The Alkaloids: Chemistry and Biology (volume 67)

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THE ALKALOIDS Chemistry and Biology VOLUME

67 Edited by

Geoffrey A. Cordell Evanston, Illinois

Amsterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Sydney Tokyo Academic Press is an imprint of Elsevier

ACADEMIC PRESS

Academic Press is an imprint of Elsevier 84 Theobald’s Road, London WC1X 8RR, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2009 Copyright r 2009 Elsevier Inc. 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 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http:// www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice 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. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made ISBN: 978-0-12-374785-3 ISSN: 1099-4831

For information on all Academic Press publications visit our website at books.elsevier.com

Printed and bound in USA 09 10 11 12 13 10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. Malgorzata Baranska (217), Department of Chemistry, Jagiellonian University, Krakow, Poland Qiao-Hong Chen (1), Department of Chemistry of Medicinal Natural Products, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of China Vinicius Ilha (79), Departamento de Quı´mica, NPPN, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil Xiao-Tian Liang (1), Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China Graciela Maldaner (79), Departamento de Quı´mica, NPPN, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil Russell J. Molyneux (143), Western Regional Research Center, Agricultural Research Service, USDA, Albany, California, USA Ademir Farias Morel (79), Departamento de Quı´mica, NPPN, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil Kip E. Panter (143), Poisonous Plant Research Laboratory, Agricultural Research Service, USDA, Logan, Utah, USA Hartwig Schulz (217), Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Quedlinburg, Germany Feng-Peng Wang (1), Department of Chemistry of Medicinal Natural Products, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of China

vii

PREFACE As each volume of The Alkaloids series unfolds, much as one might have a vision to accumulate certain chapters, the reality of life intervenes. When this happens, one recalls the fourth ‘‘rule’’ of life’s events: ‘‘Don’t anticipate the outcome.’’ The chapters here actually were planned originally to appear in two quite distinct volumes. Now they are gathered here; and actually they fit together quite well. The diterpenoid alkaloids have been studied by several major research groups over the years in a number of countries. One of the leading sites for structure elucidation has been Professor Feng-Peng Wang’s group at Sichuan University in Chengdu, People’s Republic of China. This is the first review, with Qiao-Hong Chen and the venerable Xiao-Tian Liang as co-authors, summarizing the chemistry and biology of what has developed to be a quite distinct group within the diterpenoid alkaloids, the C18-diterpenoid alkaloids. The cyclopeptide alkaloids were previously reviewed in Volume 49 of this series in 1997, and were thus due for an update. This has been provided by Professor Ademir Farias Morel, Graciela Maldaner, and Vinicius Ilha from the Universidade Federal de Santa Maria, Brazil. The review examines the isolation, chemistry, synthesis, and several of the important biological aspects of the cyclopeptide alkaloids obtained in the past 13 years. We frequently think of natural products research as being focused on the health beneficent effects for humans. The final two new chapters serve to remind us that there are nondesirable animal and human effects that also need to be studied. The chapter by Russell J. Molyneux and Kip E. Panter from laboratories of the United States Department of Agriculture focuses on the tremendous research efforts that have been undertaken on the toxic effects of several major alkaloid groups to the livestock that graze the fields in several major areas of the world. Aspects of these studies have been discussed in other chapters previously, but this is the first collection in this series. A new topic to The Alkaloids series recognizes that the detection of alkaloids, particularly those with significant social implications, such as the opioids and cocaine, is of great interest. Rather than isolate and characterize compounds through liquid or solid spectroscopic or spectrometric methods, the remote detection and characterization of alkaloids, as discussed by Malgorzata Baranska and Hartwig Schulz, has become an area witnessing significant developments in the past few years. Geoffrey A. Cordell Evanston, Illinois ix

CHAPT ER

1 The C18-Diterpenoid Alkaloids Feng-Peng Wang1,*, Qiao-Hong Chen1 and Xiao-Tian Liang2

Contents

I. II. III. IV. V.

Introduction Classification, distribution, and occurrence NMR spectroscopy Chemical reactions Pharmacological activity A. Analgesic Activity B. Antiarrhythmic Activity C. Anti-Inflammatory Activity D. Anesthetic Activity E. Miscellaneous Acknowledgments References

1 3 5 30 33 33 68 72 72 72 73 73

I. INTRODUCTION The C18-diterpenoid alkaloids constitute a small group within the diterpenoid alkaloids. The first C18-diterpenoid alkaloid, lappaconitine, was isolated from the plants Aconitum septentrionale (1–3), A. orientale (4), and A. excelsum (5) by German and Russian scientists during the period 1922–1958. The structure of lappaconitine was confirmed by extensive chemical studies (6) and single-crystal X-ray analysis of its hydrolyzed

1

Department of Chemistry of Medicinal Natural Products, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of China

2

Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China

Corresponding author.

E-mail address: [email protected] (F.P. Wang) The Alkaloids, Volume 67 ISSN: 1099-4831, DOI 10.1016/S1099-4831(09)06701-7

r 2009 Elsevier Inc. All rights reserved

1

2

Wang et al.

product, lappaconine hydrobromide (7,8). Following lappaconitine, three important C18-diterpenoid alkaloids, lappaconidine (9,10), aconosine (11), and excelsine (12,13), were isolated and identified. These alkaloids have served as the cornerstones for subsequent advancements in C18-diterpenoid alkaloids over the past 40 years. OCH3 OCH3 OH N

OCH3

OCH3 OH OH N OH

OH

OH

lappaconidine

aconosine

OCH3

N OH

OH O

HO

N

OH O

OCH3

OCH3

OCH3 OCH3

OH O

excelsine

lappaconitine

Lapaconitine, together with other C18-diterpenoid alkaloids, was structurally classified as belonging to the broad C19-diterpenoid alkaloids for a long time (4). However, Wang and Fang in 1985 (14) suggested the use of the term ‘‘C18-diterpenoid alkaloids’’ for these alkaloids, which were subsequently also named as ‘‘norditerpenoid alkaloids’’ (15) or ‘‘bisnorditerpenoid alkaloids’’ (16), distinguishing them from the C19-diterpenoid alkaloids. In order to clarify this confusing situation, Wang and Liang suggested the restoration of the original terms ‘‘C18-,’’ ‘‘C19-,’’ and ‘‘C20-diterpenoid alkaloids’’ (17). There were only 19 C18-diterpenoid alkaloids discussed in the review by Pelletier et al. in 1984 (18). However, by the end of July 2008 the number of naturally occurring C18-diterpenoid alkaloids isolated from 40 plant species had risen to a count of 78 delineated structures. A careful analysis of the chemical structures and distribution of known C18-diterpenoid alkaloids led to the conclusion that they possess the following distinctive features relative to C19-diterpenoid alkaloids: 1.

2.

Structurally, C-4 is a methine unit or an oxygenated quaternary carbon; the former is similar to the C19-diterpenoid alkaloids, but with fewer substitutions when compared with C19-diterpenoid alkaloids; while the latter often possesses 4-OH, 4-OAc, or 4-Nacetyl anthranoyl groups and their derivatives. In very few instances, it contains a 3,4-epoxy structural unit. From the distribution perspective, all of the C18-diterpenoid alkaloids isolated from Chinese Aconitum plants are thus far distributed exclusively in plants of the subgenus Lycoctonum of the genus Aconitum, which is of chemotaxonomic significance (19).

The C18-Diterpenoid Alkaloids

3

Based on these aforementioned features, we strongly believe in the separation of this type of alkaloids from the C19-diterpenoid alkaloids, and naming them as an independent group, the ‘‘C18-diterpenoid alkaloids.’’ There are relatively few studies on the biological activities of the C18-diterpenoid alkaloids. However, it is of interest to note that lappaconitine, obtained from various Aconitum plants, has been clinically developed as an antiarrhythmic drug in Uzbekistan (20,21), and as a nonnarcotic analgesic drug in China (22–24).

II. CLASSIFICATION, DISTRIBUTION, AND OCCURRENCE As indicated previously, the C18-diterpenoid alkaloids have also been classified as C19-diterpenoid alkaloids (6), norditerpenoid alkaloids (15), or bisnorditerpenoid alkaloids (16). Perhaps due to the limited number of characterized C18-diterpenoid alkaloids, no extensive investigation on their classification was conducted prior to the report by Ichinohe et al. (25). In 2004, Ichinohe et al. (25) classified the C18-diterpenoid alkaloids into two broad categories: the lappacontitine type (A) and the ranaconitine type (B, see Figure 1), based on whether an oxygencontaining functionality is attached at C-7. The lappacontitine-type C18diterpenoid alkaloids are similar to the aconitine-type C19-diterpenoid alkaloids, while the ranaconitine-type C18-diterpenoid alkaloids are similar to the lycoctonine-type C19-diterpenoid alkaloids. We absolutely concur with this classification criterion, and recognize it as an appropriate structural classification of the C18-diterpenoid alkaloids. In addition, according to whether an oxygen-containing functionality is attached at C-4, we have further subdivided the lappaconitine and ranaconitine types, respectively, into two subtypes: the aconosine (AI) and lappaconine (AII) subtypes, and the leuconine (BI) and ranaconitine (BII) subtypes (Figure 1). The aconosine (AI) and leuconine (BI) subtypes are characterized by a C-4 methine moiety, while the lappaconitine (AII) and ranaconitine (BII) subtypes feature a C-4 oxygenated quaternary carbon. It is worth pointing out that the names of the subtypes were designated by the name of the first alkaloid discovered within the corresponding subtype. 1.

Aconosine subtype (AI): few substituents; lacks a hydroxyl group at C-4; similar to the aconitine-type C19-diterpenoid alkaloids, an acetoxyl group or aromatic ester group (such as OBz, OAs) attached to C-8 or C-14 in most cases. Only a few alkaloids possess the imine (e.g., liconosine A) or the amide (e.g., piepunendine A) structural unit.

4

Wang et al.

17

2

R

3

10

1

N

12

11

13

16 14

9

15

4

6

N

R

8 5

7

4

7 19

H

R′ Lappaconine subtype (AII)

Aconosine subtype (AI)

R = H, CH3, Et

R = H, CH3, Et

R' = OH, OCOAr Lappaconitine type (A)

R

N

R

N

7

H

OR′

R′′

OR′

Ranaconitine subtype (BII)

Leuconine subtype (BI)

R = H, CH3, Et

R = H, CH3, Et

R′ = H, CH2-, CH3

R′ = H, CH2-

R′′ = OH, OCOAr Ranaconitine type (B)

Figure 1

2.

3.

4.

Classification of the C18-diterpenoid alkaloids.

Lappaconine subtype (AII): most alkaloids of this subtype contain a hydroxyl group at the C-9 position; an anthranoyl group or its derivatives is located at C-4 in most cases, if it is present in the alkaloids; a very few alkaloids, such as monticamine, excelsine, and 8-acetylexcelsine, possess a 3b,4b-epoxy group. Leuconine subtype (BI): generally contains fewer oxygenated functionalities (such as hydroxyl and ester groups); lacks a hydroxyl group at the C-9 position; almost all of the alkaloids of this subtype have an oxygenated functionality at the C-6 position; only a very few alkaloids have a C-8 methine moiety (e.g., leuconine) or an amide unit (e.g., lamarckinine). Ranaconitine subtype (BII): some alkaloids have an amine-containing aromatic ester (e.g., an anthranilic acid ester or its derivatives) located at the C-4 position in most cases; some alkaloids, such as

The C18-Diterpenoid Alkaloids

5

sinomontanine D and ranaconitine, possess a hydroxyl group at C-9; individual alkaloids, such as finaconitine and N-deacetylfinaconitine, contain a hydroxyl group at C-10; a very few alkaloids, such as sinomontanine G and monticoline, contain a 3b,4b-epoxy group. Almost all of the lappaconitine-type C18-diterpenoid alkaloids are distributed exclusively in plants of the genus Aconitum, whereas only about 65% of the ranaconitine-type alkaloids have been isolated from plants of the genus Aconitum. Very few alkaloids (e.g., hohenackeridine and 14-O-demethoxydelboxine) have been obtained from plants of the genus Cosolida; all of the remaining alkaloids have been isolated from plants of the genus Delphinium. The C18-diterpenoid alkaloids are listed in Tables I and II according to the above-mentioned criteria, including their name, code, chemical structure, molecular formula, molecular weight, melting point, optical rotation, 1H NMR, 13C NMR, MS, and plant source. Table III lists the C18-diterpenoid alkaloids occurring in the genera Aconitum, Delphinium, and Cosolida of the Ranunculaceae family. The C18-diterpenoid alkaloids listed in this chapter and their code numbers are cross-indexed in Table IV.

III. NMR SPECTROSCOPY No comprehensive, systematic summary of the NMR data of the C18-diterpenoid alkaloids has been provided thus far. Pelletier et al. (18,122) and Atta-ur-Rahman (123) have presented the NMR data for only around 20 C18-diterpenoid alkaloids in their summarizing tables, in which the NMR data of the C18-diterpenoid alkaloids were listed. Herein, the 1H NMR data of 76 C18-diterpenoid alkaloids and the 13C NMR data of 64 C18-diterpenoid alkaloids are presented in Tables V and VI, and VII and VIII, respectively, arranged in the order of the proposed classification criteria. In most cases, only the characteristic signals in the 1H NMR spectra of the alkaloids have been reported (Tables V and VI). The major signals of most of the C18-diterpenoid alkaloids were assigned based on comparison with those of similar alkaloids. Only for some of the alkaloids, such as 8-acetyldolaconine (31); piepanendine B (40); piepunendine A (40); delphicrispuline (63); kiridine (94); leucostine (97); anthriscifolcines A (102), C (102), and D (102); linearilin (106); sinomontanine D (55); tiantaishansine (118); 14-O-demethyldelboxine (120); and hohenackeridine (119), were all of the 1H (13C) NMR signals assigned according to their 2D NMR spectra. It is therefore difficult to avoid some assignment errors in the 13C signals of the individual alkaloids. However, this does not affect the correctness of the structures.

6

Table I

Lappaconitine-type alkaloids (A)

AI-2 Aconosine (2)

AI-3 Delavaconine (Episcopalisinine) (3)

OCH3 HO

OH

OH N

N

C21H33NO4 MW ¼ 363 mp 167–169 (26)

[a]D 1

H NMR (26), 13C NMR (26), MS (26) Aconitum episcopale (26)

OCH3

OCH3 OH N

OH

OH H

OH

OCH3 OCH3

H

C22H35NO4 MW ¼ 377 mp 148 (27,28), 142–143 (29), 147 (30), 149–151 (31), 149–152 (32), 150 (33,29), 147–48 (34) [a]D 21 (MeOH) (27), 25.4 (CH3OH) (29), 24.4 (EtOH) (32) 1 H NMR (27*,28,29,31,32), 13C NMR (28*,30–32,124), MS (19) A. arcuatum (34) A. campylorrhynchum (31) A. contortum (32) A. dunhuaense (37) A. fischeri (34) A. forrestii (29,30) A. napellus (33) A. nasutum (27) A. stapfianum var. pubiceps (28)

OH H

C22H35NO5 MW ¼ 393 mp 148–149 (32), 152–154 (14), 152 (35)

[a]D 5 (EtOH) (32), 3.8 (EtOH) (14), 6.4 (CHCl3) (35) 1 H NMR (14,32,35*), 13C NMR (32,35), MS (14,35) A. contortum (14,32,36) A. delavayi (35)

Wang et al.

Aconosine subtype (AI) AI-1 Scopaline (1)

AI-4 8-Deoxy-14-dehydroaconosine (4)

AI-5 8-Acetyldolaconine (5)

OCH3 HO

AI-6 Dolaconine (14-Acetyl aconosine, Episcopalitine) (6)

O

OAc N

N

OH

OCH3 OCH3

OH N

OAc H

C26H39NO6 MW ¼ 461 mp 148 (31) [a]D 1 H NMR (31),

AI-7 Delavaconitine C (7)

AI-8 Delavaconitine D (8)

13

C NMR (31), MS (31)

A. campylorrhynchum (31)

OCH3

OCH3

OH

OH N

N

N

OCH3

OBz

OBz

OAc H

C31H41NO6 MW ¼ 523

H

C30H43NO5 MW ¼ 497

OCH2CH2

OH

7

C29H39NO5 MW ¼ 481

AI-9 Piepunendine B (9) OCH3

OCH3 OCH3

C24H37NO5 MW ¼ 419 mp 44–46 (28), 81–83 (31) [a]D 0.9 (EtOH) (14), 8.7 (EtOH) (32) 1 H NMR (28*,31,32,39), 13C NMR (14), MS (32) A. campylarrhynchum (31) A. contortum (A. episcopale) (14,32,36) A. nasutum (39) A. stapfianum var. pubiseps (28)

The C18-Diterpenoid Alkaloids

C21H31NO3 MW ¼ 345 mp 142–145 (38) [a]D 1 H NMR (38), 13C NMR, MS (38) A. stapfianum (38)

H

OH

H

H

OCH3

OCH3

8

Table I (Continued )

AI-10 Delavaconitine (Episcopalisine) (10) OH

mp 185–186 (35) [a]D 45.6 (CH3OH) (35)

mp 85–87 (40) [a]D 10.6 (CHCl3) (40)

1

1

H NMR (35),

13

C NMR (35), MS (35)

H NMR (40),

C NMR (40), MS (40)

A. delavayi (35)

A. piepunense (40)

AI-11 Contortumine (11)

AI-12 Delavaconitine E (12) OAc

OH

OCH3

OCH3

OBz

OAs

OBz

N

N

N OH

OH

OH H

H

H

OCH3

OCH3

OCH3

OCH3

C29H39NO6 MW ¼ 497 mp 59–64 (42) [a]D9.56 (EtOH) (35,42), 11.7 (EtOH) (14), 14.4 (EtOH) (32) 1 H NMR (32*,35), 13C NMR (32,35), MS (32,35) A. contortum (14,32) A. delavayi (35,42–44)

13

C30H41NO7 MW ¼ 527 mp [a]D 4.8 (EtOH) (32)

C31H41NO7 MW ¼ 539 mp 118–119 (41) [a]D 30.0 (CH3OH) (41)

1

1

H NMR (32),

13

C NMR (32), MS (32)

A. contortum (32)

H NMR (41),

13

A. delavayi (41)

C NMR, MS (41)

Wang et al.

mp [a]D 0.03 (CH3OH) (41), +10.4 (EtOH) (32) 1 H NMR (32,41), 13C NMR (32,41), MS (41) A. contortum (32) A. delavayi (41)

AI-13 Liconosine A (13)

AI-14 Piepunendine A (14) OCH3

OCH3 OCH3

OCH3 OH

OH NH

N

OH

OH

H

H O

C20H29NO4 MW ¼ 347 mp 240–242 (30) [a]D 1 H NMR (30), 13C NMR (30), MS (30) A. forrest (30)

A. piepunense (40) AII-2 Lappaconidine (16)

OCH3 HO

OCH3 HO

OCH3 N

OH N

H

OH

OH ΗΟ

H

C22H35NO6 MW ¼ 409

OH ΗΟ

H

C23H37NO6 MW ¼ 423

OCH3

9

C22H35NO5 MW ¼ 393

OCH3

OCH3 OCH3 N

OH ΗΟ

AII-3 Lappaconine (17)

The C18-Diterpenoid Alkaloids

Lappaconine subtype (AII) AII-1 Dihydromonticamine (15)

C20H29NO5 MW ¼ 385 mp 107–109 (40) [a]D 14.3 (CHCl3) (40) 1 H NMR (40), 13C NMR (40), MS

10

mp 156–157 (45) [a]D 1 H NMR (45,47*), MS (47) A. monticola (45)

13

C NMR (47*),

AII-4 Sinomontanine E (18)

mp 206–207 (9) [a]D +120 (CHCl3) (9) 1 H NMR (9*,10), 13C NMR (48), MS (9,10) A. leucostomum (A. excelsum) (9,10) A. septentrionale (48,53)

mp 96 (46) [a]D +27 (CHCl3) (46) 1 H NMR (49), 13C NMR (48), MS (50), X-ray (7,8) A. orientale (51,52) A. leucostomum (54)

AII-5 Akiramidine (19)

AII-6 Akiranine (20)

OCH3 HO

OCH3 HO

OH

OCH3

N

OCH3 N

ΗΟ

H

C22H35NO7 MW ¼ 425 mp [a]D 7.8 (CHCl3) (55) 1 H NMR (55), 13C NMR (55), MS A. sinomontanum (55)

OCH3

N

OH

HO

OCH3 OCH3

OH ΗΟ

H OCH3

C23H37NO6 MW ¼ 423 mp [a]D 1 H NMR (56), 13C NMR, MS (56) A. kirinense (56)

OH ΗΟ

H OCH3

C24H39NO6 MW ¼ 437 mp [a]D 1 H NMR (57), 13C NMR, MS (57) A. kirinense (57)

Wang et al.

Table I (Continued )

AII-7 Kiramine (21)

AII-8 Akiramine (22)

AII-9 Akiran (23)

OCH3 HO

OCH3

OCH3 N

N

N OH

H

OCH3 C25H39NO7 MW ¼ 465 mp [a]D 1 H NMR (60), 13C NMR, MS A. kiriense (60)

AcO

AcO

OCH3

N

C26H41NO7 MW ¼ 479 mp 162–164 (59) [a]D 1 H NMR (59), 13C NMR, MS, X-ray (35) A. kiriense (57,59)

AII-11 9-Deoxylappaconitine (25)

AII-12 4-Anthranoyllappaconitine (26)

O

OCH3

OCH3

N O O

HO OH N

OH

H

O

O NH2

OH

H O NH2

NHCOCH3

C32H44N2O7 MW ¼ 568

OCH3

C29H40N2O7 MW ¼ 528

11

C30H42N2O6 MW ¼ 526

OCH3

OCH3

OH

H

OCH3

C25H39NO7 MW ¼ 465 mp 162–164 (58) [a]D 1 H NMR (58), 13C NMR (58), MS A. kiriense (58)

OCH3 OCH3

OH H

H

The C18-Diterpenoid Alkaloids

AII-10 Neofinaconitine (Delphicrispuline) (24) OCH3

OCH3

OCH3

OH AcO

OCH3

OCH3 HO

12

Table I (Continued )

AII-13 Demethyllappaconitine (27)a

mp 212–214 (61), 195–198 (62) [a]D 1 H NMR (61*,62), 13C NMR (61), MS (61)

mp [a]D 48.8 (CHCl3) (53) 1 H NMR (53), 13C NMR (53), MS (53)

A. finetianum (61) A. rubicundum (62)

A. septentrionale (53)

AII-14 Sinomontanine B (28)a

AII-15 Septefine (29)

OCH3 HO OH

OCH3

N O

HO OH

OCH3

H

C31H42N2O8 MW ¼ 570 mp [a]D +52.0 (CHCl3) (65) 1 H NMR (65), 13C NMR (65), MS (65) A. orientale (65)

OCH3

OH O

H O

O NHCOCH3

OH

OH O

O

HO N

N OH

H

OCH3

OCH3

NHCOCH3

NHCH3

C31H42N2O8 MW ¼ 570 mp [a]D +37.1 (CHCl3) (66) 1 H NMR (66), 13C NMR (66), MS (66)

C31H44N2O7 MW ¼ 556 mp 194–195 (64) [a]D 1 H NMR (64), 13C NMR, MS (64)

A. sinomontanum (66)

A. septentrionale (64)

Wang et al.

mp [a]D +23.8 (CHCl3) 1 H NMR (61,63*), 13C NMR (61,63*), MS (61,63) A. finetianum (61) D. crispulum (syn. D. speciosum var. linearilobum) (63)

AII-16 N-Deacetyllappaconitine (puberanidine) (30)

AII-17 Lappaconitine (31)

OCH3

OCH3

OCH3

OH

OH

OCH3

OCH3 OH

N

N H

NH OH

OH O

OH

H

O

NH2

[a]D +42 (EtOH) (67), +23.3 (CDCl3) (73), +29.4 (69)

C32H44N2O8 MW ¼ 584 mp 227 (46), 219–221 (67), 224 (71), 224–225 (72), 222–224 (73), 226–228 (74), 199–221 (75) [a]D +27 (CHCl3) (46,67,72), +28.2 (CDCl3) (46), +30.0 (EtOH) (74), +20.0 (CHCl3) (67) 1 H NMR (46,67*,71–73), 13C NMR (48*,72), MS (72) A. barbatum var. puberulum (73,76) A. excelsum (5,79,80) A. finetianum (67,73) A. leucostomum (9,10,50,81,82,79) A. nasutum (75)

NHCOCH3

C30H40N2O8 MW ¼ 555 mp

[a]D

1

H NMR (66),

13

C NMR (66), MS

A. sinomontanum (66)

13

H NMR (64,67,73*), 13C NMR (73), MS A. barbatum var. puberulum (73) A. finetianum (77,78) A. leucostomum (81,82) A. orientale (51,52,68) A. ranunculaefolium (74)

NHCOCH3

The C18-Diterpenoid Alkaloids

C30H42N2O7 MW ¼ 542 mp 120–121 (67), 212–214 (68), 213–214 (69), 209–214 (70)

H O

O

O

1

OCH3 OCH3

OCH3

OCH3

O

AII-18 Sinomontanine A (32)

14 Wang et al.

Table I (Continued ) A. septentrionale (46,53) Delphinium cashmiranum (67)

A. orientale (4,51,52,68,83) A. ranunculaefolium (74,84) A. septentrionale (1–3,48,46) A. sinomontanum (71) A. sinomontanum var. angustius (69) A. talassicum (85) D. cashmirianum (67)

AII-19 Sepaconitine (33)

AII-20 N-Acetylsepaconitine (34) OCH3

OCH3

OH

OCH3

OCH3

OH

HO

OCH3

N

N H

OH O

O

H

OH O

O NH2

C30H42NO8 MW ¼ 558

OCH3

OH

OH O

OCH3

OCH3

OH

N

AII-21 Monticamine (35)

NHCOCH3

C32H44N2O9 MW ¼ 600

C22H33NO5 MW ¼ 391

mp 250–253 (86,87) [a]D +25 (CHCl3) (86,87) 1 H NMR (86), 13C NMR (86*,87), MS (86) A. leucostomum (87) A. septentrionale (53,86)

mp [a]D 1 H NMR (87),

A. leucostomum (87)

A. karakolium (47,88) A. monticola (47)

AII-22 Excelsine (36)

AII-23 8-Acetylexcelsine (37)

AII-24 Akiradin (38)

13

C NMR (87), MS (87)

OCH3 HO OH

mp 163–164 (47) [a]D +4 (CH3OH) (47) 1 H NMR (47), 13C NMR (47), MS (47)

OCH3 HO

OCH3

OH

N

OCH3 AcO

OCH3

OH

N

N

OH O

OAc

O

C24H31NO7 MW ¼ 449 mp [a]D 1 H NMR (92), A. kiriense (92)

13

C NMR, MS (92)

O

OH

C24H35NO7 MW ¼ 449 mp 108–110 (90) [a]D 1 H NMR (90), 13C NMR, MS (90) A. kiriense (90)

The C18-Diterpenoid Alkaloids

C22H33NO6 MW ¼ 407 mp 94–96 (89), 102–104 (57) [a]D 1 H NMR (13,79*,89,91), 13C NMR (91), MS (13,89), X-ray (13,89) A. excelsum (13) A. kiriense (60) A. sinomontanum (91)

OCH3

15

16 Wang et al.

Table I (Continued ) AII-25 Akirine (39)

AII-26 Kiridine (40)

AII-27 Kiritine (41)

OCH3 HO

N O

OCH3 HO

O O

N

OH

O

O

OCH3 HO OCH3

O

N

OH

OH

O OCH3

C22H31NO6 MW ¼ 405 mp 214–217 (93), 206–208 (94) [a]D 1 H NMR (93,94*), 13C NMR, MS (93) X-ray (93) A. kiriense (93,94) Selected NMR data. a

Same structure with different

13

C NMR.

C23H31NO6 MW ¼ 405 mp 206–208 (94) [a]D 1 H NMR (94), 13C NMR (94), MS

C23H35NO6 MW ¼ 421 mp [a]D 1 H NMR, 13C NMR, MS

A. kirinense (60,94)

A. kiriense (60)

Table II

Ranaconitine-type alkaloids (B)

Leuconine subtype (BI) BI-1 Leuconine (42)

BI-2 Leucostine (43)

BI-3 Acosepticine (44)a OCH3

OCH3

OCH3

N

N

H

O

OCH3

OCH3

OCH3 N

OCH3

OCH3

OCH3

H

OH

O

OH

OH OH

H OH

OH

C23H35NO6 MW ¼ 421 mp [a]D +19.4 (EtOH) (96) 1 H NMR (96*,97), 13C NMR (96*,97), MS (96) A. leucostomum (96) A. septentrionale (97)

C23H37NO6 MW ¼ 423 mp [a]D +23.4 (CHCl3) (53) 1 H NMR (53), 13C NMR (53), MS (53)

BI-4 Umbrofine (45)a

BI-5 6-O-Acetylumbrofine (46)b OCH3

BI-6 6-O-Acetylacosepticine (47)b OCH3

OCH3

OCH3

OCH3

OCH3

OCH3 N

OCH3 N

H OH

OCH3 N

OH OH

H OAc

C25H39NO7 MW ¼ 465

OH OH

H OAc

C25H39NO7 MW ¼ 465

17

C23H37NO6 MW ¼ 423

OH OH

A. septentrionale (53)

The C18-Diterpenoid Alkaloids

C23H35NO5 MW ¼ 405 mp 195–197 (95), 193–195 (95) [a]D +10.5 (EtOH) (96) 1 H NMR (96), 13C NMR (96), MS (95) A. leucostonum (95,96) A. septentrionale (95)

18 Wang et al.

Table II (Continued ) mp 110–112 (98) [a]D 1 H NMR (98), 13C NMR (98), MS (98) A. umbrosum (98)

mp 174–175 (98) [a]D 1 H NMR (98), 13C NMR (98), MS (98)

mp [a]D 1 H NMR (99),

A. umbrosum (98)

A. septentrionale (99)

BI-7 Acoseptrine (48)

BI-8 Exceconidine (6-O-Methylumbrofine) (49)

BI-9 Anthriscifolcine B (50)

OH

OCH3 OCH3 N

OH H OH OH

C23H37NO7 MW ¼ 439 mp 105–107 (53) [a]D +19.6 (CHCl3) (53) 1 H NMR (53), 13C NMR (53), MS (95) A. septentrionale (53)

OCH3 OCH3

OCH3

N

C NMR (99), MS

OCH3

OCH3 OCH3

13

OCH3 N

H

OH OH

CH3O

C24H39NO6 MW ¼ 437 mp 145–148 (100,101) [a]D 1 H NMR (100*,101), 13C NMR (100*,101), MS (100,101) A. excelsum (100,101)

O H OH

O

C24H37NO6 MW ¼ 435 mp 75–77 (102) [a]D 27 (CHCl3) (102) 1 H NMR (102), 13C NMR (102), MS (102) D. anthriscifolium var. savatieri (102)

BI-10 Anthriscifolcine A (51) OCH3 OCH3

BI-11 Anthriscifolcine G (52)

BI-12 Anthriscifolcine C (53) OH OCH3

OCH3 N

N

OH

N H

OAc

OH

O

O H

OCH3 OCH3

OCH3

O

OAc

O H

O

OAc

O

C25H37NO7 MW ¼ 463 mp 136–138 (103) [a]D 56.7 (CHCl3) (103) 1 H NMR (103), 13C NMR (103), MS D. anthriscifolium var. savatieri (103)

C25H37NO8 MW ¼ 479 mp 222–224 (102) [a]D 11.4 (CHCl3) (102) 1 H NMR (102), 13C NMR (102), MS D. anthriscifolium var. savatieri (102)

BI-13 Anthriscifolcine E (54)

BI-14 Anthriscifolcine D (55)

BI-15 Annthriscifolcine F (56)

OCH3 OCH3

OH

OCH3

N

OCH3

OH

OCH3

OH

O

OCH3 O

O H OAc

O

C26H39NO8 MW ¼ 493 mp 185–187 (102)

H OAc

O

C25H37NO8 MW ¼ 479 mp 178–180 (103)

19

C24H37NO7 MW ¼ 451 mp 150–152 (102)

OH

N

N O H

OH

OCH3 OCH3

The C18-Diterpenoid Alkaloids

C26H39NO7 MW ¼ 477 mp 135–137 (102) [a]D 12.2 (CHCl3) (102) 1 H NMR (102), 13C NMR (102), MS D. anthriscifolium var. savatieri (102)

20 Wang et al.

Table II (Continued ) [a]D 34.5 (CHCl3) (102) 1 H NMR (102), 13C NMR (102), MS D. anthriscifolium var. savatieri (102) BI-16 Lamarckinine (57) OCH3 OCH3 OCH3 N H

OH OH

CH3O

C22H33NO6 MW ¼ 407 mp [a]D +76.6 (CHCl3) (104) 1 H NMR (104), 13C NMR (104), MS A. larnarokii (104)

[a]D 41.3 (CHCl3) (102) 1 H NMR (102), 13C NMR (102), MS D. anthriscifolium var. savatieri (102)

[a]D 27.8 (CHCl3) (103) 1 H NMR (103), 13C NMR (103), MS D. anthriscifolium var. savalier (103)

Ranaconitine subtype (BII) BII-1 Sinomontanine D (58)

BII-2 Delbine (59)

BII-3 Linearilin (60) OCH3

OCH3 OCH3 OH

OH

OCH3 N

N OH

OH

OCH3 N

OH

OH

HO

OCH3 OCH3

HO

HO

OH OCH3

HO

OH OOH OCH3

C22H35NO7 MW ¼ 425 mp 116–118 (105) [a]D +53.3 (CH3OH) (105) 1 H NMR (105), 13C NMR (105), MS (105) D. bonvalotii (105)

C24H39NO8 MW ¼ 469 mp [a]D +18.4 (CHCl3) (106) 1 H NMR (106), 13C NMR (106), MS D. linearibum (106)

BII-4 Hispaconitine (61)

BII-5 Sinomontanine H (62)

BII-6 9-Deoxy-6-methoxy-N-succinyldeacetylranaconitine (63)

OCH3

OCH3 OCH3

OCH3 OCH3 N HO

OCH3

OCH3

OCH3 N

N OAc OH

OCH3 OH

O

OH OH OCH3 O

OH OH OCH3 O NHCOCH2CH2CO2H

21

NHAc

O

The C18-Diterpenoid Alkaloids

C22H35NO8 MW ¼ 441 mp [a]D +26.6 (CHCl3) (55) 1 H NMR (55), 13C NMR (55), MS A. sinomontanum (55)

22

Table II (Continued ) C32H44N2O9 MW ¼ 600 mp [a]D 28.8 (CHCl3) (108) 1 H NMR (108), 13C NMR (108), MS (108) A. sinomontanum (108)

C35H48N2O11 MW ¼ 672 mp [a]D +34.0 (CHCl3) (76) 1 H NMR, 13C NMR, MS A. barbatum var. puberulum (76)

BII-7 Isolappaconitine (64)

BII-8 N-Deacetylranaconitine (65)

BII-9 Ranaconitine (66)

OCH3 OCH3

OCH3

OCH3 OCH3

OH N

N OH OH

O

OCH3

OCH3 OCH3

OH N

OH OH

O O

O

OH OH

O O

NH2

NHAc

OCH3

C32H44N2O8 MW ¼ 584 mp 198–200 (61), 186–188 (62)

C30H42N2O8 MW ¼ 558 mp 125–127

[a]D

[a]D +43.7 (CHCl3) (77)

NHAc

C32H44N2O9 MW ¼ 600 mp 132–134 (109), 135–137 (73), 131–132 (69), 129–132 (80), 131–133 (68), 130–131 (110), 137 (71) [a]D +33.2 (CHCl3) (109), +40.2 (CHCl3) (110)

Wang et al.

C26H41NO8 MW ¼ 495 mp 185–187 (107) [a]D +43.4 (CHCl3) (107) 1 H NMR (107), 13C NMR (107), MS A. barbetum var. hispidum (107)

1

H NMR (61*,62), 13C NMR (61), MS (61,62) A. finetianum (61) A. rubicundum (62)

1

BII-10 Puberanine (67)

BII-11 Sinomontanine F (68)

H NMR (77),

13

C NMR, MS (77)

A. septentrionale (77)

OCH3 OH

OH

O

OCH3

O

OH OH

O O

NHAc

C30H40N2O9 MW ¼ 457 mp [a]D +44.4 (CHCl3) (108) 1 H NMR (108), 13C NMR (108), MS A. sinomontanum (108)

NH2

C30H42N2O9 MW ¼ 574 mp 121–123 (77) [a]D 34.9 (CHCl3) (77) 1 H NMR (77), 13C NMR, MS (77) A. finetiaum (77)

23

C32H44N2O9 MW ¼ 600 mp [a]D +16.6 (CHCl3) (73) 1 H NMR (73), 13C NMR (73), MS A. barbatum var. puberulum (73)

OH N

OH OH O

NHAc

OH

The C18-Diterpenoid Alkaloids

O

OCH3

OCH3

NH OH OH

BII-12 N-Deacetylfinaconitine (69) OCH3

OCH3

N

H NMR (73*,109), 13C NMR (109*,110,111), MS (73,112) A. barbatum var. puberulum (73) A. excelsum (80) A. finetianum (110) A. orientale (68) A. ranunculaefolium (109) A. septentrionale (112) A. sinomontanum (113,71) A. sinomontanum var. angustius (69)

OCH3

OCH3 OCH3

1

24 Wang et al.

Table II (Continued ) BII-13 Finaconitine (10bhydroxyranaconitine) (70)

BII-14 Sinomontanine G (71)

OCH3 OCH3

BII-15 Monticoline (72) OCH3

OCH3 HO

HO OH

OH

OCH3

OH

N

OCH3

OCH3 N

N OH OH

O

OH

OH O

OH

O

OH

O NHAc

C32H44N2O10 MW ¼ 616 mp 220–221 (110) [a]D +44.7 (MeOH) (110) 1 H NMR (44,71,110*), 13C NMR (110), MS (110) A. finetiaum (110) A. sinomentanum (71)

C22H33NO7 MW ¼ 423 mp [a]D 32.0 (CHCl3) (108) 1 H NMR (108), 13C NMR (108), MS

C22H33NO6 MW ¼ 407 mp 166–167 (47) [a]D +15.0 (CHCl3) (47) 1 H NMR (47), 13C NMR (47), MS (47)

A. sinomantanum (108)

A. monticola (47) A. kakakolicum (114)

BII-16 Tuguaconitine (73)

BII-17 14-Demethyltuguaconitine (74) OCH3

OCH3

OCH3

HO

H3CO

HO OCH3

OH

OH

N

N

N OH

O

BII-18 Tiantaishansine (75)

OH

OH O

OH OCH3

O

OH OCH3

OH OH

C22H33NO7 MW ¼ 423 mp 208–210 (117) [a]D +59.5 (CHCl3) (117) 1 H NMR (117), 13C NMR (117), MS (117)

C22H33NO7 MW ¼ 421 mp 94–96 (118) [a]D +35.4 (CHCl3) (118) 1 H NMR (118), 13C NMR (118), MS

D. stapeliosum (117)

D. tiantaishanense (118)

BII-19 Delboxine (76)

BII-20 14-O-Demethyldelboxine (77)

BII-21 Hohenackeridine (78)

OCH3

OCH3

HO OCH3

OH

N

N OCH3

O

OCH3 HO

HO

OH OCH3

N OCH3

O

O

OH OCH3

O

OH OH OCH3

The C18-Diterpenoid Alkaloids

C23H35NO7 MW ¼ 437 mp 196–198 (115), 197–199 (116) [a]D 1 H NMR (115*,116), 13C NMR (116), MS (116) A. sibiricum (115,116)

25

26 Wang et al.

Table II (Continued ) C24H37NO7 MW ¼ 451 mp 200–202 (105) [a]D +43.5 (CHCl3) (105) 1 H NMR (105), 13C NMR (105), MS, X-ray (121) D. bonvalotii (105) Selected NMR data. a,b

Same structure with different

13

C NMR.

C23H35NO7 MW ¼ 437 mp [a]D +43.3 (CHCl3) (120) 1 H NMR (120), 13C NMR (120), MS

C22H31NO7 MW ¼ 421 mp 224–225 (119) [a]D +39.5 (CH3OH) (119) 1 H NMR (119), 13C NMR (119), MS

Consolida orientalis (120)

Consolida hohenaceri (syn. Aconiteda hohenckeri, Delphinium hohenaceri) (119)

The C18-Diterpenoid Alkaloids

Table III Plants

Occurrence of C18-diterpenoid alkaloids in plant species Alkaloids (references)

Ranunculaceae Aconitum spp. A. arcuatum Maxim. Aconosine (34) A. barbatum var. hispidum (DC.) Hispaconitine (107) Ser. A. barbatum var. puberulum Ledeb. N-Deacetyllappaconitine (73) 9-Deacetylranaconitine (77) 9-Deoxy-6-methoxy-N-succinyldeoxyranaconitine (76) Lappaconitine (73,76) Purberanine (73) Ranaconitine (73) A. campylorrhynchum Hand.-Mazz 8-Acetyldolaconine (31) Aconosine (31) Dolaconine (31) A. contortum Finet & Gagnep. Aconosine (32) Contortumine (32) Delavaconine (14,32,36) Delavaconitine (14,32) Delavaconitine C (32) Dolaconine (14,32,36) A. delavayi Franch. Delavaconine (35) Delavaconitine (35,42–44) Delavaconitine C (41) Delavaconitine D (35) Delavaconitine E (41) A. dunhuaense S. H. Li Aconosine (34) A. espicopale H. Le´v. Scopaline (26) A. excelsum Rchb. Exceconidine (6-Methylumbrofine) (100,101) Excelsine (13) Lappaconitine (5,79,80) Ranaconitine (80) A. finetianum Hand.-Mazz N-Deacetylfinaconitine (77) N-Deacetyllappaconitine (Puberanidine) (77,78) 9-Deoxylappaconitine (61) Finaconitine (110) Isolappaconitine (61) Lappaconitine (67,72) Neofinaconitine (61) Ranaconitine (110) A. fischeri Rchb. Aconosine (34) A. forrestii Stapf. Aconosine (29,30)

27

28

Wang et al.

Table III (Continued ) Plants A. karakolicum Rapaics A. kirinense Nakai

A. lamarckii Rchb. A. leucostomum Vorosch.

A. monticola Steinb.

A. napellus L. A. nasutum Fisch ex. Reichb. A. orientale Mill.

A. piepunense Hand.-Mazz A. ranunculaefolium (?)

A. rubicundum Fisch.

Alkaloids (references) Liconosine A (30) Monticamine (47,88) Monticoline (114) 8-Acetylexcelsine (92) Akiradin (90) Akiramine (58) Akiramidine (56) Akiran (57,59) Akiranine (57) Akirine (93,94) Excelsine (60) Kiramine (60) Kiridine (60,94) Kiritine (60) Lamarckinine (104) N-Acetylsepaconitine (87) N-Deacetyllappaconitine (81,82) Excelsine (79) Lappaconidine (9,10) Lappaconine (54) Lappaconitine (9,10,50,79,81,82) Leuconine (95,96) Leucostine (96) Sepaconitine (87) Dihydromonticamine (45) Monticamine (47) Monticoline (47) Aconosine (33) Aconosine (27) Dolaconine (39) N-Deacetyllappaconitine (51,52,68) Demethyllappaconitine (65) Lappaconine (47,51,52) Lappaconitine (4,51,52,68,75,83) Ranaconitine (68) Piepunendine A (40) Piepunendine B (40) N-Deacetyllappaconitine (Puberanidine) (74) Lappaconitine (74) Ranaconitine (109) 9-Deoxylappaconitine (62) Isolappaconitine (62) Ranaconitine (73)

The C18-Diterpenoid Alkaloids

Table III (Continued ) Plants A. septentrionale Koelle

A. sibiricum Poir. A. sinomontanum Nakai

A. sinomontanum var. angustius W. T. Wang A. stapfianum Hand.-Mazz

A. stapfianum var. pubipes W. T. Wang A. talassicum Popov A. umbrosum (Korsh.) Kom. Delphinium spp. D. anthriscifolium var. savatieri (Franch.) Munz

Alkaloids (references) 6-O-Acetylacosepticine (99) Acosepticine (53) Acoseptrine (53) 4-Anthranoyllappaconidine (53) N-Deacetyllappaconitine (Puberanidine) (46,53) N-Deacetylranaconitine (77) Lappaconidine (48,53) Lappaconitine (1–3,46,48) Leuconine (95) Leucostine (97) Ranaconitine (112) Sepaconitine (53,86) Septefine (64) Tuguaconitine (115,116) Excelsine (91) Finaconitine (71) Lappaconitine (71) Ranaconitine (71) Sinomontanine A (66) Sinomontanine B (66) Sinomontanine D (55) Sinomontanine E (55) Sinomontanine F (108) Sinomontanine G (108) Sinomontanine H (108) N-Deacetyllappaconitine (53) Lappaconitine (69) Ranaconitine (69) 8-Deoxy-14-dehydroaconosine (38) Dolaconine (14-Acetylaconosine, Episcopalitine) (28) Aconosine (28) Dolaconine (28) Lappaconitine (85) 16-Acetylumbrofine (98) Umbrofine (98) Anthriscifolcine Anthriscifolcine Anthriscifolcine Anthriscifolcine Anthriscifolcine

A (102) B (102) C (102) D (102) E (102)

29

30

Wang et al.

Table III (Continued ) Plants

D. bonvalotii Franch. D. cashmerianum Royle

Alkaloids (references) Anthriscifolcine F (103) Anthriscifolcine G (103) Delbine (105) Delboxine (105) N-Deacetyllappaconitine (puberanidine) (67) Lappaconitine (67) Delphicrispuline (63)

D. crispulum Rupr. (syn. D. speciosum Bieb. var. linearilobum Trautv.) D. linearilobum N. Busch Linearilin (106) D. stapeliosum Bru¨hl 14-Demethyltuguaconitine (117) D. tiantaishanense W. J. Zhang and Tiantaishansine (118) G. H. Chen Consolida spp. C. hohenackeri Grossh. (syn. Aconitella hohenackeri, Delphinium hohenackeri) C. orientalis (M. Gay ex Des Moul.) Schro¨dinger

Hohenackeridine (119)

14-O-Demethoxyldelboxine (120)

It is worth noting that demethyllappaconitine (27, AII-13) (65) and sinomontanine B (28, AII-14) (66) possess significantly different 13C NMR data, even though they were reported to have the same structure. Accordingly, they are presented as two alkaloids whose structures need to be further differentiated in the future.

IV. CHEMICAL REACTIONS Due to the requirement for structural determination, chemical studies on the C18-diterpenoid alkaloids [such as lappaconitine (6) and lappaconidine (6)] were extensively investigated in the early 1970s, an early phase of the chemical investigation on C18-diterpenoid alkaloids. However, there are very few reports on the chemistry of the C18-diterpenoid alkaloids during the subsequent 40-year period. When we attempted to prepare a C-10 oxygenated derivative of lappaconitine via the oxidation of lappaconitine with HIO4, followed by bromination, the unexpected product 1 was obtained. Treatment of compound 1 with bromine in acetic acid yielded compounds 2 (31%) and

The C18-Diterpenoid Alkaloids

Table IV

C18-diterpenoid alkaloids and their code number index

Alkaloids

Code numbers

14-Acetylaconosine 6-O-Acetylacosepticine 14-Acetyldihydrogadesine 8-Acetyldolaconine 8-Acetylexcelsine N-Acetylsepaconitine 6-O-Acetylumbrofine Aconosine Acosepticine Acoseptrine Akiradin Akiramidine Akiramine Akiran Akiranine Akirine 4-Anthranoyllappaconitine Anthriscifolcine A Anthriscifolcine B Anthriscifolcine C Anthriscifolcine D Anthriscifolcine E Anthriscifolcine F Anthriscifolcine G

BII-5 BI-6 BII-4 AI-5 AII-23 AII-20 BI-5 AI-2 BI-3 BI-7 AII-24 AII-5 AII-8 AII-9 AII-6 AII-25 AII-12 BI-10 BI-9 BI-12 BI-14 BI-13 BI-15 BI-11

Contortumine

AI-11

N-Deacetylfinaconitine N-Deacetylappaconitine N-Deacetylranaconitine 9-Deoxylappaconitine Delavaconine Delavaconitine Delavaconitine C Delavaconitine D Delavaconitine E Delbine Delphicrispuline Delboxine 14-O-Demethyldelboxine Demethylappaconitine

BII-12 AII-16 BII-8 AII-11 AI-3 AI-10 AI-7 AI-8 AI-12 BII-2 AII-10 BII-19 BII-20 AII-13

31

32

Wang et al.

Table IV (Continued ) Alkaloids

Code numbers

14-Demethyltuguaconitine 8-Deoxy-14-dehydroaconosine 9-Deoxylappaconitine 9-Deoxy-6-methoxy-N-succinyl-deacetylranaconitine Dihydromonticamine Dolaconine Delavaconine

BII-17 AI-4 AII-11 BII-6 AII-1 AI-6 AI-3

Episcopalisine Episcopalisinine Episcopalitine Exceconidine Excelsine

AI-10 AI-3 AI-6 BI-8 AII-22

Finaconitine

BII-13

Hispaconitine Hohenackeridine 10-Hydroxyranaconitine

BII-4 BII-21 BII-13

Isolappaconitine

BII-7

Kiramine Kiridine Kiritine

AII-7 AII-26 AII-27

Lamarkinine Lappaconidine Lappaconine Lappaconitine Leuconine Leucostine Liconosine A Linearilin

BI-16 AII-2 AII-3 AII-17 BI-1 BI-2 AI-13 BII-3

6-O-Methylumbrofine Monticamine Monticoline

BI-8 AII-21 BII-15

Neofinaconitine

AII-10

Piepunendine A Piepunendine B Puberanidine Puberanine

AI-14 AI-9 AII-16 BII-10

The C18-Diterpenoid Alkaloids

33

Table IV (Continued ) Alkaloids

Code numbers

Ranaconitine

BII-9

Scopaline Sepaconitine Septefine Sinomontanine Sinomontanine Sinomontanine Sinomontanine Sinomontanine Sinomontanine Sinomontanine

AI-1 AII-19 AII-15 AII-18 AII-14 BII-1 AII-4 BII-11 BII-14 BII-5

A B D E F G H

Tiantaishansine Tuguaconitine

BII-18 BII-16

Umbrofine

BI-4

3 (13%) (Scheme 1) (125). The structure of compound 1 was unambiguously determined based on its 2D NMR spectra and X-ray crystallographic analysis. In addition, the direct reaction of lappaconitine with NaIO4 and 1 equiv of bromine in a suitable amount of acetic acid at room temperature for 7 h produced a brominated N-deethyl derivative 4 in 71% yield. For this reaction, the NaIO4 is necessary and appears to serve as a ‘‘catalyst’’ (126).

V. PHARMACOLOGICAL ACTIVITY For centuries in many countries (e.g., China, Japan), the tubers of Aconitum plants have been used in traditional medicine for the treatment of various diseases (e.g., rheumatoid arthritis) and various pains including migraine, swelling induced by trauma and fracture, and facial paralysis. Some diterpenoid alkaloids, the characteristic components of Aconitum plants, possess various marked biological activities. As compared with the C19-diterpenoid alkaloids, there is little information on the biological actions of the C18-diterpenoid alkaloids. After extensive studies lasting many years, the C18-diterpenoid alkaloid lappaconitine, as both an analgesic and an antiarrhythmic drug, was introduced into clinical practice. Following Benn and Jacyno’s excellent review (127) of the toxicity and pharmacology of the diterpenoid alkaloids in 1983,

34

Wang et al.

Table V

1

H NMR data of lappaconitine-type alkaloids (A)*

Codes (names) (references) Aconosine subtype (AI) AI-1 (Scopaline, 1) (26)

AI-2 (Aconosine, 2) (27)

AI-3 (Delavaconine/ episcopalisinine, 3) (35) AI-4 (8-Deoxy-14dehydroaconosine, 4) (38) AI-5 (8-Acetyldolaconine, 5) (31)

AI-6 (Dolaconine/14acetylaconosine/ episcopalitine, 6) (28) AI-7 (Delavaconitine C, 7) (41)

AI-8 (Delavaconitine D, 8) (35)

dH 1.12 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.10 (1H, s, OH), 3.35 (3H, s, OCH3), 3.72 (1H, t, J ¼ 3 Hz, H-1b), 4.23 (1H, t, J ¼ 4.5 Hz, H-14b), 7.40 (1H, br s, OH) 1.05 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.26, 3.34 (each 3H, s, OCH3 2), 3.56 (1H, s, OH), 4.08 (1H, t, J ¼ 4 Hz, H-14b), 4.56 (1H, J ¼ 7 Hz, OH) 1.04 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.24, 3.40 (each 3H, s, OCH3 2), 4.04 (1H, d, J ¼ 4.5 Hz, 14b-H) 1.05 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.21, 3.14 (each 3H, s, OCH3 2) 3.06 (1H, dd, J ¼ 10 and 6.5 Hz, H-1b), 1.85 (1H, m, H-2a), 1.16 (1H, m, H-2b), 2.73 (1H, dd, J ¼ 15 and 9 Hz, H-3a), 2.04 (1H, dd, J ¼ 15 and 7 Hz, H-3b), 1.55 (1H, s, br s, H-4), 1.85 (1H, s, br s, H-5), 1.69 (1H, d, J ¼ 12 Hz, H-6a), 1.38 (1H, dd, J ¼ 12 Hz, H-6b), 1.68 (1H, br s, H-7), 2.54 (1H, s, br s, H-9), 2.29 (1H, br s, H-10), 2.13 (1H, d, J ¼ 9.5 Hz, H-12a), 1.93 (1H, d, J ¼ 9.5 Hz, H-12b), 3.20 (1H, br s, H-13), 4.74 (1H, t, J ¼ 4.6 Hz, H-14b), 1.84 (1H, m, H-15a), 2.30 (1H, m, H-15b), 3.19 (1H, m, H-16a), 2.77 (1H, s, H-17), 2.44 (1H, dd, J ¼ 11.4 and 7 Hz, H-19a), 2.49 (1H, dd, J ¼ 11.4 and 5 Hz, H-19b), 2.35 (2H, q, J ¼ 7.0 Hz, NCH2CH3), 1.00 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 1.88, 1.98 (each 3H, s, OAc 2), 3.22, 3.26 (each 3H, s, OCH3 2) 1.06 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.05 (3H, s, OAc), 3.23, 3.27 (each 3H, s, OCH3 2), 4.82 (1H, t, J ¼ 5 Hz, H-14b) 1.02 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.12, 3.22 (each 3H, s, OCH3 2), 5.08 (1H, t, J ¼ 4.5 Hz, H-14b), 7.38 (3H, m, Ar-H), 7.90 (2H, d, J ¼ 8 Hz, Ar-H) 1.04 (3H, t, J ¼ 4.5 Hz, NCH2CH3), 1.24 (3H, s, OAc-8), 3.20, 3.30 (each 3H, s, OCH3 2), 4.94 (1H, t, J ¼ 4.5 Hz, H-14b), 7.36 (3H, m, Ar-H), 7.86 (2H, d, J ¼ 8 Hz, Ar-H)

The C18-Diterpenoid Alkaloids

35

Table V (Continued ) Codes (names) (references) AI-9 (Piepunendine B, 9) (40)

AI-10 (Delavaconitine/ episcopalisine, 10) (32)

AI-11 (Contortamine, 11) (32)

AI-12 (Delavaconitine E, 12) (41)

AI-13 (Lieconosine A, 13) (30)

AI-14 (Piepunendine A, 14) (40)

dH 3.08 (1H, m, H-1a), 1.80 (1H, m, H-2a), 2.20 (1H, m, H-2b), 1.92 (1H, m, H-3a), 2.10 (1H, m, H-3b), 2.46 (1H, m, H-4), 1.82 (1H, m, H-5), 1.13 (1H, m, H-6a), 1.85 (1H, m, H-6b), 2.19 (1H, m, H-7), 2.28 (1H, t, J ¼ 6.0 Hz, H-9), 1.69 (1H, m, H-10), 1.92 (1H, m, H-12b), 2.17 (1H, m, H-12a), 2.06 (1H, m, H-13), 3.97 (1H, t, J ¼ 4.4 Hz, H-14b), 1.70 (1H, m, H-15a), 1.64 (1H, m, H-15b), 3.30 (1H, m, H-16), 2.85 (1H, br s, H-17), 2.43, 2.62 (each 1H, ABq, J ¼ 11.2 Hz, H2-19), 2.40, 2.46 (each 1H, m, NCH2), 1.05 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.26 (3H, s, OCH3-1), 3.25 (3H, s, OCH316), 6.78 (2H, AAuBBu system, J ¼ 11.2 Hz, H-3v, 5v), 7.04 (2H, AAuBBu system, J ¼ 11.2 Hz, H-2v, 6v), 2.68, 2.70 (each 1H, m, H2-7v), 3.47, 3.51 (each 1H, m, H2-8v) 1.05 (3H, t, J ¼ 7 Hz, NCH2CH3), 1.1–1.5 (4H, m, H-3, H-6), 3.28, 3.40 (each 3H, s, OCH3), 5.10 (1H, d, J ¼ 4.5 Hz, H-14b), 7.42, 7.54, 8.03 (5H, m, Ar-H) 1.09 (3H, t, J ¼ 7.1 Hz, NCH2CH3), 3.29, 3.38, 3.86 (each 3H, s, OCH3 3), 5.12 (1H, d, J ¼ 5.1 Hz, 14b-H), 6.92, 7.99 (2H, AAuBBu system, J ¼ 9.0 Hz, Ar-H) 1.10 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.00 (3H, s, OAc), 3.27, 3.30 (each 3H, s, OCH3 2), 5.45 (1H, d, J ¼ 4.5 Hz, H-14b), 7.50 (3H, m, Ar-H), 8.07 (2H, d, J ¼ 8 Hz, Ar-H) 3.26, 3.36 (each 3H, s, OCH3 2), 4.10 (1H, br s, H-17), 4.2 (1H, d, J ¼ 5 Hz, H-14b), 7.5 (1H, br s, H–19) 3.30 (1H, m, H-1b), 1.45 (1H, m, H-2a), 2.09 (1H, m, H-2b), 1.36 (1H, m, H-3a), 2.21 (1H, m, H-3b), 2.52 (1H, br s, H-4), 1.83 (1H, m, H-5), 1.82 (1H, m, H-6a), 2.20 (1H, m, H-6b), 2.08 (1H, m, H-7), 2.36 (1H, m, H-9), 2.11 (1H, m, H-10), 1.67 (1H, m, H-12b), 1.88 (1H, m, H-12a), 2.45 (1H, m, H-13), 4.17 (1H, dd, J ¼ 4.8 and 4.8 Hz, H-14b), 2.36 (1H, m, H-15a), 2.12 (1H, m, H-15b), 3.46 (1H, m, H-16), 3.64 (1H, d, J ¼ 4.4 Hz, H-17), 3.28 (3H, s, OCH3-1), 3.36 (3H, s, OCH3-16)

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Wang et al.

Table V (Continued ) Codes (names) (references) Lappaconine subtype (AII) AII-1 (Dihydromonticamine, 15) (47) AII-2 (Lappaconidine, 16) (9) AII-3 (Lappaconine, 17) (49) AII-4 (Sinomontanine E, 18) (55) AII-5 (Akiramidine, 19) (56)

AII-6 (Akiranine, 20) (57)

AII-7 (Kiramine, 21) (60)

AII-8 (Akiramine, 22) (58)

AII-9 (Akiran, 23) (59)

AII-10 (Neofinaconitine/ delphicrispuline, 24) (63)

dH 0.92 (1H, t, J ¼ 7 Hz, NCH2CH3), 3.14, 3.28 (each 3H, s, OCH3 2), 3.75 (1H, t, J ¼ 4 Hz, H-14b) 1.07 (1H, t, J ¼ 7 Hz, NCH2CH3), 3.26, 3.36 (each 3H, s, OCH3 2) 1.07 (1H, t, J ¼ 7 Hz, NCH2CH3), 3.23, 3.28, 3.38 (each 3H, s, OCH3 3) 1.11 (1H, t, J ¼ 7 Hz, NCH2CH3), 3.31, 3.40 (each 3H, s, OCH3 2) 1.07 (1H, t, J ¼ 7 Hz, NCH2CH3), 3.23, 3.30, 3.36 (each 3H, s, OCH3 3), 3.71 (1H, t, J ¼ 2.5 Hz, H-1b), 4.06 (1H, d, J ¼ 7 Hz, H-6a), 3.56 (1H, t, J ¼ 5 Hz, H-14b) 1.00 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.16, 3.25, 3.34, 3.34 (each 3H, s, OCH3 4), 3.92 (1H, d, J ¼ 7.5 Hz, H-6a), 3.49 (1H, t, J ¼ 5 Hz, H-14b) 0.98 (3H, t, J ¼ 7 Hz, NCH2CH3), 1.93 (3H, s, OAc), 3.17, 3.25, 3.33, 3.33 (each 3H, s, OCH3 4), 3.97 (1H, d, J ¼ 7.5 Hz, H-6a), 3.47 (1H, t, J ¼ 5 Hz, H-14b) 1.06 (3H, t, J ¼ 7 Hz, NCH2CH3), 1.97 (3H, s, OAc), 3.28, 3.29, 3.35 (each 3H, s, OCH3 3), 3.55 (1H, t, J ¼ 5 Hz, H-14b), 3.66 (1H, t, J ¼ 2.5 Hz), 3.72 (1H, t, J ¼ 2.5 Hz, H-1b), 4.02 (1H, d, J ¼ 7 Hz, H-6b), 4.28 (1H, s) 0.98 (3H, t, J ¼ 7 Hz, NCH2CH3), 1.93 (3H, s, OAc), 3.17, 3.25, 3.33, 3.33 (each 3H, s, OCH3 4), 3.97 (1H, t, J ¼ 7.5 Hz, H-6a), 3.49 (1H, t, J ¼ 5 Hz, H-14b) 3.20 (d, J ¼ 9.0 Hz, H-1), 1.50 (m, H-2a), 1.65 (m, H-2b), 1.62 (m, H2-3), 1.75 (d, J ¼ 8.0 Hz, H-5), 1.60 (m, H-6a), 1.90 (m, H-6b), 2.45 (d, J ¼ 7.5 Hz, H-7), 2.0 (dd, J ¼ 10 and 4 Hz, H-9), 1.85 (m, H-10), 1.60 (m, H-12a), 2.05 (dd, J ¼ 8 and 4 Hz, H-12b), 2.30 (m, H-13), 3.45 (t, J ¼ 4.5 Hz, H-14b), 2.05 (m, H2-15), 3.17 (d, J ¼ 8 Hz, H-16), 3.02 (s, H-17), 2.65, 3.60 (ABq, J ¼ 11 Hz, H2-19), 2.45, 2.75 (each m, H2-21), 1.7 (t, J ¼ 7 Hz, NCH2CH3), 3.32 (s, OCH3-1u), 3.30 (s, OCH3-14u), 3.40

The C18-Diterpenoid Alkaloids

37

Table V (Continued ) Codes (names) (references)

AII-11 (9-Deoxylappaconitine, 25) (61)

AII-12 (4Anthranoyllappaconidine, 26) (53)

AII-13 (Demethyllappaconitine, 27) (65)a

AII-14 (Sinomontanine B, 28) (66)a

AII-15 (Septefine, 29) (64)

AII-16 (NDeacetyllappaconitine/ puberanidine, 30) (73)

dH (s, OCH3-16u), 6.67 (d, J ¼ 8 Hz, H-3v), 7.28 (dd, J ¼ 8and 1.5 Hz, H-4v), 6.63 (dd, J ¼ 8 and 1.5 Hz, H-5v), 7.80 (dd, J ¼ 8 and 1.5 Hz, H-6v), 5.72 (br s, NH) 1.08 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 2.18 (3H, s, OAc), 3.24, 3.28, 3.36 (each 3H, s, OCH3 3), 3.68 (1H, t, J ¼ 4.5 Hz, H-14b), 7.00, 7.42 (each 1H, t, J ¼ 8.0 Hz, Ar-H), 7.68, 7.90 (each 1H, d, J ¼ 8.0 Hz, Ar-H), 11.04 (1H, s, NHAc) 1.15 (3H, t, J ¼ 7.4 Hz, NCH2CH3), 3.31, 3.39 (each 3H, s, OCH3), 3.46 (1H, t, J ¼ 4.8 Hz, H-14b), 3.77 (1H, br s, H-1b), 5.69 (2H, br s, NH2), 6.60 (2H, t, J ¼ 8.1 Hz, Ar-H), 7.22 (1H, d, J ¼ 7.9 Hz, Ar-H), 7.73 (1H, d, J ¼ 7.9 Hz, Ar-H) 1.10 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.20 (3H, s, NHAc), 3.28 (1H, dd, J ¼ 9.0 and 3.0 Hz, H-16a), 3.34, 3.44 (each 3H, s, OCH3 2), 3.45 (1H, t, J ¼ 4.5 Hz, H-14b), 3.70 (1H, m, H-1b), 7.06 (1H, br d, J ¼ 8.0 Hz, H-6v), 7.49 (1H, dd, J ¼ 8.0 and 1.5 Hz, H-5v), 7.95 (1H, dd, J ¼ 8.0 and 1.5 Hz, H-4v), 8.67 (1H, br d, J ¼ 8.0 Hz, H-3v), 11.05 (1H, br s, NHAc) 1.14 (3H, t, J ¼ 7.1 Hz, NCH2CH3), 2.25 (2.23) (3H, s, OAc), 3.33 (3.32) (each 3H, s, OCH3 2), 7.03 (1H, t, J ¼ 7.2 Hz, H-4v), 7.69 (1H, d, J ¼ 8.3 Hz, H-6v), 7.92 (1H, d, J ¼ 7.9 Hz, H-3v), 6.41 (1H, d, J ¼ 7.2 Hz, H-5v), 11.07 (1H, s, NH) 1.02 (3H, t, J ¼ 7.5 Hz, NCH2CH3), 2.80 (3H, d, J ¼ 5 Hz, NHAc), 2.90 (1H, s, H-17), 3.45 (1H, d, J ¼ 5 Hz, H-14b), 3.19, 3.20, 3.31 (each 3H, s, OCH3 3), 3.53 (1H, d, J ¼ 11 Hz, H-19a), 6.49 (1H, d, J ¼ 7 Hz, H-3v), 7.22 (1H, t, J ¼ 7 Hz, H-4v), 6.42 (1H, t, J ¼ 7 Hz, H-5v), 7.67 (1H, d, J ¼ 7 Hz, H-6v), 7.50 (1H, q, J ¼ 5 Hz, NH) 1.12 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.00 (1H, s, H-17), 3.20 (2H, dd, J ¼ 9.6 Hz, H-1b, H-16a), 3.30, 3.32, 3.42 (each 3H, s, OCH3 3), 3.46 (1H, d, J ¼ 5 Hz, H-14b), 2.55, 3.62 (each 1H, d, J ¼ 11 Hz, H-19),

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Wang et al.

Table V (Continued ) Codes (names) (references)

AII-17 (Lappaconitine, 31) (67)

AII-18 (Sinomontanine A, 32) (66) AII-19 (Sepaconitine, 33) (86)

AII-20 (NAcetylsepaconitine, 34)(87) AII-21 (Monticamine, 35) (47)

AII-22 (Excelsine, 36) (79) AII-23 (8-Acetylexcelsine, 37) (92) AII-24 (Akiradin, 38) (90)

AII-25 (Akirine, 39) (93)

AII-26 (Kiridine, 40) (94)

dH 5.68 (2H, br s, NH2), 6.65 (1H, d, J ¼ 7 Hz, Ar-H), 7.25 (1H, t, J ¼ 7 Hz, Ar-H), 6.62 (1H, t, J ¼ 7 Hz, Ar-H), 7.78 (1H, d, J ¼ 7 Hz, Ar-H) 1.11 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.18 (3H, t, NHCOCH3), 3.27 (6H, s, OCH3 2), 3.28 (3H, s, OCH3), 6.93–8.58 (4H, m, Ar-H), 10.90 (1H, br s, NH) 2.20 (3H, s, NHAc), 3.27 (6H, s, OCH3 2), 3.38 (3H, s, OCH3), 6.90–8.70 (4H, m, Ar-H), 11.0 (1H, br s, NH) 1.08 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.27, 3.28, 3.38 (each 3H, s, OCH3 3), 3.73 (1H, d, J ¼ 5 Hz, H-14b), 6.55–7.68 (4H, m, Ar-H) 1.11 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.27, 3.28, 3.38 (each 3H, s, OCH3 3), 3.73 (1H, d, J ¼ 5 Hz, H-14b), 6.55–7.68 (4H, m, Ar-H) 0.84 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 3.14, 3.26 (each 3H, s, OCH3 2), 3.54 (1H, t, J ¼ 5 Hz, H-14b), 3.92 (1H, m, H-1), 4.16 (1H, s, OH-8), 6.36 (1H, OH-1) 1.01 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.13, 3.18 (each 3H, s, OCH3 2) 1.05 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.00 (3H, s, OAc), 3.25, 3.32 (each 3H, s, OCH3 2), 3.88 (1H, br s) 1.01 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 2.00 (3H, s, OAc), 3.24, 3.33 (each 3H, s, OCH3 2), 3.54 (2H, br s), 5.07 (1H, dd, J ¼ 6.0 and 3.5 Hz, H-1b), 4.06 (1H, d, J ¼ 7.0 Hz, H-6a) 1.02 (3H, t, NCH2CH3), 3.24 (3H, s, OCH3), 3.46 (1H, s), 3.94 (1H, s), 5.03, 5.24 (each 1H, s, OCH2O) 4.00 (1H, br t, H-1), 1.34 (1H, ddd, J ¼ 14.3, 6.4, and 2.4 Hz, H-2b), 2.26 (1H, m, H-2a), 3.09 (1H, t, J ¼ 6.4 Hz, H-3), 2.34 (1H, d, J ¼ 9.0 Hz, H-5), 1.98 (1H, dd, J ¼ 14.9 and 9.0 Hz, H-6b), 2.15 (1H, m, H-6a), 1.56 (1H, d, J ¼ 6.3 Hz, H-7), 2.55–2.65 (1H, m, H-10), 1.72 (1H, dd, J ¼ 12.4 and 4.4 Hz, H-12a), 2.55–2.65 (1H, m, H-12b), 2.15 (1H, m, H-13), 4.03 (1H, br s, H-14b), 1.88 (1H, dd, J ¼ 15.1 and 7.8 Hz, H-15), 2.20 (1H, m,

The C18-Diterpenoid Alkaloids

39

Table V (Continued ) Codes (names) (references)

dH H-15), 3.47 (1H, t, J ¼ 7.8 Hz, H-16), 2.88 (1H, br s, H-17), 2.37, 2.99 (each 1H, ABq, J ¼ 10.0 Hz, H2-19), 2.55–2.65 (2H, m, H2-21), 1.11 (3H, t, J ¼ 7.3 Hz, CH3-22), 5.28 (2H, s, O CH3-16)

AII-27 (Kiritine, 41)

O), 3.32 (3H, s,

–

Note: * Here and below, solvent is not specified when using CDCl3. a Different 1H NMR.

several reviews in this field were reported (128–138). Here, the focus is placed on the biological activities of lappaconitine.

A. Analgesic Activity The crude drug ‘‘Bushi,’’ the tubers of Aconitum plants, has been used in some prescriptions of traditional Chinese and Japanese medicines for centuries as an important remedy for pain. However, the analgesic components of ‘‘Bushi’’ were not known until 1979 when the Japanese scientists Hikino et al. (139) first reported the analgesic effect of the aconitine and mesaconitine, the major constituents from Aconitum japonicum roots. On the other hand, the roots of A. sinomontanum Nakai growing in the northwestern region of China have been employed as an anodyne for a long time. Tang et al. (140) first reported that lappaconitine hydrobromide, the principal alkaloid of A. sinomontanum, and structurally different from the aconitine alkaloids, possesses strong analgesic action. Afterwards in China, the analgesic action of many diterpenoid alkaloids, including some C18-diterpenoid alkaloids, for example, lappaconitine, N-deacetyllappaconitine, ranaconitine, N-deacetylranaconitine, and N-deacetylfinaconitine, was investigated by oral and subcutaneous administration in various mice or rats anti-inflammatory models including hot plate test, acetic acid-induced writhing, formaldehyde-elicited continuous pain stimuli, and tail-flick response to light irradiation. In the mouse hot plate, formaldehyde, and acetic acidwrithing assays, lappaconitine and N-deacetyllappaconitine showed a marked analgesic action with sc median analgesic doses, with ED50 values of 3.8 and 7.1 mg/kg for the formaldehyde test, and 3.5 and 2.3 mg/kg for the acetic acid-writhing test, respectively (23). Analgesic activities of lappaconitine were greater than those of indomethacin and acetylsalicylic acid, but generally were about two to five times less when

40

Wang et al.

Table VI

1

H NMR data of ranaconitine-type alkaloids (B)

Codes (names) (references) Leaconine subtype (BI) BI-1 (Leuconine, 42) (96)

BI-2 (Leucostine, 43) (97)

BI-3 (Acosepticine, 44) (53)a

BI-4 (Umbrofine, 45) (98)a

BI-5 (6-O-Acetyllumbrofine, 46) (98)

BI-6 (6-O-Acetylacosepticine, 47) (99)

BI-7 (Acoseptirine, 48) (53) BI-8 (Exceconidine/6-Omethylumbrofine, 49) (100) BI-9 (Anthriscifolcine B, 50) (102)

dH 1.01 (3H, t, J ¼ 7.5 Hz, NCH2CH3), 3.25 (6H, s, OCH3 2), 3.32 (3H, s, OCH3), 3.65 (1H, t, J ¼ 4.5 Hz, H-14b) 3.14 (1H, dd, J ¼ 9.2 and 6.2 Hz, H-1), 2.07–2.28 (1H, m, H-2b), 2.30–2.50 (1H, m, H-1a), 1.53 (1H, m, H-3a), 1.8–2.05 (1H, m, H-3b), 2.07–2.28 (1H, m, H-4), 2.07–2.28 (1H, m, H-5), 1.8–2.05 (1H, m, H-9), 1.8–2.05 (1H, m, H-10), 2.07–2.28 (1H, m, H2-21), 2.30–2.50 (1H, m, H-13), 3.68 (1H, t, J ¼ 4.7 Hz, H-14b), 1.67 (1H, dd, J ¼ 14.3 and 5.4 Hz, H-15b), 2.55–2.90 (1H, m, H-15a), 3.20–3.48 (1H, m, H-16), 3.20–3.48 (1H, m, H-17), 2.55–2.90 (2H, m, H2-19), 2.55–2.90 (2H, m, NCH2), 1.03 (3H, t, J ¼ 7.1 Hz, NCH2CH3), 3.27 (3H, s, OCH31), 3.32 (3H, s, OCH3-14), 3.37 (3H, s, OCH3-16), 2.85 (1H, br s, OH-7), 2.85 (1H, s, OH-8) 1.04 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 3.24, 3.33, 3.40 (each 3H, s, OCH3 3), 3.70 (1H, t, J ¼ 4.5 Hz, 14b-H), 4.28 (H, s, H-6a) 1.08 (3H, t, J ¼ 7.5 Hz, NCH2CH3), 3.27, 3.37, 3.44 (each 3H, s, OCH3 3), 3.64 (1H, t, J ¼ 4.5 Hz, 14b-H), 4.24 (H, s, H-6a) 0.99 (3H, t, J ¼ 7.5 Hz, NCH2CH3), 1.99 (3H, s, OAc), 3.22, 3.30, 3.38 (each 3H, s, OCH3 3), 3.68 (1H, t, J ¼ 4.5 Hz, H-14b), 5.13 (1H, s, H-6a) 1.02 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.02 (3H, s, OAc), 3.23, 3.30, 3.38 (each 3H, s, OCH3 3), 3.69 (1H, t, J ¼ 4.5 Hz, H-14b), 5.13 (1H, s, H-6) 1.05 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.25, 3.33, 3.42 (each 3H, s, OCH3 3) 1.04 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.24, 3.32, 3.37, 3.40 (each 3H, s, OCH3 4), 3.57 (1H, t, J ¼ 4.6 Hz, H-14b), 3.84 (1H, s, H-6a) 2.36 (1H, d, J ¼ 5.6 Hz, H-4), 4.25 (1H, s, H-6), 3.66 (1H, t, J ¼ 4.48 Hz, H-14b), 1.05 (3H, t, J ¼ 7.2 Hz, H3-22), 3.27, 3.35, 3.43 (each 3H, s, OCH3 3), 5.06, 5.12 (each 1H, s, O

O)

The C18-Diterpenoid Alkaloids

Table VI (Continued ) Codes (names) (references) BI-10 (Anthriscifolcine A, 51) (102)

BI-11 (Anthriscifolcine G, 52) (103)

BI-12 (Anthriscifolcine C, 53) (102)

dH 3.01 (1H, t, J ¼ 9.6 Hz, H-1), 2.08 (2H, m, H2-2), 1.36, 1.74 (each 1H, m, H2-3), 2.31 (1H, t, J ¼ 5.6 Hz, H-4), 1.46 (1H, s, H-5), 5.21 (1H, s, H-6), 2.10 (1H, s, H-9), 3.47 (1H, s, H-10), 1.77, 2.58 (each 1H, m, H2-12), 2.11 (1H, m, H-13), 3.66 (1H, t, J ¼ 4.8 Hz, H-14b), 1.80, 2.44 (each 1H, m, H2-15), 3.20 (1H, m, H-16), 3.12 (1H, m, H-17), 2.70, 2.78 (each 1H, hidden, H3-21), 1.03 (3H, t, J ¼ 7.2 Hz, H3-22), 3.27 (3H, s, OCH3-1), 3.34 (3H, s, OCH3-14), 3.45 (3H, s, OCH3-16), 2.04 (3H, s, OAc-6), 4.91 (2H, s, O O) 1.06 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 2.07 (3H, s, OAc), 3.03 (1H, dd, J ¼ 10.4 and 7.2 Hz, H-1), 3.26, 3.51 (each 3H, s, OCH3 2), 3.70 (1H, t, J ¼ 10.0 Hz, H-16), 3.79 (1H, t, J ¼ 4.8 Hz, H-14b), 5.23 (1H, s, H-6) 3.62 (1H, t, J ¼ 8.0 Hz, H-1), 2.06, 2.12 (each 1H, m, H2-2), 1.35, 1.78 (each 1H, m, H2-3), 2.01 (1H, m, H-4), 1.83 (1H, s, H-5), 5.33 (1H, s, H-6), 3.22 (1H, s, H-9), 2.55 (1H, m, H-12), 1.72 (1H, m, H-13), 4.64 (1H, dd, J ¼ 10.0 and 4.8 Hz, H-14b), 1.83, 2.64 (each 1H, m, H2-15), 3.46 (1H, d, J ¼ 8.4 Hz, H-16), 3.30 (1H, br s, H-17), 2.80 (1H, hidden, H-19), 2.90 (2H, m, H2-21), 1.07 (3H, t, J ¼ 7.2 Hz, H3-22), 3.26 (3H, s, OCH3-1), 3.35 (3H, s, OCH3-16), 2.10 (3H, s, OAc-6), 4.99, 5.01 (each 1H, s, O

O)

BI-13 (Anthriscifolcine E, 54) (102)

4.28 (1H, s, H-6), 4.15 (1H, t, J ¼ 4.8 Hz, H-14b), 1.06 (3H, t, J ¼ 7.2 Hz, H3-22), 3.26, 3.35, 3.45 (each 3H, s, OCH3 3), 5.07, 5.13

BI-14 (Anthriscifolcine D, 55) (102)

3.50 (1H, t, J ¼ 8.8 Hz, H-1), 2.06, 2.12 (each 1H, m, H2-2), 1.37, 1.80 (each 1H, m, H2-3), 2.09 (1H, m, H-4), 1.84 (1H, s, H-5), 5.27 (1H, s, H-6), 3.29 (1H, s, H-9), 2.48 (2H, m, H2-12), 2.51 (1H, m, H-13), 4.10 (1H, t, J ¼ 4.4 Hz, H-14b), 1.85, 2.69 (each 1H, m, H2-15), 3.18 (1H, d, J ¼ 8.0 Hz, H-16), 3.05

(each 1H, s, O

O)

41

42

Wang et al.

Table VI (Continued ) Codes (names) (references)

dH (1H, br s, H-17), 2.75 (1H, m, H-19), 2.85 (2H, m, H2-21), 1.04 (3H, t, J ¼ 7.2 Hz, H3-22), 3.25 (3H, s, OCH3-1), 3.43 (3H, s, OCH3-14), 3.31 (3H, s, OCH3-16), 2.06 (3H, s, OAc-6), 4.92, 4.94 (each 1H, s, O

BI-15 (Anthriscifolcine F, 56) (103)

BI-16 (Lamarckinine, 57) (104) Ranaconitine subtype (BII) BII-1 (Sinomontanine D, 58) (55)

BII-2 (Delbine, 59) (105)

BII-3 (Linearilin, 60) (106)

O)

1.07 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 2.08 (3H, s, OAc), 3.26, 3.51 (each 3H, s, OCH3 2), 3.58 (1H, t, J ¼ 8.4 Hz, H-1), 3.66 (1H, m, H-16), 4.30 (1H, s, H-6) 3.17, 3.35, 3.38, 3.43 (each 3H, s, OCH3 4), 3.52 (1H, s, H-6a), 3.65 (1H, t, J ¼ 4.4 Hz, H-14b), 7.66 (1H, br d, J ¼ 6.5 Hz, H-19) 3.65 (1H, t, J ¼ 8.0 Hz, H-1), 1.98 (1H, m, H-2b), 2.36 (1H, m, H-2a), 4.05 (1H, t, J ¼ 3.5 Hz, H-3), 2.61 (1H, d, J ¼ 8.0 Hz, H-5), 1.68 (1H, dd, J ¼ 14.8, 7.5 Hz, H-6b), 3.15 (1H, dd, J ¼ 15.0 and 8.0 Hz, H-6a), 2.09 (1H, dd, J ¼ 12.4 and 4.4 Hz, H-10), 2.05 (1H, m, H-12b), 2.42 (1H, dd, J ¼ 10.8 and 4.4 Hz, H-12a), 2.39 (1H, m, H-13), 3.48 (1H, d, J ¼ 4.6 Hz, H-14b), 1.74 (1H, dd, J ¼ 14.0 and 8.0 Hz, H-15b), 2.99 (1H, m, H-15a), 3.28 (1H, d, J ¼ 8.0 Hz, H-16), 2.78 (1H, s, H-17), 2.97 (1H, hidden, H19b), 3.26 (1H, d, J ¼ 8.0 Hz, H-19a), 2.93, 3.03 (each 1H, m, NH2), 1.09 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.39 (3H, s, OCH3-14), 3.31 (3H, s, OCH3-16) 0.80 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.02, 3.12 (each 3H, s, OCH3), 3.50 (1H, m, J ¼ 7.2 Hz, H-1b), 4.03 (1H, t, J ¼ 4.8 Hz, H-14b), 4.38 (3H, br s, H-6a) 3.24 (1H, dd, J ¼ 10.5 and 6.8 Hz, H-1), 1.88, 2.34 (each 1H, m, H2-2), 1.62, 1.92 (each 1H, m, H2-3), 2.40 (1H, s, H-5), 3.97 (1H, s, H-6), 2.20 (1H, br t, J ¼ 6.3 Hz, H-9), 1.85 (1H, s, H-10), 1.65, 1.95 (each 1H, m, H2-12), 2.28 (1H, m, H-13), 3.56 (1H, t, J ¼ 4.8 Hz, H-14b), 2.54 (1H, s, H-15), 3.35 (1H, dd, J ¼ 10 and 4.6 Hz, H-16), 2.96 (1H, s, H-17), 2.80, 3.50 (each 1H, ABq,

The C18-Diterpenoid Alkaloids

43

Table VI (Continued ) Codes (names) (references)

BII-4 (Hispaconitine, 61) (107) (pyridine-d5)

BII-5 (Sinomontanine H, 62) (108)

BII-6 (9-Deoxy-6-methoxy-Nsuccinyl-deacetylranaconitine, 63) BII-7 (Isolappaconitine, 64) (61)

BII-8 (NDeacetylranaconitine, 65) (77) BII-9 (Ranaconitine, 66) (73)

BII-10 (Puberanine, 67) (73)

dH J ¼ 12 Hz, H2-19), 2.60, 2.88 (each 1H, m, NCH2), 1.05 (3H, t, J ¼ 4.5 Hz, NCH2CH3), 3.27 (3H, s, OCH3-1), 3.29 (3H, s, OCH3-16), 3.29 (3H, s, OCH3-14), 3.35 (3H, s, OCH3-16) 1.10 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 1.98 (3H, s, OAc), 2.04 (1H, br s, H-5), 2.47 (1H, br t, J ¼ 4.4 Hz, H-13), 2.57 (1H, dd, J ¼ 13.8 and 4.4 Hz, H-12), 2.72 (1H, br d, J ¼ 8.5 Hz, H-3), 2.96 (1H, dd, J ¼ 9.9 and 7.3 Hz, H-1), 3.20, 3.24, 3.32, 3.47 (each 3H, s, OCH3 4), 3.68 (1H, t, J ¼ 4.4 Hz, H-14b), 3.84 (1H, d, J ¼ 11.7 Hz, H-19), 4.25 (1H, br s, H-6), 4.36 (1H, br s, OH), 5.52 (1H, br s, OH) 1.10 (3H, t, J ¼ 7.0 Hz, NCH2CH3), 2.23 (3H, s, nHCOCH3), 3.27, 3.35, 3.42 (each 3H, s, OCH3 3), 3.73 (1H, t, J ¼ 4.8 Hz, H-14b), 4.56 (1H, s, H-6a), 7.06 (1H, t, J ¼ 8.0 Hz, H-5v), 7.50 (1H, t, J ¼ 8.0 Hz, H-4v), 8.05 (1H, d, J ¼ 8.0 Hz, H-3v), 8.65 (1H, t, J ¼ 8.0 Hz, H-6v) –

1.08 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.18 (3H, s, OAc), 3.20, 3.24, 3.36 (each 3H, s, OCH3 3), 3.68 (1H, t, J ¼ 4.5 Hz, H-14b), 6.69, 7.40 (each 1H, t, J ¼ 8 Hz, Ar-H), 7.84, 8.58 (each 1H, t, J ¼ 8 Hz, Ar-H), 10.94 (1H, s, NHAc) 1.08 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.22, 3.26, 3.36 (each 3H, s, OCH3 3), 5.60 (2H, br s, NH2), 6.50–7.66 (4H, m, Ar-H) 1.13 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.24 (3H, s, nHAc), 3.28, 3.33, 3.43 (each 3H, s, OCH3 3), 7.13, 7.53, 7.95, 8.68 (4H, m, Ar-H), 11.07 (1H, br s, NHAc) 1.15 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.1 (1H, m, H-1a), 3.50 (1H, d, J ¼ 5 Hz, H-14b), 3.10 (1H, m, H-16a), 2.85 (1H, s, H-17), 3.20, 3.65 (each 1H, d, J ¼ 12 Hz, H2-19), 2.25 (3H, s, NHAc), 3.28, 3.34, 3.44 (each 3H, s, OCH3 3), 7.90, 8.70 (each 1H, d, J ¼ 7 Hz,

44

Wang et al.

Table VI (Continued ) Codes (names) (references)

BII-11 (Sinomontanine F, 68) (108)

BII-12 (NDeacetylfinaconitine, 69) (77)

BII-13 (Finaconitine/10bhydroxyranaconitine, 70) (110)

BII-14 (Sinomontanine G, 71) (108)

BII-15 (Monticoline, 72) (47)

BII-16 (Tuguaconitine, 73) (115) BII-17 (14Demethyltuguaconitine, 74) (117)

BII-18 (Tiantaishansine, 75) (118)

dH Ar-H), 7.05, 7.50 (each 1H, d, J ¼ 7 Hz, Ar-H), 11.0 (1H, s, NHAc) 2.23 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.31, 3.33, 3.44 (each 3H, s, OCH3 3), 7.04 (1H, t, J ¼ 8.0 Hz, H-5v), 7.52 (1H, t, J ¼ 8.0 Hz, H-4v), 7.91 (1H, d, J ¼ 8.0 Hz, H-3v), 8.67 (1H, d, J ¼ 8.0 Hz, H-6v), 11.01 (1H, s, NH) 1.04 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.22, 3.24, 3.36 (each 3H, s, OCH3 3), 3.74 (1H, d, J ¼ 4.5 Hz, H-14b), 5.64 (2H, s, NH2), 6.50, 7.20 (each 1H, t, J ¼ 8.0 Hz, Ar-H), 6.56, 7.64 (each 1H, d, J ¼ 8 Hz, Ar-H) 1.08 (3H, t, J ¼ 7 Hz, NCH2CH3), 2.20 (3H, s, NHAc), 3.26 (3H, s, OCH3), 3.26, 3.30, 3.40 (each 3H, s, OCH3 3), 3.78 (1H, d, J ¼ 4.5 Hz, H-14b), 7.02, 7.60 (each 1H, t, Ar-H), 7.92, 8.70 (each 1H, d, Ar-H), 11.10 (1H, s, NHAc) 1.10 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.33, 3.40 (each 3H, s, OCH3 2), 3.48 (1H, d, J ¼ 4.6 Hz, H-14b), 3.72 (1H, d, J ¼ 9.4 Hz, H-3a), 3.92 (1H, br s, W1/2 ¼ 5.1 Hz, H-1b) 0.92 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.13, 3.26 (each 3H, s, OCH3 2), 3.56 (1H, t, J ¼ 5 Hz, H-14b) 1.08 (3H, t, J ¼ 7 Hz, NCH2CH3), 3.36, 3.41, 3.42 (each 3H, s, OCH3 3) 1.08 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.38, 3.42 (each 3H, s, OCH3 2), 3.87 (1H, s, H-1b), 3.94 (1H, br s, H-6a), 4.13 (1H, m, H-14b), 2.87 (1H, s, H-17), 2.55, 2.76 (each 1H, d, J ¼ 10 Hz, H2-19) 3.90 (1H, s, H-1), 1.26, 2.18 (each 1H, m, H2-2), 3.05 (1H, m, H-3), 1.56 (1H, s, H-5), 4.82 (1H, s, H-6), 3.12 (1H, m, J ¼ 5.6 Hz, H-9), 2.12 (1H, m, H-10), 1.58, 2.12 (each 1H, m, H2-12), 2.26 (1H, m, H-13), 4.03 (1H, m, J ¼ 4.4 Hz, H-14b), 1.97, 2.59 (each 1H, m, H2-15), 3.39 (1H, s, H-16), 2.91 (1H, d, J ¼ 1.5 Hz, H-17), 2.49, 3.36 (each 1H, hidden, H2-19), 2.98, 3.39 (each 1H, m, H2-21), 1.07 (3H, t, J ¼ 7.2 Hz, H3-22), 3.51 (3H, s, OCH3-1), 3.41 (3H, s, OCH3-16)

The C18-Diterpenoid Alkaloids

45

Table VI (Continued ) Codes (names) (references) BII-19 (Delboxine, 76) (105)

BII-20 (14-ODemethyldelboxine, 77) (120)

BII-21 (Hohenackeridine, 78) (119)

a

Different 1H NMR.

dH 1.03 (3H, t, J ¼ 7.2 Hz, NCH2CH3), 3.34, 3.43 (each 3H, s, OCH3 2), 3.37 (6H, s, OCH3 2), 3.86 (1H, m, J ¼ 6.8 Hz, H-1b) 3.93 (1H, br s, W1/2 ¼ 7.0 Hz, H-1), 1.26 (1H, ddd, J ¼ 14.0, 7.1, and 2.4 Hz, H-2), 2.22 (1H, ddd, J ¼ 14.0, 5.5, and 2.4 Hz, H-2), 3.11 (1H, dd, J ¼ 7.1 and 5.5 Hz, H-3), 1.42 (1H, d, J ¼ 2.9 Hz, H-5), 4.24 (1H, s, H-6), 3.32 (1H, t, J ¼ 5.9Hz, H-9), 2.10 (1H, m, H-10), 1.58 (1H, t, J ¼ 8.9 Hz, H-12), 2.09 (1H, m, H-12), 2.32 (1H, t, J ¼ 6.0 Hz, H-13), 4.05 (1H, ddd, J ¼ 4.6, 4.6, and 3.4 Hz, H-14b), 1.84 (1H, dd, J ¼ 16.2 and 6.0 Hz, H-15), 2.68 (1H, dd, J ¼ 16.2 and 8.9 Hz, H-15), 3.42 (1H, m, H-16), 2.93 (1H, d, J ¼ 2.9 Hz, H-17), 2.53, 3.46 (each 1H, ABq, J ¼ 9.6 Hz, H2-19), 3.02 (1H, dq, J ¼ 13.8 and 7.4 Hz, H-21), 3.03 (1H, ABq, J ¼ 13.8 and 7.4 Hz, H-21), 1.10 (3H, t, J ¼ 7.4 Hz, NCH2CH3), 3.41 (3H, s, OCH3-6), 3.50 (3H, s, OCH3-8), 3.42 (3H, s, OCH3-16) 4.04 (1H, br t, J ¼ 2.5 Hz, H-1b), 2.27 (1H, ddd, J ¼ 14.5, 6.0, and 3.0 Hz, H-2a), 1.26 (1H, ddd, J ¼ 14.5, 7.5, and 2.5 Hz, H-2b), 3.12 (1H, dd, J ¼ 7.0 and 5.5Hz, H-3a), 1.44 (1H, d, J ¼ 2.0 Hz, H-15), 4.41 (1H, s, H6a), 2.84 (1H, dd, J ¼ 8.0 and 1.0Hz, H-9), 2.18 (1H, ddd, J ¼ 12.0, 8.0, and 5.0 Hz, H-10), 1.90 (1H, dd, J ¼ 14.5 and 5.0 Hz, H-12a), 2.34 (1H, ddd, J ¼ 14.0, 11.5, and 8.0 Hz, H-12b), 2.44 (1H, br d, J ¼ 6.5 Hz, H-13), 2.70 (1H, dd, J ¼ 16.0 and 7.5 Hz, H-15a), 1.30 (1H, ddd, J ¼ 16.0, 6.0, and 1.5Hz, H-15b), 3.61 (1H, t, J ¼ 7.5 Hz, H-16a), 3.15 (1H, d, J ¼ 3.0 Hz, H-17), 2.55 (1H, dd, J ¼ 10.0 and 0.5 Hz, H-19a), 3.44 (1H, d, J ¼ 10.0 Hz, H-19b), 3.01, 3.04 (each 1H, dq, J ¼ 14.0 and 7.0 Hz, H2-21), 3.28, 3.32 (3H, s, OCH3 2)

Carbon

C NMR data of lappaconitine-type alkaloids (A) Aconosine subtype (AI) AI-1 (Scopaline, 1) (26)

AI-2 (Aconosine, 2) (28)

AI-3 (Delavaconine/ episcopalisinine, 3) (35,32)

72.5 28.7 29.7 33.3 41.2 23.8 45.4 74.4 46.6 41.0 47.9 28.7 44.1 75.7 42.5 82.4 64.0 54.0 48.6 12.9 – 56.3 – –

86.5 29.0 36.5 30.0 45.5 27.9 46.1 73.1 47.1 38.2 48.8 26.2 45.6 75.5 39.3 82.3 63.1 50.4 49.6 13.6 56.3 56.3 – –

84.7/86.3 28.9/26.0 29.9/29.8 36.3/36.4 45.7/44.9 26.0/29.0 49.0/45.9 73.2/73.4 44.9/49.1 39.7/42.5 48.5/48.6 35.4/35.6 77.1/77.2 79.7/79.8 42.4/39.9 86.4/84.8 63.4/63.4 50.2/50.4 49.7/49.7 13.6/13.7 56.6/56.4 57.7/57.8 – –

AI-4 (8-Deoxy-14dehydroaconosine, 4)

AI-5 (8-Acetyldolaconine, 5) (31) 85.8 29.8 37.8 36.6 44.8 29.8 45.3 86.1 42.3 38.6 48.7 27.0 41.6 75.3 28.9 82.9 62.3 50.0 49.4 13.4 56.3 56.4 169.5, 22.4 171.0, 21.3

Wang et al.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 16u 8-OAc 14-OAc

13

46

Table VII

Carbon

AI-7 (Delavaconitine C, 7) (41,32)

AI-8 (Delavaconitine D, 8) (35)

AI-9a (Piepunendine B, 9) (40)

86.1 26.3 36.8 35.3 49.5 28.3 48.7 73.9 46.3 35.5 50.3 29.1 44.7 77.6 41.4 81.9 62.9 56.0 50.3 13.1 56.0 56.5 –

81.8/96.1 29.4/26.6 29.8/30.0 36.5/36.7 45.1/45.4 26.4/29.6 45.2/46.9 74.0/74.2 46.7/45.5 46.7/45.3 48.8/48.9 28.6/28.8 45.2/36.9 76.9/77.1 41.1/41.4 86.0/82.0 62.7/62.7 50.2/50.4 49.5/49.6 13.4/13.6 55.9/56.0 56.4/56.5 –

82.8 29.1 29.1 36.2 44.8 26.1 41.6 75.4 42.0 38.8 48.7 28.6 44.8 75.4 38.0 85.9 62.3 50.2 49.4 13.1 56.4 56.4 169.7, 21.4

86.3 26.5 35.0 40.9 39.2 28.2 45.8 78.5 45.6 36.7 48.7 28.9 45.4 75.0 29.9 82.4 62.9 50.2 49.6 13.5 56.4 56.1 –

47

AI-6 (Dolaconine/ 14-acetylaconosine/ episcopalitine, 6) (14)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 16u 8-OAc

Aconosine subtype (AI)

48

Table VII (Continued ) Carbon

Aconosine subtype (AI) AI-7 (Delavaconitine C, 7) (41,32)

AI-8 (Delavaconitine D, 8) (35)

AI-9a (Piepunendine B, 9) (40)

– – – – –

166.5/166.6 130.4/130.6 129.4/129.6 128.4/128.4 132.6/132.7

166.6 130.4 129.5 128.3 132.8

– – – – –

AI-10 (Delavaconitineb/ episcopalisine, 10) (35,32)

AI-11 (Contortuminec, 11) (32)

AI-12 (Delavaconitine E, 12)

AI-13 (Liconosine A, 13) (30)

83.4/86.0 29.2/26.4 29.5/29.9 36.3/36.5 46.7/44.9 26.1/29.3 49.7/47.0 74.0/74.1

85.9 26.4 29.8 36.5 44.9 29.3 46.7 74.0

14-OBz O

C

O 1″

6″

2″

5″

3″

CO 1v 2v,6v 3v,5v 4v

4″

Carbon

1 2 3 4 5 6 7 8

Aconosine subtype (AI)

80.6 20.7 20.8 38.4 54.3 26.7 43.7 72.0

AI-14 (Piepunendine A, 14) (40) 84.4 25.8 29.9 49.2 37.2 25.9 55.8 71.6

Wang et al.

AI-6 (Dolaconine/ 14-acetylaconosine/ episcopalitine, 6) (14)

9 10 11 12 13 14 15 16 17 19 21 22 1u 16u

46.8/46.9 41.6/42.6 48.5/48.7 35.9/36.1 76.4/76.5 80.4/80.6 42.4/41.7 85.7/83.6 63.0/63.6 50.2/50.2 49.7/49.7 13.4/13.6 56.6/56.6 58.2/58.3

Carbon

Lappaconine subtype (AII)

45.3 37.3 48.8 26.9 43.2 75.0 38.7 80.9 60.8 175.0 – – 56.7 56.7

45.8 42.9 46.8 27.1 45.7 75.1 37.2 81.9 55.8 – – 56.6 56.1

AII-5 (Akiramidine, 19)

AII-2 (Lappaconidine, 16) (48)

AII-3 (Lappaconine, 17) (48)

AII-4 (Sinomontanine E, 18) (55)

72.0 29.9 33.4 70.3 48.0 24.6 45.3 75.0 45.7 37.1

72.5d/73.0e 28.9/30.7 33.5/34.6 70.7/70.0 48.2/48.3 27.4/27.9 47.0/47.6 76.3/75.5 77.6/78.2 36.3/37.1

85.2 26.6 36.3 71.1 50.8 26.9 47.8 75.7 78.8 37.4

70.1 40.5 74.9 79.6 44.1 26.3 45.0 75.7 78.1 48.8

49

AII-1 (Dihydromonticamtine, 15) (47)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10

46.9 42.5 48.7 36.1 76.5 80.3 41.7 83.5 63.0 50.2 49.6 13.5 56.5 58.2

50

Table VII (Continued ) Carbon

Carbon

AII-1 (Dihydromonticamtine, 15) (47)

AII-2 (Lappaconidine, 16) (48)

AII-3 (Lappaconine, 17) (48)

AII-4 (Sinomontanine E, 18) (55)

50.0 30.3 43.4 84.8 42.9 82.8 62.7 60.2 48.0 13.0 – 57.6 56.1

50.4/51.0 23.1/24.0 48.4/47.9 90.4/90.8 45.1/44.2 83.0/83.9 63.1/62.9 60.4/61.5 46.5/50.2 13.1/13.2 – 58.1/57.6 56.3/56.0

51.0 23.7 49.0 90.3 44.7 83.1 61.7 58.0 49.9 13.5 56.5 58.0 56.1

52.1 25.6 36.3 90.1 44.8 82.7 61.6 57.3 48.4 13.1 – 57.8 56.1

Lappaconine subtype (AII) AII-6 (Akiranine, 20)

1 2 3 4

AII-5 (Akiramidine, 19)

AII-7 (Kiramine, 21)

AII-8 (Akiramine, 22) (58) 71.9 29.5 30.4 79.9

AII-9 (Akiran, 23)

AII-10 (Neofinaconitine/ delphicrispuline, 24) (63) 82.5 24.4 34.6 83.2

Wang et al.

11 12 13 14 15 16 17 19 21 22 1u 14u 16u

Lappaconine subtype (AII)

49.0 84.5 51.4 75.0 44.1 44.0 49.6 30.5 37.6 81.8 39.9 83.0 64.0 59.1 48.0 12.7 – 57.6 55.7 56.2 169.6 – – – – – – –

49.0 26.2 47.2 75.4 43.2 38.4 47.2 24.0 48.2 83.4 42.2 83.8 62.0 56.2 50.1 11.2 56.2 – 57.3 59.0 21.9 168.0 110.7 151.7 116.3 135.6 116.8 130.6

The C18-Diterpenoid Alkaloids

5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 6u 14u 16u 4-OAc Ar-CO 1v 2v 3v 4v 5v 6v

51

Carbon

(Continued ) Lappaconine subtype (AII)

84.3 26.8 32.0 84.3 46.5 29.2 46.2 74.1 45.4 37.0 50.7 25.1 49.6 84.1 42.0 82.6 61.3 55.0 48.8 13.5 56.5 57.7

AII-14f AII-12 (4AII-13f Anthranoyllappaconitine, (Demethyllappaconitine, (Sinomontanine 27) (65) B, 28) (66) 26) (53) 72.0 29.7 30.3 81.0 48.2 27.1 46.4 75.9 77.2 43.9 50.0 23.4 36.1 90.1 44.8 82.7 63.0 57.9 48.0 12.9 –

74.5 26.2 27.0 84.6 48.6 26.8 47.6 75.8 78.7 36.5 51.1 24.2 49.0 90.2 44.8 83.0 61.5 55.4 49.8 13.6 –

57.8

57.9

71.9 29.7 30.2 82.6 48.2 27.1 47.0 76.0 77.3 43.7 50.1 23.5 36.1 90.1 45.1 82.7 63.0 57.8 48.2 13.0 – 57.9

AII-15 AII-16 (N(Septefine, Deacetyllappaconitine/ 29) (124) puberanidine, 30) (73) 83.2 26.3 32.1 84.5 48.9 26.9 47.7 75.8 78.7 36.5 51.0 24.1 49.1 90.4 44.9 83.1 61.7 55.8 48.6 14.4 56.3 58.0

Wang et al.

AII-11 (9Deoxylappaconitine, 25) (61) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 14u

52

Table VII

16u CO 1v 2v 3v 4v 5v 6v NAc NHCH3

Carbon

56.2 167.4 115.8 141.7 120.2 134.4 122.3 131.0 168.9 25.5

56.1 166.9 111.3 150.5 116.6 133.9 116.0 131.2 – –

56.2 167.1 115.5 141.7 120.2 134.5 122.7 131.0 169.0 25.5

56.3 169.2 115.7 141.2 120.2 134.6 122.5 131.3 169.3 25.6

Lappaconine subtype (AII) AII-17 (Lappaconitine, 31) (48)

82.2 24.4 30.0 83.2 52.1 26.3 44.2 76.0 78.0 49.0 51.0 23.7 36.6

AII-19 (Sepaconitine, 33) (86) 78.1 26.7 31.9 83.1 44.7 24.5 47.1 74.8 79.8 79.1 56.4 37.5 34.8

AII-20 (NAcetylsepaconitine, 34) (87) 77.8 26.5 31.6 81.7 44.3 24.5 46.9 74.6 78.9 79.6 56.4 37.5 34.7

AII-21 (Monticamine, 35) (47) 77.0 32.3 57.7 58.7 46.3 25.9 45.5 74.4 45.3 37.2 53.6 30.6 42.3

53

84.2 26.2 51.9 84.7 48.6 26.8 47.6 75.6 78.6 36.4 51.0 24.2 49.0

AII-18 (Sinomontanine A, 32) (66)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13

56.2 168.5 116.0 141.6 120.3 134.6 122.3 131.0 168.5 25.5

54

Carbon

Lappaconine subtype (AII) AII-17 (Lappaconitine, 31) (48)

14 15 16 17 19 21 22 1u 14u 16u CO 1v 2v 3v 4v 5v 6v HNAc

90.0 44.9 82.9 61.5 55.5 49.9 13.5 56.5 57.9 56.1 167.5 115.8 141.7 120.3 134.4 122.3 131.1 169.0 25.5

AII-18 (Sinomontanine A, 32) (66) 90.0 44.2 82.4 57.1 50.6 – – 55.8 57.8 56.1 167.2 115.4 141.6 120.1 134.4 122.2 – 168.5 25.6

AII-19 (Sepaconitine, 33) (86)

AII-20 (NAcetylsepaconitine, 34) (87)

AII-21 (Monticamine, 35) (47)

88.0 44.9 83.0 61.6 55.9 48.9 13.5 56.2 58.1 56.2 167.4 112.1 150.6 116.3 133.9 116.9 131.9 –

87.9 44.8 82.8 61.5 55.6 48.5 13.4 56.3 58.0 56.2 167.1 115.8 144.7 120.3 134.4 122.8 131.0 169.1 25.5

84.6 42.8 82.6 64.5 57.7 47.6 13.3 – 57.6 56.1 – – – – – –

Wang et al.

Table VII (Continued )

Carbon

Lappaconine subtype (AII) AII-22 (Excelsine, 36) (91) 77.3 32.1 57.7 58.7 47.3 24.2 44.1 75.6 77.1 47.3 53.7 27.3 36.2 89.9 44.8 82.7 64.7 53.8 47.6 13.3

AII-24 (Akiradin, 38)

AII-25 (Akirine, 39)

AII-26 (Kiridine, 40) (94) 77.3 32.3 57.5 58.5 16.1 (?) 30.7 45.1 75.1 75.2 39.7 53.0 24.8 49.8 84.4 42.5 85.0 65.2 53.4 47.7 13.2

AII-27 (Kiritine, 41)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22

AII-23 (8Acetylexcelsine, 37)

55

56 Wang et al.

Table VII (Continued ) Carbon

Lappaconine subtype (AII) AII-22 (Excelsine, 36) (91)

6u 14u 16u 1-OAc 8-OAc O a

O

* 8-OCH2CH2

b

AII-23 (8Acetylexcelsine, 37)

AII-24 (Akiradin, 38)

– 57.8 56.1 – – –

d

AII-26 (Kiridine, 40) (94) – – 56.2 – – 98.2

OH: 130.6 (1v), 129.8 (2v,6v), 115.4 (3v,5v), 154.8 (4v), 36.1 (7v), 62.8 (8v).

14-OBz: 166.7/167.1 (CO), 132.9/133.0 (C-1v), 130.1/130.0 (2v,6v) 129.7/129.8 (3v,5v), 128.4/128.5 (C-4v). 14-OAs: 166.7 (C ¼ O), 113.7 (C-1v), 122.6 (C-2v,6v), 131.7 (C-3v,5v), 163.4 (C-4v), 55.4 (OCH3-4v). CDCl3. e C5D5N. f Different 13C NMR. c

AII-25 (Akirine, 39)

AII-27 (Kiritine, 41)

Table VIII Carbon

Leuconine subtype (BI) BI-1 (Lauconine, 42) (96)

BI-2 (Leucostine, 43) (96)

BI-3 (Acosepticine, 44) (53)a

BI-4 (Umbrofine, 45) (98)a

BI-5 (6-OAcetylumbrofine, 46) (98)

84.9 26.3 30.0 35.0 56.8 222.6 85.0 39.1 40.2 46.0 44.8 29.0 35.8 84.9 23.0 83.2 62.4 51.1 49.5 14.1 56.2 57.2 56.3 – –

84.4 26.2 29.4 35.0 57.3 220.0 85.7 75.3 45.6 45.9 43.8 28.4 37.5 83.3 34.8 81.8 63.2 50.8 49.2 14.1 56.1 57.8 56.4 – –

84.7 25.6 29.3 37.2 53.0 82.4 87.8 78.7 45.3 44.1 48.5 29.3 35.4 84.1 36.6 83.5 66.1 50.3 51.6 14.5 – 57.7 56.1 – –

86.1 26.3 30.8 36.4 46.2 80.3 89.7 76.4 47.5 37.8 48.7 29.3 45.9 84.8 35.2 82.5 63.5 50.3 49.7 13.6 56.1 57.9 56.3 – –

86.0 26.1 30.7 35.9 45.8 77.4 88.5 76.1 47.1 38.0 48.2 30.1 46.3 84.2 35.1 81.9 63.6 50.1 49.9 13.9 56.2 56.2 57.8 169.5 21.6

57

CH3

C NMR data of ranaconitine-type alkaloids (B)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 14u 16u C O

13

Carbon

(Continued ) Leuconine subtype (BI) BI-6 (6-OAcetylacosepticine, 47) (99)

BI-7 (Acoseptrine, 48) (53)

BI-8 (Exceconidine/ BI-9 BI-10 BI-11 6-O-methylumbrofine, (Anthriscifolcine B, (Anthriscifolcine A, (Anthriscifolcine G, 49) (100) 50) (102) 51) (102) 52) (103)

84.2 26.0 29.1 37.5 50.6 84.5 89.2 76.9 45.6 43.4 48.6 29.1 35.2 84.3 37.9 82.3 66.3 49.7 51.2 14.2 55.9 –

84.1 25.6 29.1 37.2 48.1 77.4 87.1 76.9 54.1 80.6 54.1 36.9 35.1 82.7 39.3 81.9 66.4 50.2 51.5 14.3 55.6 –

84.9 26.0 29.4 38.3 43.4 94.4 88.6 77.7 48.7 36.8 48.7 29.0 46.2 83.9 33.4 82.8 65.1 51.2 50.2 14.1 56.0 57.7

83.0 26.4 29.2 37.9 51.0 81.5 92.9 84.5 47.6 40.2 50.2 28.3 34.7 83.3 33.5 81.9 64.4 50.8 50.8 13.5 55.7 56.1

82.4 26.4 29.2 38.4 50.2 81.1 92.0 83.5 48.0 39.7 49.9 28.3 34.2 83.3 33.9 81.7 64.4 50.5 50.3 13.8 55.8 –

83.8 25.8 28.9 33.9 49.6 80.8 93.6 81.6 38.6 47.9 49.3 27.1 40.1 83.7 37.0 72.1 64.8 50.7 50.6 13.9 55.9 –

Wang et al.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 6u

58

Table VIII

14u 16u OAc O

Carbon

57.6 56.2 172.4 O

21.6

57.6 56.0 –

58.3 56.2 –

– 57.7 –

–

–

92.9

57.9 – 170.3 21.6 93.7

Leuconine subtype (BI) BI-12 (Anthriscifolcine C, 53) (102)

77.0 26.0 28.9 34.3 45.5 82.0 92.0 82.3 50.5 83.0 55.3 34.4 37.4 81.5 38.7 81.6 63.6

BI-14 (Anthriscifolcine D, 55) (102) 77.1 26.1 28.3 33.5 44.7 81.5 91.3 81.6 50.1 83.6 55.1 34.9 36.0 81.5 39.5 81.5 63.9

BI-15 (Anthriscifolcine F, 56) (103) 77.1 259 28.6 33.7 44.5 81.4 92.8 80.4 47.9 83.1 54.6 37.8 40.1 82.4 37.8 71.7 64.7

BI-16 (Lamarckinine, 57) (104) 82.0 20.8 21.5 45.9 43.7 96.0 86.7 77.2 43.4 44.1 48.5 29.6 38.6 84.3 33.2 82.5 64.5

59

77.2 25.8 29.6 33.3 44.8 81.1 93.0 80.1 52.1 83.1 54.7 36.9 37.5 72.8 37.5 81.2 65.0

BI-13 (Anthriscifolcine E, 54) (102)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

56.2 57.7 170.4 21.6 93.5

60

Table VIII (Continued ) Carbon

Leuconine subtype (BI)

19 21 22 1u 6u 14u 16u OAc O

Carbon

50.6 50.6 13.9 55.7 – –

O

56.7 170.6 21.7 94.2

BI-14 (Anthriscifolcine D, 55) (102)

BI-15 (Anthriscifolcine F, 56) (103)

BI-16 (Lamarckinine, 57) (104)

50.5 50.7 13.4 55.6 – 57.7 56.1 – – 93.2

50.2 50.2 13.4 55.4 – 57.6 56.3 170.2 21.6 93.9

50.6 50.3 13.9 55.7 – 58.0 – 170.2 21.7 93.9

165.2 – – 56.3 59.1 57.7 56.2 – – –

BII-3 (Linearilin, 60) (106)

BII-4 (Hispaconitine, 61) (107)

BII-5 (Sinomontanine H, 62) (108)

Ranaconitine subtype (BII) BII-1 (Sinomontanine D, 58) (55)

1 2 3 4 5

BI-13 (Anthriscifolcine E, 54) (102)

70.2 40.9 74.4 79.7 44.5

BII-2 (Delbine, 59) (105) 76.1 28.4 37.0 69.3 55.9

84.7 29.9 37.7 70.8 44.1

83.2 27.2 31.6 81.4 54.9

82.8 26.9 31.4 83.4 56.2

Wang et al.

BI-12 (Anthriscifolcine C, 53) (102)

CO 1v 2v 3v 4v 5v 6v NHAc

34.5 84.4 78.2 77.6 48.8 52.6 25.5 36.7 90.1 38.5 82.2 62.5 56.6 50.2 14.1 – – 57.9 56.2 – – – – – – – – – –

89.3 88.6 77.6 44.9 42.7 49.6 29.7 37.9 75.4 33.7 82.9 62.9 58.5 50.6 13.6 – 58.5 – 56.2 170.1, 21.8 (C-1) 171.6, 21.4 (C-14) – – – – – – – –

90.6 109.7 78.7 45.2 37.9 49.4 30.7 43.6 84.7 33.7 83.0 66.3 57.5 49.6 14.2 56.5 59.3 57.9 57.5 – – – – – – – – – –

90.9 89.4 77.9 44.0 46.2 51.0 28.9 38.8 84.5 34.5 83.6 64.5 56.0 50.8 14.2 55.7 58.3 57.5 56.1 – – – – – – – – – –

61

79.8 87.6 78.4 45.3 44.2 50.5 28.8 37.2 84.1 36.6 83.3 64.9 56.2 51.0 14.5 55.9 – 57.7 56.1 – – 167.4 115.3 141.5 120.3 134.4 122.6 131.2 169.0 25.5

The C18-Diterpenoid Alkaloids

6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 6u 14u 16u OAc

(Continued )

Carbon

Ranaconitine subtype (BII)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 6u

BII-7 (Isolappaconitine, 64) (61) 83.7 26.7 31.8 84.2 51.6 33.5 76.2 86.6 45.1 35.7 50.9 25.6 47.4 84.1 37.2 82.4 62.7 55.4 49.7 14.3 56.3 –

BII-8 (NDeacetylranaconitine, 65)

BII-9 (Ranaconitine, 66) (109) 83.5 26.5 31.6 84.4 51.1 32.5 77.9 85.7 78.4 36.6 51.4 25.9 49.8 90.0 37.8 82.9 63.1 55.2 48.7 14.4 56.3 58.0

BII-10 (Paberanine, 67) (73) 81.4 26.5 37.0 84.0 51.1 32.5 78.0 85.5 78.4 36.8 51.4 26.0 49.8 90.1 38.4 82.9 63.1 55.3 48.6 14.4 56.3 58.0

Wang et al.

BII-6 (9-Deoxy-6-methoxy-Nsuccinyl-deacetylranaconitine, 63)

62

Table VIII

14u 16u CO 1v 2v 3v 4v 5v 6v NHAc

Carbon

57.8 56.3 167.4 115.8 141.7 120.7 134.4 122.3 131.0 169.1 25.6

56.3 – 169.2 115.7 141.2 120.2 134.6 122.5 131.3 169.3 25.6

Ranaconitine subtype (BII) BII-11 (Sinomontanine F, 68) (108)

BII-13 (Finaconitine, 70) (110) 77.1 26.5 31.5 84.6 44.0 32.9 76.6 84.9 79.5 78.5 57.0 37.1 34.8

BII-14 (Sinomontanine G, 71) (108) 77.7 32.7 57.9 59.3 44.5 32.6 86.3 77.3 78.3 47.2 54.1 27.5 36.4

63

82.5 24.9 30.3 80.9 45.4 32.3 83.1 77.6 78.0 48.5 51.9 26.1 37.0

BII-12 (NDeacetylfinaconitine, 69)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13

56.3 – 167.7 115.9 141.8 120.4 134.6 122.6 131.3 169.5 25.6

64

Table VIII (Continued ) Ranaconitine subtype (BII) BII-11 (Sinomontanine F, 68) (108) 14 15 16 17 19 21 22 1u 14u 16u CO 1v 2v 3v 4v 5v 6v HNAc

89.8 36.9 82.1 58.0 50.8 – – 56.1 57.9 55.9 167.3 115.3 141.6 120.2 134.5 122.3 130.9 169.0 25.5

BII-12 (NDeacetylfinaconitine, 69)

BII-13 (Finaconitine, 70) (110)

BII-14 (Sinomontanine G, 71) (108)

87.7 37.6 82.7 64.3 55.1 51.0 14.5 55.9 57.9 56.3 167.4 115.8 141.6 120.3 134.4 122.5 131.0 169.3 25.5

89.8 37.9 82.6 65.6 53.6 50.0 14.0 – 57.8 56.2 – – – – – – – – –

Wang et al.

Carbon

Carbon

BII-16 (Tuguaconitine, 73) (116)

BII-17 (14-Demethyltuguaconitine, 74) (117)

BII-18 (Tiantaishansine, 75) (118)

77.3 31.8 58.0 59.5 45.7 34.2 86.6 76.9 46.6 37.1 54.4 30.5 42.0 84.6 36.0 82.5 65.7 53.2 50.0 14.1 – – – 57.6 56.2

77.9 31.5 58.5 58.6 43.2 90.2 89.4 78.5 48.7 37.9 53.9 30.7 42.6 84.2 33.3 82.7 67.0 54.2 49.9 13.9 – 58.8 – 57.7 56.3

77.9 31.5 58.7 58.6 45.4 90.0 89.6 78.1 48.7 39.7 53.5 29.6 472.8 75.6 34.4 81.9 67.5 54.3 50.1 14.0 – 58.9 – – 56.4

77.7 31.6 57.8 58.3 52.2 79.8 91.3 84.4 43.0 43.5 54.0 29.5 40.4 74.3 26.7 82.3 67.2 54.3 49.9 13.9 51.7 – – – 56.4

65

BII-15 (Monticoline, 72) (47)

The C18-Diterpenoid Alkaloids

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 6u 8u 14u 16u

Ranaconitine subtype (BII)

66

Table VIII

(Continued )

Carbon

Ranaconitine subtype (BII)

a

Different

13

C NMR.

BII-20 (14-O-Demethyldelboxine, 77) (120)

BII-21 (Hohenackeridine, 78) (119)

77.0 31.9 52.3 58.8 39.2 90.5 92.7 82.2 50.8 36.8 54.0 30.6 43.7 83.9 29.6 83.1 67.0 54.4 49.9 14.0 – 60.3 57.5 57.9 56.2

77.6 31.8 58.1 58.8 52.4 90.0 92.7 81.3 39.7 44.0 53.6 28.8 40.1 74.6 31.1 82.1 67.5 54.3 50.0 14.1 – 60.3 51.4 – 56.4

77.3 31.6 58.4 58.4 48.7 89.8 89.0 82.6 52.8 39.6 54.4 27.8 46.8 214.2 34.9 86.6 67.7 54.2 50.3 14.1 – 58.9 – – 56.1

Wang et al.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 1u 6u 8u 14u 16u

BII-19 (Delboxine, 76) (105)

67

The C18-Diterpenoid Alkaloids

OCH3

OCH3 OCH3

OCH3 OH

OCH3

O

HIO4 N

r. t.

N OH

41%

H OR

OR′

OH

H OR 1 R′ = CH3

lappaconitine

3 R′ = H OCH3

OCH3 OCH3 1

Br2

CO

OH

OCH3

NH

+3

O

N

OH O

H OR

R

OH

HOAc r. t.

OCH3

O O

2

NHAc

NHAc

Br 4

Scheme 1

compared with morphine (141). However, in the rat tail immersion test, orally administered lappaconitine exhibited more potent analgesic action than morphine (141). Ono and Satoh (141) also reported that lappaconitine has strong analgesic activity with a wide therapeutic index [LD50/ ED50 (po) ¼ 3–5, in mice] (141). Lappaconitine was shown to be a centrally acting analgesic drug (141) without affinity for opioid receptors (140,142,143,144). Guo and Tang (24,145,146), Ono and Satoh (143,144,147–149), and Ameri et al. (150–152) have studied extensively lappaconitine’s analgesic mechanism, which could be summarized as follows: 1.

2.

The analgesic action mediated by lappaconitine could be abolished by reserpine, and be enhanced by elevation of brain 5-HT or norepinephrine, showing that modulation of analgesia induced by lappaconitine may involve the central catecholaminergic and serotonergic systems (145). The analgesic activity of lappaconitine was reduced with calcium chloride treatment and augmented by ethylene glycol tetraacetic acid (145,137).

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3.

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Lappaconitine and N-deacetyllappaconitine, as analgesics acting without the involvement of opioid receptors (24,145,146), operate at the spinal and supraspinal sites, especially the periaqueducted gray and nucleus raphe magnus (137,146,24,141), by inhibiting the spinal action of substance P and somatostation (137), decreasing cellular calcium availability (24), involving participation of central 5-HT (145), selectively inhibiting the orthodromic and antidromic population spikes (150,151), stimulating the noradrenergic descending pathway (152), or inhibiting hippocampal excitability (150). The analogs of lappaconitine, for example, ranaconitine, Ndeacetyllappaconitine, N-deacetylranaconitine, and N-deacetylfinaconitine, also possess the analgesic actions (Table IX) (140,142,136).

N-Deacetyllappaconitine with about half the acute toxicity of lappaconitine (Table IX) is a naturally occurring alkaloid and also a metabolite of lappaconitine in rats and humans. It also possesses a similar pharmacological action (analgesic, anti-inflammatory, antipyretic, and local anesthetic actions) as lappaconitine (63,142). After successfully experiencing various clinical trials, lappaconitine bromide, as a new analgesic drug, is now used clinically in China for treatment of various pains (153–163), especially the various chronic analgesias (163), and the post-operative analgesia with epidural injection (160–162). In addition, it is worth noting that the combination of lappaconitine with 3-acetylaconitine in a 7:3 proportion did not markedly reduce the analgesic action of Table IX mice

Analgesic effects and acute toxicity of some C18-diterpenoid alkaloids in

Alkaloids

ED50 (mg/kg, sc)

ED50 (mg/kg)

Hot plate

Writhing

Lappaconitine

0.16

0.16

N-Deacetyllappaconitine

7.10a

2.30

Ranaconitine N-Deacetylranaconitine N-Deacetylfinaconitine

5.60a NT NT

3.50 8.60 8.60

Morphine Aspirin Note: NT: not tested. a Formaldehyde test.

5.70 626

0.80 195

11.70 (sc) 10.50 (ip) 9.10 (ip) 6.1–11.5 (iv) 36.40 (sc) 23.50 (ip) 9.00 (sc) 27.50 (sc) W50.00 (sc)

The C18-Diterpenoid Alkaloids

69

3-acetylaconitine, and produced an increase in the LD50 value of 3-acetylaconitine by 50%. This implies that the combination obviously results in an enhancement of the therapeutic index and an increase in the margin of safety of 3-acetylacontine (164).

B. Antiarrhythmic Activity In 1977, Dzhakhangirov and Sadritdinov (165) first reported the antiarrhythmic actions of the diterpenoid alkaloids napelline and heteratisine. This report stimulated great scientific interest in the evaluation of the antiarrhythmic activities of a number of diterpenoid alkaloids (21,166,167), which resulted in the discovery of nine alkaloids, including the C18-diterpenoid alkaloids lappaconitine and N-deacetyllappaconitine, with the most potent antiarrhythmic and antifibrillation activities (21,168,169) (Table X). Table X lists, selectively, the toxicities and antiarrhythmic activities of the C18-diterpenoid alkaloids investigated (21). Chinese scientists (167) also reported that lappaconitine (0.5 mg/kg, ip) may prevent the arrhythmias induced by aconitine and BaCl2 in rats, and increased significantly the doses of ouabain that was used to induce arrhythmias in guinea pigs. However, 8,9-(methylenedioxy)lappaconitine only showed a moderate arrhythmic action (169). Finally, lappaconitine bromide (Allapinin) and Aclezine (the total alkaloids from Aconitum leueostomum), representing a new class of antiarrhythmic drugs, were introduced into clinical practice in Uzbekistan after extensive studies on the antiarrhythmic effects by Tashkent scientists (20,168). Extensive clinical trials showed that Allapinin and Aclezine possess very potential antiarrhythmic effects in the therapy of ventricular and supraventricular extrasystoles, paroxysms of atrial fibrillation, and flutter paroxysmal ventricular and supraventricular tachycardia, including Wolf–Parkinson–White (WPW) syndrome cases (20,168). As compared with the known antiarrhythmic drugs, Allapanin and Aclezine proved to be more potential for the treatment of chronic and dangerous ventricular and supraventricular tachyarrhythmic cases (20). They are also effective in the therapy of well-manifested sinus brachycardia, weak sinus node syndrome, and syndrome of broadening of QT interval cases during the reduced arterial pressure (21,168). The mechanistic studies showed that the antiarrhythmic action of Allapanin and Aclezine involved an inhibitory effect on the sodium channel in the heart tissue (152,170–172). Recently, Wright (173) deduced that lappaconitine irreversibly blocks hH1 channels by binding to the site receptor. However, Dzhakhangirov and coworker’s research (174) showed that lappaconitine blocks Ca2+ channels in Helix pomatia neurons, without an effect on Na+ channels. In addition, Dzhakhangirov et al. (21) also studied comparatively the structure–antiarrhythmic activities of the diterpenoid alkaloids,

70

Alkaloids

Antiarrhythmic action (rats, aconitine: 10–12 mg/kg) LD50 (mg/kg, iv)

Monticamine Monticoline Dihydromonticoline

Antifibrillatory activity (mice, aconitine: 200 mg/kg)

ED50 (mg/kg, iv)

LD50/ED50 (mg/kg, iv)

LD50 (mg/kg, iv)

ED50 (mg/kg, iv)

LD50/ED50 (mg/kg, iv)

495 430 220

20 22.1 Ineffective in doses of 20–40 mg/kg

24.7 19.4 –

W1000 W1000 W800

– – –

– – –

130 78

28 Ineffective in doses of 10–40 mg/kg

4.6

–

–

–

8.9

– –

– –

– –

Dihydromonticamine Excelsine 1,3-Diacetyl-4chloromonticamine 3-Benzoyl-4chloromonticamine Lappaconine Oxolappaconine

29 195 2900

22 –

–

Wang et al.

Table X Toxicities and antiarrhythmic activities of some C18-diterpenoid alkaloids on models of aconitine arrhythmia in anesthetized rats and lethal cardiac fibrillation in alert mice

Lappaconidine Dimethyllappaconine 4,8,9-Triacetyl-lappaconine Lappaconitine N-Deacetyl-lappaconitine Lappaconitine 8,9-diacetate 8,9-Dimethyl-lappaconitine Ranaconitine N-Acetylsepaconitine Sepaconitine Isolappaconitine 9-Deoxylappaconitine

195 230 145 5.9 7.3 25.9 205 6.2 15 16.5 – –

18 – 13 0.05 0.05 2 6.5 0.05 0.07 0.21 0.25 0.2

10.8 – 11.2 118 146 13 31.5 124 214.3 79 – –

– – 300 15.5 35 – – – – – –

– – – 0.48 0.8 – – – – – –

– – – 32.3 43.8 – – – – – –

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including the C18-diterpenoid alkaloids (e.g., lappaconitine and N-deacetyllappaconitine). The structure–activity relationship data acquired for the diterpenoid alkaloids established that an acetylanthranilic or anthranilic acid at C-4; methoxyl groups at C-1, C-14, and C-16; and a hydroxyl group at C-8 are necessary for the antiarrhythmic activity to be observed (21). The principal metabolites of lappaconitine were established as N-deacetyllappaconitine, N-deacetyl-14-O-demethyllappaconitine, and N-deacetyl-16-O-demethyllappaconitine in rats, and N-deacetyllappaconitine and N-deacetyl-16-O-demethyllappaconitine in humans, respectively (175,113).

C. Anti-Inflammatory Activity The rats’ formaldehyde-induced hind paw edema assay data showed that lappaconitine hydrobromide (4 mg/kg, qd 7 days) possessed a marked anti-inflammatory activity (140). In several anti-inflammatory animal models, lappaconitine (1–6 mg/kg) and N-deacetyllappaconitine (1–10 mg/kg) could serve as anti-inflammatory agents without stimulating the pituitary–adrenal axis (23).

D. Anesthetic Activity Some C18-diterpenoid alkaloids, such as lappaconitine, N-deacetyllappaconitine, ranaconitine, N-deacetylranaconitine, and N-deacetylfiaconitine, were shown to possess local anesthetic activities on the sciatic nerve block in mice (Table XI) (136,137,140). Lappaconitine hydrobromide was eight times more potent as an anesthetic than cocaine in the rabbit cornea model (140). The local anesthetic tests on the sciatic nerve block in mice and the intracutaneous wheal in the guinea pig showed it to be five times as active as cocaine (140). Table XI in mice

Anesthetic effects of some C18-diterpenoid alkaloids on sciatic nerve block

Alkaloids

IC50 (mg/kg)

Lappaconitine N-Deacetyllappaconitine Ranaconitine N-Deacetylranaconitine N-Deacetylfinacontine

0.040 0.076 0.100 0.100 0.250

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73

E. Miscellaneous In addition to the aforementioned pharmacological activities, some C18-diterpenoid alkaloids, such as lappaconitine and N-deacetyllappaconitine, also exhibited antithermic effects in normothermic and pyrexial rodents (140), and antiepileptiform activities (176,177), as well as anticonvulsive potential (178).

ACKNOWLEDGMENTS The authors wish to express their gratitude to Associate Professor Dong-Lin Chen and our graduate student Pei Tang for their help in the preparation of this manuscript.

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CHAPT ER

2 Cyclopeptide Alkaloids from Higher Plants Ademir Farias Morel*, Graciela Maldaner and Vinicius Ilha

Contents

I. II. III. IV. V. VI. VII. VIII.

Introduction Classification Occurrence in Nature New Cyclopeptide Alkaloids (2006–2008) Structure Elucidation and Stereochemistry Synthesis Biosynthesis Biological Activity A. Antimicrobial Activity B. Antiplasmodial Activity C. Sedative Activity IX. Conclusions Acknowledgments References

79 80 81 111 124 125 129 131 131 133 133 135 135 136

I. INTRODUCTION Over the past 40 years, considerable interest has been focused on the chemistry of cyclopeptide alkaloids, particularly on alkaloids containing a 14-membered ring (1–4), and many spectroscopic (4–7), conformational (1–3,8,9), configurational (1–3,10–13), and synthetic studies have been reported (2,3,14–20). Departamento de Quı´mica, NPPN, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil * Corresponding author. E-mail address: [email protected] (A. F. Morel). The Alkaloids, Volume 67 ISSN: 1099-4831, DOI 10.1016/S1099-4831(09)06702-9

r 2009 Elsevier Inc. All rights reserved

79

80

Morel et al.

In the 10 years since the cyclopeptide alkaloids were last reviewed by Itokawa et al. in Volume 49 of this series (21), many significant advances have been made, with respect to isolation, identification, biochemistry, stereochemistry, pharmacology, and synthesis of peptide alkaloids. In the review, Itokawa et al. described mainly cyclopeptide alkaloids from Zizyphus and Discaria plants, found up to 1995. In the period 1996–2008, a number of important developments have taken place in the area of this group of alkaloids, concerning the isolation and characterization of new cyclopeptide alkaloids, and some reviews have been published on the chemistry of this class of alkaloids. In 1998, Gournelis et al. (22) described mainly the mass spectral fragmentation of cyclopeptide alkaloids and related compounds. Joullie and Richard in 2004 (23) reported mainly on the bioactivity and the synthesis of cyclopeptide alkaloids. In 2006, Tan and Zhou (6) described the progress in the chemistry and biology of cyclopeptide alkaloids discovered from higher plants during 1959–2005. In the same year, El-Seedi et al. (24) mainly reviewed the cyclopeptide alkaloids found during 1995–2005. Cyclopeptide alkaloids [ ¼ peptide alkaloids (25), cyclic peptide alkaloids (2), basic peptides (25), ansapeptides (2,3), and phencyclopeptines (26)] are defined as polyamidic basic compounds found in many families of plants, and mainly in species that belong to the family Rhamnaceae (1). The core nuclei of these molecules are 13-, 14-, or 15-membered macrocycles, which, as general structural elements, bear two amino acids and a styrylamine unit. The exceptions are the acyclic peptide alkaloids lasiodine A (209) (27) and sanjoinine G2 (202) (28), isolated from plants. The generalized basic structures of these alkaloids are depicted in Figure 1. The numbering system indicated in this figure has been adopted throughout this review. The alkaloids, along with a key to their structure and physical data, are listed in Tables I–V and Tables VI–XII, in which the literature has been covered from the first reported structure determination of this class, that is, of pandamine (177) (29), to those reported till mid-2008. Table XIII records the cyclopeptide alkaloids and related derivatives (linear peptide alkaloids and neutral compounds), along with their structural elucidation methods and relative or absolute stereochemistry, as reported in the literature from 1996, when the last representative members, discarine L (176) (122) and discarine X (79) (76), were reviewed by Itokawa et al. in Volume 49 of this series.

II. CLASSIFICATION Cyclopeptide alkaloids are usually composed of a tyrosine-derived 4 (or 3)-hydroxystyrylamine moiety, a common amino acid (Phe, Leu, Ile,

Cyclopeptide Alkaloids from Higher Plants

R4 R4

O2

O

O O O HN

R2

NH

N

NH

N

R1

O O

14 13

12

O

4

6

HN HN R2

R3 O

O 8 7

X

11 10

Y NH 9

R3

4(14)-Type

R5

4(13)-Type

R4

3 5

HN

HN

R3

16

15 1

81

5(13)-Type HN R2

R4

H3CO

R4

O R1

O HN

HN O

R5

O

NH

R3

HN R2 5(14)-Type

R2

O HN R1

O

O

NH

N H

R3

Y = HC CH , CH2 (OH) CH2 , CH2 (OCH3 ) CH2 , C(O) CH2 R1 = Alkyl or Aryl R2 = terminal residue (basic or neutral) R3 = side chain of amino acids R4 = H, OH, or OCH3 R5 = intermediar amino acid

X

4(15)-Type

Figure 1 General structures of the 4(13)-, 5(13)-, 4(14)-, and 4(15)-membered cyclopeptide alkaloids sensu stricto.

Trp) as a ring-bonded amino acid residue, and a b-hydroxy amino acid unit (usually 3-hydroxyproline, 3-hydroxyleucine, or 3-phenylserine), which is connected to the styryl fragment via an ether bridge. Attached to the amino group of the latter component is a side chain, usually a peptidogenic amino acid with an N-mono-methyl or N,N-dimethylated terminus. Exceptions are the group of pandamine-type alkaloids that contain a 2-alkoxy-2-(4-hydroxyphenyl) ethylamine instead of a styrylamine moiety, and the 15-membered alkaloids that contain a 2-methoxy-5-(b-aminovinyl) phenylalanine group instead of the styrylamine group. Joullie and Richard (23) developed a system of nomenclature that classifies peptide alkaloids as (i) cyclopeptide alkaloids sensu stricto, (ii) linear peptide alkaloids, and (iii) neutral cyclopeptides. The cyclopeptide alkaloids sensu stricto were classified according to the number of amino acid constituents (outside) and the size of the macrocycle (inside) as 4(13)-, 5(13)-, 4(14)-, 5(14)-, and 4(15)-type of alkaloids (see Figure 1). Among these, the 13- and 14-membered cyclopeptide alkaloids sensu stricto, with 61 and 127 alkaloids, respectively, represent the largest subgroups, and have been the focus of research in this area. In the present work, approximately 209 peptide alkaloids from higher plants (Tables I–XII) are discussed. Among them, two are linear peptide

82 Morel et al.

Table I

Cyclopeptide alkaloids occurring in plants: 4(13)-nummularine C type

OR5 O O O N R4 N R3

NH HN O

R2

R1

Alkaloids

R1

R2

R3

R4

R5

Molecular formula, melting point, and optical rotation

References

Alkaloid 2 (1) Daechuine S6 (2)

CH2CH(CH3)2 CH2C6H5

CH(CH3)CH2CH3 CH(CH3)CH2CH3

CH3 CH3

CH3 CH3

CH3 CH3

(30) (28)

Daechuine S7 (3)

CH2CH(CH3)2

CH2CH(CH3)2

CH3

CH3

CH3

Daechuine S10 (4) CH2C6H4OH

CH(CH3)CH2CH3

CH3

CH3

CH3

Daechuine S26 (5) CH2C6H5 Lotusine F (6) CH2C6H5

CH(CH3)CH2CH3 CH(CH3)CH2CH3

CH3 H

CH3 CH3

H H

C28H48N4O4; mp 1601C C31H40N4O5; mp 1921C; [a]D 393.51 C28H42N4O5; mp 1581C; [a]D 648.31 C31H40N4O6; mp 126–1281C; [a]D 381.51 C30H38N4O5; mp 1141C C29H36N4O5; [a]D 2441 (CHCl3, c 0.5)

(28) (28) (28) (31)

Nummularine C (7) Nummularine R (8) Nummularine S (9) Nummularine S10 (10) Paliurine E (11)

CH2C6H5

CH2CH(CH3)2

CH3

CH3

CH3

CH2C8H6N

CH(CH3)CH2CH3

CH3

CH3

CH3

CH2CH(CH3)2

CH2C6H5

H

H

CH3

CH2C8H6N

CH(CH3)CH2CH3

CH3

CH3

CH3

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH3

CH2C6H5 CH2CH(CH3)2

CH3 CH3

CH3 CH3

CH3 CH3

Rugosanine B (12) CH2C8H6N Sativanine E (13) CH2C8H6N

CH(CH3)CH2CH3 CH(CH3)CH2CH3

CH3

CH3

CH3

Sativanine K (15) Sativanine N (16) Sativanine O (17) Subfraction I (18) Subfraction II (19) Tscheschamine (20) Xylopyrine A (21)

CH(CH3)CH2CH3 CH(CH3)CH2CH3 CH2C6H5 CH2C6H5 CH3 CH(CH3)CH2CH3

CH(CH3)CH2CH3 CH(CH3)CH2CH3 CH2C6H5 CH2CH2CH2 CH2CH2CH2 CH2C6H5

H H H CH3 H H

CHO H H CH3 H H

CH3 CH3 CH3 CH3 CH3 CH3

CH2CH(CH3)2

CH2C6H5

CH3

CH3

CH3

CH2C6H5

CH3

CH3

CH3

Xylopyrine B (22) CH2C6H5

C33H41N5O5; mp 126–1281C; [a]D 381.51 C31H40N4O5; [a]26 D 382.31 (CH3CN, c 0.94) C36H39N5O5; mp 216–2181C C33H41N5O5; mp 127–1281C; [a]20 D 991 (CHCl3, c 0.2) C28H42N4O5; mp 921C; [a]26 D 3271 (CH3OH, c 0.85) C27H38N4O6; mp 160–1621C C26H38N4O5 C32H34N4O5 C30H36N4O5; mp 751C C22H28N4O5; mp 721C C29H36N4O5; mp 197–1981C C31H40N4O5; [a]25 D 2701 (CHCl3, c 0.21) C34H38N4O5; [a]25 D 1901 (CHCl3, c 0.15)

(32) (33) (34) (28) (35) (36) (37) (38) (39) (40) (40) (41) (41) (42) (43) (43)

Cyclopeptide Alkaloids from Higher Plants

Sativanine G (14)

C31H40N4O5; mp 278–2801C; [a]20 D 3711 (CHCl3, c 0.2) C33H41N5O5; mp 134–1351C; [a]D 381.51 C29H36N4O5; mp 210–2111C

83

Table II

Cyclopeptide alkaloids occurring in plants: 5(13)-zizyphine A type

84

OR6 Morel et al.

O O O NH

N

HN O O

R5

R2

HN R1 R3 N R4

Alkaloids

R1

R2

R3

R4

R5

R6

Alkaloid 3 (23) Amphibine H (24)

CH2C6H5 CH3

CH(CH3)CH2CH3 CH2C6H5

CH3 CH3

CH3 CH3

CH2CH(CH3)2 CH(CH3)2

Daechuine S3 (25)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

CH3

CH(CH3)CH2CH3

Daechuine S8 (26)

CH2CH(CH3)2

CH2CH(CH3)2

CH3

CH3

CH2CH(CH3)2

Jubanine A (27)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH2C6H5

Jubanine B (28)

CH2C6H5

CH2C6H5

CH3

CH3

CH2C6H5

H C29H42N5O6 CH3 C33H43N5O6; mp 201–2051C; [a]20 D 5701 (CH3OH, c 0.12) CH3 C34H53N5O6; mp 192–1941C; [a]D 4401 CH3 C33H51N5O6; mp 185–1881C; [a]D 218.21 CH3 C40H49N5O6; [a]20 D 3261 (CH3OH, c 0.12) CH3 C32H46N4O5; mp 256–2581C; [a]26 D 53.61 (CHCl3, c 0.25)

Molecular formula, melting point, and optical rotation

References

(30) (44)

(28) (28) (45) (45)

Lotusine E (29)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

CH3

CH2C6H5

H

C36H49N5O6; [a]D 1061 (CHCl3, c 1.0) C37H51N5O6; mp 1151C; [a]20 D 4871 (CHCl3, c 0.12) C36H49N5O6; mp 235–2401C; [a]20 D 3971 (CHCl3, c 0.2) C32H41N5O6; mp 226–2311C; [a]20 D 3901 (CHCl3, c 0.2) C39H47N5O6; mp 194–1961C; [a]20 D 3431 (CH3OH, c 0.27) C31H41N5O6; mp 243–2451C

Mucronine D (30)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH2CH(CH3)2

CH3

Nummularine A (31) Nummularine B (32) Nummularine H (33) Nummularine N (34) Nummularine O (35) Nummularine P (36) Nummularine T (37) O-Desmethylmucronine D (38) Paliurine A (39)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH2CH(CH3)2

CH3

CH3

CH2C6H5

H

CH3

CH(CH3)2

CH3

CH2C6H5

CH(CH3)CH2CH3

H

CH3

CH2C6H5

CH3

H

CH2C6H5

CH3

CH3

CH(CH3)2

CH3

CH2C6H5

CH2C6H5

H

CH3

CH2C6H5

CH3

CH2CH(CH3)2

H

CH3

CH(CH3)2

CH3 C42H45N5O6; mp 159–1611C; (49) [a]20 D 2391 (CH3OH, c 0.2) CH3 C29H43N5O6; mp 143–1441C (50)

CH2C8H6N

CH2C6H5

CHO

CH3

CH(CH3)2

CH3 C33H41N5O7; mp 188–1901C

(51)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH2CH(CH3)2

H

(30)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

CH3

CH2C6H5

CH3

Paliurine B (40)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

H

CH3

CH2C6H5

CH3

Paliurine C (41)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH(CH3)CH2CH3

CH3

Paliurine D (42)

CH2C6H5

CH(CH3)CH2CH3

H

CH3

CH(CH3)CH2CH3

CH3

Paliurine F (43)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

CH3

CH(CH3)CH2CH3

CH3

Paliurine G (44)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

CH(CH3)2

CH3

C36H49N5O6; [a]20 D 1911 (CHCl3, c 0.3) C37H51N5O6; [a]26 D 3451 (CH3OH, c 1.0) C36H49N5O6; mp 111–1121C; [a]26 D 391.31 (CH3OH, c 0.76) C37H51N5O6; [a]26 D 3111 (CH3CN, c 1.0) C36H49N5O6; [a]26 D 1641 (CH3CN, c 1.0) C34H53N5O6; [a]26 D 3231 (CH3CN, c 1.0) C36H49N5O6; [a]30 D 3351 (CH3OH, c 0.33)

(31) (46) (32) (32) (47) (48)

(35)

(35) (35)

Cyclopeptide Alkaloids from Higher Plants

(35)

(35)

85

(52)

86

Table II (Continued ) R1

R2

R3

R4

R5

R6

Paliurine H (45)

CH2CH(CH3)2

CH(CH3)CH2CH3

H

CH3

CH(CH3)CH2CH3

Paliurine I (46)

CH(CH3)CH2CH3

CH2C6H5

H

CH3

CH2CH(CH3)2

Rugosanine A (47) Sativanine C (48) Sativanine F (49) Sativanine H (50) Sativanine M (51)

CH3 CH3 CH(CH3)2 CH3 CH3

CH2CH(CH3)2 CH(CH3)CH2CH3 CH2C6H5 CH2CH(CH3)2 CH(CH3)CH2CH3

CHO H H CH3 CH3

CH3 CH3 H CH3 CHO

CH(CH3)2 CH(CH3)2 CH(CH3)2 CH(CH3)2 CH(CH3)2

Zizyphine A (52)

CH(CH3)CH2CH3

CH2CH2CH2

CH3

CH3

CH(CH3)CH2CH3

Zizyphine B (53)

CH2C6H5

CH2CH2CH2

H

CH3

CH(CH3)CH2CH3

Zizyphine C (54)

CH2C6H5

CH2CH2CH2

CH3

CH3

CH(CH3)CH2CH3

Zizyphine F (55)

CH(CH3)CH2CH3

CH2CH2CH2

CH3

CH3

CH(CH3)CH2CH3

Zizyphine I (56) Zizyphine K (57) Zizyphine N (58)

CH(CH3)CH2CH3 CH(CH3)CH2CH3 CH(CH3)CH2CH3

CH2CH2CH2 CH2CH2CH2 CH2CH2CH2

CH3 CH3 CH3

CH3 CH3 CH3

CH2C6H5 CH(CH3)2 CH2CH(CH3)2

Zizyphine O (59)

CH(CH3)CH2CH3

CH2CH2CH2

CH3

H

CH2CH(CH3)2

Zizyphine P (60)

CH(CH3)CH2CH3

CH2CH2CH2

CH3

CH3

CH2CH(CH3)2

Zizyphine Q (61)

CH(CH3)CH2CH3

CH2CH2CH2

CH3

CH3

CH(CH3)2

CH3 C33H51N5O6; [a]30 D 4121 (CH3OH, c 0.24) CH3 C36H49N5O6; [a]30 D 374.31 (CH3OH, c 1.07) CH3 C30H43N5O7; mp 237–2401C CH3 C29H43N5O6; mp 113–1141C CH3 C34H43N5O7; mp 139–1411C CH3 C29H43N5O6; mp 191–1921C CH3 C30H43N5O6; mp 210–2121C; [a]20 D 2151 CH3 C33H49N5O6; mp 124–1261C; [a]20 D 4111 (CHCl3, c 0.086) CH3 C32H47N5O6; [a]24 D 4571 (CHCl3, c 1.0) CH3 C36H47N5O6; [a]20 D 3311 (CHCl3, c 0.1), [a]20 D 3431 (CH3OH, c 0.1) H C32H47N5O6; mp 2351C; [a]20 D 2771 (CH3OH, c 0.15) CH3 C36H47N5O6; mp 1351C H C31H45N5O6; mp 2301C CH3 C33H49N5O6; mp 117–1191C; [a]30 D 326.61 (CHCl3, c 0.18) CH3 C32H47N5O6; mp 106–1081C; [a]31 D 380.21 (CHCl3, c 0.15) H C32H47N5O6; mp 127–1291C; [a]31 D 385.41 (CHCl3, c 0.15) CH3 C32H47N5O6; mp 140–1421C; [a]29 D 3451 (CHCl3, c 0.16)

Molecular formula, melting point, and optical rotation

References

(52) (52) (53) (54) (55) (56) (57) (58) (59) (59)

(60) (61) (62) (63) (63) (63) (63)

Morel et al.

Alkaloids

Table III

Cyclopeptide alkaloids occurring in plants: 4(14)-frangulanine type

O O HN

O

NH

HN O

R2

R1 N CH3 R3

R2

R3

Molecular formula, melting point, and optical rotation

References

Adouetine X (62)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

(64)

Adouetine Yu (63)

CH2C6H5

CH(CH3)CH2CH3

CH3

Alkaloid 4 (64) Americine (65)

CH(CH3)CH2CH3 CH(CH3)2

CH2C6H5 CH2C8H6N

CH3 H

Amphibine A (66)

CH2C8H6N

CH(CH3)CH2CH3

CH3

AM-1 (67)

CH2C6H5

CH(CH3)CH2CH3

CH3

Anorldianine (68) Ceanothine A (69)

CH2CH(CH3)2 CH2C6H5

Proline CH2CH(CH3)2

CH3 CH3

C28H44N4O4; mp 279–280.51C; [a]25 D 3701 (CHCl3, c 0.205) C31H42N4O4; mp 289–290.51C; [a]20 D 3051 (CHCl3) C31H42N4O4 C31H39N5O4; mp 135.5–137.01C; [a]20 D 1981 (CH3OH, c 0.51) C33H43N5O4; mp 237–2391C; [a]20 D 3101 (CH3OH, c 0.021) C31H42N4O4; mp 2921C; [a]20 D 3601 (CHCl3, c 0.14) C28H48N4O4; mp 1601C C30H40N4O4; mp 256.0–259.51C; [a]D 2561 (CHCl3, c 0.5)

(65) (54) (66) (67)

(68) (69) (70)

87

R1

Cyclopeptide Alkaloids from Higher Plants

Alkaloids

88

Table III (Continued ) R1

R2

R3

Molecular formula, melting point, and optical rotation

References

Ceanothine B (70)

CH2CH2CH2

CH2C6H5

H

(70)

Ceanothine C (71)

CH2CH2CH2

CH2CH(CH3)2

H

Chamaedrine (72)

CH2C6H5

CH2C8H6N

CH3

Daechuine S5 (73) Discarine A (74)

CH(CH3)2 CH2C8H6N

CH2CH(CH3)2 CH(CH3)CH2CH3

CH3 CH3

Discarine B (75)

CH(CH3)CH2CH3

CH2C8H6N

CH3

Discarine E (76)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

Discarine F (77)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

Discarine I (78)

CH(CH3)CH2CH3

CH2C8H6N

H

Discarine X (79)

CH2C8H6N

CH2CH(CH3)2

CH3

Franganine (80)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

Frangufoline (81)

CH2C6H5

CH2CH(CH3)2

CH3

Frangulanine (82)

CH(CH3)CH2CH3

CH2CH(CH3)2

CH3

C29H36N4O4; mp 238.5–240.51C; [a]25 D 2931 (CHCl3, c 0.68) C26H38N4O4; mp 223.0–229.01C; [a]25 D 3681 (CHCl3, c 1.01) C36H41N5O4; mp 222–2231C; [a]25 D 122.51 (CHCl3, c 0.12) mp 233–2351C; [a]D 421.3 C33H43N5O4; mp 229–2311C; [a]D 2821 (CHCl3, c 0.05) C33H40N5O4; mp 235–2361C; [a]D 1721 (CHCl3, c 0.1) C28H44N4O4; mp 270–2731C; [a]25 D 2361 (AcOH, c 0.5) C37H42N4O4; mp 264.01C; [a]20 D 1911 (CHCl3) C32H41N5O4; mp 1401C; [a]25 D 1491 (CH3OH, c 0.1) C33H43N5O4; mp 295–2981C; [a]25 D 1841 (CH3OH, c 0.5) C28H44N4O4; mp 2481C; [a]22 D 3021 (CHCl3, c 0.1) C31H42N4O4; mp 2441C; [a]22 D 2991 (CHCl3, c 0.1) C28H44N4O4; mp 276–2791C; [a]D 2931 (CHCl3)

(70) (71) (28) (72) (72) (73) (74) (75) (76) (77) (77) (70)

Morel et al.

Alkaloids

CH2C8H6N

H

Hovenine A (84) Melofoline (85)

CH(CH3)CH2CH3 CH2CH3

H CH3

Melonovine A (86)

CH2CH(CH3)2 CH(OH)CH (CH3)2 CH(CH3)2

CH2CH(CH3)2

CH3

Melonovine B (87) Myrianthine C (88)

CH(CH3)2 CH2CH(CH3)2

CH(OH)C6H5 CH(CH3)2

CH3 CH3

N-Desmethylmyrianthine B (89) N-Desmethylmyrianthine C (90) N-Methyl-americine (91) Nummularine K (92)

CH2C6H5

CH2CH(CH3)2

H

CH2CH(CH3)2

CH(CH3)2

H

CH(CH3)2

CH2C8H6N

CH3

CH2C8H6N

CH2CH(CH3)2

CH3

Pubescine A (93)

CH(CH3)2

CH2CH(CH3)2

CH3

Sanjoinine B (94) Sanjoinine F (95)

CH2C6H5 CH2C6H5

H CH3

Scutianine B (96)

CH2C6H5

CH2CH(CH3)2 CH(OH)CH (CH3)2 CH2C6H5

Scutianine C (97)

CH(CH3)CH2CH3

CH2C6H5

CH3

Scutianine D (98)

CH2C6H5

CH(OH)C6H5

CH3

Scutianine E (99)

CH2C6H5

CH(OH)C6H5

CH3

CH3

C32H41N5O4; mp 135.5–137.51C and 142–1821C C27H42N4O4; mp 2151C C26H40N4O5; mp 305–3071C; [a]20 D 2521 (CHCl3) C27H42N4O4; mp 2951C; [a]D 2851 (CHCl3) C30H40N4O5; mp 200–2061C C27H42N4O4; mp 2941C; [a]20 D 2281 (CHCl3, c 1.0) C30H40N4O4; mp 2291C

(66)

C26H40N4O4; [a]20 D 1031 (CHCl3, c 1.0) C32H41N5O4; mp 2331C

(82)

C33H43N5O4; mp 235–2391C; [a]20 D 451 (CH3OH, c 0.04) C27H42N4O4; mp 247–2501C; [a]20 D 2301 (CH3OH, c 0.076) C30H40N4O4; mp 212–2141C C31H42N4O5; mp 228–2291C; [a]26 D 2151 (CHCl3, c 0.28) C34H40N4O4; mp 248–2501C; [a]20 D 2961 (CHCl3, c 0.1) C34H40N4O5; mp 202–2041C; [a]D 1881 (CHCl3, c 0.15) C31H42N4O4; mp 255–2561C; [a]D 2101 (CHCl3, c 0.5) C34H40N4O5; mp 1211C; [a]20 D 22.21 (CHCl3, c 0.1)

(78) (79) (80) (80) (81) (70)

(83) (47) (84) (28) (28) (85) (86) (87) (86)

89

CH2CH(CH3)2

Cyclopeptide Alkaloids from Higher Plants

Homoamerecine (83)

90 Morel et al.

Table III (Continued ) Alkaloids

R1

R2

R3

Molecular formula, melting point, and optical rotation

References

Scutianine G (100)

CH2C6H5

CH(OH)C6H5

CH3

(88)

Scutianine H (101)

CH(CH3)CH2CH3

CH(OH)C6H5

CH3

Scutianine J (102) Scutianine K (103)

CH(OH)C6H5 CH2C6H5

CH(OH)C6H5 CH(OH)C6H5

CH3 CH3

Texensine (104)

CH2CH(CH3)2

CH2C8H6N

CH3

Waltherine A (105)

CH2CH(CH3)2

CH2C6H5

CH3

Waltherine B (106)

CH2C8H6N

CH(CH3)CH2CH3

CH3

Waltherine C (107)

CH2C8H6N

CH3

CH3

C34H40N4O5; mp 1621C; [a]20 D 1121 (CH3OH, c 0.02) C31H42N4O5; mp 242–2431C; [a]20 D 2231 (CHCl3, c 0.1) C34H40N4O5 C34H40N4O5; mp 215–2171C; [a]25 D 20.91 (CHCl3, c 0.1) C33H43N5O4; mp 249–2521C; [a]25 D 1441 (CHCl3, c 0.50), C31H42N4O4; mp 234–2351C; [a]20 D 229.81 (CH3OH, c 0.24) C33H43N5O4; mp 242–2431C; [a]20 D 201.81 (CH3OH, c 0.21) C30H37N5O4; amorphous; [a]D 1821 (CHCl3, c 0.20)

(87) (89) (90) (91) (92) (92) (13)

Table IV

Cyclopeptide alkaloids occurring in plants: 5(14)-scutianine A type

O O

O

NH

HN

HN

R2

O O

CH3 R1

N R3

Alkaloids

R1

R2

R3

Molecular formula, melting point, and optical rotation

References

Lasiodine B (108) Scutianine A (109) Scutianine F (110)

CH2C6H5 CH2C6H5 CH2C6H5

CH2CH(CH3)2 CH2C6H5 CH2C6H5

H CH3 H

C35H47N5O5; mp 2211C; [a]20 D 3011 (CHCl3:CH3OH (1:1), c 1.0) C39H47N5O5; mp 196–1971C; [a]20 D 3991 (CHCl3, c 0.15) C38H45N4O5; mp 2081C; [a]20 D 1321 (CH3OH, c 0.02)

(27) (93) (94)

Cyclopeptide Alkaloids from Higher Plants

N

91

Cyclopeptide alkaloids occurring in plants: 4(14)-integerrine type

92

Table V

O O HN

HN O

R1

Morel et al.

O NH

R2

N CH3 R3

Alkaloids

R1

R2

R3

Molecular formula, melting point, and optical rotation

References

Adouetine Y (111)

CH2C6H5

CH(CH3)CH2CH3

CH3

(95)

Alkaloid 6 (112) AM-2 (113) Aralionine A (114)

CH(CH3)2 CH2C6H5 CH(CH3)CH2CH3

CH2C8H6N CH(CH3)CH2CH3 COC6H5

H H CH3

Aralionine B (115)

CH2C6H5

CH(CH3)CH2CH3

H

Aralionine C (116)

CH(CH3)CH2CH3

CH(OH)C6H5

CH3

Canthiumine (117)

CH2C6H5

Prolina

CH3

Ceanothine E (118)

CH2C6H5

CH2CH(CH3)2

CH3

Condaline A (119)

CH2C6H5

CH(CH3)CH2CH3

H

Crenatine A (120)

CH2CH(CH3)2

CH2C6H5

CH3

C34H40N4O4; mp 287–2891C; [a]D 2131 (CHCl3) C34H37N5O4 C33H38N4O4; mp 257–2581C C34H38N4O5; mp 165–1671C; [a]20 D +821 (CH3OH, c 0.2) C33H38N4O4; mp 103–1051C; [a]20 D 731 (CH3OH, c 0.1) C34H40N4O5; mp 95–971C; [a]20 D 171 (CH3OH, c 0.015) C33H36N4O4; mp 232–2331C; [a]D 2541 (CHCl3, c 1.0) C34H40N4O4; mp 238–2391C; [a]D 2851 (CHCl3) C33H38N4O4; mp 115–1161C; [a]25 D 731 (CH3OH, c 0.08) C34H40N4O4; mp 2231C; [a]20 D 292.581 (CHCl3, c 0.1)

(96) (68) (97) (96) (94) (98) (95) (99) (100)

Deoxo-aralionine A (121) Desbenzoylaralionine A (122) Discarine C (123)

CH(CH3)CH2CH3

CH2C6H5

CH3

C39H47N5O5; mp W3501C

(83)

CH(CH3)CH2CH3

H

CH3

(22)

CH2CH(CH3)2

CH2CH(CH3)2

CH3

Discarine D (124)

CH2CH(CH3)2

CH2C6H5

CH3

Hemsine D (125)

CH(CH3)2

CH(CH3)CH2CH3

H

Integerrine (126) Integerrenine (127)

CH(CH3)2 CH(CH3)CH2CH3

CH2C8H6N CH2CH(CH3)2

CH3 CH3

Integerressine (128)

CH(CH3)2

CH2C6H5

CH3

Myrianthine A (129)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

N-Desmethylintegerrenine (130) N-Desmethylintegerrine (131) Nummularine D (132)

CH(CH3)CH2CH3

CH2CH(CH3)2

H

C27H34N4O4; mp 101–1041C; [a]20 D +1001 (CH3OH, c 0.16) C31H42N4O4; mp 241–2421C; [a]20 D 2391 (CHCl3, c 0.1) C34H40N4O4; mp 2121C; [a]20 D 1481 (CHCl3, c 0.1) C29H38N4O4; [a]26 D 573.31 (CHCl3, c 0.75) C35H39N5O4; mp 2581C C31H42N4O4; mp 2781C; [a]20 D 2281 (CHCl3, c 0.2) C33H38N4O4; mp 2851C; [a]20 D 1641 (CHCl3, c 0.2) C31H42N4O4; mp 2631C; [a]20 D 2631 (CHCl3, c 1.0) C30H40N4O4; mp 2131C

CH(CH3)2

CH2C8H6N

H

C34H37N5O4; mp W3501C

(83)

CH(CH3)CH2CH3

CH2CH(CH3)2

H

(104)

Nummularine E (133)

CH(OH)CH3

CH2CH(CH3)2

CH3

Nummularine M (134) Sativanine A (135) Scutianine L (136)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

CH(CH3)CH2CH3 CH2C6H5

CH(CH3)2 CH(CH3)CH2CH3

CH3 CH3

Scutianine M (137)

CH2C6H5

CH(CH3)CH2CH3

H

C30H40N4O4; mp 265–2681C; [a]20 D 1861 (CHCl3, c 0.2) C29H38N4O5; mp 278–2791C; [a]20 D +121 (CH3OH, c 0.02) C31H42N4O4; mp 263–2651C; [a]D 46.661 (CHCl3, c 0.1) C30H40N4O4; mp 801C C34H40N4O4; mp 122–1231C; [a]25 D 721 (CHCl3, c 2.4) C33H38N4O4; mp 257–2591C;, [a]25 D +1201 (CHCl3, c 0.018)

(101) (101) (102) (103) (103)

(81) (83)

(104) (48)

Cyclopeptide Alkaloids from Higher Plants

(103)

(105) (90)

93

(106)

94 Morel et al.

Table VI

Cyclopeptide alkaloids occurring in plants: 5(14)-adouetine Z type

O O O HN

HN O O

N R1

NH

R2

CH3 N R3

Alkaloids

R1

R2

R3

Molecular formula, melting point, and optical rotation

Adouetine Z (138) Feretine (139) Hemsine C (140) Jubanine C (141) Oxyphylline A (142)

CH2C6H5 CH2C6H5 CH2C8H6N CH(CH3)CH2CH3 CH2C6H5

CH2C6H5 CH2C6H5 CH2CH(CH3)2 CH2C6H5 CH(OH)C6H5

CH3 H CH3 CH3 CH3

C42H45N5O5; C41H43N5O5; C41H48N6O5; C39H47N5O5; C42H45N5O6;

mp 140–1451C; [a]20 D 1841 (CHCl3, c 1.0) mp 1231C; [a]D 1391 (CH3OH, c 1.0) [a]26 D 1071 (CH3OH, c 1.0) mp 233–2351C mp 204–2061C

References (64) (107) (102) (108) (109)

Table VII

Cyclopeptide alkaloids occurring in plants: 4(14)-amphibine F type

R5 O O N

HN O

R1

O

NH

R2

N R4 R3

R1

R2

R3

R4

R5

Molecular formula, melting point, and optical rotation

References

Amphibine F (143)

CH(CH3)CH2CH3

CH2C6H5

H

CH3

H

(44)

Amphibine G (144)

CH2C8H6N

CH2CH(CH3)2

CH3

CH3

H

Hemsine A (145)

CH2C8H6N

CH(CH3)CH2CH3

CH3

CH3

H

Lotusine A (146)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

H

Lotusine D (147)

CH2C6H5

CH(CH3)CH2CH3

CH3

H

H

Lotusine G (148)

CH(CH3)2

CH(CH3)CH2CH3

H

H

H

C29H36N4O4; [a]20 D 1711 (CHCl3, c 0.26) C32H39N5O4; mp 182–1851C; [a]20 D 2181 (CHCl3, c 0.24) C32H40N4O4; [a]26 D 64.51 (CH3OH, c 2.0) C30H38N4O4; [a]D 2151 (CHCl3, c 1.0) C29H36N4O4; [a]D 1871 (CHCl3, c 0.5) C24H34N4O4; [a]D 142.71 (CHCl3, c 0.5)

(44) (102) (110) (110)

Cyclopeptide Alkaloids from Higher Plants

Alkaloids

(111)

95

96 Morel et al.

Table VII (Continued ) Alkaloids

R1

R2

R3

R4

R5

Molecular formula, melting point, and optical rotation

References

Mauritine C (149)

CH(CH3)2

CH2C6H5

H

CH3

H

(112)

Mucronine J (150)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

CH3

H

Nummularine F (151) Ramosine A (152)

H

CH(CH3)CH2CH3

CH3

CH3

H

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

CH3

H

Ramosine C (153)

CH2C6H5

CH(CH3)CH2CH3

CH3

CH3

OH

Spinanine A (154)

CH2CH(CH3)2

Prolina

H

H

H

Zizyphine G (155)

CH2CH(CH3)2

Prolina

H

H

H

C28H34N4O4; [a]20 D 2241 (CH3OH, c 0.11) C27H40N4O4; [a]21 D 2361 (CHCl3, c 1.0) C23H32N4O4; mp 1201C; [a]20 D 2041 (CH3OH, c 0.2) C27H40N4O4; mp 55–561C; [a]26 D 1251 (CH3OH, c 0.76) C30H38N4O5; [a]26 D 391 (CH3OH, c 1.0) C24H32N4O4; mp 175–1761C; [a]D 1211 (CH3OH, c 0.1) C24H32N4O4; mp 1301C; [a]20 D 1851 (CH3OH, c 0.19)

(113) (104) (102) (102) (114) (60)

Table VIII

Cyclopeptide alkaloids occurring in plants: 5(14)-amphibine B type

O O N

HN O

R4

O

NH

R2

N R5 O R1 R3

R1

R2

R3

R4

R5

R6

Amphibine B (156) Amphibine C (157) Amphibine D (158) Amphibine E (159) Hemsine B (160) Hysodricanine A (161)

CH2C6H5

CH2C6H5

CH3

CH(CH3)CH2CH3

H

CH2CH(CH3)2

CH2C6H5

CH3

CH(CH3)CH2CH3

H

CH2C6H5

CH(CH3)CH2CH3

CH3

CH(CH3)CH2CH3

H

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

CH2C8H6N

H

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

CH2C6H5

H

CH(CH3)CH2CH3

Prolina

CH3

CH2C6H5

H

CH3 C32H46N4O5; [a]20 D 1811 (CH3OH, c 0.08) CH3 C36H49N5O5; [a]20 D 2241 (CH3OH, c 0.075) CH3 C36H49N5O5; [a]D 2031 (CH3OH) CH3 C38H50N6O5; [a]26 D 1751 (CH3OH, c 0.14) CH3 C36H49N5O5; [a]26 D 1241 (CH3OH, c 1.0) CH3 C35H45N5O5; mp 93–961C; [a]20 D 2151 (CHCl3, c 0.05)

Molecular formula, melting References point, and optical rotation (115) (115) (115) (115) (102) (94)

97

Alkaloids

Cyclopeptide Alkaloids from Higher Plants

R6 N

98

Table VIII (Continued ) R1

R2

R3

R4

R5

R6

Lotusine B (162) Lotusine C (163) Mauritine A (164)

CH2CH(CH3)2

CH(CH3)CH2CH3

CH3

CH2C6H5

H

CH(CH3)2

CH(CH3)CH2CH3

H

CH2C6H5

CH3

CH3

CH2C6H5

CH3

CH(CH3)2

H

B

CH(CH3)CH2CH3

CH2C6H5

CH3

CH(CH3)2

H

D

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

CH2CH(CH3)2

H

E

CH3

CH2C6H5

CH3

CH(CH3)2

H

F

CH3

CH2C6H5

H

CH(CH3)2

H

Mauritine H (169)

CH3

CH2C6H5

CH3

CH2CH(CH3)2

H

Mauritine J (170) Mauritine K (171) Ramosine B (172)

CH2CH(CH3)2

CH(CH3)CH2CH3

H

CH2C8H6N

H

CH(CH3)CH2CH3

CH(CH3)CH2CH3

H

CH(CH3)CH2CH3

H

CH(CH3)CH2CH3

CH(CH3)CH2CH3

H

CH2C6H5

H

CH3 C36H49N5O5; [a]D 1791 (CHCl3, c 0.32) CH3 C35H47N5O5; [a]D 1681 (CHCl3, c 0.5) CH3 C32H41N5O5; mp 1041C; [a]20 D 3151 (CH3OH, c 0.33) CH3 C35H47N5O5; [a]20 D 1511 (CH3OH, c 0.44) CH3 C33H51N5O5; [a]20 D 2591 (CH3OH, c 0.16) CH3 C32H41N5O6; [a]20 D 2431 (CH3OH, c 0.11) CH3 C31H39N5O5; mp 222–2251C; [a]20 D 2851 (CH3OH, c 0.15) CH3 C33H43N5O5; mp 212–2151C; [a]20 D 1691 (CH3OH, c 0.013) CH3 C27H40N4O4; [a]21 D 2361 (CHCl3, c 1.0) H C31H47N5O5; mp 218–2201C CH3 C35H47N5O5; [a]26 D 181.51 (CH3CN, c 2.0)

Mauritine (165) Mauritine (166) Mauritine (167) Mauritine (168)

Molecular formula, melting References point, and optical rotation (31) (31) (116)

(116) (112) (112) (112)

(94)

(117) (118) (102)

Morel et al.

Alkaloids

Table IX

Cyclopeptide alkaloids occurring in plants: 4(14)-pandamine type

OR4 O O

O NH

HN

HN O

R1

R2

N CH3 R3

R1

R2

R3

R4

Molecular formula, melting point, and optical rotation

References

Discarine G (173)

CH2C6H5

CH(CH3)CH2CH3

CH3

H

(119)

Discarine H (174)

CH2CH(CH3)2

CH2CH(CH3)2

CH3

H

Discarine K (175)

CH(CH3)CH2CH3

CH2C8H6N

CH3

H

Discarine L (176)

CH(CH3)CH2CH3

CH2CH(CH3)2

CH3

H

Pandamine (177)

CH(CH3)CH2CH3

CH2C6H5

CH3

H

Pandaminine (178)

CH(CH3)2

CH2C6H5

CH3

H

Sanjoinine D (179)

CH2C6H5

CH2CH(CH3)2

CH3

CH3

Sanjoinine G1 (180)

CH2C6H5

CH2CH(CH3)2

CH3

H

C31H44N4O5; mp 2571C; [a]20 D 3661 (CH3OH, c 1.0) C28H46N4O5; mp 2321C; [a]20 D 2661 (CH3OH) C33H45N5O5; mp 2371C; [a]20 D 621 (CH3OH) C28H46N4O5; [a]D 301 (CH3OH, c 0.5) C33H4N4O5; mp 2561C; [a]D 1031 (CHCl3, c 0.5) C30H42N4O5; mp 2721C; [a]D 1171 (CHCl3, c 0.5) C32H46N4O5; mp 256–2581C; [a]26 D 53.61 (CHCl3, c 0.25) C31H44N4O5; mp 236–2381C; [a]20 D 68.61 (CHCl3, c 0.175)

(120) (121) (122) (123) (29)

Cyclopeptide Alkaloids from Higher Plants

Alkaloids

(28)

99

(28)

100 Morel et al.

Table X

Cyclopeptide alkaloids occurring in plants: 4(15)-mucronine A type

H3 CO R3 N R4

R5

O O O

HN R1

NH R2

N H

Alkaloids

R1

R2

R3

R4

R5

Molecular formula, melting point, and optical rotation

References

Abyssinine A (181)

CH(CH3)CH2CH3

CH2CH(CH3)2

CH3

H

H

(124)

Abyssinine B (182)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

CH3

H

H

Abyssinine C (183)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

H

H

H

C25H38N4O4; mp 237–2391C; [a]20 D +1601 (CHCl3, c 0.22), [a]20 D 581 (CH3OH, c 0.1) C25H38N4O4; mp 229–2301C; [a]20 D +1511 (CHCl3, c 0.16) C24H36N4O4; [a]20 D +1441 (CHCl3, c 0.12), [a]20 D 151 (CH3OH, c 0.13)

(124) (124)

CH(CH3)CH2CH3

CH2C6H5

CH3

CH3

H

Mucronine B (185)

CH(CH3)CH2CH3

CH2C6H5

CH3

H

H

Mucronine C (186)

CH(CH3)CH2CH3

CH2CH(CH3)2

CH3

CH3

H

Mucronine E (187)

CH(CH3)CH2CH3

CH2CH(CH3)2

CH3

H

OCH3

Mucronine F (188)

CH(CH3)CH2CH3

CH2CH(CH3)2

H

H

OCH3

Mucronine G (189)

CH(CH3)CH2CH3

CH(CH3)CH2CH3

H

H

OCH3

Mucronine H (190)

CH(CH3)CH2CH3

CH2C6H5

H

H

H

Zizyphine D (191)

CH(CH3)CH2 CH3

C[CH3(OH)]CH2 CH3

CH3

CH3

H

Zizyphine E (192)

CH(CH3)CH2CH3

C[CH3(OH)]CH2 CH3

H

H

H

C29H38N4O4; mp 2351C; [a]20 D 28.131 (CHCl3, c 0.06) C28H36N4O4; mp 222–2241C; [a]25 D +1751 (CHCl3, c 0.2) C26H40N4O4; mp 2571C; [a]20 D 39.41 (CHCl3, c 0.09) C26H40N4O5; mp 232–2341C; [a]20 D 891 (CH3OH, c 0.084) C25H38N4O5; mp 208–2141C; [a]20 D +17.41 (CH3OH, c 0.092) C25H38N4O5; [a]20 D 501 (CH3OH, c 0.084) C27H34N4O4; [a]20 D +51 (CH3OH, c 0.1) C25H38N4O5; mp 1951C; [a]20 D +2361 (CHCl3, c 0.1), [a]20 D 1211 (CH3OH, c 0.1) C24H36N4O5; [a]20 D +1501 (CHCl3, c 0.10), [a]20 D 1111 (CH3OH, c 0.1)

(125) (125) (125) (124) (124) (124) (124) (59)

(59)

Cyclopeptide Alkaloids from Higher Plants

Mucronine A (184)

101

Table XI

Neutral cyclopeptides

102

O R3

O HN O

NH Morel et al.

HN

O R2

R1

Alkaloids

R1

R2

R3

Intermediary AA

Molecular formula, melting point, and optical rotation

References

Discarene C (193)

CH(CH3)2

CH2CH(CH3)2

C6H5

–

(126)

Discarene D (194) Discarine M (195)

CH(CH3)2

CH2C6H5

C6H5

–

CH(CH3)2

CH2CH(CH3)2

CH(CH3)2

–

Discarine N (196)

C6H5

CH(OH)C6H5

CH(CH3)2

–

Lotusanine B (197) Sanjoinenine (198) Scutianene C (199)

C6H5

CH2C6H5

CH(CH3)2

Prolina

C29H35N3O4; mp 2971C; [a]20 D 51.71 (CH3OH:CHCl3 (1:1), c 0.2) C32H33N3O4; [a]25 D 1761 (CH3OH:CHCl3 (1:1), c 0.2) C26H37N3O4; mp 295–2971C; [a]20 D 176.71 (CH3OH:CHCl3 (1:1), c 0.2) C32H33N3O5; mp 233–2351C; [a]20 D +98.11 (CH3OH:CHCl3 (1:1), c 0.092) C37H40N4O5

C6H5

CH2CH(CH3)2

CH(CH3)2

–

C6H5

CH(OH)C6H5

CH(CH3)2

–

C29H35N3O4; mp 281–2821C; [a]22 D 272.51 (pyridine, c 1.6) C32H33N3O5; mp 232–2341C; [a]D +2031 (CHCl3:CH3OH (3:2), c 0.12)

(126) (127)

(127)

(128) (28) (129)

Table XII

Miscellaneous peptide alkaloids O O

HN

O

O

O O

O

NH

N

HN O O

HN

O

HN

OCH3

O

H3C N

O

NH2

HN O

O O HN

O

NH

HN

HN O

O

O NH

O

HN O

N

O O

O

NH

HN O

HN

O

NH

Ceanothine D (203) (95) C26H38N4O4; mp 227–2291C; [a]D –3471 (CHCl3)

O

HN O

OH

HN

O

NH

HN O

OH

N O

Anordianine 27-N-oxide (204) (131) C27H41N4O5

O O

OH

NH

N

CH3 N+ OH3 C

N CH3

Sanjoinine G2 (202) (28) C30H42N4O5; mp 1821C; [a]26 D –79.21 (CHCl3, c 0.275)

O

O

H3C

Hymenocardine (200) (130) Nummularine G (201) (46) C31H40N4O4; mp C37H50N6O6; mp 2611C; [a]20 D –1241 (CHCl3 or CHCl3:CH3OH (9:1), 174–1751C; [a]20 D –1331 c 1.0) (CH3OH, c 0.02)

O

O

O

O

N CH3

CH3 N N H CH3

N

O

NH

HN O

H3 C N

CHO

N

HO O O

OH

HN HO

NH

HN O

N CH3 H 3C HN H3C

O O

CH3 Sativanine B (205) (105) C30H38N4O4

Sativanine D (206) (132) C30H43N5O6; mp 119–1211C

Vignatic acid A (207) (133) C30H39N3O7; [a]D –991 (CH3OH, c 0.11)

Vignatic acid B (208) (133) C27H41N3O7; [a]D –791 (CH3OH, c 0.16)

Lasiodine A (209) (27) C39H49N5O7; mp 1951C; [a]20 D +381 (CHCl3, c 1.0)

Table XIII

Cyclopeptide alkaloids isolated from 1996 to mid-2008 Stereochemistry

Structural and spectral data

References

4(13)-Nummularine C type

Paliurine E (11)

3(9)S, 4(8)S, 7(5)S

CD, IR, UV, pos. FAB-MS [549(M+H)+], PMR, CMR, 2D NMR (COSY 45, HMQC, HMBC, NOESY); absolute configuration (CD, NMR, total synthesis) PMR, CMR PMR, CMR Colorless amorphous solid; IR, UV, HRMS [548(M)+], CMR, amino acid analysis after hydrolysis, partial hydrolysis Colorless granules, IR, UV, HRMS [582(M)+], CMR, amino acid analysis after hydrolysis, partial hydrolysis

(35)

Amorphous powder, CD, IR, UV, pos. FABMS [662(M+H)+], PMR, CMR, 2D NMR (COSY 45, HETCOR, NOED, NOESY, HMQC, HMBC); absolute configuration (CD, NMR) Colorless amorphous solid, CD, IR, UV, pos. FAB-MS [648(M+H)+], PMR, CMR, 2D NMR (COSY 45, HETCOR, NOED, NOESY, ROESY, TOCSY, COLOC, HMQC, HMBC); absolute configuration (CD, NMR) Colorless amorphous solids, CD, IR, UV, pos. FAB-MS [662(M+H)+], PMR, CMR, 2D NMR (COSY 45); absolute configuration (CD, NMR) CD, IR, UV, pos. FAB-MS [648(M+H)+], PMR, CMR, 2D NMR (COSY 45, NOED, NOESY, HMQC, HMBC); absolute configuration (CD, NMR)

(35)

Sativanine N (16) Sativanine O (17) Xylopyrine A (21)

Xylopyrine B (22)

5(13)-Zizyphine A type

Paliurine A (39)

3(9)S, 4(8)S, 7(5)S

Paliurine B (40)

3(9)S, 4(8)S, 7(5)S

Paliurine C (41)

3(9)S, 4(8)S, 7(5)S

Paliurine D (42)

3(9)S, 4(8)S, 7(5)S

(40) (40) (43)

(43)

(35)

(35)

(35)

Morel et al.

Alkaloids

104

Type

Paliurine G (44)

3S, 4S

Paliurine H (45)

3S, 4S

Paliurine I (46)

3S, 4S

Sativanine M (51) Zizyphine N (58)

3(9)S, 4(8)S, 7(5)S

Zizyphine O (59)

3(9)S*, 4(8)S*, 7(5)S*

Zizyphine P (60)

3(9)S*, 4(8)S*, 7(5)S*

Zizyphine Q (61)

3(9)S*, 4(8)S*, 7(5)S*

CD, IR, UV, pos. FAB-MS [628(M+H)+], PMR, CMR, 2D NMR (COSY 45, NOEs, HMBC); absolute configuration (CD, NMR) Amorphous powder, CD, IR, UV, pos. FABMS [648(M+H)+], PMR, CMR, 2D NMR (COSY 45, DEPT); absolute configuration (CD, NMR) Amorphous powder, CD, IR, UV, pos. FABMS [614(M+H)+], PMR, CMR, 2D NMR (COSY 45, DEPT); absolute configuration (CD, NMR) Amorphous powder, CD, IR, UV, pos. FABMS [648(M+H)+], PMR, CMR, 2D NMR (COSY 45, DEPT); absolute configuration (CD, NMR) Colorless granules, IR, UV, HRMS [585(M)+] Colorless solid, IR, UV, EI-MS [612(M+H)+], PMR, CMR, 2D NMR (DEPT, 1H–1H COSY, HMQC, HMBC, NOESY); absolute configuration (NMR, total synthesis) Colorless solid, IR, UV, EI-MS [598(M+H)+], PMR, CMR, 2D NMR (DEPT, 1H–1H COSY, HMQC, HMBC, NOESY); relative configuration (NMR, optical rotation) Colorless solid, IR, UV, EI-MS [598(M+H)+], PMR, CMR, 2D NMR (DEPT, COSY, HMQC, HMBC, NOESY); relative configuration (NMR, optical rotation) Colorless solids, IR, UV, pos. FAB-MS [598(M+H)+], PMR, CMR, 2D NMR

(35)

(52)

(52)

(52)

(57) (63)

(63)

(63)

(63)

105

3(9)S, 4(8)S, 7(5)S

Cyclopeptide Alkaloids from Higher Plants

Paliurine F (43)

106

Table XIII (Continued ) Type

Alkaloids

Stereochemistry

Structural and spectral data

References Morel et al.

(DEPT, COSY, HMQC, HMBC, NOESY); relative configuration (NMR, optical rotation) 4(14)-Frangulanine type

Chamaedryne (72)

3S*, 4S*, 7S, 22S

Scutianine J (102)

Scutianine K (103)

3S*, 4S*, 7R, 22S, 28S

Waltherine A (105)

3S*, 4S*, 7S*

Waltherine B (106)

3S*, 4S*, 7S*,

Waltherine C (107)

3S*, 4S*, 7S, 22S

White powder, IR, EI-MS [607(M)+], PMR, CMR, 2D NMR (DEPT, COSY, HMQC, HMBC, NOESY); relative and absolute configuration (NMR, CPGC) Amorphous, IR, UV, pos. FAB-MS [601(M+H)+], PMR, 2D NMR (COSY), elemental analysis Colorless crystals, pos. FAB-MS [585(M+H)+], PMR, CMR, 2D NMR (COSY, NOESY, DEPT, HETCOR); hydrogenation, amino acid analysis after hydrolysis, relative and absolute configuration (NMR, CPGC) Colorless needles, EI-MS [534(M)+], PMR, CMR, 2D NMR (COSY, NOESY, DEPT, HMQC, HMBC), relative configuration (NMR) Colorless needles, EI-MS [573(M)+], PMR, CMR, 2D NMR (COSY, NOESY, DEPT, HMQC, HMBC), relative configuration (NMR) Amorphous powder, pos. FAB-MS [532(M+H)+], PMR, CMR, 2D NMR (1H–1H COSY, NOESY, DEPT, HMQC, HMBC); elemental analysis, relative and absolute configuration (RMN, CPGC)

(71)

(89)

(90)

(92)

(92)

(13)

4(14)-Integerrine type

3R*, 4S*, 8S, 23S

(99)

(22)

Desbenzoyl-aralionine A (122) Hemsine D (125)

3S*, 4S*

CD, IR, UV, pos. FAB-MS [507(M+H)+], PMR, CMR, 2D NMR (COSY, TOCSY, NOESY, HMQC, HMBC), relative configuration

(121)

Hemsine C (140)

3S*, 4S*

CD, IR, UV, pos. FAB-MS [705(M+H)+], PMR, CMR, 2D NMR (COSY, TOCSY, NOESY, HMQC, HMBC), relative configuration Colorless granules, IR, UV, MS [665(M+)], amino acid analysis after hydrolysis, partial hydrolysis White crystals, IR, UV, pos. FAB-EI-MS [716(M+H)+], PMR, CMR, 2D NMR (COSY, HMQC, HMBC)

(102)

CD, IR, UV, pos. FAB-MS [558(M+H)+], PMR, CMR, 2D NMR (COSY, TOCSY, HMQC, HMBC), absolute configuration (CD, NMR) IR, UV, EI-MS [442(M)+], PMR, CMR, 2D NMR (COSY, HMQC, HMBC)

(102)

Jubanine C (141)

Oxyphylline A (142)

4(14)-Amphibine F type

Needles, IR, pos. LSI-MS [555(M+H)+], PMR, CMR, 2D NMR (COSY, DEPT, NOESY, HMQC, HMBC); elemental analysis, hydrogenation, amino acid analysis after hydrolysis, relative and absolute configuration (NMR, CPGC) CD, IR, UV, MS [478(M+)], PMR

Hemsine A (145)

Lotusine G (148)

3S, 4S

(108)

(109)

Cyclopeptide Alkaloids from Higher Plants

5(14)-Scutianine A type

Condaline A (119)

(111)

107

108

Table XIII (Continued ) Type

Stereochemistry

Structural and spectral data

References

Mucronine J (150)

3S, 4S

(113)

Ramosine A (152)

3S, 4S

Ramosine C (153)

3S, 4S

Colorless amorphous powder, CD, IR, UV, pos. FAB-MS [485(M+H)+], PMR, CMR, 2D NMR (1H–1H COSY, J-modulated 13C, HMQC, HMBC, NOE), amino acid analysis after hydrolysis, absolute configuration (NOE, GC), solution conformation (NOE, MM2) Colorless amorphous solid, CD, IR, UV, pos. FAB-MS [485(M+H)+], PMR, CMR, 2D NMR (DEPT, COSY, NOESY, HMQC, HMBC), absolute configuration (CD, NMR) CD, IR, UV, pos. FAB-MS [535(M+H)+], PMR, CMR, 2D NMR (DEPT, COSY, NOE, HMBC), absolute configuration (CD, NMR)

Hemsine B (160)

3S, 4S

Lotusine B (162) Mauritine J (170)

3S, 4S

CD, IR, UV, pos. FAB-MS [632(M+H)+], PMR, CMR, 2D NMR (COSY, TOCSY, HMQC, HMBC, NOE), absolute configuration (CD, NMR) IR, UV, EI-MS [631(M)+], PMR, CMR, 2D NMR (COSY) Amorphous, IR, UV, CI-MS [657(M+H)+], PMR, CMR, 2D NMR (1H–1H COSY, 1 H–13C COSY, HMBC, NOESY), absolute configuration ([a]D)

(102))

(102)

(102)

(31) (117)

Morel et al.

5(14)-Amphibine B type

Alkaloids

Mauritine K (171)

Ramosine B (173)

3S, 4S

Colorless granules, IR, UV, HRMS [569(M)+], PMR, CMR, amino acid analysis after hydrolysis, partial hydrolysis CD, IR, UV, pos. FAB-MS [618(M+H)+], PMR, CMR, 2D NMR (DEPT, COSY), absolute configuration (CD, NMR)

(118)

(119)

3R*, 4S*, 7S

White powder, IR, pos. FAB-MS [490(M+H)+], PMR, CMR, 2D NMR (COSY, HMQC, HMBC); amino acid analysis after hydrolysis, relative and absolute configuration (NMR, CPGC)

(126)

Discarene D (194)

3S*, 4R*, 7S

(126)

Discarine M (195)

3R*, 4S*, 7S

Discarine N (196)

3R*, 4S*, 7S

Amorphous powder, IR, pos. FAB-MS [524(M+H)+], PMR, CMR, 2D NMR (COSY, DEPT, HMQC, HMBC, NOESY); amino acid analysis after hydrolysis, relative and absolute configuration (NMR, CPGC) White amorphous powder, IR, pos. FAB-MS [456(M+H)+], PMR, CMR, 2D NMR (1H–1H COSY, HMQC, HMBC, NOESY); elemental analysis, amino acid analysis after hydrolysis, relative and absolute configuration (NMR, CPGC) White powder; IR, pos. FAB-MS [540(M+H)+], PMR, CMR, 2D NMR (DEPT, HMQC, HMBC, NOESY);

Neutral cyclopeptides

Discarene C (193)

(127)

(127)

109

(122)

Discarine L (176)

Cyclopeptide Alkaloids from Higher Plants

Amorphous powder, IR, EI-MS [518(M)+], PMR, CMR, 2D NMR (COSY, DEPT, spinecho experiments)

4(14)-Pandamine type

110 Morel et al.

Table XIII (Continued ) Type

Other 14-membered cyclopeptides

Alkaloids

Stereochemistry

Lotusanine B (197)

3S*, 4S*

Vignatic acid A (207)

3S*, 4S*

Vignatic acid B (208)

3S*, 4S*

Anorldianine 27-Noxide (204)

3S, 4S

Structural and spectral data elemental analysis, amino acid analysis after hydrolysis, absolute configuration (CPGC) Amorphous solid; IR, UV, EI-MS [620(M)+], PMR, CMR, 2D NMR (DEPT), relative configuration White powder; FAB-LRMS [554(M+H)+], PMR, CMR, 2D NMR (COSY, HMQC, HMBC, NOE-DF), relative configuration White powder; FAB-LRMS [520(M+H)+], PMR, CMR, 2D NMR (COSY, HMQC, HMBC, NOE-DF), relative configuration IR, UV, pos. HRFAB-MS [501(M+H)+], PMR, CMR, 2D NMR (HMQC, HMBC), amino acid analysis after hydrolysis

References

(128)

(133)

(133)

(131)

Cyclopeptide Alkaloids from Higher Plants

111

alkaloids and seven are neutral compounds that do not present a peptidogenic amino acid with an N-mono-methyl or N,N-dimethylated side chain.

III. OCCURRENCE IN NATURE Cyclopeptide alkaloids are natural products that have been found in the stem bark, roots, leaves, and seeds of 55 plants from 11 higher plant families, mainly from genera of the family Rhamnaceae. Their occurrence has also been confirmed in representatives of the families Sterculiaceae, Asteraceae, Rubiaceae, Urticaceae, Celastraceae, Euphorbiaceae, Menispermaceae, Pandaceae, Olacaceae, and Fabaceae. To date, approximately 209 peptide alkaloids from higher plants have been described, including two linear peptide alkaloids and seven neutral cyclopeptide alkaloids. Among these, more than 100 cyclopeptide alkaloids have been isolated from various plants of the genus Zizyphus, and these include fifty-two 13-membered, fifty-three 14-membered, and ten 15-membered ring cyclopeptides. Clear exceptions to these occurrences are the linear peptide alkaloids: the celenamides (134–136), which have been isolated from sponges, and the hirsutellones (24), isolated from a fungus. The sources of the cyclopeptide alkaloids sensu stricto, as well as the neutral cyclic alkaloids that do not exhibit basic properties, are listed in Table XIV in which the literature has been covered from 1964 to mid-2008.

IV. NEW CYCLOPEPTIDE ALKALOIDS (2006–2008) Only eight cyclopeptide alkaloids (Table XV) were newly isolated and characterized since the previous reviews by Tan and Zhou (6) and El-Seedi et al. (24). Singh et al., in 2006, isolated sativanine N (16) and sativanine O (17), two 3(14)-membered cyclopeptide alkaloids, from the stem bark of Zizyphus sativa (Rhamnaceae). Their structures were established by spectroscopy (40). In 2007, Morel and coworkers (71) isolated chamaedrine (72), a 4(14)frangulanine-type cyclopeptide alkaloid, from the roots of Melochia chamaedris (Sterculiaceae), along with the known cyclic peptide alkaloids adouetine X (62), frangulanine (82), scutianine B (96), and scutianine C (97). The structure of chamaedrine was elucidated on the basis of spectral analysis, especially by 2D NMR (1H–1H COSY, NOESY, HMQC, HMBC) (71). Oxyphyline A (142), a 5(14)-integerrine-type cyclopeptide alkaloid, has been isolated from the stem bark of Zizyphus oxyphylla (Rhamnaceae),

112

Table XIV

Higher plant sources of cyclopeptide alkaloids isolated during 1964–2008 Species

Alkaloids

Parts

References

Rhamnaceae

Zizyphus hutchinsonii

Hysodricanine A (161)

Bark

(94)

Zizyphus mucronata

Alkaloid 2 (1), Alkaloid 3 (23), O-Desmethylmucronine D (38) Mucronine J (150) Mucronine A (184), Mucronine B (185), Mucronine C (186) Mucronine E (187), Mucronine F (188), Mucronine G (189), Mucronine H (190) Mucronine D (30) Abyssinine A (181)

Roots

(30)

Root bark Stem bark

(113) (125)

Stem bark

(124)

Stem bark Root bark

(46) (124)

Daechuine S6 (2), Daechuine S7 (3), Daechuine S26 (5), Daechuine S3 (25), Daechuine S8 (26), Melonovine A (86), Franganine (80), Frangufoline (81), Mucronine D (30), Nummularine R (8) Paliurine E (11) Adouetine X (62), Frangulanine (82) Nummularine B (32)

Stem bark, root bark, roots

(28)

Stem bark Root bark, stem bark Stem bark

(35) (64) (32)

Zizyphus lotus

Lotusine F (6), Lotusine E (29), Lotusine C (163) Lotusine A (146), Lotusine D (147) Lotusine G (148) Lotusine B (162), Lotusanine B (197), Adouetine Yu (63), Frangufoline (81), Sanjoinenine (198), Sanjoinine F (95)

Root Root Root Root

(31) (110) (111) (31)

Zizyphus nummularia

Daechuine S10 (4) Nummularine C (7), Nummularine B (32)

Stem bark Stem bark, root bark

Zizyphus jujuba var. inermis

bark, bark bark bark, aerial parts

(28) (32)

Morel et al.

Family

Zizyphus sativa

(33) (34) (47) (28) (48) (49) (50)

Bark Stem bark

(51) (47)

Nummularine D (132), Mauritine D (166), Nummularine G (201), Mauritine F (168), Amphibine A (66), Jubanine B (28), Mucronine D (30), Nummularine A (31) Nummularine M (134), Nummularine F (151) Scutianine D (98) Mauritine A (164), Amphibine H (24)

Root bark, stem bark

(104)

Root bark, stem bark Bark Bark, root bark

(48) (87) (116)

Rugosanine B (12), Nummularine P (36), Sativanine H (50) Rogosanine A (47) Amphibine D (158)

Stem bark

(36)

Stem bark Bark

(53) (115)

Bark Bark Bark Stem bark Bark Bark Bark Bark Bark

(37) (38) (39) (42) (54) (55) (56) (131) (105)

Sativanine E (13) Sativanine G (14) Sativanine K (15) Tscheschamine (20) Sativanine C (48) Sativanine F (49) Sativanine H (50) Sativanine D (206) Sativanine A (135), Sativanine B (205), Frangulanine (82), Mucronine D (30), Nummularine B (32)

113

Root bark Stem bark Stem bark Stem bark, root bark Root bark Root bark Bark, stem bark, root bark

Cyclopeptide Alkaloids from Higher Plants

Zizyphus rugosa

Nummularine R (8) Nummularine S (9) Nummularine H (33) Nummularine S10 (10), Nummularine E (133) Nummularine N (34), Jubanine A (27) Nummularine O (35), Mauritine C (149) Nummularine P (36), Frangufoline (81), Integerrenine (127) Nummularine T (37) Nummularine K (92)

114

Table XIV (Continued ) Family

Species

Parts

References

Sativanine M (51), Nummularine P (36) Sativanine N (16), Sativanine O (17)

Stem bark Stem bark

(57) (40)

Zizyphus xylopyra

Nummularine K (92), Amphibine H (24) Mauritine D (166) Nummularine B (32) Xylopyrine A (21), Xylopyrine B (22)

Stem bark Bark Bark Root bark

(47) (112) (32) (43)

Zizyphus jujuba

Jubanine A (27), Jubanine B (28) Mucronine D (30) Nummularine A (31), Nummularine B (32) Jubanine C (141), Scutianine D (98) Frangufoline (81) Mauritine A (164), Amphibine H (24) Zizyphine A (52)

Stem Stem Stem Stem Stem Stem Stem

bark bark bark bark bark bark bark

(45) (46) (32) (90) (77) (116) (137)

Zizyphus oenoplia

Zizyphine Zizyphine Zizyphine Zizyphine

Stem Stem Stem Stem

bark bark bark bark

(58) (59) (60) (61)

Zizyphine K (57) Amphibine B (156) Zizyphine D (191), Zizyphine E (192) Frangufoline (81), Mauritine D (166) Abyssinine A (181), Abyssinine B (182)

Stem Stem Stem Stem Stem

bark bark bark bark bark

(62) (115) (59) (138) (124)

Amphibine H (24), Amphibine F (143), Amphibine G (144) Amphibine A (66) Amphibine B (156), Amphibine C (157), Amphibine D (158), Amphibine E (159)

Stem bark

(44)

Stem bark Stem bark, bark

(67) (115)

Zizyphus amphibia

A (52) B (53), Zizyphine C (54) F (55), Zizyphine G (155) I (56)

Morel et al.

Alkaloids

Zizyphus spinachristi

Zizyphus mauritiaa

(114)

Mauritine C (149), Mauritine D (166), Mauritine E (167), Mauritine F (168) Mauritine A (164), Mauritine B (165) Mauritine H (169) Mauritine J (170) Sativanine K (15), Mauritine K (171)

Bark, stem bark

(112)

Stem bark Stem bark Root bark Root bark

(116) (94) (117) (118)

(139) (140)

Sanjoinine B (94), Sanjoinine F (95), Sanjoinine D (179), Stem Sanjoinenine (198), Sanjoinine G2 (202), Frangufoline (81) Amphibine D (158) Sanjoinine G1 (180) Stem

(141)

Zizyphus hysodrica

Hysodricanine A (161) Nummularine E (133)

Bark Bark

(94) (142)

Zizyphus juazeiro

Amphibine D (158)

Bark

(142)

Zizyphus abyssinica

Abyssinine A (181), Abyssinine B (182), Abyssinine C (183) Mucronine A (184), Mucronine B (185), Mucronine C (186)

Bark

(124)

Bark

(124)

(28)

Zizyphus oenoplia var. brunoniana

Zizyphine N (58), Zizyphine O (59), Zizyphine P (60), Root Zizyphine Q (61)

(63)

Zizyphus oxyphylla

Oxyphylline A (142), Nummularine R (8)

(109)

Stem bark

Cyclopeptide Alkaloids from Higher Plants

Zizyphus vulgaris var. spinosus

Spinanine A (154), Amphibine H (24), Stem bark Jubanine A (27), Zizyphine F (55) Franganine (80), Sativanine A (135), Mauritine C (149) Stem bark, bark Amphibine A (66), Amphibine E (159), Mauritine C Stem bark, bark (149), Mauritine A (164), Amphibine F (143)

115

116

Table XIV (Continued ) Species

Alkaloids

Parts

References

Rhamnaceae

Discaria longispina

Discarine A (74), Discarine B (75), Frangulanine (82) Discarine X (79), Discarine E (76), Adouetine Yu (63) Adouetine Yu (63), Frangufoline (81)

Root bark Root bark Root bark

(72) (76) (143)

Discaria febrifuga

Discarine F (77) Discarine I (78), Discarine B (75) Discarine K (175) Discarine L (176) Franganine (80), Frangufoline (81), Scutianine B (96), Discarine C (123), Discarine D (124) Discarine G (173) Discarine H (174) Discarine K (175), Franganine (80) Discarine L (176) Discarine E (76)

Bark Root bark Root Root bark Stem bark

(74) (75) (121) (122) (101)

Root bark Root bark Root Root bark Stem bark

(119) (120) (121) (122) (73)

Discaria americana

Discarene C (193), Discarene D (194), Discarine B (75), Root bark, bark Adouetine Yu (63), Franganine (80), Adouetine Y (111), Discarine C (123), Myrianthine A (129)

(126)

Adouetine Yu (63), Discarine A (74), Discarine (76), Franganine (80), Frangulanine (82), Discarine C (123), Discarine D (124), Discarine M (195), Discarine N (196)

Root bark

(127)

Discaria pubescens

Pubescine A (93)

Root bark

(84)

Discaria crenata Scutia buxifolia

Crenatine A (120) Scutianine B (96) Scutianine B (96), Scutianine C (97), Scutianine A (109), Scutianine D (98), Scutianine E (99) Scutianine G (100)

Leaves, stem Root, bark Root

(100) (85) (86)

Bark

(88)

Morel et al.

Family

Scutianine K (103), Scutianine L (136) Scutianine B (96), Scutianine C (97), Scutianine D (98), Scutianine E (99), Scutianine H (101) Scutianine B (96), Scutianine C (97), Scutianine D (98), Scutianine E (99), Scutianine J (102) Scutianine A (109), Scutianine B (96) Scutianine F (110) Scutianine B (96), Scutianine D (98), Scutianine E (99), Scutianine F (110) Scutianine M (137) Scutianene C (199)

Rhamnaceae

Ceanothus americanus

Root bark Bark

(90) (87)

Bark

(89)

Bark Bark Root bark

(93) (94) (106)

Root

(129)

Ceanothus sanguineus

N-Desmethyl-myrianthine B (89) Root bark Adouetine Yu (63), Ceanothine B (70), Discarine B (75), Root bark Frangufoline (81), N-Methyl-americine (91)

(70) (144)

Ceanothus integerrimus

N-Methyl-americine (91), Discarine B (75) Deoxo-aralionine A (121) Integerrine (126), Integerrenine (127), Integerressine (128) N-Desmethyl-integerrenine (130), N-Desmethylintegerrine (131)

Root bark Root bark Root bark

(83) (81) (103)

Root bark

(83)

Condalia buxifolia

Candaline A (119), Adouetine Yu (63), Scutianine B (96), Scutianine C (97)

Root bark

(99)

Rhamnus frangula

Franganine (80), Frangufoline (81) Frangulanine (82)

Bark Bark

(77) (93)

Colubrina texensis

Texensine (104)

Whole plant

(91)

(95)

117

(66) (70) Cyclopeptide Alkaloids from Higher Plants

Americine (65), Homoamerecine (83) Root bark Ceanothine A (69), Ceanothine B (70), Ceanothine C Root bark (71) Adoutine X (62), Frangulanine (82), Adoutine Y (111), Root bark Ceanothine E (118), Ceanothine D (203)

118

Table XIV (Continued ) Family

Sterculiaceae

Alkaloids

Parts

References

Paliurus ramosissimus

Paliurine E (11), Paliurine A (39), Paliurine B (40), Roots, stem Paliurine C (41), Paliurine D (42), Paliurine F (43), Sativanine G (14) Daechuine S3 (25), Nummularine H (33), Paliurine A Stem (39), Paliurine B (40), Paliurine C (41), Paliurine F (43), Paliurine G (44), Paliurine H (45), Paliurine I (46) Ramosine A (152), Ramosine C (153), Ramosine B Roots (172), Lotusine A (146), Lotusine D (147), Mucronine J (150)

(35)

Paliurus hemsleyanus

Hemsine C (140), Hemsine A (145), Hemsine B (160), Hemsine D (125)

Roots

(102)

Lasiodiscus marmoratus

Alkaloid 2 (1) Lasiodine B (108), Lasiodine A (209)

Leaves Leaves

(30) (27)

Araliorhammus vaginatus

Alkaloid 4 (64) Aralionine A (114), Desbenzoyl-aralionine A (122) Aralionine B (115) Aralionine C (116)

Bark Leaves, stem bark, bark Leaves, bark Bark

(54) (96) (97) (94)

Hovenia dulcis

Frangulanine (82), Hovenine A (84)

Root bark

(78)

Hovenia tomentella

Frangulanine (82), Hovenine A (84)

Bark, root bark

(78)

Waltheria americana

Adouetine Z (138), Adouetine X (62), Adouetine Yu (63), Adouetine Y (111)

Whole plant

(65)

Waltheria douradinha

Waltherine A (105), Waltherine B (106), Adouetine Yu (63), Scutianine B (96) Waltherine A (105), Waltherine B (106), Adouetine Yu (63), Scutianine B (96), Waltherine C (107)

Root bark, bark

(92)

Bark

(13)

(52)

(102)

Morel et al.

Rhamnaceae

Species

Melochia corchorifolia

Adouetine Yu (63), Melofoline (85) Franganine (80), Frangufoline (81), Adouetine Yu (63)

Leaves, woody parts, aerial parts Bark, leaves, woody parts

(79) (145)

Leaves

(146)

Melochia tomentosa

Melonovine A (86), Melonovine B (87), Scutianine B (96)

Roots

(80)

Melochia chamedrys

Chamaedrine (72), Adouetine X (62), Frangulanine (82), Scutianine B (96), Scutianine C (97)

Roots

(71)

Urticaceae

Myrianthus arboreus

Myrianthine C (88), Myrianthine A (129), Adouetine Yu (63)

Leaves

(81)

Pandaceae

Panda oleosa

Pandamine (177) Pandamine (177), Pandaminine (178)

Root barks Root barks

(123) (29)

Rubiaceae

Plectronia odorata Feretia apodanthera Canthium anorldianum Canthium euryoides

N-Desmethyl-myrianthine C (90) Feretine (139), Adouetine Z (138) Anorldianine (68) Canthiumine (117)

Aerial parts, leaves Leaves Stem bark Stem bark

(82) (107) (69) (98)

Euphorbiaceae

Antidesma montana

Aerial parts, leaves, terminal branches Root bark

(68)

Hymenocardia acida

AM-1 (67), Adouetine Yu (63), AM-2 (113), Aralionine B (115) Hymenocardine (200)

Celastraceae

Euonymus europaeus

Franganine (80), Frangufoline (81), Frangulanine (82)

Leaves, stem, roots, root bark

(147)

Olacaceae

Heisteria nitida

Anorldianine 27-N-oxide (204), Integerrenine (127)

Bark

(131)

Asteraceae

Sphaeranthus indicus

Subfraction I (18), Subfraction II (19)

Flowers

(41)

Fabaceae

Vigna radiata var. sublobata

Vignatic acid A (207), Vignatic acid B (208)

Flowers

(133)

(130)

119

Frangufoline (81), Integerrenine (127), Adouetine Z (138)

Cyclopeptide Alkaloids from Higher Plants

Melochia pyramidata

Structures and spectral data of the new alkaloids (2007–2008)

Alkaloids

Spectral data

19

20

6

HN O

9

NH

8

7 27

21

28

30

NH2

23

10

O O 5

N

24

11 12

13

3 4

18

22

1

O

29

26

Sativanine N (16) (40)

2 17

21 20 23

26

HN O

NH2 28

11 10

O O

22

25

OCH3

13

5 6

N

19

24

12

3 4

18

16

1

O

29

27

Sativanine O (17) (40)

7

HRMS: m/z 554.2530 (M+, calcd for C32H34N4O5, 554.2529), 463.1983 (C25H27N4O5), 435.1784 (C24H25N3O5), 434.1718 (C24H24N3O5), 408.1910 (C23H26N3O4), 407.1825 (C23H25N3O4), 406.1748 (C23H24N3O4), 338.1270 (C19H18N2O4), 259.1084 (C14H15N2O3), 233.1278 (C13H17N2O2), 216.1020 (C13H14NO2), 215.1180 (C13H15N2O), 165.0795 (C9H11NO2), 120.0825 (C8H10N), 96.0455 (C5H6NO), 68.0510 (C4H6N)

38

15 14

9 8

NH

36

35 34

30 31 32

33

Morel et al.

OCH3 16

2 17

HRMS: m/z 486.2842 (M+, calcd for C26H38N4O5, 486.2841), 401.1956 (C21H27N3O5), 400 (C21H26N3O5), 374.2054 (C20H28N3O4), 373.2008 (C20H27N3O5), 372.1896 (C20H26N3O5), 259.1084 (C14H15N2O3), 233.1278 (C13H17N2O2), 216.1022 (C13H14NO2), 209.1284 (C11H17N2O2), 181.1342 (C10H17N2O), 165.0794 (C9H11NO2), 114.0920 (C6H12NO), 96.0454 (C5H6NO), 86.0971 (C5H12N), 68.0508 (C4H6N)

38

15 14

25

120

Table XV

2

O

OCH3 11 12

O O 5

N

HN O 6

19

20 21

23

31

NH 37

36 35

32

29

33

N CH3

28

25

9

8

7

22 24

10

13

4

18

16

1

3

17

13

38

15 14

34

C d: 107.0 (C-11); 122.0 (C-10); 167.2 (C-8); 60.8 (C-7); 170.0 (C-5); 65.0 (C-4); 76.8 (C-3); 152.8 (C-1); 111.8 (C-13); 118.0 (C-14); 125.0 (C-12); 114.5 (C-15); 152.0 (C-16); 33.5 (C-31); 141.0 (C-32); 129.5 (C-33); 129.5 (C-37); 128.3 (C-34); 128.3 (C-36); 125.4 (C-35); 32.5 (C-17); 46.2 (C-18); 56.0 (C-38); 171.0 (C-20); 68.2 (C-21); 32.4 (C-22); 138.0 (C-23); 129.0 (C-24); 129.0 (C-28); 128.7 (C-25); 128.7 (C-27); 42.0 (C-29, C-30). MS: m/z 582.2842 ([M]+, C34H38N4O5), 148.1126 (C10H14N, base peak), 491.2294 (C27H31N4O5), 435.1784 (C24H25N3O5), 434.1718 (C24H24N3O5), 408.1910 (C23H26N3O4), 407.1825 (C23H25N3O4), 406.1748 (C23H24N3O4), 215.1270 (C13H15N2O), 259.1084 (C14H15N2O3), 233.1278 (C13H17N2O2), 216.1020 (C13H14NO2), 338.1186 (C19H18N2O4), 165.0795 (C9H11NO2), 120.0825 (C8H10N), 96.0455 (C5H6NO), 68.0505 (C4H6N)

H3C

26

30

27

Xylopyrine B (22) (43)

OCH3

2

O

17

24

21

HN 25

10

O O 5

19N 20 22

23

11 12

13

3 4

18

16

1

HN O 6 O 26 27

8

NH

7 32 35 28

H3C N 29

30

O

31H

Sativanine M (51) (57)

9

33 34

HRMS: m/z 585.162 ([M]+, C30H43N5O7), 557.3212 (C29H43N5O6), 472.2688 (C25H36N4O5), 457.2578 (C25H35N3O5), 401.1954 (C21H27N3O5), 400.1873 (C21H26N3O5), 374.2053 (C20H28N3O4), 373.2007 (C20H27N3O4), 372.1897 (C20H26N3O4), 259.1084 (C14H15N2O3), 233.1278 (C13H17N2O2), 216.1020 (C13H14NO2), 213.1240 (C10H17N2O3), 209.1282 (C11H17N2O2), 185.1290 (C9H17N2O2), 181.1344 (C10H17N2O), 165.0795 (C9H11NO2), 114.0556 (C5H8NO2), 96.0454 (C5H6NO), 86.0608 (C4H8NO), 58.0668 (C3H8N)

Cyclopeptide Alkaloids from Higher Plants

36

15 14

121

122

Table XV (Continued ) Alkaloids

Spectral data

18

O

17

5

HN

HN 6 O

20 21

23 25 24

8

29

NH

H d: 2.27 (6H, s, N-Me2); 3.95 (1H, d, CH-25, J ¼ 8.0 Hz); 1.30, 1.92 (2H, m, CH2-26); 0.77, 1.47 (2H, m, CH2-27); 2.58 (1H, t, CH-28, J ¼ 7.0 Hz); 3.40 (1H, d, CH-28u, J ¼ 7.0 Hz); 3.34 (1H, t, CH-30, J ¼ 7.0 Hz); 5.51 (1H, d, OH at CH-40, J ¼ 5.5 Hz); 5.70 (1H, d, CH-3, J ¼ 7.0 Hz); 7.04–7.10 (5H, m, CH-42 to CH-46); 6.06 (1H, d, CH-11, J ¼ 8.0 Hz); 6.35 (1H, d, CH-10, J ¼ 8.0 Hz); 4.04 (1H, dd, CH-10, J ¼ 7.0, 5.5 Hz); 4.68 (1H, t, CH-4, J ¼ 7.0 Hz), 4.48 (1H, dd, CH-40, J ¼ 5.5, 5.0 Hz), 8.49 (1H, br, s, NH-9); 7.60 (1H, d, NH-6, J ¼ 7.0 Hz); 8.60 (1H, br, s, NH-23); 3.34 (1H, t, CH-30); 2.02 (2H, br, s, CH2-31); 7.40–7.46 (5H, m, CH-33 to CH-37); 6.94–7.02 (4H, m, CH-13, CH-14, CH-15, CH-16); 7.19–7.29 (5H, m, CH-18 to CH-22). 13C d: 127.43 (C-11); 128.24 (C-10); 168.95 (C-8); 58.19 (C-7); 168.78 (C-5); 55.82 (C-4); 80.49 (C-3); 154.44 (C-1); 120.08 (C-15); 129.68 (C-16); 131.35 (C-12); 129.82 (C-13); 120.51 (C-14); 72.01 (C-40); 142.03 (C-41); 127.15 (C-46); 129.05 (C-45); 127.29 (C-44); 129.16 (C-43); 127.15 (C-42); 167.88 (C-24); 58.55 (C-25); 25.33 (C-26); 23.38 (C-27); 45.03 (C-28); 169.75 (C-29); 68.14 (C-30); 40.13 (C-31); 140.06 (C-32); 128.93 (C-33); 130.65 (C-34); 128.04 (C-35); 130.65 (C-36); 128.93 (C-37); 40.78 (C-38, C-39); 138.11 (C-17); 1267.1 (C-18); 128.40 (C-19); 126.53 (C-20); 128.40 (C-21); 126.67 (C-22). EI-MS (high resolution): m/z 716.33922 ([M+H]+, C42H46N5O6 requires 716.34472), 715.33429 (M+, C42H45N5O6 requires 715.33694), 518.23942 (C28H32N5O5 requires 518.24030)

37

36

38

9

41

7

39 40

32 35

22

26

1

10

O O

4

H d: 2.12 (6H, m, N-Me2); 2.83 (2H, dd, CH2-32, J ¼ 4.5, 15.0 Hz); 2.90 (1H, dd, CH2-32u, J ¼ 8.0, 15.0 Hz); 0.87 (3H, d, CH3-18, J ¼ 6.7 Hz); 1.15 (3H, d, CH3-19, J ¼ 6.7 Hz); 1.85 (1H, m, CH-17); 2.60 (1H, dd, CH22, J ¼ 4.5, 8.0 Hz); 2.70 (1H, dd, CH2-23, J ¼ 4.5, 14.0 Hz); 2.81 (1H, dd, CH2-23u, J ¼ 8.0, 14.0 Hz); 4.32 (1H, m); 4.33 (1H, dd, CH-4, J ¼ 8.0, 10.5 Hz); 7.0–7.5 (4H, br, m, CH-13 to CH-16); 4.84 (1H, dd, CH-3, J ¼ 2.0, 8.0 Hz); 5.75 (1H, d, NH-6, J ¼ 8.0 Hz); 5.75 (1H, overlapping, CH-10); 5.76 (1H, overlapping, CH-11), 7.38 (1H, d, NH-20, J ¼ 10.5 Hz), 9.55 (1H, br, s, NH-33). 13C d: 126.9 (C-11); 131.4 (C-10); 167.2 (C-8); 54.0 (C-7); 171.5 (C-5); 54.9 (C-4); 81.8 (C-3); 156.0 (C-1); 120.0–129.4 (C-13 to C-16); 131.6 (C-12); 14.0 (C-18); 20.3 (C-19); 172.6 (C-21); 69.3 (C-22); 141.0 (C-24); 119.0–130.0 (C-25 to C-29); 41.5 (C-30, C-31); 39.2 (C-32); 136.0 (C-40); 126.6 (C-41). HRESIME: m/z 608.32308 ([M+H]+, C36H42N5O4 requires 608.32331). EI-MS: m/z 607 [M]+, 190, 148 (100%), 135, 130

12 1 1 15 16

3

19

NH

34

33

N CH3 31

H 3C 27

1

1

30

28

Chamaedryne (72) (71) 14 19

2

18 17

20

O

22

23HN 24 26 25

27 28 31 32 33 34 35

N 29 30

12 11 15 16

3

10

O O

4 21

13

1

5

HN O6

7

9 8

NH 46

45

40

44

41

HO O 3 N CH 39

37 CH3 38 36

Oxyphyline A (142) (109)

42

43

Morel et al.

13

14 2

14 2

O 17 18 22

N 19

21 24

20

HN 25

29 30

12 11 15 16

3

10

O O

4 23

13

13

1

5

8

HN O6 O 26 27 28

7

NH 9

34 33

36

NH2 32

35

C d: 122.4 (C-11); 125.5 (C-10); 167.0 (C-8); 58.4 (C-7); 171.6 (C-5); 64.5 (C-4); 84.0 (C-3); 156.5 (C-1); 121.9 (C-15); 114.3 (C-14); 132.2 (C-16); 132.8 (C-13); 130.1 (C-12); 37.4 (C-33); 25.0 (C-34); 10.1 (C-35); 15.8 (C-36); 33.2 (C-17); 46.4 (C-18); 172.0 (C-19); 54.0 (C-20); 35.2 (C-21); 24.6 (C-22); 12.1 (C-23); 15.3 (C-24); 169.8 (C-26); 70.4 (C-27); 34.8 (C-28); 24.2 (C-29); 12.0 (C-30); 15.2 (C-31). HRMS: m/z 569.3577 ([M]+, C31H47N5O5), 512.2873 (C27H38N5O5), 484.3142 (C26H36N4O5), 482.2529 (C26H34N4O5), 441.2627 (C25H35N3O4), 371.1845 (C20H25N3O4), 370.1767 (C20H24N3O4), 344.1974 (C19H26N3O3), 343.1896 (C19H25N3O3), 342.1817 (C19H24N3O3), 296.2338 (C16H30N3O2), 227.1759 (C12H23N2O2), 235.1083 (C12H15N2O3), 229.0977 (C13H13N2O2), 199.1810 (C11H23N2O), 209.1290 (C11H17N2O), 203.1184 (C12H15N2O), 274.1317 (C15H18N2O2), 186.0919 (C12H12NO), 181.1341 (C10H17N2O), 135.0684 (C8H9NO), 96.0449 (C5H6NO), 86.0970 (C5H12N, base peak), 68.0500 (C4H6N)

31

Mauritine K (171) (118) Cyclopeptide Alkaloids from Higher Plants

123

124

Morel et al.

together with the known 13-membered alkaloid nummularine R (8). The structure of oxyphyline A (142) was elucidated mainly by 1H NMR, 13C NMR, and 2D NMR spectral data (109). In the same year, Pandey et al. (118) reported the isolation of mauritine K (171), another 5(14)-amphibine B type alkaloid, together with the known sativanine K (15), from the roots of Zizyphus mauritiana. The structure of mauritine K was established mainly by analysis of its high-resolution mass spectra, and also by chemical evidence (118). From the root bark of Zizyphus xylopyra, two new 4(13)-nummularine C-type cyclopeptide alkaloids, xylopyrine A (21) and B (22), were isolated. The structures of both alkaloids were determined mainly by high-resolution mass spectra and chemical evidence (43). In 2008, Pandey et al. (57) isolated sativanine M (51), a 5(13)-type cyclopeptide alkaloid, together with the known alkaloid nummularine P (36), from the stem bark of Z. sativa. The structure of sativanine M (51) was determined mainly by high-resolution mass spectra and supported by deformylation into the known alkaloid sativanine C (48) (57).

V. STRUCTURE ELUCIDATION AND STEREOCHEMISTRY For the structural elucidation of cyclopeptide alkaloids, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, especially 2D NMR (COSY, NOESY, HMQC, HMBC) (4–7,71,72), are the most useful analytical techniques. Besides, UV, IR, CD, X-ray analysis, and chemical degradation (10,35,102,148) are also important tools for the structure elucidation of this class of alkaloids. Cyclopeptide alkaloids, principally those of the 14-membered ring type, contain amino acids in both the L and D forms. Whereas in most cases the L-configuration was established, many alkaloids, such as the scutianines D (98), E (99), L (136), and M (137), isolated from Scutia buxifolia (87,88,90,106), and condaline A (119), isolated from Condalia buxifolia (99), bear ring amino acid residues of opposite stereochemical configuration (namely, L/D-threo-b-hydroxyleucine, L/D-erythro-b-hydroxyleucine, L/D-threo-b-phenylserine, and L/D-erythro-b-phenylserine). The 1H NMR and 13C NMR spectral data were important tools for establishing the relative and absolute configuration of the b-OH amino acid. In the 1H NMR spectrum, the erythro form of the b-OH amino acid shows a Ja,b ca. 8 Hz, whereas the threo compounds show a Ja,b ca. 2 Hz (12,148,149). 13C NMR spectroscopy is also an important tool for the elucidation of the absolute configuration of the b-OH amino acid. For the L-erythro series, the signal of the alpha carbon appears at ca. 55.0 ppm, whereas for the D-erythro it appears at ca. 53.8 ppm. A clear difference in chemical shift is also observed for the beta carbon – in the L-erythro series

Cyclopeptide Alkaloids from Higher Plants

125

the signal appears at 81.5 ppm, whereas for the D-erythro configuration it appears at lower field, at ca. 87 ppm (5,8,87). In addition, 13C NMR spectra have proved to be a valuable tool to distinguish between the cis and trans configurations when the b-OH amino acid is proline. The derived rule is as follows: C-g-Pro: trans, d ¼ 21.2–21.6, and cis, d ¼ 23.8–24.1 (5,146). In 1974, Ruveda and coworkers (129) determined the stereochemistry of all of the chiral centers of scutianine A, with the exception of those of the b-hydroxyleucine unit, which had been previously determined for other alkaloids through degradation reactions (10,148). Continuing their structural studies of this class of alkaloids, in 1979, Ruveda and coworkers (87) determined the stereochemistry of scutianines D (98) and E (99), isolated from S. buxifolia, by analysis of the 1H NMR spectra. The erythro form of the b-hydroxyleucine unit was shown to be present in 98 and 99, and the threo form of the b-phenylserine moiety was identified among the hydrolysis products of dihydro-98 and dihydro-99. The 1H NMR of 98 showed that the signal corresponding to the a- and b-protons of the hydroxyleucine unit had d values different than those observed for the corresponding protons of 99, indicating some change in the stereochemistry of this unit in 98 compared with 99. Thus, 98 should be related to an alkaloid stereoisomeric with 99. At the same time, Tschesche and Ammermann (86) published the isolation, from the same source, of scutianine C (97) and two diastereomers, 98 and 99. Tschesche and Ammermann determined the stereochemistry of the latter compounds as L-erythro-b-hydroxyleucine, L-threo-b-phenylserine and D-erythro-b-hydroxyleucine, D-threo-b-phenylserine, respectively. In 1982, Ruveda and coworkers (11) determined the stereochemistry of the N,N-dimethylphenylalanine residues in two of these diastereoisomeric alkaloids, in the dipeptide forms, by gas chromatography (GC). Continuing the structural studies of this class of alkaloids, Morel and coworkers (12,119,126,127) analyzed the stereochemistry of the terminal N,N-dimethyl amino acid residue and of the a-amino acid within the cyclic system of the alkaloids adouetine Yu (63); franganine (80); scutianines B (96), C (97), D (98), E (99), and H (101); discarines B (75), D (124), and F (77); discarines M (195) and N (196); discarenes C (193) and D (194); and condaline A (119) by enantioselective GC (CPGC). To conduct this study, 3-Bu-2,6-Pe-g-CD (150), 3-Pe-2,6-Me-b-CD (151), and 6-Me-2,3-Pe-g-CD (152) were used as chiral stationary phases. The stereochemistry of the b-hydroxy amino acid (b-hydroxyleucine and b-phenylserine), which was not found in the hydrolysate of the alkaloids, was deduced by analysis of the NOESY spectrum. Since the stereochemistry of C-7 is absolute by enantioselective GC, the spatial position of H-7 is a starting point for the assignment of the configuration of C-3 and C-4 of the alkaloids. Selected NOE correlations, 1H NMR and

126

Morel et al.

H

HHO

L-eryt hro

D-erythro

4.37(7.4,10.0) [55.1]

S*

H N

H [122.5]

O H

S

HN O

NH4.48(6.0,8.0)

4.46 (6.0, 8.8) [53.8]

R*

H N

4.97 (6.0) H[72.5] OH

R

S 3.13 (4.5,7.2) [69.6]

H O

R*

[57.9]

H

S

H

O H

R

HN O

S

3.14 (5.2,6.2) [69.7]

L-threo

Scutianine D (98)

NH 4.21(8.1, 5.7)

O H HO

4.58( 9.2) [55.3]

H N S

HN O

L-erythro

NH

NH(CH 3)

O H HO

S

H N

HN O

6.73 (8.5) [124.9]

O H HO

6.72 (10.0 , 7.0) H [125.54]

O S

HN O

H

6.38 (14.6) [77.8]

O H HO

L-erythro S*

NH

(8.2, 3.8) H4.08 [59.52]

H N

7.35 (14.6,10.2) [58.4]

H

S

OH 4.88(dd) H[72.7]

NH

S*

4.52 (8.0,10.0) [54.2]

4.28 (5.4,10.1) [55.2]

H N

NH

S 2.69(m) [74.5]

O H HO

Scutianine L (136) 6.51(d) [119.2]

6.40 (d) [122.9]

O H

(dd)

S

NH

S*

D-threo

H

NH

H N

5.32 (14.0) [120.9]

Discarine K (175)

H [125.8]

O S

NH

HN O

H

4.06 (m) [55.2]

H E

6.29 ( 6.6,14.0) H [148.2]

N(CH 3) 2

NH(CH 3) 2

6.51(dd)

HO

R*

4.88(7.6,10.0) [55.7]

4.20 (m) [53.0]

S 2.63 (d) [71.5]

H

5.80 ( 7.6) [82.2]

6.20 H [124.7]

O

HN O

H

S

N(CH3 )2

H

S*

HN O

NH 4.55 (8.2, 4.2) [68.2]

S 3.62 (4.6, 8.2) [71.0]

2.90(9.4, 3.6) [65.82]

4.90 (2.0,8.0) [81.4] L-erythro

- 7.30 H 7.10 [126.3]

O

S*

Scutianine M (137)

2.69 (m)/4.03(3.3,12.1) [48.6]

O

S*

4.55 (1.2,8.3) [58.0]

5.39 (1.2) H[71.0] OH D-threo

Scutianine K (103)

NH(CH 3)

Condaline A (119)

4.49 (9.0,10.0)

S

2.63 (5.7,6.7) [68.5]

H

H N

4.70( 6.6, 9.2) [56.39]

S

4.86 (2.0,9.0) [80.6]

H

R

HN O

NH

N(CH 3) 2

S*

3.90 (9.0, 4.4) H [56.8]

S

S*

3.02 (5.6,13.6) [65.5]

S*

S

D-t hreo

6.16 ( 6.6) [81.83] 6.03( 9.0 , 7.6) H[131.4]

O

S*

H N

H [122.8]

O

6.41(7.6) [116.20]

H

R*

S*

[58.6]

Scutianine E (99)

5,81(d) [86.3]

6.68(7.6, 9.0)

HO

S* 4.46 (7.0,10.0) [55.0]

5.04(5.7) H [72.7] OH

6.98(7.6) [126.9]

L-threo

L-erythro

6.50 (dd) [125.3]

N(CH 3) 2

N(CH 3 )2

H

H HO

H HO

6.55( 7.7, 9.2)

HO

S*

6.73 (7.6) [118.5]

4.94(2.0, 7.0) [82.1]

6.58 (d) [118.5]

4.10 (6.0, 6.6) [87.5]

6.40 (7.7) [119.0]

4.93 (2.0, 7.4) [81.4]

Discarine B (75)

Discarene D (194)

H H OCH3 (9.0) H 5.90 [106.6]

5.40 (3.0,7.2) [76.8]

O H S H O O S

H L-trans

H 4.43 (3.0) [64.5]

N S

HN O

S

5.09 (8.8,7.4,5.4) [79.6] L-trans

H

S

H 6.92 (9.0,11.3)

H [121.5] NH

4.22 (4.3, 4.3) [60.3]

3.42 (10.3, 3.4) [68.0]

N(CH3 )2 Paliurine E (11)

4.26 (5.4) [62.4]

OCH3 5.82 (8.9) H [107.5] O H

6.79 (8.9,11.9)

S

N

4.66 ( 8.1,7.8) [47.5] S

H [121.9]

O O NH

N O O

5.61 (9.6) [109.5]

H

S 4.39 (8.9,3.5) [62.2]

(H 3C)HN

6.81 (9.6) H[120.3]

S

O

4.42 (8.2, 3.2) [59.2]

HN

HN S

H H3 CO (β-aminovinyl) phenylalanine

N(CH 3) 2 2.45 (5.4) [70.6]

Zizyphine N (58)

3.07/3.30(m) [60.0]

O

O

NH 3.07/3.30(m) S [66.2]

S

N H

Abyssinine B (182)

Figure 2 Stereochemistry and selected 1H NMR and [13C NMR] data, and H/H correlations obtained from NOESY experiments (2) of some representative cyclopeptide alkaloids. 13

C NMR data, as well the stereochemistry of some cyclopeptide alkaloids are given in Figure 2. Besides NMR and CPGC, total synthesis (19,35,153,154) and circular dichroism (35,102,155) were also important tools for elucidation of the stereochemistry of this class of alkaloids.

VI. SYNTHESIS The first total syntheses of cyclopeptide alkaloids were described in 1983, when the total synthesis of zizyphine A (52), a 13-membered

127

Cyclopeptide Alkaloids from Higher Plants

cyclopeptide alkaloid, and of mucronine B (185), a 14-membered cyclopeptide alkaloid, was carried out by Schmidt and coworkers (156,157). In 1991, the same group (16) accomplished the first total synthesis of frangulanine (82), a 14-membered cyclopeptide alkaloid. Subsequently, several methodologies and total syntheses of cyclopeptide alkaloids have been published (18,19,153,158–171). Schmidt et al. in 1985 (2), Joullie and coworkers in 1985 (3) and 2004 (23), and Tan and Zhou in 2006 (6) have reviewed the synthesis of this class of alkaloids. Recently, several new strategies have been devised to overcome some of the synthetic challenges inherent in the macrocyclization reaction. Zhu and coworkers (161–166) developed an efficient macrocyclization protocol based on endocyclic alkylaryl ether bond formation through an intramolecular SNAr reaction. On the basis of this methodology, the total syntheses of sanjoinine G1 (180) and mauritines A (164), B (165), C (149), and F (168), all 14-membered cyclopeptide alkaloids, were performed. In 2005, the Wessjohann group (174,175) employed an Ugi-four component reaction (Ugi-4CR) (176) as a rapid and powerful method for the synthesis of a variety of heterocycles and macrocycles. In 2006, Zhu and coworkers (168) employed the Ugi-4CR combined with the intramolecular SNAr reaction to obtain natural and non-natural paracyclophanes (Scheme 1). In 2007, Ma and coworkers (169,170) reported the total syntheses of the 13-membered cyclopeptide alkaloid zizyphine N (58) and the 15membered cyclopeptide alkaloids abyssenine B (182) and mucronine E (187). The synthetic convergent route is summarized in Scheme 2 for 58 including the construction of its aryl ether unit via a Mitsunobu reaction, installation of its enamide part via a Cu(I)/N,N-dimethylglycinecatalyzed coupling reaction, and macrocyclization with coupling agents such as FDDP and DPPA (169).

No2

O 2N

NO2 PGX

R2CHO + R3 NH 2

F n

XPG

+

O n

COOH CN

F

R1

N R3

R1 O R2

R1 = H; COOMe; COOEt R 2 = Ph; n-C6 H 13 ; i-Pr; CH 2CH 2Ph; C5 H 10 NHBoc R 3 = Ph; n-C4 H 9NH2 ; cy-Hex; CH2 CH 2Ph PGX = [CH2 ]NHBoc; [CH 2]3 NHBoc; [CH 2]SCPh 3; (4-OMe)Ph

NH

X O n

O

R1 NH

N R3 R2 para-cyclophanes

Scheme 1 Summary of the Zhu syntheses of para-cyclophanes in two steps, employing the Ugi-4CR and intramolecular SNAr reaction for macrocyclization. Reagents and conditions: TFA in CH2Cl2 (eventually), then K2CO3, DMF, rt.

128

Morel et al.

TBSO

OH

OH

O a

OH NHBoc

b

c

d

O

N

N

OTBS Boc (I)

OTBS

Boc OH

OCH 3

e

f

g

THPO

h

HO (II)

I OCH 3

OCH 3

+

(II)

O

j

O (I)

i I N Boc

OTBS

k

O

O

l

NH 2

Alloc

m

N

OCH 3

N Boc

O NH

n o

OH N Alloc

OCH 3

O

N

O

p

O O NH

O O

HO

N

O

Boc HN (III)

O

NH

N CH3 N CH3

O HN

N

O

CH 3 N CH 3

ziziphyne N (58)

Scheme 2 Summary of the Ma synthesis of zizyphine N (58). Reagents and conditions: (a) Meldrum’s acid, DCC, DMAP, CH2Cl2; (b) MeOH, reflux; (c) NaBH4, CH2Cl2 (40–50% yields); borane-dimethylsulfide, THF, reflux (90%); CHCl3, aq. NaOH, EtOH, 41%; MeI, K2CO3, DMF, 87%; Ph3P+(CH2I)I, LiHMDS, HMPA, THF, 80 to 251C; PPTS, MeOH, 58%; DIAD, Ph3P, THF, 801C, 54%; Cu(I), Me2NCH2CO2H, Cs2CO3, dioxane, 75%; TBAF, THF; Dess–Martin oxidation, then NaClO2/NaH2PO4; (Ph3P)4Pd, Et2NH, 56%; FDPP, i-Pr2NEt, DMF, 66%; ZnBr2, then (III), HATU, K2CO3, DMF, 22%.

The total synthesis of 187 was performed using a Cu(I)/N,Ndimethylglycine-catalyzed coupling reaction of vinyl iodide with amides in the key step (170). In the same year, the total syntheses of the 15-membered ring abyssinine A (181) and the 13-membered ring paliurine F (43) were performed by the Evano group (171,172). The synthesis of 181 was accomplished in 15 steps with excellent overall yield (35%). A notable feature in this synthetic approach includes an original and efficient copper (I)-catalyzed Ullman coupling/Claisen rearrangement sequence, which allows for both ipso and ortho functionalization of an aromatic iodide (Scheme 3). The total synthesis of 43 was accomplished by the same method.

129

Cyclopeptide Alkaloids from Higher Plants

HO

I

HO

c

a

OTBS

d

OTBS

b

H 3CO

H 3 CO

H3 CO

f

g

OTBS

OTBS

OHC

O

OTBS

BocHN

(MeO) 2P

BocHN

COOCH3

H 3 COOC

e

OTBS

COOCH 3

NHBoc (I) H3 CO

H 3CO h

Boc i

j

l N

N H 3C

COOCH3

H3 C

H3 CO

COOH

I

O H 2N

O

NH2

N H (II)

H 3 CO

Boc

Boc N

H3 C

k

CHO

Boc

I O

HN

O

O N H

NH 2

N m

H3 C

HN

O O O

H 3CO

NH n

N H

H N H 3C

HN

O O O

NH

N H

Abyssenine-A (181)

Scheme 3 Summary of the Evano syntheses of abyssenine A (181). Reagents and conditions: (a) allylic alcohol, Cu(I), 1,10-phenanthroline, Cs2CO3, toluene, 1101C; (b) 2401C, 93% (for two steps); RuClH(CO)(PPh3)3, toluene, 801C, 93%; (c) Me2SO4, K2CO3, acetone, reflux, 98%; (d) OsO4, NaIO4, CCl4, t-BuOH/H2O, rt, 88% (for two steps); (e) (I), then N,N,N,N-tetramethyl-guanidine, CH2Cl2, rt, 96%; (f) [Rh(COD) {(S,S)-Et-DuPHOS}]+TfO, H2 (5 atm.), MEOH, 251C, 89%, 98% ee; (g) NaH, MeI, DMF, rt, 97%; (h) TBAF, THF, rt, quant.; (i) Dess–Martin periodinane, 2,6-lutidine, CH2Cl2, 01C, 92%; (j) PPh+3(CH2I)I, NaHMDS, HMPA, THF, 781C to rt, 91%; (l) LiOH, MeOH/THF, H2O, rt, 97%; Bop, DIPEA, DMF, 01C, then (II) 01C to rt, 92%; Cu(I), N,N-dimethyl ethylenediamine, Cs2CO3, THF, 631C, 83%; TMSOTf, 2,6-lutidine, CH2Cl2, 20 to 01C, 95%.

In 2008, the same group reported the total synthesis of mucronine E (187) and of paulirine E (11). The new total synthesis of 187, as shown in Scheme 4, has been accomplished in eight steps and in 10% overall yield, and was achieved by the formation of the highly substituted aromatic core using an asymmetric hydrogenation–Vilsmeier formylation sequence. The macrocyclization step was accomplished using a coppercatalyzed coupling reaction (153). The total synthesis of 11, a 13-membered cyclopeptide alkaloid, is described in 14 steps and in 11% overall yield (173). The salient feature of

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OCH 3

OCH 3

OCH 3

a

H 3 COOC

(I)

OCH 3 NHBoc

OCH3 CHO d

O

H 3CO

H 3CO

OH

OCH3 Boc

CH 3

CH 3

CHO f

N NH2

O

O H 2N

N

N

OCH 3

Boc

e

O

c

O

NHBoc

NHBoc

OCH3

Boc

H 3 CO

COOCH3

(MeO) 2P

CHO

H 3CO

b

H 3 CO

O

H 3 CO

H 3C

HN

O O O

NH 2

N H

N H (II)

H 3CO

H 3CO

OCH 3

Boc I HN

O O O

NH2

N H

g

OCH 3

H

Boc N

H 3C

H 3CO

OCH 3

N

N H 3C HN

O O O N H

NH

h

H 3C HN

O O O

NH

N H Mucronine-E (187)

Scheme 4 Summary of the Evano syntheses of mucronine E (187). Reagents and conditions: (a) (I), then N,N,N,N-tetramethyl-guanidine, CH2Cl2, rt, 89%; (b) [Ph(COD){(S,S)-Et-DuPHOS}]+TfO, H2 (5 atm.), MEOH, 251C, 95%, W95% ee; (c) NaH, MeI, DMF, rt, quant.; (d) POCl3, DMF, 601C, then 3 M NaOH, rt, 68%; (e) Bop, DIPEA, DMF, 01C, then (II) 01C to rt, 47%; (f) PPh+3(CH2I)I, NaHMDS, HMPA, THF, 781C, 50%, W95 de; (g) Cu(I), N,N-dimethyl ethylenediamine, Cs2CO3, THF, 631C, 84%; (h) TMSOTf, 2,6-lutidine, CH2Cl2, 20 to 01C, 92%.

this synthetic approach includes a macrocyclization by isomerization/ ene-enamide ring-closing metathesis, as shown in Scheme 5.

VII. BIOSYNTHESIS Since no experiments with labeled precursors have been carried out, the biosynthetic pathways of the cyclopeptide alkaloids are not yet known. In 1993, Baig et al. (154) reported the first establishment of callus cell tissue cultures of Ceanothus americanus (Rhamnaceae), and a study of their secondary metabolism. They used a mixture of valine, leucine, isoleucine, tyrosine, phenylalanine, and tryptophan, and isolated the novel tetrapeptides Val-Leu-Leu-Tyr, Ile-Leu-Leu-Tyr, Phe-Phe-Leu-Tyr, and Phe-Phe-Ile-Tyr, the probable precursors of the cyclopeptide

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OCH3

OCH 3 OH

OCH 3 a

+ N Boc

OTBS

O

O b

I N Boc

c

O d

N Boc

OTBS

e O

OH

H 2N

N H (I)

OCH3

OCH 3 O

O O O N HN Boc

NH

f

OCH3 O O

N HN Mes N N Mes Boc Cl Ru Ph Cl PCy3 (GII)

NH

g

O

h

O O N

NH HN O

N CH 3 H 3C Paulirine-E (11)

Scheme 5 Summary of the Evano syntheses of paulirine E (11). Reagents and conditions: (a) Cu(I) (10%), 1,10-phenanthroline (20%), Cs2CO3, toluene, 1101C, 79%; (b) TBAF, THF, 201C to rt, 89%; (c) DMSO, (COCl)2, Et3N, CH2Cl2, 78 to 01C; (d) NaClO2, NaH2PO4, 2-methylbut-2-ene, t-BuOH/THF/H2O, rt, 86% (two steps); (e) (I) from (Boc-L-Ile-OH and steps of the isobutyl chloroformate, Nmethylmorpholine, DME, 01C, then allylamine, quant.; ZnBr2 (4.5 equiv), CH2Cl2, 01C to rt, EDC, HOBt, N-methylmorpholine, DMF, 01C to rt, 92%; (f) 1 equiv Grubbs’ second-generation catalyst (GII) (2 10%), 1,2-dichloroethane (0.005 M), reflux, 36% isolated yield; (g) TMSOTf, 2,6-lutidine, CH2Cl2, 10 to 01C; (h) N,N-dimethyl-Lphenylalanine, HATU, HOAt, DIPEA, DMF, 01C to rt, 78% (two steps).

alkaloids americine (65), frangulanine (82), ceanothine E (118), and adouetine Y (111), respectively (Figure 3). As expected, tyrosine is the basic amino acid used as a building block in the biosynthesis of the styrylamine unit in the 13- and 14-membered cyclopeptide alkaloids, while the biogenesis of 15-membered cyclopeptide alkaloids may involve a m-phenylenedialanine precursor or the corresponding dehydrocompound (Scheme 6) (2,6).

VIII. BIOLOGICAL ACTIVITY Plants from which cyclopeptide alkaloids were isolated have been used as traditional medicines in many parts of the world for centuries. For example, some Zizyphus species have been used traditionally for their sedative (177), antimicrobial (139,178), hypoglycemic (109,179), antiplasmodial (63), anti-infectious, antidiabetic, diuretic (180), analgesic and anticonvulsant (181), and anti-inflammatory (182) activities. This class of alkaloids requires special consideration due to the several different

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HO

O

R1 HN

HN O

O R1

O O

NH

HN HN R2

R3

R2

O

OH

O

NH

R3

N CH3 R4 R1

R2

R3

R4

Alkaloids

CH(CH3)2

CH (CH3)2

CHCH2C8H6N

H

Americine (65)

CH(CH3)2

CH(CH3)CH2CH3

CHCH2(CH3)2

CH3

Frangulanine (82)

CHC6H5

CH2C6H5

CH(CH3)CH2CH3

CH3

Adouetine Y (111)

CHC6H5

CH2C6H5

CHCH2(CH3)2

CH3

Ceanothine E (118)

Figure 3 The Baig proposal for the biogenetic formation of precursors of cyclopeptide alkaloids.

O

HO R1

O HN R2

O

O H

NH

O R1

R3

N HN R1

NH

O

N HN R1

NH

NH

R3

OR 3 O O O

O H

NH

N HN R1

R2

R2

R5

O

H 2N

HO

R1

O HN HN R2

O O O

O H

O

H2 N

O

R2

H 3 CO R3 N R4

O H

OR 3 O

O O

NH

R3

OR3 HO

R1

O

O HN HN R2

HN

O

O

O

O

OH

N H

R2

O H

H3 CO R3 N R4

R5

H3 CO O

HN R1

O O O N H

NH O H

R3 N R4 HN

R2

R1

R5

O O O N H

Scheme 6 The biogenetic pathway for the macrocyclization of the 13-, 14-, and 15-membered cyclopeptide alkaloids.

NH R2

Cyclopeptide Alkaloids from Higher Plants

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biological activities exhibited by many of them, for example, insecticidal (133), sedative (183,184), antimicrobial (3,4,124), antiplasmodial (63), and immunostimulant (35) activities.

A. Antimicrobial Activity The antibacterial activity of the alkaloids adouetine Yu (63); scutianines B (96), C (97), D (98), E (99), F (110), and M (137); condaline A (119); and scutianene D (199) was evaluated by means of direct bioautography using a TLC bioassay (99,106) against the standard bacterial strains Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus (gram-positive), Klebsiella pneumoniae, Salmonella setubal, and Escherichia coli (gram-negative), and two yeasts, Saccharomyces cerevisiae and Candida albicans. From these experiments, the alkaloid with the widest spectrum of activity was 119, followed by 99. Alkaloid 98, a diastereoisomer of 99, showed only modest activity against M. luteus (25.0 mg), S. epidermidis (50.0 mg), and E. coli (50.0 mg), while 137, a diastereoisomer of 119, was inactive against all of the tested strains. Alkaloid 96 was only active against E. coli (12.5 mg), while 97, 110, and 199 were inactive against all strains tested. This finding suggested that the stereochemistry and the presence of the b-phenylserine residue in the structure of the alkaloid have an influence on the bioactivity. Furthermore, the importance of the N,N-dimethyl (or N-methyl) group in the structures was demonstrated by the total lack of activity of 199, when compared to the related alkaloid 98. None of the tested alkaloids showed antifungal activity against S. cerevisiae and C. albicans. Frangulanine, isolated from Melochia odorata, exhibited moderate activity against C. albicans (25 mg), C. neoformans (W50 mg), and S. cerevisiae (50 mg), by the direct bioautographic method (185). The cyclopeptide alkaloid mauritine K (171), isolated from Z. mauritiana, exhibited significant antifungal activity against Botryitis cinerea (118). Zizyphine N (58) and P (60), isolated from Zizyphus oenoplia var. brunoniana (63), exhibited weak antituberculosis activity against Mycobacterium tuberculosis with the same MIC values of 200 mg/mL.

B. Antiplasmodial Activity In 2005, Suksamrarn et al. (63) found that the root extract of the Thai plant Z. oenoplia (L.) Mill. var. brunoniana (Cl. ex Brand) exhibited significant antiplasmodial activity against the parasite Plasmodium falciparum. From this extract, the same group (63) isolated four, new, 13-membered cyclopeptide alkaloids, the zizyphines N (58), O (59), P (60), and Q (61). All of the isolated alkaloids were tested in vitro against the parasite P. falciparum. Only 58 and 61 demonstrated significant antiplasmodial activity with the IC50 values of 3.92 and 3.5 mg/mL, respectively.

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C. Sedative Activity The cyclopeptide alkaloids known as the sanjoinines, isolated from various Zizyphus species, are the most investigated group of cyclopeptide alkaloids with respect to their sedative properties. Sanjoinine A (frangufoline) (81) and sanjoinine G2 (202) are reported to be effective inhibitors of calmodulin-induced activation of Ca2+ ATPase, while sanjoinine D was shown to act as an inhibitor of calmodulin-induced activation of phosphodiesterase (184). The inhibitory activity of the sanjoinine alkaloids on Ca2+ ATPase is in agreement with the popular use of plants of the genus Zizyphus in Traditional Chinese Medicine as a treatment for insomnia (21). In 1996, Han and coworkers (186) reported that frangufoline (sanjoinine A) was converted to the linear alkaloid sanjoinine G2 (202) under acidic conditions (2 M HCl, 551C, 10 h) (Scheme 7, pathway B). One year later, the same group reported that frangulanine was converted, via enzymatic process in rodents, to the tripeptide M1 {(S)-(N,N-dimethylphenylalanyl)-(2S,3S)-3-[(4-formylphenoxy)leucyl]-(S)-leucine} (Scheme 7, pathway A). On the basis of these

OH OH OH O

O O

O

A

HN

HN

O

NH

OH

O

O O

NH

HN

HN

O

N CH3

N CH3

N CH 3

H3 C

H 3C

OH

HN

HN

O

O

NH 2

O

H 3C

Frangufoline (81) B

O O

O

H O O HN

HN

O

NHCH 2OH

H O O

H3 C

N CH3 H 3C

OH

HN

HN

O

O

O N CH3

H O O

NH2

HN

HN

O O

N H 3C

CH 3 (M1)

Sanjoinine G2 (202)

Scheme 7 Summary of the proposed mechanism by Han and coworkers for the cleavage of frangufoline (81). Pathway A (enzymatic) and pathway B (2 M HCl, 551C, 10 h).

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results, Han and coworkers (183) suggested that frangufoline, once absorbed, rapidly undergoes metabolic conversion to M1. Nummularine H, isolated from Paliurus ramossisimus, could shorten, in mice, the methohexital-induced sleeping time (52). In 2005, the Han group (184) investigated the effects of the sedative cyclic and linear peptide alkaloids from Zizyphus species on calmodulin-dependent protein kinase II. Daechuinine S27 (IC50 2.95 mM), sanjoinine F (IC50 18.2 mM), and sanjoinine G2 (202) (IC50 19.0 mM) were the most active alkaloids. In 2008, Oh and coworkers (181) reported that the cyclopeptide alkaloid fraction of the dried seed of Zizyphus jujube Mill. var. spinosa enhanced hypnotic effects in pentobarbital-treated mice.

IX. CONCLUSIONS In the time that has elapsed since Itokowa and coworkers reviewed the cyclopeptide alkaloids in Volume 49 of this series, there have been a number of papers published on the chemistry and biology of cyclopeptide alkaloids. In the succeeding 10-year period covered in this review, significant progress has been made in all aspects of the study of this family of alkaloids. Many cyclopeptide alkaloids have been discovered, and much work has been reported on both known and new alkaloids, especially with regards to their relative and absolute stereochemistry and their biological activity. In addition, a number of synthetic approaches to the cyclopeptide alkaloid skeleton have been developed. The family Rhamnaceae continues to be a rich source of cyclopeptide alkaloids (ca. 185 isolated). The genus Zizyphus, with 19 species investigated thus far, has yielded the greatest number of cyclopeptide alkaloids (ca. 120 isolated), followed by Discaria with 6 species studied and 26 alkaloids isolated. In conclusion, significant contributions to the study of the isolation, properties, classification, structural determination, biological properties, and synthetic chemistry of cyclopeptide alkaloids continue to be made. On-going fundamental studies on the bioactivity, biological functions, stereochemistry, biosynthesis, and the development of new strategies of synthesis will have a positive impact on the chemistry and biology of this class of alkaloids.

ACKNOWLEDGMENTS The author thanks CNPq for grants. Professor Edmundo Alfreo Ruveda (IQUIOS, Rosario, Argentina) is also acknowledged for helpful discussions.

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CHAPT ER

3 Alkaloids Toxic to Livestock$ Russell J. Molyneux1,* and Kip E. Panter2

Contents

$

I. Introduction II. Pyrrolidine Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships and Mode of III. Piperidine Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships and Mode of IV. Bipiperidine Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships and Mode of V. Pyridine Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships and Mode of VI. Polyhydroxy Indolizidine Alkaloids and Related Pyrrolizidine and Nortropane Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships and Mode of VII. Quinolizidine Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships and Mode of

Action

Action

Action

Action

Action

Action

143 145 145 146 146 146 146 148 149 150 150 152 152 153 153 154 155 157 157 162 165 168 168 172 174

Dedicated to Dr. Lynn F. James in recognition of a career devoted to poisonous plant research with emphasis on the need to understand the chemistry of the toxins involved.

1

Western Regional Research Center, Agricultural Research Service, USDA, Albany, California, USA

2

Poisonous Plant Research Laboratory, Agricultural Research Service, USDA, Logan, Utah, USA

Corresponding author.

E-mail address: [email protected] (R.J. Molyneux). The Alkaloids, Volume 67 ISSN: 1099-4831, DOI 10.1016/S1099-4831(09)06703-0

r 2009 Elsevier Inc. All rights reserved

143

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VIII. Tropane Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships IX. Pyrrolizidine Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships X. Isoquinoline Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships XI. Diterpene (Larkspur) Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships XII. Diterpene (Yew) Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships XIII. Steroidal (Veratrum) Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships XIV. Steroidal (Zigadenus) Alkaloids A. Plant Species and Alkaloids B. Toxicity and Clinical Signs C. Structure–Activity Relationships XV. Conclusions and Outlook References

and Mode of Action

and Mode of Action

and Mode of Action

and Mode of Action

and Mode of Action

and Mode of Action

and Mode of Action

176 176 178 178 179 179 184 185 188 188 189 190 191 191 193 194 196 196 197 198 199 199 201 202 203 203 204 205 206 206

I. INTRODUCTION Poisonous plants are a problem for livestock producers around the globe. In developed countries they can have significant economic consequences for farmers and ranchers, while in subsistence cultures the impact can be devastating, leading to loss of income and even starvation. There are a number of excellent volumes addressing poisonous plant problems, especially those devoted to Australia (1), South Africa (2), and the United States (3). An older, but still valuable, volume on poisonous plants in the United States is that of Kingsbury (4). For other areas where livestock poisoning is likely to be a significant problem, such as South America, China, and central Asia, the information is more scattered, but an

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important source is the series of seven symposium volumes resulting from the periodic meetings of the International Society on Poisonous Plants (ISOPP) (5–11). In many cases, a situation occurring in one country may benefit from extrapolation of previous observations in another country through correspondence in signs and symptoms of poisoning, plant species, or classes of toxins. Early settlers in the United States found that their animals were exposed to plant species with which they were not familiar, but in general the grazing regimen was similar to that in Europe, with an abundance of nutritious forage, and poisoning was episodic in nature. Furthermore, the close relationship of animals with the human community meant that animals were carefully observed and correlation of cause and effect of problems was relatively easy. As the pioneers moved westward across the American continent they encountered an unfamiliar agricultural environment of increasingly arid landscapes populated by less forage and with diverse plant species that were foreign to them. Livestock were forced to graze over vast areas for adequate feed, and the close relationship to human communities was disrupted, with animals remaining unobserved for days or even weeks at a time. Moreover, annual climatic changes often resulted in enormous populations of a particular plant species in some years, which might not reappear for many years thereafter. As a result, large numbers of animals could be lost or injured by poisonous plants with the responsible species being difficult to identify because of the disjunction between time of consumption and discovery of affected animals. Losses were often so large, amounting to hundreds or even thousands of animals in a particular area, that the viability of communities was affected. This ultimately led to the involvement of the federal government, through the U.S. Department of Agriculture and the land grant universities, in efforts to establish the specific plants causing livestock poisonings and develop the means to limit such losses. Research over the last century has incriminated the majority of plant species involved, identified the toxins and their modes of action, and provided grazing management plans that have been remarkably successful. In many (if not most) of the cases the toxins have proved to be alkaloids. Much of this research has led to the discovery of new structural classes of alkaloids, or the discovery of known types in new plant genera. Furthermore, identification of their biological mode of action has provided potential leads for therapeutic drugs for treatment of human diseases (12,13). Because the subject matter of livestock poisoning is so vast on a global basis, this chapter is focused primarily on plant species of concern within the United States. In a number of cases these correspond to situations pertaining in other countries, such as Australia, and these will be discussed when appropriate. The discussion is restricted to

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poisonings resulting from animals grazing plants, and those poisonings caused by fungal toxins, as in spoiled silage, are not covered. However, it should be recognized that the alkaloids may be biosynthesized by endophytes within plants, and when this may be the case, it will be noted. Poisoning episodes due to contamination of feed by ornamental plants are not dealt with since they are idiosyncratic in nature. This chapter is organized by class of alkaloids, arranged as far as possible in order of increasing structural complexity. The occurrence and distribution of each class within plant families and genera is discussed, followed by the signs and symptoms of poisoning seen in different livestock species and the management methods adopted to prevent poisoning episodes. Features of the structure essential for toxicity and in vivo metabolic transformations required for toxicity or resulting in detoxification are dealt with. The mode of action, structure–activity relationships, and potential for therapeutic applications of particular alkaloids are also presented.

II. PYRROLIDINE ALKALOIDS A. Plant Species and Alkaloids The English bluebell (Hyacinthoides non-scripta; Scilla non-scripta) is widespread in woodlands of western Europe and has been introduced into North America as an ornamental, where it occasionally escapes from cultivation. 2R,5R-Dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) (1) and 2,5-dideoxy-2,5-imino-DL-glycero-D-manno-heptitol (homo-DMDP) (2, R ¼ H) are the major alkaloid constituents of this species and other members of the family Hyacinthaceae (14,15). Homo-DMDP-7-Oapioside (2, R ¼ apiosyl), homo-DMDP-7-O-b-D-xylopyranoside (2, R ¼ b-D-xylosyl), 1,4-dideoxy-1,4-imino-D-arabinitol (3), and 6-deoxy-6-C(2,5-dihydroxyhexyl)-DMDP (4) have also been identified. Monocyclic alkaloids of this structural type have been described as iminosugars because of structural affinities to aminosugars, but a more inclusive term is polyhydroxy alkaloids. The polyhydroxy pyrrolidine alkaloids, together with the analogous polyhydroxy piperidine alkaloids, have been extensively reviewed with particular emphasis on their glycosidase inhibitory properties (16–19). OH

HO HO

4

3

5

2

N H 1

OH

HO

R O

3

4 5

7

OH

6

2

N H 2

OH

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Alkaloids Toxic to Livestock

OH

HO 4

3

5

2

OH

HO OH

4

OH

N H

3

5

CH3

3

2

OH

N H

OH 4

B. Toxicity and Clinical Signs Grazing of bluebells by cattle in the UK has been reported to cause gastrointestinal problems and lethargy (20), but there have been no associated reports of livestock poisonings in the USA.

C. Structure–Activity Relationships and Mode of Action The gastrointestinal symptoms are consistent with disruption of normal digestive processes through inhibition of glycosidases. DMDP (1) is a potent inhibitor of a- and b-glucosidase, b-mannosidase, invertase, and trehalase, while homo-DMDP (2, R ¼ H) inhibits b-glucosidase, b-galactosidase, and trehalase. The presence of such pyrrolidine glycosidase inhibitors in the plant is therefore circumstantial evidence for their involvement in the toxicity. Although H. non-scripta poisoning is not currently a concern in North America, the potential exists for this species, or other members of the Hyacinthaceae family, to cause sporadic problems. For this reason, and also because of their structural affinities and similar biochemical mode of action to the bicyclic polyhydroxy pyrrolizidine and indolizidine alkaloids, the pyrrolidine alkaloids are included in this compilation (Section VI) (16,17).

III. PIPERIDINE ALKALOIDS A. Plant Species and Alkaloids Poison hemlock (Conium maculatum) contains a number of piperidine alkaloids, with the predominant members being (R)-()-coniine (5, R ¼ H) and g-coniceine (6). Lesser amounts of five additional, structurally related alkaloids, N-methylconiine (5, R ¼ CH3), conhydrine (7), (2S,5S)-(+)-pseudoconhydrine (8, R ¼ H) and its N-methyl derivative (8, R ¼ CH3), and conhydrinone (9), also occur. The phytochemistry of the Conium alkaloids and their toxicity to livestock has been reviewed (21). It is important to distinguish poison hemlock from water hemlocks (Cicuta spp.). Both genera are members of the family Umbelliferae (Apiaceae), resemble each other in appearance, grow in similar moist

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habitats, and are poisonous to livestock. However, the exceptional toxicity of Cicuta spp., causing violent convulsions and rapid death due to respiratory failure, is due to cicutoxin, a long-chain acetylenic alcohol, and not due to alkaloids (22).

H N

CH3

N

CH3

R 5

6

HO H CH3

N H

OH 7

H

H CH3

N R 8

CH3

N H

O

9

C. maculatum is a native of Europe and western Asia, but has become naturalized in North America, South America, Australia, and New Zealand. Livestock and human poisonings by this plant have been reported to a greater or lesser extent in all of these countries, but consistent documentation as a serious problem in animals has come mainly from the United Kingdom and the United States. Alkaloid levels and relative content vary with a number of factors, including plant part, stage of growth, environment, and geographic locations. In vegetative growth the alkaloid content is almost exclusively g-coniceine (6), but as the flowers and fruit develop the level of coniine (5, R ¼ H) increases, and the overall alkaloid content is comprised of an increasingly diverse mixture. In ripe fruits, coniine predominates, and only trace amounts of g-coniceine are found. This progression is in accord with the established biosynthetic pathway, with g-coniceine being initially formed and serving as a branch-point for the formation of coniine and the conhydrines (23). A facile reversible interconversion between g-coniceine and coniine exists, the forward reaction catalyzed by an NADPHdependent g-coniceine reductase (24,25). Conium alkaloids are moderately volatile and sun drying of the fresh plant results in significant decline in alkaloid content. Fresh plant contained 0.4% total alkaloids, whereas the dried plant contained only 0.03%, with a corresponding decline in toxicity (26).

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Piperidine alkaloids also occur in Lobelia berlandieri (Campanulaceae), the only member of the Lobelia genus that causes problems in livestock, although other species have poisoned humans, primarily through their use as herbal remedies. Three D3-piperideines (10–12) were isolated and identified, and an additional three minor constituents were detected by TLC; there was no evidence of the presence of lobeline (13), a piperidine alkaloid commonly found in other Lobelia species (27). Total alkaloid content was estimated as 0.17% of dry weight (d.w.) of the whole plant, with piperideines 10 and 12 accounting for about 90% of the total alkaloid.

OH CH3

OH H

CH3

N

CH3

N

CH3

CH3

CH3

10

11

O

OH CH3

OH

OH H

H

HO

H

H N

CH3

C6H5

H

O H

H N

CH3

CH3

12

13

C6H5

The piperidine alkaloids class, including Conium alkaloids, has been reviewed a number of times, generally in conjunction with the pyridine alkaloids (28).

B. Toxicity and Clinical Signs Consumption of fresh C. maculatum, or its seeds, causes acute toxicity of many domestic animals and chronic teratogenic effects. Clinical signs of poisoning are similar, regardless of which animal species consumes the plant, including humans (29,30). Initially, the signs are general stimulation, which includes uneasiness, frequent urination and defecation without diarrhea, rapid pulse, temporarily impaired vision from the nictitating membrane covering the eyes (pigs, sheep, and cows), muscular weakness, muscle fasciculations, ataxia, incoordination

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followed by general depression, recumbency, collapse, and death from respiratory failure if the dose is high enough. Lo. berlandieri has been the cause of sporadic but quite heavy losses of cattle, and to a lesser extent sheep and goats, in Texas and northern Mexico (31). Livestock poisoning from lobelia occurs after ingestion of 0.6–2.2% of body weight (b.w.) or lesser amounts over several days, usually in the late winter or early spring. Clinical signs generally begin with diarrhea, nasal discharge, and general depression, followed by loss of appetite, dyspnea, dilated pupils, ataxia, coma, and death from respiratory failure. If death does not occur, clinical signs may worsen over the course of a few days and elevated liver enzymes may be detected.

C. Structure–Activity Relationships and Mode of Action Comparative pharmacological studies of the primary Conium alkaloids have been conducted on laboratory animals. In mice, the oral LD50 values for g-coniceine, coniine, and N-methylconiine were 12, 100, and 204.5 mg/kg b.w., respectively (32). In cows, a maximum nonlethal single oral dose of coniine, producing acute effects, was 3.3 mg/kg b.w. (33). Because of the difficulty of obtaining sufficient pure alkaloid for oral dosing in large animals, their toxicity in livestock has been extrapolated primarily from feeding experiments with plant material in which the alkaloid content and composition was known. The lethal dose of g-coniceine, measured as 98% of total alkaloid content in fresh C. maculatum, was found to be 1.8, 21, and 39 g/kg b.w. in sows, cows, and sheep, respectively (26,29,34). Although there are similarities in the effects of the alkaloids on different animal species, relative toxicities vary markedly, particularly between ruminants and monogastric animals. However, it should be noted that the interconversion of g-coniceine and coniine could be very facile in the digestive tract. Reduction of g-coniceine to coniine may occur, especially in ruminants, whereas oxidative processes in the liver may result in the reverse transformation. It is therefore impossible to ascertain whether the measured oral doses can be attributed to the individual alkaloids or to a mixture thereof. In the latter case, there could be differences in the relative proportions between animal species, and possibly also between individual animals in a given species. N-Methylconiine (5, R ¼ CH3) is considerably less toxic than the other two primary alkaloids. The N-methyl group blocks any possibility of oxidation to a dehydro derivative, structurally analogous to g-coniceine (6), suggesting that the latter could be the ultimate toxin responsible for acute toxicity. Available quantities of the minor alkaloids have been insufficient to establish their relative oral toxicities.

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Chronic teratogenic toxicity of the piperidine alkaloids is less easily assessed than acute toxicity. Skeletal malformations differ in type, between animal species, and with time of insult, but within the first trimester of the gestational period (26,29,35,36). In general, cows, pigs, and goats appear to be more sensitive than sheep and horses. Teratogenesis has been studied only with pure coniine, or with whole plant or seeds. In one investigation with fresh C. maculatum, in which g-coniceine was the predominant alkaloid (98%), it was teratogenic in cattle, but another plant sample from which coniine was absent, but that contained 20% g-coniceine and 80% other alkaloids, caused no malformations in pigs even though it was toxic (36). Experiments in ewes with a number of natural and synthetic piperidine alkaloid analogs have permitted specific structural requirements for teratogenicity to be postulated (26). These include a propyl side chain, or larger, with partial unsaturation in the ring (e.g., g-coniceine, 6) conferring increased toxicity; however, complete aromatization as in 2-propylpyridine results in loss of activity. There is no information on the specific or relative toxicities of the individual D3-piperideine alkaloids (10–12) occurring in Lo. berlandieri. However, the symptoms are similar to those produced by Lobelia species that contain lobeline (13), and the mode of action may be the same. Lobeline is a potent stimulant of the nicotinic ganglionic receptor with atropinic activity. In rats it has similar, but lesser, effects, relative to those of nicotine, on reduction of heart rate and blood pressure, as well as on the central nervous system (37,38).

IV. BIPIPERIDINE ALKALOIDS A. Plant Species and Alkaloids Ammodendrine (14, R ¼ H), N-methylammodendrine (14, R ¼ CH3), and N-acetylhystrine (15) are widely distributed in various genera of the Fabaceae family, including Ammodendron, Baptisia, Lupinus, and Thermopsis. It is tempting to include them as members of the simple piperidine alkaloid class, but there are also obvious structural similarities to the pyridine alkaloids anabasine and anabaseine, which occur primarily in Nicotiana species (Section V), and they have also been found in genera of the families Chenopodiaceae, Fabaceae, and Berberidaceae. Furthermore, ammodendrine and its relatives co-occur with quinolizidine alkaloids (Section VI), making it difficult to classify them from a chemotaxonomic point of view. For the purpose of this review, they have been placed in a separate bipiperidine alkaloid class, primarily because of structural features that are intermediate between the piperidine and

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pyridine classes and effects on livestock that are distinctly different from those of the latter groups. An even more complex bipiperidyl alkaloid is gramodendrine (16), an indole derivative of ammodendrine, found in Lu. arbustus ssp. calcaratus (39,40).

N

N

N

H

H R

N

N

N

COCH3

COCH3

COCH3 N H

14

15

16

Disparate optical rotation values have been reported for ammodendrine and N-methylammodendrine in various Lupinus species, and within samples of the same species from different sites and years of collection. This suggests that the alkaloids occur as enantiomeric mixtures, with the proportions varying with environmental conditions. Separation of diastereomeric Fmoc-L-Ala derivatives of ammodendrine by preparative HPLC enabled the enantiomers to be separated (41). D- and L-Ammodendrine had [a]D values of +5.41 and 5.71, respectively, and values for the corresponding synthetically prepared D- and L-Nmethylammodendrines were +62.41 and 59.01. Optical rotation data were not reported for gramodendrine, while N-acetylhystrine was reported to lack a chiral center, and the enantiomeric issue is not a concern for this alkaloid. Although the presence of quinolizidine alkaloids (Section VII) is a fairly consistent feature of toxic Lupinus species, ammodendrine is much more sporadic in occurrence. Species of particular concern in which ammodendrine and its analogs are found are Lu. arbustus, Lu. formosus, Lu. sericeus, and Lu. sulphureus. However, occurrence may vary with location; for example, Lu. sulphureus from Ritzville, WA, contained exclusively ammodendrine, whereas the same species collected from Pendleton, OR, had only the quinolizidine alkaloids lupanine, 5,6dehydrolupanine, and anagyrine (42). A similar situation pertained with respect to two different collections of Lu. formosus from California, with ammodendrine and N-acetylhystrine dominating in both, but with one also containing quinolizidine alkaloids, whereas they were absent from the other sample (41). In Lu. arbustus, ammodendrine predominates, with only trace amounts of N-methylammodendrine and no N-acetylhystrine (43).

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B. Toxicity and Clinical Signs The toxicity of lupines to livestock and the relationship to the phenomenon of crooked calf disease are inextricably intertwined with the presence of both bipiperidine and quinolizidine alkaloids, therefore, discussion of this problem is dealt with under the latter class of alkaloids.

C. Structure–Activity Relationships and Mode of Action The pharmacological activity of bipiperidyl alkaloids has been scarcely investigated. In mice, at an interperitoneal dose of 300 mg/kg, ammodendrine (14, R ¼ H) and gramodendrine (16) were approximately equipotent in reducing motor coordination and spontaneous motor activity to approximately 30 and 50% of control levels, respectively (40). However, gramodendrine is relatively unstable on quaternization (39) and may be metabolized to ammodendrine in vivo. The toxicities of the enantiomers of ammodendrine and N-methylammodendrine have been compared. The LD50 values of D- and L-ammodendrine were statistically different at 94.1 and 115.0 mg/kg, respectively, whereas the corresponding values for D- and L-N-methylammodendrine were 56.3 and 63.4 mg/kg, respectively (41). Both enantiomers of N-methylammodendrine were therefore appreciably more toxic than the ammodendrine enantiomers. However, in Lu. formosus, the N-methyl compound was a relatively minor component compared to ammodendrine and N-acetylhystrine. Although there was statistical significance in the toxicity determined for D- and L-ammodendrine, the physiological effects were not large and possibly the toxicity receptor site is not particularly sensitive to chiral differences. Alternatively, the enantiomers may be metabolized into compounds in which the asymmetric center is lost, either by isomerization of the existing double bond to a position between the two piperidine rings or by oxidative processes in the liver resulting in the introduction of an additional double bond in the fully saturated piperidine ring to a structure analogous to N-acetylhystrine. Although the acute mouse toxicity model cannot be extrapolated directly to teratogenicity, the results indicate that differential activity of enantiomers should always be evaluated in predicting toxicity of plant samples to livestock. The co-occurrence of bipiperidine alkaloids with quinolizidine alkaloids in Lupinus species makes it difficult to assign specific modes of action to each individual class. However, feeding experiments with species in which any one of the two alkaloid types is essentially absent suggest that ammodendrine and its congeners act through a sedative effect on the fetus, with no overt signs of toxicity in the dam. Inhibited

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fetal movement can result in limb deformities due to contraction of the musculature or cleft palate when the tongue prevents palate closure.

V. PYRIDINE ALKALOIDS A. Plant Species and Alkaloids Although the piperidine and pyridine alkaloids are frequently considered as a common structural class, for the purpose of this review they are separated because of the different plant genera in which they are found and the type of toxicity. The Nicotiana genus (Solanaceae) is particularly common in North America, with a number of species found throughout the western and southern states, and in Mexico, and has frequently been responsible for episodes of livestock poisoning. The species most frequently incriminated in such situations are N. tabacum (tobacco, burley tobacco) and N. glauca (tree tobacco). Although the former is native to North America, it is now cultivated worldwide, either commercially or for personal use of the leaves in cigarettes, cigars, and snuff. Apart from grazing of the plant itself, the large quantities of accumulated plant residues, such as the stalks, can cause poisoning problems when animals gain access to them. The primary alkaloids in Nicotiana species are S-()-nicotine (17, R ¼ CH3), S-()-nornicotine (17, R ¼ H), and ()-anabasine (18). Several minor alkaloids have been isolated from Nicotiana, including S-()anatabine (19), anabaseine (20), formyl- and acetyl-nornicotine, and the diastereomeric N-oxides of nicotine. Other related alkaloids found in processed tobacco may be artifacts of flue-curing. In most Nicotiana species, the predominant alkaloid is nicotine, amounting to as much as 95% of the total. Nicotine content can vary highly with environment, with typical alkaloid levels for most tobacco products and Nicotiana species being 1–3% d.w. (44,45). N. tabacum varieties are also bred for either low or high levels, depending on the preferences of tobacco manufacturers. However, most analytical methods are not designed to analyze for nicotine N-oxides, which are highly water soluble and involatile, so that the overall nicotine content may be underestimated.

H

H N H

N R

N 17

N 18

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155

H N

N H N

N 19

20

In contrast to cultivated tobacco, the alkaloids in N. glauca consist almost exclusively (B99%) of anabasine (18), with a level in the bark/ cambium layer of W0.9% (46). As is apparent from its name, anabasine was first isolated from Anabasis aphylla (Chenopodiaceae) and has since been identified in many other species across a range of plant families. Anabasine occurs in N. glauca as a mixture of enantiomers, which have been separated by conversion to their Fmoc-L-Ala– diastereomers and semipreparative reversed-phase HPLC, followed by Edman degradation to the individual enantiomers (47). The isolated (S)- and (R)-anabasine enantiomers were obtained in sufficient quantities, and pure enough with respect to the complementary isomer [80% (S)- and 72% (R)-, respectively], to be used in a mouse toxicity bioassay and for pharmacological evaluation as neuromuscular nicotinic receptor (nAChR) agonists. Anabaseine (1u,2u-dehydroanabasine) (20) is a minor alkaloid of Nicotiana species and is highly unstable due to rapid ring-chain hydrolysis of the cyclic iminium ion to the keto-alkylammonium ion form (48) (Figure 1); it is more commonly found in nemertines, carnivorous marine worms that use anabaseine and other toxins as chemical defenses against predators (49).

B. Toxicity and Clinical Signs Tobaccos have been intensively studied chemically and, of the over 40 alkaloids characterized, most fall into the pyridine class, including the most studied of all, nicotine (17, R ¼ CH3). These alkaloids are all neurotoxins, and poisoned livestock, after ingesting Nicotiana tabacum,

N

+ N

H+

O

H N

N

N

20

Figure 1 Hydrolysis of cyclic iminium ion form of anabaseine (20) to keto-alkylammonium ion form.

NH3+

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N. trigonophylla, N. attenuata, or N. glauca, exhibit a variety of neurological clinical effects including muscular tremors, ataxia, muscular weakness, collapse, and recumbency, and eventually death with higher doses. Poisoning in horses from N. tabacum has occurred when horses were kept in tobacco barns, and in pigs when tobacco stalks were fed to sows following removal of the leaves for curing. Skeletal malformations and cleft palate defects were recorded in more than 300 pigs from 64 litters on five Kentucky farms where sows consumed the stalks (50,51). It was eventually determined that when pregnant sows ingested these tobacco stalks during early gestation they would deliver piglets with defects similar to those induced in sheep, goats, pigs, and cattle with poison hemlock. However, in pigs, sheep, and cattle, nicotine alone did not induce deformities and anabasine was considered a more likely candidate because of structural features consistent with teratogenicity of coniine and its analogs. N. glauca, in which W99% of the total alkaloid was anabasine, induced deformed offspring in experiments in both cows and sows (52). Isolation of a large quantity of anabasine in sufficient purity from N. glauca permitted testing for teratogenicity in pigs, and a racemic mixture of the two enantiomers showed that it was teratogenic, inducing fixed dorsal flexure of digits or carpal joints. Typical arthrogrypotic defects were induced in 21 of 26 offspring when sows ingested 2.6 mg/kg b.w. of the compound (46). These defects were indistinguishable clinically from defects induced by either N. glauca or N. tabacum. Differences in terata expression between N. glauca and N. tabacum are probably due to presence of related piperidine alkaloids such as anatabine (19) in the latter, in which it occurs at 5–15 times the level of anabasine.

C. Structure–Activity Relationships and Mode of Action Due to its presence in tobacco, the pharmacological properties of nicotine and co-occurring alkaloids have been exceptionally well studied (53). Nicotine exhibits complex activity towards a number of systems, resulting in a significant decrease in blood pressure and respiration and heart rates, while stimulating the central nervous system. It has agonistic activity towards nicotinic acetylcholine receptors (nAChRs), and is a rapidly acting ganglionic depolarizer. At low doses it stimulates depolarization of postsynaptic membranes of ganglia and at high doses causes blockade. As a result, early signs of toxicity are excitatory, followed later by depressant effects. Death is usually due to respiratory failure and cardiac arrest, but nicotine is rapidly excreted in the urine and its acute effects are of short duration. There are no antidotes for its effects on nicotinic receptors, so treatment must be palliative, but survival for a period of 12 h or longer generally leads to a favorable

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outcome. Nornicotine, the N-demethylated analog of nicotine, has effects similar to nicotine itself, desensitizing common nicotinic receptor subtypes, but having approximately 12-fold less activity (54). The alkaloid, which is a constituent of N. tabacum, and a mammalian metabolite of nicotine itself, must therefore be considered as pharmacologically relevant to the toxicity of Nicotiana species. The in vivo activity of (S)-nicotine has been compared with that of anabaseine in a rat prostration response bioassay, and the two alkaloids were found to be approximately equipotent on a micromolar basis, expressed in terms of the cyclic iminium ion form of anabaseine (55). Subsequent evaluation of these two alkaloids in a mouse toxicity bioassay, together with the substantially purified (S)- and (R)-anabasine enantiomers and synthetic (R)-nicotine, showed LD50 values in the order: (S)-nicotine (0.38 mg/kg)Wanabaseine (0.58 mg/kg)c(R)-nicotine (2.8 mg/kg)W(R)-anabasine (11 mg/kg)W(S)-anabasine (16 mg/kg) (47). The higher toxicity of anabaseine relative to anabasine is analogous to that of coniceine and coniine, with unsaturation between the 1 and 2 positions of the piperidine ring leading to enhanced toxicity and teratogenicity (56,57). The receptor binding properties of naturally occurring alkaloids have also been studied in a number of vertebrate and invertebrate cell lines, including human fetal muscle, rat a7, and rat a4b2 nAChRs (47,49,55). These results, considered in conjunction with their functional properties in the mouse bioassay suggest that the high toxicity of (S)-nicotine is due to its central convulsant action. In contrast, anabaseine and (S)-anabasine are potent agonists of the a7 neuronal receptors, but have little or no stimulatory activity on the a4b2 neuronal receptors through which nicotine is considered to produce convulsions and death. The EC50 values for activation of the fetal skeletal muscle nicotinic receptor, namely, anabaseine (0.42 mM)c(R)-anabasine (2.6 mM)W(S)-anabasine (7.1 mM), is of the same order as their relative toxicities in the mouse bioassay, and much lower than that of (S)-nicotine (26 mM). The actions of anabaseine and the anabasine enantiomers are therefore probably due to peripheral effects on skeletal muscle stimulation and subsequent block; conversely, nicotine displays low stimulatory potency in stimulating skeletal muscle-type receptors. This is consistent with feeding trials in which anabasine proved to be the teratogen, whereas nicotine is a direct-acting toxin. If the teratogenicity of the three alkaloids is due to the common mechanism of induction of sustained contracture of the skeletal muscles of the neck and back of the embryo, their different pharmacokinetic properties imply that the relative proportions of each alkaloid in the plant need to be established in order to predict the potential teratogenicity of any plant population.

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VI. POLYHYDROXY INDOLIZIDINE ALKALOIDS AND RELATED PYRROLIZIDINE AND NORTROPANE ALKALOIDS A. Plant Species and Alkaloids Poisoning of animals by certain members of the genera Astragalus and Oxytropis (Fabaceae) has been a long-standing problem in North America, preoccupying research scientists since the earliest days of poisonous plant investigations, with descriptions dating from the 1870s. These particular plants were called ‘‘locoweeds,’’ from the Spanish word ‘‘loco’’ (crazy), because of the remarkable neurological signs induced by their consumption, and distinguishes them from the common term ‘‘milk-vetches’’ applied to all Astragalus and Oxytropis species. A monograph published in 1909 (58) identified those species most commonly associated with toxicity, defined the relative susceptibility of various livestock species, and described symptoms of poisoning, pathological lesions, conditions under which poisoning occurs, possible prevention techniques, and remedies. The suggestion was also made that the toxin was a water-soluble alkaloid, but the specific compound remained unidentified for another 70 years. The situation was also confused by an acute toxicity caused by the presence of nitro-toxins in some Astragalus and Oxytropis species. It was recognized early in the twentieth century that locoweed poisoning in the United States was remarkably similar to the ‘‘peastruck’’ condition in Australia that was caused by sheep feeding on legumes of the genus Swainsona. However, it was not until 1979 that Colegate et al. (59) identified the toxin as a trihydroxy indolizidine alkaloid, swainsonine (21). Subsequently, swainsonine was isolated from Astragalus lentiginosus, together with its N-oxide derivative (60), and was then detected in other Astragalus and Oxytropis species, as well as some from South America and Asia (61,62). In addition, two minor dihydroxy indolizidine alkaloids, lentiginosine (22) and 2-epi-lentiginosine (23), were isolated and identified in A. lentiginosus (63). It is noteworthy that swainsonine has an 8a-R bridgehead configuration, whereas the lentiginosines have the opposite configuration at this position. OH H

OH

H OH

OH

H OH

OH

OH

N

N

N

21

22

23

Initially, swainsonine appeared to be characteristic of a few genera of the family Fabaceae, but it was soon identified in two unrelated

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microorganisms, Rhizoctonia leguminicola (64) and Metarhizium anisopliae (65). R. leguminicola is a pathogen of red clover (Trifolium pratense), causing excessive salivation (slobbers disease) in animals consuming the infected plant, due to the presence of the parasympathomimetic indolizidine alkaloid, slaframine (24) (66). More recently, swainsonine has been shown to co-occur with polyhydroxy nortropane alkaloids, named calystegines (vide infra), in Ipomoea species (Convolvulaceae) (67–71) and the Brazilian plant species, Turbina cordata (syn. Ipomoea martii) (72), as well as Sida carpinifolia (Malvaceae) (73). The chemotaxonomic significance of swainsonine is therefore problematic, given its widespread occurrence among species from several different, widely distributed plant families, together with its isolation from both plants and microorganisms. Furthermore, its biosynthesis has been shown to proceed by an identical pathway in both Diablo locoweed (Astragalus oxyphysus) and R. leguminicola (74).

H

OCOCH3

N H2N 24

Recent studies on the occurrence of swainsonine in white locoweed (Oxytropis sericea) have shown that it occurs in some populations, whereas others are completely devoid of the alkaloid (75). This led to the isolation of an endophyte from the locoweeds O. sericea, O. lambertii, and Astragalus mollissimus containing the alkaloid, but not from those in which swainsonine was absent (76). The fungus, subsequently identified as an Embellisia species, produced significant quantities of swainsonine when cultured in vitro. A comparative feeding study of rats fed either the fungus or a locoweed (O. lambertii) showed identical symptoms, indicating that the plant constituents are not required for toxicity (77). The major locoweed species A. lentiginosus, A. mollissimus, A. pubentissimus, A. wootoni, and O. sericea were examined, and the endophyte was shown to be present in each of them (78). An interesting question therefore arises as to the precise nature of the endophyte(s) responsible for production of the alkaloid in swainsonine-containing plant species from diverse genera and in many parts of the world, other than North America. It is unlikely that the same endophyte occurs in areas such as Australia, South America, and Asia, suggesting that there are probably several endophytic fungal species capable of swainsonine biosynthesis.

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The involvement of an endophyte explains the variability and relatively low swainsonine content of locoweeds. The alkaloid is found in all aboveground parts of the plant, and is more concentrated in the flowers and seeds, generally not exceeding 0.2% of the dry weight of the plant. However, it has been calculated that levels greater than 0.001% can cause poisoning if the plant is consumed over a sufficient period of time because of its potent bioactivity (79). The alkaloid is retained in dead plants for as long as 2 years at a sufficient level to cause locoism (80). This may be either because the alkaloid is remarkably stable or because the endophyte is still metabolically active. OH H HO 7

8

OH

OH

H

OH

HO 8a

6

6

N

HO

N

HO 25

OH H

26

OH

OH

H

OH

HO 7 6

7 6

N

HO

N

HO 27

28

Swainsonine and the lentiginosines are the only indolizidine alkaloids of concern for livestock poisoning in North America. However, the seeds of Black Bean or Moreton Bay Chestnut (Castanospermum australe, Fabaceae), a monotypic rainforest tree, contain relatively large amounts (ca. 0.3%) of the structurally analogous alkaloid, castanospermine (25) (81), together with lesser amounts of the epimers 6-epi-castanospermine (26), 7-deoxy-6-epi-castanospermine (27), and 6,7-di-epi-castanospermine (28) (82–84). It should be noted that all of the castanospermines have an S configuration at the bridgehead 8a-position, in contrast to swainsonine that has the 8a-R configuration. Two polyhydroxy pyrrolizidine alkaloids, isomeric with castanospermine, have also been isolated in minor amounts and named australine (29) (85) and 1-epi-australine (30) (86), and a rigorous re-examination of Ca. australe seeds has led to the isolation and identification of 2,3-di-epi-australine (31), 2,3,7-tri-epiaustraline (32), and 1-epi-australine 2-O-b-D-glucopyranoside (33) (87).

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In Australia, the large leguminous seeds of Ca. australe litter the ground beneath the trees and have proved toxic to livestock, especially cattle and horses, and occasionally even humans. The tree has been introduced into warmer parts of North America only as a horticultural specimen, and is not so far a threat to animals. However, the identification of the bicyclic castanospermines and australines, differing only in ring size or stereochemistry, has provided a series of alkaloids of considerable value in establishing structure–activity relationships. This possibility has been enhanced by the addition of the tetra- and penta-hydroxy pyrrolizidines, hyacinthacines B1 (34) and C1 (35), respectively, from H. non-scripta (Hyacinthaceae) (15). As weak inhibitors of b-glucosidase and b-galactosidase, the latter alkaloids may contribute somewhat to the gastrointestinal problems caused by bluebells, in a manner analogous to that of the polyhydroxy pyrrolidines (Section II). HO

OH

H

HO

OH

H

1

7 7a

1 2

N

OH

OH

N

3

OH

OH

29

HO

30

OH

H

HO

OH

H

HO

H

1

7 2

N

2

OH N

3

OH

OH

OH

32

33

OH

HO

OH

OH

OH N

OH 34

H

HO

N

HO

OβGlu

N

3

OH

31

H

OH

H3C

OH 35

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A relatively new group of alkaloids with structural affinities to polyhydroxy pyrrolizidines and indolizidines is the polyhydroxy nortropanes, otherwise named calystegines, from the bindweed Calystegia sepium (Convolvulaceae), the source of the first member to be isolated (88,89). The nortropane ring system can be conceptualized as a result of the fusion of a five-membered pyrrolidine ring with a six-membered piperidine ring, but in contrast to the indolizidines the fusion points are a to the nitrogen atom of each monocyclic system. Tropane alkaloids have a long history as plant toxins (Section VIII), but the relationship of nortropanes, in which the nitrogen atom is not methylated, to livestock poisonings has only just begun to be studied. A consistent feature of all calystegines is an aminoketal functionality due to the presence of an a-OH group at the bridgehead juncture (C1) of the bicyclic ring system. Three subclasses have been defined, namely, calystegines A, B, and C, corresponding to tri-, tetra-, and penta-hydroxylation, respectively, with the most commonly found within each of these groups being calystegines A3 (36), B1 (37) and B2 (38, R ¼ H), and C1 (39, R ¼ H). N-Methylcalystegine B2 (38, R ¼ CH3) and N-methylcalystegine C1 (39, R ¼ CH3) could be classified as tropane alkaloids, but their co-occurrence with polyhydroxy nortropanes suggests that these alkaloids result from N-methylation of the latter rather than from the normal biosynthetic route to tropane alkaloids. A review of the calystegines, with particular emphasis on their biosynthesis, has recently been published (90). H N 1 HO

5

4 2

6 7

OH

3

OH

H N HO HO

OH

37

36

R N HO OH

38

OH

OH OH

R N HO HO

OH

OH OH

39

Calystegines were initially found in Calystegia species of the family Convolvulaceae, but have now been found in 14 additional genera of this plant family (91), especially Ipomoea species (67–69,92). They also occur to some extent in the families Moraceae and Erythroxylaceae, and are quite

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widely distributed in the families Solanaceae and Brassicaceae (93–95). The discovery of a swainsonine-producing endophyte in certain locoweeds raises the question as to whether the calystegines are also endophytic metabolites, as both classes of alkaloid can co-occur in Ipomoea species at somewhat similar levels. A calystegine-catabolizing strain of Rhizobium meliloti is found in roots of Calystegia sepium producing the alkaloids, but this is not present within plants that do not produce calystegines (96). While this suggests a strong plant–fungal or fungal–fungal symbiotic relationship, the diversity of calystegine structures and the structural similarities to the tropane alkaloids suggest that they are true phytochemicals.

B. Toxicity and Clinical Signs Locoweed poisoning, caused by species of Astragalus and Oxytropis containing the indolizidine alkaloid swainsonine, was one of the first recognized poisonous plant problems reported as settlers in the United States moved their livestock westward (58). Shortly after the Civil War, the range livestock industry boomed as cattle barons moved large numbers of cattle north and west from eastern and southern states, taking advantage of the vast grasslands of the western ranges. Sheep men soon followed with large bands and competition for the ranges escalated. Long before it was officially acknowledged in 1873, poisoning of livestock was recognized and large losses were associated with locoweeds. Over 350 species of Astragalus and over 20 species of Oxytropis have been identified. Not all contain the alkaloid swainsonine; for example, a few contain nitro-toxins, some are selenium indicator species and accumulate levels sufficient to be toxic, and other species may contain multiple toxins, including all three types. The clinical signs of locoweed poisoning are numerous, but the classical syndrome from which the term ‘‘loco’’ arose is one of neurological dysfunction. Locoweed poisoning is chronic, developing after weeks of ingesting plants containing swainsonine, and the classical signs are general depression, dull appearance, incoordination, tremors, and progressive weight loss, emaciation, staggering, and aberrant behavior, including proprioceptive deficits and aggressivness, ultimately ending in death (97). Other disorders associated with locoweed poisoning, often occurring before the more severe clinical signs, include reproductive failure, embryo loss, abortion, and occasional birth defects (98). Locoweed poisoning affects all grazing livestock species and wildlife, but because of the transient nature of the poisoning, animals removed from locoweed pastures before the onset of neurological signs will recover most of their productivity. However, with horses, once poisoning occurs it is not recommended to use them for work or riding,

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as even slight neurological damage can result in horses reacting to stress situations in violent ways. The locoweed disease results from swainsonine inhibition of key enzymes (mannosidases) in cellular glycoprotein metabolism (99), resulting in a buildup of abnormal proteins in cells of multiple organ systems and subsequent functional losses. In the nervous system, the brain is especially vulnerable and severely affected animals will develop irreversible neurological lesions (100), resulting in altered synaptic function and aberrant behavior. Microscopic lesions of locoweed poisoning are found in most tissues, but the vacuolation is most pronounced in brain, pancreas, liver, thyroid, and kidney. Neurovisceral vacuolation is the general description of these lesions, and is the result of cellular ‘‘constipation’’ by abnormal glycoprotein buildup and subsequent cell damage and death (101). Toxicity and neurological damage is especially severe in horses, followed in sensitivity by cattle and sheep. Goats are also very sensitive and exhibit neurological disease similar to that seen in horses (102). Deer are quite resistant, as are rats and mice (103). Gross lesions include right heart enlargement, hydrops in pregnant animals, embryonic loss/abortion, and generalized edema and ascites. While locoweed poisoning has been known for over 100 years, research information over the past 50 years has decreased the significance of locoweed poisoning in the western United States, and large episodic losses are less frequent. For many years, research evidence demonstrated that swainsonine level in certain populations was variable, as was the risk of poisoning, and that some populations did not contain swainsonine. Only recently was it discovered that an endophyte (Embellisia) is responsible for production of swainsonine in toxic species (76). This discovery explains much of the variability in toxicoses around the world. However, more research is needed to understand the ecology of the endophyte, and determine the significance of the endophyte in relation to competitiveness and survival of the plant. Other glycosidase inhibitors (lentiginosine, 22 and its 2-epimer, 23) have been found in Astragalus species containing swainsonine, and it remains to be established whether these are also produced by the endophyte, or are plant metabolites, subsequently biotransformed into swainsonine by the fungus. There are other plant species throughout the world that contain swainsonine and cause livestock poisoning similar to locoweed poisoning. Ipomoea spp. in Africa and Brazil (68,104) and Swainsona spp. in Australia (1,105) contain swainsonine and cause livestock losses in those regions. Research has yet to determine if Embellisia or another endophyte is responsible for swainsonine production in these plant species. Other indolizidine alkaloids have been implicated in livestock poisoning (81,106). Ingestion of pods and seeds from the Australian tree Ca. australe (Moreton Bay chestnut) by horses, cattle, and people has

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resulted in clinical signs of poisoning and discovery of another set of polyhydroxylated, hydrophilic indolizidine alkaloids that inhibit key metabolic enzymes. Clinical signs of poisoning are slightly different from those of swainsonine and include emaciation, scouring, dry coat, dyspnea, dull eyes, general depression, and incontinence. Inflammation of the stomach and intestinal tract are the predominant lesions, suggestive of inhibition of metabolic enzymes affecting different organ systems than swainsonine. These castanospermines are potent inhibitors of b-glucocerebrosidase and lysosomal a-glucosidase, key enzymes in glycoprotein processing. The presence of calystegines in the Solanaceae and Brassicaceae plant families, including important vegetable and fruit species, such as potatoes, eggplant, and cabbage, together with their occurrence in mulberries and sweet potato (Ipomoea batatas) raises questions as to the overall intake and relative safety of these alkaloids in the human diet (107). Furthermore, trimmings or waste from such vegetable crops may have adverse effects on animals such as pigs, if included in their feed. A study of the calystegine content and composition in potatoes (Solanum tuberosum) from the groups Phureja and Tuberosum showed that concentrations in the peel and sprouts were up to 13 and 100 times higher, respectively, than in the flesh (108). Since potato peels discarded from human consumption are often fed to animals, their potential for toxicity should be studied.

C. Structure–Activity Relationships and Mode of Action Swainsonine was first isolated from Swainsona species (59) through the recognition that the poisoning induced in livestock by these plants was biochemically, morphologically, and clinically similar to the genetic disease mannosidosis, a consequence of insufficiency or complete absence of the enzyme a-mannosidase. The hypothesis that the toxin was an inhibitor of this enzyme led to its use as a probe for such bioactivity and ultimately the separation and purification of the inhibitor, swainsonine. a-Mannosidase is essential for the proper functioning of all animal cells, trimming mannose units from complex glycoproteins so that the smaller molecules can be targeted for retention, incorporation into the cell wall structure, or release from the cell to perform the specific functions for which they have been synthesized (16). Disruption of this trimming sequence results in an accumulation of aberrant glycoproteins within the cell that cannot be appropriately targeted, leading to vacuolation, until sufficient cellular damage has occurred for signs of poisoning to appear. Because proper functioning of a-mannosidase is an essential requirement of cells, many different organs can be damaged, including the brain, heart, reproductive system, and digestive system,

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although the neurological defects are the most obvious. Specific organs affected, and signs of poisoning, depend on the amount of swainsonine ingested, the length of exposure, and factors such as nutritional status, pregnancy, and environment (109). The gross structure of swainsonine (21), as a bicyclic indolizidine alkaloid, is quite simple, but the number of chiral centers is extraordinary for such a small molecule. The stereochemical arrangement of the hydroxyl substituents is remarkably similar to those of mannose, but the presence of a nitrogen atom appears to confer on 21 the ability to inhibit a-mannosidase in a competitive manner (110). The water solubility of swainsonine is analogous to that of sugars, and it is transported to many organs of the body, but is also rapidly excreted in the urine. In lactating animals, a portion of it is transferred to the milk, with nursing calves or lambs becoming poisoned without actually feeding on locoweeds (111). Unless consumption of locoweeds is extremely high, ingestion over short periods is therefore unlikely to have lasting adverse effects, but it is important to remove animals from locoweed-infested land in order to prevent continuous consumption over longer periods, even at relatively low swainsonine levels. As an aza-analog of D-mannopyranose, the inhibitory activity of swainsonine against a-mannosidase appeared to be predictable (110). Similarly, castanospermine has a stereochemical arrangement of hydroxyl groups consistent with inhibition of a- and b-glucosidases (112). However, testing of the minor alkaloids from locoweeds and Ca. australe showed that such predictions were too simplistic and that it was impossible to derive alkaloid structure and enzyme inhibition correlations a priori. For example, all castanospermine and australine epimers were found to inhibit amyloglucosidase regardless of the stereochemistry of their hydroxyl groups (82–86,113). Furthermore, the presence of the pyrrolizidine ring system in the australines showed that a six-membered nitrogencontaining ring was not essential. In fact, castanospermine and australine are equipotent inhibitors, with Ki values of 8 and 6 mM, respectively, their activities exceeding those of all of the other less abundant epimers. A similar situation pertains with respect to the calystegines, with most members of the group inhibiting either b-glucosidase and/or a- and b-galactosidase, regardless of the number, disposition, and stereochemistry of the hydroxyl groups (114). In contrast, calystegine C2 is an azamannose analog with respect to the six-membered ring system, and is, in fact, an inhibitor of a-mannosidase (115). Apart from the question of whether swainsonine and the calystegines are endophytic or phytochemical natural products, their co-occurrence in Ipomoea species from such diverse regions as Australia, Mozambique, and South America (67–72) raises the question as to the relative contributions of each to poisoning of livestock by such plants.

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Empirically, it would seem likely that the biochemical lesions and associated signs of poisoning should result from the specific enzymes that are inhibited and the relative potency and concentration of each alkaloid that is present. Whenever swainsonine was present in the plant, typical neurological damage and cytoplasmic vacuolation of neurons have been observed, but syndromes of Ipomoea poisoning also involve muscle-twitching, tremors, epileptiform seizures, and a ‘‘stargazing’’ attitude, which are atypical of locoweed poisoning. Histopathology of animal tissues also showed vacuolation of Purkinje cells, not observed with locoweed consumption. Such signs are typical of the human genetic defects, Gaucher’s and Fabry’s diseases, caused by deficiencies of b-glucosidase and a-galactosidase, respectively, and it could be hypothesized that inhibition of these enzymes by the calystegines induces phenocopies of these lysosomal storage diseases. In opposition to this, feeding experiments of Ipomoea asarifolia to goats produced a tremorgenic syndrome, even though calystegines were absent from the plant and swainsonine occurred only in trace amounts of less than 0.01% d.w. of the plant (116). This suggested that neurotoxins other than glycosidase alkaloid inhibitors may be present in Ipomoea species. Current evidence indicates that calystegines are not primary livestock toxins, but instead play a secondary role, possibly by altering the uptake and distribution of swainsonine or other toxins by changing the activity of digestive enzymes. The overall question of synergistic interactions between the alkaloid glycosidase inhibitors remains to be investigated. It is not known whether inhibition of a particular enzyme will potentiate or ameliorate inhibition of a different enzyme. However, some of the products of glycoprotein processing are themselves glycosidases, and reduced production levels, combined with partial inhibition, could induce effects that would not otherwise be observed. In a recent study in mice of the comparative pathology of swainsonine, castanospermine, and calystegines A3, B2, and C1, the two indolizidine alkaloids produced clinical and histological changes typical of their expected toxicity, whereas the highest dose (100 mg/kg) of calystegine A3 caused only minor hepatic changes (117). Gastrointestinal problems are the most consistent feature of field cases of livestock and occasional human poisonings by Ca. australe in Australia, a predictable outcome of the ability of castanospermine and australine, and their minor epimers, to inhibit a- and b-glucosidases. Neurological damage has not been reported, but rodent feeding experiments with castanospermine produced hepatic vacuolation and glycogen accumulation, consistent with a phenotype of Pompe’s disease or type II glycogenesis (118). Although rodents are relatively resistant to induced lysosomal storage diseases, it is important to define the effects of reduced glycosidase activity

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because the inhibitors undoubtedly affect mammalian liver enzymes in vitro (114,115), and their widespread presence in both commonly consumed fruits and vegetables and rangeland plants may have deleterious effects on both humans and animals. Another reason for elucidating the mode of action of the polyhydroxy alkaloid glycosidase inhibitors has been to investigate these compounds and certain synthetic derivatives as therapeutic drugs. Their potential application for the treatment of diseases resulting from altered glycoprotein processing in animal cells has been reviewed (12,18,19, 119,120). The alkaloids have been used to investigate treatments for human lysosomal storage defects, such as mannosidosis, Pompe’s, Fabry’s, and Gaucher’s diseases by induction in animal models, and the application of various treatments to alleviate the disease. The mechanistic rationale for using alkaloid glycoprocessing inhibitors as anticancer agents has been reviewed (121) and swainsonine and castanospermine in particular have been shown to inhibit tumor growth and metastasis in animal models. Swainsonine enhanced natural killer cell activity, and when administered to mice in the drinking water for 24 h prior to treatment, it reduced pulmonary colonization of the lungs by murine melanoma cells by 80% at 3 mg/mL and reduced human melanoma xenografts by 50% at 10 mg/mL (122). Administration for such limited time periods and exposure levels would be insufficient to produce neurological damage (123), suggesting that intravenous administration prior to tumor removal surgery could suppress metastasis. Preliminary human studies have shown promise. A Phase I study of swainsonine administered over 5 days by continuous infusion to humans with advanced malignancies produced remission of head and neck tumors in one patient and symptomatic improvement in two other patients (124). A subsequent Phase IIB clinical trial established the maximum oral tolerated dose as 300 mg/kg per day (125). Viral replication is highly dependent on the correct folding of their envelope glycoproteins that can be altered by treatment with aglucosidase inhibitors. Alkaloid glycosylation inhibitors have been tested against most of the major viral diseases affecting humans, including human immunodeficiency virus (HIV), hepatitis B, and hepatitis C (126,127). 6-O-Butanoylcastanospermine (Celgosivir) has also undergone trials and shown promise as an oral drug for patients with resistant hepatitis C (128). Castanospermine and 6-O-butanoylcastanospermine inhibit HIV in vitro, with the latter having a therapeutic index similar to the reverse transcriptase inhibitor drugs used for this condition, such as azidothymidine (AZT) (129). In addition to its antiviral activity, 6-Obutanoylcastanospermine demonstrates antiparasitic activity, preventing adhesion of Plasmodium falciparum to infected erythrocytes and providing protection against cerebral malaria (130).

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Limitations of swainsonine and castanospermine as drug candidates are their high water solubility and rapid excretion rate, but this can be overcome by synthesis of prodrug derivatives with improved pharmacokinetics. Whereas swainsonine is generally obtained from plant sources in low yield and is consequently expensive, castanospermine can be obtained from Ca. australe seeds in quite high yields, making it a cost-effective and available material for synthetic modification.

VII. QUINOLIZIDINE ALKALOIDS A. Plant Species and Alkaloids Quinolizidine alkaloids are a large group of alkaloids, most commonly found in the family Fabaceae, of which certain species of the genera Baptisia, Cytisus, Laburnum, Lupinus, Sophora, and Thermopsis have been incriminated as plants poisonous to livestock. Among these, the lupines (Lupinus spp.) are of greatest concern because they are the most widely distributed in North America and encompass more than 150 species, exhibiting a great deal of polyploidy and hybridization. This has caused problems with taxonomic classification, which has generated corresponding difficulties in predicting those species responsible for toxicity. Some lupines have little or no alkaloid content, and those having concentrations of o0.1% total alkaloid are classified as ‘‘sweet’’ lupines, and may be cultivated as forage crops or for the high protein content of their seed. The most common of these are Lu. albus and Lu. angustifolius. In North America, 55 species and varieties have been reported to be toxic (3), with high total alkaloid content, including Lu. arbustus, Lu. argenteus, Lu. formosus, Lu. leucophyllus, Lu. sericeus, and Lu. sulphureus. Other species may have high alkaloid content, but are of such limited distribution, or are not in proximity to livestock, that they rarely or never cause problems. The distribution of quinolizidine alkaloids in Lupinus species has been extensively reviewed (131,132). Structurally, these alkaloids fall into four classes: the fundamental quinolizidine ring system type, represented by lupinine (40); those consisting of two quinolizidine systems incorporated into a tetracyclic structure, such as sparteine (41) and lupanine (42); tricyclic and tetracyclic pyridone ring-containing alkaloids, exemplified by cytisine (43, R ¼ H) and anagyrine (44), respectively; and fused tetracyclic alkaloids, such as matrine (45) (133,134). Many of these alkaloids occur as esters of the various hydroxyl group constituents, or as tricyclic compounds that may be regarded as ring-degraded tetracyclic types. It should also be noted that some lupine species contain bipiperidine alkaloids of the ammodendrine (14, R ¼ H) type (Section IV),

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which have now been conclusively linked to teratogenic effects. The occurrence of two different structural types of alkaloid in lupines has complicated attempts to associate different toxic effects with any particular structural class. This problem has recently been emphasized by a comprehensive GC–FID and GC–MS analysis of 286 Lu. sulphureus collections and herbaria accessions for alkaloid composition (135). In spite of the fact that all samples were classified as Lu. sulphureus, they fell into seven distinct chemotypes, two of which contained only the bipiperidine alkaloids ammodendrine (14, R ¼ H) and N-methylammodendrine (14, R ¼ CH3). The other five chemotypes contained only quinolizidine alkaloids of the tetracyclic type, with lupanine (42) being consistently present. Each chemotype had a distinct geographical distribution, illustrating the difficulty of predicting toxicity risk based on taxonomic classification for the large area of eastern Washington and Oregon, and British Columbia, over which this species grows.

H

CH2OH

H

H

5

N N

2

N H

40

N 6

11

N H

O

41

42

O H N

R N N

H

N H

O

H

N

H N

O 43

44

45

Biosynthesis of quinolizidine alkaloids has been shown to occur in the leaves, with subsequent transport in the phloem to other plant organs, but with marked diurnal fluctuations, with two-fold higher levels in the late afternoon relative to the night (136). Dry-weight alkaloid concentrations in the foliage are highest in the early growth stages, declining as the plant matures, but with a corresponding accumulation in the seeds. In Lu. caudatus (Lu. argenteus) total alkaloid concentrations of 0.5–2.5% d.w.

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were reported for the leaves and stems and 1.2–9.5% for the seeds (137). Because of accumulation in the seed as the plant matures, concentrations in the latter can be as much as 10 times greater than in the foliage at later growth stages. Of all the quinolizidine alkaloids present in lupines, anagyrine (44) seems to be the most common, but it is also highly variable. In many species, anagyrine predominates; for example, in Lu. sulphureus it comprises 36–78% of the total alkaloid content, depending on plant part and growth stage (138). Alkaloid levels of 0–1.22% have been found in the foliage of Lu. argenteus, and 0–4.8% in the seeds (139,140). In Lu. latifolius some collections have none of the alkaloid, while others have as much as 1.14% in the foliage (141). The latter species is of particular interest because of a report of limb deformities in an infant born to a woman regularly consuming milk from goats grazing the plant (142). Several fetuses in the herd of goats also had skeletal defects and puppies born to a dog fed the same milk showed similar malformations (143). Although Lupinus species are the most common plants involved in livestock poisonings, there are sporadic episodes of quinolizidine alkaloid-related toxicity from Baptisia, Cytisus, Laburnum, Sophora, and Thermopsis. Wild indigos (Baptisia spp.) are confined to the eastern United States and are rarely eaten by animals, although the plant may be incorporated into hay. In the most widespread species, B. australis, total alkaloid levels may attain 0.5% with N-methylcytisine (43, R ¼ CH3), cytisine (43, R ¼ H), and anagyrine (44) being the predominant individual alkaloids (144). Scotch broom, Cytisus scoparius, and other brooms (Genista and Spartium spp.) are introduced species in North America that have become highly invasive in some localities. Reports of toxicity are rare, but Cyt. scoparius has been reported to contain 0.1% d.w. quinolizidine alkaloids, with 17-oxosparteine, sparteine (41), 12,13-dehydrosparteine, and lupanine (42) being the major alkaloids detected in the phloem (145). Laburnum anagyroides (golden-rain tree) is a European species, cultivated in North America as an ornamental, together with two other less common species. Toxicity has been reported for many domestic animal species and occasionally humans, with horses being the most susceptible. The greatest hazard arises from consumption of the seeds, which contain 1.23% total alkaloid, with much lower levels in the leaves; the primary alkaloid was cytisine (43, R ¼ H) with lesser amounts of N-methylcytisine (43, R ¼ CH3) (146). The most toxic Sophora species consist of 10 species, some of which are native and others introduced, growing mainly through the Rocky Mountains states and southward into Texas and New Mexico. The most widespread are S. nuttalliana (silky sophora) and S. secundiflora (mescal bean, coral bean, Texas mountain laurel, or frijolito). These plants are

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toxic to livestock, domestic animals such as dogs, and humans, with children being particularly susceptible because of the attractive red color of the seeds. The structural types of quinolizidine alkaloids can be quite diverse, falling mainly into the cytisine, sparteine, and matrine classes, but comprehensive studies of the alkaloid content and composition of those species responsible for livestock poisoning have not been done. Stems of S. secundiflora have been shown to contain cytisine (43, R ¼ H), sparteine (41), anagyrine (44), N-methylcytisine (43, R ¼ CH3), 5,6-dehydrolupanine (D5,6-42), rhombifoline (46), lupanine (42), epi-lupinine, N-acetylcytisine (43, R ¼ COCH3), and N-formylcytisine (43, R ¼ CHO) (147), while the mature seeds contain a similar array of alkaloids (148). The matrinetype alkaloid, ()-sophocarpine (47), has been isolated from S. nuttalliana (149). O H N N

H

N 11

N

H2C

H

H

N

N O

H O

46

47

48

Only one of the three species of Thermopsis found in North America is of concern for livestock poisoning. T. rhombifolia (false lupine, yellow pea or mountain pea, mountain thermopsis) is widely distributed in an arc from the Rocky Mountains to the northern Pacific Coast. The species is highly polymorphic and has often been identified as T. montana, as well as a number of other taxa. The plant induces muscle degeneration in cattle with consequent recumbency, and a purified alkaloid extract, shown to contain N-methylcytisine (43, R ¼ CH3), cytisine (43, R ¼ H), 5,6-dehydrolupanine (D5,6-42), thermopsine (48) (i.e., 11-epi-anagyrine), and anagyrine (44), produced similar effects on administration. Comparison of these effects with those produced by a Lupinus species containing mainly anagyrine, and La. anagyroides containing only cytisine, showed that these two alkaloids alone were not sufficient to induce the poisoning, suggesting that alkaloids of the A-ring a-pyridone quinolizidine type are essential to cause myopathy (150). As with many other quinolizidine alkaloid-containing species, T. rhombifolia (T. montana) is most toxic in the early growth stages and at the seed stage of growth (151). Thermopsis species have also been implicated in human poisonings, primarily children, especially on consumption of the seeds (152).

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B. Toxicity and Clinical Signs It is important to distinguish toxicity of lupines from lupinosis, caused by animals grazing lupine stubble infected with the fungus Diaporthe toxica. This is a particular problem with sheep in Australia, and is caused by fungal metabolites, the phomopsins, that inhibit microtubule assembly (153). The genus Lupinus is the most frequently implicated among the Fabaceae in livestock poisoning, including mortality in sheep and cattle, and skeletal birth defects in cattle known as ‘‘crooked calf syndrome.’’ This genus contains more than 500 classified species of perennial, annual, or shrub-like species worldwide, with over 300 species in South and North America, including 150 species in the Intermountain West of the United States (154). Twelve species have been identified in Europe and Africa (132) and over 95 species in California (155). Lupine-induced poisoning in livestock was first recognized in the western United States in the late 1800s when large numbers of sheep died acutely as a result of grazing lupines in late summer and early fall, or when lupine hay harvested with intact seedpods was fed during the winter (156). This led early investigators to determine that the seeds and seedpods were ‘‘rich in the poison.’’ Poisonings in sheep flocks still occur today, although because of research and dissemination of information, losses are much less frequent and at greatly reduced incidence. Over 150 quinolizidine alkaloids have been characterized from various species of lupine, and all are believed to possess some level of neurotoxicity, but only one quinolizidine alkaloid, anagyrine, has been linked to the ‘‘crooked calf syndrome’’ (137). ‘‘Crooked calf disease’’ was first recognized in the early 1950s (157), but was not linked to lupines until about 1960 (158,159). Soon thereafter research to understand the etiology and define the critical stage of pregnancy began and the syndrome was defined in greater detail (160–162). The defects were defined as skeletal contractures, and were specifically characterized as arthrogyposis, scoliosis, kyphosis, torticollis, and other skeletal defects associated with the joints of the skeletal system. Cleft palate was also part of the syndrome and was associated with some or all of the skeletal malformations, or it could occur as a unilateral or bilateral cleft alone. The period of insult was defined (40–70 days gestation) and later extended to 100 days gestation (138). The specific alkaloid teratogen was isolated and characterized as the quinolizidine alkaloid anagyrine (44) (137). Since then, other plants have been experimentally shown to cause the same malformations and the teratogens have been isolated and characterized as piperidine and bipiperidine alkaloids (Sections III and IV) (26,57,163). Using ultrasound monitoring (164) it was shown that fetal movement ceased during a

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critical stage of fetal development and the mechanism of action was determined to be an alkaloid-induced neuromuscular blockade of the fetus (165). T. montana induces a chronic myopathy if ingested over a period of time. Poisoning in cattle and horses has been reported and prognosis is poor once clinical effects are manifest. Clinical signs generally start with tremors, ataxia, and muscular weakness, progressing to prolonged recumbency and death. Microscopic lesions are those of generalized myopathy, hyaline degeneration of myofibers, satellite nuclear proliferation, macrophage infiltration of muscle tissue, and occasional mineralization of myofibers. Generally, animals will avoid grazing the plant unless other forage is limited, or if it is fed as a contaminant in hay or silage. A purified alkaloid extract containing N-methylcytisine (43, R ¼ CH3), cytisine (43, R ¼ H), 5,6-dehydrolupanine (D5,6-42), thermopsine (48), and anagyrine (44) induced symptoms and microscopic changes similar to those produced by T. montana plant material when administered to cattle (150). Alkaloid extracts from a Lupinus sp. and La. anagyroides, containing mostly anagyrine (44) or cytisine (43, R ¼ H), respectively, produced skeletal muscle degeneration and necrosis similar to the alkaloid extract from T. montana, but these were less severe and there was no recumbency. This indicates that neither of those two alkaloids alone induces the Thermopsis toxicity and that quinolizidine alkaloids with an a-pyridone A ring may be responsible for the lesions.

C. Structure–Activity Relationships and Mode of Action Quinolizidine alkaloids are represented by a considerable range of structural types, but the majority fall into the tetracyclic class, incorporating the fusion of two quinolizidine moieties, often with a keto group in the A or B ring. Particularly common constituents are lupanine (42) and anagyrine (44), but the great diversity of structures makes it difficult to assign specific biological activities to particular structural features. Wink (166) has remarked on the bitter taste of sparteine (41), which was probably noted because of its potential application as an antiarrhythmic agent and a sodium and potassium channel blocker. Both alkaloids are weak sedatives of the CNS at low doses, with lupanine being less toxic than sparteine (167). The bitter taste of lupine seeds has been attributed to lupanine, although it is less intense than sparteine. The relative bitterness of other quinolizidines is not established, and it is impossible to determine whether animals perceive the same sensation as humans and even if they do, if it is sufficient to inhibit plant grazing.

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Quinolizidine alkaloids, such as lupanine and sparteine, bind to acetylcholine receptors of the nicotinic and muscarinic types, respectively. The agonist effect thus induced probably accounts for the neurological symptoms produced on lupine consumption, although the signs and susceptibility to poisoning vary with animal species. The lethal dose in laboratory animals is ca. 100 mg/kg, but much greater in large animals, with the alkaloids being rapidly excreted so that recovery may occur after initial signs of toxicity. Repeated ingestion of lower doses of plant material may induce tolerance to the toxic effects (168). Teratogenic effects produced by Lupinus species are complicated by the co-occurrence of the bipiperidine alkaloid ammodendrine (14) and quinolizidine alkaloids. However, Lupinus species that induce terata (e.g., crooked calf disease), but do not contain ammodendrine, frequently appear to contain anagyrine (44) as the major quinolizidine alkaloid (169–171). The involvement of this alkaloid was confirmed by feeding trials with anagyrine-rich extracts (137). This study indicated that anagyrine must occur at a concentration of at least 1.44 g/kg and the plant be consumed for a minimum period of 30 days. It has been postulated that anagyrine, and other quinolizidine alkaloids with an a-pyridone ring moiety, undergo metabolic cleavage of this ring to the actual teratogen. This metabolite would have certain structural features similar to those of ammodendrine and the mode of action of both classes of alkaloid may therefore be through suppression of fetal movement. Sheep and goats are less susceptible than cattle to teratogenicity induced by anagyrine-rich lupines, and this could be a result of lack of transformation to the ultimate teratogen. These differences could also be accounted for by differences in absorption between animal species. In animals fed Lu. caudatus, blood levels of anagyrine and lupanine were highest in sheep, and lowest in goats, with cattle having intermediate levels. The levels were essentially undetectable 48 h after administration, after peaking at 3 h and remaining high for up to 10 h (172). Rapid excretion in the urine is facilitated by hepatic metabolism to the highly water-soluble N-oxide forms. The tricyclic alkaloid cytisine (43) is neurotoxic with activity similar to nicotine (17, R ¼ H), cytisine being a more powerful ganglion stimulant than a blocking agent, resulting in ataxia and short-lived seizures (150,173). N-Methylation to give the alkaloid caulophylline results in reduced activity because of its lower basicity. At high concentrations, cytisine induces cardiotoxicity, with heart arrhythmia. However, in spite of the presence of an a-pyridone moiety that could undergo biotransformation as hypothesized for anagyrine, there is no evidence that it is teratogenic. The tetracyclic quinolizidines, such as sparteine (41) and lupanine (42), exhibit a reverse pattern of activity to

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cytisine, being primarily cardiotoxic, with secondary neurotoxicity. Cardiac effects are decreased heart rate and increased contractions, with arrythmia at high doses (174) induced through Na+ channel blocking (175). The tetracyclic a-pyridone alkaloids appear to be the structural type most likely to be responsible for the myopathic effects observed on Thermopsis consumption, resulting in muscle degeneration and persistent recumbency (150,151). The fused tetracyclic matrine (45) class of alkaloids shows similar cardiotoxic effects, although in this case the activity has been associated with increased calcium uptake (176).

VIII. TROPANE ALKALOIDS A. Plant Species and Alkaloids The toxic tropane alkaloids, L-hyoscyamine (49) and scopolamine (50), occur in species of the genera Datura and Brugmansia (Solanaceae) (177). These plants are similar in appearance, with attractive trumpet-shaped flowers, and Brugmansia species were once classified in the genus Datura. However, Datura species are widely distributed around the world, whereas Brugmansia species occur primarily as ornamentals. The plants are known by a wide variety of common names, including angel’s trumpet, devil’s trumpet, thorn apple, and jimson weed (a corruption of Jamestown weed). In the United States, they are sometimes known by adolescents as ‘‘loco weeds,’’ because of their use to produce hallucinations, but are completely unrelated to the true locoweeds (Astragalus and Oxytropis species) (Section VI), either botanically or in their phytochemistry. These plants have been responsible for numerous episodes of human poisoning, either through deliberate misuse due to their hallucinogenic properties, or accidental ingestion, especially in children (178,179). In some cases, Datura poisonings may be mistaken for a heart attack (180). Livestock poisonings from Datura are sporadic. The fresh plant is unpalatable to most animals, but the dry plant is less so, and can become incorporated into hay. The main species of concern is D. stramonium, widely distributed in the Eastern and Midwestern States, and along the Pacific Coast. Datura inoxia, D. quercifolia, and D. wrightii are found mainly in southern Texas, New Mexico, and Arizona, and D. metel is restricted to a coastal band along the Gulf of Mexico from Louisiana to Florida. Hyoscyamine and scopolamine also occur in Hyoscyamus niger (henbane) an introduced species in North America, occurring across the northern tier of Western and Midwestern states and into southern Canada. Tropane alkaloids have also been reported in the families Proteaceae, Euphorbiaceae, Brassicaceae, Moraceae, Oleaceae,

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Rhizophoraceae, Erythroxylaceae, and Convolvulaceae. H3C

H3C

N CH2OH O

N O

CH2OH O

O

49

O

50

In most tropane alkaloid-containing species, L-hyoscyamine (49) predominates over the epoxide, scopolamine (50). On isolation, it racemizes to form a diastereomeric mixture with D-hyoscyamine known as atropine. Hyoscyamine content is therefore generally reported as ‘‘atropine,’’ however, most of the physiological effects of the latter are due to L-hyoscyamine. The principal site of alkaloid synthesis is in the roots of young plants, with subsequent transport to the leaves and stems. However, reports of the content in specific plant parts have been conflicting. GC–MS analysis of D. stramonium showed that the total alkaloid level was highest in the leaves and stems of young plants, and in the seeds in mature plants (181), with atropine/scopolamine ratios varying from 2.1:1 to 17.7:1. However, the alkaloid profile is much more complex than these two major components, which generally comprise ca. 52–92% of the total, with minor amounts of as many as 46 other tropane alkaloids having been identified (182). Highest total alkaloid was found in early root growth (0.25% d.w.), with ca. 0.2% in leaves and 0.15–0.2% in seeds. In contrast to the results of Miraldi et al. (181), the lowest alkaloid levels (0.1%) were in the stems. HPLC analysis of D. stramonium seeds from various locations in the United States showed total alkaloid contents of 0.21–0.34% and atropine/ scopolamine ratios ranging from 3.5:1 to 6.9:1 (183). A survey of four varieties of D. metel showed that scopolamine always predominated over hyoscyamine, ranging from highest in the seeds (0.29–0.63% d.w.) and flowers (0.19–0.70% d.w.) to considerably lower in the leaves (0.04–0.26% d.w.) (184). These results are consistent with those of Oshima et al. (185), which showed over two-fold higher levels of scopolamine than hyoscyamine in both the leaves and flowers. In the seeds of D. inoxia hyoscyamine (0.14%) occurred in seven-fold excess over scopolamine (0.02%), but the reverse was true in the leaves, with 0.07% scopolamine and only trace amounts of hyoscyamine (186). In the leaves of Hyoscyamus niger, hyoscyamine and scopolamine were found at approximately equal levels, amounting to ca. 0.1% total concentration (185).

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The high inter- and intraspecies variability and content and composition of these alkaloids indicate that careful analysis of plants involved in poisoning episodes is essential. A review of the chromatographic methods employed for the analysis of Datura alkaloids has been published (187). It should be noted that the leaves of D. wrightii contain calystegine B2 (188) and trace amounts of this and other calystegines have been detected in the roots and leaves of D. stramonium (189). As mentioned in the section on polyhydroxy nortropane alkaloids (Section VI), the role of the calystegines in livestock poisonings is unclear at present. However, it should be considered that they could play a role in influencing the metabolism and toxic effects of the tropane alkaloids.

B. Toxicity and Clinical Signs Most plant species containing tropane alkaloids are unpalatable to animals, and consequently they are only a risk when little forage is available. Species of Datura, Hyoscyamus, and other tropane-containing genera are of more risk to humans than to animals. For example, seeds from these plants have been abused by young people, used for religious or ritualistic functions, or occasionally ingested by accident. Cases of bizarre and aggressive behavior have been reported in people ingesting seeds or tea from some of these plants (3). A relatively recent report appeared where 17 young people ingested jimson weed seeds in a single mid-Atlantic state (190). Fifteen were admitted to the hospital, with 13 of them to the intensive care unit. Reported quantities of seed ingestion were as low as 7 seeds and as high as approximately 200 seeds. Some states have listed jimson weed as illegal to grow or have in possession. Poisoning has been reported in animals when contaminated hay, silage, or seed-contaminated feed has been ingested (3,4). Clinical effects in animals and humans include papillary dilation, increased heart rate, dyspnea, restlessness, dry mucous membranes, intense thirst, and death. In horses, colic often occurs, and in cattle, bloat is common, and both can result in death.

C. Structure–Activity Relationships and Mode of Action The tropane alkaloids hyoscyamine and scopolamine are potent anticholinergic compounds, acting as competitive antagonists of muscarinic cholinergic receptors. They act on a number of subtypes of receptor in the central and autonomic nervous systems, heart and smooth muscle, and secretory systems (191). These actions produce the typical signs of toxicity, including increased heart and respiration rates, depression, anorexia and drowsiness, dilation of the pupils, and gastric disturbances. Hyoscyamine has limited ability to penetrate the blood–brain barrier and

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its effects are primarily peripheral, whereas scopolamine produces neurological alterations. The metabolism of scopolamine in vivo has been compared in a number of laboratory animals, showing high species specificity (192). In mice, scopolamine and norscopolamine were excreted primarily as glucuronide conjugates, but in rabbits and guinea pigs the tropic acid, formed by hydrolysis of the ester bond, was found in the urine. Aposcopolamine and aponorscopolamine, dehydrated metabolites, were abundantly excreted in guinea pigs, but much less so in rabbits and rats. In the latter species, the phenolic metabolites 4u-hydroxy-, 3u-hydroxy-, and 4u-hydroxy-3u-methoxy-scopolamine were formed by hydroxylation of the aromatic ring. Similar differences in metabolism in livestock species have not been established, but may account for the high variability in the symptoms of toxicity.

IX. PYRROLIZIDINE ALKALOIDS A. Plant Species and Alkaloids Approximately 400 pyrrolizidine alkaloids have been identified, encompassing a number of structural types, which may elicit somewhat different clinical effects and associated risk to animals and humans, depending on structural characteristics. Various aspects of the chemistry, toxicology, plant and animal metabolism, biosynthesis, and ecological roles of these alkaloids have been covered in dedicated volumes (193,194), and in several chapters and reviews (195–198). These alkaloids have a fundamental core structure, the necine base, consisting of two fused, five-membered rings with a nitrogen atom at the bridgehead, and bearing one to three hydroxyl groups. Eighteen hydroxylated necine bases are known, with (+)-retronecine (51) being representative and one of the most common. These amino alcohols are rarely isolated as naturally occurring compounds, but are generally esterified with acid moieties known as necic acids. When free necine bases are found there is always a concern that they may be artifacts of the extraction, formed by hydrolysis of the esters typically found. The necine bases can be mono-unsaturated as in retronecine (51), completely saturated as in ()-platynecine (52), or with the common bridgehead bond cleaved to give a seco-necine, such as otonecine (53). HO

H

N

51

CH2OH

HO

H

N

52

CH2OH

HO O

CH2OH

HO

O

N

N

CH3

CH3 53

CH2OH

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Esterification of the necine bases with an array of necic acids enhances the diversity of pyrrolizidine alkaloid structures, and these can be classified into three fundamental types: macrocyclic diesters, nonmacrocyclic diesters, and monoesters. These broad structural categories are often associated with particular plant genera. Macrocyclic diesters are typified by 12-membered macrocycles, such as senecionine (54, R1 ¼ CH3, R2 ¼ H) and riddelliine (55), and 11-membered rings, such as monocrotaline (56, R ¼ H). When an exocyclic double bond is present, as in the Z-isomer, for example, senecionine, the corresponding E-isomer, for example, integgerimine (54, R1 ¼ H, R2 ¼ CH3), can often be found. Saturation of the necine base double bond gives alkaloids of the platynecine (57) type, while cleavage of the bridgehead bond leads to seco- alkaloids such as senkirkine (58). The necic acid moieties generally bear one or more hydroxyl groups and epoxide groups also occur. R2

HO

H

CH3

C

O C

R1 O

H O

CH3

HO

CH2OH O CH3 C

C H3C CH2

O

O

H

O

H

CH3

CH3 O C

OR O

O

H

O

N

N

54

55

56

HO

C CH3

H O

H

N

O

HO

CH3 O

C

O

C CH3 O

H

CH3 H3C

C CH3

H O

O

H

N

H

OH

O

O O

N CH3

57

58

Nonmacrocyclic diesters are represented by alkaloids such as lasiocarpine (59) and 7-angelyl heliotrine (60), and monoesters by necine bases esterified on the primary hydroxyl group such as lycopsamine (61) and 7-angelyl heliotridine (rivularine) (62); monoesters of the 7-hydroxyl group are relatively rare. The complexity of these structural types is further amplified by the occurrence of alkaloids in the plants as both free

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bases and N-oxides. The N-oxides are very water soluble and therefore not easily obtained by standard partitioning techniques used to isolate alkaloids. Depending on the plant species, the N-oxides often predominate, but this may not be recognized and extraction therefore only gives the free base component, leading to a serious underestimation of the total alkaloid content. H H3C

C C

CH3

CH3 OCO

O

O

H

H

CH3 OH H

C

C

H3C CH3

O

CH3

CH3 OCO

O

OH OCH3

H

CH3 OH OCH3

N

N

60

59

CH3

HO

CH3 H H

C H3C CH3

OCO H

H

CH3 H H

OH OH

O

C

CH3 OH O

H

N

N 61

62

Pyrrolizidine alkaloids occur worldwide in many plant species, encompassing diverse genera. It has been estimated that as much as 3% of the world’s flowering plants may contain these alkaloids (199), which have been isolated from about 600 plant species, or about 10% of the potential number. In North America, livestock poisonings are most commonly associated with genera of the Asteraceae, Boraginaceae, and Fabaceae families, with Senecio species (groundsels or ragworts; Asteraceae) predominating. Of particular concern are native species growing in the Rocky Mountains states and Great Basin region, including S. douglasii var. longilobus (syn. S. flaccidus) (threadleaf groundsel) and S. riddelli (Riddell’s groundsel), and to a lesser extent S. integerrimus (western groundsel) and S. spartioides (broom groundsel). In the Great Plains, S. plattensis (prairie groundsel) is widespread, but the availability of better forage than in drier areas makes it less likely to be a threat.

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Introduced species are also a problem, especially S. jacobaea (tansy ragwort) and to a lesser extent S. vulgaris (common groundsel), both of which thrive on the East and West Coasts. Another species classified as a noxious plant is S. madagascariensis (fireweed), originating in East Africa and subsequently introduced via Australia into ranching areas of the Big Island of Hawaii, where it has become highly invasive (200). Senecio species responsible for poisoning livestock contain primarily macrocyclic diester alkaloids, but in highly variable amounts and proportions of individual alkaloids, depending on plant part and growth stage (201). For example, S. riddelli generally contains 2–9% of a single alkaloid, riddelliine (55), while S. longilobus (S. flaccidus) contains 1–3% of a mixture of senecionine, seneciphylliine, retrorsine, and riddelliine, and S. jacobaea has 0.1–0.9% of as many as nine different alkaloids, including the epoxy- and chloro- derivatives, jacobine (63) and jaconine (64), respectively. In S. madagascariensis from Hawaii, 13 alkaloids of the macrocyclic diester type were identified, and 5 of these were secoalkaloids (200). The overall pattern corresponded closely to a sample of the same species from Australia, the suspected source of the introduction. Highest total alkaloid levels in all Senecio species are found in the inflorescences and seeds, and alkaloid levels decline markedly after seed dispersal. In some species and locations these levels can be very high, comprising as much as 18% of the dry weight of S. riddellii aboveground plant (202). The N-oxide/free base ratios also vary, with N-oxides comprising as much as 95% of the total alkaloid in S. riddellii, but only 27% in S. jacobaea. These ratios are probably very sensitive to the growth stage, as the alkaloids appear to be biosynthesized in the roots and transported to other plant tissues as the water-soluble N-oxides (203). H

H C

HO

O

O

CH3

C O

Cl

CH3

H O

CH3 H

O

HO

OH

CH3

C

O

CH3

C O

H O

CH3

O

H

N

N

63

64

In the family Boraginaceae, Cynoglossum officinale (hound’s tongue) is the species of most concern for livestock poisoning. Hound’s tongue is an introduced species, originating in Eurasia, and forming dense stands in most parts of the United States and Canada, particularly in moist areas of

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the western Great Plains and the intermountain region. The primary alkaloids in this species are the nonmacrocyclic diester, heliosupine (65, R ¼ H) and its acetyl derivative (65, R ¼ COCH3), and monoesters such as echinatine (66) and 7-angelyl heliotridine (62). The total alkaloid content in a pasture heavily infested with first- and second-year growth of Cy. officinale was found to be 0.51 and 1.18% d.w. in leaves and seeds, respectively, with corresponding N-oxide/free base ratios of 1.4 and 2.6 (204). Another collection of second-growth whole plant at the flower stage had an alkaloid content of 0.73% d.w. and N-oxide/free base ratio of 3.1 (205). A study of alkaloid production throughout the biennial growth stages of the plant showed that levels were highest in the immature leaves, attaining 2.12% d.w., declining to 0.68% in mature growth; the N-oxide form was dominant at all growth stages (206). H H3C

C C O

CH3

CH3

CH3 OR H CH3

OCO O

H

OH OH

N

CH3

HO

CH3 H H CH3

OCO H

OH OH

N

65

66

Other boraginaceous species of some concern are Amsinckia intermedia (tarweed), Echium vulgare (viper’s bugloss, blue devil), Heliotropium curassavicum (heliotrope), Symphytum spp. (comfreys), Hackelia floribunda (stickseed), Mertensia spp. (lungworts), and Myosotis spp. (3). All of these species contain monocyclic and nonmacrocyclic diester alkaloids similar in structure to those found in Cy. officinale, but they have rarely or only sporadically caused livestock poisonings in North America. In contrast, Echium plantagineum (Paterson’s curse) and E. vulgare, and various Heliotropium spp., have caused poisonings in cattle, sheep, and horses in Australia, and are regarded there as major poisonous plant problems. The prolific seed production can also present a hazard as contaminants of feed for poultry and pigs (207). Hepatotoxicity in humans resulting from contamination of grain with seeds of a Heliotropium species has been a recurring problem in Afghanistan (208). Other potential poisonings in humans can result from herbal remedies, especially Symphytum and Echium spp. (209,210), and contamination of honey and pollen from floral sources such as E. plantagineum and H. europaeum (211–213). In the Fabaceae family, pyrrolizidine alkaloids are restricted to the genus Crotalaria. Worldwide, these plants are responsible for numerous

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episodes of toxicity, but in North America C. saggitalis, a native species found in the southeastern United States, is of greatest concern. Other species have been introduced as forage crops or soil builders (C. retusa, C. spectabilis, and C. pallida), or for fiber production (C. juncea), often without consideration or evaluation for toxic potential. The alkaloids of Crotalaria are primarily 11-membered macrocyclic diester types, such as monocrotaline (56, R ¼ H) and its acetyl derivative, spectabiline (56, R ¼ COCH3), but with some species containing 12-membered macrocyclic diesters, such as the retronecine-based integerrimine (54) and usaramine (67), and the crotanecine-based anacrotine (68). In contrast to many other pyrrolizidine alkaloid-containing genera, the alkaloids in Crotalaria species, which are concentrated in the pods and seeds, occur primarily in the free base form, but with total alkaloid levels as high as 3.85% (C. spectabilis) and 2.69% (C. retusa) (214). Contamination of feed with the seeds is therefore of serious concern and has caused toxicity in pigs and poultry (215,216). Long-term consumption of contaminated cereal grains has also led to poisonings of humans (217). CH3

HO

C H H O

O

CH2OH O C CH3

O

H

H

HO

CH3

C

O C

H3C O

H O

CH3

O

H

HO N

N

67

68

B. Toxicity and Clinical Signs Ingestion of plants containing pyrrolizidine alkaloids often results in toxic effects; however, the severity of the toxicosis is dependent on many factors, including chemical form, amount and rate of ingestion, species of animal, and whether any underlying conditions occur, such as liver disease. Although acute and peracute poisonings by pyrrolizidine alkaloid-containing plants have been reported in animals, the poisonings are generally more chronic in nature and often go undiagnosed until long after the ingestion of the toxic plant has occurred (197). Animals ingesting plants containing these alkaloids are often asymptomatic at the time of ingestion and may remain so until a stress-related event such as extreme cold, transportation, vaccination, or some other stressor impacts

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the animal’s response. This stressor can elicit a ‘‘hepatic crisis’’ resulting in liver failure, rapid weight loss, and death. This same series of events can occur in humans, and has been recently reported (2008–2009) in Afghanistan, and possibly also Ethiopia, where people allegedly ingested contaminated grains. Although horses, cattle, and sheep are the livestock species most affected by plants containing pyrrolizidine alkaloids, other species naturally poisoned or used as animal models have been ranked in the following order from most sensitive to most resistant (218): pigsWchickensWcattle/horsesWratsWmiceWsheep/goatsWJapanese quail/gerbils/ rabbits/guinea pigs. While differences in species sensitivity are generally attributed to the rate of production of pyrroles in the liver (219), other differences such as disease condition, age, gender, rumen adaptation, absorption, and excretion rates may also impact toxicity. Sheep have been shown to rapidly develop resistance to toxicity of monocrotaline from toxic Crotalaria seeds and this is believed to be associated with rumen adaptation (F. Riet-Correa, personal communication). However, the exact mechanism of detoxification is not fully understood. Three stages of pyrrolizidine alkaloid poisoning have been described in animals, namely, chronic, acute, and peracute. These stages depend on the chemical constituents of the plant, the quantity of alkaloids ingested, the alkaloid types, and the profiles and rate of ingestion. In field cases, chronic poisoning is usually observed, with acute and peracute stages generally resulting from experimental administration of plant material, extracts, or purified alkaloids. While different organ systems may be involved, a common thread in pyrrolizidine alkaloid poisoning is liver disease. In some cases, especially in horses, a neurological syndrome may be manifest, yet no lesions are described. It is believed that the liver dysfunction in the horse results in abnormal protein metabolism and a buildup of ammonia in the blood. In cattle, horses, and sheep the liver disease generally progresses slowly with little overt evidence of poisoning. If ingestion of toxic plants ceases, progression of the disease may also cease, however, later stressors may trigger a hepatic crisis with no further alkaloid ingestion. This chronic form of the disease has been recognized by cattle producers for many years in the Northwest, and animals were often marketed in a relatively healthy condition, yet die at a later date from liver failure exacerbated by some stressful event.

C. Structure–Activity Relationships and Mode of Action The extraordinary diversity of pyrrolizidine alkaloid structures and their occurrence in free base and N-oxide forms complicate any understanding of the mode of action and structural-based toxicity predictions. Furthermore, the pyrrolizidine alkaloids are not toxic per se but require

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metabolic transformation for toxicity to be manifested. There are enormous differences in species susceptibility to these alkaloids, due to differential rates of absorption and excretion, monogastric and ruminal metabolism, and structural type. The metabolic activation to the proximate toxin is now well understood and differences in susceptibility due to animal species, age, sex, and numerous other factors have been comprehensively reviewed (194). Fundamentally, as illustrated for riddelliine (55), hepatic oxidation leads to formation of the highly reactive pyrrole (dehydropyrrolizidine or dihydropyrrolizine) derivative 69, subsequently undergoing rapid hydrolysis to a highly reactive necine-based pyrrole 70, which acts as a bifunctional alkylating agent, capable of reacting with nucleosides and nucleic acids, and cross-linking DNA (Figure 2). Specific cytochrome P-450 enzymes are responsible for the oxidation and activation of these alkaloids, and the liver is the primary organ affected, since these reactive metabolites are formed in situ. The changes observed are necrosis, atrophy, fibrosis and bile duct proliferation, and megalocytosis, resulting in cirrhosis and hepatic insufficiency. Long-term (2-year) studies in rats and mice resulted in high mortality in some groups and hemangiosarcomas in the liver as a consistent feature (220). As a result of this and other studies, riddelliine has been nominated as a candidate substance for listing as ‘‘reasonably anticipated to be a human carcinogen’’ (221). In contrast to most pyrrolizidine alkaloids, the metabolites of the 11-membered macrocyclic pyrrolizidine class occurring in Crotalaria species appear to be longer-lived, and are excreted into the bile and blood, ultimately causing nephro- and pneumo-toxicity. The effects of monocrotaline on the lung are particularly noticeable, with amounts insufficient to affect the liver nevertheless causing marked changes in pulmonary vasculature. In competition with metabolic activation are ester hydrolysis to the necine base and necic acid and N-oxidation, both of which produce water-soluble products that can be easily excreted. The large proportion of N-oxides present in many pyrrolizidine alkaloid-containing species raises the question as to whether these should be considered as contributing to the hepatotoxicity. Their high water solubility makes them susceptible to urinary excretion, and since they are at the same oxidation state they cannot be converted directly into the metabolically produced pyrrole. However, the potential exists for them to be at least partially reduced to the free base form in the digestive tract, especially in ruminants. An experiment with feeding Senecio riddellii, and pure free base and N-oxide forms of riddelliine at the same dosage levels as in the plant, showed that the free base content (10% of total alkaloid) did not cause toxicity, but the N-oxide content (90% of total alkaloid) was sufficient (222). This indicates that a large proportion of the N-oxide must undergo reduction to the free base in the rumen, and subsequent oxidation to the toxic pyrrole in the liver.

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Alkaloids Toxic to Livestock

H

HO

C CH3 O

CH2 O

H

CH2OH O C

HO

C CH3

O

O

H

CH2

CH2OH O C O

O

[O] Hepatic metabolism

N

N 55

69

Hydrolysis

CH2

CH2

R′O

OR

or N

N 70

Tissue-bound nucleophile (Nu-Tissue)

R′O

CH2

Nu

Tissue

Tissue

Nu

CH2

OR

or N

N

Tissue-bound pyrroles

Figure 2 Metabolic transformation of pyrrolizidine alkaloids to hepatotoxic pyrrole derivatives, illustrated for riddelliine (55).

It is difficult to assess the relative toxicity of the various structural classes of pyrrolizidine alkaloids because toxicology tests have been done with many different animal species under various conditions. From experiments conducted under similar conditions (typically in laboratory animals such as rats and mice) it appears that the macrocyclic diesters are the most toxic, followed by the nonmacrocyclic diesters, the seco-diesters, and finally, the monoesters (194). The alkaloids with a saturated necine base, and the necine bases themselves, are not hepatotoxic.

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X. ISOQUINOLINE ALKALOIDS A. Plant Species and Alkaloids Isoquinoline alkaloids are common to the poppy family (Papaveraceae). In this family, plants of most concern for livestock poisoning are Argemone spp. (prickly poppies), with A. polyanthemos and A. mexicana being the most widespread. In addition A. albiflora is found in the eastern and southeastern United States and A. munita in Nevada, Utah, Arizona, and the coastal areas of California. Other members of the family that may be potentially poisonous are Chelidonium majus (celandine), Papaver (poppy) spp., some of which are native and others introduced, Sanguinaria canadensis (bloodroot), and species of Eschscholtzia native to western North America, including E. californica, the state flower of California. O

O

N

N

CH3

O

O

O OCH3

O

OCH3

O

71

72

O

CH3O N

O

CH3

HO

5 6

O

HO

N CH3

O

CH3O 73

74

The core isoquinoline nucleus may be present as such, or as a structural moiety integrated into alkaloids classified as benzophenanthridines, morphinans, protoberberines, and protopines (223). In Argemone spp., the most common alkaloids are of the berberine and protopine types, the latter having a structure in which the nitrogen-containing ring is cleaved to give keto- and N-methyl- groups; this feature is reminiscent of the seco-pyrrolizidine alkaloids. When cut, the seedpods of A. mexicana exude a pale yellow latex containing berberine (71) and protopine (72),

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and the seeds contain dihydrosanguinarine (73) and sanguinarine (D5,6-73), which can be extracted into the seed oil; the benzylisoquinoline alkaloid reticuline (74) is also present in the plant. The chemotaxonomy of the Papaveraceae family with respect to alkaloids has been reviewed by Preininger (224).

B. Toxicity and Clinical Signs Contamination of edible mustard seed oil with Argemone seed oil has caused poisoning in humans in India (225,226). Contamination of feed with Argemone seeds appears to be the major route for animal poisoning, in particular poultry. As with almost all members of the Papaveraceae family, the plants are unpalatable and rarely eaten. The Papaveraceae family contains a large array of isoquinoline alkaloids that have been extensively characterized and have significant medicinal uses. A familiar species is P. somniferum, which is the source of opium and is cultivated in some countries for illicit drug production. The most important class of these alkaloids is referred to as the morphinoids, and includes codeine, morphine, oripavine, and thebaine. These are important in medicine and are extensively used, primarily for pain management and control. However, they are addictive and have created numerous social and associated health problems throughout the world. The plant genera listed above are generally very distasteful and toxicosis in animals, particularly livestock, is rare. However, clinical signs have been reported in cattle ingesting P. somniferum, including restlessness, loss of appetite, drowsiness, induced deep sleep, and lingering debilitation, but no death (227). In dogs, restlessness followed by ataxia, drowsiness, and a hypnotic stare were reported, but no death (228). In general, clinical signs are those to be expected from the morphinoids, namely, analgesia, drowsiness, decreased GI motility, slowed respiration, and in some cases mood alterations, nausea, and/or vomiting. When lethal doses are ingested one might expect similar signs to those reported for human overdoses, such as ataxia, restlessness, miosis, decreased respiration rate, coma, and death. There are few reports of poisoning by the other genera, and some of those are experimentally induced. For example, Argemone seeds fed as a complete diet to rats for several days induced sedation, depression, ventral edema, and death. Pathology included mild-to-moderate hepatocellular necrosis and other degenerative lesions in the GI tract, kidney, and liver (229). Similar effects have been described in cattle fed hay contaminated with Argemone (2). Ventral subcutaneous edema, ascites, hydrothorax and centrilobular, and portal fibrosis were reported. Many of these plants have been associated with folklore and early medicinal remedies, but little experimental substantiation has been

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reported. Argemone oil extracted from seeds reportedly was used as an emetic and cathartic, much like castor oil, and congestive heart failure (epidemic dropsy) and other related issues associated with this were noted (230). The alkaloids include protopines, protoberberines, and benzophenanthridines, with the protopine types being the most common (223). Extracts from Chelidonium species are reported to relieve bloat, nausea, and cramps, the effects believed to be from aporphine, protoberberine, and benzophenanthridine alkaloids (227). Extracts from Eschscholtzia species used as a herbal remedy are reported to cause sedation and increased pentobarbital-induced sleep times in mice (231). E. californica contains similar alkaloids to Argemone, including protoberberine and simple benzylisoquinoline alkaloids (224).

C. Structure–Activity Relationships and Mode of Action The subclasses of isoquinoline alkaloids produce such different toxic effects that it is impossible to establish structure–activity relationships across all structural groups. However, the toxicity in livestock appears to be related to Na+-K+-ATPase inhibition (232,233), possibly resulting in inhibition of the active transport of D-glucose, which requires a sodium pump (234). The inhibition of Na+-K+-ATPase activity of the heart by sanguinarine is due to interaction with the cardiac glycoside receptor site of the enzyme, producing degenerative changes in cardiac muscle fibers. There is evidence that the toxicity of argemone oil may also be due to the production of reactive oxygen species (ROS), subsequently causing enhancement of lipid peroxidation (LPO) in hepatic microsomes and mitochondria. This damage in hepatic membranes causes loss of cytochrome P-450 responsible for xenobiotic metabolism, thereby leading to a delay in excretion of sanguinarine, or of its metabolites, through urine and feces. Sanguinarine is retained in the GI tract, liver, lung, kidney, heart, and serum of rats, even after 96 h of exposure, indicating that these are the target sites of argemone oil toxicity. Sanguinarine has been found to be toxic in vivo on intraperitoneal injection into mice (235), but oral administration of dihydrosanguinarine produced no toxicity in rats (236). While these results are not strictly comparable because of differences in mode of administration and animal species, a similar comparison exists with respect to cytotoxicity towards HL-60 human leukemia cells, with dihydrosanguinarine showing much less cytotoxicity than sanguinarine (237). Dihydrosanguinarine is the primary metabolite in rats (238), with maximum plasma levels of sanguinarine and dihydrosanguinarine being 0.6 and 1.6 mM, respectively, after a single intragastric dose of 10 mg/kg b.w. of sanguinarine (239). However, if metabolism is inhibited due to hepatic damage, the more toxic alkaloid may remain in circulation for a longer time period, resulting in increased toxicity.

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XI. DITERPENE (LARKSPUR) ALKALOIDS A. Plant Species and Alkaloids Diterpenoid alkaloids of concern for livestock poisoning have been identified in the genera Aconitum and Delphinium (Ranunculaceae). These can be classified into two general structural types, namely, the C19 norditerpenes and the C20 diterpenes. A recent review has cataloged 360 structures on the basis of 1H and 13C NMR spectra and physical constants, together with the plant species in which they occur (240). A number of other reviews exist, among the more recent of which are those covering the chemistry (241), and the toxicology and pharmacology (242). In spite of historical associations of Aconitum species with poisonings in humans, and their use for pest control and medicinal purposes, there are few reports of livestock poisonings in North America. A. columbianum (western monkshood) is the most widespread species in areas of livestock production, being found in the Western States from Mexico to British Columbia, but the plant is rarely grazed. The alkaloids in Aconitum species are norditerpenes of the aconitine type. Aconitine (75) is absorbed through skin and mucous membranes and produces a burning sensation in the mouth of humans. A similar perception in animals may discourage grazing of the plants by animals. HO CH3O

H

OCH3

H O

C2H5

N

COC6H5

OH OCOCH3

HO H OCH3 CH3O 75

In contrast to Aconitum species, many Delphinium species have been recognized as toxic to livestock since the earliest days of research on plants poisonous to animals grazing rangelands in the United States (243). A recent publication has summarized research on this problem over the past 100 years (244). Worldwide there may be as many as 300 species of Delphinium, of which 61 species have been defined in North America (245). The taxonomy is complex because it has been difficult to define species and varieties, and numerous synonyms have been applied (246). Hybridization for horticultural purposes has further exacerbated such problems, but the application of RAPD markers to study genetic

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variability has shown promise in resolving these difficulties (247). For practical purposes the toxic species of concern have been referred to as ‘‘tall,’’ ‘‘low,’’ and ‘‘intermediate’’ on the basis of their general appearance and phenology; currently, the preference is to classify them as ‘‘tall,’’ ‘‘low,’’ and ‘‘Plains’’ larkspurs. Major species of economic significance in the tall larkspur group are D. barbeyi, D. glaucum, D. glaucescens, and D. occidentale, growing primarily in mountain meadows where they appear immediately after snow melts and can comprise dense stands, generally attaining a height of 90 cm and above. The low larkspur group, consisting of D. andersonii, D. bicolor, and D. nuttallianum, also grows in mountain areas, but tends to favor brushy, drier habitats, reaching a height of only 60 cm at maximum growth. The Plains larkspur, D. geyeri, inhabits desert shrub and shortgrass prairie, with an intermediate maximum height of 80 cm. The toxic larkspurs can produce a broad array of 40 or more diterpene alkaloids, with about 25 having been tested for relative toxicity. Alkaloids common to many species are the 7,8-methylenedioxy lycoctonine (MDL) types, such as deltaline (76), which are relatively nontoxic; lycoctonine (77) and similar structural types having moderate toxicity; and the N-(methylsuccinyl)anthranoyl lycoctonine (MSAL) types with very high toxicity, such as methyllycaconitine (MLA) (78, R ¼ CH3). Together with MLA, three other alkaloids, namely, nudicaline (78, R ¼ COCH3), 14-deacetylnudicaline (78, R ¼ H), and deltaline (76) are primarily responsible for the toxicity of most tall larkspurs (248–251). In a mouse bioassay, MLA, nudicaline, 14-deacetylnudicaline, and deltaline had LD50 values of 4.8, 2.7, 4.0, and 133 mg/kg iv, respectively. Although deltaline is relatively nontoxic, it can occur at concentrations as high as 1–3% of the dry weight of the plant, and therefore cannot be disregarded as a contributor to larkspur toxicity. At such levels, the alkaloid may be toxic per se or exacerbate the toxicity of the more potent alkaloids. A series of publications have summarized many aspects of the relationship of alkaloid content to larkspur chemotaxonomy, toxicity, and management regimens (252–255). H CH3O HO

OCH3

H CH3O

H

H

H

OCH3 C2H5

N

OCH3 C2H5

N

O H HOH2C

O OCOCH3 76

OCH3

H HOH2C

OCH3 77

OH OH

Alkaloids Toxic to Livestock

H CH3O

H

193

OR

H OCH3

C2H5

N H OCH3

OH OH

O C O O CH3 N O 78

Depending on the species and environmental conditions, variable concentrations of highly toxic and relatively nontoxic alkaloids occur. For example, D. glaucum has particularly high levels of toxic alkaloids, while D. occidentale has fluctuating levels, with toxic alkaloids being absent from some populations, and D. barbeyi is intermediate, with levels declining at the pod stage. The mean concentration over the summer growing season of MLA in D. glaucum from California was 1.95%, compared to 0.9% in D. barbeyi and 0.35% in D. occidentale (253). As a general rule, there is a trend in tall larkspurs for declining toxicity with maturity, but palatability increases as the plant matures. The most toxic period around which these trends intersect is from the elongation of flower racemes to early pod stage, encompassing a period of about 5 weeks; this has led to the concept of a ‘‘toxic window.’’ Assessment of toxicity is therefore highly dependent on rapid analytical methods for measurement of total and individual alkaloids in plant samples, and FTIR and LC–MS have been employed for this purpose, as well as for serum analysis (256,257). A class-specific ELISA assay has also been developed for the toxic MSAL alkaloids, with a limit of detection of 30.5 pg for MLA (78, R ¼ CH3) (258).

B. Toxicity and Clinical Signs Several factors apparently affect larkspur toxicity, including the stage of plant growth, alkaloid composition and concentration, soil site, weather patterns, quality and quantity of alternate forage, animal species grazing, and others. Additionally, animal-to-animal variation in susceptibility to larkspur toxicosis was documented using inbred strains of mice as a model system for cattle, wherein a potential susceptibility factor was identified (259).

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Clinical signs in cattle generally begin with uneasiness, muscular fasciculations, drooping ears, lowered head (muscular weakness) rapidly progressing to a stiff gait, straddled stance, and sudden collapse, usually forelimbs first, but with the animal trying to maintain standing until complete collapse results. If the dose is high enough the animal will progress to lateral recumbency with possible vomition. Death occurs rather quickly from aspiration, asphyxiation, or respiratory paralysis. Heart and respiration rates are elevated. Sheep tolerate two to six times more larkspur than cattle, and therefore are better adapted to heavily infested ranges. Unfortunately, a declining sheep industry in the western United States creates a situation where less utilization of these ranges may increase cattle losses as a result.

C. Structure–Activity Relationships and Mode of Action The toxic principles of the larkspurs are of the norditerpenoid alkaloid class. Over 40 of these alkaloids have been identified and relative toxicities of about over 20 are known (254). There are three basic types of norditerpenoids in larkspur including the MSAL type, the lycoctonine type, and the MDL type. The MSAL types are the most toxic. These include, in order of toxicity by mouse bioassay and in vitro assay: nudicaline, 14-deacetyl nudicaline (14-DAN), MLA, and barbinine. All of these alkaloids contain the ester function at the C4 position, which is apparently essential for increased toxicity over the lycoctonine and MDL types. Of these four, MLA is believed to be the most prevalent of the MSAL type, and a major contributing factor in cattle poisoning. Deltaline (76), an MDL-type alkaloid, is generally the alkaloid of highest concentration, and even though it is 18–25 times less toxic than MLA, it is also a contributing factor in larkspur poisoning, having an additive effect on the toxicity of larkspur plants (260). When cattle were dosed orally with plant material from three different populations of tall larkspur that contained different ratios of MDL- to MSAL-type alkaloids, the larkspurs with a lower ratio of MDL-type alkaloids relative to MSALtype alkaloids required a greater amount of MSAL-type alkaloids to elicit clinical signs of poisoning. Consequently, both the amount of MSAL-type alkaloids and the amount of total (MSAL+MDL) alkaloids need to be fully characterized to obtain a more accurate assessment of the relative toxicity of tall larkspur populations. Clinical signs of poisoning are related to the mechanism of action, whereby the alkaloids act as nicotinic antagonists inhibiting neuromuscular transmission by blocking acetylcholine binding at the postsynaptic acetylcholine receptor (261). Steep dose–response curves in vitro for the norditerpenoid alkaloids indicate that most of the receptors must be

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blocked before muscle contraction is significantly reduced, and blood alkaloid levels even slightly above those that evoke the initial toxic signs are likely to be lethal. Drug therapies for larkspur-intoxicated cattle have shown that neostigmine given im at 0.04 mg/kg b.w. is effective in reversing the toxic effects of larkspur on heart rate and muscle tone in cattle. Neostigmine is a reversible cholinesterase inhibitor that is an improvement over the cholinesterase inhibitor, physostigmine, which has been used in the past. Neostigmine, unlike physostigmine, does not effectively cross the blood–brain barrier, which results in less central nervous system toxicity, and neostigmine directly stimulates muscle-type nicotinic cholinergic receptors (262,263). In mice, doses of the pure alkaloids given iv or ip result in rapid stimulation, resulting in muscle fasciculations and involuntary jumping until recumbency and death or recovery occur. This occurs within 1–2 min after iv injection. The availability of a large number of structurally related diterpene alkaloids, obtainable in significant quantities from larkspurs, has enabled structure–activity relationships to be elucidated, particularly when tested in a mouse bioassay (249,254,261). The essential structural feature for toxicity is the diterpene alkaloid core exemplified by lycoctonine (77) (LD50 449 mg/kg), but a methylsuccinimido anthranilic ester moiety appended at the C18 position, as in MLA (78, R ¼ CH3) (LD50 4.8 mg/kg), greatly enhances toxicity. On loss of the methylsuccinimido substituent, to give anthranoyllycoctonine, strong toxicity (LD50 21 mg/kg) is still retained, but the methylsuccinimido anthranilate ethyl ester alone has low toxicity (LD50 438 mg/kg), and the clinical responses were entirely different (254). However, hydrolysis and methylation of the succinimido ring to give a mixture of the two possible straight chain esters lead to no reduction in toxicity relative to MLA (78, R ¼ CH3). Another critical feature is an N-ethyl substituent, although most of the larkspur alkaloids have this functionality. Deltaline (76) (LD50 133 mg/kg) has relatively low toxicity, and other alkaloids bearing the same 7,8-methylenedioxy substituent are even less potent, but the presence of only a methyl group at C4 may play a role in view of the presence of bulkier substituents at this position in the more toxic alkaloids. The underlying mechanism of action of the diterpene alkaloids is curare-like competitive blockage of neuromuscular transmission at a1 nAChRs. A number of cholinesterase inhibitors have been shown to reduce deaths and toxicity in rat studies of larkspur poisoning (264), but practical application is limited. The inhibitor physostigmine prevented deaths in experimentally intoxicated cattle, but repeated injections were required (265). The larkspur alkaloids affect many different receptor subtypes involving muscle, ganglia, brain, and autonomic neurons (266). With such a diversity of bioactive alkaloids in the toxic larksur species,

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each alkaloid acting on one or many receptors, it is unlikely that any single treatment will prove effective for the prevention of livestock losses. In contrast to the larkspur alkaloids, aconitine (75), the primary toxic alkaloid of Aconitum spp., acts on the type-2 Na+ channel receptor in the heart and other tissues, causing a decrease in ion selectivity. At low doses this results in decreased heart rate, whereas higher doses cause arrhythmias, including ventricular fibrillation. Toxicity of aconitine is estimated to be ca. 2–3 mg in humans, for whom it is of particular concern because many traditional Asian medicines may contain this alkaloid (242,267).

XII. DITERPENE (YEW) ALKALOIDS A. Plant Species and Alkaloids In the Northern Hemisphere, the yew family comprises five genera and 17–20 taxa, but only the Taxus genus is recognized for toxicoses in animals. The English yew, Taxus baccata (Taxaceae), has a long history in Europe as a poisonous plant, and this reputation has continued with its introduction into North America, where it has been commonly planted as a hedge or shade tree. There are three native species of yews: T. brevifolia (Pacific yew), T. canadensis (Canada yew), and T. floridana (Florida yew), but none of these presents a serious poisoning problem probably because they are not common in the vicinity of grazing livestock. Another introduced species, T. cuspidata (Japanese yew), has been responsible for some poisoning problems. OR2

R1O CH3

CH3 CH3

CH3 O CH3

N

CH3

OCO H

HO

CH2

OH 79

OR2

R1O CH3

CH3 CH3

CH3 O CH3

OCO H

H

CH2

OH 80

N

CH3

Alkaloids Toxic to Livestock

HO

O

CH3

197

OH CH3 CH3

CH3 H3CCOO CH3

N

CH3

OCO

H OH RO 81

The yew has become renowned as a source of the anticancer drug, taxol (paclitaxel), but this compound cannot be classified as an alkaloid because the nitrogen atom exists as an amide. The toxins in yew are the taxines, with as many as 11 having been identified in numerous studies, but not all of these have been characterized. The taxines have been classified into two classes, taxines A and B, and labeled as ‘‘pseudoalkaloids’’ (268), although they would be regarded as true alkaloids by most definitions. The major alkaloid in T. baccata is taxine B (79, R1 ¼ COCH3, R2 ¼ H), accompanied by structurally related compounds, consisting of polyhydroxylated diterpenoid moieties known as taxicins, esterified with b-dimethylaminob-phenylpropionic acid at the 5-OH position and acetylated at the 9-OH or 10-OH, respectively. These include isotaxine B (79, R1 ¼ H, R2 ¼ COCH3), or they are deoxygenated derivatives such as 1-deoxytaxine B (80, R1 ¼ COCH3, R2 ¼ H) and 1-deoxyisotaxine B (80, R1 ¼ H, R2 ¼ COCH3). Other minor alkaloids are taxine A (81, R ¼ COCH3) and 2-deacetyltaxine A (81, R ¼ H), which have a rearranged diterpenoid skeleton. The history of structural elucidation of the taxines, together with the pharmacology and toxicity has been reviewed recently (269). The taxine alkaloids occur in all parts of the plant, except for the arils, but the needles are of most concern because these are the part eaten by animals. The crude taxine fraction constitutes about 0.5–1% of the dry weight of the needles, and about 40% of this consists of taxine alkaloids, with taxine B being predominant and comprising 30% (270). A comprehensive understanding of yew toxicity will require complete identification of all of the alkaloids, their distribution in different parts of the plant, and variation between species. Preliminary results have shown that highperformance liquid chromatography either alone or in combination with mass spectrometry may provide the necessary information (270,271), as well as permit diagnosis of poisonings from animal tissues (272).

B. Toxicity and Clinical Signs The yew is considered one of the most toxic plants to livestock and is believed to be one of the most dangerous shrubs in North America.

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Typically the clippings from shrubs or hedges are discarded where grazing animals are kept, and curiosity, together with apparent palatability, creates a dangerous combination resulting in poisoning and death. In horses, clinical signs are usually not seen because death occurs so rapidly. Often, horses will appear to drop suddenly and die within minutes. Poisoning in cattle may be more prolonged, showing signs of depression, weakness, nervousness, tremors, ataxia, jugular distention, dyspnea, and death. The lethal dose is so small that the animal is able to readily consume enough to cause death in a few minutes. In one such case, a group of 44 heifers consumed needles from a single yew tree (T. baccata) that had been discarded and ingestion of an estimated amount of needles that one could hold in two cupped hands resulted in death of the entire herd (273). This was a catastrophic accident, resulting in over $30,000 lost in a few hours, not to mention the emotional cost to those involved. Death occurs because of peracute cardiac failure and interference with cardiac electrical conduction. Treatments with iv atropine may be beneficial if used before clinical signs appear, and lidocaine has been used in human poisoning to treat the ventricular and rhythmic abnormalities (273,274).

C. Structure–Activity Relationships and Mode of Action The yew alkaloids are prone to rearrangement, and it is difficult to obtain any individual pure taxine in sufficient quantity for feeding experiments. Estimates of oral lethal minimum doses (LDmin) have therefore been extrapolated from feeding of yew leaves and estimated crude taxine content. On this basis, the LDmin value in cows is 10.0 mg/ kg, in sheep 12.5 mg/kg, in pigs 3.5 mg/kg, and horses are particularly susceptible with LDmin 1.0–2.0 mg/kg (269). A comparison of the effects of pure taxines A and B, together with related transformation compounds, on guinea pig isolated perfused heart and papillary muscle preparations indicated that taxine B was the most toxic (275). The core taxane alcohol showed no significant cardiotoxic effect, indicating the requirement for a b-dimethylamino-b-phenylpropionic ester substituent. Further detailed structure–activity relationships will require the isolation of significant quantities of individual pure alkaloids. The primary action of taxine alkaloids is on the cardiovascular system. Crude taxine extracts induce hypotension and brachycardia, leading to cardiac arrest. In an isolated frog heart, taxines depressed atrioventricular (A-V) conduction by alteration of calcium and sodium channel conduction (276). This effect was induced by direct action on the

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myocardium, rather than through mediation of the sympathetic or parasympathetic nervous system (277). Because the cause is cardiac failure, there are few warning signs prior to sudden death and postmortem lesions are few. Diagnosis of poisoning therefore depends on the examination of plant fragments in the digestive tract, or the analysis for phytochemical constituents characteristic of Taxus spp. (273).

XIII. STEROIDAL (VERATRUM) ALKALOIDS A. Plant Species and Alkaloids European species of the family Liliaceae, Veratrum album (white hellebore) and V. nigrum (black hellebore), are well recognized as poisonous to humans, either through accidental ingestion or as components of herbal remedies. The roots of V. nigrum have been used in Chinese herbalism, but it is difficult to prepare an effective dose without adverse consequences. The toxicity of Veratrum spp. has been reviewed (278). In North America, two species are of concern for livestock poisoning, V. californicum (California false hellebore or corn lily) and V. viride (Indian poke, Indian hellebore, green false hellebore). V. californicum is a Western species found in alpine meadows and open woodlands at higher elevations of the Rocky Mountains, Cascades Range, and the Sierra Nevada, often in dense stands when conditions are favorable. V. viride is native to eastern and western North America, and has been classified into two varieties, V. viride var. viride and V. viride var. eschscholzianum, respectively. Three other species of limited distribution are of little concern. All Veratrum spp. contain steroidal alkaloids, the majority of which are of the C-nor-D-homo type, in which the C ring is contracted and the D ring expanded in relation to the usual steroid structure. Representative of these alkaloids are veratramine (82), germine (83), cyclopamine (84, R ¼ H), cycloposine (84, R ¼ b-D-glucose), and jervine (85). Alkaloids with a typical steroidal ring system, such as rubijervine (86), also occur. The levels of both total alkaloid and percent cyclopamine are highest in the leaves during early growth, in the stems in midgrowth, and in the roots and rhizomes in late growth (279). An ELISA assay has been developed for cyclopamine and jervine (280). The chemistry and pharmacology of the steroidal alkaloid constituents of Veratrum have been covered in many review articles, the most recent in 2006 (281).

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CH3 CH3 H

H CH3

H N

H N

H HO H

CH3

H

OH

H

H

CH3

CH3

H

HO

OH H

82

OH OH

O OH

HO OH

83

CH3

CH3

CH3

H

H N

H O H

CH3

H

RO 84

CH3

CH3

H

O CH3

H N

H O H

CH3

H

HO 85

CH3 HO

H

CH3 H

CH3

N

H

CH3 H

H

H

HO 86

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B. Toxicity and Clinical Signs During the 1950s, up to 25% of pregnant ewes that grazed on pastures infested with Veratrum californicum in the mountains of central Idaho gave birth to lambs with serious craniofacial malformation (282). These malformations ranged from the gross anomaly of cyclops (a single or fused double globe) to less severe deformities of the upper and lower jaws. The Basque shepherds called the cyclopic defect ‘‘chatto,’’ which translated into ‘‘monkey faced,’’ and the condition became known as monkey-faced lamb disease (283). Losses from Veratrum have long been reduced or eliminated on these ranges because of research findings and recommended management changes. Recent research using these alkaloids, isolated and identified at the Poisonous Plant Research Laboratory, as molecular probes has opened a new frontier for human medical research, and interest in the chemotherapeutic properties of cyclopamine has changed the direction of the research from agricultural to biomedical (12,283). The description of the synopthalmia defects has been more closely investigated and the kinetics of cyclopamine in sheep have been described (284). This cyclopic defect is induced in the sheep embryo during the blastocyst stage of development, when the pregnant mother ingests the plant during the 13th or 14th day of gestation (285). Early embryonic death up to the 19th day of gestation and other defects such as limb defects and tracheal stenosis occur when maternal ingestion includes days 28–33 of gestation (284,286–288). The solanidine alkaloids are also found in many Solanum spp., and are toxic and teratogenic. The cholestanes have been used as hypotensive drugs, but are much less likely to induce the birth defects (289). The structure–activity relationship is very important in relation to the potential to produce birth defects. It is now known that this structure–activity relationship is key in the mechanism of action, which is the inhibition of the sonic hedgehog signaling pathway (283). This sonic hedgehog gene pathway and the subsequent downstream regulation of the expression of other genes have now been implicated in numerous cancers, birth defects, and other anomalies. The toxin cyclopamine has become a significant tool in the study of this very complex pathway. Clinical trials have been proposed, and studies are ongoing to further identify the hedgehog complex of genes, and to understand its mechanism and function in the formation and growth of numerous cancers, childhood birth defects, and the manipulation of regulatory pathways. Clinical signs of poisoning are most likely caused by neurotoxic cevanine alkaloids that are present in most species of Veratrum (c.f. Section XIV). Typical signs begin with excess salivation with froth around the mouth, slobbering, and vomiting, progressing to ataxia, collapse, and death.

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C. Structure–Activity Relationships and Mode of Action Over 50 complex steroidal alkaloids have been identified from the Veratrum spp. Five classes of steroidal alkaloids have been characterized: veratrines, cevanines, jervanines, solanidines, and cholestanes. The veratrines and cevanines are of considerable interest in toxicology, as they are neurological toxins and hypotensive agents that bind to sodium channels delaying closure and causing cardiotoxic and respiratory effects. The cevanine alkaloids, such as zygacine, are also found in death camas. The jervanines are most significant for their teratogenic effects, the most potent inducers being cyclopamine and jervine. The teratogenic potential of steroidal alkaloids has been reviewed by Keeler (290). Cyclopamine (84, R ¼ H) is the predominant alkaloid in V. californicum, and has been established as the primary teratogenic constituent. Of the 50 or more steroidal alkaloids and their ester and glycoside derivatives reported in Veratrum spp., 13 were tested in laboratory animals and only 3 were shown to be teratogenic when pure (291,292). These were cyclopamine (11-deoxojervine) (84, R ¼ H), jervine (85), and the glycoside of cyclopamine, cycloposine (84, R ¼ glucose). A number of analogs of cyclopamine, jervine, and solanidine were synthesized and tested in a hamster bioassay (293–295). Cyclopamine and jervine analogs lacking the C5–C6 or C12–C13 double bonds were teratogenic, suggesting that unsaturation was not required for activity, while N-methyljervine was as active as jervine, indicating that a free electron pair on the nitrogen atom was required for teratogenicity. Subsequently, it was shown that unsaturation at the C5–C6 was necessary for teratogenesis, with the additional requirement that the electrons of the basic nitrogen had to be a- to the steroid plane (296). These studies identified the structural requirements for formation of terata, but did not provide a mechanism. Recently, the gene Sonic hedgehog (Shh) was found to play a role in the development of cyclopia in mice (297), suggesting a specific molecular target for cyclopamine and other steroidal alkaloids (283). Signaling molecules secreted by Shh and its downstream genes Patched (Ptc) and Smoothened (Smo) regulate embryonic patterning, such as identity and position of organs. The teratogenic effects of cyclopamine are therefore a result of its ability to block activation of the Shh response pathway by influencing the balance between active and inactive forms resulting from a conformational change in Smo (298). Cyclopamine was recently shown to inhibit Shh signaling by binding directly to Smo, and to override the consequences of oncogenic mutations in Smo and Ptc (299,300). These findings have generated potential interest in cyclopamine and its analogs as drug candidates for the treatment of many forms of cancer.

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XIV. STEROIDAL (ZIGADENUS) ALKALOIDS A. Plant Species and Alkaloids The genus Zigadenus (Liliaceae) is largely confined to North and Central America, with about 20 recognized species. The most widely distributed are Z. elegans (mountain death camas), Z. nuttallii (Nuttall’s death camas), Z. paniculatus (foothill death camas), and Z. venosus (meadow death camas). Other species are of quite limited distribution, and are rarely consumed by livestock. The frequently encountered species are found in the mountain and intermountain areas of the western United States and Canada, with the exception of Z. nuttallii that occurs in the southern Plains. The common name derives from the similarity in appearance of the bulb to the edible camas or quamash (Camassia spp.), a staple food of Native Americans. Human poisonings have occurred because of confusion between the two genera (301). Death camas species typically appear in the Spring, when little other forage may be available, with the foliage withering by early Summer. All parts of the plant are toxic, but animals mainly consume the leaves and flowers, although bulbs may also be eaten when pulled out of the ground; the dried plant may also become incorporated into hay. Individual species vary considerably in their toxicity, reflecting differences in content and composition of the toxic principles. The toxins of Zigadenus species are steroidal alkaloids of the cevanine type, with structural affinities to some of those present in Veratrum. Chief among these are zygadenine (87, R ¼ H) and its acetyl derivative zygacine (87, R ¼ COCH3), and the corresponding angeyloyl-, vanilloyl-, and veratroyl-zygadenine monoesters, together with mono- and polyesters of germine (83). The steroidal alkaloid constituents of Zigadenus were extensively investigated and structurally characterized by Kupchan and coworkers (302) and reviewed as part of chapter on the Veratrum alkaloids (303). CH3

H N H CH3

H

H

OH CH3

H OH

H

OH OH

O RO OH

87

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Scanning thin-layer chromatography has been used to determine the alkaloid content of Z. venenosus throughout the growing season, and established that the highest levels occurred in reproductive organs such as flowers and pods, with zygacine levels ranging from 0.56 to 0.85% d.w. At this stage of growth, the bulbs had only low concentrations of zygacine (o0.05%) (304). A subsequent, more detailed, study of Z. venesosus var. gramineus showed that there was no diurnal fluctuation in zygacine content, nor was there any significant effect of elevation on the alkaloid level (305). The maximum zygacine content of the whole plant (0.46–0.53% d.w.) was at the vegetative and bud stage of growth, with a slight decline at the flower stage and a more marked decrease in the pod stage. The angeloyl and veratroyl esters of zygadenine were found to become the predominant constituents at mature growth and close to senescence, ranging from 0.24 to 1.14% d.w. Zygacine was also the major alkaloid in Z. venesosus var. venenosus, but was absent from Z. elegans, in which vanilloylveracevine was the major alkaloid.

B. Toxicity and Clinical Signs All death camas species are assumed to be toxic; however, variation in toxicity exists between species, and even within species, depending on season, climate, soils, and geographical locations. Poisoning in sheep, cattle, horses, pigs, fowls, and humans has been reported (4,306). The largest losses generally occur in sheep. Sheep are primarily affected because of their tendency to select forbs, particularly in early spring, when they are turned onto the range before grasses have emerged. Death camas is generally not palatable to livestock, but is one of the earliest species to emerge in the spring. Poisoning most frequently occurs in spring when other more palatable forage is not available, or on overgrazed ranges where there is a lack of more desirable forage. Poisonings have resulted due to management errors in which hungry animals were placed in death camas-infested areas (307). Clinical signs of toxicity are similar in all livestock poisoned by Zigadenus, irrespective of the species of plant involved (4,308). Excessive salivation is noted first, with foamy froth around the nose and muzzle, which persists, followed by nausea, and occasionally vomiting, in ruminants (307). Intestinal peristalsis is dramatically increased, accompanied by frequent defecation and urination. Muscular weakness with accompanying ataxia, muscular fasciculations, prostration, and eventual death may follow. The pulse becomes rapid and weak, and the respiration rate increases, but the amplitude is reduced. Some animals become cyanotic, and the spasmodic struggling for

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breath may be confused with convulsions. The lungs fill with fluid, contributing to the respiratory distress. The heart fails before respiration, and at necropsy the heart is usually found in diastole. A comatose period may range from a few hours to a few days before death. Pathological lesions are those of pulmonary congestion. Gross lesions in sheep include severe pulmonary congestion, hemorrhage, edema, and subcutaneous hemorrhage in the thoracic regions. Microscopic lesions include severe pulmonary congestion with infiltration of red blood cells in the alveolar spaces and edema. Diagnosis of poisoning may be established by clinical signs of toxicity, evidence of death camas being grazed, histopathological analysis of tissues from necropsied animals, and identification of death camas plant fragments in the rumen or stomach contents. Death camas plant fragments have very distinct chain-like cellular patterns that can easily be identified under a microscope (307). Clinical signs of poisoning are reported to be similar, no matter which species is ingested, suggesting that similar alkaloids are present. However, toxicity from species to species does vary, but this is thought to be associated with varying concentrations of the toxin (4).

C. Structure–Activity Relationships and Mode of Action The steroidal cevanine-type class, to which the toxic Zygadenus alkaloids belong, exhibits hypotensive, and to a lesser extent emetic, properties similar to those seen with structurally related Veratrum alkaloids (309,310). These alkaloids interact with open Na+ channels in cell membranes, delaying closure and permitting increased levels of Na+, Ca2+, and K+ ions to pass. Epimyocardial receptor cells are particularly sensitive, resulting in peripheral vasodilation, slowed heart rate, and hypotension. At high dose rates, arrhythmia and EEG alterations are observed (311). Death may therefore result from either coronary failure or respiratory depression. Structure–activity studies with natural and synthetic alkaloids have shown that esterification at positions 3 and 15 is required for high activity, but positions 6 and 7 need not be esterified. The ester grouping at position 3 need not be branched, but a branched chain ester at position 15 enhances activity. Oxidation of the alcohol group at position 16 to a ketone results in the loss of activity (312,313). The overall finding is that alterations in the structure of the ester moiety affixed at position 15 greatly affect the hypotensive potency, whereas considerable alterations can be made in the structure of the ester at position 3 without significantly altering potency.

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XV. CONCLUSIONS AND OUTLOOK In the 100 years since the pioneering studies by Marsh in the United States (58,243), an enormous amount of research has been done on plants poisonous to livestock. For much of this period, the work was essentially observational, devoted to identifying the species involved, the geographical areas infested, the conditions under which poisoning occurred, the animals most affected, and the means of prevention. However, without identification of the specific toxins and methods to analyze their changes in composition and concentration with plant growth stage and environment, the ability to predict poisoning outbreaks was essentially impossible. From the 1960s until the present there has been a growing emphasis on chemistry, especially of the alkaloids, as the essential feature of toxic plant studies. This has led to management techniques that have largely averted the historic, catastrophic animal losses from the major plants of concern such as locoweeds, larkspurs, lupines, and Veratrum spp. Similarly, the primary pyrrolizidine alkaloid-containing species have received considerable attention, but this class of alkaloids are so widely distributed across diverse plant genera that many remain to be studied. Other toxic plant species causing sporadic or episodic losses require more detailed investigation, even though the general class of alkaloids may be known. Chief among these are the yews and the death camas, in which slight variation in individual alkaloid structures may alter the relative toxicity. Opportunities also exist to investigate poisoning situations that may arise when animals consume more than one alkaloid-containing plant species concurrently. The ability of chemists to isolate large quantities of the pure alkaloids may provide animal scientists and veterinarians with the ability to investigate physiological effects, mode of action, and potential treatments. In addition, more subtle manifestations of toxicity, such as reduced immune response, reproductive failure, and inability to thrive, can be studied. Most importantly, the specific alkaloids can provide invaluable leads for medical research for the treatment of animal and human diseases, as has been already established with the steroidal alkaloids, such as cyclopamine, the polyhydroxy alkaloids, such as swainsonine and castanospermine, and the quinolizidine alkaloids, such as anagyrine.

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[288] R. F. Keeler, Cornell Vet. 80, 203 (1990). [289] W. Gaffield and R. F. Keeler, Pure Appl. Chem. 66, 2407 (1994). [290] R. F. Keeler, in: ‘‘Alkaloids: Chemical and Biological Perspectives’’ (S. W. Pelletier , ed.), vol. 4, p. 389. Wiley, New York, 1986. [291] R. F. Keeler, Phytochemistry 8, 223 (1969). [292] R. F. Keeler, Teratology 3, 175 (1970). [293] D. Brown and R. F. Keeler, J. Agric. Food Chem. 26, 561 (1978). [294] D. Brown and R. F. Keeler, J. Agric. Food Chem. 26, 564 (1978). [295] D. Brown and R. F. Keeler, J. Agric. Food Chem. 26, 566 (1978). [296] W. Gaffield and R. F. Keeler, Experientia 49, 922 (1993). [297] C. Chiang, Y. Litingtung, E. Lee, K. E. Young, J. L. Corden, H. Westphal, and P. A. Beachy, Nature 383, 407 (1996). [298] J. P. Incardona, W. Gaffield, R. P. Kapur, and H. Roelink, Development 125, 3553 (1998). [299] J. Taipale, J. K. Chen, M. K. Cooper, B. Wang, R. K. Mann, L. Milenkovic, M. P. Scott, and P. A. Beachy, Nature 406, 1005 (2000). [300] J. K. Chen, J. Taipale, M. K. Cooper, and P. A. Beachy, Genes Dev. 16, 2743 (2002). [301] K. L. Heilpern, Ann. Emerg. Med. 25, 259 (1995). [302] S. M. Kupchan, J. Am. Chem. Soc. 81, 1925 (1959) and previous publications in the series. [303] S. M. Kupchan and A. W. By, in: ‘‘The Alkaloids, Chemistry and Pharmacology’’ (R. H. F. Manske , ed.), vol. 10, p. 193. Academic Press, New York, 1968. [304] W. Majak, R. E. McDiarmid, W. Cristofoli, F. Sun, and M. Benn, Phytochemistry 31, 3417 (1992). [305] D. Makeiff, W. Majak, R. E. McDiarmid, B. Reaney, and M. H. Benn, J. Agric. Food Chem. 45, 1209 (1997). [306] D. J. Wagstaff and A. A. Case, Clin. Toxicol. 25, 361 (1987). [307] K. E. Panter, M. H. Ralphs, R. A. Smart, and B. Duelke, Vet. Hum. Toxicol. 29, 45 (1987). [308] K. E. Panter, D. R. Gardner, S. T. Lee, J. A. Pfister, M. H. Ralphs, B. L. Stegelmeier, and L. F. James, in: ‘‘Veterinary Toxicology, Basic and Clinical Principles’’ (R. D. Gupta , ed.), p. 825. Academic Press, New York, 2007. [309] S. M. Kupchan and W. E. Flacke, in: ‘‘Antihypertensive Agents’’ (E. Schittler , ed.), p. 429. Academic Press, New York, 1967. [310] C. H. Mullenax, W. B. Buck, R. F. Keeler, and W. Binns, Am. J. Vet. Res. 27, 211 (1966). [311] W. B. Buck, R. F. Keeler, and W. Binns, Am. J. Vet. Res. 27, 140 (1966). [312] F. L. Weisenborn, J. W. Bolger, D. B. Rosen, L. T. Mann, L. Johnson, and H. L. Holmes, J. Am. Chem. Soc. 76, 1792 (1954). [313] S. M. Kupchan, E. Fujita, J. C. Grivas, and L. C. Weaver, J. Pharm. Sci. 51, 1140 (1962).

CHAPT ER

4 Determination of Alkaloids through Infrared and Raman Spectroscopy Malgorzata Baranska1,* and Hartwig Schulz2

Contents

I. Introduction II. General Overview of the IR Spectrum of Nitrogen Bridgehead Compounds: Bohlmann Bands III. Purine Alkaloids IV. Opioid Isoquinoline Alkaloids V. Natural Isoquinoline Alkaloid Dyes VI. Antimalarial Isoquinoline and Naphthyisoquinoline Alkaloids VII. Benzylisoquinoline Alkaloids VIII. Tropane Alkaloids IX. Pyrrolidine Alkaloids X. Piperidine Alkaloids XI. Quinolizidine Alkaloids XII. Monoterpenoid Indole Alkaloids XIII. Protoalkaloids XIV. Conclusions References

217 219 220 228 233 234 239 241 243 244 246 248 251 252 253

1

Department of Chemistry, Jagiellonian University, Krakow, Poland

2

Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Quedlinburg, Germany

Corresponding author.

E-mail address: [email protected] (M. Baranska). The Alkaloids, Volume 67 ISSN: 1099-4831, DOI 10.1016/S1099-4831(09)06704-2

r 2009 Elsevier Inc. All rights reserved

217

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Malgorzata Baranska and Hartwig Schulz

I. INTRODUCTION Over the past decades, vibrational spectroscopy methods, that is, infrared (IR) absorption and Raman spectroscopy (RS) have been applied increasingly in food and agricultural research, as well as for industrial quality control of various plant products. In this context, the main focus was laid on the authenticity of the individual plant samples to discover different forms of adulteration. Although both mentioned techniques can be observed as a consequence of molecular vibrations, the resulting spectroscopic features may differ significantly due to the different excitation conditions and the observed physical phenomena. Midinfrared (MIR) and near-infrared (NIR) spectrometers operate with polychromatic light from which the liquid plant extract or solid sample absorbs specific frequencies corresponding to the individual molecular vibrations of the analyte (mostly fundamental vibrations for MIR and overtones, as well as combination vibrations, for NIR). In Raman spectroscopy, the plant material is irradiated with monochromatic laser light excited in the UV–Vis or NIR wavelength range. This radiation moves the molecule to a virtual energy level that is located above the continuum of the vibrational states if the excitation is performed by a UV–Vis laser. Applying a Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser excitation takes place in the range of higher vibrational overtones. Falling back to other excited vibrational levels by inelastic scattering, the emitted Raman lines (the so-called Stokes lines) can be observed at a lower frequency in the spectrum. The difference between excitation and the Stokes line usually matches very well with the vibrational bands registered in the corresponding MIR spectra. In most cases, excitation in the NIR range using a Nd:YAG laser emitting at 1064 nm provides the best option for the analysis of biomaterials because the spectra are fluorescence-free and do not cause the destruction of plant samples (1–4). However, the resolution and sensitivity of such measurements is usually not as good as those obtained by dispersive instruments with excitation in the UV–Vis range. To avoid strong fluorescence and to enhance the sensitivity and selectivity, the Raman measurement can be combined with the absorption of the sample on nanometal particles. This technique is called SERS (surface-enhanced Raman scattering), and allows enhancement of the Raman signals from molecules absorbed on the metal surface by six orders of magnitude or more due to electromagnetic and chemical factors (5). Another useful technique applied for in situ plant analysis is Raman mapping that allows chemical maps of the investigated samples to be obtained, and these can be directly compared with their visual image.

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The derived two-dimensional maps provide detailed information regarding the distribution of specific compounds, for example, secondary metabolites, occurring in the surface layer of the measured sample (6). Alkaloids represent a large and structurally diverse group of nitrogen-containing metabolites, and several of them are used for medicinal purposes. Only a few are derived from purines (e.g., caffeine), pyrimidines, or terpenes (e.g., aconitine), while the large majority of alkaloids are produced from amino acids. In spite of the fact that alkaloids show a broad range of different chemical structures and are of significant biological interest, so far only a few vibrational measurements of these plants substances have been published.

II. GENERAL OVERVIEW OF THE IR SPECTRUM OF NITROGEN BRIDGEHEAD COMPOUNDS: BOHLMANN BANDS IR spectroscopy has been used to assign the stereochemistry and conformational preferences of quinolizidines, indolizidines, pyrrolizidines, and other fused 6/5 and 5/5 ring systems, as well as their derivatives for many years. During the structural investigation of a large number of compounds containing such moieties, Bohlmann found that it was possible to distinguish between the cis and trans configurations of the nitrogen bridgehead arrangement (7). Most alkaloids are built on such systems, and consequently, based on the presence or absence of the so-called Bohlmann bands, the stereochemical configuration of these compounds can be established. The first correlation between the configuration about the ring fusion of a nitrogen bridgehead compound and the position of the IR bands in the 2800–2700 cm1 region was noted by Wenkert (8) in connection with studies on the stereochemistry of the yohimbines and related alkaloids. Comparison of the spectra of various pairs of C-3 epimers showed that all of the alkaloids possessing an a-configured hydrogen at C-3 gave rise to two or more distinct peaks of medium intensity on the low wavenumber side of the symmetric C–H stretching band. Alkaloids possessing a 3b-hydrogen showed only shoulders on the low wavenumber side of the main peak at 2860 cm1. Subsequently, Bohlmann found that it was possible to distinguish between the cis and trans configurations of the quinolizidine moiety: the trans-fused systems exhibited a characteristic series of bands in the IR spectrum between 2700 and 2800 cm1, which were absent when the quinolizidine was cis-fused. A number of simple quinolizidine and deuterated quinolizidines were synthesized, and it was demonstrated that the appearance of the band was due to the axial C–H bonds a to the nitrogen atom and trans to the

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nitrogen lone pair of electrons. Two such C–H bonds were found to be necessary for the bands to be observed. In cis-fused quinolizidines only one such bond is present. Rosen and Shoolery (9) and Becket and coworkers (10) found that only those alkaloids possessing in their preferred conformation the C-3–H bond and at least one more C–H bond trans diaxial to the nitrogen lone pair will exhibit bands in IR spectrum between 2860 and 2700 cm1. Those alkaloids which, in their preferred conformation, possess a C-3–H cis to the nitrogen lone pair will not exhibit those bands. Wiewiorski and Skolik (11) suggested that the lower frequency of the trans bands compared with the normal C–H region was due to charge delocalization of the nitrogen lone pair of electrons to the a axial C–H bonds. In the trans-fused quinolizidine system all three a-axial C–H bonds should take part in this delocalization. The axial C–H bonds on both C-4 and C-6 have the same symmetry and force constants, so vibrational coupling can occur, and two bands appear at 2800 and 2761 cm1, respectively. The first is due to asymmetric, and the second due to symmetric, stretching vibrations. In cis-fused quinolizidines both the chair–chair and the chair– boat conformations are considered. The chair–chair conformation has only one a-axial C–H bond, and this gives rise to a single band between 2840 and 2600 cm1. In the chair–boat conformation three a-axial C–H bonds are present, and these give rise to two main bands at ca. 2808 and 2761 cm1. Generally, it has been found that in order for Bohlmann bands to occur, one hydrogen on a carbon atom a to a nitrogen atom situated trans and axial with respect to the nitrogen lone pair of electrons is necessary. The intensity and the complexity of the absorption was found to be roughly proportional to the number of hydrogen atoms so situated. Some theoretical studies have been also performed, and these led to the conclusion that Bohlmann bands are similar in origin to the characteristic N-methyl bands due to Fermi resonance, which appear in the same region. Moreover, it has generally been ascertained that Bohlmann’s IR criterion, originally deduced for the quinolizidine system, is applicable to other saturated systems with a bridgehead nitrogen atom (7).

III. PURINE ALKALOIDS Fourier transformed (FT)-IR and FT-Raman spectra of xanthine (1), caffeine (2), theophylline (3), and theobromine (4) have been recorded, and the individual vibrational bands discussed in detail (12). Considering the individual structure and point group symmetry of the four molecules, a normal coordinate analysis has been applied to support the qualitative analysis of the vibrational spectra. Based on these data, most

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IR and Raman bands of the analyzed alkaloids could be successfully assigned and with the calculated force constants the individual potential energy distribution was described. The strong bands observed at 1658/ 1567 cm1 in FT-IR spectra and at 1600/1560 cm1 in FT-Raman spectra have been considered to be due to asymmetric and symmetric carbonyl stretching vibrations of xanthine (1). It was found that caffeine (2), theophylline (3), and theobromine (4) present corresponding bands in the same wavenumber region. Detailed band assignments are given in Table I. Similar results were obtained in a study using ab initio Hartree– Fock and density functional theory (DFT) methods for calculation of molecular structures and vibrational assignments of xanthine (1), caffeine (2), and theobromine (4) (13). O

OH

O

N

N

CH3

1 Xanthine

2 Caffeine

O N O

N

N

N

H

H3C

N

N

N

N HO

CH3

H3C

O

H N

H

N

O

CH3 3 Theophylline

CH3 N

N N

N

CH3 4 Theobromine

Caffeine (2) is naturally present in coffee beans, and guarana seeds also contain a relatively high concentration of this alkaloid (up to 8%), as well as theophylline (3) and theobromine (4) in smaller amounts. The FTRaman spectra of these products of the Amazonian rain forest were recorded by Edwards et al. for the first time (14). Theobromine (4) was distinguished from caffeine (2) and theophylline (3) by the presence of a band at 620 cm1, whereas the other two alkaloids show a strong feature at 556 cm1, and a medium doublet for caffeine (1) was seen at 643 and 741 cm1. Contrary to that, theophylline (3) presents a characteristic single Raman band located at 668 cm1. Furthermore, the authors succeeded in the discrimination of anhydrous caffeine and its monohydrate form by comparing the intensities of the carbonyl bands occurring at 1656 and 1698 cm1.

222

Vibrational spectral assignments of caffeine (2), theophylline (3), and theobromine (4) (12)

Vibrational frequency (cm1) Caffeine (2)

Assignment Theophylline (3)

Theobromine (4)

FT-IR

FT-Raman

FT-IR

FT-Raman

FT-IR

1189 (ms) 1600 (vs) 1130 (w) 1071 (w) 1548 (s) 923 (w)

1188 (w) 1600 (s) 1131 (w) 1080 (w) 1550 (s) 925 (w)

1195 (ms) 1622 (ms) 1125 (w) 1095 (w) 1556 (s) 925 (ms)

1195 (w) 1613 (ms) 1140 (w) 1099 (w) 1570 (ms) 940 (w)

1204 1592 1072 1041 1548

(w) (ms) (w) (w) (s)

1210 1600 1065 1046 1561

(w) (ms) (w) (w) (ms)

1285 (ms) 1326 (w) 1700 (vs) 1237 (s) 973 (ms)

1288 (w) 1331 (vs) 1700 (vs) 1241 (s) 975 (w)

1285 1310 1710 1240

1280 1313 1706 1244

1295 1334 1691 1224 1041

(w) (w) (vs) (s) (w)

1302 1340 1689 1231 1046

(ms) (vs) (vs) (ms) (w)

(ms) (ms) (s) (ms)

3377 (w)

(s) (s) (vs) (s)

3120 (w)

FT-Raman

C–C stretching C ¼ C stretching C–N stretching C–N symmetric stretching C ¼ O symmetric stretching N–CH3 symmetric stretching N–H symmetric stretching C–N stretching C–N stretching C ¼ N stretching C–N stretching N–CH3 stretching N–H stretching

Malgorzata Baranska and Hartwig Schulz

Table I

2954 (vs) 450 (w) 420 (w)

642 (w) 611 (w)

645 (w) 613 (w)

3001 (w) 450 (ms) 420 (ms) 1664 (vs) 980 (s) 1220 (w) 1195 (ms) 744 (s) 670 (w) 915 (ms) 970 (ms) 500 (ms)

2980 (ms) 450 (ms) 380 (w) 200 (w) 1663 (vs) 980 (ms) 1220 (w) 1195 (w) 760 (w) 660 (ms) 920 (w) 970 (ms) 450 (ms)

850 (ms) 615 (ms) 560 (w)

860 (w) 610 (w) 550 (s)

3027 (ms) 508 (w) 423 (w) 1667 (vs) 941 (w) 1173 (w) 1140 (w) 752 (ms) 733 (ms) 865 (w) 889 (w) 456 (ms) 508 (ms)

3007 (ms) 515 (w) 415 (w) 376 (w) 1660 (vs) 952 (w) 1183 (w) 1144 (w) 782 (w) 740 (w) 860 (w) 895 (w) 465 (w) 515 (ms)

684 (ms) 617 (ms)

681 (w) 626 (s)

C–H stretching N–C–C deformation C–N–C deformation (sym) N–C–N deformation C ¼ O asymmetric stretching N–CH3 asymmetric stretching C–N asymmetric stretching C–N asymmetric stretching O ¼ C–C deformation O ¼ C–N deformation N–C–H deformation N ¼ C–H deformation C–N–C deformation (asymmetric) C–N–CH3 deformation C–N–H deformation C ¼ C–N deformation C ¼ C–C deformation

Determination of Alkaloids through IR and Raman Spectroscopy

1660 (vs) 1025 (ms) 1210 (ms) 1189 (ms) 743 (s) 700 (w) 800 (w) 862 (w) 481 (ms) 550 (w)

2963 (vs) 444 (ms) 394 (w) 225 (w) 1656 (ms) 1020 (w) 1215 (w) 1188 (w) 745 (ms) 700 (w) 800 (w) 850 (w) 488 (ms) 556 (vs)

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Several attempts have been made to determine theobromine (4) in the context of developing quality parameters of raw and roasted cocoa beans, chocolate mass, and finished chocolate products applying various NIR spectroscopy (NIRS) methods (15). The main application of NIR calibration is found in the chocolate industry in improving quality control at all stages of processing. An NIR feasibility study to predict the theobromine (4) level in chocolate was performed by Kurowski et al. (16) obtaining good predictive quality for the concentration range 0.1–0.7 g/ 100 g. Also, Raman spectroscopy studies of cacao seeds and their extracts have been successfully performed (17). Characteristic theobromine (4) bands were detected at 1682 (C ¼ N stretching), 1594 (C ¼ C stretching), 1334 (C–N stretching), 1296 (C–N stretching), 1225 (C–N stretching), 776 (O ¼ C–C deformation), and 733 cm1 (O ¼ C–N deformation), which can be used as biomarkers for this alkaloid. Because the Raman spectra of powdered seed kernels do not show any spectral shift of theobromine (4), in comparison to the spectrum of the pure standard, it is assumed that this alkaloid is present in its free form (not coordinated with phenolic substances such as tannins). Beside theobromine (4), a small amount of caffeine (2) was also found, but no theophylline (3) was detected in the dried cacao powder. A special FT-Raman method has been developed to detect small amounts of caffeine (2) in commercial energy drinks (18). For this, the alkaloid was adsorbed on C18 solid-phase material, and subsequently measured between 3500 and 70 cm1. For quantification, the peak area values between 573 and 542 cm1 were used. The described combination of FT-Raman and solid-phase retention was found to enhance the sensitivity of direct Raman measurements of caffeine (2) by a factor of 31. Raman spectroscopy has been applied to determine in situ the distribution of caffeine (2) in a tablet (19). For these studies, the caffeine band at 555 cm1 (O ¼ C–N deformation vibration) was used for integration of the Raman map. Other active substances, such as paracetamol, which are also present in the tablet, could be clearly discriminated. The structural analysis and the analysis of fundamental vibrations of the FT-Raman spectrum were done with the help of the DFT calculations in order to improve the previous assignment and for a reliable analysis of SERS spectra. The BPW91 functional gave good results with respect to the calculated harmonic vibrational wavenumbers and no scaling was required. The vibrational behavior of caffeine (2) on an Ag colloidal surface at different pH values was described. A flat orientation of mainly chemisorbed caffeine (2) through the p electrons and the lone pair of the N atom was considered to occur for normal and basic pH values. At acidic pH values, caffeine (2) was probably adsorbed on the Ag surface

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225

through one or both oxygen atoms. The surface selection rules, along with the vibrational assignment of the SERS spectra and the theoretical results, reasonably explained the adsorption structure (20,21). Vibrational spectroscopy, combined with chemometry, has been used for various quality control tests and the quantification of the main components occurring in coffee beans and coffee beverages, which is the main source of the alkaloid caffeine (2). Of more than 70 coffee species (Coffea L., Rubiaceae) found worldwide in nature, only the following three have achieved commercial importance: Coffea arabica L., Coffea canephora Pierre ex Froehn., and Coffea liberica Hiern. Commercial coffee is mainly made from the two cultivars ‘‘Arabica’’ and ‘‘Robusta’’ or blends of them. Generally, ‘‘Arabica’’ coffee beans are viewed as superior in quality to ‘‘Robusta’’ and are therefore the more expensive of the two. Therefore, it is very important to be able to identify possible adulterations and mislabeling with special regard to consumer protection. The first successful attempts to use vibrational spectroscopy in this context were described by Downey et al. (22,23) applying NIR methods. They performed studies on whole and ground coffee beans using a factorial discriminant procedure. For the whole beans, in the absence of blended samples, a correct classification rate of 96.2% was achieved. Inclusion of blended samples into the calibration model reduced this figure to between 82.9 and 87.6%. Best results were obtained with the 1100–2498 nm wavelength range by a discriminant model using eight principal components, leading to four, well-separated clusters, for roasted ‘‘Arabica,’’ green ‘‘Arabica,’’ roasted ‘‘Robusta,’’ and green ‘‘Robusta’’ coffees. It was found that the discriminant profile of factor 1 was mainly related to water bands (1466 and 1962 nm) and absorptions of lipids (1209, 2308, and 2346 nm). Guyot et al. developed efficient NIRS methods for the simultaneous determination of caffeine and dry matter content in green ‘‘Robusta’’ coffees and mixtures of roast coffees (24). Based on these calibrations, 410 green coffee samples and 155 roast coffee mixtures were successfully characterized, regardless of their different geographic origin. Generally, the statistical parameters present a good correlation (R2 values W0.95, SECV comparable to the standard deviation values obtained by chemical reference methods). The NIR spectrum of pure caffeine (2) shows a significant absorption band between 2160 and 2320 nm with a maximum at 2250 nm. Downey et al. (25) also succeeded in discriminating lyophilized coffee powders and air-dried coffee beverages. Instant coffee, mixtures of coffee substitute samples made from malt (Hordeum vulgare L.), rye (Oryza sativa L.), chicory (Cichorium intybus L.), and sugar beet (Beta vulgaris L.) were also analyzed for their caffeine (2) and chlorogenic acid (5) content by FTNIR spectroscopy (26). The calibration function of actual and calculated coffee content in various instant coffee products shows a high correlation

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(R ¼ 0.999, SEC ¼ 1.30 g/100 g, calibration range: 0–100 g/100 g). In addition, the chlorogenic acid (5), as well as the caffeine (2), content were reliably estimated by these measurements (chlorogenic acid: R ¼ 0.987 and SEC ¼ 1.13 mg/g; caffeine: R ¼ 0.997 and SEC ¼ 0.8 mg/g). In comparison to the FT-IR spectra obtained from the same sample set, the NIR technique was found to be superior. The authors suggested that this is mainly related to the fact that FT-IR is more sensitive to the inhomogeneity of the individual coffee samples. OH HO

O

O

HO

OH

O

OH

HO

5 Chlorogenic acid

The application of FT-NIR to control the decaffeination process of coffee was first described by Davies and McClure in 1985 (27). Discriminant analysis of the spectral data showed a clear separation of normal instant coffees and decaffeinated instant coffee, both in the wavelength and Fourier domains. In order to determine which spectral data contributed mainly to the discrimination process, principal component analysis was applied to the measured spectra. The authors suggest that there is a ‘‘decaffeination vector,’’ which means that all of the decaffeinated samples appear in the same quarter of the principle component plot. They also conclude that FT-NIR data could be successfully used to detect departures from the normal variability of a production line. A reliable prediction of the caffeine (2) occurring in tea (Camellia sinensis L., Theaceae) beside other major compounds, namely, polyphenols and amino acids, was performed. The authors demonstrated that the NIR analysis time can be shortened by 2–3 min per sample using the noncrushed leaf samples compared with the method containing the crushed leaf samples (28). Based on the spectral data of 40 green tea samples, Xianming and Ning (29) found characteristic bands associated with caffeine (2), total nitrogen, free amino acids, and tea polyphenols. They applied MLR analysis and verified the established equations with

Determination of Alkaloids through IR and Raman Spectroscopy

227

31 green tea samples. The chemometrical results establish that the presented NIR method can be used without any difficulties for tea quality control purposes. Goto et al. (30) obtained very good correlation coefficients and low standard errors utilizing NIRS for the simultaneous analysis of caffeine (2), together with total nitrogen, free amino acids, tannin, ascorbic acid, theanine, and moisture content in green tea samples. A new NIR reflectance spectroscopy method for the rapid prediction of polyphenol and two alkaloids in green tea was described by Schulz et al. (31). Applying the PLS algorithm to the whole measurement range from 1100 to 2500 nm, good calibration statistics were obtained for the prediction of catechins, caffeine (2), and theobromine (4). In order to fulfill the legislative requirements for decaffeinated black tea, which is mainly produced for deliveries to the US market, quality control measurements must be continuously performed during the extraction process to guarantee a caffeine content of o1 g/100 g by weight. For this purpose, a specially adapted NIR method was developed using a FT-NIR spectrometer system equipped with a fiber-optic bundle and a rotating sample cup placed on an external integrating module (32). Due to a larger sampling area and the use of a rotating sample cup, the integrating module showed some advantages over fiber-optic measurements with respect to reproducibility and sensitivity. According to the different specification limits (o0.4 g/100 g and o0.1 g/100 g, respectively) two calibration sets were established. In spite of the fact that for the latter case the relative deviation between reference data (obtained by HPLC) and NIR is somewhat larger, the spectroscopic results are accurate enough to determine whether an unknown tea sample meets this specification or not. Some attempts were made to interpret the NIR spectrum of pulverized green tea. In this context, the NIR spectra of caffeine (2) and its related alkaloids, such as xanthine (1), theophylline (3), and theobromine (4), were studied in detail and the authors also tried to assign the individual absorption bands. Major differences seen in the spectra of the pure alkaloids seem to be caused by the different number of CH3 groups in these molecules (33). Dioicine (6) is a novel prenylated purine alkaloid, identified as the major lipophilic alkaloid extracted from leaves and seeds of Gymnocladus dioicus (L.) K. Koch (Fabaceae). This alkaloid functions as a ligand, like the well-known tobacco alkaloid nicotine (7), as well as the quinolizidine alkaloids cytisine (8) and sparteine (9). The structure of dioicine (6) resembles that of a caffeine derivative, and there are historical reports of the use of the G. dioicus tree as a coffee substitute. Gas chromatographic analysis, combined with IR spectroscopy, permitted an indication of some characteristic bands of dioicine (6). The spectrum showed a carbonyl vibration at 1710 cm1, with alkene/arene absorption at 1653

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Malgorzata Baranska and Hartwig Schulz

and 1531 cm1, partly overlapping with the phenyl vibrational modes. A weak band at 3380 cm1 indicated a possible NH stretching absorption. Condensed phase spectra of a purified sample indicated significant differences. While a sharp NH adsorption band was present at 3332 cm1, no obvious carbonyl signal could be identified above 1700 cm1. On the other hand, the major absorptions were seen at 1663 and 1634 cm1, possibly due to intermolecular interaction in the condensed phase. Analysis of related species indicated that none contained dioicine (6), except for G. dioicus; extracts from the leaves and seed shells contained the highest amount of dioicine (6), leaf stems also contained dioicine (6), but in much lower amounts, while neither young nor older bark contained detectable amounts of this alkaloid (34). NH H3C

N

O

CH3 N

N

N

H N CH3

CH3

N

6 Dioicine

7 Nicotine

NH O

H

N N 8 Cytisine

N H

9 Sparteine

IV. OPIOID ISOQUINOLINE ALKALOIDS Poppy (Papaver somniferum L., Papaveraceae) is well known as a medicinal plant for the treatment of pain in various regions in the world. In Europe, the cultivation of poppy started with the extraction of alkaloids, such as morphine (10) and codeine (11), from the dry poppy capsule. Today, poppy extracts with a high morphine content are predominantly used in the pharmaceutical and chemical industry, whereas poppy seeds with a very low alkaloid content are mainly produced for bakers’ ware. Schulz et al. recorded the NIR spectra (1100–2500 nm) of numerous dried and

Determination of Alkaloids through IR and Raman Spectroscopy

229

milled poppy samples (35) and found good correlation coefficients between second-derivative spectra and the reference HPLC data. Beside morphine (10), other alkaloids, including codeine (11), papaverine (12), thebaine (13), and noscapine (14), occurring in lower amounts, could be determined with an acceptable predictive quality (Figure 1). The authors emphasized that the described method is very useful for the evaluation of poppy breeding material, for the quality control of incoming raw materials, and in the area of forensic analysis. HO

MeO

O

O H

N CH3

HO

H

NMe

HO

10 Morphine

11 Codeine

OMe H2C

MeO

OMe O

MeO

N H

MeO

NMe

HO

12 Papaverine

13 Thebaine

O NMe

O H3C O

H H O O CH3 O

O CH3

14 Noscapine

ATR-IR and FT-Raman spectroscopy methods have been also demonstrated to be very promising tools for the fast and reliable determination of the main alkaloids present in poppy plant material and in related pharmaceutical products (36). The methods were found to be

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Malgorzata Baranska and Hartwig Schulz

0.80 0.70

R2 = 0.99 SECV = 0.059 %

NIR prediction (%)

NIR prediction (%)

0.60 0.50 0.40 0.30 0.20

Morphine

Codeine

0.10 0.00 -0.10 -0.10 0.00 0.10

0.20 0.30

0.40 0.50 0.60 0.70

0.80

reference (%)

reference (%) 0.45

0.14

R2 = 0.97 SECV = 0.016 %

R2 = 0.94 SECV = 0.048 %

NIR prediction (%)

NIR prediction (%)

0.35 0.10

0.06

Thebaine 0.02

-0.02 -0.02

0.25

0.15

Noscapine 0.05

0.02

0.06

reference (%)

0.10

0.14

-0.05 -0.05

0.05

0.15

0.25

0.35

0.45

reference (%)

Figure 1 Reference HPLC values versus NIRS predictions of the individual alkaloids: morphine (10), codeine (11), thebaine (13), and noscapine (14) in dry powdered poppy capsules (results are expressed in % dry matter) (35).

very useful for the rapid characterization of raw material, as well as for the rapid, on-site control of poppy field cultivation. Raman spectra in the fingerprint range between 700 and 1500 cm1 show numerous sharp bands, which are mainly assigned to deformation and stretching vibrations of the alkaloid ring system (Figure 2). The strongest IR bands, which are predominantly due to –C–O–C– stretching modes, can be found in the 1050 cm1 region. Because of their similar molecular structures, morphine (10), codeine (11), and thebaine (13) show no significant differences in their Raman spectra. Characteristic bands are especially found in the range of ring stretching and deformation vibrations (1600–1650 cm1 and 630–650 cm1, respectively). Apart from these minor spectral variations, noscapine (14) shows comparatively strong, broad peaks at approximately 1765 cm1 in the IR, and at 1784 cm1 in the Raman spectrum, which can be described as the {C ¼ O stretching vibration of the lactone ring. The molecular similarity of morphine (10), codeine (11), and thebaine (13) is also reflected in the range between 2800 and 3100 cm1, where –C–H aromatic bands of these alkaloids can be observed. Generally, both complementary

231

1276

1455

1613

1784

2958

Determination of Alkaloids through IR and Raman Spectroscopy

642 605 362

1408 1388

631

628

1632

975

3073 3044

B

1642 1620

C

3021

2940

1632 1607

D

2962

Raman Intensity

2929

1603

E

A 3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

Figure 2 FT-Raman spectra of pure alkaloids occurring in poppy: (A) morphine sulfate (10), (B) codeine base (11), (C) thebaine base (13), (D) papaverine hydrochloride (12), and (E) noscapine hydrochloride (14). (*) –S ¼ O stretching vibration of sulfate (36).

spectroscopic methods are very sensitive tools for distinguishing the most important poppy alkaloids and they therefore allow for the detection of these active principles in different plant tissues, as well as in various alkaloid drugs, even in low concentration. The Raman spectrum obtained from the poppy milk (latex) presents very clearly characteristic morphine bands (e.g., peaks at 631, 1620, 1642, 3044, and 3073 cm1). An aqueous-ethanolic extract prepared from unripe poppy capsules also shows several specific signals that are assigned to various vibrational modes of the alkaloids. In comparison to Raman measurements, ATR-IR spectra are less resolved and the strong water absorption bands coincide with some of the key signals related to the alkaloids. Nevertheless, the strong bands in the region between 900 and 1100 cm1, resulting from –C–O–C– stretching vibrations of morphine (10), can be seen in the poppy tissue and also the aqueous-ethanolic extract. Generally, it has been found that for those alkaloids occurring in higher amounts in poppy capsules [morphine (10), codeine (11), papaverine (12), and noscapine (14)] the correlation coefficients between the spectral data and reference HPLC values demonstrate an acceptable

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predictive quality. Furthermore, the total alkaloid content of poppy samples could be determined with comparatively high accuracy (R2 ¼ 0.90 and 0.84 for Raman and IR, respectively) (36). SERS, FT-IR, and normal Raman spectroscopy methods, together with DFT computational methods, were employed to investigate the absorption and orientation of morphine (10) on a silver surface. The structure of morphine and its vibrational spectra were determined at the B3LYP/6–31 G(d) level, and allowed the assignment of the experimental bands. The results suggested that the molecule had a charge transfer adsorption on Ag island film, and both planes of its ‘‘T’’ type structure had a rather perpendicular orientation to the substrate, mainly via the lone-pair electrons on the oxygen atoms (37). Papaverine (12) hydrochloride and its neutral species were investigated by experimental methods and supported by theoretical calculations. The DFT-computed structural parameters and harmonic wavenumbers reproduced the experimental data well, and helped to characterize the vibrational behavior of the adsorbed species (38,39). A pH-dependent Raman study was possible at pH values o6.5; at higher values the solubility was dramatically diminished. The vibrational analysis of the Raman spectra allowed the two species of papaverine (12), protonated and neutral, to be distinguished. The pH-dependent SERS spectra of papaverine (12) on citrate-reduced silver colloid revealed two different chemisorbed species on the Ag colloidal surface: protonated and neutral – the protonated one being adsorbed with the phenyl group closer to the surface and the neutral one through the isoquinoline part, probably through the nitrogen atom. In either acidic or basic media, the oxygen adsorption of papaverine (12) species was excluded (21). A first approach to apply FT-IR spectroscopy for the quantitative determination of heroin (15), a semisynthetic opioid drug derived from morphine (10) by acetylation, was done by Ravreby using area integration of the intensive carbonyl band (40). Several years later, the same spectroscopic technique was successfully used for the identification of heroin (15) of different origin based on cluster analysis (41). Alternatively, Raman spectroscopy has been used as a forensic tool in the detection of street drugs, which are commonly a mixture of drugs and cutting agents (42). However, it must be mentioned that some problems may occur with the sensitivity of the detection in relationship to the complexity of diluents and the matrix of the drug. Recently, a fast and direct NIRS determination of heroin (15) in seized street illicit drugs was developed by Moros et al. (43). From a hierarchical cluster analysis of all of the samples, the authors built a PLS model working with zero-order NIR spectra in the wavelength interval between 1111 and 1647 nm. Under these conditions, only eight factors are required

Determination of Alkaloids through IR and Raman Spectroscopy

233

for calibration, therefore, the remaining samples were used to validate the proposed method. AcO

N

CH3 O

O O H AcO

N CH3

O O

15 Heroin

16 Cocaine

Raman spectroscopy of free drugs [cocaine (16), morphine (10), and heroin (15)] and their mixtures (cocaine–heroin and cocaine–morphine) was used to study the drug–drug interactions at the molecular level (44). The results were analyzed with the help of quantum–mechanical calculations (the complete conformational analysis, including frequency calculations, was conducted at the DFT level). Raman spectra reflected a clear interaction occurring between cocaine (16) and morphine (10), but not between cocaine (16) and heroin (15). Moreover, a noticeable interaction was detected for the cocaine–morphine mixture, and for the salt of the latter, whereas for the cocaine–heroin mixture no variation in the Raman bands was observed compared to the individual molecules, even 1 week after preparation of the mixture. In the fingerprint range of 50–1750 cm1, the Raman pattern of morphine (10) (especially the bands at 632, 775, 1066, 1347, 1419, and 1635 cm1 due to CH2, C ¼ C, and C–O deformation modes) was found to be strongly affected by the presence of cocaine (16). In the highwavenumber region (which contains the C–H stretching modes) new features were observed for the mixture, at 2959, 3019, 3052, and 3138 cm1, whereas the bands at 2841 and 2877 cm1 from cocaine (16), and at 2998 and 3000 cm1 from morphine (10), either disappeared or decreased markedly in intensity. The most affected bands were those assigned to the aromatic moiety of the morphine (10) molecule [n(C ¼ C) at 1635 cm1 and n(CH/CH2)ring at 2900–3100 cm1]. Consequently, for the cocaine– morphine mixture, formation of a binary complex involves the 3-phenolic group and the heterocyclic oxygen of morphine (10) and the carbonyl oxygen together with the methyl protons of the cocaine (16) methyl ester group. The obtained results provided evidence for the occurrence of cocaine–morphine interactions, both in the solid state and in solution (44).

V. NATURAL ISOQUINOLINE ALKALOID DYES Berberine (17), a quaternary ammonium isoquinoline alkaloid salt, is a yellow coloring substance present in the roots of various Berberis species

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Malgorzata Baranska and Hartwig Schulz

(Berberidaceae) shrubs, such as B. vulgaris L. in Europe and B. thunbergii DC. in China (45). Raman spectra from a pure berberine (17) standard can be recorded without any problems, and show well-resolved signals, whereas reliable Raman data from paper or silk dyed with berberine (17) are difficult to obtain due to high fluorescence and the low concentration of the analyte (46). This is the reason why the SERS technique was introduced to enhance the sensitivity of alkaloid signals (47). The authors demonstrated that SERS is a very useful approach for measuring berberine (17) in ancient textiles. They assume that silver nanoparticles, added as silver nitrate colloid to the sample, possess great affinity to the positively charged berberine (17) molecule. The most intense signals of the ordinary Raman spectrum occur at 1397 and 1520 cm1, and have been assigned as the in-plane and ring deformation vibrations, respectively. These lines are missing in the SERS spectrum, which is dominated by three bands at 729, 753, and 770 cm1, assigned as out-ofplane vibrations. Similar results for the SERS characterization of berberine (17) were described by Wang and Yu (48). Furthermore, they added DNA to the alkaloid, and observed that the SERS signals increased by a factor of 1.5, and also shifted in the spectrum. O O

N

+

OMe OMe

17 Berberine

Recently, the isolation and characterization of berberine (17) from the leaves of Naravelia zeylanica DC. (Ranunculaceae) were described (49). The alkaloid was extracted with methanol and subsequently purified by preparative TLC applying a solvent system consisting of methanol, water, and ammonium hydroxide in the ratio 8:1:1 (v/v/v). The isolated berberine (17) was characterized by IR, 1H NMR, and mass spectrometry. The IR spectrum was recorded with KBr pellets and showed the characteristic feature with a strong band at 1597 cm1 assigned to C ¼ C and C ¼ N stretching vibrations.

VI. ANTIMALARIAL ISOQUINOLINE AND NAPHTHYISOQUINOLINE ALKALOIDS The first approaches to develop antimalarial drugs were based on natural plant components such as quinine (18), isolated from the bark of different

Determination of Alkaloids through IR and Raman Spectroscopy

235

Cinchona species (Rubiaceae), especially Cinchona pubescens Vahl. Later on, other active substances, such as chloroquine and mefloquine, possessing a somewhat similar molecular structure, were synthesized (50). The bark of trees in the Cinchona genus is the source of a variety of alkaloids, the most familiar of which are the following pairs of stereoisomers: cinchonine (19) and cinchonidine (20), quinine (18) and quinidine (21), and dihydroquinidine (22) and dihydroquinine (23). Quinine (18) and cinchonine (19), beside antimalarial properties, were shown to have a potential use in reversing multidrug resistance in cancer patients and are considered as first-generation blockers. It was also suggested that they have intracellular protein targets that may be involved in drug distribution (51). Moreover, quinine (18) and quinidine (21) are bitter and due to this property are used as food additives. Despite the supposed universality of bitter taste rejection, many commonly consumed food and beverages have bitterness as a major sensory attribute, which, in the overall taste profile of a food, is often appreciated by consumers. Quinine (18) has been used for many years as a bitter agent in some tonic-type drinks and alcoholic beverages (at a concentration of approximately 80 mg/L) and to a small extent in flour confectionery. CH CH2 HO

H

CH CH2 H

H N

OH H N

MeO N

N

18 Quinine

19 Cinchonine

CH CH2

CH CH2 HO

H

H

H N

OH H N

MeO

H N 20 Cinchonidine

N 21 Quinidine

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Malgorzata Baranska and Hartwig Schulz

C2H5 H

C2H5

OH

HO

H N

MeO

H H N

MeO N 22 Dihydroquinidine

N 23 Dihydroquinine

The high potential of resonance Raman spectroscopy was demonstrated by the detection of small amounts of these alkaloids in plant materials, without any further sample preparation. The nonresonant Raman microscopy with excitation wavelengths in the visible (532 and 633 nm) and NIR (1064 nm) region was unsuccessful due to a strong fluorescence background and due to too noisy signals resulting from low laser power, respectively. The use of higher laser power resulted in destruction of the plant material. Nonresonant Raman spectroscopy suffers from low scattering cross section and the active agents are present at low concentration in the biological environment; the signals can be strongly enhanced if the excitation laser lies within an electronic absorption of the sample. Consequently, UV resonance Raman microspectroscopy (lexc ¼ 244 nm) was applied for the highly sensitive and selective localization of quinine in Cinchona bark. The applied methodology allowed Raman spectra of the active agents to be obtained selectively, and enhanced the resonance signals by a factor of up to 106 compared to the background Raman signal of the plants. It was found that the UV resonance Raman spectrum obtained from cinchona bark corresponds well with that of pure isolated quinine (18). Moreover, in situ UV-RR microspectroscopy is capable of differentiating between quinine (18) and its diastereomer quinidine (21), structurally very similar active agents. This discrimination is possible via the transition at 831 cm1 in the Raman spectrum of quinine (18), which is shifted to 843 cm1 in the case of quinidine (21). This vibration involves a banding motion within the side chain around the chiral centre of quinine (18). Vibrations belonging to the quinoline ring, important for its antimalarial activity in forming p–p interactions to hemozoin, and the vinyl group are resonantly enhanced in the UV-Raman spectra. The interpretation of the experimental spectra was based on DFT calculations combined with FT-Raman spectroscopy of the pure isolated standards. DFT calculations were performed for a quinine (18) molecule in the gaseous phase, as well as for a hydrous environment to model this alkaloid in Cinchona bark. The solvent effect was correlated with the shift of the band at 1362 cm1 to 1371 cm1 for anhydrous quinine (18). This vibration is sensitive to the presence of an aqueous environment and is assigned to a C ¼ C stretching mode within the quinoline ring where the middle is narrowed while the sides are extended and vice versa (52).

237

Determination of Alkaloids through IR and Raman Spectroscopy

In order to study aqueous solutions of quinine (18) with a variable pH, 2D FT-Raman correlation spectroscopy was applied (53). The cross peaks of synchronous and asynchronous maps were associated with the protonation process recognized to be crucial in the context of the antimalarial activity of quinine (18). The two stereoisomers, quinine (18) and quinidine (21), were determined in their mixtures by using IR spectroscopy and chemometric multivariate methods [principal component regression (PCR) and partial least squares (PLS)]. The training set of 30 synthetic binary mixture solutions in the possible combinations containing 0.0–4.0% and 4.0–0.0% (w/v) quinine (18) and quinidine (21), respectively, in chloroform was used. To validate the developed calibration, mixtures of variable ratios in the range of 0.2–4.0 and 4.0–0.2 (w/v) for quinine (18) and quinidine (21), respectively, were used. The results of the analysis were found to be 100.570.44% (RSD% ¼ 0.44) and 100.570.38% (RSD% ¼ 0.38) for quinine (18) and 100.170.67% (RSD% ¼ 0.67) and 100.170.68% (RSD% ¼ 0.68) for quinidine (21) using PCR and PLS models, respectively (54). However, the protozoan parasites (species belonging to the genus Plasmodium) responsible for malaria became more and more resistant to the pathogen, thereby making these drugs decreasingly effective against the most dangerous Plasmodium strains (55). As a consequence of this development, antimalarial alkaloids [dioncophylline A (24), dioncophylline C (25), and dioncopeltine A (26)] belonging to the group of naphthylisoquinoline alkaloids were isolated from the tropical liana Triphyophyllum pelatatum (56). In order to locate those parts of the plant containing the highest concentration of these bioactive substances, Raman microspectroscopy was found to be a very efficient tool capable of differentiating between various structurally similar naphthylisoquinoline alkaloids (57). In this context, the signals registered at 1356 and 1613 cm1 assigned to C ¼ C stretching and C–H bending vibrations were especially useful for the reliable distinction of the different alkaloid structures (58). IR and Raman spectra obtained from the individual pure alkaloid standards, as well as in vivo measurements on the plant tissue, were presented and discussed in detail. Most of the identified signals could be successfully assigned to various vibrational modes of the alkaloid molecular structures. Me

HO

Me

NH OH

MeO MeO

24 Dioncophylline A

NH

Me

OH OH Me

OMe 25 Dioncophylline C

Me

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Malgorzata Baranska and Hartwig Schulz

Me

HO

NH MeO

Me

OH

HO 26 Dioncopeltine A

Another group of naphthylisoquinolines has been analyzed by Bringmann and coworkers (59). They applied FT-Raman spectroscopy to characterize the distribution of ancistrocladine (27), ancistrocladidine (28), ancistrocladinidine (29), ancistrocladisine (30), and ancistroheynine A (31) in plant cells of tropical lianas of the family Ancistrocladaceae. The authors found that FT-Raman spectroscopy is a very sensitive method for discriminating the above-mentioned five alkaloids, in spite of the fact that their structures differ only by the linkage between the molecular moieties, by the OH/OMe pattern, and by the N-functionality in the isoquinoline part. For example, the Raman spectrum of ancistrocladisine (30) clearly shows the cyclic C ¼ N stretching vibration at 1560 cm1, whereas ancistrocladine (27) does not present this spectral feature due to the free N–H function in the molecule (59). Systematic spectroscopic studies of different parts of the plant resulted in the spectroscopic localization of different alkaloids in various plant parts. Applying Raman microspectroscopy, the authors discovered that the roots of Ancistrocladus heyneanus contain small crystalline inclusions of ancistrocladine (27) in the plant tissue. OMe OMe

Me

Me

HO N

MeO OMe OH

Me N

H

OMe Me Me 28 Ancistrocladidine

OMe Me 27 Ancistrocladine OMe OMe

MeO Me

Me

Me N

Me

HO N OMe Me

29 Ancistrocladinine

OMe Me

MeO MeO

30 Ancistrocladisine

Determination of Alkaloids through IR and Raman Spectroscopy

239

Me Me

HO N

HO OMe Me

MeO

31 Ancistroheynine A

VII. BENZYLISOQUINOLINE ALKALOIDS Sanguinarine (32) and chelerythrine (33) are classified as quaternary benzophenanthridine alkaloids. They are encountered in many plant species in the family Papaveraceae, and significant levels of these alkaloids were found in the roots of greater celandine (Chelidonium majus L.). Some new drugs have been developed on the basis of sanguinarine (32), one is used to treat Trichomonas vaginitis, and the other, with pronounced immunostimulating properties, is a component of an antitumor treatment. Chelerythrine (33) is a potent, selective, and cellpermeable protein kinase C inhibitor. O H3C

N

+

OMe

O

H3C

O

O

O

O 32 Sanguinarine

+ N

OMe

33 Chelerythrine

Electrochemical oxidation of these two benzophenanthridine alkaloids [sanguinarine (32) and chelerythrine (33)] has been characterized by SERS excited in the NIR range (60). Several narrow signals were observed in the wavenumber range 1700–100 cm1, and the positions of many signals correspond very well between the spectra of these two alkaloids. For example, bands at 1648, 1550, 1487, 1401, 1373, 1353, 734, 675, 431, and 241 cm1 of sanguinarine (32) have their counterparts at 1641, 1546, 1483, 1401, 1377, 1345, 740, 664, 436, and 246 cm1 in the spectrum of chelerythrine (33), which reflects the structural analogy of both substances. Differences were identified in the C–H stretching vibrational modes (3100–2800 cm1) due to the methylendioxy group of sanguinarine (32). On the other hand, the Raman spectrum of chelerythrine (33) shows additional signals at 2946 and 2848 cm1 attributed to C–H stretching modes of terminating methoxy groups. Based on the spectral

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Malgorzata Baranska and Hartwig Schulz

data it is assumed that electropolymerization of the alkaloids proceeds via formation of ortho-benzoquinone derivatives. In aqueous solution in acidic and neutral media, the sanguinarine (32) molecule is present in the form of the cation, while in an alkaline medium, it is present in the form of a neutrally charged molecule. Raman spectroscopy also allows monitoring of the structural changes of molecules in aqueous media; however, relatively large amounts and high concentrations are needed to record conventional spectra. SERS spectroscopy, with its inherent high sensitivity and selectivity, allows determination of the presence of a specific compound in trace amounts. SERS spectra of sanguinarine using a silver hydrosol and an electrochemical cell in aqueous salt solution at different pH values were reported (61). In order to obtain the SERS spectra, a three-electrode electrochemical cell, with a solution of sanguinarine (32) of concentration 7.2 106 M/L in a 0.1 M NaCl solution, was used. To assign the bands in the SERS vibrational spectra, computer chemistry methods were applied. It has been observed that upon the absorption on a silver surface in both acidic and alkaline media, the cationic form of sanguinarine (32) appears, where the molecule interacts with the surface through all its rings, since all the groups of atoms participate in the vibrations appearing in the SERS spectra. Only one band with frequency 1520 cm1, appearing in the SERS of sanguinarine (32) adsorbed on the silver colloid and on the surface of a silver electrode at the zero-charged potential of silver, can be satisfactorily identified with the neutral form of the sanguinarine (32) molecule. Table II gives the frequencies of bands in the SERS spectra of sanguinarine (32) adsorbed on the surface of a silver electrode and a sliver hydrosol at pH 5.6, together with the calculated frequencies and assignment of the vibrations of a sanguinarine (32) molecule in the cationic form. The SERS spectra of sanguinarine (32) adsorbed on silver colloids and on the surface of a silver electrode are very similar in shape in acidic and alkaline media; the only difference is that the SERS spectrum in alkaline medium has considerably lower intensity. Moreover, the intensity of the bands in the SERS spectra of sanguinarine (32) in cationic form strongly depends on the applied potential. The bands most sensitive to the applied potential are as follows: 1284, 1346, 1457, 1540, and 1604 cm1, they are associated with vibrations involving the nitrogen atom bearing an uncompensated positive charge (61). Isotetrandrine (34) is an alkaloid extracted from the roots of Mahonia beali Pynaert (Berberidaceae) and shows anti-influenza effects. Its structure was established, in part, based on FT-IR spectroscopy. The spectrum showed the presence of saturated C–H by the absorption at 2936 cm1; the presence of a phenyl moiety by the absorptions at 1606, 1583, 1500, and 1445 cm1; the presence of methoxy groups by the absorption at 1232 cm1; and the presence of –NCH3 by the absorptions at 1335 and 2790 cm1 (62).

Determination of Alkaloids through IR and Raman Spectroscopy

241

Table II Assignment of selected (most intense) sanguinarine (32) vibrations appearing in the SERS spectra (61) Experiment Electrode Solution

Calculations Band assignment DFT/6-31G, scaling factor 0.95

1635

1635

1641

1604

1604

1610

1585 1414 1346

1585 1414 1346

1585 1415 1351

1284

1284

1279

1181

1181

1210

1108

1108

1106

n(C ¼ C) of ring 2,4,5; n(C–C), n(C–C) of ring 3 n(C–C) of ring 4,5; n(C–N) of ring 3; n(C–C) of ring 2; d(HCH), n(C–O) of ring 1 n(C–N), n(C–C) of ring 3; d(HCH) n(C–C) of ring 2,4; n(C–C), n(C–N) of ring 3 n(C–C), n(C–N), d(NCH) of ring 3; n(C–C), d(CCH) of ring 4,5,2 n(C–C), d(CCH) of ring 5; n(C–O) of ring 6; n(C–C), d(CCH) of ring 2; n(C–C), d(NCH) of ring 3 n(C–N), d(NCH) of ring 3; n(N–CH3), d(NCH) d(CCC), n(C–C) of ring 2,3; n(C–O) of ring 1 OMe OMe

OMe

N

N

Me

Me

O OMe 34 Isotetrandrine

VIII. TROPANE ALKALOIDS Cocaine (benzoylmethyl ecgonine) (16) is a crystalline tropane alkaloid that is obtained from the leaves of the coca plant (Erythroxylum coca Lam., Erythroxylaceae). FT-Raman spectra were recorded from a series of 33 solid mixtures containing cocaine (16), caffeine (2), and glucose [containing

242

Malgorzata Baranska and Hartwig Schulz

cocaine (16) in amounts varying between 9.8 and 80.6 wt%] (63). It was found that 98% of the analyzed samples could be definitively classified according to their individual cocaine (16) concentration. Quantitative calibration models have been developed using PLS algorithms, which allowed very precise predictions (RMSEP ¼ 4.1%) of the cocaine (16) content in the solid mixture. Cocaine was also investigated by SERS on silver tetrahydroborate and citrate colloids. A high-quality SERS signal was obtained at the concentration of 1000 mg/mL (64). The characteristic IR spectra of scopolamine (35) hydrobromide was presented by Brannon and Levine (65) for the first time. The interaction of scopolamine (35) and cholesterol with shingomyelin bilayers of porcine brain has been investigated by FT-Raman spectroscopy (66). The Raman band at 717 cm1, originating from the totally symmetric C–N stretching mode of the C4N+ group (gauche conformation), remained invariant after adding scopolamine (35); no additional band at 770 cm1 associated with the backbone of O–C–C–N+ in its trans conformation was observed. These results indicate that scopolamine (35) does not change the conformation of the O–C–C–N+ backbone in the choline group of shingomyelin bilayers. ATR-IR spectroscopy has also been used to characterize the variation of scopolamine (35) in a transdermal therapeutic system (67). For identification of the alkaloid, the bands at 1251, 1165, and 853 cm1 were used. Quantification of scopolamine (35) was based on the intensive carbonyl signal at 1725 cm1. After an application time of 2 h on human skin, a significant decrease in scopolamine (35) could be measured on the transdermal patch containing the active principle. Vibrational circular dichroism (VCD) spectroscopy has been used to determine the absolute configuration of anisodamine (6b-hydroxyhyoscyamine) (36) (68), which is a well-known tropane alkaloid found in the roots and leaves of various species in the genera Physochlaina, Scopolia, Duboisia, and Datura (69). The predicted VCD and IR spectra of the two naturally occurring diastereoisomers were calculated applying DFT. It was found that the predicted IR spectra of both molecules show good agreement with the original measurements. NCH3

NCH3 OH

O O

OH

HO O

O

35 Scopolamine

O

36 Anisodamine

Determination of Alkaloids through IR and Raman Spectroscopy

243

IX. PYRROLIDINE ALKALOIDS In 1978, Hamid et al. developed a NIRS method to predict the total content of alkaloids in cured tobacco samples (70). From 68 samples, with a total alkaloid content ranging from 0.78 to 6.1%, the individual reflectance spectra were recorded. The authors concluded that their method had some potential for the tobacco industry, but they also stressed that the NIR technique should be used with caution because the utility of the developed equations heavily depends on the selection of enough representative calibration samples. Nevertheless, the prediction quality of the proposed NIRS method was found to be comparatively high (R2 ¼ 0.95). A few years ago, the FT-IR spectra of tobacco leaves were measured in the range of 12000–4000 cm1, and an optimized calibration curve for the simultaneous analysis of nicotine alkaloids, total sugars, and total nitrogen content was calculated using the PLS algorithm (71). The study demonstrated that the best predictions for nicotine (7) content could be obtained when the following wavenumber ranges of the NIR spectrum were used for calibration: 9500–4231 cm1, 7502–4246 cm1, and 7502–4597 cm1. A fully automated FT-IR method for the determination of nicotine (7) in tobacco leaves was proposed by Garrigues et al. (72,73). Tobacco samples, weighed inside empty extraction cartridges, were humidified with ammonia and subsequently extracted with chloroform. The extracts passed through a microflow cell, and the IR absorbance was measured in the wavenumber range from 1334 to 1300 cm1. The described method provided a detection limit of 0.1 mg/mL nicotine (7). Results obtained for natural samples of cut tobacco and cigars correspond very well with those obtained by a batch FT-IR procedure involving the off-line extraction of tobacco samples. Stimulated Raman scattering from liquid nicotine (7), together with IR and spontaneous Raman spectra, has been recorded (74). The lowfrequency region was of special interest in that study. Raman intensity maxima occurred near 303, 260, and 115 cm1, with the half-widths ranging from 15 to 30 cm1. Such relatively sharp Raman bands almost certainly arise from intramolecular deformation or torsional oscillations of nicotine (7). Close inspection of the spectrum below 100 cm1 revealed an inflection near 65–75 cm1 and an accompanying wide region of downward concavity. These features may result from intermolecular effects. From the intensity, position, and half-width of the low-frequency Raman band it was possible to conclude that the band arises from intermolecular effects, a restricted rotational or vibrational motion of the nicotine (7) molecule, or the closely related collisional mechanism. Of the two intermolecular mechanisms, the first has been strengthened by

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Malgorzata Baranska and Hartwig Schulz

the observed good correlation between the integrated Raman intensity and the quantity. It should be noted that the low-frequency Raman band from pyridine is centered near 85 cm1 with a half-width of 90 cm1, and that the integrated Raman intensity is B70% of that from benzene. For comparison, nicotine (7), which is a substituted pyridine, gives rise to a relatively intense band centered near 65–75 cm1, having a half-width of 60 cm1. The spectral similarities between nicotine (7) and pyridine below 100 cm1 indicate that a restricted rotational motion is also reasonable as an explanation for the low-frequency nicotine band. The nicotine (7) SERS spectrum on a silver–alumina substrate reveals a sharp band at 1032 cm1 (trigonal ring breathing), and several broad bands similar to those for nicotinamide (37). This feature is supported by the interaction of nicotine (7) with the surface through the pyridine ring, while the characteristic five-membered pyrrolidine ring of the alkaloid stays far from the surface (21,75). O NH2

N 37 Nicotinamide

X. PIPERIDINE ALKALOIDS The main pungent principle in the green berries of pepper (Piper nigrum L., Piperaceae) is the alkaloid piperine (38), which occurs within the concentration range of 3–8 g/100 g in black and white pepper. FT-Raman spectra obtained from green pepper berries, ground black pepper, and black pepper oleoresin predominantly show significant key signals of piperine (38) (76). Apart from the intense –C–H stretching vibrations between 2800 and 3100 cm1, the main Raman signals occur in the fingerprint range between 1100 and 1630 cm1. The aromatic and aliphatic –C ¼ C– as well as {N–C ¼ O stretching vibrations can be detected between 1580 and 1635 cm1. The signals observed at 1448 cm1 can be assigned to –CH2 bending vibrations, whereas the other bands in the range between 1100 and 1400 cm1 are mainly due to –C–C– stretching (1153 cm1), as well as –CH2 twisting and rocking vibrations (1295 and 1256 cm1) of piperine (38) molecules. The corresponding IR spectrum of ground black pepper (Figure 3) reveals several specific piperine (38) signals, for example, due to ¼ C–O stretching vibrations at

245

1153

1295

1256

1448

1625 1600 1584

Determination of Alkaloids through IR and Raman Spectroscopy

Raman Intensity

D

C

B

A 1800 1700 1600 1500 1400 1300 1200 1100 1000

900

800

700

Wavenumber (cm-1)

Figure 3 FT-Raman spectra of pure piperine (38) (A), intact green pepper berries (B), ground black pepper (C), and black pepper oleoresin (D) (76).

1194 and 1252 cm1 as well as wagging vibrations at 996 cm1. Based on the individual key bands detected in the range between 1580 and 1635 cm1, the distribution of piperine (38) in a peppercorn can be analyzed in situ applying FT-Raman microscopic mapping. According to these measurements the pungent principle is distributed more or less in the whole perisperm of the green fruit; only the endosperm, located in the center of the berry, contains lower amounts of piperine (38). Applying ATR-IR and Raman measurements, chemometric equations have been developed for the calibration of piperine (38) content in pepper samples, presenting a comparatively high prediction quality (R2 ¼ 0.86 and 0.84, respectively) (76).

N O

O

O 38 Piperine

246

Malgorzata Baranska and Hartwig Schulz

XI. QUINOLIZIDINE ALKALOIDS Quinolizidine alkaloids (QA), of which more than 170 structures have been reported, are distinct from other alkaloids in that they contain at least one quinolizidine ring system (77). Only the a-pyridone alkaloids with an aromatic ring A and the cinnamoyl derivatives have a reasonable chromophore, but absorption in UV–Vis range does not normally provide much information for the identification of QA. On the other hand, until 1970, IR spectroscopy was an important method for the identification of QA (78–80). QA act as a nitrogen reserve and confer resistance toward pathogens and herbivores, especially in Lupinus and other genera of the Fabaceae family (77). The principal QA are sparteine (9), lupanine (39), albine (40), 13-hydroxylupanine (41), isolupanine (42), and angustifoline (43). The first attempts to use NIRS for the determination of total alkaloid content in lupine (Lupinus leucophyllus Dougl. ex Lindl.) and tall larkspur (Delphinium occidentale S. Watson ex J.M. Coult.) were conducted by Clark et al. (81). The authors developed three calibration equations to predict the total alkaloid content: one for larkspur, another for lupins, and the final equation for randomly combined larkspur and lupin samples. The R2 values were highest for larkspur and larkspur plus lupin, followed by lupin (R2 ¼ 0.93, 0.93, and 0.90, respectively). Due to the different alkaloid profiles in the two forages (larkspur contains principally diterpenoid alkaloids, while lupines contain principally the quinolizidine class of alkaloids), the first-derivative NIR spectra of the analyzed species show some variations. However, similarities were found where the NIRS equation was deriving information for alkaloid determinations (1772, 1712, and 2052 nm).

H

N

H

O N

H

H

H N

N H

H

42 Isolupanine

40 Albine

H

N

N H

39 Lupanine

H

NH

N

O

O

H

H

H

O

H

OH

41 13-Hydroxylupanine

H NH

N H O 43 Angustifoline

H N

H N

H H 44 13-Hydroxysparteine

OH

247

Determination of Alkaloids through IR and Raman Spectroscopy

Recently, feasibility studies have been performed to analyze the total amount of alkaloids and the individual content of most important alkaloids in European lupin species (L. albus L. and L. angustifolius L.) by using two different NIR instruments with the dispersive and FT-NIR technique, respectively (82). It was found that the main alkaloids, lupanine (39) and angustifoline (43), could be reliably predicted, and generally a high correlation to reference GC data could be observed (Table III). A lower level of analytical predictive quality was reached for 13-hydroxylupanine (41), which is mainly due to the significantly lower concentration of this alkaloid (maximum 200 mg/g). The authors assumed that the high correlation quality for lupanine (39) and angustifoline (43) is mainly due to the overtones and combination bands of specific C–N–fundamental vibrations. These present characteristic absorptions that stand out against the spectral background of the cellulose matrix (83). The NIR spectrometers worked with different scanning principles and sample presentation techniques; however, multivariate calibrations with comparable accuracy were obtained for the same lupin sample set. The described NIR method is proposed for in-process control measurements in industry to monitor the elimination of alkaloids from bitter lupins, and to evaluate rapidly the wild populations, as well as the progenies of crossing experiments. Investigations focused on the structure elucidation of 13-hydroxylupanine (41) and 13-hydroxysparteine (44), which were extracted from the seeds of L. angustifolius, provided detailed information concerning the configuration and conformation of both alkaloids based on NMR and Table III NIRS correlation statistics for lupine alkaloid data sets obtained from FT-NIR and dispersive NIR spectrometer systems (82) Content

R2

SECV (mg/g)

Range (mg/g)

MPA (multipurpose analyzer) with sample rotation Total alkaloids 0.98 965 83–35810 Lupanine (39) 0.96 574 61–16060 13-Hydroxylupanine 0.35 26 1–140 (41) Angustifoline (43) 0.96 156 13–75779 NIR 5000-C (dispersive NIRS) Total alkaloids 0.99 Lupanine (39) 0.99 13-Hydroxylupanine 0.34 (41) Angustifoline (43) 0.99

Spectra Outliers

153 153 153

1 4 12

153

6

487 318 34

76–36484 153 58–15143 153 5–196 153

8 7 6

120

64–4224 153

7

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Malgorzata Baranska and Hartwig Schulz

IR data (84,85). The IR spectrum of 13a-hydroxylupanine (41) (measured in KBr) presented the following absorptions: 3293, 2802, 2762, and 1599 cm1 (N–C ¼ O). The principal bands of 13a-hydroxysparteine (44) were observed at 3364, 2828, 2798, 2777, 2753, and 2681 cm1. The corresponding b-epimers of both alkaloids show slight wavenumber shifts in the individual IR spectra, which allow for the discrimination between conformations.

XII. MONOTERPENOID INDOLE ALKALOIDS Yohimbine (45) is the principal alkaloid of the bark of the West African evergreen Pausinystalia yohimbe Pierre, family Rubiaceae, used traditionally in Africa as an aphrodisiac. There are at least 31 other yohimbane alkaloids found in yohimbe, but the active principle is yohimbine (45) hydrochloride. Several new indole alkaloids were extracted from the stem bark of Stemmadenia obovata K. Schum. (Apocynaceae) and Delphinium cardiopetalum DC. (Ranunculaceae). The molecular structures of the alkaloids were confirmed by the use of various spectroscopic techniques, including IR spectroscopy. Table IV gives the frequencies of characteristic bands in the IR spectra of the selected indole alkaloids (45–58) (8).

N

OH

CH3O

H

N

N H H H H3COOC

CH3

N OH

45 Yohimbine

46 Voacangine hydroxyindolenine

OAc CH3O

N

MeO N CH3

N COOCH3 47 Voacangine acetoxyindolenine

N H 48 Ibogaine hydroxyindolenine

Determination of Alkaloids through IR and Raman Spectroscopy

Table IV

249

Frequencies of characteristic bands of selected indole alkaloids (8)

Alkaloids

IR (cm1)

Yohimbine (45)

3342, 2931, 1719, 1438, 1314, 1201, 1150, 1096, 1055, 741 3565, 3460, 2955, 2872, 1737, 1690, 1601, 1478, 1361, 1272, 1161, 1026, 979

Voacangine hydroxyindolenine (46) Voacangine acetoxyindolenine (47) Ibogaine hydroxyindolenine (48) Obovamine (49) Obovatine (50) Cardionidine (51) Cossonidine (52) Cardiopine (53) Cardiopinine (54) Cardiopimine (55) Cardiopidine (56) Cardiodine (57) Lochnerine (58)

2955, 2860, 1754, 1731, 1601, 1474, 1435, 1367, 1242, 1200, 1170, 1110, 1087, 1029, 995 3690, 3589, 3024, 2958, 2934, 2859, 1601, 1561, 1474, 1435, 1361, 1281, 1226, 1205, 1157, 1098, 1029, 979 3378, 2966, 2931, 2860, 1731, 1601, 1466, 1437, 1349, 1302, 1243, 1155, 1090 3540, 3340, 1720 1760, 1720, 1170, 1087 3650, 2950, 2900, 1650, 1450, 1380, 1150, 1080, 1050, 1010, 990 3367, 3026, 2931, 1733, 1693, 1601, 1451, 1361, 1292, 1245, 1141, 1107, 1034, 979, 712 3411, 3025, 2980, 1734, 1719, 1657, 1450, 1370, 1272, 1237, 1149, 1109, 1069, 1034, 980, 901, 713 3367, 3029, 1729, 1657, 1272, 1239, 1151, 1110, 776 3371, 2935, 1729, 1653, 1451, 1371, 1272, 1240, 1090, 710 3400, 2900, 1745, 1740, 1730, 1725, 1650, 1370, 1270, 1240, 1140, 910 3602, 3220, 2955, 2902, 1629, 1593, 1486, 1456, 1453, 1377, 1324, 1214, 1029, 803

N CH3OOC CH3O CH3O O

N

N

H3C CH3

H N N

COOCH3 49 Obovamine

50 Obovatine

N H

OH

CH3 COOCH3

250

Malgorzata Baranska and Hartwig Schulz

O

HO OH

O H3C

N

O

O AcO AcO

O O

OH

N

N

O HO

CH3 O 51 Cardionidine

52 Cossonidine

53 Cardiopine

O

O O

O

AcO

AcO

O

AcO

AcO HO

O N

N

O O

HO

54 Cardiopinine

55 Cardiopimine

O

O

O

O

AcO

O

AcO HO

AcO

OH

O

N

O

AcO

N

O

AcO

56 Cardiopidine

57 Cardiodine

CH2OH

CH3O

N H

N

N H

H CH 3 H O

H

H

H

H H

OMe

CH CH2 OMe

O

58 Lochnerine

N

59 Ajmalicine

CH3OOC

60 Corynantheine

Determination of Alkaloids through IR and Raman Spectroscopy

251

With the assistance of IR spectroscopy, the stereochemistry of the various pairs of C-3 epimers of yohimbine (45), ajmalicine (59), and corynantheine (60) was investigated. It became apparent that the 3.4– 3.7 mm region of the C–H stretching vibration can be used for this purpose. All of the alkaloids possessing an a-hydrogen at C-3 exhibit two or more distinct and characteristic peaks of medium intensity on the high-wavelength side of the major 3.46 mm band. However, those alkaloids containing a C-3–H b-orientation show merely a shoulder on the high-wavelength side of the main peak (86).

XIII. PROTOALKALOIDS Five different capsaicinoid derivatives have so far been found in paprika and chilli pods, of which capsaicin (61) and dihydrocapsaicin (62) are the most important (87). The other three naturally occurring capsainoids – nor-dihydrocapsaicin (63), homo-capsaicin (64), and homo-dihydrocapsaicin (65) – are only present in smaller amounts. In order to develop an efficient alternative to the classical HPLC methods (88,89), a new NIR method was established for the determination of capsaicin (61) content in red pepper (Capsicum annuum L., Solanaceae) and related extracts (90). For spectral measurements the ground peels of the vacuum-dried red pepper fruits were used and spectra were recorded over the region of 1100–2500 nm. Strong bands were observed in the second-derivative spectra between the 2250 and 2350 nm, 1700 and 1760 nm, and at the 1200 nm regions that were assigned to the combination vibration, the first overtone vibration, and the second overtone vibration of the individual C–H groups, respectively. HO

HO

H N

H N MeO

MeO O

O 62 Dihydrocapsaicin

61 Capsaicin

HO

HO MeO

H N

H N

MeO O

63 Nor-dihydrocapsaicin

O 64 Homo-capsaicin

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Malgorzata Baranska and Hartwig Schulz

HO

MeO

H N O 65 Homo-dihydrocapsaicin

The bands in the 1950 and 2200 nm regions were interpreted as combination vibrations of N–H and O–H groups in the capsaicin (61) molecule. A stepwise MLR analysis was performed to select the five most suitable absorption wavelengths. The chemometric results (R2 ¼ 0.993, SEE ¼ 0.0036 g/100 g, calibration range: 0.05–0.13 g/100 g) led to the conclusion that NIR reflectance spectroscopy can be successfully used to determine the capsaicin (61) content in ground red pepper.

XIV. CONCLUSIONS This chapter presents the results of some selected published studies describing various applications of Raman and IR spectroscopic methods for an efficient measurement of alkaloids. Both methods have the potential to supplement existing standard procedures presently used for plant analysis (mainly chromatographic and NMR techniques), but particularly FT-Raman spectroscopy was found to be an extremely useful technique for the nondestructive analysis of the intact fresh plant tissue without the necessity to perform any sample cleanup steps. Raman spectroscopy combined with microequipment can provide detailed molecular information with high spatial resolution at the cellular level. Raman spectra can be obtained directly from single plant cells, even if they contain water, and a specified plant area can be mapped. Over the past decade, the rapid development of imaging techniques has occurred and, as a result, the potential to apply vibrational methods in plant research has significantly increased. Usually, the sensitivity of the discussed vibrational methods is lower in comparison to other analytical techniques, but in some cases, components occurring in low concentration can be successfully analyzed in complex matrices in a nondestructive manner. However, an increase in sensitivity can be provided by some special Raman techniques, like the SERS or ‘‘tips’’ methods, which give the signal an enhancement of six or more in magnitude. Because the Raman spectra of biological samples are very often obscured by strong fluorescence signals from other components of the complex matrix, it is very useful to excite the Raman spectra of the active agents selectively and enhance the resonance signals up to a

Determination of Alkaloids through IR and Raman Spectroscopy

253

factor 106 compared to the background Raman signal of the plants. Also, resonance Raman spectroscopy has also advantages compared to conventional Raman due to the high selectivity and sensitivity of this technique. In summary, the increased demand to solve complex problems of alkaloid detection and biochemistry requires a multidisciplinary approach, in which Raman, IR, and NIR spectroscopy can play a prominent role.

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SUBJECT INDEX Abyssinine A, 100, 112, 114–115, 128–129 Abyssinine B, 100, 114–115, 126, 127 Abyssinine C, 100, 115 14-Acetylaconosine, see Dolaconine 6-O-Acetylacosepticine, 40, 58–59 6-O-Acetylacosepticine, 17–18, 29, 31 14-Acetyldihydrogadesine, 31 8-Acetyldolaconine, 7, 31, 34, 46 plant sources of, 7, 27 8-Acetylexcelsine, 15, 31, 38 plant sources of, 15, 28 N-Acetylhystrine, 151–153 Acetyl-nornicotine, 154 N-Acetylsepaconitine, 14–15, 31, 38, 53–54 pharmacological activity of, 71 plant sources of, 14–15, 28 6-O-Acetylumbrofine, 17–18, 31, 40, 57 plant sources of, 17–18, 29 Aconitine, 191, 196 lappaconitine v., 3 Aconitum spp. diterpene alkaloids in, 191–193 C18-diterpenoid alkaloids from, 5, 27–30 Aconosine classification and structure of, 2, 6, 31, 34, 46 plant sources of, 6, 27–29 Aconosine-subtype, classification and structure of, 3–9, 34–35, 46–49 Acosepticine, 17, 31, 40, 57 plant sources of, 17, 29 Acoseptrine, 18, 31, 40, 58–59 plant sources of, 18, 29 Activity, see Structure–activity relationships and mechanisms of action Adouetine X, 87, 112, 117–119 Adouetine Y, 92, 116, 118, 131–132 Adouetine Y’, 87, 112, 116–119, 125, 133 Adouetine Z, 94, 118–119 5(14)-Adouetine Z type cyclopeptide alkaloids, 94 Ajmalicine, 250–251 Akiradin, 15, 31, 38 plant sources of, 15, 28

Akiramidine, 10, 31, 36 plant sources of, 10, 28 Akiramine, 11, 31, 36, 50–51 plant sources of, 11, 28 Akiran, 11, 31, 36 plant sources of, 11, 28 Akiranine, 10, 31, 36, 50–51 plant sources of, 10, 28 Akirine, 16, 31, 38 plant sources of, 16, 28 Albine, 246 Alkaloid 2, 82, 112, 118 Alkaloid 3, 84, 112 Alkaloid 4, 87, 118 Alkaloid 6, 92 Alkaloid glycoprocessing inhibitors, 166–168 AM-1, 87, 119 AM-2, 92, 119 Americine, 87, 117, 131–132 Ammodendrine, 151–153, 169–170, 175 Amphibine A, 87, 113–115 Amphibine B, 97, 114 5(14)-Amphibine B type cyclopeptide alkaloids, 97–98, 108–109 Amphibine C, 97, 114 Amphibine D, 97, 113–115 Amphibine E, 97, 114–115 Amphibine F, 95, 114–115 4(14)-Amphibine F type cyclopeptide alkaloids, 95–96, 107–108 Amphibine G, 95, 114 Amphibine H, 84, 113–115 Anabaseine, 154–155, 157 (–)-Anabasine, 154–157 Anacrotine, 184 Anagyrine, 169–175 Analgesic activity, of C18-diterpenoid alkaloids, 39, 69 S-(–)-Anatabine, 154–156 Ancistrocladaceae family, antimalarial alkaloids of, 238 Ancistrocladidine, 238 Ancistrocladine, 238 Ancistrocladinidine, 238 Ancistrocladisine, 238

267

268

Subject Index

Ancistroheynine A, 238–239 Anesthetic activity, of C18-diterpenoid alkaloids, 72 Angel’s trumpet, toxic alkaloids of, 176 7-Angelyl heliotridine (Rivularine), 180–181, 183 7-Angelyl heliotrine, 180–181 Angustifoline, 246–247 Anisodamine, 242 Anorldianine, 87, 119 Anorldianine 27-N-oxide, 103, 110, 119 4-Anthranoyllappaconitine classification and structure of, 11–12, 31, 37, 52–53 plant sources of, 11–12, 29 Anthriscifolcine A, 19, 31, 41, 58–59 plant sources of, 19, 29 Anthriscifolcine B, 18, 31, 40, 58–59 plant sources of, 18, 29 Anthriscifolcine C, 19, 31, 41, 59–60 plant sources of, 19, 29 Anthriscifolcine D, 19–20, 31, 41–42, 59–60 plant sources of, 19–20, 29 Anthriscifolcine E, 19–20, 31, 41, 59–60 plant sources of, 19–20, 29 Anthriscifolcine F, 19–20, 31, 42, 59–60 plant sources of, 19–20, 30 Anthriscifolcine G, 19, 31, 41, 58–59 plant sources of, 19, 30 Antiarrhythmic activity, of C18-diterpenoid alkaloids, 69–72 Anticholinergic activity, of hyoscyamine and scopolamine, 178–179 Antidesma spp., cyclopeptide alkaloids from, 119 Anti-inflammatory activity, of C18-diterpenoid alkaloids, 72 Antimalarial isoquinoline and naphthylisoquinoline alkaloids, IR and Raman spectroscopy of, 234–239 Antimicrobial activity, of cyclopeptide alkaloids, 133 Antispasmodial activity, of cyclopeptide alkaloids, 133 Arabica coffee beans, quantification of caffeine in, 225 Aralionine A, 92, 118 Aralionine B, 92, 118, 119 Aralionine C, 92, 118

Araliorhammus spp., cyclopeptide alkaloids from, 118 Argemone oil, 190 Argemone spp., toxic alkaloids of, 188–190 Astragalus spp., toxic alkaloids in, 158–160, 163–164 Atropine, 177 Australine, 160–161, 166–167 1-epi-Australine, 160–161, 166–167 1-epi-Australine 2-O-b-Dglucopyranoside, 160–161, 166–167 Baptisia spp., toxic alkaloids of, 171 Barbinine, 194 Benzoylmethyl ecgonine, see Cocaine Benzylisoquinoline alkaloids, IR and Raman spectroscopy of, 239–241 Berberine, 188 IR and Raman spectroscopy of, 233–234 Biological activity, see Pharmacological activity Biosynthesis, of cyclopeptide alkaloids, 130–132 Bipiperidine alkaloids, 151–154 Bisnorditerpenoid alkaloids, see C18-Diterpenoid alkaloids Black Bean, toxic alkaloids of, 160–161, 164–169 Bluebell, see English bluebell Bohlmann bands, 219–220 Brugmansia spp., toxic alkaloids of, 176 6-O-Butanoylcastanospermine (Celgosivir), 168 Caffeine, 220–227 Calystegia sepium, toxic alkaloids of, 162 Calystegines, 159, 162–163, 165–167, 178 Camas, toxic alkaloids of, 203–205 Candaline A, 117 Canthium spp., cyclopeptide alkaloids from, 119 Canthiumine, 92, 119 Capsaicin, 251–252 Cardiodine, 249–250 Cardionidine, 249–250 Cardiopidine, 249–250 Cardiopimine, 249–250 Cardiopine, 249–250 Cardiopinine, 249–250 Castanospermine, 160–161, 165–169

Subject Index

6-epi-Castanospermine, 160 Castanospermum australe, toxic alkaloids of, 160–161, 164–169 Ceanothine A, 87, 117 Ceanothine B, 88, 117 Ceanothine C, 88, 117 Ceanothine D, 103, 117 Ceanothine E, 92, 117, 131–132 Ceanothus spp., cyclopeptide alkaloids from, 117 Celandine, medicinal alkaloids of, 239 Celgosivir, see 6-O-Butanoylcastanospermine Cevanines, 202 Cevanine-type steroidal alkaloids, 203–205 Chamaedrine, 88, 106, 119, 122 Chelerythrine, 239–240 Chemometrics of caffeine, 225, 227 of capsaicin, 252 of cocaine, 242 of heroin, 232–233 of nicotine, 243 for piperine, 245 of quinine and quinidine, 237 of theobromine, 227 Chlorogenic acid, 225–226 Cholestanes, 201–202 Cinchona spp., alkaloids of, 235–236 Cinchonidine, 235 Cinchonine, 235 Clinical signs of poisoning, see Toxicity Cocaine, 233, 241–242 Codeine, 229–231, 233 Coffee, quantification of caffeine in, 225–226 Colubrina spp., cyclopeptide alkaloids from, 117 Condalia spp., cyclopeptide alkaloids from, 117 Condaline A, 92, 107, 124, 125–126, 133 Conhydrine, 147–148 Conhydrinone, 147–148 g-Coniceine, 147–148, 150–151 (R)-(–)-Coniine, 147–148, 150 Conium maculatum, toxic alkaloids of, 147–151 Consolida spp., C18-diterpenoid alkaloids from, 5, 30 Contortumine, 8, 31, 35, 48–49 plant sources of, 8, 27

269

Corynantheine, 250–251 Cossonidine, 249–250 Crenatine A, 92, 116 Crooked calf disease, 173 Crotalaria spp., toxic alkaloids of, 183–186 Cyclopamine, 199–202 Cyclopeptide alkaloids, from higher plants, 79–135 biological activity of, 131–135 antimicrobial, 133 antispasmodial, 133 sedative, 134 biosynthesis of, 130–132 classification of, 80–111 introduction to, 79–80 natural occurrence of, 111–119 newly isolated, 111, 120–124 structure elucidation and stereochemistry, 124–126 synthesis of, 126–131 Cycloposine, 199–200, 202 Cynoglossum officinale, toxic alkaloids of, 182–183 Cytisine, 169–172, 174–176 IR and Raman spectroscopy of, 227–228 Cytisus scoparius, toxic alkaloids of, 171 Daechuine S3, 84, 112, 118 Daechuine S5, 88 Daechuine S6, 82, 112 Daechuine S7, 82, 112 Daechuine S8, 84, 112 Daechuine S10, 82 Daechuine S26, 82, 112 Daechuine S27, 135 Datura spp., toxic alkaloids of, 176–178 N-Deacetylfinaconitine, 23, 31, 44 pharmacological activity of, 39, 68, 72 plant sources of, 23, 27 N-Deacetyllappaconitine classification and structure of, 13–14, 31–32, 37, 52–53 pharmacological activity of, 39, 68, 71–73 plant sources of, 13–14, 27–30 14-Deacetylnudicaline, 192–194

270

Subject Index

N-Deacetylranaconitine classification and structure of, 22–23, 31, 43 pharmacological activity of, 39, 68, 72 plant sources of, 22–23, 27, 29 2-Deacetyltaxine A, 197 Death camas, toxic alkaloids of, 203–205 Decaffeination, IR measurement of, 226–227 5,6-Dehydrolupanine, 170, 172, 174 Delavaconine, 6, 31–32, 34, 46 plant sources of, 6, 27 Delavaconitine, 8, 31, 35, 48–49 plant sources of, 8, 27 Delavaconitine C, 7–8, 31, 34, 47–48 plant sources of, 7–8, 27 Delavaconitine D, 7–8, 31, 34, 47–48 plant sources of, 7–8, 27 Delavaconitine E, 8, 31, 35 plant sources of, 8, 27 Delbine, 21, 31, 42, 60–61 plant sources of, 21, 30 Delboxine, 25–26, 30, 31, 44, 66 plant sources of, 25–26 Delphicrispuline, see Neofinaconitine Delphinium spp., diterpene alkaloids in, 191–193 Delphinium spp., C18-diterpenoid alkaloids from, 5, 29–30 Deltaline, 192, 194–195 14-O-Demethyldelboxine, 25–26, 31, 44, 66 plant sources of, 25–26, 30 Demethyllappaconitine classification and structure of, 12, 31, 37, 52–53 plant sources of, 12, 28 14-Demethyltuguaconitine, 25, 32, 44, 65 plant sources of, 25, 30 Density functional theory (DFT) calculation for anisodamine, 242 of opioid isoquinoline alkaloids, 232–233 for quinine, 236 for sanguinarine, 241 xanthine, caffeine, and theobromine calculations using, 221, 224–225 Deoxo-aralionine A, 93, 117 6-Deoxy-6-C-(2,5-dihydroxyhexyl)DMDP, 146–147 7-Deoxy-6-epi-castanospermine, 160

9-Deoxy-6-methoxy-N-succinyldeacetylranaconitine classification and structure of, 21–22, 32, 43 plant sources of, 21–22, 27 8-Deoxy-14-dehydroaconosine classification and structure of, 7, 32, 34 plant sources of, 7, 29 1-Deoxyisotaxine B, 197 9-Deoxylappaconitine classification and structure of, 11–12, 31–32, 37, 52–53 pharmacological activity of, 71 plant sources of, 11–12, 27–28 1-Deoxytaxine B, 196–197 Desbenzoyl-aralionine A, 93, 107, 118 N-Desmethyl-integerrenine, 93, 117 N-Desmethyl-integerrine, 93, 117 O-Desmethyl-mucronine D, 85, 112 N-Desmethyl-myrianthine B, 89, 117 N-Desmethyl-myrianthine C, 89, 119 Devil’s trumpet, toxic alkaloids of, 176 DFT calculation, see Density functional theory calculation 1,4-Dideoxy-1,4-imino-D-arabinitol, 146–147 2,5-Dideoxy-2,5-imino-DL-glycero-Dmanno-heptitol (Homo-DMDP), 146–147 2,3-Di-epi-australine, 160–161, 166–167 6,7-Di-epi-castanospermine, 160 Dihydrocapsaicin, 251 Dihydromonticamine, 9–10, 32, 36, 49–50 pharmacological activity of, 70 plant sources of, 9–10, 28 Dihydroquinidine, 235–236 Dihydroquinine, 235–236 Dihydrosanguinarine, 188–190 2R,5R-Dihydroxymethyl-3R,4Rdihydroxypyrrolidine (DMDP), 146–147 Dioicine, 227–228 Dioncopeltine A, 237–238 Dioncophylline A, 237 Dioncophylline C, 237 Discarene C, 102, 109, 116, 125 Discarene D, 102, 109, 116, 125–126 Discaria spp., cyclopeptide alkaloids from, 116 Discarine, 116 Discarine A, 88, 116 Discarine B, 88, 116, 117, 125–126

Subject Index

271

Discarine C, 93, 116 Discarine D, 93, 116, 125 Discarine E, 88, 116 Discarine F, 88, 116, 125 Discarine G, 99, 116 Discarine H, 99, 116 Discarine I, 88, 116 Discarine K, 99, 116, 126 Discarine L, 99, 109, 116 Discarine M, 102, 109, 116, 125 Discarine N, 102, 109–110, 116, 125 Discarine X, 88, 116 Diterpene alkaloids IR and Raman spectroscopy of, 246–248 larkspur, 191–196 yew, 196–199 C20 Diterpenes, see Diterpene alkaloids C18-Diterpenoid alkaloids, 1–73 chemical reactions of, 30, 33, 67 classification and structures of, 2–26, 30 code numbers of, 30–33 introduction to, 1–3 NMR spectroscopy of, 5–26, 30 occurrence of, 3–5 pharmacological activity of, 3, 33, 39, 67–73 analgesic, 39, 69 anesthetic, 72 antiarrhythmic, 69–72 anti-inflammatory, 72 plant sources of, 27–30 C19-Diterpenoid alkaloids, C18-diterpenoid alkaloids v., 2 DMDP, see 2R,5R-Dihydroxymethyl3R,4R-dihydroxypyrrolidine Dolaconine, 7, 31–32, 34, 47–48 plant sources of, 7, 27–29

a-Galactosidase, calystegine inhibition of, 166–167 b-Galactosidase, calystegine inhibition of, 166 Gas chromatography (GC), for cyclopeptide alkaloid structure elucidation, 125 GC, see Gas chromatography Germine, 199–200, 203 a-Glucosidase inhibitors, 166–168 b-Glucosidase inhibitors, 166–167 Gramodendrine, 152–153

Echinatine, 183 Echium spp., toxic alkaloids of, 183 Embellisia, swainsonine production by, 164 English bluebell, toxic alkaloids of, 146–147 Episcopalisine, see Delavaconitine Episcopalisinine, see Delavaconine Episcopalitine, see Dolaconine Euonymus spp., cyclopeptide alkaloids from, 119 Exceconidine, 18, 32, 40, 58–59 plant sources of, 18, 27

Hartree–Fock method, xanthine, caffeine, and theobromine calculations using, 221 Heisteria spp., cyclopeptide alkaloids from, 119 Heliosupine, 183 Heliotropium spp., toxic alkaloids of, 183 Hemlock, see Poison hemlock Hemsine A, 95, 107, 118 Hemsine B, 97, 108, 118 Hemsine C, 94, 107, 118 Hemsine D, 93, 107, 118

Excelsine, 15, 32, 38, 55–56 isolation of, 2 pharmacological activity of, 70 plant sources of, 15, 27–29 structure of, 2 Fabaceae family, toxic alkaloids of, 151–153, 158, 169–173 Feretia spp., cyclopeptide alkaloids from, 119 Feretine, 94, 119 Finaconitine, 24, 32, 44, 63–64 plant sources of, 24, 27, 29 Formyl-nornicotine, 154 Fourier transformed infrared and Raman spectroscopy, see Infrared and Raman spectroscopy Franganine, 88, 112, 115–117, 119, 125 Frangufoline, 88, 112–117, 119, 134–135 cleavage of, 134–135 Frangulanine, 88, 112–113, 116–119, 131–133 4(14)-Frangulanine type cyclopeptide alkaloids, 87–90, 106

272

Subject Index

Hepatotoxicity, of pyrrolizidine alkaloids, 185–187 Heroin, 232–233 Hispaconitine, 21–22, 32, 43, 60–61 plant sources of, 21–22, 27 Hohenackeridine, 25–26, 32, 44, 66 plant sources of, 25–26, 30 Homoamerecine, 89, 117 Homo-capsaicin, 251 Homo-dihydrocapsaicin, 251–252 Homo-DMDP, see 2,5-Dideoxy-2,5imino-DL-glycero-D-mannoheptitol Homo-DMDP-7-O-apioside, 146 Homo-DMDP-7-O-b-D-xylopyranoside, 146–147 Hovenia spp., cyclopeptide alkaloids from, 118 Hovenine A, 89, 118 Human poisoning, 143–206 by diterpene alkaloids larkspur, 191–196 yew, 196–199 by isoquinoline alkaloids, 188–190 by piperidine alkaloids, 147–151 by polyhydroxy indolizidine alkaloids and related pyrrolizidine and nortropane alkaloids, 158–169 by pyridine alkaloids, 154–157 by pyrrolidine alkaloids, 146–147 by pyrrolizidine alkaloids, 179–187 by quinolizidine alkaloids, 152–154, 169–176 by steroidal alkaloids Veratrum alkaloids, 199–202 Zigadenus alkaloids, 203–205 by tropane alkaloids, 176–179 Hyacinthacine B1, 161 Hyacinthacine C1, 161 Hyacinthoides non-scripta, toxic alkaloids of, 146–147, 161 13-Hydroxylupanine, 246–248 10-Hydroxyranaconitine, classification and structure of, 32 10b-Hydroxyranaconitine, see Finaconitine 13-Hydroxysparteine, 246–248 Hymenocardia spp., cyclopeptide alkaloids from, 119 Hymenocardine, 103, 119 L-Hyoscyamine, 176–179

Hyoscyamus niger, toxic alkaloids of, 176–178 Hysodricanine A, 97, 112, 115 Ibogaine hydroxyindolenine, 248–249 Indole alkaloids, see Monoterpenoid indole alkaloids Indolizidine alkaloids, see Polyhydroxy indolizidine alkaloids Indolizidines, IR spectroscopy of, 219 Infrared (IR) and Raman spectroscopy, 217–253 of antimalarial isoquinoline and naphthyisoquinoline alkaloids, 234–239 of benzylisoquinoline alkaloids, 239–241 conclusions on, 252–253 introduction to, 218–219 of monoterpenoid indole alkaloids, 248–251 of natural isoquinoline alkaloid dyes, 233–234 of nitrogen bridgehead compounds, 219–220 of opioid isoquinoline alkaloids, 228–233 of piperidine alkaloids, 244–245 of protoalkaloids, 251–252 of purine alkaloids, 220–228 of pyrrolidine alkaloids, 243–244 of quinolizidine alkaloids, 246–248 of tropane alkaloids, 241–242 Infra-red Band assignments of berberine, 234 of caffeine, 221–223 of capsaicin, 251–252 of chelerythrine, 239 of cocaine, 233 of codeine, 230–231 of dioicine, 227–228 of isotetrandrine, 240 of lupines, 248 of monoterpenoid indole alkaloids, 249 of morphine, 230–233 of naphthylisoquinoline alkaloids, 237–238 of nicotine, 243–244 of noscapine, 230–231 of opioid isoquinoline alkaloids, 230–231 of papaverine, 231–232 of piperine, 244–245

Subject Index

of quinidine, 236 of quinine, 236 of sanguinarine, 239–241 of scopolamine, 242 of thebaine, 230–231 of theobromine, 221–224 of theophylline, 221–223 Integerrenine, 93, 113, 117, 119 Integerressine, 93, 117 Integerrimine, 180, 184 Integerrine, 93, 117 4(14)-Integerrine type cyclopeptide alkaloids, 92–93, 107 Ipomoea spp., toxic alkaloids of, 159, 162–167 IR spectroscopy, see Infrared and Raman spectroscopy Isolappaconitine classification and structure of, 22–23, 32, 43, 62–63 pharmacological activity of, 71 plant sources of, 22–23, 27–28 Isolupanine, 246 Isoquinoline alkaloids, 188–190 IR and Raman spectroscopy of, 228–233 antimalarial alkaloids, 234–239 natural isoquinoline alkaloid dyes, 233–234 opioid alkaloids, 228–233 Isotaxine B, 196–197 Isotetrandrine, 240–241 Jacobine, 182 Jaconine, 182 Jervanines, 202 Jervine, 199–200, 202 Jimson weed, toxic alkaloids of, 176, 178 Jubanine A, 84, 113, 114, 115 Jubanine B, 84, 113, 114 Jubanine C, 94, 107, 114 Kiramine, 11, 32, 36 plant sources of, 11, 28 Kiridine, 16, 32, 38, 55–56 plant sources of, 16, 28 Kiritine, 16, 32, 39 plant sources of, 16, 28 Laburnum anagyroides, toxic alkaloids of, 171, 174 Lamarckinine, 20, 32, 42, 59–60 plant sources of, 20, 28

273

Lappaconidine, 9–10, 32, 36, 49–50 isolation of, 2 pharmacological activity of, 71 plant sources of, 9–10, 28–29 structure of, 2 Lappaconine classification and structure of, 9–10, 32, 36, 49–50 pharmacological activity of, 70 plant sources of, 9–10, 28 Lappaconine-subtype classification and structure of, 36–39, 49–56 classification of, 3–5, 9–16 structure of, 4 Lappaconitine classification and structure of, 13–14, 32, 38, 53–54 isolation of, 1 pharmacological activity of, 3, 39, 67–73 plant sources of, 13–14, 27–30 structure of, 2 Lappaconitine-type classification and structure of, 34–35, 46–56 classification of, 3–5, 6–16 Larkspur alkaloids, see Diterpene alkaloids Lasiocarpine, 180–181 Lasiodine A, 103, 118 Lasiodine B, 91, 118 Lasiodiscus spp., cyclopeptide alkaloids from, 118 Lentiginosine, 158, 160 2-epi-Lentiginosine, 158 Leuconine classification and structure of, 17, 32, 40, 57 plant sources of, 17, 28–29 Leuconine-subtype classification and structure of, 40–42, 57–60 classification of, 3–5, 17–20 structure of, 4 Leucostine, 17, 32, 40, 57 plant sources of, 17, 28–29 Liconosine A, 9, 32, 35, 48–49 plant sources of, 9, 28 Linearilin, 21, 32, 42–43, 60–61 plant sources of, 21, 30

274

Subject Index

Livestock poisoning, 143–206 by bipiperidine alkaloids, 151–154 conclusions and outlook on, 206 by diterpene alkaloids larkspur, 191–196 yew, 196–199 introduction to, 144–146 by isoquinoline alkaloids, 188–190 by piperidine alkaloids, 147–151 by polyhydroxy indolizidine alkaloids and related pyrrolizidine and nortropane alkaloids, 158–169 by pyridine alkaloids, 154–157 by pyrrolidine alkaloids, 146–147 by pyrrolizidine alkaloids, 179–187 by quinolizidine alkaloids, 152–154, 169–176 by steroidal alkaloids Veratrum alkaloids, 199–202 Zigadenus alkaloids, 203–205 by tropane alkaloids, 176–179 Lobelia berlandieri, toxic alkaloids of, 149–151 Lobeline, 149 Lochnerine, 249–250 Locoweeds, 158–160, 163–166, 176 Lotusanine B, 102, 110, 112 Lotusine A, 95, 112, 118 Lotusine B, 98, 108, 112 Lotusine C, 98, 112 Lotusine D, 95, 112, 118 Lotusine E, 85, 112 Lotusine F, 82, 112 Lotusine G, 95, 107, 112 Lupanine, 169–172, 174–176 IR and Raman spectroscopy of, 246–247 Lupines IR and Raman spectroscopy of, 246–248 livestock toxicity of, 151–154, 169–175 Lupinine, 169–170 Lupinus spp., toxic alkaloids of, 151–153, 169–175 Lycoctonine, 192, 194–195 ranaconitine v., 3 Lycopsamine, 180–181 Mahonia beali, medicinal alkaloids of, 240 a-Mannosidase, swainsonine inhibition of, 165–166

Mass spectrometry (MS), for cyclopeptide alkaloid structure elucidation, 124 Matrine, 169–170, 172, 176 Mauritine A, 98, 113, 114, 115, 127 Mauritine B, 98, 115, 127 Mauritine C, 96, 113, 115, 127 Mauritine D, 98, 113, 114, 115 Mauritine E, 98, 115 Mauritine F, 98, 113, 115, 127 Mauritine H, 98, 115 Mauritine J, 98, 108, 115 Mauritine K, 98, 109, 115, 122, 133 MDL, see 7,8-Methylenedioxy lycoctonine Mechanisms of action, see Structure–activity relationships and mechanisms of action Melochia spp., cyclopeptide alkaloids from, 119 Melofoline, 89, 119 Melonovine A, 89, 112, 119 Melonovine B, 89, 119 Metarhizium anisopliae, 159 N-Methyl-americine, 89, 117 N-Methylammodendrine, 151–153, 170 N-Methylcalystegine C1, 162 N-Methylconiine, 147–148, 150 N-Methylcytisine, 170–172, 174 7,8-Methylenedioxy lycoctonine (MDL), 192, 194 Methyllycaconitine (MLA), 192–195 N-(Methylsuccinyl)anthranoyl lycoctonine (MSAL) alkaloids, 192, 194 6-O-Methylumbrofine, see Exceconidine Mid-infrared spectroscopy, see Infrared and Raman spectroscopy MLA, see Methyllycaconitine Monocrotaline, 180, 184–186 Monoterpenoid indole alkaloids, IR and Raman spectroscopy of, 248–251 Monticamine, 14–15, 32, 38, 53–54 pharmacological activity of, 70 plant sources of, 14–15, 28 Monticoline, 24, 32, 44, 65 pharmacological activity of, 70 plant sources of, 24, 28 Moreton Bay Chestnut, toxic alkaloids of, 160–161, 164–169 Morphine, 228–233 MS, see Mass spectrometry

Subject Index

MSAL alkaloids, see N-(Methylsuccinyl)anthranoyl lycoctonine alkaloids Mucronine A, 101, 112, 115 4(15)-Mucronine A type cyclopeptide alkaloids, 100–101 Mucronine B, 101, 112, 115, 126–127 Mucronine C, 101, 112, 115 Mucronine D, 85, 112–114 Mucronine E, 101, 112, 127, 129 synthesis of, 129–130 Mucronine F, 101, 112 Mucronine G, 101, 112 Mucronine H, 101, 112 Mucronine J, 96, 108, 112, 118 Myrianthine A, 93, 116, 119 Myrianthine C, 89, 119 Myrianthus spp., cyclopeptide alkaloids from, 119 nAChRs, see Nicotinic acetylcholine receptors Na+-K+-ATPase inhibition, by isoquinoline alkaloids, 190 Naphthyisoquinoline alkaloids, antimalarial alkaloids, 234–239 Natural isoquinoline alkaloid dyes, IR and Raman spectroscopy of, 233–234 Near-infrared spectroscopy, see Infrared and Raman spectroscopy Necic acids, 179–180 Necine base, of pyrrolizidine alkaloids, 179–187 Neofinaconitine, 11–12, 31–32, 36, 50–51 plant sources of, 11–12, 27, 30 Neostigmine, 195 Neutral cyclopeptides, 102, 109–110 Nicotiana spp., toxic alkaloids in, 154–157 Nicotinamide, 244 Nicotine, 227–228, 243–244 S-(–)-Nicotine, 154–157 Nicotinic acetylcholine receptors (nAChRs), nicotine and related alkaloid effects on, 156–157 Nitrogen bridgehead compounds, IR spectroscopy of, 219–220 NMR, see Nuclear magnetic resonance spectroscopy 1 H NMR, see 1H Nuclear magnetic resonance spectroscopy

13

275

C NMR, see 13C Nuclear magnetic resonance spectroscopy Nor-dihydrocapsaicin, 251 Norditerpenes, 191–196 plant spp. and specific alkaloids, 191–193 structure-activity relationships and mode of action of, 194–196 toxicity and clinical signs of, 193–194 C19 Norditerpenes, see Norditerpenes Norditerpenoid alkaloids, see C18-Diterpenoid alkaloids S-(–)-Nornicotine, 154, 157 Nortropane alkaloids, 158–169 plant spp. and specific alkaloids, 158–163 structure-activity relationships and mode of action of, 165–169 toxicity and clinical signs of, 163–165 Noscapine, 229–231 1 H Nuclear magnetic resonance spectroscopy (1H NMR) for aconosine-subtype alkaloids, 6–9, 34–35 for lappaconine-subtype alkaloids, 9–16, 36–39 for leuconine-subtype alkaloids, 17–20, 40–42 for ranaconitine-subtype alkaloids, 21–26, 42–45 13 C Nuclear magnetic resonance spectroscopy (13C NMR) for aconosine-subtype alkaloids, 6–9, 46–49 for lappaconine-subtype alkaloids, 9–16, 49–56 for leuconine-subtype alkaloids, 17–20, 57–60 for ranaconitine-subtype alkaloids, 21–26, 60–66 Nuclear magnetic resonance spectroscopy (NMR), for cyclopeptide alkaloid structure elucidation, 124–126 Nudicaline, 192–194 Nummularine, 83 Nummularine A, 85, 113–114 Nummularine B, 85, 112–114 Nummularine C, 83, 112 4(13)-Nummularine C type cyclopeptide alkaloids, 82–83, 104 Nummularine D, 93, 113

276

Subject Index

Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine Nummularine

E, 93, 113, 115 F, 96, 113 G, 103, 113 H, 85, 113, 118, 135 K, 89, 113–114 M, 93, 113 N, 85, 113 O, 85, 113 P, 85, 113–114 R, 83, 112–113, 115 S, 83, 113 S10, 83, 113 T, 85, 113

Obovamine, 249 Obovatine, 249 Opioid alkaloids, 188–190 IR and Raman spectroscopy of, 228–233 Otonecine, 179 N-Oxides of nicotine, 154 of pyrrolizidine alkaloids, 181–186 Oxyphylline A, 94, 107, 115, 122 Oxytropis spp., toxic alkaloids in, 158–160, 163–164 Paclitaxel, see Taxol Paliurine A, 85, 104, 118 Paliurine B, 85, 104, 118 Paliurine C, 85, 104, 118 Paliurine D, 85, 104–105, 118 Paliurine E, 83, 104, 112, 118, 126, 129 Paliurine F, 85, 105, 118 Paliurine G, 85, 105, 118 Paliurine H, 86, 105, 118 Paliurine I, 86, 105, 118 Paliurus spp., cyclopeptide alkaloids from, 118 Panda spp., cyclopeptide alkaloids from, 119 Pandamine, 99, 119 4(14)-Pandamine type cyclopeptide alkaloids, 99, 109 Pandaminine, 99, 119 Papaveraceae family, toxic alkaloids of, 188–190, 228–233 Papaverine, 229, 231–232 Para-cyclophanes, synthesis of, 127 Partial least squares (PLS) of caffeine and theobromine, 227 of cocaine, 242 of heroin, 232–233

of nicotine, 243 of quinine and quinidine, 237 Paulirine E, synthesis of, 130–131 PCR, see Principal component regression Pepper alkaloids, IR and Raman spectroscopy of, 244–245 Pharmacological activity of cyclopeptide alkaloids, 131–135 antimicrobial, 133 antispasmodial, 133 sedative, 134 of C18-diterpenoid alkaloids, 3, 33, 39, 67–73 analgesic, 39, 69 anesthetic, 72 antiarrhythmic, 69–72 anti-inflammatory, 72 Physostigmine, 195 Piepunendine A, 9, 32, 35, 48–49 plant sources of, 9, 28 Piepunendine B, 35, 47–48 plant sources of, 7–8, 28 D3-Piperideines, 149, 151 Piperidine alkaloids, 147–151 IR and Raman spectroscopy of, 244–245 Piperine, 244–245 Plant alkaloids, IR and Raman spectroscopy of, 217–253 antimalarial isoquinoline and naphthyisoquinoline alkaloids, 234–239 benzylisoquinoline alkaloids, 239–241 conclusions on, 252–253 introduction to, 218–219 monoterpenoid indole alkaloids, 248–251 natural isoquinoline alkaloid dyes, 233–234 nitrogen bridgehead compounds, 219–220 opioid isoquinoline alkaloids, 228–233 piperidine alkaloids, 244–245 protoalkaloids, 251–252 purine alkaloids, 220–228 pyrrolidine alkaloids, 243–244 quinolizidine alkaloids, 246–248 tropane alkaloids, 241–242 Plant sources of cyclopeptide alkaloids, 111–119 of C18-diterpenoid alkaloids, 6–30

Subject Index

Platynecine, 180 (–)-Platynecine, 179 Plectronia spp., cyclopeptide alkaloids from, 119 PLS, see Partial least squares Poison hemlock, toxic alkaloids of, 147–151 Poisonous plant spp., 144–145 bipiperidine alkaloids in, 151–152 diterpene alkaloids in, 191–193, 196–197 isoquinoline alkaloids in, 188–189 piperidine alkaloids in, 147–149 polyhydroxy indolizidine alkaloids and related pyrrolizidine and nortropane alkaloids in, 158–163 pyridine alkaloids in, 154–155 pyrrolidine alkaloids in, 146–147 pyrrolizidine alkaloids in, 179–184 quinolizidine alkaloids in, 169–172 steroidal (Veratrum) alkaloids in, 199–200 steroidal (Zigadenus) alkaloids in, 203–204 tropane alkaloids in, 176–178 Polyhydroxy alkaloid glycosidase inhibitors, 166–168 Polyhydroxy indolizidine alkaloids, 158–169 plant spp. and specific alkaloids, 158–163 structure-activity relationships and mode of action of, 165–169 toxicity and clinical signs of, 163–165 Polyhydroxy nortropanes, see Calystegines Poppy family, toxic alkaloids of, 188–190, 228–233 Principal component regression (PCR), of quinine and quinidine, 237 Protoalkaloids, IR and Raman spectroscopy of, 251–252 Protopine, 188–190 (2S,5S)-(+)-Pseudoconhydrine, 147–148 Puberanidine, see N-Deacetyllappaconitine Puberanine, 32, 43–44, 62–63 Pubescine A, 89, 116 Purine alkaloids, IR and Raman spectroscopy of, 220–228 Pyridine alkaloids, 154–157

277

Pyrrole, metabolic pathway creating, 186–187 Pyrrolidine alkaloids, 146–147 IR and Raman spectroscopy of, 243–244 Pyrrolizidine alkaloids, 158–169, 179–187 plant spp. and specific alkaloids, 158–163, 179–184 structure-activity relationships and mode of action of, 165–169, 185–187 toxicity and clinical signs of, 163–165, 184–185 Pyrrolizidines, IR spectroscopy of, 219 Quinidine, 235–237 Quinine, 234–237 Quinoline ring, 236 Quinolizidine alkaloids, 152–154, 169–176, 219–220 IR and Raman spectroscopy of, 246–248 plant spp. and specific alkaloids, 169–172 structure-activity relationships and mode of action of, 174–176 toxicity and clinical signs of, 173–174 Raman mapping, 218–219 of benzylisoquinoline alkaloids, 239–241 Raman spectroscopy, see Infrared and Raman spectroscopy Ramosine A, 96, 108, 118 Ramosine B, 98, 109, 118 Ramosine C, 96, 108, 118 Ranaconitine classification and structure of, 22–23, 33, 43, 62–63 pharmacological activity of, 39, 68, 71–72 plant sources of, 22–23, 27–29 Ranaconitine-subtype classification and structure of, 42–45, 60–66 classification of, 3–5, 21–26 structure of, 4 Red pepper, alkaloids in, 251–252 Reticuline, 188–189 (+)-Retronecine, 179 Retrorsine, 182

278

Subject Index

Rhamnus spp., cyclopeptide alkaloids from, 117 Rhizoctonia leguminicola, 159 Rhombifoline, 172 Riddelliine, 180, 182, 186–187 Rivularine, see 7-Angelyl heliotridine Robusta coffee beans, quantification of caffeine in, 225 Rubijervine, 199–200 Rugosanine A, 86, 113 Rugosanine B, 83, 113 Sanguinarine, 188–190 IR and Raman spectroscopy of, 239–241 Sanjoinenine, 102, 112, 115 Sanjoinine A, see Frangufoline Sanjoinine B, 89, 115 Sanjoinine D, 99, 115, 134 Sanjoinine F, 89, 112, 115, 135 Sanjoinine G1, 99, 115, 127 Sanjoinine G2, 103, 115, 134–135 Sativanine A, 93, 113, 115 Sativanine B, 103, 113 Sativanine C, 86, 113 Sativanine D, 103, 113 Sativanine E, 83 Sativanine F, 86, 113 Sativanine G, 83, 113, 118 Sativanine H, 86, 113 Sativanine K, 83, 113, 115 Sativanine M, 86, 105, 114, 121 Sativanine N, 83, 104, 114, 120 Sativanine O, 83, 104, 114, 120 Scilla non-scripta, toxic alkaloids of, 146–147 Scopaline, 6, 33, 34, 46 plant sources of, 6, 27 Scopolamine, 176–179 IR and Raman spectroscopy of, 242 Scotch broom, toxic alkaloids of, 171 Scutia spp., cyclopeptide alkaloids from, 116–117 Scutianene C, 102, 117 Scutianene D, 133 Scutianine A, 91, 116–117, 125 5(14)-Scutianine A type cyclopeptide alkaloids, 91, 107 Scutianine B, 89, 116–119, 125, 133 Scutianine C, 89, 116–117, 119, 125, 133 Scutianine D, 89, 113–114, 116–117, 124–126, 133 Scutianine E, 89, 116–117, 124–126, 133

Scutianine F, 91, 117, 133 Scutianine G, 90, 116 Scutianine H, 90, 117, 125 Scutianine J, 90, 106, 117 Scutianine K, 90, 106, 117, 126 Scutianine L, 93, 117, 124, 126 Scutianine M, 93, 117, 124, 126, 133 Sedative activity, of cyclopeptide alkaloids, 134 Senecio spp., toxic alkaloids of, 181–182, 186 Senecionine, 180, 182 Seneciphylliine, 182 Senkirkine, 180 Sepaconitine, 14–15, 33, 38, 53–54 pharmacological activity of, 71 plant sources of, 14–15, 28–29 Septefine, 12, 33, 37 plant sources of, 12, 29 SERS, see Surface-enhanced Raman scattering Sida carpinifolia, toxic alkaloids of, 159 Sinomontanine A, 13–14, 33, 38, 53–54 plant sources of, 13–14, 29 Sinomontanine B, 12, 33, 37, 52–53 plant sources of, 12, 29 Sinomontanine D, 21, 33, 42, 60–61 plant sources of, 21, 29 Sinomontanine E, 10, 33, 36, 49–50 plant sources of, 10, 29 Sinomontanine F, 23, 33, 44, 63–64 plant sources of, 23, 29 Sinomontanine G, 24, 33, 44, 63–64 plant sources of, 24, 29 Sinomontanine H, 21–22, 33, 43, 60–61 plant sources of, 21–22, 29 Slaframine, 159 Solanidines, 201–202 (–)-Sophocarpine, 172 Sophora spp., toxic alkaloids of, 171–172 Sparteine, 169–172, 174–176 IR and Raman spectroscopy of, 227–228, 246 Spectabiline, 180, 184 Sphaeranthus spp., cyclopeptide alkaloids from, 119 Spinanine A, 96, 115 Stem bark, 113 Steroidal alkaloids Veratrum alkaloids, 199–202 Zigadenus alkaloids, 203–205

Subject Index

Stokes lines, 218 Structure–activity relationships and mechanisms of action of diterpene alkaloids, 194–196, 198–199 of isoquinoline alkaloids, 190 of piperidine alkaloids, 149–150 of polyhydroxy indolizidine alkaloids and related pyrrolizidine and nortropane alkaloids, 165–169 of pyridine alkaloids, 156–157 of pyrrolidine alkaloids, 147 of pyrrolizidine alkaloids, 185–187 of steroidal (Veratrum) alkaloids, 202 of steroidal (Zigadenus) alkaloids, 205 of tropane alkaloids, 178–179 Subfraction I, 83, 119 Subfraction II, 83, 119 Surface-enhanced Raman scattering (SERS), 218 of berberine, 234 of caffeine, 224–225 of chelerythrine, 239 of cocaine, 242 of nicotine, 244 of opioid isoquinoline alkaloids, 232 of sanguinarine, 239–241 Swainsona spp., toxic alkaloids of, 158, 164–165 Swainsonine, 158–160, 163–169 Symphytum spp., toxic alkaloids of, 183 Synthesis, of cyclopeptide alkaloids, 126–131 Taxicins, 197 Taxines, 197–198 Taxol (Paclitaxel), 197 Taxus spp., diterpene alkaloids in, 196–197 Tea, IR measurement of caffeine in, 226–227 Texensine, 90, 117 Thebaine, 229–231 Theobromine, 220–224, 227 Theophylline, 220–224, 227 Thermopsine, 172, 174 Thermopsis spp., toxic alkaloids of, 172, 174, 176 Thorn apple, toxic alkaloids of, 176 Tiantaishansine, 25, 33, 44, 65 plant sources of, 25, 30

279

Tobacco plants IR and Raman measurement of alkaloids in, 243–244 toxic alkaloids in, 154–157 Toxicity of bipiperidine alkaloids, 153 of diterpene alkaloids, 193–194, 197–198 of isoquinoline alkaloids, 189–190 of piperidine alkaloids, 149–150 of polyhydroxy indolizidine alkaloids and related pyrrolizidine and nortropane alkaloids, 163–165 of pyridine alkaloids, 155–156 of pyrrolidine alkaloids, 147 of pyrrolizidine alkaloids, 184–185 of steroidal (Veratrum) alkaloids, 201 of steroidal (Zigadenus) alkaloids, 204–205 of tropane alkaloids, 178 2,3,7-Tri-epi-australine, 160–161, 166–167 Triphyophyllum pelatatum, antimalarial alkaloids of, 237 Tropane alkaloids, 176–179 IR and Raman spectroscopy of, 241–242 Tscheschamine, 83, 113 Tuguaconitine, 25, 33, 44 plant sources of, 25, 29 Turbina cordata, toxic alkaloids of, 159 Ugi-four component reaction (Ugi-4CR), 127 Umbrofine, 17–18, 33, 40, 57 plant sources of, 17–18, 29 Usaramine, 184 UV resonance Raman microspectroscopy of quinidine, 236 of quinine, 236 VCD spectroscopy, see Vibrational circular dichroism spectroscopy Veratramine, 199–200 Veratrines, 202 Veratrum alkaloids, see Steroidal alkaloids Vibrational circular dichroism (VCD) spectroscopy, of anisodamine, 242 Vibrational spectral assignments, see Infra-red Band assignments

280

Subject Index

Vigna spp., cyclopeptide alkaloids from, 119 Vignatic acid A, 103, 110, 119 Vignatic acid B, 103, 110, 119 Voacangine acetoxyindolenine, 248–249 Voacangine hydroxyindolenine, 248–249 Waltheria spp., cyclopeptide alkaloids from, 118 Waltherine A, 90, 106, 118 Waltherine B, 90, 106, 118 Waltherine C, 90, 106, 118 Wild indigos, toxic alkaloids of, 171 Xanthine, 220–221, 227 Xylopyrine A, 83, 104, 114 Xylopyrine B, 83, 104, 114, 121 Yew alkaloids, see Diterpene alkaloids Yohimbine, 248–251 Yohimbines, IR spectroscopy of, 219

Zigadenus alkaloids, see Steroidal alkaloids Zizyphine A, 86, 114, 126–127 5(13)-Zizyphine A type cyclopeptide alkaloids, 84–86, 104–106 Zizyphine B, 86, 114 Zizyphine C, 86, 114 Zizyphine D, 101, 114 Zizyphine E, 101, 114 Zizyphine F, 86, 114–115 Zizyphine G, 96, 114 Zizyphine I, 86, 114 Zizyphine K, 86, 114 Zizyphine N, 86, 105, 115, 126, 133 synthesis of, 127–128 Zizyphine O, 86, 105, 115, 133 Zizyphine P, 86, 105, 115, 133 Zizyphine Q, 86, 105–106, 115, 133 Zizyphus spp. cyclopeptide alkaloids from, 112–115 pharmacological activities of, 131, 133–134 Zygacine, 202–204 Zygadenine, 203

CUMULATIVE INDEX OF TITLES Aconitum alkaloids, 4, 275 (1954), 7, 473 (1960), 34, 95 (1988) C18 diterpenes, 67, 1 (2009) C19 diterpenes, 12, 2 (1970) C20 diterpenes, 12, 136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, 54, 259 (2000) experimental antitumor activity of acronycine, 21, 1 (1983) Actinomycetes, isoquinolinequinones, 21, 55 (1983), 53, 120 (2000) N-Acyliminium ions as intermediates in alkaloid synthesis, 32, 271 (1988) Aerophobins and related alkaloids, 57, 208 (2001) Aerothionins, 57, 219 (2001) Ajmaline-Sarpagine alkaloids, 8, 789 (1965), 11, 41 (1986), 52, 104 (1999), 55, 1 (2001) enzymes in biosynthesis of, 47, 116 (1995) Alkaloid chemistry marine cyanobacteria, 57, 86 (2001) synthetic studies, 50, 377 (1998) Alkaloid production, plant biotechnology of, 40, 1 (1991) Alkaloid structures spectral methods, study, 24, 287 (1985) unknown structure, 5, 301 (1955), 7, 509 (1960), 10, 545 (1967), 12, 455 (1970), 13, 397 (1971), 14, 507 (1973), 15, 263 (1975), 16, 511 (1977) X-ray diffraction, 22, 51 (1983) Alkaloids apparicine and related, 57, 258 (2001) as chirality transmitters, 53, 1 (2000) biosynthesis, regulation of, 49, 222 (1997) biosynthesis, molecular genetics of, 50, 258 (1998) biotransformation of, 57, 3 (2001), 58, 1 (2002) chemical and biological aspects of Narcissus, 63, 87 (2006) containing a quinolinequinone unit, 49, 79 (1997) containing a quinolinequinoneimine unit, 49, 79 (1997) containing an isoquinolinoquinone unit, 53, 119 (2000) ecological activity of, 47, 227 (1995) ellipticine and related, 57, 236 (2001) forensic chemistry of, 32, 1 (1988) histochemistry of, 39, 165 (1990) infrared and raman spectroscopy of, 67, 217 (2009) in the plant, 1, 15 (1950), 6, 1 (1960) of the Menispermaceae, 54, 1 (2000) plant biotechnology, production of, 50, 453 (1998) toxic to livestock, 67, 143 (2009) uleine and related, 57, 247 (2001) with antiprotozoal activity, 66, 113 (2008)

257

258

Cumulative Index of Titles

Alkaloids from amphibians, 21, 139 (1983), 43, 185 (1993), 50, 141 (1998) ants and insects, 31, 193 (1987) Chinese traditional medicinal plants, 32, 241 (1988) Hernandiaceae, 62, 175 (2005) mammals, 21, 329 (1983), 43, 119 (1993) marine bacteria, 53, 239 (2000), 57, 75 (2001) marine organisms, 24, 25 (1985), 41, 41 (1992) medicinal plants of New Caledonia, 48, 1 (1996) mushrooms, 40, 189 (1991) plants of Thailand, 41, 1 (1992) Sri Lankan flora, 52, 1 (1999) Alkyl, aryl, alkylarylquinoline, and related alkaloids, 64, 139 (2007) Allelochemical properties of alkaloids, 43, 1 (1993) Allo congeners, and tropolonic Colchicum alkaloids, 41, 125 (1992) Alstonia alkaloids, 8, 159 (1965), 12, 207 (1970), 14, 157 (1973) Amaryllidaceae alkaloids, 2, 331 (1952), 6, 289 (1960), 11, 307 (1968), 15, 83 (1975), 30, 251 (1987), 51, 323 (1998), 63, 87 (2006) Amphibian alkaloids, 21, 139 (1983), 43, 185 (1983), 50, 141 (1998) Analgesic alkaloids, 5, 1 (1955) Anesthetics, local, 5, 211 (1955) Anthranilic acid derived alkaloids, 17, 105 (1979), 32, 341 (1988), 39, 63 (1990) Antifungal alkaloids, 42, 117 (1992) Antimalarial alkaloids, 5, 141 (1955) Antiprotozoal alkaloids, 66, 113 (2008) Antitumor alkaloids, 25, 1 (1985), 59, 281 (2002) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4, 119 (1954), 9, 1 (1967), 24, 153 (1985), 53, 57 (2000) Apparicine and related alkaloids, 57, 235 (2001) Aristolochia alkaloids, 31, 29 (1987) Aristotelia alkaloids, 24, 113 (1985), 48, 191 (1996) Aspergillus alkaloids, 29, 185 (1986) Aspidosperma alkaloids, 8, 336 (1965), 11, 205 (1968), 17, 199 (1979) synthesis of, 50, 343 (1998) Aspidospermine group alkaloids, 51, 1 (1998) Asymmetric catalysis by alkaloids, 53, 1 (2000) Azafluoranthene alkaloids, 23, 301 (1984) Bases simple, 3, 313 (1953), 8, 1 (1965) simple indole, 10, 491 (1967) simple isoquinoline, 4, 7 (1954), 21, 255 (1983) Benzodiazepine alkaloids, 39, 63 (1990) Benzophenanthridine alkaloids, 26, 185 (1985) Benzylisoquinoline alkaloids, 4, 29 (1954), 10, 402 (1967) Betalains, 39, 1 (1990) Biosynthesis in Catharanthus roseus, 49, 222 (1997) in Rauwolfia serpentina, 47, 116 (1995)

Cumulative Index of Titles

259

isoquinoline alkaloids, 4, 1 (1954) pyrrolizidine alkaloids, 46, 1 (1995) quinolizidine alkaloids, 46, 1 (1995) regulation of, 63, 1 (2006) tropane alkaloids, 44, 116 (1993) Bisbenzylisoquinoline alkaloids, 4, 199 (1954), 7, 439 (1960), 9, 133 (1967), 13, 303 (1971), 16, 249 (1977), 30, 1 (1987) synthesis, 16, 319 (1977) Bisindole alkaloids, 20, 1 (1981), 63, 181 (2006) noniridoid, 47, 173 (1995) Bisindole alkaloids of Catharanthus C-20u position as a functional hot spot in, 37, 133 (1990) isolation, structure clucidation and biosynthesis of, 37, 1 (1990), 63, 181 (2006) medicinal chemistry of, 37, 145 (1990) pharmacology of, 37, 205 (1990) synthesis of, 37, 77 (1990), 59, 281 (2002) therapeutic uses of, 37, 229 (1990) Bromotyrosine alkaloids, marine, 61, 79 (2005) Buxus alkaloids, steroids, 9, 305 (1967), 14, 1 (1973), 32, 79 (1988) chemistry and biology, 66, 191 (2008) Cactus alkaloids, 4, 23 (1954) Calabar bean alkaloids, 8, 27 (1965), 10, 383 (1967), 13, 213 (1971), 36, 225 (1989) Calabash curare alkaloids, 8, 515 (1965), 11, 189 (1968) Calycanthaceae alkaloids, 8, 581 (1965) Calystegines, 64, 49 (2007) Camptothecin and derivatives, 21, 101 (1983), 50, 509 (1998) clinical studies, 60, 1 (2003) Cancentrine alkaloids, 14, 407 (1973) Cannabis sativa alkaloids, 34, 77 (1988) Canthin-6-one alkaloids, 36, 135 (1989) Capsicum alkaloids, 23, 227 (1984) Carbazole alkaloids, 13, 273 (1971), 26, 1 (1985), 44, 257 (1993), 65, 1 (2008) biogenesis, 65, 159 (2008) biological and pharmacological activities, 65, 181 (2008) chemistry, 65, 195 (2008) Carboline alkaloids, 8, 47 (1965), 26, 1 (1985) b-Carboline congeners and Ipecac alkaloids, 22, 1 (1983) Cardioactive alkaloids, 5, 79 (1955) Catharanthus alkaloids, 59, 281 (2002) Catharanthus roseus, biosynthesis of terpenoid indole alkaloids in, 49, 222 (1997) Celastraceae alkaloids, 16, 215 (1977) Cephalotaxus alkaloids, 23, 157 (1984), 51, 199 (1998) Cevane group of Veratrum alkaloids, 41, 177 (1992) Chemosystematics of alkaloids, 50, 537 (1998) Chemotaxonomy of Papaveraceae and Fumariaceae, 29, 1 (1986) Chinese medicinal plants, alkaloids from, 32, 241 (1988) Chirality transmission by alkaloids, 53, 1 (2000) Chromone alkaloids, 31, 67 (1987) Cinchona alkaloids, 3, 1 (1953), 14, 181 (1973), 34, 332 (1988)

260

Cumulative Index of Titles

Colchicine, 2, 261 (1952), 6, 247 (1960), 11, 407 (1968), 23, 1 (1984) pharmacology and therapeutic aspects of, 53, 287 (2000) Colchicum alkaloids and allo congeners, 41, 125 (1992) Configuration and conformation, elucidation by X-ray diffraction, 22, 51 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4, 249 (1954), 10, 463 (1967), 29, 287 (1986) Curare-like effects, 5, 259 (1955) Cyclic tautomers of tryptamine and tryptophan, 34, 1 (1988) Cyclopeptide alkaloids, 15, 165 (1975), 67, 79 (2009) Cytotoxic alkaloids, modes of action, 64, 1 (2007) Daphniphyllum alkaloids, 15, 41 (1975), 29, 265 (1986), 60, 165 (2003) Delphinium alkaloids, 4, 275 (1954), 7, 473 (1960) C10-diterpenes, 12, 2 (1970) C20-diterpenes, 12, 136 (1970) Detection of through IR and Raman spectroscopy, 67, 217 (2009) Dibenzazonine alkaloids, 35, 177 (1989) Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8, 336 (1965) Diterpenoid alkaloids Aconitum, 7, 473 (1960), 12, 2 (1970), 12, 136 (1970), 34, 95 (1988) C18, 67, 1 (2009) C20, 59, 1 (2002) chemistry, 18, 99 (1981), 42, 151 (1992) Delphinium, 7, 473 (1960), 12, 2 (1970), 12, 136 (1970) Garrya, 7, 473 (1960), 12, 2 (1960), 12, 136 (1970) general introduction, 12, xv (1970) structure, 17, 1 (1979) synthesis, 17, 1 (1979) Eburnamine-vincamine alkaloids, 8, 250 (1965), 11, 125 (1968), 20, 297 (1981), 42, 1 (1992) Ecological activity of alkaloids, 47, 227 (1995) Elaeocarpus alkaloids, 6, 325 (1960) Ellipticine and related alkaloids, 39, 239 (1990), 57, 235 (2001) Enamide cyclizations in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in vitro, 18, 323 (1981) Ephedra alkaloids, 3, 339 (1953) Epibatidine, 46, 95 (1995) Ergot alkaloids, 8, 726 (1965), 15, 1 (1975), 38, 1 (1990), 50, 171 (1998), 54, 191 (2000), 63, 45 (2006) Erythrina alkaloids, 2, 499 (1952), 7, 201 (1960), 9, 483 (1967), 18, 1 (1981), 48, 249 (1996) Erythrophleum alkaloids, 4, 265 (1954), 10, 287 (1967) Eupomatia alkaloids, 24, 1 (1985) Forensic chemistry, alkaloids, 12, 514 (1970) by chromatographic methods, 32, 1 (1988) Galbulimima alkaloids, 9, 529 (1967), 13, 227 (1971) Gardneria alkaloids, 36, 1 (1989) Garrya alkaloids, 7, 473 (1960), 12, 2 (1970), 12, 136 (1970)

Cumulative Index of Titles

261

Geissospermum alkaloids, 8, 679 (1965) Gelsemium alkaloids, 8, 93 (1965), 33, 84 (1988), 49, 1 (1997) Glycosides, monoterpene alkaloids, 17, 545 (1979) Guatteria alkaloids, 35, 1 (1989) Haplophyton cimicidum alkaloids, 8, 673 (1965) Hasubanan alkaloids, 16, 393 (1977), 33, 307 (1988) Hernandiaceae alkaloids, 62, 175 (2005) Histochemistry of alkaloids, 39, 165 (1990) Holarrhena group, steroid alkaloids, 7, 319 (1960) Hunteria alkaloids, 8, 250 (1965) Iboga alkaloids, 8, 203 (1965), 11, 79 (1968), 59, 281 (2002) Ibogaine alkaloids addict self-help, 56, 283 (2001) as a glutamate antagonist, 56, 55 (2001) comparative neuropharmacology, 56, 79 (2001) contemporary history of, 56, 249 (2001) drug discrimination studies with, 56, 63 (2001) effects of rewarding drugs, 56, 211 (2001) gene expression, changes in, 56, 135 (2001) mechanisms of action, 56, 39 (2001) multiple sites of action, 56, 115 (2001) neurotoxicity assessment, 56, 193 (2001) pharmacology of, 52, 197 (1999) review, 56, 1 (2001) treatment case studies, 56, 293 (2001) use in equatorial African ritual context, 56, 235 (2001) Imidazole alkaloids, 3, 201 (1953), 22, 281 (1983) Indole alkaloids, 2, 369 (1952), 7, 1 (1960), 26, 1 (1985) ajmaline group of, 55, 1 (2001) biomimetic synthesis of, 50, 415 (1998) biosynthesis in Catharanthus roseus, 49, 222 (1997) biosynthesis in Rauvolfia serpentina, 47, 116 (1995) distribution in plants, 11, 1 (1968) Reissert synthesis of, 31, 1 (1987) sarpagine group of, 52, 103 (1999) simple, 10, 491 (1967), 26, 1 (1985) Indole diterpenoid alkaloids, 60, 51 (2003) Indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) 2,2’-Indolylquinuclidine alkaloids, chemistry, 8, 238 (1965), 11, 73 (1968) Infrared spectroscopy of alkaloids, 67, 217 (2009) In vitro and microbial enzymatic transformation of alkaloids, 18, 323 (1981) Ipecac alkaloids, 3, 363 (1953), 7, 419 (1960), 13, 189 (1971), 22, 1 (1983), 51, 271 (1998) Isolation of alkaloids, 1, 1 (1950) Isoquinoline alkaloids, 7, 423 (1960) biosynthesis, 4, 1 (1954) 13 C-NMR spectra, 18, 217 (1981) Reissert synthesis of, 31, 1 (1987) simple isoquinoline alkaloids 4, 7 (1954), 21, 255 (1983)

262

Cumulative Index of Titles

Isoquinolinequinones, 21, 55 (1983), 53, 120 (2000) Isoxazole alkaloids, 57, 186 (2001) Khat (Catha edulis) alkaloids, 39, 139 (1990) Kopsia alkaloids, 8, 336 (1965), 66, 1 (2008) Lead tetraacetate oxidation in alkaloid synthesis, 36, 70 (1989) Local anesthetics, 5, 211 (1955) Localization in the plant, 1, 15 (1950), 6, 1 (1960) Lupine alkaloids, 3, 119 (1953), 7, 253 (1960), 9, 175 (1967), 31, 116 (1987), 47, 1 (1995) Lycopodium alkaloids, 5, 265 (1955), 7, 505 (1960), 10, 306 (1967), 14, 347 (1973), 26, 241 (1985), 45, 233 (1944), 61, 1 (2005) Lythraceae alkaloids, 18, 263 (1981), 35, 155 (1989) Macrocyclic peptide alkaloids from plants, 26, 299 (1985), 49, 301 (1997) Mammalian alkaloids, 21, 329 (1983), 43, 119 (1993) Manske, R.H.F., biography of, 50, 3 (1998) Manzamine alkaloids, 60, 207 (2003) Marine alkaloids, 24, 25 (1985), 41, 41 (1992), 52, 233 (1999) bromotyrosine alkaloids, 61, 79 (2005) Marine bacteria, alkaloids from, 53, 120 (2000) Maytansinoids, 23, 71 (1984) Melanins, 36, 254 (1989) chemical and biological aspects, 60, 345 (2003) Melodinus alkaloids, 11, 205 (1968) Mesembrine alkaloids, 9, 467 (1967) Metabolic transformation of alkaloids, 27, 323 (1986) Microbial and in vitro enzymatic transformation of alkaloids, 18, 323 (1981) Mitragyna alkaloids, 8, 59 (1965), 10, 521 (1967), 14, 123 (1973) Molecular modes of action of cytotoxic alkaloids, 64, 1 (2007) Monoterpene alkaloids, 16, 431 (1977), 52, 261 (1999) glycosides, 17, 545 (1979) Morphine alkaloids, 2, 1 (part 1), 161 (part 2) (1952), 6, 219 (1960), 13, 1 (1971), 45, 127 (1994) Muscarine alkaloids, 23, 327 (1984) Mushrooms, alkaloids from, 40, 190 (1991) Mydriatic alkaloids, 5, 243 (1955) a-Naphthophenanthridine alkaloids, 4, 253 (1954), 10, 485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (1986), 46, 127 (1995) Narcotics, 5, 1 (1955) Narcissus alkaloids, 63, 87 (2006) New Caledonia, alkaloids from the medicinal plants of, 48, 1 (1996) Nitrogen-containing metabolites from marine bacteria, 53, 239, (2000), 57, 75 (2001) Non-iridoid bisindole alkaloids, 47, 173 (1995) Nuphar alkaloids, 9, 441 (1967), 16, 181 (1977), 35, 215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11, 205 (1968) Ourouparia alkaloids, 8, 59 (1965), 10, 521 (1967)

Cumulative Index of Titles

263

Oxazole alkaloids, 35, 259 (1989) Oxindole alkaloids, 14, 83 (1973) Oxoaporphine alkaloids, 14, 225 (1973) Pandanus alkaloids chemistry and biology, 66, 215 (2008) Papaveraceae alkaloids, 10, 467 (1967), 12, 333 (1970), 17, 385 (1979) pharmacology, 15, 207 (1975) toxicology, 15, 207 (1975) Pauridiantha alkaloids, 30, 223 (1987) Pavine and isopavine alkaloids, 31, 317 (1987) Pentaceras alkaloids, 8, 250 (1965) Peptide alkaloids, 26, 299 (1985), 49, 301 (1997) Phenanthrene alkaloids, 39, 99 (1990) Phenanthroindolizidine alkaloids, 19, 193 (1981) Phenanthroquinolizidine alkaloids, 19, 193 (1981) b-Phenethylamines, 3, 313 (1953), 35, 77 (1989) Phenethylisoquinoline alkaloids, 14, 265 (1973), 36, 172 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7, 433 (1960), 9, 117 (1967), 24, 253 (1985) Picralima alkaloids, 8, 119 (1965), 10, 501 (1967), 14, 157 (1973) Piperidine alkaloids, 26, 89 (1985) Plant biotechnology, for alkaloid production, 40, 1 (1991), 50, 453 (1998) Plant systematics, 16, 1 (1977) Pleiocarpa alkaloids, 8, 336 (1965), 11, 205 (1968) Polyamine alkaloids, 22, 85 (1983), 45, 1 (1994), 50, 219 (1998), 58, 83 (2002) analytical aspects of, 58, 206 (2002) biogenetic aspects of, 58, 274 (2002) biological and pharmacological aspects of, 46, 63 (1995), 58, 281 (2002) catalog of, 58, 89 (2002) synthesis of cores of, 58, 243 (2002) Pressor alkaloids, 5, 229 (1955) Protoberberine alkaloids, 4, 77 (1954), 9, 41 (1967), 28, 95 (1986), 62, 1 (2005) biotransformation of, 46, 273 (1955) transformation reactions of, 33, 141 (1988) Protopine alkaloids, 4, 147 (1954), 34, 181 (1988) Pseudocinchoma alkaloids, 8, 694 (1965) Pseudodistomins, 50, 317 (1998) Purine alkaloids, 38, 226 (1990) Putrescine and related polyamine alkaloids, 58, 83 (2002) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960), 11, 459 (1968), 26, 89 (1985) Pyrrolidine alkaloids, 1, 91 (1950), 6, 31 (1960), 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6, 35 (1960), 12, 246 (1970), 26, 327 (1985) biosynthesis of, 46, 1 (1995) Quinazolidine alkaloids, see Indolizidine alkaloids Quinazoline alkaloids, 3, 101 (1953), 7, 247 (1960), 29, 99 (1986) Quinazolinocarbolines, 8, 55 (1965), 21, 29 (1983) Quinoline alkaloids related to anthranilic acid, 3, 65 (1953), 7, 229 (1960), 17, 105 (1979), 32, 341 (1988) Quinolinequinone alkaloids, 49, 79 (1997)

264

Cumulative Index of Titles

Quinolinequinoneimine alkaloids, 49, 79 (1977) Quinolizidine alkaloids, 28, 183 (1985), 55, 91 (2001) biosynthesis of, 47, 1 (1995) Raman spectroscopy of alkaloids, 67, 217 (2009) Rauwolfia alkaloids, 8, 287 (1965) biosynthesis of, 47, 116 (1995) Recent studies on the synthesis of strychnine, 64, 103 (2007) Regulation of alkaloid biosynthesis in plants, 63, 1 (2006) Reissert synthesis of isoquinoline and indole alkaloids, 31, 1 (1987) Reserpine, chemistry, 8, 287 (1965) Respiratory stimulants, 5, 109 (1995) Rhoeadine alkaloids, 28, 1 (1986) Salamandra group, steroids, 9, 427 (1967) Sarpagine-type alkaloids, 52, 104 (1999) Sceletium alkaloids, 19, 1 (1981) Secoisoquinoline alkaloids, 33, 231 (1988) Securinega alkaloids, 14, 425 (1973) Senecio alkaloids, see Pyrrolizidine alkaloids Sesquiterpene pyridine alkaloids, 60, 287 (2003) Simple indole alkaloids, 10, 491 (1967) Simple indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) Simple indolizidine and quinolizidine alkaloids, 55, 91 (2001) Sinomenine, 2, 219 (1952) Solanum alkaloids chemistry, 3, 247 (1953) steroids, 7, 343 (1960), 10, 1 (1967), 19, 81 (1981) Sources of alkaloids, 1, 1 (1950) Spectral methods, alkaloid structures, 24, 287 (1985) Spermidine and related polyamine alkaloids, 22, 85 (1983), 58, 83 (2002) Spermine and related polyamine alkaloids, 22, 85 (1983), 58, 83 (2002) Spider toxin alkaloids, 45, 1 (1994), 46, 63 (1995) Spirobenzylisoquinoline alkaloids, 13, 165 (1971), 38, 157 (1990) Sponges, isoquinolinequinone alkaloids from, 21, 55 (1983) Sri Lankan flora, alkaloids, 52, 1 (1999) Stemona alkaloids, 9, 545 (1967), 62, 77 (2005) Steroid alkaloids Apocynaceae, 9, 305 (1967), 32, 79 (1988) Buxus group, 9, 305 (1967), 14, 1 (1973), 32, 79 (1988), 66, 191 (2008) chemistry and biology, 50, 61 (1998), 52, 233 (1999) Holarrhena group, 7, 319 (1960) Salamandra group, 9, 427 (1967) Solanum group, 7, 343 (1960), 10, 1 (1967), 19, 81 (1981) Veratrum group, 7, 363 (1960), 10, 193 (1967), 14, 1 (1973), 41, 177 (1992) Stimulants respiratory, 5, 109 (1955) uterine, 5, 163 (1955) Structure elucidation, by X-ray diffraction, 22, 51 (1983) Strychnine, synthesis of, 64, 104 (2007)

Cumulative Index of Titles

265

Strychnos alkaloids, 1, 375 (part 1) (1950), 2, 513 (part 2) (1952), 6, 179 (1960), 8, 515, 592 (1965), 11, 189 (1968), 34, 211 (1988), 36, 1 (1989), 48, 75 (1996) Sulfur-containing alkaloids, 26, 53 (1985), 42, 249 (1992) Synthesis of alkaloids enamide cyclizations for, 22, 189 (1983) lead tetraacetate oxidation in, 36, 70 (1989) Tabernaemontana alkaloids, 27, 1 (1983) Taxol, 50, 509 (1998) Taxus alkaloids, 10, 597 (1967), 39, 195 (1990) Terpenoid indole alkaloids, 49, 222 (1997) Thailand, alkaloids from the plants of, 41, 1 (1992) Toxicity to livestock, 67, 143 (2009) Toxicology, Papaveraceae alkaloids, 15, 207 (1975) Transformation of alkaloids, enzymatic, microbial and in vitro, 18, 323 (1981) Tremogenic and non-tremogenic alkaloids, 60, 51 (2003) Tropane alkaloids biosynthesis of, 44, 115 (1993) chemistry, 1, 271 (1950), 6, 145 (1960), 9, 269 (1967), 13, 351 (1971), 16, 83 (1977), 33, 2 (1988), 44, I (1933) Tropoloisoquinoline alkaloids, 23, 301 (1984) Tropolonic Colchicum alkaloids, 23, 1 (1984), 41, 125 (1992) Tylophora alkaloids, 9, 517 (1967) Uleine and related alkaloids, 57, 235 (2001) Unnatural alkaloid enantiomers, biological activity of, 50, 109 (1998) Uterine stimulants, 5, 163 (1955) Veratrum alkaloids cevane group of, 41, 177 (1992) chemistry, 3, 247 (1952) steroids, 7, 363 (1960), 10, 193 (1967), 14, 1 (1973) Vinca alkaloids, 8, 272 (1965), 11, 99 (1968), 20, 297 (1981) Voacanga alkaloids, 8, 203 (1965), 11, 79 (1968) Wasp toxin alkaloids, 45, 1 (1994), 46, 63 (1995) X-ray diffraction of alkaloids, 22, 51 (1983) Yohimbe alkaloids, 8, 694 (1965), 11, 145 (1968), 27, 131 (1986)