Studies in Natural Products Chemistry Volume 15 Structure and Chemistry (Part C)
Studies in Natural Products Chemistry edited by Atta-ur-Rahman
Vol. 1 Stereoselective Synthesis (Part A) Vol. 2 Structure Elucidation (Part A) Vol. 3 Stereoselective Synthesis (Part B) Vol. 4 Stereoselective Synthesis (Part C) Vol. 5 Structure Elucidation (Part B) Vol. 6 Stereoselective Synthesis (Part D) Vol. 7 Structure and Chemistry (Part A) Vol. 8 Stereoselective Synthesis (Part E) Vol. 9 Structure and Chemistry (Part B) Vol. 10 Stereoselective Synthesis (Part F) Vol. 11 Stereoselective Synthesis (Part G) Vol. 12 Stereoselective Synthesis (Part H) Vol. 13 Bioactive Natural Products (Part A) Vol. 14 Stereoselective Synthesis (Part I) Vol. 15 Structure and Chemistry (Part C)
Studies in Natural Products Chemistry Volume 15 Structure and Chemistry (Part C)
Edited by
Atta-ur-Rahman
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
1995 ELSEVIER ,Amsterdam
- Lausanne
- New
York - Oxford
- Shannon
- Tokyo
ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands
ISBN: 0-444-82083-3 91995 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Printed in The Netherlands
FOREWORD
Natural product chemistry has changed dramatically over the last 50 years. The advent of modern sophisticated instrumentation and new bioassay techniques has shifted the emphasis to the structure elucidation of minor natural products, particularly those which show bioactivity. The complex structures of many of these offer challenges to synthetic organic chemists to develop synthetic approaches to them, which often leads to the development of new synthetic methods in order to achieve specific transformations. Professor Atta-ur-Rahman has done a truly remarkable job in editing this excellent series of books on natural products chemistry which has become the world's top encyclopaedic series of volumes in the field. He should be congratulated on persuading the world's top experts in natural product chemistry, both structural and synthetic chemists, to write timely and comprehensive reviews on their various areas of expertise. Another major contribution of Professor Atta-ur-Rahman is the establishment of H.E.J. Research Institute of Chemistry, a centre of excellence in natural product chemistry. He was entrusted with the task of the planning and building this Centre which he has done admirably, first as Co-Director and later as Director, and he has succeeded in putting together one of the finest products. It is therefore in the fitness of things that this institute is now known worldwide, not only because of the many books which Professor Atta-ur-Rahman has written or edited are published internationally, but also because of the excellent research articles published from H.E.J. Research Institute of Chemistry, University of Karachi. This has been possible because of the excellent research facilities (5 superconducting NMR spectrometers, 6 mass spectrometers, X-ray etc.) in the institute, quite unique for a third world country. The present volume which is the 15th in this series should prove to be of wide interest to scientists in the field and I am confident that it will receive the same excellent reviews as its predecessors. Prof. Dr. S a l i m u z z a m ~ F.R.S.
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vii
PREFACE
Natural product chemistry covers a fascinating area of organic chemistry and its study has enriched organic chemistry in a myriad of different ways. In recent years the thrust has been in three major directions: advances in stereoselective synthesis of bioactive natural products, developments in structure elucidation of complex natural products through the applications of multidimensional NMR and mass spectroscopy, and the integration of bioassay procedures with the isolation processes leading to the isolation of active principles from the extracts. The present volume reflects these developments, and there is a growing emphasis on bioactive natural products. Articles in this volume include those on structure-activity relationships of highly sweet natural products, chemical constituents of echinoderms, diterpenoids from Rabdosia and Eremophila sp., structural studies on saponins, marine sesquiterpene quinones and antimicrobial activity of amphibian venoms. The reviews on bioactive metabolites of Phomopsis, cardenolide detection by ELISA, xenocoumacins and bioactive dihydroisocoumafins, CD studies of carbohydrate-molybdate complexes, oncogene function inhibitors from microbial secondary metabolites and Gelsemium and Lupin alkaloids present frontier developments in several areas of natural product chemistry. It is hoped that the present volume, which contains articles by eminent authorities in each field, will be received with the same enthusiasm as the previous volumes of this series. I would like to express my thanks to Miss Anis Fatima, Miss Farzana Akhtar and Mr. Ejaz Ahmad Soofi for their assistance in the preparation of the index. I am also grateful to Mr. Waseem Ahmad for typing and to Mr. Mahmood Alam for secretarial assistance.
July 1994
Prof. A tta-ur-Rahman, Editor
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CONTENTS Foreword Preface
VII
Contributors
XI
Structure-activity relationships of highly sweet natural products A.D. KINGHORN, F. FULLAS AND R.A. HUSSAIN Structural studies on chemical constituents of Echinoderms L. M1NALE, R. RICCIO AND F. ZOLLO
43
Recent advances in the chemistry of diterpenoids from Rabdosia species Y. TAKEDA AND H. OTSUKA
111
Structural elucidation of saponins G. MASSIOT AND C. LAVAUD
187
The chemistry of unusual terpenoids from the genus Eremophila E.L. GHISALBERTI
225
Marine sesquiterpene/quinones R.J. CAPON
289
Antimicrobial activity of amphibian venoms G.G. HABERMEHL
327
Bioactive metabolites of the genus Phomopsis Y.S. TSANTRIZOS
341
Detection of cardenolides by Elisa in plant sciences K. YOSHIMATSU, J. SAWADA, M. JAZIRI AND K. SIIIMOMURA
361
The Xenocoumarins and related biologically active dihydroisocoumarins B.V. Mc INERNEY AND W.C. TAYLOR
381
Circular dichroism of carbohydrate-molybdate complexes z. SHAH, M. GEIGER, Y. AL-ABED, T.H. AL-TEL AND W. VOELTER
423
Screening of oncogene function inhibitors from microbial secondary metabolites K. UMEZAWA
439
Recent advances in the chemistry of Gelsemium alkaloids H. TAKAYAMA AND S. SAKAI
465
Chemistry, Biochemistry and Chemotaxonomy of Lupine alkaloids in the Leguminosae K. SAITO AND I. MURAKOSHI
519
Subject Index
551
xi
CONTRIBUTORS
Yousef A1-Abed
Physiologisch-Chemisches Institut, der Universitat, Hoppe-Seyler Strasse 4, D-7400 Tubingen 1, Germany.
Taleb H. A1-Tel
Physiologisch-Chemisches Institut, der Universitat, Hoppe-Seyler Strasse 4, D-7400 Tubingen 1, Germany.
Robert John Capon
Department of Chemistry, University of Melbourne, Parkville, Victoria 3052, Australia.
Fekadu Fullas
The Univeristy of Illinois, College of Pharmacy, 833 South Wood Street, Chicago, Illinois 60612-7231, U.S.A.
Marcela Geiger
Physiologisch-Chemisches Institut der Universitat, Hoppe-Seyler Strasse, 4 D-7400 Tubingen 1, Germany.
Emilio L. Ghisalberti
The University of Western Australia, Department of Chemistry, Nedlands, Perth, Western Australia, Australia 6009.
Gerhard Georg Habermehl
Chemisches Institut der Tierarztlichen Hochschule Hannover Bischotscholer Damm 15-3000, Hannover-1 Bischotscholer, F.R. Germany.
Raouf A. Hussain
The Univeristy of Illinois, College of Pharmacy, 833 South Wood Street, Chicago, Illinois 60612-7231, U.S.A.
Bernie Vincent Mclnerney
Biotech Australia Pty Ltd., 28 Barcoo Street, P.O. Box 20, Roseville, N.S.W 2069, Australia.
Mondher Jaziri
Head of Breeding and Physiology Lab, Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki, 305, Japan.
A. Douglas Kinghorn
The Univeristy of Illinois, College of Pharmacy, 833 South Wood Street, Chicago, Illinois 60612-7231, U.S.A.
xii Catherine Lavaud
Laboratoire De Pharmacognosie, Universite De Reims, Faculte De Pharmacie 51, Rue Cognacq-Jay, 51096 Reims Cedex, France.
Georges Massiot
Laboratoire De Pharmacognosie, Universite De Reims, Faculte De Pharmacie 51, Rue Cognacq-Jay, 51096 Reims Cedex, France.
Luigi Minale
Dipartimento di Chimica delle Sostanze Naturali Universita degli Studi di Napoli "Federico II" via D. Montesano 49, 80131 Napoli, Italy.
Isamu Murakoshi
Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Chiba 260, Japan.
Hideaki Otsuka
Institute of Pharmaceutical Sciences, Hiroshima University, School of Medicine 1-2-3, Kasumi, Minami-ku, Hiroshima 734, Japan.
Raffaele Riccio
Dipartimento di Chimica delle Sostanze Naturali Universita degli Studi di Napoli "Federico II" via D. Montesano 49, 80131 Napoli, Italy.
Kazuki Saito
Faculty of Pharmaceutical Sciences Chiba University, Yayoi-cho 1-33, Chiba 260, Japan.
Shin-ichiro Sakai
Faculty of Pharmaceutical Sciences, Yayoi-cho, Inage-ku, Chiba 263, Japan.
Jun-ichi Sawada
Head of Breeding and Physiology Lab, Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan.
Zahir Shah
Physiologisch-chemisches Institut, der Universitat, Hoppe-Seyler Strasse 4, D-7400 Tubingen 1, Germany.
Koichiro Shimomura
Head of Breeding and Physiology Lab, Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan.
H. Takayama
Faculty of Pharmaceutical Sciences, Yayoi-cho, Inage-ku, Chiba 263, Japan.
Chiba
Chiba
University,
University,
1-33
1-33
xiii Yoshio Takeda
Institute of Pharmaceutical Sciences, Hiroshima University, School of Medicine 1-2-3, Kasumi, Minami-ku, Hiroshima 734, Japan.
Walter Charles Taylor
Department of Organic Chemistry, University of Sydney, N.S.W 2006, Australia.
Youla S. Tsantrizos
Department of Chemistry and Biochemistry, Concordia University, 1455, de Maisonneuve Blvd. W. Montreal, Quebec H3G 1M8, Canada.
Kazuo Umezawa
Department of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan.
Wolfgang Voelter
Physiologisch-Chemisches Institut, der Universitat, Hoppe-Seyler Strasse 4, D-7400 Tubingen 1, Germany.
Kayo Yoshimatsu
Head of Breeding and Physiology Lab, Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan.
Franco Zollo
Dipartimento di Chimica delle Sostanze Naturali Universita degli Studi di Napoli "Federico II" via D. Montesano 49, 80131 Napoli, Italy.
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XV
ERRATUM
Stereoselective Synthesis (Part I) Studies in Natural Products Chemistry, Vol. 14 Atta-ur-Rahman (Editor) Elsevier Science B.V., 1994
The address of Professor Yoshiharu Matsubara should read as follows: Professor Yoshiharu Matsubara Department of Applied Chemistry Faculty of Science and Engineering Kinki University Kowakae, Higashi-Osaka 577, Japan The address of Professor Tetsuo Nozoe should read as follows: Professor Tetsuo Nozoe Tokyo Research Laboratories Takasago Corporation Kamata, Ohta-ku Tokyo 144, Japan
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Structure and Chemistry
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
Structure-Activity Relationship of Highly Sweet Natural Products A. Douglas Kinghorn, Fekadu Fullas and Raouf A. Hussain
1. INTRODUCTION There is an insatiable desire by humans for sweet substances, more so for the hedonic delight of the sweet taste sensation rather than for caloric needs (1). In fact, evidence has been put forth that even a five-month-old human fetus has a liking for sweet substances (2). Sucrose, the most abundant of all sugars, has been known and used as a sweetener and food item since as far back as 2,000 B.C. It is one of 100 or so saccharides that have been demonstrated to exhibit a
sweet taste, and is by far the most widely used sugar (3). Sugar cane (Saccharum officinarum L.) and sugar beet (Beta vulgaris L.) are the two major sources of sucrose. The world production of sucrose from these two sources exceeded 100 million metric tons in 1988 (4). High-fructose corn syrup (HFCS), commercially produced from corn starch, is a recently developed product used to replace sucrose in many food systems. HFCS production in the United States alone was over 11 billion pounds in 1988, with the world production of HFCS outside of the United States being 3.8 billion pounds in 1989 (5). Sucrose exhibits a clean sweetness that is unmasked by any other taste sensation. In addition to other properties, its high solubility in water, its stability under thermal and hydrolytic conditions, and its cheap cost of production make it a widely utilized sweetener (6). Thus far, no sweetener has been found, either of natural or synthetic origin, which fulfils all of the desirable properties of sucrose. Therefore, sucrose still enjoys wide popularity as a sweetener in foods, beverages and medicines. However, large amounts of sucrose are used to sweeten these products, a situation which creates consequent nutritional and medical problems. Sucrose consumption by humans has been shown definitively to be the major cause of dental caries (7), and has been associated with cardiovascular diseases, diabetes mellitus, obesity and micronutrient deficiency (8). There is an obvious need by diabetic patients to cut down on their sugar intake. Hence, there is a continuous and growing societal demand for highly sweet non-caloric and non-cariogenic sucrose substitutes. Any synthetic or natural sucrose substitute should have a sucrose-like taste profile, should have no toxic or cariogenic effects (either in the metabolized or unmodified form), should be odorless, should exhibit a liberal water solubility, and should be thermally and hydrolytically stable. A new commercially exploitable sucrose substitute should be economical to synthesize or to extract from a readily cultivable plant source. In addition to these diverse and demanding attributes, a new sweetener should be able to be easily incorporated into different food and beverage products. Finally, it should also be extremely sweet, usually at least 100 times the sweetness potency of sucrose (9). Such compounds are often referred to as "intense sweeteners",
and are generally regarded as a separate category than the caloric or "bulk" sweeteners constituted by sugars and polyols (10). The market for non-nutritive, intensely sweet substances of use as sucrose substitutes in foods, beverages, and medicines is very large, and was estimated as $1.1 billion in the United States alone in 1989 (11). Although many synthetic (1,2,9,12-17) and natural compounds (1,2,12,17-21) have been found to be intensely sweet, only a handful have wide commercial use. The major potently sweet sucrose substitutes approved for use in countries in North America and Western Europe are synthetic compounds, inclusive of saccharin, cyclamate, aspartame and acesulfame-K (10). Saccharin is approved for use in over 90 countries around the world (7). However, in 1977, the United States Food and Drug Administration (FDA) proposed a ban on the use of saccharin largely because of findings of bladder tumors in rats fed with high doses of saccharin. This ban has been lifted by a U.S. Congressional moratorium and saccharin use has been extended five times since being first imposed. The compound has now been pronounced safe by numerous expert committees (22). Cyclamate has also been associated with the production of bladder cancer in laboratory animals, and, cyclohexylamine, one of its metabolites, has been linked to additional adverse effects. While this sweetener is still approved for use in more than 50 countries, it is presently banned in Canada, the United States and the United Kingdom (11,23). The dipeptide, aspartame, is used in about 75 countries in more than 500 different products and is regarded as a very pleasant-tasting substance (7), but is contraindicated for persons suffering from phenylketonuria (24). Acesulfame-K is now employed as a sweetener in about 40 countries, and although a stable substance, a bitter taste is sometimes perceived with this compound (25). The search for synthetic sweeteners with better temporal qualities, greater sweetness potency, and improved stability and safety is continuing, and among the most promising compounds are the dipeptide, alitame (26), and the chlorinated sucrose derivative, sucralose (27), which are awaiting approval in several countries. An example of a hyperpotent sweet compound is the N-cyclononyl guanidine derivative, sucrononic acid, the sweetest compound to have been reported in the literature to date, with a potency of some 200,000 times the sweetness of sucrose (28). Whether or not such types of extremely potent sweeteners will continue to show promise during further development, and eventually enter the market, remains to be seen. As will be seen from section 3 of this chapter, there are more than 70 known plant-derived potently sweet compounds, representing about 20 structural types of organic compounds, and these occur in species of over 20 families of higher plants. Presently, several plant-derived compounds are used as sweetening and/or flavoring agents for human consumption in one or more countries, namely, glycyrrhizin (Japan), phyllodulcin (Japan), mogroside V (Japan), stevioside (Japan, Brazil and Korea), rebaudioside A (Japan) and thaumatin (Japan and U.K.), with most of these being utilized in the form of plant extracts (29,30). Also commercially used are the semisynthetic sweeteners, perillartine (Japan) and neohesperidin dihydrochalcone (Belgium and Argentina), which are based on natural products (14,31). In Japan, extracts from the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae), containing stevioside and rebaudioside A, have
the largest share in the "intense" sweetener market (29). The plant-derived commercially available
sweetening agents will be discussed briefly in the next three paragraphs. Glycyrrhizin is the sweet principle of the roots and rhizomes of Glycyrrhiza glabra L. (Leguminosae), as well as of other species in the same plant genus, and occurs as a mixture of potassium, calcium, and magnesium salts in over 10% w/w yield in the plant. This substance, an oleanane-type triterpene glycoside, is widely utilized for the sweetening of beverages, cosmetics, foods, medicines, and tobacco in Japan (32,33). Ammonium glycyrrhizin, the fully ammoniated salt of glycyrrhizic acid, is in the GRAS (Generally Recognized As Safe) list of approved natural flavoring agents of the U.S. Food and Drug Administration, and finds broad application as a foaming agent, flavor modifier, and flavorant (32,33). Extracts from Stevia rebaudiana leaves, with stevioside and rebaudioside A as the major sweet principles, have been used in Japan as a sucrose substitute for over 15 years, and are particularly advantageous because these diterpene glycosides are heat stable, nonfermentable, and suppress the pungency of sodium chloride which is used in many Japanese foods (34,35). Products made from S. rebaudiana are approved for use in the sweetening of dietetic foods, oral hygiene products, and soft drinks in Brazil (36,37), and also have minor use in South Korea (33). A third type of natural product sweetener with commercial use in more than one country is the protein, thaumatin, of which the major principles are thaumatins I and II, and is extracted from the fruits of the West African rain forest shrub,
Thaumatococcus daniellii (Bennett) Benth. (Marantaceae). Although approved for use as a sweetening agent in Japan and the United Kingdom, thaumatin is used elsewhere as a flavor enhancer and palatability improver, including the United States (29,33). There are two other plants whose highly sweet-tasting extracts are utilized on a limited basis in Japan. The first of these is the Chinese plant "1o han kuo" [Siraitia grosvenorii (Swingle) C. Jeffrey] [synonyms Momordica grosvenorii Swingle; Thladiantha grosvenorii (Swingle) C. Jeffrey] (Cucurbitaceae), whose dried fruits contain the cucurbitane-type triterpene glycoside, mogroside V, as the most abundant sweet constituent (20,33). In addition, the crushed leaves of
Hydrangea macrophylla Seringe var. thunbergii (Siebold) Makino (Saxifragaceae) are used at certain religious festivals, and their sweetness is attributed to dihydroisocoumarin, phyllodulcin (20,33). Perillartine is the semi-synthetic a-~yn-oxime of perillaldehyde, a major constituent of the volatile oil of Perillafrutescens (L.) Britton (Labiatae), and is used in Japan as a replacement for maple syrup or licorice in the sweetening of tobacco (33). Neohesperidin dihydrochalcone, prepared by the sequential alkaline hydrolysis and catalytic reduction of neohesperidin, a flavanone constituent of Citrus aurantium L. (Rutaceae), is permitted for use in chewing gum and certain beverages in Belgium and elsewhere (31). 2. THEORIES OF SWEET TASTE RESPONSE INITIATION Since sweet natural products often co-occur in complex mixtures with bitter- and/or neutral-tasting analogs, it is germane to mention briefly some of the presently held views on the mechanism of sweet taste reception. Although sweet taste chemoreception is thought to be mediated by proteinaceous receptor sites located on the microvillus membrane of taste cells of the
tongue, such a receptor has not so far been isolated and characterized (38,39). However, indirect evidence has been provided for the involvement of proteinaceous receptors in the sweet taste response. Thus, when the rat tongue was treated with specific proteases, the response to the sweet taste of sugars but not to other tastes was selectively abolished (40). A prevailing theory put forward by Shallenberger and colleagues in the late 1960's refers to the fact that nearly all sweet compounds possess two electronegative groups designated as AH and B in their molecular structures, which act as an acid and a base, respectively. The atomic orbitals of these groups should be between 2.5 and 4.5 A apart (with 2.86 A being optimal) and be in the right spatial orientation. Such an AH,B "glucophore" is considered to form a double hydrogen-bonded complex with a reciprocal AH, B unit at the sweetness receptor sites on the papillae of the tongue (41,42). [Van der Wel and colleagues (16) point out that a sweet compound contains two units, a "glucophore" and an "auxogluc". The glucophore is defined as a group of atoms capable of forming a sweet compound when combined with any auxogluc, which would otherwise be tasteless]. Although one can often discern possible AH, B units in many sweet compounds, it is not always possible to do this reliably in more structurally complex natural products such as sweet glycosides. For potent sweeteners, a third lipophilic site (X) at distances of 3.5 and 5.5 A from the AH and B units, respectively, seems to be involved in the initiation of sweet response (43,44). However, to complicate this issue somewhat, it has been postulated that as many as eight binding sites are involved in the mediation of the sweet-taste of the exceptionally potent sweetener, sucrononic acid, which, as mentioned earlier, has been rated as being about 200,000 times sweeter than sucrose (28). It is well-known that some substances exhibit a bitter-sweet taste, while sweet substances such as saccharin have some intrinsic bitterness. It is not clear, however, how these molecules distribute themselves, either with some on sweet receptors and some on bitter receptors, or else as single molecules that can span both sweet and bitter receptor sites simultaneously. However, it appears for such molecules that the corresponding sweet and bitter receptor sites must at least be very close to each other (43). As will be seen from many examples later in this chapter, minor structural modification of highly sweet natural products frequently results in the production of either bitter or tasteless analogs. There has been considerable debate for some time as to whether a single receptor or multiple receptors is(are) responsible for the initiation of sweetness (39,41). Certain evidence with inhibitors and photoaffinity labeling ligands supports the single receptor notion (39). Other authorities have postulated the existence of multiple receptors, from evidence such as the structural diversity of sweet compounds thus far discovered, from single-nerve fiber electrophysiology data, from cross-adaptation experiments, and as a result of the demonstration of synergism in sweetener mixtures (39). The possibility of the occurrence of multiple receptors for sweet substances complicates the task of new sweetener design, as a different receptor might exist for each class of sweetener. Hence, the approach in synthetic sweetener design has been to modify structural features within a given class of compounds, acting at a common receptor (14).
3.
STRUCTURE-SWEETNESS
RELATIONSHIPS
AMONG
SOME
NATURAL
SWEETENERS In this section of the chapter, the presently known highly sweet substances of natural origin are listed in Table I, and new information on the known structure-sweetness relationships for each compound category is presented in the text. In order to focus attention on naturally occurring sweet substances, the only semi-synthetic compounds included in the table are those that represent prototype members of distinct structural types. We have reviewed the various types of natural product intense sweeteners in some detail previously (20,21), and only references published subsequently to these reviews will be provided in the table. The structures of the compounds will be interspersed in the text. The following abbreviations are used to designate the sugars present in the various glycosides included in these structures:
api = D-apiofuranosyl; ara = L-
arabinopyranosyl;
D-glucuronopyranosyl;
glc
=
D-glucopyranosyl;
glcA
=
rha
=
L-
rhamnopyranosyl; xyl = D-xylopyranosyl. There has been much activity in several laboratories in recent years leading to the isolation of many novel natural product sweeteners, and it is of interest that sweet compounds are now known in three new classes, namely, the proanthocyanidin, dibenz[b,d]oxocin, and amino acid classes. It is to be noted that to date, all of the natural product sweet substances have been found as constituents of higher plants, although it is conceivable that such compounds may also occur as constituents of microorganisms, lower plants, marine animals, or insects (21). While plants in restricted taxonomic groups often biosynthesize chemically similar secondary metabolites, the distribution of plants known to produce intensely sweet plants appears to be random throughout the angiosperms. However, there is some evidence of more than one species in the same genus producing the same sweet compounds, as in the case of Glycyrrhiza and Periandra species (21). In addition, in the last few years considerably more information has become available on this phenomenon. Prior to presenting data on the sweetness potency of each compound in Table I, it is pertinent to briefly mention how sensory data of this type are obtained in the laboratory. It is highly advisable to perform experiments using human taste panels only on compounds which are pure, and for which acute toxicity and bacterial mutagenicity studies have been performed (e.g., 48,57,76,77). Such preliminary safety testing will consume a minimum of several hundred milligrams of each sweet substance examined, which is often not available for minor sweet analogs present in plant extracts. It is mainly for this reason that several of the compounds listed in Table I are indicated as being sweet, but for which no quantitative data are available. A number of approaches to determining the sensory characteristics of sweet-tasting compounds have involved quite large numbers of human subjects, and enable hedonic attributes (indicating pleasant and unpleasant flavors) as well as sweetness intensity values relative to sucrose to be obtained (e.g., 78-80). However, in the last few years in our laboratory, we have performed sensory evaluations with small taste panels consisting of only three experienced staff personnel (48,57,76,77). In this manner, approximate threshold values of sweetness intensity for a compound under test can be determined by dilution until a sweetness level equivalent to that of an aqueous solution of 2% w/v sucrose is obtained. This method is very economical in the amount of each sample consumed, and
TABLE I PLANT-DERIVED HIGHLY SWEET COMPOUNDS Compound type/name a
Sweetness potency b
Reference
370
20
1,500
20, 21
L. dulcis
N.S. d
48
Pine tree f
1,3001,800g
20
Stevia rebaudiana (Bertoni) Bertoni
30
20
Plant name
MONOTERPENE Perillartine c (1)
Perilla frutescens Britton
(Labiatae)
SESQUITERPENES Hernandulcin (2)
Lippia dulcis Trev.
(Verbenaceae) 41]-Hydroxyhernandulcin
(3) DITERPENES Diterpene acid 4~, 10o~-Dimethyl-1,2,3,4,5,10 hexahydrofluorene-4ct,6ctdicarboxylic acid e (4) ent-Kaurene glycosides
Dulcoside A (5)
(Compositae) Rebaudioside A (6)
S. rebaudiana
242
20
Rebaudioside B (7)
S. rebaudiana
150
20
Rebaudioside C (8)
S. rebaudiana
30
20
Rebaudioside D (9)
S. rebaudiana
221
20
Rebaudioside E (10)
S. rebaudiana
174
20
Stevioside (11)
S. rebaudiana
210
20
Rubusoside (13)
Rubus suavissimus S. Lee
114
20
(Rosaceae) Steviolbioside (12)
S. rebaudiana
90
20
Steviol 13-O-13-D-glucoside (14)
Rubus suavissimus
N.S. d
50
Suavioside A (15)
R. suavissimus
N.S. d
49
Suavioside B (16)
R. suavissimus
N.S. d
50
Suavioside G (17)
R. suavissimus
N.S. d
50
TABLE I (continued) PLANT-DERIVED HIGHLY SWEET COMPOUNDS Compound type/name a
Plant name
Sweetness potency b
Reference
ent-Kaurene glycosides (continued)
R. suavissimus
N.S. d
50
Suavioside I (18)
R. suavissimus
N.S. d
50
Suavioside J (20)
R. suavissimus
N.S. d
50
Baiyunoside (21)
Phlomis betonicoides Diels (Labiatae)
5OO
20
Phlomisoside I (22)
P. betonicoides
N.S. d
21
Gaudichaudioside A (23)
Baccharis gaudichaudiana DC. (Compositae)
55
51
Bryodulcoside h
Bryonia dioica Jacq. (Cucurbitaceae)
N.S. d
20
Bryoside (24)
B. dioica
N.S. d
52
Bryonoside (25)
B. dioica
N.S. d
52
Carnosifloside V (26)
Hemsleya carnosi.flora C.Y. Wu et Z.L. Chen (Cucurbitaceae)
51
21, 53
Carnosifloside VI (27)
H. carnosiflora
77
21
Scandenoside R6 (28)
Hemsleya panacis-scandens C.Y. Wu et Z.L. Chen
54
53, 54
Mogroside IV (29)
Siraitia grosvenorii i (Swingle) C. Jeffrey (Cucurbitaceae)
233-392g
20, 54
Mogroside V (30)
S. grosvenorii
250-425g
20
Siraitia siamensis Craib (Cucurbitaceae) S. grosvenorii
N.S. d
55
84
54
S. siamensis S. grosvenorii
563
54, 55
Suavioside H (19)
Labdane glycosides
TRITERPENES Cucurbitane glycosides
11-Oxomogroside V (31)
Siamenoside I (32)
10 TABLE I (continued) PLANT-DERIVED HIGHLY SWEET COMPOUNDS Compound type/name a
Plant name
Sweetness potency b
Reference
Cycloartane glycosides Abrusoside A (33)
A brus precatorius L.; A. fruticulosus Wall et W. & A. (Leguminosae)
30
56-58
Abrusoside B (34)
A. precatorius; A. fruticulosus
100
57, 58
Abrusoside C (35)
A. precatorius; A. fruticulosus
50
57, 58
Abrusoside D (36)
A. precatorius; A. fruticulosus
75
57, 58
Glycyrrhizin (37)
Glycyrrhiza glabra L. (Leguminosae)
93
20
Apioglycyrrhizin (38)
Glycyt~hiza inflata Batal
180
59
Araboglycyrrhizin (39)
G. #~ata
93
59
Periandrin I (40)
Periandra dulcis Mart. (Leguminosae)
90
20
Periandrin II (41)
P. dulcis
95
20
Periandrin III (42)
P. dulcis
92
20
Periandrin IV (43)
P. dulcis
85
20
Periandrin V (44)
P. dulcis
N.S. d
60
Osladin (45)
Polypodium vulgare L. (Polypodiaceae)
500
20, 61
Polypodoside A (46)
Polypodium glycylT"hiza DC. Eaton
600
62, 63
Polypodoside B (47)
P. glycytT~hiza
N.S. d
63, 64
Foenicuhtm vulgate Mill. (Umbelliferae)
13
65
Oleanane glycosides
STEROIDAL SAPONINS
PHENYLPROPANOIDS trans-Anethole (48)
Illicmm verum Hook. f. (Illiciaceae) Mytwhis odorata Scop. (Umbelliferae)
TABLE I (continued) PLANT-DERIVED HIGHLY SWEET COMPOUNDS Compound type/name a
Plant name
Sweetness potency b
Reference
13
65
50
21
400
20
300
20
Citrus aurantium L.
1,000
20
Smilax glycyphylla Sm.
N.S. d
20
N.S. d
20
Symplocos microcalyx Hayata
N.S. d
20
Tessaria dodoneifo#a
400
21
PHENYLPROPANOIDS (continued)
trans-Anethole (48)
Osmorhiza longistylis DC.
(continued)
(Umbelliferae)
Piper marginatum Jacq. (Piperaceae)
Tagetesfificifo#a Lag. (Compositae)
trans-Cinnamaldehyde (49)
Cinnamomum osmophloeum Kanehira (Lauraceae)
DIHYDROISOCOUMARIN
Phyllodulcin (50)J
Hydrangea macrophylla Seringe var. thunbergii (Siebold) Makino (Saxifragaceae)
FLAVONOIDS Dihydrochalcone glycosides Naringin dihydrochalcone c
(sl)
Neohesperidin dihydro-
Citrus paradisi Macfad. (Rutaceae)
chalcone c (52) Glycyphyllin (53)
(Liliaceae) Phlorizin (54)
Symplocos lancifolia Sieb. et Zucc. (Symplocaceae)
Trilobatin (55) Dihydroflavonols and Dihydroflavonol glycosides Dihydroquercetin 3-0acetate 4'-(methyl ether) c
(s6)
(Hook. & Arn.) Cabrera (Compositae)
12 TABLE I (continued) PLANT-DERIVED HIGHLY SWEET COMPOUNDS Compound type/name a
Plant name
Sweetness potency b
Reference
Dihydroflavonois and Dihydroflavonol glycosides (continued) (2R,3R)-Dihydroquercetin 3-O-acetate (57)
T. dodoneifo#a; Hymenoxys turneri K. Parker (Compositae)
80
21
(2R,3 R )-2,3-Dihydro-5,7, 3',4'tetrahydroxy-6-methoxy-3-Oacetylflavonol (58)
H. turneri
25
66
(2R,3R)-2,3-Dihydro-5,7,4'trihydroxy-6-methoxy3-O-acetylflavonol (59)
H. tmTwri
20
66
(2R,3R)-2,3-Dihydro5,7,3 ',4'-t etrahydroxy6-methoxyflavonol (60)
H. turned
15
66
Neoastilbin (61)
Engelhat'dtia cht),solepis Hance (Juglandaceae)
N.S. d
67
Huangqioside E (62)
E. chlysolepis
N.S. d
68
Cinnamtannin B-1 (63)
Cimlamomum sieboldii Meisner (Lauraceae)
N.S. d
69
Cinnamtannin D- 1 (64)
C. sieboldii
N.S. d
69
Unnamed (65)
Arachniodes sporadosora Nakaike; A. exifis Ching (Aspidiaceae)
N.S. d
70
Unnamed (66)
A. sporadosora; A. exifs
N.S. d
70
Selligueain A (67)
Selliguea feei (Polypodiaceae)
35
71
Haematoxylon campechianum L. (Leguminosae)
N.S. d
72
PROANTHOCYANIDINS
BENZIbllNDENOII,2-d]PYRAN Hematoxylin (68)
13 TABLE I (continued) PLANT-DERIVED HIGHLY SWEET COMPOUNDS Compound type/name a
Plant name
Sweetness potency b
Reference
AMINO ACID Monatin (69)
Schlerochiton ificifolius A. Meeuse
1,2001,400g
73
(Acanthaceae)
Curculigo latoeolia Dryand.
550
74
N.S. d
21
3,000
20
500
21, 75
1,600
20
PROTEINS
Curculin
(Hypoxidaceae) Mabinlin
Capparis masaikai Levl. (Capparidaceae)
Monellin
Dioscoreophylhtm cumminsii (Stapf) Diels. (Menispermaceae)
Pentadin
Pentadiplandra brazzeana Baillon (Pentadiplandraceae)
Thaumatin
Thaumatococcus daniellii (Bennett) Benth. (Marantaceae)
aStructures of compounds are shown in the text. bValues of relative sweetness on a weight comparison basis to sucrose (= 1.0) are taken from the respective literature data, or from ref. 20. Compounds may have been converted to more watersoluble salts, prior to sensory evaluation. CSemi-synthetic derivative of natural product. dSweetness potency not given. e Synthetic sweetener. fBinomial name not given. gRelative sweetness varied with the concentration of sucrose. hComplete structure and stereochemistry not yet determined. 1 Formerly named Momordica gT~osvenorii Swingle, and Thladiantha grosvenorii (Swingle) C. Jeffrey (33). JThe plant of origin has to be crushed or fermented in order to generate phyllodulcin.
14 provides at least some information on compound taste qualities other than sweetness. The sensory data for the sweet plant-derived compounds in Table I refer to sweetness intensity comparisons with sucrose on a weight basis. However, comparison of these data is most reliable for compounds in the same structural class that have been evaluated for sweetness intensity in the same laboratory. Also, sweetness intensity values tend to vary depending upon concentration of the tastant compound. For example, the sodium salt of the newly discovered amino acid, monatin (69) exhibited relative sweetness intensities to 5% and 10% w/v sucrose of 1,400 and 1,200, respectively (73). Therefore, for this compound, and for several others listed in Table I, sweetness intensity values are expressed as ranges. 3.1 Terpenoids and Steroids 3.1.1 Monoterpenoids. It was mentioned earlier in this chapter that the semi-synthetic oxime, perillartine (1), is sweet and has some commercial use in Japan. In experiments designed to optimize the sensory attributes of the oxime sweeteners, it was found that the introduction of ether groups was advantageous, while hydroxyl groups and ring oxygen atoms tended to lower sweetness intensity and to destroy the sweet taste, respectively (16). Despite the discovery of cyclic derivatives of perillartine that are sweeter than this lead compound (20), the further development of this class of sweet substances is limited by poor water solubility and inappropriate hedonic qualities (20,21).
OH I N
) ~CH, 2
3.1.2 Sesquiterpenoids. The bisabolane-type sesquiterpene alcohol, (+)-hernandulcin (2), has been rated by a taste panel as being about 1,500 times sweeter than 0.25 M sucrose on a weight basis, but has also some bitterness and distinct off- and attertastes (20,21). This novel compound was isolated in 1985 from Lippia dttlcis, a plant recognized as being sweet by the 16th century Spanish physician, Francisco HernS.ndez. Accordingly, the compound was named in Hern/mdez' honor (20,21). Both racemic hernandulcin and the (+)-isomer have been synthesized by several other groups, and the absolute configuration of the naturally occurring form has been shown as 6S,1'S (48,81). It has also been established that 6S, l'S-hernandulcin is the only one of the four possible diastereomeric forms of this substance to be intensely sweet (81). This sweet substance has been produced from both hairy root cultures and shoot cultures ofL. dulcis, with a
15 yield of as high as 2.9% w/w dry weight being obtained in the latter case (82,83). Although hernandulcin was obtained in very low abundance (0.004% w/w) when first isolated (80), this sweet sesquiterpenoid was afforded in a much higher yield (0.15% w/w) from the leaves and flowers of a sample of L. dulcis collected in Panama (48), thereby suggesting that it occurs at high levels when the plant is flowering.
0
OH
2
R=H
3
R =OH
Attempts were made in this laboratory to synthesize sweet-tasting analogs of hernandulcin with improved hedonic characteristics, but resulted in the production of no further derivatives with a sweet taste. Thus, acetylation of the tertiary alcohol unit at C-I' and reduction of the C-1 keto group abrogated any perception of sweetness in each case (76). A series of racemic hernandulcin analogs was prepared by directed-aldol condensation between appropriate starting ketones, according to reaction conditions worked out for the synthesis of the parent compound (77,80). It was found that, even when the C-1 keto and the C-1' hydroxyl groups were kept intact, removal of the double bonds between either C-2 and C-3, and C-4' and C-5', or the methyl groups attached to C-3 or C-5', or the lipophilic side-chain, or substitution of the cyclohexenone ring with a cyclopentenone ring, led to the generation of mainly bitter-tasting analogs (77). As a consequence of this work, and as a result of accompanying molecular modeling experiments, it was concluded that the C-1 keto group, the C-I' tertiary hydroxyl group, and the double bond between carbon atoms C-4' and C-5' are three structural units involved in the binding of hernandulcin to its putative receptor (77). A compound isolated and characterized from the Panamanian collection of L. dulcis referred to earlier is a second highly sweet bisabolane sesquiterpenoid, 413-hydroxyhernandulcin (3). The sweetness potency of this substance relative to sucrose was not determined because of the very small quantity isolated. However, this isolate is noteworthy since it demonstrates that a C-4 methylene unit is not essential for the mediation of the sweet taste of the hernandulcin-type natural product sweeteners, and also provides a possible point-of-attachment for sugars or other polar moieties, in order to render more water-soluble sweet hernandulcin analogs (48). Despite its high sweetness intensity, hernandulcin is limited as a potential sweetener because of its somewhat unpleasant hedonic attributes and its thermolability. In spite of this, a
16 dentifrice formulation containing menthol and some cyclic ketones has recently been developed to both mask the taste ofhernandulcin and to afford storage stability (84). 3.1.3 Diterpenoids. Perusal of Table I shows that the known sweet diterpenoids from plants can be classified into the tricyclic resin acid (4), and ent-kaurene (5-20) and labdane (21-23) glycosides. Despite being a very promising lead because of its sweetness potency (20,21), there appears to have been no further work performed on developing analogs of resin acid 4 in recent years. Therefore, this substance will not be discussed further in the present chapter. In the following paragraphs, the sweet-tasting ent-kaurene and labdane diterpene glycosides will be discussed in turn.
,,
-',,.OO'H'COOH
Structurally closely related, potently sweet ent-kaurene glycosides are found in high concentration levels in the leaves of two taxonomically disparate species, Stevia rebaudiana (Compositae) and Rubus suavissimus (Rosaceae), which are native to the borders of Paraguay and Brazil, and the People's Republic of China, respectively (20,21). It is interesting to note that no other species in either of the genera Stevia or Rubus appear to accumulate sweet ent-kaurene glycosides in significant amounts, although these compounds have been detected in trace quantities in a Mexican species, Stevia phlebophylla A. Gray (85,86). Documentation has come to light recently indicating that the leaves of S. rebaudiana have been used by Guarani Indians, Mestizos, and local herbalists in Paraguay to sweeten beverages for at least 100 years (87). Chemical work to determine the structural nature of the sweet constituent or constituents of S. rebaudiana leaves began in the early years of the present century, but the structure of stevioside (11) was not correctly determined until some sixty years later (20,21,34). During the 1970's, additional sweet compounds were isolated and characterized from S. rebaudiana leaves by the Tanaka group at Hiroshima University in Japan, inclusive of rebaudioside
A (6), which is sweeter and more pleasant-tasting than stevioside. The sweet ent-kaurene glycosides occur at remarkably high yields in dried S. rebaudiana leaves, with the four major glycosides being stevioside (5-10% w/w), rebaudioside A (2-4% w/w), rebaudioside C (9, 1-2% w/w), and dulcoside A (5, 0.4-0.7% w/w) (34). As noted earlier, S. rebaudiana extracts, as well as stevioside and rebaudioside A, have use in Japan for sweetening purposes, and are also commercially utilized in other countries (29,30,34,35).
17
[~"~OR2 - ~ CH2 ~FOOR1
/+%H
__
R1
R2
5
I]-glc
13-glc~-rha
6
]3-glc
13-glcZ---13-glc
13
[3-glc 7
H
13-glc~13-glc
8
13-glc
~3-glc~oc-rha
13-glc2l]-glc
~3-glc [3-glc~13-glc
-glc
9
13
13
13-glc 10
13-glc~13-glc
13-glcZ---13-glc
11
13-glc
13-glc~l]-glc
12
H
13-glc~13-glc
Sweetness potency figures for the eight individual sweet diterpene glycosides (5-12) so far isolated and characterized from S. rebaudiana leaves are shown in Table I. Thus, it may be observed that the more highly branched compound, rebaudioside A (6), is somewhat sweeter than stevioside (11), and that a similar relationship holds true for the minor S. rebaudiana constituents, rebaudiosides D (9) and -E (10), both of which are highly sweet. Removal of the C-19-affixed Dglucosyl groups of rebaudioside A and stevioside to produce rebaudioside B (7) and steviolbioside (12), respectively, which can be performed by alkaline hydrolysis, results in a diminution of sweetness potency in both cases. Substitution of one of the glucose units in the C-13-attached saccharide moiety by rhamnose of rebaudioside A and stevioside, as in rebaudioside C (8) and dulcoside A (5), results in even greater reduction in sweetness potencies (Table I). In addition, rebaudioside C has been demonstrated to exhibit pronounced bitter properties. While the other S.
18 rebaudiana sweeteners are less bitter than rebaudioside C, many of them have undesirable
aftertastes (20,21). Rubusoside (13; = desglucosylstevioside) is found in the dried leaves of Rubus suavissimus in a yield of over 5% w/w, and has been rated as being about 115 times sweeter than sucrose, but, like stevioside, it has some bitterness and an at~ertaste. Recently, additional analogs of rubusoside have been isolated as minor constituents ofR. suavissimus leaves (49,50). One of these glycosides, suavioside A (15), was found to be sweet, while an analog with a keto group replacing the secondary alcohol group at the 3-position (sugeroside) was bitter (49). Compared with rubusoside and the sweet ent-kaurene glycosides from S. rebaudiana, suavioside A lacks an exomethylene functionality at C-16 and the position of the sugar moiety is translocated from C-13 to C-17 (49). Six additional minor diterpene glycoside constituents of R. suavissimus have been structurally determined recently, namely, steviol 13-O-]3-D-glucoside (14; = steviolmonoside) and suaviosides B, G, H, I and J (16-20) (50). Although quantitative sweetness potency values have not been determined for the minor sweet diterpene constituents ofR. suavissimus, suavioside B (16), which differs from rubusoside (13) only in the possession of a 913-hydroxyl group, is considerably less sweet (50). Several additional bitter- and neutral-tasting ent-kaurene diterpenoids were also obtained from R. suavissimus leaves (50).
-
,,,,
9
/OR2
~~1CH2
COOR1
]9
R1
R2
R3
13
[3-glc
]3-glc
14
H
]3-glc
H
16
[3-glc
]3-glc
OH
19
~,,,,CH2OR /"'OH HO
,,,,,,, 15
R = 13-glc
/OR2 ,~,,,, 9 CH2R1 /"OH
R1
R2
R3
17
H
13-glc
]3-glc
18
OH
H
]3-glc
There have been several attempts to improve the organoleptic properties of stevioside and rubusoside in the last ten years. In one such study, a disulfonic acid derivative of stevioside with increased hydrophobicity was found to be devoid of any bitter taste (88). The Tanaka group at Hiroshima, however, has improved the taste qualities of both stevioside and rubusoside by enzymic ~-(1-->4)-transglucosylation using cyclodextrin glucanotransferase (89-92). For example, 1,4-o~mono-, di-, tri- and higher glucosylation occurred at both the 13-O-glucosyl and the 19-COO-[3glucosyl moieties of stevioside, when treated with enzyme, leading to a mixture of glycosylated products consisting of nearly ten components (92). Most of these were less intensely sweet than stevioside, but more pleasant-tasting Sweetness intensity was optimal with three or four glucosyl units attached to the C-13 hydroxyl group (92). It appears that a sweet-tasting glycoside mixture obtained from stevioside in this manner now has commercial value in Japan, where it is known as "glucosyl stevioside" (92).
20 /OR2
:
R1
.....
/,~R1
R2
R3
19
CHO
13-glc
13_glc
20
CH2OH
13-glc
~-glc
Baiyunoside (21) is a sweet constituent of the Chinese medicinal plant, Phlomis betonicoides Diels, that was first identified by the Tanaka group at Hiroshima. This substance has
been rated as having a sweetness potency of about 500 times greater than that of sucrose, although it has a lingering aftertaste lasting for more than an hour (20,21). Phlomisoside I (22) is based on the same aglycone, baiyunol, as baiyunoside, and is also sweet-tasting. This compound bears a neohesperidyl saccharide unit, and may be contrasted with another P. betonicoides glycosidic constituent, phlomisoside II, which is bitter, and differs only from 22 in possessing a sophorosyl (13-D-glucopyranosyl-(2~ 1)-13-D-glucopyranosyl) sugar unit (20,21). Nishizawa and co-workers at Tokushima Bunri University in Japan have performed some substantial work on the preparation of analogs of baiyunoside, having initially prepared racemic baiyunol by the catalytic cyclization of 13-oxoambliofuran with a mercury (II) triflate/N,N-dimethylaniline complex (93), and then producing baiyunoside by a novel 2'-discriminated glucosidation procedure (94). Altogether, over 20 glycosides based on the baiyunoside parent molecule have been prepared, many of which were bitter while others were sweet. It was found that A7,8-baiyunoside was as sweet as baiyunoside itself, and, rather surprisingly that the sweetest compound of all derivatives made was the corresponding 13-D-glucopyranosyl-o~-D-glucopyranosyl analog of the previously mentioned bitter compound, phlomisoside II (95). The latter compound was reported as being very expensive to produce (95), which probably precludes its possible commercial development.
21
RO"
~,,,,
v
21
R = ~-glc2~3-xyl
22
R = [3-glc2ct-rha
In recent work carried out at our institution, a novel labdane diterpene arabinoside, gaudichaudioside A (23) was obtained as the sweet principle of
Baccharis gaudichaudiana
DC.
This observation is unexpected, since other species in the same genus taste very bitter rather than sweet. The plant was identified as being sweet-bitter-tasting after ethnobotanical inquiries at a medicinal plants' market at Asuncion, Paraguay, where it was referred to as "chilca melosa" and used traditionally as an antidiabetic remedy (51). The plant was collected in the field from a native population in eastern Paraguay, and found to exhibit a predominantly sweet taste, accompanied by some bitterness. In the laboratory, the sweet effect was traced to a 1-butanol extract, thereby suggesting the compound (or compounds) responsible was glycosidic. Gaudichaudioside A (Fig. 4, 23) was found to be based on a normal labdane skeleton, and to possess an L-arabinopyranosyl unit that is substituted equatorially, as well as having two double bonds, an unsaturated aldehyde affixed to C-8, and two hydroxymethyl groups attached to C-15 and C-19. Gaudichaudioside A exhibited about 55 times the sweetness intensity of a 2% w/w aqueous sucrose solution, when evaluated by a human taste panel. At the concentration at which it was tested, the compound gave only a very low perception of bitterness (51).
~
2 HOH2
,~CHO "
23
OR
R = o~-ara
CH20H
15
22 Attempts to purify the aglycone of gaudichaudioside A (23) using mineral acids and various enzymes were not successful due to apparent lability. Therefore, it has not yet proven possible to synthesize potentially sweeter analogs of this parent compound with longer saccharide moieties, in an analogous manner to the work previously performed on baiyunoside (21) that has just been described. However, a series of five diterpene arabinosides closely related structurally to the parent compound
has been isolated from ethyl acetate-
and butanol-soluble
extracts
of B.
gaudichaudiana, and were named gaudichaudiosides B through F (51,96). It was found that while only gaudichaudioside A was highly sweet, these other compounds demonstrated a range of taste effects. Thus,
substitution of the C-8-affixed aldehyde of gaudichaudioside A with a
hydroxymethyl group as in gaudichaudioside B (0.5% w/v in water) resulted in a sweetness sensation lasting for a few seconds followed by prolonged bitterness, when tasted (51). Substitution of gaudichaudioside B with an o~-substituted secondary hydroxyl group at C-2, as in gaudichaudioside C, resulted in a tasteless derivative. The other compounds in this series, inclusive of gaudichaudioside F, which is based on a novel trihomolabdane skeleton, gave either sweet-bitter or entirely bitter taste responses (51,96). Thus, the prototype labdane diterpenoid arabinoside, gaudichaudioside A (23) remains the only highly sweet compound in this class discovered to date. At one time, B. gaudichaudiana was taxonomically classified as a varietal form of
Baccharis articulata (Lam.) Pers., a widely utilized medicinal plant in several South American countries. However, when B. articulata was examined for its constituents, neither gaudichaudiosides A-F nor any labdane diterpenoids were found to be present. These observation thereby offer chemotaxonomic substantiation for classifying these two taxa as separate species (97). 3.1.4 Triterpenoids. It may be seen from Table I that more highly sweet triterpenoids are now known than any other class of natural product. Furthermore, sweet compounds of this type are now based on three distinct triterpene carbon skeletons, namely, cucurbitane (24-32), cycloartane (33-36), and oleanane (37-44). In the latter category, two distinct groups are now evident, analogs of glycyrrhizin and of periandrin I. These groups of natural sweeteners will be discussed in turn. One of the most fascinating sweet-tasting species that has been encountered thus far is the Chinese medicinal plant "1o han kuo", which has been used for centuries for the treatment of colds, sore throats, and minor gastro-intestinal complaints. The fruits of this vine are dried in large ovens before being used in commerce, a fact which testifies to the thermal stability of the sweet components. However, this plant was not studied botanically until the 1930's, and proved to be a new species when first examined in 1937 (20). Originally called Momordica grosvenorii, then later Thadiantha grosvenorii, the plant is now correctly referred to as Siraitia grosvenorii (20). Phytochemical work commenced on S. grosvenorii in the 1970's and the structures of the two major sweet constituents of "1o han kuo" as mogroside IV (29) and V (30) were determined by Takemoto and Arihara and colleagues at Tokushima Bunri University in Japan (20,21). However, the major sweet constituent is mogroside V, and it occurs in over 1% w/w yield in S. grosvenorii fruits (20). This compound is one of the sweetest natural products, and has been rated in a range of 256-425 times the sweetness potency of sucrose (20,54). Mogroside IV, with one D-glucose unit
23 less in its structure than mogroside V, is slightly less potently sweet.
OH
H
o"%0
25
R2
O
P
_
7"%,,,
RiO"
R1
R2
24
13-glcLo~-rha
25
13-glcLo~-rha
13-glc 13-glcLI3-glc
~176 ~
92
HOoso0
o
R1
,.,.,.,
i
R1
R2
R3
26
13-glc
CH20-l]-glc2--13-glc
CH3
27
13-glc
CH20-I]-glc613-glc
CH3
28
13@c
CH3
CH20-13-glc 213-glc
24
OR2 H
R
R1o
....,,,
R1
R2
R3
29
13-glc613-glc
13-glc~13-glc
oc-OH, [~-H
30
13-glc613-glc
[3-glc~13-glc
c~-OH, I3-H
31
13-glc 13-glc6--13-glc I3-glc~13-glc
32
16
16
13-glc
13-glc 13-glc~13-glc
=o
c~-OH, I3-H
13-glc
Following the discovery of the mogrosides IV and V, the cucurbitane triterpenes have emerged as a large group of natural sweeteners. Phytochemical investigation of the roots of Bryonia dioica has led to the isolation and characterization of two sweet compounds, bryoside (24) and bryonoside (25). Both of these compounds were isolated and structurally determined earlier by Hylands and Kosugi (98), but the structure of the sugar unit attached to C-25 in bryonoside was revised (52). No information was provided about the relative sweetness potencies of these substances (52). Three sweet natural product cucurbitane glycosides have been purified from two species in the genus Hemsleya by the Tanaka group at Hiroshima (26-28), and were found to co-occur with several analogs that were either bitter-or neutral-tasting (21,53,54). However, none of these compounds, nor several sweet semi-synthetic analogs were found to be of very high sweetness potency (53,54). Two minor sweet cucurbitane glycosides were isolated from the fruits of Siraitia grosvenorii, namely, l l-oxomogroside V (31) and siamenoside I (32). Siamenoside I, which is identical in structure to mogroside V (30), except for being only monoglucosylated at the C-3 position, is the sweetest compound among the cucurbitane glycosides discovered so far, in having a sweetness potency rated as 563 times that of sucrose (54). Compounds 29 through 32 were earlier isolated from Siraitia siamensis by Tanaka and co-workers (55). It has been determined that in order to exhibit a sweet taste in this class of compounds, at
25 least three sugar units must be present in the molecule, and glycosides of 1 l o~-hydroxy, 1113hydroxy, and 11-keto compounds are, respectively, highly sweet, neutral-tasting, and less highly sweet or bitter (53,54). Abrusosides A-D (33-36) are a group of recently discovered cycloartane-type triterpene sweeteners, that were first isolated from the leaves of Abrus precatorius (56,57). Although the seeds of this species are well-known to produce the ribosome-inactivating protein toxin, abrin, the leaves of A. precatorius do not appear to be poisonous, and are ingested without apparent harm in systems of traditional medicine in certain southeast Asian countries. The well-defined sweetness of the leaves has been frequently documented in the scientific literature as being due to the presence of the oleanane-type triterpene sweetener, glycyrrhizin (see below). However, analysis of a sample of the leaves ofA. precatorius collected in Florida did not reveal the presence of glycyrrhizin, but the new compounds abrusosides A-D were found to occur. These compounds are similar in polarity to glycyrrhizin, and were extracted from a 1-butanol extract of A. precatorius leaves. Abrusoside A (33), the least polar representative of this series, was found to possess a cyclopropyl ring and an tx,[3-unsaturated 8-1actone ring, as well as an unsubstituted carboxylic acid unit at C29, and was glucosylated at the C-3 position. Abrusoside A was shown to be based on a new carbon skeleton, and the structure of its aglycone was confirmed atter the performance of singlecrystal X-ray crystallography on the methyl ester (56). The three other analogs isolated from A.
precatorius leaves, abrusosides B-D (34-36), all possess a disaccharide unit affixed to C-3 and are sweeter than abrusoside A. 0
"
H
RO
0
I
_
"'COOH 29
33
13-glc
34
13-glcA-6-CH3~13-glc
35
[3-glc2--13-glc
36
J3-glcALf3-glc
26 The two sweetest compounds in this series are those possessing glucuronic acid units, namely, abrusoside B and abrusoside D, with the former compound with one D-glucuronic acid methyl ester being more potent. Abrusoside C, with a sophorosyl sugar unit, was intermediate in sweetness potency between abrusoside D and abrusoside A. In A. precatorius leaves, the most abundant of these compounds was abrusoside D (57). All four compounds have also been detected in the leaves of a second species, A. fruticulosus, with abrusoside B being the most abundant representative (58). The abrusosides are very stable to heat, and can be made water-soluble by conversion to their ammonium salts, and do not seem to have a bitter taste accompanying their sweetness (57). As mentioned earlier, the oleanane triterpene diglucuronate, glycyrrhizin (37) and its ammonium salt, are widely used for sweetening and flavoring purposes (20,21). While two previous semi-synthetic studies that modified the saccharide substitution of glycyrrhizin to improve its sweetness potency were inconclusive (99,100), a more highly sweet analog has recently been isolated from Glycyrrhiza #~ata roots (59). Thus, apioglycyrrhizin (38) was rated as exhibiting about twice the sweetness potency of the parent substance, and a further isolate from this plant source, araboglycyrrhizin (39), exhibited comparable sweetness intensity to glycyrhizin itself (59). The periandrins are a further group of oleanane triterpenes, and were first isolated from Periandra
dulcis, and they are present in P. mediterranea (20). Periandrins I-IV (40-43) were characterized in the early 1980's by Hashimoto and colleagues at Kobe Women's College of Pharmacy in Japan, and all have about the same sweetness potency of glycyrrhizin (Table I). Recently, periandrin V (44) was obtained as a further sweet constituent of P. dulcis roots, and it was found that the terminal D-glucuronic acid sugar residue of periandrin I was replaced by D-xylose. However, the sweetness intensity of compound 44 relative to sucrose has not yet been determined (60).
%
RO
%
37
13-glcALI3-glcA
38
13-glcA213-api
39
13-glcALot-ara
COOH
27
HOOC,% H
R1
R2
40
13-glcALl3-glcA
CHO
42
I]-glcALl]-glcA
CH20H
44
13-glcA2 13-xyl
CHO
HOOC,
"s
R2 RiO
R1
R2
41
]3-glcA213-glc
CHO
43
~3-glcALl3-glcA
CH2OH
3.1.5 Steroidal Saponins The fern genus,
Polypodium, has so far yielded three sweet
steroidal saponins, namely, osladin (45) and polypodosides A and B (46, 47). The first-named of these compounds was structurally determined without full stereochemistry as an isolate of P.
28
vulgare by Herout and co-workers at the Czechoslovak Academy of Sciences in Prague in 1971, with the configuration of the aglycone later determined by partial synthesis from solasodine (20,21). However, Nishizawa and co-workers have recently established the correct structure of osladin as 45, after isolation from the plant and single-crystal X-ray diffraction, thereby reversing the stereochemistry from that originally proposed at positions C-22, C-25, and C-26. In addition, the configuration of the C-26-affixed rhamnose unit was assigned for the first time for osladin (61). The same group has also established that the actual sweetness potency of osladin relative to sucrose is 500 times (61), and not the higher figure of 3,000 as widely quoted in the literature (20,21). The compound has been produced from a steroidal aldehyde by total synthesis, using a triflic acid-catalyzed 2'-discriminated and [3-selective glucosylation in addition to an a-selective thermal rhamnosylation (101). At this institution, we have examined the rhizomes of the North American fern,
Polypodium glycyl~hiza, and isolated three novel steroidal saponins, which have been called polypodosides A-C (62-64). The major sweet-tasting constituent of P. glycyrrhiza rhizomes is polypodoside A, which is based on a known aglycone, polypodogenin, a compound previously assigned by Czechoslovak workers as the A7-8-derivative of the aglycone of osladin (62). However, in view of the recent structural revision for osladin, it has been necessary to revise the stereochemistry of polypodoside A at the three asymmetric centers in the pyran ring in the aglycone (46). This was done on the basis of comparing the ]3C-NMR spectrum of polypodoside A with that of authentic osladin (63), and similar reasoning has been used to revise the structure of a second sweet constituent ofP. glycyrrhiza, polypodoside B (47).
O R2
y-
H
RIO 0
R]
R2
Other
45
13-glc~ot-rha
c~-rha
7,8-dihydro
46
]3-glc~c~-rha
ot-rha
47
[3-glc
ot-rha
--
29 Polypodoside A was rated as exhibiting 600 times the sweetness intensity of a 6% w/w aqueous sucrose solution, but it revealed a licorice-like offiaste and a lingering attertaste (62). Although the quantitative sensory evaluation of polypodoside B was not carried out, it was somewhat less intensely sweet than polypodoside A. It was found that polypodoside C, a compound which only differs structurally from polypodoside B in having an L-acofriopyranosyl (3-O-methylrhamnosyl) unit affixed to C-26 in place of an L-rhamnosyl residue, was devoid of sweetness. Alter investigating several other compounds in this series, it was concluded that steroidal saponins of this type must be bidesmosidic with saccharide substitution at both C-3 and C-26, in order to exhibit a sweet taste (64). 3.2 Phenylpropanoids In an earlier study in this laboratory, trans-cinnamaldehyde (49) was found to be responsible for the sweet taste exhibited by the leaves of Cinnamomum osmophloeum, and was rated as being 50 times sweeter than sucrose by a taste panel (21). As a result of the investigation of six plants either collected in the field in Costa Rica or cultivated at the Pharmacognosy Field Station, University of Illinois at Chicago, their sweet taste was attributed to high levels of transanethole (48), as listed in Table I. Of these species, Myrrhis odorata is documented as being a sweet-tasting plant (102), but its sweet constituent had hitherto been unknown. When purified,
trans-anethole was judged by a taste panel to exhibit a sweetness intensity of nearly 13 times that of sucrose (10,000 ppm) (65). Both cinnamaldehyde and anethole are used at low concentrations as flavoring agents in foods in the United States and elsewhere, but they both possess undesirable hedonic attributes which do not merit their further development as sweeteners (21,65). However, the realization that trans-anethole is potently sweet, and can occur commonly in plants is important from the point-of-view of selecting candidate sweet plants for study in the field. Consequently, it is necessary to use analytical methods for the dereplication of phenylpropanoids as well as sugars and polyols when working on the isolation of potently sweet natural products, so that time and resources are not wasted on re-isolating these sweet compounds (65,103).
R~2 R 1 R1
R2
48
CH 3
OCH 3
49
CHO
H
30 3.3 Dihydroisocoumarin var.
Phyllodulcin (50) is released when the newly harvested leaves of Hydrangea macrophylla thunbergii are crushed or fermented, and this dihydroisocoumarin is the sweet principle of a
ceremonial tea called "amacha" that is used in Japan (20,21). While highly sweet (400 x the sweetness of 3% sucrose), the compound is limited as a sweetener by its almost total insolubility in water, and unpleasant hedonic attributes, such as a lingering aitertaste (21). Although no further sweet-tasting natural product analogs appear to have been isolated, there is a vast literature on attempts to improve the sweetness characteristics of phyllodulcin, by producing synthetic derivatives. This has been summarized recently by van der Wel and colleagues, who also describe the
various
hypotheses
on
the
structural
elements
of the
phyllodulcin
and
other
dihydroisocoumarins that are necessary to exhibit a sweet taste (16).
~ I
OH
H OCH3 t
II
OH
0
50
3.4 Flavonoids Flavonoids are usually regarded as bitter- or neutral-tasting plant constituents. However, there are flavonoids in two structural classes for which sweet representatives are known, namely, the dihydrochalcones (51-55) and the dihydroflavonols (56-62), and these will be discussed in turn. 3.4.1
Dihydrochalcones
The
semi-synthetic
dihydrochalcone
glycosides,
naringin
dihydrochalcone (51) and neohesperidin dihydrochalcone (52), are produced from widely available by-products of the citrus industry, and compound 52, the sweeter of the two, has agreeable hedonic properties, with a lack of bitterness, although it has a slow onset of sweetness (20,21). There have been many attempts to produce dihydrochalcone analogs with taste qualities more like those of sucrose, and it is clear from these studies that highly sweet compounds in this series require a 3-hydroxy-4-alkoxy substitution in ring B (16). The effects of varying the substituents on ring A and in the pyran ring of the dihydrochalcones have been summarized by van der Wel and colleagues (16). The only dihydrochalcone currently in use as a sweetener is neohesperidin dihydrochalcone, which has particular use in chewing gum, candies, and oral hygiene products because of its long-lasting sweetness (31).
31
93 R10
OR2
OR4 0
R1
R2
R3
114
R5
51
[3-glc~c~-rha
CH3
H
H
H
52
[3-glc2---o~-rha
CH3
OH
H
H
53
H
H
H
(x-rha
H
54
H
H
H
H
[3-glc
H
H
H
H
55
~3-glc
One of the first plant constituents to be recognized as being sweet was the dihydrochalcone glycoside, glycyphyllin (53), which was isolated in 1886 from the Australian species, Glycyphylla
smilax (20,21). Related sweet-tasting compounds are phlorizin (54) and trilobatin (5fi), although it is not apparent how potently sweet any of these three naturally occurring dihydochalcones is relative to sucrose (20,21). 3.4.2 Dihydroflavonols Sweet-tasting representatives of the dihydroflavonol class of compounds were first isolated in 1988, in independent studies by Tanaka and co-workers from
Engelhardtia chrysolepis (67) and in our laboratory from Tessaria dodoneifolia (21,104). However, Delaveau and colleagues had earlier pointed to the sweetness and astringency of the dihydroflavonol constituents in the bark of Glycoxylon huberi Ducke (21,105). In a phytochemical study on T. dodoneifofia, the previously known (+)-dihydroquercetin 3-O-acetate (57) was isolated as a sweet constituent of the young shoots of this plant. This compound was rated as having 80 times the sweetness potency of sucrose. The introduction of a 4'-methyl ether group in ring B, as in synthetic (racemic) dihydroquercetin 3-acetate 4'-(methyl ether) (56), greatly increased the sweetness potency to 400 times that of sucrose. Compound 56 was synthesized from 2,4-bis(benzyloxy)-6-(methoxymethoxy)acetophenone and 3-(benzyloxy)-4-methoxybenzaldehyde, according to a known method for producing dihydroflavonols, and remains the sweetest member of the class so far found. It contains ring B 3-hydroxy-4-alkoxy substitution, like the more highly sweet dihydrochalcones, a functionality which also confers greater stability to the molecule relative
32 to 57 (105).
2'
3'
R3
R2~~,i~~ "OR1 OH
O
R1
R2
R3
R4
56
Ac
H
OH
CH3
57
Ac
H
OH
H
2R, 3R
58
Ac
CH30
OH
H
2R, 3R
59
Ac
CH30
H
H
2R, 3R
60
H
CH30
OH
H
2R, 3R
61
cz-rha
H
OH
H
2S, 3S
ct-rha~- 13-glc H
OH
H
2R, 3R
62
Other
However, removal of the acetoxyl group of 56, as in the synthetic (racemic) compound, dihydroquercetin 4'-(methyl ether), reduced the sweetness potency to a tenth of its former level. Replacement of both the 3- and 4'-substituents by hydroxyl groups leads to the tasteless compound, (+)-dihydroquercetin (105). In work performed in collaboration with Mabry and coworkers at the University of Texas, a series of sweet dihydroflavonols was reported from the above-ground parts of Hymenoxys turneri (57-60) (66). One of the isolates, (2R,3R)-2,3-dihydro5,7,3',4'-tetrahydroxy-6-methoxy-3-O-acetylflavonol (58), with a 6-methoxy substituent, was less than half as sweet as compound 57. As a result of evaluating the sweetness of additional isolates from this plant (Table I), it was concluded that in the dihydroflavonol series of sweeteners a ring-B catechol unit is not mandatory for the exhibition of sweetness, since the 3-O-acetate unit also appears to have a role in mediating the sweet effect of these compounds (66,105). Also, Kasai and colleagues have shown that naturally occurring taxifolin glycosides (61,62) with both 2S,3S- and
2R,3R- stereochemistry may exhibit a sweet taste (67,68).
33 3.5 Proanthocyanidins The proanthocyanidins (formerly known as "condensed tannins") have emerged as a rather unlikely group of sweet-tasting compounds in recent years, since this group of polyphenols and the polyesters based on gallic and/or hexahydroxydiphenic acid ("hydrolyzable tannins") are much better known for the harsh, astringent taste they produce in the mouth (a feeling of constriction, roughness and dryness) (106). However, two of twelve proanthocyanidins obtained from the roots of Cinnamomum sieboldii were reported by Nishioka and colleagues at Kyushu University in Japan to be sweet-tasting in 1985 (69). Subsequently, in a review article, Tanaka accorded these sweet compounds the trivial names cinnamtannin B-1 (63) and cinnamtannin D-1 (64), respectively (107). More recently, N. Tanaka and co-workers at the Science University of Tokyo demonstrated that a pair of proanthocyanidins in the acid (65) and the corresponding lactone (66) form were sweet-tasting These compounds were isolated from two fern species in the same genus, namely,
Arachniodes sporadosora and A. exilis (70). None of the four sweet-tasting proanthocyanidins 6366 appears to have been evaluated for its sweetness intensity relative to sucrose. In very recent work carried out at the University of Illinois at Chicago, selligueain A (67), a further sweet-tasting proanthocyanidin, has been isolated from the rhizomes of a fern, SelligT~ea
feel, collected in Indonesia. Like compounds 63-66, selligueain A possesses a doubly-linked ring A, and is trimeric. However, selligueain A differs from 63-66 in having an epiafzelechin C-unit rather than an epicatechin unit in this part of the molecule. Compound 67 has been assessed for sweetness potency, and was found to exhibit about 35 times the sweetness intensity of a 2% w/v sucrose solution. At a concentration of 0.5% w/v in water, selligueain A was judged as being pleasant-tasting rather than astringent. It may be anticipated, however, that sweet-tasting proanthocyanidins are rare, because of the stringent structural requirements necessary to elicit sweetness (71 ).
HO O ( IH OH OH
Ik 35 *OH'......J"S
HO" "~ FII
HOA.I.x.,,OH
R1
R2
63
OH
~-OH
64
OH
t~-OH
67
H
~-OH
34
......... //"
,
OH
OH
65
OH
66
3.6 Benz[b]indeno[1,2-d]pyran derivative Tanaka and co-workers at Hiroshima University have followed up on the observation that an extract of the heartwood of
Haematoxylon campechiamim is
known to taste sweet, and the
sweet principle turned out to be hematoxylin (68), which is a well-known microscopical staining reagent (72). The sweetness potency of this compound relative to sucrose was not determined. By
35 reference to several tasteless compounds also obtained in the same investigation, it was concluded that the structural requirements necessary for the attribution of sweetness of 68 are the C-4 hydroxyl group, and the cis-stereochemistry (13-OH at C-6a and 13-H at C-12) linking the pyran and cyclopentene rings (72).
HO~) OH
H
Hf
/ HO
OH
68
3.7 Amino acid Monatin [4-hydroxy-4-(indol-3-ylmethyl)glutamic acid; 69] has recently been obtained as a very sweet constituent of the root bark of the South African plant, Schlerochiton ilicifolius, by Vleggaar and co-workers at the University of Pretoria in South Africa (73). This compound was purified by ion-exchange chromatography and gel filtration. The compound was obtained in this manner as a mixture of salts, with the sodium salt predominating, and the free amino acid was produced by treatment of the salt mixture with glacial acetic acid (73). The trivial name of this compound is based on a local Sepedi name, "monate", meaning "nice". Monatin is of comparable sweetness to the synthetic amino acid, 6-chloro-D-tryptophan (1,300 times sweeter than sucrose) (16). It is of interest to note that the plant of origin of monatin was described taxonomically for the first time as recently as 1965. Furthermore, in a lengthy description of the plant, there was no mention of any sweet characteristics, which is rather surprising in view of the sweetness potency of monatin (108).
~
H O 2 / ~ ~ CO2H
H , ~~N. . OH ~ N I H 69
2
36 3.8 Protein and Peptide Sweeteners and Taste Modifiers Sweet-tasting proteins have attracted wide attention, and thaumatin, monellin, mabinlin, and pentadin have been subjected to review previously (20,21,109,110). The five presently known highly sweet proteins and their plants of origin are shown in Table I. Of these, only thaumatin is used commercially as a sweetening agent and flavor enhancing agent (21,33,111). The complete amino acid sequence of monellin was reported recently (112), and the solid-phase synthesis of this sweet protein has been undertaken (113). Attempts are continuing to delineate the sweet receptor binding sites of thaumatin and monellin, although it has been found that the two compounds have no apparent similarities in either their amino acid sequences or their backbone three-dimensional structures (114). The structure of curculin, obtained from the fruits of Curculigo latifolia collected in Malaysia, was recently reported by Kurihara and co-workers at Yokohama National University in Japan. The compound possesses 114 amino acid residues, and, in addition to eliciting a sweet taste itself, it induces the sour-tasting substance citric acid to taste sweet (74). Another taste-modifying protein is miraculin, from Richardella dulcifica (Schumach. & Thonning) DC. (Sapotaceae), a West African plant (20,21). In contrast, gurmarin, a sweet-taste-suppressing peptide of 35 amino acids has been isolated from the leaves of Gymnema sylvestre R. Br. (Asclepiadaceae), although this has a very weak effect in humans while strongly suppressing the sweet taste responses in the rat (115). 3.9 Non-Protein Sweetness-Modifying Natural Products A number of non-proteinaceous substances of plant origin are known that induce or inhibit the sensation of sweetness. Sweetness inducers and enhancers from plants include cynarin, chlorogenic acid, caffeic acid, and arabinogalactin (larch gum) (33). A synthetic compound, 2-(4methoxyphenoxy)propanoic acid, which is also a constituent of roasted coffee beans, is currently on the market as a sweetness inhibitor (33). Several oleanane-type triterpene esters with sweetness-inhibitory activity occur in Gymnema ~ylvestre leaves (33,116,117), with dammaranetype saponins with similar effects having been reported recently from the leaves ofHovenia dulcis Thunb. (Rhamnaceae) (118) and Ziziphusjujuba Mill. (Rhamnaceae) (33,119,120). 4. CONCLUSIONS It may be seen from this chapter that ongoing research activities on the isolation and characterization of naturally occurring sweet principles have continued to afford many novel molecules in several structural classes. These compounds occur in species representing a taxonomically wide range of plant families. The fact that many of these compounds are highly sweet-tasting might well be of curiosity value only, were it not for the fact that several of the known naturally occurring intense sweeteners have important commercial uses, particularly in Japan. Methodology has been developed in terms of candidate plant selection, dereplication of sugars, polyols and sweet phenylpropanoids, and in other phytochemical procedures, so that significant progress can be made in the elucidation of further highly sweet-tasting molecules with only a modest investment of capital (121). In addition to being used in an unmodified form as
37 sweeteners, plant-derived sweet-tasting molecules can serve as useful lead molecules for synthetic optimization. Therefore, a knowledge of structure-sweetness relationships of plant sweeteners, and their naturally occurring congeners and semi-synthetic analogs is of use in assisting with the rational design of new sweeteners based on natural product leads. ACKNOWLEDGMENTS Certain of the work carried out at the University of Illinois at Chicago described in this chapter was supported by grant R01-DE08937, funded by the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland. We are very grateful to many capable postdoctorals and graduate students, as well as faculty colleagues, who have participated in our sweetener research, and whose names are indicated in the bibliography section. Prof. D.D. Soejarto of this institution is thanked for providing valuable taxonomic information for some of the plants mentioned in this chapter. We thank Prof. M. Nishizawa, of Tokushima Bunri University, Tokushima, Japan, for kindly providing the 13C-NMR spectrum of osladin. We wish to acknowledge Drs. I.-S. Lee and M.-S. Chung, and Mr. R. Suttisri for helpful suggestions. REFERENCES
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8. 9. 10.
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38 21. 22.
23.
24.
25.
26. 27. 28.
29. 30. 31.
32. 33.
34. 35.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
43
Structural Studies on Chemical Constituents of Echinoderms Luigi Minale, Raffaele Riccio and Franeo Zollo
1.
INTRODUCTION The phylum Echinodermata, which comprises about 6000 living species, is divided into five
classes: Crinoidea (sea lilies and feather stars), Holoturoidea (sea cucumbers or holothurians), Echinoidea (sea urchins), Asteroidea (sea stars or starfishes) and Ophiuroidea (brittle stars). Among the echinoderms, starfishes and sea cucumbers usually contain saponins, which are responsible of their general toxicity. Chemically, saponins derived from sea cucumbers are triterpenoid glycosides whereas those from starfishes are steroidal glycosides. The presence of oligoglycosides in both Holothuroidea and Asteroidea classes gives support to the opinion that sea cucumbers and starfishes are phylogenetically closely related (1). In starfishes and sea cucumbers A7-sterols, which are probably a consequence of the presence of haemolytic saponins, are also predominant, whereas the other three classes contain the usual AS-sterols (2). According to the view of Seeman et a/.(3) haemolysis is caused by the abstraction of membrane cholesterol by the saponins. Saponins show a much lower affinity for AV-sterols (4) and this helps to explain the apparent immunity of starfishes and sea cucumbers to their own saponins. It also makes it highly reasonable to regard the presence of AV-sterols in these organisms as the result of a biochemical convergence, i.e. an adaptation to the action of inherent cytotoxins. Furthermore, the coloration of starfishes and sea cucumbers is mainly due to carotenoids, while in sea lilies, feather stars and sea urchins it is due to quinoid pigments (5), which are characteristic of these two classes of echinoderms, although they also have been identified in some representatives of starfishes and sea cucumbers. Ophiuroids, which have received moderate attention by chemists as compared to the four above classes, have been reported to contain naphtoquinone pigments (51)and also carotenoids (6). Recently we examined some species of brittle stars and found a series of sulphated polyhydroxysteroids (7-9) and two steroidal glycosides (10). These findings provide biochemical support to the opinion of Fell and Pawson (11), mainly based on fossil records, that the phylogenetic relationship of starfishes and ophiuroids is more evident than that between starfishes and holoturoids. The distribution of secondary metabolites in the different classes of echinoderms, in connection with the phylogenetic relation among them, appears complex and offers a contradictory picture. A detailed presentation of the structures of some secondary metabolites of the phylum Echinodermata and their significance as chemotaxonomic markers can be found in an article of Stonik and Elyakov (12). In the past few years a large number of metabolites have been isolated from echinoderms, mainly
44 steroidal glycosides and polyhydroxysteroids from starfishes and triterpene glycosides from sea cucumbers, with cytotoxic, antifungal and antineoplastic activity. The interest in these compounds has resulted in a number of monographs entirely or in part devoted to this subject. We should mention the works by Hashimoto (1979) (13), Burnell and ApSimon (1983) (14), Krebs (1986) (15), Minale, Riccio, Pizza and Zollo (1986) (16), Quinn (1988) (17), Stonik and Elyakov (1988) (12), Habermehl and Krebs (1990) (18), Minale, Riccio and Zollo (1993) (19) and D' Auria, Minale and Riccio (1993) (20). The present paper is devoted to the more recent developments in the field of natural products from echinoderms, with emphasis given to those metabolites recently (1989-93) isolated in the laboratory of the Authors. 2.
ASTEROIDEA
The toxic properties of starfishes have been known for many years (21), but it was only in 1960 that Hashimoto and Yasumoto recognized that the toxicity is associated with compounds similar to plant saponins. They extracted some dried Asterina pectinifera by a method developed for plant saponins and the extract, which appeared to be a mixture of saponins, proved to be toxic and haemolytic (22). Saponins also possess ichthyotoxicity (22-23) and toxicity has also been noted toward annelids, mollusks, arthropods and vertebrates (24-26). Because of their general toxicity, it is probable that saponins act primarily as chemical defense agents, rejecting infectious aquatic fungi, protists, parasites and predators. The "avoidance reactions" and the "escape responses" exhibited by many organisms, such as some sea anemones, brittle stars, sea urchins and especially many species of mollusks, when in presence of, or contacted by, starfishes, represent a fascinating biological phenomenon. Makie et al. found that at least in some species the substance eliciting the escape response was a saponin (27). Besides, these oligoglycosides, at least in some species of starfish, participate in reproduction processes. Ikegami et al. identified saponins as the spawning inhibitors in the ovaries of Asterias amurensis (28). Fujimoto et al. observed that three steroidal saponins, designated Co-ARIS I, II and III, isolated from the egg jelly of the starfish Asterias amurensis, are essential for inducing the acrosome reaction (29). Starfish extracts and purified saponin fractions have shown a variety of pharmacological activities: haemolytic activity (24, 30); in vitro cytotoxicity toward tumor cells (31-33); antiviral activity (34); blockage of neuromuscolar transmission in mammals (35); antiinflammatory, analgesic and hypotensive activities (36). Starfish saponins are also known to inhibit development of fertilized sea urchin eggs (37); an investigation on the effects of seventeen individual starfish saponins on fertilized sea urchin and starfish eggs has shown that all compounds inhibited sea urchin embryos from further development in the morula stage, that the saponins with ergostane-type side-chain were more active than those with no methyl group at C-24 and that the pentaglycosides are somewhat more active than hexaglycosides (30). Fusetani et al. also showed that higher doses of saponins were required to inhibit or kill starfish embryos (30). In an analogous study, extended to the cyclic glycosides, glycosides ofpolyhydroxysteroids (mono- and diglycosides) and polyhydroxysteroids from starfishes, we have shown that the sulphated penta- and hexaglycosides (asterosaponins) are more active than the other groups ofrelated steroids (38). Among active saponins asteroside C, a pentaglycoside with a methyl group at C-24 in the aglycone side-chain,
45 was the most active (50% inhibition at 10-7 M), while the tetrasaccharide myxodermoside (172) was the less active one (20% inhibition at 10.5 M), thus parallelling the results of Fusetani et al. (30). A more recent study of the biological activity of representative saponins and related steroids from starfishes confirmed a high incidence of cytotoxicity and inhibition of Gram-positive bacteria but only weak antiviral activity and no inhibition of Gram-negative bacteria (39). Most of the work on starfish saponins has been initially prompted by their toxic and biological properties and in recent years structural studies on these molecules have expanded rapidly, largely exceeding the biological studies on individual compounds. More than two hundred steroidal constituents, which include steroidal glycosides and polyhydroxysteroids, have been isolated from ca. fifty different starfish species, belonging to fourteen families, representative of the three major orders (Paxillosida, Valvatida and Forcipulata) of the class Asteroidea (for phylogeny and classification of the Asteroidea cfr. ref. 40). According to their chemical structures, the steroidal glycosides have been subdivided into three main groups: the asterosaponins, which are sulphated steroidal penta- and hexaglycosides; the cyclic glycosides, so far only found in two species of the genus Echinaster, and the glycosides of polyhydroxysteroids, which, although unnoticed for long time, are as widespread as asterosaponins among starfishes (41). These molecules, which usually occur in minute amounts, consist of a polyhydroxysteroidal aglycone linked to one or two sugar units and can be found in both sulphated and non-sulphated forms. Analysis of the polar extractives of the starfish Tremaster novaecaledoniae has now led to the discovery of a new class of saponins, in which the polyhydroxysteroids
also present a phosphate in conjugation to which the sugars are glycosidically attached (42). Beside steroidal glycosides, starfishes have also proved to be a rich source of non-glycosidated highly oxygenated steroids, also found in sulphated and non sulphated forms. Sulphation is commonly observed in the biosynthesis of secondary metabolites in many marine invertebrates, especially in echinoderms. 2.1 Asterosaponins This group of saponins, for which the term asterosaponin was originally coined, is present in all species examined with the exception of two Echinaster species, which contain the steroidal cyclic glycosides (,43-451). Euretaster insignis is also apparently devoid of asterosaponins and contains instead a group of disulphated 3,21-dihydroxysteroids along with the more usual sulphated 3[3hydroxysterols (44). In contrast with the preponderance of AT-sterols in starfishes, the mixture of free sterols in E. insignis includes a low level of cholest-7-en-3[3-ol (3% of the total sterols mixture) and large amounts of 5a-steroidal alcohols. This finding could be related to the apparent absence of asterosaponins in this species. The asterosaponins are quite fragile molecules and usually occur as complex mixtures, whose separation in individual components is rather difficult and elaborate (see below). For this reason most of the initial works were concerned with the analysis of aglycones obtained by acid hydrolysis of partially purified saponin mixtures. This has resulted in production of several artifacts. Asterone (313, 6~-dihydroxy-5~-pregn-9(11)-en-20-one, 1), which is an artifact obtained by retro-aldol cleavage of the genuine thornasterol A sulphate (2) aglycone, has been the most widely reported steroid obtained by acid hydrolysis of asterosaponins (14). Complete structures have begun to appear in the literature in 1978, when Kitagawa and Kobayashi determined the structure of the major saponin from
46 OH
H
-
RO
-
2 R=SO3-Na § 2a R=H
Achantaster planci, thornasteroside A (3) (47). Thornasteroside A is the most widely distributed asterosaponin, having been isolated from fifteen species representative of the three major orders of Asteroidea. In addition to A. planci (family Acanthasteridae, order Valvatida), thornasteroside A (3) has been reported from Thromidia catalai (48) (family Mithrodiidae, order Valvatida); Halityle
regularis (49), Protoreaster nodosus and Pentaceraster alveolatus (50) (family Oreasteridae, order Valvatida); Linkia laevigata (51), Nardoa gomophia (52), Ophidiaster ophidianus (53) (family Ophidiasteridae, order Valvatida); Asterias amurensis (54), Coscinasterias tenuispina (55), Pisaster
brevispinus and P. ochraceus (56), P. giganteus (57), Pycnopodia heliantoides (58) (family Asteriidae, order Forcipulata) and from Luidia maculata (59) (family Luidiidae, order Paxillosida). Later the structure of glycoside B 2 (4), firstly isolated from the ovaries of Asterias amurensis, was elucidated. It differs from thornasteroside A only in that the terminal fucose has been replaced by quinovose (60). A little later the Ikegami's group, also working on the ovaries of A. amurensis, described the structure of ovarian asterosaponin- 1 (OA- 1,5), which contains the rare D-6-deoxy-xylohexos-4-ulopyranosyl unit (61). The FAB mass spectrum (negative ion mode) of 5 gave the molecular anion peaks at m/z 1257 (hydrate form) and m/z 1239 (keto form), and on SiO 2t.l.c. 5 gave two spots, in agreement with the presence of a keto sugar easily converted into the hydrate form. On alkaline treatment OA-1 (5) gave the asterone (1), because of the lability of the glucopyranosiduloses in alkaline media, which results in the release of substituents at C- 1 and C-3, thus establishing the direct attachment of the ketosugar to the aglycone. Solvolysis with pyridine and dioxane also resulted in the loss of the ketosugar, affording the genuine aglycone thomasterol A (2a). Reduction with NaBD 4, followed by sugar analysis, led to the identification of the ketosugar as 6-deoxy-xylo-hex-4-ulose (cfr. also ref.s 62, 63). It is interesting to note that OA-1 (5), along with the ovarian asterosaponin OA-4 (64) and a third saponin, differing from 5 only for the presence of a A~V(2~
structure in
the aglycone side-chain (22), designated Co-ARIS I, II and III, respectively, were identified as essential factors for inducing acrosomal reaction in the egg jelly ofAsterias amurensis (29). After the discovery of thornasteroside A (3), glycoside B 2 (4) and asterosaponin OA-1 (5), a series of papers describing the structures of about fifty asterosaponins has appeared in the literature starting from 1983. This study has revealed that the asterosaponins present several common structural features, which include a A9(11)-3[3-6~-dioxysteroidal aglycone, often with a 23-oxo function in the side-chain, bearing a sulphate at C-3 and an oligosaccharide moiety, made up by five to six sugar units, at C-6. A close
47 = Fuc
1-2
=Gal
1-4
1-3
X 9 yl
= Qui ---
1-2
Qui thornasteroside A R
= Qu i
1-2
=Ga I
1-4
1-3
Xyl-..-,.Qu
i
1 -2
Qui
Na + O 3 S O
glycoside B2 R
1 -3 1-2 = Gal --.-.,.Fuc -----,.Ga I 1-4 X y l l - 3
Qui
1 -2
Qui
versicoside A R
1 -3
1-2
= F u c - - , . F u c - - - - ~ G a I - L I ~ Xy ! 1-3= Qu i ~ 1-2
Qui marthasteroside A1 8
R
1-4
1 -4
= F u c---,.G I c----Qu i
1,-2 1,-2
Xyl
Qui
OH
Na + O 3 S O
1-3
~- Qu i ~
pcctinioside E
...
Fuc ~-2= Fuc 1-._~4 Qu
OH
1-2
Qui
5
ovarianasterosaponin 1 (OA-1)
Fig. 1. Some representative structures of starfish asterosaponins. (All sugars are in their pyranose forms and glycosidic linkages are 13; abbreviations: Fuc=fucose, Gal=galactose, Glc=glucose, Qui=quinovose, Xyl=xylose).
resemblance is also evident in the saccharide portions of these asterosaponins: sugars occur in their pyranose form with [3-anomeric configuration (a for arabinose) and linked with a constant pattern of interglycosidic linkages. A branching point is always located on the second monosaccharide (xylose or quinovose) starting from the aglycone and a terminal quinovose is always found 2-1inked to the branched sugar. The more common sugars are D-fucose, D-quinovose, D-xylose, D-galactose and Dglucose. Other less common monosaccharides are D-6-deoxy-xylo-hex-4-ulose and L-arabinose, of
48 which the latter has been occasionally found in laevigatoside from Linkia laevigata (51) and very recently in henricioside A (45), from Henricia laeviuscola (81), and in pectinioside G (42) and patirioside A (43), fromAstetina pectinifera (65) and Patiria miniata (66), respectively. Thomasteroside A (3) is an illustrative example of the general structure of pentaglycosides, which are the more common components among asterosaponins; versicoside A (6), isolated from Asterias amurensis versicolor (54) and Astropecten latespinosus (68), and marthasteroside A 1 (7), from Marthasterias
glacialis (67), are representative of the structure of the first reported group of hexaglycosides, while pectinioside E (8), isolated from Asterina pectinifera (33), is representative of the more recently described group of asterosaponins, wherein the hexasaccharide chain has two branches. Very recently the syntheses of the pentasaccharide chain of glycoside B 2 (4) and of the doubly branched hexasaccharide chain of pectinioside E (8) have been reported by R. R. Schmidt and coworkers (69, 70). More differences are observed in the structure of the steroidal side-chain, although thornasterol A, the aglycone of thornasteroside A and of all the saponins reported in Fig. 1, is by far the more common aglycone. Non-glycosidated thornasterol A 3-sulphate has been isolated from Asterias
amurensis (63) and Asterias rubens (71). A number of asterosaponins containing aglycones with various functionalities in the side-chain have been isolated. The structures of these steroidal aglycones are shown in Fig. 2. The aglycones 9, 15, 16, 17, 18, 19, 22 and 23 have never been obtained as free steroids by hydrolysis of the native glycosides and their structures are derived from aH and 13C nmr spectral analysis of the intact saponins. Examination of the IH nmr spectra of asterosaponins allows a quick identification of the aglycone structure (Table 1)1. The 20-hydroxy steroids are recognized by the C-21 methyl singlet ranging from ~51.30 to 1.37, which also indicates the 20S configuration
(cfr. (20S)- and (20R)-20-h ydro xycholesterol: ~51.28 and 1.13, respectively), and by the C- 18 methyl singlet shifted downfield to ca.~)0.82 ppm (53, 63, 72). The 20S-configuration of thornasterol A (2) and related 24-nor thornasterol A (9), 24-methyl thornasterols A [(24R)- and (24S)-thornasterol B (11 and 12)] and 24-ethyl thornasterol A (13) has been confirmed by synthesis (73, 75), which also allowed the definition of the configuration at C-24 in 11, 12 and 13. All saponins containing thomasterol A show characteristic C-22 and C-24 methylene signals at ca. 5 2.60, AB quartet with J=15 Hz, and at ca. 8 2.40, doublet with J=6.5 Hz. Any change of these signals is a clear indication of a modification in the side-chain. They are replaced by a broad singlet at 5 2.70 (22-H2) and by a multiplet at 2.73 (25-H) in saponins containing 24-nor thornasterol A aglycone (9). Particularly characteristic in these steroids are also the isopropyl methyl doublet signals shifted downfield to ~5 1.08 and 1.10. In the 24-alkylated thornasterol A (11, 12 and 13) the C-22 methylene proton signals are observed as two well separated doublets at ca.8 2.60 and 2.70 while that of the C-24 proton in 11 and 12 appears as a clear quintet at 2.52 ppm. Differentiation between (24R)- and (24S)thornasterol B (11 and 12) can be made on the basis of the different molecular ellipticity [O] in their CD spectra, which happens to be much higher in the 24R isomer: values reported are [O]2ss= -5780 for the 24R isomer (11) and
[0]277-" -631 for the 24S isomer (12) (73). Characteristic signals due to
the epoxide protons are observed in the 1H nmr spectra of saponins containing 22, 23-epoxysteroids: 1Unlessotherwisestated ~Hrunr datareportedin this paper are takenfromsolutionin CD3ODand signalsare referred to the CHD2OD(8 3.34 ppm) central signal.
OH
A
o~~ ~
o~
A
A
2
OH
-
A
9
A
49
11
10
A
A
12
13 OH
14 OH
Na+03Ss 5H
A
A 15
A 16
ISt,,~
Ire,.
A
A 18
17
tet,, A 19
20
OH
I~
A
A 21
22
Na+'03sO 5H
23
Fig. 2.3~i-sulphated steroidal aglycones found in asterosaponins. Description of saponins containing the above aglycones can be found in the following references: 2 (47), 9 (53), 10 (72), 11 (62, 63, 73), 12 (73, 74), 13 (75), 14 (76), 15 (63), 16 (50), 17 (63), 18 (55), 19 (51), 20 and 21 (68, 77, 78), 22 (29), 23 (55). a doublet at ca. 8 2.80 with J=2.5 Hz for the 22-H signal, almost constant in all compounds, and different 23-H signals depending on the substitution pattern on C-24. Namely: add at 5 2.76 (J=2.2, 7.5 Hz) in 17; a dt at 8 2.94 (J=2.5, 6.0 Hz) in 18 and a dd at 5 2.78 (J=2.5, 7.5 Hz) in 19. The 22R, 23S configuration was suggested by 13Cnmr data and confirmed by comparing spectral data with those of appropriate 22,23-(trans)-epoxy steroidal models (51, 55, 63). The discovery of 22,23-epoxy steroidal aglycones in starfish saponins, usually present as very minor components, is of biological interest and provides sound support to the earlier suggestion that the 23-oxo-function present in so many aglycones could arise via epoxidation of the 22(23) double bond (79). Distinction between the aglycones 14 and 16 by 1H nmr spectral data only rely on the chemical shifts of the C-21 methyl protons, at 8 1.31 in 14 (76) and 8 1.25 in 16 (50), since the C-22 hydroxymethine proton signal in
Table 1. Selected ‘H nmr data from steroidal aglycones in asterosaponins. 11
18
19
21
22
5.37b
0.81
1.02
1.37 s
2.62 ABq (15)
5.37b
0.82
1.02
1.37 s
2.70 br s
5.30111 0.79
0.95
1.34 s
5.37b
0.82
1.02
1.37 s
12”
0.78
1.02
1.34s
2.45 d (16) 2.68 d (16) 2.62 d (15) 2.76 d (15) 2.60 s
13d
1.00
1.10
1.67 s
2.83 s
1.03
H’s at C 3 Aglycone 2 4.22” 9
4.22”
10 11
a
4.22”
23
24
25
26.27
2.73 m
0.93 d (6.5) 0.94 d (6.5) 1.08 d (6.8) 1.10 d (6.8) 1.92 s, 2.16 s
2.42 d (7.5)
6.03 s 2.52 m (7.5)
14 15
4.22” 4.22”
5.37b 5.37b
0.82 0.83
1.03
1.31 s 1.31 s
16 17
4.22” 4.22”
5.38b 5.37b
0.85 0.83
1.02 1.03
1.25 s 1.30 s
2.83 d (2.2)
2.76 dd (2.2, 7.5)
18 19
4.22” 4.22”
5.37b 5.38b
0.82 0.83
1.02 1.03
1.30 s 1.29 s
2.76 d (2.5) 2.75 d (2.4)
2.94 dt (2.5, 6.0) 2.78 dd (2.4, 7.5)
20 21 22 23
4.22” 4.22” 4.20“ 4.22”
5.37b 5.37b 5.40 br 5.57”
0.70 0.70 0.84 0.78
1.04 1.04 1.oo 1.05
0.94 d 0.94 d 1.70 s 1.39 s 2.55 d (15) 2.75 d (15)
5.13 t (6.5)
5.28 t (6.5)
6.20 s 6.16 s 2.43 d (7.0)
0.88 d (6.5) 0.97 d (6.5) 0.92 d (6.6) 0.85 d (6.6) 0.84 d (6.8) 0.93 d (6.8) 1.65 s, 1.70 s 1.07 d (6H)(7.0)
28
1.00 d (7.5) 0.99 d (5.5) 0.88 t (29-H,)
4.72 br s 4.78 br s
1.67 s, 1.74 s 0.98 d (6.2) 1.04 d (6.2) 1.01 d (6H) (7.0) 1.02 d (6.6) 0.93 d (6.8) 0.97 d (6.8) 1.94 s, 2.11 s 0.94 d (7.0) 1.90 s, 2.11 s 0.94 d (7.0) 0.95 d (7.0)
m W,,,=22 Hz; br d, J=5.5 Hz; free aglycone synthetic model in CDC1,; free aglycone synthetic model in d,-Pyr, ‘br d (J=5 Hz), 12P-H 6 3.94 d (J=5 Hz)
51 16 is difficult to differentiate from the sugar signals. The 20R, 22S configuration in protoreasteroside, the only known asterosaponin containing 5t~-cholesta-9(11),24(25)-dien- 3 [3,6o~,20,22-tetraolaglycone (16), was based on the chemical shift of the C-21 methyl protons signal of the intact saponin in d 5pyridine (8 1.64) and of the derived acetonide in d4-methanol (8 1.37), in comparison with reported data for 5~-cholesta-313,20,22-triol models (50). Finally, the signal for the C-18 methyl protons at 8 0.70 (shifted to higher field relative to the 8 0.82 signal in 20-hydroxysteroids) points out the presence of marthasterone (20) or dihydromarthasterone (21), aglycones lacking the 20-hydroxyl function, at a glance. The aglycone 22 found in CO-ARIS II is characterized by the methyl signals shifted downfield to 8 1.70, 1.90 and 2.11 (21-, 26- and 27-H3) and the olefinic singlet at 8 6.16 (24H) (29). (20S)-3~,6t~, 12t~,20-Tetrahydroxy-5tx-cholest-9(11)-en-23-one (23), found in tenuispinoside C from Coscinasterias tenuispina, represents a major departure from the previous aglycones, differing from the common thornasterol A (2) by placement of an extra hydroxyl group in the tetracyclic nucleus. The presence of this "extra" hydroxyl group is revealed, in the IH nmr spectrum of tenuispinoside C, by the 12~-H signal appearing as a doublet 0=5 Hz) at 8 3.94; the coupling constant with the 11-H proton (8 5.57) is also indicative of its axial (a) orientation (55). The asterosaponins usually occur as complex mixtures of very similar molecules along with other closely related polar steroids and their isolation requires a complicated series of separation steps. An illustrative example of the complexity of polar steroid constituent mixtures in starfishes can be taken from Coscinasterias tenuispina, from which a mixture of nineteen components was separated into the ten asterosaponins, the six glycosides ofpolyhydroxysteroids and the three polyhydroxysteroids shown in Figs. 3 and 4 (55). The minor saponins 24 and 25 are the asterone analogs of thomasteroside A (3) and regularoside B (26), probably formed during the extraction by retroaldol cleavage of the thomasterol A side-chain. Some stereochemical assignment can be made now, based on some more recent experimental data. The 24R-configuration in 27 can be now indicated on the basis of direct comparison ofthe ~3Cnmr data (8c.28:12.1 ppm) with those of asteroside C (8c.28:12.1 ppm), the minor thornasterol B containing saponin from Asterias amurensis, for which the configuration was determined by CD measurements (63, 73). The side-chain stereochemistry ofcoscinasteroside A (33) can be assigned as threo, following the synthesis of 24-methyl-26-hydroxy steroid models (80) and comparison of ~Hand 13Cnmr data. The absolute configuration is most probably 24R, 25S, by analogy with that detected in echinasteroside A (81) (81). The 24R configuration in coscinasteroside C (35) was assigned after stereoselective synthesis of model (24R)- and (24S)-hydroxymethyl-cholesta5,22(E)-dien-313-ols (82). Details on the elucidation of absolute configuration in the side-chains of glycosides of polyhydroxysteroids and in polyhydroxysteroids, which present a greater structure variability, are given below. The problem of isolating individual saponins and related polar steroid constituents from starfishes has been satisfactorily solved by the Authors using a combination of chromatographic techniques, which are summarized in Fig. 5, as applied in the separation of nineteen polar steroid constituents from Coscinasterias tenuispina (55). Recovery of saponins and other polar steroids from the aqueous extract is made by passing this solution through a column of Amberlite XAD-2 resin, washing out salts with distilled water and eluting the adsorbed material with methanol. The residue from this eluate is subjected to an initial gel-filtration on a Sephadex LH-60 column, eluting with a
52
A = Fuc
1-2
1-4
, Gal
1-3
=Xyl
= Qui---
1-2
Qui 1-2
B = F u c .---,, F u c
Na+-OaSO
1-4
X9y l
T
R~ ~
24,
R=A
25,
R=B
1-3 Qui..__
1-2 Qui 1-2
C = Fuc ~
1-4
Ga I--_,.Qu i
2~.G
Ic
1-2
Qui OH es~
Na+O3SO
Na+O3SO
..
~o
R/~
R = A (thornasteroside A)
2 8 9 A24 ( 25 )
26,
R = B (regularosideB)
29,
27"
(24R)-Me,
3,
R = C
,
R = C (marthasteroside B) (marthasteroside C)
R = A (thomasteroside B) OH
OH
,,.
HO ,
Rf O
30,
R = A (tenuispinosideA)
31,
R = B (tenuispinosideB)
,
~
R~ O
32,
R -- A (tenuispinoside C)
Fig. 3. Asterosaponins from the starfish Coscinasterias tenuispina (55).
methanol-water (2:1) mixture. This step allows a good separation of the asterosaponins mixture from the lower molecular weight glycosides and polyhydroxysteroids. The crude asterosaponin mixture is further fractionated by droplet counter current chromatography (DCCC), using a two phase solvent system made up by n-butanol-acetone-water in a 45:15:75 ratio (in descending mode). In a typical separation, as in the case of C. tenuispina asterosaponin mixture, this procedure afforded two major
53
Is,,
=~.
SO3Na +
LH H
"""OSO3-Na* H
H
()H
O
H
34, coscinasteroside B
33, coscinasteroside A
OH Na+O 3 S ~ ( J'O ~ O , ~ _
OH ~ ~ O 3 N a * =,.,,, -
H
6H 6H 35, coscinasteroside C
36, coscmasteroside D OH
s,.
HOI~ H
'~
=_. OH
Na§O3SO~
H
37, R = OH, coscinasteroside E
H
(~H
39
38, R = coscinasteroside F
OH
9
H
OH
""s/OSO3"Na+
H Na+"O3SO
OH 40
_ H
(~H
41
Fig. 4. Glycosides of polyhydroxysteroids and related polar steroids from the starfish Coscinasterias tenuispina (55).
54
Water extract from starfish Coscinasterias tenuispina
I
Amberlite XAD-2 column methanol eluate
I
Sephadex LH-60 column methanol/water 2/1
Glycosides of polyhydroxysteroids and polyhydroxysteroids
Asterosaponins
Droplet Counter Current Chromatography n-butanol~acetone~water 45/15/75 descending mode
Enriched asterosaponin fractions
Reverse Phase HPLC g-Bondapak C- 18 methanol/water 45-50%
I Pureasterosaponins: "1 24, 25, 3, 26, 27, 28, 29, 30 31 and 32
Droplet Counter Current Chromatography chloroform/methanol/water 7/13/8 ascending mode
Sulphated polyhydroxysteroids and glycosides in the first eluted fractions
Enriched fractions of non-sulphated polyhydroxystemids and glycosides
Droplet Counter Current Chromatography n-butanol~acetone~water 45/15/75 ascending mode
Enriched fractions of sulphated polyhydroxysteroids and glycosides
Reverse Phase HPLC g-Bondapak C- 18 methanol/water 60-70%
I
Reverse Phase HPLC g-Bondapak C- 18 methanol/water 50-60%
Pure polyhydroxysteroids I and glycosides: I 37 and 38 ]
Pure sulphated polyhydroxysteroids and glycosides: 33, 34, 35, 36, 39, 40 and 41 Fig. 5. Isolation of saponins and related polar steroid constituents from the starfish Coscinasterias tenuispina.
55 fractions, the first fraction containing the more polar tenuispinoside C (32) together with the asterone containing saponins 24 and 25 and the second fraction consisting of the remaining saponins 3 and 26 - 31. The DCCC step, even through it usually does not allow complete purification of individual asterosaponins, is, in our experience, an essential step for a successful subsequent final separation by reverse phase HPLC. Final semipreparative HPLC (l.t-Bondapack C-18 column, methanol-water 45:55 to 50:50) was then sufficient to obtain ten pure asterosaponins. The strategy for the separation of crude monoglycosides and polyhydroxysteroid fraction, as obtained from the Sephadex LH-60 column, again involves a preliminary DCCC step, performed in ascending mode with a solvent system made up of chloroform-methanol-water in a 7:13:8 ratio. The more polar sulphated compounds are recovered, still as a full mixture, from the first eluted fractions, while the non-sulphated steroidal glycosides and polyhydroxysteroids are gradually eluted in order of decreasing polarity and combined in fractions, which are then submitted to a final purification by reverse phase HPLC. The fractionation of sulphated compounds is achieved by a further two-step procedure: again a DCCC, in ascending mode with n-butanol-acetone-water (45:15:75), to give partially resolved fractions which are then submitted to final separation by HPLC. Thus, the sulphated and the non-sulphated compounds could be isolated from C. tenuispina. The structure determination of the asterosaponins has involved the use of spectral techniques such as 1H and 13C nmr spectroscopy, by which the structure of the native aglycone can be derived without degradation of the molecules, and FAB mass spectrometry (in both positive and negative ion modes), which successfully gives the molecular weight of the underivatized sulphate saponins together with useful informations on the saccharide sequence. In this regard, data from FAB mass spectra in the positive and negative ion modes complement each other. Indeed the positive spectra give generally a weak series of pseudomolecular ions, derived from combinations of cross cationization, and intense fragmentations due to the cleavage of the glycosidic bonds with the positive charge located on the sugar fragments (67, 83). The negative spectra give an intense negatively charged molecular ion (anion) and a weak fragmentation arising from the loss of sugar units, starting from the terminal one, with charge located on the aglycone-containing fragments (63). The nature of the glycosidic linkages has been generally determined by chemical methods and interpretation of 13C nmr spectral data, a technique of increasing importance as a larger number of completely assigned model compounds became available. Where required, the saccharide sequences have been determined by partial enzymatic hydrolysis and analysis of the shortened glycosides. More recent additions to the list of asterosaponins are pectinioside G (42) from Asterina
pectinifera (65), patiriosides A (43) and B (44) from Patiria miniata (66), which are further representatives of the group of doubly branched hexaglycosides, henricioside A (45) from Henricia
laeviuscola (81), solasteroside A (46) from Solaster borealis (84), nipoglycosides A, B, C and D (4750), which co-occur with the known versicoside A (6) and thornasteroside A (3) in Distolasterias
nipon (85), collected at Mutsu Bay, Japan and brasiliensoside (51), differing from the more common ovarian asterosaponin I (5) for an "in chain" fucose replaced by galactose, isolated from Echinaster
brasiliensis (94) (Fig. 6). Details of the determination of the structure of pectinioside G (42) are given as an example of the strategies followed in the structural elucidation of asterosaponins (Fig. 7). Characteristic 1H nmr
56
Na* -O3SO
side chain R/~
Ara
42, R =
1 -4
=GIc
1 -4
=Qui
Fuc Fuc
43, R =
__ Q u i . - - -
11-2
[1-2
1-4
OH
1 -3
Qu i
pectinioside G
1-4
1-3
_-G I c.--.,.Xy I
1,-2
s,,
-- Qu i
1,-2
Ara
Qu i
patiriosideA OH
44,
a
Gal
=
1 -4
=GIc
1-4
11-2
Ara
1-2
=Gal
= Qui----
11-2
Fuc
45, R =
1-3
=Qui Qu i
1-4
patiriosideB 1-3
=Xyl
= Qu i --.-
1-2
Qui 46,
a
Fuc
-
1-2
= Fu
C 1-4
henriciosideA
_-Xyl
1 -3
OH
= Qu i - - -
1-2
Qui 47, R = G I c
1-3
=Fuc
1-2
=Gal
1 -4
=Xyl
solasterosideA 1-3
OH
= Qui----
1-2
Qui
48, R =
Fuc
1-2
=GIc
1-4
nipoglycosideA 1-3
=Qui
= GIc.--
1-2
Qui 49, R =
Fuc
1-2
_-GIc
1-4
nipoglycosideB 1-3
_-Qui
_- G I c - - -
1-2
Qui 50,
R =
Fuc
1-2
_-GIc
1-4
_-Qui
nipoglycoside C 1-3
_- G I c - - -
1 -2
Qui
nipoglycosideD CH 3
51,
R =
Fuc~--L-~ Gal ~
T1-2
Qui
Fig. 6. Recently isolated asterosaponins.
OH
Q u i ~ OH
brasiliensoside
57 spectral data indicated that the aglycone was the common thornasterol A sulphate (2). This was confirmed by 13C nmr data, which also supported the usual location of oligosaccharide chain at C-6. Sugar analysis and FAB mass spectra indicated that pectinioside G is a hexaglycoside containing 1 arabinosyl, 1 fucosyl, 3 quinovosyl and 1 glucosyl units. Analysis of terminal sugars by permethylation, acid methanolysis and GLC gave permethylated methyl arabinosides, methyl fucosides and methyl quinovosides, thus implying the presence of two branches. In addition to the major peak corresponding to the molecular anion (m/z 1389), the negative ion FAB mass spectrum exhibited OH
FAB MS (-ve ion): m/z 1389 ( M - )
~ , , .
m/z 1243
Ho~
OH H s ~,
..OH CH HO
HO
H
Na+03: S
,~
-/N. r-H :,' HO/~jv"3
'/
"
~
HO
' H
~0y CH~
/
3 " 42 ,u~"ec' p m,:":os:~^ o
m/z 949 .
;~z-~?;7-"
I-E)
.
.
.
.
.
Enzymatic hydrolysis $
~
HO'~~,' H
~
O
Y
i - ' ~ (OH~ i '
i
.
.
1111
m/z 949 .
.
.
.
.
Qui II
103.3
Qui III
105.3
Glc
102.1
Fuc
106.0
Ara
104.9
I
Qui I
104.5
Qui II
103.4
Qui III Glc
105.0 104.3
Ara
104.7
.
.
.
.
FAB MS (-ve ion): m/z 1243 (M) .
42a
i. . . . . . . . . . . . . . .
m/z
.
104.4
~CH3
--"a
.
Qui I
OH
HO.~ Na+03: HO/N~jCH3 rWz 1097 v ' ~ " " " ~ ' .......... H-O A''0
.OH
13C nmr shifts of anomeric carbons
OH
FAB MS (-ve ion)" m/z 949 s H%
Na*-03 r.z
"-:
H ~ ~.......... ~ "~ CHa 42b Fig. 7. Structure determination of pectinioside G from the starfish Asterina pectinifera (65).
58 fragments arising from the separate loss of arabinose (m/z 1257), a 6-deoxyhexose (rn/z 1243) and a minor fragment at m/z 949, interpreted as being due to the loss of arabinose, glucose and a 6deoxyhexose. These data were assumed as a preliminary indication of the presence of the second branching point on the glucose unit. The sugars sequence was determined by enzymatic hydrolysis with glycosidase mixture from Charonia lampas and analysis of the shortened glycosides (42a and 42b), while the interglycosidic linkages were deduced from the 13C nmr data as summarized in Fig. 7. The triglycoside 42b is identical with a sample obtained earlier by enzymatic hydrolysis of a known asterosaponin (51). FAB ms data and sugar analysis proved that 42a is a pentaglycoside arising from pectinioside G (42) by removal of the terminal fucose. Both linkages between the terminal arabinose and glucose in 42a and between the terminal fucose and glucose in the intact saponin were determined by 13C nmr spectroscopy. The linkage T-arabinose (1->4)glucose in 42a was determined upon comparison with the spectra of the appropriate methyl glycosides, taking into account the known glycosidation shifts, while the presence of one anomeric carbon at relatively high field in the spectrum of pectinioside G can be explained in terms of a substitution effect at C-2 of the glucopyranose unit, thus locating the terminal fucose there. We note that the glycosidase mixture from Charonia lampas, which is used to cleave the oligosaccharide chain of the asterosaponins, easily removes the sugar residues linked up to C-4 of the first branched unit starting from the aglycone, leaving a triglycoside which is more resistent to further enzymatic hydrolysis. For example, after 24 h enzymatic hydrolysis, pectinioside G (42) gave the triglycoside 42b as the sole shortened prosapogenol. Recently Findlay et al. have characterized three asterosaponins, forbesides A - C fromAsterias forbesi, identical with the previous versicoside A (= forbeside A, 6), glycoside B 2 (= forbeside B, 4) and OA- 1 (= forbeside C, 5). Analysis of the oligosaccharide structures was achieved totally by nuclear magnetic resonance methods, mostly using 2D techniques as COSY, HCCORR, RECSY and NOESY (86, 87). Three minor glycosides isolated from the methanol extractives of A.forbesi were shown to be related to OA-1 (5) and to versicoside A (6). Forbesides F and G are related to OA-1 (5), by loss of the terminal fucose and of the fucosyl(1->2)fucose disaccharide unit, respectively; whereas forbeside H is a trisaccharide of thomasterol A sulphate, apparently originating from versicoside A (6), a major saponin from A. forbesi, by loss of the Gal(1->3)Fuc(1->2)Gal- trisaccharide residue attached at C-4 of the branched xylose (88). Whether these shortened glycosides originate by enzymatic hydrolysis of the major penta- and hexa-glycosides or are their biological precursors is an open question. We have also very recently isolated from the starfish Myxodermaplatyacanthum (89) a tetraglycoside of thornasterol A 3-sulphate (172) apparently originating from thornasteroside A (3) by loss of the terminal fucose. A further tetrasaccharide, santiagoside (52), has been isolated from the Antarctic starfish Neosmilaster georgianus (90). This represent an exception to the general pattern of interglycosidic linkages encountered in asterosaponins, having a 4-substituted glucose unit linked to the aglycone, instead of the 3-substituted unit present in all asterosaponins. We would note that the 13Cnmr data of santiagoside (52) are very close to those assigned to related asterosaponins, including the unusually downfield shifted signal at 91.0 ppm, which is a distinctive feature of 13C nmr spectra of all asterosaponins and has been assigned to the glycosidated carbon-3 of the monosaccharide unit (]3-glucopyranosyl or [3-quinovopyranosyl) directly attached to the aglycone
59 A further minor constituent of A. forbesi, forbeside E (53), was shown to be the 6-0quinovopyranosyl (4'-sulphate) of (20R)-5~-pregn-9(11)-en-313,6o~,20-triol (3-sulphate) (91), an aglycone also isolated as hydrolysis product of saponins fromAsterias vulgaris (92). The corresponding 20-keto analog, cheliferoside, has been reported from Lethasterias nanimensis chelifera (93).
I~
CH3
e~~
Na+-O3s
-" -
F u c ~ - 4 Qu i 1-4= G i c . . . . i o
52, sandagoside
1-2
I A~ Na+-O3S
CH3 .O
Na+ O 3 S ~
Qui
0
53
2.2 Cyclic Steroidal Glycosides Toxic saponins of a completely different structural type have been discovered in two species of the genus Echinaster (Fig. 8). They have a number of unusual features when compared to the more common asterosaponins: there is no sulphate group and the charge is due to a glucuronic acid unit in the saccharide moiety, the AT-313,613-dihydroxysteroidal nucleus is unprecedented and a most remarkable feature is that the trisaccharide chain is cyclized between C-3 and C-6 of the aglycone giving rise to a macrocyclic ring reminiscent of a crown ether. Sepositoside A (54), the major saponin from the Mediterranean starfish Echinaster sepositus (43), is accompanied by smaller amounts of three related saponins (55 - 57), which differ only in the structure of the side-chain of the aglycones, all having a 22, 23-epoxy functionality (44). The key step during structural study of sepositoside A (50) was the mild acid hydrolysis, which, by cleavage of the allylic ether linkage, gave rise to the opening of the macrocyclic ring and formation of the UV active glycoside (58). A further representative of this class of glycosides was isolated from a Pacific starfish belonging to the same genus, E. luzonicus, and accordingly named luzonicoside A (59) (45). These findings and the absence of asterosaponins in both Echinaster species led us to assign a chemotaxonomic significance to the cyclic steroidal glycosides. This view has been questioned only very recently, when we had the occasion to investigate a third species of Echinaster, the starfish E. brasiliensis, collected at Grand Bahama Island (Carribean Sea). The cyclic steroidal glycosides were completely absent from polar extracts of this echinoderm, while typical asterosaponins were isolated in quite a large amount, including the known marthasteroside A 1 (3) and brasiliensoside (51) (94). Sepositoside A (54) is moderately toxic (LDs0 = 43 mg Kg 1 by i. p. injection in mouse) (43), and showed cytotoxic activity towards bovine turbinate cells up to a level of l~tg m1-1 (39). Both sepositoside A (54) and luzonicoside A (59) were slightly effective in the inhibition of cell division of fertilized sea urchin eggs (ca. 30% inhibition at 10.5 M) and showed antifungal activity.
60 54, sepositoside A
C02.N+a ~~,~~~
55
56
H
OH
57 H+ r.t.
(D021
C02-Na*
y
OH
OH
58
59, luzonicoside A
Fig. 8. Cyclic steroidal glycosides (43 -45).
2.3 Glycosides of polyhydroxysteroids This third group of steroidal glycosides from starfishes shows a much larger degree of structural variability. Most of these compounds usually occur in minute amounts and are also widespread among starfishes as the asterosaponins, having been usually found, as complex mixtures, in almost all the species we have investigated. They are composed of a polyhydroxysteroidal aglycone and a carbohydrate portion made up from only one or two monosaccharide units, often attached at C-3 or C-24 of the aglycone. Only very recently we have isolated from the New Caledonian species Fromia monilis three cytotoxic triglycosides (95), which constitute the only examples of triglycosides among
more than one hundred different mono- and diglycosides of polyhydroxysteroids isolated so far. The most common monosaccharide s are D-xylose, often methylated at position 2 and/or 4 and occasionally at position 3, and L-arabinose, found in its furanose form. Sulphated forms are quite common, with the sulphate group located on the steroidal moiety, at position 313, 6~ or 15t~, or on the saccharide portion. The first representative of such compounds, the cytotoxic nodososide (60) was first isolated from Pacific Protoreaster nodosus (96) and later from other Valvatida species: Pentaceraster
61
alveolatus (97), Acanthaster planci (98) and Linckia laevigata (98). In the following years the structures of more than one hundred different glycosides of polyhydroxysteroids were elucidated. Structural variations originate from the hydroxylation pattern of the steroidal tetracyclic nucleus, the functionalization of the side-chain, the presence of sulphate and the nature and location of the saccharide moiety. Beside the invariable 313hydroxylation, hydroxyl groups are commonly found at positions 6(or or 13),8, 15(~ or 13)and 24 of the aglycone occasionally with additional hydroxyl group(s) at one or more of the positions 4[3, 7o~ and 16[3. A few representative examples of the more common hydroxylation patterns are shown in Figs. 9 and 10. Compounds 61 - 65 are representative of the subgroup characterized by 3[3,6o~,15~-hydroxylation pattern; amurensoside A (61) from Asterias
amurensis (63) is one of the few examples of glycosides lacking the hydroxyl group at C-8 of the aglycone, while asterosaponin P-1 (62), first isolated from Patiria pectinifera (99) and later from
Oreaster reticulatus (100) andPatiriaminiata ( 101), contains the (24S) 5 ~-cholestane-3[3,6~,8,15~,24pentaol aglycone, one of the more frequently encountered steroids among oligoglycosides from starfishes. Crossasteroside A (63) from Crossaster papposus (102) and attenuatoside C (64) from
Hacelia attenuata (103) contain steroid aglycones related to the previous one by introduction of an additional hydroxyl group at position C-7o~ and C-4~, respectively. Glycoside 65 from Patiria
pectinifera (104) contains a 29-hydroxy-24-ethylcholestane aglycone with the sugar attached at C29, a structural feature encountered in a number of starfish derived glycosides. The 24R configuration is suggested here on the basis of the reported chemical shifts of the isopropyl methyl carbons (cfr. section 2.5.4). A second subgroup of compounds presents the 313,60~,8,15[3-hydroxylation pattern (e.g. 66 - 71 ); the (24S) 5~-cholestane-3[3,6a,8,15 [3,24-pentaol aglycone, first encountered in attenuatoside A-I (66) from Hacelia attenuata (105), is the most common steroidal aglycone found in glycosides from starfishes. Indicoside A (68) from Astropecten indicus (106) is a rare example of galactofuranoside, while indicosides B (105) and C (69) (107) represent, with their uncommon 3-O-methyl-~xylofuranosyl-5-O-sulphated moiety, the first cases of steroidal glycosides from starfish having xylose in furanose form. Indicoside A (68) together with halityloside A (67), halityloside I (70), both from Halityle regularis (108), and pisasteroside A (71), from Pisaster ochraceus and P. brevispinus (56), are examples of structural variations encountered in the steroidal side-chains. Compounds 72, 73 (109) and 74 (110) represent the subgroup of glycosides whose aglycones possess the 313,6~,8,15~hydroxylation pattern; they are also examples of those oligoglycosides in which the two monosacchatides are not linked to each other. The relative positions of the two monosaccharides were determined by mild acid hydrolysis to selectively remove the arabinofuranosyl unit and subsequent acetylation of the monoxylosides. In all cases, the ~H nmr spectrum of the acetate derivative defined the substitution pattern showing the multiplet assigned at 3-H unshifted (at ca.8 3.70) and the isolated multiplet signal assigned to 24-H shifted downfield (to ca. 8 4.68) (fi 3.4 before acetylation). Halityloside F (75) and gomophioside A (76), from Hal#fie regularis (108) and Gomophia watsoni (111), respectively, represent the smallest sub group, with the 313,613,8,1513-hydroxylation pattern in the steroidal nucleus. More recent additions to the list of the glycosides of polyhydroxysteroids are the related pycnopodiosides A (77), B (78) and C (79) from Pycnopodia heliantoides (58), miniatoside A (80) and B (81) minor glycosides from Patiria miniata ( 101 ), and the twelve steroidal glycosides (Fig. 11 )
62 OH
H~
H~o ~" :
HO
o
t,,,
-= ". o
t,,,.
OH
I;L HO~
.v
v
nsoside A
Na § -O3SO
MeO
MeO1t.~
tt, o.
jO
"
-'N
I
tt,,.
P-1
H~"',,,OH
-
:
63, crossasteroside A
=
(~H
(~H
~
o
OH
H
~
-
-
H
_
64, attenuatoside C
~
v
OH
'T
saponin P-2
(:31-1
OH O
MeO
=,'1,,
"
,,i,,.
"~OH H
-
"~OH A-I
6H
A
8H
5H
Fig. 9. Some representative glycosides of polyhydroxysteroids from starfishes.
63
OMr "
OSO3o
H
tt,
e,,
HO~"~
.'J. ""'OH (~, indicosideA
s
OH
,
~SH
HI'- ~O'7~~
H ~ ~ , ~ _ ' O3S-Na O+ O H
HO~
Ho
70, halityloside I
:
H
71, pisasterosideA
.
OH (~S03-Na+
-
(~H OH
OH
72, R=H; 5-deoxyisonodososide H~_70 73, R=OH; isonodososide ~
H~=_ O ",,.
.
i
MoO
OH M
e
H ~ MeO
OH
75, halitylosideF H
M e ~ ~ . ~ ~ MeO
-
H
-
76, gomophiosideA H
Fig. 10. Some representative glycosides of polyhydroxysteroids from starfishes.
64 avle
Na+03SO ~
L "'%,OH R
HO
~SH
!
77, R=H, R'=H; pycnopodioside A 78, R=SO3Na+, R'=H; pycnopodioside B
80, miniatoside A
79, R=H, R'=CH2OSO3-Na+; pycnopodioside C OH
HO
r 81, miniatoside B
isolated from the starfish Henricia laeviuscola (81), a further example of the structural variety of steroidal glycosides co-occurring in the same organism. One is the sulphated steroid bioside named laeviuscoloside A (82); six are sulphated steroid monosides [ laeviuscoloside B - E, 83 - 86 and the previously known echinasterosides A (87) and B (88) (112)] with four types of steroid aglycones (cholestane; 24-hydroxymethyl-; 24-(~-hydroxyethyl)- and 24-methyl-26-hydroxy-cholestane), all having the same nuclear hydroxylation pattern; two are non-sulphated biosides with two monosaccharide residues attached at different positions of their aglycones (laeviuscolosides F and G, 89 and 90) and two more are the non-sulphated monosides laeviuscolosides H and I (91 and 92). The last component is the asterosaponin henricioside A (45). The major constituent, laeviuscoloside A (82), inhibited growth of the fungus Cladosporium cucumerinum at a level of less than 1 ~g and caused 50% inhibition of cell division of fertilized sea urchin eggs at 10-7 M. A group of new 3-O-~-xylopyranosides of A4-313,6lS,8,15ot,16lS-pentahydroxysteroids with different side chains and sometime a sulphate at C- 15 (Fig. 12), closely related to those encountered in the previous Henricia laeviuscola (81) and in other species of the family Echinasteridae (112), has been recently isolated fromEchinaster
brasiliensis (94), a species collected at Grand Bahama Island, Caribbean Sea. Two new sulphated steroidal glycosides, imbricatosides A (101) and B (102), both containing the same disaccharide chain,
65
H
H
HO
OSO3-Na+
82, laeviuscoloside A
45, henricioside A
/OH
',,..~ A
A ~ , ~ .
OH
83, R=Me, laeviuscoloside B
J
.,/OH
"'"StOS03_ Na+ e
84, R=H, laeviuscoloside C
OH
tt,,.~
H B 85, laeviuscoloside D
A 86, R=H, laeviuscoloside E
J tt,,.~
tt'~ H
H
""//OSO3 "Na +
OH
B
B
87, echinasteroside A
88, echinasteroside B
OH H
MeO
OH
H
OH
MeO
H
89, R=H, laeviuscoloside F
91, laeviuscoloside H
90, R=OH, laeviuscoloside G
92, 22,23-dihydro, laeviuscoloside I
Fig. 11. Asterosaponin and glycosides of polyhydroxysteroids from the starfish Henricia laeviuscola (81).
66
OH
J 93, echinasteroside C ""'OH O
I"~H
OH
94, desulphated echinasteroside B
95, desulphated 22,23 dihydro-echinasteroside A .~H
OH
H
""'OSO 3 -Na +
87, echinasteroside A
H
OH
,,. jT H
84, 4,5-dihydro laeviuscoloside C
?
96, echinasteroside D
97, echinasteroside E
98, echinasteroside F
OH
99, R=H, laeviuscoloside I 100, R=OH, echinasteroside G
Meo
Io.
Fig. 12. Glycosides of polyhydroxysteroids from the starfish Echinaster brasiliensis (94).
i.e. 2,4-di-O-methyl-l]-D-quinovopyranosyl-(1->2)-5-O-sulphate-l]-D-fucofuranosyl, have been isolated from Dermasterias imbricata (113), a Pacific starfish species from which the alkaloid imbricatine, responsible of the swimming response induced in its prey, the sea anemone Stomphia
coccinea, had been previously isolated (114). Fucose is a common monosaccharide among the asterosaponins, but has never been found before in furanose form. Along with the more common A46~-hydroxy steroidal glycoside pisasteroside D (103), Pisaster giganteus also gave pisasteroside E (104), an isomer with A4-6~-hydroxy functionality (57). From the extractives of Astropecten
67 CH 3 Na + O3SO0,,.~......m~
OSO a -Na + _-. Ire,
MeO
=
OH
HO
.o,- T y-.%. R
OH 103, pisasteroside D
~SH
101, R=OH; imbricatoside A 102, R=H; imbricatoside B OH
HO
.5 =
104, pisasteroside E
OH
O~ ..R' Us~
CH2OH
~,~
HO 6H
105, R = H ,
R'=
OSO 3 Na
_.._J H,,., r~Me~/O ,.,-....q
+
MeO
,
indicoside B
,
scoparioside
OSO 3 - Na +
106, R=H; R'=
H
A
HO
OSO 3 Na +
107, R=H, R'=
Ho ~ '
scoparioside B
N a § " O 3SO..~,. ~
108, R=H,
R'=
M
H
~ v
e
109, A22E, R=SO3Na +, R
~PE)
'
~
~,
. -
~
110, R = H, crossasteroside P1 111, R = OH, crossasteroside P2
, scoparioside C
, scoparioside D
68 OR
9176162 M
""~H OH
OH
OH
OH
112, R=
HO~
)
9forbeside J
114, forbeside K
113, R= H; forbeside I
"<)14
"',OH Na + 0 3 S 0
116, aphelasteroside A
115, forbeside L
OH
""',OSO 3 "N a
H
..I-
OH
(T)I-I
118, R=H; moniloside I 117, aphelasteroside B
119, R=CH3; moniloside G 120, R=CH3, A22E;moniloside H
scoparius we have now isolated four new sulphated glycosides (115), designated scopariosides A D (106 - 109) along with indicoside B (105) previously isolated from A. indicus. Compounds (105 - 108) have the same common (24S) 5et-cholestane-3~,6(z,8,15 ~,24-pentaol aglycone and interestingly scoparioside B (107) also contains the same rare xylofuranosyl moiety as indicoside B (105) and C (69) from A. indicus (107). The major difference in the 1H nmr spectra of 106 and 107, which allows differentiation of the two isomeric sugar moieties, arabinofuranosyl-5-O-sulphate in 106 and xylofuranosyl-5-O-sulphate in 107, is the chemical shift of 4'-H, downfield shifted to 8 4.42 in 107
69 OH _= "z
OH
R
121, R = H; moniloside A 122, R = OH; moniloside B OH
l#t,,, OH
C)I--I
125, moniloside E
126, A22E;moniloside F
cH~o
1
1
OH
OH
123, R = H; moniloside C 124, R = OH; moniloside D (8 4.15 in 106) owing to the 1,3-syn interaction between 4'-H/2'-OH in the xylofuranose structure as compared to the anti arrangement in the arabinofuranose structure. Crossasteroside P1(110) and its 413hydroxy analog crossasteroside P2 (111), both containing a rare galactofuranose unit, have been described from Crossaster papposus by Kicha et al. (116), whereas Findlay and Zheng-Quan He (117), in their continuing study of the polar constituents fromAsteriasforbesi, have isolated four new glycosides ofpolyhydroxysteroids, designated forbesides I,J, K, and L (112 - 115). Forbeside L (115) is isomeric with desulphated echinasteroside B (88), by having the 24-ethyl-26-hydroxy side-chain instead of the 24-(~-hydroxyethyl) side-chain. The Authors left the stereochemistry at C-24 and C25 undetermined; we can now suggest the (24R,25S) stereochemistry on the basis of the reported chemical shift value for carbons 26, 27 and 28 (66.83, 13.69 and 23.48 ppm) and comparison with model compounds (118) (cfr. section 2.5.6).Aphelasteriasjaponica gave two novel steroidal xyloside sulphates (116, 117), aphelasteroside A (116) is isomeric with coscinasteroside B (34), in which the sulphate is at position 15 instead of 3 (119). Three unique cytotoxic triglycosides (118 - 120) have been now isolated from the starfish Fromia monilis. They co-occur with four mono and two diglycosides (121-126), all with cytotoxic activity (95); we note that the A7(8),9(11)-steroidalskeleton in monilosides A and B (121, 122) is unique among the polyhydroxysteroids from starfishes. A reinvestigation of the polar extractives from starfish Culcita novaeguineae (120) has led to the isolation of eleven polyhydroxysteroid glycosides and five polyhydroxysteroids (Fig. 13). One of them has been identified as culcitoside C 1(128), previously isolated from the same organism by Kicha
et al. (121), nine are known compounds previously found in starfishes, the majority having been
70
OH Me ~ ~ ~ - - ~ ~ L ~ ~
%
A
M R'
e
~
OH
0
(~H
127, R=A, R'=H, halityloside E 128, R=A, R'=OH, culcitoside C 1 129, R=B, R'=H, culcitoside Ca
MeO
130, R=B, R'=OH, culcitoside C 5 131, R=H, R'=H 132, R=H, R'=OH
OR
% .= .
t~
H
H
R'
~H 135, R=C, R'=H, halityloside B
133, R=A, R'=H, halityloside F 134, R=A, R'=OH, gomophioside A
136, R=C, R'=OH, halityloside A
Ro,,~ = es,
137, R=A, R'=OH, R"=H, R"'=OH, culcitoside C 2 138, R=A, R'=H, R"=H, R"'=OH, culcitoside C 3 139, R=A, R'=OH, R"=H, R"'=H, culcitoside
R'
C6
140, R=A, R'=OH, R"=SO3-, R"'=H, culcitoside C7
OR"
9
//et,
HO~ v ulcitoside C8
T OH
"''"R
H
143, R = H 144, R---OH
Fig. 13. Glycosides and polyhydroxysteroids isolated from the starfish Culcita novaeguineae (120) 9
71
RO
H
=
HO
.
OH 145,
R = Me; borealoside A
147,
R = H; borealoside C
146,
R = H; borealoside B
148,
R = OH; borealoside D
OH
~ ~~
OH
OSO 3 -Na +
149,
R = H
150,
R =
OH
(~),S0 a N a +
152
Me
151, R = H,
A 22E
isolated from the related species Halityle regularis (108), and six are new compounds, including one polyhydroxysteroid (141) and the five steroid diglycosides culcitosides C 4 - C 8 (129, 130, 139, 140 and 142). The analysis of C. novaeguineae provides a further example of the complexity of steroidal mixtures occurring in a single organism. We failed to isolate culcitoside C 2 and C 3 (137 and 138) reported by Kicha et al. (122). The recent analyses of the starfishes Solaster borealis, collected at Mutsu Bay, Japan (84), and Nardoa tubercolata, collected at Okinawa, Japan (123), have revealed the occurrence of further novel glycosides of polyhydroxysteroids. Borealosides A - D (145 - 148) from S. borealis are based on the common (24S)-5~-cholestane-3~,6~,8,15~,24-pentaol aglycone, with an additional hydroxyl group at 4~-position in 148 (84). The glycosides 149-152 fromNardoa
tubercolata, with 24-(~-hydroxyethyl) and 24-hydroxymethyl side chains in their steroidal aglycones, co-occur with halitylosides A (136), B (135), D (128), E (127) and F (133), first isolated from Halityle
regularis (108) and then from Culcita novaeguineae (Fig. 13, 120) and other species. These compounds were tested in the sea urchin eggs assay and proved to inhibit cell division of fertilized sea uchin eggs with IDs0ranging from 10v to 10.5 M.
72
153, R=R'=H; tremasterol A 154, R=R'=Ac; tremasterol B
Na + 0 3 S 0 OR
155, R=H, R'=Ac; tremasterol C
0
We had recently the opportunity to examine a deep water starfish species, T r e m a s t e r novaecaledoniae, considered to be a living fossil and discovered at 530 m depth during exploration of the
bathial zone off New Caledonia. Analysis of the polar extracts from this organism resulted in the isolation of a new group of glycosides of polyhydroxysteroids with phosphate and sulphate conjugation, tremasterols A - C (153 - 155) (42). To the best of our knowledge this is the first reported isolation of steroids with phosphate conjugation. Their structures were essentially derived from spectral data. The FAB mass spectrum of tremasterol A (153) displayed molecular ion species at m/z 803 [Mr~a] and 781 [Mr~]- together with fragments at m/z 641 (100%) and 619 (90%), corresponding to the loss of the glucosyl residue, and at rn/z 539 and 521, interpreted as losses of SO 3 from m/z 619 and NaHSO 4 from m/z 641. In addition to the [3-glucosyl moiety, the low-field region of the 1H nmr spectrum revealed 1H signals at 8 4.06 (H-6), 4.24 (H-3) and 5.27 (H-16) ppm. The multiplet at 4.06 ppm, assigned to H-613, appeared as a dddd (J = 9.5, 9.5, 7.5 and 4.5 Hz) with one more coupling constant than what was expected for a 6t~-hydroxycholestane, in which the 615proton is usually seen as an apparent double triplet with J = 4.5 and 9.5 Hz. This was the first indication of the presence of a phosphate (Jri-c-o-e= ca. 8 Hz) also linked to C-1 of the ~-glucosyl residue (H-1' ~5 4.89, t, J=7.5 Hz). Confirming evidences came from the proton noise decoupled 13C nmr spectrum, in which the signals due to C-5, C-6, C-I' and C-2' appeared as doublets because of 31p-13Ccouplings through two and three bonds, and by 31p nmr spectrum which showed a triplet signal (J=7.5 Hz) at 8 3.54 ppm from external standard (H3PO4 85% in D20), converted into a doublet by irradiation at either 8H4.06 (H-6) and 4.89 (H- 1'). The location of acetoxy group at C- 1613was also derived from 1H and 13C nmr data. Definitive structural data were obtained through removal of glucose by very mild acid treatment, followed by solvolysis in pyridine-dioxane to remove sulphate. 2.4 Polyhydroxysteroids The glycosides of polyhydroxysteroids are often accompanied by varied polyhydroxysteroids. Polyhydroxysteroids are not uncommon in marine species, having been isolated from algae, soft corals, gorgonians, nudibranchs, sponges, ophiuroids and also fishes; however starfishes appear to be the richest source of new polyhydroxysteroids (20). They have been found in almost all species examined, quite constantly in complex mixtures and more than eighty polyhydroxysteroids from starfishes have been reported until now. The 313,6o~ (or 13),8, 15o~(or [~), 16~-pentahydroxycholestane structure is a common feature, the major structural sub-group possessing a 26-hydroxyl function usually with the 25 S-configuration, whereas in a less common sub group the side-chain is hydroxylated at C-24 with 24S-configuration. Additional hydroxyl groups are found at positions 413, 5o~, 7tx (or 13)
73 OH -2"
""OH HO--
I~.T HO
156, R=H
.,o~
OR
158, R=SO3-Na§
157, R=SOaNa +
OH
0
-'.
140••H HO
'""OH
159
t t ' ~
8H
160
OH OH
161
.~~. FI
H
162
"',o~o3Na.
OH
163, R=H 164, R=OH
C
B
Crystal packing of 156 along the a direction; oxygen atoms are represented as filled circles
Fig. 14. Higly hydroxylated steroids from the starfish Archaster typicus (124 - 126).
74 OH
166
165
""%'0S0 3 "Na* H OH
O
~'t,,.~'*V~,J~,,~~O
&
3 - Na*
H
170
167 "'',,OH
|
t t ' , ~ C O 2
H
/set.
SO 3 Na +
H OH
171
168 jCO2H
OH
169
s4..
Na + O, 3s
..
6~ Qu i 1-3 Xyl 1-4 Gal
T
1-2
172, myxodermosideA
Qui
Fig. 15. New glycosides and polyhydroxysteroids from the starfish Myxodermaplatyacanthum (89).
and occasionally at 14o~, all disposed on one side of the steroidal nucleus, giving an amphiphilic character to the molecules with an hydrophilic and an hydrophobic region. Partly they are found in sulphated forms, with the sulphate group located at position 3[3, 6~, 15t~ or 24. Figure 14 illustrates the variety of highly hydroxylated steroids encountered inArchaster typicus (124 - 126); these steroids have been isolated in relatively large amounts as compared with the very limited fraction of steroidal glycosides. The nonaols 156, 157, 158, 161 and 162 constitute, as far as we know, the most highly hydroxylated sterols isolated from a natural source. The structure 156, which has the remarkable feature of eight sequential hydroxyl groups protruding from the same side of the molecule, was confirmed by single-crystal X-ray study (125). The crystal packing is a consequence of the amphiphilic character of the molecule, the hydroxyl groups form an intricate extensive network of hydrogen bonds linking the molecules in double layers, which interact through their hydrophobic surfaces (Fig. 14). The assignment of the stereochemistry at C-24 and C-25 in 161 and at C-24 in 162
75 required comparison with synthetic model compounds (cfr. section 2.5.7). The nonaol 156 and the major sulphated steroid 164, which were tested for anticancer activity, caused inhibition of human lymphoma cells growth (25-50% inhibition at 0.05 ~g/ml). Polyhydroxysteroids isolated up to early 1988 are listed in the paper of Habermehl and Krebs (18). More recent additions are reported in the papers of Minale, Riccio and Zollo (19) and D' Auria, Minale and Riccio (20). Figure 15 shows the structures of the polyhydroxysteroids isolated from the starfishMyxoderma
platyacanthum, again an example of structural variety from the same organism (89). The novel compounds 165 - 171 have the same 3~,5,6~, 15~-tetrahydroxycholestane nucleus (165 and 166 with sulphate conjugation at C-15) with different side-chains: 26-hydroxy; 26-oic; A2L27-nor-24-methyl 26-oic; 24-carboxymethyl and 24-methyl-26-oic. In compound 166 the 26-hydroxyl group is glycosidated with a [3-xylopyranosyl residue, while in 170 and 171 the carboxylic acid function is found as the amide derivative of taurine. This is the first reported isolation from starfishes of sterols with a methyl group oxidized to carboxyl. The new compounds co-occur with three known polyhydroxysteroids and the tetraglycoside of thornasteryl A 3-sulphate, myxodermoside A (172). In contrast with the common 25S configuration of the many 26-hydroxysteroids isolated from starfishes, polyhydroxysteroids with the 25R configuration have been isolated from Tremaster novaecaledoniae (129); a polyhydroxylated sterol analog but with the 515skeleton (cis AB ring fusion) has been isolated from the same starfish. Their structures include (25R) 5~-cholestane31],5,613,15t~,1613,26-hexaol (173), its 15-sulphate derivative and (25R) 513-cholestane3t~,613,15t~,16~,26-pentaol (174). More interestingly Tremaster novaecaledoniae has also given a
"%
.
H
H
,t,,.
=
OH-
H
"''<)H HO
HO,"'
v
1 "I" H OIH
174
_OR
o
H
,,.
03
",OH
H (~SO 3 - N a
R'
+
175, R = H, A9(11)
179, R = OH, R' =-.~OH; carolisterol A
176, R = Ac, A9(11)
180, R = H, R'= ~ O ;
177, R = H
181,
178, R = Ac
R = H, R' =
carolisterol B
.......OH; carolisterol C
- Na
+
76 group of polar steroids with a 3,6-disulphate (22R)-313,6~,22-trihydroxycholestane skeleton (175 178), which completely inhibited HIV-1 induced cytopathogenicity (ECs0 13 - 48 l.tM) (130). The last additions are the carolisterols A - C (179 - 181) isolated from the starfish Styracaster
caroli collected at a depth of 2000 m offNew Caledonia. These are a striking new addition to the large number of polyhydroxysteroids isolated from marine sources, being the first bile acid-type polyhydroxysteroids isolated from marine organisms. As a further singolar characteristic the 24-oic function is found in all compounds as an amide derivative of D-cysteinolic acid (131). 2.5 Assignment of configurations in side-chains of polyhydroxysteroids The occurrence of a large variety of steroids with multiple oxygen functionalities and different alkylation patterns in the side-chains has often required the assignment of absolute configurations to chiral centers. In most cases this has involved the synthesis of appropriate models and the analysis of their 1H and 13C nmr spectra and of those of their derivatives with a chiral reagent. 2.5.1 Absolute configuration at C-24 in 24-hydroxysteroids Hydroxylation at C-24 is the most common functionality found in the aglycone side-chain of glycosides of polyhydroxysteroids and exigency of determining the absolute configuration first arose with nodososide (60). (24R)- and (24S)-hydroxy cholesterol have been synthesized (132, 133) but differencies in their ~H and ~3Cnmr data are so small that direct comparison of both stereoisomers is necessary for unequivocal determination of stereochemistry. We assigned the 24S configuration to nodososide (60) by application of the gas-chromatographic modification of Horeau's method to the (24S) 3~,5,613,15~-tetramethoxy-5t~-cholest-8(9)-en-24-ol obtained by methylation of nodososide and subsequent acid methanolysis (134). More recently we found more convenient the use of the Mosher' s method for determination of absolute configuration of secondary carbinols (135, 136). This method is based on the observation of non equivalent 1H n m r chemical shifts of selected signals from diastereotopic groups contiguous to the chiral carbinol, upon derivatization with chiral t~-methoxytx-(trifluoromethyl)phenylacetic acid (MTPA, the Mosher' s reagent). According to the nmr configurational correlation scheme developed by Dale and Mosher, in the 1H nmr spectra of diastereomeric MTPA esters of the chiral carbinol, signals due to the diastereotopic groups will be typically shifted upfield in one diastereomer and downfield in the other in consequence of the interaction with the phenyl ring of the MTPA moiety. Following this useful configurational correlation scheme, we tested its suitability to the determination of absolute configuration in 24-hydroxysteroids by measuring 1H nmr spectra of (R)-(+)-MTPA esters [the term R-(+)- or S-(-)-MTPA ester refers to an ester obtained using the acid chloride prepared from R-(+)- and S-(-)-t~ methoxy-~-(trifluoromethyl)phenylacetic acid, respectively] of authentic (24S)- and (24R)-613-methoxy-3~,5-cyclo-5t~-cholestan-24-ol. As expected the signals due to the 26 and 27 methyl protons appeared as two upfield doublets at ~i0.83 (J=6.5 Hz) and 0.85 0=6.5 Hz) in the spectrum of the 24S-isomer and as a downfield 6-H doublet at ~5 0.91 in the spectrum of the 24R-isomer. Followed the assignment of configuration at C-24 in amurensoside A (61) (Fig. 16). The (24S) 313,6o~,15o~-trimethoxy-5o~-cholestan-24-ol,obtained by methylation of 61 and successive acid methanolysis, was converted into the diastereomeric (R)-(+)and (S)-(-)-MTPA esters, which showed the signals of the isopropyl methyl protons significantly upfield (~50.84 and 0.86) in the nmr spectrum of the (R)-(+)-MTPA ester and downfield (~50.89 and 0.91) in that of the (S)-(-)-MTPA, thus establishing the 24S-configuration in amurensoside A (61)
77 Models
Natural (from amurensoside A (55)) O M T P A (+ ) _=
O M T P A (+) ~
0.85 d
!1~
0.86 d upfield
"'~ c~l PA(+)
Me
OI~PA(-)
.91 d downfield
~ 0 . 8 9 d downfield 4,, I 0.91 d
Fig. 16. Configuration at C-24 in 24-hydroxysteroids. Selected 1H nmr data of (R)-(+)- and (S)(-)-MTPA derivatives of 24-hydroxysteroids (63). (63). The same methodology has been used to confirm the 24S configuration in several 24-oxygenated steroids. Recently X-ray crystallographic analysis of desulphated asterosaponin P-1 (62) confirmed the 24-S configuration of the aglycone (137). 2.5.2 Absolute configuration at C-25 in 26-hydroxysteroids Hydroxylation at C-26 is a common feature in a large majority ofpolyhydroxylated sterols from starfishes. As in the case of 24-hydroxysteroids, the spectral data of epimeric 26-hydroxysteroids show only very small differences (138) and assignment of configuration requires derivatization with a chiral reagent which induces significant differences in the spectra of 25R and 25S diastereomeric derivatives. The absolute configuration at C-25, in this group of compounds, was firstly determined as 25S in 5ot-cholestane-315,6c~,8,15ot,16~,25-hexaol,
a polyhydroxylated sterol isolated from the
starfish Protoreaster nodosus (134), using the method developed by Yasuhara and Yamaguchi, which allows determination of the absolute configuration of primary carbinols with chiral center at C-2 on the basis of Lanthanide induced shifts of the OMe signal of the MTPA moiety in the 1H nmr spectrum of the (R)-(+)- and (S)-(-)-MTPA ester derivatives (136, 139). During this work we could observe that a clearly different pattern was shown by the 26-H 2 signals in the aH nmr spectra of the (R)-(+)- and (S)-(-)-MTPA esters, appearing as two overlapping double doublets, at 8 4.15 (J=l 1.0 and 7.0 Hz) and 4.17 (J= 11.0 and 5.5 Hz), in the spectrum of the (R)-(+)-MTPA ester and as two well separated double doublets, at 8 4.06 (J=10.8 and 6.8 Hz) and 4.25 (J=10.8 and 5.5 Hz), in the spectrum of the (S)-(-)-MTPA ester. Because an inverse behaviour had been observed by Tachibana and Nakanishi during a configurational analysis of a 25R-hydroxysteroid (140), namely the 26-H 2 signals appeared as well separated signals in the spectrum of the (R)-(+)-MTPA ester and as a doublet in the spectrum of the (S)-(-)-MTPA ester, a direct correlation could be deduced between pattern of the diastereotopic 26-H 2signals, in the spectrum of the (R)-(+)-MTPA and (S)-(-)-MTPA esters, and configuration at C-25. Thus, in the spectra of the MTPA esters of a 25S-isomer the 26-methylene proton signals appear much closer in the spectrum of the (R)-(+)-MTPA ester than in that of the (S)-(-)-MTPA derivative, while the reverse occurs for MTPA esters of a 25R isomer, the resonances being closer in the (S)(-)-MTPA ester and more separated in the (R)-(+)-MTPA ester. Such a correlation was since used as a basis for the assignment of configuration at C-25 in most of the 26-hydroxysteroids isolated from starfishes (Fig. 17).
78 4.16d
H H -,,. ~ . AcO~
~
OH
TPA(+)
4.06 dd 4.25 dd
H H CDCI 3 %. MTPA(-)
"',OAc
OAc
4.21 d
H H
TPA(+)
OH
~
4.12 dd 4.30 dd
H H
MTPA(-) 4.13 dd 4.27 dd
CD3OD
H H %. MTPA(+)
MTPAO~. HOOH9
4.18 dd 4.23 dd
H H " ' : ~ M
TPA(-) 4.15 dd 4.24 dd
H H
MTPA(+)
MTPAO~= HO(~H
"'tO 'H
~
4.13 dd 4.26 dd
CD30D
MTPA(-)
Fig. 17. Configuration at C-25 in 26-hydroxysteroids.1H nmr data of some representative 26hydroxysteroids
79
2.5.3 Absolute configuration at C-24 in 24-hydroxymethylsteroids Stereochemical analysis of such compounds required the stereoselective synthesis of (24S)- and (24R)-24-(hydroxymethyl)cholesta-5,22 (E)-dien-3[3-ols and of their side-chain saturated derivatives, which enabled the acquisition of a set of spectral data suitable for stereochemical assignments in natural steroids possessing a 24-hydroxymethyl side-chain (82). Synthesis of model compounds was performed via a Claisen rearrangement reaction on a cis-allylic C-22 alcohol, a method developed by Sucrow and co-workers (141) which has grown into a general and effective methodology for the stereospecific functionalization of steroidal side-chains (142 - 146). In the series with the saturated side-chain the two C-24 epimers can be differentiated directly by their 1H nmr spectra: the C-28 methylene protons resonate as a broad doublet (8 3.52 br d, J=5.0 Hz) in the IH nmr spectrum of the 24R synthetic model and as two well-separated signals (8 3.47 dd, J--6.5, 10.0 Hz and 3.56 dd, J-6.5, 10.0 Hz) in the spectrum of its 24S epimer. Additional useful informations can also be obtained by 13Cnmr chemical shifts of the isopropyl methyl carbons; these appear more separated in the spectrum of the 24R isomer (19.2, 20.4 ppm) than in that of the 24S isomer (19.3,19.9 ppm). The 1Hnmr spectra of the two epimeric A22-24-hydroxymethyl synthetic models were virtually identical and the 13Cnmr spectra were equally useless for differentiating between the C-24 epimers. However the IH nmr spectra of (R)-(+)- and (S)-(-)-MTPA esters showed a number of diagnostic differences in the side-chain signals which can be used for assignment of configuration (Table 2). The most noticeable feature deals with the diastereotopic C-28 methylene proton signals, which in the spectra of (R)-(+)-MTPA esters appear more separated in the (24S)- than in the (24R)-isomer. In the spectra of the (S)-(-)-MTPA esters the differences, as expected, are reversed. The chemical shift of the C-21 methyl protons is also noteworthy: the signal is significantly upfield shifted in both MTPA derivatives of the (24S)-isomer, whereas in the MTPA esters of the (24R)-isomer it is affected to a minor extent. These data made it possible to assign the 24R configuration of coscinasteroside C (35) (82), pisasteroside A (71) (56) and laeviuscolosides C (84) and D (85) (81). The 24-methyl-5o~-cholest-22(E)-ene-313,6r hexaol, derived from coscinasteroside C (35) after removing the glucose unit by enzymatic hydrolysis, was converted to a 313,6o~,28-(R)-(+)-MTPA derivative (186) (Fig. 18). In the IH nmr spectrum (CD3OD) of 186 the resonances of the C-28 protons (a doublet at 8 4.37, J=6.5 Hz) and of the 21-Me protons (a doublet at 8 1.06, J=7 Hz) occur essentially at the same chemical shifts as in the underivatized steroid, in good agreement with the corresponding resonances observed in the (R)-(+)MTPA ester of the 24R-synthetic model, thus establishing the stereochemistry 24R in coscinasteroside C (35). 2.5.4 Absolute configuration at C-24 in 24-(13-hydroxyethyl)- and 24- (carboxymethyl)steroids The configuration at C-24 in 24-(13-hydroxyethyl)steroids can be determined from the 1H and 13C nmr spectral data. It has been reported that a little but diagnostic difference can be observed for
the C-26 and C-27 proton signals in the epimeric 29-hydroxyclionasterol (24R) and 29-hydroxysitosterol (24S) (144): in the 220 MHz ~H nmr spectrum of the (24R)-isomer the isopropyl signal is observed as an apparent triplet at 8 0.84, because coincidental overlap of the low-field arm of one doublet (8 0.83) with the hig-field arm of the other (8 0.86), while in the spectrum of the (24S)-isomer the isopropyl methyl protons give rise to two overlapping doublets at 8 0.84 and 0.85. These findings were confirmed in a study aimed to investigate the diagnostic utility of 13C nmr spectroscopy of a series
80 Table 2 Selected IH nmr data for side-chain signals in (24S)-, (24R)-24-hydroxymethylsteroids and their (R)(+)- and (S)-(-)-MTPA derivatives. compound 182 183 184
184a 184b 185
185a 185b 186
21-Me 26, 27-Me
(24R) (24S) (24S) (24S), (R)-(+)-MTPA (24S), (S)-(-)-MTPA (24R) (24R), (R)-(+)-MTPA (24R), (S)-(-)-MTPA (24R), (R)-(+)-MTPA
0.98 0.99 1.07 0.96 0.96 1.08 1.03 1.01 1.06
0.91, 0.94 0.93, 0.92 0.88, 0.94 0.87, 0.94 0.88, 0.92 0.87, 0.93 0.87, 0.91 0.87, 0.93 0.87, 0.91
28-H 2 3.52 br d (5.0) 3.47 dd (6.5, 10.0) 3.56 3.49 dd (6.5, 10.0) 3.57 4.25 dd (7.5, 10.5) 4.39 4.32 br d (6.2) 3.51 dd (6.5, 10.0) 3.58 4.34 d (6.5) 4.23 br dd (7.0, 10.5) 4.43 4.37 d (6.5)
dd (6.5, 10.0) dd (6.5, 10.0) dd (5.5, 10.5) dd (6.5, 10.0) dd (5.5, 10.5)
250 MHz (CD3OD), chemical shift values are given in ~5ppm and are referred to central CHD2OD (~5H3.34) signal. The coupling constants (in parentheses) are given in Hz.
182
183
184, R=H
185, R=H
184a, R=(+)MTPA
185a, R=(+)MTPA
184b, R=(-)MTPA
185b, R=(-)MTPA
.fCMTPA ( + ) .
( + )MTP ~VITPA ( + )
186 (from natural 35)
Fig. 18. Configuration at C-24 in 24-hydroxymethyl steroids (82).
81 of 24R- and 24S-(13-hydroxyethyl)- and-(carboxymethyl)steroids, stereospecifically synthesized, for assignment of configuration at C-24 in unknown natural steroids (147). Assignment is possible on the basis of the chemical shift differences (AS) observed between C-26 and C-27 carbon signals in the 24R- and 24S-isomers. Indeed, in C-24 epimeric couples with saturated side-chains, the difference in chemical shift (AS) between C-26 and C-27 carbon resonances ranges between 1.1 and 1.4 ppm in the case of 24R-isomers and between 0.1 and 0.4 ppm for 24S-isomers (Fig. 19). In conclusion 1H nmr spectroscopy allows differentiation between couples of epimers, but comparison of spectra of both compounds is advisable. 13C nmr spectroscopy, owing to the larger difference of A8 values, permits a more confident assignment of the C-24 stereochemistry of 29-oxygenated steroids with a saturated side-chain even in the presence of a single epimer. In the case of A22side-chains the chemical shift differences between isopropyl methyl carbons are very similar and 13C nmr fails to differentiate between 24R- and 24S-epimers. However the assignment is always possible in principle by examining the spectrum of the saturated derivative. The 24R configuration was assigned to a number of 24-(13-hydroxyethyl)steroidal glycosides on the ground of the above arguments. For example, in halityloside A (67) signals due to the isopropyl methyls, at 18.1 and 20.0 ppm (AS 0.9 ppm) in the ~3C nmr spectrum, and at 8 0.88, 0.91, in the 1H nmr spectrum, allowed the 24R stereochemistry to be assigned (108). Similarly the 24R configuration was assigned to 313,5,613,15a-tetrahydroxy-5o~-stigmastan-29-oic acid (169) isolated from Myxoder-
ma platiacanthum (89). OH
OH
.....
S 19.2
.,4,..
~19.3
o , . . ~ "4"
A8 (0.1 ppm)
' 19.3
A8 (0.4 ppm)
~19.7
A8 (1.1 ppm)
.CO2H
-.,4,..
18.6
(002 H
"4"
'20.1
A8 (1.4 ppm)
Fig. 19. Configuration at C-24 in 24-(13-hydroxyethyl)- and 24-(carboxymethyl)steroids. 13C nmr data of model compounds (147).
2.5.5 Absolute configuration at C-24 and C-25 in 24-methyl-26-hydroxy- and 24-methyl. 26-oic steroidal side-chains. In order to acquire a set of spectral data allowing the assignment of configuration at C-24 and C-25 of 24-methyl-26-hydroxysteroids, model compounds with all possible configurations at C-24 and C-25 were synthesized by a scheme involving a Claisen rearrangement reaction on cis-allylic C22 alcohols (80) (Fig. 20). These were converted, by reaction with triethyl-orthopropionate, into a
82
A
EtC(OEt)3 ) H3
% ~~~C02E , t
t,,~C02
s,
Et
C02Et
,88.-
187b -
187a
.
I LiAIH
1
~ ' ~ H 189b
189a H
1
Pt/C te
1901)
190a
H 191a
A
H ~ " ~ H 191b
I s " ~ H
192a
1921)
Fig. 20. Synthesis of model 24-methyl-26 hydroxysteroids (80). Table 3. Selected nmr data for side-chain signals in synthetic and natural 24-methyl-26 hydroxysteroids. x3C
IH
C-24 C-27 C-28
26-H 2
39.7 39.5 39.2 39.3
13.8 13.8 13.6 13.6
17.1 16.8 19.0 19.2
3.28 dd, 3.29 dd, 3.34 dd, 3.34 dd,
Saturated side-chain series 191a (24R,25S) threo 35.1 192a (24S,25R) threo 35.1 192b(24S,25S) erythro 36.8 191b (24R,25R) erythro 36.1
14.8 15.1 17.5 17.4
12.0 11.6 14.4 14.1
3.38 3.38 3.36 3.37
Compound
(R)-(+)-MTPA esters 27-H 3 28-H 3
26-H 2
3.60 dd 3.59 dd 3.53 dd 3.52 dd
0.90 0.90 0.87 0.88
d d d d
0.95 d 0.97 d 1.02 d 1.02 d
4.19 4.13 4.21 4.16
dd, 4.31 dd dd, 4.38 dd br d dd, 4.21 dd
3.55 3.49 3.58 3.57
0.83 0.81 0.93 0.91
d d d d
0.81 0.81 0.92 0.91
4.23 4.14 4.22 4.16
br d dd, 4.34 dd dd, 4.32 dd dd, 4.38 dd
A22series
189a(24R,25S) threo 190a (24S,25R) threo 190b (24S,25S) erythro 189b (24R,25R) erythro
Natural steroids echinasterosideA(87) 40.4 14.5 17.5 [AE2,(24R,25S) threo] (256) 36.7 17.4 14.3 [saturated side-chain, (24S,25S) erythro]
dd, dd, dd, dd,
dd dd dd dd
d d d d
3.46 dd, 3.58 dd 0.91 d 0.97 d 4.17 [(S)-(-)-MTPA: 4.06 3.34 dd, 3.62 dd 0.92 d 0.92 d 4.24 [(S)-(-)-MTPA: 4.16
dd, 4.33 dd, 4.46 dd, 4.32 dd, 4.37
dd dd] dd dd]
250 MHz (CD3OD), chemical-shift values are given in ppm and are referred to central CHD2OD (~H 3.34) and CD3OD (8 c 49.0) signals.
83 mixture of two A22E-26-oic esters epimeric at C-25, which, upon reduction by lithium aluminium hydride followed by HPLC separation, afforded the four possible A22E-24-methyl-26-hydroxy stereoisomers, which were then converted by catalytic hydrogenation into the saturated derivatives. The Claisen rearrangement of the individual allylic alcohols gave, in both cases, a major amount of the erythro product, in agreement with an expected predominance of the more stable (E) isomers of the intermediate ketene acetals, formed during the reaction when ethanol is lost from the orthoester intermediates (148, 149). The configuration at C-24 of all synthetic models followed from the stereoselectivity of the Claisen rearrangement reaction, while the stereochemistry at C-25 was confirmed by application of the lanthanide-induced chemical shift (LIS) non-equivalence method described by Yasuhara and Yamaguchi (136, 139). The chemical shifts of the C-26, C-27 and C-28 protons and those of the carbons 24, 27 and 28 are virtually identical in the A22threo pair (189a - 190a) and significantly different from those, likewise identical, of the alternative erythro pair (189b - 190b) and the same is true in the saturated side-chain series, with a more differentiated pattern exhibited by the carbon signals (Table 3). Thus, nmr spectroscopy makes the assignment of relative stereochemistry in the side-chain straightforward, while differentiation between the individual stereoisomers of every threo or erythro pair can be achieved by analysis of 1H nmr spectra of their (R)-(+)- and (S)(-)-MTPA derivatives. The following strategy has been formulated for the stereochemical assignment of an unknown natural 24-methyl-26-hydroxy steroid: (a) identification of relative stereochemistry by comparison of nmr spectral data with those of reference models (through a typical threo or erythro pattern); (b) assignment of absolute configuration at C-25, and hence to C-24, by the shape of the C26 methylene proton signals in the 1H nmr spectrum of the (R)-(+)- and (S)-(-)-MTPA derivatives.
&e,,,
SO3"Na+ 171
HO~~
''~
"~et~o
S
03Na+
OH 171a Table 4. Selected 1H nmr data of synthetic and natural 24-methyl-26-oic steroidal side-chains. Compound
olefinic H's
methyl doublets
187a (24R,25S) threo 188a (24S,25R) threo 187b (24R,25R) erythro 188b (24S,25S) erythro Natural 171 (24R,25S) threo C-25 epimer 171a (24R,25R) erythro
5.27 5.28 5.16 5.16 5.26 5.15
1.00, 1.01, 0.99, 1.00, 0.99, 0.98,
m m dd, 5.31 dd dd, 5.33 dd m dd, 5.30 dd
1.03, 1.03, 1.05, 1.06, 1.01, 1.04,
1.10 1.11 1.09 1.08 1.10 1.09
250 MHz (CD3OD), chemical-shift values are given in 8 ppm and are referred to central CHD2OD (8. 3.34) signal.
84 In this connection it has been established that the C-26 proton signals appear always as two double doublets, but, in every pair, they are closer to each other in the spectrum of the (R)-(+)-MTPA derivative of the (25S)-isomer than in that of the (25R)-isomer and such behaviour is reversed in the (S)-(-)-MTPA derivatives, the signals being now closer in the spectra of the (25R)-isomer. Thus, a closer position of the C-26 methylene proton signals in the spectrum of the (R)-(+)-MTPA derivative will be indicative of a 25S stereochemistry while on the contrary a closer position in the (S)-(-)-MTPA derivative will point to a 25R stereochemistry. These arguments were utilised for the stereochemical analysis of echinasteroside A (87) and of the steroid 256 from the Ophiuroid Ophiolepis superba (80). Recently we have encountered in Myxodermaplatyacanthum the steroid 171 (Fig. 15) with a A=, 24methyl-26-oic side-chain and have assigned the threo-24R, 25S stereochemistry by spectral comparison with those of the synthetic models 187a, b and 188a, b (Table 4). As expected the erythroisomers can be easily differentiated from the threo isomers by 1H nmr, especially by comparing the shifts of the olefinic protons. More subtle differences are observed in the shifts of the side-chain methyl protons between each pair. In order to assign the absolute configuration more confidently, we equilibrated the natural amide 171 with 10% KOH affording the epimer at C-25 (171a). Then followed a comparison of spectral data of 171 with the threo models and of the epimer at C-25 171a with the erythro models. The methyl doublets pattern in the 1H nmr of 171 compared better with the (24R, 25S)-isomer, and accordingly that of 171a compared better with the (24R, 25R)-isomer. 2.5.6 Absolute configuration at C-24 and C-25 in 24-ethyi-26-hydroxysteroids Model 24-ethyl-26-hydroxysteroids with all possible configurations at C-24 and C-25 have been synthesized (146, 150). In the A=-series 1H and 13C nmr spectra of the pair 193a - 194a are significantly different from those of the pair 193b - 194b, major differences being relative to the chemical shifts of the C-26 methylene and C-27 methyl protons and of the C-24, C-27 and C-28 carbons. In the saturated side-chain series the 1H nmr spectra of all isomers are very similar, while the 13C nmr spectra of the pair 195a - 196a were significantly different from those of the pair 195b - 196b,
with major differences relative to the C-23 and C-28 carbon signals (Table 5). Thus, nmr analysis permits discrimination of the relative stereochemistry (24R, 25S)/(24S, 25R) from the alternative (24R, 25R)/(24S, 25S). The absolute configuration can then be derived from 1H nmr analysis of (R)(+)- and (S)-(-)-MTPA derivatives. Indeed, as expected, in (R)-(+)-MTPA esters of each pair with the same relative stereochemistry, the C-26 methylene protons of the 25S-isomer appear as signals resonating much closer than those in the corresponding 25R-isomer and the opposite is true for (S)(-)-MTPA esters. So again, more proximate signals in the 1H nmr spectrum of the (R)-(+)-MTPA ester points to a 25S configuration, while an inverse behaviour is indication of a 25R configuration. Following these arguments we have assigned the stereochemistry at C-24 and C-25 of the steroid 257 from the ophiuroid Ophiolepis superba (150). 2.5.7 Absolute configuration at C-24 and C-25 in 24-methyl-25,26-dihydroxy-steroids. A A2224-methyl-25,26-dihydroxysteroidal side-chain was found in a highly polyhydroxysteroid (161) from the extracts of the New Caledonian starfish Archaster typicus (126). Simplified side-chain models were synthesized by epoxidation of (E)-2-methyl-2-pentenol followed by reaction with lithium dimethylcuprate to give the (2R,3R)/(2S,3S)-2, 3-dimethylpentane-l,2-diol enantiomeric pair (197a, b) (Fig. 22). This was converted into the (2S,3R)/(2R,3S)-enantiomeric pair (198a, b) by
85
't
H
tt
193a
H 193b
H 195a
194a
H
i
i
195b
,
,
ts
H 194b
~
~ ~ V ~ ~
196a
H
196b
A
Ha
Fig. 21. Model 24-ethyl-26-hydroxysteroids (146, 150). Table 5. Selected nmr data for side-chain signals in synthetic and natural 24-ethyl-26-hydroxysteroids. 13C Compound
1H
C-23 C-24 C-27 C-28
A22series 193a (24R,25S) 48.7 194a (24S,25R) 48.5 194b (24S,25S) 47.1 193b (24R,25R) 47.1 Saturated side-chain series 195a (24R,25S) 28.3 43.1 196a (24S,25R) 28.4 42.8 196b(24S,25S) 27.3 43.2 195b (24R,25R) 27.0 42.5
(R)-(+)-MTPA esters
26-H 2
27-H 3
26-H 2
15.2 15.2 12.9 12.9
25.5 25.5 26.9 26.9
3.25 dd, 3.25 dd, 3.32 dd, 3.34 dd,
3.64 dd 3.63 dd 3.48 dd 3.48 dd
0.93 d 0.94 d 0.83 d 0.85 d
4.10 dd, 4.32dd 4.04 dd, 4.38 dd 4.15 br d 4.08 dd, 4.19 dd
13.6 13.0 13.5 13.0
23.6 23.7 25.2 24.9
3.37 dd, 3.38 dd, 3.37 dd, 3.39 dd,
3.56 dd 3.52 dd 3.55 dd 3.5 3 dd
0.89 0.84 0.89 0.87
4.17 dd, 4.26 dd 4.11 dd, 4.32 dd 4.22 br d 4.12 dd, 4.31 dd
Natural (257) 27.6 43.4 13.4 25.1 [saturated side-chain, (24S,25S)]
3.35 dd, 3.58 dd
d d d d
0.92 d 4.26 br d [(S)-(-)-MTPA: 4.16 dd, 4.39 dd]
250 MHz (CD3OD), chemical-shift values are given in ppm and are referred to central CHD2OD (6H3.34) and CD3OD (6c 49.0) signals. tosylation, alkaline treatment and opening of the resulting 1,2-epoxide with diluted aqueous sulphuric acid. Enantiomeric pairs of model compounds depicted typical and easily recognizable 1H nmr spectra which, when compared with that of the 22,23-dihydroderivative of the natural steroid (200), allowed recognition of its relative stereochemistry on the basis of the good agreement between the C-26, C27 and C-28 proton signals and the corresponding signals of the (2R,3R)/(2S,3S)-2,3-dimethylpen-
86 tane- 1,2-diol enantiomeric pair (197a, b). In order to recognise the absolute configuration, the optical active (2R,3R)-2,3-dimethylpentane-1,2-diol (197a) was synthesized by using the titanium tartratecatalysed asymmetric epoxidation of allylic alcohols discovered by Katsuki and Sharpless (151). (R)(+)-MTPA Esters were then prepared from the (2R,3R)/(2S,3S)-2,3-dimethylpentane-l,2-diol enantiomeric pair (197a, b), the single enantiomer (2R,3R)-2,3-dimethylpentane- 1,2-diol (197a), and the 22,23-dihydroderivative (200) and their 1H nmr spectra compared. In the 1H nmr spectrum (250 MHz, CDC13) of the (R)-(+)-MTPA ester of the (2R,3R)-2,3-dimethylpentane-1,2-diol (199a), the signal due to the C-1 methylene protons appeared as an AB quartet centred at 84.20 (J=l 1.0 Hz) with the two central lines separated by ca.4.0 Hz, closely resembling the signal for the C-26 methylene protons of the (R)-(+)-MTPA ester 201, also an AB quartet with central lines separated by ca.7 Hz. An identical signal was present in the spectrum of the (R)-(+)-MTPA ester mixture of the epimeric (2R,3R)/(2S,3S)-2,3-dimethylpentane-1,2-diols (199a, b) together with two additional well separated doublets at 8 4.06 and 4.30 (J=l 1.0 Hz, central lines separated by ca. 45 Hz), which were consequently assigned to the C- 1 methylene protons of the (2S,3S)-stereoisomer (199b). On this basis the configurations of C-24 and C-25 in 200 and accordingly in the naturally occurring 161 were determined as 24R, 25R. Models 0.89 d 3.42 d ~3.49 d
R
~
" S H "" ~OH
H
197a 0.96 d
|
~ T P A ( 4 ""OH
197b
3.45 ABq
199a
=:
-=
4.06 d 4.30 d
H
H
198a
4.18 d 4.22 d
TPA(+)
198b
199b
Natural 0.88 d 3.42 d
l %" ~
4.21 d 4.27 d
3.49d
H 1 ~1. "'OH
.,~
9 "sOH
TPA( §
200 H 2, Pd/C
201
161
Fig. 22. Configuration at C-24 and C-25 in 24-methyl-25, 26-dihydroxysteroids. 1H nmr data of model and natural compounds (126).
87 3.
HOLOTHUROIDEA
The poisonous properties of sea cucumbers have been known for centuries. Their toxins are mainly used as self defense; they are produced in the skin and also in the Cuvier' s tubules and ejected from the body cavity through the anus when the animals are disturbed. In 1942 Yamanouchi, who began research on the toxicity of sea cucumbers in 1929 when he observed that an aqueous extract of Holoturia leucospilota quickly killed his aquarium fishes, isolated a crystalline toxin, which he named "holothurin" (152 - 154). Nigrelli of the New York Aquarium also discovered that aqueous suspensions of Actinopyga agassizi were strongly ichthyotoxic (155). Nigrelli also named the toxin "holothurin". Similar toxicity was found in three Mediterranean species (156) and twentyfour Pacific species (154). Currently the term"holothurin" is used to mean any sea cucumber saponin. Chemically, these compounds are glycosides of triterpenoids based on "holostane" skeleton, which, according to Habermehl and Volkwein, is (20S)-20-hydroxy-5o~-lanostane- 18-carboxylic acid (18->20) lactone (202) (157). Common sugars are glucose, 3-O-methyl glucose, quinovose and xylose, often bearing a sulphate group. Much of the chemical work done during the 1960's and 1970's centred on the isolation and characterization of the aglycones and has been reviewed comprehensively by Burnell and ApSimon covering the literature up to 1982 (14). From that time the extensive use of 13C nmr spectroscopy and FAB mass spectrometry in the examination of intact or partially fragmented saponins provided complete structures more quickly and with lower risk of causing aglycone degradation. The study of triterpene glycosides from more than fifty sea cucumber species has been carried out to date and quite a few structural series of glycosides have been described. Glycosides from sea cucumbers of the family Holothuriidae are based on holost-9(11)-ene- 12t~-hydroxy structure [e.g. holothurin A and B (203 and 204) (158 - 160)], which, on acid hydrolysis, generates the holosta7,9( 11)-diene artificial genins. Holothurins A and B are the first complete structures reported among glycosides from sea cucumbers. Glycosides from the family Stichopodidae contain lanost-7-ene type aglycones with the 9[3-H configuration andno sulphate group on the saccharide chains. Stichlorogenol and dehydrostichlorogenol are genuine aglycones of stichlorosides A 1 (205), B 1, C 1and A 2 (206), B2, C 2, respectively, a series of antifungal hexaglycosides isolated from the sea cucumber Stichopus
chloronotus (161). The structure of stichlorogenol was confirmed by X-ray analysis, which showed the ring C with 9[3-H configuration to be in a strained boat form, a plausible reason for the ready conversion from the genuine 7-erie moieties to the artifact 8-erie and 9(11)-ene moieties (162). The C-913-H configuration, which is common in holothurins with the holost-7-ene aglycone, can be confirmed by: a) the presence of a broad doublet at 8 ca. 3.4 ppm (9~-H) and b) a strong NOE between 913-H and 19-H 3. A group of holothurins from the family Cucumariidae, cucumariosides G 1 (207), C 1 and C 2 from Cucumaria fraudatrix (163, 164), have the aglycones with a 7-ene and a 1613-acetoxy function, while cucumarioside A2-2 (208) from Cucumaria japonica (165) has the aglycone with a 9(11)-ene and a 16-keto function. A novel member of the group of cucumariosides from the holothurian Cucumariajaponica, cucumarioside A1-2 (209) has been recently described by Drozdova
et al. (166). A further group of saponins from holothurians is also characterized by a 9(11)-double bond and a 16-keto function, such as the closely related hexaglycosides holotoxins A (210), A 1, B and B 1 found in Stichopus japonicus and Parastichopus californicus of the family Stichopodidae (167, 168). Holothurins are active against a variety of fungi. A complete listing of all triterpenoid glycosides
88 21
202
tlholostan
~
t!
o
-
Na+- O a S ( ~ _10
H~~ o ~ . . , , ,
OH
/OH ! H30.~JO~H i
.................... 203, holothurin B ....................................................
OH H
~
~
.OH
~
204, holothurin A
9~ I
~
205, stichloroside A 1 206, A25, stichloroside A2
o
? H~'0 H ~ ~ - ' ~
~
o
= -
G!
.
89
Na§03S~ OH
/OH
~
~"
H3cJO~H
208, c u c u m a r i o s i d e A2-2
..=
Na+0 3 S ~ ~ . ; 0 OAc H
/OH
q "~"
H3cJO~
209, c u c u m a r i o s i d e A1-2
H He
OH
.OH -
H Me
-OH
,OH
HO ~....O
"~,
H
2111,holotoxin A
OAc
Na+-OaS
.
o
H
_OH
~ / H / ~
~176 H ''r
-
o'N 211, psolusoside B
OH
from holothurians up to 1988 may be found in the papers of Stonik and Elyakov (12) and Habermehl and Krebs (18). More recently Kalinin et al. have reported the complete structure of Psolusoside B (211) from Psolus fabricii (169), made up from an uncommon non-holostane aglycone with
a 18-> 16 lactone and an unprecedented tetrasaccharide moiety with three glucose residues. A second example of an holothurin having a non-holostane aglycone with a 18->16 lactone ring has been encountered in cucumarioside G2 (212) isolated from Eupentacta fraudatrix (170). Kuriloside A
90
.a.-o3so--~_..o F ~ ~ o
#'%
,OH .e4~ /
~
,
Me~~O
H~
]
o
--
OH
OH
I
~.,,oAo -
OH
H
-
OH
"',,,
0
~0~~ H~ Vr
213, kuriloside A
OH
sugars
sugars
214, DS-penaustroside A, R =
216, DS-penaustroside C, R = ~ - ~
215, DS-penaustroside B, R =
217, DS-penaustroside D, R =
OH sugar chain =
MeC)'-~,'~ HO
,O. OH -
.4OZ ~'~ ~.OH
HO
91 R2
He i
;
o. MeO-X/
I~
_
-
.o. OH
H ,
a~O
,,! OH ~
i
I" D
9. . . . . . . . . . . . . . . . . . . . . . . . . . . .
f~l_.l
z,,.to, . t X l = , , J , ,
D
l..l~
, ,x2=.t.t ,
bivittoside A
219, RI=OH, R2=H; bivittoside B
...............................
,
!-..................................................................
220, RI=R2=H; bivittoside C
;.....................................................................
221,RI=OH, R2=H; bivittoside D
=_
:_
4
.4
H
H 222
o,r o .-.
%,,
223, preseycheUogenin
224, seychellogenin
(213) from Duasmodactyla kurilensis (171) and DS-penaustrosides A and B (214 and 215) from Pentacta australis (172) are further new non-holostane triterpenoid oligoglycosides. These latter two
co-occur with the two holostane-type glycosides DS-penaustrosides C and D (216 and 217). The prefix DS has been used to indicate that the compounds are desulphated derivatives of the parent sulphated glycosides, having been isolated after the solvolysis of the crude saponin mixture (172). A full account of the structure elucidation of bivittosides A, B, C and D (218 - 221) from Bohadschia bivittata has appeared (173). Acid hydrolysis of the 9(11)-en- 12~-ol bivittosides A, B and D provided
three artifact sapogenols, i.e. the 1213-oi aglycone (222), a homoannular-dienic sapogenol named preseychellogenin (223) and the 7, 9(11)-diene seychellogenin (224). Bivittoside D (221)exhibited significant antifungal activity, while bivittoside C (220), which lacked the 12o~-OH function of bivittoside D, had lost the activity completely. Bivittoside B, with 12c~-OH function but with a branched tetrasaccharide moiety also didn' t show any antifungal activity, in contrast with the corresponding oligoglycoside with the linear tetrasaccharide moiety, i.e. desulphated echinoside A, which showed significant antifungal activity. The Authors were tempted to conclude that the 12~-OH
92
Na §
OH
"OaSHO~j"~, , , O ~
-
,,OH H~c.JO~
pervicoside A pervicoside B pervicoside C
227, R=
.--. R 1
RH2O~ .
"'s
"'~"OA c
228, RI=
R2= H; lefevreioside A1
229,R 1 =
R2= SO3-Na§ ; lefevreioside A2
230, RI=
R2=SO3Na + ; lefevreioside C
231, RI=
R2=SO3Na + ; lefevreioside D
function as well the linear sequence of carbohydrates may play an important role in the antifungal activity (173). The same Kitagawa' s group reported a full account of their investigation on the saponin constituents of the sea cucumber Holoturia pervicax, and described three novel 9(11)-en-12t~-ol holothurins, pervicosides A, B and C (225- 227) (174), which differ only in the triterpenoid side-chain. Pervicosides gave a single spot on TLC and the composition was clarified only after solvolytic removal of the sulphate group. Desulphated pervicosides exhibited distinct antifungal activity against pathogenic microorganisms. A triterpenoid tetraglycoside with the 9(11)-en- 12~-ol functionality has been isolated from sea cucumber Neothyone gibbosa, and named neothyoside A (175). The proposed structure is identical to that of pervicoside A (225). Three additional species of the genus Cucumaria have been investigated. Cucumaria lefevrei furnished four closely related glycosides, lefevreiosides A 1, A 2, C and D (228 - 231) (176), with 7-ene and 16t~-acetoxy functionalities in their aglycones.
Cucumaria frondosa gave a pentaglycoside with a branched oligosaccharide chain, frondoside A (232) (177). The holostane-type aglycone features an endocyclic double bond at position 7(8) and a [3-acetoxy group at C-16, like cucumariosides G 1(207), C 1 and C 2 from C.fraudatrix (177, 178). A
93
Na+O OH
3
~
~
-
H3040~~/OH
Na§
M H
S
i
3
[I'"
H~..%
C
OH ,
OR4O H
R3
232, frondoside A
H~
233, frondoside B H
,o-..4
OH
234, R=O, RI=SO3, R2=H, R3=CH2ONO3-,R4-H; cucumechinoside A 235, R=O, R1=SO3-, R2=803-, R3=H2, Rg-H; cucumechinoside D 236, R=H2, R1=SO3-, R2=H, R3=CH2OSO3-, R4=H; cucumechinoside B 237, R=O, RI=SO3, R2=H, R3=CH2OSO3-, R4=503-; cucumechinoside E 238, R=O, R I=SO3-, R2=503-, R3=Hz, R4=503-; cucumechinosideC 239, R=H2, RI=SO3-, R2=H, R3=CH2OSO3-, R4=503-; cucumechinoside F o
Na+O3S~
~
~
~
~
2~, ~,~u~os.c.o~si~e~
94 more recent investigation of Cucumariafrondosa has led to the isolation of frondoside B (233), a new disulphated pentaglycoside, and of frondecaside, a unique hexasulphated decasaccharide with a dimeric pentasaccharide structure closely related to the pentasaccharide chain of frondoside B (178). The two pentasaccharide moieties are joined through the C- 1 of the first glucose unit and bear a third sulphate group at C-6 of the last glucose. This is the first report of a complex oligosaccharide structurally related to an echinoderm saponin. From the whole body of Cucumaria echinata the Komori' s group has isolated six holostane-type triterpenoid tetraglycosides possessing two or three sulphate groups in the oligosaccharide chain, named cucumechinosides A, D, B, E, C and F (234 - 239) (179). In tests for antitumor activity against L1210 and KB cells in vitro, as well in tests for antifungal activity, desulphated cucumechinosides showed a stronger activity than the native sulphates. Kalinin
et al. have established the structure ofpseudostichoposide A (240), the main triterpene oligoglycoside from Pseudostichopus trachus (180), with a holost-7-en-22-one aglycone and a tetrasaccharide chain common to many holothurins. 4.
OPHIUROIDEA
The study of natural products from starfishes and sea cucumbers has received considerable attention in the last few years, especially because of their content of toxic saponins. On the contrary brittle stars (ophiuroids) have received moderate attention as compared to the two above mentioned classes. Only sporadic papers dealing with their sterol content have appeared in the literature (2). As a direct consequence of our efforts to isolate biologically active compounds from starfishes, we had the occasion to investigate some ophiuroid species from which we could isolate a number of sulphated polyhydroxysteroids and two steroidal glycosides (Fig. 23). The steroid glycosides, longicaudosideA (241) and B (242) have been isolated from the Mediterranean Ophioderma longicaudum (10). Both have a 5t~-cholestane-3tx,6[3,12[3,21-tetraol
aglycone bearing a sulphate group at C-3 and a
monosaccharide residue at C-12 ([3-D-xylopyranosyl in 241 and 13-D-glucopyranosyl in 242). In contrast with the hydroxylation at C-26, commonly encountered among polyhydroxysteroids from starfishes, the polar steroids from ophiuroids are characterized by the hydroxylation at C-21, only found, among starfish metabolites, in steroids from Euretaster insignis (46). 13C nmr spectroscopy was found to be a better tool for differentiation between the two hydroxylated side-chains. Noticeable differences are observed in the methyl and hydroxymethyl resonances: the C-21 hydroxylated sidechain exhibits the typical signals for C-26 and C-27 at 23.0 and 23.1 ppm and the CH2OH signal at 63.4 ppm, while the C-26 hydroxylated side-chain exhibits methyl carbon signals at 17.3 and 19.2 ppm and the CH2OH signal at 68.4 ppm. The remaining signals are reported in Table 6. We also note that the placement of an extra hydroxyl group at C- 1213is accompanied by a downfield shift of the C-21 carbon signal to 64.3 ppm in the desulphated pentaol 243 (7) and to 66.1, 66.8 ppm in the 12-Oglycosides 241 and 242 (10) and an upfield shift of the C-20 carbon signal to 42.8 in the desulphated 243 and 41.9, 41.4 ppm in 241 and 242, respectively. The narrow signal at a rather low field (8 4.72 ppm, W1/2=7 Hz) in the 1H nmr spectra of both 241 and 242, shifted to 8 4.10 in the spectra of their desulphated derivatives, was indicative for a 3txOSO3--5o~-stanol structure. The alternative 3[3-OSO3-5~-stanol structure could be eliminated mainly on the basis of the chemical shift of C-19 at 15.3 ppm. Indeed, in steroids with a cis-A/B ring fusion
95 TABLE 6. Comparison of 13C nmr data between C-21 and C-26 hydroxylated side-chains (CD3OD, ppm)
21 OH side-chain 26-OH side-chain
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
43.8 36.6
63.4 19.2
30.6 37.4
24.9 24.5
40.8 34.9
29.1 36.9
23.0 68.4
23.1 17.3
Data have been extracted from the spectra of 5 ~-cholestane-3tx,4oc,11[3,21-tetraolfrom Ophiodermalongicaudum (8) and (25S)-5ct-cholestane-313,5ct,6~,15ct,26-pentaol from Myxodermaplatyacanthum (89).
R
OH
OSO3 - Na §
o
Na +
-~176
Na+-OaSOX,,',
JJ
y
,
L.
~ v
~5 H
248, R-H
241, R=H
249, R=H, 24-Me, A 24(28)
242, R=CHzOH
250, R=H, A22E OSO3 "Na +
OSO3 Na+
Na+O3SO ~,'....~ _ - " " ~
R
245, A 5
247,24-Me,
N a + 03 SO~~ V ~ "
Na+ - OaSO'" ....
246, A 5'22E
251 NO
A 5'24(28)
H
252, mixture of 25R- and 25S- isomers 253, mixture of 25R-and 25S- isomers, A5 Fig. 23. Sulphated polyhydroxysteroids from the ophiuroids Ophioderma longicaudum (7, 10), Ophiocoma dentata, Ophiarthrum elegans and Ophiorachna incrassata (8).
96 the 19-methyl resonance is significantly downfield shifted (e.g. in 5[3-cholestan-3]]-ol the C- 19 signal appears at 23.9 ppm). Acetylation of 241 gave a pentaacetate, whose 1H nmr spectrum still contained the essentially unshifted H-12 dd at 8 3.58, thus providing evidence for location of xylose at C-12.
Ophioderma longicaudum also furnished unusual sulphated sterols together with a mixture of common AS-313-hydroxysterols (7). The more polar sulphated sterol was characterized as 513cholestane-3o~,4tz, 1113,1213,21-pentaol 3,21-disulphate (243), featuring the cis-A/B ring fusion, later also found in the polar steroids from the four further species we have analyzed (8, 9). The lowfield resonance of the angular 19-methyl carbon (27.0 ppm) in 243 was strongly indicative for the cis-A/ B ring fusion, a feature confirmed by the chemical shift of the 19-methyl protons at 8 1.18 (calc. 8 1.19; for the alternative 5o~-cholestane-3[3,4[3,11[3,12[3,21-pentaol structure calc. values are: 8 1.33 ppm for 19-H 3 and 17.9 ppm for C-19). Two 1H signals are distinctly observed as a triplet at 8 3.99 (J=3.5 Hz) and as a doublet at 8 3.28 (J=3.5 Hz), coupled to each other, thus providing evidence for a vicinal diol framework located between a methine and a quaternary carbon, i.e. 11,12-position. The downfield narrow resonance at 8 3.99 is consistent with an equatorial proton, while the upfield signal at 8 3.28 is consistent with an axial proton, i.e. 1113,12[3-dihydroxy. In the spectra of both 243 and its desulphated analog the signals for H-3 and H-4 heavily overlap. Thus the desulphated pentaol was converted into the 3, 21-di-(p-bromobenzoate) derivative, which provided an apparent first order spectrum with isolated signals at 8 4.14 (t, J=3 Hz) for H-4 and 8 4.98 (br d, J=10.5 Hz) for H-3. Analysis of the spectrum by decoupling experiments led to a structural element with vicinal benzoyloxy and hydroxy groups at 3o~,4t~-positions in a 5[3-cholestane skeleton. A second group of components consisted of a mixture of disulphated 3tz, 21-dihydroxysteroids (244 - 247), whose composition was established after solvolytic removal of the sulphate groups (7). New sulphated polyhydroxysteroids have been isolated from three species of ophiuroids, Ophiocoma dentata,
Ophiarthrum elegans and Ophiorachna incrassata, all collected off Noumeb. (New Caledonia) (8). The major compound in all three species has been shown to be 5~-cholestane-3o~,4o~,1113,21-tetraol 3,21-disulphate (248). Two minor components of Ophiocoma dentata possess the same nucleus as 248 but differ in the side-chain (249 and 250). Along with minor amounts of 250 and major amounts of the disulphated tetraol 248, Ophiarthrum elegans also contains the 11-keto derivative 251. The structural relationship between the 11-hydroxy steroid 248 and the corresponding 11-keto analog 251 was established by oxidation of 248 with Sarret reagent to give 251 and, conversely, by reduction of the latter with sodium borohydride to give 248. Ophiorachna incrassata also contains major amounts of the steroid disulphate 248 along with minor amounts of the two more polar steroids 252 and 253, bearing three sulphate groups, which represent the first occurrence of 26-hydroxylation in ophiuroids. The major support for the presence of both 25S- and 25R-epimers is found in the 13C nmr spectra, where every side-chain carbon signal appeared split into two peaks separated by 0.04 - 0.2 ppm. All 26hydroxysteroids from starfishes have been shown to have 25R configuration, with the only exception of the two recently isolated from the "living fossil" starfish Tremaster novaecaledoniae, for which the 25S configuration has been established (129). A recent investigation of the Pacific ophiuroid Ophiolepis superba, collected at Okinawa, Japan, has led to the isolation of seven new sulphated polyhydroxysteroids (254 - 260) (Fig. 24), all with 3tx,21-disulphoxy-4a-hydroxy substituents and the A/B cis ring junction (9). The 3c~,4o~,21-triol-
97 OSO a - N a +
254 [
'
~ OH
-OSO3-Na+ 1 ~
255 Na + - O3SO,,~"" _= H HO
OSO 3- Na +
256
_
[ " ~ ~ " ~ OSO3-Na +
OSO 3 N a +
"
H6
OH
258
OSO3 .Na + ~,,,OH
~t,.A~.~j
Na+ -O3SO HO
260
Fig. 24. Sulphated polyhydroxysteroids from the ophiuroid Ophiolepis superba (9).
3,21-disulphated structure with a 513-steroidal nucleus, common to compounds 254 - 257, was inferred by 1H and 13C nmr data [5H4.19 (2H, m, H-3 and H-4), 4.06 and 4.22 (each 1H, dd, J=10.0, 6.5 Hz and J=10.0, 3.7 Hz, H2-21); 8c 82.4, 75.2 and 69.8 ppm for C-3, C-4 and C-21] and comparison with 248. A confirmation of the cis A/B ring junction was obtained by oxidation of 254 to the corresponding 4-keto derivative, which showed a very weak negative CD curve (a strong negative CD curve is expected for the alternative 313-hydroxy-5ct-cholestan-4-one structure). In addition, in the 1H nmr spectrum of the ketone, the H-3 signal moved downfield to 8 4.80 ppm as add with J=12.0, 7.0 Hz
98 TABLE 7 Comparison of 1H nmr data between desulphated 258 and synthetic models 513-cholestane-3ct,4o~, 5triol and 5~-cholestane-313,413,5-triol. H-3 desulphated 258 5[3-cholestane-3o~, 4o~, 5-triol 5o~-cholestane-3[3, 4[3, 5-triol
H-4
3.89, dt, J=l 1.5, 3.7 Hz 3.90, dt, J=ll.0, 3.5 Hz 4.09, dt, J=l 1.0, 3.5 Hz
3.63, d, J=3.7 Hz 3.65, d, J=3.5 Hz 3.47, d, J=3.5Hz
250 MHz (CD3OD), signals referred to the central CHD2OD (8 H3.34) signal.
(consistent with an axial proton) and the 19-CH 3 signal was observed shifted to 8 1.16 ppm (calc. 8 1.15; for the alternative 3[3-hydroxy-5ct-cholestan-4-one structure calc. 8 0.79 ppm). The stereochemistry at C-24 and C-25 in 256 and 257 have been determined after the synthesis of model compounds
(cfr. sections 2.5.5 and 2.5.6). The 3~,4t~,513-trihydroxy cholestane structure of 258 was derived from analysis of 1H and 13C nmr data and confirmed by the synthesis of 5]3-cholestane-3~, 4t~, 5-triol and the alternative 5a-cholestane-3[3, 4[3, 5-triol models. The 1H nmr spectrum of the 5[3-cholestane3~,4t~,5-triol showed the same shifts and coupling constants as the desulphated 258, while substantial differences were observed with the spectrum of the 5o~-cholestane-313,413,5-triol (Table 7). A comparison of the 1H and 13C nmr spectra of 258 with those of its desulphated derivative allowed the placement of the sulphoxy group at C-3. The introduction of an additional hydroxyl group at C-213 in the 3ct-sulphoxy-4tx-hydroxy-513-steroidal nucleus, as in structures 259 and 260, leads to the appearance in the 1H nmr spectrum of a signal at 8 4.03 overlapping with the 3-H signal, while the 4-H and 19-CH 3 signals are observed slightly downfield shifted to 8 4.33 (t, J=3.4 Hz) and 1.02 (s), respectively. When the spectrum of the desulphated 259 was measured, the three hydroxymethine protons appeared as isolated signals at 8 3.81 (m), 3.23 (dd, J=9.5, 3.5 Hz) and 3.94 (t, J=3.5 Hz) ppm. Decoupling experiments proved that they were located in a sequential arrangement and allowed the inference of a 2[3,3~,4t~-trihydroxy-5 [3-steroidal structure. Comparison of 1H and 13Cnmr data of 259 and 260 showed conclusively that both have 213,3tz, 4t~,21-tetrol 3,21-disulphate structure. The 24S configuration was assigned to 260 after the stereoselective synthesis of (24R, 22E)- and (24S, 22E)24-hydroxymethyl-cholesta-5,22-dien-313-ol (78; cfr. section 2.5.3). Stonik and Elyakov have isolated cholest-5-ene-3c~,413,21-triol 3,21-disulphate (261) from
Ophiura sarsi (210).
OS03Na+
HO
261
99 A new 5~-steroid disulphate (262), with an unique 4oq9o~-ether bridge, has been isolated from the ophiuroid Ophiomastix annulosa, together with the minor related compound 263 (181). The unique 4(z,9o~-epoxide-5(~-steroid structure of 262 was determined by detailed spectral analysis. High resolution mass spectrometry of the desulphated derivative indicated a molecular formula (C27H4603) requiring an additional unsaturation besides those of the tetracyclic nucleus. Two carbon signals shifted downfield to 84.5 (CH) and 87.5 (C) ppm, along with the absence of signals for sp 2 carbons indicated that one oxygen was involved in an ether bridge. Further analysis of the 13C nmr data provided structural information which led to location of the ether bridge between C-4 and C-9 in a 513-steroidal skeleton. The absence of couplings between H-3/H-4 and H-4/H-5 indicated that their dihedral angles are close to 90 ~ Conformational analysis by MM2 calculations showed strain in the A and B rings. The good agreement between the calculated and observed vicinal coupling costants of the proton signals from H-2 to H-7, in the ~Hnmr spectrum of 262, gave final support to the structure and conformation of the new steroid. Two further novel polyhydroxysteroids 264 and 265 have been isolated from the Antarctic ophiuroid Ophiosparte gigas (182). The three hydroxymethine protons on ring A appeared as isolated signals at 8 3.88 m (H-2), 4.46 dd (J=5.1,2.0 Hz; H-3) and 4.31 br s (H-4) ppm, in the ~Hnmr spectrum of 264. Decoupling experiments and analysis of coupling constants established location and stereochemistry of the hydroxyl groups; the low field position of the H-3 signal and its upfield shift at 8 3.76 upon removal of the sulphate group clarified its location at C-3. In an analougus manner the sulphate group was located at the unusual 213-position in 265. The pentaol disulphate 243, the tetraol disulphate 248 and the AS-steroidal sulphates 245 and 247 have moderate cytotoxic activity (39). In the frame of a project devoted to the evaluation of the anti-HIV activity of sulphated sterols by the National Cancer Institute, Frederik, MD, USA, a selection of sulphated polyhydroxysteroids from ophiuroids (243,244,248, 254,258, 251,264) has been tested and found to inhibit the cytopathic effects of HIV-1 infections (130).
OSO3"Na*
Na+.O3SO~,,,.[~~ ~ H 262
OSOs-Na+
Na+. 0 3 S 0 ~ , , . ~ ~ ~ HO H 263
OSO3"Na+
Na
4-
~176 I'1OH
264
OSO3"Na§
HC'~"~""g
2,65
100 Two closely related carotenoid sulphates have been recently isolated from ophiuroids. Ophioxanthin (266) is the major pigment from both Ophiodermalongicaudum(6) and Ophiocominanigra(183), of which the latter has also furnished dehydroophioxanthin (267), with an acetylenic functionality. OSO 3 N a + .,,~OH
9
HOO,,'"
:
266
OSO 3 Na +
_OSO 3 - N a + 9
HO,~
-_-
,,~,,OH
267
OSO 3 Na +
5.
CRINOIDEA The crinoids are the most ancient echinoderms, appearing in the First Age of our planet 350
million years ago. They have known a great bloom at the sea bottom where they were fixed by a stalk. After the Second Age (about 150 millions of years ago) they began to disappear and their study is mainly confined to the paleontology. Abouteighty species of these attached stalked crinoids (sea lilies) still exist today and nearly 550 more recent free swimming comatulid species (feather stars) have been described. Because of the spectacular colourful appearance of many crinoids, these animals have received considerable attention, almost entirely through the efforts of Sutherland and his co-workers (184- 192), and have yielded a greater structural variety of pigments than did the other classes of echinoderms. The attention has been essentially focused on comatulid crinoids, of which about twenty species have been examined since 1950. In addition to angular and linear naphthopyrones, a number of anthraquinones, 4-acylanthraquinones and 3-alkylanthraquinones have been isolated and characterized (184-195). More recently the range of polyketide constituents in comatulid crinoids has been extended to include dimeric pigments, such as bianthrones and bianthraquinones (192). One representative example for each of the above groups is shown in Fig. 25. A listing of the quinoid pigments from crinoids can be found in the paper of Stonik and Elyakov (12). We wish to observe that anthraquinoid pigments and naphthopyrones are usually present as sulphate esters and several species of fish are deterred from eating food treated with these sulphates at the concentration usually found in crinoids (191). Sutherland and colleagues concluded that constituent sulphate esters of polyketides provide some species of crinoids with a chemical defense mechanism against fishes.
101
269
268
OH
0
0
OH
I-~
OVle
OH
270
S
o
271 HO
HO
o
HO
0
272
I-I0
0
0
OH
OH
273
Fig. 25. Some representative quinoid pigments from crinoids.
Anthraquinoid pigments were biosynthesized many millions of years ago. In support to this belief is the observation that fringelites, described by Blumer as hydroxylated phenantroperylenequinones, were found in the fossilised remains of a Jurassic crinoid (Apiocrinus) discovered near Fringeli in north western Switzerland (196). We recently had the opportunity to examine a deep water stalked crinoid, Gymnocrinus richeri, discovered by B. Richard de Forges during an exploration of the bathial zone off the coast of New Caledonia. Gymnocrinus richeri is one of the best examples of those species to which it is appropriate to apply the description "living fossil". The body of the live crinoid is saffron yellow, whereas its stalk is a darker yellow and the inside of its tentacles is dark yellow-green. However, within a few minutes ofcollection, outside sea water, the animals turn dark green. Extraction with methanol gave a dark green solution, which on very mild acidification turned violet. Five violet pigments, gymnochromes A - D (274 - 277) and isogymnochrome D (278), which constitute a novel group of brominated phenantroperylenequinones, have been isolated (197). The overlapping of the substituents at C-3 and C-4 and at C- 10 and C- 11 causes twisting of the ring system and the molecule acquires axial chirality, as revealed by the CD curves (Fig. 26). Therein the strong peaks are evidently due to the inherently dissymetric chromophore of the twisted phenantroperylenequinone system.
102
R
OH O OH ~r l~ B Br ~OH ~ ~~0
OH O
OH
.05 d
~ \1
H
OH O
5 -0.O7 d 5 0.01 d
v 60.94t
I
r ~ ~ r OH OH
Br
@
~
OH
274, R=Br; gymnochromeA
~ 0
B
r (~S03 - Na+
OH
276 gynmochromeC
275, R=Br,H; gynmochrome B
Br
OH O
OH
OH O
Br
OH
Br
5 0.89 t
-~ .......... :" OH O
BrOSO3_Na,"
5 -0.05 t
Br"
OH
-y OH
277 gymnochromeD
"~ B ~ 0
oSO~-Na"
OH
278 isogymnochromeD
0.50
10.00
Ag
-I0.00 200
Wavetenght
(rim)
gymnochrome D
10.50 600
200
Wavelenght
(run)
600
isogymnochrome D
Fig. 26. The gymnochromes, phenanthroperylenequinone pigments from the crinoid Gymnochrinus
richeri. When the major gymnochrome B (275) was heated in pyridine at 160~
it was partially converted
into a diastereomer which exhibited a CD curve opposite in sign to that of 275. This implies that the two compounds are both configurational helixes (the configurations of the side-chain chiral centres cannot be modified by the thermal isomerization). The inversion of helicity is accompanied by a change in the conformations of the side-chains. The strong upfield shift (8-0.05) of the methyl doublet of the C 3 side-chain in gymnochromes A and B indicates that the group lies sufficiently above the phenanthroperylenequinone ring system to be exposed to a strong shielding effect, whereas the C 5
103 side-chain, whose protons resonate "downfield", is outside the zone affected by the ring current. The reverse happened with the isomer obtained by thermal inversion of gymnochrome B. Gymnochrome D (277) and isogymnochrome D (278) are sulphated molecules which shed light on the stereochemical features of this group of pigments. Both are chiral molecules, but are symmetric by virtue of the presence of a C 2axis of symmetry in their structures, as the symmetry of their nmr spectra, which show only half of the expected signals, indicates. The 1H nmr spectra of the two isomers are substantially different and the major difference deals with the chemical shift values of the terminal methyl protons, strongly upfield shifted to 8 -0.05 in the spectrum of isogymnochrome D (278) and to downfield values, 8 0.89, in that of gymnochrome D (277). The 13C nmr spectra of the two isomers are also significantly different. However the CD spectrum of isogymnochrome D (278) is opposite in sign to that of gymnochrome D (277) (Fig. 26) and this finding suggests that 278 and 277 are diastereomers with opposite helicity. In confirmation gymnochrome D, after solvolytic removal of sulphate groups, was partially converted to sulphur-flee isogymnochrome D by heating in pyridine at 160~
Likewise
heating desulphated isogymnochrome D in pyridine at 160~ afforded desulphated gymnochrome D. Because heating interconverts the two diastereomers, the two corresponding chiral carbons in the sidechains of gymnochrome D (277) and isogymnochrome D (278) must have the same configuration. The axial chirality (helicity) of gymnochromes was suggested by a CD correlation with a group of mold perylenequinone pigments, axially dissymmetric because the aromatic ring system has been forced into a non planar helical shape (198). The suggested configurations of the side-chain chiral carbons were based on correlation between the conformation of the side-chains, the 1H n m r and CD data and by analogy with the above perylenequinones. The orientation of the side-chain is regulated by both the helicity of the ring system and the configurations of the chiral centres in the side-chains. That is, when the helicity of the ring system and the configurations of the side-chain chiral carbons are M and R (or P and S), respectively, the side-chain is oriented above the ring system, whereas, when the helicity of the ring system and the configurations of the side-chain chiral carbons are M and S (or P and R), respectively, the side-chain is directed away from the aromatic ring (198). The configurations of the chiral carbons of the C 5side-chain of both gymnochrome D (277) and isogymnochrome D (278), which must be of the same absolute configuration, have been confirmed by applying the empirical approach of Horeau to appropriate derivatives in which all the phenolic groups were protected. The green colour of the animals suggests that the pigments are present as phenoxide salts, probably in the form of Zn complexes, while the yellow colour of the live animals suggests that the phenanthroperylenequinone system of the gymnochrome might have been formed, at least in part, by oxidation and exposure to sunlight of structurally related bianthrones or other dimeric precursors. 6.
ECHINOIDEA Sea urchins elaborate a large number of closely related pigments based on naphtoquinone
skeleton. They occur as structural pigments (spinochromes) and in the eggs, ovaries and perivisceral fluid of the animals (echinochromes). The first research in this field dates back to 1885 (199), but it was more than fifty years later when a pure crystalline pigment (echinochrome) was isolated and identified (200). After that, nearly 25 spinochrome pigments have been identified from various laboratories. This extensive work has been reviewed by Goodwin (201), Thomson (202), Scheuer
104
~-~,,~
~.~
279
M~-~,~ 280
~IM~I~ 281
(203), Grossert (204) and more recently by Stonik and Elyakov (12). Studies on sea urchin pigments have apparently stopped around the seventies. Sea urchins are endowed with two types of venomous organs, spines and pedicellariae. Most investigations have been performed with pedicellariae venoms, which apparently are proteinaceous materials. This work has been reviewed by Habermehl and Krebs (18). Pettit and colleagues have reported the isolation of antineoplastic glycoproteins fromLytechinus variegatus (205) and Strongylocentratus droebachiensis (206). Prota and colleagues have isolated redox-reactive 4-thiohistidine compounds from the eggs of the echinoid Paracentrotus lividus and of other echinoderm species and identified them as 1-methyl-5-thio-L-histidine (or 3-methyl-4-thio-Lhistidine) and its o~N, o~N-dimethyl derivative (279, 280) (207, 208). More recently a novel 4thiohistidine compound, ovothiol (i.e. 1-methyl-aN, otN-dimethyl-4-thio histidine, 281), has been isolated from sea urchin eggs and shown that it accounts for the previously reported ability of egg cytoplasmic extracts to stimulate oxidase activity of the sea urchin ovoperoxidase (209). Turner et al. (209) have confirmed the assignment of the methyl group in ovothiol (281) to the N-1 position by comparison of the 500 MHz 1Hnmr spectrum of des-S-ovothiol, produced by desulphuration with NiRaney, with those of aN, otN-dimethylated commercial samples of 1-methylhistidine and 3methylhistidine. Very small differences could be observed for the resonances of the methyl protons only when the spectra were run on mixtures of des-S-ovothiol with the reference compounds. These Authors also express some doubt on the assignment of 3-methyl-4-thiohistidine structures to 279 and 280, for which Prota and colleagues deduced the position of the methyl group on the imidazole ring by comparison of the rotation and electrophoretic mobility of the derived des-S-N-methylhistidine with those of authentic samples of 1-methyl and 3-methyl-L-histidine. The difference of studied species could account for the isolation of different compounds, even if it is difficult to believe that ovothiol from the echinoid Strongylocentrotus purpuratus (209) has a different ring linked methyl position than the thiohistidines isolated from the echinoids Paracentrotus lividus, Sphaerechinus
granularis, Arbacia lixula, the holothurian Holothuria tubulosa and the asteroids Marthasterias glacialis and Astropecten aurantiacus (208). ACKNOWLEDGEMENTS Our researches have been supported in part by grants from Ministero dell' Universit~ e della Ricerca Scientifica e Tecnologica and in part by C. N. R., Roma (P. F. Chimica Fine II). Some of the researches described here are part of the project SMIB (Substances Marines d' Int6ret Biologique), ORSTOM - CNRS, Noum6a, New Caledonia.We thank Prof. P. Potier (ICSN, CNRS, Gif- sur-Yvette, France) for having promoted and encouraged the collaboration between our group in Napoli and the groups of CNRS and ORSTOM in Noum6a, New Caledonia. We wish to express our thanks to the following persons engaged in the echinoderm research in Napoli: Dr.s M. V. D' Auria, M. Iorizzi, I. Bruno, E. Finamore, L. Gomez Paloma and F. De Riccardis, and in Noum6a: Dr.s C. Debitus, B. Richer de Forges, T. Sevenet and J. Pusset, for their extensive intellectual, creative and technical contributions. We are also grateful to Professor's W. Fenical (Scripps Institution of Oceanography,
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
111
Recent Advances in the Chemistry of Diterpenoids from Rabdosia species Yoshio Takeda and Hideaki Otsuka
I.
Introduction Plants
perennial eastern and
parts
Japan,
species the
in
has
Hara
isolated
research main
1966
and
their
also
examined.
and
M.
on
the
Node
as
this
article
tion
was
except
1984
[2],
in the cases was
genus,
data
revised.
reported
for easy
distribution and
their
reported
as
was
isolat-
compounds,
Professors
E.
like
were Fujita
to
focus
Accordingly,
are not and
article
the
activities.
a
IH- and
elucidamainly
on
ISC-NMR
isolated
the
in
structure
focuses
No
the
included
b)
of the among
a
eluci-
been
the s t r u c t u r e
identification
Japanese
6,7-seco-ent-
we w o u l d
compounds,
of d i t e r p e n o i d s
biological
data.
a)
began
activities,
these
field.
journal
This
(1) a
have
by
same
Enmeiso
biological
review
when
three
enmein
of
so
in this
reported
the
of the
review
(Maxim.) the
plectranthin
from
having
The
in the previous
of newly
1958,
for f.)
bellyache
genus
named
In
ones
in a scientific
spectroscopic pounds,
in
of
diterpenoids
reported
structures
this
of
nice
development
mentioned
plants
al.
antitumor
published
under
structure
many
for
trtchocarpa
independently
determined.
a very
remedy
principle et
China Various
(Burm.
stomachic
a diterpenoid
then,
a
in
medicine
japonica
R.
of
Yagi
the
of
as
a bitter
enmein
and
sebsequent
previously
by
and that
Since
times
bitter
Enmeiso
Thailand,
principles.
R.
are
especially
in indigeneous Thus,
constituents
Since
was
as
isolated
antibacterial
compounds
made
the
bitter
"Enmeiso".
crystalline
structures
as
this
market
nucleus.
such
the
the
principle
in
kaurene ed
ancient
from
bitter
used
of
groups
dated
been
from
the
the Himalayas,
diseases.
name
on
when
have
several
(Labiatae)[l]
Rabdosia
in the world,
contain
the
on
Studies
1910
was
now
India,
used
under
is
name.
of
been
widely
they usually
of Rabdosia
Japan
to the genus
distributed
of Africa,
and
treatment
Hara
in
belonging herbs
com-
species
of
criticism
is
112
II.
Classification According
=enus
o
C)(4)-
are
19,20
A)
(group
E)
Fig.
I).
(5),
Compounds
skeleton
2) 3,20
4)
by
hydroxyl
group
groups,
but
ethoxyl
which
alphabetical The
reader
should AS01
=
exidonin
and E309
have
o
reference Among [49],
noids,
isolated
(A411)
reported
and
ones
to elucidate
compounds,
resentative
Based
IH-NMR
denotes
the of
or
acetalic
methoxyl
or
number, three
have
C, A526
two
rabdoA,
B203 B,
A = macrocalin
A,
= ludongnin
(group
optical
A)
into
A,
this
rotations
and
2.
ones on
of b u l l e y a n i n A (A514)[51,
being
the
inflexanin
the s t r u ct u r e s
IH-
and
ISC-NMR
for
this
data
group
and
and
56]
were
thus
shown
were 12158]
structure
14[I3](Fig.
are very
are
(A501)
50],
correlated
B (A409),
15155]
data
11148,
revised
of c h e m i c a l l y
being
de-
the number
classified
and w e i s i e n s i n reported
the structures
inflexinol
'
are
D = rabdolongin
skeleton
the structures
respectively.
order
structures
in Fig.
compounds,
(A309)[33],
(A309).
the p r e v i o u s l y In
Their
the p r e v i o u s l y
15157],
inflexin
9
C.
of 84 c o m p o u n d s
shtkok~ana
reniformin
macrocalyxin
found gibbane
However,
ludongnin
the e n t - k a u r e n e
are shown
these
inflexin
revised,
B, E306
El04
R.
7) (see
been
compounds
=
(6),
(I0)
review
and
: macrocalyxin henryin
F)
the serial
group.
C307 m a o e c r y s t a l
G = rabdosin
appeared
H), are
several
reports
numbers
~
that
having
1984,
(R
figures
(group
the
and ketalic
be aware
= rabdosin
has
letter
and CG03).
C, A408
have
represents
CG02
H = trichodonin,
Diterpenoids
Since
-OR
two
amethystonal
rabdophyllin
first
the ent-
(8),
I)
from
(CG01,
= novelrabdosin,
trichorabdal
7,20 (group
in this
-OAc
I)
3) 6 , 7 - s e c o - e n t -
which
in the c o m p o u n d ' s
glaucocalyxin
neorabdosin
=roup
order,
names"
(3)-,
groups
The
number
from
I)"
6) gibbane
isolated
-OH,
remaining
are exceptions
different sinatol
second
or acetalic
The
(A ~
(group
compound
mentioned
include
not ketalic
groups.
glucosides
III.
sole
like A201.
the
(7),
and a b i e t a n e
compounds
and
B)
to the nine
The
four c h a r a c t e r s
compound's
E304
(9)
G)
is r a b d o e p i g i b b e r e l l o l i d e
noted
E215
(group
(group
belonging
The new natural
isolated
groups
8,9-seco-ent-kaurenoids
H)
decade.
[3].
-OH,
nine
D)-epoxy-ent-kaurenoids,
(group
the past
diterpenoids
into
7,20-cyclo-ent-kaurenoids
during
and
(2),
(group
ent-isopimarane
in
structure,
classified
(group
and
kauranes 5)
to their
Rabdos~a
kaurenoids
of Rabdos~a d i t e r p e n o i d s
to
revised, 5).
identify
useful. in List
of
diterpe-
Rep1
and
113
H
20 Io ~ . ! ~ - - 1 7
~
.o- "~',,-- ~o.
19
1 i
18
7 15
2(A) i
20
A
4(C)
3 (B) H I
/
I
7 H
6 (F)
5 (E) H
/ / ~ I" ~ 7 7
H
7(G) 12
2
f--4~%J" /i ~ k 19
is
8 17
16
,~
6 9(H)
10(I)
Fig. 1. Basic skeletons of Rabdosia diterpenoids.
H
114 A101
A201 'l
A202
~ --CH2OH
0
"
liangshanin G
glaucocalyxin E
glaucocalyxin D
[ (I. ]O-142.5 o ( i e O H )
[ O. ]D "161.5~ (MeOH)
[41 A203
[5]
[51 A204
A205
!
c
,co
o- Z~v inflexarabdonin C
-o.
o- ~ , - -
liangshanin A
[61 A206
[4,]
,
[4]
OH u
A301
4~~v
liangshanin B
[ (x ]D "192.5~ (MeOH)
[ (x ]D "55-2~ (MeOH)
0"
-o.
[~
;
"OAc
liangshanin C
I v ' H OHC "
A303
"OH0
amethystonal
amethystonoic acid [ O, ]D-86.1~(PY)
[71 A305
I~ v
~
compound I [ (x ]D "94.0~ (MeOH)
compound 2
"OH 0
HO
4-epihenlyiu~; A
[ (x ]O-121.8 ~ (MeOH)
[91
"oHO
[7,81 A304
.o,
II v ~H HOOC '
[ (x ]O "75.8~ (PY)
[41
OH n
A302
[91
Fig. 2. Diterpenoids having an ent-kaurane skeleton (group A)
[ ,tll)
o.t(I 4" (l,y)
L1r
115
A306
A307
.o.:~~v
-o.
y,v
"HoH
excisanin C
[ (x ]D -112.7~ (MeOH) [11]
A c O ~
A311 AcO~m A ?H,
AcO
O inflexarabdonin E [ Ex.]D-42.8~ (ieOH) [6]
[33]
A313
HO OAc
C)H inflexaninA
[ (x ]D -108.3~ (MeOH) [13]
,o.
inflexin
A312
...o
henryine A
HO
AcO
AcO,
.o
,co
A310
o,.,y -p~
AcO
-o.
[ Or. ]O +30-8~(PY) [12]
A309 AcO~ A
'
A308
o,
OH i
inflexarabdonin G [ Ex.]D "54.9~ (MeOH) [14] A314 A
c
T
~
OH
;
HO"
O
inflexarabdonin H [ Q' ]D "64-2~(MeOH) [14] A315
./
OH
HO isodopharicin C [ (x ]O "160~(ieOH) [15] Continued
AcO" isodopharicin A [ (x ]D "154~(EtOH) [15]
A316
-o isodopharicin B [ or.]D "190~(EtOH) [15]
A317
liangshanin D [4]
liangshanin E [4]
116 OAc t
A318
A319
i
O
o- ~ , , ~ v
OH t
I~v
A322
"OH0
[17]
?-
A323
HO"
'
Z.~ V "OH
parvifoline B
pseurata A
[ (x ]D-65.96~ (MeOH)
[19]
[18]
,
macrocalyxin D
OH
~, ~ v .o, OHC
macrocalyxin E [ (x ]D +17.75~ (EtOH)
A324
~ ~ HA ~ 0 OH ~,I~v -OH0 OHC
~,H ' OAc Iongirabdosin [ (x ]D -150.0~ (MeOH) [16]
[4] A321
O A f s . , I ~ , 0 "OAc
-o,
liangshanin F
OHC
A320
0
[20]
OH I
A325
A326
"O
o~.o~~
HO pseurata C
rabdokunmin B
[20]
.co
A327
rosthornin A [ or.]D -150.98~ (CHCI3) [24] Continued
[21]
OH
~0
rabdosinatol [ (x ]D "108~ (MeOH)
[ or.]D-46.2~ (MeOH) A328
AcO
[22, 23]
HO
A401
~0 O xindongnin A [ (I ]D "44.2~ (PY) [25]
I i H~o~cO HO O adenanthin [ (I ]D "76~ (CHCI3) [26]
117
A402
A403
2 f L ~-~.
.o-Z~"
-o.
Z~"
coetsoidin B [ (x ]D "104.2~(MeOH) [27] A405
'~o"
~H
C)Ac
oHOH~
-o.
Z~"
compound 3 [ (x ]D "109.7~ (MeOH) [9] A406
H,~ ~
AeO
,co- ~
v
A407
-OH
,
"OH
dihydropseurata F
A409
( ~, ~.;OH y,, t," "OH
henryin [ (x ]D "88~ (MeOH) [31, 32] A411 A
c
O
~
H? i
~,'H ~ H ~ ' ~"O 6H
inflexinol [ (x ]D-43.0~ (MeOH) [33, 35] Continued
v
.oO H
"oHO
flexicaulin A [ (x ]D "99.43~(MeOH) [30]
[29]
A408
-o,c
compound 4 [ (x ]D "110.9~ (MeOH) [9]
O
..
dawodensin A [ (~ ]e " 34-3~(MeOH) [28]
AGO
A404
A410 AcO
HOHo',
HO , ~ ' T ' ~
H
x
AcO
OH AcO
~H
O
inflexanin B [ (x ]D "46-2~(MeOH) [13, 33] A412
A413
HO OH A
Z,~v
inflexarabdonin B [ (x ]D "16.9~ (MeOH) [34]
"OH
kamebakaurin [ (x ]D-107~(MeOH) [36]
HO.,,,,,,~
~-"
"OH~
kamebakaurinin [ (x ]D "101~(PY) [36]
118
A415
A414 AcO. AcO~
HO~
!
AcO" ~ ~ i
OAc
lushanrubescensin C [ (x]D"50~(MeOH) [37]
6H
AcO" ~ I ~ ' ~ A c
lushanrubescensin D [38] A418
A417
HO.
HO
H
0
AcO" 2 " H
A416
HO
lushanrubescensin E [ (x]D"77,5~(MeOH) [39] OH I
A419
OHC HO" y | V
"OAc
"OH
H O ~ \H
HO" ~ ~ v
OH
"OH
parvifoline A
phyllostachysin B
pseurata B
[19]
[40]
[20]
[ (x]D "23.50~(MeOH)
A4 O
0
Ho
A421
~'~~H~~o~H AcO~" y l v "OH AcO- - j ' ' H
HoHO.
A422
~"~~~~0 AcO" ~ , ~ v
O~H
~lv
, H
pseurata F
[29]
[29]
OH I
~ ~
I~~ ' O
OAc
"OHO
rabdokunmin A
[ O.]D "51"0~ (Me2CO) [21] Continued
OH
A424
~
HO
|v
~H
[~~ " ~ A ~
"OH
pseurata D
A423
~v
0
"OHO
rabdokunmin C
[ (x]D"85.7~(MeOH) [21]
OOH "OH
rabdoinfiexin B "74.9~(MeOH) [41]
[ or. ]D
A425
HO
i~v
"OH 0
rabdokunmin D -113.3~(MeOH) [21]
[ or. ]D
119
A426
OH i
OH t
A427
A428 .o
,o~.o~O
y.~v
rabdoloxin A [ (x ]o-62.9~ (PY) [42] A429
.o,
/.~v
rabdoloxin B [ r162 ]O "59.5~ (PY) [42] A430
.o,
rabdoserrin D [43] A431 o,c
,co- ~ ~ y o,c
c
,co-Z~'~,-o,c
rabyuennane A [ (x ]D + 19.1~ (MeOH) [44] ,,32
o.
,•
rabyuennane B
,,33
A435 AcO,
OH i
.o~,~
,,3,
o.
shikoccidin [ (x ]D "3.2~ (MeOH) [45, 46] OH A501 , A c ( ~ C O ~
"~176Z,,,~" "~176 xindongnin B [ (x ]D "72.5~ (PY) [25]
Continued
.o,
reniformin B [ (x ]D "37.13~ (EtOH) [32]
[44]
,co-~,~
rosthornin B [ (x ]D "156.3~ (MeOH) [24]
,o. Z ~ v
bulleyanin [48, 49, 50]
Z~v
-o.
umbrosianin [ (x ]D "81-5~ (MeOH) [47] A502
.o- ~ , , ~ T
OAc
-o,c
calcicolin A [ (x ]D-59.3~ (MeOH) [51]
120 A503
A504
A505
Ac'O
AcO inflexarabdonin A [ ~ ]D "5.5~ (MeOH) [34] A506
inflexarabdonin D [ (x ]D "17.1~ (MeOH) [6] A507
,co. ~~~-~o
OH lungshengrabdosin [ (x ]D "21-9~ (PY)
,co~,
o~O ,co. ~~y-o.
OAc lushanrubescensin [ (x ]D "62.1~ (PY) OH I
H l'~
9o
"OAc
AcO"
nervosanin [ (x ]D +37.11~ (PY) [54]
A512
.oj,~-~
OH ,
~ l . V
,co-,'.
"OH
AcO '~
pseurata E
[21]
," ~
rabdoforrestin A [ (x ]D "83~ (CHCI3) [55]
[29] A514
,co* ~ , ~ v - o , c
rabdokunmin E [ ~ ]D -110-5~ (MeOH)
.o,
,oo ,
A513
,o.
[37] A511
,
HO '~ ~
OAc lushanrubescensin B [ (X ]D "90~ (MeOH)
[53] A510
,co,C O , ~ ~
inflexarabdonin F [ ~ ]D + 13.4~ (MeOH) [6] A508
[52] A509
I~
.o-Z~'/~-o,c
rabyuennane C [44]
weisiensin A [51, 56, 57]
121 List.
I.
IH-NMR Chemical Shift Data Ent-kaurane Skeleton (group
for A).
Diterpenoids
Having
an
A101 L i a n g s h a n i n G(N) 0 . 8 5 ( s , 19-3H), 0 . 8 7 ( s , 1 8 - 3 H ) , 1 . 0 9 ( s , 3H), 2.49(t, 7, 13-H), 2.05(d, 12, 14-Ha ), 3.60(d, 12, Ha), 3.77(d, 12, 17-Hb), 4.43(t, 5, II-H)[4].
2017-
A201
Glaucocalyxin D(P) 20-3H), 3.24(br.s, 5.67(dd, II.3 4.5,
0.97(s, 19-3H), 1.02(s, I3-H), 4.96(s, 14-H), 7-H), 6.23(s, l-Hb)[5].
18-3H), 5.38(s,
A202
Glaucocalyxin E(P) 20-3Hs), 1.18(d, 4.61(dd, 9.6, 4.6,
1.00(s, 19-3H), 1.07, 1.07(each s, 8.0, 17-3H), 3.21(dd, 14.2 7.1, 7-H), 5.06(s, 14-H)[5].
A203
Inflexarabdonin C(P) 1.06, 1.08, 1.40(each s, 18, 19 and 203Hs), 1.46(br.dd, 12 4, 14-Ha), 2.07(d, 13.5, 7-Ha), 2.35(d, 12, 14-Hb), 2.50(br.s, 9-H), 2.96(s, 5-H), 3.00(m, I3-H), 3.55(d, 15.5, 7-Hb), 4.33(br.d, 4.5, II-H), 4.68(t, 2.5, 3H), 5.31(br.s, 17-Ha), 6.05(br.s, 17-Hb)[6].
1.07(s, 17-Ha), 18 and 13-H),
A204 Liangshanin A(N) 1.17(s, 18-3H) 1.17(s, 19-3H), 1.28(s, 203H), 3.12(m, 13-H), 4.40(dd, 9 5, 7-H), 4.83(br.s, 14-H), 5.47(br.s, 17-Ha), 5.88(d, I0, 2-H), 6.19(br.s, 17-Hb), 7.01(d, i0, I-H)[4]. A205
Liangshanin B(N) 1.17(s, 18-3H), 1.17(s, 3H), 3.15(m, 13-H), 4.24(dd, 9 5, 7-H), 5.90(d, 10, 2-H), 5.93(br.s, 14-H), 7.03(d, i0, I-H)[4].
19-3H), 1.40(s, 205.48(br.s, 17-Ha), 6.22(br.s, 17-Hb),
A206
Liangshanin C(N) 1.17(s, 18-3H), 1.17(s, 19-3H), 1.29(s, 203H), 3.15(m, 13-H), 4.81(br.s, 14-H), 5.42(dd, 9 5, 7-H), 5.48(br.s, 17-Ha), 5.91(d, i0, 2-H), 6.22(br.s, 17-Hb), 7.03(d, I0, I-H)[4].
AS01
Amethystonal(P) 1.13(s, 19-3H), 1.61(s, 20-3H), 3.61(d, 3, 13-H), 4.38(t, 3, 12-H), 4.97(dd, 12 4~ 7-H), 5.43(br.s, 17-Ha), 5.83(br.s, 14-H), 6.34(br.s, 17-Hb), 9.29(s, 18H)[7].
A302 Amethystonoic acid(P) 1.47(s, 19-3H), 1.71(s, 20-3H), 3.63(d, 3, 13-H), 4.41(t, 3, 12-H), 5.17(dd, 12 47-H), 5.42(br.s, 17-Ha), 5.89(br.s, 14-H), 6.34(br.s, 17-Hb))[7]. A303 Compound l(C) 0.86, 0.88, 1.30(each s, 18, 19 and 20-3Hs), 3.10(m, 13-H), 3.33(dd, i0 5.5l-H), 4.11(dd, II 5~ 7-H), 5.37(br.s, 17-Ha), 6.02(br.s, 14-H), 6.14(br.s, 17-Hbi[9]. A304
Compound 3.12(m, 5.36(dd, Hb)[9].
2(C) 0.84, 0.91, I. 19(each s, 18, 19 and 20-3Hs), 13-H), 3.33(dd, 9.5 6~, I-H), 4.90(br.s, 14-H), 12.5 4, 7-H), 5.39(br.s, 17-Ha), 6.13(br.s, 17-
A305
4-Epihenryine 12.3 4.5, 3.01(dd, 18 1, 13-H), 5 . 5 1 ( d , 1,
A306
Excisanin
A(P) 0.88(s, 19-3H), 1.23(s, 20-3H), 1.89(dd, 5-H), 2.16(s, 9-H), 2.73(dd, 18 3.4, 12-Ha), 3.3, 12-Hb), 3.29(d, 10.8, 18-Ha), 3.45(ddd, 1 1 3 . 6 6 ( d , 1 0 . 8 , 18-Hb), 4 . 9 4 ( d d d , 12 3 3, 7-H), 1 7 - H a ) , 5 . 6 9 ( d , 1, 14-H), 6 . 3 2 ( d , 1, 1 7 - H b ) [ 1 0 ] .
C(N)
0.90(s,
19-3H),
1.13(s,
20-3H),
3.34(d,
10.6,
122
A307
A308
18-Ha), 5.17(s, Henryine 12-H a ), 6, 7-H), Hb)[12].
3.66(d, 10.6, 18-Hb), 4.92(dd, 11.9 3.6, 7-H), 14-H), 5.39(br.s, 17-Ha), 6.32(br.s, 17-Hb)[ll]. A(P) 0.90(s, 18-3H), 1.26(s, 20-3H), 3.16(dd, 16 4, 3.38(d, ii, 19-Ha), 3.76(d, ii, 19-Hb), 5.06(dd, 12 5.68(br.s, 14-H), 5.85(br.s, 17-Ha), 6.50(br.s, 17-
Inflexanin A(C) 0.96, 1.28, 1.42(each s, 18, 19 and 20-3Hs), 2.27(ddd, 14.5 5 3, 12-Ha ), 2.31(dd, 14.5 3, 7-H ~ ), 2.78(d, 12.5, 14-Ha ), 3.09(m, I3-H), 4.10(br.d, 5, II-H), 4.49(dd, 5 3HI~ 6 - H ) , 4 . 6 2 ( t , 3, 3 - H ) , 5.31(br.s, 17-Ha), 5.91(br.s, 17 )[13]
A509 Inflexin(C) 0.87(s, 18-3H), 1.16(s, 20-3H), 1.33(s, 19-3H), 1.57(dm, 12.4, 14-H 8 ), 1.83(d, 12.5, 7-H a ), 2.09(br.s, 9H), 2.33(d, 12.4, 14-H a ), 2.72(br.s, 5-H), 3.12(m, 13-H), 3.14(dd, 12.5 0.6, 7-H B ), 3.99(dd, 11.7, 4.8, I-H), 4.62(t, 3.0, 3-H), 5.39(t-like, 0.8, 17-Ha), 5.97(br.d, 5.0, II-H), 5.99(t-like, 0.8, 17-Hb)[33]. A310 Inflexarabdonin E(C) 0.98, 1.13, 1.33(each s, 18, 19 and 203Hs), 1.50(br.dd, 12.5 4.5, 14-Ha), 1.89(d, 13.5, 7-Ha), 2.32(d, 12.5, 14-Hb), 2.80(br.s, 5-H), 3.09(m, 13-H), 3.21(br.d, 13.5, 7-Hb), 4.08(dd, 10.5 6, l-H), 4.60(t, 3, 3H), 5.24(br.d, 4.5, II-H), 5.37((br.s, 17-Ha), 5.96(br.s, 17-Hb)[6].
ASll
Inflexarabdonin G(C) 0 . 8 4 , 0.91, 1 . 1 5 ( e a c h s , 18, 19 and 20-3Hs), 2.45(d, 12, 1 4 - H a ), 3 . 0 7 ( m , 1 3 - H ) , 3 . 7 7 ( d d , 11.5 4.5* l-H), 4.76(t, 3, 3 - H ) , 5 . 2 6 ( b r . s , 17-Ha), 5.88(br.d, 5, l l - H ) , 5.89(br.s, 17-Hb)[14].
A312
Inflexarabdonin H(C) 0.91, 1.20, 1.35(each s, 18, 19 and 203Hs), 1.86(d, 12.5, 7-Ha), 2.23(d, 12, 14-Ha ), 2.29(br.s, 9-H), 2.90(br.s, 5-H), 3.05(m, 13-H), 3.25(d, 12.5, 7-Hb), 4.29(dd, 5 2.5~, II-H), 4.61(t, 3, 3-H), 5.33(dd, 11.5 5.5, I-H), 5.36(br.s, 17-Ha), 5.95(br.s, 17-Hb)[14].
A313
Isodopharicin A(C) 0.79(s, 18-3H), 1.01(s, 20-3H), 2.18(d, 5.0, 12-H /3 ), 2.54(d, 3.22(dd, I0.0 6.8, 3-H), 5.23(dd, 5.0 1.2, 0.7, 17-Ha), 5.98(d, 0.7, 17-Hb)[15].
19-H), 11.3, ll-H),
1 .84(s, 14-H a ), 5.37(d,
A314 Isodopharicin B(C) 0.86(s, 18-3H), 0.89(s, 19-3H), 20-3H), 2.17(d, 5.0, 12-H /3 ), 2.54(d, 11.5, 4.46(dd, 10.5 5.6, 3-H), 5.22(dd, 5.0 1 . 5 , II-H), 0.5, 17-Ha), 5.98(d, 0.5, 17-Hb)[15].
14-Ha ), 5.35(d,
i .09(s,
A315 I s o d o p h a r i c i n C(C) 0 . 9 9 ( s , 20-3H), 1.06(s, 18-3H), 2.18(d, 3.7, 12-H /3 ), 3.45(d, 9.0, 19-Ha), 3.69(d, 9.0, 19-Hb), 5.24(br.d, 4.0, II-H), 5.36(s, 17-Ha), 5.99(s, 17-Hb)[15]. A316
Liangshanin D(N) 1.13(s, 19-3H), 1.19(s, 18-3H), 1.22(s, 203H), 3.06(m, 13-H), 4.35(dd, I0 5, 7-H), 4.86(br.s, 14-H), 4.86(br.s, 15-H), 5.34(br.s, 17-Ha), 5.71(br.s, 17-Hb), 5.98(d, i0, 2-H), 7.12(d, i0, I-H)[4].
A317
Liangshanin E(N) 1.14, 1.14, i. 14(each s, 18, 19 and 203Hs), 2.14(dd, 16 2.9, 2-Ha), 3.12(m, IS-H), 3.18(dd, 16 8.8, 2-Hb), 4.32(dd, 9 5, 7-H), 4.79(br.s, 14-H), 4.91(dd, 8.8 2.9, l-H), 5.42(br.s, 17-Ha), 6.14(br.s, 17-Hb)[4].
123 A518
Liangshanin F(N) 1.12(s, 18-3H), 1.12(s, 19-3H), 1.21(s, 203H), 3.58(m, 13--H), 4.85(dd, I0 6, 7-H), 5.20(m, 12-H), 5.44(br.s, 14-H), 5.52(br.s, 17-Ha), 6.36(br.s, 17-Hb)[4].
A319
Longirabdosin(C) 1.25, 1.29(each s, 18 and 19-3Hs), 13.5, 5 - H ) , 2.70(dd, 8 3.5, 9-H), 2.78(m, 13-H), 12, 2 0 - H a ) , 4.83(d, 12, 20-Hb), 5 . 0 4 ( m , 17-2H), 13.5, 6-H), 5.86(d, 10.5, 2-H), 6.16(t, 2.5, 15-H), 10.5, 3-H)[16].
2.29(d, 4.67(d, 5.73(d, 6.39(d,
A320 Macrocalyxin D(P) 1.22(s, 19-3H), 3.28(br.s, 13-H), 4.16(d, 10, 2 0 - H a ) , 4.28 (d, i0, 20-Hb), 4.92 (dd, 12 4, 7-H), 5.42(br.s, 14-H), 5.62(s, 17-Ha), 6.34(s, 17-H), 9.32(s, 18H)[17]. A321
Macrocalyxin 9-H), 3.78(d, H), 5.66(s, 18-H)[18].
E(P) 1.15, 1.66(each s, 19 and 20-3Hs), 2.32(s, 4, 13-H), 4.32(d, 4, 12-H), 4.96(dd, I0 5, 717-Ha), 6.18(s, 14-H), 6.46(s, 17-Hb), 9.34(s,
A322
Parvifoline B(P) 0.94(s, 19-3H), 2.18(q, 12.4, 6-H a ), 2.20(br.s, 9-H), 3.46(br.d, 9.5, 13-H), 4.13(dd. 8.7 1.9, 20-Ha), 4.19 (d, 8.7, 20-Hb), 4.97 (dd, 12.1 3.3, 7-H), 5.42(br.s, 14-H), 5.42(br.s, 17-Ha), 6.25(br.s, 17-Hb), 9.39(s, 18-H)[19].
A323 Pseurata A(A) 0.88, 1.02, 1.10(each s, 18, 19 and 20-3Hs), 1.77(q, 12.6, 6-H), 2.99(br.s, 13-H), 3.17(dd, i i . 5 4.5, 3H), 4.19(dd, 13.2 4.3, 7-H), 4.83(br.s, 14-H), 5.34(s, 17Ha), 5.99(s, 17-Hb)[20]. A324 Pseurata C(A) 0.92, 0.93, 0.96(each s, 18, 19 and 20-3Hs), 1.73(q, 12.2, 6-Ha ), 3.58(br.s, 13-H), 4.43(dd, 1 2 . 5 4.2, 7-H), 4.62(dd, 4.9 2.0, 3-H), 5.04(br.s, 14-H), 5.16(s, 17Ha), 6.12(s, 17-Hb)[20]. A325
Rabdokunmin B(P) 0.84(s, 19-3H), 1.63(s, 20-3H), 3.37(d, 10.6, 18-Ha), 3.68(m, 13-H), 3.69(d, 10.6, 18-Hb), 4.46(m, 12-H), 5.38(br.s, 17-Ha), 5.59(br.s, 14-H), 6.28(br.s, ]7Hb)[21].
A326
Rabdosinatol(P) 1.06, 1.08, 1.10(each s, 18, 19 and 20-3Hs), 3.0(br.s, 13-H), 4.34(dd, 14.0 5.0, 7-H), 4.99(s, 14-H), 5.28(br.s, 17-Ha), 5.65(br.s, 17-Hb), 5.91(br.s, 15-H)[22]. 1.05, 1.07, 1.10(each s, 18, 19 and 20-3Hs), 3.04(m, 13-H), 4.27(dd, I0 6~ 7-H), 4.88(s, 14-H), 5.33(br.s, 17-Ha), 5.68(br.s, 17-Hbi, 5.96(br.s, 15-H)[23].
A327
5-H), Rosthornin A((P) 1.02(s, 20-3H), 1.05(br.d, 13.4, 14-Ha), 1.18(s, 18-3H), 1.55(br.s, 9-H), 1.78(d, 11.4, 10.7, 2.71(d, 11.4, 14-Hb), 3.62(d, 10.7, 19-Ha), 3.91(d, 6.27(d, 19-Hb), 5.45(d, 4.7, II-H), 5.75(d, 1.4, 17-Ha), 1.4, 17-Hb)[24].
A328 Xindongnin A(P) 1.00(s, 19-3H), 1.10(s, 18-3H), 1.37(s, 20-3H), 2 . 8 6 ( s , 5-H), 3 . 0 0 ( m , 13-H), 3 . 6 6 ( s , 9-H), 4.32(t, 4~ l l - H ) , 4 . 7 1 ( t , 3, 3-H), 5 . 2 5 ( b r . s , 17-Ha), 5 . 4 1 ( s , 7-H), 5.95(br.s, 17-Hb)[25]. A401 Adenanthin(C or B ?) 0.90(s, 3 . 4 1 ( d d , 3.7 2 . 4 , 3 - H ) [ 2 6 ] .
18-3H),
1.31(s,
19-3H),
124 A402 Coetsoidin B(P) 1.05(s, 19-3H), 1.20(s, 18-3H), 3.33(m, H), 3.68(br.s, 3-H), 4.34(br.s, 20-2H), 5.07(dd, 10..0 7-H), 5.39(br.s, 17-Ha), 5.72(br.s, 14-H), 6.33(br.s, Hb)[27].
136.0, 17-
A403 Compound 3 (C) 0 . 8 6 , 0 . 8 9 ( e a c h s , 18 and 1 9 - 3 H s ) , 3 . 1 1 (m, 13H), 3.57(dd, 10.5 5, l-H), 4 . 2 3 ~ 4 . 3 1 (7-H and 20-Ha), 4.47(d, 12, 20-Hb), 5.39(br.s, 17-Ha), 6.00(d, 1, 14-H), 6.13(br.s, 17-Hb)[9]. A404 Compound 4 ( C ) 0 . 8 4 , 0.93(each s, 18 and 19-3Hs), 3.13(m, 13H), 3.60(dd, 10 5, l-H), 3.96(d, 12.5~, 20-Ha), 4.43(d, 12.5, 20-Hb), 4.93 (d, 1~, 14-H), 5.41 (br. s, 17-Ha), 5.47(dd, 12 4 . 5 , 7 - H ) , 6 . 1 3 ( b r . s , 17-Hb)[9]. A405 D a w o d e n s i n A(P) 0 . 9 9 , 1 . 0 3 , 1 . 4 1 ( e a c h s , 18, 19 and 2 0 - 3 H s ) , 2.51(s, 5-H), 2.64(d, 15.0, 14-H a ), 2.99(br.s, 9-H), 4.32(br.s, ll-H), 4.74(br.s, 3-H), 5.19(s, 17-Ha), 5.28(br.s, 6-H), 5.49(d, 3.5, 7-H), 5.92(s, 17-Hb)[28]. A406
Dihydropseurata F(A) 0.92, 0.95, 1.05(each s, 18, 19 and 20-3Hs), 1.05(d, 4.6, 17-3H), 3.03(d, 7.0, 13-H), 3.57(dd, 12.1 5.5, I-H), 4.15(dd, 11.8 4.0, 7-H), 4.64(t, 3.0, 3-H), 5.02(br.s, 14-H)[29].
A407 Flexicaulin A ( P ) 0 . 8 0 ( s , 19-3H), 0 . 8 9 ( s , 18-3H), 3 . 3 9 ( m , 13H), 4.55(d, 12, 20-Ha), 4.59(d, 5, II-H), 5.01(dd, i0 6, 7H), 5.05(d, 12, 20-Hb), 5.26(br.s, 14-H), 5.46(br.s, 17-Ha), 6.34(br.s, 17-Hb)[30]. A408 H e n r y i n ( P ) 0 . 8 7 ( s , 19-3H), 0.95(18-3H), 3.30(m. 13-H), 3.60(dd, 8 6-, l-H), 4.81(dd, 12 5 - , 7 - H ) , 4 . 9 9 ( s , 20-2H), 5.38(s, 14-H), 5.38(s, 17-Ha), 6.34(s, 17-Hb)[31]. 0.88, 0.92(each s, 18 and 19-3Hs), 3.30(br.s, 13-H), 3.56(dd, 10 6, l - H ) , 3 . 5 2 ( d d , 13 5, l l - H a ), 4 . 8 0 ( d d , 13 6, 7-H), 5.00(s, 20-2H), 5.40(s, 14-H), 5.40(s, 17-Ha), 6.30(17-Hb)[32]. A409 Inflexanin B(P) 1.09, 1.58, 2.05(each s, 18, 19 and 20-3Hs), 1.67(d, i, 5-H), 2.52(br.s, 9-H), 2.64(dd, 14 3, 7-H~ ), 5.11(m, I3-H), 3.28(d, 12, 14-Ha ), 4.17(dd, 16 4, l-H), 4.72(m, 6-H), 4.95(dd, 3 3, 3-H), 5.24(br.s, 17-Ha), 6.04(br.s, 17-Hb), 6.18(br.d, 3, II-H)[13, 35]. A410 I n f l e x a r a b d o n i n B(C) 0 . 8 7 , 1 . 0 9 , 1 . 3 3 ( e a c h s , 18, 19 and 203Hs), 2.26(br.s, 9-H), 2.66(s, 5-H), 2.68(m, 13-H), 2.96(d, 12, 7-H), 3.87(t, 2.5~, 15-H), 3.96(dd, 11.5 5~, l-H), 4.60(t, 3, 3 - H ) , 5 . 0 8 ( b r . d , 3, 1 7 - H a ) , 5 . 2 0 ( b r . d , 2, 1 7 - H b ) , 6.29(br.d, 5, 1 1 - H ) [ 3 4 ] . A411
Inflexinol(C) 0.91, 1.28, 1.49(each s, 18, 19 and 2 0 - 3 H s ) , 2.91(d, 13, 14-H a ), 3.11(m, 13-H), 3.69(dd, 12 5, l-H), 4.43(dd, 5 3, 6-H), 4.71(t, 3, 3-H), 5.29(br.s, 17-Ha), 5.90(t, 3, II-H), 5.90(br.s, 17-Hb)[33, 35].
A412 K a m e b a k a u r i n ( P ) 0 . 8 0 , 0 . 9 0 ( e a c h s , 18 and 1 9 - 3 H s ) , 3.54(dd, 10 6, l-H), 4.30(d, 12~, 20-Ha), 4.62(d, 12~ 20-Hb), 4.85(dd, 9 9, 7 - H ) , 5 . 3 0 ( b r . s , 17-Ha), 5.57(d, 1.5, 14-H), 6.24(br.s, 17-Hb)[36].
125 A413 Kamebakaurinin(P) 0 . 8 2 , 0.84(each, s 18 and 19-3Hs), 4.29(s, 20-2H), 4.82(br.d, 4, II-H), 4.92(dd, II 6, 7-}{), 5.38(br.s, 17-Ha), 5.62(br.s, 14-H), 6.27(br.s, 17-Hb)[36]. A414 Lushanrubescensin C(P) 1.01(s, 19-3H), 1.16(s, 18-5H), 1.53(s, 20-3H), 5.50(s, 17-Ha), 5 . 5 9 ~ 5 . 6 8 ( m , 2, 3, 6 and ll-Hs), 6.06(s, 17-Hb)[57]. A415 L u s h a n r u b e s c e n s i n D(P) 1 . 1 4 ( s , 19-3H), 1.29(s, 18-5H), 1.54(s, 20-3H), 3.07(dd, 8 4, 13-H), 3.72(br.s, II-H), 4.37(m, 6-H), 4.47(br.s, 2-H), 5.27(s, 1 7 - H a ) , 5 . 8 0 ( d , 3, 3 H), 6 . 0 1 ( s , 17-Hb)[38]. A416 Lushanrubescensin E(P) i. I0, 1.13(each s, 18 and 19-3Hs), 1.55(s, 20-3H), 3.08(m, I3-H), 4 . 4 6 ~ 4 . 6 8 ( m , 2 and ll-Hs), 5.29(br.s, 17-Ha), 5.35(m, 6-H), 5.74(d, 3, Z-H), 6.03(br.s, 17-Hb) [39]. A417
Parvifoline A(P) 1.06, 1.15(each s, 19 and 20-5Hs), 2.11(q, 12.2, 6-Ht~ ), 2.47(br.d, 14.0, ll-Ha ), 3.31(br.d, 9.0, I3H), 3.70(d, 10.5, 18-Ha), 4.20(dd, I0.0 6.1, 3-H), 4.21(d, 10.5, 18-Hb), 4.98(dd, ii.9 4.1, 7-H), 5.19(br.s, 14-H), 5.39(s, 17-Ha), 6.52(s, 17-Hb)[19].
A418 Phyllostachysin B(P) 0.92, 1.12(each s, 18 and 19-3Hs), 4.52(d, 4.8, I1-H), 4.61(s, 17-Ha), 5.31(s, 14-H), 5.55(s, 17-Hb) , 6.25 (s, 15-H) , 6 . 7 5 (d, 12, 6-H) , I 0.75 (s, 20H)[40]. A419 P s e u r a t a B(P) 1 . 0 9 , 1 . 2 2 ( e a c h s, 18 and 1 9 - 3 H s ) , 1 . 6 8 ( s , 203H), 3 . 4 1 ( d d , 11.7 4 . 7 , 3-H), 3 . 6 4 ( d , 3 . 2 , 13-H), 4.40(t, 2.9, 12-H), 4.94 (dd, 11.8 4.7, 7-H), 5.44 (s, 17-Ha), 5.89(br.s, 14-H), 6.36(s, 17-Hb)[20]. A420 Pseurata D(A) 0.94, 1.04(each s, 2 x Me), 3.60(br.s, IS-H), 4.02(d, ii.5, 18-Ha), 4.26(d, 11.5, 18-Hb), 4.40(dd, 11.7 4.2, 7-H), 4.91(t, 3, 5-}{), 5.04(br.s, 14-H), 5.57(s, 17-Ha), 6.13(s, 17-Hb)[29]. A421 P s e u r a t a F(A) 0 . 8 8 , 0 . 9 4 , 0 . 9 8 ( e a c h s , 18, 19 and 20-3Hs), 3.57(dd, 12.1 5 . 5 , l - H ) , 3 . 6 6 ( b r . s , 13-H), 3 . 8 2 ( d , 1 8 . 0 , 11H a ), 4 . 3 8 ( d d , 11.7 4 . 4 , 7-H), 4 . 6 7 ( t , 2 . 8 , Z-H), 5 . 0 3 ( b r . s , 14-H), 5.56(s, 17-Ha), 6.08(s, 17-Hb)[29]. A422 Rabdoinflexin B(D+W) 0.84, 0.90, 1.05(each SHs), 4.75(s, 14-H), 5.10(d, 4, II-H), 5.75(s, 17-Hb)[41].
s, 18, 19 and 205.17(s, 17-Ha),
A423 Rabdokunmin A (P) 0.80 (s, 19- 3H), 0.85 (s, 18- 3H), I. 94 (s, 203H), 3.69(m, 13-H), 4.52(br.s, II-H), 4.81(d, 3.i, 12-H), 4.89(dd, 12.0 4.2, 7-H), 5.55(br.s, 17-Ha), 6.35(br.s, 17Hb), 7.23(br.s, 14-H)[21]. A424 Randokunmin C(P) 0.87(s, 19-3H), 1.67(s, 20-3H), 10.6, 18-Ha), 3.58(m, 13-H), 3.65(d, 10.6, 18-Hb), 12-H), 4.98(dd, I0.0 3.7, 7-H), 5.42(br.s, 5.82(br.s, 14-H), 6.29(br.s, 17-Hb)[21].
3.32(d, 4.35(m, 17-Ha),
A425 R a b d o k u n m i n D(P) 0 . 8 8 ( s , 19-3H), 1.12(s, 20-3H), 10.6, 18-Ha), 3.32(m, 13-H), 3 . 6 5 ( d , 10.6, 18-Hb), 3.3, 11-H), 5 . 0 2 ( d d , 11.7 3 . 6 , 7-H), 5.26(br.s, 5.41(br.s, 14-H), 6 . 2 7 ( b r . s , 17-Hb)[21].
3.30(d, 4.25(d, 17-Ha),
126
A426 Rabdoloxin A(P+W) 0.88(s, 19-3H), 1.73(s, 20-3H), 1.92(d, 12.0, 5-H), 2.Z6(d, 0.5, 9-H), 3.Sl(d, 10.5, iS-Ha), 3.67(d, 10.5, 18-Hb), 3.82(dd, 3.5 1.5, 13-H), 4.34(dd, 3.0 0.5, 12H), 5.05(dd, 12.0 4.5, 7-H), 5.26(br.s, 17-Ha), 6.26(br.s, 14-H), 6.41(br.s, 17-Hb)[42]. A427 Rabdoloxin B(P+W) 0.81(s, 18-3H), 0.82(s, 19-3H), 1.04(d, 12.0, 5-H), 1.59(s, 20-SH), 2.10(br.s, 9-H), 3.78(m, IS-H), 4.44(br.s, II-H), 4.80(d, 4.0, 12-H), 4.97(dd, 12.0 4.5, 7H), 5.49(br.s, 14-H), 5.49(br.s, 17-Ha), 6.39(br.s, 17Hb)[42]. A429 R a b y u e n n a n e A(C) 0 . 8 9 , 1.04, 1 . 4 3 ( e a c h s , 18, 19 3Hs), 4.64(t, 1.6, S-H), 4.93(d, 5.4, 7-H), 4.98(d, Ha), 5 . 1 2 ( d , 2.0, 17-Hb), 5 . 3 0 ( d d , 3.4 2.0, 6-H), 2.0, 14-H)[44].
and 202 . 0 , 175.56(t,
20A450 R a b y u e n n a n e B(C) 0 . 8 7 , 1.25, 1 . 4 4 ( e a c h s , 18, 19 and 3Hs), 4.14(dd, 5 . 4 2 0, 6 - H ) , 4 . 6 4 ( t , 1.6, S-H), 4.78(d, 3.4, 7-H), 4.96(d, 2, 17-Ha), 5.10(d, 2, 17-Hb), 5.56(t, 2.0, 15-H)[44].
A451 Reniformin B(P) 1.08, 1.14, 1.40(each s, 18, 19 and 20-3Hs), 3.27(br.s, IS-H), 3.50(dd, I0 5, S-H), 4.74(dd, ii 6, 7-H), 4.85(dd, i0 6, l-H), 5.17(s, 14-H), 5.40(s, 17-Ha), 6.30(s, 17-Hb)[S2]. A4S2 Rosthornin B(P) 0.94(s, 20-3H), 1.03(s, 18-3H), 1.08(d, 12.4, 5-H), 1.51(br.s, 9-H), 2.38(d, ii.3, 14-Ha), 2.85(d, 1 I. 3, 14-Hb), 3.98 (d, 11, 19-Ha), 4.29 (d, 11, 19-Hb), 4.48(dd, 12.4 4.0, 7-H), 5.40(d, 4.5, II-H), 5.67(d, 1.3, 17-Ha), 6.16(d, 1.3, 17-Hb)[24]. A454
Umbrosianin(P) 0.87, 0.92(each s, 18 and 19-3Hs), 1.46(s, 20-3H), 3.31(m, 1 3 - H ) , 3 . 3 4 ( d , 9, I - H ) , 3.68(m, l l - H a ), 4.08(ddd, 12 9 4 . 5 , 2-H), 4.76(dd, i1.5, 4.5, 7-H), 5.27(br.s, 14-H), 5 . 3 6 ( b r . s , 17-Ha), 6.31(br.s, 17-Hb)[47].
A455 Xindongnin B(P) 1.14(s, 19-3H), 1.34(s, 18-3H), 1.49(s, 203H), 2.75(s, 5-H), 3.05(d, 1.2, 9-H), 3.62(t, 3, S-H), 4.10(d, 3.6, 12-H), 4.44(t, 3, 6-H), 5.33(br.s, 17-Ha), 5.76(dd, 3.6 1.2, II-H), 6.02(br.s, 17-Hb)[25]. A501 Bulleyanin(C)0.82(s, 19-3H), 0.94(s, 18-3H), 1.49(s, 20-3H), 3.70(br.s, 12-H), 4.77(t, 3, 3-H), 5.10(dd, 12 8, l-H), 5.17(t, 3, 7-H), 5.27(br.s, 17-Ha), 5.31(br.s, II-H), 5.92((br.s, 17-Hb)[48, 49, 50]. A502 Calcicolin A(P) 1.14(s, 19-3H), 1.22(s, 18-3H), 1.74(s, 203H), 2.95(m, IS-H), 3.58(br.s, 9-H), 3.63(m, 3-H), 5.21(br.s, 17-Ha), 5.45(d, 6.3, 7-H), 5.46(m, 6-H), 5.74(t, 8, l-H), 5.90(br.s, 17-Hb), 5.99(d, 3.6, II-H)[51]. A505
Inflexarabdonin A(C) 0.91, 1.29, 1.46(each s, 18, 19 and 203Hs), 2.66(m, 13-H), 3.71(dd, 12 4.5~, I-H), 3.83(t, 3-, 15H), 4.39(m, 6-H), 4.69(t, S, 3-H), 5.04(br.d 3, 17 Ha) 5.16(br.s, 17-Hb), 6.23(br.d, 5, II-H)[34].
A504
Inflexarabdonin 3Hs), 2.36(d,
D(C) 0.92, 12.5, 14-Ha
1.29, 1.39(each s, ), 2.71(m, 13-H),
18, 19 and 203.86(dd, 11.5
127 4.5, Ha), 2.5,
l-H), 4.35(m, 6-H), 4.68(t, 3, 5.05(br.d, 5, l l - H ) , 5 . 1 8 ( d d , 15-H)[6].
3 - H ) , 4 . 9 5 ( d d , 2 1, 172 . 5 1, 17-Hb), 5.33(t,
A505
Inflexarabdonin F(C) 0.92, 1.29, 1.41(each s, 18, 19 and 203Hs), 2.36(d, 12.5, 14-Ha ), 2.64(m, 13-H), 3.80(t, 2.5, 15H), 3.84(dd, 11.5 4.5, I-H), 4.39(m, 6-H), 4.67(t, 3, 3-H), 5.05(br.d, 5, II-H), 5.05(br.d, 3, 17-Ha), 5.12(br.s, 17Hb), 5.26(br.d, 5, II-H)[6].
A506
Lungshengrabdosin(P) 1.06(s, 19-3H), 1.66(s, 18-3H), 1.78(s, 20-3H), 2.98(m, 13-H), 3.06(s, 5-H), 3.22(s, 9 - H ) , 4.32(d, 10w I-H), 4 . 6 9 ( b r . t , 4 . 5 ~ 6 - H ) , 5.21(br.s, 17-Ha), 5.43(d, 3, 3-H), 5.80(dd, 10 3, 2-H), 6 . 0 l ( b r . s , 17-Hb), 7 . 0 6 ( b r . t , 4, 11-H)[52].
A507
Lushanrubescensin(P) 1.07(s, 19-3H), 1.14(s, 18-3H), 1.51(s, 20-3H), 5.00(m, 13-H), 4.05(d, 4, 7-H), 5.33(d, 3, 3-H), 5.37(s, 17-Ha), 5.37-~5.80(m, 2, 6 and ll-Hs), 6.07(s, 17Hb)[53].
A508 Lushanrubescensin B(P) 1.08(s, 19-3H), 1.14(s, 18-3H), 1.53(s, 20-3H), 3.08(m, 13-H), 4.06(d, 4, 7-H), 4.42(m, IIH), 5.28(d, 3, 3-H), 5.35(s, 17-Ha), 5.40~5.68(m, 2 and 6Hs), 6.04(s, 17-Hb)[37]. A509
Nervosanin(P) 1.16(s, 20-3H), 1.40(s, 19-3H), 1.48(s, 183H), 2.56(m, 13-H), 3.05(s, 5-H), 3.54(br.s, 9-H), 3.94(m, 3-H), 4.75(s, 7-H), 4.93(br.s, 17-Ha), 4.96(br.s, 15-H), 5.83(br.d, 3.6, II-H), 5.87(br.s, 17-Hb), 5.99(dd, 11.2 4.6, I-H)[54].
A510 Pseurata E(P) 1.09(s, 18-3H), 1.66(s, 20-3H), 3.61(d, 2.8, 13-H), 4.22(d, 11.5, 19-Ha), 4.38(t, 3, 12-H), 4.50(d, 11.5, 19-Hb), 4.94(dd, 11.8 4.7, 7-H), 5.25(br.s, 3 - H ) , 5.43(s, 17-Ha), 5.81(br.s, 14-H), 6.33(s, 17-Hb)[29]. A511
Rabdoforrestin A(C) 1.097(s, 19-3H), 1.15(s, 18-3H), 1.51(s, 20-3H), 3.09(m, 13-H), 4.16(d, 4, 7-H), 4.97(d, 3, 3-H), 5.09~5.45(m, 2, 6 and ll-Hs), 5.10(s, 17-Ha), 5.82(s, 17Hb)[55].
A512 Rabdokunmin E(P) 0.91(s, 19-3H), 1.68(s, 20-3H), 3.34(d, 10.5, 1 8 - H a ) , 3.66(d, 10.5, 1 8 - H b ) , 3 . 7 4 ( m , 13-H), 4.45(br.s, II-H), 4.73(m, 12-H), 5.12(dd, 1 1 . 8 3.5, 7-H), 5.48(br.s, 17-Ha), 5.97(br.s, 14-H), 6.35(br.s, 17-Hb)[21]. A513 Rabyuennane C(C) 0.70, 0.80, 1.12(each s, 18, 19 and 203Hs), 3.91(dd, I0.0 6.0, II-H), 4.74(dd, 4.0 2.0, 7-H), 4.80(t, 3.0, 3-H), 4.97(d, 2.0, 17-Ha), 5.04(d, 2.0, 17-Hb), 5.56(t, 2.0, 15-H), 5.79(br.d, 4.8, I-H)[44]. A514
Weisiensin A(P) 1.31(s, 19-3H), 1.59(s, 18-3H), 1.74(s, 203H), 3.72(br.dd, 4.5 3.0, 3-H), 4.48(m, 6-H), 5.20(br.s, 17Ha), 5.59(d, 3.4, 7-H), 5.85(dd, 9.0 6.9, I-H), 5.91(br.s, 17-Hb),6.09(d, 4.0, II-H)[51].
wafter D20 C5D5N, A" description.
treatment. (CD3)2CO,
Solvents are D" (CD3)2S0,
in parentheses. W" D20, B"
C" CDCI 5, C6D 6 , N"
P" no
Number 1
40.8
38.0
38.1
33.4
159.6
158.0
158.0
32.3 a
36.1
80.1
40.68
2
18.6
33.7
33.8
22.5
126.7
127.8
127.8
17.1
17.2
30.2
19.34
3
42.0
215.5
215.7
77.7
205.4
38.5
38.1
38.9
36.32
33.1
46.6
46.8
36.0
204.5 44.9
205.4
4 5 6
45.5
33.2
39.25
46.6 26.6
51.7
60.1
50.7
24.9
210.8
30.2
28.8
52.0 28.8
49.7 45.1
46.2
56.5 20.2
45.5 52.0
46.5 25.2
51.7 25.1
47.43 30.43
38.2 44.7
73.9
74.1
50.5
73.9
73.6
76.6
73.7
72.8
76.6
75.67
62.0
60.9
55.0
61.2
62.6
61.7
60.1
61.9
61.92
64.6 45.3
48.9 42.0
62.6 50.4 42.6
50.4 42.6
56.5 37.6
56.2 37.1
56.1 45.5
69.99 41.10
63.6 41.2
18.6 31.2
18.7 32.8
18.7
32.l a 72.1
31.4
19.5
208.43
46.6
45.0
70.9 53.7
31.3 46.2
51.33 46.55
7 8
9
60.2
53.7
53.7
10
41.1
39.1
11 12
76.7 43.6
16.5 31.5
38.6 16.2
13
42.4
46.4
30.9 43.4
32.8 45.0
26.2
55.6
14
37.0
75.5
75.4
37.3 37.2
75.7
76.6
73.6
71.1
74.01
53.2
205.5
221.4
205.0
207.0
206.4
206.4
208.4
69.7 207.4
74.6
15
205.6
206.24
16 17 18 19 20 Ref.
89.6 65.6 34.1
148.5 117.0
49.0
150.0
149.2
146.7
112.9
119.2 29.4
146.0 117.0
147.5 117.6
149.46 121.24
26.9
118.0 29.0
147.4 117.2
27.7
9.5 27.5
146.7 119.2 29.4
205.8
179.2
32.9
72.15
22.2
20.7
21.1
21.9
22.6
23.2
23.2
16.6
16.2
18.5
18.3
18.1
18.8
22.4
22.4
5
5
6
4
4
14.1 7
15.9 7
14.5
4
22.3 4
20.69 19.24
9
10
Number 1
2 3 4 5 6 7 8 9 10 11
32.7
39.6
39.3
74.9
74.7
75.3
77.9
18.5
17.9
32.3
33.4
34.5
29.0
32.8 26.9
23.1
33.3 18.7
79.5 42.2
31.6
34.9
78.2
78.9
78.6
78.2
78.3
80.3
35.5
214.1
38.0
37.8 46.0
35.9
36.1
58.1
59.4
36.8 49.1
36.0 58.9
38.5 54.2
37.5 54.3
39.6 55.7
46.4 52.6
18.2
18.1 37.8
18.2
28.6
30.8
38.8
73.1
53.1 58.6
53.3 59.0
61.2 48.1
73.6 60.7
38.4
38.6
42.6
68.8
69.0
18.9
47.1 29.6
29.0
209.9
211.2
18.2
210.2
74.4
74.1
50.2
51.0
51.1
37.8
62.1
60.2
54.9
54.6
53.1
55.2
68.5
60.2
54.8 64.9
33.7 51.2 60.4
63.6
58.7
40.0 18.2
39.7
51 . O
44.6
49.4
71.8
65.6
38.9 69.1
207.0
70.3
50.9 65.9
38.0 34.0 215.6 47.0 51.5
51.2 38.0 23.8
12
35.7
50.0
37.5
40.8
38.2
40.8
44.0
30.8
74.1
46.7
44.9
37.6
37.3
37.4
75.3
53.6
75.7
72.2
37.6
37.1
37.5
45.4
45.5
75.6 45.4
45.9
14
36.4 36.6
44.7 75.2
44.8
13
70.9
15
207.9
204.2
205.3
149.6 115.1
147.4
150.8
208.3 151.3
204.9
16 17
205.6 148.5
207.0 152.1
206.4 152.0
206.6 152.1
75.2 207.1
115.1
111.8
111.6
112.4
113.5
113.4
113.3
147.6
207.3 145.9
117.9
119.2
18
71.3
120.1 19.4
26.3
26.5
27.9
26.2
28.3
28.2
27.1
28.6
27.3
19
18.2
70.3
21.8
21.9
17.7
65.5
20.2
21.3
18.6
17.9
14.2
15.3
21.8 15.7
17.8
20
21.7 14.2
15.5
16.5
17.9
16.5
11
12
33
6
14
14
15
15
15
14.5 4
Ref.
4 c
N W
.-
Table 1-3. "C-NMR Chemical Shifts for Ent-kauranes ( g r o u p A). .......................................................................................... ~ 3 2 0 ~ 3 2 1 ~ 3 2 2 ~323* A S ~ * ~ 3 2 5 ~ 3 2 6 A S ~ * *~ 3 2 7 ~ 3 2 a Carbon-----------------------------------------------------------------------------------Number 38.65 33.52 39.7 39.3 38.6 38.11 40.5 35.85 1 34.07 33.5 18.4 34.2 18.30 22.6 28.00 23.02 22.1 2 17.42 16.9 18.69 31.77 78.05 77.61 35.9 216.3 215.5 33.89 77.2 3 32.06 31.7 39.0 39.36 36.21 46.8 46.2 4 49.22 49.5 50.51 39.09 35.8 43.34 53.06 47.74 48.7 50.2 50.8 55.95 54.8 5 45.62 44.7 29.29 28.83 18.6 31.8 30.6 18.99 202.2 31.7 31.27 6 32.06 74.98 73.74 27.1 72.9 73.92 73.6 39.71 80.4 7 73.80 72.1 59.4 54.3 52.8 61.67 61.50 53.46 53.4 8 61.77 59.7 57.62 51.8 49.0 59.26 59.1 50.23 57.4 9 54.82 68.9 62.02 55.28 10 41.88 39.6 48.85 39.36 39.80 38.2 38.4 38.8 38.99 44.8 11 18.34 207.3 103.68 18.22 36.21 26.0 11.4 17.6 69.66 64.7 12 30.63 78.8 46.04 31.81 207.10 73.6 33.1 32.1 46.48 40.7 13 47.21 52.8 44.74 47.03 64.23 55.7 47.5 46.6 74.86 36.8 14 76.28 70.6 73.86 75.56 73.15 68.6 74.9 71.4 45.00 34.4 15 207.74 205.3 204.35 208.31 210.9 77.0 75.8 207.29 206.5 16 150.09 144.3 153.00 149.85 145.30 147.5 159.1 157.8 154.13 151.1 17 115.62 122.2 116.71 116.30 119.21 115.9 106.9 106.5 112.68 112.6 18 205.14 204.7 205.24 28.83 28.16 71.6 27.6 27.8 27.90 27.0 19 14.85 14.2 13.70 16.35 21.90 17.9 21.2 21.9 64.15 22.0 20 60.35 18.2 68.88 18.36 16.66 16.9 18.4 18.0 18.19 18.5 21 _Ref. _ _ _ _ _ _ _17 _ _ _ _ _ 18 _ _ _ _ _ _19_ _ _ _ _20 _ _ _ _ _20 ______ _ _ _ _ _22 - - - - - - - -23 - - - - - - - -24 ~ - - - - - -25 -------------------The data are for the solution in C5D5N. * (CD3)2CO, * * (CD3)zSO.
Number 1
78.43
34.4
81.5
33.83
18.5
31.7
81.4 31.5"
75.2
81.3
34.7
40.5
44.0
33.8
30.7
19.0
67.5
65.5
2
25.7
35.6 22.6
3 4
75.4 42.4
78.5
75.60
41.7
39.9
39.8
78.8
39.0
41.9
77.5
79.8
36.8
37.23
33.0
33.2
33.1
36.2
32.9
33.3
38.3
38.5
5
46.3
43.7
46.44
53.3
52.2
52.2
58.8
52.2
53.8
48.7
47.5
6 7
27.6
70.5
28.51
30.0
210.6
30.3
30.0
68.3
69.7
71.6
74.7
59.4
62.0
74.8 62.1
41.3
48.5
54.6 49.3a
40.0
60.7
74.5 61.8
75.4
8
74.53 62.29
29.6 74.7
30.2
74.1
48.9
48.9
9
54.6
59.3
52.93
56.4
56.Za
53.6
10
37.1
38.5
44.95
64.5 41.2
46.0
45.8 21.3
50.Za 71.3
56.8 4i.9
60.1 67.0
59.1 37.9
63.6 39.2
33.2
39.0
21.5 31.5
46.8
38.7
30.1
11
17.9
64.9
35.67
65.9
12
28.5
40.9
210.06
13 14
46.2 73.7
37.0
60.36
39.1 46.3
15
207.9
16 17
18 19 20 Ref.
20.3 31 . O 47.1
44.0 65.4
68.3
65.1
37.2
37.9
48.1
39.8 47.1
38.6
39.7
34.5
74.10
77.2
76.3
76.1
36.6
218.64
208.8 150.2
82.5
76.5 209.3
77.5 207.7
59.16
206.7 150.8
208.7
149.3
205.0 151.2
149.7
156.7
150.8
151.8
115.8
111.1
13.83
113.7
115.9
116.2
105.6
115.3
29.3
28.1
28.01
33.8
33.2
30.6
26.5
33.3
22.8
23.3
22.20
22.4
19.3
14.05
63.3
21.5 64.5
22.1
59.3
21.5 64.9
27
28
30
31
32
29
37.2
37.9
207.4
207.7 150.9
113.3
149.8 113.2
34.2
27.9
29.4
22.3
22.9
22.7
24.2
15.0
61.9
60.3
20.0
20.4
34
36
36
37
38
111.5
L
Chemical Shifts f o r Ent-kauranes ( g r o u p A). -5. _ _ _13C-NMR ______ _ _ _ - _ _ _ _ _ _ _ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -N -----A416 A417 A418 A419 A420* A421* A422** A423 A424 A425 A426 Carbon-_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - W
Nurnbe r 1 2 3
44.5 63.8 81.4
39.63 27.82 75.57
43.05 19.21 35.01
38.30 28.05 77.76
33.44 23.03 74.32
78.43 34.11 75.49
78.8 29.2 37.3
39.4 18.8 41.9
39.5 18.3 35.6
38.5 42.94 35.41 39.37 41.47 37.26 32.7 33.4 38.7 49.2 45.51 55.04 52.46 47.96 46.76 51.1 52.5 46.5 69.0 29.50 73.50 29.95 29.32 28.70 28.8 29.0 29.7 7 38.5 72.27 202.40 74.79 73.64 74.77 73.8 73.3 74.5 8 48.8 61.85 64.79 61.58 61.44 61.55 59.5 61.4 61.7 9 63.6 54.91 58.33 56.95 50.21 50.43 64.4 68.9 57.2 10 39.7 39.63 54.73 38.74 39.98 45.38 43.7 39.2 38.0 11 65.0 18.08 63.13 26.74 36.28 38.65 66.5 70.8 26.5 12 41.3 31.45 40.75 72.39 207.10 205.83 42.3 79.8 72.5 45.3 53.9 55.6 48.15 52.48 64.25 64.78 13 37.9 46.92 71.3 75.2 72.9 73.06 72.98 74.81 71.23 14 37.9 74.50 209.0 206.7 77.74 209.44 209.18 209.25 205.3 15 208.0 208.82 147.7 146.3 16 150.8 149.77 156.19 147.80 145.29 143.68 150.2 117.0 115.4 17 111.4 116.71 109.33 117.05 119.20 120.80 119.2 71.3 22.43 28.61 32.9 33.6 34.57 28.77 18 28.2 66.71 21.5 18.0 66.61 21.87 21.9 21.59 16.39 19 22.9 13.34 17.0 14.4 17.2 16.63 16.86 13.65 20 20.2 18.08 206.11 21 29_ _ - - - -41 - --- _ _ - _ _29 ____-_ - - - - -. -----21 -------.-- - _ _R-e-f_. - _ - _ _39 _ _ _ _ - -19 - - - - -_ - -_40 -_ - -_ _ _ _20 Unless otherwise stated, the data are for the solution in C5D5N. 4
5 6
* (CD3)2C0, * * (CD3)zSO.
39.2 18.3
39.7 18.1
35.4
35.3
40.4 46.9 29.3 76.0 60.0 64.3 38.6 66.3 37.8 45.9 74.5 207.6 150.3 114.7 71.1
41.1 46.3 29.5 73.2 60.2 70.1 38.3 208.5 78.8 53.3 71.0 206.4 144.9 122.3 71.1
18.5 17.9 21
18.I 18.9 42
Table 1-6. 13C-NMR Chemical Shifts for Ent-kauranes ( g r o u p A). .......................................................................................... A427 A429 A431 A432 A434 A435 A501* A502 A503 A504 Carbon-----------------------------------------------------------------------------------Number 1 39.5 36.9 81.2 85.8 35.3 80.5 77.6 77.3 36.20 74.6 2 18.7 34.8 34.1a 18.07 69.0 26.4 29.8 33.0 34.3 34.3 3 41.8 78.1 74.2 36.85 75.9 78.0 75.6 48.5 80.0 80.1 4 5 6
33.3 53.2 29.6
7 8 9 10
74.8 60.1 67.6 39.0
11
70.9 79.1 54.6 71.6 208.0 147.6
12 13 14 15 16
37.2 43.4 69.1 75.6 57.7 60.8 38.3
39.1 50.4 28.4 74.2 61.6 54.8 44.0
208 47.3 40.1 37.2 79.2 151.5
16.0 31.3a 46.6 75.5 208.2 149.2
37.19
34.0
38.6
36.6
38.3 40.8 70.9 70.9
38.0 49.4 67.1 47.9
37.9 49.1 66.6 47.4
52.63 29.21 69.82
52.3 29.5 74.4
41.0 74.0 41.4
40.4 24.4 74.6
59.71 58.93 38.61
62.5 56.4 45.8
50.1 59.7 38.3
50.0 56.8 43.0
48.6 55.0 43.3
44.0a 53.7 43.ga
44.1a 58.5 43.ea
69.61 47.16 75.14 39.26 206.83 154.67
20.2 31.8 47.3 75.8 208.4
65.0 72.5 35.4 38.3 214.2 151.2
72.2 79.6 43.9 29.8 205.0 147.1
69.9 38.6 36.8 35.8 204.5 150.5
73.1 39.6 39.7 38.3 83.9 158.2
66.8 43.6 39.8 38.4 83.3 154.1
17 116.0 110.3 116.8 112.25 115.5 112.2 116.2 112.6 104.6 107.3 18 33.4 28.2 28.4 27.39 33.5 29.4 27.9 29.1 28.5 28.2 19 21.8 23.2 21.5 66.80 22.4 24.2 22.0 23.8 24.0 24.2 14.1 15.6 14.5 14.1 20 17.4 20.7 15.0 18.11 16.4 19.8 Ref. 42 44 32 24 47 25 48,49 51 34 _ _ _ _ _ _ _ _ _ - - _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - - - - - - - - - - - - - - - - -6- - - - - - - - - - - - - - - - - - - Assignments with the same superscripts in each column are interchangeable. Unless otherwise stated, the data are f o r the solution in C5D5N. * CDC13.
c
W W
c
W Table Shifts _ _ _ _ _1-7. _ _ _I3C _ _ _NMR _ _ Chemical _______ _ _ _ _f_o_r _Ent-kauranes _ _ _ _ _ _ _ _ _(group _ _ _ _ -A). - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -P- - - - - - - - - -
A505
A506
A507
A508
A509
A510
A511*
A512
Carbon-----------------------------------------------------------------------------------Number 1 77.9 78.1 40.7 40.7 79.3 33.41 40.2 39.1 2 34.5 72.5 67.5 67.8 33.1 22.89 67.2 18.5
A513
A514
75.0 35.3
81.0 33.4
3 4
80.2 37.9
78.9 38.4
77.5 38.2
77.7 38.2
74.9 36.9
74.60 41.01
77.3 37.9
35.8 39.1
79.9 38.5
76.2 38.8
5 6 7
57.3 44.0a
48.7 71.1 73.0 39.9 54.9 39.7
73.6 49.8 59.1 39.4
50.5 207.5 86.8 51.2 51.9 49.1
48.15 29.51 73.06 61.55 56.87 38.44
42.8 69.5 70.8
9 10
49.0 71.9 43.0 49.6 60.4 45.1
42.1 71.4
8
49.6 67.6 48.5 44.4a
46.9 29.9 75.0 60.3 68.0 38.2
42.6 33.0 78.7a 46.1 50.3 38.7
41.6 70.3 75.8 49.1 55.5 43.5
67.0 42.9 40.8 38.7 84.0 159.5 104.8 28.4
65.8 38.9 37.8 38.3 209.2 151.1 111.7 28.4
68.2 37.3 41.9 34.5 214.0 150.0 114.5 27.9
64.9 38.0 41.2 35.2 213.5 150.9 112.7 27.9
68.3 39.8 38.2 34.1 79.7 152.1 106.8 27.3
26.55 72.20
24.0 14.7
23.4 16.2
23.2 20.5
23.2 20.6
22.9 14.9
11
12 13 14 15 16 17
47.8 57.9 39.0
65.3 41.0 55.64 36.4 71.08 35.0 208.71 204.7 147.57 149.6 117.29 112.9 22.30 27.8
71.2 79.4 54.8 71.9 208.1 147.8 116.0
69.8 69.3 36.6 38.6 39.3 37.0 37.8 37.0 78.ga 205.6 151.1 151.1 150.7 111.8
71.6 26.6 29.6 66.60 22.8 18.2 23.6 24.7 20 16.51 20.2 18.1 12.4 16.1 Ref. 6 52 55 21 44 53 37 54 29 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - ~ - ~ - - - - - - -51 --------------~--~Assignments with the same superscripts in each column are interchangeable. 18
19
Unless otherwise noted, the data are for the solution in C5D5N.
CDC13.
135 OH I
""X" | v 1',H
AcO "
H
"OAc
O
HO
~
AcO
HO
I ~ H I 4 ,,'" OAc
(12) R 1 = Ac ; R2= 0 (13) R I = A c ; R 2 : a - O H , ~- H (14) R I = H ; R 2 : a - O H , ~ - H
(11)
(15)
Fig. 3.
the
ISC-NMR
moieties,
data,
are
inflexanins [16]
were
also
nonoxygenated which
were
in
hydride
shift
(C205)
[60].
(16) 18
the was
were
rings
the p r e v i o u s
(crossed Thus
treated
obtained. this
closely
to
occur
type
located when
A
example
mass
reactions
[2],
(19)
in the
of hydride carbonyl
depending for a
hand,
was
and and
[62]
are
the
func-
to occur
kamebacetal
aldehyde
of
only
18
between
A
triacetate
carbonate,
a carbinyl
were
intramolecular
and
gave
rearrangement
[61]
The
pathways
reported
treatment
way
20-C-
[59].
on
novel
potassium
group
respective
ent-kaurenoids
(A412)
same
the
for
(A519)
twenty-one
published
reaction)
the e n t - k a u r e n e
in
of
dihydrokamebakaurin
the other
ether
data
fragmentation
groups
review
pretazettin
among
three
5% m e t h a n o l i c
[3.3.1]-nonane-S-ol-9-one first
also been
and
ISC-NMR
longirabdosin
spectra
of k e m e b a k a u r i n
On
to ester
The
assignments
Cannizzaro
when
with
due
and
B and C.
chemical
derivatives
and
has
into
A,
the
dihydrokamebacetal Although
without
on the mass rationalized
classified
of
described
and
signals
I-i~i-7.
(A409)[13],
ent-kauranes
Although
in
report
discussed
tionality
and B
reported
A
for the
in Tables
A (A508)
literature. authors
except
shown
17
and
demethyl(Fig.
a
4).
spatially
group
is
known
endo-7,7-dimethylbicyclo-
treated series
with
base,
this
diterpenoids.
is the
136 OAc H I C
OHI ~ O H / ~
~
: H' .
Hv
< < ~O.~oOH
"OAc0
/~.H
(16)
(19)
5% K2CO3 - MeOH
5% K2CO3 - MeOH
OH[--
OH ~ . O H '
9
"'OH0
v
F-"
HJ"L'~o "f "OH
'
(17)
OB) Fig. 4.
IV. D i t e r p e n o i d s
having
3,20
(group B)-,
7,20
(group
C)-
and
19,20 (group D)-epoxy-ent-kaurane s k e l e t o n s F o r t y - f o u r new d i t e r p e n o i d s c l a s s i f i e d i n t o groups B, C and D were
isolated
and t h e i r s t r u c t u r e s
elucidated.
d i t e r p e n o i d s , the r e a d e r should be aware t h a t coetsoidin
A,
has been given to two
the compound name,
different
5,20-epoxy-ent-
kaurenes,
B201 and B301 [27,
rotations
of the new d i t e r p e n o i d s are shown in Fig.
and
63].
Among t h e s e
The s t r u c t u r e s
13C-NMR data are summarized in L i s t 2 and
and
optical
5.
Tables
The
IH-
2-1~2-4,
respectively. V. D i t e r p e n o i d s having a 6 , 7 - s e c o - e n t - k a u r a n e s k e l e t o n (group E) Many papers on 44 compounds have been p u b l i s h e d . tures,
and IH- and 13C-NMR data are summarized in Fig.
and Tables 3 - I ~ 3 - 4 ,
other
has
6,
L i s t 3,
respectively.
There are two types of 6,7-seco-ent-kaurenoid One
The s t r u c -
a C-7,20-1actone moiety ( s p i r o - l a c t o n e
a C - l , 7 - 1 a c t o n e moiety.
diterpenoids. type)
The d i s c r i m i n a t i o n of
and
the
these
two
137
B201
B202
B203 o
A
A
A OAe
""
Z,~
-o
"Too. o
.
iT-o '
coetsoidin A [ or.]O "60~ (CHCI3) [63]
.
OH
maoecrystal A [ or.]O "68~ (PY) [64, 65, 66]
B301
neorabdosin [ or.]O -175.5~ (py) [67, 68] C201 H OMeA
coetsoidin A [ (x ]D - 150.1~ (ieOH)
coetsoidin C [ (x ]O "35.5~ (ieOH)
[27] C202
H
[27] C203
OEtA
H OMe
C204 MeO H
~
/\a coetsoidin E [ (x ]D "36-8~ (ieOH) [27] C205
H OEtA
l ',"
/\a kamebacetal A [ (x ]D "40~ (ieOH) [36]
C301
H OMe A
oN reniformin C
[ or. ]D "33.80~ (EtOH) [32]
Fig. 5.
c o e t so id in D [ (x ]D "27.3~ (MeOH) [27]
kamebacetal B [ o. ]D "58~ (ieOH) [36] C302
l', compound 5 [ or.]O "55-8~ (MeOH)
D i t e r p e n o i d s having 3, 20 ( g r o u p B) -, 7, 20 ( g r o u p C) - and 19, 20 ( g r o u p D) - e p o x y - e n t - kaurane skeletons.
[9]
138
C303
C304
~'~TH
"OH
C305
~'~YoH
Iongikaurin A [ (x ]D "91-1~(PY) [69]
macrocalin B [ (X ]D "42-5~ (PY)
[70]
C306
C307
o
"OH OH maoecrystal B [ (x ]D "94~(PY) [64, 65, 66]
41 \H'~'|"f
"OH
C308
,X
,X
,X
OH
OH
9 OH maoecrystal C [ (x ]D "95-5~ (PY) [64, 66]
'
C309
~T
OAc
"OH OAe
maoecrystal D [ (X]D +13~(PY) [64, 66, 71]
C310
maoecrystal G [72]
C401 H
~~1 v
"OH
~
rabdocoetsin D
rabdokaurin A [ or.]D "93.6~(MeOH)
[ Or,]D "97~(EtOAc)
[73] C403
HO H
HO" ~ . ~ 4 Y '
[27] Continued
'
"H OH coetsoidin F [27]
C404
o,,~
~
"H OH coetsoidin G
"
[74]
C402
"
HO- y , ~ y
~ T A c "OH
HO
c
~y '
"OH OAc
ganervosin A
[75]
"
'
OAc
jiuhuanin A [ (]" ]O "98.6~(MeOH)
[76]
139 C405
C406
C407
.o.~
' o.~o.
OAc
Z,~oT "~ AcO
C408
Iongikaurin B [ (x ]D -115.9~ (py) [69]
,,.r
Iongikaurin G [ (x ]D "82.0~ (aeOH) [77]
[64, 66]
C409
o,c ~
C410 OAc
OH OH
~ AcO"
~H IT " // AcO
maoecrystal F
C411
OH maoecrystal J
OAc maoyerabdosin [ ~ ]D "28.8~ (MeOH) [79]
C413
.Z~o.~
y- -o.
OAc rabdoternin C [ (x ]O "112.5~(ieOH) [80]
C501
. HO
C412
OH rabdoternin A ID-45.6~(MeOH) [801
.
"OH
[ (x ]D "49.2~(MeOH) [78]
[72]
[~
-o.
OH maoecrystal E
4f' \ H OH rosthorin A [ (x ]D "87-9~ (MeOH) [81]
C503
C502
.o
.o
.o-
/
AcO
o.
maoecrystall
[78] Continued
~,',-r
/
-o. oN
HO maoecrystal K [ (x ]D "1.3~ (MeOH) [82]
.o
,~ Y
"~
OAc rabdolongin A [ (x ]D "75.5~ (MeOH) [71]
140
C504
c5o6
c505
AcACO-,~',,,,l~
r T'o.T--C ~ ,~~o. ~
OH rabdosichuanin D [ (x ]D "32.79~(MeOH) [83]
~,,. z" -o.
OH
OAc
rabdoternin B [ (x ]D "72-5~(MeOH) [80]
ternifolin [ (x ]D "48.09~ (PY) [84]
C601
r T'o_T-,~"o. ,~~o. ~
OAc rabdophyllin H
[85, 86] CG01
CG02
CG03 OH
k OH
"
o~H "OH
HO
parvifoliside [ (x ]D +0.5~ (MeOH)
OH GIc-O
rabdoside 1 [ (x ]D "4-6~(MeOH)
[87] D201
[82] D401
HOH
"oyr /,%~
-
.
rabdoserrin A [ (x ]D "98-2~(DMF) [88, 89]
HO
l.
t~
,v .o, rabdoinflexin A [ (x ]D "115-7~(MeOH) [41]
OH GIc-O
rabdoside 2
[82]
141
~oo~ ~ ~
E001
II
E101
''o ludongnin B [103]
E1~ 4f \ H
~,o~O~1~ ~ '~~ II
I
[lO5] E104
HO,, ~
Z~o~O- o
O
glaucocalactone [ (x ]D +55~(CHCI3) [107]
[106]
i
il
dihydroisodocarpin
o-~----4
ganervosin B
O
'"-o
macrocalyxoformin B [ (x ]D "104.9~(CHCI3) [104] OAc == E103 ~ '! l!
*OEt
.O.
ludongnin [ or. ]D "94.1~ (PY)
[lO8] E107
E106
?,,, ~O ff ~, !..iO ~ H
,, O
macrocalyxoformin A [ (x ]D "79-8~(PY) [109]
macrocalyxoformin C
~,o. " ~
~,o
Me nervosin
[ (x ]D "186.7~ (MeOH)
[ Or.]D "149-8~ (PY)
[104]
II
[1101
~,,o
Z,~'c.o "~ rabdosichuanin B [ (x ]D "58.16~ (MeOH) [83] Fig. 6. Diterpenoids having a 6, 7 -
sculponeatin A [ (x ]O "139~(PY) [111] seco- ent-
sculponeatin C [ (X ]D "163~ (PY) [111]
kaurane skeleton (group E).
142 Elll
E2ol
HO,, ~
0
i
II
~
',
E202
H O , , ~
k OH
Z,~-c.o~
~~H
trichorabdal A [ (7.]D "63.9~ (EtOH) [99, 101,112] E203
OA~ArOH
dihydrorugosanin
guidongnin [ ~ ID -160~(PY) [1131
[105] E204
E205
AcoHO~~I~
.o_~, .",c.:~ isodonal [ (x ]D +91.8~ (py)
isolongirabdiol [ (x ]D +37.7~(MeOH)
[94]
macrocalyxoformin D [ (x ]D "20.4~(CHCI3)
[114]
[115] 0
E206
E2o8 ~ !i
E207
AcoHO~~,~
macrocalyxoformin E [104] 0
o li
rabdosichuanin A [ (x ]D+107.27~ (MeOH)
E2,Oo ,I, \l
OAOH rugosanin [105] Continued
I
4/'"H
~OH
rabdosichuanin C [ (x ]D "120-94~(MeOH)
[83]
OH
II
I
.O;H
OH
E2o,
Z', ~'-c.:~ " ""OH "
OH H
sculponeatin B [ (x ]D "109~ (PY) [111]
[83] E211
HO,,~
Z~CH~ , CHO o 0 trichorabdal B [ (x ]D "120.3~(EtOH) [98, 101]
143 E212
E213
E214
HO,, ~ , ~ ~
~O
HO
AcO
4" -..~.n(~ -OH trichorabdal C [ (x ]D +31.5~ (EtOH) [99, 101] E215 AcOHO" ~
trichorabdal F [100, 102, 116]
9 "'-~%6 OAc
trichorabdal G-acetate [ (x ]D "65.2~ (EtOH) [100, 102] o ;
~Oo~
E301
=
,
~,~:o~
/,,.~ ~
9
trichorabdal H [ (x ]D +19.5~ (MeOH) [100, 102]
o ,~
E 3 0 3 0 ~
I
9 O O
~
acetylexidonin
o~
OAe dihydrocarpalasionin
[117]
E3os
E304 AcoHO~~I~
[105]
O O II
II
o~
~,~:oo2 ~ 9
ememogin [ (x ]D "145-8~(PY) [118] OAc
exidonin [ ~ ]D+101.6~ (py) [92, 93, 119] E307
o~ I',H
O
E308
A c o H O ~
,co
"OH
macrocalyxin A [ (x ]D "189.97~ (CHCI3) [8, 70] Continued
isodonoiol [ (X ]D +98.6~ (PY) [79]
~o~-~~'oO2 ~ rabdokaurin B [ (x ]D +57.9~ (MeOH) [73]
~4.~ /,,~ c.o~
~
rabdolasional [ (x ]D +7.5~ (MeOH) [96, 94]
144 E310
E309
E311
AcoHO~~,,I~
o-~ rabdophyllin G
E312
[ (x ]D +80~ (PY) [90, 91,120] O , O II a.~OH II ! ~"
HO
"CHO ~O OAc
AcO
4f' ~ 0
trichorabdal D [ (x ]D "89-2~ (EtOH) [99, 101] E401 AcACO~,/~'~
"OH
trichorabdal E [ (x ]D "98.4~ (EtOH) [100, 101] E501
HOOH '
,
L 1 oq.~.o.c.O.~C Y .
~" HA"CH2OOc 9 o
",{
HO" " ~ l l " H "OH trichorabdonin [ or. ]D "75.0~ (MeOH) [118]
rabdosinate [ (x ]D "47.2~(CHCI3) [22]
H O~Xo~O/~. '
~
sculponeatin D [ (x ]D "100.0~ (MeOH) [121]
145 List 2. IH-NMR Chemical Shift Data for Diterpenoids Having 5,20Epoxy (group B)-, 7,20-Epoxy (group C)- and 19,20-Epoxy (group D)-ent-kaurane Skeletons. B201
Coetsoidin A(C) 1.22, 1.55(each s, 18 and 19-3Hs), 3, 2-2H), 2.85(m, 13-H), 3.26(br.d, 7, 9-H), 3.78(t, H), 4 . 2 5 ( d , 10, 2 0 - H a ) , 4 . 6 0 ( d , 10, 2 0 - H b ) , 5 . 0 8 ( d , Ha), 5.10(d, 3, 17-Hb), 6.34(t, 3, 15-H)[63].
2.82(d, 3, 33, 17-
B202
Maoecrystal A(P) 1.18, 1.73(each s, 18 and 19-3Hs), 2.61(m, 1 3 - H ) , 2 . 8 0 ( d , 3, 2 - 2 H ) , 3 . 3 2 ( b r . d , 7.7, 9-H), 3.79(t, 3, 3H), 4.17(dd, i0 2, 20-Ha), 4.87(d, I0, 20-Hb), 5.00(d, 8.2~, 6-H), 5 . 0 2 ( d , 3, 1 7 - H a ) , 5 . 2 6 ( d , 3, 1 7 - H b ) , 6 . 5 6 ( t , 3, 15H ) [ 6 4 , 65, 6 6 ] .
B203
Neorabdosin(P) 1.00(s, 19-3H), 1.38(s, 18-3H), 2.17(dd, 12 2, 5-H), 2.57(m, 13-H), 2.82(d, 2.5, 2-2H), 3.28(br.d, 7.6, 9-H), 3.75(t, 2.5, 3-H), 4.21(dd, I0 2, 20-Ha), 4.96(d, I0, 2 0 - H b ) , 5 . 0 0 ( d , 2, 1 7 - H a ) , 5 . 2 0 ( d , 2, 1 7 - H b ) , 5 . 9 1 ( d , 12, 6H), 6 . 4 7 ( t , 2, 1 5 - H ) [ 6 7 , 6 8 ] .
B501 Coetsoidin A(P) 1.21(s, 19-3H), 1.33(s, 18-3H), 3.18(m, I3H), 3.94(dd, 9.0 2.0, 20-Ha), 4.54(dd, 9.0 2.0, 20-Hb), 4.59(dd, 12.0 4.0, 7-H), 4.71(br.s, 14-H), 5.37(br.s, 17Ha), 6.30(br.s, 17-Hb)[27]. C201
Coetsoidin C(P) 1.06(s, 19-3H), 1.15(s, 18-3H), 3.18(d, I0, 13-H), 5.75(br.s, Z-H), 4.79(dd, 4.0 2.0, 7-H), 5.15(br.s, 14-H), 5.38(s, 20-H), 5.41(br.s, 17-Ha), 6.20(br.s, 17Hb)[27].
C202 Coetsoidin E(P) 1.07(s, 19-3H), 1.16(s, 18-3H), 3.20(d, i0, IS-H), 3.73(br.s, 3-H), 4.79(dd, 3.0 2.0, 7-H), 5.23(br.s, 14-H), 5.37(s, 20-H), 5.51(br.s, 17-Ha), 6.20(br.s, 17Hb)[ 27]. C203 K a m e b a c e t a l A(P) 0 . 7 6 , 0 . 9 3 ( e a c h s , 18 and 1 9 - 3 H s ) , 4 . 7 0 ( d d , 4 2, 7-H), 5.10(d, 1.5, 14-H), 5.36(br.s, 17-Ha), 5.46(d, l, 20-H), 6.18(br.s, 17-Hb)[36]. C204
Kamebacetal 3.19(br.d, 5.40(br.s,
B(P) 0.99, 1.23(each i0, 13-H), 4.79(t, 3, 17-Ha), 5.48(s, 20-H),
s, 18 and 19-3Hs), 7-H), 4.96(d, 1.5, 14-H), 6.23(br.s, 17-Hb)[36].
C205
Reniformin C(P) 0.80, 0.96(each s, 18 and 19-3Hs), i0 7, I-H), 4.70(dd, 5 2, 7-H), 5.21(d, I, 20-H), 14-H), 5.62(s, 17-Ha), 6.22(s, 17-Hb)[32].
3.50(dd, 5.40(s,
C301 Coetsoidin D(P) 1.13(s, 19-3H), 1.57(s, 18-5H), 3.20(d, 8.0, 13-H), 3.70(d, 2, 3-H), 4.42(dd, 7.0 3.0, 6-H), 4.77(d, 3.0, 7-H), 5.08(br.s, 14-H), 5.28(s, 20-H), 5.46(br.s, 17-Ha), 6.18(br.s, 17-Hb)[27]. C302
Compound 5(P) 0.81, 0.95(each s, 18 and 19-3Hs), 3.70(m, H), 4.86(dd, 3.5 2, 7-H), 5.42(br.s, 17-Ha), 5.57(d, I, H), 6.26(br.s, 17-Hb), 6.29(br.s, 20-H)[9].
I14-
C303
Longikaurin A(C) 1.09, 1.09(each s, 18 and 19-3Hs), 2.50(m, 12-Ha ), 3.03(br.d, 10, 13-H), 3.77(d, 7-, 6-H), 3.81(dd, i0 i*, 20-Ha) , 4.07 (d, i 0, 20-Hb), 4. 76 (d, 2, 14-H),
146 5.52(br.s,
17-Ha),
6.14(br.s,
17-Hb)[69].
C304
Macrocalin B(P) 0.94,1.03(each s, 18 and 19-3Hs), 1.67(d, !.5, 5-H), 2.82(ddd, 12 5 5.5, 12-H), 3.34(s, 9-H), 3.38(m, I3-H), 4.35(d, 1.5, 6-H), 4. 54(d, 6, II-H), 5.20(d, 7, 14H), 5.47(s, 17-Ha), 5.65(s, 20-H), 6.30(s, !7-Hb)[70].
C305
Maoecrystal B(P) 1.15, 1.43(each s, 18 and 19-3Hs), 2.68(dd, 8 . 2 1 . 5 , 5 - H ) , 4 . 2 1 ( d d , 9 . 5 1 . 5 , 2 0 - H a ) , 4 . 3 7 ( d , 8.2~ 6-H), 4.61(dd, 9.5 1.5, 20-Hb), 5.14(t, 2 . 4 , 17-2H), 6 . 0 7 ( d , 10, 2-H), 6.31(t, 2 . 4 , 15-H), 6 . 7 8 ( d , !0, 3 - H ) [ 6 4 , 65, 6 6 ] .
C306
Maoecrystal C(P) 1.14(s, !9-3H), 1.36(s, 18-3H). 2.64(dd, 8.5 1.5, 5-H), 4.2!(dd, 9.5 1.5, 20-Ha), 4.38(d, 8.5~, 6-H), 4.69(dd, 9.5 1.5, 20-Hb), 5.05(t, 3, 15-H), 5.24(t, 3, 17Ha), 5.50(t, 3, 1 7 - H b ) , 5 . 9 9 ( d , 10, 2 - H ) , 6 . 7 0 ( d , 10, 3H)[64, 66].
C307
Maoecrystal D(P) 0.92(s, 19-3H), i0 2, 20-Ha), 4.66(d, I0, 20-Hb), 8, 6-H), 6.18(t, 3, 15-H)[64, 66,
0.98(s, 18-3H), 4.19(dd, 5.18(br.s, 17-2H), 5.71(d, 71].
C308 Maoecrystal G(P) 0.86(s, 19-3H), 0.93(m, 1-Ha), 1.09(s, 183H), 1.21(m, !-Hb), 1.23(2-Ha), 1.25(2-Hb), 1.25(3-Ha), 1.26(3-Hb), 1.29(12-Ha), 1.43(ddd, 14.0 9.0 9.0, ll-Ha), 1.52(dddd, 14.0 13.0 12.0 6.0, ll-Hb), 1.60(d, 6.0, 5-H), 1.90(d, 12.0, 14-Ha), 2.03(dd, 12.0 5.0, 14-Hb), 2.13(ddd, 13.0, 9.0 9.0, 12-Hb), 2.36(ddd, 12.0 6.0 1.5, 9-H), 2 . 6 3 ( d d , 9 . 0 5 . 0 , 13-H), 3 . 9 8 ( d d , 10.0 1 . 5 , 2 0 - H a ) , 4 . 1 0 ( d d , 10.0 2.5, 20-Hb), 4.99(br.s, 15-H), 5 . 2 3 ( t , 1.5, 17-Ha), 5.47(br.s, 17-Hb), 5 . 7 0 ( d , 6.0, 6 - H ) [ 7 2 ] . C309 Rabdocoetsin D(P) 0.80(s, 18-3H), 1.13(s, 19-3H), 4.05(t, 8~ I-H), 4.60(dd, I0 2, 20-Ha), 4.78(d, 10, 20-Hb), 5.P4(br.s, 17-Ha), 6.00(br.s, 17-Hb), 6 . 0 4 ( m , 1 1 - H ) [ 7 3 ] . C310
Rabdokaurin A(P) 0.90, 1.12(each s, 18 and 19-3Hs), 1.53(m, 2-Ha), 1.73(d, 5.9, 5-H), 1.83(m 2-Hb), 1.90(dd, 11.9 4.6, 9-H), 2.87(br.d, 9.9, 13-H), 4.28(d, 9.5, 20-Ha), 4.50(d, 9.5, 20-Hb), 4.88(dd, 11.5 5.4, l-H), 5.11(br.s, 17-Ha), 5.57(d, 5.9, 6-H), 5.89(br.s, 17-Hb)[74].
C401 C o e t s o i d i n F(P) 1 . 1 2 ( s , 1 9 - 3 H ) , 1 . 6 3 ( s , 18-3H), 3 . 1 6 ( d , 9, 13-H), 3 . 8 2 ( d , 2 . 5 , Z-H), 4 . 5 5 ( d d , 6 . 9 3 . 4 , 6-H), 4.91(d, 3.4, 7-H), 5.45(br.s, 17-Ha), 5.52(br.s, 14-H), 6 . 1 0 ( s , 20H), 6.17(br.s, 17-Hb)[27]. C402
C o e t s o i d i n G(P) 1.12(s, 19-3H), 1.63(s, 18-3H), 3.25(d, I0.0, 13-H), 3.72(d, 2.5, 3-H), 4.55(dd, 6.9 3.4, 6-H), 4.89(d, 3.4, 7-H), 5.52(br.s, 14-H), 5.46(br.s, 17-Ha), 6.10(s, 20-H), 6.21(br.s, 17-Hb)[27].
C403
Ganervosin A(A) 0.95, 1.01(each s, 18 and 19-3Hs), 2.67(d, 3 . 5 , 2 - 2 H ) , 3 . 2 3 ( d , 10, 5 - H ) , 3 . 7 5 ( t , 3 . 5 , 3 - H ) , 3 . 9 1 ( d , 10, 20-Ha), 4.22(d, i0, 20-Hb), 4.87(m, 17-Ha), 5.07(m, 17-Hb), 5.20(d, I0, 6-H), 5.59(t, 1.8, 15-H)[75].
C404
Jiuhuanin A(P) 0.90, 1.14(each s, 18 and 19-3Hs), 1.60(d, 8, 5-H), 2.78(d, 5, 17-Ha), 2.92(d, 5, 17-Hb), 3.96(d, I0, 20Ha), 4.24(d, 10, 2 0 - H b ) , 5 . 1 0 ( s , 15-H), 5 . 8 4 ( d , 8, 6-H), 6.34(s, 14-H)[76].
147
C405
Longikaurin B(P) 1.37(s, 18-3H), 2.35(m, 12-Ha ), 3.17(br.d, I0, 13-H), 3.99(d, I0, 20-Ha), 4.14(d, i0, 20-Hb), 4.18(d, 6-, 6-H), 4.40(d, II, 19-Ha), 4.68(d, ii, 19-Hb), 5.10(d, I, 14-H), 5.51(br.s, 17-Ha), 6.26(br.s, 17-Hb)[69].
C406
Longikaurin G(P) 1.12(s, 19-3H), 1.33(s, 18-3H), 1.41(dd, 7.5 2, 5-H), 1.64(dd, 4.5 1.5, 9-H), 3.35(br.d, 10.5, 13-H), 4.30(dd, 8.5 1.5, 20-Ha), 4.34(d, 7.5~, 6-H), 4.42(dd, 4.5 4.5~ II-H), 5.17(dd, 8.5 2, 20-Hb), 5.52(br.s, 17-Ha), 6.29ibr.s, 17-Hb), 6.39(d, 1,14-H)[77].
C407
Maoecrystal E(P) 1.16, 1.19(each s, 18 and 6-, 6-H), 4.51(d, 12, 20-Ha), 4.60(d, 12, I0 6, I-H), 5.00(t, 5, 15-H)[64, 66].
19-5Hs), 20-Hb),
4.28(d, 4.95(dd,
C408 Maoecrystal F(P) 0.83(s, 19-3H), l.I5(s, 18-5H), 1.24(2-Ha), 1.26(3-Ha), 1.28(12-Ha), 1.50(m, l!-Ha), 1.52(m, ll-Hb), 1.74(d, 6.0, 5-H), 1.83(3-Hb), 1.86(2-Hb), 1.99(d, 12, 14Ha), 2.03(dd, 12.0 5.0, 14-Hb), 2.14(ddd, 13.0 9.0 9.0, 12Hb), 2.61(dd, 9.0 5.0, I3-H), 2.63(ddd, 12.0 6.0 1 . 5 , 9-H), 4.27(dd, I0.0 2.5, 20-Ha), 4.52(dd, i0.0 1 . 5 , 20-Hb), 4.93(dd, i0.0, 4.0, I-H), 4.99(t, 2.5, 15-H), 5.20(t, 1.5, 17-Ha), 5.42(br.s, 17-Hb), 5.70(d, 6.0, 6-H)[72]. C409 M a o e c r y s t a l J(M) 1 . 1 9 ( s , 18-3H), 1.29(br.d, 14.0, 1-Ha), 1.40(dd, 14.0 5.0, 9-H), 1.50(m, 12-Ha), 1.56(m, 2-Ha), 1.72(t, 3.0, 1-Hb), 1 . 7 9 ( d d , 15.0 5.0, 2-Hb), 1 . 9 0 ( d , 5.0, 5-H), 2.14(14-Ha), 2.25(d, 12.0, 14-Hb), 2.40(ddd, 15.5 8.0 8.0, 12-Hb), 3.14(dd, 8.0 5.0,13-H), 3.87(d, 5.0, 6-H), 3.97(br.d, 10.5, 20-Ha), 4.05(br.d, 10.5, 20-Hb), 4.43(d, 11.7, 19-Ha), 4.49(d, 11.7, 19-Hb), 5.06(br.s, Z-H), 5 . 5 3 ( s , 17-Ha), 5.99(s, 17-Hb)[78].
C410
Maoyerabdosin(P) 0.95, 1.18(each s, 18 and 19-5Hs), 5, 5 - H ) , 3 . 7 8 ( m , 1Z-H), 4 . 1 2 ( d , 11, 1 7 - H a ) , 4 . 2 4 ( d , Hb), 4 . 4 5 ( b r . s * , 14-H), 4.39(d, 11, 2 0 - H a ) , 4 . 5 2 ( d , Hb), 5.39(s, 15-H), 5.76(d, 5, 6-H)[79].
2.52(d, 11, 1711, 20-
C411
Rabdoternin A(P) 0.94, 1.12(each s, 18 and 19-3Hs), 1.74(d, 4, 5-H), 2.80(d, 8, I3-H), 2.88(dd, 13 6, 9-H), 4.26(d, 4~, 6-H), 4.84(s, 14-H), 5.52(s, 17-Ha), 5.65(s, 15-H and 17Hb)[80].
C412 R a b d o t e r n i n C(P) 0 . 9 6 , 1 . 1 7 ( e a c h s , 18 and 1 9 - 3 H s ) , 1 . 7 5 ( d d , 7.5 1.5, 5-H), 2 . 5 0 ( d d , 12.5 5 . 5 , 9-H), 2 . 7 9 ( b r . d , 10, 1ZH), 5.92(dd, 9.5 1 . 5 , 20-Ha], 4.23(dd, 9.5 i, 20-Hb), 5.00(s, 1 4 - H ) , 5 . 2 7 ( d , 2, 1 7 - H a ) , 5 . 4 3 ( b r . s , 17-Hb), 5.87(d, 7.5, 6-H), 6.70(t, 2, 15-H)[80]. C413 R o s t h o r i n A(P) 1 . 0 9 , 1 . 1 1 ( e a c h s , 18 and 1 9 - 3 H s ) , 1 . 6 3 ( d 6, 5 - H ) , 2 . 1 9 ( d , 9, 9 - H ) , 3 . 2 9 ( d , 9, 1 3 - H ) , 4 . 1 7 ( d , 10, 2 0 - H a ) , 4 . 2 7 ( d , 6, 6 - H ) , 4 . 3 5 ( d , 10, 2 0 - H b ) , 4 . 4 5 ( d d d , 9 9 9, l l - H ) , 5.21(s, 14-H), 5 . 5 4 ( s , 17-Ha), 6.28(s, 17-Hb)[81]. C 5 0 1 M a o e c r y s t a l I(M) 1.25(s, 18-3H), 1.46(ddd, 13.5 13.0 7.0, 12-Ha), 1.74(ddd, 14.0, 8.0 2.0, 11-Ha), 1.8](ddd, 14.0, 13.0, 12.5, ll-}tb), 1.93(ddd, 15.0, 3.0 3.0, 2-Ha), 1.94(ddd, 15.0 3.0 3.0, 2-Hb), 2.02(d, 5.0, 5-H), 2.03(ddd, 12.5 6.0 2.0, 9-H), 2.10(dd, 12.0 5.0, 14-Ha), 2.20(d, 12.0, 14-Hb), 2.35(ddd, 13.5 8.0 8 . 0 , 12-Hb), 3 . 1 1 ( d d , 7.0 5.0, 13-H), 3.53(br.d, 3.0 I-H), 3.71(dd, 1 0 . 5 2.0, 20-Ha),
148 3.87(d, 3.0, Hb), 4 . 3 2 ( d , Ha), 5 . 9 5 ( s ,
3-H), 3.89(d, 11.7, 19-Ha), 17-Hb)[78].
5.0, 6-H), 4.05(dd, 10.5 1.0, 4.57(d, 11.7, 19-Hb), 5.47(s,
2017-
C502 M a o e c r y s t a l K(M) 1 . 1 1 ( d d d , 15.0 5.0 2 . 0 , 1-Ha), 1 . 1 6 ( s , 185H), 1 . 2 9 ( d d d , 1 4 . 0 9 . 0 9 . 0 , l l - H a ) , 1 . 4 1 ( d d , 15.0 5 . 0 , 12Ha), 1 . 4 2 ( d d d d , 14.0 13.0 12.0 6 . 0 , l l - H b ) , 1.54(m, 2-Ha), 1.55(dd, 12.0 5 . 0 , 14-Ha), 1.57(m, 1-Hb), 1 . 6 5 ( d , 12, 14Hb), 1 . 7 2 ( d d d , 15.0 3.0 2.0, 2-Hb), 1 . 8 0 ( b r . d , 6.0, 5-H), 2.00(dd, 12.0 6 . 0 , 9-H), 2 . 0 6 ( d d d , 15.0, 9.0 9.0, 12-Hb), 2 . 5 6 ( d d , 9.0 5 . 0 , 15-H), 5 . 7 2 ( d , 11.5, 19-Ha), 5.76(d, 6.0, 6-H), 5 . 8 5 ( d d , 10.0 2.5 20-Ha), 5.85(t, 2.0, 5-H), 3.87(d, 11.5, 19-Hb), 5 . 9 1 ( d d , 10.0 1.5, 20-Hb), 4 . 4 1 ( t , 2.5, 15-H), 5.00(s, 17-Ha), 5.05(br.s, 17-Hb)[82]. C505 R a b d o l o n g i n A(P) 1 . 2 0 , 1.25(each s, 18 and 19-SHs), 2.58(br.d, 9, 1 3 - H ) , 2 . 6 5 ( d , 7, 5 - H ) , 5 . 2 1 ( d d , 12 6, 9-H), 3.72(t, 3-, I-H), 3.77(t, 3-, 5-H), 4.11(s, 20-2H), 5.09(br.s, 17-Ha), 5.21 ( b r . s , 17-Hb), 5.90(d, 7, 6-H), 6.20(t, 2, 1 5 - H ) [ 7 1 ] . C504 R a b d o s i h u a n i n D(P) 1 . 1 5 , 1.23(each s, 18 and 19-SHs), i.94(dd, 7.1 1 . 4 , 5 - H ) , 2 . 0 4 ( d d , 1 2 . 0 4 . 4 , 1 4 - H a ) , 2.42(9H), 2.69(m, 15-H), 2.72(d, 12.0, 14-Hb), 4.29(d, 8.8, 20Ha), 4 . 5 6 ( d , 7 . 1 , 6-H), 4 . 8 5 ( d d , 8.8 1.4, 20-Hb), 5 . 0 1 ( b r . s , 1 5 - H ) , 5 . 0 7 ( d d , 11.4 5 . 2 , I - H ) , 5 . 1 4 ( b r . s , 17-Ha), 5.15(11H), 5 . 4 4 ( b r . s , 17-Hb)[85]. C505 R a b d o t e r n i n B(P) 0 . 9 8 , 1 . 1 4 ( e a c h s , 18 and 1 9 - 3 H s ) , 1.78(d, 4, 5 - H ) , 2 . 8 2 ( d , 8, 1 5 - H ) , 5 . 1 2 ( d d , 12 6, 9 - H ) , 3 . 5 2 ( d d , 8 8-, I-H), 4.24(d, 4-, 6-H), 4.94(s, 14-H), 5.55(br.s, 17Ha), 5 . 6 6 ( b r . s , 15-H and 1 7 - H b ) [ 8 0 ] . C506 T e r n i f o l i n ( P ) 0 . 7 6 , 1 . 0 2 ( e a c h s , 18 and 1 9 - 3 H s ) , 2 . 5 6 ( d d , 9 5, 1 3 - H ) , 2 . 9 8 ( d d , 9 2, 9 - H ) , 4 . 1 0 ( m , l - H ) , 4 . 2 6 ( d d , 10 2, 20-Ha), 4 . 6 1 ( d , 10, 2 0 - H b ) , 4 . 9 8 ( s * , 15-H), 5 . 1 8 ( b r . s , 17Ha), 5 . 2 8 ( b r . s , 1 7 - H b ) , 5 . 6 8 ( d , 5*, 6 - H ) , 5 . 8 8 ( q , 9 9 9, 11H)[84].
C601 Rabdophyllin H(P) 0.92, 1.12(each s, 18 and 19-SHs), 1.70(br.d, 8, 5-H), 2.40(br.d, 9, 15-H), 5.95(d, 10, 20-Ha), 4.18(d, I0, 2 0 - H b ) , 4.61(d, 12, 1 7 - H a ) , 4.85(s, 14-H), 4.86(d, 12, 17-Hb), 4.92(s, 15-H), 5.75(d, 7, 6-H)[85, 86]. CG01Parvifoliside(P) 1 . 1 4 , 1 . 1 6 ( e a c h s , 18 and 1 9 - S H s ) , 1.65(d, 7, 5-H), 1.98(d, 14, 14-Ha), 2..24(dd, 14 4, 14-Hb), 2.45(dd, 12 9, 1 2 - H a ) , 2 . 7 4 ( d d , 9 4, 1 5 - H ) , 5 . 1 2 ( d d d , 12 9 9, 1 2 - t t b ) , 5 . 1 8 ( d , 9, 9 - H ) , 4 . 2 5 ( b r . d , 7, 6 - H ) , 4 . 5 0 ( d , 10, 20-Ha), 4.64(dd, 10 6, l - H ) , 4 . 8 2 ( d , 10, 2 0 - H b ) , 4.97(br.s, 15-H), 5 . 0 9 ( q 9 9 9, l l - H ) , 5 . 2 5 ( b r . s , 17-Ha), 5.55(br.s, 17-Hb)[87]. CG02 Rabdoside I(M+W) 1 . 1 8 ( b r . d , 15.0, 1-Ha), 1.21(s, 18-5H), 1.54(m, ll-Ha), 1.42(m, ll-Hb), 1.42(m, 12-Ha), 1.52(dd, 12.0 5.0, 14-Ha), 1.55(m, 2-Ha), 1.56(m, 1-Hb), 1.66(d, 12.0, 14-Hb), 1.76(d, 6.0, 5-H), 1.79(m, 2-Hb), 1.95(br.dd, 12.0 6 . 0 , 9-H), 2 . 2 0 ( d d d , 14.0 9.0 9 . 0 , 12-Hb), 2 . 6 1 ( d d , 9.0 5.0, 15-H), 5.71(d, 10.0, 19-Ha), 5.86(br.s, 5-H), 5.91(d, 6.0, 6-H), 5.92(overlap, 20-Ha), 5.96(d, 10.0, 20-Hb), 4.18(d, 10.0, 19-Hb), 4.20(t, 5.0, 15-H), 5.02(br.s, 17-Ha), 5.09(d, 5.0, 17-Hb)[82].
149 CG03 Rabdoside 2(M+W) 1.26(s, 18-3H), 1.44(m, 12-Ha), 1.55(m, llHa), 1.55(m, ll-Hb), 1.57(dd, 12.0, 5.0, 14-Ha), 1.66(d, 12, 14-Hb), 1.90(ddd, 15.0 5.0 5.0, 2-Ha), 1.98(d, 5.5, 5-H), 1.99(ddd, 15.0, 3.0 3.0, 2-Hb), 2.19(m, 12-Hb), 2.46(br.t, 9.0, 9-H), 2.58(dd, 9.0 5.0, I3-H), 3.52(t, 3.0, I-H), 3.66(d, 10.5, 19-Ha), 3.78(dd, i0.0 2.0, 20-Ha), 3.98(br.d, 10.0, 20-Hb), 3.99(t, 3.0, 3-H), 4.14(d, 10.5, 19-Hb), 4.23(d, 5.5, 6-H), 4.43(t, 2 . 5 , 15-H), 5.00(br.s, 17-Ha), 5.04(d, 3.0, 17-Hb)[82].
D201
Rabdoserrin A(P)0.64(s, 18-3H), 3.24(d, 6, 13-H), 12, 19-Ha), 3 . 8 3 ( d d , 11 6, l - H ) , 4 . 2 1 ( d d , 12 1, 4.88(d, 5, 7 - H ) , 5 . 0 9 ( b r . s , 14-H), 5 . 5 2 ( s , 17-Ha), 1, 2 0 - H ) , 6 . 3 8 ( s , 17-Hb)[88, 89].
3.50(d, 19-Hb), 6.23(d,
D401Rabdoinflexin A(P) 0.63(s, 18-3H), 1.18(br.d, 12.2, 1.42(td, 13.7 13.7 6.3, 3-Ha), 1.69(5-Hb, ll-Ha and 1.97(d, 7.8, 9-H), 2.11(2-Ha and 6-2H), 2.85(m, 2.95(m, 2-Hb), 5.27(s, 13-H), 3.43(br.d, 15, ll-Hb), I-H), 3.64(d, 11.7, 19-Ha), 3.77(dd, Ii.7 2.4, 4.63(dd, 11.7, 4.9, 7-H), 5.16(s, !4-H), 5.34(s, 5.94(br.d, 4.9, 20-H), 6.25(s, 17-Hb)[41]. ~After CDCIs,
D20 treatment. Solvents A" (CD5)2 CO' M" CD3OD,
are in W" D20 .
parentheses.
P"
List 3. IH-NMR Chemical Shift Data for Diterpenoids 6,7-Seeo-ent-kaurane Skeleton (group E).
5-H), 12-Ha), 12-Hb), 3.72(m, 19-Hb), 17-Ha),
C5D5N,
Having
E001 L u d o n g n i n B ( P ) 0 . 9 1 ( s , 1 8 - 3 H ) , 1 . 0 0 ( d , 7, 1 7 - 3 H ) , 2 . 4 2 ( s , H), 3 . 6 5 ( d , 8, 1 9 - H a ) , 3 . 9 6 ( d , 8, 1 9 - H b ) , 4 . 4 3 ( d , 12, Ha), 4 . 7 3 ( d , 12, 2 0 - H b ) [ 1 0 3 ] .
C"
a 520-
E002 M a c r o c a l y x o f o r m i n B(C) 1 . 1 4 ( s , 18-3H), 2 . 4 0 ( d , 5.2, 5-H), 3.25(m, 1 3 - H ) , 3.61(d, 9, 1 9 - H a ) , 3.91(d, I0, 20-Ha), 4.08(d, 9, 19-Hb), 4.21(d, I0, 20-Hb), 4.64(q, 6, I-H), 5.68(s, 17-Ha), 6.01(d, 5.2, 6-H), 6.28(s, 17-Hb)[104]. E101 D i h y d r o i s o d o c a r p i n ( C ) 0.95(s, 19-3H), 1.03(s, 1.15(d, 7.1, 17-3H), 1.91(s, 5-H), 2.47(br.d, 11.8, 2.60(br., 13-H), 3 . 9 8 ( d , 9 . 4 , 20-Ha), 4 . 0 4 ( d , 9 . 4 , 4 . 3 9 ( d d , 11.6 5 . 9 , l - H ) , 5 . 3 3 7 ( s , 6 - H ) [ 1 0 5 ] .
18-3H), 14-Ht] ), 20-Hb),
El03 G l a u c o c a l a c t o n e ( D ) 0.93(s, 19-3H), 1 . 1 0 ( s , 18-3H), 1.48(dd, 13.0 8.7, 12-Ha), 1.54(m, 3-Ha), 1.62(m, 3-Hb), 1.69(dd, 12.5 5 . 2 , 14-Ha), 2 . 0 5 ( d d , 13.0 4 . 2 , 2-Ha), 2.23(d, 12.5, 14-Hb), 2 . 3 4 ( d t d , 1 3 . 0 11.9 5 . 2 , 2 - H b ) , 2 . 4 3 ( d , 1 1 . 3 , 9-H), 2 . 7 1 ( d d d , 13.0, 8 . 7 , 8.3, 12-Hb), 2 . 8 6 ( s , 5-H), 3 . 0 ( d d , 8.3 5 . 2 , 13-H), 4 . 8 4 ( d d , 11.9 4 . 2 , I-H), 5 . 0 0 ( d r , 11.3, 8.7, llH), 5.01(br.s, 17-Ha), 5.28(dd, 2.5, 1.0, 17-Hb), 5.92(t, 2.5, 15-H), 1 0 . 0 0 ( s , 2 0 - H ) [ 1 0 7 ] . El04 L u d o n g n i n ( P ) 1 . 0 3 ( s , 1 8 - 3 H ) , 2 . 3 4 ( d , H), 3 . 1 4 ( d d , 9 5, 1Z-H), 3 . 5 2 ( d , 8, Hb), 3 . 6 9 ( d , 12, 1 4 - H ) , 4 . 5 3 ( t , 4~,
2, 9 - H ) , 2 . 8 4 ( b r . s , 1 9 - H a ) , 5 . 6 1 ( d , 8, l l - H ) , 5 . 2 5 ( d , 12,
51920-
150
Ha), 5.40(dd, Hb)[ 108].
12 2,
20-Hb),
5.50(s,
17-Ha),
6.16(s,
17-
El05 Macrocalyxoformin A(C) 1.14(s, 18-5H), 2.51(d, 5, 9-H), 2.48(d, 5.2, 5-H), 2.90(m, 13-H), Z.57(d, 9, 19-Ha), 5.85(d, 10, 20-Ha), 4.05(d, 9, 19-Hb), 4.10(d, I0, 20-Hb), 4.67(dd, 9 7, l - H ) , 5 . 1 7 ( t , 2.5-, 15-H), 5.Z2(m, 17-2H), 6.00(d, 5.2, 6-H) [ 109 ]. E l 0 6 M a c r o c a l y x o f o r m i n C(C) 1 . 0 8 ( s , 1 8 - 5 H ) , 5 . 1 0 ( d d , 9 4, 5.80(d, 12, 2 0 - H a ) , 4 . 0 0 ( d , 10, 1 9 - H a ) , 4 . 1 5 ( d , 12, 4.50(d, 10, 1 9 - H b ) , 4 . 7 8 ( d , 6 - H ) , 5 . 4 7 ( s , 17-Ha), [7-Hb)[ 104 ].
15-H) , 20-Hb), 6.03(s,
El07 Nervosin(P) 0 . 9 5 ( s , 19-5H), 0 . 9 7 ( s , 18-3H), 2 . 5 8 ( s , 5-H), 20-Ha) , 5.18(m, 15-H), 5 . 7 4 ( d , 11, 14-H B ), 4.50(d, 10, 4.45(d, 10, 2 0 - H b ) , 4 . 8 9 ( d d , 6 4* ll-H), 4.97(s, 6-H), 5.71(fi, 9, I-H), 5.59(s, 17-Ha), 6.05(s, 17-Hb)[ll0]. E l 0 8 R a b d o s i c h u a n i n B(P) 0 . 9 7 ( s , 19-3H), 1.01(s, 18-3H), 1.15(d, 8.0, 17-5H), 2.95(d, 4.0, 5-H), 3.55(m, 15-H), 4.6(m, ll-H), 5.1(m, 20-2H), 10.08(d, 4.0, 6-H)[85].
El09 Sculponeatin A(P) 1.06(s, 18-5H), 2.24(d, 4, 9-H), 2.46(dd, 14 9, 12-H ~ ), 2.90(d, 5, 5-H), 5.18(dd, 9 5, I5-H), 5.47(d, 8, 19-Ha), 5.62(d, 11, 14-H ~ ), 4.08(d, 8, 19-Hb), 4.18(d, I0, 2 0 - H a ) , 4.54(d, I0, 20-Hb), 4.58(dd, 5 4*, II-H), 5.62(dd, I0 6. I-H), 5.58(br.s, 17-Ha), 6.04(br.s, 17-Hb), 6.15(d, 5, 6 - H ) [ I I I ] . Ell0
S c u l p o n e a t i n C(P) 1 . 1 1 ( s , 18-5H), 1 . 8 1 ( d d , 15 5, 12-Ha), 2.10(dd, 11 4, 1 4 - H a ) , 2 . 4 7 ( d d , 15 9, 1 2 - H b ) , 2 . 6 0 ( m , 9-H), 2 . 7 5 ( d d , 4 1, 5 - H ) , 5 . 1 6 ( d d , 9 4, 1 5 - H ) , 5 . 6 1 ( d , 11, 1 4 - H b ) , 5 . 7 0 ( d , 8, 1 9 - H a ) , 5 . 8 9 ( d , 8, 1 9 - H b ) , 4 . 5 6 ( d d , 12 2, 2 0 - H a ) , 4 . 4 4 ( d d , 5 5 - , l l - H ) , 4 . 6 4 ( d d d , 3 3 1, I - H ) , 5 . 3 3 ( d , 12, 20Hb), 5.42(br.s, 1 7 - H a ) , 5 . 8 7 ( d , 4, 6-H), 6.10(br.s, 17Hb)[lll].
Elll
Trichorabdal A(P) 0 . 9 5 , 1 . 0 5 ( e a c h s , 18 and 1 9 - S H s ) , 2 . 9 0 ( d , 5, 5 - H ) , 5 . 1 2 ( d d , 10 4, 1Z-H), 5 . 4 5 ( d , 12, 14-H a ), 4.60(m, II-H), 4.71(d, 12, 20-Ha), 5.1(d, 12, 20-Hb), 5.55(s, 17Ha), 6.05(s, 17-Hb), 10.05(d, 5, 6-H)[I01].
E201 D i h y d r o r u g o s a n i n ( P ) 0.98(d, 6.6, 17-5H), 1.16(s, 18-5H), 2.54(s, 5-H), 2.44(br., 1S-H), 2.69(br.d, 11.9, 14-H), 2.84(dd, 12.9 5 . 5 , 9-H), 4 . 2 1 ( d , 11.6, 19-Ha), 4 . 3 1 ( d , 9.0, 20-Ha), 4.45(d, 9.0, 20-Hb), 4.48(d, 11.6, 19-Hb), 4.75(dd, 10.2 5 . 7 , I-H), 5 . 9 5 ( s , 6 - H ) [ I 0 5 ] .
E202 Guidongnin(P) 1.45(s, 18-5H), 2.90(dd, 8 4, 13-H), 3.16(br.s, 5-H), 3.30(d, 12, 14-Ha ), 3.47(d, i0, 19-Ha), 17-Ha), 3.68(d, i0, 19-Hb), 4.53(m, II-H), 5.24(br.s, 5.41(br.s, 20-2H), 5.52(br.s, 17-Hb), 5.63(t, 2* , 15H)[llS]. E203 I s o d o n a l ( C + M , 2 : 1) 1 . 1 0 ( s , 19-3H), 1.19(s, 18-3H), 1.35(dd, 13.6 9.5, 12-Ha), 1.62(m, 3-2H), 1.92(m, 2-2H), 1.94(d, 12.6, 14-Ha), 2 . 3 5 ( d d , 12.6 4 . 4 , 14-Hb), 2.36(d, 11.5, 9-H), 2 . 6 0 ( d d d , 13.6 9.4 7 . 4 , 12-Hb), 3.09(dd, 9.4 4.4, 13-H), 3 . 9 4 ( d d d , 11.5 9.5 7 . 4 , l l - H ) , 3 . 9 5 ( d , 2 . 2 , 5H), 4.99(d, 12.6, 20-Ha), 5.04(d, 12.6, 20-Hb), 5.07(dd,
151 11.3 4.1, l-H), 5.60(br.s, 9.82(d, 2.2, S-H)[94]. E204
17-Ha),
6.08(br.s,
17-Hb),
Isolongirabdiol(P) 0.97(s, 19-3H), 1.29~ 1.38(I-H, 2-Ha, 3Ha and 12-Ha), 1.48(m, 2-Hb), 1.60(m, ll-Ha), 1.78(m, llHb), 1.88(dt, 13.5 4, 3-Hb), 2.04(m, 12-Hb), 2.22(d, 12.5, 14-Ha), 2.57(dd, 12.5 4.5, 14-Hb), 2.91(br.dd, 9 5, 13-H), 3.16(dd, 1 2 . 5 5, 9 - H ) , 3 . 3 9 ( d , 11, 1 8 - H a ) , 3 . 8 4 ( d , 11, ]8Hb), 4 . 0 1 ( m , 6 - 2 H ) , 4 . 8 7 ( d , 1 1 . 5 , 2 0 - H a ) , 4 . 9 4 ( d , 1 1 . 5 , 20Hb), 5.31(br.s, 17-Ha), 5.95(br.s, 17-Hb)[l14].
E205 Macrocalyxoformin D(C) 1.08(s, 18-3H), 1.50(m, 5-H), 2.16(d, 12, 14-Ha), 2.44(dd, 12 4, 14-Hb), 3. i3(br.dd, i0 4, 13-H), 3.33(d, 12, 19-Ha), 3.67(dd, 12 3, 6-Ha), 3.88(dd, 12 3, 6Hb), 4.01(d, 12, 19-Hb), 4.69(d, 12, 20-Ha), 4.93(d, 12, 20Hb), 5.52(br.s, 17-Ha), 6.02(br.s, 17-Hb)[l15]. E206
Macrocalyxoformin 2.73(br., 13-H), 5.56(t, 2, 15-H),
E(P) 0.92, 1.05(each 5.10(d, 2, 17-Ha), 5.68(s, 20-H), 5.74(d,
s, 18 and 19-3Hs), 5.42(d, 2, 17-Hb), 6, 6-H)[104].
E207
Rabdosichuanin A(P) 0.99(s, 19-3H), 1.02(s, 18-3H), 1.09(d, 8.0, 17-3H), 1.36(br.s, 2-Ha), 1.40(m, 3-2H), 1.45(m, 12Ha), 1.95(m, 2-Hb), 2.00(dd, 9.6 1.2, 13-H), 2.10(dd, 12.0 1.2, 14-Ha), 2.40(qd, 8.0 2.4, 16-H), 2.55(ddd, 12.0 9.6 8.4, 12-Hb), 2.65(dd, 12.0 4.0, 14-Hb), 2.80(d, 11.2, 9-H), 4.31(d, 4.0, 5-H), 4.35(ddd, 11.2 9.2 8.4, II-H), 5.16(d, 12.0, 20-Ha), 5.31(d, 12.0, 20-Hb), 5.45(dd, 11.6 4.0, I-H), 10.02(d, 4.0, 6-H)[83].
E208 Rabdosichuanin C(D) 0.91(s, 19-3H), 0.96(s, 18-3H), 1.04(d, 7.6, 17-3H), 1.20(m, 3-2H), 1.33(m, 12-Ha), 1.43(m, 2-2H), 1.71(q, 7.6, 16-H), 1.82(m, 13-H), 1.86(dd, 12.5 3.6, 14Ha), 2.01(d, 10.6, 9-H), 2.22(dd, 12.5 3.4, 14-Hb), 2.27(dd, 15.0 8.0, 12-Hb), 2.66(br.s, 5-H), 3.52(d, 9.1, 20-Ha), 3 . 6 4 ( d , 9 . 1 , 20-Hb), 4 . 1 8 ( d d , 10.6 8 . 0 , l l - H ) , 4 . 7 5 ( d d , 11.2 6.0, I-H), 5.17(br.s, 6-H)[83]. E209 Rugosanin(P) 1.16(s, 18-3H), 2.35(s, 5-H), 2.64(br.d, 11.9, 14-H B ), 2.96(dd, 9.0 4.3, 13-H), 3.09(dd, 13.2 4.9, 9-H), 4.33(d, 9.3, 20-Ha), 4.42(d, 11.6, 19-Ha), 4.46(d, 9.3, 20Hb), 4.49(d, 11.6, 19-Hb), 4.79(dd, ii.I 6.1, l-H), 5.31(s, 17-Ha), 5.94(s, 6-}{ and 17-Hb)[105]. E210 Sculponeatin B(P) 1.09(s, 18-3H), 2.45(dd, 14 8, 12-Hil ), 2.79(d, 4, 9-H), 2 . 9 4 ( d d , 8 5, 1 3 - H ) , 3.00(d, 5, 5-H), 3.16(d, 11, 1 4 - H ) , 3 . 4 6 ( d , 8, 1 9 - H a ) , 4 . 0 5 ( d , 8, 19-Hb), 4 . 2 6 ( d , 9, 2 0 - H a ) , 4 . 5 3 ( d , 9, 2 0 - H b ) , 4 . 5 6 ( d d , 4 4~, ll-H), 5.23(br.s, 17-Ha), 5.52(br.s, 17-Hb), 5.71(br.s, 15-H), 5 . 8 8 ( d d , 10 6, I - H ) , 6 . 1 4 ( d , 5, 6 - H ) [ l l l ] . E211 Trichorabdal B(P) 1.19(s, 18-3H), 3.11(dd, 8 4, 13-H), 3.50(d, 4, 5-H), 3.50(d, 12, 14-H), 4.03(d, 12, 19-Ha), 4.16(d, 12, 19-Hb), 4.50(m, II-H), 4.50(d, 12, 20-Ha), 5.15(d, 12, 20-Hb), 5.46(s, 17-Ha), 6.05(s, 17-Hb), 10.19(d, 4, B-H)[101]. E212 Trichorabdal C(P) 1.44(s, 18-3H), 2.88(dd, 8 4, 13-H), 3.20(d, 3.5, 5-H), 3.80(t, 3, 3-H), 4.28(d, 12, 19-Ha), 4.46(d, 12, 19-Hb), 4.98(d, 12, 20-Ha), 5.04(d, 12, 20-Hb), 5.31(s, 17-Ha), 5.93(s, 17-Hb), 10.09(d, 3.5, 6-H)[101].
152 E215 Trichorabdal F(P) 0.96(s, 18-3H), 1.12(s, 18-3H'), 3.43(d, d, 8, 19-Ha), 3.71(d, 8, 19-Ha'), 3.85(d, 8, 19-Hb), 3.98(d, 8, 19-Hb'), 4.40(dd, 13 2, 20-Ha), 4.52(m, II-H), 4.86(dd, 13 2, 20-Ha'), 5.29(d, 13, 20-Hb), 5.36(d, 13, 20-Hb'), 5.90(d, 6, 6-H), 6.11(d, 5, 6-H')[I02].
E214 Trichorabdal G-acetate(P) 1.17(s, 18-3H), 2.40(d, 12, 14H a ), 2.62(dd, 5 2, 5-H), 2.92(dd, 9 5, 13-H), 3.69(d, 8, 19-Ha), 3.78(d, 8, 19-Hb), 3.90(dd, 12 2, 2 0 - H a ) , 4.24(d, 12, 20-Hb), 5.13(dd, 12 4, 3-H), 5.42(s, 17-Ha), 6.09(s, 17Hb), 6.80(d, 5, 6-H)[I02]. E215 Trichorabdal H(P) 0.86, 1.04(each s, 18 and 19-3Hs), 2.80(d, 4.5, 5 - H ) , 3.11(dd, 9.5 5, 13-H), 3.37(br.d, 12, 14-H), 4.48(br.m, II-H), 4.90(d, 12, 2 0 - H a ) , 5.36(s, 17-Ha), 5.36(d, 12, 20-Hb), 5 . 7 0 ( b r . t , 8, l-H), 5.98(s, 17-Hb), 9.95(d, 4.5, 6-H)[I02]. E301Acetylexidonin(P) 1.00, 1.07(each s, 18 and 19-3Hs), 1.42(br.td, 1 3 . 8 4.3, 12-Ha), 1.58(dt, 1 4 . 3 3.3, 3-H a ), 2.05(dd, 1 3 . 0 4.5, 14-Ha), 2.05(m, 14-Hb), 2.83(ddd, 13.6 9.2 7.5, 12-Hb), 3.03(d, 11.6, 9 - H ) , 3.14(dd, 9.2 4.5, 13H), 4.14(dd, 1 2 . 8 6.0, 6-Ha), 4.43(dd, 1 2 . 8 3.5, 6-Hb), 4.48(d, 12.3, 20-Ha), 4.63(dd, 10.8 3.8, I-H), 4.89(d, 12.3, 20-Hb), 4 . 9 7 ( d d d , 11.8 9.3 7 . 5 , l l - H ) , 5.64(br.s, 17-Ha), 6.16(br.s, 17-Hb)[l17].
E302 Dihydrocarpalasionin(P) 1.02(d, 7.0, 17-3H), 1.17(s, 18-3H), 2.88(d, 3.5, 9-H), 2.98(s, 5-H), 3.85(br.d, 12.0, 14-HB ), 4.25(d, ii.6. 19-Ha), 4.37(d, 9.3, 20-Ha), 4.48(d, 9.3, 20Hb), 4.52(d, 11.6, 19-Hb), 5.76(dd, II.3 5.8, I-H)[105]. E303 Ememogin(P) 1.00, 1.38(each s, 18 and 19-3Hs), 3.05(s, 5-H), 3.79(m, 3-H), 5.12(m, 15-H), 5 . 5 2 ( b r . s , 17-Ha), 5.55(dd, 12 6, I-H), 5 . 6 3 ( b r . s , 17-Hb), 6.12(s, 6-H)[i18]. E304 Exidonin(C) 1.01, 1.03(each s, 18 and 19-3Hs), 3.11(dd, 9.5 4.5, 13-H), 3.92(m, II-H), 4.11(dd, 1 2 . 7 5.6, 6-Ha), 4.26(d, 12, 20-Ha), 4.35(dd, 1 2 . 7 3.8, 6-Hb), 4.87(d, 12, 20-Hb), 5.13(dd, 9.6 5.5, I-H), 5.62(s, 17-Ha), 6.12(s, 17-Hb)[92,
93].
E305 Isodonoiol(P) 0.87, 0.97(each H), 3.05(t, 4, 5-H), 3.28(d, 4.40(m, II-H), 4.97(d, 12, 5.42(br.s, 17-Ha), 5.60(t, 8,
s, 18 and 19-3Hs), 3.04(m, 13II, 9-H), 4.01(d, 4~, 6-2H), 20-Ha), 5.18(d, 12, 20-Hb), l-H), 6.02(br.s, 17-Hb)[79].
E306 Macrocalyxin A(P) 1.0(s, 18-3H and 19-3H), 2.05~2.30(m, 12Ha), 2.24(s, 5-H), 2.85(q, 16 i0, 12-Hb), 3.48(d, i0, 13-H), 3.58(d, 5, 9-H), 4.46(d, i0, 20-Ha), 4.65(d, I0, 20-Hb), 5.72(dd, ii 6, l-H), 5.72(s, 17-Ha), 6.0(s, 6-H), 6.39(s, 17-Hb), 6.50(t, 6, II-H), 6.93(s, 14-H)[70]. E307 Rabdokaurin B(P) 0.83(s, 19-3H), 1.51(m, ll-Ha), 1.65(m, llHb), 2.16(d, 12.5, 14-Ha), 2.59(dd, 12.5 4.6, 14-Hb), 2.62(dd, 5.8 3.0, 5-H), 2.75(dd, 13.1 4.2, 9-H), 2.95(dd, 9.2 4.6, 13-H), 3.28(d, Ii.0, 18-Ha), 3.72(d, Ii.0, 18-Hb), 4.36(dd, 13.0 5.8, 6-Ha), 4.47(dd, 13.0 3.0, 8-Hb), 4.76(d, 12.1, 20-Ha), 5.04(dd, 10.7 4.3, I-H), 5.18(d, 12.1, 20-Hb), 5.46(br.s, 17-Ha), 6.08(br.s, 17-Hb)[73].
153 E508
Rabdolasional(C) 1.15(s, 18-5H and 19-5H), 1.26(dd, 12.6 9.5, 12-Ha), 1.49(d, 12.8, 14-Ha), 1.62(m, 3-2H), 1.91(m, 22H), 2.02(dd, 12.8 5.4, 14-Hb), ~2.50(12-Hb), 2.56(d, Ii.0, 9-H), 2.70(dd, 8.2 5.4, 13-H), 5.80(d, 2.8, 5-H), 5.88(ddd, ii.0 9.6 8.5, II-H), 4.48(t, 2.6, 15-H), 4.91(d, 12.2, 20Ha), 4.95(d, 12.2, 20-Hb), 5.11(dd, 10.2 5.5, l-H), 5.20(m, 17-Ha), 5.21(br.s, 17-Hb), 9.90(d, 2.8, 6-H)[94, 96].
E509
Rabdophyllin G(C) 0.86, 1.06(each s, 18 and 19-SHs), 2.67-~2.85(m, 5-H), 3.09(m, 13-H), 3.66(d, 11, 6-Ha), 3 . 8 2 ( d , 11, 6 - H b ) , 3 . 8 8 - ~ 4 . 1 2 ( m , l l - H ) , 4 . 3 7 ( d , 13, 20-Ha), 4 . 9 4 ( d , 13, 2 0 - H b ) , 5 . 1 2 ( t , 7, l - H ) , 5 . 5 7 ( s , 1 7 - H a ) , 6 . 0 9 ( s , 17-Hb)[90, 91, 120].
E510
Trichorabdal D(P) 1.40(s, 18-5H), 5.14(dd, I0 4, 5.50(d, 12, 14-Ha ), 5.54(d, 4, 5-H), 4.20(m, 5-H), 12, 19-Ha), 4.30(d, 12, 20-Ha), 4.52(d, 12, 19-Hb), II-H), 5.16(d, 12, 20-Hb), 5.48(s, 17-Ha), 6.06(s, I0.22(d, 4, 6-H)[101].
ESII
Trichorabdal E(C) 0.99(s, 18-3H), 1.06(s, 18-3H'), 3.23(d, Ii, 13-H), 5.31(d, 12, 13-H'), 3.53(d, 9, 19-Ha), 3.61(19Ha'), 3.73(19-Hb'), 4.01(d, 9, 19-Hb), 4.41(d, Ii, 20-Ha), 4.79(d, Ii, 20-Hb), 4.90(3-H' and 20-H'), 5.38(dd, 12 4, 3H), 5.47(t, 6, 6-H), 5.60(dd, 4 2, 6-H')[101].
E512
Trichorabdonin(P) 1.06, 1.56(each s, 18 and 19-SHs), 1.48(s, 17-5H), 2.75(s, 5-H), 5.82(m, 3-H), 4.57(d, 9, 20-Ha), 4.57(d, 9, 20-Hb), 5.46(dd, i0 6, I-H), 5.89(s, 6-H)[I18].
E401
Rabdosinate(P) 0.99, 1.05(each s, 18 and 19-SHs), 12.0, 9-H), 5.02(dd, 1.7 0.7, 17-Ha), 5.22(dd, 2.7 Hb), 5.60(t, 2.7, 15-H)[22].
15-H), 4.27(d, 4.58(m, 17-Hb),
5.25(d, 0.7, 17-
E501Sculponeatin D(P) 0.92(s, 19-5H), 1.51(m, Z-Ha), 1.68(m, 14Ha), 1.87(m, 5-Hb), 1.95(m, 2-Ha), 2.05(m, 12-Ha), 2.15(br.s, 14-Hb), 2.17(m, 12-Hb), 2.50(m, 2-Hb), 2.58(d, 5.4, 9-H), 2.61(br.s, 5-H), 2.87(br.s, I5-H), 5.81(d, 11.6, 18-Ha), 4.01(d, 11.6, 18-Hb), 4.61(dd, 11.9 4.1, I-H), 4.79(dt, 9.8 8.0, II-H), 5.09(s, 20-H), 5.18(br.s, 17-Ha), 5.49(br.s, 15-H), 5.51(br.s, 17-Hb), 5.95(d, 5.2, 6-H)[121]. ~After D20 treatment. C" CDCI3, D- (CD3)2SO.
Solvents
are
in
parentheses.
P"
C5D5 N,
e
u, Table _ _ _ - _2-1. - - _ _13C-NMR _ _ _ - - Chemical _ _ _ _ - - _Shifts - - - - - - f-o- r- -3,20(B)_ - - - - - - -and - - - -7.2O(C)-Epoxy-ent-kauranes. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -P~ - - ~ - - B201* B202 9301 B203 C201 C202 C205 C301 C302 Carbon-----------------------------------------_---------------------------------Number 1 205.5 208.8 202.8 34.2 31.9 31.9 76.1 31.4 76.2 2 41.5 42.1 42.0 29.1 22.8 22.8 30.7a 23.2 30.9 3 77.4" 77.3 77.1 97.1 73.6 73.5 39.2 74.2 39.2 4 40.3 38.1 37.6 39.6 39.5 39.4 34.0 39.7 34.0 5 143.6 51.5 47.6 47.7 43.8 43.8 53.4 48.7 48.8 6 132.3 71.9 29.9 74.5a 25.5 25.4 25.6 69.7 25.9 7 193.8 207.9 75.4 66.9 66.8 66.8 210.2 70.6 66.7 8 53.7 56.9 59.7 58.8 58.9 56.6 58.5 61.7 58.9 9 41.0 40.2 40.0 47.4 50.9 50.8 51.1 51.7 51.7 38.6 38.6 10 52.6 51.4 36.1 51.8 38.6 43.5 43.6 11 19.2 20.8 20.6 17.7 19.9 19.8 19.8 23.7 23.5 12 31.9 29.9 25.7 25.6 32.8 32.ab 32.0a 25.4 32.5 41.9 13 27.3 45.3 42.0 35.0 34.8 43.6 44.5 43.8 70.7 70.7 14 38.5 35.0b 71.7 35.9 70.3 71.0 70.5 206.1 15 76.0a 74.8 74.0a 206.2 206.1 205.9 211.1 206.4 16 150.9 151.6 151.3 148.4 154.3 154.3 154.1 153.4 154.5 115.6 115.2 17 108.9 116.2 115.6 108.3 108.2 118.4 115.3 23.2 29.5 28.9 26.6 28.2 28.1 18 32.2 29.5 32.2 21.8 23.1 22.9 18.7 21.8 21.8 19 20.7 22.8 20.8 101.9 100.2 100.2 20 67.0 62.4 62.1 66.8 101.9 94.2 27 _Ref. _ _ _ _ _ _ _63 _ _ _ _ _ 66 _ _ _ _ _67.68 _ _ _ _ _ _27 ______ _ _ _ _ _27 _ _ _ _ _ -32 - - - - - - - -27 - - - - - - - - -9- - - - - - - - - - - - - - - - - - Assignments with the same superscripts in each column are interchangeable. Unless otherwise noted, the data are for the solution in CgD5N. CDC13.
--___
198.5
208.7
72.5
75.6
31.7
31.4
210.5
31.3
2
17.3
198.3 127.7
127.8
35.7
32.4
25.4
23.5
25.7
47.0
18.7
3
42.1
160.5
161.3
39.0
37.8
74.4
75.9
76.Oa
41.4
4
34.5
35.9
36.1
38.7 32.7
34.1
33.7
39.7
39.8
33.5
5
53.1
53.1
55.2
54.5
48.9
56.2
53.5
54.6
38.0 47.7
54.4
6
74.8
73.2
73.3
75.3
29.7
74.3
69.9
74.5a
75,5a
70.9 62.8
96.5
97.5
52.7
54.3
42.0
53.4 46.5
49.7 17.5
7
98.9
97.4
97.6
96.3
96.0
96.2
69.9 71.2
8
63.2
53.2
52.2
9
61.4
52.3 43.0
41.2
58.0 49.1
62.2 52.1
10
37.1
46.6
46.6
42.8 48.8
56.0 54.2 41.6
40.5
38.7
37.4
11
18.2
18.2
17.8
67.5
18.3
20.0
17.9
12
19.6 30.
32.3
33.0
32.3
36.7
29.5
25.6
32.0
15.4 26.3
13
44.4
35.9
36.1
35.7
34.1
34.1
44.6
27.1 44.0
35.5
44.7
14 15 16 17 18
74.1
27.0
26.3
2:. 1
27.7
27.4
70.9
70.7
26.5
75.4a
75.2
74.0
205.7
203.0
211.4
203.8
76.0"
73.2
35.8
153.5
75.0 159.2
160.1
158.7
154.1
73.8
107.9
109.7
113.5
153.7 118.4
158.5
108.7
154.8 112.1
153.1
120.1
119.1
109.5
48.0
21 . o 24.0 65.5 75
32.8
209.3
34.2
30.3
30.0
29.5
31 .8
31.4
29.7
31.3
19
23.0
24.6
24.3
22.9
20.5
21.5
23.3
20
66.8
65.3
65.2
63.6
65.1
63.3
94.1
23.8 96.3
22.3 66.4
27 27 74 73 76 66 66 66 Ref. 69 ___________---____-__--_-----------------------------------------------------------_------Assignments With the same superscripts in each column are interchangeable. Unless otherwise noted, the data are f o r the solution in C5D5N.
Number 1
27.4
32.3a
31 . g a
66.5
2
17.9
19.0
25.3
22.6a
19.6
17.2
15.2
30.55 19.91
3
36.5
42.0
37.9
72.7b
34.1
40.9
41.7
41.50
71.4
30.g a
31.1
74.5a
22.1a
31.2
4
37.1
33.9
33.5
41.3
33.9
34.3
33.7
34.08
41.ga
5
52.8
56.4
55.6
57.4
50.9
54.1
59.10
52.3
6
73.8
72.4a
76.2a
73.2b
75.4
54.7 71.7
74.l b
73.46
7 8
99.0 63.1
98.8
96.0
96.0 52.8
107.8
97.9
98.22
62.9
95.5 52.4
73.6 96.5
53.6
52.3
9 10 11
62.0 38.1 19.2
59.9
42.7
49.9
42.7
46.9
60.62 61.43
45.8
37.5 65.1
39.8
44.8
36.1
37.76
43.Oa
17.3
36.3 16.7
36.8a 40.8 14.8
12
30.6a
31.8
29.4
25.9
18.9 31.7a
62.73
42.5
19.2 29.6a
41.75
16.3 29.7
13 14
44.4 74.1
43.4
36.7 27.7
35.0 26.7
36.5a
45.4 74.7
45.5
44.15
35.2
72.4
75.5
73.62
27.0
210.3
65.0
72.4
73.8b
77.6 71.1
159.1
158.8
208.68 152.17
210.9 153.8
110.4 31.4
111.1
119.13
116.3
33.1
33.32
22.6
74.9a
75.4a
60.2
15 16
209.3 153.3
209.2 153.4
161.4
153.7
17
120.2
118.7
107.4
116.7
21.6
21.7
22.9
31.2
66.5'
22.8
21.1
22.5
22.43
67.4
69.1
63.0
66.4'
65.6
175.4
66.7
67.12
66.2
77
72
78
79
80
80
18
28.0
34.5
19 20
67.qb 67.g b
Ref.
69
59.8
32.2
81
78
- ----- - - -. '7$. 3
i
25.8
25.6
3 2 . a3
18.2
--
7
30.5 39.3 34.1
--- -- --. ---
-14.0
67.5
72.9
83.2
26.0
30.9
31.7
29.5
'71.7 43.1
72.0 41.53
38.3 33.7
39.5 32.4
53.6 53.5
50.0 73.2
42.8
48.0 31.1
3 4
70.5
36.7
39.2
43.8
33.6
34.1
40.3 33.0
5
53.7
55.0
53.6
6
73.5
55.8 75.8
72.0
74.4
59.3 '74.7
7
97.9
97.2
98.0
97.5
65.8
73.5
52.6
108.4 53.4
96.9
8
97.2 52.0
52.5
53.2
52.8
52.0
59.5
62.1
9
47.0
44.5
42.8
43.0
40.8
48.4
43.3 35.2
48.8
10
42.7 36.5
41.0
36.6
43.oa
49.8 40.2
11
16.1
70.5
20.7
20.7
70.8
16.1
15.0
20.8
12
33.1
41.7
31.0"
13
37.8
33.2 37.8
32.5 37.2
26.8 75.5
45.5 74.7
41.0 37.6
14 15 16 17 18 19 20 Ref
35.7 26.6
21.1 43.2
160.9
159.3
75.8 161.1
-16.8 75.5 161.8
26.2
72.5
72.Oa 72.aa
28.7
74.9
107.8
108.2
110.1
70.0
21.9
33.9
31.1
106.4 22.2
107.8 "2.7
67.2
22.5
20.9
30.5 21 . o
32.8
75.5
66.2
65.1
82
83
161.7
76.1
176.1
75.0'' 161.2
23.5
32.0 39.1 70.2
54.1 44.8 22.2 34.7 47.5 77.2
206.0
208.6 150.5
107.0
149.0 114.8
22.0
22.6
22.9
74 . o b 66.0 82
65.0
7'-
115.4 >L.C
64.7 67.1 85.0 93.1 98.8 87 82 85 88.89 ill 83 ----------____-__---____________________-- --- - --- ---- - - - - . - - Assignments xi th the same superscripts in each column are interchangeable.
* CD30D. * * C5D5N. * * * (CDs),SO, * * * * CD30D-D20(1
: i ) ,
*I***
CDC13.
Number 1
31.8
76.68
75.50
32.6
78.1
31.7
78.2
31.7
78.1
77.5
2
17.8
23.94
18.6
22.2
29.Ea
31.5 18.6
38.47
18.6 31.4
23.9
30.3
18.4 42.1
23.0
3
23.29 37.25
37.3
40.2
28.7
30.5a
34.2
4 5 6
38.8 40.7 176.5
32.87
32.22 49.52
40.0a
41.1
37.1
31.5
40.6
54.7
52.8
41 . O 52.4
40.3
53.2
34.3 56.9
40.8
43.1
169.38a 175.0
110.3
170.4
109.1 171.5
205.4
171.3
101.54 172.30
105.9
7
110.6 174.5
171 . O
170.4
107.9 171.5
8
57.3
56.42
52.2
53.3
56.3
54.8
54.6
56.7
48.7 49.6
60.7 47.0
44.7
40.5
49.2 64.1
51 . O 66.3
47.8 42.7
39.9
41 . O
53.92
169.05a 172.0 49.7Zb
54.3
29.5
9
47.7
44.90
39.49
46.8
36.4
41.4
10 11
39.0 17.1
49.47
48.23b
38.5a
19.16a 34.57
68.62 36.24
65.1 34.2
50.4 18.7
43.0 17.4
65.7
42.1 65.0 42.9
12
29.4
32.7
29.7
41.2
13 14 15
35.5 29.4 215.2
32.51
36.59
35.0
35.5
34.8
18.9Za 214.50
31.47 80.77
27.7 200.0
32.2 75.7
25.1
35.5 34.1
199.8
16 17
50.8 16.5
48.73 10.61
152.19 111.50
150.9 118.7
156.4
150.0
18
25.0
32.87
22.24
21.4
108.9 30.6
118.0 21.1
19 20
76.2 69.0
23.12 29.43 74.09 202.26
75.9 71.6
76.9
71.2
23.2
26.1
72.1
67.8
74.2
70.9
Ref.
103
105
108
109
104
110
83
107
20 1 151.3
60.7 204.5 169.5
64.9 42.0
35.2
34.0
35.2
35.2
28.5 216.0
33.1
33.4
28.4
200.0
200.4 151.1
200.7
51.0
149.9
117.3
16.5
32.8
32.3
118.1 30.0
117.4
150.6 117.1
27.8
32.3
71.3 76.1
72.2
26.0
83.9
70.7
111
111
101
Number 1
76.41
40.0
2
23.02
19.2
76.07 24.34
27.9
39.2
34.1”
75.8
75.9
76.17
78.4
17.6
17.9
19.6b
24.4
23.1
23.02
22.4
3
30.74
37.2
40.19
36.7
44.0
34.l a
40.2
36.1
30.74
28.9
4
34.61
34.80
38.9
34.4
31.1
34.57
40.9
55.00
61 . O O
44.8
39.2 56.5
30.2
5
38.3 46.7
61 . O
52.9
100.35
176.0
204.91
58.0
57.7
204.9
101.1
55.13 100.35
52.9
6
60.9 107.4
110.2
7
172.09
175.8
171.32
171.3
171.5
173.8
170.3
171.39
174.3
56.90
50.7
58.18
58.2
57.7
59.2
170.5 55.9
56.54 46.09
50.6
8 9
46.78 49.84
46.37 44.23
58.2 42.1
50.3
10
45.8 40.6
55.2 40.2
42.6
48.9
46.8 44.3
51.4 49.6
11
19.37a
65.2
65.12
17.5
16.9
21.lb
65.4
62.2
19.78
63.7
12
34.34
33.8
40.64
29.9
29.4a
33.2a
41.6
39.5
32.69
44.0
13 14
32.69 19.13a
34.1
34.36
35.4
35.0
36.0
35.2
34.4
35.13
36.2
29.69
29.8
28.8
27.1
29.8
31.9
29.57
202.2
76.0
215.0
215.3
200.53
32.9 77.6
159.6
149.8
156.7
51.2
51.2
151.07
158.2
10.50
108.0
120.26
117.4
118.0
105.9
117.55
107.8
21.7
33.37
71.7
27.5
31.1
16.5 33.2
15.0
26.80 66.19
26.80
30.2
75.9
24.81 67.23
19.9 71.1
71.2
24.4 67.1
66.19 73.86
72.5
68.3
27.4 102.0
32.6 22.8
77.0
114
115
104
83
105
111
215.11
81.5
16 17
49.10
18
74.13
71.8
105
113
Ref
38.6 48.9
200.38 203.2 149.76 151.3
15
19 20
29.Oa
50.09
94
noted, the data are for the solution in C5D5N.
* CDC13-CD30D(2
72.3 83 : 1).
* * CDC13, * * + (CD3)2S0.
L
\o 1/1
Table . . . . . . .3-3. . . . . .13C-NMR . . . . . . . .Chemical . . . . . . . . . Shifts . . . . . . . for . . . . .6,7-Seco-ent-kauranes . . . . . . . . . . . . . . . . . . . . . . (. g. r. o. u. .p . E) . . ... . . . . . . . . . E211 E212 E215 E301 E302 E303 E304* E304** E305 Carbon----------------------------------------------------------------------------Number 1 32.3 79.7 76.47 78.31 74.5a 76.75 76.22 77.1 2 18.1 23.8 23.76 23.33 24.09 23.97 24.8 3 4 5 6 7 8 9 10
68.9 41.4 55.6 203.1 170.5 57.8 44.7 44.0
12
33.7 38.5 57.9 203.0" 170.0 56.4 46.2 41.5 65.0 42.0
32.8 40.4 61.9 204.3a 170.7 59.2 47.8 44.9 63.7 42.2
13 14
35.1 28.0
35.3
34.1 30.0
34.24 29.58
33.02
15 16 17 18
200.4a 150.6 117.5 26.7
202.4 150.6 117.8 24.2
201.8" 149.1 118.5 33.8
199.61 148.97 120.56 23.90
214.80 46.40 11.10 27.11
11
39.99 30.97 33.90 35.10 48.99 55.40 61.48 100.40 170.17 172.28 57.41 56.80 43.26 49.45 44.02 49.60 68.22 65.60 40.10
70.Ea 36.3 48.7 98.6 175.2 52.2 37.0b 46.9
39.85 34.09 48.16 61.76 170.39 57.95 45.22 44.38 64.91 41.29
39.53 34.08 48.61 62.30 169.70 57.59 45.26 44.36 65.94 41.52
40.0 34.0 52.8 58.9 170.4 58.7 46.7 45.1 65.7 42.2
37.3b
34.05 29.22
33.91 29.13
34.7 29.8
200.08 151.11 119.44 24.29
199.00 149.13 119.66 23.97
201.5 150.8 118.9 34.2
77.9 159.2 108.8 26.6
-
m
0
19 71.4b 69.6a 24.1 33.60 66.34 22.7 33.79 33.91 24.0 20 70.Eb 68.7a 68.2 66.71 74.27 176.5 67.21 66.50 68.1 Ref. 101 101 102 117 105 118 119 93 79 __-_________________--------------------------------------------------------------Assignments with the same superscripts in each column are interchangeable. Unless otherwise noted, the data are f o r the solution in C5D5N. * CDC13-C5D5N(1 : l),
* * CDC13.
161
1
ii.5
76.7
2
23.2
3
37.5
23.8 34.1
4
31.2
38.8
5
54.5
42.7
6
101.5
61.5
7
170.2
170.5
75.67 23.91
33.0 24.9
74.4
76.6 23.5
78.0 25.6
39.95
69.6
75.1
40.0
30.0
34.09
41.1
36.3
34.2
39.7
61.40
56.4
51.7
49.0
46.1
205.78 203.6 174.99 170.9
102.9 172.9
62.1 172.2
100.0 174.4
8
59.3
58.6
52.26
55.4
57.7
50.7
53.6
9
48.9
42.5
41.47
45.1
48.4
39.2
34.1
10
44.1 17.8 30.5
38.0 74.7
35.2 29.0
43.4 65.1 42.1 35.1 27.4
43.0 68.4 40.3 35.1 31.2
42.0 67.0
13 14
43.17 65.57 44.32 35.16 30.04
50.5
12
50.2 69.2 40.1
15 16
146.7
202.2 151.4
83.41 200.0 157.78 150.6
77.1 154.1
17
120.4
110.29
82.9 153.4 111.4
11
118.0
41.8 212.7 78.6
38.8 39.7 32.3
18
33.2
118.8 69.6
33.45
20.8
20.4 28.8
34.2
66.4
19
23.1
19.9
24.73
72.1a
23.7
23.9
22.5
20 Ref.
73.1 8
68.8 74
66.36 94
70.ga 101
75.0 118
66.5 22
105.9 121
108.0
changeable. Unless otherwise noted, the data are f o r the solution in C5D5N. * CDC13.
* CHO
(20) R = H (21) R = A c
R’ = CHO ;
R~ = a -OH
(23) R’ = COOH ; (24) R’ = CHO ;
R2 = a OH
(22)
Fig. 7.
R2 =
- OH
(25)
162 skeletons are
is very
employed.
is
the best
example,
For
was
results,
(=E504)[93]
(E205)
The
and
cently both
compounds
spiro-lactone 22
[95]
of isodonic and
type and
as that
A
possible
[98-102].
and
the
chair
the other
a C-9
belongs
s t r uc t u r e
H (E215)(Fig.
at Kyoto
University
conformations during
a C-9
axial
equatorial
to the
former
in
E508
(E205),
25
were
[97]
of t r i c h o d o n i n 7).
found
that on
the
structures
of isodonal being
axial
and
of
structures
the
the
and
re24
is
the
there
are
spiro-6,7-
trichorabdals
equatorial
and C-20
structures
of isodonal
studies
and C-20
group
and
re-
correlation
reported
ring A of
their
exidonin isodonal
the
results,
at C-ll
of
relatively
that
B
being
of
range
to E205
structures
revised
nucleus
One has
(E211)
latter
group
of the
on
rabdosin
structure
as that
long
co n g e n e r
an epimer
reported
The
same
on the
its type
Based
correlated
previously
For
and
20191].
the structures
revised
acid
method
reported
IH-13C
Based
of t r i c h o r a b d a l
research
seco-ent-kaurene
B
be
[94],
(=E215),
respectively.
being
by means
analysis
spiro-lactone
the c o n c l u s i o n
should
the p r e v i o u s l y
[95],
two
reached
a carboxylic
trichodonin
vised,
(E508)
25.[96].
acid,
by this
is the
re-examined
methods
two skeletons.
of the
of c h e m i c a l l y
two-dimensional
and
these
structure
structure
rabdolasional
of
(E309)
spectroscopic
crystallographic
the p r e v i o u s l y
et al.
developed
X-ray
analyzed
reported
revised
spectroscopy,
being
was
to one
revised,
NMR
same
G
the structure
Takeda
the usual
reason,
revised
was
[92].
(E504).
when
for d i s t i n g u i s h i n g
the p r e v i o u s l y
the 21
this
method
rabdophyllin
st r u c t u r e [90],
difficult
orientation,
one.
Trichorabdal
trichorabdal
C
to
the
one.
having an 8,9-seco-ent-kaurane
VI. Diterpenoids
skeleton
(group
F) Since the p u b l i c a t i o n of the previous review [2], diterpenoids umbrosanin
of t h i s type, Following
the p r e l i m i n a r y
details
of the structure
tuents
of R.
125].
marized
shs163
The
in Fig.
Shikoccin
determination var.
structures,
8, List (F204),
and
IH- and 4,
[45,
were
have
15C-NMR
also
data
new
rabdo-
124],
of the d i t e r p e n o i d
occs
4 and Table a major
reports
three
[122],
(FI02)[47] and rabdohakusin (F202) [123],
described.
[46,
r a b d o l a t i f o l i n (F205)
been full
consti-
published are
sum-
respectively.
diterpenoid
of R.
sh~koks
var.
163 F101
F102
F103
If~,~,~ AcO
~
O
AcO
"OMe
O - methylepoxyshikoccin [46, 124]
AcO
9
o
"OH
~ \ H
F202
"~
.
0
-
--0
AcO
"OMe
" OH
rabdoumbrosanin [ (x ]D "40.6~ (MeOH) [47] F203
.~,~TDo., L-~ .o.~._ d
epoxyshikoccin [ (x ]D "6.3~ (ieOH)
-
--0
O - methylshikoccin [ (x ]D "4.5~ (MeOH) [46, 124]
[ (x ]D +24.2~ (MeOH)
F201
I
__
\
H
.
.o
"OH
rabdohakusin
rabdolatifolin
[ (x ]D +78.7~ (CHCI3)
[ (x ]O "45.1~ (MeOH)
[46, 124]
[123]
[122]
F204 H
I
9
i O
AcO
\H
"OH
shikoccin [ O. ]D "37~ (CHCI3)
[45, 46, 125]
Fig. 8.
Diterpenoids having an 8, 9 - s e c o - ent- kaurane skeleton (group F).
G401
HO
G402
I" ,,
I" ,,
phyllostachysin A [ ~ ]D "30.50(PY) [127]
Fig. 10.
rubescensin D [ (x ]D "57.2~(PY) [128]
Diterpenoids having a 7, 20 - cyclo - ent-kaurane skeleton (group G).
164 List 4. IH-NMR
Chemical
Shift Data for Diterpenoids (group F).
8,9-Seco-ent-kaurane Skeleton
Having
an
FI01 O-Methylepoxyshikoccin(C) 0.97, 1.00, 1.08(each s, 18, 19 and 20-5Hs), 5.54(m, IS-H), 5.65(s, 14-H), 4.11(dd, 12 5, 7H), 4.74(br.t, 5, S-H), 5.48(d, i, 17-Ha), 6.27(s, 17Hb)[46, 124, 125]. FI02 O-Methylshikoccin(C) 0.99, 1.01(6H)(each s, 18, 19 and 205Hs), 5.66(m, IS-H), 4.17(dd, 15 6, 7-H), 4 . 7 4 ( b r . t , 5, 5H), 5.44(s, 17-Ha), 6.15(s, 17-Hb), 7.17(d, 5, 14-H)[46, 124, 125 ]. F105
Rabdoumbrosanin(C) 0.95, 0.97, 1.05(each 5Hs), 5.62(m, I5-H), 4.71(dd, 12 5, 7-H), 6.15(br.s, 17-Hb), 7.26(d, 2, 14-H)[47].
s, 18, 19 and 205.46(br.s, 17-Ha),
F201 Epoxyshikoccin(C) 1.04(6H), 1.08(each s, 18, 19 and 20-3Hs), 3.27(m, 13-H), 3.73(s, 14-H), 4.57(dd, 12 5, 7-H), 4.77(br.t, 3, 3-H), 5.55(d, I, 17-Ha), 6.28(s, 17-Hb)[46, 124, 125]. F202
Rabdohakusin(C) 1.01(s, 19-5H), 1.02(s, 18-5H), 1.07(ddd, 15 4 2, I-H a ), 1.69(ddd, 14 4 2, 2-H ~ ), 1.85(ddd, 14 15 4, 6H a ), 1.8~2.0(dd, 18 8, ll-Ha), 2.08(br.t, 4, 5-H), 2.16(d, 15, 14-H a ), 2.77(ddd, 14 11 6, 12-H a ), 2.84(15-H), 2.95(dd, 18 II, ll-Hb), 4.75(5-H), 5.02(dd, 2 1.5, 17-Ha), 5.50(t, 2, 17-Hb), 5.45(7-H)[123].
F205
Rabdolatifolin(P) 0.91, 1.12, 1.55(each s, 18, 19 5Hs), 5.54(m, 15-H), 4.24(dd, 8 7.5, I-H), 5.20(dd, H), 5.59(br.s, 17-Ha), 6.24(br.s, 17-Hb), 7.62(d, H)[122].
and 2012 7, 75, 14-
F204 Shikoccin(C) 0.98, 1.01(6H)(each s, 18, 19 and 20-5Hs), 5.65(m, 15-H), 4.64(dd, ii 5, 7-H), 4.75(br.t, 5, S-H), 5.45(s, 17-Ha), 6.15(s, 17-Hb), 7.25(d, 5, 14-H)[45, 46, 125]. Solvents
are
in
parentheses.
P:
C5D5N,
C:
CDCI 3.
List
5.
G401
Phyllostachysin A(P) 0.89, 1.32(each s, 18 and 19-SHs), 1.90(dd, 15 I0, 12-Ha), 2.28(br.d, I0, 9-H), 5.06(ddd, 15 i0 I0, 12-Hb), 5.22(d, 9, IS-H), 4 . 6 0 ( b r . s , 20-H), 5.50(m, I I H), 5.41(br.s, 17-Ha), 5.95(br.s, 14-H), 6.20(br.s, 17Hb)[ 127].
G402
Rubescensin D(P) 0.90, 1.52(each s, 18 and 19-SHs), 1.12(dd, 12.5 2.5, 3-Ha), 1.52(5-Hb), 1.60(ddd, 14.0 1 5 . 2 6.5, 12Ha), 1.84(dd, 1 2 . 5 4.5, 2 - H a ) , 1.94(ll-Ha), 1.99(2-Hb), 2.06(dd, 1 5 . 5 5.6, 9-H), 2.26(s, 5-H), 2.44(ddd, 1 4 . 0 15.2 9.2, 12-Hb), 2.86(ddd, 14.0 15.5 6.5, ll-Hb), 5.18(br.d, 9.2, 15-H), 4.79(s, 20-H), 4.90(dd, 11.5 5.5, l-H), 5.59(s, 17-Ha), 5 . 8 2 ( s , 14-H), 6.17(s, 17-Hb)[128].
Solvent
IH-NMR Chemical Shift Data for Diterpenoids 7,20-Cyclo-ent-kaurane Skeleton (group G).
is
in
parentheses.
P:
C5D5N.
Having
a
2
25.0a
25.8a
18.1
25.1a
25.Ea
28.1
25.Ea
2
19.3
30.8
3 4
77.3
76.8
41.4
76.9
76.7
37.8
76.9
37.3
38.0
34.6
38.3
37.8
34.5
38.0
3 4
41.2 33.4
33.4
35.4 27.la
36.7
43.5
35.6
36.4
42.7
27.3"
36.7
27.Oa
27.7a
39.8
37.1 27.2a
72.0
73.5
64.4
61.8 64.5
119.5 141.2
147.3
64.3 148.5
216.1
214.4
214.5
9
55.6
53.0
10 11
5 6 7
64.0
8
64.0
146.2
146.0
9 10
213.5
213.7
215.1
52.9
53.8
11
52.8 31.5
213.3 52.7
33.4
30.7
32.1
29.6"
59.6 30.4
12
21 .ga
22.oa
25.8
21.9a
23.Oa
26.0
35.8 22.oa
13 14
38.6
42.3
42.4
39.9
47.6
44.1
42.3
59.9 197.0
158.8
159.5
60.2
25.1a
159.8
194.8 145.7
195.0
195.6
75.0
145.0
115.8
148.4 116.7
15 16 17
145.8 121.9
18
28.3
28.3
33.6
19
21 . o
20.8
22.3
20
16.6
16.8
39.0
5
63.7
63.3
6 7
211.6
210.6
90.0 60.0
90.1
62.3 47.4
58.7 48.8
8
59.9
64.9
22.8
12
43.2
32.4
43.6
43.4
158.9
13 14
195.5
194.5
15
73.0 204.4
204.6
155.9
150.2
145.9
152.8
106.1
116.0
116.3
16 17
152.3
121.9
117.0
116.9
28.0
27.8
33.1
28.2
18
35.0
34.0
73.8
22.6 20.9 20.8 21.5 19 23.0 23.5 16.6 11.9 16.8 16.6 16.0 80.6 20 82.3 47 46 Ref 46 122 46 46 123 Ref. - - - - _ - - _ - - - - - - _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - - - - - - - - - - -_- _- -_-_---_ _ _ _ _ - _ _ -127 _ _ _ _ _ - _ 128 __________ Assignments with the same superscri ts in each column are The Data are f o r the solution interchangeable. Unless otherwise notes, the data are for the of CgDgN. solution in C5D5N. * CDC13.
166
3.59(sextet, 7.6, 15-H), Ii 7.6, 16-Hb), 4.46(br.s, Solvents
are
in
parentheses.
4.25(dd, ll 7.6, 16-Ha), 4.33(dd, 6-H), 4.54(d, 2, 7-H)[132]. P:
C5D5 N,
C:
CDCl 3, A:
Jonesoxidation
,co~~T-,. ,~. -~
AeO
II
/
.H
~H
OH
(F204)
(F201)
I H2/PtO2 MeOH
I Jonesoxidation =l
,co,>~,.
~ -o
i
(26) C
,
-N o
-!-
AcO
/
H
AcO
-I-
AcO
(CD3)2C0.
I
H
~H
OH
,~C02H Jonesoxidation~~ 0 AcO
/
~H
(28)
(27) Fig. 9.
167
gave
occidentaLis, (F201)
rahydroshikoccin
over
platinum
abietane type
dioxide
cleavage
Only
rubescensin having and
obtained gave
(Fig.
by c a t a l y t i c
The m e c h a n i s m
(G402)
a novel
5, respectively.
VIII.
Diterpenoids
1,3-diketone
have been
having
were eton In
as the c o n s t i t u e n t s
from two Rabdosia
1987,
(I201) having that
on the
was
reported
an abietane
report,
(I301),
F (I305)
[131]
15C-NMR
A
aldol
skeleton
(group
(G401)
eight
species,
for the
compounds,
and R.
A (I503),
were
isolated
Lophanthos
B (I401), from R.
data are s u m m a r i z e d
List
5
and
abietane glutinosin
(HS01)
[129]
an e n t - i s o p i m a r a n e and R.
skel-
parv~foLia.
16-acetoxy-7 a - m e t h o x y r o y l e a n o n e
as a d i t e r p e n o i d nucleus
structures,
I0,
(H201),
R. gLutinosa
hand,
and
examples
(group H) and
a-ol
having
[127]
as
The
in Fig.
constituent first
time
of R.
strache~i
[150].
Following
16-acetoxy-7-O-acetylhorminone
16-acetoxy-12-O-acetoxyhorminone
lophanthoidins and
other
ent-
by
ent-isopimar-15-en-6 a ,7 a ,8 a - t r i o l
reported
an
retro-aldol
followed
nucleus.
ent-isopimarane
Ent-5 a - a c e t o x y i s o p i m a r - 1 5 - e n - 8 and
having
reported
summarized
I) skeletons
(H202),
(28)
involving
tet-
of F204
[126].
7,20-cyclo-ent-kaurane are
hand,
hydrogenation
phyllostachysin
[128],
ISC-NMR data
Table
epoxyshikoccin
a 7,20-cyclo-ent-kaurane
compounds,
D
through
On the other
only a c o m p o u n d
9).
having
(26)
reagent.
of the resulting
two
IH- and
(group
Jones
was also d i s c u s s e d
Diterpenoids
G)
with
(27)
skeleton
condensation VII.
an e p o x y k e t o n e
on treatment
C (I402),
(I302),
D (I504), var.
Lophanthoides
[132].
in Fig.
The
ii,
structures, List
and
E
(I405)
gerard~ana
and
6 and Table
IH-
6,
and
re-
spectively. IX. been
Distribution Since
examined
summarized In
each
1984,
parallel
species,
and
7.
studies
some
of the genus
constituents.
on the d i s t r i b u t i o n
analytical
J. Hu et al.
of the content that
species
for d i t e r p e n o i d
in Table
the HPLC method. tion
forty-eight
studies
were
The
results
of d i t e r p e n o i d s
performed
examined
the seasonal
of r a b d o p h y l l i n
G (E309)
in
A in R. amethystoides,
are in
involving
[144]
of a m e t h y s t o i d i n
have
Rabdosia
varia-
R.
maerophyLga
and
found
that
168
H202
H201
i
n ,,CHOH
H301
F'
CH2OH
I
i
!
-o.
AcO"
glutinosin
ent-3a-acetoxyisopimar-15en-8cx-ol
[ or. ]D +20.4~(MeOH)
[ o. ]D +59.6~(CHCI3) [133]
[129]
OH
1201
CH2OAc
1301
O
CH2OAc
1302
[ (x ]D +12.3~(MeOH)
O
"OH
16-acetoxy-7- O-acetylhorminone
[130] OH
OAc CH2OAc
~~H
16-acetoxy-7~x-methoxyroy-
13
[129]
O
"OMe
leanone
OH
ent- i so pi m a r- 15-en-6 ~, 7 ~, 8~-triol [ or. ]D +7.7~(MeOH)
CH2OAc
16-acetoxy- 12- O-acetylhorminone
[131] OH
13
CH2OH
[131] OH
1305 O
~_~y "OMe " OH
~.l~Y "OEt ' OH
"
lophanthoidin D
[132] OH
1401 O
CH2OAc O
O
OH
"
lophanthoidin A
CH2OAc
lophanthoidin F
[132] OH
1402 O
CH2OH O
[132] OH
1403 O
CH2OAc O
"OAc OH
Iophanthoidin B [132]
OH
Iophanthoidin C
Iophanthoidin E
[132]
Fig. 11. Diterpenoids having e n t - isopimarane (group H) and abietane (group I) skeletons.
[132]
169
List
6.
IH-NMR Chemical Shift Data for Diterpenoids isopimarane (group H) and Abietane (group I)
Having EntSkeletons.
H201 e n t - 5 a-Acetoxyisopimar-15-en-8 a -ol(A) 0.86, 0.90, 1.05, 1.25(each s, 17, 18, 19 and 20-SHs), 0.96(dd, 12.5 2.2, 5H), 1.55(d, 13.9, 14-Ha), 1.40(d, 15.9, 14-Hb), 4.42(dd, 11.2 5.2, 5-H), 4.76, 4.85, 5.72(ABX, 17.5 10.8 1.5, 15-H and 16-2H)[129].
H202
Glutinosin(P) 5H), 1.09(s,
0.85, 17-5H),
0.86(each s, 18 and 19-SHs), 1.00(s, 5.90~4.25(15-H and 16-2H)[155].
20-
HS01
ent-lsopimar-15-en-6 a ,7 a ,8 a -triol(A) 0.89(dt, 15.0 3.6, 5-H), 0.91(br.s, 5-H), 0.95(dd, 12.1 2.6, 9-H), 0.95, 1.20, 1.25, 1.27(each s, 17, 18 19 and 20-5Hs), 1.54(d, 15.4, 14Ha), 1.45(d, 15.4, 14-Hb), 5.15(d, 5.5, 7-H), 4 . 5 2 ( b r . s , 6H), 4.81, 4.85, 5.89(ABX, 1 7 . 5 1 0 . 8 1.5, 15-H and 162H)[129].
1201 16-Acetoxy-7 a-methoxyroyleanone(C) 0.86, 0.88, 1.22(each s, 18, 19 and 20-SHs), 1.22(d, 7, 17-5H), 5 . 4 0 ( m , 15-H), 4.25(m, 16-2H), 4.27(m, 7-H)[150]. 1501
16-Acetoxy-7-O-acetylhorminone(C) 0.89(6H)(s, 18 and 195Hs), 1.24(d, 7, 17-5H), 1.25(s, 20-5H), 2.75(m, I-HB ), 5.56(sextet, 7, 15-H), 4.21(dd, II 7, 16-Ha), 4.29(dd, ii 7, 16-Hb), 5.95(t, 5, 7-H)[151].
1502
16-Acetoxy-12-O-acetylhorminone(C) 0.91, 0.98, 1.25(each s, 18, 19 and 20-SHs), 1.25(d, 7, 17-5H), 2.75(m, I-H i3 ), 5 . 5 6 ( s e x t e t , 7, 15-H), 4.21(dd, II 7, 16-Ha), 4.29(dd, ii 7, 16-Hb), 4.72(t, 5, 7-H)[151].
I505
Lophanthoidin A(C) 1.04, 1.25, 1.60(each s, 18, 19 and 205Hs), 1.24(d, 7.6, 17-5H), 2.58(m, I-HB ), 5.40(sextet, 7.6, 15-H), 4.15(d, 2.5, 7-H), 4.25(dd, ii 7.6, 16-Ha), 4.52(dd, II 7.6, 16-Hb), 4.47(br.s, 6-H)[152].
I504
Lophanthoidin 5Hs), 1.25(d, H), 5.74(dd, 4.22(d, 1.8,
I505
Lophanthoidin F(C) 1.05, 1.25, 1.60(each s, 18, 19 and 205Hs), 1.24(d, 7, 17-5H), 2.58(m, l-H t3 ), 5.40(sextet, 7, 15H), 4.25(br.s, 7-H), 4.25(dd, 11 7, 1 6 - H a ) , 4 . 5 i ( d d , 11 7, 16-Hb), 4.45(br.s, 6-H)[152].
I401
Lophanthoidin B(C) 0.95, 1.25, 1.65(each s, 18, 19 and 205Hs), 1.24(d, 7.6, 17-5H), 2.18(s, 5-H), 2.58(m, I-HB ), 5.56(sextet, 7.6, 15-H), 4.22(dd, Ii 7.6, 16-Ha), 4.50(dd, Ii 7.6, 16-Hb), 4.52(br.s, 6-H), 5.66(d, 1.8, 7-H)[152].
1402
Lophanthoidin C(P) 1.22, 1.55, 1.92(each s, 18, 19 and 205Hs), 1.58(d, 7, 17-5H), 2.09(s, 5 - H ) , 2.95(m, I-H /3 ), 5.89(sextet, 7, 15-H), 4.60(dd, Ii 7, 16-Ha), 4.75(dd, II 7, 16-Hb), 4 . 9 5 ( b r . s , 6-H), 5.45(d, 1.4, 7-H)[152].
1405
Lophanthoidin 5Hs), 1.25(d,
D(C) 1.02, 1.25, 1.59(each 7, 17-5H), 2.59(m, I-H~ ), 10.5 7, 16-Ha), 5.87(dd, 7-H), 4.45(br.s, 6-H)[152].
s, 18, !9 and 205.51(sextet, 7, 1510.5 7, 16-Hb),
E(C) 1.05, 1.26, 1.62(each s, 18, 19 and 207.6, 17-5H), 2.18(s, 5-H), 2.65(m, I-HI3 ),
-
4 Table 6. o r_ _ Ent-isopimaranes _____ _ _13C-NMR _ _ _ _ _Chemical _ _ _ _ _ _Shifts _ _ _ _ _f _ _ _ _ _ _ _ _ _ _ _ _ (group _ _ _ - - -H) - - -and - - - -Abietanes - - - - - - - - - - (group - - - - - - - I). - - - - - - - -0 ----------
H201* H202 H301* 12011 1 3 0 1 ~ I302* I303 I305** Carbon------------------------------------------------------------------------------------
Number
I401*
I403**
1
39.00
40.7
42.52
35.70
35.8
35.8
38.5
37.7
38.8
37.8
2 3
24.31 81.02
19.2 41.9
18.49 43.55
18.86 41.03
18.8 41.0
18.9 41.1
19.6 42.8
18.8 42.1
19.0 42.3
18.8 42.2
4 5
38.40 57.80
32.5 51.7
34.14 55.65
39.15 45.32
33.0 46.1
33.0 45.8
34.0 49.7
33.4 48.5
33.6 49.8
33.4 47.7
72.37
6
18.40
20.5
7 8
43.78 71.60
33.4 124.3
22.30
24.6
25.9
67.0
64.7
66.7
65.8
70.62 141.57
64.5 139.5
63.1 143.3
77.2 139.9
74.0 139.0
68.8 137.4
68.3 140.9
55.40 36.73 16.96 37.80
147.74
150.3
148.2
148.8
147.5
33.03 185.66 151.39
39.1 183.3 152.1
39.2 183.5 152.3
39.5 184.8
38.2 183.2
150.3 38.4
146.9 38.3
182.9
183.4
156.6
153.9
120.11
120.2 185.2
119.8
120.6 187.8
119.5
152.0 120.0
153.7 119.4
186.6
185.6
186.9
28.7 65.7
29.3 66.3
28.8 65.8
9
56.50
10 11
37.80 17.92
12
38.29
137.3 37.6 19.3 32.9
13 14
37.41 51.71
35.1 37.1
35.95 46.62
183.64
15
152.80
75.9
151.12
29.39
16 17
108.35 24.72
63.7
108.87
66.07
18
28.78
21.3 33.3
24.15 33.42
19
17.28
20
16.30
21.8 19.8
23.77 17.61
29.4 66.2
188.6 29.3 66.3
30.0 65.2
18.53
14.9
15.0
15.5
33.03 20.74
33.0
33.0
34.2
15.0 33.4
14.9 33.6
15.0 33.4
21.6
21.7
24.3
23.6
14.96
18.5
18.5
22.0
21.3
23.7 21.5
23.7 21.4
131 131 132 132 132 132 - Ref. _ _ _ _ _ _ 129 _ - - - - _ _133 _ _ _ _ -129 - - - - - -.-- -130 - - -- -- --- _ _ _ _ _ _ _ _ _ - _ _ _ - _ _ _ - _ _ _ _ . - - - - - - - Unless otherwise noted, t h e data are f o r the solution in C5D5N. * CDC13, * * (CD3)zSO.
171 Table
7. D i s t r i b u t i o n
species diterpenoids
R.
)
amethystotdes
but l,eyana
ooetsa
rabdoeoetsin rabdocoetsin
eoetso
49,
subeaLvus
B D(C309)
A(B201) C(C201) coetsoidin E(C202) coetsoidin G(C402) A(A405)
R. e a t i s a excisanin A excisanin C(A306) k a m e b a k a u r i n (A412) erioeaLyx
maoecrystal A(B202) maoecrystal C(C306) maoecrystal E(C407) maoecrystal G(C308) maoecrystal J(C409) rabdoside I(CG02) eryocalyxin A neorabdosin(B203) sodoponin R.
eryoeatyx
eryocalyxin maoecrystal
R.
ffexteauL
flexicaulin r a b d o 1o x i n R.
forresti
rabdoforrestin
vat. taxi ftora B B(C305) is
A(A407) B ( A427 )
B(A402) D(C301) F(C401) coetsoidin A(B301)
xindongnin
27,
excisanin kamebanin kamebacetai
63
28
A(A528)
I!
B A(C203)
64, 66, 72, 78, maoecrystal B(C305) maoecrystal D(C307) maoecrystal F(C408) maoecrystal I(C501) maoecrystal K(C502) rabdoside 2(CG03) eryocalyxin B
82,
134
odonicin
maoecrystal oridonin
henryin
A(B202)
A(A408)
65
30
55
A(A511)
gaponiea var. ffLaueoealyx acetylexidonin(E301 ) rabdosinate(E401 )
R.
73
rabdocoetsin C rosthorin A(C413) coetsoidin coetsoidin coetsoidin
50 51
A(A514)
dawoensts
dawodensin
R.
weisiensin
ides
coetsoidin coetsoidin
R.
C)
)
R. e a L e i e o L u s vat. c a l c i c o l i n A(A502)
R.
references 26
bulleyanin(A501
R.
of R a b d o s i a .
isolated
amethystonal(A301)(=macrocalyxin amethystonoic acid(A302) R.
in the G e n u s
adenantha
adenanthin(A401 R.
of D i t e r p e n o i d s
rabdophyllin epinodosin
G(E309)
i 17
172
lasiodonin R. ggutinosa glutinosin(H202)
ent-kauran-
16 ft , 1 7 - d i o l
133
henryine A(A507) henryin(A408) kamebakaurin(A412) rabdophyllin G(ES09) epinodosin
I0, 12, 31, 119, 135 4-epihenryine A(AS05) exidonin(E304) kamebacetal A(C20S) lasiokaurin macrocalyxoformin A(EI05)
R. i n f t e x a inflexanin A(AS08) inflexanin B(A409) inflexarabdonin A(A50S) inflexarabdonin C(A203) inflexarabdonin E(ASI0) inflexarabdonin G(ASII) rabdoinflexin A(D401) kamebanin rabdoserrin A(D201) excisanin A
6, 13, 14, 33, 34, 35, inflexinol(A411) inflexin(AS09) inflexarabdonin B(A410) inflexarabdonin D(A504) inflexarabdonin F(AS05) inflexarabdonin H(ASI2) rabdoinflexin B(A422) kamebakaurin(A412) rabdoserrin B rabdoloxin B(A427)
R. j a p o n i c a glaucocalactone(ElO5) rabdosinate(E401) maoyerabdosin(C410) rosthorin A(C413) rabdosin B[=exidonin(E304)] epinodosinol rabdosin C[=rabdophyllin G(ES09)] epinodosin lasiokaurinol
22, 79, 93, rabdosinatol(A526) isodonoiol(E305) oridonin rabdosin A enmenol
R.
henryi
R. t a s i o c a r p a rabdolasional
107,
120
23,
136
lasiokaurin isodonal ( E 2 0 3 )
R. j a p o n i c a v a r . g t a u e o e a t y x glaucocalyxin C[:rabdosinatol(AS26)] glaucocalyxin D(A201) glaucocalyxin glaucocalyxin A glaucocalyxin R. kunmingensis rabdokunmin A(A423) rabdokunmin C(A424) rabdokunmin E(A512) 4-epi-isopimaric acid
41
5,
E(A202) B 21
rabdokunmin B(A325) rabdokunmin D(A425) rabdoloxin B(A427) callitristic acid
94 (ES08)
R. L a t i f o L t a var. reniformis reniformin A[:henryin(A408)] reniformin C(C205)
reniformin B(A431) kamebacetal A(C20S)
R. t t a n g s h a n liangshanin liangshanin iiangshanin liangshanin
liangshanin liangshanin liangshanin
B(A205) D(A316) F(A318)
16, 69, longikaurin
71, 74, G(C406)
tea A(A204) C(A206) E(AS17) G(A101)
R. Longituba isolongirabdiol (E204)
32
77,
114
173
longirabdosin(A319) rabdolongin A(C503) longikaurin A(C303) longikaurin B(C405) rabdokaurin A(C310) rabdokaurin B(E307) oridonin lasiokaurin nodosin odonicin trichokaurin exidonin(ES04) maoecrystal D(=rabdolongin B)(C307) kamebakaurin(A412) effusanin B macrophyllin B rabdophyllin G(E309) R.
tophantho
ides
lothanthoidin lothanthoidin lothanthoidin enmein( 1 )
lophanthoidin lothanthoidin lophanthoidin
A(1303) C(I402) E(I403)
132
B(I401) D(I304) F(I305)
lophanthoides var. K e r a r d i a n a 16-acetoxy-7-O-acetylhorminone(I301) 16-acetoxy-12-O-acetylhorminone(I302) 16-acetoxy-7 a-methoxyroyleanone(I201) royleanone horminone 6,7-dehydroroyleanone
131
R.
R. loxothyrsa rabdoloxin A(A426) R.
rabdoloxin
lungshengensis
52
lungshengrabdosin(A506)
R.
lushanrubescensin
macrocalyx
macrocalin A(E306) macrocalyxoformin A(EI05) macrocalyxoformin C(EI06) macrocalyxoformin E(E206) macrocalyxin B macrocalyxin D(A320) maeroaalyx var. excisanin A 3iuhuanin A(C404)
R.
R.
42
B(A427)
macrophy
8, 17, 18, 70, 104, 109, 115 macrocalin B(C304) macrocalyxoformin B(E002) macrocalyxoformin D(E205) macrocalyxin A(E306) macrocalyxin C(A301) macrocalyxin E(A321)
j i uhua
excisanin B macrocalyxin 85, rabdophyllin oridonin lasiodonin
l la
rabdophyllin G(E309) amethystoidin A isodonal (E203) ]asiokaurin
nervosa 54, ganervosin A(C403) nervosin(E107) odonicin adenanthin(A401) effusanin A nodosin neorabdosin(B20Z)(:novelrabdosin) R.
R . p a r v tf o L ta ent-5 a -acetoxyisopimar-15-en-8
ent-isopimar-15-en-6 epinodosinol parvifoline A(A417)
C(A414)
67,
76 A(E306) 86, 90, 91, H(C601)
137
6 8 , 7 5 , 106, 110, 138, ganervosin B(EI02) nervosanin(A509) weisiensin A(A514) dehydroabietic acid efiflusanin E shikokianal acetate
139
129,
140
a -o1(H201) a ,7 a ,8 a - t r i o l ( H 3 0 1 ) lasiodonin parvifoline
19,
87,
B(A322)
174
parvifoliside(CG01 ) parvifolinoic acid I sodon
rabdoloxin
pharicus.
isodopharicin A(A313) isodopharicin C(A315) R. phg t [ostachys phyllostachysin A(G401) R.
pseudo- irrorata
R.
rosthorni
pseurata A(A323) pseurata C(A324) pseurata E(A510) dihydropseurata F(A406) rosthornin rosthorin oridonin
t
rubeseens
R.
rubescens
R.
rubeseens
R.
rugosa
R.
scuLponeata
R.
serra
lushanrubescensin(A507) xindongnin B(A435) guidongnin(E202) oridonin rubescensin C ludongnin B(E001) f.
[ushanensis
lushanrubescensin (A507) lushanrubescensin C(A414) lushanrubescensin E(A416) ludongnin
var. A(EI04)
L ushiensfs
rugosanin(E209) dihydroisodocarpin(El01 ) carpalasionin sculponeatin A(EI09) sculponeatin C(EII0) enme in ( I ) macrocalyxoformin A(EI05)
B(A314) 40, B(A418)
phyllostachysin
pseurata B(A419) pseurata D(A420) pseurata F(A421) isodomedin B(A432)
25, 53, 108, xindongnin A(A328) ludongnin(El04) rubescensin D(G402) ponicidin ludongnin A(E104)
ludongnin
setsehwanensis
29
24,
81
37, 38, B(A508) D(A415)
lushanrubescensin lushanrubescensin
sculponeatin sculponeatin epinodosin
128
39
103
B(E001)
dihydrorugosanin(E201 dihydrocarpalasionin(E302) isodocarpin
127
20,
113,
)
I05
iii, 121 , 141 B(E210) D(E501)
43, rabdoserrin D(A428) kamebakaurin(A412)
rabdoserrin A(D201) excisanin A R.
15
isodopharicin
rosthornin ponicidin
A(A327) A(C413)
R .
B(A427)
88,
89
83
rabdosichuanin A(E207) rabdosichuanin C(E208) sodoponin
rabdosichuanin rabdosichuanin
shikokiana vat. shikoccin(F204) epoxyshikoccin(F201 shikoccidin(A433)
4 5 , 4 6 , 1 2 4 , 125 O-methylshikoccin(F102) O-methylepoxyshikoccin(F101 )
R.
occidentatis
)
B(EI08) D(C504)
175 R.
130
strachewi
16-acetoxy-7 a-methoxyroyleanone(I201)
6,7-dehydro R.
roy leanone
80, 84, rabdoternin B(C505) ternifolin(C506) ponicidin effusanin B longikaurin A(C303)
ternifoLia
rabdoternin rabdoternin oridonin sodoponin effusanin
A(C411) C(C412) E
trichocarpa 98, trichorabdal A(Elll) trichorabdal C(E212) trichorabdal E(E311) trichorabdal O acetate(E214) trichorabdonin longikaurin D
R.
R.
umbrosa
99,
142,
143
100, 101, 102, 112, 116, trichorabdal B(E211) trichorabdal D(E310) trichorabdal F(E215) trichorabdal H ( E2 1 5 ) ememogin(E303)
118
umbrosianin(A434) compound I(A303) compound 3(A405) compound 5(C502) kamebanin isodomedin rabdolatifolin(F203)
rabdoumbrosanin(Fl03) compound 2(A304) compound 4(A404) umbrosin A shikoccidin(A433) shikoccin(F204) kamebakaurin(A412)
umbrosa var. h a k u s a n e n s i s rabdohakusin(F202) kamebanin umbrosin A
shikoccin(F204) mebadonin isodomedin
umbrosa vat. LatifoLia rabdolatifolin(F205) shikoccidin(A433) kamebanin
shikoccin(F204) isodomedin leukamenin E
R.
R.
R.
umbrosa
kamebakaur kamebacetal kamebanin
var. teucantha in ( A412 ) A(C203)
f.
R. w e i s i e n s i s w e i s i e n s i n A(A514) R.
wuennanens
genus, 1 ].
is
Isodon,
kamebakaur kamebacetal
trichorabdal rabyuennane
rabyuennane A(A429) rabyuennane C(A513) *The Hara[
kameba
has
been
transferred
9,
47
123
122
36
inin ( A413 ) B(C204)
A(EIII)
56,
44
B(A450) to Rabdosia
57
by
Prof.
H.
176
the rabdophyllin amethystoidin al.
[145]
also
eriocalyxin changes
G content
A did not examined
B
stage,
the diterpenoid
constituents D.
for eight Rcbdoss
Ruan
and quantitative
and
maoecrystal
et
al.
[148]
C (C306)
biological
ones,
activity
of diterpenoids
tional an
cells
active
the
antitumor
against and
reported activity Ehrlich group
carbonyl
several
group
Finally,
and
tumor
against ascites
J.
Hu
plants
by
of
cells
from
series
that
portion
tion
is closely
and
and suggested
moiety
activity
of some
against
compounds leukemia
activity
In relation
stress
activity
against which
his
to these
stress and
also
cytotoxic
of diterpenoids
the chemical
in
results
et al.
u~tro
the interior
to the antitumor
the
compounds
in
or
five-
a -methylene
lymphocytic
the interior
an addi-
increase
T. Fujita
[152].
ascites
of the
reported
of the
150]
chemically
that
an
of ent-kaurene
increases
that
found
the antitumor
that
for
a spiro-lactone
with
etc.
known
Ehrlich
of these
in mice
materials
showed
diterpenoids related
cells,
cells
plant
Y. Chen et al.
it was
such as P 388
examination
carcinoma
and their
to a synergistic
activity
and
[149.
against
also
and
growth
the antitumor
to the presence
the antitumor
cells
HeLa
[151],
The authors
leukemia
the results
rise
are
decade,
trs
conjugated
gave
a-methylenecyclopentanone kaurene
qualita-
antibacterial
an aldehyde,
in addition
site,
lymphoid
isolated
studies,
the
as to insect
from R.
As a result,
activity.
tn u~tro
L 1210
from
parameters
of Rabdos~c
the past
examined
to the cytotoxic
HeLa cells
the
A and B, odonicin
such as
occtdentaLts
was
group),
ring
During
(for example,
as an active
pertaining
on
examined
~cz~fLora
[147].
activities
isolated
in mice.
site
epoxyketone
group
possible
the HPLC
in rat mitochondria,
[2].
derivatives
membered
er~oca~yz
activities,
vat.
sh~koktana
carcinoma
odonicin,
and reported
five species
phosphorylation diterpenoids
R.
A,
constituents.
and inhibitory
Rabdoss
modified
var.
et
activities
Various oxidative
er~ocaLNx
of
for
depending
of eriocalyxins
in R.
Wang
They also
determined
[146],
analysis
distinguished
X. Biological
and
of R.
their diterpenoid
antitumor
diterpenoids
et al.
Z.
er~ocaL~z
and habitat.
diterpenoids
tive
analyzing
of these
in May and that
change.
of eriocalxin
B in R.
site of collection
this viewpoints.
a maximum
the contents
and maoecrystal
in the contents
growth
reached
show any prominent
of
the
~ -seco-ent-
reactivity of that
of these
of
por-
diter-
177 penoids
[153].
The R.
antiinflammatory
mccrocaLyx
has
administered inducers. been
The
described
enhancement dazole
to
tion. show
i.p.
of
On t h e any
were
effect
effect
the
effective oridonin Oridonin
effect
hypoxic other on
of
described
of
[155].
treat
effect
been
hand,
when Chinese the
radiosensitization
the
total
[154]. for
diterpenoids
The
total
inflammation
due
on
radiosensitization
(0.01
mM) s h o w e d
used
together
hamster same
with
V 79
amount under
of
cells
to
several has
also
supra-additive 1
mM
misoni-
under
radia-
oridonin
aerobic
from
diterpenes
did
conditions.
not
178
Table
8. Alphabetical
Compound
ent-5 a-Acetoxyisopimar15-en-8 a-ol(H201) 16-Acetoxy-7-O-acetylhorminone(IS01) 16-Acetoxy-12-O-acetylhorminone(IS02) 16-Acetoxy-7-O-methoxyroyleanone(1201) Acetylexidonin(ES01) Adenanthin(A401) Amethystonal(AS01) Amethystonoic acid(AS02) Bulleyanin(A501) Calcicolin A(A502) Coetsoidin A(B201) Coetsoidin A(BS01) Coetsoidin B(A402) Coetsoidin C(C201) Coetsoidin D(CS01) Coetsoidin E(C202) Coetsoidin F(C401) Coetsoidin G(C402) Compound I(A505) Compound 2(A504) Compound 5(A405) Compound 4(A404) Compound 5(C502) Dawodensin A(A405) Dihydrocarpalasionin(ES02) Dihydroisodocarpin(El01) Dihydropseurata F(A406) Dihydrorugosanin(E201) Ememogin(ES05) 4-Epihenryine A(AS05) Epoxyshikoccin(F201) Exidonin(ES04) Excisanin C(A506) Flexicaulin A(A407) Ganervosin A(C405) Ganervosin B(EI02) Glaucocalactone(El05) Glaucocalyxin C(A526) Glaucocalyxin D(A201) Glaucocalyxin E(A202) Glutinosin(H202) Guidongnin(E202) Henryin(A408) Henryine A(AS07) Inflexanin A(AS08) Inflexanin B(A409) Inflexarabdonin A(A505) Inflexarabdonin B(A410) Inflexarabdonin C(A205) Inflexarabdonin D(A504) Inflexarabdonin E(ASI0) Inflexarabdonin F(A505) Inflexarabdonin G(ASII) Inflexin(AS09) Inflexinol(A411)
Index. Isodonal(E205) Isodonoiol(ES05) Isodopharicin A(ASIS) Isodopharicin B(ASI4) Isodopharicin C(A515) Isolongirabdiol(E204) ent-Isopimar-15-en6 a ,7 a ,8 a-triol(HS01) Jiuhuanin A(C404) Kamebacetal A(C205) Kamebacetal B(C204) Kamebakaurin(A412) Kamebakaurinin(A413) Liangshanin A(A204) Liangshanin B(A205 ) Liangshanin C(A206 ) Liangshanin D(ASI6 ) Liangshanin E(ASI7 ) Liangshanin F(ASI8 ) Liangshanin G(AI01 ) Longikaurin A(C505 ) Longikaurin B(C405 ) Longikaurin G(C406 ) ) Longirabdosin(ASl9 L o p h a n t h o i d i n A(IS 05) Lophanthoidin B(I401) Lophanthoidin C(I402) Lophanthoidin D(I504) Lophanthoidin E(I405) Lophanthoidin F(I505) Ludongnin(El04) Ludongnin A(EI04) Ludongnin B(E001) Lungshengrabdosin(A506) Lushanrubescensin(A507) Lushanrubescensin B(A505) Lushanrubescensin C(A414) Lushanrubescensin D(A415) Lushanrubescensin E(A416) Macrocalin A(ES06) Macrocalin B(C504) Macrocalyxin A(ES06) Macrocalyxin C(AS01) Macrocalyxin D(A520) Macrocalyxin E(A521) Macrocalyxoformin A(EI05) Macrocalyxoformin B(E002) Macrocalyxoformin C(EI06) Macrocalyxoformin D(E205) Macrocalyxoformin E(E206) Maoecrystal A(B202) Maoecrystal B(C505) Maoecrystal C(C506) Maoecrystal D(C507) Maoecrystal E(C407) Maoecrystal F(C408) Maoecrystal G(C508) Maoecrystal I(C501) Maoecrystal J(C409)
179 Maoecrystal K(C502) Maoyerabdosin(C410) O-Methylepoxyshikoccin(Fl01) O-Methylshikoccin(Fl02) Neorabdosin(B203) Nervosanin(A509) Nervosin(El07) Novelrabdosin(B203) Parvifoline A(A417) Parvifoline B(A322) Parvifoliside(CG01) Phyllostachysin A(G401) Phyllostachysin B(A418) Pseurata A(A323) Pseurata B(A419) Pseurata C(A324) Pseurata D(A420) Pseurata E(A510) Pseurata F(A421) Rabdocoetsin D(C309) Rabdohakusin(F202) Rabdoforrestin A(A511) Rabdoinflexin A(D401) Rabdoinflex i n B(A422) Rabdokaurin A(C310) Rabdokaurin B(E307) Rabdokunmin A(A423) Rabdokunmin B(A525) Rabdokunmin C(A424) D(A425) Rabdokunmin E(A512) Rabdokunmin Rabdolasion al(E308) Rabdolatifo lin(F203) A(C503) Rabdolongin Rabdolongin B(C307) Rabdoloxin A(A426) Rabdoloxin B(A427) Rabdophyllin G(E309) Rabdophyllin H(C601) Rabdoserrin A(D201) Rabdoserrin D(A428) Rabdosichuanin A(E207) Rabdosichuanin B(EI08) Rabdosichuanin C(E208)
Rabdosichuanin D(C504) Rabdoside 1(CG02) Rabdoside 2(CG03)) Rabdosin B(E304) Rabdosin C(E309) Rabdosinate(E401) Rabdosinatol(A326) Rabdoternin A(C411) Rabdoternin B(C505) Rabdoternin C(C412) Rabdoumbrosanin(Fl03) Rabyuennane A(A429) Rabyuennane B(A430) Rabyuennane C(A513) Reni formin A(A408) Reniformin B(A431) Reni formin C(C205) R o s t h o r i n A(C413) R o s t h o r n i n A(A327) R o s t h o r n i n B(A432) R u b e s c e n s i n D(G402) Rugosanin(E209) S c u l p o n e a t i n A(E109) S c u l p o n e a t i n B(E210) Sculponeatin C(Ell0) S c u l p o n e a t i n D(E501) Shikoccidin(A433) Shikoccin (F204) Ternifolin(C506) Trichodonin (E215) Trichorabdal A(EII1) Trichorabdal B(E211) Trichorabdal C(E212) Trichorabdal D(E310) Trichorabdal E(E311) Trichorabdal F(E213) Trichorabdal G-acetate(E214) Trichorabdal H(E215) Trichorabdonin(E312) Umbrosianin(A434) Weisiensin A(A514) Xindongnin A(A328) Xindongnin B(A435)
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, 9 1995Elsevier Science B.V. All rights reserved.
Vol. 15
187
Structural Elucidation of Saponins Georges Massiot and Catherine Lavaud
INTRODUCTION SAPONINS ARE COMPLEX MOLECULES Saponins constitute an important class of secondary metabolites from plant and animal domains (1-3). They display a broad spectrum of biological activities that have raised the interest of phytochemists for the past forty years. With molecular weights ranging from 600 to 2000 Daltons, their structures are complex. It was only in the seventies that, with the use of chemical degradation techniques, their precise structures were established (4-5). Since then, the field has benefitted from the recent progress in instrumentation. The purpose of this article is to describe the current techniques in use for the structural elucidation of saponins. From a chemical standpoint, saponins are made of three entities: an aglycone (steroid or triterpene), sugars and sometimes acids. The determination of the structure therefore requires identification of the elements and sequencing. While the first problem is trivial, the second is generally more difficult to solve because of the large number of possible combinations. For example, let us consider the case of soyasaponin I (6), a saponin, which is made of soyasapogenol B, rhamnose, galactose and glucuronic acid. If there was only one branching point on each of the elements, there would be only 24 isomeric structures for soyasaponin I (4!). The larger number of iso
HO
COOH 0
HOOHo \
HO,~~O 0 CH3._.7.~0 ~
OHO-~H
C)H
I~ OH soyasaponin I
188 mers obtained with these four elements (35712) comes from the n u m b e r of possible branching on each of them (3 for soyasapogenol B and rhamnose, 4 for glucuronic acid and galactose). The number which is obtained after this combinatory analysis m u s t be further multiplied by 64 to account for a or ~ anomeric configurations and pyran or furan forms (table 1). Fortunately, all these possibilities are not met in nature (for example ~-L-rhamnose) but this illustrates the difficulties of saponin sequencing.
TABLE 1. N u m b e r of saponins made of soyasapogenol B, glucuronic acid, galactose and rhamnose.
Tridesmosides :
6 isomers
Bisdesmosides : 22 disaccharides:
rha-gal-
(4 isomers)
rha-glcUA-
(4 isomers)
gal-glcUA-
(4 isomers)
x 3 branching points
gal-rha-
(3 isomers)
x 2 possibilities for
glcUA-rha-
(3 isomers)
glcUA-gal-
(4 isomers) 22x3x2
the third sugar. =
132 isomers
Monodesmosides : 4 x rha-gal-
x 6 branching points
4 x rha-glcUA- x 6 branching points 4 x gal-glcUA- x 7 branching points 3 x gal-rha-
x 3 points of a t t a c h m e n t
x 6 branching points
on aglycone
3 x glcUA-rha- x 6 branching points 4 x glcUA-gal- x 7 branching points 140 x 3 =
420 isomers
Total of isomers (including anomers, furanoses and pyranoses): 64 x (6 + 132 + 420) = 64 x 558 = 35712 isomers. (this n u m b e r must be multiplied by ten if a single acid substitutes the molecule)
189 THE IDENTIFICATION OF THE ELEMENTS OF A SAPONIN - ARTEFACT FORMATION The elements of a saponin may be identified either on the intact saponin or after hydrolytic cleavage. It is always better to perform the identification on the intact saponin to eliminate the possibility of artefact formation during hydrolysis. This identification can be realized by mass spectrometry (MS) or nuclear magnetic resonance (NMR 1H or 13C). Artefacts are most often formed when the aglycone is sensitive to acid. The following equations give examples of such rearrangements (7-9).
..~
HO
..,,i
O~H,
/0
H
02H
HO ,~r
Suga
H..~ protobassie acid
v
"IOH
9
bassie add
....
~,,~ 20%H2804 10
sugars
',
+
MeOH
i|,
sU
H
Ii I Ii I
Three examples of formation of artefacts after acid hydrolysis.
190 SUGARS PRESENT IN SAPONINS IDENTIFICATION AND ABSOLUTE CONFIGURATION The sugars present in saponins are common sugars such as D-glucose, D-galactose, L-rhamnose, D-fucose, D-xylose, L-arabinose, D-glucuronic acid and D-galacturonic acid. The enantiomers of these sugars are not found in plants a fact used as a clue in the determination of the configuration of the sugars. Less common sugars from saponins are for example : D-allomethylpyranose (10), D-ribose (11), D-apiose (12), D-quinovose (13), 2-acetamido-2-deoxy glucose (8, 14), 2-acetamido-2-deoxy galactose (15) and D-arabino-2-hexulopyranose (16),. Sugars are obtained by hydrolysis of the saponins in acidic medium (1 or 2N HC1 or H2SO4) , in the free form if the solvent does not interfere (aqueous dioxane) or as methyl glycosides if the solvent contains methanol. Free sugars are separated from the aglycone by solvent extraction (CHC13) and the medium is neutralized with silver carbonate (with precipitation of AgC1) or basic resin (IRA 45, IRA OH-). They are finally identified using reference samples by paper chromatography (17) or directly by HPLC (refractive index detection) (18). The methyl glycosides may also be analyzed by GC after silylation (O,N-bis trimethylsilyl trifluoroacetamide or hexamethyldisilazane and trimethylchlorosilane). Identification is based on retention times (8), FTIR (19) or mass spectrometry. The absolute configuration of the sugars is determined by running the same operations on a larger scale with a preparative separation stage. A variant is Nakanishi's method which consists in the parabromobenzylation of the methyl glycosides (vide infra for a complete discussion of the method) (15). Several analytical methods have been introduced to determine the absolute configuration of sugars on a microscale. The principle of these methods is based on the coupling of the sugar with an optically active reagent and the comparison of the conjugates with reference samples. A selection of the reagents comprises (S)-2-butanol (19), L-cysteine methyl ester (20) or L (-)-a-methylbenzylamine / NaBH3CN (21).
ACIDS PRESENT IN SAPONINS The presence of acids in saponins further complicates the structural elucidation process and in most cases, one chooses to start the study with saponified compounds. It has been observed fortunately, that acyl groups on sugars are more labile than the hindered sugar chain esters at C-23,-24 o r - 2 8 and that they can be selectively cleaved with sodium bicarbonate (21):
191 .,i
r
h
. ..0" ~"~.,. v ara~glc I OH a ~ OH
..... / v-,, /
.,'
Fuc "rha I 0 xyl
i . . 0 ~ ara~g c (~H
~ rhaO
A
A
A
A
Fuc
+ A
A
rha I Q x y l xyl / j , -/COOH
OH
Sodium bicarbonate hydrolysis of crocosmioside H. Commonly encountered esters are angelates and tiglates, which are found, for example, in the saponins from horsechesnut Aesculus glabrus L. (22). Table 2 lists some of the acids present in saponins with their representative references. TABLE 2 Examples of acids present in saponins.
Aesculus saponins Dodonea saponins Kalopanax saponins
22
ciwujianosides
25
ginsenosides
26
malonylsafkosaponins
27
3-hydroxybutyric acid
Solidago saponins
28
dicrotalic acid
lobatosides
29, 30
tubeimoside
31
brownioside
32
Castanopsis saponins
33
phtalic acid
pseudoginsenoside
34
syringic acid
periandradulcins
35
p-coumaric acid
Tragopogon saponins
36
2, 9, 16-trihydroxyhexadecanoic acid
crocosmiosides
37
2, 6-dimethyl, 6-hydroxy-2-trans-
Gleditsia saponins Entada saponins
38
tiglic acid angelic acid acetic acid malonic acid
(hydroxymethylglutaric acid) gallic acid digalloyl acid
2, 7-octadienoic acid
23 24
33
39
In most instances, the presence of esters is detected by comparison of the mass spectrum of the saponin with its desacetylated counterpart. This is also done with use of 1H and 13C NMR spectra. Some esters, particularly malonyl esters, are labile
192 and thus special care must be taken during the isolation process (26). FULL STRUCTURAL DETERMINATION OF SAPONINS. THE CLASSICAL METHOD IDENTIFICATION OF BRANCHING POINTS Since information on branching is lost if complete hydrolysis is performed on a native saponin, the hydrolysis must be performed on a derivatized material. A general protocol proceeds as follows. The saponin is first permethylated according to Hakomori's method (Nail, DMSO, MeI) and cleaved with acid (TFA, 120~ The methylated sugars are reduced into alditols (NaBH4) , peracetylated and the mixture is analyzed by GC-MS (40). When a uronic acid is present, it is advised to reduce it into a bis-deuterated hexitol by treatment of the corresponding ester by means of LiA1D4 before hydrolysis (41). Alternatively, the methylated sugars from the hydrolysis may be directly analyzed and identified by GLC (42) or HPLC (15).
/ HO
NHAc
~
O ~,.,Ac HN-7..........~-OH HO-'~,,k_.10 /
HO~---~HO~
.Oo
o.
HO
aOH / Mel / DMSO
O
MeO-..--,
M~~M~
~o~:..-.~..o~
H
"-
O
~o=k
^
9
~o-~
OMe +,
O
M
NHAc ON
e
M
e
OMe
Derivatisation- hydrolysis of sarasinoside A 1 (15) SEQUENCING OF THE SUGARS In case where the chain of sugars has no more than two units, the permethylation-hydrolysis experiment suffices to identify the terminal sugar. In other cases, one must rely on incomplete hydrolysis experiments or on related series of compounds when they occur in the plant (43). Even though purely degradative methods are no longer used, recent literature offers judicious examples of combination of degradations (18). The arsenal of degradative methods is impressive and contains tools such as microorganisms, isolated en-
193 zymes (18) and mineral salts (for example LiI for the cleavage of hindered esters) (21). Diazomethane itself, has been used as a cleavage reagent in quillaic acid derivatives. Participation of an aldehyde to the cleavage of a proximal ether bond explains this special reactivity (12). ..,'
~~COO--ara-~ha--xyl .. v glc ~ gal-glcUA-O (~HO 1)KOH /%<' ,=~~cellulase 2)D ~ : O ~ ~ C O O ,~~~<~CO0-ara-rha OMe I, I I " , ~.~
CHO.,,
glcUA--O -
~
gal-glcUA-O CHO
'~COO-ara-rha-xyl
H
~COOH OMe ~ : ~IIcUA_O/'~/"-.. / CHO + ~ , ~ COOH HO
i CHO
quillaic acid
Combination of degradations of lucyoside P. CIRCULAR DICHROISM - NAKANISHI' S METHOD Nakanishi's method is based on the splitting of CD waves when two chromophores on a chiral molecule are close in space (the dibenzoate rule). Such is the case with dibromobenzoates of sugars which have a 1,2 or 1,3 relationship. In the first version of the method, saponins were permethylated, methanolyzed with acid and the liberated OH positions were p-bromobenzoylated (44). As terminal sugars are fully methylated and UV transparent, they need not be considered. Branched sugars (two substitutions at least) yield di-or tri-benzoates with exciton-split CD curves. The difference in Ae values of the two extrema of split CD curves is directly related to the respective positions of the benzoates (1, 2 eq-eq = 1, 2 eq-ax = 62 ; 1, 2 ax-ax = 6 ; 1, 3 eq-eq = 0 ; 1, 3 eq-ax = 16). The sensitivity of circular dichroism makes the method suitable for microassays. Typical analyses are performed on a 100 ~g scale (nanomolar levels). An improvement to the original method has recently been proposed (45). The saponin is not permethylated but is first parabromobenzoylated and cleaved with bro-
194 moacetylbromide. This operation transforms the sugars into 1-bromo-derivatives with bromoacetates on each of the linkage positions. The bromo sugars are converted into ~-O-methylglycosides (Ag2CO 3 / AgOTf) and the bromoacetates are exchanged for p-methoxy-cinnamates. After separation, one obtains derivatives of sugars bearing different combinations of benzoates and cinnamates. Their CD curves are characteristic of the sugar and of the substitution pattern.
HO
HO.,,~'~/ 0 HO~..--.~0, HO~~~.~/O~? H(~H /(9HO~k
Ho~-~,-~ _.-o
HO-K
~~o
OH
/
-0 0
I~'%"''0
~
HO
0
OH I BrR~ ~
~ r OMe
1
Br Br R.20
Me
OMe
t = p-BrC6H4C02 R1 = BrCH2CO
OMe OMe
R2 = p-MeOC6H4(CH)2CO
Preparation of derivatives suitable for CD from digitonin. (45)
......
195 MASS SPECTROMETRY AND SEQUENCING. RECENT ADVANCES Saponins are too polar, too thermally unstable and not volatile enough to directly provide ions that are suitable for analysis. Until recently, derivatives (Me or TMS ethers) had to be prepared in order to obtain spectra. In the past ten years however, new techniques of soft ionization have emerged which have proved useful in the analysis of saponins. They are : Field Desorption (FD), Fast Atom Bombardment (FAB), Laser Field Desorption (LD) and Californium Plasma Desorption (CPD). FDMS readily gives molecular ions and fragments with saponins (46, 47); in all cases cationized molecules are observed (M+Na) +, (M+K) + and the spectra are often complicated by the presence of cationized fragment ions. For these reasons, it seems that FAB MS prevails over FDMS. Negative FAB MS gives (M-H)" ions of strong intensities and fragments corresponding to losses of the sugars. Positive FAB MS does not provide (M+Na) + or (M+K) + of strong intensity but the ionization may be increased by adding NaC1 or KI to the glycerol matrix (48, 49).
iI
"
896
0 ' H
427~11 8' 0
t72J
469
r162
rha ~vN~
265
MW = 1616
ara 1483@,~
xyl
]aa
xyl
xyl
Gleditsia japonica saponin. Calculated MS fragments (50) 1655 1639 1523 1507 1493 1391 1374
[M+K]+ [M+Na]+ [M+K-132]+ [M+Na-132]+ [M+Na-146]+ [M+K-264]+ [M+Na-264]+
1243 1229 1213 936 920 839 831
[M+Na-3x132]+ [M+Na-410]+ [M+Na-426]+ [M+K-719]+ [M+Na-719]+ [M+Na+K]++ [M+2Na]++
Gleditsiajaponica saponin. Observed FD MS fragments (50)
1205 411 1351 265 1483 133
rha
196
HO-N
/
#1191
-867
I
~ H O ~~'O~o U ~>"o HoJ...--X-.V~
~21 , \
,~ ~u..-~
0
[
~
.~/J
J
I
OH OH-"~7"~O? ,OHm_ ~ \ 1207
OH ~1191
HO~-/'-'-y
OH OH
-
Negative FAB fragmentation ofAlium vineale saponin (19) Laser desorption (time of flight or Fourier transform) also yields ions and fragments with saponins (15, 51, 52). Although promising the technique is not yet widely used (for cost reasons ?). Californium Plasma Desorption MS is the least expensive of the available MS techniques; it gives molecular ions of high intensity for non volatile, polar and fragile molecules of high molecular weight including saponins (53). While the spectra of underivatized saponins show little fragmentation (54), intense fragments for sequential losses of sugars are observed for peracetylated material (55). A disadvantage of CPDMS is the relatively low resolution of the peaks. Mass spectrometry is able to provide molecular ions and fragments corresponding to sequential losses of sugars. When the chains of sugars are linear and if the sugars have different molecular weights, the combined use of MS fragmentation and of methylated alditol analysis allows the full structural elucidation of saponins. This is the case for soyasaponin A3 (from soyabean, Glycine max) where hydrolysis of the permethylate reveals the presence of soyasapogenol A, terminal rhamnose, 2-substituted galacturonic acid and 2-substituted galactose. Negative FAB MS shows sequential losses of deoxyhexose, hexose and hexuronic acid, thus allowing full structural elucidation (56). OH
COOH H ~ O ,,, HOnU~\ .0 LX'~o
HO.~~..~'
"ov
OH , ....
OH
CH3"7"'--O; HOOH'~O H Soyasaponin A3
197 A m o n g f u r t h e r developments of MS in the field of saponins, it m a y be expected t h a t LC/MS coupling will ease the tedious process of t i t r a t i o n a n d analysis of saponins in mixtures. First results obtained with the Jeol FRIT-FAB s y s t e m describe the analysis of saponins from Panax and Bupleurum (57). 13C N M R AND S T R U C T U R E S OF SUGAR CHAINS The resolving power of 13C NMR, which allows identification of individual resonances for each s u g a r carbon has rapidly contributed to the domain. The chemical shifts of carbon a t o m s in a chain of sugars m a y be recognized from the literature. The s t r u c t u r e s of the yunganosides, saponins isolated from a chinese liquorice a n d which possess the s a m e chain as glycyrrhizin (rha-glcUA-glcUA), were, in this m a n n e r , det e r m i n e d by comparison of 13C spectra (41). 13C N M R is now used routinely to determine the s u b s t i t u t i o n p a t t e r n of sugars in chains. S u b s t i t u t i o n of a sugar position by a n o t h e r sugar induces w h a t is called glycosylation shifts (see table 3 for typical examples). The m e t h o d seems perfectly reliable, its only limitations being the d e t e r m i n a t i o n of ramification in chain a n d sequencing. In highly complex molecules however, it is worth w o n d e r i n g if in the absence of real correlations (vide infra for the use of 2D techniques), a n a s s i g n m e n t is unique or not. In this a r e a of the field, it is expected t h a t computers will help provide the o p e r a t o r with definitive or alternative assignments.
TABLE 3. Typical 13C 5 values for sugars (bold characters refer to s u b s t i t u t e d positions; s t a r r e d values correspond to esters). ~-D-glucopyranose C- 1 104.3 105.5 105.1 106.4 105.5 103.7 103.5 105.6 104.4 105.7 105.5 95.4* 105.8 105.1 105.1
~-D-galactopyranose 106.4 103 106.3 101.7
ref. C-2 74.1 73.3 75.1 75.9 75.3 79,8 83,5 74 74.9 75.5 75.5 73.5 75.5 82
C-3 77.4 75.9 78.2 78 78.5 77.6 78 84.8 77.5 78.7 78.3 78.3 75.5 76.2
C-4 70.2 69.1 71.3 71.5 71.5 71.2 71.3 69.7 78.4 72.2 72.6 70.1 72.2 78.4
C-5 77.1 74.1 77.7 77.9 78.1 78.2 77.9 77.9 76.3 76.7 75.5 76.6 76.7 77.4
C-6 61.8 62 61.7 62.5 62.5 63.2 62.5 62.6 61.4 69.7 68.8 70.8 69.8 63.8
(60) (60) (58) (29) (62) (59) (29) (62) (64) (21) (59) (64) (40) (65)
81.5
87.3
78.6
77.6
62.8
(65)
74.5 72.5 74.4 77.8
74.9 74.3 74.9 73.6
70.1 69.2 69.8 71.1
77.1 75.4 77 76.9
62.1 60.7 61.7 61.6
(18) (60) (30) (63)
198
~-D-glucuronic C-1 106.1 105.5 103.4 102.2 105.5 105.6 102.6
acid C-2 75.4 75.1 83.5 81.3 78.7 74.4 77.6
C-3 78.1 78.2 77.5 76.6 76.8 85.1 85.2
C-4 73.4 73.5 73 71.7 73.7 71.9 70.1
C-5 77.8 77.4 77.5 76.1 77.6 76.8 76
~-D-xylopyranose 103.8 74.8 105.2 75.5 106.8 74.9 106.8 76 107.6 75.6 106 79.5 102.6 79.4 106.4 75.2 107 75.8 105.3 74.5 106.2 75 106.6 76
77.9 78.4 78.6 78.6 78.5 77.5 78.1 83 82.9 85.8 73.8 76.4
70.1 71.3 71.7 71 71.1 71.5 70.8 69.2 69.7 67.8 76.7 76.5
66 67.3 67.5 67.4 67.5 67 66.8 67.2 67.3 65.7 64.3 64.5
(60) (18) (18) (58) (43) (43) (63) (62) (43) (6O) (43) (40)
a-L-arabinopyranose 105.1 72.6 105.6 71.9 106.5 73.8 101.3 79.6 94.3* 74.9 93.3* 76.1 93.8* 75.7 93.4* 75.6 105.6 72 104.1 79.6
74.4 73.4 74.3 72.1 71.4 70 69.6 70 73.1 73.2
69.2 68.3 69.2 66.9 67.6 66 66 65.9 77.4 75.8
66.5 65.9 67.2 63.3 64.9 62.9 63.1 62.8 65.4 64.3
(21) (60) (29) (59) (29) (62) (18) (58) (60) (17)
a-L-rhamnopyranose 101.6 72.3 101.9 72.3 102.3 72.4 101.5 71.8 101.5 71.7 101.5 71.5 101.2 72.1 101 72.7 99.9 70.8 102.2 72.6 101 71.6 101.1 71.7 100.8 69.8
72.6 72.5 72.1 82.7 80.3 83.5 72.6 72 71.2 72 82.3 82.8 81.3
73.8 74 73.5 72.9 73.6 72.7 83.4 83.5 83 84.2 77.9 79 77.7
70.6 69.9 69.4 69.6 71.7 70.1 68.3 68.6 67.1 67.9 71.2 68.9 67.8
18.7 18.6 18.1 18.5 18.5 18.4 18.6 18.5 17.3 18.5 18.5 18.8 17.7
(18) (61) (64) (43) (58) (30) (21) (18) (60) (43) (58) (18) (60)
~-D-fucopyranose 93.7* 73.1 95* 73.5 95* 72.5
75.3 76.9 85.6
72 73.4 72.5
71.6 72.7 72.2
15.6 17 17
(60) (21) (40)
79.5 80.4 80.3
74.5 75.4 74.9
62.2 65.3 64.8
75.4
68.4
78.7
3-D-apiofuranose 111.7 77.5 109.1 77.7 111.2 77.6 3-D-mannopyranose 102.3 71.6
ref. C-6 172.9 172.8 172.5 171 170.5 172.8 170.7
(16) (16) (18) (60) (63) (16) (60)
(58) (21) (62) 62.3
(16)
199 13C RELAXATION TIMES AND SEQUENCING It is well known that long relaxation times T 1 correspond to fast correlation times and this fact has been used to determine the position of a carbon in a chain. The more mobile sugars (end of chains) are therefore expected to have longer T 1 than those whose motions are restricted. This was first demonstrated with k-strophantoside (66) but only very few examples of sequence determination with this technique are found in the literature (67).
CONTRIBUTIONS OF 1H NMR TO SAPONIN STRUCTURAL ELUCIDATION An 1H NMR spectrum of a saponin is characterized by a 3 to 5 ppm highly entangled area where most of the sugar proton resonances overlap. Until recently, these spectra were used to identify the aglycone by its methyl resonances (in analogy with the Zurcher steroid increments) or olefinic protons. The advent of high magnetic field and of 2D NMR has recently allowed the exploration of the sugar proton resonances, which had been hiding a wealth of information on sugar nature, configuration and linkage. Basically, there are two approaches for the use of 1H NMR spectroscopy of saponins which consist either in working on the underivatized (in deuterated MeOH, pyridine or DMSO) or on derivatized material (peracetates). Each of the methods has its advantage, and inconveniences, which will be discussed below. The first article in the domain was authored by Breitmaier in 1984 (59) and described a full strategy for the structural elucidation of saponins using 2D NMR techniques. The saponin chosen as an example was muscaroside C which contains glucose, arabinose and apiose in a 2:1:1 ratio (HPLC titration). The 1H NMR spectrum was determined in pyridine d 5 at 500 MHz and assigned by means of a COSY-45. Despite the high field, only one of the glucose proton system could be directly assigned with COSY; the second glucose had H-2, H-3 and H-4 (5 4.16, 4.14, 4.22) too close to give clear off diagonal signals and arabinose had H-2, H-3 and H-4 strongly coupled (5 4.52, 4.55, 4.54). In fact, the COSY spectrum was mostly used as an aid to assign the 13C spectrum by means of one-bond heteronuclear C-H correlations. Combination of 1H-1H and 1H-13C correlation provided a set of assignments for the 13C resonances, which were then compared to literature reference resonances (methyl-~-Dglucopyranoside, methyl-a-L-arabinopyranoside and methyl-~-D-apiofuranoside). Deshielding of C-6 of one glucose (+ 6.3 ppm), of C-2 of the other glucose (+ 5 ppm) and
200 of C-2 of arabinose (+ 7.4 ppm) could tell that these positions were substituted but this was not sufficient to allow full sequencing of the saponin. Sequence was determinated by negative FAB MS which showed peaks at (M-H-pentose)-, (M-H-pentosehexose)-, (M-H-pentose-hexose-pentose)" and (M-H-pentose-hexose-pentose-hexose)" at m/z 929, 767, 635, and 473. Apiose was determined to be the terminal sugar of the chain by its 13C chemical shii~s. The glucose substituted at position C-6 showed an nOe effect between its anomeric proton and H-3 of the aglycone. This allowed the structural determination of muscaroside C as :
O
HO
HO HO H O ~
..... '
0 0 H~O
OH
~
!
HO HO OH Muscaroside C In 1986, three articles were published which presented important improvements of methodologies; two concerned the NMR of peracetates (68, 69) and one the NMR of a saponin in DMSO (17). The latter article dealt with the structures of saxifragifolins A and B from Androsace saxifragifolia. Combination of positive and negative FAB MS and of chemical degradation left two possibilities for the sugar chain of saxifragifolins A and B. They differ in the substitutions of an inner arabinose :
xylr or
xylr
~4,arap~aglycOne
glcp ~---2Tiap~aglycOne glcp
201 The point was settled by taking advantage of the special feature of NMR spectra run in DMSO that, as chemical exchanges are frozen, show individual lines for OH. OH protons are identified at low field and can be exchanged with D20; they show three bond couplings with CH or CH 2. The absence of couplings of CHO with OH means that the oxygen atom is not substituted by an hydrogen atom and COSY experiment verifies this. It was thus found that arabinose was linked through its 2- and 4positions, that a glucopyranose was linked in turn through its 2- position and that xylopyranose and glucopyranose were terminal. A NOESY experiment, which showed transfers of magnetization from terminal glucose H-1 and arabinose H-2 and from the other glucose H-1 and arabinose H-4 allowed final proposals for the structure of saxifragifolins :
CHO
O O H ~ ( H HO-----m.. ~0 L HO ~-~ OH HO
P L
0
H'o
0
0
iIl
"~'v~'"OH =R-H R OAc
R
=
OH Saxifragifolins The articles referenced (68) and (69) describe the advantages of running 1H NMR spectra of peracetates : - molecules are soluble in CDC13 and give spectra with better resolution -
-
sugar proton resonances are spread over 2.5 ppm protons a to acetates have chemical shifts larger than 4.5 ppm (except CH2OAc which resonate between 4 and 4.5 ppm)
- protons a to ether linkages are shielded 5 < 4 ppm - anomeric protons appear between 4.2 and 4.7 ppm except for those linked to acid functions. In general, assignments of 1H NMR spectra of peracetates are easily performed with a COSY experiment; an example of such an analysis will be outlined below. Branching points, which correspond to the most shielded protons and interglycosidic
202 linkages are determined in two fashions : -
long range couplings
- Overhauser effects. When the geometry is favourable (W or sickle paths), there exist 4 j between anomeric protons and protons situated on the other side of the glycosidic bond (69). These couplings do not split resonances because they are smaller than the proton natural lines (J < 1 Hz) but they are detectable in delayed COSY. In the delayed COSY experiments, a delay is introduced before and aider the "read" pulse. To observe these long range effects, the delays must be of the order of 300 to 400 ms. Delayed COSY are complicated by a number of long range couplings (70) but it is unnecessary to examine the whole map to determine the sugar sequence : the information stands at the cross points of the rows corresponding to the anomeric protons and of the columns corresponding to the protons a to linkages (or vice-versa). With this technique, we were able to solve the structure of a four sugar saponin from Medicago sativa (69). The second article on peracetyled saponins (68) describes the complete analysis of the 1H NMR spectrum of a four sugar saponin using a COSY experiment. Little use was made in this article of 1H chemical shii~s to determine substitutions (except for H-2 of a galactose) which were established instead according to 13C glycosylation shii~s. Nuclear Overhauser effects were detected between H-3 of the aglycone and H-1 of the uronic acid, between H-4 of the uronic acid and H-1 of the 2- substituted galactose and between the anomeric proton of glucose and H-2 of this galactose. No nOe could substantiate the linking of the terminal galactose to C-2 of the uronic acid. The chemical shift of the anomeric proton of this galactose (5 = 5.49 ppm) rather makes us think of an ester linked sugar (to the 6- position of galacturonic acid ?). In this case position 2 would have escaped acetylation for steric hindrance or for any other reason.
Ro R,~~O
COOH
n'o---,
~
H
.
R'O
_ R ' ( ~ ' ~
?R~0
0
R=Ac
R'=H
R = R'= H
camellidinl camellidin II
203 STRATEGY FOR 1H NMR ASSIGNMENT OF SAPONINS. RELAYED COSY AND HOHAHA The anomeric protons are most often doublets (or broad singlets for rhamnoses) readily detectable in the spectra of either native saponins (in CD3OD, C5D5N or C2D6SO) or of peracetates. Starting from these protons, the H-2s are located by means of a COSY experiment, and so on (H-3, H-4..) until the terminal positions of the sugar. Unless the spin systems are especially well behaved, it is not possible to obtain an unambiguous assignment with this sole technique owing to crowding in the vicinity of the diagonal. To circumvent the difficulty, one uses the relayed COSY experiment in which the coupling from H-2 to H-3 rebounds on H-1 through the H-1 to H-2 J relay. In this case there is a "relayed correlation" between H-1 and H-3 in the absence of genuine coupling between these protons.
f
Relayed COSY
"x
H I ~
COSY
The experiment may be extended to two, three, and more relays and if there is a continuous path of couplings, one can read H-4, H-5 and H-6 from H-1 in a single experiment. These experiments must be performed in sequence (in order of increasing complexity of maps). One must remember however that each of the relays brings an attenuation of the signal and more scans must be recorded for multi-relayed experiments.
3 relays
H1
H2
~'i3
H4
4 relay
H5
J
H6
204 To alleviate sentivity problems, Homo Hartmann-Hahn spectroscopy (HOHAHA or TOCSY, Total Correlation Spectroscopy) may be used to obtain correlations for all the protons of an isolated spin network (71). Glycosides are ideal systems for this experiment since they represent 5, 6 or 7 closed spin systems. This experiment was used
inter alia in the structural elucidation of Gypsophila saponins (60). A variant of this experiment, which will surely become popular once NMR users are able to master it, is 1D-HOHAHA (71, 72). Based on the spinlock principle and difference spectroscopy techniques, 1D-HOHAHA yields a subspectrum of complex spectra each time a proton may be selectively excited (shaped pulses or DANTE). The longer the spin-lock (usually up to 400 ms), the larger the spin system which is explored. Reference (58) presents 1D-HOHAHA spectra showing H-l, H-2 and H-3 (length of the spinlock is not precise). The 1D-HOHAHA signal may be utilised in other experiments such as 1D-HOHAHA-COSY in order to give selective COSY of selected sugars (73). Reference 74 presents impressive results based on selective experiments but with no experimental details unfortunately.
THE OVERHAUSER EFFECTS IN SAPONINS . SPECIFIC PROBLEMS
It is well known that nuclear Overhauser effects arise from dipole-dipole relaxation mechanisms depending on r "6 relationship (75). There are three ways of observing nOes: -
saturate a frequency while measuring the integral of the spectrum. Changes are related to nOes.
- perform a subtraction between spectra or FIDs with the decoupler alternatively on and off (NOEDS) - perform a 2D experiment called NOESY. The first two means of observations are clean and devoid of artefacts (with the exception of Bloch-Seegert effects which are less important at higher magnetic fields). The third technique is sometimes more difficult to interpret, given that strong and, sharp lines often give rise to off-diagonal peaks at the point of encounter of their "ridges": weak nOe cross peaks are not easily distinguished from noise. As an example, reference 76 shows a series of COSY, relayed COSY and NOESY on a five sugar
205 saponin from Blighia welwitschii. |11 I
COC)H
OAc H
AcO ~ ,
H.__OAc I I / CHa---
Oes"'-OAc
AcoLl H
2nee
nOe NOe and rOe correlations used for sequencing of a saponin from Blighia welwitschii.
It is not so well known that nOes also depend on molecular mobility (correlation times ~c) and observation frequency (co) in a 1-cozc fashion. This factor is almost null when one observes molecules of ca 1000 Daltons at 400 MHz. Saponins are thus not ideal molecules for the observation of strong nOes. NOes may be negative even though in some articles the nOe stand for enhancement (29, 30)! The first negative nOes of saponins were recognized as such in a 1989 article by I. Kitagawa et al.(61). The best way to avoid the 1 - ~ c problem (and weak nOes) is to use a rotating frame experiment, for instance ROESY (Rotating frame Overhauser Effect Spectroscopy) also named CAMELSPIN by its inventors (77). The co~c dependence of rOes is complex and one may simply remember that rOes are always positive, never null. The ROESY sequence is similar to the sequence of HOHAHA; the main difference is the power of the spinlock which is generated by a long soft pulse rather than by a WALTZ sequence. In ROESY experiments, rOe cross peaks may be accompanied by Hartmann-Hahn correlations which are easily distinguished by their opposite sign (in phased experiments) (78). There is no doubt that the ROESY experiment will become the best experiment to sequence chains of sugars by 1H NMR. Many examples of this experiment are published in the recent literature, either 1D (58, 79) or 2D (62, 73, 76, 77, 80), even though reference 77 describes a ROESY on a triterpene !
206 OVERHAUSER EFFECTS - SUGAR SEQUENCING AND CONFORMATIONS When an Overhauser effect is detected between an anomeric proton and the proton of another sugar, a partial sequence is immediately deduced. With the spectra at hand, one sees that things are less simple and that generally two or three effects appear instead of a single one. For example in the xylose-arabinose part of acutoside H (79), there are ROEs between xylose H-1 and rhamnose H - 2 , -3 and -4. It is possible to admit, in accordance with the authors, that one of the effects is larger t h a n the other ones but this is not as obvious as stated. Molecular models of rhamnose show t h a t H-2 and H-3 are on the ~ side of the mean plane while H-4 is on the a face. Why does a proton displays proximity effects with partners so far apart ? There is no answer with a single model and one has to admit that there exists at least two conformations around the glycosidic bonds (not to speak of freely rotating systems).
su0a o ~ o],_,
su0ars
~ H . 0H3"7"~O,~' -H
"
Rotating frame Overhauser Effects between H-1 of xylose and H-2, 3 and 4 of the neighbouring rhamnose ! (79) Interestingly, these multiple nOes systems are only met with underivatized saponins. Peracetylated material where steric hindrance is more important do not show these phenomenoms. To further comment on nOes, there is at least one example of a molecule where the expected immediate effect (xyl H-l---> gluc H-2) does not appear and where instead one sees an effect with the second neighbour (xyl H-l---> gluc H-l). H
" o O--0enin sugar ~ Observation of a nOe between anomeric protons in wistariasaponin B1 (63)
207 SUGAR ANOMERIC CONFIGURATION Sugar anomeric configurations were formerly derived from chiroptical properties such as the Klyne-Hudson rule. They are now determined by 1H or 13C NMR spectroscopy. When pyranoses bear a substituent at position 5 (methyl or hydroxymethyl), they are locked in a single conformation with the C-5 substituent equatorial and the anomeric configuration can be determined from H1-H2 coupling constants. The pentoses (arabinose and xylose), which do not bear a C-5 substituent, do not behave so well and they may exist as interconvertible chairs in the 1C 4 and 4C 1 conformation depending on the substitution.
OR 13-D-xylose
RORo~~~,~OR""~"~Q OR RO I
a - L-arabinose
RO
OR
OR
_
R O T O R '
-
k
OR
-_ ,NO 10 4
4C 1
Table 4 lists some values found in the literature for JH1-H2" These 3 j are Karplus dependent and they also vary according to the electronegative character of the oxygen substituents. TABLE 4 . A selection of J H1-H2 found in recent literature. Variability may arise from 1C 4, 4C 1 equilibrium or strong couplings. 3-D-glucopyranose J 5.3 5.4 6 7 7.1 7.3
ref. (19) (19) (19) (12-19-29-43) (65) (19-59)
J 7.5 7.7 7.8 8 8.1 8.3
ref. (29-32) (17-31-32) (17-19-21-32-37-59) (18-28-29-30-36-37-60-31-83) (65) (15)
208 a-L-arabinopyranose <1 2.3 2.8 2.9 3 3.3 4 4.1 4.5 4.9 5 5.2 5.5 5.8 6 6.2 6.5 6.8 6.9 7 7.5 8 8.5
(29) (81) (81) (81) (18-29-30-79-81-83-85) (81) (29-30-85) (59) (81) (81) (84) (81) (17) (81) (25-30-85) (81) (81) (37-81) (31) (28-79) (28) (28-30-36) (36)
~-D-xylopyranose 6 7 7.5 7.6 7.8 8 8.3 8.5
(13-34-61) (13-18-21-35-37-85) (43) (17-31) (21-37) (12-60-62) (15) (36)
~-D-fucopyranose 7 8
(12-21-37-60) (12)
a-L-rhamnopyranose <1 1 1.5 2
(13-18-28-43-85) (12-35-60-61-79-85) (21-37) (12-60-79-85)
~-D-quinovose 3
(13)
~-D-galactopyranose ~-D-apiofuranose 7 7.5 8
(18-30) (30) (30-60-83)
2 3 4
(59) (21-37) (60)
~-D-glucuronic acid 7 7.5 8 8.5
(34) (18) (35-60) (36)
Before translating 3j into configurations, one must make sure that what is measured is really coupling and not splitting. A ~-D-glucose with 3JH1.H2 = 5.4 Hz has been measured under conditions where 5H2 = 4.15 ppm and 5 H3 = 4.12 ppm (at 400 MHz) (19); this is a case of second order effects even though in ABX system the apparent splitting of the X part equals the sum of JAX + JBXWhen the C(2)-O bond is axial (rhamnose and mannose for instance), it is not easy to distinguish 3JHlax.H2 from 3JHleq.H2 because the typical values of these couplings are similar and because of the antiparallelism of the C-O and C-H bonds in the a-L isomer decreases Jax-eq (Booth rule). As a consequence, L-rhamnose is char-
209 acterized as a with signal shapes ranging from singlets to doublets with J = 1.7 Hz (70). In those cases, one relies instead on 13C chemical shifts and 1Jc. H (76). Xylose and arabinose must be analyzed more carefully. In principle, xylose should exist in the 4C 1 chair with all the substituents equatorial but there exist examples where the anomeric effect forces them all in axial positions. Arabinose should always be discussed in terms of chairs equilibrium and the first thorough examination of arabinose in saponins is due to Tori et al. (81). 8C values range from 90.6 to 95.8 ppm, in esters JH1-H2 from 2.3 Hz to 5 Hz and 1JCH from 165 to 177 Hz. Of course these values depend on temperature and solvent. TABLE 5. Typical 1Jc. H values for some sugars in saponins. 3-D-glucopyranose 157.5 159.4 161.8
(19) (19) (19)
~-D-xylopyranose 161 170
(15) (82)
a-L-arabinopyranose 162 169 173
(30) (30-81-85) (81-83)
a-L-rhamnopyranose 168 172 174.9
(21-82) (13) (19)
The interested reader is referred to a series of recent articles of Okabe et al. (8284) where the configurations of xylose and arabinose are examined in detail. In the xylose ester of A s t e r saponin A (82) JH1-H2 is measured equal to 4 Hz and 1JCI_H1 = 170 Hz suggesting a 4C 1 conformation with a-configuration. These values may also fit a ~-linkage in the 1C4 conformer. The only way to decide which configuration is correct, is to analyze the full spin system; this is done by 1D-HOHAHA. The vicinal coupling constants are found equal to 2 Hz (H2-H3), 5 Hz (H3-H4) and 5 and 4 Hz (H4-H5). These values support the ~-configuration in the 1C 4 conformation (with problably a slight contribution of the 4C 1 conformation to account for the somehow large J values). The same analysis and deduction were done on xylose in chrysanthellin (73) and the spin-spin analysis was supported by 1D-HOHAHA and ROESY.
210
H O o H ~ OH ~ /OJ
OH CH3
?H [k'l~O]'~/ HO HO~ ~0 /
HO A HOHO~O"
~~_
....: ~HO H
~HOH
_"f':
OH CH3 H /CH3 0 OH CHa. ..-~-~J4~OH N h a ~ ~ o H OH
HO HO~o"
Glu OH
~ I
I
T (~H3
Xyl
C000~0HXyl ....OH
_
~-D-Xyloses in 1C4 conformation in astersaponin A and chrysanthellin B. A s t e r saponin H (84), the scaberosides (85) and the dubiosides (83) are examples
of arabinoside esters with L-arabinose in a a-configuration and 1C4 conformation. It is worth noting that in all of these examples, arabinose and xylose are linked to hindered C-28 acids and that they are substituted by one or several sugars at C-2. The steric congestion introduced by the equatorial chains at positions -1 and -2 of the sugar are relieved by a full inversion of the chair and a predominant all trans configuration.
211 OH
H O ~ O
H~~o~
~
,o~
OH Aster saponin H d
OH
....
HOHo~,~X~/O OH
o
OH
=: scaberoside B 5
OH
HO"~OH OH
OH 3
~
O.~._
~o_o_~o.~ -o~
o .... ~ O H
HO~~~~ H~--~% O OH dubioside F a-L-Arabinoses in 1C4 conformations
212 CONTRIBUTIONS FROM C-H 2D NMR EXPERIMENTS The domain is concerned with mainly two varieties of CH 2D experiments : the one-bond correlation and the long range (two- or three-bond) correlation. These experiments were formerly run in the so-called normal mode (13C acquisition with 1H modulation) and were named CH COSY (XH CORR or HETCOR) and COLOC (long range). These acronyms are now being replaced with HMQC ( Heteronuclear Multiple Quantum Correlation) and HMBC (Heteronuclear Multiple Bond Correlation) and they are run in the reverse mode (1H acquisition with 13C modulation). The differences between normal and reverse experiments are sensitivity (a factor of 5) and resolution (in the dimension of acquisition ). HMQC (and CH COSY) have been used to assign 1H and 13C spectra or at least to assign one spectrum when the other is known. Two types of determinations have been made with these experiments : the count and assignment of 1H and 13C anomeric signals and therefore the count of the number of sugars. This contribution may seem trivial but in some cases overlap in 1H or 13C NMR spectra makes the determination hazardous. In one of the first examples of the structural determination of a saponin with 2D NMR, CH COSY was used to assign sugar carbon resonances and hence, to deduce branching points with use of glycosylation shifts (59). The main use of HMBC (2j and 3 j CH) is in the determination of glycoside linkages and it is one of the rare experiments which allow passing through osidic bonds. For ether bound sugars, there are two CH correlations going from one sugar to the other; both are Karplus dependent and usually one of them at least, yields a correlation. It must not be forgotten that in the tuning of HMBC (and HMQC) experiments, there is one delay which must be introduced in the pulse sequence and which is J dependant (0.25 j-1 or 0.5 j-l). The adjustment of this delay is crucial for some correlations, such as the COOCH (values must be adjusted stepwise from 70 to 200 ms). When the assignment of a CH direct correlation is ambiguous owing to proton overlap, the correlation may be relayed to another proton which couples to the first. Reference 60 reports the use of such relayed CH experiments in the assignment of the spectra of 7 sugar saponins. Modern variants of these experiments include CH - HOHAHA where the information is relayed to the entire proton system via a spin lock (86) and which constitutes a multiple check of 13C and 1H resonances. The resolution problem in 2D NMR may be alleviated through phased experiments - this must always be considered before running them.
213
CONTRIBUTIONS FROM X-RAY CRYSTALLOGRAPHY AND MOLECULAR MODELING Saponins are usually isolated as white solids with high melting points but it is uncommon to obtain high quality monocrystals suitable for X - ray analysis. To the best of our knowledge, there is only one three sugar saponin whose structure has been determined by crystallography : asiaticoside (64) and a mono glycoside (87). It would be interesting to obtain more saponin atom coordinates to verify if solid state conformation of chains of sugars are similar to the ones deduced from nOe data.
,,0 C '
HO,, HO
OH CH3
O
~
OH
~
o
OH ~
H
0
= CH2OH _
Asiaticoside Molecular modeling is another means of determination of the r and r angles linking two sugars. Some efforts are being made in view of obtaining data on sugar chain conformation and the method is starting to be applied to the saponin field (64, 88). This work is related to the important field of molecular recognition. THE PROBLEM OF ESTER LOCALIZATION Amongst saponins, those with ester functions are the source of redoubtable structural problems. They can be solved chemically, i.e. by derivatization followed by hydrolysis and identification of the fragments. Reduction reactions may be used to complement hydrolysis reactions in the fragmentation of saponins. A reagent of choice is LiA1H 4 which reduces esters into diols; prior protection of alcohol functions as ethers is necessary to avoid solubility problems (5). The chemoselective reduction of acids in the presence of esters by diborane is an excellent method of distinguishing these two functions in multifunctional compounds (55). The effects of esterification on chemical shifts are well known and have been used to localize esters. It is worth noting, given the fact that a effects (+ 2 ppm) and effects (- 2 ppm) have a small amplitude in a congested area of the spectra, that early examples of ester sequencing by NMR relied on 13C chemical shifts. Although popular, the method is relatively inaccurate as witnessed by references 24 and 25 in which acylations of position C-6 of glucose induce a and ~ shifts equal to 1.9 and 3.1 ppm
214 (24), 3.3 and 2.3 ppm (25). 1H acylation shifts on the other hand have a magnitude in the 1 ppm range which displaces protons in relatively unencombered region of the spectra (4.5 - 5 ppm). Pattern recognition suffices then to determine points of acylation of a sugar residue (21). Complications arise when two esters of different acids are present or when acylation occurs on the aglycone. One must then rely on partial hydrolysis. The tactics are examplified by the Entada saponins which contain C 2 and C 10 acids (89). The acetate was selectively removed by 0.025 % K2CO 3 while both acids were removed by 1 % KOH. Comparison of 13C NMR spectra of the parent compound and of derivatives allowed determination of the points of acylation. The dicrotalic (3-hydroxy 3-methyl glutaric acid) esters of the tubeistemosides and related compounds (29-32) provide more complicated examples where the double anchoring of the diacid transforms a prochiral carbon atom into a center of chirality. The only general solution to the problem of ester localisation is the recently introduced 2D NMR experiment HMBC. The nature of the CO 2 groups prevents observation of interproton couplings beyond the C - 0 bond. The problem is made even more difficult by the fact that acid functions on triterpenes are planted on quaternary carbon atoms. Three bond C - H couplings can be observed through ester bonds and this is becoming the technique of choice to determine esters (60, 80, 90). CONCLUSION The recent advances in MS and NMR instrumentation provide chemists with tools which enable them to rapidly solve the structure of complex saponins. This will hopefully open new explorations into the vast structure-activity relationship domain. To those who would be tempted to use their instrumental skills to illuminate the field, it is reminded, as a last remark, that the separation of saponins remains a major and primordial task which brings little glory to the actors.
APPENDIX
In order to illustrate some of the points raised above, the following pages display typical examples of some important NMR experiments presently in use to solve saponin structures. They all concern a saponin from Medicago sativa, isolation of which is reported in reference 55.
215
1D 1H a n d 13C N M R A. 1H N M R of a non derivatized saponin in CD3OD (recorded at 300 MHz). At high field, one distinguishes the methyl resonances. The large p e a k at 4.7 p p m is OH (from solvent a n d saponin). The 5.3 ppm signal is from the t r i t e r p e n e double bond; o t h e r signals in this a r e a are anomeric protons from arabinose (5.7), r h a m n o s e (5), xylose (4.5) a n d glucose (4.4 ppm).
~". . . . .
, ....
f .........
5.1~
~. . . . . . . . . . .
4.6
I''"''
.....
3.~
PPM
I '~" 9" , . . . . . . . .
2.0
1,0
~
o O
H
.
OH
..~
O....~
u,,,,,,,,,J
OOOH
B. The 1H N M R of the same derivatized saponin in CDC13. The p a r t of the spect r u m corresponding to the s u g a r resonances is only shown. The r h a m n o s e H-1 reson a n c e is now buried a m o n g other resonances (5ppm) b u t it can be detected with a COSY e x p e r i m e n t .
|
....
I
'
5
'1~
"'r
(
4
,
1
"~
.....
3
'i
"
216 C. COSY-45 experiment performed on the underivatized saponin (256 experiments of 1K). Arrows point H-1 to H-2 correlations in sugars. Note that the experiment is symmetrized (same information on each side of the diagonal).
L H-2
H-1
9
, ~
,. :"..-
L t.~
2
m
I
~
,
if"
.
I,I
.
Q
3.0
I Rha-5 -> 6 1
~.0
! '
-
,Ir~-~Ir
:IdP i
m
0
'
II
ss
II $
I H-2-> H-1 I S.O
o
$
I H-12-> H-11 I
Oe
4.0
3,~ PPH
2.0
1,0
PH
G.Q
217 D. HOHAHA experiment performed on the underivatized saponin (256 experiments of 2K, spin lock : 250 ms MLEV16 sequence; reverse mode; experiment is phased). Horizontal lines cross the correlation peaks of each sugar. On the column corresponding to rhamnose H-6 are indicated the correlations observed between Me-6, H-l, 2 and 3 (superimposed), H-4 and H-5.
2 0
!
i
t
I
1
II
Iit
8
_
3.~
_
5.~
H-4
,
!
H-2,3 ( o
it
Glc
Xyl Rha o
I
Rha-1
Ara 5.0
~,.0
3, B ?PM
2.0
PPM
218 E. ROESY experiment performed on the underivatized saponin (256 experiments of 2K, spin lock : 200 ms single long pulse generated through the decoupling channel; normal mode with phase coherence between transmitter and decoupler; experiment is phased). Heavy lines correspond to solvent peaks. Roes have phase opposite to the phase of diagonal peaks (here positive and negative levels are plotted). Arrows point correlations which were useful for sequencing. The H-12 to H-18 correlation is indicative of the 18 ~-H configuration.
e
!
. . . . - '"~ i
9
1.8
H-18-> Me-30
i I /
e
,
$.
_
2.0
.
3,0
_
r
H-12-> H-18 O
'
m u
!,',
,
, o
,~
, O
~
"
"
'1~
:'
0
J
"
.
s,
,
,,
,
, / -
~
t
.
Xyl-l->Rha-4
,e
:~
o
'
5.0
,,
0~."'
,
==.n~
OA~II
'.~ ' i~~ ' . ',~
.
t
I
4.0
S.8 u
3,B PPM
2.0
1.0
PPM
219 F. HMQC experiment performed on the derivatized saponin (256 experiments of 2K, reverse mode with decoupling in the carbon dimension; experiment is not phased). The part of the map corresponding to the high field resonances (methyls) is shown on top. At the bottom is the part of the map corresponding to anomeric resonances (plus H,C-3 a n d - 1 2 of the triterpene).
Acetates
~.,, 25
24
.
25. ~1 ."~~e. ~n '' ~, n a - ~-" ~ ~
,
,,
~o
3
~'
-"OR
;
!
-
20
-
30
-
40
.7
m.
29
27~7
,
.
"~" W
I
?
i
S'
COOH
#
'r
"$
. . . . .
f
2
- Ara-1
'
!
!
i
....
l
1
-
90
-
110
Rha-1
9
9
Glc, Xyl-1 ~ ,
0
10
'
I -
0
~
o_ _ _ . -/ ~ j R
,
,,,,29
30.
H
. : ~] ~ ~. ~
-
12
i
5
220 G. HMBC experiment performed on the derivatized saponin (256 experiments of 2K, reverse mode without decoupling in the carbon dimension; experiment is not phased). P a r t of the map corresponding to the high field resonances (methyls) is shown below. The observed correlations allow assignment of most of the carbons of the triterpene (medicagenic acid). Carbon assigned by means of HMQC appear as squares on the formula and carbon assigned by means of HMBC appear as circles.
30
29
o=
C:i~.,,O
.
,=u
I,
Aj
29, 30
.
6 2
16
2:
20 84
tb
o
1
15
o ~
7 ~
I0,,, 9o ~5"
.8
o
14
6
8~
40
14~ 9o 60
Rha-5 *
Rha-4
80
,, 3
100
120
o-
140
13
160
28
180
=
='PM PPM
221 H. HMBC e x p e r i m e n t performed on the derivatized saponin (256 e x p e r i m e n t s of 2K, reverse mode without decoupling in the carbon dimension; e x p e r i m e n t is not phased). P a r t of the m a p corresponding to the low field resonances (sugars) is shown below. The observed correlations allow sequencing of the chains of sugars.
~Ara '
60
H-1 -> Ara-C-5 I 9 Rha H-1 -> Ara-C-2
'
s# ~
,
)'
4
"'
|~I._
~
'
'
)~
' ~
80
Xyl H-1 -> Rha-C-4
,%
Gle H-1 -> C-3 100
:,•
,dl
Xyl H-2-> Xyl-C-1
' '.
]
Glc H-2 -> Glc-C- 1
120
140
160
4 I '
,
~ ~"~ i
!
f
~
9
Ara "H- 1 - > C- 28 .
5.5
i
!
!
!
I
5.~
i
!
!
-I
' f
~.5 PPM
i
i
, i
i
. f.
4.0
!
!
180 !
3
PM
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights rcservcd.
225
The Chemistry of Unusual Terpenoids from the Genus
Eremophila
Emilio L. Ghisalberti
1.
INTRODUCTION The genus Eremophila is one of three genera, together with Myoporum and Bontia, which
constitute the small Myoporaceae family. This family is restricted largely to Australia except for species of Myoporum which extend north to China and Japan and to the islands of the Indian and Pacific Oceans, and Bontia, a monotypic genus found only in the West Indies. The taxonomy of the Myoporaceae has a long and confused history. The genus Myoporum was founded by George Foster in 1786 and Eremophila by Robert Brown in 1810.
Brown
considered EremophiIa to be one of three genera in the order Myoporinae. Ferdinand Mueller, who had published on most of the species then known, found it more and more difficult to maintain the division and, in 1859, reduced them to the single genus Eremophila R. Br. The majority of Australian authors have followed Mueller in adopting the two genera, Myoporum and Eremophila, to embrace all of the Australian Myoporaceae. Members of the EremophiIa genus are confined to Australia, being distributed throughout all mainland states, and total over 200 species. Cytogeographic studies suggest that the genus may have colonised the Australian Eremaea from the west (1). A revision of the taxonomy of Eremophila, made necessary by the many new species individuated and the existence of many species complexes, has been undertaken (2). Species of Eremophila (from the Greek: eremos, desert; phileo, to love) consist of woody shrubs and trees, most exhibiting attractive flowers and some having attractive foliage. The flowers show wide variations, their colour ranging from blue, mauve, pink, red, yellow to white. Since the flowers are displayed for extended periods, many species are highly ornamental and appear to have horticultural potential. The majority of species are essentially plants of the arid land, occurring typically as undershrubs in mulga country where the annual rainfall is below 200 cm, and some can survive periods of up to two years without rain.
There are many adaptations developed by
Eremophila species to cope with the harsh conditions. Several species produce resin on the leaves and stem which, presumably, protects them from the sun and drying winds. Others have a coveting of silver hairs which reflect the sun's rays and reduce water loss from the stomata. The genus is also represented by many species in temperate habitats. In the past, species of Eremophila have been referred to as "poverty bushes", the implication being that they are unpalatable to stock and, in fact, only a few species have found limited use as fodder plants. A number of Eremophila species have been listed as poisonous (3). Although few details are known, records note that 18 species were used by the Aboriginal people for medicinal purposes, E.
alternifolia and E. longifolia being the most widely used. E. freelingii was used for the treatment of headaches, E. elderi for colds and E. gilesii, E. dalyana, E. duttonii for body sores (4).
226
Eremophila species have attracted attention from several quarters in recent years. From the horticultural point of view, they seem to have some merit as ornamental plants adapted to a semi-arid environment. Several Eremophila species are restricted to small areas within agricultural regions and therefore are at serious risk of becoming extinct (2,5). Many species have leaves covered with glandular hair (trichomes) that are considered to be responsible for the production of resin (6). Since many compounds produced by trichomes of flowering plants are biologically active, one hypothesis is that these provide 'a first line of defense' for the plant (7). This suggests that such compounds may exhibit bioactivity as insecticidal, antifungal, antibacterial or antiviral agents. There has been renewed interest in the study of plants which have adapted to arid or semiarid habitats. This interest extends from the individuation of tolerant species for land reclamation to the cultivation of species for the production of renewable materials. Many Eremophila species produce abundant quantities of resin or material (up to 20% of dry weight) extractable with organic solvents. From the chemical point of view the genus produces a diversity of metabolites of which the most studied are the terpenoids. It seems timely to review the various terpenoid compounds produced by this genus and to highlight their uniqueness and the novel chemistry they have disclosed. 2.
SECONDARY METABOLITES The interest in the chemical constituents of Australian native plants arose from casual
observations. The fatal effects of a plant to grazing stock suggested the action of toxins, the scent associated with the leaves and flowers indicated the presence of essential oils, and the accumulation of resin on the leaves or bark pointed to a source of renewable material. All of these factors appear to have stimulated the interest of chemists, pharmacists and agriculturalists of the late 1800s in the plants of the Myoporaceae family.
E. maculata was classified as a stock poison in 1887 and was later (1910) shown to contain a cyanogenetic glycoside, which, in 1929, was identified as prunasin (1)(8). E. mitchelli, first described in 1847 by the explorer and botanist Sir Thomas Mitchell, gained the reputation as one of the strongest scented woods of western New South Wales. The oil derived from the wood was shown (9) in 1932 to contain the sesquiterpene ketone eremophilone, now known to have the structure depicted in 2. The structure, stereochemistry and biogenesis of eremophilone was to attract the attention of some of the foremost organic chemists of the time and to provide new insights into the biosynthesis of terpenes. Several observations by the early European settlers indicated that Aboriginal people used the exudate of many native plants, including Eremophila, as sealants and as natural adhesives for cementing spear heads to spear shafts (10). A number of Eremophila species
OH
0
HO
H
1
2
3
H
227 produce an abundance of resin which is composed mainly of diterpenes. One of these, eremolactone (3), was one of the first diterpenes found (11, 12) which contained a ring system differing from the bi- tri- and tetracyclic systems typical of the then known diterpenes from pine resin. 3.
MONOTERPENES Monoterpenes do not appear to accumulate to any great extent in Eremophila species and their
occurrence has been described for only a few cases. Thus, geranyl acetate (4) was isolated from E.
abietina (13), 1,8-cineole (5) from E. scoparia (14) and E. dalyana (15), (+)-verbenone (6) from E. dempsteri (17) and (+)-fenchone (7) from E. caerulea (14). This last monoterpene is responsible for the scent which characterises this species in its normal habitat. From E. cuneifolia, m- and pcamphorene (8, 9) can be isolated from the steam distillate of the leaf resin (14) and, presumably, arise from dimerisation of the monoterpene myrcene during the isolation. Since most monoterpenes are volatile, they would not be expected to accumulate in the resin exudate of those species which are exposed to strong sunlight and dry winds. Secondly, as these terpenes often occur as complex mixtures, they may easily have been overlooked during attempts to isolate more polar major metabolites.
4.
4
5
7
8
6
9
SESQUITERPENES Sesquiterpenes are well represented in the Eremophila genus although only a few species
accumulate them in significant quantities. With some exceptions, they have been found as the major constituents of the essential oil fraction which typically represents between 1 to 3% of the dried weight of the plant. The Eremophila sesquiterpenes can be divided into two groups. On the one hand, there is a large group of furanosesquiterpenes, and on the other, bi- and tricarbocyclic compounds representing the eudesmane, eremophilane, aromadendrane, cadinane and zizane classes. 4.1
Furanosesquiterpenes A group of sesquiterpenes which can be regarded as oxygenated farnesols characterise the
essential oils of a number of Eremophila and Myoporum species. Although the majority of these
228 furanosesquiterpenes appear to be more characteristic of Myoporum than Eremophila species, many have been included in this discussion. To some extent, this is necessitated by the fact that the taxonomy of the family is under revision. For example, M. deserti, which has been a major source of these compounds was transferred in 1986 to the Eremophila genus by one authority (17). To avoid confusion, the botanical binomials used in the original literature have been maintained. Myoporone would appear to be, structurally, the simplest example of the group of furanosesquiterpenes. Despite this, the determination of its absolute configuration did not prove to be a simple matter. Myoporone was first isolated as an oil, [0t]D 0 ~ in 1957 from the essential oil of
M. bontioides, the only Myoporum species found in Japan (18). The plane structure (10) assigned was based on degradation to the semicarbazone of 6-oxo-4,8-dimethylnonanoic acid (11) which showed no "remarkable depression" in melting point on admixture with a sample of the (+)-11 (19). The bis-dinitrophenylhydrazone derivative of myoporone showed significant optical rotation, [O~]D -28 ~, but apparently showed no depression of melting point on admixture with the same derivative from synthetic (+)-myoporone which was prepared as shown in Scheme 1.
O
O O
HN
/ N
I
CONH2
10
00202H5
~
o
00202H5 ~O
11
H502020
CH2Br
00202H5 O ""-O
H2SO4 O
O
|.
H2(Pd/C)
O
( COfl)2
..dr
O
~
O
2. Cd[CHzCH(CH3)2]2
"--O 10
SCHEME
1
From chiroptical studies, separate groups arrived at conflicting configurational assignments for the single asymmetric centre. Sutherland et al. (20) resolved the ambiguity and showed that (-)myoporone (12) could be degraded to methyl S-(+)-2-methylglutarate (13) by oxidation of the
229 myoporone ozonide with nitric acid followed by methylation of the products. (-)-Myoporone therefore possesses the S-configuration. In a similar way, the co-occurring (-)-dehydromyoporone (14) was shown to have the S-configuration by oxidation of the corresponding ozonide with hydrogen peroxide to yield R-(+)-3-methyladipic acid (15).
H ,.,
0 OMe
0
12
H ,o,
13
0 0 ~H ....
0 14
0
15
Samples of myoporone obtained from different Myoporaceous species show wide variations of optical purity between the two extremes, with M. deserti producing the S-enantiomer and M. bontioides, M. montanum and E. inflata producing the R-enantiomer (14,20,21). A number of Myoporaceae, including E. latrobei, E. maculata and E. miniata produce varying amounts of the two enantiomers. The simplest explanation for these observations invokes the presence of two enzyme systems capable of reducing precursors of myoporone with 6,7- or 7,8-unsaturation. The relative activity of these enzymes in a particular plant would determine the enantiomeric excess. Alternatively, different precursors with 6,7- or 7,8-unsaturation could be reduced by one enzyme of low specificity to give both configurations at C-7 (21). An explanation which attempts to rationalise the racemisation of myoporone during extraction, steam distillation or vacuum fractionation, has been given by Sutherland and Rodwell (21). This mechanism (Scheme 2) relies on the presence of an acid catalyst to replace the proton at the asymmetric centre which is removed by the y-carbonyl group. Abstraction of the unactivated methine proton, however, would seem unlikely even if participation of the ~-carbonyl, leading to the cyclopropanol (16), is invoked. Syntheses of R-(+)-myoporone (ent-12) and R-(+)-10,11-dehydromyoporone (ent-14) have been published recently (22). These rely on the use of methyl S-citronellate as the source of the asymmetric carbon (Scheme 3). Of more general interest and application is an approach (23) which leads to the stereospecific synthesis of 9-dihydromyoporone, which contains two asymmetric centres. This synthesis formally constitutes a synthesis of myoporone. Still and Darst (23) have shown that hydroboration of E-2,6-dimethylhepta-l,4-diene with thexylborane leads, with high diastereoselectivity, to the diastereomeric 1,4-diols (Scheme 4). Appropriate transformation of the syn-diol to the electrophilic iodo compound, followed by alkylation of the ~-keto sulphoxide, gives
230 the coupled product which can be converted to (+)-9-dihydromyoporone. This sequence was employed in the preparation of (-)-9-dihydromyoporone (24). In this approach (Scheme 5), the hydroboration of the chiral ene-ol (17) produced a diastereomeric mixture of diols, with the synisomer (18) predominating (76:24). This was then submitted to a sequence essentially the same as that devised by Still and Darst to produce 7S,9R-(-)-9-dihydromyoporone, a compound previously identified as a stress metabolite of potato.
H26~--H ~ , , .....
H20
oooo
0 +
a~
HO
H26~H ~
"xz----O"3 SCHEME
H20
2
16
- H 03, (CH3)2S
Me02
Me02C
H 1.13-1ithiumfuran 2. t-butyldimethyl silylchloride
=
H 0
ent- 12
0
1. dibah 2. (CH3)2CHCH2Li 3. Bu4N+ FMe02C 4. PDC/CH2CI2
SCHEME
I
~0
3
An interesting chemical and possible biosynthetic link is that between myoporone and the
231
1. I-!-..2
OH ~,.
OH
+
2. NaOOH
OH 1. p-TsC1, C5HsN 2. BuMe2SiCI imidazole, DMF 3. NaI, acetone
O
I
Nail, DMF
O
Cl3i- "
1. Al(Hg), THF-H20 2. HOAc-H20 O
9-DIHYDROMYOPORONE
SCHEME
4
I +
~"SiMe
3
TiCI4
~
,,.
~
Ti(Oi-Pr)4
OH .
"
1 2.1. aq. pccKOH, MeOH
OH 2. NaOOH 18
17 SCHEME
5
monocarbocyclic furanoterpenes represented by the myodesmanones (eg 1 9 ) a n d the myomontanones (eg 21) (25,26). These sesquiterpenes contain skeletons derivable from the two formal intramolecular aldol condensations available to myoporone. The structures and configuration of (-)-myodesmone (19) and its double bond isomer (20), which were interelated by base treatment, were separately elucidated on the basis of spectroscopic evidence and chemical degradation. Thus, ozonolysis of 20 followed by treatment of the ozonide with hydrogen peroxide gave S-(+)-2-methylglutaric acid (12). Similar treatment of 20 yielded the keto acid (22) which was isolated as the ester. Wolff-Kishner reduction of this ester gave R-(+)dihydrocitronellic acid (25). These results showed that C-7 in both compounds have the Sconfiguration. The
cis-arrangement of the cyclopentenyl substituents in 20 was deduced from the
232 fact that on base treatment it provides the more stable trans-isomer. S-(+)-myomontanone (21) is also obtained as a natural product. The structure follows from its preparation from S-(-)-myoporone by treatment with methanolic KOH (26). Appropriate chemical studies have shown that the myomontanones are the kinetic products, and the myodesmones the thermodynamic products, of in vitro base-catalysed cyclisation of myoporone. The expected intermediates in these condensations are the corresponding [3-ketols (eg 23 and 24).
All the four possible diastereoisomers leading to the myodesmoids from S-(-)-
myoporone, and three of the four leading to the myomontanoids from R-(+)-myoporone, have been identified as natural products. As yet, a definitive assignment of their relative stereochemistry has not been achieved. The apparent correlation of the myodesmoid series of compounds with the C7-S configuration and the myomontanoid series with the CT-R-configuration, although tempting, is not valid since at least one ]3-ketol with the myodesmoid CT-R-configuration is known (21).
7
"7
o
o
19
20
0
~ o
21
=
H,,
22
OH
0
23
24 SCHEME
u
6
The finding that the myodesmonoid and the myomontanoid [3-ketols are readily converted to myoporone during distillation or gas chromatography is significant. The widespread occurrence of these carbocyclic sesquiterpenes in members of the Myoporaceae, including E. alternifolia, E. latrobei var glabra and E. scoparia, was previously concealed by this facile ring opening reaction. This raises the question of the status of myoporone as a natural product and the intensive study, extending over 35 years, of its occurrence and chemistry has not yet clarified its role. The
233 production of myoporone of varying enantiomeric excess, the presence of diastereomeric 13-ketol precursors of the myodesmones and myomontanones, and the in vitro and in vivo relationship between all of these compounds are as interesting as they are disturbing. Distinguishing the biogenetic pathways and compounds from the artefacts generated by extraction and isolation is a significant challenge. Of minor chemical and biosynthetic significance are dendrolasin (25), 4S-hydroxydendrolasin (26) and the isomeric dihydrophymaspermones (27) which co-occur in E. rotundifolia. The carbocyclic diene (28), also isolated from this plant, is optically inactive and is probably an artefact of the isolation procedure. A plausible explanation for its formation invokes acid-catalysed electrophilic aromatic substitution of a cationic species (29) derived from a 4-hydroxy derivative of 27, a transformation which has been achieved in the laboratory (27).
25R=H 26 R = OH
28
\
27 (E and Z)
29
Freelingyne (30), isolated from E. freelingii and E. rotundifolia, was the first naturally occurring acetylenic terpenoid to be found (28). Its gross structure was determined largely from 1Hnmr studies, and chemical degradation (Scheme 7) established the substitution around the lactone ring. Thus, hydrogenation gave a saturated 7-1actone (1770 cm -1) which on treatment with phenyl magnesium bromide provided a diol as indicated by the formation of a diacetate. The diol, on treatment with iodine in benzene, afforded a tetrahydrofuran which on reduction with lithium in ammonia yielded a secondary alcohol. Oxidation of this with chromium trioxide produced a ketone whose 1H-nmr spectrum indicated the presence of 4or-methylene protons. Treatment of the diacetate in acetic acid with p-toluenesulphonic acid yielded an unsaturated acetate whose UV spectrum showed the presence of a styrene system and its 1H-nmr spectrum showed a resonance signal attributable to a vinylic methyl group. The outstanding point in the structural determination concerned the stereochemistry of the two trisubstituted double bonds. A tentative assignment (4E,6E-) had been attempted from an interpretation of chemical shift for the C-6 methyl and the magnitude of the H-3, H-5 coupling constant from the low-field 1H-nmr spectrum then available (1966). Synthesis of some model compounds, and of a mixture of isomers from which freelingyne itself could be isolated, supported the 4Z,6E-stereochemistry, but this evidence was not unequivocal (29,30). Finally, the problem was solved by X-ray diffraction methods using a crystal of synthetic freelingyne which had been shown to be identical to the natural compound (31). Two syntheses of freelingyne have been
234 described (29,32), both however lead to the formation of mixtures of double bond isomers. The simpler one is shown in Scheme 8 (32).
7
5
3 Pd/C
I PhMgBr
~
h
~
I2/C6H6
Ph
HO Ph
AcO Ph TsOH
H Ph
H Ph SCHEME
I2
31
Ph3P~_ 0
~
7
I 3-furylcopper
0 R = THP
F----~R= H
30
(2:3) SCHEME
8
It is worthwhile noting that the stereochemistry of the double bond in (31) was assigned from an X-ray study of the 0~-naphthylurethane derivative. Further stereochemical ambiguity arose in the final step. The reaction between the enyal (32) and the phosphorous ylide gave a 2:3 mixture of isomers, with the minor, and lower polarity, isomer being identical with freelingyne (30).
235 Comparative 1H-nmr data alone could not be used to distinguish between the isomers because of the dearth of suitable model compounds (32). Freelingyne undergoes an interesting reaction on treatment with aqueous methanolic sodium hydroxide to give the phenol (33) in which, however, the stereochemistry of the double bond has not been established (33). The lack of a shielded methyl carbon (-15-178) in its 13C-nmr spectrum argues in favour of an E-configuration, a point that could be confirmed by NOE measurements. The formation of 32 can be rationalised as proceeding via the allene (34) to the dioxo acid (35) which then undergoes an intramolecular aldol reaction.
33
34
35
Circumstantial evidence for the biosynthetic events leading to freelingyne comes from three related sesquiterpenes which have been isolated from Eremophila species. Dihydrofreelingyne (36) co-occurs with freelingyne in E. freelingii, has one less degree of unsaturation, and gives the same hydrogenation product (29). Again, the stereochemistry associated with the double bonds could not be assigned unambiguously from nmr data, although the 4Z,6E-configuration seemed likely. A synthesis of the 4Z,6E,8E-isomer established the identity of dihydrofreelingyne. Freelingnite (37), the third furanosesquiterpene present in E. freelingii, was the first example of a 4-alkylbut-2-enolide to be isolated from higher plants (34). The assignment of structure followed from an interpretation of spectral data, but the stereochemistry at C-4 was not established.
36
38
37
39
The isolation of the bis-butenolide (38) from a new species of Eremophila provides a likely precursor of freelingnite and freelingyne (35). Its structure was deduced from spectroscopic data, the stereochemistry of the double bond at C-6 being inferred from the well known shielding effect on a methyl carbon by asyn-alkyl group. The configuration of the single asymmetric carbon was
236 tentatively assigned as R- from a comparison of the optical rotation ([O~]D +21.5 o) with that of the marine furanoterpene (39), ([t~]D -16.8~
of known S-configuration.
It is worth noting that
freelingnite has the same sign for the optical rotation as 38 and, probably, the same configuration. Another furanoterpenoid which has been the subject of protracted structural studies is (-)ngaione (40). This metabolite was first isolated from a New Zealand sample of M. laetum (ngaio tree) in 1925 (36) and, subsequently, from E. latrobei (37). Its structure remained uncertain until it was recognised (38) as the enantiomer of the dextrarotatory ketone (+)-ipomeamarone (41), a phytoalexin formed by the sweet potato under challenge from fungi, insects or mercuric chloride.
40
41
42 H~
-CO2Me
MeO2C's'"OAc 43
41
1. LiNPri2
44
~-
/ ~ OAc~ r . ~ ~ A ~,,.,.~. ~
2. LiAIH4 ~O~i~ t,.) ~ 3. Ac20/pyridine 46
45
,.O3;2. LiAIH4~. 3. Me2CO,p-TsOH 4. PDC
SCHEME 9
/CO2H 1. LiA1H4 J 2. MezCO, HO2C "OH p-TsOH
,..
3. NBS, Ph3P
Br
1.2-methyl-2-1ithio-l,3-dithian lip
2. N-chlorosuccinimide, NaIO4, H20
SCHEME 10
48
Although the structure and relative stereochemistry were easily determined by classical degradative and, later, synthetic studies, the absolute stereochemistry of the two enantiomers proved more challenging. Oxidative ozonolysis of (+)-ipomeamarone afforded a lactone, [C~]D+7.4 ~ (39), which had similar rotation to the same lactone (42) ([Ot]o +6.3 ~ prepared from R-linalool (43) (In this work (39) the sign of the optical rotation quoted was wrong, because, as determined later, the lactone prepared in this way has a negative rotation). On this basis, C-4 of (+)-ipomeamarone was
237 assigned (erroneously) the R-configuration. Since the furyl and methyl substituents had been established on indirect chemical evidence to be trans-, the S-configuration was deduced for C-1. However, Sutherland et al. (40) found that oxidation of isongaione acetate (44), a mixture of stereoisomers, with ozone afforded partially racemized dimethyl S-acetyl malate (45). On the assumption that ring opening occured with retention of configuration at C-l, the S- configuration was assigned to this carbon in (-)-ngaione, clearly in conflict with the enantiomeric relationship established between the two. This group also prepared lactone (ent-42) from (-)-ngaione but found negligible rotation ([~]D +0.05~ a tenuous result in support of their assignment. They were later to report that the optical rotation of the lactone (ent-42) shows a solvent dependency ([~]D 0~ to 7.7~ In the meantime, degradation of (-)-ngaione to the 6-desoxolactone corresponding to ent-42 supported the R-assignment of absolute configuration at C4 (41). Thus (-)-ngaione was shown to have the 1S,4R-configuration and (-)-ipomearone the 1R,4S-configuration.
1. m-CPBA 2. H5IO6 3. NaBH4
EEO
1. EtOCH=CH2, p-TsOH; 2. B2H6, H202 HO
"
3. PDC; 4. MezCHCHzMgBr
K2CrO7, H2SO4 SCHEME
_-
Q ) ~ ~ . . ~ ent-42
11
Final confirmation came independently from the work of Schneider et al. (42). They repeated the preparation of the lactone (42) which showed substantially the same rotation as that reported by Kubota et al. (39). They also degraded (+)-ipomeamarone via 46 to the ketone (47) which retains the configuration of C-1 (Scheme 9), and showed 47 to be enantiomeric to the ketone (48) prepared from S-malic acid (Scheme 10). Finally, the configuration at C-4 was established by synthesis of the laevorotatory lactone (ent-42) (Scheme 11), from R-linalyl acetate. Thus, the structure of ngaione was fully described some 56 years after its isolation. In a variety of E. latrobei, the yield of (-)-ngaione can reach 6.8% of the dry weight of the plant (40). This compound is usually accompanied by greatly differing proportions of the transisomer (-)-epingaione (epimeric at C4). The two isomers have been interrelated by thermal and basecatalysed equilibration.
The corresponding 7,8-dehydro analogue, also known as (-)-10,11-
dehydrongaione, (49) and (-)-10,11-dehydroepingaione (50) occur in E. rotundifolia (27). ,
0
49
50
0
238 Eremoacetal (51), an unique sesquiterpene containing the 2,8-dioxabicyclo[3.2.1]octane skeleton (43), is structurally related to 10,11-dehydrongaione. The structure, suggested from interpretation of spectroscopic data, was confirmed by correlation with 10,11-dehydrongaione (49). Treatment of (51) with aqueous pyridine gave an equilibrium mixture of eremoacetal and the dihydroxyketone (52) (3:7). Selective reduction of the acetate (53) with zinc borohydride provided a mixture of diastereoisomers at C1, the configuration of the 1R-isomer (54) being determined by the Horeau method. Treatment of this isomer with p-toluenesulphonyl chloride in pyridine and removal of the acetate gave the alcohol which was identical to one of the C9-epimers (55) obtained by hydride reduction of 10,11-dehydrongaione (49). Oxidation of 55 with dimethyl sulphoxide-oxalyl chloride yielded 49, thus establishing the structure of eremoacetal. The configuration at C3 in eremoacetal was established as S- by application of the Horeau method on the diol (56), obtained by standard methods from the acetate of 55. It follows that the configuration at C 1 in eremoacetal is R.
0
0
1
3
~
r 52R=H
51
~ 53 R = AC
ZnBH4
-"
l. p-TsC1 2. LiA1H4
55
~
~
54R=Ac
l
~
o~~~,,,.~/ '-.'
56
49 SCHEME
4.2
~0/
12
Bi- and tricarbocyclic sesquiterpenes
Eremophila species elaborate a number of these sesquiterpenes belonging to several different classes. The single most significant Eremophila metabolite, in terms of impact on the development of terpene chemistry and biosynthesis, is eremophilone. Eremophilone (2) was isolated from the wood oil of E. mitcheUi, a tall shrub which attains a height of up to 10 metres and, in spring, bears a profusion of white scented flowers. This species
239 was often confused with sandalwood and was popularly referred to as bastard sandalwood, buddah wood or budtha. Steam distillation of the wood shavings afforded a dark reddish oil (2 to 3%) with a lasting, soft odour and "marked blending and fixative properties" (9). Fractional distillation of this oil indicated the presence of at least three compounds, which, however, could not be separated in this way. A method originally developed for the separation involved dissolution of the oil in hot sodium bisulphite which provided unreacted hydroxydihydroeremophilone (57). Treatment of the bisulphite solution with increasing amounts of NaOH led to the liberation of hydroxyeremophilone (58) and, subsequently, eremophilone. In a second method, a preliminary distillation gave an oil which, when treated with semicarbazide acetate, afforded eremophilone semicarbazone (9). Apart from the original literature (44-48) on the structural elucidation of eremophilone, details of the more relevant results have been published (49,50) and are summarised below. Eremophilone, C15H220, was shown to be laevorotatory and to contain an ct,13-unsaturated ketone from its UV spectrum. Reduction with sodium in alcohol gave an alcohol which on dehydrogenation with selenium afforded 7-isopropyl-1-methylnaphthalene, with loss of a tertiary methyl group, thus establishing its bicyclic skeleton (Scheme 13). The presence of two double bonds was indicated by the formation of a tetrahydroderivative on catalytic hydrogenation. The presence of an isopropenyl group was deduced from the fact that ozonolysis of dihydroeremophilol yielded formaldehyde and a methyl ketone, inferred from a positive iodoform test. The formation of a hydroxymethylene derivative of eremophilone indicated a methylene group o~-to the ketone. On the basis of this evidence the structure 59 was assigned to eremophilone.
H 0
0
2
OH
57
0
58
OH a/EtOH
~
Se
Hz/cat.
HO
SCHEME
13
0
240 However, reaction of tetrahydro-eremophilone with methyl magnesium iodide followed by dehydrogenation gave 7-isopropyl-1,5-dimethylnaphthalene instead of the expected 1,3-dimethyl isomer.
Thus, the partial structure of eremophilone should be represented by 60, with the
remaining methyl group at one of positions 4, 5 or 7. In earlier experiments, it had been observed that the 1,10-epoxide derivative of eremophilone on treatment with sodium acetate in acetic anhydride gave hydroxyeremophilone, thus establishing the same skeleton for the two compounds.
Ozonolysis of the benzoate ester of
hydroxyeremophilone gave a C10 keto acid which on Clemensen reduction yielded a cyclohexyl acetic acid different to 2,2-dimethylcyclohexyl acetic acid, excluding C4 as the location of the tertiary methyl group (Scheme 14). By this time, a careful consideration of the published data had led Robinson to suggest to Simonsen that eremophilone and its congeners had the structure displayed in 60 with the tertiary methyl group located at C5. The suggestion by Robinson was capable of simple proof since, if correct, the acid obtained by ozonolysis should be 1,2-dimethylcyclohexyl acetic acid. This was indeed the case (Scheme 14) and so, after a prolonged series of investigation spanning 7 years, the conclusion reached was that eremophilone had the gross structure depicted in 2.
0
59
60
61
OBz -----
C02H
SCHEME
,.,.....~C02H
14
The determination of the structure of eremophilone occurred at a period when the isoprene rule had been propounded and appeared to have general application. Eremophilone challenged the rule. Ruzicka wrote "Whereas fifteen years earlier such a formula would have been advanced without further comment, in 1937 eremophilone caused a sensation" (51). It was the insight of Sir Robert Robinson that resolved the conflict. He suggested to Simonsen, who was fully aware of the biosynthetic anomaly, that the simplest rationalisation would be to assume that the migration of a methyl group, induced by a carbonium ion, had taken place in a precursor of the normal, and biogenetically regular, eudesmane series (eg. 61). This proposal is of great historical significance in the development of subsequent theories of terpene biosynthesis since it emphasised the possibility of methyl migration, and more generally, skeletal rearrangement in the elaboration of terpenes. Barton
241 was to write "Although the idea that carbon-skeleton re-arrangement can take place during biogenesis is now generally accepted..the suggestion was of startling novelty when it was originally made" (50). As mentioned above, evidence for the gross structure of hydroxyeremophilone (58) came from its synthesis from eremophilone. In addition, 58 showed the properties of an enolic ~diketone, forming both carbonyl derivatives, esters and ethers. Ozonolysis gave acetone, proving the presence of an isopropylidene group. The position of the carbonyl group was established by treatment of the tetrahydro ketone ether, obtained by methylation and hydrogenation, with methyl magnesium iodide. The methyl carbinol thus produced, on dehydrogenation, gave 7-isopropyl-l,6dimethylnaphthalene. This allowed the carbonyl group to be placed at C8 and the hydroxyl at C9 on a skeleton with the same absolute stereochemistry as eremophilone. The third member of this group, hydroxydihydroeremophilone (HDE) (57), was characterised as a keto alcohol containing one double bond in an isopropenyl group.
The
hydrogenation product on reduction with Na amalgam yielded tetrahydroeremophilone, a compound obtained by similar hydrogenation of eremophilone, thus establishing the gross structure of HDE. The elucidation of the stereochemistry of eremophilone is bound up with that of HDE and is summarised below. The relative configuration of HDE was obtained from an X-ray diffraction study which showed the cis-decalin system with the methyl, isopropyl and hydroxyl substituents all in an equatorial arrangement as depicted in 57A (52,53). Since pyrolysis of the acetate of HDE yielded eremophilone, both have the same relative and absolute stereochemistry. Attempts to assign the absolute stereochemistry of eremophilone by chiroptical methods led to contradictory results (5456). The origins of these lay in the use of unsuitable model systems and, therefore, a chemical solution was needed. H
H
O
57
O
OH
57 CH 3
H
H
H
0
62
SCHEME
15
63
The hexalone (62), of known absolute configuration, was converted to the decalone (63) (Scheme 15) which proved identical to that obtained from degradation of hydroxyeremophilone (56). This marked the culmination of the chemical effort on the structure and stereochemistry of
242 eremophilone which had involved several organic chemists, most notably Simonsen, Robinson, Geissman, Klyne and Djerassi. Although Penfold and Simonsen had recognised the presence of other sesquiterpene components in the essential oil of E. mitchelli (9), the lack of suitable separation techniques precluded their isolation at that time. With the application of chromatographic techniques, a number of other eremophilanes were detected in, and isolated from, the wood oil.
The chemical
interrelations of these congeners are shown in Scheme 17. 8o~-Hydroxyeremophilone (64) was converted to eremophilone by calcium/ammonia reduction of its acetate (57). The location of the hydroxyl group and its stereochemistry was indicated by a signal in its 1H-nmr spectrum which appeared as a doublet at ~54.37, J = 12 Hz. 8cx-Hydroxy-7ot(H)-eremophila-l,11-dien-9-one (65) was hydrogenated to give a compound identical to that obtained by similar hydrogenation of HDE (57), thus establishing its stereochemistry. The position of the double bond, not definable by low resolution 1H-nmr spectroscopy, was established by reduction of its acetate and equilibration with sodium methoxide to yield eremophilone (57). As for 64,the stereochemistry of the secondary hydroxyl was evidenced by the coupling constant observed for the methine proton, J = 12.4 Hz.
H
0
0
57
o
66
2 2" acetylate
/
acetylate
67 ~ ' x , x 1. acetylate
65
64 SCHEME
16
243 The structure of isoeremophilone 66 was established by correlation with the compound derived from calcium/ammonia reduction of the acetate of 1,11-diene (58). Alloeremophilone (67), also reported as a minor constituent of E. mitchelli, can be generated from the acetate of HDE (57) by pyrolysis, which also yields equivalent amounts of eremophilone (59). Despite the fact that eremophilone was the first and best known example of these group of sesquiterpenes, no successful synthesis was achieved until 1974. The synthetic challenge lies in establishing the required cis-relationship of the vicinal methyl groups and the isopropenyl group, which is in the thermodynamically less favourable stereochemistry (axial in the chair-chair conformer). The first synthesis of (+)-eremophilone was described by Ziegler and Wender (60). A key step involves a Claisen rearrangement of the vinyl ether (68).
This proceeds without
stereoselectivity to generate a 55:45 mixture of the isomers, that required, 69, being the minor component. In 1975, a completely stereoselective synthesis of (+)-eremophilone was achieved starting from (+)-[3-pinene, loss of optical activity arising in a step in which the cyclobutane ring is opened by acid treatment (61). A number of other syntheses have been described (62-64). The rearrangement of eudesmenes using mineral or Lewis acids have been disappointing from the point of view of achieving a biomimetic synthesis of the eremophilanes. The complexity of the products obtained indicates the great degree of stereochemical control involved in the in vivo conversion (65).
0
0
0 1. A
0 I
~
0 ,,,. J~
2. H+ -
0
+
T 68
69 SCHEME
17
O
~
70
CHO
71
H
72
H
Eremophilane sesquiterpenes are not widely distributed in Eremophila species. In fact, the only other example so far known is the aldehyde (70) which occurs in E. rotundifolia (66). The structure of 70 was deduced largely from 1H- and 13C-nmr spectral data and, the absolute, stereochemistry by correlation with eremophilone. The keto aldehyde was reduced with sodium borohydride-cerium trichloride to give a mixture of diastereomeric diols. The major compound was assigned the 9o~-configuration. The derived diacetate, on treatment with lithium-diethylamine, gave mainly the 9-allylic alcohol which was oxidised to eremophilone with Collin's reagent.
244 Given the relatively rare occurrence of eremophilanes in Eremophila species, it is perhaps of little surprise that compounds displaying the putative precursor skeleton, eudesmane, have also been rarely observed. 13-Eudesmol (71) has been isolated or detected in a number of species, sometimes co-occurring with elemol (72) (14,15,67). Three eudesmane sesquiterpenes are the major components of an ether extract from the leaves of E. scoparia (68). These were easily interelated since the dihydroxy ketone (73), on acetylation, gave the monoacetate (74) and readily epimerised to a mixture of 73 and 75. The trans-,ciseudesmane relationship between these two was evident from nmr data. The tertiary methyl group showed resonance signals at 5H 0.9 and 8C 17.6 in the trans-fused system and 5H 1.2 and 5C 27.4 for the cis-fused system. The dihydroxy ketone was interelated with ptericarpol by the sequence shown in Scheme 18, thus establishing the absolute sterochemistry of the three eudesmane metabolites. Finally, the location and stereochemistry of the secondary hydroxyl group was deduced from the multiplicity of H7 which appeared as a dt at 5 4.0 with one equatorial-axial (J = 5Hz) and two diaxial coupling constants (J = 10Hz).
o
H
I H
73R=H 74 R = Ac
75
H2/PtO2
LiAIH4/THF
I H
I-OH
(.=M
I H
R = Ts
I~oH
I-OH H2]PtO2
HO ....~ 1. H 2 ~ t O 2 0 ~ ~ C o l l i n s ~
II H
i-oH
I
H
SCHEME
LiA1H4/Et20HO =
I'OH
~"OH
18
Another sesquiterpene class represented in the Eremophila genus is that of the bicyclic calamenenes. Two sesquiterpenes, (+)-calamenene (76) and its 7-hydroxy derivative (77), were isolated from E. drummondii (69). At that time, contradictory claims regarding the absolute configuration of the known (-)-calamenene had been made. This confusion was resolved by an
245 X-ray diffraction study of p-bromobenzoate derivative of 77 which established the 1R,4Rconfiguration for the compound. Deoxygenation of 77 yielded (+)-calamenene, identical with the naturally occurring sample, indicating that the two compounds had the same configuration and were enantiomeric with the (-)-calamenenes then known. The diastereoisomeric 1R,4S-calamenene (78) was also prepared from a diterpene of known absolute configuration (see below) (70). In this way, values for the optical rotations of the four stereoisomers of calamenene were obtained. Since the cisand trans-diastereoisomers can be distinguished by nmr spectroscopy, the ratio and chirality of the naturally occurring calamenenes, which often occur as diastereoisomeric mixtures, can now be determined. The calamenene skeleton contains only two asymmetric carbon atoms 1,4-disposed on a tetralin system. Until recently, the synthesis of this skeleton had only been achieved starting from chiral pool agents with the two asymmetric carbon atoms of interest of predetermined configuration (71,72). Recent developments in synthetic methodology now allow the elaboration of the 1,4-transor 1,4-cis- disubstituted system starting from 5-methoxytetralone or its equivalent (73-75).
H
....
76
H
77
78
[o(,]D +37 ~ 41 ~
[OqD-77 ~ 80 ~
Although the resins accumulated by Eremophila species are most often composed of a mixture of diterpenes and flavones, in the case of the taxonomically related E. virgata and E.
interstans the resin is a complex mixture of sesquiterpene acids (76,77). A portion of the methylated mixture, by glc, showed the presence of four compounds, later shown to be the acids 79 (52%), 80 (31%), 81 (8%), and 82 (9%). Although 79 and 80 could be separated with difficulty from 81 and 82 by RP chromatography, they could not be separated from each other either as the acids, methyl esters or acetate derivatives. The spectroscopic properties of 79 and 80 were distinguishable and suggested that the two compounds were diastereomers based on the cadinane skeleton.
H,,
,,
H _-_
H,,
CO2H
H,,
H CO2H
H
,
H,,
H
CO2H
CO2H
~_.,.....~ OH H
79
80
81
82
Treatment of the mixture of the methyl esters of 79 and 80 with sodium methoxide in methanol gave
246 the corresponding ethers (83 and 84) which could now be separated (Scheme 19). The structures assigned to the ethers arose directly from nmr measurements on the two compounds. For example, in 84 the connectivity C4-C5(C10)-C6-C7 on the one hand, and C7-C8-C9-C10-C1-C11 on the other, could easily be established by 2D-nmr techniques. This information, together with NOE measurement, secured the relative stereochemistry of each (Scheme 20). Separate treatment of 83 and 84 with LDA generated the methyl esters of the original acids (Scheme 19). To determine the absolute stereochemistry of 83 and 84 the corresponding nor-ketones were prepared by ozonolysis (Scheme 19). The p-bromobenzenesulphonyl hydrazone derivative of the nor-ketone from 83 gave crystals suitable for X-ray diffraction measurements. These confirmed the stucture assigned to the compound and established the stereochemistry shown. The hydrazone derivative of the nor-ketone derived from 84 proved to be unstable and did not yield crystals suitable for X-ray diffraction. Thus, only the relative stereochemistry of 80 can be depicted.
"" -..-
NaOMe ~.
,,,,,~.i1.",,,,,~--'CO2Me LDA
HH~ .~.,L .E-. "H H~ ' C O H 2I M e ' I
Cir..Co. 79
83
H ~
_
~
NaOMe
e
9~ C 0 2 M
,,,~OH
H ~ ~H, , " "
,
"
LDA
03
H
CO2Me
.
H"" H I
80
H,, H "
~
"
H
CO2Me
-
84 SCHEME
3
12 ' , , ~ 13
o/~,,,,,,/o
H
19 8
G-H3
4
10
~2He"~
H l l C
,.j
,
i z.,,,,~
-
H
3
12
83
SCHEME
20
84
Of the minor metabolites, the epoxide (81) was the more easily purified. The spectroscopic parameters clearly indicated the epoxide structure. Treatment of this with Zn-NaI-NaOAc-acetic
247 acid gave the deoxygenation product and a hydroxy acid whose methyl ester was identical to that obtained from 79. The fourth metabolite (82) was shown to be the 12,13-dihydro derivative of 79. This was achieved by conversion to the corresponding ether which was identical to the minor product obtained from hydrogenation of 83. The taxonomically closely related E. interstans produces a mixture of 79, 80 and the minor metabolite 85, whose structure and stereochemistry were deduced from spectroscopic data (77). The metabolites 79, 81, 82 and 85 belong to the same stereochemical set and represent a new stereochemical class of the cadinene group. Four classes have been identified in this group, based on the nature of the ring fusion and the orientation of the substituent at C4, but ignoring the orientation of the secondary methyl at C1. If the orientation of this methyl is fixed, 13for example, there are eight diastereomeric classes possible.
Of these, four have been named cadinane,
muurolane, bulgarane, and amorphane. The fungal sesquiterpene, panal (86) (78), and the diketone (87) from Eupatorium trapezoideum (79), represent type 5 and 6, respectively, and compounds 79, 81, 82 represent the seventh class. The remaining metabolite (80) could have either the muurolane or ent-type 5 class stereochemistry. In this context it is interesting to note that 85 shows the same stereochemistry as (+)-oplopanone (88), a metabolite produced by E. miniata (35). This compound, together with the calamenenes, represents another case in which Eremophila species elaborate the opposite enantiomer to that obtained from other terrestrial sources.
-
:
Cadinane
=
Muurolane
Bulgarane
SCHEME
,,,,,
"3-.." 85
CO2H
=
_-=
Amorphane
21
H,,
H
H U ~
.p.... L . 4 - J
c.oCO .o 86
H
87
o
88
Another example of this comes from the tricyclic sesquiterpene metabolites isolated from E.
georgei. The steam volatile fraction of an acetone extract of the leaves contained four tricyclic sesquiterpenes displaying the zizaene skeleton (tricyclo[6.2.1.01,5]undecane) (80). The major component (55%) was assigned structure 89 on the basis of spectroscopic data.
This was
248 confirmed by dehydration with phosphorous oxychloride to yield a hydrocarbon identical with the known prezizaene (90), but with opposite sign for the optical rotation. The nor-ketone produced from the latter by oxidative cleavage, on treatment with methyl lithium, regenerated 89 exclusively, securing the stereochemistry at C7. A smaller amount of the rearranged alkene (91) is produced on dehydration of 89. (-)-Prezizaene (90) was also present in the essential oil as a minor metabolite. The second major metabolite (92) (25%) was a secondary alcohol isomeric with 89. Jones oxidation gave the ketone (93), also isolated as one of the minor metabolites, which appeared to lack a-hydrogens. Assessment of the spectroscopic data led to the tentative structure shown in 92. A single crystal X-ray diffraction study of the p-bromobenzoate derivative confirmed the structure and provided the absolute configuration (81). The tricyclo[6.2.1.01,5]undecane skeleton has been the target of synthetic efforts by several groups (summarised in ref. 72,82) and syntheses of (-)prezizanol and (-)-prezizaene have been described (82, 83).
H
_=_H IO 89
H H
H
H 90
91
H
~H OH H
92
93
94
The only other class of sesquiterpenes isolated from Eremophila is the aromadendrane type which is represented by a single compound, (+)-spathulenol (94), found in E.cuneifolia (67), E.
paisley, E. racemosa and E. drummondii var brevis (14). First isolated from Eucalyptus spathulata (84), this compound has since been found to occur in a broad spectrum of plant genera (85). 4.3
Biosynthetic aspects No direct biosynthetic studies have been carried out on the sesquiterpenes from Eremophila.
The difficulty of studying the biosynthesis of terpene metabolites of woody plants is well recognised and, until recently, little was known about the propagation of Eremophila species from seeds. Plant tissue cultures of many Eremophila species have been established, but an exhaustive examination of the metabolites produced by undifferentiated callus tissue failed to reveal the accummulation of terpenoid metabolites (86). The dominant metabolite found to be produced was the phenylpropanoid glycoside verbascoside, not previously detected in Eremophila species. Fatty acids and sterols were also produced. The formation of sterols may explain an earlier observation that tissue cultures of E.
fraserii were capable of metabolising mevalonic acid (87).
249 The biosynthesis of ipomeamarone has been much investigated and the results obtained can be taken to apply to the biosynthesis of ngaione in particular and to the furanosesquiterpenes in general (88). Apart from radiolabelled acetate and mevalonate, farnesol, 9-hydroxyfarnesol, 6-oxodendrolasin and dehydroipomeamarone have been shown to be incorporated into ipomeamarone. Interestingly, in the biosynthesis of ipomeamarone, 6-oxodendrolasin undergoes a 1,3-hydrogen transfer in the isomerisation of the double bond at 3,4- to the 4,5-position. The migrating proton from C-5 is transferred with retention to C-3. The more likely biosynthetic interelationships for the furanosesquiterpenes are illustrated in Scheme 22.
(R=OH
/
R AND S-MYOPORONE
f
f
~
O
H
~
EPINGAIONE
MYODESMOIDS MYOMONTANOIDS SCHEME
22
EREMOACETAL
The furanosesquiterpenes from the Myoporaceae and other genera, Anthanasia, Eumorphia and Gymnopentzia, which are more geographically widespread, have been intensely studied because of their toxicity to grazing stock. They are hepatotoxic compounds and are precursors of a group of low molecular weight 3-substituted furans which exhibit potent lung toxicity (89). For the carbocyclic sesquiterpenes, three main biosynthetic pathways can be distinguished (Schemes 23 and 24). The first presumably arises from the germacrene carbonium ion derived from
250 cyclization of 2E,6E-farnesyl pyrophosphate. Further cyclization of this putative ion, followed by the appropriate rearrangement, allows the formation of the eudesmanes, eremophilanes and aromadendranes to be rationalised. The biosynthesis of eremophilane sesquiterpenes has attracted considerable attention. Although these studies involve systems other than Eremophila, they provide insights into the likely mode of formation of these carbobicyclic sesquiterpenes. For example, using cell free extracts of Aspergilleus terreus the biosynthesis of (+)-aristolochene, a 7-epi-eremophilene, has been shown to involve the cyclisation of 2E,6E-farnesyl pyrophosphate to the macrocyclic germacratriene. Proton-initiated cyclisation leads to the eudesmane cation which can rearrange by sequential 1,2-hydride and methyl migrations followed by deprotonation. The hydrogen lost is that originally syn- to the migrating methyl group (90). The second pathway involves the intermediacy of a carbocation derived from 2Z,6E-farnesyl pyrophosphate. Following a 1,3-hydride shift, the resulting allylic carbocation can cyclize to the decalin ring system which characterises the calamenenes (76,77) and the cadinenes (79-82,85). The derivation of oplopanone (88) by ring contraction of the cadinene system seems likely.
1.
=
+
EUDESMANES
H
EREMOPHILANES
,
" 11
SCHEME
76
23
88
251 For the biosynthesis of the calamanenes, some information is available from the studies carried out on the cadalene dimer, gossypol. In this case, it has been shown that the bicarbocyclic ring system is assembled by cyclisation of 2Z,6E-famesyl pyrophosphate (91). Of the carbocyclic sesquiterpenes found in Eremophila, the most elaborate are the tetracyclic ent-zizaenes (89,90,92,93). One possible sequence for the assembly of such a nucleus is given in Scheme 24. Cyclization of 2E,6Z-farnesyl pyrophosphate between the 1 and 6 position would generate the bisabolonium cation equivalent which, after a hydride shift, could further cyclize to a spiro carbocation. The tricyclic ring system can then be assembled by invoking alkylation of the cyclohexene double bond. The tertiary carbocation generated incorporates a bicyclo[3.2.1]octane system which can rearrange in two ways leading to the tricyclic sesquiterpenes metabolites found in
Eremophila. From a consideration of the steps involved, it seems likely that more sesquiterpene skeletons remain to be found in Eremophila species. In principle, these could reflect branching points along the pathway leading to the zizaenes and result from neutralization of the putative carbocations in the sequence.
r o
<
a
(
/
H
/
H
~
H
/
/ 89
H
/ 92
SCHEME
24
Sesesss ~ l ~
252 5.
DITERPENES Research into the resin constituents of Eremophila species originated as an extension of the
interest in the components of the resins from Western Australian desert-adapted plants. White (92) and Jefferies (93) carried out major surveys of the chemotaxonomy of the local Euphorbiaceae which, in the main, were shown to be excellent sources of ent-labdane, beyerane and kaurane diterpenes and flavones. Initial investigations of apparently similar resins from Eremophila species were restricted by the problem of separating what were often complex mixtures. Later developments in analytical and preparative methods provided suitable tools for the separation and purification of these mixtures and, in combination with advances in physical methods, allowed access to the unusual diterpenoids elaborated by this genus. An indication that Eremophila species might be a source of novel diterpenes was given by the isolation of eremolactone (3) from E. fraseri and E.freelingii in 1962 (11,12). Subsequent investigations have amply demonstrated this and the sequel describes the diverse structural and stereochemical types of diterpenes so far isolated from this genus. These are described in order of perceived biogenetic complexity. 5.1
Acyclic diterpene From a biosynthetic perspective, the least elaborate of these diterpenes are the acyclic
metabolites which have been isolated from varieties of E. glutinosa, E. exilifolia (94,95) and E.
petrophila (14). These diterpenes occur as complex mixtures of carboxylic acids, separable only with difficulty, and often require methylation to facilitate their separation. The first example isolated (94) was assigned the structure shown in 95 on the following evidence.
Reduction of the
corresponding dimethyl ester with aluminium hydride, followed by hydrogenolysis/hydrogenation of the triol obtained, yielded phytane, thus establishing the terpenoid nature of 95. The location of the functional groups and double bonds was determined from nmr and mass spectral data.
HO2Q
~CO2H/7~--002H\\_ _ ~/ CO2H \ CH2OH :3~ / '
Ho c-#
95
96
R
/
//
\--// CO2H
""
Ho c-/
97 R = C H O 98 R = C O O H
The stereochemistry of the internal double bonds was inferred, principally, from the absence of signals for allylic methylene carbons near 8C 40, indicating that these carbons, associated with each double bond, were cis-related. The configurations of the remaining double bonds were assigned by comparison of the 13C-nmr spectral parameters with those of model systems. In these studies, the remarkable shielding effect of carbon atoms which are cis-arranged about a double bond was evident. Allylic methylene carbons resonated near 5(2 32 and methyl carbons below 20, compared to values >40 and >23 ppm, respectively, for trans-disposed carbons. Interestingly, the triacid (96) displaying the double bond at C2 in the E-configuration was produced by E. glutinosa, and the
253 corresponding Z-isomer by E. exilifolia. Of the nine acyclic diterpenes isolated, only two (97,98) contained an asymmetric carbon atom. The S-configuration at this centre was established by oxidative degradation to S-3-methyl adipic acid. The occurrence of these acyclic diterpenes is interesting on two counts. Geranylgeraniol, a central intermediate in the biosynthesis of diterpenes, and oxygenated metabolites derived from it are rarely significant components of terrestrial plants, although several examples have recently been found in algal species. In most cases, where internal double bonds occur in these compounds, they have been shown, or assumed, to be trans- with respect to the carbon chain. The significance of the
Eremophila acyclic diterpenes lies in the presence of two internal cis-double bonds. Since in every case, only one of the two double bonds is conjugated to a formyl or a carboxylic acid group, this suggests that inversion from the biosynthetically expected trans-configuration is not necessarily mediated by these groups. This is of some significance in connection with the occurrence in
Eremophila species of the unique cembranoids with cis-double bonds. 5.2
Monocyclic diterpenes Cembranoids: The first macrocyclic diterpene isolated from an Eremophila species was the
triol (99) produced by E. clarkei (96). The resin obtained from this plant was a complex mixture of compounds from which the crystalline triol could be obtained with difficulty from the neutral fraction. Larger quantities of triol were available after methylation and lithium aluminium hydride reduction of the acidic fraction. The plane structure of 99 was established by conversion of the triol to a crystalline stereoisomer of cembrane (100) as shown in Scheme 25. The two dihydrotriols (101 and 102) obtained in this sequence also served to prove the location of the hydroxymethylene groups on the cembrane skeleton. Since both 101 and 102 are optically active, the symmetrical
4,12-cis arrangement of the primary alcohol groups can be excluded. Furthermore, the 4,12-transdisposition of these groups can also be excluded since elimination of the asymmetry at C1 leads to two different olefins (103 and 104). The location of the double bond was established by dehydration of the hydroxy-ene (105), followed by ozonolysis, to give a compound which behaved as a 1,4-dione (106). Confirmation for the structure assigned and evidence for the relative stereochemistry came from an X-ray diffraction study on the triol (97). The absolute configuration was not determined for 99 but, in view of later results on Eremophila cembranes, that shown can confidently be predicted. The cembradiene hydroxy ether (107) was isolated as the major metabolite of the neutral portion of the resin from E. georgei (98). This species is a highly variable one which is widely distributed in the Murchison district of Western Australia. To date, six individual chemovarieties have been distinguished (14) although members of the complex are morphologically very similar. The gross structure of the new cembrenoid (107) was established by conversion to a mixture of cembrane diastereoisomers. Dehydration of the tertiary hydroxyl group gave a separable mixture of triene isomers, one of which (A4,20) was converted to the mixture of hydrocarbons as shown in Scheme 26. The structure and relative stereochemistry, including the configuration of the double bonds, but excluding that of the tertiary hydroxyl at C4, were determined from analysis of spectral parameters. These conclusions were supported by an X-ray diffraction study of the hydroxy ether (107) which also revealed the stereochemistry at C4 (99). The absolute stereochemistry was
254
Hz/Pt iiii
CH2OH
\ HOHzC
'"CH2OH+ ~ / 0 ~
99
~"
101
,#
HOH2C I 1. Benzoylate I 2. POCI3 3. Hydrolysis
1. TsC1 2. LiA1H4
102 / 1. Acetylate ~ 2. POCI3 3. Hydrolysis
HOH2C
~
,,,CH2OH
\
,,,CH2OH ~ " ' C H e O H ]101
IIIII
g-"
105 POCl 3
HOH2C""" + 1 0 3
HOHzg _
,•, •, ,CH20
104
H
o
i,
H ~ HOH2d
"~11'H2~2tT C1 ~iA1H4
~----~ .....
g'"
106
/ SCHEME
25
1 0 0(CEMBRANE)
determined by degradation of the hydroxy ether (107)to R-(-)-homoterpenyl methyl ketone (Scheme 26). The spectral information accumulated from the foregoing examples made easier the recognition of cembrane compounds in extracts of Eremophila species. Subsequent investigation uncovered several other cembrane derivatives (13,16). Of these, two merit special attention. E. dempsteri produces the triene (108) in which all double bonds were shown to have the cisconfiguration (with respect to the carbon ring) (16). This compound represents a biosynthetic link between the cembranes and the acyclic diterpenes with an all cis-arrangement of double bonds. The ether acid (109) from E. granitica shows an ether linkage with a configuration at C3 different to that observed in 99, perhaps reflecting the intermediacy of acyclic precursors with different double bond geometries (13). This is also consistent with the situation observed for the acyclic diterpenes in which the double bond at C2-C3 can have either configuration.
255
POCI3=
,,,,, 107
/
OH
CrO3 H2SO4
~ ~
~)SH20 ~)SH
OsO4 = Nal O4
/
x ~ 2H2/Pd/C . POCI3 3. H2/Pd/C
'~
~~
CEMBRANE
==0
H O
SCHEME
26
H
15~"~3Z _ ~ ~ \ " H CO2H
108
109
110
The availability of the triene (108) prompted the preparation of all cis-cembrene A (110) which was achieved by standard methods.
Thus, five of the eight possible 3,7,11,15-
cembratetraenes have been prepared. Of these, the 3E,7E,11E,15- and the 3Z,7E,11E,15-isomers have been found as natural products and prepared by synthesis.
The 3E,7Z,11E,15-,
3E,7E,11Z,15- and the 3Z,7Z,11Z,15-isomers have been synthesized only (16). Bisabolene diterpenes"
Although this class of diterpenes was the last one to be
discovered in Eremophila, its existence was predicted because of the occurrence of bicyclic and tricyclic diterpenes in this genus which, formally at least, could be derived from further cyclization of a putative intermediate containing this ring system (100). The resin of E. foliosissima contained three carboxylic acids as the major metabolites which could be isolated more conveniently as their methyl ester derivatives. The major component was the unstable acetoxy ester (111) which displayed an allylic tertiary alcohol. Transesterification of the acetate with NaOMe, followed by heating the derived hydroxy compound in a solution of CDC13, led to the formation of the tetrahydropyran (112). This compound proved identical to the second major metabolite isolated from the methylated acidic fraction. This interrelationship was fortunate
256 because, whereas 111 was suitable for degradative elucidation of the structure of the two compounds, the more rigid 112 was instrumental in establishing the relative stereochemistry. Application of modern nmr spectroscopic measurements provided substantial support for structure 111 as a working hypothesis. However, apart from the E-configuration of the conjugated double bond, no stereochemical inferences could be made from these measurements. The presence of a tertiary allylic hydroxyl group was evidenced when PCC oxidation yielded a cyclohexenone (113) and acid-catalysed dehydration of 111, in the presence of oxygen, provided the 1,4disubstituted benzene (114). Degradation of 111 by the standard methods shown in Scheme 27 to the known 3-(4-methylphenyl)-butanoic acid yielded the R-enantiomer (115). This sequence also allowed the generation of the secondary alcohol (116) which, by application of Horeau's method, was shown to have the R-configuration at C9. NOE measurements on the tetrahydropyran (112) showed interactions between the protons at C1, C2, C7 and C9 which, therefore, must be in a
syn-
arrangement. The only outstanding point of stereochemistry refers to the configuration at C1 in 111. Since the hydroxyl group could be esterified by phenylbutyric anhydride, it was tentatively assigned the
pseudo-equatorialdisposition in which the two alkyl groups in the cis-relationship.
1,4-disubstituted
cyclohexene ring are in a
H,,, H
H,,
__
H,,
Is
" i~I
MeO2C 112
p-TsOHJ o2/ /
H,,,
1.03
c
MeO2C
.
2. Zn
'''OH
HO~o ~ 115
II OAc
/
MeO2CJ~~~~<~
~ 111
113
....
J..
H3 CH,,,~ , ,
~ LiAIH4
114 H,,,
H,,,, H
PCC ~
1.NaOMe H , , , , ~ ~2.& ~OAc~
MeO2C
H,,
H
1. NalO 4
<2.Jones
H.... ~ H , , ~
S
p-TsCI 116 2. LiAIH4
HOH2C
SCHEME
27
The gross structure of the methyl ester derivative of the third metabolite was taken to be that shown in 117 from extensive nmr spectral measurements. Evidence for the 8-oxy-conjugated ester functionality was obtained when LDA treatment yielded the E,E-conjugated diene (118). Further evidence for the structure came from degradation of 117 (Scheme 28). The 1-(1-methylcyclohex-3en-l-yl)-ethanol (119) obtained was shown to be enantiomeric to a sample of the 1R,l'R-isomer
257
H,,,
MeO2C~ v " ' ~ ~ ~~ 117
H_ .3
MeO2C 118
Li/NH3
0~,.~H,,, H R = SitBuMe2 PDC/CH2CI2 ROH2C#-~v'*"~~ mCPBA
R=H H.... H
(+)-LIMONENE
2. KOH,MeOH O
119 SCHEME
(~'~
1. Zn, Nal
28
prepared from (+)-limonene by a stereochemically unambiguous route, thus securing the stereochemistry at C1 and C7.
A NOESY spectrum of 117 showed two sets of significant
interactions. The protons at C1, C2 and C7 were syn-related, as were the hydrogen at C8 and the methyl protons at C19. 5.3
Bicarbocyclic Diterpenes Serrulatanes:
The most common diterpene skeleton found in Eremophila is that
represented by the bicarbocyclic serrulatane group of compounds (101-104). Essentially, these are isoprenologues of the calamenene sesquiterpenes in which the dimethylallyl derived unit of the acyclic precursor does not participate in cyclisation. The first example of this skeleton was found in dihydroxyserrulatic acid (120) which was isolated from E. serrulata (101). As observed for the calamenenes, the presence of the serrulatane skeleton can be inferred from nmr spectroscopic data.
20
OH
1
8
OCH3
CO2H
H3
18 H 120
CH2QH
121
122
258 Apart from the aromatic ring protons, the 1H-nmr spectra clearly show signals for benzylic protons at 8 2.6 (C4, C1) and, if a peri-hydroxyl group is present, 8 3.2 for the C1 methine. Furthermore, the mass spectra often show a distinctive loss of the side chain, by cleavage of the C4C11 benzylic bond, allowing inferences to be made regarding the functional groups contained on it. The structural elucidation of dihydroxyserrulatic acid (120) required evidence for the tetralin ring and the relative arrangement of the substituents around it. To this end, the dimethoxy derivative was deoxygenated at C16 and the resulting compound was dehydrogenated with DDQ to provide a naphthalene (121). The orientation of substituents on the aromatic ring and the stereochemistry of the side chain double bond was inferred from spectroscopic analysis. Confirmation of the structure and the relative stereochemistry of the three asymmetric carbons was obtained by an X-ray diffraction study of 120. The absolute stereochemistry was determined by conversion of the methoxy naphthalene (121) to S-(-)-4-(l',5'-dimethylhexyl)- 1,6-dimethylnaphthalene (122) which proved identical to, except for the sign of optical rotation, a synthetic sample of the R-enantiomer (123) prepared from R-citronellal (Scheme 29) (102).
O CHO 9 MVK
_-
1. M e M g I
9NaOAc AcOH
2. PCC
v
H
O
H
1. H2/Pd/C 2. Pyrrolidine 3. MVK 4. KOH
1. Pd/C/ cinnamic acid 2. DDQ
MeMgI 0
-Z.
H 123
SCHEME
29
Many other serrulatanes have been isolated from different species of Eremophila (77,103105). A number contain an asymmetric carbon atom at C15 (eg 124), the configuration of which was determined by X-ray diffraction methods (106).
Apart from the oxygenation sites in
dihydroxyserrulatic acid, others are observed at C2, C5, C7, C13, C18 and C20. The structurally more interesting serrulatanes include biflorin (125) from E. latrobei (104), an antibiotic originally
259
I
~OH 9
~
OH
124
0
0
125
OH
OH ~
126
O l W
0
127
isolated from Capraria biflora (Scrophulariaceae) (107). From a phytochemical perspectives, this is an interesting observation since the two families, Myoporaceae and Scrophulariaceae, have been considered to be closely related botanically. Biflorin represents the first case of a diterpene common to the two. Interestingly, the methyl ester derivative of the serrulatane (126), from E. drummondii var brevis, on treatment with Fremy's salt yielded the ortho-quinone analogue of biflorin (127). The dihydroquinone corresponding to 127 was also isolated from the same plant (104). Total syntheses of the serrulatane skeleton have recently been achieved.
This is a
consequence of two apparently unrelated factors. Firstly, the development of synthetic methodology utilising (q6-arene) chromium complexes, which allowed the regio- and stereoselective synthesis of the calamenenes, has been adapted for the synthesis of (+)-dihydroxyserrulatic acid (74). The introduction of the extra asymmetric carbon atom at C11 can be achieved with reasonable selectivity. Secondly, the recent discovery of the pseudopterins (128) and their seco-analogues (129), classes of compounds with potent anti-inflammatory and analgesic activities from the marine sea whip
Pseudopterogorgia elisabethae, has made these compounds rewarding synthetic targets (108,109). The secopseudopterosin skeleton is the 11-epimer of the serrulatane or ent-serrulatane skeleton. Since the related tricyclic pseudopterosins are claimed to occur in either enantiomeric form from different sources, the same can be anticipated for their seco-counterparts.
Interestingly, the
cycloserrulatane (130), which is essentially a 3-epi-pseudopterosin, has recently been found in E.
serrulata, the original source of the serrulatane diterpenes (110).
/
128
O-13-L-Fucose OH
O-Arabinose ~.OH
OH3
/7*'CH3
1
129
OH CO2
130
All serrulatanes so far described contain the 1R,4S-stereochemistry in contrast to the calamenenes (1R,4R) from EremophiIa species with which, in at least two cases, they co-occur.
260
03, Zn
11113 Q
~ _ -
1. LiA1H4_ I H II
H
:HO
~1 ~/ H
"CH2OH
1. PDC 2. PhMgBr 3. PDC
1. BBr3 C
I
hv, benzene ~
2. 5-chloro1-phenyl tetrazole 3. H2/Pd/C SCHEME
H3
H3 -CH 3
~,/V ~
H
P
h
O
30
The availability of the serrulatanes made a correlation with the 1R,4S-calamanenes possible and this was achieved by stepwise removal of the side chain in the former as shown in Scheme 30 (70). This sequence was profitably used for the correlation of other diterpene skeletons with their sesquiterpenoid counterparts (vide infra). Spiro diterpenes: E. viscida, as the name implies, is a species whose branches and leaves carry a sticky resin and this appearance gave rise to the vernacular name of varnish bush. The resin consists largely of material extractable into base. The major components are two diterpene acids (131,132) which disclosed a new diterpene skeleton (111). Although a small amount of the crystalline dihydroxy acid (131) could be obtained after tedious separation, as often found with
Eremophila resins, the process was simplified by methylation of the crude acidic fraction and separation of the methyl esters. The presence of a 2,3,5,5-tetrasusbtituted cyclohexene in 131 was inferred from nmdr experiments which revealed all the protons on this ring. In the mass spectrum, a peak attributable to loss of C8H170 circumstantially indicated a side chain containing the primary alcohol group. With this in mind, interpretation of the 1H- and 13C-nmr spectra required the allocation of a secondary methyl group and two methylene carbons. These could be conveniently accomodated by assuming that a cyclopentane ring was part of the spiro-system. Confirmation of the structure proposed, the location of the primary hydroxyl group and the relative stereochemistry of 131 was provided by an X-ray diffraction study which showed that the viscidane skeleton was isoprenologous with tx-acorene (ent-133). For the determination of the absolute configuration, a correlation with this sesquiterpene ring system seemed convenient using the method developed for the serrulatane-calamenene correlation. To this end, the methyl ester was
261 -
pH
pH /~ CO2H
"-
O2R
H'
q,
f
lilt
- I ~
CH2OH
I:!
131
~ :
132
pH CO2CH3
,f-F! v "CHO
~
pH A/~CH2OH
"~),._--"{ CH20H
-CH20 H
"CHOH
H~. v H 135
134
133
-
136
.
~ ~ ~~ v 137
g'~
OH 3
"CH2OH
~
OH3 CH2OH
138
converted to the trisnor-aldehyde (134). Lithium aluminium hydride reduction led to the expected triol (135) but also provided equivalent amounts of the diol (136) and the alcohol (137) resulting from allylic deoxygenation (67). The formation of the deoxygenated compounds, although not unexpected, merited further investigation since only one of the two double bond isomer anticipated from literature precedents was obtained. In fact, treatment of the triacetate of the triol (135) with lithium-ammonia yielded the fully deoxygenated products (137,138) as a mixture of double bond isomers which were distinguishable by ]3C-nmr. To determine the position of the double bond in 136, it was decided to generate the isomer in which the double bond had been transposed, using 131 as the model system (Scheme 31). Hydrogenation of the methyl ester gave the trans-isomer whose stereochemistry was deduced from 1H-nmr analysis of the corresponding acetate which showed J4,5 = 10Hz. Elimination of the secondary acetate with KtOBu and reduction of the conjugated ester with A1H3 yielded the allylic alcohol identical to that obtained by similar reduction of 131. It is noteworthy that the deoxygenation reaction requires the presence of an o~,]3-unsaturated ester. When the system represented in 135 is subjected to the same reduction conditions, no reaction is observed. Since the deoxygenation involved in the conversion of 134 to 136 and 137 occurs with complete isomerisation of the double bond, this meant that the correlation target now had to be [3acoradiene. The successful sequence undertaken is as shown in Scheme 32. In the event, the
262
Methyl ester of
131
H2/Pd/C
,OR
@
21LiAIH4/A1C~ H ....
M ....
I ~ c H 2 O R H
" I ~ c H 2 O H
H
H
H
R=H
c
R=Ac
SCHEME
31
sequiterpene derived from the viscidane skeleton was shown to be the enantiomer (139) of the known (+)-~-acoradiene. Mutatis mutandis, it follows that the absolute configuration of the viscidane diterpenes is enantiomeric to that of the naturally occurring tx-acoradiene (ent-133) (67).
:
--
CH 3 ~.- - v H
C
""
PhMgBr
"R
J"~"-.~-"'~..F OH
"A-
37 R = CH2OH
CH 3
1
PDC
R = CHO
139
CH 3
Ph
CH2C12
hv
~
300nm
O
CH3
Ph SCHEME
32
A number of other viscidanes have been isolated and all contain oxygenation at C5 in the cyclohexene ring (112,113). One such compound (140) provided some interesting chemistry. On storage or on heating under reduced pressure, in attempts to obtain a sample for elemental analysis, the ether (141) was produced (112). From molecular models, it was clear that the alternative (142) could not be excluded and, since a distinction on spectroscopic grounds did not appear unambiguous, a chemical proof for the structure was investigated. Conjugate reduction of the ester derivative of 141 with NaBH4 gave, inter alia, the tetrahydroester (143) which, on treatment with LDA, produced the A4,5-conjugated ester (144) (Scheme 33). A sample for comparison was
263 prepared from the methyl ester of 140. This, on hydrogenation, gave products arising from hydrogenolysis and the tetrahydro derivative (145). Protection of the primary hydroxyl group with t-butyldimethylchlorosilane, acetylation of the secondary hydroxyl group, elimination with KOtBu and deprotection afforded a sample identical to 144 (Scheme 34). It can be concluded that the formation of 141 proceeds by an SN2-type mechanism rather than an SN2'. As yet, no synthesis of the viscidane skeleton has been reported. However several synthetic approaches to the various acorane sesquiterpenes have been investigated (72). The extension of these methods to the elaboration of a viscidane requires sterecontrol in the introduction of the asymmetric centre at C 11.
CH2OH -:
:-'-~-O
,,,OH
~ ' / ~ H""~ I '
.~
A
CO2H
,a
OR
"..-'~ O
C02H
CO2H
=
"
140
141
A
" ~
142
R = H R = OH 3
NaBaB~.~
0
._4 .....
.
c.
H~CO2CH3
LDA
o.
0020H3
_~
/ 143
SCHEME
HOH2C
33
ButMe2SiO -=
,,,,,OAc
1.H2/Pd
H3 2.ButMe2SiCl
1. KtOBu
CO20H3 2. Bu4NF ---- 1 4 4
3. Ac20 /145 SCHEME
5.4
34
Tricyclic diterpenes Decipianes: The unique decipianes, containing the tricyclo[5,3,1,05, ll]undecane skeleton,
were first isolated from the widespread sticky shrub, E. decipiens (114-117). The resin was a complex mixture of diterpenes from which the triol (146) and the two acids (147,148) could be isolated. These compounds were interelated by standard transformations (Scheme 35) allowing the
264 independent use of each compound in determining the skeletal structure of the decipianes. The three compounds isolated represented only a small portion of the resin and, although no other compounds were completely separated from this mixture, there were strong spectroscopic and chromatographic indications for the presence of more than ten related compounds which appeared to arise from permutation of the oxidation level of the three pendant groups in the triol (146). The neutral portion of the extract could be simplified by acetylation followed by reduction with Li/NH3 and then Pdcatalysed hydrogenation. Alumina chromatography of the products thus formed gave useful quantities of decipi-14-ene (149), the alcohol (150) and the ether (151). This last compound gave a six-membered ring lactone on oxidation with RuO4. This information, combined with nmr and ms
2O =
1
[
HO
-
-6
14
CH2OH
%,
-"
1. Acet, la e
2. Hz/Pd/C
H CH3
I'~CH2OH
"'CH2R R = OAc 149 R = H 150 R = O H
146 N2
4. H2/Pd]C H
H
" ,H
HO2C
I~
147 R = O H 148 R = H
CH 3
H'CH2R ~'CH20H SCHEME
0
151
.....J_.~OH 35
evidence, implicated the presence of a six-carbon atom side chain linked to a tetrasubstituted carbon in the decipianes. Exploration of the tricyclic nucleus was initiated by oxidative cleavage of the cyclohexene double bond in the monoacetate (Scheme 36). The series of reactions shown served to prove the relationship of the 14-ene to the tetrasubstituted carbon atom. A similar oxidative sequence on decipi-14-ene (149) was employed to generate the ketone (152), which was shown to be a cyclobutanone with a single proton or- to the carbonyl group. Evidence for the presence of a cyclohexene ring, suggested from nmdr studies, was obtained by formation of the cyclohexenone
265
LH~
O2Me
H CO2Me
-,H O
1.0 3, Z n 2. CH2N2
i_i+
"J
O
"" JL, , ~ H CH3
" '....H CH3
%CH2OAc
H2OAc
I m_CPA
I p-TsOH
~~__~ ? O2Me
~ . , . 7 9 O2Me
OH , H O ~Jones R R=H 152
~~___=CO2Me
,,," .... OH2.
CH2N2
SCHEME
H
-
CrO3.PY2 = k,~ '-' ..,.~-
..~
"",H H OAc
36
H __o
i
"
1. Li/NH3 2. m-CPA =
/O ,H
o
%CH2OAc I HO
153
121CH2N2
~..,, O ~ : ~,
CO2Me 'I
1. ~-bromination
H23"LC i I/DMF
O ,,,,H , SCHEME
CO2Me
H,,OH ,,,,,CO2Me
1. BzCI
:~ 3~ 2"JOnes
~CH3 ",CH20H
37
(153). This ketone also allowed exploration of the third ring (Scheme 37) which was shown to be six-membered. The relative stereochemistry was partly deduced from hoe experiments which showed the double bond in the side-chain to have the E-configuration and the protons on C18, C-16 and C-17 to be cis-related. The CA-ring fusion was shown to be cis- since the hydroxy acid (154) resisted lactonisation, whereas the C16 epinaer, generated by NaBH4 reduction of the cyclobutanone, lactonised readily to 155. The cis -nature of the BC-ring fusion follows. The stereochemistry of the secondary methyl group at C11 was inferred from the deshielding effect attendant on the introduction of a carbonyl at C13 (eg in 152). The conclusions from these results
266 and other self-consistent circumstantial evidence regarding the relative stereochemistry were confirmed by X-ray diffraction analysis of the hydroxy acid (148) (116).
__ CO2H
r , ~ . . .-" CO2H " ,H
NaBH4
0
"'OH ~H 3
"~
154 SCHEME
38
H O
H~ ,,,, "~
0
H ca 3 155
,cH2R,oH OH
_.=
CH3
\ L,o.2R 0.3 OHq --t.- 9 I
[~CH SCHEME
CH3 O H Q "t"
3 39
In the ms of decipiene derivatives containing the cyclohexenone system, the dominant fragmentations could be rationalised as arising by the sequence shown in Scheme 39. It seemed probable that a similar [2+2] cycloreversion might be induced photochemically. Although this reaction eliminates five asymmetric centres, the compound generated would allow simple degradation of the decipiene skeleton and provide a means of establishing the configuration of the remaining asymmetric centre (115). Since the relative configuration of the decipiane skeleton was known, this determination would provide the absolute stereochemistry. To this end, the keto diacetate (157) was irradiated with uv light (254nm) to yield a 2:3 mixture of starting material and the phenolic photoisomers (159) which, as shown in the Scheme 40, were degraded to the 4-arylpentanoic acid (160). This proved to be identical to the 4R-enantiomer synthesised from R-(+)citronellal, thus establishing the R-configuration at C11 in the decipiane diterpenes (115). In a preliminary attempt to assemble the tricyclic skeleton of the decipianes, the photochemically-induced [2+2] cycloaddition of the readily available enone (161) was investigated (118).
267
H
O CrO3.pyr2
RO
,,,H I
.-,
.....H CH3
CH3
R
"%C. OAc
156 R = H
158
157 R = AC OR OsO4 NaIO4
hv;EtOH 254nm
~.CO2Hv
H3
159R=H R =
"CH3
160
1. OSO4,NalO4 2. Jones 3. Pd/C/NaOAc
CH 3
OH Pyridinium bromide perbromide
=
--
O
CH3
SCHEME
H
40
H
H
hv; 300nm=
~O
4-
O
161
162
163
m-CPA
O2 H =Jones
O2H= HO; EtOH .....OH
164
SCHEME
41
268 At this stage, although this type of adddition was well known, the photochemistry of the 1,7-diene variant had not been studied. In the event, irradiation (300nm) of 161 yielded a complex mixture from which the two diastereoisomeric ketones (162 and 163) could be isolated (Scheme 41). The formation of two isomers was expected since the starting enone was a mixture of the 1R,I'R- and 1S,I'R- isomers. Evidence that direct addition, in contrast to crossed addition, had occurred came from the spectroscopic properties of the compounds generated in the sequence leading to the cyclobutanone (164). The two diastereomeric products resulting from an ene-reaction were also produced.
Since the spectroscopic data revealed little information regarding the relative
configuration of the tricyclic photoisomers, this point was resolved by X-ray diffraction studies on derivatives of the two tricyclic ketones (162 and 163) (118). The results showed that both ketones had a trans-AB ring junction suggesting that in the triplet state of the cyclohexenone the singly occupied p-orbitals at the o~- and [3-carbons are essentially axial and equatorial respectively as shown in 165. Interaction of this biradical with the ene-component could occur on either the Re-or Si-face. Only attack on the Si-face can lead to the decalone biradical which can ring close to a cyclobutane with an all-cis-hydrogen arrangement. The ene-products could also arise by hydrogen transfer in this biradical (166) or from the biradical generated by attack on the Re-face of the ene component which cannot ring close to the trans-substituted cyclobutane system. Circumstantial evidence for the tricyclic diastereoisomers with the desired all-cis arrangement was obtained, but clearly this approach was not promising.
~
H
0
H
166 SCHEME
42
OH
OH hv
'1 "~
0
OH H~0
1. L-Selectride _-2. Acetylate 3. OsO4/Pb(OAc)4
OAc O
167
168 SCHEME
43
In a more extended study Dauben et al. (119) found that a similar photoaddition involving the allene (167), instead of the erie component, yielded the all-cis product in 60% yield (Scheme 43). This approach has the added merit of generating an exocyclic ene which can be used to introduce the
269 side chain present in the decipiane diterpenes. That the allene function confers the stereoselectivity observed has been demonstrated and it has been suggested that the initial biradical reacts by attack of C2 on the allene. In a different approach to the decipiane nucleus (Scheme 44), an intramolecular aldol reaction on the bicyclic ketone (169) gave the tricyclic alcohol (170) in 70% yield. Hydrogenation occurs from the convex or-face to yield the saturated ketone (171). This compound was
H H
CHO
H_
1.t-BuMe2SiCl
0.1M BaO
.....OH 2.H2/Pd
MeOH
.....OTBS H
H
"H
O
O
170
169
H eeeee
9
H H
(Me2N)2CHO-tBu eeeee
H
.~
,,,, "H OBz O
H"
171
1.1-Li cyclopropyl sulphide 3" Dowex resin Benzoic anhydride, DMAP i SnCI4
,,, ' ....O Bz
H
172
I H ...."
,,,
rl"'
' ....OBz
,
H
H eeee
H
rl " ~ " , c o H 2 M e e SCHEME
44
f
"H
CH2OH H
~CH20 H
subjected to a spiroanellation procedure and the resulting cyclobutanone was elaborated to the triol as shown in Scheme 44 (120). C e d r e n e isoprenologues: In a detailed examination of the several varieties that constitute the E. georgei complex, two were found to produce diterpenes with a C5 extended cedrene skeleton, eg 173 (121,122).
Nmdr and 2D-nmr measurements on this compound indicated the presence of a
1-methyl-4,4,6-trisubstituted cyclohexene component which is not contained in the two other tricyclic diterpene skeletons, decipiane and eremanes, previously isolated from Eremophila species. On the assumption that 173, in common with other bi- and tricyclic diterpenes from this
270 genus, contained a 6-carbon side chain, it was decided to convert 173 to the hydrocarbon (174) and to compare the 13C-nmr spectrum of this with that of ot-cedrene (175). A good correlation was observed between 12 of the 15 carbons. The exceptions involved C6,C7 and C12, but these could
20
H O2C.,._
10~ ' ' ' ~
,''~
H
18
H3
1.,,,~',",,I
CH20 H
H
173
174
be accomodated by allowing for the presence of the alkyl side chain in 174. The dihydro diol (176), obtained as a single isomer by hydrogenation of the diol, proved suitable for X-ray diffraction study (121). The results thus obtained confirmed the nature of the skeleton, and divulged the relative stereochemistry, of this new class of diterpenes. The relative location of the primary alcohol and carboxylic acid groups was easily deduced from the 13C-nmr spectra of the hydroxy acid and its parent hydrocarbon. The differences between the chemical shifts of the carbons assigned to the side chain were those expected if the former contained a primary alcohol at C16.
HO
H =
-
H
HOH2C,,,,
H 175
H HO - H
CH2OH
H
176
177
HO2C,% C02Me
H 178
179
Given the structure and relative stereochemistry of 173, determination of the absolute configuration was simplified by reference to the known chemistry of 0~-cedrene. Hydroboration of ot-cedrene produces isocedranol (177) in which the carbinol carbon has been shown to have the Sconfiguration by application of Horeau's method. A similar sequence carried out on hydrocarbon (174) yielded the alcohol (178) which was shown to have the 9S-configuration, thus establishing the absolute stereochemistry for the
2-epi-c~-cedrene isoprenologue.
The corresponding
271 sesquiterpene nucleus has been found previously in the terpenes of lac resin.
Several other
analogues of 173 were detected in the extract from E. georgei. One could be isolated in a pure form and was assigned structure 179 from spectroscopic and chemical evidence (122).
The other
metabolites appeared to be the exocyclic bond isomers of 173 and 179, and the corresponding side chain dihydro analogue of 179. Significantly, in an undescribed Eremophila species the bisabolene and cedrene isoprenologues are found to co-occur (122). Although a number of syntheses of cedrol and cedrenes have been achieved (72), extension of these to the synthesis of the cedrene isoprenologues requires the introduction of an extra asymmetric centre at C6.
19 18
15 9~ 7.j15
O
3
~H
HO2C
HO2C ~
6 2
~
180 O~
181
\
H
~ 182
E r e m a n e diterpenes: From a structural and biogenetic point of view, the most complex diterpene system elaborated by Eremophila species is that represented by eremolactone (3), the first diterpene isolated from this genus in 1962 from E.fraseri (11) and E.freelingii (12). Also isolated from E. fraseri were the isomeric diene-dioic acids which were later shown to have structures (180,181). Early spectroscopic and chemical studies on eremolactone led to structure 182 being suggested as a working hypothesis (11). Ir absorption at 1760 cm -1 suggested the presence of an unsaturated 3t-lactone whereas uv absorption at 292nm indicated extension of conjugation to the diene system. The nature of the side chain was deduced from the observation that alkali treatment of eremolactone yielded pyruvic acid and a trisnor-ketone, formulated as a methyl ketone from its positive iodoform reaction, and later shown to be 183. However, subsequent studies did not support the structure proposed for the tricarbocyclic portion (123-125).
By this time, pmr
spectroscopy had became available and the spectrum of eremolactone provided support for the presence of the side chain previously deduced and eliminated structure 182 from further consideration. The vinylic proton of the isolated double bond appeared at 85.87 as a d d with vicinal (J = 5Hz) and an allylic coupling constant (J = 2Hz). Treatment of eremolactone with neutral permanganate gave the keto dicarboxylic acid (184) which was found to have only one
272 exchangeable proton ct- to the ketone, thus supporting the structure 3 for eremolactone (123). Furthermore, isoeremolactone, the isomer produced by exposure of 3 to acids and which cocrystallizes with it, was shown by X-ray diffraction studies to have structure (185) (126). The stereochemistry of the C4 methyl was established in 1983 by the total synthesis of (_)eremolactone and its C4-epimer (127). An X-ray diffraction study of a new eremane (186) lent further support for the assignment (128). The absolute stereochemistry of the eremanes was finally elucidated by the synthesis of (+)-isoeremolactone (185) from tricyclovetivene (129). A number of eremanes with different levels of oxidation in the side-chain (eg 187) or in the nucleus (eg 188) have been isolated (77,125,128).
HO2C "~CO2 H
183
=
A
184
A
H O 2 C ~ o
'
186 HO2C
H
HO2C
~"
188
H
Interesting synthetic approaches for the construction of the tricyclo[5.2.2.01,5]undecane skeleton of the eremanes have been developed, but only two have been successful. The synthesis of (_+)-eremolactone relied on an acid-catalysed double Michael addition on the silyloxydiene (Scheme 45) (127). This on treatment with mesityl oxide in the presence of titanium (IV) chloride gave, inter
alia, an inseparable 1:2 mixture of diastereisomers (189) in 64% yield. Reduction with NaBH4 gave the separable hydroxy ketones (190 and 191, 1:2), the relative stereochemistry of which was secured from an X-ray study of 190. Following the introduction of the double bond, the side chain was elaborated on each of the two diastereoisomers as shown in Scheme 45. This synthesis has a number of problems. A complex mixture of isomers is generated in the first step, the cyclohexene
273 double bond introduced early in the sequence shows a tendency to isomerise and the introduction of the 4-ylidenebutenolide moiety leads to mixtures of 10E- and 10Z-isomers. A partial synthesis of (+)-isoeremolactone, patterned after a biosynthetic proposal, was instrumental in determining the absolute configuration of the eremanes (Scheme 46). Furthermore, this synthesis interelates the vetiver oil sesquiterpene, zizaenes, with the eremanes (129). O
~
~
mesityloxide TiCI4
O
..~
O
NaBH4,MeOH
OSiMe3 189
(_-'-)- 3
].~OSiMe3 ~
190
0
R
0
< (PhO)3PCH3I
+
HMPA
SnC14;-78~ 2. DBU,CH3CN
R =CH3 [--~
191
R=H
SCHEME
45
HOH2C~ ~ > ~
H
m-cPA
7 = (30~ I PCC CH2C12 OR
(.)-185
~OSiMe
Et3N
~
3
SnCI4;-78~
R = SiMe3 = S02Me SCHEME
46
Degradation of the hydroxy acid (186) to the sesquiterpene (192), using the sequence developed previously for the serrulatane-calamenene correlation, generated a new sesquiterpene skeleton whose occurence in nature is likely (128).
274
0
H
0
0
/
....-
192
183
o
o
193
194
,O
HO
5__._ ..
...~ ""
HO
iI ~
HO
195
196
197
198
The propensity for eremolactone to isomerize to the more stable isoeremolactone has been mentioned previously. Under more forcing acidic conditions, the cyclopentene double bond in the iso-compound can be protonated, generating a carbocation which undergoes rearrangement to the thermodynamically more stable isozizaene ring system. Treatment of eremone (183) leads to a mixture of double bond isomers from which the keto-enes (193 and 194) can be separated as the diols (195-197), whose structures were determined by X-ray diffraction methods (130). Evidence was also obtained for the presence of the postzizaene isomer (198) (131). In view of this, the isolation of the isoeremane (181) and the cyclic ether (199) from collections of E. fraseri poses interesting chemical and biogenetic questions. These compounds would appear to be artefacts of the isolation/purification procedures. Certainly, the eremaneisoeremane conversion is difficult to prevent. However, the cyclic ether (199), whose chemical precursor (187) is known as a natural product, could not be obtained by subjecting the hydroxy acid (187) to conditions used in the isolation of the ether. Thus prolonged exposure of the hydroxy acid to acidified charcoal or silicic acid resulted in the formation of the isomer (iso-187) only (125). In any event, the formation of 199 suggests that the eremanes do not necessarily represent a biosynthetic terminus in Eremophila species.
HO20.., 199
HO20 ",.._
ISO-187
275 5.5
Biosynthetic aspects As mentioned previously, the acyclic diterpenoids and cembranoids from Eremophila species
are unique in containing internal cis-double bonds. The derivation of the two classes of compounds from a common precursor seems reasonable. Thus, the acyclic hydroxy diacid (95) and the all-cis cembratriene (108) must branch from the same intermediate. Nevertheless, at least one acyclic diterpene (96) contains a trans-double bond and cyclization of its precursor would lead to cembrenes displaying a 3,4-trans double bond. No such examples have yet been found but a clue to their formation might be obtained from a comparison of the two cembrenoids, 107 and 109, which differ in configuration at C3 and C4. Their possible origins from acyclic precursors which differ in the geometry of the 2,3-double bond are shown in Scheme 47.
a PPO
)
H20
'
' HO
/
l~ )
.20
oxldalaon
___•H
HO '"
CO2H
II20 epoxidation
--~ii
_
epoxidadofi
OH
1
107
H
Hydroxylation pinacol oxidation
109 SCHEME
47
The second group of diterpene metabolites from Eremophila consists of those which are sesquiterpene isoprenologues. For these compounds, the cyclization processes involved in the elaboration of their nucleus engage only three of the four isoprene units present in an acyclic precursor. Their formation can be rationalized by application of those concepts derived from studies of sesquiterpene biosynthesis (132) and a consideration of the differing structures and absolute configuration of the various metabolites produced. Such an analysis has been attempted (133) and has resulted in the generation of many hypothetical biosynthetic sequences.
The following
discussion presents a summary of this analysis and includes only the most economical sequences which rationalize the formation of the various diterpene skeletons.
276
OPP
OPP
,
I/a
6~ ~
.
~\ )
CH3 ~R
H3C~-
200 (3R,6E)
201
(3R,6Z)
IS
i//I J
R
I~CH 3 7 -'%,_,
H3C~ X
I
<
H
=o~ ~,s,,s~ III
H
H
R
R
CH3
J
H otnnoo
OH2
H aaaa
205
204
1o
....OH 206 SCHEME
48
277 Considering the formation of the bisabolene isoprenologues (111,112,117), the interaction of a A6-double bond with a terminal allylic pyrophosphate moiety in an acyclic precursor is required (for convenience the tertiary pyrophosphate is considered). Given that the prerequisite ~-bond overlap can be achieved with either the A6-E or Z-configuration and either configuration at C3, and considering the two likely conformations, eight potential precursors can be generated. Cyclization of these, with resultant stabilization of the carbocations generated by a hypothetical nucleophilic Xgroup, leads to eight monocyclic structures. As shown in Scheme 48, cyclization of the 3R,6E(200) and the 3R,6Z-arrangements (201) gives rise to the 1R,7S- and 1S,7S-monocyclic pair (202 and 203) (similar stereoisomers are generated from the 3S,6E- and 3S,6Z-precursors but, although these are structurally identical, they are biosynthetically different. A distinction can be made by following the fate of the hydrogens at C1 of the precursor which arise from diastereotopic hydrogens at C5 in mevalonic acid). The bisabolene isoprenologues can be derived either from 202 or 203. Two 1,2-hydride shifts on 202 provides 204 with the electron deficent lobe of the secondary carbocation residing on the ]3-face of the cyclohexene ring. Alternatively, a 1,3-hydride shift on 203 provides the intermediate 205. Net syn-addition of water to the electron deficent r~-system of 204, or to 205 with displacement of the X group, generates the tertiary allylic alcohol (206). The remaining oxidative processes can readily be envisaged. Importantly, the double bond at C10 in these compounds is cis- with respect to the carbon chain. Although its geometry might have been inverted on route to the formation of the conjugated acid function, the possible involvement of such a geometric isomer is an important consideration in the derivation of the other carbocyclic diterpenes.
H
"H
X
x
R
c
~ ' /
207 CH3
205
:_: R
CH3
+
H3c SCHEME
49
208
The serrulatanes possess the same configuration at C7 as the bisabolene isoprenologues and can reasonably be derived from elaboration of the bisabolonium intermediate (205) as shown in
278 Scheme 49. Attack of the re-electrons from the Si-face of C 10 of the stabilized carbocation leads to the bicyclic intermediate (207) with the side chain in the equatorial orientation. A 1,3-hydride shift with displacement of X- initiates the process which results in the aromatic serrulatane nucleus (208). An alternate route to the serrulatanes involves formation of a macrocyclic intermediate (a germacrene isoprenologue) analogous to that implicated in the biosynthesis of the cadinane sesquiterpenes (91).
X
R D,
205
H
209
H,,,
Illl I
H""
It
,,
"H
H""
SCHEME
50
,,,, "H
210
The decipiane diterpenes can be envisaged to arise from the same bisabolonium intermediate (205) but, in this case, by attack of X- on the Re-face of C11.
This leads to the bicyclic
intermediate (209) with the side chain in an axial orientation. If this is allowed to interact with the cyclohexene double bond, the decipiane tricyclic skeleton (210) can be generated as shown in Scheme 50. The viscidanes exhibit an antipodal configuration at C7 compared to the bisabolane, serrulatane and decipiane diterpenes. This difference may reflect the involvement of a 3R,6E-acyclic precursor (211), or 3S,6E-, which cyclizes to the 1S,7R-intermediate (212) (Scheme 51). A 1,2hydride shift with displacement of X- would generate the tertiary carbocation (213) which could alkylate the Re-face of C10.
The spiro-ring system produced (214) contains the 1,4-trans-
disubstitution on the cyclopentane ring observed for the viscidane nucleus. A 1,5-hydride shift of the quasi-axial allylic hydrogen in 214 and allylic rearrangement, with net syn-addition of water, completes the elaboration of the nucleus. Circumstantial support for the last step can be enlisted
279 from the observation that all the viscidanes isolated so far contain allylic oxygenation in the cyclohexene ring.
OPP
H3C~ R/
OPP
6
H3 R~'~~ X
211 (3R,6E)/ ~ . ,
~
~H
H3C,~ 6 R"
212 (1S,7R)
H
3S,6E
~ Ha~
~~~_CH a
21 ..
X
H3C%.
%sIsI
/
. . No. R / ""CH 3
'~
-.
RX
-
H3C"
H
OH
214
SCHEME 51
The cedrene isoprenologues can also be generated from the carbocation (213).which, in this case, alkylates the Si-face of C10 in a 10Z-ene. The spiro-ring system produced displays the 1,4-
cis- disubstituted cyclopentane ring. Further cyclization of this intermediate generates the tricyclic ring system of the 2-epi-cedrene metabolites as shown in Scheme 52. The same carbocation (213) can be invoked as an intermediate in the generation of the eremanes. Cyclization with engagement of the C10 Re-face provides the spiro-intermediate (214) seen in the viscidanes. Direct alkylation of the cyclohexene-ene provides the tricyclic carbocation
280
H
H
,H
R
213
H3C..
.,
1 H3C... ,, a
SCHEME
52
(215) which initiates a series of C-C bond, methyl and hydride shifts leading to the bicyclooctene ring system of the eremanes as shown in Scheme 53. There are two other plausible pathways that can lead to the eremanes. One involves cyclization of the 10E-analogue of 213. This results in the tricyclic intermediate equivalent to 215, but with opposite orientation of the methyl and side chain. The other requires starting from the 3S,6Z-acyclic precursor to generate the 1R,TR-monocyclic stabilized carbocation with the E-configuration of the 10-ene. In all three sequences equilibration steps are required for at least one carbocation in the sequence. This can be taken to suggest that other ring systems, resulting from dissociation of the intermediate from the enzyme active site, remain to be found. Most likely, such ring system would be those derived from the carbocations 215-217. Although the sequences presented above are entirely hypothetical, they have proven useful in suggesting the type of skeleton that a newly discovered ring system might contain. This has been the case for the bisabolene isoprenologues and, more importantly, the cedrene isoprenologues. The intermediate role of this skeleton on the sequence leading to the eremanes is predictable. In summary, the bisabolenes, serrulatanes and decipianes all possess the R-configuration at C7 and the viscidanes, cedrenes and eremanes, the S-configuration. This difference may result from acyclic precursors with different geometries of the 6-ene, or simply from different conformations of a single precursor with a A6-E geometry. In view of the fact that, so far, attempts to produce tissue cultures which elaborate diterpenes have proved unsuccessful, the isolation of diterpenes with new skeletons remains the only method available which will provide information on the complex biogenetic pathways operating in Eremophila species.
281
H
,,,
lee S
H
H
~~~
H
CH3 X
214
X
H
-I-
H
CH 3
_
' ....CH 3
H
R
215
%
216
H SCHEME
6.
217 53
TRITERPENES Remarkably few triterpenes have been isolated from Eremophila species. Oleanolic acid and
ursolic acid were isolated from E. caerulea (134) and 3-epi-oleanolic acid from E. platycalyx (16). Two unusual triterpenes (218 and 219) have been isolated from the wood oil of Eremophila
mitchelli. Their structures were determined from a combination of spectroscopic techniques, aided by an X-ray diffraction study of the tetrahydro-derivative ( 2 2 0 ) o f the dione (218). The
282 stereochemistry of the hydroxyl group in 219 and the absolute configuration remain to be established (135,136). The proposed biogenetic origin of the dimers (Scheme 54) from 8othydroxyeremophilone (64) strongly suggests the absolute configuration depicted.
w
0
0
64
i .
-
-
,,
,,H
i
--
OH
i o
--
,,
0
219 SCHEME
7.
-
,,H
H 0
0
218
-
0
220 54
CONCLUDING REMARKS In the past, Eremophila species have been of cultural importance to the Aboriginal people of
Central Australia. They have also been regarded by agriculturalists as invasive woody species, sometimes poisonous, and of limited use as fodder. More recently, the ecological importance of
Eremophila species has been recognized and their potential in horticulture and revegetation programmes is being evaluated. The interest in the chemistry of Eremophila species, spanning the last ninety years, has been rewarded by the discovery of some of the intriguing bioynthetic processes at work in this genus. Two hundred and eight species of Eremophila are currently documented (137) and approximately one half of these have been investigated chemically. The genus is characterized by the variety and uniqueness of the diterpene skeletal types it can produce, nine to date, and the ability of a high proportion of its members to accummulate large quantities of these metabolites. The use of
Eremophila as a source of renewable material is an attractive possibility.
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
289
Marine Sesquiterpene/Quinones Robert John Capon
Introduction
1.
Over products
the last two decades discoveries
chemistry
metabolites.
yielded
a remarkable
array
pathways,
environment. considerable
novel
secondary
One
some
class
of
of
which
marine
amount of attention,
both
are
unique
to
that
has
metabolite through
the
marine
received
the abundance
a
of structural
and the biological properties ascribed to specific examples, is that
involving quinone.
the mixed biogenesis of a sesquiterpene This
review
sets
out
to present
A
Survey
Although
of
survey
of the
(1-52).
Sesquiterpene/Quinones
Marine
marine
unit with a quinol or a
a comprehensive
known examples of this structure class, 1 to 108 2.
of
These have included structure classes arising from a variety of
biosynthetic
variants
have
in the field of marine natural
sesquiterpene/quinones
have
predominantly
been
isolated from sponges, some of the earliest examples were reported from brown algae, and at least one example has been described from a gorgonian. The
following
survey
with
related
natural
along
with
a Table.
reflecting subunits.
the
bicyclic
sesquiterpene/quinones comprises
structure
diagrams
complexity
order
of
a listing
of
are
their
presentation
and
of
quinols
structure
presented respective
progresses
and tricyclic sesquiterpene carbon
in
(along
diagrams an
order
sesquiterpene
through
skeletons.
acyclic, An effort
been made, wherever possible, to present the aromatic subunits in
increasing trivial
The
structural
Thus, t h e
monocyclic, has also
of marine analogues),
order
names
are
of substitution indicated,
and/or
along
with
oxidation
state.
references
to
Where literature
appropriate, associated
with the issue of isolation and structure elucidation (1-52). These references are listed that
in the bibliography
address
biological
in chronological
(54-99)
and/or
order.
synthetic
Additional
investigations
references (100-109),
but do not contribute to the structure elucidation, are acknowledged later in this review.
Although a number of synthetic analogues and derivatives have
290 been prepared and reported, this survey is restricted exclusively to naturally occurring examples. The Table represents a consolidated listing highlighting source organism, along with physical and optical properties, plus a c o m m e n t - o n the current status of absolute stereochemistry. In
compiling
this
sesquiterpene/quinones
survey were
it
became
poorly
apparent
documented
that
with
many
respect
marine to
their
stereochemistry. Indeed, in several instances the failure to characterise marine natural products with respect to their chiroptical properties (measurement of an [O~]D) has made it impossible for future workers to properly
address
the issue
of absolute
early
assignments
of
sesquiterpene/quinones
were
stereochemistry.
absolute either
Furthermore,
stereochemistry
incorrect,
or
at best
to
many
marine
ambiguous.
In
a
number of cases the absolute stereochemistry issue was not even addressed in
the
primary
literature,
a point
that
several
subsequent
reviews
have
overlooked. This has the effect of perpetuating a "stereochemical bias" with no experimental foundation. The proper stereochemical characterisation of natural products is all the more important given the not infrequent reports of enantiomeric pairs of metabolites being reported from separate collections of the same, or related, source organisms. In order to appreciate the biosynthetic stereochemical versatility of marine organisms it is essential that the absolute stereostructures of new marine metabolites be considered, or at the very least their chiroptical properties suitably documented. This is all the more significant given the influence that stereochemistry is known to play with regards to biological activity. A brief account of some of the "problems" that have evolved with regards to stereochemical assignment among
marine
sesquiterpene/quinones,
together
with
some
strategies
that
we have pursued in chemical correlation, are outlined later in this review. Every effort has been made to include all known naturally occurring marine sesquiterpene/quinones
as of September
1992. While compounds
90
to 93 appear not to satisfy the criteria of the survey, they are in fact aromatic ring contracted analogues. Likewise, although compounds 97 to 1 0 8 have received recognition in the literature as polyketides, in the absence
of
contention
any definitive of
this
norsesquiterpene/quinones.
biosynthetic evidence author Some
that
they
anomalies
compiling of this survey are presented below;
to the contrary be
it is the
considered
highlighted
during
as the
291 8-epichromazonarol
[37]
A s s i g n m e n t of the trivial name 8-epichromazonarol to the metabolite 3 7 isolated from S m e n o s p o n g i a a u r e a is misleading since it implies a stereochemical has in
not this
be
relationship
established.
review
metabolite
is
more
with chromazonarol
The
arbitrary
closely
related
ent-chromazonarol,
chromazonarol.
In
fact
the
(an
absolute to
rather
absolute
algal
metabolite)
stereochemistry
that
of
than
the
the
known
algal
stereochemistry
of
that
depicted sponge
metabolite 37
remains
ortho
quinone
unknown. smenorthoquinone Although
smenorthoquinone
structure,
both
transformations shown
in this
than p a r a has
both cases for
was
initially
spectroscopic more
review.
data
consistent
The principal
quinone
structure
was
assigned and
with
the
an
results para
from
chemical
quinone
structure
evidence cited
for an o r t h o
the
smenorthoquinone
ability
of
109 with o r t h o - p h e n y l e n e d i a m i n e .
been clearly established
similar reaction para
the are
form an adduct which
[77]
Ilimaquinone
to be a p a r a - q u i n o n e ,
with o rtho-phenylenediamine
rather
to yield
this is possible due to the tautomerization
undergoes
the adduct between
to
[75], a
110. In
ortho
and
forms (see Figure 1). Most telling however is the spectroscopic data smenorthoquinone,
ilimaquinone.
which
.is
very
similar
indeed
to
that
of
Such a correlation would not be likely if s m e n o r t h o q u i n o n e
was an o r t h o rather than p a r a
quinone as originally
OC2H5
OCH 3
N H
109
-
proposed.
{
H
110
N I
292
u
OR
OR
H +
-?
+H+
O
0
(9
~" -H +
-H+ [ +H+ OR
OR
O -~
J
OH
..
_H+
--
H+
+H +
F i g u r e 1 " T a u t o m e r i z a t i o n between ortho a n d p a r a f o r m s
of ilimaquinone (75)
dictyoceratin-A
and/or
smenospondiol [86]
Although the literature suggests that these two c o m p o u n d s are enantiomers, this interpretation is doubtful given that both compounds possess [CZ]D measurements that are +ve and of comparable magnitude. Indeed, the absolute stereochemistry initially assigned to dictyoceratin-A on the basis of c.d. measurements on a degradation product (25) is very likely incorrect. The magnitude of this c.d. m e a s u r e m e n t was exceptionally small, and earlier studies (29) on the absolute stereochemistry of ilimaquinone have shown that c.d. measurements on 4 - k e t o - 5 - m e t h y l - t r a n s - d e c a l i n s are unreliable with respect to assigning
293 absolute
st e r eo ch em is try .
It
would
appear
that
dictyoceratin-A
and
smenospondiol are one in the same. siphonodictyoic
acid [87]
The aromatic subunit in siphonodictyoic acid is closely related to that in dictyoceratin-A
[86]
and
srnenodiol
[27]
(along
with
a number
of
terrestrial and synthetic compounds) and yet there exist significant discrepancies in the 13C n.m.r, shifts for several aromatic carbons. Our investigations on synthetic model compounds confirm that this is not due to
unexpected
distinctly
solvent
possible
shifts,
that
nor
either
abnormal
the
substituent
aromatic
effects.
substitution
It
pattern
is in
siphonodictyoic acid is incorrect, or the 13C n.m.r, data is in error. The substitution aromatic based
pattern
was
initially
13C n.m.r, chemical
on
the
additivity
rules
Extrapolation
of these
fraught
with
difficulties,
authors
in
metabolite
re-assigning
rules
highly
from
predicting
substitution
t h e expected
substitution
monosubstituted
substituted
observation
the
siphonodictyal-B
by
for alternative
derived to
an
assigned
shifts
aromatic
acknowledged pattern
to
patterns, benzenes.
systems
by
the
the
is
original
co-occurring
[30] (26).
s i p h o n o d i c t y o l - G [88] Although dispute
there the
is
assigned
siphonodictyol-G,
no
specific
substitution
spectroscopic pattern
it is worth noting
about
or
chemical
the
aromatic
that the same method
evidence
to
subunit
in
was used to
determine this pattern as in siphonodictyoic acid [87] and siphonodictyalB [30]. That some doubt exists about this assignment in the latter two compounds
must raise questions
siphonodictyol-G.
about the validity of the assignment in
294
~
O
H
2-(3,7,1 l-trimethyl-2,6,1 0-dodecatrienyi)-hyd r~ uin~
( 1)
OCH 3 HO
OH
6-(3,7,1 l-trimelhvl-2,6,10-dodecatrienyl)-2"nleth~
-hydr~176
(2)
OCH 3
O
6-(3,7,1 l-trimeihvl-2,6,10-dodecatrienvi)-2-nlethoxy-p -quinone s3 (3)
CHO
H C02tt siphonodictyaI-E 26 (4)
OAc OAc
I ....~oAc 0"~'"0
H
. O~'~~
~
~
moritoside 23 (S)
H3
~0 H
diciyochromeno119 (6)
295
O
O
)
o
smenochromene-A
smenoch
48 (7)
~
O - ~ ~ O C H3
smcnoch
romene-C
O
C
H
3
( 1 1 ) 13
panicein-A1(12)
OHC
B 4s (8)
OCH3
s men oc h ro me n e- I)48(I0)
48 (9)
OCHa O
romene-
0 OH panicein-B21 (13)
296
HO.~~....
/.... O.~,~
O H C / ~ V ~ /
v
.v
"0
panicein-B31 (15)
panicein-Bl I (14)
/
OH
paniccin-C I (16)
OCH3
metachromin-A3t (17)
/
OCH3
OCH3
H3 OH metachromin-C33 (19)
metachromin-F,31 (1~)
~
O
zonarol 2'6's3 (20)
H
~
O
zonarone 2A9(21)
297
~
O
H
O
O
isozonarone2'19 (23)
isozonarol2'53 (22)
~
/ ~
H
/••CO2H
_
z(,naric acid6 (25)
vahazunoll~ (24)
_
CO2CH
CO2CH
3
3
smenodio148 (27)
(_) dactylosponolsl (26)
0020H3
~
H
~vLr
(.) dac|yiospontriolsl (28)
O o . ~"~
siphonodictyaI-A14 (29)
H
298 CHO
CHO
H
s i p h o n o d i c t y a I - B 14'26 (30)
siphonodictyaI-C 26 (31)
CH2OH
OCH 3
siphonodictyol-l126 (32)
s p o n g i a q u i n o n e s'53 (33)
1
OCH
OCH 3
3
~
spongiaquinone potassium salt s3 (34)
OH
precyclospongiaq uinone- I s3 (35)
OH
0
c h r o m a z o n a r o l 4'6113'19 (36)
8-epicllromazonarol 13 (37)
299
OCH 3
OH
~ ). ~
ent_chromazonarol 5 (38)
OCH 3 O.
dehyd rocyclospongiaquinone- 18 (39)
OH
K~ O
"Oo
cyclospongiaquinone- 18 (40)
OH
l)romol)t~ upehenone 9
(42)
OH
o
puupehenone 9'16'28 (41)
c~o OH
chloropuupehenone 9 (43)
OCH 3
/5 -
_
0
isochromazonarol 4'19 (44)
cyclospongiaquinone-28 (45)
300
OCH3
OCH3
~
o
dictyoceralidaquinone 44 (46)
CHO
~
o.
J
nlamanuthaquinone 47 (47)
OH
H ,~
zt
siphonodictval-l) 26 (48)
au reoll3 (49)
H O ~ _ OCH3 ~
sirongylin-A 46 (50)
O
H
avarol 3'7 (51)
Ac0~ ~OAc
avarol monoacelale 37 (52)
v
diaceloxyavaro142 (53)
OAc
301
OH
~
O
3'_hydroxyavarone4s
avarone3 (54)
(55)
~ ~ ~
O ....,
OAc
4'-meihoxyavarone4~ (57)
6'-acetoxyavaroneas (56)
~o~ ~
CH3
O
H
J
6'-hvdroxyavarolas (58)
Ho~
~o~ OAc /,,
6'-aceloxyavarol4-~ ( 6 0 )
~ ~ O Ao~c J
6'_hvdro•
vl-avaro142 (59)
~~••1
~cH3
J
6,_hydroxv_4'_melhoxyavarone 45 (61)
302 OH
OO1-13
~~ J
J
3',6'-di hyd rox va va tone 45 (62)
is~
s'39 (63)
OCH3
OC2H5
J
S-epi-is~
uinone 52 (64)
5-epi- homoisospongiaq u inone s2 (65)
OCH3 ~
~
HCH3
J
OH
hyaloquitlone 3~
(66)
(67) 15
NHCH3
~
O
(68) is arenarol Is (69)
H
303
~
~
0
O
H
neoavarol 4~ (isoavarol)41 (71)
arenarone Is (70)
~/~~~
4'-methoxyneoava rone 4~ (73)
neoavarone 4~ (72)
-
OH
OCH
OH
OH
smenoquinone 35 (74)
J
o~
5.epi_ilimaquinone 2~
3
ilimaquinone 12'29 (75)
OCH3
~
CH3
(76)
OC2H
~
J
5
o~
smenorthoquinone 3s's2 (77)
304
N.~
NH2
~
J
smenospongine 27'3s (78)
o.
J
smenosl)ongidine 35 (79)
NH
~o.
J
(+) epi_smenospongidine sl (80)
-~j
smenosi)ongorine 35 (81)
-~j
OH
smenospongiarine 3s (82)
OH
(+) epi_smenospongiarine sl (83)
CO2CH3
.NH ~
~ ~o OH
( 8 4 ) 44
t
~
J
o.
d i c t y o c e r a ( i n - C 36 (85)
305 CO2H
CO2CH 3
f~?-o.
~ o.
J
J
.
dictyocerat~n-A
25
siphonodictyoic
acid 26 (87)
( s m c n o s p o n d i o l ) 38 (86)
CO2H
CO2CH3
j0-o.
~ o~ OH
OSOaNa
-
s i p h o n o d i c t y o I - G 26 (88)
d i c l y o c e r a t i n - B 2s (89)
OCH3
O
~o~ J 'H~ ' ~ .
OH
CH302C~\
O2CH3
~
o
J
.J
d a c t v l o s p o n g e n o n e - A 36 (90)
O
OCH 3
_
dact vlosl)on genone-l~ 36 (91)
OCH3
Hll,. ~~O2CH
~o~
d a c t y l o s p o n g e n o n e - C 36 (92)
OH
C H 3020",..~\
3
~ _
OCH3
o
J
d a c t y l o s p o n g e n o n e - D 36 (93)
306
u\ i| O ",,,0. O H
HO Oj~
bispuupehenone 16 (94)
O
O
H e
J
j
I1,,
Ii,,,
p o p o l o h u a n o n e - A 43 (96)
popolohuanone-B 43 (95)
HO
o,
OH
o~
I
o O
h a l e n a q u i n o n e 17"21'22'32'34 (97)
Na03SO~/~ OH
_
0
halenaquinol
0
sulfa(e 21"22"34 (99)
O
halenaquino121"22'34
(gg)
~ ~~o 0
x e s t o q u i n o n e 32'24 (100)
307
HO ~OH
_ "~~~J"oH
0
O
xestoquinol sulfate 5~ (101)
13,14,.15,16-tel ra hyd r o x e s t o q uino134 (102)
O,
0"~~0 H
0H o
3 , 1 3 - d i d e o x o - 1 , 2 , 1 4 , 1 5 - t e t r a h vd ro3 , 1 3 - d i h y d r o x y h a l e n a q u i n o n e 32 (103)
x c s t o s a i ) r o l - A 5~ (104)
HO
OH
O ~
0 x e s t o s a p r o l - B s~ (105)
a d o c i a q u i n o n e - A 32 (106)
0 0
0
0
a d o c i a q u i n o n e - B 32 (107)
0 ~
0
0
3 - k e t o a d o c i a q u i n o n e - A 32 (108)
308
Sou'ce
Organism Phaeophyta
Dictyopteris undulata
I "~ i (Brown
P"ysica'
Properties
I
Absolute Stereochemical
Status
Algae)
1 Colourless viscous oil. Colourless oil
24
Solid, mp 127-129~ 2 5 Amorphous powder, mp 81-85~ 3 6 Gum 4 4 Gum
i
Dictyopteris zonarioides
O0"ca'
Properties
2 0 Non-crystalline solid 2 1 Long yellow needles, mp 125-127~ 2 2!Gum 2 3 Bright yellow plates, mp 111-112~
[O~]D +4 ~ (no solvent Unknown reported) Chemical correlation to zonarol [a]D -12 ~ (CHCI3) [OC]D -5.4 ~ (CHCI3)
Chemical correlation to zonarol, plus degradation
[~]D "50 ~ (CHCI3) Chemical correlation to zonarol [O~]D +110 ~ (CHCI3) Unknown (co-occurs with chromazonarol) [CC]D +15.1 ~ Chemical correlation to zonaric acid, and degradation (CHCI3) [(~]D +88 .70 Chemical correlation to zonarol (MeOH) [C~]D +29.7 ~ (CHCI3) [C{]D +95.2 ~ (MeOH)
Unknown (co-occurs with zonarol) Unknown (co-occurs with zonarol)
[O~]D +22.6 ~ (CHCI3)
C9 stereochemistry unknown
[C~]D +22.2 ~ (CH2CI2) [(:Z]D +17.2 ~ (CH2CI2) [O~]D not reported
Cd analysis
Gorgonian
Euplexaura sp. Porifera
Adocia sp
5 I Colourless oil
(Sponges) 9 7 Yellow solid, mp >250~ 1 0 0 Yellow powder, mp 212-214~ 1 0 2 White solid, mp 224-229~ 1 0 3 White solid, mp 235-245~ 1 0 6 Yellow solid, dec >300~ 1 0 7 Yellow solid, dec >300~ 1 0 8 Yellow solid, ] dec >300~
[C~]D not reported [O~]D +25 ~ (no solvent reported) [OC]D +22 ~ (no solvent reported) [O~]D +65.4 ~ (no solvent reported)
Unknown Unknown relative stereochemistry Unknown Unknown Unknown Unknown
309
Source
....
Organism
Dactylospongia
sp
! "~ I
P"ys'ca'
Properties 8 5 IWhite solid
I
90 9 1 9 2 9 3
Dactylospongia elegans
2 6 mp 145-147~ 2 8 mp 167-169~ 8 01Oil I ! 8 3 Oil
Dysidea sp
|.
5 7 Yellow crystals, mp 150-152~ 7 1 Colourless needles, 9 mp 151-153~ 7 2 Yellow crystals, mp 7 8 - 7 9 ~ 7 3 Yellow crystals, mp 161-163~ 9 5 Purple dust 9 6 Purple dust
Dysidea arenaria Dysidea avara
6 9 ! Solid, I mp 128-130~ 7 0 LYellow oil 51 r 5 2 Solid, mp 151-153~ 5 3 Solid, mp 92-94~ 54 5 9 i Solid, , mp 113-115~ 6 7 Solid, mp 160-163~ 6 8 Solid, mp 9 153-155~
Absolute Stereochemical Status Biogenetic grounds (co-occurs with ilimaquinone) [O~]D -167 ~ (MeOH) Biogenetic grounds (co-occurs with ilimaquinone) [O~]D +96 ~ (MeOH) 'Biogenetic grounds (co-occurs with ilimaquinone) [O~]D +26 ~ (MeOH) i Biogenetic grounds i(co-occurs with ilimaquinone) [O~]D -121~ (MeOH) Biogenetic grounds , (co-occurs with ilimaquinone) [oc]546 -14 ~ Biogenetic grounds (co-occurs with ilimaquinone) (CH2CI2) [o~]546 -18 ~ Biogenetic grounds (co-occurs with ilimaquinone) ; (CH2CI2) [C~]D +37.5 ~ Biogenetic grounds (co-occurs with .ilimaquinone) (CHCI3) [O~]D +96.7 ~ Biogenetic grounds (co-occurs with ilimaquinone) (CHCI3) [C~]D +16.4 ~ Biogenetic grounds (co-occurs with avarol) (CHCI3) [O~]D -38.6 (CHCl3) Chemical correlation to avaroi ,
Optical Properties [oc]D not reported
!
J
i
[O~]D -55.2 ~ (CHCI3) [~]D -8-1~ (CHCl3) [O~]D not measurable due to intense colouration [a]D not measurable due to intense colouration [C~]D +19 ~ (CHCI3) [~]D +8-3 ~ (CHCI3) [O~]D +6.1 ~ (CHCl3) [O~]D +11.1 ~ (CHCI3) [C~]D +12.5 ~ (CHCI3) [C~]D not reported [O~]D +16 ~ (CHCI3) [O~]D not reported [O~]D not reported
Chemical correlation to avarol Biogenetic grounds (co-occurs with avarol) Unknown Unknown Unknown Unknown Cd analysis Chemical correlation to avarol Chemical correlation to avarol Chemical correlation.to avarol Biogenetic grounds (co-occurs with avarol) Biogenetic grounds (co-occurs with avarol) Biogenetic grounds (co-occurs with avarol)
. ' . i
i
310
Source Organism
Dysidea cinerea
|
, |
Dysidea pallescens Fasciospongia sp Fenestraspongia
sp
Halichondria panicea
Heteronema sp
Hippospongia sp
Hippospongia metachromia
Hyatella sp Hyrtios (=lnodes) eubamma Hyrtios eubamma
Optical Properties [a]D +45 ~ (CHCI3) 5 5 ! Oil [~]D -4.2~ (CHCI3) 5 6 , Oil [~]D -60.0 ~ (MeOH) 5 8 ! Oil [~]D +18.9~ 6 0 Oil (CHCI3) , [~]9 +75~ (CHCI3) 6 1 1 Oil [CC]D +65 ~ (CHCI3) 6 2 Oil i [C~]D not reported 3 8 Gum (acetate, [O~]D +39 ~ (CHCI3)) 4 7 Pale yellow solid, [c~]546 -31~ mp 108-109.5~ (CHCI3) [O~]D not reported 76 (acetate, [~]D +22.6 (CHCI3)) 12
I N~ I
Physical Properties
1 3i 14 15 i 16 4 1 Yellow solid, , mp
129-130~
4 2. 4 3 8 6 Colourless solid, mp 180-181.5~ 1 8 9 Amorphous solid, mp 154.5-155~ 1 7 Orange crystals, , mp 80-82~ 1 8 Oil 1 9 Yellow solid, mp 90-91~ , 7 5 Amorphous orange solid, mp 113-114~ 6 6 Light.orange needles, mp 68.569.5~ 9 4 Solid, mp 234-240~ 4 1 Yellow solid, mp 129-130~
1
[CZ]D 0 ~ (MeOH)
i
,
i
! i !
Absolute Stereochemical Status Biogenetic grounds Derivatization and cd analysis Derivatization and cd analysis Derivatization and cd analysis
!
, Biogenetic grounds Biogenetic grounds ! Comparison to chromazonarol
Chemical correlation to ilimaquinone Chemical correlation to ilimaquinone
i
Unknown
!
[CZ]D +315 ~ (CCI4)
Unknown
[CZ]D not reported [CZ]D not reported [C~]D +12.8 ~ (CHCI3) [a]D -1.22 ~ (CHCI3) [CZ]D -11~ (CHCI3)
. Unknown Unknown Biogenetic grounds (co-occurs with ilimaquinone) Biogenetic grounds
[OC]D +8 ~ (CHCI3)
Biogenetic grounds (co-occurs with metachromin-A), and cd analysis ~Biogenetic grounds (co-occurs with metachromin-A), and cd analysis Degradation
i[CC]D -29.7 ~ (CHCI3) j [O~]D -24 ~ (CHCI3)
Degradation
[O~]D +34 ~ (EtOH)
Unknown
[~]D -98~ (CHCI3)
Unknown
[cz]o +315 ~ (CCI4)
Unknown
311 Source Organism
Siphonodictyon coralliphagum
N~ I
Physical Properties
2 9 Solid, mp 192-193~ 3 0 Yellow crystals, mp 145-147~ 3 1 Solid, mp 192-193~ 32 4 8 Solid, mp 131-132~ 87 88
Smenospongia
sp
Smenospongia aurea Smenospongia echina
7
Colourless crystals, mp 98~ 8 Colourless crystals, mp 8082~ 9 Colourless crystals, mp 52~ 1 0 Colourless glass
Optical Properties [O~]D not reported
Absolute Stereochemical Unknown
[OC]D +3.2 ~ (MeOH)
Unknown
[O~]D not reported
Unknown
[ ~ ] 5 7 8 -23 .60 (MeOH) [~]D no rotation observed [o~]546 -67.6~ (MeOH) [(x ] 578 - 5.7~ (MeOH) [o~]578 -24.9 ~ (MeOH) [O~]D 0 ~ (0H2012)
Unknown
[OC]D +6.4~ (CH2Cl2)
Status
Unknown Unknown Unknown Unknown ! Unknown Unknown
[(X]D -217 ~ Unknown (CH2CI2) [O~]D -68.5 ~ Unknown (CH2CI2) [(X]D -53.5 ~ (MeOH) I Unknown
2 7 Colourless crystals, mp 185~ 7 4 Solid, mp >350~
[O~]D not reported
7 7 Yellow needles.
[O~]D not reported
7 8 Red crystals, mp 9 153-155~ 7 9 Solid, mp 168-170~ 8 1 8 2 Solid, mp 170-172~ 8 6 Colourless solid, mp 180-181.5~ 3 7 White solid, mp 132-134~ 4 91Solid, , mp 144-144.5~ 1 1 Oil
[O~]D not reported [o~]D not reported [O~]D not reported [O~]D not reported [(X]D +5.8 ~ (CHCI3) [O~]D-2 ~ (CCI4) [C~]D +65 ~ (CCI4) '[O~]D +3.3 ~ (CCI4)
Chemical correlation to ilimaquinone Biogenetic grounds (co-occurs with ilimaquinone), and by cd. Biogenetic grounds (co-occurs with ilimaquinone), and by cd. Biogenetic grounds (co-occurs with ilimaquinone), and by cd. Unknown Unknown Biogenetic grounds {co-occurs with ilimaquinone) Unknown X-ray analysis of a brominated derivative Relative stereochemistry unknown
312
Source Organism Spongia sp
Spongia hispida
N~ I
Physical
Properties
3 4 Purple solid, mp >350~ decomp 3 5 Yellow oil 8 4 Red needles, mp 131-134~ 6 4 Yellow/orange oil I
6 5 Pale yellow oil
Stelospongia conulata
Stronglyophora hartmani Thorecta choanoides Xestospongia exigua Xestospongia sapra
3 3 Red plates, mp 159-160~ 3 9 Deep red needles, , mp 90.5-92.0~ 4 0 Yellow needles, mp 196-196.5~ 4 5!i Orange prisms, imp 196-197~ 6 3 Yellow crystals, mp 135.5-136~ 4 l j Yellow solid, mp 129-130~ i 50 2 Pale yellow oil. 3 Pale yellow oil. 9 7 Yellow solid, mp >250~ 9 8 Unstable yellow solid 9 9 1 0 1 i Yellow solid 1 0 4 Yellow solid J 1 0 5 Yellow solid
Unidentified Unidentified
1 A colourless viscous oil. 4 6 Yellow needles, mp 159-161~
Optical Properties
Absolute Stereochemical
[C~]D not measurable (methyl ether, [C~]D -82 ~ (CHCl3)) [O~]D not reported (methyl ether, [O~]D +37 ~ (CHCI3)) [O~]D not reported
Chemical correlation to spongiaquinone
Status
Biogenetic grounds (co-occurs with spongiaquinone) Relative Stereochemistry unknown Chemical correlation to isospongiaquinone Biogenetic grounds (co-occurs with 5-epi-isospongiaquinone)
[o~]O -41.2 ~ (CHCI3) [CC]D -28.8 ~ (CHCI3) Degradation [O~]D not reported (methyl ether, [O~]D -82 ~ (CHCI3)) [O~]D -140 ~ (CHCI3) 'Complete relative stereochemistry unknown [O~]D -2.1 5 ~ Complete relative stereochemistry unknown (CHCI3) ! I Unknown [C~]D +11.68 ~ , (CHCI3) Degradation [C~]D +64.8 ~ (CHCI3) [a]D +315 ~ (CCI4) Unknown
I
i
[C~]D +72 ~ (CH2CI2)
[O~]D +22.2 ~ (CH2CI2) [o~]577 +179~ (acetone) [cc]577 +106 ~ , (UeOH) , [O~]D +27 ~ (MeOH) :[O~]D -42 ~ (MeOH) i i[O~]D-49 ~ (MeOH)
[O~]D -12.8 ~ (CHCI3)
I
Unknown
Cd analysis Cd analysis Cd analysis I Unknown I Complete relative r i ;reochemistry unknown stereocher[ t !Complete relative r stereochemistry unknown Relative stereochemistry unknown
313 3.
Biological
Activity
I n c r e a s i n g l y the search for novel natural products has b e c o m e f o c u s e d on the quest for metabolites that possess interesting and/or useful b i o l o g i c a l p r o p e r t i e s . I n t e r d i s c i p l i n a r y c o l l a b o r a t i o n between chemists,
biochemists,
bioassay
directed
"bioactive"
metabolites.
biologically it
is
potent
also
frequently
pharmacologists,
isolation,
true not
have
etc ....
the
together
discovery
the
pursued
of many
to a meaningful
of these
conclusion.
The
many
to, reveal
from both marine and terrestrial
significance
with
of
While this approach has, and continues
metabolites
that
ecologists
facilitated
sources,
discoveries marine
is
natural
products literature abounds with claims for new antibiotic, anticancer and anti-inflammatory agents etc .... with the literature on marine sesquiterpene/quinones being no exception. Among the many and varied claims
for
"biological
sesquiterpene/quinones
activity" listed
attributed
above
to
some
of
the
marine
are;
9 Anticancer (lung, colon, mammary, leukemia) 9 Antibiotic
(against numerous
bacteria and fungi)
9 Antiviral (against HIV and Influenza PR-8) 9 Inhibition of cell division (starfish and sea urchin) 9 Inhibition
of tubulin
polymerisation
9 Toxicity (against Killifish and Brine Shrimp) 9 Coronary
vasodilating
Some describing
of
these
claims
the
initial
structure
subsequently
(54-88),
applications biological
properties
are
made
within
elucidation
and of these several
(89-99).
the
(1-52),
context others
of
properties
noting
concern
to
specific
that overwhelmingly
themselves
papers
been
have formed the basis
made
of. patent
Rather than attempt to correlate these latter reports compounds,
a
comprehensive
references (54-99) are listed in the bibliography in chronological worth
the
have
with
studies
these latter reports focused
on avarol
set
of
order. It is
on biological [51]
on
activity
and avarone
[54].
Early reports on the effect of these two compounds against HIV have clearly been
responsible
for
much
of
this
interest.
This
phenomenon
is
amply
314 demonstrated by the frequent appearance of these compounds in the patent literature (89-98). As a cautionary note it should be observed that although patent
applications,
and
awarded
patents,
appear
as
entries
in
Chemical
Abstracts, patents are not peer reviewed documents and are awarded on legal rather than scientific accuracy. One measure of the significance of a patent in d o c u m e n t i n g valuable and worthwhile biological properties is whether
the
respect
it
awarded
patent is
to
interesting marine
the application early claims could
is maintained note
that
several
sesquiterpene/quinones
initially
of
have
the
not
granted.
patent
been
In this
applications
pursued
beyond
stage. Further to this, it has recently been reported (51) that
attributing anti-HIV properties to avarol
not
substantial
to
beyond the date
be
substantiated.
body
of research
Despite
this
documenting
[51] and avarone [54]
controversy
avarol
there
remains
[51] and avarone [54]
altering the biochemistry of cells infected with HIV. The question
a as
appears to
be whether these properties constitute an ability to act as an anti-HIV agent. In contrast
to the research effort directed towards
of marine
sesquiterpene/quinones,
109) regarding
their synthesis.
sesquiterPene/quinones
and
no
naturally
little
has
been
Perhaps a more accessible
structural
c o m p r e h e n s i v e structure/activity r e v i e w it would appear that activities
relatively
the biological
analogues
properties
published supply
would
(100-
of known
encourage
more
investigations. At the time of writing this despite an array of p r o m i s i n g biological
occurring
marine
sesquiterpene/quinone
has
evolved
in any real sense towards a pharmaceutical agent, or a useful research drug (the dilemma of avarol [51] and avarone [54]
4.
Stereochemical As
reviewed
stereochemistry dichroism for
X-ray
Investigations
in
an earlier
attributed
measurements
ilimaquinone
aureol
[49].
was
[75]
a
the correct
monobromo
ilimaquinone
was
by
the
on the basis absolute
absolute
of circular
stereochemistry to
of
established
(110)
via
analysis
source
ilimaquinone
series
correlation
establishing
new
in this
was incorrect. The correct ultimately
to
the
to
volume
The absolute stereochemistry of aureol had been determined
assignment Ilimaquinone
not withstanding).
derivative.
important
This
stereochemical
reasons
than
re-
simply
marine
metabolite. have been
of
as ilimaquinone
a
variety have
of
been
new
for this particular
other
is a common co-metabolite in sponges that in turn
compounds
stereostructure
for
degradative
sesquiterpene/quinones.
attributed
the
same
"absolute
Frequently
these
stereochemistry"
[75], principally on the basis of biosynthetic considerations.
315 Careful examination of these stereochemical assignments revealed that in m a n y instances a simple chemical transformation could have provided a more secure correlation. Over the last few years we have pursued several chemical correlations between marine sesquiterpene/quinones with interesting and useful consequences. Some of these results are highlighted below. Our early investigations into the absolute s t e r e o c h e m i s t r y of ilimaquinone [75] suggested that the acid catalysed rearrangement of both ilimaquinone [75] and isospongiaquinone [63] would yield the same p r o d u c t s , I l l and 112 (see Scheme 1). Thus the absolute stereochemistry of isospongiaquinone could be u n a m b i g u o u s l y correlated to that of ilimaquinone. Not only was this approach successful but we have used it to chemically correlate the absolute stereochemistry of both isospongiaquinone [63], as well as 5-epi-isospongiaquinone [64] and 5-epi-ilimaquinone [76], to ilimaquinone [75] (see Scheme 1). A related approach was reported (47) to successfully correlate mamanuthaquinone [47] with ilimaquinone [75]. In determining the structures of the acid catalysed rearrangement products, 111 and 112, we came to examine the mechanistic nature of this reaction in some detail. In doing so it became apparent that the structure tentatively assigned (18) to the product obtained on acid catalysed rearrangement of arenarol [69] was very likely incorrect. This issue was subsequently resolved when X-ray analysis identified the acid catalysed rearrangement product of arenarol as 113 (see Scheme 2), formed via heterocyclic ring closure to C10 rather than C5 as originally supposed. Unfortunately, the chiroptical properties of 113 were not documented at that time, and the absolute stereochemistry of arenarol [69] and arenarone [ 7 0 ] remained unassigned. In an attempt to redress this situation we obtained a generous gift of authentic arenarol (D. J. Faulkner) and intended to examine its acid catalysed chemistry. To our surprise the sample of arenarol, which was several years old, had undergone complete conversion to aureol [49]. Presumably during storage the intrinsic acid character of arenarol
had
autocatalysed
confirm
that
aureol
was
its
conversion
indeed
to
a possible
aureol acid
(see-Scheme
catalysed
3).
To
rearrangement
product of arenarol we turned to avarol. Not having an authentic supply of arenarol it was argued that acid catalysed re-arrangement of avarol should yield
intermediate
carbocations
identical
to those
expected
(see Scheme 3), and thus should ultimately be capable Careful
analysis
of the reaction
products
revealed
from
arenarol
of forming aureol.
that aureol
had
indeed
316
OCH 3
OCH3
_~o.
O -?
OH
5-epi-ilimaquinone (76)
ilimaquinone (75)
~, H+ OCH3 0 -
OH
(Ill)
0
O~ Q
7
(112)
H+
~ H+
o
isospongiaquinone (63)
Scheme 1
OCH3
_~o 5-epi-isospongiaquinone (64)
9 Acid catalysed correlation of marine sesquiterpene/quinones
317
(51)
avarol
H+ OH I
OH
~
H.
HO
(113)
aureol (49)
(114)
H+
~
O
arenarol (69)
Scheme 2
9 Acid catalysed correlation of a v a r o l / a r e n a r o l / a u r e o l
318 been formed. This result was particularly satisfying in that it closed the chemical correlation loop; [arenarol; avarol] to [aureol] to [ilimaquinone] to [isospongiaquinone; 5-epi-isospongiaquinone; 5-epi-ilimaquinone]. In doing so this result re-confirmed the observation that these two historica.lly important marine metabolites, avarol [51] and ilimaquinone [75], belong to the same enantiomeric series. Also noteworthy were the results from acid cataysed rearrangement of aureol. This experiment suggested that under acid conditions aureol could undergo ring opening to yield an intermediate carbocation that in turn could reform aureol, or through a process of deprotonation and re-protonation (see Scheme 3 ) c o u l d yield the trans fused analogue
(113).
During the course of acid catalysed investigations on avarol we observed an unexpected electrophilic aromatic ring closure to yield 1 1 4 . Although the yield of 1 1 4 was low, and variable, the corresponding dimethyl ether 116 was obtained in almost quantitative yield on acid catalysed rearrangement of avarol dimethyl ether [ 1 1 5 ] . Our tentative explanation for this process is described in Scheme 3. This ring closure suggested a possible biosynthetic mechanism to the more highly cyclized marine norsesquiterpene/quinones 97 to 108, via electrophilic cyclization to a corresponding protonated double bond in ring-B, and served to reinforce the contention that these compounds should be considered as sesquiterpene/quinones rather than polyketides. We are currently investigating the synthetic versatility of this ring closure on suitable model compounds.
. ~ _11
o
CH3
CH30
(115)
More
recently
CH3 m
(116)
we
completed
a
re-examination
of
the
marine_
sesquiterpene/quinone spongiaquinone ( 3 3 ) . This i n v e s t i g a t i o n was prompted by our discovery from a southern Australian marine sponge of the
319
H
~0
~
~~'~O
O
:
avaroi/arenaro!
ot - H+ attack//~ ~A/'_H+
j
1,2 H shift
OH
-
~ c~- H+ attack
-H
O
~ -
_.+
13- H+ attack
1,2 H s h i f t / /
/
cyclization
cyclization
r
OH
OH
I cyclization
OH (113)
:
H
=
aureoi
(49)
(114)
Scheme 3
" Proposed mechanism for acid catalysed cyclization
320 naturally occurring potassium salt (34) of spongiaquinone, and our desire to c h e m i c a l l y correlate this salt to spongiaquinone, and hence determine is c o m p l e t e stereostructure. In examining the literature on spongiaquinone, which
was
became
one
of the earliest known
apparent
that
the
reported
examples
of this
stereochemical
structure
class,
determination
was
it not
entirely complete. After securing the 9,11 double bond geometry as E on the basis
of n.O.e,
measurements, we were surprised
to note
that although
the
absolute stereochemistry of spongiaquinone had supposedly been secured by degradation, no [C~]D measurements were reported for either spongiaquinone or any our
of its derivatives.
re-isolated
problematic.
This made
spongiaquinone
Furthermore,
spongiaquinone
a stereochemical
with
although
that the
shown
for
reported
absolute
between somewhat
stereochemistry
of
was said on the basis of c.d. measurements on a degradation
product to be the same as that of zonarol (20), zonarol
ambiguities
comparison
initially
we
was
in
undertook
fact an
incorrect.
the As
independent
absolute a
stereochemistry
consequence
analysis
of
of
the
these
absolute
stereochemistry of spongiaquinone, and in doing so came to acknowledge the curious
optical
neither
of
properties
which
measurements
of both
gave
were
a
spongiaquinone
stable
performed
optical
at
a
and
rotation
range
of
its
potassium
measurement!
concentrations,
salt, These
and
the
transmission of light deemed not to be a problem. They were also run in two solvents
(CH2C12
wavelengths,
and EtOH), at acid, neutral and basic pH, and at several
all to no avail. The methylated derivative
(117)
did
however
yield a stable measurement, thus allowing the stereochemical character of both s p o n g i a q u i n o n e and its salt to be characterised through the optical properties of
of (117).
spongiaquinone
although
At this point we cannot satisfactorily explain the failure or
its
salt
to
generate
stable
it seems likely that the 9,11 double
optical
measurements,
bond and the phenol/phenoxy
anion are involved. Only one other marine sesquiterpene/quinone contains these same structural units, siphonodictyal-B (30), and curiously the [O~]D for J
this
compound
was
not
reported.
Oxidative
degradation
of spongiaquinone
yielded the decalone (118) which was in turn converted into the more stable stereoisomer
(119).
These
two
degradation
products
were
known
d
compounds,
and
there
optical
properties
stereochemistry of spongiaquinone (and its salt).
confirmed
the
absolute
321 OCH 3 0 0
0
0
OCH3
(117)
,,,jl
(118)
(119)
Additional opportunities exist to chemically inter-relate naturally occurring marine sesquiterpene/quinones, and in doing so to both resolve their absolute stereostructures and to explore their chemical reactivity.
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324 68. W. E. G. Muller, C. Sobel, B. Diehl-Siefert, A. Maidhof and H. C. Schroder, Biochemical Pharmacology, 36, 1489 (1987). 69. W. E. G. Muller, D. Sladic, R. K. Zahn, K. Bassler, N. Dogovic, H. Gerner, M. J. Gasic and H. C. Schroder, Cancer Research, 47, 6565 (1987). 70. I. Tabokovic, A. Davidovic, W. E. G. Muller, R. K. Zahn, D. Sladic, N. Dogovic and M. J. Gasic, Bioelectrochemistry and Bioenergetics, 17, 567 (1987). 71. W. E. G. Muller, H. C. Schroder, P. Reuter, P. S. Sarin, G. Hess, K. Meyer zum Buschenfelde, Y. Kuchino and S. Nishimura, Aids Research and Human Retroviruses, 4, 279 (1988). 72. E. Batke, R. Ogura, P. Vaupel, K. Hummel, F. Kallinowski, M. J. Gasic, H. C. Schroder and W. E. G. Muller, Cell Biochemistry and Function, 6, 123 (1988). 73. Y. Kuchino, S. Nishimura, H. C. Schroder, M. Rottmann and W. E. G. Muller, Virology, 165, 518 (1988). 74. R. Voth, S. Rossol, G. Hess, H. P. Laubenstein, K. Meyer zum Buschenfelde, H. C. Schroder, M. Bachmann, P. Reuter and W. E. G. Muller, Jpn J. Cancer Res (Gann), 79, 647 (1988). 75. M. J. Gasic, J. Serb. Chem. Soc., 53, 229 (1988). 76. H. C. Schroder, P. S. Satin, M. Rottmann, R. Wenger, A. Maidhof, K. Renneisen and W. E. G. Muller, Biochemical Pharmacology, 37, 3947 (1988). 77. H. C. Schroder, R. Wenger, H. Gerner, P. Reuter, Y. Kuchino, D. Sladic and W. E. G. Muller, Cancer Research, 49, 2069 (1989). 78. S. Loya and A. Hizi, FEBS, 269, 131 (1990). 79. R. Cozzolino, A. de Giulio, S. de Rosa, G. Strazzullo, M. J. Gasic, D. Sladic and M. Zlatovic, J. Nat. Prod., 53, 699 (1990). 80. N. Dogovic, D. Sladic, M. J. Gasic, I. Tabakovic, A. Davidovic and E. Gunic, Gazz. Chim. ltal., 121, 63 (1991). 81. V. V. Sova and S. A. Fedoreev, Chemistry of Natural Compounds, 420 (1991). 82. M. Kobayashi, H. Nakamura, J. Kobayashi and Y. Ohizumi, The Journal of Pharmacology and Experimental Therapeutics, 82 (1991). 83. M. Kobayashi, A. Muroyama, H. Nakamura, J. Kobayashi and Y. Ohizumi, The Journal of Pharmacology and Experimental Therapeutics, 90 (1991). 84. W. E. G. Muller and H. C. Schroder, Int. J. Sports. Med, 12, $43 (1991). 85. M. Bourguet-Kondracki, A. Longeon, E. Morel and M. Guyot, Int. J. I m m u n o p h a r m a c . , 13, 393 (1991). 86. M. A. Belisario, R. Pecce, A. R. Arena, A. de Giulio, G. Strazzullo and S. de Rosa, Toxiology Letters 57, 183 (1991). 87. H. C. Schroder, M. E. Begin, R. Klocking, E. Matthes, A. S. Sarma, M. Gasic and W. E. G. Muller, Virus Research, 21, 213 (1991). 88. M. A. Belisario, M. Maturo, R. Pecce, S. de Rosa and G. R. D. Villani, Toxicology, 72, 221 (1992). Patents 89. W. E. G. Muller, R. K. Zahn, E. Eich" Avarone derivatives, Ger. Often., DE 3427383 A1, 30 Jan 1986.
325 90. W. E. G. Muller; Use of avarone and avarol and derivatives thereof for the preparation of a pharmaceutical composition for the control of AIDS and ARC, Eur. Pat. Appl., 0 252 304 A2, 13 Jan 1988. 91. W. E. G. Muller; Use of avarone and avarol and derivatives thereof for the preparation of a pharmaceutical composition for the control of adult T-cell leukemia/lymphoma, Eur. Pat. Appl., 0 252 305 A2, 13 Jan 1988. 92. W. E. G. Muller, R. K. Zahn, E. Eich; Extraction and purification of avarone a n d avarol from Dysidea avara, Ger. Often., DE 3621032 A1, 28th January 1988. 93. W. E. G. Muller; Application of avarol and avarone derivatives in combination with 3'-azido-3'-deoxythymidine for antiviral therapy, G er. Offen., DE 3821676 A1, 8th February 1990. 94. W. E. G. Muller; Use of avarol for the control of AIDS and ARC, U.S. 4939177A, 3rd July 1990. 95. W. E. G. Muller; Use of avarol for the control of adult T-cell leukemia/lymphoma, U.S. 4939178, 3rd July 1990. 96. A. Bach, W. R. Shanahan; Neoplasia treatment compositions containing antineoplastic agent and side-effect reducing agent, Eur. Pat. Appl., 0 393 575 A1, 24th October 1990. 97. W. E. G. Muller, H. C. Schroder, P. Langen, E. Matthes; Preparation of combinations of avarone derivatives and 2',3'-dideoxy-3'flurothymidine as antitumor agents, Ger. (East), DD 289 706 A5, 8th May 1991. 98. W. E. G. Muller, H. C. Schroder; Synergistic neoplasm inhibitors comprising avarone derivatives and 3'-deoxy-3'-flurothymidine, G e t . Offen., DE 3939701 A1, 6th June 1991. 99. A. E. Wright, N.S. Buttes, F. Koehn; Novel antiviral and antitumor terpene hydroquinones and methods of use, PCT Int. Appl. WO 91 12250, 22 August 1991. Synthesis 100. S. C. Welch and A.S.C.P. Rao, Tetrahedron Lett., 505 (1977). 101. G. L. Trammell, Tetrahedron Lett., 1525 (1978). 102. S. C. Welch and A. S. C. P. Rao, J. Org. Chem., 43, 1957 (1978). 103. A. S. Sarma and P. Chattopadhyay, J. Org. Chem., 47, 1727 (1982). 104. A. S. Sarma and A. K. Gayen, Tetrahedron Lett., 3385 (1983). 105. N. Harada, T. Sugioka, Y. Ando, H. Uda and T. Kuriki, J. Am. Chem. Soc., 110, 8483 (1988). 106. S. Inayama, N. Shimizu, T. Ohkura, H. Akita, T. Oishi and Y. Iitaka, Chem. Pharm. Bull.,37, 712 (1989). 107. N. Harada, T. Sugioka, H. Uda and T. Kuriki, J. Org. Chem., 55, 3158 (1990). 108. S. N. Suryawanshi, A. Mukhopadhyay, T. S. Dhami and D. S. Bhakuni, Ind. J. Chem., 29B, 1001 (1990). 109. K. Kanematsu, S. Soejima and G. Wang, Tetrahedron Lett., 4761 (1991). 110 "Studies in Natural Products Chemistry", Vol. 9 "Structure a n d Chemistry (Part B)", edited by Atta-ur-Rahman, Chapter 2 "Marine
326 norterpene cyclic peroxides" a stereochemical paper chase." Capon, Elsevier, Amsterdam, 1991.
by R. J.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
327
Antimicrobial Activity of Amphibian Venoms Gerhard G. Habermehl
1.
INTRODUCTION Amphibians (toads,
frogs,
salamanders, newts) are a worldwide d i s t r i b u -
ted class o f animals comprising about 2.600 species.
During t h e i r
they have developed skin glands covering most parts o f t h e i r
body
From these glands small amounts of a mucous slime are secreted Frequently
mucus glands
morphologically.
These
central
nervous
system,
substance classes as w e l l , Until
hypotensive
contain as
such as
steroids,
This i s ,
Investigations (ref.
hypertensive a
wide
alkaloids,
have
of
indolalkylamines, secretions
however, not the case.
such predators
were
act as
a
not
are
Before yet
protection
R e s p i r a t i o n in amphibians i s
done
by
on
against the
Many amphibians do not even possess
primitive
sacks
filled
with
air
for
i s performed by means o f
the
skin
lungs the
secretions.
t h e r e f o r e would be a p e r f e c t s u b s t r a t e f o r b a c t e r i a and f u n g i , did not share one common p r o p e r t y , f u n g i or yeasts,
agents,
variety
at
diving
To p e r m i t the exchange o f oxygen and carbon d i o x i d e the skin
has to be moist which
concentrations.
the
1).
and not by the lungs.
procedure o n l y .
on
in the l a b o r a t o r y of the author a l r e a d y in 1969 showed
The reason i s simple: others j u s t
different
acting
years ago, when the e v o l u t i o n o f
w i t h o u t any doubt t h a t these t o x i n s p r i m a r i l y
all,
with
was commonly held t h a t these
the amphibians was f i n i s h e d more or l e s s ,
mainly,
differentiated
substances as
surface.
permanently.
and peptides mostly o f low molecular w e i g h t .
twenty years ago i t
microorganisms
be
neurotoxins
well
one must consider t h a t some 50 m i l l i o n
earth.
may
Chemically they belong to
used only a g a i n s t n a t u r a l p r e d a t o r s . all,
glands
such as c a r d i o t o x i n s ,
and many o t h e r s .
catecholamines,
venom
venom glands
pharmacologic a c t i v i t i e s hemolysins,
and
evolution
namely they act as
Some of them are a c t i v e
against
and again others act as f a i r l y
if
The
the t o x i n s
antibiotics
bacteria,
skin
others
in
low
against
broad spectrum a n t i b i o t i c s .
328 Poisoning in man i s very rare and w i t h o u t f a t a l i t y animals may occur o c c a s i o n a l l y ,
and an o c c a s i o n a l
so f a r .
Poisoning in
case i s r e p o r t e d in which
a h u n t i n g dog played w i t h a toad or a salamander, got s i c k and f i n a l l y Cats on the other hand avoid to touch these animals,
died.
and stay away, p o s s i b l y
due to a s p e c i a l smell. 2.
ANURA (Toads and Frogs) 2.1 Bufonidae (Common Toads) Our knowledge of the t o x i c i t y
of toads dates back to
the a n c i e n t Asian medicine -as e a r l y as
3.000
powdered s e c r e t i o n was used as heart drug. I t
years
times.
In
ago-
the
dried
and
be
found
in
may s t i l l
early
pharmacies as a remedy f o r dropsy. Such substances were also Europe and were used u n t i l
digitalis
introduced
g l y c o s i d e s came i n t o use f o r
purpose. The composition of the skin gland s e c r e t i o n substances may be d i v i d e d i n t o three groups:
is
Biogenic
local
the
complex, amines,
to same
and
the
bufogenins,
and b u f o t o x i n s . The b i o g e n i c amines ( r e f . 2 ) partly
indolalkylamines.
nor-adrenaline
are p a r t l y
derivatives
The most i m p o r t a n t compounds
(as catecholamines),
of
are
w e l l known from the
brenzcatechin, adrenaline,
animal
and
kingdom
as
s u p r a r e n a l hormones, as w e l l as i n d o l a l k y l a m i n e s o f the b u f o t e n i n type.
All
i n d o l a l k y l a m i n e s possess v a s o c o n s t r i c t i v e
all
and hypotensive a c t i v i t y ,
and
o f them are strong a n t i b i o t i c s . The inotropic
bufogenins effect,
responsible
be mentioned f u r t h e r
in t h i s
remarkable growth i n h i b i t i o n field,
however, i s s t i l l
2.2 L e p t o d a c t y l i n a e
context
America,
derivatives, leptodactylin histamin,
like
heart
activity
with
effect
(ref.3) on
contain
and
they
do
microorganisms.
L.
ocellatus
considerable
N-methylserotonine,
not
not
possess
Research
(ref.
4).
amounts
native of
to
in
South
a
this
Of
a
new
type
and
5-hydroxytryptamine
b u f o t e n i n and b u f o t e n i d i n as
and a c e t y l - h i s t a m i n s ,
s y n t h e s i z e d in v i t r o
as
positive need
( B u l l Frogs)
and c a n d i c i n
methyl-,
the
limited.
Leptodactylus pentadactylus Central
for
and the b u f o t o x i n s a c t i n g as c o n v u l s i v e t o x i n s ,
are
well
substances
as like
and spinaceamin. Spinaceamin can be
by a biomimetic r e a c t i o n o f histamin
in aqueous s o l u t i o n at pH 6.8, and room temperature
(ref.
and 5).
formaldehyde
329 .o. //o
S HO"
v ~ vH Bufotalin
I
H /NH1 CO-(CH1)~-CO-NH-CH-(CH2)3-NH-C-I "~NH COOH Bufotoxin
HOrN
I~N(CH,)2 H Bufotenin
eO~NJ~N(CH3) H Bufotenidin
J
H3C\ /CH3
H~C\ /CH3 eO3SO.,,,.~NE-----~
Lkr
H
H
Bufothionin
Leptodactylin
Dehydrobufotenin
eo~(CH Candicin i-IN~
HN~N
NH
NHa
l-listamin
Spinaceamin
~)~
330
Deka-, undeka-, and dodeka-peptide amides of the t a c h y k i n i n family found in the n e o t r o p i c a l l e p t o d a c t y l i d frogs of the genus of these peptides is physalaemin ( r e f .
Physalaemus.
are One
6).
Pyroglutamyl-Ala-Asp-Pro-Asp (NH2) -Lys-Phe-Tyr-Gly-Leu-Met (NH2) It
acts as a
strong
hypotensive e f f e c t
v a s o d i l a t o r and t h e r e f o r e
(ref.
possesses a
long
lasting
7).
Closely r e l a t e d to physalaemin i s u p e r o l i n from Uperoleia rugosa: Pyr-Pro-Asp-Pro-Asn-Ala-Phe-Tyr-Gly-Leu-Met-NH2 as w e l l
as
litorin
leptodactylinae
from
(ref.
the
same species
and
from
other
Australian
8) :
Pyr-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2 Steroids or a l k a l o i d s have not Leptodactylidae; activity
it
been
found
so
far
in
the
skin
of
would c e r t a i n l y be w o r t h w i l e to look f o r the a n t i b i o t i c
of the peptides.
2.3 Ranidae (True Frogs) In the skin of Rana temporaria and Rana nigromaculata,
bradykinin,
a
hypotensive and smooth muscle e x c i t i n g substance has been found (7): Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg The skin gland s e c r e t i o n of the water f r o g ,
Rana esculenta, d i s t r i b u t e d
over Europe, contains a high molecular weight peptide with strong activity
(ref.
9).
2.4 Discoglossidae
all
hemolytic
This secretion also acts as a skin i r r i t a n t . (Disc Tongues, Unks)
Among the discoglossidae the red b e l l i e d unk, Bombina bombina, and y e l l o w b e l l i e d unk, Bombina variegata, H.Michl and co-workers ( r e f .
10).
bombina contains 10% s e r o t o n i n ,
The
have been i n t e n s e l y dried
skin
free amino acids,
the
investigated
secretion
of
Bombina
and basic peptides l i k e :
Ala-Glu-His-Phe-Ala-Asp (NH2)
by
331
From
the
serotonin,
secretion
of
Bombina
variegata,
gamma-amino-butyric
acid,
and nonapeptides of the f o l l o w i n g type have been i s o l a t e d : Ser-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe
and Gly-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe H. M i c h l could unambiguously show t h a t these
peptides
are
active
against
microorganisms. In a d d i t i o n ,
Barberio et a l .
(ref.
m o l e c u l a r weight from the skin of shows a n t i b a c t e r i a l
11) have i s o l a t e d a Bombina
protein
variegata
of
pachypus
6700
which
D
also
properties.
2.5 Dendrobatidae
(Tree Frogs, Coloured Frogs)
These b r i g h t and c o l o u r f u l
f r o g s are endemic t o C e n t r a l America and
the Northwestern p a r t of South America. The skin s e c r e t i o n s are used by
to the
indigenous people as arrow poison f o r hunting e s p e c i a l l y in the Choco region o f Columbia ( r e f .
12).
Most of the chemical work of i s o l a t i o n been done at the N . I . H . their
co-workers.
and
in Bethesda, USA,
by
They were able to i s o l a t e
several different
structure B.Witkop
by mass spectrometry
per kg body weight substance at a l l . irreversably tool
derivatives.
(mouse, i . m . ) ,
being
elucidation, (ref.
i n the s t u d i e s of sodium channels.
c a r b o x y l i c acids. qualitatively
(refs. under
Pumiliotoxin C class. f a r as
the
toxicity
most
batrachotoxinin
differs
it
is
without
toxic
has become the
A,
with
o f the d i f f e r e n t
quantitatively
first
of
any 2
ug
nonprotein t o keep open
an
important
batrachotoxins different
are
pyrrol
batrachotoxins is
according t o the acid p a r t
14, 15).
Others are the f a m i l i e s More i n t e r e s t i n g
the
Chemically
Although the a c t i v i t i e s
the same, i t
o f the molecule
at
of
13).
s p e c i a l pharmacologic a c t i v i t y
the sodium channels of nerve c e l l s
e s t e r s o f a 20-hydroxy s t e r o i d ,
and
alkaloids
B a t r a c h o t o x i n has a LD50
thus
Because o f i t s
J.W.Daly,
of
The most famous of the many a l k a l o i d s i s o l a t e d so f a r doubt b a t r a c h o t o x i n and i t s
has
and
a vast amount
s t r u c t u r e types and to do s t r u c t u r e
by X ray c r y s t a l l o g r a p h y l a t e r
elucidation
o f the g e p h y r o t o x i n s and the h i s t r i o n i c o t o x i n s .
antimicrobial
aspects
The expression " t o x i n " against
higher
are
the
i s somewhat
animals
is
low
compounds misleading in
of
the
in
so
comparison
to
332 O .CH3
CIH3 ~ H H.,,J I HO. ~
.or
I __1-.6 II 6
CH3/'\N/\H H
Batrachtoxin
NCH3
H
O ?H30/~,~CH3 H
!
HO. ~
H Homobatrachotoxin
I. . . . !--6 II 6
NCH3
tt
.
H H"
H H/
/
H
H - ~ ~ N ~ ..C~_/H \ C
C~ H C
\ / H H Histrionicotoxin
CH3 !I .N~..,~ZH 2-CH 2-CH:I H H H Pumiliotoxin C
%
/.~(/ H \\ H'" 0 HH~c~C~ I
\
/H
\ c
\
H Dihydro-iso-histrionicotoxin
CHa-CHa -OH I;I
C
H
CHa
~CI-I
I /c ~/ HC=CH Gephyrotoxin
333 H3C
I~I
CH~
H~C~ /CH2 /C=C~ H
H,C
N" "'~ ~x.~
CHOH I CH2 I
CH~OOH ~ H I CHOH I
CH3
Pumiliotoxin A Br
[~
Pumiliotoxin B
Br
H~
Pr
CH3
H 3 C ~ c = C~ C H a
CH~
EtOzC
lit
Et
H
Br
Pr
Pr
Et
a
Pr 7oz
92z
EtOzC
H H
49
Pr .f.
l't, mtl t o t o x t n
C
Br
H=N
H "" Pr
~
~ ""Pr
51 (s)
( - ) l'umtl t o t o x t n
C
lal o - -13.1" (natural
O
OK " ~
= -1~.9 ~
H
OH
~H
H
Synthesis of a 5-hydroxy-cis-decahydroquinoline analog of pumiliotoxin C A i) KOH, ii) NH.~, iii) Urushibara nickel. B i) PtO2, H2
334
b a t r a c h o t o x i n , ranging in the order of 1 to 100 mg per kg the other hand they are a c t i v e against microorganisms, t h i s respect t o x i c i t y
body
weight.
so t h a t at
least
i s observed. A complete survey of these a l k a l o i d s
been published by Daly ( r e f .
has
16).
There are several ways to synthesize these a l k a l o i d s . in the a u t h o r ' s l a b o r a t o r y ,
On in
The way developed
an i n t r a m o l e c u l a r enamine c y c l i z a t i o n ,
allows the synthesis of the n a t u r a l l y
not
only
occurring stereoisomers but also
others by v a r i a t i o n of the s t a r t i n g m a t e r i a l s
and/or
reaction
the
conditions,
and, by the same procedure, the a l k a l o i d s of the gephyrotoxin class may also be obtained ( r e f s .
17, 18, 19). Results on the a n t i m i c r o b i a l a c t i v i t y
of the
gephyrotoxins are,however, not yet a v a i l a b l e . 2.6 Pipidae (Tongueless Frogs) Xenopus l a e v i s i s the only frog i n v e s t i g a t e d in t h i s respect.
Serotonin
and b u f o t e n i d i n have been i s o l a t e d fom i t s skin glands ( r e f . 2 0 ) . R e c e n t l y the magainins, a n t i m i c r o b i a l peptides w i t h 23 isolated
(ref.
amino
acid
residues
have
been
21):
Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Gly-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-GluIle-Met-Lys-Ser and Gly-Ile-Gly-Lys-Phe-leu-His-Ser-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Gly-GluIle-Met-Asn-Ser 2.7 Hylidae (Leaf Frogs) Hyla arborea and other h y l i d frogs are d i s t r i b u t e d in wide parts of the Old World, the New World and Asia. Low their
skin secretions are s e r o t o n i n ,
molecular
bufotenin,
compounds i s o l a t e d
and histamin.
Hyla arborea moreover contains a hemolytic a c t i v e structure,
still
a small peptide,
peptide
of
a c t i v e in s o l u t i o n s of 1:200~000, while from Hyla caerulein,
has been obtained ( r e f .
7):
Pyr og1 utamy 1-G 1 u- Asp- Tyr- Thr-G 1y - Try p- Met- Asp- Phe S03H
from
unknown caerulea
335 3.
URODELA (Salamanders, Newts) 3.1 Salamandra (Salamanders) Like
the
toads, the salamanders played a big r o l e in mythology. For i n s t a n c e i t
Salamanders are known t o be poisonous since a n c i e n t
times.
was
considered as an animal capable o f e x t i n g u i s h i n g f i r e , a n d
even k i l l i n g
not
only a s i n g l e man but complete people. The animal t h e r e f o r e played i t s in w i t c h c r a f t , it
and i t
was assumed t h a t l o o k i n g at a salamander
role
or
putting
i n t o a d r i n k or a soup would cause death. Thus the most curious
stories
are r e p o r t e d t h a t i n d i c a t e t h a t the w r i t e r s themselves.
Laurentius,
the source of t o x i c i t y , Zalesky i n 1866, research
on
but the f i r s t
these
experiments
amphibian
toxins
pharmacology, c h e m i s t r y , (ref.
23), The
so t h a t j u s t biologically
had
never
seen
a
salamander
in 1768 discovered the skin gland s e c r e t i o n detailed being
experiments
the
(ref.22).
were
starting
A
point
review
on
active
compounds
be
for
by all
toxicology,
and b i o c h e m i s t r y has been published the main r e s u l t s
to made
by
Habermehl
s h a l l be described here. of
the
(Salamandra salamandra salamandra) are s t e r o i d a l
European
alkaloids
of a
salamander new
type
in so f a r as the basic n i t r o g e n atom i s p a r t o f an enlarged r i n g A, and the oxygen bound to carbon atom 3 i s not a hydroxy or a keto but forms t o g e t h e r w i t h the n i t r o g e n ,
o x a z o l i d i n r i n g system o f remarkable s t a b i l i t y . some o f the minor a l k a l o i d s i s replaced by a ding the n i t r o g e n ,
results
The o x a z o l i d i n carbinolamine
as
usual, 3
an
system
in
system
inclu-
carbon atoms 1, 2 and 10, as w e l l as C-19 o x i d i z e d to an
aldehyde group. Samandarin, the main a l k a l o i d , t h a t acts
group
and the carbon atoms 1, 2, and
on the c e n t r a l nervous system,
from primary r e s p i r a t o r y
i s a very potent
and
causes
paralysis,without
neurotoxin
convulsions.
Death
damage of the h e a r t .
It
moreover possesses remarkable h y p e r t e n s i v e and l o c a l a n e s t h e t i c p r o p e r t i e s . Besides the a l k a l o i d s , they have not is toxic for
to
yet been all
hemolytically examined
a c t i v e peptides have been
regarding
animals, the l e t h a l
X-ray c r y s t a l
and s t r u c t u r e
structure.
doses f o r the f r o g
the mouse o.0034 gm, and f o r the r a b b i t The i s o l a t i o n
their
being
found, 0.019
gm,
o . o o l gm per kg body w e i g h t .
d e t e r m i n a t i o n by i . r .
spectroscopy and
s t r u c t u r e a n a l y s i s have been described elsewhere ( r e f . 2 3 )
Sch~pf, and by Habermehl and coworkers. The s t r u c t u r e s are shown on the next page.
but
Samandarin
represented
by by as
336 H3
CH
CH~
CHj
CH~.~
.~~~~J.~-.~ o.
H H
tt
Samandenon
Samanin OH
CH 3 !,-I
Cit
OH
CH.~
o
H
tt
Cycloneosamandion
Cycloneosamandaridin ('1t.~_ C'll~ II H
Samandarin
OH, ~ C)II
HN
~ .Xs
H
tl
I~ H
Samandaridin
~0
337
Samandarin and remarkable a c t i v i t y
the
other
alkaloids
of
a g a i n s t microorganisms
the
(refs.
Geotrichum candidum, Saccharomyces c e r e v i s i a e , viride
as w e l l as b a c t e r i a l i k e
Escherichia coli, of their
f o r s e v e r a l weeks u n t i l l infections
Staphylococcus
in local lesions, been
aureus,
infections
and f i n a l l y
such animals are held under s t e r i l e
enough venom has
a
Trichoderma
D e t o x i f i e d animals get
skin w i t h i n a few days, r e s u l t i n g
death. On the other hand, i f
possess
including
Candida c r u s e i ,
Bacillus subtilis,
and Proteus m i r a b i l i s .
same type
1,24,25)
regenerated
in
conditions again,
such
are not observed, even when put back i n t o a t e r r a r i u m .
The b i o s y n t h e s i s of
salamander
alkaloids
Insertion
o f n i t r o g e n between the carbon atoms
skeleton,
and h y d r o x y l a t i o n of carbon
d e g r a d a t i o n of
the
side
chain
atom
to
form
starts 2
16
from
and is
3
cholesterol.
of
followed
alkaloids
like
the
steroid
by
stepwise
samandinin
or
samandaridin. The c l a s s i f i c a t i o n still
controversial
as
subspecies had been maculosa
(black
regards the
described
animals
maculosa t a e n i a t a
o f the European salamander among h e r p e t o l o g i s t s different
under
with
Austria,
s t r i p e d subspecies i s endemic
to
Germany.
that
S.m.taeniata
is
found, and
interesting
contains
samandaron and
orange
cycloneosamandion is
as
replaced
by
& Taricha
differ
the
and
and
main
bigger
their
alkaloid,
samandaridin.
In
maculosa Salamandra
two on the back
Belgium, in
is two
than
the
whereas
the
and
Northern
venom, followed
S.m.maculosa
too. by we
The main a l k a l o i d here i s samandaron,
O-acetyl-samandarin
investigation,
been described also in o t h e r amphibians 3.2 T r i t u r u s
Salamandra
dots),
yellow stripes,
France,
they
samandarin
o b s e r v a t i o n deserves f u r t h e r
of
Formerly,
Slovenia and C r o a t i a ,
Spain,
however, no samandarin at a l l .
samandarin
names
or
The dotted animals which are
s t r i p e d ones occur i n Bavaria, It
the
yellow
(black w i t h f o u r p a r a l l e l
and one each on the s i d e s ) .
subspecies.
since
(ref.
such
(ref.
26).
differences
This have
20).
(Newts)
The European newts T r i t u r u s v u l g a r i s have been s t u d i e d .
cristatus,
Most s t r i k i n g
Tr i t u r u s
alpestris
and T r i t u r u s
i s the strong h e m o l y t i c
of the s e c r e t i o n o f up to a c o n c e n t r a t i o n o f 1" 1.000.000.000.
In
activity addition
338 amylases, phosphatases and arylamidases have been found, but no s t e r o i d s or alkaloids
(refs.
27,
28).
The
occurrence
of
tetrodotoxin
debatable, obviously due to r e g i o n a l subspecies ( r e f . Tetrodotoxin, however, has been i s o l a t e d from Taricha torosa, Taricha r i v u l a r i s , been c a l l e d
tarichatoxin
but
bacterial toxin
(ref.
29)
was l a t e r
(ref.
30)
We now
still
Californian
found to be know t h a t
has
newts
formerly
identical
tetrodotoxin
with is
a
but we have no idea about t h i s kind of symbiosis
between bacteria and amphibia. I t bactericide
the
and Taricha granulosa; i t
t e t r o d o t o x i n from Tetraodontidae.
is
27).
but i t
c e r t a i n l y does not act as a fungicide
or
might act as a r e p e l l e n t against predators of
the animals themselves or of t h e i r egg c l u s t e r s as
has
b e e n observed
in
Tetrodotoxin containing f i s h .
0 ~
0
H2N
H H
OH
Tctrodotoxin
4.
CONCLUSION During e v o l u t i o n a l l
amphibians have developed skin glands in which
great v a r i e t y of d i f f e r e n t peptides,
steroids,
chemical compounds i s
alkaloids,
i n t e r e s t i n g pharmacological system, as neurotoxins,
produced.
and biogenic amines. Most
activities
on
the
heart,
or on smooth muscle preparations.
T h e y comprise
of
them
on
the
possess vascular
Some of them
by blocking or opening sodium channels, and others as hallucinogens. d e t a i l e d review see r e f s .
31 and 32. I t
i s f a s c i n a t i n g t o see
in chemistry as w e l l as in pharmacological or t o x i c o l o g i c a l these compounds, and i t
is
species or subspecies d i f f e r These a c t i v i t i e s ,
also
fascinating
that
even
substances ( r e f . 1 ) ,
the
act For a
variety
properties closely
of
related
in the compositions of t h e i r secretions.
however, cannot be the
original
purpose
development. Most of them possess a n t i m i c r o b i a l a c t i v i t y , the crude skin gland
a
secretion
is
more active
i n d i c a t i n g t h a t the secretion
animals against i n f e c t i o n s by microorganisms.
than as
of
their
and in many cases the
whole
single
pure
protects
the
339
REFERENCES 1 G. Habermehl and H.J. Preusser, Z.Naturforschg., 24B (1969) 1599-1601. 2 V. Deulofeu and E.A. R~veda, "The Basic Constituents of Toad Venoms" in "Venomous Animals and Their Venoms (W.B~cherl & E.Buckley, Eds.), Vol. I I Academic Press, New York, 1971, Chapter 38. 3 K. Meyer and H. Linde, "Collection of Toad Venoms and Chemistry of the Toad Venom Steroids", i b i d . , Chapter 40. 4 V. Erspamer, M.Roseghini, and J.M. Cei, Biochem.Pharmacol. 13 (1964), 1083. - V.Erspamer, T . V i t a l i , M.Roseghini, and J.M.Cei, Arch. Biochem. Biophys. 105 (1964), 620. - J.M.Cei, V.Erspamer, and M.Roseghini, Syst.Zool. 16 (1967) 328. 5 G. Habermehl and W. Ecsy, Heterocycles, 5 (1976) 127-134. - G.Habermehl and H.J. Preusser, Z. Naturforschg. 25 B (1970) 1451-1452. 6 A. Anastasi, V. Erspamer and G. Bertaccini, Comp. Biochem. Physiol. 14 (1965) 43 f f . 7 V.Erspamer, and P.Melchiorri, Pure Appl.Chem. 35, (1973), 463-494. 8 V.Erspamer, P.Melchiorri, M.Brocardo, G.Falconieri, P.Erspamer, P.Falaschi, G.Improta, L.Negri, and T.Reuda, Peptides 2,(1981), Supp. 2, 7-16. 9 G.Kiss and H.Michl, Toxicon, 1, (1962), 33-36. 10 A. Csordas and H. Michl, Toxicon, 7 (1969) 103-108. 11 C.Barberio, G.Delfino and G.Mastromei, Toxicon 25, (1987), 899-909. 12 B. Witkop, Experientia (Basel),27 (1971) 1121 f f . 13 J.W. Daly, T. Tokuyama, G. Habermehl, I . L . Karle und B. Witkop, Liebigs Ann. Chem.,729 (1969) 198-204. 14 J.W. Daly, B. Witkop, T. Tokuyama, T. Nishikawa, and I . L . Karle, Helv. Chim. Acta,60 (1977) 1128 15 J.E. Warnick, E.X. Albuquerque, R. Onur, S.E. Jansson, J.W. Daly and B. Witkop, J. Pharm. Exp. Ther.,193 (1974) 232. 16 J.W. Daly, "Alkaloids of Neotropical Poison Frogs" in "Fortschr.d.Chemie Org. N a t u r s t . , Springer-Verlag, Wien, New York, 1982. 17 G. Habermehl and W. Kissing, Chem. Ber.,107 (1974) 2326-2328. 18 G. Habermehl, H. Andres und K. Miyahara, Liebigs Ann. Chem.,(1976) 15771583. 19 G. Habermehl and O. Thurau, Naturwissenschaften, 67 (1980) 193. 20 J.W.Daly and B.Witkop, "Chemistry and Pharmacology of Frog Venoms" in "Venomous Animals and Their Venoms" (W.BOcherl and E.Buckley, Eds.) V o l . I I , Academic Press, New York, 1971, Chapter 38. 21 M. Z a s l o f f , Proc. Natl. Acad. Sci. USA, 84 (1987) 5449-5453. 22 S. Zalesky, Med.Chem Untersuch. Hoppe-Seyler, 1 (1866) 85 f f . 23 G. Habermehl, "Toxicology, Pharmacology, Chemistry and Biochemistry of Salamander Venom" in "Venomous Animals and Their Venoms" (W. BOcherl & E. Buckley, Eds.), Vol. I I , Chapter 42, Academic Press, New York, 1971. 24 H.J.Preusser, G.Habermehl, M.Sablofski and D.Schmall-Haury, Toxicon, 13, (1975), 285-289. 25 G. Habermehl and H.J. Preusser, Abh. Dtsch. Akad. Wiss.(Berlin) (1972) 447-449. 26 G. Habermehl, Liebigs Ann. Chem. 679 (1964), 164-167. 27 G.Habermehl, unpublished. 28 R. Jaussi, Ph.D. Thesis, Basel, 1977. 29 U.Simidu, T.Noguchi, D.F.Huang, Y.Shida and K.Hashimoto, Appl. Envir. M i c r o b i o l . 53 (1987), 1714. 30 H.C.Krebs and G.Habermehl, in preparation 31 G.Habermehl, Naturwissenschaften 56 (1969), 615-622. 32 T.Foorden, Doctoral Thesis, T i e r ~ r z t l i c h e Hochschule, Hannover, 1990.
This Page Intentionally Left Blank
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
Bioactive Metabolites of the Genus
341
Phomopsis
Youla S. Tsantrizos
The Phomopsis fungi are plant pathogens often associated with diseases of agricultural crop plants and produce. Among the best known examples are fungi which cause post-harvest fruit-rot, such as P. viticola, a pathogen of grapes and vines, P. citri, a pathogen of citrus fruits and trees, and the seedborn pathogens of soybeans (P. Iongicolla and P. phaseolO and lupines (P. leptostomiformO. Phomopsis fungi are also known to infect ornamental shrubs and perennial flowering plants causing leaf necrosis and stem cankering. These include the host-specific pathogens of sunflower and field bindweed, P. helianthi and P. convolvulus respectively. A complete list of all the known pathogens in this genus is beyond the scope of the present chapter and only those organisms which represent recent examples of interest in chemistry will be discussed in some detail. Some of the chosen examples are fungi which produce phytotoxic metabolites which are often associated with the disease symptoms caused by the pathogen on its host plant. Others produce metabolites which are toxic to insects, animals and occasionally humans. The chemical structures and biosynthetic origin of these compounds are just as diverse as their biological activity. Thus, some representative examples of general interest in natural product chemistry will be described.
Phomo_Dsis convolvulus Phomopsis convolvulus, 1 is a host-specific pathogen of the perennial plant Convolvulus arvensis, better known as field bindweed or wild morning glory. Infestations of this plant are encountered in both city green spaces and cultivated lands, and they represent a serious agricultural problem around the world with the exception of the tropics. 2 Phenoxy herbicides which are routinely used in agriculture for the control of common weeds, are generally ineffective in suppressing the spread of bindweed. 3 Consequently, a number of independent studies on its possible biological control have recently been initiated. One of these efforts led to the isolation and identification of the host-specific pathogen P. convolvulus, whose infection results in necrotic lesions and wilting of the leaf tissues of bindweed. Initially, it was also noted that the lesions formed on the infected leaves were often surrounded by yellow
342
halos, characteristic of symptoms caused by phytotoxins. 4 The same necrotic spots could be reproduced on the leaves of young bindweed plants when exposed to cellfree extracts of P. convolvulus in leaf-puncture bioassays, 5 further supporting the production of phytotoxins by this organism. Subsequent investigation into the extracellular metabolites of P. convolvulus led to the isolation of the known steroids, ergosterol (1) and ergosterol peroxide (2) and four novel phytotoxins; the phthalides, convolvulanic acid A (3), convolvulanic acid B (4) and convolvulol (5), and the o~pyrone convolvupyrone (6).7
HO
0 (1)
(2)
OCH30
R2
,,o /
O
R1
(3),
R 1 = OH,
R 2 = COOH
(4),
R1 = H ,
R 2 = COOH
(5),
R 1 = H,
R 2 = CH2OH
c
'T' H
z~,,,,COOH H3C 1
(6)
The cultures of P. convolvulus were grown on moist barley grains at room temperature for a period of 28-30 days. Methanol extraction of the infected grains, followed by solvent partitioning of the metabolite mixture, led to the separation of the steroid compounds from metabolites 3, 4, 5 and 6 which dissolve readily in basic aqueous solvent. While the final purification of metabolites 1 and 2 was easily achieved by silica gel chromatography, the purification of the polar metabolites 3-6
343
was much more difficult and could only be accomplished by reverse-phase flash column chromatography 6 and HPLC using a C-18 reverse phase column. 7 IR, high resolution MS and extensive NMR analysis of metabolites 3, 4 and 5 led to the structural assignments of 4-carboxy-3-hydroxy-7-methoxy-6-methyl-l(3H)isobenzofuranone (3) and 4-carboxy-7-methoxy-6-methyl-l(3H)-isobenzofuranone (4) for convolvulanic acids A and B respectively, while convolvulol (5) was shown to be the reduced analogue of compound 3, 4-(hydroxymethyl)-7-methoxy-6-methyll(3H)-isobenzofuranone, e Extensive analysis by 1H-13C coupled NMR experiments and selectively decoupled experiments provided final confirmation for both the proposed structures and the chemical shift assignments for each carbon signal. For example, a chemical shift difference of only 0.7 ppm was observed between the two carbonyl signals of metabolite 3 (5 166.4 and 165.7); both of which appeared as doublets due to 3j coupling in the 1H_13C coupled spectrum, making their assignment difficult. Selective de-coupling of the aromatic H5 proton led to the collapse of the signal at 5 165.7 into a singlet, while leaving the doublet at 5 166.4 uneffected. Similarly, selective irradiation of H3 led to the collapse of only the doublet at ~ 166.4 confirming the assignment of the lactone and carboxylic acid carbons at 5 166.4 and 165.7 respectively. During NMR analysis of metabolites 3 and 4, an interesting complication was encountered in the acquisition of the 130 NMR data. At room temperature, the signals associated with the carbonyl of the carboxylic acids (3, 5 166.4 and 4, 168.4) and the aromatic C-4 carbons (3, 5 121.9 and 4, $120.5) were extremely broad in the normal 130 NMR or completely non-observable in the DEPT and APT NMRs. However, at temperatures of -45~ to-55~ these signals were sharp and clearly visible. Such an effect is often characteristic of aromatic carboxylic acids and it has been observed before with structurally related metabolites of the fungus Aspergillus duricaulis. 9 The most likely explanation for this effect is the degree of dissociation of the acidic proton, thus the existence of an equilibrium between the acid and its carboxylate ion, which causes the broadening of both the carbonyl carbon and the carbon to which it is directly attached. Similar spectral analysis of metabolite 6 led to its assignment as 3-[4methoxy-3-methyl-o~-pyron-6-yl]-2-methyl-2-butenoic acid. The trivial name of convolvupyrone was given to this novel ~-pyrone and molecular modeling calculations were carried out in order to establish its most favorable conformation. A nearly planar conformation which would permit extensive conjugation between the two carbonyl groups and the double bonds as shown in structure 6' (placing the methyl group at C-2 away from H5' in order to avoid any steric hindrance), was initially assumed. However, NOE NMR experiments did not show any positive effect
344
between H3 and H5' ((5 6.62, 6.91 respectively) while a strong positive effect was observed between H5' and the C-2 methyl ((~ 2.42). Molecular modeling calculations ~~ for both structures 6' and 6 gave an energy difference of ~3.2 kcal in favor of the latter, which translates to an equilibrium ratio of about 1/99 for these two conformers, providing adequate support for the NOE NMR data obtained. All other spectral data, high resolution MS, IR and 2D-NMR were consistent with the proposed assignment for convolvupyrone.
0 H3C= 'J~O
c. o" T
H
(6')
OH3 rl
0 H3C~o
c
H
Z~COO H3C 1 4
r
(6)
Biological testing of all metabolites isolated from P. convolvulus was at first carried out using the small aquatic macrophyte Lemna, commonly called duckweed. Lemna plants consist of a leaf-like front with a single root, they propagate vegetatively and many of their species have been used in bioassays due to their high sensitivity to phytotoxins. 11 Biological testing of ergosterol (1) and its peroxide (2) was limited by the poor solubility of these compounds in aqueous solvents. In Lemna assays, a suspension of 0.5 mg of each compound in a solution of 0.5% ethanol to 95% Lemna growth medium showed no effect for ergosterol and only 10% inhibition of growth for its peroxide. The phytotoxic effects of ergosterol peroxide have been reported before 12 and it is conceivable that this metabolite may have a much more pronounced effect when produced in vivo in the tissues of infected bindweed plants. Metabolite 4 was found to be the most potent phytotoxin of the other four metabolites, causing total inhibition of growth and 100% chlorosis of the Lemna plants within 12 hr at concentrations of 5.9x10 -4 M and within 24 hr at concentrations of 3.5x10 -4 M. At 5.9x10 -4 M concentrations, metabolites 3 and 5 were also found to inhibit the growth of Lemna plants by approximately 80% and 50% respectively. In contrast, the phytotoxic activity of metabolite 6 was very weak at all concentrations tested. The relative potency observed in Lemna bioassays was consistent with the results obtained in bioassays using leaf-cuttings of young bindweed plants. Ergosterol (1) was found to be completely non-toxic and only minor effects were
345
observed with its peroxide (2), convolvulol (5) and convolvupyrone (6). The most intense toxicity symptoms were induced by the two convolvulanic acids, A (3) and B (4). At concentrations of 3.5x10 -4 M, convolvulanic acid B (4) caused wilting and browning of the leaf tissues after only four hours, while A (3) caused the same symptoms twelve hours later. Based on these biological properties, it is reasonable to assume that compounds 2, 3, 4, 5 and 6 act as the chemical mediators of the disease inflicted by P. convolvulus on its host plant, field bindweed. Phytotoxic phthalides are fairly uncommon fungal metabolites. Interestingly, in spite of strong structural similarities between the metabolites of P. convolulus and those of A. duricaulis (eg. metabolite 3 of P. convolvulus and metabolite 7 of A. duricaulis), there is no analogous similarity in their biological activity. For example, metabolite 7 exhibited strong antibiotic activity against Bacillus subtilis, causing an 8 mm inhibition zone on a paper-disk diffusion assay at a concentrations of 0.1 lag,9b while metabolite 3 failed to cause any inhibition to B. subtilis even at concentrations of 50 l~g under similar bioassay conditions. 7
OCH30
OCH30 ~
CHO (3)
PhomoDsis
(7)
helianthi
P. helianthi (Diaporte helianthi, perfect stage) is a pathogen causing leaf necrosis and steam canker of sunflowers. It was first discovered in 1980 in Yugoslavia 13 and since then it has been found in other countries, severely effecting sunflower crops. 14 Recently, the phytotoxic compound phomozin (8), an ester analogue of orsellinic acid (9), was isolated from the liquid culture of P. heliantht15 and shown to cause brown lesions on the leaves of sunflowers, extending from the point of application of the toxin. The overall effects were closely related to those observed on sunflowers infected with P. he/ianthi, implicating the involvement of this metabolite in the disease symptoms caused by the fungus. The phytotoxic activity of phomozin was also evaluated on melon, soybean, corn, pea and tobacco plans and some host-selectivity was observed. 15
346
, j-CH, HO
HO
(8)
(9)
The structure elucidation of phomosin (8) was primarily based on its NMR data and X-ray crystallography. The latter suggested an erythro absolute configuration for the dimethylglyceric acid part of the molecule. However, comparison of the 1H NMR chemical shifts for the two equivalent methyls in an authentic sample of erythro dimethylglyceric acid with those of phomosin [-CO0-CH(CH3)-COH(CH3)-COOH] did not confirm this assignment. Thus, the absolute stereochemistry of metabolite 8 requires further investigation. PhomoDsis _
iuniDerovora
P. juniperovora Hahn is the causal agent of the most damaging disease of eastern red cedar (Juniperus virginiana L.) and many other Cupressaceae trees. Although its metabolites are known more for their bright colouration than for their biological activity, the orange-red pigments are believed to be diagnostic of P. juniperovora infection. 16 The chemical structure of the main compound was identified to be the red pigment 7-methoxy-2-methyl-1,2,3,4,5-pentahydroxy-1,2,3,4tetrahydroanthraquinone (10), 17 previously isolated from Altemaria solani and named altersolanol A. 18 A number of other known anthraquinones have also been isolated from the cultures of P. juniperovora.
OH
0
OH
0
OH
(lo)
347
PhOmODSiS leDtostromiformis
Phomopsis leptostromiformis occurs in nature as a parasite and saprophyte of certain lupin plants, which are in turn associated with the animal disease lupinosis, a hepatotoxic condition characterized by severe liver damage and jaundice. Field outbreaks of lupinosis have been reported in grazing sheep, cattle, horses and pigs in Europe, Australia, New Zealand and South Africa where lupins are cultivated extensively for livestock feeding. Phomopsin A (11) and several other related metabolites are produced by P. leptostromiformis, in cultures grown on lupin seeds, 19 liquid media 2~ or maize kernels. 21 The isolation of 11 from P. leptostromiformis grown on sterilized maize was achieved via extraction of the infected kernels with methanol, followed by partial purification by chromatography through a macroreticular polystyrene resin (XAD-2) and a Sephadex LH-20 column. 21 Phomopsin A was obtained in its pure form after further purification on a DEAE cellulose column, using an ammonium hydrogen carbonate buffer, and crystallization from a mixture of methanol/ethanol/water. FAB MS of 11 suggested an empirical formula of C36H45CIN6012 while the IR data was consistent with the presence of amide bonds. Seven carbonyl and four methine carbon signals were observed in the 130 NMR of 11 indicating the possible involvement of several modified amino acids. However, some of the key structural features of phomopsin A (11) were identified by extensive GC-MS analysis. Catalytic hydrogenation and NaBH4 reduction of 11, followed in each case by acid hydrolysis and analysis of the amino acid mixture by GC-MS was carried out. The acid hydrolysate of the otherwise chemically unchanged metabolite 11 was also subjected to GC-MS analysis and the combined results led to an amino acid composition of 3,4-didehydroproline, E-2,3-didehydroisoleucine, E-2,3-didehydroaspartic acid, N-methyl-3-(3-chloro-4,5-dihydroxyphenyl)-3-hydroxyalanine, 3,4didehydrovaline, and 3-hydroxyisoleucine. Similar results had been suggested earlier from biosynthetic studies showing the incorporation of L-[U-14C]valine, L-[U14C]isoleucine, L-[U-14C]proline and L-[U-14C]phenylalanine into the structure of phomopsin A (11).22 Detailed NMR studies of 11, using extensive homonuclear decoupling, 13C1H selective population inversion (SPI) 23 and 1H-13C selective decoupling experiments, provided further support for the proposed amino acid content and defined their sequence in a cyclic peptide. 2~ However, the correct structure of phomopsin A was later shown to be the 13-membered macrocyclic ring 11, and not that of a cyclic hexapeptide. 24 Phomopsin A is known to have a tubulin-binding action, interfering with the formation of mitotic spindles thus blocking cell division. 2s
348
Based on its structure, an additional function as an ionophore-type of compound has also been suggested. 24
cO,. " o
' o.
COOH
9
COOH
(11)
PhomoDsis
oblonoa
P. oblonga (Desm.) Trav. is a saprophyte frequently found in the outer bark of healthy trees of the Ulmus species [particularly wych elms (U. glabra)] and in the phloem of stressed trees infected by Ceratocystis ulmi, the causative agent of Dutch elm disease. 26 In 1981, it was discovered that P. oblonga interferes with the breeding process of bark beetles of the Scolytus species, which are the vectors of C. uIm/. 27 Since this effect could potentially be useful in the biological control of Dutch
elm disease, the phenomenon was further investigated. During the initial study, it was noted that the inner bark of wych elm trees, suffering from Dutch elm disease, contained zones infected with P. oblonga which showed very limited evidence of Scolytus breeding.
It was subsequently
demonstrated that the presence of P. oblonga caused abnormalities in the development of the beetle's larvae and retarded their growth. In some cases, it was also observed that larval galleries swerved away from Phomopsis infected tissue into uninfected areas and in bioassays where S. scolytus and S. multistriatus beetles
were forced to breed on Phomopsis infected elm bark, it was observed that female beetles attempting to cut maternal galleries usually abandoned their efforts.
In few
cases where galleries were completed and eggs laid, the number of resulting larvae was dramatically lower than in the control and only a few of them developed into adult beetles. 27
349
Two morphologically distinct types of P. oblonga fungi were later isolated from infected elm trees 2eb and investigated for the production of biologically active metabolites having feeding deterrent activity against the Scolytus beetles. 2e Each isolate was grown on both malt extract and natural elm phloem medium, in both surface and shake cultures. A bioassay-guided isolation scheme was developed using female S. scolytus beetles for assessing the feeding deterrent activity of different extracts. Among the bioactive compounds isolated from the more commonly occuring type of P. oblonga2ebwas the novel norsesquiterpene 7-1actone oblongolide (12). 28 The culture filtrates of a P. oblonga, grown on a malt extract medium in surface culture, were extracted with ethyl acetate and the crude metabolite mixture obtained was purified by column chromatography in order to isolate metabolite 12 in yields of 1-2 mg/L. The spectral data of oblongolide indicated the presence of a disubstituted double bond [1H: 8 5.56 m, 5.61 <3(J4,5 10Hz); 130:5 134.6 (CH), 121.4 (CH)], a 7-1actone (130:5 180.3; IR: 1763 cm-1), two methyls attached to a tertiary and quaternary carbon atom (1H: <5 0.91, 1.14 respectively) and a CH2 group attached to the bridge oxygen of the lactone [1H: 6 3.84, 4.38 (Jab = 8.7 Hz); 13C: 8 70.3 (CH2)]. In addition, the mass spectrum of 12 gave a base peak of 013H19 + after loss of CO2H. However, the combined spectral data alone was not sufficient to allow a clear structural assignment to be made and only after X-ray crystallography was the structure of oblongolide confirmed to be as shown (12). O
H3c~"
O
o
H3C~t" (12)
Catalytic hydrogenation of 12 gave a negative Cotton effect at 225 nm in the predicted by the lactone sector rule for a (13).29 Thus, it was concluded that
(13) its dihydro derivative 13, which displayed circular dichroism spectrum, as would be compound of such absolute configuration structure 12 represents the absolute
configuration of oblongolide. Interestingly, oblongolide was not produced by cultures grown on medium containing elm phloem (either surface or shake cultures) and the less common strain of P. oblonga did not produce 12 in any medium. However,
350
similar Phomopsis species associated with ash and sycamore trees were found to produce 12 but in malt medium only. 28 An additional set of metabolites were produced by the same strain of P. obionga as oblongolide, in both surface and shake cultures of malt extract medium and they were found to act as feed deterrent of the scolytid beetles. 3o For one of these compounds, a composition of C15H2006 was obtained by chemical ionization (NH3) mass spectrometry and its oxygen atoms were shown to be associated with two CHOH (1H" 5 3.62, 3.93; 13C'6 76.2, 70.6) and two ester or lactone moieties (13C" 6 162.6, 166.7; IR: 1728, 1710 cm-1). The remaining of the 1H and 130 NMR data suggested the presence of two methyl groups (CMe and CHMe) attached to trisubstituted double bonds and two additional cis and trans double bonds. Taking into account both 1H and 13C chemical shifts and coupling constants for each resonance of the 1H NMR, the structure of the o~-pyrone 15 and the trivial name phomopsolide B was assigned to this compound.
Its structural assignment was
consistent with the allylic coupling observed between HI'-H3' and between H2'-H6 (J = 1.1 Hz in both cases) and homoallylic coupling between H6-H3' (J = 0.9 Hz). The cis arrangement between H5 and H6 was deduced on the basis of their coupling constant which are similar to those reported for phomolactone (16) 31 and asperline (17); 32 the cis configuration of the latter fungal metabolite has been confirmed by X-ray crystallography. 32 Similarly, the 613 assignment of the C-6 substituent in structure 15 was based on the lactone sector rule (a positive Cotton effect in the circular dichroism curve at 264 nm) and close similarity with phomolactone (16). 32 The threo-o~-glycol absolute configuration(3'S, 4'S) was
via application of the exciton chirality method on its bis-anisyl
determined derivative, 33
O j
Ht, Oc~
O
,,11 3.~0 1' H OH 4~ C H 3
0 o
H3
OH c
OH3 H3
(14)
Hl*~ "H 2' H~e~"H
0
H3C
"OH3 (15)
351
0 ~, Hr
(16)
0 ~, H
.~. OH3
0"3 H eOAr |
H
(17)
The NMR spectral data of the second feed deterrent metabolite was also consistent with the structure of a tiglic ester but it had one of the CHOH moieties oxidized to a ketone and the C-1' double bond isomerized from tran to cis ( J H I ' - H 2 ' = 11.8 Hz). Thus, the chemical structure 14 was assigned to phomopsolide A.30 Phomopsolide B (but not A) was also produced by the Phomopsis species associated with sycamore and ash, however, neither phomopsolide A nor B was produced by the less common strain of P. oblonga. Although esters of tiglic acid are common plant metabolites (found as glycerides in croton oil, butyl esters in the oil of Roman camomile, as tiglylpseudotropeine in some Solanaceae plants and as geranyl esters in the oil of geranium), phomopsolides A and B are rather unique examples of such compounds produced by fungi. Further investigation into the metabolites of both strains of P. oblonga led to the isolation of several other compounds of known chemical structure which deter feeding in the elm bark beetle. 34 Among them was the compound 5-methylmellein (18), a metabolite of the almond pathogen Fusicoccum amygdah~s and numerous species of Hypoxylon and Numularia. 3s This compound was produced by both strains of P. oblonga, under different fermentation conditions, in addition to the mellein-5-carboxylic acid derivative (19) which was produced only by the less common P. oblonga strain. In contrast, the Phomopsis species associated with ash and sycamore did not produce 18 but did produce (+)-mellein having the opposite configuration at C-3. 37 An analogous observation was made with the compound 2furoic acid (20), a metabolite produced by both of the P. oblonga fungi but not by the Phomopsis spp. of ash and sycamore; its 2,5-dicarboxylic acid derivative (21) was isolated from the latter two organisms. 2-Furoic acid was previously known as a metabolite of Cercospora beticola 3e and presumably of Penicillium chrysogenum. 39 Finally, nectriapyrone (22), a known antibiotic of Gyrostroma missouriense, 4o 4-hydroxyphenylethanol (23), a phytotoxin of the plant pathogens Ceratocystis fimbriata 41 and Gloeosporium lacticolor 42 and the common fungal metabolites orsellinic acid (9) and 3-nitropropanoic acid (24) were also isolated from P. oblonga and found to be effective feeding deterrents of the elm bark beetle.
352
OH
R1
0 0 ~
3 R2
R (18)
R = OH 3
(20)
R 1 = H,
(19)
R = COOH
(21)
R 1 = R 2 = COOH
O
R 2 = COOH
CH2CH2OH O
CH30
OH3 (22)
CH 3
OH (23)
COOH HO
CH 3
O2N-CH2CH2-COOH (24)
OH (9)
Phomo_Dsis DasDalli An important class of potent mammalian toxins is associated with the pathogen P. paspalfi and some other unidentified Phomopsis fungi. The toxic effects of P. paspalli were most likely first reported in the ancient text Arthasastra (300 B.C.) which describes the consumption of kodo millet grains as "a poison for tigers" .43 Kodo millet (Paspalum scrobiculatum) is a crop grain cultivated in India, particularly
353
at the coastal areas, which is known to cause severe toxicity to humans and animals (known as kodrava poisoning) when infected with P. paspalli. The main symptoms of kodrava poisoning are difficulty in swallowing, vomiting, unconsciousness, violent muscle tremors and delirium. In the mid 1970s, two cytochalasin metabolites of P. paspalli were isolated from toxic kodo millet grains and shown to induce toxicity symptoms characteristic of kograva toxicity; metabolite 25 was reported to induce death in mice within 20 min. at a dose concentrations of 5 mg/kg or within 45 min. at concentrations of 2 mg/kg. 44 Compound 25 and its desacetyl derivative 26 were initially given the trivial names kodo-cytochalasin-1 and kodo-cytochalasin-2, respectively. Metabolite 25 was later also isolated from an unidentified Phomopsis fungus, known to infect weevildamaged pecans and it was named Cytochalasin H. 45 In the latter study, strong inhibition of growth and floral development was observed when tobacco plants were exposed to metabolite 25 at concentrations of 10 -2 to 10 -4 M. In addition, the previously known animal toxicity was confirmed (LD5o = 12.5 mg/kg was reported for one day-old male chickens). 45 The toxic metabolite of P. paspalli (kodocytochalasin-1) and the compound isolated from the Phomopsis pathogen of pecans (cytochalasin H) were finally shown to be the same compound. The details of its complex molecular structure were confirmed by two independent X-ray studies. 46 Recently, ten cytochalasin compounds were isolated from yet another unidentified Phomopsis fungus.
Six of these metabolites were shown to have novel
structures and they were given the trivial names cytochalasins N (27), O (28), P (29), Q (30), R (31) and S (32). 47 The remaining four compounds were identified as the known epoxycytochalasins H and J (33, 34), and cytochalasins H and J (25, 26). In addition, the compounds trans-and cis-(3S,4S)-4-hydroxymellein, were also isolated from the same extract; the latter is a known fungal metabolite of Lasiodiplodia theobromae48 and the genus Cercospora. 49 The structural assignment of the above cytochalasins was based primarily on their characteristic spectral data and direct comparison with previously known compounds. The mass spectrum of all metabolites showed a tropylium ion as the base peak (91 m/z) which is characteristic of 10-phenylcytochalasins and the molecular formulae of the six new compounds were established via high resolution MS and elemental analyses. Extensive 1H and 130 NMR analysis using COSY, HETCOR and DEPT NMR experiments, was also carried out in order to establish the reported structures.
354
",.s~OH
~ / , ~ O_OH HS'~
11
' O OR
*"-~H
O OH
(25) R = AC
e OH TM
(31)
(26) R = H
OH "~ ,~
HO,,
OH "~
""el
O OH (27) R = Ac
..,q
~3H
(32)
(28) R = H
.o... /
e,,,~j~O H
~ -O ...,,
(29) R = A c
(33) R = A c
(30) R = H
(34) R = H
The glycol moiety of metabolites 29 and 30 is unique among cytochalasins. The reported absolute stereochemistry was based on their NMR data and chemical modifications. 47 For example, metabolite 33 was converted to 29 upon treatment with trifluoroacetic acid in acetonitrile, strongly suggesting the existence of a transglycol moiety in the structure of 29. A 13configuration was assigned to the C-7 hydroxyl group based on the coupling constants observed (J7-8 = 11.4 Hz) and a positive NOE effect between the C-6 methyl of 29 and the acetate group of a 7-0acetate derivative. Furthermore, the dibenzoate chirality rule was applied to the 7-
355
mono, 7,18-di-, and 6,7,18-tri-benzoates of cytochalasin P (29) which showed a positive first Cotton effect for the 6,7-dibenzoate, indicating an o~ configuration for the C-6 hydroxyl. Similar investigations of the remaining five metabolites led to their detailed NMR data analysis and structural assignment as shown. 47 Cytochalasins are a very important family of natural products. They were first isolated and characterized in the mid 1960s by Aldrich and Turner so and independently by Tamm. 51 Since then, more than fifty such compounds have been isolated from diverse fungal sources including the Phomopsisfungi. Their biological properties include phytotoxicity, 45 antibiotic activity, s2 inhibition of sugar transport in plasma membranes s3 and selective antitumor activity; s4 however, cytochalasins are best known for their potent and unique toxicity on animal cells. This latter effect is mostly attributed to their interaction with the common target protein actin and cell processes which have been shown to be inhibited by cytochalasins are also known to be mediated by microfilaments of actin. 5s Actin is found primarily in skeletal muscle tissues, although it also plays an important role in many other mammalian cells. In cells other than those of muscles, it participates in localized contractive events in the cytoplasm and in cell-aggregation phenomena. Some of the cell processes which depend on microfilaments related to actin, include phagocytosis (engulfment of foreign particles by cell membranes), cytokinesis (the formation of a contractile ring around a dividing cell), exocytosis (the extrusion of materials from the cells) and various other cell and organelle movements associated with membrane transport processes. The structure-activity relationship of the known cytochalasins has been examined by a number of research groups, s8 A common structural feature of all these metabolites is expressed by the perhydroisoindol-l-one moiety bearing either a benzyl (cytochalasins), p-methoxybenzyl (pyrichalasin), 5~ (indol-3-yl)methyl (chaetoglobosins) s8 or 2-methylpropyl (aspochalasins) s9 moiety at the C-3 position, in addition to a 11-,13- or 14-membered carbocyclic (or oxygen-containing) ring, connecting atoms C-8 to C-9. It has been generally observed that the perhydroisoindol-3-one nucleus is the most important factor with regard to activity. Compounds with an aromatic substituent at C-3 (phenyl or indolyl) exhibit similar magnitudes of biological activity, while those possessing an isopropyl substituent at that position are relatively inactive. The macrocyclic ring (connecting C-8 to C-9) is also essential for activity, however, its functional groups or ring size over C17 appears to be unnecessary. It is believed that the conformation of the central core structure (primarily the cyclohexane ring moiety) assumes major importance in the toxicity of cytochalasins, while the large macrocycle seems to be essential only in its bulk and the amount of lipophilicity it contributes to the compound. It is noteworthy,
356
that the Phomopsis metabolites P (29), Q (30), R (31) and S (32) exhibit a relatively weak cytotoxicity to mammalian cells. Although the conformation of cytochalasins in biological systems is uncertain, the 1H NMR data of metabolites 29, 30, 31 and 32 suggested that their cyclohexane ring adopts a boat or half-boat conformation, as in the case of most other cytochalasins.
It has been proposed, however, that the
presence of an additional oxygen substituent at the C-5 or C-6 positions adjacent to that at C-7 has an unfavorable effect on the binding properties of these compounds causing a decrease in their cytotoxicity. Although the biosynthesis of cytochalasins in Phomopsis fungi has not yet been investigated, their formation is most likely analogous to that of the structurally related cytochalasin D (35). The biosynthesis of 35 in cultures of Zygosporium masonii was investigated by Tamm and Vederas and it was shown to involve the condensation and cyclization of the amino acid phenylalane and a C16 polyketide, so Unlike many metabolites which are formed from a methylated polyketide chain which have propionate as their biosynthetic precursor, 6~ the exoclyclic carbons at C-6, C16 and C-18 of cytochalasins were shown to originate from methionine. 6o
0 0
0
HO HO N
0
0
0
0
0
0
I I !
12 4~ 9 I" 13 14 15
>6 ~9 "
o 6ff (35)
m l l
---o
OH
357
Acknowledaements
The author is indebted to Professor David N. Harpp, Chemistry Department McGill University, Montreal, Que, Canada and Ms Alexandra Glashan, Biology Department, Vanier College, Montreal, Que, Canada for their help in the preparation of this manuscript.
References
9
1
.
4. 5. ,,
7.
B
I
10. 11. 12. 13.
Ormeno-Nunez, J.; Reeleder, R.D.; Watson, A.K. (1988) Can. J. Bot. 66, 2228. a) Alex, J.F. (1982)in: Holzner, W. and Numata, M. (Eds), Biology and Ecology of Weeds, W. Junk Publishers, The Hague, pp. 309. b) Holm, L.G.; Plunknett, D.L.; Pancho, J.V.; Herberger, J.P. (1977), The World's Worst Weeds, University Press of Hawaii, Honolulu, Hawaii, Chapter 12. Rosenthal, S.S. (1983) Calif. Agric. 37, 16. Morin, L.; Watson, A.K.; Reeleder, R.D. (1989) Weed Science, 37, 830. Sugawara, F.; Strobel, G.; Strange, R.N.; Siedow, J.N.; Van Duyne, G.D.; Clardy, J. (1987) Proc. Natl. Acad. Sci. USA 84, 3081. Evans, M.B.; Dale, A.D.; Little, C.J. (1980) Chromatographia 13, 5. (a) Tsantrizos, Y.S.; Watson, A.K.; Ogilvie, K.K. (1992) Can. J. Chem. 70, 2276. (b) Tsantrizos, Y.S.; Ogilvie, K.K.; Watson, A.K. USA Patent Number 5,100,456. Date of Patent: March 31, 1992. a) The synthesis of compounds 4 and 5 were reported in 1953 by Brown and Newbold, 8b unfortunatly, the amount of spectral data available from that report (m.p. and uv) was of limited help in the structural assignment of these natural products. b) Brown, J.J.; Newbold, G.T. (1953) J. Chem. Soc., 1285. a) Achenbach, H.; Muhlenfeld, A.; Weber, B.; Kohl, W.; Brillinger, G.-U. (1982) Z. Naturforsch37B, 1091. b) Achenbach, H.; Muhlenfeld, A.; Brillinger, G.U (1985) Liebigs Ann. Chem., 1596. A PCMODEL program was used which was obtained from Serena Software, P.O. Box 3076, Bloomington, Indiana, USA 47402-3076 a) Einhellig, F.A.; Leather, G.R.; Hobbs, L.L. (1985) J. Chem. EcoL 11, 65. b) Datko, A.H.; Mudd, S.H.; Giovanelli, J. (1980) Plant PhysioL 65,906. Otomo, N.; Sato, H.; Sakamura, S. (1983) Agric. BioL Chem. 47, 1115. a) Muhaljcevic, M.; Muntanola-Cvetkovic, M.; Petrov, M. (1980) Savremenapoljopriveda 28, 531.
358
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b) Muntanola-Cvetkovic, M.; Muhaljcevic, M.; Petrov, M. (1981) Nova Hedwigia 34, 417. a) Henning, A.A. (1983) Campinas 9, 26. b) Herr, L.J.; Lipps, P.E.; Watter, B.L. (1983) Plant Dis. 67, 911. c) Lamarque, C.; Perny, R.A. (1985) Cultivar179, 57. Mazars, C.; Rcssignol, M.; Auriol, P.; Klaebe, A. (1990) Phytochemistry29, 3441. Hahn, G.G. (1926) Phytopathology16, 899 Wheeler, M.M.; Wheeler, D.M.S.; Peterson, G.W. (1974) Phytochemistry14, 288. Stoessl, A. (1967) Chem. Commun., 307. Culvenor, C.C.J.; Beck, A.B.; Clarke, M.; Cockrum, P.A.; Edgar, J.A.; Frahn, J.L.; Jago, M.V.; White, R.R. (1977) Aust. J. BioL Sci. 30, 269. Lanigan, G.W.; Payne, A.L.; Smith, L.W.; Wood, P.Mc.; Petterson, R. (1979) AppL Environ. MicrobioL 37, 289. Culvenor, C.C.J.; Cockrum, P.A.; Edgar, J.A.; Frahn, J.L.; Gorst-AIIman, C.P.; Jones, A.J.; Marasas, W.F.O.; Murray, K.E.; Smith, L.W.; Steyn, P.S.; Vleggaar, R.; Wessels, P.L. (1983) J. Chem. Soc., Chem. Commun. 1259. Payne, A.L. (1983) AppL Environ. MicrobioL 45, 389. Pachler, K.G.R.; Wessel, P.L. (1977) J. Magn. Reson. 28, 53. Mackay, M.F.; Donkelaar, A.V.; Culvenor, C.J. (1986) J. Chem. Soc., Chem. Commun, 1219. Tonsing, E.M.; Steyn, P.S.; Osborn, M.; Weber, K. (1984) Eur. J. Cell Biol. 35, 156. a) Gibbs, J.N.; Smith, M.E. (1978) Ann. AppL BioL 89, 125. b) Webber, J.F.; Giggs, J.N. (1984) Trans. Brit. MycoL Soc. 82, 348. Webber, J. (1981 ) Nature 292, 449. Begley, M.J.; Grove, J.F. (1985) J. Chem. Soc. Perkin Trans. I, 861. Jennings, J.P.; Klyne, W.; Scopes, P.M. (1965) J. Chem. Soc., 7211. Grove, J.F. (1985) J. Chem. Soc. Perkin Trans. I, 865. Evans, R.H.; Ellestad, G.A.; Kunstmann, M.P. (1969) Tetrahedron Lett., 1791. Fukuyama, K.; Katsube, Y.; Noda, A.; Hamasaki, T.; Hatsuda, Y. (1978) Bull. Chem. Soc. Jpn 51, 3175. Harada, N.; Nakanishi, K. (1972) Acc. Chem. Res. 5, 257. Claydon, N.; Grove,F.J.; Pople, M. (1985) Phytochemistry24, 937. Ballio, A.; Barecellona, S.; Santurbano, B. (1966) Tetrahedron Let., 3723. Anderson, J.R.; Edwards, R.L.; Whalley, A.J.S. (1983) J. Chem. Soc. Perkin Trans 1,2185. Grove, J.F.; Pople, M. (1979) J. Chem. Soc. Perkin Trans 1,2048. Sakaki, T.; Ichihara, A.; Sakamura, S. (1981) Agric. Biol. Chem. 4,5, 1275. Cram, D.J.; Tishler, M. (1948) J. Am. Chem. Soc. 70, 4238. Nair, M.S.R.; Carey, S.T. (1975) Tetrahedron Let., 1655. Stoessl, A. (1969) Biochem. Biophys. Res. Commun. 35, 186.
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Atta-ur-Rahman(Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 ElsevierScience B.V. All rights reserved.
361
Detection of Cardenolides by ELISA in Plant Sciences Kayo Yoshimatsu, Jun-ichi Sawada, Mondher Jaziri and Koichiro Shimomura
1.
INTRODUCTION Cardenolides are composed of a steroid or an aglycone portion which contains a five-
membered lactone ring and from one to four sugar molecules. They naturally occur among several plant families, and some of them such as digoxin, lanatoside C and deslanoside derived from genus Digitalis (Scrophulafiaceae) (Fig. 1) are frequently prescribed for congestive heart failure. A patient
requires long term treatment with about 1 mg of digoxin a day and several tons of drug in a year are required for world-wide use (1). Although many cardiac monoglycosides have been synthesized, all the medicinal preparations are still derived from plant sources (1). In vitro cell and tissue cultures of Digitalis, which is a main source of pharmaceutically important cardenolides, have so far been investigated extensively by many researchers to obtain cardenolides more efficiently including clonal propagation of the plants, production and biotransformation of cardenolides (2, 3). Improvement of productivity through the manipulation of culture conditions and/or the screening of high-producing clones requires simple and sensitive analytical methods, especially if the target compound is not colored or fluorescent, as is the case with cardenolides.
Several
analytical methods have been reported for the determination of cardenolides in plant materials, including chromatographic (4-8) and immunological assays (9-15). Immunoassay is the most powerful analytical method to handle a large number of plant materials because of its accuracy and simplicity. Immunoassays are based on the specific interaction between an antibody and its antigen. When the affinity of the antibody for its antigen is sufficiently high, the antibody can recognize its antigen even in a crude extract solution, so that sample pre-purification steps generally employed in chromatographic analysis are not necessary and the analytical time might be shortened. Since radioimmunoassay (RIA) for the determination of cardenolides in plants was first demonstrated by Weiler and Zenk in 1976 (9), several researchers have been carrying it out and proving its advantages to analyze a large number of plant materials. However, RIA requires special facilities and precautions to use radio-labeled compounds which involve unavoidable risks of exposure to radiation and problems concerning disposal. Then an alternative enzyme immunoassay (EIA) method which uses stable, nonisotopic enzyme labels instead of a radio isotope, have been introduced to overcome these problems of RIA (16-18). EIA methods do not suffer from the troublesome handling problems and instability of radioisotopes associated with RIA, have sensitivity comparable
to RIA, and are now extensively used in biological and medical fields
especially where RIA cannot be employed for technical reasons.
Competitive enzyme-linked
immunosorbent assay (ELISA) is one of the most suitable EIA techniques for the screening of plant materials because of moderate costs of the apparatus and the reagents, the high sensitivity and the simplicity (19).
362 Aglycone _O
O
R2 R30~
OH
Sugar residue CH2OH
OH B-D-Glucose (G)
CH 3
OH B-D-Digitoxose (D)
CH3
OCOCH3 3-acetyl-B-D-digitoxose (AcD)
Aglycones
R1
R2
R3 (Sugar moiety)
Cardenolides
Digitoxigenin
H
H
G-AcD-D-DG-D-D-DD-D-D-
Lanatoside A Purpurea glycoside A Digitoxin
Gitoxigenin
H
OH
G-AcD-D-DG-D-D-DD-D-D-
Lanatoside B Purpurea glycoside B Gitoxin
Digoxigenin
OH
H
G-AcD-D-DG-D-D-DD-D-D-
Lanatoside C Deslanoside Digoxin
Diginatigenin
OH
OH
G-AcD-D-DD-D-D-
Lanatoside D Diginatin
Gitaloxigenin
H
OCHO G-AcD-D-DG-D-D-DD-D-D-
Fig. 1. Cardenolides
Lanatoside E Glucogitaloxin Gitaloxin
ingenusDigitalis.
363 2. METHODOLOGY OF ELISA FOR PHYTOCHEMICALS The preparation of specific antibodies against a target phytochemical is an indispensable requirement in order to carry out ELISA. However, compounds of low molecular weight such as cardenolides themselves are not immunogenic. However, they become immunogenic after they are coupled to a suitable macromolecule, i.e. a cartier protein, and the small molecules which are specifically recognized by antibodies but cannot induce immune responses are called hapten. Thus a target phytochemical must be conjugated covalently to a carrier protein. The phytochemical-protein conjugate will induce specific antibodies against the hapten moiety as well as the carrier protein i n vivo. The coupling of the small molecule with a carrier protein is therefore a crucially important factor to be taken into account as it may determine the specificity of the antibodies. Indeed, antibodies which recognize a distant part of hapten from the conjugated site show lower crossreactivity with the carrier protein. Additionally, the linkage between the hapten and the carrier protein should be stable and non-hydrolyzable. The general conjugation methods are reviewed by Erlanger (20) and a typical method to prepare digoxin-protein conjugates will be described in detail in section 3.1. Among the various EIA methods (19), a solid-phase EIA using an immobilized antibody (Fig. 2) and a competitive ELISA can be easily applied, and especially the latter might suit phytochemical assays. In this article, we mainly refer to the methods of competitive ELISA.
ifl~)O
9
: I m m o b i l i z e d antibody Q
: Hapten in the sample : E n z y m e - l a b e l e d hapten
S
\?
P S
: Substrate
P
:Product
Fig. 2. Solid-phase enzyme immunoassay (EIA) using immobilized antibody.
364 (1) Coating 'Antigen
0
0
0
(2) Blocking A
: Irrelevant protein
(3) Competitive reaction between the solid-phase antigen and the hapten in the sample solution with first antibody 9 ~J,,,
: Hapten in the sample 9First antibody (specific antibody)
(4) Wash out of the unbounds thoroughly
(5) Reaction of enzyme-labelled second antibody with first antibody :Enzyme-labeled second antibody
(6) Detection of enzyme activity proportional to the amount of second antibody on the solid phase S
: Substrate
p
:Product
Fig. 3. Diagram of the competitive ELISA on a microtitration plate.
365
2.1. Principle of the ELISA The determination of target phytochemicals is performed by using a competitive reaction between pre-adsorbed antigen and free hapten with their specific antibody. In order to avoid a nonspecific reaction between the antibody and the protein used as a coating conjugate, the protein carrier used for immunization must be different from the protein used for the synthesis of the coating conjugate. For this purpose, two different hapten-protein conjugates are needed. A diagram of the competitive ELISA is shown in Fig. 3. The antigen (coating conjugate) is adsorbed onto the solid-phase (step 1). The surface of the solid-phase is saturated with an irrelevant protein to avoid non-specific binding (step 2). A mixture of the sample containing free haptens and the specific antibody (first antibody) is added. The targets (haptens) in the sample competitively inhibit the binding reaction between the specific antibody and the antigen adsorbed to the solid-phase (step 3).
After washing out the unbound first antibodies (step 4), the enzyme-labeled second
antibody which recognizes IgG of the animal species used for immunization (antibody against first antibody) is added and allowed to bind to the first antibody (step 5). After washing, the substrate is added and the color or fluorescence developed in proportion to the amount of the second antibody bound to the solid-phase is measured (step 6). The amount of the first antibody (and also the second antibody) bound to the solid-phase is inversely proportional to the amount of the target substances in the sample. 2.2. Materials and reagents The materials and reagents needed are listed in Table 1. Table 1. Materials and reagents for ELISA.
(a) Materials 1) 96 well microtitration plates 2) Pipettes and pipette tips 100 - 200 btl single channel (for sample or standard solutions) 50 - 200 l,tl multichannel (for blocking, antibody, substrate solutions)a 3) Test tubes (for standard or sample dilution) 4) Solvent vats (for multichannel pipettes)a 5) Microplate washera 6) Microplate reader 7) Timer 8) Incubatora (b) Reagents 1) Coating buffer (50 mM bicarbonate buffer, pH 9..6)b 2) 10raM phosphate buffered saline (PBS), pH 7.2b 3) Blocking, sample and antibody diluent [PBS containing 0.1% (w/v) _casein] (C-PBS)b 4) Washing solution [PBS containing 0.05 % (w/w) Tween 20](T-PBS)b 5) Standard solutions (for makingr standard curve)C 6) Antigen for coating (hapten-protein conjugates)d 7) Specific antibody (first antibody)d 8) Enzyme-conjugatedspecies-specific anti-IgG (second antibody)e 9) Substratee 10) Stop solution a: not necess~uilybut convenient if prepared b: examples used in our laboratory c: see section 3.5. Data processing and sensitivity of the assay. d: set section 3.4. Establishment of the assay conditions. e: commercially available
366 2.2.1. Materials Although physico-chemical characteristics of the adsorption of the antigen onto the solid-phase have not been clarified, it is well known that proteins are passively adsorbed onto plastic materials and are not removed by washing.
Among several materials employed as the solid-phase,
polystyrene, polycarbonate, polypropylene, or polyvinyl chloride are usually used. Since the condition of the solid-phase surface is crucially important, it is desirable to test every rot of the plates for its ability of protein adsorption (see section 3.4.). We obtained satisfying results using commercial polystyrene microtitration plates for ELISA. An 8 or 12-channel micropippet is convenient for adding or stepwise dilution of the antigens and antibodies on the plates. In every step, washing has to be carried out thoroughly and carefully with suction or decantation so that the solutes are completely removed and they do not interfere with the next step. A microplate washer is useful for this purpose. An incubator (37 ~C) can be used for the incubation with the first or second antibody, and for the enzymatic reaction, but we obtained satisfactory results by performing all the procedures at room temperature (ca. 25 ~C). An optical observation of enzyme activity is possible if the selected substrate develops color after the reaction, which is a simple and easy way of selecting positive samples and one of the advantages not provided by the RIA method. A photometric or fluorometric microplate reader is generally employed depending on the labeled enzyme and substrate. 2.2.2. Reagents The passive adsorption of antigens to the solid-phase depends both on the pH and the ion strength of the buffer. Most antigens are adsorbed well under alkaline conditions with low ion strength. Fifty mM of bicarbonate buffer (pH 9.6) is generally used, but phosphate buffered saline (PBS) is suitable in some cases. The microtitration plates coated with the antigen are then saturated with an irrelevant protein to avoid nonspecific adsorption of the antibody onto the solid-phase. Proteins such as bovine serum albumin are commercially available for this purpose but they are costly. In our laboratory, good results are obtained using casein which is dissolved in a small amount of PBS at 60 ~ and diluted to a final volume. The enzymes used as labels are horseradish peroxidase, alkaline phosphatase, 13-galactosidase, glucose-6-phosphate dehydrogenase, etc. Usually the enzyme-labeled second antibody and the corresponding substrates are commercially available and should be selected according to one's microplate reader or purpose of the assay. The stop reagent which is generally prescribed in the substrate kit is not necessary if the measurement can be immediately carried out after the enzymatic reaction, however, it is helpful to manipulate a large number of samples. In addition, there are avidin/biotin reagents and kits for the enhancement of the sensitivity. The avidin/biotin labeling system is based on the high affinity of egg white avidin or streptavidin for biotin. One molecule of avidin will bind four molecules of biotin by non-covalent interaction which is essentially irreversible, so that the number of enzymes per one second antibody will increase by using biotinylated antibody with enzyme complexes which are previously coupled with both of avidin and biotin. This system may not be quantitative but it is preferable for the rapid screening of secondary metabolite production.
367 3.
C O M P E T I T I V E ELISA FOR THE S E M I - Q U A N T I T A T I V E D E T E R M I N A T I O N OF CARDENOLIDES IN DIGITALIS LANATA PLANT AND TISSUE CULTURES The production of cardenolides by Digitalis plants has been of great interest because of their
pharmaceutical importance (2, 3). The development of a simple and sensitive assay technique has been desirable in order to investigate the productivity of a large number of strains. We have established an ELISA technique using monoclonal antibodies against digoxin for cardenolide determination and it was employed to evaluate cardenolide production by transformed root cultures of D. lanata (15). In this chapter, the details of ELISA for cardenolides used in our laboratory are described.
3.1. Preparation of Digoxin-protein conjugates Digoxin human serum albumin conjugate (Dig-HSA) and ovalbumin conjugate (Dig-OVA) were prepared by a periodate oxidation method (21, 22) and used as an immunogen and a coating antigen, respectively. This method includes the following three steps and the proposed mechanism is shown in Fig. 4. D=Digoxigenindigitoxoside
H
.
step 1 IO4-
OH
step 2 pH 9-9.5
~ - HO
step 3 NaBH4
O
NHz
d9 Fig. 4. Proposed mechanism for conjugation of digoxin to human serum albumin (after Smith et al. 1970). step 1. Cleavage of the terminal digitoxose moiety by oxidation with sodium metaperiodate (NaIO4). step 2. Cyclization of two aldehyde portions of a sugar moiety and the amino group of lysine or Nterminal residue in human serum albumin (HSA) or ovalbumin (OVA) in alkaline conditions (pH 9-9.5). step 3. Stabilization of the protein-digoxin conjugate by reduction with NaBH 4. The procedure is as follows. 1. Dissolve digoxin (436 mg) in 20 ml absolute ethanol at room temperature. 2. Add 20 ml of 0.1 M NaIO 4, dropwise with magnetic stirring and after 25 min add 0.6 ml of 1 M ethylene glycol and wait 5 min (solution A). 3. Dissolve 280 mg HSA or OVA in 20 ml H20 and adjust pH to 9.5 with 0.4 ml of 5% K2CO 3 (solution B). 4. Add 10 ml of solution A to solution B twice (total 20 ml) with stirring and adjust pH to 9-9.5
368 with 5 % K2CO 3. 5. Allow to stand for 45 min. (Check pH. Its pH will become stable within 45 min, and the HSA solution will be brown. If pH is over 9.5, adjust to 9.3 with HCI.) 6. Add 10 ml of 0.3 g NaBH 4 in 20 ml H20 (freshly and carefully prepared), and allow to stand for 3 hours. 7. Add 3.8 ml of 1 M formic acid dropwise to lower the pH to 6.5 (check with pH paper) and stand for 1 hour. (Be careful of gas bubbles. Precipitation will occur in the case of OVA mixture.) 8. Add 0.75 ml of 1 M NH4OH to raise pH to 8.5 (check with pH paper). 9. Dialyze the entire reaction mixture against tap water for 2 hours. 10. Dialyze against normal saline twice a day for 5 days (total 10 times). 11. Collect the Dig-HSA dialysate and measure the optical density (OD) at 280 nm after 50-fold dilution with PBS. Approximately 1 mg/ml conjugate solution will give an OD of 0.53 for HSA. In the case of Dig-OVA, centrifuge the dialysate at 1500g for 15 min to collect the supernatants. Dilute the supernatant 10-fold with PBS and measure OD (1 mg/ml solution: an OD of 0.74). The supernatant may contain enough concentration of conjugate for coating. Keep the dialysate and diluted dialysate a t - 20~ until use. For long-term preservation, lyophilization after dialysis against H20 is recommended. Digoxin or Dig-HSA turns yellowish-brown color (absorption maxima are 388 and 475 or 466 nm, respectively) after mixing with concentrated H2SO 4. Number of digoxin residue per molecule of HSA is calculated by comparing their molar extinction coefficients at 388 nm (molar of Dig-HSA can be estimated by its absorbance at 280 nm). In our case, an average of 4 digoxin residue/HSA was prepared by the method mentioned above. 3.2. Production of antisera (poIyctonaI antibodies) Production of antisera does not require specialized immunological techniques and cell culture facilities. Therefore it may be a simpler, faster and cheaper way than production of monoclonal antibodies (see section 3.3.). However, it must be noted that antisera-based immunoassays are sometimes less selective than monoclonal antibody-based immunoassay because of the polyclonality of antibodies in the antisera, and good antisera are not reproduced. The production of high affinity antisera may be helped by long-term immunization schedules from the mechanism of antigendependent B lymphocyte differentiation and selection (23). A typical method for the production of antisera against digoxin is described below (22); Animal to immunize: Japanese albino rabbits (3 rabbits for an experiment) Preparation of immunogen: Mix 1 ml of Dig-HSA (5 mg/ml in PBS) and 1 ml of Freund's complete adjuvant (FCA). Make complete w/o emulsion using syringes and a connecter. Immunization: Inject 0.5 ml of freshly prepared immunogen (1.25 mg/rabbit) into the footpads or back four times at three week interval. Preparation of Antisera: Bleed at day 10 after final immunization.
Keep the blood at room
temperature for a while and then centrifuge at 1500g for 20 min twice. Collect the supernatants (antisera) and keep at - 20 ~C.
369 Monitoring of the antiserum titer before killing rabbits is important to obtain specific antisera. It is recommended that the titer of a small volume of serum bled from ears is monitored periodically by ELISA (Follow the same procedure described in section 3.7. using 2 lag/ml DigOVA as a coating conjugate). Collecting antisera should be carried out when the titer reaches the stationary phase. 3.3. Production of monoctonal antibodies During the immune response against an antigen in vivo, B lymphocytes differentiate towards the terminal plasma cells, which produce specific antibodies against particular parts of the antigen and secrete antibodies in large quantities in a soluble form. Though we can obtain the clone of B cells which produce monoclonal antibody, B cell itself cannot be maintained in vitro. Therefore hybrids (hybridoma cells) between B cells and myeloma cells must be prepared by somatic cell fusion to obtain the cloned cells continuously producing antibodies (23, 24). Specialized laboratory equipments and immunological training are required to generate the hybridoma clones which have the characteristics of producing specific monoclonal antibody and proliferating sufficiently in vitro. The procedures for this are time consuming, sometimes frustrating and definitely expensive. However, monoclonal antibody still has many advantages as follows: 1, its production is unlimited and reproducible; 2, the production against specific antigen is possible even when the antigen cannot be obtained in a pure form; 3, a small amount of antigen is sufficient as a starting material for immunization; 4, contaminant antibodies which are a major source of cross reactivity are excluded by definition (25). In this section, we briefly describe the conditions to generate hybridoma producing monoclonal antibodies against digoxin, and expect one to consult professional literatures (23, 26-28) for details of sophisticated techniques for establishing hybridoma clones including taking out spleen cells from animals, preparing both lymphocyte and myeloma cells for fusion, maintenance of hybridoma cells etc. A typical method for the production of antisera against digoxin is described below (22); Animal to immunize: Female BALB/c mice (4 mice for an experiment) Preparation of immunogen: A, Mix 1 ml of Dig-HSA (5 mg/ml in PBS) and 1 ml of Freund's complete adjuvant (FCA). Make complete w/o emulsion using syringes and a connector. B, Prepare 1 mg/ml Dig-HSA with saline. Immunization: Inject 50 ~tl of immunogen A (125 ~tg/mouse) into footpads six times at intervals of three weeks and inject 50 gl of immunogen B (50 gg/mouse) intraperitoneally 3 days before fusion. Fusion partner: P3/NS 1/1-Ag4-1 (NS 1) myeloma cells. A systematic protocol for establishing hybridoma is presented in Fig. 5 and additional information is described below;
370 Grow and maintain NS-1 cells [
[ Harvest NS-1 cells I
Immunize mice with antigen ]
Prepare a single cell suspension of spleen cells ]
i Fuse spleen and NS-1 cells (cell density ratio, 1:2-4) using 45 % polyethylene glycol 4000
Plate fused cells into 24-well tissue culture plate I
Select hybrids by growth in HAT (hypoxanthine-aminopterin-thymidine) medium I a
] Screen cultures for production of relevant antibodies by ELISA ] b
Freeze cells for possible ,,ll 9 ..................................... use in cloning Clone cells in positive cultures by limiting dilution using 96-well tissue culture plates containing thymocytes
d
Expand cultures which show monoclonal growth by transferring cells to 12 or 24-well tissue culture plates
Check the antibody titer by ELISA I f |
~-]Freeze cells for safe keeping ]
Produce a large amount of antibody in vitro or in vivo ]g
Fig. 5 Systematic protocol for establishing hybridoma cell lines a-g: explanations appear in the text.
c
371 (a) To survive in the presence of aminopterin, which blocks the de novo biosynthesis of purines and pyrimidines, cells must synthesize these nucleotides by utilizing an exogenous source of hypoxanthine and thymidine via alternate nucleotide biosynthetic pathways, i.e. salvage pathways. However, NS- 1 cells and NS- 1:NS- 1 fused cells which lack hypoxanthine-guaninephosphoribosyltransferase (HGPRTase) are not capable of growing in HAT medium. On the other hand, normal spleen cells and spleen:spleen fused cells have a limited growth potential in culture and most of them die within two weeks in culture. Thus the NS-1 cells provided HGPRTase by fused spleen cells, the desired NS-1 :spleen cell hybrids, can selectively grow in HAT medium. (b) Undiluted culture supernatant is directly subjected to ELISA using the same procedure described in section 3.7. with 2 lag/ml Dig-OVA as the coating conjugate. In our result, the cultures in 22 out of 288 wells tested were positive for both growth in HAT medium and ELISA. (c) The cells suspended in RPMI 1640 medium containing 50 % v/v fetal calf serum and 10 % (v/v) DMSO are gradually cooled to-80~ (-lq2/min) and then immediately to-196~ in liquid nitrogen. They are kept at- 196 ~ until use. (d) Viable cells in ELISA positive wells are diluted and placed in 96-well tissue culture plates at an average of 0.5 cell/well. The thymocyte cells are used as feeder cells. Most hybridoma cells will not grow without thymocyte feeders at a very low cell density. Thymocytes can be replaced with commercially available hybridoma growth factors. (e) The procedures for screening and cloning (subcloning) are repeated.
In our case, three
subcloned lines for each clone were established. (f) The culture supernatants collected just before the cultures become confluent are checked their ELISA titer (see 3.4.) and used as source of monoclonal antibody. (g) For the propagation of cells in vivo, 0.2 ml of cloned cells (approximately 1,000,000-5,000,000 cells in number) in serum-free medium is injected intraperitoneally into pristane treated BALB/c mice. Ascitic fluids are collected when the abdominal expansion is observed. The titer of ascitic fluids are generally much higher (ca. 100-fold) than those of the culture supernatants. 3.4. Establishment of the assay conditions The sensitivity of an ELISA depends on both the concentration of the coating conjugate and the dilution of the antibody used. In order to determine these parameters, a range of coating conjugate concentrations for optimal coating of the polystyrene surface should be tested at different dilutions of the antibody. The procedure for this is given in Fig. 6. The titer of the antibody can be defined as the reciprocal of the working dilution where the antibody gives 80 % of the maximal binding at a fixed concentration of the coating conjugates (from our experience). In this condition, the concentration of coating conjugate which gives a measurable response (absorbance approximately 1.5) should be chosen. In our case, coating conjugate at 25 rig/well and antibody dilution of 1/40 (of the supernatant) were selected as suitable (Fig. 6). Under these conditions, the maximum antibody-binding activity (B100) is fixed.
372 Coating conjugate concentration v
9169169169169169169 Q 9169169169169 0 ng/well
1/1 ........... 1/'3 ........... 1/9
1/2"/ ........... ~
1/81
1/243 ........... o~ M
1/729 .......... [1/2187
25 n g / w e l l
50 n g / w e l l
1 O0 ng/well
2 0 0 ng/well
800 n g / w e l l
..O.....O..D.O..D.. 9 ...O.......O....Q.O..D....O....D...O....D....O....D....O.... ...O.......O....D...O....D....O....9169 ...0.......0.... 99169169169169169169
91691699169
9169169169
O0DODODO 9
A- Determination of the coating conjugate concentration and first antibody dilution The procedure is the same as section 3.7.
I!
[ ]."'"
E =
"-"O"--
U
--
1
[] . . . . . D
~ .
"- *---,---.i, 2 ,~x-. . . . A- . . . . A , I. .........
. . .
A--
25
50
--i--1oo ~.
,,
0 ng/well Dig-OVA
...... 9 .....
,,,,.
, ~....
-"-r'l- -
200
- -I"
800
-
9......... 9.......I'A,, _~'&\
.,,,ll .................................
i.O..:;.,'".-'\k i"-../'k ' , :
"..,~
,<
0
' 1/1
1/10
1/100
1/1000
1/10000
Antibody dilution
B 9Antibody titer determination for ELISA using anti-digoxin monoclonal antibody (DIG 64.2B.5) Fig. 6. Establishment of the assay conditions.
373 3.5. Sensitivity of the assay and data processing Additional free digoxin competes with the coating conjugate for antibody-binding and results in a displacement of the equilibrium of the reaction. For the semi-quantitative analysis by ELISA, the unknown concentration of digoxin in crude plant extract can be determined as digoxin equivalents from a reference dose-response curve constructed with known concentrations of standards. To make a standard curve standard solutions should be included on each plate. In our laboratory, standard solutions comprising at least 6 concentrations along with a non-specific binding (blank, without either the standard solution or the first antibody) and B0 ( 100 % binding, without standard solution) are prepared for each assay. If linear absorbance versus log concentration is plotted on semi-log paper, it presents a sigmoid curve where the concentration of the sample solution can be obtained from its absorbance. However, drawing the best fit sigmoid curve is difficult.
Computer softwares for graphical
transformation, statistical analysis and data reduction have been developed and are commercially available. A typical standard curve for digoxin is shown in Fig. 7. The regions of the curve near the limit absorbance values (non-specific binding and B0) have very shallow slopes, where small differences in absorbance result in large differences. Thus the reliable region, near the center of the curve which gives linearity after transformation into logit absorbance versus log concentration, should be used to determine the sample concentration values. If B is the absorbance of the sample in question and B0 is the absorbance of zero response, logit absorbance can be obtained by the following equation; Logit y = Ln [y/(1-y)], where y = B/B0.
1 O0
9
2"3t
,i ............................................... 80 ..........................................
o.]
60 Reliable region
i
40 A
~
Dgioxni
(nM)
B
20
I! ...............
_
................
i
0.0005
0.]5
A" lower limit of detection B: higher limit of detection
i
|
4.52 Digoxin (nM)
Fig. 7. Typical standard curve of digoxin.
|
32.9
!
134
400
374 The lower limit of detection of the assay is defined as a concentration of standard where the maximum antibody-binding activity is significantly reduced.
The working range of the assay
extends from 0.2 nM to 2 nM (higher limit of detection) of digoxin. 3.6. Specificity of the assay The immunoassay is disturbed by the cross-reaction with compounds which are structurally related to the hapten against which the antibody has been raised. If the assay is designed for the analysis of crude plant and tissue culture extracts, it is particularly important to test the relative binding ability of pure compounds other than target molecule to the antibody. Otherwise, one will misjudge the results obtained by ELISA because plants generally contain structurally related compounds formed through common biosynthetic pathways. commercially available for the clinical use.
Anti-digoxin antibodies are now
In this case, it should be required to check their
specificity as well as their sensitivity before their application to the field of plant sciences. Table 2 shows a summary of the cross-reactivities of the monoclonal anti-digoxin antibody (DIG 64.2B.5) with compounds structurally related to digoxin. The results clearly demonstrate that the binding specificity of the antibody is directed towards C-and D-tings of the aglycone, and the antibody is extremely specific for cardenolides having a B-OH group at the C12 position which are characteristic substituents of the digoxin series (see Fig. 1). All compounds which have a modification at the C12 position (e.g. digitoxin) show a remarkable reduction in recognition. Furthermore, cardenolides with the same aglycone and different sugar moieties (e.g. lanatoside C and deslanoside) are not distinguished by the antibody from the aglycone alone (digoxigenin). It is well established that the specificity of the antibodies produced against a hapten is affected by the binding site of the hapten to the carrier (see section 2.). In our case, the digoxin molecule was attached to the HSA via the third digitoxose molecule of digoxin. The antibody did not recognize different carbohydrates linked to the C3 position of digoxigenin. Therefore the cross-reactivity study with pure compounds shows that the monoclonal anti-digoxin antibody used reacts selectively with several digoxigenin glycosides.
Table 2.
Cross-reactivity of monoclonal anti-digoxin antibody (64. 2B.5) with compounds structurally related to digoxin in ELISA (after Yoshimatsu et al. 1990).
Inhibitor
Relative IC50 value*
digoxin digoxigenin lanatoside C deslanoside gitoxin digitoxin digitalin digitoxigenin gitoxigenin digitonin ouabain digitoxose
1.0 1.2 1.2 1.8 46 2800 16000 23000 >540000 >6000000 >6000000 > 120000000
* Conentration required to give 50 % inhibition relative to digoxin. The concentration of digoxin was set at 1.0.
375 This was also confirmed by the correlation study of cardenolide contents in Digitalis lanata plants between ELISA and HPLC. The cardenolide contents in leaves of D. lanata cultivated in the field or hydroponic facilities were quantitatively analyzed by both ELISA and HPLC (Fig. 8). The content of digoxin equivalents in the leaves by ELISA correlated well to the total content of digoxin, deslanoside and lanatoside C as analyzed by HPLC.
This indicates that the digoxin equivalents
obtained by ELISA represent the total amount of digoxigenin glycosides present in the crude extracts of D. lanata. 0.5
~J o=., ra~
,., r,.) my
0.4 r= 0.957
"2 + ~
0.3
"~
0.2
+
=
o~
0.1-
~
.0
0.0
|
|
|
0.5
1.0
1.5
digoxin equivalents
% dry wt. by E L I S A
Fig. 8. Correlation of cardenolide contents between ELISA and HPLC. The correlation coefficient (r) was calculated from fitting a straight line by least squares (after Yoshimatsuet al. 1990). In addition, we attempted to examine the occurrence of specific interfering materials in crude extracts from other scrophulariaceous plants (Table 3). In the Scrophulariaceae, cardenolides have been found only in the genus Digitalis (1). Indeed all plants tested which do not belong to the genus Digitalis showed values slightly above the lower limit of detection of the assay. The lower values
observed in D. purpurea extracts can be attributed to cross-reactivity of cardenolides which do not contain a g-OH group at the C12 position (digitoxingenin glycosides principally). Table 3. Evaluation of cross-reacting materials in Scrophulariaceae leaf crude extracts using anti-digoxin monoclonal antibody (DIG 64.2B.5).
Plant species
condition
Digitalis lanata D. purpurea D. purpurea Veronicastrum sibiricum Rehmannia glutinosa var. purpurea Rehmannia glutinosa var. hueichigensis
in vitro in vitro
field field field field
LD: Value slightly above the lower limit of detection.
Digoxin equivalents ~tg/g fresh weight 25.3 0.007 0.05 LD LD LD
376 3.7. Protocol of ELISA for cardenolide detection in plant extracts ELISA procedure 1) Put the antigen (coating conjugate) solution into the wells of a microtitration plate (50 gl/well) and allow to be adsorbed overnight at 4 o C, or for 2 hours at room temperature. 2) Wash the plate with T-PBS, add C-PBS (200 gl/well) and incubate for 30 minutes at room temperature (blocking). 3) After washing the plate with T-PBS, add the specific antibody (50 gl/well, first antibody) and CPBS (50 gl/well), and allow to react for 30 minutes at room temperature. 4) After washing the plate with T-PBS, add the second antibody-enzyme conjugate (50 ~tl/well) and allow to react for 30 minutes at room temperature. 5) After washing the plate thoroughly with T-PBS (over 5 times), add the substrate solution (50 gl/well) and allow to react for 30 minutes at room temperature. 6) Stop the reaction by adding stop reagent (50 gl/well) and measure the absorbance using a microplate reader. For the inhibition study, add the sample or standard solutions (50 gl/well) together with the first antibody instead of C-PBS at step 3. Preparation of standard solutions 1) Dissolve digoxin in 70 % methanol to make 1 mM stock solution and keep in a refrigerator until use. 2) From the stock solution make standard solutions in C-PBS (at least 6 different concentrations). Sample preparation 1) Weigh fresh or lyophilized plant material. 2) Extract with an appropriate volume of 70 % EtOH by sonication for 20 min. 3) Centrifuge the solution (1 min at 18500g) and dilute the supernatant with C-PBS (at least ten times) or evaporate the combined extract in vacuo, redissolve in a small volume of 70 % EtOH, and dilute with C-PBS (at least ten times). Less than 10 % EtOH dose not affect the reaction between the cardenolides and the first antibody. Diluted sample solutions will be subjected to ELISA. However, 3 to 5 different dilutions of each sample are usually required for the assay, because the reliable range is narrow as shown in Fig. 7. If ELISA is employed for the screening of the samples which contain higher concentrations of target compound, the stepwise dilution will not be needed. 4. DETERMINATION OF CARDENOLIDES IN HAIRY ROOT CULTURES OF DIGITALIS
LANATA Transformed root culture (i.e. hairy root culture) is one of the most attractive tools for the production of secondary metabolites. The plant cells which are transformed with Agrobacterium
rhizogenes harboring Ri (root-inducing) plasmid develop "hairy roots", and they have several advantages compared to the normal tissue, organ and cell cultures, i.e. rapid growth, phytohormone independency and genetic stability. A number of researches have appeared since 1985 for the successful production of secondary metabolites by hairy root cultures (29, 30). Most of successful products have been strictly limited to those which are normally produced in roots of intact plants. In
377 order to expand the application of hairy root cultures, we established the green hairy root cultures of
D. lanata and elucidated that even the hairy roots could produce the secondary metabolites which normally appear in the aerial part of plants when it became green (15). By infection of D. Ianata leaf segments with A. rhizogenes A4, we established five hairy root clones and investigated their growth and productivity of cardenolides under the two cultural conditions, i.e. dark and 16 hr light/day. The cultures grown under light became green and showed a consistently higher growth rate than those in the dark (Fig. 9). The roots cultured in the dark accumulated only small amounts of cardenolides.
In contrast, light significantly promoted the
production of cardenolides (Table 4) which were also detectable by HPLC.
Fig. 9. Hairy roots of D. lanata cultured under 16 h/day light (left) and in the dark (right). Table 4. Growth and cardenolides production in hairy root cultures (after Yoshimatsu et al. 1990).
clone
10
condition
growth rate (fr. wt.)
digoxin equivalents mg/g dry wt.
16 h light dark
38.8 38.7
11.56 0.02
16 h light dark
4.4 4.0
0.12 0.07
16 h light dark
19.5 14.3
16.47 0.04
16 h light
32.5
3.34
dark
16.3
0.03
16 h light dark
65.9 30.5
0.23 0.05
Hairy roots were cultured in 1/2 MS liquid medium at 25 ~ for 1 month on a rotary shaker (100 rpm).
378 These contents were still lower (one sixteenth) than those in the shoot culture, however, it is noteworthy that cardenolides which production were mainly reported to be produced in aerial parts or in organs related to aerial parts (e.g. shoot-forming calli or somatic embryo) (2, 3) were produced in hairy root cultures at relatively high contents. This study might be helpful in investigating the factors which affect cardenolide biosynthesis. 5. FUTURE ASPECTS OF ELISA FOR PLANT SCIENCES The evaluation of the biosynthetic capability of cultured plant cells is a key step for the improvement of secondary metabolite production as well as for the investigation of culture conditions (31-33). Analytical techniques have made remarkable progress in these decades. Various chromatographic methods, in particular, have been improved remarkably and made it possible to determine the concentrations of much lower amounts of substances accurately than ever before. Plant secondary metabolites can be analyzed by bioassays (34) or by chromatographic methods only after prepurification (or extensive fractionation for bioassay) of plant extracts, and in some cases, the quantitative analysis from small amounts of samples is often impossible.
However,
immunoassay methods, which exploited the intrinsic selectivity of a high affinity antibody for its antigen, have enough potential to detect low amounts of the compound in crude plant extracts. Indeed, the number of researches which employed ELISA for the detection of phytochemicals in crude plant extracts has been increasing and these researches are proving the sensitivity and simplicity of this technique (35-39). Recently the application of immunoassay in plant sciences has expanded widely. Strnad et al. (40) succeeded in the detection and identification of a new naturally occurring cytokinin using HPLC fractionation combined with polyclonal antibody-based immunoassay. The exploitation of a HPLC fractionation method combined with immunoassay is specially attractive for the biogenetic localization and the isolation of compounds closely related to the antigens (free and conjugated forms as well as immediate or distant biosynthetic precursors). On the other hand, certain enzymes involved in secondary metabolite biosynthesis can be studied with antibodies specific for their substrates or products. Treimer and Zenk (41) studied the bioconversion of tryptamine and secologanin to corynanthe-type alkaloids using RIA and further characterized the properties of the converting enzyme. Shr6der et al. (42) investigated the function of gene 2 in the T-region of Ti plasmid using ELISA for IAA and elucidated that the gene 2 codes both in bacteria and in plants for an amidohydrolase which is involved in the biosynthesis of indole-3-acetic acid. The extension of this approach to biosynthetic pathways must be most promising to facilitate the purification of enzymes and elucidation of their properties even if spectrophotometric or radioassays are unavailable. In addition, tissue and organ-specific distribution and subcellular fractionation of secondary products using immunohisto- and immunocyto-chemical techniques should give us further information regarding the biosynthesis of secondary metabolites, as the subcellular compartmentation of plant secondary metabolites is believed to play an important role in the regulation of their biosynthetic pathways. Although immunocytochemical localization of a hapten generally presents a number of technical problems, especially those related to leaching of the haptens during chemical fixation and dehydration of the material, the use of this approach (43) will increase along with the development of biochemical technology.
379 Antibody-based immunoassays may become widely accessible and might turn into routine procedures in physiological, biochemical and molecular biological studies on plant secondary metabolites. Future development in this field will provide a detailed knowledge of tissue and organ distribution, intracellular localization and translocation of secondary products. REFERENCES
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
26 27 28 29 30 31 32
W. C. Evans, Pharmacognosy, 13th Ed. Bailliere Tindall, London, 1989, pp. 504-513. W. Rticker, in Y. P. S. Bajaj (Ed.), Biotechnology in agriculture and forestry 4, Medicinal and aromatic plants I. Digitalis spp.: In vitro culture, regeneration, and the production of cardenolides and other secondary products, Springer-Verlag, Berlin Heidelberg, 1988, pp. 388-418. M. Luckner and B. Diettrich, in I. K. Vasil (Ed.), Cell culture and somatic cell genetics of plants 5. Cardenolides, Academic press, San Diego, 1988, pp. 193-212. C. Brugidou, M. Jacques, L. Cosson, F. X. Jarreau, T. Ogerau, Planta Med., (1988) 262265. W. Kreis, U. May, E. Reinhard, Plant Cell Rep., 5 (1986) 442-445. S. Sch6ner and E. Reinhard, Planta Med., (1986) 478-481. T. Stuhlfauth, K. Klaus, P. H. Fock, Phytochem., 26 (1987) 2735-2739. M. Wichtl, M. Mangkudidjojo, W. Wichtl-Bleiner, J. Chromatogr., 234 (1982) 503-508. E. W. Weiler, M. H. Zenk, Phytochem., 15 (1976) 1537-1545. E. W. Weiler and P. Westekemper, Planta Med. 35 (1979) 316-322. R. Garve, M. Luckner, E. Vogel, A. Tewes, L. Nover, Planta Med., 40 (1980) 92-103. M. Hagimori, T. Matsumoto, T. Kisaki, Plant Cell Physiol., 21 (1980) 1391-1404. E. Vogel, M. Luckner, Planta Med., 41 (1981) 161-165. K. Saito, M. Yamazaki, K. Shimomura, K. Yoshimatsu, I. Murakoshi, Plant Cell Rep., 9 (1990) 121-124. K. Yoshimatsu, M. Satake, K. Shimomura, J. Sawada, T. Terao, J. Nat. Prod., 53 (1990) 1498-1502. E. Engvall, P. Perlmann, Immunochemistry (Chemical Abstracts: 76), 8 (1971) 871-874. E. Engvall, K. Jonsson, P. Perlmann, Biochem. Biophys. Acta, 251 (1971) 427-434. B. K. Van Weemen and A. H. W. M. Schuurs, FEBS Leters, 15 (1971) 232-236. H. A. Kemp, M. R. Morgan, in F. Constabel and I. K. Vasil (Eds), Cell culture and somatic cell genetics of plants 4. Use of immunoassays in detection of plant cell products, Academic Press, San Diego, 1987, pp. 287-302. B. F. Erlanger, in H. Van Vunakis and J. J. Langone (Eds), Methods in enzymology 70 Part A. The preparation of antigenic hapten-carrier conjugates: A survey, Academic Press, New York, 1980, pp. 85-103. T. W. Smith, V. P. J. Butler, E. Haber, Biochemistry, 9 (1970) 331-337. J. Sawada, N. Janejai, T. Terao, Bull. Nat. Inst. Hyg. Sci., 108 (1990) 29-33. G. Galfr6 and G. W. Butcher, in T. L. Wang (Ed.), Immunology in plant science. Making antibodies, Cambridge Univ. Press, 1986, pp. 1-25 G. K/3hler and C. Milstein, Nature (London), 256 (1975) 495-497. E. W. Weiler, J. Eberle, R. Mertens, R. Atzorn, M. Feyerabend, P. S. Jourdan, A. Arnsheidt, U. Wieczorek, in T. L. Wang (Ed.), Immunology in plant science. Antiseraand monoclonal antibody-based immunoassay of plant hormones, Cambridge Univ. Press, 1986, pp. 27-58. V. T. Oi, L. A. Herzenberg, in B. B. Mishell and S. M. Shiigi (Eds), Selected methods in cellular immunology. Immunoglobulin-producing hybrid cell lines, WH Freeman and Company, San Francisco, 1979, pp. 351-372. J. W. Goding, J. Immunol. Methods, 39 (1980) 285-308. R. H. Kennett, T. J. Mckearn, K. B. Bechtol, in Monoclonal antibodies. Hybridomas: A new dimension in biological analyses, Plenum Press, New York and London, 1980. K. Saito, M. Yamazaki, I. Murakoshi, J. Nat. Prodc., 55 (1992) 149-162. M. Sauerwein, K. Yoshimatsu, K. Shimomura, Plant Tissue Culture Letters, 9 (1992) 1-9. D. K. Dougall, in F. Constabel and I. K. Vasil (Eds), Cell culture and somatic cell genetics of plants 4. Cell cloning and the selection of high yielding strains, Academic Press, San Diego, 1987, pp. 117-124. M. Sakuta, A. Komamine, in F. Constabel and I. K. Vasil (Eds), Cell culture and somatic
380
33 34 35 36 37 38 39 40 41 42 43
cell genetics of plants 4. Cell growth and accumulation of secondary metabolites, Academic Press, San Diego, 1987, pp. 287-302. J. M. Widholm,in F. Constabel and I. K. Vasil (Eds), Cell culture and somatic cell genetics of plants 4. Selection of mutants which accumulate desired secondary compounds, Academic Press, San Diego, 1987, pp. 125-137. H. Arens, H. O. Borbe, B. Ulbrich, J. St6ckigt, Planta Med., 46 (1982) 210-214. M. R. A. Morgan, S. Bramham, A. J. Webb, R. J. Robins, M. J. C. Rhodes, Planta Med., 51 (1985) 237-241. M. R. A. Morgan, D. T. Coxon, S. Bramham, H. W. Chan, W. M. J. van Gelder, M. J. Allison, J. Sci. Food. Agric., 36 (1985) 282-288. M. Jaziri, Phytochem., 29 (1990) 829-835. Y. Kikuchi, M. Irie, K. Yoshimatsu, K. Ishimaru, K. Shimomura, M. Satake, S. Sueyoshi, M. Tanno, S. Kamiya, J. Sawada, T. Terao, Phytochem., 30 (1991) 3273-3276. M. C. Cibotti, C. Freier, J. Andrieux, M. Plat, L. Cosson, C. Bohuon, Phytochem., 29 (1990) 2109-2114. M. Strnad, W. Peters, E. Beck, M. Kaminek, Plant Physiol., 99 (1992) 74-80. J. F. Treimer, M. H. Zenk, Phytochem., 17 (1978) 227-231. G. Schr6der, S. Waffenschmidt, E. W. Weiler, J. Schr6der, Eur. J. Biochem., 138 (1984) 387-391. L. Brisson, P. M. Charest, V. De Luca, R. K. Ibrahim, Phytochem., 31 (1992) 465-470.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
381
The Xenocoumarins and Related Biologically Active Dihydroisocoumarins Bernie Vincent Mc Inerney and Walter Charles Taylor
1.
INTRODUCTION
The
xenocoumacins
are
3,4-dihydroisocoumarin and
co-workers
Xenorhabdus but well
were
from
The
in
as are
pests. role for
The in
As
and
the
which
would
be
insect
compound from
the
are
nematodes
authors
the
genus
compounds
activity
as
of
and
the
but
also
growth
These
spp.)
not
enhances other
of
only
the
and
their
of
control
nutrients of
nematodes for
genera
the
with
families
studied
Several for
particularly
associated
(ref.2-4) . media
providing
these insect
plays
a
conditions
by
producing
microbial
flora
in
(ref.6,7) . and
Nature
a number
Xenorhabdus
hydroxystilbene
of
antiulcer
spp.)
(Xenorhabdus
host by
expected,
isolated
antimicrobial
(ref.5) .
symbiont
inhibit
antibiotic
the
as
symbiotically
commercially
reproduction
cadavers
pyrrothine
are
artificial
used
bacterial
by
bacteria
potent
Heterorhabditae on
being
killing
insect
They
bioinsecticides
nematode
antibiotics the
exhibit
insect-pathogenic
cultured
nematodes
isolated
isolated
of
(Xenorhabdus
bacteria
Steinernematidae potential
to
broth
methylbutylamino-
activities.
themselves.
soil-dwelling, been
found
biological
source
were
occurring
recently
culture
They
subsequently
interesting
have
derivatives the
(ref.l) .
as o t h e r
naturally
not
depend
of d i f f e r e n t species,
derivatives
derivatives
does
solely
antibiotics
including
(ref. 8) ,
(xenorhabdins,
on
have
one been
indole
and
dithiolopyrrolone ref. 9)
as
well
or
as
the
xenocoumacins. We
present
structure (i)
and
here
a
elucidation 2
compounds
(2). that
particular, structures
the and
xenocoumacins,
As
review
and
well,
contain
the
AI-77
and
of
we
review
be
studies
on
activities the
work
amicoumacin
of
discussed
with
are
respect
isolation,
xenocoumacin
others
compounds,
activity
the
of
3,4-dihydroisocoumarin
pharmacological will
our
biological
very to
ring
on
related
system.
whose
In
chemical
similar their
1
to
the
structure
382 determination,
biological
relationships
and chemical
Presented
that
have
interesting all
below
been
is an overview
isolated
activities.
compounds,
summary
of
activities,
synthesis.
their
most
of
OH
0
from
sources the
of the
natural
It is not and
structure-activity 3,4-dihydroisocoumarins
sources
and
some
of
their
intended as a complete
listing
activities
provides
important
but
rather
findings
on
N.H2
.
this
of
class
a of
compound.
7r,'/
.y
-O 2
.OH
6
N
4'
2'
7,,
8,~
O
9,, ,
10, ,
,
/
,
'
OH
NH 2
NH
1
OH 8
7
0
I
0 2
6
2:.~3.
4'
O
,,I
2.
AN
OVERVIEW
OF
THE
OH
H
11'
OH
12'
"
2
NATURALLY
OCCURRING
3, 4 -
DIHYDROISOCOUMARINS
Compounds occur
widely
containing distributed
microorganisms organisms,
and
from m y c o t o x i n s growth into alkyl
of
a
wide
Structurally, classes
derivatives,
(asperentins),
fungi) ,
such
have
been
of
and
system
isolated and
biological
compounds
mellein
ring
insects
to pheromones,
these as
3-phenyl
ochratoxins
They plants,
spectrum
and a n t i m i c r o b i a l s
promoters.
a number
3,4-dihydroisocoumarin
in Nature.
(mainly display
the
activities
phytoalexins can
its
be
from
marine and
subdivided
derivatives,
derivatives,
and the 3 - m e t h y l b u t y l a m i n o
3-
cladosporins derivatives.
383 2.1
Mellein
By
far
and
the
most
dihydroisocoumarin Mellein from
(also
the
in
commonly
compounds
called
is
ochracin)
1933.
Its
has
since
structure
Together
with
isolated
f r o m A.
(+)-mellein been
oniki
as
boring
Helicascus
and
kanaloanus
from
5-carboxymellein
also
include and
insects,
termites
mandibular
J ~l~
mellein and
gland
Myrmecocystus
02
R3
implicated
in
(ref.22,23) .
of
defensive
the
weaver
of
elm
bark
Fusarium
produced
fuckeliana
Cercospora ascomycete
by
the
apple
(3) , c i s (8)
(-)-mellein
ant
H
H
H
H
OH
H
5
H
H
OH
6
Me
CO2H
H
H
H
8
OH
H
H
by
the
has
and
was
of
where
it
has
honey
of
several
is
of
thought
gland
mellein
obtained
secretions
phenolic
compounds
(ref.25) .
together
been the
This
of
ant to
secretion
?doddi
ant a
be
a
of sex
swarming as
gland
a of
(ref.24) .
Crematogaster
mixture
was
of the
species
identified
mandibular
the
with
in
species
synchronised
Polyrhachis(cyrtomyrma)
metapleural
glands
identified
the
of
also
H
exocrine
been
males
secretions
deformis
was
R3
4
co-ordination It
R2
3
workers
spp.)
the
sexes
Australian the
the from
(R)-(-)-mellein
produced
(ref.21)
(Componotus
ants
From
have
(R)-(-)-5-hydroxymellein
Mellein
secretions
carpenter
the
is
moths.
testaceus
pheromone both
it was
oblonga (7)
(ref.16) ,
Hawaiian
7
component
to
isolated
R1
6
of
(5)
Phomopsis
fungus
deterrents
0
8
Z
In
sources.
(ref.20) .
OH
ants,
(ref.12) .
mangrove
Nectria
fungus
Newbold different
missouriense
(ref.19) .
the
(-)-8-hydroxy-3-
the
(4)
From
and
isolated
ochraceus
3-hydroxymellein
was
(3R, 4 R ) - ( - ) - 4 - h y d r o x y m e l l e i n isolated
and
Phytotoxins
obtusa
be
3, 4-
derivatives.
A.
and
many
the
feeding
(ref.18) .
Botryosphaeria
pathogen
(6)
Gyrostroma
(ref.17)
and
the
its
originally
to
Blair
From
(S)-(+)-mellein
(ref.15) ,
taiwanensis
and
shown
from
(4)
(ref.13) .
(3) , 5 - m e t h y l m e l l e i n
(ref.14) .
larvarum
by
of
and
(ref. i0)
was
isolated
4-hydroxymellein
isolated
beetle
been
(3)
a metabolite
melleus
methyl-3,4-dihydroisocoumarin Mellein
occurring
mellein is
Aspergillus
fungi
(ref.ll)
Derivatives
shown
of to
other be
a
384 powerful
repellant
and
from
body
identified
Rhytidopenera has
shown
been by
molesta
wing
sociella
of
from
the
sociella
and
the
pheromone
has
been
is
raised
defensive (ref.31) .
Carrot exposure
low
metals,
produce
a to
in
as
(ref.35) . nucleotide and
fungi
cells
indole
(ref.40) . and
acetic
be
to
a compound It
has
factor
In
together
and
is
Azadirachta with
7-methoxymellein
~2
indica
OH I
to has
O II .,J~
as
animal
3
4
Me
also
(9)
plants
of
inhibit
cyclic
bacteria,
been
yeast
and
plant
isolated
from
derivatives
6-methoxymellein
7-hydroxymellein
R2
9
H
OMe
10
H
OH
OH OMe
is
carrots
chlorinated
twigs
(10) ,
11 12
2,4,5-T)
stored
(ref.32)
R1 02
fungi,
ethylene,
mechanisms
to
to
well
two
(Neem)
Apsena
6-methoxymellein
(ref.41) .
6
5
higher
defence
shown as
with
6-hydroxymellein (12)
R~7.,~
along
and
off-flavour
toxic
6-methoxymellein
2,4-D
the
6-methoxymellein
the
been
of
with as
identified
(ref.36)
8-hydroxy-
(such
in
A.
insect
beetle,
infection
was
of
this
compound
agents
in
viewpoint
larvae of
of
Aphomia
moth
a constituent
herbicides
a bitter
also
(ref.39,40)
by
and
subsequently
(ref.37)
as
phytoalexins
isolated
giving
origin
phytoalexin
phosphodiesterase
fungi
the
component
wax
tenebrionid
stress
of
important first
the
released
ochraceus W i l h e l m
related
chemical
acid,
accumulation
an
a major
of has
Grapholitha
moth,
a biosynthetic
fungal
which
be p a r t
pheromone
last-instar
The
of
or
to
be
identified
dihydroisocoumarin
(ref.38) .
Sporormia found
subjected
Sondheimer
1957
been
temperature
(ref.32-34) . The plants.
(ref.30).
secretions
roots
to
the
ant
(R)- ( - ) - m e l l e i n
Aspergillus of
also
termite
appears
bumble-bee from
of
mellein
fruit
to
the
canals of
glands
a courtship
found
fungus
has
and
was
ponerin.e
produce
Further,
interesting
possibility
abdominal
cephalic
Oriental
of
Mellein
Australian
ants
of
also
alimentary
pubescens
thought
was
pheromone
3,4-dihydroisocoumarin
The
to
male
mellein-producing
isolated
heavy
the
and
gland
ants.
the
(ref.27) .
a constituent
(ref.30) . It
the
(ref.26) .
a deterrent
(ref.28,29)
male
that
as
hairpencils
enemy of
ovatus a n d C. pugnax
defences
identified
the
the
to be
chemical
of
extract
metallica
of
been
toxin
Cornitermes
soldiers their
a
H H
was (ii)
385 The
presumed
hydroxymellein stored from
(i0),
under found
produced
minor
the
the
(ref.45) .
the
fungus
the
genus
of
more
other
common
almond
ceratosperma
bark
terreus
(ref.42)
Tabebuia
of
Pyricularia the
Discula
oryzae
have
(6).
It
Fusicoccum
amygdali Phomopsis
and
~R2
been
of
Semecarpus
spp.
heartwood
of
Alternaria
3,6-dihydroxymellein and
stem
structure
2.2
elongation may
be
a
of
the
from
the
Hypoxylon
(ref.52) , the
and
Valsa fungus-
Fijian
R2
R3
,OH
13 Me
Me
Me
Me
15 14 Me H
Me H
H
from
the
as
sclerotinin culture
species
sclerotiorum
kikuchiana (15) of
was
rice
sufficient
Asperentin
Tanaka
isolated seedlings
for
(14) the and
OH
O
4'
16
of
(ref.55) .
respectively.
plant shown
growth-promoting
(13)
and
B
the Their
7-trimethylmellein growth to
indicating
(Cladosporin)
A
filtrate
3, 6 - d i h y d r o x y - 4 , 5 ,
3, 6 - d i h y d r o x y - 5 , 7 - d i m e t h y l m e l l e i n
fungus
been
from
(ref.14) ,
R1
Sclerotinia
fungus
identified
the
One
isolated
(ref.48) ,
metabolites,
isolated
structures and
found.
been
the
~R3
phytopathogenic (13)
has
and
oblonga
O
growth-promoting
were
isomer
(ref.54) .
HO"
have
and
Ceratocystis
and
(ref.46)
been
has
R I ~ ~-L,~ ~ , ~
Plant
has
(ref.43)
(ref.47) .
OH
(14) ,
roots
isolated
subsequently
(ref.44)
siamea
(ref. 49, 50) ,
fidjiana
and
6-
carrot first
(+)-6-hydroxymellein
derivatives
(ref.53) ,
from was
avellanedae
Cassia
of
(ref.51) , p l a n t s
wood
Euphorbia
compound
5-methylmellein
spp.
infected
isolated
This
flowers
pathogen
Numularia
been
mellein
is
6-methoxymellein,
also
Recently,
from
of
(ref.32) .
fungi
isolated Many
precursor
Aspergillus
of
in
by
has
stress
a mutant
been
biosynthetic
promoter
stimulate that
activity
From
this
root basic
(ref.56) .
386 Asperentin entomogenous 6-methyl same
ethers
and
shows
activity
a review
2.3
and
metabolite
and
an and
58).
The
It has
subsequently
spp.
(ref.60-62)
properties growth been
as
well
inhibitory
the
subject
of
(ref.65) .
3-Alkyldihydroisocoumarins OH
0
R1
a2J'~ OH
R3
R2
R3
17
H
OH
n-C11H23
18
H
H
n-C11H23
19
Br
OH
n-C3H 7
O
OH
0
CH30,,'"~
HO
21
R1 = H
22
R 1 =CH30
the
isolation
20
There
have
been
numerous
reports
8-hydroxy-3,4-dihydroisocoumarins.
of
Ononis natrix
From
6,8-dihydroxy-3-undecyl-3,4-dihydroisocoumarin together
with
(ref. 66, 67) .
cephalornithos (18)
8-
Cladosporin
plant has
of
its
from
antibacterial
(asperentin)
with
(ref.57,
(Aspergillus)
(ref.63)
Cladosporin
by Scott
minor
(ref.59) .
Eurotium
antifungal
(ref.64).
a
independently
cladosporin
several
specific
as
flavus t o g e t h e r
5'-hydroxyasperentin
obtained
and n a m e d from
insecticidal
effects
isolated
4'- and
was
cladosporiodes isolated
was
of Aspergillus
and
compound
been as
(16)
strain
the
6-methoxy,
An
extract
contained
(ref. 68) .
The
dihydroisocoumarin brominated
compound
metabolite
of
fusamarin
(ref.70)
and
the from
and
the
(Leguminosae)
(17)
was
6'-hydroxy
brown
3-alkylisolated
derivatives
Caulocystis
algae
8-hydroxy-3-undecyl-3,4-dihydroisocoumarin
only
other
compound
from
hiburipyranone
3,4-dihydroisocoumarin metabolite
6'-oxo of
of
(19))
sponge (20)
which
Mycale was
Fusarium
reported a
finding
marine
of
organism
a
3,4-
was
the
(7-bromo-6,8-dihydroxy-3-propylwas
isolated
adhaerens
isolated
larvarum
from
as
Lambe a
Fuckel
a
(ref.69) .
species the
cytotoxic
of
The
Fusarium
insecticidal
387 metabolites dimethyl
fusarentin
ether
(ref. 15, 71) .
were
ether
isolated
Asperentin
biogenetically 2.4
(22)
6-methyl and
(21)
along
and
fusarentin
with
compounds
6,7-
(+) - m e l l e i n
17-22
are
(3)
clearly
related.
3-Phenyldihydroisocoumarins
OH
0
8
7
I
02
o
,)__ OR 2 4
\ R1
23 R 1 = R2= H 24 R 1 = OH, R 2 = Me Hydrangea
species
dihydroisocoumarins.
H.
(Saxifragaceae)
macrophylla,
phenyl-3,4-dihydroisocoumarins and 77),
its 8 - ~ - g l u c o s i d e and
its
times
activity
as
does
phyllodulcin
was
selective mast
derivative
8-~-glucoside
thousand
sweeter
than
(ref.74),
sugar
hydrangenol
Recently,
CO2H O
OH
ochratoxins
originally including
release
with
from
rat
are
isolated
from
subsequently
A. melleus
(ref.85-89)
.
hepatotoxic
whilst
a small
family
Aspergillus
been
reported
(ref. 83, 84)
Ochratoxins ochratoxin
and
are A
O
CI
25
have
monomethyl
leucadendron
histamine
is a
antifungal
Ochratoxins
O
They
3-
(ref. 72, 73)
(24) (ref.75-
(ref.80) . against
produces
Phyllodulcin
and p o s s e s s e s
Melaleuca
in
(23)
phyllodulcin
(ref.78) .
rich
(ref.81) .
2.5
The
particular,
(ref.79)
from
activity
in
as h y d r a n g e n o l
derivative
isolated
inhibitory
cells
such
are
of toxic
ochraceus
from
other
several
known is
fungal
to
Wilhelm
(ref.82) .
Aspergillus
species
species be
a teratogen
metabolites
of Penicillium
nephrotoxic (ref.90) .
and
Because
388 ochratoxin-producing (including health al
grain)
hazard.
in
1965 to
B and
C were of
were
Ochratoxin
moiety,
identified of
which
hydroxyochratoxin Several
It
as
A
A
published
D)
van
ethyl
der
Merwe
et
7-carboxyl Ochratoxin
ester
derivatives
Subsequently,
the
as
methyl
well
7-carboxymellein
the
human
7-carboxy-
the
(ref.92)
foods,
and
the
through
and
and
on
by
contains
isolated
reviews
stored
animal
configuration.
(ref.91) .
B were
(ochratoxin
comprehensive
3R
dechloro
A
and
deduced (25)
linked
the
of
a potential
A
is
has
the
ochratoxin
ochratoxin
contaminants
structures
L-phenylalanine.
respectively
common are
Their
5-chloromellein
are
ochratoxins
(ref.82,91) .
group
esters
fungi
the
as
4-
(ref.93) .
ochratoxins
have
been
(ref.94-98) .
2.6
3-Methylbutylaminodihydroisocoumarins
OH
0
0
OH
26 There
are
a
dihydroisocoumarin to
the
an
amino
(30), C
acid
m
E,
remainder
is
F
of
the
not
fit
suspect
as
(26) .
identical
review Note,
the
G
below should
amicoumacin
A
amicoumacins
and
but
pharmacologically
3.
are
also
ISOLATION The
of
xenocoumacins
OF
antibiotic
and be and are very
XENOCOUMACINS
xenocoumacins metabolites
were of
(27),
A
on
these
but
of
this
our
proposed that
B
only
similar
AND
isolated
(27),
Xenorhabdus
is C
(28)
compounds
and
baciphelacin
and The
structurally as d i s c u s s e d
and The the (34)
structure
structure
AI-77-A
RELATED as
B
R
(28),
(35) .
respectively.
not
B
reported
noted
similar
where
Y-05460M-A
structure (26)
very
AI-77-A
are
concentrate
are (26)
amicoumacin
reported
occurring
structure
substance
structure
discussed it
They
(33),
naturally
structurally
general
and
will the
general
Also,
to
and (34) ,
of
which
the
fragment.
(32)
baciphelacin
xenocoumacins.
with
having
derived
(31),
number
metabolites,
xenocoumacins,
(29) ,
does
small
agrees B
are
AI-77s, analogous
below.
COMPOUNDS
part
of
the
spp.
by
ourselves
investigation and
co-
389 workers. broth
of
Xenocoumacin an
subsequently
was
2
antibacterial
initially
is shown
based
in S c h e m e
strain All
Fractionation
the Q1
culture and
together
was
Micrococcus
on
from
species
X.nematophilus
from
(ref.l) .
assay
isolated
Xenorhabdus
isolated
xenocoumacin procedure
1
undescribed
guided
luteus.
The
was with
by
an
isolation
i.
Fermentation
culture
broth
1
F l o ~ h chromatocrra~hv _
_
discarded ~
_
(5 L)
(ODS-silica)
H20 w a s h I:I M e C N : NH4OAc (0.2M, pH 4.5)
Crude
mixture
(purity
3%)
1
Sephadex
G-10
chromatography
0.5 % HOAc Crude x e n o c o u m a c i n s
Preparative
(purity
20%)
RP-HPLC (Cls)
I 30"70 MeCN 9 NH4OAc (0.2M, pH 4.5) 1, Scheme
i.
Isolation
of x e n o c o u m a c i n
X. nematophilus Reversed-phase powerful useful of the
tool
in the
in
spp.
purification of We
dihydroisocoumarins
detector
at
254
nm
and
culture
utilised by 314
1
(i) and
2
(2)
from
All.
high-performance
the
analysis
Xenorhabdus
2
liquid
broths the
for
It high
also
with
Absorbance
a
UV
was
proved
producing
characteristic
monitoring nm.
chromatography
protocol.
a
very
strains
spectrum
of
multi-wavelength
ratioing
techniques
in
390 combination
with
retention
xenocoumacins,
even
Alternatively, array)
a more
could
be
identification This high
of the
strains
study
AI-77
from
the
culture
This
organism strong
77-A
products
biological
isolated
by
layer
had much
weaker
the
of the
column (A
new
culture
vacuo,
to
find
analogues
from
our
work
however,
species
(34)
in
1982
found
and
G
and et al
(ref.99) . activity
and
(33).
AI-77-A
later
were
named
AI-
Note
that
and
B
fermentation
minor
products
isolation
stages less
employed
adsorption
whereas,
were
with
procedure
by
column
AI-77-B
to
G were
and
silica
gel
column
(29))
were
chromatography B
(28)
and
broth a
C
of
B. in
series
exchange,
of
activity
A
(ref.100,101)
column
as
(reported
was
well
isolated BN-103
pumilus
1981
activated
Amicoumacin
as
and
major
a diverse
latter).
The
in
a
. The
chromatography
carbon the
by
other
size
component range
of
compounds
(ref.101) .
was
filtrate
(ref.102) . filtrate
were
assigned. and
Shimojima
antibacterial
(32)
antibiotics
activities
isolated of
step u s i n g
B.
The
with
precipitation
chromatography
diode-
(ref.99) .
involved
activity
culture
us
of
by
AI-77
followed
culture
for
F
resin),
(27),
antibacterial
Baciphelacin antibiotic
E,
not
XAD-2
cation
biological
by
other
sample
showing
AI-77-A
chromatography.
exhibited
.
accurate
indicators,
isolated
compounds
AI-77s
The
the
procedure
including
(e.g.
indications
pumilus
earlier
other
chromatography
isolation
other
was
the
program
exclusion
were
m(31),
AI-77-E
screening steps
as
matrices.
identify
larger
peaks
solutions the
employed
to
are
a
chromatography
from
in
taxonomic
soil
Seven
adsorption
al
on
from
(30),
amicoumacins
et
there useful
Bacillus
(Amberlite
and t h i n
and
compounds
of
activity.
chromatography
Itoh
C
The
exchange
The
of
isolated
in
respectively. cation
be
required
broth
of
successfully
assign
detector
aid
in c o m p l e x
strains
to
broth
this.
(28),
structure
ma jot
in
been
may
series was
B
methods
metabolites
fluorescence.
(27),
the
from
Such
Further,
is
to c o n f i r m
The
an
scanning
has
metabolites
additional
and
sophisticated
of m i n o r
xenocoumacins.
these
used
culture
used.
technique
were
crude
xenocoumacin-producing
that
and
behaviour
in
several
thiaminolyticus
isolation
ethyl
with
years
scheme
acetate
ether
a chloroform-methanol
a
IFO 9.5,
in
1975,
3967/B-I-7
employed
at pH
then
earlier,
as
extraction
concentration
final gradient.
silica
gel
0 0 0- r
Z'T"
Z n-
~ \7-~~ o-~~
"1"
0
0
0
\ Z~
0
"1"'1-
0 -r0
H
o0-1O0
H
~~ ~
Zl
~
0
Co
I
0 0
~Z-r
o
0
Z
0 0
z-r
\
o
o
0
392 Substance
Bacillus
sp.
ant itumour It
Y-05460M-A
05460M
(35)
(ref.103).
was This
isolated compound
from
has
a
culture
of
antimicrobial
and
activity.
is
interesting
compounds,
with
from Bacillus
the
that
exception
all of
of the
these
structurally-related
xenocoumacins,
were
isolated
and
alkaline
species.
OH
O
HG./
N
0
OH
N I--I,2
34 OH
O ,OH
H
NH,2
35
4.
STRUCTURAL 4.1
UV/IR
The
Spectroscopy
xenocoumacins
solutions the
STUDIES
similar
occurs
at
247
and
shift
reported
for the
all
AI-77
the
(33), ring
to that
UV
spectra
of m e l l e i n
the in Of
Upon
(MeOH)
to
298 nm.
at
in 1660
the
IR
The
has
cm-1 w h i c h
of
The
been
of
kmax (MeOH)
alkali
there
UV s p e c t r a
(ref.99) .
is
were
(ref.102) With
hydrolysed
a
also and
AI-77-G
causing
a
8-hydroxy-3,4-dihydroisocoumarin
a characteristic spectra
characteristic
baciphelacin
AI-77-G
ring
and
Similar
(ref.101),
except
neutral
chromophore.
addition
to 347 nm.
amicoumacins
compounds
also exhibits
note
absorption
nm. 314
dihydroisocoumarin
Imax
system
314 from
in
(ref.12)
8-hydroxy-3, 4-dihydroisocoumarin
bathochromic
shift
exhibit
of
supports
fluorescence
spectrum.
the
xenocoumacins,
the
presence
of
a
is
an
lactone
393 carbonyl
group
function.
A
the
.
Upon
hydrogen
absorption
(ref.99) ,
expected.
showed
carbonyl
bonded
band
is
amicoumacins
acetylation
AI-77-B
lactone
is
similar
AI-77s
(ref.102)
that
group
a
a shift from
to
a
found
phenolic
in
the
(ref.104)
IR
and
shift
in
IR a b s o r p t i o n
to
spectra
absorption
1730
cm-1
of
baciphelacin
in the
1680
hydroxy
would band
be
of
the
upon
acetylation
in the
elucidation
(ref. i05) . 4.2
IH
n.m.r.
Studies
IH n . m . r . s t u d i e s of
the
structures
advent
of
the
effort
which
structure
by
includes
the
(ref.40), C,
was
following
d6-DMSO,
confirm
of
(H4)2 will on
the
(H4)2
D20)
are
the
isocoumarin available
dihydroisocoumarins
(ref.104),
(ref.l) .
polarity
been
such
used
that
(amicoumacin
(ref.102), series
and
s o c o u m a r ins
AI-77-B
The
of
to
of
and
most
derivatives
studies
the
provides
chemical
and
allow
and
2D-COSY
a
with of
are
not
recently
protons been
nuclear
effects and to used
spectra
of
vicinal be
made
widely
Overhauser
IH-IH c o r r e l a t i o n
of
CD3OD,
necessarily
geminal
have
range
(CDCl3,
deshielding
taken
experiments
more
spectra
shifts
assignments
and
compounds
obtain
Nevertheless,
Decoupling
to
effect
have
been
(ref.l) . feature
from the
H3
with
be a p p a r e n t
resonates
of
attached
substituent
vicinal
C
the
with
data
3-methyl-dihydroi
baciphelacin
2
consistent
easily.
singular
signal
the
structure-activity
varying has
experiments
also
because
for
the
assignments
(n.O.e.)
the
of
constants
relatively
and
1 and
comparable.
coupling
distinguishing occurring
since
contrasts
(ref.12, 35) . IH n.m.r,
(ref.105),
solvents
substituents
A
B
derivatives This
(ref.106,107) .
CD3COCD3,
directly
for
(ref.59) ,
A,
G
extensively
1960's.
naturally
prepared
Because
used
and
data
the
methods
xenocoumacin
AI-77-B
deuterated
in
necessary
chemical
F
used
dihydroisocoumarin
amicoumacin
additional
been
cladosporin
D,
recently, of
of
technique
derivatives" B) ,
have
coupling
a
with
5 3, H3,
spectra
chemical
lactonic further
attached
near
to n o n - e q u i v a l e n c e
and
the
at
dihydroisocoumarins in
as
the
group.
multiplicity
or as the
of the p r o t o n s .
shift
ester
C3.
either
of
range The
will
The
benzylic
a two
proton
AB p o r t i o n
of
6 4.50-4.9
coupling arise
an A B X
with
depending
methylene doublet
is
group
from
the
system
due
394 The
three
Sporormia
simple
fungus
(ref.40)
system,
(60 MHz,
CH3
deshielded
is
derivative The with
(39)
hydroxyl
CDCI3)
shows
and
isocoumarin
fragment.
shielded
and
conformation
group.
and
/
OH
H
3
0
the
from for
the
The
C3-
dichloro
shift
from H - b o n d i n g structures
patterns
aromatic
ring
H5
been
reported
derivative and
H4B
ring
is
(4 in
and a
(ref.47) .
~5 3.83 ~CH3~ ) ~
8 3.90 CH30
4.45, m
CH3 5 1.42, d, J 6-7 Hz
~t H H 8 6.33, d, q5 2.80, d, J 2-3 Hz
J 6-7 Hz
37
ii
6The Hz
half-chair
CH3 8 1.54, d, J 6-7 Hz
H
for
(41) .
8 6.33, d, ~ 2.88, d, J 2-3 Hz J 6-7 Hz 36
5 6.33, d, J 2-3 Hz H~
H7
have
..~) i 8 H 4.80, m H
and
meta c o u p l i n g of 2Hz
O
~
of
for the
with
II
H
a
non-equivalence.
complex
equatorial
J 2-3 HzH~~ , , ~~.,~~ T ~5 3.87 C
H4A
lactone
group
11.33
6.33, d,
and
the
with the m e t h y l
data
In
IH n.m.r,
monoacetate
H3
that
6 6.3,
n.m.r,
its
between
indicate
more
data
(36-40).
of
downfield
dioxygenated
highfield
n.m.r,
degree
similar
at about
isolated
function.
The
show
IH
structures
slight
typical
In the
(I0)
couplings
respectively)
a
the
resonate
basic
the
h-oxygen
derivatives
(ref.59) . Recently, hydroxymellein
on
shows
peri-carbonyl
the
vicinal
the
(H4) 2
at C8
provide
shown
by
cladosporin are
3-methyldihydroisocoumarins
395
6 11.23
6 4.70, m
6 3.97 CH!30
CH3 6 1.50, d, J 6-7 Hz
6 6.33'
6 2.88, d, J 6-7 HZ
38
6 11.17
6 4.58, m
6 3.92 CH30 CI H
CH3 6 1.55, d, J 6-7 Hz H 6 2.95, m
39
6 3.95
6 4.47, m 6 3.95 C&O
CH3 6 1.45, d, J 6-7 HZ
6 6.63
6 2.83, d, J 6-7 HZ 40
Me2CO-d6
6 9.5 OH
0
6 4.40, ddq, J 11, 7, 3 Hz 6 2.09 C&COO
CH3 6 1.24, d, J 7 Hz
dd, J 16, 11 Hz d, J 2 Hz
6 2.82, dd, J 16, 3 Hz 41
396
In the amicoumacins, AI-77 compounds, and xenocoumacins, where there is no oxygen function at C6, H6 resonates at ca. 6 7.5 as a triplet J 7-8 Hz, and H5, H7 as doublets, J 7-8Hz at c a . 6 6.8. In the spectra of these compounds the aminoisopentyldihydroisocoumarin system and the aminoacid moiety attached through amide formation with the 5'-amino group give rise to two distinct coupling systems whose connectivities can be established completely in favourable cases.
42 The connectivity pattern of the isocoumarin system is seen fully in the spectrum of the amine hydrolysis product ( 4 2 ) although the reported data are of doubtful quality. Okazaki et a l (ref.102) assign the diacetate as follows (frequency, solvent, J values not given) : 6 3.00, dd, (sic!), (H4)2; 4.46, m, H3; 4.28, m, H5'; 1.34, m, (H4')z; 1.65, m, H3'; 0.91, d, (Hl')s, t, H6; 7.08, d, H7.
(H2')3; 6.97, d, H5; 7.47,
In CD30D solvent at unspecified MHz, Shimojima
(ref.105) report as follows: 6 0 92, 0.97, 2 x d, J 6Hz, (H1')3, (H2')3; 1.90-2.0, m, H3', (H4')z; 2.98, m, (H4)2; 4.26, m,
et a l
H5'; 4.50, m, H3; 7.04, d, J 8 Hz, H5; 7 56, t, J 8 Hz, H6; 7.21, d, J 8 Hz, H7. Chemical shift character sation of the amine ( 4 2 ) is reported (ref.106) in CD30D: 0.92, 0.99, 2 x d, J 7 Hz, (H1')3, (H2')3; 1.35-2.16, m, H3', (H4')2; 3.5, m, H5'; 3.35-3.04, m, (H4)2; 4.97, m, H3; 6.85, d, J 8 Hz, H7; 6.95, d, J 8 Hz, H5; 7.59, t, J 8 Hz, H6.
When the 5'-amino group is acylated, and a non-exchanging
solvent is employed, the NH proton will give a doublet resonance at about 6 8 ( J ca. 8 Hz); this resonance can provide additional characterization of H5' through decoupling (by double resonance or by deuterium exchange).
Due to intrinsic asymmetry, methyl groups
(H1')3 and (H2')3 are chemically non equivalent, and (H4')2 can show both chemical and magnetic non-equivalence. Because of the heteroatom substituents present in the acyl portion in the amicoumacins, AI-77's and xenocoumacins the proton
397
resonances arising from this moiety are quite well dispersed and the connectivity patterns can be determined fairly readily.
A good
starting point is the resonance of H8' which occurs at ca. 6 4.2 as a d o u b l e t since it is flanked by the C=O and only H9'. resonance is at ca. 6 4.1, a dd pattern
The H9'
from the two vicinal
Because of the associated amino couplings 8'-9' and 1 0 ' - 9 ' . function, H10' resonates at slightly higher field, ca. 6 3.5. The multiplicity of this signal depends on the adjoining chain normally containing
at least one methylene
CH2
( o f t e n magnetically
and
chemically non-equivalent) . The data reported for the AI-77-B Table 1.
(ref.105) are shown in
Tables 2 and 3 contain the data for xenocoumacin 1 (1)
and 2 (2) and their acetate derivatives ( 4 3 - 4 5 ) , MHz
spectra
(ref.1); assignments
derived from 400
were confirmed by
decoupling
experiments and 2D-COSY 'H-'H correlation spectra.
TABLE 1 IH-nmr data for AI-77-B (amicoumacin B) (28)a (ref.105) . Proton H-3 H-4A H-4B H- 5 H- 6 H-7 H-1' H-2 ' H-3 ' H-4 'A H-4 'B H-5 ' H-8
'
H-9' H-10' H-11'A H-11'B 6'-NH
Mu Itip1i city ,J ( H z)
6
4.68 2.88 3.06 6.84 7.48 6.80 0.89 0.92 1.67 center 1.67 center 1.38 center 4.22 3.99 3.73 3.34 2.22 2.38 7.76 ~~
aAt 400 MHz in DMSO-d6 at 6OoC.
~
m dd, 16,4 .5 dd, 11,4.5 d, 8 dd,8,8 dr 8 d,6.5 d, 6.5 m m m m, 4 d,7.5 dd,7 .5,4 m dd, 18,4.5 dd, 18,9 dr 9 ~~~
398
1'
I
1 43
R=H R=Ac
TABLE 2
'H-nmr
d a t a f o r x e n o c o u m a c i n 1 (1) a n d h e x a a c e t a t e (43) ( r e f . 1)
43b
1" Proton
6
Multiplicity, J(Hz)
6
Multiplicity, J(Hz)
3
4.59
ddd, 8 . 2 , 4 . 5 , 4 . 0
4.09
ddd, 1 2 . 6 , 3 . 5 , 2 . 8
4A 4B 5
2.94 2.98 6.80 7.45 6.82 0.82 0.90 1.58 1.41 1.64 4.20
dd, 1 6 . 8 , 8 . 2 dd, 16 . 8 , 4 . 5 d,8.0 t,8.0 d,8.0 d,6.6 d, 6.6
dd, 1 6 . 8 , 2 . 8 dd, 1 6 . 8 , 1 2 . 6 d,8.0 t,8.0 d,8.0 d,6.6 d,6.6
4.21 4.11 3.47 1.70 1.83 1.1 1.1 3.19) 3.19)
d,6.1 dd, 6 . 1 , 4 . 0 d d d , 8 . 4 , 4 . 0 , 3 .5
2.53 2.97 6.84 7.20 6.86 0.89 0.91 1.66 1.20 1.81 4.24 1.06 5.32 5.09 4.43 1.37 1.50 1.50 1.50 3.39J 3.39J
6
I
1' 2' 3' 4'A
4'B 5'
6' 8' 9' 10' 11'A 11'B
12'A 12'B 13'A 13'B
-
mc
dd, 1 3 . 2 , 7 . 2 , 4
.
mc
dt, 9.8,4,4 -
mc mc
mc mc
t,6.4
mc
ddd, 1 3 . 5 , 9 . 1 ,4 . 2 ddd, 1 3 . 5 , 9 . 1 , 4 . 2 tdd, 9 . 1 , 9 . 1 , 3 . 5 , 4 . 2 , 1 . 4 d,9.1 d,1.7 dd, 1 . 2 , l . l qd, I . 4 ,I . 4 , I . 4 , 2 mc mc mc mc
91 6
d
a B . C l i n D20; CDC13-C6Dh
dioxan, 63.70. ( 3 :2 ) . 'Overlapping s i g n a l s . ( 6 9 . 0 0 , t , 6 ) , NH-10' dother signals: H-14'
Ac
(61.81, 1.86,
1.89, 1.94, 2.13,
2.23).
(66.59,d,9.8),
399
TABLE 3
1H-nmr data for xenocoumacin 2 (2) and derivatives (ref.1). 2"
Proton 6
Multiplicity, J (Hz)
3
td,7,4.8 d, 7 d, 7 d, 8
4.76 2.96 2.96 6.78 7.44 6.81 0.82 0.89 1.56 1.39 1.69 4.17
4A 4B
5 6 7 1' 2'
3' 4'A
4'B 5' 6' 8' 9' 10'
4.16 4.10 3.70 1.92 2.10 1.92 2.06 3.271 3.271
11'A 11'B
12'A 12'B 13'A 13'8
6
4.60 2.83 3.11 6.71 t r 8 7.92 d, 8 6.71 d,6.5 0.93 d,6.6 0.96 m 1.68 ddd, 1 4 , 9 . 5 , 3 . 6 1.50 ddd, 1 4 , l O . 5 , 4 . 5 1 . 7 8 ddd, 1 0 . 5 , 4 . 8 , 3 4.35 7.9 d,6.6 3.9 dd, 6 . 6 4 3.70 ddd, 9 , 7 , 4 4.21 m 1.91 m 2.27 m 1.88 m 2.04 dd, 8 , 5 3.53 3.63 C
aIn D,O;
45b
44b
Multiplicity, 6 J (Hz) dt,12.7,2.4,2.0 dd, 16 .8,2. 4 dd, 1 6 . 8 , 1 2 . 7 d, 8 t t 8 d, 8 d,6.6 d,6.6 m ddd, 1 3 . 5 , 8 . 6 , 5 m tdd,lO, 1 0 , 5 . 2 d, 1 0 d,7.8 dd, 7 . 8 , 4 . 4 ddd, 9 , 7 , 4 . 4
m m m
m ddd ddd
Multiplicity, J (Hz)
ddd, 1 2 . 5 , 2 . 5 , 1 . 5 dd, 1 6 . 8 , 1 3 dd, 16 . 8 , 2 . 5 d, 8 t,8 d, 8 d,6.6 d,6.6 m ddd, 1 3 . 5 , 8 . 5 , 5 . 5 1.88 ddd, 1 3 . 5 , 1 0 , 5 . 5 4.34 m,8.5,8.5,5,1.5 8.69 d,8.5 5 . 2 1 d,1.5 5 . 1 4 dd, 9 . 9 , l . 5 4 . 5 6 t (br), 7 . 5 , 2 1.63 m m 1.7 2.01 m m 1.7 3 . 4 5 q(b)10 3.54 td, 10,10,3 d
4.50 3.30 2.89 7.03 7.52 7.12 0.93 0.97 1.7 1.48
dioxan 63.70.
CDC13,
'Other signals: AC ( 6 2 . 1 2 ) , 8'-OH, 9'-OH (broad envelope,63.6), 8-OH ( 6 10.8) . dother signals: Ac (61.95, 2.10, 2.11, 2.40).
400 The
spectra
of
acetate
derivatives
spectra
of
DMSO
solvents,
acid
used
and
in the
useful signal were
and
then It
acetic
mainly
spectra
were
present
procedure
in
CDCI~
assigning
the
Addition
narrowing
was
found by
sample
such that
protons
of CD30D
was
salt)
gave
spectrum
gave
also
the
because
of
basic 6
the
envelope;
of the
acetic
guanidino of
d6-DSMO
2.9-3.9
was
spectrum
was
dihydroisocoumarin
removal
d6-
for analysis.
drops
region
The
portion
amidic
decoupling
proton
experiments
baciphelacin reported,
H9'
structure,
which
(46)
which
would
acid
precursor. The
can't
really
obtained
[in
free
containing
IH n.m.r,
a doublet is A
of
HCI.
spectrum
in
by means
of
(3"2)
for
assigned CDCI3-C6D6
IH-~H c o r r e l a t i o n
In be
by of
to
as
H8'
with
the
would reported
support
seem that
incorrect. from
also other
HI0'
(H3"
despite
and
from
the
the the in the
therefore
two
vicinal
from
a
more
satisfactory
known
isoleucine
of
course
in ref.102 (34)
biogenetic
structures,
of
structure
In
H9'
a doublet, and
doublets
strange
HS'
results
used
be signal
incorporation
(46) ,
it w o u l d
biogenetically
in k e e p i n g
involve
may
is d e s c r i b e d
below) . is
here,
the
flanked
(34)
decoupling
(46) .
is
to be
structure (see
be
solution
A 2D-COSY
discussed
of
tetracetate
(34)
viewpoint
data.
data
in
expected
could
peracetate
of the data
n.m.r,
The
1
aqueous
well-resolved
additional
in the
be
a
The
ref.102) that
an
obtained.
light
structure
structure
the
could be c o m p l e t e l y
IH
they
of
caused
of
partial
signal.
highly few
the
D20 and
retention
a
and
presumably
Nevertheless
xenocoumacin
experiments.
In the proposed
that
the
dispersion]
J's.
the
that
of the O H / N H
evaporation
(HCI
decoupling
could
by
in
suitable of
D20
studies.
possible.
The
fact
broad, not
containing
except
in
obtained
because
envelope.
acid
spectrum
were
the
OH/NH
D20,
best
peaks
isolation
obtained resolution
originally
spectra
HI'-H6'.
were
high
a large
by
for
2
for
1 were
also
taken
satisfactory
obscured and
the
were
Spectra
gave
CDCI3
effects;
peaks
group.
in
xenocoumacin
of a s s o c i a t i o n Spurious
xenocoumacin
as
give are
an a
is
amino
doublet
equivocal;
or to
rule
out
401
OH
0 H
O
OH
,
3,~4, 0 3' I
OH
NH 2
34
0
( (•H.ko
QH 46
4.3
13C
n.m.r.
13C n.m.r, for
the
work
B
and
(ref.104) B
(28)
spectral
structures
earlier (28)
the C
and
(29) ,
were as
correlation
decoupling
B)
well with
(Table were
made
~H-coupled
assignments
determined
assignments for C3'
and
values made
a n d C4'
for
IH
(32)
(Table were
at
on
the
spectra,
and
The of
and
earlier C4'
in
the
compounds
data MHz
multiplicities in
except
reported
In b o t h
through i00 HETCOR
spectra selective for
the
spectra. DEPT
spectra.
The
with
a reversal
amicoumacins
(ref.105) .
cases effect
from
agreement for
(27),
for A I - 7 7 -
off-resonance newer
In
A
substituent
from
~H-~3C 2D
essentially
are also
MHz.
spectra
4) .
basis
antibiotics.
and in
support
amicoumacin data
25
shift
derived
further
products
report
n.m.r,
5)
are
in
degradation
multiplicities
in the A I - 7 7
to g i v e
carbons
chemical
reported C4
of
AI-77-F
on as
used
(ref.105)
al
the
experiments, corresponding
the
et
been
dihydroisocoumarin
shifts
and
based
have
the
experiments
xenocoumacins Assignments
of
Shimojima
considerations
data
chemical
(amicoumacin
assignments and
Studies
the
of
the
(ref.104)
and
402
The chemical shifts and 'JCH values reflect consistently the heteroatom substituent effects operating.
The presence of the
guanidine group in xenocoumacin 1 is indicated by the singlet at 6 158.0 due to C15'.
TABLE 4
13C-nmr data for amicoumacin A ( 2 7 ) and C ( 2 9 ) (28)
(ref.l05), and the amine ( 4 2 )
Carbon
27b
170.5 82.1 39.4 140.5 119.8 137.6 116.2 160.8 108.6 21.7 23.6 25.2 30.0 51.2' 173.8 71 . 2 f 73.2f 50.2' 32.3 175.1
1 3 4 4a 5 6 7 8 8a 1' 2' 3' 4' 5' 7' 8' 9' 10' 11 ' 12 '
(ref.l04), AI-77-B
(ref.104)a.
2 9'
28'
169.4 81.6 39.3 140.2 119.3 137.2 116.6 160.9 108.5 22.2 24.0 25.1 29.8 49.6' 171.1 71.gh 85.6h 48.7' 35.3 175.6
168.gi 80.9 28.9 140.6 118.4 136.2 115.2 160.9 108.3 21.5' 23.3' 38.6 24.0 48.1 172.7' 71.4' 71.6' 50.4 33.4 174.6i
"25 MHz. D~O.
'In DMSO-d6. CD~OD. e , f , g , h , i , j.kAssignments may be interchanged
42d
169.2 79.3 39.3 139.9 119.5 137.5 116.9 162.8 108.9 22.0 23.2 25.2 30.5 53.2
403
TABLE 5 I3C-nmr data for xenocoumacins 1 and 2" (ref.1) .
2
1
~
1 3 4 4a 5 6 1 8
170.9 82.5 30.4 141.2 120.5 138.3 116.8 161.4 109.1 22.2 23.6 25.5 40.0 50.9 174.5 13.1 12.1 55.1 25.1 26.0 42.0 158.0
8a
1' 2' 3' 4' 5' 7' 8' 9' 10' 11' 12 ' 13' 15 '
171.1 82.6 30.4 141.2 120.4 138.3 116.9 161.6 109.3 22.2 23.8 25.6 40.0 50.8 114,4 74.4 10.7 62.8 25.8 24.6 46.9
S
d 151 t 132 S
dd 162,6 d 161 dd 162,7 d 9.6 S
q 126 q 126 d 126 t 124 d 141 s (br) d 144 d 144 d 141 t 126 t 126 t 138 t (br)3
5
d 152 t 130 5
dd 157,4 d 161 dd 164,7 d 9.6 5
q 125 q 125 d 126 t 126 d 140 s (br) d 141 d 145 d 145 t 130 t 130 t 148
"100.62 MHz, D20 solution; dioxan 6 67.8. Mass
4.4
Spectrometry
No molecular
electron
impact
ion was found for xenocoumacin (EI) or
chemical
ionization
1 or 2 using
(CI) techniques,
however, some useful fragment ions were observed which supported the structures. In particular, the EI mass spectrum of xenocoumacin 1
had
ions
of
m / z
163,
dihydroisocoumarin moiety
206
and
(ref.1).
249
which
arise
from
the
Peracetylation was found to
increase the volatility and stability of these compounds and more useful structural information was obtained from the EI and positive and negative ion CI spectra of xenocoumacin 1 hexaacetate ( 4 3 ) and the xenocoumacin 2 tetraacetate ( 4 5 ) . A principal fragment ion of m/z
184
in
the
EI
and
PCI
mass
spectra
of
xenocoumacin
1
hexaacetate has the composition C8H14N302 and contains the guanidino group (ref.1) .
404 For mass ionization
spectral
bombardment spray)
(FAB)
with
[M+H] § ion their
good
(ref.108)
(ref.ll0,111) use
of
this
sensitivity with
mainly
MS
Figure
with
i.
was
A mass reports similar with
on
the
doubly
of
AI-77-B
use
of
MS
xenocoumacins
(MS-MS)
MS-MS
158 and 70.
analysis
on
have been
such as c l a d o s p o r i n
ion
experiments, ion of
(m/z 466) fragment of
both
using
the
field
been
no
other
underivatised
EI MS has
in
[M+2H]2+ at
spectra
obtained There
MS
ion-
molecular
a series
the
of
spectra
ion
215,
though
dihydroisocoumacins
molecular
ions yields
tandem
use
the
in
was
it
by
on the by
in
molecules femtomolar
obtained
232,
(28)
interest
processes
to give
(ref.105) .
The
biomolecules
of
of the m o l e c u l a r
argon
liquid
~i/min).
large
protonated
tandem
(CID)
with
common
technique
the
by
matrix-derived
studies
charged
is
atmospheric
organic
demonstrated
For
at m/z 250,
spectrum (FD)
to the
simpler
ions
can be
that
limited
polar
glycerol
ions
at
(i-i0
with
ionization
experiments
dissociation
occur
indicated
introduced
advantage
of our
1 are
obtained.
Fragment
the
soft
results
were
2
thermally-labile
has been
small,
interfering other
by b o m b a r d m e n t
xenocoumacins
desorption
MS
of the
and
technique
rates
been
there
has
and the
measurements 1
are
flow
has
Fragment
The
single
low
MS
with
466.4
induced
effected
ions.
ions.
atom (ion-
evaporation
typically,
recently
xenocoumacin
By
233.6
collision
of
Like
(MS-MS).
[M+H] + at m/z
m/z
lack MS.
molecular
techniques spray
and FAB
soft
fast
electrospray
non-volatile, ion
at very
Ion-spray
mass
ionization
by
technique
assisted
both
C22H35N506 and C21H30N206 r e s p e c t i v e l y .
polar, is
xenocoumacins
used
xenocoumacin
new
ion-spray
and until
of
of
samples,
(ref.109)
(ref. I12, 113) . found
to
and
application
the
a
have
resolution
MS
to be
uses
Ionization
chromatography main
High
FAB
formulae suited
compounds.
We
pneumatically
by
MS
of u n d e r i v a t i s e d
required.
results.
molecular
particularly
are
and
obtained
Ion-spray
pressure
analysis
techniques
used
compounds extensively
( 1 6 ) (ref.59) .
405
466.4
1 O0
75
>,,
233.6
O3 tO1) ,,i-,
~
50
n-
25
158.8
1 ''~176 "'ii
1 O0
150
200
158.2
12.5
vo~
Y
251.0
9.0
250
300
350
400
450
232.2 250.3
200.2
1
[M+H] +
9.4
u~ C
181
~
70.1
6.2
60.
-~-...
rr
122.1142.1
3.1
11:2.1 lO I
o.o
"
.
430.9
215.: .,
371.4
jl,
-
lOO
,0,-91,4491 t.
2
I
.,..,,...1. .....
Ll..|,.ll
300
200
41
9
11
56o
400
m/z
Fig. i. I o n - s p r a y m a s s s p e c t r a of x e n o c o u m a c i n 1 (I) . (A) F u l l scan (m/z 100-800) s i n g l e MS s h o w i n g the [M+H] + at m/z 466.4 and the [ M + 2 H ] 2 + at m/z 2 3 3 . 6 . (B) M S - M S spectrum from the collision induced dissociation (CID) of the m/z 466 m o l e c u l a r ion.
4.5
X-ray
X-ray
structure centres was
of
had
AI-77-B to
be
structure
dihydroisocoumarin adopts
a
(28)
analysis
and
S configurations
confirmed
crystal
Crystallography
crystallographic
half-chair
(ref.ll4) .
The
hydrangenol
(23)
S
by
to
was
indicate
of
it
that
form
(ref.ll5)
that
five
shown and
the
structure and
all
absolute
degradation
determination
crystal
to
(ref.105) . The chemical
was
employed
confirm
the
asymmetric
configuration
studies.
In
the
3,8-dihydroxy-3-methyl-3,4the
dihydropyranone
hydroxyl has
group
also
cladosporin
been
(16)
at
C3
is
determined
(ref.64) .
ring axial for
406 5.
BIOGENESIS
The mellein with
biosynthesis
(3)
and
13C-labelled
acetate
derivatives acetate
(ref.118) .
reduction
of
the
cyclization
formation, the
through
formation
(cladosporin) made
from
derived
one
a
an
to
group of
a
gives
the can
(3)
to
be
units
0
of the
central allows
group
(Scheme
initiating
unit
by
diphenolic
pentaketide give
formed
(47)
followed
the
reduction in
studies
chain
(48)
give
hydroxyl
mellein shown
by
as
doubly-labelled
pentaketide
alcohol
Cyclization
been
building
2H , 13C
Selective end
acetate-derived
two-carbon
0
the
(51) . has
of
such
established
and
formation
(49).
as
then
(16)
firmly
to
h-elimination
deoxypentaketide
lactone
been
group
lactone
well
dihydroisocoumarins
formation
system
as
has
ketone
allows
group
simple
(ref 9116,117)
After
end
dihydroisocoumarin carbonyl
of
(50),
of
monophenol;
2).
Asperentin
from and
in
a an
octaketide
seven
malonate-
(ref.ll9) .
0
0
OH
O
OH v
0
0
47
0
0
0
HO
O
0
49
OH
0
OH
50
OH
48
O
0
OH
51
Scheme
2
In contrast no studies have yet been reported on the amicoumacins/AI77's, to
or
the
arise
xenocoumacins.
from
(52) .
leucine
Selective
and
the
isocoumarin
reduction be
would
less
at
likely
give C4
in v i e w
and
at
of
some
the
be
considered
units
to
orsellinic point
(53). results
would
Scheme
(54)
pathway studies
would on
3)
polyketide give
cyclization acid
deoxygenation
(This of
(see
form the
cyclization
Alternatively,
acylated
produces
can
acetate
(53). the
(55) ;
C3,
four
reduction
aminodihydroisocoumarin polyketide
They
and at
of
the
thence C6
appear
mellein
the
and
and to 6-
407 hydroxymellein) derived
from
arginine
in
xenocoumacin 77's
The
acetic the
2.
can
oxidation
of
(or
from at
(57)
C8'
and
(53)
malonic
is
acylated
acid) i,
by
a
and
an
amino
proline
in
the
as
acid, the
and
amino
reduction
C9'
substance acid.
(cf.
56)
to
all
~
NH2
OH 2
O
0
H
...__ v
HO
s4 OH
OH
[O]
O
55
]
O
the
O
>~
OH O
cases
produce
NH 2
C
of
Y-05460M-A
In
OH OH
j
acid"
case
3).
O
,
unit
the formation of the amicoumacins/AI-
aspartic
valine
(Scheme
of
xenocoumacin
involves
occurs
O
acid
group
On the other hand,
arise
system
amino
case
presumably
(35) diol
.
N.H ~A i 1-12
~
O
O
NH,~~H2 R
=
[H]
O
56
57 Scheme
The the
series
of
Bacillus
spp.
transformations spp.
Xenorhabdus It
OH
is
and for
the
this
3 is
remarkably
taxonomically reason
that
the
consistent remote
within
organisms,
structure
proposed
408
for b a c i p h e l a c i n
(34)
the
the
formation
CI0'
in
of
(34) .
A
seems
more
containing
a CHCH3 unit
isoleucine
and
(58)
and
an
thence
baciphelacin
doubtful.
C-methylated
attractive, is
(46).
unit
The
as
n.m.r,
has been d i s c u s s e d
and
is
difficult
the
diol
biogenetic,
(46) which
acetate
OH
It
C8'
can be
the
as
N-acylating for
explain at
structure
derived
evidence
to
system
the
still
above
unit,
C9', with
to
give
structure
of
above.
0
58
OH
0
0
OH
=
46
6.
6.1
BIOLOGICAL
ACTIVITIES
Xenocoumacins
Pharmacological both
compounds
(ref.l) . positive
They
against moderate species
exhibit
organisms,
Xenocoumacin
1
studies
possess
has
and
weak was
has no a n t i f u n g a l
xenocoumacins and
xenocoumacin antifungal
Cryptococcus neoformans to
the
antibacterial
with
also
on
antibiotic
activity
inactive activity.
potent
activity 1 being activity
(MIC 0 . 1 2 5 - 0 . 5
against
against
have
shown
antiulcer mainly the
against
more
being
Candida species.
and
Gram-
effective.
highly
~g/ml)
Aspergillus
that
activity
but
active has
only
Trichophyton
Xenocoumacin-2
409 Xenocoumacin experimental, shown
in
ulcers
low
both
at
may
therapeutic
be
gastric
2 has
the
I0 m g / k g a
potent
compounds
concentrations
toxicity
TABLE
6,
Xenocoumacin
inhibits at
2 display
stress-induced
Table
orally.
1 and
ulcers
are better
problem
like
activity
rats and
the
use
of
As
administered significantly
xenocoumacins AI-77s
against
(ref.l) .
when
activity
the
with
in
effective
(po) . B o t h
and,
antiulcer
are
and
cytotoxic
amicoumacins,
these
compounds
as
agents.
6
A n t i u l c e r a c t i v i t y of x e n o c o u m a c i n 1 (1) a n d i n d u c e d g a s t r i c u l c e r s in rats (ref.l) . Compound
Dose mg/kg,
Xenocoumacin
1
Xenocoumacin
2
2
po
(2)
against
Protective (%)
25 i0 25 i0 5
stress-
Value
74 a 8 NS 70 b 61 b 26 NS
~p<0.01. bp<0 05
6.2
Baciphelacin
Baciphelacin positive
bacteria
antiviral
activity
(ref.102) . 1
mechanism in
HeLa
~g/ml) .
of cells This
was
only
of In
is
was
toxic
effective
was
also
disease
against
against
reported
virus)
P388
inactive
found
and
herpes
Gram-
to
have
to h a v e
lymphatic
against
in
study
at
of
animal and/or related
methyl-3, 4-dihydroisocoumarin cells
but
not
in
HeLa
that A 50% a
yeast
cells
it
was
and
cells
and a
inhibition
concentration
is u n u s u a l
systems.
eukaryotic a
to
revealed
(ref.121) .
compound coli
HeLa
mainly It
Newcastle
activity
action
synthesis
Escherichia only.
(against
Baciphelacin
translation blockers
antibiotic
in
leukemia
simplex
virus
(ref.120) .
Baciphelacin protein
an
(ref.102) .
anti-tumour
vivo type
is
and
prokaryotic
not
study
potent around
of
its
inhibitor
of p r o t e i n
interesting
Translation
a
of
synthesis
10 -7 M
(0.042
because
it b l o c k s
plant,
yeast
in
inhibitors
are
systems
animal
not
or
usually cells
compounds
4-acetyl-6,8-dihydroxy-5-
was
to
cells.
found
block
Interestingly,
translation the
in
saturated
410 isocoumarin was
analogue,
inactive
cladosporin
6.3
gastric
has
ulcers
has
antibacterial
greater
activity
in rats
of
than
reported
paw
A
(ref.99) .
AI-77-B
importantly
potent
have
significant
179 m g / k g
6.3.1
activity
62 m g / k g
(ip),
800 mg/kg
et
while
for
antiulcer Nine
for p r o t e c t i v e
gastric
ulcers
rats
summary,
both
required
can
significantly
for
activity.
activity
and
inactive,
conversion but
resulted
in
the
of
isocoumarin to
moiety,
a small
ring r e s u l t e d As
di-
was
(ref.107) .
the with
evaluated
alkyl
of the d i d AILD50 7
an LD50 of
a
carboxyl
y-lactone
chains potent
The
moiety
side-chain
moiety
considerably
derivatives
group in
of the
In
side-chain
to
an
the
amide
With
of
and
side-chain
phenolic
hydrolysis
were
activity
activity.
the
hydroxyl
the
lactone
in activity. only
active
Another to
six
of m e t h y l
stress-induced,
increased
ring
in
but
of A I - 7 7 - B of
the
by
were
intraperitoneally.
of
methylation
series
AI-77-B
N-acetylation
decrease
is
investigated
of
against
considerably
AI-77-B
The structure-
were
triacetylated
in a c t i v i t y
(ref.107,123) .
exhibited
toxic,
s y s t e m and the
larger d e c r e a s e
conversion A
activity
and
intraperitoneally.
studied
derivatives
selective
previously,
active
derivatives
of
decrease
given
is quite
activity
terminal A)
small
in a much
shown
when
a
was
activity
nor
derivatives
activity;
the
formation only
for
spider
None
so with
the
doses A
activities
Studies.
Modification
to A I - 7 7 - A / a m i c o u m a c i n
toxicity,
amicoumacin
is less
administered ring
influence
decreased (i.e.
when
isocoumarin
are
seen
(ref.99) .
AI-77-A
AI-77-B
and t e s t e d in
were
the t w o - s p o t t e d
biological
prepared
the
at
(po) (ref.99) .
(ref.106) .
al
rats
gastroprotective
Structure-Activity
relationships
Shimojima
potent
activity.
(po),
AI-77-B
addition, against
AI-77-A
inhibiting in
activities
intraperitoneally
antiinflammatory
(ip),
has
stress-induced,
(ref.99) .
carrageenin
In
also
orally
Similar
especially
administered
analogues
77-B have
and
as was
significantly
by
(ref.100, i01) .
(ref.122) .
mg/kg
induced
to be mitocidal,
only when
other
drug
cells
experimental,
administered activity,
oedema
50mg/kg
amicoumacin
led
or yeast
a c t i v i t y but more
against
when
antiinflammatory
development
but
in HeLa
(ref.121) .
gastroprotective
mite
translation
AI- 77s/Amicoumacins
AI-77-A
also
4-acetyl-6,8-dihydroxy-5-methylisocoumarin,
in b l o c k i n g
as
an
chemical
prodrugs
that
N-monoalkylated to
n-hexyl
N-ethyl
antiulcer
and
were
N-propyl
activity
antiulcer
modification are
orally
7-1actone synthesised 7-1actone
against
stress
411
ulcers
when
dosed
antiinflammatory 1250
and
activity
1075
mg/kg
antiinflammatory The
for
activity
in
w i t h most potent
ulceration
models
induced
by
models
action
indometacin-induced
water
immersion,
acid-induced
general
toxicity
the t h e r a p e u t i c
upon
repeated
OH
the
a
administration
use of this
Most
of in
AI-77of
ulcer,
ligation was,
rat
stress
ulcer
and
however,
of A I - 7 7 - C 2
interesting
non-
activity
range
gastric
There
was
effect
Further,
wide
pylorus
ulcer.
(59)
and a n t i u l c e r
side
drugs.
- the
prevent
(AI-77-C2)
ulcerogenic
in
(LD50
displaying
is novel.
the
low
showed
toxicity
drug
(ref.124) .
have
antiinflammatory
prophylactic
acetylsalicylic
A
derivative
relatively
other
acute
activity
drugs
has
low
also
of the u l c e r o g e n i c i t y
experimental
AI-77-C2
showed
the
assessment
antiinflammatory
comparison
ulcers
relatively
they
respectively) .
y-lactone-N-ethyl
further
ulceration. C2
and
(po) ,
several
steroidal
Interestingly,
as well as a n t i u l c e r
AI-77-B
selected
orally.
some
w h i c h may
compound.
0 O
H
OH
I~I-"C2H 5
N
59
6.3.2
Mode
combination
of
77s and a m i c o u m a c i n s to be unique.
class.
With
regard
investigated recognised
the key
that
biosynthesis it
did
however,
an
have
that
nor
membrane
generation
In general,
was
that
on
Inouye some
a the
the mode
from other n o n - s t e r o i d a l
major
previously
or
A2.
in
of the
Further,
There may
on the
inhibition
amicoumacins antiinflammatory
They
influence
involved
factor.
of a c t i o n
and K o n d o
(ref.122) .
on p h o s p h o l i p a s e stabilisation
formation
to a n e w drug
significant
enzymes
novel
of the AI-
and u l c e r
mediators no
their
belong
activity,
amicoumacins
5-1ipoxygenase,
indication
i.
compounds
chemical
amicoumacins
and
appear
is d i f f e r e n t
of
and
with
of a c t i o n
inflammation
these
antiinflammatory
effect
the
anion
interleukin
to
is,
of p r o s t a g l a n d i n s ,
not
superoxide
keeping the mode
That
enzymes
cyclooxygenase
In
activities,
in i n h i b i t i n g
appears
concluded
Action.
of b i o l o g i c a l
of
was,
inhibit
amicoumacins agents.
412
With
regard
tested
for
ATPase
and
that
the
central
found
to
and
suggested
SYNTHESIS
fragments, acid
and
(ref.122).
of
et
AI-77-B
effect
effects
side-chain
in
the
can
be
considered
to
moiety
approaches
(28)
have been
synthesis
be
(MOM)
generated
to
was
(2.6
the
constructed the
from
hydroxy
NI-I2
in
and
(63)
by
(42)
two
amino
CO2H
situ,
Hamada
was
prepared of
(61)
manner.
was
in
(TMEDA)
of
This
afforded
in 32%
yield. an
(61)
the
in THF
64b)
equiv.)
prepared
anion
Briefly, from anion with , and
a separable Upon
use
6-
process
ethyl (62)
the 6was
lithium
presence
(-78~
81:19
The
ethyl a
benzylic
4).
The b e n z y l i c
reaction
from by
the
synthesis
groups.
(ref.125,126) .
al
(S) - l e u c i n a l
(63). (1.4
et
(Scheme
by
total
several
formation
ester
usual
by
1,2-addition
lactone
(LDA)
(64a and
stereoselective
reported
methyl
in the
Boc-(S)-leucinal equiv.)
mechanism
OH
recently
N-protected
diisopropylamide
diastereomers
(ref.124).
in the
HO_ . ~ ~ .
reported
tetramethylethylenediamine with
model
and
OH
diastereoselective
methylsalicylate then
and
by spontaneous
methoxymethyl
(mainly g a s t r i c
(60).
aminodihydroisocoumarin
followed
effects AI-77-C2
60
methylsalicylate involving
that
with
0
Successful
The
rat
(42)
42
total
found
involved
O
first
reported
AI-77-B
NH2
of A I - 7 7 - B
been
was
H+/K §
associated
secretion
are
aminodihydroisocoumarin
OH
has
not
studies
on gastric
movement
these
It is
A
against
and a n t i h i s t a m i n e r g i c
in their
al
amicoumacin
activity
activity.
(28)
the
inactive
gastric
that
OF
AI-77-B
activity,
activity
anticholinergic,
inhibitory
of its a n t i u l c e r 7.
be
Urushidani
secretion)
was
antiulcer
activity
suppressive,
had a potent It
the
antiulcer
(ref.106) . acid
to
anticholinergic
of reacted
mixture
of
of excess
LDA
stereoselectivity
was
413
obtained (64a) . boron
with
the
Removal
tribromide
~
major
of the gave
product
Boc
being
and m e t h y l
the h y d r o c h l o r i d e
MOMO
the
correct
ether of
N HBoc CO2C1-%
LDA/TMEDA/,..__ THF/-74~ Y-
CH2Li
61
62
63
O
MOMO
ll-OH
~
with
O
CO2CH 3
MOMO
groups
(42) .
MOM(
Me
diastereomer
protecting
O
+
NHBoc
N HBoc
64a
64b
I BBr3 42.HCI Scheme Construction Scheme
5)
of the
commenced
(R)-pyroglutaminol the
synthetic
unsaturated
presence the
of
diol
lactam
(66)
of
conversion
with the
amino
from
ozone;
was
then
removed MeOH
of the
to
using give
alcohol
as 5%
(68) .
in
(65)
of
features
of
of
Key (65)
selenylation
group
protected
(shown
acid.
conversion
by
(60)
derivative
fragment
D-glutamic
the
cis-diol
acid
N,O-benzylidene
the by
highly
catalytic
N-methylmorpholine-N-oxide
was in
prepared were
group
function hydrate
the
sequence
deselenoxylation introduction
hydroxy
from
4
(NMO)
the
to the n i t r i l e
unit (69).
~, ~-
followed
by
stereoselective
to
give
acetonide Cl
the
osmylation
palladium-carbon A
to
was
(67).
the and
in
the
After
benzylidene hydrazine
introduced
The N - l a c t a m
was
by
414
HO
1.LDA/PhSeBr 2. ozone "-
p~~O
G
G--,.., ~o oso~
~~-o
~,~~
66
67
65
1. 2,2-dimethoxy [~ro[~ane/PPTS 2. 5% Pd-C / NH2NH2 .H20
KCN/Bu3P/CCI4/~ 18-crown-6 "-
...,,,jOH
O
OH
o'~o ..,,,,~CN
O
H
H
68
69 OH
1.(Boc)20/DMAP 2. LiOH/70% aq,THF
0
J~oH
NH2
-~"'Y"""'~ C N
HO2 C ~
~
NHBoc
70
4
DEPC/ Et3N
OH 1. Me3orthoformate/5% HCI-MeOH 2. H20/12 h; 0.1N NaOH (pH 9)/3 h; 0.1N HCI (pH 6.5)
Scheme 5
O H N
o>~o " ~ i O i
71
NHBoc
415 protected
with
conditions The
a Boc
using
contains
the
converted
to
the
full the
Selective There
fragment
and
the
et
one
was
the
starting
(72)
and and
(74)
85-15
with
boron
lithiated
acid moiety 7) .
prepared bis
out
(80)
hydroxyl
in
being
-78~
and
the was
DCM
to the
with
osmylation
found
be
to
was acid.
correct
a
small
were in
moderately in the
to
from
the
presence
(79)
(11%)
introduced
the
by
-78~
a in
of
separable ratio
diastereoisomer groups
under
al
(42). hydroxy
(ref.127)
(78)
with
afforded
benzyl
the
(Z)The
cis-hydroxylation
using
gave
a
the
major
product
(81).
(78)
(E)-isomer.
and
with
(see
aldehyde
stereoselective 80"20
with
acidic
NMO.
ca.
to were
the
of
ratio
diastereomer
amount
of
-78~
1 equivalent
give
5-1actam
Condensation
(74) . at
of the p r o t e c t e d et
the
products
hydrogenolysis
synthesis
route
give
at
with
protecting
was also d e s c r i b e d by B r o a d y
substituents
to
added
correct
cbz
the
oxazoline
reaction
the
and
of AI-
2-methoxy-6-
gave the amine h y d r o c h l o r i d e
approach
catalytic
of
aminodihydroisocoumarins ether
L-aspartic
along
diastereomers
was
described
preferred
treated
with
silica
approach
n-butyllithium
(75) The
synthesis
oxazolone to
previously
methyl
at
the
(2,2,2-trifluoroethyl)phosphonoacetate
ester
which
derivative.
by n e u t r a l i s a t i o n
chloride
using
chloride.
Construction
from
in MeOH
aminodihydroisocoumarin
with
oxazoline
product
the
respectively,
A different Scheme
the
anhydrous
y-lactone
they
acid
cyclising
(76),
major
of
tribromide
amino
5% HCI
under
total
the
Instead
carried
treatment
the
conditions
with
was
using
intermediate
the
found
of
from the
then
cbz-protected
(77) . R e m o v a l
(71)
was
which
group
conditions
formate
of the
reacting
was
leucinal
by
mild
(42) (71)
nitrile
9 followed
They
unreliable.
6, (73)
of
report
synthesis
tert-butylmagnesium
hydrolysed
the
other
(ref.127) .
al
for
generated
mixture
at pH
to give
The
give
mild
(70).
lactone
to
opening
under
.
of
cbz-protected of
ring
opened
(DEPC)
under
t rimethylortho imino
4)
hydroxyamide
function
H20
methylbenzoate Deprotonation
acid
(28) .
an
been
(42)
of
of
in S c h e m e
ring
aminodihydroisocoumarin
gave
by B r o a d y (Scheme
amino
the
addition
(28)
has
lactam
r e a c t i o n by r e a c t i n g of
y-lactone
AI-77-B
shown
the
phosphorocyanidate
carboxyl
This by
to
skeleton
Pinner
conditions.
77-B
(70)
presence
hydrolysed
above
of
diethyl
intramolecular
gave
and
to give the p r o t e c t e d
coupling
achieved
in
group
This
reaction mixture
The diol group was p r o t e c t e d
was of
being as the
416
MeO
O
O
MeO
HN'~O
CI
\
/
O
._
N
~
~
73
72
MeO 1. SOCI2 2. NaOH/aq.EtOH
N~O
r
uLi/THF/-78~
74 O NHcbz t-BuMgCI/THF/ -78uC
76.MgCI
-i-
CH2Li 76
75 1.-78~ h 2. silica/DCM/18 h
MeO 42.HCI
0
1. BBr3
2. H2/Pd-C/HCI
Scheme 6
NHcbz
77
417
C02H
CHO
0
0 OBzl
SiBd Me,
CO2H
L-aspartic acid
78
79
K&O3/18-c-6
OH
$CO2BzI
OsOd NMO
SiBtf Me,
0
SiBd Me2
9
80
1. 2,2-dimethoxy
81
,H
propane 2. H2/lO% Pd-C/ EtOH 0
*
N\
SiBd Me,
+ I
82
+
42
DCCI DMAP
83 Scheme I acetonide and the free acid was obtained by hydrogenolysis to give the protected amino acid ( 8 2 ) . The aminodihydroisocoumarin
fragment
( 4 2 ) was coupled with
dicyclohexylcarbodiimide (DCC) and 4dimethylaminopyridine (DMAP) to give ( 8 3 ) . Deprotection under acidic conditions and ring opening resulted in the desired AI-11-B (82)
(28).
using
418
Other routes for the synthesis of the protected hydroxy amino acid moiety of AI-77-B have been described (ref.128-130).
8.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. A. Jones (Centre for Drug Design and Development , University of Queensland) for the ion-spray mass spectra and Beverley Smallbone, Sheila Yong and Catherine NelsonSmith for their assistance in preparing the manuscript.
9. 1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
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97
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111
112 113 114 115 116
of Animal, Plant and Microbiol Toxins, San Jose, Costa Rica, 1976, Pergamon, Oxford, 1978, pp. 739-758. K.J. van der Merwe, P.S. Steyn and L. Fourie, J. Chem. SOC. 1965 (1965) 7083-7088. P.S. Steyn and C.W. Holzapfel, J. South African Chem. Inst., 20 (1967) 186. R.D. Hutchinson, P.S. Steyn and D.L. Thompson, Tetrahedron Lett. , 43 (1971) 4033-4036. P.S. Steyn, in: A. Ciegler, S. Kadis and S.J. Ajl (Eds.), Microbiol Toxins. Vol. VI: Fungal Toxins, Academic Press,New York, 1971, pp. 179-205. F.S. Chu, Crit. Rev. Toxicol., 2 (1974) 499-524. P.M. Scott in: T.D. Wyllie and L.G. Morehouse (Eds.), Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. Vol. I: Mycotoxic Fungi and Chemistry of Mycotoxins, Marcel Dekker, New York, 1977, pp. 283-291. P. Krogh, in: D. Eaker and P. Wadstrom (Eds.), Natural Toxins. Proceedings of the 6th International Symposium on Animal, Plant and Microbial Toxins, Uppsala, Sweden, August 1979, Pergamon, Oxford, 1980, pp. 673-680. P.S. Steyn, in: V. Betina (Ed.), Developments in Food Science. Vol. 8: Mycotoxins - Production, Isolation, Separation and Purification, Elsevier, Amsterdam, 1984, pp. 183-216. Y. Shimojima, H. Hayashi, T. Ooka and M. Shibukawa, Agric. Biol. Chem., 46 (1982) 1823-1829. J. Itoh, S. Omoto, T. Shomura, N. Nishizawa, S. Miyado, Y. Yuda, U. Shibata and S. Inouye, J. Antibiot., 34 (1981) 611613. J. Itoh, T. Shomura, S. Omoto, S. Miyado, Y. Yuda, U. Shibata and S. Inouye, Agric. Biol. Chem. 46 (1982) 1255-1259. H. Okazaki, T. Kishi, T. Beppu and K. Arima, J. Antibiot., 28 (1975) 717-719. T. Sato, M. Morioka, N. Tsunoda and K. Suzuki, CA113(3) (1988) 23457~. J. Itoh, S. Omoto, N. Nishizawa, Y. Kodama and S. Inouye, Agric. Biol. Chem., 46 (1982) 2659-2665. Y. Shimojima, H. Hayashi, T. Ooka, M. Shibukawa and Y. Iitaka, Tetrahedron, 40 (1984) 2519-2527. Y. Shimojima and H. Hayashi, J. Med. Chem., 26 (1983) 13701374. Y. Shimojima, T. Shirai, T. Baba and H. Hayashi, J. Med. Chem., 28 (1985) 3-9. B.A. Thompson and J.V. Iribarne, J. Chem. Phys., 71 (1979) 4451-4463. C.M. Whitehouse, R.N. Dreyer, M. Yamashita and J.B. Fenn, Analyt. Chem, 57 (1985) 675-679. R.D. Smith, J.A. Loo, R.R. Loo, M. Busman and H.R. Udseth, Mass Spectrom. Rev., 10 (1991) 359-452. J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, Science, 246 (1989) 64-71. R.D. Voyksner, Nature, 356 (1992) 86-87. R.D. Voyksner and T. Pack, Rapid Commun. Mass Spectrom., 5 (1991) 263-268. K. Kawai, H. Ito, H. Nagase, R. Yamaguchi and S. Nakajima, Acta Crystallogr., Sect. C: Cryst. Struct. Commun., C41 (1985) 415-417. H.W. Schmalle, O.H. Jarchow, B. M. Hausen and K.H. Schulz, Acta Crystallogr., Sect. B. B38 (1982) 2938-2941. J.S.E. Holker and T.J. Simpson, J. Chem. SOC., Perkin Trans. 1 (1981) 1397-1400.
422 117 118 119 120 121 122
123 124 125 126 127 128 129 130
J.S.E. H o l k e r a n d K. Young, J. Chem. Soc., Chem. C o m m u n . , (1975) 525-526. C. A b e l l , D.M. Doddrell, M.J. Garson, E.D. Laue a n d J. Staunton, J. Chem. Soc., Chem. Commun., (1983) 694-696. L. Cattel, J.F. G r o v e a n d D. Shaw, J. Chem. Soc., P e r k i n Trans. i, (1973) 2626-2629. B. Alarc6n, J.C. Lacal, J.M. F e r n ~ n d e z - S o u s a and L. Carrasco, A n t i v i r a l Res., 4 (1984) 231-244. L. Carrasco, Biochimie, 69 (1987) 797-802. S. Inouye and S. K o n d o in" A.L. Demain, G.A. Somkuti, J.C. Hunter-Cevera a n d H.W. Rossmoore (Eds.), Novel Microbiol P r o d u c t s for M e d i c i n e and A g r i c u l t u r e , E l s e v i e r , A m s t e r d a m , 1989, pp. 179-193. T. U r u s h i d a n i , Y. K a s u y a and S. Yano, Exp. Ulcer, i0 (1985) 215-218. Y. K a s u y a a n d S. Yano,, A r z n e i m . - F o r s h / D r u g T. U r u s h i d a n i , Res., 36 (1986) 1383-1390. Y. Hamada, A. Kawai, Y. Kohno, O. Hara, T. Shioiri, J. Am. Chem. Soc., iii (1989) 1524-1525. Hara, A.Kawai, Y. Kohno and T. Shioiri, Y. H a m a d a , O. Tet rahedron, 47 (1991) 8635-8652. S.D. Broady, J.E. R e x h a u s e n and E.J. Thomas, J. Chem. Soc., Chem. Commun., (1991) 708-710. J.P. Gesson, J.C. J a c q u e s y and M. Mondon, T e t r a h e d r o n Lett., 30 (1989) 6503-6506. A. Kawai, O. Hara, Y. H a m a d a a n d T. S h i o i r i , Tetrahedron Lett., 29 (1988) 6331-6334. Y. H a m a d a , A. Kawai, T. M a t s u i , O. H a r a a n d T. S h i o i r i , Tetrahedron, 46 (1990) 4823-4846.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
423
Circular Dichroism of Carbohydrate-Molybdate Complexes Zahir Shah, Marcela Geiger, Yousef AI-Abed, Taleb H. AI-Tel and Wolfgang Voelter
i.
INTRODUCTION recent
times,
chiral molecules
(1-3).
CD records
the
left-
circularly
polarized
light,
to determine
the
emerged
In
as
Basically,
the
and
result
technique
rightof
Most
be employed
which
nucleic
acids
immense
sugars
biological
improvements
in
interest
using
in
CD
stereochemistry
of
intersaccharide
and
the
Earlier
thereby
include,
was
cobalt
among (14),
(24,25)
powerful
of
According
to
of
which
(15),
or
(i0),
as
CD
in
but the
recent
CD
as
the
of
(9).
other
involved
molecule
effects.
azides
(16-22),
well
sugar
Cotton
the
of the polymers
by
the
is
stimulated
investigating
i.e.
with
and
configuration
other
exibit
However,
have
structure
into
the
nm,
bands,
hand
carbohydrates
molybdate
complexes
for
the
each
(4-8)
ions
one
other
technique
xanthates
form
on
of
as well
1000-190
absorption
the
chromophores
metal
chromium
on
hence
can measure
of
in this region.
sugars
with
studies
derivatives
some
have
groups;
has
between
spectrum
conformations
range
monomers,
sugars
suitable
ions
the
the secondary
These
Later
it
copper
(11,13),
nickel
(23)
carbohydrates,
or
which
Cotton effects.
CD - BASIC PRINCIPLES
proportional
of
and the
instruments
proteins
sugar
others,
that
show a p p r e c i a b l e 2.
this
structural
of
in
instrumentation
linkages,
affording
found
vanadate
and
bands
investigation
in absorption
neighboring
are transparent
molecules.
introduction
among
importance
interaction
biological
( C D ) spectroscopy
structural
difference
available
absorption
unfortunately
for
of asymmetric molecules.
commercially
electronic
dichroism
technique
interactions
can
as the c o n f i g u r a t i o n of
circular
a powerful
to Beer,s the
law,
path
the
length
absorption of
the
(A) of a sample
cell
(i)
and
is
the
424
concentration
(c)
proportionality usually
molecule exibits
~(~)
is
to
its
expressed
(CD)
of
into
elliptically
optically
light passes
ellipse.
extinction
~(k)
the
left-
the
The
characteristic
spectrum.
When
right-
polarized
and
substance.
active medium
light.
transmitted
(expressed
The
tip
light
traces
in
degrees)
whose
length [~]:
[~]
and
length
Similarly
defined
where
of
to
by
ellipticity substance
the
in
CD
rotation
examined,
of
in
a
Thus,
left
diagram
for
intense
polarizer light.
unit
The
light
right
(S)
(P)
plate
light
passing
CD
becames
wave
that
is
left
through
a
the
between
stressed
right
sample
are
is
of
the
and c
this relationship
Fig.
1
absorption
spectrometer
light
shows
usually
(M)
to
block
by
and
an
a
A quarterpolarized
modulated
unit
polarization. partially
a
emitted
polarized.
modulator
polarized
in absorption
monochromator
electronically
is
[8]
observed
ellipticity
circularly
The
1 the
g/ml.
(1,2,3,26): ~ = k ( A L-
electronic
linearly
CD
path
rotation
in
this light into circularly
A
the
weight
the difference
retarders
and
on
in centimeters
a
the
The specific
is an absorption
through
to
length
instrument.
materials.
with
molecular
polarizations.
and
equal
ellipticity is
an
defined as the
~
optics to produce
passes
case
where
of
of
electrical
in millidegrees,
is given by
a
dichroism
concentration
the
kind
beams
specific
molar
to detect
(R) transforms
quarter
the
the
a CD instrument
typical
electric
isotropic
produce
a
source
wave retarder piezo
and
the
is
in extinc-
this
dependence
The relationship
particular
and the electronics
between
to
of
polarized
the
is
the molar ellipticity
equipped with the adequate light
M
in absorption
[8]=3298,~.
represents
c
1 the path
in mg/ml.
Considering
spectroscopy.
and
[8]=~*M/10*l*c,
and the difference
the
analogy
millidegrees,
being
AR)=32,98*AA.
in
cell
molar
(26):
account
and
it is transformed
tangent
9 is the ellipticity
the
concentration
becomes:
into
concentration,
[~]=~/l*c
path
the
takes
of
molecule
liniarly
ratio of the minor to the major axis of the ellipse. ellipticity
a
the circular
When
CD is also often reported as ellipticity,
~
constant
coefficient
a
which represents
active
polarized
of
is
absorption
through an optically
vector
angle
called
A(k)=E(~)*l*c.
they arise from the difference
from
~E(~)=EL(~)-~R(~), an
sample:
I/cm*mol.
Cotton effects,
coefficients
field
in
related
tion
light:
of the
(MD)
using
is
an
alternately
This
absorbed
modulated and
the
425
transmitted
light
produces
photomultiplier component
(PM) .
produced
a current
The
small
in
in the PM is m e a s u r e d
I
I
I
the d e t e c t o r
alternating by a
unit,
current
lock-in
a
(AC)
amplifier.
R
I
ISamplel I I PSI IOCCTRI I PMI
IC!MP I
Fig. i. B l o c k d i a g r a m of a typical CD instrument. PS = p o w e r supply, S = source, M = m o n o c h r o m a t o r , P = polarizer, R = retarder, MD = modulator, DC CTR = d i r e c t c u r r e n t controller, PM = p h o t o m u l t i p l i e r , AM = amplifier, COMP = c o m p u t e r The
intensity
dichroism light
of
of the
produces
of
the
in
absorption
light
CD
is
CD
(28) has
magnitude
thus
permits
to
of
value
the
measure
are
value
(ACS)
for
of -4.8
was
molecular
the
of
the
used
also ellipticity
for
acid
(CSA) of
for
a
soluble
used
as
CD
of a 0.06%
of the
PM.
circular
transmitted
The
electronics
the
difference
circularly
polarized
the
intensity
Therefore,
the
was
and
This
two-point
at 192.5
calibration
CD
using
sign
of
standard component
290.5
l-mm p a t h
CD band
ammonium
at
AC
correct
a primary
used.
is
the
signal
magnitude As
of
the
the AC
absorption
nm.
length nm,
The cell
with
(Fig.
a
2).
d-10-camphor-sulfonate
calibration aqueous
of
If the DC c o m p o n e n t
of CSA in a
a negative
the
the AC and DC c o m p o n e n t s
~A=k(AC/DC).
maximum
solution
water
CD.
of
determine
right-
by c a l i b r a t i n g
calibrated
CSA also has
Non-hygroscopic,
in
PM voltage,
simply
for a 1 mg/ml mdeg.
and
between
k in the e q u a t i o n 2.36
(DC)
left-
(+)-10-camphorsulfonic aAE
is 33.5 ~E
direct
sample.
constant
measure
average
by v a r y i n g
instruments
a
AL-AR=AA=k(AC/DC).
can be r e c o r d e d
a standard
is
The
the ratio
(27):
a
signal
current
between
by m e a s u r i n g
signal
the
sample.
a direct
(A)
constant
value
AC
spectrometer
the PM c u r r e n t
kept
the
standard
solution
(29).
at 290.5
The
nm is
426
+7910. cell
The CD peak value of this standard solution
is +190.4 mdeg.
or visible region
'
,
(29) in a 1 cm
Other standards were reported
for the far UV
(30, 31).
,
i
,
i
20 A ~
-2
-4 200
220
Fig. ref.
2. CD 28) .
3.
CARBOHYDRATE-MOLYBDATE
forms
till
Although
a
the
Richtmyer Kiang
spectrum
240 260 280 Woveiength (nm)
complex 1950s, and
(37) and
of
Gernez with
when
COMPLEXES
reported
D-mannitol
(33,34),
Voelter
320
(+)-10-camphorsulfonic
systematic
Hudson
300
et
as
early
(32),
1891
et
al.
work
were
(35,36),
(17,18,19,22,38)
structure of carbohydrate-molybdate
(similar
that
much
investigations
Weigl
al
as
not
acid
to
complexes.
to
molybdate was
done
initiated Spence
elucidate
by
and
the
3.1 Relationship Between pH and Optical Rotation of Sugar-molybdate Complexes
Following paper ionophoresis
lybdate solution
pyranose
sugars,
(pH 5.0), e.g.
in a cis-cis- l(ax),
appreciable
Weigl et ai.(35,36)
D-ribose(1),
2(eq),
mobility.
They
3(ax)
3 shows variations
having
that
that only
hydroxyl
(chair form) only
such
groups
exibit sugars
system complex with molybdate.
of specific rotations
lybdate complexes with changes
in acidic mo-
observed
three
arrangement
concluded
possessing a cis-cis-l,2,3-triol Fig.
studies of sugars
of sugar(1,2,3)-mo-
in pH where the optimum pH for the
complex formation lies near 5.5.
427
Based
on
these
studies,
(37)
formulated
a
1
al.
(35,36)
which
H
H
OH
H
1
OH
[
1
" Affinity
H
1
et
al. for
OH
[
3
/
o -
--10
o
--20
L.
Spence
0
H
H
2
+1o
A
and
stated:
0o0o> >o, _
OH
i
HO
et
hypothesis
oC>~
H
Weigl
'
/
/
& -3o -40
1
2
3
4 pH
5
6
7
8
9
Fig. 3. E f f e c t of pH on s p e c i f i c r o t a t i o n [~] of s u g a r - m o l y b d a t e c o m p l e x e s . R a t i o of m o l y b d a t e to sugar, i:i. Temp. 2 5 + 0 . 5 ~ D - r i b o s e (i) , D - m a n n o s e (2) , - .... D - l y x o s e (3) . (similar to ref. 37). molybdate compound
can
formation However,
be
regarded
possessing with
sugar-molybdate incorrect.
For
as
a
diagnosis
cis-cis-l,2,3-triol
a l(ax),
Voelter
potentiometric,
a
et al.
2(eq), (16,
3(ax)-triol
17,
polarimetric complexes instance
and and
,
19,
it
38) CD
proved was
shown
for
the
system
to
ability adopt
of a
a
con-
system". conducted studies the (16)
a of
series a
number
hypothesis (contrary
of of
to
be
to
the
428
observation
by
D-xylose(6), of s p e c i f i c
Spence
et
al.)
D-galactose(7) rotations
and
OH
HO~C OL ~ H3
I
4-8
OH OH
I
OH
I
OH OH
7 4 shows the m a x i m u m
4).
6
OH
I
variations
(Fig.
H
5
]
Fig.
exhibit
H
OH
CH20 HH O.'~V Hoi~ ko 1 , 1 OH
D-glucose(5),
in the pH v a l u e s
H
I
4
D-arabinose(4),
L-rhamnose(8)
with changes
HO"~, OH HOkJ.__..._j/ H
'o.
that
8 change
(under the e x p e r i m e n t a l
in
specific
conditions)
rotation
in the pH r a n g e
of s u g a r s 5.7-6.0.
- 80~
80 ~
[Cr
D
i~'lo --I00 ~
60~
1
I
3
6
I
pH
9
[C'-ID
57 ~
40~
56 ~ 55 ~
20~
54 ~ D 53 ~ '
I
;
6
pH
:~
I
I
3
6
l
'pH
9
Fig. 4. E f f e c t of pH on the s p e c i f i c r o t a t i o n s [~] of s u g a r - m o lybdate complexes. A: L - r h a m n o s e ( 8 ) , B: D - x y l o s e ( 6 ) , C: D - g a l a c t o s e ( 7 ) , D: D - g l u c o s e ( 5 ) , E: D - a r a b i n o s e ( 4 ) (similar to ref. 16).
429 Surprisingly, specific
Spence
rotation
reported
by
Tanret
et
by
al.
( 3 7 ) could
compounds
(39)
4-7,
as well
as
that these sugars exibit changes of
molybdate
complexes.
verified
titration
and
concluded
not
by
detect
although
formation
of a sugar-molybdate
curve
solution
potentiometric of
a
studies
of
in
already
Murgier
(40)
by addition
sugar-molybdate
complex was
(16).
of
change
was
and
in specific r o t a t i o n
the
The formation
by
it
Darmois
the
Fig.
D-galactose(7)
5
further
shows
the
and sodium
pH
2
Fig.
5.
molybdate
4 ml I~ t1(1
Potentiometric
(A)
in
titration
comparison
molybdate
(C)
(similar to ref.
mo l y b d a t e
(A)
which
formation
of
D-galactose
(B) a
and
and
which
beyond
proved
by
be
16).
compared
sodium
with
molybdate
potentiometric
CD
studies
doubt
of
the hypothesis
The
isopolyanions
solutions
of
is generally
known,
its p o l y m e r i s a t i o n products
the
(C).
Ion
by
titration This
sodium
sodium
curve
of
the
(16,17,19,38,39,40)
were
The
results
complexes
by Weigl
acidifying Fig.
+
and
proves
although there
(41-43).
(B)
complex.
studies
(37) to be erroneous.
3.2 The Complexinq Molybdate
D-galactose
s ugar-molybdate
and Spence et al. formation
of
D-galactose
D-galactose-molybdate
polarimetric
substantiated
can
curves
with
et al.
neutral
of
(17,19)
(35,36)
molybdate
is no agreement
over
6 shows the dichroitic
430 E
16
E
..."..
-a)
12
oi @
8
4J ,,~
L,
_
.-
.
.-'"
/
:
:
/
/--\
t
U -10 O
f
u -&O
--
&5
/
L
-20
-
-~0
-
/
\ \ \ \
I
\
J
-I00
-120 .." "....." " " 1 5 55pH 6
-50 --
/
-80
/-
".
o~
/
-60
II! : \.
/
J
J
9
9
-30
L
\\
I
-20
.E
_
..'""~" /
E
b}
~0
i
1
&
~
&5
i
5
i
55pH 6
Fig. 6. p H - D e p e n d e n c e of the d i c h r o i t i c a m p l i t u d e s of s u g a r - m o l y b d a t e complexes. Sugar and m o l y b d a t e c o n c e n t r a t i o n s : 0.02 and 0.04 mol/l, a) ..... D - x y l o s e ( 6 ) , D-glucose(5), Dg a l a c t o s e (7) b) L - r h a m n o s e (8) , D - m a n n o s e (2) . amplitudes function
of of
two pH
D-galactose(7) pH
range
5-6,
sugars.
In
many
(45)
pH
range
sugar-molybdate Voelter
between
a sugar
MOO42-
+ H+
follows:
molecule ~
7[HMoO 4
pH range, 9 C6H1206]-
of
exists al.
that
(formed
at
curves
show
neither
the
nor
low
maximum
hexa-
pH
exibit proposed
and the HMoO 4
mobility the
in
monomeric
values)
of
and H M o O 4-
as
as
complex
the the
following
the
-[HMoO 4
electric
ion:
complex
decomposes
with
reaction
9 C6HI206]-
sugar-molybdate
ion well
Since
- H M o O 4~
the
pyranose
ions(45). in
a
D-xylose(6), maxima
(44)
stability
as M o 0 4 2 -
(17)
complexes
D-glucose(5),
The
pH>7)
complexes
et
H M o O 4- + C 6 H 1 2 0 6 In a c i d i c
indicates range
molybdate
field,
sugar-molybdate
D-mannose(2),
ions
the
of
L-rhamnose(@).
which
(stability
complexes,
bands
for
and
MOO42-
heptamolybdate
CD
as
-7C6H1206 +H ~ 7 H M o O 4- ~--------Mo 02 6- + 4H 0 +7C6H1206 -H + 7 4 2
431
3.3 Confiqurational Analysis of Suqars by CD can
On the basis
be
divided
of their molybdate
into
two
groups.
complexes,
Compounds
have two adjacent hydroxyl groups at C-2 and C-3 Group trans
II compounds
position.
belonging
to
The
have
group
CD
their hydroxyl
spectra
groups
of molybdate
I generally
show
3-4
aldehyde
belonging at
to
I
in cis position. C-2
complexes
bands
sugars
group
in
and
the
C-3
in
range
of
of
sugars
220-350 nm and
much more intensive Cotton effects as compared to
show
bands
the sugar-molybdate complexes of group II. The group II compounds only
lacking
two
free
in
hydroxyl
pyranose ring do not
groups
same at
wavelength C-I
and
region.
C-3
methyl
Thus,
S-D-glucopyranoside(10),
2,3,4,6-tetra-O-methylglucose(12),
methylgalactose(13),
arabinopyranoside(15), u-D-xylopyranoside(17)
Compounds
position
form complexes with molybdate.
~-D-glucopyranoside(9),
thylglucose(ll),
the
3-O-me-
methyl o-D-
methyl B-D-arabinopyranoside(16),
not
Tables 1 and 2 show CD data for the sugar-molybdate complexes
of
CH20H
(19).
CH,zOH
0
0~ ~0
~
OH
9
CH20H
0~c.~
~ 0~0. '
OH
i0
oCi-~
CH3
6-D-xylopyranoside(18)
methyl
do
complex with molybdate
methyl
the
methyl
2,3,4-tri-O-
methyl ~-D-ribopyranoside(14), and
of
c.~ [~OH 1 OCH ' 3
I~C.~ I
12
ii
O.
15
I
' OH
13
' OH
16
I
oc.,
H
' 3 OCH
' OH
14
o
' OH
OH
1
' OH
17
.o O, 1
' OH
18
432 TABLE
i.
CD s p e c t r a l Sugar
name
D-Ribose i
data
of s u g a r - m o l y b d a t e
Structure
pH
Band 1 ~ax [e]a
H0~ 0~OH5.4 I
complexes
312
of g r o u p
Band 2 k m a x [e]a
I.
Band 3 k m a x [e]a
-1400
269
+6600
233
-23100
294 + 2 0 0 0
263
-21800
230 +5900
298 +2300
265-23800
237-19800
297 -1500
262
+8900
233
317-1600
272
+8200
237-28700
305 -910
266 +27600 237 -31200
308 + 9 6 0
,.70-1200
I
OH OH
CH20H D-Mann~
'
0
Ho~OHHO~ OH 5"4 I
D-Lyxose
3
1
.o4O. 1
OH
1
5.4
0
L-Rhamnose c
D-Allose 19
D-Gulose 20
H H3 OH 5.5 OH OH CH20H HO~ ~~OH~ 5.4 OH OH CH2OH HO~ 1
O/~OH 5.4
-
1
OH OH
CH20H
D-Talose 21
a
H0/~--0\.
~o/~OH
I
5.s
231 §
1
[8] v a l u e s in d e g * m o l - l * c m 2, sugar conc. 0.02 M, m o l y b d a t e conc. 0.04 M. b T h e D - m a n n o s e - m o l y b d a t e c o m p l e x shows an a d d i t i o n a l n e g a t i v e C o t t o n e f f e c t at 339 nm ([8]=36). c T h e L - r h a m n o s e - m o l y b d a t e c o m p l e x shows an a d d i t i o n a l p o s i t i v e C o t t o n e f f e c t at 331 nm ([8]=150).
433 TABLE 2. CD spectral data of sugar-molybdate complexes of group II. sugar name
pH
Band 1 kma x [e] a
Band 2 kma x [e] a
5.3
280
-200
247
+1500
5.5
278
+1400
244
-6100
OH
5.5
275
+3000
244
-9100
OH
5.2
272
+500
242
-4600
5.2
270
+700
236
-2000
5.4
275
-3400
242
+15400
5.4
280
+300
246
-1600
5.4
275
+2100
243
-5000
Structure
"0
D-Arabinose 4
OH
OH ' CH2OH
D-Glucose 5
D-Xylose 6
' OH
1
0~0 H H
0 V
OH
CH20H D-Galactose 7
H040H 0 ' OH
I
CH20H 2-Desoxy-Dglucose 22
Ho~OH ~ 1
OH
I
CH20H D-Altrose
23
OH
I
l
L-Arabinose 24
6-O-Methyl-Dglucose b 25
HO
OH
1
OH CH2OCH3 OH
oOH~ ~
H
'
OH
a [8] values in deg*mol-l*cm 2, sugar conc. 0.02 M, molybdate conc. 0.04M. b sugar conc. 0.01 M, molybdate conc. 0.04 M.
434
sugars of
belonging
molybdate
belong
to
297-294,
to
group
complexes
group
I
263-262
I and
of
(Table
respectively.
D-mannose(2) i)
show
and 233-230
the D - m a n n o s e - m o l y b d a t e
II
nm.
Fig.
complex.
and
four
The
CD
spectra
L-rhamnose(8)
signals
7 shows
at
the
which
339-331,
CD spectrum
of
@230(5.9}
Ol
+5
339(3.61
-5 10
320 nm 360 '=
-15
,--"'-I
-20 -25 %,\
\\
<~) 263121.81
1.5 - 1.0
X
\\
220
0.5
250
300
350
klnmJ
L~00
Fig. 7. C D - s p e c t r u m of the D - m a n n o s e - m o l y b d a t e complex. m o l y b d a t e concentrations: 0.02 and 0.04 mol/11 pH 5.4. Band 1 (Fig.
most
intense
this
group
signals
7) appears band
2 and
3
characteristic intense
in
group
237-231
and nm.
positive contrast,
as
narrow As
well
270-260
The
Cotton
D-lyxose(3)
at C-2
C-3
reversal
of
Cotton
effects
(band
have and
D-talose(21) image
1-3).
ratio
(positive,
of
most
D-allose(&9), signal
have
in
orientation.
In
the
two
which
positive)
sugar
L-
a negative,
at C-2 and C-3
orientation
negative,
the
of D-ribose(1),
cis
of
therefore
intense
traverses
groups
required
The
L-rhamnose(8)
most
1-3)
pyranoses
i,
and
the
The hydroxyl
The
Table
D-ribose(1),
(bands
230 nm.
are
show
D-gulose(20) the
other
and
while
in mirror
signs
in
in
4 at
D-lyxose(3)
effects
of signs.
pyranoses and
nm,
and
as
region
shown
D-talose(21)
groups
by the
I.
D-allose(19)
sequence
these
case,
a
and
2 at 294 nm, while the
at 263 nm and band
for D-mannose(2),
in the range
rhamnose(8), all
of
signal
appears at
in this
occur
D-gulose(20)
at 339 nm and band
3 appears
Sugar
to
hydroxyl
is shown in their
molybdate
in
435
complexes
belonging
sugar-molybdate
effects shows
to group
complexes
are observed
CD
spectra
I compounds
belonging
in the region
of
molybdate
is always
to
group
280-270
i:i
II,
and 247-236
nm.
8
Fig 8. CD spectra of D-glucose(a) and 2 - d e s o x y - D - g l u c o s e m o l y b d a t e complexes. Sugar and molybdate concentrations: 0.02 0.04 mol/l; pH 5.5 (a) and 5.2 (b).
(b) and
exibiting
effects.
f
a
+2
-2
a positive
D-glucose(5)
Fig.
2-
their Cotton
of
For
Cotton
and
desoxy-D-glucose(22)
complexes
(i0).
two
, negative
in
0270(0.71
(~ 236{2.0) \
A-"'+2
sequence
\
~
1.5 - 1.0
\
\
t
0.5
~
L,,
~-'t----.~L
L
I
L
I
r
L
o
L._ fD z'-',
1.5
\\~)
-6
16.1) - 1.0
\ \
220
[
:
- 0.5
;
250
i
l
~
300
'
350
l
l
~
1
/.(]0
),(nm)
As
shown
complexes
positive effects
in table of
2,
sequence.
for
the
signs
D-arabinose(4) On
molybdate
g a l a c t o s e (7) ,
the
other
complexes
L-arabinose (24) ,
methyl-D-glucose(25)
are
thus
hydroxyl
mirror
indicating image
the
orientation
and D-altrose(23). group
II is 2:1
as
Cotton
effects
D-altrose(23) hand,
of
the
for
show
sign
D-glucose(5),
of
a
(positive,
groups compared
of sugar
of to
these those
molybdate negative,
the
Cotton
D-xylose(6),
2 - d e s o x y - D - g l u c o s e (22)
reversed
The rate
(16,19).
of
and
negative compounds in
to molybdate
and
D6-0-
sequence) to
have
D-arabinose(4) in complexes
of
436
4.
MOLYBDATE COMPLEXES CD provides
linkages
in
monstrated
dithat
4-O-pyranosyl saccharides,
]
the et
trisaccharides.
6-O-pyranosyl to
show
T
can
be
The essential
Cotton
C-2 and C-3.
maltose(28)
Voelter
sugars effects
namely that complex
at C-l,
biose(27),
a powerful mean to determine and
sugars by CD.
trisaccharides groups
from DI- and T R I S A C C H A R I D E S
requirement
is
the
same
formation requires
However,
(46,47)
differentiated
although
and maltotriose(29)
possess
CH2OH
l
for
[ OH
mono-
free hydroxyl cello-
free hydroxyl
o
OH
defrom
for di- and as
lactose(26),
o
r OH
intersaccharide al.
,
OH CH2OHO
26
27
CH2OH o~O I
CHzOH
0
l
0
' OH
I
' OH
OH
28
CH2OH
CH2OH
CH20H OH
1
1
' OH
1
' OH
' OH
29
groups due
to
at CI-C3, steric
these do not react with molybdate. hindrance
and/or
on
r e a c t i o n between molybdate and such sugars 6-O-pyranosyl melibiose(32) Band
sugars,
unfavourable
(46).
gentiobiose(30),
isomaltose(31)
and
complex with molybdate and show two Cotton effects.
1 for the
gentiobiose(30) melibiose(32)
e.g.
This could be
energetically
three
compounds
appears
at
242
lies nm
at 243 nm. Band 1 has a
at and
275
nm,
for
positive
while
band
isomaltose(31) sign
while
2 for and the
437
CH20H 0 0" ,
0 'cO 'OH
CH~OH
""0
o
H
]
OH
I
OH
/
OH
0--
30
OH
31
0
CH20H
1 !
I
OH
OH
OHc~
0
,
OH
32
most is
intense 2 : i,
complexes of
a
band
which
di-
of or
differentiate
is
group
2 is negative. the
same
II
(47).
as
The for
Thus,
ratio the
of
sugar
by d e t e r m i n i n g
trisaccharide-molybdate
to m o l y b d a t e
monosaccharide-molybdate complex,
b e t w e e n the 6-0- and 4 - O - p y r a n o s y l
the
CD
one
can
spectrum easily
linkages.
REFERENCES 1 2 3 4 5 6 7 8 9 i0 ii 12 13
C. Djerassi, Optical R o t a t o r y Dispersion, M c G r a w Hill, N e w York, Toronto, London, 1960. G. Snatzke, Optical R o t a t o r y D i s p e r s i o n and C i r c u l a r D i c h r o ism in O r g a n i c Chemistry, Heyden & Son Ltd, London, 1967. P. Crabb~, Optical R o t a t o r y D i s p e r s i o n and C i r c u l a r D i c h r o ism in O r g a n i c Chemistry, Holden-Day, San Francisco, London, A m s t e r d a m , 1965. B. Sj~berg, D.J. Cram, L. Wolf and C.Djerassi, A c t a Chem. Scand., 16 (1962) 1079. Y. T s u z u k i and K. Tanabe, Bull. Chem. Soc. Japan, 35 (1962) 1614. C. Djerassi, H. Wolf and E. Bunnenberg, J. Am. Chem. Soc., 84 (1962) 4552. Y. Tsuzuki, K. Tanabe, M. Akagi and S. Tejima, Bull. Chem. Soc. Japan, 37 (1964) 162. Y. Tsuzuki, K. Tanaka, K. Tanabe, M. Akagi and S. Tejima, Bull. Chem. Soc. Japan, 37 (1964) 730. H. Paulsen, Chem. Ber., i01 (1968) 1571. W. V o e l t e r A Malik, F.L. Ansari and J. T s c h a k e r t in : ' H K l e i n and G . Snatzke (Eds), 4 t h Int. C o n f e r e n c e ' on CD, Bochum, Germany, S e p t e m b e r 9-13, 1991, pp. 212. H. Vergin, H. Bauer, G. K u h f i t t i g and W. Voelter, Z. N a t u r forsch. 27b (1972) 1378. W. Voelter, H. Bauer and G. Kuhfittig, Chimia 27 (1973) 274. W. Voelter, H. Bauer and G. Kuhfittig, Chem. Ber., 107 (1974) 3602.
438
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
H. Bauer, J. Brun, A. R. Hernanto and W. Voelter, Z. N a t u r forsch. 44b (1989) 1464. D.H. Brown, W.E. Smith, M.S. El-Shahawi and M.F.K. Wazir, Inorg. Chimica Acta, 124 (1986) L 25. E. B a y e r and W. Voelter, Liebigs Ann. Chem., 696 (1966) 194. W. Voelter, E. Bayer, R. Records, E. B u n n e n b e r g and C. Djerassi, Liebigs Ann. Chem, 718 (1968) 238. W. Voelter, H o p p e - S e y l e r ' s Z. Physiol. Chem., 350 (1969) 15. W. Voelter, G. Kuhfittig, G. Schneider and E. Bayer, L i e b i g s Ann. Chem., 734 (1970) 126. V. Bilik, W. Voelter and E. Bayer, Angew. Chem., 83 (1971) 967. V. Billk, W. Voelter and E. Bayer, Liebigs Ann. Chem., 759 (1972) 189. W. V o e l t e r and H. Bauer, T e t r a h e d r o n Lett., 40 (1974) 3597. S. Takizawa, H. Sugita, S. Yano and S. Yoshikawa, J. Am. Chem. Soc.,102 (1980) 7969. S. Bunel and C. Ibarra, P o l y h e d r o n 4 (1985) 1537. S. Bunel, C. Ibarra, V. Calvo and E. Moraga, P o l y h e d r o n 8 (1989) 2o23. L. Velluz, M. Legrand and M. Grosjean, Optical C i r c u l a r Dichroism, Principles, M e a s u r e m e n t s and A p p l i c a t i o n s , V e r l a g Chemie, Weinheim, 1965. M o d e l J - 7 1 0 / 7 2 0 S p e c t r o p o l a r i m e t e r , I n s t r u c t i o n Manual, J a p a n S p e c t r o s c o p i c Co., Ltd, Ishikawa-Cho, H a c h i o j i City, Tokyo, Japan, 1990. G.C. Chen and J.T. Yang, Anal. Lett., i0 (1977) 1195. T. Takakuwa, T. Konno and H. Meguro, Anal. Sci., 1 (1985) 215. P.H. Scippers and H.P.J.M. Dekkers, Anal. Chem., 53 (1981) 778. B. N o r d e n and S. Seth, Appl. Spectrosc., 39 (1985) 647. D. Gernez, C. R. hebd. S~ances Acad. Sci., 112 (1891) 1360. N.K. R i c h t m y e r and C.S. Hudson, J. Amer. Chem. Soc.,72 (1950) 3880. N.K. R i c h t m y e r and C.S. Hudson, J. Amer. Chem. Soc., 73 (1951) 2249. E.J. Bourne, D.H. Hutson and H. Weigl, J. Chem. Soc., (1960) 4252. E.J. Bourne, D.H. Hutson and H. Weigl, J. Chem. Soc., (1961) 35. J.T. Spence and S.-C. Kiang, J. Org. Chem., 28 (1963) 244. W. Voelter, Ph.D. Thesis, T H b i n g e n U n i v e r s i t y (1966). G. Tanret, Bull. Soc. Chim. France 29 (1921) 670; C. R. hebd. S~ances Acad. Sci., 172 (1921) 1363. E. D a r m o i s and M. Murgier, C. R. hebd. S~ances Acad. Sci., 195 (1932) 707. J.B. G o e h r i n g and S.Y. Tyree, Seventh I n t e r n a t i o n a l C o n f e r e n c e on C o o r d i n a t i o n Chemistry, Stockholm, Uppsala, 25-29 June 1962; Abstracts, pp. 172. O. G l e m s e r and W. Holznagel, R e f e r e n c e 41, pp. 172. H. H e i t n e r - W i r g u i n and R. Cohen, R e f e r e n c e 41, pp. 172. G. J a n d e r and K.F. Jahr, Kolloid-Beih., 41 (1935) i. Y. Sasaki, I. Lindqvist and L.G. Sill~n, J. Inorg. Nucl. Chem. 9 (1959) 93. W. Voelter, G. K u h f i t t i g and E. Bayer, Angew. Chemie, 82 (1970) 985. W. Voelter, G. Kuhfittig, O. Oster and E. Bayer, Chem. Ber., 104 (1971) 1234.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
439
Screening of Oncogene Function Inhibitors from Microbial Secondary Metabolites Kazuo Umezawa
i.
INTRODUCTION Microorganisms
low m o l e c u l a r past
it
was
substances 1960's
inhibitors
of
as and
show
and
no
that
these
role
in the
enzyme
example,
of r e m n a n t
inhibitors origin
antipain,
are
antibacterial producing
genes
that
not
very
to
that
their
they
late
antibacterial
enzyme it
inhibitors
and
lost
the
of e n z y m e
Streptomyces, such
Therefore,
enzyme
strains have
any
from
specific
in
and m o s t
show
of
In the
antibacterial
However,
inhibitors
effect.
and
produce
started,
did
substances
streptomycin.
competitors.
protease
antibiotics
and
microorganisms
their
microbial
For
leupeptin
of
of a n t i b a c t e r i a l
as p e n i c i l l i n
that
suppress
screening
a number
such
believed to
activity.
produce
weight
is
have
are
more
likely
absolutely
only
relevance
inhibition
over
the the
no
products course
of
evolution. Therefore, having
various
microorganisms structures
are
the
specific
bioactive
products
the
testing
method.
Since
developed oncogene
in the
field
function
a treasury
and b i o l o g i c a l in c u l t u r e
oncogene
inhibitors
broths
theory
of c a r c i n o g e n e s i s as a new
of o r g a n i c
activities.
is d e p e n d e n t
has
been
research,
group
compounds
Isolation
of
upon
extensively
we have
of m i c r o b i a l
screened secondary
metabolites. All
normal
are
at
are
considered
growth
least and
oncogenes,
cells
70 d i f f e r e n t to play then
The m e c h a n i s m
and
and myc,
However,
and gene
in Fig.
viruses
may
I.
in the
role
in the
they
neoplastic
of a c t i v a t i o n
as shown
oncogenic
oncogene.
important
induce
as in myc,
in t h e i r
proto-oncogenes
an
differentiation. which
amplification abl
have p r o t o - o n c o g e n e s
can
Such
regulation be
mutation
or c h r o m o s o m e Chemical the
cell.
There genes
of cell
activated
into
transformation.
includes
induce
nucleus.
as
in ras,
translocation
carcinogens, activation
of
gene as in
radiation, a
proto-
440
I
!
A
B
t
--.
I
B Activation
9 Src
C
I
~N.~fication
A' i.
F//zt
ranslocati on
proto-oncogenes I -t
I mutation
Fig.
C'
I
B
I
B
of p r o t o - o n c o g e n e s .
0
Osis 0 0
Lck
Erb B2
0 Fps
\
/
o
0
Fos
Ab[
0
Jun
ErbB
i
,
I
Fins
I
/
9
9 Fig.
2.
Cellular
TABLE
ER
Ras
localization
of oncogene
products.
1
Oncogene
functions.
Function
Oncogene
Growth
factor
Growth
factor
Tyrosine
Sis receptor
kinase
G protein Transcription
product
ErbB Src H-Ras
regulator
Fos
ErbB2
Fms
Trk
etc.
Fps
Lck
Abl
etc.
K-Ras
N-Ras
Jun
Myc
etc.
44 1
An a c t i v e oncogene e n c o d e s t h e p r o t e i n p r o d u c t h a v i n g s p e c i f i c activity t o
induce transformation.
The o n c o g e n e p r o d u c t s a r e The s r c , y e s ,
l o c a t e d either i n t h e cytoplasm o r i n t h e n u c l e u s . a b l , e r b B, (Fig. 2 ) .
and r a s p r o d u c t s a r e l o c a t e d i n t h e cytoplasm,
erb BZ,
while t h e fos,
j u n , myc, and myb p r o d u c t s a r e f o u n d i n t h e n u c l e u s
The oncogene f u n c t i o n s a r e summarized i n T a b l e 1 .
The s i s oncogene e n c o d e s a p r o t e i n t h a t i s c l o s e l y r e l a t e d t o p l a t e l e t - d e r i v e d growth f a c t o r t o a cell
binds
(PDGF) i n primary s t r u c t u r e .
surface receptor t o activate
t y r o s i n e kinase a s s o c i a t e d with t h e r e c e p t o r . growth f a c t o r - l i k e p r o t e i n .
an i n t r a c e l l u l a r
Thus, s i s p r o d u c e s a
The e r b B p r o d u c t h a s homology w i t h
t h e e p i d e r m a l g r o w t h f a c t o r (EGF) r e c e p t o r .
EGF b i n d s t o t h e EGF
receptor t o a c t i v a t e tyrosine kinase i n t h e l a t t e r . product
is
an
activated
PDGF
EGF
receptor
a c t i v i t y i n t h e a b s e n c e o f EGF.
having
The s r c , y e s ,
The e r b B
tyrosine ros,
kinase
a b l , t r k and
fms p r o d u c t s a l s o h a v e t y r o s i n e k i n a s e a c t i v i t y . The r a s o n c o g e n e i s i m p o r t a n t , b e c a u s e i t i s o f t e n f o u n d i n human t u m o u r s , elucidated.
but
i t s mechanism o f
I t i s a k i n d o f G p r o t e i n t h a t b i n d s t o GTP and a c t i -
vates c e l l u l a r phosphatidylinositol oncogene p r o d u c t s
such a s FOS,
regulatory proteins.
AP-I
itself. as
screening.
The n u c l e a r
a n d Myc a r e t r a n s c r i p t i o n
element
(TRE).
J u n was f o u n d t o
Fos forms a heterodimer w i t h Jun,
and t h i s dimer
W e can t h i n k of t y r o s i n e k i n a s e a c t i v i t y and P I
b i n d s t o TRE. turnover
( P I ) turnover.
Jun,
is the transcription regulatory protein
t h a t b i n d s t o t h e TPA-responsive be A P - I
a c t i o n h a s n o t been f u l l y
oncogene
functions
Especially,
for
the
tyrosine kinase
target
of
inhibitor
i s e s s e n t i a l f o r many
oncogene f u n c t i o n s . 2.
TYROSINE K I N A S E I N H I B I T O R S 2.1 Erbstatin
C u l t u r e f i l t r a t e s o f Streptomyces were s c r e e n e d f o r i n h i b i t o r s o f t y r o s i n e k i n a s e i n t h e membrane f r a c t i o n o f human e p i d e r m o i d
c a r c i n o m a c e l l l i n e A431.
T h e s e c e l l s c o n t a i n a l a r g e number o f
EGF r e c e p t o r s w i t h t y r o s i n e k i n a s e a c t i v i t y on t h e i r membrane. strain
was
found t o produce
tyrosine kinase
(Fig. 3 ) .
a
novel
compound t h a t
I t was named e r b s t a t i n
One
inhibited
( 1 ) . Taxonomic
s t u d i e s i n d i c a t e d t h a t t h e s t r a i n producing e r b s t a t i n i s c l o s e l y r e l a t e d t o Streptomyces v i r i d o s p o r u s .
442
OH
!
R1
ORe erbstatin methyl 2,5-dihydroxycinnamate
R1 NHCHO COOMe
R2
NHCHO
Me
5'- O-methylerbstatin Fig.
3.
Erbstatin
H H
and r e l a t e d
compounds.
Culture broth (5 1)
I
Filtrate
I
BuOAc extraction
I
Silica gel chromatography
I
Reversed-phase HPLC (Nucleosil 5Cla)
I
Crystallization with CHCla-MeOH
I
Erbstatin (60.0 rag) Fig.
4.
Isolation
Erbstatin
was
of e r b s t a t i n . isolated
4.
with
5 liters
of b u t y l
acetate,
reduced
pressure
to
under
Erbstatin
dried material
gel
column
active vacuo
culture
to give
eluted
a yellow
reversed-phase
crystals
were
Erbstatin
is
obtained soluble
and
the
After
a
extract
and then wash
(ii0 mg) .
ethanol,
The
to silica
CHCI3-MeOH,
the
concentrated
Erbstatin
was
then
and
in
puri-
Sixty m i l l i g r a m s from
in
concentrated
(370 mg) .
and
recrystallization
in m e t h a n o l ,
shown
extracted
subjected
with
5C18) .
as
was
was
powder
CHCI3-MeOH
(Nucleosil
after
filtrate
(5 liters)
a yellowish
with
powder
HPLC
culture
in CHCI3,
chromatography. was
the
filtrates
give
was d i s s o l v e d
fraction
fied by
in
from
Fig.
of
MeOH-CHCI3.
acetone,
slightly
443
soluble in chloroform and ethylacetate, and insoluble in H20 and nhexane. The 50% inhibition concentration of erbstatin against tyrosine kinase was 0.55 pg/ml, when it was examined as follows: The reaction mixture contained 1 mM MnC12, 100 ng EGF, 40 pg protein of A431 membrane fraction, I 5 pg of albumin, 3 pg of histone, and HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer
(20 mM, pH 7.4) in a final volume of 50 pl. The reaction tubes were placed on ice and incubated for 10 minutes in the presence or absence of erbstatin. The reaction was initiated by the addition of labeled ATP (10 pl), and the incubation was continued for 30 minutes at 0°C. Then aliquots of 50 pl were pipetted onto Whatman 3MM filter paper and put immediately into a beaker of cold 10% TCA containing 0.01 M sodium pyrophosphate.
The filter papers were
washed extensively with TCA solution containing 0.01 M sodium pyrophosphate at room temperature, extracted with ethanol and ether, and then dried.
Radioactivity was measured by a scintil-
lation counter. Erbstatin was found to inhibit tyrosine kinase associated with Src, Erb B2, Abl, and Fyn. Erbstatin inhibited tyrosine kinase competitively with the peptide (2), but did not inhibit protein kinase A or C. Erbstatin was chemically synthesized by several groups. One of the
effective
syntheses
includes
the
reaction
of
diethyl(isocyanomethy1)phosphonate with dihydroxybenzaldehyde by a modified Schollkopf's procedure ( 3 ) . This procedure was used for preparation of various erbstatin analogues (3). Erbstatin easily decomposes in calf serum and in cell culture medium in the presence of oxygen and ferric ion (4). We isolated 2,5-dihydroxybenzaldehyde by overnight incubation of erbstatin with ferric chloride. The decomposition of erbstatin in calf serum was prevented by addition of foroxymithine, a ferric ion chelator
Streptomyces. We synthesized methyl 2,5(2,5-MeC, Fig. 3) as a stable analogue of erbstatin (5). This analogue is about 4 times more stable than erbstatin in calf serum, and its inhibition of EGF receptor tyrosine kinase in vitro and i n s i t u is comparable to that of isolated
from
dihydroxycinnamate
erbstatin.
We also prepared additional erbstatin analogues that
are even more stable than 2,5-MeC, as well as 5'-O-methylerbstatin (Fig. 3).
All except the latter inhibited src oncogene functions in cell culture (6).
as an inactive analogue of erbstatin
444
Recently,
we
prepared
biosynthetic
manner
cell
medium.
culture
min.
On
the
cells
reached
(7),
radioactively
and
using
Erbstatin
other
hand,
the
half
maximal
labeled
it we
was
erbstatin
assessed
degraded
intracellular
its
about
in the
stability 50%
in
within
concentration
30
in A 4 3 1
in 2 h o u r s .
OH N ~
R1
COOH OH
R1 lavendustin A
R2
-~
OH
OH
B
H OH
C6 Fig.
5.
2.2
n-C6H13
OH
Lavendustins. Lavendustin
We
screened
tyrosine
kinase
utilizing
inhibitors
an
A431
RRLIEDAEYAARG.
As
structure
from
lavendustin The
MeOH; active of
to
the
fractions
Sephadex
of t h e
twice
active
by
HPLC
collected The
mixture
gel
were
15%
extracted
was
vacuo
liters) pH
give
an
oily
The then
was
eluted
the
residue
MeCN/H20.
with to
two
butyl
more
potent
assay
system
tridecapeptide
compounds
(8) .
We
of
novel
named
them
5) .
the
were
the
lavendustrin
(20
for
kinase
and
strain (Fig.
of
to give
applied
tyrosine
isolated
concentrated
which
with
in
for
column.
compounds
fractions,
and
concentrated
to
vacuo
LH-20,
B
filtrate
a silica active
we
Streptomyces
procedure
filtrates
fraction
result,
adjustment in
a new
lavendustin
broth
after
concentrated applied
a
isolation
acetate
in
membrane
a
A and
Fermentation
culture
Streptomyces
A is s h o w n
was
to
extracted
2.4.
residue
column
(2.1 g) with (1.03 The
a preparative
washed
with and
charged After
g)
with g)
was
that
with
CHCl 3-
on a c o l u m n
and
the
purified
fractions
lavendustins gel
The
evaporation
further
silica
butyl was
was
active
6.
extract
CHCI3-MeOH.
MeOH.
acetate, of
(7.05
was
eluted
a mixture
The
in Fig.
were
extract
was
(105.2
mg) .
TLC
plate
to
445
obtain
lavendustin
compounds
were
insoluble that
in
the
A
(37.0
soluble
CHCI3,
H20,
producing
griseolavendus
in
mg)
MeOH,
and
lavendustin
and
Et20.
Taxonomic
strain
EtOH,
was
B
Me2CO,
related
(29.7
to
Culture broth (20 I)
I
Filtrate
I
BuOAc extraction (pH 2.4)
I
Silica gel chromatography
I
Sephadex LH-20 chromatography
I Reversed-phase HPLC (Nucleosil 5C18)
I Preparative TLC (CHCI 3:MeOH=3:I)
I
I
Sephadex LH-20 chromatography
I
I
kavendustin A
kavendustin B
(42.3 mg)
(29.7 rag)
Fig.
6.
[
Isolation
NH2
of l a v e n d u s t i n
1)
OH OH
~CO2H OH 5-amino-2-hydroxy benzoic acid 1)
2) NaCNBH 3/MeOH
OH
OH
I ~ 'CliO
Total
synthesis
CH2" NH [~002 OH OH
O.cH - 2.0 OH
7.
[~ OH
2) NaCNBH3/MeOH
Fig.
A and B.
OH
I~CHO
of
~CO2 H OH lavendustin A
lavendustin
A.
mg) .
DMSO,
features
.
I
and
Strept
Both but
indicated omyces
446
TABLE 2 Inhibition of kinases by lavendustins A and B. Kinase Tyrosine
Sample Lavendustin A Lavendustin B
0.0044 0.49
Erbstatin Staurosporine
0.20 0.35
IC5o (pg/ml) C A >lo0
100
-
-
100 0.0011
>179 <0.01
PIa 6.4 25.0 26.0
~~
a Phosphatidylinositol Lavendustin A inhibited EGF receptor tyrosine kinase with an IC50 of 4.4 ng/ml, which was about 50 times greater than that of erbstatin.
It did not inhibit protein kinase C or A but weakly
inhibited phosphatidylinositol kinase, as shown in Table 2. Lavendustin B showed much weaker activity than lavendustin A. The EGF receptor-associated tyrosine kinase was reported to act in the sequential ordered bi bi mechanism, in which the peptide comes first and ATP second to the active site of the enzyme. Kinetic studies by Lineweaver-Burk plotting indicated that lavendustin A inhibits the tyrosine kinase competitively with ATP (8). Thus, the mechanism of inhibition is different from that of erbstatin (9), but similar to that of orobol and genistein.
The total synthesis (8) of lavendustin A was accomplished by use of successive reductive alkylation of aminohydroxybenzoic acids and hydroxybenzaldehydes, as shown in Fig. 7. The synthetic lavendustin A gave exactly the same results as the natural product in terms of Rf value, spectral data, and inhibition of tyrosine kinase. Although lavendustin A is a potent inhibitor of tyrosine kinase in v i t r o , it did not have any biological effect on cultured cells.
Assuming that the molecule is so polar that it cannot pass
through the plasma membrane, we therefore prepared lavendustin A methyl ester. effect
on
The methyl ester derivative had a weaker inhibitory tyrosine
kinase,
but
inhibited
EGF
receptor
autophosphorylation and internalization in cultured A431 cells (10). Recently, we prepared lavendustin C6 (Fig. 5) as a new permeative analogue of lavendustin A . It inhibited PDGF and
447
bombesin-induced
phospholipase
differentiation Erbstatin cells.
The
in
them
induces
kidney
within
4
rat
organized, poor. the
kidney but
in
greatly
MeC,
by
actin
4
irreversible
or
in
continuation
the
respectively, Shibuya cells. actin
cells
or
of
with DNA
(14) .
also at
EGF-induced
2,5-
cells.
also
Thus,
rat
kidney
induced
normal
expression
in
synthesis
in
temperature.
erbstatin
DNA
cell
culture
to by
the Src
delayed
at
was
observed.
peak
without
was p u l s e - a d d e d ,
phase
and
appears
in
to
mid-S
be
the
permissive
the
respectively,
erbstatin
can
inhibited normal 20 for
showing
inhibited
analogue,
such
shifted
activity
prepared EGF
in
blotting
of
DNA
showing it was phase.
essential
for
synthesis.
a peak
al.
stable
src-expressing
synthesis
When
synthesis
without et
its
Western
and f i b r o n e c t i n
induction
In q u i e s c e n t
fiber organization,
inhibited
DNA
kinase
of D N A
(5) .
2,5-MeC
is
2,5-MeC
16 hours,
and 2,5-MeC
synthesis
delayed
of
the
tyrosine
in NRK cells
expression
cells.
quiescent was
cytotoxicity.
Erbstatin
in
product-induced
a
8 ~g/ml
8 hours
both
Especially,
src
When
induction
for
is
organization
at the p e r m i s s i v e
inhibited
In
extensively
organization
in
fiber organization,
cells
are
Fibronectin
kinase.
induce
fiber
expression
tyrosine
not
cells.
fiber
and
phenotypes
temperature
of
effective
(ERI2)
normal
cells.
synthesis
erbstatin
fibronectin
nonpermissive
DNA
both
did
fibers
the
the
transformed
that
it
at
morphology
actin
8 hours.
Erbstatin
Addition
cells
normal
in
v-abltS-NIH3T3
temperature,
stress
in
inhibiting
Rsvts-NRK
about
small
normal
ras-transformed
cells
induces
morphology,
actin
virus-infected
and
flat
however,
or
src-expressing brought
increased
cells
(13),
the
thin
induced
normal
Inhibitors
sarcoma
are
suppressed
showed
erbstatin
hours in
cells
src-expressing
analysis
8
changes
Erbstatin
often
the
to
Erbstatin
induced
in src o n c o g e n e - e x p r e s s i n g
Rous
cells
and
(12).
Kinase
phenotypes
(RSVtS-NRK)
(ii)
cells
of T y r o s i n e
normal
temperature.
morphological normal
Activity
temperature-sensitive
rat
permissive
activity
in rat p h e o c h r o m o c y t o m a
2.3 B i o l o g i c a l
normal
C
EGF-induced
rat k i d n e y
hours.
Erbstatin
about
3
irreversible EGF
induce
hours
EGF and
and
8
transformed
phenotypes
transformation
and f i b r o n e c t i n
induces 2,5-MeC hours,
toxicity.
receptor-overexpressing
EGF-induced
DNA synthesis
DNA synthesis
cells
expression
in q u i e s c e n t
ERI2
NIH3T3 in ERI2
of morphology, (15) . cells.
It also Pulse
448 addition the
of 2 , 5 - M e C
G0-GI
indicated
Phospholipase Recently, factor
C
whether
the
So we
(16) .
stimulates
EGF
human
carcinoma
block
the
looked
phospholipase
C.
in the
The
Erbstatin
and
of
oligopeptide
having
development The
used
cell
cells,
tyrosine
Since
into
was
phosphorylation tried
to
erbstatin
did not
erbstatin
itself
lavendustin formation inactive induced
kinase
C6 in
the
found with
of
to
EGF,
Thus,
involved
be and the
in
the
inhibited
we
found
the
as to
that
in
to
erbstatin of PLC
G
and
about
is i m p l i c a t e d
receptor.
for
the rat
mechanistic
cells
(NGF)
they
having
NGF
by
easily
neurites.
intracellular
high-affinity
study
pheochromocytom~
factor
neuron-like increase
an
autocrine
linked
kinase
are
growth
differentiation
an
both
is
involved
be
activation
tyrosine
bombesin
of n e r v e to
It may be
carcinoma
cells
PDGF-induced
Bombesin
tyrosine
receptor
c-TrK,
erbstatin.
we
However,
inhibit
the d i f f e r e n t i a t i o n ;
and on the
contrary,
induced
neurite
Erbstatin,
2,5-MeC,
transient
neurite
and
PCI2h
analogue
was
is
considered
inhibitors PC12
sympathetic
through
inhibit
erbstatin
activation
(ii) .
lung is
of the
reported
block
C
also
Therefore,
differentiation.
NGF
d i d not
erbstatin.
bombesin-induced
and in the p r e s e n c e
blocked
treatment
functions.
surprisingly, (ii) .
in c u l t u r e d d i d not
activity by
enzyme
erbstatin
Therefore,
by
cells
cell
transduction
differentiate
C6
receptor
inhibited
the
not
analogue,
kinase-induced C
growth was
completely
erbstatin
phospholipase
factor
small
cells
signal
We of
of
hand,
by it
activity.
NIH3T3
growth
bombesin
C6
in N I H 3 T 3
in the
in
However,
lavendustin
of
was
inactive
cells
lavendustin
PLC
an
inhibited
enzymic
activation
70%
in
turnover.
phosphates
formation.
of A431
phosphorylation of t h i s
other
also
PI
activates
inositol
phospholipase
was
in
role by u s i n g
stimulation
tyrosine
cytoplasm
enhancement
proteins.
of
phosphate
inhibited
factor.
is e s s e n t i a l
However,
functional
The
On the
inositol
regulation
kinase.
phosphorylation
into the
specifically
the
kinase
involved
tyrosine
cells.
formation.
tyrosine
enzyme
5'-O-methylerbstatin,
ATP-induced
enhanced
tyrosine
f o u n d to be p h o s p h o r y l a t e d
formation
A431
erbstatin.
this
an
C was
tyrosine
activity.
by
is
phospholipase
receptor-associated
known
that
phase.
herbimycin (a
of
subline erbstatin
acetylcholinesterase
formation. all of
induced
PC12)
did
in P C I 2 h
not
cells do
cells
so. like
(12) ,
whereas
Erbstatin NGF
(12) .
an also
449 Human
chronic
activated Ethyl
abl
myelogenous
oncogene,
leukaemia
the p r o d u c t
2,5-dihydroxycinnamate erythroid
differentiation
tyrosine
kinase.
Interestingly,
only
of K562
cells,
Erbstatin transplanted have
EGF
mice ion an
receptors.
when
that
inhibited
foroxymithine
antitumour
along
R/~S F U N C T I O N
cells
by
induced
with
on
breast
inhibited
human
the
erbstatin
effect
Human
the
oesophageal
growth
of
K562
growth
cells.
carcinomas MCF-7 of
which
nude
cells
MCF-7
in
is a ferric
Erbstatin
in
of
differentiation
carcinoma
MCF-7
unpublished
kinase.
inhibition
human
in serum.
an
analogue,
carcinoma
foroxymithine,
erbstatin
on
(M. Toi,
K562
have
is a t y r o s i n e
an
2,5-EtC
(17).
protects
effect
in
cells
of a d r i a m y c i n - r e s i s t a n t
Erbstatin
administered
antitumour
3.
an
in nude mice
chelator
2, 5 - M e C
and not
showed
of w h i c h
(2,5-EtC) ,
induced
K562
also
EH-4
mice
had
(17) .
without
result) .
INHIBITORS
3.10xanosine Oxanosine
Streptomyces weak
(18) .
guanosine.
was
isolated
activity
Oxanosine
also
that has
normal ~g/ml
was
morphology
infected
normal cell
K-rasts-NRK oxanosine
antibiotic analogue
antagonized
a weak
alter
by
antitumour
for 2 days blotting
mRNA. 48
into
the
from
and
shows
guanine
effect
on
In W e s t e r n
at
the
hours
permissive
at the n o n p e r m i s s i v e
of o x a n o s i n e .
in K - r a s - N R K For
the
In the
to XMP by
and LI210
Oxanosine
or K - r a s - N I H 3 T 3
oxanosine
competitively
did not
cells
having
expression
of ras
oncogene
metabolic
pathway
of GMP
Oxanosine
5'-monophosphate with
the
does
with
wild
ras
proteins. must
IMP
is
is c o n v e r t e d inhibit IMP
IMP it
bind
to
converted
to GMP by GMP dehydrogenase,
dehydrogenase
Therefore,
in the
phenotypes
synthesis,
not
of
content
change
normal
of
content
fibronectin
did not
induce
level
2 ~g/ml
fibronectin
Ras
inhibits
substrate.
the
incubation
function,
and the XMP
itself
analysis,
The
of 2
changed
morphology.
increased
temperature
cells.
the
virus-
Addition
temperature
cell
temperature
into
sarcoma
(19).
cellular
of n o r m a l
IMP d e h y d r o g e n a s e ,
synthetase.
Kirsten
oxanosine
blotting
increased
level
morphology
cells
normal
analysis,
to the
presence
cell
at the p e r m i s s i v e
in the
cells
tumour
(K-rastS-NRK)
dramatically
but
an
nucleoside
is
in t e m p e r a t u r e - s e n s i t i v e
cells for
to
morphology
In N o r t h e r n fibronectin
found
rat k i d n e y
of o x a n o s i n e
tumour
GTP.
as
leukemia. Oxanosine
of
8)
It is an u n u s u a l
antibacterial
mouse
the
(Fig.
is
almost
likely
that
450 oxanosine
is p h o s p h o r y l a t e d
dehydrogenase of GTP, level
to reduce
GDP, of
and
GMP
is
not
ATP
dehydrogenase
NRK
cells
in the
synthesis
are
inhibitor,
also
(19).
and
of GTP.
actually
affected
cells
The
lowered
(19) . induces
thereby
intracellular
by
oxanosine,
Mycophenolic normal
inhibits
levels
while
acid,
morphology
IMP
an
the IMP
in K-ras ts-
O
H2N N HOH2C~ HO OH
Fig.
8.
3.2
Oxanosine.
Compactin
Post-translational removal
of
the
esterification addition
of
of
is
to
the is
is
cell
catalyzed
which
by
with
the
linked
(Fig.
i0)
ras
be
oncogene.
N-ras
to
(21) .
mevalonate.
Addition
matured
in
Ras
lovastatin, in
the
the
a large
cytosol
from
(20) .
of
simvastatin
differentiate
of
amount
fraction,
Induction in was
these
no
Ras Ras
by
reduce as
an
such
as
clinically
induced In
precursor was
found
transfection
MMTV
of
cells
antagonized
fraction.
of the
will
are
transfected
dexamethazone
but
by
mevalonate
body. al.
membrane
the
analogues
et
inhibition
pyrophosphate,
a microorganism
Mendola cells.
of
of Ras
synthesized
enzyme
Compactin
and
Especially,
is
to
dexamethazone The
this
and
translocation
farnesyl
induced
PC12
by
of
methyl
acid,
Farnesylation
enzyme
isolated
in the
9.
for
Mevalonate
pravastatin
cholesterol
differentiation lovastatin
was
reductase
can
with
acids,
amino
Fig.
functions.
rate-limiting
i0),
cells
Ras
in
farnesylation,
amino
essential
inhibition
(Fig.
reduce
PC12
be
mevalonate.
a
includes
terminal
shown to
transferase
from
of H M G - C o A
lovastatin
as
farnesyl
of Ras.
Compactin inhibitor
carboxyl
and
Therefore,
farnesylation
to
new
membrane
reductase,
pathway.
used
the
of Ras
carboxyl-terminal
considered
derived
HMG-CoA
3
palmitate,
farnesylation Ras
modification
first
promoter-
morphological was
blocked
by
by
addition
of
accumulation the
presence
protein in
the
was
of of
found
membrane
451 fraction. This
induction
was
also
We s h o w e d that NRK cells induced
normal
and
Addition
actin
filament
showed
that
fibronectin
expression
in
both
K-rasts-NRK
of
compactin
Compactin
inhibitors
and were
that
of
of m e v a l o n a t e
S~ ,SH
synthesis
, SH
,~ S I
N
I N
SH
S I
S I
S I
- -
PLASMA
~
3"-.
MEMBRANE
"~
~'
9 '~
OUT
Fig.
9.
Post-translational 0
0
Fig.
.OH 0
compactin
R=H
Iovastatin
R=CH 3
i0.
modification
HMGCoA
reductase
inhibitors.
increased cells.
blocked
suppress
of Ras.
in
by
normal Ras
K-
blotting
of r a s - e x p r e s s i n g
~N
~ e
was
counterpart
culture.
C~-C-AAX ~
Western
K-ras-NIH3T3
their
for 3 days
organization
completely
i n h i b i t e d the g r o w t h
than
H-Ras.
in K-ras ts-
of c o m p a c t i n
temperature.
effects
by
(22) .
normal p h e n o t y p e s
permissive
effectively
Thus, cell
morphology
cells.
induced
compactin
at the
mevalonate. more
i n h i b i t e d by
cells
compactin cellular
oocyte can be
compact in induces
and K - r a s - N I H 3 T 3
rastS-NRK analysis
of Xenopus
Maturation
by
These i00
~M
cells cells.
functions
in
452
4.
PI TURNOVER INHIBITORS 4.1 Psi-tectoriaenin Many oncogenes such as ras, src, sis, fms, and fes enhance
cellular PI turnover.
Phosphatidyl breakdown by phospholipase C
generates two second messengers,
diacylglycerol and inositol
trisphosphate. The formed directly activates protein kinase C, and the latter binds to the receptor on endoplasmic reticulum to mobilize calcium ions. Therefore, we screened culture filtrates of microorganisms for inhibitors of PI turnover and thus isolated psitectorigenin (Fig. 11, ref. 23). Phosphatidylinositol turnover was assayed by incorporation of labeled inositol into phospholipids. A431 cells were preincubated in HEPES-buffered saline containing [3H]inositol at 37 " C for 30 min.
Then, test chemical and EGF were added, and the incubation
was continued for containing 0.01 M insoluble fraction was extracted from
60 min. Subsequently, 10% trichloroacetic acid sodium pyrophosphate was added, and the acidwas scraped off from the dish in H20. The lipid it by the addition of CHCl3 and CH30H (l:l), and
[3H]inositol-labeled lipids were counted by liquid scintillation spectrophotometry. A culture filtrate from a Nocardiopsis strain found in a river near Shanghai showed strong inhibition against phosphatidylinositol turnover. The active principle was purified (Fig. 12), and its structure was found to be identical with that of psi-tectorigenin by nmr. Psi-tectorigenin inhibited EGF-induced inositol incorporation with an IC50 of about 1 yg/ml. The inhibitory activity of psi-tectorigenin was several times stronger than that of orobol or genistein, related isoflavonoids that inhibit tyrosine kinase. Psi-tectorigenin, orobol, and genistein all showed similar
in v i t r o inhibitory activity against EGF receptor tyrosine kinase, with an IC50 of about 0.1 yg/ml. However, only psi-tectorigenin did not inhibit EGF receptor tyrosine kinase at 50 pg/ml in s i t u (24). Phospholipase C is a rate-limiting enzyme of phosphatidylinositol turnover.
Psi-tectorigenin does not inhibit phospholipase
C, but we recently found that it inhibits activation of the enzyme by EGF (24). The phospholipase C activity in a homogenate prepared from EGF-treated A431 cells was 4-fold higher than that from untreated cells, and EGF failed to activate the enzyme in the presence of psi-tectorigenin.
453 OMe
OH
Fig.
11.
psi-tectorigenin.
Culture broth (3.5 I)
Filtrate BuOAc extraction Silica gel chromatography
I
Toyopearl HW-40 chromatography Reversed-phase HPLC (Nucleosil
Psi-tectorigenin (6.0 mg) Fig.
12.
I s o l a t i o n of p s i - t e c t o r i g e n i n
4 . 2 Inostamycin
By c o n t i n u a t i o n o f t h e s c r e e n i n g p r o c e d u r e w e f o u n d a n o v e l inhibitor
of
inostamycin
phosphatidylinositol
(Fig. 13, r e f . 25)
turnover,
and
named
For i s o l a t i o n of inostamycin
( F i g . 14), t h e c u l t u r e b r o t h
l i t e r s ) was c e n t r i f u g e d a t 5 , 0 0 0 rpm f o r 1 0 min, were e x t r a c t e d w i t h 1 . 0 l i t e r o f a c e t o n e .
(5
and t h e mycelia
The a c e t o n e e x t r a c t was
i n v a c u o and combined w i t h t h e s u p e r n a t a n t ,
concentrated
it
.
m i x t u r e was t h e n e x t r a c t e d w i t h 3 . 5 l i t e r s o f E t O A c .
which
This e x t r a c t
w a s c o n c e n t r a t e d i n vacuo, a p p l i e d o n t o a s i l i c a g e l column ( 5 0 g ) , and
eluted
with
CHC13-MeOH
(1OO:l).
p r e c i p i t a t e d with a c e t o n i t r i l e ,
The
active
f r a c t i o n was
crystallized
were
washed
f r o m hexane/CH2C12
with
was
a n d t h e p r e c i p i t a t e was f u r t h e r
p u r i f i e d by c e n t r i f u g a t i o n p a r t i t i o n c h r o m a t o g r a p h y . crystals
fraction
1N H C 1 ,
followed
The a c t i v e
solution,
by
and t h e
1N N a O H
and
454
concentrated
component
mg
of
NaCl
was
to obtain
from the
inostamycin.
Inostamycin
and insoluble
in hexane
Me2CO,
inostamycin confirm
was
determined
the p r o p o s e d
we c o n d u c t e d
..A~,
HOOCh, O . OH Et
13.
,
Me
by
structure
Me 9 Et ~ I ,
Me
the
was
IH-
sodium
same
and
in MeOH,
13C-nmr
and e l u c i d a t e
The
to give
The tentative
analysis.
Me
salt.
solution
soluble
and H20.
crystallographic
OH Et
Me~
Fig.
solution
recrystallized
active
CHCI3,
112.4
structure
spectroscopy.
its
and of To
stereochemistry,
, Me
H
OH
O
Et.
Inostamycin
(relative
OH
structure) .
Culture broth (5 I)
I
I
I
Supernatant
Mycelia
I
Acetone extraction
I
I EtOAc extraction
I
Silica gel chromatography
I Toyopearl HW-40 chromatography
I Precipitation with acetonitrile
I
Crystallization with hexane-dichloromethane
I
Inostamycin (112.4 mg) Fig.
14.
Isolation
Thus, to
the
14873A,
of inostamycin.
inostamycin
polyether and
was
antibiotic
related
shown to be a novel group,
compounds
from
which
polyether
includes
Streptomyces.
belonging
lysocellin,
X-
Inostamycin
455
inhibited EGF-induced inositol incorporation into inositol lipids with an Ic50 of about 0.5 yg/ml in the A431 cell assay system. Lysocellin, which has a closely related structure, also inhibited phosphatidylinositol turnover; however, monensin, a polyether antibiotic having a considerably different structure, did not inhibit phosphatidylinositol turnover. Inostamycin did not inhibit EGF receptor tyrosine kinase, phospholipase C, or phosphatidylinositol kinase, but inhibited CDPDG:inositol transferase (26). The drug inhibited cellular inositol phosphate formation only when it was added at the same time as labeled inositol. It was found to inhibit i n vitro CDP-DG: inositol transferase activity of the A431 cell membrane, the IC50 being about 0.02 yLg/ml.
Therefore, inhibition of PI turnover must
be due to the inhibition of CDP-DG:inositol transferase. Thus, inostamycin is a novel inhibitor of CDP-DG:inositol transferase.
\\
teleocidin 6
pendolmycin
Fig. 15.
k
lyngbiatoxin
Indole alkaloid tumour promoters.
456
Culture broth (36 I)
I
I
I
Filtrate
Mycelium
I
Acetone extraction
I
I EtOAc extraction
I
Silica gel chromatography
I
Toyopearl HW-40 chromatography
I
Reversed-phase HPLC
I
Pendolmycin (8.8 mg) Fig.
16.
4.3
Isolation
of pendolmycin.
Pendolmvcin _
In novel
the
same
indole
Pendolmycin produces
screening
alkaloid, was
procedure,
was
isolated
(Fig.
psi-tectorigenin. with
an equal
volume
extracted
with
acetone.
The
in H20,
and
concentrated
silica
MeOH mg),
gel
in
column,
(i00:i) . which
column was
and e x t r a c t e d
was
yielding
of
15).
tumour
Like
PI
of
was
give
applied
HPLC
strain
with
was
were
eluted
to
that was
cake
dried,
a
(27) .
filtrate
was
dissolved
combined
applied
a yellow
(40.8 mg) .
pendolmycin
is
promoters an
EGF
phosphorylation
and
material
was
extracts
material
to
same
liters)
and the m y c e l i a l
fraction
dried
tumour-promoting
and
suppresses
the
(36
extract
dried
15),
with
to
a
CHCl 3-
powder
(81.6
a Toyopearl
HW-40
Further
purification
50% M e C N / H 2 0
as eluent,
of p e n d o l m y c i n .
structures
induced
was
in M e O H
purified
structure
receptor,
active
(Fig.
Nocardiopsis
The EtOAc
out by r e v e r s e d - p h a s e
8.8 mg
The
eluate
dissolved
to obtain
carried
the
from
broth
acetone The
vacuo.
from
of EtOAc,
with EtOAc.
and
The
16)
The
extracted
pendolmycin
obtained
of
EGF
turnover
of
A431
in
to
lyngbiatoxin
kinase
(28) .
related
to the
kinase
resulting cells
and
binds
protein
tyrosine
receptor, in
closely
teleocidin
pendolmycin
activator
receptor
is
phorbol
C. by
(Fig. ester
Pendolmycin stimulating
inhibition Thus,
the
of
EGF-
actually,
457
pendolmycin is not an inhibitor of PI turnover, but an activator of protein kinase C. 4.4 Piericidins Piericidins are insecticidal compounds isolated from mycelia of Streptomyces mobaraensis and Streptomyces pactum. They are toxic to several species of insects, aphids, and mites. Piericidin A is known to block electron transport between NADH dehydrogenase and coenzyme Q. In the course of our screening for phosphatidylinositol turnover inhibitors, we isolated the novel antibiotics piericidin B1 N-oxide (29), as well as B5 and B5 N-oxide ( 3 0 ) , frcm Streptomyces (Fig. 17). The fermentation broth (12 liters) was filtered, and the mycelia were extracted with acetone (Fig. 18). After removal of
the acetone, the extract was combined with the filtrate; and the mixture was next extracted with EtOAc. The EtOAc extract was concentrated in v a c u o to give an oily material, which was mixed with silica gel and applied to a silica gel column. The column gave two active fractions, Fractions 1 and 2.
Fraction 1 was
purified through a silica gel column, Sephadex LH20 column and subjected to centrifugal partition chromatography to give 6.3 mg of piericidin Bg. Fraction 2 was purified through silica gel column, Sephadex LH20 column, centrifugal partition chromatography and HPLC to give 136.8 mg of piericidin B1 N-oxide and 39.9 mg of piericidin
Bg N-oxide. OH
CH,
CH,
A
piericidin B,
B1 N-oxide
Me
0
N B, N-oxide N+ 0 B5
Fig. 11.
CH,
R
N
N+
CH,
Me Et Et
New piericidins from Streptomyces.
known compound.
Piericidin B1 is a
458
Whole broth (12 I)
I
Mycelia
FiItrate
I
I
Acetone extraction
EtOAc extraction
Silica gel column
Fr. 1
Fr. 2
Silica gel column
Silica gel column
I
I
Sephadex LH-20
Sephadex LH-20
I
I
CPC
CPC
I Preparative HPLC
Piericidin 6 (6.3 mg) Fig. 18.
5
Piericidin 6 N-oxide (136.8 mg)
Piericidin B N-oxide (39.9 mg)
Isolation of piericidin B5, B1 N-oxide, and B5 N-oxide.
Piericidin B1 N-oxide showed antibacterial activity against Gram-positive and Gram-negative bacteria and fungi, while piericidin B l , a known piericidin, did not. Piericidin B1 N-oxide inhibited the phosphatidylinositol turnover with an I C 5 0 of 1.5 pg/ml, while piericidin B1 showed weaker activity ( I c 5 0 , 4.0 pg/ml) . Piericidin B 5 and B5 N-oxide inhibited EGF-stimulated [3Hlinositol incorporation into phospholipids with I C 5 o s of 10.0 pg/ml and 1.1 pg/ml, respectively, in the A431 cell assay system. Piericidin B 5 N-oxide showed antimicrobial activity against Grampositive and some Gram-negative bacteria and fungi, although piericidin B 5 did not. Both in B 1 and B5, N-oxidation increased the inhibitory effect on PI turnover and antibacterial activity. 4.5 Echisuanine Phosphatidylinositol kinase is involved in the PI turnover pathway
and
may
phosphatidylinositol
be
important
for
the
4,5-bisphosphate levels.
of Therefore, we
regulation
459 screened and
microbial
isolated
We
also
culture
filtrates
2,3-dihydroxybenzoic
isolated
novel
for
acid
and p o t e n t
inhibitors
33) .
OH H2N,~N~~
N4
H
U
CO~NH ~CH2~CH2~R
echiguanine A:
B:
Fig.
19.
R=C-NH2 II NH R=CH2-NH2
Echiguanines.
Broth filtrate (27 I)
I
Diaion HP-20 column
i
Reversed-phase silica gel column
I CM-Sephadex (Na § type) column
I
Diaion CHP-20 column
I
Neutralization by Amberlite IR-45 (OH-type)
I
Mixture of echiguanines A and B (73 mg)
I
Centrifugal partition chromatography
I
I
Echiguanine A (28.6 mg) Fig.
20.
Isolation
I Echiguanine B (6.7 mg) of e c h i g u a n i n e s .
kinase,
( 3 1 ) and t o y o c a m y c i n
inhibitors
of PI
kinase
A
B
Streptomyces s t r a i n and n a m e d t h e m e c h i g u a n i n e
ref.
of PI
and
(32). from
(Fig.
a
19,
460
For the assay of PI kinase, A431 cell membrane and [y-32P]ATP were mixed in 20 mM HEPES buffer (pH 7.2).
The reaction mixture
was incubated for 20 minutes at 20’C and stopped by the addition of CHC13, MeOH, and 1N HC1 (4:1:2). After vigorous vortexing, the lower phase was applied to a silica gel column for the separation of phospholipid and unreacted [y-32P]ATP (31). The phosphorylated lipid was eluted with CHC13, MeOH, and 4N NH40H (9:7:2); and the radioactivity was quantified by liquid scintillation counting. For the isolation of echiguanines A and B (Fig. 20), the broth filtrate (27 liters) was adsorbed on Diaion HP-20 resin; then the resin was washed with distilled water, and the active components were eluted with 50% aqueous methanol. The eluate was concentrated
in v a c u o ,
and the concentrate was passed through a column of
reversed-phase silica gel. The column was successively washed with distilled water and aqueous methanol. After evaporation to dryness, the residue was dissolved in distilled water and applied to a CM-Sephadex column; and elution was conducted with a gradient of 0.4 M-1.0 M NaC1. The combined eluates were subjected to a column of Diaion CHP-20 and eluted with aqueous methanol. After neutralization of the eluate with Amberlite IR-45 (OH-) anionexchange resin, the active fraction was concentrated in V ~ C U Oto give a mixture of echiguanines A and B (73 mg).
The mixture was
further partitioned in the solvent system EtOAc-BuOH-water (4:7:10) by centrifugal partition chromatography to give purified echiguanine A (28.6 mg) and B (6.7 mg) . Echiquanines A and B inhibited PI kinase of the A431 cell membrane with IC5o’s of 0.04 pg/ml and 0.11 pg/ml, respectively. Structurally related compounds such as 7-deazaguanine showed only weak activity (IC50, 35 pg/ml) ; and adenine, hypoxanthine, guanine and isoguanine did not inhibit the enzyme. Echiguanines A and B showed no antimicrobial activity. The LD50’s of echiguanine A and
B, when administered intravenously to mice, were >lo0 mg/kg. Echiguanine A is the most potent phosphatidylinositol kinase inhibitor discovered. It is stronger than toyocamycin (IC50, 3.3 pg/ml) , 2,3-dihydroxybenzaldehyde (IC50, 0.45 pg/ml) , quercetin (IC50, 1.8 pg/ml), or orobol (IC50, 0.25 pg/ml). Thus, we have isolated PI turnover inhibitors acting at various points. The diagram of PI turnover pathway and inhibition of each step by microbial secondary metabolites are shown in Fig. 21.
46 1
echiguanine, to,yocarnyci n
V
\\
ATP
PI
\ ADP
ATP
ADP
f*PIP-
"-pendolrnycin
PIP2
pki- t e c torigenin I
CDP-DG<-PA
CTP
DG ADP
I p3
ASP
V
V
Protein kinase C
IP3 recep'tor
'I Ca++release Fig. 21.
PI turnover pathway and inhibitors.
4.6 Bioloqical Activitv of PI Turnover Inhibitors
Inostamycin did not show antitumour activity on L1210 mouse leukemia in v i v o , but inhibited the growth of Ehrlich carcinoma in mice (our unpublished result) . The relation between antitumour activity and inhibition of PI turnover is being studied. In the presence of Ca2+, EGF induces elongation of A431 cells in 30 min. The cell elongation was shown to be accompanied by a reorganization of actin filaments.
These phenotypical changes were specifically inhibited by a tyrosine kinase inhibitor, erbstatin, and inhibitors of PI turnover such as psi-tectorigenin and inostamycin (34). The amount of filamentous actin was increased by EGF, which was also inhibited by these compounds. Long-term treatment of A431 cells with EGF induced the disappearance of cytoskeleton and aggregation of the cells, which was again inhibited by the P I turnover inhibitors. On the other hand, in Ca2+-free medium, A431 cells exhibit rapid rounding in response to EGF.
Psi-tectorigenin and
inostamycin again inhibited the EGF-induced rapid rounding of A431 cells.
Thus PI turnover inhibitors were shown to inhibit the
signaling pathways of EGF-induced cytoskeletal organization of A431 cells. Inostamycin is an inhibitor of CDP-DG:inositol transferase, the enzyme catalyzing the synthesis of PI.
Inostamycin was shown
462
to inhibit serum-induced DNA synthesis in quiescent NRK cells. Therefore, PI synthesis may be one of the signals for S-phase induction. Unexpectedly, inostamycin suppressed multidrug resistance induced by P-glycoprotein in human carcinoma KB cells (36). Unlike verapamil the effect of inostamycin is long-lasting (37). But suppression of multidrug resistance by inostamycin is not due to the inhibition of PI turnover. Pendolmycin was shown to have tumour-promoter activity in cell culture (38) and in mice (39). REFERENCES 1
2 3 4 5 6 7 8 9 10 11 12 13 14
15 16 17 18 19 20 21
H. Umezawa, M. Imoto, T. Sawa, K. Isshiki, N. Matsuda, T. Uchida, H. Iinuma, M. Hamada and T. Takeuchi, J. Antibiot., 39 (1986) 170-173. M. Imoto, K. Umezawa, K. Isshiki, S. Kunimoto, T. Sawa, T. Takeuchi and H. Umezawa, J. Antibiot., 40 (1987) 1471-1473. K. Isshiki, M. Imoto, T. Takeuchi, H. Umezawa, T. Tsuchida, T. Yoshioka and K. Tatsuta, J. Antibiot., 40 (1987) 1207-1208. M. Imoto, K. Umezawa, K. Komuro, T. Sawa, T. Takeuchi and H. Umezawa, Jpn. J. Cancer Res. (Gann), 78 (1987) 329-332. K. Umezawa, T. Hori, H. Tajima, M. Imoto, K. Isshiki and T. Takeuchi, FEBS Lett. , 260 (1990) 198-200. T. Hori, T. Kondo, T. Tsuji, M. Imoto and K. Umezawa, J. Antibiot. , 45 (1992) 280-282. Y. Tabata, M. Imoto and K . Umezawa, J. Antibiot., 45 (1992) 1382-1384. T. Onoda, H. Iinuma, Y. Sasaki, M. Hamada, K. Isshiki, H. Naganawa, T. Takeuchi, K. Tatsuta and K. Umezawa, J. Nat. Prod., 52 (1989) 1252-1257. K. Umezawa and M. Imoto, Meth. Enzymol., 201 (1991) 379-385. T. Onoda, K. Isshiki, T. Takeuchi, K. Tatsuta and K. Umezawa, Drugs Exptl. Clin. Res., 16 (1990) 249-253. M. Imoto, I. Sujikai, H . Ui and K. Umezawa, Biochim. Biophys. Acta, 1166 (1993) 188-192. E. Ikoma, M. Naoi, T. Nagatsu and K. Umezawa, Biogenic Amines, 9 (1992) 153-162. K. Umezawa, K. Tanaka, T. Hori, S. Abe, R. Sekizawa and M. Imoto, FEBS Lett., 279 (1991) 132-136. H. Yamazaki, H. Ohba, N. Tamaoka and M. Shibuya, Jpn. J. Cancer Res., 81 (1990) 773-779. K. Umezawa, D. Sugata, K. Yamashita, N. Johtoh and M. Shibuya, FEBS Lett., 314 (1992) 289-292. M. Imoto, N. Shimura, H. Ui and K. Umezawa, Biochem. Biophys. Res. Commun., 173 (1990) 208-211. M. Toil H. Mukaida, T. Wada, N. Hirabayashi, T. Toge, T. Hori and K. Umezawa, Eur. J. Cancer, 26 (1990) 722-724. N. Shimada, N. Yagisawa, H. Naganawa, T. Takita, M. Hamada, T. Takeuchi and H. Umezawa, J. Antibiot., 34 (1981) 1216-1218. 0. Itoh, S. Kuroiwa, S. Atsumi, K . Umezawa, T. Takeuchi and M. Hori, Cancer Res., 49 (1989) 996-1000. A. Endo, In: Methods in Enzymology, Vol. 72, Academic Press, 1981, pp. 684-689. C. E. Mendola and J. M . Backer, Cell Growth & Differentiation, 1 (1990) 499-502.
463
T. S. Vincent, E. Wulfert and E. Merler, Biochem. Biophys. Res. Commun., 179 (1991) 1284-1289. 23 M. Imoto, T. Yamashita, T. Sawa, S. Kurasawa, H. Naganawa, T. Takeuchi, Z. Bao-quan and K. Umezawa, FEBS Lett., 230 (1988) 43-46. 24 M. Imoto, N. Shimura and K. Umezawa, J. Antibiot., 44 (1991) 915-917. 25 M. Imoto, K. Umezawa, Y. Takahashi, H. Naganawa, Y . Iitaka, H. Nakamura, Y. Koizumi, Y. Sasaki, M. Hamada, T. Sawa and T. Takeuchi, J. Nat. Prod., 53 (1990) 825-829. 26 M. Imoto, Y. Taniguchi and K. Umezawa, J. Biochem., 112 (1992) 299-302. 27 T. Yamashita, M. Imoto, K. Isshiki, T. Sawa, H. Naganawa, S. Kurasawa, B. Zhu and K. Umezawa, J. Nat. Prod., 51 (1988) 11841187. 28 B. Friedman, A. R. Frackelton, Jr., A. H. ROSS, J. M. Connors, H. Fujiki, T. Sugimura and M. R. Rosner, Proc. Natl. Acad. Sci., USA, 81 (1984) 3034-3038. 29 H. Nishioka, T. Sawa, K. Isshiki, Y. Takahashi, H. Naganawa, N. Matsuda, S. Hattori, M. Hamada, T. Takeuchi and K. Umezawa. J. Antibiot., 44 ( 991 i 1283- 1285'. 30 H. Nishioka, T. Sawa, Y. Takahashi, H. Naganawa, M. Hamada, T. Takeuchi and K . Umezawa, J. Antibiot., in press. 31 H. Nishioka, M Imoto, T. Sawa, M. Hamada, H. Naganawa, T. Takeuchi and K. Umezawa, J. Antibiot., 42 (1989) 823-825. 32 H. Nishioka, T . Sawa, M. Hamada, N. Shimura, M. Imoto and K. Umezawa, J. Ant biot., 43 (1990) 1586-1589. 33 H. Nishioka, T Sawa, H. Nakamura, H. Iinuma, D. Ikeda, R. Sawa, H. Naganawa, C. Hayashi, M. Hamada, T. Takeuchi, Y. Iitaka and K. Umezawa, J. Nat. Prod., 54 (1991) 1321-1325. 34 N. Johtoh and K. Umezawa, Drugs Exptl. Clin. Res., 18 (1992) 1I. 35 M. Imoto, N. Johtoh and K . Umezawa, J. Cell Pharmacol., 2 (1991) 49-53. 36 M. Kawada, M. Imoto and K. Umezawa, J. Cell Pharmacol., 2 (1991) 138-142. 37 M. Kawada and K. Umezawa, Jpn. J. Cancer Res., 82 (1991) 11601164. 38 K. Umezawa, M. Imoto, T. Yamashita, T. Sawa and T. Takeuchi, Jpn. J. Cancer Res., 80 (1989) 15-18. 39 S. Nishiwaki, H. FuJiki, S. Yoshizawa, M. Suganuma, S. FuruyaSuguri, S. Okabe, M. Nakayasu, K. Okabe, H. Muratake, M. Natsume, K. Umezawa, S. Sakai and T. Sugimura, Jpn. J. Cancer Res., 82 (1991) 779-783. 22
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Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
465
R e c e n t A d v a n c e s in the C h e m i s t r y of Gelsemium Alkaloids H. Takayama and Shin-ichiro Sakai
Introduction In the tribe Gelsemiae, which belongs to the family Loganiaceae, there are two genera: Mostuea, which consists of eight species, and Gelsemium, which comprises three species. The first Gelsemium species, Gelsemium elegans Benth. (Kou-Wen, or Hu-Man-Teng), which is distributed over southeastern Asia, has been used in traditional Chinese medicine, as well as a remedy for certain kinds of skin ulcers, and more recently has been used as an analgesic for the palliation of various acute cancer pains. The second species, Gelsemium sempervirens Ait. (yellow jasmine), is native to s o u t h e a s t e r n United S t a t e s and the h i g h l a n d s of central America. Although this plant causes death and abortion in livestock which feed upon its leaves, it has been used in the t r e a t m e n t of neuralgia, migraine, and spasmodic disorders such as a s t h m a and whooping cough. The third species, Gelsemium rankinii Small grows in the s o u t h e a s t e r n United States. All of the species are rich sources of indole alkaloids. Numerous chemical studies related to these structurally complex alkaloids have been conducted since the end of the last century. In 1988, an excellent chemical review on the Gelsemium alkaloids was published by Liu and Lu (1). However, recent intensive research led to significant progress in the field of Gelsemium alkaloid chemistry, which we will review in this chapter. In the first section, alkaloids isolated from the Gelsemium species up to now are presented in tabular form, and the structure elucidation of the r e p r e s e n t a t i v e alkaloids using m a i n l y spectroscopic m e t h o d s are described. The second p a r t of this c h a p t e r covers the biogenetic speculation of the structurally unique Gelsemium alkaloids as well as the biomimetic transformation of the known indole alkaloids leading to the various skeletal types of Gelsemium alkaloids. Concerning a number of studies directed toward total synthesis of the Gelsemium alkaloids, only literature citations are given.
466
I.
Isolation
and
Structure
I-1. Sarpagine-Type Indole Alkaloids A genuine indole nucleus is present only in the sarpagine group and sempervirine (1) among the many skeletal classes of the Gelsemium alkaloids. The sarpagine group found in the Gelsemium species consists of six indole alkaloids, 19(Z)-akuammidine (koumicine) (2), koumicine Noxide (3), koumidine (4), 16-epi-voacarpine (5), 19(Z)-anhydrovobasinediol (6), and Na-methoxy-19(Z)-anhydrovobasinediol (7) (Table 1). The structure elucidation of the indole alkaloid, the so-called akuammidine isolated from Gelsemium elegans, was published in 1982 (4, 5). The configuration of the ethylidene side chain was considered to be the 19(E) form as generally found in the monoterpenoid indole alkaloids (12). The so-called akuammidine from G. elegans showed the same mass spectral fragment pattern as authentic akuammidine (8) obtained from other plant sources (13); however, the 1H- and 13C-NMR spectra of the alkaloid were similar but not completely identical to those of the authentic akuammidine (8). The 1H- and 13C-NMR assignments were reinvestigated by the present authors using two-dimensional NMR techniques, such as 1H-1H COSY (correlation spectroscopy), phase sensitive HSQC (heteronuclear single q u a n t u m coherence) (14) and HMBC (heteronuclear multiple bond connectivity) (15) spectra. As a result, the assignments of C-2, C-13, C-20, C-9, and C-10 in the literature (7) should be revised as shown in Table 2. The 13C-NMR chemical shifts of C-15 [5.9 ppm lower field than 19(E) form] and C-21 [3.0 ppm higher field than 19(E) from] of the newly isolated akuammidine compared with those of authentic akuammidine (8) can be reasonably interpreted in terms of the ~/-gauche effect due to C-18 on the double bond having the (Z) configuration. A differential nuclear Overhauser effect (NOE) experiment also gave evidence of the configuration of the side chain. Irradiation of H-15 in 8 led to enhancement (12%) of H3-18, indicating that the methyl group on the double bond lies syn to H-15. On the other hand, 23% enhancement was observed between H-15 and H-19 in the 19(Z)-akuammidine (2) (Fig. 1). The structure of 2 including the 19(Z) configuration was finally confirmed by single crystal X-ray analysis. The CD spectra of both akuammidines exhibited very similar curves, and therefore 19(Z)-akuammidine (2) has the same absolute configuration as the common indole alkaloids. The ethylidene configuration of koumidine (4), which was first proposed as having a 19(E) form (4) as do conventional indole alkaloids, was also revised by the NMR techniques (6, 7, 10). Comparison of the 13C. NMR spectra of koumidine ( 4 ) a n d 19(E)-koumidine (9), which was
467
TABLE 1 Sempervirine and Sarpagine-type Alkaloids from G e l s e m i u m Species Alkaloid
Structure
Species
em ervirine 1
Ref.
Osem e ,e a nservirens
MeO2~~CH20H 19(Z)-Akuammidine 2 (Koumicine)
Koumicine N-oxide 3
G. elegans
Nb-oxide
4-8
G. elegans HOH2C
Koumidine 4
G. elegans G. sempervirens
~~ 16-epi-Voacarpine 5
19(Z)-Anhydrovobasinediol 6 Na-Methoxy- ] 9(Z)-anhydrovobasinediol 7
HOH2C
CO M,e G. elega ns
R=H R=0Me
4,6,7 10
6-8
G. elegans G. elegans
11
468
TABLE 2 13C NMR Assignments of the Alkaloids 2 , 8 , 4 , 9 , 6 , and 16 Carbon
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
co
OMe NMe
2a
8a
1 3 8 .7 51.7 59.3 25.0 1 0 5 .8 1 2 8 .0 118.6 1 1 9 .7 1 2 2 .0 1 1 2 .0 138.5 31.2 37.0 53.0 68.9 12.6 1 1 8 .3 138.9 53.9 174.6 51.5
139.3* 52.7 59.8 25.9 106.9 128.8 119.4 120.5 122.8 112.8 138.8 29.7 31.1 53.5 69.8 14.1 118.1 139.4" 56.9 175.6 52.4
___
4a
___
138.4 51.0 54.0 23.4 105.9 127.5 118.7 119.8 122.1 111.9 138.2 29.3 35.1 44.3 61.2 12.5 115.3 142.3 54.6
___
9a
6b
16b
138.2 51.3 53.8 23.4 106.2 127.5 118.7 119.8 122.2 11 2.0 138.0 27.8 27.5 43.7 61.1 12.9 115.0 140.7 57.0
131.9 67.6 60.5 18.0 110.9 128.3 118.2 119. 3 122.3 110.9 135.3 29.7 33.5 37.5 61.9 12.8 119.8 136.2 45.9
131.8 67.5 60.5 17.6 111.0 128.3 118.2 119.3 122.3 110.9 135.3 28.0 26.5 37.0 62.0 12.4 119.8 136.3 52.8
___
___ ___
___
___
___ ___
___
- - _
43.0
42.7
Chemical shift in ppm downficld from TMS. aRecordcd in CD30D; bRecordcd in CDC13. *Signals may be interchanged.
HO*-
OH
H H..+'
H H..**' $5
2
NOE
Me
$5
H
8
23%
Fig. 1
H
12%L M e 18
469
prepared from gardnerine (10) by removal of the aromatic methoxy group in five steps (Section II-1), afforded the same conclusion as in the case of the akuammidines described above. Thus, the signal due to C-15 of koumidine (4) was observed downfield (7.6 ppm) and, on the contrary, t h a t of C-21 was observed upfield (2.4 ppm) compared to the corresponding signals of 19(E)-koumidine (9) (Table 2). Irradiation of H19 enhanced H-15 with 13% NOE. The stereochemistry at C-16 in koumidine (4) is unambiguous from the chemical reactions in which 4 produces the indolenine derivative (11) having a C-7/C-16 bond (7) and the epoxy derivative (12) having an ether linkage with the C-6 position (4) (Fig. 2). The significantly different 13C-NMR shifts of C-6, C-14 and C-17 were observed (Table 3) between 4 and its 16-epimer (13) (Section II-1). This is an alternative method for the assignment of the stereochemistry at C-16 in this type of compound. Koumidine (4) was stereoselectively prepared from ajmaline (Section II-1). A new sarpagine-type indole alkaloid, 16-epi-voacarpine (5), was isolated from G. elegans (6, 7) which is native to Thailand. The presence of a hydroxyl group at C-3 as well as the stereochemistry at C-16 in 5 were demonstrated by the formation of 14 and 15 from 16-epi-voacarpine (5) by acetylation with acetic a n h y d r i d e in pyridine (Fig. 3). The stereochemistry at C-19 in 5 was determined by the NOE observation at H3-18 (10%) from H-15. The structure was later confirmed by single crystal X-ray analysis (8). Interestingly, 16-epi-voacarpine (5) is the one and only compound among the Gelsemium alkaloids having a 19(E) ethylidene side chain. Two biogenetically interesting sarpagine-type indole alkaloids 6 (7) and 7 (11), characterized by the bond cleaving between the C3 and Nb position, were isolated from G. elegans as minor components. The spectroscopic data of 6 showed similarity to that of reported anhydrovobasinediol (taberpsychine) (16) (16, 17), but a significant NOE observed between H19 and H-15 in the 1H-NMR s p e c t r u m of 6 suggested t h a t the configuration of the ethylidene side chain was the (Z) form. The stereochemistry at C-19 was also elucidated by comparison of the 13CNMR spectra of 6 and anhydrovobasinediol (16), which was prepared from 19(E)-koumidine (Fig. 4) and was identified by direct comparison with an authentic sample provided by M. Hesse. The signal due to C-15 of 6 was observed downfield (7.0 ppm) and , on the contrary, that of C-21 was observed upfield (6.9 ppm) compared to the corresponding signal of 16 (Table 2). The structure of 6 inferred by spectroscopic analysis to be 19(Z)-anhydrovobasinediol was confirmed by chemical synthesis from ajmaline (Section II-1).
470 HOH2C
17
~
NOE~ H ~ 4
4
13%
7
o
~
19
N 6
H
H 12
HOH2CN
HOH2C
II-I
MeOA~N
NOE~.~...~ ~e
9
3%
IH
N
Gardnerine 10
H e
Fig. 2 TABLE 3 13C NMR A s s i g n m e n t of 4 and 13
~
~
6
R'~ 16 Re
Koumidine 4 RI=CH2OH, R2=H
16-epi-Koumidine 13 RI=H, R2=CH20H
~II
-1
Ajmaline
C
4
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
138.4 51.0 54.0 23.4 105.9 127.5 118.7 119.8 122.1 111.9 138.2 29.3 35.1 44.3 61.2 12.6 115.3 142.7 54.6
13 136.3 51.6 56.8 27.9 104.0 128.8 118.7" 119.9 122.1 112.0 138.2 35.9 35.7 45.3 65.3 12.6 118.6" 139.0 54.1
Chemical shift in ppm downfield from TMS. Solvent; CD3OD. *Signals may be interchanged.
471
HOH2C ~N~N~ HHO"~ N O E 10% ~
AcOH2C
..y Me
Ac20 ~ ~ L ~ ~ Pyridine
~8
COCH3
+
LLJLcoc. ~~
14
cO2Me
15
16-epi-Voacarpine 5
Fig. 3
HOH2C
H 1. C1C02Me MgO, aqTHF~. ~
Gardnerine 10
'
>
2. LiA1H4 THE, rt.
II-1
e
~HN~H~ 16
I
Anhydr ovoba sine diol Fig. 4
A new alkaloid (7) is the first example of a Gelsemium alkaloid having an Na-methoxyindole moiety in the molecule. This alkaloid may be an early biogenetic intermediate to the Na-methoxyoxindole alkaloids and their related compounds. Full assignments of the 1H- and 13C-NMR spectra of Na-methoxy-19(Z)-anhydrovobasinediol (7) were conducted mainly by CSCM 1D (decoupled selective population transfer experiment) (18) and selective INEPT (insensitive nuclei enhanced by polarization tansfer) (19) experiments. The s t r u c t u r e was finally determined by single crystal X-ray analysis (11). As demonstrated above, comparison of the 13C-NMR spectra is a secure method for determination of the stereochemistry of the ethylidene chain in the sarpagine-type indole alkaloids when pairs of isomers at C-19 are available. Alternatively, the differential NOE technique is also an efficient method for this purpose.
472
I-2. Humantenine-Type Oxindole and the Related Alkaloids Only three humantenine-type N a - m e t h o x y o x i n d o l e alkaloids, h u m a n t e n i n e (17), humantenirine (18) and rankinidine (19), from the G e l s e m i u m species have been reported up to 1988. Recently, nine h u m a n t e n i n e - r e l a t e d new alkaloids ( 2 0 ) - (28) were isolated from Gelsemium elegans Benth (Table 4). A characteristic strong peak produced by loss of the aromatic moiety of the molecule in the mass spectrum of the humantenine-type compounds provides important information for the structure elucidation (Fig. 5) (27). Compounds (21), determined by single crystal X-ray analysis (24), and 22 are the first oxindole alkaloids with an 11-hydroxy group, although some other oxindole alkaloids have an 11-methoxy group. Two Nadesmethoxy humantenine-type alkaloids (23) (24) and (24) (25) were elucidated by spectroscopic data. These compounds were synthesized from ajmaline via koumidine (4) by the stereoselective transformation to oxindoles (Section II-2). 20-Hydroxydihydrorankinidine ( 2 6 ) ( X - r a y analysis) (25) and 15-hydroxyhumantenine ( 2 5 ) ( 2 5 ) a r e the very few members with a hydroxy group at the C-20 or C-15 positions among the Gelsemium oxindole alkaloids. Two novel seco-oxindole alkaloids, gelsemamide (27) and 11methoxygelsemamide (28), were isolated from G. elegans (26). The 1HNMR spectrum of 28 showed the signals for an amine proton, an olefinic proton, three aromatic protons, and two methoxy groups. The HETCOR (heteronuclear correlation) (28) and selective INEPT (19) spectra of 28 permitted the unambiguous assignment of the 13C-NMR signals. The presence of an NOE between H-18 and Ha-21 or H~-21 distinguished the H-21 signals from those of H-17, the former of which are unusually downfield (5 4.76) compared with t h a t in h u m a n t e n i n e s , and also established the Z-configuration for the 19,20-double bond. Magnetization transfer in a selective INEPT experiment via irradiation of H-9 resulted in enhancement of carbons assignable as C-13, C-11, and C-7. The structure was established unequivocally by single crystal X-ray analysis. The absolute configuration was determined by assuming that C-15 is the biogenetically controlled asymmetric center with the same R absolute stereochemistry as the h u m a n t e n i n e s . This is supported by the observation that 11-methoxygelsemamide (28) has a CD curve identical to those of the humantenines. The spectral data of gelsemamide (27) closely resemble those of 28, except that 27 has no aromatic methoxy group at C-11. Therefore, the structure of gelsemamide was determined to be that of formula 27.
473
TABLE 4 H u m a n t e n i n e - t y p e Alkaloids from G e l s e m i u m Species Alkaloid
Structure
Species
Ref.
O
OMe
Humantenine 17
RI=Me, R2=H
G. elegans G. rankinii G. sempervirens
Humantenirine 18
RI=H, R2=0Me
G. elegans G. rankinii G. sempervirens
5, 20-22 23 23 20, 22 23 23
Rankinidine 19
RI=H, R2=H
G. rankinii
23
Ra
11-Methoxyhumantenine 20
RI=Me, R2=0Me, R3=0Me, R4=H
G. elega ns
24
11-Hydroxyrankinidine
RI=H, R2=0H, R3=0Me, R4=H
G. elegans
24
11-Hydroxyhumantenine 22
RI=Me, R2=0H, R3=OMe, R4=H
G. elegans
24
Na-Desmethoxyrankinidine 23
RI=H, R2=H, R3=H, R4=H
G. elegans
24
Na-Desmethoxyhumantenine 24
RI=Me, R2=H, R3=H, R4=H
G. elegans
25
15-Hydroxyhumantenine 25
RI=Me, R2=H, R3=0Me, R4=0H
G. elega ns
25
21
474
TABLE 4 (Continued) Humantenine-type Alkaloids from G e l s e m i u m Species Alkaloid
Structure
Species
Ref.
0
"'"~OH
20-Hydroxydihydrorankinidine 26
G. elegans
25
R=H
G. elegans
26
R=0Me
G. elegans
26
N OMe
O 9
R
Gelsemamide
27
1 1-Methoxygelsemamide 2 8
I
19
N-H 0 OMe
21
18
-It
R3
R3
base peak
Fig. 5
475
I-3. Koumine-Type Alkaloids Koumine (29) is a representative alkaloid of Gelsemium elegans Benth (29-33). Because of its structural novelty, many chemical and synthetic studies have been done. They were summarized in the previous review (1) and section II-3. The presence of koumine Nb-oxide (30) in the leaves of G. elegans was reported (7). Oxidation of koumine (29) with m-chloroperbenzoic acid (mCPBA) produced two diastereomeric Nb-oxides, one of which was identical with the natural N-oxide (30) (Fig. 6). The configuration on the Nb atom was deduced by 1H-NMR spectral analysis (Table 6). In the n a t u r a l N-oxide (30), the signals due to H-15 and H-16 are observed significantly downfield, respectively, compared to those of koumine (29), while Ha-6 in the diastereomeric N-oxide (34) is deshielded downfield compared with t h a t of koumine (29). These p h e n o m e n a may be interpreted by the anisotropy of the N-O function. Therefore, the configuration on Nb can be deduced to be (S) in natural (30) and (R) in 34, respectively. U n n a t u r a l N-oxide (34) gave crystals suitable for X-ray analysis. The results obtained from X-ray analysis agreed with the conclusion obtained from 1H-NMR analysis. The isolation of a pair of 19-hydroxydihydro derivatives of koumine from G. elegans was independently reported by two research groups (Table 5). The Beijing group (34) elucidated the structure of the two alkaloids by comparison of the 1H- and 13C-NMR spectra with those of koumine (29) as well as by the CrO3-pyridine oxidation of kouminol, which produced the 19-keto derivative (8 1.79, 3H s). The stereochemistry at the C-19 of each alkaloid was deduced by examination of molecular models to elucidate the difference in the chemical shifts in the 1H-NMR spectra between both diastereomers. Thus, the H-18 in the so-called 19(S)kouminol exhibited an upfield shift of 0.16 ppm (8 0.77 vs. 0.93) as a result of the positive shielding by the aromatic ring. The H-9 of 19(R)-kouminol had a downfield shift of 0.5 ppm (8 7.33 vs. 7.23) because of its closer proximity to the hydroxyl group. The result of Horeau's method supported the conclusion from the spectroscopic analysis that the isomer having [a]D -153.8 ~ (MeOH-CHC13) was 19(R) and the other one having [a]D -209 ~ (EtOH) was the 19(S) configuration. The Illinois group succeeded in the crystallization of both compounds suitable for X-ray analysis (35). One diastereomer (31) (rap 198-200~ {[aid -232.7 ~ has the 19(R) configuration, and the isomer (32) (mp 270-272~ having [a]D -184.6 ~ has the 19(S) configuration. The NMR spectra of 19(S)-hydroxydihydrokoumine (32) reported by the Illinois group were recorded in DMSO-d6 and those of other three 19-hydroxy isomers, 31, the so-called 19(S)- and 19(R)-
476
TABLE 5 Koumine-type Alkaloids from Gelsemium Species Alkaloid
Structure
Species
Ref.
NMe
Koumine 29
(
"'H
G. elegans
29-33
MeI~~N~O "'~H
Koumine N-oxide 30
OH
G. elegans
NMe
19(R)-Hydroxy18,19-dihydrokoumine 31
19(R)
G. elegans
34, 35
19(S)-Hydroxy18,19-dihydrokoumine 32
19(S)
G. elegans
34, 35
G. elegans
36
,
1,2-Dihydrokoumine 33 H
I~NMe
477
kouminols, were taken in CDC13. However, the physical and spectroscopic similarities between 1 9 ( R ) - h y d r o x y d i h y d r o k o u m i n e (31), which was determined by X-ray analysis, and the so-called 19(S)-kouminol reported by the Beijing group are greater than those between 31 and the so-called 19(R)-kouminol. 1,2-Dihydrokoumine (33) was first isolated as a natural product (36), although it has been prepared reductively from koumine (29) during the s t r u c t u r e elucidation of 29 (37). The 1 H - a n d 13C-NMR s p e c t r a l a s s i g n m e n t s of 33 were reinvestigated. In this report, the result of modified Hofmann degradation of koumine derivative (35) was presented (Fig. 7). The structure and stereochemistry of the main product (36) (1520% yield) were deduced by spectral methods and confirmed by X-ray diffraction analysis.
-Koumine 29
M__ee,...~.~N//~H 0H
/
O ~ N / MI ~{'~
mCPBA 30
Fig. 6
34
TABLE 6 Selected 1H NMR data (5) of 29, 30 and 34
30 H-15 H-16 Ha-6
"H
2.72 4.10 2.94
2.23 2.96 3.62
Koumine 29 2.34 2.8 2.41
Br + ~NMe
_NMe I
N-oxides 34
CH2Br2
Koumine 29
~
~'" N 35
Fig. 7
D
HO .,,,,.. ,NMe "'H
~
"'H 36
478
I-4. Gelsemine-Type Oxindole Alkaloids Gelsemine (37) is one of the principle alkaloidal constituents of the Gelsemium species. The structure was fully elucidated in 1959 (38, 39). 21-Oxogelsemine (38), gelsevirine (39), and 21-oxogelsevirine (40) were the members of the gelsemine group. Recent investigation led to the isolation of five new alkaloids structurally related to gelsemine (Table 7). From the leaves of G. elegans Benth, an Nb-oxide derivative of gelsemine was found (7). The stereochemistry of the quaternary nitrogen was determined by 1H-NMR spectral comparison of two diastereomeric N-oxides prepared from gelsemine by m-CPBA oxidation (Fig. 8). In the 1H-NMR spectrum of the synthetic N-oxide (41), which was identical with that of natural product, H-6 was remarkably shifted downfield (A 1.43 ppm) compared with that of gelsemine (37) (Table 8), and 9% NOE was observed between the N-Me group and H-16. On the other hand, H16 in the isomeric N-oxide (46) was shifted to downfield (A 1.96 ppm) compared with t h a t of gelsemine (37). These phenomena can be interpreted by the anisotropic effect of the oxygen on Nb, as in the case of koumine N-oxides (30) and (34). Therefore, the natural N-oxide has the R configuration on Nb. Both stereoisomers of gelsevirine derivatives having a secondary hydroxy group at the C-19 position were isolated from the same species. One of the isomers first isolated from G. elegans (7) showed the characteristic resonances at 8 5.13 (1H, q, J 6.6 Hz) and at 5 1.09 (3H, d, J 6.6 Hz) due to the secondary hydroxy group in place of the vinyl group of gelsevirine (39) in the 1H-NMR spectrum. In the 13C-NMR spectrum, which was very similar to that of gelsevirine (39), the appearance of a new doublet at 8 64.3 and a new quartet at 8 19.4, and the absence of vinyl carbons (C18-19) in gelsevirine (39) also revealed the presence of a secondary hydroxy group on C-19. By the Swern oxidation, the 19hydroxylated alkaloid (44) gave the ketone derivative (47) that was identical with the sample prepared from gelsevirine (39) in three steps including the Wacker oxidation (1. m-CPBA, 2. PdC12, 02 in aq. DMF (48), 3. NaHSO3) (Fig. 9). On reduction with NaBH4 in MeOH, the ketone (47) afforded the diastereomeric alcohol as the major product, accompanied by a trace amount of the natural compound. From the mechanistic consideration of this stereoselective reduction using the Dreiding model, the stereochemistry of the C-19 position has been deduced as follows. In the transition state during the reduction, the ketone (47) may assume the conformer A rather than B, C, or D due to the dipoledipole repulsion and/or steric hindrance (Fig. 9). Hydride would approach from the less hindered side (anti to the oxindole nucleus), resulting in the
479
TABLE 7 G e l s e m i n e - t y p e Alkaloids from G e l s e m i u m Species Alkaloid
Structure
Species
Ref.
,C
I
R1 Ri=H, R2=H2
G. sempervirens G. elegans
38-44 4,7
21-Oxogelsemine 38
Ri=H, R2=O
G. sempervirens
45
Gelsevirine 39
Ri=OMe, R2=H2
G. elegans G. sempervirens G. rankinii
7, 20 43 46
21-Oxogelsevirine 40
Ri=OMe, R2=O
G. rankinii
46
Gelsemine
37
G. elega ns
Gelsemine N-oxide 41 H 0~
N I
R1
19
I_ -~OR2
Me
19(R)-Hydroxydihydrogelsemine 42
Ri=H, R2=H, 19(R)
G. sempervirens G. rankinii
47
19(R)-Hydroxydihydrogelsevirine 43
Ri=OMe, R2=H, 19(R)
G. elegans
47
19(S)-Hydroxydihydrogelsevirine 44
Ri=OMe, R2=H, 19(S)
G. elegans
7, 47
19(R)-Acetoxydihydrogelsevirine 45
Ri=OMe, R2=COCH3, 19(R) G. sempervirens
G. rankinii
47
480
O-~.~N/Me ~ N O E ~-, 9% Gelsemine 37
mCPBA
H~
"" 6
H
Me.,~NsO .-~--,,, +
~I 6
41
Fig. 8
I~
H
46
TABLE 8 S e l e c t e d 1H N M R d a t a (5) of 37, 41 a n d 46
N-oxides H-6 H-16
Gelsemine
41
46
37
3.41 2.59
2.28 4.26
1.98 2.30
o e
"N"Me ii, iii, iv
i
-oMo" E ~
6Me
19(S) 44 A
III
Gelsevirine 39
~
B
[-N"Me
C
"N"Me ~N OMe
OMe Me
'Me
O ~N 6
O~....N.Me
D
"N"Me
Me ~N''~0 OMe 0
Rotational isomers at the C19-20 bond (Conformer A may be more stable than others due to the dipole-dipole repulsion or steric hindrance in B, C, or D).
19(R) 43
Reagents; i,
(COC1)2, DMSO, t h e n Et3N, CH2C12. ii, mCPMA, CH2C12. iii, PdC12, 02, DMF. iv, NariS03, CH2C12. v, NaBH4, MeOH.
Fig. 9
481
predominant formation of the 19(S) alcohol. However, the same alkaloid with the major reduction product of 19-oxo-gelsevirine (47) was later isolated from G. elegans, and its 19(R) configuration was established by single crystal X-ray analysis (47). Therefore, the stereochemistry of the first isolated compound initially proposed as the 19(R) configuration clearly became that of (S). The fact that NaBH4 reduction of the ketone (47) gave the 19(R) isomer (43) predominately can be now interpreted by the initial complexation of the boron reagent with the carbonyl function in oxindole, followed by the hydride attack from the oxindole side in conformer A. A new alkaloid having an acetoxy group at the C-19 position in the gelsevirine skeleton was isolated from G. sempervirens and G. rankinii (47). The 1H- and 13C-NMR spectra of 45 were similar to those of 19(R)hydroxydihydrogelsevirine (43), except for the presence of an acetyl group in 45, suggesting that 45 had the 19(R) configuration. That was proved by hydrolysis of 45 to produce 43. 19(R)-hydroxydihydrogelsemine (42) was also obtained from G. sempervirens and G. rankinii (47). The stereochemistry at C19 in 42 was deduced by the similarity of the 1H- and 13C-NMR spectra of 42 and 19(R)-hydroxydihydrogelsevirine (43). I-5. Gelsedine-Type Alkaloids The gelsedine-type alkaloids (Table 9), which included gelsedine (48), 14-hydroxygelsedine (49), gelsemicine (50), 14-hydroxygelsemicine (51), gelsenicine (52), and 14-hydroxygelsenicne (humantenidine) ( 5 3 ) u p to 1988, are oxindoles with a novel skeleton similar to that of humanteninetype oxindole alkaloids but lacking their C-21 carbon. The presence of 19-oxo-gelsenicine (54) in the leaves of G. elegans was reported (7). The IR spectrum of 54 displayed two carbonyl bands at 1715 and 1695 cm-1. The 13C-NMR spectrum of 54 was similar to that of gelsenicine (52) except for the signals due to C-18 and C-19, which were shifted downfield at 8 26.1 and 8 197.6, respectively, suggesting the presence of a ketone function on C-19. The structure was established by X-ray analysis and by comparison of the CD spectra between gelsenicine (52) and 54. A novel gelsenicine-related oxindole alkaloid, named gelsemoxonine (55), was isolated from G. elegans (25). The UV absorption of 55 at 209.5 and 257.5 nm suggested a 3,3-disubstituted Na-methoxyoxindole skeleton. The 1H-NMR spectrum displayed a methyl group as a triplet at 8 1.07 (H18), which was coupled to the H-19 methylene group multiplets. The COSY spectrum of 55 showed two methine doublets (8 3.78, d, H-3 and 8
482
TABLE 9 G e l s e d i n e - t y p e Alkaloids from G e l s e m i u m Species Alkaloid
Structure
Species
Refi
R2 O~
a
N,
OMe
Gelsedine 48 14-Hydroxygelsedine
49
RI=H, R2=H
G. sempervirens G. elegans
49, 50 4
Rz=H, R2=0H
G. sempervirens G. elegans G. sempervirens
51 7 52, 53
G. sempervirens
54, 55
Gelsemicine 50
RI=0Me, R2=H
14-Hydroxygelsemicine 51
RI=0Me, R2=0H
a1 0~
14
N
dMe Gelsenicine 52
RI=H, R2=H2
G. elegans
5, 20
14-Hydroxygelsenicine 53
Rz=OH, R2=H2
G. elegans
20, 56
19-Oxogelsenicine 54
R I=H, R2=0
G. elegans
7
17 0 ~ 1 6
Gelsemoxonine 55
.I.... G. elega ns 18 OMe
25
483
4.99, d, H-14a), which were coupled only to each other, indicating that C14 had only one proton and C-15 had no proton. Therefore, both carbons should be oxygenated. The sequential connectivity of the protons, starting from H2-17 to H2-6 via H-16 and H-5, such as that in gelsenicine (52), was elucidated by the COSY spectrum. Unambiguous assignments of the protonated carbons was obtained by a HETCOR spectrum. The remaining quaternary carbons were assigned by the selective INEPT technique. For example, irradiation of H-16 enhanced C-14, C-6, and C-20, which was also enhanced by irradiation of H-18. The C-20 signal (5 211.67) of 55 is in the range for a ketonic carbonyl resonance, indicating the existence of a carbonyl and an amine group in 55 instead of the N-4/C-20 double bond in the gelsenicine (52). Selective INEPT irradiation of H-14 with a different 3JCH value (3-7 Hz) more strongly enhanced the C-15 signal than the C16 signal, indicating an epoxide oxygen bridge between the C-14 and C-15. Gelsemoxonine (55) is the first Gelsemium oxindole alkaloid without a N4/C-20 bond and might be formed from the corresponding gelsenicine derivative by cleavage of the C=N double bond owing to the strain by the epoxide at the C14-15 position. I-6. Gelselegine-Type Oxindole Alkaloids The isolation of gelselegine (56) and 1 1 - m e t h o x y - 1 9 ( R ) hydroxygelselegine (57) (Table 10), new types of oxindole alkaloids having a hydroxymethyl group at the C-20 position in the gelsedine skeleton, was repo rted (57). The UV, IR and 1H-NMR spectra of gelselegine (56) indicated the characteristic of an Na-methoxyoxindole nucleus. Beside the C-17 methylene in the 13C-NMR spectrum, the chemical shift of the other methylene signal at 5 63.52 indicated the presence of a hydroxymethyl carbon. This is supported by the mass spectral fission pattern, a molecular ion peak at m/z 358 (C20H26N204) with a base peak at m/z 327 [MCH2OH]+ and a principal fragment ion at m/z 296 [M-CH2OH-OMe]+, corresponding to successive losses of the hydroxymethyl and Na-methoxy groups. The HETCOR and selective INEPT spectra of 56 permitted the unambiguous assignment of the 13C-NMR signals. Selective INEPT irradiation of H-5 enhanced three carbon signals at C-15, C-7, and C-20, the latter of which was also enhanced by the irradiation of H-18. This suggested that a rearrangement and ring contraction in the aliphatic portion had occurred to form a quaternary carbon at the C-20 position. The structure was established unequivocally by single crystal X-ray analysis.
484 TABLE 10 Gelselegine-type Alkaloids from Gelsemium Species
Alkaloid
Structure
Species
Ref.
17
5 ~ ~ ~ R
N OMe
Me
R2
Gelselegine 56
RI=H,R2=H
G. elegans
57
11-Methoxy-19(R)hydroxygelselegine 57
RI=OMe, R2=OH
G. elegans
57
TABLE 11 Gelsemium Alkaloids with Two Monoterpene Units
Alkaloid
Structure
Species
Ref.
0
N
19Me
HO ~.,
Elegansamine 58
9
G. elegans
58
G. elegans
59
"Me
0
_~
Gelsamydine 59
AO N 19'"Me NI OMe H O' ~l H -" Me,.....
- ~- o"'*'
485
The second gelselegine-type new alkaloid, 11-methoxy-19(R)hydroxygelselegine (57), has two more oxygen substituents compared with gelselegine itself. Including the new asymmetric center at the C-19, the structure of 57 was determined by X-ray analysis. Biogenetically, gelselegine-type alkaloids might be derived from the c o r r e s p o n d i n g h u m a n t e n i n e s by r e a r r a n g e m e n t of a z i r i d i n i u m intermediates. According to this biogenetic hypothesis, the gelselegine skeleton was synthesized from the sarpagine-type alkaloid (Section II-5). I-7. Oxindole Alkaloids Containing Two Monoterpene Units From the methanol extracts of the branches of G. elegans Benth, a new indole alkaloid, named elegansamine, was obtained as colorless prisms (mp. 172-173~ (Table 11) (58). Elegansamine (58) showed the UV spectrum characteristic to the Na-methoxyoxindole nucleus. The high resolution mass spectrum showed the M+ 508.2572, corresponding to the formula C29H36N206, and gave the base peak at m/z 326, corresponding to the molecular weight of gelsenicine (52), C19H22N203, indicating that elegansamine (58) was constructed from gelsenicine (52) or its isomer and a monoterpene unit containing three oxygen atoms. In the 1H-NMR spectrum, in addition to some readily assignable signals due to the gelsenicine moiety such as four aromatic protons, Na-OMe, H-3, H-15, and H-16, characteristic signals of a doublet on the C-18 protons and a multiplet due to H-19 were observed in place of the ethyl group in 52, suggesting that the monoterpene unit might be connected at the C19 position. From the 13C-NMR spectrum (Table 12), the composing indole alkaloid portion and the m o n o t e r p e n e u n i t were respectively demonstrated to be gelsenicine and an iridoid skeleton, which possessed a lactone, C-Me, and a secondary hydroxy group. The connectivity of H-18 and the methylene carbon (C-11', 5 33.1) in the monoterpene portion shown by the HMBC spectrum as well as the magnetization transfer from H-18 to the methylene protons (H2-11') via H-19 in the differential HOHAHA (homonuclear Hartmann-Hahn) (60) spectrum demonstrated the linkage position between both units. Finally, the structure of elegansamine including the stereochemistry of the iridoid moiety and the C-19 position was determined with the aid of single crystal X-ray analysis. The CD spectrum of 58 closely resembles that of gelsenicine (52), and therefore the gelsenicine part and the iridoid residue in 58 have the same absolute configuration as the conventional indole alkaloids and iridoid monoterpenes, respectively.
486
T ABLE 12 13C NMR assignments of the
~
o
alkaloids 52, 58, and 59 C
N
52
58
59 a
171.5 75.0 71.2 37.4 56.1 131.9 124.7 123.4 128.1 106.6 138.0 27.7 42.5 40.1 61.9 18.7 37.7 186.2 63.2 70.4 177.9 48.7 37.7 72.5 43.7 35.1 45.1 19.3 33.1
171.3 72.0 74.7 37.0 56.0 131.7 125.0 123.4 128.2 106.8 138.3 26.5 39.3 40.1 61.8 20.2 35.7 187.8 63.2 60.1 182.0 52.5 48.5 81.9 42.4 33.0 33.1 17.2 37.5
Me
d)Me Gelsenicine 5 2
o
'~
Me O
1,
'~~'H 7' 8' '~Me
Elegansamine 58
10'
o
19
N
OMeH O ~ !
,,Me
N
Me,,, ,t,:( " ,,;,I "
~o' 8 ~ o ' 3 ' 7' Gelsamydine 5 9
fi
11'
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe 1' 3' 4' 5' 6' 7' 8' 9' 10' 11'
171.2 74.8 72.5 37.7 55.8 132.2 124.6 123.3 128.0 106.5 138.0 27.0 42.5 39.8 62.0 10.0 25.6 184.2 63.3
)===0
Chemical shift in ppm downfield from TMS. Recorded in CDC13. a: data taken from ref.59. The assignment of 52 was reinvestigated by the H-H and C-H COSY, and HMBC spectra.
487
A new oxindole alkaloid gelsamydine (59) (59) isolated from the same species is structurally related to elegansamine (58). The structure was deduced by a spectroscopic method and then established by X-ray analysis. It is also constructed from gelsenicine and a monoterpenoid unit. The monoterpene parts in 58 and 59 are structurally interconvertible with each other by ring-opening/ring-closure of the lactone function and the residual hydroxy group and by epimerization at the C-4' position. However, interestingly, the stereochemistry of the connection part at the C-19 is (S) in elegansamine (58) and (R) in gelsamydine (59), respectively. These alkaloids are a new class of alkaloids composed of the carboncarbon linkage of a monoterpenoid indole alkaloid and a monoterpene unit.
II.
Synthetic
Studies
Based
on
Biogenetic
Considerations
II-1. Sarpagine-Type Alkaloids It is well recognized that dehydrogeissoschizine (64) is formed from strictosidine (62)via an intermediate (63), and compound 64 is further converted to the yohimbine class of indole alkaloids (Fig. 10) (61). It can be easily imagined that the biosynthesis of the Gelsemium alkaloids also starts from the condensation of tryptamine (60) and secologanin (61), because the in vivo transformation of [6-14C]strictosidine to gelsemine (37) in Gelsemium sempervirens was already demonstrated by Zenk et al. (62). An intermediate (65) formed by the intramolecular carbon-carbon bond formation between the C-5 and C-16 position in strictosidine (62) will serve as a precursor for ajmaline (66) and sarpagine-type alkaloids. The olefin migration in the hypothetical intermediate (65) would afford two geometrical isomers ( 6 7 ) a n d 68). They would be respectively transformed into 16-epi-voacarpine (5) having the 19(E) configuration, and 19(Z)-akuammidine (2) and koumidine (4) having the 19(Z) form. By C/D ring-cleavage and simultaneous ether linkage formation between C-3 and the primary alcohol at C-17, one of the biogenetically key intermediates, 19(Z)-anhydrovobasinediol (6), would be generated from koumidine (4). (Fig. 10)
488 6
OHC 9
NH2
H Tryptamine
60
5
t~
9
9 MeO2c~O
Secologanin 6 1
Hr MeO2CA~,,IO Strictosidine
MeO2C6~"~..~OH ] Intermediate
62
MeO2C~""=~_...--OH
63
Dehydrogeissoschizine
64
OH
OHC R
[~N
J~N
,~OH
H H ' ~ Ajmaline 6 6 _
Intermediate
65
! ,
~CO2Me nu v
" ~ 19
19(E) 67
16-epi-Voacarpine
5
MeO2C~
19(z) 68 19(Z)-akuammidine
O
.@1.,
H
19(Z)-anhydrovobasinediol
Fig. 10
Tentative
biogenetic
2
HOH2%H
6
koumidine 4
route of the G e l s e m i u m
19
alkaloids-1
489
Ajmaline (66) is one of the major Rauwolfia alkaloids, and its total synthesis and the absolute configuration were already established (63). Compound 66 exists as an amino acetal form at the Nb/C-21 position. The equilibrium isomer (69) chemically corresponds to the hypothetical intermediate (65). Then, starting from commercially available ajmaline, the synthesis of koumidine (4) and 19(Z)-anhydrovobasinediol (6) was planned along the above biogenetic sequence (Fig. 11). Ajmaline (66) was converted into the hydrazone derivative ( 7 0 ) b y t r e a t m e n t with N,Ndimethylhydrazine and a catalytic amount of sulfuric acid followed by methyl chloroformate in 79% overall yield to liberate the masked aldehyde existing as an amino acetal function and to protect the Nb as the carbamate. The hydrazone was hydrolyzed with copper(II) chloride in aq. tetrahydrofuran (pH 7) (64) to afford the aldehyde (71) in 75% yield. In the 1H-NMR spectrum, the signals due to the aldehyde group were observed at 5 9.62 and 9.59 (ajmaline derivatives possessing a carbamate function in the molecule are often shown by their 1H and 13C-NMR spectra to occur as a mixture of rotational isomers). After the protection of the 17-hydroxy group in compound (71) as the methoxyethoxymethyl (MEM) ether, bromine was selectively introduced to the c~ position of the
OH
OH
'~
/ Ajmafine 66
i. H2N-NMe 2, cat.H2SO4, MS, EtOH ii. C1COOMe, 1N-NaOH / CH2C12 y. 79% iii. CuCI 2, aq. THF (pH 7)
y. 75% iv. MEMCI, i-PrzNEt, CH2C12 y. 81%
69
0R2
~N
,,
Me
ii. NBS, THF 70: 71: 72:
OMEM
i. TBSOSO2CF3, Et3N, CH2C12 y. 71%
DBU DMF
y. 76%
RI=CH=NNMe2,R2=H RI=CHO,R2=H RI=CHO,R2=MEM OMEM
~,,
i. NaBH4, MeOH y. 88% ii. NaOH, HOCH2CH2OH, H20
~--CO2Me ':HIS. Me if ~ "OH3 CHO ~..
Z - f o r m 74 y. 60% +
73
OH y. 87%
iii. MsC1, Py. y. 62% iv. cat.HC1, MeOH y. 95% 76
19
same reaction conditions as above
19(E): T e t r a p h y l l i c i n e 77
E - f o r m 75 y. 12%
Fig. 11-1
490
aldehyde by treatment with N-bromosuccinimide (NBS) of the tbutyldimethylsilyl (TBS) enol ether derivative. To create the double bond at the C19-20 position, the bromide (73) was treated with 1,8diazabicyclo[5,4,0]undec-7-ene (DBU) to give two a , ~ - u n s a t u r a t e d aldehydes, the desired 19(Z)-olefin (74) and 19(E)-olefin (75) in 60% and 12% yield, respectively. The geometry of the two olefins was confirmed by NOE experiments. Irradiation of the 18-methyl protons in the Z-isomer (74) led to enhancement (17%) of the C-21 aldehyde proton, while 25% enhancement was observed between the C-19 olefinic proton and the C21 aldehyde proton in the E-isomer (75). The major cz,~-unsaturated aldehyde (74) was reduced with NaBH4 to give the primary alcohol. After deprotection of the carbamate by alkaline hydrolysis, the amine was treated with mesyl chloride in dry pyridine to afford the ring-closure product by bond formation between Nb and C-21. By removal of the MEM ether, the deoxyajmaline derivative (76) was obtained. By the same sequential treatment, the minor 19(E)-olefin ( 7 5 ) g a v e the Rauwolfia alkaloid tetraphyllicine (77) (65). The indoline-to-indole transformation for 76 was accomplished as follows. In order to prevent epimerization at
76
OSiMe2-R
i. TBSOTf, Et3N, CH2C12 or TMSOTf, Et3N, CH2C12
a (R=tBu): Bu4NF, THF b (R=Me): AcOH-THF-H20=3"I-1 I11
ii. Pb(OAc)4, CH2C12 78: R=tBu '79: R=Me
~ I
~
H
N
8O
",•aCNBH3
--,,
i. C1COOCH2CC13,MgO THF-H20 y. 58% N H
ii. LiAIH4, THF
~ I Koumidine 4
Fig. 11-2
CH2OH
16-epi-Koumidine 13
y. 86%
19Z-Anhydrovobasinediol 6
H. 16
H
N
491
C-16 during this process, mild removal of the protective group was required. The MEM group at the C-17 hydroxy group was substituted with TBS ether, which was then converted to the indolenine (78) by lead tetraacetate [Pb(OAc)4] oxidation (66). After deprotection of the TBS ether by use of tetrabutylammonium fluoride in THF at room temperature, the resulting aldehyde (80) was immediately reduced with NaBH4 in MeOH, however, to afford 16-epi-koumidine (13) as the sole product. Strong basicity of the fluoride anion presumably led to the epimerization at C-16 in the aldehyde intermediate (80). To prevent epimerization at C-16, the 17-hydroxy group of the compound (76) was protected as the trimethylsilyl (TMS) ether, which could be easily removed under mild conditions. The TMS ether derivative was then oxidized with Pb(OAc)4 in dry CH2C12 at -70~ to -10~ to give the unstable indolenine (79). The indolenine (79) was successively treated with aq. AcOH-THF at 0~ and then with sodium cyanoborohydride to afford koumidine (4). The 1HNMR, IR, UV, mass spectral and [a]D data and mp. were identical with those of natural koumidine. Koumidine (4) was treated with 2,2,2-trichloroethyl chloroformate in aq. THF in the presence of a large excess of magnesium oxide (MgO) to give the carbamate, which was then reduced with lithium aluminum h y d r i d e (LiA1H4) to afford 1 9 ( Z ) - a n h y d r o v o b a s i n e d i o l [19(Z)taberpsychine]. The 1H-NMR, IR, UV and mass spectral and [a]D data were identical with those of natural 19(Z)-anhydrovobasinediol. Since the absolute configuration of ajmaline has already been established, the structure including the absolute configuration of koumidine (4) and 19(Z)-anhydrovobasinediol (6) was determined (67, 68). Gardnerine (10), one of the major indole alkaloids of Gardneria nutans (69), was converted into koumidine (4) by demethoxylating the indole ring and inverting the configuration of the ethylidene side chain in the following manner (Fig. 12) (70). Gardnerine (10) was first converted to 17-O-acetyl-Na-tosyl derivative (81), which was then treated with a l u m i n u m chloride in ethanethiol and CH2C12 to give the phenolic compound (82). The triflate (83), prepared from 82 with triflic anhydride and triethylamine, was treated with a catalytic amount of palladium acetate [Pd(OAc)2], 0.4 equiv of 1,1'-bis(diphenylphosphino)-ferrocene (DPPF), triethylamine, and formic acid in DMF (71) to produce the deoxygenated compound (84) in good yield. Reductive deprotection of the Na-tosyl and 17-O-acetyl group in 84 with LiA1H4 gave rise to 19(E)koumidine (9). On the other hand, by treating compound (84) with a large
492 17
Gardnerine 10
X=OMe X=OH X=OTf X=H
9al
Koumidine 4 19(E)-Koumidine
982 983 984
Reagents and conditions i, Ac20, pyridine, r.t., 8 h, 97%; TsC1, n-Bu4NHSO4, 50%KOH-benzene, r.t., 3 h, 98%. ii, A1C13, EtSH, CHzC12, -18~ 3 h, 91%. iii, (CF3SO2)20, Et3N, CH2C12, -20~ 10 rain, 97%. iv, Pd(OAc)2, DPPF, Et3N, HCOOH, DMF, 60~ 2 h, 98%. v, Mg, PdC12, PPh 3, MeOH, r.t., 50 h, 48% (4), 34% (9).
Fig.
o
12
H
X
0
J]-oxidation=
~e
N
H
19 6 19(Z)-anhydrovobasinediol
8 5
I rearrangement Na-oxidation
I Na-oxidation o
13-oxidation ~ rearrangement'-
,:
~ OMe
7
Fig.13
" If
N
s
17 humantenine" R=Me 19 rankinidine: R=H
Tentative biogenetic route of the G e l s e m i u m
alkaloids-2
9
493
excess of magnesium (turnings) in dry MeOH in the presence of a 0.1 equiv of palladium chloride and 0.2 equiv of triphenylphosphine, deprotection and olefin inversion proceeded simultaneously to afford koumidine (4) as the major product (48% yield) along with the isomer (9) (34% yield) and 19,20-dihydrokoumidine (7% yield). The plausible mechanism of this olefin inversion reaction would involve the metal hydride 1,2 addition/f3-hydride elimination process (72). Total syntheses of koumidine (4) (73) and Na-methyl-A18-isokoumidine (74) were accomplished. II-2. Humantenine-Type Oxindole Alkaloids Biogenetically, the humantenine-type oxindole alkaloids would be g e n e r a t e d from the s a r p a g i n e - t y p e compounds such as 19(Z)anhydrovobasinediol (6) through ~-oxidation of the indole portion and subsequent rearrangement to the oxindoles (Fig. 13). Initially, using the C / D ring-cleaved compound (86), oxidative transformation of sarpagine-type indole alkaloids into the corresponding oxindole derivatives was investigated (75). Oxidation of the indole (86) by the conventional method with t-butylhypochlorite (t-BuOC1)in the presence of triethylamine (76) gave the unstable chloroindolenine (87), which was directly treated with aq. acetic acid in methanol to afford two oxindoles (88) and (89) in 9% and 37% yield, respectively. These two products are diastereomers at the C-7 spiro position. The minor product (88) has the same s t e r e o c h e m i s t r y at C-7 as t h a t of n a t u r a l humantenine-type alkaloids that was demonstrated by comparison of the CD spectra. In turn, treatment of the chloroindolenine (87) with sodium methoxide in MeOH gave two imino ethers in 41% and 23% yield, respectively. The configurations at the spiro center of these products were determined by their hydrolytic conversion into the corresponding oxindoles (88) and (89), respectively. The stereochemical course of the rearrangement reaction, in which the products having opposite configuration at the spiro center were predominantly generated from the common intermediate (87) under two different conditions, was speculated as follows, t-BuOC1 should approach the ~-position of the indole portion from the less hindered side (anti to the bridged e t h e r linkage), r e s u l t i n g in the f o r m a t i o n of C7-achloroindolenine (87) (Fig. 14). In treating 87 with NaOMe, the reagent may attack the C-2 position from the c~ side because of the steric hindrance of the ether linkage to generate the intermediate (90), and then
494
O
HO
MeO
N H
N.
l~---~
C1CO2M~e/
~
N~/__CO2Me ~ O ~ N A ~
7S ,, ,"
~ L
CO2Me
~eO" "-~ " ~ ' ' ~ ' L ~ .
~'1 i'~
y.37%
8 8 =
N~://CO2Me
7~[i~,~N,"/CO2Me
Me~
§
y. 9%
1) NaOMe 2) H30*
~.V"....~
86
10 gardnerine
AcOH,McOH,H20
CI, O ~
'suoo, ~
y.
y. 41%
89
11%
Or
NaOMe
C~---
r~.~o.
88: C7S
Me
MeO" ~
MeO~
-
90
H3O+
89: C7R
I(~1 MeO~N
MeO/
91
Fig. 14
"
o2R 2eq
2"~.~'~ 20
S, ,'
o~~
OsO4~
93:R=CH2CC~3 ~-~19 Py.-THF
92
MeO
as . 1. so3-Py N./N./ l. HC(OMe)3, PPTS Et3N, DMSO F - r ~ 1 9 O,H~ ..... OH 2. Ac20, A. 3. NaOH 2. NaBH4
96
~N
COOR
94
/" 97
H
CH3
AcOH
MeO
98: Na-demethoxyhumantenirine
Fig. 15
495
the anti-periplanar C2-C3 bond to the leaving group (C1) will rearrange to the C-7 position to form the (S)-isomer preferentially. On the other hand, the chloroindolenine derivative (87) may react with H20 in aqueous acidic media to form C2-[3-hydroxylated intermediate (91) due to the affinity of the ether oxygen atom for H20. After the formation of a carbocation on C7 by the elimination of CI- under SN1 condition, the C2-C3 bond in the intermediate (92) will move to the C-7 position from the a-side to form the (R)-isomer (89) predominantly. From the mechanistic consideration above, it is predictable that the preparation of an intermediate such as 90, in which both the leaving group on C7 and the oxygen function on C2 possess a-orientation, would provide the C7-(S) spiro-isomer in a highly diastereoselective manner. As anticipated, treatment of 93 with 2.0 equiv of OsO4 in pyridine-THF afforded the oxindole (95) as a sole product in 77% yield (Fig. 15). It is interesting to note that all of the oxindole alkaloids found from Gelsemium spp. (humantenine-, gelsemine-, and gelsedinetype alkaloids) possessed the (S )-configuration at the spiro position. Oxidative r e a r r a n g e m e n t of the indole alkaloids into the oxindole derivatives in the Gelsemium plant may occur enzymatically via an intermediate similar to that of osmylation process (94) described above. Next, the olefin inversion utilizing the vicinal diol function in 95 was conducted. The configuration at C-19 in 95 was initially inverted by the oxidation-reduction sequence. Thus, compound 95 was oxidized with SO3pyridine complex to afford the C19-keto derivative, which was then reduced with NaBH4 in MeOH at -75~ to give the desired C 19-(S) alcohol (96), predominantly (96:95=16:1). A vicinal diol in 96 was converted into the corresponding 2-methoxy-l,3-dioxolane by t r e a t m e n t with trimethyl orthoformate in the presence of pyridinium p-toluenesulfonate in THF (77). The resulting 2-methoxy-l,3-dioxolane was refluxed in acetic anhydride, and then the Na-acetyl group was removed by alkaline hydrolysis to produce the desired olefinic compound (97). The configuration of the ethylidene side chain in 97 was confirmed by the differential NOE experiments. Thus, irradiation of H-21 and H-15 enhanced H3-18 and H-19 with 6% and 8% NOE, respectively. Finally the protecting group on Nb was removed with Zn in AcOH to give the desired Na-demethoxyhumantenirine (98) (mp. 262~ A correlation of CD the spectra of 98 with corresponding data for d e m e t h o x y h u m a n t e n i n e allowed unambiguous assignments to the structure (98). Methoxylation of the nitrogen in the oxindole moiety is now underway in the present author's laboratory.
496
Synthesis of Na-demethoxyrankinidine (23), recently isolated from
Gelsemium elegans as a minor component, was achieved (Fig. 16) (68) from koumidine (4) that was prepared from ajmaline (66) as described in section II-1. An efficient method developed for the stereoselective oxidation-rearrangement of sarpagine-type indole alkaloids using OsO4 was applied to the C/D ring-cleaved compound (99). In the case of the gardnerine series such as compound (93), which has a methoxy group at the C-11 of the indole ring, the oxidation with OsO4 p r o c e e d e d predominantly on the C2-C7 bond (indole moiety) rather than on the C19C20 (ethylidene side chain). However, under the same conditions, the demethoxy indole (99) gave the oxindole diol (100) and the diol (101) in 38% and 37% yield, respectively. Attempts at the regioselective oxidation by increasing the reactivity of the indole nucleus using the Na anion or Na-trimethylsilyl derivative were ineffective. Furthermore, application of the general procedure using t-BuOC1 to compound (99) gave the
0 Koumidine
OCH2CCI3
:
4
N H
99 i. OsO4, T H F - P y , -20 ~
40 m i n
ii. NaHSO3,rt, 1.5 h
~
~
~
i. OsO4, Py.-THF -28 ~ to -15 ~
,' '
OOCH2CCI3
~
"N" o ~ 10 0
1.5 h
~1 I1~ -,
~
COOCH2CCl3
ii" NaHSO3' 55% rt'2hy.
~
6."~
N 101
OH
~ 6H "1"
i. HC(OCH3)3, PPTS,THF,rt. ii. Ac20, reflux iii. NaOH,MeOH,rt, y. 74%. iv. Zn, AcOH,rt, y.78% o
.-~ I-J "' H H"" O Na-Demethoxyrankinidine 23 R=H --~ HCHO,AcOH Na-Demethoxyhumantenine 24 R=Me j NaCNBH3
Fig. 16
OH
497
undesired oxindole having the opposite configuration at C-7. In turn, the indole (101) was subjected to OsO4 oxidation to give the compound (100) that was identical with the oxindole derived directly from 99. The oxindole thus obtained stereoselectively had the natural 7S configuration, which was confirmed by comparison of the CD spectrum with t h a t of h u m a n t e n i n e - t y p e alkaloids. The 19Z ethylidene double bond was regenerated by a three-step sequence. Thus, the diol (100) was treated with t r i m e t h y l orthoformate in the presence of p y r i d i n i u m ptoluenesulfonate to give the corresponding 2-methoxy-l,3-dioxolane, which was refluxed in acetic anhydride. The Na-acetyl group was then removed by alkaline hydrolysis to afford the desired olefinic compound in 74% yield from diol (100). Finally the protecting group on Nb was removed with Zn in acetic acid to furnish Na-demethoxyrankinidine (23) in 78% yield. The synthetic compound (23) had spectral properties (1H NMR, 13C NMR, IR, UV, high resolution mass and CD) in accord with those of the natural product. On Nb-methylation of 23 with formalin and N a C N B H 3 in the presence of AcOH, a new indole alkaloid N a demethoxyhumantenine (24) was obtained (78). The appearance of a new indole alkaloid N a - m e t h o x y - 1 9 ( Z ) anhydrovobasinediol (7) (Section I-1) suggests the possibility that the Namethoxyindole would be a biogenetic precursor of Na-methoxyoxindoles such as the humantenines in the Gelsemium alkaloids. Alternatively, by the introduction of an oxygen function into the oxindoles such as Nademethoxyrankinidine (23), Na-methoxyoxindoles would be generated. The preparation of Na-methoxyindoles and Na-methoxyoxindoles was achieved by the oxidation of the dihydroindole derivatives as follows (79). Indoloquinolizidine (102) was reduced with NaBH4 in trifluoroacetic acid to give the indoline derivative (103) in 94% yield. On oxidation with 10 equiv, of aqueous hydrogen peroxide (H202) in methanol-H20 (10:1) in the presence of 0.2 equiv, of sodium tungstate (Na2WO4o2H20) and successive t r e a t m e n t with ethereal diazomethane (CH2N2)(80), the indoline afforded the Na-methoxyindole (104) and Na-methoxyoxindole (105) in 49 and 14% yields, respectively. The Na-methoxyindole (104) could be converted to the corresponding Na-methoxyoxindoles (105 and 106) in 30 and 13% yields, respectively, by treating with t-BuOC1 in aqueous THF in the presence of MgO. The stereochemistry of the oxindole (105) was determined by the NOE observation in the N-oxide derivative (107) (Fig. 17). Dihydroyohimbine (109) was also oxidized with aqueous H202 in the presence of Na2WO4 "2H20 and then treated with CH2N2 to afford three products, Na-methoxyyohimbine (111, 45% yield) and two Na-methoxyoxindole derivatives (112, 2% yield and 113, 2% yield) (Fig.
498
8
NOE
H
7
NaBH4 CF3COzH 94%
2
Indoloquinolizidine 102
OMd,N"%0 fi ~2b 107
~
121Na2WO4~ 31% H20 2 CHzN2
CPBA
.~H $$t,o-
MeO 105 (14%) (30%) $
104(49%)
MeO
106 (13%) )
-(
t-BuOC1 (1.3 eq.), MgO (5 eq.)k,. H20 (4 eq.), THF
Fig. 17
H
H NaBH4 N H~"
MeO2C
CF3COzH
-L OH
10 9
Yohimbine 108
11:H
H f H ~
MeOzC:''~
OH
OH
1
1. Na2WO4o2H20 31% H202 2. CH2N2
(
,.~
~ Pyridine, A
IN••
MeO" 7"J" " H."~f N ~ ) 111 (45%) MeO2C~JY OH
112 (2%)
MeO
+
N
113
H
MeO2C~*
MeO2C~" ~ OH 38%
k, t-BuOC1 (1.3 eq.), MgO (5 eq.) H20 (4 eq.), THF
Fig. 18
29%
y~ l
T
J
OH
499
18). The stereochemistry of the spiro position in 112 and 113 was deduced by the comparison of their CD spectra with those of authentic yohimbineoxindoles (81). The isomer (113) was predominantly epimerized to 112 in hot pyridine. A similar behavior was observed in two yohimbine-oxindoles, also supporting the assumption that 112 has the 7S and 113 has the 7R c o n f i g u r a t i o n , respectively. N a - m e t h o x y y o h i m b i n e ( 1 1 1 ) was successfully converted into Na-methoxyoxindole derivatives (112 and 113) in 38 and 29% yields, respectively, by treating with t-BuOC1 in aqueous THF in the presence of MgO.
116
W06
~
I
/
H
H
OH
,,.--
(O)H
I
I H
O
H
114
W06
115 I
-
Fig. 19
HOH2C. ]
N
N
118
117
HOH2C_
HOH2C NaCNBHa
1. Na2WO4 ~ ~ ( ' H202 ._
CF3C02 H
2. CH2N2
93% 9
OH
OH 2
119
oN '
49% 120
Fig. 20
D.
NazWO4, H202
I
aq. MeOH
H 121
I
OH 118
Fig. 21
H
500
The plausible mechanism of the direct formation of Na-hydroxyindoles from the dihydroindoles by the oxidation with H202/Na2WO4 system can be considered as follows. On attempts at the oxidation of the Namethoxyindole (104)with the H202/Na2W04 system, the formation of the corresponding Na-methoxyoxindoles (105) and/or (106) was not observed at all. Therefore, onto the benzylic position of the nitrone intermediate (115) (82), an OH or OOH function might be oxidatively introduced, and subsequent pinacol-type rearrangement in 117 might give the Nahydroxyoxindole 118 (Fig. 19). The procedure thus developed was applied to the sarpagine type alkaloid, 19(E)-koumidine (9) giving Na-methoxy-19(E)-koumidine (120) in 49% yield from the corresponding dihydroindole (119) (Fig. 20) (83). Oxidation of 3,3-disubstituted indoline derivatives (121)with 5 equiv of H 2 0 2 . H 2 N C O N H 2 complex in the presence of a catalytic amount of Na2WO4 (82b) also affords the Na-hydroxyoxindole derivatives (118) in moderate yield (84a). By applying this procedure, the synthesis of a new humantenine-type alkaloid, 20-hydroxydihydrorankinidine (26), starting from ajmaline (66) was achieved (84b). II-3. Koumine-Type Alkaloids The biosynthetic route of the novel cage structure of koumine (29), the principal indole alkaloid of Gelsemium elegans Benth, would diverge from a key intermediate, 19(Z)-anhydrovobasinediol (6). Oxidation of the allylic C-18 position in 6 would give an unnatural (not yet isolated) 18-hydroxy19(Z)-anhydrovobasinediol (122), and subsequent intramolecular coupling between the C-7 and C-20 position would produce koumine (29) (Fig. 21). Originally, Lounasmaa and Koskinen proposed the biogenesis of koumine starting from 18-hydroxy-desoxysarpagine (85). Based on the biogenetic hypothesis, the synthesis of koumine (29) was accomplished by three groups independently. The present author's group has initially realized the biogenetic concept above using 18-hydroxygardnerine (123) isolated from Gardneria nutans. Thus, the C / D ring-fission compound (124) was converted to the 18-Oacetate (125), which was then treated with Nail and Pd(PPh3)4 to undergo a transannular SN2' cyclization to give the hexacyclic compound (126). This was transformed into unnatural 11-methoxykoumine (128) by two steps. The 1H- and 13C-NMR as well as the CD spectra are comparable with those of koumine except for the aromatic portion (86). Next, natural koumine was prepared from the alkaloid (129) (Fig. 24) (87). Removing the methoxy group from the indole nucleus was achieved
501
-
O
O
18 19
122
19(Z)-Anhydrovobasinediol
6
~
O
e
K o u m i n e 29
Fig. 22
T e n t a t i v e b i o g e n e t i c r o u t e o f t h e Gelsemium a l k a l o i d s - 3
9....,)'~N ~M,....fik,.. / N . I
Meu
~
-
"
-N-
THF-H20 MeO" ~ rt, 3.5 h y . 85%
~
~/
-,,,~
125
"~,,,
~
ii. Pd(OAc) 2, PPh3 90 ~ 5 h y. 56%
7
;e
COOMe
O
N'Me
o
Pb(OAc)4 Benzene MeO rt, l h
9 l]
y. 78%.
7 v
m
Me , N" -~- , / , ~ v
MeO,~~
.~ N~.,,..,,,,,~O/
-~ l l-Methoxykoumine 128
Fig. 23
LiA1H4 THF rt, 1.5 h y. 67%.
126
o
127
%H
~~~_~~
[~.
18 up, c
MeO
rt, 15 h y. 77%.
124
COOMe aeO" ~
-~!/
L,,,.oH
123
Ac20, Py.
COOMe
ClC02Me, MgO ~
502
o
0
I
i-~ii _
MeO
I~1
II
--RI,"/~'~'~" 7 ~ y ~ 18~OH
18-hydroxygardnutine 129
.Me
x NH Koumine 29
I/
viii-x I
"
18 ~OR3 RI=OMe, R2=H, R3=H
] 2,3
RI=OMe, Rz=Ts, R3=Ac RI=OH, R2=Ts, R3=Ac RI=OTf, R2=Ts, R3=Ac RI=OTf, R2=Ts, R3=H RI=H, R2=Ts, R3=H RI=H, R2=H, R3=H
130 131 132 133 134 135
N
7
"OR2 RI=CO2Me, R2=H 136 R 1=Me, R2=H 137 RI=Me, R2=Ac 138
7
~
9
H H
RO---~ zo I H 137u
HFI~ 137s
R
Reagents and conditions i, LiA1H4, THF, reflux, 4 h. ii, Ac20, pyridine, r.t., 15 h; TsC1, n-ButNHSOt, 50%KOH-benzene, r.t., 2h, 86% from (129). iii, AICI3, EtSH, CH2CI2, -18~ 7 h, 95%. iv, (CF3802)20 , Et3N, CH2C|2, -18~ 15 min, 82%. v, aq. 5%K2C03, MeOH, r.t., 10 min, 86%. vi, Pd(OAc)2, DPPF, Et3N, HCO2H, DMF, 60~ 2 h, 97%. vii, LiA1H4, THF, reflux, 11 h, 96%. viii, C1C02Me, MgO, THF-H20, r.t., 2.5 h. ix, LiA1H4, THF, r.t., 6.5 h, 41% from (135). x, Ac20, pyridine, r.t., 1 h, 96%. xi, Nail, DMF, r.t. 10 min, then Pd(OAc)2, PPh3, 90~ 1 h, 80%.
F i g . 24
503
by the same three-steps operations shown in section II-1, demethylation of the aryl methyl ether, trifluoromethanesulfonylation of the resultant phenol, and reductive deoxygenation assisted by a palladium catalyst. The 11-demethoxy derivative (135) thus obtained was converted to 18hydroxyanhydrovobasinediol (137) by C / D ring-opening with methyl chloroformate followed by reduction of c a r b a m a t e with LiA1H4. Compound (137), a hypothetical precursor to koumine (29), could form two conformational isomers ( 1 3 7 s ) a n d (137u). Carbon-carbon bond formation between C7 and C20 could happen via the ~-orbital overlapping in the folded form (137u). Based on the calculation by the MNDO method (88), the extended form (137s) (DHf=12.3 kcal/mol) is more stable than (137u) (DHf-17.8 kcak/mol). However, the energetic difference is only 5.5 kcal/mol, so that facile conformational change from (137s) to (137u) could be expected. Actually, to the indole anion prepared from the 18-O-acetate (138) by treating with sodium hydride in DMF was added 0.1 eq. of Pd(OAc)2 and 0.5 eq. of triphenylphosphine and the mixture was stirred for 1 h at 80-90~ After purification with silica gel column chromatography, koumine (29) was obtained in 80% yield. The synthetic koumine (29) showed no depression of mixed mp. and had spectral properties (1H-NMR, UV, High resolution MS, and CD) in accord with those of natural koumine. Another biomimetic partial synthesis of koumine (29) was reported in 1986 by Liu et al (Fig. 25) (89, 1). Vobasine (139) on reduction with LiA1H4 gave vobasinediol which was dehydrated with aqueous sulfuric acid to afford anhydrovobasinediol (16). Allylic oxidation of 16 with SeO2/H202 gave koumine (29) in a modest (25%) yield. Application of this procedure to 19(Z)-anhydrovobasinediol (6) led to the formation of a trace amount of koumine (29) detectable by HPLC analysis (90). Thirdly, total synthesis of antipodal koumine was achieved by Magnus et al. In the final stage for coupling the C-7 and C-20 position, they utilized the Mitsunobu condition (diethyl azodicarboxylate, Ph3P, a catalytic amount of imidazole, and Nail) and obtained koumine in moderate yield (73). HO
0
Se02 H202 y. 25% Vobasine 139
Anhydrovobasinediol 16 F i g . 25
Koumine 29
504 II-4. Gelsemine-type Oxindole alkaloids A biogenetic speculation previously proposed for gelsemine (37) included a crucial carbon-carbon bond formation between C-6 and the allylic C-20 position in the oxindole alkaloid, humantenine (1, 12a, 39). A new hypothetical biosynthesis of gelsemine involves the ene-type reaction between C-6 and C-20 in the indolenine intermediate (140) generated from (85) by the elimination of HX. Through the second ~-oxidation of the indole portion in 141 and successive rearrangement to the oxindole, gelsemine (37) would be formed. Since the structure elucidation in 1959, a number of synthetic approaches to the fascinating alkaloid gelsemine (37) having a compact hexacyclic skeleton have been published (91). Very recently, a formal total synthesis of 37 accomplished by Johnson et al. was introduced into a review by Saxton (9 lq).
06 N
~
H
-HX r_-- ~
85
140
141
~ ~~/~'o
H
II
'-~Me ~
37 gelsemine
H o
"-NMe ~ i ~ 39 gelsevirine
"--NMe Me
19-hydroxydihydrogelsevirines
Fig.26 Tentativebiogenetic route of the Gelsemium alkaloids-4
505 II-5. Gelselegine- and Gelsedine-Type Oxindole Alkaloids Gelsedine-type alkaloids have a novel oxindole skeleton missing the C21 carbon from the humantenines. A biogenetic route initially speculated for gelsedines involves the release of the C-21 carbon in the early stage of the biogenesis (Fig. 27 route A). Thus, a hypothetical intermediate, Dnorsarpagine type indole compound (142), would be generated by the loss of the C21-aldehyde carbon from the common intermediate (65) followed by ring closure between Nb and C-20. Compound 142 would be converted to the C / D ring-opening compound (143) and then transformed into the gelsedine series via the oxidative rearrangement to oxindole (92). Ajmaline (66), which could be considered as a synthetic nearly equivalent with the hypothetical biogenetic intermediate (65), was chosen as a starting material in the synthetic study of gelsedines based on the above biogenetic speculation (Fig. 28). The first task was removing the CStrictosidine 62
1
OH:" 16 H
HO
O~ ~
-C21 Koumidine4
Humantenine 17
~
HOl,.
Fig. 27 Tentative biogenetic route of the Gelsemium alkaloids-5
~ /
~
OMe Gelsedine48 Gelsenicine(ANb'20)52
0,~,
Elegansamine58
506
21 aldehyde carbon from ajmaline (66) (92). Masked aldehyde existing as an amino acetal function in 66 was converted to the aldehyde (145) in 64% overall yield by the three-step operations, i.e. formation of N,Ndimethylhydrazone, protection of the liberated Nb as benzyl carbamate under Schotten-Baumann condition, and hydrolysis of the hydrazone with CuC12 (pH 7), as already described in section II-1. The aldehyde function in 145 was converted to the silyl enol ether in 71% yield by t r e a t m e n t with two equivalents of TBS-trifluoromethanesulfonate, and then a hydroxy group was introduced onto the a-position of the aldehyde in 81% yield by exposure of silyl enol ether to OsO4 in pyridine-THF. The aldehyde function in 146 was reduced with NaBH4 in MeOH, and the CC bond in the resultant glycol was cleaved with NaIO4 in MeOH to yield the C20-keto compound (147) in 76% overall yield from 146. The carbonyl group at C-20 was reduced with NaBH4 in iso-propanol/H20/CH2C12 at -20~ to give two diastereomers (148) and (149) in the ratio of 3.7:1 in a quantitative isolated yield. In order to determine the configuration at C-20 of these alcohols, ring closure between C-20 and Nb was performed. Each alcohol (148) and (149) was respectively converted to mesylate and then subjected to hydrogenolysis to give the tertiary amines ( 1 5 1 ) a n d (152). The stereochemistry at C-20 in 151 and 152 was d e t e r m i n e d by spectroscopic analysis, as follows. In the 13C-NMR spectra, the signal due to C-5 of compound (151) was observed downfield (5.8 ppm) and, on the contrary, that of C-14 was observed upfield (4.9 ppm) compared to the corresponding signal of the C20-epimer (152). This phenomenon can be reasonably interpreted in terms of the ~,-gauche effect due to the a-ethyl group on C-20 in 151. Differential NOE experiments also supported the configuration of the C-20 position in both compounds. Thus, irradiation of H-20 (5 2.97) in 151 led to enhancement (21%) of H-5, indicating that the ethyl group lies anti to H-5. From these results, compound (151) had the desired configuration at C-20 for the synthesis of gelsedines. Based on a mechanistic consideration, the alcohol (148) has the S configuration at the C-20 position. The (R)-alcohol (149) was converted to the (S)-isomer by repeating the oxidation (Swern method)-reduction (NaBH4 at-20~ sequence. The next requirement of this work was the transformation of the indoline moiety to the indole. After the acetylation of the C-20 hydroxy group in 148, the TBS ether on the C-17 hydroxy group was substituted for TMS ether to afford 150 in 95% overall yield from 148. On oxidation of Na-methyl indoline 150 with Pb(OAc)4 in CH2C12 at-70~ indolenine derivative 153 was obtained in 68% yield. Compound 153 exhibited the characteristic indolenine absorption [208, 221(s), 228(s), 253 nm] in the UV spectrum. The TMS ether in 153 was cleaved in AcOH/THF/H20
507 OR1
OH
OR iii, iv
66 ajmaline
_
~eH
21 CHO
20 ii
L 145: R~=Re=H 146: RI=TBS, R2=OH vii,viii,ix
R 1 0 - - ~ 16 H
OTMS
--Cbz
R~ "R2
v (~ 147: R=TBS, RI+R2=O 148: R=TBS, RI=H, R2=OH~ _ __.149: R=TBS, RI=OH, R2=H"'~ 150: R=TMS, RI=H, R2=OAc I
~
OTBS
/
N t:1
H#' "'OR2
14 15 151: Ri=H, R2=Et 152: Rl=Et, R2=H
153
154: R~=H, R2=Ac -3 xii,xiii,xiv 155: RI-TBS, R2-Ms~
I XV
\ 156: R=TBS -~ . 142:R=H ~)xvl / J
/
158
157
14
. XVlll OsO4
F
~ N -f-~H-~
/ 159A L
extended form
160
R CO2tBu
161
Reagents and conditions: i, H2N-NMe2, cat.H2S04, 3A-MS, EtOH; CBZ-C1, 1N-NaOH/CH2C12, 80% from (66). 9CuCl 2, aq. THF(pH7), 80%. ii, TBSOSO2CF3, Et3N, CH2CI2, 71%; 0s04, THF-Py., NariS03 aq, 81%. iii, NaBH4, MeOH, 92%. iv, NaI04, MeOH, 83%. v, L-Selectride, THF, -70~ (148) 60%, or NaBH4, iso-Propanol/H20/CH2C12,-20~ (148) 78%, (149) 21%. vi, MsC1, Py, then H2, Pd-C, AcOH, EtOH, 83% (151) from (148), 48% (152) from (149). vii, Ac20-Py., 96%. viii, n-Bu4NF, THF, quant, ix, TMSOSO2CF3, CH2C12, 90%. x, Pb(OAc)4, CH2C12,-70~ 68%. xi, AcOH-THF-H20 then NaBH4, MeOH, 80%. xii, TBSOSO2CF3, Et3N, CH2C12, 80%. xiii, 5% aq.KOHIMeOH, 90%. xiv, MsC1, Et3N, DMAP, CH2C12, 93%. xv, H2, Pd-C, AcOH, EtOH, 66%. xvi, n-Bu4NF, THF, 90%. xvii, BrCN, MgO, benzene, (157) 48%, (158) 30%. xviii, (BOC)20, DMAP, CH2C12, 97%. xix, Os04, THF-Py., NariS03 aq. xx. AcOH, H20, MeOH, 42% from (159).
Fig. 28
508 (3:1:1) at rt, and after the evaporation of the solvent under reduced pressure, the resultant unstable aldehyde was immediately reduced with NaBH4 in MeOH at 0~ to give the indole (154) in 80% yield without the epimerization at C-16. After the protection of the primary alcohol in 154 with TBS ether, the acetate was converted to mesylate 155, which upon hydrogenolysis (Pd/C, H2, EtOH/AcOH) provided the D - n o r s a r p a g i n e derivative (156), mp 244-246~ in 66% yield by ring closure between Nb and the C-20 position. The stereochemistry at the C-16 and C-20 position was unambiguously determined by the differential NOE spectra of 156. Thus, the irradiation of the H-16 (5 2.46) led to enhancement (9% and 7%) of the H-5 (5 3.74) and H-20 (5 3.05), respectively. Furthermore, 7% enhancement was observed between the H-3 (5 4.24) and H2-19 (5 1.63 and 1.53). By removing the TBS ether in 156 with n-Bu4N+F- in THF, the D-norsarpagine type indole compound (142), mp 269-270~ was obtained in 90% yield. On treatment with BrCN and MgO in benzene under reflux conditions, compound 142 afforded the C/D ring-cleaved product (157) in 48% yield accompanied by the 2-vinyl derivative (158). Treatment of NaBOC derivative (159) successively with OsO4 in pyridine/THF and then with A c O H / M e O H / H 2 0 at 80~ gave the oxindole (161) as the sole product in 70% overall yield from 159. However, the CD spectrum of 161 demonstrated that 161 had the opposite configuration at the spiro C-7 position compared with that of natural gelsedine-type oxindoles. The stereochemical course of this oxidation-rearrangement reaction was speculated as follows, assuming that, in the transition state 159 took the folded conformation A rather than the extended conformation B (Fig. 28) from careful 1H-NMR analysis. OsO4 might attack from the less hindered ~-side to generate the diol (160). Subsequent treatment of this intermediate with aqueous acetic acid provided oxindole (161) having the C7(R) configuration via a stereoselective pinacol-type rearrangement. The formation of the C7(R) isomer from 159 using Os04 oxidation is in contrast to the results obtained in the humantenine-type series (75). Thus, the C/D ring-opening derivatives such as 93 and 99 from the compounds having six membered D - r i n g gave the oxindoles with the C7(S) configuration using the same oxidizing reagent (Section II-2, Fig. 15 and 16). This knowledge based on the chemical reactions as well as the appearance of a new Gelsemium alkaloid gelselegine (56) (57) led to a consideration of the possibility of an alternative biogenetic pathway for gelsedine-type alkaloids (93). Thus, enzymatic oxidation of sarpagine-type indole alkaloids, such as koumidine ( 4 ) a n d 19-(Z)-anhydrovobasinediol (6), would first provide the humantenine-type oxindole alkaloids having the
509
C7(S) configuration, and subsequent ring contraction of the ninemembered ring through the elimination of the C21 carbon would furnish gelsedine-type alkaloids (Route B in Fig. 27). Base on the second biogenetic speculation, chemical transformation of the sarpagine-type alkaloid, gardnerine (10), was carried out. The C/D ring cleaved compound (93) readily available from (10) by t r e a t i n g with 2,2,2-trichloroethyl chloroformate in THF was subjected to OsO4 oxidation (1 equiv.) at-70~ for 1.5h (Fig. 29). The reaction proceeded stereoselectively to yield the oxindole (162) possessing the C7(S) configuration in 52% yield, accompanied by the 19,20-diol derivative (95) (19%) and the recovered starting material (18%). The stereochemistry at C-7 was confirmed by the CD spectrum. Treatment of 162 with trimethylsilyl chloride and sodium iodide in acetonitrile at rt for 10 min resulted in the olefin migration to provide the enecarbamate (163) (93) (5 6.52, s, H-21) in 95% yield. Oxidation of the double bond in 163 with OsO4 afforded the diol (164) (5 5.65, d, J=2.4Hz; +D20, s, H-21) and the aldehyde (165) (5 9.46, CHO) in 84% and 15% yield, respectively. Reduction of both 164 and 165 gave the same diol derivative (166). The C-21 carbon was removed in 97% yield by the oxidative cleavage of the glycol system in 166 with sodium periodate in MeOH. The resultant ketone in 167 was reduced with NaBH4 to give two diastereomers 168 and 169 in 79% and 20% yield, respectively. The major isomer 168 was subjected to ring closure between the C-20 and Nb position. The mesylate (170) prepared from 168 in 59% yield was treated with sodium hydride in dry THF to yield the carbamate (171). The protecting group in 171 was removed with Zn in AcOH to afford the secondary amine ( 1 7 2 ) ( m p 242-243~ 84% yield from 170. The stereochemistry at the C-20 position in 172 was determined by the differential NOE experiments. Irradiation of H-20 (5 3.03, t, J=7.4 Hz) led to enhancement (4.2%) of 14-Ha (5 2.01, br. d, J=15.1 Hz). This indicates that the configuration at C-20 in 172 is opposite to that of the natural gelsedine series. In turn, treatment of 167 with Zn in AcOH gave the imine (173) in 88% yield. Compound (173) showed a definite absorption at 1630 cm-1 due to the C=N function. The catalytic reduction (PtO2/H2)of 173 afforded stereoselectively the desired amine (174) (mp 248~ in 98% yield. The stereochemistry at the C-20 position was unambiguously d e t e r m i n e d by the comparison of 13C-NMR spectra and NOE experiments. By irradiation at one of the H2-19 (5 1.72, m) and H-20 (5 2.94, m) in 174, 4.5% and 5.9% enhancements of H~-14 (5 2.20, br. d, J=15.4Hz) and H-16 (5 2.46, quint, like J 4.5Hz) were observed, respectively. Furthermore, in the 13C-NMR spectra the signal of C-14 in 174 was observed upfield (8.9 ppm) compared to the corresponding signal
510
;~~C02
R
1. leq. OsO4, THF-Py.
TMSC1, NaI
2R
MeCN, rt y. 97%
2. NaHSO3 93 R=CH2CCla
162
y. 52%
MeO,~~
02R
1.OsO4, THF-Py.
+
R02CH
2. NaHSO3 163
!
x
164 (84%)
165 (15%) NN~NaBH4 MeOH
~ . t ~ ~
Zn, AcOH
O~~20
0 NaIO4 aq. MeOH 97%
\'-"'% o" ,.,s,,,
167
173
RO2uN BH4~ Na MeOH ~
/
/
[ PtO2,H2 { EtOH 98%
"~0
98% Me
~'~
~.
2o/
168: R,=OH, R2=H(79%) 169: RI=H, R2=OH (20%) ~ 170 R10Ms R2 H J : = ' =
NaH, THF -~ ~ Zn, AcOH, 84%
..fiN
MeO" v
166
R02C"I~ nl
.N,~0 ~I ~R2
172: RI=R2=H, R~=Et 174: R~=R3=H, R2=Et
.,,,OH
"a, l, " ~ ~ ' , , ~ -%~ "N "D m2
/ \ 2 0 ~, f,,! ..-,~,.,.,~.,"~3
171: RI=CO2R,R2=H, R3=Et
-
~ _
~
O~ 14~'~~
H ~k
1
50 Gelsemicine
"N~ ~'0 OMe
Fig. 29
MsC1 Et3N CH2C12
511
of the diastereomer (172), due to the y-gauche effect of the ethyl group. The 1H-NMR spectrum of 174 closely resembled that of gelsedine (48) except for the signals of the oxindole moiety. Other physical and s p e c t r o s c o p i c d a t a also s u p p o r t e d the s t r u c t u r e of N a desmethoxygelsemicine (174). Conversion of 174 to the natural alkaloid, gelsemicine (50), by introduction of a methoxy function onto the Na group using the method developed above was accomplished (94). Gelselegine 56 and l l-methoxy-(19R)-hydroxygelselegine 57 are new skeletal types of oxindole alkaloids having a hydroxymethyl group at the C-20 position, meaning that the C-21 carbon is rearranged to the exo position on the D-ring (57). Biogenetically, the alkaloid 56 might be derived from the humantenine-type oxindole rankinidine (19)via the formation of an aziridinium intermediate such as 177 formed from 20hydroxydihydrorankinidine (26) or by ring-closure at C-19 in rankinidine (19), and then ring-opening by the attack of water at the C21 position in 177 (Fig. 30). ll-Methoxy-19-hydroxygelselegine (57) would be g e n e r a t e d v i a the formation of epoxide ( 1 7 8 ) a n d rearrangement to the aziridinium intermediate (179). Oxidative cleavage of the 1,2-aminoalcohol function in gelselegine (56) would afford gelsenicine (52), a member of the gelsedine-type alkaloids, and subsequent reduction of the C=N bond in (52) would give gelsedine (48) in the plant. By addition of the monoterpene unit (144) to gelsenicine (52) at the nucleophilic C-19 position, a novel alkaloid elegansamine (58) would be formed (Fig. 27). The gelsedine skeleton was alternatively prepared from a sarpaginetype alkaloid via the gelselegine type compound (183) (Fig. 31) (94). As already described above, gardnerine (10) was converted to the oxindole (162). Attempts at the formation of aziridine (181) from the amine (180) were unsuccessful. The glycol in (166) was converted to the epoxide (182) by the conventional method. On deprotection of the Nb carbamate, the resultant free nitrogen attacked the epoxide at the C-20 position (5-exot e t r a h e d r a l system) (95) regio- and stereoselectively to give the gelselegine skeleton (183) in 82% yield. The stereochemistry at the C-20 was elucidated by the NOE observation between H-16 and H-21 and comparison of the 13C-NMR between n a t u r a l gelselegine ( 5 6 ) a n d synthetic (183). The 1,2-aminoalcohol in 183 was oxidatively cleaved with NaIO4 to give the gelsenicine congener (173), which was identical with the sample prepared by a different route (Fig. 29). In turn, an other member of the gelselegine-type compounds having 19-hydroxy group was also prepared stereoselectively from the same
512
O
O N-N ' ~
I
20-Hydroxydihydrorankinidine 26
O
R
O
N
N I OMe
20 19
H
~"'
H--N
20
21 176
R=OMe: Humantenirine 18 R=H: Rankinidine 19
177
~" OH2
/
l
m
~,~
O 21 ,---OH
o
~\ ~ o
.o
due Gelselegine 56
1
1
O
die 2~.._OH
Gelsenicine 52
"~
Me
N ~Me
11-Methoxy-19(R)-hydroxygelselegine57
C)Me Gelsedine 48
Fig. 30
Tentative biogenetic route of the Gelsemium alkaloids-6
513
7SOIr ~ 2 ~/ ~
.,
AcOH ~
Gardnerine 1
MeO
N H
H
0
R=CH2CCI3
162
21
180
,
! I !
i o_~
"'~O .
i. MsC1 PY'-CH2C12 ~'~N ~ ii.
K2CO3 MeOH
182
~,
MeO
H
N
H
21
181
166
Zn, AcOH 16 xH .
NOE
~.~CH2OH
~.
_
i
.~" 21
NaI04
r
H
H
183
173
~"/~-----~ Fig. 29
Gelsemicine 5 0
Fig. 31
intermediate (93) (Fig. 32) (96). The diol function at C19-C20 in 95 was converted t o the epoxide 185 (1. MsC1, Et3N in CH2C12, 2. K2CO3 in MeOH) in 76% overall yield. Using this procedure, the epoxide could be obtained, which has the same relative stereochemistry between the C-19 and C-20 positions as that derived by the direct epoxidation of the (19Z) ethylidene side chain in Gelsemium alkaloids such as 19. Removal of the Nb protecting group in 185 with zinc in AcOH gave secondary amine 186 in 95% yield. The differential NOE experiment between H-19 and H-15 suggested the desired (19R) configuration. The amine-epoxide 186 was then heated in dioxane at 120~ for 11h to give aziridine 187 (mp 243248~ in 60% yield, whose structure, including the stereochemistry at C19 and C-20, was established by single crystal X-ray analysis. Treatment of 187 with trifluoroacetic acid in THF at 85~ for l h furnished Nademethoxy- 11-methoxy-(19R)-hydroxygelselegine 188 (rap 279-283~
514
in 81% yield by regioselective ring opening at C-21. The 1H- and 13CNMR spectra of 188 strongly resembled that of natural 57 except for the signals of the Na-methoxy portion in the oxindole moiety. Comparison of the CD spectrum of 188 with that of 57 confirms the absolute configuration of the new alkaloid 57. A synthetic approach to gelsemicine (50) was reported (97).
i. CIC02CH2CCI3 MgO, aq.-THF rt, h (93)
O N e / OH OMs ?S /,, " ~ _~ M e
I
G a r d n e r i n e 10
L
O
K2C03
MeO
MeOH 0 ~ 15 min y. 92%
184
:'% 19R
[ N H
IE
L
MeO~NI'~O
ii. OsO4 (2 eq.), Py.-THF, rt, I h iii. NariS03, rt, 4 h y. 78% (95) iv. MsC1, Et3N, CH2C12 rt, y. 83%
Zn, AcOH
Me
185
rt, 8 h y. 95%
i NI/20 kH19S
/OYR
'"
R=CH2CC13
C ~ 9 R
~MeO~N, % H
H:V
~H-~Me
186
%9% NOE
Dioxane 120~ 11 h sealed tube y. 60%
%~-~ x ~k.~ ~
MeO" v
j~,,~
~19R
"N" -u H
H/~~OH E,le
187 ORTEP Drawing of the Compound 187 O
CF3COOHy._ ~ reflux,i h y.81% THF
MeO
~,,,~
N
H 188
NOE 3 and 4 %
H"
OH
H r.
Me
Fig. 32
515 REFERENCES 1 Z. J. Liu and R. R. Lu, in : A. Brossi (Ed.), The alkaloids. Vol. 33: Gelsemium
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518 82 a) H. Mitsui, S. Zenki, T. Shiota, and S. Murahashi, J. Chem. Soc., Chem.
85 86
Commun., 1984, 874-875. b) S. Murahashi, T. Oda, T. Sugahara, and Y. Masui, J. Org. Chem., 55 (1990) 1744-1749. H. Takayama, N. Seki, and S. Sakai, to be published. a) H. Takayama, N. Seki, and S. Sakai, to be published, b) C. Phisalaphong, H. Takayama, and S. Sakai, to be published. M. Lounasmaa and A. Koskinen, Planta Medica, 44 (1982) 120-121. S. Sakai, E. Yamanaka, M. Kitajima, M. Yokota, N. Aimi, S.
87 88 89 90 91
Wongseripipatana, D. Ponglux, Tetrahedron Lett., 27 (1986) 4585-4588. H. Takayama, M. Kitajima, and S. Sakai, Heterocycles, 30 (1990) 325-327. M. J. S. Dewar and W. Thiel, J. Am. Chem. Soc., 99 (1977) 4899-4907 and 4907-4917. Z. J. Liu and Q. S. Yu, Youji Huaxu, 1 (1986) 36-37. M. Kitajima, unpublished result. a) A. Ashimori and L. E. Overman, J. Org. Chem., 57 (1992) 4571-4572. b) A.
83 84
92 93 94 95 96
Madin and L. E. Overman, Tetrahedron Lett., 33 (1992) 4859-4862. c) L. E. Overman, M. J. Sharp, J. Org. Chem. 57 (1992) 1035-1038. d) C. J. Flann, L. E. Overman, and A. K. Sarkar, Tetrahedron Lett., 32 (1991) 6993-6996. e) W. G. Earley, E. J. Jacobsen, G. P. Meier, T. Oh, L. E. Overman, Tetrahedron Lett. 29 (1988) 3781-3784. f) W. G. Earley, T. Oh, L. E. Overman, Ibid, 29 (1988) 3785-3788. g) M. M. Abelman, T. Oh, L. E. Overman, J. Org. Chem. 52 (1987) 4130-4133. h) D. J. Hart, S. C. Wu, Tetrahedron Lett., 32 (1991) 4099-4102. i) J. K. Choi, D. C. Ha, D. J. Hart, C. S. Lee, S. Ramesh, S. Wu, J. Org. Chem. 54 (1989) 279-290. j) G. Stork, G. Nakatani, Tetrahedron Lett. 29 (1988) 2283-2286. k) G. Stork, M. E. Krafft, S. A. Biller, Ibid, 28 (1987) 1035-1038. 1) I. Fleming, R. C. Moses, M. Tercel, J. Ziv, J. Chem. Soc., Perkin Trans. 1, 1991 617-. m) C. Clarke, I. Fleming, J. M. D. Fortunak, P. T. Gallagher, M. C. Honan, A. Mann, C. O. Nfibling, P. R. Raithby, J. J. Wolff, Tetrahedron 44 (1988) 3931-3944. n) I. Fleming, M. A. Loreto, J. P. Michael, I. H. M. Wallace, Tetrahedron Lett. 23 (1982) 2053-2056. o)H. Hiemstra, R. J. Vijn, W. N. Speckamp, J. Org. Chem. 53 (1988) 3882-3884. p) R. J. Vijn, H. Hiemstra, J. J. Kok, M. Knotter, W. N. Speckamp, Tetrahedron, 43 (1987) 5019-5030. q) J. E. Saxton, Nat. Prod. Reports, 9 (1992) 393-446. H. Takayama, M. Horigome, N. Aimi, and S. Sakai, Tetrahedron Lett., 31 (1990) 1287-1290. H. Takayama, H. Odaka, N. Aimi, and S. Sakai, Tetrahedron Lett., 31 (1990) 5483-5486. M. Kitajima, H. Takayama, and S. Sakai, to be published. J. Baldwin, J. Chem. Soc., Chem. Commun., 1976, 734-736. H. Takayama, M. Kitajima, K. Ogata, and S. Sakai, J. Org. Chem., 57 (1992)
4583-4584. 97 N. K. Hamer, J. Chem. Soc., Chem. Commun., 1990, 102-103.
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 15 9 1995 Elsevier Science B.V. All rights reserved.
519
Chemistry, Biochemistry and Chemotaxonomy of Lupine Alkaloids in the Leguminosae Kazuki Saito and Isamu Murakoshi
1. Introduction The lupin(e) alkaloids are a group of alkaloids possessing quinolizidine ring or piperidine ring in molecules. This group of compounds comprises ca. 200 structurally related alkaloids that represent 2% of the 10000 known alkaloids in nature [1 ]. These alkaloids are distributed in the family Leguminosae, subfamily Papilionoideae (Lotoideae, Faboideae, Papilionatae), in particular, in the species in the more primitive tribes of the Papilionoideae, but not in the other subfamilies Caesalpinioideae or Mimosoideae. These alkaloids are of importance to mankind because they are toxic to human and livestock, and some of these alkaloids show pharmacological activities. Indeed, some lupin-alkaloid containing plants have been used as sources of crude drugs in Chinese and Japanese traditional medicine. In these traditional medicinal plants, lupin alkaloids are proven to be the principal constituents responsible for the pharmacological activities found in these herbal medicines. Besides their pharmacological activities, inhibitory activities for larval feeding of insects and for plant germination have been also demonstrated. Numerous original research articles have been published on various aspects of the chemistry and biochemistry of lupin alkaloids. Some excellent reviews and chapters in books have attempted to provide comprehensive lists of known lupin alkaloids [2-6]. Recent advances, specially on the chemistry of these alkaloids, have appeared in the annual reviews published by the Royal Society of Chemistry [7]. Therefore, in this chapter we summarize the natural occurrence and structural characteristics of newly discovered alkaloids in our recent investigations with brief comments on the spectroscopy of the alkaloids. We also describe the summarized results of biochemical aspects including plant tissue culture and works on isolated enzymes. Chemotaxonomic and pharmacological significance of the alkaloids will be given briefly. 2. Isolation and structure of new lupin alkaloids
Lupin alkaloids can be classified into seven structural groups according to tile carbon skeleton and the oxidative states (Fig. 1). The alkaloids of lupinine- and
520 dipiperidine-types are formed with two units of cadaverine which is the decarboxylated product of lysine; whereas the alkaloids of sparteine-, anagyrine-, matrine-, and tsukushinamine-types are constructed with three units of cadaverine. Camoensidine is presumably formed with two cadaverine units and one putrescine unit which is a decarboxylated diamine of ornithine. Our recent investigations on the plants mainly grown in Japan have led to the isolation of a number of new alkaloids and the confirmation of the structures including absolute configurations of previously isolated bases. These forty four alkaloids are listed in Table I. 1. Lupinine type 11
C
5 H~7
2. Sparteine/lupanine type 17
H2OH
5 H 7 ./~ 4
15
{ 6~ 1 4 N 13
98
3
3
(-)-Lupinine (1)
,tl0"
~i-2
(-)-Sparteine (2)
3. Anagyrine/cytisine (o~-pyridone) type
0
O (-)-Anagyrine (4)
(-)-Cytisine (5)
6. Dipiperidine type
5. Matrine type
O (+)-Lupanine (3)
4. Camoensidine type
0 (-)-Camoensidine (6)
7. Tsukushinamine type
14
m
12
3%.,,N (+)-Matrine (7)
N
coc. (+)-Ammodendrine (8)
O (-)-Tsukushinamine-A (9)
Fig. 1 Types of structure of lupin alkaloids in Leguminous plants
Table I List of newly isolated lupin alkaloids in recent studies on the plants mainly grown in Japan Compound
Structure
Name
Absolute configuration
Source
Reference
(-)-(iruns-4'-hydroxycinnamoy1)lupinine (Rl=H, R,=H)
Lupinus hieus L. (seedling)
11
(-)-(iruns4'-rhamnosyloxycinnamoy1)lupinine (R,=a-L-Rha, R2=H)
Lupinus hieus L. (seedling)
12
(-)-(nuns-4'-rhamnosyloxy3-'methoxycinnamoyl)lupinine (Rl=a-L-Rha, R2=OMe)
Lupinus Juieus L. (seedling)
13
(-)-(nuns-4'-P-D-glucopyranosyloxycinnamoy1)lupinine (R1=a-D-Glc, R2=H)
Lupinus iuteus L. (seedling)
14
(-)-(irans4'-B-D-glucopyranosyloxy3'-methoxycinnamoyl)lupinine (Rl=a-D-Glc, R,=OMe)
Lupinus iuieus L. (seedling)
12
15
(-)-(irans4'-a-L-rhamnosyloxycinnamoy1)epilupinine @=a-L-Rha)
Lupinus hirsuius L. (aerial parts)
13
16
(-)-(cis4'-a-L-rhamnosyloxycinnamoy1)epilupinine @=a-L-Rha)
Lupinus hirsuius L. (aerial parts)
13
17
(+)-(iruns-4'-acetoxycinnamoy1)epilupinine @=Ac)
Lupinus hirsuius L. (seedling)
14
8
R2
A"
: iram
A7'8 : trans AT8' .si.,
(continued)
Vl
2
ul
N N
Table I (conrinued) Structure
Compound
Name
Absolute configuration
Source
Reference
Maockia amurensis var. buergeri and M . tashiroi Makino (fresh stems)
15
Sophora chrysophylla (arerial parts)
16
H NHCOCH3
0
lR, 6R
18
(-)-lusitanine
19
(-)-mamanine N-oxide
20
(-)-epilamprolobine
5R, 6s
Sophora tomentosa L. (leaves)
17
21
(+)-epilamprolobine N-oxide
lR, 5R, 6s
Sophora tomentosa L. (leaves)
17
22
5-(3'-rnethoxycarbonylbutyroyl) aminomethyl-trans-quinolizidine N-oxide
Sophora tomentosa L. (leaves) (possibly artifact)
17
+ - + f o
23
tashiromine
Maackia rashiroi Makino (stems)
18
24
(+)-13P-hydroxymamanine
Maackia amurensis var. buergeri (stems)
19
Cyrisus scoparius Link (aerial parts)
20
Thermpsis lupinoides Link (seeds)
21
Thermopsis chinensis Benth (aerial parts) (Determination of absolute configuration)
22
OH
CH20H
J$p%H ($p
HO
25
(-)-3, 13a-dihydroxylupanine
26
(+)-lupanine N-oxide
27
(+)-5,6-dehydrolupanine
3s. 6R, 7s. 9s. 11S, 13s
0
0
0
0
l R , 9R, I l R
(continued)
Table I (continued) Cornpu nd
Name
Absolute configuration
Source
Reference
Lygos raetam var. sarcocarpa (aerial parts)
(in preparation)
6S, I R , 9R, 11R
LYWS raetam Var. sarcocarpa (aerial part)
23
6 ~is,, 9s, 11s
Lupinus hirsutus L. (seedlings)
24
(-)-13a-tigloyloxymultiflorine
Lupinus hirsurus L. (seedlings)
14
(-)-A5-dehydroalbine
Lupinus rermis Forsk (Seeds)
25
28
(-)-&-hydroxylupanine
29
(+)-12a-hydroxylupanine
30
(-)-multiflorine N-oxide
0
32
33
v3N> 0
(-)-A5-dehydromuItiflorine
34
N-(3-0xobutyl)cytisine
Echinosophora koreensis Nakai (aerial pans)
27,28
35
(-)-N-ethylcytisine
Echinosophoru koreensis Nakai (fresh flowers)
29
36
(-)-12-cytisineacetic acid
Euchresta japonicu Benth (aerial parts)
30, 31
37
(-)-N-fomylcytisine
Thermopsis chinensis Benth (air-dried and
32
0
0
0
0
finely ground roots)
(continued) v, N
v,
tn
Table I (conrinued) Structure
@$0COCH3
Compound
Name
38
(-)-0-acetylbaptifoline
39
(+)-11-oxmytisine
40
(-)-12-cytisineacetamide
41
42
Absolute configuration
I R , 9R, 11R
Source
Reference
Thermopsis chinensis Benth 33 (roots)
0 Sophora secundiflora (leaves)
34
Sophora exigua Craib (dry roots)
35
(-)-teuahydrocytisine
Thermopsis chinensis Benth (roots)
(in preparation)
(-)-camoensidineN-oxide
Maackia tashiroi (stems)
I R . 9s
0
36
43
(+)-5a, 9a-dihydroxymatrine
Euchresta horsfeldii (E.formosana cv riukiensis) (leaves)
37
44
(+)-5,17-dehydromatrine N-oxide
Euchresta japonica Benth (areial parts)
38
45
(-)-sophoridine N-oxide
Euchresia japonica (aerial parts)
39
46
leontalbinine N-oxide
Sophora flavescens var. angustifolia
40
(See&)
(continued)
Table I (continued) Structure
d? N
I
Me
Compound
Name
Absolute configuration
Source
Reference
47
(-)-5a-hydroxysophocarpine 5R, a, 7R, 1 1 ~Sophoraflavescens Solander ex &ton var. angustifolia Kitagawa (seeds)
48
(-)-7,11 -dehydromatrine ((-)-leontalbinine)
Sophora flavescens (fresh flowers)
42
49
(+)-maackiamine
Maackia murensis var. buergeri Rupr. et Maxim (flowers)
43
50
(+)-kuraramine
Sophora flavescens (flowers)
44
51
isokuraramine
Sophora flavescens (flowers)
42
41
9
(-)-tsukushinamine-A
6R, 7R. 9s. 14R
Sophora franchetiana Dunn
45,46
~arts)
0
52
(-)-tsukushinamine-B
6R, 7R, 9S, 14s
Sophorafranchetiana Dunn (epiged parts)
47
Sophorafranchetiana Dunn (epigeal parts)
47
0
VI
N W
530 2.1 Lupinine-type alkaloids Several esters (10-17) of (-)-lupinine (1) and (+)-epilupinine (54) were isolated from Lupinus species. The carboxylic acid moieties of these esters are the derivatives of hydroxycinnamic acid and its glycosides. Lusitanine is a rare alkaloid only found in Maackia species and Genista lusitanica [48], and each of the enantiomeric bases were isolated from these two genera. (-)-Mamanine N-oxide (19) and (+)-1313hydroxymamanine (24) are classified in the lupinine group by their structures; however, these alkaloids are likely biosynthesized from t~-pyridone-type alkaloids (see section 4.3). (-)-Epilamprolobine (20) and its derivatives (21, 22) are assumed to be tricyclic quinolizidine alkaloids derived from a possible bicyclic intermediate (55) formed with three units of cadaverine [17]. Tashiromine (23), an analogue of 54, has the indolizidine ring instead of an quinolizidine ring. These two analogous alkaloids, 23 and 54, co-exist in the same plant, Maackia tashiroi.
H CliO
(+)-Epilupinine (54)
55
H
(-)-Multiflorine (56)
2.2 Sparteine/lupanine-type alkaloids A number of oxygenated alkaloids of lupanine and (-)-multiflorine (56), positional analogues of oxo-group in ring A, were isolated from the seeds and seedlings of several plants. The absolute configurations of multiflorine-type alkaloids are all 7S:9S. The ~-pyridone alkaloids, representated by anagyrine (4) and cytisine (5), are derivatives of lupanine. The alkaloids of this group exhibit characteristic features in the ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra, due to the ~pyridone ring. These spectroscopic characters are useful for identification of the newly isolated alkaloids. All t~-pyridone alkaloids so far isolated belong to an enantiomeric series of 7 R : 9 R , although both antipodal alkaloids, 7R:9R and 7S:9S, were found in sparteine/lupanine-type alkaloids. This may imply an interesting biogenetic significance as discussed in section 4.2. Lygos raetam var. sarcocarpa is a desert shrub growing in the desert of the Sinai Peninsula. This plant contains the oxygenated sparteine-type alkaloids at unusual positions, i.e., (+)-aphylline (57), (+)-retamine (58) and the derivatives. The structure of 58 was confirmed by X-ray analysis (in preparation).
531 (-)-Camoensidine (6) and its derivatives (42) were isolated from genus Maackia together with tashiromine. These alkaloids possessing indolizidine ring are presumably formed with two cadaverine units and one putrescine unit. H
-
,%
N
H -
,,,,,
%,
OH
O
(+)-Aphylline (57)
N
(+)-Retamine (58)
N
.CH3
O
(-)-N-Metylcytisine (59)
2.3 Matrine-type alkaloids The new alkaloids recently isolated in this category are the oxygenated or dehydrogenated forms of (+)-matrine (7), which is assumed as a structural counterpart of lupanine in the sparteine/lupanine group (see section 4.1). The flowers and seeds of Sophora species accumulate a variety of oxygenated matrine-type alkaloids, suggesting the particular physiological functions of the oxygenated alkaloids in the reproductive organs of these plants. These matrine-type bases are contained in the plants of primitive tribes, i.e., Sophoreae and Dalbergieae, in the subfamily Papilionoideae. 2.4 Dipiperidine-type alkaloids (+)-Maackiamine (norammodendrine) (49) is a pyrrolidinyl analogue of ammodendrine (8). This alkaloid co-exists with camoensidine (6), which possesses an indolizidine ring, in the flowers of Maackia amurensis. (+)-Kuraramine is a possible oxidative metabolite of N-methylcytisine (59), as discussed in section 4.3. 2.5 Tsukushinamine-type alkaloids Tsukushinamine A-C are novel cage-type lupin alkaloids isolated only from Sophora franchetiana, which is locally native and a very rare shrub in Japan. The absolute configuration of tsukushinamine A was determined by the X-ray analysis of its hydrobromide salt. Tsukushinamine A and B were effectively derived by means of intramolecular photocyclization from a common lupin alkaloid (-)-rhombifoline (60), which co-exists in S. franchetiana [49] (Fig. 2). This suggests the possible biosynthetic mechanism involving photo-mimetic activation of the o~-pyridone precursors for the formation of these unique cage-type alkaloids. 3. Spectroscopy for structure determination of lupin alkaloids Common techniques of spectroscopy in natural product chemistry, e.g., NMR,
532 9,,,,
N-- CH2CH2CH=CH2 hv
~--
9+52
O (-)-Rhombifoline(60) Fig. 2 An effective phototransformationof (-)-rhombifoline(60) into tsukushinamine-A(9) and B (52) mass, infrared (IR), UV, circular dichroism (CD) etc., are useful for determination of structures of lupin alkaloids. Since general methods for interpretation of each spectra of these physicochemical techniques are extensively described in books and reviews [5056], some useful comments on the characteristic spectral features for the structure determination are briefly given in this section. For the interpretation and assignment of individual signals in the spectra of the various alkaloids, the readers can consult the original papers published.
3.1 13C- and 1H-NMR spectroscopy Since, in the 1H_NMR spectra of lupin alkaloids, most signals of alicyclic hydrogens appear in the range of S 1-2.5, it is not easy to assign all overlapping signals. However, several characteristic features in the spectra are helpful for structural elucidation. In the 1H-NMR spectra of lupanine-type alkaloids, only the signal of H10c~
(equatorial) resonates downfield ca. $4.5, because of the deshielding effects of the amidocarbonyl residue. This downfield shift is even more remarkable in the signals of both H10c~ and H10 ~ of ot-pyridone alkaloids. These two methylene hydrogens appear as the AB part of ABX-type system by spin-coupling with H 9. This signal pattern is common in the spectra of ~-pyridone alkaloids and can be ascribed to the steric relation of the C-10 hydrogens and the carbonyl residue, which is positioned at the center of the angle between H10ot and H1013 (Fig. 3).
Heq HeC~;IN~-'~L~ql..Iax ~eq ~_~ Hax .. ,
.~.~C ~ ,
O11H~
Ca" "-'~, 5a
O H 17HL-~N~
H
Ho~ 7a
Fig. 3 Conformation of (-)-cytisine (5) and (+)-matrine (7)
533 In the 1H-NMR spectrum of (+)-matrine (7), three hydrogens of H17ot, H1713 and H l l are observed in the downfield region ((54.5-3.1). The hydrogen at 17~ is in the same electronic and steric environments as the H 10c~ of lupanine and it is deshielded by the adjacent amido group as mentioned above. The hydrogens at 1713 and 11 are affected by the axial lone pair of N 1 and consequently shifted downfield (Fig. 3). These are characteristic features of the spectra of alkaloids having the stereostructure of matrine-type bases. These downfield shifts are more pronounced in the spectra of Noxides of matrine-type alkaloids. 13C-NMR spectroscopy is also an important technique for structural study of lupin alkaloids. Experiments using various standard pulse techniques provide us much information on the structures. The substitution effects of hydroxy and N-oxide groups are observed in chemical shifts of carbon signals, and they are, therefore, useful for determination of the positions of substitutions. In these days, 2D-NMR techniques on superconducting high-resolution NMR instruments (1H, ~600MHz) are becoming common in laboratories. Among these 2D-techniques, NOESY is quite useful for the determination of configuration and conformation of lupin alkaloids.
3.2 Mass spectroscopy In most cases, the molecular ions of lupin alkaloids are detectable in electron impact (El) ionization technique, and, therefore, useful for determination of molecular mass and composition by the combination with high resolution mass spectrometry. However, N-oxides usually exhibit very small molecular ion peaks in the E1 mode. Inbeam ionization technique provides relatively strong molecular ions of the alkaloid Noxides. However, fast atom bombardment (FAB) mass spectrometry has been recently proved very useful for the determination of the molecular masses of N-oxides. In the El mass spectra of ~-pyridone alkaloids, the fragment ions at m/z 146 and 160 appear generally. These ions are assigned as 61 or 62 and 63 or 64, respectively. In lupinine-type alkaloids, the characteristic ions at m/z 169 (65) and 152 (66) are useful for structural elucidation. In the E1 mass spectra of matrine-type alkaloids, the ions M +o and [M-H] + are dominant. Additionally the fragment ions with loss of CH 2 (14) or CH (-13) units from M +o also appear sequentially with almost equal intensities in the spectra of matrine-type bases. Combined gas chromatography and mass spectrometry (GC/MS), in particular, equipped with high-resolution capillary GC, is quite powerful for identification and quantification of known alkaloids from small quantities of plant materials.
3.3 Miscellaneous methods In the IR spectra of lupin alkaloids, Bohlmann bands are characteristic for molecules having trans-quinolizidine rings and are useful for structural elucidation. The UV spectra of a-pyridone and multiflorine-type alkaloids show absorptions at
534
.C,. H2
(+)
+
(+) or
c 89
0
(§
0
61
o
62
o
63
m/z 160
64
m/z 146
4-. OH
65
m/z 169
66
m/z 152
310 nm and 326 nm, respectively. These UV absorptions are useful for monitoring the eluates by high performance liquid chromatography (HPLC) [57, 58]. The CD spectra provide information about the absolute stereochemistry of these alkaloids. The signs of Cotton effects due to the chirality of structurally similar chromophore predicts the absolute configuration of the alkaloids by comparison to that of known compounds. For the quantification of each known alkaloid in the plant materials, the most reliable data are obtained by the combination of normal-phase HPLC, reverse-phase HPLC and capillary GC/MS. The N-oxides of lupin alkaloid can be normally detected by HPLC. 4. Biochemical and physiological aspects of alkaloid biosynthesis 4.1 Production of matrine in cell culture and proposed enzymatic mechanism of biosynthesis In vitro tissue and cell cultures of lupin plants are not appropriate systems for the study of biosynthesis of lupin alkaloids, because the production ability by in vitro culture is rather low, i.e., 10 -2 to 10-3 times compared with that of differentiated plants. The production of the alkaloids of lupinine- and sparteine-groups by cell culture have been reported by us [59] and by Wink's group [60]. We have also successfully produced matrine in green callus culture and in multiple shoots of Sophora flavescens [61]. The producibility of matrine was positively correlated with the chloroplast formation. This indicates that the formation of carbon skeleton of matrine-type alkaloids also likely takes place in chloroplasts in plant cells as postulated in that of sparteine-type alkaloids [62].
535 We propose the biosynthetic pathway of the carbon framework of matrine as shown in Fig. 4. This scheme also indicates the pathway for the formation of sparteine and lupanine. The former part of this scheme was proposed by Wink et al. [63], with minor modification by Leete [64], from the in vitro experiments using isolated chloroplasts of leaves of Lupinus. They postulated the presence of 17-oxosparteine as the first key intermediate for the formation of lupanine and sparteine [63]. However, this hypothesis involving 17-oxosparteine synthase was not confirmed by the tracer experiments using 2H and 13C independently conducted by the groups of Spenser [65, 66] and Robins [67]. They, in turn, hypothesized the pathway involving the diiminium cation (73) as the tetracyclic intermediate [68, 69]. The postulation of the presence of this reactive intermediate is consistent with the results of isotope incorporation into lupanine and sparteine. The biosynthetic scheme of matrine can be also drawn by involving the electronically equivalent diiminium cation (76) preceded by additional 1,3-hydride shift or imine-enamine isomerization (74 ~ 75). All these reactions take place via enzyme-bound intermediates possibly binding to pyridoxal 5'-phosphate.
4.2 Absolute configuration in biosynthesis of enantiomeric alkaloids Some interesting relations are observed in the absolute configuration and biosynthesis of the alkaloids. Firstly, in nature, there are enantiomeric series of sparteine/lupanine-type alkaloids (Table II). Both antipodal alkaloids, 7S:9S and 7R:9R alkaloids, exist in the group of saturated-ring A alkaloids, e.g., sparteine and lupanine; whereas the alkaloids of ot-pyridone-ring A, e.g., anagyrine, cytisine and their derivatives, have only 7R:9R configuration. Secondly, the plants in genus Thermopsis accumulate both enantiomeric alkaloids, i.e., (7S:9S)-lupanine and (7R:9R)-c~-pyridone alkaloids [33, 70]. A few species of Lupinus contain racemic lupanine and (7S:9S)-multiflorine [25, 26]. These results can be explained as follows. The absolute configuration of C-6, C-7, C-9 and C-11 are determined at the ring-cyclization steps (69 ~ 70 and 71 72) in Fig. 4. As shown in Fig. 5, two steps of double-bond migration to the monocyclic imminium intermediate are crucial for the stereochemistry of tetracyclic rings. in the plants of Thermopsis, the enzyme(s) responsible for the cyclization of rings can catalyze the formation of racemic intermediates. Thus, the two series of intermediates in ring cyclization are enantiomeric. Then (7R:9R)-antipodal intermediate is converted to (-)-lupanine (3a) as the initial product released from the enzyme complex. This 3a is dehydrogenated on ring A to afford sequentially (+)-5,6dehydrolupanine (27), (-)-anagyrine (4) and (-)-cytisine (5) (Fig. 6). On the contrary, the (7S:9S)-intermediate gives (+)-lupanine (3), which, however, is not dehydrogenated, but a part of 3 is converted to (+)-17-oxolupanine (77) and (+)-lupanine N-oxide (26). The enzyme(s) responsible for the dehydrogenation of ring A of lupanine to ~-pyridone
Fig. 4 Tentative biosynthetic mechanism of the sparteine- and the matrine-type alkaloids via enzyme-bound intermediates
Enz I
8
attack from 7-Siface to 6-Si face
attack from 9-Si face to 11-Re face
Cj5" N 1
( 7 s :9 s )
9
69
/
69b
70b
attack from 7-Re face to 6-Re face
Enz
-
Enz
E nz
71b
attack from 9-Re face to 11-Si face
Fig. 5 The ring-cyclization steps in biosynthesis of sparteineflupanine-type alkaloids
72b
(7R : 9R)
ul W W
co2 8
( 7 R : 9 R ) series
[ry+ 1
78
i 2
(-)-Lupanine (3a)
0
27 0
(-)-Tetrahydrocytisine (41)
0
0
26
(+)-17-Oxolupanine (77)
38
Fig. 6 Possible pathway for the formation of enantiomeric alkaloids in genus Thermopsis
539 Table II Stereochemical aspects of the sparteine/lupanine- and the oc-pyridone-type alkaloids in leguminous plants. Genus
(7S :9S)-Alkaloid
( 7R :9R )-Alkaloid
o
o
Sparteine/ lupanine
c~-pyridone
Sophora
-
-
Sparteine/ lupanine
+
(z-pyridone
+
Thermopsis
+
-
-
+
Baptisia
-
-
+
+
Maackia
-
-
+
+
Euchresta
-
-
-
+
Lupinus
+
-
+
-
Cytisus
+
-
-
-
can convert only the ( 7 R : 9 R ) (-)-lupanine (3a) but not the antipodal ( 7 S : 9 S ) ( + ) lupanine (3). In the plants of L u p i n u s , the enzyme(s) responsible for the formation of unsaturated ring A of (-)-multiflorine (56), in turn, accept only the ( 7 S : 9 S ) intermediate as the substrate but not the ( 7 R : 9 R ) - i n t e r m e d i a t e . 4.3
Oxidative
cleavage
of N1-ClO
b o n d in b i o s y n t h e s i s
The structures of (+)-mamanine (80) and (+)-13]3-hydroxymamanine (24) correspond to oxidative products derived from the N l-C10 cleavage of (-)-anagyrine
o._Q. 4 " R=H 79"R=OH
NI+Clo
HOH2~C (+)-Mamanine (80) . R-H 24 " R=OH
Fig. 7 Oxidative cleavage of N1-C10bond in (-)-anagyrine-type compounds
540 (4) and (-)-baptifoline (79), respectively (Fig. 7). Similar relationships in the structures involving oxidative bond cleavage have been found between (+)-kuraramine (50) and ()-N-methycytisine (59), and (-)-pohakuline (81) and lupanine (3). The possible precursors and the corresponding oxidative products in this proposed pathway of transformation always co-exist in the same plants, suggesting the validity of this hypothesis. During the development of flowers of Sophora flavescens, the concentrations of 80 and 50 increase with the concomitant decrease of those of possible precursors, 4 and 59, suggesting that this conversion is a common pathway of the metabolism of lupin alkaloids in floral development.
|
H
CH20H (-)-Pohakuline (81)
4.4 Possible involvement of aza-Cope rearrangement in biosynthesis of albine-type alkaloids Lupinus termis and related species accumulate a group of alkaloids possessing an unusual carbon skeleton, albine-type bases. In L. termis, (-)-albine (88) co-existed with (-)-multiflorine (56), (-)-13ot-hydroxymultiflorine (82) and (-)-ll,12-seco-12,13didehydromultiflorine (83). The hypothetical biosynthetic mechanism is proposed to explain the substitution pattern of the propenyl side chain of 88 in relation to the coexistence of 82 and 83 (Fig. 8). In this proposed pathway, aza-Cope rearrangement is involved as the key step for formation of the allylic side chain at the unusual position, C-13. 83 can also be derived from the same pathway of biosynthesis (84 --> 83). This is a unique example of a biosynthetic mechanism in which the aza-Cope rearrangement is involved.
4.5 Acyltransferase for biosynthesis of ester alkaloids: Study with isolated enzymes Lupinus plants usually contain ester lupin alkaloids. Although the precise functions of these ester alkaloids in plants are not completely understood, these alkaloids are assumed to be end products of biosynthesis and storage forms of alkaloids. A potent activity as larval feeding deterrents for spruce budworm was reported in cinnamoyloxyand tigloyloxylupanine [71]. The most characteristic physiological feature of these alkaloids is that the formation of ester alkaloids is specifically induced during seedling growth up to 2-3 weeks after germination in several Lupinus plants.
541 O
N
(-)-Multiflorine (56)
O
1
i (-)-11,12-Seco-12,13didehydromultiflorine (83)
N r
,
"~o. (-)-13o~-Hydroxymultiflorine (82)
1
-
84
O
N
85
85
O
H2OH
OH
87
86
'~O2 002+H20
~
13
(-)-Albine (88) Fig. 8 A possible biosynthetic pathway involving aza-Cope rearrangement for the formation of albine-type alkaloid
542 In Lupinus hirsutus, (-)-13ct-tigloyloxymultiflorine (31) and (+)-(trans-4'hydroxycinnamoyl)/(trans-4'-hydroxy-3'-methoxycinnamoyl)epilupinine (89, 90) are formed by the different acyltransferases, which require tigloyl-CoA and 4'hydroxycinnamoyl (p-coumaroyl)/4'-hydroxy-3'-methoxycinnamoyl (feruloyl)-CoAs, respectively, as acyl donors (Fig. 9). Partial purification of these acyltransferases revealed some interesting features of these enzymes [72]. The tigloyltransferase showed a single peak on ion exchange chromatography, while the activities of 4'hydroxycinnamoyl- and 4'-hydroxy-3'-methoxycinnamoyltransferase showed two peaks each possessing both activities. The tigloyltransferase could transfer the tigloyl residue not only to 13ot-hydroxymultiflorine (82) but also to 13o~-hydroxylupanine (91). This was indicated from the chromatographic pattern in the enzyme purification and from the results of distributions of ester alkaloids and responsible acyltransferases (Table III). This table also indicates the fact that the specific acyltransferases exist in the plants in which the corresponding products, ester alkaloids, accumulate. In Lupinus luteus, the activity of acyltransferase was detected for (-)-lupinine (1), a diastereomer of epilupinine (54) [73]. 4.6 Enzymatic synthesis of N-substituted derivatives of (-)-cytisine
The N-substituted derivatives of (-)-cytisine (5) are widely distributed in leguminous plants as minor components. The enzymatic activity of S-adenosyl-Lmethionine: (-)-cytisine methyltransferase was detected in the young seedlings of two
82
~
Tigloyl-CoA CoA-SH
~
31
CoA-SH
~20-CO-CH=CH-
~
OH
p-Coumaroyl-CoA (+)-(trans-4'-Hydroxycinnamoyl)epilupinine (89)
O-CO-CH=CH-~OH OCH3
54
Ferul CoA-SH
(+)-(trans-4'-Hydrox y- 3'-methox y-
cinnamoyl)epilupinine (90)
Fig. 9 Acyltransferase-catalyzedformationof the lupin alkaloidesters in Lupinus hirsutus
543 Table III Distribution of acyltransferase activities and ester alkaloids in lupin plants.
Plant species
Enzyme activity (pkat/mg protein)
Alkaloid accumulation
HMTase HLTase ECTase EFTase 13OH-multi. 13OH-lupa. Epilupinine Lupinine deriv,
deriv,
Lupinus hirsutus
1.1
7.5
0.94
+
-
L. termis
3.8
99.8
-
+
+
0.7
26.1
-
+
+
0.56
deriv,
deriv.
L. luteus L. polyphyllus x L. arboreus Thermopsis
N.D.
N.D.
N.D.
lupinoides Baptisia australis
N.D.
N.D.
N.D.
Echinosophora
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
koreensis Sophora flavescens
Key: HMTase, tigloyl-CoA: 13ct-hydroxymultiflorine O-tigloyltransferase; HLTase, tigloyl-CoA: 13othydroxylupanine O-tigloyltransferase; ECTase, p-coumaroyl-CoA: epilupinine O-coumaroyltransferase; EFTase, feruloyl-CoA: epilupinine O-feruloyltransferase; 13OH-multi. deriv., 82 and its derivatives; 13OH-lupa. deriv., 91 and its derivatives; Epilupinine deriv., 54 and its derivatives; Lupinine deriv., I and its derivatives; +, present; -, absent; N.D., not determined.
H
N
"eOH 0 (+)- 13~-Hydroxylupanine (91)
Thermopsis species [74] (Fig. 10). Tile seedlings of Sophora tomentosa and S. flavescens, on the contrary, exhibited the activity of acetyl-CoA:(-)-cytisine Nacetyltransferase [75, 76]. These two enzymatic activities are expressed in the course of seedling development of the specific plant species. 5. Chemotaxonomy and phylogenic relations of lupin plants based on structure and distribution of alkaloids
544
,,,,,
N--COCH3
58
S-AdenosylL-methionine
Acetyl-CoA
in Thermopsis sp.
in Sophora sp.
0 N-Acetylcytisine (92)
Fig. 10 Enzymatic biosynthesis of N-substituted dervatives of (-)-cytisine (5) Table IV
The distribution of lupin alkaloids in plants of the Leguminosae.
Plant
Sophora S. flavescens S. tomentosa S. chrysophylla S. franchetiana S. mollis S. secundiflora S. exi g ua Euchresta E. japonica E. f o r m o s a n a Echinosophora E. koreensis Maackia M. amurensis M. tashiroi M. pubescens M. floribunda Thermopsis T. lupinoides T. chinensis Baptisia B. australis Cytisus C. scoparius Lupinus L. luteus L. hirsutus L. termis Lygos L. raetam
Matrine
+++ +++ ++
Sparteine/ lupanine
+ +++ ++ + +
+++ +++
t~-Pyridone Lupinine
++ ++ + +++ +++ +++ +++ +
++ +++ +
+
++ -t-++ ++ +++
+ +
+++ +++
+
+++
+ ++ +++ +
+++ ++ +++ +++
+++
+++ +++
+
Symbols denote relative amounts of the alkaloids in total alkaloids: +++, >30%; ++, <30%; +, <5%.
545 The distribution of lupin alkaloids in the plants Leguminosae investigated by our recent study is summarized in Table IV. As mentioned before, the unusual types of alkaloids such as kuraramine (50), tsukushinamine (9) and albine (88) are assumed to be biogenetically derived from cytisine (5), anagyrine (4) and multiflorine (56), respectively. Thus, it is reasonable to classify the alkaloids simply into four groups: (i) lupinine, (ii) sparteine/lupanine, (iii) ~-pyridone (anagyrine/cytisine), and (iv) matrine. According to this classification, the lupin plants are divided into three major categories" (i) plants containing lupinine group, (ii) plants containing sparteine and anagyrine groups but no matrine group, and (iii) plants containing matrine group. Kinghorn and Balandrin [3] have discussed the phylogenic relationships of tribes and genera containing lupin alkaloids in subfamily Papilionoideae. By extending their discussion, we can draw the phylogenic tree of the genera investigated extensively by our study (Fig. 11). The genera containing matrine are the most primitive followed by the genera accumulating sparteine- and ct-pyridone-type alkaloids but no matrine bases. The most advanced genera are those containing lupinine-type alkaloids.
I
Thermopsis Baptisia \
Sparteine type I I I
I EUCHRESTEAE
Euchresta
9
9%
ot-Pyridone type
iI
f
O
O
L
""'- /$OPHQREAE
"| Sophora | Echinosophora k Maackia Matrine type Papilionoideae
Fig. 11 Phylogenicrelationships of tribes and genera containing lupin alkaloids in Papilionoideae 6. Biological activity of lupin alkaloids Some pharmacological activities of lupin alkaloids were briefly summarized by Kinghorn and Balandrin [3]. Recently, some additional interesting activities have been revealed by our study (Table V).
By in vivo pharmacological screening, the activities of hypoglycemic, inhibition of edema, inhibition of natural killer cell growth and antiulcerogenic were shown. The inhibitory activities against acetylcholinesterase and 13-glucuronidase were
546 Table V Example of recently examined biological activities of lupin alkaloids. Type of activity
Compound
Reference
Hypoglycemic
56 59 N-oxide of 54 5 59 N-oxide of 54 2
K. Saito et al., unpublished.
Inhibition of edema Inhibition of natural killer cell growth Inhibition of acetylcholinesterase
Inhibition of l]-glucuronidase Antiulcerogenic Larval feeding deterrent Nemoticidal
4 8 13 89 9O 3 89 7 N-oxide of 7 Cinnamoyl- and tigloyllupanine 4 5
K. Saito et al., unpublished. K. Saito et al., unpublished. K. Saito et al., unpublished.
K. Saito et al., unpublished. 77 71 78
demonstrated by in vitro enzymatic assays. The deterrent activity against nematode was also shown with several lupin alkaloids. This activity suggests the possible role of lupin alkaloids for selective advantage in natural selection. 7. Conclusions and future prospects Forty four alkaloids were newly isolated from the 22 lupin plants and the structures were elucidated in our recent study. The structures were determined by spectroscopic investigations, in particular, by extensive 2D-NMR experiments in the recent works, and by chemical transformation. Biosynthetic pathways are also proposed, in some cases, by the preparation of isolated enzymes and by in vitro cell culture system. Chemotaxonomic relationships of the lupin plants are discussed based on the structural classification of alkaloids. In future, it is absolutely necessary to investigate the precise mechanism and regulation of biosynthesis of these interesting alkaloids at the molecular biological level using the technology of gene cloning. The resulting molecular biological data can be applicable to sophisticated engineering of alkaloid production for pharmaceutical and agricultural purposes.
547
Acknowledgments The authors wish to thank H. Suzuki for preparation of the manuscript, and Drs. S. Ohmiya, T. Sekine, H. Kubo and S. Takamatsu for sharing unpublished data. References .
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,
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8. ,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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548 22. K. Saito, S. Takamatsu, T. Sekine, F. Ikegami, S. Ohmiya, H. Kubo, H. Otomasu and I. Murakoshi, Phytochemistry, 28 (1989) 958-959. 23. O. B. Abdel-Halim, T. Sekine, K. Saito, A. F. Halim, H. Abdel-Fattah, and I. Murakoshi, Phytochemistry, 31 (1992) 3251-3253. 24. S. Takamatsu, K. Saito, S. Ohmiya and I. Murakoshi, Heterocycles, 32 (1991) 1167-1171. 25. M. H. Mohamed, K. Saito, H. A. Kadry, T. I. Khalifa, H. A. Ammar and I. Murakoshi, Phytochemistry, 30 (1991) 3111-3115. 26. M. H. Mohamed, K. Saito, I. Murakoshi, H. A. Kadry, T. I. Khalifa and H. A. Ammar, J. Nat. Prod., 53 (1990) 1578-1580. 27. I. Murakoshi, K. Fukuti, J. Haginiwa, S. Ohmiya, and H. Otomasu, Phytochemistry, 16 (1977) 1460-1461. 28. I. Murakoshi, E. Kidoguchi, M. Kubota, J. Haginiwa, S. Ohmiya and H. Otomasu, Phytochemistry, 21 (1982) 2385-2388. 29. I. Murakoshi, M. Watanabe, J. Haginiwa, S. Ohmiya and H. Otomasu, Phytochemistry, 21 (1982) 1470-1471. 30. S. Ohmiya, H. Otomasu, J. Haginiwa and I. Murakoshi, Phytochemistry, 18 (1979) 649-650. 31. I. Murakoshi, M. Watanabe, T. Okuda, E. Kidoguchi, J. Haginiwa, S. Ohmiya and H. Otomasu, Phytochemistry, 24 (1985) 2707-2708. 32. S. Ohmiya, H. Otomasu, I. Murakoshi and J. Haginiwa, Phytochemistry, 13 (1974) 643-644. 33. K. Saito, S. Takamatsu, I. Murakoshi, S. Ohmiya and H. Otomasu, J. Nat. Prod., 52 (1989) 1032-1035. 34. I. Murakoshi, H. Kubo, M. Ikram, M. Israr, N. Shaft, S. Ohmiya and H. Otomasu, Phytochemistry, 25 (1986) 2000-2002. 35. S. Takamatsu, K. Saito, S. Ohmiya, N. Ruangrungsi and I. Murakoshi, Phytochemistry, 30 (1991) 3793-3795. 36. S. Ohmiya, H. Kubo, Y. Nakaaze, K. Saito, I. Murakoshi and H. Otomasu, Chem. Pharm. Bull., 39 (1991) 1123-1125. 37. S. Ohmiya, K. Higashiyama, H. Otomasu, I. Murakoshi and J. Haginiwa, Phytochemistry, 18 (1979) 645-647. 38. S. Ohmiya, H. Otomasu, J. Haginiwa, and I. Murakoshi, Phytochemistry, 17 (1978) 2021-2022. 39. S. Ohmiya, H. Otomasu, J. Haginiwa and I. Murakoshi, Chem. Pharm. Bull., 28 (1980) 546-551. 40. T. Sekine, K. Saito, N. Arai, H. Suzuki, Y. Koike and I. Murakoshi, Yakugaku Zasshi, (in Japanese) in press (1992). 41. K. Saito, N. Arai, T. Sekine, S. Ohmiya, H. Kubo, H. Otomasu and I. Murakoshi, Planta Med., 56 (1990) 421-429. 42. I. Murakoshi, E. Kidoguchi, J. Haginiwa, S. Ohmiya, K. Higashiyama and H. Otomasu, Phytochemistry, 21 (1982) 2379-2384. 43. K. Saito, T. Yoshino, T. Sekine, S. Ohmiya, H. Kubo, H. Otomasu and I. Murakoshi, Phytochemistry, 28 (1989) 2533-2534. 44. I. Murakoshi, E. Kidoguchi, J. Haginiwa, S. Ohmiya, K. Higashiyama and H. Otomasu, Phytochemistry, 20 (1981) 1407-1409. 45. S. Ohmiya, H. Otomasu, J. Haginiwa and I. Murakoshi, Chem. Pharm. Bull. 27 (1979) 1055-1057. 46. J. Bordner, S. Ohmiya, H. Otomasu, J. Haginiwa and I. Murakoshi, Chem. Pharm. Bull., 28 (1980) 1965-1968. 47. S. Ohmiya, K. Higashiyama, H. Otomasu, J. Haginiwa and I Murakoshi, Phytochemistry, 20 (1981) 1997-2001.
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551
SUBJECT INDEX Abrus fruticulosus 26 Abrusosides A-D 25 from Abrus precatorius 25 Abrus precatorius 25 abrusosides A-D from 25 Absolute configuration 76-86, 190 in 24-ethyl-26-hydorxysteroids 84 in 24-(~-hydorxyethyl)-and 24-(carboxymethyl)steroids 79-81 in 24-hydroxymethyl steroids 79 in 24-hydroxysteroids 76, 77 in 26-hydroxysteroids 77, 78 in 24-methyl-25, 26-dihydroxy-steroids 84-86 in 24-methyl-26-hydroxy and 24-methyl26-oic steroidal side- chains 81-84 of sugars saponins 190 Acanthaster planci 61 Acesulfame-K 4 16-Acetoxy-12-O-acetoxyhorminone 167 16-Acetoxy-7-O-acetyl-horminone 167-170, 173 13C_nmr of 170 from Rabdosia lophanthoides var. gerardiana 173 IH-nmr of 169 16-Acetoxy-12-O-acetyl-horminone 168-170, 173 13C_nmr of 170 from Rabdosia lophanthoides var. gerardiana 173 IH-nmr of 169 6'-Acetoxyavarol 301 6'-Acetoxyavarone 301 (+)-(trans-4"-Acetoxy-cinnamoyl) epilupinine 521 from Lupinus hirsutus 521 19(R)-Acetoxydihydrogelsevirine 479 from Gelsemium rankinii 479 from G. sempervirens 479 ent-3ct-Acetoxyisopimar-15-en-8ot-ol 167-170, 173 13C_nmr of 170 from Rabdosiaparvifolia 173 1H-nmr of 169 16-Acetoxy-7ot-methoxyroyleanone 167-170, 173, 175 13C_nmr of 170 from Rabdosia lophanthoides var. gerardiana 173 from R. stracheyi 175 IH-nmr of 169 3-Acetyl-~-D-digitoxose 362 4-Acetyl-6,8-dihydroxy-5-methyl-3,4-dihydro-
isocoumaxin 409 Acetylexidonin 143, 152, 160, 171 13C.nmr of 160 from Rabdosia gaponica vat. glaucocalyx 171 IH-nmr of 152 Acetyl-histamins 328 Achantaster planci 45 t~-Acoradiene 262 13-Acoradiene 261,262 ct-Acorene 260 Actinopyga agassizi 87 Adenanthin 116, 123, 171, 173 from Rabdosia adenantha 171 from R. nervosa 173 1H-nmr of 123 Adrenaline 328 Aesculus glabrus 191 Aesculus saponins 191 Aglycone 362 Agrobacterium rhizogenes 376, 377 AI-77-A to G 390 AI-77-B 393,396,397,402,404, 412-418 synthesis of 412-418 AI-77-G 392 Ajmaline 469 Akuammidine 466, 467 from Gelsemium elegans 466, 467 19 (Z)-Akuammidine 466, 467 Alium vineale 196 24-Alkylated thomasterol A 48 3-Alkyldihydroisocoumarins 386, 387 3-Alkyl-8-hydroxy-3,4-dihydroisocoumarins 386 D-Allomethylpyranose 190 Alternaria kikuchiana tanaka 385 Alternaria solani 346 altersolanol A from 346 Altersolanol A 346 from Alternaria solani 346 Amberlite IR-45 460 Amethystoidin A 173, 176 from Rabdosia macrophylla 173 Amethystonal 112, 114, 121, 128, 171 13C.nmr of 128 from Rabdo sia atnethystoides 171 IH-nmr of 121 Amethystonoic acid 114, 121, 128, 171 13C_nmr of 128
552 from Rabdosia amethystoides 171 IH-nmr of 121 Amicoumacin A, B and C 388 Amicoumacins 410-412 biological activities of 410-412 (+)-Ammodendrine 520 Ammonium d-10-camphor-sulfonate 425 Ammonium glycyrrhizin 5 Amorphane 247 (-)-Anagyrine 520
Androsace saxifragifolia
200
saxifragifolins A and B from
200
trans-Anethole 29 Anhydrovobasinediol 469 19 (Z)-Anhydrovobasinediol 466, 467, 491 Antibacterial substances 439 Antibiotic activity 479 Anti-digoxin antibodies 374 Antimicrobial activity 327-339, 460 of Amphibian venoms 327-339 Antisera 368 Antitumour activity 461 Aphelasteriasjaponica 69 Aphelasteroside A and B 68 Aphomia meUeus 387 Aphomia oniki 383 Aphomia socieUa 384 D-Apiofuranosyl 7 Apioglycyrrhizin 26 Apsena pubescens 384 Arabinogalactin 36 D-Arabino-2-hexulopyranose 190 L-Arabinopyranosyl 7 L-Arabinopyranosyl unit 21 Arabinoside gaudichaudioside A 21 from Baccharis gaudichaudiana 21 Araboglycyrrhizin 26 Arachniodes exilis 33 Arachniodes sporadosora 33
Arbacia lixula 104 Archaster typicus 74, 84 Arenarol 315 (+)-Aristolochene 250 biosynthesis of 250 Aromadendrane 227 Asiaticoside 213 Asperentin 382, 385-387
from Aspergillusflavus 386 Aspergillusduricaulis 343, 345 Aspergillusflavus 386 asperentin from
386
AspergiUus melleus 383 Aspergillus ochraceus 387 ochratoxins from
387
Aspergillus ochraceus wilhelm 384 Aspergillus terreus 385 Asperline 350,351 Aspochalasins 355
Asterias amurensis 44 Asteriasforbesi 58, 69 forbesides A-C from
58
Asterias rubens 48 Asterias vulgaris 59 Asterina pectinifera 44, 48, 55 pectinioside E 48 Asteroidea 43-45 Asterone 45 Asterosaponins 45-58 Aster saponin A 209 Aster saponin H 210 Aster saponin Hd 211
Astropecten aurantiacus 104 Astropecten indicus 61 indicoside A from
61
Astropecten latespinosus 48 Astropecten scoparilis 68 Attenuatoside C 61 from Hacelia attenuata Aureol 300, 314 Avarol 238, 252 Avarol monoacetate 300 Avarone 301,314
61
Azadirachta indica 384
Baccharis articulata 22 Baccharis gaudichaudiana
21 arabinoside gaudichaudioside A from Bacillus pumilus 390 Bacillus subtilis 345 Bacillus thiaminolyticus 390 baciphelacin from 390 Baciphelacin 388, 390, 409, 410 biological activities of 409, 410
21
553 from Bacillus thiaminolyticus 390 Baiyunoside 20, 22 from Phlomis betonicoides 20 Beer's law 423
Beta vulgaris 3 Beyerane 252 Biflorin 258 from Eremophila latrobel 258 Bioactive metabolite 341-356 of genus Phomopsis 341-356 Biogenesis 406-408 of xenocoumacins 406-408 Biogenic amines 328 Bioinsecticides 381 Biological activities 313,314, 408-410, 447, 449, 461,462 of amicoumacins 410-412 ofbaciphelacin 409,410 of PI turnover inhibitors 461,462 of tyrosine kinase inhibitors 447,449 of xenocoumacins 408, 409 Biomimetic reaction 328 Biosynthesis 249, 250, 272, 273,406 of (+)-aristolochene 250 of (+)-eremolactone 272 of ipomeamarone 249 of (+)-isoeremolactone 272, 273 of mellein 406 Bisabolene diterpenes 255-257 Bisabolene isoprenologues 277 Bisdesmosides 188 Bivittosides A, B, C and D 91 from Bohadschia bivittata 91 Blighia welwitschii 205 Bohadschia bivittata 91 bivittosides A, B, C and D from 91 Bombesin-induced phospholipase C activity 447 Borealosides A-D 71 Botryosphaeria obtusa 383 Brasiliensoside 55 Brenzcatechin 328 7-Bromo-6,8-dihydroxy-3-propyl-3,4-dihydroisocoumarin 386 Bromopuupehenone 299 Brownioside 191 Bryonia dioica 24 bryoside from 24
bryonoside from 24 Bryonoside 24 from Bryonia dioica 24 Bryoside 24 from Bryonia dioica 24 Bufogenins 328 Bufotenidin 328 Bufotenin 328 Bufotoxins 328 Bulgarane 247 BuUeyanin 119, 126, 133, 171 13C-nmr of 133 from Rabdosia bulleyana 1H-nmr of 126
171
Cadinane 247 Cadinane sesquiterpenes 278 Caffeic acid 36 Calamanenenes 251 biosynthesis of 251 1R, 4S-Calamanenes 260 (+)-Calamenene 244, 247 from Eremophila drummondii 244 Calamenenes (1R, 4R) 259 Calcicolin A 119, 126, 133, 171 13C-nmr of 133 from Rabdosia calcicolus 171 1H-nmr of 126 Callitristic acid 172 from Rabdosia kunmingensis 172 Camellidin I 202 Camellidin II 202 (-)-Camoensidine 520 m and p-Camphorene 227 from Eremophila cuneifolia 227 (+)-10-Camphorsulfonic acid 425 Candicin 328 Capraria biflora 259 Carbohydrate-molybdate complexes 426-435 5-Carboxymellein 383 Carcinogenesis research 439 Cardenolides 361-380 from Digitalis 361,367 Carolisterol A-C 75, 76 from Styracaster caroli 76 Carpalasionin 174
554 from Rabdosia rugosa 174 Cassia siamea 385 Castanopsis saponins 191 Caulocyctis cephalornithos 386 a-Cedrene 270 Cedrene isoprenologues 269-271 2-epi-a-Cedrene isoprenologue 270 Cellobiose 436 Cembradiene hydroxy ether 253 Cembrane 253 Cembranoids 253-255 cis-Cembratriene 275 cis-Cembrene A 255 Ceratocystis fimbriata 351 Ceratocystis minor 385 Cercospora beticola 351 Cercospora taiwanensis 383 Chaetoglobosins 355 Charonia lampas 58 Cheliferoside 59 from Lathasterias nanimensis chelifera 59 Chemical constituents 43-100 of Echinoderms 43-100 Chlorogenic acid 36 Chloropuupehenone 299 6-Chloro-D-tryptophan 35 5a-Cholesta-9(11), 24(25)-dien-31], 60, 20,22tetraol aglycone 49 5a-Cholestan-3~, 60, 8, 150, 1613,25-hexaol 77 from Protoreaster nodosus 77 (25R) 5a-Cholestane-3~, 5, 6[~, 150, 161326-hexaol 75 (25R) 513-Cholestane-30, 6~, 150, 1613,26- pentaol 75 (24S) 5a-Cholestane-31], 60, 8, 1513,24-pentaolaglycone 61, 68 513-Cholestane-30, 40, 1113, 12~, 21-pentaol 3, 21-disulphate 96 513-Cholestane-30, 40, 1113,21-tetraol 3, 21-disulphate 96 Cholest-5-ene-30, 413, 21-triol 3, 21- disulphate 98 from Ophiura sarsi 98 Chromazonarol 291,298 ent-Chromazonarol 291,299 Chrysanthellin 209 Chrysanthellin B 210 1,8-Cineole 227 from Eremophila dalyana 225,227 from E. scoparia 227
Cinnamomm osmophloeum 29 Cinnamomum sieboldii 33 Cinnamtannin B-1 33 Cinnamtannin D-1 33 Circular Dichroism 193, 194, 423-438 Nakanishi's method 193, 194 of carbohydrate-molybdate complex 423-438 R-(+)-Citronellal 258, 266 S-Citronellate 229 Citrus aurantium 5 Ciwujianosides 191 Cladosporin 382, 385-387 Cladosporin cladosporiodes 386 Cladosporium cucumerinum 64 Claisen rearrangement 243 Claisen rearrangement reaction 79, 81 Classification 112 of Rabdosia diterpenoids 112 Clemensen reduction 240 13C-nmr and structures 197, 198 of sugar chains 197, 198 Coetsoidin A 137, 145, 154, 171 13C-nmr of 154 from Rabdosia coetsoides 171 IH-nmr of 145 Coetsoidin B 117, 124, 131,171 13C-nmr of 131 from Rabdosia coetsoides 171 1H-nmr of 124 Coetsoidin C 137, 145, 154, 171 13 C_nmr of 154 from Rabdosia coetsoides 171 1H-nmr of 145 Coetsoidin D 137, 145, 154, 171 13C-nmr of 154 from Rabdosia coetsoides 171 IH-nmr of 145 Coetsoidin E 137, 145, 154, 171 13C-nmr of 154 from Rabdosia coetsoides 171 1H-nmr of 145 Coetsoidin F 138, 146, 155, 171 13C-nmr of 155 from Rabdosia coetsoides 171 IH-nmr of 146 Coetsoidin G 138, 146, 155, 171 13C-nmr of 155
555
from Rabdosia coetsoides 171 1H-nmr of 146 Compound I 114, 121,175 from Rabdosia umbrosa 175 1H-nmr of 121 Compound 2 114, 121, 128, 175 13C_nmr of 128 from Rabdosia umbrosa 175 1H-nmr of 121 Compound 3 117, 124, 175 from Rabdosia umbrosa 175 IH-nmr of 124 Compound 4 117, 124, 175 from Rabdosia umbrosa 175 1H-nmr of 124 Compound 5 137, 145, 154, 175 13C-nmr of 154 from Rabdosia umbrosa 175 IH-nmr of 145 Convolvulanic acid A and B 342 Convolvulol 342 Convolvulus arvensis 341 Cornitermes ovatus 384 Cornitermes pugnax 384 Corynanthe-type alkaloids 378 Coscinasterias tenuispina 46, 51, 52 Coscinasteroside A 53 Coscinasteroside B 53 Coscinasteroside C 53 Coscinasteroside D 53 Coscinasteroside E 53 Coscinasteroside F 53 Cotton effect 434, 435 Crematogaster deformis mellein 383 Crinoidea 100-103 Crocosimioside H 191 Crossasteroside A 61 from Crossaster papposus 61 Crossasteroside Pl and P2 69 Crossasterpapposus 61, 69 crossasteroside from 61 Crossed cannizzaro reaction 135 Cryptococcus neoformans 408 Cucumaria echinata 94 Cucumaria fraudatrix 87,92 cucumariosides G1, C1 and C2 from
Cucumaria frondosa
92
Cucumaria japonica 87 cucumarioside A2-2 from 87 Cucumaria lefevrei 92 Cucumarioside A2-2 87 from Cuscumariajaponica 87 Cucumarioside G2 89 from Eupentacta fraudatrix 89 Cucumariosides G1, C1 and C2 87, 92 from Cucumariafraudatrix 87, 92 Culcita novaeguineae 69 Culcitoside C1 69 Culcitoside C4-C8 71 Curculigo latifolia 36 Curculin from 36 Curculin 36 from Curculigo latifolia 36 Cyanogenetic glycoside 226 Cyclic steroidal glycosides 59, 60 [2+2] Cycloaddition 266 Cyclohexylamine 4 7,20-Cyclo-ent-kaurane skeleton 167 7,20-Cyclo-ent-kaurenoids 112 Cyclospongiaquinone-I 299 Cyclospongiaquinone-II 299 [2+2] Cycloreversion 266 D-Cysteinolic acid 76 (-)-Cytisine 522 Cytisus scoparius 525 (-)-3, 13ot-dihydroxylupanine from 525 Cytochalasin H and J 353 Cytochalasins N, O, P, Q, R and S 353 Cytopathogenicity 76 Cytotoxicity 356
87, 92
(-)-Dactylosponol 297 (-)-Dactylospontriol 297 Dawodensin A 117, 124, 131,171 13C-nmr of 131 from Rabdo sia dawoensis 171 1H-nmr of 124 Decipiances 263-269 from Eremophila decipiens 263 Dehydroabietic acid 173 from Rabdosia nervosa 173 (-)-AS-Dehydroalbine 524 from Lupinus termis 524
556 Dehydrocyclospongia quinone-I 299 (-)-10,11-Dehydroepingaione 237 Dehydrogeissoschizine 486-488 from strictosidine 487, 488 Dehydroipomeamarone 249 (+)-5,6-Dehydrolupanine 523 from Thermopsis chinensis 523 (-)-AS-Dehydromultiflorine 525 from Lupinus termis 525 R-(+)-10,11-Dehydromyoporene 229 (-)-Dehydromyoporone 229 (-)-10,11-Dehydrongaione 237 Dehydroophioxanthin 100 6,7-Dehydroroyleanone 173, 175 from Rabdosia lophanthoides vat. gerardiana 173 from R. stracheyi 175 Na-Demethoxyhumantenirine 494, 495 Na-Demethoxy-11-methoxy-(19R-hydroxygelselselegine 513 Na-Demethoxyrankinidine 496 from Gelsemium elegans 496 Demethyldihydrokamebacetal A 135 Dendrolasin 233 2-Deoxy-D-glucose 435 Dermasterias imbricata 66 2,4-di-O-methyl-I]-D-quinovopyranosyl-(l>2)-5O- sulphate-13-D-fucofuranosylfrom 66 Desglucosylstevioside 18 from Rubus suavissimus 18 Deslanoside 361, 362 Diacetoxyavarol 300 Diaporte helianthi 345 Dibenz [b,d] oxocin derivative 34, 35 Dictyoceratidaquinone 300 Dictyoceratin-A 292, 293 Dictyochromenol 294 E-2,3-Didehydroaspartic acid 347 E-2,3-Didehydroisoleucine 347 3,4-Didehydroproline 347 3,4-Didehydrovaline 347 7,9(11)-Diene seychellogenin 91 Diginatigenin 362 Diginatin 362 Digitalis lanata 367, 375, 377 cardenolides from 367, 375, 377 Digitalis purpurea 375 Digitoxigenin 362
Digitoxin 362 Digoxigenin 362 Digoxin 362 Digoxin-protein conjugates 366-368 Diglucuronate 26 Dihydrocarpalasionin 143, 152, 160, 174 13C-nnu"of 160 from Rabdosia rugosa 174 IH-nmr of 152 Dihydrochalcones 30,31 R-(+)-Dihydrocitronellic acid 231 Dihydroflavonols 30,31 Dihydrofreelingyne 235 Dihydroisocoumaxins 5, 30, 31, 381-418 3,4-Dihydroisocoumarins 382 Dihydroisodocarpin 141, 149, 158, 174 13C-nrnr of 158 from Rabdosia rugosa 174 IH-nmr of 149 Dihydrokamebakaurin aldehyde triacetate 135 1,2-Dihydrokoumine 476 from Gelsemium elegans 476 Dihydromarthasterone 51 7S, 9R-(-)-9-Dihydromyoporone 230 9-Dihydromyoporone 229 Dihydrophymaspermones 233 DihydropseurataF 117, 124, 131,174 13C-nmr of 131 IH-nmr of 124 from Rabdosiapseudo-irrorata 174 Dihydroquercetin 3-acetate 4'-(methyl ether) 26 (+)-Dihydroquercetin 3-O-acetate 31 Dihydrorugosanin 142, 150, 159, 174 13C-nmr of 159 from Rabdosia rugosa 174 1H-nmr of 150 (2R, 3R)-2,3-Dihydro-5,7,3',4'-tetrahydroxy6-methoxy-3-O- acetylflavonol 32 3,6-Dihydroxy-5,7-dimethylmellein 385 (-)-3,13a-Dihydrox2vlupanine 523 from Cytisus scoparius 523 313,6ot-Dihydroxy-5ot-pregn-9(11)-en-20-one 45 Dihydroxyscrrulatic acid 257 from Eremophila serrulata 257 3,6-Dihydroxy-4,5,7-trimethylmellein 385 6,8-Dihydroxy-3-undecyl-3,4-dihydroisocoumarin 386 from Ononis natrix 386
557 Dihydroyohimbine 487 endo-7,7-Dimethylbicyclo- [3.3.1 ]-nonane3-ol-9-one 135 (2R, 3R)/(2S, 3S)-2,3-Dimethylpentane- 1,2diol-enantiomeric pair 86 2,4-Di43-methyl-13-D-quinovopyranosyl(1>2)-543- sulphate-13-D-fucofuranosyl 66 from Dermasterias imbricata 66 bis-Dinitrophenylhydrazone derivative 228 of myoporone 228 Directed-aldol condensation 15 Distolasterias nipon 55 Diterpenes 252-260 Diterpenoids 111-185 from Rabdosia species 111-185 with ent-kaurene skeleton 112-136 with 6,7-seco-ent-kaurane skeleton 136-162 Dodonea saponins 191 Duasmodactyla kuriIensis 91 kufiloside A from 89 Dubioside F 211 Dulcoside A 16
Echiguanine 458-461 Echiguanine A and B 459 Echinaster brasiliensis 55 Echinaster sepositus 59 Echinoidea 43, 103, 104 Effusanin A 173 from Rabdosia nervosa 173 Effusanin B 173, 175 from Rabdosia langituba 173 from R. ternifolia 175 Effusanin E 173, 175 from Rabdosia nervosa 173 from R. ternifolia 175 Elegansamine 483, 484 from Gelsemium elegans 484 Elemol 244 ELISA 363-366 methodology of 363-366 Ememogin 143, 152, 160, 175 13C-nmr of 160 from Rabdosia trichocarpa IH-nnu" of 152 Ene-reaction 268
175
Engelhardtia chrysolepis 31 Enmein 173, 174 from Rabdosia lophanthoides 173 from R. sculponeata 174 Enmenol 172 from Rabdosiajaponica 172 Entada saponins 191,214 8-Epichromazonarol 291,298 from Smenospongia aurea 291 Epidermal growth factor 441 4-Epihenryine A 114, 121,128, 172 13C-nmr of 128 from Rabdosia henryi 172 1H-nmr of 121 4-Epi-isopimaric acid 172 from Rabdosia kunmingensis 172 (-)-Epilamprolobine 524 from Sophora tomentosa 524 (+)-Epilamprolobine N-oxide 524 from Sophora tomentosa 524 Epinodosin 171, 172, 174 from Rabdosia gaponica var. glaucocalyx from R. henryi 172 from R. japonica 172 from R. sculponeata 174 Epinodosinol 172, 173 from Rabdosmjaponica 172 from R. parvifolia 173 3,20-Epoxy-ent-kaurenes 136 Epoxyketone 167 Epoxyshikoccin 167 Epoxyshikoccin 163-165, 174 13C-nmr of 165 from Rabdosia shikokiana var. occidentalis lH-nmr of 164 22,23-Epoxysteroids 48 Erbstatin 441 Eremane diterpenes 271-274 Eremoacetal 238 Eremolactone 226, 227, 252, 271, 272 from Eremophilafreelingii 252, 271 from E.fraseri 252, 271 (+)-Eremolactone 272 synthesis of 272 Eremophila 225-282 Eretnophila abietina 227 geranyl acetate from 227
171
174
558
Eremophila alternifolia 225, 232 Eremophilacaerulea 227,281 (+)-fenchone from 227 oleanolic acid from 281 ursolic acid from 281 Eremophila clarkei 253 Eremophila cuneifolia 227, 248 m and p-camphorene from 227 (+)-spathulenol from 248 Eremophila dalyana 225, 227 1,8-cneole from 227 Eremophila decipiens 263 decipianes from 263 Eremophila dempsteri 227, 254 (+)-verbenone from 227 Eremophila drummondii 244, 248, 259 (+)-calamenene from 244 serrulatane from 259 (+)-spathulenol from 248 Eremophila duttonii 225 Eremophila eMeri 225 Eremophila exilifolia 252 Eremophilafoliosissima 255 Eremophilafraserii 248, 252, 271 eremolactone from 252, 271 Eremophilafreelingii 233,252, 271 eremolactone from 252, 271 freelingyne from 233 Eremophila georgei 269, 271 Eremophila glutinosa 252 Eremophila gilessi 225 Eremophila granitica 254 Eremophila inflate 229 R-enantiomer from 229 Eremophila interstans 245,247 Eremophila latrobei 229, 232, 258 R and S-enantiomers from 229 biflorin from 258 Eremophila longifolia 225 Eremophila maculata 226, 229 R and S-enantiomers from 229 Eremophila miniata 229 R and S-enantiomers from 229 Eremophila mitchelli 226, 281 Eremophilanes 227, 243 Eremophila paisley 248 (+)-spathulenol from 248
Eremophila petrophila 252 Eremophilaplatycalyx 281 3-epi-oleanolic acid from
281
Eremophila racemosa 248 (+)-spathulenol from
248
Eremophila rotundifolia 233 freelingyne from
233
Eremophila scoparia 227, 232, 244 1,8-cineole from eudesmane from
227 244 Eremophila serrulata 257, 259 dihydroxyserrulatic acid from Eremophila virgata 245
257
Eremophila viscida 260 7-epi-Eremophilene 250 Eremophilone 238-248 from Eremophila mirchelli 238 (+)-Eremophilone 243 synthesis of 243 stereoselective synthesis of 240 Eriocalxin A 176 Eriocalyxin B 176 Eryocalyxin A 171 from Rabdosia eriocalyx 171 Eryocalyxin B 171 from Rabdosia eriocalyx 171 from R. eriocalyx var. laxiflora 171 (-)-N-Ethylcytisine 525 from Echinosophora koreensis 525 24-Ethyl-26-hydroxysteroids 84 absolute configuration of 84 24-Ethyl thomasterol A 48 Eucalyptus spathulata 248 (+)-spathulenol from 248 Eudesmane 227, 244 from Eremophila scoparia 244 13-Eudesmol 243,244 Eupatorium trapezoideum 247 panal from 247 Eupentactafraudatrix 89 cucumarioside G2 from 89 Euphorbiafidjiana 385 5-methylmellein from 385 Euretaster insignis 94, 45 Excisanin A 171, 172, 173, 174 from Rabdosia excisa 171 from R. inflexa 172
559 from R. macrocalyx var. jiuhua from R. serra 174 Excisanin B 171, 173 from Rabdosia excisa 171 from R. macrocalyx var. jiuhua Excisanin C 115, 121,129, 171 13C-nmr of 129
173
173
from Rabdosia excisa 171 1H-nmr of 121 Exidonin (rabdosin B) 143, 152, 160, 172, 173 13C-nmr of 160 from Rabdosia henryi 172 from R. japonica 172 from R. longituba 173 1H-nmr of 152
(+)-Fenchone 227 from Eremophila caerulea 227 Flavones 252 Flavonoids 30 Flexicaulin A 117, 124, 131, 171 13C-nmr of 131 from Rabdosiaflexicaulis 171 IH-nmr of 124 Forbesides A-C 58 from Asteriasforbesi 58 Foroxymithine 449 Freelingyne 233 from Eremophilafreelingii 233 from E. rotundifolia 233 Fromia monilis 60 Frondoside A 92 Frondoside B 94 Furanosesquiterpenes 227-238 Fusarentin 6,7-dimethyl ether 387 Fusarentin 6-methyl ether 387 Fusarium larvarum 381,386 Fusicoccum amygdali 385 5-methylmellein from 385
D-Galactose-molybdate complex 429 Ganervosin A 138, 146, 155, 173 13C-nmr of 155 from Rabdosia nervosa 173 1H-nmr of 146
Ganervosin B 141,173 from Rabdosia nervosa 173 Gardneria nutans 491 gardnerine from 491 Gardnerine 491 from Gardneria nutans 491 Gaudichaudiosides B 22 Gaudichaudioside C 22 Gaudichaudioside F 22 Gelsamydine 483-486 from Gelsemium elegans 484 Gelsedine 481,482 from Gelsemium elegans 482 from G. sempervirens 482 Gelsedine-type alkaloids 481-483 Gelselegine 483,484 from Gelsemium elegans 483 Gelselegine and gelsedine-type oxindole alkaloids 506-515 Gelselegine-type oxindole alkaloids 483-485 Gelsemamide 472, 473 from Gelesemium elegans 472, 473 Gelsemicine 482 from Gelsemium sempervirens 482 Gelsemine 478, 479 from Gelsemium elegans 479 from G. sempervirens 479 Gelsemine N-oxide 479 from Gelsemium elegans 479 Gelsemine-type oxindol alkaloids 478-481, 504, 505 Gelsemium alkaloids 465-515 chemistry of 465-515 Gelsemium elegans 465-476 akuammidine from 466 gelsemamide from 472, 473 humantenine from 472 koumine from 475,476 11-methoxygelsemamide from 472, 473 16-epi-voacarpine 469 Gelsemium rankinii 465 Gelsemium sempervirens 465-467 Gelsemoxonine 481,482 from Gelsemium elegans 482 Gelsenicine 481,482 from Gelsemium elegans 482 Gelsevirine 478, 479 from Gelsemium elegans 479
560 from Gelsemium rankinii 479 from G. sempervirens 479 Gentiobiose 436 Geranyl acetate 227 from Eremophila abietina 227 Geranylgeraniol 253 Germacrene isoprenologue 278 Ginsenosides 191 Gitaloxigenin 362 Gitaloxin 362 Gitoxigenin 362 Gitoxin 362 Glaucocalactone 141, 149, 158, 172 13C_nmr of 158 from Rabdosiajaponica 172 IH-nmr of 149 Glaucocalyxin A 172 from Rabdosia japonica var. glaucocalyx 172 Glaucocalyxin B 172 from Rabdosia japonica vat. glaucocalyx 172 Glaucocalyxin C 112 Glaucocalyxin D 114, 121, 128, 172 13C-nmr of 128 from Rabdosia japonica vat. glaucocalyx 172 IH-nmr of 121 Glaucocalyxin E 114, 121, 128, 172 13C-nmr of 128 from Rabdosia japonica vat. glaucocalyx 172 1H-nmr of 114 Gleditsia japonica 195 Gleditsia saponins 191 Gloeosporium lacticolor 351 Glucogitaloxin 362 (-)-(trans-4 ' -~-Glucop yranos ylo x y cinnamo yl) lupinine 521 from Lupinus luteus 521 (- )-(trans-4 ' -~-D-Glucopyranos ylo x y- 3 ' methoxycinnamoyl) lupinine 521 from Lupinus luteus 521 Glucosyl stevioside 19 Glucuronic acid 26 D-Glucuronopyranosyl 7 Glutinosin 167-170, 172 13C-nmr of 170 from Rabdosia glutinosa 172 1H-nmr of 169 Glycine max 196
soyasaponin A3 196 Glycosides of polyhydroxysteroids 60-72 Glycoxylon huberi 31 Glycyphyllasmilax 31 Glycyphyllin 31 Glycyrrhiza glabra 5 Glycyrrhiza inflata 26 Glycyrrhizin 4, 22, 25 Gomophia watsoni 61 gomophioside from 61 Gomophioside Al9 61 from Gomophia watsoni 61 from Halityle regularis 61 Gossypol 251 Granulatoside A 63 Grapholitha molesta 384 Guidongnin 142, 150, 159, 174 13C_nmr of 159 from Rabdosia rubescens 174 1H-nmr of 150 Gymnema sylvestre 36 Gymnochromes A-D 105-107 Gymnocrinus richeri 105 Gyrostroma missouriense 351,383
Hacelia attenuata 61 attenuatoside C from 61 Haematoxylin 34 Haematoxylon campechainum 34 Hakomori's method 192 Halityle regularis 46, 61 Halityloside D and I from 61 gomophioside A from 61 Halityloside A, I 61 from Halityle regularis 61 Helicacus kanaloanus 383 Henricia laeviuscola 48, 55 henricioside from 48 Henricioside A 48, 64 from Henricia laeviuscola 48 Henryin (reniformin A) 112, 117, 124, 131,172 13C-nmr of 131 from Rabdosia henryi 172 from R. latifolia var. reniformis 172 IH-nmr of 124 Henrycine A 115, 122, 129, 171,172
561
13C_nmr of 129 from Rabdosiaflexicaulis 171 from R. henryi 172 tH-nmr of 122 (+)-Hemandulcin 14-16 6S, l'S-Hernandulcin 14 Hexahydroxydiphenic acid 33 Hexalone 241 Hiburipyranone 386 Histamin 328 HMG-Co A reductase 450 Holothuria leucospilota 87 Holothuria pervicax 92 Holothuria tubulosa 104 Holothurin 87 Holothuroidea 43, 87-94 Holotoxins A, Al, B and B I 87 from Parastichopus californicus 87 from Stichopusjaponicus 87 Horeau's method 76, 270, 475 Horminone 173 from Rabdosia lophanthoides var. gerardiana Horseradish peroxidase 366 Hovenia dulcis 36 Humantenine 472 from Gelsemium elegans 472 Humantenine-type oxindole alkaloids 472-474 Humantenirine 472 Hydrangea macrophylla 5, 30, 387 phylloduclin from 30 Hydrangenol 387 6'-Hydroxy-5'-acetyl-avarol 301 3-Hydroxy-4-alkoxy substitution 30 18-Hydroxyanhydrovobasinediol 503 18-Hydroxy- 19(Z)-anhydrovobasinediol 500 4'-and 5'-Hydroxyasperentin 386 6'-Hydroxyavarol 301 3'-Hydroxyavarone 301 (-)-(trans-4'-Hydroxy-cinnamoyl) lupinine 521 from Lupinus luteus 521 4S-Hydroxy-dendrolasin 233 Hydroxydihydroeremophilone 239, 241 19(R)-Hydroxydihydrogelsemine 481 from Gelsemium rankinii 481 from G. sempervirens 481 19(S)-Hydroxydihydrogelsemine 479 from Gelsemium elegans 479
173
from G. rankinii 479 19(R)-Hydroxydihydrogelsevirine 479 from Gelsemium elegans 479 19(S)-Hydroxydihydrogelsevirine 479 from Gelsemium elegans 479 8-Hydroxy-3, 4-dihydroisocoumarin 384 19(R)-Hydroxy-18, 19-dihydrokoumine 476, 477 from Gelsemium elegans 476 X-ray analysis of 477 19(S)-Hydroxy-18, 19-dihydrokoumine 476 from Gelsemium elegans 476 19(S)-Hydroxydihydrokoumine 475 20-Hydroxydihydrorankinidine 472, 473 X-ray analysis of 472 8a-Hydroxy-7a(H)-eremophila-l,11-diene-9-one 242 8ot-Hydroxyeremophilone 242 24-(I]-Hydroxyethyl)-and 24-(carboxymethyl) steroids 79-81 absolute configuration of 79-81 (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer 443 18-Hydroxygardnutine 502 14-Hydroxygelsedine 482 from Gelsemium elegans 482 from G. sempervirens 482 14-Hydroxygelsemicine 481,482 from Gelsemium sempervirens 482 14-Hydroxygelsenicine 481,482 from Gelsemium elegans 482 4l]-Hydroxyhernandulcin 15 15-Hydroxyhumantenine 472, 473 3-Hydroxyisoleucine 347 (-)-6t~-Hydroxylupanine 524 from Lygos raetam 524 (+)- 12ot-Hydroxylupanine 524 from Lygos raetam 524 (+)- 13~-ttydroxymamanine 523 from Maackia amurensis 523 3-Hydroxymellein 383 4-Hydroxymellein 383 (R)-(-)-5-Hydroxymellein 383 cis-(3R, 4R)-(-)-4-Hydroxymellein 383 trans and cis-(3S, 4S)-4-Hydroxymellein 531 6'-Hydroxy-4'-methoxyavarone 301 24(S)-Hydroxymethyl-cholesta-5, 22(E)-dien313-ols 51 (24S, 22E)-24-Hydroxymethyl-cholesta-5, 22-dien-
562 313-ol 98 (-)-8-Hydroxy-3-methyl-3,4-dihydroisocumarin 383 24-Hydroxymethyl steroids 79 absolute configuration of 79 4-Hydroxyochratoxin A 388 4-Hydroxyphenylethanol 351 24-Hydroxysteroids 76, 77 absolute configuration of 76, 77 26-Hydroxysteroids 77, 78 absolute configuration of 77, 78 5-Hydroxytryptamine derivatives 328 8-Hydroxy-3-undecyl-3,4-dihydroisocoumarin 386 Hymenoxys turneri 32 Hypoxanthine 460 Hypoxanthine-guanine-phosphoribosyltransferase 371
Ilimaquinone 291,314 5-epi-Ilimaquinone 315, 319 Imbricatosides A 64 Immunological assays 361 Immunosorbent assay 361 Indicoside A 61 from Astropecten indicus 61 Indoloquinolizidine 497 Inflexanin A 112, 115, 122, 172 from Rabdosia inflexa 172 IH-nmr of 122 Inflexanin B 112, 117, 124, 172 from Rabdosia inflexa 172 1H-nmr of 124 Inflexarabdonin A 120, 126, 133, 172 13C-nmr of 133 from Rabdosia inflexa 172 1H-nmr of 126 Inflexarabdonin B 117, 124, 131, 172 13C-nrnr of 131 from Rabdosia inflexa 172 IH-nmr of 124 Inflexarabdonin C 114, 121, 128, 172 13C-nmr of 128 from Rabdosia inflexa 172 1H-nmr of 121 Inflexarabdonin D 120, 126, 133, 172 13C-nmr of 133 from Rabdosia inflexa 172 1H-nmr of 126
Inflexarabdonin E 115, 122, 129, 172 13C-nmr of 129 from Rabdosia inflexa 172 IH-nmr of 122 Inflexarabdonin F 120, 127, 134, 172 13C_nmr of 134 from Rabdosia inflexa 172 IH-nmr of 127 Inflexarabdonin G 115, 122, 129, 172 13C_nmr of 129 from Rabdosia inflexa 172 IH-nmr of 122 Inflexarabdonin H 115, 122, 129, 172 13C-nmr of 129 from Rabdosia inflexa 172 IH-nmr of 122 Inflexin 112, 115, 122, 129, 172 13C.nmr of 129 from Rabdosia inflexa 172 1H-nmr of 122 Inflexinol 112, 117, 124, 172 from Rabdosia inflexa 172 IH-nmr of 124 Inostamycin 453-456, 461 Intramolecular aldol condensation 231,235 Intramolecular aldol reaction 269 Intramolecular pinner reaction 415 Introduction 381,382 of xenocoumacins 381,382 Ipomeamarone 249 biosynthesis of 249 (+)-Ipomeamarone 236, 237 (-)-Ipomearone 237 Irreversible cytotoxicity 447 Isocedranol 270 Isochromazonarol 299 Isodocarpin 174 from Rabdosia rugosa 174 Isodomedin 174, 175 from Rabdosiapseudo-irrorata 174 from R. umbrosa 175 from R. umbrosa vat. hakusanensis 175 from R. umbrosa var. latifolia 175 Isodonal 142, 150, 159, 162, 172, 173 13C-nmr of 159 from Rabdosiajaponica 172 from R. macrophylla 173
563
IH-nmr of 150 Isodonoiol 143, 152, 160, 172 13C-nmr of 160 from Rabdosiajaponica 172 IH-nmr of 152 Isodopharicin A 115, 122, 129, 174 13C_nmr of 129 from Isodonpharicus 174 1H-nmr of 122 Isodopharicin B 115, 122, 129, 174 13C_nmr of 129 from Isodonpharicus 174 IH-nmr of 122 Isodopharicin C 115, 122, 129, 174 13C-nrnr of 129 from Isodonpharicus 174 IH-nmr of 122 Isoeremolactone 210 (+)-Isoeremolactone 272, 273 synthesis of 272, 273 Isoeremophilone 243 Isoguanine 460 Isogymnochrome D 105-107 Isolation 388-392 of xenocoumacins 388-392 L-[U-C 14] Isoleucine 347 Isolongirabdiol 142, 151,159, 172 13C_nrnr of 159 from Rabdosia longituba 172 IH-nmr of 151 Isomaltose 436 Isongaione acetate 237 ent-Isopimarane 112 ent-Isopimar-15-en-6ct, 7ct, 8ct-triol 167-170, 173 13C-nmr of 170 from Rabdosiaparvifolia 173 1H-nmr of 169 Isospongiaquinone 315,418 5-epi-Isospongiaquinone 315, 318 Isozonarol 297 Isozonarone 297
Jiuhuanin A 130, 146, 155, 173 laC-nmr of 155 from Rabdosia macrocalyx var. jiuhua IH-nmr of 146
173
Jones oxidation 248 Juniperus virginiana 346 Kalopanax saponins 191 Kamebacetal A 135, 137, 145, 171,172 from Rabdosia excisa 171 from R. henryi 172 from R. latifolia var. reniformis 172 from R. umbrosa var. leucantha 175 IH-nmr of 145 Kamebacetal B 137, 148, 175 from Rabdosia umbrosa var. leucantha 175 1H-nmr of 148 Kamebakaurin 117, 124, 131,171-175 13C_nmr of 131 from Rabdosia excisa 171 from R. henryi 172 from R. inflexa 172 from R. longituba 173 from R. serra 174 from R. umbrosa 175 from R. umbrosa var. leucantha 175 1H-nmr of 124 Kamebakaurinin 117, 125, 131,175 13C_nmr of 131 from Rabdosia umbrosa vat. leucantha 175 IH-nmr of 125 Kamebanin 171,172, 175 from Rabdosia excisa 171 from R. inflexa 172 from R. umbrosa 175 from R. umbrosa var. hakusanensis 175 from R. umbrosa vat. latifolia 175 from R. umbrosa var. leucantha 175 ent-Kauran-16[5, 17-diol 172 from Rabdosia glutinosa 172 Kaurane diterpenes 252 6,7-seco-ent-Kauranes 112 8,9-seco-ent-Kaurane 162 6,7-seco-ent-Kaurane skeleton 136, 162 ent-Kaurene 16, 135 ent-Kaurene glycoside 16-18 from Stevia rebaudiana 16-18 ent-Kaurenoids 112 6,7-seco-ent-Kaurenoid diterpenoid 136 8,9-seco-ent-Kaurenoids 112 Kemebakaurin 135
564 Liangshanin A 114, 121,128, 172 13Conml"of 128 from Rabdosia liangshanica 172 IH-nmr of 121 Liangshanin B 114, 121, 128, 172 13C-nmr of 128 from Rabdosia liangshanica 172 IH-nmr of 121 Liangshanin C 114, 121, 128, 172 13C_nmr of 128 from Rabdosia liangshanica 172 IH-nmr of 121 Liangshanin D 115, 122, 172 from Rabdosia liangshanica 172 IH-nmr of 122 Liangshanin E 115, 122, 129, 172 13C_nmr of 129 from Rabdosia liangshanica 172 IH-nmr of 122 Liangshanin F 116, 123, 129, 172 13C.nmr of 129
Klyne-Hudson rule 207 Kodo-cytochalasin-1 and 2 353 Kograva toxicity 353 Koumicine 466, 467 Koumicin N-oxide 466, 467 Koumidine 466, 467 19(E)-Koumidine 466, 467 from gardnerine 469 Koumine 475, 476 from Gelsemium elegans 475, 476 Koumine Nb-oxide 475 from Gelsemium elegans 475 Koumine-type alkaloids 475-477, 500-503 synthetic studies of 500-503 Kouminol 475 Kuriloside A 89 from Duasmodactyla kurilensis 89
ent-Labdane 252 Laeviuscolosides F and G 64 Laeviuscolosides H and I 64 Lanatoside A 362 Lanatoside B 362 Lanatoside C 361,362 Lanatoside D 362 Lanatoside E 362 Lasiodiplodia theobromae 477 Lasiodonin 172, 173 from Rabdosia gaponica var. glaucocalyx from R. macrophylla 173 from R. parvifolia 173 Lasiokaurin 172, 173 from Rabdosia henryi 172 from R. japonica 172 from R. longituba 172 from R. macrophylla 172 Lasiokaurinol 172 from Rabdosiajaponica 172 Lavendustin 444-447 Lavendustin A and B 445 Lefevreiosides AI, A2, C and D 96 Lemna assays
344
Leptodactylin
328
Lethasterias nanimensis chelifera 59 Leukamenin E 175 from Rabdosia umbrosa var. latifolia 175
172
from Rabdosia liangshanica 172 1H-nmr of 123 Liangshanin G 114, 121,128, 172 13C.nmr of 128 from Rabdosia liangshanica 172 IH-nmr of 121 (+)-Limonene 257 Linckia laevigata 46, 61 Lipohilicity 355
Lipria dulcis
14
Lobatosides 191 Longicaudoside A and B 94 from Ophioderma longicaudum 94 Longikaurin A 138, 145, 155, 173, 175 13C-nmr of 155 from Rabdosia longituba 173 from R. ternifolia 175 1H-nmr of 145 Longikaufin B 139, 147, 156, 173 13C_nmr of 156 from Rabdosia longituba 173 1H-nmr of 147 Longikaurin D 175 from Rabdosia trichocarpa 175 Longikaurin G 139, 147, 156, 172 13C_nmr of 156
565 from Rabdosia longituba 172 1H-nmr of 147 Longirabdosin 116, 123, 135, 173 from Rabdosia longituba 173 1H-nmr of 123 Lophanthoidin A 167, 168-170, 173 13C-nmr of 170 from Rabdosia lophanthoides 173 1H-nmr of 169 Lophanthoidin B 168-170, 173 13C-nmr of 170 from Rabdosia lophanthoides 173 1H-nmr of 169 Lophanthoidin C 168, 169, 173 from Rabdosia lophanthoides 173 1H-nmr of 169 Lophanthoidin D 168, 169, 173 from Rabdosia lophanthoides 173 1H-nmr of 169 Lophanthoidin E 168-170, 173 13C-nmr of 170 from Rabdosia lophanthoides 173 1H-nmr of 169 Lophanthoidin F 168-170, 173 13C_nmr of 170 from Rabdosia lophanthoides 173 1H-nmr of 169 Ludongnin 112 Ludongnin A 112, 141,149, 158, 174 13C-nmr of 158 from Rabdosia rubescens 174 from R. rubescens var. lushiensis 174 1H-nmr of 149 Ludongnin B 141,149, 158, 174 13C-nmr of 158 from Rabdosia rubescens 174 from R. rubescens vat. lushiensis 174 IH-nmr of 149 Luidia maculata 46 Lungshengrabdosin 120, 127, 134, 173 13C_nmr of 134 from Rabdosia lungshengensis 173 IH-nmr of 127 (+)-Lupanine 520 (+)-Lupanine N-oxide 523 from Thermopsis lupinoides 523 (-)-Lupinine 520
Lupin alkaloids
519-549
Lupinus hirsutus 521 (+)-( trans-4 ' -acetylox y-cinnamo yl )
epilupinine from
521
(-)-( cis-4 "-ot-L-rhamnos ylo x y-cinnamo yl)
epilupinine from 521 (- )-( trans -4 ' -t~-L-rhamnos ylo x y-cinnamo yl )
epilupinine from 521
521
Lupinus luteus
(-)-( trans-4 ' -~-D-glucop yranos ylo x y-
cinnamoyl) lupinine from 521 (- )-( t rans -4 ' -[I-D-g l ucop yranos yl ox y-
3'-methoxycinnamoyl) lupinine from
521
(-)-( trans-4 ' -h ydro x y-cinnamo yl)
lupinine from 521 (- )-( trans-4 ' -rhamnos y lo x y-cinnamo yl )
lupinine from 521 (-)-( trans-4 ' -rhamnos ylo x y- 3 '-methox y-
cinnamoyl) lupinine from 521 524, 525 (-)-AS-dehydroalbine from 524 (-)-AS-dehydromultiflorine from 525 Lushanrubescensin 120, 127, 134, 174 13C_nmr of 134 from Rabdosia rubescens 174 from R. rubescens var. lushanensis 174 IH-nmr of 127 Lushanrubescensin B 120, 127, 134, 174 13C_nmr of 134 from Rabdosia rubescens var. lushanensis 174 1H-nmr of 127 Lushanrubescensin C 118, 125, 131,173, 174 13C-nmr of 131 from Rabdosia lungshengensis 173 from R. rubescens var. lushanensis 174 1H-nmr of 125 Lushanrubescensin D 118, 125, 131,174 13C-nmr of 131 from Rabdosia rubescens var. lushanensis 174 IH-nmr of 125 Lushanrubescensin E 118, 125, 132, 174 13C_nmr of 132 from Rabdosia rubescens vat. lushanensis 174 IH-nmr of 125 (-)-Lusitanine 522 from Maackia amurensis 522 Luzonicoside A 59, 60 Lupinus termis
566
Lygos raetam 524 (-)-6a-hydroxylupanine from 524 (+)-12a-hydroxylupanine from 524 Lymphocytes 369 Lysocellin 455 Lytechinus variegatus 104
Maackia amurensis 521, 522 (+)-1313-hydroxymamanine from 522 (-)-lusitanine from 521 Maackia tashiroi 523 tashiromine from 523 Mabinlin 36 Macrocalin A 112, 173 from Rabdosia macrocalyx 173 Macrocalin B 138, 146, 173 from Rabdosia macrocalyx 173 IH-nmr of 146 Macrocalyxin A 112, 143, 152, 161,173 13C-nmr of 161 from Rabdosia macrocalyx 173 from R. macrocalyx var. jiuhua 173 1H-nmr of 152 Macrocalyxin B 112, 173 from Rabdosia macrocalyx 173 Macrocalyxin C 112, 173 from Rabdosia macrocalyx 173 Macrocalyxin D 116, 123, 130, 173 13C_nmr of 130 from Rabdosia macrocalyx 173 IH-nmr of 123 Macrocalyxin E 116, 123, 130, 173 13C-nmr of 130 from Rabdosia macrocalyx 173 1H-nmr of 123 Macrocalyxoformin A 141,150, 158, 172-174 13C-nrnr of 158 from Rabdosia henryi 172 from R. macrocalyx 173 from R. sculponeata 174 1H-nmr of 150 Macrocalyxoformin B 141,149, 173 from Rabdosia macrocalyx 173 IH-nmr of 149 Macrocalyxoformin C 141,150, 158, 173 13C-nmr of 158
from Rabdosia macrocalyx 173 IH-nmr of 150 Macrocalyxoformin D 142, 151,159, 173 13C-nmr of 159 from Rabdosia macrocalyx 173 IH-nmr of 151 Macrocalyxoformin E 142, 151,159, 173 13C-nmr of 159 from Rabdosia macrocalyx 173 IH-nmr of 151 Macrophyllin B 173 from Rabdosia longituba 173 Malonylsaikosaponins 191 Maltotriose 436 (-)-Mamanine N-oxide 522 from Sophora chrysophylla 522 Mamanuthaquinone 300, 315 D-Mannitol 426 D-Mannose-molybdate complex 434 Maoecrystal A 137, 145, 154, 171 13C-nmr of 154 from Rabdosia eriocalyx 171 from R. eriocalyx vat. laxiflora 171 IH-nmr of 145 Maoecrystal B 138, 146, 155, 171,176 lac_nmr of 155 from Rabdosia eriocalyx 171 from R. eriocalyx vat. laxiflora 171 IH-nmr of 146 Maoecrystal C 138, 146, 155, 171 13C-nmr of 155 from Rabdosia eriocalyx 171 1H-nmr of 146 Maoecrystal D (rabdolongin B) 112, 138, 146, 155, 171, 173 13C_nmr of 155 from Rabdosia eriocalyx 171 from R. longituba 173 IH-nmr of 146 Maoecrystal E 139, 147, 171 from Rabdosia eriocalyx 171 IH-nmr of 147 Maoecrystal F 139, 147, 156, 171 lac-nmr of 156 from Rabdosia eriocalyx 171 IH-nmr of 147 Maoecrystal G 138, 146, 171
567 from Rabdosia eriocalyx 171 1H-nmr of 146 Maoecrystal I 139, 147, 156, 171 13C_nmr of 156 from Rabdosia eriocalyx 171 1H-nmr of 147 Maoecrystal J 139, 147, 156, 171 13C-nmr of 156 from Rabdo sia eriocalyx 171 IH-nmr of 147 Maoecrystal K 139, 148, 157, 171 13C-nmr of 157 from Rabdosia eriocalyx 171 1H-nmr of 148 Maoyerabdosin 139, 147, 156, 172 13C.nrnr of 156 from Rabdosiajaponica 172 IH-nmr of 147 Marine quinones 289-326 Marine sesquiterpene 289-326 Marthasterias glacialis 48, 104 Marthasteroside A 47 (+)-Matrine 520 Mebadonin 175 from Rabdosia umbrosa vat. hakusanensis 175 Medicago sativa 202 Melaleuca leucadendron 387 phyllodulcin from 387 Mellein 406 biosynthesis of 406 (+)-Mellein 351 (R)-(-)-Mellein 383 (S)-(+)-Mellein 383 Mellein and derivatives 383-385 Metachromin-A 296 Metachromin-B 296 Metachromin-C 296 Na-Methoxy-19(Z)-anhydrovobasinediol 466, 467 4'-Methoxyavarone 301 p-Methoxybenzyl (pyrichalasin) 355 5-(3'-Methoxycarbonylbutyroyl)aminomethyltrans-quinolizidine N-oxide 522 from Sophora tomentosa 522 11-Methoxygelsemamide 472,473 from Gelsemium elegans 472, 473 11-Methoxy-19(R)-hydroxygelselegine 483,484 from Gelsemium elegans 484
Na-Methoxyindole 497 11-Methoxykoumine 501 6-Methoxymellein 384 from Sporormiafungi 384 Na-Methoxyoxindoles 497 2-(4-Methoxyphenoxy) propanoic acid 36 5-Methoxytetralone 245 Na-Methoxyyohimbine 497 R-(+)-3-Methyladipic acid 229 Methyl-ot-D-arabinopyranoside 431 3-Methylbutylamino derivatives 382 Methylbutylamino-3,4-dihydroisocoumarin 381 3-Methybutylaminodihydroisocoumarins 388 N-Methyl-3-(3-chloro-4,5-dihydroxyphenyl)-3hydroxyalanine 347 24-Methyl-5ot-cholest-22(E)-ene-313,6ot,8,15~, 1613,28-hexaol 79 24-Methyl-25,26-dihydroxy-steroids 84-86 absolute configuration of 84-86 ot-Methylenecyclopentanone moiety 176 O-Methylepoxyshikoccin 163,164, 165, 174 13C_nmr of 165 from Rabdosia shikokiana vat. occidentalis 174 IH-nmr of 164 5'-O-Methylerbstatin 448 3-O-Methylgalactose 431 Methyl-ot-D-glucopyranoside 431 Methyl ~-D-glucopyranoside 431 6-O-Methyl-D-glucose 435 (S)-(+)-2-Methylglutarate 228 Methyl-histamins 328 24-Methyl-26-hydroxy and 24-methyl-26-oic steroidal side chains 81-84 absolute configuration of 79-81 5-Methylmellein 383,385 from Euphorbiafidjiana 385 from Fusicoccum amygdali 385 from Hypoxylon and Numularia spp. 385 from Phomopsis oblonga 385 from Valsa ceratosperma 385 Methyl-ot-D-ribopyranoside 431 N-Methylserotonine 328 O-Methylshikoccin 163-165, 174 13C.nmr of 165 from Rabdosia shikokiana var. occidentalis 174 IH-nmr of 164 24-Methyl thornasterols A 48
568 Methyl-13-D-xylopyranoside 431 Micaculin 36 from RichardeUa dulcifica 36 Michael addition 272 Microbial secondary metabolites 439-463 screening of oncogene function inhibitor from 439-463 Micrococcus luteus 389 Miniatoside A and B 61 from Patiria miniata 61 Modified Schollkopf's procedure 443 Mogroside V 22 Molar Ellipticity 424 Molecular Modeling 213 Molecular modeling calculations 344 Molybdate complexes 436, 437 from di and trisaccharides 436, 437 Momordica grosvenorii 22 Monatin [4-hydroxy-4-(indol-3-ylmethyl) glutamic acid 35 Monellin 36 Monodesmosides 188 Monosaccharide-molybdate complexes 437 Moritoside 294 Mosher's method 76 (-)-Multiflorine N-oxide 524 from Lupinus hirsutus 524 Muscaroside C 200 Mutatis mutandis 262 Muurolane 247 Mycale adhaerens 386 Myodesmanones 231 Myodesmonoid 232 Myomontanoid I]-ketols 232 S-(+)-Mymontanone 232 Myomontanones 231 Myoporone 228 synthesis of 228 R-(+)-Myoporone 229 Myoporum species 227 Myoporum bontioides 229 R-enantiomer 229 Myoporum deserti 228, 229 S-enantiomer 229 Myoporum laetum 236 (-)-ngaione from 236 Myoporum montanum 229
R-enantiomer from 229 Myrmecocystus testaceus 383 Myrrhis odorata 29 Myxodermaplatiacanthum 58, 75, 81 3~,5,6~, 15a- tetmhydrox y-5a-stigmastan-29-oic acid from 81 Myxodermoside A 74
Nakanishi's method 190, 193, 194 Nardoa gomophia 46 Nardoa tubercolata 71 Nectria fuckeliana 383 Nectriapyrone 351 Neohesperidin dihydrochalcone 5, 30 Neohesperidyl saccharide 20 Neorabdosin (novelrabdosin) 112,137,145,154,171 13C.nmr of
154
from Rabdosia eriocalyx 171 from R. nervosa 173 lH-nmr of 145 Neosmilaster georgianus 58 Neothyone gibbosa 92 neothyoside A from 92 Neothyoside A 92 from Neothyone gibbosa 92 Nervosanin 120, 127, 134, 173 13C_nmr of 134 from Rabdosia nervosa 173 IH-nmr of 127 Nervosin 141,150, 158, 173 13C-nmr of 158 from Rabdosia nervosa 173 1H-nmr of 150 (-)-Ngaione 236 from Myoporum laetum 236 Nipoglycosides A, B, C, D 55 3-Nitropropanoic acid 351 Nodosin 173 from Rabdosia longituba 173 from R. nervosa 173 Nodososide 76 Non-Adrenaline 328 Nonaols 74 Northem blotting analysis 449 24-Nor thomasterol A 48
569
Oblongolide 473 Ochratoxin A and D 388 Ochratoxins 382, 387, 388 from Aspergillus ochraceus 382 Odonicin 171, 173, 176 from Rabdosia eriocalyx 171 from R. longituba 173 from R. nervosa 173 9(ll)-en-12ot-Ol bivittosides A, B and D 91 Oleanolic acid 281 from Eremophila caerulea 281 3-epi-Oleanolic acid 281 from Eremophila platycalyx 281 Oncogene function inhibitor 439-463 from microbial secondary metabolites 439-463 screening of 463 Oncogenic viruses 439 Ononis natrix 386 6,8-dihydroxy-3-undecyl-3,4-dihydroiso coumarin from 386 Ophiarthrum elegans 96 Ophiocoma dentata 96
Ophiocomina nigra 100 Ophioderma longicaudum 94, 96 longicaudoside A and B from 94
Ophiodiaster ophidianus 46 Ophiolepis superba 84, 96 Ophiomastix annulosa 99 Ophiorachna incrassata 96 Ophiosparte gigas 99 Ophiura sarsi 98 cholest-5-ene-3ot,4~, 21-triol 3,21- disulphate from 98 Ophiuroidea 43, 94-100 Oplopanone 250 (+)-Oplopanone 247 Oreaster reticulatus 61 Oridonin 171-175 from Rabdosia eryocalyx vat. laxiflora 171 from R. japonica 172 from R. longituba 173 from R. macrophylla 173 from R. rosthornii 174 from R. rubescens 174 from R. ternifolia 175 Osladin 27 Overhauser effects
204-206
in saponins 204-206 Oxindole alkaloid 485-487 N-(3-Oxobutyl) cytisine 525 from Echinosophora koreensis 525 6-Oxo-4,8-dimethylnonanoic acid 228 21-Oxogelsemine 478, 479 from Gelsemium sempervirens 479 19-Oxogelsenicine 481,482 from Gelsemium elegans 482 19-Oxo-gelsevirine 481 from Gelsemium elegans 481 21-Oxogelsevirine 478, 479 from Gelsemium rankinii 479 11-OxomogrosideV 24
Panal 247 from Eupatorium trapezoideum Panicein-A 295 Panicein-B1 296 Panicein-B2 295 Panicein-B3 296 Panicein-C1 296
247
Paracentrotus lividus 104 Parastichopus californicus 87 holotoxins A, A1, B and B l from Parvifoline A 118, 125, 132 13C_nmr of 132 from Rabdosiaparvifolia 173 IH-nmr of 125 Parvifoline B 116, 123,130, 173 13C_nmr of 130 from Rabdosia parvifolia 173 lH-nmr of 123 Parvifolinoic acid 174 from Rabdosiaparvifolia 174 Parvifoliside 140, 148, 157, 173 13C_nmr of 157 from Rabdosia parvifolia 173 IH-nmr of 148 Paspalum scrobiculatum 352-356 Patiria miniata 48, 55 miniatoside A and B from 61 patirioside A from 48 Patiria pectinifera 61 Patirioside A 48 Pectinioside G 57
87
570 Pectinioside E 47, 48 from Asterina pectinifera 48 DS-Penaustrosides A and B 91 from Pentacta australis 91 Pendolmycin 456, 462 tumour-promoter activity of 462 Penicillium chrysogenum 351 Pentaceraster alveolatus 46,61 Pentacta australis 91 DS-penaustrosides A and B from 91 Pentadin 36 A4-3~, 6~, 8, 15~ 1613-Pentahydroxysteroids 64 Perhydroisoindol- 1-one 355 Periandradulcins 191 Periandra dulcis 26 Periandra mediterranea 26 Periandrin I 22 Periandrins I-IV 26
PeriUafrutescens
5
Perillartine 14 Pervicosides A, B and C 92 L-[U-C 14] Phenylalanine 347 10-Phenylcytochalasins 353 3-Phenyldihydroisocoumarins 387 3-Phenyl-3,4-dihydroisocoumarins 387 Phenylpropanoids 29 Phlomis betonicoides 20 baiyunoside from 20 Phlomisoside I 20 Phlomisoside II 20 Phomolactone 350, 351 Phomopsis citri 341 Phomopsis convolvulus 341-345 Phomopsis helianthi 341,345, 346
Phomopsisjuniperovora 346 Phomopsis leptostromiformis 341,347 Phomopsis longicolla 341 Phomopsis oblonga 383, 385, 388-392 5-methylmellein from
385
Phomopsis paspaUi 352 Phomopsis phaseoli 341 Phomopsis viticola 341 Phomosin
346
Phosphatidylinositol 441 Phosphatidylinositolturnover 452 Phospholipase C 448 Phyllodulcin 5, 30, 387
from Hydrangea macrophyUa 30 from Melaleuca leucadendron 387 Phyllostachysin A 163-165, 167, 174 13C-nmr of 165 from Rabdosiaphyllostachys 174 IH-nmr of 164 Phyllostachysin B 118, 125, 132, 174 13C-nmr of 132 from Rabdosia phyllostachys 174 tH-nmr of 125 Phytotoxic activity 345 Phytotoxicity 479 Phytotoxins 342 Piericidin B l N-oxide 457 Piericidins 457,458 Pinacol-type rearrgement 500 Pisaster brevispinus 61 pisasteroside A from 61 Pisaster giganteus 66 Pisaster ochraceus 61 pisasteroside A from 61 Pisasteroside A 61 from Pisaster brevispinus 61 from Pisaster ochraceus 61 PI turnover inhibitors 452 biological activity of 461,462 echiguanine 458-461 inostamycin 453-456 pendolmycin 456 piericidins 457, 458 psi-tectorigenin 452, 453 Platelet-derived growth factor 441 Polyclona antibodies 368 Polyhydroxysteroids 72-76 Polypodium glycyrrhiza 28 Polypodium vulgare 28 Polypodogenin 28 Polypodosides A and B 27 Ponicidin 174, 175 from Rabdosia rosthornii 174 from R. rubescens 174 from R. ternifolia 175 Pravastatin 450 Precyclospongiaquinone-I 298 Preseychellogenin 91 Pretazettin 135 Prezizaene 248
571 synthesis of 248 (-)-Prezizanol 248 synthesis of 248 Proanthocyanidins 33, 34 Proanthocyanidin dibenz [b,d] oxocin 7 L-[U-C 14] Proline 347 Protoreaster nodosus 46, 60, 77 5a-cholestan-3~, 6 ~ 8, 15a, 1613,25-hexaol from 77 Pseudoginsenoside 191 Pseudopterins 259 Pseudopterogorgia elisabethae 259 3-epi-Pseudopterosin 259 Pseudostichoposide A 94 from Pseudostichopus trachus 94 Pseudostichopus trachus 94 pseudostichoposide A from 94 Pseurata A 116, 123, 130, 174 13C-nmr of 130 from Rabdosia pseudo-irrorata 174 1H-nmr of 123 Pseurata B 118, 125, 132, 174 13C-nmr of 132 from Rabdosiapseudo-irrorata 174 1H-nmr of 125 Pseurata C 116, 123, 130, 174 13C.nmr of 130 from Rabdosiapseudo-irrorata 174 1H-nmr of 123 Pseurata D 118, 125, 132, 174 13C.nmr of 132 from Rabdosiapseudo-irrorata 174 IH-nmr of 125 Pseurata E 120, 127, 134, 174 13C_nmr of 134 from Rabdosiapseudo-irrorata 174 IH-nmr of 127 PseurataF 118, 125, 132, 174 13C-nmr of 132 from Rabdosiapseudo-irrorata 174 1H-nmr of 125 Psi-tectorigenin 452, 453,456 Psolus fabricii 89 psolusoside B from 89 Psolusoside B 89 from Psolusfabricii 89 Purpurea glycoside A 362
Purpurea glycoside B 362 Puupehenone 299 Pycnopodia heliantoides 46, 61 pyenopodiosides A, B and C from 61 Pyenopodiosides A, B and C 61 from Pycnopodia heliantoides 61 4-O-Pyranosyl sugars 436 6-O-Pyranosyl sugars 436 Pyricularia oryzae 385 a-Pyrone 474 a-Pyrone convolvupyrone 342
Quercetin 460 D-Quinovose 190
Rabdocoetsin B 171 from Rabdosia coetsa 171 Rabdocoetsin C 171 from Rabdosia coetsa 171 Rabdocoetsin D 138, 146, 155, 171 13C-nmr of 155 from Rabdosia coetsa 171 1H-nmr of 146 Rabdoepigibberellolide 112 from Rabdosia shikokiana 112 Rabdoforrestin A 120, 127, 134, 171 13C_nmr of 134 from Rabdosiaforresti 171 1H-nmr of 127 Rabdohakusin 162-165, 175 13C_nmr of 165 from Rabdosia umbrosa var. hakusanensis IH-nmr of 164 Ralxloinflexin A 140, 149, 157, 172 13C_nmr of 157 from Rabdosia inflexa 172 IH-nmr of 149 Rabdoinflexin B 118, 125, 132, 172 13C_nmr of 132 from Rabdosia inflexa 172 1H-nmr of 125 Rabdokaurin A 138, 146, 155, 173 13C-nmr of 155 from Rabdosia longituba 173 1H-nmr of 146
175
572 Rabdokaurin B 143, 152, 161,173 laC-nmr of 161 from Rabdosia longituba 173 1H-nmr of 152 Rabdokumin A 118, 125, 132, 172 13C-nmr of 132 from Rabdosia kunmingensis 172 1H-nmr of 125 Rabdokunmin B 116, 123, 130, 172 13C-nmr of 130 from Rabdosia kunmingensis 172 IH-nmr of 123 Rabdokunmin C 118, 125, 132, 172 13C.nmr of 132 from Rabdosia kunmingensis 172 IH-nmr of 125 Rabdokunmin D 118, 125, 132, 172 13C-nmr of 132 from Rabdosia kunmingensis 172 1H-nmr of 125 Rabdokunmin E 120, 127, 134, 172 13C-nmr of 134 from Rabdosia kunmingensis 172 1H-nmr of 127 Rabdolasional 143, 153, 161,162, 172 13C-nmr of 161 from Rabdosia lasiocarpa 172 IH-nmr of 153 Rabdolatifolin 162-165, 175 13C-nmr of 165 from Rabdosia umbrosa 175 from R. umbrosa vat. latifolia 175 1H-nmr of 164 Rabdolongin A 139, 148, 173 from Rabdosia langituba 173 lH-nmr of 148 Rabdolongin B 112 Rabdoloxin A 119, 126, 132, 173 13C-nmr of 132 from Rabdosia loxothyrsa 173 IH-nmr of 126 Rabdoloxin B 119, 126, 133, 171,172 13C-nmr of 133 from Rabdosiaflexicaulis 171 from R. inflexa 172 from R. kunmingensis 172 from R. loxothyrsa 173
from R. parvifolia 173 1H-nmr of 126 Rabdophyllin G (rabdosin C) 112, 144, 153, 162, 171, 172, 173, 176 from Rabdosia gaponica var. glaucocalyx 171 from R. henryi 172 from R. japonica 172 from R. longituba 173 from R. macrophylla 173 lH-nmr of 153 Rabdophyllin H 140, 148, 157, 173 13C_nmr of 157 from Rabdosia macrophylla 173 IH-nmr of 148 Rabdoserrin A 140, 149, 157, 172, 174 13C_nmr of 157 from Rabdosia inflexa 172 from R. serra 174 IH-nmr of 149 Rabdoserrin B 172 from Rabdosia inflexa 172 Rabdoserrin D 119, 174 from Rabdosia serra 174 Rabdosia amethystoides 167 Rabdosia diterpenoids 112 classification of 112 Rabdosia eriocalyx 176 Rabdosia gerardiana 167 Rabdosia glutinosa 167 Rabdosia japonica 111 Rabdosia laxiflora 176 Rabdosia lophanthoides 167 Rabdosia macrophylla 167 Rabdosia occidentalis 176 Rabdosia parvifolia 167 Rabdosia shikokiana 112, 162, 176 rabdoepigibberellolide from 112 Rabdosia species 111-185 diterpenoids from 111-185 Rabdosia stracheyl 167 Rabdosia trichocarpa 135, 176 Rabdosichuanin A 142, 151, 159, 174 13C_nmr of 159 from Rabdosia setschwanensis 174 IH-nmr of 151 Rabdosichuanin B 141,150, 158, 174 13C-nmr of 158
573 from Rabdosia setschwanensis 174 IH-nmr of 150 Rabdosichuanin C 142, 151, 159, 174 13C_nmr of 159 from Rabdosia setschwanensis 174 IH-nmr of 151 Rabdosichuanin D 140, 148, 157, 174 laC.nmr of 157 from Rabdosia setschwanensis 174 IH-nmr of 148 Rabdoside 1 140, 148, 157, 171 13C_nmr of 157 from Rabdosia eriocalyx 171 1H-nmr of 148 Rabdoside 2 140, 149, 157, 171 13C_nmr of 157 from Rabdosia eriocalyx 171 IH-nmr of 149 Rabdosin A 172 from Rabdosiajaponica 172 Rabdosin B 112, 162 Rabdosin C 112 Rabdosinate 144, 153, 161, 171, 172 13C-nmr of 161 from Rabdosia gaponica vat. g laucocalyx 171 from R. japonica 172 IH-nmr of 153 Rabdosinatol (glaucocalyxin C) 112, 116, 123, 130, 172 13C-nmr of 130 from Rabdosiajaponica 172 from R. japonica var. glaucocalyx 172 1H-nmr of 123 Rabdoternin A 139, 147, 156, 175 13C-nmr of 156 from Rabdosia ternifolia 175 IH-nmr of 147 Rabdoternin B 140, 148, 157, 175 13C.nmr of 157 from Rabdosia ternifolia 175 IH-nmr of 148 Rabdoternin C 139, 147, 156, 175 13C-nmr of 156 from Rabdosia ternifolia 175 IH-nmr of 147 Rabdoumbrosanin 162-165, 175 13C-nmr of 165 from Rabdosia umbroba 175
IH-nmr of 164 Rabyuennane A 119, 126, 133, 175 13C-nmr of 133 from Rabdosia yuennanensis 175 1H-nmr of 126 Rabyuennane B 119, 126, 175 from Rabdosiayuennanensis 175 IH-nmr of 126 Rabyuennane C 120, 127, 134, 175 lac-nmr of 134 from Rabdo sia yuennanensis 175 IH-nmr of 127 Radioimmunoassay 361 Rahydroshikoccin 167 Rankinidine 472 Ras function inhibitors 449-451 compactin 450,451 oxanosine 449 Ras oncogene 441 Rebaudioside A 16 Rebaudioside B 17 Rebaudioside C 16 Regularoside B 51, 52 Reniformin A 112 Reniformin B 119, 126, 133, 172 13C-nmr of 133 from Rabdosia latifolia var. reniformis 172 IH-nmr of 126 Reniformin C 137, 145, 154, 172 13C-nmr of 1.~ from Rabdosia latifolia var. reniformis 172 1H-nmr of 145 L-Rhamnopyranosyl 7 (-)-( cis -4 ' -a-L-Rhamnos ylo x y-cinnamo yl) epi- lupinine 521 from Lupinus hirsutus 521 (-)-( trans-4 ' -a-L-Rhamnos ylox y-cinnamo yl) epilupinine 521 from Lupinus hirsutus 521 (- )-( t rans-4 ' -Rhamnos ylo x y-cinnamo yl ) lupinine 521 (- )-( t rans-4 ' -Rhamnos y lo x y- 3 ' -metho x y cinnam o yl ) lupinine 521 from Lupinus luteus 521 Rhytidopenera metaltica 384 Richardella dulcifica 36 miraculin from 36
574 Rosthornin A 116, 123, 130, 174 13C-nmr of 130 from Rabdosia rosthornii 174 1H-nmr of 116 Rosthornin B 119, 126, 133, 174 13C-nmr of 133 from Rabdosia rosthornii 174 IH-nmr of 126 Rosthorin A 139, 147, 156, 171,172, 174 13C-nmr of 156 from Rabdosia coetsa 171 from R. japonica 172 from R. rosthornii 174 1H-nmr of 147 Royleanone 173 from Rabdosia lophanthoides var. gerardiana 173 Rubescensin C 174 from Rabdosia rubescens 174 Rubescensin D 163-165, 167, 174 13C-nmr of 165 from Rabdosia rubescens 174 lH-nmr of 164 Rubusoside 18 from Rubus suavissims 18 Rubus suavissimus 16, 18 desglucosylstevioside from 14 rubusoside from 14 Rugosanin 142, 151,159, 174 13C_nmr of 159 from Rabdosia rugosa 174 1H-nmr of 151
Saccharum oflicinarum 3 Saponins 187-224 structural elucidation of 187-224 Sarasinoside A1 192 Sarpagine-type indole alkaloids 466-471 Saxifragifolins A and B 200 from Androsace saxifragiifolia 200 Scaberoside B5 211 Schlerochiton ilicifolius 335 Sclerotinia sclerotiorum 385 Sclerotinin A and B 385 Scopadosides A-D 68 Sculponeatin A 141,150, 158, 174
13C-nmr of 158 from Rabdosia sculponeata 174 IH-nmr of 150 Sculponeatin B 142, 151, 159, 174 13C_nmr of 159 from Rabdosia sculponeata 174 IH-nmr of 151 Sculponeatin C 141, 150, 158, 174 13C-nmr of 158 from Rabdosia sculponeata 174 1H-nmr of 150 Sculponeatin D 144, 153, 161, 174 13C-nmr of 161 from Rabdosia sculponeata 174 IH-nmr of 153 Sect-analogues 229 Secologanin 487 Secondary metabolites 226, 227 Selective antitumor activity 355 l-Selective glucosylation 28 a-Selective thermal rhamnosylation 28 Selliguea feei 33 Selligueain A 33 Semenochromene-A 295 Sempervirine 466, 467 Sepositoside A 59, 60 Serrulatane 259 from Eremophila drummondii 259 ent-Serrulatane 259 Sen'ulatanes 257-260 Sesquiterpene ketone eremophilone 226 Sesquiterpenes 227-251 Shikoccidin 119, 174, 175 from Rabdosia shikokiana var. occidentalis from R. umbrosa 175 from R. umbrosa var. latifolia 175 Shikoccin 162-165, 174, 175 13C_nmr of 165 from Rabdosia shikokiana var. occidentalis from R. umbrosa 175 from R. umbro sa var. hakusanensis 175 from R. umbrosa vat. latifolia 175 IH-nmr of 164 Shikokianal acetate 173 from Rabdosia nervosa 173 Siamenoside I 24 Simvastatin 450
174
174
575 Siphonodictyal-A 297 Siphonodictyal-B 293, 298 Siphonodictyal-C 298 Siphonodicytal-D 300 Siphonodicytal-E 294 Siphonodictyoic acid 293 Siphonodictyol-G 293 Siphonodictyol-H 298 Siraitia grosvenorii 5, 22 Siraitia siamensis 24 Smenochromene-B 295 Smenochromene-C 295 Smenochromene-D 295 Smenodiol 293, 297 Smenorthoquinone 291, 292 Smenospondiol 292, 293 Smenospongia aurea 291 8-epichromazonarol from 291 Sodoponin 171, 174, 175 from Rabdosia eriocalyx 171 from R. setschwanensis 174 from R. ternifolia 175 Solasodine 28 Solaster borealis 55 solasteroside from 55 Solasteroside A 55 from Solaster borealis 55 Solidago saponins 191 Sophora chrysophylla 522 (-)-mamanine N-oxide from 522 Sophora tomentosa 522 (-)-epilamprolobine from 522 (+)-epilamprolobine N-oxide from 522 5-(3'-methoxycarbonylbutyroyl) aminomethyl-trans-quinolizidine N-oxide from Sophorosyl ([3-D-glucopyranosyl-(2---~1)-13-Dglucopyranosyl) sugar unit 20 Soyasapogenol B 187 Soyasaponin A3 196 from Glycine max 196 Soyasaponin I 187 (-)-Sparteine 520 (+)-Spathulenol 248 from Eremophila cuneifolia 248 from E. paisley 248 from E. racemosa 248 from E. drummondi 248
522
from Eucalyptus spathulata 248 Sphaerechinus granularis 104 Spinaceamin 328 Spiro-diterpenes 260-263 Spongia hispida 312 Spongiaquinone 298, 318-321 Spongiaquinone potassium salt 298 Sporomia fungi 384 6-methoxymellein from 384 SRE oncogene 443 Stelospongia conulata 312 Stereoselective pinacol-type rearrangement 509 Stereoselective synthesis 243 of (+)-eremophilone 243 Steroidal saponins 27-29 Stevia phlebophylla 16 Stevia rebaudiana 4, 5 Steviol 13-O-13-D-glucoside 18 Steviolmonoside 18 Stevioside 16 Stichlorosides A1, B1, Cl and A2, B2, C2 87 from Stichopus chloronotus 87 Stichopus chloronotus 87 stichlorosides Ah B l, CI and A2, B2, C2 from 87 Stichopusjaponicus 87 holotoxins A, Al, B and B l from 87 Stomphia coccinea 66 Streptomyces griseolavendus 445 Streptomyces mobaraensis 457 Streptomyces pactum 457 Streptomyces viridosporus 441 Strictosidine 486-488 dehydrogeissoschizine from 487, 488 Strong lyophora hartmani 312 Strongylin-A 300 Strongylocentratus droebachiensis 104 Strongylocentrotus purpuratus 104 Structural elucidation 187-224 of saponins 187-224 Structural studies 392-408 of xenocoumacins 392-408 Styracaster caroli 76 carolisterol A-C 75, 76 Sucrononic acid 6 Synthesis 228, 243, 248, 251,412-418 of AI-77-B 412-418 of calamanenenes 251
576 of (+)-eremophilone 243 of myoporone 228 of prezizanol 248 Synthetic studies 487-503 of humantenine-type oxindole alkaloids of koumine-type alkaloids 500-503 of sarpagine-type alkaloids 487-493 Scolytus scolytus 348, 349 Scolytus multistriatus 348
493-500
Tabeduia avellanedae 385 Tashiromine 523 from Maackia tashiroi 523 Tenuispinoside A, B, C 51, 52 Ternifolin 140, 148, 175 from Rabdosia ternifolia 175 1H-nmr of 148 Tessaria dodoneifolia 31 Tetrahydro-eremophilone 240 3 [3,6ix,12ix,20- Tetrahydroxy-5tx-cholest-9(11 )-en23-one 51 313,5,613,15or- Tetrahydroxy- 5tx-stigmastan-29oic acid 81 from Myxoderma platiacanthum 81 (24S) 3~, 5, 613, 15ot-Tetramethoxy-5tx-cholest-8(9)-en-24-ol 76 2,3,4,6-Tetra-O-methylglucose 431 Thadiantha grosvenorii 22 Thaumatin 5, 36 Thaumatins I 5 Thaumatins II 5 Thaumatococcus daniellii 5 Thermopsis chinensis 523 (+)-5,6-dehydrolupanine from 523 Thermopsis lupinoides 523 (+)-Lupanine N-oxide from 523 Thladiantha grosvenorii 5 Thorecta choanoides 312 Thornasteroside A 45, 52 from Thromidia catalai 46 24R-and (24S)-Thoronasterol A 48 Thromidia catalai 46 thornasteroside A from 45 (-)-13ct-Tigloyloxymultiflorine 524 from Lupinus hirsutus 524 Total synthesis 493
of koumidine 493 of Na-Methyl-DlS-isokoumidine 493 Toyocamycin 459 TPA-responsive element 441 Tragopogon saponins 191 Transannular SN2 cyclization 500 Tremaster novaecaledoniae 45, 72, 75, 96 Tremasterol A, B and C 72 Trichodonin 112, 162 Trichokaurin 173 from Rabdosia longituba 173 Trichorabdal A 142, 150, 158, 175 13C-nmr of 158 from Rabdo sia trichocarpa 175 from R. weisiensis 175 IH-nmr of 150 Trichorabdal B 142, 151,160, 175 13C.nmr of 160 from Rabdosia trichocarpa 175 IH-nmr of 151 Trichorabdal C 143, 151,160, 175 13C.nmr of 160 from Rabdo sia trichocarpa 175 IH-nmr of 151 Trichorabdal D 144, 153, 161,175 13C-nmr of 161 from Rabdo sia trichocarpa 175 ltl-nmr of 153 Trichorabdal E 144, 153, 175 from Rabdo sia trichocarpa 175 IH-nmr of 153 Trichorabdal F 143, 152, 175 from Rabdo sia trichocarpa 175 IH-nmr of 152 Trichorabdal G-acetate 143, 152, 175 from Rabdo sia trichocarpa 175 IH-nInr of 152 Trichorabdal H 112, 143, 152, 160, 162, 175 13C-nmr of 160 from Rabdosia trichocarpa 175 IH-nmr of 152 Trichorabdonin 144, 153, 161,175 13C-nmr of 161 from Rabdosia trichocarpa 175 lH-nmr of 153 Tricyclic resin acid 16 Tridesmosides 188
577 Trilobatin 31 2-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)hydroquinone 294 6-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)-2methoxy-p- hydroquinone 294 6-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)-2methoxy-p-quinone 294 2,3,4, Tri-O-methylgalactose 431 Triterpenes 281,282 Tryptamine 487 (-)-Tsukushinamine-A 520 Tubatosides 191 Tumour-promoter activity 462 of pendolmycin 462 Tyrosine kinase activity 441 Tyrosine kinase inhibitors 441-447 biological activity of 447-449
Umbrosianin 119, 126, 133, 175 13C-nrnr of 133 from Rabdosia umbrosa 175 IH-nmr of 126 Umbrosin A 175 from Rabdosia umbrosa 175 from R. umbrosa var. hakusanensis Ursolic acid 281 from Eremophila caerulea 281
175
L-[U-C 13] Valine
347 Valsa ceratosperma 385 5-methylmellein from 385 (+)-Verbenone 227 from Eremophila dempsteri 227 Versicoside A 47 Vetiver oil sesquiterpene 273 16-epi.Voacarpine 466, 467, 469 from Gelsemium elegans 469 Vobasine 503
WALTZ sequence 205 Weisiensin A 112, 120, 127, 134, 171,173, 175 13C-nmr of 134 from Rabdosia calcicolus from R. nervosa 173
171
from R. weisiensis 175 IH-nmr of 127 Western blotting analysis 449 Wistariasaponin BI 206 Wolf-kishner reduction 231
Xenorhabdus nematophilus 389 xenocoumacins from 389 Xenocoumacins 381-472 biological activities of 408-412 from Xenorhabdus nematophilus 389 introduction of 381,382 isolation of 388-392 structural studies of 392-408 Xenopus oocyte 451 Xenorhabdus spp. 381 X-ray crystallography 213 Xestospongia exigua 312 Xestospongia sapra 312 Xindongnin A 116, 123, 130, 171, 174 13C.nmr of 130 from Rabdosia dawoensis 171 from R. rubescens 174 IH-nmr of 123 XindongninB 119, 126, 133, 174 13C.nmr of 133 from Rabdosia rubescens 174 IH-nmr of 126 3- O-13-Xylopyranosides 64 D-Xylopyranosyl 7
Yahazunol 297 Yunganosides 297 Zizaenes 273 ent-Zizaenes 251 Zizane 227 Ziziphus jujuba 36 Zonaric acid 297 Zonarol 296, 320 Zonarone 296 Zygosporium masonii
356
577 Trilobatin 31 This Page Intentionally Left Blank 2-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)hydroquinone 294 6-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)-2methoxy-p- hydroquinone 294 6-(3,7,11-Trimethyl-2,6,10-dodecatrienyl)-2methoxy-p-quinone 294 2,3,4, Tri-O-methylgalactose 431 Triterpenes 281,282 Tryptamine 487 (-)-Tsukushinamine-A 520 Tubatosides 191 Tumour-promoter activity 462 of pendolmycin 462 Tyrosine kinase activity 441 Tyrosine kinase inhibitors 441-447 biological activity of 447-449
Umbrosianin 119, 126, 133, 175 13C-nrnr of 133 from Rabdosia umbrosa 175 IH-nmr of 126 Umbrosin A 175 from Rabdosia umbrosa 175 from R. umbrosa var. hakusanensis Ursolic acid 281 from Eremophila caerulea 281
175
L-[U-C 13] Valine
347 Valsa ceratosperma 385 5-methylmellein from 385 (+)-Verbenone 227 from Eremophila dempsteri 227 Versicoside A 47 Vetiver oil sesquiterpene 273 16-epi.Voacarpine 466, 467, 469 from Gelsemium elegans 469 Vobasine 503
WALTZ sequence 205 Weisiensin A 112, 120, 127, 134, 171,173, 175 13C-nmr of 134 from Rabdosia calcicolus from R. nervosa 173
171
from R. weisiensis 175 IH-nmr of 127 Western blotting analysis 449 Wistariasaponin BI 206 Wolf-kishner reduction 231
Xenorhabdus nematophilus 389 xenocoumacins from 389 Xenocoumacins 381-472 biological activities of 408-412 from Xenorhabdus nematophilus 389 introduction of 381,382 isolation of 388-392 structural studies of 392-408 Xenopus oocyte 451 Xenorhabdus spp. 381 X-ray crystallography 213 Xestospongia exigua 312 Xestospongia sapra 312 Xindongnin A 116, 123, 130, 171, 174 13C.nmr of 130 from Rabdosia dawoensis 171 from R. rubescens 174 IH-nmr of 123 XindongninB 119, 126, 133, 174 13C.nmr of 133 from Rabdosia rubescens 174 IH-nmr of 126 3- O-13-Xylopyranosides 64 D-Xylopyranosyl 7
Yahazunol 297 Yunganosides 297 Zizaenes 273 ent-Zizaenes 251 Zizane 227 Ziziphus jujuba 36 Zonaric acid 297 Zonarol 296, 320 Zonarone 296 Zygosporium masonii
356
