(d, C-3) and 67.73ppm (d, C-8). Compare spectrum with trichothecinol A. Mass Spectrum: HR-CIMS: 351.1805m/e; MH +, calcd mass, 351.1807.

14. Trichothecenes and Related Metabolites

307

Reference A. Iida, K. Konishi, H. Kubo, K. Tomioka, H. Tokuda, and H. Nishino; Trichothecinols A, B, C, Potent anti-Tumor Promoting Sesquiterpenoids from the Fungus Trichothecium roseum; Tetrahedron Letters, Vol. 37, pp-9219-9220 (1996).

308

14. Trichothecenes and Related Metabolites

Common/Systematic Name Trichodermone 4-Keto- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2oO3, M W -- 248.14124 ~o H

H

,% 15

14

General Characteristics Crystals from ether-hexane; mp., 154-155~

[ a ] D z0 -

42.3 ~ (c=l.0, in CHC13).

Fungal Source Obtained from the oxidation of trichodermin. Spectral Data UV:

maxE~O" 205nm (e=2,800). IR.:

(KBr) 1735cm"1. ~H NMR: (CDC13) 4.22(1H, H-2); 2.80(2H, H-3); 2.01(2H, H-7); 2.01(2H, H-8); 5.41(1H, H10); 4.38(1H, H-11); 3.15(2H, H-13); 0.84(3H, CH3-14); 0.39(3H, CH3-15); and 1.70ppm (3H, s, CH3-16). 13CNMR: 76.2, C-2; 42.0, C-3; 214.9, C-4; 55.3, C-5; 42.7, C-6; 23.9, C-7; 27.9, C-8; 140.9, C9; 118.7, C-10; 71.4, C-11; 65.3, C-12; 50.1, C-13; 5.4, C-14; 15.4, C-15; and 23.4ppm, C- 16. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 224(1981).

14. Trichothecenes and Related Metabolites

309

Common/Systematic Name Nivalenol 3tz,413,7tt,15-Tetrahydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula]Molecular Weight C15H2007; M W -- 3 1 2 . 1 2 0 9 0

~o H

.1o

0

"12

I

,,o.

~ ......~=. ,~I

~ - :~ H(3

H

-

~,

OH

CH2OH 15

General Characteristics Crystals from methanol; mp., 80-90~ dried in presence of P205 in reduced pressure; mp., 222-223~ (dec.); [tZ]D24 + 21.54 ~ (C=1.3, in EtOH); tetraacetate; mp., 168-170~ Funsal Source

Fusarium nivale (Fn-2B) = F. sporotrichioides, F. graminearum, and F. crookwellense.

Biolozical Activity LDs0 in mice dosed IP was 501~g/g (injured proliferating cells) and in chick embryo, 4.0ktg/egg Minimum SC dosage to induce vomiting in ducklings was 1.0 mg/kg. 110t,tg/ml inhibited uptake of [14C]leucine and [14C]thymidine into protein and DNA in Ehrlich ascites tumor cells. However, uptake of [~4C]uracil into RNA was not affected. Inhibition of protein synthesis occurred before inhibition of DNA synthesis. It inhibited the initiation step of protein synthesis on polyribosomes. IDs0 in rabbit reticulocytes was 3.0~g/ml (whole cell); 0.5~g/ml (cell free); rat liver was 8.0ktg/ml (whole cell); and tumor cell was 6.0~tg/ml (whole cell). Dermally toxic in rabbit and guinea pig. Emetic to laboratory animals; in ducklings emetic at lmg/kg (S.C.). Caused cell degeneration of bone marrow, lymph nodes, intestines, testes and thymus. Soectral Data ~

UV:

~, m~M"~ 218nm (E=7,500); tetraacetate, 227nm (e=7,900).

~H NMR: (CDC13) 3.61(1H, d, d=4.8Hz, H-2); 4.14(1H, dd, J=4.8, 3.4Hz, H-3); 4.43(2H, d, J=3.4Hz, H-4); 4.81(1H, s, H-7); 6.57(1H, dd, J=6.0, 1.SHz, H-10); 4.72(1H, d, d=6.0Hz, H-11); 3.01(1H, d, J=4.4Hz, H-13); 2.97(1H, d, J=4.4Hz, H-13); 1.08(3H, s, CH3-14); 3.74, 3.79(2H, d, J=12.1Hz, CH2OH-15); and 1.84ppm (3H, d, d=!.5I-Iz, CH3-16).

310

14. Trichothecenes and Related Metabolites

13C NMR: 81.6, C-2; 81.3, C-3; 80.8, C-4; 50.6, C-5; 54.5, C-6; 75.1, C-7; 201.5, C-8; 137.1, C9; 139.2, C-10; 71.0, C-11; 66.0, C-12; 46.3, C-13; 8.3, C-14; 61.5, C-15; and 15.2ppm, CH3-16. TLC Data A: Adsorbent, Kieselgel G; solvent, chloroform-methanol, 5:1 v/v; Rf, 0.45; detection, brown color after H2SO4 spraying and heating at 110~ for 5 minutes. B: Adsorbent, silica gel G; solvent, ethyl acetate-toluene, 3:1 v/v; Rf, 0.09; detection; brown color after H~SO4 spraying and heating at 110~ for 5 minutes. GLC Data Support, Shimalite W; liquid phase, OV-17 (1.5%); retention time, 3.55 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 206-207( 1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).

14. Trichothecenes and Related Metabolites

311

Common/Systematic Name 4-Deoxynivalenol; Rd Toxin; Vomitoxin 3 c~,7tt,15-Trihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C]5H2006; M W = 2 9 6 . 1 2 5 9 9

10

H

H

16~O~1

J8 HO

2 3.,,,OH

6J13~1 ,,0 -

14

(~H2OH 15

General Characteristics Needles from ethyl acetate-petroleum ether; mp., 151-153~ and 176-178~ (bound water); semicarbazone derivative; mp., 199-201 ~ triacetate, colorless needles from ethyl acetate-petroleum ether; mp., 155-157~ [t~]Dz5 + 6.35 ~ (C=0.07, in EtOH). Fungal Source Fusarium graminearum, F. culmorum, and F. roseum. Isolation/Purification Contaminated corn was extracted with aqueous methanol, concentrated and allowed to sit for 4 hours at 4~ The supernatant liquid was recovered,and basic and acidic components were removed from the neutral components. The neutral material was chromatographed on a Florisil column eluted with chloroform-methanol, 50:50 and 25:75, v/v. The deoxynivalenol containing fractions were further fractionated by flash chromatography, preparative TLC (silica gel F254developed with chloroform-methanol-water, 80:20:0.1, v/v/v) and Sephadex LH-20 column chromatography. Biological Activity LDso in male mice (ddY strain) dosed IP was 70mg/kg; females, 49.4mg/kg; ducklings (10 day old) 27mg/kg. Also, inhibited the multiplication of culture cells of Tetrahymena pyriformis at same level as fusarenon-X (4.6ktg/ml); IDs0 in rabbit reticulocytes was 2ktg/ml(whole cells); caused emesis in swine (7mg/601b via intubation), duckling (10 day old, 10mg/kg), dogs (0. lmg/kg, s.c.) and food refusal in swine at 40mg/kg. Spectral Data UV:

~, ~m~

218nm(e=4,500); semicarbazone derivative, 268nm (6=18,200).

312

14. Trichothecenes and Related Metabolites

IR~

(KBr) 3470, 3430, 3350, and 1680cm-1. 1H N1V[R:

(CDC13) 3.62(1H, d, J-4.5Hz, H-2); 4.53(1H, dt, J=10.7, 4.5Hz, H-3); 2.21(1H, dd, Jt~=14.8, 4.5Hz, H-4); 2.07(1H, dd, J[]=14.8, 10.7Hz, H-4); 4.83(1H, d, J=2.0Hz, H7); 6.61(1H, dq, J=5.9, 1.5Hz, H-10); 4.80(1H, d, J=5.9Hz, H-11); 3.07(1H, d, J=4.3Hz, H-13); 3.15(1H, d, J=4.3Hz, H-13); 1.13(3H, s, CH3-14); 3.73, 3.89(2H, d, J=l 1.7Hz, CH2OH-15); and 1.86ppm (3H, br s, CH3-16). 13CNMR: 80.6, C-2; 68.5, C-3; 43.0, C-4; 46.0, C-5; 52.1, C-6; 74.3, C-7; 200.1, C-8; 135.7, C9; 138.3, C-10; 70.2, C-11; 65.7, C-12; 47.3, C-13; 14.1, C-14; 61.4, C-15; and 15.01ppm, CH3-16. TLC Data A: Adsorbent, silica gel G; solvent, chloroform-methanol, 97:3, v/v; Rf, 0.09; detection, H2SO4 spraying and heating at 110~ for 5 minutes. B: Adsorbent; silica gel G; solvent, ethyl acetate-toluene, 3:1, v/v; Rf, 0.21; detection, H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 202-203(1981 ). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; Plant Research Centre, Agriculture Canada, Ottawa, Ontario, K1A 0C6. R. F. Vesonder, A. Ciegler, and A. H. Jensen; Isolation of the Emetic Principle from Fusarium-Infected Corn; Applied Microbiology, Vol. 26, pp. 1008-1010(1973).

R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusafi'um Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).

14. Trichothecenes and Related Metabolites

313

Common/Systematic Name 3-Acetyl-4-deoxynivalenol 3a-Acetoxy-7a, 15-dihydroxy-12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2207, M W -- 3 3 8 . 1 3 6 5 5

16

~ O ' ~10l J8 HO

H

H

2

3

,,,OAc

6|13~ .... ,0 -

14

(~H2OH

15

General Characteristics Colorless needles from ethyl ether-n-pentane; mp.,185.5-186~ EtOH); [t~]D25 + 40.5~ tetraacetate; mp., 168-170~

[0g]D20 q-

43.0 ~(c=1.3, in

Isolation/Purification Fungal mycelium was extracted with methylene chloride, concentrated and the residue dissolved in methanol. The methanol phase was extracted with petroleum ether and evaporated to dryness. The residue was chromatographed on a silica gel column eluted with chloroform-ethanol (50:1, v/v) and then chloroform-ethanol (400:1, v/v). 3-Acetyl-4deoxynivalenol was recrystallized from hot ethyl ether. Fungal Sour.ce Fusarium culmorum (HLX 1503; ATCC 28114), F. graminearum (HLX 1506), and F. roseum. Biological Activity LDs0 in male ddS strain of mice dosed IP was 76.7mg/kg; females, 49.9mg/kg; ducklings, 37mg/kg. Minimum SC dosage to induce vomiting in ducklings was 10.0 mg/kg; dogs, S.C., 0.2mg/kg. Inhibited Tetrahymena pyriformis, 29.0~g/ml. Spectral Data UV:

~m~

219nm (e=5,900).

IR:

(KBr) 3480, 3400, 1720, and 1680cm].

314

14. Trichothecenes and Related Metabolites

1H NIVIR: (CDC13) 3.87(1H, d, Jr= 4.5Hz, H-2); 5.18(1H, dt, J=l 1.2, 4.5Hz, H-3); 2.35(1H, dd, Ja=15.1, 4.5Hz, H-4); 2.12(1H, dd, 313=15.1, 11.2Hz, H-4); 4.79(1H, d, J=2.1Hz, H7); 6.56(1H, dq, J=5.9, 1.5Hz, H-10); 4.66(1H, d, J=5.9Hz, H-11); 3.08(1H, d, Ja=4.3Hz, H-13); 3.14(1H, d, J=4.3Hz, H-13); 1.12(3H, s, CH3-14); 3.75(a), 3.79(13) (2H, d, J=l 1.7Hz, CH2OH-15); 1.85(3H, d, J=l.5Hz, CH3-16) ;and 2.10ppm (3H, s, CH3COO-). 13C NMR: 79.1, C-2; 71.3, C-3; 40.5, C-4; 45.9, C-5; 52.0, C-6; 74.5, C-7; 199.8, C-8; 135.8, C9; 138.4, C-10; 70.2, C-11; 65.1, C-12; 47.5, C-13; 14.1, C-14; 62.2, C-15; 15.2, CH316; 20.9, CH3-Ac; and 170.3ppm (CH3C=O). Mass Data: LREIMS: 338(M+), 278, 290, 248, 241,231,223(100), 203, 189, 181, and 175re~e; anal. calcd for C17H2207: C, 60.3; H, 6.6; O, 33.1%; found C, 60.1; H, 6.9; O, 32.9%. TLC Data A: Adsorbent, silica gel G; chloroform-methanol, 93:7 v/v; Rf, 0.40; detection, H2SO4 spraying and heating at 110~ for 5 minutes. B: Adsorbent, silica gel G; solvent, ethyl acetate-toluene, 3:1 v/v; Re, 0.43; detection, H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 209(1981). R. Greenhalgh, A. W. Hanson, J. D. Miller, and A. Taylor; Production and X-ray Crystal Structure of 3a-Acetoxy-7a, 15-dihydroxy-12,13-epoxytrichothec-9-en-8-one; J. Agric. Food Chem., Vol. 32, pp. 948-952(1984). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).

14.

T r i c h o t h e c e n e s and Related M e t a b o l i t e s

315

Common/Systematic Name 413, 15-Diacetylnivalenol 4[3,15-Diacetoxy-3 a,7a-dihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2409; MW

--- 3 9 6 . 1 4 2 0 3

16

o4

lo H

J: :l

H

I

HO ~ A ''OAo ~H2OAc 15

General Characteristics Crystals from acetone-n-hexane; mp., 135-136~ [a]D25 + 72 ~ (in acetone).

[a]D 26 -I- 64.3 ~ (c--1.0,

in EtOH);

Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed by a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase. Fungal Source

Fusarium sporotrichioides, F. crookwellense, F. scirpi (CMI 45490), and Gibberella mtricans = F. equiseti.

Biological Activity Caused skin necrosis and emesis in laboratory animals (emetic in ducklings); S. C., 0.4mg/kg; LDs0 in male mice (ddY strain) dosed IP was 3.5mg/kg; chick embryo, 0.9mg/egg; IDs0 in rabbit reticulocytes, 0.10~g/ml (whole cell). Spectral Data UV: ~, M~oa 220nm (e=6,200). max IR:

(KBr) 3420-3450, 2980-2920, 1735, 1680, 1235, and 1040cm1.

316

14. Trichothecenes and Related Metabolites

1H NMR: (CDCI3) 3.82(1H, d, J=4.7Hz, H-2); 4.22(1H, ddd, J=4.8, 3.1, 2.7Hz, H-3); 3.25(1H, d, J=2.7Hz, OH-3); 5.16(1H, d, J=3.1Hz, H-4); 4.86(1H, d, J=2.0Hz, H-7); 3.80(1H, d, J=2.0Hz, OH-7); 6.63(1H, dd, J=5.9, 1.6Hz, H-10); 4.72(1H, d, J=5.9Hz, H-11); 3.07(1H, d, .]=4.3Hz, H-13); 3.08(1H, d, J=4.3Hz, n-13); 1.09(3H, s, CH3-14); 4.19, 4.32(2H, d, J=12.3Hz, CH2OAc-15); 1.88(3H, d, J=l.6Hz, CH3-16); 1.90(3H, s, CHaAc- 15); and 2.14ppm (3 H, s, CHaCOO-). 13C NMR: 79.7, C-2; 78.4, C-3; 83.9, C-4; 49.4, C-5; 52.2, C-6; 73.2, C-7; 198.9, C-8; 136.0, C9; 138.3, C-10; 69.3, C-11; 64.3, C-12; 46.1, C-13; 7.6, C-14; 61.8, C-15; 15.3, CH316; 20.6, CH3-Ac-15; 21.0, CH3-Ac-4; and 170.0, 172.7ppm (CH3C=O). Mass Spectrum: [,REIMS: 396(M+), 366, 336, 294, 277, 247, 189, 179(100), 151,137, and 123m/e. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 221(1981). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).

14. T r i c h o t h e c e n e s and Related Metabolites

317

Common/Systematic Name 413, 15-Diacetyl-7-deoxynivalenol 413,15-Diacetoxy-3 t~-hydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2408; M W " - 3 80.14712 16

~o H

: .--

0

H "~

"

2

i ~5

:_ 14

,OH

L,

3,,"

' OAc

~H2OAc 15

Fungal Source Fusarium sporotrichioides and F. crookwellense. Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed by a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase. Biological Activity The trichothecenes, as a group, show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-flee systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans.

318

14. Trichothecenes and Related Metabolites

Spectral Data 1H NMR: (CDCI3) 3.77(1H, d, J=4.9I-Iz, H-2); 4.21(1H, ddd, J=4.9, 2.9, 2.6Hz, H-3); 3.21(1H, d, J=2.6Hz, OH-3); 5.06(1H, d, J=2.9Hz, H-4); 2.46(1H, dd, J=15.9, 1.6Hz, H-7); 2.92(1H, dd, J=15.9, 1.6Hz, H-7); 6.59(1H, dd, J=5.9, 1.5Hz, H-10); 4.51(1H, d, J=5.9Hz, H-11); 2.79(1H, d, J=3.9Hz, H-13); 3.07(1H, d, J=3.9Hz, H-13); 0.80(3H, s, CH3-14); 4.10, 4.17(2H, d, J=12.4Hz, CHaOAc=15); 1.82(3H, d, J=l.5Hz, CH3-16); 1.97(3H, s, CH3Ac-15); and 2.14ppm (3H, s, CH3COO-). 13CNMR: 79.0, C-2; 78.5, C-3; 83.8, C-4; 47.5, C-5; 48.8, C-6; 38.2, C-7; 196.7, C-8; 138.9, C9; 136.9, C-10; 68.4, C-11; 64.4, C-12; 46.7, C-13; 6.1, C-14; 64.5, C-15; 15.3, CH316; 20.8, CH3-Ac-15; 20.5, C__H3-Ac-4;and 170.2, 172.7ppm (CH3C=O). Mass Spectrum: LREIMS: 380(M+),338, 320, 292, 278, 247, 189, 173, 121,109, and 43m/e (100). References D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

319

Common/Systematic Name 15-Acetyl-4-deoxynivalenol 15-Acetoxy-3 tt,7tt-dihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17Hz207, ~

= 338.13655

10 H

16~O~1

H

2

3.,-"OH

.... ,o

HO

: 14 ~H2OAc

15

General Characteristics Crystals; mp., 142-145~

[a]D 19 +

79 ~ (in CHC13).

Fungal Source Fusarium grammearum (DAOM 17; 8148). Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosome and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans. Spectral Data 1H N]VIR:

(CDCI3) 3.60(IH, d, J=4.4Hz, H-2); 4.50(IH, ddd, J=I0.6, 4.4, 4.51--Iz,H-3); 2.20(IH, dd, Ja=14.8, 4.SHz, H-4), 2.07(IH, dd, J[3=14.8, 10.6Hz, H-4); 4.81(IH, d, J=l.9Hz, H-7); 6.58(IH, dq, J=5.8, 1.31--Iz,H-10), 4.87(IH, d, J=5.81--Iz,H-I I); 3.11(1H, d, Jtt=4.2Hz, H-13); 3.06(1H, d, J=4.2Hz, H-13); 1.04(3H, s, CH3-14); 4.21(2H, s, CH2OAc-15); 1.86(3H, br s, CH3-16) and 1.85ppm (3H, s, CH3COO-).

320

14. Trichothecenes and Related Metabolites

13C NMR: 80.7, C-2; 68.9, C-3; 43.3, C-4; 46.4, C-5; 51.4, C-6; 73.5, C-7; 199.4, C-8; 135.5, C9; 138.6, C-10; 70.1, C-11; 65.4, C-12; 47.3, C-13; 13.8, C-14; 62.2, C-15; 15.3, CH316; 20.6, CH3-Ac; and 170.1ppm (CH3C=O). References M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed., Elsevier Pub. Co., New York, pp. 1323(1989).

14. Trichothecenes and Related Metabolites

321

Common/Systematic Name 3,15-Diacetyl-4-deoxynivalenol 3a, 15a-Diacetoxy-7tt-hydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2408; M W -- 380.14712

10 H

1 6 ~ O " ~

H

2

3.,,,OAc

'~

CH2OAc 15

General Characteristics Crystals from ethyl acetate-hexane; mp., 119-120oC. Fungal Source Fusarium roseum (ATCC 28114) and F. grammearum.

Spectral Data 1H NIVIR:

(CDC13) 3.90(1H, d, J=4.5Hz, H-2); 5.22(2H, ddd, J=l 1.2, 4.5, 4.4Hz, H-3); 2.17(1H, dd, J=l 1.2, 15.1Hz, H-4); 2.30(1H, dd, ,/--15.1, 4.4Hz, H-4); 3.81(1H, d, J=l.6Hz, H-7), 3.76(1H, d, J=l.6Hz, OH-7); 6.57(1H, dd, J=5.8, 1.6Hz, H-10); 4.70(1H, d, J=5.8Hz, H-11); 3.10(1H, d, J=4.3Hz, H-13); 3.15(1H, d, J=4.3Hz, H13); 1.09(3H, s, CH3-14); 4.22, 4.28(2H, d, J=12.0Hz, CH2OAc-15); 1.89(3H, d, J=0.8Hz, CH3-16); 1.88(3H, s, CH3Ac-15); and 2.13ppm (3H, s, CH3COO-). 13CNMR: 78.9, C-2; 71.1, C-3; 40.4, C-4; 45.8, C-5; 51.5, C-6; 73.4, C-7; 199.2, C-8; 135.6, C9; 138.4, C-10; 70.1, C-11; 64.9, C-12; 47.4, C-13; 13.6, C-14; 62.1, C-15; 15.3, CH3-16; 20.9, CH3-Ac-15; 20.6, CH3-Ac-4; and 170.2, 170.7ppm (CH3C=O). Mass Spectrum: LREIMS: 380(M+), 320, 291, 231,203, 181, and 163m/e. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Part 2. Minor Metabolites o f Fusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 115-118(1986).

322

14. Trichothecenes and Related Metabolites

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. Trichothecenes and Related Metabolites

323

Common/Systematic Name Fusarenon-X; Nivalenol monoacetate; Fusarenon 413-Acetoxy-3 a,7 a, 15a-trihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2208; ~

-- 3 5 4 . 1 2 1 4 5

~o H O

~

~

i

H ~

....,o I

- 12

HO i 1{5 CH2OH 15

4\OAc

General Characteristics Hexagonal bipyramid crystals from dichloromethane-n-pentane; mp., 91-92~ crystals from dichloroethane; mp., 181-182~ [a]o 25 + 58 ~ (c=l.0, in MeOH.) Fungal Source

Fusarium nivale, Fn-2B = sporotrichioides, F. crookwellense, F. graminearum, and F. equiseti.

Isolation/Purification The toxin in the fungal broth was absorbed on active carbon, followed by elution with methanol. After evaporation of the solvent, 5 volumes of chloroform were added to the methanol extract. The methanol-chloroform soluble fraction, herein referred to as crude toxin, was chromatographed on Kieselgel. Development with chloroform-methanol (97:3 to 5:1, v/v) yielded a highly toxic fraction that eluted before the nivalenol fraction. Rechromatography on Kieselgel with chloroform-acetone (5:1, v/v) gave a white powder. This material, when crystallized from dichloromethane-n-pentane, gave hexagonal bipyramid crystals. Biological Activity Application of 0.2-1.0~g to dehaired back skin of rabbit, guinea pig, and mouse caused cytotoxic effects on epidermis, dermis, and hair follicles; no change in the subcutaneous tissues or muscle fibers. LDs0 in male mice (ddY strain) dosed IP (0.9% saline) was-3.3 mg/kg; nonlethal doses produced mucoidal secretion around the eye one week after treatment. LDs0 in chick embryos, 2.6mg/egg; IDs0 in rabbit reticulocytes, 0.25~g/ml (whole cell); 0.51~g/ml (cell flee); tumor cells, 6.01~g/ml (whole cell); and rat liver, 8.0~tg/ml (cell flee). Emetic, minimum S.C. dose to induce vomiting in ducklings was 0.40.5 mg/kg; in cats, 0.3-0.5 mg/kg; and in pigeons, 0.5-1.0 mg/kg (IV and PO).

324

14. Trichothecenes and Related Metabolites

Spectral Data UV: X m~,M~~ 220nm (e=6,500). 1H NMR: (CDC13) 3.79(1H, d, J=4.7Hz, H-2); 4.32(2H, m, H-3); 5.53(1H, d, J=3.4Hz, H-4); 4.83(1H, d, J=2.0Hz, H-7); 3.71(1H, d, J=2.0Hz, OH-7); 6.66(1H, d, J=6.0Hz, H-10); 4.81(1H, d, J=6.0Hz, H-11); 3.04(1H, d, J=4.2Hz, H-13); 3.09(1H, d, J=4.2Hz, H-13); 1.08(3H, s, CH3-14); 3.59(1H, dd, J=12.5, 4.7Hz, H-15); 4.01(1H, br d, J=12.5Hz, H-15); 1.88(3H, br s, CH3-16); and 2.14ppm (3H, s, CH3COO-). 13CNMR: 77.5, C-2; 80.0, C-3; 83.5, C-4; 49.4, C-5; 52.9, C-6; 75.8, C-7; 199.6, C-8; 135.9, C-9; 138.3, C-10; 69.1, C-11; 64.7, C-12; 46.3, C-13; 7.8, C-14; 61.6, C-15; 15.2, CH3-16; 21.0, CH3-Ac-15; and 172.9ppm (CH3C=O).

Mass Data: Found: C, 57.62; H, 6.22; O, 36.16%; calcd for C, 57.62; H, 6.22; O, 36.16%. TLC Data A. Adsorbent: silica gel G; Solvent: chloroform-methanol, 97:3, v/v; Rf, 0.19; detection: purple to yellow-green with H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: silica gel G; solvent: toluene-ethyl acetate, 1:3, v/v; Rf, 0.36; detection: purple to yellow-green with H2SO4 spraying and heating at 110~ for 5 minutes. GLC Data Support: Shimalite W; liquid phase: OV-17 (1.5%); retention time: 3.95; relative retention time (relative to nivalenol): 1.11. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 213 (1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from F u s a r i u m Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). Y. Sugiura, Y. watanabe, T. Tanaka, S. Yamamoto, and Y. Ueno; Occurrence of Strains That Produce Both Nivalenol and Deoxynivalenol; Appl. Environ. Microbiol.; Vol. 56, pp. 3047-3051 (1990). Gibberella zoeae

Y. Ueno, I. Ueno, T. Tatsuno, K. Okubo, and H. Tsunoda; Fusarenon-X, A Toxic Principle o f F u s a r i u m n i v a l e - Culture Filtrate, Experientia, Vol. 25, pp. 1062 (1969).

Modified Trichothecenes 2-Deoxy- 11-epi- 12-acetyl-3~t-hydroxysambucoin 2-Deoxy- 11-epi-3ct-hydroxysambucoin Trichodiol A Trichodiene FS-1 FS-2 FS-3 FS-4 3-Ketoapotrichothecene 3ct-Hydroxyapotrichothecene 313-Hydroxyapotrichothecene

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15.

ModifiedTrichothecenes

327

Common/Systematic Name 2-Deoxy- 11-epi- 12-acetyl-3 a-hydroxysambucoin Molecular Formula/Molecular Weight C17H2604; M W = 294.18311 Me

_H

OAc

M~Mel4~//2 14

OH General Characteristics Isolated as an oil. Fungal Source

Fusarium sporotrichioides isolated from Ethiopian wheat.

Isolation/Purification

F. sporotrichioides was cultured on ground corn grits and the fermented corn grits were blended with chloroform-acetone (85:15, v/v) and the mixture allowed to stand overnight. After filtration the remaining solid residue was blended with acetone; the mixture was filtered and the acetone extract and the chloroform-acetone extract were combined and concentrated under reduced pressure, which yielded a dark green oil. The oil was dissolved in 2 liters benzene-n-hexane (2:1, v/v) and aliquots were applied to Florisil columns. Columns were eluted successively with benzene-n-hexane (2:1, v/v), methylene chloride, chloroform-acetone (9:1, v/v), and acetone. The residue from the chloroformacetone fraction was recrystaUized from acetone-n-hexane to yield a mixture of trichothecenes. Reversed-phase flash column chromatography of the solid trichothecene mixture using MeOH-H20-acetic acid (35:20:1, v/v/v) yielded neosolaniol, NT-1, 8acetylneosolaniol, 8-propionyl-neosolaniol, 8-isobutyrylneosolaniol, 8-nbutyrylneosolaniol, T-2 toxin, 8-n-pentanoyl-neosolaniol, and 8-n-hexanoylneosolaniol. The acetone fraction was dissolved in benzene-acetone (2:1, v/v) and subjected to Florisil column chromatography. The column was eluted successively with benzene-acetone (2:1, v/v), chloroform-acetone (4:1, v/v), and acetone. The residue from the acetone fraction was chromatographed using flash column chromatography, eluting with solvent mixtures of increasing polarity: benzene-acetone (65:35, v/v), benzene-acetone (57:63, v/v), acetone, acetone-methanol (1:1, v/v), and MeOH. Fractions were collected and similar fractions were combined. The polar fractions of the silica gel column chromatography were combined, concentrated at reduced pressure, and subjected to reversed-phase column chromatography eluting with methanol-water-acetic acid (35:20:1, v/v/v). Similar fractions were combined. Acuminatin, FS-1, scirpenol, T-2 tetraol, T-2 triol, 15acetylscirpenol, DON (deoxynivalenol), 8a-hydroxytrichothecolone, verrucarol, 2-deoxy11-epi-3 a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3 a-hydroxysambucoin were

328

15.

ModifiedTrichothecenes

isolated in pure form. Biological Activity 2-Deoxy- 11-epi-3a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3 ~-hydroxysambucoin were screened for relative cytotoxicity in cultured baby hamster kidney (BHK' 21) cells and found to be non-toxic (LC~00=1000 ng/ml). Spectral Data IR:

(NaCI) 1736(acetyl group) and 3408cm"1 (OH group). 1H NMR:

(CDC13) 1.40(1H, dd, J=14.9, 2.0Hz, H-2); 2.32(1H, dd, J=14.9, 8.4Hz, H-2); 4.4(1H, m, H-3); 1.91(1H, m, H-4); 2.12(1H, m, H-4); 1.68(1H, dd, J=5.5, 2.0Hz, H7); 1.95(1H, dd, J=9.0, 2.0Hz, H-7); 1.95-2.0(2H, m, 2H-8); 5.45(1H, dd, J=6.0, 1.0Hz, H-10); 3.6(1H, bs, H-11); 3.58(1H, d, d=15.1Hz, H-13); 3.75(1H, d, d=15.1Hz, H-13); 0.96(3H, s, H-14); 0.73(3H, s, H-15), 1.68(3H, s, H-16); and 2.05ppm (3H, s, COCH3). 13C NMR: (CDC13) 45.9, t, C-2; 67.5, d, C-3; 44.8, t, C-4; obscured by solvent, C-5; 35.2, s, C6; 24.5, t, C-7; 29.2, t, C-8; 140.4, s, C-9; 120.8, d, C-10; 75.9, d, C-11; 86.9, s, C-12; 74.5, t, C-13; 16.8, q, C-14; 16.3, q, C-15; 23.0, q, C-16; and 22.4ppm, q, COCH3. (COCH3, not observed). Mass Spectrum: CIMS (TFA derivative): 391(M + + 1, 10%), 331(17), 301(38), 277(22), 217(55), and 111role (100). Reference D. M. Fort, C. L. Barnes, M. S. Tempestra, H. H. Casper, E. Bekele, A. A. Rottinghaus, and G. E.Rottinghaus; Two New Modified Trichothecenes from Fusarium sporotrichioides; J. Natural Products, Vol. 56, pp. 1890-1897(1993).

15. ModifiedTrichothecenes

329

Common/Systematic Name 2-Deoxy- 11-epi-3 a-hydroxysambucoin Molecular Formula/M01ecular Weight C15H2403; MW

Me 16

-- 2 5 2 . 1 7 2 5 4

H .= O .

9

is M6 E..~. 14

OH

General Characteristics Colorless crystals from methanol-H20, mp., 177-178 oC. Funsal Source v

Fusarium sporotrichioides isolated from Ethiopian wheat.

Isolation/Purification

F. sporotrichioides was cultured on ground corn grits at 10~ for 28 days and the fermented corn grits were blended with chloroform-acetone (85:15, v/v) and the mixture allowed to stand overnight. After filtration, the remaining solid residue was blended with acetone. The mixture was filtered and the solid autoclaved and discarded. The acetone and the chloroform-acetone extract were combined and concentrated under reduced pressure, which yielded a dark green oil. The oil was dissolved in 2 liters benzene-n-hexane (2:1, v/v), and aliquots were applied to Florisil columns. Columns were eluted successively with benzene-n-hexane (2:1, v/v), methylene chloride, chloroform-acetone (9:1, v/v), and acetone. The residue from the chloroform-acetone fraction was recrystallized from acetone-n-hexane to yield a mixture of trichothecenes. Reversed-phase flash column chromatography of the solid trichothecene mixture using methanol-water-acetic acid (35:20:1, v/v/v) yielded neosolaniol, NT- 1, 8-acetylneosolaniol, 8-propionyl-neosolaniol, 8-isobutyrylneosolaniol, 8-n-butyrylneosolaniol, T-2 toxin, 8-n-pentanoylneosolaniol, and 8-n-hexanoylneosolaniol. The acetone fraction was dissolved in benzene-acetone (2:1, v/v) and subjected to Florisil column chromatography. The column was eluted successively with benzene-acetone (2:1, v/v), chloroform-acetone (4:1, v/v), and acetone. The residue from the acetone fraction was chromatographed using flash column chromatography, eluting with solvent mixtures of increasing polarity: benzene-acetone (65:35, v/v), benzene-acetone (57:63, v/v), acetone, acetone-methanol (1:1, v/v), and MeOH. Fractions were collected and similar fractions were combined. The polar fractions of the silica gel column chromatography were combined, concentrated at reduced pressure, and subjected to reversed-phase column chromatography eluting with methanol-water-acetic acid (35:20:1, v/v/v). Similar fractions were combined. Acuminatin, FS-1, scirpenol, T - 2 tetraol, T-2 triol, 15-acetylscirpenol, DON (deoxynivalenol), 8a-hydroxytrichothecolone,

330

15.

ModifiedTrichothecenes

verrucarol, 2-deoxy- 11-epi-3a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3tthydroxysambucoin were isolated in pure form. Biological Activity 2-Deoxy-11-epi-3a-hydroxysambucoin and 2-deoxy-11-epi-12-acetyl-3a-hydroxysambucoin were screened for relative cytotoxicity in cultured baby hamster kidney (BHK21) cells and found to be non-toxic (LC100= 1000ng/ml). Spectral Data IR:

(NaC1) 3412cm1 (OH group). 1H ~ :

(CDC13) 1.46(1H, dd, J-15.0, 2.4Hz, H-2); 2.28(1H, dd, .]=15.0, 9.4Hz, H-2); 4.44(1H, ddd, J=15.6,7.8, 2.4Hz, H-3); 1.85(1H, m, H-4); 2.02(1H, m, H-4); 1.53 (1H, dd, J-13.3, 2.0Hz, H-7); 1.91(1H, dd, J=8.0, 2.0Hz, H-7); 1.95-2.0(2H, m, H8); 5.52(1H, dd, J=5.6, 0.8Hz, H-10); 3.69(1H, bs, H-11); 3.66(1H, d, J=12.6Hz, H13); 3.95(1H, d, J=12.6Hz, H-13); 0.97(3H, s, 3H-14); 0.71(3H, s, H-15); and 1.70ppm (3H, s, H-16). 13C NMR: (CDC13) 44.1, t, C-2; 67.0, d, C-3; 44.2, t, C-4; 48.6, s, C-5; 35.0, s, C-6; 24.3, t, C-7; 28.2, t, C-8; 139.5, s, C-9; 119.5, d, C-10; 75.1, d, C-11; 77.6, s, C-12; 74.0, t, C-13; 16.6, q, C-14; 16.1, q, C-15; and 23.0ppm, q, C-16. Mass Spectrum: LREIMS (TMSi derivative): 396(NY, 1%), 378(4), 258(100), 108(63), and 73m/e (60); ElMS (TMSi derivative): 397(M++ 1, 18%), 381 (15), 307(22), 291 (100), 263(42), 217(53), 199(32), 73role (21); and CIMS (TFA derivative): 445(M § + 1, 45%), 445(45), 331(100), 217(13), 115(67), and 93role (80). Reference D. M. Fort, C. L. Barnes, M. S. Tempestra, H. H. Casper, E. Bekele, A. A. Rottinghaus, and G. E. Rottinghaus; Two New Modified Trichothecenes from Fusarium sporotrichioides; J. Natural Products, Vol. 56, pp. 1890-1897(1993).

15. ModifiedTrichothecenes

331

Common/Systematic Name Trichodiol A Molecular Formula/Molecular Weight C15H2403,1VIW' = 252.17254

Me

Me~/.O

H

General Characteristics Colorless crystals from ether-hexane; mp., 81-830C;

[tg]D =

+52 ~ (CHC13).

Isolation/Purification The ethyl acetate extract of the fermentation broth from Trichothecium r o s e u m was saponified with 10% ethanolic KOH at room temperature. The mixture was concentrated to ca. 1/3 volume below 40~ under reduced pressure. This material was extracted with ether and the extract washed with water, dried and the solvent evaporated to dryness. Repeated column chromatography of the nonsaponifiable material using Wakogel C-200 (60% ether-benzene) gave trichodiol A which crystallized from ether-hexane to give a pure sample. Fungal Source T r i c h o t h e c i u m roseum.

Spectral Data UV:

E~H

End absorption, 210nm (e = 360).

IR:

(CHCI3) 3460cm1 (OH). 1H NMR: (CDC13) The NMR spectrum revealed the presence of three tertiary methyl groups (singlets at 0.86, 0.96, and 1.15ppm, 3H each), a CHzOH group (3.48, 4.06, 2H, Abq, J = 12.0Hz) attached to a non-hydrogen atom, and a-CH=CHgroup (5.46, 5.73ppm, 2H, Abq, J = 10Hz). Both carbon atoms are attached to quaternary carbon atoms and a methine proton (3.17ppm, 1H, bs). The signals at 3.48 and 4.06ppm shifted to 4.33 and 4.54ppm upon acetylation. Mass Data: HREIMS: 234.162(C15H2202, M § - 1-120, calcd. 234.162), 127.074(C7Hl102, calcd 127.076), 125.099(CsHx30, ealcd 125.097), 108.095(CsH12, calcd 108.094), 107.088(C8H11, calcd 107.086), 109.065(C7H90, calcd 109.065), 9 6 . 0 5 6 m / e (C6HsO, calcd 96.058). Found: O, 18.84; C15H2403 requires O, 19.02%.

332

15.

ModifiedTrichothecenes

Reference S. Nozoe and Y. Machida; The Structures of Trichodiol and Trichodiene; Tetrahedrort, Vol. 28, pp. 5105-5111(1972).

15.

ModifiedTrichothecenes

333

Common/Systematic Name Trichodiene Molecular Formula/Molecular Weight C15H24; M W --" 204.18780

CH2

General Characteristics Colorless oily material; [a]D--" q- 21 ~ (CHCI3). Isolation/Purification The myeelium from the fermentation of T. r o s e u m was suspended in warm acetone for 3 hours. After filtration, the mycelium was resuspended in acetone and allowed to stand overnight at room temperature and then filtered. The combined acetone extracts were concentrated to ca. ~/3 volume, to which water was added. Extraction with ethyl acetate followed by successive washing with water, drying, and removal of solvent afforded a brown residue, which was saponified with 10% KOH at room temperature to obtain the nonsaponifiable material. Column chromatographic separation was first afforded using ether-benzene. The first fraction (100% benzene) was further chromatographed using a AgNO3 impregnated silica gel column (hexane elution) to obtain pure trichodiene. Fungal Source Trichothecium roseum.

Spectral Data IR:

(CHC13) 3065, 1645, and 890cm"]. ]H NMR: (CDCI3) The NMR spectrum revealed the presence of ringlets at 0.85 and 1.04 ppm due to two tertiary methyl groups, a broad ringlet at 1.63ppm due to an olefinic methyl group, a multiplet at 5.23ppm due to an olefinic proton, and two broad singlets at 4.71 and 4.92ppm due to olefinic protons. Mass Spectrum: EIMS: 204(M +, C15H24); intense peaks at 189, 161,133, 121, 119, 109 (base peak CsHI3), 95 (Cyril0, 93, and 67m/e.

334

15.

Mo d i fi ed T r i ch o th ecen es

Reference S. Nozoe and Y. Machida; The Structures of Trichodiol and Trichodiene; Tetrahedron, Vol. 28, pp. 5105-5111(1972).

15. ModifiedTrichothecenes

335

Common/Systematic Name FS-1 Molecular Formula/Molecular Weight C15H2203; MW = 250.15689 16

~,'

,,OH

13CH20H

0 General Characteristics Isolated as an oil. Fungal Source

Fusarium sporotrichioides (MC-72083) and F. sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil column chromatography eluted with benzene-hexane, 2:1 (v/v), methylene chloride, and chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil was further purified by normal phase preparative HPLC using benzene-acetone followed by preparative reversed-phase TLC to yield several trichothecenes including FS-1. Spectral Data IR~

(thin film) 1684(enone) and 3352cm"1 (OH). 1H NMR:

(CDC13) 6.24(1H, t, J-1.5Hz, H-2); 3.10(1H, d, J-18.2Hz, H-4), 2.03(1H, d, J=18.2Hz, H-4); 1.95(1H, dd, ,/=15.0, 4.6Hz, H-7); 1.79(1H, d, J=15.0Hz, H-7); 1.45(2H, m, J-4.6Hz, H-8); 5.05(1H, br s, H-10); 4.11(1H, br s, H-11); 4.55(1H, d, J= 17.3I-Iz, H-13); 4.34(1H, d, J-17.3Hz, H-13); 1.24(3H, s, CH3-14); 0.84(3H, s, CH3-15); and 1.61ppm (3H, br s, CH3-16). 13C NMR: (CDC13) 129.5, C-2; 208.4, C-3; 50.4, C-4; 53.1, C-5; 41.9, C-6; 29.9, C-7; 27.4, C-8; 135.7, C-9; 125.0, C-10; 71.3, C-11; 188.0, C-12; 61.0, C-13; 22.4, C-14; 13.1, C-15; and 22.5ppm; C-16.

336

15.

ModifiedTrichothecenes

Mass Spectrum: HREIMS: 250.150m/e (M+); calcd for C15H2zO3,250.156; LREIMS: 250, 232, 219, 204, 167(base peak), 126, 107, 84, and 55m/e. References D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene Mycotoxins of Fusarium sporotrichioides (MC- 72083); Tet. Lett., pp. 4133-4136 (1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

ModifiedTrichothecenes

337

Common/Systematic Name FS-2 Molecular Formula/Molecular Weight C15H2403, M W = 2 5 2 . 1 7 2 5 4 16

9 All

HO,,,"~ "~

13CH20H

OH Fungal Source Fusarium sporotrichioides (MC-72083). Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by acetone. The combined extracts were concentrated to yield a dark red oil which was treated with n-hexane to remove non-polar materials. The resulting orange-red oil was further purified by Florisil column chromatography eluted with benzene-hexane, 2:1 (v/v), methylene chloride, and chloroform-methanol (95:5, v/v) followed by acetone. The latter two were combined to give an oil highly enriched with trichothecenes. Flash chromatography followed by normal phase preparative HPLC using toluene-acetone and preparative reversed-phase TLC yielded several purified triehotheeenes including FS-2. Spectral Data UV: max

194.5nm (e=12,000; two ene 7: to re* transitions).

:H NMR: (CDCI3) 5.75(1H, dd, J=l.5, 3.3Hz, H-2); 4.79(1H, m, H-3), 2.10(1H, m, H-4); 1.75(1H, m, H-7); 1.31(1H, m, H-7); 1.98, 1.73(2H, m, H-8); 5.58(1H, d, J=10.EHz, n-10); 5.67(1H, dd, J=l.7, 10.2Hz, n-11); 4.35(1H, br d, J=14.5Hz, n-13); 4.23(1H, br d, J=14.5Hz, H-13); 1.09(3H, s, CH3-14); 0.95(3H, s, CH3-15); and 1.27ppm (31-1, s, CH3-16). 13C N M R :

(CDCI3) 133.0, C-2, 73.4, C-3; 47.4, C-4, 54.6, C-5, 40.0, C-6; 27.8, C-7, 35.0, C-8; 65.7, C-9, 135.6, C-10, 133.2, C-11,154.0, C-12; 60.7, C-13; 21.3, C-14, 22.0, C-15; and 30.9ppm (C- 16).

338

15.

ModifiedTrichothecenes

Mass Spectrum: HREIMS: 252.173m/e (M+); calcd for C15H2403,252.173. CIMS: 253(M § + 1, 2%), 235(M + - OH, 50), 127(cleavage ofC-6/C-6 bond, 85), 125(90), 109(127 - I410, 100), and 107m/e (125 - 1-/20, 50), References D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene Mycotoxins ofFusarium sporotrichioides (MC- 72083); Tet. Lett., pp. 4133-4136 (1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

Modified Trichothecenes

339

Common/Systematic Name. FS-3 Molecular Formula/Molecular Weight C 1 5 H 2 0 0 3 ; M W -- 248.14124 16

0

13CH20H

General Characteristics Isolated as a colorless glass. Fungal Source

Fusarium sambucmum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit overnight, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum, and the nonpolar material~were removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1"1 (v/v), acetone, and acetone-methanol, 11 (v/v). The toluene-acetone (4:1, v/v) fraction was further purified by flash chromatography eluting with toluene-acetone (4:1v/v). This resulted in purified diacetoxyscirpenol, 3,15-diacetoxyscirpenol, 3,4,15triacetoxyscirpenol, neosolaniol, sambucoin, 4-monoacetoxyscirpenol, 15monoacetoxyscirpenol, and FS-3. Spectral Data UV:

max^~176 229nm (e = 13,000). IR:

(film) 3470(OH)and 1675crn1 (C=O). 'H NMR:

(CDCI3) 6.29(IH, t,J=1.5I-Iz,H-2); 2.32(11-1,d, J=18.8, H-4), 2.60(IH, d, J=I8.8Hz, H-4); 1.73(IH, dd, J=I2.0, 2.5Hz, H-7); 1.87(IH, dd, J=I2.0, 2.51-Iz,H-7); 2.20(IH, dd, J=19.0, 2.51-1z,H-8); 1.63(IH, dd, J=19.0, 2.SHz, H-8); 5.75(IH, br s, H-10), 4.49(IH, dd, d=I7.0, 1.5Hz, H-13), 4.56(IH, dd, J=17.0, 1.5Hz, H-13), 1.50(3H, s, CH3-14); 1.17(3H, s, CH3-15); and 1.93ppm (3H, s, CH3-16).

340

15.

ModifiedTrichothecenes

13C NMR: (CDC13) 129.6, C-2; 207.0, C-3; 50.6, C-4; 53.3, C-5; 47.1, C-6; 30.9, C-7; 28.3, C8; 160.1, C-9; 126.4, C-10; 202.5, C-11; 186.3, C-12; 61.6, C-13; 23.8, C-14; 18.2, C15; and 29.0ppm; C- 16. Mass Spectrum: HREIMS: 248.141m/e (M+); calcd for C15H2003,248.154; LREIMS: 248(M+, 1%), 181(14), 131(31), 124(26), and 69m/e (100). References D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

Modified Trichothecenes

341

Common/Systematic Name FS-4 Molecular Formula/Molecular Weight C15H2203; M W -- 2 5 0 . 1 5 6 8 9

16

HO"~

13CH2H 0 0

General Characteristics Isolated as a colorless glass. Fungal Source

Fusarium sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit overnight, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by preparative reversed-phased HPLC with water-acetonitrile (5:1, v/v) as mobile phase. This resulted in purified FS-4. Spectral Data UV: ,~ maxACa~ 198nm (e = 10,400 rr - ~:* transition). IR:

(film) 3450(OH) and 1690cm1 (C--O). 1H N~IR: (CDCla) 6.27(1H, t, J=l.5Hz, H-2); 2.15(1H, d, ,/--19.0, H-4), 2.73(1H, d, J=19.0Hz, H-4); 1.40(1H, m, n-7); 1.70(1H, m, n-7); 1.70(1H, m, n-8); 1.85(1H, m, n-8); 5.25(1H, d, J=10.0Hz, H-10); 5.61(1H, dd, J=10.0, 1.5Hz, H-11); 4.69(1H, d, J--17.0, n-13); 4.35(1H, d, J=17.0, n-13); 1.29(3H, s, CH3-14); 0.98(3H, s, CHa-15); and 1.25ppm (3H, s, CH3-16).

342

15.

ModifiedTrichothecenes

13C NMR: (CDC13) 128.1, C-2; 205.0, C-3; 49.0, C-4; 28.2, C-7; 32.5, C-8; 133.6, C-10; 130.9, C-11; 183.0, C-12; 62.0, C-13; 19.2, C-14; 18.0, C-15; and 29.0ppm (C-16). Mass Spectrum: EIMS: 235(4%, M + - Me), 125(86), 108(100), and 81m/e (83); exact mass for C8H130 ( M + - C7H902) calcd. 125.097; found 125.096m/e; C7H902 ( M + - C s n l 3 0 ) calcd 125.060, found 125.059; CsH11 (CsH130 - 1-120)calcd. 107.086, found 107.086. References D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

ModifiedTrichothecenes

343

Common/Systematic Name 3-Ketoapotrichothecene Molecular Formula/Molecular Weight C15H2203; M W -" 2 5 0 . 1 5 6 8 9 13

H

CH2OH

15

14

General Characteristics Colorless glass. Fungal Source Fusarium sambucmum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (2:1, v/v) fraction was further purified by preparative normal-phase TLC which resulted in purified 3ketoapotrichothecene. Spectral Data UV: ~. maxA'~~176 197nm(e=8,600 rr to n* transition). Im: (film) 3420(OH) and 1737cm1 (C=O). 1H NMR: (CDC13) 2.60(1H, dd, J=20.0, 2.2Hz, H-2); 2.79(1H, dd, J=20.0, 1.4Hz, H-2); 2.14(1H, dd, J=19.2, 1.4Hz, H-4), 2.68(1H, dd, J=19.2, 2.2Hz, H-4); 1.48(1H, m, H7); 1.63(1H, m, H-7); 2.05, 2.15(2H, m, H-8); 5.55(1H, m, H-10); 4.23(1H, m, H-11); 3.53(1H, d, J=l 1.3Hz, H-13); 3.83(1H, d, J=l 1.3Hz, H-13); 1.16(3H, s, CH3-14); 0.76(3H, s, CH3-15); and 1.68ppm (3H, s, CH3-16).

344

15.

ModifiedTrichothecenes

13CNMR: (CDC13) 49.6, C-2; 215.5, C-3; 49.6, C-4; 51.3, C-5; 46.2, C-6; 28.8, C-7; 26.8, C-8; 135.7, C-9; 210.4, C-10; 80.1, C-11; 90.8, C-12; 65.0,.C-13; 19.2, C-14, 15.3, C-15; and 22.6ppm (C- 16).

Mass Spectrum: HR IMS: 250.158m/e (M+); calcd for C15H2203,250.156. LREIMS: 250(M+,15%), 235(38), 124(45), 107(50), and 43m/e (100). References D. R. Sanson, D. G. Codey, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

ModifiedTrichothecenes

345

Commort/Systematic Name 3 a-Hydroxyapotrichothecene 3a, 13-Dihydroxy- 11-epiapotrichothec-9-ene Molecular Formula/Molecular Weight C15H2403; MW = 252.17254 13

H

CH2OH

15

14

General Characteristics Crystals from 2-propanol-hexane; mp., 167-169~

[a]D - 33.2 ~ (in ~EtOH).

Fungal Source Fusarium sambucmum, F. graminearum (ATCC 28114), F. crookwellense, F. culmorum, and F. sporotrichioides. Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which resulted in purified 3a-hydroxyapotrichothecene. Alternatively, the crude extract from F. graminearum was chromatographed on Florisil; after removal of 3-aeetyldeoxynivalenol, an oil remained which was chromatographed by medium-pressure liquid chromatography using LiChroprep silica gel column eluted with the following: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. The later fractions eluting with ethyl acetate contained the 3a and 3 [3-isomers; these were separated by preparative HPLC using a CSC-S nitrile column with a 6% 2-propanol-hexane mobile phase and a flow rate of 4 ml/minute. Spectral Data UV~ ~ Aoetonilrilr max

196nm (e=7,200 ~ to rr* transition).

346

15. ModifiedTrichothecenes

IR:

(film) 3400cm1 (OH). 1H NMR:

(CDC13) 1.74(1H, m, H-2); 2.62(1H, ddd, J-1.7, 6.1, 12.3Hz, H-2); 4.30(1H, b m, H-3), 1.64(1H, m, H-4); 2.15(1H, dd, J=10.3, 13.0Hz, H-4); 1.40(1H, dddd, J=2.0, 4.0, 5.8, 13.0Hz, H-7); 1.58(1H, m, H-7); 2.02(1H, m, H-8); 1.62(1H, b m, H-8); 5.55(1H, b sptet, H-10); 4.18(1H, b sptet, H-11); 3.18(1H, b t, J=l 1.1Hz, H-13); 3.77(1H, dd, J=8.0, ll.lHz, H-13); 1.02(3H, s, CH3-14); 0.95(3H, s, CH3-15); and 1.50ppm (3H, q,J=l.4Hz, CH3-16). 13C NMR: (CDC13) 44.4, C-2; 72.8, C-3; 43.5, C-4; 52.5, C-5; 44.7, C-6; 27.8, C-7; 29.3, C-8; 135.5, C-9; 21.4, C-10; 81.3, C-11; 92.5, C-12; 63.3, C-13; 19.2, C-14; 17.9, C-15; and 22.6ppm (C-16). Mass Spectrum: HREIMS: 252.173m/e (M+); calcd for C15H2403, 252.186. LREIMS: 252(M § , 28%), 237(17), 140(100), and 124role (51). References R. Greenhalgh, D. A. Fielder, L. A. Morrison, J-P Charland, B. A. Blaekwell, M. E. Savard, and J. W. Apsimon; Secondary Metabolites of Fusarium species: Apotriehothecene Derivatives; J. Agile. food Chem., Vol. 37, pp. 699-705(1989). D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi, In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

ModifiedTrichothecenes

347

Common/Systematic Nam_e 313-Hydroxyapotrichothecene Molecular Formula/Molecular Weight C15H2403; M W = 252.17254

H

15

13

,H2OH

14

General Characteristics Colorless glass. Fungal Source

Fusarium sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which resulted in purified 3[3-hydroxyapotrichothecene. Spectral Data UV:

~. maxA~176176196nm(e=7,800 z~to n* transition). IR:

(film) 3420 and 1048cm1 (-C-O-C-). 1H NMR: (CDC13) 1.38(1H, m, H-2); 2.45(1H, ddd, 3"=1.3, 5.8, 6.4Hz, H-2); 4.51(1H, b m, H3), 2.10(1H, H-4); 2.23(1H, H-4); 1.36(1H, m, H-7); 1.57(1H, m, H-7); 1.99(2H, m, H-8); 5.50(1H, H-10); 4.11(1H, H-11); 3.56(1H, d, J=l 1.4Hz, H-13); 3.78(1H, d, J=l 1.4Hz, H-13); 1.07(3H, s, CH3-14); 0.52(3H, s, CH3-15); and 1.63ppm (3H, b s, CH3-16).

348

15.

ModifiedTrichothecenes

13C NMR: (CDC13) 47.0, C-2; 74.2, C-3; 45.7, C-4; not observed, C-5; not observed, C-6; 27.5, C-7; 29.3, C-8; not observed, C-9; 122.5, C-10, 81.3, C-11; not observed, C-12; 65.5, C-13; 20.0, C-14; 16.5, C-15; and 22.5ppm (C-16). Mass Spectrum: HREIMS: 252.181m/e (M+); calcd for C15H2403,252.186. LREIMS: 252(M~, 15%), 237(15), 124(38), 107(92), and 83m/e (100). Reference D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989).

Macrocyclic Trichothecenes and Related Metabolites Trichodermadiene Trichodermadienediol A Trichodermadienediol B

12,13-Deoxytrichodermadiene

Verrol Tfichoverrol A (C6'R,C7'R)-Isotrichoverrol A (2'E, C6'R, C7'R)-Isotrichoverrol A Trichoverrol B (C6'R,C7'S)-Isotrichoverrol B (2'E, CCR,C7'S)-Isotrichoverrol B Trichoverrin A (2'E,4'Z)-Isotrichoverrin A (C6'R,C7'R)-Isotrichoverrin A 913,1013-Epoxyisotrichoverrin A 8~-Hydroxyisotrichoverrin A Trichoverrin B (C6'R,C7'R)-Isotrichoverrin B (2'E,4'Z)-Isotrichoverrin B 913,10j3-Epoxyisotrichoverrin B (2'E)- 12,13-Deoxyisotrichoverrin B (9'E)-Roridin L-2 Roridin L-2 Verrucarin A Verrucarin A 13-D-glucoside 2'-Dehydroverrucarin A Verrucarin B Verrucarin J Verrucarin K Verrucarin L Verrucarin L Acetate Roridin A Roridin A 1313-D-glucoside 8a-Butoyloxyroridin A 8a-Crotonyloxyroridin A Isororidin A Rofidin D Roridin D 13-D-glucoside Roridin E Roridin E 13-D-glucoside Isororidin E Epiroridin E Epiisororidin E 713,813-Epoxyisororidin E 349

350

16. MacrocyclicTrichothecenes and Related Metabolites

Roridin K Acetate Roridin H (Verruearin H) Roridin J 7J3,8J3-Epoxyroridin H 7j3,Sj3,2',3'-Diepoxyisororidin H Satratoxin F Satratoxin G Isosatratoxin F Isosatratoxin G Satratoxin H S-Isosatratoxin H 12'-Hydroxy-2'-isoverruearin J (PD 113,325) M-Isosatratoxin H (PD 113,326) Vertisporin

16.

Macrocyclic Trichothecenes and Related Metabolites

351

Common/Systematic Name Trichodermadiene Molecular Formula/Molecular Weight C23H3005; ~

"- 3 8 6 . 2 0 9 3 2

~o H

16

~s

H

j

14

[;,~,,0

g

H 0

Me

' 'bCCH=CHCH=CH---Ca'C:. II 2' 0

3'

4'

s'

6'

r

General Characteristics Crystallization from ether gave needles; mp., 145-146~ [~]D 27 -I-17.7~

in CHC13).

Fungal Source

Myrothecium verrucaria (ATCC 24571).

Isolation/Purification Trichodermadiene was isolated by a methanol extraction of the mycelium of a fermentation ofMyrothecium verrucaria. Chromatography on silica gel gave a series of the less polar trichothecenes (roridin H and verrucarins B and J) and a previously uncharacterized trichothecene which had an Rf value higher than any heretofore reported macrocyclic trichothecene. Crystallization from ether gave needles. Biological Activity Trichodermadiene showed a T/C = 143 at 26mg/kg in vivo against p388 mouse leukemia (PS). Spectral Data UV:

~, ~f~

264nm (log e = 4.41).

IR:

(CHC13) 1700, 1640, and 1600cm"1. 1H NMR: (CDC13) 3.83(1H, d, J=5.0Hz, H-2); 2.1(1H, m, H-313); 2.66(1H, dd, J=7.5, 15.0Hz, H-3a); 5.7(1H.dd, J=4.0, 7.5Hz, H-4); 5.43(1H, d, J=5.5Hz, H-10); 3.64(1H, d, J=5.5Hz, H-11); 2.97(2H, JAB=4.0Hz, H-13); 0.74(3H, s, H-14); 1.0(3H, s, H-15); 1.72(3H, s, H-16); 5.7(1H, d, J=l 1.0Hz, H-2'); 6.57(1H, t, J=l 1.0Hz, H-3'); 7.83(1H, dd, J=ll, 15.5Hz, H-4'); 5.7(1H, dd, J=8, 15.5Hz, H-5'); 3.21(1H, dd, J=2.0, 8.0Hz,

352

16. MacrocyclicTrichothecenes and Related Metabolites

H-6'); 2.97(1H. dq, J= 2.0, 5Hz, H-7'); and 1.36ppm (1H, d, Jr- 5.0Hz, H-7'). 13CNMR: (CDC13) 79.2 d, C-2; 36.9 t, C-3; 75.0 d, C-4; 49.2 s, C-5; 40.5 s, C-6; 24.6 t, C-7; 28.1 t, C-8; 140.0 s, C-9; 118.9 d, C-10; 70.6 d, C-11; 65.5 s, C-12; 47.8 t, C-13; 6.0 q, C-14; 16.1 q, C-15; 23.2 q, C-16; 165.8 s, C-1'; 118.4 d, C-2'; 143.1 d, C-3'; 130.3 d, C-4'; 140.4 d, C-5'; 58.8 d, C-6'; 56.8 d, C-7'; and 17.5ppm q, C-8'. Mass Data: EIMS: 386m/e (M+); anal. calcd for C23H3005: C, 71.48; H, 7.82%; found: C, 71.38, H, 8.07%. Reference B. B. Jarvis, J.O. Midiwo, G. P. Stahly, G. Pavanasasivam, and E.P. Mazzola; Trichodermadiene: A New Trichothecene; Tetrahedron Letters, Vol. 21, pp. 787788(1980).

16.

Macrocyclic Trichothecenes and Related Metabolites

353

Common/Systematic Name Trichodermadienediol A Molecular Formula/Molecular Weight C23H3206; M W -" 4 0 4 . 2 1 9 8 9

16

10 H

Me

H

I

0

I~Me

i~i Me

General Characteristics Crystallization from dichloromethane-hexane; mp., 184-185~ [a]D28 +26.3 ~ (C=0.39, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol in methylene chloride), II (eluted with 1-5% methanol in methylene chloride), III (eluted with 5-7.5% methanol in methylene chloride), and IV (eluted with methanol). Fraction II was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 1-5% dichloromethane in petroleum ether to give a fraction rich in the trichothecenes. This was chromatographed to yield an oil, Re 0.15-0.20 (silica gel, 3% methanol in methylene chloride). This material was subjected to HPLC (1-4% methanol-methylene chloride gradient) followed by HPLC with 70% ethyl acetate in hexane to yield triehodermadienediol A and triehodermadienediol B.

354

16. MacrocyclicTrichothecenes and Related Metabolites

Spectral Data UV;

maxMeOH260nm (log e=4.53). 1HN]VIR: (CDCI3) 0.76(3H, s, 14-H); 0.98(3H, s, 15-H); 1.13(3H, d, J=6I-Iz, 8'-H); 1.74(3H, s, 16-H); 2.60(1H, dd, J=8 and 15Hz, 3a-H); 3.03(2H, AB, d=4Hz, 13-H), 3.66(1H, d, J=5 Hz, 1l-H); 3.87(1H, d, J=5Hz, 2-H); 4.06(1H, m, 6'-H); 5.44(1H, d, J=5Hz, 10H), 5.73(1H, d, J=l 1Hz, 2'-H), 6.09(1H, dd, J=6, 16Hz, 5'-H); 6.64(1H, dd, J=l 1Hz, 3'-H); and 7.65ppm (1H, dd, J =11.0, 16Hz, 4'-H.

13CN M ~ :

(CDCI3) 79.1 d, C-2; 37.0 t, C-3; 75.0 d, C-4; 49.3 s, C-5; 40.5 s, C-6; 24.5 t, C-7; 28.1 t, C-8; 140.2 s, C-9; 118.7 d, C-I0; 70.6 d, C-11; 66.1 s, C-12; 48.1 t, C-13; 6.2 q, C-14; 16.1 q, C-15; 23.2 q, C-16; 166.0 s, C-I'; 118.7 d, C-2'; 143.4 d, C-3'; 127.9 d, C-4'; 141.9 d, C-5'; 76.6 d, C-6'; 70.6 d, C-7'; and 18.9ppm q, C-8'.

Mass Spectrum: HRCIMS (methane gas reagent): 4 0 5 . 2 2 6 3 m / e 4 8 9 . 2 4 5 5 m / e (M+ + H, calcd 489.2488).

(M ++

H, calcd 405.2277); diacetate,

Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verruearins, J. Org. Chem.,Vol. 47, pp. 1117-1124(1982).

16. Macrocyclic Trichothecenes and Related Metabolites

355

Common/Systematic Name Trichodermadienediol B Molecular Formula/Molecular Weight C23H3206; M W -- 4 0 4 . 2 1 9 8 9 16

Me~.---

I-

1o H

�9 13

'~./'~--7

H

OJ2

I

3

.... ,o

o

~

HO

I1~'

.

Me

General Characteristics Isolated as an oil; [a]D 28 - 15.8 ~ (C=0.80, in CHC13).

Fungal Source

Myrothecium verrucaria (ATCC 24571).

Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction II was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 1-5% dichloromethane in petroleum ether to give a fraction rich in the trichothecenes. This was chromatographed to yield an oil, Re, 0.15-0.20 (silica gel, 3% methanol-methylene chloride). This material was subjected to HPLC (1-4% methanol-methylene chloride gradient) followed by HPLC with 70% ethyl acetate in hexane to yield trichodermadienediol A and trichodermadienediol B.

356

16.. Macrocyclic Trichothecenes and Related Metabolites

Spectral Data UV~

~, maxM~~ 260rim(log C=4.55). 1H NMR: (CDCI3) 0.74(3H, s, 14-H); 0.98(3H, s, 15-H); 1.16(3H, d, J=6Hz, 8'-H); 1.73(3H, s, 16-H); 2.62(1H, dd, J=8,15Hz, 3a-H); 3.04(2H, AB, J=4Hz, 13-U); 3.66(1H, d, J=5Hz, 1l-H); 3.88(1H, d, J=5Hz, 2-H), 4.30(1H, m, 6'-H), 5.44(1H, d, J=5Hz, 10-H); 5.72(1H, d, d=l 1Hz, 2'-H); 6.16(1H, dd, J=6, 16Hz, 5'-H); 6.66(1H, dd, J's=l 1Hz, 3'-H); and 7.64ppm (1H, dd, J=l 1, 16Hz, 4'-H); trichodermadienediol B diacetate, 0.74(3H, s, 14-H); 0.98(3H, s, 15-H); 1.23(3H, d, J=6Hz, 8'-H); 1.74(3H, s, 16-H); 2.06 and 2.12(3H each, s, acetates); 2.60(1H, dd, J=8, 15Hz, 3a-H); 3.00(2H, AB, J=4Hz, 13-H); 3.64(1H, d, J=5Hz, 1l-H); 3.86(1H, d,J=5 Hz, 2-H); 5.16(1H, dq, J=4, 6Hz, 7'-H); 6.60(1H, dd, 3's=l 1Hz, 3'-H); and 7.68ppm (1H, dd, J=l 1, 16Hz, 4'H). 13C NMR: (CDC13) 79.1 d, C-2; 36.9 t, C-3; 74.9 d, C-4; 49.2 s, C-5; 40.5 s, C-6; 24.5 t, C-7; 28.0 t, C-8; 140.3 s, C-9; 118.6 d, C-10; 70.6 d, C-11; 66.1 s, C-12; 48.1 t, C-13; 6.1 q, C-14; 16.0 q, C-15; 23.2 q, C-16; 166.0 s, C-I'; 118.2 d, C-2'; 143.7 d, C-3'; 128.0 d, C-4'; 141.3 d, C-5'; 75.6 d, C-6', 70.3 d, C-7'; and 17.7ppm q, C-8'. Mass Spectrum: HRCIMS (methane gas reagent): 405.2252m/e (M§ + H, calcd 405.2277); diacetate, 489.2436role (M+ + H, caled 489.2488). Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola, and R. F. Creoghegan; Isolation and Characterization of the Tdchoverroids and New Roridins and Verrucarins; J. Org. Chem,Vol. 47, pp. 1117-1124(1982).

16.

Macrocyclic Trichothecenes and Related Metabolites

357

Common/Systematic Name 12,13-Deoxytrichodermadiene Molecular Formula/Molecular Weight C23H3004; MW = 370.21441

16

10H

3

H 13

O

,,,Z'.~ 0 8' General Characteristics Isolated as an oil;

[ a ] D 25 -

H

5.6 ~ (c=0.95, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction I was passed through a silica gel flash column and eluted with dichloromethane to yield several fractions. The first fraction was subjected to preparative TLC (20% hexane in methylene chloride) on 2-mm silica gel plates to yield an oil. Further purification was done on a Magnum-9 column (silica gel) under gradient conditions (90-100% methylene chloride in hexane) to g ive 12,13deoxytrichodermadiene as an oil.

358

16.

MacrocyclicTrichothecenes and Related Metabolites

Spectral Data UV:

k maxMeOH265nm (log r 1H NMR: (CDC13) 0.98(3H, s, 14-H); 0.98(3H, s, 15-H); 1.40(3H, d, J=5Hz, 8'-H); 1.72(3H, br s, 16-H); 2.00(1H, m, H-3[3); 2.62(1H, dd, J=7 and 15Hz, 3a-H); 4.75, 5.16(1H each, s, 13-H); 3.68(1H, d, J=5 Hz, 1I-H); 4.47(1H, d, J=5Hz, 2-H); 3.21(1H, dd, J=2.0, 8.0Hz, 6'-H); 5.40(1H, d, J=5Hz, 10-H); 5.80(1H, d, J=l 1Hz, 2'-H); 5.70(1H, dd, J=8, 15Hz, 5'-H); 6.60(1H, t, J-11Hz, 3'-H); 7.75(1H, dd, J=l 1.0, 15Hz, 4'-H); 5.70(1H, dd, J=8, 15Hz, 5'-H); 3.21(1H, dd, J=2, 8Hz, 6'-H), and 3.01ppm (1H, dq, J=2, 5Hz, 7'-H). 13CNMR: (CDC13) 75.7, C-2; 38.0, C-3; 75.1, C-4; 52.1, C-5; 40.4, C-6; 23.6, C-7; 28.1, C-8, 139.8, C-9; 119.0, C-10; 70.6, C-11; 152.7, C-12; 104.9, C-13; 10.2, C-14; 16.2, C15; 23.3, C-16; 169.4, C-I'; 118.3, C-2'; 143.2, C-3'; 129.9, C-4'; 140.3, C-5'; 58.8, C6'; 57.0, C-7'; and 17.5ppm, C-8'. Mass Spectrum: HRCIMS (methane gas reagent), 371.2216m/e (M+ + H, calcd 371.2222). Reference B. B. Jarvis, V. M. Vrudhula, J. O. Mikiwo, and E. P. Mazzola; New Trichoverroids from Myrothecium verrucaria: Verrol and 12,13-Deoxytrichodermadiene; J. Org. Chem., Vol. 48, pp. 2576-2578 (1983).

16.

MacrocyclicTrichothecenes and Related Metabolites

359

Common/Systematic Name Verrol Molecular Formula/Molecular Weight C21H3006; M W = 3 7 8 . 2 0 4 2 4

~ 0

~o H

H

O,J~

I

n

~ 6'

CH2CH2OH 5'

General Characteristics Isolated as an oil. Fungal Source

Myrothecium verrucaria (ATCC 24571).

Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to flash chromatography (silica gel, 0-8% methanol in methylene chloride) to yield three major fractions rich in roridin A, verrol, and trichoverrins, respectively. The verrol containing fraction was subjected to flash chromatography again under the conditions described above to yield a fraction which was mostly verrol and trichoverrols. This was further purified by column chromatography on silica (10-25% 2-propanol in hexane) to yield verrol as an oil.

360

16. Macrocyclic Trichothecenes and Related Metabolites

Spectral Data 1HNMR: (CDCI3) 0.87(3H, s, 14-H), 1.70(3H, br s, 16-H); 2.60(1H, dd, J=7.5, 15.7Hz, 3a-H); 2.00(1H, m, 31~-H);2.96(2H, AB, J=3.9Hz, 13-H); 5.42(1H, d, J=5.4Hz, 10-H); 5.73(1H, q, J=l.2Hz, 2'-H); 2.42(1H, t, J=6.1, 4'-1-1);3.82(1H, d, J=5.1Hz, 2-H); 1.9(2H, m, 7-H); 2.0(2H, m, 8-n); 3.64(1H, d, J=5.4Hz, 1l-H); 2.96(2H, AB, J=3.9Hz, 13-H); 4.05(2H, AB, J=12.4Hz, 15-H); 3.80(2H, t, J=6.1Hz, 5'-H); and 2.21ppm (3H, d, J=l.2Hz, 6'-H). 13CNMR: (CDCI3) 78.8, C-2; 39.9, C-3; 74.5, C-4; 49.2, C-5; 42.8, C-6; 21.4, C-7; 28.1, C-8; 140.7, C-9; 118.6, C-10; 66.7, C-11, 65.6, C-12; 47.6, C-13; 7.1, C-14; 62.9, C-15; 23.1, C-16; 166.0, C-I'; 117.2, C-2'; 157.3, C-3'; 43.9, C-4'; 60.3, C-5'; and 18.8ppm, C-6'. Mass Spectrum: HRCIMS (methane gas reagent): 379.2112m/e (M++ H, calcd 379.2120). Reference B. B. Jarvis, V. M. Vrudhula, J. O. Mikiwo, and E. P. Mazzola; New Trichoverroids from Myrothecium verrucaria: Verrol and 12,13-Deoxytrichodermadiene; J. Org. Chem., Vol. 48, pp. 2576-2578(1983).

16. MacrocyclicTrichothecenes and Related Metabolites

361

Common/Systematic Name Trichoverrol A Molecular Formula/Molecular Weight C23H3207; M W = 4 2 0 . 2 1 4 8 0

H H M e ~ . , , ~. ~ O ~- l _. _ II....iO ~"~I/~'/'~'HOH2(~ ~Me

0

"~0,~

I Me 7-~---. OH General Characteristics Crystals from ethyl acetate-petroleum ether; mp., 177-179~ [a]D2s +37.7 ~ (C=0.45, in CHCI3). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Re, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Re, 0.30 in 90% ethyl acetatepetroleum ether). After filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6% methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin B from fraction III-B.

362

16.

MacrocyclicTrichothecenes and Related Metabolites

Spectral Data

UV~ ~" maxMeOH260nm (log e=4.56). 1H NMR: (CDC13) 0.84(3H, s, 14-H); 1.19(3H, d, J=6I-Iz, 8'-H); 1.72(3H, s, 16-H); 2.46(1H, dd, ,/=8, 15Hz, 3a-H); 3.00(2H, AB, J=4H_z, 13=H); 5.49(1H, d, J=5Hz, 10-H); 5.73(1H, d, J--11Hz, 2'-H); 6.66(1H, dd, d's=l 1Hz, 3'-H); 7.62(1H, dd, J-11, 16Hz, 4'-H). 13CNMR: (CDCI3) 79.0d, C-2; 36.3 t, C-3; 75.5 d, C-4; 49.1 s, C-5; 44.4 s, C-6; 21.3 t, C-7; 28.2 t, C-8; 140.6 s, C-9; 118.8 d, C-10; 66.9 d, C-11; 66.1 s, C-12; 48.3 t, C-13; 6.8 q, C-14; 62.7 t, C-15, 23.3 q, C-16; 166.9 s, C-I'; 118.2 d, C-2'; 144.2 d, C-3'; 127.8 d, C-4'; 142.5 d, C-5'; 76.5 d, C-6'; 70.7 d, C-7'; and 19.0ppm q, C-8'. Mass Spectrum: HRCIMS (methane gas reagent): 421.2226m/e

(M + +

H, calcd 421.2226).

Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and g. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

16.

Macrocyclic Trichothecenes and Related Metabolites

363

Common/Systematic Name (C6'R, C7'R)-Isotrichoverrol A Molecular Formula/Molecular Weight C23H3207; M W -- 4 2 0 . 2 1 4 8 0

~o H I

~I

H ~3

i14

I

.~

~,~,"

I

O

~.~ 2

HO' ~ : ~8' H General Characteristics Isotrichoverrol A was recrystallized from methylene chloride-hexane to give crystals; mp., 180-183~ [a]D2~+54.0 ~ (C=1.65, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol in methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give (C6'R, C7'R)-isotrichoverrol A. Spectral Data 1H NMR: (CDC13) 0.81(3, s, H-14); 1.19(3H, d, J=6.3 Hz, H-8'); 1.54(1H, brd, J=7.6Hz, H-7A); 1.70(3H, s, H-16); 1.98(3H, m, H-7B, H-8); 2.08(1H, ddd, J=4.0, 5.1, 15.4I~, H-313), 2.49(1H, dd, J=8.0, 15.4Hz, H-3a); 2.83(1H, d, J=4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.63(1H, d, J-12.3Hz, H-15A); 3.67(1H, dq, J=6.3, 6.3Hz, H-7'); 3.80(1H, d, J=12.3Hz, H-15B); 3.83(1H, d, J=5.1Hz, H-2'); 3.88(1H, br d, J=5.0Hz, H-11); 4.03(18, m, H-6'); 5.47(1H, br d, J=5.0Hz, H-10); 5.70(1H, d, J=l 1.5Hz, H-2'); 6.06(1H, dd, J=5.7, 15.5Hz, H-5'); 6.08(1H, m, H-4); 6.61(1H, dd, J=11.3, 11.5Hz, H-3'); and 7.59ppm (1H, dd, J= 11.3, 15.5Hz, H-4').

364

16.

MacrocyclicTrichothecenes and Related Metabolites

13C NMR: (CDC13) 79.0d, C-2; 36.3 t, C-3; 75.5 d, C-4; 49.1 s, C-5; 44.4 s, C-6; 21.3 t, C-7; 28.2 t, C-8; 140.6 s, C-9; 118.8 d, C-10; 66.9 d, C-11; 66.1 s, C-12; 48.3 t, C-13; 6.8 q, C-14; 62.7 t, C-15; 23.3 q, C-16; 166.9 s, C-I'; 118.2 d, C-2'; 144.2 d, C-3'; 127.8 d, C-4'; 142.5 d, C-5'; 76.5 d, C-6'; 70.7 d, C-7'; and 19.0ppm q, C-8'. Mass Spectrum: HRCIMS: calcd for C23H3307 ([M + + H] +) 421.2226; found 421.221 lm/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins, J. Org. Chem.,Vol. 47, pp. 1117-1124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16. MacrocyclicTrichothecenes and Related Metabolites

365

Common/Systematic Name (2'E, C6'R, C7'R)-Isotrichoverrol A Molecular Formula/Molecular Weight C23H3207, MW = 420.21480 16

10

H -

~l

H

2

3

NO

.17'

8'

_

General Characteristics Isolated as an oil. Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative CCC (V~=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3 1.2, v/v/v/v), flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) (ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S1Fla, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. Fraction S1F2 contained mainly trichoverrols according to TLC analysis. The sample was subjected to preparative CCC with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min to give six fractions: S 1F2a, S 1F2b, S 1F2c, S1F2d, S 1F2e, and S 1F2f. S1F2e was further chromatographed on CCC with a solvent system of of chloroformhexane-methanol-water (3:1:3:2, v/v/v) and a flow rate of 2.0ml/min. The chromatogram

366

16.

MacrocyclicTrichothecenes and Related Metabolites

appears as a single peak; however, the eluent was collected into two parts, A and B. Fraction A was subjected to CCC (analytical column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (3:5:2:6:4, v/v/v/v/v) and a flow rate of 1 ml/min (ca. 40mg/injection) to give (2'E, C6'R,C7'R)-isotrichoverrol A and isotrichoverrol B as oils. Spectral Data UV~

~, M~o, 262nm (log e=4.36). max

IR~

(CHCI3) 3600, 3470(OH), 1703(C=O), 1644(C=C), and 1620cm~ (C=C). :H NMR: (CDCI3) 0.80(3H, s, H-14), 1.19(3H, d, J=6.3Hz, H-8'); 1.70(3H, s, H-16); 2.47(1H, dd, J=8.1, 15.3Hz, H-3a),(1H, d, J=4.0Hz, H-13A); 3.12(1H, d, J=4.0Hz, H-13B); 3.64(1H, d, J=12.5Hz, H-15A); 3.67(1H, dd, J-6.3Hz, H-7'); 3.80(1H, d, ,/=12.5Hz, H-15B); 3.83(1H, d, J=5.0Hz, H-2); 3.92(1H, d, J=5.2Hz, H-11); 3.97(1H, dd, J=6.1, 6.3Hz, H-6'); 5.48(1H, bd, J=5.2Hz, H-10); 5.94(1H, d, J=15.4Hz, H-2'), 6.10(1H, dd, J=6.1, 15.4Hz, H-5'); 6.12(1H, dd, J=3.7, 8.2Hz, H-4); 6.45(1H, dd, J=l 1.3, 15.4Hz, H-4'); and 7.29ppm(1H, dd, J=l 1.3, 15.4Hz, H-3'). ~3CNMR: (CDCI3) 6.4, C-14; 19.1, C-8'; 21.2, C-7; 23.3, C-16; 28.0, C-8; 35.9, C-3; 44.3, C-6, 48.2, C-13; 48.9, C-5; 62.9, C-15; 65.6, C-12; 66.8, C-11; 70.6, C-7'; 75.6, C-4; 76.5, C-6'; 79.0, C-2; 118.7, C-10; 121.6, C-2'; 129.6, C-4'; 140.4, C-9; 141.5, C5'; 144.4, C-3'; and 167.7ppm, C-I' Mass Spectrum: HREIMS calcd for C23H3207(M§ 420.2148; found, 420.2164m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261(1996).

16.

Macrocyclic Trichothecenes and Related Metabolites

367

Common/Systematic Name Trichoverrol B Molecular Formula/Molecular Weight

Meoj

C23H3207; M W = 4 2 0 . 2 1 4 8 0

H

H

.....iO

O

Me ~0~

HOH26 I

General Characteristics Isolated as an oil; [a]D 25 -3.3 ~ (c=0.39, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate/hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Rf, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Re, 0.30 in 90% ethyl acetatepetroleum ether). After filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6%methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin B from fraction III-B.

368

16. MacrocyclicTrichothecenes and Related Metabolites

Spectral Data

UW: ~bmaxMeOH260nm (log e=4.53). 1H NMR: (CDC13) 0.84(3H, s, 14-H); 1.16(3H, d, J=6Hz, 8'-H); 1.74(3H, 8, 16-H); 2.47(1H, dd, J=8, 15Hz, 3a-H); 3.07(2H, AB, J=4Hz, 13-H); 4.18 (1H, m, 6'-H); 5.50(1H, d, J=5Hz, 10-H); 5.74(1H, d, J=l 1Hz, 2'-H); 6.67(1H, dd, J=l 1, 11Hz, 3'-H); 7.61(1H, dd, J=l 1, 16Hz, 4'-H). 13C NMR: (CDC13) 79.0 d, C-2; 36.2 t, C-3; 75.5 d, C-4; 49.1 s, C-5; 44.4 s, C-6; 21.2 t, C-7; 28.12 t, C-8; 140.6 s, C-9; 118.8 d, C-10; 66.9 d, C-11; 66.0 s, C-12; 48.3 t, C-13; 6.8 q, C-14; 62.6 t, C-15; 23.2 q, C-16; 167.0 s, C-I'; 117.9 d, C-2'; 144.3 d, C-Y; 128.0 d, C-4'; 141.6 d, C-5'; 75.5 d, C-6'; 70.3 d, C-7'; and 17.8ppm q, C-8'. Mass Spectrum: HRCIMS (methane gas reagent): 421.2223m/e

(M ++

H, calcd 421.2226).

Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem.,Vol. 47, pp. 1117-1124(1982).

16.

Macrocyclic Trichothecenes and Related Metabolites

369

Common/Systematic Name (C6'R, C7'S)-Isotrichoverrol B Molecular Formula/Molecular Weight C23H3207; M W -- 4 2 0 . 2 1 4 8 0

10 H

~

I-

111 13

H

I ,iO

~

0

HOH2~ ,,

HI0 H1

3'

General Characteristics Isotrichoverrol B was isolated as an oil; [a]D2~- 4.0 ~ (C=1.50, in CHel3). Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermemation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative high speed countercurrent chromatography (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Spectral Data 1H NMR: (CDCI3) 0.80(3H, s, H-14); 1.12(3H, d, J-6.41-Iz, H-8'); 1.57(IH, m, H-7A); 1.70(3H, s, H=16); 1.98(31-1, m, H-7B, H-8); 2.08(IH, ddd, d=4.0, 5.2, ]5.41-Iz, H-313); 2.49(1H, dd, J=7.8, 15.4Hz, H-3tt); 2.82(1H, d, J=3.9Hz, H-13A); 3.12(1H, d, J=3.9Hz, H-13B); 3.61(1H, d, J=12.3Hz, H-15A); 3.81(1H, d, J=12.3Hz, H-15B); 3.83(1H, d, J=5.2Hz, H-2); 3.86(1H, br d, J=5.1Hz, H-11); 3.90(1H, dq, J=3.5, 6.5Hz, n-7'); 4.25(1H, m, n-6'); 5.45(1H, br d, J=5.1Hz, H-10); 5.70(1H, d,

370

16. MacrocyclicTrichothecenes and Related Metabolites

J=l 1.4Hz, H-2'); 6.09(1H, m, H-4); 6.11(1H, dd, ,/-6.0, 15.4Hz, H-5'); 6.63(1H, dd, ,/--11.3, 11.4Hz, H-3'); and 7.55ppm (1H, dd, J=l 1.3, 15.4Hz, H-4'). 13C NMR: The 13C NMR data are identical to those reported earlier for (C6'S,C7'R)-trichoverrol B.

Mass Spectrum: HRCIMS: calcd for C23H3307; [M+ + H] +, 421.2226; found, 421.2206m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins, J. Org. Chem.,Vol. 47, pp. 1117-1124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16.

MacrocyclicTrichothecenes and Related Metabolites

371

Common/Systematic Name (2'E, C6'R,C7'S)-Isotrichoverrol B Molecular Formula/Molecular Weight C23H3207; M W -- 4 2 0 . 2 1 4 8 0 ~o

H

H

~ ~

''"

HOH2l,, "

5

15

0

4

II

IL HO ....i 7'

8'

H General Characteristics Isolated as an oil. Fungal Source

Myrothecium verrucaria (ATCC 20540).

Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S1 was subjected to semipreparative CCC (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v; ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S1Fla, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. Fraction S1F2 contained mainly trichoverrols according to TLC analysis. The sample was subjected to preparative CCC with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min to give six fractions: S 1F2a, S 1F2b, S 1F2c, S 1F2d, S 1F2e, and S 1F2f. S1F2fwas subjected to CCC (analytical column) with a solvent system of methylene chloride-carbon tetrachloride-methano!-wat~r (2:3:3:2, v/v/v/v) and a flow rate of 1

372

16. MacrocyclicTrichothecenes and Related Metabolites

ml/min to give a mixture of (2'E)-isotrichoverrol B and another unknown trichoverroid. This mixture was chromatographed on CCC (analytical column) with a solvent system of methylene chloride-carbon tetrachloride-hexane-methanol-water (3:5:2:6:4, v/v/v/v/v) and a flow rate of lml/min to give pure (2'E)-isotrichoverrol B, as an oil. Spectral Data UV~ ~, M~o. 262nm (log e = 4.44). max

IR~ (CHCI3) 3600, 3470(OH), 1705(C=O), 1645(C=C), and 1621cm1 (C=C). 1H NMR: (CDC13) 0.79(3H, s, H-14); 1.12(3H, d, J=6.5 Hz, H-8'); 1.69(3H, s, H-16); 2.47(1H, dd, J=8.0, 15.3Hz, H-3a); 2.80(1H, d, J=4.0Hz, H-13A); 3.11(1H, d, J=4.0Hz, H13B); 3.63(1H, d, J=12.ZHz, H-15A); 3.80(1H, d, J=lZ.ZHz, H-15B); 3.82(1H, 3, J=5.1Hz, H-2); 3.88-3.93(2H, m, H11 and H-7'); 4.21(1H, m, H-6'); 5.47(1H, bd, J=4.5Hz, H-10); 5.93(1H, d, J=15.3Hz, H-2'); 6.10(1H, dd, J=3.6, 8.2Hz, H-4); 6.13(1H, dd, J=5.9, 15.9Hz, H-5'); 6.42(1H, dd, J=10.9, 15.9Hz, H-4'); and 7.29ppm (1H, dd, J=10.9, 15.3Hz, H-3').

13C NMR: (CDC13) 6.4, C-14; 17.6, C-8'; 21.2, C-7; 23.2, C-16; 28.0, C-8; 35.9, C-3; 44.3, C-6; 48.1, C-13; 48.9, C-5; 62.8, C-15; 65.6, C-12; 66.8, C-11; 70.1, C-7'; 75.3, C-6'; 75.6, C-4; 79.0, C-2; 118.8, C-10; 121.5, C-2'; 129.6, C4'; 140.5, C-5'; 140.5, C-9; 144.5, C3'; and 167.7ppm, C-I'. Mass Spectrum: HR IMS: calcd for C23H3207 (M+), 420.2148; found, 420.2188m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16. MacrocyclicTrichothecenes and Related Metabolites

373

Common/Systematic Name Trichoverrin A Molecular Formula/Molecular Weight C28I--I3809; M W -- 5 1 8 . 2 5 1 5 8

H

LA.y

{)l

H

.. ,o i ,

Me

'

.CH2 HO--~8' (~H20H Me. !;',OH _

General Characteristics Crystals from ethyl acetate-petroleum ether; mp., 78-79~ [t~]D2s -21.5 ~ (C=0.39, in CHC13). Fungal Source

Myrothecium verrucaria (ATCC 24571).

Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Rf, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Re, 0.30 in 90% ethyl acetatepetroleum ether). Atter filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6% methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin

374

16. MacrocyclicTrichothecenes and Related Metabolites

B from fraction III-B. Spectral Data

Lv~ ,~ maxMeOH 260nm (log e=4.60). ~H NMR:

(CDCI3) 0.82(3H, s, 14-H); 1.21(3H, d, J=6I-Iz,8'-H); 1.74(3;H, s, 16-H); 2.53(I;H, dd, ,/=8, 15Hz, 3(x-H);3.04(2H, AB, J=4Hz, 13-H); 4.18(2H, s, 15-H); 5.52(IH, d, J=5Hz, 10-H); 5.75(IH, d, J=l IHz, 2'-H); 5.90(IH, s, 2'-H);6.67(IH, dd, Js=l IHz, 3'-H); and 7.63ppm (IH, dd.,J=l I, 16Hz, 4'-H). 13C NMR: (CDC13) 79.1 d, C-2; 36.9 t, C-3; 75.1 td, C-4; 48.6 s, C-5; 42.9 s, C-6; 21.9 t, C-7; 27.9 t, C-8; 140.5 s, C-9; 118.5 d, C-10; 66.7 d, C-11; 65.8 s, C-12; 48.2 t, C-13; 6.7 q, C-14; 63.5 t, C-15; 23.2 q, C-16; 166.0 s, C-I'; 118.1 d, C-2'; 144.1 d, C-3'; 127.5 d, C-4'; 142.4 d, C-5'; 76.5 d, C-6'; 70.5 d, C-7'; 18.9 q, C-8'; 166.0 s, C-l"; 116.9 d, C2"; 157.3 s, C-3"; 43.6 t, C-4"; 59.7 t, C-5"; and 19.1ppm q, C-6". Mass Spectrum: HRCIMS (methane gas reagent): 533.2754m/e (M § + H, calcd 533.2754). Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

16.

Macrocyclic Trichothecenes and Related Metabolites

375

Common/Systematic Name (2'E,4'Z)-Isotrichoverrin A Molecular Formula/Molecular Weight C29H4009;

"

Me

MW

,0

-- 5 3 2 . 2 6 7 2 3

U

H

~;;

I

I-

O.3

0

,,,0

H2~~Me ~ 0 14

"0~

I

07~~~'H Me'~CH2

1'

,

HO i, HO3,J:L-.7 Me _

~H2OH 5" General Characteristics Isolated as an amorphous solid. Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative CCC (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400mg/injeetion, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v; ca. 400mg/injection). The major fraction of this sample was eluted with. little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S IF 1a, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. S1Fle was subjected to C18 reversedphase TLC (70% methanol in 0.5 M aqueous NaC1) to give (2'E,4'Z)-isotrichoverrin A.

376

16. MacrocyclicTrichothecenes and Related Metabolites

S.pectral Data UV:

), mx,M~~ 261nm(log e=4.49). (CHC13) 3600(OH), 1710(C=O), and 1652cm-1 (C=C). 1H NMR: (CDC13) 0.78(3H, s, H-14); 1.12(3H, d, J=6.3Hz, 8'-H); 1.69(3H, s, 16-H); 2.17(3H, d, J-1.0Hz, 6"-H); 2.40(2H, m, H-4"); 2.55(1H, dd, J=7.9, 16.0Hz, H-3~); 2.81(1H, d, J-4Hz, H-13A); 3.13(1H, d, J=4.0Hz, H-13B); 3.66(1H, dq, J values-~ 6.6Hz, H-7'); 3.83(1H, d, J=5.3Hz, H-2); 3.94(1H, bd, J=4.8Hz, H-11); 4.10(2H, s, H-15); 4.37(1H, dd, J=6.7, 8.1Hz, H-6'); 5.46(1H, bd, J-=4.8Hz, H-10); 5.79(1 H, dd, J=8.3, 10.7Hz, H-5'); 5.81(1H, d, J=l.0Hz, H-2"); 5.95(1H, d, J=15.2Hz, H-2'); 6.13(1H, dd, J=3.4, 7.9Hz, H-4); 6.24(1H, dd, J=10.7, 11.9Hz, H-4'); and 7.64ppm(1H, dd, J= 11.9, 15.2Hz, H-3'). 13CNMR: (CDC13) 6.6, C-14; 18.6, C-8'; 19.0, C-6"; 21.9, C-7; 23.2, C-16; 27.8, C-8; 36.7, C3; 43.0, C-6; 43.7, C-4"; 48.1, C-13; 48.6, C-5; 59.7, C-5"; 63.5, C-15; 65.5, C-12; 66.8, C-11; 70.7, C-7'; 72.4, C-6'; 75.7, C-4; 79.1, C-2; 117.1, C-2"; 118.5, C-10; 123.1, C-2'; 129.2, C4'; 138.6, C-5'; 139.8, C-3'; 140.5, C-9; 157.0, C-3"; 165.9, C-I"; and 166.9ppm, C- 1' Mass Spectrum: HRCIMS: caled for C29H4109

( M + d-

1), 533.2751; found, 533.2772m/e.

References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; J. Org. Chem., Vol. 47, pp. 1117-1124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261(1996).

16. MacrocyclicTrichothecenes and Related Metabolites

377

Common/Systematic Name (C6'R, C77~)-Isotrichoverrin A Molecular Formula/Molecular Weight C29H4009; M W --- 5 3 2 . 2 6 7 2 3

H

H

,,,,0 |

0

i

5

General Characteristics Isolated as an amorphous solid; [~]D2~ + 5.6 ~ (C=2.10, in CHel3). Fungal Source

Myrothecium verrucaria (ATCC 20540).

Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 2 ($2) contained mainly trichoverrins according to TLC analysis. This sample was chromatographed on CCC (semipreparative column, Vc=355ml) with a solvent system of chloroform-hexane-methanol-water (31:3:2, v/v/v/v) and a flow rate of 3.2ml/min (ca. 400mg/injection). Like fractions were combined to give seven fractions: S2F 1, $2F2, $2F3, $2F4, $2F5, $2F6, and $2F7. A portion of $2F2 was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 2.8ml/min. The components of the mobile phase (organic layer, methylene chloride-carbon tetrachloride) were varied from 2:3 to 5:2 (v/v), starting at t = 120 min and going to t = 160 min. Like portions were combined to give seven fractions: I, (C6'R, C7'R)-isotrichoverdn A; II, (C6'R, C7'S)-isotrichoverrin B; III, a mixture of isotrichoverrin B and trichoverdn C); IV, V, VI, 2~E-isotrichoverdn A; VII, 2'E-isotrichoverrin B.

378

16.

MacrocyclicTrichothecenes and Related Metabolites

Spectral Data 1H NMR: (CDC13) 0.79(3H, s, H-14); 1.19(3H, d, J=6.3Hz, H-14'); 1.70(3H, s, H-16); 2.17(3H, d, J= 1.0Hz, H-6"); 2.40(2H, m, H-4"); 2.56(1H, dd, J=7.6, 15.5Hz, H-3a); 2.82(1H, d, J=4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.66(1H, dq, ,/=6.3, 6.3Hz, H-7'); 3.80(2H, m, H-5"); 3.84(1H, d, J=5.4Hz, H-2); 3.98(1H, d, J=4.7Hz, H11); 4.03(1H, m, H-6'); 4.07(1H, d, J=12.5Hz, H-15A); 4.14(1H, d, J=12.5Hz, H15B); 5.46(11-1, d, J=4.7Hz, H-10); 5.67(1H, d, J=l 1.3Hz, H-2'); 5.85(1H, d, J=l.0Hz, H-2"); 6.07(1H, dd, J=4.7, 15.5Hz, H-5'); 6.20(1H, dd, ,/=7.6, 15.5Hz, H-4), 6.59(1H, dd, J= 11.3, 11.3Hz, H-3'); and 7.54ppm(1H, dd, J= 11.3, 15.5Hz, H-4'). 13CNMR: (CDC13) 6.7, C-14; 18.9, C-8'; 19.1, C-6"; 21.9, C-7; 23.2, C-16; 27.9, C-8; 36.9, C3; 42.9, C-6; 43.6, C-4"; 48.2, C-13; 48.6, C-5; 59.7, C-5"; 63.4, C-15; 65.8, C-12; 66.6, C-11; 70.6, C-7'; 75.0, C-4; 76.1, C-6'; 79.1, C-2; 117.0, C-2"; 118.2, C-2'; 118.5, C-10; 127.1, C-4'; 140.4, C-9; 142.1, C-5'; 143.7, C-3'; 157.0, C-3"; 165.9, C1"; and 166.0ppm, C- 1'. Mass Spectrum: HRCIMS: calcd for C29H4109 (M q- H) +, 533.2751; found, 533.2759role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16. Macrocyclic Trichothecenes and Related Metabolites

379

Common/Systematic Name 913,1013-Epoxyisotrichoverrin A Molecular Formula/Molecular Weight C29H40010; MW = 548.26215

o. Yo3

.,,,,o

I

?

Me

0/

"i~2'

',/

y)l,

'e/\cH2CH225

General Characteristics Isolated as an oil; [~]D2~ - 16~ (C=0.12, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 3 ($3) fromM, verrucaria isolate ATCC 20540, which was more polar than those that contained trichoverrols (S 1) and trichoverrins ($2), was partitioned between chloroform and methanol-water mixture (50%). The organic fraction was subjected to preparative TLC (Chromatotron, 2 mm, silica gel) with methanol-methylene chloride (310%). The most polar fraction was subjected to high speed countercurrent distribution (CCC) with a solvent system of chloroform-hexane-methanol-water (12:8:15:5, v/v/v/v) at a flow rate of 1.8ml/min to give 913,1013-epoxyisotrichoverrin A and 913,1013epoxyisotrichoverrin B.

380

16. MacrocyclicTrichothecenes and Related Metabolites

Spectral Data 1H NMR: (CDC13) 0.72(3H, s, H-14); 1.20(3H, d, J=6.5Hz, H-8'); 1.34(3H, s, H-16); 1.602.00(5H, m, H-7, H-8, S-3~); 2.20(3H, d, J=0.9Hz, S-6"); 2.41(2H, t, J - 6.0Hz, H-4"); 2.53(1H, dd, J=7.9, 15.5Hz, H-3a); 2.78(1H, d, J=4.0Hz, H-13A); 3.11(1H, d, J=5.5Hz, H-10); 3.19(1H, d, J=4.0Hz, H-13B); 3.67(1H, dq J=6.5, 6.5Hz, H-7'); 3.81(2H, m, H-5"); 3.89(1H, br d, J=5.5Hz, H-11);3.93(1H, d, J-5.1Hz, H-2); 4.03(1H, m, H-6'); 4.04(1H, d, J=12.5Hz, H-15A); 4.15(1H, d, J=12.5Hz, H-15B); 5.68(1H, d, J-11.3Hz, H-2'; 5.82(1H, d, J=0.9Hz, H-2"); 6.03(1H, dd, J=3.5, 7.5Hz, H-4); 6.08(1H, dd, ,]=5.8, 15.4Hz, H-5'); 6.60(1H, dd, J=l 1.3, 11.3Hz, H-3'); and 7.55ppm (1H, dd, J=l 1.3, 15.4Hz, H-4'). 13CNMR: (CDC13) 6.8, C-14; 18.9, C-8'; 19.2, C-6"; 19.4, C-7; 22.4, C-16; 26.5, C-8; 26.5, C8; 36.6, C-3; 42.6, C-6; 43.6, C-4"; 48.0, C-13; 48.5, C-5; 57.3, C-10; 57.5, C-9; 59.7, C-5"; 63.1, C-15; 65.2, C-12; 66.9, C-11; 70.6, C-7'; 74.8, C-4; 76.2, C-6'; 78.7, C-2; 116.5, C-2"; 118.1, C-2'; 127.5, C-4'; 142.2, C-5'; 143.9, C-3'; 158.1, C-3"; 165.8, C1'; and 166.0ppm, C- 1" Mass Spectrum: HRCIMS: calcd for C29H41Olo(M + H)+, 549.2700; found, 549.2732m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261(1996).

16. MacrocyclicTrichothecenes and Related Metabolites

381

Common/Systematic Name 8tt-Hydroxyisotrichoverdn A Molecular Formula/Molecular Weight C29H400]0; MW = 548.26215

Me~

HO'

H

H ....,0

H ?IoMe

V,Jl:

J

M~> General Characteristics Isolated as an oil;

0

HO,,, ~,

,

HO-4.

~

"CH2CH20H H ,, 5

[ ~ ] D 20 -

22.0 ~ (c=0.37, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carded out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 4 ($4) was triturated with methanol. The soluble portion was concentrated and dissolved in 50% aqueous methanol solution, and the solution was washed with carbon tetrachloride-hexane, chloroform-hexane (7:3, v/v), and chloroform. The aqueous phase was concentrated by rotary evaporation and extracted with chloroform. The chloroform extract was subjected to high-speed countercurrent distribution (CCC) with a solvent system of chloroform-hexane-methanol-water (7:3:5:5, v/v/v/v) to give 16hydroxyisotrichodermadienediols A and B and 8a-hydroxyisotrichoverrin A.

382

16. Macrocyclic Trichothecenes and Related Metabolites

Spectral Data 1H NMR: (CDC13) 0.81(3H, s, H-14); 1.19(3H, d,J=6.3Hz, H-14'); 1.69(1H, br d,J=14.4Hz, H-7~); 1.84(3H, s, H-16); 2.02(1H, m, H-313); 2.17(3H, s, H-6"); 2.32(1H, dd, J=6.5, 14.4Hz, H-7t~); 2.39(2H, m, H-4"); 2.58(1H, dd, J=7.8, 15.5Hz, H-3t~); 2.85(1H, d, J=4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.66(1H, dq, ,/=6.3, 6.3Hz, H-7'); 3.78(2H, m, H-5"); 3.83(1H, d, J=5.3Hz, H-2); 4.03(1H, br d, J=5.7Hz, H-11); 4.09(1H, m, H-6'); 4.11 (1H, br d, J=6.5Hz, H-8); 4.24(1H, d, Jr=13.0Hz, H- 15A); 4.39(1H, d, J=13.0Hz, H-15B); 5.58(1H, br d, J=5.7Hz, H-10); 5.68(1H, d, J=l 1.3Hz, H-2'); 5.85(1H, s, H-2"); 6.07(1H, dd, J=5.1, 15.5Hz, H-5'); 6.30(1H, dd, J=3.1, 7.8Hz, H-4); 6.60(1H, dd, J= 11.3, 11.3Hz, H-3'); and 7.53ppm (1H, dd, J= 11.3, 15.5Hz, H-4'). 13C NMR: (CDC13) 6.6, C-14; 18.9, C-8'; 19.2, C-6"; 20.4, C-16; 31.3, C-7; 36.8, C-3; 42.9, C6; 43.5, C-4"; 48.3, C-13; 48.5, C-5; 59.6, C-5"; 64.6, C-15; 65.8, C-12; 66.5, C-8; 66.8, C-11; 70.5, C-7'; 74.8, C-4; 76.4, C-6'; 79.0, C-2; 116.9, C-2"; 118.1, C-2'; 120.9, C-10; 127.3, C-4', 139.8, C-9; 142.2, C-5'; 144.0, C-3'; 157.2, C-3"; 165.2, C1'; and 166.0ppm, C- 1" Mass Spectrum: HRCIMS: calcd for C29H41010(M + H)+, 549.2700; found, 549.2710m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod.,Vol. 59, pp. 254-261 (1996).

16.

Macrocyclic Trichothecenes and Related Metabolites

383

Common/Systematic Name Trichoverrin B Molecular Formula/Molecular Weight C29I--I4009; M W = 5 3 2 . 2 6 7 2 3

H

H

M e,,,,,,,.,,,~,,~/..~ O.,,,J " I

0

iO

I Me

U '1 M e ' 7 " " C H2 (L H20H ~" General Characteristics Isolated as an oil;

HO HO,--+~. Me 8' H

[a]D 28 - 3 2 . 2 ~

(c=0.57, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Re, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Rf, 0.30 in 90% ethyl acetatepetroleum ether). After filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6% methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin

384

16. MacrocyclicTrichothecenes and Related Metabolites

B from fraction III-B. Spectral Data UV;

maxM~O" 260nm (log e=4.53). 1H NMR: (CDCI3) 0.82(3H, s, 14-H), 1.15(3H, d, J=6Hz, 8'-H); 1.70(3H s, 16-H); 2.48(1H, dd, J=8, 15Hz, 3a-H); 3.04(2H, AB, J=4Hz, 13-H); 4.18(2H, s, 15-H); 5.51(1H, d, J=5Hz, 10-H), 5.73(1H, d, J=l 1Hz, 2'-H); 5.93(1H, 8, H-2'); 6.68(1H, dd, d's=l 1Hz, H-3'); and 7.58ppm (1H, dd, J= 11, 16Hz, 4'-H).

13C NMR: (CDC13) 79.2 d, C-2, 36.9 t, C-3, 75.1 d, C-4; 48.6 s, C-5; 42.9 s, C-6, 21.9 t, C-7; 27.9 t, C-8; 140.5 s, C-9; 118.6 d, C-10; 66.7 d, C-11; 65.8 s, C-12; 48.3 t, C-13; 6.7 q, C-14; 63.5 t, C-15; 23.2 q, C-16; 166.1 s, C-I'; 117.9 d, C-2'; 144.1 d, C-3'; 127.6 d, C-4'; 141.4 d, C-5'; 75.4 d, C-6'; 70.3 d, C-7'; 17.9 q, C-8'; 166.0 s, C-I"; 116.9 d, C2"; 157.2 s, C-3"; 43.6 t, C-4"; 59.7 t, C- 5"; and 19.2ppm q, C-6". Mass Spectrum: HRCIMS (methane gas reagent):

5 3 3 . 2 7 2 2 m / e ( M ++

H, ealed 533.2754).

Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

16.

MacrocyclicTrichothecenes and Related Metabolites

385

_Common/Systematic Name (C6'R,C7'S)-Isotrichoverrin B Molecular Formula/Molecular Weight C29H4009; MW

16

= 532.26723

10 H

H

H2Cl 15 14 :

I'

5

..

CH2CH2OH

~

"

~J

3'

Ho..IJ

HO" 1. "8' H

General Characteristics Isolated as an oil; [tZ]D2~ - 25.0 ~ (C=2.20, in CHC13). Fungal Source

Myrothecium verrucaria (ATCC 20540).

Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carded out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 2 ($2) contained mainly trichoverrins according to TLC analysis. This sample was chromatographed on CCC (semipreparative column, V~=355ml) with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min (ca. 400rag/injection). Like fractions were combined to give seven fractions: S2F 1, $2F2, $2F3, $2F4, $2F5, $2F6, and $2F7. A portion of $2F2 was subjected to CCC (semipreparative column) with a solvent system of of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 2.8ml/min. The components of the mobile phase (organic layer, CH2C12/CCIa) were varied from 2:3 to 5:2 (v/v), starting at t = 120 min and going to t = 160 min. Like portions were combined to give seven fractions: I, (C6'R, CT'R)-isotriehoverrin A, II, (C6'R, C7'S)-isotrichoverrin B, III, a mixture ofisotrichoverrin B and trichoverrin C), IV, V, VI, 2'E-isotrichoverrin A, VII, 2'E-isotrichoverrin B.

386

16. MacrocyclicTrichothecenes and Related Metabolites.

Spectral Data

1HN/VIR: (CDC13) 0.78(3H, s, H-14); 1.12(3H, d, J=6.5Hz, H-14'); 1.69(3H, s, H-16); 2.16(3H, d, J=l. 1Hz, H-6"); 2.39(2H, t, J=6.0Hz, H-4"); 2.55(1H, dd, ,/--7.7, 15.5Hz, H-3a); 2.82(1H, d, J= 4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.75(1H, dt, J=6.0; 11.5Hz, H-5"A); 3.83(1H, dt, J=6.0, 11.5Hz, H-5"B); 3.84(1H, d, J=5.1Hz, H2); 3.89(1H, dq, J=3.6, 6.5Hz, H-7'); 3.97(1H, d, J=5.4Hz, H-11); 4.10(2H, s, H-15); 4.23(1H, rn, H-6'); 5.46(1H, d, J=5.4Hz, H-10); 5.66(1H, d, J=l 1.3Hz, H-2'); 5.83(1H, d,J=l.OHz, H-6"); 6.11(1H, dd, J=5.2, 15.5Hz, H-5'); 6.19(1H, dd, J=-3.3, 7.7Hz, H-4); 6.60(1H, dd, J= 11.3, 11.3Hz, H-3'); and 7.52ppm(1H, dd, J=l 1.3, 15.5Hz, H-4'). 13CNMR: (CDCI3) 6.7, C-14; 17.9, C-8'; 19.2, C-6"; 21.9, C-7; 23.2, C-16; 27.9, C-8; 36.9, C3 42.9, C-6; 43.6, C-4"; 48.2, C-13" 48.6, C-5; 59.7, C-5"" 63.4, C-15 65.8, C-12; 66.6, C-11" 70.2, C-7'; 75.0, C-4; 75.4, C-6'; 79.1, C-2; 116.9, C-2"; 118.0, C-2'; 118.5, C-10; 127.6, C-4'; 140.4, C9; 141.1, C-5'; 143.9, C-3'; 157.0, C-3"; 165.9, C-I"; and 166.0ppm, C- 1'. Mass Spectrum: HRCIMS: calcd for C29H4109(M + H) +, 533.2751; found, 533.2786role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16.

Macrocyclic Trichothecenes

and Related Metabolites

387

Common/Systematic Name (2'E, 4'Z)-Isotrichoverrin B Molecular Formula/Molecular Weight C29H4009; M W = 532.26723

16

~

~o H

H

-

0 . 1 2

" 13

1"

~;'~

O

I 15

0

3

14

6"

3'

H

HOWl',

HO" ,I, "8' CH2CH2OH

I"1

5"

General Characteristics Isolated as an amorphous solid. Fung.al Source

Myrothecium verrucaria (ATCC 20540).

Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 2 ($2) contained mainly trichoverrins according to TLC analysis. This sample was chromatographed on semipreparative countercurrent chromatography (CCC; Vc=355ml) with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min (ca. 400mg/injection). Like fractions were combined to give seven fractions: S2F1, $2F2, $2F3, $2F4, $2F5, $2F6, and $2F7. A portion of $2F2 was subjected to semi-preparative CCC with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 2.8ml/min. The components of the mobile phase (organic layer, methylene chloride-carbon tetrachloride) were varied from 2:3 to 5:2 (v/v), starting at t = 120 min and going to t = 160 min. Like portions were combined to give seven fractions: I, (C6'R, C7'R)-isotrichoverrin A, .II, (C6'R,C7'S)-isotrichoverrin B, III, a mixture of isotrichoverrin B and trichoverrin C, IV, V, VI, (2'E)-isotrichoverrin A), VII, (2'E)-isotrichoverrin B). Fractions IV and V were subjected to CCC (semi-preparative column) separately with the solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 1.8ml/min to yield fractions rich in (2T, 4'Z)-isotrichoverrin B. These fractions were

388

16. MacrocyclicTrichothecenes and Related Metabolites

combined and purified on TLC (silica gel, 5% methanol in methylene chloride, developed three times) to give pure (2'E, 4'Z) isotrichoverfin B. Spectral Data UV~

~, maxM~~ 262nm (log 6=4.38). IR;

(CHCI3) 3600(OH), 3470(OH), 1710(C=O), and 1646cm~ (C=C). ~H NMR: (CDC13) 0.77(3H, s, H-14); 1.1 l(3H, d, J=6.4Hz, H-8'); 1.69(3H, s, H-16); 2.17(3H, s, H-6"); 2.40(2H, t, J=5.7Hz, H-4"); 2.55(1H, dd, J=7.8, 15.5Hz, H-3a), 2.81(1H, d, J=4.0Hz, H-13A); 3.13(1H, d, J=4.0Hz, H-13B); 3.75-3.92(3H, m, H-7', H-5"); 3.83(1H, d, J=5.1Hz, H-2); 3.95(1H, bd, J=4.8Hz, H-11); 4.1 l(2H, s, H-15); 4.60(1H, dd, J=3.3, 8.6Hz, H-6'); 5.46(1H, bd, J=4.8Hz, H-10); 5.83(1H, s, H-2"); 5.88(1H, dd, J=8.6,11.0Hz, H-5'); 5.94(1H, d, J=15.1Hz, H-2'); 6.16(1H, dd, J=3.5, 7.8Hz, H-4); 6.26(1H, dd, J=l 1.0, 11.8Hz, H-4'); and 7.59ppm(1H, dd, J=l 1.8,

15.1Hz, H-3').

13C NMR: (CDCla) 6.6, C-14; 17.3, C-8'; 19.0, C-6"; 21.9, C-7; 23.2, C-16; 27.9, C-8; 36.7, C3; 42.9, C-6; 43.7, C-4"; 48.1, C-13; 48.7, C-5; 59.7, C-5"; 63.5, C-15; 65.5, C-12; 66.8, C-11; 70.4, C-7'; 71.6, C-6'; 75.6, C-4; 79.1, C-2; 117.2, C-2"; 118.5, C-10; 122.7, C-2'; 128.9, C-4; 138.1, C-5'; 139.9, C-3'; 140.5, C-9; 156.9, C-3"; 165.9, C-I"; and 167.0ppm, C-I'. Mass Spectrum: HRCIMS: calcd for C29H4109,(M + H) +, 533.2751; found, 533.2768role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod.,Vol. 59, pp. 254-261 (1996).

16. MacrocyclicTrichothecenes and Related Metabolites

389

Common/Systematic Name 913,1013-Epoxyisotrichoverrin B Molecular Formula/Molecular Weight C29H40010; M W = 548.26723

01o

H

H

.....

I

....

H CI :

15

5

o

4 %

14

CH2CH2OH

HO'"lx ' H

5"

General Characteristics Isolated as an oil;

[ ( g ] D 20

-

21.0 ~ (c= 0.13, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 3 ($3) fromM, verrucaria isolate ATCC 20540, which was more polar than those that contained trichoverrols (S 1) and trichoverrins ($2), was partitioned between chloroform and methanol-water mixture (50%). The organic fraction was subjected to preparative TLC (Chromatotron, 2mm, silica gel) with methanol-methylene chloride (310%). The most polar fraction was subjected to high speed countercurrent distribution (CCC) with a solvent system of chloroform-hexane-methanol-water (12: 8:15:5, v/v/v/v) at a flow rate of 1.8ml/min to give 913,1013-epoxyisotrichoverrin A and 9[},1013epoxyisotrichoverrin B. Spectral Data 1H NMR: (CDC13) 0.74(3H, s, H-14); 1.13(3H, d, J-6.4Hz, H-8'); 1.34(3H, s, H-16); 1.702.03(5H, m, H-3~, H-7, H-8); 2.20(3H, d, d=0.9Hz, H-6"); 2.41(2H, t, J=6.1Hz, H-

390

16. MacrocyclicTrichothecenes and Related Metabolites

4"); 2.53(1H, dd, J=7.9, 15.5Hz, H-3a); 2.78(1H, d, J=4.0Hz, H-13A); 3.10(1H, d, J=5.6Hz, H-10); 3.19(1H, d, J=4.0Hz, H-13B); 3.81(2 H, m, H-5"); 3.90(2H, m, H11, H-7'); 3.93(1H, d, J=5.2Hz, H-2); 4.04(1H, d, J=12.6Hz, H-15A); 4.15(1H, d, J=12.6Hz, H-15B); 5.67(1H, d, J=l 1.3Hz, H-2'); 5.81(1H, d, J=0.9Hz, H-2"); 6.03(1H, dd, J=3.4, 7.9Hz, H-4); 6.12(1H, dd, J=5.3, 15.5Hz, H-5'); 6.62(1H, dd, J=l 1.3, 11.3Hz, H-3'); and 7.53ppm (1H, dd, J=l 1.3, 15.5Hz, H-4'). 13CNMR: (CDC13) 6.7, C-14; 17.9, C-8'; 19.2, C-6"; 19.4, C-7; 22.3, C-16; 26.4, C-8; 36.6, C3; 42.6, C-6; 43.6, C4"; 48.0, C-13; 48.5, C-5; 57.3, C-10; 57.5, C-9; 59.7, C-5"; 63.1, C-15; 65.2, C-12; 66.7, C-11; 70.2, C-7'; 74.7, C-4; 75.4, C-6'; 78.7, C-2; 116.5, C-2"; 118.0, C-2'; 127.7, C-4'; 142.1, C-5'; 143.9, C-3'; 158.4, C-3"; 165.8, CI'; and 165.9ppm, C- 1" Mass Spectrum: HRCIMS: calcd for C29H41010 (M d- H)§ 549.2700; found, 549.2754m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16. MacrocyclicTrichothecenes and Related Metabolites

391

Common/Systematic Name (2'E)- 12,13-Deoxyisotrichoverrin B Molecular Formula/Molecular Weight C29H40Os; MW = 516.27230

MemO.,,]

H

H

LL Lc I H2~ Mel 0 I O=~1"~ H

o "~

'

M~'~CH2 I CH2OH ~,,

OH H

GeneralCharacteristics Isolatedasan oil.

Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative CCC (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/); flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v; ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S IF 1a, S 1F 1b, S 1F 1c, S 1F 1d, S 1F 1e, S 1F 1f, and S 1F 1g. S 1F 1f was subjected to reversed-phase TLC on Cs (55% methanol in water) to give (2'E)12,13-deoxyisotrichoverrin B as an oil.

392

16. MacrocyclicTrichothecenes and Related Metabolites

Spectral Data UV~

maxM~~ 260rim(log e=4.44). IR~

(CHC13) 1707(C=O) and 1652cm~ (C=C). 1H NMR: (CDCI3) 1.02(3H, s, H-14); 1.12(3H, d, J-6.5Hz, H-8'); 1.66(3H, s, H-16); 2.18(3H, d, J=l.0Hz, H-6"); 2.39(2H, t, J=5.6Hz, H-4"); 2.57(1H, dd, J=7.7, 15.5Hz, H-3~); 3.76-3.90(3H, m, H-7', H-5"); 3.96(1H, bd, J=5.7Hz, H-11); 4.1 l(2H, s, H-15); 4.18(1H, m, H-6'); 4.42(1H, d, J=5.1Hz, H-2); 4.71(1H, s, H-13A); 5.13(1H, s, H13B); 5.41(1H, bd, J=5.7Hz, H-10); 5.83(1H, d, J=15.4Hz, H-2'); 5.84(1H, d, J=l.lHz, H-2"); 6.06(1H, dd, J=3.0, 7.7Hz, H-4); 6.11(1H, dd, J=5.9, 15.6Hz, H-5'); 6.39(1H, dd, J=10.9, 15.6Hz, H-4'); and 7.21ppm(1H, dd, J=10.9, 15.4 Hz, H-3'). 13C NMR: (CDC13) 11.1, C-14; 17.6, C-8'; 19.0, C-6"; 20.9, C-7; 23.2, C-16; 28.0, C-8; 37.9, C3; 42.8, C-6; 43.8, C-4"; 51.6, C-5; 59.8, C-5"; 63.7, C-15; 66.6, C-11; 70.2, C-7'; 75.4, C4; 75.6, C-6'; 78.8, C-2; 105.4, C-13; 117.3, C-2"; 118.8, C-10; 121.8, C-2'; 129.8, C-4'; 140.2, C-5'; 140.1, C-9; 143.9, C-3'; 152.3, C-12; 156.7, C-3"; 166.2, C1"; and 166.5ppm, C- 1'. Mass Spectrum: HRCIMS: calcd for C29I-'I4109, (M+d " 1), 517.2801; found, 517.2849role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16. MacrocyclicTrichothecenes and Related Metabolites

393

Common/Systematic Name (9'E)-Roridin L-2 Molecular Formula/Molecular Weight C29H3809, ~

= 530.25158

~~ 0 ~ ~o H

13

H 3 I

HOH2(~ i~, 15

General Characteristics Isolated as an oil. Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative high-speed countercurrent chromatography (CCC, V~=355ml) with a solvent system of methanol-waterchloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400 mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) (ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S1Fla, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. S1Fld was subjected to reversed-phase TLC on Cs plates (60% methanol in water) to give isotrichoverrin A and (9'E)-roridin L-2.

394

16. MacrocyclicTrichothecenes and Related Metabolites

Spectral Data 1H N1VIR: (CDC13) 0.81(3H, s, H-14); 1.1 l(3H, d, J=6.3Hz, H-14'); 1.71(3H, s, H-16); 2.49(1H, dd, J=7.9, 15.5Hz, H-3a); 2.70(2H, t, J=6.0Hz, H-4'); 2.81(1H, d, J=4.0Hz, H-13A), 3.13(1H, d, J=4.0Hz, H-13B); 3.83(1H, d, J=5.1Hz, H-2); 3.92(1H, bd, J=5.7Hz, H-11); 4.77(2H, d, J=l.5Hz, H-12'); 5.48(1H, bd, J=5.7Hz, H-10); 5.90(1H, d, J=l.5Hz, H-2'); 5.90(1H, dd, J=8.2, 15.3I-Iz, H-7'); 5.97(1H, d, J=15.4Hz, H-10'); 6.12(1H, dd, J=3.6, 7.9Hz, H-4); 6.36(1H, dd, J=l 1.0, 15.3Hz, H-8'); and 7.27ppm (1H, dd, J=l 1.0, 15.4Hz, H-9'). 13CNMR: (CDCI3) 6.6, C-14; 18.4, C-14'; 19.0, C-12'; 23.2, C-16; 28.0, C-8; 29.3, C-4'; 31.3, C-7; 35.9, C-3; 44.2, C-6; 48.2, C-13; 49.0, C-5; 63.0, C-15; 65.6, C-12; 66.2, C-5'; 66.8, C-11; 71.4, C-13'; 75.8, C-4; 79.0, C-2; 85.9, C-6'; 116.7, C-2'; 118.8, C-10; 122.6, C-10'; 132.4, C-8'; 138.6, C-7'; 140.5, C-9; 143.4, C9'; 166.8, C-11'; 167.4, C3'; and 173.2ppm, C- 1'. Mass Spectrum: HREIMS: calcd for C29H3sO9(1W), 530.2516; found, 530.2516m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod.,Vol. 59, pp. 254-261 (1996).

16. MacrocyclicTrichothecenes and Related Metabolites

395

Common/Systematic Name Roridin L-2 Molecular Formula/Molecular Weight C29H3809; ~ 16

lo

= 530.25158 H

~ O . , , J

H

2

HOH2~s 11s, O

3

~O'~

0

IJg'

HO ~; General Characteristics Isolated as a white, homogeneous solidi rap., 93-97~

[a]D + 83.6~ (c= 1.0, in CHC13).

Fungal Source Myrothecium roridum. Isolation~urification Fermented beer was extracted with ethyl acetate, concentrated, and chromatographed by silica gel high performance liquid chromatography eluted with methylene chloride-ethyl acetate, 50:50 (v/v) and by C]8-reversed phase chromatography eluted with methanolwater, 50:50 (v/v). Spectral Data UV: )i, M~H

~x

259nm (e=24,650).

IR:

(CC14) 3500, 3500, 1785, 1750, 1710, 1640, and 1600cm"1. ]H NMR: (CDC13) 0.84(3H, s, H-14); 1.14(3H, d, J=6.0Hz, H-14'); 1.72(3H, s, H-16); 2.50(1H, dd, J=8.0, 15.5Hz, H-3a); 2.0(1H, m, H-313); 2.70(2H, m, H-4'); 2.81(1H, d, J=4.0Hz, H-13A); 3.13(1H, d, J=4.0Hz, H-13B); 3.70(1H, m, H-2); 3.70(1H, m, H-11); 4.78(1H, dd, J=l.5, 17.5Hz, H-12'A); 4.84(1H, dd, J=l.5Hz, 17.5Hz, H-12'B); 5.48(1H, d, J=5.0 Hz, H-10); 5.92(1H, br, J=l.3Hz, H-2'); 5.86(1H, dd, J--7.0, 15.5Hz, H-7'); 5.78(1H, d, J=l 1.5Hz, n-10'); 6.10(1H, dd, J=3.5, 8.0Hz, n-4);

396

16. MacrocyclicTrichothecenes and Related Metabolites

7.61(1H, dd, J=l 1.5, 15.SHz, H-8'); and 6.61ppm (1H, t, J=l 1.SHz, H-9'). 13CNMR: (CDCI3) 6.7, C-14; 18.5, C-14'; 73.6, C-12'; 23.3, C-16; 28.1, C-8; 29.3, C-4'; 21.2, C-7; 36.3, C-3; 44.3, C-6; 6.7, C-13; 48.9, C-5; 62.7, C-15; 65.7, C-12; 66.4, C-5'; 66.9, C-11; 69.8, C-13'; 75.5, C-4; 79.0, C-2; 85.4, C-6'; 116.7, C-2'; 118.9, C-10; 118.8, C-10'; 130.6, C-8'; 139.1, C-7'; 140.6, C-9; 143.5, C9'; 166.5, C-11'; 167.8, C3'; and 174.2ppm (C- 1'). Mass Spectrum: EIMS: 530m/e (M+). References R. J. Bloem, T. A. Smitka, R. H. Bunge, J. C. French, and E. P. Mazzola; Roridin L-2, A New Trichothecene; Tet. Lett., Vol. 24, pp. 249-252(1983). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

16.

Macrocyclic Trichothecenes and Related Metabolites

397

Common/Systematic Name Verrucarin A Molecular Formula/Molecular Weight C27H3409; M W = 502.22028

~o H

H2r o

H

! 15

4 "~O"'~

, & "

HO

II O

O

12'

General Characteristics Crystals; mp., >360 ~ (dec.);

[a]D 22 + 2 0 6 ~

(c=1.012, in dioxane); +260 ~ (in CHC13).

Fungal Source

Myrothecium verrucaria and M. roridum.

Biological Activity Rats given verrucarin A developed creatinuria; m vitro it inhibited ATP-creatine phosphotransferase. It caused severe edema in body cavities. Major clinical signs in several different experimental animals were diarrhea, hematuria, vomiting, anorexia, loss of weight, ataxia, and thirst. At relatively low dosages, it caused leukocytosis followed by severe leukopenia. The LDs0 values in mice were 1.5mg/kg (IV) and 0.5mg/kg (IP). It caused dermal toxicity: necrosis of epidermis with suppuration and extensive necrosis of dermal tissue with damage extending into subcuticulis. IDs0 of protein synthesis in rabbit reticulocytes was 10-15ktg/ml. Spectral Data UV:

~.~a 260nm (6=17,700).

max

~H NMR: (CDCI3) 5.83(H-4); 5.46(H-10); 2.97(H-13a); 0.87(H-14); 1.79(H-16); 4.20(H-2'), 6.06(H-7'); 8.08(H-8'); 6.70(H-9'); 6.17(H-10'); and 0.89ppm (H-12').

398

16. MacrocyclicTrichothecenes and Related Metabolites

13CNMR: (CDC13) 78.6, C-2; 34.6, C-3; 75.3, C-4; 49.2, C-5; 43.9, C-6; 19.7, C-7; 27.2, C-8; 140.7, C-9; 117.7, C-10; 66.6, C-11; 64.9, C-12; 47.4, C-13; 7.0, C-14; 63.1, C-15; 22.6, C-16; 174.3, C-I'; 73.8, C-2'; 32.9, C-3'; 31.9, C-4'; 60.8, C-5'; 165.8, C-6'; 127.2, C-7'; 138.6, C-8'; 138.6, C-9'; 125.5, C-10'; 165.1, C-11'; and 9.8ppm, C-12'. TLC Data A. Adsorbent: silica gel G; solvent system, chloroform-methanol, 98:2 v/v; Rf, 0.70; detection, H2SO4spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina; solvent system, chloroforrrl-methanol, 98:2 v/v; Re, 0.28; detection, HzSO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 247-248(1981 ). J. Gutzwiller and Ch. Tamm; Uber die Struktur von Verrucarin A; Helvetica Chimica Acta, Vol. 48, pp. 157-176(1965). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

16.

Macrocyclic Trichothecenes and Related Metabolites

399

Common/Systematic Name Verrucarin A [~-D-glucoside Molecular Formula/Molecular Weight C33H44014; MW

"-

664.27311

H

H 13 ~ ; ,,it) I

o

H2(~ i s

O

~0/'~

I ,,

II

1

~-glucose

0

General Characteristics Amorphous solidi [~]D + 98~ (c=0.63, in CHC13). Fungal Source Baccharis coridifolia. Isolation/Purification The crude extract (10% methanol-chloroform) of female B. coridifolia plant was partitioned (hexane-10% water in methanol) and the aqueous methanol, atter solvent removal,was chromatographed (silica gel) with ethyl ether-hexane, ethyl ether-hexane, and methanol-methylene chloride. The 20% methanol-methylene chloride was subjected to further flash chromatography, preparative TLC (Chromatotron), and finally preparative C ls reversed phase HPLC to give verrucarin A ~-D-glucoside. Biological Activity Cytotoxic: IC60 ca. 100 ng/ml. Spectral Data IR:

(CH2C12) 3440, 1720, 1660, 1640, 1595, 1270, 1195, and 1080cm"1. 1H N]h/[R: (CDCls) 7.99(1H, dd, J=12.0, 15.5Hz, H-8'); 6.65(1H, dd, ,/--12.0, 15.5Hz, H- 8'); 6.65(1H, dd, J=l 1.1, 12.0Hz, H-9'); 6.14(1H, d, J=l 1.1Hz, H-10'); 6.02(1H, d, J=15.5Hz, H-7'), 5.77(1H, dd, J=3.9, 8.1Hz, H-4); 5.42(1I-I, br d, J=5.0Hz, H-10); 4.75 and 4.20(1H each, AB, J=12.1Hz, H-15); 4.46(1H, ddd, J=H-5~B); 4.20(1H, d,

400

16. MacrocyclicTrichothecenes and Related Metabolites

J=2.0Hz, H-2'); 4.12(1H, d, J=7.6Hz, H-I"); 3.97(1H, ddd, J-11.7, 11.7, 3.5Hz, H5'A); 3.90(1H, dd, J=3.6, 11.8Hz, H-6"B); 3.84(1H, d, J=5.1Hz, H-2); 3.79(1H, d, J=5.1, 11.SHz, H-6"A); 3.54-3.57(3H, m, H-11, H-3", H-4"); 3.40(1H, dd, ,]-7.6, 9.2Hz, H-2"); 3.36(1H, ddd, J=3.6, 5.1, 7.SHz, H-5"); 3.11 and 2.78(1H each, AB, J=3.9Hz, H-13); 2.46(1H, dd, J-8.1, 15.4Hz, H-3Gt); 2.44(1H, m, H-3'); 2.20(1H, ddd, J=3.9, 5.1, 15.4Hz, H-313); 1.99(1H, m, H- 8B); 1.80-1.90(4H, m, H-4', H-7B, H-8A); 1.75(3H, s, H-16); 1.69(1H, m, H-7A); 0.94(3H, d, J=5.9Hz, H-12'); and 0.82ppm (an, s, n-14). 13CNMR: (CDC13) 78.9(C-2), 34.9(C-3), 75.6(C-4), 49.5(C-5), 44.1(C-6), 20.1(C-7), 27.5(C8), 141.0(C-9), 118.0(C-10), 66.8(C-11), 65.2(C-12), 47.8(C-13), 7.4(C-14), 63.6(C15), 23.4(C-16), 172.4(C-1'), 80.6(C-2'), 32.4(C-3'), 32.1(C-4'), 61.0(C-5'), 166.1(C6'), 127.3(C-7'), 138.8(C-8'), 138.7(C-9'), 125.9(C-10'), 165.4(C-11'), 11.2(C-12'), 102.6(C-1"), 73.6(C-2"), 76.5(C-3"), 70.0(C-4"), 75.7(C-5"), and 62.3ppm (C-6"). TLC Data Adsorbent: Silica gel, solvent system: 10% methanol-methylene chloride; Re, 0.38. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

16. MacrocyclicTrichothecenes and Related Metabolites

401

Common/Systematic Name 2'-Dehydroverrucarin A Molecular Formula/Molecular Weight C27H3209; M~V = 500.20463

H

I.

H

~l'~',~i ....,u I

_~v"T~, ~

H27,~!,5

o~,

I

o

II~,'

''0%

/, ,

Id

o

General Characteristics Needles from acetone-ethyl ether; mp., 233-240~

[~]D 25 +

118~ (in CHCI3).

Fungal Source Myrothecium roridum. Biological Activity Antibiotic activity similar to that of verrucarin A. Spectral Data UV:

X maxEtO" 262nm (e=23,400). IR:

(KBr) 1710, 1725, 1590, and 1630cm1. Reference B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

402

16.

Macrocyclic T r i c h o t h e c e n e s and Related Metabolites

Common/Systematic Name Verrucarin B Molecular Formula/Molecular Weight C27H3209; MW = 500.20463 ~

O I

l~

1o

H 2

H

~1 13

I

d

61

~

3 r

....i " l

I

0

Fungal Source Myrothecium verrucaria and M. roridum.

General Characteristics Crystals; mp., >330~

[a]D + 94 ~ (in CHCI3), + 101 ~ (in dioxane); + 147~ (in benzene).

Biological Activity Typical trichothecene type activity: dermal toxicity, antifungal, cytostatic, and acute toxicity to vertebrate animals. The LDs0 in mice dosed IV was 7.0mg/kg. The EDs0 for cytostatic activity in vitro against Ehrlich ascites tumor cells was 0. 003 ~tg/ml. Spectral Data UV: maxE~O" 258.5nm (e=23,400). 1H NIV[R: (CDCI3) 5.90(H-4); 5.47(H-10), 3.00(H-13a); 0.88(H-14); 1.74(H-16), 3.41(H-2'); 6.10(H-7'); 7.98(H-8'), 6.69(H-9'); 6.19(H-10'); and 1.56ppm (H-12'). ~3CNMR: (CDCI3) 78.7,C-2; 34.8, C-3; 75.4, C-4; 49.0, C-5; 43.6, C-6; 19.8, C-7; 27.4, C-8; 140.4, C-9; 118.0, C-10; 66.9, C-I I; 64.9, C-12; 47.5, C-13; 7.3, C-14; 63.6,C-15; 22.8, C-16; 167.4, C-I'; 58.0, C-2'; 61.1, C-3'; 36.9, C-4'; 60.4, C-5'; 165.8, C-6'; 127.2, C-7'; 138.0, C=8'; 138.0, C-9'; 125.6, C=I0'; 164.8; CI I', and 15.8ppm, C-12'.

16.

MacrocyclicTrichothecenes and Related Metabolites

403

TLC Data Absorbent: Kieselgel G; solvent, ethyl ether (two consecutive runs); Re, 0.37; detection, iodine vapors. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 253-254(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991).

404

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematie Name Verrucarin J Molecular Formula/Molecular Weight C27H3208; M W - 4 8 4 . 2 0 9 7 2 ,0 H

HC

H

2l,sa4

0

'

15

,~0

'~

,~'

.

Jl

j~r

0 Fungal Source Myrothecium verrucaria. General Characteristics Colored needles from chloroform-ether; acetone-ether; mp., >315~ (dec.); (c=l.011, in CHCI3); [a]D23 + 41 ~ (C=0.784, in benzene).

[a]D 22 +22 ~

Biological Activity Antibiotic activity similar to that of verrucarin A. Spectral Data UV:

~, ~m~ ~

196(e = 15,500), 219(19,900), and 262nm (14,500).

IR:

(CH2C12) 2810, 1710, 1650, 1630, 1588, 1352, 1221, 1180, 1147, 1070-1088, 1042, 995, 968, 877, and 820cm "~. ~H NMR: (CDCI3) 5.90(H-4); 5.47(H-10); 2.98(H-13a); 0.83(H-14); 4.30(H-lSa); 1.72(H-16); 3.50(H-2'); 2.50(H-4'a); 3.82(H-5'); 6.05(H-7'); 8.12(H-8'); 6.60(H-9'); 6.10(H-10'); and 2.28ppm (H- 12'). TLC Data Adsorbent: Kieselgel G; solvent was ethyl ether (two consecutive runs); Rf, 0.42; detection by iodine vapors.

16.

MacrocyclicTrichothecenes and Related Metabolites

405

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 259 (1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

406

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Verrucarin K 12,13-Deoxyverrucarin A Molecular Formula/Molecular Weight C27H3408; M'W -- 486.2253 7 ,0

H

H

'

'

0

I

,

H

Fungal Source

Myrothecium verrucaria.

General Characteristics Crystals from dichloromethane-ether; mp., >320~ (dec.); [a]D23 +218 ~ (C=0.58, in CHCI3). Monoacetyl derivative, colorless needles from acetone-ether-petroleum ether; mp., 199-202~ [a]D23 + 143~ (C=0.83, in CHC13). Spectral Data UV:

~, maxS~n 259nm (6=15,500). IR:

171O, 3550, 1630, and 1585cm~. 1H NMR:

(CDCI ) 5.810-1-4),5.a9(H-10), 4.71(H-13a); 5.18(H-13b); 1.09(H-14); 3.66(H-15); 1.70(H-16); 6.05(H-7'); 8.05(H-8'); 6.67(H-9'); 6.08(H-10'); and 0.89ppm (H-12'). 13C NMR:

(CDCIa) 78.5, C-2; 35.9, C-3; 75.5, C-4; 52.0, C-5; 44.2, C-6; 18.7, C-7; 27.4, C-8; 140.5, C-9; 118.2, C-10; 66.5, C-11; 151.6, C-12; 106.3, C-13; 12.1, C-14; 63.5, C15; 23.2, C-16; 174.5, C-I'; 74.0, C-2'; 61.1, C-3'; 32.1, C-4'; 61.1, C-5'; 165.9, C-6'; 127.4, C-7'; 138.7, C-8'; 138.7, C-9'; 125.6, C-10'; 165.3, C-11'; and 10.0ppm, C-12'.

16. MacrocyclicTrichothecenes and Related Metabolites

407

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 260(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

408

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Verrucarin L Molecular Formula/Molecular Weight C27H3209; MW

16

= 500.20463

1o H

I

H

~I

~

I ,,I I,r~ I v

1

o,

o9 0 -/

i

g

'C----C(CN2)2OC"

H

~

General Characteristics Crystals from dichloromethane-hexane, mp., 230-23 5~ [tt]D 27 + 15.0 ~ (c = 0.92 in chloroform). Acetate derivative, crystals from dichloromethane-ether, m.p. 132-135~ [a]D27 + 29.7 ~ (C=0.52, in CHC13). Isolation/Purification Spores ofMyrothecium verrucaria grown on N-Z amine agar were added to Czapek-Dox media and allowed to grow at 28~ in a shake culture. After 2 days, the solution was divided into three equal portions and added to three separate Fernback flasks containing 1L of production media. After 3 days of growth, the mycelium in each flask was separately centrifuged, washed, and resuspended in 1L of sterile water. Trichoverrin A and trichoverrin B were added separately to flasks one and two; flask three was used as the control. After 7 days, the mycelium was removed and extracted with ethyl acetate. The mycelium extract was subjected to partition chromatography on 500g Celite impregnated with 250ml of 18% water in methanol. The column was eluted with petroleum ether followed by increasing amounts of dichloromethane in petroleum ether up to 40% dichloromethane in petroleum ether. Fractions were combined on the basis of TLC analysis to give a total of eight fractions: A, B, C, D, E, F, G, and H (methanol wash). Flash chromatography of fraction G (3% MeOH in CH2C12) gave a fraction rich in roridin A, which upon crystallization from dichloromethane-hexane yielded roridin A. The mother liquor was subjected to HPLC (2% methanol-methylene chloride) to give verrucarin L which elutes just before roridin A. Fungal Source

Myrothecium verrucaria (ATCC 24571).

16.

MacrocyclicTrichothecenes and Related Metabolites

409

Spectral Data UV:

~E,o. max

262nm (log e = 4.42).

1H NIV[R: (CDCI3) 0.86(3H, s, 14-H); 1.87(3H, s, 16-H); 2.26(3H, d, J = l H z , 12'-H); 2.99(2H, AB, J=4Hz, 13-H); 3.80(1H, d, J=5Hz, 1 I-H), 4.45(2H AB, J=12 Hz, 15-H); 5.2(1H, m, 8-H); 5.58 (1H, d, J=5Hz 10-H); 6.00 (1H, d, J=16Hz, 7'-H); 6.12 (1H, d, J=l 1Hz, 10'-H); 6.65(1H, dd, d's=l 1Hz, 9'-H); and 8.10ppm (1 H, dd, J = l l , 16Hz, 8'H). Verrucarin L acetate, 0.86(3H, s, 14-H); 1.80(3H, s, 16-H); 1.94(acetate); 2.27(3H, d, J=lHz, 12'-H); 2.97(2H, AB, d=4Hz, 13-H); 3.75(1H, d, J=5Hz, 1 I-H); 4.40(2H, AB, J=12 Hz, 15-H), 5.70(1H, d , J = 5.0Hz, 10-H); 5.96(1H, d, J=16Hz, 7'H); 6.07(1H, d, J=l 1Hz, 10'-H); 6.61(1H, dd, 3's=l 1Hz, 9'-H); and 8.01ppm (1H, dd, J=l 1, 16Hz, 8'-H). 13C NMP~: (CDCI3) 79.0 d, C-2; 35.3 t, C-3; 75.1 d, C-4; 48.8 s, C-5; 42.5 s, C-6; 30.1 t, C-7; 66.8 d, C-8; 139.7 s, C-9; 120.9 d, C-10; 67.2 d, C-11; 65.5 s, C-12; 48.1 t, C-13; 6.9 q, C-14; 65.0 t, C-15; 20.6 q, C-16; 165.7 s, C-I'; 118.2 d, C-2'; 156.6 C-3'; 40.3 t, C4'; 60.5 t, C-5'; 165.5 s, C-6'; 127.2 d, C-7'; 139.3 d, C-8', 139.3 d, C-9'; 125.6 d, C10', 165.7 s, C-11'; and 17.3ppm q, C-12'. Mass Spectrum: HRCIMS (methane gas reagent), 501.2112m/e (M § + H, calcd 501.2124). Verrucarin L acetate (chemical ionization, methane gas reagent), 543.2217m/e (M ++ H; calcd for 543.2230). Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

410

16. Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name Verrucarin L Acetate Molecular Formula/Molecular Weight C29I-I34010;MW = 542.21520 16

10 H

ACO

;I

H

5

4%~

2 "C--C(CH2)20~ , 2 /

0 ~"

H

O

General Characteristics Crystals from ether-hexane, mp., 132-135~

[a]D27+ 29.7 ~ (c= 0.52 in CHCI3).

Isolation/Purification Spores ofMyrothecium verrucaria grown on N-Z amine agar were added to Czapek-Dox media and allowed to grow at 28~ in a shake culture. After 2 days, the solution was divided into three equal portions and added to three separate Fernback flasks containing 1L of production media. After 3 days of growth, the mycelium in each flask was separately centrifuged, washed, and resuspended in 1L of sterile water. Trichoverrin A and trichoverrin B were added separately to flasks one and two; flask three was used as the control. After 7 days, the mycelium was removed and extracted with ethyl acetate. The mycelium extract was subjected to partition chromatography on 500g Celite impregnated with 250ml of 18% water in methanol. The column was eluted with petroleum ether followed by increasing amounts of dichloromethane in petroleum ether up to 40% dichloromethane in petroleum ether. Fractions were combined on the basis of TLC analysis to give a total of eight fractions: A, B, C, D, E, F, G, and H (methanol wash). Fraction E was subjected to MPLC using 30-40% ethyl acetate in hexane to give verrucarin B, verrucarin L acetate, and a mixture of verrucarin A and roridin J. Fungal Source

Myrothecium verrucaria (ATCC 24571).

Spectral Data

UV: maxE~n

261 nm (log e = 4 .28 ).

16. Macrocyclic Trichothecenes and Related Metabolites

411

1H NMR: (CDC13) 0.86(3H, s, 14-H); 1.80(3H, s, 16-H); 194 (acetate); 2.27(3H, d, J=lHz, 12'-H), 2.97(2H, AB, J=4 Hz, 13-H); 3.75(1H, d, J=SHz 1 l-H); 4.40(2H, AB, J=12Hz, 15-H); 5.70(1H, d, J=5.0Hz, 10'-H); 5.96(1H, d, J=16Hz, 7'-H); 6.07(1H, d, J=l 1Hz, 10'-H); 6.61(1H, dd, J's=l 1Hz, 9'-H); and 8.01ppm (1H, dd, J=l 1, 16Hz, 8'-

H).

~3C NMR: (CDCls) 79.0 d, C-2; 34.9 t, C-3; 74.1 d, C-4; 49.0 s, C-5; 42.2 s, C-6; 26.5 t, C-7; 68.8 d, C-8; 136.5 s, C-9; 123.9 d, C-10; 67.0 d, C-11; 65.3 s, C-12; 47.9 t, C-13; 7.0 q, C-14; 64.5 t, C-15; 21.0 q, C-16; 165.5 s, C-I'; 117.8 d, C-2'; 156.9 C-3'; 40.2 t, C4'; 60.4 t, C-5'; 165.4 s, C-6'; 127.8 d, C-7'; 138.8 d, C-8'; 139.9 d, C-9'; 125.2 d, C10'; 165.8 s, C-11'; and 17.1 q, C-12'; 170.9, s, CH3C=O; and 20.4ppm q, CH3C=O. Mass Spectrum: HRCIMS (methane gas reagent), 543.2217m/e (M + + H, calcd 543.2230). Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

412

16. Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name Roridin A Molecular Formula/Molecular Weight C29H4009; M W ~- 532.26723

1 6 ~H

2 3 ,;,,0 ]

H

0

I " 0 .....

h

14'

General Characteristics Crystals from ether; mp., 198-204~ [aiD 22 +130 ~ (c=1.36, in CHCI3); [~]D 22 + 192~ (c=1.38 in dioxane); di-O-acetylroridin A, needles from ether-petroleum ether; mp., 165166~ [tt]D22 +168.5 ~ (C= 1.108, in CHCI3). Fungal Source Myrothecium verrucaria and M. roridum. Biological Activity The LDs0 value of roridin A in mice dosed IV was 1mg/kg. In mouse tumor cells, it caused 50% inhibition of cell growth at 1ng/ml. Spectral Data UV~

m,~E~~ 263nm (e=18,600). IR: (Kbr) 3546, 3472, 1742, 1709, 1704, 1701, 1637, 1631, and 1597cm "1. 1H NMR:

(CDCI3) 5.85(IH, H-4); 5.44(IH, H-10); 2.96(IH, H-13a); 0.80(3H, H-14); 4.44(IH, H-15a); 1.74(3H, H-16); 4.09(IH, H-2');6.00(IH, H-7');7.68(IH, H-8');6.66(IH, H9'0;5.78(IH, H-10'); 1.08(3H, H-12'); and 1.17ppm (3H, H-14').

16.

Macrocyclic Trichothecenes and Related Metabolites

413

13C NMR: (CDC13) 78.8, C-2; 34.6, C-3; 74.2, C-4; 49.1, C-5; 43.6, C-6; 20.0, C-7; 27.5, C-8; 140.4, C-9; 118.2, C-10; 66.9, C-11; 64.9, C-12; 47.4, C-13; 7.2, C-14; 64.2, C-15; 22.9, C-16; 174.5, C-I'; 75.3, C-2'; 36.7, C-3'; 33.0, C-4'; 69.5, C-5'; 83.7, C-6'; 139.0, C-7'; 126.0, C-8'; 143.6, C-9'; 117.2, C-10'; 166.3, C-11', 14.4, C-IT; 70.4, C-13', and 18.0ppm C- 14'. TLC Data A. Adsorbent: silica gel G; solvent: chloroform-methanol, 98:2 v/v; Re, 0.70; detection: H2SO4spraying and heating at 110~ for 5 minutes; B. Adsorbent: alumina; solvent: chloroform-methanol, 98:2 v/v, Rf, 0.18, detection: H2SO4 spraying and heating at 110~ for 5 minutes. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 230-231 (1981 ).

414

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Roridin A 13' [3-D-glucoside Molecular Formula/Molecular Weight C35H50014; MW = 694.32006 H

H

3

o

!.

Ho'" ~

-o '

0

I H

General Characteristics Amorphous solid; [a]D + 90 ~ (c=1.6, in CHCI3). Fungal Source Female Baccharis coridifolia. Isolation/Purification The crude extract (10% methanol-chloroform) of female B. coridifolia plant was partitioned (hexane-10% water in methanol) and the aqueous methanol, after solvent removal was chromatographed (silica gel) with ethyl ether-hexane, ethyl ether-hexane, and methanol-methylene chloride. The first methanol fraction from the chromatography of the crude extract of the plant (see verrucain A glucoside isolation) was slurried with 70% methanol-water and filtered through a pad of C-18 silica (ca. 10 g, 40pm), washed with 70% methanol-water. The filtrate was concentrated by rotary evaporation to give a gum. The gum was subjected to CCC (semi-preparative column, Vc = 3 55ml) with a solvent system of methanol-water-chloroform-hexane (6:4: 7:3, v/v/v/v) and the lower organic phase as the mobile phase to give five fractions A, B, C, D, E. Fraction E from a reversed phase filtration chromatography was subjected to C18 reversed phase semipreparative HPLC (250 x 10 mm, 63% methanol-water, 4.0mL/min) to yield roridin A 13-glucoside. Biological Activity Cytotoxic: IC90ca. lng/ml.

16. Macrocyclic Trichothecenes and Related Metabolites

415

Spectral Data IR:

(CH3CI) 3406, 1732, 1718, 1637, 1600, 1181, and 1081cmq. 1H NMR:

(pyridine-ds) 7.92(1H, dd, J-- 11.5, 15.7Hz, H-8'); 6.58(1I-I, dd, J=-11.1, 11.5Hz, H-9'); 6.41(1H, dd, J=2.5, 15.7Hz, H-7'); 6.03(1H, dd, J=4.0, 8.1Hz, H-4); 5.81(1H, d, d=l 1.1Hz, H-10'); 5.49(1H, br d, J=4.3Hz, H-10); 4.96(1H, d, J=7.7Hz, H-I"); 4.66 and 4.60(1H each, AB, J=12.2Hz, H-15); 4.57(1H, rn, H-6"B); 4.55(1H, m, H-6'); 4.51(11-1, d, J=3.5Hz, H-2'); 4.40(1H, dd, J=5.4, 11.8Hz, H-6"A); 4.37(1H, dq, J=6.2, 6.2Hz, H-lY); 4.27(1H, dd, J=8.7, 8.9Hz, H-4"); 4.25(1H, dd, J=8.5, 8.9Hz, H-3"); 4.03(11-1, dd, J=7.7, 8.5Hz, H-2"); 3.97(1H, m, H-5"); 3.93(1H, d, d=4.9Hz, H-2); 3.70(1H, d, J=4.9Hz, H-11); 3.55(1H, m, H-5'B); 3.48(1H, m, H-5'A); 3.10 and 2.86(1H each, AB, d=4.1Hz, H-13); 2.51(1H, m, H-3'); 2.43(1H, dd, ,/--8.1, 15.2Hz, H-3a); 2.26(11-1, ddd, d=4.0, 4.9, 15.2Hz, H-313); 2.00(1H, m, H-4'); 1.90(2H, m, H-4', H-7B); 1.83(1H, m, H-8B); 1.77(2H, m, H-7A, H-8A); 1.53(3H, s, H-16); 1.42(3H, d, J=6.6Hz, H-12'); 1.192(3H, d, d=6.2Hz, H-14'); and 1.08ppm(3H, s, H-14). 13C NMR:

(pyfidine-ds) 79.1, C-2; 35.3, C-3; 75.3, C-4; 49.8, C-5; 44.2, C-6; 20.5, C-7; 27.8, C-8; 139.6, C-9; 119.7, C-10; 67.3, C-11; 65.8, C-12; 47.7, C-13; 7.6, C-14; 63.6, C15; 23.1, C-16; 175.0, C-I'; 76.2, C-2'; 36.8, C-3'; 34.7, C-4'; 69.5, C-5'; 80.9, C-6'; 141.6, C-7'; 126.5, C-8'; 144.3, C-9'; 117.0, C-10'; 166.9, C-11'; 14.7, C-12'; 76.6, C13'; 14.8, C-14' 104.1, C-I"; 75.2, C-2"; 78.6, C-3"; 71.6, C-4"; 78.6, C-5"; and 62.8ppm, C-6". TLC Data Adsorbent: Silica gel; solvent system: 10% methanol-methylene chloride; Re, 0.20. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

416

16. Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name 8a-Butoyloxyroridin A; YM-47525 Molecular Formula/Molecular Weight C33H46Oll; M W --" 618.30401 16

~ o . j10~H

o

H

"ol .~.~I ..o I

Io

u , . u , . ,"'. , , ,. , 'r,.~ i 5 4" n2bn2Hv MeC ,

:l~

o

/ ..,,,I,Lll ., 4'~,--0"'0

.I/ 12 General Characteristics Crystalline solid;

[a]D

--

HO" ~I ~ 4 '

+ 152.4 ~ (C=0.84, in MeOH).

Fungal Source Unidentified fungal species (likely a deuteromycete in the Agonomycetes) isolated from a deadwood sample ofAcer sp. Isolation/Purification The filtrate from a submerged culture was extracted at pH 3 (ethyl acetate) and the extract subjected to silica gel chromatography (methanol in chloroform). The active fraction was crystallized from hexane and the solid subjected to reversed phase HPLC (wateracetonitrile) to give 6.5mg of 8a-crotonyloxyroridin A and 10.6mg of 8a-butoyloxyroridin A. Biological Activity Inhibited growth of Candida albicans at 6.25mg/mL. Spectral Data UV:

~M~O.m~x 262nm (e =18,100). IR:

(CHCI3) 3450, 2970, 1720, 1640, 1600, and 1440cml.

16.

Macrocyclic Trichothecenes and Related Metabolites

417

1HNMR: (CDC13) 0.80(3H, s, H-14); 0.93(3H, t, J=7.6Hz, H-4"); 1.10(3H, d, J=6.8Hz, H12'); 1.19(3H, d, J=6.1Hz, H-14'); 1.60(1H, tq, J=7.6 and 7.6Hz, H-3"); 1.74(2H, m, H-4'); 1.74(3H, br s, H-16); 1.99 br dq, ,/=2.7 and 6.8Hz, H-3'); 2.11(1H, br d, J=14.0Hz, H-7B); 2.14(1H, t, J=7.6Hz, H-2"); 2.21(1H, dd, ,/--5.5 and 14.0Hz, H7A); 2.22(1H, ddd, J=4.8, 8.2", and 15.5Hz, H-313); 2.47(1H, dd, J=8.2 and 15.5Hz, H-3tt); 2.81 and 3.09(1H each, AB, J=3.6Hz, H-13); 3.54(2H, t, J=5.8Hz, H-5'); 3.60(1H, br q, J=6.1Hz, H-13'); 3.64(1H, br d, J=3.4Hz, H-6'); 3.69(1H, d, J=5.5Hz, H-11); 3.84(1H, d, J=4.8Hz, H-2); 4.02(1H, dd, ,/--2.7 and 7.6Hz, H-2'); 4.36 and 4.58(1H each, AB, J=12.2Hz, H-15); 5.28(1H, br d, J=5.5Hz, H-8); 5.67(1H, br d, J=5.5Hz, H-10); 5.77(1H, d, J=l 1.6Hz, H-10'); 5.78(1H, dd, ,/--8.2 and 8.2Hz*, H-4); 5.98(1H, dd, ,/=3.4, 15.9Hz, H-7'); 6.64(1H, dd, J=l 1.6 and 11.6Hz, H-9'); and 7.63(1H, dd, J=l 1.6, 15.9Hz, H-8'). * As reported, but value should be closer to 5Hz.

13CNMR: (CDC13) 78.8, C-2; 34.8, C-3; 73.7, C-4; 49.3, C-5; 42.3, C-6; 26.2, C-7; 67.8, C-8; 136.7, C-9; 123.5, C-10; 66.6, C-11; 65.1, C-12; 47.6, C-13; 7.4, C-14; 65.5, C-15; 20.4, C-16; 174.3, C-I'; 75.5, C-2'; 37.4, C-3'; 33.5, C-4'; 70.2, C-5'; 84.0, C-6', 139.4, C-7'; 126.0, C-8'; 144.1, C-9'; 117.3, C-10'; 166.3, C-11'; 14.5, C-12'; 70.8, C-13'; 18.2, C-14'; 173.5, C-I"; 36.0, C-2"; 18.5, C-3"; and 13.6ppm, C-4". Reference T. Sugawara, A. Tanaka, K. Nagai, K. Suzuki, and G. Okada; New Members of the Trichothecene Family; J. Antibiotics, Vol. 50, pp. 778-780(1997).

418

16.

Macrocyclic

Trichothecenes

and

Related

Metabolites

Common/Systematic Name 8a-Crotonyloxyroridin A YM-47524 Molecular Formula/Molecular Weight C33H46Oll; M W -- 618.30401 lo H

H 2

3

o

. .

"C /

H.-.(~ Is

1'

Me 4

"~,",""~

,

H

~2'

"

H

i

0,,, HO ~

-

H

~

,

~'

General Characteristics Crystalline solid; [a]D -k-100.5~ (c=0.58, in MeOH). Fungal Source Unidentified fungal species (likely a deuteromycete in the Agonomycetes) isolated from a deadwood sample ofAcer sp. Isolation/Purification The filtrate from a submerged culture was extracted at pH 3 (ethyl acetate) and the extract subjected to silica gel chromatography (methanol in chloroform). The active fraction was crystallized from hexane and the solid subjected to reversed phase HPLC (wateracetonitrile) to give 6.5mg of 8t~-crotonyloxyroridin A and 10.6mg of 8ttbutoyloxyroridin A. Biological Activity Inhibited growth of Candida albicans at 6.25mg/mL. Spectral Data UV; ~b MeOH max

207(e = 18,100) and 262nm (17,300).

IR:"

(CHCI3) 3500, 2980, 1710, 1640, 1600, and 1440cm"l.

16. Macrocyclic Trichothecenes and Related Metabolites

419

1HNMR: (CDCI3) 0.79(3H, s, H-14); 1.08(3H, d, J=7.3Hz, H-12'), 1.19(3H, d, J=6.1Hz, H14'); 1.7(2H, m, H-4'); 1.76(3H, br s, H-16), 1.88(3H, dd, J=6.7 and 1.3Hz, H-4"), 2.19(2H, br s, H-7), 2.23(1H, ddd, J=4.9, 4.9, and 15.SHz, H-3fl); 2.47(1H, dd, J= 7.9 and 15.SHz, H-3a); 2.82 and 3.09(1H each, AB, J=3.9Hz, H-13), 3.5(2H, m, H- 5'); 3.59(1H, br q, J=6.1Hz, H-13'); 3.63(1H, br d, J=3.0Hz, H-6'), 3.71(1H, d, J - 5.5Hz, H-11); 3.84(1H, d, J=4.9Hz, H-2); 3.91(1H, dd, J=2.4 and 7.3Hz, H-2'), 4.40 and 4.57(1H each, AB, J=12.2Hz, H-15); 5.25(1H, br s, H-8); 5.67(1H, dd, J = 1.3 and 15.3Hz, H-2"); 5.70(1H, br d, d=5.5Hz, H-10), 5.77(1H, d, J=l 1.3Hz, H - 10'), 5.78(1H, dd, J=4.9, 7.9Hz, H-4); 5.98(1H, dd, J=3.0, 15.2Hz, H-7'), 6.64(1H, dd, J=l 1.3 and 11.6Hz, H-9'), 6.94(1H, dq, J=6.7 and 15.3Hz, H-3"); and 7.62ppm (1H, dd, J-11.6, 15.2Hz, H-8'). 13CNMR: (CDC13) 78.8, C-2; 34.8, C-3; 73.7, C-4; 49.3, C-5; 42.4, C-6; 26.2, C-7; 68.0, C-8; 136.8, C-9; 123.5, C-10; 66.6, C-11; 65.0, C-12; 47.6, C-13; 7.5, C-14; 65.6, C-15; 20.5, C-16; 174.6, C-I'; 75.7, C-2'; 37.6, C-3'; 33.2, C-4'; 70.3, C-5'; 84.0, C-6'; 139.3, C-7'; 125.9, C-8'; 144.0, C-9'; 117.4, C-10'; 166.3, C-11'; 14.6, C-IT; 70.8, C-13'; 18.2, C-14'; 166.1, C-I"; 121.8, C-2"; 146.7, C-3"; and 18.1ppm, C-4". Reference T. Sugawara, A. Tanaka, K. Nagai, K. Suzuki, and G. Okada; New Members of the Trichothecene Family; J. Antibiotics, Vol. 50, pp. 778-780(1997).

420

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Isororidin A Molecular Formula/Molecular Weight C29H4oO9; M W = 532.26723

10 H '~

-

H o.,j~

.l: . o ......

5

HO/~.~3'

General Characteristics Clear spherical crystals from methylene chloride-hexane; mp., 183-185~ [a]D25 +6.7 ~ (C=3.3, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification Partition chromatography of the mycelial extract gave a fraction rich in roridin A. This fraction was subjected to flash chromatography on silica gel eluting with 3% methanol in methylene chloride to give three principal fractions: A, B, and C, in the order of elution. Fraction A was composed of roridin D and roridin K acetate. Fraction C was composed mostly of trichothecenes of lower Rf than that of roridin A. Fraction B was crystallized from methylene chloride-hexane to give roridin A. The mother liquor was subjected to preparative HPLC (2% methanol-methylene chloride) to give verrucarin L, roridin A, and isororidin A in their order of elution. Spectral Data ~H NMR: (CDCI3) 0.83(3H, s, H-14); 1.09(3H, d, J=7Hz, H-IT); 1.16(3H, d, J=7Hz, H-14'); 1.75(3H, s, H-16); 2.48(1H, dd, J=8 and 15Hz, H-3a); 2.96(2H, AB, J=4Hz, H-13); 3.60(1H, d, J=5Hz, H-11); 3.86(1H, d, J=5Hz, H-2); 4.11(1H, dd, J=3 and 6Hz, H-2'); 4.44(2H, s br, H-15); 5.41(1H, d, J=SHz, H-10); 5.80(1H, m, H-4); 5.80(1H, d, J=l 1Hz, H-10'); 6.00(1H, dd, J=2 and 15.5Hz, H-7'); 6.66(1H, dd, J's=l 1Hz, H-9'); and 7.64ppm (1H, dd, J= 11 and 15.SHz, H-8').

16. MacrocyclicTrichothecenes and Related Metabolites

421

13C NMR: (CDCI3) 7.5, q, C-14; 14.4, q, C-12'; 17.9, q, C-14'; 20.3, t, C-7; 23.3, q, C-16; 27.7, t, C-8; 33.3, t, C-4'; 34.9, t, C-3; 37.0, d, C-3', 43.8, s, C-6; 47.8, t, C-13; 49.4, s, C-5; 64.4, t, C-15; 65.3, s, C-12; 67.2, d, C-11; 69.4, t, C-5'; 70.0, d, C-13'; 74.4, d, C-4; 75.5, d, C-2'; 79.1, d, C-2, 82.6, d, C-6'; 117.3, d, C-10'; 118.3, d, C-10; 126.4, d, C8', 139.0, d, C-7'; 140.9, s, C-9; 143.9, d, C-9'; 166.6, s, C-11', and 174.9ppm, s, C-I' References B. B. Jarvis, Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. W. Jarvis, J. O. Midiwo, J. L. Flippen-Anderson, and E. P. Mazzola; Stereochemistry of the Roridins; J. Nat. Prod., Vol. 45, pp. 440-448(1982).

422

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Nam.e Roridin D Molecular Formula/Molecular Weight C29H3809, ~

l ~ ~ o ,~o ,j

= 530.25158

H

H2(~ 15

I ,, o..~.o

H

~ 4"~O~

.7

II

H 117

-"

........

o1 lz

1r / | ~OH H

General Characteristics Needles from acetone-ether; mp., 232-235~ [a]D 23 +29~ = 2.71, in CHC13). Fungal Source

Myrothecium verrucaria and M. roridum.

Biological Activit_y Antibiotic activity. Spectral Data UV~

Z EmtO~ 260(~=21,400). IR~

(KBr) 3540-3440,2970, 1747, 1711, 1641, 1601, 1420, 1190, 1105, 1083, 968, 818, 753, and 660cm1. 13C NMR:

(CDC13) 78.8, C-2; 34.9, C-3; 74.3, C-4; 49.0, C-5; 43.1, C-6; 20.4, C-7; 27.4, C-8; 140.1, C-9; 118.4, C-10; 66.9, C-11; 65.1, C-12; 47.4, C-13; 6.8, C-14; 64.3, C-15; 22.9, C-16; 167.8, C-I'; 57.9, C-2'; 62.9, C-3'; 39.4, C-4'; 67.3, C-5'; 85.3, C-6'; 138.1, C-7'; 126.2, C-8'; 142.9, C-9'; 117.8, C-10'; 166.1, C-11', 17.2, C-12'; 70.5, C-13'; and 17.9ppm, C- 14'.

16.

MacrocyclicTrichothecenes and Related Metabolites

423

TLC Data Absorbent: Kieselgel G; solvent system, ethyl ether (two consecutive runs); Rf, 0.18; Detection, iodine vapors. References B, Bohner, E. Fetz, E. Harri, H. P. Sigg, and C. Tamm; Helv. Claim. Acta., Vol. 48, pp.

1079(1965).

R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 237-238(1981). B. B. Jarvis, Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991).

424

16.

MacrocyclicTrichothecenes

and Related Metabolites

Common/Systematic Name Roridin D 13-D-glucoside Molecular Formula/Molecular Weight C35H48014; M W -- 692

~6

~o H

---

0

H

2

o ,o 2

I.' /

~.'0 H

l]'glucose

General Characteristics Amorphous solid; [0~]D q- 27* (c=0.80, in CHC13). Fungal Source Female Baccharis coridifolia.. Isolation~urification The first methanol fraction from the chromatography of the crude extract of the plant (see verrucain A glucoside isolation) was slurried with 70% methanol-water and filtered through a pa~ of C-18 silica (ca. 10 g, 40 ~tm), washed with 70% methanol-water. The filtrate was concentratec by rotary evaporation to give a gum. The gum was subjected to CCC (semi-preparative column, Vc = 355 ml) with a solvent system of methanol-water-chloroform-hexane (6:4:7:3 v/v/v/v) and the lower organic phase as the mobile phase to give five fractions A, B, C, D, E. Fraction C was subjected to PTLC (Chromatotron, silica gel, 2 mm) with 3-10% methanol-methylene chloride tt give three portions CI, CII and CIII. Roridin D glucoside was isolated by semipreparative HPLC (silica gel, 250 xl 0 mm, 6% methanol-methylene chloride, 4.0 mL/min) from all three of these fractions. Biological Activity Cytotoxic: ICs5 ca. 1 ng/ml.

Spectral Data IR: (CH2C12) 3418, 1750, 1718, 1650, 1600, 1181, and 1081cm"l.

16.

MacrocyclicTrichothecenes and Related Metabolites

425

1H NMR: (pyridine-d5) 7.63 (1H, dd, d= 11.9, 15.4Hz, H-8'); 6.50 (1H, dd, J= 11.2, 11.9Hz, H-9'); 6.38 (1H, dd, J= 2.3, 15.4Hz, H-7'); 6.00 (1H, dd, J= 3.9, 7.9Hz, H-4); 5.79 (1H, d , J = 11.2Hz, H-10'); 5.49 (1H, br d, J= 4.1Hz, H-10); 4.99 (1H, d , J = 7.THz, H-I"); 4.71 and 4.40 (1H each, AB, J = 12.4Hz, H-15); 4.58 (1H, m, H-6"B); 4.56 (1H, m, H-6'); 4.37 (1H, m, H-6"A); 4.35 (1H, dq, J= 6.2, 6.2Hz, H-13'); 4.26 (1H, dd, J= 8.0, 7.7Hz, H-4"); 4.24 (1H, dd, J= 8.6, 7.7Hz, H-3"); 4.04 (1H, dd, J= 7.7, 8.6Hz, H-2"); 4.00 (1H, m, H-5"); 3.95 (1H, d, J= 4.9Hz, H-2); 3.73 (1H, d , J = 4.THz, H-11); 3.82 (1H, dd, d= 10.0, 10.3Hz H-5'B); 3.73 (1H, d, J= 4.7Hz, H-11); 3.62 (1H, s, H-2'); 3.26 (1H, m, H-5'A); 3.12 and 2.89 (1H each, AB, J= 4.0Hz, H13); 2.47 (1H, dd, J= 8.3, 15.3Hz, H-3ot); 2.42 (1H, dd, J= 10.0, 15.0Hz, H-4'B); 2.26 (1H, ddd, J = 3.9, 4.9, 15.3Hz, H-313); 1.88-1.95 (4H, m, H-7, H-8); 1.90 (3H, s, H-12'); 1.57 (3H, s, H-16); 1.37 (1H, dd, J= 3.9, 15.0Hz, H4'A); 1.20 (3H, d, d= 6.2Hz, H-14'); and 1.07ppm (3H, s, H-14). 13C NMR: (CDC13) 79.1, C-2; 35.3, C-3; 75.3, C-4; 49.8, C-5; 43.7, C-6; 20.6, C-7; 27.7, C-8; 139.5, C-9; 119.8, C-10; 67.2, C-11; 65.8, C-12; 47.7, C-13; 7.4, C-14; 64.7, C-15; 23.1, C-16; 168.7, C-I'; 58.6, C-2'; 63.8, C-3'; 40.6, C-4'; 68.4, C-5'; 82.6, C-6'; 141.4, C-7'; 125.9, C-8'; 143.9, C-9'; 117.2, C-10'; 169.9, C-11'; 17.6, C-12'; 77.4, C-13'; 17.9ppm, C- 14'. TLC Data Adsorbent: Silica gel; solvent system: 10% methanol-methylene chloride; Rf, 0.26; detection with iodine vapors. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71 (1996).

426

16.

MaerocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Roridin E Molecular Formula/Molecular Weight C29H3808, M ~ r = 5 1 4 . 2 5 6 6 7

10 H

H

o

'

12'

1; /

1 "OH H

General Characteristics Colored needles from ether-pentane; mp., 177-1780C or 211-212~ needles from ether, 220-221~ [tt]D23 -27 ~ (C=1.3, in CHCI3); [tt]D25 -21" (C=1.852, in CHCI3). Fungal Source Myrothecium verrucaria and M. roridum.

Biological Activity Antibiotic activity. Spectral Data UV:

~, ~~

195(e=15,800), 223(25,100), and 263nm (19,900).

IR:

(CH2C12) 3570, 3050, 2975, 1712, 1647, 1603, 1365, 1220, 1180, 1148, 1142, 1096, 1090, 1080, 966, and 814cm4. 1H NMR: (CDCI3) 3.82, (1H, d , J = 5.0Hz, H-2); 2.04, (1H, ddd, J= 4.2, 5.0, 15.5Hz, H-313; 2.53, (1H, dd, J= 8.2, 15.0Hz, H-3a); 6.20, (1H, dd, J= 4.0, 8.2Hz, H-4); 5.47 (1H, br d, Jr= 5.0Hz, H-10); 3.89 (1H, d, J=- 5.0Hz, H-11); 2.81 and 3.12(1H each, AB, J= 4.1Hz, H-13); 0.79 (3H, s, H-14); 3.93 and 4.32 (1H each, AB, J= 12.5Hz, H-15); 1.71 (3H, br s, H-16); 5.95(1H, q, d=- 1.5Hz, H-2'), 3.70 (1H, m, H-6'); 5.89 (1H, dd, or-- 2.0, 16.0Hz, H-7'); 7.51(1H, dd, Jr= 11.2, 16.0Hz, H-8'); 6.56 (1H, dd, J= 11.0,

16.

MacrocyclicTrichothecenes and Related Metabolites

427

11.2Hz, H-9'); 5.73 (1H, d, J= 11.0Hz, H-10'); 2.25 (3H, d, Jr= 1.5Hz, H-12'); 3.70 (1H, m, H-13'; and 1.19ppm (3H, d, Jr= 6.0Hz, H-14'). 13CNMR: (CDCla) 79.3, C-2; 35.8, C-3; 74.2, C-4; 48.4, C-5; 42.8, C-6; 21.6, C-7; 27.7, C-8; 140.0, C-9; 117.8, C-10; 67.2, C-11; 65.6, C-12; 48.1, C-13; 6.7, C-14; 63.7, C-15; 23.2, C-16; 165.8, C-I'; 119.0, C-2'; 159.0, C-3'; 41.3, C-4'; 69.8, C-5'; 83.8, C-6'; 138.1, C-7'; 126.6, C-8'; 143.7, C-9'; 117.2, C-10'; 166.4, C-11'; 20.2, C-12'; 70.5, C13'; 18.3, C-14'. TLC Data Adsorbent: Kieselgel G; solvent system: ethyl ether (two consecutive runs); Re, 0.24; detection with iodine vapors. References B. Bohner, E. Fetz, E. Harri, H. P. Sigg, and C. Tamm; Helv. Chim. Acta.; Vol. 48, pp. 1079(1965). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 237-238(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto; Structures oflsororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon; J. Sci. Hinoshima; University serial A; Vol. 43, pp. 107-118 (1979).

428

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Roridin E [3-D-glucoside Molecular Formula/Molecular Weight C35I-I48013;MW = 676.30949 H

18

H

01 13~.;,,,io

I

o O ~ ~ ~ 9'

'3

~'

O

[7'

glucose General Characteristics Amorphous solid; [a]D+ 37 ~ (C=2.0, in CHC13). Fungal Source Female Baccharis coridifolia. Isolation/Purification The first methanol fraction from the chromatography of the crude extract of the plant (see verrucain A glucoside isolation) was slurried with 70% methanol-water and filtered through a pad Of Cls silica (ca. 10 g, 40 lam), washed with 70% methanol-water. The filtrate was concentrated by rotary evaporation to give a gum. The gum was subjected to CCC (semi-preparative column, Vc = 355 ml) with a solvent system of methanol-waterchloroform-hexane (6:4:7:3 v/v/v/v) and the lower organic phase as the mobile phase to give five fractions A, B, C, D, E. Fraction B was subjected to Cls reversed phase HPLC (250 x 10 mm, 68% methanol-water, 4.0 mL/min) to yield roridin E 13-glucoside. Biological Activity Cytotoxic: ICs5 ca. 1 ng/ml. Spectral Data IR:

(CH2CI2) 3412, 1706, 1643, 1600, 1181, and 1081cm~.

16.

Macrocyclic Trichothecenes and Related Metabolites

429

1H NMR: (CDCI3) 7.39(1H, dd, ,/--12.0, 15.0Hz, H-8'); 6.59(1H, dd, J=l 1.2, 12.0Hz, H-9'); 6.08(1H, dd, J=3.9, 7.6Hz, H-4); 5.98(1H, dd, J=2.7, 15.0Hz, H-7'); 5.86(1H, s, H-2'); 5.69(1H, d, J=l 1.2 Hz, H-10'); 5.44(1H, d, J=4.4Hz, H-10); 4.38(1H, d, J=7.4Hz, HI"); 4.31 and 3.90(1H each, AB, J=12.4Hz, H-15); 4.10(1H, m, H-6'); 3.98(1H, dq, d =5.5, 6.0Hz, H-13'); 3.80-3.83(3H, m, n-6", n-11); 3.80(1H, d, J=5.2Hz, H-2); 3.70(1H, m, H-5'B); 3.56(1H, dd, ,/--8.6, 9.4Hz, H-4"); 3.51(1H, dd,J=8.0, 9.4Hz, n3"); 3.48(1H, m, H-5'A); 3.33(1H, dd, ,/--7.4, 8.0Hz, n-2"); 3.28(1H, bd, J=8.6Hz, n5"); 3.09 and 2.78(1H each, AB, J=3.7Hz, H-13); 2.48(1H, dd, ,/--7.6, 15.0Hz, H-3'); 2.43(2H, m, n-4'); 2.21(3H, s, U-12'); 2.04(1H, ddd, ,/--4.0, 5.0, 15.0Hz, H-3B); 1.98(4H, m, n-7, n-8); 1.68(3H, s, n-16); 1.08(3H, d, J=6.0Hz, n-14'); and 0.76ppm(3H, s, H-14). (pyridine-d5) 7.71(1H, dd, J=l 1.8, 15.5Hz, H-8'); 6.49(1H, dd, J=l 1.1, 11.8Hz, H-9'); 6.34(1H, dd, ,/=2.8, 15.5Hz, H-7');6.23(1H, dd, ,1=4.0, 8.0Hz, H-4); 6.02(1H, s, U-2'); 5.74(1H, d, J=l 1.1Hz, H-10'); 5.5 I(1H, br d, `/=5.0Hz, H-10); 4.96(1H, d, J=7.8Hz, H-I"); 4.75 and 4.09(1H each, AB, J=12.5Hz, H-15); 4.57(1H, dd, J=2.2, 11.8Hz, H-6"B); 4.40(1H, dq, J=6.3, 6.3Hz, H-13'); 4.37(1H, m, H-6"A); 4.26(2H, m, H-3", H-4"); 4.03(1H, dd, ,/--7.8, 8.0Hz, H-2"); 3.99(1H, m, H5"); 3.92(1H, d, J=5.0Hz, H-2); 3.83(1H, br d, J=5.0Hz, H-11); 3.48(1H, m, H-5'B); 3.38(1H, m, H-5'A); 3.10 and 2.85(1H each, AB, J=4.0Hz, H-13); 2.47(1H, dd, J=8.0, 15.0Hz, H-3a); 2.41(3H, s, H-12'); 2.31(2H, m, H-4'); 2.19(1H, ddd, J=4.0, 5.0, 15.0Hz, H-313); 1.92-1.99(3H, m, H-7B, H-8); 1.79(1H, br d, J=7.5Hz, H-7A); 1.56(3H, s, H-16); 1.12(3H, d,J=6.3Hz, H-14'); and 1.05ppm (3h, s, H-14). 13CNMR: (CDC13) 79.2, C-2; 35.6, C-3; 74.3, C-4; 48.5, C-5; 42.8, C-6; 21.3, C-7; 27.6, C-8; 140.2, C-9; 117.2, C-10; 67.2, C-11; 65.6, C-12; 48.1, C-13; 6.8, C-14; 63.6, C-15; 23.3, C-16; 166.5, C-I'; 118.8, C-2'; 159.0, C-3'; 41.4, C-4'; 69.0, C-5'; 80.4, C-6'; 138.6, C-7'; 126.9, C-8'; 143.9, C-9'; 117.2, C-10'; 166.2, C-11'; 19.5, C-12'; 76.7, C13'; 15.0, C-14' 101.8, C-I"; 73.2, C-2"; 76.2, C-3'; 69.7, C-4"; 75.5, C-5"; and 61.7ppm, C-6". TLC Data Adsorbent: Silica gel; solvent system: 10% methanol-methylene chloride; Rf, 0.26; detection with iodine vapors. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

430

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Isororidin E Molecular Formula/Molecular Weight C29H3808; M W = 5 1 4 . 2 5 6 6 7

10 H

H

HO"

1 ~ 14'

H

General Characteristics Colorless prisms from ethyl acetate; mp., 200-202~

[tt]D - 65.1 o (in CHC13).

Fungal Source

Cylindrocarpon spp.

Spectral Data UV~

~

EtOH max

223(e=24,000) and 262nm (16,000).

IR~

(CHaCI) 3757, 1713, 1644, and 1598cm"l. ~H NMR: 3.84(H-2); 2.03(H-3a); 2.58(H-3b); 6.35(H-4); 5.50(H-10); 4.09(H-11); 2.83(H-13a); 3.15(H-13b); 0.80(H-14); 4.06(H-15a); 4.16(H-15b); 1.71(H-16); 5.83(H-2'); 3.74(H6'); 5.71(H-7'); 7.55(H-8'); 6.60(H-9'); 5.82(H-10'); 2.22(H-12'); 3.70(H-13'); and 1.17ppm (H- 14'). 13CNMR: (CD3C1) 79.2, C-2; 36.6, C-3; 75.3, C-4; 48.5, C-5; 42.6, C-6; 22.7, C-7; 27.7, C-8; 140.1, C-9; 118.9, C-10; 66.7, C-11; 65.7, C-12; 47.7, C-13; 6.4, C-14; 64.5, C-15; 23.2, C-16; 166.3, C-I'; 119.5, C-2'; 158.0, C-3'; 40.0, C-4'; 67.0, C-5'; 83.2, C-6'; 135.3, C-7'; 131.0, C-8'; 142.0, C-9'; 117.1, C-10'; 166.3, C-11'; 19.8, C-12'; 67.6, C-13'; and 18.5ppm, C-14'.

16. MacrocyclicTrichothecenes and Related Metabolites

431

References R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 242 (1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds., CRC Press, Boca Raton, Florida, pp. 361-421 (1991). M. Matsumoto, H. Minato, K. Tori, and M.Ueyama, Structures oflsororidin E, Epoxyisororidin E, and Epoxy- and Diepoxyroridin H, New Metabolites Isolated from Cylindrocarpon Species Determined by Carbon-13 and Hydrogen-1 NMR Spectroscopy. Revision of C-2':C-3' Double Bond Configuration of the Roridin Group; Tet. Lett.; pp. 4093-4096(1977).

432

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Epiroridin E Molecular Formula/Molecular Weight C29H3808; M W = 5 1 4 . 2 5 6 6 7

10 H

H

~,-r162 "OH H General Characteristics Amorphous solid; [~]D 30-~- q- 0.70 ~ (c--1.8, in chloroform). Fungal Source Myrothecium verrucaria (ATCC 24571 ). Isolation/Purification A chromatography fraction from an earlier fermentation ofM. verrucaria ATCC 24571 [Jarvis et al.; J. Org. Chem.; Vol. 47,pp. 1117-1124 (1982)] was subjected to CCC (Vc = 850ml) with a solvent system of carbon tetrachloride-methanol-water (5:3:2, v/v/v), the lower organic phase was the mobile phase and the flow rate was 3.3ml/min, to give verrucarin A (23mg), roridin D (1 lmg), a fraction rich in roridin E (400mg), and roridin A (67mg). The roridin E fraction was subjected to CCC (Vc 355mL) with a solvent system of carbon tetrachloride-hexane-methanol-water (4:1:3:2, v/v/v/v), the lower organic phase was the mobile phase, and the flow rate was 1.8ml/min, to give 310mg of a mixture of roridin E and isororidin E and a later fraction of pure epiroridin E (50mg). Spectral Data IR:

(CHCI3) 2491,1713, 1652, and 1601cm"1. 1H NMR: (CDCI3) 0.76(3H, s, H-14); 1.13(3H, d, J=6.0Hz, H-14'); 1.67(3H, s, H-16); 1.92-2.20(5H, m, H-7, H-8, H-313); 2.24(3; H, s, H-12'); 2.42-2.54(3H, m, H-4', H3tt); 2.83(1H, d, J=4.0Hz, H-13B); 3.15(1H, d, J=4.0Hz, H-13A); 3.50(1H, dt,

16.

MacrocyclicTrichothecenes and Related Metabolites

433

J=l 5.0, 6.0Hz, H-5B); 3.69(1H, dt, J=15.0, 6.0Hz, H-5A); 3.87(1H, d, J=5.0Hz, H-2); 3.79-3.90(2H, m, H-6', H-13'), 3.89(1H, d, J=12.0Hz, H-15B); 3.91(1H, d, J=5.0Hz, H-11); 4.31(1H, d, J=12.0Hz, H-15A); 5.44(1H, d, J=5.0Hz, H-10); 5.70(1H, d, J=l 1.0Hz, H-10'); 5.86(1H, dd, J=3.0, 15.0Hz, H-7'); 5.98(1H, s, H-2'); 6.15(1H, dd, J=4.0, 8.0Hz, H-4); 6.54(1H, dd, J=l 1.0Hz, H-9'); and 7.45ppm (1H, dd, J=l 1.0, 15.0Hz, H-8'). 13CNMR: (CDC13) 6.7, C-14; 17.8, C-14'; 19.8, C-12'; 21.5, C-7; 23.2, C-16; 27.2, C-8; 35.7, C-3; 41.4, C-4'; 42.7, C-6; 48.1, C-13; 48.4, C-5; 63.6, C-15; 65.6, C-12; 67.2, C-11; 69.1, C-13'; 69.5, C-5'; 74.2, C-4; 79.2, C-2; 82.5, C-6'; 117.2, C-10'; 117.6, C-10; 118.9, C-2'; 126.8, C-8'; 137.8, C-7'; 140.8, C-9; 143.6, C-9'; 159.0, C-3'; 165.9, C-I'; and 166.4ppm, C- 11' Reference B. B. Jarvis and S. Wang; Stereochemistry of the Roridins. Diastereomers of Roridin E; J. Nat. Prod., Vol. 62, pp. 1284-1289(1999).

434

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Epiisororidin E Molecular Formula/Molecular Weight C29H3808; ~

= 514.25667

lo H

16

H

2

3

, , ' / 1 "OH H General Characteristics Amorphous solid;

[ a ] D 30 --

-29.2 ~ (c=1.5, in CHCI3).

Fungal Source Myrothecium verrucaria (ATCC 24571).

Isolation/Purification Extraction of a submerged culture ofM. verrucaria [see Jarvis, B. B., Armstrong, C. A., Zeng, M. J.; Antibiotics; Vol. 43, pp. 1502-1504 (1990)] with organic solvent gave 7.5g of crude extract atter solvent removal. A series of chromatographies (silica gel and CCC) gave 353mg of a mixture of roridin E and isororidin E, 17mg of mixture of roridin E, isororidin E and epiisororidin E and 20mg of pure epiisororidin E. The roridin E diastereomers can be separated by RP-HPLC on a phenyl column (Phenomenex, 4.6 x 250mm) with 40% acetonitrile in water at a flow rate of 1.2ml/min. The observed retention times under these conditions were: roridin E (27.7 min), isororidin E (23.3 min), epiroridin E (26.1 min), and epiisororidin E (22.1 min). Spectral Data IR:

(CHC13) 3486,1713, 1647, and 1599cm q. 1H NM:R: (CDC13) 0.77(3H, s, H-14); 1.12(3H, d, J-6.5Hz, H-14'); 1.68(3H, s, H-16); 1.962.13(5H, m, H-313, H-7, H-8); 2.21(3H, d, J=l.2Hz, H-12'); 2.29(1H, m, H-4'B); 2.502.56(2H, m, H-3~, H-4'A); 2.81(1H, d, J=4.0Hz, H-13B); 3.13(1H, d, J-4.0Hz, H-

16.

Macrocyclic Trichothecenes and Related Metabolites

435

13A); 3.56(1H, ddd, J=5.0, 7.4, 10.1 Hz, H-5'B); 3.73(1H, ddd, J=7.4, 7.5, 10.1Hz, H-5'A); 3.83(1H, d, J=5.0Hz, H-2); 3.89(1H, m, H-6'); 3.98(1H, dq, J=3.0, 6.5Hz, H13'); 4.01(1n, d, J=12.5Hz, H-15B); 4.03(1H, d, J=5.5Hz, n-11); 4.17(1H, d, J=12.5Hz, H-15A); 5.47(1H, d,J = 5.5Hz, H-10); 5.80(1H, d,J=l 1.0Hz, H-10'); 5.82(1H, d, J=l.2Hz, H-2'); 5.87(1H, dd, J=6.1, 15.6Hz, H-7'); 6.29(1H, dd, J-4.1, 8.0Hz, H-4); 6.60(1H, dd, J=l 1.0, 11.0Hz, H-9'); and 7.54ppm (1H, dd, J = 11.0, 15.6Hz, H-8'). 13CNMR: 6.5, C-14; 17.9, C-14'; 19.2, C-12'; 22.3, C-7; 23.2, C-16; 27.7, C-8; 36.4, C-3; 40.3, C-4'; 42.6, C-6; 47.8, C-5; 48.4, C-13; 64.3, C-15; 66.3, C-5'; 66.6, C-12; 67.0, C-11; 68.5, C-13'; 75.0, C-4; 79.2, C-2; 81.9, C-6'; 117.2, C-10'; 117.6, C-10; 119.0, C-2'; 131.0, C-8'; 134.7, C-7'; 140.2, C-9; 142.3, C-9'; 157.9, C-3'; 166.3, C-I'; and 166.5ppm, C-11'. Reference B. B. Jarvis and S. Wang; Stereochemistry of the Roridins. Diastereomers of Roridin E; J. Nat. Prod., Vol. 62, pp. 1284-1289(1999).

436

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name 713,813-Epoxyisororidin E Molecular Formula/Molecular Weight C29H3609; MW

-- 5 2 8 . 2 3 5 9 3

~oH

H

~~

0 H271~!~ -" .,0,~11i 0=:~--~' , / 01

3

H General Characteristics Colorless prisms from acetone; mp., 216-219~

[ a ] D 24

-

69.9 ~ (c= 0.438 in CHC13).

Fungal Source

Cylindrocarpon spp.

Spectral Data UV: ~, E~

221 (C=26,300) and 262nm (16,700).

IR: (CH3C1) 3567, 1712, 1644, and 1598cm1. 1H NMR: (CDCI3) 3.90(1H, d , J - 4.7Hz, H-2), 2.06(1H, ddd, J-- 4,3, 4.7, 15.0Hz, H-3~), 2.55(1H, dd, J = 8.1, 15.0Hz, H-3tx), 6.30(1H, dd, J= 4.3, 8.1Hz, H-4); 3.34(1H, dd, Jr-- 3.2, 4.0Hz, H-7); 3.17(1H, dd, J-2.1, 4.0Hz, H-8); 5.75(1H, ddd, J = 1.5,2.1, 7.0Hz, H-10); 4.21(1H, dd, J= 3.2, 7.0Hz, H-11); 2.99 and 3.21(1H each, AB, J 3.4Hz, H-13); 1.02(3H, s, H-14), 3.79 and 4.07(1H each, AB, J = 12.5Hz, H-15), 2.00(3H, d,J = 1.5Hz, H-16); 5.80(1H, q , J - 1.0Hz, H-2'), 3.75(1H, m, H-6'); 5.77(1H, dd, J - 5.0, 15.9Hz, H-7'), 7.54(1H, dd, J = 11.0, 15.9Hz, H-8'); 6.62(1H, dd, J= 11.0, 11.1Hz, H-9'); 5.83(1H, d, Jr-- 11.1Hz, H-10'); 2.24(3H, d, Jr- 1.0Hz, H-12'); 3.70(1H, m, H-13'); and 1.16ppm (3H, d, J= 6.0Hz, H-14').

16. MacrocyclicTrichothecenes and Related Metabolites

437

13C NMR: (CDCI3) 79.2, C-2; 36.6, C-3; 74.6, C-4; 47.0, C-5; 44.0, C-6; 50.8, C-7; 56.1, C-8; 138.2, C-9; 123.0, C-10; 66.7, C-11; 65.7, C-12; 48.3, C-13; 7.2, C-14; 62.9, C-15; 21.9, C-16; 165.9, C-I'; 119.1, C-2'; 159.0, C-3'; 40.1, C-4'; 67.0, C-5'; 83.3, C-6'; 135.5, C-7'; 130.5, C-8'; 142.4, C-9'; 116.7, C-10'; 166.2, C-11'; 20.0, C-12'; 69.7, C-13', and 18.5ppm, C-14'. References R. J. Cole and R. H. Cox; Handbook of Toxic Fun_galMetabolites; Academic Press, New York, pp. 243(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto, H. Minato, K. Toil,and M. Ueyama; Structures oflsororidin E, Epoxyisororidin E, and Epoxy- and Diepoxyroridin H, New Metabolites Isolated from Cylindrocarpon Species Determined by Carbon-13 and Hydrogen-1 NMR Spectroscopy. Revision of C-2': C-3' Double Bond Configuration of the Roridin Group, Tet. Lett.; pp. 4093-4096(1977).

438

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Roridin K acetate Molecular Formula/Molecular Weight

C31H40010;M W ~o H

-- 5 7 2 . 2 6 2 1 5

H

,

, -

,.

13' 14'~07'H H General Characteristics Crystals from dichloromethane-hexane, rap., 255-257~

[a]D2~ + 2.1 ~ (C= 5.6 in CHel3).

Isolation/Purification Spores ofMyrothecium verrucaria grown on N-Z amine agar were added to Czapek-Dox media and allowed to grow at 28~ in a shake culture. After 2 days, the solution was divided into three equal portions and added to three separate Fernback flasks containing 1L of production media. After 3 days of growth, the mycelium in each flask was separately centrifuged, washed, and resuspended in 1L of sterile water. Trichoverrin A and trichoverrin B were added separately to flasks one and two; flask three was used as the control. After 7 days, the mycelium was removed and extracted with ethyl acetate. The mycelium extract was subjected to partition chromatography on 500g Celite impregnated with 250ml of 18% water in methanol. The column was eluted with petroleum ether followed by increasing amounts of dichloromethane in petroleum ether up to 40% dichloromethane in petroleum ether. Fractions were combined on the basis of TLC analysis to give a total of eight fractions: A, B, C, D, E, F, G, and H (methanol wash). Fraction F was subjected to MPLC (30-60% ethyl acetate in hexane) to yield a fraction composed principally of a mixture of roridin E and isororidin E in a ratio of ca. 1:4, v/v. The following fraction was subjected to HPLC (20% ethyl acetate in hexane) to give roridin D and roridin K acetate. Fungal Source

Myrothecium verrucaria (ATCC 24571).

Spectral Data UV:

max

~. E~.

263nm (log e= 4.21).

16.

Macrocyclic Trichothecenes and Related Metabolites

439

~H N M R : (CDCI3) 0.78(3H, s, 14-H); 1.19(3H, d , J = 6.0Hz, 14'-H); 1.76(3H, s, 16-H); 1.19(acetate); 2.30(3H, d, J=l.2Hz, 12'-H); 2.52(1H, dd, J=7, 15Hz, 3tt-H); 2.97(2H, d=4Hz, 13-H); 3.84(11-1, d, J=5Hz, 2-H); 3.90(1H, d, J=5Hz, 1 l-H), 4.31(2H AB, d=12Hz, 15-H); 5.75(1H, d, J=l 1Hz, 10'-H), 5.78(1H, d, d=16Hz, 7'-H); 6.10(1H, dd, J=4, 8Hz, 4-H); 6.57(1H, dd, Js=llHz, 9'-H); and 7.47ppm (1H, dd, J=ll, 16Hz, 8'H). 13C NMR: (CDCIa) 79.1 d, C-2; 35.5 t, C-3; 73.8 d, C-4; 48.6 s, C-5; 42.2 s, C-6; 27.4 t, C-7; 68.8 d, C-8; 136.4 s, C-9; 124.0 d, C-10; 67.0 d, C-11; 65.4 s; C-12; 48.0 t, C-13; 6.8 q, C-14; 64.7 t, C-15; 21.1 q, C-16; 165.9 s, C-I'; 117.6 d, C-2'; 159.7 s, C-3'; 41.2 t, C-4'; 70.3 t, C-5'; 84.1 d, C-6'; 138.5 d, C-7', 126.5 d, C-8'; 143.0 d, C-9'; 116.7 d, C10'; 166.2 s, C-11'; 20.2 q, C-12'; 70.7 d, C-13'; 18.3 q, C-14'; 170.8 s, COCH3; and 20.7ppm q, COCH3. Mass Spectrum: HRCIMS (methane gas reagent): 573.2692m/e(M + + H, calcd 573.2700). Reference B. B. Jal~S, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

440

16. Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name Roridin H; Verrucarin H Molecular Formula/Molecular Weight C29H3608, M W -- 5 1 2 . 2 4 1 0 2 ~o H

H

,,,o

,

General Characteristics Crystals from acetone-ether, mp >325~ (dec.), [~]D 23 +31 ~ (c=1.16, in CHC13), [{~]D23 + 40.3 ~ (c=1.065, in benzene); [~]Dz3 +31.5 ~ (C=0.855, in dioxane). Fungal Source

Myrothecium verrucaria.

Biological Activity Antibiotic activity. Spectral Data UV:

195(e=15,800), 224 (24,500), and 260nm (18,200). IR;

(CH2CH2) 2970,2910, 1710, 1645, 1600, 1380, 1355, 1220, 1175, 1115, 1101, 1087, 1071, 1033, 1005, 992, 971, and 832cm 1. 1H NMR: (CDC13) 3.80(1H, d, H-2); 5.90(H-4); 5.42(1H, d, J=4.0Hz, H-10); 3.64(H-11); 2.96(2H, AB, J=4.0Hz, H-13); 0.85(H-14); 4.15(2H, AB, J=12.0Hz, H-15); 1.69(H16); 5.67(H-2'); 2.74(1H, m, H-4'); 5.58(1H, J= 3.5, 8.0Hz, H-5'); 4.03(1H, m, H-6'); 5.90(1H, m, H-7'); 7.68(1H, dd, J=l 1.0, 15.5Hz, H-8'); 6.55(1H, t,J=l 1.0Hz, H-9'); 5.79(1H, d, J=l 1.0Hz, H-10'); 2.27(3H, d, J=l.5Hz, H-12'); 3.65(1H, m, H-13'); and 1.32ppm(3H, d, J-6.0Hz, H-14').

16. Macrocyclic Trichothecenes and Related Metabolites

441

13CNMR: (CDC13) 79.0,C-2; 34.8,C-3; 74.0, C-4; 48.9,C-5, 43.2, C-6; 20.5, C-7; 27.6, C-8; 139.9, C-9; 118.6, C-10; 67.6, C-11; 65.3, C-12; 47.3, C-13; 7.0, C-14; 63.0, C-15; 22.9, C-16; 166.0, C-I'; 119.0, C-2'; 154.4, C-3'; 47.7, C-4'; 100.8, C-5'; 81.9, C-6'; 134.6, C-7'; 126.2, C-8'; 142.5, C-9'; 118.9, C-10'; 166.0, C-11'; 18.2, C-12'; 76.8, C13'; and 16.3ppm, C-14'. TLC Data Absorbent: Kieselgel G; solvent, ethyl ether (two consecutive runs); Rf, 0.51; detection: iodine vapors. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 239-240(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto; Structures of Isororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon; J. Sci. Hinoshima, University serial A, Vol. 43, pp. 107-118 (1979).

442

16.

Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name Roridin J Molecular Formula/Molecular Weight C29H3609; M W -- 5 2 8 . 2 3 5 9 3

~o H

H

Ii

i,o H2C I s

O ~ OH

1

o

L

U,,,

4~ 0 ~ ~ .

J

12

General Characteristics Crystals from dichloromethane-hexane; mp., 281-285~ [a]D2s + 21.8 ~ (in CHCla). Acetate derivative, crystals from diehloromethane-ether; mp., 230-235~ [a]D2s -40.6 ~ (in CHC13). Fungal Source

Myrothecium verrucaria (ATCC 24571).

Isolation/Purification The metabolite was isolated by a combination of column chromatographies (adsorption and partition) and recrystallizations from an ethyl acetate extract of the fermentation broth. Roridin J, is closely related to roridin H, but can be separated by careful chromatography using alumina with hexane-methylene chloride as eluant. B.i..ological Activity Exhibited substantial in vivo activity against P388 mouse leukemia (PS). It was toxic at 10mg/kg and exhibited the following T/C activities (dose level) in PS: 158 (Smg/kg), 149 (2.5mg/kg), 140 (1.25mg/kg), and 125 (0.62mg/kg). Spectral Data UV"

~.~H

max

26 lnm (log 6=4.28).

16.

MacrocyelicTrichothecenes and Related Metabolites

443

IR:

(KBr) 3535(OH), 1715(C=O), 1645, and 1595cm1 (diene); acetate, 1745 and 1715(C=O's), 1655, 1605cm"1 (diene). 1H ~ : (CDCI3) 3.85(1H, d, J=5.0Hz, H-2); 2.1(1H, m, H-313); 2.48(1H, dd, J=8.0, 15Hz, H3ct); 6.0(1H, dd, J=4.0, 8.0Hz, H-4); 2.0(2H, m, n-7); 2.0(2H, m, n-8); 5.44(1H, d, J=5.0Hz, H-10); 3.63(1H, d, J=5.0Hz, n-11); 2.97(2H, AB, J=4.0Hz, H-13); 0.87(3H, H-14); 4.21(2H, AB, J=2.0Hz, n-15); 1.74(3H, H-16); 5.84(1H, d, J=l, 2Hz, n-2'); 3.85(1H, d, J=7.0Hz, H-4'); 5.24(1H, d, J=7.0Hz, n-5'); 3.87(1H, n-6'); 5.8(1H, d, J=15, 5.0Hz, H-7'); 7.70(1H, dd, J=l 1.5, 15.5Hz, H-8'); 6.54(1H, t, J=l 1.5Hz, H-9'); 5.9(1H, d, J=l.5Hz, n-10'); 2.28(3H, d, J=l, 2.0Hz, H-12'); 3.70(1H, q, J=6.0Hz, H13'); and 1.36ppm (3H, d, J=-6.0Hz, H-14'). 13CNMR: (CDC13) 79.2, d, C-2; 34.7, t, C-3; 73.9, d, C-4; 49.2, C-5; 43.3, C-6; 20.4, t, C-7; 27.6, t, C-8; 140.4, C-9; 118.6, d, C-10; 67.9, d, C-11; 65.6, C-12; 47.9, t, C-13; 7.4, q, C-14; 63.4, t, C-15; 23.3, q, C-16; 165.9, C-I'; 119.8, d, C-2'; 155.4, C-3'; 79.8, d, C-4'; 103.4, d, C-5'; 82.3, d, C-6'; 134.5, d, C-7'; 126.1, d, C-8'; 143.1, d, C-9'; 118.9, d, C-10'; 166.2, C-11'; 13.1, q, C-12'; 76.5, d, C-13' and 16.0ppm, q, C-14'. Mass Spectrum: EIMS: 528m/e (M~). Reference B. B. Jarvis, G. P. Stahly, and G. Pavanasasivam, E. P. Mazzola; Structure ofRoridin J. A New Macrocyclic Trichothecene from Myrothecium verrucaria; Joumal of Antibiotics; Vol. 33, pp. 256-258(1980).

444

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name 7[3,8[3-Epoxyroridin H Molecular Formula/Molecular Weight C29H3409; MW - 526.22028

10 H

16

H

141 '

(D 011~*

a. ~ General Characteristics Colorless, amorphous powder. Fungal Source

Cylindrocarpon spp.

Spectral Data IR:

(CHCI3) 1717, 1646, and 1601cmq.

IH N~/[R: (CDCIs) 5.91 (IH, d, J= 4.5Hz, H-2), 2.15, (IH, ddd, ~-- 4.0, 4.5, 14.0Hz, H-3~; 2.40 (IH, dd, ~= 8.0, 14.0I-Iz,H-3c0, 5.87 (IH, dd, J= 4.0, 8.0Hz, H-4), 3.75 (IH, dd, J= 3.0, 4.0Hz, H-7); 3.17 (IH, rid,J= 2.1, 4.0I-Iz,H-8); 5.69 (IH, m, H-10); 4.08 (II-I,m, H-11); 2.98 and 3.18(IH each, AB, J= 3.6I-Iz,H-13), 1.12 (3H, s, H-14), 3.41 and 4.43 (IH each, AB, J= 12.3Hz, H-15), 1.98 (3H, d , J = 1.4Hz, H-16), 5.69(1H, q,J= 1.0Hz, H-2'); 2.32 and 2.41 (1H each, m, H-4'), 5.54 (1H, dd, J= 3.4, 8.4Hz, H-5'), 4.06 (1H, ddd, J = 2.0,2.3, 8.0Hz, H-6'); 5.95 (1H, dd, J = 2.3, 15.4Hz, H-7'), 7.76(1H, ddd, J = 2.0, 11.4, 15.4Hz, H-8'), 6.58 (1H, dd, J = 11.1, 11.4Hz, H-9'), 5.79 (1H, d,J-- 11.1Hz, H-10'), 2.28 (3H, d, J= 1.0Hz, H-12'), and 1.34ppm (3H, d, J - 5.9Hz, H-14').

16.

Macrocyclic Trichothecenes and Related Metabolites

445

13C NMR: (CDC13) 79.3, C-2; 35.0, C-3; 73.6, C-4; 48.2, C-5; 44.2, C-6; 50.9, C-7; 57.2, C-8; 138.0, C-9, 123.0, C-10; 67.9, C-11; 65.5, C-12; 47.9, C-13; 8.2, C-14; 65.8, C-15; 22.0, C-16; 165.9, C-I'; 118.7, C-2'; 155.6, C-3'; 47.7, C-4'; 101.0, C-5'; 82.0, C-6'; 135.4, C-7'; 126.2, C-8'; 143.4, C-9'; 118.4, C-10'; 165.9, C-11'; 18.4, C-12'; 77.1, C13'; and 16.5ppm, C-14'.

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 244(1981). B. B. Jarvis, Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). M. Matsumoto; Structures of Isororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon;J. Sci. Hinoshima, University serial A, Vol. 43, pp. 107-118 (1979).

446

16.

Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name 7 p,813,2',3'-Diepoxyisororidin H Molecular Formula/Molecular Weight C29H3401o; M W = 5 4 2 . 2 1 5 2 0

lo H

H

';I o

,,o I

o

I10,

12

General Characteristics Colorless prisms from ethyl acetate, mp 291-293~ (dec.). Fungal Source

Cylindrocarpon spp.

Spectral Data IR:

(CHCI3) 1755, 1710, 1645, and 1601cm ~. ]H NMR: (CDCI3) 3.90 (1H, d, J= 4.5Hz, H-2), 2.26 (1H, ddd, J= 4.5, 5.0, 15.0Hz, H-313; 2.45 (1H, dd, J = 8.0, 15.0Hz, H-3ot); 5.89 (1H, dd, J = 5.0, 8.0Hz, H-4), 3.62 (1H, dd, J = 3.0, 3.9Hz, H-7), 3.18 (1H, dd, J= 1.9, 3.9Hz, H-8), 5.67 (1H, ddd, J= 1.5, 1.9, 6.3Hz, H-10); 3.75 (1H, dd, J = 3.0, 6.3Hz, H-11); 2.97 and 3.17(1H each, AB, J = 3.5Hz, H13); 1.12 (3H, s, H-14), 4.44 (2H, s, H-15), 2.01 (3H, d , J = 1.5Hz, H-16), 3.29(1H, s, H-2'), 1.57 (1H, dd, J = 9.2, 14.0Hz, H-4'A), 2.32 (1H, dd, J = 2.7, 14.0Hz, H-4'B); 5.37 (1H, dd, J = 2.7, 9.2Hz, H-5'); 4.17 (1H, ddd, J = 1.8, 2.2, 8.0Hz, H-6'), 5.98 (1H, ddd, J = 2.2, 3.0, 15.5Hz, H-7'); 7.60(1H, ddd, J = 1.8, 11.3, 15.SHz, H-8'); 6.62 (1H, dd, J= 11.3, 11.7Hz, H-9'), 5.88 (1H, d, J - 11.7Hz, H-10'), 1.60 (3H, s, H-12'), 3.70 (1H, m, H-13'), and 1.33ppm (3H, d, J= 6.0Hz, H-14').

16. MacrocyclicTrichothecenes and Related Metabolites

447

13C NMR: (CDC13) 79.2, C-2; 34.8, C-3; 73.9, C-4; 48.5, C-5; 44.7, C-6; 50.9, C-7; 56.3, C-8; 138.6, C-9; 122.4, C-10; 67.3, C-11; 65.3, C-12; 48.0, C-13; 8.5, C-14; 65.6, C-15; 22.0, C-16; 167.2, C-I'; 59.2, C-2'; 60.8, C-3'; 44.0, C-4'; 101.1, C-5'; 82.7, C-6'; 134.6, C-7'; 126.8, C-8'; 142.7, C-9'; 119.0, C-10'; 166.9, C-11'; 17.1, C-12', 76.3, C13'; and 15.8ppm, C-14'.

References R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 245 (1981). B. B. Jarvis; Macrocyclic Trichothecenes, In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto; Structures of Isororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon;J. Sci. Hinoshima, University serial A, Vol. 43, pp.107-118 (1979).

448

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Satratoxin F Molecular Formula]Molecular Weight C29H3401o; M W -- 5 4 2 . 2 1 5 2 0

O32

3

ok, o

o

,o. o

General Characteristics Crystals; m.p., 140-143~ Fungal Source Stachybotrys chartarum (S. atra) ATTC 26303. Isolation/Purification A fraction from the isolation of satratoxin H (see R. M. Eppley et al.; J. Org. Chem, Vol. 42, p. 240, 1977) was crystallized from CHCl3-hexane. Biological Activity Cytotoxic. Spectral Data IR~

(CHaC1) 3460(OH), 1748(C=O), 1715, and 1183cm1. 1H NMR: (CDC13) 0.83(3H, s, H-14); 1.73(3H, s, H-16); 2.0(5H, m, H-313, H-7 and H-8); 2.5(1H, m, H-3a); 2.98(2H, center of AB system, J=4Hz, H- 13); 3.38(1H, s, H-2'); 3.59(1H, d, J=5Hz, H-11); 4.24(1H, s, H-12'); 3.85(1H, d, J=5Hz, H-2); 4.15(2H, m, H-5'); 3.88(2H, center of AB system, H- 15); 5.43(1H, d, J=5Hz, H-10); 5.60(1H, d, J=15.5Hz, H-7'); 5.9(1H, m, H- 4); 5.92(1H, d, J=10.5Hz, H-10'); 6.57(1H, dd, J=6

16. Macrocyclic Trichothecenes and Related Metabolites

449

and 10.5Hz, H-9'); and 6.81ppm (1H, dd, 3--6 and 15.5Hz, H-8'). 13C NMR: 79.2, C-2; 34.6, C-3; 74.3, C-4; 49.5, C-5; 43.2, C-6; 20.1, C-7; 27.5, C-8; 140.4, C-9; 118.6, C-10; 67.9, C-11; 65.3, C-12; 48.0, C-13; 8.0, C-14; 65.1, C-15; 23.3, C-16; 166.1, C-I'; 58.9, C-2'; 63.9, C-3'; 22.7, C4'; 61.2, C-5'; 87.1, C-6; 130.2, C-7'; 130.5, C-8'; 143.2, C-9'; 121.2, C-10'; 166.9, C-11'; 73.7, C-12'; 217.8, C-13'; and 29.7ppm, C-14'. Reference R. M. Eppley, E. P. Mazzola, M. E. Stack, and P. A. Dreifuss; Structures of Satratoxin F and Satratoxin G, Metabolites of Stachybotrys atra: Application of Proton and Carbon-13 Nuclear Magnetic Resonance Spectroscopy; J. Org. Chem., Vol. 45, pp. 2522-523(1980).

450

16.

Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name Satratoxin G Molecular Formula/Molecular Weight C29H36010; M W --- 5 4 4 . 2 3 0 9

~o H

H

~ ~ , ~ , , O

H2~. 14

0.

~

0"-~

14'

General Characteristics Crystals; m.p., 132-136~ Fungal Source Stachybotrys chartarum (S. atra) ATTC 26303. Isolation/Purification A fraction from the isolation of satratoxin H (see R. M. Eppley et al.; J. Org. Chem, Vol. 42, p. 240, 1977) was crystallized from CHCls-hexane. Biological Activity Cytotoxic. Spectral Data IR:

(CHCIs) 3450(OH), 1747(C=O), 1710, and 1185cm1. ~HNMR: (CDCIs) 0.87(1H, s, H=14); 1.12(3H, d, J=7Hz, H=14'); 1.74(3H, s, H-16); 2.0(5H, m, H-3[}, H-7 and H-8); 2.5(1 H, m, H-3a); 2.5(2H, m, H-4'); 2.98(2H, center of AB system, J=4Hz, H-13); 3.61(1H, d, J=5Hz, H- 11); 3.43(1H, s, H-2'); 3.90(1H, d, J=5Hz, H-2); 3.9(2H, m, H-5'); 4.02(2H, center of AB system, J=12Hz, H-15); 4.35(1H, s, H-12'); 4.45(1H, q, J=7Hz, H-13'); 5.46(1H, d, J=5Hz, H-10); 5.90(1H, d,

16. Macrocyclic Trichothecenes and Related Metabolites

451

J-16.5 Hz, H-7'); 5.93(1H, d, J=10.5Hz, H-10'); 6.0(1H, m, H-4); 6.68(1H, dd, J=7.5 and 10.5Hz, H-9'); and 7.00ppm (1H, dd, J=7.5 and 16.5Hz, H-8'). 13C NMR: (CDC13) 79.3, C-2; 34.4, C-3; 73.7, C-4; 49.3, C-5; 43.3, C-6; 20.2, C-7; 27.5, C-8; 140.3, C-9; 118.8, C-10; 67.1, C-11; 65.4, C-12; 48.1, C-13; 8.0, C-14; 64.9, C-15; 23.3, C-16; 166.9, C-I'; 61.0, C-2'; 65.4, C-3'; 22.7, C-4'; 60.3, C- 5'; 81.5, C-6'; 132.0, C-7'; 131.5, C-8'; 144.2, C-9' 120.0, C-10'; 166.9, C-11'; 72.6, C-12'; 70.1, C13'; and 16.1ppm, C-14'. Reference R. M. Eppley, E. P. Mazzola, M. E. Stack, and P. A. Dreifuss; Structures of Satratoxin F and Satratoxin G, Metabolites of Stachybotrys atra: Application of Proton and Carbon-13 Nuclear Magnetic Resonance Spectroscopy; J. Org. Chem., Vol. 45, pp. 2522-523(1980).

452

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Isosatratoxin F Molecular Formula/Molecular Weight C29H34010; M V ~ -- 542.21518

~o H I

H

" I 13

I

r~

~ ~ ' ~ " "

0

") 0

~'

~'

,~r

14'

General Characteristics Crystals; m.p., 153-155~

[a]D = + 46.4 ~ (C---0.40, in acetone).

Fungal Source Stachybotrys atra (S. chartarum) ATTC 26303. Isolation/Purification Methanol extraction of a culture of S. atra (JS5106) grown at room temperature for 30 days gave 7.5g of crude extract. This material was subjected to MPLC over silica gel. Elution with dichloromethane gave 520mg in the first fraction which was subjected to high speed countercurrent chromatography (methanol-water-carbon tetrachloride-hexanemethylene chloride, 6:4:8:1:1, v/v/v/v/v) to give five fractions. Fraction 4 was recrystallized from methylene chloride-hexane to give 20mg of isosatratoxin F. Spectral Data IR:

(CHCl3) 3478(OH), 1747(C-O), 1713, and l188cm "1. 1H NMR: (CDC13) 0.80(3H, s, H-14); 1.70(3H, s, H-16); 1.80-2.00(4H, m, H-7 and H- 8); 2.20(ddd, 1H, J=4.7, 5.1, and 15.5Hz, H-3~), 2.50(1H, dd, J-8.0, 15.5Hz, H-3a); 2.80 and 3.12(1H each, AB, J= 4.0 Hz, H-13); 3.38(1H, s, H-2'); 3.54(1H, d, J=5.0Hz, H-11); 3.62(1H, s, H-12'); 3.83(1H, d, J=5.1Hz, H-2); 4.14(2H, m, H-5'); 4.20(2H, s, H-15); 5.39(1H, d, J=5.0Hz, H-10); 5.55(1H, dd, J=l.6 and 16.4Hz, H-7');

16. Macrocyclic Trichothecenes and Related Metabolites

453

5.84(1H, dd, d=4.7 and 8.0Hz, H-4); 5.90(1H, dd, ,]=1.6 and 11.6Hz, H-10'); 6.54(1H, ddd, J=l.6, 5.7, and 11.6Hz, H-9'); and 6.72ppm (1H, ddd, ,]--1.6, 5.7, and 16.4Hz, H8'). 13CNMR: (CDCI3) 79.1, C-2~ 34.4, C-3, 74.1, C-4~ 49.4, C-5~ 43.0, C-6, 20.0, C-7~ 27.4, C-8; 140.5, C-9~ 118.5, C-10, 67.7, C-11, 65.3, C-12, 47.9, C-13~ 7.9, C-14~ 65.0, C-15~ 23.3, C-16~ 166.0, C-I', 58.8, C-2', 63.8, C-3', 22.5, C4'~ 61.1, C- 5', 87.0, C-6, 129.8, C-7'~ 130.5, C-8', 143.2, C-9', 121.1, C-10', 166.9, C-11', 73.5, C-12', 208.6, C-13'~ and 27.4ppm, C-14'. Reference B. B. Jarvis, W. G. Sorenson, E.-L. Hintikka, M. Nikulin, Y. Zhou, J. Jiang, S. Wang, S. Hinkley, R. A. Etzel, and D. Dearborn; Studies of Toxin Production by Isolates of Stachybotrys chartarum and Memnoniella echinata Isolated During a Study of Pulmonary Hemosiderosis in Infants; Appl. Environ. Microbiol., Vol. 64, pp. 3620-3625(1998).

454

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Isosatratoxin G Molecular Formula/Molecular Weight C29H36010; M~W = 5 4 4 . 2 3 0 8 5

10 H 16~O ~

~

H

o,

2 3 .~,,O

H2(7-'~A O'~~U2'~

~O~7,

O OH~OH 14'

General Characteristics Crystals; mp., 136-137~ Fungal Source

Stachybotrys chartarum.

Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexanemethanol-methylene chloride, and collected in 124 fractions. Fractions 83/84 (3% methanol-methylene chloride) were combined to give A; fractions 85-87 (3% methanolmethylene chloride) were combined to give B, fractions 88-89 (3% methanol-methylene chloride) and 90-99 (4% methanol-methylene chloride) were combined to give C, fractions 100-103 (5% methanol-methylene chloride) and fractions 104-110 (6% methanolmethylene chloride) were combined to give D; fractions 111-112 (6% methanol-methylene chloride) were combined to give E; fractions 113-115 (10% methanol-methylene chloride) were combined to give F; and fractions 116-119 were combined to give G. Fraction A was subjected to CCC (Vc = 850ml, methanol -water-carbon tetrachloride-methylene chloridehexane(3: 2: 3.5:0.5:1 v/v/v/v/v) organic mobile phase at 3.2ml/min) to give 10 fractions (F1-F10). Fraction F5 was subjected to TLC (Chromatotron, 2 mm plate, 20% hexane in ethyl acetate) to give 2tt-acetoxystachybotrylactone acetate. The stationary phase of this CCC was concentrated to dryness to give a gum which upon preparative TLC (Chromatotron, 2 mm plate, -~ 20% hexane in ethyl acetate) yielded fractions from which

16.

Macrocyclic Trichothecenes and Related Metabolites

455

were isolated satratoxin G and isosatratoxin G. Biological Activity Cytotoxic. Spectral Data UV: ~, maxMeOH255nm. IR~ (CH3C1) 3450(OH), 1745(C=O), and 1712cm1 (C=O). 1H NMR: (CDC13) 0.80(1H, s, H-14); 1.23(3H, d, J-6.5 Hz, H-14'); 1.70(3H, s, H-16); 1.84(2H, m, H-7); 2.03(2H, m, H-8); 2.17(1H, dt, J=5.0 and 15.2 Hz, H-313; 2.44(1H, dd, J-8.5 and 15.2Hz, H-3~); 2.08(1H, dd, J=5.2 and 14.1Hz, H-4'A); 2.35(1 H, bd, J=14.1Hz, H-4'B); 2.81 and 3.12 (1H each, AB, J-4.0Hz, H=13); 3.55(1H, d, J=5.0 Hz, H-11); 3.74(1H, s, H-2'); 3.82(1I-I, d, J=5.0 Hz, H-2); 3.83(1H, m, H-5'B); 4.08(1H, dt, J=2.0 and 11.4Hz, H-5'B); 3.87(1H, q, J=6.5Hz, H=13'); 4.16 and 4.28(1H each, AB, J=12.5Hz, H-IS); 4.30(1H, s, H-IT); 5.40(1H, d, J-5.0Hz, H-10); 5.80(1H, d, J-16.SHz, H-7'); 5.90(1H, d, J-11.4Hz, H-10'); 5.90(1H, dd, ,/-5.0 and 8.5Hz, H-4); 6.65(1H, dd, J-6.8 and 11.4Hz, H-9'); and 6.88ppm (1H, dd, J-6.8 and 16.8Hz, H-8'). 13C NMR: (CDC13) 79.2, C-2; 34.5, C-3; 73.5, C-4; 49.5, C-5; 43.2, C-6; 20.0, C-7; 27.4, C-8; 140.5, C-9; 118.5, C-10; 67.8, C-11; 65.3, C-12; 48.0, C-13; 8.1, C-14; 64.7, C-15; 23.4, C-16; 166.5, C-I'; 54.2, C-2'; 65.0, C-3'; 27.4, C-4'; 60.9, C-5'; 81.5, C-6'; 130.9, C-7'; 130.1, C-8'; 144.0, C-9'; 120.3, C-10'; 168.5, C-11'; 68.7, C-12'; 73.0, C-13'; and 17.3ppm, C-14'.

Mass Spectrum: HREIMS: 544.2301m/e for C29H36010;calcd., 544.2308. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

456

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Satratoxin H Molecular Formula/Molecular Weight C29H3609, M~IV = 528.23 593

2'. ?o. 14'

General Characteristics Crystals; mp., 162-166~ Fungal Source

Stachybotrys atra.

Biological Activity Toxic to brine shrimp and positive in rabbit skin tests. Details not reported. Spectral Data UV:

Z ~n

225(c=14,700 ) and 255nm (10,400).

~HNMR: (CDCI3) 3.90(1H, m, H-2); 2.20(1H, ddd, J=4.0, 5.0, 15Hz, H-313);2.45(1H, dd, J=7.5, 15Hz, n-3a); 5.90(1H, m, n-4); 1.90(2H, m, n-7); 2.10(2H, m, n-8); 5.46(1H, d, J=5.0Hz, H-10); 3.62(1H, d, J=5.0Hz, H-11); 2.81 and 3.12(1H each, AB, J=4.0Hz, H-13); 3.12(1H, d, J=4.0Hz, H-13b); 0.83(3H, s, H-14); 2.88 and 4.56(1H each, AB, J=12.0Hz, H-15); 1.74(3H, s, n-16); 5.85(1H, d, J=2.0Hz, n-2'), 2.60(1H, m, U-4'a); 3.74(1H, dt, J=3.0, 10.0Hz, H-4'b); 3.90(2H, m, H-5'); 6.09(1H, d, J=17.5Hz, H-7'); 7.36(1H, dd, J--10.5, 17.5Hz, H-8'); 6.63(1H, t, J=-10.5Hz, H-9'); 5.91(1H, d, J=10.5Hz, H-10'); 3.97 1n, s, H-12') 4.38(1H, q, J=7.0Hz, H-13'); and 1.16ppm (3H, d, J=7.0Hz, H- 14').

16. Macrocyclic Trichothecenes and Related Metabolites

457

13C NMR: (CDC13) 79.1, C-2; 34.4, C-3; 74.2, C-4; 49.0, C-5; 43.4, C-6; 20.4, C-7; 27.6, C-8, 140.2, C-9; 119.0, C-10; 68.2, C-11; 65.4, C-12; 48.0, C-13; 7.6, C-14; 64.2, C-15; 23.3, C-16; 166.2, C-I'; 119.0, C-2'; 155.1, C-3'; 25.3, C-4'; 60.4, C-5'; 81.4, C-6'; 134.2, C-7'; 132.2, C-8'; 143.0, C-9'; 120.4, C-10'; 167.0, C-11'; 73.7, C-12'; 69.7, C-13'; and 15.7pprn, C-14'. TLC Data Adsorbent: silica gel; solvent: isopropanol-chloroform, 2:98, v/v; Re, not reported; detection method: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 227-229(1981). R. M. Eppley, E. P. Mazzola, R. J. Highet, and W. J. Bailey; Structure of Satratoxin H, a Metabolite ofStachybotrys atra. Application of Proton and Carbon-13 Nuclear Magnetic Resonance; J. Org. Chem., Vol. 42, pp. 240-243(1977). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991).

458

16.

Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name S-Isosatratoxin H Molecular Formula/Molecular Weight C29H3609; M ' W -- 5 2 8 . 2 3 5 9 3

lo H H ~O.,,12

~

~~I,0 . H2~

!~

0

General Characteristics Crystals, m.p., 180-183~ Fungal Source

Stachybotrys chartarum.

Isolation/Purification In a manner similar to that described for extraction of S. chartarum, a rice culture of S. chartarum Egypt I gave a crude extract that was taken up in 10ml of methanol and filtered through a pad Of Cls silica gel in a Biichner funnel. Elution with methanol gave, aRer solvent removal, a black gum. This material was dissolved in a small amount of methylene chloride and loaded onto a PEI silica gel column and eluted with methylene chloridehexane(4:1, v/v), methylene chloride and 10% methanol in methylene chloride. A combination of preparative HPLC and TLC gave pure satratoxin H, S-isosatratoxin H, stachybotrylactone, and stachybotrylactam. Biological Activity Cytotoxic. Spectral Data UV:

)t maxM~O" 228 and 255nm.

16.

Macrocyclic Trichothecenes and Related Metabolites

459

IR:

(CH3C1) 3440(OH) and 1712cm1 (C=O). 1H N]VIR:

(CDCI3) 0.80(1H, s, H-14); 1.35(3H, d, J=6.5 Hz, H-14'), 1.70(3H, s, H-16); 1.92(2H, m, n-7); 2.01(2H, m, n-8); 2.18(1H, dt, d=5.0 and 15.2Hz, H-313);2.45(1H, dd, J=8.0 and 15.2Hz, H-3a); 2.30(1H, m, H-4'A), 4.15(1H, bd, d=14.1Hz, H-4'B); 2.80 and 3.12(1H each, AB, d=4.0Hz, H-13); 3.57(1H, d, J=5.0Hz, H-11); 3.80(1H, d, J=5.0Hz, H-2); 3.83(2H, m, H-5'); 3.95(1H, q, J=6.SHz, H-13'); 3.85 and 4.55(1H each, AB, d=12.5 Hz, H-15); 4.65(1H, s, H-12'); 5.41(1H, d, d=5.0Hz, H-10); 5.70(1H, d, d=17.0Hz, H-7'); 5.90(1H, d, J=l 1.4Hz, H-10'); 5.87(1H, dd, J=4.5 and 8.0Hz, H-4); 6.10(1H, s, H-2'), 6.52(1H, t, J=l 1.4Hz, H-9'); and 7.45ppm (1H, dd, J= 11.4 and 17.0Hz, H-8'). 13CNMR: (CDCI3) 79.2, C-2; 34.5, C-3; 74.1, C-4; 49.0, C-5; 43.4, C-6; 20.3, C-7; 27.7, C-8; 140.3, C-9; 119.0, C-10; 68.2, C-11; 65.5, C-12; 48.0, C-13; 7.5, C-14; 63.9, C-15; 23.3, C-16, 166.8, C-I'; 112.9, C-2'; 158.4, C-3'; 29.3, C-4', 60.5, C-5'; 80.6, C-6'; 133.8, C-7'; 134.5, C-8'; 142.5, C-9'; 120.6, C-10'; 166.8, C-11'; 73.8, C-12'; 71.6, C13'; and 17.3ppm, C-14'. Mass Spectrum: HREIMS: 528.2374m/efor C29H3609,calcd 528.2359. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

460

16.

Macrocyclic Trichothecenes and Related Metabolites

Common/Systematic Name 12'-Hydroxy-2'-isoverrucarin J PD 113,325 Molecular Formula/Molecular Weight C27H3209; M W = 500.20463

H

I~

H

~1 '~-~i~ u I

, L

H2C,s l '

~

o

U,,,

4" ~ 0 ~ ' ~ ,

~

i, I'

General Characteristics Crystals; mp., >250~ Plant Source

Myrothecium roridum (ATCC 20605).

Isolation/Purification A large scale submerged fermentation was extracted (ethyl acetate) and concentration of the organic layer gave 650g of gum. Large scale preparative HPLC of 118g of this gum over silica gel (Prep-500) gave 18 fractions. A portion (7.2g) from the 25% ethyl acetatemethylene chloride fraction (12g) was further chromatographed (MPLC, isopropanolhexane) to give a fraction (1.37g) from which 568mg of 12'-hydroxy-2'-isoverrucarin J was crystallized (methylene chloride-cyclohexane). Biological Activity In vim P388 mouse anti-leukemic activity: T/C x 100 at 5mg/kg = 129. Cytotoxic and skin irritant. Spectral Data UV:

~, M~,

217(e = 23,500) and 262nm (18,900).

IR:

(CHCI3) 3640, 2980, 2920, 1745, 1660, 1270, 1210, and l l90cm "l.

16. Macrocyclic Trichothecenes and Related Metabolites

461

1H NMR: (CDCI3) 0.64(1H, s, H-14); 1.72(3H, s, H-16); 1.95(2H, m, H-7);-~2.05(2H, m H- 8); 2.19(1H, dt, J=6 and 17Hz, H-3~3);2.45(1H, dd, J=8 and 17Hz, H-3a); 2.3(1H, m, H4'A); 4.14(1H, m, H-4'B); 2.83 and 3.14(1H each, AB, J=4Hz, H-13); 3.7(1H, d, J=5 Hz, H-11); 3.86(1H, d, J=6Hz, H-2); 4.20 and 4.27(1H each, AB, J=16Hz, H-12'); -~4.47(2H, m, H-5'); 3.7 and 5.07(1H each, AB, J=12Hz, H-15); 5.44(1H, dd, J=5 and 2Hz, H-10); 5.9(1H, d, J=16Hz, H-7'); 5.98(1H, dd, J=4 and 8Hz, H-4); 6.10(1H, d, J=l 1Hz, H-10'); 6.1(1H, d, J=2 Hz, H-2'); 6.65(1H, t, J=l 1Hz, H- 9'); and 8.2ppm (1H, dd, Jr=11 and 16Hz, H-8'). 13CNMR: 79.2, C-2; 34.9, C-3; 75.2, C-4; 49.4, C-5; 43.7, C-6; 20.7, C-7; 26.9, C-8; 143.3, C-9; 118.3, C-10; 67.3, C-11; 65.2, C-12; 48.1, C-13; 7.0, C-14; 62.7, C-15; 23.2, C-16; 165.3, C-I'; 116.7, C-2'; 157.4, C-3'; 28.1, C-4'; 61.8, C-5'; 165.9, C-6'; 127.3, C-7'; 139.4, C-8'; 140.0, C-9'; 125.0, C-10'; 166.1, C-11'; and 64.4ppm, C-12'. Reference T. A. Smitka, R. H. Bunge, R. J. Bloem, and J. C. French; Two New Trichothecenes, PD 113,325 and PD 113,326; J. Antibiotics, Vol. 37, pp. 823-828(1984).

462

16. MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name M-Isosatratoxin H; PD 113,326 Molecular Formula/Molecular Weight C29H3609; ~

= 528.23593

,o H

H O.,,]

~

r

6

[

0

II

TM

6H

~t~-OH

General Characteristics Crystals mp., 168-171 ~ Fungal source Myrothecium roridum (ATCC 20605). Isolation/Purification A large scale submerged fermentation of M. roridum was extracted with ethyl acetate. Concentration of the organic layer gave 650g of gum. Large scale preparative HPLC of 118g of this gum over silica gel (Prep-500, Waters Assoc.) gave 18 fractions. A portion (7.2g) from the 25% ethyl acetate-methylene chloride fraction was further chromatographed (MPLC, isopropanol-hexane) to give a fraction rich in M-isosatratoxin H. This fraction was further purified by reversed phase HPLC (C~8, water-acetonitrile) to give, after crystallization from methylene chloride-cyclohexane, 139mg of M-isosatratoxin H. Biological Activity In vivo P388 mouse anti-leukemic activity: T/C x 100 at 5mg/kg = 198. Cytotoxic and skin irritant. S,.pectral Data UV:

~, ra~. 227(e = 19,500) and 255nm (12,500).

16.

Macrocyclic Trichothecenes and Related Metabolites

463

IR~

(CHC13) 3560, 3440,2975,2930, 1730, 1660, 1595, 1195, 1165, 1145, 1080, and 970cm-a. 1H NMR:

(CDCI3) 0.83(1H, s, H-14); 1.35(3H, d, J=7Hz, H-14'); 1.72(3H, s, H-16); 1.92(2H, m, H-7); -~2(2H, m H-8); 2.18(1H, dt, J=5 and 15Hz, H-3~); 2.45(1H, dd, J=8 and 15I-Iz, H-3tt); 2.7(1H, m, H-4'A); 3.78(1H, m, H-4'B); 2.83 and 3.14(1H each, AB, J=4 z, H-13); 3.59(1H, d, J=5Hz, H-11); 3.9(1H, m, H-2); 3.9(2H, m, H-5'); 4.0(1H, q, J=6.5Hz, H-13'); 4.22(1H, s, H-12'); 4.54 and 5.10(1H each, AB, J=13Hz, H-15); 5.39(1H, d, J-17Hz, H-7'); 5.43(1H, d,J--5 Hz, H-10); 5.93(1H, d, J=12Hz, H-10'); 5.9(1H, m, H-4); 5.80(1H, d, J=2Hz, H-2'); 6.52(1H, dd, J=10 and 12Hz, H-9'); and 7.26ppm (1H, dd, J= 10 and 17Hz, H-8'). 13CNMR~ 79.1, C-2; 34.5, C-3; 74.2, C-4; 49.0, C-5, 43.4, C-6; 20.3, C-7, 27.6, C-8; 140.4, C-9, 118.9, C-10, 68.2, C-11; 65.5, C-12, 48.1, C-13; 7.5, C-14; 64.0, C-15; 23.3, C-16, 165.4, C-I'; 118.2, C-2'; 154.9, C-3'; 25.5, C-4'; 61.1, C-5'; 78.7, C-6'; 133.6, C-7'; 134.2, C-8'; 142.4, C-9'; 121.0, C-10'; 166.7, C-11', 75.4, C-12', 74.7, C-13'; and 15.8ppm, C-14'. Reference T. A. Smitka, R. H. Bunge, R. J. Bloem, and J. C. French; Two New Trichothecenes, PD 113,325 and PD 113,326; J. Antibiotics, Vol. 37, pp. 823-828(1984).

464

16.

MacrocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Vertisporin Molecular Formula/Molecular Weight C29H36010; M W -- 544.23085

10 H

H

~.~o.,J

1-8 "61

~

/

~ ~ ' ~ H2r

Is

I

9

4 k Al1'

d~oH .

_

OH General Characteristics A colorless amorphous powder; mp., 176-183~ [a]D26 + 62.5~ diacetate; mp., 145155~ Fungal Source Verticimonosporium diffractum. Biological Activity Cytotoxic and antibiotic activity: EDs0 to HeLa cells was 0.00 l mg/ml; limited antifungal activity against Trichophyton asteroides. Spectral Data UV:

~, maxE~"216rim (e=19,500). IR:

(CHCI3) 1723 and 1711cm"l. 1H NMR: (diacetate derivative) (C6Dr) 3.82(H-2); 5.80(H-4); 5.40(H-10); 3.57(H-11); 2.77(H-13a); 3.09(H-13b); 0.80(H-14); 3.97(H-15a); 4.24(H-15b); 1.70(H-16); 5.81(H-2'); 6.41(H-9'); 5.82(H10'); 4.19(H-12'); 5.10(H-13'); and 6.61ppm (H-14').

16. Macrocyclic Trichothecenes and Related Metabolites

465

13C NMR: (CDC13) 79.0, C-2; 35.0, C-3; 74.0, C-4; 49.7, C-5; 43.2, C-6; 20.7, C-7; 27.7, C-8; 138.4, C-9; 119.9, C-10; 67.6, C-11; 65.3, C-12; 47.2, C-13; 8.0, C-14; 64.7, C-15; 23.0, C-16; 165.7, C-I'; 118.5, C-2'; 152.6, C-3'; 22.8, C-4'; 64.9, C-5'; 86.7, C-6'; 23.6, C-7'; 26.4, C-8', 149.4, C-9'; 121.1, C-10'; 166.0, C-11'; 86.0, C-12'; 75.7, C-13'; and 97.5ppm, C- 14'. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 241 (1981). 0 B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). H. Minato, T. Katayama, and K. Toni: Vertisporin, A New Antibiotic from Verticimonosporim diffractum; Tetrahedron Lett., Vol. 16, pp. 2579-2582 (1975)

Miotoxins Miotoxin A Miotoxin A 13' 13-D-glucoside Miotoxin B Miotoxin C Miotoxin D Isomiotoxin D Miotoxin E Miotoxin F Miotoxin G

467

This Page Intentionally Left Blank

17.

Miotoxins

469

Common/Systematic Name Miotoxin A Molecular Formula/Molecular Weight C29H3809; M W -- 5 3 0 . 2 5 1 5 8

~o H

,,

H

,.1.o

.j

~.'/_-" "OH H

General Characteristics Crystals mp., 159-161~ [(~]D = +100.0 ~ (c=1.40, in CH2C12). Rf=0.52 (CH3-MeOH, 100:4) and 0.66 (ethyl acetate). Plant Source Baccharis coridifolia. Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-rrflOtOXJnD. Spectral Data ~,Em~x~H220 and 263nm. IR: (KBr) 3410, 2960, 1710, 1635, 1220, and 1185cm"1. 1H N]VIR: (CDCI3) 0.78(3H, s, H-14); 1.20(3H, d, J=6.0Hz, H-14'), 1.71(3H, s, H-16); 2.01(2H, m, H-7), 2.03(2H, m, H-8), 2.10(1H, ddd, no Jvalues given, H-313), 2.30(3H, d, J=l.5Hz, H-12'); 2.51(1H, d, J=8Hz, H-3a); 2.81, 3.13(1H each, AB pattern, J=4Hz, U-13), 3.72(1H, d, J=6.7Hz, n-5'), 3.74(1H, t, J=7Hz, n-6'), 3.84(1H, m, H-11), 4.32(1H, br s, H-4'); 3.84(1H, d, no Jvalue given, H-2), 3.93, 4.38(1H each,

470

17.

Miotoxins

AB pattern, J=12nz, H-15); 3.74(1H, t, J=7Hz, H-6'); 5.47(1H, d, J=5Hz, H-10); 5.75(1H, d, J=l 1Hz, H-10'); 5.88(1H, dd, J=3 and 16Hz, H-7'); 6.07(1H, d, J=l.5Hz H-2'); 6.11(1H, dd, J=4 and 8Hz, H-4); 6.55(1H, dd, Js.,9~J9,,~o~=l1Hz, H- 9'); and 7.43 ppm (1H, dd, Jr--11 and 16Hz, H-8'). 13CNMR: (CDC13) 79.1, C-2; 35.6, C-3; 74.4, C-4; 48.6, C-5; 42.8, C-6; 21.3, C-7; 27.7, C-8; 140.1, C-9; 118.9, C-10; 67.2, C-11; 65.5, C-12; 48.0, C-13; 6.7, C-14; 63.7, C-15; 23.2, C-16; 165.9, C-I'; 115.2, C-2'; 160.0, C-3'; 74.4, C-4'; 73.7, C-5'; 83.9, C-6'; 137.5, C-7'; 126.8, C-8'; 143.3, C-9'; 118.0, C-10'; 166.4, C-11'; 15.8, C-12'; 70.8, C13'; and 18.6ppm C-14'. Reference G. G. Habermehl, L. Busam, and J. Stegemann; Miotoxin-A: A Novel Macrocyclic Trichothecene from the Brazilian Plant Baccharis coridifolia; Z. Naturforsch., Vol. 39c, pp.212-216(1983).

17.

Miotoxins

471

Common/Systematic Name Miotoxin A 13' 13-D-glucoside _Molecular Formula/Molecular Weight C35H48014; M W -- 6 9 2 . 3 0 4 4 1

~o H

H

0~

H2~s 15

o@:

4 ~0 ~

o

fi-glucose 0

= H

14'

General Characteristics An oil; [~]D + 50 (C--1.13, in CHC13). Plant Source Female Baccharis coridifofia. Isolation/Purification Fraction D from a reversed phase filtration chromatography (see isolation of roridin E glucoside) was subjected to CCC (Vc = 355mL) with a solvent system of methanol-waterchloroform-carbon tetrachloride-hexane (6:4:6:1:3, v/v/v/v/v). The lower organic phase was the mobile phase and the flow rate was 1.8ml/min to yield roridin A glucoside and a fraction containing miotoxin A glucoside, which was further purified on C18 reversed phase HPLC (250 x 10mm, 60% methanol-water, 4.0mL/min) to give miotoxin A 13-D-glucoside Biological Activity Cytotoxic: ICs5 ca. 1 ng/ml. Spectral Data IR:

(KBr) 3431, 1712, 1650, 1600, 1181, and 1081cm1. 1H NMR: (pyridine-d5) 7.73(1H, dd, J=12.0, 15.6Hz, H-8'); 6.51(1H, dd, J-11.1, 12.0Hz, H-9'); 6.35(1H, dd, J-2.9, 15.6Hz, H-7'); 6.18(1H, s, H-2'); 6.10(1H, dd, J = 4.2, 8.1Hz, H-4);

472

17.

Miotoxins

5.76(1H, d, J=l 1.1Hz, H-10'); 5.48(1H, d, J=4.5Hz, H-10); 4.90 and 4.06(1H each, AB, J=12.5Hz, H-15); 4.89(1H, d, J=7.7Hz, H-I"); 4.62(1H, m, H-6'), 4.52(1H, rn, H6"B); 4.51(1H, rn, H-4'); 4.39(1H, dq, J=6.3, 6.3Hz, H-13'), 4.37(1H, m, H-6"A); 4.26(1H, dd, J=8.5, 9.6Hz, H-4"); 4.23(1H, dd, J=8.5, 9.6Hz, H-3"); 4.01(11-1, dd, J=7.7, 8.5Hz, H-2"); 3.95(2H, m, H-5'B, H-5"); 3.91(1H, d, J=5.1Hz, H-2); 3.76(1H, dd, J=4.3, 8.1Hz, H-5'A); 3.72(1H, d, J=5.0Hz, H-11); 3.08 and 2.84(1H each, AB, J=4.1Hz, H-13); 2.72(3H, s, H-12'), 2.42(1H, dd, J=8.1, 15.1Hz, H-3); 2.22(1H, ddd, J=4.2, 5.1, 15.1Hz, H-3), 1.86-1.93(4H, m, H-7, H-8); 1.56(3H, s, H-16); 1.11(3H, d, J=6.3Hz, H-14'); and 1.09ppm (3H, s, H-14). 13CNMR: (pyridine-d5) 79.2, C-2; 35.2, C-3; 74.9, C-4; 49.4, C-5; 43.6, C-6; 20.7, C-7; 27.7, C-8; 139.0, C-9; 120.1, C-10; 67.7, C-11; 66.0, C-12; 47.8, C-13; 7.5, C-14; 63.7, C15; 23.1, C-16; 166.7, C-I'; 116.9, C-2'; 161.7, C-3'; 75.5, C-4', 72.9, C-5'; 81.3, C-6'; 141.3, C-7'; 126.7, C-8'; 144.7, C-9'; 116.9, C-10'; 166.7, C-11'; 14.2, C-12'; 75.7, C13'; 14.6, C-14'; 104.1, C-l"; 75.2, C-2"; 78.5, C-3"; 71.5, C-4"; 78.5, C-5"; and 62.7ppm, C-6". Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

17. Miotoxins

473

Common/Systematic Name Miotoxin B Molecular Formula/Molecular Weight C29H3809; M W - 530.25158

~o H

~~: ~ O . . J

13

H

2

3

I"o

L

0

~,11'

0

12'

HO" I " 1; H

General Characteristics Amorphous solidi Rf=0.45 (chloroform-methanol, 100:6) and 0.38 (ethyl acetate-hexane; 3:1, v/v). Plant Source Baccharis coridifolia. Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHCI3and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-miotoxJn D. Spectral Data UV: ~,max~~" 252nm (log e=3.9). IR:

(KBr) 3460, 2960, 1725, 1680, 1630, 1590cm]. 1H Nlh4R: (CDCI3) 0.77(3H, s, H-14); 1.17(3H, d,J=6Hz, H-14'); 1.19(3H, d,J=7I-Iz, H-12'); 1.75(3H, s, H-16); 1.84(2H, m, H-7); 1.95(2H, m, H-8); 2.09(1H, dt, J=5 and 15Hz, H-3I}); 2.3 I(1H, dd, J=5 and 17Hz, H-3a); 2.60, 2.98(1H each, d ofAB, J=8 and 15I-Iz, H-2'); 2.83, 3.14(1H each, AB pattern, J=4Hz, H-13); 3.38(1H, m, H- 3'); 4.26(2H, AB, J=l 8Hz, H-5'); 3.79(2H, m, H-6' and H-13'); 3.70(1H, d, J=6Hz, H-11);

474

17.

Miotoxins

3.51(1H, d, J=5Hz, H-2); 3.73, 4.78(1H each, AB pattern, J=12Hz, H-15); 3.79(1H, m, H-6'); 5.44(1H, d, J=6Hz, H-10); 5.58(1H, dd, J=4.5 and 8Hz, H-4), 5.80(1H, d, J=l 1Hz, H-10'), 5.87(1H, dd, J=6 and 15Hz, H-7'); 6.61(1H, dd, Jg.,9,=J9,,lo,=l1Hz, H9'); and 7.52ppm (1H, m, H-8'). 13C NMR: (CDC13) 79.0, C-2; 36.5, C-3; 76.1, C-4; 48.9, C-5; 43.9, C-6; 20.6, C-7; 28.0, C-8; 141.0, C-9; 119.7, C-10; 67.3, C-11; 65.3, C-12; 48.0, C-13; 7.5, C-14; 63.0, C-15; 23.2, C-16; 172.1, C-I'; 37.1, C-2'; 38.1, C-3'; 211.1, C-4'; 73.2, C-5'; 85.1, C-6'; 136.2, C-7'; 129.8, C-8'; 141.5, C-9'; 118.3, C-10'; 166.5, C-11'; 18.7, C-12'; 70.2, C13'; and 16.8ppm C-14'. References G. G. Habermehl, L. Busam, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl and L. Busam; Miotoxins B and C, Two New Macrocyclic Trichothecenes from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 17461754(1984).

17.

Miotoxins

475

Common/Systematic Name Miotoxin C Molecular Formula/Molecular Weight C31H42011; MW --" 590.27271

~

H

H

~ O ~ J 2

0

~

3

I~ ,,0

, L~ 0~1'' 0

0

12

r

HO"

I

H

~ 14'

General Characteristics Amorphous solid; Rf=0.45 (chloroform-methanol, 100:6) and 0.30 (ethyl acetate-hexane; 3:1, v/v). Plant Source

Baccharis coridif o lia.

Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-nfiotoxdn D. Spectral Data UV:

~,maxEt~ 271nm (log e=4.2). IR:

(KBr) 3400, 2960, 1735, 1712, 1630, 1590cm-1. 1H NMR: (CDC13) 0.79(3H, s, H-14); 1.27(3H, d, J-6Hz, H-14'); 1.20(3H, s, H-12'); 1.76(3H, s, H-16); 1.85-2.12(4H, m, H-7 and H-8); 2.09(3H, s, acetate); 2.22(1H, dt, J=5 and 15Hz, H-3~); 2.49(1H, dd, J=8 and 17Hz, H-3tt); 2.49, 2.67(1H each, AB, J=5Hz, H2'); 2.86, 3.18(1H each, AB pattern, J=4Hz, H-13); 3.60(2H, m, H-5'); 4.05(1H, m, H6'); 3.83(1H, br s, H-2); 3.90(1H, d, J=5Hz, H-11); 4.08, 4.55(1H each, AB pattern,

476

17.

Miotoxins

J=12Hz, H-15); 5.47(1H, d, J=5Hz, H-10); 5.88(1H, dd, J=4.5 and 8Hz, H-4), 5.86(1H, d, J=l 1Hz, H-10'); 5.91(1H, dd, J=3 and 16Hz, H-7'); 6.74(1H, dd, Js, 9,=J9,~o,=11Hz,H-9'); and 7.65ppm (1H, m, H-8'). 13CNMR: (CDCI3) 79.0, C-2; 34.9, C-3; 74.4, C-4; 49.1, C-5; 43.4, C-6; 21.1, C-7; 27.7, C-8; 140.7, C-9; 118.4, C-10; 67.2, C-11; 65.2, C-12; 47.9, C-13; 7.1, C-14; 64.0, C-15; 23.2, C-16; 171.6, C-I'; 43.4, C-2'; 72.2, C-3'; 74.2, C-4'; 72.1, C-5'; 80.8, C-6'; 137.8, C-7'; 126.9, C-8'; 143.7, C-9'; 117.5, C-10'; 166.6, C-11'; 23.2, C-12'; 71.2, C-13'; 15.4, C-14'; 170.3(acetate C=O), and 20.5ppm (acetate Me). References G. G. Habermehl, L. Busam, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl and L. Busam; Miotoxins B and C, Two New Macrocyclic Trichothecenes from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 17461754(1984).

17.

Miotoxins

477

Common/Systematic Name Miotoxin D Molecular Formula/Molecular Weight C29H4009; MW

I-

I:

: 532.26723

~o H

H

"113

I

,~

o

_

H2C I s 0

~,

11'

4~0"~

M

H General Characteristics Amorphous solid. Rf=0.56 (chloroform-methanol, 100:4) and 0.71 (ethyl acetate). Plant Source

Baccharis coridifolia.

Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-rrfioto~dn D. Spectral Data IR:

(KBr) 3420, 1730, 1710, 1640, and 1600cm~. IH NMR: (CDC13) 0.77(3H, s, H-14); 0.98(3H, d, d=7Hz, H-12'); 1.20(3H, d, 3=6Hz, H- 14'); 1.71(3H, s, H-16); 1.82-2.04(4H, m, H-7 and H-8); 2.16(1H, dt, J=5 and 15Hz, H-313); 2.23-2.39(3H, m, H-2' and H-3'); 2.44(1H, dd, d=8 and 15Hz, H-3a); 2.81, 3.13(1H each, AB pattern, J-4Hz, H-13); 3.4-3.6(2H, m, H-5'); 3.70(1H, m, H-6'); 3.6(1H, m, H-11); 3.90, 3.84(1H, m, H-2); 3.90(1H, m, H-4'); 4.62(1H each, AB pattern, J=12Hz, H-15); 5.43(1H, dd, 3=1.1 and 5Hz, H-10); 5.84(1H, dd, 3=5.0, 8.0I-Iz, H-4); 5.77(1H, d, 3=11Hz, H-10'); 6.0(1H, dd, J=3 and 16Hz, H-7'); 6.65(1H, dd, ds. 9~J~,10~11Hz, H-9'); and 7.75ppm (1H, m, 8'-H).

478

17.

Miotoxins

13C NMR: (CDC13) 79.2, C-2; 35.1, C-3; 74.5, C-4; 49.2, C-5; 43.5, C-6; 20.6, C-7; 27.9, C-8; 140.7, C-9; 118.6, C-10; 67.5, C-11; 65.4, C-12; 48.0, C-13; 7.2, C-14; 63.8, C-15; 23.3, C-16; 172.7, C-I'; 37.5, C-2'; 32.9, C-3'; 73.5, C-4'; 71.7, C-5'; 84.8, C-6'; 138.8, C-7'; 126.9, C-8'; 144.0, C-9'; 117.7, C-10'; 166.6, C-11'; 16.1, C-12'; 70.9, C-13'; and 18.5ppm C- 14'. References G. G. Habermehl, L. Busam, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl, L. Busam, and M. Spraul; Miotoxin D and iso-MiotoxJn D, Two New Macrocyclic Trichothecene from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 633639(1985).

17.

Miotoxins

479

Common/Systematic Name Isomiotoxin D Molecular Formula/Molecular Weight C29H4009; M W -- 532.26723

~o H

16"~~~~'~

OJ

I~1 13 I~~J~~;,'"

o

H

2

I

3 r~

!' ~,

0

o-],

H

9

O, 12'

HO"

: " 1; H

General Characteristics Amorphous solid. Rf=0.56 (chloroform-methanol, 100:4) and 0.65 (ethyl acetate). Plant Source Baccharis coridifolia. Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and isomiotoxin D. Spectral Data IR:

(KBr) 3460, 1725, 1710, 1640, and 1600cm~. 1H N M R : (CDCI3) 0.77(3H, s, H-14); 0.98(3H, d, J=7Hz, H-12'); 1.20(3H, d, J=6Hz, H- 14'); 1.71(3H, s, H-16); 1.84-2.04(4H, m, H-7 and H-8); 2.14-2.22(1H, m, H- 3~3);2.382.49(4H, m, H-3a, H-2', and H-3'); 2.81, 3.12(1H each, AB pattern, J=4Hz, H-13); 3.57-3.70(2H, m, H-5'); 3.57-3.70(1H, m, H-11); 3.90, 3.84(1H, m, H-2); 3.743.79(2H, m, H-4' and 6'); 4.62(1H each, AB pattern, J=12Hz, H-15); 5.42(1H, dd, d=l.2 and 5Hz, H-10); 5.80-5.82(1H, rn, H-4); 5.79(1H, d, J=llHz, H-10'); 6.04(1H, dd, J=3 and 16Hz, H-7'); 6.66(1H, dd, Js,9.=J9.lO.=11Hz, H- 9'); and 7.81ppm (1H, m, H-8').

480

17.

Miotoxins

13C NMR: (CDC13) 79.2, C-2; 35.0, C-3; 74.7, C-4; 49.3, C-5; 43.5, C-6; 20.6, C-7; 27.9, C-8; 140.8, C-9; 118.6, C-10; 67.5, C-11; 65.4, C-12; 47.9, C-13; 7.0, C-14; 63.6, C-15; 23.3, C-16; 172.7, C-I'; 38.3, C-2'; 32.4, C-3'; 73.0, C-4'; 74.7, C-5'; 86.0, C-6'; 139.5, C-7'; 126.7, C-8'; 143.8, C-9'; 117.9, C-10', 166.8, C-11'; 14.7, C-12'; 71.0, C-13'; and 18.6ppm C-14'. References G. G. Habermehl, L. Busarn, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl, L. Busam, and M. Spraul; Miotoxin D and iso-Miotordn D, Two New Macrocyclic Trichothecene from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 633639(1985).

17.

Miotoxins

481

Common/Systematic Name Miotoxin E Molecular Formula/Molecular Weight C29H3sO10; MW = 546.24650

~.

~0 H

H

,,iO

O

~2

,

II,

o

0 12

HO" ~1 ~ 1,'

General Characteristics An oil; [0~]D-25 ~ (c=0.40, in CHCI3). Plant Source Female Baccharis coridifolia. Isolation/Purification The 70-100% ethyl acetate in hexane and 10% methanol-methylene chloride fractions from the initial column chromatography of the crude extract (see description under verrucarin A 13-glucoside) were subjected to flash chromatography (silica, 80g, methanolmethylene chloride) to yield various trichothecene-containing fractions which were combined, based on TLC analysis, and subjected to further chromatographic procedures including preparative TLC, filtration reversed phase chromatography, preparative HPLC, and countercurrent chromatography (CCC). From these operations were isolated miotoxins E, F, and G. Spectral Data IR;

(CHC13) 3493, 1743,1706, 1637, 1600, 1175, 1081 cm4. 1H NMR: (CDC13) 7.57(dd, 1H, J=ll.5, 15.5Hz, H-8'), 6.60(dd, 1H, J=ll.5, ll.5Hz, H-9'), 5.80(dd, 1H, J=5.7, 15.5Hz, H-7'), 5.76(d, 1H, J=l 1.5Hz, H-10'), 5.53(dd, 1H, J=4.5, 8.0Hz, H-4), 5.39(br d, 1H, J=5.0Hz, H- 10), 5.09 and 3.47(AB, 1H each, J=12.3Hz, H-15), 4.72 and 4.42(AB, 1H each, J=18.4Hz, H-5'), 3.79(d, 1H, J=4.8Hz, H-2),

482

17.

Miotoxins

3.73(m, 2H, H-6', H-13'), 3.53(br d, 1H, J=5.0Hz, H-11), 3.11 and 2.80(AB, 1H each, J=4.0Hz, H-13), 2.94 and 2.59(AB, 1H each, J=17.6Hz, H-2'), 2.57(dd, 1H, J=8.0, 15.4Hz, H-3a), 2.07(ddd, 1H, J=4.5, 4.8, 15.4Hz, H-3b), 1.80-2.00(m, 4H, H-7, H-8), 1.71(s, 3H, H-16), 1.27(s, 3H, H-12')l.15(d, 3H, J=5.9Hz, H-14'), and 0.71ppm (s, 3H, H-14). 13CNMR: (CDC13) 79.0, C-2; 36.7, C-3; 76.4, C-4; 48.8, C-5; 44.0, C-6; 20.5, C-7; 28.0, C-8; 141.3, C-9; 119.6, C-10; 67.2, C-11; 65.1, C-12; 48.0, C-13; 7.5, C-14; 63.6, C-15; 23.2, C-16; 172.3, C-I'; 43.0, C-2'; 77.0, C-3'; 211.0, C-4'; 69.2, C-5'; 85.7, C-6'; 135.9, C-7'; 130.7, C-8'; 141.6, C-9'; 118.0, C-10'; 166.4, C-11'; 26.0, C-12'; 70.2, C13'; and 18.7ppm, C-14'. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: livestock poisoning and the isolation of macrocyclic trichothecene glucosides; Natural Toxins, Volume 4, pp. 58-71(1996).

17.

Miotoxins

483

Common/Systematic Name Miotoxin F Molecular Formula/Molecular Weight C29H40010; MW = 548.26215 ~o H

I"OH

12

H

~

HL

H O " ~ ~ 14'

General Characteristics An oil; [t~]D+29 ~ (c=0.87, in CHC13). Plant Source Female Baccharis coridifolia. Isolation/Purification The 70-100% ethyl acetate in hexane and 10% methanol-methylene chloride fractions from the initial column chromatography of the crude extract (see description under verrucarin A ]3-glucoside) were subjected to flash chromatography (silica, 80g, methanolmethylene chloride) to yield various trichothecene-containing fractions which were combined, based on TLC analysis, and subjected to further chromatographic procedures including preparative TLC, filtration reversed phase chromatography, preparative HPLC, and countercurrent chromatography (CCC). From these operations were isolated miotoxins E, F, and G. Spectral Data IR: (CHC13) 3475, 1712, 1636, 1600, 1418, 1375, 1181, and 1081cm"l. IH NMR: (CDC13) 7.78(dd, 1H, J=l 1.3, 15.6 Hz, H-8'), 6.66(dd, 1H, J=l 1.3, 11.3, H-9'); 5.98(dd, 1H, J=2.9, 15.6 Hz, H-7'); 5.81(dd, 1H, J=4.5, 8.1 Hz, H-4); 5.76(d, 1H, J=l 1.3 Hz, H-10'); 5.40(br d, lI-I, J=4.8 Hz, H-10), 4.58 and 3.97(AB, 1H each, J=12.3 Hz, H-15), 4.08(dd, 1H, J=2.0, 9.5 Hz, H-4'), 3.83(d, 1H, J=5.0 Hz, H-2),

484

17.

Miotoxins

3.66(m, 1H, H-6'), 3.62(dq, 1H, J=6.0, 6.9 Hz, H-13'), 3.59(br d, 1H, J=4.8 Hz, H-11), 3.59(dd, 1H, J=2.0, 9.5 Hz, H-5'B); 3.48(dd, 1H, J=-9.5, 9.5 I-~ H-5'A), 3.11 and 2.79(AB, 1H each, J=4.0 Hz, H-13), 2.65 and 2.44(AB, 1H each, J=15.7 Hz, H-2'), 2.41(dd, 1H, J=8.1, 15.4 Hz, H-3a), 2.15(ddd, 1H, J=4.5, 5.0, 15.4 Hz, H-3b), 1.802.00(m, 4H, n-7, H-8), 1.70(s, 3H, H-16); 1.19(d, 3H, J=6.0 Hz, H-14'), 1.12(s, 3H, H-12'); and 0.72ppm (s, 3H, H-14). 13C NMR: (CDC13) 79.1, C-2; 34.9, C-3; 74.6, C-4; 49.1, C-5; 43.9, C-6; 20.6, C-7; 27.8, C-8; 140.8, C-9; 118.2, C-10; 67.3, C-11; 65.2, C-12; 48.0, C-13; 7.2, C-14; 64.4, C-15; 23.3, C-16; 171.9, C-I'; 43:9, C-2'; 72.4, C-3'; 74.4, C-4'; 70.9, C-5'; 85.3, C-6'; 138.8, C-7'; 126.2, C-8'; 144.1, C-9'; 117.5, C-10'; 166.8, C-11'; 22.7, C-12'; 71.4, C-13'; and 18.4ppm, C-14'. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: livestock poisoning and the isolation of macrocyclic trichothecene glucosides; Natural Toxins, Volume 4, pp. 58-71(1996).

17. Miotoxins

485

Common/Systematic Name Miotoxin G Molecular Formula/Molecular Weight C29H4009; MW = 532.26723

~oH 0 . ~ H2

~6",,,~~/~

3

I, ':1 "-.J...~ I -

l~

12'; , ~ a , 0//'~0

o

' "~o~'~

HI

....

HO%

General Characteristics An oil; [a]D +31 o (C=0.27, in CHCI3). Plant Source Female Baccharis coridifolia. Isolation/Purification The 70-100% ethyl acetate in hexane and 10% methanol-methylene chloride fractions from the initial column chromatography of the crude extract (see description under verrucarin A 13-glucoside) were subjected to flash chromatography (silica, 80g, methanolmethylene chloride) to yield various trichothecene-containing fractions which were combined, based on TLC analysis, and subjected to further chromatographic procedures including preparative TLC, filtration reversed phase chromatography, preparative HPLC, and countercurrent chromatography (CCC). From these operations were isolated miotoxins E, F, and G. Spectral Data IR:

(CHC13) 3481, 1781, 1700, 1643, 1606, 1187, and 1081cm"1. 1H N]VIR:

(CDC13) 7.59(dd, 1H, J-11.3, 15.5 Hz, H=8'); 6.60(dd, 1H, J-11.3,11.3 Hz, H-9)); 6.10(dd, 1H, J=3.8, 8.0 Hz, H-4); 5.83(dd, 1H, J=7.5, 15.5 Hz, H-7'); 5.77(d, II-L d-11.3 Hz, H-10'); 5.47(br d, lI-I, J=5.0 Hz, H-10); 4.16(ddd, lI-I, J=3.3, 5.2, 6.8 Hz, H-4'); 3.89(br d, 1H, ,/=5.0 Hz, H-11); 3.83 and 3.65(AB, 1H each, J=12.5 Hz, H-15);

486

17.

Miotoxins

3.82(d, 1H, J=5.2 Hz, H-2); 3.72(dd, 1H, J=3.3, 11.0 Hz, H-5'B); 3.71(m, 1H, H-13'); 3.66(m, 1H, H-6'); 3.51(dd, 1H, J=5.2, 11.0 Hz, H-5'A); 3.11 and 2.80(AB, 1H each, J=4.0 Hz, H-13); 2.73(dd, 1H, J=8.5, 17.5 Hz, H-2'B); 2.48(dd, 1H, J=8.0, 15.3 Hz, H3a); 2.43(m, 1H, H-3'); 2.17(dd, 1H, J=8.4, 17.5 Hz, H-2'A); 2.07(ddd, 1H, J=3.8, 5.2, 15.3 Hz, H-ab); 1.92-1.99(m, 3 H, n-7a, n-8); 1.70(s, 3H, n-16); 1.54(m, 1n, H-7A); 1.15(d, 3H, J-6.8 Hz, H-12'); 1.13(d, 3H, J=6.2 Hz, H-14'); and 0.81ppm (s, 3H, H14). 13C NMR: (CDC13) 79.0, C-2; 36.0, C-3; 75.4, C-4; 48.9, C-5; 44.2, C-6; 21.2, C-7; 28.0, C-8; 140.6, C-9; 118.7, C-10; 66.8, C-11; 65.6, C-12; 48.1, C-13; 6.8, C-14; 62.8, C-15; 23.3, C-16; 176.3, C-I'; 36.7, C-2'; 32.1, C-3'; 85.8, C-4'; 69.4, C-5'; 85.5, C-6'; 139.0, C-7'; 130.8, C-8'; 143.7, C-9'; 118.7, C-10'; 166.8, C-11'; 18.4, C-12'; 69.7, C-13'; and 18.2ppm, C- 14'. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis Toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Volume 4, pp. 58-71(1996).

Roritoxins Roritoxin Roritoxin Roritoxin Roritoxin

A B C D

487

This Page Intentionally Left Blank

18.

Roritoxins

489

Common/Systematic Name Roritoxin A Molecular Formula/Molecular Weight C29H340]0, MW = 542.21520 ~o H

H

O-x~OH OH General Characteristics Crystals; mp., 220-225~

[0[,]D 25 -at- 16~ (c = 0.51, in chloroform).

Fungal Source Myrothecium roridum (CL-514; ATCC 20605).

Isolation/Purification At the end of 3 weeks of rice fermentation with M. roridum the culture was extracted with methanol in a sonicator. The methanol extracts were combined, washed with hexane, and concentrated in vacuo to give an aqueous solution which was extracted with ethyl acetate. The ethyl acetate extract was dried and solvent was removed by rotary evaporation to give a brown gum. The crude extract was subjected to filtration chromatography (silica gel) with methanol in dichloromethane as the eluting solvent. Fractions obtained with 1-5% methanol in dichloromethane were combined to give a yellow material. This fraction was subjected to flash chromatography with a step gradient ofisopropyl alcohol in dichloromethane as eluting solvent to obtain five fractions. Fractions 2, 3, and 4 obtained with 3-10% isopropyl alcohol were shown to contain trichothecenes by TLC analysis. These were subjected to further purification. Fraction 2 was triturated with dichloromethane and filtered. The white solid precipitate was recrystallized from methanol to give roritoxin D. The mother liquor from the recrystallization and the filtrate from the previous step were combined and purifed on the Chromatotron (eluting solvent 0-3% methanol in dichloromethane). An additional amount of roritoxin D was obtained, and fractions which were collected after the roritoxin D band were added to fraction 3 for further purification. Fraction 3 was triturated with dichloromethane and filtered. The

490

18.

Roritoxins

dichloromethane insoluble white solid obtained was recrystallized from methanol to yield crystals of a mixture of roritoxin C and D. The filtrate of the dichloromethane solution and the mother liquor of the recrystallization were combined and purified on the Chromatotron, (0-5% methanol in dichloromethane). The roritoxin C containing fraction was recrystallized from ethyl acetate to yield pure roritoxin C. The fractions which, by TLC, contained trichothecenes more polar than roritoxin C were combined with fraction 4. Fraction 4 was crystallized from 95% ethanol to yield roritoxin B. The mother liquor was purified on the Chromatotron (0-7% methanol in dichloromethane as eluting solvent). The roritoxin B containing fraction was recrystallized from ethyl acetate to give pure roritoxin B. Roritoxin A was isolated from a more polar fraction from this chromatography. Biological Activity The roritoxins were acutely cytotoxic against L1210 leukemia cells and they exhibited the following IDs0's: roritoxin A, 0.0011 lag/ml; roritoxin B, 0.00171ag/ml; roritoxin C, 0.00421ag/ml; and roritoxin D, 0.00211xg/ml. Roritoxin B was toxic at 801xg~g in mice against P388 leukemia cells; roritixin B also exhibited toxicity (LDs0 ca. 1001.tg&g) when tested against B 16 melanoma in mice. Spectral Data UW:

X ~"

231 (log e= 4.35) and 260nm (log e= 4.70).

1H NMR: (CDCI3) 3.57(1H, J=5.1Hz, H-2); 2.46(1H, dd, J=8.4, 15.4Hz, H-3ct);- 5.9(1H, H4 ) ; - 1.9(1H, n-7); - 1.9(2H, H-8); 5.45(1H, J=4.8Hz, H-10); 3.85(1H, J=4.8Hz, H11); 3.14(1H, J=a.9Hz, H-13A); 2.83(1H, J=3.9Hz, n-13a); 0.80(3H, s, n-14); 4.50(1H, J=12.4Hz, H-15A);- 3.9(1H, n-15a); 1.73(3H, s, n-16);-5.9(1H, n-2'); 2.2(2H, n-4'); 3.9(2H, n-5'); 6.06(1H, J=16.6Hz, H-7'); 7.34(1H, J=10.6, 16.6Hz, n8'); 6.60(1H, d=10.6, 10.6Hz, H-9'); 5.98(1H, J=10.6Hz, U-10'); 4.48(1H, s, H- 12');5.9(1H, H-13'); and 5.22ppm (1H, J=5.0Hz, H-14'). 13C NMR: (CDC13) 79.2, C-2; 34.6, C-3; 74.4, C-4; 49.1, C-5; 43.5, C-6; 20.4, C-7; 27.7, C-8; 140.2, C-9; 119.0, C-10; 68.1, C-11; 65.4, C-12; 48.0, C-13; 7.5, C-14; 65.3, C-15; 23.2, C-16; 165.5, C-I'; 120.8, C-2'; 151.3, C-3'; 25.7, C-4'; 64.0, C-5'; 81.8, C-6'; 133.4, C-7'; 129.0, C-8'; 141.4, C-9'; 121.9, C-10'; 166.9, C-11'; 81.8, C-12'; 81.0, C13'; and 96.7ppm, C-14'. Mass Spectrum: HREIMS: 542.2145role (M § calcd. 542.2151). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum; J. Org. Chem., Vol. 51, pp. 2906-2910(1986).

18.

Roritoxins

491

Common/Systematic Name Roritoxin B Molecular Formula/Molecular Weight C29H34Oll, M W = 558.21011 lo

H ---

0

H 2

3

Io,, O~OH OH General Characteristics Crystals, mp. 262-265~ (dec.); [0I,]D 25 + 2.6~ (c=0.60, in chloroform). Fungal Source Myrothecium roridum (CL-514; ATCC 20605). Isolation/Purification See roritoxin A. Biological Activity See roritoxin A. Spectral Data UV:

L maxMO ' H254nm(log e =4.99). IH NMR: (CDC13) 3.84(1H,J=4.8Hz, H-Z); 2.45(1H, dd, J=8.5, 15.0Hz, H-3a); --~5.9(1H, H4); ~ 1.9(1H, H-7); ~ 1.9(2H, H-8); 5.41(1H, J=5.5Hz, H-10); 3.57(1H, J=5.5Hz, H11); 3.14(1H, J=3.9Hz, H-13A); 2.81(1H, J=3.9Hz, H-13B); 0.81(3H, s, H-14); 4.02(1H, J=12.0Hz, H-15A); 3.94(1H, J=12.0Hz, H-15B); 1.71(3H, s, n-16); 3.3 l(1n, s, n-2'); -~ 2.2(2H, n-4');-3.9(2H, n-5'); 5.87(1H, J=16.4Hz, n-7'); 7.01(1H, J=8.3, 16.4Hz, n-8'); 6.67(1H, J=8.3, 12.2Hz, n-9'); 5.96(1H, J=12.2Hz, H10'); 4.25(1H, s, H-12'); 5.22(1H, J=5.0Hz, H-13'); and 5.93ppm (1H, J=5.0Hz, H-

492

18.

Roritoxins

14'). 13CNM~: (CDC13) 78.4, C-2; 33.9, C-3; 73.9, C-4; 48.7, C-5; 42.8, C-6; 19.2, C-7; 26.8, C-8; 138.8, C-9; 118.9, C-10; 66.6, C-11; 65.3, C-12; 47.3, C-13; 7.7, C-14; 64.2, C-15; 22.8, C-16; 167.3, C-I'; 56.4, C-2'; 62.0, C-3'; 22.8, C-4'; 60.1, C-5'; 82.2, C-6'; 131.2, C-7'; 129.7, C-8'; 143.3, C-9', 120.1, C-10'; 166.5, C-11'; 81.9, C-12'; 78.1, C-13'; and 100.4ppm, C-14'. Mass Spectrum: HREIMS: 558.2106m/e (M +calcd. 558.2139). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum, J. Org. Chem., Vol. 51, pp. 2906-2910(1986).

18.

Roritoxins

493

Common/Systematic Name Roritoxin C Molecular Formula/Molecular Weight C29H32012; M W = 572.18938

~

,. . . . .

H2C,

t

~ J]'"'

O~/~--OH 0 General Characteristics Crystals; mp., 288-290~ (dec.); [0l]D25 + 8.9~ (c= 0.40, in chloroform). Fungal Source Myrothecium roridum (CL-514, ATCC 20605). Isolation/Purification See roritoxin A. Biological Activity See roritoxin A. Spectral Data UV:

~, maxMeOH254nm (log e = 4.97). 1H NMR: (CDCI3) 3.93(1H, J=4.8Hz, H-Z),-- 2.4(1H, H-3~);-- 6.1(1H, H-4),-- 1.9(1H, H-V), --~1.9(2H, H-8); 3.06(1H, J=5.0Hz, H-10), 3.56(1H, J=5.0Hz, H-11); 3.14(1H, J=3.8Hz, H-laA); 2.81(1H, J=a.8Hz, H-laB); 0.82(3H, s, H-14); 4.41(1H, J=12.6Hz, H-15A); 4.32(1H, J=12.6Hz, H-15B); 1.55(3H, s, n-16); 3.36(1H, s, n-2'),- 2.4(2H, n-4');-~a.9(En, n-5'); 5.82(1H, J=16.0Hz, n-7'); 7.10(1H, J-8.0, 16.0Hz, n-8'); 6.68(1H, J=8.0, 11.0Hz, H-9'); 5.97(1H, J=l 1.0Hz, H-10'); 4.24(1H, s, H-12'); and

494

18.

Roritoxins

5.40ppm (1H, s, H- 13'). 13C NMR: (Roritoxin C Acetate) (CDC13) 78.8, C-2; 34.2, C-3; 73.1, C-4; 49.0, C-5; 42.9, C-6; 17.4, C-7; 26.2, C-8; 57.4, C-9; 57.7, C-10; 68.2, C-11; 64.8, C-12; 48.0, C-13; 8.0, C-14; 64.7, C-15; 22.2, C-16; 166.8, C-I'; 57.2, C-2'; 60.7, C-3'; 22.3, C-4'; 61.4, C-5'; 80.6, C-6'; 132.5, C-7'; 127.3, C-8', 142.2, C-9'; 122.0, C-10'; 166.6, C-11'; 79.5, C-12'; 74.1, C-13'; 169.7, C14'; 20.4, CH3COO-; and 168.6ppm, CH3COO-.

Mass Spectrum: HREIMS: 572.1955m/e (M +calcd. 572.1974). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum; J. Org. Chem.; Vol. 51, pp. 2906-2910(1986).

18.

Roritoxins

495

Common/Systematic Name Roritoxin D Molecular Formula/Molecular Weight C29H32Oll, MW = 556.19446 v

~o H

H 0

o'"

IS O,~OH 0

General Characteristics Crystals; mp., 294-297~ (dec.); [G/,]D25 Jr- 3 0 . 0 ~ ( C - - 0 . 1 0 , in chloroform). Fungal Source Myrothecium roridum (CL-514; ATCC 20605). Isolation/Purification See roritoxin A. Biological Activity See roritoxin A. Spectral Data UV: ~,maxM~~ 253nm (log e = 4.98). IH N]VIR: (CDC13) 3.84(1H, J=4.6Hz, H-2); 2.45(1H, J-8.6, 15.2Hz, H-3a); ~5.8(1H, H- 4); ~2.0(1H, H-7); ~2.0(2H, H-8); 5.41(1H, J-5.0Hz, H-10); 3.57(1H, J-5.0Hz, H-11); 3.14(1H, J-3.9Hz, H-13A); 2.81(1H, J=3.9Hz, H-13B); 0.82(3H, s, H-14); 4.32(1H, J=12.5Hz, H-15A), 4.20(1H, J=12.5Hz, H-15B); 1.71(3H, s, H-16); 3.36(1H, s, H-2'); ~2.0(2H, H-4'); ~4.0(2H, H-5'); 5.88(1H, J=16.4Hz, H- 7'); 7.10(1H, J=8.2, 16.4Hz, H-8'); 6.67(1H, J-8.2, 11.1Hz, H-9'); 6.01(1H, J=l 1.1Hz, H-10'); 3.87(1H, s, H-12');

496

18.

Roritoxins

and 5.27ppm (1H, s, H-13'). 13CNMR: (Roritoxin D Acetate) (CDCI3) 79.1, C-2; 34.3, C-3; 73.2, C-4; 49.3, C-5; 43.3, C-6; 20.4, C-7; 27.4, C-8, 140.5, C-9; 118.5, C-10; 67.8, C-11, 65.3, C-12; 48.0, C-13; 7.9, C-14, 65.2, C-15, 22.2, C-16; 166.7, C-I'; 57.3, C-2'; 60.4, C-3'; 23.3, C-4'; 61.3, C-5'; 80.5, C-6'; 132.5, C-7'; 127.0, C-8'; 142.0, C-9'; 122.1, C-10'; 166.4, C-11'; 79.4, C-12'; 74.0, C-13'; 169.6, C- 14'; 20.0, CH3COO-; and 168.8ppm, CH3COO-. Mass Spectrum: HREIMS: 556.1940m/e (M +calcd. 556.1980). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum; J. Org. Chem., Vol. 51, pp. 2906-2910(1986).

Myrotoxins and Mytoxins Myrotoxin A Myrotoxin B Myrotoxin C Myrotoxin C Hydrate Myrotoxin D Myrotoxin D Hydrate Mytoxin A Mytoxin B Mytoxin C

497

This Page Intentionally Left Blank

19. Myrotoxins and Mytoxins

499

Common/Systematic Name Myrotoxin A Molecular formula./Molecular Weight C27H3209; ~

~o H

-- 5 0 0 . 2 0 4 6 3

H

O.,J

I

~

,,:o I o

OH General Characteristics Colorless solid; mp., 220-222~

[(~]D +122.0 (c-0.50, in CH2C12).

Fungal Source

Myrothecium roridum (ATCC 60379).

Isolation/Purification An aqueous shake culture ofMyrothecium roridum (ATCC 60379) was filtered and supernatant extracted with ethyl acetate and the mycelium with acetone. The extracts were combined, concentrated, and subjected to filtration chromatography (silica gel, ethyl acetate-hexane) followed by MPLC (ethyl acetate-hexane through 10% MeOH/CH2C12). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% MeOH/CH2C12) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate/hexane followed by alumina, 20-60% ethyl acetatel-hexane) giving myrotoxins A and B. Biological Activity Cytotoxic: IDs0 = 5 x 10"4 pg/mL, L-1200 cells. Spectral Data IR~

(CHEC12) 3590(OH), 1760, and 1715cm1 (C=O's). 1H NMR:

(CDCI~) 0.80(3H, s, H-14); 1.sa(aH, s, H-16); 1.90(2H, m, H-4'B); 2.0 - 2.1(4H, m,

500

19. Myrotoxins and Mytoxins

H-7 and H-8); 2.17(1H, ddd, J=3.8, 5.0, 15.5Hz, H-3); 2.48(1H, dd, J=8.2, 15.5Hz, H3); 2.60(1H, ddd, 3'=2.0, 9.0, 14.6Hz, H-8q3); 2.84(2H, m, H-4'A); 3.36(1H, s, H-2'); 3.63[1H, s(atter D20 exchange], H-12'); 3.69(1H, d, 3"=4.2Hz, H-11); 3.85(1H, d, 3"=5.0Hz, H-2); 2.79 and 3.13(1H each, AB,3"=4.0Hz,H-13); 3.85 and 4.68(1H each, AB, 3'=12.0Hz, H-15), 4.00(1H, m, H-5'B); 4.12,(1H, dddd, 3'=1.5, 8.8, 9.8, 14.6Hz, H-8'A); 4.25(1H, m, H-5'A); 4.65(1H, dd, 3"=2.0, 9.8Hz, H-7'); 5.42(1H, d, 3"=4.2Hz, H-10), 5.85(1H, dd, 3"=3.8 and 8.2Hz, H-4); 5.93(1H, dd, ,/=1.5, 11.2Hz, H-10); and 6.59ppm (1H, ddd, 3"=8.8, 9.0, 11.2Hz, H-9'). laC NMR: (CDCI3) 79.1, C-2; 35.1, C-3; 74.8, C-4; 49.5, C-5; 43.2, C-6; 20.2, C-7; 27.4, C-8; 140.6, C-9; 118.5, C-10; 67.4, C-11; 65.4, C-12; 47.6, C-13; 7.8, C-14; 64.2, C-15; 23.3, C-16; 164.7, C-I'; 57.1, C-2'; 65.3, C-3'; 23.5, C-4'; 67.2, C-5'; 150.9, C-6'; 109.8, C-7'; 24.3, C-8'; 148.7, C-9'; 121.9, C-10'; 167.1, C-11'; and 72.1ppm, C-12'. References B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclie Trichothecenes from Myrotheciumroridum;Bull. Soe.Chim. Belg., 95, pp. 681-697(1986). B. B. Jarvis, Y.-W. Lee, F. T. Comezoglu, S. N. Comezoglu, and G. A. Bean; Myrotoxins: A New Class ofMacrocyclic Trichothecenes; Tetrahedron Lett., Vol. 26, pp4859-4862(1985).

19.

Myrotoxins and Mytoxins

501

Common/Systematic Name Myrotoxin B Molecular Formula/Molecular Weight C29H34Oll; M W = 558.21011

16

10 H

H

AcO,,,~,~"2"04 I H2(X, j

.O ~ k L 11'

~

HO' ""~ General Characteristics Colorless solid; mp., 195-197~

[c~]D = +10117~ (c=0.60 in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379 and ATCC 52485). Isolation/Purification An aqueous shake culture ofMyrothecium roridum (ATCC 60379) was filtered and the supematant extracted with ethyl acetate and the mycelium extracted with acetone. The extracts were combined, concentrated, and subjected to filtration chromatography (silica gel, ethyl acetate-hexane) followed by MPLC (ethyl acetate-hexane through 10% methanol-methylene chloride). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% methanol-methylene chloride) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane followed by alumina, 20-60% ethyl acetate-hexane) giving myrotoxins A and B. Biological Activity Immunotoxirg cytotoxic, LDs0 in mice = 1.0mg/kg, inhibition ofblastogenesis; LDs0 = 1l~g/rat intracerebrally. Spectral Data IR~

(CH2CI2) 3600 (br, OH), 1710, 1730, and 1755cm~ (C=O's).

502

19. Myrotoxins and Mytoxins

1H NMR: (CDC13) 0.79(3H, s, H-14); 1.76(3H, s, H-16); 1.89(2H, m, H-4'B); 2.1(2H, m, H-7); 2.17(1H, ddd, J=3.6, 5.2, and 15.7Hz, H-313);2.49(1H, dd, J=8.1 and 15.7Hz, H-3a); 2.61(1H, ddd, J=2.0, 9.0, and 14.6Hz, H-8'B); 2.86(2H, m, H- 4'A); 3.32(11-1, s, H-2'); 3.63(1H, s(atier D20 exchange), H-12'); 3.68(1H, d, J=5.4Hz, H-11); 3.85(1H, d, J=5.2 Hz, H-2); 2.80 and 3.12(1H each, AB, J=4.0 Hz, H-13); 3.92 and 4.89(1H each, AB, J=12.0Hz, H-15); 3.98(1H, m, H-5'B); 4.11(1H, dddd, J=l.6, 8.7, 9.8, and 14.6Hz, H-8'A); 4.28(1H, m, H-5'A); 4.65(1H, dd, J=2.0 and 9.8Hz, H-7'); 5.28(1H, d, J=5.2Hz, H-8); 5.66(1H, d, J=5.4Hz, H-10); 5.83(1H, dd, J=3.6 and 8.1Hz, H-4); 5.92(1H, dd, J=l.6 and 11.2Hz, H-10'); 6.60ppm (1H, ddd, J=8.7, 9.0, and 11.2Hz, H9'). 13CNMR: (CDCI3) 78.9, C-2; 35.0, C-3; 74.8, C-4; 49.5, C-5; 42.0, C-6; 23.4, C-7; 68.1, C-8; 136.7, C-9; 164.5, C-I'; 56.7, C-2'; 65.2, C-3'; 23.2, C-4'; 67.1, C-5'; 150.9, C-6'; 109.7, C-7'; 26.7, C-8'; 149.0, C-9'; 121.9, C-10'; 166.8, C-11'; 72.1, C-12'; 20.5, acetate methyl; and 170.8ppm, acetate C=O. References B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrotheciumroridum;Bull. Soc.Chim. Belg.,Vol. 95, pp. 681-697(1986). B. B. Jarvis, Y.-W. Lee, F. T. Comezoglu, S. N. Comezoglu, and G. A. Bean; Myrotoxins: A New Class of Macrocyclic Trichothecenes; Tetrahedron Lett., Vol. 26, pp4859-4862(1985).

19. Myrotoxins and Mytoxins

503

Common/Systematic Name Myrotoxin C Molecular Formula/Molecular Weight C27H3209; M W -- 5 0 0 . 2 0 4 6 3

~~

~o H

H

~

~~)~,,;,,0 H2(~

l'

0

4~ J'L'"

OH General Characteristics Colorless solid; mp., 240~ (dec.);

[~]D =

+124.5 ~ (c=0.98, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification An acetone extract of a rice culture ofMyrothecium roridum (ATCC 60379) was concentrated and partitioned between aqueous NaHCO3 and ethyl acetate; the ethyl acetate solubles were subjected to filtration chromatography (silical gel, ethyl acetatehexane) followed by MPLC (ethyl acetate-hexane through 10% MeOH-CH2C12). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% MeOH-CH2C12) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane) to give myrotoxin C. A later MPLC fraction gave myrotoxin D upon PTLC (alumina, 20-60% ethyl acetate-hexane). Biological Activity Cytotoxic. Spectral Data IR~

(CH2C12) 3580, 2900, 1750, 1715, 1642, 1405, 1190, 1160, 1110, 1095, 1035, 992, 970, and 670 cm~

504

19. Myrotoxins and Mytoxins

:H NMR: (CDCI3) 0.Sl(3H, s, H-14); 1.73(3H, s, H-16); 1.8(1H, m, H-SB), 1.9(2H, m, H- 7); 2.0(2H, m, H-SA and H-4~B); 2.15(1H, ddd, J=3.9, 5.2, 15.5Hz, H-3A); 2.36(1H, dd, J=8.2, 15.SHz, H-3); 2.57(1H, m, H-8~B); 2.80, 3.13(1H each, AB, J=4.1Hz, H-13); 3.57(1H, d, J--4.1Hz, H-11); 3.79(1H, s, H-2H); 3.84(1H, J=12.1Hz, n-15a); 3.85(1I-I, d, J=5.2Hz, n-2); 3.93(1H, ddd, J=2.4, 11.1, 13.1Hz, H-5~B); 4.15(1H, m, H-5'A); 4.20(1H, m, H-8'A); 4.43(1H, s after D20 exchange, H-12'); 4.58(1H, d, J=12.1Hz, H-15A); 4.84(1H, ddd, J=-l.8, 1.8, 10.0Hz, H-7'); 5.41(1H, d, J=4.1Hz, H10); 5.86(1H, dd, J=3.9, 8.2Hz, H-4), 5.91(1H, dd, J=l.5, 11.2Hz, n-10'), 6.62ppm (1H, ddd, J=8.9, 8.9, 11.2, H-9'). 13CNMR: (CDCla) 79.1, C-2; 35.0, C-3; 72.5, C-4; 49.5, C-5; 43.1, C-6; 20.1, C-7; 27.4, C-8; 140.6, C-9; 118.4, C-10; 67.4, C-11; 65.3, C-12; 47.6, C-13; 7.7, C-14; 63.9, C-15; 23.2, C-16; 164.7, C-I'; 52.3, C-2'; 66.5, C-3'; 23.9, C-4'; 66.8, C-5'; 151.1, C-6'; 104.1, C-7'; 28.5, C-8'; 149.4, C-9'; 121.4, C-10'; 168.4, C-11'; and 67.Sppm C-12'. Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrotheciumroridum;Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).

19. Myrotoxins and Mytoxins

505

Common/Systematic Name Myrotoxin C hydrate Molecular Formula/Molecular Weight C27H34010; M W -- 5 1 8 . 2 1 5 2 0

0=4

No~ OH

~' OH

General Characteristics Colorless solidi mp., 178-182~

[a]D =

+29.5 ~ (c=0.44, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379 and ATCC 52485). Isolation/Purification A fraction from a rice culture ofM. roridum (ATCC 60379) rich in myrotoxins C and D was allowed to stand at room temperature in a mixture of acetone and chloroform. The solvent was removed, and the mixture subjected to MPLC (150g of 13-25gt silica gel, elution with 40-60% ethyl acetate-hexane) followed by PTLC (2mm alumina plate, elution with 40-70% ethyl acetate-hexane) to give 80mg of myrotoxin C hydrate and 60mg of myrotoxin D hydrate. Spectral Data IR:

(CHECI2) 3590(OH), 1750, and 1710cml(c=O's). 1H ~ : (CDCl3) 0.82(3H, s, H-14); 1.71(3H, s, H-16); 1.6-2.4(10R m, H-313, H-7, H-8, H4', H-7', H-8'A); 2.43(1H, dd, J=8.5 and 15.4Hz, H-3A); 3.00(1H, m, H-8'B); 2.80 and 3.12(1H each, AB, J=4.0Hz, H-13); 3.54(1H, d, ,/--5.4 Hz, H-11); 3.80(1H, s alter D20 exchange, H-12'); 3.83(1H, s, H-2'); 3.85(2H, m, H-5'); 3.85(1H, d, J=5.3Hz, H2); 4.03 and 4.56(1H each, AB, J=12.3Hz, n-15); 5.40(1H, d, J=5.4Hz, H-10); 5.90(1H, dd, ,/--1.8 and 11.4Hz, H-10'); 5.93(1I-I, dd, J=4.5 and 8.5Hz, H-4); and 6.60ppm (1H, ddd, J=8.8, 8.8, and 11.4Hz, H-8').

506

19. Myrotoxins and Mytoxins

13CNMR.: (CDCI3) 8.0, C-14; 19.8, C-7'; 20.1, C-7; 23.2, C-16; 25.7, C-4'; 27.4, C-8; 27.7, C8'); 34.4, C-3; 43.2, C-6; 47.8, C-13; 49.8, C-5; 53.5, C-2'; 60.3, C-5'; 64.1, C-15; 64.3, C-3; 65.2, C-12; 67.3, C- 11; 72.8, C-4; 74.8, C-12'; 79.1, C-2; 100.6, C-6', 118.3, C-10; 120.8, C-10'; 140.5, C-9; 150.9, C-9'; 166.5, C-I'; and 168.2ppm C-11'. References B. B. Jarvis, F. T. Comezoglu, S. Wang, and H. L. Ammon; Mycotoxins from a Plant Pathogenic Isolate ofMyrotheciumroridum;Mycotoxin Res., Vol. 7, pp. 73-78(1991). F. T. Comezoglu; Triehothecenes: The Isolation of Myrotoxins and Mytoxins; Mechanistic Studies of Rearrangement and Biosynthesis; Ph.D. Thesis, University of Maryland, College Park, MD.

19.

Myrotoxins and Mytoxins

507

Common/Systematic Name Myrotoxin D Molecular formula/Molecular Weight C29H34Oll; M W -- 558.21011

~0.,,~ 2 3 Ac ~'~"04~ H~, 16

10

H

H

'

ON General Characteristics Colorless solid; mp., 232-236~

[~]D--

+ 106.0~ (C=0.10, in CHC13).

Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification An acetone extract of a rice culture ofMyrothecium roridum (ATCC 60379) was concentrated and partitioned between aqueous NaHCO3 and ethyl acetate; the ethyl acetate solubles were subjected to filtration chromatography (silical gel, ethyl acetatehexane) followed by MPLC (ethyl acetate-hexane through 10% methanol-methylene chloride). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% methanol-methylene chloride) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane) to give myrotoxin C. A later MPLC fraction gave myrotoxin D upon PTLC (alumina, 20-60% ethyl acetate-hexane). Biological Activity Cytotoxic. Spectral Data IR:

(CHC13) 3580,2900, 1750, 1710, 1640, 1370, 1160, 1115, 1095, 1000, and 975cm1. 1H NIVIR: (CDC13) 0.78(3H, s, H-14); 1.73(3H, s, H-16); 1.95(3H, s, CH3-acetate); 2.06(1H, m,

508

19. Myrotoxins and Mytoxins

H-4~B); 2.07(1H, m, H-7B); 2.17(1H, m, H-7A); 2.15(1H, m, H-313); 2.32(1H, m, H4'A); 2.46(1H, dd, J=8.1 and 15.6Hz, n-3tt); 2.55(1H, m, H-8~B); 2.79(1H, d, J=4.0Hz, H-13B); 3.08(1H, d, J--4.0Hz, H-13A); 3.65(1H, d, J=5.4Hz, H-11); 3.74(1H, s, H-2'); 3.82(1H, d, J=5.3Hz, H-2); 3.86, 4.87(1H each, AB, J=l 1.9Hz, H15); 3.87(1H, m, H-5~B);4.12(2H, m, H-5'A and H-8'A); 4.40(1H, s after D20 exchange, H-12'); 4.80(1H, ddd, J=1.9, 1.9, and 9.9Hz, H-7'); 5.25(1H, d, J=5.0Hz, H8); 5.63(1H, d, J=-5.4Hz, n-10); 5.80(1H, dd, J=-3.6 and 8.1Hz, H-4); 5.88(1H, dd, ,/=1.6 and 11.2Hz, H-10'); 6.60ppm (lI-I, ddd, J=8.8, 8.8, and 11.2Hz, H-9'). 13C NMR: (CDCI3) 79.0, C-2; 35.0, C-3; 72.0, C-4; 49.5, C-5; 42.0, C-6; 24.0, C-7, 68.1, C-8; 136.8, C-9; 123.7, C-10; 66.8, C-11; 65.2, C-12; 47.5, C-13; 7.7, C-14; 65.9, C-15; 21.1, C-16; 164.6, C-I'; 51.9, C-2'; 66.9, C-3'; 26.8, C-4'; 66.9, C-5'; 151.2, C-6'; 104.2, C-7'; 28.2, C-8'; 149.7, C-9'; 121.5, C-10'; 166.2, C-11'; 67.9, C-12'; 20.5, acetate Me; and 170.8ppm, acetate C=O.

Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrothecium roridum;Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).

19.

Myrotoxins and Mytoxins

509

Common/Systematic Name Myrotoxin D hydrate Molecular Formula/Molecular Weight C29H36012; M W -- 5 7 6 . 2 2 0 6 8

6

10 H

H

AcO,,.~"2"O I H2C I

ol,,

O kLl"

General Characteristics Colorless solidi mp., 225-229~

[a]D

--

+31.5~ (c=0.92, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379 and ATCC 52485). Isolation/Purification A fraction from a rice culture ofM. roridum (ATCC 60379) rich in myrotoxins C and D was allowed to stand at room temperature in a mixture of acetone and chloroform. The solvent was removed, and the mixture subjected to MPLC (150g of 13-251x silica gel, elution with 40-60% ethyl aeetate-hexane) followed by PTLC (2mm alumina plate, elution with 40-70% ethyl aeetate-hexane) to give 80mg of myrotoxin C hydrate and 60mg of myrotoxin D hydrate. Spectral Data IR:

(CH2C12) 3590 (OH), 1750, 1730, and 1710cm1 (C=O's).

IH ~ : (CDC13) 0.80(1H, s, H-14); 1.75(3H, s, H-16); 1.93(3H, s, acetate CH3), 1.6-2.4(9H, m, H-313, H-7, H-4', H-7', and H-8A); 2.45(1H, dd, J=8.3 and 15.3H_z, H-3a); 2.97(1H, m, H-8~B); 2.81, 3.10(1H each, AB, J=4.0Hz, H-13); 3.65(1H, d, J=5.4I-Iz, H-11); 3.80(1H, s after D20 exchange, H-12'); 3.80(1H, s, H-2'); 3.85(1H, m, H-5'); 3.83(1H, d, J=-5.3Hz, H-2); 4.09, 4.82(1H each, AB, J=12.3Hz, H-15); 5.30(1H, d, J=4.2Hz, H-8); 5.66(1H, d, J=5.4 Hz, H-10); 5.89(1H, dd, J=l.8 and 11.4Hz, H-10'); 5.93(1H, dd, J=4.5 and 8.3H~ H-4); and 6.60ppm (1H, ddd, Js.9,s.9=l1.4Hz, H-9').

510

19. Myrotoxins and Mytoxins

13C NMR: (CDC13) 8.0, C- 14; 20.2, C-7'; 20.4, acetate CH3; 20.9, C- 16; 25.8, C-7; 26.4, C-4'; 27.4, C-8'; 34.5, C-3; 42.0, C-6; 47.7, C-13; 49.8, C-5; 53.0, C-2'; 60.6, C-5'; 64.3, C3'; 65.1, C-12; 66.0, C-15; 66.9, C-11; 68.2, C-8; 72.5, C-4; 75.4, C-12'; 79.1, C-2; 100.5, C-6'; 120.9, C-10'; 123.7, C-10; 136.7, C-9; 150.9, C-9'; 166.5, C-I'; 167.9, C11'; and 170.7ppm, acetate C=O. References B. B. Jarvis, F. T Comezoglu, S. Wang, and H. L. Ammon; Mycotoxins from a Plant Pathogenic IsolateofMyrotheciumroridum;Mycotoxin Res., Vol. 7, pp. 73-78(1991). F. T Comezoglu; Trichothecenes: The Isolation of Myrotoxins and Mytoxins; Mechanistic Studies of Rearrangement and Biosynthesis; Ph.D. Thesis, University of Maryland, College Park, MD.

19. Myrotoxins and Mytoxins

511

Common/Systematic Name Mytoxin A Molecular Formula/Molecular Weight C29H36010; M W = 544.23085

~o H

H ~,~,,0

I \

0

'11.

0

~

r

H" OH ~2-Me

General Characteristics Amorphous colorless solid; [C~]D+22.9 ~ (c--0.96, in CH2C12). .Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification An aqueous shake culture ofMyrothecium roridum was filtered and the supernatant extracted with ethyl acetate; the mycelium extracted with acetone. The two extracts were combined, concentrated, and subjected to filtration chromatography (silica gel, ethyl acetate-hexane) followed by MPLC (ethyl acetate-hexane through 10% methanolmethylene chloride). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% methanol-methylene chloride) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane followed by alumina, 20-60% ethyl acetatehexane) to give mytoxins A and B. Mytoxin C was isolated by PTLC (silica gel, ethyl acetaate-hexane followed by silica gel, methanol-methylene chloride) of an earlier MPLC fraction. Biological Activity Cytotoxic.

512

19. Myrotoxins and Mytoxins

Spectral Da.ta IR;

(CH2C12) 3580(OH), 1750, and 1710cm~(C=O). 1H NMR:

(CDCI3) 0.79(3H, s, H-14), 1.58(1H, m, H-8'B), 1.67(1H, m, H-7'B), 1.67-2.00(6H, m, H-7, H-8, H-4B and H-7'B); 1.71(3H, s, H-16); 2.16(1H, ddd, J=4.5, 5.0, 15.4Hz, H-3~); 2.29(3H, s, H-14'); 2.33(1H, m, H-7'A); 2.56(1H,ddd, ,/-9.6, 9.6, and 14.1Hz, H-4'A); 2.79(1H, d, J=4.0Hz, H-13B), 3.02(1H, m, H-8'A), 3.12(1H, d, J=4.0Hz, H13A); 3.23(1H, s(aiter D20 exchange), H-12'); 3.42(1H, s, H-2'); 3.54(1H, d, d=5.1Hz, H-11), 3.84(1H, d, J=5.0Hz, H-2); 4.03(2H, d, J=9.6Hz, H- 5"s), 4.04(1H, d, J=12.3Hz, H-15B); 4.54(1H, d, J=12.3Hz, H-15A), 5.40(1H, d, J=5.1Hz, H-10), 5.85(1H, dd, J=l.8, 11.5Hz, H-10'); 5.92(1H, dd, J=4.5, 8.4Hz, H-4); and 6.44 ppm (1H, ddd, J=7.6, 9.1, 11.5Hz, H-9'). 13CNMR: (CDC13) 79.0, C-2; 34.4, C-3; 72.9, C-4; 49.6, C-5; 43.2, C-6, 19.9, C-7; 27.3, C-8; 140.5, C-9; 118.3, C-10; 67.3, C-11; 65.2, C-12; 47.8, C-13; 7.9, C-14; 64.4, C-15; 23.3, C-16; 166.4, c-r; 58.7, C-2'; 63.9, C- 3'; 22.5, C-4'; 60.3, C-5'; 87.2, C-6'; 22.3, C-7'; 26.4, C-8'; 149.7, C-9'; 120.7, C-10'; 166.7, C-11'; 75.1, C-12'; 212.6, C-13'; and 28.6ppm, C-14'. Mass Spectrum: HRNICIMS: 544.2295m/e for C29H36010;calcd 544.2308. Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrothecium roridum; Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).

19.

Myrotoxins and Mytoxins

513

Common/Systematic Name Mytoxin B Molecular Formula/Molecular Weight C29H3609, M W = 528.23 593 10

H -

O.J

13

~1~

~ ~

H

~

!

n

illi 1~'j

0

o=4: ,/--Xo H" OH Xl~Me 0 14'

General Characteristics Colorless solid, mp., 158-161~ [a]D25= +15.4~ (c=0.78, in CH2C12). Fungal Source Myrothecmm roridum. Isolation/Purification See mytoxin A. Biological Activity Cytotoxic. Spectral Data IR:

(CH2C12) 3600, 2925, 1720, 1655, 1640, 1355, 1230, 1180, 1165, 1085, 1055, 1030, 1000, 910, and 820cm"1. 1H ~ : (CDC13) 0.77(3H, s, H-14); 1.58(1H, ddd, J-3.3, 13.4, 13.4Hz, H-7'B), 1.70(3H, s, H-16), 1.73-2.02(5H, m, H-7, H-8 and H-8'B); 2.11(1H, ddd, J=3.6, 5.3, 15.6Hz, H313); 2.29(3H, s, H-14'), 2.30(1H, m, H-7'A), 2.45(1H, dd, J=8.1,15.6Hz, H-3t~), 2.69(1H, m, H-4~B); 2.72(1H, m, H-8'A), 2.78, 3.10(1H each, AB, J=4.0Hz, H-13); 3.57(1H, d, J=4.0 Hz, H-11); 3.63(1H, d, J-13.6Hz, H-4'A), 3.82(1H, d, J=5.3Hz, H2), 3.96, 4.21(1H each, AB, J=12.6Hz, H-15), 4.02(1H, s after D20 exchange, H-12');

514

19. Myrotoxins and Mytoxins

4.04(2H, dd, J=2.0, 8.1Hz, H-5'); 5.41(1H, d, J=4.0Hz, H-10); 5.75(1H, s, H-2'); 5.76(1H, dd, J=3.6, 8.1Hz, H-4); 5.80(1H, dd, J=2.0, 11.7Hz, H-10'); and 6.37ppm (1H, ddd, J=6.6, 9.1, 11.7Hz, H-9'). 13CNMR: (CDCIa) 79.1, C-2; 35.0, C-3; 73.4, C-4, 49.5, C-5, 42.9, C-6, 20.6, C-7; 27.5, C-8, 140.3, C-9, 118.8, C-10, 67.7, C-11; 65.3, C-12, 47.6, C-13, 7.7, C-14, 64.3, C-15, 23.3, C-16, 165.9, C-I'; 117.3, C-2'; 154.3, C-3'; 25.7, C-4'; 63.2, C-5'; 87.7, C-6'; 22.0, C-7'; 27.9, C-8'; 148.7, C-9'; 121.3, C-10', 166.3, C-11', 77.3, C-12', 213.1, C13'; and 28.6ppm, C-14'. Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichotheeenes from Myrothecium roridum; Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).

19. Myrotoxins and Mytoxins

515

Common/Systematic Name Mytoxin C Molecular Formula/Molecular Weight C29H36010; ~

-" 5 4 4 . 2 3 0 8 5

~~ ~~,,;'0 ~0

H

H

0

,/--Xo f \

0

~

r

H"OH Me 14'

General Characteristics Colorless solid; mp., 181-185~

[{g]D --

+ 4-0~ (C=0.83, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification See mytoxin A. Biological Activity Cytotoxic. Spectral Data IR: (CH2C12) 3510, 2900, 1750, 1710, 1650, 1360, 1200, 1170, 1100, 1090, 1040, 995, 960, 910, 860, 820, and 650crn"1. 1HNIVIR: (CDCI3) 0.81(3H, s, H-14); 1.58(1H, m, H=8'B); 1.70(3H, s, H-16); 1.85(1H, m, H= TB); 1.9=2.1(6H, m, H=7, H-8, and H=4'); 2.17(1H, ddd, J=4.5, 5.2, 15.3Hz, H-3A); 2.29(1H, m, H-7'A); 2.31(3H, s, H-14'); 2.42(1H, dd, J-8.4, 15.3Hz, H-3); 2.79, 3.11(1H each, AB, J=4.0, H=13); 3.07(1H, m, H-8A); 3.53(1H, d, J=5.2Hz, H-11); 3.82(1H, d, J-5.2Hz, H-2); 3.84(1H, s after D20 exchange, H-12'); 3.86(1H, s, H- 2'); 3.91(1H, ddd, J=l.3, 5.2, 11.8Hz, H=5'B); 4.00(1H, ddd, J=2.1, 11.8, 11.8Hz, H=5'A);

516

19. Myrotoxins and Mytoxins

4.02, 4.54(1H each, AB, J=12.3Hz, H-15), 5.39(1H, d, J=-5.2H~ H-10); 5.86(1H, dd, J=l.8, 11.4Hz, H-10'); 5.92(1H, dd, J=4.5, 8.4Hz, n-4); 6.47(1H, ddd, J=7.7, 9.0, 11.4Hz, H-9'). 13CNMR: (CDC13) 79.1, C-2, 34.5, C-3, 73.0, C-4; 49.7, C-5, 43.2, C-6, 19.8, C-7, 27.4, C-8, 140.6, C-9, 118.3, C-10, 67.4, C-11, 65.3, C-12; 47.8, C-13, 8.0, C-14, 64.1, C-15, 23.2, C-16, 166.5, C-I'; 53.7, C-2', 63.3, C- 3', 23.6, C-4', 60.2, C-5', 85.7, C-6', 22.4, C-7'; 27.8, C-8', 149.9, C-9', 120.8, C-10', 168.3, C-11', 69.2, C-12', 214.2, C-13'; and 26.6ppm C 14'. Reference B. B. Jarvis, F. T. C6mezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Maeroeyelie Triehotheeenes from Myrothecium roridum;Bull. Soe. Claim. Belg., Vol. 95, pp. 681-697(1986).

Baccharinoids Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baceharinoid Baccharinoid Baccharinoid Baceharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid

B1 B2 B3 B4 B5 B6 B7 B8

B9 B10 B12 B13 B14 B16 B17 B20 B21 B23 B24 B25 B27

517

This Page Intentionally Left Blank

20.

Baccharinoids

519

Common/Systematic Name Baccharinoid B 1 Molecular Formula/Molecular Weight C29H40010; M W = 5 4 8 . 2 2 6 2 1 5

H -

o.j

H ....

HO"

v

- v

,o I

OHO..... v 0,,~ HO"-H~ General Characteristics Melting point, 158-161~

[ a ] D --

+100.0 ~ (c=1.40, in CHzCI2).

Plant Source Baccharis megapotamica. Isolation/Purification A portion (200mg) of fraction 9D-4 (see isolation ofbaccharinoid B25) was subjected to semi-preparative HPLC (amino column, 1.5% MeOH-CH2C12) to give two fractions, the first of which gave 60mg ofbaccharinoid B 1, recrystallized from ethyl acetate-hexane. The second fraction gave 100mg of B2, recrystallized from ethyl acetate-methylene chloride. Spectral Data UV: maxE~" 263nm (e=20,100). 1H NMR: (CDC13) 0.77(3H, s, H-14), 1.02(3H, d, J=6.4Hz, H-12'), 1.20(3H, d, J=6.0Hz, H14'); 1.83(3H, s, H-16); 2.20(5H, m, H-2', H-3, and H-7); 2.44(2H, m, H-3' and H-3); 2.85, 3.13(1H each, AB pattern, J=4.0Hz, H-13); 3.6(2H, m, H-5'); 3.63(1H, d, J=5.3Hz, H-11); 3.70(2H, m, H-4' and H-13'); 3.86(1H, d, J=4.5Hz, H-2); 4.01(1H, m, H-8); 3.76, 4.68(1H each, AB pattern, J=12.4Hz, H-15); 4.10(1H, m, H-6'); 5.50(1H,

520

20.

Baccharinoids

d, J=5.3Hz, H-10); 5.77(1H, m, H-4); 5.78(1H, d, J=l 1.3Hz, H-10'); 6.05(1H, dd, J=3.1 and 15.5Hz, H-7'); 6.67(1H, dd, Js,9~J~,lo~l 1.3Hz, H-9'); and 7.70ppm (1H, dd, J-11.3 and 15.5Hz, H-8'). 13CNMR: (CDCla) 79.3, C-2; 34.9, C-3; 74.1, C-4; 49.4, C-5; 45.3, C-6; 30.7, C-7; 68.3, C-8; 143.0, C-9; 120.8, C-10; 67.3, C-11, 65.3, C-12, 47.9, C-13; 7.0, C-14; 64.1, C-15; 18.8, C-16; 172.7, C-I'; 38.3, C-2'; 32.4, C-3'; 73.1, C-4'; and 74. lppm C-5'. Reference B. B. Jarvis, S. N. C6mezoglu, H. L. Ammon, C. K. Breedlove, R. F. Bryan, R. W. Miller, M. K. Woode, D. R. Streelman, A. T. Sneden, R. G. Dailey, Jr., and S. M. Kupchan; New Macrocyclic Trichothecenes from Baccharis megapotamica; J. Nat. Prod., Vol. 50, pp. 815-828(1987).

20.

Baccharinoids

521

Common/Systematic Name Baccharinoid B2 Molecular Formula/Molecular Weight C29H40010, M W = 5 4 8 . 2 2 6 2 1 5

H -

HO"

H

o~j

.

.

.

.

I

.

I

H General Characteristics Crystals; m.p., 177-180~ [IX]D -- +116.0 ~ (c=l.0, in CH2C12). Plant Source Baccharis megapotamica. Isolation/Purification See isolation of baccharinoid B 1 Spectral Data UV: X EtOHmax

263nm (e=20,100).

1H NMR: (CDCI3) 0.77(3H, s, H-14); 1.02(3H, d, J=6.4Hz, H-12'); 1.20(3H, d, J=6.0Hz, H54'); 1.83(3H, s, H-16); 2.20(5H, m, H-2', H=3, and H-7); 2.44(2H, m, H=3' and H=3); 2.85, 3.13(1H each, AB pattern, J=4.0Hz, H-13); 3.6(2H, m, H-5'); 3.63(1H, d, J=5.3Hz, H-15); 3.70(2H, m, H-4' and H-13'); 3.86(1H, d, J=4.5Hz, H-2); 4.01(1H, m, H-8); 3.76, 4.68(1H each, AB pattern, J=12.4Hz, H-15); 4.10(1H, m, H-6'); 5.50(1H, d, J=5.3Hz, H-S0); 5.77(1H, m, H-4); 5.78(1H, d, J=l 1.3Hz, H-10'), 6.05(1H, dd, J=3.1 and 15.5Hz, H-7'); 6.67(1H, dd, Js.,9~J~,lo.=l 1.3Hz, H-9'); and 7.70ppm (1H, dd, J=l 1.3 and 15.5Hz, H-8').

522

20.

Baccharinoids

13CNMR~ (CDCI3) 79.3, C-2, 34.9, C-3, 74.1, C-4, 49.4, C-5, 45.3, C-6, 30.7, C-7, 68.3, C-8, 143.0, C-9, 120.8, C-10; 67.3, C-11; 65.3, C-12; 47.9, C-13, 7.0, C-14; 64.1, C-15; 18.8, C-16, 172.7, C-I', 38.3, C-2', 32.4, C-3', 73.1, C-4', and 74. lppm C-5'. Reference B. B. Jarvis, S. N. CSmezoglu, H. L. Ammon, C. K. Bmedlove, R. F. Bryan, R. W. Miller, M. K. Woode, D. R. Strcdman, A. T. Sne,den, R. G. Dailey, Jr., and S. M. Kupchan, New Macrocyclic Tdchothecenes from Baccharis megapotamica; J. Nat. Prod., Vol. 50, pp. 815-828(1987).

20.

Baccharinoids

523

Common/Systematic Name Baceharinoid B3 Molecular Formula/Molecular Weight C29H40010; M W -- 5 4 8 . 2 6 2 1 5

H

H~ O

H

o..j

....,0

H General Characteristics Melting point, 172-180~

laiD-"

+164.0 ~ (c=0.58, in CH2C12).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B7. The mother liquor from the first crystallization of baccharinoid B7 was subjected to PTLC (10% MeOH-CH2C12, SiO2, 2mm plates) to yield baecharinoid B3. Spectral Data UV2 ~bEtOH

max 263nm (6=20,200).

IR:

(CHCIa) 3590, 2450, 1720, 1650, and 1605cm"l. 'H NMR: (CDCI3) 0.79(3H, s, H-14); 1.12(3H, d, J=6.9Hz, H-12'-H); 1.19(3H, d,J=6.0Hz, H 14'); 1.55(2H, m, H-4'); 1.83(3H, s, H-16); 2.10(21-I, m, H-7); 2.20(1H, rn, H-313); 2.28(1H, m, H-3'); 2.44(1H, dd, J=8.4 and 15.3, H-3a); 2.85, 3.13(1H each, AB

524

20.

Baccharinoids

pattern, J=4.0 Hz, H-13); 3.30(1I-1, s, H-2'); 3.40-3.65(5H, m, H-11, H-5', H-6' and H13'); 3.87(11-1, m, H-2); 4.01(1H, dd, J=5.3 and 10.3Hz, H-8); 3.91, 4.85(1H each, AB pattern, J=12.4Hz, H-15); 4.16(1H, d, J=4.2Hz, H-2'); 5.49(1H, d, J=5.2Hz, H-10); 5.75(1H, dd, ,/--4.5 and 8.4Hz, H-4); 5.78(1H, d, J=l 1.6Hz, H-10'); 6.01(1H, dd, J=2.6 and 15.4Hz, H-7'); 6.67(1H, dd, Js-9~J~,10~l 1.6Hz, H-9'); and 7.60ppm (1H, dd, jr=l 1.6 and 15.4Hz, H-8'). 13CNMR: (CDC13) 79.3, C-2; 35.0, C-3; 74.3, C-4; 49.4, C-5; 45.7, C-6; 30.8, C-7; 68.2, C-8; 143.3, C-9; 120.6, C-10; 67.2, C-11; 65.1, C-12; 47.8, C-13; 7.1, C-14; 64.7, C-15; 18.8, C-16; 172.6, C-I'; 76.9, C-2'; 35.0, C-3'; 31.0, C-4'; 68.3, C-5'; 82.6, C-6'; 138.8, C-7'; 127.0, C-8'; 143.8, C-9'; 117.3, C-10'; 166.8, C-11'; 16.1, C-12'; 69.7, C-13'; and 18. lppm C-14'. Reference B. B. Jarvis, S. N. CSmezoglu, H. L. Ammon, C. K. Breedlove, R. F. Bryan, R. W. Miller, M. K. Woode, D. R. Streelman, A. T. Sneden, R. G. Dailey, Jr., and S. M. Kupchan; New Macrocyclic Trichothecenes from Baccharis megapotamica; J. Nat. Prod., Vol. 50, pp. 815-828(1987).

20.

Baccharinoids

525

Common/Systematic Name Baccharinoid B4 Baccharinol Molecular formulamolecular Weight C29H38Oll; M W -- 5 6 2 . 2 4 1 4 2

H H - 0., 3 HO"

v -

y

'"

J

0 HO/- x H General Characteristics Melting point, 259-263~

[aiD =

+ 165.0~ (C=0.50, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B5. The 100% methanol fraction from the initial alumina column was subjected to silica gel (MeOH-CHC13) chromatography and the 10% MeOH fraction crystallized (MeOH-CHC13) and recrystallized (acetone-hexane) to give 3.5g of baceharinoid B4. Spectral Data UV: EtOH

~,m= 260nm (e=20,400). IR:

(CHCI3) 3360, 1750, 1715, 1170, 1640, and 1600cmq.

526

20.

Baccharinoids

~HNMR: (CDCI3) 0.83(3H, s, H-14); 1.18(3H, d, J=6.6Hz, H-14'); 1.59(3H, s, H-12'); 1.83(31t, s, H-16); 2.50(1H, dd, ,/--15 and 8Hz, H-3a); 2.88, 3.13(1H each, AB pattern, J=4Hz, H-13); 3.44(1H, s, H-2'); 4.24, 4.44(1H each, AB pattern, J=12Hz, H15); 5.46(1H, d, J=5Hz, H-10); 5.8(1H, rn, H-4), 5.83(1H, d, J=l 1Hz, H-10'), 6.02(1H, dd, J=3 and 15Hz, H-7'); 6.63(1H, dd, Js,,e=Je,lo~l 1Hz, H-9'); and 7.42ppm (1H, dd, J= 11 and 15Hz, H-8'). 13CNMR: (CDC13) 78.6, C-2; 34.5, C-3; 73.8, C-4; 48.9, C-5; 44.7, C-6; 29.6, C-7; 66.7, C-8; 143.2, C-9; 119.4, C-10; 66.4, C-11; 65.0, C-12; 47.2, C-13; 6.5, C-14; 64.4, C-15; 18.3, C-16; 167.3, C-I'; 55.9, C-2'; 64.8, C-3'; 74.9, C-4'; 72.0, C-5'; 85.7, C-6'; 138.1, C-7'; 125.3, C-8'; 142.2, C-9'; 117.7, C-10'; 166.2, C-11'; 11.8, C-12'; 71.0, C-13'; and 17.8ppm C-14'. Reference S. M. Kupchan, D. R. Streelman, B. B. Jarvis, R. G. Dailey, Jr., and A. T. Sneden; Isolation of Potent New Antileukemic Trichothecenes from Baccharis megapotamica; J. Org. Chem., Vol. 42, pp. 4221-4225(1977).

20. Baccharinoids

527

Common/Systematic Name Baceharinoid B5; Baccharin Molecular Formula/Molecular Weip.ht C29H38Oll; ~

,6, .... .~~

= 562.24141

_15

H

3

1~ Is H2C I .

,1

i

9'

H General Characteristics Melting point, 238-240~

diacetate, mp., 254-256~

[a]D = + 41.5 ~ (C=2.2, in CHCI3).

Plant Source

Baccharis megapotamica.

Isolation/Purification An ethanol extract of 54kg of plant material (leaves and twigs) was concentrated to a black tar and then partitioned between water and ethyl acetate. The ethyl acetate solubles were partitioned between hexane and 10% water in methanol. The methanol solubles were taken up in methanol-ethyl acetate (1:4, v/v) and the mixture filtered through a column of alumina (activity H-HI), washing with 6L of 20% methanol in ethyl acetate. The filtrates were combined, concentrated, and subjected to column chromatography over alumina (activity II-III, elution with increasing methanol in ethyl ether). The 10% methanol-ethyl ether (48g) was subjected to column chromatography on silica gel (methanol-chloroform) and an early 2% methanol fraction (3g) upon crystallization from methanol-chloroform gave a solid which upon recrystallization from acetone-hexane gave 1. lg ofbaccarinoid B5. Spectral Data UV:

Eto. 26Ohm.

max

528

20.

Baccharinoids

IR:

(CHC13) 3580,2880, 1755, 1720, 1170, and ll00cm q. 1H (CDCI3) 0.77(3H, s, H-14); 1.23(3H, d, J=6.5Hz, H-14'); 1.37(3H, s, H-12'); 1.66(3H, s, n-16); 2.19(1H, ddd, J2,ap=Jap,4=4.6Hz and Js~--15.3Hz, H-313); 2.42.6(3H, m, H-3 and H- 4'); 2.76, 3.17(1H each, AB pattern, J=3.9 Hz, H-13); 3.09(1H, d, J=5.4Hz, n-10); 3.30(1H, s, n-2'); 3.32-3.38(1H, m, H-5'B); 3.57(1H, d, J=5.4Hz, n-11); 3.84-3.92(1H, m, H-H-5'A); 3.94(1H, d, J=5.0I-Iz, n-2); 4.20, 4.43(1H each, AB pattern, J=12.6Hz, H-15); 5.76(1H, dd, ,/-=4.6 and 8.2Hz, H-4); 5.80(1H, d, J=l 1.2Hz, H-10'), 5.97(1H, dd, J=3.0 and 15.6Hz, H-7'); 6.60(1H, dd, Js,,a,=dr~,lo~l1.2Hz, H-9'); and 7.54ppm (1H, dd, ,/=11.2 and 15.6Hz, H-8'). 13CNMR: (CDC13) 78.1, C-2; 34.0, C-3; 73.8, C-4; 48.5, C-5; 42.3, C-6; 16.7, C-7; 25.7, C-8; 57.7, C-9; 56.9, C-10; 66.6, C-11; 65.2, C-12; 47.1, C-13; 6.5, C-14; 63.1, C-15; 21.6, C-16; 167.4, C-I'; 56.0, C-2'; 64.4, C-3'; 75.3, C-4'; 72.1, C-5'; 86.8, C-6'; 138.2, C-7'; 125.1, C-8'; 142.6, C-9', 117.4, C-10', 166.3, C-11', 11.6, C-12'; 71.0, C-13'; and 17.7ppm C-14'. Reference B. B. Jarvis, S. N. CSmezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

529

Common/Systematic Name Baccharinoid B6; Isobaccharinol Molecular Formula/Molecular Weight C29H38Oll, M W = 5 6 2 . 2 4 1 4 1

H -

H

o...I

v~"- 1 " H20 I

HOv

|

O, HC~'" " ~ 0 ~ H General Characteristics Melting poim, 249-251~

[a]D = + 149.0 ~ (C=0.66, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B4. The 10% methanol fraction from which baccharinoid B4 was isolated was crystallized from methanol-chloroform followed by recrystallization from acetone-hexane to give 0.20g ofbaccharinoid B6. Spectral Data UV:

EtOH

~.~

260nm (6=20,400).

IR:

(KBr) 3420, 1750, 1720, 1170, 1650, and 1605cm"l.

530

20.

Baccharinoids

~HNMR: (CDCh) 0.83(3H, s, H-14); 1.16(3H, d, J--6.6Hz, H-14'); 1.65(3H, s, H-12'), 1.83(3H, s, H-16); 2.84, 3.13(1H each, AB pattern, J=4Hz, H-13); 3.38(1H, s, H-2'); 4.24, 4.46(1H each, AB pattern, J=12Hz, H-15); 5.52(1H, d, J=5I-Iz, H-10); 5.8(1H, m, H-4); 5.81(1H, d, J=l 1Hz, H-10'); 5.92(1H, dd, J=3 and 15Hz, H-7'); 6.59(1H, dd, Js, ~=Jv,~o~l1Hz, H-9'); and 7.40ppm (1H, dd, J=l 1 and 15Hz, H-8'). 13CNMR: (CDCI3) 78.8, C-2; 34.5, C-3; 73.9, C-4; 49.1, C-5; 44.8, C-6, 29.7, C-7, 67.0, C-8; 143.3, C-9, 119.6, C-10, 66.7, C-11; 65.4, C-12; 47.5, C-13; 6.7, C-14, 64.6, C-15; 18.5, C-16; 167.4, C-1'; 56.3, C-2', 65.0, C-3', 75.5, C-4', 72.3, C-5', 85.2, C-6', 138.4, C-7'; 125.3, C-8', 142.5, C-9', 117.5, C-10', 166.4, C-11'; 11.9, C-12'; 69.0, C-13'; and 15.Sppm C-14'. Reference S. M. Kupchan, D. R. Streelman, B. B. Jarvis, R. G. Dailey, Jr., and A. T. Sneden; Isolation of Potent New Antileukemic Trichothecenes from Baccharis megapotamica; J. Org. Chem., Vol. 42, pp. 4221-4225(1977).

20.

Baccharinoids

531

Common/Systematic Name. Baccharinoid B7 Molecular Formula/Molecular Weight C29H40010; MW = 548.26215 H

H

o.,j

H

0

0

H

General Characteristics Melting point, 229-231 ~

[ a ] D --

+ 150~ (C=0.66, in CH2C12).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B 17. Fraction F8 from the first silica gel column of the large scale extract was taken up in methylene chloride and the solution carefully washed with 2.5% aqueous NaOH. The methylene chloride soluble material was subjected to filtration chromatography over alumina (activity III, 15 % isopropyl alcohol-hexane, 100 % isopropyl alcohol, and 100 % MeOH). Crystallization of the 100 % isopropyl alcohol fraction from ethyl acetate-hexane and recrystallization from ethanol gave baccharinoid B7. Spectral Data UVz ~ ~.~o,max 263nm (e=20,100). IR:

(KBr) 3590, 2450, 1720, 1650, and 1605cm"1. 1H NIVIR: (CDCI3) 0.78(3H, s, H-14); 1.12(3H, d, J=6.9Hz, H-12'); 1.19(3H, d, d=6.0Hz, H14'); 1.60(2H, m, n-4'); 1.83(3H, s, n-16); 2.10(2R rn, n-7); 2.22(1H, m, H-313);

532

20.

Baccharinoids

2.28(1H, m, H-3'), 2.44(1H, dd, J=8.1 and 15.2Hz, H-3t~); 2.85, 3.13(1H each, AB pattern, J=4.0Hz, H-13), 3.30(1H, s, H-2'); 3.40-3.65(5H, m, H-11, H-5', H-6' and H13'); 3.87(1H, d, J=4.0Hz, H-2), 4.01(1H, dd, J=4.9 and 9.4Hz, H-8), 3.91, 4.89(1H each, AB pattern, J=12.3Hz, H-15); 4.18(1H, d, J=4.0Hz, H-2'); 5.50(1H, d, J=5.4Hz, H-10); 5.76(1H, dd, J=4.7 and 8.1Hz, n-4); 5.79(1H, d, d=l 1.5Hz, 10'-H); 6.00(1H, dd, J=2.5 and 15.5Hz, H-7'); 6.66(1H, dd, Js,9_Jg,,lo~11.5Hz, H-9'), and 7.65ppm (1H, dd, J=l 1.5 and 15.5Hz, H-8'). 13CNMR.: (CDC13) 79.2, C-2; 35.0, C-3; 74.3, C-4, 49.4, C-5; 45.7, C-6; 30.9, C-7; 68.2, C-8; 143.3, C-9; 120.9, C-10; 67.2, C-11; 65.1, C-12; 47.8, C-13; 7.1, C-14; 64.8, C-15; 18.8, C-16; 172.5, C-I'; 76.9, C-2', 34.8, C-3'; 30.8, C-4'; 67.9, C-5'; 83.9ppm, C-6'; 139.1, C-7', 126.8, C-8', 143.7, C-9', 117.6, C-10'; 166.7, C-11'; 16.1, C-12', 70.7, C13'; and 18.3ppm, C-14'. TLC Data Rf=0.54 (5% MeOH-CH2CI2), 0.29 (EtOAc), and 0.55 (40% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica, J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

533

Common/Systematic Name Baccharinoid B8; Isobaccharin Molecular Formula/Molecular Weight C29H38Oll; MW - 562.24141

|111|

3 13-

-

s

I

.

I ,0

4

14

U

,

iii I

HO,,"~

12'

O,.....6.~ r H

General Characteristics Melting poim, 228-230~

[a]D = + 42.0 ~ (C=0.36, in CHC13).

Plant Source

Baccharis megapotamica.

Isolation/Purification See baccharinoid B5. Continued elution of the silica gel column with 2% MeOH-CHC13 gave a baccharinoid B8-containi'ng fraction which upon PTLC (25% isopropyl alcoholbenzene) followed by recrystallization from acetone-hexane gave baccharinoid B8. Spectral Data UV:

X~" max 260nm (e=21,300). IR: (KBr) 3470, 1755, 1710, 1170, 1650, and 1605cm-1. 1H NIV[R:

(CDCI3) 0.76(3H, s,H-16); 1.17(3H, d, J=6.6 Hz, H-14'); 1.34(3H, s,H-16), 1.68(3H, s,H-12');2.47(IH, dd, J=16 and 8 Hz, H-3(x);2.75, 3.16(IH each, AB pattern,J=4 Hz, H-13), 3.09(IH, d, J=6 Hz, H-10); 3.35(IH, s,H-2');4.22, 4.47(IH each, AB pattern,J=12.2 Hz, H-15), 5.8(IH, m, H-4), 5.80(IH, d, J=l I Hz, H-10');

534

20.

Baccharinoids

each, AB pattern, J=12.2 Hz, H-15), 5.8(1H, m, H-4); 5.80(1I-I, d, J-11 Hz, H-10'); 5.93(1H, dd, J=3 and 15.5 Hz, n-7'); 6.60(1H, dd, Js,,~ =J~,lo. =11 Hz, n-9'); and 7.44ppm (1H, dd, J=l 1 and 15.5 Hz, 8'-H). laC NMR: (CDCI3) 78.2, C-2; 34.0, C-3, 73.9, C-4, 48.5, C-5; 42.4, C-6, 16.7, C-7, 25.8, C-8, 57.7, C-9, 57.0, C-10, 66.7, C-11, 65.4, C-12, 47.2, C-13, 6.6, C-14, 63.1, C-15, 21.6, C-16; 167.4, C-I', 56.1, C-2', 64.4, C-3', 75.6, C-4', 72.1, C-5'; 85.3, C-6'; 138.7, C-7', 125.0, C-8'; 142.7, C-9'; 117.1, C-10'; 166.3, C-11'; 11.6, C-12', 68.8, C-13'; and 15.6ppm, C-14'. Reference B. B. Jarvis, S. N. CSmezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

535

Common/Systematic Name Baeeharinoid B9 Molecular F0rmula/Mo!ecular Weight C29I-/38010; MW = 546.24650 0

H

H

I0

I

o

N General Characteristics Crystals; mp., 216-218~

[tt]D = + 2.4 ~ (C= 0.68, in CH2C12).

Plant Source

Baccharis megapotamica.

Isolation/Purification See baeeharinoid B 10 isolation. On the basis of TLC analysis, two of the later chromatography fractions from fractions E1 and E2 were combined and crystallized from ethyl ether-methylene chloride to give baecharinoid B9. Spectral Data IR"

(CHCI3) 2930, 2870, 1720, 1650, 1610, and 1095em"1. UV: ~.~

220 and 262nm.

536

20.

Baccharinoids

1HNMR:

(CDC13) 0.69(3H, s, H-14), 1.18(3H, d, J=6.0Hz, H-14'), 1.33(3H, s, H-16), 2.102.25(1H, m, H-3]3); 2.31(3H, d, J=l.2Hz, H-12'); 2.45(1H, dd, J=8.5, 15.8Hz, H3t~); 2.74, 3.14(1H each, AB pattern, J=4.0Hz, H-13); 3.07(1H, d, J=5.2Hz, H-10); 3.63-3.73(6H, m, H-4', HS', H-6', H-11, and H-13'); 3.90(1H, d, J=5.0Hz, H-2); 3.76, 4.46(1H each, AB pattern, J=12.7Hz, H-15); 5.71(1H, d, J=l 1.3Hz, H-10'); 5.87(1H, dd, J=2.5, 16.1Hz, H-7'), 5.98 (2H, m, H-2', H-4), 6.53 (1H, dd, Js,,9,=Jg,lo,=ll.3Hz, H-9'); and 7.38ppm (1H, dd, J=l 1.3, 16.1Hz, H-8'). 13CNMR: (CDC13) 78.8, C-2; 35.1, C-3; 74.1, C-4; 47.8, C-5; 42.5, C-6; 18.3, C-7; 26.3, C-8; 57.5, C-9; 57.5, C-10; 67.7, C-11; 65.0, C-12; 48.5, C-13; 6.9, C-14; 63.1, C-15; 22.3, C-16; 166.4, C-I'; 115.0, C-2'; 161.0, C-3'; 74.8, C-4'; 74.7, C-5'; 84.2, C-6'; 138.0, C7', 126.6, C-8'; 143.7, C-9'; 117.8, C-10'; 166.0, C-11'; 15.7, C-12'; 70.9, C-13'; and 18.6ppm, C-14'. TLC Data Re= 0.40 (4% methanol-methylene chloride), 0.43 (ethyl acetate), and 0.48 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from A Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

537

Common/Systematic Name Baccharinoid B 10 Molecular Formula/Molecular Weight C29H38010; M~V = 546.24650 0

o

H

H

oJ)

H General Characteristics Crystals; mp., 157-158~

[(g]D-- + 8.1 (C=0.62, CHC13).

Plant Source

Baccharis megapotamica (original trichothecene nucleus suspected to be of fungal origin).

Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 7 (1.125kg) from this column was chromatographed over SiO2 with increasing MeOH in CH2C12 to give five fractions: F7A-F7E. Fraction F7E (290g) was taken up in CH2C12 and carefully washed with 2.5% aqueous NaOH to give 12 lg of CH2C12 material which was subjected to filtration chromatography over alumina: 20% i-PrOH/hexane (E 1), 100 % i-PrOH (E2), and 100% MeOH (E3) fractions. The 20% i-PrOH/hexane fraction (80g) was subjected to filtration chromatography (A12Os) and MPLC (SiO2) and several PTLC's (Chromatotron) to give baccharinoids B1 - B5 and two additional fractions Ela and Elb that contained additional baccharinoids. Fraction E2 (100% i-PrOH) was subjected to Sephadex chromatography (CH2C12) to give four fractions. Fraction E2b (5.4g) was subjected to flash chromatography (EtOAc/hexane) and PTLC (A12Os, i-PrOH/CH2C12, Chromatotron). A fraction from this was combined with E lb and crystallized from Et20/CHC13 to give 65mg of B 10.

538

20.

Baccharinoids

Spectral Data IR;

(CHC13) 3420, 1720, 1650, 1610, and 1170cm"1. UV:

~.ma~ 220 and 260nm. 1H NMR:

(CDCI3) 0.72(3H, s, 14-8); 1.19(3H, d, J=6.4Hz, 14'-H); 1.33(3H, s, 16-H); 2.102.25(1H, m, 3~3-H); 2.33(3H, d, J-1.0Hz, 12'-H); 2.43(1H, dd, J=8.0 and 15.5Hz, 3czH); 2.74, 3.18(1H each, AB pattern, J=4.0Hz, 13-I-I); 3.08(1H, d, J-5.2I-Iz, lO-H); 3.60(1H, d, J=5.2Hz, 1l-H); 3.90(1H, d, J=4.9Hz, 2-H); 3.81, 4.45(1H each, AB pattern, J=12.7Hz, 15-H); 5.71(1H, d, J-11.4Hz, 10'-H); 5.87(1H, dd, J-3.0 and 15.6Hz, 7'-H); 5.97(1H, br s, 2'-H); 5.98(1H, dd, J-4.2 and 8.0Hz, 4-H); 6.53(1H, dd, Js.~=J~,lo~=l1.4Hz, 9'-H); and 7.34ppm (1H, cld, J=l 1.4 and 15.6Hz, 8'-H). 13CNMR: (CDC13) C-2, 78.7; C-3, 34.9; C-4, 74.2; C-5, 48.4; C-6, 42.5; C-7, 18.2; C-8, 25.2; C-9, 57.4; C-10, 57.5; C-11, 67.7; C-12, 65.0; C-13, 47.6; C-14, 6.9; C-15, 62.9; C16, 22.2; C-I', 166.3; C-2', 115.0; C-3', 161.3; C-4', 75.1; C-5', 74.3; C-6', 83.0; C-7', 137.9; C-8', 126.6; C-9', 143.5; C-10', 117.5; C-11', 166.2; C-12', 15.5; C- 13', 68.9; and C-14', 17.1ppm. TLC Data Rf = 0.40 (4% MeOH/CH2CI2), 0.36 (EtOAc), and 0.43 (30% i-PrOH/hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M.Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56 (1987).

20.

Baccharinoids

539

Common/Systematic Name Baccharinoid B 12 Molecular Formula/Molecular Weight C29H38Oll; M W -- 5 6 2 . 2 4 1 4 2

H 03

..,,OH .i,,,0

HO"

v

-v

o

I

iiii j

H

HO H Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 7 (1.125kg) from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions: F7A-F7E. Fraction F7B (290g) was taken up in CH2C12 and carefully washed with 2.5% aqueous NaOH to give 172g of methylene chloride material which was subjected to filtration chromatography over alumina: 15% isopropanol-hexane, 100% isopropanol, and 100% methanol fractions. The 100% isopropanol fraction was triturated with ethyl ether-methylene chloride to give a precipitate which upon recrystallization from acetone-hexane gave 430mg ofbaccharinoid B12. Spectral Data UV:

~m~ H 260nm. IR: (CHC13) 3500,2990, 1730, 1715, 1180, and ll00cm 1.

540

20.

Baccharinoids

1H NMR.: (CDCI3) 0.85(3H, s, H-14); 1.19(3H, d, ,/=6.41--Iz,H-14'); 1.46(3H, s, H-12'); 1.75(3H, s, H-16); 2.79, 3.08(1H each, AB pattern, J=4.0Hz, H-13); 3.30(1H, s, H-2'); 3.4-3.5 (1H, m, H-5'B); 3.62-3.75 (5H, m, H-5'A, H-6'A, and H-13'); 4.18 (1H, d, J=5.6Hz, H-11); 4.19, 4.43(1H each, AB pattern, J=12.6Hz, H-15); 4.35 (1H, dd, J=2.7 and 5.0Hz, H-313); 5.76(1H, dd, J=4.6 and 8.2Hz, H-4); 5.80(1H, d,J=ll.2Hz, H-10'); 5.57(1H, d, J=5.6Hz, H-10); 6.02(1H, dd, J=2.7 and 15.6Hz, H-7'); 6.63(1H, dd, ffs,,9,=J~,lo,=l1.5Hz, H-9'); and 7.50ppm(1H, dd, J=l 1.5 and 15.6Hz, H-8'). 13CNMR: (CDCI3) C-2, 79.4; C-3, 75.5; C-4, 83.0; C-5, 49.2; C-6, 44.1; C-7, 20.0; C-8, 25.5; C-9, 139.0; C-10, 119.1; C-11, 67.4; C-12, 64.4; C-13, 46.3; C-14, 6.8; C-15, 63.7; C16, 23.0; C-I', 167.9; C-2', 57.8; C-3', 63.1; C-4', 39.8; C-5', 67.9; C-6', 84.7; C-7', 140.6; C-8', 125.2; C-9', 143.7; C-10', 116.7; C-11', 166.2; C-12', 17.2; C- 13', 69.1; and C- 14', 17.9ppm. Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation of Macrocyclic Trichothecenes from a Large Scale Extract of Baccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

541

Common/Systematic Name Baccharinoid B 13 Molecular Formula/Molecular Weight C29H38Olo; M ~

H

= 546.24650

H iO

H O"

v

- v

I ,

o

H General Characteristics Crystals, m.p., 218-219~

[ ~ ] D --

"+"130~ (C=0.74, in MeOH).

Plant Source Baccharis megapotamica. Isolation~urification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 9 from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions 9A-9E. Fraction 9D was dissolved in methylene chloride and hexane was added slowly to give a precipitate. This material was dissolved in methylene chloride and the solution washed with 2.5% aqueous NaOH, concentrated to dryness, and subjected to preparative HPLC using alumina and eluting with isopropyl alcohol-methylene chloride to give six fractions. From these fractions by successive chromatographic procedures baccharinoids B 13, B 14, B23, B24, B25, and B27 were isolated. Spectral Data UV: EtOH

max 220 and 262nm. IR:

(CHC13) 3600, 2880, 1720, and 1180cm ~

542

20.

Baccharinoids

1H NMR: (CDCI3) 0.76(3H, s, H-14); 1.21(3H, d, J=6.4Hz, H-14'); 1.81(3H, s, H-16), 2.052.15(1H, m, H-313); 2.22(3H, d, J=l.2 Hz, H-12'); 2.51(1H, dd, J=8.3, 15.7Hz, H3~); 2.83, 3.14(1H each, AB pattern, J=4.0Hz, H-13); 3.62-3.69 (3H, m, H-13', H5'B, H-6'); 3.85(1H, d, J=5.2Hz, H-2); 3.90-3.96(2H, m, H-11 and H-5'); 3.93, 4.23 (1H each, AB pattern, J=12.5Hz, H-15); 4.05(1H, br s, H-8); 4.37(1H, br s, H-4'); 5.54(1H, d, J=5.6Hz, H-10); 5.75(1H, d, J=l 1.5Hz, H-10'); 5.85(1H, dd, J=3.0, 15.7Hz, H-7'); 6.21(1H, dd, J=3.9, 8.1Hz, H-4); 6.24(1H, br s, H-2'); 6.55(1H, dd, Js,,9,=Jg,,1o,=l1.SHz, H-9'); and 7.48ppm (1H, dd, J=l.5, 15.7Hz, H-8'). 13C NMR: (CDC13) 79.3, C-2; 35.6, C-3; 73.9, C-4; 48.7, C-5; 45.2, C-6; 31.7, C-7; 68.1, C-8; 142.4, C-9; 118.0, C-10; 67.1, C-11; 65.5, C-12; 48.0, C-13; 6.8, C-14; 64.2, C-15; 18.6, C-16; 166.3, C-I'; 121.1, C-2'; 160.5, C-3'; 74.3, C-4'; 73.6, C-5'; 84.0, C-6'; 137.6, C-7'; 126.8, C-8'; 143.5, C-9'; 115.0, C-10'; 165.9, C-11'; 18.9, C-12'; 70.8, C13'; and 16.0ppm, C-14'. TLC Data Rf 0.19 (4% methanol-methylene chloride), 0.28 (ethyl acetate), and 0.43 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract of Baccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

543

Common/Systematic Name Baccharinoid B 14 Molecular Formula/Molecular Weight C29H3801o; M W

H

HO ~"

= 546.24650

H

--__ o,,,]

....,0

o

H General Characteristics Crystals from ethyl acetate-hexane; m.p., 149-151~

[tt]D = + 72.4 ~ (C=0.76, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 9 from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions 9A-9E. Fraction 9D was dissolved in methylene chloride and hexane was added slowly to give a precipitate. This material was dissolved in methylene chloride and the solution washed with 2.5% aqueous NaOH, concentrated to dryness, and subjected to preparative HPLC using alumina and eluting with isopropyl alcohol-methylene chloride to give six fractions. From these fractions by successive chromatographic procedures baccharinoids B 13, B 14, B23, B24, B25, and B27 were isolated. Spectral Data UV~

~EtoH max 220 and 260nm. IRz

(CHC13) 3590, 2870, 1715, and l l80cm "1.

544

20.

Baccharinoids

1H NMR: (CDCI3) 0.79(3H, s, H-14); 1.20(3H, d, J=6.4Hz, H-14'); 1.83(3H, s, H-16); 2.052.20(1H, m, H-313); 2.27(3H, s, n-12'); 2.52(1H, dd, J=8.0, 15.4Hz, n-3ct); 2.86, 3.16(1H each, AB pattern, J=3.9Hz, H-13); 3.66, 3.77(1H each, d of AB pattern, J=3.9, J4,,S=--4.7Hz,Jgem=9.8Hz, H-5'); 3.87(1H, d, J=5.0Hz, H-2); 3.86-4.04(4H, m, H-11, H-4', H-6', and H-13'); 3.99, 4.26(1H each, AB pattern, d=12.7Hz, H- 15); 5.57(1H, d, J=6.0Hz, H-10); 5.76(1H, d, J=l 1.2Hz, H-10'); 5.89(1H, dd, J=3.0, 15.5Hz, H-7'); 6.17-6.18(2H, m, H-2' and H-4' overlapping); 6.57(1H, dd, ds,,9_Jg,,lo~l 1.5Hz, H-9'); and 7.41ppm (1H, dd, J=l 1.5, 15.4Hz, H-8'). 13CNMR: (CDC13) 79.3, C-2; 35.6, C-3; 74.0, C-4; 48.6, C-5; 45.2, C-6; 31.5, C-7; 68.1, C-8; 142.4, C-9; 117.7, C-10; 67.1, C-11; 65.5, C-12; 48.0, C-13; 6.8, C-14; 64.1, C-15; 17.1, C-16; 166.3, C-I'; 121.1, C-2'; 161.3, C-3'; 74.8, C-4'; 73.1, C-5'; 82.7, C-6'; 137.3, C-7', 126.9, C-8'; 143.5, C-9'; 114.9, C-10'; 166.3, C-11'; 19.0, C-12'; 68.5, C13'; and 15.9ppm, C-14'. TLC Data Rf=0.19 (4% methanol-methylene chloride), 0.21 (ethyl acetate), and 0.42 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

545

Common/Systematic Name Baccharinoid B 16 Molecular Formula/Molecular Weight C29H38010; M W -- 5 4 6 . 2 4 6 5 0

HOH2C~O,,,~ H

H

H2~ I o 6

~) o I

H General Characteristics Crystals from ethanol-hexane, mp., 160-161~

[a]D = + 63.5 ~ (C=0.47, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation for baccharinoid B25. Fraction 9E in methylene chloride was washed with 2.5% aqueous NaOH, concentrated, and chromatographed by silica gel MPLC eluting with ethyl acetate-hexane to give two fractions. The second fraction was fractionated using preparative PTLC (60% ethyl acetate-hexane, Chromatotron) which gave baccharinoid B 16 which crystallized from methylene chloride-hexane. Spectral Data UV;

~.m~x 220 and 260nm. IR:

(CHC13) 3600, 2860, 1710, and 1180cm"1.

546

20.

Baccharinoids

1H NIVIR: (CDCI3) 0.78(3H, s, H-14); 1.16(3H, d, J=6.4Hz, H-14'); 2.15-2.20(IH, m, H-313); 2.22(3H, d, J=1.0Hz, H=I2'); 2.54(IH, dd, J=8.2, 15.5Hz, H-3a); 2.81, 3.12(IH each, AB pattern, J=4.0I-Iz, H-13); 3.62, 3.76(IH each, d of AB pattern, J=3.7, 9. IHz, H5'); 3.63(IH, d overlapping with H-5', H-I I); 3.84(IH, d, J=4.9Hz, H-2); 3.964.07(IH, m, H-6'); 4.01(2H, br s, H-16); 4.26(IH, br s, H-4'); 4.04, 4.29(IH each, AB pattern, J=12.5Hz, H-15); 5.74(IH, d, J=l 1.4Hz, H-10'); 5.82(IH, d, J= 5.4Hz, HI0); 5.86(1H, dd, J=2.8, 15.8Hz, H-7'); 6.08(1H, br s, H-2'); 6.55(1H, dd, Js,,9~19,.lo~11.4Hz,H-9'); and 7.38ppm (1H, dd, J=l 1.4, 15.8 Hz, H-8').

13CNMR: (CDC13) 79.4, C-2; 35.4, C-3; 74.5, C-4; 48.9, C-5; 43.5, C-6; 20.8, C-7; 23.2, C-8; 143.7, C-9; 117.6, C-10; 67.1, C-11; 65.7, C-12; 48.1, C-13; 6.9, C-14; 65.7, C-15; 63.5, C-16; 166.8, C-I'; 118.6, C-2'; 161.4, C-3'; 75.1, C-4'; 74.0, C-5'; 83.2, C-6'; 138.1, C-7'; 126.8, C-8'; 143.5, C-9'; 115.0, C-10'; 165.9, C-11'; 18.9, C-12'; 70.8, C13'; and 16.0ppm, C-14'. TLC Data Rf0.10 (4% methanol-methylene chloride), 0.17 (ethyl acetate), and 0.29 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

547

Common/Systematic Name Baccharinoid B 17 Molecular .Formula/Molecular Weight C29H38Olo, M~W -- 5 4 6 . 2 4 6 5 0

1~, .0~1o H IIIIiill

H

H2.~1~

H General Characteristics Crystals from ethyl ether-methylene chloride, m.p., >300~ CH2C12).

[a]D = + 11.0 ~ (C= 0.68, in

Plant Source Baccharis megapotamica. Isolation/Purification A large scale extract of 1800 kg of plant material was extracted with a total of 6000gal of isopropyl alcohol, which was concentrated to a black tar and partitioned between hexane and 10% water in methanol. The methanol solubles were treated by ferric gel precipitation, filtered, and the "pudding-like" insolubles washed with 50% aqueous methanol. The resulting filtrate was extracted with methylene chloride, and concentrated to give a total of 4.7kg of a dark oil. This material was chromatographed over silica gel, eluted with methylene chloride in hexane, methanol in methylene chloride, and 100% methanol to give 10 fractions (F 1 - F 10). Fraction F6 was taken up in methylene chloride and the solution carefully washed with a solution of 2.5% aqueous NaOH. The methylene chloride soluble material was subjected to filtration chromatography using alumina eluted with 10% isopropyl alcohol-hexane, 100% isopropyl alcohol, and 10% methanol in isopropyl alcohol). The 100% isopropyl alcohol fraction was chromatographed over Sephadex LH-20 eluted with methylene chloride and then chromatographed by flash chromatography over silica gel eluted with ethyl acetate-hexane). Final purification was by PTLC (Chromatotron) to give baccharinoid B 17 after recrystallization from ethyl ethermethylene chloride.

548

20.

Baccharinoids

Spectral Data UV:

~E~On max 260nm. IR:

(CHC13) 3580,2880, 1755, 1720, 1170, and ll00cm 1. 1H N/V[R:

(CDCI3) 0.77(3H, s, H-14); 1.23(3H, d, J=6.5Hz, H-14'); 1.37(3H, s, H-12'); 1.66 (3H, s, H-16); 2.19(1H, ddd, Jz3p=J3p,4=4.6Hz and Jgem=15.3Hz, H-313); 2.4- 2.6(3H, m, H-3ot and H-4'); 2.76, 3.17(1H each, AB pattern, J=3.9Hz, H-13); 3.09 (1H, d, J=5.4Hz, H-10); 3.30(1H, s, H-2'), 3.32-3.38(1H, m, H-5'B); 3.57(1H, d, J= 5.4Hz, H- 11); 3.84-3.92(1H, m, H-5'A); 3.94(1H, d, J=5.0Hz, H-2); 4.20, 4.43(1H each, AB pattern, J=12.6Hz, H-15); 5.76(1H, dd, J=4.6, 8.2Hz, H-4); 5.80(1H, d, J=l 1.2Hz, H-10'); 5.97(1H, dd, J=3.0, 15.6Hz, H-7'); 6.60(1H, dd, Js,,9,=Jg,,lo,=l1.2Hz, H-9'); and 7.54ppm (1H, dd, J= 11.2, 15.6Hz, H-8'). 13CNMR: (CDC13) 78.8, C-2; 34.8, C-3; 74.3, C-4; 49.1, C-5; 42.9, C-6; 17.6, C-7; 26.4, C-8; 57.7, C-9; 58.2, C-10; 67.5, C-11; 64.9, C-12; 47.6, C-13; 7.1, C-14; 63.7, C-15; 22.2, C-16; 168.2, C-I'; 57.4, C-2'; 63.7, C-3'; 40.1, C-4'; 67.9, C-5'; 86.0, C-6'; 138.6, C-7'; 126.3, C-8'; 143.5, C-9'; 117.9, C-10'; 166.5, C-11'; 17.4, C-12'; 70.8, C13'; 18.3, C-14'. TLC Data Rf0.66 (4% methanol-methylene chloride), 0.74 (ethyl acetate), and 0.54 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

549

Common/Systematic Name Baccharinoid B20 Molecular Formula/Molecular Weight C29H40010; M W -- 5 4 8 . 2 6 2 1 3

i

IIIIil, '

;

I,,'"~ J

o

H General Characteristics Melting point, 170-172~

[s

"-

q- 39.9 ~ (C=1.44, in CHC13).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B9. Fractions E1 and E2 were combined and crystallized from acetone-hexane to give 210mg baccharinoid B20. Spectral Data UV:

~EtOH

max 262nm.

IR:

(CHCI3) 940, 1730, and 1370cm1. 1H NMR: (CDC13) 0.69(3H, s, H-14); 0.98(3H, d, J=6.6Hz, H-12'); 1.16(1H, d, J=6.5Hz, H-14'); 1.34(3H, s, H-16); 2.05-2.21(1H, m, H-313); 2.35-2.50(2H, m, H-3a and H-3'); 2.74, 3.14(1H each, AB pattern, J=4.0Hz, H-13); 3.06(1H, d, J=5.1Hz, H-10); 3.54(1H, d, ,/--5.1 Hz, H-11); 3.60-3.68(3H, m, H-4' and H-5'); 3.80-4.00(3H, m, H-2,

550

20.

Baccharinoids

H-6', and H-13'); 3.78, 4.64(1H each, AB pattern, J=12.3Hz, H-15), 5.74(1H, d, J=l 1.1Hz, H-10'); 5.74(1H, m, H-4); 6.05(1H, dd, J=3.4 and 16.4Hz, H-7'); 6.71(1H, dd, Js,,9,=J9,,lo~l 1.1Hz, H-9'); and 7.77ppm (1H, dd, J=l 1.1 and 16.4Hz, H-8'). 13CNMR: (CDCI3) 78.6, C-2; 34.6, C-3; 74.4, C-4; 48.8, C-5; 42.9, C-6; 17.6, C-7; 26.4, C-8; 57.4, C-9; 57.8, C-10; 67.6, C-11; 64.8, C-12; 47.6, C-13; 6.9, C-14; 63.7, C-15; 22.1, C-16; 172.7, C-I'; 38.3, C-2'; 32.5, C-3'; 73.4, C-4'; 74.2, C-5'; 84.4, C-6'; 139.6, C-7'; 126.3, C-8'; 144.1, C-9'; 117.1, C-10'; 166.7, C-11'; 14.9, C-12'; 69.8, C-13'; and 17.9ppm C- 14'.

Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

551

Common/Systematic Name Baccharinoid B21 Molecular Formula/Molecular Weight C29H38Olo; M W -- 5 4 6 . 2 4 6 5 0

H

H

.-: o~d

H2dl

....'~

HO ~- - ~ H General Characteristics Melting point, 259-260~

[{g]D =

q- 73.5 ~ (C=0.68, in CH2C12).

Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the second column. Fraction 7C was subjected to preparative HPLC (3.5% methanol-methylene chloride, Waters Prep. LC/500) to give 10 fractions. The third fraction was sequentially subjected to MPLC (ethyl aeetate-hexane) and PTLC (50-100% ethyl acetate-hexane, Chromatotron) to give a fraction which upon crystallization from ethyl ether-methylene chloride gave 140mg of baccharinoid B21. Spectral Data UV~ EtOH j~max 2 6 0 n m . IR;

(CHCI3) 2880, 1750, 1715, 1170, and ll00cm "1.

552

20.

Baccharinoids

1H ~ : (CDCIa) 0.82(3H, s, H-14); 1.18(3H, d, J--6.0Hz, H-14'); 1.55(3H, s, H-12'); 1.81(3H, s, n-16); 2.10-2.22(3H, m, n-3~, and n-7); 2.44(1H, dd, Ja~.4=8.3Hz and Js~=15.3Hz, H-3a); 2.82, 3.11(1H each, AB pattern, J--4.0Hz, H-13); 3.29(1H, s, H2'), 3.33-3.37(1I-I, m, H-5~B), 3.58-3.64(2H, m, H-5'A and n-11); 3.64-3.80(2H, m, H6' and H-13'); 3.85(1I-I, d, J--4.9Hz, H-2); 3.82-3.99(1H, m, n-8), 4.19, 4.40(1H each, AB pattern, J=-12.3Hz, H-15); 5.48(1H, d, J=5.3Hz, H-10); 5.75(1I-I, dd, J=4.3 and 8.3Hz, n-4); 5.78(1H, d, J=-I 1.4Hz, n-10'); 5.95(1H, dd, J=-2.9 and 15.7Hz, H-7'); 6.58(1I-I, dd, Ja. ~=J~,~o~l1.4Hz, H-9'); and 7.48ppm (1H, dd, J=l 1.4 and 15.7Hz, H8'). 13CNMR: (CDCI3) 79.2, C-2, 35.1, C-3, 74.0, C-4, 49.4, C-5, 45.2, C-6; 30.7, C-7; 67.0, C-8, 142.9, C-9; 120.8, C-10, 68.0, C-11, 65.2, C-12, 47.8, C-13; 7.0, C-14; 65.0, C-15; 18.8, C-16; 168.1, C-I', 58.2, C-2'; 63.4, C-3'; 39.7, C-4', 67.6, C-5', 85.8, C-6'; 138.5, C-7'; 126.3, C-8'; 142.8, C-9', 118.0, C-10', 166.4, C-11'; 17.4, C-12'; 70.9, C-13'; and 18.3ppm C-14'. Reference B. B. ]arvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis meg~potamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

553

Common/Systematic Name Baccharinoid B23 Molecular Formula/Molecular Weight C29H40010; M W -- 5 4 8 . 2 6 2 1 5

H H -.-- O~r_~__ ~ ....,0

HOH2C

H2 I o

HO'~ H General Characteristics Colorless glass; [a]D = + 113.5~ (C=0.90, in MeOH). Plant Source Baccharis megapotamica. Isolation/Purification See isolation procedure for baccharinoid B25. Spectral Data UV:

EtOH

~,m,x 261nm. IR:

(CH2C12) 3600, 2870, 1730, 1715, and l175crn1. :H NMR: (CDC13) 0.77(3H, s, H-14); 1.00(3H, d, J=6.4Hz, H-12'); 1.21(3H, s, H-14'); 2.102.25(3H, m, H-3~, and H-2'); 2.40-2.53(2H, m, H-3a and H-3'); 2.82, 3.12(1H each, AB pattern, J=4.0Hz, H-13); 3.60-3.75(4H, m, H-4', H-5', and H-13'); 3.73(2H, s, H16); 3.84(1H, d, J=4.SHz, H-2); 4.06(1H, br s, H-6'); 3.92, 4.67(1H each, AB pattern, J=12.SHz, H-15); 5.77-5.85(2H, m, H-10 and H-4); 5.79(1H, d, J=l 1.3Hz, H-10');

554

20.

Baccharinoids

6.06(1H, dd, J=3.3 and 15.7Hz, H-7'); 6.58(1H, dd, Jg.,9~J9.,10,=l1.3Hz, H-9'); and 7.81ppm (1H, dd, J=l 1.3 and 15.7Hz, H-8'-H). 13C NMR: (CDC13) 79.2, C-2; 34.9, C-3; 74.5, C-4; 49.2, C-5; 43.9, C-6; 20.1, C-7; 23.2, C-8; 143.6, C-9; 118.3, C-10; 66.8, C-11; 65.3, C-12; 47.9, C-13; 7.0, C-14; 66.0, C-15; 63.5, C-16; 172.8, C-I'; 38.2, C-2'; 32.4, C-3'; 73.0, C-4'; 74.3, C-5'; 86.0, C-6'; 139.8, C-7'; 126.5, C-8'; 144.0, C-9'; 117.6, C-10'; 166.8, C-11'; 15.0, C-12'; 71.0, C-13'; and 18.6ppm C-14'. Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation of Macrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

555

Common/Systematic Name Baccharinoid B24 Mol.eeul~ Formula/Molecular Weight C29H40010; M W - 548.26215 H

H

"

I

HOH2C,~.- O,,J .... ,o i

OJ) H General Characteristics Colorless glass from methylene chloride-hexane; [a]D = + 90.8 ~ (C=1.36, in MeOH). Plant Source Baccharis megapotamica. Isolation/Purification See isolation for isolation ofbaeeharinoid B25. Spectral Data UV:

~.~ 260nm. IR:

(CHCh) 3600, 2875, 1720, 1715, and llS0em "~. 1H N/VIR:

(CDCI3) 0.79(3H, s, H-14); 1.01(3H, d, J=6.5 Hz, H-12'); 1.17(3H, d,J=6.4Hz, H14'); 2.10-2.50(5H, rn, H-3, H-2', and H-3'); 2.82, 3.12(1H each, AB pattern, d=4.0 Hz, H-13); 3.62-3.74(4H, m, H-11', H-5', and H-13'); 3.72(2H, s, H-16), 3.84(1H, d, J---4.8H~ H-2); 4.04(1H, br s, H-6'); 3.94, 4.63(1H each, AB pattern, J=-12.4I-!z, H15); 5.69(1H, d, J=5.3Hz, n-10), 5.78(1H, d, J=l 1.3H~ H-10'); 5.84(1H, dd, ,/---4.5

556

20.

Baccharinoids

and 8.2Hz, H-4); 6.06(1H, dd, ,/=3.0 and 15.6Hz, H-7'); 6.69(1H, dd,

Js,,9,=J9,,1o,=l 1.3Hz, H-9'); and 7.79ppm (1H, dd, J=l 1.3 and 15.6Hz, H-8'). 13CNMR: (CDC13) 79.3, C-2; 35.0, C-3; 74.7, C-4; 49.4, C-5; 44.0, C-6; 20.2, C-7; 23.3, C-8; 143.6, C-9; 118.5, C-10; 66.9, C-11; 65.3, C-12; 47.9, C-13; 7.0, C-14; 66.1, C-15; 63.4, C-16; 172.8, C-I'; 38.4, C-2'; 32.6, C-3'; 73.2, C-4'; 74.7, C-5'; 84.7, C-6'; 139.3, C-7'; 126.8, C-8'; 144.0, C-9'; 117.5, C-10'; 166.8, C-11'; 14.8, C-12'; 70.1, C-13'; and 18.0ppm C- 14'. Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

557

Common/Systematic Name Baccharinoid B25 Molecular Formula/Molecular Weight C27H32Olo; M W = 516.19955 H

H

-

HO"

v

0

-

....,oI

y

0

0 General Characteristics Melting point, 205~

[a]D -- + 214 ~ (C=0.70, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 9 from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions 9A-9E. Fraction 9D was dissolved in methylene chloride and hexane was added slowly to give a precipitate. This material was dissolved in methylene chloride and the solution washed with 2.5% aqueous NaOH, concentrated to dryness, and subjected to preparative HPLC using alumina and eluting with isopropyl alcohol-methylene chloride to give six fractions. From these fractions by successive chromatographic procedures baccharinoids B 13, B 14, B23, B24, B25, and B27 were isolated. Spectral Data UV: ~EtOH max

220 and 260nm.

IR:

(KBr) 1740, and 1165cm"1.

558

20.

Baccharinoids

1H NMR: (CDCI3) 0.81(3H, s, H-14); 1.83(3H, s, H-16); 2.10-2.30(1H, m, H-313); 2.53(1H, dd, J3~4=8.1Hz, Jg =15.5Hz, H-3ot); 2.86, 3.17(1H each, AB pattern, J=4.0Hz, H13); 3.88(1H, d, J=5.0Hz, H-2); 3.91(1H, d, J=5.4Hz, n-11); 4.03-4.14(3H, m, H5'A, H-8-H, and H-15B); 4.40-4.48(2H, m, H-4' and H-5'A); 4.17(1H, AB pattern, J=12.7Hz, H-15A); 5.56(1H, d, J=5.4Hz, n-10); 5.99(1H, dd, J=2.9, 15.7Hz, n-7'); 6.07(1H, br s, n-2'); 6.06-6.1 l(1n, m, n-4); 6.12(1H, d, J=l 1.3Hz, H-10'); 6.63(1H, dd, Js,,9,=Jg,,ltr=ll.3Hz, H-9'); and 8.1 lppm (1H, dd, J=l 1.3, 15.8 Hz, n-8'). 13CNMR: (CDC13) 78.3, C-2; 35.4, C-3; 75.1, C-4; 48.7, C-5; 45.4, C-6; 31.6, C-7; 68.3, C-8; 142.4, C-9; 121.2, C-10; 66.9, C-11; 65.4, C-12; 48.1, C-13; 6.8, C-14; 64.2, C-15; 18.9, C-16; 165.8, C-I'; 116.8, C-2'; 158.3, C-3'; 74.3, C-4'; 63.7, C-5'; 165.1, C-6'; 126.5, C-7'; 139.1, C-8'; 139.7, C-9'; 125.9, C-10'; 165.8, C-11'; and 13.9ppm, C-12'. TLC Data Rr 0.31 (4% methanol-methylene chloride), 0.64 (ethyl acetate), and 0.58 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

559

Common/Systematic Name Baccharinoid B27 Molecular Formula/Molecular Weight C29I-I36010; M W = 544.23085 H

H

=- o~I

~

H General Characteristics Melting point, 165~

[tX]D= + 5.4~ (C----0.40,in methanol).

Plant Source Baccharis megapotamica. Isolation/Purification See description for isolation ofbacchafinoid B25.

Spectral Data UV"

~.~.tOH 223 and 263nm. IR:

(KBr) 3600, 2900, 1720, and 1685crn"1. 1HNMR: (CDCh) 0.76(3H, s, 1H-14); 1.18(3H, d, J--6.4Hz, H-14'); 1.83(3H, s, H-16); 2.10(1H, ddd, Jz3f4.9Hz, J~p,4~3.9Hz, Jg~=l 5.5Hz, H-313);2.24(1H, d, J=l. 1Hz, H12'); 2.60(1H, dd, J=-8.1, 15.5Hz, n-3ct); 2.42, 2.94(1H each, AB pattern, J=-16.3I~ H-7); 2.85, 3.15(1H each, AB pattern, J--4.0Hz, 13-H); 3.64, 3.82(1H each, d of AB pattern, J=4.0, 9.1I~ n-5'); 3.93(1H, d, J=4.9Hz, H-2); 4.00-4.3 l(6H, m, H-11, H15, n-4', H-6', and H-lY); 5.75(1H, d, J=l 1.5Hz, H-10'); 5.91(1H, dd, J=2.6, 15.4Hz, n-7'); 6.18(1H, br s, n-2'); 6.20(1H, dd, J=3.9, 8. lI-~ U-4); 6.57(1H, d, J=5.0Hz, H-

560

20.

Baccharinoids

10), 6.58(1H, dd, ,]8,9,=.]9,]o_11.5Hz, H-9'), and 7.48ppm (1H, dd, J=l 1.5, 15.4Hz, H8'). 13C NMR: (CDC13) 79.4, C-2; 36.1, C-3; 73.6, C-4; 48.3, C-5; 46.5, C-6; 38.8, C-7; 197.1, C-8; 136.6, C-9; 138.5, C-10; 66.4, C-11; 65.7, C-12; 47.9, C-13; 6.4, C-14; 64.6, C-15; 17.4, C-16; 166.4, C-I'; 114.6, C-2'; 161.1, C-3'; 74.6, C-4'; 71.8, C-5'; 82.3, C-6'; 137.2, C-7'; 127.3, C-8'; 143.4, C-9'; 117.7, C-10'; 165.8, C-11'; 15.5, C-12'; 68.2, C13'; and 16.1ppm, C-14'. TLC Data Rf=0.44 (4% methanol-methylene chloride), 0.44 (ethyl acetate), and 0.51 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

Fumonisins, AAL Toxins, and Related Metabolites Fumonisin B1 Fumonisin B2 Fumonisin B3 Fumonisin B4 Fumonisin A1 Fumonisin A2 Fumonisin C~ Hydroxylated Fumonisin C1 Fumonisin C3 Fumonisin Ca Fumonisin AK1 AAL Toxin TA~ AAL Toxin TA2 AAL Toxin TB 1 AAL Toxin TB2 AAL Toxin TC~ AAL Toxin TC2 AAL Toxin TD~ AAL Toxin TD2 /X~L Toxin TE~ AAL Toxin TE2 Sphingofungin A Sphingofungin B Sphingofungin C Sphingofungin D Myriocin

561

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21.

Fumonisins, AALToxins, and Related Metabolites

563

Common/Systematic Name Fumonisin BI, Macrofusin Molecular Formula/Molecular Weight C34I-I59NO15; MW = 721.38847 30 O H 0

o.o.

2

1 20

o.

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source

Fusarium moniliforme Sheldon (M-2326), (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50~ The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The main fumonisin B1 fraction was again fractionated on a silica gel 60 column eluted with chloroform-methanol-water-acetic acid (55:36: 8:1, v/v/v/v) but without the anhydrous sodium sulfate on top of column. Fractions containing only fumonisin B1 were combined and finally purified using RP-C18 column using methanol-water (1:1,v/v) as eluant. The pH of the sample was adjusted to 3.5 with 1N HC1 prior to application to the column. Fractionation was achieved using a linear gradient from methanol-water (1:1, v/v) to methanol-water (4:1, v/v) to produce purified fumonisin B1.

564

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly-phytotoxic. Spectral Data 13C NMR: (D20) 17.7, C-I; 55.5, C-2; 71.8, C-3; 42.2, C-4; 69.9, C-5; 39.5, C-6; 27.7, C-7; 27.6, C-8; 39.8, C-9; 71.4, C-10; 45.1, C-11; 27.7, C-12; 37.5, C-13; 74.7, C-14; 80.6, C-15; 35.9, C-16; 34.3, C-17; 30.8, C-18; 25.1, C-19; 16.3, C-20; 17.3, C-21; 22.4, C22; 175.2, C-I'; 37.8, C-2'; 40.0, C-3'; 38.1, C-4'; 179.6, C-5'; 179.1, C-6'; 175.1, C1"; 37.8, C-2"; 39.9, C-3"; 38.0, C-4"; 178.0, C-5"; and 177.6ppm, C-6". (Hydrolysis product) 16.8, C-I; 53.8, C-2; 70.4, C-3; 42.9, C-4; 68.5, C-5; 39.3, C-6; 26.8, C-7; 26.9, C-8; 39.6, C-9; 70.7, C-10; 44.6, C-11; 26.9, C-12; 41.6, C-13; 70.4, C-14; 80.8, C-15; 35.9, C-16; 31.7, C-17; 30.6, C-18; 24.2, C-19; 14.5, C-20; 16.0, C21; and 21.4ppm, C-22. Mass Spectrum: FABMS: (M+H) § 722m/e and (M+Na) + 744m/e. References S. C. Bezuuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun. pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991).

21.

Fumonisins, AALToxins, and Related Metabolites

565

Common/Systematic Name Fumonisin B2 Molecular Formula/Molecular Weight C34H59NO14; M W -- 7 0 5 . 3 9 3 5 6

0

OH OH NH2

OH

O/ CH3 O.,,,,~O OH

OH General Characteristics Fumonisins are highly funetionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source

Fusarium rnoniliforme Sheldon (M-2326); (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue alter filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50 ~ C. The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The fumonisin B2 fraction that coeluted with B1 and B3 was again fractionated on a silica gel 60 column eluting with chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v) but without the anhydrous sodium sulfate on top of column. Fractions containing only fumonisin B2 were combined and finally purified using RP-C 18 column using methanol-water (1:1, v/v) as eluant. The pH of the sample was adjusted to 3.5 with IN HC1 prior to application to the column. Fractionation was achieved using a linear gradient from methanol-water (1:1, v/v) to methanol-water (4:1, v/v) to produce purified fumonisin B2 (92% purity).

566

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leueoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13CNMR: (D20) 16.0, C-l; 53.8, C-2; 70.4, C-3; 41.8, C-4; 68.7, C-5; 39.3, C-6; 27.7, C-7; 29.7, C-8; 26.7, C-9; 30.7, C-10; 36.1, C-11; 30.2, C-12; 36.2, C-13; 73.1, C-14; 78.9, C-15; 34.9, C-16; 33.2, C-17; 30.8, C-18; 23.9, C-19; 14.4, C-20; 16.0, C-21; 20.9, C22; 173.0, C-I'; 36.5, C-2'; 38.6, C-3'; 36.1, C-4'; 276.8, C-5'; 175.2, C-6'; 172.9, C1"; 36.5, C-2"; 38.5, C-3"; 36.1, C-4"; 176.6, C-5"; and 175.0ppm, C-6". (Hydrolysis product) 16.8, C-l; 53.8, C-2; 70.9, C-3; 41.9, C-4; 68.6, C-5; 39.3, C-6; 26.8, C-7; 27.9, C-8; 31.2, C-9; 30.8, C-10; 36.9, C-11; 30.5, C-12; 40.7, C-13; 70.4, C-14; 80.9, C-15; 36.0, C-16; 31.9, C-17; 30.6, C-18; 24.2, C-19; 14.5, C-20; 16.0, C21; and 21.5ppm, C-22. Mass Spectrum: FABMS (M+H)+ 706m/e and (M+Na) + 728role. References S. C. Bezuidenhout, W. C. A. Gelderblom, C. P. Gorst-AUman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun.; pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agile. Food Chem.;Vol. 39, pp. 1958-1962(1991). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.

Fumonisins, AALToxins, and Related Metabolites

567

Common/Systematic Name Fumonisin B3 Molecular Formula/Molecular Weight. C34H59NO14; MW

-- 7 0 5 . 3 9 3 5 6

% / OH

NH2

0/

OH

CH3

O ~

OH

i

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source

Fusarium moniliforme Sheldon (M-2326); (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50~ The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The fumonisin B2 fraction that coeluted with B~ and B3 was fractionated on an Amberlite XAD-2 column equilibrated with methanol-water (1:1, v/v). The pH of the sample was adjusted to 3.5 with 1N HC1 prior to application to the column. The column was washed with methanolwater (1:1, v/v). Fractionation was achieved using acetonitrile-methanol (1:1, v/v) to produce purified fumonisin B3.

568

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C lX~iR: (D20) 15.9, C-I; 53.5, C-2; 73.1, C-3; 34.6, C-4; 26.2, C-5; 26.1, C-6; 26.8, C-7; 29.6, C-8; 39.3, C-9; 69.9, C-10; 44.5, C-11; 26.9, C-12; 36.4, C-13; 73.1, C-14; 78.8, C-15; 34.9, C-16; 33.1, C-17; 30.7, C-18; 23.8, C-19; 14.4, C-20; 16.0, C-21; 20.6, C22; 173.1, C-I'; 36.6, C-2'; 38.6, C-3'; 36.1, C-4'; 177.0, C-5'; 175.2, C-6'; 173.0, C1"; 36.6, C-2"; 38.6, C-3"; 36.1, C-4"; 176.6, C-5"; and 175.0ppm, C-6". (Hydrolysis product) 16.8, C-I; 53.5, C-2; 73.1, C-3; 34.7, C-4; 26.3, C-5; 30.7, C-6; 30.8, C-7; 26.9, C-8; 39.6, C-9; 70.1, C-10; 44.6, C-11; 27.0, C-12; 41.6, C-13; 70.4, C-14; 80.8, C-15; 35.9, C-16; 31.7, C-17; 30.7, C-18; 24.2, C-19; 14.5, C-20; 16.0, C21; and 21.4ppm, C-22. Mass Spectrum: FABMS: 706role (M+H) § and MS/MS ofM + from FABMS (706rn/e). References S. C. Bezuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun.; pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991 ). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.

Fumonisins, AALToxins, and Related Metabolites

569

Common/Systemati_c Name Fumonisin B4 Molecular Formula/Molecular Weight C34H59NO13~, U W

-- 6 8 9 . 3 9 8 6 4

0===~O 0H,~~--OH / O"

OH NH2

Me

OH

0 ~ . ~ 0

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source Fusarium moniliforme Sheldon (M-2326); (MRC-826). Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue aider filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50~ The dried extract was dissolved in methanol-water (1:1, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:1, v/v). The column was eluted with methanol-acetonitrile (1:1, v/v). The fractions containing fumonisin B4 were further chromatographed on a silica gel 60 column eluted with chloroformmethanol-water-acetic acid (55:36: 8:1, v/v/v/v). The fumonisin B4 was finally fractionated through a Sep-Pak ClS cartridge. Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high

570

21.

Fumonisins, AALToxins, and Related Metabolites

levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. References S. C. Bezuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun.; pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.

Fumonisins, AALToxins, and Related Metabolites

571

Common/Systematic Name Fumonisin A1 Molecular Formula/Molecular Weight C36H61NO]6; M W = 763.39904

O

OH

OH

NHCOMe

0~~//

OH Me O~OoH O''?

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fumonisin A1 was isolated as the tetramethyl derivative as a colorless oil. Funsal Source v

Fusarium moniliforme Sheldon (M-2326), (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50 ~ C. The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). Fractions containing fumonisin A1 and A2 were further purified using an Amberlite XAD-2 column. The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. Fumonisins ml and A2 were further purified on a silica gel column eluted with chloroform-methanol-acetic acid-water (65:25:6:4, v/v/v/v) which resulted in separation of fumonisins A] and A2. Final purification was achieved on a RP C1, column equilibrated with methanol-water (1:1, v/v) and eluted with methanol-water (3:1, v/v).

572

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C NMR: Tetramethyl derivative; (CDC13) 18.03, C-I; 49.72, C-2; 71.49; C-3; 40.22, C-4; 68.83, C-5; 38.55, C-6; 25.63, C-7; 25.48, C-8; 37.22, C-9; 68.78, C-10; 43.08, C-11; 25.17, C-12; 35.39, C-13; 71.24, C-14; 77.79, C-15; 33.64, C-16; 31.84, C-17; 28.48, C-18; 22.73, C-19; 13.92, C-20; 15.36, C-21; 20.36, C-22; 175.48, C-23; 2332, C-24; 170.88, C-25; 35.27, C-26; 37.28, C-27; 35.10, C-28; 173.41, C-29; 171.69, C-30; 52.31, C-31; and 51.82ppm, C-32. Mass Spectrum: Tetramethyl derivative; FABMS: (M+H) + 820m/e and (M+Na) + 842m/e. References S. C. Bezuuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allmart, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Soc., Chem. Commun.; pp. 743-745 (1988). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991).

21.

Fumonisins, AALToxins, and Related Metabolites

573

Common/Systematic Name Fumonisin A2 Molecular Formula/Molecular Weight C36H61NO15, MW = 747.40412

0

OH

NHCOMe

OH

OH

0/

Me

0.,,,,,~0

L

OH

I. o OH

General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fumonisin A2 was isolated as the tetramethyl derivative as a colorless oil. Fungal Source

Fusarium moniliforme Sheldon (M-2326), (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50 ~ C. The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). Fractions containing fumonisin A1 and A2 were further purified using an Ambedite XAD-2 column. The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. Fumonisins A1 and A2 were further purified on a silica gel column eluted with chloroform-methanol-acetic acid-water (65:25:6:4, v/v/v/v) which resulted in separation of fumonisins A~ and A2. Final purification was achieved on a RP C18 column equilibrated with methanol-water (1:1, v/v) and eluted with methanol-water (3:1, v/v).

574

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data Mass Spectrum: Tetramethyl derivative; FABMS: (M+W) 84m/e. References S. C. Bezuuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun. pp. 743-745 (1988). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: a quantitative approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991).

21.

Fumonisins, AALToxins, and Related Metabolites

575

Common/Systematic Name Fumonisin C1 Molecular Formula/Molecular Weight C33H57NO15, MW = 707.37282

\

OH OH NH2

0==~

OH

0/ OH

Me

0~0

OH

OH General Characteristics Obtained as a colorless liquid. Fungal Source

Fusarium moniliforme Sheldon (M-2326); (MRC-826).

Isolation/Purification Method 1. The crude culture material was separated on preparative reversed-phase HPLC using a ClS column eluted with a gradient from 100% water to 100% methanol. The fraction containing a solvent concentration of approximately methanol-water (60:40) was taken to dryness and chromatographed on a C~s column using an isocratic mobile phase of 0.05 M NaH2PO4 (adjusted to pH 3.35)-acetonitrile (72:24, v/v). Fractions containing both fumonisins B~ and C1 were rechromatographed on a C~s column using a mobile phase of 0.05 M NaH2PO4 (adjusted to pH 3.35)-acetonitrile (74:26, v/v). Fractions containing fumonisin C~ were combined, taken to dryness, and desalted on a Cls mini-column. Method 2. Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographed on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1:1, v/v), (3:1, v/v), and finally with methanol. Fumonisin C1, hydroxylated C1, C3, and C4 were eluted in the methanol-water (3:1) and methanol fractions. These fractions were combined, concentrated to dryness and chromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was

576

21.

Fumonisins, AALToxins, and Related Metabolites

chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisin Ca or C4were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak Cis column eluted with a gradient from methanol-water-acetic acid (20: 80:1, v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C~ or hydroxylated C~ were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3:1, v/v) and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol. Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C NMR: (D20) 47.5, C-l; 71.2, C-2; 43.6, C-3; 67.4, C-4; 39.7, C-5; 27.6, C-6; 27.5, C-7; 39.7, C-8; 69.8, C-9; 42.5, C-10; 27.9, C-11; 37.6, C-12; 74.7, C-13; 80.5, C-14; 35.8, C-15; 34.0, C-16; 30.5, C-17; 24.9, C-18; 15.9, C-19; 17.4, C-20; 22.2, C-21; 175.4, C-I'; 38.4, C-2'; 41.0, C-3'; 39.0, C-4'; 180.4, C-5'; 180.0, C-6'; 175.4, C-I"; 38.4, C-2"; 41.0, C-3"; 38.9, C-4"; 179.0, C-5"; and 178.6ppm, C-6". (CDaOD) 46.6, C1; 70.0, C-2; 43.1, C-3; 66.3, C-4; 37.9, C-5; 26.7, C-6; 26.6, C-7; 39.0, C-8; 68.6, C9; 44.6, C-10; 26.9, C-11; 34.9, C-12; 72.8, C-13; 78.8, C-14; 34.9, C-15; 33.0, C-16; 29.7, C-17; 23.9, C-18; 14.4, C-19; 16.0, C-20; 20.8, C-21; 173.5, C-22; 37.0, C-23; 39.7, C-24; 37.6, C-25, 178.7, C-26; 178.0, C-27, 173.3, C-28; 36.8, C-29; 39.6, C-30; 37.2, C-31; 176.7, C-32; and 176.7ppm, C-33. 1H ~: (CDaOH) 5.14(IH, dt,d=11.3, 3.7Hz, H-13); 4.93(IH, dd, J=8.3, 3.9I-Iz,H-14); 4.00(IH, m, H-2); 3.79(IH,m, H-4); 3.66(IH, l'n,H-9); 3.14(2 x IH, m, H-24 and H30); 3.03(IH, dd, J=13.0, 3.4I-Iz,H-Ib); 2.80(IH, dd, J=12.9, 7.6I-Iz);2.76(IH, H-la); 2.72(IH, dd, J=7.6, 3.0I-Iz);2.72(IH, dd, 7.6,3.0Hz);2.69-2.66(3 x IH, m); 2.532.41(3 x IH, m); 1.81(IH, m, H-I I); 1.67(IH, H-15); 1.58(IH, H-12b); 1.351.54(16H,m); 1.30(IH, m, H-17a); 1.16(IH, I0~); I.II(IH, H-16a); 0.95(3H, d,J= 6.3Hz, H-21); 0.93(3H, d, J=6.81-Iz,H-20); and 0.88ppm (3H, t,J=7. II-Iz,H-19). Mass Spectrum: FABMS: (M+H) § 708role and MS/MS of 708role from FABMS 708role. 708(parent ion), 690(25), 532(7), 514(5), 374(5), 356(30), 338(100), and 320m/e(55). TFA derivative of hydrolyzed fumonisin C1 (molecular weight 981), 740(0.1), 626(6),

21.

Fumonisins, AALToxins, and Related Metabolites

577

568(2), 542(3), 541(3), 528(7), 512(6), 414(3), 264(4), 211(20), 180(60), 126(59), 97(100), 55(66), and 43m/e (43). TLC: Rf = 0.29 on Cls reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% panisaldehyde solution in methanol-sulfuric acid-acetic acid (85: 5:10, v/v/v) and heating at 110~ for 10 rain. References B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993). J-A Seo, J-C. Kim, and Y-W Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 1003-1005 (1996).

578

21.

Fumonisins, AALToxins, and Related Metabolites

Common/Systematic Name Hydroxylated Fumonisin C1 Molecular Formula/Molecular Weight C33H57NO16; M W = 723.36774

O=:~OOH~OH OH OH NH2 OH

O/ OH Me 0.~0 ?H0

,r,~ OH General Characteristics Obtained as a colorless liquid. Fungal Source Fusarium oxysporum.

Isolation/Purification Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographed on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1:1, v/v), (3:1, v/v), and finally with methanol. Fumonisin C~, hydroxylated CI, C3, and C4 were eluted in the methanol-water (3:1) and methanol fractions. These fractions were combined, concentrated to dryness and chromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisin C3 or C4 were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak C]s column eluted with a gradient from methanol-water-acetic acid (20: 80:1 v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C1 or hydroxylated C~ were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3:1, v/v) and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol.

21.

Fumonisins, AALToxins, and Related Metabolites

579

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 1H N-IVIR: (CD3OH) 5.14(1H, dt, d=l 1.0, 3.0Hz, H-13); 4.95(1H, dd, J=8.3, 3.2Hz, H-14); 3.88(1H, td, d=8.0, 3.4Hz, H-2); 3.79(1H, td, J=7.8, 3.4Hz, H-4); 3.63(1H, m, H-9); 3.36(1H, m, H-3); 3.28(1H, dd, J=13.4, 3.4Hz, H-lb); 3.15(2 x 1H, m, H-24 and H30); 3.00(1H, dd, J=12.7, 8.3Hz, H-la); 2.78(1H, dd, J=16.6, 7.1Hz); 2.74(1H, dd, 11.8, 3.9Hz); 2.69(1H, dd, J=7.1, 4.3Hz); 2.63(1H, dd, J=7.6, 4.9Hz); 2.57 (1H, dd, J=13.9, 6.6Hz); 2.56 (1H, dd, J=l 1.2, 5.4Hz); 2.50 (1H, dd, J=l 1.7, 6.1Hz); 2.46 (1H, dd, J=10.5, 6.3Hz), 2.46 (1H, dd, J=10.5, 6.3Hz); 1.81(1H, m, H-11); 1.698(1H, H-15); 1.64(1H, H-12b), 1.35- 1.57(14H,m); 1.31(1H, m, H-17a); 1.16(1H, H-10a); 1.09(1H, H-16a); 0.96(3H, d, J=6.3Hz, H-21), 0.94(3H, d, J=6.8Hz, H-20); and 0.90ppm (3H, t, J=7.1Hz, H-19). 13CNMR: (CD3OH) 44.5, C-l; 70.9, C-2; 76.0, C-3; 69.0, C-4; 37.6, C-5; 26.9, C-6; 26.8, C-7; 39.2, C-8; 70.0, C-9; 44.0, C-10; 26.9, C-11; 34.8, C-12; 72.9, C-13; 78.7, C-14; 34.6, C-15; 34.6, C-16; 33.0, C-17; 23.9, C-18; 14.4, C-19; 16.0, C-20; 20.7, C-21; 173.4, C-22; 36.8, C-23; 39.5, C-24; 37.3, C-25; 178.6, C-26; 178.0, C-27; 173.2, C-28; 36.7, C-29; 39.5, C-30; 37.1, C-31; 176.9, C-32; and 176.5ppm, C-33. Mass Spectrum: FABMS: (M+H)§ 336m/e (4).

(100%) 706(6), 688(4), 548(9), 372(12), 354(6), and

TLC: Rf = 0.26 on C18 reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36: 8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% p-anisaldehyde solution in methanol-sulfuric acid-acetic acid (85:5:10, v/v/v) and heating at 110~ for 10 min.

580

21.

Fumonisins, AALToxins, and Related Metabolites

References B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 16301633(1993). J-A Seo, J-C. Kim, and Y-W Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 10031005(1996).

21. Fumonisins, AALToxins, and Related Metabolites

581

Common/Systematic Name Fumonisin C3 Molecular Formula/Molecular Weight C33H57NO]4; MW = 691.37791

% / OH

NH2

0/

OH Me 0~0

?H0

OH General Characteristics Obtained as a colorless liquid. Fungal Source

Fusarium moniliforme Sheldon (M-2326); (MRC-826).

Isolation/Purification Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographed on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1" 1, v/v), (3 1, v/v), and finally with methanol. Fumonisin C1, hydroxylated C1, C3, and C4 were eluted in the methanol-water (3"1) and methanol fractions. These fractions were combined, concentrated to dryness and ehromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisin C3 or C4 were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak C18 column eluted with a gradient from methanol-water-acetic acid (20:80:1, v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C1 or hydroxylated C1 were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3-1_ v/v] and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol.

582

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 1H NMR: (CD3OD) 5.13(1H, dt, d-11.3, 2.9Hz, H-13); 4.95(1H, dd, J=8.6, 2.9Hz, H-14), 3.77(1H, m, H-2), 3.61(1H, m, H-9); 3.16(2 x 1H, m, H-24 and H-30); 3.03(1H, dd, d=12.7, 2.7Hz, H-lb); 2.80(1H, dd, J=l 1.7, 7.3Hz); 2.76(1H, H-In); 2.74-2.64(3 x 1H, m); 2.61-2.52(3 x 1H, m); 2.48(1H, dd, d=16.6, 6.6Hz); 1.79(1H, m, H-11); 1.70(1H, H-15); 1.66(1H, H-12b); 1.27-1.53(19H, m), 1.17(1H, H-10a); 1.09(1H, 16a); 0.95(3H, d, d=6.6Hz, H-21); 0.94(3H, d, J=6.6Hz, H-20); and 0.88ppm (3H, t, J=6.8Hz, H-19). 13C NMR: (CD3OD) 46.1, C-l; 69.9, C-2; 39.2, C-3; 30.6, C-4; 30.5, C-5; 26.7, C-6; 26.2, C-7; 39.0, C-8; 68.7, C-9; 44.5, C-10; 26.9, C-11; 35.8, C-12; 73.0, C-13; 78.7, C-14; 34.8, C-15; 33.0, C-16; 29.5, C-17; 23.8, C-18; 14.4, C-19; 15.9, C-20; 20.6, C-21; 173.3, C-22; 36.6, C-23; 39.0, C-24; 36.8, C-25; 177.7, C-26; 177.3, C-27; 173.1, C-28; 36.6, C-29; 39.0, C-30; 36.8, C-31; 176.0, C-32; and 175.7ppm, C-33.

Mass Spectrum: FABMS: (M+H)§692(100), 674(7), 516(8), 340(15), 322(14) and 304(7), 512(6), 414(3), 264(4), 211(20), 180(60), 126(59), 97(100), 55(66), and 43role (43). TLC: Rf = 0.36 on C~s reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% panisaldehyde solution in methanol-sulfuric acid-acetic acid (85:5:10, v/v/v) and heating at 110~ for 10 rain. Reference J-A. Seo, J-C. Kim, and Y-W. Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 1003-1005 (1996).

21.

Fumonisins, AALToxins, and Related Metabolites

583

Common/Systematic Name Fumonisin C4 Molecular Formula/Molecular Weight C33H57NO13, MW = 675.38299

OH

NH2

ohm_ 0/

Me 0...~0 OH

General Characteristics Obtained as a colorless liquid. Fungal Source Fusarium oxysporum (strain CAR isolated from carnation in Korea; F. moniliforme Sheldon). Isolation/Purification Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographer on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1:1, v/v), (3:1, v/v), and finally with methanol. Fumonisin C1, hydroxylated C1, C3, and C4 were eluted in the methanol-water (3:1) and methanol fractions. These fractions were combined, concentrated to dryness and chromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisins C3 or Ca were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak C18 column eluted with a gradient from methanol-water-acetic acid (20: 80:1, v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C1 or hydroxylated C1 were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3:1, v/v) and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol.

584

21.

Fumonisins, AALToxins, and Related Metabolites

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C NMR: (CD3OD) 46.1, C-l; 68.9, C-2; 39.0, C-3; 30.7, C-4; 30.5, C-5; 27.5, C-6; 26.3, C-7; 30.5, C-8; 30.5, C-9; 30.1, C-10; 20.9, C-11; 35.9, C-12; 73.0, C-13; 78.8, C-14; 34.9, C-15; 33.0, C-16; 29.6, C-17; 23.8, C-18; 14.4, C-19; 16.0, C-20; 20.9, C-21; 173.1, C-22; 36.3, C-23; 38.9, C-24; 36.7, C-25; 177.5, C-26; 177.3, C-27; 173.0, C-28; 36.1, C-29; 38.9, C-30; 36.6, C-31; 175.9, C-32; and 175.6ppm, C-33. 1H NMR: (CD3OD) 5.17(1H, dt, J-11.1, 3.2Hz, H-13); 4.92(1H, dd, J=8.5, 3.4Hz, H=14); 3.76(1H, m, H-2); 3.16(2 x 1H, m, H-24 and H-30); 3.03(1H, dd, J=12.7, 2.9Hz, Hlb); 2.80(1H, dd, J-12.7, 7.1Hz); 2.76(1H, H-l,); 2.74-2.64(3 x 1H, m); 2.59(1H, dd, ,/=10.3, 6.4Hz); 2.56-2.472(3 x 1H, m); 1.71(1H, H-15); 1.62(1H, H-12b); 1.141.54(23H, m); 1.06(1H, 16,); 0.93(3H, d, J=7.1Hz, H-21); 0.91(3H, d, J-6.4Hz, H20); and 0.89ppm (3H, t, J-7.1Hz, H-19). Mass Spectrum: FABMS: (M+H)+ 676(100), 658 (3), 500 (5), 324 (30), and 306m/e (11). TLC: Rf = 0.44 on C~s reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% panisaldehyde solution in methanol-sulfuric acid-acetic acid (85:5:10, v/v/v) and heating at 110~ for 10 min. References J.-A. Seo, J.-C. Kim, and Y.-W. Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 1003-1005 (1996). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.

Fumonisins, AALToxins, and Related Metabolites

585

Common/Systematic Name Fumonisin AK1 Mole.cular Formula/Molecular Weight C3oi-I45NO11; M W --- 6 0 4 . 3 6 9 6 9

OH

OH

MeC~NH2 II O

0

OH

Me

O..

tq

_..

O

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in non-polar solvents. Fungal Source v

Fusarium proliferatum (M- 1597).

Isolation/Purification Solid culture material was extracted with methanol-water (75:25, v/v) and filtered. The filtrate was diluted with water to a final methanol concentration of 30%. The diluted solution was chromatographed on a Bondapak Cls preparative column washed with water, methanol-water (50:50, v/v), and eluted with methanol-water (70:30, v/v). The methanol was removed from this fraction and the aqueous portion was applied to a Dynamax Cls column (Rainin Corp.) washed with acetonitrile-water (25:75, v/v). The fumonisin containing fractions were eluted with acetonitrile-water (70:30, v/v). The acetonitrile was removed in vacuo and the aqueous fraction was applied to a pBondapak cyano columrL The fumonisins were eluted with 1% pyridine in water. Final purification and separation of fumonisin B1 and fumonisin AK~ was achieved on a Cs column using a gradient from acetonitrile containing 0.1% acetic acid-water (20: 80, v/v) to aeetonitrile-water (45:55, v/v). Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoeneephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic.

586

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 13C NMR: (D20) 18.4, C-I; 52.3, C-2; 72.5, C-3; 42.1, C-4; 70.3, C-5; 39.2, C-6; 27.1, C-7; 27.3, C-8; 27.1, C-9; 71.4, C-10; 44.6, C-11; 28.6, C-12; 39.0, C-13; 79.4, C-14; 219.5, C-15; 44.8, C-16; 33.9, C-17; 31.3, C-18; 24.4, C-19; 15.6, C-20; 21.6, C-21; 19.7, C-22; 181.6, C-23; 38.2, C-24; 41.4, C-25; 39.3, C-26; 179.1, C-27; 176.2, C28; 175.9, C-33; and 24.5ppm, C-34'. Mass Spectrum: Negative-ion ESMS: Showed only a molecular anion at 603 daltons. References S. M. Musser, R. M. Eppley, E. P. Mazzola, C. E. Hadden, J. P. Shockcor, and G. E. Martin; Identification of an N-Acetyl Keto Derivative of Fumonisin B 1 in Corn Cultures ofFusarium proliferatum; J. Natural Prod., Vol. 58, pp. 1392-1397(1995).

21.

Fumonisins, AALToxins, and Related Metabolites

587

Common/Systematic Name AAL Toxin TAI Molecular Formula/Molecular Weight C2sHaTNO10; MW = 521.32000

OH OH

OH

1

NH2

OH

Me O~o O ~ ~ ~H

OH General Characteristics AAL toxin TAI was ninhydrin positive. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness m vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

588

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 1H NMR: (CD3OD) 3.05(1H, H-l); 2.82(1H, n-l'); 4.01(1H, H-2); 1.72(1H, n-3); 1.51(1H, H-3'); 3.67(1H, H-4); 3.44(1H, H-5), 1.36(1H, H-6); 1.68(1H, H-12), 5.12(1H, dd, J=3.5, 3.THz, H-13); 3.89(1H, dd, J=3.5, 3.THz, H-13); and 1.34ppm (1H, H-15). 13CNMR: (CD3OD) 47.9, C-l; 64.97, C-2; 70.46, C-4; 74.70, C-5; 74.46, C-13; and 76.30ppm C-14. Mass Spectrum: FABMS: (M+H)§ 522m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).

21. Fumonisins, AALToxins, and Related Metabolites

589

Common/Systematic Name AAL Toxin TA2 Molecular Formula/Molecular Weight C25I-I47NO10; MW = 521.32000

OH

NH2

O~ \

OH

O

OH

Me

OH

OH

/

Me

General Characteristics AAL toxin TA2 was ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

590

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 1H NMR: (CD3OD) 3.05(1H, H-l), 2.82(1H, H-I'); 4.01(1H, H-2); 1.72(1H, 5.2Hz, H-3); 1.51(1H, n-3'), 3.67(1H, n-4); 3.44(1H, n-5); 1.36(1H, n-6); 1.81(1H, H-12); 3.75,(1H, dd, J=3.5, 3.7Hz, H-13); 4.76(1H, dd, J=2.9, 8.0Hz, H-14); and 1.77ppm (1H, n-15). 13CNMR" (CD3OD) 47.9, C-l, 64.97, C-2, 70.46, C-4, 74.70, C-5, 69.16, C-13, and 81.72ppm C-14. Mass Spectrum: FABMS: (M+H)+ 522m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).

21. Fumonisins,AALToxins, and RelatedMetabolites

591

Common/Systematic Name AAL Toxin TB1 Molecular Formula/Molecular Weight C25H47NO9; MW = 505.32508

OH

NH2

OH

OH

Me 0 . . ~ 0

OH General Characteristics AAL toxin TB1 was ninhydrin positive. All five AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. Atter 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

592

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 1H NMR: (CD3OD) 3.03(1H, H-I); 2.82(1H, H-I'); 4.02(1H, H-2); 1.57(1H, H-3); 1.44(1H, H-3'); 3.79(1H, H-4); 1.58(1H, H-5); 1.68(1H, H-12); 5.11(1H, dd, J=3.5, 3.7Hz, H13); 3.39(1H, dd, J=3.5, 3.7I-Iz, H-14); and 1.37ppm (1H, H-15). 13CNMR: (CD3OD) 46.50, C-l, 66.27, C-2, 68.58, C-4; 74.62, C-13; and 77.30ppm C-14. Mass Spectrum: FABMS: (M+H) § 506m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).

21.

Fumonisins, AALToxins, and Related Metabolites

593

Common/Systematic Name AAL Toxin TB2 Molecular Formula/Molecular Weight CzsH47NO9; MW = 505.32508

\ OH

NH2

OH

0

Me

OH

/

/

Me

General Characteristics AAL toxin TB2 was ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. A_~er 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

594

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Dat.a. 1H NMR: (CD3OD) 3.03(1H, H-I); 2.82(1H, H-I'); 4.02(1H, H-2); 1.57(1H, H-3); 1.44(1H, H-3'); 3.79(1H, H-4); 1.58(1H, H-5); 1.81(1H, H-12); 3.76(1H, dd, J-3.5, 3.7Hz, H13); 4.77(1H, t, J=5.7I-Iz, H-14); and 1.74ppm (1H, H-15). 13CNMR: (CD3OD) 46.50, C-l 66.27, C-2; 68.58, C-4; 69.75, C-13; and 82.12ppm, C-14. Mass Spectrum: FABMS: (M+H)§ 506m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALT0xins Produced by Alternaria alternata f. sp. Lycopersici, J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).

21. Fumonisins, AALToxins, and Related Metabolites

595

Common/Systematic Name AAL Toxin TC1 Molecular Formula/Molecular Weight C25I--I4708; M W -- 4 8 9 . 3 3 0 1 7

OH

NH2

OH

Me

0

~

0

OH General Characteristics AAL toxin TC1 was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

596

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 1H ~ : (CD3OD) 3.01(1H, H-l); 2.75(1H, H-I'); 3.77(1H, H-2); 1.70(1H, H-3); 1.46(1H, H-3'); 1.67(1H, H-12); 3.39(1H, dd, .]=3.5, 3.7Hz, H-13); 5.02(1H, dd, J=3.5Hz, H14); and 1.37ppm (1H, H-15). Mass Spectrum: FABMS: (M+H)§ 490m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agile. Food Chem., Vol. 42, pp. 327-333(1994).

21. Fumonisins, AALToxins, and Related Metabolites

597

Common/Systematic Name AAL Toxin TC2

0 = = ~ \

Molecular Formula/Molecular Weight C25I--I47N08; M W -- 4 8 9 . 3 3 0 1 7

OH

NH2

OH

0/

Me

OH

Me

General Characteristics AAL toxin TC2 was not ninhydrin positive. All five AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay. Spectral Data 1H NMR: (CD3OD) 3.01(1H, H-I); 2.75(1H, H-I'); 3.77(1H, H-2); 1.70(1H, H-3); 1.46(1H, H-3'); 1.81(1H, H-12); 3.74(1H, H-13); 4.75(1H, t, J-5.7Hz, H-14); and 1.75ppm (1H, H-15).

598

21.

Fumonisins, AALToxins, and Related Metabolites

Mass Spectrum: FABMS: (M+H) + 490m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAI_Toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agile. Food Chem., vol. 42, pp. 327-333(1994).

21.

Fumonisins, AALToxins, and Related Metabolites

599

Common/Systematic Name AAL Toxin TD1 Molecular Formula/Molecular Weight C27Ha9NO10; MW = 547.33565

OH OH NHCMe II 0

OH Me O ~ j ~ O

OH General Characteristics AAL toxin TD1 was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp.. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

600

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 1H NMR: (CD3OD) 3.01(1H, H-l); 2.75(1H, n-l'); 3.77(1H, H-2); 1.70(1n, H-3); 1.46(1H, H-3'); 1.67(1H, H-12); 5.02(1H, dd, J=3.5, 3.7Hz, H-13); 3.39(1H, dd, .]=3.5, 3.7Hz, H-14); and 1.37ppm (1H, H-15). Mass Spectrum: FABMS: (M+H) § 548m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAL Toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., vol. 42, pp. 327-333(1994).

21.

Fumonisins, AALToxins, and Related Metabolites

601

Common/Systematic Name AAL Toxin TD2 Molecular Formula/Molecular Weight C27H49NO10; M W -- 5 4 7 . 3 3 5 6 5

ON

OH

NHCMe II 0

OH

0

Me

OH

Me

General Characteristics AAL toxin TD2 w a s not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source A lternaria alternata f. sp. Lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biologica/. Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

602

21.

Fumonisins, AALToxins, and Related Metabolites

Spectral Data 1H NMR: (CD3OD) 3.25(1H, dd, J=4.8, 5.2Hz,H-1); 3.12(1H, H-I'); 3.82(1H, m, J=2.2 2.8Hz, H-2); 1.48(1H, H-3); 1.46(1H, H-3'), 3.76(1H, H-4); 1.39(1H, H-5), 1.31(1H, H-12); 8.78(1H, H-13); 4.77(1H, t, J=5.7Hz, H-14); and 1.74ppm (1H, H-15). 13CNMR: (CD3OD) 46.13, C-l; 68.37, C-2; 69.49, C-4; 68.94, C-13; and 82.24ppm C-14. Mass Spectrum: FABMS: (M+H)+ 548m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilehrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata s sp. Lycopersici; J. Agile. Food Chem., vol. 42, pp. 327-333(1994).

21. Fumonisins, AALToxins, and Related Metabolites

603

Common/Systematic Name AAL Toxin TEl Molecular Formula/Molecular Weight C27H49NO9; MW-- 531.34073

OH

NHCMe

II 0

OH

Me

0...,~0

OH 0

OH General Characteristics AAL toxin TEl was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay. Spectral Data 1H NMR:

604

21.

Fumonisins, AALToxins, and Related Metabolites

(CDaOD) 3.26(1H, dd, J=4.0, 4.5Hz, H-l); 3.06(1H, H-I'), 3.60(1H, dd, J=2.9, 8.0Hz, H-2); 1.36(1H, H-3); 1.38(1H, n-3'); 1.60(1H, n-12); 5.1 l(1n, n-13), 3.36(1H, dd, J=3.5, 3.7Hz, H-14); and 1.36ppm (1H, H-15). 13CNMR: (CD3OD) 46.71, C-l; 68.49, C-2; 74.89, C-13, and 77.58ppm C-14. Mass Spectrum: FABMS: (M+H) § 532m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAL Toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agile. Food Chem., Vol. 42, pp. 327-333(1994).

21. Fumonisins, AALToxins, and Related Metabolites

605

.Common/Systematic Name AAL Toxin TE2 Molecular Formula/Molecular Weight C27H49NO9; M W -- 5 3 1 . 3 4 0 7 3

\

0=~

OH)OH

/

OH

0II

NHCMe

0

Me

OH

Me

General Characteristics AAL toxin TE2 was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic aeid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system.After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

606

21.

Fumonisins, AALToxins, and Related Metabolites

Soectral Data 1H NMR:

(CD3OD) 3.26(IH, dd, ,/=4.0,4.5I--Iz,H-I); 3.06(IH, H-I'); 3.60(IH, dd, J=2.9,

8.0Hz, H-2); 1.36(1H, H-3); 1.38(1H, H-3'); 1.81(1H, H-12); 3.77(1H, H-13), 4.78(1H, dd, J=2.9, 3.0Hz, H-14); and 1.74ppm (1H, H-15). 13CNMR: (CD3OD) 46.71, C-l; 68.49, C-2; 71.33, C-13 and 82.30ppm C-14. Mass Spectrum: FABMS: (M+H) + 532role. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAL toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., vol. 42, pp. 327-333(1994).

21. Fumonisins, AALToxins, and Related Metabolites

607

Common/Systematic Name Sphingofungin A Molecular Formula/Molecular Weight C21H41N306; M W = 431.29954

OH _

OH

_

HOOC

I

OH

OH

NHO(NH)NH2 Fungal Source

Aspergillusfumigatus Fres. (ATCC 20857 = MF 5038) was isolated from soil collected from a pasture in Young, Departmento Rio Negro, Uruguay.

Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase C~s HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. After elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column ofDowex 1 (CI) resin. The Dowex 1 eluant, containing sphingofungins B and C was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; A New Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Journal of Antibiotics, Vol. 45, pp. 861867(1992).

608

21.

Fumonisins, AALToxins, and Related Metabolites

F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

21.

Fumonisins, AALToxins, and Related Metabolites

609

Common/Systematic Name Sphingofungin B Molecular Formula/Molecular Weight C20H39NO6; M W -- 3 8 9 . 2 7 7 7 4

OH _

OH

_

HOOC

_

NH2 OH

OH

Fungal Source

Aspergillusfumigatus Fres. (ATCC 20857 = MF 5038) was isolated from soil collected from a pasture in Young, Departmento Rio Negro, Uruguay.

Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase ClS HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. Alter elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column ofDowex 1 (CI) resin. The Dowex 1 eluant, containing sphingofungins B and C was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; A New Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Journal of Antibiotics, Vol. 45, pp. 861867(1992). F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

610

21.

Fumonisins, AALToxins, and Related Metabolites

Common/Systematic Name Sphingofungin C Molecular Formula/Molecular Weight C22H43NOs; ~

OH

-- 4 0 1 . 3 1 4 1 2

OAc

_

_

NH2

OH

OH

Fungal Source

Aspergillusfumigatus Fres. (ATCC 20857 = MF 5038) was isolated from soil collected from a pasture in Young, Departmento Rio Negro, Uruguay.

Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase ClS HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. After elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column of Dowex 1 (C1-) resin. The Dowex 1 eluant, containing sphingofungins B and C, was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general, sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; ANew Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Journal of Antibiotics, Vol. 45, pp. 861867(1992). F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

21.

Fumonisins, AAL Toxins, and Related Metabolites

611

Common/Systematic Name Sphingofungin D Molecular Formula/Molecular Weight CzzH41NOs; MW = 447.28322

OH

OH _

HOOC

"

OH NHAc Fungal Source

Aspergillusfumigatus Fres. (ATCC 20857 = MF 5038) was isolated from soil collected from a pasture in Young, Departmento Rio Negro, Uruguay.

Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase Cls HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. After elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column ofDowex 1 (CI) resin. The Dowex 1 eluant, containing sphingofungins B and C,was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general, sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; A New Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Joumal of Antibiotics, Vol. 45, pp. 861867(1992). F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

612

21.

Fumonisins, AAL Toxins, and Related Metabolites

Common/Systematic Name Myriocin Molecular Formula/Molecul~ Weight C21H39NO6; M W -~- 401.27774

.oo 2 A 1

OH

HOH2C~ /

28

NH2 OH

O

General Characteristics Crystals from methanol; mp., 180-181~ ninhydrin test.

[a]D 24 W 10.3~ (c=0.386,

in MeOH). Positive

Fungal Source Myriococcum albomyces. Isolation/Purification See D. Kluepfel et. al., Journal of Antibiotics, Vol. 25, p. 109 (1972). Biological Activity Antifungal activity. Spectral Data IR:

(Nujol) Broad band in OH region, 1702, 1665, and 962cm"1. 1H NMR: (CDCI3) (Tetraacetate derivative) 6.30(1H, NH); 4.51(s); 5.79(d); 4.74(m); 5.5(q) and 0.88ppm. Mass Spectrum: LREIMS: 383(M + - 18) and 256m/e (base peak, M § - [ 127 + 18]). Reference J. F. Bagli, D. Kluepfel, and M. St-Jacques; Elucidation of Structure and Stereochemistry ofMyriocin. A Novel Antifungal Antibiotic; J. Organ. Chem., Vol. 38, pp. 1253-1260 (1973).

Ochratoxins and Related Metabolites Ochratoxin A Ochratoxin B 4-Hydroxyochratoxin A Ochratoxin C Mellein 4-Hydroxymellein

613

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22.

Ochratoxins and Related Metabolites

615

Common/Systematic Name Ochratoxin A 7-Carb•xy-5-ch••r•-8-hydr•xy-3•4-dihydr•-3•-methy•is•c•umarin-7-L-•3-pheny•a•anine Molecular FormulaJMolecular Weight C20H]sO6NC1, MW = 403.0822

~ 17

22

COOH I 0

OH

N"~"~ ,'~ l 6L I.,L H "~1o~

CI General Characteristics Crystals from benzene; mp., 94-96~ (c = 1.1, in chloroform).

0 "O ~ 3..L'"'Me "~H

Crystals from xylene; mp., 169~

[~]D25 -118 ~

Fungal Source Aspergillus ochraceus (NRRL 3174); A. sulphureus (NRRL 4077); A. melleus (NRRL 3519; 3520); Penicillium viridicatum (ATCC 18411). Biological Activity LDs0 in weanling rats dosed orally was 22mg/kg, in trout IP, 3.0mg/kg. Spectral Data UV: Em~ 215 (36,800) and 333nm (6,400). 1H NMR:

(CDC13) 4.76 (1H, H-3), 2.97 (2H, H-4); 8.07 (1H, H-6); 8.58 (1H, d, J= 7.3Hz, NH12); 4.76 (1H, H-13), 3.18 (2H, H-14); 7.25 (5H, H-16-H-20); and 1.46ppm (3H, d, J= 6.1Hz, H-21). 13CNMR: (CDC13) 169.1, s, C-l; 76.2, d, C-3; 32.4, t, C-4; 122.3, s, C-5; 136.8, d, C-6; 120.9, s, C-7; 163.5, s, C-8; 112.1, s, C-9; 142.6, s, C-10; 159.1, s, C-11; 54.3, d, C-13; 37.4, t, C-14; 137.7, s, C-15; 129.1, d, C-16, C-20; 130.0, d, C-17, C-19, 127.4, d, C18; 20.8, q, C-21; and 173.1ppm, s, C-22. Mass Spectrum: LREIMS: 256m/e (M) +. TLC Data Silica gel; benzene-acetic acid(3:1, v/v), Rf = 0.50; detected as a green fluorescent spot under UV light.

616

22.

Ochratoxins and Related Metabolites

References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, S. Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox, Ochratoxins; In Handbook of Toxic Fungal Metabolites, Academic Press, Inc., New York, N.Y., pp. 137-140 (1981).

22.

Ochratoxins and Related Metabolites

617

Common/Systematic Name Ochratoxin B 7-Carboxy-8-hydroxy-3,4-dihydro-3R-methylisocoumarin-7-L-13-phenylalanine Molecular Formula/Molecular Weight C20H1906N; ~

-- 3 6 9 . 1 2 1 2 4 22

1,

COOH I

,-----,

H

0

OH

0

~ , o ~

General Characteristics Crystals from acidic methanol; mp., 221 ~ (c=0.29, in MeOH).

~H [•]D25 -35 ~

(c=0.15, in EtOH);

[a]D 25 - 5 6 ~

Fungal Source

Aspergi llus ochraceus.

Biological Activity Ochratoxin B is considerably less toxic than ochratoxins A or C. The LDs0 in day-old chicks was dosed orally was 54mg/kg while ochratoxin A was 3.3-3.9mg/kg in the same assay. Spectral Data UV: EtOH max

218(37,200) and 318nm (6,900).

1H N-IV[R:

(CDC13) 4.90(1H, H-3); 3.10(2H, H-4); 8.21(d, J=6.8Hz, H-5); 7.02(1H, d, J=6.0Hz, H-6); 8.64(1H, d, J=6.1Hz, NH-12); 4.90(1H, H-13); 3.10(2H, H-14); 7.28(5H, H-16H-20); and 1.45ppm (3H, d, J=5.4Hz, H-21). 13C NMR: (CDCI3) 168.7, s, C-l; 75.1, d, C-3; 33.1, t, C-4; 117.3, d, C-5; 135.5, d, C-6; 117.9, s, C-7; 162.0, s, C-8; 107.7, s, C-9; 142.7, s, C-10; 158.9, s, C-11; 52.8, d, C-13; 36.3, t, C-14; 137.0, s, C-15; 128.2, d, C-16, C-20; 127.0, d, C-17, C-19; 125.5, d, C-18; 19.3, q, C-21; and 171.6ppm, s, C-22. Mass Spectrum: LREIMS: 369role (M) +.

618

22.

Ochratoxins and Related Metabolites

TLC Data Silica gel; benzene-acetic acid (4:1, v/v), Re = 0.35, detected as a blue fluorescent spot under UV light. References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 (1981).

22. Ochratoxins and Related Metabolites

619

Common/Systematic Name 4-Hydroxyochratoxin A 7-Carboxy-5-chloro-4,8-dihydroxy-3,4-dihydro-3R-methylisocoumarin-7-L-13phenylalanine Molecular Formula/Molecular Weight C20HlaOTNC1, MW = 419.07718 22

17

COOH

is

13

12 0 l

8iHO ,L,

I..L Cl

o

3~.,,,,Me OH

General Characteristics Colorless crystals from benzene; mp., 216-218~ Fungal Source Penicillium viridicatum (ATCC 18411).

Biological Activity 4-Hydroxyochratoxin A was excreted in urine of male Wister rats dosed with ochratoxin A (IP). 4-Hydroxyochratoxin A had no effect when dosed to rats at a level of 40mg/kg; ochratoxin A at this level caused 100% mortality. Therefore, 4-hydroxyochratoxin A may be a detoxification product in animals dosed with ochratoxin A Spectral Data UV: ~k EtOH max

213 (32,500) and 334nm (6,400).

IR:

(CH3C1) 3380, 3000, 2500, 1723, 1678, 1655, and 1535cm-1. 1H NMR:

(CDC13) 4.80(1H, d, J=7.2Hz, H-3); 5.11(1H, d, J=2.0Hz, H-4); and 8.70ppm (1H, H-6). Mass Spectrum: LREIMS: 419m/e (M) +. TLC Data Silica gel, benzene-acetic acid (3:1, v/v), Re= 0.25; detected as a green fluorescent spot under UV light.

620

22.

Ochratoxins and Related Metabolites

References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 (1981).

22.

Ochratoxins and Related Metabolites

621

Common/Systematic Name Ochratoxin C Molecular Formula/Molecu!ar W~/ight C22H2206NCI; M'W = 431.11357 22

COOC2H5 '~

is

13

12 0

8OH

1 oL E

H

y~o~

0

~H

Cl General Characteristics Amorphous compound. Fungal Source Aspergillus ochraceus. Biological Activity Originally thought to be relatively nontoxic; however, it was later reported to be comparable to ochratoxin A. Spectral Data UV:

~

EtOH max

214 (30,000), 333 (7,000) and 378nm (2,050).

IR:

(CH3C1) 1730 and 1680em1. Mass Spectrum: LREIMS: 43 lm/e (M) +. TLC Data Silica gel; benzene-acetic acid (25:1, v/v), Re = 0.55; detected as a light green fluorescent spot under UV light. References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 (1981).

622

22.

Ochratoxins and Related Metabolites

Common/Systematic Name Mellein; Ochracin (-)3,4-Dihydro-8-hydroxy-3-methylisocoumarin Molecular Formula/Molecular Weight CloHloO3; MW

OH 8

~

-- 1 7 8 . 0 6 2 9 9

0

9

0 g

,, lo g

A,

H

General Characteristics Crystals (subl.); mp., 54-55~ Fungal Source Aspergillus melleus; A. ochraceus. Biological Activity Biological activity unknown. Spectral Data UV: Ef~ 212 (20,000), 246 (6,500) and 314nm (4,100). 1H NMR: (CDCI3) 4.76(1H, dd, J-7.0, 7.0Hz, H-3); 2.95 (2H, d, J=7.0Hz, H-4); 6.72 (1H, d, J-7.5Hz, H-5). 7.44 (1H, dd, J-7.5, 8.0Hz, H-6); 6.91 (1H, d, J=8.0Hz, H-7); 1.55 (d, J-7.0Hz, H-11); and 11.07ppm (1H, OH-8). ~3CNMR: (CDC13) 169.8, C-I; 76.1, C-3; 34.7, C-4; 117.8, C-5; 136.0, C-6; 116.2, C-7; 162.1, C-8; 108.2, C-9; 139.2, C-10; and 20.8ppm, C-11. Mass Spectrum: LREIMS: 178(M)+ (100%), 161,149, and 134m/e. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid (5:4:1, v/v/v), Rf = 0.82; detected as a blue fluorescent spot under UV light. References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 ( 1981).

22.

Ochratoxins and Related Metabolites

623

Common/Systematic Name 4-Hydroxymellein (-)3,4-Dihydro-4,8-dihydroxy-3-methylisocoumarin Molecular Formula/Molecular Weight CloHloO4;

OH

C

~

L

MW'

= 194.05791

0

0 -

~.,,,,Me

ox. ". General Characteristics Crystals from chloroform-methanol; mp., 131-132~

[ a ] D 25 -

40~ (c: 1.0, in CHC13).

Fungal Source Aspergillus melleus; A. ochraceus; A. oniki; Lasiodiplodia theobromae (cis-4hydroxymellein); Apiospora camptospora. Biological Activity Biological activity unknown. Spectral Data UV: EtOH ~ max

247(5,300) and 315nm (4,200).

1H NMR: (CDC13) 4.63(1H, H-3); 4.63(1H, H-4), 7.04(1H, d, J=7.5Hz, H-5), 7.57(1H, H-6); 7.02(1H, d, J=8.2Hz, H-7); 1.52(d, J=6.5Hz, H-11); 2.16(1H, H-4OH); and 11.01ppm (1H, H=8OH). 13CNMR: (CDC13) 168.1, C-I; 79.7, C-3; 69.1, C-4; 117.7, C-5; 136.4, C-6; 115.9, C-7; 161.7, C-8; 106.5, C-9; 140.9, C-10; and 29.6ppm, C-11.

Mass Spectrum:

LREIMS: 194(M)+ (100%), 161,149, and 134m/e.

TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid (5:4:1, v/v/v), Re = 0.74; detected as a blue fluorescent spot under UV light.

624

22.

Ochratoxins and Related Metabolites

References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox, Ochratoxins; In Handbook of Toxic Fungal Metabolites, Press, Inc., New York, N.Y., pp. 137-140 (1981).

Miscellaneous Metabolites

4,5,10,11-Tetrahydroxybisboline 24-Ethyllanosta-8,24(24')-diene-313,22~-diol 25-Methylpisolactone 24-Methyllanosta-8,24'-diene-313,22~-diol 24-Methyllanosta-8,24(24')-diene-313,22~-diol 3a-Acetoxylanosta-8,24-dien-21-oic acid 21-Hydroxylanosta-8,24-dien-3-one 3a-(4-Carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oicacid Isofunicone Xylobovatin Deacetyl-19,20-epoxycytochalasin Q 19,20-Epoxycytochalasin Q Deacetyl-19,20-epoxycytochalasin C 19,20-Epoxycytochalasin C Fusaproliferin Retigeranic Acid Moniliformin Cyclopiazonic Acid

625

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23.

Miscellaneous Metabolites

627

Common/Systematic Name 4,5,10,11-Tetrahydroxybisboline Molecular Formula/Molecular Weight

H~

C15H2804; M W -- 272.19876

OH

.........

OH "*"Hil~~o H

General Characteristics Colorless glass. Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit overnight, filtered, and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum, and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated, and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which resulted in purified 4,5,10,11-tetrahydroxybisboline. Fungal Source

Fusarium sambucinum.

Soectral Data UV:

~, maxA~176 195nm (e=5,000, ~ to ~* transition). IR:

(film) 3376(OH), 1370, 1158, 1124, 1048, and 1050cm-1 (-C-O-C-). 1H NMR: (CDC13) 1.17(3H, s, H-12), 1.22(3H, s, H-13), 1.27(3H, s H-14), 1.47(1H, m, H-9a), 1.57(1H, m, H-3a), 1.59(1H, m, 2a), 1.64(1H, m, H-9b), 1.69(1H, m, H-6a), 1.74(1H, m, H-2b), 1.78(1H, m, H-3b), 1.92(11-1, m, H-6b), 2.10(1H, m, H-8a), 2.30(1H, m, H-l); 2.36(1H, m, H-8b); 3.38(1H, dd, J=l.7, 10.4Hz, H-10), 3.64(1H, b t, J=3.0Hz, H-5); 4.79(1H, b m, H-15a); and 4.84ppm (1H, m, H-15b).

628

23.

Miscellaneous Metabolites

13CNMR: (acetone-d6) 25.1 (q, C-12); 25.9(q, C-13); 27.7(t, C-2); 28.1(q, C-14); 31.0(t, C-6); (t, C-8), 34.6(t, C-3), 35.8(t, C-9), 37.1(t, C-l); 70.8(s, C-4), 72.8(s, C-11), 74.1(d, El0); 78.7(d, C-5); 107.2(t, C-15); and 156.1ppm (s, C-7). Mass Data: LREIMS: 254(M + - H20, 1%), 239(2), 236(3), 218(2), 125(21), 108(64), 81(89), and 43m/e (100); exact mass for C~5H2603 (M + - 1-120); calcd 254.188; found 254.190m/e. Reference D. R. Sanson, D. G. Codey, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucmum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989).

23.

Miscellaneous Metabolites

629

Common/Systematic Name 24-Ethyllanosta-8,24(24')-diene- 313,22~-diol Molecular Formula/Molecular Weight C32H5402; M W = 470.41238

OH

24'

21 .......

s HO

/28

29

General Characteristics Crystals; mp., 170-180~

[ a i D 24 + 3 9 . 2 7 ~

(c=0.45, in CHC13).

Fungal Source

Pisofithus tinctorius, a commercially important ectomycorrhizal fungus that is considered

to have a broad host range. Isolation/Purification Freeze-dried P. tinctorius myeelia from submerged cultures were powdered in liquid N2 and extracted three times with ethyl ether. The combined extracts were evaporated to dryness yielding a pale yellow solid containing mainly a mixture of triterpenoids. The solid was redissolved in 2-propanol and centrifuged. Separation and purification of the triterpenoids was carried out by repeated chromatography on a reversed-phase (Cs) HPLC column (PrepPack Bondapak 25 x 200 mm) using 80% aqueous methanol (adjusted to pH 3 with formic acid) as eluting solvent at a flow rate of 10ml/minute and detection at 210nm. Spectral Data UV:

End absorption.

630

23.

Miscellaneous Metabolites

1H NE/IR: (CDC13) 1.72, 1.23(2H, H-I); 1.67, 1.58(2H, H-2), 3.24(1H, dd, J=l 1.6, 4.6Hz, H3); 1.05(1H, H-5); 1.68, 1.50(2H, H-6); 2.04, 2.04(2H, H-7), 2.01, 2.01(2H, H-11); 1.78, 1.67(2H, n-12); 1.61, 1.20(2H, n-15); 2.00, 1.35(2H, n-16); 1.92(1H, n-17), 0.70(3H, s, n-18); 0.99(3H, s, n-19); 1.42(1H, H-20); 0.91(3H, d, J=6.6Hz, n-21); 3.77(1H, ddd, J=8.6, 4.6, 1.SHz, H-22); 2.05, 2.05(2H, H-23); 2.85(1H, septet, J=7.0Hz, H-25), 1.06(3H, d, J=7.0Hz, H26"); 1.00(3H, d, J=6.9Hz, H-27"); 5.25(1H, q, J=6.8Hz, H-24'); 1.00(3H, s, H-28); 0.81(3H, s, H-29); 0.91(3H, s, H-30); and 1.65ppm (3H, d, J=6.8Hz). * Assignmems may be reversed. 13CNMR: (CDC13) 35.6, C-l; 27.9, C-2; 79.0, C-3; 38.9, C-4; 50.4, C-5; 18.3, C-6; 26.5, C-7; 134.4, C-8; 134.5, C-9; 37.0, C-10; 21.0, C-11; 21.0, C-12; 44.5, C-13; 49.9, C-14; 30.8, C-15; 27.7, C-16; 47.2, C-17; 15.6, C-18; 19.1, C-19; 41.3, C-20; 12.0, C-21; 71.0, C-22; 38.7, C-23; 142.4, C-24; 28.6, C-25; 21.2, C-26; 21.0, C-27; 120.6, C-24'; 28.0, C-28, 15.4, C-29; 24.4, C-30; and 12.9ppm C-31. Mass Spectrum: HREIMS: 470.4096(M+, calcd for C32H5402,470.4124, 16%), 455[M - Me]+ (6%), 437[M - Me - H20] + (8), 419(3), 372.3037[calcd for C25H4002,372.3029] (36), 357.2782[calcd for C2,I-I3702, 357.2794] (100), 344(8), 339.2664[calc for C24I-I350, 339.2688] (32), 329(5), 321(6), 314(9), 311(11), 299(12), 281(9), 215(6), 187(8), 161(7), and 109role (9). Reference A. Baumbert, B. Schumann, A. Porzel, J. Schmidt, and D. Strack; Triterpenoids from Pisolithus tinctorium Isolates and Ectomycorrhizas; Phytochemistry, Vol. 45, pp. 499504(1997).

23.

Miscellaneous Metabolites

631

Common/Systematic Name 25-Methylpisolactone (22S)-24-Methyllanosta-8-ene-22,24'-epoxy-313-01-24'-one Molecular Formula/Molecular Weight C32H5203; M W -- 484.39165 0

0

27 18 .....

" 26

28

29

General Characteristics Colorless needles; mp., 313-316~

[a]D 24 +38.7 ~ (c=0.23, in CHC13).

Fungal Source

Pisolithus tinctorius, a commercially important ectomycorrhizal fungus that is considered

to have a broad host range. Isolation/Purification Freeze-dried P. tinctorius mycelia from submerged cultures were powdered in liquid N2 and extracted three times with ethyl ether. The combined extracts were evaporated to dryness yielding a pale yellow solid containing mainly a mixture oftriterpenoids. The solid was redissolved in 2-propanol and centrifuged. Separation and purification of the triterpenoids was carried out by repeated chromatography on a reversed-phase (Cs) HPLC column PrepPack Bondapak (25 x 200 mm) using 80% aqueous methanol (adjusted to pH 3 with formic acid) as eluting solvent at a flow rate of 10ml/minute and detection at 210nm. Spectral Data UV~

End absorption.

632

23.

Miscellaneous Metabolites

~HNMR: (CDCI3) 1.72, 1.23(2H, H-I); 1.68, 1.58(21-1,H-2); 3.24(11-1, dd, J=l 1.4, 4.6Hz, H3); 1.05(1H, H-5), 1.68, 1.50(2H, H-6); 2.03, 2.03(2H, H-7), 2.03, 2.02(2H, H-11); 1.77, 1.65(2H, H-12); 1.61, 1.23(2H, H-15); 2.01, 1.34(2H, H-16); 1.96(1H, H-17); 0.70(3H, s, H-18); 0.98(3H, s, n-19); 1.58(1H, H-E0); 0.94(3H, d, J=6.7Hz, H-E1), 4.39(1H, ddd, J=10.7, 6.1, 1.5Hz, H-22); 2.01, 1.92(2H, H-23); 2.46(1H, dd, J=12.6, 8.9Hz, H-24); 1.07(3H, s, H-26"); 1.07(3H, s, H-27"); 1.00(3H, s, H-28); 0.81(3H, s, H-29); 0.89(3H, s, H-30); and 1.07ppm (3H, s, H-31). * Assignments may be reversed. 13CNMR: (CDC13) 35.6, C-I; 27.9, C-2; 78.9, C-3; 38.9, C-4; 50.4, C-5; 18.2, C-6; 26.5, C-7; 134.2, C-8; 134.7, C-9; 37.0, C-10; 21.0, C-11; 30.9, C-12; 44.6, C-13; 49.8, C-14; 30.8, C-15; 27.9, C-16; 47.4, C-17; 15.6, C-18; 19.1, C-19; 39.8, C-20; 12.2, C-21; 79.4, C-22; 28.5, C-23; 50.6, C-24; 31.8, C-25; 29.6, C-26; 29.6, C-27; 177.2, C-24'; 28.0, C-28; 15.4, C-29; 24.2, C-30, and 29.6ppm, C-31. Mass Spectrum: HREIMS: 484.3905[calcd for C32H5203,484.3917] (28), 469[M - Me] + (80), 451.3542[M-Me- H20] § [calcd for C3~H4702,451.3576] (100), 329(7), 299(9), 281(14), 227(9), 215(8), 213(8), 187(10), 161(10), 159(9), 147(6), 135(17), 121(10), 95(15), and 57m/e (18). Reference A. Baumbert, B. Schumann, A. Porzel, J. Schmidt, and D. Strack; Triterpenoids from Pisolithus tinctorium Isolates and Ectomycorrhizas; Phytochemistry, Vol. 45, pp. 499504(1997).

23.

Miscellaneous Metabolites

633

Common/Systematic Name 24-Methyllanosta-8,24(24 ')-diene-313,22~-diol Molecular Formula/Molecular Weight C31H5202, M W ' = 456.39673 24'

21 ....

28

OH ZZ~

j

27

2g

General Characteristics Crystals from isopropanol, mp. 177-179~

[ a ] D 24 + 4 9 . 7 ~

(c=0.20, in CHCI3).

Fungal Source

Pisolithus tinctorius, a commercially important ectomycorrhizal fungus that is considered

to have a broad host range. Isolation/Purification Freeze-dried P. tinctorius mycelia from submerged cultures were powdered in liquid N2 and extracted three times with ethyl ether. The combined extracts were evaporated to dryness yielding a pale yellow solid containing mainly a mixture oftriterpenoids. The solid was redissolved in 2-propanol and centrifuged. Separation and purification of the triterpenoids was carried out by repeated chromatography on a reversed-phase (Cs) HPLC PrepPack Bondapak column (25 x 200 mm) using 80% aqueous methanol (adjusted to pH 3 with formic acid) as eluting solvent at a flow rate of 10ml/minute and detection at 210nm. Spectral Data UV:

End absorption.

634

23.

Miscellaneous Metabolites

1H N1VIR: (CDC13) 1.72, 1.23(2H, H-I); 1.66, 1.58(2H, H-2); 3.239(1H, dd, J=l 1.7, 4.5Hz, H3), 1.04(1H, H-5), 1.68, 1.49(2H, H-6); 2.03, 2.03(2H, H-7); 2.03, 2.01(2H, H-11); 1.79, 1.68(2H, H-12); 1.61, 1.21(2H, H-15), 2.00, 1.36(2H, H-16); 1.91(1H, H-17); 0.70(3H, s, H-18); 0.99(3H, s, H-19); 1.43(1H, H-20), 0.92(3H, d, J=6.6Hz, H-21); 3.81(1H, ddd, J=9.4, 3.9, 1.4Hz, H-22); 2.26(1H, dd, J=14.3, 9.4Hz, H-23); 2.10(1H, dd, J=14.3, 3.9Hz, H-23); 2.24(1H, septet, J=6.8Hz, H-25); 1.06(3H, d, J=6.8Hz, H26"); 1.04(3H, d, J=6.9Hz, H-27"); 4.89(1H, dd, ,/=1.3, 1.3Hz, H-24'); 4.79(1H, dd, J=2.4, 1.3Hz, H-24'); 1.00(3H; s, H-28); 0.81(3H, s, H-29); and 0.92ppm (3H, s, H-

30). * Assignments may be reversed. 13CNMR: (CDC13) 35.6, C-I; 27.9, C-2; 79.0, C-3; 38.9, C-4; 50.4, C-5; 18.3, C-6; 26.5, C-7; 134.4, C-8; 134.6, C-9; 37.0, C-10; 21.0, C-11; 31.0, C-12; 44.5, C-13; 49.9, C-14; 30.8, C-15; 27.7, C-16; 47.1, C-17; 15.6, C-18; 19.1, C-19; 41.1, C-20; 12.0, C-21; 70.8, C-22; 41.2, C-23; 153.4, C-24; 33.5, C-25; 21.7, C-26; 22.0, C-27; 109.2, C-24'; 28.0, C-28; 15.4, C-29; and 24.3ppm C-30. Mass Spectrum: LREIMS: 456[M] § (32%), 441[M-Me] § (5), 423[M-Me-H20] § (9), 405(4), 372(8), 357(100), 344(6), 339(35), 329(5), 321(9), 314(6), 311(13), 299(11), 281(10), 215(6), 187(7), 161(6), 135(6), and 95role (8). Reference A. Baumbert, B. Schumann, A. Porzel, J. Schmidt, and D. Strack, Triterpenoids from Pisolithus tinctorium Isolates and Ectomycorrhizas; Phytochemistry, Vol. 45, pp. 499504(1997).

23.

Miscellaneous Metabolites

635

Common/Systematic Name 3 t~-Acetoxylanosta-8,24-dien-21-oic acid Molecular Formula/Molecular Weight C32H5004; MW = 498.37091

HO0

:

AcO

General Characteristics White powder; mp., 192-194~

[~]D 21 + 1 4 ~

(c=l.0, in CHC13).

Fungal Source Fomitopsispinicola, a wood-rotting fungus growing on coniferous and broad-leafed trees in Europe. Isolation/Purification Lyophilized fruiting bodies of F. pinicola were extracted successively with methylene chloride and methanol (3x). The methylene chloride extract was separated on a silica gel column Si 60 with a step-gradient of petrol-ethyl acetate (95:5-9:1-7:1-5:1-3:1-1:1, v/v), ethyl acetate and finally methanol to give 19 fractions (A-S). Fraction I was submitted to low pressure liquid chromatography (LPLC) on silica gel with chloroformisopropanol (98:2, v/v) giving two fractions (I1 and 12). Fraction I1 was further purified on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) to yield the non-UV absorbing metabolite 3a-acetoxylanosta-8,24-dien-21-oic acid. Fraction H was submitted to LPLC with petrol-ethyl acetate (5:1, v/v) which gave five fractions (H1-H5). Further separation of fraction H4 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) afforded 21-hydroxylanosta-8,24-dien-3-one. Fraction N was submitted to medium pressure liquid chromatography on DIOL with a stepwise gradient of n-hexanechloroform (2:1-1:1, v/v) affording 3 fractions (N1-N3). A further purification of fraction N3 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) yielded 3tt-(4carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oic acid as a white non-UV absorbing powder. Biological Activity Antimicrobial activity against Bacillus subtilis in a TLC bioassay.

636

23.

Miscellaneous Metabolites

Spectral Data IR:

3600-2750, 2920, 1715, and 1680cmq. 1H NMR:

(CDC13) 0.74(3H, s, H-18); 0.83(3H, s, H-29); 0.88(3H, s, H-28); 0.92(3H, s, H-30); 0.95(3H, s, H-19); 1.56, 1.65(each 3H, s, H-26, H-27); 2.04(3H, s, acetoxy); 4.65(1H, br s, H-3); and 5.07ppm (1H, t, H-24). 13C NMR: (CDC13) 30.3, C-l; 23.4, C-2; 77.9, C-3; 36.8, C-4; 45.2, C-5; 17.9, C-6; 26.9, C-7; 134.4, C-8; 133.8, C-9; 36.9, C-10; 20.8, C-11; 28.9, C-12; 44.2, C-13; 49.5, C-14; 30.8, C-15; 27.0, C-16; 47.1, C-17; 16.0, C-18; 18.8, C-19; 47.7, C-20; 183.3, C-21; 32.4, C-22; 25.9, C-23; 123.6, C-24; 132.2, C-25; 17.6, C-26; 25.7, C-27; 21.8, C-28; 27.5, C-29; 24.3, C-30; 171.0, CH3C=O; and 21.3ppm, CH3C=O. Mass Spectrum: LREIMS: 498(M) + (45%), 483(18), 437(17), 423(100), 281(27), 187(23), and 69m/e (23). D/CI-MS: 516[M + NH4]§ (100%), 499[M + H] § (6), and 472m/e (21). TLC Data Ethyl acetate-petrol (1:1, v/v), Rf= 0.54; ethyl acetate-petrol (1:3, v/v), Re = 0.24. Reference A. C. Keller, M. P. Maillard, and K. Hostettmann; Antimicrobial Steroids from the Fungus Fomitopsis pinicola; Phytochemistry, Vol. 41, pp. 1041-1046(1996).

23.

Miscellaneous Metabolites

637

Common/Systematic Name 21-Hydroxylanosta-8,24-dien-3-one Molecular Formula/Molecular Weight C30H4sO2; MW = 440.36543 HOH2C .....

General Characteristics White powder; mp., 92-96~

[a]D 21 + 5 9 ~

(c=l.0, in CHC13).

Fungal Source

Fomitopsis pinicola, a wood-rotting fungus growing on coniferous and broad-leafed trees in Europe.

Isolation/Purification Lyophilized fruiting bodies ofF. pinicola were extracted successively with methylene chloride and methanol (3x). The methylene chloride extract was separated on a silica gel column Si 60 with a step-gradient of petrol-ethyl acetate (95:5-9:1-7:1-5:1-3:1-1:1, v/v), ethyl acetate and finally methanol to give 19 fractions (A-S). Fraction I was submitted to low pressure liquid chromatography (LPLC) on silica gel with chloroformisopropanol (98:2, v/v) giving two fractions (I1 and 12). Fraction I1 was further purified on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) to yield the non-UV absorbing metabolite 3a-acetoxylanosta-8,24-dien-21-oic acid. Fraction H was submitted to LPLC with petrol-ethyl acetate (5:1,v/v),which gave five fractions (H1-H5). Further separation of fraction H4 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) afforded 21-hydroxylanosta-8,24-dien-3-one. Fraction N was submitted to medium pressure liquid chromatography on DIOL with a stepwise gradient of n-hexanechloroform (2:1-1:1, v/v) affording 3 fractions (N l-N3). A further purification of fraction N3 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) yielded 3a-(4carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oic acid as a white non-UV absorbing powder. Biological Activity Antimicrobial activity against Bacillus subtilis in a TLC bioassay.

638

23.

Miscellaneous Metabolites

Spectral Data IR:

3250, 2905, and 1685cm"1. 1H NMR: (CDC13) 0.69(3H, s, H-18); 0.86(3H, s, H-30), 1.01(3H, s, H-28), 1.04(3H, s, H-29); 1.07(3H, s, H-19); 1.56, 1.63(each 3H, s, H-26, H-27); 3.65(2H, m,H-21); and 5.07ppm (1H, t, H-24). 13CNMR: (CDC13) 35.9, C-l; 34.4, C-2; 217.0, C-3; 47.2, C-4; 51.0, C-5; 19.3, C-6; 26.2, C-7; 133.0, C-8; 135.1, C-9; 36.8, C-10; 20.9, C-11; 29.6, C-12; 44.1, C-13; 49.8, C-14; 30.7, C-15; 27.5, C-16; 44.2, C-17; 16.0, C-18; 18.6, C-19; 42.7, C-20; 62.3, C-21; 30.3, C-22, 24.8, C-23; 124.8, C-24; 132.2, C-25; 17.6, C-26; 25.6, C-27; 21.2, C-28, 26.0, C-29; and 24.3ppm, C-30. Mass Spectrum: LREIMS: 440[M]+ (100%), 425(61), 407(54), 271(16), 257(19), 245(25), 109(63), and 69m/e (23). D/CI-MS: 458[M + NH4]+ (100). TLC Data Ethyl acetate-petrol (1:3, v/v), Re= 0.25. Reference A. C. Keller, M. P. Maillard, and K. Hostettmann; Antimicrobial Steroids from the Fungus Fomitopsis pinicola; Phytochemistry, Vol. 41, pp. 1041-1046(1996).

23.

Miscellaneous Metabolites

639

Common/Systematic Name 3 tt-(4-Carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21 -oic acid Molecular Formula/Molecular Weight C37H5807; M - ~ = 614.41825

HOOC .....

.0 Me

OH .0

M e O ~ ~ ~ ' ~ O

General Characteristics White powder; mp., 147~

,.....

[~]D 21 + 5 ~

(c=l.0, in CHC13).

Fungal Source

Fomitopsispinicola, a wood-rotting fungus growing on coniferous and broad-leafed trees in Europe.

Isolation/Purification Lyophilized fruiting bodies ofF. pinicola were extracted successively with methylene chloride and methanol (3x). The methylene chloride extract was separated on a silica gel column Si 60 with a step-gradient of petrol-ethyl acetate (95:5-9:1-7:1-5:1-3:1-1:1, v/v), ethyl acetate and finally methanol to give 19 fractions (A-S). Fraction I was submitted to low pressure liquid chromatography (LPLC) on silica gel with ehloroformisopropanol (98:2, v/v) giving two fractions (I1 and I2). Fraction Ilwas further purified on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) to yield the non-UV absorbing metabolite 3t~-acetoxylanosta-8,24-dien-21-oic acid. Fraction H was submitted to LPLC with petrol-ethyl acetate (5:1, v/v) which gave five fractions (H1-H5). Further separation of fraction H4 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) afforded 21-hydroxylanosta-8,24-dien-3-one. Fraction N was submitted to medium pressure liquid chromatography on DIOL with a stepwise gradient of n-hexanechloroform (2:1-1:1 v/v) affording 3 fractions (N1-N3). A further purification of fraction N3 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) yielded 3t~-(4carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oic acid as a white non-UV absorbing powder. Biological Activity Antimicrobial activity against Bacillus subtilis in a TLC bioassay.

640

23.

Miscellaneous Metabolites

Spectral Data 1HNMR: (pyridine-ds) 0.86(3H, s, H-18); 0.91(3H, s, H-29); 0.95(3H, s, H-28); 0.99(3H, s, H30); 1.05(3H, s, H-19); 1.62, 1.66(each 3H, s, H-26, H-27); 1.66(3H, s, 3'-methyl); 2.97(2H, s); 3.01(d, J=13.9Hz); 3.61(3H, s, 5'-methoxy); 4.93(1H, s, H-3); and 5.32ppm (1H, t, H-24).

13C NMR: (pyridine-ds) 30.7, C-l; 23.5, C-2; 78.1, C-3; 36.8, C-4; 45.8, C-5; 18.2, C-6; 26.7, C-7; 134.2, C-8; 134.1, C-9;.37.1, C-10; 21.1, C-11; 29.2, C-12; 44.8, C-13; 49.8, C14; 30.9, C-15; 27.4, C-16; 47.6, C-17; 16.3, C-18; 19.0, C-19; 49.0, C-20; 178.6, C21; 33.2, C-22; 26.2, C-23; 124.8, C-24; 131.7, C- 25; 17.6, C-26; 25.7, C-27; 21.8, C-28; 27.5, C-29; 24.2, C-30; 171.8, C-I'; 45.9, C-2'; 69.8, C-3'; 28.4, C-3' methyl; 46.3, C-5'; 171.2, C-6'; and 51.2ppm C-5'-O-methyl. Mass Spectrum: LREIMS: 614[M] + (0.4%), 540(1.6), 438(56), 423(100), 296(32), 281(24), 187(29), 177(60), 159(39), 117(30), and 69m/e (17). D/CI-MS: 632[M + NH4]+ (50%), 615[M + H] + (16), 474[M-side chain + NH4]+ (17), 458(22), 439[M-side chain + H] § (68), 194[side chain + NH4]§ (100). TLC Data Chloroform-isopropanol (9:1 v/v), Re = 0.60. Reference A. C. Keller, M. P. Maillard, and K. Hostettmann; Antimicrobial Steroids from the Fungus Fomitopsis pmicola; Phytochemistry, Vol. 41, pp. 1041-1046(1996).

23.

Miscellaneous Metabolites

641

Common/Systematic Name Isofunicone (E)•3•Meth•xy•2•pr•peny•-5-(2'-carb•meth•xy-4'-hydr•xy-6'-meth•xybenz•y•)-4-pyr•ne Molecular Formula/Molecular Weight C19HIsOs; MW = 374.10017 8'

O~

OMe r

0

0

10

z

HO 4 <

s

3

H Me 9

General Characteristics Recrystallized from methanol to give plates; mp., 215-218 oC. Isolation/Purification The culture broth was grown at 24~ without shaking for 21 days and then filtered to separate the mycelium from the broth. The filtrate was adjusted to pH 2.0 with dilute HC1 solution, and successively extracted with ethyl acetate. After evaporating the solvent, the residue was fractionated by silica gel column chromatography with hexane-ethyl acetate mixtures. Fractions containing 50% ethyl acetate were bulked and further purified by silica gel column chromatography with benzene-ethyl acetate mixtures. The active fraction (inhibited tea pollen growth) was evaporated to dryness and recrystallized from methanol, to give plates of isofunicone Fungal Source Unidentified Penicillium sp. Biological Activity Inhibited tea pollen (Camellia sinensis) growth by 84% at a concentration of 3mg/ml and 100% at 10mg/ml. Spectral Data UV:

~EtOH

m~x

293,285, and 249nm.

IR:

(KBr) 3228,2950,2852, 1715, 1681, 1653, 1611, and 1443cm1.

642

23.

Miscellaneous Metabolites

1H NMR: (DMSO) 1.94(3H, s, H-9); 3.64(3H, s, H-10); 3.66(3H, s, H-8'); 3.71(3H, s, H-9'); 6.54(1H, br, d, J--15Hz, H-7); 6.62(1H, m, H-8); 6.64(1H, d, J=2Hz, H-5'); 6.81(IH, d,J=2Hz, H-3'); 8.51(1H, s, H-6); and 10.21ppm (1H, br s, H-4'). 13CNMR: (DMSO) 18.4(q, C-9); 52.0(q, C-8'); 55.9(q, C- 10); 60.0(q, C-9'); 102.9(d, C-5'); 107.7(d, C-3'); l18.1(d, C-8); 122.6(s, C-I'); 126.8(s, C-5); 130.6(s, C-2'); 135.1(d, C7); 143.2(s, C-6'); 153.7(s, C-2); 157.9(s, c-a); 159.0(d, C-6); 159.3(s, C-4'); 166.3(s, C-7'); 171.1(s, C-4); and 189.7ppm (s, C-11). Mass Spectrum: 374(M) § (82%), 343(M- OMe)+ (39), 315(M-COOMe) § (42), 209(M-165) § (40), 192(M-182)+ (100); found: C, 60.99%; H, 4.75, C19HlsOs requires: C, 60.96; H, 4.85% Reference Y. Kimura, T. Yoshinari, A. Shimada and T. Hamasaki; Isofunicone, A Pollen Growth Inhibitor Produced by the Fungus Penicillium sp.; Phytochemistry, Vol. 40, pp. 629631(1995).

23.

Miscellaneous Metabolites

643

Common/Systematic Name Xylobovatin 18-Deoxy- 19,20-epoxycytochalasin Q Molecular Formula/Molecular Weight C 3 o H 3 7 N O 6 ; M W -- 507.26209 O

6' u,-,o

I

-o

Fungal Source Xylaria obovata (ADA-228), a wood-inhabiting tropical fungus collected from the Munesa Forest in southern Ethiopia. Isolation/Purification Cultured on a rice medium; after 20-40 days the moldy rice was extracted with chloroform, dried over Na2SO4, filtered and evaporated to dryness. The crude extract was separated on a Si-60 column eluted with a gradient from n-hexane-ethyl acetate (19:1, v/v) to ethyl acetate. When necessary, the collected fractions were further purified by preparative TLC [n-hexane-ethyl acetate (1:2 v/v); Re 0.71]. Biological Activity Cytotoxic. Spectral Data (KBr) 3430, 1745, 1710, and 1685cm-1. CD: [a] 27 589nm, + 44.5~ 578nm, + 45.4~ 546nm, + 49.6~ 436nm, + 79.6~ 365nm, + 139.6 ~(c=1.3, in MeOH). 1H NMR: (CDC13) 3.60(1H, ddd, J=7.0, 7.0, 2.5Hz, H-3); 2.12(1H, dd, J-5.7, 2.5Hz, H-4); 1.6 I(1H, qd, ,/--7.2, 5.7Hz, H-6); 2.74(1H, J=5.5Hz, H-7); 2.38(1H, J= 10.0, 5.5Hz, H-8); 2.83(2H, m, H-10); 0.79(3H, d, J=7.2Hz, H-11); 1.20(3H, s, H-12); 6.27(1H, dd, J=15.0, 10.0, 1.0Hz, H-13); 5.57(1H, ddd, J=15.0, 10.5, 5.0Hz, H-14); 2.50(1H, td, J-13.0, 10.5Hz, H-15); 2.17(1H, dddd, J=13.0, 5.0, 3.5, 1.0Hz, H-15); 2.97(1H, dqd, J=13.0, 6.7, 3.5Hz, H-16); 2.26(3H, dq, J=9.0, 7.0, H-18); 2.94(1H, dd, J=9.0,

644

23.

Miscellaneous Metabolites

2.0Hz, H-19); 3.55(1H, dd, J=2.0, 1.0Hz, H-20); 5.55(1H, s, H-21); 1.14(3H, d, J=6.7Hz, H-22); 1.30(3H, d, J=7.0Hz, H-23); 7.20(1H, m, H-25); 7.33(1H, m, H-26); 7.25(1H, m, H-27); and 2.12ppm (3H, s, acetate).

13CN M R : (CDCI3) 170.0,C-I; 54.5, C-3; 50.9, C-4; 36.7, C-5; 57.2, C-6; 62.5, C-7; 44.5, C-8; 54.1, C-9; 45.8, C-10; 12.7,C-II; 19.7, C-12; 131.4, C-13; 130.8, C-14; 37.5, C-15, 43.6, C-16; 215.9, C-17, 50.6, C-18; 58.6, C-19; 57.4, C-20; 72.8, C-21; 18.6,C-22; 14.8, C-23; 136.9, C-24; 129.2, C-25; 129.0, C-26; 127.2, C-27, 174.7, COMe; and 20.6ppm COMe. Mass Spectrum: HREIMS: 507.2586, calc. for C30H37NO6,507.2621 (17%), 489.2557, calc. for C3oH35NOs, 489.2515[M- H20] + (7), 448.2484, talc. for C2sH34NO4, 448.2488[MAc] § (6), 416.2032, talc. for C23H3oNO6, 416.2073[M - benzyl]§ (100), 3 56.1854, talc. for C21H26NO4, 356.1862[416-HOAc] § (59), 338.1779, calc. for C21H24NO3, 338.17561356-1-120] § (60), 328.1923, talc. for C20H26NO3,328.19131356-CO] § (13), 320.1672, talc. for C21H22NO2 320.16511338-H20] § (9), 310.1770, talc. for C20H24NO2, 310.18071338-CO] § (18), 296.1651, calc. for C19H22NO2, 296.1626[310CH2]§ (9), 91 (21), and 43m/e (12). Reference D. Abate, W-R. Abraham, and H. Meyer; Cytochalasins and Phytotoxins from the Fungus Xylaria obovata; Phytochemistry, Vol. 44, pp. 1443-1448(1997).

23.

Miscellaneous Metabolites

645

Common/Systematic Name Deacetyl- 19,20-epoxycytochalasin Q Molecular Formula/Molecular Weight C28H35NO6,1V[W = 481.24644 ',,,

!

O

General Characteristics Crystals; rap., 119-121~ Fungal Source Xylaria obovata (ADA-228), a wood-inhabiting tropical fungus collected from the Munesa Forest in southern Ethiopia. Isolation/Purification Rice was allowed to soak for 3 hr. in sterile distilled water, and then sterilized and inoculated with X. obovata. When all the seeds were invaded and the mycelial mat had turned black (18 days after inoculation), the moldy rice was ground with a mortar and pestle. The ground moldy rice was soaked overnight in a total volume of 2L of MeOHwater (55:45, v/v). It was then filtered and the filtrate extracted with chloroform. The crude chloroform extract was partitioned between aqueous methanol and hexane and the aqueous portion extracted with chloroform. Removal of the chloroform yielded an extract, which was applied to a silica gel column eluted with a hexane-acetone gradient system. Ten 300ml fractions were collected, and fractions 4-5 were further purified by preparative TLC [hexane-isopropyl alcohol (8:2, v/v)] which yielded pure 19,20-epoxycytochalasin Q (Re 0.68) and deacetyl- 19,20-epoxycytochalasin Q(Rf 0.74). Biological Activity Cytotoxic. Spectral Data IR;

(KBr) 1370, 1450, 1685, 1740, 2970, and 3400cm "1.

646

23.

Miscellaneous Metabolites

1H NMR: (pyridine-ds) 3.91(1H, br t, J=7.0Hz, H-3); 2.10(1H, m, H-4); 1.96(1H, m, H-5); 3.09(1H, d, J=5.6Hz, H-7); 2.75(1H, m, H-8); 2.85(1H, dd, J=13.2, 7.0Hz, n-10); 3.06(1H, dd, J=13.2, 5.8Hz, U-10); 0.82(3H, d, J=7.2Hz, H-11); 1.20(3H, s, n-12), 6.66(1H, dd, J=15.4, 9.6Hz, H-13); 5.85(1H, sept, J=15.6, 10.0, 4.0Hz, H-14); 1.97(2H, m, n-15), 3.21(1H, m, H-16); 3.98(1H, d, J=2.0Hz); 4.27(1H, t, J=l.6Hz, n-20); 4.97(1H, br s, H-21), 1.06(3H, d, J=6.4Hz, H-22); 1.67(3H, s, H-23); 9.25(1H, s, Nit); and 7.20ppm (2H, m, H-3', -5'). ~3CNMR: (pyridine-ds) C-I, 177.2.s; C-3, 54.6 d; C-4, 52.8 d; C-5, 37.5 d; C-6, 57.6 s; C-7, 63.6 d; C-8, 44.7 d; C-9, 55.7 s; C-10, 46.2 t; C-11, 12.7 q; C-12, 19.8 q; C-13, 132.9 d; C-14, 131.1 d; C-15, 38.1 t; C-16, 42.2 d; C-17, 216.8 s; C-18, 77.7 s; C-19, 60.8 d; C-20, 56.1 d; C-21, 73.3 d; C-22, 19.2 q; C23, 22.8 q; C-I', 138.3 s; C-2', -6', 130.1 d; C-3', -5', 128.9 d; and C-4', 126.1ppm d. Mass Spectrum: HREIMS: 481.2464[M] § (5%)calc. for C28H35NO6,481.2467; 390[M- 91] + (100), and 338role [M- H20-C7H7] § (96). Reference E. Dagne, A. A. L. Gunatilaka, S. Asmellash, D. Abate, D. G. I. Kingston, G. A. Hofmann, and R. K. Johnson; Two Toxic Cytochalasins from Xylaria obovata; Tetrahedron, Vol. 50, pp. 5615-5620(1994).

23.

Miscellaneous Metabolites

647

Common/Systematic Name 19,20-Epoxycytochalasin Q Molecular Formula/Molecular Weight C30H37NO7; M~W = 523.25700

IIii ,,onallll

General Characteristics Crystals; mp., 266-268~

[a]D - 73* (c=l.1, in CHCI3).

Fungal Source Xylaria obovata (ADA-228) a wood-inhabiting tropical fungus collected from the Munesa Forest in southern Ethiopia. Isolation/Purification Rice was allowed to soak for 3 hr. in sterile distilled water, and then sterilized and inoculated with X. obovata. When all the seeds were invaded and the mycelial mat had turned black, 18 days after inoculation, the moldy rice was ground with a mortar and pestle. The ground moldy rice was soaked overnight in a total volume of 2L of MeOHwater (55:45, v/v). It was then filtered and the filtrate extracted with chloroform. The crude chloroform extract was partitioned between aqueous methanol and hexane and the aqueous portion extracted with chloroform. Removal of the chloroform yielded an extract, which was applied to a silica gel column eluted with a hexane-acetone gradient system. Ten 300ml fractions were collected, and fractions 4-5 were further purified by preparative TLC, which yielded pure 19,20-epoxycytochalasin Q and deacetyl-19,20-epoxycytochalasin Q. Biological Activity Cytotoxic. Spectral Data IR~

(KBr) 1220, 1370, 1450, 1690, 1740, 2970, and 3440crn~.

648

23.

Miscellaneous Metabolites

1H NMR: (pyridine-ds) 3.92(1H, br t, J=7.0Hz, H-3); 2.52(1H, dd, J=5.8, 2.0Hz, H-4); 1.89(1H, dq, J=7.2, 5.8Hz, H-5); 3.02(1H, d, J=5.6Hz, H-7); 2.75(1H, m, H-8); 2.87(1H, dd, J=13.2, 8.0Hz, H-10); 3.08(1H, dd, J=13.2, 6.0Hz, H-10); 0.63(3H, d, J=7.2Hz, H-11); 1.17(3H, s, H-12); 6.64(1H, dd, J=15.2, 10.0Hz, H-13); 5.83(1H, sept, J=15.6, 10.0, 6.0Hz, H-14); 2.00(2H, m, H-15); 3.24(1H, m, H-16); 3.72(1H, d, J=2.0Hz); 4.30(1H, dd, J=2.0, 8.0Hz, H-20); 6.18(1H, br s, H-21); 1.07(3H, d, J=6.8Hz, H-22); 1.64(3H, s, H-23); 9.58(1H, s, NH); 2.18(3H, s, OAc); 7.40(2H, d, J=6.7Hz, H-2', -6'); 7.34(2H, t, J=7.8Hz, H-3', -5'); and 7.23ppm (1H, d, J=6.7Hz, H4'). (CDC13) 3.60(1H, br t, J=6.0, 8.0Hz, H-3); 2.21(1H, m, H-4); 1.60(1H, m, H-5); 2.71(1H, d, J=5.6Hz, H-7); 2.32(1H, dd, H-8); 2.80(2H, d, J=7.2Hz, H-8); 0.80(3H, d, J=7.2Hz, H-11); 1.18(3H, s, H-12); 6.08(1H, dd, J=15.2, 10.0Hz, H-13); 5.65(1H, m, H-14); 2.61(2H, dd, J=12.0, 11.4Hz, H-15); 3.20(1H, m, H-16); 3.15(1H, br s, H19); 3.52(1H, d, J=l.2Hz, H-20); 5.65(1H, m, H-21); 1.19(3H, d, J=6.4Hz, H-22); 1.52(3H, s, H-23); 5.80(1H, br s, NH); 2.10(3H, s, OAc); and 7.20ppm (2H, m, H-3', 5'). 13CNMR: (pyridine-ds) C-I, 175.4 s; C-3, 54.9 d; C-4, 52.2 d; C-5, 37.3 d; C-6, 57.4 s; C-7, 63.1 d; C-8, 45.7 d; C-9, 55.6 s; C-10, 46.1 t; C-11, 12.4 q; C-12, 19.6 q; C-13, 132.5 d; C14, 131.6 d; C-15, 37.9 t; C-16, 42.2 d; C-17, 216.3 s; C-18, 77.4 s; C-19, 60.8 d; C20, 53.9 d; C-21, 73.7 d; C-22, 19.2 q; C-23, 22.7 q; C-I', 138.2 s; C-2', -6', 130.2 d; C-3', -5', 128.9 d; C-4', 127.1 d; CHACO, 40.4 q; and CH3CO, 170.8ppm s. Mass Spectrum: HREIMS: 523.2648[M]*(8%) calc. for C3oH37NO7323.2569, 495[M - CO] § (17), 480[M- CHACO]§ (62), 464[M -OAc] § (90), 432[M - C7H71+ (37), 338[M- HOAc C7H7]§ (77), 9 lm/e [C7H7]§(100). Reference E. Dagne, A. A. L. Gunatilaka, S. Asmellash, D. Abate, D. G. I. Kingston, G. A. Hofmann, and R. K. Johnson; Two Toxic Cytochalasins from Xylaria obovata; Tetrahedron, Vol. 50, pp. 5615-5620(1994).

23.

Miscellaneous Metabolites

649

Common/Systematic Name Deacetyl- 19,20-epoxycytochalasin C Molecular Formula/Mol..ecular....Weigh_t C28HssNO6; MW = 481.24644

H,,, "'

H II~ .,,111111

,-Olo -o Fungal Source Xylaria obovata (ADA-228) a wood-inhabiting tropical fungus collected from the Munesa Forest in southern Ethiopia. Isolation/Purification Cultured on a rice medium; after 20-40 days the moldy rice was extracted with chloroform, dried over Na2SO4, filtered and evaporated to dryness. The crude extract was separated on a Si-60 column eluted with a gradient from n-hexane-ethyl acetate (19:1, v/v) to ethyl acetate. The collected fractions were further purified by preparative TLC [nhexane-isopropanol (7:3, v/v); Rf 0.46]. Biological Activity Cytotoxic. Spectral Data IR: (KBr) 3423, 3347, 1708, and 1684cm "1. CD: [~]27 589nm, +11.6" and 578nm, +14.8~

in MeOH).

1H ~ : (CDCI3) 3.35(1H, m, H-3); 2.84(1H, s, H-4); 3.75(1H, d, d=10Hz, H-7); 2.40(1H, t, J=10.0Hz, H-8); 2.96(1H, dd, J=13.0, 6.0Hz, H-10); 2.88(1H, dd, d=l.0,8.0Hz, H10); 1.42(3H, s); 1.43(3H, s, H-11); 1.66(3H, s); 1.67(3H, s, H-12); 5.98(1H, dd, J=15.5, 10.0Hz); 5.65(1H, ddd, J=15.5, 10.0, 6.0Hz, H-14); 2.09(1H, dddd, J=12.0, 6.0,2.0, 1.0Hz, H-15); 2.60(1H, td, d=12.0, 10.0Hz, H-15); 3.21(1H, dqd, d=12.0, 7.0, 2.0Hz, H-16); 3.47(1H, d, J=2.0Hz, H-19); 3.3 I(1H, m, H-20); 4.13(1H, s, H-21); 1.19(3H, d, d=7.0Hz, H-22); 1.56(3H, s, H-23); 7.20(1H, m, H-25); 7.32(1H, m, H26); and 7.26ppm (1H, m, H-27).

650

23.

Miscellaneous Metabolites

Mass Spectrum: HREIMS: 481.2450 talc. for C2sH35NO6,481.2464(10%), 463(6), 453(9), 435(28), 321 (48), and 9 lm/e (100). Reference D. Abate, W.-R. Abraham, and H. Meyer, Cytochalasins and Phytotoxins from the Fungus Xylaria obovata; Phytochemistry, Vol. 44, pp. 1443-1448(1997).

23.

Miscellaneous Metabolites

651

Common/Systematic Name 19,20-Epoxycytochalasin C Molecular Formula/Molecular Weight C30H37NO7; M W -- 523.25700

H ,,.

.,,ll ii ||11|||||

6' OAC H "O/"011 "O General Characteristics [a]n - 6.8 ~ (c=0.25, in CHCls). Fungal Source

Xylaria obovata (ADA-228), a wood-inhabiting tropical fungus collected from the Munesa Forest in southern Ethiopia.

Isolation/Purification Cultured on a rice medium; after 20-40 days the moldy rice was extracted with chloroform, dried over Na2SO4, filtered and evaporated to dryness. The crude extract was separated on a Si-60 column eluted with a gradient from n-hexane-ethyl acetate (19:1, v/v) to ethyl acetate. The collected fractions were further purified by preparative TLC [nhexane-isopropanol (7:3, v/v), P~ 0.52]. Biological Activity Cytotoxic. Spectral Data IR:

(KBr) 3438, 1745, 1708, and 1685cm1.

:H NMR:

(CDCI3) 3.35(1H, dd, J=8.0, 6.0, 1.0Hz, H-3); 2.54(1H, s, H-4); 4.01(1H, dd, J-7.0, 2.0Hz, H-5); 3.81(1H, d, J-10.0Hz, H-7); 2.26(1H, t, J=10.0Hz, H-8); 3.08(1H, dd, J-13.0, 6.0Hz, H-10); 3.01(1H, dd, J=13.0, 8.0Hz, H-10); 6.16(1H, dd, J=16.0, 10.0Hz, H-13); 5.72(1H, ddd, J-16.0, 10.0, 6.0Hz, H-14); 2.12(1H, dddd, J-12.0, 6.0, 2.0, 1.0Hz, H-15); 2.68(1H, td, J=12.0, 10.0Hz, H-15); 3.24(1H, dqd, J=12.0, 7.0, 2.0Hz, H-16); 3.21(1H, d, J=2.0Hz, H-19); 3.47(1H, dd, J-2.0, 1.0Hz, H-20); and 1.22ppm (3H, d, J=7.0Hz, H-22).

652

23.

Miscellaneous Metabolites

Mass Spectrum: HREIMS: 523.2560, talc. for C30H37NOT,523.2570(15%), 480(10), 477(13), 463(10), 435(14), 432(13), 420(22), 405(20), 372(34), 362(31), 334(13), 316(9), 312(15), 252(40), 238(22), 187(16), 185(17), 174(24), 169(16), 163(9), 162(16), 161(19), 160(20), 133(18),120(43), 98(9), 91(61), and 43m/e (100). FABMS: 524.2[M + H]+ and 546.2[M + Na]§ Reference D. Abate, W.-R. Abraham, and H. Meyer; Cytochalasins and Phytotoxins from the Fungus Xylaria obovata; Phytochemistry, Vol. 44, pp. 1443-1448(1997).

23.

Miscellaneous Metabolites

653

Common/Systematic Name Fusaproliferin; Proliferin 18-[ 1-(Acetoxymethyl)-2-methylethyl]- 10,17-dihydroxy-3,7,11,15- tetramethylbicyclo[ 13.3. O]octadeca-trans, trans, trans, cis-2,6,11,17-tetraen- 16-one Mol_ecular Formula/Molecular Weight C27H4005; M W -- 4 4 4 . 2 8 7 5 7

OCOMe

OH

General Characteristics Crystals from methanol; melting point not reported; [a] 2s -35~ (c=0.7, in CHC13). Fungal Sour.ee Fusarium proliferatum isolated from earrot-infected corn in northern Italy and the strain no. 1494 deposited at the collection of the IstitutoTossine Micotossine da parassiti vegetali (ITEM), Bari, Italy. Isolation/Purification Fusaproliferin was obtained by inoculation of autoclaved yellow corn kernels with F. proliferatum. Purification was performed as follows: 300g oflyophilized culture filtrate was resuspended in 1.3L of MeOH- 1% aqueous NaC1 solution (55..'45, v/v). Fusaproliferin was extracted from the aqueous residue with 3.250L of n-hexane and concentrated under reduced pressure. Successive steps of the purification were carded out as previously reported (A. Ritieni et al., 1995). Spectral Data lH NIVIR: (CDCI3) 1.71(1H, s, J=6.6, 13.6Hz, H-I'); 2.38(1H, m, J=10.6, 13.6Hz, H-l"); 5.24(1H, m, n-2); 2.30(1H, m, n-4'); 2.01(1H, m, H-4"); 2.30(1H, m, J= 8.8Hz, n-5'); 2.13(1H, m, J= 4.5 Hz, n-5"); 5.12(1H, m, H-6); 2.1 l(1H, m, H-8'); 1.78(1H, m, n-8"); 1.77(1H, m, J= 4Hz, n-9'); 1.68(1H, m, J=10Hz, n-9"); 4.05(1H, dd, n-10); 5.38(1H, m, H-12); 2.40(IH, m, J=3, 17, 2.5Hz, H-13'); 1.92(1H, m, d=6-7, 17, ll.lHz, H-13"); 2.67(1H, dd, J=l 1.1Hz, H-14); 2.78(IH, m, H-19); 1.64(3H, s, 1-1-20); 1.64(3H, s, H-21); 1.56(3H, s, H-22); 0.99(3H, s, H-23); 4.28(2x1H, m, J=7.6, 10.6Hz, H-24'); 4.25(1H, m, J=6.9Hz, H-24"); 1.31(3H, d, J=-2Hz, I-1-25); 2.02(3H, s, H-27); and 5.56ppm (IH, s, OH-17).

654

23.

Miscellaneous Metabolites

13CNMR: (CDC13) 207.86, C-16; 170.91, C-26; 147.27, C-17; 146.71, C-18; 138.20, C-3; 136.54, C-11; 132.93, C-7; 128.89, C-12; 124.31, C-6; 121.38, C-2; 76.51, C-10; 66.43, C-24; 49.56, C-14; 49.01, C-15; 40.33, C-4; 39.14, C-l; 34.93, C-8; 33.71, C19; 29.72, C-9; 28.72, C-13; 23.83, C-5; 16.19, C-23; 15.55, C-20; 15.32, C-21; 14.52, C-25; and 10.38ppm, C-22. References A. Ritieni, V. Fogliano, G. Randazzo, A. Scarallo, A. Logrieco, A. Moretti, L. Mannina, and A. Bottalico; Structure and Absolute Stereochemistry of Fusaproliferin, A Toxic Metabolite from Fusarium proliferatum; Natural Toxins; Vol. 3, pp. 17-19(1995). A. Santini, A. Ritieni, V. Fogliano, G. Randazzo, L. Mannina, A. Logrieco, and E. Benedetti; Structure and Absolute Stereoehemistry ofFusaproliferin, a Toxic Metabolite from Fusarium proliferatum; J. of Nat. Prod.; Vol. 59, pp. 109-112(1996).

23.

Miscellaneous Metabolites

655

Common/Systematic Name Retigeranic Acid Molecular Formula/Molecular Weight C25H3802; M W -- 3 7 0 . 2 8 7 1 8

H

.r-

'" H

iii

H

COOH

General Characteristics Crystals; mp. 218-221~

[ a ] D 23 -

59~ (CHC13).

Fungal Source Lobaria isidiosa (a lichen) collected in the eastern Himalayas, Bhutan. Isolation/Purification Retigeranic acid was obtained from the chloroform-soluble fraction of the ethereal extracts ofLobaria isidiosa. Spectral Data UV:

X max

242nm (log e = 3.91).

(KB 0

1662 and 1608cmq.

IR:

Mass Spectrum: HREIMS: 370.286re~e; calcd, for C25H3sO2, 370.287; LREIMS: 370(M +) and 325m/e (M +- COOH). Reference M. Kaneda, R. Takahashi, Y. Iitaka, and S. Shibata; Retigeranic Acid, a Novel Sesterterpene Isolated from the Lichens of Lobaria retigera Group; Tetrahedron Letters; No. 45, pp. 4609-4611(1972).

656

23.

Miscellaneous Metabolites

Common/Systematic Name Moniliformin 3-Hydroxycyclobut-3-ene-1,2-dione Molecular Formula/Molecular Weight C4HO3Na 119.9823 (sodium salt) C4HO3K 136.0945 (potasium salt) C4H203 98.0081 (free acid); MW = 98.00039 ,

H/

2

4

0

OH

General Characteristics Crystals from aqueous methanol, decomposes without melting at temperatures up to 350~ (Na or K salts). Crystalline acid, mp., 158~ (dec.). pI~ 1.70. Fungal Source Gibberellafufikuroi (ATTC 12763, original isolation), Fusarium moniliforme (= F. subglutinans), F. proliferatum, F. nygamai, F. oxysporum, F. anthophilum, F. graminearum, F. avenaceum, F. acuminatum, "F. concolor, F. equiseti, F. semitectum, F. fusarioides (F. chlamydosporum), F. sporotrichioides, F. culmorum, and F. reticulatum. Isolation/Purification Original isolation involved extraction with aqueous methanol followed by column chromatography using Sephadex LH-20 and elution with increasing amounts of methanol in water. Subsequent isolations involved ion-exchange chromatography. Biological Activity Moniliformin had an LD50of 4.0 mg/kg in day-old cockerels(1601ag/40g cockerel). Gross and histologic lesions in cockerels were ascites with edema of the mesenteries and small hemorrhages in the proventriculus, gizzard, small and large intestine, and skin. The LDs0 in mice was 20.9mg/kg (females) and 29.1 mg/kg (males). Exceeding low concentrations of moniliformin (less than 5~tm) selectively inhibited mitochondrial pyruvate and t~ketoglutarate oxidations by 50%. Moniliformin produced plant-growth regulating and phytotoxic effects in plant systems. It affected mitosis in Allium root assays. Spectral Data UV~

~"

229 (e = 19,100) and 260nm(5,600).

1H NMR: (D20) 8.23ppm (1H, s, H-4) (nonexchangeable with D20).

23.

Miscellaneous Metabolites

657

~3C NMR: (D20) 202.9, C-l; 207.4, C-2; 202.9, C-3, and 168.8ppm, C-4. References J.P. Springer, J.Clardy, R.J. Cole, J.W. Kirksey, R.K. Hill, R.M.Carlson, J.L. Isidor; J. Am. Chem. Soc.; Structure and Synthesis ofMoniliformin, A Novel Cyclobutane Microbial Toxin; Vol.96, pp. 2267-2268(1974). H. G. Cutler, R. J.Cole, B. R. Blankenship, J. W. Kirksey, B. Doupnik,Jr.;Growth Regulating and Phytotoxic Effects of A Metabolite Produced by Fusarium moniliforme; Phytopathology; Vol.62, pp. 752-754(1972).

658

23.

Miscellaneous Metabolites

Common/Systematic Name Cyclopiazonic Acid Molecular Formula/Molecu.lar Weight C 2 o H 2 o N 2 0 3 , M~TV -- 3 3 6 . 1 4 7 3 9 99

,,I H

,,~~

I

OH 0

16

General Characteristics Crystals from methanol; mp., 245-246~

[a]D2~ - 36.1 ~ (C=0.36 in pyridine).

Fungal Source Penicilliun cyclopium; P. viridicatum; P. camemberti; P. grieseofulvum; P. urticae; P. patulum; P. biforme; P. puberulum; P. chrysogenum; P. commune; P. hirsutum; P. nalgiovense; Aspergillus flavus; A. tamarii; A. oryzae; and A. versicolor. Isolation/Purification The original isolation/purification of cyclopiazonic acid was accomplished using chloroform-methanol extraction partitioned between chloroform-water. The dried chloroform extract was distributed between 95% methanol and hexane. The methanol residue was chromatographed on cellulose powder impregnated with 50:3, HCONH2(COOH)2. Lipids were eluted with hexane and the column further eluted with mixtures of hexane-benzene. The biologically active fraction in ethyl ether was extracted with 0.5M aqueous sodium bicarbonate followed by extraction of the acidified aqueous phase with chloroform. The active fraction was chromatographed on Dowex 1 X 8 using aqueous methanol (1:1, v/v). Final purification was crystallization from methanol. More recently advanced analytical procedures for cyclopiazonic acid include thin-layer chromatography, HPLC, enzyme-linked immunosorbent assay, capillary electrophoresis, and immunoaffinity column chromatography (see Dorner, 2002). Biological Activity Cyclopiazonic acid is a potent, specific, and reversible inhibitor of the sarcoplasmic and endoplasmic reticulum Ca 2+ activated ATPase. It has been demonstrated to efficiently partition into various tissues including eggs and milk. See Burdock and Flamm, 2000 for detailed discussions on the biological effects of cyclopiazonic acid.

23.

Miscellaneous Metabolites

659

Spectral Data UV~

k mMH ~ 225 (e = 39,810), 253 (16,595), 275 (sh)(19,054), 284 (20,417), and 292nm(sh)(17,378). IR~

(CHC13) 3478, 3200-2600, 1708, and 1618cm"1. 1H NMR: (See Holzapfel, 1968 for detailed discussion of proton NMR). Mass Spectra: HREMS: 336.1463m/e (M+) C20H20N203requires 336.1474. References C. W. Holzapfel; The Isolation and Structure of Cyclopiazonic Acid, A Toxic Metabolite of Penicillium cyclopium Westling; Tetrahedron, Vol. 24, pp. 2101-2119(1968). G. A. Burdock and W. G. Flamm; Review Article: Safety Assessment of the Mycotoxin Cyclopiazonic Acid; International Journal of Toxicology, Vol. 19, pp. 195-218(2000). J. W. Domer; Recent Advances in Analytical Methodology for Cyclopiazonic Acid; in Mycotoxins and Food Safety; L. S. Jackson, M. W. Trucksess, and J. W. DeVries (eds.); Kluwer Academic/Plenum Publishers, New York, N.Y.(2002).

This Page Intentionally Left Blank

Secondary. Metabolite Index Secondary metabolite

Page

Secondary metabolite

Page

Secondary metabolite

Page

A A L Toxin TA1 A A L Toxin TA2 A A L Toxin TB1 A A L Toxin TB2 A A L Toxin TC1 A A L Toxin TC2 A A L Toxin TD1 A A L Toxin TD2 A A L Toxin TEl A A L Toxin TE2 15-Acetoxy-3ot,4l]dihydroxy-12,13epoxytrichothec-9ene 3t~-Acetoxy-70t,15dihydroxy-12,13epoxytrichothec-9en-8-one

587 589 591 593 595 597 599 601 603 605

15-Acetoxyscirpentriol 20~Acetoxystachybotrylactam acetate 2t~Acetoxystachybotrylactone acetate 4~-Acetoxy-30~,7t~,15txtrihydroxy-12,13epoxytrichothec-9en-8-one 3-Acetyl-4deoxynivalenol

235

Austalide Austalide Austalide Austalide Austalide

H I J K L

33 35 37 39 41

Baccharin Baccharinoid B1 Baccharinoid B2 Baccharinoid B3 Baccharinoid B4 Baccharinoid B5 Baccharinoid B6 Baccharinoid B7 Baccharinoid B8 Baccharinoid B9 Baccharinoid B 10 Baccharinoid B12 Baccharinoid B13 Baccharinoid B 14 Baccharinoid B16 Baccharinoid B 17 Baccharinoid B20 Baccharinoid B21 Baccharinoid B23 Baccharinoid B24 Baccharinoid B25 Baccharinoid B27 Baccharinol 8-Butrylneosolaniol 8r hydroxy-413,15diacetoxy-12,13epoxytrichothec-9ene 8tx-Butryloxyroridin A

527 519 521 523 525 527 529 531 533 535 537 539 541 543 545 547 549 551 553 555 557 559 525 273

235

286, 313

15-Acetoxy-30~,7t~dihydroxy-12,13epoxytrichothec-9en-8-one 319 3t~-Acetoxy-12,13epoxytrichothec-9ene 207 4fI-Acetoxy-12,13epoxytrichothec-9ene 205 30~-Acetoxy-15\ hydroxy-12,13epoxytrichothec-9en-8-one 284 3tx-Acetoxy-15hydroxy-12,13epoxytrichothec-9ene 223 3o~-Acetoxylanosta-8, 24-dien-21-oic acid 635 8-Acetoxyneosolaniol 269 Acetoxyscirpentriol 237 4-Acetoxyscirpendiol 233

15-Acetyl-4deoxynivalenol 41]-Acetyl-3tx,15dihydroxy-12,13epoxytrichothec-9ene 4-Acetyl-T-2 tetraol Acetyl T-2 Toxin Acuminatum Altertoxin I Altertoxin II Altertoxin III Anguidine Antibiotic T Atranone A Atranone B Atranone C Atranone D Atranone E Atranone F Atranone G Austalide A Austalide B Austalide C Austalide D Austalide E Austalide F Austalide G

189

183

323 286, 313 319

233 279 255 267 45 47 49 237 295 3 5 7 9 11 13 15 19 21 23 25 27 29 31

273 416

219 Calonectrin 30~-(4-Carboxymethyl3-hydroxy-3methylbutanoyloxy)lanasta-8,24-dien212 oic acid 639

661

662

Secondary Metabolite Index

Secondary metabolite

Page

Secondary metabolite

Cercophorin A Cercophorin B Cercophorin C 3-Chloro-5hydroxyspiro [napthalene-l(4H), 2'-naphtho[1,8de][1,3]dioxin]-4one

53 55 57

3t~,15-Diacetoxy-7t~,813dihydroxy-12,13epoxytrichothec-9ene 413,15tx-Diacetoxy-3ct, 8~-dihydroxy-12,13epoxytrichothec-9ene 8ct,15-Diacetoxy-3ct, 4~-dihydroxy-12,13epoxytrichothec-9ene 413,150~-Diacetoxy-30~, 7tx-dihydroxy-12,13epoxytrichothec-9en-8-one 413,15t~-Diacetoxy-3t~, 8t~-dihydroxy-12,13epoxytrichothec-9ene 413,80~-Diacetoxy-30t,15dihydroxy-12,13epoxytrichothec-9ene 3t~,15-Diacetoxy-12,13epoxytrichothec-9ene 413,15-Diacetoxy-12,13epoxytrichothec-9ene 3t~,l 5-Diacetoxy- 12,13epoxytrichothec-9en-8-one 413,15-Diacetoxy-70thydroxy-12,13epoxytrichothec-9ene 3ct,15-Diacetoxy-7t~hydroxy-12,!3epoxytrichothec-9ene 3t~,15-Diacetoxy-8hydroxy-12,13epoxytrichothec-9ene 3t~,150~-Diacetoxy-7t~hydroxy-12,13epoxytrichothec-9en-8-one

(4aRS,8aRS)3-

Chlorospiro[4a,8aepoxynapthalene- 1 (4H),2'-naphtho[1,8de][1,3]dioxine]4,5,8-trione Crotocin Crotocol 413-Crotonoyloxy-12,13epoxytrichothec-9en-8-one 8ct Crotonyloxy roridin A Culmorin Cyclopiazonic Acid

77

83 295 297

300 418 201 658

3-Deacetylcalonectrin 225 15-Deacetylcalonectrin 223 Deacetyl-19,20epoxycytochalasin C 649 Deacetyl-19,20e po xycytoch al asin Q 645 4-Deacetylneosolaniol 279 15-Deacetylneosolaniol 277 2'-Dehydroverrucarin A 401 2-Deoxy-11-epi-12acetyl-30thydroxysambucoin 327 2-Deoxy-11-epi-30thydroxysambucoin 329 (2E')12,13-Deoxyisotrichoverrin B 391 4-Deoxynivalenol 311 12,13-Deoxytrichodermadiene 357 12,13-Deoxyverrucarin A 406

Page

231

265

267

315

265

215

219

294

221

245

227

229

321

Secondary metabolite 413,15-Diacetoxy-3cthydroxy-12,13epoxytrichothec-9en-8-one 4,15Diacetoxyscirpenol 3,15-Diacetoxyscirpentriol 80~,15-Diacetoxy-T-2 tetraol 3,15-Diacetyl-4deoxynivalenol 413,15-Diacetyl-7de oxynivalenol 413,15-Diacetyl-30thydroxy-12,13epoxytrichothec-9ene 413,15Diacetylnivalenol 4,15Diacetylverrucarol 713,813,2',3'Diepoxyisororidin H (4R*)-3,4-Dihydro-4methoxyspiro[2,3epoxynapthalene-1, (2H)2'-naphtho[1,8de][1,3]dioxine]5,8-dione 7,8-Dihydroxy-3txacetoxy-12,13epoxytrichothec-9ene 3ct,41~-Dihydroxy-15acetoxy-8o~-[3hydroxy-3methylbutyryloxy]12,13epoxytrichothec-9ene 7t~,8t~Dihydroxycalonectrin 3t~,7t~-Dihydroxy-4~, 15-diacetoxy-12,13epoxytrichothec-9ene

Page

317 237 245 267 321 317

237 315 294

446

85

213

261

231

241

Secondary Metabolite Index

Secondary metabolite 7a,8a-Dihydroxydiacetoxyscirpenol 3{x,13-Dihydroxy-11epiapotrichothec-9ene 413,7a-Dihydroxy-12,13epoxytrichothec-9ene 413,15-Dihydroxy-12,13epoxytrichothec-9ene 7,8-Dihydroxyisotrichodermin (2S*,3R*)-5,8Dihydroxyspiro [2,3epoxynapthalene-1 (2H),2'-naphtho [1,8-de][1,3]dioxin]4(3H)-one 4,7-Dihydroxyspiro [1H-inden-l,2'naphtho[1,8de][1,3]dioxin]3(2H)-one (8R*,8aS*)-4,8Dihydroxy-6,7,8,8atetrahydrospiro [napthalene-l(5H), 2'-naphtho[1,8de][1,3]dioxin]-5one Epiisororidin E Epiroridin E 19,20Epoxycytochalasin C 19,20Epoxycytochalasin Q 713,813-Epoxyisororidin E 9~,10~lEpoxyisotrichoverrin A 9~l,10~-Epoxyisotrichoverrin B 7~,8~-Epoxyroridin H

Page 243

345

290

292 213

81

87

75 434 432

651

647 436

379 389 444

663

Secondary metabolite 12,13-epoxytrichothec9-ene 24-Ethyllanosta-8,24 (24')-diene-3~,22~diol

Page 203

629

Fiscalin A Fiscalin B Fiscalin C FS-1 FS-2 FS-3 FS-4 Fumiquinazoline A Fumiquinazoline B Fumiquinazoline C Fumiquinazoline D Fumiquinazoline E Fumiquinazoline F Fumiquinazoline G Fumonisin A1 Fumonisin A2 Fumonisin AK1 Fumonisin B1 Fumonisin B2 Fumonisin B3 Fumonisin B4 Fumonisin C1 Fumonisin C3 Fumonisin C4 Fusaproliferin Fusarenon Fusarenon-X

61 63 65 335 337 339 341 117 119 121 123 125 127 129 571 573 585 563 565 567 569 575 581 583 653 323 323

Ganomastenol A Ganomastenol B Ganomastenol C Ganomastenol D

133 135 137 139

HT-2 Toxin 30~-Hydroxy-15acetoxy-12,13epoxytrichothec-9ene 7{~-Hydroxy-30~acetoxy-12,13epoxytrichothec-9ene

253

225

209

Secondary metabolite 8-Hydroxy-30~-acetoxy12,13epoxytrichothec-9ene 30~-Hydroxyapotrichothecene 31~-Hydroxyapotrichothecene 7{x-Hydroxycalonectrin 8a-Hydroxcalonectrin 3a-Hydroxy-413,15diacetoxy-8a-[3hydroxy-3methylbutyryloxy]12,13epoxytrichothec-9ene 3{x-Hydroxy-413,15diacetoxy-8a-[3methylbutyryloxy]12,13epoxytrichothec-9ene 30~-Hydroxy-413,15diacetoxy-8a-[(2methylpropionyl) oxy]-12,13epoxytrichothec-9ene 70~-Hydroxydiacetoxyscirpenol 413-Hydroxy-7,8,12,13diepoxytrichothec9-ene 5-Hydroxy-2,3dihydrospiro [napthalene-l(4H), 2'-naphtho[1,8de] [1,3]dioxin]-4one 41~-Hydroxy-12,13epoxytrichothec-9ene 813-Hydroxy-12,13epoxytrichothec-9ene 41~-Hydroxy-12,13epoxytrichothec-9en,8-one

Page

211 345 347 227 229

259

251

275 241

297

71

288

204

298

664

Secondary Metabolite Index

Secondary metabolite

Page

3'-Hydroxy HT-2 toxin 7o;-Hydroxyisotrichodermin 80t-Hydroxyisotrichodermin 80~-Hydroxyisotrichoverrin A 12'-Hydroxy-2'isoverrucarin J 21-Hydroxylanosta8,24-dien-3-one Hydroxylated Fumonisin C1 4-Hydroxymellein 4-Hydroxyochratoxin A 5-Hydroxyspiro [napthalene-l(4H),2'naphtho[1,8-de][1,3] dioxin]-4-one

261 209 211 381 460 637

Isobaccharin Isobaccharinol 8-Isobutrylneosolaniol Isofunicone Isomiotoxin D Isoneosolaniol Isororidin A Isororidin E Isosatratoxin F Isosatratoxin G S-Isosatratoxin H M-Isosatratoxin H Isotrichodermin (2'E,4'Z)

Page

Secondary metabolite

Page

Isotrichoverrin A

375

469

Isotrichoverrin A (C6'R C7'R) Isotrichoverrin A (2'E,4'Z) Isotrichoverrin B (C6'R,C7'S) Isotrichoverrin B (C6'R,C7'S) Isotrichoverrol B (C6'R,C7'R) Isotrichoverrol A

377

363

Isotrichoverrol A

365

Isotrichoverrol B

371

Miotoxin A Miotoxin A 13'13-Dglucoside Miotoxin B Miotoxin C Miotoxin D Miotoxin E Miotoxin F Miotoxin G M-Isosatratoxin H Moniliformin Myriosin Myrotoxin A Myrotoxin B Myrotoxin C Myrotoxin C hydrate Myrotoxin D Myrotoxin D hydrate Mytoxin A Mytoxin B Mytoxin C

471 473 475 477 481 483 485 462 656 612 499 501 503 505 507 509 511 513 515

Neosolaniol Nivalenol Nivalenol monoacetate NT-1 Toxin NT-2 Toxin

265 309 323 215 279

Ochracin Ochratoxin A Ochratoxin B Ochratoxin C

622 615 617 621

Palmarumycin C1 Palmarumycin C2 Palmarumycin C3 Palmarumycin C4 Palmarumycln C5 Palmarumycln C6 Palmarumycm C7 Palmarumycln C8 Palmarumycln C10 Palmarumycln Cll Palmarumycln C12 Palmarumycln C13 Palmarumycm C14 Palmarumycln C15 Palmarumycm C16 Palmarumycm CP1

77 79 81 83 85 87 89 91 93 95 97 99 101 103 105 69

(C6"R,C7"R)

578 623

(2"E,C6"R,C7"R)

619

(2"E,C6"R,CY'R)

69

(2S*,3R*)-5Hydroxyspiro[2,3epoxynapthalene- 1 (2H),2'-naphtho[1,8de][1,3]dioxin]-4 (3H)-one 20~-Hydroxystachybotrylactone 3'-Hydroxy T-2 toxin 3'-Hydroxy T-2 triol 70t-Hydroxytrichodermol 813-Hydroxytrichothecene

Secondary metabolite

79 181 259 263 290 204 533 529 275 641 479 267 420 430 452 454 458 462 207

K-76 8-Keto-3a-acetoxy12,13-epoxytrichothec-9-ene 3-Ketoapotrichothecene 8-Ketocalonectrin 4-Keto-12,13-epoxytrichothec-9-ene 8-Ketoisotrichodermin Macrofusin Mellein Memnobotrin A Memnobotrin B Memnoconol Memnoconone (22S)-24Methyllanosta-8en-22,24'-epoxy313-ol-24'-one 24-Methyllanosta-8,24 (24')-diene-313,ZZ13diol 25-Methylpisolactone (E)-3-Methoxy-2propenyl-5-(2'carbomethoxy-4'hydroxy-6'methoxybenzoyl)-4pyrone

363 387 385 369

177

217 343 221 308 217 563 622 143 145 149 147

631

633 631

641

Secondary Metabolite Index

665

Secondary metabolite

Page

Secondary metabolite

Page

Secondary metabolite

Palmarumycin CP: Palmarumycin CP3 Palmarumycin CP4 PD 113,326 Phomosine A Phomosine B Phomosine C Proliferin 4-Propanyl HT-2 413-Propanyl-3t~hydroxy-15acetoxy-8o~-[3methylbutyryloxy]12,13-epoxytrichothec-9-ene 8tx-Propanyl-30thydroxy-413,15diacetoxy-12,13epoxytrichothec-9ene 8-Propionyl neosolaniol

71 73 75 462 109 111 113 653 257

Roridin C Roridin D Roridin D 13'13-Dglucoside Roridin E Roridin E 13'13-Dglucoside Roridin H Roridin J Roridin K acetate Roridin L-2 (9'E)Roridin L-2 Roritoxin A Roritoxin B Roritoxin C Roritoxin D

288 422

415,80~,15-Triacetoxy3ct,7a-dihydroxy12,13epoxytrichothec-9ene 3t~,4~,15-Triacetoxy12,13epoxytrichothec-9ene 30t,413,15-Triacetoxy-8ct[3-methylbutryloxy]12,13epoxytrichothec-9ene 3,4,15-Triacetoxyscirpentriol Trichodermadiene Trichodermadienediol A Trichodermadienediol B Trichodermin Trichodermol Trichodermone Trichodiene Trichodiol Trichothecene Trichothecin Trichothecinol A Trichothecinol B Trichothecinol C Trichothecolone Trichoverrin A Trichoverrin B Trichoverrol A Trichoverrol B 3ot,70t,8ct-Trihydroxy41],150~-diacetoxy12,13-epoxytrichothec-9-ene 30~,413,15-Trihydroxy12,13-epoxytrichothec-9-ene 3ct,413,15-Trihydroxy8t~-[3-hydroxy-3methylbutyryloxy]12,13-epoxytrichothec-9-ene Tsugicoline A

Radicinin Radicinin Diastereomer (2S*, 3R*)

2S,3S,4S-epi-Radicinol

257

271 271 163

165 169 171

Radicinol Radicinol diasteromer (2S* 3S'4S*) Rd toxin

167 311

Trihydroxycadina4,10(15)-diene

133

Trihydroxycadina4,10(15)-diene

135

Trihydroxycadina10(15)-ene

137

rel-3o~,8,9o~-

rel-3~i,8~,9t~rel-3~,8~,9t~-

rel-8~i,9t~-Dihydroxy-4hydroxymethylcadina-4,10(15)diene Retigeranic Acid Roridin A Roridin A 13'13-Dglucoside

139 655 412 414

Satratoxin F Satratoxin G Satratoxin H Scirpenetriol S-isosatratoxin H Solaniol Sphingofungin A Sphingofungin B Sphingofungin C Sphingofungin D Sporotrichiol Stachybotramide Stachybotrin A Stachybotrin B Stachybotrydial Stachybotrylactam Stachybotrylactam acetate Stachybotrylactone Stachybotrylactone acetate Stemphylone 4,5,10,11-Tetrahydroxybisboline 3tx,413,7tx,15Tetrahydroxy-12, 13-epoxytrichothec9-en-8-one 3t~,4[3,8t~,15Tetrahydroxy-12, 13-epoxytrichothec9-ene

424 426 428 440 442 438 395 393 489 491 493 495 448 450 456 239 458 265 607 609 610 611 281 191 195 197 193 185 187 175 179 163 627

309

249

Page

283

247

255 247 351 353 355 205 288 308 333 331 203 302 304 306 298 373 373 383 361 367

243

239

263 153

666

Secondary Metabolite Index

Secondary metabolite

Page

Secondary metabolite

Page

Tsugicoline B Tsugicoline C Tsugicoline D T-2 Tetraol T-2 Toxin

155 157 159 249 251

Verrol Verrucarin A

359 397

Verrucarin A I]-Dglucoside Verrucarin B Verrucarin H Verrucarin J Verrucarin K Verrucarin L Verrucarin L acetate

399 402 440 404 406 408 410

Secondary metabolite

Page

Verrucarol Vertisporin Vomitoxin

292 464 311

Xylobovatin

643

YM-47524 YM-47525

418 416

Molecular Formula Index Molecular formula

C4 C4H203 C10 CloHlo03 CloHloO4 Ca2 C12H1205 C12H1405 C12H1406 C15 C15H2o03 CasH2oO4

C15H2o06 C15H2007 ClsH2202

C15H2203

C15H2204 C15H2205 C15H2206 C15H24 C15H2403

C15H2404 C15H2602 C15H2603 C15H1804

C17 C17H1607 C17H2205 C17H2206 C17H2207 C17H2208 C17H2404 C17H2405

Page

Molecular formula

C17H2406 656 622 623 163, 165 167, 171 169 159, 308, 339 297, 298 311

309 203 155,204, 288, 335, 341,343 153,290, 292 239

249 333 133, 135, 139, 329, 331,337, 345,347 157

201 137 627 113 217 284 286,313, 319 323 205,207 209,211, 223,225

C17H2407 C17H2604 C18 C18H1807 C19 C19H1205

C19H1808 C19H2207 C19H2405 C19H2406 C19H2407 C19H2408 C19H2409 C19H2605 C19H2606 C19H2607 C19H2608

C19H2609

Page

Molecular formula

Page

213, 233, 235 277, 279 327

C2oH18NO6C1 C2oH18NO7CI C2oH1807 C2oHa9NO6 C2oH2oN203 C2oH3o06 C2oH3o08 C20H39NO6 C21 C21H1605 C21H18N402 C21H2808 C21H2809 C21H2801o C21H3o06 C21H39NO6 C21H41N306 C22 C22He2NO6C1 C22H3009 C22H3208 C22H3209 C22H41NO8 C22H43NO5

615 619 105 617 658 281 263 609

109 87 641 111 295,300 302 221 317,321 315 304 219,294, 306 227,229, 237,245 215,231, 241,265, 267 243

C2oHllCIO4 C2oHlaC106 C2oH1204 C2oH1205 C2oH1206 C2oH1207 C2oH13C106

C2oH1404 C2oH1405 C2oH1406 C20H1605 C2oH1606 C2oH1607 C2oH1609 C2oH1601o

621 271 253 261 611 610

C23

C/o

C2oH9C106

85 127,129 247 269 283 359 612 607

83 77 89 69 79 49,81 93 91 71 73,95 47,97 75 45 99,101, 103 53 55,57

C23H22N402 C23H3o04 C23H3o05 C23H3006 C23H31NO4 C23H31NO5 C23H3206 C23H3206 C23H3207

C23H3209 C24 C24H21N504 C24H23NsO4 C24H25N505

63 357 147,175, 193,351 177,181 185,197 195 149,353 355 361,363, 365,367, 369,371 273,275 121,123 117,119 125

667

668

Molecular formula

C24H3206 C24H3207 C24H3404 C24H3409 C24H34Olo

Molecular Formula Index

Page

Molecular formula

3, 7 13 9, 11 251

C28 C28H35NO6 C28H3609 C28H36010 C28H3809 C29 C29H32Oll C29H32012 C29H3409 C29H34010

259

C25

C25H3205

C25H33NO5

39 41,179 37 143, 187

C25H3407 C25H3408 C25H3609 C25H3802

5 15 257 655

C25H47NO8 C25H47NO9 C25H47NO1o

595,597 591,593 587,589

C29H3608 C29H3609

154 21 29 33 255

C29H36Olo

65 404

C29H3809

C25H3206

C25H3207

C26 C26H27NsO4 C26H3408 C26H3409 C26H3608 C26H36010 C27 C27H29NsO4 C27H3208 C27H3209

C27H32Olo C27H3408 C27H3409 C27H34010 C27H35NO7 C27H37NO6 C27H4oO5 C27H49NO9 CzyH49NO10

401,402, 408, 460, 499, 503 557 35, 183, 406 397

505 189 145 653 603,605 599, 601

C29H34Oll

C29H36012 C29H3808

C29H38010

C29H38Oll C29H4008 C29H4oO9

Page

Molecular formula

C29H4oOlo 645,649 19 25, 27 31,373 495 493 444 410,446, 448,452, 489 491,501, 507 440 436,442, 456,458, 462,513 450,454, 464,511, 515,559 509 426,430, 432,434 393,395, 422,469, 473 481,535, 537,541, 543,545, 547,551 525,527, 529,533, 539 391 375,377, 383,385, 387,412, 420,477, 479,485

C30 C30H37NO6 C30H37NO7 C30H38011 C30H45NOll C30H4802 C31 C31H40010 C31H42Oll C31H5202 C32 C32H5oO4 C32H5203 C32H5402 C33 C33H44014 C33H46Oll C33H57NOI3 C33H57NO14 C33H57NO15 C33H57NOI6 C34 C34H59NO13 C34H59NOI4 C34H59NOI5 C35 C35H48013 C35H48014 C35H5oO14 C36 C36H61NO15 C36H61NOI6 C37 C37H5807

Page 379,381, 389,483, 519,521, 523,531, 549,553, 555 643 647, 651 23 585 637 438 475 633 635 631 629 399 416,418 583 581 575 578 569 565, 567 653 428 424, 471 414 573 571 639

Molecular Weight Index Molecular weight 98.00039 178.06299 194.05791 204.18780 234.16198 236.06847 238.08412 238.19328 248.14124 250.15689 252.17254

254.07904 254.18819 264.13616 266.15181 268.16746 272.19876 282.14672 292.16746 294.18311 296.12599 298.14164 306.14672 308.16237 312.12090 316.07356 318.08921 320.06847 322.14164 324.15729 332.06847 332.08960 332.16237 334.08412 334.17802 336.09977 336.14739

Page 656 622 623 333 203 163,165 167,171 201 159, 308, 339 155, 204, 288, 335, 341, 343 133, 135, 139, 329, 331, 337, 345, 347 169 137 297, 298 153, 290, 292 157 627 239 205,207 327 311 249 217 209,211,223,225 309 69 71 87 284 213,233,235 79 113 295,300 73,95 304 75 658

Molecular weight 338.13655 340.15220 346.10525 348.06339 348.09977 348.15729 350.03459 350.07904 350.17294 352.09469 354.12145 358.14298 362.13655 364.05830 364.15220 366.07395 366.16785 366.20424 367.08178 368.08960 369.09743 369.12124 370.21441 370.28718 374.10017 378.20424 380.00877 380.14712 382.02442 382.16277 384.04007 385.22531 386.17428 386.20932 386.24571 389.27774 396.14203 398.15768 398.19407

Page 286,313,319 277,279 109 49,81 85 302 77 47,97 219,294,306 45 323 127,129 111 93 221 99 227,229,237,245 281 103 101 105 617 357 655 641 359 83 317,321 89 215,231,241,265, 267 91 185,197 63 147,175,193,351 9,11 609 315 243 263

Molecular weight 400.07943 401.22022 401.27774 401.31412 402.20424 403.0822 404.21989 408.17842 412.22497 416.07435 416.21989 419.07718 420.21480 424.17333 424.20972 427.23587 428.21989 429.25152 431.11357 431.29954 432.21480 438.18898 440.16825 440.20463 440.36543 443.15935 444.21480 444.28757 445.17500 446.23045 447.28322 452.20463 456.39673 462.22537 466.22028 470.41238 471.26209 473.20630 474.22537

Page 53 195 612 610 177,181 615 149,353,355 247 39 55,57 3,7 619 361,363,365,367, 369,371 269 253 143,187 41,179 191 621 607 13 271 283 261 637 121,123 37 653 117,119 5 611 273,275 633 15 251 629 145 61 125

669

670

Molecular weight 475.18557 476.24102 480.23593 481.24644 482.21520 484.20972 484.39165 485.24135 486.22537 487.22195 489.33017 490.22028 498.37091 500.20463 502.22028 505.32508 507.26209 508.23085 512.24102 514.25667 516.19955 516.23593 516.27230 518.21520

Molecular Weight Index

Page 23 33 257 645,649 259 404 631 189 35,183,406 65 595,597 29 635 401,402,408,460, 499,503 397 591,593 643 255 440 426,430,432,434 557 19 391 505

Molecular weight 518.25158 521.32000 523.25700 526.22028 528.23593 530.25158 531.34073 532.23085 532.26723

542.21520 544.23085 546.24650 547.33565 548.22645 548.26215 548.26723 556.19446

Page 31,373 587,589 647,651 444 436,442,456,458, 462,513 393,395,422,469, 473 603,605 25,27 375,377,383,385, 387,412,420,477, 479,485 410,446,448,452, 489 450,454,464,511, 515,559, 481,535,537,541, 543,545,547,551 599,601 519,521 379,381,483,523, 531,549,553,555 389 495

Molecular weight 558.21011 562.24142 572.18938 572.26215 574.24141 576.22068 590.27271 604.36969 614.41825 618.30401 664.27311 675.38299 676.30949 689.39864 691.37791 692.30440 694.32006 705.39356 707.37282 721.38847 723.36774 747.40412 763.39904

Page 491,501,507 525,537,529,533, 539 493 438 23 509 475 585 639 416,418 399 583 428 569 581 424,471 414 565,567 575 563 578 573 571

Fungal/Plant Source Index A

Alternaria alternata f. sp. Lycoperisci, 587, 589, 591,593,595,597, 599, 601,603, 605 A. chrysanthemi, 163 A. helianthi, 163 A. mali, 45, 47 A. radicina, 163 A. tenuis, 45, 47, 49 Apiospora camptospora, 623 Aspergillus fischerianus, 61, 63, 65 A. flavus, 658 A. fumigatus, 117, 119, 121,123, 125,127, 129, 607,609, 610, 611 A. melleus, 615,622, 623 A ochraceus, 615,617, 621,622, 623 A. oniki, 623 A. oryzae, 658 A. sulphureus, 615 A. tamarii, 658 A. ustus, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 A. versicolor, 658 B

Baccharis coridifolia, 399, 414, 424, 428, 469, 473,475,477, 479 B. coridifolia (female), 471,481,483,485 B. megapotamica, 519, 521,523,525,527, 529, 531,533,535,537, 539, 541,543,545,547, 549, 551,553,555, 557,559 Bipolaris coicis, 163, 165, 167, 169

12 Calonectria nivalis, 219, 223 Cephalosporium crotocinigenum, 295 Cercophora areolata, 53, 55, 57 Cochliobolus lunata, 163, 171 Coniothyrium palmarum, 69, 71, 73, 75 Coniothyrium sp., 77, 79, 81, 83, 85, 88, 90, 92, 94, 96, 98, 99, 101,103, 105 Cylindrocarpon spp., 430, 436, 444, 446

F

Fomitopsis pinicola, 635,637, 639 Fusarium. Acuminatum, 215,261,263,267, 279, 656 F. anthophilum, 656 F. averaceum, 265,277, 656 E Chlamydosporum, 656 E compactum, 267, 269, 277 E concolor, 656 F. crookwellense, 201,207, 209, 211,213, 217, 309, 315,317, 323,345 E culmorum, 201,207,209, 211,219, 221,223, 225,229, 231,251,253,265,286, 311,313, 345,656 E diversisporum, 237 E equiseti, 215,237, 239, 265,267,283,315, 323,656, E fusarioides, 656 E graminearum, 201,207, 223,225,229, 231, 237, 286, 309, 311,313, 321,323,345,656 E lateritium, 237,241,243,265 E moniliforme, 563,565,567, 569, 571,573, 575,581,656 E nivale, Fn-2B = sporotrichioides, 309, 323 F. nygamai, 656 E oxysporum, 578, 583,656 E poae, 249, 251,253,265 E proliferatum, 585,653,656 E reticulatum, 656 E roseum, 201,203,207,209, 211,219, 223, 227, 229, 231,233,236, 237,239, 269, 284, 286, 298, 301,311,313,321 E sambucinum, 233,236, 237,245,257,335, 339, 341,343,345,347,627 E scirpi, 237,315 E semitectum, 237, 656 E solani, 251,265 E sporotrichioides, 204, 215,249, 253,257, 259, 265,271,273,275,277,281,315,317, 323, 327, 329, 335,337, 345 Fusarium. sp., 241,243 F. subglutinans, 656 E sulphureum, 233,237,247 F. tricinctum, 215,237, 251,267, 269, 277

671

672

Fungal/Plant Source Index

G

Ganoderma mastoporum, 133, 135, 137, 139 Gibberella fujikuroi, 656 G. intricans, 237,315 It Hypocrea austro-grandis, 288 L

Lasiodiplodia theobromae, 623 Laurilia tsugicola (Echinodontium tsugicola), 153,155, 157, 159 Lobaria isidiosa, 655

P. chrysogenum, 658 P. commune, 658 P. cyclopium, 658 P. grieseofulvum, 658 P. hirsutum, 658 P. nalgiovense, 658 P. patulum, 658 P. puberulum, 658 Penicillium sp., 641 P. urticae, 658 P. viridicatum, 658 Phompsis sp., 109, 111, 113 Pisolithus tinctorius, 629, 631,633 S

M

Memnoniella echinata (JS6308), 143, 145, 147, 149 Myriococcum albomyces., 612 Myrothecium roridum, 288, 290, 351,395,397, 401,402, 412, 422, 426, 460, 462,489, 491, 493,495,499, 501,503,505, 507, 509, 511, 513,515 Myrothecium. spp., 294 M. verrucaria, 292, 351,353, 355,357, 359, 361, 363,365, 367,369, 371,373, 375,377, 379, 381,383, 385,387,389, 391,393,397,402, 404, 406, 408, 410, 412, 420, 422,426, 432, 434, 438, 440, 442

Stachybotrys atra, 452, 456 S. chartarum, 3, 5, 7, 9, 11, 15, 175,179, 181,183, 185,187, 189, 191,193,448, 450, 454, 458 S. complementi, 177 S. cylindrospora, 175, 191, 193, 205 Stachybotrys sp., 195, 197 Stemphylium radicinum, 163 T Trichoderma lignorum, 205,251 T. polysporum, 288 T. sporulosum, 288 T. viride, 205 Trichothecium roseum, 295,298, 302, 304, 306, 331,333

N

Neosartorya fischeri, 61, 63, 65

V

Verticimonosporium diffractum, 464

P

Penicillium biforme, 658 P. camemberti, 658

X

Xylaria obovata, 643, 645,647, 649, 651