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THE ALKALOIDS Chemistry and Pharmacology VOLUME 39

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THE ALKALOIDS Chemistry and Pharmacology Edited by Arnold Brossi National Institutes of Health Bethesda. Maryland

VOLUME 39

Academic Press, Inc. Harcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ COPYRIGHT 0 1990 BY ACADEh4IC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

ACADEMIC PRESS, INC. San Diego, California 92101 United Kingdom Edition published by ACADEMIC P R E S S LIMITED 24-28 Oval Road, London NW17DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 50-5522

ISBN 0-12-469539-6 (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 9 0 9 1 9 2 9 3

1 0 9 8 1 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS .............. PREFACE. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . ..............................

ix xi

Chapter 1. Betalains WOLFGANG STEGLICH A N D DIETERSTRACK I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural Elucidation and Chemistry of Betanidin . . . . . . Betacyanins ...................................... Betaxanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Muscapurpurin . .................... Betalamic Acid . .................... Muscaflavin ......................... ............... Syntheses of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemotaxonomy and Distribution of Betalains . . . . . . . . . . . ........................ Biosynthesis of Betalains References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

3 8 19 26 26 21 28 35 53 59

Chapter 2. Benzodiazepine Alkaloids W. Ross 1. Introduction

111. IV. V. VI. VII.

..............................................

robial Producers . . . . . . . . . . . ......... Structural Elucidation and Related Chemistry ............................ Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic Conversion of Benzo Physiological Aspects . . . . . . . ........... Biological Activity of Naturally References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 66 66 73 79 81 93 94

Chapter 3. Phenanthrene Alkaloids LUISCASTEDOAND GABRIELTOJO I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Occurrence and Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

99 100

vi

CONTENTS

111. Synthesis . . . . . . . . . . . . . V. Biosynthesis . . . . . . . . . . . . . . . . . . . . VII. Pharmacology ............................................. References . . . . . . . . . . . . . . . . . . .

121 128 132 134 135 135

Chapter 4. The Alkaloids of Khat (Carha edulis)

L. CROMBIE, W. M. L. CROMBIE, AND D. A. WHITING 1. Introduction ....................................................... 11. The Phenylalkylamine Alkaloids (Khatamines) . . . . . . . . . . . . . . . . 111. Synthesis of the Khatamines . . . . IV. Pharmacological Action of the Khatamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. The Cathedlin Alkaloids of Khat VI. Triterpenoid Extractives of Khat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. The Catvaalens Sesquiterpenoids of Cuthu trunsvuulensis . . . . . . . . . . . . . . . . . . .... VIII. Synthetic Work Relevant to the Cathedulin Alkaloids References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I

139 I 40 141 144 145 157 159 159 162

Chapter 5. Histochemistry of Alkaloids AND MOMOYO ICHIMARU YOHEIHASHIMOTO, KAZUKO KAWANISHI,

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Histochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Histochemical Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165

167 180 191

Chapter 6. Taxus Alkaloids A N D DANIEL GUENARD SIEGFRIED BLECHERT

I. Introduction . . . . . . . . . . . .............................. 11. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Isolation . . . ............................................ V. Hemisynthesis ...................................................... .............................. VI. Synthesis . . . . . . . . . . . . . . VII. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

195 196 197 202 203 208 229 234

Chapter 7. Synthesis and Antitumor Activity of the Ellipticine Alkaloids and Related Compounds GORDONW. GRIBBLE 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Occurrence and Structural Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 240

CONTENTS 111. Synthesis of Ellipticine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Synthesis of Olivacine . . . . . . . . . . . . . . . . . . . . . , . . , . , . . . . . . . . . . . . . . , . . . . . ...... cine Derivatives . . . . . . . . . . V. Synthesis of Modif

VI. VII. VIII. IX. X. XI.

Biological Detectio ........................................... mental Models . . . . . . , . , . . . . . . . . . . . . . . . . . . . . Antitumor Activity Mechanism of Action . . . . . . . . . . . . . . . . . Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolism and Microbial Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure-Activity Relationships . . . . . . . . . .

XIII. Clinical Trials , . . . . . . , . XIV. Conclusion

CUMULATIVE INDEX OF

............................... ............................... ............................... . . . . . . . . . . .....................

TITLES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 242 250 254 305 307 31 I 325 325 328 340 340 343 343

353 36 I


CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

SIEGFRIED BLECHERT (195), Institut fur Organische Chemie Universitat Bonn, D-5300 Bonn 1, Federal Republic of Germany LUISCASTEDO(99), Department of Organic Chemistry, University of Santiago de Compostela, Santiago, Spain L. CROMBIE(139), Department of Chemistry, The University of Nottingham, Nottingham, NG7 2RD, England W. M. L. CROMBIE(139), Department of Chemistry, The University of Nottingham, Nottingham, NG7 2RD, England GORDONW. GRIBBLE(239), Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755 DANIELGUENARD(195), Institut de Chimie des Substances Naturelles, C.N.R.S., F-91190 Gif-sur-Yvette, France YOHEIHASHIMOTO (165), Kobe Women’s College of Pharmacy, MotoyamakitaMachi, Higashinada-Ku, Kobe 658, Japan MOMOYO ICHIMARU (163, Kobe Women’s College of Pharmacy, MotoyamakitaMachi, Higashinadu-Ku, Kobe 658, Japan KAZUKOKAWANISHI (163, Kobe Women’s College of Pharmacy, Motoyamakita-Machi, Higashinadu-Ku, Kobe 658, Japan W. Ross (63), Biotechnikum, Martin-Luther-Universitat Halle-Wittenberg, 4050 Halle (Saale), German Democratic Republic, Federal Republic of Germany WOLFGANG STEGLICH (l), Institut fur Organische Chemie und Biochemie, Universitat Bonn, D-5300 Bonn 1, Federal Republic of Germany DIETERSTRACK (l), Institut fur Pharmazeutische Biologie, Technische Universitat Braunschweig, D-3300 Braunschweig, Federal Republic of Germany GABRIEL TOJO(99), Department of Organic Chemistry, University of Santiago de Compostela, Santiago, Spain D. A. WHITING(139), Department of Chemistry, The University of Nottingham, Nottingham, NG7 2RD, England

ix


PREFACE

With the exception of “The Ellipticines,” briefly discussed in Volume 25 as interesting antitumor agents and updated here with regard to chemistry and pharmacology, all other presentations appear in this text for the first time. “The Betalains,” present as pigments in red beets, are noteworthy because of their red color and their chemical relationship with L-dopa. “The Benzodiazepine Alkaloids,” with anthramycin as the best known representative and chemically related to the tranquilizers librium and Valium, are potent but toxic antitumor agents. “The Phenanthrene Alkaloids,” biogenetically derived from aporphines, and “The Alkaloids of Khat,” used in Arab countries as a stimulant that causes physical dependence, also are discussed here for the first time. The same applies to “Taxus Alkaloids,” which includes the interesting experimental antitumor drug taxol. Its presentation is timely and may help scientists to coordinate its large-scale production. Last but not least is the chapter on “Histochemistry of Alkaloids,” that presents microtechniques used to locate and to identify alkaloids in plant tissues with alkaloid reagents, most useful in biosynthetic studies. The authors listed in this volume come from six different countries, which attests once more that alkaloid research is a multidisciplinary and multinational science. Arnold Brossi National Institutes of Health

xi


-Chapter

1-

BETALAINS WOLFGANG STEGLICH Institut fur Organische Chemie und Biochemie Universitat Bonn 5300 Bonn I , Federal Republic of Germany

DIETERSTRACK Institut fur Pharmazeutische Biologie Technische Universitat Braunschweig 3300 Braunschweig, Federal Republic of Germany

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 11. Structural Elucidation and Chemistry of Betanidin .......................... 3 111. Betacyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 A. Isolation and Structural Elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 B. Individual Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 IV. Betaxanthins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 A. Isolation and Structural Elucidation 19 B. Individual Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 V. Muscapurpurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 VI. Betalamic Acid VII. Muscaflavin . . . VIII. Syntheses of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 A. Syntheses of Betalamic Acid _ . _ . . _28 B. Syntheses of Cyclodopa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 C. Syntheses of Betalains by Reaction of Betalamic Acid with Amino Acids 31 D. Syntheses of Betalain Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 E. Synthesis of Muscaflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 IX. Chemotaxonomy and Distribution of Betalains ............................ 35 X. Biosynthesis of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 References . . . . . 59

I. Introduction

Betalains constitute a class of structurally closely related chromoalkaloids which are characteristic of the plant order Caryophyllales (Centrospermae). They may be divided into two main structural types, the betacyanins and betaxanthins. The former are derivatives of the aglycone betanidin (1)which can be considered 1

THE ALKAlDIDS. VOL. 39 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

WOLFGANG STEGLICH AND DIETER STRACK

as a condensation product of cyclodopa with betalamic acid (3). Similarly, reaction of 3 with proline and other a-amino acids or amines yields yellow betaxanthins, for example, indicaxanthin (2). Early investigations showed that the pigments of red beet and pokebeny were different from the common anthocyanins. In contrast to the progress which was quickly achieved in the structural elucidation of the anthocyanins, the chemical nature of the betalains remained obscure until the middle of the twentieth century. Dreiding has given a careful account of these early studies (1,2). A major breakthrough was the crystallization of betanin (3,4),which led to the structural elucidation of betanidin (1) by Wyler et al. ( 5 ) in 1963. A year later, Piattelli et al. ( 6 ) published the structure of indicaxanthin (2), an orange-yellow pigment from fig cactus fruits. The development of new isolation and separation techniques allowed the Italian group to characterize several new betalain pigments. In parallel with these investigations, the biosynthesis of betalains was studied, and the chemotaxonomic value of these pigments was amply demonstrated (7). In 197 1, Dopp et al. (8) reported the unexpected finding that the pigments of the fly agaric belong to the betalains. Later, muscaflavin (4) an isomer of betalamic acid (3), was discovered as a new type of betalain pigment, and it was shown that 4 occurs in the form of amino acid derivatives in the brightly colored fruit-bodies of Hygrocybe species (9). With the enormous progress in the development of spectroscopic techniques and methods for separation achieved in the 1980s, a renaissance in the study of betalains appears timely. Recent studies on betalain pigments clearly demonstrate the new possibilities offered by FAB MS, high-field NMR, and HPLC (10). The first total synthesis of betalains was announced in 1975 by Hermann and Dreiding (II), and it was later considerably improved by the same group. An independent synthetic approach to betalain pigments was developed by Buchi and co-workers (12). The enzymatic synthesis of betalains has not yet been investigated. Also, the physiological aspects of their formation are still poorly understood, despite the fact that numerous investigations have been carried out, for example, on their photoregulation and hormonal control (13). Betalains have recently received much attention by the food industry as the red to violet beta-

1

2

3

1.

3

BETALAINS

4

cyanins are evidently suitable as nonmutagenic natural color additives for some food systems (Z4,Z5). As the chemistry, biochemistry, and chemotaxonomic relevance of betalains have been reviewed by several authors (Z,13,16-24), the present chapter only briefly summarizes earlier results. Here we concentrate on more recent findings and give a critical survey of the present state of betalain chemistry and the distribution of betalains in plants.

11. Structural Elucidation and Chemistry of Betanidin

Betanidin (1) constitutes the basic structural unit of all betacyanin pigments. The compound occurs as its glucoside betanin (5) in red beets and is therefore easily accessible. The chemistry of 1 has been intensively studied by Dreiding's

HCI

5

Ho

1

6

7

0

4


group in Zurich, and because it has served as the key compound for the structural elucidation of the other betacyanins, it is discussed here in some detail. Controlled hydrolysis of betanin (5) with hydrochloric acid yields betanidin (1)and glucose (1,25).Depending on the reaction conditions, 1 is accompanied by varying amounts of isobetanidin (6), which could be separated by paper chromatography (25).Alkaline degradation of betanidin under careful exclusion of oxygen afforded formic acid and two fragments which were identified as 2,3dihydro-5,6-dihydroxyindole-2-carboxylic acid (7)and 4-methylpyridine-2,6-dicarboxylic acid (8) (26). When the alkaline degradation of betanidin was carried out in the presence of air, 5,6-dihydroxyindole-2-carboxylicacid was obtained instead of 7 (25). An important step toward the structural elucidation of betanidin (1) was the observation that on treatment of its hydrochloride in methanol with a large excess of diazomethane the yellow di-O-methylneobetanidin trimethyl ester (1 1) was obtained, whereas esterification of the pigment with methanolic hydrochloric acid yielded the expected violet betanidin trimethyl ester hydrochloride (9) (27,28).A compound related to 11, di-O-acetylneobetanidin trimethyl ester (lo), was formed on acetylation of 9 with acetic anhydride in pyridine. Compound 10 can be deacetylated to neobetanidin trimethyl ester hydrochloride (12) on standing in methanolic hydrochloric acid (28).The neobetanidin derivative 11exhibits a major absorption maximum at 403 nm (E 32,500)which is shifted toward 513 nm (E 44,500)on addition of acid, producing a deep violet color. In contrast to those of betanidin, the 'H-NMR signals of 10 and 11 are nicely resolved, and, taking into account the spectra of several model compounds, structures could be assigned to the neobetanidin compounds (27,28). On treatment of 11with palladium, an interesting disproportionation reaction takes place which yields the colorless indole derivative 14 (27,28).Reaction of betanidin with acetyl chloride in trifluoroacetic acid afforded di-O-acetylbetanidin (13), which could be hydrolyzed back to 1 with concentrated hydrochloric acid (5). This demonstrated the presence of two phenolic hydroxyl groups in betanidin, and from consideration of the 'H-NMR data formula 1was assigned to betanidin. The (2s)configuration of betanidin has been determined by the identification of degradation product 7 as (S)-cyclodopa (5). For the determination of the absolute configuration at C- 15, studies on the stereochemical relationship of betanidin (1) and isobetanidin (6) were of importance. It was found that on hydrolysis of betanin (5) under drastic conditions (18% HCl, 87°C) the ratio of 6 to 1 formed increased with the reaction time, whereas with concentrated HCl at room temperature the aglycone contained only 5% of 6 (29). On treatment of either pure 1 or 6 with 0.4 N KOH at 25°C with exclusion of oxygen, a 1 : 2 equilibrium mixture of the two isomers was formed. That the epimerization takes place at C-15is indicated by the fact that both isomers gave the same optically

1.

5

BETALAINS

H HO w C

AcO

O

2

C

H

3

AcpO

AcO

PY

10

9

MeoH

HO

A~CI\ \ q . TFA

HCI

11 Pd-C

1 C 0 2 CH3

12

\

H3c0m H3CO

C o p CH3

H3C02Cb C O p C H 3

13

14

active neobetanidin derivative 11 on treatment with diazomethane. In an elegant experiment, Dreiding and co-workers (29) reacted betanidin (1) with (R)cyclodopa (R-7) in the presence of ammonia and obtained ent-isobetanidin (15) which exhibited an ORD curve which was the mirror image of that from isobetanidin (6). In contrast, when the same reaction was carried out with 6, a product was obtained which was identified in the same way as ent-betanidin (16). This provided additional proof for 1 and 6 being epimers at C- 15. The absolute configuration at C-15 was finally determined by peracid degradation of indicaxanthin (2), which had been previously correlated with betanidin (1)

6


HO

HO~ . . . . c o 2 0

H 02C x

C H

0

2H

16

15

(R)-7

6

1

H~CCOSH

2

17

by amino acid exchange with (S)-cyclodopa (7) (29,30). The isolation of (9aspartic acid (17) after peracid oxidation allowed the assignment of the (15s) configuration to betanidin (29). It is important to note that the amino acid exchange in aqueous ammonia proceeds without epimerization of betanidin. In contrast, indicaxanthin (2) is labile under these conditions. Compound 17 had previously been obtained by direct oxidation of betanidin (31). This result is ambiguous, however, because it could be demonstrated that (S)-cyclodopa (7) is degraded to 17 under the same conditions (29). 'H-NMR investigations indicate that betanidin and isobetanidin are present in trifluoroacetic acid as a 3 : 1 mixture of the 12E and 122 stereoisomers l a / l b and 6a/6b, respectively (32). The interconversion of these stereoisomers at room temperature is so fast that they can not be separated from each other. In accord

1. HO

b7.W

HO \ 1 7.38

7

BETALAINS

s 5.56 dd

P

la: 15a

( ' H NMR data in TFA)

lb: 15p 6b: 15a

6a: 15p

with the behavior of other 1,7-diazaheptamethiniurnsystems, the protons at C-12 and C-18 are exchanged with deuterons on dissolving the pigments in deuteriotrifluoroacetic acid. Betanidin hydrochloride is smoothly decarboxylated in boiling ethanol (33).In order to investigate the mechanism of this reaction, the decarboxylation of 1 was performed in monodeuterioethanol (34). The resulting decarboxybetanidin (19),

I

-He

U-b

1 + 6

8


characterized in the form of its dimethyl ester, showed deuterium incorporation at C-15 and not at the olefinic position C-17. This can only be explained by the mechanism given, which leads to loss of the carboxyl group at the original C-15. Proton loss from intermediate 18 followed by reprotonation at C-17 and double bond migration would explain the easy epimerization of betanidin and isobetanidin. This mechanism, however, could be excluded by labeling experiments which ruled out an equilibration of the two carboxyl groups during epimerization at C-15.

111. Betacyanins

A. ISOLATIONAND STRUCTURAL ELUCIDATION Progress in the chemistry of betacyanins depended on the development of efficient methods for their isolation and separation. Betalains often occur as complex mixtures and are easily decomposed during the purification steps, which render the isolation of larger amounts of material for structural studies difficult. It is therefore understandable that some of the compounds described during the 1960s need reinvestigation by modem techniques. The first separations of individual betacyanins were camed out by paper chromatography (35).The observation that betacyanins migrate on paper electrophoresis at pH 2 to 7 toward the anode was used for analyses of the betalain pigments from red beet (36,37)and other plants (38). This method was subsequently applied by two groups (3,4)for the isolation of crystalline betanin from a crude pigment preparation from red beet (39). A major step forward was the introduction of polyamide adsorbents for the column chromatographyof betalain pigments (4). This technique was successfully applied by Piattelli and Minale (40) for the isolation of betalains. The Italian group developed a standard procedure in which the aqueous plant extract is stirred with a strongly acid ion-exchange resin that adsorbs the betalain pigments nonionically. After washing the resin with 0.1% aqueous HCl, the chromoalkaloids are eluted with water. In a second step, the pigments are chromatographed on a polyamide column, which leads to good separation in most cases (41). To avoid losses, all steps have to be carried out at 5°C. The individual pigments are characterized by means of their absorption spectra and their behavior on electrophoresis, paper chromatography, or TLC. In some cases, electrophoretic analysis of apparently pure chromatographic fractions indicated the presence of mixtures. Modem developments in the separation of betacyanins include the use of Sephadex ion exchangers (42-45) and the application of HPLC (46) for a fast and efficient analysis of the pigments.

1. BETALAINS

9

A number of general methods have been developed for the structural elucidation of betacyanins. In almost all cases the betacyanins are hydrolyzed with 1 N HC1 in order to identify the aglycone. Derivatives of betanidin (1) yield a mixture of 1 and isobetanidin (6) under these conditions, whereas isobetanidin derivatives yield only 6 (40).Betacyanins that are epimers at C-15 can be partially interconverted by treatment with dilute alkali under the exclusion of oxygen (25) or with 5% aqueous citric acid at room temperature (41). The position of the sugar residue in the dihydroindoleportion can be determined by methylation of the pigment with diazomethane and subsequent alkaline degradation of the resulting neobetanin derivative. In the case of 5-O-glycosides, 5hydroxy-6-methoxyindole-2-carboxylicacid is obtained, whereas the 6-0-glycosides yield 6-hydroxy-5-methoxyindole-2-carboxylic acid (47,48). The indole carboxylic acids are easily identified by TLC. The configuration at the glycosidic linkage can be determined by enzymatic hydrolysis with P-glycosidases (e.g., emulsin) or by NMR techniques (49). Pigments which possess acyl residues at the sugar moiety are often resistant to enzymatic cleavage. Acyl groups are removed by treatment of the pigments with dilute aqueous alkali in the absence of oxygen. The organic acids and deacylated betacyanins formed are then identified in the usual way (50). The position of the acyl or sulfate groups in the sugar moiety has been determined by permethylation of the pigments with MeI/AgO in dimethylformamide (DMF) followed by acid hydrolysis and identification of the methylated sugar. In some cases, the acylated glycoside is subjected to periodate cleavage followed by acid hydrolysis of the resulting dialdehyde. After reduction of the product with sodium borohydride a mixture of polyols is obtained from which the original position of the acyl residue in the sugar can be deduced. Finally, for the identification of oligosaccharide residues present in betacyanins, the usual methods of carbohydrate chemistry have been applied. Thus, partial hydrolysis and methylation studies served to elucidate the structure of the branched trisaccharide part of two epimeric pigments from Bougainvillea glabra (51,52). Most of the betalain pigments described in the 1960s have not been characterized by mass or NMR spectra. With FAB MS now at hand, the molecular ions of underivatized betalains can be easily determined, and the sensitivity of highfield NMR spectrometers allows the complete structural assignment, even of small samples. For the structural determination of oligosaccharide moieties, modem two-dimensional (2D)NMR techniques are now the method of choice. Recent enzymatic studies in betacyanin synthesis have led to corrections of several structures which were based on insufficient chemical evidence. In the following, only the betacyanins that have been well characterized and whose structures have been elucidated are discussed in detail. In most cases, the betacyanins are accompanied by small quantities of their C-15 epimers (“iso” compounds), which are not treated under separate headings but are considered with the main pigment.

10


B. INDIVIDUAL PIGMENTS

1. Betanin Group a. Betanin. Betanin (S),the main pigment of red beet and pokebeny, is a monoglucoside of betanidin (1) (1,25). The position of the sugar residue at the aglycone was determined by methylation of betanin with diazomethane and consecutive acid hydrolysis (49). The resulting 0-monomethylneobetanidin trimethyl ester (20) was purified as its 0-acetyl derivative 21, which on carefully controlled degradation with exclusion of oxygen yielded a mixture of compounds which were esterified with methanolic hydrochloric acid and oxidized with potassium nitrosodisulfonate. From the resulting mixture, 5-hydroxy-6-methoxyindole-2-carboxylic acid (22) was isolated and identified by comparison with synthetic material. This proved the point of attachment of glucose in betanin at the 5 position of the indole ring. Betanin is cleaved four times more easily with emulsin than with cellulase. This establishes the p configuration of the glucoside linkage, which is also in accord with the 'H-NMR spectrum of betanin in trifluoroacetic acid (Jc., ,,c.2, 5 Hz) (49). Detailed studies on the stability of betanin in aqueous solutions have shown that the pigment is hydrolyzed on heating to betalamic acid (3) and cyclodopa-5-0-glucoside (53).The latter compound has been obtained on a preparative scale by base exchange of betanin with proline in the presence of dilute aqueous ammonia (43). b. Phyllocactin. Phyllocactin (23) and isophyllocactin (23') were isolated from flowers of Phyllocactus hybridiis (Cactaceae) by adsorption on an acidic ion-exchange resin followed by column chromatography on polyamide (50).On acid hydrolysis, 23 gave a mixture of betanidin and isobetanidin, whereas 23' afforded only isobetanidin. On treatment with alkali in the absence of oxygen, a mixture of 23 and 23' yielded malonic acid. Titration of the pigments with alkali indicated that only 1 mol equiv of malonic acid is bound, in the form of its half ester.

HO H3CO

H

22

5

20 (R = H)

21 (R = Ac)

C02H

1.

11

BETALAINS

In order to determine the position of the malonyl group, the pigment mixture was subjected to periodate oxidation followed by borohydride reduction, mild acid hydrolysis, and a second borohydride reduction. The identification of glycerol and 1,2-ethanediol as degradation products indicated the attachment of the malonyl group to C-6 of the glucose residue. This was further confirmed by permethylation of the pigments with MeI/AgO in DMF, which yielded 2,3,4tri-0-methyl-D-glucose after acid hydrolysis (50). These findings are in accord with formulas 23 and 23' for phyllocactin and isophyllocactin, respectively. It has been pointed out, however, that the possibility of an acyl migration during the work-up procedure cannot be completely excluded (50).

c. Lampranthin I. Two betalain pigments, lampranthin I (24) and isolampranthin I (24'), were isolated from the flowers of a Lumprunthus species (54). That both compounds are epimeric at C-15 was determined in the usual way: alkaline hydrolysis of 24 yielded a mixture of betanidin (1) and isobetanidin (6), whereas pure 6 was obtained from 24'. A 1 : 1 mixture of (E)-ferulic and (E)-p-coumaric acid was obtained in both cases, and it was claimed that the pigments were p-coumaroylferuloylbetanidin and the corresponding isobetanidin derivative, respectively. Recently, enzymatic studies have cast serious doubt on the correctness of this finding (55). Protein preparations from petals of Lamprunfhus sociorum catalyze the formation of monoacylated glycosides from betanidin and 1-0-feruloyl-pglucose or 1-0-(p-coumaroy1)-p-glucose. The products were shown by HPLC comparison to be identical with the pigments present in the plant. It is therefore highly probable that lampranthin I and isolampranthin I are the 5-0-[6'-0-(E)-(pcoumaroy1)-p-glucosides] of betanidin and isobetanidin, respectively. The position of the acyl residue remains to be rigorously established by NMR methods. d. Lampranthin 11. Lampranthin I1 (25) and isolampranthin I1 (25') were first described by Piattelli and Impellizzeri (54), who concluded that these pigments are derivatives of betanin and isobetanin, respectively, esterified with two molecules of (E)-ferulic acid and one molecule of (E)-p-coumaric acid in the sugar residue. In a recent enzymatic investigation of betacyanin formation in

R

1501 -

HO

15

158

23

HO2CCH2CO-

23'

24

(E)-p-coumaroyi

2 4'

25

(E)-feruloyi

25'

28

eo3s-

12


Lumprunthus, it was demonstrated that the pigments are in fact monoacyl derivatives of betanin and isobetanin, respectively, in which an (E)-feruloyl residue is attached to the 6 position (55). Structure 25 for lampranthin I1 has been unambiguously established by an NMR study (56).

e. Prebetanin. Extracts of red beet contain the violet pigment prebetanin (26) that exhibits an exceptionally high electrophoretic mobility and is adsorbed strongly on polyamide during chromatography (57,58). It was shown by acid hydrolysis and elemental analysis that the compound is a sulfuric acid half-ester of betanin. Acetylation yields a peracetyl derivative which exhibits the signal of an aromatic acetoxy group at 6 2.48 in the 'H-NMR spectrum. This excludes the attachment of the sulfate group at the indole moiety. The location of the sulfate group at C-6 of the glucosyl residue was deduced from comparison of the 'HNMR spectra of betanin and prebetanin in trifluoroacetic acid. The signals of the glucose C-6 protons experience a down-field shift from 6 4.23 to 4.70, indicative of sulfonation at this position. Structure 26 for prebetanin has been further corroborated by its synthesis from betanin (6) (58). Reaction of 6 in trifluoroacetic acid with chlorosulfonic acid yielded an orange-yellow complex which was hydrolyzed with 1 N HCl to a mixture of products from which prebetanin was separated and identified by UV comparison and cochromatography with an authentic sample. The detailed course of the degradation of prebetanin in methanol has been studied by following the reaction by TLC and electrophoresis (58). f. Rivinianin. The red fruits of Riviniu humilis contain a red-violet pigment, rivinianin (27), which was isolated by preparative electrophoresis (52). The identification of betanin (5)and sulfuric acid after alkaline hydrolysis indicated that the pigment is a sulfate ester of 5. Attachment of the sulfate residue at the indole hydroxyl group was excluded by diazomethane treatment and subsequent alkali fusion of the methyl derivative, which yielded 5-hydroxy-6methoxyindole-2-carboxylicacid (22). The position of the sulfate residue was

QO$O HO

om L

Ho

C02H

H02C"" h C 0 , H 27

1.

13

BETALAINS

finally determined by permethylation of rivinianin with MeI/AgO in DMF followed by acid hydrolysis. This treatment afforded 2,4,6-tri-0-methyl-~-glucose, confirming structure 27 for the pigment. 2. Amaranthin Group

a. Amaranthin. Amaranthin (28) and isoamaranthin (28’) have been isolated from leaves of Amaranthus tricolor (47). Amaranthin (28) forms dark red 536 nm) and yields glucuronic acid, glucose, betanidin, and crystals (A, isobetanidin on acid hydrolysis. Enzymatic cleavage of 28 with P-glucuronidase leads to a mixture of glucuronic acid and betanin, which indicates that the pigment is an aldobiuronide of betanidin. The structure of the sugar part was elucidated by oxidative degradation of 28 with 36% hydrogen peroxide followed by permethylation of the resulting disaccharide (59). Acid hydrolysis of the permethyl derivative furnished a mixture of 2,3,4-tri-0-methyl-~-glucuronic acid and 3,4,6-tri-0-methyl-~-glucose.This proves the 1 + 2 linkage of the two hexose units and leads to structure 28 for amaranthin. The structure of its C-15 epimer isoamaranthin (28‘) was established in a similar fashion. b. Celosianin I. Two pigments, celosianin I (29) and isocelosianin I (29’), isolated from the violet inflorescences of Celosia cristata, were shown to be C-15 epimers (41). Hydrolysis of the pigments yielded a mixture of amaranthin (28) and isoamaranthin (28’) and a fraction of aromatic acids from which p coumaric acid and ferulic acid were identified (50). Recent enzymatic studies on the formation of celosianins in cell suspension cultures of Chenopodium rubrum provided strong evidence that celosianin I differs from amaranthin by an (E)-pcoumaric acid residue at 0-2”of the glucuronic acid moiety (29) ( 5 3 , by analogy with the respective ferulic acid ester (celosianin 11).

c. Celosianin 11. Recently, Strack et al. (56) established the structure of a closely related betacyanin, celosianin II (30). The compound was isolated from cell suspension cultures of Chenopodium rubrum and its structure identified by spectroscopic methods. In the positive-ion FAB mass spectrum of 30 the protonated molecular ion is visible at mlz 903, and the 2D COSY ‘H-NMR spectrum

Ho22L0, HO

h

28 OR

R

15a H

29

(E)-p -co urna royi

30

(E)-ferulo$

28’ 29’

14


reveals the connectivities of the individual protons in the sugar moieties. The low-field shift of 2”-H in the ‘H-NMR spectrum indicates the presence of the feruloyl residue at C-2” of the P-glucuronic acid, and the NOE difference spectra are in accord with an attachment of the P-glucuronic acid moiety to C-2‘ of the glucose residue, which in turn is bound to C-5 of the betanidin moiety. Furthermore, the IH-NMR spectra revealed the presence of two major stereoisomers in the material isolated from cell suspension cultures of Chenopodium rubrum (56).

d. Iresinin I. Several new betaqyanins have been isolated from leaves of Iresine herbstii (41),of which only the structures of iresinin I(31) and iresinin I1 (31’) could be elucidated (50). Iresinin I(31) was obtained in the form of violet crystals, ,,,A 537 nm (in H,O), [01];9~ 160. That both compounds are C-15 diastereomers was established in the usual way. Alkaline hydrolysis of 31 afforded amaranthin (28), isoamaranthin (28’), and 3-hydroxy-3-methylglutaric acid (HMG). Treatment of 31 and 31’ with P-glucuronidase yielded glucuronic acid and a red-violet pigment, deglucuronosyliresinin I , which was not attacked by emulsin. This pigment on acid hydrolysis afforded 1, 6, glucose, HMG, and 6-0-(3-hydroxy-3-methylglutaryl)-~-glucose. From the results of periodate degradation and permethylation, the attachment of the HMG residue at 0-6 of the glucose unit could be firmly established.

+

e. Acyl Derivatives of Undefined Structure. Iresinin 111 and iresinin IV from leaves of Iresine herbstii (50)yielded amaranthin (28), isoamaranthin (28‘), and a mixture of hydroxycinnamic acids on alkaline hydrolysis. Suaedin has been isolated from leaves of Suaedafruticosa (60). On alkaline deacylation, 28, caffeic acid, p-cournaric acid, and citric acid were obtained, and hydrolysis with 0.03 N HCl at 25°C afforded “celosianin” and citric acid. The elucidation of the structures of these pigments awaits further investigations.

I -“Y

31: 15a

; 31’: 158

1.

15

BETALAINS

3. Bougainvillein r-I Group

a. Bougainvillein r-I. The spectacular colors of the bracts of Bougainvillea species were first studied by Robinson (6/-63), who concluded that the pigment present in Bougainvillea glabra ( “bougainvillein”) resembled betanin. Wyler and Dreiding (64) later isolated four betacyanins from the same species and two others from B. spectabilis. A reinvestigation of the pigments with improved isolation techniques later revealed the presence of complicated betacyanin mixtures in these plants (41,65). Seven new red-violet betacyanins were isolated from purple bracts of a horticultural variety of Bougainvillea (“Mrs. Butt”) (65). Two of these, named bougainvillein r-I (32) and isobougainvillein r-I (32’), were shown to be the 5-0-P-sophorosides of betanidin and isobetanidin, respectively. An important clue to their structure was the formation of sophorose on partial acid hydrolysis with 10% acetic acid (3.5 hr, reflux). Mild acid hydrolysis of the pigment mixture with 1 N HCI (10 min, 80°C) yielded betanin and isobetanin, establishing the p configuration of the glycosidic linkage between the disaccharide moiety and the aglycone. b. Acyl Derivatives of Undefined Structure. A preliminary investigation of the Bougainvillea pigments bougainvillein r-11, bougainvillein r-111, bougainvillein r-IV, and bougainvillein r-V indicated that they constitute esters of bougainvillein r-I with hydroxycinnamic acids (65).Bougainvillein r-I1 and isobougainvillein r-I1 are epimeric at C- 15. 4. Betanidin-5-O-~-cellobiosideGroup ( “DO 1 Group”)

a. Acyl Derivatives of Undefined Structure. Two red-violet betacyanins, oleracin I and oleracin 11, have been found in Portulaca oleracea ( 4 f ) .The pigments exhibit identical spectral (A, 548 nm) and electrophoretic properties and were shown to be epimeric at C-15 (66). On alkali treatment the pigment mixture yielded ferulic acid and two diastereomeric pigments, DO1 and D02,

HO HO A

1 \

OH

32: 15a ; 32‘: 158

16


33: 15a ; 33': 158

which were separated on polyamide. Controlled acid hydrolysis of these pigments with 10% acetic acid afforded a mixture of glucose and cellobiose. Since the pigments yielded 5-hydroxy-6-methoxyindole-2-carboxylic acid after methylation and alkaline degradation, the cellobiose is bound to the 5-hydroxyl group of the indole moiety. Careful acid hydrolysis of DO1 and DO2 yielded betanin and isobetanin, which indicates the P configuration of the sugar linkage to the aglycone. Oleracin A and B are therefore acyl derivatives of betanidin-5-O-Pcellobioside (DO 1) (33) and isobetanidin-5-0-P-cellobioside(D02) (33'), respectively. The site of attachment of the acyl residue is unknown. 5 . Gomphrenin I Group

a. Gomphrenin I. Piattelli and Minale (41) isolated a family of structurally closely related betacyanins from the violet inflorescences of Gomphrena globosa. The principal pigments, gomphrenin I (34) and gomphrenin-I1 (34'), are isomers of betanin and isobetanin, respectively (48), as revealed by diazomethane treatment followed by alkaline degradation, which yielded 6-hydroxy-5-methoxyindole-2-carboxylicacid. Both compounds are therefore 6-0monoglucosides of betanidin and isobetanidin, respectively. Compounds 34 and 34' are not cleaved by emulsin nor by maltase. There is strong evidence, however, that the configuration at the glucoside linkage is p, as treatment with dilute alkali yielded cyclodopa-6-O-P-~-glucoside, which can be hydrolyzed with

( z ) - p - cou rnaroyi 36'

1.

BETALAINS

17

emulsin. Conclusive evidence is provided by the IH-NMR spectrum of a mixture of 34 and 34’ in CF,CO,H; the signals of the anomeric protons at 6 5.18 have an 8-Hz coupling, in accord with a p configuration.

b. Gomphrenin 111. Gomphrenin 111 (35), on treatment with aqueous alkali in the absence of oxygen, yielded gomphrenin I (34), gomphrenin I1 (34’), cyclodopad-O-~-~-glucopyranoside, and (a-p-coumaric acid (48). The exclusive existence of the latter in betacyanins is uncommon and needs reinvestigation in the intact molecule. The attachment of the acyl residue to the 6 position of the glucose unit was determined in the usual way. c. Gomphrenin V and Gomphrenin VI. Structures 36 and 36’ have been assigned to gomphrenin V and its C-15 epimer gomphrenin VI, respectively (48). The techniques used were the same as in the case of other Gomphrena betalains. d. Acyl Derivatives of Undefined Structure. There is insufficient experimental evidence to ascribe structures to gomphrenin VII and gomphrenin VIII, two additional acyl derivatives of gomphrenin I which yield (E)-ferulic acid and (E)-p-coumaric acid on alkaline hydrolysis, respectively (48). 6. Betanidin-6-0-6-sophorosideGroup ( “DP3 Group”)

a. Acyl Derivatives of Undefined Structure. During studies on the distribution of betacyanins in the Caryophyllales, Piattelli and Minale (41) detected several new betacyanins in the violet bracts of Bougainvillea glabra var. sanderiana. Because it was difficult to obtain individual pigments in quantities necessary for structural elucidation, the unfractionated betacyanin mixture was treated with alkali to yield four deacylated pigments which were separated by chromatography on polyamide and high-voltage electrophoresis (51). Pigments DP3 (37) (A,,, 541 nm) and DP4 (37’) were shown to be C-15 epimers. On total acid hydrolysis (22% HC1,5 min, 80°C) glucose was obtained, whereas reflux in 10% AcOH yielded sophorose. Hydrolysis of the pigment mixture with 1 N HCl (10 min, 80°C) afforded small amounts of gomphrenin I and gomphrenin 11, consistent with structures 37 and 37‘.

37: 15a ; 37’: 15p

18

WOLFGANG STEGLICH A N D DIETER STRACK

38: 15a ; 38’: 158

7. Betanidin-6-0-2G-glucosylrutinosideGroup (“DPI Group”)

a. Acyl Derivatives of Undefined Structure. The gross structures of two deacylated pigments DP1 (38) and DP2 (38’) from Bougainvillea glabra var. sanderiana, isolated as described above, were reported in 1970 (51). Degradation experiments revealed that the pigments were 6-0-glycosides of betanidin and isobetanidin, respectively. The structure of the trisaccharide part has been clarified more recently (52). Controlled acid hydrolysis afforded, besides rhamnose, glucose, rutinose, and sophorose, a sugar that was shown by methylation and hydrolysis experiments to be the branched trisaccharide 0-P-D-glucopyranosyl-( 1+2)-[ O-P-~-rhamnopyranosyl-(1+6)-~-glucopyranose] ( “2G-glucosylrutinose”). As controlled hydrolysis of the pigment mixture with 1 N HCl yielded small amounts of gomphrenins I and I1 with the P configuration at the sugaraglycone linkage, DPI and DP2 possess structures 38 and 38’, respectively. 8. Modified Betacyanins

a. Neobetanin. In 1975, Alard et al. (67) reported the isolation of an orange-colored minor pigment from the fresh extract of a red beet cultivar (Beta vulgaris) that proved to be neobetanin (39) (44). This finding has been questioned by Wyler, who attributed it to artifact formation during the isolation process (68). Strack et al. (69),however, provided unequivocal evidence for the natural occurrence of 39 in a red beet cultivar and, in addition, isolated neobetanin as a major constituent from fruits of the fig cactus (Opuntiaficus-indica). The betanin-neobetanin ratio in fresh extracts from fruit flesh could be determined by means of HPLC and was found to be 1 : 2.5. Neobetanin (39) ,A( 267, 306, 470 nm) gives a yellow color ,A( 401 nm) in alkaline solution, which changes to pink ,A( 500 nm) on addition of acid. This behavior is

1.

BETALAINS

19

HO

HO

39

typical for “neo” compounds (23). The molecular formula of neobetanin was determined by positive-ion FAB MS, and structure 39 is fully supported by the IH- and I3C-NMR data (67). The attachment of the sugar moiety at C-5 of the aglycone was deduced unambiguously from the IH NOE difference spectra.

b. Decarboxybetanidin. The purple flowers of Carpobrotus acinaciformis (Aizoaceae) contain a mixture of betacyanins that on cellulose column chromatography yielded a very minor one as the last fraction (70). The violet pigment (A, 542 nm) experiences a bathochromic shift of 10 nm on addition of borate, a behavior similar to that of betanidin, indicating the presence of a free catechol group. On treatment with aqueous SO, solution the pigment is degraded to betalamic acid, which was identified by the formation of indicaxanthin (2) on addition of proline. The formation of a monomethyl and finally a dimethyl ester on reaction of the compound with methanol in the presence of boron trifluoride suggested its identity with decarboxybetanidin (19). This was substantiated by direct comparison with an authentic sample prepared by reaction of indicaxanthin with 2,3-dihydro-5,6-dihydroxyindole(33).

IV. Betaxanthins

A. ISOLATION A N D STRUCTURAL ELUCIDATION Betaxanthins can be isolated by the same techniques as described for betacyanins (6). In general, betaxanthins are more susceptible to degradation during the work-up procedure and must be handled with special care. Dopp et al. (71) separated the complex pigment mixture present in the fly agaric by chromatography on DEAE-Sephadex (sodium chloride gradient) followed by desalting with

20


Sephadex G-10. In this case, the separation steps had to be repeated several times in order to achieve complete resolution of the individual betaxanthins. Most of the betaxanthins could not be obtained in crystalline form. As with the betacyanins, epimerization at C-1 1 of the betalamic acid moiety may lead to the formation of is0 compounds (29). The complete stereochemistry of the betaxanthins has not been determined in many cases. It can be assumed, however, that the natural compounds are derived from L-amino acids and (S)-betalamic acid (3). Thus, the betalamic acid dimethyl ester obtained from betaxanthins of the fly agaric is always dextrorotatory, which proves its (S) configuration (71). Betaxanthins are immediately recognized by their characteristic absorption spectra, which show a maximum at 475 nm. On alkaline or acid hydrolysis, they yield the corresponding amino acids. In a more recent approach (71)the betaxanthins were cleaved with 0.6 N aqueous ammonia to yield betalamic acid (3) and the corresponding amino acids. Compound 3 was then extracted with organic solvents and characterized as its dimethyl ester. Unknown amino acids were identified by GC-MS of their N-trifluoroacetyl methyl ester derivatives. After identification of the amino acid, the structure of the betaxanthin can be verified by partial synthesis from betanin (5). The amino acid exchange is carried out by treating 5 with a large excess of the amino acid in 0.6 to 1.2 N aqueous ammonia (72). The reaction can be followed by monitoring the decrease of the violet betanin absorption at 540 nm and the increase of the betaxanthin maximum at 475 nm. Separation of the pigments from unreacted betanin is achieved by chromatography on Sephadex. Only betaxanthins that have been obtained in pure form and sufficiently characterized are listed in this review. Certainly, a number of additional betaxanthins await identification. Purely synthetic betaxanthins that have served for comparison purposes are not covered here (71).

B. INDIVIDUAL PIGMENTS 1. Betaxanthins Derived from Proteinogenic Amino Acids

a. Indicaxanthin. Piattelli er al. (6) isolated the first crystalline betaxanthin, indicaxanthin (2), from the yellow-orange fruits of Opuntiaficus-indica. It forms orange crystals which decompose at 160-162°C and show an optical rotation ([a]bo)of +394". The UV spectrum in water exhibits maxima at 485 (log E 4.63), 305 (3.19), and 260 nm (3.73). The structure and stereochemistry were deduced by alkaline degradation to 4-methylpyridine-2,6-dicarboxylic acid and by peracid oxidation to yield L-aspartic acid (17). Structure 2 is supported by the NMR spectrum in CF,CO,H, which shows signals of the proline moiety and the characteristic AB doublets (J 12 Hz) for the protons in the 7 and 8 position at 6 8.65 and 6.35, respectively. The signals of the dihydropyridine moiety are in close agreement with those reported for betanidin (5).

1.

21

BETALAINS

Q C 0 2 H

2a

2b

A detailed NMR analysis revealed that indicaxanthin is present in CF,CO,H solution as a 65 : 35 mixture of its 8E and 82 stereoisomers (%aand 2b, respectively), which causes a partial doubling of the NMR signals (32). In CF,CO,D an exchange of the protons at positions 8 and 14 takes place in accord with theoretical considerations and observations on related 1,7-diazaheptamethinium systems. Indicaxanthin can be prepared from betanin (5) by amino acid exchange with L-proline in aqueous ammonia (72). The product obtained by this method is partially epimerized at C-1 I , in contrast to betanidin which is not affected under these conditions (29). Unlike betanidin and isobetanidin, the two epimers of indicaxanthin have not yet been separated by chromatographic or electrophoretic methods.

b. Portulacaxanthin. A betaxanthin closely related to indicaxanthin (2) has been isolated from flowers of Portulaca oleracea (73). After hydrolysis, hydroxyproline was identified as the amino acid component, suggesting structure 40 for portulacaxanthin. c. Miraxanthin 11. Miraxanthin I1 (41) has been isolated in small quantities from flowers of Mirabilis jafapa (30). Its absorption maximum at 475 nm (sh 462) is shifted to 465 nm on addition of HCl, which suggests the presence of a betaxanthin chromophore. This is supported by the formation of 4-meth-

40

22


C02Me I

C02H

I

C02Me I

CH2 I

41

43

42

ylpyridine-2,6-dicarboxylic acid on alkaline degradation of the pigment. On methylation of 41 with diazomethane, a neo derivative (42) is formed which exhibits an absorption maximum at 359 nm, shifted to 435 nm on addition of acid. This can be explained by the formation of an extended cation (43).The same behavior is known from betanidin derivatives and has been discussed in Section 11. On acid hydrolysis, miraxanthin LI yields aspartic acid. The fact that the pigment has nearly the same electrophoretic mobility as the glutamic acid derivative vulgaxanthin I1 (44) indicates the presence of four carboxylic groups and excludes asparagine as part of the molecule.

d. Vulgaxanthin 11. Vulgaxanthin I1 (44) is one of the major betaxanthins present in red beet (74). Its isolation required several detrimental separation steps and yielded only a small amount of the chromatographically pure pigment in amorphous form. The usual degradation reactions led to structure 44 which, however, awaits experimental confirmation of the stereochemistry. COR I

44 , R = OH 45 , R = NH2

46

23

1 . BETALAINS

e. Vulgaxanthin I. Vulgaxanthin I (45)cooccurs with 44 in red beet (74). The pigment has been obtained in crystalline form and is hydrolyzed under mild conditions ( 1 N HCl, 25"C, 24 hr) to glutamine. f. Muscaaurin VII. Muscaaurin VII (46) has been isolated in pure from from the complex pigment mixture present in the cap skin of the fly agaric (Arnanira rnuscariu) (42,71). Its structure follows from the UV/VIS spectrum with maxima at 476, 460 (sh), and 260 nm and from results of acid hydrolysis which leads to betalamic acid (3) and histidine (71). 2. Betaxanthins Derived from Nonproteinogenic Amino Acids

a. Humilixanthin. Humilixanthin (47),a recent addition to the betaxanthin family, was isolated from the yellow- and orange-red colored fruits of Riviniu hurnilis (Phytolaccaceae) (10). It was obtained in pure form after three consecutive column chromatography steps including semipreparative HPLC on a reversed-phase column. The negative-ion FAB mass spectrum gave an ion at mlz 325 corresponding to the deprotonated molecular ion. The 'H-NMR spectrum of humilixanthin (CD,OD and trace of DCl) indicated the existence of four stereoisomers in this solvent. Besides the E,Z isomerization at the 8,9 double bond already known from other betalains (32), the presence of another pair of stereoisomers owing to E,Z isomerism at the 1,7 double bond is discussed to explain this phenomenon. From an analysis of the NMR data, 5-hydroxynorvaline was identified as the amino acid moiety. This was confirmed by direct comparison of a synthetic sample with the amino acid obtained by acid hydrolysis.

b. Miraxanthin I. Miraxanthin I (48) is another betaxanthin pigment from Mirubilis julapu (30).Its structure was established in the usual way. Esterification on a microscale with methanolic hydrochloric acid yielded a mixture of

0 FH2 S CH3

qoH

y 2

47

40

49

24


mono-, di-, and triesters, which indicates that the compound is a tricarboxylic acid. On acid hydrolysis methionine sulfoxide is formed. Structure 48 was confirmed by partial synthesis of miraxanthin I from betanin.

c. Dopaxanthin. A few milligrams of dopaxanthin (49) has been isolated from petals of Glottiphyllum longum (Aizoaceae) (75). The compound exhibits absorption maxima in water at 483 (sh at 470 nm), 267,and 225 nm (inflection). On addition of borate, the peaks at lower wavelengths experience bathochromic shifts to 292 and 237 nm, respectively, which indicates the presence of ortho phenolic hydroxyls. The pigment yields 3,4-dihydroxyphenylalanine on acid hydrolysis in accord with structure 49, which was subsequently confirmed by partial synthesis. d. Muscaaurin I. Muscaaurin I (50) from Amanita muscaria incorporates ibotenic acid (51), which was identified as its N-trifluoroacetyl methyl ester derivative after acid hydrolysis of the pigment (71). Ibotenic acid is a common constituent of the fly agaric (76). Structure 50 for muscaaurin I was confirmed by partial synthesis.

e. Muscaaurin 11. Another unusual amino acid is present in muscaaurin I1 (52) from Amanita muscaria (71). The pigment exhibits the usual UV maximum at 478 nm and yields betalamic acid and stizolobic acid (53) on acid hydrolysis. The latter gives a red-brown ninhydrin reaction on TLC plates and was identified by its spectroscopic data. Compound 53 had been isolated previously from Amanita pantherina (77). Structure 52 for muscaaurin I1 was confirmed by partial synthesis (71).

3. Betaxanthins Derived from Biogenic Amines a. Miraxanthin 111. Flowers of Mirabilis jalapa contain, besides other derivatives, two unusual betaxanthins in which betalamic acid is condensed with primary amines instead of amino acids (30). One of these compounds, mira-

yb

HO

51

50

1. BETALAINS

25

Ho2cTo Ho2cTo y 2

O/CH, HN

0

C02

CH2

H 02C'"' b HC 0 2 H

52

53

xanthin 111 (54), yielded tyramine on acid hydrolysis and gave only monomethyl and dimethyl esters on treatment with methanol-BF,, in accord with structure 54. The structure was confirmed by partial synthesis of the pigment from betanin and tyramine.

b. Miraxanthin V. Miraxanthin V (55) from Mirubilis j a l a p is closely related to miraxanthin 111 and incorporates betalamic acid and dopamine in its formula (30). Structure 55 has been confirmed by the 'H-NMR data of the pigment and by partial synthesis of miraxanthin V from betanin and dopamine. 4. Betaxanthins of Undetermined Structure

During investigation of the major betaxanthin pigments, a number of compounds have been isolated for which no structure could be established because of the paucicity of material. Examples are miraxanthin IV and miraxanthin VI from Mirubilis julupu (30) and some pigment fractions from Amunitu muscuriu (71). OH

@pH2 HN

e H 54

@/k

HN

e02C"" b

HC 0 2 H

55

26

WOLFGANG STEGLlCH AND DIETER STRACK

The latter point to the natural occurrence of betaxanthins derived from a-aminoadipic acid, asparagine, leucine, and valine. Some of these pigments were prepared from betalamic acid and the corresponding amino acids (71).

V. Muscapurpurin

One of the fly agaric pigments was named muscapurpurin (56) because of its intense purple color (A, 540 nm) (42). The compound was investigated by the late Professor Musso, who tentatively suggested a formula which appeared to be derived from betanidin via an oxidative fission of the benzene ring (78). Later, this proposal was withdrawn in favor of the interesting structure 56 in which an unsaturated cyclic amino acid, muscapurpurinic acid (57),forms a betalain type pigment with betalamic acid. As in the betacyanins, the extended chromophore in 56 causes a bathochromic shift of the long-wavelength absorption maximum in the UV/VIS spectrum (79).

VI. Betalamic Acid

Betalamic acid (3) is an essential structural element of all natural betacyanins and betaxanthins. It had been suggested as an intermediate in the amino exchange reaction between different members of this group (72,80) before its natural occurrence was established by Mabry and co-workers (81,82).In a chemotaxonomic investigation, Reznik (83)demonstrated the wide distribution of 3 in plants of the order Caryophyllales (Table I). Compound 3 can also be obtained by mild base-catalyzed hydrolysis of betaxanthins (42,84). In contrast to the beOH I

OH I

56

57

1.

27

BETALAINS

6 10.00 d 8Hz

10.10 d 8

5.00 d 0

3.0-4.0 rn

3.70

S

2.0-4.0 rn

6.15 s

5.67

6.93 s

3.86 s

58a

talains, betalamic acid can be extracted from acidified aqueous solutions into organic solvents. Its light-yellow sodium salt exhibits UV/VIS maxima at 428 and 250 nm, and the IH-NMR data of the dimethyl ester indicate the presence of a 5 : 3 mixture of the two stereoisomers 58a and 58b (42,84).Reduction of this mixture with sodium borohydride affords the alcohol 59. On treatment of 3 with semicarbazide a crystalline semicarbazone is formed (85), and the reaction with aniline yields a salmon-pink Schiff base (A, 506 nm) (81). The formation of betalains by reaction of 3 with amino acids and amines is discussed in Section VII1,C.

VII. Muscaflavin Muscaflavin (4) [A, 420, 238 nm (in H,0)] was discovered by Dopp and Musso (42,84)during studies on the pigments of the fly agaric (Amanitu muscuria). The first spectroscopic investigation revealed a close resemblance of muscaflavin to betalamic acid (3), which led the Karlsruhe group to suggest a dihydropyridine structure. Independent investigations on the bright yellow and red pigments of Hygrocybe toadstools carried out by Steglich et al. revealed that a compound could be obtained from the acidified aqueous extracts of these fungi which appeared to be closely related to muscaflavin. From a careful inspection of its 'H-NMR data and biogenetic considerations, the dihydroazepine structure 4 was advanced for the Hygrocybe pigment, and its identity with muscaflavin was established by direct comparison (9).Compound 4 forms a crystalline dimethyl ester (60) (A, 394 nm, in MeOH) on treatment with methanolic hydrogen

28


OHC

HOH2C NaBH4

60

L 61

chloride or diazomethane. On reduction of 60 with sodium borohydride, the alcohol 61 is obtained which exhibits a UV maximum at 336 nm in accord with the disappearance of the merocyanin chromophore (9,86). Structure 4 is fully supported by the I3C-NMR spectrum of muscaflavin (87). From CD studies and biosynthetic considerations (Section X) the (7s) configuration can be assigned to the pigment. On treatment of a methanolic solution of muscaflavin with glutamic acid and a catalytic amount of p-toluenesulfonic acid, the UV maximum of the solution is shifted from 405 toward 456 nm, in agreement with the formation of Schiff bases (87). Muscaflavin is accompanied in Hygrocybe species by pigments which are muscaflavin analogs of the betaxanthins (87,88). These pigments, named hygroaurins (9), appear to be more labile than the corresponding betaxanthins, which has so far prevented their isolation in pure form and their unambiguous spectroscopic characterization. In a chemotaxonomic survey, several hygroaurin fractions were separated by gel chromatography on Sephadex and partially characterized by the amino acids obtained after hydrolysis (89). The distribution of muscaflavin and hygroaurins in toadstools is described in Section IX.

VIII. Syntheses of Betalains

A. SYNTHESES OF BETALAMIC ACID

Two syntheses of racemic betalamic acid have been camed out so far. In Dreiding's approach (Scheme 1) (11,90,91), chelidamic acid (62) was used as the starting material. Hydrogenation of 62 with a rhodium catalyst yielded an all-cis piperidine derivative, which was converted to the dimethyl ester 63. The conditions used for the hydrogenation step kept the concomitant removal of the hydroxyl group to a minimum. The oxidation of alcohol 63 to the corresponding piperidone derivative 64 required careful control of the reaction conditions to avoid overoxidation to pyridine derivatives. This was accomplished by use of a polymeric carbodiimide in the Pfitzer-Moffat oxidation, which afforded the desired product 64 in 90% yield. For the introduction of the side chain, a new

1.

Me

I Me2NCON,

29

BETALAINS

Me

I

Me2NCON,N

N

iv 2

Me02C.'"

N ""C02Me H

H

65

66

SCHEME 1 . Synthesis of betalamic acid derivative 66. i, H2, Rh-A1203; MeOH-HCI; ii, DMSOpolymeric carbodiimide-Py-CF3C02H; iii, (Et0)20PCH2CH=NN(Me)CONMe2-NaH;iv, tBuOCI.

Homer-Wittig reagent had to be developed. In the first version of the synthesis (11,90) the olefination was carried out with a reagent derived from simple semicarbazide. It was found later (91), however, that the fully methyl-protected reagent gave much better yields. The trimethylsemicarbazone 65 was isolated in 55% yield and appeared to be the pure anti compound ('H NMR: 6 7.52, d, 8-H). On treatment with tert-butyl hypochlorite followed by addition of triethylamine, 65 yielded a 7 : 3 mixture of the 4(7)E and 4(7)Z stereoisomers of the corresponding betalamic acid derivative 66, which on recrystallization afforded the pure 4(7)E compound. Buchi's synthesis of betalamic acid (Scheme 2) (12,92) commences with Nbenzylnorteleoidine (67), which had been obtained by a Robinson-Schopf synthesis. After protection of the glycol moiety as the cyclic ortho ester, the benzyl group was removed by hydrogenolysis. The resulting aminoketone 68 was reacted with ally1 magnesium bromide to yield the tertiary alcohol 69 with high stereoselectivity. In an interesting reaction, the imino group in 69 was converted to an 0-benzoylhydroxylamine moiety by means of dibenzoyl peroxide and potassium carbonate in DMF. The resulting hydroxylamine derivative was acetylated to yield ester 70,which was converted subsequently to diol71 by acid

30

WOLFGANG STEGLICH A N D DIETER STRACK

67

71

70

72

69

68

73 3 (E/Z-mixture)

SCHEME 2. Synthesis of betalamic acid (3). i , (Me0)3CH-CF3C02H-Me0H, H2-Pd/C; ii, H2C=CHCH2MgBr; iii, Bz202-DMF-K2C03; Ac20-DMAP; iv, H02C-C02H, HlO; NaOHH,O; v, NCS, Me2S-NEt3; vi, 03;Pb(OAc)4-PhH-MeOH; vii, silica gel Chromatography.

cleavage of the ortho-ester protecting group. Oxidation of the diol moiety with N-chlorosuccinimide (NCS) and dimethylsulfide gave the 1 ,Zdiketone 72 which on ozonolysis was transformed into aldehyde 73. On treatment with lead tetraacetate in benzene and methanol, the diketone moiety was oxidatively cleaved, and the resulting unstable dicarboxylic acid yielded (+)-betalamic acid as a mixture of E and Z isomers after silica gel chromatography.

B . SYNTHESES OF CYCLODOPA (S)-Cyclodopa (7) is an essential part of all betacyanins. Compound 7 is prepared by oxidative cyclization of @)-dopa (93) or its methyl ester (94) with potassium ferricyanide followed by immediate reduction of the unstable dopachrome intermediate 74 with sodium dithionite. The first synthesis (Scheme 3) is suitable for scale-up and allows an easy access to the more stable 0 , O diacetyl methyl ester derivative 75.

1.

31

BETALAINS

L

7 H2Pd C02CH3 AcO

Z

AcO

C02CH3 H

75 Z=PhCHZOCO-

SCHEME 3 . Syntheses of (S)-cyclodopa (7) and its 0.0-diacetyl methyl ester (75)

C. SYNTHESES OF BETALAINS BY REACTION OF BETALAMIC ACIDWITH AMINOACIDS The betalamic acid derivatives obtained by total synthesis have been used for the preparation of natural and unnatural betalains following the procedures developed for amino acid exchange described earlier (Scheme 4). Reaction of the 9 : 1 EIZ mixture of semicarbazone 66 with the p-toluenesulfonate salt of (S)cyclodopa methyl ester in dilute methanolic HCl afforded betanidin trimethyl ester (9), which was hydrolyzed with aqueous HCl to yield a 4 : 6 mixture of betanidin and isobetanidin (9f).In the case of L-proline, the reaction had to be carried out in dilute aqueous ammonia because of the sensitivity of the resulting indicaxanthin (2) toward acids. Most likely, an inseparable mixture of 2 and its C- 13 epimer 2' was obtained under these conditions. Reaction of indoline with 66 yields a more stable condensation product (76), which was isolated as its crystalline perchlorate salt (91)(Scheme 5). Saponification of 76 with concentrated HCl afforded the dicarboxylic acid (77), which was cleaved with aqueous ammonia to (?)-betalamic acid (3) and then characterized as its dimethyl ester (58). Interestingly, it was not possible to cleave ester 76 directly because of smooth oxidative aromatization to the neo derivative 78. This proves that the catechol unit is of no importance for the formation of neo compounds from betalains.

32


9

66

(E/Z

= 9:l)

aqu. NH3

2

+

2" (E/Z = 65: 30)

SCHEME 4. Syntheses of betalains from betalamic acid derivative 66.

D. SYNTHESES OF BETALAIN ANALOGS An elegant synthesis of decarboxybetalains has been developed by Dreiding 's group (95) (Scheme 6). It is based on the photolytic ring opening of 3-(4pyridy1)alanine (79) in weakly basic methanolic ammonia. Under these conditions, the imine of decarboxybetalamic acid (81) is formed in high yield. The reaction proceeds via a Dewar pyridine intermediate (80) which undergoes ring opening by attack of the side chain amino group at the imino function present in the bicyclus. The imine 81 has been transformed into several decarboxybetalains. With (S)cyclodopa (7) the reaction takes place in 1 N HCI and affords the blue-violet 17decarboxybetanidin (19) in 34% yield. For the reaction with L-proline, neutral conditions have to be used because of the instability of the resulting yellow 13decarboxyindicaxanthin (83). Indoline reacts in a weakly acidic medium and affords the red 14-decarboxyindobetalain (82) in 78% yield. Compounds 19 and 83 were identical with the compounds obtained from thermolysis of the natural products (33,34).It is noteworthy that the highest wavelength absorption maxima are shifted toward shorter wavelengths in comparison to the corresponding be-

1.

66

(E’Z

=

33

BETALAINS

e

-

/ N

c. HCI

1 % HC104,

9: 1) H20

H3C02C

/

C02CH3

76

/

(E/Z

77

= 7: 3)

!

aqu. NH3

1) aqu. NH3

2) CH2N2

58 78

(E/Z

=

5:3)

SCHEME5. Syntheses of 2-decarboxybetanidin derivatives.

talains. Like the latter, the decarboxy derivatives occur as mixtures of E and Z isomers. Betenamine (85), the simplest derivative carrying a betacyanin chromophore, has been synthesized from N-tert-butoxycarbonyl-4-(2-aminoethyl) pyridine (84) in three steps (Scheme 7) (96).The perchlorate of 85 forms deep orange microcrystals (Arnm 494 nm). It is, in contrast to betanidin, not converted to a neo derivative on treatment with diazomethane.

E. SYNTHESIS OF MUSCAFLAVIN Musso el al. (87,97) have developed an elegant synthesis of muscaflavin dimethyl ester (61) (Scheme 8). It is conceptionally similar to the synthesis of decarboxybetalains described before. The synthesis starts with conversion of pyridylalanine derivative 86 to the dimethyl ester 87 by esterification and consecutive N-protection with the base-labile p-toluenesulfonylethoxycarbonyl group. For cleavage of the pyridine ring, 87 was transformed into the N-methoxypyridinium salt (88) which was then reacted with pyrrolidine in tetrahydrofuran

H02C

h NH2 80

I

A H02C

HO

8

N

8

H

19

€3

SCHEME 6. Synthesis of 17-decarboxybetanidin (19), 2,17-bisdecarboxybetanidin (82). and 13decarboxyindicaxanthin (83).

x

NHC02tBu

4

NHC02tBU

i

-

N

84

ii t

NO2

NHC02tBu

iii

H

85

SCHEME7. Syntheses of betenamine (85).i, I-Chloro-2.4-dinitrobenzene; ii, indoline; iii, CF3C02H.

1. 1) CH30H. HCI P H02C

C

O

z

H

b

2) C I C O Z C H ~ C H ~ T S

NH2

35

BETALAINS

H3C02C

p

1) rn-CPBA C02CH3

b

2) dimethyl-

NHCO~CH~CHZTS

sulfate

3) N0C104

86

87 CIO~'

,

y C H 3

p C O z C H 3

H THF, 0 'C

H3C02C

NHCO~CH~CH~TS

NHCO~CH~CH~TS

89

88

H CO C

&

H

1) 0,5 N KOH

N H *z

+

2) 70 % HC104 3 ) CHZNZ

1) HC104, HC02H 2) CH2N2

90

C02CH3

H3C02$ H3C0NH%

b

3) chromatography on potato starch

H3CO2C."'

C02CH3

COzCH3 61

91

SCHEME 8. Synthesis of (+)-muscaflavin dimethyl ester (61).

(THF) at 0°C to yield the oxime ether 89. The latter was N-deprotected with 0.5 N aqueous KOH, and the resulting mixture on acidification with 70% perchloric acid followed by treatment with diazomethane yielded a mixture of two stereoisomeric pyrroline derivatives (90 and 91) in 22% yield. Finally, these products could be rearranged to (+)-muscaflavin dimethyl ester (61) by consecutive treatment with perchloric acid/formic acid and diazomethane in low overall yield. It was possible to resolve 61 by means of chromatography on a starch column.

IX. Chemotaxonomy and Distribution of Betalains

In the 1950s betalains were shown to be useful as chemotaxonomic markers for members of the Caryophyllales (Caryophyllidae), even before their actual structures were known ("N-anthocyanins") (35,38).These pigments have since

36


been found to be of high taxonomic significance for this group of plants (17,27), and there is a mutual exclusion of betalains and anthocyanins (98). However, other flavonoid classes, for example, flavonols and flavones, are commonly produced in leaves and flowers of the Caryophyllales and coexist with the betalains. This phenomenon seems to be a consequence of the absence of the enzymes that lead from dihydroflavonols via flavan-3,Ccis-diol derivatives (leucoanthocyanidins) to anthocyanidins (99), whereas the dehydrogenation reactions of dihydroflavonols leading to flavonols still exist. On the other hand, the formation of leucoanthocyanidins seems to be possible as documented by the occurrence of leucocyanidin in Carpobrotus edulis (Aizoaceae) (98). The absence of anthocyanins may have favored the appearance of betalains, which have similar functions (13). Since all families of the Caryophyllales produce flavonoids, the absence of anthocyanins and the presence of betalains may be a coincidental appearance during evolution of these systematic groups (100). For arguments concerning the subclass relationship between the Caryophyllidae and the Magnoliidae or Dilleniidae (Scheme 9), the chemistry of the Caryophyllales may be considered. Whereas on the basis of several flavonoid types a closer relationship of the Caryophyllales to the Dilleniidae might be proposed (101), the absence of ellagic acid and the rare occurrence of myricetin support a possible relationship of the Caryophyllales with the Magnoliidae (102). This becomes especially apparent when the betalains are treated as merely tyrosine-derived alkaloids (103) and compared to the aromatic amino acid-derived benzylisoquinoline-producing Magnoliidae (100). The order Caryophyllales embraces families which have a characteristic ultrastructure of their sieve-element plastids, namely, the P-I11 subtype (104). In addition, the widespread occurrence of C, photosynthesis as well as DNA-RNA hybridization data support this taxonomic treatment (23). Within this order, the occurrence of betalains is restricted to nine of the eleven families of the Caryophyllales. The two exceptions, Caryophyllaceae and Molluginaceae, produce anthocyanins instead. There is controversy regarding the phylogenetic importance of this phenomenon (105). It has been suggested that a division of the Caryophyllales into two phylogenetic lines is possible, the betalain-producing Chenopodiineae and the anthocyanin-producing Caryophyllineae (103) (see Scheme 9). The presence of betalains has been an important criterion in the classification of questionable taxa as demonstrated by various examples (13). Further systematic classification at the level of families and subfamilies using the patterns of betalains is possible only in a few cases (see below). The majority of chemotaxonomic work on members of the Caryophyllales has been concentrated on flavonoids (101). More investigations, especially on the basis of sound phytochemical work as well as extensive screening data, for example, with the aid of HPLC, are needed. Table I summarizes the distribution of betalains in the Caryophyllales (only well-characterized compounds are listed).

1.

37

BETALAINS

ASTERIDAE

DlLLENllDAE

CARYOPHYLLIDAE (Caryophyilales)

HAMAMELIDAE

MAGNOLIIDAE

MAGNOLIOPSIDA

I I I I

I

PROTOANGIOSPERMS

Caryophyilales Chenopodiineoe: Aizoaceae, Arnaranthaceae, Basellaceae Cactaceae, Chenopodiaceae, Didiereaceae, Nyctaginaceae. Phytolaccaceae, Portulacaceae Caryophyllineae: Caryophyilaceae, Molluginaceae

SCHEME 9. Possible phylogenetic relationships of the subclasses of the Magnoliopsida (dicotyledons) (100). The Caryophyllales, belonging to the subclass Caryophyllidae, are divided into two phylogenetic lines, the betalain-producing Chenopodiineae and the anthocyanin-producing Caryophyllineae.

Betalamic acid (3) is widespread among members of the Caryophyllales and is also particularly abundant in yellow and red flowers and fruits of the Aizoceae and Cactaceae (83).It is obviously correlated with the production of betaxanthins if these are among the main components of betalain patterns. Organs with betacyanins as the only pigments do not contain 3. As a rule, yellow organs regularly contain betaxanthins and 3, orange-red and deep red ones contain betaxanthins, betacyanins, and often 3, whereas violet-red and mauve organs contain only betacyanins without 3. Those plants listed in Table I, for which accumulation of

+ +

. + +

106

46. lfk5

106

I06

1 46. 106

I I2 * . + + + + . + * + + - + +

100

+

16. I06 I 1M

.

I 1m 106 106

112

6. I 0 6

om

,

. +

+ +

. + +

+

+ + + . . + + .

. . . . . . . +

. . . . + .

+ . + . . . +

. . . . . . . . . . + . . . +* . . . + . . * . . . . . . . . . . . . . . . . . . . . . . . . . +

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . + . - + . . . + . . . .

*

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

. . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . + . . . . . . .

rn,

. . . . . . . . . . . . . . *

c . brew C . cuuliferum lfl) C . conrudii l f l )

. . . . . . . . . . . . . . . . C . uurrflorum (11) C . hilohum lfl)

(fl) C apiutum In) C . apprnrimurum C . out'lum I l l ) C. uureum (fl)

. . . . .

. . . . . +

C. ulrum Ill) C. unduusunum (fl) C . ongusturn ffl)

. . . . .

. . . . . +

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

C . nelir ( I l l Coniwsia C . robusru In) Conophwdm C . udvenum c f l )

i

+ +

1om

C . corculum Ill)

. . . . .

38

C. ulsronii (flj Chusmurophyllum

a"

-

112

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . ++ . + + + + +++.++++++++++

I I2

C . peers;; lfl) Cephulophphllum

I

I12

m W

v1

70 C . acinutformis (fl) Curruunrhus

6

I12 I12

B. multicrpps lfl) 6.vespertinus f fl ) Curpobrorui

U

w

m

I

41 A. ocrophyllum In) Bergerunrhus

Ref.

47

48

49

54 55

Betalamic acid (3) TaXOIl

I12 A. orpenii ffl) Arprodemu A . uureum lfl)

Bemanthin Belacyaninb

45 41 44

40 2 39 35 36

30 31 32 34 29 28 26 27 24 25 19 23

5 1

(order Caryophyllalesc)

-1

TABLE I BETALAINS FROM THE CARYOPHYLLALES~

Airoaceae

Aloinopsis

+

I 1m -

+

+

I 1M

+

106, 1117 46. 106 +

+

-

+

+

A

Ill6 116 116 46. I116 106 46. 116 46, I06 r

107 +

i (m +

+

I06 46. 106 46, 106 +

107 .

64 .

46. 106 +

I06

I 1m 46. IW 107 106

I 1m

46. 106

iim

106 106

I06

106 106

- + + + + + . + + + - + + + + +

lorn I16

. . . . . . .

+

. . . . . . .

.

-

t

i

-

. . . . .

.

. . . . .

+

-

+

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . .

+

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . .

. . . . .

.

. . . . .

r

. . . . .

+ + .

. . . . .

+

. . . . .

-

. . . . .

+

. . . . .

. . . . .

*

. . . . .

.

. . . . .

+

. . . . .

.

. . . . .

.

t

i

+

. . . . . . . . . . . . . . . . . . . . . . . . .

+

. . . . . .+. . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . *.. . . . . . . + . . . . . . . . +

+

C. nanum (fl) C. nelianum lfl) C. ohscurum (fl) C. ohrusum (fl) C. ornurum (fl) C. oiigerum ttll C. peursonii (fl) C . percrassurn (fl) C. plenum (fll C . pok-evansii (tl) C. polyondrum 111) C. puherulum (Ill C. rumoxurn (fl) C. rerusum (fl) C. sororium (fl)

. . . . . . . . . C . frurernum ( f l l

. . +

c./lavum cn,

. . +

C. ,frurescen.sIfl C. immeculurum cfl) C. incurrum ( I l l C. karvaranum (11) C. k1ipbdbcrgm.w cfl, C. kuhusunum (ill C . Iariperulum cfl) C. murkoerrerae (fl) C. mewrue (ll) C. mewri (fl) C. minurum cfl) C. mirabile (tl) C. muscuripapilluruni

39

cn,

. . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

*

+ + . + + T - + + - + + . + . + - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * . + - . . f * + . + + + . . * + + + . . .

+ . . +

. . . . + . + + . . . + + . . + . . . . . + . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

++++++++++++

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' + + + + . ' + ++++++.++++++++

'

46. I M

C. uirnijivum I11 ) C. diformr ( I l l C. ecnpum I l l ) C. elishoe (flr C . exrracrum 1I1l

.

TABLE I (Conrinued)

+ + + " +

46. 106

106

106 "

'

107 /(I7 I12

112 112

+ + + +

I I2 I12

'

7s

' . ,

'

107 107 I07 107

'

112 112

+ + + + + + +

I12 I12

112 112 112

112

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

+ + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + +

. . . . . . . .

. .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. + . . . . . . . . . . . . . . . + . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . .

F . ourunriaca (fl)

. . . . . . . .

F . cradmkensis (fl) F . longidens (n) F . ruberculosu (fl) Fenesrrarru

. . . . . .

. . .

. . . . . . . .

F . ucuriperolus cn) F . urmsrrongii (fl) F . bosscheanu (IT F . brirrmiue (fl)

. . . . . .

Foucurio

. . . . . .

E. inclaudens (Ill E. luceru (fl) E. piiiunsii cn) E . rumosu (fl)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . + + + + + . . . . . + + . . . . . + ++++ . . . . . D . Jlorihundum (fl) Erepsiu

. + .

40

D . microspermus (fl) D . pole-evunsir (n) D . vunzijlir (fl) D . wilmorianus (n) Drosunrhemum

.

D . brunnrhuleri (n) D . luvisiue (n) D . leendenziue (fl) Dinrerunthus

. . + . .

C . ruvloriunum cn) C . rnrucurpum (n) C. violucrflorum (fl) Delosperma

+

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + ' + + . . + ++++ . . . . . +++++++ .

106 107

C.subriscum (fl)

.

cn) C. selosum

.

rfl,

Ref Betalamic acid (3)

54 55 49

48 47

106

c. springbokbergense

Betaxanthin Betacyaninb

40 41 44 45 2 39 35 36

30 31 32 34 29

28 26 27 24 25 19 23

5 1

Taxon (order Caryophyllales~)

++++++++++++

112 112 112 112 75. 112 112 112 112 112 112 112 112

++

112 112

+ . . . ' . .

10. 112 107 107 107 107 107 10. 56. 107 I I2 56. 107 54 107 106 106 106 106 106

. . . .

.+ . . . . . .

. . . . +. . . . . . . . . . . . . . . . . . . . . . . +

. +. . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + . . . . . . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . .

. . . .

. .

.

.

I

.I

. . . . . . . I . .

. . + . .

. . . .

. . . .

. . . . . . . + . . + . . . + . .

. . . . . . . . . . . .

. . . . . . . . . . . . .

. . . .

. . . . .

(garden hybrid?)

. . . . .

A

L. zeyheri (fl) Lirhups L. U1PiM (fl) L. uucampiae (fl) L. bromjieUii (fl) L. dendririca (fl) L. diverpens (fl)

. . . . .

41

+

L. polyunrhon (fl) L. sociorum (fl) Lumprunrhus sp.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .+.++++ .++ + . . . . . . . . . . . . . . . .+ . + + + + . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '++++++ ..+ + . . . . . . . . . . . . . . . .++.+++ . . . + . . . .

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+ ' .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +++++

+

112 Frirhiu F. pulchru (fl) Glorriphy11um G.erecrum (fl) G. compressurn (fl) C. davirii (fl) C. diflorme (fl) C. longum (fl) C. linguiforme rfl) G. murlorhii (fl) G. muiri tfl) G.p l o ~ r a t p u r n(fl) G.pruepinguae (fl) G. srurkeae (fl) G. suuve (Il) Hererou H. acuminmu (fl) H . graci1ir (fl) Lumprunthus L. auranriucus (fl) L. bicolor (fl) L. emarginarus (fl) L. fa1curus (fl) L. furvus (fl, L. mu1tiserium (fl) L. peersii (fl)

(continued)

TABLE I (Continued)

3

106 106

I06

106 106

I06

106

I06

I06

106

I06 106 106 I06

I 12 41 41 41 64

. . . t . . .

+++++++++++

. . . . . . + . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

+

. . . .

. . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

. . . . . . . . . . .

42

. . . . . . . .

. . . . . .

. . . .

. . . . . .

. . . .

. . . . . .

. . . .

. . . . . .

. . . .

. . . . . .

. . . .

. . . . . .

. . . .

. . . . . .

. . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . .

. . . . . . . .

. . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . .

+

. . . .

. + . +

. . . . +++.

. . . .

. . . .

. . . . . . . .

. . . .

. . . .

. . . . . . . . . . . .

. . . .

. . . .

. . . .

. . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . + + . . . + . + . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+++++++

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +

106 106

++++++++

106 106 106

I06 I06 I06 (fl) L . rugosa (fl) L. rusrhinrum lfl) L . schwunresii Ill) L. ferriculor lfl) L . furbin~ormi,~ (fl) L. urikusensis Ill) L. vallis-mariue (fl) L. vulkii lfl) Malephora M. cram (fl) hlesemhryanrhemum M . conspimum In) M . edule (fl) M . floribundum (flJ M. roseum lfl)

-J l.

106

L . dororheue (fl) L. edirhue lfll L . elisabethue ifl) L. fossul$eru lfl) L. Reyeri lfl) L. goais (fl) L . grucilidelineuru

Ref. Betalamic acid (3) 54 55

49 48

I06 I06 I06

tfl) L. helmurii lfl) L . herrei In) L . kuibisensis ifl) L. kunjasensis (fl) L . lesliei lfl) L. lineam In) L. 1ocali.r (fl) L. mormurufu (fl) L. mennellii ifl) L . ot:eniuna (fl) L. pseudurrunrarellu

Betaxanthin Betacyaninb

47

40 41 44 45 2 39 35 36

30 31 32 34 29

28 26 27

24 25

19 23 5

I Taxon IorderCaryophyUalesr)

+ + + - + + + + * + + + +

. + + + '

+

112 112

. . . .

. . . .

. . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . C

. . . .

.

+++ . ++

. . . . . . . +

.

+

+

. . . . . .

. . . . . . . . . . . . . .

.

. . . .

. . . .

.

. . . .

. . . .

.

.

.

.

.

. . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . . . . . + . .

. . . . . . . . . . . . . .

. . . . . . .

. . . .

. . . . . . .

. . . . . . .

. . . .

. . . . . . . . . . . . . .

.

. . . . . . .

. . . . . . .

. . . .

. . . .

. . . . . . . .

. . . .

. . . .

. . . .

. . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

. . . +

. . . . . . . . . . . . .

. . . .

. . . .

. . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . .

. . . . . . .

107

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+

112 112

112

. . . . . . . . . . . . . . . . . . . . . . . . . . .

+

(fl)

I I2

. . . . . . . . . . . . . . . . . . . . . . . . . . . +++++++++ ++++

107

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +

*

. . . .

++

. . . .

. +

. . .

+

. . .

. . .

. . . .

+

. . . . . . . . .

. . .

. . .

. . . . . . . . .

. . .

. . . . . .

. . . . . .

. . .

. . .

. . . . . .

. . .

41 41

Amurunrhus A. ruudarus (inIlvls0 A. firarrizuns (in)

112 112 112

I I2 112 112 I 12

Rhinrph?llum R. broomii (fl) Rhombophdlum R. dolobriformc (fl) R. nelii (fl) R. rhomboidcum ( f l l SmicrosriRma S. viride (fll Tischleria T.pmdidpns ( f l l T. prersii (fl)

41

Alrernanrheru A. wrsicolur (Iv)

112

112 112 112 112 112

P w

107

Ophrhulmoph~llum 0. pruerecrum Osruluriu 0.delroidrs (Ill Pleiospilos P. artheri (fl) P. bolusii P. brevisepulur (fl) P. dimidiarus (fl) P . framesii (fl) P. hilmurii (fl) P. kinfiiue (fl) P. leipoldrii (fl) P. longibrucreuru cfl, P. oprorus (fl) P . rouri (11) P. srmulans (fll P. wrllowmorm,i~

Amaranthacreac

(conhued )

TABLE TABLE II (Continued) (Continued) Betacyanins Betacyanins Taxon Taxon (order (orderCaryophyllalesc) Caryophyllalesc) A. A. hybridus hybridus (Iv) (Iv) A. A. hyporondriacus hyporondriacus

11

55

19 19 23 23 24 24 25 25

26 26 27 27 28 28

Betaxanthin Betaxanthin

-

29 29 30 30 31 31 32 32 34 34 35 35 36 36 39 39

22

40 40 41 41 44 44 45 45

47 47 48 48

49 49 54 54 55 55

Bekdamic Bekdamic acid acid (3) (3)

Ref. Ref. 41 41 41 41

(in) (in) A. A. rerroJlexus rerroJlexus tn) tn) A. A. rricolor rricolor (Iv) (Iv) A. A. rricolor rricolor CCVV.. "Re "Re del del fuoco" fuoco" (IV) (IV) Celoma Celoma (in) C C.. crisrara crisrara (in)

C. C. crisrara crisrara cv. cv.

41 41 41. 41. 47. 47.

59 59 41 41

41. 41. 50. 50. 106 106 41 41

"Toreadof' "Toreadof'(in) (in) C C.. plumosa plumosa cv. cv.

41 41

44

"Forest "Forest lire" lire" (in) (in) Gomphrem Gomphrem G. G. glohsa glohsa (in) (in) Iresine Iresine 1. 1. herbstii herbstii (Iv) (Iv) 1. 1. lindpnii lindpnii (Iv) (Iv) 1. 1. lindenii lindenii var. var. formosa formosa (Iv) (Iv)

41. 41. 48 48 41. 41. 50 50 41. 41. 106 106 41 41

Cactaceae Cactaceae Apororacrus Apororacrus A. A. J7agellijormis J7agellijormis(fll (fll Bonrcacrus Bonrcacrus B. B. sepium sepium (fl) (fl) Cereus Cereus C. C. comarapanus comarapanus(fl) (fl) C. C. stemgonus stemgonus (fl) (fl) Chnnrrpcpreus Chnnrrpcpreus C. C. silvrsrrii silvrsrrii (fl) (fl) Cleistoracrus Cleistoracrus

110 110 110 110 110 110 110 110

106 106

?

gs

?

+ .

..

$5 5PSE

5

. . . . . .

+ + + +

. . . .

+ +

. . . .

. . . . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. .

. +

. . . . . . . .

. .

. . . .

. .

. .

. . . .

. . . . . . . .

. . . . . . . . . .

. . . .

. .

. . . . . . . .

. . . .

. . . .

.. . . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . .

. .

. .

. . . .

. . . .

+ + + +

. . . . . . . .

. .

++

+ +

. . . .

++++

. . . .

. .

. . . .

. . . . . . . .

+ . . + . . . .

. . . .

+ . . .

. . . .

. . . .

. . . .

. . . . . . . .

+ '

106 106

+ . + '

rfl,

+ + + +

. . + .

--

5E

45

555

. . .

. . .

. . .

. . . . . .

. . . . . .

. . .

. . .

. . .

. . . . . .

. . .

. . . . . .

. . .

+ + +

. . .

. . .

+++

. . .

. . .

. . . . . .

. . .

+ + +

106 106 106

. . .

+++

106

. . . . .

. .

+ +

..

+ +

. . . .

. .

. . . .

. .

. .

. .

. . . . .

+ +

. .

. .

. .

+ +

. .

. .

. .

. .

. .

. . . . . .

+ .

. .

. . .

. . . . . .

. . . . . .

. . . . . .

. . . . . . . .

. . . . .

+ . ' + .

. . . . . . . . . .

. . . . . . . .

. . . . .

. . . . . .

. . . . .

. . . . .

+++++

. . . . . . . . . .

. . . . .

+ .

. . . . .

+ . .

. . . . .

.....

. . . . .

$5 106

++++'

myosorus

+ +

106 106

46. I06 106 106 Lobivia L. arachnacantho (fl) L. cornea (fl) L . chlorogona (fl) L . h g e a M (fl) L. hermannia

106. 110 106

C. straussii (fl) C. tarijensis (fl)

++++

C. smaragdiflorus

106 Heliocereus H. speciosus (fl) Lepismium L . cruciforrne (fl) L . cruciforme var.

106 106

110

110

6. 106 106

110

Echinocereus E . durangensis (fl) E. ochorerenae (fl) E. radians (fl) E . subinemis (fl) Epicactus E . cu1rivar.s Eriocereus E. guelichii (fr) Epiphvllum E. strirtum (fr) Gymnocdycium G. venturionum (fl) Haageocereur H . acranthus (fl) Hariora H . bambusoides (fl) H . salicornioides (fl) H . salicornioides var

u P

106. I10 110

106

C.azerensis (fl) C.jujuyemis C. pawiflorus (fl)

snicta

(continued)

~

TABLE I (Continued)

Ref. acid (3)

54 55 49 48 47

106

106 I06 106 106 106 106

I06

106

. . . . . . . . . . . . . . . . .

64

+

I10 106 110

+

I06

I06

110 106 106 I10 I10

'

/If1

+

I06

' + ' +

106

106 106

+

+

+

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

+

. . . . . .

+

. . . . . .

*

. . . . . .

+

. . . . . .

.

. . . . . .

. . . . . .

+

.

. .

. .

+

. . . .

.

.

+ . + +

. . .

A

+-+. . + . . . . . . . . . . . . . . . . . . . . . . . . . . . + . + . +

. . . . . .

t

. . . .

. . . . . .

i

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . + + + + + + + + + . +++++++ + . . . . . . . . . . . . . . . . . . . . . . . . . . +

+ + + .

26 27

24 25

106

mummulosur (fl) orronis (fl) mriluns lfl) rubuluris (fl) N. N. N. N.

+

. . . . . . . . . . . . . . . . . . . . . . . . . .

46

cfl,

+

L. marimiliunu (fl) L. penrlundii (fl) L. pusillu (fl) L. pusilla var. fluvfloru (fl)

+ + + + + + + + +

19 23

30 31 32 34 5

L. scopuriu (fl) Mumilloria M. renrricirrha (fr) M. gooldii lfr) M . longiflora tfr) M. ma,qnrmummu (fr) M. neumanniunu (fr) M. prrcuyensir (fr) M . seirirrunu (fr) M. resopacensis lfr) M. zuccuriniunu lfr) Monvilka M. speguz:ini lfl) Neoporreriu N. rhvaru (fl) N. litoralis (fl) N. nigrihorrida (fl) N. pluniccps (fl) N. vullenarensis (fl) N. villosu In) N. wagenknechrii cfl) Nopulea N. dejecru Ill) Norococrus N. rarambabionsi.$

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

+ + + + . . + . . . + . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + . . . . . . . . . . . . . . . . . . . +. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

-

. * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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T

46, I06 106

2 29

I

Bctaxanthin Betacyaninb

Betalamic

Taxon

40 41 44 45 39 35 36 28 (order Caryophyllaler~)

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106 58

de Huler” ( f l )

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46, 106 46. lorn P . soinr-pieono lfl) P. sruemrri var. rilcarensis (fl) Ph.vllixacrus P. hybridus (fl)

+

46. llh5 P . microspermu var. marrancisrru ( 11) P . murabilis ( I l l Purodia “Quebradd

+ + + +

-

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-

IZa

\ 204

Ar

l5

k4

R2 R2 205 a: R I = CONH2: R2= OCH, b: RI= CtI2N(CH3)C02CH2CH~;R2= OCll, c: R I = CONH2: Rz= H

'"

SCHEME 4. Reagents: i, (a) NaBH,, (b) HCI; ii, methanol, HCI; iii, (a) hv, 12. 0 2 , (b) methanol, NH3; iv, (a) LiAIH,, tetrahydrofuran, reflux, (b) ethyl chloroformate, (c) NaH, methyl iodide, tetrahydrofuran; v, KOH, ethanol, reflux; vi, LiAIH,, AIC13, tetrahydrofuran, reflux.

titanium, resulted in the formation of phenanthrene 201c in 50% yield. Reduction of 201c afforded thalictuberine 201d (29). Treatment of keto acid 202a, obtained by self-condensation of 3,4-dimethoxyphenylacetic acid, with sodium borohydride followed by acid, afforded lactone 203a, which was converted to 204a by means of methanolic hydrochloric acid (Scheme 4). Compound 204a can be transformed into amide 205a by oxidative photocyclization followed by reaction with ammonia. Sequential treatment of 205a with lithium aluminum hydride, ethyl chloroformate, and methyl iodide gave urethane 205b, which was converted to secoglaucine (15) by basic hydrolysis and to N-methylsecoglaucine (12a) by reduction with lithium aluminum hydride. The same synthetic approach used to obtain N-methylsecoglaucine (12a) was employed to get atherosperminine (4). Starting from keto acid 202b, 4 was obtained via compounds 203b, 204b, and 20% ( 2 9 ~ ) .

IV. Phenanthrenes as Intermediates in the Synthesis of Other Alkaloids

Ozonolysis of the phenanthrene alkaloid 206, easily obtained by base treatment followed by acetylation of the aporphine magnoflorine, gives dialdehyde 207 (Scheme 5). Aldehyde oxidation with silver oxide, followed by hydrolysis

3.

131

PHENANTHRENE ALKALOIDS

CH3 N. CH r CH3C02

:

, z

F

'

CH,O

206

201

H CH30

-

CI-

0

2ox

SCHEME 5. Reagents: i , (a) 03.methanol, (b) NaI; ii, (a) Ago, (b) HCI, reflux, (c) NaHCO,, (d) HCI.

and acid-catalyzed lactone formation, affords taspine hydrochloride (208). This overall transformation of magnoflorine into taspine is biogenetically patterned (91). c H 3 0 F N ' CH, R

,

c H 3 0 F N ' CH3 R

-

CH,O

CH,O

CH30

'

CH,O

OCH,

A

' OCH3 210 a: R = H b: R= COCF3

ZOY a: R = 14 b: R= COCF3

OCH3 21 I

OCH, 212

SCHEME6. Reagents: i , HIO,, acetic acid, H20, reflux; i i , sodium carbonate, methanol, reflux

132

LUIS CASTEDO A N D GABRIEL TOJO

213

214

SCHEME7. Reagents: i, (a) H103, acetic acid, H,O, reflux, (b) sodium carbonate, methanol, reflux.

Phenanthrene 209a, which is obtained from the aporphine glaucine by treatment with ethyl chloroformate in the presence of base followed by basic hydrolysis of the resulting urethane, is oxidized by iodic acid to 9,lO-phenanthrenedione 210a (Scheme 6). Compound 210a is transformed by sodium carbonate in methanol into 60% U-methylatheroline (211) and 10% corunnine (212). This overall transformation may be done more efficiently through the intermediacy of 210b by sequential treatment of 209b, obtained by reacting glaucine with tri-

-

CH30 CH30 215

CH30 il

R= CI

I) R = l l

3

216 I"

SCHEME8. Reagents: i, chloroform, NaOH (50%), tetra-n-butylammonium chloride; ii, methanol, sodium carbonate; iii, toluene, reflux; iv, LiAIH4, AIC13, tetrahydrofuran; v, HS,10% Pd/C, sodium acetate. ethanol.

3.


133

fluoroacetic anhydride and pyridine, with iodic acid and sodium carbonate. This allows an overall yield of 62% 0-methylatheroline and 4% corunnine. Similarly, liriodenine (213) may be obtained from secoroemerine (214), which is prepared by degradation of the aporphine roemerine via treatment with iodic acid followed by methanolic sodium carbonate (Scheme 7) (39).The main interest in the above transformations is that, whereas phenanthrene alkaloids are usually obtained from aporphines, they suppose a unique way of obtaining aporphine alkaloids from phenanthrenes. Dichlorocarbene adds to the central double bond of phenanthrene 209b to give adduct 214a (Scheme 8). Heating in toluene of 214b, obtained by basic deprotection of 214a, gives rise to 215a. Dechlorination of 215a with lithium aluminum hydride followed by reduction of the olefinic double bond of 215b by catalytic hydrogenation affords homoaporphine homoglaucine (216) (106).

V. Biosynthesis

Although no labeling experiments have yet been done, there is complete consensus concerning the origin of phenanthrene alkaloids from aporphines (5.79,117). In fact, quaternary aporphine salts undergo Hoffmann elimination to give phenanthrenes with a 2-dimethylaminoethyl side chain so easily that some doubts were cast about the real existence of phenanthrerie alkaloids in plants (5,79). Shamma and Rahimizadeh found that boldine methiodide (176) is partly decomposed to boldine methine (177) on a silica column. However, these authors cautioned against the straightforward deduction that phenanthrene alkaloids are artifacts, pointing out that, for example, whereas magnoflorine (217) is one of the most widely occurring aporphines, its Hoffmann elimination product has not yet been found in nature (79). It could be argued that magnoflorine does not easily suffer this elimination because of the presence of a substituent at C - 1 1, but

217

2111

I34

LUIS CASTEDO A N D GABRIEL TOJO

nature can accomplish such difficult eliminations, as in the case of coridinemethine (6). Furthermore, the alkaloid taspine (218) is most probably formed from the Hoffmann elimination product of magnoflorine. We may safely assume that phenanthrene alkaloids with a 2-dimethylamino side chain derive from quaternary aporphinium salts by in vivo Hoffmann elimination. These phenanthrene alkaloids may later be oxidized to the corresponding N-oxides or methylated to give trimethyl 2-( l-phenanthry1)ethylammonium salts. Far more intriguing is the biosynthesis of phenanthrene alkaloids with a 2monomethylaminoethyl side chain, for which we have four examples: N-noratherosperminine (13), noruvariopsine (14), secoglaucine (15), and secophoebine (16). One possibility would be direct elimination after protonation of the nitrogen of an aporphine. This transformation would have some similarity to the formation of 167 by acid treatment of 166 (18).

VI. Spectroscopy

As with aporphines, the C-5 proton appears quite upfield relative to the other aromatic protons in 'H-NMR spectra. Very characteristic is the presence of two doublets arising from the protons at C-9 and C-10. They appear close to each other at 7.9-7.3 ppm, with a coupling constant of 9-10 Hz. The 2-aminoethyl side chain gives rise to two multiplets at 3.6-3.2 and 3.0-2.6 ppm, each derived from two protons. The mass spectra of phenanthrene alkaloids are very informative regarding the nature of the side chains. The two main fragmentations correspond to the loss of *CH,NR,R, and to the CH,=N+R,R, cation. This cation is usually the base peak and allows the identification of R, and R,. Thus, phenanthrenes with a 2dimethylaminoethyl side chain present the base peak at 58 mass units, whereas ones with a 2-monomethylaminoethyl side chain show the base peak at 44 mass units. In the case of the N-oxides there is a Cope elimination in the mass spectrometer, giving rise to a strong peak corresponding to a vinyl phenanthrene.

VII. Pharmacology

Some antimicrobial activity was found in thaliglucinone (23) (64,67,69) and thalictuberine (20) (51),but this activity was not found in phenanthrene 106 (18). Phenanthrene 63 (98) and uvariopsine (30) (76) show cytotoxicity in vitro. Cell

3.


135

CH30

219

lines resistant to the protein synthesis inhibitor (-)-emetine (219) were found not to have cross-resistance to uvariopsine (76). Thaliglucinone (23) possesses hypotensive activity in dogs and rabbits (62,64,68). Phenanthrenes 91, 104, 145, and 147 produce sedation in rats

220

221 K = CH? 222 K=CH:C‘H?

(103,118),whereas 220 is active in pigeons (22). Dihydrophenanthrenes 221 and 222 and phenanthrene 61 induce compulsive gnawing in mice and pecking and emesis in pigeons (89). A comparative pharmacological study of the aporphine nuciferine (163) and its Hoffmann degradation product atherosperminine (4) shows that, whereas the former has a pharmacological profile associated with blockade of dopamine receptors, the later produces effects associated with dopamine receptor stimulation (27). REFERENCES H. Guinaudeau, M. Leboeuf, and A. CavC, Llovdiu 38, 275 (1975); H. Guinaudeau, M. Leboeuf, and A. CavC, J . Nut. Prod. 42, 325 (1979); H. Guinaudeau, M. Leboeuf, and A. Cave, J . Nut. Prod. 46, 761 (1983); H. Guinaudeau, M. Leboeuf, and A. CavC, J . Nut. Prod. 51, 389 (1988). T. Kametani, “The Chemistry of the Isoquinoline Alkaloids,” Chap. 9. Hirokawa, Tokyo, and Elsevier, Amsterdam, 1969; T. Kametani, “The Chemistry of the Isoquinoline Alkaloids.“ Vol. 2, Chap. 10. Kinkodo, Sendai, 1974. M. Shamma and H. Guinaudeau, Nut. Prod. Rep. 3 , 345 (1986). M. Shamma and H. Guinaudeau, “Specialist Periodical Reports, The Alkaloids.” The Royal Society of Chemistry, London, 1976- 1983.

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77. J. Gadamer, M. Oberlin, and A. Schoeler, Arch. Phurm. (Weinheim. G e r . ) 263, 81 (1925). 78. S. M. Kupchan, J. L. Moniot, R. M. Kanojia, and J. B. O'Brien, J. Org. Chem. 36, 2413 ( 1971). 79. M. Shamma and M. Rahimizadeh, J. Nut. Prod. 49, 398 (1986). 80. D. S. Bhakuni, S. Jain, and R. S. Singh, Terruhedron 36, 2525 (1980). 81. K. Warnat, Chem. Ber. 58, 2768 (1925). 82. T.-H. Yang. S.-T. Lu, and Ch. Y. Hisao, Yukuguku Zasshi 82, 816 (1962); Chem. Absrr. 58, 7991h (1963). 82a. G. Barger and R. Silberschmidt, J. Chem. Soc., 2919 (1928). 82b. M. Tomita and F. Fusada, Chem. Phurm. Bull. 1, 5 (1954). 83. D. S. Bhakuni and S . Jain, Tetrahedron 37, 3175 (1981). 84. R. Robinson and I. Shinoda, J. Chem. SOC., 1987 (1926). 85. V. V. Kiselev and R. A. Konovalova, Zh. Obshch. Khim. 19, 148 (1949):V. V. Kiselev and R. A. Konovalova, J. Gen. Chem. USSR (Engl. Trunsl.) 19, 135 (1949). 86. W. Klee, Arch. Phurm. (Weinheim, G e r . ) 252, 21 1 (1914). 87. P. Gorecki and H. Otta, Monutsh. Chem. 113, 201 (1982). 88. J. Comin and V. Deulofeu, J . Org. Chem. 19, 1774 (1954). 89. J. G . Cannon, R. J. Borgman, M. A. Aleen, and J. P. Long, J. Med. Chem. 16, 219 (1973). 90. R. Pschorr, B. Jaeckel, and H. Fecht, Chem. Ber. 35, 4377 (1902). 91. M. Shamma and J. L. Moniot, J. Chem. SOC., Chem. Commun., 1065 (1971). 92. I. Weiss, E. Valencia, A. J. Freyer, and M. Shamma, Heterocycles 23, 301 (1985). 93. W. Fleischhacker, R. Hloch, and F. Viebijck, Monutsh. Chem. 99, 1568 (1968). 94. W. Fleischhacker, W. Passl, and F. Viebock, Monutsh. Chem. 99, 300 (1968). 95. M. Tomita, Y. Watanabe, and H. Furukawa, Yukugaku Zusshi 81, 942 (1961). 96. S. M. Kupchan and N. Yokoyama, J . Am. Chem. SOC. 86, 2177 (1964). 97. T. Kitamura, Yukuguku Zasshi 80, 219 (1960). 98. Y.-F. Liou, K.-H. Lin, and S.-T. Lu, J. Taiwan Phurm. Assoc. (T'ui-wan Yuo Hsueh Tsa Chih) 31, 28 (1979). 99. P. Gorecki and H. Otta, Monutsh. Chem. 112, 1077 (1981). 100. Kh. G. Pulatova, Z. F. Ismailov, and S. Yu. Yunusov, Khim. Prir. Soedin. 2, 426 (1966): Chem. Nut. Prod. 2, 349 (1966). 101. H. Doshi, A. B. Cardis, J. K. Crelling, S. I. Miller, and D. R. Dalton, J. Org. Chem. 52,2604 (1987). 102. A. C. Allen, D. A. Cooper, J. M. Moore, and C. B. Teer, J. Org. Chem. 49, 3462 (1984). 103. E. E. Smissman, A. C. Makriyannis, and E. J. Walaszek, J. Med. Chem. 13, 640 (1970). 104. C. Bertgen, W. Fleischhacker, and F. Viebijck, Chem. Ber. 100, 2992 (1967). 105. W. M. Whaley and M. Meadow, J. Org. Chem. 19, 661 (1954). 106. J. L. Castro, L. Castedo, and R. Riguera, J. Org. Chem. 52, 3579 (1987). 107. S. R. Johns, J. A. Lamberton, and A. A. Sioumis, Ausr. J . Chem. 19, 2339 (1966). 108. S. R. Johns, J. A. Lamberton, and A. A. Sioumis, J . Chem. Soc., Chem. Commun., 480 (1966). 109. M. Tiffeneau and M. Porcher, Bull. SOC. Chim. Fr. 17, 114 (1915). 110. J. G. Cannon, J. F. Hensiak, and A. M. Burkman, J. Phurm. Sci. 52, 11 12 (1963). I l l . M. Freund, Chem. Ber. 32, 168 (1899). 112. A. Waefelaer, Ing. Chim. (Brussels) 63, 12 (1981). 113. H. Bach, W. Fleischhacker, and F. Viebijck, Monatsh. Chem. 101, 362 (1970). 114. W. M. Hanis and T. A. Geissman, J. Org. Chem. 30,432 (1965). 115. J. R. Cannon, G. K. Hughes, E. Ritchie, and W. C. Taylor, Ausr. J. Chem. 6, 86 (1953). 116. F. Faltis and M. Krausz, Monutsh. Chem. 42, 377 (1922). 117. M. Shamma and H. Guinaudeau, Tetrahedron 40, 4795 (1984). 118. A. Waefelaer, Ing. Chim. (Brussels) 63, 12 (1981).

-Chapter 4-ALKALOIDS OF KHAT (CATHA EDULIS) L. CROMBIE,W. M. L. CROMBIE, A N D D. A. WHITING Department qf Chemistry The Urziversio qf Nortingharn Nottingham NG7 2RD. England

............................. 1. Introduction . . . . . . . . . . . . . . . . . . 11. Phenylalkylarnine Alkaloids (Khata ............................. 111. Synthesis of Khatamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Pharmacological Action of Khatarnines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Cathedulin Alkaloids of Khat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139 140 141 144 145 150 A. Cathedulins of Lower Molecular Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cathedulins of Medium Molecular Weight . . . . . . . . . . . . . . . . . . . . . . . . 151 . . . . . . . . . . . . . . . . 152 C. Cathedulins of Higher Molecular Weight . . . . . . . . . .............................. 157 VI. Triterpenoid Extractives of Khat . . . lensis . . . . . . . . . . . . . VII. Catvaalens Sesquiterpenes of Catha 159 VIII. Synthetic Work Relevant to Cathedulin Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction

The vegetable drug khat, much used in Arab lands, consists of the fresh leaves and shoots of the tree Catha edulis (Forsk). Catha edulis is a member of the family Celastraceae which appears to have originated in Ethiopia and is now widely cultivated in the southern parts of Arabia and in East Africa (from Ethiopia and the Sudan to South Africa, including Madagascar) ( I ) . There are about 100 known names for the drug including many phonetic variants of the Somali name khat (quat, chat, jat, tschatt, etc.) (2,3).Administration is by chewing the plant, with buccolingual or enteral absorption of the active materials. Long periods of chewing large amounts of vegetable material are required to obtain the stimulating and euphoric effects, which are similar to those produced by amphetamine administration, and because psychic dependence is developed, khat chewing dominates and disrupts the lives of habitual users (3-6). Khat also acts as an appetite suppressant, leading to malnutrition and proneness to disease (4). Other economic consequences are the displacement of coffee growing by more lucrative khat crops which are often freighted by air to their destinations 139

THE ALKALOIDS. VOL. 39 Copyright 0 1990 hy Academic Press. Inc. All npht, d reproduction in any lorn reserved.

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L. CROMBIE ET A L .

since fresh material of good quality is necessary for satisfactory amphetaminelike effects. Despite its stimulant nature, the use of khat is acceptable to Islam and indeed has, historically, certain theological approval (4).

11. Phenylalkylamine Alkaloids (Khatamines)

The chemistry of the khat alkaloids mainly concerns two groups, the phenylalkylamines and the complex polyesters of polyhydroxylated dihydroagarofurans (cathedulins). Chemical studies of khat date back to at least 1887 (7), but, although the presence of alkaloids was established, including a basic fraction having stimulant properties (8-1 I), structurally characterized pure substances were not obtained until Wolfes (12) showed that (+)-norpseudoephedrine (cathine) (1) was present. Evidence was also obtained for the presence of waterinsoluble weak bases probably representative of the cathedulin-type polyester alkaloids (8,9,12). Pharmacological study has concentrated on the phenylalkylamine types (13)and early suggested that, in addition to cathine, a more This was powerfully acting stimulant was present in the fresh plant (10,Il). isolated in the United Nations Narcotic Laboratory from fresh plant material by Szendrei (14) and shown to be (-)-a-aminopropiophenone (cathinone) (2): it has the (S) configuration. Cathinone is the major active component and in some samples of khat accounts for up to 70% of the phenylalkylamine bases. Cathinone shows, as does amphetamine, a strong potency in stimulating the central nervous system (CNS) and exhibits a similar mechanism of action. Cathine, like cathinone, shows CNS activity, but is weaker in action, and there are both quantitative and qualitative differences (15-17). At the time of its discovery in khat, cathinone had long been known in racemic form (18), but it had not been optically resolved. During the isolation of cathinone, 3,6-dimethyl-2,5-diphenyl pyrazine (3) was also found (14). This is doubtless an artifact, the product of oxidative dimerization of cathinone which involves the formation of condensation product 4 as an intermediate (18). Also found was the diketone (5) and the cinnamoyl compound (6) (14), later shown to have the (S) stereochemistry illustrated and named merucathinone (6,19,20). Merucathine (7) has also been isolated (6,21,22), and this and merucathinone are minor components. (-)-Norephedrine (8) also occurs in khat (Id),and its N-formyl derivative (9) has been found in plant material of South Arabian origin (23). Despite earlier positive reports, ephedrine and pseudoephedrine have not been verified as components of khat (14). HPLC has been employed to separate and quantify mixtures of the khatamines cathinone, (+)-norpseudoephedrine, and (-)-norephedrine in connection with a study of their distribution in different parts of the khat plant and in specimens of different geographical origin (20,21).

4.

ALKALOIDS OF KHAT ( C A T H A EDULIS)

5% Me

Ph

-N

Me

Ph

141

$7 Me

Ph

N

Ph

Me

(3)

(4)

111. Synthesis of Khatamines

Since the isolation of cathinone and the other khat phenylalkylamines from fresh material, and their structural and stereochemical characterization, there has been interest in producing these compounds synthetically in chiral form. Buckley and Rapoport (24) have shown that N-ethoxycarbonyl-L-alanine,as its acid chloride (lo), can successfully acylate benzene in the presence of aluminum

142

L. CROMBIE ET A L .

chloride without loss of optical purity to give I1 (Scheme 1). Reduction of the latter with various reagents gave 12 in erythro/threo ratios of I : I to 4 : 1 . Hydrolysis of the carbamate mixture (12)with aqueous methanolic potassium hydroxide then gave a mixture of norpseudoephedrine (I) and norephedrine (8), separable by HPLC. Tests using amide formation with Mosher's acid chloride showed over 99% retention of configuration. A similar Friedel-Crafts acylation of N-methoxycarbonyl-D-alaninehas been described by McClure and colleagues (25).In the case of aryl ethers, complexation with aluminum chloride inhibits the reaction, but the difficulty can be overcome by the use of arylmetallo reagents

(241. Optically active cathinone has been obtained through the optical resolution of racemic norephedrine using O,O-dibenzoyl-D-tartaric acid (26). Each enantiomer was formylated (9) and oxidized with chromium trioxide in pyridine, and the product (13)was then deformylated by heating with 20% hydrochloric acid at 40°C (Scheme 2). Estimation of the optical purity by formation of a urea (14) with (-)- 1 -phenylethyl isocyanate and HPLC scrutiny, showed an optical purity exceeding 98% for each enantiomer. Cathinone as the free base racemizes and dimerizes readily in hydroxylic media, and similar behavior, at a somewhat reduced rate, is also observed for solutions of the oxalate salt sometimes used for isolation purposes. Cathinone base is fairly stable in dilute solution in nonhydroxylic, nonpolar media. It readily decomposes during drying of the leaf, hence the desire to use fresh material for hedonistic purposes (14,27). Adapting Rapoport's general approach, Wolf and Pfander (28) synthesized merucathine (7)in a manner that establishes its correct stereochemistry (Scheme 3 ) . N-Ethoxycarbonyl-L-alaninewas treated first with 2 equiv of n-butyllithium at -78°C and then with 1 equiv of styryllithium at - 120°C to give 15. Reduction

a (10)

NHC0,Et

+

L

Ph

(11)

NHC0,Et

b

*

Ph

L

(12)

iHC02Et

IC

t Ph

L

SCHEME I . Reaction conditions: a, benzene, AICI,: b, reduction; c. kOH/MeOH-H20

4.

Ph

(9)

L

NHCHO

143

ALKALOIDS OF KHAT (CATHA EDULIS)

P

hL

b

+

(13)

NHCHO

P

h

(2)

NH2

0

(14)

HNyo p h Y N H

SCHEME2. Reaction conditions: a, Cr03/pyridine; b, 20% HCI at 40°C.

SCHEME3. Reaction conditions: a, CICO,Et/NaOH; b, 2 equiv BuLi, I equiv P h C H S H L i ; c, DIBAL; d, KOH/MeOH, chromatography; e, KOH/aq. MeOH.

144

L. CROMBIE ET A L

SCHEME 4. Reaction conditions: a, 2 equiv BuLi, 1 equiv PhCH=CHLi: b. CF~COZH, (C02H)z; c, 3 equiv PhLi; d , CF3C02H, HCIIEt20.

with diisobutyl aluminum hydride (DIBAL) (-78°C) then gave a 1 : 1 mixture of difficultly separable diastereoisomers (16) which were converted into a pair of oxazolidinone diastereoisomers (17 and 18) by treatment with 1 M methanolic potassium hydroxide. Compounds 17 and 18 could be separated chromatographically (flash) and stereochemical identities assigned to each by NOE difference spectroscopy. Hydrolysis of oxazolidinone 17 by refluxing aqueous methanolic potassium hydroxide gave, with decarboxylation, the (3R,4S) stereoisomer (7), which was isolated as the hydrochloride and was identical with natural merucathine from khat. Prior to this synthesis it had been thought that the natural compound was the (3S,4S) form (22). As a check that inversion had not occurred during hydrolysis, 7 could be reconverted to 17 by treatment with phosgene. Merucathinone was synthesized by Wolf and Pfander (19) from tert-butoxycarbonyl-L-alanine (Scheme 4). Treatment with n-butyllithium and styryllithium as before formed 20, which was deblocked with trifluoroacetic acid to give merucathinone (6), isolated as the oxalate. As assessed by treatment with (+)-1phenylethyl isocyanate and HPLC, the optical purity of 6 was greater than 97%. Treatment of t-BOC-L-alanine with 3 equiv of phenyllithium gave ketone 21, which was deblocked as before to provide a short synthesis of cathinone (2), isolated as its hydrochloride (enantiomeric excess >95%) (19).

IV. Pharmacological Action of Khatamines (-)-Cathinone has been the subject of a number of pharmacological studies and comparisons with (+)-amphetamine which emphasize their similarity of

4.


145

action (13,27,29-33);indeed, there is cross-tolerance between the two (34).The alkaloid acts by facilitating release at physiological catecholamine storage sites (30,31)and is viewed as the major source of the symptoms of khat chewing (32). In contrast to the case for (+)-amphetamine, there are fewer reports of psychotic states after khat ingestion, but this is probably due to the large amount of plant that has to be chewed, making the activity self-limiting.

V. Cathedulin Alkaloids of Khat

As mentioned above, earlier workers recognized that, apart from phenylalkylamines, khat contained less basic alkaloids, though little progress was made on their separation or structures. However, the progress made on certain other Euonymus alkaloids (35)stimulated interest. Kupchan and colleagues elucidated the structures of maytoline (22) and maytine (23) (36.37)from Mayrenus ovarus, whereas the Hirata group, along with Pailer and colleagues, cleared up the structures of the alkaloids of Euonymus sieboldianus and spindle (Euonymus europaeus), in particular evonine (24) and neoevonine (25) (38-41). A singlecrystal X-ray structure of bromoacetylneoevonine (26) as its monohydrate was successfully obtained, clearing up all stereochemical details including the absolute configuration (42). These structures are based on a dihydro derivative of the sesquiterpene agarofuran (27) which occurs in agar wood oil (43); dihydroagarofuran itself is found in galbanum resin. In early work Stockman (8,9)had obtained an alkaloid preparation from khat which he called “cathidine,” and mass spectrometric examination of an old preparation led Luftmann and Spiteller (44) to the view that it was a mixture based on a reduction product of the evonine sesquiterpene core esterified with acetic, benzoic, trimethoxybenzoic, nicotinic, and evoninic acids. Ginsburg and colleagues (45) also examined “cathidine” which they had reisolated from khat and found that it contained at least four compounds. One of these, cathidine D, was studied in some detail and formulated as being either 28 or 29 (45). As a result of a detailed collaborative investigation between the Chemistry Department of the University of Nottingham (Crombie, Whiting) and the U . N . Narcotics Laboratory (Szendrei), at least 14 cathedulin alkaloids have been isolated and formulated (46,47). Plant material collected in Kenya, Ethiopia, and the Yemen Arab Republic was extracted as fresh material, as freeze-dried material, and as sun-dried material. A number of extraction procedures were also employed, with methods directed toward the isolation of weak bases. In one procedure fresh material was extracted with ethanol in the presence of sodium bicarbonate, and the diluted extract was adjusted to pH 5.5 and extracted with

I46

L . CROMBIE E T A L . OAc

OAc

(24) R = A c

(22) R = OH

(25) R = H

(23) R = H

(26) R = BrAc

(28) R = B z o . R ' = A c (29) R = AC

HO

OH

~

Me

W

Me

OH H

Me

/

(30)

R' = Bzo

(30)

Me OH

OH

OH

4.


147

benzene followed by extensive chromatography. In another, dried material was treated with ammonia and then extracted with ether, followed by dilute acid treatment and chromatography. Examination by TU3 indicated that there were quantitative differences in alkaloid content between samples of fresh and dried plant material and between samples of differing ages, but there appeared to be no gross differences between the number and types of alkaloid. There were, however, substantial differences in the khat specimens from different geographic areas, and this is probably indicative of the existence of a range of Catha rdulis chemotypes (48). Plant material from the Yemen Arab Republic yielded five alkaloids: cathedulin Y I (which was identical with cathedulin Kl), Y7, Y8, Y9, and Y 10, but on account of the very limited supplies only cathedelin Y 1 could be studied in detail. However, cathedulin Y8 appears to be identical with E8 and Y7, Y8, and Y10 were shown by mass spectrometry to have cores similar to those of the other cathedulins. Khat from Kenyan sources, which was available in more plentiful supply, yielded pure samples of cathedulin K I , K2, K6, K I 1, K12, and K15.Continued investigation has recently led to the isolation of three further new cathedulins from this source, K17, K19, and K20. Ethiopian khat provided cathedulins E2, E3, E4, E6, and E8. The only alkaloid common to the Kenyan and Ethiopian groups was E3, which was the same as K11 (48). All the cathedulins isolated up to the present are polyesters of two sesquiterpene polyol cores, 30 and 31. The latter, euonyminol, is much the more common, and the polyol core reported for cathidine D (cf. 28) was not encountered. The sesquiterpene cores themselves are highly hydroxylated derivatives of the eudesmane type, or, more specifically, of dihydroagarofuran. Cathedulins (Table I) can be conveniently divided into three groups based on molecular weight (46). Those of lower molecular weight (-600-700), E2 and E8, are based on core 30. The medium molecular weight group (750-900), K1, K2, K6, and K15, has structures based on the euonyminol core (31), and two of its nine hydroxyl groups are spanned by one dilactone bridge. The high molecular weight ( 1 1001200) group, E3, E4, E5, E6, K12, K17, K19, and K20, is also based on euonyminol, and they contain one or two dilactone functions together with other esterifying acids. The esterifying acids encountered (Scheme 5) are acetic, 2hydroxyisobutyric (32), 2-acetoxyisobutyric (33), benzoic, nicotinic (34), and tri-0-methylgallic (35) acids, and the dilactone spans are formed from (S,S)evoninic (36), edulinic (37), and cathic (38) acids. Two groups of the cathedulin alkaloids, E3, E4, E5, and E6 and K l , K2, K6, and K15, form subsets which differ within each other only in the acetylated or nonacetylated state of certain hydroxyl groups. In consequence, certain alkaloids could be artifacts caused by deacetylation induced by isolation or chromatography, or acetyl migration might have unexpectedly occurred. This cannot be ruled out with certainty in all cases, but there is evidence which indicates in general

TABLE I CATHEDULIN ALKALOIDS OF KHAT

Designation

Molecular formula

E2 E8

C38H40N201

K1 (Yl) K2 K6 K15

C42HS3N020

E3 (Kl1) E4 E5 E6 K12 K17 K19 K20

C,4HmNzO23 C,*H,,N2O22 C59H64N2O23

I

C32H37N0,0

CmH5I NO I9 C38H49N018 C36H47N017

C57H62N2022

C.54H62N2023 CS!2H62N2023

54

58 2

CS9H62N2023

23

MoIecu1ar weight

mp ("C)

Structure

700 595

149-151 Amorphous

39

40

30 30

2 Ac, 2 Nic, I Bzo 2 Ac, I Nic, 1 Bzo

89 I 849 807 765

Amorphous 183- I84 176-180 191-194

41 42 48 49

31 31 31 31

6 Ac, 1 HyBu', 5 Ac, 1 HyBu', 4 Ac, 1 HyBu', 3 Ac, 1 HyBu',

1 Evon 1 Evon

1104 1062 I168 I126 I106 1166 1102 1166

245-248 Amorphous Amorphous Amorphous 268-272 Crystals 249-25 1 Amorphous

50 51 57 58 59 62 61 60

31 31 31 31 31 31 31 31

4 Ac, 3 Ac, 3 Ac, 2 Ac, 4 Ac, 3 Ac, 4 Ac, 3 Ac,

1 Evon, 1 Cath I Evon, 1 Cath I Bzo, 1 Nic, 1 MGall, 1 Evon 1 Bzo, 1 Nic, 1 MGall, 1 Evon 1 Nic, 1 MGall, I Evon 1 Bzo, 1 Nic, I Edul 1 Cath, 1 Edul I Bzo, 1 Cath, 1 Evon

Triterpenoid core

Esterifying acidsU

1 HyBu', 1 HyBu', 1 HyBu', 1 HyBu', 1 HyBu', 1 HyBu', 1 HyBu', 1 HyBu',

1 Evon 1 Evon

QAC,Acetic; Nic, nicotinic: Bzo, benzoic: HyBu', 2-hydroxyisobutyric: MGall. tri-0-methylgallic: Evon, evoninic: Cath, cathic; Edul. edulinic acid

4.

149


OMe

(34)

(35)

H

Me

H

10

(37) 14

17

OMe

15

HOZC 3

SCHEME 5. Esterifying acids of the sesquiterpene cores of khat alkaloids. (The numbering indicated is as transferred from the intact alkaloids).

that this is not so and that the compounds isolated are not artifacts. Thus, TLC of fresh khat plant shows an alkaloid pattern essentially the same as that in the bulk extracts from which the pure alkaloids are individually isolated chromatographically with TLC monitoring. Further, partial hydrolysis or ethanolysis of K2 leads to a set of deacetylated products isomeric with, though different from, the deacetylated compounds of natural occurrence (K6 and K15).

150

L. CROMBIE ET A L .

At the outset of the structure investigation it was hoped that single-crystal Xray methods might be successful, particularly with the large alkaloids; however, as a number of trials were not promising, the approaches to structures have relied on spectroscopic and chemical methods. Spectroscopic examination involved electron impact, chemical ionization, and field desorption mass spectrometry, 'H- and 13C-nuclear magnetic resonance study (sometimes NOE and correlation spectroscopy), and this was followed by isolation of the polyhydroxylated core by alcoholysis or hydrolysis. Core 30 was novel and its structure established spectroscopically (48), whereas euonyminol was characterized as its octaacetate and compared with an authentic sample. The acids which esterify the core were isolated as esters from the alcoholysis. Both cathic (38) and edulinic (37)acids were new acids, and their structures were determined from chemical and spectroscopic studies; evoninic acid was already known from previous work on celastraceous alkaloids. Simpler acids were identified and quantified by NMR and GLC means. 2-Hydroxyisobutyric acid had not been found previously as an esterifying acid in celastraceous alkaloids, and its high water solubility and steam volatility made it difficult to isolate: an added difficulty was its tendency to react by a B,,2 mechanism in alcoholysis, giving the free acid when the ester was expected. The positioning of the esterifying acids on the hydroxylated sesquiterpene core involved graded hydrolysis and alcoholysis experiments on a small (milligram, sometimes submilligram) scale, followed by spectroscopic structure work on the partially degraded products. Characteristic chemical shifts, coupling constants, specific long-range shieldings, and nuclear Overhauser effects, together with some chemical evidence, were the main tools used. Sometimes chemical interconversions were useful, for example, the conversion of cathedulin E4 to E3 by acetylation. Little is known about the pharmacological activity of the cathedulin alkaloids. However, Kubo et al. (49) have reisolated cathedulins E3, E4, and E5 by HPLC and report that all three compounds are potent growth inhibitors for the pink bollworm at approximately 1 ppm. This is nearly as strong as azadirachtin, one of the strongest known natural growth inhibitors. A. CATHEDULINS OF LOWER MOLECULAR WEIGHT(50,51) Both cathedulins E2 and E8 (39 and 40, respectively) are based on the same pentahydroxylated dihydroagarofuran core (30). Cathedulin E2 is esterified with 2 mol of acetic, 1 mol of benzoic, and 2 mol of nicotinic acids (NMR and GLC). The positioning of the esters on the core was assigned by deacylation techniques accompanied by NMR assignments using chemical shifts, coupling constants, and nuclear Overhauser effects. Treatment with methanolic triethylamine at 5°C gave the 8-denicotinoyl compound, which was shown to be identical with cathe-

4.


151

dulin E8. The ease of removal of this 8-ester must, however, raise uncertainty as to whether E8 could be an artifact of isolation. Deacylation with the same reagent at 25°C gave the 8,15-bisdenicotinoyl derivative along with a little E8, and, finally, the use of sodium hydrogen carbonate in aqueous methanol at room temperature for 4 days stripped off all the ester functions except the benzoate. The 8,15-bisdenicotinoyl compound formed a cyclic carbonate (41), using the newly unmasked and axially oriented 8- and 15-hydroxyls, when treated with phosgene.

B. CATHEDULINS OF MEDIUM MOLECULAR WEIGHT(52,53) The cathedulin alkaloids of medium molecular weight (750-900) comprise four members, cathedulins K I , K2, K6, and K15 with K 2 (42) being the most intensively studied. It was shown by mass spectral and 'H- and 13C-NMR

152

L. CROMBIE ET A L .

methods, together with ethanolysis, that the latter (C,,H, ,NO,,) is compiled from euonyminol, one evoninic acid residue, five acetic acid residues, and one 2hydroxyisobutyric acid residue. It was possible to position some of the esterifying residues from the spectral information. The remainder were positionally assigned as a result of controlled treatment of cathedulin K2 with methanolic diethylamine. This experiment, carried out on a 79-mg sample, led to the isolation of four partially deesterified products, 43-46, which were studied and formulated spectroscopically. Cathedulin K1 has one acetate more than K2 and is structure 47, whereas K6 has one acetate less but a free primary hydroxyl and is thus 48. Cathedulin K15 has one acetate less than K6, and NMR chemical shift data, as well as mass spectrometric information, identify its absence from the hydroxyl of the hydroxyisobutyrate residue, leading to structure 49. It is noteworthy that the deacetylation of K2 did not produce K6 or K15 but the isomeric structures 46 and 45, thereby providing evidence that K6 and K15 are not artifacts.

c. CATHEDULINS OF HIGHER MOLECULAR WEIGHT(54-56) Cathedulins E3 (C54H60N2023)(50) and E4 (C,,H,,N,O,,) (51) are closely related; acetylation of E4 gives E3 whereas partial hydrolysis of E3 gives E4. There is also mass spectrometric evidence of two further cathedulins corresponding to E3 and E4 but in which an acetate is replaced by one benzoate (i.e., M + 62). Methanolysis of cathedulin E4 gave dimethyl evoninate (cf. 36), ethanolysis of E3 gave its diethyl ester, whereas treatment of E4 with lithium aluminum hydride gave evoninyl alcohol (52). The euonyminol formed was isolated as its octaacetate. A third product formed in the alcoholysis was cathic acid diester (cf. 38). The structure of the crystalline dimethyl ester of the latter was shown by NMR means and formed an N-oxide (53), whereas on hydrogenation over Raney nickel it cleaved to give methyl syringate (54). Reduction of E4 itself by lithium aluminum hydride also gave syringyl alcohol among the products. 2-Hydroxyisobutyric acid was found as its ester among the alcoholysis products. In sum, cathedulin E3 is made up from the nona-ol core euonyminol esterified by the two diacids cathic and evoninic, along with 4 mol of acetic acid and 1 mol of hydroxyisobutyric acid. Using data from other alkaloids, the NMR data enabled substantial progress to be made in the placement of the esterifying ester groups, but in these compounds the cathate bridge provided a novel feature. In the cases of both E3 and E4 this dilactone bridge could be opened by hydrogenolysis over palladium or by Raney nickel treatment to form 55 and 56, respectively. A new methyl resonance appeared in the NMR spectrum, and the new phenolic hydroxyl was characterized by a reversible bathochromic shift in the UV spectrum on the addition of

4.


153

H .H

alkali. NMR data indicate that the cathate residue spans the 8-axial oxygen and the 15-oxygen, whereas the long-range shielding and deshielding effects can only be satisfactorily explained on the basis of formulas 50 for E3 and 51 for E4. Cathedulins E5 (C,,H,,N,O,,) (57)and E6 (C57H62N2022)(58)are related to each other as is E3 to E4, and, indeed, E5 is produced on acetylation of E6. The

154

L . CROMBIE ET A L .

OH

H

core of E6 was again euonyminol (octaacetate), and the esterifying acids, as detected by NMR and methanolysis, were evoninic, benzoic, nicotinic, trimethylgallic, and hydroxyisobutyric acid in equimolar amounts, along with acetic acid (2 mol). NMR data along with biogenetic considerations lead to the proposal of structures 58 for E6 and 57 for E5. Two further alkaloids having an evoninate 3,13 span have been isolated; both occur in only small amounts. The first of these, K12 (Cs4H6zN20z3),shows 'HNMR data very similar to those for E3, but with trimethylgallate and nicotinate residues replacing the cathate span; it is formulated as structure 59 (55). The conversion A r - O - C H , R + A r - O - C H , R ' is well known in the biosynthesis of the methylenedioxy group, rotenoids, and homoisoflavonoids, and alkaloid K12 could be the biogenetic precursor of E3. Cathedulin K20 was studied by 'H and 13C nmr, 13C-'H two-dimensional (2D) (C,,H,,N,O,,) correlation, and 2D COSY 90 spectroscopy, which permitted its formulation as structure 60 (56). The ester positionings are similar to those of E3 but with the C-2 hydroxyl group esterified by a benzoate residue, the high-field C-2 acetoxy methyl seen characteristically in the spectrum of E3 being absent in that of K20.

+

4.

155

ALKALOIDS OF KHAT ( C A T H A EDULZS)

H

H

OH

H

OH

Me02C

OMe

0 Me0

Me0

Cathedulin K19 (C,,H,,N,O,,) showed, in the NMR spectrum, all the carbon and proton resonances appropriate to the euonyminol core, the cathic acid residue, three acetate units, and an acetoxyisobutyric acid residue. Shifts and coupling constants simulate those for E3 over a large part of the molecule, but in place of the evoninic acid fragment there were the signals of a new acid, edulinic acid. The proton connectivity pattern for this was established by decoupling, and

\=/

OMe

the Z configuration of the olefinic hydrogens was demonstrated by NOE difference spectroscopy and by a coupling constant (J7,*)of 11 Hz, leading to structure 61 (56). Cathedulin K17 (C59H,2N202,) (62), isolated as crystalline but only as a 2.5mg sample, revealed in its 'H-NMR spectrum an euonyminol core carrying eight ester groups, with the usual free tertiary 4-hydroxyl. The same 3,13-bridging edulinic residue was present as was found in K19. Two acetate units, a benzoate,

4.


157

a nicotinate, and a trimethylgallate (4-methylated syringate) completed the esterification pattern. The residues were placed using the information gathered from the spectra of cathedulins K19, E3, E5, and E6, with the trimethylgallate and nicotinate moieties at C-8 and C-15, respectively. The benzoate is placed at C-2 where it replaces the shielded acetate of K19 (56). The edulinic acid residue found in K17 and K19 may share a similar biogenetic origin to evoninic acid. Both may be viewed as products of coupling of (at a different oxidation level) nicotinic acid (or quinolinic acid) at C-2 with C-4 or C-5 of isoleucine as in 63. The absolute configuration at C-3 of isoleucine is the same as at C-8' of evoninic acid, but that of edulinic acid is not yet known.

VI. Triterpenoid Extractives of Khat

The roots of Ethiopian khat contain cathedulins E2, E3, E4, E5, and E6. The root bark itself is orange-red, and this was found to be caused by the presence of triterpenoid quinones (48). Those identified were celastrol (64), pristimerin (65),

158


iguesterin (66), and “tingenone,” known to be a difficultly separable crystalline mixture of tingenin A (67) and tingenin B (68). Such triterpenoid quinones are known elsewhere in the family Celastraceae. Other neutral products from khat leaf are reported to be p-sitosterol, p-sitosterol glycoside, friedeline, and certain of its hydroxylated relatives.

(65) R = M e

I

(67) R = H (68) R = O H

VII. Catvaalens Sesquiterpenes of Catha transvaalensis (57)

Until 1966 khat, Catha edulis (Vahl.) Forssk. ex Endl., was thought to be the sole member of the genus Catha Forssk. ex Scop., but in that year a new species confined to a relatively small area of northeastern Transvaal was identified and at first placed in a new genus as Lydenburgia cassinoides N. K . B . Robson, but later reclassified as Catha transvaalensis Codd (58). Recently, a third, rare species Catha abbotti Van Wyk et Prins, occurring in southern NataVPondoland, has been described (59).

4.

159


TABLE I1 CATVAALENS SESQUITERPENES

Designation

Molecular formula

Molecular weight ~~

Vaalens Vaalens Vaalens Vaalens

I 3 5 7

C2xH3,Ox C32H4,PI 2 C2&36O, Cu,H3xO, I

mp ("C) ~~

500 616 516 574

~

214-21 8 79-82 195-197 181-185

The dried leaves of Cuthu trunsvualensis have been shown to contain at least four dihydroagarofuran-based sesquiterpenes belonging to the lower molecular weight range (Table 11). The structure of only one of the compounds has thus far been reported, namely, that of vaalens 5, and this has very recently been revised to 69 in agreement with X-ray evidence (57). It contains a cinnamate and a glycolate residue not hitherto found among the esterifying acids of the cathedulin series. The hydroxylation pattern of the sesquiterpene core also differs from those of the known khat types. OH

VIII. Synthetic Work Relevant to Cathedulin Alkaloids

Although none of the known triterpene cores of the cathedulin group of alkaloids has been synthesized, progress has been made in the broader area of synthesis of Celastraceae sesquiterpenes, which is relevant to this problem. Thus, a completely stereoselective synthesis of the trihydroxylated dihydroagarofuran isocelorbicol (70) has been reported by Huffman and Raveendranath (60,61), which improves upon an earlier synthesis (62). The synthesis (Scheme 6) starts from the ketoagarofuran 71, which is available in six steps and 10% overall yield from carvone and methyl vinyl ketone.

160


SCHEME 6. Reaction conditions: a, 3-chloroperoxybenzoic acid (MCPBA)/dichloromethane/aq. bicarbonate, then lithium diisopropylamide (LDA); b, diimide; c, POC13/pyridine; d, LiAIH4, BuLi/PhCOCI; e, MCPBA; f, (PhSe)2/NaBH4/EtOH, then MCPBA/Pr2NH; g, Os04/pyridine; h, acetonide formation, then Barton deoxygenation; i, Ba(OH)2, then H . +

Useful information is also provided by Yamada’s resynthesis (63) of evonine (24) from evoninol (72) and evoninic ester (73), both formed by degradation of evonine. It provides a solution to the problem of correctly orienting the dilactone bridge. Dimethyl evoninate was converted to the trityl protected compound (75) with its pyridyl acid grouping activated as a mixed anhydride. This was then allowed to react with evoninol carrying pentamethyl and acetonide blocking

4.

161


Me *OH

(73)

(72)

E'

. -

CHZOCPh,

Me

(74)

(75) pMe

Me

CH20CPh3

Me

groups (74) to give 76. The latter was detritylated, and the exposed primary hydroxyl was oxidized to the carboxylic acid. The acetonide protection was removed, the carboxylic acid methylated, and the product converted to the bislactone (77) by treatment with sodium hydride in dimethylformamide (DMF). Demethylation and reacetylation then produced evonine. A synthesis of the racemic diastereoisomers of evoninic acid has been reported by Pailer and Pfleger (64).2-Ethyl nicotinate was brominated to form 78, and the

162


latter was treated with methyl cyanoacetate to give 79. This was hydrolyzed and decarboxylated to form evonic acid as a mixture of diastereoisomers, which were separated, after methylation, by GU7 and rehydrolyzed to the acids. The acids are the (RRISS) (80) and the (RSISR) (81) pairs.

REFERENCES

I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

A. Nordal, Bull Narc. 32(3), 51 (1980). United Nations Laboratory, Document MNAR13I 1979. R. A. Heacock and J. E. Forrest, Can. J . Pharm. Sci. 9, 64 (1974). A. D. Krikorian and A. Getahun, Econ. Bot. 27, 353, 378 (1973). H. X. Schomo, Pharm. Ztg. 11(3), 65 (1982). R. Brenneisen and S. Geisshuesler, Pharm. Acta Helv. 60, 290 (1985); R. Brenneisen and S. Geisshuesler, J . Nat. Prod. 50, I188 (1987). F. A. Flueckiger and J. E. Gerock, Pharm. J . Trans. 18. 221 (1887). R. Stockman, Pharm. J . Phnrm. 89, 676 (1912). R. Stockman, J . Pharm. Exp. Ther. 4, 251 (1912). F. Th. von Brucke, Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol. 198, 100 (1941). H. Friebel and R. Brilla, Naturwissenschafen 50, 354 (1963). 0. Wolfes, Arch. Pharm. (Weinheim. Ger.) 268, 81 (1930). H. Halbach, Bull. W.H.O. 47, 21 (1972). K. Szendrei, Bull. Narc. 32(3), 5 (1980).

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15. J. L. Zelger, H. X . Schomo. and E. A. Carlini, Bull. Narc. 32(3), 67 (1980). 16. D. W. Peterson, C. K. Maitai, and S. B. Sparber, Life Sci. 27, 2143 (1980). 17. C. E. Johanson and C. R. Schuster, J . Pharmacol. Exp. Ther. 219, 355 (1981). 18. S. Gabriel, Chem. Ber. 41, I127 (1908). 19. J-P. Wolf and H. Pfander, Helv. Chim. ACIU69, 1498 (1986). 20. X . Schorno, R. Brenneisen, and E. Steinegger, Pharm. Acta Helv. 57, 168 (1982). 21. S. Geisshuesler and R. Brenneisen, J . Ethnopharmacol. 19, 269 (1987). 22. R. Brenneisen, S . Geisshuesler, and X. Schorno, Planta Med. 50, 531 (1984). 23. I. A. Al-Meshal, M. Nasir, and F. S. El-Feraly, Phytochemistry 25, 2241 (1986). 24. T. F. Buckley 111 and H. Rapoport, J . Am. Chem. Soc. 103, 6157 (1981); see also C. G. Knudsen, and H. Rapoport, J . Org. Chem. 48, 2260 (1983); P. J. Maurer, H. Takahata. and H. Rapoport, J . Am. Chem. Soc. 106, 1095 (1984). 25. D. E. McClure, B. H. Arison, J. H. Jones, and J. J. Baldwin, J . Org. Chem. 46, 2431 (1981). 26. B. D. Berrang, A. H. Lewin, and F. I. Carroll, 1.Org. Chem. 47, 2643 (1982). 27. X . Schomo and E. Steinegger, Experientia 35, 572 (1979). 28. J-P. Wolf and H. Pfdnder, Helv. Chim. Acta 69, 918 (1986). 29. P. Kalix, Gen. Pharmacol. 15, 179 (1985). 30. I . Khan and P. Kalix, Trends Pharmacol. Sci.. 326 (1984). 31. P. Kalix, Alcohol Alcohol. 19, 319 (1984). 32. P. Kalix and 0. Braenden, Pharmacol. Rev. 37, 149 (1985). 33. G. C. Wagner, K. Preston, G. A. Ricaurte, C. R. Schuster, and L. S. Seiden, Drug Alcohol Depend. 9(4), 279 (1982). 34. R. Foltin and C. Schuster, J . Pharmacol. Exp. Ther. 222, 126 (1982). 35. For a review, see R. M. Smith, in “The Alkaloids” (R. H. F. Manske. ed.), Vol. 16, p. 215. Academic Press, New York, 1977. 36. S. M. Kupchan, R. M. Smith, and R. F. Bryan, J . Am. Chem. Soc. 92, 6667 (1970). 37. R. F. Bryan and R. M. Smith, J . Chem. Soc. B , 2159 (1971). 38. H. Wada, Y. Shizuri, K . Yamada, and Y. Hirata, Tetrahedron Lett.. 2655 (1971). 39. Y. Shizuri, H. Wada, K . Sigura, K . Yamada, and Y. Hirata, Tetrahedron Lett.. 2659 (1971);Y. Shizuri. H. Wada, K. Sigura, K. Yamada, and Y. Hirata, Tetrahedron 29, 1773 (1973). 40. K. Sigura, Y. Shizuri, H. Wada, K. Yamadd, and Y. Hirata, Tetrahedron Lett.. 2733 (1971); H. Wada, Y. Shizuri. K. Sigura, K. Yarnada, and Y. Hirata, Tetrahedron Lett.. 3131 (1971). 41. M. Pailer, W. Streicher, and J. Leitch, Monatsh. Chem. 102, 1873 (1971). 42. K. Sasaki and Y. Hirata, J . Chem. Soc., Perkin Truns. 2, 1268 (1972). 43. H. C. Barrett and G. Buechi, J . Am. Chem. Soc. 89, 5665 (1967). 44. H. Luftmann and G. Spiteller, Tetrahedron 30,2577 (1974). 45. M. Cais, D. Ginsburg, A. Mandelbaurn, and R. M. Smith, Tetrahedron 31, 2727 (1975). 46. L. Crombie, Bull. Narc. 32(3), 37 (1980). 47. L. Crombie, Pure Appl. Chem. 58, 693 (1986). 48. R . L. Baxter, L. Crombie, D. J. Simmonds, D. A. Whiting, 0. J. Braenden, and K . Szendrei, J . Chem. Soc., Perkin Trans. I , 2965 (1 979). 49. I. Kubo, M. Kim, and G. De Boer, J . Chromatogr. 402, 354 (1987). 50. R. L. Baxter, L. Crombie, D. J. Simmonds, and D. A. Whiting, J . Chem. Soc., Chem. Commun., 465 (1976). 5 I . R. L. Baxter, L. Crombie, D. J. Simmonds, and D. A. Whiting, J . Chem. Soc.. Perkin Trans. I , 2972 (1979). 52. L. Crornbie, W. M. L. Crombie, D. A. Whiting, 0. J. Braenden. and K. Szendrei, J . Chem. Soc., Chem. Commun., 107 (1978). 53. L. Crombie, W. M. L. Crombie, D. A. Whiting, and K. Szendrei, J . Chem. Soc., Perkin Trans. I , 2976 (1979).

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54. R. L. Baxter, L. Crombie, D. J. Simmonds, and D. A. Whiting, J . Chem. Soc.. Chem. Commun., 463 (1976). 55. R. L. Baxter, L. Crombie, W. M. L. Crombie, D. J. Simmonds, D. A. Whiting, and K . Szendrei, J . Chem. SOC.. Perkin Trans. 1 . 2982 (1979). 56. L. Crombie, D. Toplis, D. A. Whiting, Z. Rozsa, J. Hohmann, and K . Szendrei, J . Chem. Soc.. Perkin Trans. I , 531 (1986). 57. L. Crombie, R . A. Fleming, W. M. L. Crombie, D. Toplis, and D. A. Whiting, J . Chem. Soc.. Perkin Trans. I , 1700 (1989); M . I. Begley, L. Crombie, W. M. L. Crombie, D. Toplis, and D. A. Whiting, J. Chem. SOC., Perkin Trans. 1, in press. 58. L. E. Codd, Borhalia 9, 123 (1966); L. E. Codd, Borhalia 10, 363 (1971). 59. A. E. Van Wyk and M. Prins, S. Afr. J . Bot. 53(3), 202 (1987). 60. J. W. Huffman and P. C. Raveendranath, J . Org. Chem. 51, 2148 (1986). 61. J. W. Huffman and P. C. Raveendranath, Tetrahedron 43, 5557 (1987). 62. J. W. Huffman, R. C. Desai, and G . F. Hildebrand, J . Org. Chem. 49, 982 (1984). 63. K. Sugiura, K. Yamada, and Y. Hirata, Chem. Lett., 579 (1975). 64. M. Pailer and K . Pfleger, Monatsh. Chem. 107, 965 (1976).

-CHAPTERC HISTOCHEMISTRY OF ALKALOIDS YOHEIHASHIMOTO, KAZUKOKAWANISHI, AND MOMOYO ICHIMARU Kobe Women's College of Pharmacy Motoyamakitu-machi, Higashinada-ku Kobe 658, Japan

.......

. . . . . . . . . . . . . . . . . . . 165 167 .............................. A. Papaveraceae 168 ............................................. 172 c. Leguminosae (Lupinus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 D. Rutaceae (Rue) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Apocynaceae (Periwinkle) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 111. Histochemical Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 A. Comparison of Histochemical Chromatography and Histochemistry . . . . . . . . 180 B . Microaspiratoscope ................................ 180 C. Apparatus for Autom .................................. 181 D. Macleuya cordata ................................... 182 E. Sanguinaria cana ................................ 190 F. Phellodendron amurense . . . . . . . . . . . . . . . . . 190 191 G . Kidney: An Application for Animal Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction . . . . . . . . . . . . . . . . . . . .

11. Histochemistry

.................

..............................

I. Introduction

Life is maintained by a good balance between structure and function. The external characteristics of plants, their parts, and organs are observed macroscopically to understand their structure. This is the morphologic approach. Plant morphology has helped us distinguish useful plants from toxic ones on the basis of the appearance of aboveground parts including flowers. Based on the detailed structures revealed morphologically plants have been classified systematically by the taxonomic method; however, the underground parts of plants have not been well characterized by the morphological method. Microscopic study enables us to discuss plant structures at the level of tissues and cells. This is the histologic approach. By these two approaches, taxonomic criteria began to include underground parts along with more minute structures such as fibers, glands, cortex, xylem, phloem, and medullary rays. Morphological and histological studies of plants have contributed not only to comparative study of plant species in taxonomy but also to biological, 165

THE ALKALOIDS. VOL. 39 Copynght Q 1990 by Academic Press. Inc. All nghrs of reproduction in any form reserved.

166

YOHEI HASHIMOTO

et al.

physiologic, and ecologic sciences. Genetics which subsequently flourished has promoted the understanding of morphogenesis and histogenesis. Crystals, oil droplets, liquids, and other cell contents in tissues and cells have been examined microscopically and chemically to determine their chemical components. This histochemical approach reveals not only their chemical components but also the chemical process occurring in tissues and cells of plants. Histochemistry is advantageous in investigating the distribution of chemical substances and their biological functions in plants. There are various methods to identify chemical compounds, such as fluorescent microscopy and autoradiographic microscopy by administering labeled compounds. The cytological approach to investigating cellular organelles such as mitochondria, Golgi bodies, liposomes, and plastids is aided by electron microscopy. These extensive methods have revealed fine structure, biological processes, and functions. Morphology and histology in combination with pharmacognosy, pharmaceutics, pharmacology, physiology, genetics, and chemistry have contributed much to distinguish useful plants from nonuseful ones, to develop more useful plants by modem hybridization techniques, and to obtain important organic chemicals, medicines, and dyes from various organs or the entire plant. Certain alkaloids manifest physiological activities in animals. They are distributed in certain species of plants, and the alkaloid content varies depending on species organs, age, growing stage, season, etc. The same plants sometimes contain different alkaloids in different organs. Quantities of alkaloids usually change according to life cycle stages such as germinating, growing, flowering, fruiting, and death, demonstrating that alkaloids are not waste products but metabolites. They do not seem to be predominant metabolites, however, as they constitute only a small portion of the total nitrogen contained in plants and are not found in all plants. Plant enzymes that have been isolated would be concerned in production of alkaloids by complex, sequential, and stereospecific reactions at particular locations in the plant body. Alkaloids, once formed, usually move through the plant and then accumulate in specific tissues and cells. Histochemical studies have revealed that alkaloids exist mostly in the cell vacuoles. Vacuoles are filled with cell sap and become enlarged with plant growth, thus occupying a majority of the space in many cells. The bigger vacuoles probably contain more alkaloids. Living cells are likely to contain more alkaloids than dead cells. Alkaloids are basic and form salts with acids, and in plants they are stored in the free state, usually as salts. Certain alkaloids form salts with certain acids, such as meconic acid, quinic acid, veratric acid, aconitic acid, and tiglic acid. Some other alkaloids are present in the plants as N-oxides, such as nupharidine, as glycosides such'as solanine, amides such as piperine, and esters such as cocaine. The alkaloids in the tissues and cells are detected as colored liquid, crystals, or precipitates by staining the section with alkaloid reagents and viewing with a microscope.

5 . HISTOCHEMISTRY

OF ALKALOIDS

167

The authors have developed a new technique to modify a conventional method of histochemistry, referred to as histochemical chromatography (HC) (1). After removing cell components such as crystals, oil droplets, and colored liquid from tissue sections with an aspiratoscope, chemical analysis is accomplished by gas chromatography (GC), high-pressure liquid chromatograpy (HPLC), and gas chromatography-mass spectrometry (GC-MS). The microscope we use for HC is equipped with an aspiratoscope to withdraw the components from the cells. It was originally developed by Yamamoto and Furusawa as an injectoscope to inject substances into cells (2) and later devised by Hashimoto. Histochemical chromatography is superior to histochemistry for determination of the composition of the compounds in cells in situ. Biosynthesis and localization of the alkaloids in plants have also been studied by HC after administration of isotopically labeled compounds into the plants (3,4). It is still unclear whether there are specific tissues and cells in which alkaloids are formed and stored. Alkaloids can be suggested to have the following functions: protection of the plant against insects and herbivores with poisonous agents, detoxification of harmful metabolites in the plant, regulation of the growth of the plant, and reservation of substances for supplying nitrogen and other elements. Light and electron microscopy, historadiography, and biological study by techniques for fractionation of cellular organelles, cell fusion, and cell culture have all promoted better understanding of plant alkaloids. Histochemical chromatography can be linked to these approaches. This chapter reviews various biological and biochemical investigations, using histochemical and other techniques, of plants from five families. We also describe our apparatus and methods for histochemical chromatography and its application to plant and animal tissues.

11. Histochemistry

Alkaloids form crystalline salts by reacting with acids, such as hydrochloric acid, sulfuric acid, and oxalic acid. They also form metallic salts with mercuric chloride, platinic chloride, and gold chloride. They precipitate with alkaloid reagents or alkaloid precipitants, such as phosphomolybdic acid, potassium mercuric iodide, picric acid, tannin, Dragendorff‘s reagent, Mayer’s reagent, and Reifer’s reagent. When alkaloids are reacted with these reagents, the products are often crystallized. The crystal forms and habits can be identified microscopically. In histochemistry, plant slices are treated with the above-mentioned reagents, and the crystals thus formed are examined microscopically for detection, identification, and localization of alkaloids in the tissues and cells.

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YOHEI HASHIMOTO

et al.

It is sometimes difficult and unpractical to detect alkaloids, as crystals, precipitates, or colors may be affected by the chemicals used for fixation and the alcohols used for dehydration. Montes et al. prepared frozen tissues to preserve the alkaloids in situ without any chemical changes (5). White and Spencer used freeze-dried tissues to study the location of alkaloids in plant tissues (6). Their freezing-drying method prevented alkaloid diffusion during sectioning. Proteins, amino acids, and other compounds in the tissues and cells of the plants, however, can be precipitated with alkaloid reagents. This is confirmed by adding alkaloid reagents to the tissue after extraction of the alkaloids with alcohols or acids (7,8). It is sometimes impossible to detect minute amounts of alkaloids among the excess of proteins in plant tissues and cells. James suggested that one drop of ether applied to the tissues could break down the cellular membrane to separate alkaloids from proteins (7). After removal of ether from the tissues, Bouchardat’s reagent is used to detect alkaloids as reddish cloudiness. The density of cloudiness in the tissues depends on the quality of alkaloids. Errera examined the distribution of alkaloids in plant tissues by histochemistry and found that alkaloids were present in active tissues near the vegetative points, ovule, epidermis and the layer just inside of it, hair, peripheral layers of fruits and seeds, vascular bundle, cork cambium, cork tissues, and latex tube (9). Molisch microscopically investigated 15 kinds of alkaloids as distinguishable crystal forms after treatment with acids or alkaloid reagents, and then histochemically examined them in plant tissue and cell sections following treatment with acids or alkaloid reagents (9). Tunmann and Rosenthaler observed histochemically the distribution of alkaloids in tissues and cells of 36 families of plants (10). A. PAPAVERACEAE Plants which belong to the family Papaveraceae contain latex tissues. The latex is sometimes in vessels, as observed in Papaver and Corydalis, and sometimes in latex sacs, as in Macleaya and Sanguinaria. The latex is a biological fluid containing alkaloids and proteins. The capsules and stems of Papaver somniferum contain opiate alkaloids essential in medicine. They are classified into two groups, phenanthrene types (morphine, codeine, thebaine) and benzylisoquinoline types [papaverine and noscapine(narcotine)]. These two types of alkaloids show sharply specific pharmacological properties. It is noteworthy that morphinane alkaloids are formed from (-)-(R)-reticuline, whereas most other alkaloids derive from (+)-(S)-reticuline (11). The laticiferous vessels in stems of P . somniferum were first illustrated by Leger (1895), and those of the capsule were studied by Tschirch and Oesterle (1900) and k d d e (1936) (12). Esau (1953) reported that the laticifers developed from single vertical files of cells. Horizontal end walls of these cells were

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absorbed, and subsequent lateral anastomoses occurred to join neighboring tubules (12). Fairbairn and Kappor found laticiferous vessels in the phloem of pedicles. These vessels had slightly thickened and highly refractive walls and were larger in diameter than the surrounding cells (12). They were stained deep blue with Calco-oil Blue in 50% alcohol and recognized by the granular contents. Laticiferous vessels were also found in young ovaries, sepal traces, hypocotyls, rootlets, and veins of the first foliage leaf. Laticiferous vessels were characterized as tubes formed by joining parallel elongated narrow cells, with their common end walls and adjacent lateral walls being absorbed. In sepal traces protuberances appeared as short bulges by joining up with corresponding bulges in the opposite tube. The granular contents in the laticiferous vessels became dense with growth of the plant. On transverse section of the pedicel, two kinds of laticifers, a convex arc and a concave one, were observed in the peripheral bundle and in the central one, respectively. The placenta traces (placentae) and the valve traces (valves) through the capsule wall appeared light microscopically as dense masses of branching vascular tissues in chlorenchyma within the epicarp. The former were fewer in number and smaller in diameter than the latter. The placenta traces contained laticifers but no fibers. The valve traces became the main source of latex. No laticifers were detected in the ovule; however, the terminals of the laticifers were observed near the junction of the placenta and ovule. Laticifers appeared in the phloem of the sepals and petals during blooming. Laticifers were detected in the stamen traces in the thalamus but not in the stamens. The ends of laticifers were detected in ovule traces. The diameters of laticifers in the ovary 2 weeks before petal fall were as large as those in the mature capsule. The presence of alkaloids in organs was examined by phytochemical and histological methods. No alkaloids were detected in extracts of stamens and seeds. The alkaloid reaction with Dragendoms reagent was examined at three stages during seedling development: the reaction was negative before (first stage) and after (second stage) the cotyledons opened; after the first foliage leaf appeared, the alkaloidal reaction became slightly positive. This was confirmed by histochemical methods. In other words, seedlings at the second stage stained red with iodine/potassium iodide, and this staining persisted even after treatment with 5% tartaric acid to remove any alkaloid, confirming that no alkaloids were present at the second stage. On the other hand, alkaloids were detected in seedlings at the last stage. Alkaloids were detected in sepals, petals, and capsules but not in stamens or seeds by paper chromatography. The localization of enzymes and alkaloidal metabolites in Papaver somniferum was studied by Roberts er al. by using latex from capsules and stems (13). The expelled latex from the cut end of the capsule or stem was centrifuged at 1000 g for 30 min. The pellet was then layered onto a continuous sucrose gradient. Two fractions sedimenting at 30 and 55-60% sucrose contained a

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soluble enzyme, which was readily released on organelle plasmolysis, and membrane-bound catecholase ( polyphenoloxidase), respectively. The latter also contained dopamine as well as alkaloids including morphine and thebaine. The latex of P . somniferum takes part in biosynthesis as well as accumulation of alkaloids enzymatically (13-16). Fairbairn and DjotC centrifuged the capsule and stem latex of P . somniferum at 1000-3000 g, and morphine synthesized from dihydroxyphenylalanine (dopa) was detected in the pellet in vitro (14). Roberts et a l . , however, observed dopa decarboxylase activity in the supernatant (13,17). Fairbairn et al. proposed that alkaloid biosynthesis and storage occurred in the heavy fraction of the organelles. They further suggested that the alkaloids were stored in the vacuole sap of the vesicles rather than being membrane bound, that the latex and vesicles were translocated into the capsule after petal fall, and that the metabolites passed out of the latex into the pericarp in ovules (15). Two types of vesicles were distinguished by electron microscopy, one with a capped structural zonation for biosynthesis of alkaloids and another with smooth or slightly granulated walls for alkaloid storage (16). The latex of P . bracteatum contains thebaine, which can be converted synthetically to codeine, a compound essential in medicine. Nessler and Mahlberg examined laticifers of P . bracteatum by electron microscopy (17). Laticifers were not detected in ungerminated embryos but were first detected among the differentiating vascular tissue of the elongating radicle associated with phloem about 72 hr after seeds were sown. Seedlings of P . bracteatum were morphologically similar to those of P . somniferum, but germination of the former occurred more slowly than that of the latter; consequently, the initial appearance of laticifers in the former was delayed. Thus, the alkaloid spectra that varied between them are due to unknown protoplasmic components rather than to structural differences. The laticifers filled with closely packed vesicles were distinguished from adjacent parenchyma in which a large central vacuole developed and adjacent sieve tube members with parietal cytoplasm. Vesicle caps which appeared in growing laticifers decreased in number after maturation as vesicles were enlarged, irregularly shaped, and packed closely. Enlargement of laticifers may occur with simple fusion of several vesicles or with their growth in size by derivation of endoplasrnic reticulum. Fairbairn and Williamson studied P . bracteatum anatomically and compared it to P . somniferum (18).They found the two plants to be very similar in structure. The laticifers of P . bracteatum were usually more closely packed and anastomose more frequently. The subcellular fraction of protoplasts from cultured P . bracteaturn cells (organelles sedimenting at 1000 g ) was the major site where thebaine and sanguinarine accumulated (19). It also contained dopamine as a precursor and the vacuolar enzyme a-mannosidase. Dopamine also appeared in the supernatant. Dopamine compartmentalization in vacuoles of cultured cells was observed by histofluorescence microscopy. Dopamine, sanguinarine, and thebaine occurred in vacuoles of different densities. This result is consistent with

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the concept that in vacuoles within the same plant, heterogeneous populations exist which play specific roles for alkaloid synthesis and storage. This is also consistent with differences of optimum pH for the different enzymes, subcellular localization, and uptake of thebaine and sanguinarine into vacuoles. Verz6r-Petri and Hoang studied the anatomy of tubers, stems, leaves, flowers, and green and ripe fruits of Corydulis cava and examined the localization of alkaloids (20). Idioblasts containing alkaloids in brown secretions were observed in the cortex of tuber. In the stem, vessels with brown-colored secretions were present in the phloem part and trachea with brown substances in the wood part. Intercellular areas of the parenchyma with mucous secretion were observed in the ground tissue of the stem. Secretory vessels containing brown cells were observed sporadically in ground tissues and in the vascular sheaths of leaf. The secretory vessels occurred individually or in groups of three to four cells. The distribution of alkaloids was confirmed by using Dragendoms reagent and a bromic reagent (4-bromobenzenediazonium tetrafluoroborate). Treatment with 5% tartaric acid to remove alkaloids was performed in parallel sections. In C . cava alkaloids were localized within the cell wall. The intercellular areas and secretory vessels in the parenchyma of the ground tissue were the places where alkaloids were transported and accumulated, respectively. Cell walls of the tracheas of the vascular tissues, secondary cells occurring in the bast part and bast parenchyma, can be considered to accumulate as well as to form alkaloids, owing to tissue activity, the presence of living plasma in the elements, and their role in transportation of several organic materials. Alkaloid cells filled with yellow liquid were formed in sterile tissue cultures of Mucleuyu cordutu (21). The location of protopine, sanguinarine, and allocryptopine mainly in the alkaloid cells was revealed by light and electron microscopy. Radioactive alkaloids were observed in the cells by electron microscopic autoradiography after administration of [3H]phenylalanine. The latex in the stem was filled with yellow to orange-red liquid, the color being dependent on the kind and amount of alkaloids. Neumann and Miiller also produced alkaloids in callus cultures of Macleaya (22). The amount of alkaloids produced was reciprocally related to the growth rate of the culture. It was shown by autoradiography that alkaloids accumulated in the region of the leukoplasts after application of labeled precursors. Neumann and Muller also studied Chelidonium majus and Sunguinaria canudensis (23). In leaves of C . majus the alkaloids were present beside the small vacuoles and along the cell walls of the latex vessels. Leaf tissues of C . majus accumulated [3H]protopine supplied exogenously. About 70% of the alkaloid was bound within thickenings of the xylem vessels 1 hr after feeding. Three hours after feeding, the thickenings of the xylem and latex vessels contained radioactive protopine, In rhizomes of S. canadensis isodiametric cells containing alkaloids were observed as rather large cells (150-200 pm), colored with sanguinarine. Alkaloids were demonstrated by fixation with glutaraldehydel

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chloroplatinic acid/osmium tetroxide to be present inside the central vacuoles. Autoradiography revealed that the alkaloid cells contained 4 times as many silver grains per unit area as the neighboring cells. The cell walls were radioactive, too. B . SOLANACEAE

The family Solanaceae is one of the important and interesting plant families. It may be classified into four groups based on their usefulness and chemical composition of alkaloids contained: (1) Nicotiana spp. such as N . tabacum and N . rustica, which provide tobacco and contain nicotiana alkaloids such as nicotine and nornicotine; (2) Hyoscyamus, Scopolia, Atropa, and Datura species, which are pharmacologically important and contain tropane alkaloids such as hyocyamine, scopolamine, and tropine; ( 3 ) Solanum spp., which are sometimes the starting material for production of synthetic steroids for medical uses and which contain solanum alkaloids; and (4) Capsicum, Solanum, and Lycopersicum species, which serve as foods and are alkaloid-free. Atropa belladonna has been investigated histochemically since the beginning of the twentieth century (24). James reviewed the distribution of alkaloids in Atropa belladonna and their variation according to the developmental stage (24). The alkaloids in A . belladonna were located mostly in young and soft stems. The alkaloids were found more frequently in undifferentiated cells at stem apexes and less in the newest cells. With aging, cells became lignified and alkaloids decreased and finally disappeared from the vascular strands and from cells in the pith. After complete differentiation alkaloids were present in xylem parenchyma and medullary rays. The epidermis and outer cortical layers just below it, the parenchyma within and adjacent to the phloem, and the periphery of the pith just inside the xylem contained alkaloids abundantly. In the leaf blade alkaloids were found in the epidermis and parenchyma adjacent to the phloem strands along the veins. The epidermis, root cap, and new tissues just behind it in young roots were sites of alkaloid accumulation. Alkaloids localized in the central tissues moved to the piliferous layer and outer cortex, parenchyma around the outer phloem, and the periphery of the pich. As the plant grows older, alkaloids in the root were distributed more in parenchyma cells in the outer tissues, the vascular parenchyma and phellogen, and young cork derived from it. Alkaloids accumulated in the epidermis of the calyx and corolla. Alkaloids in the anthers were found in the epidermis, the tapetal layers around the developing pollen sacs, and the bundle sheath of the filament but not in the vascular bundles. Alkaloids accumulated in all parts of the ovules, but they decreased during development of the ovules into seeds, remain only in a single layer inside the testa. The fluorescent color arising from alkaloids located in plant cells may be observed under a microscope by irradiation of tissue sections with UV light (365

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nm). The colors correspond to the authentic alkaloids in solution irradiated with the same light (25).Solanum aviculare and S. tuberosum, which contain solanine, were examined by this method (25).Solanine was distributed in the cork and gave a bluish white fluorescence on fluorescence microscopy. Hyoscyamine contained in Hyoscyamus niger was also revealed as bluish white in color (25).Localization of alkaloids in seeds and fruits of Datura innoxia was also examined by fluorescence microscopy (26). Scopolamine, hyoscyamine, norhyoscyamine, and metholidine were present in all parts of the seeds, especially in the membranous parenchyma of the seed cover. Alkaloids decreased in the seeds during ripening and disappeared from the seed epidermis. In the fruits, alkaloids were observed in the calyx in significantly large quantities and in pedicels in small quantities. James examined the production of alkaloids in germinating seedlings of three species of Datura (0.stramonium, D. stramonium var. inermis, and D. tatura)by using ether for accurate detection of alkaloids in the tissues (5). Roots 1-2 mm in length contained no alkaloids, roots 3 mrn in length contained a slight amount of alkaloids, and those longer than 3 mm contained alkaloids conspicuously. Cloudy alkaloid reactions were observed on the surface of the root just behind the root cap, owing to its meristematic properties, but not on the root cap or the elongation zone. The smallest apex and rudiments of the first two layers reacted positively for alkaloids, but the cotyledons and the hypocotyl did not. The roots had more alkaloids than the shoot apex in the three varieties of Datura, whereas Atropa belladonna seedlings with six leaves showed reverse results. From these findings it was concluded that alkaloids were very rapidly synthesized by cells during the phase of active metabolism and growth. VerzBr-Petri examined the distribution of alkaloids in stems and roots of Datura innoxia under a microscope by using Dragendoms reagent, Mayer’s reagent, picric acid, and platinium chloride, and they also assessed alkaloid deposition points in the tissues under a microscope after injecting isotopic compounds into seedlings (27). With the former method, alkaloids were observed as huge crystals in parenchyma of the pith of stems and roots. Histoautoradiograms obtained following [3H]atropine injection showed the radioactivity concentrated in the heads of glandular hairs, the epidermis of the entire seedling, and in midribs of the leaves. After incorporating [3H]atr~pine,both radioactive and nonradioactive alkaloids were detected in the primary bark, which showed that atropine (1-hyoscyamine) was already stored there and also suggested the excretory nature of alkaloids. Injection of sodium [3H]acetate into young shoots revealed the distribution of alkaloidal crystals in the root bark, stem hypocotyl, and also in palisade parenchyma of the cotyledons. Radioactive compounds were demonstrated in the epidermis and hypodermis of roots of young shoots by histoautoradiograms. Radioactive crystals were also observed. In cell walls and intercellular spaces of the parenchyma, strong radioactivity was observed, indicating that the radioactive alkaloids appeared as elements in fundamental tissues.

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By incubation of plants with 3-~~-[N-~~CH~]tropylteloidin, Verzir-Petri found radioactive 6-hydroxyhyoscyamine and 7-hydroxy-3,6-ditigloyloxytropane(27). Following incubation for 2 more days meteloidine was activated. Ditigloylteloidine might not have been produced in the root. In teloidin metabolism in D . innoxia, the time required for esterification of tiglic acid was different from that required for decomposition of the hydroxyl group. Study showed that the acid part of teloidine had affinity for tiglic acid. No radioactive scopolamine was formed during the 3-day experiment, suggesting that a longer time may be required to build scopolamine or that teloidin may not be a precursor of scopolamine. In the leaf mesophyll more active compounds were found in the palisade tissues than in spongy tissues. The radioactive alkaloids were transported in xylem and also in phloem in the stem. Montes et al. investigated alkaloid localization in situ in Nicotiana glauca by preparing frozen tissues (7). The vegetal tissues in various parts of the plant and at various stages of growth of the same plant were prepared by a freezing substitution procedure to examine the distribution of alkaloids without any disturbance in situ (7). The plants were collected at 1.5, 3, and 7 months and 1 year after seedling germination, which were called the first, second, third, and fourth stages, respectively. Alkaloids were found in stomatic cells of the leaves at the first and second stages; in the first stage especially stomas were almost occluded with alkaloids. In leaf chloroplasts alkaloids were detected for an entire year after seedling development. The amount detected was conspicuously large at the second stage but very small at the third stage. The leaves were not quite hairy at the first stage, but alkaloids were detected in any hair. At the second stage alkaloids were contained in the small glandular heads of hairs, but at the third stage no hairs existed. Conductive root tissues were found to contain alkaloids through 1 year, abundantly at the third and fourth stages judging by analysis with alkaloid reagents. Cellular membranes that appeared greatly thickened during the elapsed time were highly stained with alkaloid reagent. Stems and limbs were not found to be separated at the first stage. The stems at the second stage were still less woody, and alkaloids were detected in chloroplasts and phloem cells. At the third stage xylematic rays, phloem cells, and membranes of the cortical cells in several zones showed positive alkaloid reactions in cross sections. At the fourth stage alkaloids were detected in stomas, epidermal cells, one or two layers of palisade cells directly under the conductive vessels, and in membranes of cortical cells next to the central cylinder. To investigate the localization of secondary plant products, organelles such as vacuoles have been isolated by dilution of protoplasts obtained by incubating the tissue in digesting solution (28). Discontinuous Ficoll gradients may be used to produce a large number of vacuoles. It was demonstrated that the vacuole was the site of accumulation of cyanogenic glucosides in Sorghum. Saunders applied this technique to tobacco leaf (N. rustica) (29). Protoplasts prepared from leaf

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tissues were suspended in buffer and loaded on top of a four-step discontinuous gradient consisting of Ficoll in buffer. After centrifugation, mature intact vacuoles which banded at the Ficoll interface were collected. Nicotine, known to accumulate in the leaf, was found in the vacuoles at high concentration. This result corresponded well with findings on alkaloids in vacuoles in Chelidonium majus (30) and cyanogeic glucosides in vacuoles in Sorghum bicolor (28). The vacuole is the site of accumulation of several kinds of secondary metabolites. Grating is one of the techniques to determine which plant organs produce alkaloids. In the Solanaceae species from different genera, either containing different alkaloids or being alkaloid-free, can be combined relatively easily by grafting. Waller and Nowacki reviewed the production of alkaloids by grafting in the Solanaceae (31). It was reported when Nicotiana rustica or N. tabacum was grafted on potato or tomato, no alkaloids or considerably reduced amounts of alkaloid were found in the scions. Nicotiana alkaloids with pyrrolidine rings can be synthesized predominantly in the root, whereas anabasine alkaloids with piperidine-type rings can be produced in the roots and aerial parts of the plant. However, contrary results were obtained in that the tobacco scions produced some nicotine when grown on tomato. It was demonstrated that nicotine was formed predominantly in the root from the many available precursors. In all grafts between plants producing nicotiana-type alkaloids and those producing tropane alkaloids, the alkaloids were accumulated in the root. C. LEGUMINOSAE (Lupinus)

Waller and Nowacki described grafting in Lupinus (32).Lupinus albus, bitter and sweet varieties, L. angustifolius, bitter and sweet varieties, L. pilosus, L. mutabilis, L . arboreus, L . hartwegi, and L. polyphyllus, all bitter varieties, were examined by grafting. The alkaloid content in bitter varieties was 1-2%, whereas that in sweet varieties was 0.03-0.11% (33). When a bitter scion was grafted on a low-alkaloid stock or on a low-alkaloid stock with a side shoot, the shoot produced alkaloids at the beginning of the experiment. After the two kinds of grafting, plants were later found to contain alkaloids in the whole body as normal plants. The part of the plant growing from the side shoot was transformed into a bitter shoot. On the other hand, grafting of either a low-alkaloid scion on bitter stock or on one with a side shoot containing alkaloids, produced plants having alkaloids in all parts except the shoots at the beginning of the experiment. Later, a small amount of alkaloids was detected in the former shoot, but in the latter grafting alkaloids were detected in whole the plants, with fewer alkaloids in the main inflorescence. These experiments showed that lupine alkaloids are synthesized and accumulate in the aerial parts of the plant. Staining with Dragendorff's reagent and Reifer's reagent revealed alkaloids near the outer surface of green stems of Lupinus luteus. A smaller amount of

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alkaloids was found in the vascular tissues; very few alkaloids in the cortical tissues and none in the root tissues were observed (8). Plants growing in light accumulated more alkaloids in the stems around the outer surface than plants growing in the dark. Although some alkaloids were present in the stem near the outer surface, most alkaloids were distributed in young leaves, but the apical bud did not contain much. No alkaloids existed in the epidermis. The palisade mesophyll tissues in green cotyledons contained higher concentrations of alkaloid than spongy mesophyll tissues. These results confirmed that lupine alkaloids are synthesized only in green parts of the plants and increase only during the period when plants are illuminated. Alkaloid metabolism in lupine was proved by Wink and Hartmann to be associated with chloroplasts (34). A series of enzymes involved in the biosynthesis of lupine alkaloids were localized in chloroplasts isolated from leaves of Lupinus polyphylls and seedlings of L. albus by differential centrifugation. They proposed a pathway for the biosynthesis of lupanine via conversion of exogenous 17-oxosparteine to lupanine with intact chloroplasts. The biosynthetic pathway of lupinine was also studied by Wink and Hartmann (35).Two enzymes involved in the biosynthesis of alkaloids, namely, lysine decarboxylase and 17oxosparteine synthetase, were found in the chloroplast stoma. The activities of the two enzymes were as low as one-thousandth that of diaminopimelate decarboxylase, an enzyme involved in the biosynthetic pathway from lysine to diaminopimelate. It was suggested that these differences are not caused by substrate availability (e,g., lysine concentration) as a critical factor in the synthesis of alkaloids. Feedback inhibition would play a major role in the regulation of amino acid biosynthesis but not in the control of alkaloid formation. Lupine alkaloids localized in stems of Lupinus polphyllus were investigated by Wink er al., who employed laser microprobe mass analysis with a laser desorption mass spectrometer (LAMMA 1000) (36). The LAMMA is an instrument combining a laser microscope for specimen observation, which focuses a UVpulse laser beam onto the specimen, and a time-of-flight (TOF) mass spectrometer for analysis of positive and negative atomic and molecular ions (LMMS). In the analysis, the magnification of the light scope was 250x, and the laser focus had a diameter of 2-3 Fm. Lupine alkaloids were found to be stored in peripheral cell layers, which corresponded well with Jacquemin’s histochemical results. This report showed that peripheral cells were protected from attack by microbes or herbivores and that the alkaloids were chemical defense compounds. The LAMMA technique facilitates accurate quantitative analysis as well as qualitative analysis in histochemical studies, even without the need for extractions. This is advantageous in conventional histochemistry by allowing direct staining of alkaloids in the tissues. However, the following points must be noted. Only high local concentrations of organic compounds (usually 5 mmol/kg) and intensive molecular ions in the electron-impact mass spectrum with molecular

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weights higher than 100-150 mass units can be detected satisfactorily. Thus, glycosides and esters and compounds with molecular weights less than 100-150 mass units are poorly detected. Lupine alkaloids were revealed to be accumulated in epidermal tissues of leaves, petioles, and stems of Lupinuspolyphyllus, L . albus, and L. mutabilis by staining with potassium iodide/iodine solution (37). In leaves, dark brown precipitates were observed in the epidermal tissue layer whereas lighter brown precipitates were present in mesophyll and phloem cells. The first subepidermal cell layer and phloem cells in stems were positively stained in bitter, alkaloidrich ecotypes but less positively in sweet varieties. These results were confirmed by GC analysis on each part of the plants. Extracts from the epidermis of sweet varieties were almost free of alkaloids. On the other hand, extracts from nonepidermal tissues of petioles of L. polyphyllus were relatively rich in alkaloids, because alkaloids were transported to phloem tissues. Wink had previously reported that alkaloid storage cells were acidic, and he subsequently studied their acidic nature in Lupinus by staining with methyl red. Red-stained cells were observed in the epidermis of leaflets of L . polyphyllus and in stems of L . albus. The epidermis of stems of sweet varieties stained red with methyl red and less positively with potassium iodide/iodine. Thus, it is assumed that the acidic nature of the epidermis is essential for strong alkaloid retention, and the free base forms are used for transporting alkaloids across the tonoplast membrane. However, Wink found that the pK, value of lupine alkaloids (e.g., sparteine) was more than 11.8 and that of the free bases only 0.17 and 0.02% at pH 9 and 8, respectively. Therefore, nearly 100% of the alkaloids would be present as charged molecules in epidermal as well as nonepidermal cells. Wink and Mende further studied uptake of lupanine into the epidermis as lupine alkaloids are synthesized mainly in the green mesophyll, then transported into the epidermal tissues (38).They suggested that the alkaloids would be transported with the aid of special carrier proteins. D. RUTACEAE (RUE)

Genus Ruta contains acridone alkaloids, which are lipophilic and occur as droplets or as amorphous grains. They are usually weak bases, which do not form picrate or perchlorate salts. The methoxy group in the acridine ring is easily demethylated in acid (39). However, the alkaloids are fluorescent, emitting a bright yellowish color following excitation with UV light (40).Verz&-Petri et al. utilized these fluorescence characteristics for histochemical studies in rue, Ruta graveolens (41). They prepared freeze-dried tissues and detected alkaloids by the fluorescence arising from excitation at 400-420 nm. Acridone alkaloids were observed as droplets or grains because of their lipophilic nature. They were localized in calyptra of the root cap on longitudinal sections of roots a few

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months old. They were also scattered in rhizodermic cells and intensively accumulated in hypodermic cells and parenchymatous cells in the root cortex. In roots 1-3 years old, acridones were localized in the meristematic cambium, in many cells of the secondary xylem, and in cells of the pith. Location of the fluorescence in the rhizomes of old roots was similar to that in the young ones as described above. Yellow fluorescence was observed in inner cells of hypodermis, which consisted of many layers under the epidermis, and also in the primary cortex. Strongly fluorescent alkaloids were detected as grains on idioblasts in the center of the roots. In the stems acridones were accumulated in differentiated cells within the epidermis. Primary cortex contained fewer acridones, and medullary rays and pith did not contain any. Chloroplasts in leaf palisade tissues showed a strong reddish fluorescence because of chlorophylls. Homogeneously granulated acridones were distributed in the cell walls of fibrous cells and the intercellular space near parenchyma cells in the xylem inside the leaves. Eilert et al. also examined roots of R . graveolens using light and electron microscopy to detect acridones by the fluorescence arising from excitation at 330-500 nm and by staining with permanganate (42). They found fluorescent droplets in the root hairs, especially toward the tips. A conspicuous cluster of vacuoles in the root were identified electron microscopically as acridone alkaloid-containing cells by fixation with glutaraldehyde-osmium. The idioblasts distributed in the outer cortex of young roots were visible as cells containing fluorescent droplets and cells stained with permanganate. These cells were easily distinguished from cortical cells with vacuoles by their color, refraction, and electron densities. The idioblasts also differed from the surrounding parenchyma cells because the many vacuoles were scattered and variable electron-dense precipitations were observed in the idioblasts. In cell cultures of roots, idioblasts identical with those in the original plants were detected. Some acridone alkaloids have been reported to have strong antimicrobial activity (43). Elilert et al. added fungal elicitors to culture suspensions to increase alkaloid accumulation without affecting the structure of the idioblasts (44). They proposed that acridones were defensive chemicals.

E. APOCYNACEAE (PERIWINKLE) Periwinkle, Carharanthus (Vinca) roseus contains the pharmacologically important indole alkaloids vinblastine and vincristine, which are now available commercially. Vinblastine sulfate and vincristine sulfate are used for the treatment of generalized Hodgkin's disease and chorionepithelioma and for the treatment of leukemia in children (45). Vinblastine and vincristine were detected with Dragendorffs reagent, iodinelpotassium iodide, and ceruric ammonium sulfate as well as by fluorescence (6). Jaffrey reagent was applied to purified vindoline on chromatograms

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and in aqueous solutions to form a red reaction product. Jaffrey reagent was added to stem sections to produce a red color reaction in protoplasts of laticifers and certain parenchyma cells. Alkaloids were not found in very young shoots or root apexes but increased with age until positive parenchyma cells appeared about 20% in the parenchyma cells in old stems and roots. The color reaction in the cells gradually became strong. The greatest intensity was observed 10-15 min after reaction and then faded in 30 min. The Alkaloid content of various organs of this plant was examined by the color response with Jaffrey reagent and ceric ammonium sulfate. The content was dependent on the age, location, and type of cells. Laticifers in mature embryos were unreactive 4 days after germination. This result corresponded to a report that the seeds contained no alkaloids (46). Laticifers in seedling hypocotyls and cotyledons up to 4 weeks old, except for first 4 days, showed a blue-green color response, whereas other cells showed no response. All laticifers in plants older than 4 weeks showed a red precipitate, and a few short laticifers and parenchyma cells in callus formed at the ends of cuttings which were more than 2 years old showed a blue-green reaction. No difference in laticifers of immature and more mature plants was observed. In roots the presence of reactive laticifers was associated with vasculature of the secondary tissue and in stems with internal and external phloem as well as ground meristem of the pith and outer cortex. Laticifers in the leaf, corolla, style, and ovary of flowers all reacted with Jaffrey reagent. Positive reactions in the laticifers and certain parenchyma cells were also observed with other alkaloid reagents described above, which corresponded to the reactions with Jaffrey reagent. Fats and lipids in laticifers and certain parenchyma cells were visualized with Sudan IV, Sudan black, Nile blue, and osmium tetroxide. No starch or other polysaccharides were detected by iodine/potassium iodide, periodic acid-Schills reagent, and an aniline blue fluorochrome procedure under a fluorescence microscope. Eilert et ul. observed laticifers in fruits of Catharunthus roseus as cells filled with droplets, using light and electron microscopy (47). Their method to analyze alkaloids was as follows. The excluded latex in fruits was collected on a glass rod and immersed in a mixture of ethyl acetate and methanol. The latex suspension was centrifuged, and the supernatant was reduced. The drained fruits were extracted and analyzed by chromatography. The cytoplasm of laticifers and neighboring parenchyma cells showed various degrees of degradation of organelles, which affected the nuclei, plastids, and mitochondria. The latex contained electron-dense droplets and some vesiculated cytoplasm interspersed with highly degraded organelles. In comparisons of alkaloids in the excluded latex versus drained fruits, the former was found to contain prominent amounts of vindolinine and 19-epivindolinine and a trace of stricosidine lactam and the latter, strictosidine lactam and considerable amounts of vindolinine and 19-epivindolinine. It was concluded that the latex is a storage compartment only for certain alkaloids. Alkaloids in the latex and fruits are differentially accumulated.

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111. Histochemical Chromatography

A. COMPARISON OF HISTOCHEMICAL CHROMATOGRAPHY AND HISTOCHEMISTRY In histochemistry localization of alkaloids in plant tissues and cells is recognized by microscopically observing crystals, precipitates, or colored materials that are formed or stained when acids or alkaloid reagents are added to tissue slices. The alkaloids localized in tissues and cells may be pure or a mixture of related compounds. In order to determine the composition, the alkaloids are usually obtained by extraction from large portions of the plants, not from the local tissues and cells because amounts there are too small to analyze. Alkaloids might be changed, however, during the extraction process. By means of HC such problems can be solved. When cells containing alkaloids are distinguished from cells containing no alkaloids microscopically without any reagents, alkaloids in the cells can be picked up as being intact and their chemical constituents analyzed immediately. Even when cells containing alkaloids cannot be distinguished from other cells, the cell contents may be aspirated freely and analyzed afterward. Consequently, cells with and without alkaloids could be distinguished. B . MICROASPIRATOSCOPE A micromanipulator was first made by Schmidt in 1859 to manipulate an amoeba under a microscope (2). Since then, several microscopes having micromanipulation or microinjection apparatuses have been developed, but they all use micromanipulators (2). Yamamoto and Furusawa developed a microinjectoscope to inject proteins, genes, etc. into cell cytoplasm and nuclei without using a micromanipulator (2). They rebuilt a condenser with a perpendicular hole into which a glass micropipette was inserted. The hole was made through the optical axis of the condenser lens. There was no significant difference in illumination of the visual field between the conventional and modified condensers. Hashimoto converted the injectoscope with the modified condensers to an aspiratoscope in order to aspirate target materials in plant tissues and cells for histochemical chromatography (Fig. 1) ( I ) . The tissue slices are first focused by an objective (lo-, 20-, or 40-fold magnification) and a binocular lens (10-fold magnification). The condenser is moved upward until the tip of the capillary is seen in the visual field. The target in the cells to be aspirated is positioned under the tip of the capillary by moving the horizontal stage. The vertical position of the tip of the capillary, which is seen at almost the center of the visual field, is confirmed with a side scope by moving the condenser downward until the tip approaches very near to the surface of the slices on the stage. The horizontal stage should be moved carefully to avoid breaking the tip of the capillary by touching the slice.

5.


181

FIG. I . Suction apparatus of the rnicroaspiratoscope.

When the tip of the capillary is attached to the target by adjusting the screw to control vertical movement, the target is aspirated through the capillary by a micropump and collected in a microtrap. Then the condenser is moved upward. The procedure is repeated until the amount of target material, such as cell contents, collected in the microtrap is sufficient for analysis by chromatography or GC-MS.

c. APPARATUS FOR AUTOMATIC ASPIRATION In order to facilitate repeated aspiration, an automatic system for the process was required (4). A Braun tube was connected to the aspiratoscope through the camera, so that the operator can handle the microscope by watching the Braun tube instead of looking through the ocular of the microscope. In this automatic system, determination of the position of the capillary tip and aspiration of the cell contents can be operated with buttons on the controller (Fig. 2). There is another button on the control panel to operate a cursor, which moves on the Braun tube. A joy stick circuit (“table JS-l”), moves the stage to eight horizontal levels at variable speeds controlled with the “table speed VR 1” button. Another joy stick circuit, “cursor JS-2”, moves the cursor on the Braun tube to eight horizontal levels at variable speeds controlled with “cursor speed VR 2”. The capillary tube is moved vertically by the “up/down” switch at variable speeds controlled with “capillary speed VR 3”. In the first procedure, the tip of the capillary should be adjusted to coincide with the cursor on the Braun tube by operating JS-2. Positionings of the tip of the capillary to be placed slightly above the slice for home set and the lower level for

182 tablespeed

YOHEI HASHIMOTO

et al. capillary speed

c u r s o r speed

@o

0

home s e t

0

(sec. 1

cont

0 asijlration

timer

FIG. 2 . Control panel for the microaspiratoscope.

aspiration are determined with the “home set” and the “lower set” buttons after adjusting with up/down switch, respectively. The target in the tissue for analysis is attached to the cursor by operating JS- 1. Aspiration of the target at the definite position is completed by pushing the button “aspiration”. The aspiration speed can be set with “timer (sec)”, when “cont/timer” is switched to timer. After aspiration, the capillary tube is automatically moved back to the home set position. D. Macleaya cordata

Macleaya cordata (Papaveraceae) contains alkaloids in alkaloid cells filled with yellow to orange-red liquid (21). Alkaloid cells were observed as bluish yellow fluorescence in transverse and longitudinal sections of roots of M . cordata with a fluorescence microscope (48). Four main alkaoloids are known to be present in roots of M . cordata, namely, sanguinarine and chelerythrine, which are benzo[c]phenanthridine alkaloids red and yellow in color, respectively, and protopine and allocryptopine, which are colorless protopines. The liquid from colored cells in M . cordata roots was aspirated into the microtrap under a microaspiratoscope. The liquid remaining in the capillary and the connector tubes between the capillary and microtrap was washed into the microtrap with the solvent used for HPLC analysis. It was shown that the liquid of colored cells contained all four alkaloids, judging by comparison with authentic samples on HPLC. Therefore, HC proved the colored cells to be the alkaloid cells named by Neumann and Muller. The red-colored cells were located primarily in the center (pith) of the root, and the orange-colored cells were found primarily near the cambium. There were orange-red colored cells between the cambium and center. The red cells, orangered cells, and orange cells contained more sanguinarine, equal distributions of colored and colorless alkaloids, and less sanguinarine but more protopine, re-

5.

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spectively (Table I) (48). Only the ratios of the four alkaloids listed on Table I were calculated from the areas of each peak on the HPLC chromatogram. Peak areas for the benzo[clphenanthridine-type alkaloids were greater than those counted for protopine type alkaloids because of stronger absorbances at 254 nm. Therefore, the sanguinarine and chelerythrine contents in Table I exceed the true contents. The alkaloid contents in a single alkaloid cell from M . cordara roots of three different thicknesses (age 1-2 years) were determined as follows. The liquid from various numbers of alkaloid cells (Table 11) was removed uniformly and collected in the microtrap. The liquid in the capillary and connection tubes was washed into the microtrap to make a certain definite volume for analysis. Quantitative analysis of each alkaloid was carried by preparing a calibration graph (4). Figure 3 shows the HPLC chromatogram of alkaloids from the alkaloid cells. The content of each alkaloid per single alkaloid cell in tissues from three different thickness of roots and its ratio are shown in Table 11. The liquid in colorless cells contained only a minute amount of protopine and allocryptopine (Fig. 3). The thicker the roots, the more alkaloids were contained in a single alkaloid cell. In any thickness of root, the content of protopine-type alkaloids exceeded that of benzo[c]phenanthridine-type alkaloids. The ratio of the former to the latter was almost steady over 5 mm of root thickness (86-87%). The ratio of alkaloids in methanol extracts of the same fresh samples (thickness -5 mm) was determined by HPLC (Table 111). The ratio of protopine-type alkaloids in the methanol extracts (-80%) was less than that in the liquid from the alkaloid cells (-87%). This was because the liquid in alkaloid cells near the cambium were picked up more than that in center cells (pith). Thus, intracellular components scattered in different places are analyzed qualitatively and quantitatively in situ by HC. The sequence of alkaloid biosynthesis in M . cordara was reported as shown in Scheme 1, and it was proved in both intact plants and callus cultures (49). Histochemical chromatography was applied for examination of the biosynthetic

RATIOOF FOURALKALOIDS IN

TABLE I LIQUIDOF ALKALOID CELLS OF Mucleuyu Cordutu ROOT^' THE

Color of cells Alkaloid Protopine Allocryptopine Sanguinarine Chelerythrine a

,I

Red (n = 1 1 ) 19.7%b 10.1% 41.9% 28.3%

Orange-red (n 28.8% 13.8% 34.8% 22.6%

Diameter of root 15 mm. Calculated by peak area on the HPLC chrornotogram.

=

4)

Orange (n = 9) 32.9% 17.0% 26.4% 23.8%

184

YOHEI HASHIMOTO

et al.

TABLE I1 ALKALOID CONTENTA N D RATIOPER SINGLECOLORED CELLI N Macleaya cordata ROOT Root diametep

-3 mmb

5-7 mm"

-14 mmd

Amount (ng)

Ratio

(%I

Amount 0%)

Ratio

(%)

Amount 0%)

Ratio

Alkaloid Protopine Allocryptopine Sanguinarinee Chelerythrinee

9.7 11.4 0.6 0.4

43.9 51.6 2.1 1.8

16.0 26.3 3.2 2.9

33.1 54.3 6.6 6.0

25.9 40.6 4.9 5.5

33.7 52.8 6.4 7. I

Q

b c

d

e

(%)

Root age 1-2 years. Average of 1000 cells (n = 5). Average of 2800 cells (n = 14). Average of 2000 cells (n = 10). As the chloride.

(a)

b

d C

.:

I

1;

0

A I . 0

5

L

10 15

(min )

0

5

10 15 (min)

FIG. 3. HPLC chromatograms of cell liquid in Macleaya cordata root. Column: NOVA-pack C i 8 X 10 cm; eluent: 0.1 N tartaric acid (containing 0.125% sodium dodecyl sulfate)acetonitrile, 45 :55 at 2.0 ml/min; detection: UV, 285 nm. (a) Alkaloid (colored) cell. (b) Colorless cell. Peaks: a, protopine; b, allocryptopine; c, sanguinarine; d, chelerythrine. ( 5 ~ ) 8. m m

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TABLE 111 ALKALOID CONTENTA N D RATIOOF FOURALKALOIDS I N METHANOL EXTRACTS OF FRESHROOTSQ OF Macleayu cordata Alkaloid

Amount (mg)

Ratio (%)

2.3 3.1 0.7 0.6

43.3 46.3 10.4 9.0

Protopine Allocryptopine Sanguinarineb Chelerythrineb a b

Root age 1-2 years, weight I . I g, diameter 5 mm As the chloride.

sequence (I1 + I11 + IV) after administration of labeled compounds to the whole plant or root pieces (3) (50). To examine the sequence I1 * 111, two groups of whole plants of M. cordutu were used, namely, ones grown under hydroponic culture in the presence of cis-[N-C2H3]tetrahydroberberiniumsalt and reference plants grown under hydroponic culture but without the radioactive compound. Every few days after administration, the liquid in alkaloid cells of the root was removed by a microaspiratoscope and subjected to mass fragmentography (MF). Four peaks, namely, at mlz 369 (M+ for allocryptopine), 370 ([M 1]+ for allocryptopine), 372 ( M I for [N-C*H,]allocryptopine), and 373 ([M + 11 for [N-C2H,]allocrypto-

+

+

Rl+5=C%

(-)-Tetrahydrocoptisine

I

tetrahydroberberinium salt

L

L

R1+ %= Cl$ (-)-Cis-N-Methyltetrahydrocoptisinium salt

@ c R1 =

Chelirubine

%=C 5

Allocryptopine R,

+ %= C 3

P r o t o p in e

SCHEME1 Biosynthetic sequence for the alkaloids of Macrleuvu

cordata.

186

YOHEI HASHIMOTO et

al.

pine) were monitored. Alkaloid cells of roots fed the labeled compound showed all four peaks, whereas alkaloid cells of the reference roots showed only two peaks at mlz 369 and 370 (Fig. 4). As shown in Table IV, deuterium-labeled allocryptopine was produced on the eighth day after administration of cis-"C2H,]tetrahydroberberium salt, and by the twenty-first day the amount became nearly 3 times that on the eighth day. The sequence I1 + 111 was confirmed by means of HC using the whole plant. In the following experiment only pieces of root (6 mm thickness, 4 cm length, and 0.9 g weight; 1-2 years old) were immersed in an aqueous solution containing cis-[N-C*H,]tetrahydroberberinium salt (35 mg in 5 ml) at 5°C in the dark. Roots immersed in the aqueous solution not containing the radioactive compound were also examined as a reference. The liquid in the alkaloid cells of the roots was aspirated and analyzed by MF (Table V). In this experiment labeled allocryptopine was remarkably detectable on the twenty-third day after immersion of the root, but more was not produced after that. Smaller amounts of cis-[NC*H,]tetrahydrobeberium salt, namely, 0.2, 2, and 20 mg in 5 ml (0.1, 1 .O, and 10 mM) were then administered to roots (1-2 years old) at 15°C in the dark, separately (Table VI). Labeled allocryptopine was detected following administration of 2 and 20 mg by the fourth day but not with 0.2 mg. On the seventh day labeled allocryptopine was still detected with both 2 and 20 mg administrations.

,

m/z, 3 7 3

L

c 0

1. in ject

.-

.1

I

2

3

0

7 inject

(min)

1

2

3

(min)

FIG.4. Mass fragmentograms of the liquid from alkaloid cells in Murleuva cordatu root after administration of ris-[N-C2H3]tetrahydroberberiniumsalt to whole plants. (a) Feeding for 2 I days. (b) Reference. Conditions: 3% SE-52 column (250°C constant), 5 mm X 50 cm, He 30 mllmin. Selected ions: r n l z 369, M + of allocryptopine; mlz 370 [M + I]+ of allocryptopine; mlz 372, M + of [N-C2H3]allocryptopine;m l z 373, [M + I ] of [N-C2H3]allocryptopine. In this experiment ( 2 )cis-[N-C2H3] tetrahydroberberinium salt was used. +

5.


187

TABLE IV ENRICHMENT OF ALLOCRYPTOPINE IN T H E LIQUID OF ALKALOID CELLSOF Macleaya rordata ROOT AFTER ADMINISTRATION^^ OF cis-[N-cZH,]TETRAHYDROBERBERINIUM SALTTO WHOLEPLANTS

ISOTOPE

Feeding period (days)

Enrichment (%) of allocryptopine”

8 9

4.5 4.3 4.7 8.0 14. I

11

14 21

Administration to three whole plants (60 mg), at room temperature h Isotope enrichment was calculated from the ratio of peak areas monitored at mlz 372 to 369 in the mass fragmentogram.

The enrichment for allocryptopine after 2 mg of administration was the same as that after 20 mg of administration. Biosynthesis of allocryptopine from cis-Nmethyltetrahydroberberinium salt occurred faster at higher ( 15°C) than at lower temperatures (5°C). The sequence I1 + 111 was thus confirmed by HC using only a piece of the root. The next sequence 111+ IV (Scheme 1) was demonstrated with two groups of root pieces, ones immersed in a buffer solution containing [N-C2H,]allocrypto-

TABLE V ENRICHMENT OF ALLOCRYPTOPINE IN THE LIQUID OF ALKALOID CELLSOF Macleaya cordata ROOT AFTER ADMINISTRATION“ OF

ISOTOPE

Ck-[N-C*H,]TETRAHYDROBERBERINIUM

SALTTO PIECESOF ROOT^

0


Enrichment (%) of allocryptopinec

13 23 30 33 55

0.6 6.1 7.8 7.8 7.4

Administration to two pieces of root (35 mg), at 5°C in the

dark. Root age of 1-2 years. See Table 1V.

188

YOHEI HASHIMOTO

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TABLE VI ISOTOPE ENRICHMENT OF ALLOCRYFTOPINE I N THE LIQUIDOF ALKALOID CELLSOF Macleaya cordata ROOT AFTER ADMINISTRATIONO OF DIFFERENT AMOUNTS OF C~S-[~-C2H,]TETRAHYDROBERBERlNIUMSALT TO PIECES OF ROOT^ Amount (mg) of administration


Enrichment (%) of allocryptopinec

0.2 (0.1 mM)

4 7 4 7 4 7

-

2 (1 mM) 20 (10 mM)

0

b (.

2.5 2.8 2.2 2.9

Administration to pieces of root, at 15°C in the dark Root age of 1-2 years. See Table IV.

pine hydrochloride at 15°C in the dark and others immersed in the buffer solution not containing the compound described above as a reference sample. Sodium borohydride was added to the sample solution in the microtrap, which contained the liquid aspirated from alkaloid cells and the solvent for washing out the liquid in the capillary and connection tubes. The reductant is necessary because the chelerythrine which would be produced in this sequence is observed at mlz 333 (M+ - 15) but not at mlz 348 (M+) on EI-MS. After hydrogenation of chelerythrine with sodium borohydride, the alkaloid was identified as dihydrochelerythrine at mlz 349 (M+) on EI-MS. Mass fragmentograms of the alkaloids treated with sodium borohydride in the sample solutions obtained from the both groups are shown in Fig. 5. Peaks of mlz 349 and 350 for the M + and [M + 11 ions of dihydrochelerythrine, and 352 and 353 for M and [M + 11 of [N-C*H,]dihydrochelerythrine, respectively, were detected in the liquid obtained from roots administered labeled alkaloid, whereas only two peaks of mlz 349 and 350 were found in the reference sample. Isotopic enrichments of dihydrochelerythrine after feeding [N-C2H,]allocryptopine was obtained as shown in Table VII. The maximum enrichment of dihydrochelerythrine was detected 13-16 days after feeding. Thus, the sequence 111 + IV was confirmed by HC using only a piece of the root. The biosynthetic sequence of alkaloid production in M. cordata was also determined by means of HC using both whole plants and root pieces. It is advantageous in the HC study of metabolic sequences in plants to feed small amounts of compounds (2 mg in 5 ml of solution in this experiment) and stable isotopically labeled compounds instead of radioisotopes. +

+

+

5.

189


(b)

(a)

,peak

peak I

I

nI(

0

r

1

3

2

4 (min)

0

r

1

2

3

4

(inin)

inject

inject

FIG. 5 . Mass fragmentograms of the liquid from alkaloid cells in Macleaya cordara root after administration of [N-C2H3]allocryptopine to pieces of root. (a) Feeding for 13 days. (b) Reference. Conditions: See Fig. 4. Selected ions: mlz 349, M + of dihydrochelerythrine; m l z 350, [M + I ] + of dihydrochelerythrine; m l z 352, M + of [N-C2H3]dihydrochelerythrine; m l z 353, [M + I ] + of [N-

C2H3]dihydrochelerythrine. TABLE VII ISOTOPE ENRICHMENT OF DIHYDROCHELERYTHRINE I N THE LIQUIDOF ALKALOID CELLS OF Macleaya cordata ROOT AFTER ADMINISTRATIONO OF [N-C*H,]ALLOCRYPTOPINE TO PIECESOF ROOT^ Enrichment (%) of dihydrochelerythrinec Feeding period (days)

Sample I

Sample I1

Sample 111

Sample IV

4 7 9 11

2.3 2.2 4.4 6.5

13

11.1

3.8 4.2 5.3 4.9 6.1

16 20 25 29

6.2 4.2 3.3 4.2

5.5

2.6 2.8 9.2 3.8 6.3 5.4

5.6 4.7 3.8

4.6 3.9

2.8 3.4 4.7 6.9 7.6 7.8 6.5 4.2 3.9

5.1

* Administration to four root pieces (6.5 mg), at 15°C in the dark. Root age 1-2 years. Isotope enrichment was calculated from the ratio of peak areas monitored at mlz 352 to 349 in the mass fragmentogram. b

c

190

YOHEI HASHIMOTO

et al.

TABLE VIII ALKALOID CONTENT A N D RATIO PER SINGLE COLORED CELLI N RHIZOMES OF Sanguinaria Canadensis

Alkaloid

Amount (ngp

Ratio ( 8 p

Protopine Allocryptopine Sanguinarineb Chelerythrineh

9.3 12.7 136.9 68.3

4. I

b

5.6 60.2 30. I

Average of 2800 cells (n = 14) As the chloride.

E. Sanguinaria canadensis The alkaloids in rhizomes of Sanguinaria canadensis were observed inside the central vacuoles with the electron microscope (23).The reddish cells containing alkaloids were scattered evenly in the rhizome tissues. Known alkaloids from S. canadensis include sanguinarine, chelerythrine, and other benzo[c]phenanthridines, protopine and allocryptopine as protopine types, and coptisine and berberine as protoberberine types (51-53). To examine the constituents in the liquid from alkaloid cells, the liquid from 200 reddish cells was removed using the automated apparatus and collected into microtrap (4).Quantitative analysis of the liquid was followed by analysis of the liquid from M . cordata (see Table 11); specifically, the four alkaloids which were analyzed for M. cordata were examined. Two minor peaks eluted after chelerythrine on HPLC chromatograms were not further examined. The major alkaloid was sanguinarine, followed by chelerythrine, with protopine and allocryptopine being minor components (each -5%), a reversal of the ratios in the case of M . cordata (Table VIII). It was shown that benzo[c]phenanthridine-type alkaloids were dominantly present in the alkaloid cells of S. canadensis. The total amount of alkaloids per single alkaloid cell in S. canadensis was greater than that in the case of M. cordata. Therefore, HC demonstrated why the liquid in S. canadensis was more reddish than that in M . cordata.

F. Phellodendron amurense The bark of Phellodendron amurense (Rutaceae) is known as an important Japanese medicine for stomach and intestinal diseases. The bark is yellowish in color and contains two major alkaloids, berberine and palmatine. Yellow alkaloids do not usually have distinguishing characteristics in tissues when observed microscopically. The bark was soaked in 5% nitric acid solution for 3 days and sectioned for microscopical studies ( I ) . The alkaloids were crystallized as nitrate salts in the phloem, medullary rays, and cortex. Crystals were removed by

5.


191

means of the microaspiratoscope and analyzed by HPLC. The crystals were composed of about 95% berberine and 3% palmatine in both fresh and dried bark samples. Traces of an unknown compound were also shown to present in both samples analyzed.

G. KIDNEY: AN APPLICATION FOR ANIMAL TISSUE Application of HC to animal tissues was carried out for renal stones in kidneys. Rats were freely fed a laboratory ration containing 3% uric acid and 2% potassium oxonate (54). After 3 weeks on this diet, the rats were sacrificed to obtain the kidneys. The left kidney was frozen, and the right one was fixed in absolute alcohol. Both kidneys were sectioned to observe amorphous and crystalline deposits in the tubules and collecting tubes with the microscope. Amorphous and crystalline deposits in both kidneys were removed by the microaspiratoscope, separately, for analysis by HPLC (55). To determine the constituents of the deposits, uric acid, known as a potential component of kidney stones, xanthine and hypoxanthine as precursors, and potassium oxonate were used for reference on HPLC. Only uric acid, probably urate or both, was detected in both kidneys on HPLC.

Acknowledgments

The authors thank Mrs. T. Urabe for assistance with the English translation of this chapter.

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5.


193

51. R. H. F. Manske and W. R. Ashford, in “The Alkaloids” (R. H. F. Manske and H. L. Holmes, eds.), Vol. 4, p. 84. Academic Press, New York, 1954. 52. R. H. F. Manske, in “The Alkaloids” (R. H. F. Manske, ed.), Vol. 10, p. 471. Academic Press, New York, 1968. 53. F. Sintavg, in “The Alkaloids” (R. H. F. Manske, ed.), Vol. 12, P. 338. Academic Press, New York, 1970. 54. J. Waisman, R. Bluestone, and J. R. Klinenberg, Lab. Invesr. 30, 716 (1974). 5 5 . K. Kawanishi, Y. Hashimoto, H. Tomita, and M. Moriyasu, J . Pharrnacobio-Dyn. 5, s-37 (1982).


-CHAPTER

I;

TAXUS ALKALOIDS SIEGFRIED BLECHERT Institut fur Organisch Chemie und Biochemie der Universitat Bonn 0-5300Bonn I , Federal Republic of Germany

DANIEL GUENARD Institut de Chimie des Substances Naturelles C.N.R.S. F-91190 Gif-sur-Yvette, France 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Compounds Having an Exo Methylene at C-4 . . . . . . . . . . . . . . . . . . . . . . . . . . B. Compounds Having an Epoxide at Positions 4-20 . . . . . . . . . . . . . . . . . . . . . . . C. Compounds Having an Oxetane Ring at 4-5 . . . . . . . . .......

IV. V.

VI.

VII.

I95 I96 I97 198 200 20 I D. Compounds in Which C-20 Is Part of Ring B . . . . . . . . . . . . . . . . . . . . . . . . . . 202 Biogenesis . . . ... . . . . . . . . . . . . . . . . . . . 202 Hemisynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 A. Reactivity of Taxol and Derivatives . . . . . . . . 204 B. Hemisynthesis of Taxol . . . . . . . . . . . . . . . . . . 206 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 A. Cyclization Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 B. [3,3] Sigmatropic Rearrangements . . . 216 218 C. Ring Expansions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Fragmentations . . .............................................. 226 E. Taxane Building Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

I. Introduction

The genus T a u s has been the subject of study since the beginning of the twentieth century, especially as regards the toxic activities associated with different compounds derived from various parts of the tree. The complex nature of the diterpenes present in this plant, as well as the relative instability of certain of these, retarded for some time the structural elucidation of these novel tricyclic systems referred to as taxanes. 195

THE ALKALOIDS VOL 19 Copynght 0 19W by Acddemic h e % , Inc All nghh of reproduction in my form re\ervcd

196

SIEGFRIED BLECHERT A N D DANIEL GUENARD

Since the first review by Lythgoe ( I ) , which appeared in this treatise in 1968, the subject of taxane-type molecules has developed predominantly around taxol (l),discovered in 1971 by Wani et al. (2). This is clearly borne out by the 1985 review by Suffness and Cordell ( 3 ) ,also in The Alkaloids, wherein the chemistry and pharmacology of taxol are the principal subjects. Since then, the major part of the work concerned with this family of substances has centered almost exclusively on (1) hemisyntheses based on the taxol molecule and on deacetylbaccatin 111; (2) total synthesis of the taxane skeleton as well as certain polyfunctional derivatives, and (3) development of the pharmacology of taxol and its derivatives.

11. Structure

Taxane-type substances possess a totally original skeleton which may be described as a diterpene composed of three [9.3.1.03v8]pentadecene rings (2). The nomenclature first proposed for this ring system was modified in 1969 (4,5),the accepted numbering of the carbon atoms now being that shown in Fig. 1.

OBz

Ph

1_:

2

TAXOL

FIG. 1

6.

T A X U S ALKALOIDS

197

This tricyclic system possesses a highly folded conformation, not obvious from its two-dimensional representation. The six-membered A ring, a distorted boat, is cis-fused to the eight-membered B ring which, in turn, has a boat-chair conformation. In the case of taxinine or baccatin derivatives, the remaining sixmembered ring C has a disturbed chair conformation and is trans-fused to ring B (6-8). This structural unit, though having a bridgehead double bond, is stabilized both by conformational effects and by the presence of substituents in the neighborhood of the double bond, as shown by MM2 calculations performed on a bicyclo[5.3. llundec- 1(10)-ene (9).This conformational preference is confirmed by all X-ray diffraction studies done to date, by NOE NMR techniques ( l o ) ,and by conformational calculations (11).

111. Isolation

The first extractions performed on different parts of the yew (leaves, bark, roots, or wood) employed the usual techniques for the isolation of alkaloids: 1% sulfuric acid applied directly to the leaves or after their extraction with methanol (12). This rather rough treatment did not allow isolation of the very labile substances present in this plant. However, the use of petroleum ether for the wood (13) and alcohol for the trunk bark ( 2 ) or leaves (14) led to the isolation of several interesting products such as the baccatin derivatives. These observations explain why a certain number of taxanes have been identified only by way of their derivatives, artifacts of the extraction process. In the same way that the toxic properties of the constituents of the yew tree have encouraged investigation of all its parts, practically all Taxus species have also been studied: T . baccata L. in Europe, T . brevifolia in North America, T. cuspidata, T. wallichiana, and T . mairei in Asia, and Austrotarus spiccata Compton in New Caledonia. Although taxanes may be considered as the typical constituents of the genus Taxus, other natural products, such as ecdysones, triterpenoids, lignans, glycosides, and flavonoids have also been identified [these are described in a recent review by Ud-Din Khan and N . Parveen (IS)]. Besides X-ray crystallography, NMR and mass spectrometry are the two principal methods used for identifying taxanes. For instance, the proton-NMR spectra constitute a good fingerprinting of the substitutions present on the skeleton, as has been shown in Miller’s excellent review of the NMR of taxane-type derivatives (16). Carbon-13 NMR has also been applied to the specific study of taxanes having an epoxide or oxetane functionality (17). As for mass spectrometry, only chemical ionization (for the various fragmentations) or FAB (fast-atom bombardment for the molecular weight) may be used for structural resolutions. Circular

198

SIEGFRIED BLECHERT AND DANIEL GUENARD

dichroism has helped to determine the absolute configuration in certain cases owing to the Cotton effect (18). In addition to the compounds described by Suffness and Cordell in their 1985 review, the principal structures of which will be mentioned here, many new taxane-type substances have been discovered in extracts of yew since 1968. Each of these compounds may be classed as a particular type of taxane derivative characterized either by substitutions on certain carbon atoms (C-1, C-7, C-13) or by a particular functionality at C-20 (ex0 methylene, epoxide, oxetane, or direct linkage to C-2). A. COMPOUNDS HAVING A N Exo METHYLENE AT C-4

Many derivatives of taxusin (3h, Fig. 2) [whose structure was confirmed in 1987 by X-ray diffraction (19)]possessing various hydroxylated or acetylated functional groups have been extracted from leaves, bark, or wood. In 1969, derivatives 3a-g were found in the heartwood of T. buccutu (4). Another example of X-ray structural confirmation appeared in 1984 with that of one of the

*. \Comp. l

i h

a &

%--

3g

14

U

“RZ

a

RI

2

H H

3

O H H

OAc OAc OAc OAc OAc C O @ I OAc CO.@ OH

OAc OAc OAc

CO-$iBu OAc

OAc

3.l

{

OAc

2k FIG. 2

H

OAc

H

4

5

6

OAc OAc OAc OAc

OAc OAc OAc OAc OAc OAc

OH OAc OAc OAc H

H OAc OAc

OAc

H H

OAc OH OH

H

OAc

OAc

OH OH

OAc OAc

OAc OAc

OAc OAc

OAc OAc OH

OAc

OH

H

OAc

OAc

H

C@iBu on C-14

6.

199

TAXUS ALKALOIDS

constituents of the mixture referred to as taxine: 0-cinnamoyltaxicine I triacetate (3k) (20). An isomer (4, Taiwanxan) (21)of compound 3e was found in T. mairei and identified by X-ray diffraction. From T. cuspidata (22) two taxanes, 8a and taxagifine (Sb, Fig. 3), previously extracted from T. baccata (23), have been isolated, each having a cinnamate group at C-5 (no doubt originating from a Winterstein acid-type ester) and functionalized methyl groups at positions 17 and 19 with formation of an oxirane bridge between C-12 and C-17. From the leaves (24) or bark (25)of the genus Austrotaxus have been identified other taxane-type alkaloids (3i,j, 5a-f, 6a-c, 7a-c) possessing a similar sidechain Winterstein acid, at position 5. It should be noted that compounds 7a-c

comx

'

OH Ac Ac Ac AcO- -

hl h 5.f

Ac Ac

Ac H Ac A c H H

O H H H H Ac Ac

AcO--

Comp.

a

R OAc

Z h O H Z r H

OAc

FIG. 3

k

R=OCOPh

&

R=H

200


have, on one hand, a ketone at position 10 in contrast to that of ring B of baccatin 111 and, on the other hand, a functional group at C-14 as in Taiwanxan. Other minor coniponents, related to 5a, possess a ketone at position 13 with or without a 3-11 bond [a linkage previously mentioned by Lythgoe ( I ) in other compounds].

B. COMPOUNDS HAVINGA N EPOXIDE AT POSITIONS 4-20 Relatively few compounds having an epoxide at positions 4-20 have been extracted (Fig. 4). I-P-Hydroxybaccatin 1 (Sc), accompanied by 9a and 9b (26), was isolated from T . baccata, and 9d from T . mairei (27). However, several alkaloids of this type have been isolated from Austrotaxus (25), in contrast to the other Taxus species. One group, 10a-c, possesses an isophenylalanine-type chain at C-5 and is accompanied by cinnamates corresponding to the loss of dimethylamine. A second group, l l a , b and 12a-c, in which a nicotonate moiety is present at C-9, had never before been obtained in extracts of Taxus. Two of these were named nicaustrine ( l l a ) and nicotaxine (12c). Taxol was not found in this plant; however, the presence of various taxanes confirms the close relationship of Taxus and Austrotaxus. It is also worth noting the unusual presence of derivatives having a side chain at C-5 with a partially or totally demethylated amine .

Aco--m: AcO

OAc

RZO

QRI

OAc

R 1 Ac

H

2

1921 H

Ac

Ph

' OR,

R,

R 1

A

c

H o

2 H H

-OAc : ~

o

.

.

AC Ac

N(CH3h ~ v ulr Ac

1

Ac OH H OAc

2

H

3

CH3 CH, OAC H CH3 OAc H CH, =O H H =O OHatC-5 =O

FIG. 4

6.

20 1

T A X U S ALKALOIDS

C. COMPOUNDS HAVING A N OXETANE RINGAT 4-5 Compounds having an oxetane ring at positions 4-5 are presently the most closely studied class of taxanes owing to their promising therapeutic potential. Present in fairly small quantities in various parts of the tree, they are characterized, in addition to the oxetane ring fused at positions 4-5 of the C ring, by a ketone group at C-9 and by the frequent presence at C- 13 of a relatively complex and easily hydrolyzed side chain of the type already seen at C-5. These taxol-type compounds have already been described by Suffness and Cordell (3).The various substances extracted from the bark of T. baccata, T . wallichiana, or T . brevifolia differ only in the nature of the substituents R , , R,, and R,; R, may be a free hydroxyl group or a xylose derivative (14), R, a hydroxyl, acetyl (taxol), or hydroxybutyrate group ( I d ) , and R, a phenyl (taxol) (2), tiglate (cephalomannine) (28), or hexanoate group (14). The first compound of this type was extracted from the heartwood of T. bacccra in 1966 by Chan er al. (29) and was shown to be, after several structure revisions, baccatin 111 (13b, Fig. 5) (30). In this latter work were published several new derivatives (14a-d) possessing an oxetane ring and an acetate group

2 CIAc OH OAc H OCOPh OH

3 OAc OAc OAc

OCOC-jHIlOH OAc H

OAC OH

E 'e l&

AcO---

&&

14d FIG. 5

202


ACO

FIG. 6

at C-9. In this same series, taxane 14e, having a tertiary hydroxyl group at the 4 position, has recently been isolated (27). A chemical degradation product of taxol (2), 10-deacetylbaccatinI11 (13a), was also isolated from the leaves of T. baccata in 1981 (31), the sole example of this class of products in the leaves of this plant, as well as from the bark of T. brevifolia in 1982 (32).Among the more recent taxol-type compounds extracted from T. brevifolia should be mentioned the product of C- 10 hydroxyl oxidation of 10-deacetyltaxol (33). IN WHICHC-20 IS PARTOF RINGB D. COMPOUNDS

The only example of a compound in which C-20 is part of ring B is taxine A (15), extracted from T. baccata (34), and its structure is shown in Fig. 6.

IV. Biogenesis

In light of the different compounds that can be extracted from yew, a classification into four major groups, having a common biogenetic origin, may be made. In 1966 Lythgoe et al. proposed a biogenetic scheme starting from geranylgeraniol (35), by way of a series of cyclization reactions, by which the four basic taxane-type skeletons A, B, C, and D may be obtained (see Scheme 1). Taxanes having a 4-20 epoxide may originate from derivatives with an exocyclic double bond at these positions, whereas the oxetane ring may be the result of opening of the epoxide followed by rearrangement (36). In the latter case, the rearrangement could give rise to the 7a-hydroxyl group of baccatin V, though this is more likely due to a retroaldol reaction, as discussed below. As far as the origin of taxol itself is concerned, Gueritte-Voegelein et al. have proposed, starting from the product of epoxide ring opening, an intramolecular reaction

6.

203

TAXUS ALKALOIDS

A

n

SCHEME1

between an ester group at C-5 and the hydroxyl at C-13 as a way of explaining the difficulty in esterifying this alcohol (37).

V. Hemisynthesis In view of the novelty of the structure of taxol and the biological and therapeutic interest in taxol-type substances, a considerable amount of work has been devoted to study of the stability of this class of molecules. This research has allowed access both to numerous analogs and to hemisynthesis such that the availability of compounds is no longer dependent on extraction. This last point is especially pertinent in the case of the yew owing to the low levels of taxanes found (40-100 mg/kg of bark) as well as to the relative scarcity and slow growth rate of this tree. Except for a few results concerning type A and B taxanes in which either esterification or saponification was used to confirm structures by comparison with known compounds, studies of opening of the epoxide rings (36),or determination of the structure of autoxidation products (38), most of the published chemistry has been effected on baccatin 111-type compounds (13b). Thus, two synthetic approaches for structural modifications have been described in the literature using, in one case, taxol itself as starting material or, instead, 10deacetylbaccatin 111 (13a), extracted from leaves (easily collected without causing damage to the tree).

204


A . REACTIVITY OF TAXOL AND DERIVATIVES In weakly basic media such as aqueous sodium hydrogen carbonate (2, I I ,28,39),the most labile groups of taxol (1) are the esters found at C- 10 and C - 13 which may either hydrolyze to give the corresponding alcohols (hydrolysis at C-13 being more rapid than that at C-10) or epimerize at C-7 via a retroaldol reaction leading to 7-epitaxol (23a) (39). Similarly, epimerization of 13b gives baccatin V (16) stabilized by an interaction with the acetate group at C-4 (40) (see Scheme 2). In acidic or electrophilic (Lewis acid) media, rearrangements are mainly centered at the oxetane ring (41,42);thus, taxol rearranges to the ortho ester 17 in the presence of ZnBr, and to 18 with BF,. In the presence of ZnCl,, a Wagner-

04

Ph

'Ph

I ZnBrmeOH

if5 UR=Ac

OH

\

12

a

L6

3

1

-

7-epilaxol

OH

ZnClZ

HO

__c

Toluene OAc

OBz

L8

OAc

R=COOCH2CC13 SCHEME

2

HO

6.

205

TAXUS ALKALOIDS

Meenvein-type rearrangement has also been reported (37) for an analog of baccatin (13c) which yields, after elimination of the tertiary alcohol at C-1, compound 19. Under oxidizing conditions (Jones reagent), taxol is converted to the 7-keto derivatives 20a and 20b (43) (see Scheme 3). Under these same conditions (2,11,28), the free 13-hydroxyl group of baccatin 111 (13b) and 10-deacetyl baccatin 111 (13a) is oxidized to form 13-oxobaccatin 111 (21b) and the deacetyl derivative (21a), respectively. Treatment of compound 20c, derived from the oxidation product 20a after protection of its 2'-hydroxyl group, with base leads to rearrangement products such as D-seco taxane 22. Under acylating conditions (acetic anhydride or other protecting agent and pyridine) taxol is preferentially acylated at the 2' position (giving the acetate 23b, shown in Scheme 4, for example). Further acylation occurs at the 7hydroxyl group (e.g., the diacetate 23c) ( 3 9 , 4 2 4 4 ) . In the case of 10-deacetylbaccatin 111 (13a), the order of reactivity is 7 > 10 > 13 (ZI); the difference in reactivity between positions 7 and 10 is minor except when certain protecting groups are used ( 4 3 , in which case only the hydroxyl at position 7 reacts. Esterification of the hydroxyl group at 13, though easily achieved in the

HO.

H\ N

l,&

R=H R=Ac

2Lh

R=H R=Ac

DBU

o=(

'Ph Ph

N

20a

Rl=OH, Rz=O

a

R,=Rz=O

05

Ph

SCHEME 3

'Ph

22

R-Ac

206


R=COCH=CH-Ph RXO-CHOH-CHOH-Ph

SCHEME 4

case of acetylation (11,39)using Ac,O/pyridine at 80°C for 24 hr, can only be obtained under more drastic conditions when less reactive acids, such as cinnamic acid, are used (acid chloride-AgCN/toluene) (I1,46). These protecting and esterification reactions were advantageously employed for the synthesis of an analog of taxol (23d) radioactively labeled on the acetate group at C-7 (47). A few derivatives of taxol and of deacetylbaccatin 111 have been synthesized in which the different hydroxyl groups have been modified for structure-activity studies; these are described later in Section VII, which deals with pharmacology. However, three reactions bear special mention: (a) the intramolecular cyclization of the side chain of taxol leading to the oxazolone (24) (43),(b) an approach to the functionalization of the side chain via vicinal dihydroxylation (11,46) of the cinnamate (25) to give 26, and (c) Sharpless oxyamination leading to mixtures of 2'- and 3'-oxyaminated products (see below).

B. HEMISYNTHESIS OF TAXOL 10-Deacetylbaccatin 111 (13a), extracted from yew leaves in good yield [2001000 mg/kg of leaves (31,48,49)], is an obvious choice of starting material for the hemisynthesis of taxol. Two approaches (Scheme 5) have been developed.

6.

207

TAXUS ALKALOIDS

1) protection at 7 and 1 0 C1COOCH2CCI,

2)HOCOCH=CHPh.DCC,DMAPfloluece, 70°C HO 3) Na+tBuOCONC1~.0s0,.CHlCN,48~ OH

OAc

'

OBZ Ph

l3.a R=H

Z h R,=OH(R), Rz=NHBOC(S). R=CooCH~CCl3 21b Rl=OH(S), RFNHBOC(R), R=COOCH~CCl3

5 steps

Ilp R=Ac

21E Rl=NHBOC(R). R2=OH(S). R=COOCHzCCl~

XU

1

Ri=NHBOC(S). R#H(R).

R=COOCHzCCl3

1) deprotection

desacetyl-lOtaxol

m

4

2)hCocI

EI I

0

%

1) DCC. DMAP. Toluene

* I 2) AcCVPy

NHcoph

:

OBz

2) AcOHMeOH

OAc

m

(2'R,3S)

SCHEME 5

The first, reported in 1986 (46) utilizes Sharpless oxyamination of the C-13 cinnamate derivative (25), leading in one step to both the correct substitutions at C-2' and C-3' and to the threo configuration present in taxol. This reaction yields 10-deacetyltaxol (230, after deprotection and benzoylation of the oxyamination product, in five steps starting from 13a with an overall yield of about 10% and to taxol (1) itself starting from 13b. When the standard conditions described for oxyamination are used, the reaction is nonspecific and leads to two regioisomers and their associated diastereoisomers (27a-d). Use of asymmetric catalysts (50) in the reaction leads to an improvement in the yield of the desired isomer 27a, the precursor of natural deacetyltaxol. A second approach to the hemisynthesis of taxol, published in 1988 (45), relies on the esterification of the hydroxyl group at position 13 of 13a (selectively protected at C-7 and acetylated at C-10) with the separately synthesized side chain 30 (51).This coupling reaction was performed under conditions previously

208


Ph

w

-

Ti'"isopropoxide diethyl-L-tartrate

0

BuOOH CHzClz/-23"C

m

2%

-

I

1) Methylvinyl ctha

PhOCHN

kc

2)NazC03

PhocHN

1)RuCl3,IO4Na 2) CHzNz 3) N3N3 4) PhCOcl 5) H#dC

Ph

0 ~

OH

SCHEME 6

developed for 25 [ 1,3-dicyclohexylcarbodiimide (DCC), equimolar 4-dimethylaminopyridine (DMAP) in toluene at 70"Cl. In this way, the formation of taxol (1) in four steps from 10-deacetylbaccatin 111 was realized with an overall yield of 26% from 13a. The necessity of extracting yew bark to obtain taxol was circumvented. The enantioselective synthesis (51) of the side chain 30 of taxol had been achieved by way of stereospecific Sharpless epoxidation of cis-cinnamyl alcohol (29a), giving 29b (see Scheme 6). Following oxidation of the alcohol group, protection of the resulting carboxylic acid, regioselective opening of the epoxide with azide, benzoylation, and reduction, a suitably substituted moiety (28) was available which, after protection and deprotection of the acid function to form 30, was coupled to baccatin 111.

VI. Synthesis

The biological activity of some taxanes, especially that of taxol, together with the synthetic challenge of the strained and highly functionalized tricycles has led to constantly increasing interest, resulting in much preparative work. Partial structures as well as various concepts concerning the buildup of the tricyclic

6.

209

TAXUS ALKALOIDS

system have been the subjects of research. In this section the different synthetic approaches and the synthesis of some structural elements are presented. Besides the vast number of functionalities which have to be installed stereoselectively, the buildup of the highly strained middle ring system with two stereochemically correctly annealed six-membered rings A and C is already a synthetic problem. MM2 calculations give a hint that the double bond of ring A decreases rather than increases the strain energy of the tricycles, whereas the geminal dimethyl group causes extreme stress in the taxane nucleus (9). Derivations of natural products also prove the remarkable stability of the bridgehead olefin (12,26,35). These perceptions should be useful for further synthetic planning. In spite of numerous attempts by many teams, only a very few were successful in synthesizing a tricyclic taxane framework with the stereochemically correct ring linkage (62,71,76,80,97). A synthesis of the unnatural enantiomer of taxusin has recently been reported by Holton et al. (104). In order to build up the eight-membered ring, various strategies have been investigated, namely, cyclization reactions, ring enlargements or ring contractions, rearrangements, and fragmentations. A. CYCLIZATION REACTIONS 1. Biomimetic Approaches

Biomimetic cyclization reactions have often been used successfully to synthesize terpenoids. Nevertheless, taxane frameworks have not been reached by this strategy so far. In 1978 Kato and co-workers had already synthesized the secotaxane derivative 34 (shown in Scheme 7), but a transannular cyclization forming the eight-membered ring could not be achieved (52). Verticillene (38), corresponding to verticillol, a constituent of the conifer

1) PPh,, CC14

2) LDA

u

224 ( 4 5 % )

2.2 SCHEME7

210


Sciadopitys verticillata (Taxodiaceae) (53),is the putative biogenetic precursor of the taxane alkaloids. The first synthesis of 38 was reported by Pattenden and Jackson (54). They started from 3-isobutoxycyclohexenone and reached 38 in 10 steps (see Scheme 8). The main product 37 of the reductive coupling of the bisaldehyde 36 results from a 1,5-H shift of a primarily built olefin. The following Birch reduction gives the target molecule. Nevertheless, attempted transannular cyclizations of 38 or epoxides thereof, aided by Lewis acids, failed (55). 2. Ring Connections Some teams followed the obvious strategy of connecting previously functionalized rings A and C by a C, or C, bridge in order to reach the ABC system by a suitable eight-membered ring closure. Starting from 3-methylcyclohexenone, Fetizon and co-workers synthesized building blocks for the C ring of type 40 (see Scheme 9) which were connected with the ring A unit by a Michael addition (56). However, subsequent attempts to cyclize failed. Coupling of silylenol ether 40 (R = OSiMe,) with 41 by the Mukaiyama reaction did not succeed. At an early stage Kitagawa and co-workers followed a path which, unfortunately, did not reach the goal (57). Starting from d-camphor, building block 43 (Scheme 10) was reached via various steps (58). Resolution of diastereomeric ketals of cyclopropanated 3-methylcyclohexanone likewise yielded the chiral

34

(78%)

SCHEME 8

6.

21 I

TAXUS ALKALOIDS

1) LDA, THF, -7OOC

,THF,HMPA

41 411

2.2

& R=H(66%)

a

R=Si(CH,), (40%)

SCHEME 9

sulfone 44. Following alkylation of the deprotonated sulfone and reductive sulfur extrusion, an additional protected carbonyl unit was established in the C ring. But, after having invested that much work into the synthesis of 45, it was regrettable that the expected formation of the eight-membered ring by displacement of the mesylate did not take place. The strategy to fix the centers to be connected within a larger and thus less strained ring system also failed (59). The centers to be coupled may successfully be pre-fixed whenever sterically much less strained bicycles of type 48 (see Scheme 11) are synthesized (60,61). The protected thiophenol derivative 46 was derived from a-ionone by 21 steps. Release of the highly nucleophilic sulfur leads to the favored 12-membered ring. Oxidation to the sulfoxide augments the CH acidity and thus permits the desired ring contraction. So far the only successful direct cyclization of the eight-membered ring was realized by Kende et al. (62) via the McMurry reaction, which is very suitable for the formation of strained frameworks. Starting from 2,6-dimethylcyclohexenone, acetal49 (Scheme 12) was synthesized in 10 steps. Mukaiyama coupling and subsequent acid treatment yielded a 2 : 1 mixture of two Z and two E isomers (51). Via vinyl cleavage by N-methylmorpholine N-oxidelOs0, and NaIO,, stereoisomeric diesters were derived. Chromatography on silica gel and hydro-

MEMO--

-++

7.:b

+ +

P

MEMO--

0

OMS

45 SCHEME 10

212


SCHEME1 1

3

+ l'p

1) TiCI,,-50°C ~)TsOH/C~H~ ___)

80°C

\

0

OSi(CH3)3

sa

42

4

1) modified Tebbe (60%) 4

2) 6eq. iBuAlH (90%)

3) Swern oxidation (85%)

H

H (via separation of stereoisomers)

54

55 (44%)

(20%) SCHEME12

6.

213

TAXUS ALKALOIDS

genation on Pd/C led to 52 and the stereoisomeric C-3 p product (taxane numbering), which could luckily be isomerized by K,CO,/MeOH to a 4 : 1 mixture favoring the desired C-3 a isomer. Methylenation by Zn/CH,Br,/TiCl,, reduction, and subsequent oxidation with dry dimethyl sulfoxide (DMSO) (thus preventing the formation of significant amounts of alkyl halide) yielded key product 53. The somewhat unstable dialdehyde was added over 24 hr to a refluxing suspension of Zn-Cu and TiCl, in ethylene glycol dimethyl ether (DME) and refluxed an additional 18 hr. Thereby the first direct cyclization of the taxane B ring from a bicyclic seco-B intermediate could be accomplished in 20% yield. Another product was isolated in 10% yield, arising from the vinylogous reductive coupling of dialdehyde 53. Compound 54 seems to offer attractive possibilities of introducing additional functionalities and of synthesizing natural products. Selective introduction of the C-13 carbonyl group could be realized, as 55 shows. However, functionalizing the double bond of the eight-membered ring presents a significant problem, as the n system is shielded at the upper side by the geminal dimethyl group and at the lower side by the annealed six-membered rings. It would be desirable to stop the reductive coupling at the diol stage, but here stereochemical problems have to be taken into consideration. 3. Cycloaddition Reactions Intramolecular cycloaddition reactions generally offer attractive possibilities of building bicyclic or tricyclic compounds. In this case the size of the ring can be easily guided by the chain length between the reacting centers. To synthesize the problematic eight-membered ring, two strategies are therefore at hand. Either the middle ring could be created with the help of a suitable chain length in the course of a [4 + 21 cycloaddition, or the cycloaddition reaction itself could yield the eight-membered ring.

a. [4 + 41 Cycloaddition. The latter possibility was investigated in model reactions carried out by Wender and co-workers. Well-known nickel-catalyzed cycloadditions of dienes were primarily employed in intramolecular variants (63,64).The bisdiene 56 (Scheme 13), which was synthesized from myrcene and OSi(Me),tBu

OSi(Me),tBu

H

SCHEME 13

!

214


vinylacetylene, can be cyclized to a mixture of stereoisomers 57. Under these conditions, the Diels-Alder reaction can already be observed. It is not clear, however, whether AB ring systems with a geminal dimethyl group can be reached likewise. On the contrary, an analogous ring closure yielding a BCtaxane model (58) can be achieved much easier. b. Diels-Alder Reactions. The second way to reach the goal, by 14 + 21 cycloadditions, has been investigated by various teams. In one of the first examples, by Sakan and Craven, a tricyclic taxane model (shown in Scheme 14) was synthesized by the transformation 59 + 60 (65). In order to force the system into the stereochemically correct although sterically less suitable BC ring connections, ring A should assume a boat conformation as in the case of the natural products, and the substituent fixed to it including the diene part has to take an axial orientation. These requirements are fulfilled by the bicyclo[2.2.2]octene system. Nothing is known about transformations of this bicyclic structure into the taxane A ring. The synthesis of 59 was accomplished via some steps from Hagemann’s ester, which was transformed into a cyclohexadiene and added to methylacrylate to yield the bicycle. Manipulation of the side chain finally led to 59. At the same time Shea and co-workers dealt with intramolecular Diels-Alder reactions, in the course of which ring A was reached by cycloaddition reactions. To circumvent stereochemical problems, compounds of type 61 (see Scheme 15), which can be cyclized without any problems thermically or via catalysis by Lewis acids, were synthesized from disubstituted aromatic compounds (66-69). The extremely mild reaction conditions of the catalysis are remarkable. Obviously 62 or even the corresponding 6-methoxyaryl compound were not apt to undergo a transformation into the saturated ring C, as the same team recently reported the use of nonaromatic ring C building blocks in the synthesis of the tricyclic taxane framework (70). The initial strategy to fix both side chains containing the Diels-Alder reactants to a sp2 center was maintained. Compound 63 was synthesized by alkylation and esterification. The second chain with the diene unit was introduced by a Grignard reaction. Ketalization and transformation of the vinyl bromide with rert-

se

b~ (70%) SCHEME14

6.

215

T A X U S ALKALOIDS

xylene, 155"C,93h (70%) or EtzA1C1,CHZCl2,-7OoC (9W)

0

61

0

fi2

butyllithium and dimethylformamide (DMF) into the aldehyde gave key product 64, which was then cycloadded to form the taxane framework 65 in much smaller yields than previously obtained. Experiments to employ a saturated ketone corresponding to 64 in the Diels-Alder reaction failed. So far the closest approach to the natural carbon framework via the DielsAlder reaction has been reached by Bonnert and Jenkins (71,72). Their strategy was based on the buildup of a trans-disubstituted cyclohexane ring containing a 2-substituted diene unit and an electron-poor dienophile in order to guarantee the desired stereochemistry at C-3 and C-8. Following initial syntheses leading to tricycles without the geminal dimethyl group (73,74) synthesis of 70 (shown in Scheme 16) could be achieved. In order to build up a suitably functionalized cyclohexane building block, they selected the easily accessible (by Robinson annealing) trans-decalin derivative 66. This could be transformed into aldehyde 68 by oxidative ring opening and further simple transformations. Conversion of the aldehyde to a 2-substituted butadiene, on the other hand, required the development of a new procedure. Reaction with 2-propenylmagnesium bromide followed by oxidation afforded an a,P-unsaturated ketone. Subsequent reaction of the ketone with LiCMe,SePh and elimination with thionyl chloride gives the highly substituted butadiene in acceptable yields, from which 69 is reached by release of the silyl protecting group and oxidation. The key reaction 69 + 70 can be carried out under remarkably mild conditions and yields (as already seen in the case of the model compounds) exclusively one diastereomer with the desired C-1 configuration. The

216


~ - ? p - -

(Me)3Si0

H

H

H

0

66

tBu(CH3)2Si0

62

qp-

11

BF,-OEt,

toluene,40"C, 24h.

0

0

7 0 (>55%)

62 SCHEME16

high stereoselectivity results from the preferred boat-chair conformation versus an alternative twist boat-chair conformation of the eight-membered ring in the transition state. B . [3,3] SIGMATROPIC REARRANGEMENTS [3,3] Sigmatropic rearrangements offer important synthetic possibilities and can be employed for ring enlargements or ring contractions. Thus, their applicability for solving the problems caused by the eight-membered ring was investigated. Obviously those rearrangement variants that employ only mild conditions are of special interest, for instance, the anionic oxy-Cope systems. Martin et al. made use of this reaction principle aiming at a C, ring enlargement (75). By a Wittig reaction and addition of an vinyl anion, 71 (see Scheme 17) is generated. The low stereoselectivity of this reaction is problematic because the stereoisomer is not apt to rearrange. At room temperature, a [3,3] sigmatropic rearrangement yields tricycle 72, the stereochemistry of which was not clarified. Further synthesis should be difficult as regards missing substituents and the unsuitable position of the double bond. Taking advantage of the likewise mild conditions of the Ireland version of the Claisen rearrangement, easily accessible ester or lactone bonds may be transformed via their enolates into C-C bonds. In the case of cyclic systems, the ring size may be varied by the position of the double bonds. Recently Funk et al. made use of this often applied principle for the synthesis of tricycle 76 (shown in Scheme 18) (76). The strategy aims at the connection of rings A and B by a C , unit in order to

6.

217

T A X U S ALKALOIDS

0

z1

22

SCHEME 17

realize the previously discussed problematic formation of the eight-membered ring by way of a less strained ten-membered lactone followed by a ring contraction. Ring C building block 73 needed for this purpose was reached by oxidative cleavage of a corresponding enol ether. Reaction with a vinyl anion leads to the connection of both six-membered rings. Lactonization with N-methyl-2-chloropyridinium iodide according to Mukaiyama gives the desired lactone along with its chromatographically separable C- 10 stereoisomer (taxane numbering) in 63% yield. Silylation gave rise to a single ketone acetal, presumably the E stereoisomer. The consecutive unproblematic rearrangement has to pass a chairlike transition state and yields the ring-contracted tricycle, the stereochemistry of

O

HCOZCH, C

p

--

23

dp 24

toluene

reflux. 8h.

SCHEME 18

COzCH,

4

218


J z1

UL SCHEME19

which was verified by single-crystal X-ray analysis. It is not yet clear whether the selective transformation of the ex0 methylene group into a dimethyl group and the removal of the inevitable ester will be accomplished. Kanematsu and co-workers investigated an example in which the [3,3] sigmatropic profits by the strain of a cyclobutane derivative (77). The propargyl ether 77 (Scheme 19) derived in 13 steps from the Wieland-Miescher ketone is isomerized by base into the allene. Allenes of this type have often been used as well-suited dienophiles in intramolecular Diels-Alder reactions. However, in this case the (s) cis conformation of the diene necessary for a [4 21 cycloaddition is not formed, for steric reasons, so that the alternative [2 + 21 cycloaddition giving 78 takes place. Under these reaction conditions the molecule undergoes rearrangement, closing the preformed ring A and leading to ring B by opening of the annealed cyclobutane. Compound 79 gives a hint to a taxane skeleton, the formation of which using this strategy would require various transformations as far as the AB system is concerned.

+

C. RINGEXPANSIONS Different teams followed the strategy mentioned above, namely, that of building up the eight-membered ring by cyclobutane annealing to a six-membered ring. In a simple model, Kraus el al. made use of the high reactivity of a bridgehead enone for such a two-carbon ring expansion (see Scheme 20) (78). The enone generated from 80 with triethylamine at 0°C is captured immediately by an electronically rich alkene. The following fragmentation leads to bicyclo[5.3. Ilundecene 82. This, however, is still very far from a taxane skeleton. 1. Intermolecular Photocycloadditions Photochemical cyclobutane annealings are much more promising and find their way into synthesis to a greater extent. Photochemical cycloaddition reactions to enolized 1,3-diketones and consecutive ring openings via retroaldol reactions have been applied in ring expansion reactions numerous times.

6.

219

TAXUS ALKALOIDS

A modified de-Mayo reaction (shown in Scheme 21) also served as a tool in the first synthesis of a saturated taxane framework with a geminal dimethyl group (79,80). Photocycloaddition of cyclohexene (functioning as a ring C model) to a bicyclic 1,3-diketone derivative from 85 is the key step. In the course of the cyclobutane annealing, the C-8 (Y ring connection is established; contrary to C-3, 1) (CH,),CuLi (85%)

0

3) KH / xylene reflux (85%)

84

SZ (92%)

!U (70%) SCHEME 21

"1

220


it cannot be corrected at a later stage. The necessary p-exo attack, that is, attack

from the side disfavored by the geminal dimethyl group, is forced on the molecule by the even stronger shielding effect of an ethylene ketal. At the same time, this ketal is needed for later functionalizations of the taxane A ring. By double cyanide addition to 83, ketalization, saponification, and formation of the anhydride, 84 was generated. Smooth opening by a cuprate reagent and subsequent esterification led to a keto ester, the Diekmann cyclization of which, yielding 85, could only be achieved with an excess of KH. The efficiency of the photocyclization is dependent on the enol derivative applied. Allylcarbonates are best suited. Compound 86 is reached with high stereoselectivity, but it gives the C-8 a cis-connected tricycle 87 after Pd-catalyzed cleavage of the protecting group and retroaldol reaction. Complete epimerization at C-3 can only be achieved by unproblematic stereoselective reduction of the C- 10 carbonyl group to the “naturally configurated” carbinol, owing to the rigid conformation of the eight-membered ring. Hydrolysis of the ketal and reduction by LSelectride make the functionalized taxane skeleton 88 accessible. In the course of a planned synthesis of taxol analogs it could be seen that unambiguous differentiation between both carbinols could not be achieved. As a result of a flexible synthetic strategy though, this problem could be solved by a modified reaction sequence (81). Cleavage of the allylcarbonate, deketalization by aqueous acid, and reduction with NaBH, in ethanol led to the transformation 86 + 89. Complete isomerization at C-3 takes place in the course of a retroaldol reaction with potassium tert-butoxide in tert-butanol owing to the higher conformational flexibility of the tricyclic carbinol. Esterification with cinnamic acid and DCC gave 90. Reduction by NaBH,/citric acid in methanol led to a single stereoisomer, the cis-hydroxylation by OsO, of which gave two chromatographically separable taxol analogs (91). Nearly all naturally occurring taxanes are hydroxylated at C-9 or possess a carbonyl group at this position as taxol does. All efforts to insert oxygen at the stage of various C-10 ketones were not successful. The desired functionalization, however, could be achieved by oxidative ring opening of a cyclobutene instead of by retroaldol reaction (see Scheme 22) (82). Enone 92 was reached from 86 by mild elimination using K,CO, in methanol and consecutive cleavage of the ketal. The stereoselective reduction giving the “naturally” configurated carbinols was unproblematic at C-13 but was not simple at C-10 (taxane numbering). Most reducing agents gave the 10-a-carbinol exclusively. At present the best reaction conditions have led to a mixture of 93 and 94, with the desired product prevailing to a small extent. Compound 93 could be transformed into 94, however, by ally1 oxidation and reduction. Acetylation and ozonolysis with reductive work-up led to the first taxane skeleton containing three oxygen atoms in ring B (95). Synthesis of corresponding 10-epi compounds from 93 was much smoother owing to steric reasons.

6.

22 1

TAXUS ALKALOIDS

-

LiAIHflHF -200-1 ooc 2) H30f

HO-

+

1) AcOAc, DMAP (62%)

HO -

AcO - 2) 03.CHJOH/CH2C12 then (CH3)2S(36%)

es

-& 4-

SCHEME 22

The importance of the ethylene ketal described above with respect to stereocontrol of the de-Mayo reaction is emphasized by later published works of two other teams. Fetizon and co-workers obviously followed a similar concept and carried out photocycloaddition reactions with 96a in model studies (Scheme 23) (83). As can be seen from retroaldol product 98, exclusive a attack of the cyclohexene has taken place. Thus, the relative stereochemistry of the BC ring connection is opposite to that of taxane. Totally comparable results were obtained by Berkowitz et al. in the course of cycloaddition reactions of cyclopentene, cyclohexene, or those of a cyclohexenone ketal to the camphor derivative 96b (84).

Naturally occurring taxanes may be very different with respect to the C ring

OAc

R=H

96b R=CH,

H

AcO 0

222


substitution. For this reason strategies were kept in mind that involved the photochemical buildup of an AB system that allows the desired C ring annealings. Short sequences leading to functionalized bicyclo[5.3. llundecane systems are shown in Scheme 24. The enol carbonate of 85 yields exclusively the desired regioisomer 99 when irradiated with allene. Removal of the protecting group and retroaldol reaction leads to the conjugated enone 100 (85). Later Etizon et al. reported on similar research (86,87). Even more highly oxidized AB systems were synthesized by oxidative ring opening of cyclobutenes (88). By basic elimination and selective hydrogenation of the exocyclic double bond, the enone 101 was easily accessible from 99. Reduction with diisobutylaluminum hydride, acetylation, and cleavage using OsO,/NaIO, leads to 102. Compound 103 was prepared from the enone by ozonolysis followed by reductive work-up. 2 . Intramolecular Photocycloaddition Reactions Intramolecular photocycloadditions have often been taken advantage of in order to synthesize polycyclic compounds. By chosing a suitable chain length between the reacting IT systems, especially five-membered rings but also sixmembered rings can be built up in a simple way. After bimolecular photocycloadditions carried out in analogy to the conversion 85 + 86 had failed with I-methylcyclohexene, an attempt to bring along the missing C-8 methyl group in an intramolecularized variant was tried (80). Com-

u12 SCHEME 24

uu

6.

T A X U S ALKALOIDS

223

pound 104 was prepared by opening of the anhydride 84 with 6-methyld-heptenyllithium, esterification, and cyclization. Neither the 1,3-diketone nor several enol derivatives were suited for the photoaddition (see Scheme 25). The sterically less pretentious dimedone derivative 105 reacted smoothly, but as a result of undesired regiochemistry it led to the annealed seven-membered ring 106 in the cycloaddition reaction (89). The desired cyclization leading to the six-membered ring was achieved by intramolecular photocycloaddition of dioxolenone 107 as shown in Scheme 26. Consecutive cleavage of the ketal followed by retroaldol reaction augmented the initial ring system by a C, unit to trans-bicycle 108 (90). Attempts to apply this reaction principle to a trans-decalin system in order to reach a taxane skeleton failed (91). Other groups tried to connect both 7~ reactants via the enol oxygen of the 1,3diketone. Berkowitz et al. (84,92) esterified a homocamphorquinone with a functionalized ring C building block and reached the desired de-Mayo precursor 109 by simultaneous elimination of HBr (see Scheme 27). By a modified Rylander oxidation the product of the photocycloaddition, 110, was transformed into a lactone, the hydrolysis of which initiated retroaldol reaction and led to tricycle 111. The “unnatural” ring connection at C-8 demonstrates the endo addition caused by the geminal dimethyl group even in the intramolecular case. In a similar study, published a short time earlier by Inouye and co-workers (93), 112 was employed in an analogous reaction sequence. In this case only one stereoisomer out of the 1 : 1 mixture reacted when irradiated. Here, the missing

84--

SCHEME 25

224


1) hv, Pyrex b

2) TsOH, CH,OH, reflux H

107

u 8 (80%) SCHEME 26

dimethyl group allowed exo addition and thus led to tricycle 113 containing a C-8 p methyl group. The stereochemical problems mentioned above arising in the course of the photochemical approach to the taxane tricycle with a geminal dimethyl group was circumvented by Swindell et al. They did not introduce a bicycle leaving the exo/endo possibility open, but instead built up the BC skeleton first and then annealed ring A at a later stage (94-97). Taking a pattern from the works of Schell ef u1. (98), the group made use of an intramolecular photocycloaddition during which the future ring C is connected to a dimedone derivative via the nitrogen atom. The stereochemistry of the cycloaddition of vinylogous imides of type 114 (see Scheme 28) depends on the kind of N-acyl group involved. In the case presented here it smoothly yielded 115 containing the desired BC ring connection.

U (25.30%)

lL2 SCHEME 27

6.

225

TAXUS ALKALOIDS

O-Si-tBuPhz

JJNm-Q-& I

COzCHzCC13

N H

I I

COzCHzCC13

I

NH

I I

COzCHZCC13

SCHEME28

The carbonyl group in 115 allowed the introduction of the C, unit essential for ring A later via the dimethylhydrazone. The primarily a-alkylated product was epimerized and transformed into mesylate 116, which was needed for the key reaction. The B ring-forming fragmentation 116 + 117 was effected by reductive trichloroethyl urethane cleavage and leads to a masked C-2 ketone (taxane numbering). The amine released by hydrolysis was transformed into a C-8 p methyl group by dissolving metal reduction via isocyanide. Here the ketone had to be protected. Hydrogenation and deketalization gave 118, which yielded the saturated taxane skeleton 119 by intramolecular enolate alkylation. The “natural” stereochemistry at C-1 and C-3 is preferred for thermodynamical reasons. 3. C, Ring Expansions

Instead of carring out C, ring expansions by opening a cyclobutane, Trost and co-workers worked on a strategy to reach the problematic eight-membered ring by opening an annealed cyclopentane. During initial investigations (99) ring opening was accomplished between two five-membered rings by a reversible retroaldol process using carbanions stabilized by a sulfone. In later work (100) diol cleavage was employed that led to a suitably functionalized eight-membered ring. The bifunctional annealing reagent 120 (see Scheme 29) was synthesized from 2-methylcyclohex-2-en01 and coupled with 121 in the presence of a Lewis acid,

226


0

120

u

L22

la

SCHEME29

yielding two diastereoisomeric a-silyloxy ketones. Subsequent cyclization with the ally1 silane was carried out by treatment with ethylaluminum dichloride and gave the two stereoisomeric cis-diols (122), which were transformed into 123 by sodium metaperiodate. D. FRAGMENTATIONS The Grob fragmentation is an important reaction principle by which cyclic compounds may be cleaved while at the same time an olefin and other functionalities are generated. The cleavage of a suitable bond in polycyclic systems leads to ring expansions and thus comes in handy for the synthesis of the taxane skeleton. Yamada and co-workers applied such a strategy to the synthesis of bicyclo[3.3.l]undecane 126 (101) as shown in Scheme 30. By double Michael addition with ethyl crotonate, a bicyclo[2.2.2]octanone was made from 124. Introduction of a side chain and intramolecular cyclization led to the buildup of a tricycle which could be transformed stereoselectively by numerous steps into key product 125. Treatment with potassium hydride in toluene at 100°C for 10 min gave 126 in good yield. However, the dimethyl group that augments the strain on the framework was still missing. Successful cyclization leading to ring C has not yet been reported.

124

125 SCHEME30

l a

6.

T A X U S ALKALOIDS

227

The idea of building up the AB system via fragmentation and later annealing the C ring was also followed by Holton. In the course of a retrosynthetic analysis Holton noticed a structural relationship between patchouli alcohol and a tricycle apt to undergo fragmentation. It should be easily accessible by a way similar to Buchi’s natural product synthesis. Thus, the natural product was converted to ppatchouline oxide (130) in two steps and was opened following the Buchi procedure to tertiary alcohol 127 by BF,-Et,O (Scheme 31) (102). Epoxidation, which for steric reasons can only occur from the a side, gave the unstable compound 128 which surprisingly fragmented in refluxing methylene chloride to the AB skeleton 129. Studies on related bridged bicyclic systems showed that a syn periplanar relationship between breaking bonds is necessary for successful fragmentation (103). An anti orientation led only to intramolecular opening of the epoxide. A first-generation synthesis of the taxane ring was based on the buildup of ring C by a Michael addition of the thermodynamic silylenol ether of 129 protected as the MOM ether to trimethylsilyl methyl vinyl ketone, coming out with the desired C-8 stereochemistry. The subsequent Aldol condensation could not be carried out as planned, however; at the end only the “unnatural” cis-annealing to the C-3 carbinol was achieved. During a second-generation synthesis this problem was solved by application of a different reaction sequence. Even the introduction of additional hydroxyl functionalities could be accomplished, thus guiding the development from a naturally occurring terpene to the unnatural enantiomer of taxusin (104). This first synthesis of (-)-taxusin is presented in Scheme 32. p-Patchouline oxide (130) was transformed by tert-butyllithium into an allylic alcohol, which was then converted to a diol related to 127 by epoxidation and treatment with BF,-Et,O and CF,SO,H in CH,Cl,. This additional C-10 alcohol (taxane numbering) allows further functionalization at C-9 and C-8. To solve the ring C problems described above, a C, moiety was introduced stereoselectively at C-8 before the fragmentation step by connection with the tertiary alcohol and radical C-C formation with an enone. Eventually, 131 could be reached by various standard transformations. Epoxidation and treatment with titanium (IV)

SCHEME31

228


l a SCHEME 32

isopropoxide led to fragmentation product 132 as desired. Introduction of the acetyl group was achieved by addition of a-methoxyvinyllithium in hexane followed by in situ hydrolysis. The directing effect of the MEM (methoxyethoxymethyl) ether protecting group is supposed to be the reason for high stereoselectivity. From 133 the thermodynamically stable ketone could be reached by reduction of the alcohol with SmI,. It could be cyclized to the taxane framework by way of tosylate 134. Stereoselective oxidation at C-5 was accomplished with 3chloroperoxybenzoic acid via the silylenol ether. After acetylation and a Wittig reaction 135 was finally reached, by about 30 reactions altogether. E. TAXANE BUILDING BLOCKS Besides the investigations treated so far concerning the synthesis of the taxane skeleton, some groups have also reported on the synthesis of particular building blocks. Synthesis of the taxol side chain 30 (51) has already been introduced in Section V,B. A Swedish group presented an enantiospecific synthesis of a functionalized ring A derivative (105,106). Starting from L-arabinose, 136 (Scheme 33) was made in 20 steps and could be transformed by three further steps into 137. The A building block 138 was reached by Clark and co-workers (107) by 1,3-butaDiels-Alder reaction of 1-ethoxy-3-[(trimethylsilyl)-oxyl]-2-methyldiene with ethyl-(E)-2-acetoxyacrylate.

6.

229

T A X U S ALKALOIDS

%6 CHO

0

0

MOM0

ue SCHEME 33

Much more attention has been paid to ring C. Trost and Fray reported an enantiomerically pure access to such a fragment in the course of their efforts toward taxanes (108). The CD structural element of taxol has been treated by several groups. Clark and co-workers (107,109) constructed oxetane 140 by substitution of a primary mesylate. Berkowitz and Amarasekara made use of the Mitsunobu procedure (110) in the course of studies leading to such four-membered ring ethers. However, model reactions of Swindell and Britcher to reach the ring D unit corresponding to biogenetic assumptions by an a-oxygenated epoxide did not succeed in building up the four-membered ring (111).

VII. Pharmacology

The yew tree has long been known for its toxicity as well as for other biological activities, which have been studied since 1968. Such activities include (a) the antiovulatory effects of leaf extracts of T. baccata, described in 1970 (112), (b) the tranquilizing effects on the central nervous system produced by extracts

230


(113). and (c) the acute toxicity of taxine in mice, particularly with respect to the heart where this substance may act as a calcium antagonist (114). Despite the existence of a large number of taxol derivatives, only taxol itself has given rise at present to significant pharmacological and biological studies. However, some work employing the mixture taxol-cephalomannine (115) (more easily extracted from the bark of T . baccata than taxol itself) as well as derivatives having a xylose residue attached to the taxane skeleton may be cited (116). Since 1985, when the last review in this treatise concerning taxanes was published (3), several new elements concerning the pharmacology and therapeutic use of taxol have appeared. Moreover, knowledge of the structure-activity relationships of this class of compounds has been broadened with the discovery of promising analogs. Since the discovery (117) of its action on tubulin (118) (the protein which, in the form of microtubules, constitutes the mitotic spindle), taxol has been of great utility to biologists. Numerous publications, not all of which need be cited here, describe the use of taxol for the isolation of tubulin from cellular preparations in which the concentration of this protein is too low to permit its polymerization, as in the pancreas (119) or the vegetal domain (120). Taxol has permitted not only the discovery of new microtubules in the Xenopus oocyte cortex (121) but also the study of the role of microtubules in certain cellular processes owing to its lack of destructive effects, in contrast to other known spindle poisons such as colchicine or vinblastine (122). Among other problems, taxol has helped in studies of the influence of the tubulin-microtubule equilibrium on the fluidity of platelet membranes (123) and of the function of the meiotic spindle in spermatocytes (124). Microtubules are implicated not only in cellular division but also in other cellular activities such as those occurring in the cytoskeleton, where taxol has been shown to have very interesting properties (125). Microtubules are especially important in axonal transport where, again, taxol may have a less destructive role than colchicine, which is often used as a blocking agent (126). The effect of taxol on neurons appears to be related to its interaction with both tubulin and its associated proteins (127), the latter being so-called owing to their copurification with tubulin during its preparation. These proteins can interact among themselves in the presence of taxol (128). Recently, a study of the importance of the microtubule network in the regeneration of nerves (129) (destroyed by crushing) has appeared (130,131) in which the role that taxol may play in this process is described. Though taxol may be considered as an inhibitor of axonal transport, it has nevertheless been used to distinguish slow, fast, and retrograde axonal transport (132,133). The mechanism by which tubulin assembles and disassembles itself continues to be a major problem in this field, and taxol, by virtue of its unique ability to inhibit disassembly, remains the tool of choice for this study (134). As for the

6.

T A X U S ALKALOIDS

23 1

interaction between taxol and tubulin, several articles are concerned with (a) the role of ring B of colchicine in the induction of polymerization by taxol (135), (b) the connections between GTP, colchicine, and taxol by which the conformation of tubulin remains unchanged during the process of taxol-promoted polymerization (136), and (c) thermodynamic aspects of taxol-induced polymerization, as demonstrated by a physicochemical approach ( 1 3 3 , which seems to be governed by entropy factors (138). Other results suggest that taxol alters the hydrodynamic properties of the microtubules owing to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins (139). It should, however, be noted that this substance has affinity not for tubulin itself (140) but, rather, for a certain number of polymerized structures such as microtubules, zincinduced plaques, or other drug-induced spirals (141). In vivo, taxol has been tested on numerous cell cultures in order to evaluate their sensivity to this drug or to study the mechanism by which resistance to these types of compounds often develops. Thus, taxol has been studied in cultured human prostatic cancer cells (142) (in which, even at nontoxic concentrations, it provokes ultrastructural alterations) as well as in ovarian tumor cells xenografted into nude mice (115). The experiments on cell cultures, which allow investigation of the physicochemical factors affecting microtubules, will be of great assistance in the evaluation of potential anticancer agents (143). As for problems of resistance, two types are possible: a specific resistance owing to a mutation in the gene coding for tubulin, leading to a decreased affinity of the drug for its receptor (144), and a nonspecific resistance (145) related to an overproduction of a family of P-glycoproteins (146,147). Although tubulin is the most probable target within the cell, it is possible that inhibition of mitotic spindle formation is, in fact, not essential to the antimitotic activity of taxol. Another mechanism has been proposed by Gupta and Dudani (148) whereby taxol, by interacting with membrane-bound tubulins of cytoplasmic organelles, alters calcium regulation. This is supported by observations on mitochondria1 matrix proteins of taxol-resistant mutants. This alteration of calcium regulation would, as a side effect, lead to mitotic spindle dysfunction (calcium being implicated in the tubulin-microtubule equilibrium). The study of a therapeutic agent requires knowledge of its pharmacokinetics and metabolism. Pharmacokinetic studies, generally associated with phase I clinical evaluations, in turn require a method by which a drug is identified and quantified. In the case of taxol, HPUJ appears to be the only method (149) which allows determination of the kinetics of elimination of this drug in treated patients: only 5% of the taxol can be recovered in urine after 24 hr (150). Concerning the metabolism of taxol (l),only one report (151) mentions its unsurprising transformation, in culture media, to 7-epitaxol(23a) by way of the retroaldol mechanism described above. Although no metabolites of taxol have been found in the urine of treated rats (152), a more recent study (153) uncovered the presence, in rat

232


bile, of taxol derivatives in which the aromatic groups were hydroxylated (141a,b, Fig. 7), as well as baccatin I11 (13b), resulting from hydrolysis of the side chain at position 13. One of the major problems with water-insoluble compounds such as taxol which must be administered intravenously is obtaining a stable, homogeneous, nontoxic solution of the product. Currently, taxol is dissolved in ethanol, and Cremophor, a polyoxyethylated castor oil, is added (3). This is somewhat less than ideal as the adjuvant is toxic and can provoke serious allergies (154).As an alternative, use of a triacetin-based emulsion has been suggested (155). Numerous phase I clinical trials have already been conducted in the United States in which, using various protocols (156-160), the effects of taxol on solid tumors and on adult leukemia [a single study (161)l were evaluated. These studies have permitted determination of the doses (200-250 mg/m2) and the protocol (30 mg/m2/day preceded by treatment with glucocorticoids and antihistamines) recommended for phase I1 evaluation, keeping in mind the main side effects caused by the solubilizing agent as well as leukopenic and neurotoxic effects. These phase I1 trials are currently underway, and only the results of one study (on a renal carcinoma) are presently available (162).It should be noted that one of the main obstacles to more rapid development of therapeutic uses of taxol is the very limited quantities of this drug available, for reasons already discussed. In addition to the toxicity owing to Cremophor, taxol itself is mainly neuropathic, both upon injection ( 1 6 3 , as a result of problems arising from nerve regeneration, as well as 1 to 3 days after injection (164). Other authors (165) have detected epithelial necrosis in the gastrointestinal tract as a result of the accumulation of polymerized microtubules and consequent mitotic arrest. Since the discovery of taxol in 1971, a large number of analogues have been extracted or synthesized. An attempt to tabulate these derivatives in order to extrapolate structure-activity relationships would be pointless as the structural

FIG. I

6.

233

TAXCJSALKALOIDS

modifications are all quite minor and, in most cases, the primary baccatin 111 framework (13b) is found. With regard to the biological tests used to determine the activity of these analogs, two major techniques are available which give satisfyingly comparable results: the in vitro test on tubulin [promotion of polymerization (166) or inhibition of depolymerization (167)] and cytotoxicity tests on various cell cultures (2,166,168). These studies have led to the conclusion that, whereas the nature of the substituents carried by the hydroxyl groups at positions 7 and 10 may be varied considerably, a taxol-type side chain at position 13 is essential for activity (2,3,166). Though some variability is allowed within the side chain itself, a free hydroxyl group at the 2' position is important (especially in vitro)as in the taxol-type configuration at 2' and 3' (11,32).Kingston et al. have demonstrated the importance of the oxetane ring both in assuring binding of these derivatives to tubulin and in maintaining the conformation of the taxane skeleton (41). An interesting analog (142, Fig. 8) suitable for photoaffinity labeling studies has recently been described (169); having a good affinity for tubulin, this compound could greatly help in elucidating the interactions between taxol and its binding site on tubulin. Although taxol itself continues to be widely studied both biologically and clinically, new analogs are currently being hemisynthesized with the double aim of improving therapeutic activity as well as ease of administration. The latter implies finding a compound which is soluble in water. Application of these two approaches has led to (a) the synthesis of a derivative of 10-deacetyltaxol (143) in which the benzoyloxy group on the nitrogen at C-3' has been replaced by a tert-butoxycarbamate (47) and (b) the synthesis of a series of taxol analogs in which the alcohol at C-2' is esterified by acids having an amino group, thereby allowing formation of water-soluble salts (144) (170). These latter molecules no doubt act as taxol prodrugs. The activities reported for these derivatives appear to be very interesting, being even greater than taxol in vivo. However, because 144

1 142

H

2

3

4

Ac

H

Ph

H

Ph

H

OtBu

% N o N CF3

H,

N

04

u.3 \

Ph

R4

FIG. 8

H

H

234


is synthesized from taxol, the usual problem of obtaining adequate supplies of these materials persists. This is not the case for analog 143, which is synthesized from the easily obtained 10-deacetylbaccatin 111.

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in press. M. Lassus, D. Scott, and B. Leyland-Jones, Proc. ASCO 4, 268 (1985). B. D. Tarr, T. G. Sambandan, and S. H. Yalkowsky, Phurm. Res. 4, 162 (1987). J. L. Grem, K. D. Tutsch, and K. J. Simon, Cancer Trear. Rep. 71, 1179 (1987). P. H. Wiemik, E. L. Schwartz, A. Enzig, J. J. Straumann, R. B. Lipton, and J. P. Dutcher, J. Clin. Oncol. 5 , 1232 (1987). 158. S. S. Legha, D. M. Tenney, and I. R. Krakoff, J. Clin. Oncol. 4, 762 (1986). 159. M. S. Kris, J. P. O'Connell, R. J. Gralla, M. S. Wertheim, R. M. Parente, P. B. Schiff, and C. W. Young, Cancer Treat. Rep. 70, 605 (1986). 160. R. C. Donehover, E. K. Rowinsky, and L. B. Grochow, Cancer Treat. Rep. 71, 1171 (1987). 161. E. K. Rowinsky, P. J. Burke, J. E. Karp, D. S. Ettinger, R. W. Tucker, andR. C. Donehover, Proc. Am. Assoc. Cancer Res. Annu. Meet. 29, 215 (1988). 162. A. I. Einzig, E. Gorowski, J. Sasloff, and P. H. Wiemik, Proc. Am. Assoc. Cancer Res. Annu. Meet. 29, 222 (1988). 163. M. Roytta and C. S. Raine, J. Neurocyrol. 15, 483 (1986). 164. R. B. Lipton, S. C. Apfel, J. P. Dutcher, R. Rosenberg, J. Kaplan, A. Berger, A. I. Einzig, P. Wiernik, and H. H. Schaumburg, Neurology 39, 368 (1989). 165. R. H. Hruban, J. H. Yardley, R. C. Donehover, and J. K. Boinott, Cancer 63, 1944 (1989). 166. J. Parness, D. G. I. Kingston, R. G. Powell, C. Harracksingh, and S. B. Horwitz, Eiochem. Eiophys. Res. Commun. 105, 1082 (1982). 167. H. Lataste, V. Senilh, M. Wright, D. Guenard, and P. Potier, Proc. Narl. Acad. Sci. U.S.A. 154. 155. 156. 157.

81, 4090 (1984). 168. D. B. McKay, J. Phurmucol. Exp. Ther. 248, 1302 (1989). 169. G. Samaranayake, D. G. I. Kingston, and P. Wong, Absrr. Pap. Am. Chem. SOC.5271 (1988). 170. H. M. Deutsch, J. A. Glinski, M. Hernandez, R. D. Haugwitz, V. L. Narayanan, M. Suffness, and L. H. Zalkow, J. Med. Chem. 32, 788 (1989).

-CHAPTER7SYNTHESIS AND ANTITUMOR ACTIVITY OF ELLIPTICINE ALKALOIDS AND RELATED COMPOUNDS GORDONW. GRIBBLE Deparhnent of Chemistry Dartmouth College Hanover, New Hampshire 03755

I. Introduction ................................................. 11. Occurrence and Structural Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............. 111. Synthesis of Ellipticine . . . . . . . . . . . . . . . . . . . . IV. Synthesis of Olivacine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Synthesis of Modified Ellipticine Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Synthesis of Substituted Ellipticines . . . . . . . . . . . . . . . . . . . B. Synthesis of Isoellipticines .................................... C. Synthesis of Azaellipticines . . . . . . . . . . . . . . . . . . . . . D. Synthesis of Nonlinear Pyridocarbazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Synthesis of Oxazolopyridocarbazoles . . ................ F. Synthesis of Tricyclic Analogs . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . .

VI. VII. VIII. IX. X.

XIII. XIV.

239 240 242 250 254 255 266 27 1 274 219 283 29 1 . . . . . . . . . . . . . 294 H. Synthesis of Ellipticine Conjugates . . . . . 300 I. Synthesis of Miscellaneous Analogs . . . . . . . . . . 305 Biological Detection .................................... Antitumor Activity in . . . . . . . . . . . . . . . . . 307 31 I Mechanism of Action . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 ............................... Mutagenicity . . . . . . . . . . . . . . 325 Metabolism and Microbial Transformation . . . . . . . . . . . . . . . . . . . . 328 340 340 Clinical Trials .................... . . . . . . . . . . . . . . . . . . . 343 Conclusion . . . . . . . . . . . . . . . . . . . . . . . 343 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction

This chapter deals with the synthesis and biological properties of the relatively small family of pyrido[4,3-b]carbazole alkaloids, exemplified by ellipticine (l), 9-methoxyellipticine (2), 9-hydroxyellipticine (3), and olivacine (4), and of the much larger number of structural analogs that have been synthesized and studied 239

THE ALKALOIDS, VOL. 39 Copyright 0 1990 by Academic Press, Inc. All rights of reproduclion in any form reserved.

240

GORDON W. GRIBBLE

following the initial discovery of the antitumor properties of these alkaloids (15 ) . Indeed, although synthetic activity in this area has been intense and constant in the 30-year period since the original isolation of these alkaloids (6,7),the introduction into the cancer clinic of 9-methoxyellipticine lactate in 1969 (8) and, especially, "elliptinium" (2-methyl-9-hydroxyellipticinium acetate) (5) in 1977 (9) has triggered an explosion of activity, both in the synthesis of pyridocarbazoles and related ring systems and in their biological evaluation. Thus, even though this area was comprehensively reviewed by Suffness and Cordell (10) in Volume 25 of this treatise [with coverage through December 1984 and some additional references to April 1985, and including much unpublished data from the National Cancer Institute (NCI)], the wealth of new material justifies the present review. This chapter covers the literature from 1985 through most of 1989. In addition to the excellent Suffness and Cordell review (lo),there are several other important articles that provide coverage of the synthesis andlor biological profile of the ellipticine alkaloids (11-21). Owing to the large number of pyridocarbazole structural variants to be discussed, the Chemical Abstracts pyridocarbazole numbering system (l),rather than the alkaloid biogenetic pathway numbering system, is used throughout this chapter.

11. Occurrence and Structural Determination Ellipticine (1) and 9-methoxyellipticine (2)have been isolated from Ochrosia acurninata stems (22) and from in vitro callus cultures derived from the stems of Ochrosia elliptica (23,24). It is found that the in vitro production of these alkaloids can be increased by cloning small cell aggregates. A noteworthy development is the isolation and characterization of the first

1

2

R=H R=OCH3

3 R=OH

7.ELLIPTICINE

ALKALOIDS AND RELATED COMPOUNDS

24 1

naturally occurring bisellipticine alkaloid, strellidimine (8), from the African tree Strychnos dinklugei Gilg. (Loganiaceae) (Scheme 1) (25). it is evident that this optically inactive alkaloid is formed in vivo by the coupling of 9-hydroxyellipticine (3) and 3,4-dihydroellipticine (7), both of which are present in S. dinklugei (26). This biogenesis was demonstrated by the biomimetic synthesis of 8 shown in Scheme 1. Oxidation of 3 to the ellipticine quinone imine 6 with horseradish peroxidase (HRP) and hydrogen peroxide in the presence of 7 gave strellidimine (8) in quantitative yield (25). A method for the separation of ellipticine (l),9-methoxyellipticine(2), and 9hydroxyellipticine (3) using cellulose adsorption chromatography (thin layer or paper) has been developed (27). The technique involves a solvent system consisting of a 50 : 50 mixture of 1.3 M ammonium sulfate and 96% acetic acid, followed by iodine vapor detection. An X-ray crystal structure of 9-methoxy-l l-demethylellipticine (9) (28) reveals little difference in geometry from that previously observed in ellipticine and its derivatives (29,30). Thus, the absence of a methyl group at C-11 and the presence of the 9-methoxyl substituent does not alter the pyrido[4,3-b]carbazole structure. An X-ray crystal structure study of the charge-transfercomplex formed between 9-methoxyellipticine (2) and 7,7,8,8-tetracyano-p-quinodimethane

yJ--& 0 6

CH3

+

Q @

pH 7.4

30 rnin

0

- 100%

H 7

CH3

SCHEME1. Structure and biomimetic synthesis of strellidirnine (8) (25)

*

242

GORDON W. GRIBBLE

(TCNQ) (10) reveals a stacking sequence between electron acceptor (10) and electron donor (2) as follows: 10-2-2-10 (31). In what, it is hoped, will be the final word on the NMR chemical shift assignments of 9-methoxyellipticine (2), Commenges and Rao (32) have revised several of the I3C-NMR assignments published earlier by Sainsbury and coworkers (33). In the present work, a combination of two-dimensional (2D) NMR techniques ('H-IH homonuclear and 'H- 13C heteronuclear chemical shift correlations, including long-range 'JCHdeterminations) and the reinvestigation of model compounds(5-methoxyindole and 1,4-dimethylcarbazole)has required the reassignment of C-4, C-5, C-7, C-8, C-10, C-lOa, C-lob, C-11, and C-1 la of 9methoxyellipticine (2). The full set of 'H and I3C chemical shifts is given in Scheme 2.

111. Synthesis of Ellipticine

In addition to the reviews cited earlier (10-14,16,19), several other reviews covering the previous synthetic efforts toward ellipticine are available (34-37). This section deals with new synthetic approaches to the ellipticine skeleton only; 123.3 7.89

7.20 7.49

11.2

3.27

9.69

CH,

H

2.77 7.91

153.1

123.6

8.41

137.3

107.8

SCHEME 2. 'H- and I3C-NMR chemical shifts of 9-methoxyellipticine (2) in parts per million downfield from tetramethylsilane in (methyl sulfoxide)-d6 (32).

7.

ELLIPTICINE ALKALOIDS AND RELATED COMPOUNDS

243

derivatives of ellipticine as well as variations of the pyrido[4,3-b]carbazole ring system are covered in Section V. Differding and Ghosez (38) have reported a novel and highly convergent construction of the 6H-pyrido[4,3-b]carbazolering system, involving, as the key step, an intramolecular Diels-Alder cycloaddition of a vinylketenimine (18 --., 19) (Scheme 3). Piperidone 11 was converted to the unsaturated ester 12 by an Emmons-Wadsworth reaction, and then deconjugation and saponification gave acid 14. Conversion to acid chloride 15 and acylation of aniline 16 gave the amide 17. In situ formation of the vinylketenimine 18 was accomplished with triphenylphosphine dibromide to yield, after the facile Diels-Alder cycloaddition, tetracycle 20 after tautomerism of the initially formed 19. Reduction of the ester group in 20 gave the (unnamed) alkaloid 21 which had been previously converted to ellipticine (1) by dehydrogenation/demethylation(39). The overall yield of 1 from 11 is 5%, although the final step obviously reduces the overall yield drastically. Surprisingly, this strategy-wherein more than one ring of the final tetracycle is formed in a single step-apparently has not been previously pursued in the construction of pyridocarbazoles. Miyake and co-workers (40) have published a synthesis of ellipticine that features a novel reductive phenylation of nitroarenes (41) (Scheme 4). Nitration of 5,g-dimethyl- 1,2,3,4-tetrahydroisoquinoline(22) gave an inseparable mixture of nitro compounds 23. Treatment of this mixture with iron pentacarbonyl and triflic acid in the presence of benzene gave a 2 : 1 mixture of amines 24 and 25. Separation of these isomers and diazotization of each with nitrous acid, conversion to the azide, and thermolysis yielded ellipticine (1) and “isoellipticine” (27) (5,l I-dimethyl- 10H-pyrido[3,4-b]carbazole),respectively, following Pd/C dehydrogenation of the initially formed nitrene insertion product (e.g., 26). The overall yield of ellipticine is 9%. Ketcha and Gribble (42) have adapted the earlier Saulnier-Gribble synthesis (43) of isoellipticine quinone to the synthesis of ellipticine quinone 33 and hence to ellipticine (Scheme 5). A refinement of the earlier 3-lithioindole technology (43) involves the direct Friedel-Crafts acylation of 1-( phenylsulfony1)indole (31) to introduce the 3-acyl group. Thus, the inherent regioselectivity of 3,4pyridinedicarboxylic anhydride (28) (cinchomeronic anhydride) was reversed by conversion of the known ester acid 29 to acid chloride 30. Acylation of indole 31 with 30 gave keto ester 32. Final closure to quinone 33 was accomplished using tandem in situ carbonyl protection and deprotonation at C-2, followed by cyclization. Since quinone 33 had been previously converted to ellipticine by Joule and co-workers (44),this work represents a formal synthesis of 1, in 13% overall yield from anhydride 28. The identical protocol applied to keto acid 34, obtained via Friedel-Crafts acylation of 1-( phenylsulfony1)indole (31) with pyridine anhydride 28, provided isoellipticine quinone 36 and, hence, isoellipticine (27) in 13% overall yield from the N-protected indole 31 (42) (Scheme 6).

6

COZCH,

1. LDA

( E10)2POCH(CH3)C02Me

NaHA € 1 2 0

I

THF -68°C

CH3

12

13

HzN

H3cYC02H 1. HCI

MeO,C-CaC

CHpClp 2. (CH,),C I

75%

I

-100%

CH3

75%

2 h 60°C

1h

2. NHiCl

I

CH3 11

1N KOH

-4 H3C

CH3

14

16

Ei3N

=C(CI)NMe, CHzCIz 20°C

I CH3

75%

15

Ph3PBr2 t

C I

I CH3

__c

ET3N CH2CIp A

50%

2.5 h

17

H3c?F) COZCH,

I

CH3

COZCH,

-

I

~ c H 3

/

/

20

€120

CH3

t

A

1.25 h

71Yo

10% Pd/C

/

AICl3

NH

19

*0cH3 /

LiAIH4

l

36%

CH3

21 SCHEME 3. Differding-Ghosez synthesis of ellipticine (1) (38).

*

7.


iH3

CH3

22

23

245

CH3 25 (22%)

I 10% Pd/C 1A

46‘70

2. ” N$02 NaN3

WAC /

/

H

CH3 26

SCHEME4. Miyake et al. synthesis of ellipticine (1) and isoellipticine (27) (40).

Ketcha and Gribble (42) have also used this methodology to convert phthalic anhydride (37)to the benzo[b]carbazole quinone 38. In two papers, Miller and co-workers (45,46) have extended their intramolecular ring B cyclization strategy (47) to the use of aryl nitrenes in the synthesis of pyridocarbazoles. Thus, in the first paper, isoquinoline azide 39 was heated at 180-200°C to afford ellipticine (1) as the minor product (20%) (Scheme 7). The major product was the isomeric pyrido[3,4-~]carbazole40 (60%).This result is consistent with the relative nucleophilicitiesof C-6 and C-8 of isoquinoline. The isomeric azido isoquinoline 41 exhibited comparable regioselectivity in the cyclization of the corresponding nitrene to yield isoellipticine (27)as the minor product (Scheme 7).

246

GORDON W . GRIBBLE

7

0 % 0

0

37

38

The second paper (46) describes an improvement in the nitrene strategy by merely switching the location of the azide group to the C-6 position of the isoquinoline ring (Scheme 8). Using their previously developed isoquinoline synthesis (48),Miller and Dugar prepared 47 in several steps from acetanilide 43 via indene 46. A Suzuki reaction with 47 gave the requisite amine 48, which, upon diazotization and trapping, afforded azide 49. Heating 49 gave ellipticine (1) in excellent yield and in 41% overall yield from 43.

1. NaOMe

MeOH THF -70°C + rt

C0ZCH3

PhH A 78%

._

73%

28

-

-

2. aqHCI

29

&cocl

QCTJfyl I

AICl3 CH2C12 25°C 50%

30

COZCH,

0

C0ZCH3

SOZPh

32

1. LiN(Me)CH2CH2NMe2 THF -75°C 2 h

*fJ--& - 1. 1 CHBLi

2. (TMS)*NLi THF -75'C+rt

47%

2. NaBH4 0

94%

33

SCHEME 5 . Ketcha-Gribble synthesis of ellipticine quinone 33 and ellipticine (1) (42).

07

+

0

-

N*O

25°C 2 h

S0,Ph

J

J IN SOzPh

69% 28

31

34

0 EtOH

@

COZH

COZEt 1. LiN(Me)CHzCHzNMez M F -75°C 2 h

OTMN

pTsOH

PhH A 3days 75%

*

2. (TMS)2NLi THF -75°C + rt

I

SOzPh

37%

35

36

27

0-w

SCHEME 6. Ketcha-Grihhle synthesis of isoellipticine quinone 36 and isoellipticine (27) (42).

0

/

180 dodecane - 200°C

(20%) 1+fJ--QH3

\

CH3

39

40 (60%)

dodecane

CH3 41

w

2 7 +

1

.

N

07gH 3

\ 42

SCHEME7. Miller et al. synthesis of ellipticine (1) and isoellipticine (27) (45).

N

248

GORDON W. GRIBBLE

1. CICH2CH2C02H CS2 AIC13

2. AIC13, NaCl 180°C

AcHN CH3 43

- @ H2N

CH3

3. 2NHCI A 84%

2. 40%H2SO4 THF A 3. Ac20 NaOAc

45 CH3

2. CH3COCI Et3N THF rt 94%

-78°C MeOH CH2CI2

1. 0 3

Bf*

t

AcHN CH3

75%

2. (CH&S NaHC03 3. NH40H 4. 2N HCI A

46

85%

1. NaN02 dil HCI 0°C t

PhB(OH)2

Br@

H2N

____)

44

1. NaBH4 MeOH 0°C E rt AcHN Br*

1. Br2, l2cat CH2C12 rt

\ CH3 47

/

Pd(PPh3)4(cat) PhH A 2M Na2C03 99%

/

\

2. NaN3

85%

H2N CH3 48

180°C -

CH3

1

96%

49

SCHEME8. Miller-Dug= synthesis of ellipticine (1) (46).

Zee and Su (49) have modified the original Woodward et al. (7) synthesis of ellipticine, as improved by Sainsbury and Schinazi (50) and Berman and Carlsson (51), to achieve a convenient and reasonably efficient synthesis of ellipticine (1)(Scheme 9). Bergman’s improved method (51) was used to prepare 3-vinylindole 53, which, after catalytic hydrogenation to 54, was reductively acetylated directly to afford 2-acetyl-1,2-dihydroellipticine(55). Hydrolysis and aromatization completed the synthesis of ellipticine (1) in 12% overall yield from indole (50).

7.

249


CH3

CH3 I

I

< 1 torr

H 50

50%

H

10h 81%

52

51

Zn

HZ 20psi CH30H 5‘X0 P 6dc ’h ,

o : - i NH

0

7 H -

i

N

Ac20

A48YO2 h 53

90%

54

CH3 I

10% H2S04 ____t

CH30H air A 6h

68%

SCHEME9. Zee-Su synthesis of ellipticine (1) (49).

May and Moody (52) have reported a full account of their Diels-Alder cycloaddition route to ellipticine (1) and isoellipticine (27) (Scheme 10). Conversion of indole (50) to 3-indole-2-propionic acid (56) with lactic acid was followed by a Plieninger cyclization to the pyranoindole 57. Reaction of 57 with 3,4-pyridyne (59), as generated from triazene 58, afforded equal amounts of ellipticine (1) and isoellipticine (27). Although the overall yield of 1 from indole is only 3%, the sequence involves only three steps. In an effort to overcome the lack of regioselectivity in the cycloaddition of 3,4pyridyne (59) with dienes, such as 57 (Scheme lo), or furo[3,4-b]indoles (e.g., 60) (53),Davis and Gribble (54) have utilized unsaturated lactams 61 and 62 as

250

GORDON W. GRIBBLE

lactic acid 250°C

@--j---jCOzH -0 7 BF3 Et20

H

32%

H

43%

56

50

59

57

CH3

1 0 ‘ H

/

N H

CH3 1 (20%)

CH3 27 (20%)

58

SCHEME 10. May-Moody synthesis of ellipticine (1) and isoellipticine (27) (52)

3,4-pyridyne surrogates (Scheme 11). Work with model dienophiles and a frontier molecular orbital analysis of furo[3,4-b]indole 60 led to the prediction that the “ellipticine orientation” would obtain (54). Thus, the dimethylfuroindole 60, prepared from 3-ethylindole as previously described (53),was treated with unsaturated lactam 61 (prepared from 8-valerolactam in three steps) in the presence of trimethylsilyl triflate to give lactam 63 as a single product. Difficulty in removing the benzyl group forced these workers to synthesize the p-methoxybenzyl analog 62. The Diels-Alder cycloaddition reaction yielded the adduct 64, which was converted to ellipticine (1) by reduction and dehydrogenation. Control experiments with mixtures of 1 and isoellipticine (27) revealed that the Diels-Alder cycloaddition leading to 64 was at least 99% regioselective. However, the overall yield of l from 60 thus far is a disappointing 18%, owing to the difficulty in manipulating the D ring.

IV. Synthesis of Olivacine As we see in later sections, olivacine (4), the oft forgotten cousin of ellipticine, is receiving renewed attention as the search for improved antitumor pyridocarbazoles continues. Nevertheless, the number of new synthetic routes to olivacine is few.

0 N

7.


25 1

0

2.

61 62

60

CH3

aqNaHC03

R = PhCH2 (76%) R = p-CH3OPhCH2 (88%)

0 1 . LiAIH4

THF 2. PdIC

decalin

A

20%

63 R = P h C H 2 R = p-CH30PhCH2

64

SCHEME1 1 . Davis-Gribble synthesis of ellipticine (1) (54)

Using Husson’s method (39), Maftouh and co-workers (55)have described syntheses of 7-hydroxy- (71) and 9-hydroxyolivacine (73). Thus, 7-methoxyindole (65) was condensed with enamine ketal66 (prepared from the corresponding pyridine 67) to give carbazole 68 (Scheme 12). Standard ring D construction involving a Bischler-Napieralski cyclization (39) gave the tetrahydroolivacine derivative 69. Dehydrogenation and demethylation completed the synthesis of 71. In identical fashion, the synthesis of 9-hydroxyolivacine (73) was accomplished (55)by the demethylation of the alkaloid 9-methoxyolivacine (72), which had been previously synthesized by Besselievre and Husson (39). Using the Cranwell-Saxton synthesis (56)of ellipticine, as modified by Birch et al. ( 5 3 , Sainsbury and co-workers (58),have described a new 3-acylcarbazole synthesis and its application to a synthesis of olivacine (4) (Scheme 13). Reaction of gramine (74) with the appropriate biscyano ketone gave 75. Cyclization in acetic acid afforded 76, which, upon treatment with hot silica gel, underwent dehydrocyanation and tautomerization to give cyanocarbazole 77. Reduction to aldehyde 78 was followed by imine 79 formation. The addition of methyllithium and p-toluenesulfonyl chloride gave carbazole acetal80. Ring D was crafted by the standard hydrogen chloride ring closure. The final dephenylsulfonylation was performed with sodium to give olivacine (4). Surprisingly, sulfonamide 81 was very stable in acid, but, more importantly, no cyclization to the alternative pyrido[3,6c]carbazole 82 was detected. Although the methyl ketone 83 could be easily prepared from nitrile 77, condensation of the latter with amino

1. CH31 CH&N A 2. NaBH4 81%

I

OCH, 65

66

67

50% HOAc 82%

A

56 h

t

pyr 65%

1. Ac20 l h rt

WHCH3 \

/

OCH3

*

2. POC13 CHC13 A 10h 3. NaBH4

CH3

68

CH30H

CHCl3

39% 7H3

9

-

-

g

C

H

OCH,

y

A

3

CH3

3

9-q

24h 22%

/

H

OCH,

69

/

CH3

70

48% HBr l h

A

70% OH

CH3 71

SCHEME 12. Maftouh et al. synthesis of 7-hydroxyolivacine (71) (55)

cH30n--""n48% HBr

'

/

N

H CH3

/

A l h

70 o/'

'

/

N

H CH3

73

72 252

/

o - q c : N

250°C silica gel

0--qCN 5 94%

55% 76

77

CH3

SCHEME 13. Synthesis of olivacine (4) by Sainsbury and co-workers (58).

254

GORDON W . GRIBBLE

oq

r

2. LiBHEt3

85

'

-1 00°C

I L

OH

1

-Njl'H ,

86

J

NaBH4 EtOH A 57%

SCHEME14. Gribble-Obaza-Nutaitis synthesis of olivacine (4) (60).

acetaldehyde acetal to the olivacine precursor 84, in contrast to a prior report (59), could not be effected (58). In unpublished work, Gribble and Obaza-Nutaitis (60) have adapted the Saulnier-Gribble ellipticine synthesis (61) to the synthesis of olivacine (Scheme 14). Keto lactam 85, available from indole in four steps (71% yield) (61), was treated sequentially with methyllithium and lithium triethylborohydride to give diol 86, which, without isolation, was reduced with sodium borohydride to give l-demethylolivacine (87). This had been previously converted to olivacine (4) by Kutney and co-workers (62).The success of this synthesis of 87 was due to the fact that Saulnier and Gribble (63) had previously established that the ketone carbonyl of keto lactam 85 is more reactive than the lactam carbonyl group.

V. Synthesis of Modified Ellipticine Derivatives

Most of the new synthetic work on ellipticine in the late 1980s has dealt with modified ellipticine derivatives. Several French groups have made enormous strides in the design and synthesis of ellipticine analogs. The material in this section is divided into nine areas: A. Synthesis of Substituted Ellipticines; B. Synthesis of Isoellipticines; C. Synthesis of Azaellipticines; D. Synthesis of Nonlinear Pyridocarbazoles; E. Synthesis of Oxazolopyridocarbazoles; F. Syn-

7.

255


thesis of Tricyclic Analogs; G. Synthesis of Bispyridocarbazoles; H. Synthesis of Ellipticine Conjugates; and I. Synthesis of Miscellaneous Analogs. OF SUBSTITUTED ELLIPTICINES A. SYNTHESIS

Although direct substitution on the ellipticine nucleus is rare, a number of important such developments have been reported since 1984. Pandit and coworkers (64,65)have achieved excellent success in the important introduction of a hydroxyl group into the C-9 position of the pyrido[4,3-b]carbazole nucleus (Scheme 15). Thus, 6-methylellipticine (88), prepared from 1 in 81% yield

88

89

CH3COCI

CICH20CHC12 AIC$ 0°C 3 h CH2C12

91%

96% CH3

0

CH3

CH3

CH3

91

90

93

92

CH,

SCHEME 15. C-9 hydroxylation of 6-methylellipticine (88) by Pandit and co-workers (64,65).

256

GORDON W. GRIBBLE

[NaH, dimethylformamide (DMF), CH,I], undergoes electrophilic nitration (89), Friedel-Crafts acylation (90), and alkylation (91) at the C-9 position. Although attempts to effect a Baeyer-Villiger oxidation of ketone 90 were successful, the route was laborious since oxidation to amine oxide 92 preceded oxidation of the methyl ketone 90. However, a Dakin reaction of aldehyde 91 gave 9-hydroxy-6methylellipticine (93) in excellent yield. It remains to be seen if this methodology can be extended to an N-unsubstituted ellipticine. In attempting to functionalize the C-1 1 position of ellipticine-in the reasonable belief that it resembles electronically the C-2 methyl group of 2-methylpyridine N-oxide-Pandit and group (66,67) prepared 6-methylellipticine Noxide (94) (Scheme 16). However, treatment of 94 with acetic anhydride led not to the anticipated 97 but rather to pyridones 95 and 96, in what represents a new functionalization of the C-1 position. However, the fact that the C-11 methyl group is in potential conjugation with the pyridine nitrogen allowed Pandit and co-workers (66,67) to deprotonate selectively this position with lithium diisopropylamide (LDA), and, after quenching 98 with formaldehyde, they were able to prepare several novel glycosides (e.g., 99-101) (Scheme 17). Honda and team (68-70) at the Suntory Institute have reported a simple synthesis of N-2 ellipticine glycosides (e.g., 104) (Scheme 18), which have high water solubility and extraordinary antitumor activity (see Section XI). A key feature in the preparation of these quaternary glycosides is the use of cadmium carbonate, which seems to enhance the remarkable 1 ,2'-trans stereoselectivity of the condensation step.

88

rn-CPBA CH2Cl2 rt 75%

nT@RoAc20

/

/

NaOAc A 40%

CH,

CH, 94

0 II

95 R = H 96

97

R=COCH3

SCHEME16. C-l functionalization of 6-methylellipticine (88) by Pandit and co-workers (66,67).

7.

257


-

CH,

CH3

Q)98

1 P HCHO

56%

CH3

/

CH3

CH3

/

CH,

q-% "O H /

1. AcO& AcO

OAc OAc

OH I

/

CH,

'

CH,

CH3 99

25%

CH3 100

/

CH,

CH, 101

SCHEME 17. Synthesis of ellipticine glycosides (99-101) by Pandit and co-workers (66,67).

Werbel and co-workers (71) have synthesized several N-6 (88, 105-109) and some N-2 (110, 111) amino and alkyl derivatives of ellipticine (1) by straightforward alkylation methods. Paoletti and co-workers (72) have reported the synthesis of a variety of 1-amino-substituted 9-methoxyellipticines (Scheme 19) in a continuing study of the antitumor properties of these compounds. The starting 1-

258

GORDON W . GRIBBLE

102

103

104

SCHEME 18. Honda er al. synthesis of quaternary ellipticine glycosides (e.g., 104) (69).

chloroellipticine (112) is available from the corresponding pyridone by treatment with phosphorus oxychloride (73). The subsequent reactions with amines or ammonia were typically carried out neat. The 1-amino substituted 9-hydroxyellipticine derivatives were synthesized by starting with the 9-benzyloxy derivative 119 as shown below for the preparation of 120 (72). Bisagni and co-workers (74) have synthesized the 1-chloroellipticine 126 in unique fashion (Scheme 20). Condensation of the readily available aldehyde 122 with lithiated chloropyridine 124 gave alcohol 125. Ionic hydrogenation and cyclization afforded 126 in 35% yield from aldehyde 122. This route is much shorter than an 11-step procedure reported earlier (75).

1. NaH

DMF rt 0.5 h DMF

R

CH3

tt 17h 2.5- 55%

88 R=CH3 R = (CH&NMez R = CHZCH~N(CH~CHZ)~O R = CH2CH2N(CH2)5 R = CH2CHzNEtz R = CH2CONEt2

105 106 107 108 109

RX

Et3N MeOH

rt 20 - 100%

H 110 111

X-

CH3

R=CH3,X=l R = CHzCHzNEtz,X = Br

7.


H

259

H CH,

CH, 114

120°C 7 days

150°C 3days

CH3

CI

112

23 - 54%

CH,

NHR

H 115 116 117

CH, R = CH2CHzCH3 R = CHzCH(CH3)z R = CH2CHzCH(CH&

H CH, 118

SCHEME 19. Synthesis of 1-amino-substituted 9-methoxyellipticines 113-118 by Paoletti and coworkers (72).

Gansser ef al. (76) have employed a Cranwell-Saxton synthesis (56) to prepare 9-(dimethylamino)ellipticine (130) from 5-(dimethy1amino)indole (127) (Scheme 21). To avoid formylation of the carbazole N-9 position, it was necessary to use the hydrochloride of 128. However, the yield of the desired aldehyde 129 was still very poor (3%) as formylation at C-5 was a side reaction. Finally, the Dalton modification ( I ) was used to form the D ring. Gansser and co-workers

260

GORDON W . GRIBBLE

CH,

CI

1. H2NCH2CH2CH(CH&

90°C 2days

*

2. H2 Pd/C

EtOH 60°C 24h 45% 119

H

CH3 120

cH3073-I

CH,

DMF A 87%

>-

I

121

122 CH3

Co '

LDA P

THF -70°C

0

CH3

CH, 123

124

OH

CI

Cl 1. Et3SiHKF3C02H*

c

H

3

0

0

y

@

/

2. 50%H2S04

4 h 60°C 54% 125

CH, 126

SCHEME20. Bisagni et al. synthesis of 1-chloropyridocarbazole 126 (74).

CH,

/

MezNa~ i57.

26 1


+

Bh’Et20

MezNf&.--

CH3

1. HCI EtOH

dioxane A 5h

H CH3

127

39%

*

/ 2. DMF PCCI~

H

9%

CH3

128

aT-cHo

/

1. H2NCH&H(OEt)z M *ezNQT&

/

2h A

CH3 129

2. H3W4 165°C

30 rnin

CH3 130

SCHEME21. Gansser et al. synthesis of 9-dimethylaminoellipticine(130) (76)

(77) have also reported a synthesis of 8-methoxyellipticine (134) using Miller’s strategy (47) (Scheme 22), although both the Goldberg coupling leading to 131 and the final pyrolysis proceeded in very poor yields. The Gansser group (78) also described the synthesis of 9-methoxy-4-hydroxy-1,2,3,4-tetrahydroellipticine (137) (Scheme 23) using the Cranwell-Saxton method (56). Thus, the Dalton intermediate 135 ( I ) , which was prepared from 6-methoxy-1,4-dimethylcarbazole in two steps, was reduced with sodium borohydride and cyclized with mild acid to give a mixture of 137 and the imine 138. Gribble and Saulnier (79)have extended their ellipticine synthesis (43)to the synthesis of 9-methoxyellipticine (2) (Scheme 24). One of the key features of this approach is the regioselective nucleophilic addition to the C-4 carbonyl group of pyridine anhydride 28. The other noteworthy transformation is the conversion of keto lactam 142 to the diol 143 with methyllithium, a process that presumably involves cleavage of the initial adduct to a methyl ketone which undergoes cyclization at the C-3 position of the indolyl anion. Reduction of 143 with sodium borohydride completes the synthesis of 2, in 47% overall yield from 5methoxyindole (139). Gribble and students (80) have also used this method to synthesize 8-methoxyellipticine (134), 9-fluoroellipticine (144), and the previously unknown 7,8,9,10-tetrafluorellipticine(145), each from the appropriate indole. In an improvement of the earlier use of keto lactam 85 to synthesize the alkaloid 17-oxoellipticine (148) (63) [alkaloid numbering ( S I ) ] ,Obaza-Nutaitis and Gribble (82) have found that vinyllithium is an excellent alternative to the more conventional acyl anion equivalents (Scheme 25). Thus, the addition to

N2H4 RaNi

NaN02

*

95% EtOH

/

CH,O

/

-

aq HOAc

H

88%

CH3 132

7H3

500°C CH30

CH30

30/0

H CH3 134

133

SCHEME 22. Gansser et al. synthesis of 8-rnethoxyellipticine (134) (77).

H

CH3

OEt

CH3

88%

135

6N HCI EtOH 28 h

OEt

136

cH300-& :H30n /

H

'

/

N

\

H

N

H CH3 137 (59%)

OH

CH3 138 (28%)

SCHEME 23. Martin-Onraet er al. synthesis of 9-methoxy-4-hydroxy- I ,2,3,4-tetrahydroellipticine (137) (78).

7.

263


-

L

~

cH30Q-

aq MeOH A 5h

H

lOO~/O

139

-

3 g 3 0 7 3 - 4

cH3073--J-+

COZH

-1MeLi 00°C

98% 0

’

141

\

N

cH3073,142

HO

cH30QN@

I

CH3

I HO 143

;aN

’ /

aq EtOH

H

CH3 75% from 142

CH3 2

SCHEME24. Gribble-Saulnier synthesis of 9-methoxyellipticine (2) (79).

keto lactam 85, sequentially, of vinyllithium and methyllithium gave, after reduction of the intermediate diol 146, vinyl ellipticine 147. The cleavage of the vinyl group was surprisingly difficult but was finally achieved with chromic acid and a dispersing agent. Ross and Archer (83,84) have also synthesized 17-oxoellipticine (148) (Scheme 26). Using the Weller-Ford methodology (85), these workers prepared ester 149 and, in a clever maneuver, effected debenzylation via the Krohnke

264

GORDON W. GRIBBLE

1. H,C=CHLi

% 85

\

M F -100°C 2. CH3Li -1 00°C 4 rt 3h

-

Q);p NaBH4

OH

EtOH A 23h

78% from 85 146

N

SCHEME25. Obaza-Nutaitis-Gribble synthesis of 17-oxoellipticine (148) (82).

aldehyde synthesis (86) to afford ellipticine ester 150. Subsequent standard manipulation of the carbomethoxy group gave 17-oxoellipticine (148). Reaction of alcohol 152 with methyl isocyanate (MIC) gave carbamate 153, which has important antitumor activity (see Section VII). Archer and co-workers (84) have used the original Stillwell ellipticine synthesis ( 8 3 , as later exploited by Gouyette et al. (88) to prepare the simple 9hydroxy-6H-pyrido[4,3-b]carbazole(158) (Scheme 27). N-Benzyl-4-piperidone was converted via enamine 154 to the enone 155. Hydrogenation gave a mixture of cis- and trans-ketones 156 which were separately converted to indole 157 by Fischer indolization. Some of the nonlinear pyrido[3,4-~]carbazole (1 7%) was formed from the cis-ketone. Dehydrogenation and demethylation gave the desired 158. Using their earlier developed methodology (89), which is similar to that described by Weller and Ford (85) but developed independently, Pandit and coworkers (90) have synthesized the 3-methyl derivatives of 6-methylellipticine and l-demethyl-6-methylolivacine,165 and 166, respectively (Scheme 28). Acylation of indole ester 159 with 6-methylnicotinoyl chloride hydrochloride (160) in hot sulfolane gave keto ester 161. Alkylation and cyclization gave dihydropyridine 162, which was oxidized with N-benzylacridinium bromide to give salt 163. Reductive debenzylation gave the key intermediate ketone 164. Conversion of 164 to the ellipticine derivative 165 was accomplished by the addition of methylmagnesium iodide, followed by hydroxide-induced decarbox-

7.

265


1. BrCH,

QpJ$TJ

acetone 24 h

/

CH2C6H,NO2

92%

/

2. NaOCH3

149

O N e N ( C H 3 1 2

5 h 25°C

Br-

150

80%

NaOCH3 CH30H

/

C02CH3

CO2CH3

LiAIH4

*)-& \

* / H

/

THF l h rl 56% from 150

C02CH, 151

MIC

rl 3days

CH20H 152

61%

CHZOCNHCH,

II

153

0

148

CHCb A 3.5 h 79%

SCHEME26. Ross-Archer synthesis of 17-oxoellipticine (148) and carbamate 153 (83).

ylation-dehydration. The olivacine derivative 166 was prepared by the reduction of 164 with Red-Al. Using the Birth modification (57) of the Cranwell-Saxton (56)methodology, Narasimhan and Dhavale (91) have described a synthesis of 6-methyl-1 l-demethylellipticine (171) (Scheme 29). Carbazole aldehyde 167 underwent the usual condensation to give imine 168. Direct cyclization of 168 with phosphoric acid gave a mixture of 171 and 172, although only the former could be obtained in pure form (by repeated crystallization). However, reduction of 168 to 169, followed by tosylation and cyclization, gave 171 exclusively. Sainsbury er al. (92) have also employed the Cranwell-Saxton strategy (56)to prepare ring A- and ring D-substituted 9-methoxyellipticine derivatives. The synthesis of amine derivative 177 was accomplished as shown in Scheme 30. An

266

GORDON W. GRIBBLE

(p-ph LUPh 1.

q (

H2 5% RtVA1203

-

dioxane

A 17h 2. H 2 0

154

0

A l h

C H 3 0 0 HCI N H N H 2

*

95% EtOH 4h

155

*c H 3 0 Phn 7

EtOH rt 24 h

0

38% (cis) 61% (trans)

H 156

H

157

I

___)

Ph20 A 2h

H

H

52%

H

158

73%

SCHEME 27. Archer et al. synthesis of 9-hydroxy-6H-pyrido[4,3-b]carbazole(158) (84).

aza Cope rearrangement afforded mainly the desired 174. Some C-3 ally1 product (24%) was obtained along with 7% of the hydrogen chloride addition product. This could be converted to 174 on treatment with sodium hydride (DMF, O°C, 86%). Palladium-catalyzed double-bond isomerization, followed by ozonolysis, gave the aldehyde 175. A Wadsworth-Emmons olefination, followed by formylation, gave aldehyde 176. Hydrogenation of the unsaturated amide, followed by the standard ring D formation and amide reduction, gave the target 177. In related chemistry Sainsbury et al. (92) prepared 178, but ring D formation was thwarted, giving instead the unusual dimer 179. Attempts by this group (92) to prepare C-3-substituted ellipticines by the modified Cranwell-Saxton approach were foiled by the decomposition of the side chain during the Pomeranz-Fritsch reaction. B.

SYNTHESIS OF ISOELLIPTICINES

By “isoellipticine,” we refer to a derivative in which the pyridine nitrogen is in a different ring D position. Compared to the other structural variations described in later sections, the isoellipticines have received little attention. A few

7.

267


0

n ___)

90-1OO"c CH3

CH3 CH3 CH3

CH3

74%

160

159

161

V"/

+I

1. PhCH2Br

90°C 30min 2. Et3N EtOAc

rt 54%

,

'N-Ph

-sD$cA

'Ph

w

CH3

CH3CN rt

95%

I H3C CO2Et H3C 162

'' CH3

CH3

165

Br-

CH3

CH3 CH3

CH3

166

SCHEME 28. Synthesis of 3,6-dimethylellipticine (165) and I-demethyl-3,6-dimethylolivacine (166) by Pandit and co-workers (90).

new syntheses of the so-called isoellipticine (27) have already been noted. A short synthesis of 4-hydroxy-2,5,11-trimethyl-6H-pyrido[3,2-b]carbazole (183) has been published by Viossat et al. (93) (Scheme 31). An X-ray crystal structure reveals that the lactam structure 182 exists in the solid state but the hydroxypyridine form (183) predominates in solution. Using their earlier methodology to synthesize ellipticine (l),Gribble and

268

GORDON W. GRIBBLE

mcHo T L C \

CH,

H 3

HzNCHZCH(0Me)z PhH A 2 h *

/

I

I

86%

CH3

CH,

CH,

20 rnin

OCH,

45%

167

F

I CH3

CH3

171

\

38%

\

N

I

CH, 6NHC' dioxane 1o-2o0c 20h

CH, CH, 170

CH,

172

OCH, OCH,

aq THF PTsCl 83%

CH,

CH,

OCH, OCH,

169

SCHEME29. Narasimhan-Dhavale synthesis of 6-methyl-1 I -demethylellipticine (171) (91).

students (94) developed a synthesis of the lOH-pyrid0[2,3-b]carbazolering system (Scheme 32). Once again, excellent, if not complete, regioselective acylation of 2-lithioindole 184 with pyridine anhydride 185 was attained. The subsequent conversion to keto lactam 186 and the addition of methyllithium, followed by reduction, gave 187, in 60% overall yield from indole. The 7- and 8-methoxyl derivatives of 187 were similarly prepared. The 5H-pyrido[4,3-b]benzoV]indolering system (e.g., 192) represents another type of isoellipticine, and its synthesis has been explored by Bisagni and co-workers (95) (Scheme 33). Azaindole 188 was elaborated by means of conventional lithiation methodology to alcohol 189. A sequence of dehydration, hydrogenation, and chlorination gave 190. Either Vilsmeier-Haack conversion to aldehyde 191 and polyphosphoric acid (PPA) cyclization to the desired ring system 192 or direct cyclization to 192 completed the synthesis. The side chain amines were introduced by heating the components neat to provide 193-195. The methoxyl derivatives 196 and 197 were also synthesized (95). Bisagni and team (96) have also reported a synthesis of the related 5Hpyrido[4,3-b]benzo~indolo-6,ll-quinone ring system (e.g., 200) (Scheme 34). Using a modification of the Watanabe-Snieckus synthesis (97) of ellipticine, the

7.

-

-

NaH DMF

N H

269


CH3

9 0 ° C +II 7h 62%

50°C 6 h 92% 173

1. PdCl2 CH3CN PhH

c

H

3

o

w

1.NaH DMF (E10)2POCH2CONEt2 98% 24h rt t

t

50°C 24h

2. TFAA

89% 2.03 -20°C MeOH CHgI2

"

174

lmkhzole CHO

CH3

:H3 175

79%

C H S N A 3.5h 3. aq NaOH EtOH A 15min 83%

c

H

3

0

~

c

A

NEt,

1. H2NCH2CH(OMe)2 CHo 2.H2 80°CPt02 mlsieve c

latrn rt

H CH3

/

0

H HZ0z l ' d l c

CH3

82% ~ONEI,

176

EtOH latrn 4 h II 90% 3.pTsCl pyr 24h rl 85%

1.HCI doxane 100% 3h 56% OCH3 2. BH3 Me2S THF A 75min OCH, 3.6MHCI I

CONEt,

H

51% 'NEt2

SCHEME 30. Sainsbury et al. synthesis of ring A-substituted 9-methoxyellipticine (177) (92)

French group condensed indole aldehyde 198 with the appropriate metalated aromatic to give, after spontaneous oxidation of semiquinone 199, the desired quinone 200. Conventional amination yielded the target compounds ( e . g . , 201). Many such derivatives were prepared by Bisagni and co-workers (96),including the disubstituted compounds 202 and 203.

270

GORDON W . GRIBBLE

1. rrBuLi

M F -78°C

CH,O

53% 2. pTsCI

ww3

CH,

aq MF

OCH,

CH3

OCH3

178

78%

f

CH3

-

OCH,

5M HCI

dioxane

ti 1 2 h

179

NHz

CH3COCH2C02C2H5 to1 8h A

H

Q QNycH3 \

/

N

CH,

CH,

180

Ph20

C02C2H5

H 181

-

A 65% from 180

H

CH3 182

0

CH3

OH

183

SCHEME 31. Viossat et al. synthesis of 4-hydroxy-2,5,1 l-trimethyl-6H-pyrido[3,2-b]carbazole (183) (93).

7.

27 1


0

07 I

LDA THF

-

0 J

Li

SOZPh

O0

R 185

-1

00°C 83%

S02Ph 184

31

Ac~O

2 eq MeLi

@ J o

-1oo"c-trt

*

NaBH4

H3C

OH

EtOH A 96% from 185 187

SCHEME 32. Gribble et al. synthesis of the lOH-pyrido[2,3-b]carbazolering system (e.g., 187) (94).

c. SYNTHESIS OF AZAELLIPTICINES The importance of azaellipticines is illustrated by the fact that 204 (BD-40) is undergoing clinical trials (20,98).Using their new 1-chloroellipticine synthesis (Scheme 20), Bisagni et al. (74) have described an extremely concise route to 10chloro-5,6-dimethyl-5~-pyrido[3',4':4,5]pyrrolo[2,3-g]isoquinoline (208) and the side-chain amine derivatives 209-211 (Scheme 35). Formylation of 1 -methyl -5-azaindole (205), followed by reaction with the lithiochloropyridine 124, gave

CH,

CH,

CH, (23%)

CH, (53%)

SCHEME33. Synthesis of the SH-pyrid0[4,3-b]benzov]indolering system (192) by Bisagni and co-workers (95).

Et,N(CH2)3HN N '

J

N

w

/ c H /3

o

I CH3

Ph

CH,

196

197

272

LifiH,

-

EI2NOC

OCH,

E1,NOC

OCH,

POCI,

DMF

1004: 2h 94%

OLi

198

I CH,

OCH,

OLi

OCH,

OCH,

OH

CH,

OCH,

0

CH,

199

0 200

201

SCHEME 34. Synthesis of the SH-pyrido[4,3-b]benzov]indolo-6,1I-quinone ring system ( e . g . , 200) by Bisagni and co-workers (96).

EtzN(CH2)JiN

NH(CH,),NEt,

& 7 & CH,

0

f J 7 @ NH(CHz),NEt,

CH3

0

203

202

273

NH(CHz),NEtz

274

GORDON W . GRIBBLE

NH(CH,),NEt2 I

204 ("BD-40")

alcohol 207. Reduction and acid-induced cyclization gave 208 in 28% overall yield from aldehyde 206. Displacement of chloride occurred upon heating 208 neat with the appropriate diamine to give 209-211.

D. SYNTHESIS OF NONLINEAR PYRIDOCARBAZOLES An obvious structural modification of ellipticine is the fusion of the pyridine ring to the a or c bond of carbazole. Indeed, pursuit of this idea has led to the

NaJ

1. Hexamethylenetetramine

TFA

A

3h

2. 3NHCI

59%

..

I

A

1

3h

CH3 205

CH3

72%

206

OH

CI

1. Et3SiH TFA 2. 50%H2SO4 24h rt CH3

48% 207

RNH2

208

~

NaT@ /

140 - 160°C 5-48h 44 - 89%

CH3

CH3

/

210 R 209 R=(CH2)3N(CH3)2 = (CH2)3N(C2H5)2 21 1

R = (CH2)3NHC2H5

CH,

SCHEME 35. Bisagni er al. synthesis of I-amino-substituted 9-azaellipticines 209-211 (74).

7.

275

ELLIPTICINE ALKALOIDS A N D RELATED COMPOUNDS

H

H 21 2

CH,O

N-NH N

H2S04

10% P&C

HOAc A 15min

decalin A 6h

42 - 50%

\ =-

cH30=%

4

2

'N

48%

14 - 20%

HBr

120°C 5h

215 216 217

1N 3N 4N

,

218 1N 219 3N 220 4N

40°C

1N 3N 4N

'\

H o = %

27 - 40%

221 222 223

*

N

214

cH30y-qJq) 1 \\3

N

213

-.

NCH,

f

SCHEME 36. Lescot et al. synthesis of 11H-pyrido[a]carbazoles(215-223) (100)

N

276

GORDON W. GRIBBLE

discovery of the clinically active drug “ditercalinium” (212) (99), a bis-7Hpyrido[4,3-c]carbazole derivative. Lescot and co-workers (100) have described two routes to the 11Hpyrido[a]carbazoles. In the first (Scheme 36), a Fischer indole cyclization of the naphthylhydrazone 213, followed by dehydrogenation, gave the fully aromatic pyrido[a]carbazoles 215-217. Methylation or demethylation completed the preparation of the desired target compounds. A different route was used to synthesize the 5-methyl-2-aza derivatives 226 and 227 (Scheme 37) (100). Condensation of aldehyde 224 with 4-ethylpyridine gave the vinylindole 225. Deacylation and a variation of the Snieckus pyrido[c]carbazole synthesis (101) gave the desired compounds 226 and 227. Roques and co-workers (102) have described a general route to the 7Hpyrido[c]carbazole ring system (Schemes 38 and 39). The preparation of the 5methyl isomer 233 was performed by first converting 5-methoxy-2-indole carboxylic acid (228) to aldehyde 229. Condensation with 4-ethylpyridine and a Snieckus oxidative photocyclization (101) of the heterocyclic stibene 230 gave 231 in low yield. A somewhat better procedure utilized the methiodide of 4ethylpyridine in the coupling step, although photocyclization of 232 was still poor. The 6-methyl derivatives (238-241) were prepared by converting isogramine methiodide 234 to nitrile 235 (Scheme 39) (102). Aldol condensation with the three isomeric pyridine aldehydes afforded the expected products 236. Photocyclization proceeded in much higher yields than before (Scheme 38) to

‘~-JCHo H

+

cb

A 18h

21 - 25%

Ac

224

225

aq NaOH EtOH 15rnin rt 90 - 94%

95% EtOH 11 -14%

226 227

R=H R=CH3

SCHEME 37. Lescot el al. synthesis of the 5-methyl-1 1H-pyrido[3,4-a]carbazole ring system (226, 227) (ZOO).

SOCI2 A 4h

cH30Q-1 C02H

H

228

EtoH 89%

Et20 THF A 1.5 h

H

98%

CrO3 H

LiAIH4

CHO

cH30Q-~

AC20 A

rt 19h

H

60h

60%

229

10%

95% EtOH 28%

I

Ac

H

230

231

'

cH3073-7-

CHO

9""""' *cH3

H3C' IE t+2 0N piperidine 0

H

rt 21h

H

229

63%

232

'

CH3 I-

hv

EtOH 14%

I-

H

233 SCHEME 38. Synthesis of the 5-methyl-7H-pyrido[4,3-c]carbazolering system (e.g., 231, 233) by Roques and co-workers (102).

278

GORDON W . GRIBBLE

cH3073---&+

KCN

-cH3073-*-L

N(CH3)3 CH30H

H 234

I-

CN

A 18h

H

67%

235

CH30H NaOCH3

20%

l h

51 - 70"/0

95% EtOH

236

CN

31 - 75%

237

CN

NaN02

H2

H20 HBr

RaNi NH3 HMPT lobar 3.5 h

II 30rnin

59 - 83%

57 - 95%

2

H2

I

CH,Br

RaNi CH30H latrn 4 h 20 - 52%

238 239 240 241

1-aza 2-aza 3-aza 4-aza

SCHEME 39. Synthesis of the 6-methyl-7H-pyrido[c]carbazolering system (e.g., 238-241) by Roques and co-workers (102).

N

279

7 . ELLIPTICINE ALKALOIDS AND RELATED COMPOUNDS NaH THF COCO2CH3 PhO2S

S0,Ph

T ’243 H2P(olPh rt 16h

242

244

79%

hv 12

LiAIH4

MeOH 24h

M F 0°C 20min

4

52%

H

95%

COZCH,

245

&OH

246

62%

’.

I

CH20CNHCH3

II

0

247 69%

Mn02 CHC13 A 6h

Ph3P=CH,

DMSO rl 16h

CHO

I

CH=CH2

65%

248

H2 10% Pd/C CH3W 3atm 2 h 80%

249

SCHEME40. Synthesis of the 7H-pyrido[4,3-c]carbazolering system (e.g., 247, 249) by Archer and co-workers (103).

280

GORDON W. GRIBBLE

give the tetracyclic nitriles 237. Reduction, diazotization, and debromination gave the desired 1-, 2-, 3-, and 4-aza derivatives (238-241). Archer and co-workers (103) have also employed the Snieckus oxidative photocyclization in the key step of their synthesis of the 7H-pyrido[4,3-c]carbazole ring system (Scheme 40). Thus, a Wittig condensation between pyruvate 242 and pyridine 243 gave the unsaturated ester 244. Photocyclization gave the tetracyclic ester 245. Reduction and reaction with methyl isocyanate led to carbamate 247. Oxidation of alcohol 246 to aldehyde 248, followed by a standard one-carbon homologation, gave the desired ethyl derivative 249. These chemists also synthesized the 10-methoxyl derivative of each compound. In an extension of their earlier work on the use of furo[3,4-b]indoles to construct ellipticine (Scheme ll), Gribble and Saulnier (79) have utilized an intramolecular Diels-Alder reaction to prepare 6-methylbenzo[c]carbazole(255) as a model for the 6-methylpyrido[c]carbazolering system (Scheme 41). Addition of the unsaturated Grignard reagent 251 to aldehyde 250 gave alcohol 252. The usual C-2 functionalization, oxidation, and cyclization furnished furo[3,4-b]indole 253. This underwent a smooth intramolecular Diels-Alder cycloaddition to give 254. Hydrolysis and dehydrogenation furnished the target compound 255. E. SYNTHESIS OF OXAZOLOPYRIDOCARBAZOLES As seen in Section VIII, the facile oxidation of 9-hydroxyellipticine (3) and elliptinium (5) to the corresponding quinone imines (6 and 256, respectively) with the enzyme HRP and H,O, may represent an important facet of the mechanism of antitumor action of these compounds. In the presence of amino acids, the quinone imine 256 formed adducts that were assigned structures 257 (104,105). However, numerous discrepancies between the expected and observed chemical and physical behavior led the original group (106) and, independently, Potier and co-workers (107) to reassign these amino acid adducts as having the oxazolopyridocarbazole structure 258. For example, the mass spectra of these adducts displayed parent ion peaks that were 46 mass units lower than expected for structure 257, and the infrared “carbonyl” band at 1670 cm-1 seemed dubious (107).The adducts did not react with acetic anhydride, did not undergo electrochemical oxidation, but, unlike 9-hydroxyellipticine (3), were strongly fluorescent in water (106). Moreover, the ‘H- and 13C-NMR data seemed more consistent with the oxazole structure 258. Finally, quinone imine 256 reacted with alanine and ethylamine to give the same adduct 258 (R = CH,) (106)! Potier and co-workers (107,208) have proposed a mechanism for this reaction leading to the oxazolopyridocarbazole structure (Scheme 42). Potier and coworkers (107,108) have also demonstrated that this oxidation and interception with amines can be performed using manganese dioxide as the oxidant and

7.

28 1


7 MgBr_

251 -1 00%

..

..

I SOZPh

I

SO2Ph

250

252

OH 1. t-BuLi

1. MnO;!

2. CH3CHO

2. TFA

94%

OH

PhOZS

*

CH2C12 A 36%0

I

Ph02S

6H3

253

1.

NaOH

--

2. DDQ

41%

H

I CH3 255

SCHEME4 1. Gribble-Saulnier synthesis of 6-methylbenzo[c]carbazole(255) (79).

simple carbazoles (e.g., 259) as the substrate (Scheme 43). In the absence of a trap, quinone imine 260 was isolated in nearly quantitative yield. Meunier and co-workers (109) also studied the oxidation of elliptinium (5) in the presence of aminocarboxylic acids with HRP/H,O, as a preparative route to the novel oxazolopyridocarbazole acids 267. Photooxidation of 5 in the presence of leucine also gives the oxazopyridocarbazole adduct (110). Archer and colleagues (84) used this facile oxidation-amine trapping protocol to prepare oxazole 268.

282

HOW GORDON W . GRIBBLE

,CH,

‘

/

N H

/

peroxidase horse radish (HRP) H2Q

amino acids

CH3

CH3 256

5

CO,H

U

II

SCHEME 42. Reassignment of ellipticine quinone amino acid adduct 258 and proposed mechanism of formation by Potier and co-workers (107).

7.


283

R

F. SYNTHESIS OF TRICYCLIC ANALOGS In an unquestioned tour de force, Bisagni and co-workers (111-113,115I 17) have published an extensive account of the syntheses and biological properties of a series of novel tricyclic analogs in which the C ring of ellipticine or 9-azaellipticine has been deleted to give y-carbolines or 5H-pyrido[3’,4’:4,5]pyrrolo[3,2-~]pyridines, respectively. Bisagni’s first approach to the diazacarbazole ring system (SH-pyrido[3’,4’:4,5]pyrrolo[3,2-c]pyridine)involved the buildup and cyclization of the pyridine ring onto an azaindole (Scheme 44) (111). Metalation of indole 269, followed by quenching with acetaldehyde, gave alcohol 270. Oxidation and an Emmons-Wadsworth reaction gave acid 272, after saponification. Formation of acyl azide 273 was followed by thermolysis to effect cyclization of the isocyanate to the indole C-3position, to give pyridone 274. Subsequent manipulation gave the target molecules 275 and 277. Similar chemistry starting with N-methylindole 188 afforded 278. Bisagni’s second approach to these tricyclic molecules involved a Fischer indolization strategy (Scheme 45) (112) and was improved over the previous synthesis (Scheme 44). Hydrazinolysis of hydroxypyridone 279 followed by condensation with N-acetylpiperidone gave hydrazone 280. The subsequent Fischer cyclization was accomplished in refluxing diphenyl ether. Dehydrogenation and chlorination gave the target ring system 277. Interestingly, methylation conditions gave, in addition to the expected 278, a substantial quantity of 283.

284

GORDON W. GRIBBLE

Mn02 CH2C12 H o Q $ -

CH3

98% rt

259

260

CH3

R

RCH2NH2 DME rt 4h

00 - 85%

261 262

R=mPr R=Ph

R

H

3

CH3

RCH2NH2 DME EtOH rt 4h 65 - 88%

'

/

N

/

H CH3 263 R =mPr 264 R=mBu 265 R=mPen 266 R=IFCgH13

SCHEME43. Oxidation and amine trapping reactions of 6-hydroxy- 1,4-dimethylcarbazole (259) and 9-hydroxyellipticine (3) (108).

7.


H 158

DME EtOH rt 7 h

285

H

268

55%

Similarly, hydroxypyridone 284 was converted to 285. As before, each of these 2-chloropyridine compounds was heated neat at 160- 170°C with the appropriate amine to afford the target side-chain amine derivatives, the exact structures of which are presented and discussed in Section XI. Bisagni and co-workers (113,116) also explored several synthetic routes to the tricyclic y-carbolines (5H-pyrido[4,3-b]indole ring system). Unfortunately, the attractive one-step Nenitzescu reaction (114) proceeded in only 6% yield to afford 286 (113). The Fischer indolization sequence was far more efficient (Scheme 46) (113). Thus, condensation of phenylhydrazine with 279 in boiling diphenyl ether gave in one step the desired y-carboline 287 in excellent yield. Chlorination of the pyridone functionality gave chloropyridine 288, which was converted to the target amine-substituted y-carbolines 289-291 by heating with the appropriate amines. The same condensation-cyclization sequence with 4-methoxyphenylhydrazine (292), however, proceeded in only 17% yield (113), so an alternative synthesis was devised for the important 8-oxygenated derivatives, such as 293 and 294, which employed a more conventional Fischer indole reaction (Scheme 47) (113). In the event, condensation of keto ketal 295 with hydrazine 280 gave hydrazone 296. Cyclization in hot diphenyl ether gave ketone 297, which, upon dehydrogenation, protection of the phenol as the benzoate, and chlorination gave 298. Deprotection and/or methylation afforded the target chloro-y-carbolines (299-301). To avoid the expense of keto ketal 295, Bisagni et al. (116) devised an alternative synthesis that began with 4-methoxycyclohexanone 302 (Scheme 48). The usual Fisher indolization, dehydrogenation, and chlorination gave methoxy derivative 303. A sequence of demethylation and/or methylation provided the target 8-methoxy- and 8-hydroxy-y-carbolines, which were transformed into the amine derivatives (e.g., 304, 305). By the same route (Scheme 48), Bisagni and team (116) converted hydrazine 306 to the 4-demethyl-y-carbolines (307-310).

286

GORDON W. GRIBBLE

&I

Ph

1. t-BuLi Mn02 2. CH3CHO Et20 -65”c

N

*“c

48%

A

T

I

OH

Ph

269

~

ACHC13 ~20h

~

770/0

270

CI 1. NaH DME ( Et0)2 POCH2CQ Et

ti 48h 2. KOH aq EtOH Ph

98%

Ph

271

272

-

1. EtOCOCl Et3N acet P

2. NaN3

Ph

Ph 274

CI 1

H2 10% Pd/C w

Et3N EtOH latrn rt 2 h

92% 275

SCHEME 44. Bisagni-Hung synthesis of diazacarbazoles 274-277 (I If).

~

CI

CI

I CH3

I CH3

188

278

DAc 0

0

-

0

N2H4

OH CH3

A 4h 76%

HN+

NHNH,

-

EtOH A 1.5 h

CH3

279

84%

280

281

~a-4 ~a-4 ’’tfaHuFLi

\

/

N

4%

2. CH31

H CH3 277

HMPT

~

\

/

N

‘

CH3 CH3 278 (46%)

/ CH3

283 (39%)

0

284

N

I

Cl

H 285

SCHEME 45. Hung-Bisagni improved synthesis of the diazacxbazole ring system (e.g., 278, 285) (112).

288

GORDON W . GRIBBLE

0 PhpO NHNH,

88%

CH3 279

287 NHR

-Q)-+$=Q)--Q 160-1 4h 65°C

16-96 h

CH3

81%

CH3

289 (57%) R = (CH2)3N(C2H& 290 (50%) R = (CH&N(CH3)2 291 (47%) R = (CH2)2NH(CH2)20H

288

cH3073-NHNH2 -cH3073--Q CH3

292

293 R = (CH2)3N(CH$H3)2 294 R = (CH2)3NHCHzCH3

SCHEME 46. Nguyen-Bisagni synthesis of the SH-pyrido[4,3-b]indole ring system (e.g., 289291) (113).

HOAc

A 18h 6%

CH3

H 286

CH3

7.

295

2. 1. Ph20 HCI 40 rnin A

280

'

~

289


W

71Oo/

296

H

loo/opd/c PhO A

-

"QT-H

30 rnin

H CH3

297

H

81%

CH,

286

nT-~ - nT0

(PhCOhO

PhCo2

pyridine A 2h

I

CI

I

CH3

74%

I

PhCo2

A 70h

I

75%

298

NH3

CH3

2. NH3

CH30H It 18h

80%

299

300 DMF CH31

57%

CH3

CH3

301

SCHEME47. Nguyen-Bisagni synthesis of 8-oxygenated SH-pyrido[4,3-b]indoles 299-301 (113).

290

GORDON W . GRIBBLE

EtOH

NHNH,

0

302

A 4h

86Yo CH3

CH3 280

48% A75% lHBr h

*Hoqi$ 200 - 210% 4h

H2NR2gg

CH3 301

1

I

CH3

NHR I

CH3

I

48%HBr l h A

65yo NHR H2NR 200 - 210°C 4h

CH3

300

CH3

CH3

CH3

305

SCHEME48. Bisagni er al. synthesis of 1-amino-substitutedSH-pyrido[4,3-b]indoles304 and 305 (116).

7.


29 1

CI

0

306

R2

307 R 1 = CH3, R2 = H 308 R1 =R2=CH3 309 R i = R 2 = H 310

R1

=H, R2 = CH3

G . SYNTHESIS OF BISPYRIDOCARBAZOLES

Although the potential importance of bis-intercalators of DNA was recognized long ago (17), the discovery of high antitumor activity for ditercalinium (212) has prompted renewed interest in the synthesis and study of potential polyintercalators of DNA. illustrative of the general method for the synthesis of bispyridocarbazoles tethered through the pyridine nitrogens is the preparation of the ethyl ditercalinium analog 315 as reported by Roques and co-workers (118) (Scheme 49). The same general approach was used to prepare the ethylpyridocarbazole 314, but lithiation methodology was employed to attach the pyridine grouping to the indole ring. This reaction (311 + 312 + 313) proceeded in poor yield because of competing enolization at the (very) acidic methylene group ( pyridine nitrogen and carbonyl anion stabilization). Bisalkylation to yield 315 is typically performed in hot DMF. These conditions have been used to tether other pyridocarbazoles (102). The more flexible tether embodied in bispyridocarbazoles 321 and 322 was synthesized by Roques and colleagues (119) as shown in Scheme 50. The bischloro tether 319 was prepared from 4-bromopyridine (316) by halogenmetal exchange, condensation with 4-cyanopyridine, and Wolff-Kishner reduction of the resulting ketone 317. Catalytic hydrogenation, chlorination, and then alkylation of 320 with 319 gave the desired bispyridocarbazoles (321, 322). An important discovery in this research is that the methosulfate salts impart excellent water solubility to the bispyridocarbazoles. Roques and co-workers (120) have described the preparation of asymmetric bispyridocarbazoles (325, 326) in which the linking chains are of different lengths (Scheme 51). By a sequence of alkylation and hydrogenation, they converted 4,4’-bipyridine (323) to 324. Coupling with pyridocarbazoles 320 gave the desired compounds 325 and 326. The same group (120) designed and constructed novel potential bis-intercalators (327,328) in which the two intercalative rings are different, one being a pyrido[4,3-~]carbazoleand one an acridine (Scheme 52).

292

GORDON W . GRIBBLE

1. 1-BuLi

cH3073--

KOH/DME

H

139

THF 5°C 20min

PhS02CI 20°C 40min 80%

TH F I

SOzPh

31 1 312

14%

hv 12

2HCI

35h 71yo

DMF 85°C 15h CHzCH,

31 4

f) +

40%

CHZCH, I -+=! -

cH3oQ \

*

N

H2CHzC I

4CI -

/

DOCHS \

Et

/

N Et

H

31 5 SCHEME 49. Synthesis of ditercalinium analog 315 by Roques and co-workers (118).

*

7.

293


0 1. N2H4

KOH A

1. NaOH 0°C

-

t

H+N>E3r

2. CICH2CH2OH 3. H2 P I 0 2

2. mBuLi

317

316

4. H30+ CH,O 1.

I

R

+

320

2. 4CH3S03AS

HO 3 18

319

I+

..

I R

I R 321 R = H 322 R=CH3

SCHEME 50. Synthesis of bispyridocarbazoles 321 and 322 by Roques and co-workers (119).

Sainsbury and co-workers (121) have synthesized several ellipticine dimers tethered through the C-5 methyl group (333) (Scheme 53) or the C-9 position (334). The 9-methoxy derivative of 333 was also prepared. The nitrile 329 was available from the Sainsbury ellipticine synthesis (122) and was transformed into the alkaloid 17-oxoellipticine (148). A clever maneuver was to add nitric acid to protonate the pyridine nitrogen of 330. This precluded N-oxide formation during dithiane hydrolysis. Reductive amination in two steps afforded the amine 332. Coupling with adipic acid gave the target bisellipticine 333.

t

294

GORDON W . GRIBBLE

BrCHzCH20H

*

HO(CH,),N\

\

H2 ROZ

N,

aq EtOH 90%

Et20 rt 18h

323

+3€ /

80Y0 Br(CH2)sOH

HO~cH2)2N3--CNICH2)30H

EtOH Na2CO3 A 4days

HO(CHN ), =NH

83%

soc12

320 (R = H, CH3)

CI(CH,),N

CHC13 A 2h

33-3(cH2)3cl . 2 HCI

aq DMF 80% 24h

324

14 - 20%

72%

CH3S03Ag

* aq EtOH

95%

ct ..

I R

R

325 R = H 326 R=CH3

SCHEME5 I . Synthesis of unsymmetrical bispyridocarbazoles 325 and 326 by Roques and coworkers (120).

H. SYNTHESIS OF ELLIPTICINE CONJUGATES

In order to direct ellipticine and derivatives to specific biological targets, a number of ellipticine conjugates have been synthesized and evaluated for tissue specificity and antitumor activity. Roques and group (123) have synthesized several ellipticine conjugates that were designed to have strong affinity for breast tissue and also be DNA intercalators. The preparation of the ellipticine-estradiol derivative 337 is shown in Scheme 54. A Reformatsky reaction on estrone (335) gave hydroxy acid 336. Amide formation and coupling with ellipticine gave 337. This group of researchers (123) also synthesized several ellipticine-clomiphene

-

7.

295


N HCHZCHZOH OCH3

H~NCH~CHPOH

PhoH 120°C 1.5 h

CI

90%

EtOH A 3days

77% 1. SOCI, to1 A 6h

320 e

2. NH40H 47%

CH3S03H b

DMF 80°C 20h 17 - 27%

+

CH30H

99%

CI

3 CH3SO3-

327 R = H

N I R

/

328 R=CH3 SCHEME 52. Synthesis of acridine-pyridocarbazole bis-intercalators 327 and 328 by Roques and co-workers (120).

296

GORDON W. GRIBBLE

n H

-78°C

NC

-+

a--@

* - aqHOAc A 2h

sys THF Li

Q J - h N

rt

/

93%

su

329

oT-

HNO3

330

CH3NH2 PhH

0

AgNO3 THF aq acetone 40-50°C 20h

148

63%

0

93%

CHO

NaBH4

MeOH 0°C 3 h 60%

331

332 CH3

aJp I

H02C(CH2)&02H Ph2P(O)N3 DMF Et3N -1O"C-trt 37%

*

H

L

333 0

/

2

SCHEME53. Synthesis of bisellipticines (e.g., 333) by Sainsbury and co-workers (121).

7.

297


& - &15H2c0 & 0

OH

”

\

HO

?:fog:5,

2. aqNaOH EtOH rt 4 h

335

\

HO

336

76%

HzC ~ N H C 2CH2Br H

H2NCH2CH2Br EbN M F

ellipticine (1)

* EtN=C=N(CH,),NMe,

b

HO

\

DMF 90°C 7 h

93%

42%

H2co

@ HO

CH3

\ 337

SCHEME 54. Synthesis of ellipticine-estrone derivative 337 by Roques and co-workers (123).

conjugates, for example, 338 and 339. To probe the S-opioid receptor, Roques and co-workers (124,125) have synthesized several ellipticine-enkephalin conjugates, one of which is shown in Scheme 55. Straightforward peptide chemistry afforded first the activated enkephalin 340 and then the ellipticine conjugate 341. The 9-hydroxyellipticine derivative was also prepared. Meunier and co-workers (126-128) have reported the construction of several ellipticine-porphyrin molecules, which are potentially capable of both intercalation and chelation (Scheme 56). The linking chain is first connected to the N-2 position of‘9-methoxyellipticine (2) and the resulting ester 342 is attached to an amino porphyrin to give 343. The metal is introduced by letting 342 react with FeCl,, MnOAc, or ZnOAc in boiling 2,4,6-collidine to give 344-346, respectively, in 57-76% yield.

Hoo;&

/

q?

/ ,cH,cONH(cH,),NH(cH,),O

CH3

/ \ -

338

339

BOC-Tyr-D-Ala-Gly-Phe-D-Leu

Qq *

OH

BOC-T~~-D-A~~-G~~-P~E+D-L~U-NH(CH~)~ 340

76%

CHC13 THF DCC

93%

0%

45min

+

H3N-Tyr-D-Ala-Gly-Phe-D-Leu-NH-(CH2)3

0 - II 2 OCCF3 341

SCHEME55. Rigaudy et al. synthesis of ellipticine-enkephalin conjugate 341 (124).

"""w / 7.

Br(CH2)&02Et

'

/

N

/

87%

CH3

(CHz)&O&t

@ .H ,c,3 7 0

DMF 120°C 4 h

H

2

299


'

'

N

H

/

Br-

CH3 342

1. 1MHCI

2. EtOCOCl 3. amino porphyrin

47%

A(347) 3 h

343 A = H 2

I

A = Fe(lll)-OAc A = Mn(lll)-OAc 346 A=Zn(ll)

CH,

344 345

SCHEME 56. Synthesis of metalloporphyrin-ellipticine hybrid molecules 344-346 by Meunier and co-workers (127).

An obvious means by which to increase the affinity of a molecule for DNA is to link the molecule to a short segment of nucleic acid. Such a plan has been pursued by Paoletti and co-workers (129,130). To prepare the tetrathymidylateellipticine conjugate 348, these workers synthesized the appropriate oxazolopyridocarbazole carboxylic acid, as described previously (i.e., 267), and coupled it to the appropriate tetradeoxynucleotide. A second method of linking ellipticine to a nucleic acid involves condensation of the aldehyde moiety of 3'apurinic octathymidylate with 9-aminoellipticine, followed by reduction of the irnine with sodium cyanoborohydride (130). This reaction is depicted in a different context in Scheme 66 (see Section VIII).

300

GORDON W. GRIBBLE

0

OYO

348

I. SYNTHESIS OF MISCELLANEOUS ANALOGS Although the exploration of novel ellipticine analogs continued in the late 1980s, in general, the further one deviates from the fundamental ellipticine structure, the less will be the antitumor activity. This section delineates a potpourri of such analogs.

7.

30 1


Bergman and Pelcman (131) have discovered a one-step synthesis of the dimethylbenzo[b]carbazole ring system (Scheme 57) that is remarkable in its simplicity. An acid-promoted Michael reaction between 2-ethylindole (349) and enone 350, followed by cyclization of the methylene group onto the carbonyl leads to a tertiary alcohol. In situ dehydration and ring C (D) oxidation completes the sequence of events. It remains to be seen if this procedure can be applied to the synthesis of pyridocarbazoles. Hayler and Sainsbury (132) have synthesized the oxyphenylsulfonyl derivative 360 of the indeno[2,l-g]isoquinolinering system (Scheme 58). The key step in the preparation of the tricyclic precursor 355 was a Diels-Alder reaction between indenone 353 and hexadiene 354. Dehydrogenation and Wolff-Kishner reduction gave fluorene 356, which underwent Vilsmeier-Haack formylation ortho to the methoxyl group at C-7 instead of at the desired C-2 position. This was circumvented by converting the methoxyl group to oxyphenylsulfonylfluorene 357. Although 357 did not undergo formylation, it did react under chloromethylating conditions to yield 358. Subsequent conversion to amide 359 was followed by Bischler-Napieralski cyclization and dehydrogenation to give the target compound 360. Using Cranwell-Saxton (56) technology, Sengupta and Anand (133) have synthesized the known dibenzofuran analog 361 (134) of ellipticine [and of didemethylellipticine (362)l (Scheme 59). In the same paper ( 1 3 3 , the interesting and apparently new pyrido[4,3-b]phenoxathiin (363) and pyrido[4,3-b]phenothiazine (364) ring systems were reported. None of the compounds reported in this paper were cytotoxic. Anand and co-workers (135) have also described the preparation of several C-seco analogs of ellipticine (Scheme 60).

a2cH2cH: 10% P&C

H

349

HOAc 3A m01Asieve 48h

*

350

H CH3 351 (38%)

352 (22%)

SCHEME 57. Bergman-Pelcman synthesis of 6,l I-dimethyl-5H-benzo[b]carbazole(352) (131).

302

GORDON W . GRIBBLE

353

354

355

=p

1. demethylation

h

s

0

3

chloromethylation ~

2. phenylsulonylation

CH3 357

’.

NaCN

PhSO,

2. B2H6

CHZCI

-

3. formylation

CH3

CH3

358

359

-PPE

PdIC

120°C

diglyme

27%

A

I

from 359

CH3 360

SCHEME 58. Hayler-Sainsbury synthesis of 6-deazaellipticine analog 360 (132).

7.

0-6 '

0

/

a--cHo

CI~CHOBU

H2NCH2CH(OEt)2

\

Sa14 CH2C12 rt 45rnin

CH3

'N

58%

105% superphosphoric acid

/

PhH A 30rnin

CH3

99%

~

oT/&oEt

/

140°C

CH,

303


OEt

43%

/

CH3

361

36 2 SCHEME59. Sengupta-Anand synthesis of the pyrido[4,3-b]dibenzofuranring system (e.g., 361, 362) (133).

They utilized the reaction between indole Grignard reagents and either 3-acetylpyridine or nicotinoyl chloride to give the target structures 54 and 365, after the appropriate reduction. None of these compounds displayed antitumor activity anywhere comparable to that of ellipticine. Nantka-Namirski and co-workers (136) have described the synthesis and antitumor properties of a series of benzo-annulated iso-a-carbolines, some of which have significant antitumor activity. Coupling of 2-bromopyridine with naphthotriazole 366 gave 367 (Scheme 61). Heating this material with polyphosphoric acid effected the Graebe-Ullmann carbazole synthesis to give 368, reminiscent of Miller's earlier strategy (47). Methylation afforded the iso-a-carboline 369. Similar reaction sequences led to the higher order linear iso-a-carbolines 370372. A slightly different approach was used by these workers (136)to synthesize the nonlinear analogs shown in Scheme 62. The benzotriazole 375 was prepared by diazotization of amine 374, which was synthesized in a straightforward manner. Similarly, the 8H-benzo[g]-a-carboline (380)ring system was prepared.

304

GORDON W. GRIBBLE

1. EtMgl

Ng - Q)--+J COCH3

H

2.

50

8%

H

100°C 48h

53

10%H2 Pd/C

latm 12h

H

70%

54

NGcoc'

1. EtMgl

H

CH2CH3 2.

349 11%

B2H6 ___)

THF A 3h

OyJp

72%

CH3 365

SCHEME 60. Synthesis of C-seco ellipticines (e.g., 54, 365) by Anand and co-workers (135).

I

CH2CH3 363

364

7.

305


160 - 170°C

PPA e

110" --f 200°C 50 min

15 rnin

H

61%

366

28%

367 1. CH31 EtOH

100°C 12h

55%

H 368

I

CH3

369

SCHEME 61. Synthesis of l-methyl-IH-benzo[5,6]indolo[2,3-b]pyridine (369) by NantkaNamirski and co-workers (136).

VI. Biological Detection The synthesis of N-2 trideuteriomethyl elliptinium (5) in 96% isotopic purity has been reported by Gouyette (237) for use in a study of the metabolism of 5 in rats. This compound was prepared in 90%yield by allowing 9-hydroxyellipticine (3) to react with CDJ in DMF at room temperature. A combination of liquid

370 R = H 371 R=CH3

306

GORDON W. GRIBBLE

-

R NaN02

*

b

N

*

N

PPA

320 - 350°C 20 m'n 41% (28%)

20% H2SO.4

H

374

hyQ R

:

rOyNaOH EtOH 100°C 12h

55% (59%)

\ 376

+yq 375

*

\

CH3

377 R = H 378 R=CH3

SCHEME62. Synthesis of the lOH-benzo[i]-a-carbolinering system (e.g., 377,378) by NantkaNamirski and co-workers (136).

chromatography (LC)and mass spectrometry was used to analyze the metabolites (Section X). These techniques [high-performance liquid chromatography (HPLC) and fast-atom bombardment (FAB) mass spectrometry] have also been employed by Gouyette et al. (138) to identify metabolites of 5 in human cancer patients. HPLC has been used by the same group (139) to measure the uptake of

379

CH,

380

7.


I

CH,

381

307

CH, CH,

382

2,6-dimethylellipticinium (381) and 2-methyl-6-n-propylellipticinium(382) by NIH 3T3 cells. A fluorodensitometric assay was developed by Montague and co-workers (140)to analyze cultures from Ochrosiu ellipticu for ellipticine (l),9-methoxyellipticine (2), and 9-hydroxyellipticine (3) by thin-layer chromatography (TLC) without the need for prior purification. Using silica gel impregnated with dimethyl sulfoxide and a mobile phase of EtOAc-water-1-octanol (17 : 2 : 2), these workers were able to achieve good separation of these alkaloids and to assay the resulting chromatograms by fluorodensitometry (40-300 fmol detection limits of alkaloid).

VII. Antitumor Activity in Experimental Models Several significant developments have occurred during the late 1980s in the study of the antitumor activity of ellipticine and its derivatives, which are highlighted in this section. Further discussion on the antitumor activity of these compounds is expounded upon in Section XI. In attempts to deliver ellipticine and derivatives to specific biological targets, a number of investigators have combined an ellipticine drug with the appropriate carrier molecule. Of the several ellipticine-estradiol receptor conjugates synthesized by Roques and co-workers (123), only 337 had good antitumor activity against the L1210 mouse leukemia system in vitro [IC,, 0.5 pkf; ellipticine, IC,, 0.85 pkf; elliptinium (9,IC,, 0.08 pkf], which is generally considered to provide a good indication of the efficacy of drugs against human cancer (141). The IC,, refers to the inhibitory concentration that reduces by 50% the growth rate of the cells after 24 (or 48) hr of drug exposure. The triarylethyleneellipticine hybrid molecules (338, 339) were essentially devoid of cytotoxicity (123), even though they retain their affinity for DNA and estrogen receptor. Compound 337 also had activity against the human breast cancer cell line MCF-7.

308

GORDON W. GRIBBLE

The ellipticine-enkephalin conjugates (e.g., 341) exhibit in vitro binding properties both to DNA and to opioid receptors in NG108-15 mouse tumor cells that are similar to those of the parent molecules, although the conjugates did not exhibit the expected selectivity when tested on the opioid-receptor containing NG108-15 cells and Lfibroblasts as controls (125). The lack of specificity was explained in terms of an intracellular overconcentration of drug. A 9-methoxyellipticine (2)-low density lipoprotein (LDL) complex was formulated by Soula and co-workers (142) and found to be 10 times more active than 2 against L1210 and P388 leukemia in vitro. This activity seems to depend on the LDL high-affinity receptor since LDL reduces the antitumor activity. The complex was prepared by adding 2 to a dimyristoyl phosphatidylcholine, cholesteryl oleate-stabilized microemulsion and then fusing with human LDL. In a very significant study, Alberici et al. (143) synthesized several elliptinium (5)-monoclonal antibody conjugates, one of which (Fab AFO1-5) is at least 100 times more cytotoxic in vitro against human hepatocarcinoma cell lines than is 5 or doxorubicin. The conjugates were prepared by oxidizing 5 with HRP/H,O, (to give quinone imine 256) in the presence of the monoclonal antibody. Arteaga and co-workers (144) have utilized a human tumor cloning system to evaluate in vitro the effects of elliptinium (5) against 282 tumor lines, in order to determine which human tumors should be clinically treated with 5. The results indicated that phase I1 trials in patients with renal cell carcinoma, breast cancer, non small-cell lung cancer, and small-cell lung cancer should be pursued. Elliptinium (5) has been encapsulated within phospholipid vesicles by Sautereau et al. (14.9, although, as such, the drug is less cytotoxic against L1210 cells in vitro and in vivo than when it is free. However, if the onset of leukemia is delayed in mice, then the entrapped drug has higher antitumor activity than the free form. An investigation by Ali-Osman et al. (146) has shown that 5 is able to cross the blood-brain barrier in rats and is cytotoxic in vitro against three human glioma cell lines (SF126, SF375, SF407). In a study on the effects of various agents, including ellipticine (l), on the initiation of skin tumors in mice by polycyclic aromatic hydrocarbons (PAH), Alworth and Slaga (147) have observed that, depending on the dose of 1 and the nature of the PAH, 1 can either stimulate or inhibit skin tumorigenesis. Thus, high doses of 1 inhibited the tumorigenesis by 7,12-dimethylbenz[a]anthracene but low doses of 1 stimulated it. In contrast, treatment of mouse skin with 1 at all doses tested stimulated dibenzo[a,h]anthracene tumorigenesis. Ditercalinium (212) and elliptinium (5) have been studied as agents against small cell lung cancer in bone marrow in vitro. Thus, Benard and co-workers (148) have found that 212 has high activity against NCI-H449 and NCI-N417 human cells (I& 1.2 X l o p 3 and lo-, pM, respectively). By comparison, 5 is much less active (1 and 0.25 pM, respectively). In the late 1980s, several new ellipticine derivatives and modified ellipticines

7.


309

have been found to possess antitumor activity. One such class of compounds, developed by Honda et al. (68-70), are the ellipticine glycosides (e.g., Scheme 18). Several of these compounds have excellent antitumor activity against L1210, P388, B16 melanoma, and colon 38 carcinoma in vivo. Two compounds, 104 (SUN4599) and 383 (SUN5073), which have been selected for preclinical studies, were curative against several of the above tumors as well as Ehrlich ascites carcinoma (EAC) and sarcoma 180. Additional details on the remarkable antitumor activity of these simple ellipticine sugars can be found in Section XI. Another extraordinarily simple ellipticine derivative that has excellent antitumor activity is carbamate 153 (“RPI-6”). As reported by Ruckdeschel and Archer (149), 153 has high activity (1000 times higher than doxorubicin) against two human small-cell lung cancer lines (NCI H69c, N417) and two human nonsmall cell lung cancer lines (H460, H358). This compound was previously reported by Archer and co-workers (84) to have better activity against P388 leukemia in vivo than does ellipticine. In addition to elliptinium (5) and ditercalinium (212), three new ellipticine derivatives have been entered into clinical trials. The first is a simple modification of the N-2 methyl group of 5 to give 2-[(2-diethylamino)ethyl]-9-hydroxyellipticinium chloride hydrochloride (datelliptium) (384), which shows better in vivo activity than elliptinium (5)toward L1210, P388, B16, colon 38, and M5076 reticulosarcoma (150). The in vitro L1210 IC,, is 0.076 phf (5, 0.13 phf). The increased antitumor potency is believed to be due to increased diffusion across cellular membranes and a more favorable biodistribution in vivo. An azaellipticine derivative, “pazellipticine” (PZE or BD-40) (385) { 10-[(3-diethylamino)propylamino]-6-methyl-5H-pyrido-[3 ’ ,4’:4,5]pyrrolo[2,3-g] isoquinoline}, is also in clinical trials (151). This derivative has excellent in vitro activity against L1210 cells (&, 3.1 CLM). The third new clinical candidate is l-[(3-diethylamino)propylamino]-9-methoxyellipticinium chloride hydrochloride (BD-84) (386) (152). This drug has high activity against P388, L1210, B16, M5076, and colon 38 in vivo. Although the oxazolopyridocarbazoles (i.e., 258) have good activity against

383

310

GORDON W. GRIBBLE

2 CI H

H

CH3 385 ("pazellipticine") ("PZE")

CH3

384 ("datelliptiurn") ("DHE)

c

H

3

0

n

CH3

NH(CH,),NHEt,

y/ -

/ H

("ED-40")

+

2c1-

CH3

386 ("BD-84)

w),

tumor cells in virro (e.g., L1210, IC,, 0.2-0.6 these compounds in general have no antitumor activity in vivo (153,154). One compound, 387, shows some antileukemic activity in vivo. Despite the fact that the 11H-pyridocarbazoles (Schemes 36, 37) have DNA binding affinities close to those of 6H- and 7Hpyridocarbazoles, these compounds have no measurable L1210 cytotoxicity (ZOO).

As discussed in more detail in Section XI on structure-activity relationships, the tricyclic analogs of pyridocarbazole display some powerful antitumor activity (115,117,136). For example, compounds 388 and 389 have L1210 in virro ID,, values of 0.13 and 0.01 respectively. These compounds also exhibit in vivo activity against L1210, P388, B16, and colon 38 (116). Thus, 389 gives a T/C value of 236% and 40% survivors with a 100 mg/kg dose. The TIC value refers to the median day of survival of treated animals at a given dose/median day of survival of control mice ( X 100%). Significant activity is present when the TIC value exceeds 125%.

w,

7.


NH(CHZ),NEtz N \

3

N

G I

CH,

388

NH(CH,),NMe, ‘

/

31 1

O

n \

-

N

I

CH,

CH,

/

CH,

389

The interesting benzo-annulated iso-a-carbolines (e.g., Scheme 62) display good in vitro cytotoxicity against human tumor KB cells (e.g., IC,, 1 pM for 371 and 10 pM for ellipticine) and in vivo antitumor activity against P388, L1210, and B16 (e.g., TIC 224% at a dose of 100 mg/kg of 371) (136). Compound 371 can, of course, be viewed as an isoellipticine. Finally, of the several dimers prepared by Sainsbury and co-workers (121), only 334 and the 9-methoxyl derivative of 333 show significant activity against L1210 in vitro (IC50 -2 and 2.6 pM, respectively).

VIII. Mechanism of Action

As Suffness and Cordell (10) discussed in their review, there is increasing evidence that the enzyme topoisomerase I1 may play an important role in the mode of action of ellipticine and its derivatives. The late 1980s bear witness to continued activity in this regard. Ross (155) has concisely summarized the importance of topoisomerase 11 as a potential target for anticancer drugs, and Riou and co-workers (156) have written an excellent minireview on the functions of this remarkable enzyme and the mechanistic details of its interaction with DNA. The rationale for pursuing topoisomerase I1 as a drug target is that the activity of this enzyme is thought to be higher in malignant cells than it is in normal cells, obviously leading to improved selectivity. In performing its role in the cleavage and rejoining of DNA strands (catenation, decatenation, relaxation, unknotting), topoisomerase I1 bonds to the 5 ‘ phosphate on adjacent DNA strands four base pairs apart to form an enzymeDNA complex. It is the interaction between this complex and certain drugs, such as ellipticine (l),that results in the stabilization of the complex and the formation of a “cleavable complex” which leads eventually to the cleavage of doublestranded DNA. Pommier et al. (157,158)have shown that low concentrations of elliptinium (5) ( < l o pM) produce DNA double-strand breaks in mammalian cells, but higher concentrations ( > l o I.M) produce no such breaks and, in fact,

312

GORDON W. GRIBBLE

inhibit those induced by ellipticine (1). It is believed that these DNA breaks occur from topoisomerase 11-DNA complexes. This group (159,160) also found that cells which are resistant to 9-hydroxyellipticine (3) have fewer DNA doublestrand breaks than normal cells, suggesting that these breaks play a role in the antitumor activity of topoisomerase I1 inhibitors. Elliptinium ( 5 ) and other ellipticine-derived topoisomerase I1 inhibitors also lead to chromosomal abnormalities in Chinese hamster ovary cells (161) and in mouse bone marrow cells (162). These abnormalities include chromosome clumping, micronuclei formation, sister chromatid exchanges, and chromatid aberrations. There is observed a good correlation between antitumor activity and topoisomerase I1 inhibitory activity in vitro for 3, 5 , 9-aminoellipticine, and 9-fluoroellipticine (142) (162). The interaction of ellipticine derivatives with topoisomerase I1 enzymes in Plasmodium berghei (163), a parasite of mouse red blood cells, mouse lymphoma L5178Y cells (164), simian virus 40 CV-1 cells (165), Trypanosoma cruzi (166,167), and the human small-cell lung cancer cell line NCI N417 (168,169) has been studied. In the latter study, the highest in vitro activity in the topoisomerase 11-DNA cleavage reaction and decatenation was observed for elliptinium (5) and datelliptium (384) (169). Another aspect of the mode of action of ellipticine and its derivatives that has been intensely scrutinized in recent years is the chemistry of ellipticine quinone imines 6 and 256. The oxidation product of 9-hydroxyellipticine (3), formed by horseradish peroxidase-hydrogen peroxide or chemical (e. g ., manganese dioxide) oxidation of 3, undergoes a rich array of chemical reactions. Meunier et al. ( 1 70) have discussed in detail the oxidation parameters, chemical properties, and biological activities of several oxygenated ellipticine derivatives. In particular, molecular obital calculations support the fact that the C-10 position of 256 (and 6) is the preferred site of nucleophilic attack, as discussed earlier for the reaction of quinone imine 256 with amino acids (Scheme 42). It has been hypothesized (171) that this quinone imine is involved in the observed covalent binding in vivo to DNA in L1210 cells exposed to elliptinium (5). This DNA damage is not readily repaired, and 2-methylellipticinium (110) acetate is 20-30 times less active than 5 in terms of this binding. When elliptinium (5) was oxidized with HRP/H,O, to quinone imine 256 in the presence of DNA in v i m , a fluorescent compound irreversibly bound to the DNA was observed. The fluorescent properties of this complex were consistent with binding between C-10 of the quinone imine 256 and a primary amine group (N-2 guanine, N-6 adenine, or N-4 cytosine) of DNA (summarized in the hypothetical 390). With excess H,O,, the major product was the C-9,lO o-quinone (172,173). Quinone imine 256 also reacts very easily with the sugar groupings of nucleosides or nucleotides. Meunier and co-workers (174-1 76) have continued their studies of these reactions with simple nucleosides (Scheme 63 and 393) and diribonucleoside monophosphates (394). The 2'-deoxy diribonucleoside mono-

7.


0

313

*.

390

phosphate (dApdG) did not yield a detectable adduct (175), and purine sugars seemed to be more reactive than pyrimidine sugars (176). The cytotoxicity of these spiro derivatives of elliptinium (5)was less than that of 5 itself. However, C- 10 thioelliptinium adducts retained the high cytotoxicity of 5 (176). Potier and co-workers (and some members of the Meunier group) (177,178) have explored the chemistry of quinone imines 6 and 256 and oxygen nucleophiles including sugars. They found that 256 can be generated from elliptinium with Cu,Cl,/pyridine/air, as well as with HRP/H,O,. The structure of the product formed between 256 and methanol has been revised as 396 instead of

3 14

GORDON W. GRIBBLE

5

256 NHZ

I

cordycepin

*

60%

CH3

392 SCHEME63. Arylation of purine nucleosides by elliptinium (5) (174-176).

7.

393


315

394

395. These ketals can also be synthesized from 9-hydroxyellipticine(3)and lead tetraacetate/methanol/pyridine [room temperature (rt), 3 hr] . In similar fashion, the ribonucleotide adduct 397 was synthesized and characterized by an exhaustive NMR study including nuclear Overhauser effect (NOE) measurements to establish the precise stereochemistry (178). To rationalize the remarkable regioselectivity and stereoselectivity of these alkylation reactions, Potier and co-workers (I 77,178) proposed that a stacking interaction occurs between quinone imine 256 and the nucleic acid base prior to covalent bond formation. Moreover, the appropriate intermolecular NOE is observed to support this contention. The fact that these ribonucleotide adducts form so easily may suggest that ellipticine quinone imines could alkylate at the 3’ end of transfer RNA or at similar sites on other RNA molecules to inhibit protein synthesis. Thus, RNA would seem to be a reasonable target for elliptinium and related ellipticines (178). It has been found by Dugue and Meunier (179) that the combination of Fe(II1)-EDTA-H202 in the presence of elliptinium (5)is capable of degrading deoxyguanosine apparently by generating hydroxyl radical (Scheme 64).The isolated products are guanine (398)and 8-hydroxydeoxyguanosine(399).Other nucleosides and nucleotides behave similarly, but Cu(II) is much less effective

316

GORDON W . GRIBBLE

OCH3

CH3 395

than Fe(III), and 2-methylellipticinium (110) acetate does not participate in such chemistry. Auclair (110) has reported the generation of superoxide when elliptinium (5) is photolyzed in the presence of leucine to form the oxazolopyridocarbazole 387, and Kovacic ef al. (180) have presented a detailed proposal that many anticancer drugs, including ellipticines, operate by charge transfer resulting in the formation of oxygen radicals that can cleave DNA or other cellular constituents. Several studies have reported on ellipticine- or elliptinium-DNA interactions. The effects of elliptinium on chromatin in v i m or in the nuclei are an unfolding of the overall structure and a disorganization of the partial structure of the core, leading to an unwrapping of the DNA from the histone core (181). The kinetics and thermodynamics of ellipticine and ellipticinium (protonated ellipticine) binding to calf thymus DNA have been carefully investigated (182). It was

7.

317


Fe(Ill)

Fe(ll)

-

1. EDTA

H

2.

Fe(ll)-EDTA

+

-

H2Q

Fe(ll1)-EDTA

I CH3

+

OH

+

OH-

OH

398

OH

399

SCHEME 64. Proposed mechanism for the generation of hydroxyl radical and degradation of nucleosides (179).

concluded that the main force behind the DNA binding of ellipticine is hydrophobic and/or dipolar. At pH 9, the affinity constant of 1 ( 3 . 3 X lo5 M - I ) is only slightly less than that of the ellipticinium cation (8.3 X lo5 M - I ) . The base specificity of elliptinium (5) and 2-methylellipticinium (110) has been reinvestigated (183). In contrast to earlier work, it was demonstrated that the 9-hydroxylated ellipticine derivatives, such as elliptinium (9,express a guanine-cytosine (G-C) base-pair preference, with the preferred binding site being a doublet sequence of two adjacent G-C base pairs flanked by either another G-C or an adenine-thymine (A-T) base pair. In contrast, 2-methylellipticinium (110) acetate expresses no preference.

318

GORDON W. GRIBBLE

The effect of ellipticine derivatives on membranes and model membranes continues to be of interest to Sautereau and co-workers (184-186), who included 31P-NMRtechniques in their study (185). The ellipticine derivatives, such as 5, are deeply embedded in the acyl chain region of cardiolipin-containing model membranes. Sautereau et al. (186) studied the effects of elliptinium (5) on Streptococcus pneumoniae and concluded that the toxicity of 5 is related to its intracellular concentration. The interaction of elliptinium with numerous other biological targets has been studied in recent years. Elliptinium (5) is a potent inhibitor of fetal thymidine kinase and other enzymes that are induced by estradiol(187). Thus, 5 could bind the acceptor sites for estradiol receptor and, therefore, inhibit the activity of estradiol-regulating genes. Ellipticine (1)is the most potent inhibitor, of several compounds tested, of microsomal cholesterol 5,6-oxide hydrolase (188), an enzyme that converts cholesterol epoxide to the corresponding 3,5,6-triol. This work suggested that cholesterol epoxide could be a carcinogen involved in liver cancer. Elliptinium ( 5 ) and 9-hydroxyolivacinium are potent muscarinic antagonists and demonstrate pronounced affinity for muscarinic receptors (189).These compounds are only one-hundredth as active as atropine in their antagonism, but they show no interaction with three other neurotransmitter receptors. 9-Hydroxyellipticine (3) also binds to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in the rat lung (190). In another study of TCDD binding sites in rat liver cytosol, ellipticine had very weak binding affinity, but 5H-benzo[b]carbazole (400)and, especially, 5H, 11H-indolo[3,2-b]carbazole(401) had binding that was comparable to that of TCDD (191). Ellipticine and several derivatives were able to displace 7,12-dimethylbenz[a]anthracene from binding to bovine and human serum albumin using the fluorescence-quenching technique (192,193). The site of binding of ellipticines appears to be on the hydrophobic regions of the enzyme. A fast kinetic experimental technique, namely, temperature-jump spectroscopy, has been developed in order to study the interaction of elliptinium, and other molecules, with biological macromolecules (194). Several ellipticine derivatives were evaluated for their effects on Escherichia coli strains (195).There does not appear to be a correlation between the physiological effects of the ellipticines and their physiochemical behavior in vitro.

H

H

H 400

401

7.

ELLIF'TICINE ALKALOIDS AND RELATED COMPOUNDS

319

Whereas the quaternarized ellipticines have no bactericidal activity, the nonintercalating 9-bromoellipticine is a strong bactericidal agent, which apparently causes the lysis of the bacteria. When malignant liver cell cultures are treated with elliptinium, the level of spermidine increases, apparently as a result of decreased nuclear-bound polyamines connected to RNA (196). Ellipticine inhibits poly(ADP-ribose) glycohydrolase activity (297) and decreases DDT-induced tremors in rats (198). In the latter study, it was postulated that ellipticine acts directly on nerve or muscle tissue. A study of the Ir-stacking and edge-to-edge associations in several ellipticine derivatives using 'H-NMR techniques has appeared (199). As would be expected, methyl substitution at the N-6 and C-1 positions of ellipticine (1)significantly reduces the association constant, but, interestingly, the authors attribute this to electronic effects at the nitrogen atoms rather than to steric effects of the methyl groups. A quantum mechanical study on the intermolecular interaction energies of ellipticine with G-C and A-T base pairs has attempted to correlate these energies with the site of drug binding (200). A substantial number of reports have appeared since 1984 describing the effects of ellipticines on the cytochromes P-448 and P-450. The structural requirements for the substrate binding sites of these cytochromes have been studied and discussed at length by Lewis, Ioannides, and Parke in several excellent papers (201-204). Ellipticine (1) has been shown to inhibit rat embryo tissue cytochrome P-450 that is involved in the detoxification of the teratogen diphenylhydantoin (205). Thus, ellipticine enhances the in vitro toxicity of diphenylhydantoin. Ellipticine (1) also protects cells against the cytotoxicity of mitomycin C by inhibiting NADPH-cytochrome P-450 reductase (206). Ellipticine (1)and 9-hydroxyellipticine(3) also inhibit cytochrome P-450 in its role in the metabolism (hydroxylation, epoxidation) of pentachlorophenol (207), aflatoxin B-1 (208), 2-amino-3,8-dimethylimidazo[4,5-flquinoxaline and other protein pyrolysates (209), ecdysone (210), halogenated biphenyls (221), and coumarin (212). Ellipticine (l),9-hydroxyellipticine (3), and other derivatives have been studied with regard to their effects on the estrogen receptor (213), cytochrome-c oxidase in plant mitochondria ( 2 1 4 , and Ah receptor proteins and 4-S proteins in rodents (215). In the latter study, it was found that ellipticines are powerful binders of the 4-S carcinogen-binding proteins (stronger than benzo[a]pyrene) (215).The technique of microspectrofluorimetrywas used to probe the effects of ellipticine on the metabolism of benzo[a]pyrene in intact cells (216,217). The binding characteristics of oxazolopyridocarbazolestoward bacterial DNA have been studied (218). It was found that these ellipticine derivatives invariably exhibit DNA intercalation but with no sequence specificity. The new clinical candidate datelliptium (384) shows increased lipophilicity but no difference in binding or intercalation to DNA compared to elliptinium (5) (250). This new

320

GORDON W. GRIBBLE

derivative also has the same effect on topoisomerase I1 as does 5 but shows a pronounced increase in antitumor activity. Although the DNA binding properties of a series of N-2 and N-6 side-chain amine ellipticines 105-109, 111 were increased over that of ellipticine (l),the in vivo antitumor activity was less than desired (71). The excellent antitumor activity of ellipticine carbamate 153 led Archer and co-workers (84) to propose a new mechanism for the antitumor effects of ellipticine in general (Scheme 65). It is suggested that the C-5 methyl group is enzymatically hydroxylated and then converted either to the sulfate (402)or phosphate ester. This can now react with a cellular nucleophile (e.g., DNA, topoisomerase 11), by an SN1 or SN2 mechanism, to give the covalent adduct 403. This type of mechanism has been invoked to explain the antitumor activity of lucanthone (404)and hycanthone (405)(219,220). A study of the azaellipticines 204 and 406 showed that both compounds are active on topoisomerase I1 and initiate the cleavage of DNA (151,221). However, unlike ellipticine, these azaellipticines did not cleave DNA in isolated nuclei. Several papers have described the physicochemistry and biological activity of the oxazolopyridocarbazoles (i.e., 258). These interesting compounds behave as

1

* 3

metabolism

"Q7& -" Nuc:-

QyI

I

/

/

CH,Nuc 403 SCHEME 65. Archer et al. proposal for the mechanism of action of ellipticine (84).

7.


32 1

reversible intercalators and are less cytotoxic than elliptinium ( 5 ) (153,154,222), and they are frameshift mutagens (223,224).With regard to their low antitumor activity, it is such that the oxazolopyridocarbazoles cannot undergo biological conversion to quinone imines and are unable to generate “cleavable complexes” in DNA by interacting with topoisomerase I1 (154). The most frequent spontaneous mutation in DNA is depurination, occurring in mammalian cells at a rate of 10,000 per cell per day (225).It has been proposed that these sites can be trapped by condensation with aldehyde condensing reagents, such as primary amines, leading to DNA cleavage (226). Indeed, it has been found that 9-aminoellipticine (407) is remarkably effective at causing DNA cleavage at apurinic sites but not apyrimidinic sites (227-230). The concentration of 407 required to cause such breaks is the lowest of any chemical known to be active in this reaction (227). Molecular models (CPK) show that an apurinic site is ideally arranged for an insertion of 407 into the minor groove of DNA but that this is not feasible for an apyrimidinic site (228). One can quantify the number of such apurinic sites by fluorescence (229). The mechanism for the reaction of 9-aminoellipticine with apurinic DNA is shown in Scheme 66 (230). The intermediate imine (Schiff base) 408 has been trapped with sodium cyanoborohydride to give 409. Interestingly, this reductive amination reaction of apurinic DNA with 9-aminoellipticine in the presence of sodium cyanoborohydride has been used to synthesize an ellipticine-octathymidylate conjugate (130). The azaellipticine 204 has been reported in preliminary form (231) to break DNA at apurinic sites. As we have seen, the 11H-pyridocarbazoles (Schemes 36 and 37) are not cytotoxic, yet some of these derivatives have high DNA binding affinities and are

NH(CH,),NEtz

CH3

I

I

204

406

322

0

A

GORDON W. GRIBBLE

0

A

SCHEME 66. Hypothesis for the reaction of 9-aminoellipticine (407) with apurinic DNA (230).

true intercalators (100). Even more intriguing is the observation that some 11Hpyridocarbazoles are not intercalators but, nevertheless, have high affinities for DNA. For example, 410 and 411 have high DNA affinities but are not intercalators, but 412 and 413 are true intercalators. The 7H-pyridocarbazoles(Schemes 38 and 39) have been exhaustively studied by Roques and co-workers (102). It is seen that, for highly active antitumor compounds, it is necessary to have high DNA binding and intercalation. The structure-activity relationships of these nonlinear pyridocarbazoles are discussed in Section XI. Some very elegant theoretical (232) and 'H-NMR studies (233) have shown that the concave side of 7H-pyridocarbazoles, such as 414, protrudes into the major groove of a minihelical self-complementary tetranucleotide

7.


CI -

41 0

41 2

323

tH3

411

41 3

[d(CpGpCpG),] and hexanucleotide [d(CpGpApTpCpG),] . The quinoxaline analog 415 has also been studied by 'H-NMR techniques to evaluate its interaction with the self-complementary decanucleotide d(GpCpApTpTpApApTpGpC), (234). The binding sites appear to involve only the A-T base pairs. In a monumental piece of research, Roques and co-workers (235-239) have employed exceedingly sophisticated and difficult high-field 'H- and 31P-NMR experiments to examine the interaction of nonlinear bispyridocarbazoles, such as ditercalinium (212), with small polynucleotides. As in their earlier work on monomers (233), the self-complementary tetra- and hexanucleotides were used. It was found that ditercalinium is a bis-intercalator with a preference for alternating sequences, pyrimidine-purine or purine-pyrimidine, and, in agreement with the classic pyrimidine-purine model (240,241), it was found that the linking chain lies in the major groove of the helix. Although ditercalinium (212) has since been withdrawn from clinical trials

I

CH,CH,NMe, 414

41 5

324

GORDON W. GRIBBLE

because of unacceptable liver toxicity (242), it exhibits very interesting biological properties. Ditercalinium (212) seems to act as a DNA condensing agent by altering chromatin structure in vivo (L1210), but it does not cause DNA strand breaks or DNA-DNA or DNA-protein cross-links (243). Thus, the cytotoxicity of 212 may be due to the condensation of DNA rather than to the initiation of topoisomerase 11-associated DNA strand breaks. It has been shown that ditercalinium (212) forms a high-affinity yet reversible and noncovalent DNA adduct in E. coli (243). Nevertheless, cell death results (although delayed for five or six generations) because a conformational change is induced in the DNA that is similar to the changes induced by covalent adducts, thus triggering the DNA repair system. Although similar bispyridocarbazoles, but with longer linking chains, form high-affinity reversible DNA adducts, they do not induce conformational changes in the DNA, and, thus, the complex is not recognized by the repair system. Why this recognition of essentially normal DNA by the repair system leads to cell death in E. coli is unclear. A study of ditercalinium on leukemic cells indicates that it increases the sensitivity of DNA to denaturation induced by acid (244). Furthermore, 212 exhibits no cell cycle phase specificity, unlike most DNA intercalators which arrest cells in G , phase. A kinetic and thermodynamic investigation of ditercalinium and its interactions with anions and DNA has been reported (242). This study reveals the important fact that intermolecular and intramolecular stacking does not occur in 212. Although the unsymmetrical bispyridocarbazoles (Scheme 5 1) and the pyridocarbazole-acridine hybrids 327 and 328 have high DNA binding and seem to be bis-intercalators, they display little or no antitumor properties in vitro or in vivo (120). The position of the pyridine nitrogen in the D ring of ditercalinium (212) and the presence or absence of methyl groups play a very important role in the antitumor activity of the resulting derivatives (102). These are presented in Section XI. The tricyclic analogs of ellipticine and 9-azaellipticine (Schemes 46 and 45, respectively) are poorer DNA intercalators than their tetracyclic analogs but, nevertheless, have interesting and significant antitumor activity (Section XI) (115-117). Moreover, some derivatives have high DNA affinity but are essentially inactive in vitro or in vivo (L1210). Although there is no direct relationship between DNA affinity and cytotoxicity in vitro of the tricyclic analogs, high DNA affinity is necessary for antitumor activity. The observed close correlation between in vitro cytotoxicity and the induction of DNA cleavage in cells suggests that these breaks are responsible for cell death. These authors further conclude that the DNA breaks are probably induced by an interaction between drug and topoisomerase 11-DNA complex (117). In conclusion, although it is fair to say that the mechanism of the antitumor activity of ellipticines and related compounds remains unproved, metabolic activation of an ellipticine to a quinone imine or related species of high elec-

7.


325

trophilicity, DNA intercalation (not just binding), and topoisomerase I1 as a critical cellular target all seem to be important factors in this mechanism.

IX. Mutagenicity

Several reports have appeared recently that describe the mutagenicity of ellipticine and derivatives. Moore and co-workers (245) have shown that ellipticine (1) is both mutagenic and clastogenic at the tk locus of mouse lymphoma cells, and the major mechanism is chromosomal cleavage. Similar effects were seen when 1 was applied to rat bone marrow cells an? human peripheral blood lymphocytes (246).Interestingly, the source of ellipticine for this latter research was natural, Aspidosperma williansii (Apocynaceae). Ellipticine was also mutagenic in bacteriophage T4, whereas 9-aminoellipticine (407) and 9-methoxyellipticine (2) were not (247). The reversible DNA intercalators oxazolopyridocarbazoles (258) induce frameshift mutations of the mismatch-repairable type in Salmonella typhimurium and E . coli (223,224,248,249).

X. Metabolism and Microbial Transformation

The metabolism of any drug is invariably of theoretical and practical importance, and the metabolic behavior of the ellipticine family of antitumor alkaloids and synthetic derivatives is no exception. A number of new developments have been described since the Suffness and Cordell review (10).The pharmacokinetics of elliptinium (5) have been studied in a human brain tumor clonogenic cell assay (250) and in metastatic breast cancer patients (251). In the latter study, the drug was mainly excreted in the feces (I4C-labeled 5 ) The metabolism of elliptinium has been investigated in rat kidney cells and yields four metabolites: 10(S)-N-acetylcysteine 416 (major), lO(S)-glutathione 417 (minor), lO(S)-cysteine 418 (minor), and 9(0)-glucuronide conjugate 419 (minor) (252). From the bile of human cancer patients treated with elliptinium, there have been isolated, in addition to unchanged 5, the 0-glucuronide 419 and the 10(S)-cysteine 418 adducts (253). From the urine of such patients, the glutathione 417 conjugate can be isolated (138). In contrast, elliptinium ( 5 ) is metabolized in rats such that the glutathione 417 is found in bile, along with unchanged drug, whereas N-acetylcysteine 416 is found in bile and urine, along with unchanged drug (137). Red blood cells also provide a medium for the

326

GORDON W. GRIBBLE

metabolism of elliptinium, giving rise apparently to the glutathione adduct 417, after incubation of the cells in the presence of glutathione and hydrogen peroxide or tert-butyl peroxide (254). This study would suggest that red blood cells may be a significant site of bioactivation of ellipticines into their quinone imine intermediates. Rats metabolize 9-methoxyellipticine (2) into 9-hydroxyellipticine (3), the glucuronide 419, the 9(O)-sulfate, and the glutathione conjugate 417, all isolated from the bile (255). This unexpected demethylation had been previously observed when 2 was exposed to HRP/H,O, (256) or rodent liver microsomes (257). The latter study demonstrated the demethylation of BD-84 (386)as well. The mechanism of this HRP/H,O, demethylation has been studied (258,259), and it is clear from elegant 180-labelingexperiments that the aryl-oxygen bond is cleaved (Scheme 67). When 2 was incubated in H2l80, there was observed 100% inclusion of the l 8 0 label into the product quinone imine 6, which was isolated in 64% yield. Moreover, the methanol could be isolated by gas chromatography, and it was found to be devoid of l80.A proposed mechanism is shown in Scheme 67. A similar demethylation of the 9-methoxyl derivative of elliptinium has also been observed in the presence of HRP/H,O, to yield the 0

II

Hob

H02cy"HccH3 ~

@

0CH3

/

\

/ H

I

CH3

CH3 416

4 17

HozcTNHz 418

41 9

7.

-

["I

H20

HoQ--d]

CH30

327


-CH30H

~

on--+

64% C"3 6

SCHEME 67. Possible mechanism for the demethylation of 9-methoxyellipticine with HRP/H202

C-10 N-acetylcysteine adduct 416 (40% yield) when the incubation is performed in the presence of N-acetylcysteine (259). The metabolism of 6-methylelliptinium (420) in rats (bile and urine) gives rise to the 0-sulfate and 0-glucuronide conjugates, but no demethylation of the N-6 methyl group (260) (Scheme 68). Likewise, the HRP/H,O, system gives rise to the orrho-quinone 421 and the oxazolopyridocarbazole 422, when alanine is present, but not to N-6 demethylation (261). The metabolism of olivacine (4) in rats and microsomes is faster than that of ellipticine, and leads to hydroxylation at the C-7 and C-9 positions (as conjugates) (55). In very preliminary work, the metabolism in vitro and in vivo of the new clinical candidate datelliptium (384) has been reported to involve oxidative

328

'

GORDON W. GRIBBLE

-

N I

CH3

CH3

OAc-

alanine

/

/

I

CH3

CH3

420

421

422

SCHEME 68. Peroxidase oxidation of 6-methylelliptinium (420) and reaction with alanine (261).

degradation of the amine side chain and glucuronide formation of the corresponding products, as well as ortho-quinone production (262).

XI. Structure-Activity Relationships

An extensive study by Meunier et al. (170) of the electrochemical, biochemical, theoretical, and antitumor properties of a series of ellipticines and their quinone imines strongly implicates the latter species in the mechanism of the antitumor action of ellipticines. As Table I reveals, there are strong correlations between the ease of formation of quinone imines, their reactivities with nucleophiles, and their antitumor potency. Although 7-hydroxy-2-methylellipticinium (425) undergoes oxidation to the corresponding quinone imine, the latter intermediate apparently is extremely susceptible to polymerization. It is interesting that the presence of a C-10 methyl group does not seem to block the formation of a covalent adduct (as yet unidentified) with the corresponding quinone imine. Indeed, 9-hydroxy-2,8,l0-trimethylolivacinium (428) has good antitumor activity.

7.


329

The kinetics of decomposition of benzoyl peroxide in the presence of various hydroxypyridocarbazoles have been studied by Auclair and co-workers (263). These data were then used to determine the bond dissociation energies of the 0-H bond. A reasonable correlation is seen between the bond dissociation energy and the cytotoxicity of the compound (Table 11). The effects of amine-substituted ellipticine derivatives 88 and 105-111 on L1210 and the human colon tumor (HCT8) in vitro (Table 111) indicate that these compounds are not more cytotoxic than ellipticine (1) (71). Furthermore, only a low order of activity was revealed in vivo against P388 for 110 and 111. The N-6 derivatives were inactive as antitumor agents. However, the DNA binding properties of all of these amine-substituted ellipticines were superior to 1, as determined by the ethidium displacement assay. The antitumor activities and DNA affinities of several 1-alkylamino derivatives 113-117 of 9-methoxyellipticine (2) have been measured (Table IV) (72). These compounds are cytotoxic and have some antitumor properties, but there does not appear to be a correlation between their cytotoxicity and DNA intercalating ability. Moreover, these derivatives tend to accumulate in the cytoplasm rather than in the nuclei of the cells. Compound 118, with a longer alkyl chain, is not taken up by the cells, presumably owing to the hydrophobic nature of the decyl chain. From the DNA binding data, the authors conclude that only 113115 behave as true intercalators. Table V lists a few of the 49 ellipticine glycosides that have been prepared and evaluated for anticancer activity by Honda and co-workers (69). From these data, it can be summarized that the 9-hydroxyl group is essential for high activity. Peracylated glycosides are less active than the hydroxylated counterparts, and the introduction of an amide group dramatically lowers the activity. The counterion X - (chloride, bromide, acetate) makes no difference. In a series of pyranosides, those having three hydroxyl groups are more active than those having four hydroxyl groups. Several 9-hydroxyellipticine 1’ ,2’-cis-glycosides also showed good activity, but a relationship with the 1’,2’-trans sugars could not be established. Also, no clear relationship between enantiomeric pairs, or between furanosides and pyranosides, could be identified. Nevertheless, these simple ellipticine derivatives show extraordinary antitumor activity against L12 10 in vivo (also against P388, B16, colon 38, EAC, and sarcoma 180 in vivo), and they seem to be much more active than ellipticine (l), 9-hydroxyellipticine (3), and elliptinium (5) against these mouse tumors. Several ellipticines and 7Hypyrilo[4,3-c]carbazoleswere examined for their effect on topoisomerase I and I1 from trypanosomes (Table VI) (163). The activity of 9-bromoellipticine on topoisomerase I1 is especially interesting since it is not a DNA intercalator. Several other bis-7H-pyrido[4,3-c]carbazoleswere strongly active in this assay. As indicated in Table VII, a study of the cytotoxicity and uptake by TBL CL2 mouse sarcoma cells of several oxazolopyridocarbazoles

330

GORDON W. GRIBBLE

TABLE I REACTIVITY A N D BIOLOGICAL ACTIVITY OF ELLIFTICINE DERIVATIVES (170) 10

11

1

SubstituentsO Compound

C-1

9-Methoxy-6-methylellipticine(423) 2-Methylellipticinium (110) 9-Methoxy-2-methylellipticinium(424) Ellipticine (1) 9-Methoxyellipticine (2) 6-Methylelliptinium (420) 7-Hydroxy-2-methylellipticinium (425) 9-Hydroxy-6-methylellipticine (93) 9-Hydroxy-2-(2-diethylamino)ethylellipticinium(384) 9-Hydroxy-2-methylolivacinium(426) CH, Elliptinium (5) 7-Methylelliptinium (427) CH, 9-Hydroxy-2,8,lO-trimethylolivacinium (428) 9-Hydroxyellipticine (3) 8,1O-Dimethylelliptinium(429)

N-2

N-6

C-7 C-8

CH3 CH3 CH,

CH, CH,

CH,

C-10 C-11

OCH,

CH3 CH3 CH, CH, CH3 CH3 CH3

OCH, OCH, OH

CH, OH CH,

DEAEf CH3 CH, CH, CH,

C-9

CH, CH, CH,

OH OH OH OH OH OH OH OH

CH, CH, CH3 CH3 CH,

CH3 CH3

Substituents not indicated are hydrogen (or unmethylated pyridine for N-2). Horseradish peroxidase turnover number (10-6M HRP) as micromoles H,O, consumed per minute per micromole HRP. c Anodic sweep in volts. Dose which reduces by SO%, after 48 hr, the L1210 cell growth relative to controls. Antitumor activity symbols: -, no determination; 0, no activity (TIC < 125%); f , TIC > 125% and therapeutic index 5 2; TIC 125- 170% and therapeutic index 2 2; +++, T / C > 170%. I DEAE, (2-Diethylamino)aminogroup.

++,

(387,434-438) and elliptinium (5)revealed that, although uptake was rapid, consistent with a diffusion mechanism, the cytotoxicity of these amino acid conjugates is less than that of elliptinium (5) (153,154). This lower cytotoxicity is believed to be due to the absence of the 9-hydroxyl group. However, the isoleucine adduct 387 is unusually cytotoxic for the series. An extensive study by Roques and co-workers (102) of the cytotoxicity, antitumor activity, and DNA binding affinities of a series of 7H-pyridocarbazole monomers and dimers is presented in Tables VIII and IX.These data reveal the importance of methyl substitution at positions C-6 or C-7 in the N-2 monomers

7.


Oxidation HRP6 0 18

25 30 710 26,000 13,500 38,500 13,000 28,000

-

12,000 33,500 11,OOO

Electrochemicalc I .o 0.820 0.625 0.600 0.500 0.420 0.295 0.280 0.260 0.205 0.180 0.160 0.130 0.100 0.035

Adduct formation Alanine No No Yes No No Yes No No Yes Yes Yes No No No

Biological activity

Nucleoside

IC,,, phfd

In vivo L121Oe

No No Yes No Yes Yes

-

-

1.03 1.14 1.45 1.80 0.089 4.26 0.076 0.050 0.29 0.11 1.19 0.86 0.11 6.04

0 0

No Yes Yes Yes Yes

Yes? Yes Yes?

33 1

+ + ++ 0 -

+++ ++ ++ + ++ ++ 0

In vivo P388e -

0

+ +++ + +++ ++ ++ ++ ++ -

for antitumor activity, although this is relatively weak. In the dimers, several highly active compounds belonging to the N-2 series were discovered, but none in the N-3 series. No clear and consistent correlation between antitumor potency and DNA affinity is found, although the inactive N-3 dimers are unable to bisintercalate with DNA. The cytotoxicity of several benzo-annulated iso-a-carbolines (369-372,377, 378, 380)has been studied in vifro on human tumor IU3 cells (Table X) (136). The linear ellipticine analog 371 is 10 times more active than ellipticine (1)in this screen. As was mentioned earlier, this derivative also displays significant in vivo activity against P388, L1210, and B16 implanted mouse tumors (TIC values 190, 175, and 224%, respectively). The resemblance of the imine grouping of these iso-a-carbolines to ellipticine quinone imine 6 is obvious. It will be of interest to see how the electrophilic behavior of these compounds compares to that of the quinone imines .

332

GORDON W. GRIBBLE

TABLE I1 CORRELATION BETWEEN ( t H BONDDISSOCIATION ENERGIES A N D CYTOTOXICITY (263) 11

1

Substituentsa Compound

C-1

9-Hydroxyolivacine (73) 9-Hydroxyellipticine (3)

N-6

C-7

CH,

6-Methyl-9-hydroxyellipticine(93) 1 1-Demethyl-9-hydroxyellipticine(430)

CH,

7-Hydroxyellipticine (431) Phenol a-Tocopherol a b

OH

C-9

C-11

OH OH OH OH

CH, CH,

Bond dissociation energy, kcal/rnol 79.4 79.6 81.5 81.8 86.3 88.2 78.2

CH,

IC,,, pMb -

0.015 0.022

5.44 -

Substituents not indicated are hydrogen. Dose that reduces by 50%. after 48 hr, the L1210 cell growth relative to controls

ACTIVITY OF N-2

AND

TABLE I11 N-6 AMINE-SUBSTITUTED ELLIPTICINES (71)

I

CH,

In

Substituents Compound 88 105 106 107 108 109 110 111 Ellipticine (1) 0

N-2

N-6

vitro IC,,,

L1210 0.43 0.11 0.32 0.17 0.19 Inactive 0.6 0.041 0.1

Dose that reduces by 50% the cell growth relative to controls, 2 days after drug exposure.

pMa

HCT8 -

0.39 0. I 0.082 0.08

Inactive 0.5 0.26

TABLE IV ACTIVITY OF ~-(ALKYLAM1NO)-9-METHOXYELLIF'TlCINES (72)

In virro IC,,, p,W C-l Substituent

Compound

L1210

NIH 3T3

In vivo ILS, (L 12 10)

DNA binding

%b

K,,,,

113

114 115 116

117 9-Methoxyellipticine (2)

NH2 NHCH,CH, NHCH,CH,CH, NHCH2CH(CH,), NHCH,CH,CH(CH,), H

~

0.1 0.05 0.1 0.09 0.8 0.1

0.3 0.3 0.3 0.3 0.5 0.3

93 42 -

20

* Dose that reduces by 50% the L1210 cell growth relative to controls, 48 hr after drug exposure Increase in mean life span.

Unwinding angle,

M-l

~

-1

2.15 2.00 2.35 3.75 0.9

~~~

24 16 15 12 7 10

O

334

GORDON W . GRIBBLE

TABLE V In vivo ACTIVITY OF ELLIPTICINE ~',~'-~?xI~S-GLYCOSIDES ON L1210 LEUKEMIA (69)

Substituent c-9

R

H P-D-Ribofuranoside OCH, P-D-Ribofuranoside OH P-D-Ribofuranoside OH P-L-Ribofuranoside OH P-L-Ribopyranoside a-D- Arabinopyranoside OH a - L - Arabinopyranoside (104) OH a-LArabinopyranoside OCH, OH D-Lyxofuranoside (a-P 76 : 24) OH a-L-Lyxopyranoside OH P-D-Xylofuranoside(383) OH L-Rhamnopyranoside(a-P 92 : 8) Ellipticine (1) 9-Hydroxyellipticine(3) Elliptinium 5) Doxorubicin a b

Optimal dose, mgm 20 30 10 10 30 20 30 30 30 30 30 30 120 60 5

2.5

ILS,

%a

80 76 138 >391 >944 >606

>860 56 >967 >786 >682 >693 128 79 48 90

Increase in mean life span. Number of survivors/totalat 80 days.

TABLE VI ACTIVITY OF ELLIPTICINES AND 7ff-PYRIDo[4,3-C]CARBAZOLES ON TOFTIISOMERASE I AND I1 (163) Activity, IC, p M Compound Ellipticine (1) 9-Hydroxyellipticine(3) Elliptinium (5) Ditercalinium (212) 6-Methylelliptinium (420) 2,7-Dimethyl-10-methoxy-7H-pyrido[4,3-~]carbolinium(432) 9-Aminoellipticine (407) 9-Bromoellipticine(433)

Decatenation (Topo 11)

Relaxation (Top0 I)

26 7 7 5 8 21

I70 170 35

38 31

150

-

Curesb 016 016 016 216 616 316 516 016 616 516 416 416 -

-

7.

335


TABLE VII OF OXAZOLOPYIUDOCARBAZOLES (253,254) ACTIVITY

R

Activity (L1210) Compound

R

434 435 436 437 387 438 Elliptinium (5)

H CH, CH,CH, CH(CH3)2 CH,CH(CH& CH(CH,)CH,CH,

a b L.

Uptakea nmol/l06 nuclei

In vitro IC,,, )WUb

In vivo TIC, %c

1.28

0.54 0.20 0.36 0.31 0.28 0.58 0.10

123 I24 131 135 118 157

1.40

1.35 0.88 0.13 0.15 1.14

100

By isolated TBL CL2 mouse sarcoma nuclei. Dose that reduces by 50% the cell growth relative to controls, 48 hr after drug exposure Treated mean survival time per untreated mean survival time; TIC % > 125:activity.

The effect of the linker chain on the activity of bis-7H-pyridocarbazoles(Table XI) (119) reveals that the degree of flexibility inherent in the bipiperidine linker is crucial for activity. Thus, when n is 1, the dimer (321,322)can adopt a kinked structure, decreasing the tendency for intramolecular stacking and increasing the propensity for DNA bis-intercalation. However, when n is 0, 2 , or 3, the dimers are suggested to prefer a parallel arrangement of the pyridocarbazole rings in which intramolecular r stacking can occur, reducing DNA bis-intercalation. Although C-9 hydroxylation (73)of olivacine increases the in vitro activity against L1210 over that of olivacine (4), the in vivo antitumor activity is unchanged (Table XII) (55). This appears to be the result of a rapid elimination of drug. Hydroxylation at C-7 (71)leads to an inactive compound in vitro, reminescent of the low degree of cytotoxicity of 7-hydroxyellipticine (IDso 5.44 pM) (55,170). The effect of several ellipticines and 9-azaellipticines on the cell cycle progression and survival of NIH 3T3 mouse cells was studied (Table XIII) (264). The effects of both series of compounds are identical, leading to growth arrest and blockage in G , phase. The most interesting conclusion from this study is that BD-40 (385)may require metabolic activation prior to acting on the cells.

TABLE VIII ACTIVITY OF 7ff-PYRIDOCARBAZOLES (102)

Substituent

Activity (L1210)

Compound

N-R

C-5

C-6

N-7

In v i m IC,,, p M

439

2-NCH, 2-NCH, 2-NCH, 2-NCH3 3-NCH3 4-NCH3 2-NCH$H,N(CH2),

H CH3 H H H H H

H H CH, H CH, CH, CH,

H H H CH, H H H

0.95 >2.5 0.06 0.22 0.30 >2.5 0.11

233 440 441 442

443 444 a b

In vivo TIC,

Dose that reduces by 50% cell growth relative to controls, 24 hr after drug exposure. Treated mean survival time per control mean survival time; NT, not tested; NS, not significant,

NS NT 125 122 NT NT 123

%b

DNA binding ( X 105 M-1) 2.9 0.6 1.8 7.0 3 0.61 9.3

337

7 . ELLIPTICINE ALKALOIDS AND RELATED COMPOUNDS TABLE IX ACTIVITY OF 7ff-PYRIDOCARBAZOLE DIMERS (102)

Substituent

Activity (L12 10)

Compound

N

C-5

C-6

N-7

212 445

2 2 2 2 2 3 3 3

H CH, H H H H H H

H H CH, H CH, H CH, H

H H H CH, CH, H H CH,

446 447

448 449 450 451 0

In virro IC,,, pM' 0.19

>I 0.37 0.36 3.27 >I

In vivo T / C , % 182 120 172 178

0.5 50 2

-

10

130 -

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The Alkaloids: Chemistry and Pharmacology, Volume 52

The Alkaloids: Chemistry and Pharmacology, Volume 29

The Alkaloids: Chemistry and Pharmacology, Volume 37

The Alkaloids: Chemistry and Pharmacology, Volume 41

The Alkaloids: Chemistry and Pharmacology, Volume 36

The Alkaloids: Chemistry and Pharmacology, Volume 31

The Alkaloids: Chemistry and pharmacology volume 26

The Alkaloids: Chemistry and pharmacology volume 28

The Alkaloids: Chemistry and pharmacology Volume 32

The Alkaloids Chemistry and Pharmacology, Volume 04

The Alkaloids: Chemistry and Pharmacology, Volume 30

The Alkaloids: Chemistry and Pharmacology, Volume 43

The Alkaloids: Chemistry and Pharmacology, Volume 49

Alkaloids: Chemistry and Pharmacology, Volume 12

Alkaloids: Chemistry and Pharmacology, Volume 9

The Alkaloids: Chemistry and Pharmacology, Vol. 39

The Alkaloids: Chemistry and Pharmacology, Vol. 24

The Alkaloids: Chemistry and Pharmacology, Vol. 21

The Alkaloids: Chemistry and Pharmacology, Vol. 33

The Alkaloids: Chemistry and Pharmacology, Vol. 45

The Alkaloids: Chemistry and Pharmacology, Vol. 46

The Alkaloids: Chemistry and pharmacology Volume 38

The Alkaloids: Chemistry and Pharmacology, Volume 15

The Alkaloids: Chemistry and Pharmacology, Volume 44

The Alkaloids Chemistry and Pharmacology, Volume 27

The Alkaloids: Chemistry and Pharmacology, Volume 51

The Alkaloids: Chemistry and Pharmacology, Volume 22

The Alkaloids: Chemistry and pharmacology Volume 34

The Alkaloids: Chemistry and Pharmacology, Volume 37

The Alkaloids: Chemistry and Pharmacology, Volume 40

The Alkaloids: Chemistry and Pharmacology, Volume 35

The Alkaloids: Chemistry and Pharmacology, Volume 52

The Alkaloids: Chemistry and Pharmacology, Volume 29

The Alkaloids: Chemistry and Pharmacology, Volume 37

The Alkaloids: Chemistry and Pharmacology, Volume 41

The Alkaloids: Chemistry and Pharmacology, Volume 36

The Alkaloids: Chemistry and Pharmacology, Volume 31

The Alkaloids: Chemistry and pharmacology volume 26

The Alkaloids: Chemistry and pharmacology volume 28

The Alkaloids: Chemistry and pharmacology Volume 32

The Alkaloids Chemistry and Pharmacology, Volume 04

The Alkaloids: Chemistry and Pharmacology, Volume 30

The Alkaloids: Chemistry and Pharmacology, Volume 43

The Alkaloids: Chemistry and Pharmacology, Volume 49

Alkaloids: Chemistry and Pharmacology, Volume 12

Alkaloids: Chemistry and Pharmacology, Volume 9

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