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Alkaloids: Chemical and Biological Perspectives


Related Titles of Interest Books GAWLEY & AUBE Principles of Asymmetric Synthesis LEVY & TANG The Chemistry of C-Glycosides PELLETIER Alkaloids: Chemical & Biological Perspectives Volume 9 Alkaloids: Chemical & Biological Perspectives Volume 10 Alkaloids: Chemical 8L Biological Perspectives Volume 11 RILEY & ROSANSKE Development & Validation of Analytical Methods WONG & WHITESIDES Enzymes in Synthetic Organic Chemistry Major Reference Works Comprehensive Heterocyclic Chemistry II Comprehensive Natural Products Chemistry* Comprehensive Organic Functional Group Transformations *In preparation Journals Bioorganic & Medicinal Chemistry Bioorganic & Medicinal Chemistry Letters Carbohydrate Research Heterocycles (distributed by Elsevier) Phytochemistry Tetrahedron Tetrahedron Asymmetry Tetrahedron Letters


ALKALOIDS: CHEMICAL AND BIOLOGICAL PERSPECTIVES Volume Twelve

Edited by

S. WILLIAM PELLETIER Institute for Natural Products Research and Department of Chemistry The University of Georgia, Athens

1998 ELSEVffiR Amsterdam - Lausanne - New York - Oxford - Shannon - Singapore - Tokyo


Elsevier Science Ltd The Boulevard, Langford Lane Kidlington, Oxford OX5 1GB, U.K. Copyright Š1998 Elsevier Science Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without permission in writing from the publishers. First edition: 1998 Library of Congress Cataloging in Publication Data A catalog record for this serial is available from the Library of Congress. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN: 0 08 0428053 Ž The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Transferred to digital printing 2005


Dedicated to the memory of

Walter Abraham Jacobs (1883-1967) After receiving a Ph.D. with Emil Fischer (Nobel Prize, 1902) in Berlin in 1907, Jacobs joined the staff of the Rockefeller Institute for Medical Research in New York City, working with P. A. Levene in the area of nucleic acids and chemotherapeutic agents. He soon was invited to establish a Laboratory of Chemotherapy. This was later changed to a Laboratory of Chemical Pharmacology. At Rockefeller he carried out important research on the isolation and elucidation of structures of the complex Ergot alkaloids. In 1934 he and his collaborators isolated a hydrolysis product from the alkaloids to which they gave the name Lysergic Acid and showed it to be present in each member of this large family of alkaloids. The structure of lysergic acid and the major features of the structures of several alkaloids were established in Jacobs' laboratory. The ergot work was concluded with the synthesis of dihydrolysergic acid. Jacobs then turned his attention to two other large classes of toxic plant alkaloids of unknown structure, the Veratrum alkaloids from Veratrum species, and the Aconite alkaloids from Aconitum and Delphinium species. The Veratrum alkaloids proved to be modified sterol derivatives, while members of the Aconite group were shown to be diterpenoid and norditerpenoid alkaloids.


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Contributors Detlev Belder, Johannes Gutenberg-Universitat, Institut fiir Pharmazie, Lehrstuhl fiir Pharmazeutische Biologic, Staudinger Weg 5, D-55099 Mainz, GERMANY Michael F. Clothier, Animal Health Discovery Research, Pharmacia and Upjohn Inc., Kalamazoo, Michigan 49001, U.S.A. Gabe I. Kornis, Animal Health Discovery Research, Pharmacia and Upjohn Inc., Kalamazoo, Michigan 49001, U.S.A. Byung H. Lee, Animal Health Discovery Research, Pharmacia and Upjohn Inc., Kalamazoo, Michigan 49001, U.S.A. Sylvie Michel, Universite Rene Descartes-Paris V, Faculte de Pharmacie, Laboratoire de Pharmacognosie, 75270 Paris Cedex 06, FRANCE Helmut Ripperger, Institute of Plant Biochemistry, D-06120 Halle (Saale), GERMANY Alexios-L6andros Skaltsounis, Universite Rene Descartes-Paris V, Faculte de Pharmacie, Laboratoire de Pharmacognosie, 75270 Paris Cedex 06, FRANCE Michael B. Smith, Department of Chemistry, The University of Connecticut, Storrs, Connecticut 06269-4060, U.S.A. Detlef Stockigt, Institute fiir Pharmazie, Lehrstuhl fur Pharmazeutische Biologic, Johannes Gutenberg-Univcrsat Mainz, Staudinger Weg 5, 55099 Mainz, GERMANY Joachim Stockigt, Institute fiir Pharmazie, Lehrstuhl fur Pharmazeutische Biologic, Johannes Gutenberg-Univcrsat Mainz, Staudinger Weg 5, 55099 Mainz, GERMANY Fran9ois Tillcquin, Universite Rene Descartes-Paris V, Faculte de Pharmacie, Laboratoire de Pharmacognosie, 75270 Paris Cedex 06, FRANCE Matthias Unger, Institute fiir Pharmazie, Lehrstuhl fur Pharmazeutische Biologic, Johannes Gutenberg-Univcrsat Mainz, Staudinger Weg 5,55099 Mainz, GERMANY Peter Wipf, Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, U.S.A.

vii


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Preface Acronycine, a potent antitumor agent with a broad spectrum of activity, was discovered in 1948 in the bark of the small Australian Rutaceous tree, Acronychia baueri Schott. Since then, many derivatives and structural analogues have been isolated from various Rutaceae species and prepared by synthesis. Chapter 1 by Fran9ois Tillequin, Sylvie Michel, and Alexios-Leandros Skaltsounis presents a comprehensive survey of the isolation, structure determination, methods of synthesis, and the biological properties of acronycine, as well as an account of natural and synthetic analogs of acronycine, and their biological properties. Since the last review on the Solanum alkaloids in 1990, there has been substantial progress in the field concerning isolation procedures and structure elucidation methods. Chapter 2 by Helmut Ripperger, provides a brief survey of new developments and critically updates earlier reviews. Chapter 3 by Peter Wipf reviews the interesting chemistry and synthesis of cyclopeptide alkaloids characterized by an alternating sequence of five-membered heterocycles and hydrophobic amino acid residues. These cyclopeptide alkaloids have been isolated from ascidians, sea hares, and cyanobacteria. A common synthetic strategy for constructing natural products is to use a chiral, nonracemic starting material. The availability of amino acids have made them popular starting materials for such applications. Chapter 4 by Michael B. Smith summarizes the use of the functionalized lactam, pyroglutamic acid, as a chiral templete for the synthesis of alkaloids. The chapter focuses exclusively on compounds derived from L-, D-, or D, L- glutamic acid. Chapter 5 by Joachim Stockigt, Matthias Unger, Detlef Stockigt, and Detlev Belder presents a brief review on the on-line coupling of capillary electrophoresis (CE) and mass spectrometry (MS) for the analysis of alkaloid mixtures. Because of particular physical and chemical properties of alkaloids, their analytical separation and identification are frequently not easily carried out. This chapter demonstrates that the CE-MS technique provides a rapid and efficient screening procedure for alkaloid mixtures. Parasitic nematodes cause substantial health problems in humans and in domestic animals. None of the drugs currently used for control of gastrointestinal nematodes is ideally suited for all therapeutic situations. Thus expansion of the anthelmentic arsenal is an urgent goal. Chapter 6 by Byung H. Lee, Michael F. Clothier, and Gabe I. Kornis treats oxygenated analogs of Marcfortine A, an alkaloid with potent antiparasitic activity. These analogs were prepared by chemical synthesis and by microbiological hydroxylation. Each chapter in this volume has been reviewed by at least one expert in the field. The editor thanks these reviewers for the very significant contributions they have made to this volume. Indexes for both subjects and organisms are provided. ix


X

Preface

The editor invites prospective contributions to write him about topics fcÂť^ review in future volumes in this series. S. William Pelletier Athens, Georgia September 3,1997


Contents of Previous Volumes Volume 1 1. The Nature and Definition of an Alkaloid S. William Pelletier 2. Arthropod Alkaloids: Distribution, Functions, and Chemistry Tappey H. Jones and Murray S. Blum

33

3. Biosynthesis and Metabolism of the Tobacco Alkaloids Edward Leete

85

4, The Toxicology and Pharmacology of Diterpenoid Alkaloids M. H. Benn and John M. Jacyno 5. A Chemotaxonomic Investigation of the Plant Families of Apocynaceae, Loganiaceae, and Rubiaceae by Their Indole Alkaloid Content M. Volkan Kisabiirek, Anthony J.M, Leeuwenbergy and Manfred Hesse

153

211

Volume 2 1. Some Uses of X-ray Diffraction in Alkaloid Chemistry Janet Finer-Moore, Edward Arnold, and Jon Clardy 2. The Imidazole Alkaloids Richark K. Hill 3. Quinolizidine Alkaloids of the Leguminosae: Structural Types, Analyses, Chemotaxonomy, and Biological Properties A. Douglas Kinghom and Manuel F. Balandrin

1

49

105

4. Chemistry and Pharmacology of Maytansinoid Alkaloids Cecil R. Smith, Jr. and Richard G. Powell 5. ^^C and Proton NMR Shift Assignments and Physical Constants of Ci9-Diterpenoid Alkaloids S. William Pelletier, Naresh V. Mody, Balawant S. Joshi, and Lee C. Schramm

149


xii

Contents of Previous Volumes

Volume 3 1. The Pyridine and Piperidine Alkaloids: Chemistry and Phannacology GaborB. Fodor md Brenda Colasanti 2. The Indolosesquiterpene Alkaloids of the Annonaceae Peter G. Waterman

1

91

3. Cyclopeptide Alkaloids Madeleine M. Joullie and Ruth F. Nutt

113

4. Cannabis Alkaloids MahmoudA. ElSohly

169

5. Synthesis of Lycopodium Alkaloids Todd A. Blumenkopf and Clayton H. Heathcock

185

6. The Synthesis of Indolizidine and Quinolizidine Alkaloids of Tylophora, Cryptocarya, Ipomoea, Elaeocarpus, and Related Species /?. B, Herbert

241

7. Recent Advances in the Total Synthesis of Pentacyclic Aspidosperma Alkaloids Larry E. Overman and Michael Sworin

275

Volume 4 1. Amphibian Alkaloids: Chemistry, Phannacology and Biology John W. Daly and Thomas F. Spande 2. Marine Alkaloids and Related Compounds William Fenical 3. The Dimeric Alkaloids of the Rutaceae Derived by Diels-Alder Addition Peter G. Watermann 4. Teratology of Steroidal Alkaloids Richard F.Keeler

1

275

331

389


Contents of Previous Volumes

xiii

Volume 5 1. The Chemistry and Biochemistry of Simple Indolizidine and Related Poly hydroxy Alkaloids Alan D. Elbein and Russell J. Molyneux 2. Structure and Synthesis of Phenanthroindiolizidine Alkaloids and Some Related Compounds Emery Gellert

1

55

3. The Aporphinoid Alkaloids of the Annonaceae Andre Cave, Michel Leboeuf, Peter G. Waterman

133

4. The Thalictrum Alkaloids: Chemistry and Pharmacology Paul L Schiff, Jr.

271

5. Synthesis of Chephalotaxine Alkaloids Tomas Hudlicky, Lawrence D. Kwart, and Josephine W. Reed

639

Volume 6 1. Chemistry, Biology and Therapeutics of the Mitomycins William A. Remers and Robert T. Dorr

1

2. Alkaloids of Tabemaemontana Species Teris A. van Beek and Marian A.J.T. van Gessel

75

3. Advances in Alkaloid Total Synthesis via Iminium Ions, a-Aminocarbanions and a-Aminoradicals David J. Hart 4. The Biosynthesis of Protoberberine Alkaloids Christopher W. W. Beecher and William J. Kelleher 5. Quinoline, Acridone and Quinazoline Alkaloids: Chemistry, Biosynthesis and Biological Properties Michael F. Grundon

227

297

339


xiv

Contents of Previons Volumes

Volume? 1. Homoerythrina and Related Alkaloids /. Ralph C Bick and Sirichai Panichanum 2. Carbon-13 NMR Spectroscopy of Steroidal Alkaloids Pawan K. Agrawal, Santosh AT. Srivastava, and William Gaffield 3. Carbon-13 and Proton NMR Shift Assignments and Physical Constants of Norditeipenoid Alkaloids S. William Pelletier and Balawant 5. Joshi

1

43

297

Volume 8 1. Curare Norman G. Bisset 2. Alkaloid Chemistry and Feeding Specificity of Insect Herbivores James A. Saunders, Nichole R. O'Neill, and John T. Romero

1

151

3. Recent Advances in the Synthesis of Yohimbine Alkaloids Ellen W, Baxter and Patrick 5. Mariano

197

4. The Loline Group of Pyrrolizidine Alkaloids Richard G. Powell and Richard J. Petroski

320


Contents of Previous Volumes

Volume 9 1. Taxol M, E. Wall and M. C. Want 2. The Synthesis of Macroline Related Sarpagine Alkaloids Linda K. Hamaker and James M. Cook

23

3. Erythrina Alkaloids Amrik Singh Chawla and Vijay K, Kapoor

85

4. Chemistry, Biology and Chemoecology of the Pyrrolizidine Alkaloids Thomas Hartmann and Ludger Witte

155

5. Alkaloids from Cell Cultures of Aspidosperma Quebracho-Bianco P. Obitz, /. Stockigt, L. A. Mendonza, N, Aimi and S.-i. Sakai

235

6. Fumonisins Richard G. Powell and Ronald D. Plattner

247

Volume 10 1. Alkaloids from Australian Eora /. R. C. Bick 2. Pyridine and Piperidine Alkaloids: An Update Marilyn J. Schneider

155

3. 3-Alkylpiperidine Alkaloids Isolated from Marine Sponges in the Order Haplosclerida Raymond J. Andersen, Rob W. M. Van Soest and Fangming Kong

301

4. p-Carboline and Isoquinoline Alkaloids from Marine Organisms Bill J. Baker

357


xvl

Contents of Previous Volumes

Volume 11 1.

The Thalictrum Alkaloids: Chemistry and Pharmacology (1985 - 1995) Paul L Schiff, Jr.

1

2.

Taxine Giovanni Appendino

237

3.

The Alkaloids of South American Menispermaceae Mary D. Menachery

269

4.

The Chemistry and Biological Activity of Calystegines and Related M^rtropane Alkaloids Russell J. Molyneux, Robert J, Nash, and Naoki Asano

303

5.

Polyhydroxylated Alkaloids that Inhibit Glycosidases Robert J, Nash, Naoki Asano, arid Alison A. Watson

345


Contents 1.

Acronycine-type Alkaloids: Chemistry and Biology Francois Tillequin, Sylvie Michel, and Alexios-Leandros Skaltsounis

2.

Solatium Steroid Alkaloids - an Update Helmut Ripperger

3.

Synthesis and Structure-Activity Studies of Peptide Alkaloids Peter Wipf

1 103

Lissoclinum 187

4.

Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids Michael B. Smith

5.

Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis - Electrospray Mass Spectrometry Joachim Stockigt, Matthias linger, Detlef Stockigt, and Detlev Belder

289

Oxidation of Anthelmentic Marcofortine A, an Indole Alkaloid Byung H. Lee, Michael F. Clothier, and Gate /. Komis

343

6.

229

Subject Index

375

Organism Index

385

xvii


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Chapter One

Acronycine-Type Alkaloids : Chemistry and Biology Fran9ois Tillequin, Sylvie Michel, and Alexios-Leandros Skaltsounis University Rene Descartes - Paris V Faculty de Pharmacie Laboratoire de Pharmacognosie 75270 Paris Cedex 06, France

CONTENTS 1. INTRODUCTION 2. ACRONYCINE 2.1. Isolation, Chemical Properties and Structural Elucidation

2 3 3

2.2. Spectral Data 2.3. Synthesis 2.3.1. Syntheses by alkylation of a preformed 1,3-dioxygenated acridone 2.3.2. Syntheses including the construction of the acridone skeleton

9 11 11 21

2.3.2.1. Syntheses involving a carboxylic diphenylamine intermediate 2.3.2.2. Syntheses involving an aminobenzophenone intermediate 2.3.2.3. Syntheses involving a quinoHne or quinolone intermediate 2.4. Biological properties 2.4.1. Antitumor activity 2.4.2. Other biological activities 2.4.3. Pharmacokinetics and metaboHsm

21 28 31 38 38 42 42

3. NATURALLY OCCURING ACRONYCINE ANALOGS 3.1. Acronycine analogs modified at C(6), N(12), and their derivatives substituted on C ring 3.2. Alkaloids modified on the A aromatic ring 3.2.1. Alkaloids monosubstituted at C(ll) 3.2.2. Alkaloids disubstituted at C(10) and C(l 1) 3.2.3. Other alkaloids with modified A aromatic ring 3.3. Alkaloids with oxidized pyran D ring 3.3.1. Acronycine derivatives with oxidized pyran D ring 3.3.2. 12-Demethylacronycine derivatives with oxidized pyran D ring

49 49 50 50 54 57 58 58 60


2

F.Tilieqiiiii, S. Mkhel and A-L. Skaltsoimis

3.3.3. Citracridone I derivative with oxidized pyran D ring 3.4. Dimeric alkaloids 3.4.1. Dtmeric acridone alkaloid containing an ether linkage 3.4.2. Dimeric acridone alkaloids containing a carbon-carbon linkage 4. SYNTHETIC ACRONYCINE ANALOGS 4.1. Acronycine analogs modified at C(6) and/or N(12) 4.2. 3,12-Dihydro-7//-pyrano[2,3-c]acridin-7-thiones 4.3. Acronycine analogs modified at the A ring 4.3.1. Acronycine analogs substituted at the A ring 4.3.2. 11-Azaacronycine 4.4. Acronycine analogs modified at the D ring 5. CONCLUSION Acknowledgements References

61 62 62 63 66 67 72 73 73 85 87 94 95 95

1. INTRODUCTION Acronycine (3,12-dihydro-6-methoxy-3,3,12-trimcthyl-7//-pyrano[2,3-c]acridin-7-one) (1) is a natural alkaloid which was first isolated in 1948 by the group of Hughes and Lahey [l] from the baiic of the small Australian Rutaceous tree Acronychia baueri Schott The structure of acronycine has long been discussed, mainly to ascertain whether the pyranringwas fused lineaiiy or angularly on the acridone skeleton. It was only in 1966 that the angular structure 1 could be unambiguously assigned to acronycine on the basis of oxidative degradation evidence [2] and of ^H nmr data [3]. Final proof of the structure was obtained in 1970 from X-ray crystallographic data of 5-bromo-l,2-dihydn>acronycine (2) [4]. O

OCH3


Acronycine-Type Alkaloids: Chemistry and Biology

3

The biological interest of acronycine was revealed in 1966 by Svoboda and co-workers in the Eli Lilly Laboratories [5,6]. Acronycine is a potent antitumor agent whose main interest lies in its broad spectrum of activity, including numerous solid tumors resistant to other chemotherapeutic agents [5-8]. In contrast, acronycine exhibits only marginal activity against leukemias [5-8]. Since the discovery of the antitumor properties of acronycine, numerous derivatives and structural analogues have been both isolatedfromvarious Rutaceae species and prepared by total synthesis. A survey of natural alkaloids and synthetic analogues derivating from the pyrano[2,3c]acridin-7-one skeleton is presented below.

2. ACRONYCINE 2.1 Isolation, Chemical Properties and Structural Elucidation Isolation,Tht first isolation of acronycinefroma methanolic extract of Acronychia baueri bark relied only on solubility differences between the various alkaloids contained in the plant material [9]. Further isolations from the same source involved both crystallizations and chromatography on alumina and/or silica gel [5.6.10]. A chemical study of the leaves of the same plant resulted in the isolation of various acridone and furo[2,3-b]quinoline alkaloids but no acronycine could be detected [11]. Since the first isolation of acronycine, the status of Acronychia baueri Schott within the Rutaceae family has been revised severaltimesby Hartley at the Herbarium Australiense, in the course of successive taxonomic studies of genera Acronychia [12], Bauerella [13] and Sarcomelicope [14.15]. Hartley now considers this taxon belongs to the genus Sarcomelicope and should be named Sarcomelicope simplicifolia (Endl.) Hartley subsp. simplicifolia [14]. ApartfromSarcomelicope simplicifolia, all the other species belonging to that genus are endemic in New Caledonia [14,15], and most of them have been studied for their alkaloid contents [16-27]. Acronycine was obtained from the bark of Sarcomelicope simplicifolia (Endl.) Hartiey subsp. neo-scotica (P.S. Green) Hartley [16, 18], from the bark of Sarcomelicope argyrophylla Guill. [20],fromthe bark of Sarcomelicope glauca Hartley [21], and from the leaves and bark of Sarcomelicope dogniensis Hartiey [22,25] and Sarcomelicope pembaiensis Hartley [23]. In addition, acronycine has also been isolated from the aerial parts of Melicope leptococca (Baill.) Guill. [28]. Chemical properties and structural elucidation, Acronycine crystallizes from alcohol as yellow needles, m.p. 175-176째C [9]. It also readily crystallizes from methanol and acetone [5]. A dilute alcoholic solution of the base is yellow with a bright green fluorescence [9]. Acronycine forms an orange picrate, m.p. 150-154**C, a red hydrochloride which easily separates from 10


4

F.Tillequin, S. Michel and A-L. Skaltsovids

per cent hydrochloric acid in red needles, m.p. 125-130^0 (dec.)* and a sulfate which crystallizes as red needles from alcoholic sulfuric acid, m.p. 158-159째C [9]. Treatment of acronycine (1) with hot alcoholic hydrochloric acid yields polymeric amorphous products. In contrast, heating acronycine hydrochloride in the dry state brings demethylation with the fcMtnation of noracronycine (3) [29] (Scheme 1). The hydroxy group of this latter compound is chelated by the neighbouring carbonyl group at C(7), as shown by uv spectroscc^y [30] and therefcxre cannot be methylated upon treatment with diazomethane [29]. However, treatment with dimethylsulfate and potassium carbonate in acetone converts it back to acronycine, whereas hot acetic anhydride in the presence of pyridine gives a monoacetate (4) [29]. Acronycine contains a reactive double-bond since catalytic reduction in the presence of Raney nickel yields dihydroacronycine (5) [29]. Hydrochloric or hydrobromic acid converts dihydroacronycine nordihydioacronycine (6) without polymerization [29].

(5) to

Oxygenation patterns on A and C rings of acronycine, as well as fusion of a dimethylpyran unit with the latter ring, were early deduced from degradative experiments [29. 31].

Concentrated nitric acid in alcohol yields a mononitroacronycine whose structure was tentatively described as l-nitroacronycine [31]. The same reaction was then claimed to yield 2-nitroacronycine (7) [S] and this latter structure could finally be assigned to the mononitration product of acronycine on the basis of nOe experiments in ^H nmr spectroscopy [32]. With hot concentrated nitric acid, acronycine is converted to an orange trinitroacronycine, to which the structure of 2,5,9-trinitroacronycine (8) should reasonably be assigned.

7 R=H 8 R = N02

By prolonged heating with concentrated nitric acid, trinitroacronycine dissolves and gives rise to 6-nitro-l-methyl-4-quinolone-3-carboxylic acid (9) identical with a synthetic sample. Similarly, both noracronycine (3) and nordihydioacronycine (6) lead, upon treatment with nitric acid, to l-methyl-4-quinolone-3-carboxylic acid (10) whose structure was identical with that of the methylation product of 4-hydroxyquinoline-3-carboxylic acid synthetized by the GouldJacobs method [31].


Acronyciiie>Type Alkaloids: Chemistry and Biology

O

O

OCHj

OH

HCl, heating

Me2S04/K2C03

AC2O

H2/RaneyNi

O

OCH3

O

HClorHBr

Scheme 1

CXX)H 9 10

R =N02 R=H

OCOCHa


6

F.Tilieqtiin, S. Michel and A-L. Skaltsow^

Oxidation of acronycine with potassium permanganate in acetone gave rise to the dicarfooxylic add 11, easily decarboxylated to acronycinic acid (12) upon crystallizatkmfrcrni10 per cent hydn)chloric acid [29]. OCH, 11

R = CXX)H

12

R=H

COOH Pyrolysis of 12 yields three products : a-hydroxyisobutyric acid (13), 1,3-dihydroxy10-methyl-9(10//)-acridinone (14) and l-hydroxy-3-methoxy-10-methy]-9(10//)-acridinone (15). Treatment of 14 with diazomethane gives 15, which in turn can be converted by use of dimethylsulfate into-l,3-dimethoxy-10-methyl-9(10//)-acridinone (16). Compounds 14,15 and 16 were identified by comparison with authentic synthetic samples [29,31].

HO^-J^ CXX>H

13 Similarly, noracronycine yields, upon permanganate oxidation followed by decarboxylation, noracionycinic acid (17). Pyrolysis of this latter only gives a-hydroxybutyric acid (13) and l,3-dihydroxy-10-metiiyl-9(10//)*acridinone (14) [29,31].

0

8

ORi

ll

9U

M

li

II j u 4

5

CH3 14 R, = R2= H 15 Ri = H R2 = CH3 16 Ri = R2 — Cfl3 19 R, = CH3 R2 = H

COOH


Acronycine-Type Alkaloids: Chemistry and Biology

7

The results of these experiments can be explained only if acronycine contains a dimethylpyran ring fused with an acridone system. They permit assignment of either the angular structure 1 or the linear structure 18 (= isoacronycine) to acronycine.

18 The only apparent inconsistency in these reactions is found in the obtention of methyl ether 15, which is presumably formed during the degradation reaction of 12 by remethylation of the 1,3-dihydroxy derivative 14, or by intermolecular transmethylation of the 3-hydroxy-lmethoxy-10-methyl-9(10//)-acridinone (19) initially formed. Ozonolysis of acronycine gives a phenolic aldehyde, which can be methylated on its free phenolic group, oxidized to the corresponding carboxylic acid, and finally esterifled using diazomethane [2,31]. If acronycine had a linear structure, this product would be 1,3-dimethoxy2-methoxycarbonyM0-methyl-9(10//)-acridinone (20), whereas if it had an angular structure, the product would be l,3-dimethoxy-4-methoxycarbonyl-10-methyl-9(10f/j-acridinone (21). Unambiguous structure determination of the degradation product would therefore define the structure of acronycine.

20

RI = COCX:H3 R2 = H

21 R i = H R2 = COOCH3 OCHa

The chemical proof of acronycine structure was finally obtained in 1966 by Macdonald and Robertson who achieved the regioselective synthesis of 20 according to Scheme 2 [2]. The synthetic compound was not identical with the degradation product of acronycine, which was therefore l,3-dimethoxy-4-methoxycarbonyl-10-methyl-9(10//)-acridinone (21), indicating the angular structure of acronycine. Subsequently, Govindachari isolated noracronycine (3) from Glycosmis pentaphylla in 1966 and provided independendy a proof of the angular structure of the latter compound [3].


a + OCH3

F.Tillequin, S. Michel and A-L. Skaltsouiris

COOH OCH3 CCKXH3

a

l)Cu/K2C03 2) CH2N2

CCKXH3

OCH,

OCH,

H,N

a

OCH, CCXX:H3

poa.

MeOH/MeONa

CXH3 OCH3 OCH3

o

CX:H3

COOCH^

CH3i/io(y'c 0CH3 CH3

20

Scheme 2 Noracronycine (3) was converted into tosylate 22 upon treatment with tosyl chloride. Desulfurization of the tosylate 22 in a current of hydrogen did not yield the expected deoxydihydroderivative 23 but the deoxy-compound 24, saturated on both the chromene double-bond and the aromatic A ring, in agreement with further observations on the catalytic hydrogenation of acridone alkaloids [33].

1 R = CH3 3 RÂŤH 22 R= SO2

r\

CH,


Acronycine-Type Alkaloids: Chemistry and Biology

9

The iR ntnr. spectrum of 24 exhibited two aromatic doublets at 5 6.75 and 8.13 ppm with an ortho coupling constant J = 9Hz, indicating that the two protons were attached to adjacent carbon atoms. Hence, the structure of noracronycine is 3 and that of acronycine 1 [3].

ctx 2.2 Spectral Data The uv spectrum of acronycine was first recorded in the course of thorough study of the spectra of acridone alkaloids in 1950 [30]. Acronycine exhibits, in ethanolic solution, absorptions at Xmax flog e) : 281(4.57), 293(4.55), 309(sh.)(4.27), 376(sh.), (3.72) and 392(3.83) nm. The latter maximum is a characteristic feature of the uv spectrum of acridone alkaloids [34]. The only typical point on the ir spectrum of acronycine [2,6,18] is the presence of a band at ca Vmax = 1635 cm-1, typical for the carbonyl frequency of 9-acridanone [35,36]. The mass spectrum of acronycine has been recorded and studied in electron impact with spectrometers operating at 75 eV [37] or 70 eV [38]. Ions are observed at m/z (%): 322(21), 321(68)(M+), 307(26), 306(100), 293(10), 292(30), 291(11), 277(10), 276(12), 265(12), 264(30), 263(12), 248(10), 209(9), 204(7), 160.5(7), 77(6) and 63(7) [37]. The parent ion appears at m/z = 306 (25) [38] and can be linked to the corresponding fragment ion observed in the spectrum of 2,2-dimethylchromene [39,40]. Other prominent fragment ions correspond to successive losses of 15 (CH3) and 28 (CO) m.m.u.

The iR nmr spectrum of acronycine has been reported several times [2.18,41], but it is only with the advent of high resolution nmr that a complete assignment of the aromatic signals


10

F.TUIequin, S. Michel and A-L. SkaltsouiUs

was possible in 1984 [42]. It should be iK>ted that a study of the nOe effects had been faeviously published in 1970 [43]. irradiation of the N-Cf/3 signal caused a 17 % selective increase in the integrated intensity of the C(l)-H doublet, giving further evidence of the angular structure of acronycine. The l^C nmr spectrum of acronycine was reported by Cordell et al. in 1983 [44]. TaWe I summarizes the ^H and ^% nmr data of acronycine [41,43]. Table I : iH and l^C nnra- data (CDQa, TMS) of acronycine (1) according to refs 42 and 44. Assignments of C(4a) and C(6) have been reversed from those of ref.44 to take into account recent results oi COLOC and HNfBC experiments [45]. Position

1 2 3 4a 5 6 6a 7 7a 8 9 10 11 11a 12a 12b (CH3)2(3) NCH3 (12) OCH3(6)

H

C

6.55, d, J=9.3Hz 5.51, d, J=9.3Hz 6.32, s 8.40, dd, J = 1.4, 8.0 7.25, ddd, J ÂŤ 0.7. 7.3, 8.0 7.70, ddd, J = 1.4, 7.3. 8.4 7.36, dd, J = 0.7, 8.4 1.54, s 3.84, s 3.98. s

121.6 122.7 76.0 159.0 94.2 162.7 110.4 176.8 125.3 126.8 121.5 132.2 115.7 144.3 146.5 102.8 26.5 43.9 56.0


Acronycine-Type Alkaloids: Chemistry and Biology

11

2.3 Synthesis Since the first three historical interrelated syntheses completed by the Eli Lilly Research Laboratories in 1967-68 [46.47], around ten other total syntheses of acronycine have been reported. Up till now, two major approaches have been explored. One implies alkylation of a preformed 1,3-dioxygenated acridone comprising the ABC tricyclic portion of acronycine by a C5 unit, and elaboration of the pyran D ring from that unit by simultaneous or subsequent cyclization. A second approach involves construction of the acridone nucleus in the course of synthesis. Three main strategies have been used for the elaboration of the ABC basic skeleton. The first implies synthesis of the acridone nucleus through the intermediacy of carboxylic diphenylamine which gives the A and C rings of the final compound. The intermediate is usually prepared by the Ullmann reaction [48]. The second strategy, which has been much less explored, involves synthesis of the acridone nucleus by cyclization of an aminobenzophenone derivative which gives the A and C rings of acronycine. Finally, in a third strategy, the two A and B or B and C rings of the acridone skeleton arise from a quinoline or quinolone derivative. The only syntheses which rely on this scheme are those of Winterfeldt [49.50] and Anand [51.52].

2.3.1 Syntheses by alkylation of a preformed 1,3-dioxygenated acridone The starting dioxygenated acridones which have been used in these approaches are 1,3dihydroxy-9(10//)-acridinone (26) and l,3-dihydroxy-10-methyl-9(10f/)-acridinone (14).

26

R=H

14

R = CH3

Synthesis of 1,3-dioxygenated acridones 26 and 14. The first record of the synthesis of l,3-dihyroxy-9(10//)-acridinone (26) is that of Baczynski and Niementowski [53] by condensation of anthranilic acid (27) and phloroglucinol (28).


12

F.Tiliequin, S. Michel and A-L. Skaltsounis

a„ * NHo

27 R = H 3 0 R = CH3

The yield of the reaction was initially very poor but was increased by subsequent modifications. Beck et al. [47] noted that the same condensation when carried out in 1-butanol in the presence of zinc chloride gave 26 in 20 to 25 % yield. The product was still difficult to handle and purify. However, Hlubucek et al [43] found that acetylation of the crude reaction product with acetic anhydride and anhydrous sodium acetate gave the readily purifiable di-0acetylderivative (29), which could be quantitatively hydrolyzed to 26 by potassium carbonate in methanol [43, 54]. More recenUy, Smolders et al. [55] described an efficient condensation of methyl anthranilate (30) with phloroglucinol (28) in the presence of 1-heptanol and 4-toluene sulfonic acid which gave 26 in 80 % yield.

COOCH3

OCOCH3

NH2 30

29

The first syntiiesis of l,3-dihydroxy-10-mediyl-9(10//)'acridinone (14) (Scheme 3) was performed by Drummond and Lahey [31] in order to ascertain the structures of the degradation products of acronycine. Ullmann condensation of 3,5-dimethoxyaniline (31) with 2H;hlorobenzoic acid (32) gave the carboxylic diphenylamine 33 which readily cyclized to 1,3dimethoxy-9(10//)-acridinone (34) upon treatment with phosphorus oxychloride. The potassium salt of 34 on methylation with dimethyl sulfate gave 1,3-dimethoxy-lO-methyl9(10//)-acridinone (16) which was converted into the required l,3-dihydroxy-10>methyl9(10//)-acricliiione (14) by hydix>tÂťx>mic acki.


Acronycine-Type Alkaloids: Chemistry and Biology

a-

,CXX)H

32

13

OCH3 QCH3 CU/K2C03 amylic alcohol OCH3

OCH3

H2N

O

OCHa

i)pcx:i3

l)KOH/EtOH 2) (CH3)2So/

2) NH3 - H2O

OCH3 34

O

OCH3

HBr OCH^

Scheme 3

Two independent syntheses of 14 were described by Hlubucek et al. [43]. One (Scheme 4) utilized the Pfitzinger reaction between isatin (35) and phloroglucinol (28) to yield the zwitterion 36 which was converted to methyl l,3-dimethoxyacridin-9-carboxylate (37) on treatment with ethereal diazomethane. The ester 37 was transformed into the corresponding quaternary methosulfate 38. Oxidation of 38 with hot alkaline ferricyanide yielded 1,3dimethoxy-10-methyl-9(10//)-acridinone (16) which was 0-demethylated into 14 by hydrobromic acid as previously. The second synthesis of 14 by Hlubucek et al. [43] was directly attendant upon the synthesis of l,3-dihydroxy-9(10//)-acridinone (26) by the same authors. Treatment of 1,3diacetoxy-9(10//)-acridinone (29) by dimethylsulfate in dimethylformamide in the presence of sodium hydride afforded l,3-diacetoxy-10-methyl-9(10//)acridinone (39) whose hydrolysis with potassium carbonate in methanol quantitatively gave rise to 14.


F.Tillequin, S. Michel and A-L. Skaltsounis

14

CXXP OH

0;:il-x>

OH

OCH3

Scheme 4

2 9 RI = CXX::H3

R2 = H

39

R2 = CH3

Ri = COCH3

1 4 Ri=:OH

R2 = CH3


Acronycine-Type Alkaloids: Chemistry and Biology

15

Reaction of 1,3-dioxygenated acridones with l-halo-S-methylhut-l-ene. In one of the first syntheses of acronycine [46.47], Beck et at. (Scheme 5) allowed 13-dihydroxy-9(10f/)acridinone (26) to react with l-chloro-3-methylbut-2-ene (40) in trifluoroacetic acid with zinc chloride as catalyst. Under such conditions, l,2-dihydro-12-demethylnoracronycine (41) was obtained in 18 % yield, accompanied by the bischromane 42. Methylation of 41 with methyl iodide and potassium carbonate in refluxing acetone gave l,2-dihydn)noracronycine (6). Dehydrogenation of 6 with 2,3-dichloro-5,6-dicyanobenzo-l,4-quinone in refluxing toluene gave noracronycine (3) in 40-45 % yield. Finally, noracronycine (3) was methylated into acronycine (1) by methyl sulfate and potassium carbonate in refluxing acetone.

CFjCCXDH

*- 1

Scheme 5

Similarly, in 1990 Grundon and Reisch investigated the biomimetic reaction of 1,3dihydroxy-10-methyl-9(10//)-acridinone (14) with one equivalent of l-bromo-3-methylbut-2ene (42) in tetrahydrofuran at 20째C in the presence of alumina [56]. The major product was the monoalkylated derivative at 4-position, glycocitrine-II (43), whereas its isomer at 2-position 44 and the dialkylated compound 45 were isolated in lesser amount.


16

F.Tiilequin, S. Michel and A-L. Skaitsounis

O

(M

Oxidative cyclization of glycocitrine-II (43) with S-chloroperbenzoic acid gave rise to 2hydroxy-l,2-dihydronoracronycine (47) accompagnied by its dihydrofuran isomer 48, presumably via epoxide 46 (Scheme 6). In addition, more recendy Furukawa et al. isolated the novel oxidation product 49 when repeating the reaction [57]. Dehydration of 47 with concentrated sulfuric acid finally gave noracronycine (3) [56].


Acronycine-Type Alkaloids: Chemistry and Biology

Scheme 6

17


18

F.Tiilequin, S. Michel and A-L. Skaltsounis

Reaction of 1 J-dioxygenated acridones with S-hydroxyisovcderaldekyde dimethylacetal, Crombie introduced 3-hydroxy-3-iiicthyl-l,l-dinncthoxybutanc (= 3-hydroxyisovaleraldchyde dimethylacetal) (50) as a new reagent for dimethylchromenylation of phenols [58], and described the application of this method to prepare acronycine [54, 59] (Scheme 7). Condensation of l,3-dihydroxy-9(10//)-acridinone (26) with Ae hydioxy-acetal 50 in pyridine at 150째C gave a 1:3 mixture of the linear and angular chiomenes 51 and 52. Crystallization afforded the major angular isomer 12-demethylnoracronycine (52) which was methylated to acronycine (1) (24 % overall yield from 26) with excess methyl iodide and anhydrous potassium carbonate in acetone. Similarly, methylation of 51 gave the linear isoacronycine (18) which can be prepared by other routes [60,61].

C6H5N OCH3 150PC

OH OCH3

50

26

O

OH

OH

51 Scheme 7

Reaction ofl,3'dioxygenated acridones with S-chlorO'S-methylbut-l-yne, The use of 3chloro-3-methylbut-l-yne (53) [62,63] for the synthesis of 2,2-dimethylchromenes by Oalkylation of phenols followed by Claisen rearrangement was introduced by Hlubucek, Ritchie and Taylor in 1969 [64]. These authors described several interrelated syntheses of acronycine based on that methodology [43,65]. Starting from l,3-dihydroxy-10-methyl-9(10//)-acridinone (14) (Scheme 8), the acetylenic ether 54 was obtained in a 70 % yield upon treatment with 3-chloro-3-methylbut-lyne in dimethylformamide in the presence of potassium carbonate and sodium iodide at 52째C.


Acronycine-Type Alkaloids: Chemistry and Biology

19

Etherification of the hydroxyl group at C(l) was precluded due to hydrogen bonding to the carbonyl group at C(9). When the reaction was carried out at 70**C, the crude product obtained was shown to consist of a 2:3 mixture of ether 54 and noracronycine (3) because of the rapid rate of Claisen rearrangement at that temperature. Refluxing the mixture in iV^-diethylaniline yielded noracronycine in almost 90 % from 14. In a similar way, heating pure ether 54 provided noracronycine (3). Starting from l,3-dihydroxy-9(10//)-acridinone (26) (Scheme 8), a facile Claisen rearrangement occured during the etherification by 3-chloro-3-methylbut-l-yne and the pure ether 55 could not be isolated. Heating the crude reaction product in dimethylformamide at \3(fC afforded 12-demethylnoracronycine (52) in 85 % yield from 26. In crude products from the above cyclization reactions, no traces of corresponding linear isomers could be detected by tic. Later, Fryer et al. [66] when repeating the same reaction nevertheless isolated minute amounts of linear products belonging to the isocronycine series. Final alkylations of 3 with dimethylsulfate and potassium carbonate in dimethylformamide or of 52 with dimethyl sulfate and sodium hydride in dimethylformamide gave acronycine (1) in almost 90 % yield.

14 R = CH3 26 R = H

53

5 4 R = CH3 55 R = H OCH^

3 52

R = CH3 R=H Scheme 8


20

F.Titiequin, S. Michel and A-L. Skaltsomris

When acronycine itself is considered as the final target, the syntheses by Hlubucek, Ritchie and Taylor give the best results, as far as overall yields are concerned. This is most probably the reason why several modifications and/cM* improvements of these syntheses were published later on. A legioselective synthesis of acronycine (1) based on the same Claisen rearrangement was reported by Reisch et al. [67] (Scheme 9). lodination of l,3-dihydioxy-10-methyl-9(10//)acridinone (14) by iodine in 85 % perioidic acid gave l,3-dihydroxy-2-iodo-10-methyl-9(10//)acridinone (56) in 38 % yield. The iodo derivative 56 was then treated with 3-chloro-3methylbut-1-yne (53) in the presence of potassium carbonate, and potassium iodide in dimethylformamide at lOO^C to give regioselectively noracronycine (3) in 40 % yield. Conq)ound 3 was then methylated to acronycine (1).

O

OH

O

OH

KI/K2CO3/DMF

Scheme 9

In order to avoid the use of possible health hazardous 3-chlQio-3-methylbut-l-yne (53) for etherization, Reisch et al, [68] prepared noracronycine via a Mitsunobu reaction (Scheme 10). Thus, treatment of l,3-dihydroxy-10-methyl-9(10//)-acridinone (14) with 2-methyl-3butyn-2-ol (57) carried out in tetrahydrofuran in the presence of triphenylphosphine and azodiethyldicarboxylate afforded edier 54 which was cyclized into noracronycine (3) by heating in dimethylformamide at \2XfQ for 5 hours.


Acronycine-Type Alkaloids: Chemistry and Biology

O

21

OH

OH

P(Ph)3 THF

Azodiethyldicarboxylate

DMF 130째C

Scheme 10

2.3.2. Syntheses including the construction of the acridone skeleton. 2.3.2.1. Syntheses involving a carboxylic diphenylamine intermediate Synthesis of Beck et al. via a carbostyril intermediate. In thefirstsynthesis of acronycine performed by the Eli Lilly Research Laboratories [46,47] (Scheme 11), 5,7-dimethoxy-3,4dihydrocarbostyril (58) was chosen as starting material. The aromatic ring of that compound contained the correct substitution pattern for the C ring of acronycine, wheareas carbons 2, 3 and 4 featured the future pyran D ring. Carbostyril 58 was prepared in two steps from 3,5dimethoxyaniline (31) which was amidified by 3-bromopropionic acid (59) in the presence of dicyclohexylcarbodiimide. The resulting amide 60 submitted to cyclization by heating with zinc chloride and sodium chloride, yielded 58. Reaction of 58 with 2-iodobenzoic acid in the presence of cuprous iodide in nitrobenzene gave l-(2-carboxyphenyl)-5,7-dimethoxy-3,4-dihydrocarbostyril (61). Cyclization of carboxylic diphenylamide 61 to the corresponding acridone was obtained by heating with polyphosphoric acid at 90째C. The latter reaction gave a mixture of the free acid 62 and of the tetracyclic lactam 63. Both compounds were converted to the same intermediate, methyl 1,3dimethoxy-9(10//)-acridinone-4-propionate (64), upon treatment with methanolic hydrogen chloride.


22

F.Tilleciuiii, S. Michel and A-L. Skalteouiris

OCH3

Zid^aQ ^ 155^C

H.N

.CXX)H Cu2l2/Ph-N02 ff^^^^Y^ r OCH3 ^.v^^CDOH

cc,

0CH3

PPA/WC

OCH3

CHaOH/Ha O

OCH3

OCH3

COOCH3 Scheme 11 A


Acronycine-Type Alkaloids: Chemistry and Biology

O

23

OCH3

(CH3)2S04 ^

1

Toluene/Rx

Scheme 11 B Methylmagnesium iodide was used in the first attempts to introduce geminal methyl groups by alkylation at the ester carbonyl of 64 without alkylating the acridone carbonyl function. Under those conditions, the desired alkylation into tertiary alcohol was not observed but methyl l-hydroxy-3-methoxy-9(10//)-acridinone-4-propionate (65) was obtained in high yield. Alternately, 65 could be prepared by demethylation of 64 with boron trichloride in methylene chloride. Attempted alkylation of 64 with methyllithium was unsuccessful, probably due to its weak solubility in ethereal solvents at low temperature. In contrast, treatment of 65 with excess methyllithium in tetrahydrofuran at -18째C yielded the required l-hydroxy-4-(3hydroxy-3-methylbutyl)-3-methoxy-9(10//)-acridinone (66). Fusion of 66 with pyridine hydrochloride at 200째C gave l,2-dihydro-12-demethylnoracronycine (41).


24

F.THIequiii, S. Michel and A-L. Skaltsomiis

Compound 41 was converted as previously to acronycine (1) by successive Nmethylation with methyl iodide, dehydrogenation with 2,3-dichloro-5,6-dicyanobenzo-l,4quinone andfinalmethylation using mediyl sulfate. Synthesis of Beck et zl.from a 4-chromanone. Another synthesis peformed by the Eli Lilly group [47] utilized the readily available 7-hydroxy-2,2-dimethyl-4-chromanone (67) [6971] as starting material (Scheme 12). Methylation of 67 gave 7-methoxy-2,2-dimethyl-4chromanone (68) in 80 % yield. Hydrogenation c^ 68 in the presence of copper chromic gave 7-methoxy-2,2-dimethylchroman (69) in 60 % yield. Alternately, 69 was obtained by catalytic hydrogenation of 7-methoxy-2,2-dimethylchromene (70) which was prepared from 68 by lithium aluminium hydride reduction followed by dehydration with phosphoryl chloride in pyridine. Bromination of 69 with bromine in carbone tetrachloride gave 6-bromo-7-methoxy2,2-dimethylchroman (71) which was converted into 5-amino-7-methoxy-2,2-dimethylchroman (72) upon treatment with sodium amide in liquid ammonia. Reaction of 72 with 2bromobenzoic acid (73) under classical Jourdan-Ullman conditions [48] afforded the corresponding carboxylic diphenylamine 74 which was converted without isolation to 1,2dihydro-12-demethylacronycine (75) by treatment with polyphosphoric acid at 90^C. Methylation of 75 with methyl iodide and potassium carbonate in acetone gave dihydroacronycine (5) whose dehydrogenation with 2,3-dichloro-5,6-dicyanobenzo-l,4quinone yielded only traces of acronycine (1). Synthesis cfLaughhead, The general scheme of this recent and efficient synthesis is very close to that of the preceding one, but the use of 5-amino-7-methoxy-2,2-dimethylchromene (76) instead of the corresponding chromane 72 avoids the difficult final dehydrogenaticm step [72]. Chromene 76 was prepared by the method of Winterfeldt et al. (Scheme 13) [49], starting from 7-methoxy-2,2-dimethyl-4-chromanone (68) which was reduced with lithium aluminium hydride to the corresponding benzyl alcohol 77. Bromination of crude 77 with bromine in carbon tetrachloride afforded 78 which was dehydrated without purification to 6bromo-7-methoxy-2,2-dimethylchromene (79) using phosphoryl chloride in pyridine. Treatment of 79 with sodium amide in liquid ammonia gave the required 5-amino-7-methoxy2,2-dimethyl chromene (76) in 40 % yield from 68. Loughhead allowed chromene 76 to react with 2-bn>mobenzoic acid (73) under Ullmann conditions (Scheme 14). After purification, the corresponding carboxylic diphenylamine 80 was obtained in 58 % yield of analytical recrystallized material. Cyclization of 80 to 12demethylacronycine (81) was performed in 62 % yield by treatment with 5 equivalents of trifluoroacetic anhydride in dichloromethane for three days at room temperature. Final iVmethylation of 81 by methyl iodide under phase-transfer conditions smoothly afforded acronycine (1) in 96 % yfeld.


Acronycine-Type Alkaloids: Chemistry and Biology

25

l.AlLiHi/ether 2.POCI3/C5H5N

H2 / Raney Ni

H2 Copper chromite

NaNHj/NHj

P.P.A. •

K2C03/CU/ Amyl alcohol

9(fC

"^COf

O CH3I K2CO3 (CH3)2CO

Scheme 12

OCH3


Scheme 13

a

OOOH

e

Cu(OAc)2 i-PiOH

e

Ph-CH2-NEt3 CI aq.NaOH/MEK Scheme 14


Acronycine-Type Alkaloids: Chemistry and Biology

27

Synthesis ofWatanabe ct al.. It was known that small amounts of acridones were formed when benzynes were generated by diazotation of anthranilic acids [73-75]. In these cases, acridones arise from the reaction of benzynes with undiazotized anthranilic acids. Based on these findings, a new route to the acridone skeleton was developped by Watanabe et al. (Scheme 15) [76], through tandem metallation synthesis. Thus, the lithium salt of methyl ^Vmethylanthranilate (82) could be easily coupled with the benzyne generated by treatment of 6bromo-7-methoxy-2,2-dimethylchromene (79) with lithium N-isopropylcyclohexylamide in tetrahydrofiiran, to give acronycine (1) directly in 41 % yield [76].

COOCH3

LilCA -78°C/THF

LaCA •78°C/THF

a_., • .COOCH,

N—U CH3

OCHa

Scheme 15


28

F.TiHeqttiii, S. Michel and A-L. Skaltsoimb

2.3.2.2. Syntheses involving an aminobenzophenone intennediate The itMites to acronycine via an aminobenzophenone intennediate which have been develc^iped by Lewis et al. [77,78] are based on the biosynthetic pathway of acridone alkaloids [79.80]. The biogenesis of acridones involves anthranilic acid and acetate units and it has been suggested that a key-intennediate is an aminobenzophenone [81]. Indeed, 2-methylamino2*,4',6*-trimethoxybenzophenone is easily and quantatively converted into 1,3-dimethoxy-lOmethyl-9(10//)-acridinone [82,83] and these two compounds co-occur in several Rutaceae species [84]. The key-intermediates for these syntheses were the benzophenones 83,84 and 85 which can be considered as putative precursors to 12-demethylacronycine and acronycine. OCH3

83 84 85

R«H R«COCH3 R«CH3

In a first route through 83 (Scheme 16), Friedel Crafts condensation of 2-nitrobenzoyl chloride (86) with 3,5-dimethoxyphenol (87) gave two products, 4,6-dimethoxy-2-hydroxy2*-nitrobenzophenone (88) in 15 % yield and the required 2,6-dimethoxy-4-hydroxy-2*nitrobenzophenone (89) in 5 % yield only. Phenolic benzq)henone 89 was converted into the nitrochromene 90 by use of the method introduced by Hlubucek et al, [43,64] involving Oalkylation with 3-chloro-3-methylbut-l-yne followed by Qaisen rearrangement Reducti(Mi by zinc dust of the nitro group of 90 afforded the required aminobenzc^henone 83. Cyclization of amine 83 with sodium hydride in dimethylsulfoxide gave 12-demethylacronycine (81) in 27 % yield accompanied by its linear isomer 12-demethylisoacronycine (91) in 39 % yield. Usual methylation of 81 with methyl iodide in acetone yielded acronycine (1). The second route described by Lewis et al (Scheme 17) [77] involved amidobenzophenone 84 as key intermediate. Alkylation of 3,5-dimethoxyphenol (87) with 3-chloro-3methylbut-1-yne (53) according to Hlubucek et al [64] led to 5,7-dimethoxy-2,2dimethylchromene (92). This chromene could be regioselectively lithiated at 6-position to 93 using butyllithium in ether and reaction of the lithioderivative with 2-methyl-3,l-benzoxazin-4one (94) smoothly afforded the required amidobenzophenone 84. Cyclization of the latter compound with sodium hydride in dimethylsulfoxide occured together with loss of the acetyl group and gave 12-demethylacronycine (81) in 43 % yield and 12-demethylisoacronycine (91) in 46 % yield.


Acronycine-Type Alkaloids: Chemistry and Biology

1 -

K2CO3/CH3I Scheme 16

29


30

F.TiHequin, S. Michel and A-L. Skaltsou^s

OCH3

HaCX)^

^'^ 87

^OH

K2CO3/KI (CH3)2CO

„^Q^ ^

Scheme 17

A third route explored by Lewis et aL [77] was the cyclization of aminobenzophenone 85 (Scheme 18). Compound 85 was only obtained in poor yield when 84 was methylated by methyliodide and sodium hydride in dimethylsulfoxide and the resulting derivative hydrolyzed in mild alkaline medium. iV-methylaminobenzophenone 85 was more efHciently prepared by condensation of N-methylisatoic anhydride (95) with the lithiated chromene 93. Treatment of 85 with sodium hydride in dimethylsulfoxide afforded acronycine (1) in 38 %, accompanied by isoacronycine (18) in 38 % yield.


Acronycine-Type Alkaloids: Chemistry and Biology

Q

^^^

N

OCH3

31

O

OCH3

^ O H3CO

CH3 95

NaH ^ DMSO

Scheme 18 2.3.2.3. Syntheses involving a quinoline or quinolone intermediate Synthesis ofAnand and Sinha. The key-intermediate of the regioselective synthesis of acronycine and glycocitrine II by Anand and Sinha [51.52] is 3-acetyl-4-chloro-2-cyanomethylquinoline (96) which was prepared by two independent routes.


32

F.Title4llil^ S. Michel and A-L. Skaltsowiis

The fk^t (Scheme 19) involved reaction of tiie carbanion of ethyl cyanoacetate (97) with phenylisothiocyanate (98), followed by addition of methyl iodide to give the ketene 5, ^-ketal 99 in 84 % yield. Substitution of the methylthio group of 99 by the carbanion of ethyl acetoacetate in refluxing isopn^anol gave the keto ester 100 in 71 % yield. Cyclization of the ethoxycarbonyl group onto the phenyl ring by refluxing in 1,2-dichlorobenzene affcmied quinoline 101 in 55 % yield. Treatment of 101 with pho^hoiyl chknide at 120-125''C fen* 5 hrs transformed the 4-hydroxy substituent into a 4-chloro group with simultaneous demethoxycarbonylation. Thus, the desired quinoline 96 was obtained in 53 % yieki.

^COOCiHs l-CHjONa/CHaOH

SCTI3

CN

2-CH3I f/ H

^COCKHj

99

N=C=S 98 H5C2OOC

COCH^

CH3COCH2CO(x:::2H3 i-C3H70Na i-C3H70H 100 1,2-dichlorobenzenc^ Rx

Scheme 19

(M

O


Acronycine-Type Alkaloids: Chemistry and Biology

33

Alternately (Scheme 20), the ethoxygroup in the ethyl enol ether of acetylacetone (102) was substituted by heating with methylanthranilate (30) in 1,2-dichlorobenzene to give the enaminone 103 in 70 % yield. Cyclization of 103 catalyzed by sodium methoxide gave 3acetyl-4-hydroxy-2-methylquinolone (104) in 87 % yield. The corresponding 4-chloro derivative 105 was obtained in 89 % yield by heating 104 with phosphoryl chloride. Functionalization of 105 by bromination with N-bromosuccinimide afforded 106 in 73 % yield. Final conversion of 106 to the required 96 was obtained in 42 % yield upon treatment with sodium cyanide in dimethylformamide.

1,2-dichlorobenzene NH, 30

a^

H5C20

y

- CH3

102 OH

.COOCH^

O

COCH3 CHsONa/CHaOH

N H 103

a POCI3

CH3

NBS CCI4

NaCN

DMF

o


34

F.Tilief|ttiii, S. Michel and A-L. Skattsowtfs

Alkylati<m oi quinoline 96 (Scheme 21) with l-bromo-3-inethylbut-2-eiie (42) in the presence of potassium carbonate in dimethylformamide gave 107 in 48 % yield. Methanolysis of the nitrile group of 107 with hydrochl<»ic acid in methanol led to 108 in 70 % yield. Cyclization of ketoester 108 was carried out by refluxing with sodium hydride in tetrahydrofuran followed by heating with phenol at 1(X)°C and by refluxing the crude product obtained with hydrochlcMic acid in methanol to provide 10-demethylglycocitrine U (109) in 47 % yield. Acetylation of the hydroxy phenolic groups of 109 by acetic anhydride and sodium acetate yielded the diacetate 110. This compound was methylated with methyl iodide and sodium hydride in dimethylforaoamide. Hydrolysis of the crude product obtained in the presence of potassium carbonate gave glycocitrine 11 (43) in 52 % yield from 110. Alternately, 10-demethylglycocitrine 11 (109) was refluxed with 2,3-dichl(»t>-5,6dicyanoben2X>-l,4-quinone in toluene to give 12-demethylnoracronycine (52) which could be methylated to acronycine (1). Synthesis ofWinterfeUU et al.The originality of the method of Winterfeldt [49] lies in the building of the aromatic A ring at the final steps of the synthesis. This explains its versatility as far as substitution on the A ring is concerned. For instance, this method provided later on an entry to the synthesis of various acronycine metabolites bearing phenolic hydroxy substituents on the A ring [50]. The starting material was 5-anuno-7-methoxy-2,2-dimethylchromene (76) whose synthesis is described in section 2.3.2.1. (Synthesis cf Loughhead). Alternately (Schen^ 22), 76 could be obtained from 5-hydroxy-7-methoxy-2,2-dimethylchroman (111) [85-87] by conversion of the phenolic to an amino group according to method of Scherrer and Beatty [88] and dehydrogenation. Thus, treatment of 111 with 4-chloro-2-phenylquinazoline in the presence of potassium carbonate in acetone gave the 4-aryloxyquinazoline 112 in 85 % yield. This compound was dehydrogenated with 2,3-dichloro-5,6-dicyanobenzo-l,4-quinone in refluxing dioxane into chromene 113 in 85 % yield. Transposition into 114 was obtained in 74 % yield by heating for 20 hrs in parafHn oil under nitrogen. Alkaline hydrolysis of 114 provided the required chromene 76 in 85 % yield. Treatment of 5-amino-7-methoxy-2,2-dimethylchromene (76) with dimethyl acetylenedicarboxylate (115) gave the adduct 116 which was converted to 117 upon treatment with allyl bromide in alkaline medium (Scheme 23). The key stq) of the synthesis of Winterfeld et cd. was a Cope rearrangement of 117 into 118 by heating in dha to create the B ring of the final compound. The carbomethoxy group of 118 was reduced with lithium aluminium hydride to alcohol 119 which was oxidized to aldehyde 120 by manganese dioxide. Lewis acid catalyzed oleHn aldehyde cyclization of 120 using titanium tetrachloride afforded the chlorocarbind 121 as a stereoisomeric mixture, which couki be acetylated to the diacetate 122.


Acronycine-Type Alkaloids: Chemistry and Biology

35

Treatment of 122 with potassium rerr-butoxide in ethyleneglycol dimethylether led to 12demethylacronycine (81) which could be methylated in the usual way to acronycine (1).

Scheme 21


36

F.Tillequin, S. Michel and A-L. SkaHsounis

113

glycd 150PC

Scheme 22

HjN


37

Acronycine-Type Alkaloids: Chemistry and Biology

H3COOC-C ^ C-COOCH3 115 HoN COOCH 116

.Br Et^O

t-BuOK H3CO(CH2)20CH3

r

190째C

^N

COOCH3

117

n^cooc

N


38

F.TiHequin, S. Michel and A-L. Skaltsoiu^

3

9

CXIH3

H3CO(CH2)20CH3

Scheme 23

2.4. Biological properties The interest of acionycine as a broad spectrum antitumor agent was demonstrated in 1966 in the Eli Lilly Laboratories [6]. The formulation of preparations suited for parenteral use has been a major challenge, due to the very low solubility of acronycine in water. Up to now, the phase I-II clinical evaluations have only relied upon oral administration. This nnost probably explains their limited success, in relation to bad gastro-intestinal tc^erance. Despite much effort, the mechanism of action of acronycine renudns unclear, both at cellular and nx>lecular level. Other biological activities of acronycine have been evaluated, including its activity against Plasmodium strains and against various viruses, its endocrine and hormone agonistic activities, and its carcinogenicity. Finally, the metabolism and pharmacokinetics of acronycine in mammals have been studied, as well as its microbial transformations.

2.4.1. Antitumor activity The in vivo activity of acronycine on a panel of experimental tumors was first described by the Eli Lilly group [6,8]. Acronycine was tested in seventeen experimental models and possessed significant activity against twelve of them. The alkaloid was found active both against various leukemias and against numerous solid tumors including sarcoma, myeloma, carcinoma and melanoma. Of particular interest was the activity against the C1498 murine myelogenous leukemia which is resistant to most of the known chemotherapeutic agents. The activity against X-5563 myeloma was also interesting since this plasma cell tumor has several properties that relate to those of multiple myeloma in human patients. It should be emphasizedtiiatacronycine


Acronycine-Type Alkaloids: Chemistry and Biology

39

was active not only when given by intraperitoneal injection, but also when administred orally. In contrast, only minimum activity was observed when acronycine was administred intravenously, most probably due to its insolubility which did not allow sufficiently high blood levels [6]. Later on, the alkaloid was tested at the N.C.I. and discrepancies between the latter results and those of the Lilly group were observed as far as C1498, AKR and L 5178Y leukemia systems were concerned [7], The question of the actual spectrum of acronycine remains open. Nevertheless, it is clear that its main interest lies in the activity against solid tumors, assessed by the N.C.L results against B16 melanoma and Ridgway osteogenic carcinoma [7]. Phase I-II clinical evaluation of acronycine was performed by Scarffe et al. [89] in patients with refractory multiple myeloma. Oral acronycine capsules produced one clear response in sixteen patients. The remission was maintained for 72 weeks, using a daily dose of 300 mg/m^. Clinical toxicity observed in that study were dose-limiting nausea, vomiting and anorexia, and cumulatively, neurotoxicity which was manifested by ataxia. The use of drugs causing nausea and vomiting after oral administration is undesirable since the amount of drug in the gastrointestinal tract, as well as the absorption, should be altered. Nevertheless, it should be noted that antiemetics were not used in Scarffe trial, therefore the estimated gastrointestinal toxicity may be excessive in light of current practice. A parenteral form of acronycine was however highly desirable. In this respect, a formulation in which the concentration of acronycine is at least 500-fold greater than the solubility value of ca 2-3 mg per liter of water at 25**C had to be secured. In thefirstattempts to overcome the very low water solubility of acronycine, complexes resulting from hydrophobic bonding were prepared. Thus, an acronycine polyvinylpyrrolidone coprecipitate was shown to be more active than acronycine itself. The solubility of acronycine as the coprecipitate was nevertheless only fifteen times that of the non-coprecipitated alkaloid [90]. Complexetion and solubilization of acronycine in the presence of alkylgentisates has also been studied [91]. Better improvement was obtained using acetylacronycinium salts, exemplified by the perchlorate 123, as soluble prodrugs of the antineoplastic agent [92]. Simple acronycinium salts were first prepared by dissolving acrcmycine in acetone and adding the appropriate aqueous acid. The acronycinium salt precipitated and was then converted into the corresponding acetylacronycinium salt by heating with acetic anhydride. Several acetylacronycinium salts could be prepared by this way, i.e. chloride, bromide, phosphate and sulfate, but the perchlorate 123 was found to be the most interesting due to the easiness of its purification and to its greater stability. These compounds showed greatly enhanced aqueous solubility, but they were hydrolyzed to acronycine in pH 7 buffer with a half life of about 25 min. at room temperature [92. 93]. This rapid hydrolysis resulted in the formation and precipitation of the parent compound. Substantial increases in the stability of the prodrugs were observed when dihydroxybenzoic acids were employed as complexing agents [94]. Similarly O-methyl acronyciniumfiuorosulfonate(124) was also claimed as an efficient and water soluble prodrug


40

F.Tiliequin, S. Michel and A-L. Skaltsounis

of acionydne [95]. Nevertheless none of these salts has been tested clinically so far, most probaUy due to their fast hydiolysis into insoluble acronycine in water at neutral pH.

Recently, a more promising approach has been developped by DOIT et al [96,97]. Acronycine could be dissolved in the cosolvent currendy used in human therapeutics to imepare etoposide dilutions. This solvent consists of polyethyleneglycol 300, polysorbate 80 (tween SO) citric acid, benzyl alcohol and absolute ethanol. Resulting acronycine solutions could be further diluted with 5 % aqueous dextrose, 0.9 % aqueous sodium chloride or RPMI1640 culture medium. The dilutions were stable for 4-72 hrs at temperatures from 0^ to 37^C. Aqueous formulation of acronycine was active in vitro against L-1210 leukemia and against fiesh human tumors fiom patients with renal cell cancer, ovarian cancer, uterine cancer, and metastatic tunxirs of unknown primary. It was also active against P-glycoprotein-positive multidrug-resistant (MDR) Chinese hamster ovary cells, but not against multidrug-resistant L1210 murine leukemia cells, 8226 human melanoma cells, or human CXRF-CEM lymphoblasts. In mice, acronycine in the cosolvent was highly toxic by the intravenous route, but well tolerated using intraperitoneal administration. In die latter conditions, it produced significant tumor growth delays in nude mice bearing human MCF-7 breast cancer xenografts and in mice bearing colon 38 tumor. In MOPC315 plasmocyta bearing mice, it was found as effective as melphalan in prolonging life span, suggesting it couki exhibit activity against human multiple myeloma. Despite the promising antitumor activity of acronycine, the mechanism of its action at both cellular and molecular level has not yet been unambiguously established. First observations suggested that the drug did not interact with DNA and did not affect DNA function at a concentration at which it affected RNA [98,99]. In agreement with these early statements, inhibition of mammalian cells growth by acronycine was reported not to be due to an arrest in mitosis on the basis of experiments conducted on Chinese hamster lung cells, polyploid HeLa cells [6], L 5178 Y mouse lymphoma cells and IRC 8 rat monocytic leukemia cells [98]. Mcme recentiy, both acronycine (1) and 2-nitroacronycine (7) were shown to induce cultured cells to accumulate in the phase of the cell cycle wherein the DNA content ranged from 2n to 4n (S + G2 / M) [100]. Acronycine inhibited the growth of L 1578 Y leukemia cells at concentrations that


Acronycine-Type Alkaloids: Chemistry and Biology

41

inhibited incorporation of labeled uridine and other nucleotides into RNA [99]. Further tests showed that the reduced incorporation of nucleosides into RNA resulted from an inhibition of nucleoside transport across plasma membrane and across the membranes of subcellular organelles such as mitochondria and Golgi apparatus [10M03]. Delayed effects included cellular swelling, binucleation, reduced adhesion to substrata and to other cells and cessation of mitotic activity. These various effects have been postulated to be related with the interference of acronycine with the structure, function and/or turnover of cell-surface components [101-104]. In contrast with these data, it should be noted that Plagemann et aL found no inhibition of nucleoside transport in Novikoff cells, even at a concentration four times higher than that yielding 80 % inhibition in L 5178 Y cells [105]. More recently. Dorr and Liddil [96] reinvestigated the DNA-binding property of acronycine, using solutions obtained with the cosolvent used to prepare etoposide dilutions. Under such conditions, acronycine solutions displayed classic non-covalent binding patterns on DNA thermal degradation. In previous experiments [99], acronycine was dissolved in 5 % dimethylsulfoxide and no DNA thermal degradation could be observed as a consequence of artifactic dimethylsulfoxide induced quenching. The same quenching effect, although to a lesser extent, was demonstrated for the classical DNA intercalator ethidium bromide when dissolved in dimethylsulfoxide. These recent experiments strongly suggest that acronycine should interact with DNA, either by intercalation or by some other non-covalent process able to stabilize the double helix against thermal denaturation. This hypothesis seems in good agreement with the approximately flat structure of 5-bromo-l,2-dihydroacronycine (2) established by X-ray diffraction [4]. It is also consistent with the recent demonstration of the DNA binding activity of acronycine azine (125), a dimeric analogue of acronycine which exhibits increased cytotoxic activity [100].

125


42

F.TiHequin, S. Michel and A-L. Skaitsouiris

2.4.2. Otho- biological activities Acronycine was devoid oi antibacterial activity when tested against representative Grampositive and Gram-negative bacteria [6,66]. It was also inactive against the yeast Candida albicans and the dermatophytes Trichophyton mentagrophytes and Microsporum ardouini [66]. As far as antiprotozoal activities are concerned, acronycine itself was without effect against local Trichomonas vaginalis infection in mice. It was moderately active in vitro apinst Plasmodium yoelii [106] and against chloroquine-resistant and sensitive strains oi Plasmodium falciparum [107]. Acronycine as well as several of its derivatives (acronycine azine, 1,2dihydroacronycine, 2-nitroacronycine and thioacronycine) were inactive when tested for HTV-l reverse transcriptase inhibitory activity [106]. Carcinogenicity of acronycine was first tested by the pulmonary tumor response in strain A mice [109]. Applying this technique, the alkaloid was negative under the conditions of the experiment (5 intraperitoneal injections over 24 weeks) at three different dose levels (total doses : 0.53, 1.3 and 2.6 g/kg nx>use). Further tests were conducted by the NCI on mice and rats [110]. The drug was administred f(Âťr 52 weeks by intraperitoneal route at doses of 3.75,7.5 and 15 mgA^g. The low survival of control and treated animals did not allow for the carcinogenicity of acronycine to be determined in mice. However, sarcomas and related tumors of the peritoneum were observed in both males and females rats administered IP acronycine. Tumors of the mammary gland were noted in females and osteosarcomas in males. 2.4.3. Pharmacokinetics and metabolism According to Liu and Ji [111], the pharmacokinetic parameters of acronycine following intragastric administration of 200 mg/kg in rats were assessed by a two-compartment open model. The half-lives of the a and p phases of acronycine elimination were 0.92 and 38 hrs, respectively. The maximum blood concentration was 25 ^g/ml. After 72 hrs, 59 % of the dose had been excreted in the urine and 4.5 % in the feces. These results are not in agreement with the metabolic studies previously perfcmned in die Eli Lilly Research Laboratories [112] which indicated that the feces were the preponderant route of elimination of acronycine after oral or intraperitoneal administration in rats. In that study, the metabolism of acronycine was investigated in four mammalian species: mice, rats, guinea pigs and dogs. The metabolites present in the urine, bile and blood of these species, as well as those present in the urine of patients receiving orally administred acronycine, were extracted after incubation with a mixture of ^-glucuronidase and sulfatase. Most of the metabolites were hydroxylated derivatives of acronycine : 9-hydroxyacronycine (126), 11-hydroxyacronycine (127), 3-hydroxymethylacronycine (128), 9,11-dihydroxyacronycine (129) and 11-hydroxy3-hydroxymethylacronycine (130). It is only in the case of the guinea pig that 0-demethylation


Acronycine-Type Alkaloids: Chemistry and Biology

43

was an important metabolic pathway, leading to the formation of ll-hydroxynoracronycinc (131). The acronycine metabolites observed in the various mammalia are summarized in Table II. Table II: Acronycine metabolites in mammalian species [112] Metabolite

Rat

Guinea Pig

Dog

Mouse

Man

1

126

+

+

+

+

+

1 1

127 128

+

+

+

-1-

+

+

+

1

129

+

+

+

1

130

+

+

+

131

+ +

126 Ri = OH R2 = R3 = H 127 R2 = OH Ri=R3 = H 128 R3 = 0H Ri=R2 = H 129 Ri= R2 = OH R3 = H 130 R2= R3 = OH Ri=H Two of these metabolites were obtained on large scale by microbial hydroxylation of acronycine. Addition of acronycine to a metabolizing culture of Aspergillus alleaceus [113] or Cunninghamella echinulata [114] produced 9-hydroxyacronycine (126) whereas Streptomyces spectabilicus gave 3-hydroxymethylacronycine (128). Both 126 and 128 were devoid of antitumor activity when tested in mice implanted with X 5563 plasma cell myeloma or C-1498 myelogenous leukemia [113]. The main metabolite of acronycine in the guinea pig, 11-hydroxynoracronycine (131) is a natural alkaloid which occurs in serveral Rutaceae species, i.e. Atalaniia ceylanica Oliver [115] and Citrus depressa Hayata [116]. Several total syntheses of this compound have been achieved.


44

F.Tillequiii, S. Michel and A-L. Skaltsoanis

The method used by Lewis et al, [117] (Scheme 24) is very close to diat employed by Hlubucek et al, [43] for the synthesis of acronycine itself. Thus, condensaticm of phloroglucinol (28) with S-methoxyanthranilic acid (132) gave l,3-dihydroxy-5*methoxy-9(10//)-acridifioiie (133). Treatment of 133 with 3-chloro-3-methylbut-l-yne (53) resulted in the formaticm of 11methoxy-12-demethylnoracronycine (=ll-0-methylatalphyllidine) (134) and of a secondary product whose structure was later established as 135 [118]. This latter may be considered as arising by cyclization in alkaline medium of a product of C-alkylation of 133 by 3-chl(nx>-3methylbut-1-yne [118, 119]. Methylation of 134 with dimethylsulfate gave U-methoxyacronycine (136). Demethylation using pyridine hydrochloride or hydrogen bromide readily provided 11-methoxynoracronycine (sbaiyumine A) (137) which was further demethylated into 131 by use of boron tribromide in methylene chlmde [117]. A similar approach was used by Kapil et al. [120] (Scheme 25) for the synthesis of 11hydroxyacronycine (127), 11-hydroxynoracronycine (131) and of the naturally occuring alkaloid atalphyllidine (138). Condensation of 3-hydroxyanthranilic acid (139) with phloroglucinol (28) gave l,3,5-trihydroxy-9(10//)-acridinone (140) [121] which was selectively benzylated into 5-benzyloxy-l,3-dihydroxy-9(10//)-acridinone (141) by use of benzyl chloride in the presence of sodium bicaibonate and sodium iodide in refluxing acetone. Refluxing 141 in pyridine with 3-hydroxyisovala^dehyde dimediylacetal (50) according to tiie procedure previously developped by Crombie et al, [58] yielded the angular pyranoacridone 142. Debenzylation of 142 using 10 % Pd on charcoal in absolute ethanol containing NaOEt under reflux provided atalphyllidine (138). Methylation of 142 with methyl iodide and potassium carbonate in refluxing acetone gave a mixture of N-methylacridone 143 and N,0' dimethyl-acridone 144 which was separated by chromatograpy. Removal of the benzyl protecting group in the same conditions as those used for 142 gave 11-hydroxynoracronycine (131) from 143 and 11-hydroxyacronycine (127) from 144.


Acronycine-Type Alkaloids: Chemistry and Biology

45

PH

132

OH

0

28

0

OH

H 134

135

0

136

137

OH

CH3 131

Scheme 24

\

OH


46

F.Tillequin, S. Michel and A-L. Skaltsounis

O

cocm

ZnG2

Q^-

n-BuOH

HO

HO 139

OH

28

Q

CH

^J;^oa,3 C6H5N/15(r-16(yÂťC C6H5CH2O

Scheme 25

OH


Acronycine-Type Alkaloids: Chemistry and Biology

47

Finally, an elegant entry to 9 and/or 11-hydroxy derivatives in this series was designed by Winterfeldt et al, [50] on the basis of their previous synthesis of acronycine [49] (Scheme 26). Lewis acid catalyzed olefin aldehyde cyclization of 120 gave 121 which could be converted into 11-hydroxy-12-demethylacronycine (145) upon Pfitzner-Moffat oxidation. For identification purposes, 145 was methylated into the known 11-methoxyacronycine (136).

120

oxidation DMSO/C5H5N/ CF3COOH/DCCI

OCH^

H3CO

Scheme 26 Base catalyzed cyclization of the oxo-ester 146 (Scheme 27) gave the chloroacridine 147 which was solvolyzed in phenol to 148. Hydrolysis of 148 yielded the 9,11-dihydroxy derivative 149. Similarly (Scheme 28), the corresponding oxo-aldehyde, obtainable by reduction of the enol-lactone 150, gave rise, through the intermediacy of 151 and 152, to 9-hydroxy-12demethylacronycine (153).


F.Tinequin, S. Midiel and A-L. Skaltsouiris

48

a

OCH3

H3OOCO l)LDA/THF/-2(y»C AcjO

146

147 OC5H5 OCH3 RiO,

^1^

C6H5OH

N

locrc

2NHC1 ^

0R2 Rj =s H R2=:C0CH3 or Rj = C^CXJrl3 R2 — H

k^

R | ss H R2 *• C^OUrl3

or Rj = COCH3 R2 = H

148

149

Scheme 27

i(xrc

Scheme 28


Acronycine-Type Alkaloids: Chemistry and Biology

49

3. NATURALLY OCCURING ACRONYCINE ANALOGS Angular pyranoacridonc alkaloids derived from the 3,12-dihydro-7//-pyrano[2,3c]acridin-7-one basic skeleton constitute a small group of natural products. Their occurence is restricted to plants of the Rutaceous family, where such compounds have only been isolated so far from species belonging to the genera Sarcomelicope (including former Acronychia and Bauerella species), Murraya, Glycosmis, Boenninghausenia, Severinia, Pleiospermum, Atalamia and Citrus,

3.1. Acronycine analogs modified at C(6), N(12), and their derivatives substituted on C ring. Noracronycine. Noracronycine (3) was first isolated by Govindachari et aL from Glycosmis pentaphylla (Retz.) CorreS [3]. It also occurs in Murraya paniculata (L.) Jack [122], Boenninghausenia albiflora Reichb. [123. 124], Glycosmis citrifolia (Willd.) Lindl. [125], Glycosmis waMrinVwfl(Lam.) Tanaka [126], and Sarcomelicope simplicifolia (Endl.) Hartley subsp. simplicifolia (= Acronychia baueri Schott) [127]. As most naturally occuring acronycine derivatives, it bears a free phenolic OH group at C(6) which is chelated by the neighbouring carbonyl function at C(7). It is devoid of significant cytotoxic activity in vitro [44,128,129] and antitumor activity in vivo [5,6]. 12-Demethylacronycine. 12-Demethylacronycine (81) was obtained from Glycosmis pentaphylla (Retz.) CorreS [3], Murraya paniculata (L.) Jack [122], Glycosmis citrifolia (Willd.) Lindl. [125], Glycosmis mauritiana (Lam.) Tanaka [126], and Sarcomelicope simplicifolia (Endl.) Hartley ssp. simplicifolia [127]. It exhibited in vitro a cytotoxic activity of the same order of magnitude as acronycine itself, when tested against KB [44] and HL-60 [126,127] cell growth. 12-Demethylnoracronycine. 12-Demethylnoracronycine (52) was isolated from Glycosmis pentaphylla (Retz.) Correa [3], Murraya paniculata (L.) Jack [122], and Glycosmis citnfolia (Willd.) Lindl. [125]. Derivatives substituted on C ring. The root bark of the Chinese Rutaceae Severinia buxifolia (Poir.) Tenore provided two angular pyranoacridone alkaloids substituted by a prenyl group at C(5), namely severifoline (154) and iV-methylseverifoline (155) [130]. This latter compound was later isolatedfromGlycosmis citrifolia (Willd.) Lindl.[l25]. The structures were deduced from uv, ir and ^H nmr data, and confirmed by chemical correlation [130]. N-Methylseverifoline (155) yielded the 2//,4//-dipyrano[2,3-a:2',3'-c]-


50

F.Tillequiii, S. Michel and A-L. Skaltsoimis

iicridin-14-<Hie derivative 156, when heated with 85 % formic acid, whereas both 154 and 155 gave the A^,0-methyl derivative 157 upon prolonged treatment with methyl iodide and potassium carbonate in refluxing acetone.

154 R = H 155 R = CH3

157 As all other acronycine derivatives unsubstituted on the A ring and bearing a free OH phenolic group at C(6), 154 and 155 showed no significant cytotoxic activity against HL-60 cells in vitro [128,129]. A full account of the l^C nmr data of 3,52, 81,154,155 and several of their derivatives has been published by Furukawa et ai, [131].

3.2. Alkaloids modified on the A aromatic ring 3.2.1. Alkaloids monosubstituted at C(ll) 11'Hydroxynoracronycine . 1 l-HydroxyuOTacronycine (131) was first isolatedfromthe wood ofAtalantia ceylanica Oliver by Eraser and Lewis [115]. The structure was confirmed by total syntheses performed by the same group [117] and by others [120]. This alkaloid was obtained later on, from the leaves of the same species [132] and also from Glycosmis citrifolia (Willd.) Lindl. [125], Poncirus trifoliata (L.) Raf. [133], Pleiospermium aiatum (Wight ct Am.) Swingle [134,135], and numerous Citrus species, e.g. Citrus depressa Hayata [116,136], Citrus


Acronycine-Type Alkaloids: Chemistry and Biology

51

grandis Osb. [137-140], Citrus natsudaidai Hayata [141], Citrus nobilis Lour. [142], Citrus limonia Osb. [143]. ll'Methoxynoracronycine . ll-Methoxynoracronycinc ( = baiyuminc A ) (137) was first obtained but not described by Lewis et al. [117] in course of the synthesis of 131. It was later isolated from Citrus grandis Osb. [144] and from Citrus junos Tanaka [141]. It co-occurs in the latter species with its linear isomer, junosidine (158) [145].

j< H3CO

Atalaphyllidine . Atalaphyllidine (= 11-hydroxy-12-demethylnoracronycine) (138) was isolated from the root bark of Atalantia monophylla CorreS [146]. Its structure, deduced from spectral data, was confirmed by total synthesis [120].

159 R = H 162 R = CH3

160

Atalaphyllinine . Atalaphyllinine (159) derivates from atalaphyllidine by prenylation at C(5). It was obtained from Atalantia monophylla CorreS [147] and from Severinia buxifolia


52

F.Tiitequin, S. Michel and A-L. Skaitsounis

(Poir.) Tenore [130]. Evidence to the structure of atalaphyllinine (159) was obtained by cyclization with formic acid followed by catalytic hydrogenation, which led to bicycloatalaphylline (160), identical with the cyclization product of atalaphylline (161) [147149], 5'(3'methylbui-2-enyl)'ll-hydroxynoracronycine, 5-(3-methylbut-2-enyl)-l 1-hydroxynoracronycine (~N-methylatalaphyllinine s ll.hydroxy-12-methylseverifoline) (162) was isolated from Atalantia ceylanica Oliver by Fraser and Lewis [115]. It also occurs in Atalantia wightii Tan. [150], Atalantia monophylla Correa [151], Severina buxifolia (Poir.) Tcnorc [130], and Gfycosmis citrifolia (Willd.) Lindl. [125]. The structure was first established through uv, ir and ^H nmr spectral analysis and methylation into 163 upon treatment with diazomethane [115]. It was later ensured by ^^C nmr and fcnmic acid cyclization into 164 [150].

163

164

Il-Hydroxynoracronycine (131), 11-methoxynoracronycine (137), atalaphyllidine (138), atalphyllinine (159) and iV-methylatalaphyllinine (162) significantly inhibited macromolecule biosynthesis and HL-60 promyelocytic leukemic cell growth in vitro [128,129]. Compounds 138,159, and 162 also showed anti-herpes virus activity [152] and interesting activities when tested against the rodent malaria agent Plasmodium yoelii in vitro [106] and against Pneumocystis carinii in culture [153]. Acridone-coumarin dimers. Recently, two acridone-coumarin dimers with a C-C linkage between the two units involving the C(5) position of a 11-hydroxynoracronycine ccxnponent have been isolated from Citrus plants. Acrimarine I (165) was obtained from a Citrus hybrid as a dextrorotatory yellow oil [154]. The signal patterns and the results of nOe experiments in ^H nmr spectroscopy suggested the presence of 11-hydroxynoracronycine (131) and suberosin (166) moities in the structure. The mode of linkage between the two units was elucidated by ^H detected heteronuclear multiple bond connectivity (HMBC) experiment, which depicted the structure of the dimer as 165.


53

Acronycine-Type Alkaloids: Chemistry and Biology

H3CO

166 Neoacrimarine D (167) was obtained as an optically inactive yellow oil from Citrus hassaku Hort. ex Tanaka [155]. As previously, the angular orientation of the pyran ring fused to the acridone nucleus was deduced from IH nmr nOe experiments. The linking positions between the two moieties and the orientation of the 2,2-dimethylpyran ring of the coumarin unit were resolved by means of an HMBC experiment.

167


54

F.Tilleqiiiii, S. Micliel and A-L. Skaltsounis

3.2.2. Alkalmds disubstituted at C(10) and C(l 1) Citracridone I. Citracridone I (168) was first isolated from Citrus depressa Hayata [116, 136] and was later obtained from Gtycosmis citr^olia (Willd.) Lindl. [125] and from numerous Citrus species and natural hybrids, e.g. Citrus sinensis Osb. var. brasiliensis Tanaka [156], Citrus grandis Osb. [137-140], Citrus nobilis Lour. [142], Citrus limonia Osb. [143], Citrus funadoko Hort. ex. Y.Tanaka [157]. Its structure was deduced from its spectral data. Of particular interest were nOe observed in iH mxa spectroscopy between the signal of the N(12)Cf/3 group and those (tf the C( 1)-// on the pyran ring on one hand, and the O-C//3 on the other. These effects determined both the angular orientation of the D ring and the substitution pattern on the A ring [116,136]. Citracridone I (168) gave a methoxymethyl ether 169 when treated with chloromethyUnethylether and sodium hydroxide under phase-catalysis conditions, a monomethyl etho* 170 upon treatment with diazomethane, and a dihydro derivative 171 when submitted to catalytic hydrogenation [116.136].

168 169 170

RsH

171

K * dT20CH3

RÂŤCH3

Citracridone 11. Citracridone 11 (170) is the monomethyl ether of citracridone I (168) and was isolated from Citrus decumana L. [158] and Citrus grandis Osb. [139]. 1 l-Hydroxy-lO-methoxynoracronycine . ll-Hydroxy-lO-methoxynoracronycine (172) is isomeric with citracridone I (168). It was obtained from Citrus decumana L. [158]. The structure determination of this compound relied mainly on ^H nmr chemical shift comparisons between the native alkaloid 172 and its diacetylderivative 173 prepared by heating with acetic anhydride and pyridine [158]. When treated with ethereal diazomethane, 172 gave citracridone II (170).


55

Acronycine-Type Alkaloids: Chemistry and Biology

o

H3C0

cm 172

R=H

173

R = CCX:H3

Citracridone III. Citracridone III (174) was isolated from the root bark of Citrus yuko Hort ex. Tanaka [159]. The ^H and ^^c nmr data of 174 showed close ressemblance with those of citracridone I (168) and citracridone II (170). Treatment of citracridone III (174) with diazomethane gave citracridone n (170) [159].

Acrignine A, Acrignine A (175) is the first example of a naturally occuring acridonolignoid. It was recently isolated from Citrus yuko Hort. ex. Tanaka and Citrus grandis Osb. f. Hirado [160]. It derives from phenol oxidative coupling of citracridone III (174) and 3,5-dimethoxy-4-hydroxy-cinnamylalcohol (176). The complete structure and relative stereochemistry of 175 have been obtained from single-crystal X-ray analysis [160].

H3CO

HO


56

F.Tiilequin, S. Michel and A-L. Skaltsounis

Acrimarine J. Acrimarine J (177) isolatsed from a Citrus hybrid is an acridone-coumarin dimer closely related to acrimarine I (165) [154]. It derives from the union of citracridone I (168) and suberosin (166) moieties.

177 Neoacrimarine C. Neoacrimarine C (178) is an acridone-coumarin dimer of a different type isolated from the root of Citrus hassaku Hort. ex. Tanaka as (Really active cubes [155]. Spectroscopic data suggested the presence of citracridone and pyranocoumarin units. Hie angular orientation of the pyran ring on the acridone skeleton was deduced from ^H nmr nOe experiments. The linking position of the acridone and coumarin moieties was established through COSY, HETCOR and HMBC nmr spectroscopy. Furtfier confirmation of the structure was obtained from nmr examination of the monomethylether 179 obtained by treatment of neoacrimarine C (178) with ethereal diazomethane. Observation of nOe between the newly generated methoxy group and the signal of C(9)-H on the acridone moiety ascertained the location of the free hydroxy] group at C(10) and of the linkage with the coumarin unit at C(l 1) in the natural product Position of thefreealcoholic hydroxy] group on the coumarin moiety was established by ^H nmr chemical shift comparisons between 178 and its diacetyl derivative 180 obtained by treatment with acetic anhydride and pyridine at room temperature. Finally, the cis relative stereochemistry of the substituents on the pyran ring of the coumarin unit was deduced from the coupling constant (J = 4.4Hz) between C(10')-H and C(l r)-H. Only a few experiments on the biological activities of 10,11-disubstituted angular pyranoacridone alkaloids have been conducted. Citracridone I (168) was shown to be cytotoxic towards the HL-60 cell growth test [128,129] and to possess antiherpetic [152] and antimalarial [106] activities in vitro . In contrast, citracridone n 170 exhibited no significant cytotoxic, antiheipetic and antimalarial activities when tested under the same conditions.


Acronycine-Type Alkaloids: Chemistry and Biology

O

57

OH

(relative stereochemistry)

178

Ri=R2 = H

179

Ri = CH3 R2 = H

180

Ri=R2 = COCH3

3.2.3. Other alkaloids with modified A aromatic ring Two alkaloids which do not derive from the 3,12-dihydro-7//-pyrano[2,3-c]acridin-7one but whose structures are closely related to that of acronycine were isolated from Citrus plants. Azacridone A . Azacridone A ( = 10-azanoracronycine) (181) was obtained from the roots of Citrus paradisii Macf. [161]. The presence of two nitrogen atoms in the empirical formula was established by high resolution mass spectrometry. Remarkable low field resonances observed for the signals of C(ll)-H (5 9.19, s), C(9)-H (8 8.54, d, J = 4.9Hz) and C(8)-H (5 8.06, d, J = 4.9Hz) in the ^H nmr spectrum of 181 demonstrated the presence of a 3,4-disubstituted pyridine A-ring. The angular orientation of the dimethylpyran ring was deducedfromnOe observed in ^H nmr spectroscopy between the signals of the iV-methyl group and of C(l)-H. Azacridone A (181) is isomeric with 11-azanoracronycine (182), a synthetic compound which had been prepared previously by Reisch et al. [162].

N^


58

F.Tillequiii, S. Michel and A-L. Skaltsoviiis

Citropone-A . Citr(^x>ne-A (183) is an hcmioaciidone alkaloid which was isolated from the root bark of Citrus graruUs Osb. f. huntan Hayata and Citrus natsudaidai Hayata [163,164]. Its structure which includes a unique seven-membered tropolone A-ring was elucidated by single-crystal X-ray analysis. A characteristic feature is the presence of two intramolecularly hydrogen-bonded hydroxy groups.

183 3.3. Alkaloids with oxidized pyran D ring 3.3.1. Acronycine derivatives with oxidized pyran D ring Acronycine epoxide, Acronycine epoxide (184) was isolated in minute amounts fix>m Sarcomelicope argyrophylla Guill. and Sarcomelicope simplicifolia (Endl.) Hartley subsp. neoscotica (P.S. Green) Hartley [24]. Its main spectroscopic features were the high field resonances in IH nmr of the signals of C(l)-H ( 8 4.50, d, J ÂŤ 5.5Hz) and C(2)-H ( 8 2.81, d, J = 5.5Hz) when compared with the corresponding signals of acronycine (1). The high unstability and reactivity of 184 led to speculation that it may be the biologically active form of acronycine in vivo [24]. The first attempts towards the synthesis of 184 by treatment of acronycine (1) with 3chloroperbenzoic acid resulted only in hydroxylation at the aromatic ring, giving 5hydroxyacronycine (185) [165]. Methylation of 185 yielded 5-methoxyacronycine (186) [165]. When acronycine (1) was oxidized with dimethyldioxirane, acronycine epoxide (184) and the diol resulting from the opening of the epoxide, l,2-dihydro-l,2-dihydroxyacronycine, were obtained as an unseparable mixture [166]. Finally, when oxidadon with dimethyldioxirane was carried out in the presence of potassium carbonate, acronycine epoxide (184) was isolated from the reaction mixture in 14 % yield, accompagnied by 185 obtained in 13 % yield [167].


Acronycine-Type Alkaloids: Chemistry and Biology

0

59

OCH3

OCHa

184

1,2"dihydro'l,2'dihydroxyacronycines. Cis-1,2-dihydro-1,2-dihydroxyacronycine (187) and rrflÂŤj-l,2-dihydro-l,2-dihydroxyacronycine (188) were obtained as optically active compounds from the bark of Sarcomelicope giauca Hartley [21] and Sarcomelicope dogniensis Hartley [25]. The main difference between these two diols lay in the coupling constants observed between the signals of C(l)-H and C(2)-H in ^H nmr spectroscopy. A small constant (J = 4.5Hz) characterized the ci5-diol 187, whereas the trans isomer 188 exhibited a large diaxial coupling constant (J = 8Hz). The structures of the two diols were ensured by chemical correlation. Oxidation of acronycine (1) with osmium tetroxide [168] gave the racemic cw-diol 187 in 97 % yield [21]. Treatment of acronycine (1) with chromium trioxide in acetic acid [169] gave a trans-diol monoacetate which was converted into racemic trans-diol 188 upon saponification with sodium methoxide [21]. More recently, d5-l,2-dihydro-l,2dihydroxyacronycine (187) was shown to be the majorreactionproduct, isolated in 37 % yield when acronycine (1) was oxidized with aqueous potassium permanganate in acetone [170].

187

OH

^^^

(relative configurations)

OH


F.TtHequitt, S. !Vf ichd and A-L. Skaltsoui^s

60

3.3.2.12-Deinethylacronyciiie derivatives with oxidized pyran D ring / ^ 'Dihydro'l ^ -dihydroxy-ll -demethylacronycine, Cis-1,2-dihydro-1,2-dihydroxy12-deiiiethylacronycine (189) was isolated in an c^tically active form from the leaves of Sarcomelicope dogniensis Hardey [22]. Its spectral data were closely related to those of 1S7. Confirmation of the structure was obtained by oxidation of 12-demethylacronycine (81) by osmium tetroxide in pyridine, leading to racemic ds-diol 189 in 95 % yield. It should be noted that further oxidation of 189 with sodium periodate led to the D ring opened dialdehyde 190 which was also isolatedfromthe plant material [22].

HO'*'

CHO

m 189 (relative configuration)

190

/ ^-Dihydro'l -hydroxy-ll -demethylacronycine, 1,2-Dihydro-1 -hydroxy-12-demethylacronycine (191) was also obtained as an optically active compound from Sarcomelicope dogniensis Hartley leaves [22]. The structure deduced from spectral data was ensured by synthesis. Hydroxybromination of 12-demethylacronycine (81) by A^-bromosuccinimide in aqueous tetrahydrofuran yielded racemic rraff5-2-bromo-l,2-dihydro-l-hydroxy-12demethylacronycine (192) which was snnoothly debrominated into the racemic benzylic alcohol 191 by treatment with tributyltin hydride [22].

192 (relative configuration)


Acronycine-Type Alkaloids: Chemistry and Biology

61

12-Dihydro-l -oxo-12'demethylacronycine. 1,2-Dihydro- 1-oxo-12-dcmethylacronycine (193) was isolated from the leaves of Sarcomelicope dogniensis Hartley [22]. Confirmation of the structure established from spectral data was obtained by chromic oxidation of 191 which led to 193.

193 1,2'Dihydro'12'hydroxy-l -oxo-12 -demethylacronycine. 1,2-Dihydro-12-hydroxy-1 oxo-12-demethylacronycine (194) is an alkaloid also isolated from the leaves of Sarcomelicope dogniensis Hartley [27]. The structure was deduced from spectral data. The tautomerism between lO-hydroxyacridan-9-one and 9-hydroxyacridine-lO-oxide forms in this series has been extensively discussed [171],

194

3.3.3. Citracridone I derivative with oxidized pyran D ring 1,2'DihydrO'l,2-dihydroxycitracridone I. rrfl/iy-l,2-dihydro-l,2-dihydroxycitracridone I (195) was recently isolated from Citrus paradisi Macf. [172]. Its structure was established through spectral data analysis. Of particular interest was the large coupling constant (J = 7.9Hz) observed between the signals of C(l)-H and C(2)-H in iH nmr spectroscopy which ensured the trans relative orientation of the two hydroxy] groups, in good agreement with the data previously published for 188.


62

F.Tiiieqiiin, S. Michel and A-L. Skaltsounis

OH

195 (relative configmatioii)

3.4. Dimeric alkaloids Four dimeric acridonc alkaloids whose structure include a 3,12-dihydro-7//-pyrano[2,3' c]acridin-7-one unit have been isolated from Rutaceous plants. Two types should be recognized, depending on the mode of linkage between the two moieties, ether bond or caibon-caibon bond.

3.4.1. Dimeric acridonc alkaloid containing an edier linkage The single alkal<nd of this kind, atalinc, was isolated in 1973 by Fiaser and Lewis from Atalantia ceykuiica Oliver [173]. It has been assigned the tentative structure 196 on the basis of uv, ir, ms and ^H nmr data. No final proof of the structure has been provided and alternative sites for ether linkage between the two moieties cannot be excluded.


Acronycine-Type Alkaloids: Chemistry and Biology

63

3.4.2. Dimeric acridone alkaloids containing a carbon-carbon linkage Three binary alkaloids of this type have been isolated by Funikawa et al.fromGfycosmis citrifolia (Willd.) Lindl. [174.175]. All include a 1,2-dihydronoracronycine moiety attached by the C(l) position to a prenyl acridone moiety. Glycobismine A . Glycobismine A (197) is a racemic C-C dimer whose second unit is glycocitrine-n (43) linked by the C(2) position to the dihydronoracronycine unit. The structure has been unambiguously determined by spectroscopic studies including ^H - ^^c long range nmr correlation experiments [174,175]. Both noracronycine (3) and glycocitrine-H (43) co-occur in the plant from which the dimer was isolated. Glycobismine A (197) exhibited interesting activities when tested in vitro &gmnst Plasmodium yoelii [106] and Pneumocystis carinii [153].

Glycohismines B and C. Glycobismine B and Glycobismine C were also obtained as racemates from Glycosmis citrifolia (Willd.) Lindl. [175]. The two compounds were shown to be diastereoisomers. Structure 198 could be assigned to both of them from spectroscopic data, including ms fragmentation and ^^c-lH HMBC nmr correlations. Nevertheless, the relative stereochemistries of the two isomers could not be determined. Glycobismines A, B and C most probably arise by acid-catalyzed reaction between the corresponding monomeric acridone alkaloids. A remarkable fact is the racemic character of the three dimers which does not exclude the possibility of a coupling reaction during the extraction and isolation procedures. This hypothesis would be in agreement with the polymerization and


64

F.TiHequin, S. Mkhel and A-L. Skattsounis

reanangement leactions of acronycine andrelatedcompounds in acidic medium. These reactions have been extensively explored [176-185] and thoroughly reviewed [186] by Funayama and CordeU.

198

When acronycine (1) wasrefluxedin methanolic hydrochloric acid, the fcmnation of many products apart from noracronydne (3) was observed. Starting from noracronycine (3) the same pixxlucts were obtained in better yield. Eight products were isolated and named AB-1, AB2, AB-3, AB-4, AB-5A, AB-5B, AB-6A and AB-6B by Funayama and CordeU [176-184]. The structures were established as dimeric for compounds AB-1 (199) and AB-2 (200), trimeric for compound AB-3 (201), tetrameric for compounds AB-4 (202) and AB-5B (203), pentameric for compounds AB-5A (204), AB-6A (205) and AB-6B (204). The planar structure of coumpound AB-6B was identical with that of AB-5A since these two compounds differed only in stereochemistry. In each of those oligomers, dihydronoracronycine or dihydronorisoacronycine units are linked by their benzylic pyran position (i.e. C(l) in the noracronycine series) to the C(5) position of the following unit. Mechanisms of the polymerization reaction and of the rearrangement process from the acronycine to the isoacronycine scries were postulated, based on experiments involving 1,2-dihydronoracronycine and deuterium labelled reagents [186]. Some of the oligomers, e.g. 199, 200 and 201 were also isolated as thermal rearrangement products when noracronycine (3) was heated at \S(fC [187]. At 210**C, however, a completiy different range of products was obtained. Norisoacronycine (206) was isolated in moderate yield, accompanied by tworegioisomericDiels-Alder adducts (207 and 208) formed from 206 and from the putative diene 209 followed by cyclization. Natural dimeric alkaloids


Acronycine-Type Alkaloids: Chemistry and Biology

65

derived by Diels-Alder addition are well known natural products of the Rutaceae in the prenyl tyrosine, prenyltryptophane, prenylindole, and prenylquinolone scries [188]. Such compounds have not been isolated so far from natural sources in the acridone series.

n=0

200

n=2

203

n=3

205


66

F.Tilleqirin, S. Michel and A-L. Skaltsounis

O

OH

O

OH

209

4. SYNTHETIC ACRONYCINE ANALOGS Numerous derivatives of the 3,12-dihydio-7//-pyrano[2,3-c]acridin-7-one basic skeleton have been synthetized, with the aim of obtaining compounds more potent or moie water soluble dian acronycine itself and to establish structure-activity relationships in diat series.


Acronycine-Type Alkaloids: Chemistry and Biology

67

Readily accessible acronycine analogs variously substituted at C(6) and N(12) were the first to be prepared. Conversion of the carbonyl group at C(7) into a thiocarbonyl one was also explored soon after the discovery of the antitumor properties of acronycine. Most of the derivatives substituted on the aromatic A ring or modified at the pyran D ring have been described more recently. 4.1. Acronycine analogs modified at C(6) and/or N(12) 6'Alkoxy and d-acyloxy analogs. The 6-alkoxy derivatives of noracronycine 210-214 were obtained by treating noracronycine (3) with an appropriate alkyl sulfate or alkyl halide in the presence of sodium hydride in refluxing tetrahydrofuran [66].

210

R = C2H5

211

R = CH2-CH=CH2

212

R = CH2-O-CH3

213 214

R == CH2-CH2-N(CH3)2 R = CH2-COOC2H5

Similarly, treatment of 12-demethylnoracronycine (52) with ethyl sulfate and anhydrous potassium carbonate in acetone afforded the N-O-diethyl derivative 215 [66]. OC2H5


68

F.Tillequin, S. Michel and A-L. Skaltsoiinis

The 6-acyloxy derivatives 216 and 217 were prepared by reaction of excess acetic anhydride in pyridine at 8S^C with noracronycine (3) and 12-deniethylnoracronycine (52), respectively [66].

216

R Âť CH3

217

RÂŤH

Among the derivatives 210-217, only 213 exhibited significant antitumor activity causing a 52 % reduction in tumor size in mice bearing the solid form of sarcoma 180 and a 60 % prolongation of the lives of mice injected with Ehrlich ascites [66]. 6-Demethoxyacronycine, 6-Demethoxyacronycine (218) was first prepared in 1984 by Coppola [189] (Scheme 29). Reaction of N-methylisatoic anhydride (95) with the enolate derived from 2,6,7,8-tetrahydro-2,2-dimethyl-5A/-l-benzopyran-5-one (219) was the key step of the synthesis. The compound 219 was easily prepared by condensation of cyclohexane-1,3dione (220) with 3-methyl-2-butenal (221) [190]. The enolate of 219 obtained by deprotonation with lithium diisopropylamide was allowed to react with N-methylisatoic anhydride (95). The product of the reaction was heated in toluene without isolation, to give 3,5,6,12-tetrahydro-3-3,12-trimethyl-7//-pyrano[2,3-c]acridin-7-one (222) in 56 % yield from 95. Oxidation of crude 222 with 2,3-dichloro-5,6-dicyanobenzo-l,4-quinone finally led to 6deme^xyacronycine (218) in 88 % yield. A different and potentially more versatile synthesis of 6-demethoxyacronycine (218) was more recentiy achieved by Elomri et aL [191] (Scheme 30). Alkylation of 3-nitrophenol (223) with 3-chlon>-3-methylbut-l-yne (53) gave the propargylic ether 224. The nitro group of 224 was selectively reduced by iron powder to yield 3-(3-aminq)henoxy)-3-methylbut-l-yne (225). Claisen rearrangement of 225 was highly regioselective. It provided the required 5-amino-2,2dimethylchromene (226) in 60 % yield. UUmann condensation of 226 with 2-bromobenzoic acid (73) gave the carboxylic diphenylamine 227 in 75 % yield. The last steps of the synthesis were essentially similar to those described by Loughhead for the synthesis of acronycine [72]. Thus, cyclization of 227 using trifluoroacetic anhydride gave rise to 6-demethoxy-12demethylacronycine (228) in 85 % yield. Methylation of 228 with methyl iodide under phasetransfer catalyzed conditions gave 218 in 83 % yield.


69

Acronycine-Type Alkaloids: Chemistry and Biology

Q

\ â&#x20AC;˘"

MgS04

/ ^ ^ C5H5N / CHO

220

221

219

LDA THF/-65*^0

^

'

219

Q^s'^^s

DDQ

'?CH3 222

J 1 ^

.

Reflux

?

J<

CH3 218

Scheme 29 The cytotoxic activity of 6-demethoxyacronycine (218) was determined in vitro, in comparison with acronycinc, against wild type (DC-3F) and actinomycine D-resistant (DC3F/ADX) Chinese hamster fibroblastic lung cell lines. 6~Demethoxyacronycine (218) was found to have a potency within the same order of magnitude as acronycine itself against both cell lines [191].


70

F.Tilleqilin, S. Michel and A-L. Skaksouirfs

(CH3)2CO

^^^^

K2CO3/KI 02N'^ 223

^^"^

Ffe/HCl CH3OH/H2O ^O

53

cxxw

Cu(OAc)2 iPiOH CH2C12

EtjN KOAc 227

^^^^-^^

CHal PhCH2-NEt3 a aqNaOH/MEK

Scheme 30

6'Demethoxy'6'methylacronycine, The synthesis of 3,12-dihydro-3,3,6,12tetramethyl-7//-pyrano[2,3-c]acridin-7-one ( = 6-demethoxy-6-iiiethylacronycine) (229) was described by Smolders et al. (Scheme 31). Condensation of orcinol (230) with methyl anthranilate (30) in the presence of 4*toluenesulfonic acid in refluxing heptanol gave a 1:3 mixture of the regioisomeric acridinones 231 and 232. Methyladon of that mixture using methyl sulfate under phase-transfer catalyzed conditions yielded the two N,0-methylated


71

Acronycine-Type Alkaloids: Chemistry and Biology

TsOH heptanol

30

230

232

(CH3)2S04

©

e

PhCHj-NEtaCl CHjClj/NaOH/HjO

235

233

A^

O

CH,

53

K2CO3/KI DMF/100°C/72h

Scheme 31


72

F.Tillequin, S. Michel and A-L. Skaitsounis

acridiiKMies 233 and 234, which could be easily separated by column chromatography on silica gel. Treatment of the minor isomer 233 with hydrobromic acid gave the corresponding phenol 235. Condensation of 235 with 3-chlcH*o-3-methylbut-l-yne (53) according to the method develc^>ed by Hlubucek et a!, [43] led to 6-demethoxy-6~methylacronycine (229). The angular structure of 229 was unambiguously established by X-ray crystallographic analysis [192].

4.2. 342-Dihydro-7H-pyrano[2,3-c]acridin-7-thiones Thefirstattempts towards the conversion of the carbonyl group at C(7) of acronycine (1) into a thiocaibonyl group by Dinunock et al, [193] involved treatment of 1 with tetrapho^h(Âťiis decasulfide in benzene (Scheme 32). Under these conditions, conversion was accompanied by 0-demethylation to 7-thionoracronycine (236).

2.MeOH/Rx,

Scheme 32

In contrast. Smolders et ai succeeded in preparing thioacronycine (237) by carrying out the reaction of acronycine (1) with tetraphosphorus decasulfide in hexamethylphosphoric triamide [194].


Acronycine-Type Alkaloids: Chemistry and Biology

73

4.3. Acronycine analogs modified at the A ring 4.3.1. Acronycine analogs substituted at the A ring Acronycine and noracronycine analogs substituted at various positions on the aromatic A ring by halogen atoms, nitro groups and methyl groups have been described. Haloacronycities, 9-Chloronoracronycine (238) and 9-chloroacronycine (239) were synthetized in 1972 by Fryer et al. [66] (Scheme 33). The general scheme of the synthesis was essentially similar to that used by Hlubucek et al. [43] for the synthesis of acronycine. 7-Chloro-l,3-dihydroxy-9(10//)-acridinone (240) was treated with 3-chloro-3-methylbut-lyne (53) to give the angular pyranoacridone 241. Methylation of 241 with dimethylsulfate in the presence of potassium carbonate in acetone gave a mixture of 238 and 239 which were separated by column chromatography. 11-, 10-, and 9-Fluoroacronycine (242, 243 and 244) were prepared by Smolders et al. [196] (Scheme 34). The first steps of the synthesis was a series of Pfitzinger reactions between 7-, 6-, and 5-fluoroisatine (245, 246 and 247) on one hand and phloroglucinol (28) on the other, yielding 5-, 6-, and 7-fluoro-l,3-dihydroxyacridine-9-carboxylic acid (248, 249 and 250), respectively. Methylation with ethereal diazomethane gave the corresponding methyl esters 251, 252 and 253, which were converted into 5-, 6- and 7-fluoro-l,3-dimethoxy-9methoxy-carbonyl-10-methylacridinium methosulfate 254, 255 and 256 by use of excess dimethylsulfate in refluxing benzene. Oxidation with potassium ferricyanide gave 5-, 6-, and 7fluoro-l,3-dimethoxy-10-methyl-9(10//)-acridinone (257, 258 and 259) which were Odemethylated with hydrobromic acid into 5-, 6-, and 7-fluoro-l,3-dihydroxy-10-methyl9(10//)-acridinone (260, 261 and 262). Treatment with 3-chloro-3-methylbut-l-yne (53), according to Hlubucek et al. [43] led to 11-, 10-, and 9-fluoronoracronycine (263, 264 and 265) with excellent regioselectivity, since the corresponding linear isomers were not detected in the reaction mixtures. Final methylation with methyl iodide and potassium carbonate in acetone yielded 242, 243 and 244.


F.Tttlequin, S. Michel and A-L. Skaltsowiis

74

ÂŤ QJJ

53

K2CO3/DMF

240

o

239 Scheme 33

on


75

Acronycine-Type Alkaloids: Chemistry and Biology

2 4 8 Ri = F, R2 = R3 = H 2 4 9 R2 = F, R i = R 3 = H 2 5 0 R3 = F, R, =: R2 = H 245 Ri = F, R2 = R 3 = H 246 R2 = F, R i = R 3 = H 247 R3 = F, R i = R 2 = H H3COOC

H3COOC (CH2)3S04 CfiHg/Rx OH

OCH3

^^2 CH3SO?

Rj = F, R2 — R3 = H R2 ~ F, Rj s= R3 = H 2 5 3 R3 = F, R i = R 2 = H

2 5 4 Ri = F, R2 = R 3 = H 2 5 5 R2 = F. R i = R 3 = H 2 5 6 R3 = F. R i = R 2 = H

K3Fe(CN)6 OCH^ M CH3 257 Ri = F, R2 = R3 = H 2 5 8 R2 ~ F, Rj = R3 = H 259 R3 = F, R i = R 2 = H

Scheme 34 A


76

F.Tillequin, S. Michel and A-L. Skaltsounis

O HBr OCH, CH31 Rl 2 6 0 R, - F , R2 - R a ^ H 2 6 1 R2 - F , Rl «:Rj = H 2 6 2 R3 = F. Rl = R2 =»H

Rl CH3 257 Ri«F, R2 = R3 = H 258 R2=F, Ri«R3 = H 259 R3=:F. Ri=R2 = H

a

R3V 53

KjCXJj/DMF

Rj''

0 Rl

CH3I K2CO3 CH3

k:vs^

2 6 3 Rl « F , R2«R3 = H 264 R2 = F, Rl = R3 = H 265 R3 = F, Rl = Rj = H

242 Ri = F, R2 = R3 = H 243 R2 = F, Ri = R3=:H 244 R3 = F, Ri«R2 = H

Scheme 34 B

Nitronoracronycines. The four isomeric noracronycine analogs bearing a nitro substituent onringA have been synthetized by Reisch et al, [196-198] (Scheme 35, Scheme 36, Scheme 37, and Scheme 38). The synthetic strategies which led to 9-nitronoracronycine (266) [196], 10nitronoracronycine (267) and 11-nitronoracronycine (268) [197] were essentially similar. Ullmann condensation of conveniently nitro substituted 2-chlorobenzoic acids (269-271) with 3,5-dimethoxyaniline (31) afforded carboxylic diphenylamines (272-274) [199] . Depending


Acronycine-Type Alkaloids: Chemistry and Biology

77

on the substitution pattern, the iV-methylation step preceeded (e.g. 272) or followed (e.g. 273 and 274) cyclization with polyphosphoric acid to nitrodimethoxyacridone. The various nitrol,3-dimethoxy-10-methyl-9(10//)-acridinones (278-280) were converted into the corresponding nitro-l,3-dihydroxy-10-methyl-9(10//)-acridinones (281-283) by classical treatment with hydrobromic acid. Finally, the potassium salts of the nitrodihydroxyacridones were treated, according to a modified Hlubucek procedure [67] with 3-chloro-3-methylbut-l-yne (53) to yield the corresponding desired nitronoracronycines. This reaction was fully regioselective in the case of the formation of 11-nitronoracronycine (268). In contrast, 9-nitronoracronycine (266) and 10nitronoracronycine (267) were accompanied by small amounts of the corresponding linear isomers, 284 and 285, respectively. Schemes 35, 36, and 37 summarize the exact order of the reactions, the reagents used and the yields obtained at each step for the three individual syntheses. Attempts made towards the synthesis of 8-nitronoracronycine (286) involving a modified Ullmann reaction at the early steps failed, most probably due to the strong deactivating influence of the nitro group at the 6-position of methyl 2-amino-6-nitrobenzoate (287) [200]. A different approach was therefore developped, in order to obtain 286 [198] (Scheme 38). Fusion of phloroglucinol (28) with methyl 2-amino-6-nitrobenzoate (287) gave the required 1,3dihydroxy-8-nitro-9(10//)-acridinone (288) accompanied by side-products including methyl 2(3,5-dihydroxyphenylamino)-6-nitrobenzoate 289 which was considered as the intermediate giving rise to 288 in the course of the reaction. Methylation of 288 with methyl iodide and potassium carbonate in acetone yielded 290, which was selectively O-demethylated into 1,3dihydroxy-10-methyl-8-nitro-9(10fO-acridinone (291) with hydrobromic acid. Reaction of the potassium salt of 291 with 2-chloro-3-methylbut-l-yne (53), potassium iodide and potassium carbonate in dimethylformamide at 120*^C for 48 hrs in a sealed glass ampoule gave 8nitroacronycine (286) as major reaction product, isolated in 21 % yield. When the same reaction was carried out under classical Hlubucek conditions [43], 8-nitroacronycine (286) and its linear isomer (292) were obtained in 16 % and 25 % yield, respectively. Compounds 266, 267 and 268 were tested by the N.C.I, against P 388 leukemia transplanted in mice [196. 197]. At lower doses, no significant antitumor activity could be detected, whereas higher doses proved to be toxic.


78

F.TiHequin, S. Michel and A-L. Skaltsounis

Synthesis of 9-iiitronoracronycine (266)

D^D OCH3

OCH3

02N>

OCH2

I-CH3I/KOH (CH3)2CO^

O2N llOX

2-CH3COOH

275 (42%) 0,N

1 . HBr / CH3CXX)H / ZnQa/4 hrs OCH

2-H2O/Na2CO3/50^C/6hrs

CH3 278 (98%) O OH l-KOH/EKÂťl

^ ^ a 53 K2CXb/KI/DMF.72hre/100^C (sealed tube) O OH

CH3 284 (0.5 %)

266 (16%) Scheme 35


79

Acronycine-Type Alkaloids: Chemistry and Biology

Synthesis of 10-nitronoracronycine (267) Q97| OCH, OCH, .COOH 02N CX^H3

2i^ 270

OCH,

31 O

(DCHs CH3l/(tBU)4N^Br aq.KOH/Toluene OCH3 O2N

OCHa

276 (95O

OH

2 7 9 (60%)

l-46%HBr/H20/Rx/2hrs ^. 2-H20/2hrs CH3 2 8 2 (82%)

o

OH

OoN 267 (13%) CH3

1-KOH/EtOH

O

< .

2-

^

^

a 53 K2CO3/K1/DMF (sealed tube)

O9N

2 8 5 (1 %) CH3 Scheme 36

OH


80

F.Tiileqtiiii, S. Michel and A-L. Skaltsounis

Synthesis of ll-nitronoracronycine (268) Q92|

OCH3 OCH3

OCH3 I

H^N"^ ^ ^

^0CH3

NO2

274 (34%)

271

OCH3

a 53 KjC^a/KI/DMF (sealed tube) Scheme 37


Acronycine-Type Alkaloids: Chemistry and Biology

Synthesis of 8-nitronoracronycine (286) Q^S

'NH2 HO

CH3I/K2CO3 (CH3)2CO 6hrs/RT

Scheme 38

81


82

F.Tiilequin, S. Michel and A-L. Skaltsounis

Metkylacronycines, The synthetk; schemes which permitted Reisch et a/. [201] to prepare 8-, 9-, 10- and 11-methylacronycine (293, 294, 295, and 296) were similar. Condensations of 2-amino-6-, 5-, 4-, and 3-methyl benzoic acids (297, 298, 299, and 300) with phloroglucinol (28) (Scheme 39) performed in it-heptanol at reflux in a Dean-Staiic apparatus in the presence of 4-toluene sulfonic acid gave l,3-dihydroxy-8-, 7-, 6-, and 5methyl-9(10//)-acridinones (301, 302, 303, and 304) in 12 %, 47 %, 34 % and 15 % yield, respectively. Methylation of 301 with methyl iodide and potassium carbonate in refluxing acetone gave l-hydroxy-3-methoxy-8,10-dimethyl-9(10//)-acridinone (305) in 89 % yield. Treatment of 302 and 303 under the same conditions gave l,3-dimethoxy-7,10-dimethyl9(10//)-acridinone (306) and l,3-dimethoxy-6,10-dimethyl-9(10//)-acridinone (307), in 78 % and 72 % yield, respectively. Methylation of l,3-dihydix>xy-5-methyl-9(10//)-acridinone had to be performed using potassium hydroxide as alkaline agent. After 24 hrs reflux in acetone with methyl iodide and potassium iodide, l,3-dimethoxy-5,10-dimethyl-9(10f/)-ftcridinone (308) was obtained in 84 % yield. 0-Demethylation of compounds 305,306, 307, and 308 with 48 % hydrobromic acid gave the corresponding l,3-dihydn>xy-8,7,6 and 5,10-dimethyl-9(10//)acridinones (309, 310, 311, and 312) in 75 %, 79 %, 67 % and 81 % yield, respectively. Treatment of the potassium salts of 309,310, and 311 with 3-chloro-3-methylbut-l-yne (53), potassium carbonate and potassium iodide in dimethylformamide at 80째C for 6 hn. led to 8-, 9and 10-methylnoracronycine (313, 314, and 315) in 14 %, 31 % and 43 % yield, respectively. Compounds 313 and 315 were accompanied by the linear isomers 316 and 317, isolated in 2.6 % and 17 % yield, respectively. The samereactionapplied to the potassium salt of 312 led to the corresponding propargyl ether 318 (Scheme 40). This latter could be converted almost quantitatively into 319, by heating at 120^C in dimethylformamide for 8 hrs. Final methylation of 313,314,315, and 319 was obtained by treatment with methyl iodide in the presence of sodium hydride in refluxing tetrahydrofuran. The desired 8-, 9-, 10- and 11metiiylacronycine (293, 294, 295, and 296) were obtained in 57 %, 33 %, 49 % and 51 % yield, respectively. Metiiylation products at C(5), 320,321, and 322 accompanied, in smaller amounts the compounds 293,295, and 296, respectively (Scheme 39 and 40).


Acronycine-Type Alkaloids: Chemistry and Biology

Rl

83

OH TsOH n-heptanol OH*^^

297 298 299 300

Rl = CH3. R2=CH3. R3=CH3, R4=CH3,

R3

301 302 303 304

Rj = R3 = R4 = H Ri=:R3 = R4 = H Ri = R2 = R4 = H R i = R 2 = R3 = H

Ri = CH3, R2=CH3, R3=CH3, R4=CH3,

CH^I (CH3)2CO KI/K2C03orKOH

OCH3

305 306 307 308

Rj =s Crl3, R2 ~ R3 — R4 ~ R5 ~ H R2=R5 = CH3, R i = R 3 = R4 = H R3 = R5 = CH3, Ri=:R2 = R4 = H R4 — R5 = Cri3, R| = R2 = R3 ~ H

48 % HBr

309 310 311 312

Ri = CH3, R2=CH3, R3 = CH3, R4 = Cri3,

R2 = R3 = R4 = H R i = R 3 = R4 = H Ri = R2 = R4 = H Rj = R2 — R3 = H

Scheme 39 A

R2 = R3 = R4 = H R i = R 3 = R4=:H R i = R 2 = R4 = H Ri=R2 = R3=H


84

F.Tiliequin, S. Michd and A-L. Skaltsoui^

CH3 3 0 9 R i « CH3, R2 = R3 = H 3 1 0 R2«CH3. R i = R 3 = H 3 1 1 R3=CH3. R i = R 2 = H

3 1 3 R| = CH3» R2 * R3 — H 314 R 2 - C H 3 , R i = R 3 « H 315 R3-CH3, Ri=R2 = H CH3l/NaH THF/Rx OCH3

2 9 3 Ri = CH3, R2 = R3 = R4 = H 2 9 4 R2=CH3, R i « R 3 = R4 = H 2 9 5 R3 = CH3. R i = R 2 = R4 = H

3 2 0 Ri = R4=CH3. R2 = R3 = H 3 2 1 R3=:R4=CH3. Ri = R2 = H

Scheme 39 B


Acronycine-Type Alkaloids: Chemistry and Biology

O

85

OH â&#x20AC;˘

a

^

53

K2C03/KI/DMF 80*ÂťC

312

319

296 R = H 3 2 2 R = CH3

Scheme 40

4.3.2. ll-Azaacronycinc Reisch et al. prepared 11-azaacronycine (= 6-niethoxy-3,3,12-trimethyl-3,12dihydrochromeno[5,6-b][l,8]naphtyridin-7-one) (323) with the aim of obtaining an acronycine analog with increased water solubility [162,202] (Scheme 41). Ullmann condensation between 2chloronicotinic acid (324) and 3,5-dimethoxyaniline (31) yielded 325 which could be cyclized into 326 by heating with polyphosphoric acid. Usual methylation with methyl iodide in alkaline medium yielded 327 which was 0-demethylated to 328 with hydrobromic acid. Hlubucek condensation of 328 with 3-chloro-3-methylbut-l-yne gave ll-azanoracronycine (182) together with the linear isomer 329 and the dimethylpropargyl ether 330. Refluxing 330 in iV,iV-diethylaniline permitted Qaisen rearrangement into 182 and 329 [162]. Methylation of 182 with dimethyl sulfate in tetrahydrofuran in the presence of sodium hydride gave finally 11azaacronycine (323) [202]. The solubility of 323 in water was 15-fold that of acronycine itself. Both compounds 182 and 323 were tested against P 388 leukemia transplanted in mice and were found to be inactive.


F.Tillequin, S. Mkhel and A-L. Skaitsounis

86

OCH3

OCH3

100*»C

3hrs

^ ,

^

N^

N H O

OH

-9-

47%HBr OH

^

53

K2CO3/KI/DMF/70*»C/22hrs

O

'^ OH

N

N

^

CH3

O-

^

330

U/CfiHsNEtj

O

OH

CH3

329

182 CH3 I (013)2804/NaH/THF 0

323 CH3

QCH3

Scheme 41


Acronycine-Type Alkaloids: Chemistry and Biology

87

4.4. Acronycine analogs modified at the D ring Acronycine analogs modified at the 1,2-doubie bond. The alcohols resulting from the addition of water to the double bond of the pyran ring of acronycine were prepared with the aim of obtaining compounds with increased water solubility. In addition, these compounds were also considered as possible acronycine prodrugs and as starting materials for the synthesis of glycosides in the pyranoacridone series [203]. The benzylic alcohol 331 was obtained in two steps from acronycine (1) (Scheme 42) Treatment of 1 with iV-bromosuccinimide in aqueous tetrahydrofuran led to racemic trans-lbromo-1-hydroxy-1,2-dihydroacronycine (332) in 70 % yield. In a second step, the bromohydrin 332 was smoothly debrominated to the desired 1-hydroxy-1,2-dihydroacronycine (331) obtained in 35 % yield using tributyltinhydride [203].

332 Br relative configuration

Bu3SnH/AIBN ^. Toluene/Rx CH3 HO 331 Scheme 42 A good precursor of 2-hydroxy-1,2-dihydroacronycine (333) was ci5-l,2-dihydroxy1,2-dihydroacronycine (187) easily prepared by osmium tetroxide oxidation of acronycine [168] (Scheme 43). A first reaction sequence involving sulfuric acid dehydration of diol 187 to the homobenzylic ketone 334 followed by borohydride reduction of 334 to 333 was successfully


88

F.TiHeqiiin, S. Michel and A-L. Skaltsounis

applied, but the yields remained very low. From a quantitative point of view, better results were obtained by converting 187 with N^-thiocarbonyldiimidazole into the corresponding cyclic thiocarbcmate 355. Benzylic reduction of 335 with tributyltinhydride affOTded 333 in a second step [203].

187 OH relative configuration H2SO4/H2O 18%

NJV'.tiiiocarbonyldiimidazDle jButanone /Rx/20hrs QCHj

BuaSnH/AIBN Toluene/20 *ÂťC


Acronycine-Type Alkaloids: Chemistry and Biology

89

(Âą)-2-Hydroxy-l,2-dihydroacronycinc (333) was used to prepare glycosides in the acronycine series (Scheme 44). For instance, treatment of 333 with l,4-di-0-acetyl-3-chloro and 3-bromo-2,3,6-trideoxy-L-arflW/io-hexapyranoses (336 and 337) in the presence of tin tetrachloride gave stereoselectively the glycosides 338-339 and 340-341, respectively [204]. In each series, the diastereoisomers could be easily separated by column chromatography. The absolute configuration at C(2) on the aglycone part of each glycoside was deduced from ^H and 13c nmr data, compared with those of related angular hydroxydihydropyranocoumarin hexopyranosides of known configuration [205].

H3COCX)

336 X=C1 337 X = Br

OCHa

339 X = C1 341 X = Br

338 X = C1 340 X = Br

Scheme 44


90

F.TiHequin, S. Michel and A-L. SkaltsfNims

Several other 2-hydroxy-l,2-dihydroacronycine glycosides have been recently synthetized. The cytotoxicity of those compounds was determined against the [m>liferati(Hi of L 1210 cells in vitro [206]. The activity of 2-hydroxy-i;2-dihydroacronycine glycosides seems related with the lipq)hilicity oi the sugar mdety of the molecule [206]. Acronycine analogs modified ai C(3). Noracronycine and acronycine analogs variously substituted at C(3), by hydrogen atoms or alkyl residues, were synthetized by Reisch et al, [68, 207.208]. 3,12-Dihydro-6-hydroxy-12-methyl-7//-pyrano[2,3-c]acridin-7-one (342) was obtained together with the linear isomer 343 in the course of studies related to the synthesis of furacridone [207] (Scheme 45). Treatment of 13-dihydroxy-10-methyl-9(10//)-acridin(Hie (14) with propargyl bromide in alkaline oniedium afforded the ether 344 in 79 % yield. Smaller amounts of compounds di-0-alkylated 345 and 0,C-alkylated 346 were also isolated from the reaction mixture. Qaisen rearrangement of 344 by heating at reflux in ^^-diethylaniline gave 44 % overall yield of a 6:1 mixture of compounds 342 and 343 which were not separated [207]. Dramatic difference observed in the ^^C nmr chemical shifts of N-CH3 signals between the angular isomer 342 (Sc - 43.1 ppm) and the linear isomer 343 (ScÂť 33.9 ppm) permitted deduction of the relative proportions of the two compounds in the mixture from the relative intensities of the two signals. Acronycine analogs 347,348, and 349 were recently prepared in 55-70 % yield by methylation of the corresponding noracronycine analogs 350, 351 and 352 using methyl iodide in tetrahydrofuran in the presence of sodium hydride [206] (Scheme 46). The starting noracronycine derivatives 350,351, and 352 had been previously obtained in 20-30 % yield by Mitsunobu reaction between l,3-dihydroxy-10-methyl-9(10//)-acridinone (14) and the propargylic alcohols 353, 354, and 355, followed by Claisen rearrangement [68] (Scheme 46). Compounds 347-352 were tested in vitro on sixty human tumor cell lines of various types of cancer by the N.C.I.[209]. Except 347, all substances were determined to be inactive. Compound 347 was inactive against most of the sixty cell lines but showed weak cytostatic activity against the renal cancer UO-31 cell line.


Acronycine-Type Alkaloids: Chemistry and Biology

91

O

Br-CH2-C=CH

OH

K2CO3/KI/DMF/70째C/18hrs 3^

?l

O

?"'^

342 Scheme 45

OH


92

F.Tiilequin, S. Michel and A-L. Skaltsounis

P(Ph)3/THF/ Azadiethy]dicaiix>xylate

R ^ HO

14

3 5 3 Ri = R2«(CH2)5 354 Ri = R2 = (CH2)4 355 R j « CH3, R2 * ^ ^ " 3

DMF 130 ^C

3 5 0 Ri = R2 = (CH2)5 3 5 1 Ri = R2»(CH2)4 3 5 2 Rj = CH3, R2 — C2H3 CHjI/THF/NaH

Scheme 46


Acronycine-Type Alkaloids: Chemistry and Biology

93

4'Azaacronycine. The acronycine isoster at the D ring, 4-azaacronycine (356) was synthetized by Reisch et al. in 1993 [210] (Scheme 47). Permanganate oxidation of 1-methoxy3-methyl-9(10//)-acridinone (234) prepared following the method of Smolders et aL [192], gave l-methoxy-10-methyl-9(10//)-acridinone-3-carboxylic acid (357). Conversion of the carboxylic function of 357 to the iV-substituted aniline 358 was carried out in a one-pot Curtius rearrangement using diphenyl phosphoric azide in the presence of rerr-butanol and triethylamine. Acidic hydrolysis of 358 yielded 3-amino-l-methoxy-10-methyl-9(10//)-acridinone (359). Prolonged heating of 359 with 3-chloro-3-methylbut-l-yne (53) in dimethylformamide, in the presence of potassium carbonate and potassium iodide gave 4-azaacronycine (356) [210].

KMn04 ^> 18%

cxxm

tBuOH/NEt3 DPPA

CF3COOH Ihr/RT

O /C(CH3)3

Toluene/100 째C 40%

H 358

d

^ 53

^. K2CO3/KI/DMF NH2 120**C/8hrs 20% 359 Scheme 47

67 %


94

F.TiNequin, S. Michel and A-L. Skaltsounis

5. CONCLUSION In the foregoing pages, the isolation, structure determination, methods of synthesis, and the biological properties of acronycine have been reviewed. An account has been given of acronycine natural and synthetic analogs and of dieir Inolpgical properdes when studied. Despite the vast amount of research that has been carried out since the discovery of the broad experimental antitumor spectrum of acronycine in 1966, the present account demcmstrates that much remains to be done. From a biological point of view, the activity manifested by acronycine, after oral administration, against the B-lymphocyte-derived disease multiple myeloma in humans, should stimulate further research. Important biologic experiments for this class of agents should include: (i) - Definitive mechanistic studies of DNA intercalation, topoisomerase-I or topoisomerase-n inhibition, and possibly apoptosis. (ii) - Repetition of the therapeutic trials with oral acronycine, together with oral bioavailability studies. (iii) - New therapeutic trials with a reformulated parenteral formulation. (iv) - Studies to define a potential role for this group of compounds in patients with multidrug resistant tumor. Difficulties also prevail when trying to draw conclusions on structure-activity relationships within the pyranoacridone series. Many compounds which have been isolated or synthetized have not been studied from a biological point of view. Only a few have been tested in vitro for cytotoxic activity. Even fewer are those which have been examined in vivo for antitumor properties. In addition, the cell lines used for the tests greatly vary from one compound to an other. It nevertheless seems possible to draw some limited conclusions in this field: (i) - When the A ring is not substituted, only compounds bearing 0-allcyl substitution or no substitution at all at C(6) exhibit cytotoxic or antitumor activity. In contrast, all the compounds bearing a free OH phenolic group at C(6) have been considered as inactive. (ii) - When the A ring bears hydroxy or alkoxy substituents at position 11 or at positions 10 and 11, compounds bearing either an alkoxy or a free hydroxy group at C(6) have been claimed to be cytotoxic. (iii) - The 1,2-double bond on the pyran D ring appears as an indispensible structural requirement to observe cytotoxic or antitumor activity. This should argue in favor either of a mechanism of action at molecular level which involves the 1,2-double bond, or of a metabolic


Acronycine-Type Alkaloids: Chemistry and Biology

95

bioactivation of acronycine derivatives at those positions. The effect of substitution on that double bond remains less clear. It is puzzling to note that 2-nitroacronycine which was initially considered as inactive has been more recently selected as a promising agent in the series, on the basis of in vitro experiments. The greatest problem which remains open is the real target of acronycine at both cellular and molecular levels. In the near future, the synthesis or isolation from natural sources of new active compounds in the series, may help to determine this with certainty.

Acknowledgements The authors wish to thank the many colleagues and students who have stimulated their interest in the acronycine series for the past few years, and more particularly Pr. M. Koch and Drs G. Baudouin, M. Brum-Bousquet and A. Elomri (University Paris V, France), Prs. S. Mitaku and E. Mikros (University of Athens, Greece), and Pr Gh. Atassi and Dr A. Pierr^ (Institut de Recherches Servier, Issy-les-Moulineaux, France). Dr. H. Trinh Van-Dufat is also gratefully aknowledged for her help in the preparation of the manuscript.

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Chapter Two

Solarium Steroid Alkaloids - an Update Helmut Ripperger Institute of Plant Biochemistry D-06I20 Halle (Saale) Germany

CONTENTS 1. 2. 3. 4.

INTRODUCTION GENERAL OCCURRENCE STRUCTURES 4.1. Spirosolanes 4.1.1. Xylosyl-P-solamarine 4.1.2. Tomatidine Glycosides 4.1.3. (255)-5a,22pA^-Spirosolan-3-one 4.1.4. (23/?)-23-Acetoxytomatidine and Lycoperoside A 4.1.5. (255')-22pA^-Spirosol-5-en-3a-amine 4.1.6. Solasodine Glycosides

105 107 109 120 120 120 121 123 123 124 125

4.1.7. (25/?)-5p,22aA^-Spirosolan-3-one 4.1.8. Soladulcidine Glycosides Soladulcine A and B 4.1.9. 2a-Hydroxysoladulcidine 4.1.10. 3-0-(p-Solatriosyl)solanaviol 4.1.11. (23S)-23-Acetoxysoladulcidine, (23S,255)-23-Acetoxy-5a,22aA^spirosolan-3P-ol, Lycoperoside B and C 4.1.12. (23/?)-23-Hydroxysoladulcidine 4.1.13. (235)-23-Hydroxysolasodine and its Glycosides 4.1.14. 25-Acetoxyrobustine

130 131 132 132

4.1.15. Solaparnaine 4.1.16. iV-Hydroxysolasodine and its Glycosides 4.1.17. 12p,27-Dihydroxysolasodine, its 3-O-P-Chacotrioside and (25/?)-12P-Hydroxy-22aA^-spirosol-5-en-27-oic Acid

138 138

4.1.18. Solaverine III 4.1.19. (25/?)-22aiY-Spirosol-5-en-3a-amine, (25/?)-22aA^-Spirosol-5-en-

141

133 134 135 137

140

103


104

H. Ripperger

3p-amine and (25/?)-5a,22aiV-Spirosolan-3P-amine 141 4.2. Epiminocholestanes 142 4.2.1. (20/?)-, (205)-Verazine and Verazinine 142 4.2.2. Radpetine 143 4.2.3. Ebeietinone 143 4.2.4. Cordatine B 143 4.2.5. 3-0-(P-D-Glucopyranosyl)etioline, Havanine and Etiolinine 144 4.2.6. 3-0-(P-D-Glucopyranosyl)veramiline 145 4.2.7. Oblonginine 145 4.2.8. Puquietinone 146 4.2.9. Veramivirine 146 4.2.10. Teinemine, Muldamine, 22-lsoteinemine, Isocapsicastrine and Capsicastrine 147 4.2.11. Hapepunine Glycosides 148 4.2.12. Stenophylline B and its 3-O-P-D-Glucopyranoside 149 4.2.13. Vertaline B 149 4.2.14. Isosolacapine 150 4.2.15. 25-Isoetioline 150 4.2.16. 20,25-Bisisoetioline and 20-Isosolafloridine 150 4.2.17. (25/?)-22,26-Epimino-3p-(P-D-glucopyranosyloxy)-5a-cholest-22 (^-en-6-one and Cordatine A 151 4.2.18. Solaquidine 152 4.2.19. Deacetoxysolaphyllidine 152 4.2.20. Solanudine 153 4.2.21. Solacapine and Episolacapine 153 4.2.22. Pingbeinine and Pingbeininoside 154 4.2.23. Capsimine and its 3-(9-P-D-Glucoside 154 4.2.24. Solamaladine 155 4.2.25. (25/?)-23,26-Epimino-3p-hydroxy-5a-cholest-23(A0-ene-6,22dione, (20/?,25/?)-23,26-Epimino-3p-hydroxy-5a-cholest-23(A9-ene-6, 22-dione and their P-D-Glucopyranosides 155 4.2.26. Solaspiralidine 157 4.2.27. Veracintine and Rhamnoveracintine 157 4.3. Solanidanes 158 4.3.1. Solanidine Glycosides 158 4.3.2. 3-0-(P-Lycotriosyl)-, 3-0-(P-Lycotetraosyl)leptinidine and their 5a,6-Dihydro Derivatives 161 4.3.3. 22a//,25p//-Solanidane-3p,5a,6p-triol 162 4.3.4. Rubivirine 162 4.3.5. Solanogantamine (Solanopubamine), Isosolanogantamine, Solanopubamide A and B 162


Solanum Steroid Alkaloids - an Update

4.4. Solanocapsine Group 4.4.1. 3-Deamino-3P-hydroxysolanocapsine, Aculeamine and O-Methylsolanocapsine 4.4.2. Pimpifolidine, 22-Isopimpifolidine, Solanocardinol and 22,26Epimino-16p,23-epoxy-23a-ethoxy-5a,25a//-cholest-22( A^-ene-3 p, 20a-diol 4.4.3. 7p-Hydroxy-0-methylsolanocapsine 4.5. 3-Aminospirostanes 4.5.1. Antillaridine, Antillidine and Juripidine 4.6. Further Alkaloids 4.6.1. Abutiloside A and B 4.6.2. Korsiline, Petisine, Petisinine, Petisidine, Petisidinone, Petisidinine, Verdinine and Sevkorine 4.6.3. Valivine 4.6.4. Solanocastrine 4.6.5. 3P-[0-P-D-Glucopyranosyl-(l->4)-P-D-xylopyranosyloxy]-15, 16-seco-22a//,25p//-solanida-5,14-diene 5. SYNTHESES AND CHEMICAL TRANSFORMATIONS 6. BIOCHEMISTRY AND BIO ACTIVITIES 7. TABLES OF PHYSICAL CONSTANTS Alkamines (Table 4) Glycoalkaloids (Table 5) REFERENCES

105

163 163

163 165 165 165 166 166 166 167 167 167 167 172 173 173 176 179

1. INTRODUCTION The chemistry of Solanum steroid alkaloids and their occurrence in the plant kingdom have been reviewed in 1953 and 1960 by Prelog and Jeger in Volumes 3 [1] and 7 [2], in 1968 by Schreiber in Volume 10 [3] and 1981 by Ripperger and Schreiber in Volume 19 [4] of 77?^ Alkaloids (Academic Press). Since then there has been considerable progress in this field, especially concerning isolation procedures, e.g. the use of reverse-phase chromatography, and structure elucidation methods, e.g. the application of two-dimensional nuclear magnetic resonance spectroscopy. Interesting results have been obtained when the hitherto more or less neglected plant roots were studied. Our present purpose is to describe further advances in a short form and to critically update the earlier reviews. Solanum steroid alkaloids generally occur as glycosides, the aglycones of which possess the C27-carbon skeleton of cholestane and belong to the following five groups: the spirosolanes, e.g. solasodine (1); the epiminocholestanes, e.g. solacongestidine (2); the solanidanes, e.g. solanidine (3); the solanocapsine group, e.g. solanocapsine (4); the 3-aminospirostanes, e.g. jurubidine (5). These compounds occur in Solanaceae and some in Liliaceae. Alkaloids with Cnor-D-homo ring skeleton or other alterations of the cholestane ring system found in Liliaceae were not included.


H. Ripperger

106

1 Solasodine

2 Soiacongestidine

â&#x20AC;&#x201D; 27

3 Solanidine

4 Solanocapsine

5 Juaibidine


Solarium Steroid Alkaloids - an Update

107

The present review includes a survey of the occurrence of Solatium steroid alkaloids isolated since 1981 (Table 3) as well as of the physical constants of new aglycones (Table 4) and alkaloid glycosides (Table 5). These Tables are supplements to the corresponding compilations in previous Volumes of The Alkaloids (Academic Press) [3,4]. Recent papers have shown that Solarium species often contain complex mixtures of steroid alkaloid glycosides, which still present separation difficulties. Therefore, many of the older publications using less sophisticated technique are worth repeating.

2. GENERAL Progress in the isolation procedures rested essentially on the development of chromatographic techniques. Whereas normal-phase chromatography separated glycoalkaloids mainly according to their carbohydrate portions, retention of reverse-phase chromatography [5-8] strongly depended on the aglycones. This means that we have available two independent separation methods, which can be applied consecutively. Especially useful is high-performance liquid chromatography. Some selected methods are listed in Table 1. Even saturated and 5,6unsaturated compounds could be separated [9]. Table 2 indicates the dependence of the retention times of glycoalkaloids on the structure of the aglycones.

Table 1.

High Performance Liquid Chromatography of Solanum Steroid Glycoalkaloids and Aglycones

Column

Mobile phase

Ref

Zorbax NH2 "Carbohydrate Analysis" (alkylamine packing) Supelcosil LC-18-DB Alltech Cjg

Tetrahydrofuran-acetonitrile-water (11:5:4) Acetonitrile-water (pentanesulphonic acid) (83:17)

[10] [11]

Acetonitrile-methanol (ethanolamine) (3:2) Acetonitrile-water (sodium dodecyl sulphate, with H3PO4 to pH 2.3-2.5) (17:3) Resolve C18, Ultrasphere C18, Acetonitrile-ammonium phosphate buffer pH 3.5 Pecosphere CIS, Supelcosil C18 (7:13) or pH 2.5 (3:2) Nucleosil 125/8/4 RP-18 Acetonitrile-phosphate buffer pH 3.4 (27:73 to 33:67) Eurosil Bioselect C-8 Methanol-ammonium phosphate buffer pH 3 (3:2 or 1:1), acetonitrile-phosphate buffer pH 3 (1:3)

[9] [12] [13] [14] [7,15]


108

H. Ripperger

Table 2.

Relative Retention Times of Solanum Steroid dycoalkaloids (Relative to Solanine, Eurosil Bioselect C-8, Methanol-Ammonium Phosphate Buffer pH 3^)

Alkaloid type

Compound

RR^

23-Hydroxyspirosolanes

(235)-23-Hydroxyisoanguivine (235)-23-Hydroxyanguivine 3-0-(P-D-Glucopyranosyl)etiolme Solanine Demissine Chaconine Xyiosyt-p-solamarine Xylosylsolamar^ne Solasonine Isoanguivifie Robustine Solamargine Tomatine P|-Solamargine Anguivine AMiydroxyrobustine AMIydroxysolamargine 25-Acetoxyrobustine

0.51 0.55 1.00 1.00 1.00 1.11 1.29 1.35 1.46 1.58 1.69 1.71 1.80 1.81 1.82 3.27 3.58 6.70

22,26-Eptminocholestanes Solanidanes

Spirosolanes

AMIydroxyspirosolanes 25-Acetoxyspirosolanes

^Experimental conditions for analytical HPLC see ref. [7]. Especially useful for structure elucidation was the application of nuclear magnetic resonance methods, in particuhu* two-dimensional measurements. The complete ^H and ^^C spectral assignments have been reported for e.g. tomatidine, tomatine [16], solasodine [17] and many other alkaloids mentioned in Chapter 4. One-dimensional ^ C studies have been described for epiminocholestanes with a 4-keto function [18] and solanidanes [19]. Detailed reviews of NMR data of steroidal alkaloids are available [20,21]. These measurements now offer a potent nondegradative method for establishing the whole structure of a glycoalkaloid. But it should be mentioned that the structure elucidation of the carbohydrate moiety cannot be regarded as rigorous, if it is based only on the comparison of the C NMR signals with those of (substituted) monosaccharide units, because the chemical shifts not only depend on the sugar itself and the type of branching, but also on the neighboring sugar moieties; e.g. for C(l) of a terminal a-rhamnopyranose the values 6 100.3 (robustine [22]) or 102.9 (solamargine [7]), for C(5) of this moiety 6 69.1 (robustine [22]) or 70.5 (solamargine [7]), for C(l) of a terminal pxylopyranose 5 104.8 (tomatine [16]) or 107.5 (xylosylsdamargine [15]), for C(2) 5 74.6 (anguivine [23]) or 75.6 (xylosylsolamargine [15]) were detected. For C(4) of a 2,3-branched Pgalactopyranose 6 70.4 (solasonine [7]) or 71.0 (isoanguivine [23]) were found (all measurements in pyridme-â&#x201A;Ź/5). If, however, the signals of the whole carbohydrate ensemble are


Solanum Steroid Alkaloids - an Update

109.

known, then identical shifts of an unknown glycoside indicate identical oligosaccharide structure. Mass spectral methods suitable for polar compounds such as steroid alkaloid glycosides are fast atom bombardment (FAB) [24], liquid secondary ion (LSI), laser desorption/Fourier transform (LD/FT) [25] and electrospray ionization (ESI) mass spectrometry [14]. FAB or LSI mass spectra of glycoalkaloids exhibit strong [MH]"'", [aglycone â&#x20AC;˘\- H]^ and ions arising by sequential loss of sugars, whereas ESI mass spectra are described to display mainly [MH] and [aglycone + H] ions [14]. Electron impact (EI) mass spectra of aglycones display useful fragments for the different alkaloid types: m/z 138 and 114 for spirosolanes [26], m/z 125 for 22(A0-unsaturated 22,26-epiminocholestenes [27], m/z 98 for saturated 22,26epiminocholestanes [28,29], m/z 140 and 111 for 23,26-epiminocholest-23(A')-en-22-ones [30,31], m/z 204 and 150 for solanidanes [26], m/z 130, 112 and 84 for alkaloids of the solanocapsine type [32] and m/z 139 and 115 for 3-aminospirostanes [33,34]. Further mass spectrometric studies dealt with solanidane ^-oxides [35] or with the investigation of tomatine at the femtomole level by means of four-sector tandem mass spectrometry and scanning-array detection [36]. X-ray analysis has confirmed the structure of solasodine [37]. Further studies were mentioned in Chapter 4. The circular dichroism of some azomethines has been reported [38].

3. OCCURRENCE Table 3 compiles the distribution of alkamines with nonaltered C27-cholestane skeletons and their glycosides isolated since 1981 and represents a supplement of the corresponding Tables in former reviews [3,4].

Table 3. Occurrence QX Solanum Glycoalkaloids and Alkamines^ Plant species Solanum species (Solanaceae) S. abutiloides (Griseb.) Bitt. et Lillo

S. acaule Bitt. S, acaule Bitt. ssp. acaule S. acaule Bitt. ssp. punae

Alkaloid (aglycone)

Ref

Abutiloside A (16a-hydroxy-26-isobutyrylamino-5a-cholestan-22-one), solamargine (solasodine) Abutiloside B (26-acetylamino-16a-hydroxy5a-cholestan-22-one) (20-Isosolafioridine, solafloridine, solasodine) Solamargine (solasodine) (Demissidine, solanidine, tomatidine) (Demissidine)

[39]

(Demissidine, tomatidine)

[40] [41] [42] [43,44] [43] [43]


no

H. Ripperger

Table 3. Occurrence of Solanum Glycoalkaloids and Alkamlnes^ (cont.) Plant species

Alkaloid (aglycone)

Ref.

S, acaule Bitt. xS,x ajanhuiri Juz. et Buk.

Conunersonine (demissidine), sisuntne (tomatidine) (Demissidine, tomatidine) Conunersonine (demissidine), sisunine (tomatidine) Solasodine Solasodine (Solanidine) Solasodine (3-Deamino-3P-hydroxysolanocapsine, 25-isosolafloridine) (Aculeamine) Solasodine Conunersonine (demissidine), a-, P-solamarine (tomatidenol), tomatine (tomatidine) (Solanidine, tomatidenol) Chaconine, solanine (solanidine)

[45]

a-, P-Solamarine (tomatidenol)

짜6]

S, acaule Bitt. \S.x ajanhuiri Juz. et Buk. sisu S. accrescens Standi, et Mort. S. acerosum Sendt. S. achacachense Card. S, aculeastrum Dun. S. aculeatum St. Lag.

5. adoense Hochst. S, X ajanhuiri Juz. et Buk.

S. X ajanhuiri Juz. et Buk. ajawiri S, X ajanhuiri Juz. et Buk. yari S. alandiae Card. S. anguivi Lam. 1^. anomalum Thonn. S. antiiiarum 0. E. Schuiz iSl arboreum Humb. et Bonpl. ex Dun. 5. ctmezii Card. .S. asperum Vahl 5. aviculare Forst. f. 5. av/fe5/i Hawk, et Hjert. S.berihauitiiH9wk. S. boliviense Dun.

[43] [46] [47] [47] [43] [48] [49] [50] [51] [46]

[43] [46]

(Solanidine) [43] Anguivine, isoanguivine, solamargine [23] (solasodine) (Solasodine) [52,53] Antillaridine, antillidine [54] 3-0-(P-D-Glucopyranosyl)tomatidine, [55] 3-0-[0-P-D.xylopyranosyl-(l->6)-p.Dglucopyranosyl]tomatidine (tomatidine) (Tomatidine) [43] Solapamaine, solasodine [56] Solasodine [47,57] [58] Solamargine (solasodine), 5P-solasodan-3-one, solasodenone, solasodine (Solanidine) [43] [44] (Solanidine, solasodine) (Demissidine) [43] Conunersonine, demissine [46] (demissidine), (solanidine)


Solanum Steroid Alkaloids - an Update

Table 3. Occurrence of Solanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

Ref.

S. brachycarpum (Correll) Correll

Chaconine, solanine (solanidine), a-, p-solamarine (tomatidenol) (Solanidine, tomatidenol) Chaconine, solanine (solanidine) (Solanidine) Chaconine, solanine (solanidine) (Solanidine) Dehydrocommersonine, chaconine, solanine (solanidine), demissine (demissidine) (Solanidine) Demissine (demissidine), tomatine (tomatidine) (Solanidine) (25-Isoetioline, solasodine, tomatidenol) [(23ÂťS)-23 -Hydroxysolasodine, solasodine. tomatidenol] (20,25-Bisisoetioline) Capsicastrine (isoteinemine), etioline, isoteinemine Capsimine, isocapsicastrine (teinemine) 3 -0-(P-D-Glucosyl)capsimine (capsimine) (7p-Hydroxy-0-methylsolanocapsine) (Solanocastrine) Isosolacapine (Solanidine) Demissine (demissidine) Chaconine, solanine (solanidine), leptines I, II (23-0-acetylleptinidine), leptinines I, II (leptinidine) (Solasodine) (Tomatidenol, tomatidine) (Solasodine, tomatidenol) Isoanguivine, solamargine, solasonine, xylosylsolamargine (solasodine), xylosyl-P-solamarine (tomatidenol) Dehydrocommersonine (solanidine) (Demissidine, solanidine) (Demissidine, solanidine)

[59]

S. brevicaule Bitt. 5. bukasovii Juz. S. canaseme Hawk.

S. candolleanum Berth. S. canense Rydb.

S, capsicastrum Link

S. cardiophyllum Lindl. S. chacoense Bitt.

S. chloropetalon Schlecht. S, circaeifolium Bitt. S. cleistogamum Symon S. coccineum Jacq.

S. commersonii Dun. S, commersonii Dun. ssp. malmeanum S. cristalense Amsh.

(Isojurubidine, jurubidine, solasodine. tomatidenol)

[43] [60] [43,44] [60] [43,44] [60] [43,44] [42] [43] [52,61] [62] [41] [63] [64] [65] [66] [67] [68] [43] [60] [59]

[52,53] [43] [53] [15]

[59] [43] [43] [69]


112

H. Ripperger

Table 3. Occurrence of Solanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

Ref.

S. dasyphyllum Schum. et Thonn. S. decipiens Opiz 5. decurrens Wall. S, demissum Lindl.

Solamargine, solasonine (solasodine), solasodine, solanine (solanidine), tomatidenol (Solasodine) (Solasodine) Commersonine (demissidine), neotomatine (tomatidine) (Demissidine, solanidine, tomatidine) Soladulcine A, B (soladulcidine) Demissine (demissidine)

[70]

Solanelagnine (solasodine) Solamargine, solasonine (solasodine), solanine (solanidine) Chaconine, solanine (solanidine) (Solanidine) Chaconine, solanine (solanidine) (Solanidine) Solamargine, solasonine (solasodine) (Solasodine) (25-Isoetioline, solasodine, tomatidenol) [(235)-23-Hydroxysolasodine, 25-isoetioline, solasodine, tomatidenol] (20,25-Bisisoetioline) Solasodine Solanogantamine, isosolanogantamine (Solasodine) Solasodine Solanocapsine (Solanidine) (Solanidine)

[74] [75,76]

5. dulcamara L. S, ehrenbergii (Bin.) Rydb. ( = iSl cardtophyllum Lindl. ssp. ehrenbergii Bitt.) S, elaeagnifolium Cav. 5. erianthum D. Don. S. fendleri Grvy S. fendleri Gtdiy ssp. arizonicum S./!accidum Well S, fontanesianum Schrank S.fraxinifoliumDun.

.SI gayanum Phil. f. 5. giganteum Jacq. .S. ^7o Raddi iS. glaucophyllum Desf. iSl glaucum IXin. iSl gourlqyi Hawk. 5*. gourlayi Hawk. ssp. pachytrichum S havaneme Jzcq.

S. hqyesii Fernald iS. hazenii Britton S. hispidum Pers. 5. hjertingii Hawk. 5. hondelmanii Hawk, et Hjert.

Havanine (16-0-acetyletioline) Etiolinine (etioline), Ypsolamarine (tomatidenol) Solasodine Solasodine Juripidine, jurubidine Chaconine, solanine (solanidine) (Solanidine) (Solanidine)

[71] [71] [72] [43] [73] [60]

[59,60] [43] [60] [43] [77] [71] [52,61] [62] [41] [78] [79] [53] [80,81] [53] [43,44] [43] [82] [83] [47] [84] [85] [59] [43] [43]


Solanum Steroid Alkaloids - an Update

113

Table 3. Occurrence i^f Solanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

Ref.

S. hougasii Correll

Chaconine, solanine (solanidine) (Solanidine) (Solanidine) Deacetoxysolaphyllidine, deacetylsolaphyllidine Deacetoxysolaphyllidine (Solanidine)

[59] [43] [43] [86]

S, huancabambense Ochoa S. hypomalacophyllum Bitt.

S. incamayoense Okada et Clausen S, incanum L.

S, infundibuliforme Phil. S. intermedium Sendt. S. intrusum Soria S. jamaicense Mill. S. jamesii Torr. S. japonense Nakai

S. kurtzianum Bitt. et Wittm. S. laciniatum Ait. S. leptophyes Bitt.

S. luteum Mill. ssp. alaium Mill. S. lycopersicoides Dun. S. lyratum Thun.

S. macrocarpum (Maxim.) Koidz. S. maglia Schlecht.

Solamargine, solasonine (solasodine), solasodine Incanumine (solasodine) (Solanidine) Solasodine (Solasodine) Solasodine Tomatine (tomatidine) (Solanidine, tomatidine) 3-0-(p-Lycotetraosyl)solasodine, solamargine (solasodine), 3-0-(P-lycotetraosyl)soladulcidine (soladulcidine) Chaconine, solanine (solanidine) (Solanidine) Solaradinine, solaradixine, solashabanine (solasodine) Chaconine, dehydrocommersonine, solanine (solanidine), demissine (demissidine) (Solanidine) (Solanidine) (Solasodine) Tomatine (tomatidine) 3-0-(P-Lycotetraosyl)dihydroleptinidine, 3-0-(P-lycotriosyl)dihydroleptinidine (dihydroleptinidine), 3-0-(P-lycotetraosyl)leptinidine, 3-0-(P-lycotriosyl)leptinidine (leptinidine) Solasodine, tomatidine Chaconine, solanine (solanidine) (Solanidine)

[87] [43] [88] [89] [43] [47] [71] [47] [60] [43] [90]

[59] [43] [91] [60] [92] [43,44] [71] [93] [94,95]

[96] [60] [43]


114

H. Ripperger

Table 3. Occurrence of Solanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

5. medians Bin.

Chaconine, solanine (solanidine) [59] (Solanidine) [43] Commersonine, demissine (demissidine), [46] tomatine (tomatidine) (Solanidine, tomatidine) [43] Solamargine, solasonine (solasodine). [97] solasodine (Solanidine) [43] (Solanidine) [43] Chaconine, solanine (solanidine) [42] [43,44] (Solanidine) [60] Chaconine, solanine (solanidine), tomatine (tomatidine) (Tomatidenol, tomatidine) [43] [98] 3-0-(P-Lycotetraosyl)solanocardinol (solanocardinol) [43] (Solanidine) " 12P-Hydroxysolasodine" ^ ^-methylsolasodine, [99] solanocapsine, solasodine, tomatidenol [100] â&#x20AC;˘*23-0-Acetyl-12P-hydroxy-solasodine'* [101] Solanudine

S. megistacrolobum Bitt.

S, melongena L. var. esculentum Nees 51 microdontum Bitt. S. microdontum Bitt. ssp. gigantophyllum S. multidissectum Hawk. S, multi-interruptum Bitt. S. nqyaritense Rydb. S. neocardenasii Hawk, et Hjert. S. neorossii Hawk, et Hjert. S. nigrum L.

S. nudum Humb. et Bonpl. ex Dun. S. ochraceo-ferrugineum Femakl S. okadae Hawk, et Hjert. S olgae Pojark. S. oplocense Hawk. 51 paludosum Moric. 51 panduraeforme Dr^ge

51 papita Rydb. 5. pennellii Correll

51 pinnatisectum Dun. 51 platanifolium Sendt.

Solasodine (Solanidine) (Solasodine) (Solanidine) Solamargine (solasodine) Solasodine [Dihydroleptinidine, (23/?)-23-hydroxysoladulcidine, leptinidine, soladulcidine, solasodine, tomatidenol, tomatidine] (Solasodine, tomatidenol) (Solanidine) Tomatine (tomatidine) (Soladulcidine, solasodine, tomatidenol. tomatidine) Tomatine (tomatidine) (Tomatidine) Khasianine, ravifoline, solamargine, solasonine (solasodine)

Ref.

[47] [43] [71] [43,44] [102] [57] [95]

[103] [43] [104] [52] [59] [43] [105]


Solanum Steroid Alkaloids - an Update

115

Table 3. Occurrence oiSolanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

S. polytrichum Moric.

[59] Chaconine, solanine (solanidine) (Solanidine) [43] [79] Episolacapine, isosolacapine, O-methylsolanocapsine, solacapine [68] Isosolacapine [106] Solasodine [52,53] (Solasodine) [12] (Solasodine) [107] (Solanopubamine) [108] (Solanopubamide A, B) [52] (Soladulcidine, solasodine, tomatidine) [53] (Tomatidine) [60] Chaconine, solanine (solanidine) [43] (Solanidine) [46] Chaconine, solanine (solanidine) [71] (Solasodine) [22] 25-Acetoxyrobustine(25-acetoxysolasodine), A^-hydroxyrobustine(A'-hydroxysolasodine), robustine, pj-solamargine (solasodine) A^-Hydroxyrobustine, ^-hydroxysolamargine [7] (^-hydroxysolasodine), solamargine. solasonine (solasodine) (A/-Hydroxysolasodine, solasodine) [109] [47] Solasodine Solasonine (solasodine) [110] (Demissidine, tomatidine) [43] [46] Commersonine, demissine (demissidine). tomatine (tomatidine), (solanidine) (Solasodine) [111] Solamargine, solasonine (solasodine). [112] (solanidine, tomatidenol) [43] (Demissidine, solanidine) Demissidine, solanine (solanidine), tomatidine, [113] tomatine (tomatidine) (Soladulcidine) [114] [47] Solasodine Solasodine [115,116] (Tomatidenol) [43] [46] a-, P-Solamarine (tomatidenol)

S. pseudocapsicum L.

S. pseudolulo Heiser S. ptychanthum Dun. S. pubescem Willd. S. racemigerum Zodda S. raphanifolium Card, et Hawk.

S. retroflexum Dun. S. robustum Wendl.

S. rugosum Dun. S. sanctae-katharinae Dun. S. sanctae-rosae Hawk.

S. sanitwongsei Craib S. scabrum Mill. ssp. nigericum S. X semidemissum Juz. ex Buk. S. sepicula Dun. .S". shanesii F. Muell. S, siparunoides Ewan 51 sisymbrifolium Lam. S. sogarandinum Ochoa

Ref.


116

H. Ripperger

Table 3. Occurrence of Solanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

S. sparsipilum (Bitt.) Juz. et Buk.

Chaconine, solanine (solanidine) (Solanidine) (Solanidine) (Solanidine) (Etioline, 15a-hydroxytomatidenol, tomatidenol) 3-0-(P-D-Glucopyranosyl)etioline (etioline), etioline, solaspiralidine Chaconine, solanine (solanidine) (Solanidine, tomatidine)

S, spegazzinii Bitt. 5. spirale Roxb.

.SI stenotomum Juz. et Buk. S. stoloniferum Schlecht. et Bouch^ S. suaveolem Kutith et Bouch^

Solamargine, solasonine, xylosylsolamargine (solasodine) Solasodine S, subirerme Jacq. 5. sublohatum Willd. (Solasodine) (Solasodine) S, sycophanta Dun. Solasodine (Solanidine) S, tarijense Hawk. Commersonine, demissine (demissidine), S, toralapanum Card, et Hawk. dehydrocommersonine (solanidine) (Demissidine, solanidine) S, toxicarium Rich. Solaverine I, solaverine II (solaverol A) S, tridynamum Dun. Solasodine S, trilobatum L. P-Solamarine (tomatidenol) Solasodine S. triste Jacq. (25/?)-5a,22aA^Spirosolan-3p-amine, (25/{)-22a^-spirosol-5-en-3 P-amine (255)-22p^^Spirosol-5-en-3a-amine, (25/?)-22<x^-spirosol-5-en-3a-amine S. tuberosum L. Chaconine, solanine, p2~chaconine (solanidine) S. tuberosum L. x Lycopersicon Chaconine, solanine (solanidine), esculentum Mill, (somatic hybrids) tomatine (tomatidine) 51 tuberosum L. x 51 chacoense Chaconine, solanine (solanidine), a-, PBitt. solamarine (tomatidenol) 5. tuberosum L. ssp. andigena (Solanidine) (Juz. et Buk.) Hawk. (Solanidine, tomatidine) 5. tucumanense Griseb. Solanocapsine 5. umbelliferum Eschs. 0-Acetylsolasodine, 3-0-(p-Dglucopyranosyl)solasodine (solasodine), solasodine

Ref. [60] [92] [43,44] [43,44] [117] [118] [46] [43] [42] [47] [71] [52] [84] [43] [46] [43] [5] [119] [120] [121] [122] [123] [124] [125] [126] [92] [43] [127] [128]


Solanum Steroid Alkaloids - an Update

117

Table 3. Occurrence oiSolanum Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

S. unguiculatum Rich.

3-0-(0-a-L-Rhamnopyranosyl-(l-^2)-P-Dgalactopyranosyl)solasodine, 3 -O- {0-aL-rhamnopyranosyl-( 1 ->2)-0-[a-Lrhamnopyranosyl-( 1 -^4)]-P-Dgalactopyranosyl} solasodine, 3-0-(a-Lrhamnopyranosyl)solasodine (solasodine) Anguivine, isoanguivine, solamargine (solasodine), (235)-23-hydroxyanguivine, (23.S)-23-hydroxyisoanguivine (solaverol A) Solasodine (Solanidine) Solaverine I, solaverine II (solaverol A), solaverine III (solaverol B) (Solanidine, tomatidenol) (Solanidine, solasodine) [(22i?,25/?)-Solanid-5-en-3P-ol, solanidine, solasodine, tomatidenol] (Solanidine)

S. uporo Dun.

S. valdiviense Dun. S. venturii Hawk, et Hjert. S. verbascifolium L. S. vernei Bitt. et Wittm.

S. vernei Bitt. et Wittm. ssp. ballsii S. villosum Mill. ssp. alatum (Solasodine) (Moench) Edmonds (Solanidine) S. virgultorum (Bitt.) Card, et Hawk. S. zelenetzkii Pojark. (Solasodine) Lycopersicon species (Solanaceae) L esculentum Mill. iV-Nitrosotomatidine, tomatidine

L esculentum Mill. xL. hirsutum Humb. et Bonpl.

Ref [129]

[130]

[78] [43,44] [5] [43] [44] [131] [43] [71] [43] [71] [132] [8]

Lycoperoside A [(23i?)-23-acetoxytomatidine], lycoperoside B [(23.S)-23-acetoxysoladulcidine], lycoperoside C [(235,255)-23-acetoxy-5a,22aA^spirosolan-3p-ol], tomatine, y-tomatine (tomatidine) [133] (23*S)-23 - Acetoxy soladulcidine, (23iy,255)-23-acetoxy-5a,22otAr-spirosolan3P-ol, (23^)-23-acetoxytomatidine, 22,26-epimino-16p,23-epoxy-5a,22p/f,25a^cholestane-3 P,23a-diol, 22,26-epimino-16p,23-epoxy-23a-ethoxy5a,25a^-cholest-22(AO-ene-3P,20a-diol, 5a,22pA^-spirosolan-3-one, tomatidine


118

H. Ripperger

Table 3. Occurrence of Solanum dycoalkaloids and Aikamines^ (cont.) Plant species

Alkaloid (aglycone)

L hirsutum Humb. et Bonpl. ÂŁ glabratum (Mull.) Rick, Fobes et Tanks!. L pimpinellifolium Mill.

Tomatine (tomatidine)

[134]

(22-Isopimpifolidine, pimpifolidine, soladulcidine, tomatidine)

[135]

Solanidine, solanine (solanidine) (2a-Hydroxysoladulcidine, soladulcidine, solasodine)

[136] [137]

Other Solanaceae Capsicum annuum L. Lycianthes biflora (Lour.) Bitt. Liliaceae Fritillaria camtschatcemis (L.) Ker-Gawl. f. Fritillaria dela\fqyi Franch. Fritillaria ebeiensis G. D. Yu et G. Q. Ji var. purpurea G. D. Yu etP. Li Fritillaria maximowiczii Freyn

Fritillaria persica L.

Fritillaria puqiensis G. D. Yu et G. Y. Chen Fritillaria raddeana Rgl.

Ref.

[138] 3-0-{ Oa-L-Rhamnopyranosyl-(l->2)-0-[pD-glucopyranosyl-( 1 ->4)]-p-D-glucopyranosyl} solanidine (solanidine) 22a//,25p^-Solanidane-3p,5a,6p-triol [139] Ebeietinone [140]

3-0-(P-Cellobiosyl)hapepunine (hapepunine), [141] 3P-[0-P-D-Glucopyranosyl-(l->4).p-Dxylopyranosyloxy]-15,16-seco-22(x^,25p//solanida-5,14-diene (15,16-seco22a^/,25p/^.solanida-5,14-dien-3p.ol) (20/t257?)-23,26-Epiniino-3P-(p-D-glucopyra[142] nosyloxy)-5a-cholest-23(7V)-ene-6,22-dione, (25/?)-23,26-epiniino-3P-(P-D-glucopyranosyloxy)-5a-cholest-23(iV)-ene-6,22-dione, (20/?,25/?)-23,26-epimino-3P-hydroxy-5acholest-23(^-ene-6,22-dione, (25/?)-23,26-epimino-3p-hydroxy-5acholest-23(^ene-6,22-dione, (25/?)-22,26-epimino-3P-(P-D-glucopyranosyloxy)-5a-cholest-22(^-en-6-one Puqietinone [143,144] Petisine, petisinine Petisidine Petisidinone Radpetine Petisidinine Petilidine, petiline, petilinine. petisidine, petisine

[145] [146] [147] [148] [149] [150]


Solanum Steroid Alkaloids - an Update

119

Table 3. Occurrence of 5o/ii#iffm Glycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

Fritillaria thunbergii Miq. [=F. verticillataWxM. var. thunbergii (Miq.) Baker]

[151] P j -Chaconine, 3 -O- {O-a-L-rhamnopyranosyl (1 ->2)-0-[p-D-glucopyranosyl-( 1 ->4)]-p-Dglucopyranosyl} solanidine (solanidine), 3-0-{ 0-a-L-rhamnopyranosyl-( 1 ->2)-P-Dglucopyranosyl }hapepunine (hapepunine) [152] Pingbeinine, pingbeininoside (pingbeinine) [153] Valivine [154] Rhinolidine, rhinoline, rhinolinine (rhinolidine), solanidine [155] Solanidine [156] Korsiline [157] ^evkorine (sevkoridine) 3-0-{0-a-L-Rhamnopyranosyl-(1^2)-<9[6] [P-D-glucopyranosyl-(l-> 4)]-P-Dglucopyranosyl} solasodine, P|-solamargine (solasodine) Cordatine A, B [(25/?)-, (255)-22,26[158] epimino-5a-cholest-22(7V)-en-3P,6P-diol) PI-Chaconine, hyacinthoside. [159,160]

Fritillaria usuriensis Maxim. Fritillaria walujewi Rgl.

Korolkowia sewerzowi Rgl.

Lilium brownii Poit.var. colchesteri

Lilium cordatum (Thunb.) Koidz. Notholirion hyacinthirmm (Wils) Stapf

Rhinopetalum karelini Fisch. ex Sweet Rhinopetalum stenantherum Rgl. Veratrum album L. Veratrum califomicum Durand Veratrum escholtzii Gray Veratrum grandiflorum Loes.

neohyacinthoside, 3-0-{ Oa-L-rhamnopyranosyl(1 ->2)-0-[p-D-glucopyranosyl-( 1 ->4)]-P-Dglucopyranosyl} solanidine (solanidine) Rhinoline, rhinolinine (rhinolidine), solanidine p2-Chaconine, stenanthidine, stenanthine (solanidine) Veralobine Muldamine Solanidine Isorubijervine, isorubijervosine (isorubijervine), rubijervine Etioline, rubijervine

Ref.

[161] [162] [163] [164] [163] [ 163] [163]


120

H. Ripperger

Table 3. Occurrence of Solanum Giycoalkaloids and Alkamines^ (cont.) Plant species

Alkaloid (aglycone)

Veratrum lobelianum Bemh.

Rhamnoveracintine (veracintine) Isorubijervine, isorubijervosine (isorubijervine), veralosine Deacetylveralosine (etioline?), etioline. isorubijervine, rubijervine, solanidine, Y-solanine (solanidine), veralodinine. veralodisine, veralosidinine, veralosine. veralosinine, veramiline Verdinine Verazine, (20jR)-verazine Isorubijervine Verazine

Veratrum maackii Rgl. Veratrum nigrum L. Veratrum nigrum L. var. ussuriense Veratrum oblongum Loes. Veratrun oxysepalum Turc. Veratrum stenophyllum Diets Veratrum taliense Loes.

Veratrum viride Ait. Zygadenus sibiricus A. Gray

Ref. [165] [166] [163]

[167] [168] [169] [170]

Oblonginine [171] [163,172] Rubijervine [173] P|-Chaconine (solanidine), stenophylline D Etioline, stenophylline B [174] 3-0-(P-D-Glucopyranosyl)stenophylline B [175] (stenophylline B), 3-0-(P-D-glucopyranosyl)veramiline (veramiline) Stenophylline B, vertaline B [176] [177] Rubivirine, veramivirine [178] Verazinine (verazine)

^Isolated from plants since 1981; supplement of the corresponding Tables in former compilations [3,4]. Plant names according to Index Kewensis; some names have been corrected. ^ ^^C NMR data not identical with those of solanaviol [4].

4. STRUCTURES 4.1. Spirosolanes 4.1.1. Xylosyl-P-solamarine The ESI mass spectrum (positive ions) of the alkaloid suggested a glycoside structure. A peak at m/z 414, [C27H43NO2 + H]"^, was in agreement with the assumption of a spirosolenol as the aglycone. An intense [MH]"*" peak {m/z 1000) was observed. The *^C NMR spectrum indicated that tomatidenol was the aglycone. The ^^C signals of the oligosaccharide portion had the same values as that of xylosylsolamargine (23), indicating identical structures. In this way the


Solanum Steroid Alkaloids - an Update

121

Structure of (255)-3p-{0-P-D-xylopyranosyl-( 1 ->2)-0-a-L-rhamnopyranosyl-( 1 ->4)-0-[a-Lrhamnopyranosyl-(l-^2)]-P-D-glucopyranosyloxy}-22PAr-spirosol-5-ene (6) has been assigned to the alkaloid. This Solanum steroid alkaloid is remarkable because it contains an inner rhanmose moiety [15].

I HO.

4.1.2. Tomatidine Glycosides 3-0-(P'D-Glucopyranosyl)tomatidme. 3-0-(P-D-Glucopyranosyl)tomatidine was studied as the peracetyl derivative. The ^H and ^^C NMR data indicated that it was a steroidal glycoalkaloid containing 7V-acetyltomatidine as the aglycone and a single sugar, the ^H and ^"^C NMR shifts of which revealed that it was the tetraacetate of P-D-glucopyranose. Protons and carbons of the glucosyl moiety were assigned using ^H-*H COSY and HMQC spectra. Assignment of the resonances of the protons and carbons of the iV-acetyltomatidine aglycone was achieved by a combination of *H-^H COSY, HMQC and HMBC experiments. The high-resolution FAB mass spectrum indicated a molecular formula C43H^5NO|2. Further fragments confirmed the structure as the pentaacetate of 7 [55]. 3'0-fO-j3-D-Xylopyranosyl-(J -^)'^D'glucopyranosyl]tomatidine, Analogously, the structure of 3-(9-[0-P-D-xylopyranosyl-(lâ&#x20AC;&#x201D;>6)-P-D-glucopyranosyl]tomatidine was elucidated in the form of the peracetate. ^H and *^C NMR data indicated that the aglycone was A^acetyltomatidine. Hydrolysis afforded xylose and glucose. All the protons of both sugar units were identified from the TOCSY spectrum and then all their protons and carbons assigned with the aid of ^H-*H COSY and HMQC spectra. The l->6 connection was detected by the cross peak observed in the HMBC spectrum between C(l) of xylose and the H(6) protons of glucose. The data supported the structure as the heptaacetate of 8 [55].


122

H. Ripperger

y^Tomatine. y-Tomatinc (lycoperosidc D) showed a quasi-molecular ion pesk [MH]"*" at m/z 740 in the FAB mass spectrum (positive ions). The ^^C NMR data indicated the presence of one terminal p-glucopyranosyl and one P-galactopyranosyl moiety substituted at C(4). The aglycone moiety signals corresponded with those of tomatidine. Consequently, the structure was shown to be 9 [8]. Sisunine (Neoiomatine). Sisunine (neotomatine) gave tomatidine, galactose and glucose on hydrolysis. The structure was investigated using FAB mass spectrometry and permethylation followed by identification of the products of hydrolysis. Structure 10 was proposed on the basis of these results [45,72].

8 R=

y-Tomatine

9 R= ""

HO


Solanum Steroid Alkaloids - an Update

123

10 R = Sisunine (Neotomatine)

4.1.3. (255)-5a,22p7V-Spirosolan-3-one Structure 11 of this alkaloid was established by comparison of its *^C NMR signals with those of tomatidine (rings B - F) and 3-oxo-5a-steroids [133].

11 4.1.4. (23/{)-23-Acetoxytoinatidine and Lycoperoside A High-resolution mass spectrometry indicated the molecular formula C29H^'7N04 for (23/?)-23acetoxytomatidine. IR and NMR spectra suggested the presence of an acetoxy group. The EI mass spectrum exhibited characteristicfragmentsat m/z 196 and 172 instead ofm/z 138 and 114 from ring F unsubstituted spirosolanes indicating this group in ring F. The ^^C NMR signals of rings A - E were in good agreement with the corresponding data of tomatidine. The coupling constants in the % NMR spectrum proved an equatorial acetoxy group at C(23). In the NOE diflFerence spectrum, irradiation of H3(Ac) and H(23) gave positive enhancements of the signals for H(16) and H3(21), respectively, corroborating the 22pAr-configuration. Couplings between H(26e), H(26a) and H(25) indicated an equatorial methyl group at C(25) [(25S)-configuration]. Thus, the structure was determined to be 12 [133]. Lycoperoside A showed quasi-molecular ion peaks [MH]'*' and [M + Na]"*" in the FAB mass spectrum. The % and *^C NMR spectra were similar to those of tomatine, except for the carbon atoms assignable to an acetoxy group, C(16) and C(20) to C(27). The ^H NMR


124

H. Ripperger

spectrum was in accordance with an equatorial acetoxy group at C(23). Comparison of the ^-^C NNfR spectra of (23jR)-23-acetoxytomatidine (12) and lycoperoside A indicated structure 13 for the latter alkaloid [8].

12 R = H

(23R)-23-Acetoxytomatlcllne

13 R =

4.1.5. (2S5)-22P^-Spirosol-5-eii-3a-aiiiine High-resolution mass spectroscopy indicated the molecular formula C27H^N20. Fragments at m/z 138 and 114 proved a spirosolane structure; a fragment at m/z 56 suggested a 3-amino group. By comparison with NMR spectra of a 3P-aminospirosol-5-ene a 3a-configuration was assigned to the amino group. The chemical shifts of C(22), C(23) and C(24) corresponded with those of (255)-22PA^spirosolanes which proved the stereochemistry of ring F. Consequently, the structure was shown to be 14 [123].

H2N''


Solanum Steroid Alkaloids - an Update

125

4.1.6. Solasodine Glycosides 3'0-(a-L-Rhamnopyranosyl)solasodme. Acidic hydrolysis of the alkaloid gave solasodine and rhamnose. The FAB mass spectrum (positive ions) was described to show peaks at m/z 559 ([M]"^) and 413 ([aglycone]''"). On the basis of further NMR data structure 15 was proposed [129]. pj'Solamargine. Hydrolysis of Ppsolamargine produced solasodine, glucose and rhamnose. The LSI mass spectrum showed a quasi-molecular ion peak [MH] at m/z 722. The structure of the oligosaccharide moiety was deduced from the ^ C NMR spectrum. Thus, structure 16 was assigned. This was the first unequivocal identification of P|-solamargine [6]. 3'0'[0'a-L-Rhamnopyranosyl'(l ->2)'^D'galactopyranosy I Jsolasodine. Acidic hydrolysis of the alkaloid gave solasodine, galactose and rhamnose. In the FAB mass spectrum the [M]"^ ion was claimed to occur. On the basis of * C NMR data structure 17 was proposed without rigorous proof [ 129]. Anguivim. The LSI mass spectrum of anguivine suggested a glycoside structure. A peak at m/z 414, [C27H43NO2 + H]"^, was in agreement with the assumption of a spirosolenol as aglycone. An intense [MH] peak was observed. The *^C NMR spectrum indicated that solasodine was the aglycone. Structure 18 was proved by means of ^H-^H COSY, ROESY and HMBC spectra. All *H and *^C signals of the oligosaccharide portion were assigned. This Solanum steroid alkaloid is remarkable because it contains both L-rhamnose and D-xylose [23]. Isoanguivine. Analogously, structure 19 was established for isoanguivine [23]. Ravifoline. Aglycone NMR assigments for ravifoline (20) were established using homonuclear correlation techniques (COSY and TOCSY) for the ^H NMR signals, and HMQC was then used for the ^^C NMR signals. COSY, TOCSY and DQF COSY spectra were used to determine all the protons on the same sugar moiety. With the help of the HMQC data the corresponding carbon chemical shifts were assigned. From the H-^H coupling pattern the sugar residues were identified. The sugar sequence and the point of attachment of the oligosaccharide to the aglycone were recognizedfi-omthe NOESY spectrum [105]. 3'0-fO'a'L-Wiamnopyranosyl'(J'->2)-0-fa-L-rhamnopyranosyl'(J-H)J'/3-D'galactopyranosyl}solasodine. Hydrolysis of the glycoalkaloid produced solasodine, galactose and rhamnose. In the FAB mass spectrum the [MH] ion was recognized. On the basis of *^C NMR data structure 21 was proposed without rigorous proof [129]. 3'0'{0'a'L'Rhamnopyranosyl-(l'->2)'0-[P-D-glucopyranosyl'(l-^)]'P-D-glucopyranO' syljsolasodine. Acid hydrolysis of the alkaloid produced solasodine, glucose and rhamnose. The LSI mass spectrum showed a [MH] ion at m/z 884. Comparison of the ^ C NMR spectrum with NMR data of similar glycosides indicated structure 22 [6]. Xylosylsolamargine, The ESI mass spectrum of xylosylsolamargine suggested a glycoside structure. A peak at m/z 414, [C27H43NO2 + H]"*", was in agreement with the assumption of a spirosolenol as the aglycone. An intense [Nffl] peak {m/z 1000) was observed. The C NMR spectrum indicated that solasodine was the aglycone. All *H and *^C NMR signals of the oligosaccharide portion were assigned and the connections of the sugar components determined by means of ^H-^H DQF COSY, ROESY, HMQC and HMBC spectra establishing structure 23 [15].


126

H. Ripperger

15 R

16 R = Pi-Soiamargine

17 R =

18 R = Anguivine HO

OH


Solanum Steroid Alkaloids - an Update

127

HO HO

?H OH OH

19 R

HO

OH

Isoanguivine

HO HO^ HO-

^ HOA^

20 R =

O O

^째~H0

OH Ravifoline

OH OH

21 R =

HO

OH


128

H. Ripperger

.OH

H< HO,

—0

OH

OH 22 R =

0

^^^^~m

OH

OH

23 R -

""

HO

OH

HO^^

\

OH Xylosylsolamargine

24 R =

3-0-(P-Lycotetraosyl)solasodine

'


Solatium Steroid Alkaloids - an Update

129

HO

25 R =

OH

^lyjJLJ HO

un

HO

Robustine rMj

OH

26 R =

Solashabanine HO

OH

.OH

"^ii^

OH OH

27 R

HO Solaradinine

OH


130

H. Ripperger

3'0'(/3'Lycotetraosyl)sokisodine. 3-0-(P-Lycotetraosyi)so]asodine was isolated in a mixture with the 5a,6-dihydro derivative, soladuicine B. Methanolysis gave solasodine, soladulcidine, methyl a-D-glucopyranoside, methyl a-D-galactopyranoside and methyl P-D-xylopyranoside. Structure 24 was assigned by means of the ^''c NMR spectrum [90]. Robustine. Surprisingly, in the LSI mass spectrum (positive ions) of robustine the most abundant peak in the molecular ion region corresponded to the [M - H]'*' ion. Solasodine as the aglycone was identified by the ^^C NMR spectrum, the structure of the carbohydrate portion by ^H-^H COSY, TOCSY, ^H-^^C chemical shift correlation and ROESY spectra. The signals for all protons and carbon atoms have been assigned. The connections of the four sugar components were established by ROESY and HMBC spectra. An analogous investigation of the peracetyl derivative led to the same result. Structure 25 has characteristic features: (1) The presence of L-arabinose which has up to now only one time been detected in Solanum alkaloids, viz. in soladulcamarine, the structure of which has not been elucidated [3]. (2) The kind of branching of the oligosaccharide part with glucose directly bound to the aglycone and to three ftirther sugar residues [22]. Solashabanine and SolaracUnine. The number of glucose units contained in solashabanine and solaradinine [4] has been corrected. Their structures 26 and 27 were proposed on the basis of their ^^C NMR spectra, which were compared with those of solasonine, 3-gentiobiose and P-sophorose. Although tlus does not seem to be a rigorous proof, the structures were in accordance with the results of the action of P-glycosidase and Aspergillus japonicus: Solashabanine was converted to solasonine, solaradinine to solaradixine and solasonine [91]. Incanumine. Hydrolysis of incanumine, C^gHjgt^O^g, yielded solasodine, glucose, rhamnose and xylose. The proposed structure [89] left open doubts concerning the sequence of the sugars and their anomeric configurations. Solanelagnine. Hydrolysis of solanelagnine, C45H'^3NO|5, ftimished solasodine, glucose and rhanmose. The proposed structure [74] was notrigorouslyproved.

4.1.7. (25JQ-Sp,22a^^Spirosotan-3-one This alkaloid has been isolated as an antifungal stress metabolite together with (25/?)-22a^spirosol-4-en-3-one from leaves of Solanum aviculare L. Structure 28 was identified by comparison of the ^^C NMR spectrum with those of solasodine and appropriate steroidal model compounds [58].

28


131

Solanum Steroid Alkaloids - an Update

4.1.8. Soladulcidine Glycosides Soladulcine A and B Soladulcine A gave a quasi-molecular ion [MH]"^ at m/z 870 in the FAB mass spectrum. The *^C NMR spectrum exhibited forty-five signals, among which twenty-seven were assigned to the aglycone soladulcidine, while the rest could be assigned to the P-chacotriosyl moiety. Consequently, structure 29 was established for soladulcine A [73].

29 R

HO Soladulcine A

30 R

Soladulcine B

OH


132

H. Ripperger

Soladulcine B showed a quasi-molecular ion [MH]"^ at m/z 1034 in the FAB mass spectrum and fragment ion peaks at m/z 138 and 114 with EI mass spectrometry characteristic for spirosolanes. The ^^C NMR spectrum revealed fifty carbons, among which twenty-seven were attributable to soladulcidine, while the remainder were assignable to the P-lycotetraosyl moiety. Therefore, soladucine B was concluded to have structure 30 [73].

4.1.9.2a-HydrozysoUdulcidiiie This alkaloid has been isolated from the roots of Lycianthes biflora (Lour.) Bitt. The ÂŁ1 mass spectrum showed the diagnosticfragmentsof spirosolanes at m/z 138 and 114. The molecular ion indicated a saturated structure with two hydroxy groups. Comparison of the ^H NMR spectrum with those of soladulcidine and neogitogenin was in agreement with structure 31 [137].

31

4.1.10. 3-0-0-Solstriosyl)solaiiaviol

Structure 32 was assigned by the ^^C NMR spectrum. This alkaloid has been isolatedfromthe uncrushed berries of Solanum nigrum L., inunersed in cold methanol for two years [179].

3~0-(P-Solatriosyl)sotanaviol


Solatium Steroid Alkaloids - an Update

133

4.1.11. (235)-23-Acetoxysoladulcidine, (23*^,255)-23-Acetoxy-5<x,22a/V-spirosolan-3p-ol, Lycoperoside B and C The molecular formula of (23.S)-23-acetoxysoladulcidine was determined to be C29H4'7N04 by high-resolution mass spectrometry. The fragmentation in the EI mass spectrum was very similar to that of (23i?)-23-acetoxytomatidine suggesting an isomeric structure. Coupling constants in the *H NMR spectrum indicated equatorial 23-acetoxy and 25-methyl groups. In the NOE difference spectra, irradiation at the signal of 113(18) gave a positive enhancement for the singlet of H3(Ac), irradiation at the signal of H(16) gave a positive enhancement for the double doublet of H(26a) and irradiation at the signal of H(23) gave a positive enhancement for the double quartet of H(20). These data suggested the 22aN-configuration. Thus, the structure was determined to be 33 [133]. Lycoperoside B showed quasi-molecular ion peaks [MH] at m/z 1092 and [M + Na]"*" at m/z 1114 in the FAB mass spectrum. The ^â&#x20AC;˘'C NMR spectrum was similar to that of tomatine except for the C(20) to C(27) signals, which were superimposable on those of (235)-23acetoxysoladulcidine (33). Accordingly, the alkaloid has structure 34 [8].

33

R=H

Lycoperoside B The molecular formula of (235,255)-23-acetoxy-5a,22cxN-spirosolan-3p-ol was determined to be C29H4yN04 by high-resolution mass spectrometry. The fragmentation in the EI mass spectrum was very similar to that of (23i?)-23-acetoxytomatidine suggesting an isomeric structure. Coupling constants in the *H NMR spectrum indicated an axial 25-methyl group. In


J34

H. Ripperger

the NOE difference spectrum, irradiation at the signal of H(23) gave a positive enhancement for the singlet of H3(27). Hence, structure 35 was proposed for this alkaloid [133]. The FAB mass spectrum (positive ions) of lycoperoside C suggested it to be an isomer of 34. Coupling constants in the ^H NMR spectrum indicated an equatorial 23-acetoxy and an axial 25-methyl group. By comparison of the chemical shift of C(20) with those of the corresponding signals of lycoperoside A and B the 22aM<x)nfiguration was determined. The ^^C NMR spectrum was similar to that of tomatine except for the C(20) to C(27) signals which were superimposable on those of 35. Consequently, structure 36 was proposed for lycoperoside C [8].

36

R

Lycoperoside 0

4.1.12. (23i?)-23-Hydroxy8oladulcidine The elemental composition of (23/?)-23-hydroxysoladulcidine was shown to be C27H45NO3 by high-resohition mass spectrometry. The diagnostic fragments of ring E and F at m/z 138 and 114 were absent. Instead, an ion at m/z 154 (138 + 16) could be recognized, indicating the presence of a hydroxy group in ring E or F. The base peak at m/z 387 was presumed to have structure 39, formed via 38. This interpretation limited the position of one hydroxy group to C(23) or C(24). The ^H spin system of ring F was recognized from the ^H-% COSY NMR


Solatium Steroid Alkaloids - an Update

135

spectrum and a 2D HOHAHA experiment. This proved a 23-hydroxy group, the axial conformation of which followed from the coupling detected in the ^H NMR spectrum. The '^C NMR spectrum was in accordance with structure 37. All carbon and proton signals could be unequivocally assigned from a *H-*-^C one-bond heteronuclear shift correlation NMR spectrum and the information extracted from the *H-*H COSY and the *-^C APT spectrum, as well as the known shifts of the '"'c signals of soladulcidine. The C(26) signal agreed with a (25/?)- and not with a (255)-configuration. y-Gauche interactions were observed between the 23-hydroxy group and C(20) as well as C(25) [95].

4.1.13. (23^-23-Hydroxysolaso(iine [Solaverol A] and its Glycosides (23S)'23'Hydroxysolasodme (Solaverol A), The elemental composition of (23.S)-23hydroxysolasodine (solaverol A, 40) was shown to be C27H^3N03 by high-resolution mass spectrometry. The fragmentation pattern corresponded with that of (23/?)-23hydroxysoladulcidine (37), indicating a 23- or 24-hydroxy group. The proton coupling recognized in the NMR spectrum was in accordance with an equatorial 23-hydroxy group. From the chemical shift of C(26) the 22(x^-configuration was recognized. The C(20) signal showed a remarkable upfield shift compared with the corresponding signal of solasodine explained by ygauche interaction between C(20) and the 23-hydroxy group [5,62]. Structure 40 was confirmed by catalytic hydrogenation of 40 to the known tetrahydro derivative (22/?,235',25i?)22,26-epimino-5a-cholestane-33,16p,23-triol (45) [62]. Solaverine I. Solaverine I (41) was hydrolyzed to yield solaverol A (40). The FAB mass spectrum (negative ions) showed a [M - H ] ' ion at m/z 882. The *^C NMR signals were compared with those of solamargine and indicated a p-chacotriosyl moiety [5].


136

H. Ripperger

40

41

R=

42

R=

43

R=

R=H

Solaverol A

Solaverine I

Solaverine II

HO

OH


Solatium Steroid Alkaloids - an Update

137

H OH

44

R=

HO

OH

Solaverine 11. The staicture of solaverine II (42) was elucidated analogously. Comparison of the ^-^C NMR signals of the sugar part with those of solasonine indicated a P-solatriosyl residue [5]. (23S)-23-Hydroxyanguivme cmd (23S)'23'Hydroxyisoanguivme. The structures of (235)-2313r hydroxyanguivine (43) and (23iS)-23-hydroxyisoanguivine (44) were assigned by their ^â&#x20AC;˘^C NMR spectra. The aglycone signals corresponded with those of (235)-23-hydroxysolasodine (40), when the shifts caused by 3-0-glycosidation were considered, and the sugar signals with those of anguivine (18) and isoanguivine (19) [130].

4.1.14. 25-Acetoxyrobustine In the ^H and ^^C NMR spectra of 25-acetoxyrobustine (46) an acetoxy group could be recognized. The H3(27) signal was a singlet localizing the acetoxy group at C(25). The 20position was excluded by the ^H-^H COSY spectrum. The 22a/V-configuration followed from the chemical shift of C(23). Because of an equilibrium with the corresponding azomethine [double bond N=C(22)] spirosolanes always adopt a thermodynamically preferred structure of ring F. As an equatorial methyl group is unequivocally preferred to an equatorial acetoxy group, for 46 a (255)-configuration had to be expected. The *^C NMR signals of the oligosaccharide portions of 25-acetoxyrobustine and robustine (25) had the same values indicating identical oligosaccharide structures [22].


138

HO 46

OH

25-Acetoxyrobustln6

4.1.15. Solaparnaine The mass spectrum of solapamaine (47) showed, in addition to the molecular ion at m/z 429, two characteristic spirosolanefragmentsat m/z 154 (138 + 16) and 130 (114 + 16) indicating a hydroxy group inringF. The *^C NMR spectrum was in agreement with structure 47 [56].

.

HN-

47

Solapamaine

4.1.1d 7V-Hydroxysolasodine and its Glycosides N'Hydroxysolasodine. The elemental composition of A^hydroxysolasodine (48) was C27H43NO3 according to high-resolution mass spectrometry. The diagnostic fragments of spirosolanes were shifted to m/z 154 (138 -f 16) and 130 (114 + 16) indicating a hydroxy group in ring F. Catalytic hydrogenation gave the known (225,25/?)-22,26-epimino-5a-cholestane3p,16P-diol (51). The elimination of the hydroxy group inringF during this reaction limited its position to N or C(26). NMR investigations were in agreement with structure 48. Surprisingly, the ^''c NMR spectrum indicated an equilibrium between 48 and the nitrone 52 in CDCI3. In pyridine or in CDCl3/acetic acid the nitrone was absent. The structure was confirmed by


Solatium Steroid Alkaloids - an Update

139

synthesis [109,180]. N-Hydroxysolamargim and N-Hydroxyrohustim, The structure of A^-hydroxysolamargine (49) was assigned by comparison of its ^'^C NMR spectrum with those of 7^-hydroxysolasodine (48) and solamargine [7], the structure of iV-hydroxyrobustine (50) by an analogous comparison with A^-hydroxysolasodine (48) and robustine (25) [22].

s HON-

49

50

R=

HO A/-Hydroxysolamargine

OH

HO

OH

R=

A/-Hydroxyrobustlne


H. Ripperger

140

51

52

4.1.17.12p,27-DihydrosysoUsodiiie, its 3-O-P-Chacotrioside and (25iI)-12p-Hydroxy22aAr-spirosol-5-en-27-oic Acid Mass and '^C NMR spectra of 12P,27-dihydroxysolasodine were in accord with stmcture S3. The '^C NMR spectnim of its glycoside 55 indicated the P-chacotriosyt moiety [179].

Rio 53 R1 = H, R2 = CH2OH 54 Rl = H. R2 = CO2H

55 R1 =

HO R2 = CH2OH

OH


Solatium Steroid Alkaloids - an Update

141

(25i?)-12P-Hydroxy-22a^-spirosol-5-en-27-oic acid (54) showed absorption due to the carboxylate in the IR spectrum and peaks due to [M]"^ and m/z 168 (138 -â&#x20AC;˘- 30) in the mass spectrum. Comparative studies of the ^H and *^C NMR spectra with those of solasodine (1) and its 12p-hydroxy derivative revealed structure 54 [179]. The alkaloids 53, 54 and 55 have been isolated from the uncrushed berries of Solanum nigrum L., immersed in cold methanol for two years [179].

4.1.18. Solaverine DDE Solaverine III (56) showed a peak due to [M - H]" in the FAB mass spectrum (negative ions). The ^-^C NMR spectrum displayed a signal due to one additional oxygenated carbon compared with that of solaverine I (41). This signal was attributed to C(27) since the ^H NMR spectrum showed no signal for a 25-methyl group and the ^-^C NMR signals ascribed to C(24), C(25) and C(26) were shifted by -4.9, +9.3 and -3.5, respectively. The ^-^C NMR sugar signals of solaverine I and III were almost identical [5].

56

Solaverine III

4.1.19. (25/{)-22(x/V-Spirosol-5-en-3a-aiiiine, (25/{)-22(x^-Spirosol-5-eii-3p-aiiiine and (25/{)-5a,22aiV-Spirosolan-3P-aiiiine High-resolution mass spectroscopy of (25/?)-22otAr-spirosol-5-en-3a-amine indicated the molecular formula C27H44N2O. Fragments at m/z 138 and 114 proved a spirosolane structure, a fragment at m/z 56 suggested a 3-amino group. H and *^C NMR spectra were in accordance with structure 57 [123]. The complete ^H and *"^C NMR assignment has been described for (25/?)-22ocA^-spirosol-5en-3P-amine (58) and (25/?)-5a,22aA^-spirosolan-3P-amine (59) by using ^H-^H COSY, HMQC, HMBC and NOESY experiments in agreement with the indicated structures [122]. (25jR)-5a,22GLAr-Spirosolan-3|3-amine has already been synthesized [181].


142

H. Ripperger

67 R = a-NH2.A5 68 R = P-NH2, A6 69 R = P-NH2, 6aH

4.2. Epiminocholestanes 4.2.1. (20JR)-, (2a5)-Verazinc and Verazinine A mixture of (20/?)- (60) and (205)-verazine has been isolated from Veratrum maackii Rgl. The structure was determined based on mass spectrocopic data, where a molecular ion at m/z 397 and a base peak at m/z 125 [22(^unsaturated 22,26-epiminocholestene] was observed. A complete and unambiguous assignment of the ^H and ^''C NMR parameters of both alkaloids was provided based mainly on the use of homo- and heteronuclear two-dimensional chemical shift correlation spectroscopy. From the experiment using proton detection the patterns of proton multiplets, and qualitatively, also the magnitudes of homonudear coupling constants could be derived. The ^^C NMR signals of rings C and D and the side chain for both compounds showed the largest deviations. Hence, they should differ in the configurations of C(17) or C(20). The ^-configurations of the side chains were concluded from the NOE between H3(18) and H(20). The coupling constants â&#x20AC;˘%(17)H(20) ^^ ^^^^ alkaloids indicated considerable populations with antiperiplanar positions or'H(17) and H(20). The assignment of the configurations at C(20) was derived from a cross peak in the NOESY spectrum between H(12P) and H3(21) in one stereoisomer, which therefore should have the (20/5)-configuration [168].

60

(20R)-Verazine


Solarium Steroid Alkaloids - an Update

143

Hydrolysis of verazinine yielded verazine and glucose. Therefore, structure 61 was proposed, but the ring size of glucose was not studied [178].

61

Verazinine

4.2.2. Radpetine Structure 62 was established by X-ray analysis of its hydrochloride [148].

62

Radpetine

4.2.3. Ebeietinone Structure 63 was established by some preliminary spectroscopic studies and finally by X-ray analysis [140].

4.2.4. Cordatine B In the FAB mass spectrum the highest peak at m/z 600 was ascribed to the ion [M + Na]^. Acid hydrolysis yielded the aglycone and glucose. The elemental composition of the aglycone was shown to be C27H45NO2 by high-resolution mass spectrometry. A peak at m/z 125 indicated a


144

H. Ripperger

64

Cordatine B

22(7y)-unsaturated 22,26-q)iininocholestene structure. On the basis of ^H and ^"^C NMR data and a negative Cotton effect of the aglycone at 255 nm as well as of the ^H NMR spectrum of the triacetyl derivative of the aglycone structure 64 was assigned to cordatine B [158].

4.2.5.3-0-(P-D-Glucopyranosyl)etioline, Havanine and Etiolinine

65 R = H 66 R = Ac

67

Havanine

Etiolinine

The glycoside 65 had the empirical formula C33H53N0'7 according to high-resolution mass spectrometry. Its characteristic fragment at m/z 125 was in accordance with a 22(^unsaturated 22,26-epiminocholestene structure. The [M - C^HnO^]"^ ion indicated the presence of a hexose. Structure 65 was proved by comparison with the ^â&#x20AC;˘'c NMR data [C(l) to C(10), sugar


Solanum Steroid Alkaloids - an Update

145

signals] of its 16-0-acetyl derivative havanine (66) and with the values of etioline [C(ll) to C(27)]. The ^H NMR signals of the sugar portion were assigned by ^H-^H DQF COSY measurements. The coupling constants were in agreement with a glucopyranose moiety [118]. Alkaloid 65 is possibly identical with deacetylveralosine [163], the aglycone of which, veralosidine, corresponded with etioline in neariy all properties [4]. The hexopyranose structure of deacetylveralosine was not proved. In the FAB mass spectrum (positive mode) of havanine (66) an ion at m/z 618, [MH]"*", and in the EI spectrum a fragment at m/z 125 typical for a 22(7V)-unsaturated 22,26-epiminocholestene structure were observed. Comparison of the *^C NMR spectrum with those of 3-0-(p-Dglucopyranosyl)cholesterol and (9,(9-diacetyl-25-isosolafloridine indicated structure 66 [82]. Havanine is very probably identical with veralosine [163]. Hydrolysis of etiolinine (67) yielded etioline and glucose. Permethyletiolinine on hydrolysis gave 2,3,4,6-tetra-O-methylglucose and 2,3,6-tri-O-methylglucose in accordance with structure 67. The p-configurations of the glucose moieties followed from the coupling constants Jjj/jx jj/2) ^^^ position of the sugar chain with respect to the aglycone was not proved [83].

4.2.6. 3-0-0-D-Glucopyranosyl)veramiline Acidic hydrolysis yielded veramiline [4] and glucose. The ^-^C NMR spectrum indicated a 3-0P-D-glucopyranosyl residue establishing structure 68. The formula given in ref [175] does not correspond with a veramiline glycoside.

68

3-0-(P-D-Glucopyranosyl)veramiline

4.2.7. Oblonginine Structure 69 was assigned, especially by analysis of the ^H-% COSY, ^H-^^C COSY, ^H-^^C long-range COSY and NOE spectra. The new alkaloid was shown to be identical with a compound obtained by degradation of solanidine [171].


146

H. Ri^erger

69

Obtonginine

70

Puqietinone

4.2.8. Puqietinone X-ray analysis revealed structure 70 [144]. The previous assignments of the configurations at C(22) and C(25) [143] were incorrect.

4.2.9. Veramivirine Thermospray liquid chromatography mass spectrometry displayed a [MH]'^ ion at m/z 416 suggesting the molecular formula C27H45NO2. The ^H and ^^C NMR data showed a close resemblance to those of veramiline and its 3-0-P-D-glucopyranoside 68. These data together with HMQC, HMBC, ^H-^H COSY, TOCSY and NOESY experiments were in accordance with structure 71 [177].

71 Veramivirine


Solarium Steroid Alkaloids - an Update

147

4.2.10. Teineitiine, Muldamine, 22-Isoteineinine, Isocapsicastrine and Capsicastrine Deacetylation of muldamine (73) gave teinemine, for which structure 72 was established by comparison of its C NMR spectrum with the data of a wide variety of 22,26-epiminocholestanes. An X-ray analysis confirmed this structure [164] which was identical with one proposed eariier [4] for isoteinemine. Structures 72 and 74 for teinemine and 22-isoteinemine respectively, were confirmed by synthesis (Chapter 5). The position of the Oacetyl group in muldamine (73) was determined by inspection of '^C NMR data [164].

72 R = H 73 R = Ac

Teinemine Muldamine

75

76

74

22-lsoteinemine

Isocapsicastrine

Capsicastrine


148

H. Ripperger

Acidic hydrolysis of isocapsicastrine (75) gave teinemine (72) and glucose. The structure was assigned on the basis of EI mass and NMR spectroscopy. The *^C NMR data were compared with those of teinemine (72) and havanine (66) [64]. Acidic hydrolysis of capsicastrine (76) yielded isoteinemine (74) and galactose. The structure was assigned usii^ mass and NMR spectra. Especially the comparison of the ^-^C NMR data with those of 3-0-(P-D-galactopyranosyl)cholesterol and isoteinemine proved structure 76 [63].

4.2.11. Hapepunine Glycosides 3'0'(fi-Cellobiosyi)hcf}epunine. Hydrolysis yielded hapepunine [4]. Structure 77 was proposed on the basis of FAB and EI mass as well as NMR spectrometry, but not rigorously proved [141].

3-0-(p-Celiobiosyl)hapepunine

77 R

78

R

HO

OH

S-0'[0'a'L'RhamrK}pyranosyl'(l->2)'P'D'glucopyranosyl]hapepunm^ The field desorption mass spectrum showed peaks for [M -Âťâ&#x20AC;˘ Na]"*", [MH]"*", [M + Na - 146]"^, [M + H - 146]"^ and


Solatium Steroid Alkaloids - an Update

149

[M + H - 308]"^, indicating that the glycoalkaloid had a methylpentosyhexose moiety linked to an aglycone of molecular weight 429. Elemental analysis and these data gave the molecular formula C4QHg'7NOj2. Incubation with hesperidinase afforded hapepunine [4], glucose and rhamnose. Comparison of the ^-^C NMR spectrum with that of Pj-chaconine (102) proved structure 78 [151].

4.2.12. Stenophylline B and its 3>(7-P-D-Glucopyranoside Stenophylline B was isolated from Veratrum species [174,176].

79

3-0-(P-D-Glucopyranosyl)stenophylline B

Acidic hydrolysis of 3-0-(P-D-glucopyranosyl)stenophylline B yielded stenophylline B and glucose. Spectroscopic studies of the glycoside, especially comparison with 3-0-(P-Dglucopyranosyl)veramiline (68), were claimed to indicate structure 79 (position of the steroid side chain corrected by the present author) [175], but the formula given in ref [175] for 3-0-(pD-glucopyranosyI)veramiline is incorrect.

4.2.13. Veitaline B Structure 80 was determined by X-ray analysis [176].

> \ J\â&#x20AC;&#x201D;'

80

Vertaljne B


150

H. Ripperger

4,2.14. Isosolacapine For isosolacapine a (25/?)-configuration was proposed mainly on the basis of NMR analysis [79] and later because of its conversion to solanogantamine [68]. But the configuration of solanogantamine at C(25) was later corrected [95]. Therefore, isosolacapine should have structure 81.

H2N 81

Isosolacapine

4.2.15.25-Isoetioline The base peak in the mass spectrum at m/z 125 indicated the structure of a 22(^-unsaturated 22,26-epiminocholestene. The molecular peak suggested a further double bond. The ^H NMR spectrum of the alkaloid and its comparison with that of solafloridine (5a,6-dihydro-25isoetioline) were in accordance with structure 82. As expected, catalytic hydrogenation gave the known (21^,25/;)-22,26-epimino-5a-cholestane-3P,16a-diol (83) [61].

82

25-lsoetiotine

83

4.2.16.20,25-Bbisoetioline and 20-Isosolafloridine The ^^C NMR spectrum of 20,25-bisisoetioline showed that its structure differed fi-om that of 20-isosolafloridine (85) by a A^-double bond and, therefore, the alkaloid had structure 84 [41]. The elemental composition of 20-isosolafloridine (85) was shown to be C27H43NO2 by high-resolution mass spectrometry. The diagnosticfi-agmentat m/z 125 was in accordance with


Solatium Steroid Alkaloids - an Update

151

a 22(^-unsaturated 22,26-epiminocholestene structure. The positive circular dichroism at 246 nm displayed the (257?)-configuration. The C NMR signals of 20-isosolafloridine and the known solafloridine were assigned by comparison with literature data, and the H NMR signals by HMQC, ^H-^H DQF COSY and NOESY measurements. From the NMR spectra could be concluded that both structures disagreed in the configurations at C(16), C(17) and/or C(20). Especially comparisons of NOEs of both alkaloids indicated the unusual (20/?)-configuration of isosolafloridine (85) [41].

84 A 5 85 5aH

20,25-Blsisoetioline 20-lsosolafloridine

Experiments showed that 84 and 85 were not formed from the corresonding 20-isomers during the isolation procedure (acidic hydrolysis) [41].

4.2.17. (25/?)-22,26-Epiinino-3P-0-D-glucopyranosyloxy>-5a-cholest-22(7V)-eii-6-one and Cordatine A High-resolution mass spectrometry of (257?)-22,26-epimino-3P-(P-D-glucopyranosyloxy)-5acholest-22(^-en-6-one indicated that the molecular formula was C33H53N0'7. A prominent peak at m/z 125 was in agreement with a 22(A0-unsaturated 22,26-epiminocholestene structure. Acidic hydrolysis yielded glucose. The ^H and *^C NMR spectrum proved a P-Dglucopyranosyl unit. The ^^C NMR signals of the rings A and B of the aglycone corresponded to those of 3P-hydroxy-6-oxo-5a-steroids. A positive Cotton effect at 255 nm due to the azomethine group indicated a (25/?)-configuration [182]. Thus, the structure was elucidated as 86 [142], but it should be mentioned that the Cotton effect was relatively small and a (25/?)configuration is unusual for a steroidal alkaloid from Liliaceae. The FAB mass spectrum of cordatine A (87) gave peaks at m/z 600 ([M + Na]"*") and 578 ([MH] ). Acidic hydrolysis gave glucose and an aglycone, the high-resolution mass spectrum of which indicated the molecular formula C27H45NO2. A fragment at m/z 125 was typical for a 22(JV)-unsaturated 22,26-epiminocholestene structure. NMR comparisons with solacongestidine and 5a-androstane-3p,6P-diol as well as the positive Cotton effect of the aglycone at about 255 nm was in accordance with structure 87 [158],


152

H. Ripperger

86

R1+R2 = 0

87

R1 = OH. R 2 = H

Cordatine A

4.2.18. Solaquidine A 22,26-epiinino-3,3-dimethoxycholestane structure was proposed for solaquidine, however, the absolute configurations at C(5), C(22) and C(25) remained undetermined [4]. (22S,25Ry 22,26-acetylepimino-5a-cholestan-3-one, a derivative of solaquidine, was synthesized from solasodine indicating the complete stereochemistry of this alkaloid to be 88 [183].

88 Solaquidine

MeO OMe

4.2.19. Deacetoxysolaphyilidine On the basis of the IR, the ^H NMR and the mass spectrum of this alkaloid and its N,0,0'triacetyl derivative structure 89 was proposed [86], which was confirmed by X-ray analysis [87].


Solanum Steroid Alkaloids - an Update

153

HN HO

OH 89

Deacetoxysotaphyliidine

90 Solanudine

4.2.20. Solanudine No molecular ion peak was observed in the mass spectrum. The base peak at m/z 114 (98 -i- 16) indicated a hydroxy-methyl-piperidine side chain. IR and UV absorption were in accordance with an enone containing an exocyclic double bond and a hydroxy group at the a-carbon. The *H NMR spectrum did not show any olefinic protons. The ^â&#x20AC;˘'C NMR spectrum exhibited a carbonyl and two olefinic carbons. The other *-^C NMR signals agreed quite well with those found for deacetoxysolaphyllidine (89) in accordance with the stereochemistry given in formula 90 for solanudine. The mass spectrum of the ^,0,0-triacetyl derivative showed the molecular ion at m/z 555 (C33H49NO6) [101].

4.2.21. Solacapine and Episolacapine Reduction of the masked carbonyl group of solanocapsine (4) with sodium borohydride yielded solacapine as the major and episolacapine as the minor product. Coupling of H(23) recognized in the *H NMR spectra proved equatorial or axial conformations of the 23-hydroxy groups. Both these observations indicated structures 91 and 92 for both alkaloids, respectively. Chemical reactions (iV-methylation, 0-acetylation), mass spectrometry, circular dichroism of the 3-A^-salicylidene derivatives, and *H and ^^C NMR spectra were in accordance with these structures [79].


154

H. Ripperger

91 R1 = OH. R 2 = H 92 R1 = H, R 2 = OH

Sotacapine Episolacapine

4.2.22. Pingbeinine and Pingbeininoside The molecular formula of pingbeinine (93), C2gH4'7N03, was obtained by high-resolution mass spectrometry (measurement of the [M - H]'*' peak). The base peak at m/z 128.1064 corresponding with C'^Hj^NO was ascribed to the hydroxydimethylpiperidine fragment. The methyl groups in ring F were localized by singlets in the ^H NMR spectrum at 5 1.31 and 2.35 assigned to C(OH)Me and NMe. A 16P-hydroxy group and a (22/{)-configuration were assumed by ^^C NMR comparison with literature values. NOE difference spectral studies established the configuration at C(25): On irradiation of the H3(27) methyl resonance enhancements of the H(23a), H(24e) and H(26e) signals were observed [152]. Hydrolysis of pingbeininoside afforded pingbeinine (93) and glucose. On the basis of mass and NMR spectrometry the alkaloid was established to be 3-0-(P-D-glucosyl)pingbeinine [152].

93

Pingbeinine

4.2.23. Capsimine and its 3-O-P-D-Glucoside The ÂŁ1 mass spectrum of capsimine showed a molecular ion peak at m/z 415 and a base peak at m/z 98, produced as a result of a bond fission between C(20) and C(22) of a 22,26epiminocholestane. By comparison of the ^^C NMR signals with those of related compounds a


155

Solatium Steroid Alkaloids - an Update

(227?,25/?)-configuration and thus structure 94 was tentatively assigned by exclusion of other stereostructures [64]. Acidic hydrolysis of 3-0-(P-D-glucosyl)capsimine yielded capsimine (94) and glucose. The FAB mass spectrum displayed the [MH] ion peak at m/z 578. The ^â&#x20AC;˘'C NMR spectrum indicated that the glucosyl function was located at the 3-position. The size of the sugar ring was not established [65].

94 Capsinnine

Solamaladine

4.2.24. Solamaladine Structure 95 was established for solamaladine by X-ray analysis [184].

4.2.25. (25/{)-23,26-Epimino-3p-hydroxy-5a-cholest-23(A^-ene-6,22-dione,(20/;,25/?)23,26-Epimino-3p-hydroxy-5a>cholest-23(^-ene-6,22-dione and their P-DGlucopyranosides The elemental composition of (25/?)-23,26-epimino-3P-hydroxy-5a-cholest-23(A0-ene-6,22dione (96) was shown to be C27H4JNO3 by high-resolution mass spectrometry. Diagnostic peaks at m/z 140 and 111 indicated a structure of the tomatillidine type [23,26-epiminocholest23(A0-en-22-one]. The ^H NMR chemical shifts of H3(18) and H3(19) agreed with those of (25/tr)-22,26-epimino-3P-hydroxy-5a-cholest-22(7V)-en-6-one (aglycone of 86). Furthermore, the ^"^C NMR signals for the A - D rings were almost superimposable on those of this compound. (25/?)-22,26-Epimino-3p-hydroxy-5a-cholest-22(A0-en-6-one was converted to 96 in agreement with the given structure including the configuration at C(25) [142] [cf some doubts concerning the (25/?)-configuration of (25i?)-22,26-epimino-3P-(P-D-glucopyranosyloxy)-5a-cholest-22(iV)-en-6-one(86)]. Structure 98 was assigned to (25/?)-23,26-epimino-3P-(P-D-glucopyranosyloxy)-5a-cholest23(A^-ene-6,22-dione on the basis of NMR spectra and enzymatic hydrolysis [142].


H. Ripperger

156

96

97

R= H

R=H

PH 99 R =

R= OH

H< HO, OH

\

NMR data of (20/{,25/?)-23,26-epiniino-3P-hydroxy-5a-choiest-23(JV)-ene-6,22-dione suggested that it was the 20-epinier of 96, which was confirmed by comparison of the NOESY spectra of both compounds. In 96 and 97 the NOE correlation between 113(18) and H(20) (Figure 1) was observed, but no NOE between H(17) and H(20), indicating that H(20) preferred to lie toward the methyl group C(18). H3(21) showed NOE correlations with H(12p), H(17) and H3(18) in 96, but with H(16a), H(16P) and H(17) in 97. Thus, the absolute configuration of 97 at C(20) was determined to be (R) [142]. Structure 99 was assigned to (20/{,25/?)-23,26-epimino-3P-(P>D-glucopyranosyloxy)-5acholest-23(7V)-ene-6,22-dione on the basis of NMR spectra and enzymatic hydrolysis [142].

Figure 1. NOEs of 96 and 97


157

Solanum Steroid Alkaloids - an Update

4.2.26. Solaspiralidine The elemental composition of solaspiralidine (100) was shown to be C27H41NO3 by highresolution mass spectrometry. Diagnostic peaks at m/z 140 and 111 indicated a structure of the tomatillidine type [23,26-epiminocholest-23(A0-en-22-one]. The ^^C NMR signals were assigned by comparison with literature values, the *H NMR signals by HMQC, ^ H - % DQF COSY and NOESY measurements. The spectra indicated 3P- and 16-hydroxy groups as well as a A^-double bond. A NOE between H(16) and H3(18) (Figure 2) was in agreement with a 16ahydroxy group, between H3(18) and H(20) indicated a 17p-side chain. NOEs between H(20) on the one hand and H(16) and H3(18) on the other, as well as a missing effect between H(17) and H(20) displayed, at least approximately, antiperiplanar positions of H(17) and H(20). A NOE between H(16) and H3(21) indicated the (20^)-configuration. The configuration at C(25) was not established [118].

100

Solaspiralidine

Figure 2. NOEs of 100

4.2.27. Veracintine and Rhamnoveracintine By X-ray analysis of its 25,iV^dihydro derivative structure 101 was established for veracintine [185]. Enzymatic or acidic hydrolysis of rhamnoveracintine furnished veracintine (101) and rhamnose, which could be attached through the SP-hydroxy group of the aglycone [165].

101

Veracintine


158

H. Ripperger

4.3. Solanidanes 4.3.1. Solanidine Glycosides fij-Chaconine. The field-desorption mass spectrum of Pi-chaconine (102) showed [M + Na]"^, [MH]^ and the fragment ions [M + Na - 146]'*', [MH - 146]"^, [M + Na - 308]''" and [MH 308]^, suggesting a glycoside having a methylpentosylhexose moiety attached to an aglycone of molecular weight 397. Based on these results and the elemental analytical data the molecular formula was C39H^3NO}Q. Enzymatic hydrolysis yielded solanidine, glucose and rhamnose. Structure 102 followed from NMR comparison and permethylation studies. The H(l)-C(l) coupling constant of the rhanmose unit indicated an a-configuration for the L-rhamnose [151,cf 159]. P2^h(womne. The EI mass spectrum of ^2'^^^'^^^^* named '*p-chaconine'* in [162], showed the [M]*^ and the [M - 308]'*' peaks. Acidic hydrolysis afforded solanidine, glucose and rhamnose. Partial hydrolysis yielded y-chaconine [3-0-(P-D-glucopyranosyl)solanidine]. '*P-Chaconine'* was obtained by partial hydrolysis of stenanthine (105) indicating the ring sizes of the sugar units and the position of rhamnose at either the 4- (103) [162] or 6-hydroxy group of glucose. Stenanthidine. The EI mass spectrum of stenanthidtne (104) showed the [M]"^ ion peak. Hydrolysis yielded solanidine and glucose, partial hydrolysis frimished solanidine and ychaconine. Stenanthidine was obtained by partial hydrolysis of stenanthine (105) and, therefore, was in accord with structure 104 [162] or with an alternative structure with 4-position of the terminal glucose. Stenanthine. The EI mass spectrum of stenanthine (105) showed the [M]"^ ion peak. Hydrolysis yielded solanidine, glucose and rhamnose, partial hydrolysis furnished solanidine, ychaconine, P2~ch^^i^i^ (\^^) ^<1 stenanthidine (104). Hydrolysis of the permethyl derivative afforded 2,3,4,6-tetra-O-methyl-D-glucose, 2,3,4-tri-O-methyl-L-rhamnose and 2,3-di-Omethyl-D-glucose. These resuhs together with molecular rotation differences are in accordance with structure 105 [162] or with an alternative structure with 4-position of the terminal glucose and 6-position of rhamnose. S-O'fOa-L'Rhamnopyranosyl-fJ '^2)'0'[P'D'glucopyranosyl'(l -^JJ-p-D-glucopyranosyl}solanidine. The field-desorption mass spectrum of this alkaloid showed the ions [M -f Na]"^, [M + Na - 146]"*", [M + Na . 162]"^ and [M + Na - 308]"^. These data suggested that the glycoside had a branched-chain trisaccharide, methylpentosylhexosylhexose, combined with an aglycone of molecular weight 397. From the elemental analytical data, the molecular formula was C45H'^3NO|5. Hydrolysis furnished solanidine, glucose and rhamnose. Partial hydrolysis removed rhamnose. ^^C NMR spectrometry proved that the resulting dihexoside had a pcellobiosyl sugar chain. Unequivocal evidence for structure 106 was provided by the methanolysis of its permethyl derivative, yielding the methyl pyranosides of 2,3,4-tri'-0-methyla-L-rhamnose, 2,3,4,6-tetra-O-methyl- and 3,6-di-O-methyl-a-D-glucose, and by the molecular rotation difference between 106 and the P-cellobioside [151]. Hyacinthoside. The structure of hyacinthoside (107) was proposed based on mass and ^^C NMR spectrometry as well as acidic and enzymatic hydrolysis [159]. Neohyacinthoside. The structure of neohyacinthoside (108) was proposed based on mass spectral and ^^C NMR data [160].


Solanum Steroid Alkaloids - an Update

159

102 R

HO

OH

Stenanthidine 104

Stenanthine

105

R=

^^

HO

OH


160

H. Ripperger

106

R=

107

R

Hyacinthoside ™"^H0

OH

" 108

OH

"\

^

^

,0H

ohT

0—^J

R=

Neohyacinthoside

"^^'~HO

OH


Solanum Steroid Alkaloids - an Update

161

4.3.2. 3-OO-Lycotriosyl)- and 3-00-Lycotetraosyl)leptiiiidine and their 5a,6-Dihydro Derivatives 3-C>-(P-Lycotriosy!)leptinidine (109) and its 5a,6-dihydro derivative 111 were isolated as a mixture. The aglycones and the sugar chain were identified by ^â&#x20AC;˘'C NMR comparison [94,95]. 3-0(P-Lycotetraosyl)leptinidine (110) and its 5a,6-dihydro derivative 112 were also isolated as a mixture. The structures were elucidated on the basis of ^"^C NMR comparison and permethylation studies [94,95].

109 A5. R = R1 110 A5 R = R2 111 5aH, R = R1 112 5aH, R = R 2

R1 =

R2:


162

H. Ripperger

4.3.3. 22aff,25p^-Solaiiidaiie-3P,5a,6p-triol Structure 113 was established on the basis of its synthesis from solanidine, which was treated with performic add followed by alkaline treatment [139].

114

113

Rubivirine

4.3.4. Rubivirine Structure 114 was proposed for rubivirine on the basis of mass spectral and NMR data [177].

4.3.5. Solanogantamine (Solanopubamine), Isosolanogantainine, Solanopubamide A and B The structures 3P- and 3a-amino-5a,22(xf/-solanidan-23P-ol were originally assigned to solanogantamine and isosolanogantamine [4]. Later, 25(x^-configurations were assumed for both alkaloids [79]. The NMR chemical shifts of the carbon atoms of rings ÂŁ and F of solanogantamine and isosolanogantamine [79] were in close agreement with those of 5a,6dihydroleptinidine. Thus, the originally assigned configurations of solanogantamine and isosolanogantamine at C(25) must be reversed and their structures are 115 and 116, respectively [95]. Despite contrary statements [107], solanogantamine and solanopubamine should be identical. The ^H NMR spectra of solanogantamine, isosolanogantamine, solanopubamine and 5a,6-dihydroleptinidine as well as of their acetyl derivatives (^,(9-diacetylisosolanogantamit^ not measured) agreed with regard to the signals of 113(21), H(23) and H3(27), as expected from identical steric structures of the indolizidine ring moiety [95]. Solanogantamine (115) was synthesized from isosolacapine (SI) [68].

Solanogantamine. Solanopubamine Isosolanogantamine 116 R = a-NH2 Solanopubamide A 117 R = p-NHCHO Solanopubamide B 116 R = p-NHAc 116 R = p.NH2


Solanum Steroid Alkaloids - an Update

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Solanopubamide A and B gave solanopubamine (115) on acidic hydrolysis. Spectroscopic studies indicated the structures 117 and 118, respectively, for both alkaloids [108].

4.4. Solanocapsine Group 4.4.1. 3-Deamino-3P-hydroxysolanocapsine, Aculeamine and 0-Methylsolanocapsine 3-Deaniino-3P-hydroxysolanocapsine (119) was identical with a synthetic sample [4] obtained earlierfromsolanocapsine [49]. The 23-0-methyl derivative 120 was described as aculeamine and its structure determined by X-ray analysis [50]. It seems, however, doubtful that 120 could survive under the conditions of the hydrolysis (reflux with 1 N HCl in EtOH for 2.5 hours) which were used for the isolation of this compound. Perhaps 120 was formed from 119 during the chromatography with methanol. 0-Methylsolanocapsine was also described as a natural product and its structure established by synthesisfromsolanocapsine (4) [79].

119

R=H

120

R = Me

3-Deamino-3p-hydroxysoianocapsine Aculeamine

4.4.2. Pimpifolidine, 22-Isopimpifolidine (22,26-£piinino-16p,23-epoxy-S(x,22p//,2SaHcholestane-3P,23a-diol), Solanocardinol and 22,26-£piniino-16p,23-epoxy-23aethoxy-5a,25(xff-cholest-22(iV)-ene-3p,20a-diol By high-resolution mass spectrometry pimpifolidine (121) and 22-isopimpifolidine (122) were shown to be isomers with the elemental composition C27H45NO3. Diagnostic fragments at m/z 130, 112 and 84 in the mass spectra indicated the presence of alkaloids of the solanocapsine type. In order to elucidate the stereostructures, their NMR spectra were compared with those of 3-deamino-3p-hydroxysolanocapsine (119). The signals of 113(18) of 121 and 122 showed considerable downfield shifts with regard to the corresponding signal of 119. This indicated a 16P-oxygen bridge. The hitherto known natural alkaloids of the solanocapsine type possessed a 16a-oxygen ftinction. Only for solanocardinol, C27H45NO3, a 22,26-epimino-16^,23epoxycholestane structure was assumed. However, this alkaloid was neither well characterized nor its structure completely determined [98]. Because of the chirality of 121 and 122 at C(22), C(23) and C(25), we had to consider eight stereoisomers and furthermore in most cases different conformers with the same configurations. The coupling constants •/ji(25) H(26a) ^^ both alkaloids proved the equatorial conformations of the methyl groups at C(25), thus reducing the number of possible stereostructures. Pimpifolidine (121) displayed a coupling constant


164

H. Ripperger

'%(20),H(22) '^ ^^^ ^ revealing an H(20)-C(20)-C(22)-H(22) torsional angle of ca 180% and furthermore an upfield shift of the C(24) and C(26) signals compared with the corresponding signals of 119 and 122. These shifts could be rationalized by assumption of y-gauche interactions of C(24) and C(26) with C(20) [C(20) axial with regard to ring F]. An equatorial 25-methyl group, an H(20)-C(20)-C(22)-H(22) torsional angle of ca 180** and the y-gauche interactions were only in agreement with stereostructure 121 [with H(22) equatorial with regard to ring F] [135].

121

Pinfipifolidine

122

22-lsopinfipifolicline

22-Isopimpifolidine (122) displayed a coupling constant Jyu^o) H(22)' '- 6.2 Hz, indicating a torsional angle of 30-60^ and no y-gauche interactions of af C'(20) err' with * C(24) and C(26). Considering in addition the equatorial conformation of the 25-methyl group, these resuhs were in accordance with three steric structures: 22p//,25aff-cholestan-23a-ol (122), ll^H^lSoHcholestan-23p-ol [with H(22) axial with regard to ring F] and 22af^,25p//-cholestan-23a-ol [with H(22) axial with regard to ring F as well]. In the NOE difference spectrum irradiation at the H3(18) signal gave positive enhancements for the H(20) and the H(22) signals. These observations were only in accordance with stereostructure 122 with ring E in chair conformation [135]. Structure 122 was independently established under the designation 22,26epimino-16p,23-epoxy-5a,22P^,25oif^-cholestane-3p,23a-diol[133].

OMe

The molecular formula for 22,26-epimino-16P,23-epoxy-23a-ethoxy-5a,25a^-cholest22(^-ene-3P,20a-diol (123) was determined to be C29H4'7N04 by high-resolution mass spectrometry. The % NMR spectrum indicated the presence of an ethoxy group. The ^^C NNfR signals corresponding to rings A-D were very similar to those of tomatidine. The data for


Solanum Steroid Alkaloids - an Update

165

the three quaternary carbon atoms of rings E and F suggested that the structure was 123, except for the configurations of rings E and F. Coupling constants suggested an equatorial conformation for the 25-methyl group. NOE correlations between H3(18) and H3(21) revealed a 20p-methyl group. Further NOE correlations between H(16) and H(17), H(Et) (5 3.38), H3(Et) as well as between H(25) and H'(Et) (6 3.18) were in accordance with stereostructure 123 (all a-configurations for H(16), OEt and H(25) [133].

4.4.3. 7P-Hydroxy-0-methylsolanocapsine The *H NMR spectrum of 7P-hydroxy-0-methylsolanocapsine (124) revealed one methoxy group which could have been introduced, because this alkaloid was obtained after acidic hydrolysis in methanol. The mass spectrum exhibited the molecular ion peak at m/z 460 which is 30 mass units higher than the co-occurring solanocapsine (4). When treated with formaldehydeformic acid a trimethyl derivative was formed. Acetylation yielded an iViTST'.O-triacetate. These results were in accordance with a primary and secondary amino and a hydroxy group. The secondary nature and equatorial orientation of the hydroxy group followed from the *H NMR spectrum. The signals for H(16), 113(18), H3(19), H3(21) and H3(27) were very close to those of O-methylsolanocapsine. A peak in the mass spectrum at m/z 98, 16 mass units higher than expected for a 3-amine with an unsubstituted A/B ring system, suggested a location of the hydroxy group at C(4), C(6) or C(7). Comparison of the ^^C NMR spectrum with that of Omethylsolanocapsine indicated a 7P-hydroxy group [66].

4.5. 3-Aminospirostanes 4.5.1. Antillaridine, Antillidine and Juripidine For antillaridine and antillidine the structures 125 and 126 have been deduced [54]. The structure of juripidine (127) was established through its 7\f,0-diacetate. Mass spectral, ^H and ^-^C NMR data were in agreement with the structure of the diacetyl derivative of 127, which finally was confirmed by its conversion to 0,0-diacetylneochlorogenin with sodium nitrite in acetic acid-acetic anhydride [85].

126 R = 3p-NH2 126 R = 3a-NH2

Antillaridine Antillidine

127 Juripidine


166

H. Ripperger

4.6. Further Alkaloids

4.6.1. Abutlloside A and B According to high-resolution FAB mass spectrometry abuliloside A (128) had a molecular formula C49Hg3NO|'j. When hydrolyzed in acid, 128 yielded glucose, xylose and rhamnose. m, ^H-% COSY, HMQC and HMBC techniques led to the assignment of structure 128. A NOE between H(16) and H3(18) indicated a 16a-hydroxy group [39]. Analogously, structure 129 was deduced for abuliloside B. The configurations of both compounds at C(25) were assumed to be (R), however, this remains to be solved. Abutiloside A and B seem to be connected with the biosynthesis ofSolcamm steroid alkaloids [cf 4]; Ring F cannot be formed owing to acylation of the 26-amino group [40].

AbutHoaide A Abutiloside B

4.6.2. Korsiliney Petisine, Petbinine, Petisidine,Petisidinone, Petisidininet Verdinine and Sevkorine According to spectroscopy korsiline, C27H43NO2, seemed to be a stereoisomer of petiline [156]. Petisine, C27H41NO3, was synthesized from petiline by treatment with manganese dioxide. Petisinine, C33H5|NOg, was identified as the glucoside of petisine [145]. Petisine was isomerized to petisidine, C27H41NO3, [146]. Petisidinone, C27H39NO3, was obtained from petisidine by oxidation with chromium trioxide [147]. Dehydrogenation of petisine with palladium afforded petisidinine, C27H39NO3 [149]. Verdinine, C29H4JNO4, was identified as 3-0-acetylpetisidinine [167]. As the structure of petiline was not unequivocally proved [4], this has also to be concluded for the alkaloids the structures of which were correlated with petiline. Hydrolysis of sevkorine, C34H57NO7, yielded sevkoridine, C28H47NO2, and glucose. Sevkoridine seemed to be a stereoisomer of edpetilidinine [157], the structure of which was not completely determined [4].


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4.6.3. Valivine Insufficient data were available for a complete structure elucidation of this alkaloid with the molecular formula C2gH47N03 [153].

4.6.4. Solanocastrine The published arguments for the assignment of the structure of this alkaloid with the molecular formula C27H^2^2^ t^^J ^^^ "째^ convincing.

4.6.5. 3P-{0-P-D-Glucopyranosyl-(l->4)-P-D-xylopyranosyloxy]-15,16-seco-22aff,25pHsolanida-5,14-diene The FAB mass spectrum showed a quasi-molecular ion peak at m/z 692 and fragment ions at m/z 530 [MH - hexose]"*" and 398 [530 - pentose]"^. Enzymatic hydrolysis yielded glucose and xylose. On the basis of NMR data structure 130 was proposed [141], but not rigorously proved.

5. SYNTHESES AND CHEMICAL TRANSFORMATIONS Soladunalinidine (132) was synthesized from tomatidine (131) via its 3-ketone 134 and the oxime 135 which was reduced with sodium in w-propanol to soladunalinidine (132) as well as to its ring E opened (225)- and (22/?)-dihydro products 136 and 138, the iV(22,26)-chloro-iV(3)(2-hydroxybenzylidene) derivatives 137 and 139 of which were recyclized with sodium methanolate to ^(3)-(2-hydroxybenzylidene)soladunalidinine (133). The 5a-spirosolan-3-amines (25/?)-5a,22ocM-spirosolan-3a-amine (141) and -3P-amine (59) were obtained in an analogous sequence of reactions from soladulcidine (140) [181]. The 16a-hydroxylated steroid alkaloids teinemine (72), 22-isoteinemine (74), etioline (147) and 25-isoetioline (82) were sjmthesized from the abundant alkaloids tomatidenol (142) and solasodine (1), respectively. The crucial stages of these syntheses were the conversions of 142


168

H. Ripperger

131 R = OH Tomatldlne 132 R = NH2Soiadunatinidin6

134 R = 0 136 R = NOH

133 R

136

R1=NH2, R 2 = H

137 R 1 = N = C H â&#x20AC;&#x201D; . T ^

R2 = CI

136

R1 = NH2.

139

Rl=N =

R2

=H

CH - ^ . R 2 HO

140 R = p-OH Soladulcidjne 141 R = a-NH2 69 R = P-NH2

= CI


Solanum Steroid Alkaloids - an Update

142

/

169

Tomatjdenol

.

\

s ^ 143

144

145

146 Z = C02CH2C6H5

72

74

147

Etiotine


170

H. Ripperger

149

150 R Âť H 151 R = CI

Z = C02CH2C6H5 or 1 into the A^protcctcd (225.255)-, (22R,25S)- and (22S,25/?)-22,26-cpimino-3p. hydroxycholest-5-en-16-ones 145, 146 and 149 via the ring E opened epiminocholestanes 143, 144 and 148 as well as the reductions of 145,146 and 149 with sodium/2-propanol to teinemine (72), 22-isoteinemine (74) and (22iS,25/?)-22,26-epiminocholest-5-ene-3p,16a-diol (150), respectively. Treatment of the A^chloro derivatives of 72 and 74 with sodium methanolate afforded etioline (147). In an analogous manner, the ^-chloro derivative 151, obtained from 150, was converted into 25-isoetioline (82) [186]. (22/?,235,25/{)-22,26-Epiminocholest-5-ene-3p,16p,23-triol was prepared from solasodine (1) as ah-eady described [4]. Its ^-chloro derivative 152 yielded, on treatment with sodium methanolate, the 22,23-secoaldehyde 153 as major product (Grobfragmentation)uid (235)-23hydroxysolasodine (40) in poor yield (Ruschig reaction). 153 was transformed into the known acetyldiosgenin lactone (154) [187]. ^-Hydroxysolasodine (48) and i^-hydroxytomatidine have been prepared in low yield by oxidation of solasodine (1) and tomatidine (131), respectively, with hydrogen peroxide in the presence of selenium dioxide [180]. Further syntheses of solacapine (91), episolacapine (92), (25/?)-23,26-epimino-3p-hydroxy5a-cholest-23(^-ene-6,22-dione (96) and solanogantamine (115) were mentioned in Chapter 4. 22,26-Epiniino-16P-hydroxycholestanes have been cyclized to spirosolanes by manganese dioxide [188,189]. Ring F opened spirosolanes have also been transformed by photochemical reactions to spirosolanes [190]. 21,27-bis-nor-Demissidine has been synthesized from epiandrosterone and dehydroepiandrosterone acetate [191-193]. Preliminary synthetic experiments have been carried out to introduce a 4-keto group into epiminocholestanes [194].


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Nitrogen analogs of ecdysones starting from solasodine [195] as well as nitrogenous brassinolide analogs with solanidane [196], 22,26-epiniinocholestane and spirosolane skeleton [197] have been synthesized. Tomatidine deuterated and tritiated in positions 2 and 4 was prepared via the 3-ketone [198].

153

AcO 154 Configurations and conformations of iV-methyl derivatives of tomatidine and solasodine were studied by NMR [199]. Whereas the spectra of iV-methyltomatidine pointed to the unchanged tomatidine structure with an equatorial M-methyl group, the NMR data of the two isomers obtained from solasodine are difficult to correlate with structures in the opinion of the present author. Similar problems arose when the structures of two isomeric A'^O-diformylsolasodines [200] or two iV-cyanosolasodines [201] were investigated by NMR. The structures of two isomeric iV-formates, prepared from 0-acetylsolasodine, were determined by X-ray crystallographic analyses, which indicated (22/?,25/?)- and (22.S',25/?)-configuration with chair conformations of theringsF, respectively [202].


172

H. Ripperger

Solasodenoiie was degraded to progesterone [203]. Solasodine was oxidized with hydrogen peroxide [204], potassium permanganate [205] or periodic acid [206] and the compounds formed were studied The products of anodic oxidation of solanidanes [207] or on heating of 5a,6a-epoxy-3P-hydroxysolamdane A^oxide [208] were investigated. Glycoalkaloids were cleaved to aglycones by sodium iodide and acetic acid , iodine and acetic acid or iodine and alumina [209], to aglycones and prosapogenins by thermal degradation [210].

6. BIOCHEMISTRY AND BIOACTIVITIES The main reaction steps of the biosynthesis of Solanum steroid alkaloids from cholesterol are known [4]: 26-hydroxylation, 26-transamination, formation of the nitrogen-containing ring F, formation of spirosolanes via 16P-hydroxylation or synthesis of solanidanes via 16ahydroxylation. Abutiloside A (128) and B (129) connected with intermediates of the biogenesis have been isolated [39,40]. In fruits from Solanum melongena L. grafted upon Solanum viarum Dun. the presence of solasodine could not be detected. But solasodine was contained in the fruits harvested from scions of S, viarum grafted upon S, melongena. These results indicated that the synthesis of solasodine is scion-specific [211]. Chemotaxonomy of the tuber-bearing Solanum species, subsection Potatoe^ in relation to steroid alkaloid content was described [43]. A HPLC assay was used to measure the chaconine and solanine content of conmiercid and new potato varieties, difTerent parts of the potato plant and conmiercial potato products. The significance of the results to food safety was discussed [124]. When solasodine was incubated with the fungus Cunninghamella elegans Ledner, (25Ry 22a^-spirosol-5-en-3p,7a-diol, (25/?)-22a/V-spirosol-5-en-3P,7P-diol and (25/?)-3P-hydroxy22a^-spirosol-5-en-7-one were produced, wheras incubation with Penicillium patulum Bain gave (25/{)-22aA^spirosol-4-en-3-one [212]. A microsomal fraction prepared from leaves of Solanum chacoense Bitt. after addition of NADPH converted solanidine to a leptinidine glycoside of unknown structure of the sugar moiety [213]. Potato glycosidases (fixiits, blossoms) hydrolyzed chaconine to ^2^^^'^^^'^* ^ ^ ^^^ solanine unchanged [214]. Analogously, hydrolysis of solamargine to solasodine, but not of solasonine by Aspergillus niger was reported [215]. ThefringusBotrytis cinerea Pers. hydrolyzed tomatine to tomatidine and converted it to a structurally unknown P-lycotetraoside [216]. Solanum nigrum L. is used in folk medicine in Israel (toothache, sedative for external pains, constipation etc) [217]. Solasodine, solamargine, solasonine, capsicastrine, capsimine, 3-0-(pD-glucosyl)capsimine, etioline and khasianine exhibited strong activity against liver damage induced by carbon tetrachloride [64,65,218]. Numerous papers dealt with the cytotoxic activities of Solanum steroid alkaloids, e.g. 3-0-(Plycotriosyl)- and 3-0-(P-lycotetraosyl)leptinidine and their 5a,6-dihydro derivatives were observed to inhibit the growth of a human cervical cancer cell line [94], the mixture of glycoalkaloids isolated from Solarmm sodomaeum L. containing mainly solamargine and solasonine to exhibit antineoplastic activity against Sarcoma 180 in mice [219], solamargine, solasodine, capsimine, capsicastrine and etioline to exhibit significant inhibition of human hepatoma PLC/PRF/5 cells [65,89]. The structure-cytotoxic activity relationships were studied


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173

with 15 Solanum alkaloids and various cell lines: PC-6 (lung cancer), MCF-7 (breast cancer), NUGC-3 (stomach cancer), P388 (mouse leukemia) and SW620 (colon cancer). Solamargine, chaconine and solasodine were the most potent alkaloids [220]. A cream has been developed constituted of solasodine glycosides which is claimed to be effective against malignant skin cancer [221]. A preparation containing spirosolanes was protected as anticancer agent by patent [222]. The acetylcholinesterase-inhibitoiy activity of chaconine, P2"^^^^^™"® ^"^ solanine was described [223]. Solacongestidine, solafloridine and solasodine exhibited considerable inhibitory effects on the synthesis of cholesterol from 24,25-dihydrolanosterol [224]. Embryotoxicities of Solanum alkaloids using the fi'og embryo teratogenesis 2iSs^y'Xenopus (FETAX) were examined. Chaconine, solanine, solasonine and tomatine showed concentrationresponse curves. The data showed that glycoalkaloids are more toxic than corresponding aglycones, that the nature of the carbohydrate moiety of glycosides strongly influenced potency and that the nitrogen of the steroid was required for embryotoxicity [225]. One of the proposed mechanisms of the toxic action of glycoalkaloids is disruption of membranes by complexing with 3p-hydroxy sterols. Tomatine was found to disrupt liposome membranes containing 3P-hydroxy sterols, liposome membranes containing 3a-hydroxy sterols were resistent [226]. Chaconine or solamargine caused significant disruption of phosphatidylcholine/cholesterol liposomes, whereas solanine or solasonine were ineffective, but the latter showed synergistic interactions with the first mentioned compounds [227,228]. Chaconine or solanine, but not solanidine, altered the membrane potential of frog embryos [229]. Strong antifungal activities were described for solacongestidine (Candida albicans, Trichophyton rubrum, Cryptococcus albidus), solafloridine (C albicans, T. rubrum) and verazine (C. albicans, T. rubrum), while solasodine, tomatidine, tomatillidine and solanocapsine showed much lower activities. Solacongestidine also prolonged the survival time of mice infected with C. albicans [230]. (25i?)-5P,22aA^-Spirosolan-3-one and (25/?)-22ocA^-spirosol-4en-3-one were isolated as antifungal stress metabolites from leaves of Solanum aviculare Forst. f [58]. Plant growth was described to be retarded by solamargine or solasonine [231]. Tomatine induced stomatal closure in epidermal peels of Commelina comunis L. [232].

7. TABLES OF PHYSICAL CONSTANTS (Compiled according to the molecular formulae) Table 4. Alkamines^ Compound

Formula; melting point (°C); [a]£) (solvent) [ref ]

Petisidinone

C27H39NO3; 217-219 ; 0° (CHCI3) [147]

Petisidinine C27H39NO3; 290-292 [149] (25/?)-22aA^-Spirosol-4-en-3-one C27H4JNO2; 178-180 [212]; 177-179 [58] (25i?)-23,26-Epimino-3P-hydroxy5a-choIest-23(A^-ene-6,22-dione C27H4JNO3; +18.4° (CHCI3) [142]


H. Ri^erger

174

Table 4. Alkainines^ (cont.) Compound

Formula; melting point (°C); [a]Q (solvent) [ref. ]

(20/?,25/?).23,26.Epimino-3p. hydroxy-5a-cholest-23(A0-cne6,22-dione Solaspiralidine (25/?)-3P-Hydroxy-22aAr.spirosol. 5-en-7-onc Petisine

C27H41NO3; +1.6*' (CHCI3) [142]

Petisidtne (25/?>.12p-Hydroxy-22aA^spiro. sol-5-en-27-oic acid Solanocastrine (20/?)-Vcra2inc (25iS)-5a,22P^^Sptrosolan-3-onc (25/?)-5p,22a^-Spirosolan-3-onc 25-Isoetioline 20,25-Bisisoetioline Korsiline 7p-Hydroxysolasodine 7a-Hydroxysolasodine (23iS)-23-Hydroxysolasodine, Solaverol A Solaparnaine ^-Hydroxysolasodine Solanudine Ebeietinone Rubivirine 12P,27-Dihydroxysola8odine (255)-22pAr.Spirosol-5-en-3aamine (25/?)-22a^-Spirosol-5-en-3aamine (25/?)-22aAr-Spirosol-5-en-3pamine Oblonginine Veramiline Capsimine Veramivirine Teinemine 20-Isosolafk>ridine

C27H41NO3; 212-214; -63.r (CHCI3) [118] C27H41NO3 [212] C27H41NO3; 221-222 [145]; 220-222; +34.4° QAeOH) [150] C27H41NO3; 150-152 [146]; 150-152; -20.5'* (MeOH) [150] C27H41NO5; 276-278; -71.2*» (pyridine) [179] C27H42N2O; 258-260; 0° (CHCI3) [67] C27H43NO [168] C27H43NO2; 195-198; +19« (CHCI3) [133] C27H43NO2; 169-170; -49° (CHCI3) [58] C27H43NO2; 141-143; +73.6° (CHCI3) [61] C27H43NO2; 158-161; -11.8° (CHCI3) [41] C27H43NO2; 194-196; 0° (EtOH) [156] C27H43NO3; 244-246 [212] C27H43NO3; 210-212 [212] C27H43NO3; -67.r(CHCI3) [5]; 190-195 (dec); -93.2°(CHCl3)[62] C27H43NO3; 228-230; -77.8° (MeOH) [56] C27H43NO3; 206-209(dec); -119.5°(CHCI3) [109] C27H43NO3; 225; +352° (dioxane) [101] C27H43NO3; 199-203; -53.5° (CHCI3) [140] C27H43NO3; 239-241; +28.5° (CHCI3) [177] C27H43NO4; 233-237; -54.7° (MeOH) [179] C27H44N2O; 166-170 [123] C27H44N2O; 147-154 [123] C27H44N2O; 165-167; -52° (MeOH) [122] C27H45NO; 219-220; -40.7° (CHCI3) [171] C27H45NO; 198-201 [175] C27H45NO2; 264-267; -104° (MeOH) [64] C27H45NO2; 229-231; -81° (CHCI3) [177] C27H45NO2; 205-207; -38.7° (CHCI3) [164] C27H45NO2; 136-137; +45.1° (CHCI3) [41]


Solatium Steroid Alkaloids - an Update

175

Table 4. Alkainines^(cont.) Compound

Formula; melting point (°C); [ajj) (solvent) [ref]

Stenophyiline B 2a-HydroxysoIadulcidine (23/?)-23-Hydroxysoladulcidine Vertaline B Pimpifolidine 22-Isopimpifolidine, 22,26epimino-16P, 23 -epoxy5a,22p//,25otf/-cholestane3p,23a-diol Deacetoxysolaphyllidine

C27H45NO2; 225-227; -25.7° [176]; 225-228 [175] C27H45NO3; 252-255 (dec); -62.5° (CHCI3) [137] C27H43NO3; 192-193; -65.4° (MeOH) [95] C27H45NO3; 271-273 [176] C27H45NO3; 200-203; -1.9° (pyridine) [135] C27H45NO3; 200-204; -13.6° (pyridine) [135]; 215-219: +2.0°(EtOH)[133]

22a//,25p;/-Solanidane3p,5a,6p-triol 3-Deamino-3 P-hydroxysolanocapsine Solanocardinol Juripidine Solanopubamine (25/?)-5a,22aA^-Spirosolan3p-amine Solacapine Episolacapine Isosolacapine Solanopubamide A Sevkoridinine Puqietinone Pingbeinine Aculeamine Valivine 0-Methylsolanocapsine 7P-Hydroxy-0-methylsolanocapsine Verdinine Radpetine Muldamine (23/?)-23 - Acetoxytomatidine (235,255)-23.Acetoxy-5a,22aA^ spirosolan-3P-ol

C27H45NO3; 215-218; +19.5° (MeOH) [86]; 204-209 [87] C27H45NO3; 250-256; +0.01° (CHCI3) [139] C27H45NO3; 204; +20.1° (CHCI3) [49] C27H45NO3 [98] C27H45NO3 [85] ^27^46^2^^ 263; +30.5° (MeOH) [107] C27H46N2O; 178-182; -57.3° (CHCI3) [181]; 177-185 [122] ^27^48^2^2^ 286-288; +47.1° (CHCl3-MeOH 1:1) [79] ^27^48^2^2; 258-260; -41.5° (CHCl3-MeOH 1:1) [79] ^27^48^2^2^ 238-240; -12.5° (CHCl3-MeOH 1:1) [79] ^28^46^2^2; 209-210; +26.1° (CHCI3) [108] C28H47NO2; 241-243; -43.2° (EtOH) [157] ^28^47^02; 240-245; +29.4° (CHCI3) [143,144] ^28^47^03; 223-235; -32.8° (MeOH) [152] ^28^47^03; 205-207; +50.8° (CHCI3) [50] C28H47NO3; 256-258; -48° (CHCI3) [153] ^28^48^2^2' 183-185; +44.4° (CHCI3) [79] C28H48N2O3; +65.2° (CHCI3) [66] C29H4JNO4; 265-267 [167] C29H43NO3; 229-231 [148] C29H47NO3; 209-210; -95° (EtOH-CHCl3 3:1) [233] C29H47NO4; -2.8° (CHCI3) [133] C29H47NO4; -36.7° (CHCI3) [133]


176

H. RIpperger

Table 4. Alluiiiiines^(cont.) Compound

Formula; melting point (X); [a]]^ (solvent) [ref]

22,26-Epimino-16p,23-epoxy-23a- C29H47NO4; .38.3*» (CHCI3) [133] ethoxy-5a,25a^-cholest-22(JV)ene-3p,20a-diol (235)-23.Acetoxysoladulcidine C29H47NO4; -46^ (CHCI3) [133] Solanopubamide B C29H4gN202; 255-256; +40*' (CHCI3) [108] ^Some references do not contain melting points or specific rotations. Nevertheless, they are cited here, because they contain spectroscopic data.

Table 5. Glycoaikaioids^ Compound

Formula; melting point (°C); [a]j^ (solvent) [ref.]

Rhamnoveracintine ^32^51^^5' '^^'^ ^^^ ""^ MeOH) [165] (25/?)-23,26.Epimino.3P-(p-D. C33H5 jNOg; -19.6*» (MeOH) [142] glucopyranosyloxy)-5a-cholest23(iV)-ene-6,22-dione (20/?,25/?)-23.26-Epimmo-3p-(P-D- C33H5,NOg; 214-215; -13.2*» (MeOH) [142] glucopyranosyloxy)-5a-cholest23(^ene-6,22-dione C33H5 jNOg; 232-234; -35^ (CHCI3) [145] Petisinine Stenophylline D C33H53NO5; 248-252 [173] Verazinine C33H53NO^; 259-261; -112.6*» (CHCI3) [178] 3-0-(a-L.Rhamnopyranosyl)C33H53NO5; 179-181; -42*» (MeOH) [129] solasodine (25/?)-22,26.Epimino-3P-(p-DC33H53NO7; -44^ (MeOH) [142] glucopyranosyloxy)-5a-cholest22(^-en-6-one 3-0-(p-D-GIucopyranosyl)etioline C33H53NO7; 225-227(dec); -78.8**(pyridine) [118] Deacetylveralosine C33H53NO7 [163] 3-0-(P-D-Glucopyranosyl)C33H55NO5; 303-305; -41.9^ (MeOH) [175] veramiline 3-0-(p-D-Glucopyranosyl)C33H55NO7; 286-288; -42.5*» (MeOH) [175] stenophylline B Capsicastrine C33H55NO7; 220-221; -25.5*» (CHCI3) [63] Isocapsicastrine C33H55NO7; +202** (pyridine) [64] Cordatine A C33H55NO7; -3.5** (MeOH) [158] Cordatine B C33H55NO7; 187-190; -11.2** (MeOH) [158] 3-(9-(P-D-Glucopyranosyl)C33H55NO7 [55] tomatidine


Solatium Steroid Alkaloids - an Update

177

Table 5. Glycoalkaloids^ (cont) Compound

Formula; melting point (°C); [ajj^ (solvent) [ref]

3-0-(p-D-Glucosyl)capsimine

C33H33NO7; 214-216; -68.8** (cyclohexane-EtOAcMeOH 1:1:2) [65] C34H57NO7; 236-238; -41. r (MeOH) [157] C34H57NO8; 244-246; -4.6° (MeOH) [152] C35H55NO8 [163] C35H55NO8; 186-187; -110.8° (MeOH) [82] C38H61NO10; -20.5° (MeOH) [141]

Sevkorine Pingbeininoside Veralosine Havanine 3p-[Op-D-Glucopyranosyl(1 -^4)-P-D-xyIopyranosyIoxy]15,16-seco-22oc//,25p//solanida-5,14-diene 3-0-[0-P-D-Xylopyranosyl-(1^6)- C38H63NOn [55] p-D-gIucopyranosyl]tomatidine Pj-Chaconine C39H63NO10; [173]; 252-258; -62.6° (pyridine) [159]; 287-292 (dec); -52.5° (pyridine) [151] P2-Chaconine C39H53NO10; 253-255; -61.4° (pyridine) [162] P|-Solamargine C39H63NO1 j; -86.5° (MeOH) [6] 3 -0-[0-a-L-RhamnopyranosylC39H^3NOj 1; 263; -89° (MeOH) [129] (1 ->2)-P-D-galactopyranosyl]solasodine Stenanthidine C39H53NO11; 269-271; -47.5° (pyridine) [162] Etiolinine C39H63NO12; 229-230 [83] y-Tomatine C39H^5NOi2; -10.8° (MeOH) [8] 3 -0-[0-a-L-Rhamnopyranosyl C40H67NO11; 269-274 (dec); -67.2° (pyridine) [151] {1 ^2)-P-D-glucopyranosyl]hapepunine 3 -0-(P-Cellobiosyl)hapepunine C40H67NO12 29.1° (MeOH) [141] Ravifoline C44H71NO14; 280-282; -104° (pyridine) [105] Anguivine C44H71NO15; -83.2° (pyridine) [23] Isoanguivine C44H7JNOJ5; 286-291 (dec); -88.8° (pyridine) [23] (235)-23 -Hydroxyanguivine C44H71NO15; 215-222 (dec); -94.0° (pyridine [130] (235)-23 -Hydroxyisoanguivine C44H7JNO15; 225-230 (dec); -90.1° (pyridine) [130] 3 "O- {O-a-L-RhamnopyranosylC45H73NO15; 306 (dec); -110° (pyridine), (1 ->2)-0-[a-L-rhanmopyranosyl-102° (MeOH) [129] (1 ->4)]-P-D-galactopyranosyl} soiasodine 3-(9- { O-a-L-RhamnopyranosylC45H73NO15 [138]; 271-273; -53.1° (pyridine) [159]; (1 ->2)]-0-[p-D-glucopyranosyl278-283 (dec); -58.4° (pyridine) [151] (1 ^4)]-P-D-gIucopyranosyl} solanidine Stenanthine C45H73NO|5; 262-264; -46.5° (pyridine) [162] Solanelagnine C45H73NO15; 210-215 [74]


178

H. Ripperger

Table 5. Glycoalkaloids^ (cont.)

Compound

Formula; meiting point (°C); [a]j) (solvent) [ref]

JV^Hydroxysolamargine Solaverine I 3-0-( O<x-L-Rhamnopyranosyl(1 -â&#x20AC;˘2)-(9-[P-D-glucopyranosyl(1 ->4)]-P-D-glucopyranosyI}solasodine Solaverine II 3-0-(P-So]atriosyl)solanaviol 3-0(P-LycotriosyI)Ieptinidine 3-0-(p-Chacotriosyl)12p,27-dihydroxysolasodine Solaverine III Soladulcine A 3-0-(p-Lycotriosyl)dihydroleptinidine Abutiloside B Incunamine Abutiloside A Robustine Xylosyl-p-solamarine Xylosylsolamargine ^-Hydroxyrobustine 3-C><p-Lycotetraosyl)solasodine 3-0(P-Lycotetraosyl)leptinidine Soladulcine B 3-0-(P-Lycotetraosyl)dihydroleptinidine 3-0-(P-Lycotetraosyl)solanocardinol Hyacinthoside Solashabanine Sisunine, neotomatine 25-Acetoxyrobustine Lycoperoside A Lycoperoside B Lycoperoside C Neohyacinthoside Solaradinine

C45H73NOJ6; .82.8^ (pyridine) [7] C45H73NO16; -75.70 (pyridine) [5] C45H73NO15; -77.30 (MeOH) [6]

C45H73NO17; 74.60 (pyridine) [5] C45H73NO17; .53.30 (pyridine) [179] C45H73NO17P4] C45H73NO17; -64. r (pyridine) [179] C45H73NO17; -66.00 (pyridine) [5] C45H75NO15; 256-258; -91.8o (pyridine) [73] C45H75NOi7[94] C45H77NO17; -41.90 (MeOH) [40] C49H79NO19; >300; +780 (MeOH) [89] C49H83NO17; -49.20 (MeOH) [39] C5oH8,NO|9; 217-223; -73.70 (pyridine) [22] C5oHgiNOi9; -54.8o (pyridine) [15] C50H81NO19; -57.50 (pyridine [15] ^50^81^020; -^3.40 (pyridine) [22] C50H81NO21 [90] C50H81NO21 [94] C5QH83NO21 [90]; 264-266; -58.4o (pyridine) [73] C50H83NO2, [94] C50M83NO22P8I C51H83NO20; 265-268; -49.7o (pyridine) [159] C51H83NO21; 270-273 [91] C51H95NO22 [45,72] C52H83NO21 71.40 (pyridine) [22] C52H85NO23 31.60(MeOH)[8] C52H85NO'23, -40.30 (MeOH) [8] C52H85NO23 26.30 (MeOH) [8] C57H93NO25; 262-265; -25.9o (pyridine) [160] C57H93NO26; 227-230 [91]

^Some references do not contain melting points or specific rotations. Nevertheless, they are cited here, because they contain spectroscopic data.


Solanum Steroid Alkaloids - an Update

179

ACKNOWLEDGEMENT Figure 1 was reprinted in slightly modified form from Phytochemistry, Vol 3 1 , K On, Y Mimaki, Y Sashida, T Nikaido and T Ohmoto, Steroidal Alkaloids from the Bulbs of Fritillaria persica, pp 4337-4341, Copyright 1992, Figure 2 from Phytochemistry, Vol 4 3 , H Ripperger, Steroidal Alkaloids from Roots of Solanum spirale, pp 705-707, Copyright 1996, both with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0 X 5 1GB, UK.

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184 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203.

H. Ripperger EM Taskhanova and R Shakirov, Khim Prir Soedin 404 (1981); Chem Abstr 95:147138 (1981). R Shakirov, W Kul'kova and I Nakhatov, Khim Prir Soedin 100 (1995). Xitiwen Han and H Rflegger, Planta Med 58:449 (1992). NV Bondaienko, Khim Prir Soedin 527 (1981); Chem Abstr 96:65653 (1982). W Zhao, Y Tezuka, T Kikuchi, ^ n Chen and Yongtian Guo, Chem Pharm Bull 39:549 (1991). S Kadota, Shi Zhong Chen, Jian Xin Li, Guo-Jun Xu and T Namha, Phytochemistiy 38:777 (1995). NV Bondarenko, Khim Prir Soedin 243 (1983); Chem Abstr 99:3054 (1983). Liang Guang-yi and Sun Nan-jun,Yaoxue Xud)ao 19:431 (1984); Chem Abstr 103:3677 (1985). Guangyi Liang and Nanjun Sun, Yaoxue Xuebao 19:131 (1984); Chem Abstr 101:97524 (1984). M Mizuno, Tan Ren-Xiang, Zhen Pei, Min Zhi-Da, M linuma and T Tanaka, Phytochemistiy 29:359 (1990). ZD Min, RX Tan, (?r Zheng and CH He, Yaoxue Xuebao 23:584 (1988); Chem Abstr 110:92057 (1989). KA El Sayed, JD McChesney, AF Halim, AM Zaghloul and M Voehler, Phytochemistiy 38:1547 (1995). EM Taskhanova, R Shakirov and SYu Yunusov, Khim Prir Soedin 368 (1985); Chem Abstr 103:157290(1985). K Yoshita, S Yahara, R Saijo, K Murakami, T Tomimatsu and T Nohara, Chem Pharm Bull 35:1645(1987). H Ripperger, Liebigs Ann Chem 1091 (1992). Le thi Qayen, H Ripperger and K Schreiber, Liebigs Ann Chem 519 (1990). H Ripperger, K Schreiber and G Snatzke, Tetrahedron 21:1027 (1%5). G Meccia and AN UsiMlaga, J Nat Prod 50:642 (1987). A Usi^illaga, V ZatotX and WH Watson, AcU Ciystallogr, Sect B 38:966 (1982). F Paveldk and J Tomko, Chem ZvesU 37:145 (1983); Chem Abstr 99:140242 (1983). Le thi Quyen, H Ripperger and K Schreiber, Liebigs Ann Chem 143 (1991). Le thi Qaytn, H Ripperger, G Adam and K Schreiber, Liebigs Ann Chem 167 (1993). G Adam and HTh Huong, Tetrahedron Lett 21:1931,4996 (1980). G Adam and HTh Huong, J Prakt Chem 323:839 (1981). GM^jetich and KWheless, Tetrahedron 51:7095 (1995). D Miljkovic and K Gasi, Chim Chron, New Ser 9:325 (1980); Chem Abstr 95:187519 (1981). DA Miljkovic and KM Gasi, Glas Hem Dnis Beograd 46:263 (1981); Chem Abstr %:143154 (1982). D Miljkovic, K Gasi, M Kindjer, S Stankovic, B RiUbr and G Argay, Croat Chem Acta 58:721 (1985); Chem Abstr 106:50549 (1987). E Viloria, G Meccia and AN Usi4>illaga, J Nat Prod 55:1178 (1992). RC Cambie, GJ Potter, RW Read, PS Rutiedge and PD Woodgate, Aust J Chem 34:599 (1981). U thi Qvycii, G Adam and K Schreiber, Tetrahedron 50:10923 (1994). Le thi ( ^ e n , G Adam and K Schreiber, Liebigs Ann Chem 1143 (1994). D DoUer and EG Gros, J LabeUed (>>mpd Radiopharm 23:109 (1986); Chem Abstr 105:153393 (1986). HE GoCUieb, I Belie, R Komel and M Meivic, J Chem Soc, Peridn Trans 1 1888 (1981). W Gaffield, M Benson, WF Haddon and RE Lundin, Aust J Chem 36:325 (1983). S Siddiqui, BS Siddiqui and S Faizi, Z Naturforsch 38b: 1236 (1983). G Kusano, C Sekimoto, M Abe, Y In, H Ohishi, T Ishida and N Aimi, Heterocycles 34:1957 (1992). G Adam, Hoang Thanh Huong and M Lischewski, Z Chem 26:369 (1986).


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Chapter Three

Synthesis and StructureActivity Studies of Lissoclinum Peptide Alkaloids Peter Wipf Department of Chemistry University of Pittsburgh Pittsburgh, Pennsylvania 15260 U.S.A.

CONTENTS 1. INTRODUCTION

188

2. SOURCES OF LISSOCLINUM PEPTIDE ALKALOIDS

194

3. TOTAL SYNTHESIS OF LISSOCLINUM PEPTIDE ALKALOIDS 3.1. Synthesis of Heterocyclic Building Blocks 3.2. Total Synthesis of Dolastatin E 3.3. Total Synthesis of Lissoclinamide 4 3.4. Total Synthesis of Lissoclinamide 7 3.5. Total Synthesis of a Structural Isomer of Cyclodidemnamide 3.6. Total Synthesis of Nostocyclamide

196 196 203 206 208 211 213

4. SECONDARY STRUCTURES OF LISSOCLINUM PEPTIDE ALKALOIDS 215 5. BIOLOGICAL ACTIVITY OF LISSOCLINUM PEPTIDE ALKALOIDS

221

6. OUTLOOK

224

7. REFERENCES

225

187


188

P. WIpf

1. INTRODUCTION Marine flora and fauna continue to provide a rich source of pharmacologically active and structurally unique secondary metabolites [1]. Due to the difficulties in the isolation of sufficient quantities of marine natural products, synthetic chemistry serves a crucial role in structural assignment and biological evaluation [1,2]. In this review, I will cover the chemistry of cyclopeptide alkaloids characterized by an alternating sequence of five-membered heterocycles and hydrophobic amino acid residues. The ascidian genus Lissoclinum has proven to be a particularly rich source of these unique peptide derivatives, and, in an obvious generalization, I will therefore refer to the entire structural class as lissoclinum peptide alkaloids. In addition to ascidians and sea hares, cyanobacteria have yielded these structurally well defmed cyclopeptides. The about 30 members of this class can be further subdivided according to their ring sizes as (A) 18-membered cyclohexapeptide, (B) 21-membered cycloheptapeptide, and (C) 24membered cyclooctÂŁ4)eptide analogs. Classes A and C show an intrinsically greater tendency for pseudosymmetrical disposition of amino acid mid heterocycle residues, ^ereas in die 21membered macrocycles the proline residue appears to mimic the conformational properties of the missing heterocyclic moiety (Figure 1).

Figure 1. Structural classification of lissoclinum peptides. A. 18-Membered cyclohexapeptide analogs

O

BistntanOdeA (1) [3]

BtstratanOde B (2) [3]


Synthesis and Structure-Activity Studies of LissocliiMim Peptide Aliialoids

189

Figure 1. (continued)

o O

/N

I H

N ^ N

N--

N

Bistratamide C (3) m

Bistratamide D (4) [4]

V-

""'>V"'^'S'째> o'

N

H

N-

N- ^

,N

d Dendroamide A (6) [7]

Cycloxazoline (Westiellanude, 5) [5,6]

O H

AN^H

N ^

J

N H

N^

H.^/UO N.

,/^s-

Dendroamde B (7) [7]

N.

w -s' ^

-"^S'

Dendroamide C (8) [7]

o


190

p. WIpf

Figure 1. (continued)

o AJ

Y

H

NV

1

Doiastaiin E (9) [^,9]

â&#x20AC;&#x201D;O

Nostocyclamide (10) [10]

Ph

RaocyclamUeA (11) [\\] B. 21-Membered cycloheptapeptide analogs

Lissociinamide 1 (12) [12]

LissocUnamide 2 (13) [12]


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

191

Figure 1. (continued)

>=N

..H-N^r

\=u

H

)=0 H-N

H

N

I

/

N H

I

o

XÂť5ÂŁ>c///fflmiV/e-/(75> [13-15]

Lissoclinamide 3 (14) [12]

o ^t^^s N I

>=N =N

.. ,r H

N

>=N .Ph

N

H H-N

N

H

/"".,/

Ph

oV>-Vs Ph

Lissoclinamide 5 (16) [13,14,16]

O

\

Lissoclinamide 6 (17) [14]

.S>

"iZ-s-io H-N

H

= N N

V / Ph

Ph Lissoclinamide 7(18) [17,18]

)=:0 H-N Ph

H N=r

II

O

Lissoclinamide 8 (19) [17]


192

P. WIpf

Figure 1. (continued)

V^2 N4 H-N N

H

)-.../^^

A- o Vtbyclamlde (20) [12,19]

I

CyclodidemnanOde (21) [20,21]

C. 24-Membered cyclooctapeptide analogs

, \ /

O Y .8 ^ /~N

"^ ^ o

H

HN

NH

'"tN " S

V^

"

\_r

-

HN

N:^

o

I

S

AscUlmydamUe (32) [22,23]

),

O

PalettanMeA (23) [24,25]

Ph

~=N NH

H

I

Ph

"^

):sO

pN

HN

HN NH

S Patellandde B (24) [24,26,27]

1 0

I

PalellanMe C (25) [24,26]


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alicaloids

193

Figure 1. (continued)

I

O ^

=N

K

oV s

S

. ^t^'

N HN

NH

'^

HN

NH

o S

^

O

I

Palellamide D (26) [13,14]

PateUamideE(27)[2%]

o V s. \)=N

H

\ O

I

S

H

N=(^

)=N

\

<5

\ \ s H-N N-H \ - - ^ ^ \ \ 째 ^ N 'l N=r/

J o Ulithiacyclamide (29) [30,31]

Pateliamide F (28) [29]

Ph

5

N yo HN

VN

O

H

N

"^ HN

Tawicyclamide A (30) [32]

.

NH

H

fN l HN

Tawicyclamide B (31) [32]


IW

p. Wipf

Since the first report on the isolation of uiithiacyclamideand ulicyclamideby Ireland and Scheuerin 1980 [30], the majority of the cyclopeptide alkaloids listed in Figure 1 have been isolated from ascidians, marine invertebrates that form the major class of the subphylum Urochordata (Tunicata). Ascidians are sessile filter-feeders and are also sometimes referred to as **sea squirts'" because they can contract and spray water when disturbed. Then* size varies from microscopic colonial forms to large (1-30 cm) solitary species [33,34]. A remaiicable preference for nitrogenous metabolites derived from amino acids is a characteristic feature of natural products isolated from ascidians [33].

2. SOURCES OF LISSOCLINUM PEPTIDE ALKALOIDS In the 18-membered cyclohexapeptide series, bistratamides A-D have been isolated from I. bistratum in Australia and the Philippines [3,4]. At least the Australian species of I. bistratum contains the same prokaryotic algal symbiont as L patella^ namely Prochloron [3]. Indeed, it has been suggested that this obligate prokaryotic symbiont contributes to the biosynthesis of lissoclinum peptides [5,6]. This hypothesis has been supported by the isolation of cycloxazoline (5, also known as westiellamide) from both an ascidian (I. bistratum) [5] and a terrestrial bluegreen alga (Westiellopsis proUfica) [6]. Indeed, more recently terrestrial blue-green algae (cyanobacteria) have provided several bistratamide-type cyclic hexapeptides. Dendroamides A-C were isolated in 1996fromthe cyanobacterium Stigonema dendroideum [7], and nostocyclamide (10) and raocyclamide A (11) were obtained from the cyanobacteria strains Nostoc sp. 31 [10] and Oscillatoria raoi [11], respectively. Even dolastatin E (9), isolated from the sea hare Dolabella auricularia [8], could be a cyanobacterium metabolite since Dolabella spp. feed on marine algae [11,35]. Accordingly, there is considerable circumstantial evidence that links the biosynthesis of 18-membered Lissoclinum peptides to cyanobacteria and related ascidian symbionts. In the structurally very homogeneous 21-membered cycloheptapeptide series, lissoclinamides 1-8 were all isolated from the aplousobranch ascidian Lissoclinum patella [12,13,14,17]. Since Z. patella is in symbiosis with unicellular algae [36], the actual source for the biosynthesis of the lissoclinamides has not yet been established. Lissoclinamide 6 (17) had originally been isolated as a trace compound, and was undetectable in more recent L patella collections. Upon standing in CDCI3, a solution of lissoclinamide 4 (15) exhibited signals characteristic for 17 [14]. Accordingly, it is possible that lissoclinamide 6 is an artifact of sample isolation or purification. Ulicyclamide (20), reported in 1980 in an extract from L patella from Palau [30], was among the first characterized examples of lissoclinum peptides and displays the


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alltaloids

195

same broadly conserved backbone structure motif that is typical for the lissoclinamides. The only slight variation of this motif is apparently foimd in the recently identified cyclodidemnamide (21) [20]. This cyclic heptapeptide was isolated from the marine ascidian Didemnum molle collected in the Philippines. However, a recent attempt toward its total synthesis came to the conclusion that the assigned structure of 21 was incorrect [21]. Due to the known configurational lability of thiazoles and thiazolines [18,28,31,37], an epimeric stereostructure such as 32 might be representative for natural cyclodidemnamide. Alternatively, an exchange of thiazoline and oxazoline heterocyclic moieties as shown for 33 would bring the sequence of cyclodidemnamide more in line with the other members in this structural class (Figure 2).

Figure 2. Alternative structural assignments for cyclodidemnamide.

N/

24-Membered cyclooctapeptide analogs are probably the structurally and synthetically most broadly investigated lissoclinum peptides. Patellamides A-F and ulithiacyclamide (29) have been isolated from L patella between 1980 and 1995 [13,14,24,28,29,30]. In addition, ascidiacyclamide (22) was isolatedfroman unspecified ascidian [22]. The potent cytotoxicity of the disulfide-bridged ulithiacyclamide, reported with ulicyclamide in the first paper on lissoclinum peptides in 1980 [30], has inspired much of the subsequent considerable analytical, biological, and synthetic work in this field. It is worthwhile to note that ascidiacyclamide and ulithiacyclamide are completely C2-symmetric compounds. The tawicyclamides (30, 31) are also L patella isolates [32], but their structural features are slightly different from the other 24membered cyclopeptide derivatives. Rather than the standard alternating sequence of fivemembered oxazole, oxazoline, thiazole, or thiazoline heterocycles and hydrophobic amino acid residues, an azole ring is replaced by a proline residue in the tawicyclamides. Since this type of


196

P. Wipf

Structural replacement maintains, however, the heterocyclic motif, and since prolines fulfill an apparently similar structural function in the 21-memberedlissoclinum peptides, tawicyclamides can still be considered to be members of this class. In addition to the 31 lissoclinum peptides shown in Figure 1, a large number of related azole-containing cyclopeptides, but with greater structural modifications than tawicyclamides, have been isolatedfrommarine and terrestrial sources. Among them, dolastatin 3 [38], keenamide A [39], mollamide [40], patellins 1-6 and trunkamide A (isolated from L patella) [41], raocyclamide B (a potential hydrolysis product or biosynthetic precursor of raocyclamide A) [11], and waiakeamide[42] are particularly noteworthy. Minor constituents of I. patella, e.g. prelissoclinamide-2, prepatellamide-B-formate, and preulicyclamide contain a threonine side chain in place of the oxazoiine heterocycle of the parent compound [43]. These compounds are considered to be biosynthetic intermediates in the peptide pathway rather than hydrolysis products, smce the conditions for acidic hydrolysis of the macrocycle-embeddedoxazolines are quite harsh [44]. A recent review on ascidian metabolites provides further background information on structurally related metabolites [33].

3. TOTAL SYNTHESIS OF LISSOCLINUM PEPTIDE ALKALOIDS Among the fourteen synthesized lissoclinum peptides to date, in nine cases the structure, in particular the configuration, was assigned or had to be corrected upon completion of the total synthesis. Accordingly, 65% of all structural assignments based on analytical methods of investigation, mostly NMR and chemical degradation, have been partially unsolved or misleading. This percentage has basically not changed during the past fifteen years, and is most likely due to the unusual structural characteristics, small available sample sizes, and the conflgurational lability of major segments of the natural products. Total synthesis remains the most general source for defmitive structural assignments of lissoclinum peptides, even though synthetic methods and strategies are also not completely immune to stereochemical errors [18]. Not surprisingly, synthetic progress in this field has been extensively reviewed [45,46,47], most recently in 1995 [48]. This review will concentrate therefore on the most current accomplishments.

3.1. Synthesis of Heterocyclic Building Blocks Oxazolines, oxazoles, thiazolines, and thiazoles C*azoles**) fonn the characteristic heterocyclic building blocks of lissoclinum peptide alkaloids. For each of these compounds, a


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

197

large number of published synthetic procedures is available. However, most of the pre-1980 protocols are not suitable for the preparation of functionalized or enantio- and diastereopure compounds, and several newer methods have addressed azole synthesis specifically in the context of the incorporation of these heterocycles in peptide sequences. The current methods allow heterocycle formation late in the synthesis, very often via the cyclodehydration of the cyclopeptide backbone in the last step to minimize epimerization problems. Other challenges for the synthesis of lissoclinum peptides present themselves in the development of effective macrocyclization strategies and convergent protocols. The preparation of oxazolines by condensation of amino alcohols and imidates (e.g. 34-f35->36) [49] has been virtually supplanted by the cyclodehydration of P-hydroxy-a-amino acid segments under Mitsunobu conditions [50,51], and, especially, with Burgess reagent [52,53] (Scheme 1) [54]. Peptidyl oxazoles can be formed by dehydrogenation of oxazolines, or by cyclodehydration of in situ prepared p-keto-a-amino acids [55,56] (Scheme 2). Suitable agents for the oxidation of oxazolines include Ni02 [57], CuBr2/DBU [58], CuBr/peroxide [59], and phenylselenylation/elimination [60]. Oxazolines with substituents at C(5) react sluggishly and in low yield, however.

Scheme 1. Oxazoline preparations [18,61,62].

C02Me

COaMe Ph "^

Ph

^ S

\.

\

l.TFA,0째C, I h

.NH O=/

BOCHN'I'

34 2. CH2CI2, t i , 2 d; 63%

^^IT" BocHN 35

^Y OMe

째v NHBoc


198

P. WIpf

Scheme 1. (continued)

O

o

'

\ H

I N

yP ^OH H'

y=0

H H.

).....

HO

P\\

i THF..60-»0°C

O

\

\_N'

H

I N

37

O, ^ O

H

).38

Ph^

o o Y

H O I

M

°

JI

Y

Burgess-Reagent

CbzNH^^Lj X ^ O M e 5"s/ H 3» / T)H

THF,70X.2h 81%

O 1

N

Jl

CbzNH-'^V X OMe 0—L^ 40 |S<

Scheme 2. Oxazole preparations [63,64].

O \ OH 9 H V ° " SesHN>.j^^X^OMe A H I \

OBn

^j

T

O

II

BOCNH-SV^OM, ^^

O

,.

^"s^l-OAc ACO OAC

iPhjP.ij.EtjN, CH2CI2, Ih

ono ^"? .• ^ sesHN

60%

Ni02, benzene. A, 22-53%

;^ Mn02, benzene, A, 34-36%

I

^,

_-^ .,

H^\^y=°^ ^

^


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alicaloids

199

Formation of thiazolines and thiazoles follows parallel strategies to the oxygenheterocycle syntheses but is more severely plagued by epimerization at several stereocenters (Figure 3) [37]. Besides the condensation of cysteine esters and imino ethers [65], cyclodehydration of P-hydroxy-a-thioamide segments under Mitsunobu conditions [50,51] and with Burgess reagent [37,53] represents a reliable route even to complex thiazoline products (Scheme 3) [66]. The necessary thiopeptides can be prepared by thionation of amides with Lawesson reagent [67], and, sequence specifically, by thioacylation [68] or thiolysis of oxazolines [69]. Oxidative conversion of thiazolines to thiazoles is relatively straightforward and often proceeds without loss of stereochemical integrity [66]. The popular Hantzsch synthesis of thiazoles from primary thioamides and a-halo ketones is more problematic. The use of modified reaction conditions at low temperature minimizes racemization [70,71,72], but not all amino acid sequences lead to high enantiomeric purities [73]. The oxidation of thiazolidines, obtained from amino thiols and aldehydes, to thiazoles with Mn02 [74] can also lead to racemization [73].

Figure 3. Thiazoline epimerization [18].

R-

R'

R' o

Acid/Base

^ R H N ' ^

RHN

Acid/Base

>

:

2 S

' RHN-"^

HN-C

^

N-C

CO'-'

^ CO-~Base

Base

R-

R2 g

RHN' ^

Acid/Base

Acid/Base

>

••

RHN

RHN

N-

HN

CO-

R' X 2 .S. 4 00-^

CO-

Scheme 3. Thiazoline preparations [16,18,75].

NH

BocNH^A,GEt 45

Cys-OMe«HCl, EtOH, 25 "C

BocNH 46

^••••COgMe


200

p. Wipf

Scheme 3. (continued)

V o

o L^s

0 0 X - s+

OH

O

Burgess-Reagent

^

>^

H-N ,Ph

PK

HO

THF. 22^55 "C 76%

. ^

. „

47

.OMe

Burgess-Reagent, THF,21 °C, 1 h, 54% or EtaNSOjNCOjCHjCHjOPEG, THF,21X, lh,63%

PMe

Scheme 4. Thiazole preparations [16,64,73].

NH BocNH Ph^

l.Cys-OMe-HCl, EtOH, 25 *»C 2. Mn02, CH2CI2

BocNH ^S^***^^^^ „ 51


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alloloids

201

Scheme 4. (continued)

.OMe NHTr

EtO HO

^

.OMe

1. Burgess-Reagent 2. Mn02, CH2CI2 ^ 73%

H

Me02C—4. A

53

54

COpEt

l.KHC03,DME,-15°C BrCH2COC02Et ^

S^N

2. TFAA, 2,6-lutidine, DME,-15°C 96%

NHBoc 55

56

NHBoc

^

Figure 4. Oxazoline—^thiazoline conversion [55].

H Rv^N

R

S R / HO path a HS' RvLjN R

^

R^^N R

rJ ^ y. "R

pathb \ R'S" H

T X O

R / R'S

R

H N

R

n rA o R/ HS


202

P. Wipf

An attractive new protocol is the oxazoline->thiazoline conversion. Thiolytic opening at C(2) or C(5) of an oxazoline provides intermediates that can be converted to thiazolines in high yield (Figure 4) [55,69]. Both pathways have successfully been applied in the total synthesis of natural products [18,63,75], but pathway b is only stereocontroUed for C(4)-disubstituted oxazolines. The major advantages of this method are (a) the use of synthetically much more readily available and configurationally more stable serine- or threonine-derived oxazolines as starting materials, (b) an essentially epimerization-free thiazoline preparation via cyclodehydration of intermediate thioamides, (c) thioamides are obtained sequence-selectively, (d) no side-chain protective groups are required, and (e) a chemoselective differentiation between substituted oxazolines is possible [18]. The investigation of structure-activity relationships of oxazolmeand thiazoline-containing compounds is considerably simplified with these protocols. Triple conversions have been carried out in high yield (Schemes 5 and 6).

Scheme 5. Triple oxazoline-^thiazoline conversion via path a [18].

N/ O

ツーCr

\

H

>窶年

Burgess-Reagent THF,22->55*^C

76%

V P^

/ O

''A=o

i*""^"- s^_7

_

36X,36h

/

q

>"^^,

th'^'l

}ツォ_^S r-OH

^ H^^iA^O ,,H-N./


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

203

Scheme 6. Triple oxazoline-Âťthiazoline conversion via path b [63].

N^^O

AcSH ( t i )

60

SAc

l.NH3,MeOH 2. TiCl4, CH2CI2 40%

3.2. Total Synthesis of Dolastatin E The 2-D NMR analysis of the natural product established the gross structure of dolastatin E, but was inconclusive regarding its stereochemistry [8]. A total of eight possible stereoisomers were subsequently synthesized, and the spectroscopic, chromatographic, and chiroptical properties including bioactivity of structure 9 were identical with the natural material [9]. The successful synthesis used Mitsunobu reactions for thiazoline and oxazoline preparations, and oxazole and thiazole segments were obtained by oxazoline oxidation via phenylselenylation/elimination and modified Hantzsch synthesis, respectively. Condensation of Boc-L-alanine with L-serine methyl ester in the presence of diphenylphosphoryl azide (DPPA, [76]) followed by cyclodehydration [50,51] with diisopropyl azodicarboxylate (DIAD) and triphenylphosphine provided oxazoline 64 (Scheme 7).


204

P. Wipf

Aromatization of 64 with Ni02 was low-yielding (6-22%), but phenylselenylation/eliminatioii [60] led to oxazole 65 in 42% yield. DPPA coupling was also used for the synthesis of the DAla-D-Ser peptide 67 (Scheme 8). Segment condensation with the a//o-D-isoieucine-derived thiazole 68 with diethyl phosphorocyanidate (DEPC, [77]) proceeded to givetetrapeptide 69 in 78% yield. Selective thionation of 69 with Belleau's reagent [78] provided the desired thioamide in 44%, along with ca 5% of other thionation products. A second segment coupling and macrocyclization was successfully performed with DPPA. Macrolactamization via a C-terminal thiazole is generally efficient and avoids racemization problems [48]. The final thiomnide cyclodehydration, a key step in the synthesis, was performed under Mitsunobu conditions to provide dolastatin E in 20% yield in addition to the elimination product 72. Based on Boc-Lserine, the overall yield in this total synthesis amounted to 0.9%, mostly due to low efficiencies in the final two steps.

Scheme 7. Dolastatin E segment preparation [9].

OH L-Ser-OMe, DPPA, TEA

y

y 1

OH

DMF,ox,94%

^

Y I

f

N ^

n dr\

OMe

63 DIAD,Ph3P

o

uKi

^"^

BocHN

THF, 0 X , 78%

O -TV

^ ^

\ - - ^ . . > ^ i

5 ^'

|V|'''^C02Me

NaOH, H20/MeOH

l.Boc20,DMAP,MeCN,rt 2. KN(TMS)2, THF/toluene; PhSeCl, -78-Âť0 X 3.30%H2O2,CH2Cl2,0X 42%

O -rv


Synthesis and Structure-Activity Studies of Lissocfinum Peptide Allcaloids

205

Scheme 8. Dolastatin £ segment condensation [9]. EtOgC

V-N 68

OTBS BocHN

// V \ « )>^....NH2 '

BocHN ^ ^ ^ N^.,.-^OTBS

OH DEPC, TEA, DMF, 0 "C, 78%

K^/^O

O 67

EtOgC^^N^. 69

1. Belleau's reagent, THF, It, 44% 2.TFA,CH2Cl2.0X 3. 66, DPPA, TEA, DMF 0°C,65%

°^2'^ E t O a C ^ ^,N ^

^N

l.NaOH,H20/MeOH,rt ^. 2. TFA, CH2CI2, 0 °C 3. DPPA, TEA, DMF 0 °C, 22%

70

/ ^ O^N T

u H

M /-OH Nw L DIAD, PhaP, THF, 0 X

O.X.N

H

N^/

o^N

H

^M-J'

A - " "~N^° * X^'» "-.H-^O 20%

Dolastatin E (9)

72


206

P. Wipf

33. Total Synthesis of Lissoclinamide 4 The initial structure identification [13,14] of the L patella metabolite lissoclinamide 4 assigned both phenylalanine residues the 5-configuration, and the valine residue as R [14]. This stereostructure was proven to be incorrect by total synthesis [15]. A preparation of the (S)' valine-(i^)-phenylalanine isomer 15 provided synthetic material that was spectroscopically identical with the natural compound [15]. Condensation of imino ether and cysteine ester followed by oxidation with activated Mn02 was used for thiazole synthesis, and both oxazoline and thiazoline heterocycles were formed simultaneously late in the syntl^sis by cyclodehydration with Burgess reagent. iV-Deprotection of the phenylalanine-derived thiazole 73 [66], followed by coupling with serine provided tripeptide 74 in 91% yield (Scheme 9). Thioacylation with reagent 75 [79] led to thioamide 76 in 74% yield. S'eco-lissoclinamide 4 (79) was readily obtained by sequential couplings of 76 with a//<7-threonine and dipeptide 78. After basic and acidic protective group cleavages, macrolactamization with pentafluorophenyl diphenylphosphinate (FDPP, [80,81]) gave the desired 21-membered macrocycle 80 in 32% yield.

Scheme 9. seco-Lissoclinamide 4 synthesis [15].

HO BocHN>

Me02C

1.50%TFA/CH2Cl2,25X NHBOC

Ph

1.50% TFA/CH2CI2,25 "C

2.Boc-L-Ser.OH(2.5eq),' DCC (1.25 eq), TEA, CH2CI2,0 X, 91%

MeOzC-^fe

BocHN

2.NaHC03,CH2Cl2/H20 3.DMF.0''C,74Âť/o

Ph 74

1.50%TFA/CH2Cl2,25°C O

O

MeOgC-^j^S

wo

H-N \

^^

2. Boc-flr//o-L-Thr-OH, DCC, HOBt, /-Pr2NEt, DMF, 0->25 X , 71%


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Ailtaloids

207

Scheme 9. (continued)

V.OH BocHN-\

W

S

V-N

o /

//

H'

OH ^ ^

1.50%TFA/CH2Cl2, 25 °C

r^

2. 78, DCC, HOBt, /-Pr2NEt, DMF, 0-»25 °C, 77%

\AV=0

" H ~N

MeOgC-^S

^

''

CO2H

6. N \

L J^ NHBoc

^"^ V

/"""^ NHBoc

N-

s 79

The final step, simultaneous ring closure of oxazoline and thiazoline heterocycles, was performed in 61% yield by exposure to 2.2 equiv of Burgess reagent in refluxingTHF (Scheme 10). Minor traces (ca. 5%) of other diastereomers formed in this reaction were separated by HPLC. Overall, the yield of this linear total synthesis was 7% from thiazole 73.

Scheme 10. Lissoclinamide 4 cyclizations [15].

l.NaOH,MeOH/H20 79

• 2. 50% TFA/CH2CI2, 25 °C 3. FDPP, f-PrjNEt, DMF, 25 °C, 72h, 32%

oVvO /

O

Ph

^


268

P. Wipf

Scheme 10. (continued)

Burgess-Reagent, THF, 65 *»C, 30 min

•ys-^^^ V=:N >—"^

r^^

^ _|Sj

Lissoclinamide4(15)

3.4. Total Synthesis of Lissoclinsmide 7 Lissoclinamide? is the only lissoclinum peptide that contains two epimerization-prone thiazolineringsin addition to the oxazoline heterocycle and thus presents formidable challenges to an asymmetric total synthesis. Structural characterization of the L patella metabolite was inconclusive at the stereocenters next to the thiazolines [17], and a total of four stereoisomers could be formulated for the natural product. A recent total synthesis of lissoclinamide 7 imambiguously established the stereochemistry as shown for 18 [18]. An efficient macrocyclization strategy was combined with the use of Burgess reagent for multiple simultaneous oxazoline and thiazoline formations and an oxazoline-^thiazoline conversion, hi addition to the natural product, several isomers and analogs were prepared. The x-ray structure of lissoclinamide 7 secured all stereochemical assignments and provided the first secondary stricture information for 21-membered lissoclinum peptides. Tripeptide 81 was obtained from L-phenylalanine by mixed anhydride couplings with Lproline and L-threonine (Scheme 11, [18]). The natural (i?)-stereochemistry of the side chain of the threonine residue in 81 was selectively inverted via the intermediate oxazoline, hydrolysis, and 0-^N acyl shift [82]. Segment coupling with tetrapeptide 83, prepared by standard mixed anhydride strategy in 51% overall yield from D-phenylalanine, gave seco-lissoclinamide7 (84) in 81% yield with FDPP as a coupling agent. FDPP was also successfully used for the macrolactamizationof 84 in 48% yield (Scheme 12). A sequence of selective cyclodehydration and thiolysis steps was used to convert the peptide backbone of 85 into the heterocyclic lissoclinamide 7. After silylation of the secondary hydroxyl group in 85, the primary TBS-ethers


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

209

were hydrolyzed and the resulting diol 86 was converted to the bis-oxazoline with Burgess reagent. Thiolysis with hydrogen sulfide in methanol/triethylamine followed by deprotection of the TIPS-ether provided bis-thioamide 87 in 41% yield. A triple cyclodehydration simultaneously installed an oxazoline and two thiazoline rings and completed the total synthesis of lissoclinamide 7 in 90% yield. The overall yield for 18 based on Cbz-phenylalanine was 4.6%. Scheme 11. seco-Lissoclinamide 7 synthesis [18].

H

Cbz

, N,

l./-BuOC(0)Cl,NMM, L-Pro-OMe, CH2CI2

.CO2H

2. LiOH, THF/H2O 0-->22 °C 3./-BuOC(0)Cl,NMM, L-Thr-OMe, CH2CI2

Ph^

M

JI / "•^-'"""^N \ "i \ / p^-^ ^ ^

Cbz ^

81

66%

7(S) 1. Burgess-Reagent, THF, A

O

I

2. 0.3 M HCl, THF/H2O; then 2 M K2CO3, pH 9.5 80%

l.H2,Pd/C,MeOH 2. 83, FDPP, EtsN, CH2CI2, 81%

^V-NH

Cbz ^ ^ - ^ N A

82

Ph .Ph H

^VKVT^MV

HN Cbz O

"OTBS 84

HO... COaMe

OTBS Cbz-

.OTBS CO2H


210 Scheme 12. Lissoclinamide 7 cyclizations [18].

P. Wipf

H9 O 1. H2, Pd/C, McOH 2. NaOH, THF/MeOH/H20

O

"

r \

3.FDPP,NaHC03 DMF(3:1),40*»C

\J>

-H

r

^OTBS

H-N H

V.. ^ P h H H }....,

HN

H

°VrS °..

'0 48%

Ph^

l-OTf.2,6.|utidine, l.TIPS-OTf,2,6-lutidine, CH2CI2 !C>2 2.TsOH, ^,THF/H20 61%

\ >0 TIPSO O _ 1I ^^ L^^ ,/ ^ / — \\ ^ ^ "^ "^ • o" " ^ 7^^ ili ."^ >\ N " H

^OH C \ ^\ /^ ) ^ ^

r ^

\

. . . .

>^

N^

P\{

HO O V-N

f M ^N H

Ph

\

^S "

r.^

/

\L

HO

M

).....

I THF, 40-»70 "C

^/-

1.Burgess-Reagent, THF,65X 2.H2S,McOH/NEt3 22^C,4d

p h 3.TBAF,THF, •^^ 37 "C

OH

/ ^ H-NT

H.

OTBS

O

L_/*^

>=:N

/ p N

r

H

H-N )..

87 UssocUnonMe 7 (IS)


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

211

3.5. Total Synthesis of a Structural Isomer of Cyclodidemnamide Cyclodidemnamide has been isolated from the marine ascidian Didemnum molle and an (5)-stereochemistry has been proposed for the valine-derived stereocenter of 21 [20]. Based on a recent total synthesis of this isomer that provided material spectroscopically significantly different from the natural product [21], this stereochemical assignment has to be revisited. Similar to the strategy used for lissoclinamide 4, a linear coupling and thioacylation sequence was employed for the preparation of macrocycle 95. Chain extension of L-valinederived thiazole 88 via DCC-mediated coupling with L-serine, thioacylation with L-proline derivative 90, and condensation with L-valine, a//o-threonine, and D-phenylalanine provided heptapeptide 93 in 26% yield (Scheme 13). Attempts to cyclize a derivative of 93 with unprotected hydroxyl groups failed. After protection as the bis-acetate, macrocyclization occurred smoothly in the presence of DPPA to give 94 (Scheme 14). Saponification followed by simultaneous thiazoline and oxazoline ring formation with Burgess reagent provided the cyclodidemnamide isomer 95 in an overall yield of 6% from thiazole 88.

Scheme 13. Seco-95 synthesis [21].

Me02C \ _ N

^ • 5^^/° TFA/CH2CI2,0 ° c

\QA--|^'^'^^^^

I -"""^

2. Boc-L-Ser-OH, D C C ,

HOBt, DIEA, CH2CI2, 0 °C-»rt, 80%

S

1.50%TFA/CH2Cl2,0°C

OH

1.50%TFA/CH2Cl2,0°C

Boc H >=^0 H-N

2. Boc-L-Val-OH, DCC, HOBt, DIEA, CH2CI2, 0 °C-»rt, 79%

^ 2. NaHCOa, CH2CI2/H2O 3. DMF, 0 °C-->rt, 77%

o VNH


212

p. Wipf

Scheme 13. (continued) 1.50%TFA/CH2Cl2,0X 2. Boc-L-flr//o-Thr-OH, DCC, HOBt,DIEA,CH2Cl2, 0 ^'C^rt, 72%

BocHN

3.50%TFA/CH2Cl2,0X 4.Boc-D.Phe-OH,DCC, HOBt,DIEA,CH2Cl2, 0 **C->rt, 74%

H-N

OH

Me02C-<^S

O /

>i

93

H-N

\

NHBoc

^/ Ph

Me02C

N=^r

Scheme 14. Cyclization steps toward 95 [21].

AcO O

O X

>.Sv,

1. NaOH, MeOH/H20,0 째C 2. AC2O, TEA, DMAP, DMF 93

)=0

3.50%TFA/CH2Cl2,0X 4. DPPA, i-PrjNEt, DMF, 0-^25 X , 72 h, 74%

H-N

0^ /

l.K2C03,MeOH/H20 0 ^C, 1 h ^ 2. Burgess-Reagent, THF, A, 2 h

' \

H I

AcO

Ph

)" N=r/

30%

95

^^


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

213

3.6. Total Synthesis of Nostocyclamide The bisthiazole-oxazole nostocyclamide (10) has been characterized as an allelochemical from the cyanobacterium Nostoc sp. [10], A recent total synthesis uses modified Hantzsch conditions for thiazole preparation, and provides an example for the use of rhodium-catalyzed A^H insertion for oxazole synthesis [83]. Rhodium(III)-acetate catalyzed coupling of Cbz-L-alanine amide with methyldiazoacetate 96 [84] provided dipeptide 97 in 71% yield (Scheme 15). Oxazole 98 was obtained in 66% by cyclodehydration with electrophilic phosphonium reagent and hydrogenolysis. For the synthesis of thiazole 99, Boc-protected D-valine amide was converted to the thioamide, treated with ethyl bromopyruvate, dehydrated with trifluoroacetic acid anhydride, and saponified.

Scheme 15. Segment synthesis [83].

CbzHN

1

NHCbz .NHg

••••V

H

v^COgMe

Rh2(OAc)4, CHCI3, A, 71%

l.Ph3P,l2,NEt3 CH2CI2,66%

HgN

^ 2. H2, Pd/C, MeOH 96%

1. Lawesson Reagent, 78% 2. BrCHjCOCOjEt, KHCO3, DME,-15°C •

NHBoc

3. TFAA, 2,6-lutidine, 73% 4. LiOH, MeOH>ai20,100%

0-4

98

NHBoc


214

P. WIpf

Segment condensation was initiated by activation of 99 as the mixed anhydride and coupling with amine 98 (Scheme 16). Boc-deprotection and coupling with thiazole segment 101, which was prepared analogously to 99, i»ovided seco-nostocyclamide 102 in 72% yield. The natural product was obtained by conversion of the carboxyl-terminus into the pentafluorophenyl ester, ^-deprotection in HCl/dioxane,and macrocyclization in a two-phase chloroform-aqueous bicarbonate system. This efficient total synthesis provided (+)-nostocyclamide in 16% overdl yieldfromCbz-alanine amide.

Scheme 16. Nostocyclamide synthesis [83].

W'

^C02Me

l./'BuOjCCLNMHTHF 0''C;then98,71% 2.ACC1.MCOH.100V.

O ^N ^

/-BuOjCCI, NMM, THF,0"'C,72%

I ^

^

I,!^ 100

« ^

r HO2C

/^NHBOC *•*

NHBoc

V<^oMeSr\ iss^X'Sfcu Vr^*^^^^/)

102

Nostocyclamide (10)


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

215

4. SECONDARY STRUCTURES OF LISSOCLINUM PEPTIDE ALKALOIDS Upon introduction of flve-membered heterocycles into the cyclopeptide sequence, the conformationally flexibility of the 18-24 membered macrocycles is considerably reduced, and mostly a single secondary structure is preferred in the solid state and in solution. Accordingly, a better understanding of the effects of amino acid-derived thiazoline and oxazoline heterocycles on the conformation of cyclopeptides can facilitate the de novo design of 3-dimensionally preorganized macrocycles. Structural analysis of lissoclinum peptides has focused almost exclusively on 18- and 24membered macrocycles. These derivatives assume molecular "triangle", "square", and "twisted eight" conformations in solution and the solid state. No detailed conformational analyses of the solution structure of 18-membered lissoclinum peptides by NMR methods have been published. However, several solid state conformations are known from x-ray analyses. Cycloxazoline (westiellamide, 5) shows a typical planar ring with less than 1 A deviation from planarity [5]. Noteworthy is the disposition of the peptide N-H groups into the ring, and the peptide carbonyl functions toward the outside, which places the valine isopropyl side chains into an axial arrangement (Figure 5). A molecular dynamics simulation of bistratamide C established an analogous planar three-dimensional structure for 3 [4]. Since the x-ray analysis of the closely related nostocyclamide (10) also showed a planar macrocycle with a maximum deviation of any backbone atom from its ring plane of less than 0.2 A (Figure 6, [10]), one can conclude that irrespective of the oxidation state of the azoles and the nature of the side chain substituents, 18-membered lissoclinum peptides have a strong preference for a planar, porphyrin-like macrocycle geometry with all nitrogen atoms oriented toward the center of the molecule.

Figure 5. Stereoview of the x-ray structure of cycloxazoline (westiellamide, 5) [5].


216

P. Wipf

Figure 6. Stereoview of the x-ray structure of nostocyclamide (10) [10].

Considerable information about solution and solid state conformations of 24-membered lissoclinum peptides is available. Most prominent is a distorted **molecular square" or saddleshaped conformation with heterocycles placed at the comers of the rectangle. All nitrogen atoms are positioned at the mside of the ring in this conformation, and carbonyl groups are pointing outwards. This motif is illustrated by x-ray structures of ascidiacyclamide (22) [85,86,87] and patellamide A (23) [88,89] (Figures 7 and 8). The pseudo-axial attachment of the side-chains on the macrocycle is clearly visible in the stereoviews. The C2-symmetric structure for 22 and 23 observed in the crystal appears to be essentially the same as the solution structure based on NMR experiments [86]. The solvent interaction with H2O, EtOH, and benzene has been studied for ascidiacyclamide [87]. Very similarly, patellamide A also displays a saddle-shaped rectangular form wrapped around the H2O and MeOH solvent molecules (Figure 8) [88,89]. The typical pseudo C2-symmetric square was also shown by ^H NNfR analysis to be predominant for ulithiacyclamide in solution in nonpolar solvents [90]. This group of 24-membered lissoclinum peptides resembles therefore most closely the conformation of the 18-membered macrocycles. At least ulithiacyclamide, however, is thought to undergo some conformational change at the amide groups in a more polar DMSO solution [90].


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alicaloids

217

Figure 7. Stereoview of the x-ray structure of ascidiacyclamide (22) [85].

Figure 8. Stereoview of the x-ray structure of patellamide A (23) [88,89].

In a drastic departure from the conformations observed for ascidiacyclamide, patellamide A, and ulithiacyclamide, the x-ray structure of patellamide D (26) shows a severely folded, twisted "figure eight" backbone (Figure 9) [14]. Four transannular hydrogen bonds stabilize this compact geometry, and only the phenylalanine side chain protrudes away from the backbone, whereas the isoleucine side chain clusters onto the macrocycle. The isoleucyloxazoline segments form a pair of type II P-tums [91] stabilized by intramolecular hydrogen bonding. Two additional hydrogen bonds are formed between the isoleucine amide nitrogens and the oxygen atoms of the oxazoline rings. The two thiazole rings are oriented nearly parallel, with a separation distance of 4.1 A.


218

P. Wipf

Figure 9. Stereoview of the x-ray structure of patellamide D (26) [14].

The distinctly different solid state conformation of patellamide D compared with diat of ascidiacyclamide and patellamide A demonstrates that there are at least two preferred conformations for 24-membered lissoclinum peptides. A decrease in the C2-symmetry of the macrocycle has been suggested as responsible for a transition from the ''molecular square" to the 'twisted eight** conformation [88,92]. Patellamide D contains a phenylalanine residue opposite an alanine residue, whereas ascidiacyclamide is completely symmetrical. In patellamide A, the only deviation from C2-symmetry is the replacement of a threonine-derived (C(4) methylated) oxazoline with a serine-derived (C(4) unsubstituted) oxazoline. Patellamide B and C, which also have non-C2-symmetrical side chain substituents, were recently shown to have twisted solution conformations that resemble patellamide D [92]. An alternative interpretation focuses on the steric properties of ^-branched amino acid side chains [93]. Both ascidiacyclamide and patellamide A contain exclusively P-branched valine and isoleucine amino acid building blocks, whereas patellamides B, C, and D have two isoleucines or valines next to alanine and phenylalanine residues. The severe steric hindrance induced by ^-branched side chains prevents collapse of the macrocycles into the 'twisted eight* conformation [93]. In ulithiacyclamide, the disulfide bridge prevents the hydrophobic collapse sterically in a related fashion. The exclusive presence of P-branched amino acid side chains appears to be a stringent requirement for 'molecular square'-like geometry. Tawicyclamide B (31), which lacks this feature, assumes the familiar 'twisted-eight' conformation (Figure 10) [32]. Interestingly, upon oxidation of the thiazoline ring to the diiazole, the valine-proline amide bond isomerizes from cis to trans [32].


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Allcaloids

219

Figure 10. Stereoview of the x-ray structure of tawicyclamide B (31) [32].

One of the major attractions of lissoclinum peptides is the use of their unique structural features for the design of conformationally preorganized peptide and peptidomimetic sequences. Oxazoline and thiazoline heterocycles impose considerable conformational restrictions onto the macrocycles. A recent 2D NMR study has demonstrated that conversion of two cysteine residues in the flexible cyclooctapeptide c[Ile-Thr-D-VaI-Cys-IIe-Thr-D-VaI-Cys-] (a sequence derived from ascidiacyclamide) results in a single, pseudochair solution conformation [94]. Com ersely, rearrangement of the two threonine residues in this sequence to amino esters (in practice, this compound was obtained by hydrolysis of ascidiacyclamide), provides a more shallow, less constrained pseudo-boat conformation than ascidiacyclamide itself [94]. An x-ray structure of the potassium complex of the amino ester is shovm in Figure 11 [95].

Figure 11. Stereoview of the x-ray structure of ascidiacyclamide hydrolysis product-potassium complex [95].


220

P. Wipf

The almost symmetrical array of oxazolines, oxazoles, thiazolines, and thiazoles in lissoclinum macrocycles is reminiscent of ring-extended porphyrins [96] and aza-crown ethers [97] and has nurtured considerable speculation about potential metal-ion chelation properties of these natural products [98]. The fundamental validity of this hypothesis has been confirmed by the characterization of three metal complexes of lissoclinum peptides to silver(I) and copper(II) ions. Cycloxazoline (westiellamide, 5) binds silver cations strongly and selectively over Cu(I), Cu(II), Hg(II), Co(I), and other metal ions [99]. In the solid state and in solution, an unprecedented sandwich complex is formed which stabilizes a cluster of four silver cations (Figure 12). Noteworthy here is also the conformation of the macrocyclic rings compared to tl^ unchelated structure shown in Figure 5: Upon binding to Ag(I), the macrocycle flips inside-out with carbonyl groups pointing toward the center of the molecule and side chains positioned in a pseudo-equatorial orientation. This conformational switch reveals another interesting feature of lissoclinum peptides, i.e. the possibility to induce transitions between well-defined conformational states by external binding events. Since the association constant for formation of the cycloxazoline-silver cluster averages2.8 x 10^^ M"5 in d4-methanol/E)20 (9:1), the lack of conformational organization does not appear to be detrimental to tight, selective binding.

Figure 12. Stereoview of the x-ray structure of [(cycloxazoline)2Ag4](C104)4 complex [99].

A greater degree of preorganization of the parent macrocycle toward metal ion chelation is shown in the crystal structure of the bis(copper(II)) complex of ascidiacyclamide (Figure 13) [100]. The presence of a bridging carbonate in the complex is remarkable and offers the intriguing possibility that a biological function of the natural product might involve CO2 transport [100]. The detailed characterization of a related copper(II) patellamide D complex has been complicated by the occurrence of several solution equilibria [101].


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Alkaloids

221

Figure 13. Stereoview of the x-ray structure of a biscopper ascidiacyclamide complex [100].

It is clear from the present data that the lissoclinum peptide alkaloids provide a fascinating range of structural features that might well become useful for the design of conformationally preorganized macrocycles or linear strands with tailored physical or biological properties. Among areas for future investigations, the exploration of conformational switches and the determination of the structural characteristics of 21-membered lissoclinum peptides appear particularly attractive.

5.BIOLOGICALACTIVITYOFLISSOCLINUMPEPTIDEALKALOIDS In the previous chapter, a possible link between metal ion chelation properties of lissoclinum peptide alkaloids, carbon dioxide transport and biological function was already mentioned. However, to date the most extensively documented biological effect of these marine natural products is cytotoxicity. Tables 1-3 summarize the reported cytotoxic activity for 18-, 21-, and 24-membered lissoclinum peptide alkaloids, respectively. Quite frequently, mouse leukemia cells (LI210), SV40 transformed fibroblasts (MRC5CV1), transitional bladder carcinoma cells (T24), and human colon cancer cells (HCT-116) were used in these evaluations. The most active compounds appear to be lissoclinamide 7 and ulithiacyclamide; however, cell types and assay conditions have


222

P. Wipf

been widely varied for different natural products and research groups. The presence of contaminating cytotoxic impurities is a major concern with natural product samples. In the absence of any information on the biologicalmechanism of action of lissoclinum peptides, any conclusions as to the actual potency of these compounds are preliminary at best. The only pharmacological studies that are currently available focus on ulithiacyclamide and ulicyclamide. Ulithiacyclamide was found to have a strong inhibitory effect on protein synthesis and can potentiate the cytotoxicity of anticancer drugs such as bleomycin [102]. Interestingly, ulithiacyclamide self-distnicts in the process of inhibiting cell growth. Ulicyclamide, in contrast, was shown to inhibit DNA and RNA syntheses [103]. Several lissoclinum peptides show only moderate levels of cytotoxicity in cell assays. In spite of sub-micromolar activity against bladder carcinoma and SV40 transformed fibroblast cells, cycloxazoline (westiellamide) had no activity in solid tumor assays [6].

Table 1. Cytotoxic activity of 18-membered lissoclinum peptides (IC50 [mg/mL]). T24 cells

MRC5CV1 cells

1 BistratamideA

50

50

[3]

BisiraiamideB

>100

>100

[3]

HCT-116 cells

other cell lines

reference

1 BistratamldeC

125

[4]

I BistratamideD

125

[4]

0.5

0.5

2

[5,6]

22^0

[8]

1 Nostocyclamide

12

[10]

1 Raocyclamide A

<30

_ _ill]

Cycloxazoline Dolastatin E


223

Synthesis and Structure-Activity Studies of Lissoclinum Peptide Allcaloids

Table 2. Cytotoxic activity of 21-membered lissoclinum peptides (IC50 [mg/mL]). L1210 ceils

T24 cells

MRC5CV1 cells

HCT-116 cells

lymphocytes

reference

1 Lissoclinamide 1

>10

[12]

1 Lissoclinamide 2

>10

[12]

1 Lissoclinamide 3

>10

[12]

1 Lissoclinamide 4

1

1

12

[13,14]

1 Lissoclinamide 5

10

15

10,20

[14,17]

7

[14]

1 Lissoclinamide 6 1 Lissoclinamide 7

0.06

0.04

0.08

[17]

1 Lissoclinamide 8

6

1

8

[17]

1 Ulicyclamide

7

[19] 16

1 Cyclodidemnamide

[20]

1

Table 3. Cytotoxic activity of 24-membered lissoclinum peptides (IC50 [mg/mL]). L1210 cells

T24 cells

MRC5CV1 cells

HCT-116 cells

1 Ascidiacyclamide

other cell lines <10

reference 1

[22]

1 Patellamide A

3.9

[19]

Patellamide B

2.0

[19]

Patellamide C

3.2

[19]

Patellamide D 1 Ulithiacyclamide

0.35

0.15

0.2

11

[13,14]

0.01

[13,19]

1 Tawicyclamide A

31

[32]

1 Tawicyclamide B

31

[32]

1


224

P. Wipf

Nostocyclamide (10) was shown to exhibit growth inhibitory activity against diatoms, chlorophyceae, and cyanobacteria {Anabaena P-9 and others) at 0.1 ^M concentration [10]. These data support the notion that chemical defense directed against predators or competitors is a potential biological function of these secondary metabolites. Dendroamide A (but not B and C) was active in reversing multidrug-resistance due to inhibition of drug transport by P-glycoprotein [7]. Patellamide D (26) has also been reported to reverse multidrug-resistance in a human leukemia cell line [104].

A comparison of the cytotoxic effects of naturally occurring lissoclinum peptides, synthetic cyclic peptides and relatively short linear segments served as the basis for the hypothesis that the oxazoline function is essential for cytotoxicity and that a cyclic skeleton might not be needed [105]. While this hypothesis has found considerable support [32], no conclusive evidence for or against it, and no molecular rationalization for an oxazoline-induced cytotoxicity is available to date [106].

6. OUTLOOK Where are the future challenges and opportunities for research on the lissoclinum peptide alkaloids? They will certainly ciystallize in the triangle of synthesis, conformational analysis, and biological activity. Synthesis will continue to be a critical tool for structure assignment and supply of material, but further improvements in strategy and synthetic methodology will be needed to increase overall yields from commercially available starting materials by an order of magnitude consistently to the double-digit level. More conformational studies in the solid state and especially in solution are needed to understand and predict the subtle effects of structure and metal binding on the secondary structures of the macrocycles. The role of metal ion binding in the biological function of lissoclinum peptides may hold many surprises, and the investigation of the molecular mechanism causing cytotoxicity should not be neglected. Last but not least, the continued exploration and isolation of marine and other natural products is a crucial source for the structural diversity pool that inspires so many new discoveries in chemistry and biology, and one can hope that new members and classes of lissoclinum peptides will be revealed in due course.


Synthesis and Structure-Activity Studies of Lissoclinum Peptide Aiicaloids

225

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56.

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58.

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74.

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75.

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76.

T Shioiri, K Ninomiya, and S Yamada, J Am Chem Soc 94:6203 (1972).

77.

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80.

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CJ Moody, and MC Bagley, Synlett 1171 (1996).

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85.

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86.

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87.

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89.

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90.

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91.

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T Ishida, Y In, F Shinozaki, M Doi, D Yamamoto, Y Hamada, T Shioiri, M Kamigauchi, and M

93.

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94.

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99.

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Chapter Four

Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids Michael B. Smith Department of Chemistry The University of Connecticut Storrs, Connecticut 06269-4060 U.S.A.

CONTENTS 1.1. 1.2. 1.3. 1.4. 1.5.

Introduction Preparation of Pyroglutamic Acid Derivatives from Glutamic Acid Structure and Properties of Pyroglutamate Derivatives Functional Group Manipulations of Pyroglutamate Derivatives Synthesis Using Pyroglutamate Derivatives as a Starting Material 1.5.1. Synthesis of Amino Acids 1.5.2. Synthesis of Alkaloids and Other Natural Compounds 1.5.3. Synthesis of Antibiotics, Antibacterials, and Other Pharmaceuticals 1.5.4. Synthesis of Other Important Compounds

REFERENCES

230 230 235 237 252 253 257 267 271 279

229


230

M. B. Smith

1.1. Introduction One of the most common synthetic strategies for constructing natural products and other important molecules is to use a chiral, nonracemic starting material. The availability and utility of amino acids Imve made them popular stalling materials in a variety of applications. For moie than 50 years, glutamic acid has been an important starting material using the ^-form as well as D-glutamic acid and the more common Lglutamic acid. This review will focus on an important derivative of glutamic acid, 5-oxoproline (also known as pyroglutamic acid or pyrrolidin-2-one, 5-caiboxylic acid). This functionalized lactam is very useful and can be converted to a wide variety of structurally different products. The review will begin by defining the properties of pyioglutamate, move on to the impoitant reactions of pyroglutamate, and then focus on actual syntheses using pyroglutamate. Rigo recently reviewed the chemistry of pyroglirtamic acid, with an emphasis on reactions of heterocyclic derivatives.! There will some overlap but the emphasis of this current review will be on synthetic applications, patticulariy as applied to the preparation of amino acids, alkaloids, and other molecules of interest. There are methods for preparing pyroglutamate derivatives, patticulariy chiral, racemic derivatives, which do not involve the use of glutamic acid. In (Hder to highlight the use of pyroglutamate derivatives in synthesis, this review will focus exclusively on compounds derived from />, D-, or AL-glutamic acid.

1.2. Preparation of Pyroglutamic Acid Derivatives from Glutamic Acid L-Glutamic acid (1) is an inexpensive and readUy available amino acid. If it is heated between 160180째C, usually for a few hours, a cyclization reaction occurs to give 5-oxoproline (pyroglutamic acid, 2) in good yield.2 This is a very old and commercially useful process.^ Temperatures as high as 200^C have

H2Nl*^

CO2H 1

^ 2

been reported for the formation of 2,^ but Beecham reported that racemization occurred at C5 when the reaction was done at a temperature greater than 190X.^ In a preparation of ^^-labeled 2, however, retention of configuration was observed when the reaction was done at 205째C, in vacuo (0.3 mm).^ If 1 is heated with acetic anhydride, 2 is formed quantitatively.^ When L-1 is refluxed in water for 15 hours and


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

231

then passed through a Dowex AG-500X8 ion exchange resin, 2 is isolated in 86% yield. When 1 is lefluxed with hexamethyldisilazane in xylene and then treated with ethanol, 2 is formed in 93% yield. It is clear that enantiopure 2 is easily formed from the readily available and enantiopure starting material, glutamic acid. ^^\^^'J<^\^ O

PCI5. ether ^ ^ v Y ^ V

1^

^

3

SOClj^heat

T

|'^'^^C02H TsHN^'^^COjH

4

5

There are several simple derivatives of 2 such as esters and amides that are also readily formed from glutamic acid, usually by traditional methods. Esters and amides can be formed by direct conversion of 2 or 1 (and ester derivatives of 1) to an ester or amide, and amides can be formed from the corresponding ester. Formation of the acid chloride of 2 is straightforward. When the N-tosyl lactam (3) is treated with phosphorus pentachloride, for example, acid chloride 4 was isolated.^ This acid chloride can also be prepared directly from a glutamic acid derivative. When N-tosylglutamic acid (5) was treated with hot thionyl chloride, 4 was produced,^ in yields up to 93%.^^

"V^o O

^

NaH

'ツー""\^\

J窶能

HO-

3

6

Once the acid chloride is in hand, it can be used to prepare an ester or amide. Aryl esters such as 6 have been prepared using this method, in 60% yield, by reaction with sodium hydride and 4-r-butylphenol.^ Often, the acid chloride is prepared in situ and then immediately reacted with an alcohol. An example of this is treatment of 2 with thionyl chloride in methanol, which gave the methyl ester (7) in 86% yield. *^ The ethyl ester (see 11) was produced by direct treatment of glutamic acid with thionyl chloride and ethanol.* * The /-butyl ester was similariy prepared in 93% yield, but 2 was first treated with oxalyl chloride in acetonitrile and the reacted with /-butanol.12


232

M. B. Smith

HO.

I

MeOH^° SOCI2, MeOH*"

y

H

1

: — • cat.H*

1

H

2

7

An older method of fornung the ester involves direct treatment of the acid with an alcohol. When methanol and an acid catalyst reacted with 2, as shown, a 98% yield of 7 was obtained (the reacticm can be done on kilogram scale).!^ Alternatively, methanol and HCl reacted with die acid (2) to give 7 in 95% yield. 1^ The methyl ester (7) was also fonned by reaction of 2 with diazomethane, but no yield was repotted.^^ The butyl ester was prepared by reaction with butanol and sulfuric acid,^^ and simi^y heating 2 and methanol together at 150^C gave 1)^ The benzyl ester was prepared in 69% yield by refluxing benzyl alcohol and 2 in xylene for eight hours.^^ Alternatively, the benzyl ester was prepared in 56% yield by refluxing 2 with benzyl alcohol in the presence of tosic acid.^^ The identical reaction in neat refluxing ethanol, with tosic acid, produced the ethyl ester. ^9 j)^ r-butyl ester of 2 was prepared in 65% )4eid by reacting that acid with isobutylene and sulfuric acid in an autoclave for 48 hours at 25X.l^ An additional method for ester formation involves the conversion of 8 to 9 in 93% yield by reaction of the acid widi triethylamine and benzyl chloride.^

VC^o ^^^^^^^ -"vC^o o

»

S

^3%

Cbz

• Cbz

8

9

It is noted that pyroglutamate esters can be prepared from a diester of glutamic acid. When 10 was mixed with pancreatic porcine lipase, the ethyl ester of pyroglutamate (11) was fonned in up to 80% yield.2l Diester 10 could be stored for weeks in dioxane, at 25^C, without conversion to 11 but when 10 was exposed to carbon dioxide in dioxane at 25°C, 11 was formed with a half life of about 10 hours.^^ The same was true for the methyl ester, ethyl ester, propyl ester and the isopropyl ester. O OEt

5-^yC^c « 7

porcine pancreatic lipase

4d 80%

H

11


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

233

Once the acid chloride (3) is isolated, amides can be formed by reaction with amines, or amines can react with 3 that is generated in situ. Thereactionof 2 and thionyl chloride gives 3, which reacts with phenethylamine to give the corresponding amide, 12, in 68% yield.23 A wide variety of amides can be generated in this manner, including many N-aryl amides.24

O

I H

68%

r ^ N ^ O Ij

O

3

12

An interesting variation in this approach treats N-tosylglutamic acid (5) with PCI5 to give the bis-acid chloride, 13. When 13 is treated with an amine, the N-tosyl amide of pyroglutamic (14) is formed.25 CI

r

COjH PCI5, ether

— ^

/

RNH^

{jo TsHN

5

^

"^"^V-^/"^

y^N^o

—-^

*,j

*^

12

13

Amides are also produced by treating the initially formed ester with an amine. Reaction of 11 with ammonia, for example, gave amide 14 in 75% yield.^^ Anmionia dissolved in methanol converted the methyl ester (7) to 14 in 84% yield.27 Similarly, several 2-arylethylamines were reacted with 7 to give the corresponding amide in good yield.28

°

i 11

° A 14

Amide-like derivatives can be prepared by this method. When 11 was reacted with aqueous hydrazine, the hydazide derivative (15) was formed.27 Hydrazide 15 was also prepared by the reaction of ester 7 with hydrazine.2^ Similariy, reaction of 7 with hydroxylamine gave N-hydroxy-amide 16 in good yield.^^


M. B. Smith

234

IS R = N H z

7

16 R=OH

d directly from ppglutamic acid but the conditionsare much more vigorous, as

Amides can be f

with any other dircd acid-to-amide mversiOn. Heating 2 with a 17% aqi~emssolution of methylamine for

-Me

10 days in a sealed tube gave a low yield of 17?1 Several other amides were prepandby this method;the

N-phenyl amide was preparedin 46% yield by heating 2 with aniline at 195-2ooOC.32 There are alternative 17% aq. MeNH2 O

H

37OC. 10 d sealed tube

2

0

O

H

17

rcagents that m v e r t the acid to the amide. When (2) was treated with the ammonium salt of l-hydroxy-

benzotriazok m DMP (at O째C in the Dnsenoe of DCC). an 84% yield of 14was obceined.33

10

1 8 . R = Bn 18b R = c-C&~ 18s R = CH2CHBH

Just as ppglutamate esters were prepand h.om the diacid chloride or diesterof glutamic acid, amides

can also be prepared. When 10was heated with various amines, the cornsponding amide was formed.34 Heating 10 with benzylamine for thne burs at IOOOC. for example, gave a 74% yield of 18a.stirring cyclohexylamiae for three days at 25OC gave a 60% yield of 18b. and stirring amiwcthanol with 18 for 18

hours at 254.2 gave a 59% yield of 18e. Many other amides were prepand by this method. Heating 10

with aniline gave the N-phenylamide?s and heating the N-Cbz half-mthyl ester of glutarnic acid with phmthylamine gave the comspondig a m i d e . ~


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

235

1.3. Structure and Properties of Pyroglutamate Derivatives The structure of pyroglutamic acid is important for any reaction, particularly those involving chiral, nonracemic substrates. The position of the C5 side-chain relative to the substituent on nitrogen is clearly an important consideration. One method for determining this relationship is by examining the x-ray crystal structure of various pyroglutamate derivatives. Several structures have been reported in the literature. The x-ray of racemic pyroglutamic acid was determined, and found to form a lattice of hydrogen bonded units. The x-ray of the monomeric unit was determined to be that shown for 19.^6 The x-ray structure of glutamic acid was also determined as a mixed crystals of L-glutamic acid and L-pyroglutamic acid.^^ The x-ray

19

20

structures of several derivatives have also been determined. Structure 20 for L-pyrrolidin-2-one-5caiboxamide (14) was shown to be 19,^^ and that for the N-methyl derivative of this molecule was also reported.^^ The structure of 5-iodomethyl-2-pyrrolidinone (derived from ethyl pyroglutamate - vide infra) was reported to be 21.^^ In 20 and 21, the substituent at C5 is anti- to the hydrogen attached to the lactam nitrogen, but in 19 that hydrogen appears to be closer to co-planarity with the C5 substituent. It is noted that the x-ray for the lithium salt of pyroglutamic acid"^ had a structure similar to the amide. The position of the group attached to the cariK)nyl is important for many reactions that occur elsewhere in the molecule. This is particulariy true when pyroglutamic acid derivatives are used to make peptides. Although this review will not deal with peptide derivatives, the conformational aspects of the group are of


236

M. B. Smith

interest. If we examine a pyroglutamyl amide unit such as 22, the lactamringgeometry restricts the ^ torsion angle (angle N-O^C-N) to -120®. Two low energy regions for y have been calculated; the lowest at about -40® and a higher one at +100®.^^ The authors of that woik ini|>ly that a L-pyroglutamic acid residue is confomiationally equivalent to a standard D-amino add in terms of its bacldxxie conformational

22 requirements. The lactamringis essentially planar. Because of non-bonded interactions,tiievalue of q> torsion angle (angle C^-N-C^*) has two minima; '*a shallow one near the extended conformation and a higher minimum near +60® .^2 Torsional angle y has also been calculated to have two minima, at -40® and dA •1-170® .42 jiie value fof the torsion angle in the crystalline form is taken to be +169® and that for the torsion an^e in solution is -20® .43 Table 1. Location and molar ellipticity values of amide n~+ic* transition of 2 in different solvents.43 Solvent hexafluoroacetone trihydrate hexafluoroisopropyl alcohol water waterimethanol (1:1) watentrimethyl phosphate (1:1) watendioxane(l:l) methanol tiimethyl phosphate dioxane

X(mn) 209 209.5 210 211 213.5 214 216 219.5 223

[9] X 10±-3 +18.5 +20.7 +19.5 +16.4 +20.0 +16.2 +17.2 +17.3 +6.8

The characteristics of the n-^n* and ic-^iC* transitions in pyroglutamic acid can be probed by examining its ORD and CD. Pyroglutamic acid (2) is a strong acid,43 and the CD band in pyroglutamic acid measured


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

237

at pH 2 is similar to that in pure water."*^ The location and molar ellipticity values for 2 are shown in Table 1. The authors of this table expected a negative Cotton effect for the n->K* transition, but they observed a positive Cotton effect, which they explained by the static charge term in the expression of the rotational strength of the n-^n* transition for a single chromophore and perturbing group.^^ xhis supports the idea that charge and electronegativity are important for determining the magnitude and sign of rotational strength.^5 Other workers measured the CD in water and in HCl and found values for Ae = +7.18 m and X = 207 nm in water; Ae = +3.91 m and A. = 210 nm in HCl and the Xmax in water (UV) was <196 nm.'*^ The molar ellipticity was measured to be 2.5x10^ and 11x10^ with Rexp = 2.0x10^ and 12x10^.^ This led to the conformational prediction that the pyrrolidin-2-one ring is slightly non-planar with C4 about 0.2A out of the plane defined by the 0=C-N atoms.^ Molar ellipticity was measured between pH 1 and pH 10 for 2 and ranged from 14.7 to 20.6, and that for the methyl ester in methanol was 17.2.^^ Substitution at C4 was shown to move C4 further out of the plane .^"^ The absolute configuration of carboxamide 14 was estimated from a CNDO/S calculation of optical rotatory strengths."^ As mentioned above, 2 is a strong acid. The pKa in DMSO was measured to be 6.55 and estimated to be 3.45 in water.^9 The isoelectric point of 2 was shown to be 3.35, determined by paper electrophoresis of a 0.1 M solution of the acid.^O The equilibrium constant for formation of 2 from glutamic acid was also determined to be 2.148, with AS = -8.1, Acp-25° = -35.3, and pKioo> = 2.27.^1 There is a relationship between the pH of a solution of 2 and the stability of the chiral center at C5. At pH 1,2 and 7 racemization is not significant and aqueous solutions of 2 are stable for hours.^^ At pH 12, however, racemization occurs rapidly and at 142**C is complete in two hours.^2 1.4. Functional Group Manipulations of Pyroglutamate Derivatives To be synthetically useful as a chiral template, or as a staiting material in any synthesis, the functionality of pyroglutamate and its derivatives must be manipulated. This means that the carboxyl unit at C5 of 2 must be converted to other functional groups; it must be possible to attach groups to nitrogen; it must be possible to functionalize the ring carbons of the lactam moiety; and, in some cases the lactam carbonyl must be modified orremovedaltogether. This section will briefly discuss the most common functional group transformations involving pyroglutamate.


238

M. B. Smith

The first area to be discussed is stftachment of groups to nitrogen, N-alkylation. There are many exan^les of reactions that attach a simple alkyl or a fimctionalized alkyl substituent to the lactam nitrc^oi of pytoglutamate. Inspection of the literature reveals that slightly different reaction conditions were required as the alkylating agent chsmged. Pyroglutamate must react with a base to form the corresponding anion, but this is resonance stidnlized with the charge dispersed on oxygen, which diminishes the nucleophilic character of the lactam nitrogen in alkylation reactions. Non-the-less, alkylation can be accon^lished in moderate to good yield. When methyl pyroglutamate (7) was treated with sodium hydride in DMF and then with ethyl 4-bromobutanoate and sodium iodide, a 25% yield of 23 was obtained.^^ When the t-bvAy\ est^ of 7-bromobutanoic acid was used, however, the yield of N-alkylated product was 70% uncter essentially the same conditions.^ Similsu* reaction of 7 with NaH in THF, followed by reaction widi 1-bromdieptane in THF gave the N-heptyl derivative in 29% yield,^^ whereas reaction of 7 with NaH followed by reaction

H

2.NaI Brx,_x>s_^C02Et ^

^

L

25%

^^

^—C02Et

7

23

with benzyl chloride gave the N-benzyl derivative in 84% yield.^ As seen by these examples, the yields of these reactions can be quite low, as in the reaction of benzyl pyroglutamate with NaH and iodoethane in DMF, which gave on a 23% yield of the N-ethyl derivative.^^ The solvent plays a significant role in this reaction. When 7 reacted with allyl bromide, benzyl bromide, or iodomethane in refluxing benzene, the Nallyl, N-benzyl, and N-methyl derivatives were formed in 71%, 68%, and 82% yield respectively.^® An E«V 7 ~ \ \ - — v ..^^

NaOH.aq.THF somcation ^_

r ^ N ^ O

o

l 11

^

^

^

^

^

//

r

O

I

^^^^

O ^

81%

^ 24

alternative procedure reacted pyroglutamic acid (2) directly with allyl bromide and NaH in DMSO to produce the allyl N-allyl ester in 71% yield.^^ When ethyl pyroglutamate (11) was treated with allyl bromide and powdered KOH in aqueous THF, in the presence of tetrabutylammonium bromide as a phase


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

239

transfer agent, the yield of N-allyl ester (24) was less than 5%. Only when this reaction was done in an ultrasonic bath did the yield improve to 81%.^ There are several variations of this basic reaction. In one, the butyl ester of pyroglutamate (25) was treated with potassium metal in benzene, and then with 2-iodopropane to give the N-isopropyl derivative 26 in 40% yield.^^ Similariy, the N-methyl derivative of 7 was prepared using sodium metal and bromomethane.^^ An alternative alkylation method relies on formation of an N-trimethylsilyl derivative as a Bn(0 .

I

\

f^N^^O

O

I H

K^ PhH, reflux

ICHMea, 1 h

l / ^

o'

N^O

I

"A

25

40%

26

reactive intermediate. When pyroglutamic acid (2) was treated first with pyridine and hexamethyldisilazane and then with chlorotrimethyl silane, the product was the silyl N-trimethylsilyl ester 27 (formed in 24% yield), which was very unstable in air.^^^ When this procedure was modified to first treat an ester such as benzyl pyroglutamate (25) with chlorotrimethylsilane and then with benzyl bromide (130°C for 71 hours). /—\ HO,, \-.--Sw J ^

O

H

NaOH.aq.THF

sonication _ Br^ v s ^

" ^ ' ' ^ ' ^ V ^ M ^ O

//

f* ^ 24% SiMea

^ \ ^ ^

27

^"^ ^ / ^ \ w

V^N^^O 5

I H 25

l.Me3SiCl(65%)

*r^N^^O

5

L

2.PhCH2Br,130°C 71h(62%)

'-^pj, 28

the N-benzyl derivative 28 was formed in 62% yield. ^3 Several derivatives were made in moderate to good yield by this procedure, mostly with benzylic derivatives, although aliphatic bromides also gave moderate yields.^ Arecentstudy compared several different methods for the N-alkylation of pyroglutamate. The conclusion was that treatment with sodium hydride in scrupulously dry THF for 5-10 minutes, followed by


240

M. B. Smith

addition of the alkylating agent gave superior yields in most cases,^^ and when that method failed the phase transfer technique mentioned above works well. Bodi dimethyl sulfme^^ and diethyl sulfate^ have been used to form N-methyl derivatives, and N-alkenyl derivatives can be generated by reaction of pyroglutamate with aldehydes in the presence of an ackl catalyst^'7 N-Alkenyl lactams have also been prepaied by the reaction of pyroglutamate dÂťiv^v^ with sodium hydride in DNfF followed by reaction with epoxide and subsequent treatment with aque(His acid.^ N-Benzyl derivatives have been formed directly firom L-glutamic acid by reaction with benzaldehyde and sodium borohydride,^ and a similar reaction with indole and formaldehyde gave the N-indolylmeth^ derivatives^ L-Glutanuc acid was also alkylated by reaction with l-phenyl-3-(N,N-diethylamino)-lpropanone n 6N HCl and the product was the N-(3-(3-oxo-3-phenyl-l-propyl) pyroglutamic acidJ^ c*n

I

V

PhMe.catpTsOH

^ ^

/

V 83%

I H 11 J

^^^^ ^CHO

i \

EtQ ^ /

PhMe,cat.p-TsOH reflux, Dean-Stark trap

If^ir^o H 11

â&#x20AC;˘ ^ ^

EtO

I \ iJ^"'^N'^

5

I

^CHO 30

The N-alkenyl derivatives just mentioned are quite interesting compounds. When ethyl pyroglutamate (11) was treated with butanal and a catalytic amount of tosic acid in refluxing toluene, in aflaskfittedwith a Dean-Stark trap, N-butenyl lactam 29 was formed in 83% yield.^^ This group makes an excellent Nprotecting unit forfive-memberedringlactams,'^^ gnd the C5 proton did not epimerize under several different reaction conditions. Reduction of the ester group in 29 to hydroxymethyl followed by Mofliatt oxidation gave the C5 carboxaldehyde, which is stable to various conditions including Wittig reactions to give the C5 alkene derivative without loss of chirality.'^^ When 2-pynolidinone was treated with a conjugated aldehyde such as 2-hexenal under the same conditions, the N-dienyl derivative was formed in


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids COiEt

241

Etc

^

50% aq. EtOH reflux

Jvy^COaEt \ X

91%

^j

93:7 syn:anti

30

EtO.

Ph-- ^ OH (73 30

:

Ph窶能 OH 27) 46%

32

72% yieldJ"^ This reaction also woiics with pyroglutamate, although the reaction of 11 with CFotonaldehyde gave 30 in poor yield. Most other conjugated aldehydes (such as 2-hexenal and 2-octenal) gave good to excellent yields of the corresponding diene but the volatility of crotonaldehyde leads to variable and generally poor yieldsJ^ EMene 30 undergoes the Diels-Alder reaction cleanly, as do other N-dienyl lactams^^ to give the Diels-Alder the chiral, nonracemic adduct 3lJ5 Diene 30 was also shown to react with a chiral, nonracemic nitrone to give a mixture of diastercomers, with 32 as the main product7^ It is noted that a,P-unsaturated pyroglutamate derivatives react as dienophiles to give bicyclic derivatives.^^ There are many reactions of pyroglutamate and its derivatives, with a variety of reagents. Perhaps the most common reaction is reduction of the carbonyl unit at C5 to a hydroxymethyl unit, and subsequent transformation of that group to a variety of other functional groups. Silverman reduced the ester unit of ethyl pyroglutamate (11) with lithium borohydride to give 5-hydroxymethyl-2-pyrrolidinone (33) in 88% yield. 11 The hydroxyl unit was then converted to chlorine by reaction with carbon tetrachloride and triphenylphosphine, to bromide with CBr4/PPh3, to fluoride with silver fluoride, and to a cyano group with NaCN and alumina. This reduction wasreportedby Adkins, who obtained 33 in 93% yield via hydrogenation with a Copper-chromium oxide catalyst at 210-220ツーC.78


242

M. B. Smith

rsAo Cl

I H

86%

ca4 PPh3 CHCI3

C^o ^"^"^"'^ rC^o

CBr4. PPha McCN

Et02C«***

I

THF

11

HO

I H

««*

^^F :_MeCN /

y

33 NaCNV AI2O3

85%

rsAo

Alcohol 33 was also converted to the chloride byreactionwith methanesulfonyl chloride in DMF (65% yield) J^ An alternative prepafation of the nitrile converted 33 to the tosylate by reaction with tosyl chloride in pyridine, which was thenreactedwith KCN in ethylene glycol.^ The reduction of lactams to a cyclic amine with lithium aluminum hydride is well known, as illustrated by the reaction of benzyl lactam 26 (vide supra) with LiAlH4 to give N-isopropyl-2-hydroxymethylpyrrolidifie.6l When 33 was treated with HN3 under Mitsunobu conditions, a 75% yield of the azidomethyl derivative (34) was formed, and catalytic hydrogenation gave a quantitative yield of the 5-aminomethyl derivative, 35.^^ Reduction of the N,Ndimethyl amide of pytoglutamic acid also was accompanied by reduction of the lactam unit, to give 2-N,Ndimethylaimnomethylpyrrolidine.^^ When LiAlH4 was mixed with silica gel, however, the lactam unit was

/ ""

V

DEAD,HN3.THF

J

\

H

H

33

34

H2,10%Pd-C 6h <"»»•

J

\ H 35


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

243

not reduced. Addition of LiAltU to an ether slurry of silica gel and then treated with ethyl N-allyl pyroglutamate (24), only the ester unit was reduced and 36 was isolated in 87% yield.^^ it is interesting to note that when 24 was treated with LiBH4, less than 10% of 36 was formed.

24 II

36 ]

Reduction of the ester to a hydroxymethyl unit allows other transformations. When ethyl pyroglutamate (11) was reduced with sodium borohydride, the product was 33, and subsequent treatment with benzaldehyde and an acid catalyst gave an 86% yield of the bicyclic lactam 37.84 jy^^ compound has been useful in several applications, including its conversion to an a,P-unsaturated derivative that reacts with organocuprates.85 Synthetic targets that utilize this approach include (+)-lactacystin8^ and castanadiol.^ EtQ^v

O

J

L

NaBH4

"\

J

V

I

I

H

H

11

33

PhCHO,H^

.XNN(^

86%

o—L

^ ^

- ^ p , 37

The alcohol unit in 33 orrelatedcompounds can be oxidized to an aldehyde. Reduction of 38 with LiBH4 and oxidation with chromium trioxide-pyridine gave a poor yield of aldehyde 39.55 ^ Swem l.LiBH4,THF MeOl / \ 2.HCl/MeOH 81% / ~ \ O

I C7H15 38 r—i I Me 40

3.Cr03-2Py 0°C,CH2Cl2 4.aq.NaHS04 36%

| ^7Hi5 39

l.DMSO,(COCl)2 2.Ph3P=CH2 THF,-78X

/—\ //

I Me 41

44%

oxidation converted 40 to the aldehyde and a subsequent Wittig reaction gave 41 in 44% overall yield.88 in most of these oxidation reactions, the aldehyde easily racemizes. If a butenyl group is attached to nitrogen as in 42 (formed by reduction of 29), aldehyde 43 is formed by Moffatt oxidation and the proton at C5 of


244

M. B. Smith

the lactamringdoes notracemize.'^*^This butenyl group also functions as a useful protecting group for the five-nien^ied lactam ring.'^ HQ

/

\

DMSO,DCC. cat.a2CHC02H

/

V

t:^

43

81%

I

\ The hydroxymethyl unit of 33 can be reduced to a methyl group to give 5R-methyl-2-pyrrolidinone, 44. The 4-amino-3-methylbutanoic acid precursor to this lactam was prepared by Silverman by enzymatic hydrolytic resolution of 3-methylglutarate followed by conversion of the remaining ester group to an amine unit.^ Ringdahl prepared 44 via tin hydride reduction of 5-bromomethyl-2-pyrrolidinone, prepared from 33.^^ We prepared 44 from 33 by initial conversion to the tosylate (54% yield), exchange with sodium iodide and then reduction with tributyltin hydride and AIBN (53% yield).^2 jhig compound has been prepared by reaction with diisobutylaluminum hydride and sodium potassium tattrate,^^ and by catalytic hydrogenation.^^ . . HQ / \ N , i u < ^ ^ I 33

l.TsCl.KOH , . BU4NHSO4 54% / V â&#x20AC;˘ MeÂŤ****Sj^O 2.NaI,DME I 3.Bu3SnH.AroN 53% ^ 44

As seen above, the hydroxymethyl unit of 33 can be converted to a bromomethyl, iodomethyl, or a tosylmethyl unit. These halide or sulfonate ester groups can then react with organocuprates to give an alkyl or aiyl-substituted product. Tamm showed that 5-iodomethyl-N-Boc-2-pyFrolidinone (45) reacted with the higher order cuprate derivedfromphenyllithium to give a 56% yield of 46, but die analogous bromomethyl and 0-tosylmethyl derivatives gave 0% yield.'^ This contrasted sharply with the unprotected lactam where the tosylmethyl derivative (47) reacted with five equivalents of the higher order cuprate to give a %% yield of 48.*^ In addition, the iodomethyl derivative gave an 80% yield of 48 and the bromomethyl derivative gave an 83% yield of 48, although the O-mesyloxymethyl derivative gave only 3% under the same conditions.'^'^ Tosylate 47 also reacted with simple Oilman reagents such as lithium diisopropyl cuprate to give the 5-butyl derivative,^ and with lithium dibutyl cuprate to give the 5-pentyl derivative.^^


-

Ph

5 Ph2Cu(CN)Liz

Ph

Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids PhzCu(CN)Liz

I \,,I\,,

0

0

I

Boc 45

245

0

I

Boc 46

\,,It\,

T ~ \ , , \ W Q 0 -

I

0

H

0

I

H

47

48

There are two additional reactions of 5-hydroxymethylderivatives that are useful. In one, it was observed that an alkoxymethyl group at Cg of the lactam enhances N-alkylation. The reaction of 49 with phenyllithiumand then 1-bromopentane, for example, gave 50 in 75% yield.% Without the alkoxymethyl unit at Cs, the alkylation requires much more vigorous reaction conditions and the yields are poorer. When

33 is hydrogenated at 250째C in the presence of a copper-chromiumoxide catalyst, a dimeric species (51) was fonned.16 It is interestingto note that when pyroglutamic acid (2) is heated with acetic anhydride, a similar dimer (52) was formed.97 t-BuMezSiO

Qo

\,\I\\\

1.PNi

) .

2. CsH11Br

I

BOC

~-BUMQSi\,,,,,,(--J+ 758

C5H11

49

50 H2 ,Cu-Cr oxide

I H 33

2

I

dioxane , 5 . 5 h 250째C quant. 51

52

0


246

M. B. Smith

An interesting reaction of pyroglutamate involves treatmem of the ester at C5 with Grigniod reagents or organolithium reagents. In the normal reaction, the methyl ester (7) reacts with an excess of phenylmagnesium bromide to give 53 in 70% yield. ^^ This is typical of behavior for many esters, and is not unusual. In a related reaction, the diethyl ester of glutanuc acid is heated with magnesium metal and bromoethane in ether to give the corresponding S-(l-ethyM-hydroxypropyl)-2-pyirolidinone.^^ When the N-Boc ethyl ester (54) was treated with phenylmagnesium bromide at -ACfC in THF, however, the lactam ring is opened to give an a-amino acid derivative (55), in 83% yield.^

/

V

PhMgBr,THF

Ph

/

\

™ I. 70%

I. 7

53 O

B02C«»«Sj^O

L

^^^'-'^

BOiC-n

-^^

Ph

^ ~

54

83%

55

Organolithium reagents are known to react with caiboxylic acids to form ketones, although Grignard reagents do not usually give the same transformation. The reaction of N-benzylpyroglutamic acid (5<>) and two equivalents of phenylmagnesium bromide, however, gave a 40% yield of the ketone (57), along with 50% of recovered staiting material. ^^ Pyroglutamic acid reacted with three equivalents of phenyllitfaium to give the corresponding ketone, but in only 40-55% yield. ^^^ In both of these examples, the chiral center at C5 was racemized during the reaction. We recently showed that the reaction of ethyl N-benzyl pyroglutamate and two equivalents of phenyllithium lead to the ketone is moderate to good yield, with small amounts of the alcohol. ^^ The interesting fact of this transformation is that no racemization occurred at C5. A previous report showed that methyl N-methyl pyroglutam^e reacted with a dilithium hydroxylamine derivative to generate an isoxazole unit at C5 of the lactam ring.'^

Ph 56

Ph 57


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

247

We have seen that the hydroxymethyl unit of 33 can be converted to a halomethyl unit by standard reagents. ^1 There are other haiogenation reactions of pyroglutamate derivatives that are quite different. When methyl N-benzyl pyroglutamate (58) is treated with an excess of phosphorus pentachloride (PCI5), chlorination occurred via the lactam carbonyl and the product was the chlorinated dihydropyrrole 59, in 82% yield.63 A similar reaction occurred when methyl N-methyl pyroglutamate was heated with thionyl chloride, except that the products were chlorinated pyrrole derivatives and chlorinated pyrrolo-disulfides.^^ .CI 3 PCI5

Ph 58

• CH2CI2,22 h 50°C

Me02C

-0^ 82%

59

When methyl pyroglutamate (7) was treated with oxalyl chloride, the product was the chlorinated derivative 60, but this lost HCl when warmed to greater than 20°C to give 61.1^5 interestingly, when 7 was treated with phosphorus oxychloride (POCI3), a chloro-imine was formed which reacted with a second molecule of 7 to give 62.105 ^ related dimerization occurred when 7 was treated with NBS. Initial formation of a radical at C5 led to dimerization to a W^-lactam.!^ Rnally, when 7 was treated with AcOBr, methyl Nbromo-pyroglutamate was isolated in greater than 95% yield. 1^

Me02C****

/ \

y ^

(COCl)2 CH2CI2

I

H

Me02C^***

OX CI 61

60

Me02C»*''* ^

N ^ O I

POCI3 CH2CI2 •

Me02C»»*^

H

Me02C 7

62

There are several reactions of pyroglutamate derivatives that either involve reaction with an enolate anion or formation of an enolate anion. There are several variations of the Knoevenagel reaction, illustrated by the


248

M. B. Smith

condensatjon of the thiocarbonyl derivative of ethyl pyrogjlutamate (63) with bromo keto-ester 64 to give a 61% yield of 65.^^ A similar reaction occurred with 2-broino diethyl malonate.^^ The thidactam was

I—y

O

NaHCOa

J

V COaEt

I

I

H

Br

63

reflux, 36 h

L 61%

A^^ (y^^

64

65

prepared by the reaction of ethyl pyroglutamate with phosphorus pentasulfide (P2S5) in caibon disulficte (70% yield), which is a roore-or-less standard method for its preparation.^^ Lawesson's reageirt has also been used to convert pyroglutamate derivatives to the thiolactam.^^ A similar condensation of thiocaibcmyl CN NC

^^V

Me02C

I

H

75* MeOaC

Y^C02/-Bu

^Ih

•'^-N MeOjC^

7

66

^^

\

7 ^

67

pyroglutamate occurs with a-triflate esters ^ ^^ and a-keto esters J ^ ^ a-Cyano esters reacted wkli the lactim ether derivative of pyroglutamate. The reaction of 66 and r-butyl a-cyanoacetate gave 67 in 79% yield.^^^ Lactim ether derivatives such as 66 are readily formed by reaction with Meerwein's regent, as shown, ^^^^ or by reaction with dimethyl sulfate.^^^ The OEt derivative can be isolated in moderate to good yidd.

/ I

H

7

2.KHCO3 3. Meldrum's acid NEt3, PhH

\

O

Me02C«»*

I

H 68

using Meerwein's reagent.^^^ Aromatic hydrocarixHis such as benzene, toluene and anisole react with the iminium salt derivedfix)mtreatment of N-methyl pyroglutamic acid with P2Q5 and methanesulfonic acid to give 5-aryl 2-pyrrolidinone derivatives in low to good yield. ^ ^^ These lactim ethers also react with Meldrum's acid to give the corresponding derivative, illustrated by the conversion of 7 to 68.^1^


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

249

Meldnim's acid derivatives of this type can react further to give alkylidene-ester derivatives. An enolate derivative of the lactam can be generated at C3 that reacts with a variety of reagents. Simply heating 9 in DME, in the presence of 69, led to a 95% yield of the enamide product 70.^0 More traditional Me2N

/~V

P^

Bn02C»***^ N ^ ^ ^ O y Cbz

+

DME,70X,3.5h

Me2N——H ht-Bn

9

^ ~ ^ "

• 95%

/

\

BnOjC*^*^

69

70

enolate-forming conditions can also be used. When N-Boc ethyl pyroglutamate (54) was treated with lithium diisopropylamide or lithium hexamethyldisilazide, as well as other amide bases, the lactam enolate was formed and reaction with aldehydes such as benzaldehyde gave 66 in 62% yield. ^ ^^ Under conditions where benzyl bromide reacted to give a 51% yield of the 3-benzyl derivative, however, iodomethane and HO /

V I Hoc

l.LDA,THF 2.PhCHO

yT^Ph Et02C**»**S,,^n N ^ Hoc

54

62%

71

allyl bromide gave no alkylation at all.l^^ The r-butyl ester (72) gave a lower yield of both condensation and alkylation products,! ^^ although benzyl N-caibomethoxypyroglutamate reacted with LHMDS and then methyl bromoacetate to give a 56% yield of the 3-carix)methoxymethyl derivative.^^^ Long-chain allyl 1. LDA , THF, -78°C , /C02Bn y \ I

,

Boc

^

BnO^^^Cl

fl11 O

72

'-"""^^

"N^ " O

ni

^^ 73

52%

bromides react to give good yields of the corresponding 3-allyl derivatives. ^^^ Enolates can also react with a variety of specialized reagents to produce pyroglutamate with new functionality at C3. When 72 reacted with LDA and then benzyl chloroformate, the 3-carbomethoxy derivative 76 was formed in 52% yield. ^20 Similarly, reaction of the N-Boc methyl ester (73) with LDA and then isopropyl formate led to a 31% yield


250

M. B. Smith

of the 3-caiboxaldehyde <terivative, 75. In this case, using LHMDS as the base rather than LDA led to a 46% yield of 75.^21 j h e enolate anion of phenyl N-Boc pyroglutamate (76) reacted first with LHMDS and JCHO

r

\

l.LDA,THF,.78X

Me02C****S.,,,>^o

Hoc

^-

IT

I

Hoc »«^

O 74

31%

75

then with the oxaziridine shown to give a 61% yield of the 3-hydroxy derivative, 77.^22 \ similar reaction with a S-silyloxyniethyl derivative using MoOPh gave a 52% yield of the corresponding 3-hydroxy derivative. ^23 Hydroxy-lactams such as this have been converted to the 3-fluoro derivative by reaction with diethylamino sulfur trifluoride (DAST).l24 >Y)ien 76 was treated with LHNfDS and then with Eschenmoser's salt, the 3-N,N-dimethylaminomethyl derivative 78 was formed in 75% yield.^25 .OH 1. LiNCTMSh. THF,-78*C BnOiC***

C^o

<^c I Boc

Bn02C***

2. Ph

I Boc

Ts

61%

77

76

< ^ c

1. LiN(TMS)2, THF. -78*C

y C

/

NMc2

BnOaC***

Boc 76

2

"^

®

5

^

2. 5=?NMe2 I

Bn02C****\j^0 I Boc

75%

78

Pyroglutamate derivatives can be conveited to the corresponding (x,p-unsaturated lactam, which can then react with a variety of reagents including organocuprates and dihydroxylation reagents. When 79 was treated with LDA and then phenylselenyl chloride, the 3-selenophenyl derivative was formed in 86% yield, and subsequent treatment with hydrogen peroxide and pyridine gave a 90% yield of the conjugated lactam 80.123 When this was treated with lithium dimethylcuprate, an 84% yield of the 4-methyl derivative (81) was isolated, but treatment of 80 with osnuum tetroxide and N-methylmorpholine N-oxide gave a 57% yield of the 3,4-diol 82.^23 A variety of organocuprates have been shown to react with 80 to give 4substituted lactams in good yield.^26


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

251

/.««<^o /-BuPh2SiO Boc l.LDA,THF; PhSeCl 86% 2. H2O2, Py CH2CI2 90%

79

M^V_

/ r-BuPh2SiO

LiMe2Cu,-78°C

^ivr ^ O I ^oc 34^^ 81

r=.

r.BuPh2SiO

I Boc

OSO4

" ^ _ / H

/ r-BuPh2SiO 57%

80

^ N ' ^ O I Boc 82

There are many highly specialized transformations of pyroglutamate derivatives that lead to interesting compounds including ethers, amines, nitriles, aryl derivatives, and dimers. The Cs-carboxyl group of Nacyl pyroglutamic acid derivatives such as 83 was transformed to the methoxy compound (see 84) in 95% yield by electrolysis in the presence of methanol. ^27 xhis transformation can be done with the free acid, y y MeOH, THF i y y V 0.01 MKOH / \ H02C--Xj^x*^0 • MeO-^j^X^O I electrolysis I Ac 83

(2F/mol)

Ac 84

without an N-acyl group,^^^ and several alcohols can be used to generate other alkoxy derivatives. In another variation, the electrolysis was carried out in acetic acid-acetonitrile, in the presence of sodium acetate, and the product was the 5-O-acetoxy group where the COOH unit was transformed to an OAc •O PhH ,, nv hv ,, RT *—» riun IV1 r-BuOOr-Bu, 48 h Me02C NH / V Me02C^ ^ y Me02C NH H

0=0

7

85

unit. 129 It is interesting to note that the Cs-OMe unit can be converted to an alkyl group by treatment with butylcopper and BFa.^^^ In a previous section, dimeric compounds 51 and 52 were formed from


252

M. B. Smith

pyroglutamate derivatives. A diffeient type of dimer was fonned when methyl pyroglutamate (7) was photolyzed in the presence of di-r-butyl peroxide. Generation of a radical at C5 led to coupling and formation of the dimeric lactam 85. ^^^ A useful transfomiation converted the amide unit in 14 to the S-amino unit in 87 by reaction widi a hypervalent iodine derivative (86), but in poor yield.^^ The same transformation was accomplished using a phosphoryl azide.^^ When done in the presence of an alcohol, the amine was trapped as a caibamstte in good yield. 1^2 When the amide unit in 14 was treated with chlorotrimethylsilane and zinc chlcmde, the amide unit was dehydrated to nitrile 88 in 91 % yield. ^^^

^—\

%»«»«'\„^0 H,N I H

CF3

2__^ MCCN.H2O 30%

CFjCOOH.

H i N ^ ^ ^ O I H

14

H2N

87

I

91%

I

H

H

14

88

1.5. Synthesis Using Pyroglutamate Derivatives as a Starting Material

H-N \

I "

Ph ^ . ^

2.5% MeOH, 1 h aq. KOH, reflux

12

'

'

89

Pyroglutamate has been the statting material for the synthesis of many natural products and biologically important molecules. In many cases, the chirality inherent to glutamic acid, and thereby to pyroglutamate, leads to a final target with high asynunetric induction so pyroglutamate functions as a chiral tenq)late staiting


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

253

material. In other cases, pyroglutamate is an integral part of one unit of the final target. This section is divided into several sections to illustrate specific types of synthetic targets. Many of the transformations discussed above will be seen again, and there will be some new transformations as well. l.S.l.Synthesis of Amino Acids Hydrolysis of pyroglutamate under acid or base conditions leads to the amino acid precursor, glutamic acid. There are many ways to manipulate pyroglutamate to produce substituted glutamic acid derivatives. A simple example seen in a previous section converted pyroglutamic acid (1) to benzyl amide 12. Acylation of the lactam nitrogen with 2-methylbutanoyl chloride and treatment with methanolic KOH led to julocrotic NMe2 H02< Bn02CÂť***\j^>^0

'

Cbz

41% ^

2. BnOH,NEt3 62% 3. H2, MeOH 10% Pd-C 93%

'^^

HO2C

Me02C^,.^^"2)4Br JL

\ ^

^ ^ . ^ \ _n

Me02C

\

I

SiMej

I

y KF,MeCN

MeOzC'^N'^O

18-crown-6

/ ^ \

91

i

^"2C)4

Jl

P

92 J^ CO2H -X^c

â&#x20AC;˘

^

93

I (H2C)4 H02C-^^<, NH2 94

acid A, 89.23 Substituted derivatives can also be produced. When enamide 70 {yide supra) was acylated, esterified and hydrogenated, the product was L-y-carbomethoxyglutamate, 90.20'120 j]^ reaction of pyroglutamate 91 with methyl 2-phthalimido-6-bromohexanoate (92) gave the N-substituted product 93, which was a key step in the preparation of allosaccharopin, 94.^^^ 3-Aryl glutamic acid derivatives have


254

M.B. Smith

been prepared via a conjug^ed pyroglutamate derivative. When 80^^^ was txealtcd with an excess of the organocupiate derived from 4-lithiochlorobenzene, the 4-aryl lactam was formed, and hydrolysis led to formation of 95, baclofen.^^^*^^^ Baclofen is an important amino acid used to treat epilepsy.

r"^Ao

r-BuPh2SiO

I Boc

1.5(4-ClC6H4)2LiCu 2 MesSiCl • 2. hydrolysis

95

80

Hydroxylation of pyroglutamate led to another substituted a-amino acid derivative that could be converted to (-)-bulgecinine. Benzyl ester 76 was converted to a-hydroxypyroglutamate 77 (vide supra), and this was elaborated to amino acid 96. Reduction and refiinctionalization led to formation of a new lactam that JOH 1. LiN(TMS)2, THF, -TS^'C y ^

"NAC Boc

2.

yi

\

Ph-'^^r^ 7«

61%

Boc 77

^*

^OBt ^^^OBz

HO

rVA ^^2L^ » \ x x

BnOiC***

NH

I

O

_.-,.... 32% yield

Boc 96

., CO2H ^hT^C

I

H 97

was converted to (-)-bulgecinine, 97 (a proline derivative).^^ It is noted that a cyclic renin inhibitcM* was prepared from a fiinctionalized amino acid, prepared from an N-Boc 4-hydroxy-5-cyclohexylmethyl-2> / y l.Ph(XX:H2Li,THF / S^\x\/P**

Et020^<^0 '"''^"^'^^ >

Et02cA^ jf

I ^ 54

, ^^

T 0

0

78%

98

pyrrolidinone derivative derived from pyroglutamate. 1^ Both 5,7-dioxo-2-aminoheptanoic acid^^'^ and 5oxo-6-sulfinylhexanoic acid^^^ derivatives have been prepared by the reaction of the lactam carbonyl of


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

255

pyroglutamate derivatives with caibanions. The enolate of acetophenone reacted with 54, for example, to give a 78% yield of 98. Another biologically important a-amino acid is TAN-950A, 99, which was prepared from 74 via conversion to the 5-caiboxaldehyde (75; vide supra), which allowed conversion of .CO2 l.LDA / >^2H / V V V 2.iPrOCHO / ( 4 steps I

31%

^^^^^

^N^^O

ÂŤ-

L

74

75

45^,

yield

/

\

H-^-c/^O 99

the lactam to the isoxazolone ring in 99.^^^ Several 5-alkyl derivatives of 99 have been prepared by formation of an enolate anion at C3 of the lactam unit and subsequent reaction with acyl imidazoles to give 3-acyl lactams. Subsequent conversion to the isoxazolone required three steps. ^^^ It is noted that glutamate is a terminal unit on several peptides ^'â&#x20AC;˘^ and has been used to prepare the oligopeptide (4S)-(+)-anthelvencin A, which terminates in glutamic acid.^^l

p

COiMe 29% overall yield from 102

Several highly specialized a-amino acids can be prepared from pyroglutamate. The target of diphtheria toxin catalyzed ADP-ribosylation (diphthamide, 103) was prepared from 100 (prepared from D-glutamic acid), which was coupled to 101 (prepared from L-glutamic acid) to give 102 in 89% yield. ^^2 This amino acid derivative was converted to 103 in 12 steps.^^^ (+)-Lactacystin (106) is a thioserine derivative


256

M. B. Smith

prepaied from 37 (obtained in three steps from D-glutamic acid; vide supra) via convosion to 104 (5 steps; 32% yield). This was converted to 105 in four steps (in 72% yield from 104). ^^3 xhig lactam was converted to (+)-lactacystin (10<») in six steps (in 39% yieldfrom105). Me

cA/*^

'""OAc

^ steps 72% yield

105

""OH

= rw CO2H

39% yield

NHAc

106 In addition to the a-amino acids and highly fimctionalized derivatives just discussed, several non-aamino acids can also be pcepared by using pyroglutamate as a starting material. A simple example is 4,5-diaminopentanoic acid, 107) which was prepared from 5-hydroxymethyl lactam 33 (prepared by reduction of J I OH

\ I H

l.MeS02Cl,DMF

65%

2.KPhthalimide 60% 3.aq.HCl 70%

33

|

^^^H

f^^*^^^NH2 ^^ 107

the ester group in 11; vide supra) by chlorination of the hydroxyl unit, reaction with phthalimide and add hydrolysis to give 107.^^ Another simple transformation converted methyl pyroglutamate (7) to 5Rmethyl-2-pyrrolidinone (44), and this was converted to 4<aminopentanoic acid, 108.^^^ . . l.NaBH4 78% . . l.NaBH4 78% 2.CBr4.PPh3 73% / V aq. / V 2. CBr4. PPh3 73% J V aq. HCl r-^^^C02H Me02CA f A o • Mc^^^^^^Ao — I 3. BuaSnH Me^^^^'^NH2 H H AIBN 44

108


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

257

A synthesis of vigabatrin (109), used to treat epilepsy, began with ethyl pyroglutamate (11) as a starting material. Initial conversion to the N-butenyl derivative (29) allowedreductionof the ester unit to an alcohol and oxidation back to the aldehyde, 43. A Wittigreactionfollowed by hydrolysis with aqueous acid gave 109.67b I . butanal,cat.P205 / \ l.NaBH4 80% , x c \ ^ ^L \>^ toluene, reflux Et02C^' ^ N ^ ' ^ ' O OHC!^' ^ N ^ ^ O EtOz^^^N^^O • I ^ ' I 83% ' ^ 2.DMSO,DCC H ^ catTFA 77% II

29

l.Ph3P=CH2 2.5%aq.HCl

\ ,CO2H

I

77% 77%

43

NH2

81%

109 1. allyl bromide Et02C:'

N I

o

I

• 2. LiAlH4/Si02

\

I

I

^"

^

85%

" 11

I

I

\

I

2. Nal, acetone ^

'-^

W 36

\\ 110

AIBN, BuaSnH , PhH ^70% Me

111

1.5.2. Synthesis of Alkaloids and Other Natural Compounds Pyroglutamate has been an important part of the synthesis of several important alkaloids, as well as other heteroatom-containing compounds. One of the more simple classes of alkaloids are the bicyclic amines, typified by the pyrrolizidine alkaloids (l-azabicyclo[3.3.0]octanes) and the indolizidine alkaloids (1-azabicyclo[3.4.0]nonanes). A non-natural pyrrolizidine alkaloid was prepared from ethyl pyroglutamate (11) by N-allylation andreductionof the ester group to give 36. Conversion of the hydroxyl unit to a mesylate, exchange with iodide gave 110, and radical cyclization under standard conditions gave a 70% yield of 111.^ This basic approach was extended to include naturally occurring pyrrolizidine alkaloids such as


258

M. B. Smith

:^^^^Ac I

1. NaBH4 2.TsCl.NEt3 3.NaI,MeCN

1.2-Ac-butyiolactoiie ^i^w' Ni(acac)2,110X ^速

MCO2C

H 7

OMc 2.3NHCl,60X 3. NaBH4

4.H2,Pl02 5. Me2S04.

eorc

33%

l.CbzQ 2.PCC 3. w>C7Hi5MgBr

112

113

C7H15

4.PCC 5.H2.Pd-BaS04

24% 114

(-)-heliotridaiie and (-^trachelanthamidine, by converting pyroglutamate to 5-ethenyl-2-pyiTolidtne (x 5ethynyl-2-pynolidine. Subsequent fiinctionalization and radical cyclization gave the targeted alkalmds.^ Knapp also reported the synthesis of pynolizidine alkaloids via radical cyclization, using pyroglutamate as a chiral, nonracemic starting material. ^^^ OMe

OMe

H

O

Pyroglutamate has also served as a precursor to ant venom alkaloids such as pyrrolizidine 114. Methyl


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

259

pyroglutamate (7) was first converted to 5-niethyl-2-pyrrolidinone (44), which was converted to lactim ether 112 by reaction with dimethyl sulfate. Condensation with a lactone and ring opening and catalytic hydrogenation gave 113, which was converted to the final target (114) in five steps. ^^'^^ BnOH allyl bromide

/

\

GDI

BnOiC

HO2C

S

3. Mg[OCOCH2C02Me] 3. H2, Pd-C Me02< CHO

3. PdCl2.02 CuCl

I

H

XX

l.CrO

119 e" (2 F/mol) â&#x20AC;˘ NaOAc MeCN, AcOH

120

TiCU, CH2CI2 71% Me02C 122

The indolizidine-type alkaloid 118 was prepared from pyroglutamate via a pyrrolizidine-type alkaloid (117). The enolate alkylation reaction of 115 with the tosylate of 33 (see 47) gave 116. Cyclization was accomplished by reduction of the ketone unit to an alcohol, followed by treatment with acid to give a mixture of diastereomers, 117. This pyrrolizidine-type molecule was converted to 118 in seven steps.^^^ ^ steps

J.I

HO

H

NHCbz

61%

33

5 steps

125

124 Me

5 steps N^

NHOH

67% 123

,OH

30%

5 steps

Co 126

Indolizidine alkaloids are also important synthetic targets. In one synthesis, the carboxyl group in pyroglutamic acid (2) was converted to caitK)xaldehyde derivative 119, and the aldehyde unit was


260

M. B. Smith

fiiiictionalized to give 120. Electtochemical conversion of the C5-<:aiboxyl unit on the lactam ting to an Oacetoxy unit (see 121), was followed by coupling with titanium tetrachloride to give a 71% yield of 122. l^^ 1. LiCuPra 1. H2. Ni(R), 100 bar 2.Me2S04 HCl.MeOH 3. Meldrum's acid r"^ 2. LiAlH4 /•"^ Meldrum s F"^ 3.Cbza I V H 47

TsO

Bu»***V

4.NaOMe.MeOH 4.NaOMe,MeOH

^ S

Cbz

O

^^-^.^^'^

TI H 127

H2,10% Pd-C

\ N COiMe

^-^^ 5.AcCH=PPh3 6.H2.Pt02

,/^

50%

\

^JL

128

J

129

A synthesis of indolizidine 209B (12<Q also used pyroglutamate as a starting material. Initial conversicHi to the hydroxymethyl derivative 33 was followed by a seven-step conversion to amino-aldehyde 123. The aldehyde was converted to an alkene and the Cbz-amine to an oxime (124) in five steps, which allowed formation of the indolizidine unit (125) in five steps. Final elaboration to 16 required anodier five steps.^ /

\

130

/

C02Et

y

2

jjj OH

/

V

1. BrCH2C02r-Bu. Na2C03 / 2.NaH 49% r ~ V 34% V ^ ^ > ^ 10% H a1,85% ,

iv'

"

^^^Me 132

1.15%Ha,75*»C 80%

\

j

133 ^

^

1. AC2O. Py 2. NH20H-HC1, Py Py.95^C 3. 36.5% H Q , H2 PtOi.EtOH 134

The 0-tosylate of 33 (see 47) was converted to 127 via initial reaction with lithium dipropylcupr^, followed by reaction with dimethyl sulfate, and then Meldrum's acid in the presence of a nickel catalyst. Hydrogenation (a 96:4 cis:trans ratio of diastereomers was produced) and elaboration of the side chain to 128 allowed removal of the N-Cbz group by hydrogenation and reductive amination in situ gave a 50%


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

261

yield of (+)-monomorine 1,129.^^ MeO MeO C02iPr

PriOjC*''''^ - N H 2

OMe

OMe l.gl.AcOH 2. NaCNBHs, AcOH • 3. AcOH, MeOH 4. 2N KOH 88% overall

CHO 135

137

HO2C OMe

MeQ 1. oxalyl chloride 2. Sna4 3. Pd(OH)2-C_

1. oxalyl chloride l.SnCU

4. SOCI2 M( 5. H2,10% Pd-C 6. LiAlH4

Me<

MeO

139

Slaframine (134) is an important indolizidine alkaloid with anti-AIDS properties. It was synthesized from pyroglutamate derivative 130 via a Dieckmann condensation to give racemic 131, which was Me Me 1. TBDMSiCl, imid. ^C02Me 2. Boc20,DMAP f^^ Me-^^ \u-O2CEt ^ N ^ O 3.LHDMS,BuLi _. . , 140°C, o-xylene I Me02CCl / f HO H A. PhSeCl ^BuMe2SiO Boc 50% 5. H2O2 70% 140

^jAo

"''02CEt C02Me

l.TsOH 2. CBr4, PPh3 3.TFA

36% 02CEt

141 hydrolyzed and hydrogenated to give 132. Cyclization to the indolizidinone ring was initiated by N-alkyl-


262

M. B. Smith

ation followed by EHeckmaiin cyclization and decarboxylation to give 133. Conversion of the ketone unit to an amine unit gave slaframine, 134. ^^ The anti-cancer alkaloid tylophorine (139) also contained the indolizidine unit and involved a slightly different use of pyroglutamate. The pyroglutamate was prepared by coupling the diisopropyl ester of glutamic acid (135) with aldehyde 136 to give pyroglutamic acid 137 in four steps. Ftiedel-Crafts cyclization generated the indolizidine ring system in 138 and seven steps were required to convert this intermediate to S-(+)'tylophorine, 139.^^^ The stereochemistry of the ring juncture was set at 24:1 P:a via the series of reactions shown beginning with 138. The structurally related alkaloid cryptopleurine was also prepared by a similar route.

r-BuOaCI^^^^JAO

I.P4S10 2. BrCH2C02Me 3. H2,5% Pd-C

Bn 143

r-BuOaC:^^^^N^*'v

70%

Bn 144

l.LiBH4 2. oxalyl chlcmde DMSO ^C02Me 3."X" ^ 4.H2,5%Pt-C 5.AcOH,q.PrOH BOCH

POa3,95X O

H02C^*^^^^N^H. I Bn 37% 145

50%

^N

1. (Boc)20 2.KH,THF â&#x20AC;˘ 3. MeaSiCl, NEt3 4. TFA, CH2CI2

â&#x20AC;˘

28%

I

Another bicyclic lactam derivative was prepared from pyroglutamate as an intermediate for the syndiesis of cytochalasans. Hydroxymethyl derivative 33 was converted to conjugated lactam derivative 140, as shown, and subsequent Diels-Alder reaction gave a 50% yield of 141 (along with 32% of the regioisomeric cycloadduct). This was converted to 142, a key intermediate in the synthesis of cytochalasan derivatives.^ Anatoxin A (147) is a bridged bicyclic alkaloid, but it was also prepared from pyroglutamate. The


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

263

sequence used the r-butyl ester of pyroglutamate (143), which was converted to 144 via the reaction of methyl bromoacetate and the thiolactam analog of 143 followed by hydrogenation. Elaboration of the carbomethoxymethyl side chain gave 145, which was cyclized to give the 8-aza[4.2.1]octane derivative (146) as a 1:4 a:p mixture of diastereomers. Protection of nitrogen, incorporation of the conjugated ketone

^^^'^^N^o' N

2. MeC0CH(Br)C02Et[\! 3.0.1 NKOH,EtOH CN

I H 148

CN

8 steps

,xxt^\^A%

nOT BnC

Ii H 149

I C02Et

J ACS, 1980, 102,7154 >

JACS,1980, 702,7154 ^ OH

151

O

152

unit and deprotection gave anatoxin A (147). ^^ The derivatives anatoxinal and anatoxinic acid were both prepared by similar procedures.*^ l.LHDMS,BnOCOCl 2. H2,10% Pd-C NaBH4. HCl â&#x20AC;˘ oxalyl chloride j ^ D p s o

fV^°!

TBDPSO

NEt3,CH2Cl2,0X >Me02C

Boc 70%

80

p TBDPS(: < / Boc

N Bn*^ N

90%

7

7 steps

C02Me

C02Me

155

Polycyclic alkaloids can be prepared by using pyroglutamate as a starting material. Using cyanomethyl derivative 148, prepared from pyroglutamic acid {vide supra), 149 was prepared and catalytic hydrogenation of the enamino-ester unit gave a mixture of two diastereomers. *51 Diastereomer 150 was


264

M. B. Smith

converted to 151, which was a key intermediate in the preparation of 152. This constituted a formal synthesis since in previous work, 152 was converted to gephyrotoxin (153). ^^^ The tetracylie fragment of manzamine A was preparedfixMnmethyl pyroglutamate (7) by initial conversion to the protected hydroxyl methyl derivative 80, which was then converted to the enamino acid chloride 154. Formation of amide 155 was followed by a seven-step sequence that included an Q

1. saponify 2.TFAA,BF30Et2 •

EtOid^* 3.H2,Pd-C

157

64%

isg

9 steps

75%

O 159

160

OMe

intramolecular Diels-Alder reaction to give the ABCE tetracyclic ring system (156) found in Manzamine A.1^3 A synthesis of cephalotaxinamide (160) was also repotted that used 157 (prepared from ethyl 1.

C02Ba C6H,3—( OTf

l.H2,PtO2.50psi 2.BnBr,K2C03 2. N-Me piperidine C7H,5 3.LDA.THF 3. IM H3PO4 r-Bu02C I 4. cyclohexene, 4.BnBr Bn 10% Pd-C 162 l.TFA 161 2.pH7 C4H9 3. POQa. lOOX • C7H15 r-Bu02C C4H9^ 4.1% Pd-C, H2 I H Bn 55 psi, MeOH 163 164

:-^^NAS

r-Bu02C

'0'>

,A/^

^''\}'^%H,s

pyroglutamate) in a coupling reaction that was followed by removal to the ketone unit to give 158. Saponification of the ester allowed Friedel-Crafts type cyclization to give 159 (75% yield), and this was conveited to cephalotaxinamide (160) in nine steps. ^^ In addition to the complex alkaloids just discussed, pyroglutamate is also a chiral precursor to alkaloids


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

265

that are essentially highly substituted proline derivatives. One example is the conversion of thiolactam 161 (prepared form N-benzyl pyroglutamic acid) to imine 162. After alkylation at the carbon a- to the ester l.Ac20,Py l.CpHigMgBr . . 1. AC2O, Py 1. CpHigMgBr J V 2. Lawesson's reagent J A 2. NaBH(OAc)3 'O MeOiC^^^ "N N ^ 3. -x Mel, xM^iNaOMe }^^nxMf^ r N SMe o R^-pi ^" OH

H

176

7

,X^X, C9H19 N OH

l.TsCl,Py —^ 2. LiCu(C5H9)2 3. MesSil

H

X < ^ ^ 'N' \ K A ^'C7H15 H

177

178

moiety,reductionled to proline derivative 163» and this was converted to the ant venom alkaloid 164.^55 Another ant venom alkaloid was prepared from methyl pyroglutamate (7), which was converted to 165. BnOaC BnOaC NHBoc 1.03,MeOH; H02(^ Boc NaBH4 OBn

l^.^ '

l.NaBH4,Cea3 2. MsCl, NEt3 BnO

56%

168 l.NaH-KI ClCOaMe / V 2. LiHDMS , '-8"^«^S'«\X^O PhSeCl r-BuMe2Si<

I

3. H2O2

H

59%

/

\

C02r-Bu 2. Ni(R)

I

C02Me 170

169 r-Bu02C

f-BuMe2Si

I 87%

2. IN NaOH ^ 3. TFA 4. Dowex 50W

C02Me 171

l.BH3«SMe2 2. TFA 3. H2C1O4-H2SO4 • 4. KOH, MeOH 5.IRN-77-IR-120

H02C^

H02C^^''^N^

I

H

48%

172

Reaction with the appropriate Grignardreagentled to a 70:30 mixture of diastereomers and the major


266

M. B. Smith

diastereomer (166) was converted to the target, 167.^^^ A synthesis of (-)-bulgecinine (98) from 76 (preparedfrombenzyl pyroglutamate; vide supra) included initial conversion to amino acid 96 {vide supra). Cyclization to pyrrolidine 168 wasfr)llowedby a four-step conversion to 97.^^2 1. (Me2N)2CHOr-Bu OMOM 2. IN HCl

• ^ " ^ o """"°'. > 4. MOMCl. iPriNEt I

Hoc 173 ^'^

OMOM

Boc 175 A Kainic acid analog was prepared from 169 by conversion to the conjugated lactam 170. Conjugate addition of the lithium enolate of a-phenylthio acetate gave 171, and this was converted to the target (172) in five steps.^^'^ A similar sequence was used to prepare the D-ringfragmentof (-)-quinocardn. Lactam 173 was prepared in six steps from glutamic acid, and conveited to 174. Conversion of the lactam carbonyl to a cyano group led to a 73:27 anti:syn mixture of diastereomeric nitriles, with 175 as the major o C02Et

NaHCOa®' EtOid^'>^^NAS

I

H 177

2.H2.Pt02 '^ EtOaC? AcOH, TFA

/

V I H 178

y )

l.KOH,EtOH 2.B0C2O

I 3.aCOC6H4.N02(p-) "^^^^ (c-C6Hn)2NH.KI DMF,100X

I COiEt «v<J V Boc ^ X CO2PNB 179 180 "X' = l-(3'-diinethylaininylpropyl)-3-«diylcaibodiinide«HCl


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

267

product. The minor product was reduced with Dibal to give the targeted 176, but conversion of 175 to 176 required two steps which included inverting the stereochemistry of the cyano group. *^^ l.Me2S04 l.BnOH,BF3 2. KHCO3 NaBHaCN, MeOH 3. Meldrum's acid pH 3-4 Me02C Me02C:^^^'^N^o N 5% Pd-C I 61% H2, MeOH H 68 7

.A X,

PPh3,(PyS)2

Me02C

N I ca. 23% H

CO2H

52%

C02Me 182

181

1.5.3. Synthesis of Antibiotics, Antibacterials, and Otiier Pharmaceuticals In addition to the alkaloids just described, several antibiotics have been prepared using pyroglutamate as a stalling material. Other important pharmaceuticals have also been prepared, including caibapenams which are a useful class of antibiotics. The thiocarbonyl analog of ethyl pyroglutamate (177) was condensed with O

"^..-C^o I

V^^*^^^^o I

1. hydrolysis 2. GDI

H

3.

H Mg

O2N

148

NO2

NO2

4.

H O , C ^ ^ ^ SO2N2

Rh(OAc)2 CH2CI2 85%째

'cOiPm 184

o

VojH 185

an a-bromo keto-ester and then hydrogenated to give 178. This was converted to 179, which allowed cyclization to form the ^-lactam unit in 180.'"^ Another approach to caibapenams converted methyl pyroglutamate (7) to Meldrum's acid derivative 68 (vide supra), which served as a surrogate for a


268

M. B. Smith

caiboxymethyl unit. Conversion to 181 (formed in 24% yield along with 23% of the 5yn-diastereonier) was followed by cyclization to carbapenam 182.^^^ l.p-anisdaldehydedimethyl acetal, BF3 ,MDC McOH 4.2CH2=CHMgBr

l.c.MeOH 2.3 CH2=CHMgBr HO2C I H

H 186

OAc

187

O

'OMe

CO2PMB 188

Another approach generated a y-lactam analog of a carbapenam (essentially a pyrrolizidinone dmvative). Initial formation of cyanomethyl lactam 148 from pyroglutamic acid (see reactions of 33; vide supra}, was followed by conversion to diazo-keto-ester derivative 183, allowing a rhodium catalyzed carbene inserticm to give 184. This was converted to pyrrolizidinone 185 in eight steps. ^^ Another pyrroli^dine derivative l.H2NCH2CH2CN,EEDQ jj 2. EtaO* BF4" / \ J^C02H > " EtO^

(ArA(

44%

H

O

kj,j,

189 i.NH4a 2. MeOH, HC. 3.NHa ^

H2N

f\

^^)Y^' 190

NH2ÂŤ2HC1 NH

37%

EEDQ = N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoliiie was prepared from pyroglutamic acid (2) via electrolytic conversion of the catboxyl group to a methoxy, which allowed reaction with vinylmagnesium bronude to give 186. This was converted to 187, which was followed by ring closure and elaboration to 188. Deprotection of the ester and hydroxy! units in 188 gave


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

269

the targeted compound.*^^ It is interesting to observe that a simple p-lactam (3-amino-l,4-diphenylazetidin2-one) was prepared from pyroglutamate J ^2 Pyroglutamic acid served as a precursor to the anti-microbial noformycin (190). Conversion of 2 to 189 was followed by manipulation of the cyano group to give 190. ^^^* Methyl pyroglutamate (7) was the l.NaH,KI ci r

\

S.

H

/^Me

2. o ^ ' k l ^ O M e

3.Dibal

y

^

V

o^^V.^^'^^OMe

4. cat. H*, aq. THF

11 J O2N" ^"^ "OBn

191

192 l.KMn04 2. reduction 3. deketalization 4, gftpQniOipatioly,

i^''''^M''^''^OH

'it 193

starting material for a synthesis of ntothramycine A (193), where 7 was converted to the acetal derivative 191163 (derived from hydroxymethyl lactam 33; vide supra). N-Acylation and reduction of the lactam unit led to 192, which was converted to 193'^ along with the diastereomeric alcohol (isomeric at C2 in the five-membered ring). A relatively simple synthesis converted ethyl pyroglutamate (11) to (5S)-methyl-2-pyrrolidinone (44), which was converted to 194, an oxotremorine analog that shows activity with muscarinic receptors.^'*

^Ao

MeÂŤÂŤ"'

I 44

194

o


270

M. B. Smith

Antibiotic peptides have also been piepaced from pyroglutamate. Methyl pyroglutamate (7) was conveited to 195 (^so see 87, vide supra) via the ethoxy derivative. Coupling with pyrrole derividve 196 led to the targeted dipeptide, dihydrc^cikumycin B (197). 1 ^^ An identical approach was used f(x the preparation of the structurally related antibiotic oligopeptide anthloencin AM^ l.EtOa^BFV . . J \. 2.NH4Cl,MeOH / V 0<^N^CX,3Me H 7

3.,o*Ha.50«^

HQ . H . N - ^ A c X ) . H 195

"W-^v

,NH2*Ha

O

j ^

NH

DCC.DMAP.DMF

HQ •H2N^

,^

'

|.

X

^

^

> > s ^ ^ N ^ ^ . ^ s ^ ^ N H 2 • HCl

"^r'

NH

197 There have been several syntheses of aza-prostaglandin derivatives that used pyroglutamate as a precursor. The reaction of pyroglutamic acid (2) with an amino alcohol led to conversion of the caiboxyl unit to an oxazoline, and this was reduced to an oxazolidine derivative, 198. N-Alkylation followed by hydrolysis led to 199, where the oxazolidine functioned as an aldehyde surrogate (a Meyers' aldehyde synthesis). A Homer-Wadswoith-Emmons olefination followed by manipulation of the functional groups led to 1 l-deoxy-8-azaprostaglandin Ei (200) as a 1:1 mixture of diastereomeric alcohols.^^^ Zoretic used this fundamental approach to synthesize 8-aza-l l-deoxy-15-hydroxyprost-13-en-l-ols,l^ and 9-oxo-15hydroxy-8-azaprost-13-ene.^^ Bolliger had prepared 199 prior to Zoretic's woric from methyl pyroglutamate, but the N-alkylation was done first,. The ester unit was hydrolyzed to the acid, reduced to a hydroxymethyl unit, and then oxidized using the Collins' reagent. 1^*^ This approach led to racemization at the Cs-position bearing the aldehyde unit. At about the sametimeKoning used an essentially identical approach to prepare racemic 8-azaprostaglandin Ei and £ 2 . ' ^ Saijo and co-woricers used pyroglirtamate derivative 191 (vide supra) and a protecting group for the hydroxymethyl unit, and prepared 1 l-deoxy-8azaprostaglandin Ei with high enantiopurity using essentially the same approach as Bolliger after N-alkyl-


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

271

ation and removal of the alcohol protecting group. ^^^ Wang reported a synthesis of racemic 8-aza-l 1deoxy-15-deoxy-16-hydroxy-16-methyl prostaglandin using a BoUiger-type approach, but Wittig olefmation of the aldehyde (199) was followed by reaction of 2-butyl-2-methyloxirane in order to incorporate the requisite methyl and hydroxyl groups on the side chain.^ 1.

; OH

r ~ \

/

2.MeI.MeN02

V

r i i ^ y ^ ^ ^ \

H

32%

2

^ N ^ ^ ' Br(CH2)6C02Me 2. aq. TFA

o

•'

II

y

^ Me Me

I'W

J3.

t O^ ^ N CHO I 2.NaBH4 k . ^ ^ \ X ^ 3.10%HC1 38% I 4. saponify C02Me 199

Qr

^N'

I OH '^v.XVsx-^ 45% ] 200 CO2H

1.5.4. Synthesis of Other Important Compounds Apart from the antibiotics and natural products reported above, there arc many important compounds that have been synthesized using a pyroglutamate strategy. Some have important biological activity, and others are compounds produced during structure-activity studies of important drugs. This final section will sunmiarize the synthesis of many of these important compounds.

I \ 0^^N''^C02Me

NH2NH2

1 •

\

CS2, KOH

O^^^N'^Y

Bn

Bn

58

201

8" 202

NHNH2 O

V

SOaMe 203

Many of these useful compounds convert the carboxyl unit of pyroglutamic acid to a heterocyclic


272

M.B. Smith

species. Rigo has reported many such transformations, and his recent review is a good place tofinda discussion of these compounds.^ In one synthesis, methyl N-benzylpyroglutamate (58) was ccmverted to the acyl hydrazide (201) by reaction with hydrazine, and subsequent treatment with KOH in caibon disulfide gave the oxadiazole-2-thione derivative, 202. When this reacted with iodomethane andtenwith potassium permanganate, thefinalproduct was 203.^^^ When hydrazide 201 was treated with acetic anhydride and then dehydrated with phosphorus pentoxide, the product was oxadiazole 2M™ md this approach was expanded to include substituted oxadiazoles as well as benzimidazoles.^'^l When 207 was treated with the thiocaibonyl derivative of semi-carbazide, the product was a triazole-3-thi(»)e, 112}'^'^

? T

I

201

W 80%

204 S

A.

XX... ::^^- ^^^j I Bn

2.H^

Bn „.^"\^

58

56%

205

Related to this is the synthesis of 207, (ABT-418, a cholinergic channel activator), preparedfit>mNbenzyl pyroglutamic acid (56) by initial conversion to methyl N-methylpyroglutamate (206), followed by conversion to 207.1^3 It is noted that a 1 ^ and ^^S-labeled thiazolidine-4-caiboxylic acid derivative was prepared by a DCC coupling of the labeled-pyroglutamic acid with thiazolidine-4-catboxylic acid.^ ^ > . O^^N CO2H t

2.NaH,MeI •

^ > . O ^ ^ r T ^COiMe I

Bn

Me

56 l.LiCH2NOLi(Me) 2.H2SO4 ^ 3.BH3*THF 4. CsF

206

j^^^^ ^ ^

J^^..^^K^Mc I 'o-N ^^ 46% 207

'ji%


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

273

l.TFA

„/>^e..^rxr ^^^^.^

'CO2H

I Boc

I H

83%

54

55

67%

208

Pyroglutamate has been converted directly to a pyrrole derivative. When 54 was treated with phenylmagnesium bromide, the product was amino acid 55 (vide supra). This amino acid was converted to pyrrole-2-carboxylic acid 208 in three steps.^^ O

1. SOCh 2. AICI3

^

CO2H

^

I.NH2OH 2. PPA , 100°C

-N

I

Bn 82%

O

56

62%

209

210

NH2

, CO 37%

O ^ N - ^ C02Me

Pd(OAc)2, PPha

I

H

C02Me 211

Other heterocyclic derivatives have also been prepared that involve conversion to polynuclear heterocycles. In a simple example, the reaction of N-benzyl pyroglutamic acid (56) with thionyl chloride and then OEt PEt KJtll

C

C02iPr

PnU2e

1.

» ^ - ^ rOEt

EtO-'^

2. MeOH, AcOH 3. 2N KOH, MeOH

Mri2

55% inn CO2H 212

( EtO-^ TsOH 97% 213

tryptanune


274

M. B. Smith

aluminum chloride gave an 82% yield of 1,2,3,5,10,10a-tiexahydrobenz[f]indolizine-3,10-dione (209) via an intemal Friedel-Cfafts acyladon. When this was treated with hydroxylamine and then PPA, aromatization and rearrangement led to a 63% yield of l,4-dihydrobenzo[c]-l,5-napthyridin-2-(3H)-one, 210J73 /â&#x20AC;&#x201D;I H2, Cu-Cr oxide J V 250X,dioxane /^Nr^''N^^

o H 215

^"^'-

51

Other polynuclear heterocycles can be easily prepared. When methyl pyroglutamate (7) was coupled with 2-iodoaniline, in the presence of palladium acetate and caifoon monoxide, quinazoline 211 was formed in 37% yield.174 l.NaH.DMF 2. PhCOCHiBr

o^iA'^ H

CO2MC

3. io%Pd-C.H2 4. KOH

^

6. AICI3

5.soa2

NaNa, H2SO4 ^ OX

40%

217 A more involved synthesis involved coupling a pyroglutamate derivative with tryptamine to produce an aza-yohimbine analog. The reaction of the diisopropyl ester of glutamic acid (135) and bromoacetalctehyde diethyl acetal led to formation of pyroglutamate derivative 212 after basic hydrolysis of the ester. Condensation with tiyptamine led to 213 and treatment with tosic acid effected cyclizadon to give a 2:1 mixture (PH:aH) of 214 in 97% yield. ^^^ A similar strategy involving the reaction of a pyroglutamic acid derivative and tryptamine was used to prepare 1-amino-indoloquinolizidine derivatives. ^76 Reduced forms of heterocycles can also be prepared. Hydrogenation of butyl pyroglutamate (215) led to a 77% yield of decahydrodipyrrolo[a,d]pyrazine, 51 (vide supra)A^ A closed related synthetic route


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

275

converted methyl pyroglutamate (7) to hexahydroisoquinoline derivatives. The first step was the conversion of 7 to 216, albeit in only 8% for the last three steps. This was subjected to a Schmidt rearrangement to give a 40% yield of 217. ^^"^ Other aromatic units can be appended to the lactam unit of pyroglutamate. The N-alkylation of methyl pyroglutamate (7) with 2-bromomethyl thiophene gave 218, which was converted to 219.^^^

"C02Me

O^^N'^'^COzMe

^

I 3. AICI3

NaH, PhH

I H

1. IMNaOH 2.SOCI2

58% 7

AQOL

218

\s=J

219

In other synthetic schemes, pyroglutamate is used to prepare one fragment used in the synthesis of diphthamide, which contains a pyrazole ring. The initial conversion of the protected hydroxymethyl derivative 80 to 220 allowed that fragment to be coupled to 221, giving 222. This was converted to diphthamide (223) in twelve steps. 1^2 l.LiCX)H 2. r-BuCOCl, NEt3 â&#x20AC;˘ 3. xp Li

^

Bn

OTBDPS

Y

BocHN O

OTBDPS

80

220

Bn-'^^^^^^^CI NHBoc 221

Wo lllll^

.OTBDPS

C Yi^" N^y

223

NiiiiiNHAc

12 steps

O

Bn

O

29% NHBoc

O

y

222

Me02C Pyroglutamic acid also served as a precursor for the synthesis of semicorrin ligands. Conversion of


276

M. B. Smith

pyroglutamic acid (2) to lactim ether 66 gave one fragment for the synthesis. A second fragment was prepared by conveiting 66 to 67. Subsequent reaction of 66 and 67 with trifluoroacetic acid led to a 75% I \ COir-Bu -CN < Me02C*^*%j^>^ C02r-Bu I CN

locrc l.H*,MeOH

67

H

^-OEt

2.Et30^BF4" I \ • ( ^ N

COaMe TFA

C02Me 66

COiMe

223

yield of the semiconin ligand 223. ^ 12b, 179 Several of these semi-corrin derivatives have been made by manipulating die ester side chain of pyroglutamate.^^ 1. BSTFA-Me-oxirane . .

2.AcSCH2CN(Me)CXX:i / 0^^r/^C02H 1 "

3. DCHA-S 4.10%KHSO4

O s : s \ ^

2

.

\

i Me

.

V l.Hg(02CCF3)2 "SAc^"2^

224

V

0^=\^^^ i Me

"SH

225

Pyroglutamate can be used to prepare functionalized derivatives that often contain the pyroglutamate uiut or a close relative. Pyroglutamic acid (2), for example, was converted to 224, and then to 225.^^^ This l.H2S04.MeOH 2. Me2S04

I—y J. L I H

2.Me2S04 3.KHC03_

/"^

Bn02C ^^C02Me

HOzQ^I 228

(f^^cT^o

I—y ^r^cr^n 227 3^ B n 0 2 C ^ _ ^ ) w CO2MC

4. Meldrum's acid 5. BnOH. BF3*OEt2

2

XX

| "

PhH.relfux


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

277

latter compound is an angiotensin-converting enzyme inhibitor. Pyroglutamic acid was also the precursor for a synthesis of the angiotensin-converting enzyme inhibitor A58365A (229), based on condensation of an intermediate lactim ether with Meldrum's acid. A five-step conversion to 226 and this was followed by condensation with glutaric anhydride derivative 227 to give 228. This was then converted to the target, 229, in seven steps. ^^^^ I V J \ ^^N^^CO^Me I Bn

1. NaBH4, MeOH 2.TsCl,DMAP I y 11 3. Nal, acetone J. \^ \ • d ^ n ' ^ ^ ^ 4.CH2=CHMgBr I T r^ ^1 Bn

1. O3 , MeaS 2. Ph3P=CHC02Et 3. ethyl aetoacetate, NaOEt , ^ ^ „ ^,^„ > ^ ^ ^ " ; , f ^^" 5. aq. HCl

L12CUCI4

58 l.TsOH O 2. 9 steps

I

Bn 231 Pyroglutamic acid was also converted to N-substituted derivative substituted at C3 of the lactam that proved to be renin inhibitors.^^ A peptide mimetic was prepared from methyl N-benzyl pyroglutamate (58) via conversion of the ester unit to an aldehyde unit in 230. Ozonolysis of the double bond led to an aldehyde an subsequent Wittig olefination allowed elaboration to 231. Treatment with acid and nine additional steps wererequiredto give thefinaltarget, 232.^^ Pyroglutamate was also converted to a substituted proline derivative via thereactionof 37 {vide supra) with lithium diisopropylamide and cyclohexenyl bromide to give 233. Reduction of the oxazolidine ring and protection of the nitrogen gave 234, a key intermediate in a synthesis of fosenopril.^^

"O Ph»««'" h,.

«<^

k.^^ = I

l.LDA

2. / ~ ) - B r

^-^

\^_/

4. Jones ox.

I

S.Hz.Pd-C

\

Ph'»"*\Q/ 37

l.LiAIH4

233

L

92.H2,Pd-C H, PH-r

J

^ r

\

_}

P~\ Cbz

CO2H 234


278

M. B. Smith

It is clear that pyroglutamate is an impoitant staiting material for the synthesis of a wide range of in^rtant molecules. To conclude this review, two additional uses of pyroglutamate are included to show that there are many more possibilities for using this important compound. In the first, 5-methyl-2pyrrolidinone (44; vide supra) was converted to the N-chloromethyl derivative, 235, via reaction with chlorotrimethylsilane and paraformaldehyde.^ When 235 reacted with racemic 2-butanol, in the presence of sodium hydride, adduct 236 was formed. The methylene protons in the N-CH2-O unit are diastereotqpic and the resulting AB quartet in the proton NMR is cleariy separated allowing 236 to be used for determining the enantiomeric composition of chiral alcohols.^

NaH, CH2a2 â&#x20AC;˘ 2-butanol, 1 h 82% 44

*NAO

Me**^

k.

"CI 235

236

i

Finally, pyroglutamate can be used to prepare impoitant chiral catalysts. Methyl pyroglutamate was used by Doyle to prepare the dirtKxIium catalyst MEPY (237), which catalyzed an enantioselective cyck^ropanation reaction of diazo esters to give, in this case, 238. ^^^ This catalyst has been used for a variety of

O ^ J A C02Me H Rh2(OAc)4

Me020ii" Me02Q

^ C02Me

^ â&#x20AC;&#x201D; r ^ ' " " ""C02Me co2]

X

237

0-JU N2 238

cyclopropanation reactions. ^^^ Corey also used pyroglutamate derivative 239 as a precursor for the preparation of amino-alcohol 240. When this reacted with diethyl zinc, an important new catalyst (241) was formed that is useful in several reactions.^^


Pyroglutamate as a Chiral Template for the Synthesis of Alkaloids

279

The variety of transformations and the scope of the reactions discussed here clearly point to pyroglutamate as an important chiral template starting material. It has been used synthetically for more than fifty years since its early use for the preparation of polyamides. As enantiopure materials become more and more important, pyroglutamate derivative will retain its place as a key member of the short list of chiral starting materials.

C02Et

MejN

239

H

OH

240

| Et 241

References 1

B Rigo, P Cauliez, D Fasseur, and F Sauvage, Trends Heterocyclic Chem 2:155 (1991).

2

(a) N Lichtenstein, J Am Chem Soc, 64:1021 (1942); (b) JH Billman and JL Rendall, J Am Chem Soc 66:745 (1944); (c) AF Beecham, J Am Chem Soc 76:4613 (1954).

3

G Braun, U.S. Patent 2,112,329; Chem Abstr 32:37739 (1938).

4

U Schmidt, R Scholm, Synthesis 752 (1978).

5

P Ferrabooschi, P Grisenti, E Santaniello, C Giachetti, G Zanolo, G Signorelli, and G Coppi, J Labeled Compounds & Radiopharm 31:973 (1992).

6

EA Bell, J Chem Soc 2423 (1958).

7

AF Beecham, J Am Chem Soc 79:3257 (1957).

8

(a) JM Swan and V du Vigneaud, J Am Chem Soc 76:3110 (1954); (b) J Rudinger, Coll Czech Chem Conmiun 19:365 (1954); (c) RJ Stedman, J Am Chem Soc 79:4691 (1957).

9

J Arimura and S Shinkai, Bull Chem Soc Jpn 64:1896 (1991).

10

O Provot, JP C616rier, H Petit, and G Lhommet, J Org Chem 57:2163 (1992).

11

RB Silverman and MA Levy, J Org Chem 45:815 (1980).

12

JS Petersen, G Fels, and H Rapoport, J Am Chem Soc 106:4539 (1984).

13

P Cauliez, B Rigo, D Fasseur, and D Couturier, J Heterocyclic Chem 28:1143 (1991).

14

EJ Corey, RK Bakshi, S Shibata, C-P Chen, and VK Singh, J Am Chem Soc 109:7925 (1987).

15

E Hardegger and H Ott, Helv Chim Acta 38:312 (1955).


280

M. B. Smith

16

E Scgel, J Am Chem Soc 74:851 (1952).

17

(a) N Kolocouris, Bull Soc Chim Fr 1053 (1973); (b) T Wieland and H Fritz, Chem Ber 86:1186 (1953).

18

AL Johnson, WA Price, PC Wong, RF Vavala, and JM Stump, J Med Chem 28:1596 (1985).

19

JW Ralls, J Org Chem 26:66 (1961).

20

S Danishefsky, E Berman, LA Qizbe, and M Hirama, J Am Chem Soc 101:4385 (1979).

21

AL Gutman, E Meyer, X Yue, and C Abell, Tetrahedron Lett 33:3943 (1992).

22

A Hubert, R Buijile, and B Haigitay 1958,182:259 (1958).

23

T Nakano, C Djerassi, RE Corral,and OO Orazi, J Org Chem 26:1184 (1961)

24

M De Nardo, Farmaco Ed Sci 32:522 (1977).

25

RJ Stedman, J Am Chem Soc 79:4691 (1957).

26

RB Angler, CW Waller, BL Hutchings, JH Boothe, JH Mowat, J Semb, and Y Subba Row, J Am Chem Soc 72:74 (1950).

27

E Roth, J Altman, M Kapon, and D Ben-Ishai, Tetrahedron 51:801 (1995).

28

LA Walter, RJ Gyurik, W Chang, and A Bamett, J Med Chem 16:735 (1973).

29

HL Yale, K Losee, J Martins, M Holsing, FM Perry, and J Bernstein, J Am Chem Soc 75:1933 (1953).

30

T Wieland and H Fritz, Chem Ber 86:1186 (1953).

31

(a) N Lichtenstein, J Am Chem Soc 64:1021 (1942); (b) N Lichtenstein and N Grossowicz, J Biol Chem 171:387 (1947).

32

S Iriuchijima, Synthesis 684 (1978).

33

A Sisto, AS Vetdini and A Virdia, Synthesis 294 (1985).

34

RB Angier and VK Smith, J Org Chem 21:1540 (1956).

35

J-F Peyronel, O Samuel and J-C Fiaud, J Org Oicm 52:5320 (1987).

36

E van Zoeien, HAJ Oonk and J Kroon, Acta Cryst B34:1898 (1978).

37

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Chapter Five

Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis - Electrospray Mass Spectrometry Joachim StoCkigt, Matthias Unger, Detlef Stockigt, and Detlev Belder Institute fur Pharmazie, Lehrstuhl fiir Pharmazeutische Biologie Johannes Gutengberg-Universitat Mainz Staudinger Weg 5 55099 Mainz Germany This chapter is dedicated to Professor W.E. Court (Mold) for his outstanding phytochemical research and in particular to his achievements in the exciting field of Rauwolfia alkaloids.

CONTENTS 1. INTRODUCTION

290

2. PRINCIPLES OF CAPILLARY ELECTROPHORESIS WITH UV AND MASS SPECTROMETRIC DETECTION

292

2.1. Capillary Electrophoresis with UV Detection

292

2.2. Electrospray Ionization: Mass Spectrometry out of Solution

294

2.3. Capillary Electrophoresis-Mass Spectrometry

295

3. DEVELOPMENT OF A GENERALLY APPLICABLE CE-MS SYSTEM FOR THE ANALYSIS OF ALKALOIDS

296

3.1. Optimization of CE Parameters for Analysis of Alkaloids

296

3.2. CE-MS Coupling Device

296

3.3. CE-MS Electrospray Conditions

298

3.4. MS Settings

298

3.5. Resulting Data in CE-MS

299

3.6. General CE-MS Optimization Procedure

299

289


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J. Stdckigt, M.Uiiger, D. Stockigt and D. Beider

4. RECENT CE AND CE-MS ANALYSIS O F ALKALOIDS

3(M)

5. CE-ANALYSIS OF ALKALOID STANDARDS

301

5.1. Indole Alkaloids

301

5.2. Protoberberines/Benzophenanthridines

305

5.3. P-Carboline Alkaloids

308

5.4. Opium Alkaloids

308

6. CE-MS ANALYSIS OF ALKALOID STANDARDS

312

6.1. CE-MS Analysis of Indole Alkaloids

312

6.2. CE-MS Analaysis of Some Isoquinoline Alkaloids

316

7. CE-MS ANALYSIS OF CRUDE ALKALOID EXTRACTS

320

7.1. Rauwolfia Alkaloids from Roots

320

7.2. Rauwolfia Alkaloids from Cell Suspension Cultures

324

7.3. Alkaloids from Cortex Quebracho

328

7.4. Analysis of Tinctura Opii

332

8. PERSPECTIVES AND CONCLUSIONS

335

REFERENCES AND NOTES

337

1. INTRODUCTION The separation of a crude mixture of natural compounds is most often necessary for further chemical analyses. During the separation process a fast on-line positive identification of analytes, e.g. by spectroscopic methods, is highly desirable in order to decide whether the respective fraction should be submitted to further evaluation or whether it should be discarded. In this chapter we will present a short review on the on-line coupling of capillary electrophoresis (CE) and mass spectrometry (MS) for the analysis of alkaloids. Owing to the particular physical and chemical properties of alkaloids, their analytical separation and identification are in general not easily performed. These compounds may differ strongly in solubility, volatility, polarity, charge densities and isoelectric points. Additionally, their molecular weights may range from ICX) to 1000 Da. It will be shown that CE-MS provides a rapid and efficient screening procedure for alkaloids in crude mixtures. Concerning the analysis of alkaloids by chromatographic separation techniques, thin layer chromatography (TLC) [1] or column chromatography using various supports (silica gel.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

291

aluminum oxide) are well applicable for isolation and crude separation, but less successful with respect to high resolution analysis of alkaloid mixtures. Nowadays, support media based on alkylsilane-derivatized silica gel, for example RP8 or the most common RP18 material, are widely used for analysis of crude extracts by high performance liquid chromatography (HPLC) [1,2,3]. The combination of gas chromatography and MS can be applied successfully to the analysis of compounds characterized by a sufficient volatility [4]. This prerequisite is fulfilled only for a few groups of alkaloids or for particularly derivatized alkaloids. Although derivatization is connected with a number of well-known disadvantages, impressing results in the GC-MS analysis of alkaloids have been obtained [5,6,7]. Reversed phase-HPLC turned out to represent a robust analytical method for the qualitative and quantitative determination of alkaloids. The HPLC-MS on-line coupling has been reported for alkaloid analysis applying (i) a moving belt interface under chemical ionization [8], fast atom bombardment (FAB) conditions [9] and even with MS/MS [10], (ii) a thermospray interface [2,11,12,13], (iii) a continuous-flow FAB interface [14], (iv) an electrospray interface [15,16], and (v) atmospheric pressure chemical ionization (APCI) [17]. In addition, supercritical fluid chromatography-mass spectrometry (SFC-MS) with a moving belt-interface and a modified thermospray deposition device has been applied to analyze an alkaloid extract from Claviceps purpurea [18]. During the last years CE has been developed to a promising tool for the analysis of a broad range of chemicals including secondary plant metabolites. Recently, the CE-applications of nearly all groups of plant natural products have been reviewed [19]. In particular, this excellent review summarizes CE analyses of various alkaloid groups among them opium alkaloids [20,21,22], pilocarpus alkaloids [23], a range of isoquinolines [24,25], purine alkaloids [26], oxindole standard compounds isolated from Uncaria [27] or some protoalkaloids from Ephedra [28,29]. For these analyses quite different separation conditions were employed and the question remained, whether a set of parameters can be developed allowing adequate separation of any arbitrary alkaloid class. Moreover, an advanced identification procedure of single components is beneficial, e.g. by coupling CE and MS. The use of this combination provides high resolution capabilities as demonstrated for solving complex biological problems [30,31,32]. On the one hand CE provides high separation efficiencies of a broad variety of charged [33,34,35] and uncharged compounds [36,37,38]. On the other hand MS delivers immediately information on the molecular mass and gives potentially important structure information [10,39,40]. A major demand in CE-MS is, however, that volatile buffer systems need to be developed in order to facilitate the MS ionization process. In addition, ready-to-use and generally applicable CE-MS systems are required, i.e. a broad range of alkaloid classes should be analyzed with minor operative modifications. In the present article such a system is described and a number


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of examples is discussed in which different alkaloid classes - pure standard mixtures and even more challenging, crude alkaloid extracts - were separated by CE under almost identical analytical conditions. CE and CE-MS will be demonstrated to be effective alternatives with regard to LC and LC-MS in alkaloid analysis.

2. PRINCIPLES OF CAPILLARY ELECTROPHORESIS WITH UV AND MASS SPECTROMETRIC DETECTION This part is concerned with a short description of the characteristics underlying CE mid the on-line CE-MS coupling. Basic principles on CE have already been reviewed among others by Jorgenson and Lukacs [41], Wallingford and Ewing [42], Grossman and Colbum [43], Guzman [34] and Engelhardt [44]. In 1990 the first review on CE appeared focusing on the development of this analytical technique as an instrumental method [45]. 2.1. Capillary Electrophoresis with UV Detection The migration of electrically charged particles in solution due to an applied potential difference is termed electrophoresis [46]. One breakthrough development represents the highvoltage electrophoresis in small-diameter capillaries called capillary electrophoresis (CE). Virtanen [47], Mikkers et al. [48] and Jorgenson and Lukacs [49] showed the advantages of using small innerdiameter glass tubes and electrical fields in the range of £,ep = 0.3 - 0.4 kV/cm. In 1984 Terabe et al. [36] introduced a modification of CE, called micellar electrokinetic capillary chromatography (MECC). It can be regarded as a type of liquid-liquid partition chromatography without a solid support, in which the analyte retardation is achieved by a micelle-electrolyte phase exchange [50]. Performing electrophoresis in small-diameter capillaries facilitates the application of high electric fields, Esep « 0.6 kV/cm, because the heat of the *CE resistor' is effectively dissipated. In turn, these high electric fields produce separations up to a million theoretical plates within 10 min. In Figure 1, the principle of a CE instrument is shown. The instrument consists of a fused-silica capillary (e.g. 400 ^m O.D., 50 ^m I.D., and 50 - 100 cm length), an inlet and an outlet vial filled with the running buffer, additional vials containing solutions for purging and sample injection, a high-voltage source as well as an on-column UV detector. The capillary is flushed with the running buffer and its ieft end' is in electrical contact with the inlet vial held at a separation potential Vjcp = db5 - 30 kV. The *right end' is in contact with the outlet vial held at ground potential. UV detection is performed ca. 5 - 10 cm in front of the capillary outlet (Figure 1). The sample injection is performed after replacing the inlet vial with a sample-solution vial, either (i) by applying a pressure p,„j for a certain time, e.g. 345 mbar sec.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

high-voltage power supply, ^sep = 1 0 - 3 0 k V

293

fused-silica capillary filled with buffer electrolyte UV detector

electrode held at separation potential \/sep

vials filled with buffer electrolyte grounded electrode Figure 1: Schematic drawing of the basic components of a capillary electrophoresis (CE) system. The vials are replaceable. The fused-silica capillary can be thermostated.

A

stainless-steel heated tube spray tip for desolvation of Ions

^Es. = ^8kV +5kV

mass spectrometer

stainless-steel syringe needle

Ar<0

ions electrospray process

fused-silica electrospray lens system, capillary process ion guide Coulomb explosion

stainless-steel spray tip analyte solution

energy (e.g. heat) Âťâ&#x20AC;˘ droplet desolvation formation of charged droplets

Coulomb repulsion direct ion ejection

smaller-sized charged Vn5A droplets repetitive process

1*

[M+H]

Figure 2: (A) Pictorial of an electrospray (ES) source for the introduction of a liquid analyte pumped through the fused-silica capillary in the mass spectrometer. (B) Detailed view of the basic components of the ES source. Note the position of the fused-silica capillary end in the syringe needle, i. e. Ar < 0. (C) Detailed view of the ESI process.


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(ii) by a voltage pulse, i.e. the charged analytes migrate electrophoretically into the capillary (also termed electrokinetic injection) [51], or (iii) via hydrodynamic injection accomplished by siphoning. Afterwards the sample-solution vial is again substituted by the inlet vial and the separation potential Vsep is turned on. Thus, the analytes migrate due to (i) their electrophoretic mobility (jij) and (ii) the electroosmotic flow (EOF) from the capillary inlet to the detector and further up to the capillary outlet. In general, the separation performance is increased with increasing electrolyte concentration. Particularly useful in CE are phosphate buffers because of their high buffering capacity over a broad pH range. Beside the most common direct UV detection method for CE, instruments with diode array (DAD) or scanning UV detectors (SUD) are available similar to LC. Although the DADs and the SUDs provide a range from 190 to 800 nm, structural evidence for a substance based on the corresponding UV spectra cannot be given for certain. In addition, the UV spectra depend on the electrolyte composition and differ by pH values [52] and are thus not easily searched for in libraries of spectra. Other detection methods have been successfully applied for CE, among them indirect UV detection [53], fluorescence [54], chemiluminescence [55], radiometric [56], raman-based techniques [57], electrochemical [47,48,58], as well as mass spectrometry (see section 2.3). 2.2. Electrospray Ionization: Mass Spectrometry out of Solution Electrospray ionization (ES or ESI) mass spectrometry has been reported by Dole et al. in the late 1960s for the flrst time [59]. Later it was demonstrated that in ES-MS the molecules are indeed taken directly from the liquid phase into the gas phase in an ionized state [60,61]. As a result, molecular masses of up to several kDa can be measured routinely by ES-MS. A schematic drawing of an ES ion source is given in Figure 2A. The end of a capillary (mack of glass, fused silica or metal; 10 - 200 ^m I.D.) is placed in front of a small *hole' of a vacuum interface to a mass spectrometer. An electrical fleld of ca. 1 kV/cm is applied between the capillary and the oriflce of the vacuum interface (Figure 2B: the heated capillary). The electric contact between the end of the fused silica capillary and the power supply (VESI) can be provided by a coaxial sheath-flow of an electrolyte as indicated in Figure 2A. The capillary is fllled with the analyte*s solution in a concentration of ca. 10 nmol ^1'^ to 100 pmol \i\'\ depending on the I.D. of the capillary's end. Due to the differences in electrical potentials between the capillary and a counterelectrode (Figure 2B: VESI = +8 kV vs +5 kV of the heated tube), charged droplets are formed. By dissipation of energy, e.g. a counter-current drying gas (not shown) or a heated tube as in Figure 2B, solvent molecules evaporate from the initially formed droplets. Due to the Coulomb repulsion with a single charged droplet either (i) in a repetitive mechanism droplet explosions take place and solvent molecules evaporate again or (ii) ions are directly ejected out of a charged droplet (Figure 2C). In order to obtain a stable


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electrospray process, analyte solution-flow rates of 0.5 - 1000 |LI1 min* are typically delivered. Several groups [62] have achieved an increased sensitivity by using 10 - 50 nl min"' flow rates and accordingly, the latter processes are referred to as *nanospray*. The electrospray mass spectra of alkaloids [15,63] are dominated by signals resulting from [M+H]"", [M+2H]^"', [M+Na]"", [IM+H]"". Fragmentation reactions can be observed but the respective signal intensities are in general < 3% compared with the [M+H]"*^ peaks. In order to provide an efficient ionization process, there are a number of characteristics of the ESI process to be mentioned [64]. The sensitivity is deteriorated with increasing ionic strength in the spray, e.g. the buffer concentration has to be kept at a minimum. The spraying solutions should contain volatile buffers only, e.g. NH4ACO, NH4HCO3. The analyte's and sheathflow's solvents should be volatile, in order to facilitate the desolvation process of the analyte

2.3. Capillary Electrophoresis-Mass Spectrometry In most cases of a direct CE-MS coupling [30,32,65], modified low flow-rate LC-MS interfaces are applied [38], e.g. continuous-flow fast atom bombardment, ion spray or electrospray. Furthermore, nanospray devices have been reported for CE-MS [66]. A general problem in CE-MS are the opposite requirements with regard to electrolyte contents in CE and MS. A high electrolyte concentration is advantageous in the case of a CE separation, but disadvantageous for MS (cf. section 2.2). As described above (section 2.2), flow rates of 0.5 - 1000 ^1 min'* are typically delivered for ESI in order to obtain a stable electrospray process. Since the liquid bulk flow in the CE process is less than 250 nl min', a make up flow may be admixed in the CE-MS coupling facilitating the spray process. However, the amount of make up flow has to be kept at a minimum in order to minimize the dilution of the sample. Two different interfaces have been described for ESI as a CE-MS interface in the literature, (i) the liquid junction interface [67] and (ii) the coaxial sheath-flow [68]. The latter uses a concentric series of capillaries and will be described below (section 3.2) in more detail. It was reported by the group of Smith in 1987 representing the first CE-MS interface [68]. Direct spraying without the addition of a make up flow can be accomplished via nanospray devices [66]. The liquid junction interface consists of a tee in which a cross flow of make-up fluid mixes with the electrolyte from the down-stream end of the CE capillary. The slightly diluted analytes move forward into a stainless-steel or a fused-silica capillary for electrospraying. Similar to this approach is the operation of a continuous-flow FAB CE-MS interface [69]. A make-up flow of the FAB matrix is admixed to the analytes. One disadvantage, however, is the increased amount (> 5 [il min"^) of make-up fluid compared with the ES interfaces (0.5-5


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^1 min'^) resulting in a diminished sensitivity.

3. DEVELOPMENT OF A GENERALLY APPLICABLE CE-MS SYSTEM FOR THE ANALYSIS OF ALKALOIDS Having mentioned a number of possible CE-MS interfaces as well as the basics of the electrospray process in the preceding sections, a detailed description of the development of the CE electrolyte will be given in the following paragraphs. Emphasis is put on establishing a set of CE parameters for the analysis of a broad variety of alkaloid classes. 3.1. Optimization of CE Parameters for Analysis of Allcaloids In the following, the term *CE-UV only' designates the experimental set-up described in this section (section 3.1). In the case of the on-line CE-MS coupling, the UV electrophorogram is referred to as the *UV-electrophorogram in CE-MS*. This semantic distinction is essential owing to the different effective CE capillary lengths in the CE-UV only and the CE-MS mode, amounting to ca. 50 cm and ca. 20 cm, respectively. For CE-UV only a BioFocus 3000 CE apparatus (Bio-Rad Laboratories GmbH, Munich, Germany) equipped with a fast scanning UV detector was used. The fused silica capillary had a total length of 55 cm (50 cm to detector) and an inner diameter of 50 fim (Polymicro Technologies, Phoenix, AZ, USA). Samples were introduced by applying 345 mbar pressure for one second. Details concerning instrumentation and operation are published [70a]. In order to optimize the CE separation of alkaloids, we used a standard mixture consisting of 15 indole alkaloids and biogenic amines in a concentration of ca. 0.1 mg ml'* in methanol. Since the CE separation parameters were developed for using the CE-MS coupling routinely, only volatile and low-concentrated buffers have been tested due to the limitations in the ESI process (see section 2.2) [64, 70]. It turned out that with a 100 mmol 1* ammonium acetate buffer adjusted with acetic acid to pH 3.1, a good separation could be obtained in terms of resolution and migration times. This buffer system was then diluted with organic solvents. The addition of acetonitrile (10 to 80%) and its influence on the peak separation was investigated. A 1:1 (v/v) dilution of the buffer with acetonitrile gave results with a maximum of resolution in less than 30 min separation time [70a]. 3.2. CE-MS Coupling Device The CE-MS separations were performed on a BioFocus 2000 CE apparatus (Bio-Rad GmbH Munich, Germany) with a liquid-cooled fused silica (FS) capillary. Similar CE conditions as


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

297

inlet vial

capillary electrophoresis

B

sheath flow stainless-steel

Perspex

spray tip

cooling liquid in a plastic tube K_y

stainless-steel syringe needle

CE capillary Ar>0

Figure 3: (A) Schematic drawing of the CE-MS coupling. It consists of the CE system (left), the ES interface (middle) and the mass spectrometer (right). (B) Detailed view of the threeway fitting for the backflow of the CE capillary cooling liquid. (C) Detailed view of the spray tip of the ES interface. Note the position of the CE capillary outlet inside the syringe needle, i. e. AT > 0 (cf. Figure 2B).

B UV-Absorption after ca. 20 cm

mass spectra

RIC electrophorogram after ca. 70 cm

825 [M+Hf 413[M-h2H]

*-'n-'^^i> jjr-rijL^r-

0

03

IK?

rfn^T^dV"-*"'y,ÂŤ<*-\nvw>/

-r-

time / min

m/z

c

900

background electrolyte

m/z 825 electrophorogram extracted from each mass spectrum T^-Mr^f^

time/min10

time / min

100

m/z

900

Figure 4: The UV electrophorogram (A), the RIC electrophorogram (B), a selected m/z electrophorogram (C) and two mass spectra (D) obtained from a single CE-MS run. The RIC electrophorogram measured by the MS is the sum of all ion intensities of a single MS scan at a particular time.


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described in section 3.1 have been utilized. Only the running buffer was modified, it consisted of 80 mmol 1* aqueous NH4ACO pH 3.4 and acetonitrile 50% (v/v). In Figure 3 a scl^matic drawing of our CE-MS coupling with the sheath-flow interface is depicted. The mass spectrometric detection in the positive ion mode was performed on a Finnigan MAT 95 (Finnigan MAT GmbH, Bremen, Germany) forward-geometry sectorfield mass spectrometer upgraded with the API-II ion source [71] operating in the electrospray ionization (ESI) mode. A three-way fitting has been constructed connecting the liquid-cooled CE capillary to the ESsource (Figure 3A). Furthermore, the stainless-steel spray tip of the API ion source has been modified to 800 ^im I.D. The ESI needle has been replaced by a commercially available syringe needle (500 ^im O.D., 400 ^im I.D.) in order to fit in the CE capillary (375 ^im O.D., 50 ^im I.D.). The spraying of analyte ions occurs directly from the CE capillary into the ion source. When performing ESI via LC or in the infusion mode without a make up flow, the spraying end of the FS capillary has to be positioned inside the stainless-steel needle for achieving electrical contact between the stainless-steel needle and the capillary outlet (cf. Figure 2B, Ar < 0). During the CE-MS measurements, however, the end of the FS capillary has to point out of the stainless-steel needle by Ar = ca. 0.1 mm (Figure 3B) because the electrical contact results from the make-up flow. If in CE-MS the capillary is positioned such that Ar > 0.1 mm, an unstable spray process is observed and if Ar < 0, i.e. the normal position under ESI conditions (Figure 2B), a peak tailing of the total ion electrophorogram resulted due to the analyte*s back diffusion into the sheath flow. 3.3. CE-MS Electrospray Conditions The standard ESI conditions were as follows: ES-needle potential VESI ==3.0 kV, spray current /ESI = 3 - 4 ^A (CE on), temperature of the aluminum capillary Tcap = 200째C, sheath liquid (methanol : 10 mmol 1'* acetic acid in water, 9:1) flow rate 1 - 2 ^il min* delivered by a Harvard Apparatus (South Natick, Mass., USA) 22 syringe pump. An increase of the spray current was observed in CE-MS experiments because in this case both power supplies, the CE and that of the ES ion source, worked in parallel. Preceding sample injection, the ES unit was switched off and the potential of the ES-needle was held at ground potential. 3.4. MS Settings The mass spectrometer was run at a mass resolution m/Am 2000, with an accelerating voltage of ca. 5 kV. Scanning was performed from m/z 150 to 1000 with a scanrate of 2 - 5 dec sec'\ The electron multiplier was set at 2.5 kV. Data acquisition and presentation were performed


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

299

using the standard proprietary Digital DECSXATlON-based ICIS-2 system provided by Finnigan MAT and the MassLib V 8.2 mass spectra evaluation program package (Max-Planck-Institut fur Kohlenforschung, Mulheim, Germany). 3.5. Resulting Data in CE-MS If a charged UV-active analyte migrates from the CE capillary inlet to the UV detector (Figure 3), it induces a peak in the UV electrophorogram (Figure 4A) after ca. 20 cm of electrophoretic migration due to a change in the UV absorbance. The analyte moves further and passes the 'right end' of the CE capillary that is mounted in the stainless-steel spray tip. Due to the electrospray conditions the analyte will be desolvated until a 'naked' analyte ion is present (cf. Figure 2 and section 2.2). This analyte ion is focused by a lens system into the mass spectrometer, where the detection of all arriving ions is performed. One scan of the mass spectrometer results in a single mass spectrum of a certain mass-to-charge (m/z) range. Of course, the ion's flight time from the spray tip to the detector and the scanning must occur much faster than analyte migration in order to achieve a high resolution [72]. The reconstructed ion current (RIC) electrophorogram is obtained as follows (Figure 4). Every point of it results from a single mass spectrum. If a mass spectrum has been recorded during five seconds, every point of the RIC electrophorogram is spaced by five seconds. Of course, severe problems arise if a peak is narrower than five seconds. The intensity in the RIC electrophorogram is proportional to the total number of ions detected in the respective mass spectrum during the scan time, i.e. the ions' intensities are summed. Conversely, one point in the RIC electrophorogram (Figure 4B) can be deconvoluted to a complete mass spectrum. In addition, the RIC electrophorogram contains signals from the background electrolytes as well as from noise of the complete scanned mass range. The selected m/z electrophorograms can be obtained by monitoring the intensity of a particular m/z value vs. the time after which the mass spectrum has been recorded (Figure 4C). Accordingly, a m/z electrophorogram indicates the presence of a particular m/z value at the capillary outlet after a certain migration time. In contrast to the single-ion-monitoring procedure [73], these selected m/z electrophorograms have been obtained from the full range mass spectra. Furthermore, the RIC electrophorogram is the sum of all m/z electrophorograms. 3.6. General CE-MS Optimization Procedure For setting up the CE-ESI-MS coupling, a solution of 0.1 mg m l ' ephedrine in the CE running buffer was introduced continuously from the CE system by electrophoretic migration at Vsep = 30 kV (effectively ca. 22 kV, vide supra). The instrumental parameters such as


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J. Stockigt, IVI.Uiiger, D. Stockigt and D. Belder

capillary positioning (Ar in Figure 3), sheath flow rate, ES-source voltages and MS ion optics, have been optimized to produce a signal of maximum sensitivity and stability. The potentials of the aluminum capillary, the tube lens, the skimmer, and the rf-only octapole ion-guide were adjusted in order to induce 10 - 20% nozzle-skimmer dissociation [38,39] products of the protonated ephedrine.

4. RECENT CE AND CE-MS ANALYSIS OF ALKALOIDS In the preceding section some technical details of capillary electrophoresis and mass spectrometry have been briefly discussed. The following part is concerned with a (by no means complete) listing of some notable CE and CE-MS applications in the fleld of alkaloids during the last two years. The emphasis is put more on the separation and identiflcation of raw materials as well as additional new topics. Trenerry et al. [74] have analyzed morphine and related alkaloids in crude morphine, poppy straw and opium preparations by micellar electrokinetic capillary chromatography. They reported on a separation of the well-known opium-related alkaloids within less than 10 min applying uncoated fused-silica capillaries (70 cm x 50 ^m I.D.) with a particular electrolyte (10% dimethylformamide, 90% 50 mmol l ' aqueous cetyltrimethylammonium bromide, 10 mmol r^ potassium dihydrogen orthophosphate and 10 mmol l* sodium tetraborate, pH 8.6) at V^sep = 25 kV and 28째C. The coefflcients of variation for area calculation were found to be slightly greater than for HPLC. Chu et al. have reported on the quantiflcation of vincristine and vinblastine in Catharanthus roseus plants by capillary electrophoresis [75]. A buffer pH of 6.2 resulted in the best resolution of the two alkaloids and the addition of organic modiflers (methanol and ethanol, 15% each) was disadvantageous concerning the separation efflciencies. The influence of an organic modifler on the CE separation of biogenic amines (histamine, benzylamine, 2-phenylethylamine, tryptamine, tyramine, serotonin) has been investigated by Lin et al. [76]. The background electrolyte consisting of aqueous NH4ACO (100 mmol r \ pH 7.5) was admixed with methanol (40%, v/v) or acetonitrile (30%, v/v). The amines could be effectively separated employing Vscp = 10 kV separation voltage in a 44 cm x 50 p,m I.D. capillary. A method for the determination of alkaloids in tobacco has been reported by Yang and Smetana [52]. Nicotine has been determined in aqueous tobacco extracts within 100 sec. Bjoemsdottir and Hansen have developed a CE method based on guest-host complexation for analyzing major alkaloids in opium and drugs [77]. Alkaloids in Evodiae fructus have been separated by Lee et al. applying MECC and capillary zone electrophoresis (CZE) [50,78].


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

301

Linearity over one order of magnitude of concentration was generally obtained and the limits of detection for the alkaloids were found to be in the range of 35 - 47 |Lig m l ' . Different electrolyte buffer compositions have been investigated, e. g. the influence of pH-values and organic modifier concentrations on the migration behavior. Ergot alkaloids have been employed in CE as chiral selectors towards the separation of racemic hydroxy organic acids [79]. The effects of pH and MeOH added to the background electrolyte were investigated: (i) Low pH proved to have an adverse effect on enantioseparation. (ii) The addition of 50% MeOH (v/v) to the background electrolyte altered the stereoselectivity and increased the solubility of the chiral selector. MECC has been utilized for the analysis of aporphine alkaloids in a standard mixture as well as for quantitative determination in Lauraceous plants [80]. The latter results with regard to run time, resolution and limit of detection appeared to be similar to those obtained with HPLC. Henion and coworkers have applied CE-MS for the first time to the analysis of trace impurities in alkaloids [81]. The goal of their investigation was the determination of the practical CE-UV-MS detection limits for representative minor components in simple synthetic mixtures. Palmatine could be detected with UV and single-ion monitoring even if the relative amount of palmatine to berberine was only 0.15%. Henion's group also pioneered in the quantitative determination of isoquinoline alkaloids by CE-MS in standard mixtures as well as extracts from Phellodendron wilsonii bark and a herbal tablet [82]. In addition, nozzleskimmer dissociation (NSD) was applied for fragmenting and identifying berberine.

5. CE-ANALYSIS OF ALKALOID STANDARDS The above developed system (section 3.1) was applied to a more general separation of alkaloids employing different alkaloid standard mixtures. They consisted of indole alkaloids (section 5.1), protoberberine and benzophenanthridine alkaloids (section 5.2), B-carboline alkaloids (section 5.3), and opium alkaloids (section 5.4) representing a selection of alkaloids within the mass range of 150 to 800 Da. 5.1. Indole Alkaloids First we analyzed a mixture containing 13 monoterpenoid indole alkaloids and two biogenic amines. The structures of these compounds are illustrated in Scheme 1. The corresponding CE analysis showed that 13 of the 15 components were baseline separated and only two compounds showed the same retention time (vincaleucoblastine (7) and corynanthine (8)). The CE-UV only electrophorogram (Figure 5) clearly indicated that three distinct alkaloid


302

J. Stockigt, M.Unger, D. Stockigt and D. BeMer

11

UVAbsorption

10

13

1

1

r

20

time / min

30

Figure 5; The electrophorogram of the CE-UV only analysis of the indole alkaloids and the biogenic amines 1-15 given in Scheme 1.

ay

CH2N(CH3)2

C0

CHaCHjNHj

H Tiyptamine (2) M:160

H Gramine (1) M:174

,.CH3

19 O H3CO2C

Serpentine (3) M:348

Scheme 1: The chemical formulars and nominal molecular masses of the IS indole derivatives.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

303

Alstonlne (4) M:348 B-Methylajmallne (5) M:341

H3CO2C

—C2H5 CpHi 2*^5

^

/

"COaCH,

CO2CH3 Tabersonlne (6) M:336 OH I H3C0 Vinblastine (7) M:810

V ". CH3

CjHs OCOCH,

CO2CH3

H3CO2C

— C.H, OH

Corynanthine (8) M:354

Vincristine (9) M: 824

OH H.CO (Scheme 1, cont.)

^C2H 2' »5 OCOCH,

CHO

CO2CH3


J. Stockigt, M.Uiiger, D. Stockigt and D. Beider

304

OH

HgCO ,CH. Raufloridine (10) M:382 Ajmaline (11) M: 326

H3CO2C

HOgC OH

H3CO2C

Yohimblnic acid (12) M:340

Deserpldlne(13) M:578

HXO

CH3O

R=

Reserpine (14) M:608

CH3O' H3CO2C

^0CH3 OCH3

OCH,

H3CO

Rescinnamine(15) M:634 (Scheme l,cont.)

^

H3CO2C OCH,


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

305

groups can be observed; one group containing the biogenic amines gramine (1) and tryptamine (2); a slower migrating second group between 14 and 21 min consisting of the ten alkaloids serpentine (3), alstonine (4), p-methylajmaline

(5), tabersonine (6), vinblastine (=

vincaleucoblastine) (7) and corynanthine (8), vincristine (9), raufloridine (10), ajmaline (11) and yohimbinic acid (12). An incomplete peak resolution was observed for the alkaloids 3 and 4 which only differ in the stereochemistry at the C(20) position. Both alkaloids are also very difficult to be separated by other techniques, e.g. thin layer chromatography. The third alkaloid group reached the detection window between 24 and 27 min. These alkaloids belong to the reserpine group. They are baseline separated and appear in the following order of electrophoretic mobility: deserpidine (13), reserpine (14) and rescinnamine (15). 5.2. Protoberberines / Benzophenanthridines Employing a sodium acetate/acetonitrile buffer, some protoberberines have been well resolved by CE [25], recently. We used a mixture of ten components (Scheme 2), i.e. sanguinarine (16), coptisine (17), berberine (18), palmatine (19), chelidonine (20), columbamine (21), jatrorrhizine (22), stylopine (23), canadine (24), and scoulerine (25) for the CE-analysis of protoberberine and benzophenanthridine alkaloids. From the obtained electrophorogram (Figure 6), these alkaloids can be classified as follows. The first group appears at a migration time around 12 min and consists of the benzophenanthridine type, sanguinarine (16) and the protoberberines coptisine (17) and berberine (18); all three compounds are excellently separated within one minute. The second group consisted of four alkaloids migrating between 13.5 and 14.2 min, which are nearly baseline separated. Palmatine (19) and chelidonine (20) are well resolved, but the protoberberines columbamine (21) and jatrorrhizine (22) appear at almost identical migration times. Only a slight peak broadening indicates that the two alkaloids comigrate and can not be separated under the employed analytical conditions. Comparing the structure of both compounds, the only difference results from the position of the phenolic hydroxy group in ring D; both protoberberines are bearing a quaternary nitrogen atom. In the third group the three tetrahydroprotoberberines are baseline separated in the following order: stylopine (23), canadine (24), and scoulerine (25). In the later series the hydrophilicity of the alkaloids increases. The hydrophilicity seems to influence significantly the migration times leading to the highest migration time for the most hydrophilic alkaloid scoulerine (25). This alkaloid appears in the electrophorogram after about 17 min. Similar to the results from the CE separation of indole alkaloids (section 5.1), the separation performance of the alkaloids 16 - 25 turned out to be again excellent, except the comigration of two alkaloids, 21 and 22, in this group (see Figure 6). Therefore it seems that the same electrolyte applied to the indolic alkaloids 1 - 15 is also well applicable to the CE separation


J. Stockigt, M.Uiiger, D. Stockigt and D. BeMer

306

21,22 uvAbsorption

WA10

1

0

r

â&#x20AC;&#x201D;I

20

time / min

Figure 6: The electrophorogram of the CE-UV only analysis of the protoberberine/benzophenanthridine alkaloids 16-25 given in Scheme 2.

?-\ Sanguinarine (16)

Oâ&#x20AC;&#x201D;-\

Coptisine (17) H3CO

CX^Hg

Berberine (18)

Scheme 2: The chemical formulars of the protoberberine/benzophenanthridines 16 - 25.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

307

0CH3

P-A

Palmatine (19)

H,CO OCH,

Chelidonine (20) OCH, OCH,

Columbamlne (21)

H3CO

OH

Jatrorrhizlne (22)

H3CO OCHo

OCH^

?-\

p-^

Vâ&#x20AC;&#x201D;O

Styloplne (23)

^^^3

Canadine (24)

OCH^

H3CO Scoulerlne (25) (Scheme 2, cont.)


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J. Stockigt, M.URger, D. Stockigt and D. BeMer

of the protoberberine- and benzophenanthridine alkaloids. 5.3. P-Carboline Alkaloids To prove, however, the more general application of the developed electrolyte system we investigated the CE separation of a completely different alkaloid group, the structurally rather simple P-carboline alkaloids. As depicted in Scheme 3 the following six P-carbolines 2 6 - 3 1 were analyzed: norharmane (26), harmane (27), harmaline (28), harmine (29), harmalol (30), and harmol (31). Within less than 14 min all six alkaloids were baseline separated under the same CE conditions (Figure 7). The substitution pattern of these alkaloids again influences the migration behavior significantly. The mobility of methoxylated compounds like harmaline (28) and harmine (29) is more pronounced compared with the hydroxylated alkaloids harmalol (30) and harmol (31). Thus, the alkaloids 28 and 29 appeared around 12 min, whereas the more polar hydroxy compounds 30 and 31 were detected after 1.5 min later. Furthermore, the increased basic character of the alkaloids leads to shortened elution times, because harmaline (28) and harmalol (30) migrate clearly faster than the corresponding 29 and 31, respectively. This was the first example in the field of p-carboline alkaloids, that CZE has been used for the separation of this type of natural products. Moreover, it is worth noting that excellent peak resolution is observed without special optimization of CE conditions, but with the same buffer system. 5.4. Opium Alkaloids Because of the high significance of Opium the analysis of opium alkaloids has been often described. HPLC is indeed an important technique for the separation and quantification of these compounds [83]. Since the major alkaloids of opium differ in structure, basicity and lipophilicity it is expected that they should be easily separated by CE and that structuremobility relationships could be rather simply predicted. A mixture of the following six isoquinoline alkaloids (Scheme 4) was investigated and the single compounds can be detected with increasing migration times: thebaine (32), codeine (33), papaverine (34), morphine (35) and narcotine (36). The amphoteric narceine (37) appears much later at 24 min. In fact, baseline separation was observed for the first five alkaloids within the range of 14 -17 min (Figure 8). In the series of morphinanes the migration properties can easily be explained: The dimethoxylated and more lipophilic thebaine (32) migrates fastest, followed by the monomethoxylated codeine (33) and the more polar dihydroxylated morphine (35). It is worth noting that in this case die structure-mobility relationship is readily explained, too, as it has been discussed above for the 6-carbolines 26 - 31 (see section 5.3). Up to this point, the given experimental results indicate that the applied buffer system based


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

309

29

26 27

30

31

uvAbsorption

28

u

Vj

^

0

10

1

1

r

time / min

20

Figure 7: The electrophorogram of the CE-UV only analysis of the 6-carboline alkaloids 26 â&#x20AC;˘ 31 given in Scheme 3.

Harmane(27)

^^3

3 Harmaline (28)

Norharmane (26)

Harmalol (30)

Scheme 3: The chemical formulars of the B-carboline alkaloids 26-31.


310

J. Stockigt, M.Unger, D. Stockigt and D. BeMer

34 32 36 33 35

UVAbsorption

uu*u 0

10

T

-H

Nn*mdn ^^m^k ^^^un^ r*^

-T

r

20

M-l^'

1

1

time / min

Figure 8: The electrophorogram of the CE-UV only analysis of the isoquinoline alkaloids 32 â&#x20AC;˘ 37 given in Scheme 4.

HXO

H3CO

N-CH.

N-CH,

H3CO Thebaine (32) M:311

Codeine (33) M:299

Scheme 4: The chemical formulae and nominal molecular masses of the isoquinoline alkaloids 32 - 37.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

311

H3CO.

H3CO OCH, Papaverine (34) M:339

OCH«

N-CH-,

Morphine (35) M:285

NarcotJne (36) M:413

OCH« O

OCH,

N-CH3 CH3

Narceine (37) M:445

OCH, OCH,

(Scheme 4, cont.)


312

J. Stockigt, M.Unger, D. Stockigt and D. Belder

on aqueous ammonium acetate (50 mmol l ' , pH 3.1) and acetonitrile (v/v : 1/1) may be used generally to separate alkaloidal natural products [70]. As a matter of fact, this system obviously represents a general approach for analyzing a number of chargeable secondary plant metabolites [84]. In the following sections, this special CE electrolyte will be shown to be applicable in direct combination with electrospray mass spectrometry, allowing a more advanced identification of natural products.

6. CE-MS ANALYSIS OF ALKALOID STANDARDS In the preceding sections we have described the development (section 3) and application (section 5) of a buffer electrolyte for the CE-UV only analysis of alkaloid standards. This buffer electrolyte was developed keeping the limitations of electrospray mass spectrometry (see section 2.2). Herein, we are going to describe the results on the analysis of two of the alkaloid standard mixtures by the CE-MS on-line coupling. 6.1. CE-MS Analysis of Indole Alkaloids As a first example of the application of CE-MS we analyzed the same mixture of monoterpenoid indole alkaloids as described in section 5.1. In Figure 9 the analysis of the alkaloids 1 - 1 5 monitored by UV (Figure 9A) and by the corresponding reconstructed total ion current (RIC, Figure 9B) electrophorograms is presented. A comparison of the data in both parts of the figure shows that the signals of the CE-UV electrophorogram (Figure 9A) indicate reduced migration times as was found in the CE-MS electrophorogram (Figure 9B). The observed differences are due to the instrumental set-up: In the CE-MS coupling, the UV detection occurs on capillary at a distance of 21 cm, whereas the ES-MS detector is situated at the end of capillary, i.e. after 78 cm. Furthermore, the signal to noise ratio (S/N) is different for the electrophorograms in Figure 9A and B. This is due to higher background signals in electrospray MS arising from ions in the scanned mass ranges (m/z 100 - 1000) originating from the NH4ACO electrolyte and from the sheath liquid. Consequently, the S/N in CE-MS strongly depends on the composition but also on the concentration of both the electrolyte and the sheath liquid [85]. In fact the S/N of the RIC can be optimized in a certain range if the rate of the sheath flow is reduced, e.g. reduction of the flow from 2 to 1 ^1 min' approximately doubles the S/N of the RIC. Comparing the signal intensities in Figure 9A and B, the differences result (i) from the alkaloid's extinction coefficients at a selected UV wavelength and (ii) from the alkaloid's proton affinities important in the ES-MS detection. As a striking example, the signals originating from 1 and 2 vs 3 and 4 will be discussed in brief The intensity of the UV


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

313

UVAbsorption

1

1

1

time / min

r

10

B RIC

\NJWUWV>^

0

10

20

time / min

Figure 9: Results of the CE-MS analysis of the indole alkaloids and biogenic amines 1-15 given in Scheme 1. (A) UV electrophorogram at 200 nm obtained after ca. 21 cm of analyte migration. Note the decreased resolution compared to the data in Figure 5. (B) Corresponding RIC electrophorogram characterized by a lower S/N compared with part A.


314

J . Stockigt, lVl.Uiiger, D. Stockigt and D. BeMer

m/z 161

2 2J^

m/z 175

^

m/z 327

'*'*[

m/z 337

^ I

m/z 341

^

m/z 349

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m/z 609

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m/z 635 m/z 811

. K

91

m/z 825 1

0

1

1

10

1

1

1

20 time/min

Figure 9: (C) Corresponding selected nominal mass-to-charge electrophorograms. The S/N is similar to the one in part A. Note the different migration behavior of yohimbinic acid (12) as compared to the data in Figure 5.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

1

175[M+Hr

3,4

161 [M+Hf 321 [2M+H]^

100

m/z

900

100

349 [M+H]

697 [2M+H]^

m/z

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315

900

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7

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m/z

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100

m/z

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900

825 [M+H]* [M+K]*

100

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m/z

[M+K]^ 900

383 [M+H]^

413[M+2H]^'^ 100

11

709 [2M+H]* X m/z 900

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m/z

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m/z

900

100

15

100

m/z

900

635 , [M+H]

m/z

900

Figure 10: Summed ES mass spectra of the indolic compounds 1-15 given in Scheme 1 as obtained from the same CE-MS analysis as in Figure 9. Note the different S/N ratios, e. g. in the mass spectra of 5 and 12.


316

J. Stockigt, M.Unger, D. Stockigt and D. BeMer

absorption is similar within ca. +/-10% for the four components as expected at 200 nm. The ES-MS response, however, differs by a factor of ten, probably due to the different pKa values of 1 and 2 vs 3 and 4. The single-ion electrophorograms displayed in Figure 9C are characterized by a much higher S/N when compared with the RIC in Figure 9B. Because serpentine (3) and alstonine (4) are epimers differing only in the stereochemistry at carbon 19, they are difficult to separate. Also an acceptable resolution of the signals for vincristine (9) and raufloridine (10) was not observed under CE-MS conditions. In contrast to the comigration of vinblastine (7) and corynanthine (8) in the CE-UV only system (Figure 5), both could be distinguished in the RIC electrophorogram. All the other alkaloids have been separated clearly and gave the expected quasi-molecular ions as illustrated in Figure 10. For each of the 15 components the protonated molecule [M+H]*^ leads to the dominating signal, e.g. m/z 175 for gramine (1) or m/z 825 for the dimeric alkaloid vincristine (9) (see Figure 10). Only 6-methylajmaline (5) is detected as M^ since it is already bearing one positive charge on the quaternary nitrogen atom (cf. Scheme 1). It is worth noting that all the spectra have been recorded with signal to noise ratios of at least 1(X):1, since for each MS three to five scans have been accumulated. Most of the spectra gave further signals in addition to the molecular ions. These signals arise from the sodium and potassium adducts [M+Na]"^ and [M+K]^ with relative signal intensities up to 20%, cf. vinblastine (7) and vincristine (9). In some cases also solvent clusters with methanol, acetonitrile or water can be observed ([M+Na+S]*, S = solvent), but the relative intensity of these ions is less than 3%. Further evidence on the nature of the single compounds could be obtained from alkaloid dimer cluster ions [2M-fH]^ and doubly protonated molecular ions like [M-f2H]^'*', cf. ajmaline (11) and vincristine (9). This additional MS information can be important for the identification of unknown alkaloids in complex mixtures (see below). 6.2. CE-MS Analysis of Some Isoquinoline Alkaloids A further example of CE-MS analysis of an alkaloid mixture is the separation of opium alkaloid standards as illustrated in Figure 11. UV detection with the CE-MS apparatus is displayed in Figure llA and the reconstructed total ion current is shown in Figure IIB. As already observed for the above mentioned analysis of the indole alkaloids mixture in CE-MS, the in-capillary UV monitoring resulted in reduced migration times and decreased resolution of signals when compared with the RIC detection mode. The broad signal between 8-10 min in Figure llA is due to the bulk flow in the capillary. Moreover, these results of the measurement of the opium alkaloids demonstrated also a lower S/N in the RIC trace compared with the UV trace at 2(X) nm. Considering the selected m/z electrophorograms of the protonated molecules obtained from the CE-MS on-line coupling (Figure 1IC), the S/N is


317

Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

EOF UVAbsorption

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-i

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5

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r

time / min

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Figure 11: Results of the CE-MS analysis of the isoquinoline alkaloids 32 - 36 given in Scheme 4. (A) UV electrophorogram at 200 nm obtained after ca. 21 cm of analyte migration. Note the decreased resolution compared with the data in Figure 8. (B) Corresponding RIC electrophorogram characterized by a lower S/N compared with part A.


318

J. Stdckigt, M.U^er, D. Stockigt and D. Bdder

35

m/z 286

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Figure 11: (C) Corresponding selected nominal mass-to charge electrophorograms. The S/N is similar to the one in part A.

32

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900

414 [M+H]* [2M+Na]* [2M+HJ! I2M+K]*

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900

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m/z

900

Figure 12: Single-scan ES mass spectra of the isoquinoline alkaloids 32 - 36 as obtained from the same CE-MS analysis as in Figure 11. Note the presence of the dimeric clusters, e. g. [2M+Nal , in the mass spectra of 32 and 36.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

319

significantly higher than that of the RIC (Figure 1 IB). As expected, the alkaloids 32 - 36 migrate under the CE-MS conditions in the same sequence as in the CE-UV only mode (Figure 8). The appropriate single scan mass spectra are displayed in Figure 12. Their signal to noise ratios vary between about 20:1 up to more than 100:1 as measured for morphine (35) and papaverine (34), respectively. All dominating signals correspond to the protonated molecules e.g. m/z 312 for thebaine (32) or m/z 414 for narcotine (36). In addition the corresponding cluster ions [2M+H]^, [2M+Na]^ and [2M+K]^ are observed with different but lower intensities as compared with [M+H]^, evidencing the molecular masses of the alkaloids. The presently described results indicate that both CE-UV and CE-MS can be used elegantly for the analysis of complex alkaloid mixtures. Comparing both detection methods one needs to consider several points (cf. section 2.1 and section 2.2). The locations of compound detection are different: Detection in CE-UV only takes place at ca. 50 cm of migration in the capillary, whereas UV detection in the CE-MS coupling occurs at ca. 20 cm and the electrospray-MS detection at ca. 75 cm. Therefore, different migration times of the same alkaloid are observed. In addition, siphoning occurs because of (i) the electrospray process, (ii) the sheath-flow, and (iii) the differently leveled inlet and outlet vials (CE-MS: outlet vial = ES needle). As a result the bulk-flow in the capillary is increased and the analyte's migration velocity is higher. Comparing the qualities of different electrophorograms of the same CE run, the reconstructed total ion current shows always a smaller S/N than the UV and the individual mass traces. For the latter two the signal to noise ratio is comparable. Concerning the separation efficiencies, the data from the RIC are inferior to the UV because of dead volumes in the ES-MS interface as well as the diffusion of analyte ions from the capillary's end into the sheath flow. For CEUV the detection limit of a selected alkaloid depends on its extinction coefficient and in case of the ES-MS detection on its proton affinity. The CE-detection limit for gramine (1) has been found to be ca. 250 ng ml"' corresponding to only 2.5 pg in CE-UV only and in the selected m/z electrophorograms, respectively, illustrating an impressive sensitivity of the method. It has, however, not yet been proved from these experiments on alkaloid standard mixtures whether the developed conditions would also allow the analysis of crude alkaloid mixtures, for example alkaloid extracts from various plant material. In the following part of this article we are going to concentrate on the CE-MS analyses of more complex alkaloid mixtures obtained from crude extraction of different plant materials.


320

J. Stockigt, M.Unger, D. Stockigt and D. BeMer

7. CE-MS ANALYSIS OF CRUDE ALKALOID EXTRACTS The application of CE to the separation of alkaloids within particular classes has already been described in sections 4 and 5. In section 3 a more general method developed for the coupling of CE to electrospray MS has been discussed and the corresponding results have been summarized in section 6. In the following part, alkaloid identification from crude mixtures will be outlined employing the established CE-MS technique with modest changes of the instrumental parameters. 7.1. Rauwolfia Alkaloids from Roots Rauwolfia serpentina Bent, ex Kurz is an old medicinal plant from India applied for the treatment of various diseases in former times. In addition, the roots are the industrial source for the isolation of prominent alkaloids like reserpine, rescinnamine, yohimbine, ajmalicine or ajmaline which are of pharmacological-therapeutically interest. Therefore, the chemical analysis of the root material has been carried out many times [86]. Using a crude extraction of commercially available Rauwolfia roots, the developed CE-MS method (section 3) was tested for its performance concerning resolution and required time of analysis [70b]. For sample preparation, the powdered roots (25 g) were stirred in the dark for 48 h with ethyl acetate containing a trace of ammonia. After filtration and concentration of the solution, extraction with 2% sulfurk: acid was carried out. The aqueous layer was adjusted to pH 9 with ammonia and extracted with dichloromethane. The crude alkaloid mixture obtained after evaporation of the dichloromethane solvent was dissolved in methanol and subjected directly to CE-MS analysis. In Figure 13 the results of the CE-MS measurements are illustrated. Figure 13A represents the CE-UV only electrophorogram of the mixture, whereas part 13B displays the reconstructed total ion current-electrophorogram of the root extract revealing the presence of about eight signals between 22 and 28 min with quite a poor resolution. A selection of the extracted single mass electrophorograms given as m/z values is summarized in Figure 13C. At least 15 components can be extracted from these electrophorograms within 30 min. The data in Figure 13B also demonstrate the diminished resolution in CE-MS compared with CE-UV only (cf. Figure 5). As shown in Figure 13, the protonated molecular ions, [M+H]*, of the major Rauwolfia alkaloids like ajmaline (m/z 327), ajmalicine (m/z 353), and reserpine (m/z 609) have been detected with dominating signal intensities. Since a number of Rauwolfia alkaloids exhibit a molecular weight of 354 Da, the assignment of the m/z 355 peaks is not straight forward. This signal may be due to corynanthine, isorauhimbine, yohimbine, related isomers or a superposition of any of them. A possibility for clear isomer distinction can be achieved by


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

321

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0

10

20

time / min

30

10

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time / min

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Figure 13: Results of the CE-UV only (A) and the CE-MS analysis (B and C) of a root extract from Rauwolfla serpentina. The UV electrophorogram (A) has been obtained at 224 nm. The S/N is reduced in the RIC electrophorogram (B) compared with the one in part A.


322

J. Stiickigt, M.Unger, D.Stkkigt and D. Belder

m/z 309

C

mlz 327 m/z 349 1

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1

20

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time / min

Figure 13: (C) Selected m/z electrophorograms of the CE-MSanalysis of a root extract from Rauwowa serpentina.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

23:42 min

323

24:46 min

m/z 355

m/z 327 m/z 653

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m/z

900

24:41 min

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27:45 min m/z 635

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m/z

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27:22 min

m/z 349

200

m/z

900

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m/z

900

Figure 14: Electrospray mass spectra of some main alkaloids as obtained from the CE-MS analysis presented in Figure 13, part B and C.


324

J. Stockigt, M.Unger, D. Stockigt and D. Bekier

employing CE-MS/MS [39,87]. In addition, mass spectrometric evidence for the presence of a number of well-known Rauwolila alkaloids in the crude mixture has been found (Figure 14). However, components with the same electrophoretic migration behavior and the same nominal m/z data could not be resolved. With increasing molecular weight the respective signals result probably from tetraphyllicine (m/z 309), serpentine or alstonine (3 or 4, m/z 349, see section 5.1), acetylajmaline (m/z 369), reserpinine (m/z 383), acetyl-corynanthine (m/z 397), reserpinic-acid methyl ester or seredine (m/z 415), raunescine (m/z 565), deserpidine (13, m/z 579), renoxydine (m/z 625), and rescinnamine (15, m/z 635). Indications for the characteristic Rauwolfia alkaloid sarpagine which should appear as a peak in the m/z 311 electrophorogram have not been obtained. It remains unproved whether the second, smaller signal in the m/z 353 electrophorogram corresponds with acetyl-sarpagine, which could result from the work-up procedures used on the root powder. Also for the signal at m/z 405 an appropriate explanation is not yet present, because none of the typical Rauwolfia alkaloids exhibit such a molecular weight. In fact, more detailed MS investigation is necessary for a satisfying answer to this question [87]. Because of these promising results on die CE-MS identification of alkaloids in Rauwolfia roots, we have applied the same procedure to analyze the alkaloids formed in Rauwolfia in vitro cultures, as outlined in the next section. 7.2. Rauwolfia Alkaloids from Cell Suspension Cultures Several studies on alkaloidal constituents of Rauwolfia serpentina callus tissue [88,89] and cell suspension cultures [90,91] have been published in the past. We have repeated the extraction of the Rauwolfia alkaloids from cell suspension cultures in order to analyze these crude extracts by CE-MS [70b]. Firstly, the cells were grown for ten days in Linsmaier and Skoog medium [92] followed by culturing them in AP II medium [93]. The tissue was freezedried and extracted as described for the Rauwolfia roots in section 7.1. The crude alkaloid mixture obtained was analyzed by CE-MS as described above. The UV-electrophorogram and the reconstructed ion current of a CE-MS measurement of an extract from cell suspensions of Rauwolfia serpentina is shown in Figure 15A and B. The latter one illustrates that the migration times of the alkaloids amount to 19 - 23 min. As shown by the peaks of the numerous single mass electrophorograms (Figure 15C) at least 20 constituents were present in the cell extract. Because of the extraction procedure and the migration behavior of these constituents, these constituents should possess basic character. Accordingly, they are most probably Rauwolfia alkaloids. In fact most of the detected quasimolecular ions, M* (for 5) or [M+H]*, result from typical alkaloids of cultivated Rauwolfia cell suspensions [91].


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

325

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20

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0

10

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Figure 15: Results of the CE-UV only (A, at 224 nm) and the CE-MS analysis (B and C) of an alkaloid extract obtained from cell suspension cultures of Rauwolfia serpentina.


326

J. Stockigt, M.Uiiger, D. Stockigt and D. BeMer

m/z 295

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Figure 15: (C) Selected m/z electrophorograms of the CE-MS analysis of an alkaloid extract obtained from cell suspension cultures of Rauwolfia serpentina.


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

Q

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Figure 15: (D) Comparison of the m/z electrophorograms resulting from the [M+H]^ and the [M+Na]^ quasi-molecular ions of ajmaline (11) characterized by the same migration time (indicated by the dashed line). In addition, a faster migrating alkaloid most probably with the nominal molecular mass of 348 ([M+HJ*: m/z 349) has been detected.


328

J. Stockigt, M.Unger, D. Stockigt and D. Belder

The major alkaloids of the cells are known to be nortetraphyllicine, tetraphyllicine, ajmaline (11) and 17-O-acetylajmaline (given in the order of increasing molecular weight) [1,90,91]. The nominal m/z values of their [M+H]* signals correspond to m/z 295, m/z 309, m/z 327 and m/z 369. Indeed, the latter signals have been detected as the most intense ones in the CE-MS analysis (Figure 15B and C). Furthermore, the CE-MS data point to other characteristic *cell culture alkaloids' [1,90,91], e.g. norajmaline (m/z 313) and vomilenine, acetyltetraphyllicine or perakine (m/z 351). But also m/z data of alkaloids occurring in significant lower amounts are present, like those of ajmalicine (m/z 353, or its isomers), 17-(9-acetyl-yVa-norajmaline (m/z 355) etc. Because of a lack of additional MS data on the structure of these components, a straight forward identification of these components is not yet possible in CE-MS. The broad signal of the m/z 349 electrophorogram co-migrating with ajmaline (11, m/z 327) results from the sodium adduct of ajmaline (Figure 15C and D). The nominal mass of ajmaline is 326. The nominal m/z value of [11+H]^ amounts to m/z 327 and the nominal m/z value of the sodium adduct, [11+Na]*, comes to m/z 349. As mentioned in section 2.2, the formation of cation adducts (Li, Na, K, NH4,...) can be observed very often in the electrospray process. The admixing of Cu or Ag salts to the sheath flow may be helpful since the signal of the quasi-molecular ion [M+Cu]* or [M+Ag]*, may be identified according to the n^taPs typical isotopic pattern. 7.3. Alkaloids from Cortex Quebracho In a further example concerning CE-MS analyses of crude alkaloid mixtures we have investigated commercially available samples from the cortex of Aspidosperma quebrachobianco Schlecht [70c]. The sample was extracted with methanol and directly applied to CEUV only and CE-MS analysis under the same conditions as described above (section 3). As depicted in Figure 16, the CE-UV electrophorogram of a sample from Cortex Quebracho is obtained in less than 30 min. Besides three major signals several other basic constituents can be detected amounting to a total of about 20 components, most probably alkaloids. When the same sample was measured in the CE-MS mode about 16 different peaks were observed in the RIC electrophorogram (Figure 16B). As already mentioned in the preceding sections, the S/N of the latter is reduced by a factor of ca. 5 compared with that of the CE-UV only electrophorogram. Based on the data of the CE-MS analysis, 28 different quasi-molecular ions, [M+H]*, have been recognized. Selected m/z electrophorograms are given in Figure 16C. More than one peak is detectable in some of them, e.g. m/z 295, m/z 329, and m/z 341, indicating most probably distinct alkaloids with a quasi-molecular ion of the same nominal m/z value. With regard to these results, the investigated crude extract of Cortex Quebracho may consist of ca. 50 alkaloids, many of which have not yet been identified [70c].


Analysis of Alkaloids by Capillary Electrophoresis and Capillary Electrophoresis

329

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r 0

10

20

time / min

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Figure 16: Results of the CE-UV only (A, at 224 nm) and the CE-MS anaivcU rn o„^ ns


330

J. Stockigt, iVf .Unger, D. Stockigt and D. Belder

m/z 267 m/z 273 m/z 283 L. m/z 295

jy m/z 297 m/z 299 m/z 309 m/z 313 m/z 315 m/z 325 I

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