The Alkaloids: Chemistry and Pharmacology, Vol. 46

THE ALKALOIDS Chemistry and Pharmacology VOLUME 46

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THE ALKALOIDS Chemistry and Pharmacology Edited by Geoffrey A. Cordell College of Pharmacy University of Illinois at Chicago Chicago, Illinois

VOLUME 46

ACADEMIC PRESS San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1995 by ACADEMIC PRESS, INC. All Rights Reserved. N o part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX International Standard Serial Number: 0099-9598 International Standard Book Number: 0-12-469546-9 PRINTED IN THE UNITED STATES OF AMERICA 95 96 9 7 9 8 99 O O Q W 9 8 7 6

5

4

3 2 1

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

vii ix

Chapter I . Biosynthesis of Pyrrolizidine and Quinolizidine Alkaloids DAVIDJ. ROBINS I. 11. 111. IV.

Introduction ........... ........ Pyrrolizidine Alkaloi ........ Quinolizidine Alkaloids ................................................................... Conclusions ......... ................................................................ Addendum .................................................. ............................ References ....................................................................................

1

3 36 55 56 57

Chapter 2. Pharmacology of Polyamine Toxins from Spiders and Wasps ROELOFFS,A N D HUNTER JACKSON ALANL. MUELLER,ROSEMARIE I. 11. 111. IV. V.

Introduction and Ecological Aspects ................................................. Pharmacological Effects of Polyamine Toxins in Invertebrates ............... Pharmacological Effects of Polyamine Toxins in Vertebrates ................. Structure-Activity Relationship Studies ............................................. Perspectives .................................................................................. References ....................................................................................

63 61 12 86 90 91

Chapter 3. Epibatidine A N D CSABA SZANTAY, JR. CSABASZANTAY, ZSUZSANNA KARDOS-BALOGH,

I. 11. 111. IV. V.

Introduction .................................................................................. Occurrence .......................... .......... Structure and Syntheses .................................................................. NMR Spectroscopy ........................................................................ Pharmacology ................................................. Addendum ..................................................... References ......... ................................................................. V

95 % %

I16 I I9 123 I24

vi

CONTENTS

Chapter 4. The Naphthylisoquinoline Alkaloids GERHARD BRINCMANN A N D FRANK POKORNY

I. Introduction .................................................................................. 11. Isolation and Structure Elucidation of Naphthylisoquinoline Alkaloids: Dioncophylline A ("Triphyophyll 111. Other Alkaloids from the Dioncophyllaceae 1V. New Alkaloids from Asian Ancistrocladace V. Alkaloids of African Ancistrocladaceae Species.. ............. VI. The Michellamine: A New Class of Naturally Occurring .................... Quateraryls and Related Compounds .............. VII. Stereocontrolled Synthesis of Mono- and Dimeric Naphthylisoquinoline Alkaloids ........................................................ VIII. Biogenetic Origin of Naphthylisoquinoline Alkaloids ..... IX. The Chemo-ecological Context of Naphthylisoquinoline X. Tables of Known Natural Naphthylisoquinoline Alkaloids ..................... XI. Summary and Outlook ..................... ................... ...... XII. Addendum .................................................................................... ............................................... References .......... .....................

128 130 146 156 158

170 180 200 21 1 216 254 255 263

Chapter 5 . The Biotransformation of Protoberberine Alkaloids by Plant Tissue Cultures KINUKOIWASA

I. Introduction .................................................................................. 11. The First Pathway .......................................................................... 111. The Second Pathway .................................................. ............ IV. The Third Pathway ......................................................................... ................... ................... References ...........

CUMULATIVE INDEX OF TITLES................................................................ INDEX ..................................................................................................

273 277 329 333 345

347 355

CONTRIBUTORS

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

GERHARD BRINGMANN (127) Institut fur Organische Chemie der Universitat Wurzburg Am Hubland, D-97074 Wurzburg, Germany KINUKOIWASA(273) Laboratory of Pharmaceutical Chemistry, Kobe Pharmaceutical University, Higashinada, Kobe 658, Japan HUNTER JACKSON (63) NPS Pharmaceuticals, Inc., Salt Lake City, Utah 84108 ZSUZSANNA KARDOS-BALOGH (95) Central Research Institute for Chemistry of the Hungarian Academy of Sciences, H-1525 Budapest, Hungary ALANL. MUELLER(63) NPS Pharmaceuticals, Inc., Salt Lake City, Utah 84108 FRANKPOKORNY (127) Institut fur Organische Chemie der Universitat Wurzburg Am Hubland, D-97074 Wurzburg, Germany DAVIDJ. ROBINS (1) Department of Chemistry, University of Glasgow, Glasgow G12 SQQ, United Kingdom ROSEMARIE ROELOFFS (63) NPS Pharmaceuticals, Inc., Salt Lake City, Utah 84108 CSABASZANTAY (95) Central Research Institute for Chemistry of the Hungarian Academy of Sciences, H- 1525 Budapest, Hungary CSABAS ~ A N T AJR. Y , (95) Spectroscopic Department, Chemical Works of Gedeon Richter, H-1475 Budapest, Hungary

vii

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PREFACE

This volume of The Alkaloids recognizes that a primary goal of alkaloid research is making compounds available for potential therapeutic or biological use for the benefit of humankind. The motivation that leads to the discovery of novel, biologically active compounds occasionally yields “hot” topics, areas of research that attract substantial attention and a flurry of scientific activity. Three of the chapters in this volume of the series reflect that level of current interest, spider toxins, naphthylisoquinoline alkaloids, and epibatidine. Three different sources of biologically significant compounds, arachnids, higher plants, and frog skin, have yielded these alkaloids, which, in the future, may have a significant effect in three quite different areas of biological focus, CNS disorders, anti-HIV, and analgesia. Jackson and co-workers review the progress that has been made on understanding the biology of certain spider toxins and their derivatives, while Bringmann and Pokorny delineate the progress made in the structure elucidation, synthesis, and biological activity of the monomeric naphthylisoquinoline alkaloids. SzAntay reviews the very interesting, non-narcotic analgesic epibatidine, particularly the advances that have been made in developing effective synthetic procedures for analog work. The two remaining chapters reflect our innate desire, as an integral part of alkaloid chemistry, to understand how alkaloids are produced from their simple amino acid precursors and to discern how one group of metabolites may serve as a point of structural diversification for many other metabolites. Robins reviews the biosynthesis of the pyrrolizidine and quinolizidinealkaloids showing how these structurally analogous alkaloids have quite different biosynthetic pathways, while Iwasa comments on the progress made in understanding the precursor relationships and enzymatic control of the molecular acrobatics of the protoberberine alkaloids in yielding other important alkaloid classes. Geoffrey A. Cordell University of Illinois at Chicago

ix

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-CHAPTER 1-

BIOSYNTHESIS OF PYRROLIZIDINE AND QUINOLIZIDINE ALKALOIDS DAVIDJ. ROBINS Department of Chemistry University of Glasgow Glasgow GI2 8QQ

I. Introduction ....... ........................................................................... 1 11. Pyrrolizidine Alkal ........................................ ........................... 3 A. Structures and Biological Activity ........ B. Biosynthesis of Necines from Radioactive Ornithine, Arginine, and Putrescine .................................... ............... C. Biosynthesis of Necines from Putrescine Stable Isotopes ................................................................................ 7 ..................... 13 D. Biosynthesis of Necines from Homospermidine . E. Biosynthesis of Necines Involving Iminium Ions F. Biosynthesis of Necines Involving I-Hydroxymethylpyrrolizidines ...........20 G. Stereochemistry of the Enzymic Processes in Necine Biosynthesis ..........23 H. Biosynthesis of Necic Acids .............................................................. 31 111. Quinolizidine Alkaloids ........................................................................ .36 A. Structures and Biological Activity ...................................................... 36 B. Biosynthesis of Lupinine ................................................................. .36 C. Stereochemistry of the Enzymic Processes Involved in Lupinine Biosynthesis ..................................................................... .40 D. Biosynthesis of Tetracyclic Quinolizidine Alkaloids .............................. .43 E. Stereochemistry of the Enzymic Processes Involved in the Biosynthesis of Tetracyclic Quinolizidine Alkaloids ................................................ .47 IV. Conclusions ........................................................................................ .55 Addendum ......................................................... ........................... .56 References ......................................................................................... .57

I. Introduction

This series has mainly concentrated on the occurrence, structure elucidation, chemistry, synthesis, and pharmacology of the many groups of alkaloids. Yet knowledge of the biosynthesis of alkaloids is a tremendous help in dividing up alkaloids into manageable groups of biosynthetically 1

THE ALKALOIDS. VOL. 46 Copyright 0 1995 by Academic Press, Inc. All nghts of reproduction in any form reserved.

DAVID J . ROBINS

2

related compounds. Despite the complexity of many alkaloids, careful examination of their structures has shown that their origins can usually be traced back to just five of the common a-amino acids, namely ornithine (l),lysine (2), phenylalanine (3), tyrosine (4), and tryptophan (5).

;iJ-. H

C02H

1

H2N

I

H

5

I

OH 4

Sir Robert Robinson (I) was the first to suggest that pyrrolizidine alkaloids containing the 1-hydroxymethylpyrrolizidinesystem (6) were derived from two molecules of ornithine (1).He also proposed that quinolizidine alkaloids such as lupinine (7) containing the 1-hydroxymethylquinolizidine system are formed from two molecules of lysine (2).

6

6

4

7

Experimental verification of these proposals became possible only in the 1950s when precursors containing radioisotopes of I4C and 3Hbecame available. Labeled forms of ornithine (1)and lysine (2) were fed to plants

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

3

that produce pyrrolizidine and quinolizidine alkaloids, respectively; then the alkaloids produced were isolated and their radioactivity was measured. The all-important location of radioactive labels within the alkaloids was partly determined by degradation, but labeling was usually limited to just one or two of the carbon atoms. Nevertheless, Robinson’s original proposals were substantiated, and it was generally accepted that the pathways to isoretronecanol (6) and lupinine (7) would turn out to be very similar. Progress in this area of biosynthetic study was greatly accelerated by the advent of compounds labeled with stable isotopes (2H, I3C, and 15N), coupled with the ability to determine complete labeling patterns in alkaloids by high-field NMR spectroscopy. This advance has produced information that could never have been obtained using radioisotopes, and it has clearly demonstrated that the two biosynthetic pathways to isoretronecanol (6) and lupinine (7) are quite different.

11. Pyrrolizidine Alkaloids

A. STRUCTURES A N D BIOLOGICAL ACTIVITY More than 200 different pyrrolizidine alkaloids have been isolated and their structures established. This process of discovery has been well documented in The Alkaloids in 1950 (2), 1960 (3), 1970 (4,and 1985 (5). The many synthetic routes to these alkaloids have also been covered in this series, and progress in this area is reviewed annually by Robins (6). The widespread occurrence of pyrrolizidine alkaloids is discussed in a recent review (7) and is evident from the fact that they have been shown to be present in 15 plant families-Apocynaceae, Asteraceae (formerly Compositae), Boraginaceae, Celastraceae, Ehretiaceae, Euphorbiaceae, Fabaceae (formerly Leguminosae), Graminae, Linaceae, Orchidaceae, Ranunculaceae, Rhizophoraceae, Santalaceae, Sapotaceae, and Scrophulariaceae. Within these families, the most widely studied genera are Senecio (about 200 species, Asteraceae), Crotalaria (about 80 species, Fabaceae), and Heliotropium (about 30 species, Boraginaceae). Typical alkaloids are retrorsine (8) from Senecio isatideus, rosmarinine (9) from Senecio pleisrocephalus, monocrotaline (10) from C. retusa, and echinatine (11)from H. indicurn (8). Pyrrolizidine alkaloids contain a base portion (necine), as in 6, with hydroxy groups generally present at the 1- and 9-positions. The alkaloids are usually present in plants as ester derivatives, such as echinatine (11).

4

*

DAVID J . ROBINS

Me

O

b

-

5 9

0

Diesters are also frequently found, particularly macrocyclic diesters with ring sizes of 11, as in monocrotaline (lo), or 12, as in retrorsine (8) or rosmarinine (9). The esterifying acids (necic acids) have interesting structural features. They have branched chains, often contain oxygen functions or unsaturation, and usually occur with ten carbon atoms. CH,--O

& N

-CO

H HO

o

e

Me

11

C H 2 4 -CO Me&W%H

10

Me

N+

I

O-

12

Many pyrrolizidine alkaloids exhibit a range of biological activities, particularly hepatotoxicity. In fact, many livestock deaths have resulted from ingestion of plant material containing pyrrolizidine alkaloids (9). These alkaloids also contribute to human liver disease when they are consumed by humans either by accident when foodstuffs are contaminated, or deliberately through injudicious use of herbal remedies prepared from plants known to contain pyrrolizidine alkaloids, e.g., comfrey (Symphytum spp., Boraginaceae). The hepatotoxicity is observed only in pyrrolizidine alkaloids that contain 1,2-unsaturationin the necine component, e.g., retrorsine (8) or monocrotaline (10).It is now believed that the

5

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

alkaloids are not toxic per se, but are oxidized in the liver to the corresponding pyrroles, which are potent alkylating agents (9). Some N-oxides do have antiof pyrrolizidine alkaloids, particularly indicine N-oxide (U), tumor properties, and this area has been reviewed in The Alkaloids (10). One section of the animal kingdom that generates much benefit from these alkaloids is found within the Lepidotera. Some butterflies of the family Danaidae and moths from the Arctiidae feed on plants containing pyrrolizidine alkaloids and store them as a defense against predators that do not like the bitter taste of the alkaloids. Furthermore, the base portions of some of these pyrrolizidine alkaloids can be converted by these Danaid butterflies into pheromones, which are used to attract mates (6). B. BIOSYNTHESIS OF NECINESFROM RADIOACTIVE ORNITHINE, A N D PUTRESCINE ARGININE, The first biosynthetic studies on necines were carried out more than 30 years ago. Nowacki and Byerrum studied the formation of monocrotaline (10)in Crotalaria spectabilis ( 1 1 ) . They showed that ornithine was incorporated specifically into retronecine (W), which is the base portion of monocrotaline. Warren and co-workers observed similar results in their work on Senecio isatideus and S . sceleratus (12).Bottomley and Geissman (13) also studied the biosynthesis of retronecine (13). They worked with S . douglasii, which produces a mixture of pyrrolizidine alkaloids, all of which produce retronecine on alkaline hydrolysis. [2-I4C]Ornithinegave an incorporation of 0.30% into the alkaloid mixture, and 94% of the radioactivity was present in retronecine. [5-14C]Ornithinewas also incorporated well (0.75%) into the mixture of alkaloids, and again 94% of the activity was located in the base portion. Furthermore, the retronecine (13)samples were treated with osmium tetroxide and sodium periodate, and the formaldehyde liberated, corresponding to C-9 of retronecine (13),was trapped as the dimedone derivative (Scheme 1). When the radioactivity of these derivatives was determined, it was found that in both cases (after feeding

c;l2

MH

1

7

c0fl2so4

6

5

3

7 N

6

5 13

-

OsOJNa1O4

4

SCHEME 1. Degradation of retronecine (13).

9 HCHO

6

DAVID J . ROBINS

[2-14C]ornithineand [5-14C]ornithine)about 25% of the total radioactivity of the retronecine samples was present in the dimedone derivatives. This suggested that C-2 and C-5 of ornithine become equivalent in the pathway (at least in the formation of the right-hand ring) possibly through decarboxylation of ornithine to give 1,Cdiaminobutane (putrescine) (14).In support of this theory, [ 1,4-'4C]putrescinewas incorporated to a reasonable extent (0.18%) into the mixture of alkaloids in Senecio douglasii, and basic hydrolysis showed that 98% of the radioactivity was present in retronecine. Further degradation demonstrated that 25% of this activity was present in the formaldehyde dimedone derivative (13). Arginine (15)has also been shown to act as a precursor of senecionine (16)in Senecio magnificus by Bale and Crout (14). These workers had obtained low and varying incorporations of precursors in different feeding experiments using plants growing under hydroponic conditions. They therefore devised a double isotope technique using 'H and I4C to provide an internal comparison between different feeding experiments. This technique relies on the fact that the energies of the p particles emitted by 'H and I4C differ and that 3H/'4C ratios can be measured in a mixture of the two radioisotopes. Bale and Crout fed ~-[3-~H]arginine and L-[UI4C]arginine (U = uniformly labeled) with an initial 'H/14C ratio of 4.84 to Senecio magnificus plants in a number of different experiments. The senecionine samples were then shown to have an average 3H/'4Cratio of 3.0. However, when ~-[3-'H]argininewas fed with ~-[U-'~C]ornithine with an initial 3H/'4Cratio of 3.62 in a similar series of experiments, the average 'H/I4C ratio fell to 2.2 in the senecionine samples. It was concluded that ornithine is a slightly more efficient precursor than arginine for the biosynthesis of retronecine in Senecio magnificus (14). Using a similar double isotope technique, Robins and Sweeney showed that only the Lisomers of ornithine (1)and arginine (15)were incorporated into retronecine (13),the base portion of retrorsine (8)in Senecio isatideus plants (15). The use of hydroponic solutions to carry out feeding experiments had proved very unsatisfactory for Bale and Crout, resulting in low and variable incorporations of precursors. Robins and Sweeney studied a variety of feeding methods for the introduction of precursors into Senecio isatideus plants, which produce retrorsine (8)(16). The best incorporations of precursors were obtained by making stem punctures in plants growing normally in soil and allowing droplets of sterile aqueous solutions of the precursors to be drawn directly into the stems of the plants. They continued the technique of double labeling by feeding various 14C-labeledprecursors along with ~-[S-'H]arginineas an internal reference. Large total incorporations of 1.6, 2.0, and 5.2% were obtained for [1,4-'4C]putrescine, spermidine (17),and spermine (18)(both labeled [ 1,4-14C]in the tetramethylene portion) into retrorsine in Senecio isatideus (16). Spermidine and

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

7

spermine probably act as sources of putrescine, and these three precursors were incorporated into retrorsine much more efficiently than ornithine or arginine; almost all of the radioactivity was present in retronecine. When oxidative degradation was carried out on these labeled retronecine samples using osmium tetroxide and sodium periodate (Scheme l ) , about 25% of the retronecine activity was located in the dimedone derivative. A further degradation of retronecine was carried out involving oxidation with chromic acid. This generated p-alanine containing fragment C-(5 + 6 + 7) of retronecine (Scheme 1). In each experiment with putrescine, spermine, and spermidine, again approximately 25% of the retronecine activity was present in the p-alanine. These results added to the growing evidence that retronecine is formed from two molecules of putrescine (16). NH

II

NHCNH,

H 14

CQH

15 16

&N(CH&NH(CHd4NHR

17 R = H 18 R = (CH2)3NH2

Hartmann and co-workers reported that putrescine (14) is formed entirely from arginine (15)in Senecio vulgaris (17), whereas Birecka et al. found that the source of putrescine varied in different plant families. They used decarboxylase inhibitors to show that arginine is the source of putrescine in Heliotropium species (18),but that putrescine (14) is formed from ornithine (1)in Senecio and Crotalaria species (19). Clearly, further work is required to resolve the apparent contradiction with regard to the origin of putrescine in Senecio species. c .

BIOSYNTHESIS OF NECINES FROM PUTRESCINE CONTAINING STABLE ISOTOPES

PRECURSORS

Attempts to solve biosynthetic problems in the biosynthesis of necines were severely limited by the difficulty of establishing the positions of all the labeled atoms in the alkaloids by degradation. With retronecine (13),

8

DAVID J . ROBINS

all that could be achieved was the isolation of C-9 as formaldehyde and a composite fragment of C-(5 + 6 + 7) as p-alanine (Scheme 1). It was clear, however, that the improved total incorporation (1.6%) obtained on feeding [ 1,4-'4C]putrescine to Senecio isatideus by Robins and Sweeney (16) should allow the preparation of some precursors specifically labeled with stable isotopes; and then determination of complete labeling patterns in the labeled alkaloids could be achieved by using NMR spectroscopy. Accordingly, [ 1,4-'3C2]putrescinedihydrochloride (19)was prepared from 1,2-dibromoethaneby SN2 displacement with i3C-labeledcyanide followed by reduction and acidification (Scheme 2) (20). This precursor was fed by the improved feeding technique (16) to freshly rooted Senecio isatideus cuttings. It was necessry to use very young plants to avoid dilution of the labeled alkaloid with endogenous unlabeled material. Specific incorporations of I3C were estimated by comparison of the enriched signals in the "C-NMR spectrum of labeled retronecine hydrochloride with those in unlabeled material run under the same conditions. The I3C-NMRassignments for retronecine hydrochloride in D 2 0 were made by standard techniques before the feeding experiments were carried out. The estimated I3C-specificincorporations were also compared with I4C-specificincorporations since [ I ,4-'4C]putrescinedihydrochloride was always fed together with the 13C-labeledmaterial to provide an internal reference. A number of these experiments were carried out under different conditions, and the 13C {'H}-NMR spectra for the samples of retronecine hydrochloride showed enhanced signals for C-5 and C-8 (20) plus C-3 and C-9 (211,thus

c: 20

2u

1. BH9THF

*

2. HCI 19

21

22

SCHEME2. Preparation of [ 1 ,4-'3C2]putrescinedihydrochloride (19) and its incorporation into retronecine.

1.

PYRROLIZIDINE AND QUINOLIZIDINE ALKALOIDS

9

providing the first complete labeling pattern for a necine and confirming that two molecules of putrescine are required to form retronecine (20). However, considerable broadening of the enriched signals was noticeable, probably due to I3C-N-I3C coupling from C-5 to C-8 (20) together with couplings arising from combination of two labeled molecules in the formation of retronecine, i.e., C-3 to C-5 or C-8, and C-8 to C-9 (22). This made I3C enrichments difficult to measure. In order to produce I3C-NMRspectra free of these additional couplings, [ l-'3C]putrescine dihydrochloride (23)was made by Khan and Robins from N-protected 3-bromopropylamine by introduction of the 13C label using cyanide displacement, followed by catalytic hydrogenation to reduce the nitrile and remove the protecting group, then acidification (Scheme 3) (20).Feeding experiments with this precursor gave much sharper enriched signals, and enhancements of ca. 100% of the four signals for C-3, C-5, C-8, and C-9 of retronecine (24) hydrochloride were observed (20). Obtaining good enhancements of 13C-NMR signals in the foregoing experiments was difficult and required using many young plants under carefully controlled feeding conditions. A better approach when "C enrichments are low is to use I3C-l3Cdoubly labeled precursors and to determine labeling patterns in enriched alkaloids by detecting the 13C-13Cdoublets around the natural abundance signals in the 13C-NMRspectra. [2,3-13C21Putrescine dihydrochloride (25) was therefore made by Khan and Robins (20) from [ 1,2-I3C2]-I,2-dibromoethane (Scheme 4). Use of this doubly labeled precursor (25) with Senecio isarideus gave a sample of retronecine (26) hydrochloride with a distinctive labeling pattern consisting of two

f Br

H2. Pd-C

K%N

EtOH. HCI

U H C 0 2 C H z P h

NHC02CHzPh

24 is a composite representation of all the labelled species present 24

SCHEME 3. Preparation of [ l-13C]putrescinedihydrochloride (23) and its incorporation into retronecine.

10

DAVID J. ROBINS

a I

TH20H

OH

composite labelling pattern

26

SCHEME4. Preparation of [2,3-13C2]putrescine dihydrochloride(25) and its incorporation into retronecine.

pairs of doublets of about equal intensity for C-1 and C-2 and for C-6 and C-7, which were easily distinguished by their different coupling constants (J 71 and 34 Hz, respectively (20). A particularly attractive precursor for studying putrescine metabolism is [ 1,2-'3C2]putrescinedihydrochloride (30),which should show couplings in metabolites between all pairs of carbon atoms derived from putrescine. ,Zdibromoethane (22) as Precursor 30 was synthesized from [ 1,2-13C2]-1 shown in Scheme 5 . The mono N-phthalimide derivative 27 was made, and the remaining bromine was displaced with ethyl cyanoacetate to give the ester 28. Removal of the ester group yielded the nitrile 29, which was reduced and the phthalimide hydrolyzed to give the doubly labeled putrescine dihydrochloride (30).This material was fed to Senecio isatid e w , and as expected the labeled retronecine contained four pairs of doublets in its 13C{'H}-NMR spectrum with four different coupling constants. The precursor 30 should be particularly useful for studying metabolic pathways involving putrescine, leading to labeling patterns similar to those encountered when using [ 1 ,2-13C2]acetatein feeding experiments to form labeled polyketides. All of these feeding experiments with I3C-labeledputrescines gave equal amounts of label in both parts of the retronecine derived from different putrescine molecules. This indicated that a later intermediate with C2" symmetry formed by combination of two putrescine molecules could be involved in the biosynthetic pathway to retronecine. The precursor chosen to test this theory by two groups led by Robins and by Spenser was the ['3C,'5N]-doublylabeled putrescine dihydrochloride (32).This precursor should show which C-N bonds remain intact in retronecine by observa-

1.

PYRROLIZIDINE AND QUINOLIZIDINE ALKALOIDS

11

Br EtO2CCH2CN

NPhth

NaH. DMF H20, 165 OC, 2h

27

s " MH-c;; /

NPhth

1. PQ-AcOH

2. HCI, heat

+

31

XI'

30

composite labelling pattern

SCHEME 5. Preparation of [l ,2-13Cz]putrescinedihydrochloride (30)and its incorporation into retronecine.

tion of I3C-I5N doublets around the natural abundance signals in the 13C-NMR spectrum of retronecine. Thus labeled retronecine could be obtained which showed either C-3 or C-5 coupled to 15N, or both or neither. The existence of a later C4-N-C4 symmetrical intermediate in the biosynthetic pathway would be supported by observing coupling from both C-3 and C-5 to I5N. Accordingly, Khan and Robins (22) treated N-protected 3-bromopropylamine with [13C,'SN]cyanideto introduce the double label. Reduction of the nitrile, with acidic hydrolysis of the protecting group, generated the doubly labeled putrescine dihydrochloride (32)(Scheme 6). When this material was incorporated into retrorsine in Senecio isatideus and the retronecine was obtained by basic hydrolysis, two pairs of doublets were observed in the 13C-NMRspectrum with different coupling constants indicative of coupling between C-5 and "N (33) and C-3 and I5N (34).The fact that equal amounts of these doublets were present is good evidence that there is a later C4-N-C4 symmetrical intermediate in retronecine biosynthesis. Similar results were obtained by Grue-Sorensen and Spenser when they fed the same precursor (made in a similar fashion from 1-bromo-3-phthalimidopropane)to Senecio vulgaris plants and isolated a mixture of alkaloids that gave retronecine on alkaline hydrolysis (23).

12

c'" DAVID J. ROBINS

~1%15~,

Hp, PQC

EtOH, HCI

NHCOzCHZPh

NHCOpCHpPh

32 A/

33

34 composite labelling patterns

SCHEME6. Preparation of ['3C,15N]putrescinedihydrochloride (32)and its incorporation into retronecine.

In order to extend the range of necines available for biosynthetic study, Kelly and Robins (24) acquired Senecio pleisrocephalus from the Royal Botanic Garden, Edinburgh. This species produces rosmarinine (9) as the only alkaloidal constituent. Rosmarinine does not contain 1,Zunsaturation in the necine and therefore is not hepatotoxic. When [ I-'3Clputrescine dihydrochloride (23)was fed to freshly rooted cuttings of this species by the wick method, an enormous specific incorporation of 22% per C4 unit into rosmarinine was observed (24),and the signals for C-3, C-5, C-8, and CHpOH

- - -OH

36

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

13

C-9 of rosmarinine were greatly enriched. A feeding experiment with [2,3-13C,]putrescine dihydrochloride (25) gave an analogous labeling pattern to retronecine sample 26; and ['3C,15N]-labeledputrescine (32), when fed to S . pleistocephalus, afforded a labeled sample of rosmarinine in which one-half of each expected doublet around C-3 and C-5 was obscured by the natural abundance signals (24). Nevertheless, it was clear that rosmarinecine (35) is formed from two molecules of putrescine probably via a later C4-N-C4 symmetrical intermediate, as with retronecine.

D. BIOSYNTHESIS OF NECINES FROM HOMOSPERMIDINE Evidence has been presented that a C4-N-C4 intermediate with CZv symmetry is involved in the biosynthesis of retronecine (W) and rosmarinecine (35). The most obvious candidate for this role was N-(Caminobuty1)-1,Cdiaminobutane (homospermidine) ( X ) ,which is known to occur in sandalwood and other plants (25). Preliminary experiments with homospermidine were carried out with l4C-labeled material (26) to assess its status as a precursor of necines. [ 1,9-'4C]Homospermidine trihydrochloride (39) was synthesized from N-protected 4-aminobutanoic acid and 3bromopropylamine (Scheme 7). Coupling of these two components by a mixed anhydride method afforded the bromoamide 37, which was treated with I4C-cyanide to introduce the radiolabel into the nitrile 38. Removal of the protectinggroup, reduction of the nitrile and amide, and acidification gave the I4C-labeledhomospermidine trihydrochloride 39. This was incorporated fairly well (0.5%) into retrorsine in Senecio isatideus plants. When the alkaloid was hydrolyzed and retronecine subjected to the degradations described earlier (Scheme l), 44% of the retronecine radioactivity was present at C-9 and only 2% was located in the composite fragment C(5 + 6 + 7). The partial labeling pattern for retronecine is consistent with the labeling pattern shown in 40. [See (24)l. In order to provide additional evidence for homospermidine as an intermediate in retronecine biosynthesis, the [4,6-I4C]-labeledmaterial 43 was prepared (Scheme 8) (26). [ l-'4C]-4-Aminobutanoic acid was N-protected and activated as the 4-nitrophenyl ester 41. Coupling of this ester with 4aminobutanamide gave the labeled amide 42. Removal of the protecting group, reduction of the amide functions, and acidification yielded [4,6''Clhomospermidine trihydrochloride (43).This material gave a similar incorporation (0.7%) into retrorsine, and hydrolysis to retronecine and degradation showed the complementary labeling pattern to 40, with 3% of the radioactivity at C-9 and 46% in the C-(5+6+7) fragment, in agreement with the proposed labeling pattern 44.

14

DAVID J . ROBINS NHCO2CH2Ph \

0 37 K14CN

+

-

1. H p d - C 3. HCI 2.BH9THF

XI'

2 YN VHd NHC02CHZPh

0

39

1 J

38

f"

b3 40

composite labelling pattern

SCHEME7. Reparation of [ I ,9-'4C]hom~~pemidine trihydrochloride and its incorporation into retronecine (40).

The presence of homospermidine in Senecio isatideus was established using an intermediate trapping experiment (26). After ~~-[5-'~C]ornithine had been fed to one Senecio isatideus plant for one day, the plant was extracted and inactive homospermidine (36) trihydrochloride was added to the extract. The mixture was derivatized using isothiocyanatobenzene, and the homospermidine derivative 45 was recrystallized to a constant

1.

PYRROLIZIDINE A N D QUINOLIZlDlNE ALKALOIDS

I

I5

1. HZ-PdlC

2. BH3.THF 3. HCI

+ HCHO

\

CHzOH

HO

/

--

44

43

composite labelling pattern

SCHEME8. Preparation of [4,6-14C]homospermidinetrihydrochloride (43)and its incorporation into retronecine (44).

specific radioactivity corresponding to 0.5% of the activity fed to the plant. This shows that homospermidine is present in the plant and can be formed from ornithine. Homospermidine has been shown to be present in Heliotropiurn indicum by Birecka and co-workers (27). Although the total incorporations obtained with homospermidine were generally lower than those using putrescine. Rana and Robins decided to carry out an experiment using '3C-labeled material to try to obtain a complete labeling pattern in retronecine (28). The experiment was designed to position two separate labels in the homospermidine so that they unit would show a geminal coupling in the retronecine if the C4-N-C, stayed intact during the biosynthesis. The doubly labeled material was prepared as outlined in Scheme 9. [ 1-'3C]-4-Chlorobutanenitrile (46)was made from 1-bromo-3-chloropropane by displacement with I3C-cyanide. Treatment of two molecules of the chloro compound 46 with benzylamine gave the dinitrile 47 (29). Reduction of the nitrile groups and cleavage of the benzylamine gave [I ,9-I3C,Jhomospermidine trihydrochloride (48) (28). When this material was fed to Senecio isnfideus,the sample of retronecine

16

DAVID J . ROBINS

JNHPh CNHCSNHPh 45

1

doH 47

:J+?

-f-

NH2

49

48

SCHEME9. Preparation of [ I ,9-'3C2]hom~~permidine trihydriochloride (48) and its incorporation into retronecine (49).

(49) obtained showed two doublets of about equal intensity around the

natural abundance signals for C-8 and C-9 in the I3C {'H}-NMR spectrum. The geminal coupling constant of 6 Hz demonstrates that homospermidine is incorporated intact into retronecine. When they fed [ 1 ,9-'3C,]homospermidine trihydrochloride (48)to Senecio pleistocephalus, Kelly and Robins (24) obtained a better specific incorporation in rosmarinine of about 1%. This was observed as two enriched singlets of equal intensity with about 100% enhancement of the natural abundance signals in the I3C {'H}-NMR spectrum of rosmarinine. In this case, the geminal coupling constant is zero, but no other enriched signals are evident; so it is clear that rosmarinecine is also formed from homospermidine. Hartmann and co-workers (30)have isolated and partially purified an enzyme, homospermidine synthase, from root cultures of Senecio vulgaris and Eupatorium cannabinum. This enzyme catalyzes the formation of homospermidine from two molecules of putrescine in a NAD+dependent reaction. The enzyme is inhibited strongly by NADH at low

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

17

concentrations, indicating that the NADH formed in the first step of the oxidative deamination of the putrescine is bound to the enzyme and serves as a hydride donor in the reduction of the presumed intermediate imine. Diamine oxidases or transaminases are not believed to be involved, and free 1-pyrroline is not a biosynthetic intermediate. Consideration was next given to the likely course of events from homospermidine (36)to form the pyrrolizidine ring system (31).It seemed likely that diamine oxidases were involved. Oxidation of one of the primary amino groups would lead to an aldehyde 50 in equilibrium with the iminium ion 51. Oxidation of the remaining primary amino group to the aldehyde 52 and nonenzymic cyclization would generate the thermodynamically more stable em-pyrrolizidine ring system 53 and a final reduction step would lead to the 1-hydroxymethylpyrrolizidine 54. This theory was tested by Robins by incubating homospermidine (36)with pea seedling diamine oxidase. This was left for one week, then addition of a dehydrogenase or chemical reduction gave trachelanthamidine (54) in 27 and 40% yields, respectively (31) (Scheme 10). No optical activity could be detected in the product 54. This demonstration of the facile conversion of homospermidine into a 1-hydroxymethylpyrrolizidine under physiological condi-

50

36

11 51

I 1

52 H

53

CHO I

H

CH,OH I

54

SCHEME 10. Conversion of homospermidine (36) into trachelanthamidine (54).

18

DAVID J. ROBINS

tions suggested that the reactions described are likely to occur in pyrrolizidine alkaloid biosynthesis. It also indicated the next candidates for assessment as intermediates in the biosynthetic pathway-namely , iminium ions and 1-hydroxymethylpyrrolizidines.Their involvement is discussed in the next two sections. E. BIOSYNTHESIS OF NECINES INVOLVINGIMINIUM IONS In order to test the iminium ion 51as an intermediate in necine biosynthesis, a synthesis of I4C-labeledmaterial 57 was undertaken (32). The chlorine in [ 1-'4C]-4-chlorobutanenitrile was displaced with pyrrolidine to yield the labeled nitrile 55. Catalytic hydrogenation and acidification gave the saturated salt 56 (Scheme 11). Introduction of the double bond was achieved by oxidation with mercuric acetate. The location of the double bond was established by reducing the unlabeled material 5 1 with sodium cyanoborodeuteride to give a monodeuterated product. Comparison of the 'H-NMR spectrum of this material (multiplet for three protons at 6 2.95 and two protons at 6 3.55) with that of the salt corresponding to 56 (four proton multiplet at 6 2.95 and two protons at 6 3.55) demonstrated that the double bond was endocyclic as required. When the I4C-labeled iminium ion 57 was fed to Senecio isatideus plants, together with [1,4'Hlputrescine dihydrochloride as a reference with a starting 'H/14C ratio of 12.3, retrorsine (8) was isolated with a I4C-specific incorporation of 4.5% and a lower 'H/I4C ratio of 9.8. A similar experiment with Senecio pleistocephalus plants starting with a 'H/I4C ratio of 5.0 afforded rosmarinine (9) with a higher specific incorporation of 6.5% and a lower 'H/I4C

55

I

1 n

56

SCHEME1 1 . Preparation of iminium ion (57).

1. H2-Pt02.AcOH 2. HCI

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

19

ratio of 2.9. These ratios were maintained in the necines. These results demonstrate that the iminium ion 51 is a more efficient precursor for these two necines than putrescine. The iminium ion 51 was also shown to be an efficient precursor of the base portion [heliotridine (58)] of echinatine (11)formed by Cynoglossum of$cinale and of cynaustraline (59) and cynaustine (60)in C. australe. (33). The base portion of cynaustraline is (+)-isoretronecanol (61)and that of cynaustine is (+)-supinidine (62). C H 2 4 -CO

Me 11

58

CH2OH

61

CH,OH

& 62

The iminium ion 51 was also shown to be formed from putrescine and found to be present in Senecio pleistocephalus by an intermediatetrapping experiment (32). [ 1,4-'4C]Putrescine dihydrochloride was administered to one Senecio pleistocephalus plant. One day later, inactive iminium ion 51 was added to the methanolic extract of the plant, followed by sodium borohydride, and the products were derivatized using isothiocyanatobenzene. The derivative was recrystallized to a constant radioactivity corresponding to 0.4% of the activity fed.

20

DAVID J. ROBINS

F. BIOSYNTHESIS OF NECINES INVOLVING

1-HYDROXYMETHYLPYRROLIZIDINES

Evidence for 1-hydroxymethylpyrrolizidinesas biosynthetic intermediates was provided by Birecka and Catalfamo (34). They carried out pulsed labeling experiments with I4CO2on Heliotropium spathulatum, which produces trachelanthamidine (54), ( - )-supinidhe (63), and retronecine (W). Their observations of the appearance of labeled necines were consistent with the sequence of formation shown in Scheme 12. Kunec and Robins decided to make 3H-labeled I-hydroxymethylpyrrolizidines to study their incorporation into more complex necines (33, utilizing the synthetic route of Pizzorno and Albonico (36). The starting material was ~-[5-~H]proline, which was N-formylated and the product subjected to 1,3-dipolar cycloaddition with ethyl propiolate to yield the dihydropyrrolizine ester 64 (Scheme 13). The endo-ester 65 was obtained by cis-addition of hydrogen to 64, and reduction of the saturated ester 65 afforded ( +)-[5-3H]isoretronecanol(66).The endo-ester was epimerized under acidic conditions to afford the thermodynamically more stable exoacid which was re-esterified to give 67, reduction which gave ( 2 ) - [ 5 3H]trachelanthamidine(68).These 3H-labeledracemates were fed to Senecio isatideus together with [ 1,4-’4C]putrescinedihydrochloride with an initial 3H/14Cratio of 10.0. With trachelanthamidine (68) the 3H-specific incorporation was 2.8% into retrorsine (8) with a 3H/’4Cratio of 14.3, whereas isoretronecanol (66)was incorporated to a lesser extent (0.3% specific incorporation) and the 3H/14Cratio fell to 0.7. Trachelanthamidine is therefore a more efficient precursor for retrorsine than isoretronecanol. Hydrolysis of retrorsine showed that the radioactivity was almost entirely in the necine portion, and chromic acid oxidation gave a sample of palanine (Scheme 1) containing most of the radioactivity. Leete and Rana provided independent evidence for the incorporation of the exo-alcohol 68 into riddelliine (69) in Senecio riddellii (37). Kunec and Robins went on to show that the endo-alcohol66 is incorporated efficiently into rosmari-

54

63

13

SCHEME 12. Pulsed labeling of “CO2 to Heliotropium spathulatum.

1. PYRROLIZIDINE

A N D QUINOLIZIDINE ALKALOIDS

p COPE1

/-fCoZH 3H

21

I

64

/

I

3H

HCI 2.EIOHIH'

65

1.

UAIH,

SCHEME 13

nine (9) (2.4% specific incorporation) with a 3H/14Cratio of 17.0 compared to the starting ratio of 10.0. The exo-alcohol 68 was poorly incorporated into rosmarinine (
22

DAVID J . ROBINS

and cynaustine, whereas (+)-supinidine is a precursor only of cynaustine, indicating that (+ )-isoretronecanol is converted into (+)-suphidine in the biosynthetic pathway. A number of pyrrolizidine alkaloids, such as senkirkine (69), contain otonecine (70) as the base portion. Hartmann and co-workers (40) showed that the amounts of senkirkine in root cultures of Senecio uernulis in-

69

71

creased at the expense of the amounts of the N-oxide of senecionine (16). Barbour and Robins isolated emiline (71) from Emilia flammea (Asteraceae) and provided evidence for a structure revision of the alkaloid (41). Feeding experiments (42) with radiolabeled precursors on E. flammea then established that otonecine is formed from putrescine via homospermidine (36),the iminium ion 51, trachelanthamidine (S) and , retronecine (W). The C-4-N-8bond must be broken at a late stage in the biosynthetic pathway, possibly by hydroxylation at C-8and N-methylation of a retronecine derivative, followed by cleavage of the pyrrolizidine system (Scheme 14). An overall summary of the published evidence concerning the biosynthesis of necines is given in Scheme 15 with known intermediates in rectangular boxes (43). h t r e s c i n e (14) can be formed from L-ornithine (1) or L-arginine (15).Oxidation of one of the primary amino groups of putrescine catalyzed by homospermidine synthase gives an enzyme-bound species which condenses with another molecule of putrescine to give an

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

23

Me SCHEME 14. Formation of alkaloid containing otonecine from retronecine diester.

imine that is reduced by NADH to yield homospermidine (36).The iminium ion 51 is produced by oxidation of one of the primary amino groups; then the aldehyde 52 is generated by oxidative deamination of the remaining primary amino group. Cyclization of the iminium ion can occur in two different ways leading to the endo-alcohol 6 or the em-alcohol 54 after reduction of the aldehydes. Two hydroxylations of isoretronecanol (6) lead to rosmarinecine (35). It is possible that retronecine (13) and heliotridine (58) are formed from trachelanthamidine (54) by two hydroxylations followed by a dehydration, and otonecine is likely to be formed from retronecine (diester) as shown in Scheme 14.

G . STEREOCHEMISTRY OF THE ENZYMIC PROCESSES IN NECINEBIOSYNTHESIS In order to elucidate the mechanistic details of biosynthetic pathways, determination of the stereochemistry of the enzymic processes is essential once the main precursors have been established (44).The first stereochemical detail has already been mentioned: namely, that only the L-isomers of ornithine (1)and arginine (15)are precursors of necines. The stereochemistry of the decarboxylation of L-ornithine to putrescine and of Larginine to putrescine via agmatine was established by several groups of workers ( 4 5 4 7 ) . They showed that both decarboxylations proceed with retention of configuration. Further progress in the determination of the stereochemistry of the enzymic processes involved in the biosynthesis of a range of necines required the use of specifically deuterated putrescines as precursors, and determination of complete labeling patterns using 2H-NMRspectroscopy. Rana and Robins (48)began this work by preparing [2,2,3,3-2H,]putrescine dihydrochloride (72) by base-catalyzed exchange of the protons in succinonitrile in 2H20, followed by reduction of the dinitrile and acidification (Scheme 16). This precursor was fed to Senecio isatideus and the *HNMR spectrum was recorded. It was found that spectra had to be recorded at 60°C in chloroform or at 90°C in pyridine in order to obtain well defined

24

DAVID J . ROBINS

I

,

I

36 1

m /

QyHo II

51

CH20H

54

52

\

CH20H

(53 13

58

1 CHpOH

1

---OH

35

70

SCHEME 15. Biosynthesis of Necines.

CHpOH

1.

PYRROLIZIDINE AND QUINOLIZIDINE ALKALOIDS

25

72

73

composite labelling pattern

SCHEME16. Preparation of [2,2,3,3-*H4]putrescinedihydrochloride(72)and its incorporation into retrorsine (73).

signals. The 'H incorporation of retrorsine was estimated by comparison of the integrals of the signals of a known concentration of 2H-labeled retrorsine with the natural abundance signals of 2H in chloroform or pyridine. This value was compared with that obtained by feeding [ 1,4-'4C]putrescine dihydrochloride in admixture with the 2H-labeledmaterial. Thus [2,2,3,3-2H4]putrescinedihydrochloridegave a 2.4% specific incorporation per C4 unit into retrorsine, and four labeled positions of about equal intensity were observed corresponding to H-2, H-6a, H-6P, and H-7a of retrorsine (73).The retention of 2H at H-7a indicates that no keto or enol intermediates are formed as part of the overall hydroxylation process (48). Rana and Robins (48) gained further interesting information by making [ 1,l ,4,4-'H4]putrescine dihydrochloride (74) and feeding it to Senecio isatideus. The precursor 74 was made by carrying out the catalytic hydrogenation of succinonitrile under an atmosphere of deuterium gas, followed by acidification (Scheme 17). The 2H-NMR spectrum of the labeled sample of retrorsine was run in pyridine at 90°C in order to obtain sharp signals. The three major signals were attributed to retrorsine (76) labeled with 'H at H-3a, H-3/3, and H-9 pro-S. Only very small signals could be ascribed to the deuterium atoms at the H-5 and H-8 positions. The explanation for the accumulation of most of the 'H in one-half of the necine put forward was that combination of [2,2,3,3-2H4]putrescinewith unlabeled material would produce the 'H,-labeled homospermidine (75) as the major labeled species. If this labeled homospermidine undergoes an intramolecular 2H-

26

DAVID J. ROBINS

75

SCHEME17. Preparation of [ 1.1 ,4,4-*H4]putrescinedihydrochloride (74) and its incorporation into retrorsine (76).

isotope effect during its oxidation to the aldehyde, the unlabeled portion will form the left-hand ring of 76 with most of the 'H in the right-hand portion. Furthermore the observation of 'H in the pro-S position of retrorsine (76) demonstrates that reduction of the pyrrolizidine aldehyde to the alcohol occurs by addition of the incoming hydrogen (hydride) to the re-face of the carbonyl group. This is the stereochemistry expected for a coupled dehydrogenase system (48). In order to confirm this stereochemical outcome and provide more information about the stereochemistry of processes involving loss or retention of deuterium from the 1- or 4-positions of putrescine, samples of (R)- and (S)-[ I-'H]putrescine dihydrochloride were required. These were prepared independently by the groups of Spenser (49) and Robins (50) making use of the known stereospecificity of the decarboxylation of Lornithine (51). Carrying out this reaction in *H,O on L-ornithine with L-ornithine decarboxylase afforded (R)-[I-2H]putrescine isolated as the dihydrochloride77, whereas the enantiomer 78 was prepared by decarboxin water using L-ornithine ylation of the L-isomer of [2-*H]-~~-ornithine decarboxylase. Grue-Sorensen and Spenser (49) fed these precursors to obtain the mixture of pyrrolizidine alkaloids found in Senecio vulgaris, whereas Rana and Robins (50) used Senecio isatideus to obtain labeled retrorsine (Scheme 18). The sample of retrorsine (79)obtained from the (R)-isomer 77 gave a *H-NMR spectrum containing four signals of equal

1.

-

PYRROLlZlDlNE A N D QUINOLIZIDINE ALKALOIDS

G31,+

27

- v

2a

H

'D

I

77

H \ D 79 -OH

composite labelling patterns

81

+ NH3

I

* d

I

80

composite labelling patterns 02

SCHEME 18. Incorporation of (R)- and (S)-[ l-2H]putrescine dihydrochloride into retrorsine and rosmarinine.

intensity and showed that retrorsine was labeled with 2H at H-3/3, H-5a, H-8a, and H-9 pro-S. The 2H-NMR spectrum of retrorsine (80) obtained after feeding the (S)-isomer78 contained only two signals of equal intensity due to H-3a and H-5/3. These labeling patterns indicate that all of the

28

DAVID J . ROBINS

oxidation processes taking place on the primary amino group of putrescine and homospermidine occur with stereospecific loss of the p r o 4 hydrogens; the same stereospecificity has been observed for reactions catalyzed by diamine oxidase. Furthermore, reduction of the imine to give homospermidine (Scheme 15) must occur by addition of the hydride equivalent to the C-si-face of the imine. Grue-Sorensen and Spenser (49) also observed that the ’H incorporation at C-3 and C-5 into retronecine after feeding (S )-[ l-’H]putrescine was 69% of the I4C incorporation, whereas a value of 50% would normally be expected. They attribute this finding to a primary deuterium isotope effect in the oxidation of putrescine which favors oxidation at the unlabeled end of putrescine, leading to an excess of deuterium over I4C at the carbons destined to become C-3 and C-5 of retronecine. The same pattern of incorporation was observed by Kelly and Robins (52) when (R)-and (S)-[ 1-’Hlputrescine dihydrochlorides 77 and 78 were fed to Senecio pleistocephalus and the labeling patterns were determined by ’H-NMR spectroscopy. The respective labeling patterns 81 and 82 for rosmarinine are shown in Scheme 18 and confirm the stereochemical conclusions made earlier. In order to resolve the remaining stereochemical questions, it was necessary to prepare ( R ) - and (S)-[2-’H]putrescines. Arigoni and Eliel menin a review tioned an unpublished route to (R)-[2-’H]succinic acid (W) (53).The modification of this route used by Kunec and Robins (54)is shown in Scheme 19. L-Aspartic acid (83)was converted into (S )-2-chlorosuccinic acid (84) with retention of configuration. Conversion of the diacid into the diester and selective reduction yielded (S)-2-chlorobutane-1,4-diol (85). Deuterium was introduced with inversion of configuration and some of the (R)-2-deuterobutane-l ,Cdiol (86) was oxidized to (R)-[2-’H]succinic acid (87) for literature comparison (53).The rest of the material was converted into (R)-[2-’H]putrescine dihydrochloride (88)via the dibromide and diazide. (S)-[2-’H]Putrescine dihydrochloride (89) was made in an analogous fashion from D-aspartic acid. These two precursors were fed to Senecio isatideus plants and the ’H-NMR spectra showed two labeled sites in each case. Deuterium was present at H-2 and H-6a in retrorsine (90) after feeding the (R)-isomer 88, and at H-6P and H-7a in retrorsine (91) after incorporation of the (S)-isomer 89 (Scheme 20). It is clear that hydroxylation at C-7 of retrorsine occurs with retention of configuration. This stereospecificity is usually observed for direct hydroxylation at sp3 carbon atoms. The formation of the double bond in retrorsine must therefore occur with loss of the pro-S hydrogen at C-2 of the necine and retention of the pro-R hydrogen. Feeding of (R)- and (S)-[2-’H]putrescine dihydrochloride by Kelly and

1. PYRROLIZIDINE

A N D QUINOLIZIDINE ALKALOIDS

COpH

29

CO2H

I

I

HCI, HNO,

CH,CO,H

CH,CO~H

83

CHpCH20H

LAID,

CHpOH 1. HBr

I

CH2CH20H

/ Chromic acid

2. NaN3 3. LiAIH, 4. HCI

\

+ CHpNH3

H+o CHpNH3 CH2COzH 88

87

SCHEME 19

Robins (52) to Senecio pleistocephalus produced some additional information. The labeling patterns obtained are shown in Scheme 20. *H was present at H-2P and H-6a in rosmarinine (92) after feeding the (R)-isomer 88, and three signals were observed at H-la, H-6& and H-7a in rosmarinine (93) produced by feeding the (S)-isomer 89. It can be seen that hydroxylation at C-2 of rosmarinine also proceeds with retention of configuration, and that formation of the pyrrolizidine ring involves removal of the pro-R and retention of the p r o 4 hydrogen at C- 1. It is interesting to note that dehydration of rosmarinine would require cis-elimination of

30

DAVID J . ROBINS

+

iH3

I

I

H>GH:c'- - k& D'

DH

N

90

I

I D

92

di3- +

O\ H'

f-----T

2ct-

H-

89

91

93

SCHEME20. Incorporation of (R)and (S)-[2-*H]putrescine dihydrochloride into retrorsine and rosmarinine.

the elements of water, whereas if a-hydroxylation at C-2 of trachelanthamidine (54) occurs, trans-elimination of the elements of water would then lead (with hydroxylation at C-7) to retronecine (13). The presence of the 1,2-double bond is an essential feature for the observation of hepatotoxicity, as noted previously.

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

31

Robins and co-workers (55) have also studied the stereochemistry of the enzymic processes involved in the formation of the otonecine (70) portion of emiline in EmiliaJlammea. The labeling patterns are exactly analogous to those obtained for retronecine, apart from the necessary loss of deuterium on introduction of the 8-keto group of otonecine. It is noteworthy that introduction of the keto group and cleavage of the bicyclic system does not involve loss of ?H at any of the surrounding sites. Studies of the stereochemistry of the enzymic processes in a variety of necines have been carried out successfully and have shown that the stereochemical outcome of most of the enzymic reactions is consistent with the known stereospecificity of enzymic reactions believed to be involved, including the decarboxylases, diamine oxidases, and hydroxylases (44).These studies have produced the information necessary to undertake a detailed examination of individual enzymes involved in pyrrolizidine alkaloid biosynthesis, and a start has been made in this direction with the recent work on homospermidine synthase (30).

H. BIOSYNTHESIS OF NECK ACIDS The acids and diacids that esterify necines (necic acids) have been of considerable interest biosynthetically because of their unusual structures. Most of them contain ten carbon atoms, thereby encouraging the idea that they might be derived from mevalonate. It was clear that the number of oxygen functions and mode of coupling of the two putative C, units were quite atypical for monoterpenes; therefore, it was no surprise when Hughes and Warren showed that [2-~4C]mevalonolactone was not incorporated into retrorsine (8)in Senecio isatideus (56). Several groups of workers fed labeled acetate to plants to obtain labeled pyrrolizidine alkaloids, and limited degradations were carried out; but no clear conclusion was possible from the partial labeling patterns established (56-58). Crout et al. (57) showed that the amino acids threonine (94) and isoleucine (95) were incorporated into the C,, acids present in alkaloids produced by Senecio douglasii. Partial labeling patterns obtained by degradation were difficult to interpret. Clearer results were obtained by Crout (59,60) who studied the formation of echimidinic acid and angelic acid, which are the esterifying acids of heliosupine (96), in Cynoglossum officinale. DL[4-14C]Valinewas incorporated well into echimidinic acid, and periodate cleavage of the trio1 generated acetone and ethanal (Scheme 21). The acetone was trapped by forming iodoform and the amount of radioactivity present in the iodoform was consistent with the formation of echimidinic acid from valine (971, with incorporation of an additional C2unit (Scheme 21 ) (59).Crout (60) also demonstrated that ~-[U-'~C]isoleucine is efficiently incorporated into angelic acid. Angelic acid is biosynthesized from

32

DAVID J . ROBINS

7

2

Me2CHCH 'COpH

MeCH2CHCH 95 \CO2H

7

2

MeCHOHCH \COpH 94

I

C2 unit

96

NalO, H2S04

Me,co

-

CHI,

+ MeCHO

SCHEME21. Biosynthesis of esterifying acids of heliosupine (W).

isoleucine, again with loss of the carboxyl carbon. McGaw and Woolley (61) observed the incorporation of [l-'4C]tiglic acid into the angelic acid portion of heliosupine (96) and concluded that tiglic acid can isomenze to angelic acid in heliosupine biosynthesis. It was already known that isoleucine is a precursor of tiglic acid in the tiglate ester portions of tropane alkaloids in Datura meteloides (62). Crout and co-workers (63) have carried out a detailed study of the biosynthesis of senecic acid (981, the acid portion of senecionine (16) produced by Senecio magnifcus. Using I4C-labeledprecursors, they obtained partial labeling patterns by degradation and showed that senecic acid is formed from two molecules of isoleucine (95) via its biosynthetic precursor threonine (94), accompanied by loss of both carboxyl carbons (Scheme 22). Davies and Crout (64)established that of the four possible

1. PYRROLIZIDINE

A N D QUINOLIZIDINE ALKALOIDS

33

tI /NH2 MeCHOHCH 'C02H

94

98

SCHEME22. Biosynthesis of senecic acid (98).

stereoisomers of isoleucine, only L-isoleucine is incorporated efficiently into senecic acid. The identity of the five-carbon unit formed from isoleucine in senecic acid biosynthesis has proved to be elusive. Crout and co-workers (65) showed that angelate, 2-methylbutanoate, and 2-methyl-3-oxobutanoate were unlikely to be contenders. Stereochemical questions concerning the joining of the two C, portions were addressed by Crout and co-workers (66). They prepared isoleucine stereospecifically labeled with 3H at the 4-position of isoleucine and showed that H-4 pro-R is retained while H-4 p r o 3 is lost from both of the isoleucine molecules needed to form senecic acid (98). Although the stereochemistry of the joining process involving the two isoleucine molecules is known, the mechanism for uniting the halves of senecic acid is still a mystery. One possible intermediate suggested by Crout and co-workers is p-methylenenorvaline (99)formed from isoleucine by overall dehydrogenation. p-[3H2]Methylenenorvalinewas incorporated

MeCH2CCH

99

'C02H

well into senecic acid in Senecio magniJicus (65), but it was not possible to show whether this material labeled either or both halves of senecic acid. The use of a 2H-labeled precursor might resolve this incomplete picture.

34

DAVID J . ROBINS

Me OH

/

I

/NH2 MeCH2CHCH

95 C ‘ O2H

--.Me CO2H

t

COzH

N H 2 ,

100

MeCHOHCH

‘

9 ‘C 402H

SCHEME 23. Biosynthesis of monocrotalic acid (100).

The biosynthesis of other necic acids has been studied in Crotalaria species. ~-[U-~~C]Isoleucine and ~-[U-’~C]threonine were both incorporated into the monocrotalic acid (100) portion of monocrotaline (10) in C. retusa (67). Partial labeling patterns were established by degradation, and it appears that the right-hand C, unit, which is similar to that in senecic acid, is derived from isoleucine (Scheme 23). It is not clear how the remaining C3 unit is biosynthesized. Trichodesmic acid (101), the acid portion of trichodesmine, formed in Crotalaria globifera has a structure and stereochemistry similar to monocrotalic acid (isopropyl group in place of methyl). Devlin and Robins (68) carried out experiments with I4C-labeled amino acids and showed that, as expected, threonine (94) and isoleucine (95) are specifically incorporated into the right-hand portion (Scheme 24), whereas the left-hand portion is derived from valine (97) and leucine (102). 3-Hydroxy-3-methylglutaric acid (dicrotalic acid) (103) is the esterifying portion of dicrotaline (104) and is the simplest necic diacid known. Obvious

/

-

,NH2 101

Me C H O H C 6

94 102

SCHEME 24. Biosynthesis of trichodesmic acid (101).

\C02H

1. PYRROLIZIDINE

A N D QUINOLIZIDINE ALKALOIDS

35

precursors are acetate and mevalonate, but Denholm and Robins (69) showed that neither of these, nor dicrotalic acid itself, was incorporated specifically into dicrotalic acid when fed to Crotalaria lachnosema. Specific incorporation into dicrotalic acid was observed with ~-[U-'~C]threonine and ~-[U-'~C]isoleucine. Furthermore, 3H from [4,5-3H]isoleucine was specifically incorporated into the methyl group of dicrotalic acid. Thus isoleucine provides a MeC(OH)CH,CO,H portion of dicrotalic acid with loss of the C-6 methyl and carboxyl groups of isoleucine in one of two possible ways (Scheme 25). The origin of the remaining two carbon atoms in dicrotalic acid remains to be established. Further progress in understanding how the biosynthesis of necic acids from branched-chain amino acids is accomplished, and how they arejoined to the necines to form macrocvclic pyrrolizidine alkaloids, may result

103

104

from the use of precursors labeled with stable isotopes coupled with establishment of complete labeling patterns by NMR spectroscopy. Pyrrolizidine alkaloids are often converted into N-oxides within the plants (70). In root cultures of Senecio vulgaris no turnover of the accumulating senecionine (16)N-oxide occurred (71). Radioactive pyrrolizidine alkaloids with high specific activities are

SCHEME25. Biosynthesis of dicrotalic acid portion of dicrotaline.

36

DAVID J. ROBINS

required for studies of their metabolism and disposition in plants and tissue cultures. They have been prepared by pulsed feeding of I4CO2to Senecio vulgaris (72), and by feeding labeled precursors to S. jacobaea and S. vulgaris (71).

111. Quinolizidine Alkaloids

A. STRUCTURES A N D BIOLOGICAL ACTIVITY Quinolizidine alkaloids are found in the Papilionoideae, one of three subfamilies of the Fabaceae (Leguminosae). They are thus much more restricted in distribution than pyrrolizidine alkaloids. Often referred to as lupin(e) alkaloids, their occurrence, structure elucidation, and synthesis have been well documented in The Alkaloids (73) and elsewhere (74). Regular reviews also appear in Natural Product Reports (75). Many plants in the legume family are important sources of food for humans and livestock, and they help to conserve soil and fix nitrogen. Some quinolizidine alkaloids are toxic to grazing animals and can act as feeding deterrents. The main structural types of quinolizidine alkaloids are exemplified by the bicyclic alcohol (-)-lupinine (7),tricyclic pyridones such as cytisine (105) and (-)-N-methylcytisine (106), and tetracyclic alkaloids such as (-)-sparteine (107)and matrine (108).Although the Lythraceae alkaloids contain a quinolizidine ring, they are formed by a different biosynthetic pathway and are not considered here (76).

B. BIOSYNTHESIS OF LUPININE By analogy with the proposed derivation of pyrrolizidine alkaloids from ornithine (1)and putrescine (14),Robinson ( I )suggested that quinolizidine alkaloids such as lupinine (7)are formed from two molecules of lysine (2) via cadaverine (109).The first studies on lupinine biosynthesis were carried out by Schutte and co-workers. It was shown that I4C-labeled lysine and [ 1 ,5-14C]cadaverine(110)were incorporated into lupinine in Lupinus luteus plants. Furthermore, on degradation of the labeled lupinine (111) from the latter experiment, one-fourth of the total radioactivity was located in the hydroxymethyl group (79, and one-half could be accounted for in the C-4 and C-6 atoms obtained by exhaustive methylation and Hofmann degradation of lupinine followed by ozonolysis of the products (Scheme 26) (78).

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

7

37

105 R = H 106 R = Me

107

108

N ,H2 HzN(CH3,CH ‘COzH 2

109

Rana and Robins (79)obtained a complete labeling pattern for lupinine by I3C-NMR spectroscopy after feeding ~ ~ - [ 4 , 5 - ’ ~ C ~ ] l y(112) s i n eto Lupinus luteus. The same extent of labeling was observed in both rings of lupinine, and the distinctive labeling pattern 113 was consistent with the formation of lupinine from two molecules of lysine via the symmetric intermediate cadaverine (Scheme 27). Further support for this pathway came from the synthesis of [ 1,2-”C,] cadaverine dihydrochloride (116) and the study of its incorporation into lupinine (80). For the least expensive preparation of the doubly labeled precursor, the 13Clabels were added consecutively (Scheme 28).

38

DAVID J . ROBINS

p;

composite labelling pattern

---c

\

1. Me1

111

110

">-=.

(C-4 H and C-6)

03

2. Hofmann

doH do

SCHEME 26. Incorporation of [ I ,5-'4Clcadaverine (110) into lupinine (111) and partial degradation.

3-Chloropropanol was converted into the mesylate, and the first I3C label was introduced using sodium cyanide to afford [ l-13C]-4-chlorobutanenitrile. The nitrile was hydrolyzed and the acid reduced to the alcohol, then converted into the mesylate 114. The second I3Clabel was introduced as before, and the doubly labeled nitrile 115 was converted into the diamine dihydrochloride 116 via the azide. Feeding this precursor to Lupinus luteus gave the distinctive labeling pattern 117 for lupinine.

C "112

COZH

I

113

composite labelling pattern SCHEME 27. Incorporation of ~~-[4,5-"C~]lysine (112)into lupinine (113).

1.

PYRROLIZIDINE AND QUINOLIZIDINE ALKALOIDS

39

114

115

116

117

composite labelling pattern

SCHEME28. Synthesis of [ 1 ,2-13C2]cadaverinedihydrochloride (116)and its incorporation into lupinine (117).

These labeling patterns for lupinine [113 and 1171 are analogous to those obtained for retronecine [26 and 311 (Schemes 4 and 9, and it should be recalled that use of [‘3C,15N]-labeledputrescine (32) demonstrated that a later intermediate with C,, symmetry [homospermidine (3611 was involved in the biosynthetic pathway to retronecine (Scheme 6). Two groups, independently led by Robins (81) and Spenser (82),decided to see if a similar situation existed in lupinine biosynthesis. Accordingly, [‘3C,15N]-labeled cadaverine dihydrochloride (118) was synthesized by both groups from 1-phthalimido-4-bromobutaneby treatment with KI3C”N followed by catalytic hydrogenation and hydrolysis (Scheme 29). Feeding this material to Lupinus luteus afforded a sample of lupinine (119), which showed four enriched carbon signals for C-4, C-6, C-10, and C-11 in its I3C {‘H}-NMR spectrum. Furthermore, the resolution-enhanced spectrum of lupinine showed that only one signal (for C-6) was flanked by a doublet (J 3.2 Hz) due to the presence of a 13C-”N species 119. This result indicates that there is no later symmetric intermediate of the type C5-N-C5 involved in lupinine biosynthesis, and provides a distinct contrast with retronecine biosynthesis (Section IIC). As a further check, N-(5-aminopentyl)-l,5diaminopentane was prepared (81) with 14C at each terminal carbon by reaction of benzylamine with two equivalents of [ l-14C]-5-chloropentanenitrile followed by catalytic reduction. This material was incorporated very poorly (0.04% specific incorporation) into lupinine, suggesting that

40

DAVID J . ROBINS

s

A

118

1

119

composite labellingpattern

SCHEME29. Synthesis of ['3C,15N]cadaverinedihydrochloride (118) and its incorporation into lupinine (119).

this triamine is unlikely to be an intermediate in lupinine biosynthesis. An intermediate trapping experiment for this triamine also gave a negative result (83). C. STEREOCHEMISTRY OF THE ENZYMIC PROCESSES INVOLVED I N LUPININE BIOSYNTHESIS Golebiewski and Spenser (84) demonstrated that the biosynthesis of and lupinine starts from L-lysine by feeding a mixture of ~-[4-~H]lysine ~ ~ - [ 6 - ' ~ C ] l y s to i n eLupinus luteus. The 'H/14C ratio in lupinine increased from 4.1 to 8.4, indicating that lupinine is derived entirely from L-lysine. The next step in the pathway, the decarboxylation of L-lysine to form cadaverine (109)is known to proceed with retention of configuration (45, 85-88). Golebiewski and Spenser (89) answered the stereochemical questions concerning the steps from cadaverine (109)to lupinine (7)involving retention o r removal of hydrogen from the terminal carbons of cadaverine by making (R)- (120)and (S)-[ l-2H]cadaverine dihydrochloride (121)and feeding them to Lupinus luteus, then determining the complete labeling patterns in lupinine by 2H-NMR spectroscopy. The assignment of the *HNMR spectrum of lupinine (7) was carried out by Golebiewski (90),but some revisions were made by Rycroft e t a / . (91) after more detailed examination. Samples of the (R)-and (S)-[ l-2H]cadaverineswere made enzymically utilizing the knowledge that the decarboxylation of L-lysine catalyzed by L-lysine decarboxylase proceeds with retention of configuration

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

41

(45,85-88). Therefore, treatment of L-lysine in 2H20with lysine decarboxylase generated the (R)-isomer 120, whereas enzymic decarboxylation of the L-form of [2-2H]-~~-lysine in H 2 0 and treatment with HCI afforded (S)-[ l-2H]cadaverine dihydrochloride (121)(92). Feeding of these precursors to Lupinus luteus gave 2H-labeled lupinine samples (Scheme 30). The 2H-NMR spectrum of lupinine obtained after feeding the (R)-isomer 120 showed that lupinine (122) contained 2H at C-6a, C-10, and C-11 pro-S, whereas the (S)-isomer 121produced a sample of lupinine (123) labeled at C-4p and C-6p. Similar results were obtained subsequently by Fraser and Robins (93). The labeling patterns 122 and 123 show that 2H is retained with no change of stereochemistry at C-6 of lupinine after feeding each 2H-labeled precursor (Scheme 30), and follows from the results of feeding [i3C,i5N]cadaverine (118), which established that the N(S)-C(6) bond remains intact (Scheme 29). It is clear that 2H is lost after feeding the (S)-isomer 121 from the carbons destined to become C-10 and C-11 in lupinine. This may occur as a result of transamination processes, and Golebiewski and Spenser (89) proposed that two molecules of 1-piperideine could be

i/"" H

D

+

D 121

123

SCHEME30. Incorporation of (R)-(121)and (S)-[ I-2H]cadaverine dihydrochloride (122) into lupinine.

DAVID J . ROBINS

42

generated from cadaverine, which could combine to form tetrahydroanabasine (124)en route to lupinine as shown in Scheme 31. This scheme can be used to explain the loss of the pro-R hydrogen from the carbon of

CRO

cr"i' 1

attack from C-3-9 face at C-2-si face

R

S

loss of H,

R

124

0N C P

H' attack on C-si face

H

125

H- attack on C-re face

s k

s

R denotes 'H present after feeding (R)-[1 -'H]cadaverine S denotes 'H present after feeding (S>[l-2H]cadaverine

composite labelling patterns

SCHEME31. Proposed biosynthesis of lupinine via I-piperideine.

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

43

cadaverine, which becomes C-4 of lupinine, and the retention and overall inversion of configuration of the p r o 3 hydrogen at this center. Reduction of the aldehyde 125 to give lupinine must occur by attack of a hydride donor on the C-re face of the carbonyl group, which is the usual stereospecificity observed for a coupled dehydrogenase reaction. It should be noted that nearly half of the 60 plant species examined for quinolizidine alkaloids contained ammodendrine (126)(94). Ammodendrine is formed from tetrahydroanabasine (124)following dimerization of 1-piperideine produced from cadaverine (Scheme 31).

I

COCH, 126

Support for the proposed biosynthesis of lupinine (7) via 1-piperideine was supplied by Golebiewski and Spenser (84) when they fed [2-2H]-1piperideine (127) to Lupinus luteus (Scheme 32). The *H-labeled lupinine (128)contained 2H at C-10 and C-11 pro-S, as predicted by the route shown in Scheme 3 1.

D. BIOSYNTHESIS OF TETRACYCLIC QUINOLIZIDINE ALKALOIDS Studies by Schutte and co-workers with I4C-labeled lysine and cadaverine involving the establishment of partial labeling patterns in alkaloids by degradation indicated that tetracyclic quinolizidine alkaloids, such as (-)-sparteine (107), (+)-lupanine (129) ( 9 3 , and subsequently matrine (108) (96), are derived from three units of these precursors. Cadaverine

SCHEME 32. Incorporation of [2-*H]-l-piperideine (U7) into lupinine (U8).

44

DAVID J . ROBINS

(109)appeared to be incorporated in a symmetric fashion and with equal efficiency into each of the three parts of the tetracyclic quinolizidine alkaloids, as shown for sparteine (130)and matrine (131).

s

129

130 131

Exposure of Lupinus arboreus and L . angustifolius to 14C02led to the conclusion that sparteine (107)and lupanine (129) might be synthesized independently (possibly via a 1,2-didehydrosparteinium species) (97) rather than by oxidation of sparteine to give lupanine, as had been previously assumed. A range of tetracyclic quinolizidine alkaloids and tricyclic compounds such as cytisine (105)were shown to be labeled by DL[2-'4C]lysine in five species of the Fabaceae (98). The way in which three units of cadaverine combine to form the tetracyclic quinolizidine alkaloids has been a subject for speculation for many years. Golebiewski and Spenser (99) put forward the hypothesis that cadaverine is oxidized to Saminopentanal, which is in equilibrium with 1piperideine (first two steps of Scheme 31). 1-Piperideine is known to trimerize readily, and the tetracyclic quinolizidine alkaloids might be modified trimers of 1-piperideine (Scheme 33). Labeling experiments carried out with [2-I4C]- and [6-'4C]-l-piperideine on Lupinus angustifolius, as well as consideration of partial labeling patterns established by degradation, were in accord with this theory (99). Spenser and co-workers also fed [6-I4C]-1-piperideineto Sophora tetraptera and S . microphylla to obtain labeled matrine. A partial labeling pattern was established by degradation, which could be explained by a modification of the trimer theory (96). Subsequently, Golebiewski and Spenser (100)found that the labels from

1.

PYRROLIZIDINE AND QUINOLIZIDINE ALKALOIDS

45

SCHEME33. Proposed derivation of (-)-sparteine (107)from a trimer of 1-piperideine.

~ ~ - [ 6 - ' ~ C ] l y s and i n e [6-'4C]-1-piperideine did not enter the three units of lupanine (129) with equal efficiency and that one of the outer Cs units was labeled to a different extent from the other two. Some modifications to the tripiperideine theory were suggested based on the involvement of two

46

DAVID J . ROBINS

units of piperideine, initially to give tetrahydroanabasine (W),as in the route to lupinine shown in Scheme 31, then combination with a third C, unit. Careful new evidence from Perrey and Wink (101) suggests that 1piperideine may not be involved in quinolizidine alkaloid biosynthesis. In short-term experiments, cadaverine was found to be a much better precursor than 1-piperideine or the trimer (a-tripiperideine) for lupanine (129) in leaf disks of Lupinus pofyphyffusand similarly for (-)-sparteine (107) in L . arboreus. Further key experiments on the formation of tetracyclic quinolizidine alkaloids were carried out by the groups of Robins and Spenser. They fed [13C,'5N]-labeledcadaverine (118)to Lupinus futeus to obtain labeled (-)-sparteine (82,102).Complete labeling patterns were obtained for sparteine (132)by I3C-NMRspectroscopy. The presence of six enriched carbon signals, two of which were present as pairs of 13C-15N doublets ((2-2 and C- 1% confirmed that three cadaverine units are required to form sparteine and that two of these units are incorporated into the outer rings of sparteine in a specific fashion. As with lupinine (Section IIB), it was shown that 14C-labeledN-(5-aminopentyl)-l,5-diaminopentaneis not a precursor of sparteine (83).Similar labeling patterns were observed by Rana and Robins (103)in (+)-lupanine(133),(+)-13-hydroxylupanine(134),and(+)-angustifoline (135)after feeding ['3C,'5N]cadaverinedihydrochloride (118)to Lupinus polyphyflus (Scheme 34). The labeling pattern in angustifoline (135)

cry-+

-

+I I

118

132

134 R = O H

Composite labelling patterns

SCHEME34. Incorporation of ['3C,'5N]cadaverinedihydrochloride (118)into quinolizidine alkaloids l32-l35.

1. PYRROLIZIDINE

.

A N D QUINOLIZIDINE ALKALOIDS

47

is consistent with its formation from three units of cadaverine via a tetracyclic intermediate (103). When [ 1,2-"C2]cadaverine dihydrochloride (116) was fed to Lupinus futeus and L . polyphylfus, distinctive labeling patterns were established by 'T-NMR spectroscopy for sparteine (136), lupanine (137), (+)-13hydroxylupanine (1381, and (+)-angustifoline (139). (Scheme 35) (80). Again, the labeling pattern in angustifoline is consistent with the formation of the ally1 group by degradation of one of the rings of a tetracyclic precursor. By using Buptisiu uusirufis, Robins and co-workers (104) were able to isolate labeled N-methylcytisine (140)after feeding [ 1,2-'3C,]cadaverine dihydrochloride (116).The labeling pattern 140 was also consistent with the degradation of a tetracyclic precursor.

E. STEREOCHEMISTRY OF THE ENZYMIC PROCESSES INVOLVED I N THE BIOSYNTHESIS O F TETRACYCLIC QUINOLIZIDINE ALKALOIDS No intermediates have been firmly established between cadaverine (109) and tetracyclic quinolizidine alkaloids. Wink and co-workers showed that cadaverine was a substrate for crude enzyme preparations isolated from +

116

139 'R 137R=H 138R=OH

composite labelling patterns 140

SCHEME 35. Incorporation of [ 1,2-13C,]cadaverine (116) into quinolizidine alkaloids

136140.

48

DAVID J . ROBINS

cell suspension cultures of Lupinus polyphyllus, and that, in the presence of pyruvic acid, 17-oxosparteine(141)was isolated (105). These workers concluded that transamination reactions were occurring in which pyruvic acid acted as a receptor for the amino groups in cadaverine which were undergoing transamination. No intermediates could be detected in the enzymic process, and Wink et al. postulated a series of enzyme-linked intermediates in the enzyme complex. It was suggested that sparteine (107)and lupanine (129) are derived from 17-oxosparteine (141).Fraser and Robins (93,106) and Golebiewski and Spenser (107) fed samples of (R)- (120)and (S)-[1-*H]cadaverinedihydrochloride (121)to L. luteus to obtain (-)-sparteine (107).The former group also used L . polyphyllus to yield lupanine (129) and (+)-angustifoline, and the latter group fed L . angustifolius to afford (+)-lupanine (129). The 'H-labeling patterns in sparteine (142 and 143), lupanine (144 and 145), and angustifoline (146 and 147) were obtained by 'H-NMR spectroscopy (Scheme 36). Consideration of the labeling patterns 142 and 143 for (-)-sparteine shows that 'H is retained with the same stereochemistry where the C-N bonds remain intact at C-2 and C-15. The pro-R hydrogen is lost from one end of each of the three units of cadaverine, probably via transamination processes. The presence of 'H at C-17a in all three alkaloids 142, 144, and 146 after feeding (R)-[I-'Hlcadaverine (120) shows clearly that 17oxosparteine (141)cannot be an intermediate in the biosynthetic pathway

141

from cadaverine to any of these quinolizidine alkaloids. The postulate that sparteine (107)is formed by reduction of lupanine (108) or a 1,2didehydrosparteinium ion (97) is also disproved because 'H is present at C-2a (142)and C-2p (143)in sparteine after feeding (R)- and (S)-[I'Hlcadaverine dihydrochloride, respectively (Scheme 36). The labeling patterns shown in Scheme 36 are consistent with the suggestion that tetracyclic quinolizidine alkaloids are formed by extensive modification of a trimer of 1-piperideine. A late postulated intermediate in this pathway is the bis(iminium) ion 148 (100). Stereospecific attack of a hydride donor

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

49

+

dNH3 142

NH

II

0

0

146

144

145

composite labelling patterns

147

SCHEME 36. Incorporation of (R)- (l20)and (S)-[1-*H]cadaverinedihydrochloride (121) into quinolizidine alkaloids.

50

DAVID J . ROBINS

on the C-re faces of both iminium ions would lead to the three quinolizidine alkaloids sparteine, lupanine, and angustifoline labeled with 2H at C-17a after feeding the (R)-isomer 120 and with *H at C-lOa from the (S)precursor 121.

148

Fraser and Robins (109)fed ( R ) -(120) and (S)-[ l-2H]cadaverinedihydrochloride (121)to Baptisia australis to obtain 2H-labeled(+)-sparteine and (-)-N-methylcytisine (106).The labeling patterns obtained for these alkaloids after feeding the (R)-isomer (149and 150, respectively) and from the (S)-isomer (151and 152,respectively), are shown in Scheme 37 with those for (-)-sparteine for comparison. The labeling patterns in (+)- and (-1sparteine are not mirror images. Although most of the labels are in mirrorimage positions, those for C-2 and C-15 have the same stereochemistry as in the precursors where the 2H is retained on the C-N bonds that remain intact during the biosynthesis. Comparison of the labeling patterns for (+)-sparteine and (-)-N-methylcytisine (Scheme 37) suggest that it is ring A of a tetracyclic precursor that must be degraded and ring D that is converted into a pyridone. The 2H present at C-l 1 of (-)-N-methylcytisine (150)after feeding the (R)-isomer 120 is retained on cleavage of ring A, but with inversion of stereochemistry. This could arise by reduction of an intermediate C( I I)-N( 12) iminium ion stereospecifically from the C-re face. The absence of *H label in the N-methyl group of (-)-A'methylcytisine (150 and 152) after feeding the (R)- and (S)-precursors (Scheme 37) indicates that the N-methyl group is not formed from C-1 of a cadaverine precursor. This is consistent with the existence of an N-methyltransferase in Laburnum anagyroides, which can convert (-)-cytisine (105)into (-)-N-methylcytisine (106)(110). (-)-Anagyrine (153)is another tetracyclic quinolizidine alkaloid found in several species of Lupinus and Genista. Comparison of its structure and stereochemistry with those of (+)-sparteine indicated that if they are formed from the same tetracyclic intermediate with identical absolute configurations at C-6 and C-l I , then it is likely that ring A of the tetracyclic intermediate would be converted into a pyridone in order to form anagyrine (153).Robins and co-workers ( I 11 ) tested this theory by feeding (R)-(120)

1.

51

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

T-JJ H

D

H

D

H

151

\

D

MeN D

H

152

D

H

H

D

145

composite labelling patterns

SCHEME37. Incorporation of (R)- (120)and (S)-[ l-2H]cadaverine dihydrochloride (121) into (+)-sparteine and (-)-N-methylcytisine.

52

DAVID J . ROBINS

and (S)-[ l-2H]cadaverinedihydrochloride (121) to Anugyrisfoctidu, which produces (-)-anagyrine (153) and (-)-N-methylcytisine (106). Assignment of the 'H-NMR spectrum of anagyrine was established by the same group (112). The 2H labeling patterns for anagyrine (154 and 155, respectively) were established by *H-NMR spectroscopy and compared with those obtained for (+)-sparteine (149 and 151, respectively) (Scheme 38). It was immediately clear from these labeling patterns that if (+)-sparteine and (-)-anagyrine are formed from the same tetracyclic intermediate, then it must be ring D that is converted into a pyridone [this is the same +

ANH3 120

\

u

4

.'

SCHEME38. Incorporation of (R)- (l20)and (S)-[1-*H]cadaverinedihydrochloride (121) into (-)-anagyrine and ( +)-sparteine.

1.

PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

53

orientation as that required for the formation of the pyridone in ( - 1 4 methylcytisine (106)]. Thus the tetracyclic intermediate involved in (-)anagyrine formation has a different stereochemistry than (+)-sparteine at C-6, and enantiomerically deuterated precursors have been used to distinguish between possible ways of forming anagyrine (153).

153

Robins and Sheldrake (113) prepared [3,3-'H2]cadaverine dihydrochloride (156) as shown in Scheme 39. This was incorporated into a range of quinolizidine alkaloids, and the ?H labeling patterns were established by 'H-NMR spectroscopy. Four 2H atoms were retained in lupinine (157) and 'H was retained at C-8a and C-8P in (-)-sparteine (158), lupanine (1591, 13-hydroxylupanine (160), angustifoline (161) (113). (+)-sparteine (1621, and (-)-N-methylcytisine (163) (114).This shows that no hydrogen atoms are removed from this position in the biosynthesis, as previously proposed (105). The presence of 'H at C-13 in 13-hydroxylupanine (161) shows that introduction of oxygen at this position does not involve keto or enol intermediates, and is again consistent with its derivation from a tetracyclic precursor. The 'H label present at C-4 of (-)-N-methylcytisine (163) shows that no keto or enol intermediate is involved in the formation of the double bond at C-4 in the pyridone. Hemscheidt and Spenser (115) prepared [3,3-*H2]cadaverine by a different route and obtained similar labeling patterns in lupanine (159) and 13-hydroxylupanine (160) after carrying out feeding experiments with Lupinus angustifolius. [2,2,4,4,-'H4]Cadaverinedihydrochloride (164) was prepared by Robins and Sheldrake (114) (Scheme 40). The interesting feature in the 'H-NMR spectra of (+)-sparteine (165) and (-)-N-methylcytisine (166) is that no 'H is present at the bridgehead positions (C-7 and C-9). This suggests that enamine-imine equilibria are involved in the biosynthetic pathway to remove 'H from these positions in both alkaloids. The 'H labels at C-3 and C-5 in (-)-N-methylcytisine (166) demonstrate that no keto or enol units are involved during the formation of the pyridone at these carbon atoms. Further information about the stereochemistry of the formation of the pyridone system in anagyrine (153) and (-)-N-methylcytisine (106) should

54

DAVID J . ROBINS

1. MsCI. Et,N

CHzOH

2. NaCN

D

\

1

3. BH3.THF 4. HCI-MeOH

+

157

162

composite labelling patterns

SCHEME39. Synthesis of [3,3-2H2]cadaverinedihydrochloride and its incorporation into quinolizidine alkaloids.

be obtainable by making (R)-and (S)-[2-*H]cadaverinesand feeding them to Baptisia australis and Anagyris foetida to obtain complete labeling patterns of the quinolizidine alkaloids using *H-NMR spectroscopy. As stated at the beginning of this part of the review, quinolizidine alkaloids are widespread throughout the Fabaceae and are assumed to be

1. PYRROLIZIDINE A N D NC(CHd3CN

DZO

QUINOLIZIDINE ALKALOIDS

55

NCCD,CH,CD&N

DBU

1. BH9THF

2. HCI

165

SCHEME40. Synthesis of [2,2,4,4-ZH,]cadaverinedihydrochloride (164)and its incorporation into (+)-sparteine (165) and (-)-N-methylcytisine (166).

specific for this plant family. However, Wink and Witte (116) managed to induce the biosynthesis of quinolizidine alkaloids in cell cultures of plants not in this family. These plants usually produce either different alkaloids or no alkaloids. The intriguing suggestion is therefore made that the genes required to make quinolizidine alkaloids are not restricted to the Fabaceae, but have a far wider distribution and are not expressed outside the Fabaceae (116). IV. Conclusions

Our understanding of the biosynthesis of pyrrolizidine and quinolizidine alkaloids has dramatically increased over the past dozen years, particularly with the development of precursors labeled with stable isotopes

56

DAVID J. ROBINS

coupled with determination of complete labeling patterns by high-field NMR spectroscopy. The use of '3C,'5N-doubly labeled precursors has shown that the pathways to retronecine (13)and lupinine (7)are fundamentally different. The routes to retronecine and the other necines are now well characterized with a series of well-defined intermediates; however, details of the processes involved in the construction of lupinine and the tetracyclic quinolizidine alkaloids are poorly understood (intermediates may well be enzyme-bound), and theories are being continually revised. The stereochemistry of the enzymic processes involved in necine biosynthesis has been carefully and completely elucidated using precursors specifically deuterated in combination with 2H-NMR spectroscopy. Similar studies with quinolizidine alkaloids have revealed a number of stereochemical details and have established some of the later steps in the formation of pyridones in tetracyclic and tricyclic quinolizidine alkaloids, The field is now open for the study of individual enzymes involved in pyrrolizidine and quinolizidine alkaloid biosynthesis.

Addendum PYRROLIZIDINE ALKALOIDS Hartmann and co-workers (1 17) have shown that root cultures of Senecio vulgaris accumulate homospermidine(36)instead of pyrrolizidine alkaloids when putrescine is fed together with an inhibitor of diamine and polyamine oxidases (P-hydroxyethylhydrazine). The enzyme catalyzing the formation of homospermidine (36)has been isolated and partially purified from root cultures of Eupatorium cannabinum (30). This enzyme requires NAD+ as a cofactor. The NAD+ acts as a hydride acceptor in the first step of the reaction and then as a hydride donor in the second stage. Recent work suggests that homospermidine may be formed from putrescine and the four-carbon unit of spermidine (17),rather than from two molecules of putrescine. QUINOLIZIDINE ALKALOIDS Robins and Sheldrake (118) have now made possible the study of enzymic processes in quinolizidine alkaloid biosynthesis involving the removal of @hydrogens from cadaverine (109).They prepared (R)- (167) and (S)-[2-2H]cadaverine(168)from L- and D-glutamic acid, respectively,

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS

57

+

ANH3 d

\N/ D’ 167

H

169

170

168

SCHEME41. Incorporation of (R)- (167) and (S)-[2-2H]cadaverinedihydrochloride (168) into lupinine.

and fed these to Lupinus luteus plants to obtain labeled lupinine. The labeling patterns 169 and 170 were determined by ’H-NMR spectroscopy, and they show that the quinolizidine ring system is formed by retention of the pro-R hydrogen and removal of the pro-S hydrogen at C- 1 of lupinine (Scheme 41).

References

1. R. Robinson, “The Structural Relations of Natural Products.” Oxford Univ. Press, Oxford, 1955. 2. N. J. Leonard, in “The Alkaloids” (R. Manske and H. Holmes, eds.), Vol. 1, p. 107. Academic Press, New York, 1950. 3. N. J. Leonard, in “The Alkaloids” (R. Manske, ed.), Vol. 6, p. 35. Academic Press, New York, 1960. 4. F. L. Warren, in “The Alkaloids” (R. Manske, ed.), Vol. 12, p. 245. Academic Press, New York, 1970. 5. J. T. Wrobel, in “The Alkaloids” (A. Brossi, ed.), Vol. 26, p. 327. Academic Press, Orlando, FL, 1985.

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6. D. J. Robins, Nat. Prod. Rep. 1,235 (1984); 2,213 (1985); 3,297 (1986); 4,577 (1987); 6,221,577 ( 1989);7,377 ( 1990);8,213 ( 1991);9,3 13 (1992);10,487 ( 1993); 11,613 ( 1994). 7. D. J. Robins, Methods Plant Biochem. 6 , 175 (1993). 8. D. J. Robins, Fortschr. Chem. Org. Naturst. 41, 115 (1982). 9. A. R. Mattocks, “Chemistry and Toxicology of Pyrrolizidine Alkaloids.” Academic Press, London, 1986. 10. M. Suffness and G. A. Cordell, in “The Alkaloids” (A. Brossi e t a / . . eds.), Vol. 25, p. I . Academic Press, Orlando, FL, 1985. 11. E. Nowacki and R. U. Byerrum, Life Sci. 1, 157 (1962). 12. C. A. Hughes. R. Letcher, and F. L. Warren, J. Chem. Soc., 4974 (1964). 13. W. Bottomley and T . A. Geissman, Phytochemistry 3, 357 (1964). 14. N. M. Bale and D. H. G. Crout, Phytochemistry 14, 2617 (1975). 15. D. J. Robins and J. R. Sweeney, Phytochemistry 22, 457 (1983). 16. D. J. Robins and J. R. Sweeney, J. Chem. Soc.. Chem. Commun.. 120 (1979);J. Chem. Soc.. Perkin Trans. I , 3083 (1981). 17. T . Hartmann, H. Sander, R. Adolph, and G. Toppel, PIanta 175, 82 (1988). 18. H. Birecka, M. Birecki, and M. W. Frohlich, Plant Physiol. 84, 42 (1987). 19. H. Birecka, M. Birecki, E. J. Cohen, A. J. Bitonti, and P. P. McCann, Plant Physiol. 86, 224 (1988). 20. H. A. Khan and D. J. Robins, J. Chem. Soc.. Chem. Cornmun.. 146 (1981);J . Chem. Soc.. Perkin Trans. 1 , 101 (1985). 21. D. J. Robins, J. Chem. Res., Synop.. 326 (1983). 22. H. A. Khan and D. J. Robins, J. Chem. Soc., Chem. Commun., 554’(1981). 23. G. Grue-Sorensen and 1. D. Spenser, J. Am. Chem. Soc. 103, 3208 (1981); Can. J. Chem. 60,643 (1982). 24. H. A. Kelly and D. J. Robins, J. Chem. Soc., Perkin Trans. I , 177 (1987). 25. R. Kuttan, A. N. Radhakrishnan. T. Spande, and B. Witkop, Biochemistry 10, 361 (1971). 26. H. A. Khan and D. J. Robins, J. Chem. Soc., Perkin Trans. I , 819 (1985). 27. H. Birecka, T . E. DiNolfo, W. B. Martin, and M. W. Frohlich, Phytochemistry 23, 991 (1984). 28. J. Rana and D. J. Robins, J. Chem. Res., Synop., 146 (1983). 29. R. J. Bergeron, P. S. Burton, K. A. McGovern, and S. J. Kline, Synthesis. 732 (1981). 30. F. Bottcher, R.-D. Adolph, and T. Hartmann, Phytochemistry 32, 679 (1993). 31. D. J. Robins, J. Chem. Soc.. Chem. Comrnun., 1289 (1982). 32. H. A. Kelly and D. J. Robins, J. Chem. Soc.. Chem. Commun.. 329 (1988). 33. A. A. Denholm, H. A. Kelly, and D. J. Robins, J. Chem. Soc.. Perkin Trans. I . 2003 (1991). 34. H. Birecka and J. L. Catalfamo, Phytochemistry 21, 2645 (1982). 35. E. K. Kunec and D. J. Robins, J. Chem. Soc.. Chem. Commun., 250 (1986). 36. M. T. Pizzorno and S. M. Albonico, J. Org. Chem. 39, 731 (1974). 37. J. Rana and E. Leete, J. Chem. Soc., Chem. Commun.. 1742 (1985); E. Leete and J. Rana, J. Nat. Prod. 49, 838 (1986). 38. E. K. Kunec and D. J. Robins, J. Chem. Soc., Perkin Trans. I , 1437 (1989). 39. D. B. Hagan and D. J. Robins, J. Chem. Res.. Synop.. 292 (1990). 40. G . Toppel, L. Witte. B. Riebesehl, K. von Borstel, and T. Hartmann, Plant Cell Rep. 6, 466 (1987). 41. R. H. Barbour and D. J. Robins, Phytochemistry 26, 2430 (1987). 42. H. A. Kelly, E. K. Kunec, M. Rodgers, and D. J. Robins, J. Chem. Res.. Synop., 358 (1989).

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43. D. J. Robins, Chem. Soc. Rev. 18, 375 (1989). 44. D. J. Robins, Experienria 47, 1 I I8 (1991). 45. G. R. Orr and S. J. Gould. Tetrahedron Lerr. 23, 3139 (1982); G. R. Orr, S. J. Gould, A. E. Pegg, J. E. Seely, and J. K. Coward, Bioorg. Chem. 12, 252 (1984). 46. I. D. Wigle, L. J. J. Mestichelli, and 1. D. Spenser, J . Chem. Soc., Chem. Commun.. 662 (1982). 47. D. J. Robins, Phyrochemisrry 22, 1133 (1983). 48. J. Rana and D. J. Robins, J. Chem. Soc.. Chem. Commun.. 1222 (1983). 49. G. Grue-Sorensen and I. D. Spenser, J. Am. Chern. Soc. 105, 7401 (1983). 50. J. Rana and D. J. Robins, J. Chem. Soc., Chem. Comrnun., 517 (1984);J . Chem. Soc., Perkin Trans. I, 983 (1986). 51. J. C. Richards and I. D. Spenser, Can. J. Chem. 60,2810 (1982). 52. H. A. Kelly and D. J. Robins, J. Chem. Soc., Perkin Trans. I , 2195 (1987). 53. D. Arigoni and E. L. Eliel, Top. Stereochem. 4, 200 (1969). 54. E. K. Kunec and D. J. Robins, J. Chem. Soc., Chem. Commun., 1450 (1985);J. Chem. Soc., Perkin Trans. I , 1089 (1987). 55. 1. K. A. Freer, J. R. Matheson, M. Rodgers, and D. J. Robins, J. Chem. Res., Synop.. 46 (1991). 56. C. Hughes and F. L . Warren, J. Chem. Soc.. 34 (1962). 57. D. H. G. Crout, M. H. Benn, H. Imaseki, and T. A. Geissman, Phyrochemisrry 5, 1 (1966).

58. C. G. Gordon-Gray and F. D. Schlosser, J. S. Afr. Chem. Insr. 23, 13 (1970). 59. D. H. G. Crout, J . Chem. Soc. C , 1968 (1966). 60. D. H. G. Crout, J. Chem. Soc. C , 1233 (1967). 61. B. A. McGaw and J. G. Woolley, Phytochemistry 18, 1647 (1979). 62. W. C. Evans and J. G. Woolley, J. Pharm. Pharmacol. 17, Suppl. 37s (1965). 63. D. H. G. Crout, N. M. Davies, E. H. Smith, and D. Whitehouse, J . Chem. Soc., Chem. Commun., 635 (1970); J. Chem. Soc., Perkin Trans. I, 671 (1972). 64. N. M. Davies and D. H. G. Crout, J . Chem. Soc., Perkin Trans. I, 2079 (1974). 65. N . M. Bale, R. Cahill, N . M. Davies, M. B. Mitchell, E. H. Smith, and D. H. G. Crout, J. Chem. Soc., Perkin Trans. I, 101 (1978). 66. R. Cahill, D. H. G. Crout, M. B. Mitchell, and U. S. Muller, J. Chem. Soc., Chem. Commun., 419 (1980); R. Cahill, D. H. G. Crout, M. V. M. Gregorio, M. B. Mitchell, and U. S. Muller, J. Chem. Soc.. Perkin Trans. I, 173 (1983). 67. D. J. Robins, N. M. Bale, and D. H. G. Crout, J . Chem. Soc., Perhin Trans. 1. 2082 ( 1974). 68. J. A. Devlin and D. J. Robins, J. Chem. Soc., Perkin Trans. I, 1329 (1984). 69. A. A. Denholm and D. J. Robins, J. Chem. Soc., Chem. Commun., 19 (1991). 70. T. Hartmann and G. Toppel, Phytochemisrry 26, 1639 (1987). 71. T. Hartmann, H. Sander, R. Adolph, and G. Toppel, Planta 175,82 (1988);T. Hartmann and G. Toppel, Phyrochemisrry 26, 1639 (1987). 72. H. J. Segall, C. H. Brown, and D. F. Paige, J. Labelled Compd. Radiopharm. 20,671 (1983). 73. Kh. A. Aslanov, Yu. K. Kushmaradov, and S. S. Sadykov, in "The Alkaloids" (A. Brossi, ed.), Vol. 31, p. 16. Academic Press, Orlando, FL, 1987; A. J. Howard and J. P. Michael, ibid.,Vol. 28, p. 183 (1985); F. Bohlmann and D. Schumann, ibid. (R. Manske, ed.), Vol. 9, p. 175. Academic Press, New York, 1967; N. J. Leonard, ibid., Vol. 7, p. 253 (1960); Vol. 3, p. I19 (1953). 74. J. P. Michael,Nar. Prod.Rep. 10,51 (1993);8,553(1991);7,485(1990);M. F.Grundon, ibid. 6, 523 (1989), 4, 415 (1987); 2, 236 (1985); 1, 349 (1984).

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75. M. Wink, Methods Plant Biochem. 6, 197 (1993). 76. K. Fuji, in “The Alkaloids” (A. Brossi, ed.), Vol. 35, p. 155. Academic Press, San Diego. CA, 1989; W. M. Golebiewski and J. T. Wrobel, ibid. (R. Rodrigo, ed.), Vol. 18, p. 263. Academic Press, New York, 1981. 77. H. R. Schutte, Arch. Pharm. (Weinheim, Ger.) 293, 1006 (1960). 78. M. Soucek and H. R. Schutte, Angew. Chem., Int. Ed. Engl. 1, 597 (1962). 79. J. Rana and D. J. Robins, J . Chem. Res., Synop., 164 (1984). 80. D. J. Robins and G. N. Sheldrake, J. Chem. Res., Svnop.. 256 (1987); J. Chem. Res., Miniprint, 2101 (1987). 81. J. Rana and D. J. Robins, J . Chem. Soc., Chem. Commun., 81 (1984). 82. W. M. Golebiewski and I. D. Spenser. J. Chem. SOC.. Chem. Commun., I509 (1983). 83. J. Rana and D. J. Robins, J. Chem. Soc., Perkin Trans. I , 1133 (1986). 84. W. M. Golebiewski and I. D. Spenser, Can. J. Chem. 63, 2707 (1985). 85. E. Leistner and 1. D. Spenser, J. Chem. SOC.. Chem. Cornmun.,378 (1975). 86. H. J. Gerdes and E. Leistner, Phytochemistry 18, 771 (1979). 87. A. R. Battersby. R. Murphy, and J. Staunton, J. Chem. SOC., Perkin Trans. I , 449 (1982). 88. D. J. Robins, Phytochemistry 22, 1133 (1983). 89. W. M. Golebiewski and 1. D. Spenser, J. Am. Chem. SOC. 106, 1441 (1984). 90. W. M. Golebiewski, Bull. Pol. Acad. Sci., Chem. 34, 191 (1986). 91. D. S. Rycroft, D. J. Robins, and I. H. Sadler, Magn. Reson. Chem. 30, S15 (1992). 92. J. C. Richards and I. D. Spenser, Tetrahedron 39, 3549 (1983). 93. A. M. Fraser and D. J. Robins, J. Chem. SOC., Perkin Trans. I , I05 (1987). 94. M. Wink and L. Witte. Z. Naturforsch., C: Biosci. 42C, 197 (1987). 95. H. R. Schutte, and H. Hindorf. Justus Liebigs Ann. Chern. 685, 187 (1965); H . R. Schutte, H . Hindorf. K. Mothes, and G. Hubner, ibid. 680, 83 (1964). %. F. J. Leeper, G. Grue-Sorensen, and I. D. Spenser, Can. J . Chem. 59, 106 (1981). 97. Y.D. Cho, R. 0. Martin, and J. N. Anderson, J. A m . Chem. SOC.93, 2087 (1971). 98. E. K. Nowacki and G. R. Waller, Phytochemistry 14, 155 (1975). 99. W. M. Golebiewski and I. D. Spenser, J. A m . Chem. SOC.98, 6726 (1976). 100. W. M. Golebiewski and I. D. Spenser, Can. J. Chem. 66, 1734 (1988). 101. R. Perrey and M. Wink, Z . Naturforsch., C: Biosci. 43C, 363 (1988). 102. J. Rana and D. J. Robins, J . Chem. SOC.. Chem. Cornmun.,1335 (1983). 103. J. Rana and D. J. Robins, J. Chem. Res., Synop., 1% (1985). 104. A. M. Fraser, D. J. Robins, and G. N. Sheldrake, J. Chem. SOC., Perkin Trans. I , 3275 (1988). 105. M. Wink, T. Hartmann, and H. M. Schiebel, Z. Naturforsch., C: Biosci. 34C, 704 (1979). 106. A. M. Fraser and D. J. Robins, J . Chem. SOC.. Chem. Commun.. 1477 (1984). 107. W. M. Golebiewski and 1. D. Spenser, J. A m . Chem. SOC.106,7925 (1984). 108. M. Wink, L. Witte, and T. Hartmann. Planta Med. 43, 342 (1981). 109. A. M. Fraser and D. J. Robins, J. Chem. SOC..Chem. Commun.,545 (1986); J. Chem. SOC., Perkin Trans. I , 3275 (1988). 110. M. Wink, Planta 161, 339 (1984). I 11. A. M. Brown, D. S. Rycroft, and D. J. Robins, J . Chem. Soc., Perkin Trans. I , 2353 (1991). 112. D. S. Rycroft, D. J. Robins, and 1. H. Sadler, M a p . Reson. Chem. 29, 936 (1991); D. J. Robins and D. S. Rycroft, ibid. 30, 1125 (1992). 113. D. J. Robins and G. N. Sheldrake, J. Chem. Res.. Synop. 159 (1987);J. Chern. Res.. Miniprint, 1427 (1987).

1. PYRROLIZIDINE A N D QUINOLIZIDINE ALKALOIDS 114. 115. 116. 117. 118.

D. J. Robins and G . N . Sheldrake, J . Chem. Res., Synop., 230 (1988). T. Hemscheidt and I. D. Spenser, Can. J . Chem. 65, 170 (1987). M. Wink and L. Witte, FEBS L e f t . 159, 196 (1983). F. Boettcher, D. Ober, and T. Hartmann, Can. J . Chem. 72, 80 (1994). D. J. Robins and G . N. Sheldrake, J . Chem. SOC., Chem. Commun., 1331 (1994).

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--CHAPTER2-

PHARMACOLOGY OF POLYAMINE TOXINS FROM SPIDERS AND WASPS ALAN L. MUELLER, ROSEMARIE ROELOFFS, A N D HUNTER JACKSON NPS Pharmaceuticals, Inc. Salt Lake City,UT 84108

I. Introduction and Ecological Aspects ....................................................... 11. Pharmacological Effects of Polyamine Toxins in Invertebrates

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

A. Historic Background .............. B. Site and Mechanism of Action S s ............................................... C. Summary ...................................................................................... 111. Pharmacological Effects of Polyamine Toxins in Vertebrates ...................... A. Glutamate Receptor Subtypes in the Mammalian CNS .......................... B. Site and Mechanism of Action Studies ............................................... C. Summary ............................................... .................................. IV. Structure-Activity Relationship Studies ........... .................................. V. Perspectives .......................................................................................

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

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

63 67 69 70 72 72 73 85 86

90 91

I. Introduction and Ecological Aspects Spiders belong in the taxonomic order Araneae, one of the major taxa of the class Arachnida. They are among the most widespread and diverse groups of animals and are abundant in virtually all terrestrial ecosystems. In number of described or anticipated species, the spider order Araneae ranks seventh in global diversity, surpassed only by the five largest insect orders and the arachnid order Acari (mites and ticks) (I). Approximately 34,000 spider species have been described, forming about 3,000 genera within 105 families. Spiders, however, are not a well studied group, and it is likely that the number of undescribed and as yet undiscovered species is substantial. Coddington ( 1 ) speculates that there may be as many as 170,000 spider species. Spiders, which normally prey exclusively on insects, are considered among the dominant predators of most terrestrial ecosystems (2). Spiders are generalist predators: they rarely show prey specificity and usually attack different insect types relative to the rate at which they are 63

THE ALKALOIDS. VOL. 46 Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

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ALAN L. MUELLER E T A L .

encountered (3).The few instances in which prey specificity is observed are in habitats affording high numbers of particular types of prey (4). Spider venoms, together with spider webs, have determined the predatory efficiency of spiders, thus ensuring their evolutionary success. By hindering escape and preventing counterattacks, the use of venom allows spiders to prey upon animals that are often faster and larger than themselves. All spiders, except for the small family Uloboridae and some Liphistiidae, possess venom glands (5). These venom glands produce venoms that are complex mixtures of many biologically active components. As spiders evolved from a primitive general form to animals with specialized webs and prey-capture behaviors, corresponding modifications of their venom glands occurred. In the “primitive” mygalomorphs the venom glands are fairly small and are located in the chelicerae, whereas the more specialized labidognath spiders (“true” spiders) have relatively large venom glands that often extend out of the chelicerae into the chephalothorax. In some spider species, the venom glands have undergone extensive modification-in Filistata species, the glands are exceptionally large and subdivided into lobes; and in the spitting spider Scytodes, the anterior part of the venom gland produces venom while the posterior portion of the gland produces a gluey substance that aids in prey capture. The venoms of different spider species vary greatly in their chemical components, as well as in their toxicity. One of the largest American wolf spiders has venom glands proportionately much smaller than those of many small spiders. Because the wolf spider is a wandering hunter with large, powerful chelicerae and sturdy legs to control its prey, it is less reliant on its venom for prey capture (2). More delicate spiders have the problem of subduing large, often dangerous, insects and in some cases may compensate for their lack of physical strength by producing a greater or more potent amount of venom. While there is no direct evidence to show that the quantity and toxicity of the venom are correlated with physique or other factors, it is clear that spider venoms are quite diverse and work through a variety of mechanisms. Venom constituents can be divided simply into proteins and nonproteins. The proteinaceous toxins range in size from a few thousand to several thousand kilodaltons; extensive research in this area in the last several years has demonstrated that many peptide toxins target voltagesensitive ion channels (6-8). The nonproteinaceous component of spider venom is a complex mixture of amino acids, inorganic salts, biogenic amines, and nucleic acids. In 1957, Fischer and Bohn (9) reported on the existence of free polyamines, such as spermine in the venom of tarantulas in the spider family Theraphosidae. Similarly, polyamines were later discovered in the venom of the Australian funnel-web spider, Atrax robustus (10). The venom of more highly evolved spiders, notably the orb-weaving

2.

PHARMACOLOGY OF POLYAMINE TOXINS

Aromatic Chromophore

-

Polyamine Amino Acid -kckbone Linker

65

Acid - Amino Tail

JSTX-3

H

H Agel-489

FIG. 1. Structures of selected polyamine spider and wasp toxins.

spiders in the family Araneidae, contains low-molecular-weight (<1000 daltons) polyamine toxins (Fig. 1). These secondary metabolites, present only in the venom glands and in no other organs, seem to have evolved specifically as tools with which to paralyze prey (6-8,11-13). Similarly,

'

'

These toxins have been referred to as pol yamine-containing toxins, polyamine-amide toxins, acylpolyamine toxins, arylamine toxins, and arylalkylamine toxins. We shall refer to them as polyamine toxins in this review.

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ALAN L. MUELLER ET A L .

the venoms of certain parasitic wasps contain nonproteinaceous toxins that are utilized to paralyze the insect host for the wasp’s eggs. The venom of the Philanthus rriungulurn wasp has been studied most extensively (14), and has been shown to contain several low-molecular-weight polyamine toxins. In an effort to identify spider taxa that are sources of polyamine toxins, Balandrin and Roeloffs (15) examined more than 110 whole spider venoms by means of thin-layer chromatography (TLC). These venoms represent more than 100 spider species in at least 50 genera distributed among 23 families. The results of the survey indicate that certain spider families, such as the Araneidae (12 of 12 genera are polyamine-rich)and the Agelenidae (3 of 4 genera are polyamine-rich),are prolific producers of polyamine toxins. Other spider families, such as the Lycosidae, appear to be devoid of pol yamine toxins. Certain families appear to contain different species that either do or do not produce these toxins (e.g., Amaurobiidae, Pisauridae, Salticidae, and Thomisidae). It is unclear whether these “mixed” results represent a situation requiring taxonomic clarification or whether polyamine toxins could be arising independently in various spider taxa by means of convergent evolution. No clear correlation seems to exist between web-building behavior and the presence of polyamine toxins in spider venom. The Agelenidae, Araneidae, and Amaurobiidae are all web-building spiders that rely upon both their webs and venoms for prey capture, while the Pisauridae, Salticidae, and Thomisidae do not use webs for prey capture. Yet polyamine toxins are present in the venoms of spiders of all of these families. Spiders exhibit different predatory strategies as well as different venom chemistry and toxicity. For instance, ground hunting spiders, such as the theraphosids, clubionids, and salticids, do not use silken webs in prey capture, relying solely upon physical strength and venom toxicity as they grasp and bite their prey. Agelenid spiders spin nonsticky webs on the ground which are used to signal the presence of prey. Once prey is detected, the agelenid spider will rush in, grasping and biting, relying upon its venom to kill the prey. Aerial webs are built by araneid, theriid, and linyphiid spiders. These webs, which may or may not be sticky, trap and/ or knock down flying insects, which are quickly envenomated and either eaten immediately or bound in silk to be stored. In all cases, spiders use their venom to paralyze or kill insects, thus immobilizing them before they can escape or damage the spider or its web. Unfortunately, there is little information about the effects of a spider’s ecology on its venom, and how different spider venoms affect, and are affected by, predatory behavior and evolution. It is, however, known that spider venom toxicity varies with age, sex, and population (16,17). Most

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studies of spider venoms have concentrated on the medically important venoms, that is, the spider venoms that are known to be toxic to humans Only recently have there been investigaor vertebrates in general (18,f9). tions of the potency of various spider venoms against insects, the normal prey of spiders, and these studies indicate that spider venoms vary widely in potency. In one study, Quistad et al. (20) injected the venoms from 59 spider species into a number of larval crop pests. They found that large spiders, such as tarantulas and wolf spiders, as well as aerial-web-building spiders (i.e., Araneidae), typically have weak venoms. Similarly, agelenid and thomisid venoms were found to be weak against larval pests. Venoms from several primitive hunting spiders, however, were found to be relatively potent, as were the venoms of several ground hunting spiders (Salticidae and Gnaphosidae). On the other hand, a separate investigation of the potency of 18 spider venoms against cockroaches demonstrated that the venom of a wandering spider species that lives in vegetation is more potent than the venom of a ground-living and a web-building spider (21). The characteristics of the venom of a given spider, therefore, remain difficult or impossible to predict. Recent advances in the care and handling of spiders and wasps (e.g., the ability to milk small spiders repeatedly and so obtain sufficient quantities of venom), coupled with new chemical techniques for the separation and purification of individual toxins from a complex mixture of compounds present in whole venom, as well as the ability to synthesize native polyamine toxins and analogs, have allowed tremendous progress in the research on polyamine toxins. We now know that polyamine toxins from spiders and wasps are potent antagonists of receptors for the excitatory amino acid (EAA) glutamate in invertebrates and vertebrates. As such, pol yamine toxins represent new chemical leads for the development of insecticide and human therapeutic products. The pharmacology of polyamine toxins is reviewed in the present chapter. The chemistry of polyamine toxins (21a ) was the topic of a separate article in a previous volume of The Alkaloids.

11. Pharmacological Effects of Polyamine Toxins in Invertebrates

A. HISTORICBACKGROUND The first clear demonstration of glutamate antagonists in spider and wasp venom was the principal event leading to a surge of interest in these venoms that continues to this date. This demonstration was provided

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A L A N L. MUELLER ET AL.

in the early 1980s by the laboratories of Kawai in Japan, Piek in The Netherlands, and Usherwood in the United Kingdom. The evidence arose from studies of the effects of these venoms on invertebrate preparations. It has been recognized for many years that chemical transmission at the insect neuromuscular junction is mediated by glutamate. The pharmacological and physiological properties of these glutamate receptors have been well characterized by Usherwood and his colleagues (22-25). The glutamate receptor of primary importance in mediating neuromuscular transmission is activated selectively by quisqualate, and as such is termed the quisqualate receptor (QUIS-R) (26). Kawai and his colleagues reported that crude JSTX (Joro spider toxin), from the venom of the orb-weaving spider Nephila clavata, antagonized glutamate-mediated transmission at the lobster neuromuscular junction (27,28). The chemical identity of JSTX, actually a mixture of several homologous polyamine toxins, was not known at the time these studies were performed. The molecular weight of JSTX was assumed to be approximately 500 daltons. Joro spider toxin was found to block excitatory postsynaptic potentials (EPSPs) while having no effect on inhibitory postsynaptic potentials (IPSPs). The blockade of glutamate-mediated neurotransmission by JSTX was highly potent (effective concentrations were suggested to be in the 1-10 nM range). The effect of a low concentration of toxin was reversible, whereas high concentrations produced an essentially irreversible blockade (no recovery was noted during washout periods a few hours in duration). The presynaptic action potential, as well as the postsynaptic resting membrane potential and input resistance, were unaltered. In studies employing iontophoretic or bath application of glutamate agonists, JSTX preferentially inhibited responses to glutamate and quisqualate and had little effect on aspartate-evoked depolarizations. Kawai’s group carried out similar experiments regarding the activity of crude JSTX on the giant synapse of the squid stellate ganglion (29). In this preparation, JSTX produced a potent and apparently irreversible blockade of excitatory synaptic transmission and glutamate-evoked depolarizations, and had no effect on postsynaptic membrane potential or input resistance, the presynaptic action potential, or the postsynaptic action potential evoked by antidromic stimulation. On the basis of these findings, Kawai concluded that L-glutamate, as was previously suspected, is the neurotransmitter at the squid giant synapse. Data from Kawai’s laboratory dealing with two other orb-weaving spiders, Araneus ventricosus and Neoscona nautica, served to broaden the scope of their discoveries. These investigators demonstrated in 1983 that those spiders, also from the family Araneidae, produced specific antagonists of glutamate receptors at the lobster neuromuscular junction (30).

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These findings prompted Kawai to speculate that, “It is thus possible that a number of spider venoms contain a blocker of the glutamate receptor” (30, p. 440). Tashmukhamedov et al. (31) reported in 1983 that the venom of the orb-weaving spider Argiope lobata contained at least four active fractions, originally believed to be in the range of 6-7 kilodaltons, that blocked transmission at the locust neuromuscular junction. These initial findings were extended by Usherwood and his co-workers, who reported that several orb-weaving spiders (Argiope trifasciata, Araneus gemma, Argiope aurantia, Araneus andreusi) contained low-molecular-weight toxins (
B. SITEA N D MECHANISMOF ACTIONSTUDIES These initial studies revealed previously unsuspected biological activity

in spider venom with potential human therapeutic and agrochemical importance. This, in turn, spurred the chemical isolation and characterization of the responsible toxins. Additionally, chemical syntheses of active toxins,

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ALAN L. MUELLER ET A L .

coupled with technical advances, such as the routine utilization of patchclamp electrophysiological techniques, led to rapid gains in the understanding of the pharmacology of polyamine toxins. In particular, studies on invertebrate preparations using purified or synthetic toxins have provided much information with regard to site and mechanism of action of the polyamine toxins (Table I) (41-46). As is obvious even from a cursory examination of the data presented in Table I, no clear consensus has been reached as to the site and mechanism of the polyamine toxins at the invertebrate QUIS-R. The reversibility of action of all toxins examined has been reported to range from readily reversible to slowly reversible to essentially irreversible, and is, at least in part, dependent upon the concentration utilized (27-29,42,44-46). The philanthotoxins and argiotoxins generally behave as noncompetitive openchannel blockers, with their actions characterized in most (but not all) cases as use- and voltage-dependent(35,42-45,47).Additionally, the inhibitory effects of these toxins are both concentration- and time-dependent; concentrations of Arg-636 (argiotoxin-636) as low as M have been reported to produce complete blockade of glutamatergic responses if applied for long periods of time (44,45). This latter effect, coupled with the generally slow reversibility of the block, has been interpreted as evidence that polyamine toxins may be concentrated in some compartment (perhaps intracellular? see Section II,C) of the muscle. As further evidence in support of this idea, it has been reported that one can relieve the blockade elicited by either 6-PhTX (philanthotoxin-433) or Arg-636 by extensive washout or by altering the muscle membrane potential, and that the blockade then redevelops even when the toxin is absent from the recording buffer (42,45).

C. SUMMARY Overall, the studies of 6-PhTX and Arg-636 strongly suggest that these toxins antagonize glutamate responses by a use-dependent, open-channel blocking mechanism. This conclusion, however, is undoubtedly an oversimplification, as several observations illustrate. First, the voltage dependence of blockade is not straightforward, with various studies showing voltage-independent blocks (41,42,46)or blockades either increased (35) or decreased (43,45) upon hyperpolarization of the membrane. Second, these studies also suggest additional mechanisms of blockade, such as closed-channel block ( 4 3 - 4 3 , the functional significance of which is unclear. Third, Usherwood and his colleagues (46) reported that the intrucellular application of PhTX-343, a synthetic analog of &PhTX, resulted in the antagonism of glutamate responses recorded in locust muscle fibers.

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OF POLYAMINE TOXINS

TABLE 1 PHARMACOLOGICAL EFFECTSOF POLYAMINE TOXINSI N INVERTEBRATES Toxin

Preparation

JSTX (crude)

Lobster neuromuscular junction (nmj)

JSTX (crude) PhTX (crude) argiotoxins (crude)

Squid giant synapse

JSTX (purified)

Lobster nmj

8-PhTX (purified)

Locust nmj

Arg-636 (purified)

Blowfly larva nmj

Arg-636 (purified)

Locust nmj

Arg-636 (purified?)

Crayfish nmj

PhTX-343 (synthetic analog)

Locust nmj

Locust nmj Locust nmj

Features of blockade

Ref.

Selective for QUIS 27,28 Reversible (low concentrations) Irreversible (high concentrations) Irreversible 29 Presynaptic and postsynaptic components of blockade Use-dependent Voltage-dependent (relieved by depolarization) Open-channel block Voltage-independent Irreversible Acts at glutamate recognition site or mouth of channel Use-dependent Vol tage-independent Open-channel block Tissue compartmentalization Voltage-dependent (relieved by hyperpolarization) Open-channel block Additional closed channel block? Extremely potent M) Slow recovery ( > I hr) Open-channel block Tissue compartmentalization Low-dose potentiation Open-channel block: potent M) voltage-dependent (relieved by hyperpolarization) slowly reversible Flickering block: less potent M) voltage-independent Extracellular application: reversible open-channel block use-dependent voltage-independent initial potentiation Intracellular application: irreversible blockade use-independent voltage-independent no potentiation

39.40 35

41

42

43

44

45

46

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ALAN L. MUELLER ET AL.

In contrast to the use-dependent and voltage-independent blockade produced by extracellular toxin application, that produced by intracellular injection of toxin was neither use- nor voltage-dependent, suggesting another, possibly intracellular, site of action. Fourth, with regard to 6-PhTX, substantial evidence exists that a major component of the action of this toxin is presynaptic inhibition of high-affinity glutamate reuptake, presumably leading to increased levels of glutamate within the synaptic cleft and postsynaptic receptor desensitization (39,40,48).The philanthotoxins appear unique in this regard, as a similar mechanism of action has not been proposed for other pol yamine toxins. Fifth, several laboratories have reported that 6-PhTX (49,50) antagonizes not only glutamate-activated channels, but insect nicotinic receptors and associated ion channels as well, again most probably by an open-channel blocking mechanism. Sixth, low concentrations of Arg-636 (44) or 6-PhTX (51) often produce an initial potentiation of glutamate-activated currents prior to antagonism. The significance of this finding will be discussed in more detail in Section I11 with regard to the actions of polyamine toxins in the vertebrate CNS. Finally, it should be noted that Kawai’s group has claimed that JSTX does not produce open-channel blockade, but rather may act at the glutamate recognition site or the mouth of the associated ion channel (41).

111. Pharmacological Effects of Polyamine Toxins in Vertebrates

A. GLUTAMATE RECEPTORSUBTYPES IN

THE

MAMMALIAN CNS

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Those glutamate receptors that trigger the influx of cations are termed ionotropic receptors and have been classified pharmacologically (i.e., by responsivity to selective agonists) into N-methyl+-aspartate (NMDA) receptors, kainic acid (KA) receptors, and a-amino-3-hydroxy5-methylisoxazole-4-propionicacid (AMPA)receptors. An additional subtype of glutamate receptor, termed the metabotropic receptor, is coupled via G proteins to intracellular phosphoinositide metabolism and/or adenylate cyclase. Several subtypes of each of these receptors have been cloned (5233). The NMDA receptor is composed of a binding site for glutamate and an associated ion channel (ionophore), both of which appear to exist on a single molecular subunit (52,53). The NMDA receptor-ionophore complex has been implicated in a variety of neurological pathologies, including stroke, head trauma, spinal cord injury, epilepsy, and neuro-

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degenerative disorders such as Alzheimer’sdisease (54).Recent data using selective antagonists suggest a role for non-NMDA receptors in certain of these pathologies as well (55). N-Methyl-D-aspartatereceptor antagonists additionally may have potential therapeutic utility as analgesics (56) and anxiolytics (57). Finally, NMDA receptors are believed to be involved in the cellular events underlying various forms of learning and memory (54). The NMDA receptor has been the most studied and best characterized of the several subtypes of glutamate receptors. The activity of the NMDA receptor-ionophore complex is regulated by a variety of modulatory sites that can be targeted by selective antagonists (58).Competitive antagonists, such as the phosphonate APS, act at the glutamate binding site, whereas noncompetitive antagonists, such as phencyclidine (PCP), MK-801, or magnesium (Mg2+),act within the associated ion channel. There is also a glycine binding site that can be blocked selectively with compounds such as 7-chlorokynurenic acid. There is evidence demonstrating that glycine acts as a co-agonist, so that both glutamate and glycine are necessary to elicit full NMDA receptor-mediated responses. Other potential sites for modulation of NMDA receptor function include a zinc (Zn”) binding site and a sigma ligand binding site. Of particular relevance to the topic of polyamine toxins is the discovery that free polyamines such as spermine and spermidine appear to bind to a specific polyamine binding site on the NMDA receptor-ionophore complex and so potentiate the activity of the complex (59). Subsequent studies have identified compounds possessing agonist, antagonist, or inverse agonist pharmacological properties at this polyamine binding site (60-64). Whether polyamine toxins act at this pol yamine binding site on the NMDA receptor-ionophore complex is currently under intensive investigation (see Section 111,B). Because of the myriad roles of glutamate in the mammalian CNS, and the therapeutic importance of disease states in which glutamate is thought to play a major role, much effort is being put forth by researchers at academic institutions and pharmaceutical companies to identify new chemical entities that interact selectively with the various subtypes of glutamate receptors. Consequently, a number of studies have examined the interactions of the polyamine toxins with glutamate receptors in the CNS.

B. SITEA N D MECHANISM OF ACTION STUDIES Kawai’s group was the first to report that polyamine toxins had activity in the mammalian CNS. Specifically, these investigators demonstrated initially that crude JSTX suppressed postsynaptic potentials recorded extracellularly at a presumed glutamatergic synapse in area CA3 in guinea pig hippocampal slices maintained in uitro (65). In subsequent in uitro

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studies utilizing purified toxin (66-69), these investigators demonstrated that JSTX blocked the synaptic response in CAI pyramidal cells evoked by Schaffer collateral stimulation without affecting the antidromic action potential. In addition, JSTX was reported to block the response to exogenously applied glutamate, quisqualate (an agonist at the AMPA receptor) and KA, while having little effect on responses to the NMDA receptor agonist, aspartate. In a few cases, however, JSTX did not discriminate between NMDA and non-NMDA responses (68).The inhibitory effects of JSTX were reported to be at best only partially reversible. While these initial studies provided the impetus to further research on the actions of polyamine toxins in the mammalian CNS, they did not themselves directly address the issue of site and mechanism of action. However, numerous such mechanistic studies, summarized in Table 11, have been undertaken in recent years. As was the case with the invertebrate QUIS-R, no clear consensus has arisen from these studies. Several generalizations may be made, however. First, researchers have generally fallen into one of two camps as the knowledge base has increased. Kawai and his colleagues have reported repeatedly that JSTX, or more appropriately now, synthetic JSTX-3, is a highly potent and selective competitive antagonist of non-NMDA receptors. This conclusion follows not only from extensive in uitro characterization of JSTX-3 (66-7Z), but from in uiuo studies on the pharmacological effects (e.g., specific antagonism of quisqualate-induced seizures in mice) of this toxin as well (72).As reported in a recent publication, these investigators apparently have succeeded in isolating an AMPA receptor from rat brain using an affinity column prepared with JSTX-3 (73). In support of Kawai’s findings, several laboratories have independently reported a selective antagonism of non-NMDA receptors by polyamine toxins (74-78). In the other camp are numerous researchers who have demonstrated that polyamine toxins generally are selective NMDA-receptor antagonists, and that this inhibition of NMDA receptors is via a noncompetitive, open-channel blocking mechanism (79-85)(Table 11). Even within this camp, however, investigators are not able to agree upon particular features of the blockade, such as the degree of use or voltage dependence. A general problem is that the methodologies employed by the various investigators are quite diverse. Specifically, different neuronal preparations, different polyamine toxins, different species and even strains of laboratory animals, and different assay conditions all combine to make it very difficult, if not impossible, to interpret the data and draw valid conclusions that can be generalized across studies. Over the past several years, our laboratory has been involved in conducting systematic studies of the actions of polyamine toxins on the mammalian CNS. It was hoped that such a study would provide us with results which

2. PHARMACOLOGY

TABLE 11 PHARMACOLOGICAL EFFECTSOF POLYAMINE TOXINSI N Toxin JSTX (purified) JSTX (purified)

Preparation CAI pyramidal cells in guinea pig hippocampus CAI pyramidal cells in rat hippocampus

botoxins (crude)

Chick cochlear nucleus

Arg-636 (purified)

Frog spinal cord motoneurons

Arg-636 (purified)

Isolated rat hippocampal neurons

A%-636 (synthetic)

CAI pyramidal cells in rat hippocampal slices

Arg-636 (synthetic)

Cultured rat cortical neurons

Arg-636 (synthetic)

Xenopus

Arg-636 (synthetic)

Xenopus

Arg-636 (synthetic) and Agel-489 (synthetic)

Isolated rat hippocampal neurons

oocytes injected with rat brain mRNA

oocytes injected with rat brain mRNA

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VERTEBRATE CNS

Features of blockade'

Ref.

Selective for QUIS and KA Only partially reversible Use-independent Voltage-dependent (relieved by depolarization) Irreversible Open-channel block Non-NMDA receptor blockade Use-independent Not open-channel block Selective for non-NMDA receptors Irreversible Non-NMDA receptor blockade Noncompetitive Voltage-dependent (relieved by depolarization) Poorly reversible Open-channel block Selective for non-NMDA receptors Reversible (I-hr washout) Selective for NMDA receptors Use-dependent Voltage-dependent (relieved by depolarization) Selective for NMDA receptors Use-dependent Voltage-dependent (relieved by depolarization) Poorly reversible Reversed by depolarization plus agonist Not selective for NMDA or non-NMDA Uncompetitive Use-dependent Voltage-independent Selective for NMDA Use-dependent Voltage-dependent (relieved by depolarization) Poorly reversible Reversed by depolarization plus agonist

66-49

86

74

75

76

77

79

80

81

82

(continues)

TABLE I1 (Continued) Toxin

Preparation

6-PhTX (synthetic)

Xenopus oocytes injected with rat brain mRNA Xenopus oocytes injected with rat brain mRNA

PhTX-343 (synthetic)

6-PhTX (purified?)

Rat brainstem neurons in vivo

6-PhTX (synthetic)

Rat hippocampal slices

PhTX-343 (synthetic)

Rat hippocampal slices

Agel-505 (synthetic)

Xenopus oocytes injected with rat brain mRNA

PhTX-343 (synthetic)

Xenopus oocytes injected with rat brain mRNA

Arg-636 (synthetic)

Xenopus oocytes injected with rat brain mRNA

Features of blockade" Selective for NMDA Readily reversible Low concentration of toxin: potentiates responses to NMDA, KA, nicotine,GABA Higher concentration of toxin: inhibits responses to NMDA and KA Poorly reversible Selective for non-NMDA Slowly reversible (30 min) Rate of recovery enhanced by increased rate of agonist administration Open-channel block Antagonism of AMPA receptor-mediated synaptic transmission at 650 pM Noncompetitive blockade Presynaptic component Poorly reversible Antagonism of NMDA receptor-mediated responses at 2 p M Irreversible Transient potentiation often seen Blockade of induction of LTP Highly potent Markedly voltage-dependent Readily reversible Open-channel block Selective for NMDA Equipotent against NMDA and KA Partly use-dependent Partly voltage-dependent Block of KA responses reversed by depolarization plus agonist Selective for NMDA Partly use-dependent Partly voltage-dependent Block of NMDA responses readily reversible

" GABA, y-aminobutyric acid; LTP, long-term potentiation.

Ref. 83

87

78

88,89

90,91

84

85

85

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PHARMACOLOGY OF POLYAMINE TOXINS

77

are more broadly applicable. Our findings are presented in the following several paragraphs. We have taken advantage of the fact that activation of the NMDA receptor-ionphore complex elicits a rise in intracellular calcium ([Ca2+Ii) in mammalian central neurons to develop a relatively high-throughput functional assay by which to assess the inhibitory activities of polyamine toxins (92). Briefly, primary cultures of rat cerebellar granule cells loaded with the fluorimetric indicator fura-2 were used to measure changes in [Ca2+Iielicited by NMDA and its co-agonist glycine. This assay provides an extremely sensitive and precise index of NMDA receptor activity. Increases in [Ca2+Iievoked by NMDA are dependent on the presence of glycine and are blocked by extracellular Mg2+or antagonists acting at the glutamate, glycine, or MK-801 binding sites (Fig. 2). Increases in [Ca2'Ii elicited by NMDA/glycine are readily distinguished from those resulting from depolarization by their refractoriness to inhibition by blockers of voltage-sensitive Ca2+channels. Increases in [Ca"], were elicited by the addition of NMDA/glycine (50 p M / I p M ) in the presence or absence of different concentrations of each test compound. The IC,, values were derived from 3- 10 separate experiments and the standard error level was less than 10% of the mean value for each compound. Our initial studies were carried out utilizing native polyamine toxins purified from whole venom; subsequently, these findings have been confirmed and extended using synthetic toxins. All of the polyamine toxins tested blocked increases in [Ca2++li in cerebellar granule cells elicited by NMDA/glycine (Fig. 3) (93,941. Certain polyamine toxins such Arg-636 or Arg-659 (argiotoxin-659) were nearly as potent as MK-801 (IC,, = 34 nM) (Fig. 4). Many of the polyamine toxins tested were more potent than competitive antagonists such as AP5 (IC,, = 15 p M ) . The inhibitory effects of the polyamine toxins were not overcome by increasing the concentrations of NMDA or glycine; no change was observed in the IC,, for either NMDA (Fig. 5 ) or glycine. The polyamine toxins are thus noncompetitive antagonists at the NMDA receptor-ionophore complex and act neither at the glutamate nor the glycine binding site. Measurements of [Ca?'], in cerebellar granule cells can also be used to monitor activation of KA or AMPA receptors (95). Although the increases in [Ca2'Ii evoked by these agonists are of lower magnitude than those evoked by NMDA/glycine, such responses can be used to assess the specificity of action of polyamine toxins on glutamate receptor subtypes. Comparative measurements of [Ca2+Iirevealed a clear distinction in the receptor selectivity of the polyamine toxins. Polyamine toxins within the two structural classes defined by Arg-636 and Agel-489 (from Agelenopsis

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ALAN L. MUELLER ET AL.

330 165

115 250 160

h

F

v

+

90

280

N

m

V

160 110

290 175 100

290 175 100

u

0

1

2

3

4

5

Time (min)

FIG.2. Differential inhibitory effects of various agents on cytosolic CaZtresponses elicited by NMDAIglycine. NMDA (50 p M ,filled arrowheads) and glycine ( I p M ,open arrowheads) were used to increase [Caz+Ii.Test substances were added as indicated to obtain final concentrations of the following: MgCI?, 1 mM; AP5, 300 p M ; CPP, 1 mM; MK-801, 100 nM; nifedipine, 5 pM. The traces shown are representative of the pattern seen in each of four or more trials using three or more different cell preparations.

aperta) were found to be more potent antagonists of responses evoked by NMDA (Fig. 6 and Table 111). Others, like JSTX-3 and 6-PhTX, were either nonselective or more potent antagonists of responses elicited by KA (100 puM) or AMPA (30 p M ) . To further evaluate the selective inhibitory actions of the polyamine toxins, their effects on synaptic transmission mediated by NMDA or AMPA receptors were assessed (94,96).Glutamate-mediatedtransmission

2.

79

PHARMACOLOGY OF POLYAMINE TOXINS

at synapses of Schaffer collateral fibers and CAI pyramidal cells was measured in slices of rat brain containing the hippocampus. This electrophysiological assay can readily distinguish synaptic transmission mediated by NMDA or AMPA receptors (97). Polyamine toxins like Arg-636 and Agel-489 were again found to exert preferential inhibitory effects on NMDA receptor-mediated responses (field EPSP recorded in nominally Mg2+-freebuffer containing 20 pM DNQX [6.7-dinitroquinoxaline-2,3dione]. an AMPA receptor antagonist), and depressed responses mediated by AMPA receptors (population spike recorded in normal buffer) only at much higher concentrations (Fig. 7 and Table IV). For example, Arg-636 had an IC5, for the NMDA receptor-mediated response of 20 pM, but an IC,, for the AMPA receptor-mediated response of 647 pM. These results show that polyamine toxins can selectively inhibit synaptic transmission mediated by NMDA receptors. Other naturally occurring pol yamine toxins present in the venom of Agelenopsis aperra (Agel 489. Agel 505, and others) likewise exert potent and selective inhibitory effects on NMDA receptor-mediated responses in the rat hippocampus (94). Patch-clamp electrophysiological studies on isolated hippocampal neurons from adult rat brain have provided additional insight into the mechanism of action of Arg-636 and Agel-489 (82).These studies revealed potent and selective inhibitory effects of these toxins on responses mediated by NMDA receptors; Arg-636 and Agel-489 blocked responses to NMDA at nanomolar concentrations without affecting the responses to KA. 15

Agel-505 (nM)

+ 0 214 105

[ NMDA

0

2

4

6

8

10

12

14

Time (min) FIG.3. Concentration-dependent inhibition of NMDA/glycine-induced increase in cytosolic Ca?' in cultured rat cerebellar granule neurons by Agel-505.

80

ALAN L. MUELLER ET A L .

0

4

-1 0

.

I

-9

.

,

-8

.

I

-7

.

I

-6

.

I

-5

.

1

-4

.

1

-3

Log I D w I (M) FIG.4. Concentration-dependent inhibition of NMDA/glycine-induced increases in cytosolic Ca2+in cultured rat cerebellar granule neurons by polyamine toxins, the competitive NMDA receptor antagonist APS, and the noncompetitive NMDA antagonist MK-801.

Moreover, it was found that the inhibitory effects of these compounds were use- and voltage-dependent. The inhibitory effects of these toxins were poorly reversible upon washing in normal buffer; reversibility was achieved, however, with depolarization in the presence of agonist. Taken together, these findings strongly suggest that these compounds are blocking the open channel and, by this action, behave as noncompetitive NMDA receptor antagonists. Similar results with Arg-636 were obtained independently using cultured cortical neurons by Priestley el al. (79). Radioligand binding studies carried out by independent investigators have lent support to our hypothesis that polyamine toxins like Arg-636, Agel-489 and Agel-505 have a unique site and mechanism of action at the NMDA receptor-ionophore complex. Reynolds (98) reported that Arg636 decreased [3H]MK-801 binding with an ICso value of approximately 3 pM.This inhibitory effect of Arg-636 was insensitive to the concentration of glutamate, glycine, or spermidine, precluding an action at the glutamate, glycine, or polyamine binding sites. While Arg-636 did not simply mimic the effect of Mg2+ on r3H1MK-801 binding, and alone had no effect on

2. PHARMACOLOGY

OF POLYAMINE TOXINS

81

the dissociation of ['H]MK-801. it reversed the actions of Mg?+ on the dissociation of [3HlMK-801 by decreasing the apparent potency of Mg?'. This complex interaction of Arg-636 with Mg2+led Reynolds to conclude that Arg-636 may bind to one of the Mg?+ sites on the NMDA receptorionophore complex. Working with different toxins, Williams (84) determined that Agel-489 and Agel-505 have biphasic stimulatory-inhibitory effects on [3H]MK-801binding, similar to the effects seen with spermine. The stimulatory effect of low concentrations of Agel-505 (3-30 p M ) is inhibited by polyamine site antagonists, suggesting an interaction at the positive allosteric polyamine binding site on the NMDA receptorionophore complex. Higher concentrations of Agel-489 and Agel-505 (>30 p M ) inhibited [3H]MK-801 binding. In contrast to the Agelenopsis polyamine toxins, Arg-636 (1-1000 p M ) was found by Williams to inhibit only [3H]MK-801binding; no stimulatory effect was observed. Using electrophysiological methods, Williams found that, in Xenopus oocytes ex= 13 nM) pressing rat brain NMDA receptors, Agel-505 potently and reversibly inhibited NMDA-induced inward currents; this blockade was markedly voltage-dependent, with no blockade observed at membrane

-6.5

Log NMDA (M)

-6

-5.5

-5

-4.5

-4

-3.5

-3

Log NMDA (M)

FIG.5 . Noncompetitive antagonism of NMDA-induced increases in [Ca?'], by Agel-489. Leji: Increasing concentrations of Agel-489 produced a downward shift in the concentration-response curve for NMDA. Right: The curves in the left panel were normalized and each one plotted according to its own maximum. Increasing concentrations of Agel-489 did not shift the concentration-response curve for NMDA rightward. indicative of noncompetitive antagonism.

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ALAN L. MUELLER ET AL.

-

-C- KA(n=3)

O i -8

NMDA(n=6) AMPA(n=3)

I

I

I

I

I

-7

-6

-5

-4

-3

Log Agel-489 (M) FIG. 6. NMDA selectivity of Agel-489. Increases in [Ca?'], (mean ? S.E.M.) elicited by NMDAlglycine (50 p M I I p M ) were more potently antagonized by Agel-489 than were responses elicited by KA (10 p M )or AMPA (30 pM).

potentials more positive than -30 mV. These electrophysiological results are consistent with an open-channel blocking mechanism. Agel-SO5 was reported to be highly selective for NMDA over KA. In attempting to synthesize the results of these binding and electrophysiological studies, it is noteworthy that the inhibitory effects of polyamine toxins in oocytes are observed at concentrations 4-5 orders of magnitude TABLE I11 COMPARATIVE INHIBITORY POTENCIES OF I N CULTURED RAT POLYAMINE TOXINS CEREBELLAR GRANULEN E U R O N S ~ Toxin

NMDA

Arg-636 Agel-489 Agel-505 6-PhTX JSTX-3

0.085 0.245 0.229

a

15 1.7

ICm values given in p M . Not tested.

KA

AMPA

10.7

8.5

20 1 20 3.3 0.8

38 14 ntb nt

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51

l.*1

"'1 0

wash

wash 10 pM JSTX-3

10

20

30

40

50

0

1 0 2 0 3 0 4 0 5 0

Time (min)

Time (min)

FIG.7. Antagonism of the NMDA receptor-mediated field EPSP and the AMPA receptormediated population spike by JSTX-3. Left: A 20-min superfusion with 10 p M JSTX-3 produced approximately a 50% suppression of the NMDA receptor-mediated field EPSP. Right: To achieve a similar suppression of the AMPA receptor-mediated population spike, 300 pM JSTX-3 was required. In both cases, complete recovery of the synaptic response was obtained during a 20-min washout period.

lower than those required for modulation of [3H]MK-801 binding. This is one of several indications leading us to suggest that the high-affinity, noncompetitive inhibitory effect of these toxins is the predominant factor in the physiological regulation of NMDA receptor-ionophore function. TABLE IV ANTAGONISM OF EAA RECEPTOR-MEDIATED SYNAPTIC TRANSMISSION I N RAT HIPPOCAMPAL SLICESBY POLYAMINE TOXINS' Toxin

NMDA EPSP

Arg-636 Arg-659 Agel-489 Agel-505 8-PhTX JSTX-3

20 24 35 19 I48 12 ~

lCs0 values given in pM.

AMPA population spike

647 612 -100

-100 -300 878

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In the aggregate, the results of these various studies are complementary, and together they identify the argiotoxins and Agelenopsis pol yamine toxins as compounds with potent and selective inhibitory activity on NMDA receptors in the mammalian CNS. Additionally, these toxins appear to target a unique site on the NMDA receptor-ionophore complex. Support for our findings and conclusions can be found in very recent publications that examine the ability of polyamine toxins to antagonize cloned glutamate receptors expressed in Xenopus oocytes (85,99-101). First, Arg-636 was found to antagonize NMDA receptors composed of NRl/NR2A, and NRl/NR2B subunits with IC,, values of 9 nM and 4.5 nM, respectively (99).The potency of Arg-636 for NRl/NR2C receptors was considerably less (IC,, = 460 nM). Arg-636 was also reported to antagonize certain subtypes of AMPA/KA receptors, specifically those with a high Ca2+permeability (GluRl, GluR3, and GluR4), with IC,, values ranging from 230 nM-430 nM (100). GluR2 receptors were not affected by Arg-636 at concentrations up to 10 p M . Second, JSTX-3 was found to antagonize GluRl and GluR3 receptors with high potency ( IC5, values of 40 nM and 30 nM,respectively), while having no effect on GluR2containing receptors at concentrations up to 1 p M (101). Unfortunately, these investigators did not determine the potency of JSTX-3 for antagonizing expressed NMDA receptors. Third, Arg-636 was confirmed to be selective for expressed NMDA receptors (IC,, values for inhibition of NRl and GluRl of 90 nM and 3.4 p M , respectively), while PhTX-343 was equipotent at non-NMDA receptors ( IC,, values for inhibition of NRl and GluRl of 2.19 p M and 2.8 p M , respectively) (85). These results using cloned and expressed glutamate receptors not only confirm the findings that certain polyamine toxins such as Arg-636 are potent and selective NMDA receptor antagonists, but also extend these findings to demonstrate that polyamine toxins are unique in antagonizing Ca2+-permeableAMPA/KA receptors as well, albeit at somewhat higher concentrations. Importantly, other noncompetitive NMDA antagonists such as Mg2+ or TCP, an analog of PCP, do not share the ability of polyamine toxins to antagonize Ca2+-permeable AMPA/KA receptors. Finally, results such as these provide a possible explanation for the myriad potencies and selectivities reported in the literature for the polyamine toxins at NMDA and non-NMDA receptors (Table 11); the presence or absence of specific glutamate receptor subunits in varying stoichiometric proportions in the diverse range of biological preparations utilized by different investigators most certainly is an important determinant for toxin potency, receptor selectivity, and pharmacological profile, such as the degree of voltage dependence in functional assays. We next investigated whether the unique biochemical and molecular profile of polyamine toxins might translate into a unique pharmacological

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effect on the induction of LTP (long-term potentiation) in rat hippocampus. A major role for NMDA receptors in memory and learning is indicated by cellular studies on LTP in the rat hippocampus. Long-term potentiation is a long-lasting increase in the magnitude of synaptic responses produced by brief, yet intense synaptic stimulation. Since the discovery of this phenomenon, it has become the preeminent cellular model of learning in the vertebrate brain (102). Transmission at synapses formed by Schaffer collaterals onto CAI pyramidal cells is mediated by NMDA and AMPA receptors. Following a brief tetanizing stimulus, the magnitude of the population spike (a measure of synaptic transmission) is greatly increased and remains so for hours. It has been shown that all known competitive and noncompetitive antagonists of NMDA receptors block the induction of LTP in the rat hippocampus, whereas antagonists of non-NMDA receptors are without effect (103). The effects of selected polyamine toxins and literature standards were examined for effects on the induction of LTP in slices of rat hippocampus (104,105). Slices of rat hippocampus were superfused for 30-60 minutes with a test compound before delivering a tetanizing stimulus consisting of three trains, separated by 500 msec, of 100 Hz for 1 second each. The response amplitude was monitored for an additional 15 minutes posttetanus. The tetanizing stimulus caused a mean 95% increase in the amplitude of the synaptic response. The induction of LTP was significantly blocked ( p < 0.05) by competitive (AP5, AP7) o r noncompetitive (MK801, ifenprodil) NMDA receptor antagonists (Fig. 8). Quite surprisingly, none of the polyamine toxins tested (Arg-636, Agel-489, Age1 505, JSTX3,6-PhTX) blocked the induction of LTP ( p > 0.05) (Fig. 8), even when used at high concentrations (100-300 p M ) that caused some inhibition of the control response. These results highlight yet another unique and important feature of polyamine toxins. Polyamine toxins are the first and only organic compounds that are selective and potent antagonists of the NMDA receptor and yet d o not block the induction of LTP. This probably reflects the novel mechanism and site of action of polyamine toxins and suggests that drugs which target the novel site on the NMDA receptor will similarly lack effects on LTP. As LTP is the primary cellular model for learning and memory in the mammalian CNS, it additionally suggests that such drugs may lack deleterious effects on cognitive performance. C. SUMMARY The bulk of the available evidence suggests that the polyamine toxins are noncompetitive open-channel antagonists of glutamate receptor function. The argiotoxins and Agelenopsis toxins appear to be more potent and

A L A N L . M U E L L E R E T AL.

86

-

control

t

TET O !

0

I

1

10

20

1

30

Time (min)

FIG.8. Lack of effect of the polyamine toxin Arg-636 on the induction of hippocampal LTP. The induction of tetanization-induced LTP was prevented by the noncompetitive NMDA receptor antagonist, MK-801 (30 pM),but not by Arg-636 (300 pM).

selective at antagonizing NMDA receptors than non-NMDA receptors, although Ca2+-permeableAMPA receptors constitute another high-affinity site of action. Other polyamine toxins, such as JSTX-3 and 6-PhTX, are both less potent and selective for glutamate receptor subtypes. Our findings, together with those of other investigators, suggest that these toxins identify a unique site on glutamate receptor-operated calcium channels. The novel site and mechanism of action of the polyamine toxins at vertebrate glutamate receptors distinguishes them from other known classes of glutamate antagonists. This provides the impetus for the continuing development of polyamine toxins as human therapeutic agents. This latter point will be expanded upon below.

IV. Structure-Activity Relationship Studies The vast majority of structure-activity relationship (SAR) studies on pol yamine toxins have been carried out on the philanthotoxins owing to their relative ease of synthesis. Anis et af. (106) examined more than 35

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analogs of PhTX-343 on rat brain NMDA receptors ([3H]MK-80I binding) and Torpedo nicotinic receptors ([-'H]H,,-HTXbinding). 6-PhTX and the synthetic analog PhTX-343 were equipotent on NMDA receptors ( IC,, values approximately 50 p M ) , and 6-PhTX was 2.5 times more potent than PhTX-343 on nicotinic receptors (lC5(, values of 1.09 p M and 2.60 p M , respectively). In general, increasing the lipophilicity or the polyamine chain length led to an increase in potency at both receptors. The tyrosine moiety appeared to be critical for activity at NMDA receptors. Overall. these philanthotoxin analogs were more potent, and showed a greater divergence of activity, at nicotinic receptors, with potencies ranging from 0.14 pM to 150 p M , than at NMDA receptors. with potencies ranging from 4.7 p M to >I00 pM. More than 50 analogs of PhTX-343 were tested on the locust QUIS-R by Bruce et a / . (107). Again, the naturally occurring toxin. 6-PhTX, was slightly more potent than its synthetic analog, PhTX-343. presumably owing to the different spacing of the positive charges along the polyamine chain. As is true for nicotinic receptors and NMDA receptors, an increase in potency was produced by increasing either the hydrophobicity of the tyrosyl and butyryl moieties or the number of protonated groups in the polyamine chain. A positive charge at the end of the polyamine chain was important, and replacement of the terminal amine with a guanidino group enhanced potency. These investigators postulated that the more hydrophobic aromatic group might be involved in anchoring the toxin in a hydrophobic pocket of the receptor to support the binding of the protonated polyamine chain to the channel wall. Structure-activity relationship studies of the activity of philanthotoxin analogs on glutamatergic transmission in housefly larvae have been carried out by Piek's laboratory (108). Again. the polyamine chain appeared to be critical in terms of potency: the length and/or the numbyr of positive charges was most important, while the precise position of the positive charges was not. The presence of an aromatic moiety also was required for high potency. A number of synthetic analogs of JSTX-3 have been synthesized and tested on the glutamate receptor of lobster muscle (109). A reduction in potency was observed when the asparagine residue was removed or replaced and when the aromatic residue was replaced with saturated moieties. Piek's laboratory tested synthetic PhTX analogs on glutamatergic transmission at the housefly neuromuscular junction (110). Again, the polyamine chain was the essential and most critical part of the molecule for determining potency. The activity present in certain analogs led Piek to conclude that hydrophobicity in the region of the aromatic headpiece was not as important a determinant of potency as was the volume of the

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moiety. A second finding to come out of Piek’s studies is SAR data with regard to the differentiation of the presynaptic and postsynaptic effects of philanthotoxins on glutamatergic transmission (88,89,108,1 1 I ) . Methyl6-PhTX and methyl-PhTX-343, in which the hydroxyl group on the tyrosine was replaced by a methyl group, possessed only postsynaptic blocking activity. On the other hand, dideaza-PhTX-12, in which the polyamine chain was replaced with a straight carbon chain possessing a terminal amine, blocked the actions of glutamate almost entirely by presynaptic mechanism involving inhibition of glutamate uptake. This compound also was the most active analog tested at inhibiting AMPA receptor-mediated synaptic transmission in rat hippocampal slices, again presumably due to inhibition of glutamate uptake and eventual desensitization of the postsynaptic glutamate receptors. Unfortunately, these researchers did not test the effects of these philanthotoxin analogs specifically on NMDA receptormediated synaptic transmission. Blagbrough and Usherwood have synthesized a number of hybrid polyamine toxin analogs by combining chemical moieties from the native toxins Arg-636 and $-PhTX, and have tested these analogs for potency at blocking the locust muscle QUIS-R (112). The most potent analogs, N-(4-hydroxyphenylpropanoyl)spermine ( IC5, = 6.0 p M ) and N (4-hydroxyphenylacetyl)spermine(IC,, = 8.7 p M ) , were slightly more potent than PhTX-343 (IC5, 10 p M ) , but less potent than Arg-636 (IC5, - 1 pM).On the basis of these and other findings, tentative models for the binding of polyamine toxins to the QUIS-R have been developed. One such model exhibits either “three- or four-point’’ binding in which the positively charged nitrogens in the polyamine chain interact with negatively charged sites along the channel wall. According to this model, Arg636, with four positive charges, is a more potent antagonist than 6-PhTX, with only three positive charges. These investigators concluded that the L-asparagine and L-arginine residues of Arg-636 were not essential for activity, but were of importance in terms of the increased potency observed. These studies were extended by Nakanishi and his colleagues (1131, who demonstrated that Arg-636 was 7-fold more potent than PhTX343 at blocking the insect QUIS-R. Furthermore, increasing the hydrophobicity or bulk in the aromatic headpiece again led to enhanced potency. The most recent SAR study, in which more than 100 PhTX-343 analogs were prepared and tested for activity against the locust QUIS-R, the vertebrate NMDA receptor, and the Torpedo nicotinic cholinergic receptor, has been published by Kalivretenos and Nakanishi (114). Of particular importance was the finding that the attachment of a hydrophobic n-butyl group to the hydrophilic polyamine chain led to an increase in potency at all receptors. This latter finding, coupled with the synthesis of radioactive,

-

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photolabile analogs of PhTX-343 and their activity at the Torpedo nicotinic receptor, has given rise to the model in which the aromatic headpiece of the polyamine toxin binds to the cytoplasmic surface of the nicotinic receptor while the pol yamine tail inserts itself into the open channel from the cytoplasmic side (115). This model incorporates not only the use dependence of polyamine toxin activity (i.e., the channel must be open for the polyamine toxin to permeate to its cytoplasmic binding site), but the intracellular site of action, as originally proposed by Usherwood and his colleagues more than 10 years ago (42). How well this model of polyamine toxin interaction with the Torpedo nicotinic receptor generalizes to glutamate receptors, and to vertebrate NMDA receptors in particular, remains to be determined. Overall, the following general conclusions can be drawn with regard to activity of polyamine toxins and analogs at glutamate receptors. First, an aromatic headpiece is required, and increasing the bulk or hydrophobicity of this part of the molecule enhances activity. Second, a polyamine chain with at least three sites for protonation, one of which is at the end of the polyamine chain, is also required; lengthening the chain increases potency. Third, the a-amino acids within or at the end of the polyamine chain are not required, but do appear to enhance potency. Unfortunately, these published studies have done little to provide relevant data as to the SAR around vertebrate NMDA receptors. First, analogs of PhTX-343 or JSTX-3 do not appear to be the best compounds with which to obtain such data. There does not appear to be much structural stringency around PhTX-343 as its analogs are relatively impotent at NMDA receptors compared to both the insect QUIS-R and nicotinic acetylcholine receptors. Furthermore, modification of the PhTX-343 molecule leads to only minor differences in potency at NMDA receptors. Second, we suggest that the [3H]MK-801binding assay is inappropriate for demonstrating NMDA antagonist activity. We and others have shown that substantially higher concentrations of polyamine toxins are required for activity in this assay relative to other functional assays of NMDA receptor function (e.g., fura-2 studies in cultured rat cerebellar granule neurons and patch-clamp studies in rat hippocampal neurons or in oocytes expressing rat NMDA receptors) (84,85,94,99-/0/).Taken together, these concerns have led us to conclude that appropriate SAR studies of polyamine toxin potency on vertebrate NMDA receptors remain to be performed. Our results, as presented in the previous section, would strongly suggest that such studies include the argiotoxins and the Agelenopsis toxins, both classes of which are much more potent at NMDA receptors than at nicotinic acetylcholine receptors and the insect QUIS-R. Additionally, such studies should specifically examine vertebrate NMDA receptor function

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in assays where a wide range of potencies exist, rather than in the [3H]MK801 binding assay. V. Perspectives We conclude with a discussion of what the future may hold for polyamine toxins. These compounds certainly are fascinating from an academic point of view; they possess a complex and very interesting biological profile, and, while a few large SAR studies have been undertaken, the chemistry suring polyamine toxins is an area ripe for further study. One possible utility for polyamine toxins is as leads for the development of environmentally safe biopesticides (112,116-118). Progress in this area is severely hampered, however, by the fact that at present these toxins must be injected into the insect (i.e., they do not possess contact activity) and, additionally, that the flaccid paralysis produced is reversible (119,120; R. Kral, NPS Pharmaceuticals, unpublished observations). A more promising application is in the realm of human therapeutics. N-methyl-D-aspartate receptors have been implicated in a variety of CNS disorders, including epilepsy, ischemic cell death, neurodegeneration, analgesia, and anxiety. While the investigation of glutamate-mediated events is currently one of the most intensely studied areas in CNS research, it must be remembered that this field is still in its infancy. Despite the controversy surrounding the exact details of the site and mechanism of action of the polyamine toxins, there is little doubt that these compounds are effective glutamate antagonists in in vitro systems. Importantly, we have demonstrated that such compounds are effective following systemic administration as anticonvulsants and neuroprotectants in several relevant animal models of epilepsy and ischemic stroke (121). However, modem medicinal chemistry must continue to play a leading role in this process of drug development. The polyamine toxins are complex, highly charged molecules. The challenge will be to exploit these lead compounds in the development of viable drug candidates, whether by the use of chemistry to synthesize analogs with the desirable properties (e.g., enhanced permeability across the blood-brain barrier) or by developing a high-throughput assay to identify small organic molecules with activity at the novel polyamine toxin binding site on the NMDA receptor-ionophore complex. In any case, we believe that the unique biological profile of the polyamine toxins at the vertebrate NMDA receptor and the profound medical need for clinically useful NMDA antagonists provide strong motivation for pursuit of this line of drug development.

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67. N. Akaike, N. Kawai, N. I. Kiskin, E. M. Kljuchko, 0. A. Krishtal, and A. Ya. Tsyndrenko, Neurosci. Lett. 79, 326 (1987). 68. M.Saito, Y. Sahara, A. Miwa, K. Shimazaki, T. Nakajima, and N. Kawai, Brain Res. 481, 16 (1989). 69. Y.Sahara, H. P. C. Robinson, A. Miwa, and N. Kawai, Neurosci. Res. 10,200 (1991). 70. H. Pan-Hou and Y. Suda, Brain Res. 418, 198 (1987). 71. H. Pan-Hou, Y. Suda, M. Sumi, M. Yoshioka, and N. Kawai, Neurosci. Lett. 81, 199 ( 1987). 72. T.Himi, H. Saito, N. Kawai, and T. Nakajima, J . Neural Transm. 80, 95 (1990). 73. K. Shimazaki, H. P. C. Robinson, T. Nakajima, N. Kawai, and T. Takenawa. Mol. Brain Res. 13, 331 (1992). 74. H.Jackson, M. R. Umes, W. R. Gray, and T. N. Parks, SOC. Neurosci. Absrr. 11, 107 (1985). 75. S. M. Antonov, E. V. Grishin, L. G. Magazanik, 0. V. Shupliakov, N. P. Vesselkin, and T. M. Volkova, Neurosci. Lett. 83, 179 (1987). 76. N. I. Kiskin, 0. A. Kryshtal, A. Ya. Tsyndrenko, T. M. Volkova, and E. V. Grishin, Neurophysiology 21, 748 (1989). 77. J. H. Ashe, C. L. Cox, and M. E. Adams, Brain Res. 480, 234 (1989). 78. M. G. Jones, N. A. Anis, and D. Lodge, Br. J . Pharmacol. 101,968 (1990). 79. T.Priestley. G. N. Woodruff, and J. A. Kemp, Br. J. Pharmacol. 97, 1315 (1989). 80. A. Draguhn, W. Jahn, and V. Witzemann, Neurosci. Lett. l32, 187 (1991). 81. M. S. Davies, M. P. Baganoff, E. V. Grishin, T. H. Lanthom, T. M. Volkova, G. B. Watson, and R. G. Wiegand, Eur. J. Pharmacol.: Mol. Pharmacol. Sect. 227,51(1992). 82. N. I. Kiskin, I. V. Chizhmakov. A. Ya. Tsyndrenko, A. L . Mueller, H. Jackson, and 0. A. Krishtal, Neuroscience 51, I I (1992). 83. D. Ragsdale, D. B. Gant, N. A. Anis, A. T. Eldefrawi, M. E. Eldefrawi, K. Konno, and R. Miledi, J . Pharmacol. Exp. Ther. 251, 156 (1989). 84. K. Williams, J. Pharmacol. Exp. Ther. 266, 231 (1993). 85. P. T. H. Brackley, D. R. Bell, S.-K. Choi, K. Nakanishi, and P. N. R. Usherwood, J . Pharmacol. Exp. Ther. 266, 1573 (1993). 86. N. 1. Kiskin, E. M. Klyuchko, 0. A. Kryshtal, A. Ya. Tsyndrenko, N. Akaike, and N. Kawai, Neurophysiology 21, 152 (1989). 87. P. Brackley, R. Goodnow, Jr., K. Nakanishi, H. L. Sudan, and P. N. R. Usherwood. Neurosci. Lett. 114, 51 (1990). 88. N. C. M. Schluter, J. Van Weeren-Kramer, T. Piek, and J. Van Marle. Comp. Biochem. Physiol. C lOlC, 35 (1992). 89. N. C. M. Schluter, T. Piek, and F. H. Lopes d a Silva, Comp. Biochem. Physiol. C lOlC, 41 (1992). 90. N. B. Fedorov, V. G. Screbitsky, and K. G. Reymann. Eur. J. Pharrnacol.: Enuiron. Toxicol. Pharmacol. Sect. 228,201 (1992). 91. H . Matthies, Jr., P. T. H. Brackley, P. N. R. Usherwood, and K. G. Reymann, NeuroReport 3, 649 (1992). 92. T. N. Parks, L. D. Artman, N. Alasti. and E. F. Nemeth. Brain Res. 552, 13 (1991). 93. T. N. Parks, R. A. Volkmann, N. A. Saccomano, L. D. Artman, and E. F. Nemeth, SOC.Neurosci. Abstr. 15, I169 (1989). 94. T. N. Parks, A. L. Mueller, L. D. Artman, B. C. Albensi, E. F. Nemeth, H. Jackon, V. J. Jasys, N. A. Saccomano, and R. A. Volkmann, J . Biol. Chem. 266,21523 (1991). 95. M. J. Courtney, J. J. Lambert, and D. G. Nicholls, J. Neurosci. 10, 3873 (1990). %. A. L. Mueller, B. C. Albensi, A. H. Ganong, L. S. Reynolds, and H. Jackson, Synapse 9,244 (1991).

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97. J. F. Blake, M. W. Brown, and G. L. Collingridge, Neurosci. Leu. 89, 182 (1988). 98. 1. J. Reynolds, Br. J. Pharmacol. 103, 1373 (1991). 99. M. Raditsch, J. P. Ruppersberg, T. Kuner, W. Gunther, R. Schoepfer, P. H. Seeburg, W. Jahn, and V. Witzemann, FEES Lerr. 324, 63 (1993). 100. S. Herlitze. M. Raditsch, J. P. Ruppersberg. W. Jahn, H. Monyer. R. Schoepfer, and V. Witzemann, Neuron 10, 1131 (1993). 101. M. Blaschke, B. U. Keller, R. Rivosecchi, M. Hollmann, S. Heinemann, and A. Konnerth, Proc. Narl. Acad. Sci. U . S . A . 90, 6528 (1993). 102. T. V. P. Bliss and G. L. Collingridge, Nature (London)361, 31 (1993). 103. G . L. Collingridge and S. N. Davies, in “The NMDA Receptor” ( J . C. Watkins and G. L. Collingridge. eds.), p. 123. IRL Press, Oxford, 1989. 104. A. L. Mueller, L. D. Artman. B. C. Albensi, E. F. Nemeth, and H. Jackson, “Excitatory Amino Acids: Basic Science and Therapeutic Implications.” IBC Technical Services, London, 1991. 105. E. F. Nemeth, A. L. Mueller, L. D. Artman, and H. Jackson, in “Neuroreceptors, Ion Channels and the Brain’’ (N. Kawai. T. Nakajima, and E. Barnard, eds.), p. 21. Elsevier, New York. 1992. 106. N. Anis, S. Sherby. R. Goodnow, Jr., M. Niwa, K. Konno, T. Kallimopoulos, R. Bukownik, K. Nakanishi, P. Usherwood, A. Eldefrawi. and M. Eldefrawi. J. Pharmacol. Exp. Ther. 254, 764 (1990). 107. M. Bruce, R. Bukownik, A. T. Eldefrawi. M. E. Eldefrawi, R. Goodnow, Jr.. T. Kallimopoulos, K. Konno, K. Nakanishi. M. Niwa, and P. N. R. Usherwood, Toxicon 28, 1333 (1990). 108. T. Piek, N. C. M. Schluter, and H. Karst, Pol. J. Pharmacol. Pharm. 42, 573 (1990). 109. T. Asami, H. Kagechika, Y. Hashimoto. K. Shudo, A. Miwa. N. Kawai, and T. Nakajima, Biomed. Res. 10, 185 (1989). 110. J . A. Benson, F. Schurmann. L. Kaufmann, L. Gsell, and T. Piek, Comp. Biochem. Physiol. C lOZC, 267 (1992). I 1 1 . H . Karst, T. Piek, and J. Van Marle, Neurosci. Res. Commun. 7 , 69 (1990). 112. I . S. Blagbrough. P. T . H. Brackley, M. Bruce, B. W. Bycroft, A. J. Mather, S. Millington, H. L. Sudan, and P. N. R. Usherwood, Toxicon 30, 303 (1992). 113. S.-K. Choi. K. Nakanishi, and P. N. R. Usherwood, Tetrahedron 49, 5777 (1993). 114. A. G. Kalivretenos and K. Nakanishi, J. Org. Chem. 58, 6596 (1993). 115. K. Nakanishi, S.-K. Choi, D. Hwang, K. Lerro, M. Orlando, A. G. Kalivretenos, A. Eldefrawi, M. Eldefrawi, and P. N. R. Usherwood, Pure Appl. Chem., 66,671 (1994). 116. D. Quicke, New Sci.. Nov. 26, p. 38 (1988). 117. W. Nentwig, Toxicon 31, 233 (1993). 118. T. Piek, Toxicon 31, 235 (1993). 119. M. E. Adams, R. L. Carney, F. E. Enderlin. E. T. Fu, M. A. Jarema. J. P. Li, C. A. Miller, D. A. Schooley, M. J . Shapiro. and V. J. Venema, Biochem. Biophys. Res. Commun. 148, 678 (1987). 120. W. S. Skinner, M. E. Adams, G. B. Quistad, H. Kataoka. B. J. Cesarin, F. E. Enderlin, and D. A. Schooley, J. Biol. Chem. 264, 2150 (1989). 121. A. Mueller, A m . Chem. Soc. Abstr., San Diego. Part I, Medi I50 (1994).

-CHAPTER 3-

EPIBATIDINE CSABASZANTAY ,* ZSUZSANNAKARDOS-BALOGH,* A N D CSABA SZANTAY, JR.?

*Central Research Institute .for Chemistty of the Hungarian Academy of Sciences H-I525 Budapest, Hungary and fChemical Works of Gedeon Richter, Spectroscopic Department H-1475 Budapest, Hungary

I. Introduction ........... 11. Occurrence .........................................................................................

H I . Structure and Syntheses A. Diels-Alder React B. Intramolecular Nu IV. NMR Spectroscopy ...... V. Pharmacology ......... Addendum ........................................................................................ References

96

123 I24

I. Introduction

In a recently published chapter in this series Daly et al. (I) discussed the amphibian alkaloids but did not deal in depth with their syntheses. Among those compounds, a base isolated from the Ecuadorian poison frog Epipedobates tricolor was mentioned as representing a new class of amphibian alkaloids. The name epibaridine has been coined for this natural product, which has a novel, chlorine-containing structure. The rather exciting biological properties of epibatidine (see below), combined with its unique structure, has aroused the interest of synthetic chemists. Since less than 0.5 mg of alkaloid was isolated from the skin extracts of 750 frogs, further biological studies required synthetic epibatidine. This need triggered an unprecedented competition among laboratories around the world, and in a relatively short time several synthetic approaches to the target compound were reported. The aim of this chapter is to offer an overview of the published reports in order to give a menu to the chemists interested in this new area. 95 THE ALKALOIDS. VOL. 46 Copynght 0 199s by Academic Press. Inc. All rights of reproduction in any form reserved.

96

CSABA

SZANTAY

ET AL.

11. Occurrence

Epibatidine has so far been isolated only from the skin extract of an Ecuadorian poison frog of the family Dendrobatidae (2).

111. Structure and Syntheses

The structure elucidation revealed that epibatidine (1)has a 7-azabicyclo[2.2. llheptane ring system containing a chloropyridyl substituent. The three asymmetric centers in this molecule should provide, as a first approximation, eight stereoisomers. However, the strained nitrogen bridge allows for only four stereoisomers. The chloropyridyl substituent can be attached to the 7-azabicyclo[2.2.llheptane moiety either in the pseudo-equatorial or pseudo-axial position; these isomers, together with their respective enantiomeric forms, are denoted as the exo isomer (1) and endo isomer (17,respectively. The natural alkaloid contains the substituent in the pseudo-equatorial position (1)(Scheme 1). Because of the small quantities of the isolated material, its absolute stereochemistry was investigated subsequently ( 3 , 4 ) .The optical rotations of the base and the salts are opposite in sign (see Section 111,B,4) (3-5). The endo isomer l',which contains the bulky substituent in the pseudoaxial position, is thermodynamically less stable; for this reason it is expected to be equilibrated to the thermodynamically more stable natural product 1. Two different strategies were used for building up the target molecule. The 7-azabicyclo[2.2.llheptane ring system was synthesized (1) in a Diels- Alder reaction on treatment of an activated pyrrole with an electronpoor acetylene derivative and (2) by an intramolecular nucleophilic substitution reaction. The synthetic approaches will be discussed according to reaction type, and not in chronological order of appearance. First the retrosynthetic analyses, then the detailed descriptions of the syntheses will be given. A. DIELS-ALDER REACTIONS The Diels-Alder reaction seems to be the most convergent method to construct the desired 7-azabicyclo[2.2. llheptane ring system. It is well known, however, that even activated pyrroles with reduced aromaticity

3.

97

EPlBATlDlNE

exo-Epibatidine

errdo-Epibatidine

1

1'

SCHEME I

of the pyrrole ring readily undergo substitution reactions with electrophilic olefins (6). To obtain the Diels-Alder adduct 4, the activated pyrrole 2 has to be reacted with electron-poor acetylenes 3 (7,8) (Scheme 2). In the synthesis of Huang and Shen (9) the Diels-Alder reaction was carried out with electron-poor acetylene containing the chloropyridyl moiety (3, R = chloropyridyl), while Clayton and Regan (10)first constructed the N-protected 7-azabicyclo[2.2.llheptene (4, R = H), then introduced the substituent by a palladium-catalyzed coupling. Two other publications describe the use of the Diels-Alder reaction for constructing the substituted ring system; however, epibatidine itself was not synthesized via these routes, but rather its analogs. Scheeren et al. (11) applied high pressure in the course of the Diels-Alder reaction in

1

5

'E 2

4

E and R' = electron withdrawinggroups

SCHEME 2

3

R = chloropyridyl group or H

98

CSABA SZANTAY ET A L .

order to avoid substitution on treatment of the N-protected pyrrole (6) with phenyl vinyl sulfone (7). Harman et al. (12) have shown that the aromaticity in the osmium (111) complex of the pyrrole derivative 8 is disrupted, and therefore it easily undergoes the addition reaction with methyl pyridylacrylate (9).

C02Me

+

I-

6 % Na-Hg , MeOH

80-85 OC, 24 h

- 2OOC. rt, 3h

(50-70 %)

(36-42%)

702

Ph

13/13'

14/14'

(28 4 %)

(25 Yo)

I (-)-I

[aID = - 5 2 0

12

I1

10

+

resolution with di-p-toluoyl tartaric acid

(+)-I

[a],= + 5.4 0

SCHEME 3

w

3.

EPlBATlDINE

99

1. Huang and Shen

Huang and Shen (9) reacted the N-carbomethoxy pyrrole (10) and phenylsulfonyl 6-chloro-3-pyridyl acetylene (11). The acetylene component was synthesized from the dilithiated phenyl methyl sulfone and nicotinic acid chloride in a three-step procedure. Adduct 12, whose structure was confirmed by X-ray crystallographic analysis, underwent reductive desulfonation supplying the 1 : 2 mixture of the exo (13) and endo (13') isomers of N-carbomethoxy dehydroepibatidine. The remaining double bond was saturated by quick catalytic hydrogenation, which gave again the 1 : 2 mixture of the protected exo (14) and endo (14') isomers of epibatidine. The protecting group was removed with hydrobromic acid in acetic acid, and the racemic exo (1) and endo (1') isomers of epibatidine were separated by silica gel chromatography. Resolution of the exo isomer 1 was also performed via the di-p-toluoyl tartaric acid salts furnishing the levorotatory, [(Y]D - 5.2", and dextrorotatory, [a]D + 5.4" (in chloroform), epibatidine enantiomers (Scheme 3). Diels-Alder reactions were also carried out between phenylsulfonyl phenyl acetylene and N-carbomethoxy pyrrole (10) giving adduct 15, as and phenylsulfonyl well as between N-carbomethoxy-2,5-dimethylpyrrole 6-chloro-3-pyridyl acetylene (11) yielding adduct 16 (Scheme 4).

2 . Clayton and Regan Clayton and Regan (10) built up the N-methoxycarbonyl 7-azabicyclo[2.2. llheptene ring system (17) by the known method of Altenbach et al. (7)(Scheme 5 ) . In this case, the Diels-Alder reaction was performed between the N-carbomethoxy pyrrole (10) and p-toluenesulfonylacetylene (18). Adduct 19 underwent selective catalytic hydrogenation to 20, then the p-toluenesulfonyl group was reductively cleaved using 6% sodium

C02Me

I

SCHEME 4

I00

CSABA

SZANTAY

ET AL. COzMe I

1s

h 18

10

19

C02Me

A

(YY 7 0 ,

C02Me

vL

6% Na-Hg

MeOH, 'IHF

Ts Na2HPO4, NaHzP04

20

-

-78 OC rt (30-40 %)

17

SCHEME 5

amalgam, yielding the N-protected 7-azabicyclo[2.2. llheptene (17),which is required for the crucial coupling step. The other component necessary for the coupling was the 2-chloro-5iodopyridine (21).This compound was prepared from 2-aminopyridine (22)through iodination (23)and subsequent diazotization in concentrated hydrochloric acid to afford 2-chloro-5-iodopyridine (21)(Scheme 6). The coupling reaction was catalyzed with bis(tripheny1phosphine)palladium( II)acetate, prepared in situ from palladium( 1I)acetate and triphenylphosphine; the reaction proceeded stereoselectively and furnished the desired protected exo isomer of epibatidine (14).The protecting group was removed by Shen's method (9) using hydrobromic acid in acetic acid, giving epibatidine (1)(Scheme 7).

3. Scheeren et al. Scheeren et al. ( I f ) pointed out that at high pressure the activated pyrroles give Diels-Alder adducts with electron-poor olefins as well:

(53

22

Yo) 23

SCHEME 6

21

3.

101

EPIBATIDINE

(Ph3P)zPd(OAc)2, DMF, D

1J

Y

I

piperidine. HCOzH 70 OC, 6.5 h

(35 Yo) 17

21

14

HBr-AcOH

c22h

*

(74 %)

H 1

SCHEME 7

Namely, I-methoxycarbonyl-3-phenylthiopyrrole (6) reacted with phenyl vinyl sulfone (7) at a pressure of 12 kbar to yield adduct 24. The phenyl sulfonyl group was removed by reduction with sodium amalgam to give 25 and the tricyclic isomer 26. The phenyl substituent was introduced to the 7-azabicyclo[2.2. llheptene (25) via palladium-catalyzed coupling with bromobenzene producing 27. Raney nickel reduction of 27 gave the endo7-carbomethoxy-2-phenylazabicyclo[2.2. llheptane 28. The protecting group was removed with trimethylsilyl iodide, furnishing 29 as its HI salt (Scheme 8). 4. Harman et al.

Harman et al. (12, 13) decreased the aromaticity of the 23-dimethylpyrrole (30) by forming its osmium(111) complex. The osmium-coordinated pyrrole 8 reacts like an activated diene with methyl pyridylacrylate (91, giving the Diels-Alder adduct 31, which contains the epibatidine skeleton (Scheme 9).

B. INTRAMOLECULAR NUCLEOPHILIC SUBSTITUTION REACTIONS The 7-azabicyclo[2.2. llheptane ring system can also be constructed by an intramolecular nucleophilic substitution reaction. The trans position of the amino group or the protected amino group and the leaving group is the main prerequisite of ring closure. If all substituents of the cyclohexane ring 34 are in equatorial positions, the ring closure leads to the formation of the exo isomer of epibatidine (1) (Scheme 10).

102

CSABA SZANTAY E T AL.

C0,Me

Phs

C

+

N -CO,Me

iH2

12 kbar *

A

(80 Yo)

I SO, Ph

(30 %)

24

C02Me I

C02Me I

25

I

6%Na-llg

S02Ph

PhS

I

6

H

CH

26

Pd(OAc)2, PPh3 PhBr, T M D A (35-40 Yo)

I ,H

H\

1 TMSI

-%

2 MeOH.CHZClZ (65 Yo)

Phs 21

28

29

SCHEME 8

30

8

H

31

SCHEME 9

e

9

3.

103

EPIBATIDINE R'

1

34

33

R' = H or protectinggroup R"= H o r O R = chlolpyridyl group or H or 0 -

x = leavlng group

R' = H or protectinggroup R = chloropyridyl or pyridyl group or H

SCHEME 10

In the synthetic approaches of Broka (14) and Corey et al. (31, the aminocyclohexane precursor already contained the chloropyridyl substituent (34,R = chloropyridyl) before the ring closure. Daly et al. (15) synthesized a pyridyl-containing N-protected ring system (34, R = Py), then performed a radical chlorination to obtain epibatidine (1).In the synthesis of Fletcher et al. (16) the chloropyridyl substituent was introduced into the protected 7-azabicyclo[2.2. Ilheptan-2-one (33, R, R = 0)via condensation with 5-lithio-2-chloropyridine (79). The 7-azabicyclo[2.2.l]heptane can be also built up if the amino and the leaving groups are interchanged in the cyclohexane 35, but in this case the reaction leads to the formation of the endo isomer of epibatidine (1') which can be equilibrated to the natural product (Scheme 11). This principle was used in the synthesis of Szhntay et al. (17). There is one other synthesis for epibatidine (1)performed by Speckamp et al. They started from succinimide, but their results have not been published yet (18).

x = lea\lng group R = chloropyndyl g o u p

SCHEME 11

104

CSABA

SZANTAY

ET A L .

1 . Broka

Broka (14) performed the first synthesis of epibatidine (1)starting from 6-chloronicotinaldehyde (36), which was vinylogated with (triphenylphosphorany1idene)acetaldehyde to supply the enal37. Diels-Alder reaction of 37 with 2-(trimethylsilyloxy)-1,3-butadiene followed by a dilute acidic treatment gave ketoaldehyde 38 as a single stereoisomer. After

(P~)JP=CH-CHO CI

rCH ].neat, 15OOC. 1 0 h

2. dil. HCI H70, - IHF, MeOH

CI 36

31

(75 %)

1. TsCL pyridine

20 OC. 2 h c 2. PhSK. DMF. THF 20 OC, 30 min 3. (t-Bu)Ph2SiCL imidamle.DMF 20OC. 1 day (73 Yo)

dH0 HO 39

38

% qSiF'b(t-Bu)

QSiPh&Bu)

1. MCPBA, CH2Cb

20OC. 15 lr6n 2. 0.02 200 OC, M inxylem 2h

*

2. Pb(OAc)4 benzene CI

(86 Yo)

/

CI

PhS 40

1 . 0 ~ 0 4NMh4O , acetone, H20

41

OSiq(t-Bu) I . BXL pyridine

4-

2. TBAF, THF

(84 Yo)

CI 42

(63 % 60m 41 ) 43

SCHEME 12

3.

105

EPIBATIDINE

reduction of both carbonyl groups with L-selectride, the diol39 was transformed into the monoprotected diol43, thus eliminating one carbon atom. To remove the unwanted carbon atom, diol39 was reacted with 1.1 equivalents of tosylchloride, the tosyl substituent was replaced with thiophenyl, and the remaining hydroxy group was silylated with ferf-butyldiphenylsilyl chloride to yield 40. The thiophenyl substituent was oxidized with 3chloroperoxybenzoic acid, and the sulfoxide obtained was eliminated on heating in xylene at 200°C to generate the olefin 41. Cleavage of the methylene unit was followed by immediate borohydride reduction of 42, yielding the hydroxy group in an equatorial position, as in 43, along with its axial epimer in a ratio of 5 : 1 (Scheme 12). The monoprotected diol 43 was acylated with benzoyl chloride and desilylated with tetra-n-butylammoniumfluoride to give the monobenzoylated diol44. The unprotected hydroxy group was replaced with azide to

45

44

1 . 0 . 3 M NaOH in H2O. THF,MeOH 2OOC. 3.5 h 2. MsCI, NEt3, CHzCh OOC.45nin 3. SnCb, MeOH, THF 200C. I h (59 %)

- %

CHCI3

v

55 OC. 4 days (84 Yo)

MsO

CI

46

1

SCHEME 13

106

CSABA

SZANTAY

ET A L .

give 45 through the unisolated mesylate. The benzoyl substituent in 45 was hydrolyzed, the alcohol mesylated, and the azide reduced with tin(I1) chloride to afford the amino mesylate 46, which was refluxed in chloroform for 4 days to give epibatidine (1) (Scheme 13).

(CF-jCH20)2POCH2C02Me KHMDS, I S - c r o w 6 CH3CN complex, THF

CI

- 78 O C ,

36

1h

(89 %)

47 1 Et3N, (PhO)2PON3 Ioliunc, 85 OC

LiOH, I H F 23 OC, 5 h

C

(100%)

2 TMSCH2CH20H 85 OC, 12 h c1 (95%) 49

I 'IBAF,THF,55 OC, 4 h C

2. (CF3CO)zO

R3N. CH~CIZ. 23 OC, 30 m (80 %)

50

NHCOCF3 KOI-BU THFC -78 OC - 4 OC 18 h (75 %)

CI Bu-jSnH,AIBN

benzene, red.

Br

(95 %)

H

CI 23

[a], 23 + 3 1 2 0

[a], +200

52

Lflc'

53

H NaOMe MeOH, 23 OC, 2 h (96 %) H

23

[a], + 3 2 0

[a];' - 5 0

54

1

SCHEME 14

3.

107

EPIBATIDINE

2. Corey et al. Corey et al. (3) performed the first stereocontrolled synthesis of ( + )and ( - )-epibatidine (1).In their method, 6-chloropyridine-3-carboxaldehyde (36) was converted stereospecifically to the (Z)-a,@-unsaturated ester 47 using methyl bis(trifluoroethy1)phosphonoacetate. Thermal Diels-Alder addition of 1,3-butadiene to 47 at 190°C gave the cis ester 48 as a single adduct. Saponification of 48 with lithium hydroxide afforded quantitative yields of the corresponding carboxylic acid 49, which was transformed to the acyl azide. Curtius rearrangement of the azide in the presence of 2-(trimethylsily1)ethanol gave the cis carbamate 50. Treatment of 50 with tetra-n-butylammonium fluoride in THF at reflux resulted in carbamate cleavage to form the corresponding primary amine, which was acylated with trifluoroacetic anhydride to afford the cis trifluoroacetamide 51. This amide underwent stereospecific bromination at - 78°C with bromine in the presence of a bromide ion source to form a single dibromide 52. The formation of the 7-azabicyclo[2.2. llheptane ring system was performed by intramolecular nucleophilic displacement with potassium tertbutoxide, the monobromide 53 was debrominated with tri-n-butyltin hydride (54), which after deacylation gave (+)-epibatidine (1)(Scheme 14). Resolution was completed at the stage of N-(trifluoroacety1)epibatidine (54) using chiral HPLC. The levo and dextro enantiomers of 54 were hydrolyzed in the same way as the racemate to yield the dextro and lev0 enantiomers of epibatidine (l),[a]D2, + 5" and - 5" (c 0.35, CHCI,), respectively. The absolute configuration of ( - )-epibatidine was also determined as follows. The enantiomers of trifluoroacetamides 5 1 were separated by chiral HPLC, the dextrorotatory enantiomer of 5 1 was converted to the dextrorotatory dibromide 52 whose structure and absolute stereochemistry were established by single-crystal X-ray crystallographic determination. After cyclization [( +)-531 and debromination, (+)-54 proved to be identical to the dextro enantiomer obtained by the chiral HPLC method. Deacylation of ( + )-54 gave ( - )-epibatidine, [..IDz3- 5", whose absolute configuration is shown in Scheme 14.

% NHCOCF,

NBS. AcOH

OOC-23OC,I h (85%)

CI

CI

51

55

SCHEME I5

108

CSABA SZANTAY ET AL.

Corey's method is also suitable for the synthesis of azabicyclo[3.1.13heptane analogs of epibatidine. When treating the trifluoroacetamide 51 with N-bromosuccinimide in acetic acid, the trans bromonium ion was formed, and in the absence of bromide ion the amide nitrogen reacts as a nucleophile, furnishing the azabicyclo[3.1. llheptane derivative 55 (Scheme 15). Other derivatives from N-(trifluoroacety1)epibatidine(54) and its epimer have been synthesized for biological evaluation. These transformations are summarized in Scheme 16.

Lqc' L q l OCCF,

OCCF,

Nal, 120 OC, AcCI, 10 CH3CH2CN h

(84 %)

54

NaI, AcCl

56

, BqSnH, AIBN benzene, reflux

(95 %)

LqCH3

OC,CF,

OCCF,

58

57

LGx OCCF,

H

NaOMe

H

MeOH, 23 OC, 2 h H

q

:T

61

58 Me

SCHEME 16

Me

3.

109

EPIBATIDINE

3. Daly et al. Daly et al. (25) published the first patent claiming the synthesis of (+)-epibatidine and its derivatives. Their starting material was cyclohexan-l,2-dione (62)which was treated with trimethyl orthoformate in acidic medium to obtain 2-methoxycyclohex-2-enone (63).Grignard reaction of 63 with 3-pyridylmagnesium bromide furnished cyclohexenol 64, which was dehydrated with phosphorus oxychloride and hydrolyzed, subsequently, to give 65, followed by sodium borohydride-cerium chloride reduction to produce the allylic alcohol 66, which was dehydrated again with phosphorus oxychloride to give 3-pyridyl-2-cyclohexa-1,3-diene (67) (Scheme 17). 3-Pyridyl-2-cyclohexa-1,3-diene(67)was reacted with tert-butyl nitrosoformate (a), generated in situ from tert-butyloxycarbonylhydroxylamine and tetraethylammonium periodate. Two regioisomers of adduct 69 were formed, and both of them gave the same cyclized product 72. During catalytic hydrogenation, adduct 69 supplied amino alcohol 70, which was treated with thionyl chloride to give the chloro amide 71. Base-catalyzed cyclization of 71 provided the 7-azabicyclo[2.2.llheptane ring system 72. Free-radical chlorination supplied the desired chlorine substituent in 73, and compound 73 was hydrolyzed with trifluoroacetic acid in methylene chloride to give (+)-epibatidine (1)(Scheme 18).

HC(0MehRI: 63

62

P

CH3

1. POCl3 pyridine 2. di

N I

U 64

g 65

POCIJ,pyndine OH

OW

c

66

61

SCHEME 17

110

CSABA SZANTAY ET A L .

5 % Pd-C

MeOH 68

67

69

13

72

/

H 1

SCHEME 18

Daly and co-workers used not only Cirignard derivatives, but also alkyl lithium compounds to alkylate 2-methoxycyclohex-2-enone (63) to obtain other derivatives with the same 7-azabicyclo[2.2. llheptane ring system (Scheme 19). The N-acyl and N-alkyl derivatives of 75 are shown in Scheme 20.

3.

63

111

EPIBATIDINE

I5

74

R = phenyl (a orb) substibded phenyl (a orb) cyclohexyl (a) wpentyl (a) 3-pyridyl (a) 2-thienyl (a) 3-thienyl (a) 2 - f i J r . 4 @) v h w alkyl, cycbalkyl, aryl, heteroaryl, alkylaryl (a or b)

SCHEME 19

4. Fletcher et al.

Fletcher et al. ( 4 , 16) based their synthetic strategy on the condensation reaction of the protected 7-azabicyclo[2.2.l]heptan-2-one78 with 5-lithio2-chloropyridine (79). The protected 7-azabicyclo[2.2.l]heptan-2-onederivative was synthesized from N-trifluoroacetylaminocyclohex-3-ene(M), which was first benzylated. Treatment of the benzyl derivative 81 with m-chloroperbenzoic acid supplied a mixture of epoxides 82. This mixture could be separated, but more conveniently, was hydrolyzed without sepa-

AR H

76

15

X=CI OCOW

R' = nielliyl vinyl ally1 propard ti-pentyl cyclohexyl phenyl phenethyl

SCHEME 20

77

112

CSABA

SZANTAY

ET AL.

ration under mild conditions to give the amino epoxides 83. Cyclization of the isomeric mixture of 83 was performed in N-methylpyrrolidinone to give the 7-azabicyclo[2.2. Ilheptane ring system 84. The benzyl group was removed by catalytic hydrogenation using Pearlman's catalyst in hydrochloride acid-ethanol, then the amino group was converted into the N-Boc derivative 85. Alcohol 85 underwent Swern oxidation to give ketone 78 (Scheme 21). Ketone 78 was coupled with the 5-lithio derivative of 2-chloropyridine 79, obtained from 2-chloro-5-iodopyridine (86) with n-butyllithium. The resulting tertiary alcohol 87 was dehydrated in a three-step process furnishing the olefin 88. Hydrogenolysis of 88 in the presence of Adams' catalyst produced a 4 : I mixture of exo and endo isomers 89 and 90. The undesirable endo isomer 90 was epimerized to the exo isomer 89 in good yield. Deprotection of 89 with trifluoroacetic acid gave (2)-epibatidine (1) (Scheme 22). Resolution was carried out by the chiral HPLC method at the stage of the Boc derivative 89 to afford the (+)- and (-)-isomers, which were converted to ( + 1- and ( - )-epibatidine, and [a],, values were given for their

-0

mCPBA. CHzCI;! OOC-CI, 4 h (69 %)

BnBr. Cs2CO3, DMF 70 OC. 40 h (66 %)

BnNCOCF3

NHCOCF3

K2CO3, MeOH, H20

82

81

80

3 days (88 %)

BnNCOCF3

-6

Bn I

N-methylpymlidinone I80 OC. 16 h (61 %)

NHBn

83

1. H2. EtOH, 5 M HCI, Pd(O%/C 40 psi, 40 OC 2. (BochO, 1 M NaOH dioxane, rt, 18 h (79 %)

84

A;"

(COC1)2, Me2S0,

-CH2CIz,E13N 70 OC (89 %)

85

SCHEME 21

~

I8

3.

113

EPlBATIDlNE

nc'

BW

I

BOC

n-BuLi B20. THF

- 70 OC

I

(67 %)

86

78

1. KH, THF. 0 OC

- rt

87

'

flc' H2. EtOAc. PtOz

N

40 psi 110 OC (68%)

2. CS2, Me4 0 OC 3. toluene, 110 OC (73 %) 88

BW

I

89

HCI, W A C

90

KOf-Bu, f-BuOH 100 OC, 30 h (>SO %)

(loo %)

1

SCHEME 22

hydrochloride salts: ( + )-epibatidine-HCI [alD24 + 34.7"(c 0.36, methanol), (-)-epibatidineaHCI [a]D24 - 33.7" (c 0.16, methanol). Chemical resolution was also achieved at the stage of alcohol 85 by formation of a diastereomeric ester with (-)-Mosher's acid chloride 91. The diastereomers (92 and 93) were separated; then, after saponification

114

CSABA

SZANTAY

ET A L .

with potassium hydroxide in ethanol, both enantiomeric alcohols (94 and 95) were transformed to ( + )- and ( - )-epibatidine (1)following the reaction sequence described in Scheme 23. The products were identical to the materials obtained by preparative HPLC. BOC

I

H 85

CFJ'"

Ph 91

Boc

I

(42 %)

(43 Yo)

92

93

I

KOH, EtOH , 2 h (85 Yo)

KOH, EtoH, 2h (97 Yo)

BOi

BOC

HO H 94

95

(+>epbatidii hydrogen oxalate (+>1 . hydrogen oxalate

(-tepibatidine hydrogen oxalate (->I hydrogen oxalate

SCHEME23

3.

115

EPlBATlDlNE

The following [ a ] D values were given for their hydrogen oxalate salts and for the free bases: - 37.4" ( C

(-)-Epibatidine hydrogen oxalate Free base (+)-Epibatidine hydrogen oxalate Free base (natural product)

0.419, MeOH)

+ 6.5"(C 1-09 CHCI,) + 37.3" (C 0.442, MeOH)

[a]DZ3 [a]D24

- 6.7" (C 0.87, CHCIJ

Watt et al. (5) separated the ( + ) and (-)-enantiomen of the synthetic epibatidine hydrochlorides (1. HCI) and the synthetic N-acetylepibatidine (102) using Chiral-AGP and Chiralcel OD chiral stationary phases, respecBr2. MeOH, rt, Jh e O~N-(CH~)J-CO-CHJ 02N-(CH2h-CO-CH2-P(Ph)3 Ph3P. b q rt, 24 h Bre (50 %)

96

97

I . CH2CIZ'

NaoE

2.6-chloropyridine-

3 -carboxaldehyde CH2Ch. r e 5 . 8 h (58 %) 1. NaBHq, EtOH

O W , 1.5 h

CH%HCO-(CHZ)~-NOZ

2.CH3SO2Cl, CHzClz

CI

98

(59%)

CI (61 %)

99

toluene

m0.. 24 h

(80 %)

CI 100

101

H N

+ i KOI-Bh I-BuOH re5.30 h

I

(50 %)

1'

H

1

SCHEME 24

116

CSABA

SZANTAY

ET AL.

tively. Comparison of the synthetic material with natural epibatidineSHCI using retention times and UV spectra revealed that the hydrochloride salt of the natural product corresponds to the ( + )-isomer. 5 . Szantay et al.

Szantay et al. (17) worked out a practical synthesis according to which nitromethane was allowed to react with methyl vinyl ketone to give nitropentanone 96. After bromination and subsequent quaternization with triphenylphosphine, salt W was obtained. Wittig reaction of the appropriate phosphorane with chloropyridine aldehyde 36 gave rise to 98, and treatment with potassium fluoride/alumina furnished the cyclohexane derivative 99. Reduction of the keto group followed by mesylation (100) and subsequent reduction of the nitro group gave amine 101, which on heating resulted in the epimer of epibatidine (1').On boiling the latter compound in tert-butanol in the presence of potassium tert-butoxide, epimerization occurred, and racemic epibatidine 1was obtained (Scheme 24). The yield of the last step can be enhanced through Boc-protection of 1' and subsequent epimerization, as described previously (16). The aim of this synthesis was to create a practical route to epibatidine (1) on a large scale for biological investigations. The last synthesis uses commonly available starting materials, well known and well controllable chemical reactions; therefore, it should be suitable for producing the desired compound.

IV. N M R Spectroscopy Over the course of the work involving exo- and endo-epibatidine, several laboratories reported the NMR spectral features of these compounds in varying degrees of detail. The first and tentative assignment of the 500-MHz 'H-NMR spectrum for the natural compound 1 (recorded in D,O/DCI solution) was provided by Daly et al. (I). However, that article gave a detailed 'H-NMR analysis of N-acetylepibatidine (102),which allowed the stereochemical identification of epibatidine itself as the exo isomer. It was pointed out that, in accordance with previous theoretical estimates and experimental observations of suitable analogs, the coupling between the bridgehead and adjacent endo protons is characteristically small (typically on the order of 1 Hz), and in most cases these couplings are not resolved in the 'H spectra. However, vicinal couplings between the bridgehead and the respective exo protons are around 4-5 Hz. This difference in couplings formed the basis of identifying the exo stereoposi-

3.

117

EPIBATIDINE

tion of the chloropyridyl substituent in N-acetylepibatidine, and from this, the structure of 1could be directly deduced. Subsequently, 'H- and ',C-NMR data (CDCI,) for 1were provided by Broka (Z4), and for 1and 1' by Huang and Shen (9). The identity of the exo and endo isomers for the N-Boc analogs 89 and 90 (Scheme 22) was TABLE I 1 A N D 1' 'H A N D I3C NMR DATA"FOR COMPOUNDS I'

1

3.57 d J( 1,6e) == 1.7; J( I ,6") J( I ,2,) < I 2.77 d d J(2,,3,) = 9.0; J(2,,3& 5.1; J(1.2,) < I 1.92 dd J(2,,3,) = 9.0; J(3,,3,9) = 12.2; J(3,,4) < 1 I .48-1.70 rn (overlapping)

H-I:

%

H-2: H-3,:

H-3,9:

3.78 t J( I.6B) J(I.28) == 4.4; J(1.6,) < I 3.32 ddd J(2p3,) = 5.6; J(1,2,) = 4.4; J(2B.3,) 12.0; J(2p.6,) 1 1.52 dd J(2fi.3,) = 5.6; J(3,,3,9) = 12.5; J(3,,4) < 1 2.13 dddd J(2,,3,) = 12.0; J(3,,3,) = 12.5; J(4.38) 4.0; J(3,9,5,9) 3 3.79 t J(4,3,9) 5(4,5,9) = 4.0; J(4.5,) < 1 J(4.3,) 1.31-1.48 rn (overlapping) 1.66 rn 1.31-1.48 rn (overlapping) I .31-1.48 rn (overlapping) I .88 brs 8.25 d 7.48 dd 7.28 d 61.1 JC.I.H.I = I50 44.9 J C . ~ . H .= ~ 132 34.8 57.5 JC-4.H-4 = 150 31.0 24.1 149.6 135.8 138.3 123.7 149.5 %

i--

H-4:

3.81 t J(4.34 = J(4,5@)== 4.0; J(4.3,) = 443,) < 1 1.48- I .70 rn (overlapping) 1.48-1.70 rn (overlapping) 1.48- 1.70 rn (overlapping) 1.48- I .70 rn (overlapping) 2.01 brs 8.28 d 7.78 dd 7.23 d 62.6

H-5,: H-5,:

Hha: H-66

NH: H-2':

H-4':

H-5': c-I:

JC.I.H.I =

c-2:

151;

Jc.I.H-~

44.4 J c . ~ . H=. ~132 40.2 56.4 J c - 4 ~ 4= 151; J c - ~ . H . I 3 I .2' 30.e 148.7 140.9 137.6 123.8 148.8

c-3: C-4: c-5: C-6: c-2': c-3': C-4': c-5 ' : C-6':

=

i=

=i

9.5

= 9.5

~

'H:300 MHz, CDCI,. $, Interchangeable.

=

0.00 ppm, J (Hz).

118

CSABA SZANTAY

ET AL.

established from the pertinent vicinal couplings as well as 'H{'H}-NOE measurements by Fletcher et al. (4,16). The most detailed NMR data for compounds 1' and 1 were given by Szantay et al. (17). It was noted that the previous assignments (9) of H3,(,,, and H-3p(eq,in epibatidine (l),as well as those of C-5' and C-4' in 1' and 1should be reversed. For completeness Table I lists the experimental NMR data according to Szantay et al. (17) for both 1 and 1'. The upfield sections of the 300-MHz 'H-NMR spectra are shown in Fig. 1. The depicted assignments were confirmed by 2D homo- (COSY) and heterocorrelation (HETCOR), as well as by detailed 'H{'H}-NOE experiments using various solvents to circumvent some overlap difficulties. In 1' a relatively large (ca. 3 Hz) W coupling was observed between H-3,

4 1 I

l

3s

4.0

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

5s

1.6

M 1.4

1'

1.2

PPM

4.0

3.E

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

PPM

FIG. I . Upfield section of the 'H-NMRspectra (300 MHz, CDC13) of exoepibatidine (1)and endo-epibatidine (1').

3.

119

EPIBATIDINE

H

H

I

I

5'

H H

H

H

1 5'

CI SCHEME 25

and H-5,, which could be used to identify these protons; this had been pointed out before in connection with the pertinent N-acyl analogs (16). Due to the aromatic ring anisotropic effect, the relative spectral positions of H-3, and H-3, in 1' are reversed compared to 1. In 1, C-6 and C-3 show characteristic upfield shifts due to steric interaction with the pyridyl ring (Scheme 25).

V. Pharmacology Preliminary biological investigations (2,19) revealed that epibatidine

(1)has very interesting effects. It proved to be a 200 times more potent analgesic than morphine in the hot-plate and Straub-tail tests. Intriguingly, epibatidine seemed to operate via a nonopioid mechanism because naloxone, a generally used opioid antagonist, did not reverse its effect (Table 11). TABLE 11 COMPARISON OF ACTIVITY OF MORPHINEAN D EPIBATIDINE (2) Dose Eliciting Marked Straub-tail (mglkg) Morphine EDibatidine

10

0.020

Hot-plate Analgesia ED, (mdkg) 1

0.005

ICm Inhibition ['HIDihydromorphine Binding (nM) 1.1

8800

120

CSABA SZhNTAY ET AL.

H

ex0

endo

R=H R=COCH3

1 102

R = CH3

103

I'

SCHEME 26

Shen et al. (20) investigated the analgesic effect of epibatidine (1) and its isomers and analogs. They compared the activity of the synthetic racemic epibatidine (l), ( +)-epibatidine [( + )-l],( - bepibatidhe [( - )-l], racemic endo-epibatidine (l'),racemic 7-acetylepibatidine (102), and racemic 7-methylepibatidine (103)(Scheme 26). Their results as to the analgesic potency of epibatidine and its analogs are summarized in Table 111. The analgesic effect of the selected compounds was also investigated. The results are summarized in Table IV. The detailed examinations supported the results that naloxone does not reverse the effect of epibatidine (l),as is shown in Table V. According to the results of the more detailed pharmacological investigations, the potent analgesic properties of epibatidine were confirmed. Surprisingly, there was no essential difference between racemic epibatidine (1)and its enantiomers [( +)-11and [( -)-11. The endo isomer (1') proved TABLE 111 ANALGESIC POTENCIES OF EPIBATIDINE A N D ISOMERS B Y SUBCUTANEOUS INJECTION I N THE MOUSETAIL-FLICK ASSAY(20) Compound (+)-I ( -)-I (?)-l (*)-I' (*)-I02 (+)-I03 Morphine sulfate

Dose range (pglkg, SC) 5-50 5-50 5-50 50-1000 1000 10-50 1000-10,000

ED%at 5 min ( p d k g ) 7 9 10

> 1000 > 1000 9 4524

3.

121

EPIBATIDINE

TABLE IV TIMECOURSE O F T H E ANALGESIC EFFECTO F EPIBATIDINE A N D ISOMERS BY SUBCUTANEOUS INJECTIONI N THE MOUSETAIL-FLICK ASSAY' (20) Maximum possible effect (%) Compound

Dose ( p g k

+

( )-I ( - )-I

10 lo00 lo00 10 ?

5 min 892 II 58 2 18 47 +. 12 5 2 1 4*3 64 2 20

10 10

(2)-1 (2)-1' (+)-lo2 (+)-lo3 n = 5, mean

sc)

15 min

32 46 23

1 6 II 10 2 3 3*3 16 2 4 2 2

*

30 min 3 2 7 1+3 5 2 2 I1 + 2 0 2 1 8 2 6

SEM

to be less active than the exo isomer, which is the natural product. Alkylation of the bridge nitrogen atom (e.g., 103) did not affect the activity, but basicity seems to be crucial because acylation of the nitrogen atom (e.g., 102)resulted in decreased activity. Naloxone does not influence the activity, confirmingagain that epibatidine may exert its potent CNS effects via a mechanism different from that of the opioid analgesics. It was also determined that the analgesic effect of epibatidine and its active isomers was accompanied with a marked, long-lasting (3-4 hours) sedative effect. The Straub-tail response and labored breathing were commonly observed. On elevating the dose (3-10 times the effective dose), respiratory vocalization, tremors and convulsions developed. At higher doses, epibatidine caused death. The inactive endo isomer 1' and the Nacetylepibatidine 102 did not produce sedation or other visible side effects even at high doses. Qian et al. (21) pointed out that the structure of epibatidine has some

EFFECTO F NALOXONE ON

TABLE V ANALGESIC EFFECTO F ( + ) - E P I B A T I D I N E (1) A N D MORPHINESULFATE (20)"

THE

Maximum possible effect (%) Treatment Saline/( + )-1 Naloxone/( + )-1 Saline/morphine Naloxone/morphine n = 5, mean 2 SEM.

Dose of analgesic (pglkg, SC) 10 10 1o,o00 1o,o00

5 min

15 min

30 min

86 2 14 77 2 14 7 2 6 0 5 2

48 + 14 49 2 22 51220 3 2 2

4 2 3 0 3 94'3 4 2 2

*

122

CSABA

SZANTAY

ET AL.

resemblance to nicotine (104) (Scheme 27). They compared the analgesic with that of (-)-nicotine (104) and effect of (-)-epibatidine [( -)-(l)] determined that epibatidine was about 120 times more potent and had longer duration than nicotine in analgesia. Since nicotine is a nicotinic receptor agonist in the central nervous system and ganglia, they investiand ( - )-nicotine (104)after gated the effect of ( - )-epibatidine [( - )-(l)] pretreatment with mecamylamine, which is a centrally active nicotinic blocker. The results suggest that the analgesia induced by both epibatidine and nicotine is mediated through a nicotinic acetylcholine receptor agonisrn in rats and mice. The quaternary nicotinic receptor blocker hexamethonium did not show antagonism to ( - kepibatidhe. It was verified again that naloxone did not influence the effect of ( -)-epibatidine. Yohirnbine and atropine were also inactive in antagonizing ( - bepibatidhe antinociception in rats. The results are summarized in Table VI. Furthermore, epibatidine competed with high-affinity (IC,, = 70 pM, K i = 43 pM) for {3H}cysteinebinding in rat-brain preparations, and with low-affinity (IC50 = 8.9 p M , Ki = 6.1 p M ) for {3H}pirenzepine,an M,muscarinic receptor ligand. It did not replace (up to 10 p M ) a variety of other known receptor ligands, such as a,-and a,-adrenoceptors, BK2bradykinin, benzodiazepine, opioid, serotonin, MI-muscarinic, or D,- and D,-dopamine receptors. All these results indicate that epibatidine is a selective and very potent nicotinic receptor agonist. Since the nicotine acetylcholine receptor plays a very important role in the mediation of several human diseases, including Parkinson's disease, Alzheimer's disease, ulcerative colitis, and tobacco dependence, epibatidine may be a useful tool for the further investigation of the role of the nicotinic acetylcholine receptor in human diseases.

Nicotine (104)

Epitltidii (1)

S c m m 27

3.

I23

EPIBATIDINE

TABLE V1 OF ( - )-EPIBATIDINE-~NDUCED ANTINOCICEPTION IN MICE(21)" ANTAGONISM ~

( - )-Epibatidine

Effect

Pretreatment

Dose (mg/kg)

EDso (pg/kg)

CV (%)

0.9 % NaCl Mecamylamine Hexamethonium Naloxone Yohimbine Atropine

1 3 10 3 10

13.6 289.2 10.3 13.5 8.3 13.6

2.9 4.2 1.7 1.3 8.6 7.7

P value <0.001 >0.05 >0.05 >0.05 >n.o5

' Four to five doses of epibatidine were used in each pretreatment of antagonist and each dose group included five mice.

Addendum After completing the manuscript the following relevant papers were published. Syntheses

K. Sestanj et al. (22) published a paper in which they described a new approach to epibatidine. The chlorine atom is introduced in the last step by displacement of a methoxy group. M. Natsume et al. (23) synthesized the methoxy derivative of epibatidine starting from the 2,3-dimethyl-7-(p-toluenesulfonyl)-7-azabicyclo[2.2.1]hept-Zene-dicarboxylate. The substitution of methoxy group for chlorine was completed in a single operation using the Vilsmeier reagent. S.Y. KO et al. (24) employed a singlet oxygen reaction with 1-(2-chloro5-pyridyl)cyclohexa-2,4-dieneas a key step in their epibatidine synthesis. H. Nakai et al. (25) used the endo-7-methyl-7-azabicyclo[2.2. llheptane2-01,as starting material, which was transformed into the corresponding ketone, then reacted with 3-bromo-pyridine. The chlorine substituent of the pyridine moiety was formed in the last step. G. Pandey et al. (26) constructed the epibatidine ring system via [3 + 21 cycloaddition of non-stabilized azomethine ylide and substituted 6-chloro3-vinyl pyridine. E. Albertini et al. (27) built up the epibatidine structure from (+)-la-nitro2~-[3-(6-chloropyridyl)]-cyclohexanone, prepared either by Diels-Alder reaction or tandem Michael reaction from 5-(2-nitrovinyl)-2-chloropyridine and 2-trimethylsilyloxy-1,3-butadieneor ethyl 3-0x0-bpentenoate.

124

CSABA SZANTAY E T AL.

Pharmacology A. Barth et al. (28) performed the conformational analysis of epibatidine using the SYBYL molecule modelling program. M. Fisher et al. (29) revealed that epibatidine is a potent agonist of ganglionic nicotinic receptors and that the alkaloid elicits cardiorespiratory effects similar to those of nicotine. B. R. Martin et al. (30) using radioligand binding studies established that epibatidine binds at nicotine receptors with very high affinity (Ki = 0.055nM). Their molecular modelling studies indicate that although epibatidine can mimic the structure of (-)-nicotine, its N-to-N distance is somewhat longer than that found in nicotine. G. Bejeuhr (31) published a short review with 3 references of the pharmacological properties and synthetic aspects of epibatidine. B. R. Martin et al. (32) investigated the pharmacological effects of epibatidine enantiomers in different behavioural tests in mice and rats. The two enantiomers were 200 x more potent than L-nicotine as an antinociceptive agent in mice after S.C. administration. The authors did not observe significant enantioselectivity in binding results for the investigated effects. J. P. Sullivan et al. (33) revealed that (2)-epibatidine elicits a diversity of in vitro and in vivo effects mediated by nicotinic acetylcholine receptors. N. M. J. Rupniak et al. (34) published the antinociceptive and toxic effects of ( + )-epibatidine oxalate attributable to nicotinic agonist activity.

References

1. J. W. Daly, H. M. Garraffo, and T. F. Spande, in "The Alkaloids" (G. A. Cordell, ed.), Vol. 43, p. 255. Academic Press, San Diego, CA, 1993.

2. T. F. Spande, H. M. Garraffo, M. W. Edwards, H. J. C. Yeh, L. Pannell, and J. W. Daly, J. Am. Chem. Soc. 114, 3475 (1992). 3. E. J. Corey, T. P. Loh, S. AchyuthaRao, D. C. Daley, and S . Sarshar, J . Org. Chem. 58, 5600 (1993). 4.

S. R. Fletcher, R. Baker, M. S. Chambers, R. H. Herbert, S. C. Hobbs, S. R. Thomas,

H. M. Vemer, A. P. Watt, and R. G. Ball, J. Org. Chem. 59, 1771 (1994). 5 . A. P. Watt, H. M. Vemer, and D. O'Connor, J. Liq. Chromatogr. 17, 1257 (1994). 6. 0. Diels and K. Alder, Justus Liebigs Ann. Chem. 498, 1 (1932). 7. H. J. Altenbach, D. Constant, H. D. Martin, B. Mayer, H. Miiller, and E. Vogel, Chem. Eer. W, 791 (1991). 8. T. P. Toube, in "Pyrroles" (R.A. Jones, ed.). Part 2, p. 92. Wiley, New York, 1992. 9. D. F. Huang and T. Y. Shen, Tetrahedron Lett. 34,4477 (1993). 10. S. C. Clayton and A. C. Regan, Tetrahedron Lett. 34,7493 (1993).

3. EPIBATIDINE

125

11. R. W. M. Aben, J. Keijsers, B. Hams, C. G. Kruse, and H. W. Scheeren, Tetrahedron Lett. 35, 1299 (1994). 12. H. Y . Chen, D. F. Huang, J. Gonzalez. T. Y.Shen, and W. D. Harman. Abstr. Pap., 205th Narl. Meet., A m . Chem. Soc., Denver, CO, 1993, ORG, 347 (1993). 13. S. Stinson. Chem. Eng. News. Nov. 9, 70, p. 27 (1992). 14. C. A. Broka, Tetrahedron Lett. 34, 3251 (1993).

IS. J. W. Daly, T. F. Spande, and H. M. Garraffo, U.S. Pat. 7,845,042 (1993). 16. S. R. Fletcher, R. Baker, M. S. Chambers, S. C. Hobbs, and P. J. Mitchell, J. Chem. Soc., Chem. Commun., 1216 (1993). 17. Cs. Szhntay, Zs. Kardos-Balogh, I. Moldvai, Cs. Szantay, Jr., E. Temesvari-Major, and G. Blask6, Tetrahedron Lett. 35, 3171 (1994). 18. N. Speckamp, et a / . , oral communication. 19. D. Bradley, Science 261, I I17 (1993). 20. T. Li, C. Qian, J. Eckman, D. F. Huang, and T. Y . Shen. Bioorg. Med. Chem. Lett.

3, 2759 (1993). 21. C. Qian, T. Li, T. Y.Shen, L. Libertine-Garahan, J. Eckman, T. Biftu, and S. Ip, Eur. J . Pharmacol. 250, R13 (1993). see also B. Badio and J. W. Daly, Mol. Pharmacol. 45, 563 (1994). 22. K. Sestanj, E. Melenski, and I. Jirkovsky, Tetrahedron Lett. 35, 5417 (1994). 23. K. Okabe, and M. Natsume, Chem. Pharm. Bull. 42, 1432 (1994). 24. S. Y. KO, J. Lerpiniere, 1. D. Linney, and R. Wrigglesworth, J. Chem. Soc., Chem. Commun. 1994, 1775. 25. K. Senokuchi, H. Nakai, M. Kawamura, N. Katsube, S. Nonaka, H. Sawaragi, and N. Hamanaka, Synlerr 1994, 343. 26. G. Pandey, T. D. Bagul, and G. Lakshmaiah, Tetrahedron Lett. 35, 7439 (1994). 27. E. Albertini, A. Barco, S. Benetti. C. De Risi, G. P. Pollini, R. Romagnoli, and V. Zanirato, Tetrahedron Len. 35, 9297 (1994). 28. W. Brandt, and A. Barth, SAR QSAR Environ. Res. 1, 345 (1993). 29. M. Fisher, D. Huangfu, T. Y.Shen, P. G. Guyenet, J. Pharmacol. Exp. Ther. 270,702 (1994). 30. M. Dukat, M. I. Damaj. W. Glassco, D. Dumas, E. L. May, B. R. Martin, R. A. Glennon, Med. Chem. Res. 4, 131 (1994). 31. G. Bejeuhr, Pharm. Unserer Zeif 23, 105 (1994). 32. M. 1. Damaj, K. R. Creasy, A. D. Grove, J. A. Rosecrans, and B. R. Martin, Brain Res. 664, 34 (1994). 33. J. P. Sullivan, M. W. Decker, J. D. Brioni, D. Donnelly Roberts, D. J. Anderson, A. W. Bannon, C. H. Kang, P. Adams. M. Piattoni Kaplan, M. J. Buckley et a / . , J. Pharmacol. Exp. Ther. 271, 624 (1994). 34. N. M. J. Rupniak, S. Patel, R. Marwood, J. Webb, J. R. Traynor, J. Elliott, S. B. Freedman, S. R. Fletcher, and R. G. Hill, Br. J . Pharmacol. 113, 1487 (1994).

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-CHAPTER 4-

THE NAPHTHYLISOQUINOLINE ALKALOIDS* GERHARD BRINGMANN A N D FRANK POKORNY Institut fur Organische Chemie der Universirar Wurzburg Am Hubland 0-97074 Wurzburg, Germany

I. Introduction ................................................................................... 128 11. Isolation and Structure Elucidation of Naphthylisoquinoline Alkaloids: Dioncophylline A (“Triphyophylline”) ................................................ 130 A. Early Problems in the Field of Dioncophyllaceae Alkaloids: “Isotriphyophylline” ................................................. B. Isolation of “Triphyophylline” .... C. Determination of the Constitution D. Elucidation of the Full Stereostructure of Dioncophylline A .............. 133 E. The Methods: A Summarizing Overview ........................................ 144 111. Other Alkaloids from the Dioncophyllaceae (West Africa) ............... A. Triphyophyllum peltatum ...................................................... B. Other Dioncophyllaceae Species ................................................... 152 C. Joint Structural Properties of Dioncophyllaceae-Type Alkaloids ......... 153 IV. New Alkaloids from Asian Ancistrocladaceae Species ........................... 156 A. Ancistrocladus heyneanus ............................................................ 156 B. Ancistrocladus hamatus .............................................................. 156 C. Ancistrocladus tectorius .............................................................. 157 D. Joint Structural Properties of Asian Ancistrocladaceae-Type Alkaloids .................................................................................. 157 V. Alkaloids of African Ancistrocladaceae Species .................................... 158 A. Ancisfrocladus abbreuiafus: A Chemotaxonomic Link between the 158 Dioncophyllaceae and the Ancistrocladaceae? ................................. B. Ancistrocladus barteri ......................... 165 C. Ancisfrocladus robertsoniorum ..................................................... 169 D. Ancisfrocladus korupensis ........................................................... 170 VI. The Michellamines: A New Class of Naturally Occurring Quateraryls and Related Compounds ........................................................................ 170 A. Constitution and Relative Configuration of Dimeric Naphthylisoquinolines ........................................................ B. Elucidation of the Absolute Configuration at Centers and Axes . C. Base-Catalyzed Interconversion of Michellamines ............................ 177 D. Korupensamines: The Monomeric Michellamine Halves .......... .. 178 E. Chemotaxonomic Position of the New Species A . korupensis ............. 180

* Dedicated to Prof. L. Ak6 Assi (Centre National de Floristique, Abidjan, Ivory Coast), our scientific partner and friend, who, by his competence and engagement, has enormously contributed to this field. 127 THE ALKALOIDS. VOL. 46 Copyright 0 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

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GERHARD BRINGMANN A N D FRANK POKORNY

VII. Stereocontrolled Synthesis of Mono- and Dimeric Naphthylisoquinoline Alkaloids ................................................................................... A. Partial Syntheses of Naphthylisoquinoline Alkaloids ..................... B. Total Synthesis of the Alkaloids: Access to the Molecular Moieties ..................................................................... C. Directed Preparati D. Intermolecular Co E. Total Synthesis of V111. Biogenetic Origin of Naphthylisoquinol A. The Concept of Acetogenic Isoquin B. Biomirnetic Cycliz C. Isolation of Biogenetic Precursors or D. The Plants and Their Botanical Environment .................................. E. Cultivation of the Plants ........................................ ............... F. Biosynthetic Experiments ............................................................ IX. The Chemo-ecological Context of Naphthylisoquinoline Alkaloids ........... A. Biological Activities against Microorganisms ................................... B. Activities against Herbivores: Insect-Growth Retardation and Antifeedant Activity ........................................ C. Interaction with Herbal Parasites: Cuscuru .......... X. Tables of Known Natural Naphthylisoquinoline Alkaloids ...................... ........................................ XI. Summary and Outlook ....................... XII. Addendum ... ........................................ ..................... A. New Alkal African Ancistrocladaceae S .................... B. Synthesis of Dirneric Naphthylisoquinolines C. Further Confirmation of the Absolute Stereo Dioncophylline A .................... ........................................ D. Concluding Remarks .................................. References ...............

181

206 207 208 21 I 21 1

216 254 255 255

261

I. Introduction The naphthylisoquinoline alkaloids (I ,2), such as ancistrocladine (la), comprise a rapidly growing class of intriguing natural products that are remarkable in many respects: 0

structurally, because of their unusual substitution pattern, includingan unprecedented methyl substituent at C-3, a meta oxygenation pattern at C-6 and C-8, and a stereochemically interesting biaryl linkage-an axis that connects the isoquinoline part to the naphthalene moiety and is, in the case of ancistrocladine (la), configurationally stable until above 200°C (34;even at this temperature, l a will not be converted into its atropo diastereomer hamatine (lb) (see Scheme l), a similar naturally occurring compound (5,6);

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

l a (Ancistrocladine)

129

1b (Hamatine)

SCHEME1. Ancistrocladine (la) the “most unusual of all the isoquinoline alkaloids” (7), and its naturally occurring atropisomer hamatine (lb). 0

0

0

0

biosynthetically, because this unusual structure must arise from an unprecedented biogenetic origin, not from aromatic amino acids as is normal for alkaloids, but from acetic acid units (cf. Section VII1,A); pharmacologically, because from such unusual structures remarkable biological activities may also be expected, all the more so as the plants from which these compounds are isolated are used in the folk medicine of tropical countries and in many different applications (cf. Section IX,A); ecologically, with respect to the biological/chemical interaction of the plants with their environment (cf. Section IX); and chemotaxonomically, with respect to the position of the alkaloidproducing organisms in the plant kingdom (cf. Section VII1,D).

All these interdependent factors together form a remarkable overall view of a challenging new class of natural biaryls, the study of which is currently undergoing a rapid development that will certainly continue or even accelerate dramatically in the next few years. A formal measure of this development may be deduced from the reviews on this topic. In 1977, Govindachari (I), the pioneer in this field, published a short summary on the six Ancistrocladus alkaloids that he had isolated and structurally elucidated in an excellent and reliable way. Subsequently, a larger review, which appeared in this series (2) in 1986, still included only eight such naphthylisoquinoline alkaloids that were completely certain with respect to their full stereostructures. In the literature, a couple of additional alkaloids had been reported, whose structures appeared to be incomplete, uncertain, or even obviously wrong (cf. Section II,A and Table V). The present paper, however, describes nearly 50 naphthylisoquinoline alkaloids and related compounds. Apart from the number of new alkaloids that have been isolated, this field has developed in many more respects. Meanwhile, more than 20

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GERHARD BRINGMANN A N D FRANK POKORNY

naphthylisoquinoline alkaloids have been prepared by highly selective total syntheses (see Sections II,D,2 and VII), and the first clear results have been obtained on the biosynthetic origin (see Section VIII) of these compounds. Moreover, a number of interesting biological activities were found to be exhibited by the alkaloids, including fungicidal, antimalarial, and remarkably high anti-HIV activities (see Section IX). And very recently, novel dimeric naphthylisoquinoline alkaloids have been isolated-the michellamines (see Section VI), unprecedented natural quateraryls-giving this field additional future-oriented impetus. All these fruitful developments were possible only by the elaboration of a solid basis of efficient and reliable procedures for the isolation and structure elucidation of these compounds, the development of a novel synthetic methodology for the regio- and stereoselective construction of highly hindered biaryl axes, the first cultivation of the very delicate tropical plants, and the bioassay-guided search for new pharmacologically active constituents. Because of their crucial importance, these methods will be described thoroughly in the beginning of this chapter (see Section 11). This review concentrates predominantly on the methods and results obtained after the appearance of the last comprehensive article on the naphthylisoquinoline alkaloids in 1986 (2). One main reason for this is that many of the structures published before that time have turned out to be incomplete or incorrect (cf. Table V).

11. Isolation and Structure Elucidation of Naphthylisoquinoline Alkaloids: Dioncophylline A (“Triphyophylline”)

A. EARLYPROBLEMS I N T H E FIELDOF DIONCOPHYLLACEAE ALKALOIDS: “ISOTRIPHYOPHYLLINE” Outside the family of the Ancistrocladaceae, such naphthylisoquinoline alkaloids have so far been found only in the extremely small, botanically related family of the Dioncophyllaceae, which comprises only three genera, with but a single species each: Triphyophyllum peltatum (Hutch. et Dalz.) Airy Shaw , Dioncophyllum tholloni Baill., and Habropetalum dawei Airy Shaw. Previous work in the late 1970s revealed the presence of naphthylisoquinolinealkaloids in T. peltatum and D . tholloni. Eight such representatives were isolated and structurally investigated (8-JJ). A detailed analysis of the published data showed that their reported structures 2-9 (see Fig. 1) cannot be considered fully established, a relative exception

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

2 (“Triphyophylline”)

5 (‘KMethyltriphyophylline’)

3 (‘lsotriphyophylline’)

4 (“OMethyltriphyophyIline’)

6 (“Triphyopeltine’)

7 (“5-QMethyltriphyopelIine’)

8 (“OMethyl-1.2-didehydrotriphyophylline‘)

131

9 (‘OMethyl-1.2.3.4-tetra dehydrotriphyophylline’)

FIG.I . Reported (8-11) structures of some alkaloids from Triphyophylhtm peltmum and Dioncophyllurn rholloni (Dioncophyllaceae).

being triphyophylline(“2”), for which at least the constitution and relative configuration at C-1 versus C-3 seemed certain. By contrast, the absolute configuration at the centers had been proposed only arbitrarily, based upon “biogenetic” considerations (10). Furthermore, the possibility that the biaryl axis might exhibit restricted rotation and hence might constitute an additional stereogenic element, consequently leading to additional stereoisomers, was not taken into consideration. Moreover, for some of the other isolated alkaloids, even the constitutions and the relative configurations at the centers appeared uncertain. The first total synthesis (12-14) of naphthylisoquinolines, the preparation of the postulated (10) structure 3 of “isotriphyophylline” (see also Section VII,C,2), showed that none of its two possible (racemic)atropodiastereomeric forms 3a or 3b was identical, in its physical or spectroscopic data, to those reported for isotriphyophylline. As no authentic sample of the natural product is available any more, it remains unknown what natural “isotriphyophylline” really was. This, and several inconsistencies within the reported data (8-11), demonstrated the necessity of investigating the

132

GERHARD BRINGMANN A N D F R A N K POKORNY

3a

3b

(stable atropisorners)

FIG.2. Synthetic (racernic) atropodiastereorners of 3 (“isotriphyophylline”).

alkaloids again, based upon reliable methods for the unambiguous structure elucidation of genuine alkaloids, as isolated from fresh plant material. B. ISOLATION OF “TRIPHYOPHYLLINE” From several harvesting expeditions performed by Professor L. Ake Assi, Abidjan, T. peltarum from the Parc de Tai‘ in West Ivory Coast became available in sufficient amounts. From this plant material, triphyophylline was isolated (15) by standard procedures, facilitated by the availability of an authentic sample of this alkaloid (16). The typical isolation procedure involved extractions of dried and ground root bark material, consecutively with petroleum ether and dichloromethane/NH,, followed by chromatography of the alkaloid-containing CH,CI, extracts on SiOz (typical eluents: CH,CI,, 0 +-10% MeOH; MeO-tBu, 0 +. 10% MeOH; MeOH). The isolated alkaloid was identical with the authentic sample. C. DETERMINATION OF THE CONSTITUTION The determination of the constitution of the isolated alkaloid, using modern ID- and 2D-NMR techniques (IH; IH-COSY; IH,I3C-COSY;1DNOE, etc.) combined with other usual analytical methods (combustion analysis, MS, IR, UV, etc.), was straightforward and confirmed the gross structure 2, as published for triphyophylline (8). Characteristic spectroscopic features are, among others, the (M+ - 15) fragment in mass spectrometry due to the loss of the benzylic methyl group at C-1, the characteristic upfield shift (6 = 2.16 ppm) of the 2’-CH, group caused by the anisotropic ring current effect of the adjacent aryl substituent and, vice versa, the normal chemical shift of the protons at C-4 (see Fig. 3).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

-

2.73 2.83 ppm

a

133

b

2.16 ppm 6.74 ppm

H3CO

FIG. 3. Constitution of triphyophylline from (a) 'H-NMR chemical shifts (in ppm) and (b) selected NOE interactions (arrows) (IS).

D. ELUCIDATION OF THE FULLSTEREOSTRUCTURE OF DIONCOPHYLLINE A 1. NMR Spectroscopy: Relative Configuration at the Stereocenters

NMR spectroscopy is also a useful key to the elucidation of the relative configuration at the two stereocenters at C-1 versus C-3 (15). For triphyophylline (Fig. 4), the two methyl groups were confirmed to be transoriented 0

0

by an unambiguous nuclear Overhauser effect between H-3 and the likewise pseudo-axial methyl group at C-1; by the small size of the homoallylic long-range coupling between H1 and the two protons at C-4; this value is significantly larger for the cis isomer, due to its pseudo-axial proton at C-1; and compare:

bans

trans

0 6 n ~

i4nz

FIG. 4. Relative trans configuration of triphyophylline at C-1 versus C-3, as deduced from NMR data (15).

134 0

GERHARD BRINGMANN A N D FRANK POKORNY

by the characteristic chemical shift of H-3 (rule of thumb: 6 < 3 ppm: cis configuration; 6 > 3 ppm: trans configuration);this value may be modified by the naphthalene substituent if it is located at C-5.

Consequently, the structure of triphyophylline could be fully confirmed with respect to the constitution and the relative configuration at the stereocenters, as published in the literature (8). 2 . Total Synthesis: Absolute Configuration at the Stereocenters (and Confirmation of the Constitution)

For the elucidation of the absolute configuration at C-1 and C-3, a first (now enantioselective) total synthesis of this alkaloid was developed which followed, in its principal conception, the prepa(Scheme 2) (I7,f8), ration of racemic material previously described (2). Again, as was already the case for the synthesis of 3, the postulated (IO)structure of “isotriphyophylline” (see above), the two molecular moieties (i.e., the naphthalene part 10 and the now enantiomerically pure (lS,3S)-configured tetrahydroisoquinoline 11)were pre-fixed, this time via an ester-type auxiliary bridge. This again allowed very good to excellent yields in the subsequent intramolecular coupling step. The resulting lactone-bridged biaryl 12 is of high stereochemical interest. Although already disposing of the required biaryl axis, it is not (yet) split up into stable atropisomers, but rather is configurationally unstable due to a rapid rotational process at the bridged biaryl system (see also Section VII,C,l). This opens up the remarkable and unprecedented possibility of performing such a ring-opening reaction to the configurationally stable alcohols 13 atropisomer-selectively. According to the choice of the hydride transfer reagent, one can obtain l3a or, alternatively, the M-atropisomer 13b in very high diastereoselectivities (18). This completely novel approach to the regio- and stereoselective synthesis of biaryls attains the two formal goals separately-first the actual CC-bond formation to give a stereochemically labile axis, then the asymmetric induction at this axis by a stereoselective “torsion” of the biaryl system. This first total synthesis of enantiomerically pure triphyophylline established the absolute configuration at the stereocenters of this most prominent Dioncophyllaceaealkaloid. Although neither of the two atropodiastereomers synthesized was completely identical with the natural product, compound 2a at least had identical physical and chromatographic properties, except for the opposite sign of the optical rotation ([a],+14.9”, instead of -14”). This showed that natural triphyophylline must be the enantiomer ent-2a of the synthetic compound, i.e., 14a, with the (1R,3R) configuration.

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

135

Me0 MeO

/ iii

0

12

78 %

13a

iv

1

En%[ iv

82%

Me Me0

Me0

2a mp 214”C, [a10+14.9’

mp 245”C, [a10+11.3’

natural ‘Triphyophylline’: mp 215OC, [a10-14’ (8)

SCHEME 2. Regio- and stereoselective total synthesis of stereochemically pure (lS,3S,7M)-enantiomer 213of triphyophylline and its atropodiastereomer2b (17.18). Reaction conditions: (i) (C0C1)2, NEt,; (ii) Pd(PPh3)2C12,NaOAc; (iii) AIMe3+RedAl+2 N HCI; (18)-+LAH+H21Pd-C. (iii’) RedAI-2 N HCI; (iv) PPh,, (CBICI~)~

136

GERHARD BRINGMANN A N D FRANK POKORNY

A subsequent analogous total synthesis of the correct enantiomer (see Scheme 3), now starting from the (lR,3R)-configuredprecursors (see also Section VII,A), led to fully authentic, correctly configured triphyophylline (17), which is therefore the first totally synthetic authentic naphthylisoquinoline alkaloid. Consequently, in the literature ( 8 ) , the structure of triphyophylline had been published correctly with respect to its constitution and relative trans configuration, but the total synthesis now revealed the absolute configuration at the centers to be (1R,3R) (13,not (1S,3S) as previously (8) assumed. 3. CD Spectroscopy: Absolute Configuration at the Axis

The occurrence of distinct, configurationally stable atropisomers in the course of the total synthesis of triphyophylline had shown that the biaryl axis is, in addition to the stereocenters, another element of chirality in this natural biaryl. Yet, in contrast to the centers, its absolute configuration did not automatically become manifest by the enantioselective synthesis described above, but rather by an investigation of its circular dichroism (15,20). For this purpose, in order to avoid possibly disturbing complications by the likewise present stereocenters, and for a “prolongation” of the isoquinoline chromophore, the heterocyclic part was planarized by catalytic dehydrogenation. Due to harsh reaction conditions, early attempts in this field (10) had delivered only optically inactive material, which was interpreted to be achiral. By contrast, very cautious catalytic dehydrogenation (see Scheme 4), e.g., with Pd-C in refluxing toluene, gave an optically active, enantiomerically nearly pure naphthylisoquino-

qyMeBZl

OH

Me

Me0

w::

Me0

0

\

ent-10

0

Me

ent-12

Me0 14a (= ent-za)

synthetic: mp 214”C, [a]DZ0 -14.9” natural: mp 215”C, [ a ]-14“ ~ ~ ~

SCHEME 3. Analogous total synthesis of the correct enantiorner enr-29 (149) of triphyophylline (dioncophylline A) (17).

4.

137

T H E NAPHTHYLISOQUINOLINE ALKALOIDS

Ii A

absolute configuration at the axis by CD

16a 9a

R=H R=Me

SCHEME4. Synthesis and stereochemistry of an optically active tetradehydrotriphyophylline, named dioncophylleine A (16a). and its methyl ether 9a (15.20). Reaction conditions: (i) Pd-C (5%), toluene, 120°C.

line 16a with only axially chiral information in the molecule, thereby permitting analysis by circular dichroism (15,2O). Figure 5 (solid line) shows the CD spectrum of the optically active dehydrogenation product 16 thus obtained, with a positive couplet at ca. 225 nm (i.e., a first positive and a second negative Cotton effect). From this, a so-called “positive chirality,” as in the P-configured stereostructure 16a, can be deduced by application of the exciton chirality method (21,22), as well as by empirical comparison with related axially chiral natural products (4,23). More recently, methods have been developed to overcome such empirical or semi-empirical procedures by learning to calculate, and hence reliably predict, the CD spectra of axially chiral biaryls ( 2 0 , 2 4 2 6 ) .Figure 5 likewise shows the calculated CD spectrum (dashed line) of the dehydrogenation product 16, which in a very satisfactory way reproduces the crucial experimental positive couplet of the biaryl chromophore. The computational CD spectrum was generated by calculating the single spectra of a series of relevant conformations of the molecule with respect to different dihedral angles along the biaryl axis, and subsequent Boltzmann-weighted

138

GERHARD BRINGMANN A N D FRANK POKORNY

200

230

260

290

320

wavelength h (nm)

FIG.5. Experimental (---

350

(+)-Dioncophylleine A (16a)

1 and calculated [AM1 -+ CNDO-S] (-------) CD spectra of

168 (20).

addition to deliver the overall spectrum. This is a very practical and less time-consuming alternative to molecular dynamics (MD) calculations ( 2 3 , which, moreover, are based on force-field parameters, not (as here) on more reliable semiempirical AM 1 calculations. A practical procedure for the construction of such a theoretical CD spectrum is illustrated in a simplified, schematic way in Fig. 6. The great diversity within the single spectra (left bottom) to be added up to the overall spectrum (right) shows the importance of considering more than just a single conformation (e.g., that one obtained from an X-ray structure analysis). This method of reproducing or predicting CD spectra by computational methods has become an efficient and reliable tool for the determination of the absolute configuration at the biaryl axis, even in the presence of stereogenic centers (see the example of ancistrocladine), because the chiroptical behavior of these compounds is strongly dominated by the biaryl chromophore. Still, the very best agreement between theoretical and experimental spectra is obtained in those cases where the molecule is conformationally clearly defined-not too flexible, e.g., for dehydrogenated representatives and those with higher steric demands of the ortho substituents next to the axis, thereby leading to a steep potential curve.

FIG.6. Schematic procedure for the computational prediction of CD spectra, exemplified for ancistrocladine (la) (25). The calculation of the individual CD spectra of a series of axial conformers (left bottom), and their Boltzmann-weighted addition according to their energies (left top), leading to (right side) the calculated (---) CD curve as compared with the experimental spectrum (-------)

>

. \ '

0

-

l

0

. o0,

u

0 0 0 0 0 0 0 0 0u l o u l o u l - I - - " 1 1 1 1

o l n o l n

N

0

I:>

140

GERHARD BRINGMANN A N D FRANK POKORNY

4 . The Structure of Dioncophylline A (“Triphyophylline”)

Summarizing, triphyophylline has three stereo elements-the two centers and the axis. Consequently, at least three pieces of stereo information are required for its complete structure elucidation, which are available at this point, namely: 0 0 0

the relative configuration at the stereocenters by NMR spectroscopy, the absolute configuration at these centers by total synthesis, and the absolute configuration at the axis by empirical and theoretical CD spectroscopy.

Consequently, natural triphyophylline has structure 14a, i.e., a (1R,3R,7P(= 7s))-configuration (Fig. 7 ) . It is thus the very first fully elucidated structure of naphthylisoquinolinealkaloid from a Dioncophyllaceae species. In addition to the fact that the structure for triphyophylline was not completely correct in the literature (a), a much more critical point was that even among themselves the structures of the alkaloids had not been attributed correctly. As an example, the 0-methylation product 15a of triphyophylline, as prepared, e.g., via the corresponding formamide, starting from either synthetic or natural material, was shown (15) not to be identical in its spectroscopic and physical properties with those published (9) for the natural product named “0-methyltriphyophylline” (cf. Fig. 1). On the other hand, the transformation of that natural product into an authentic derivative of triphyophylline was reported, hinting at a stereochemical identity of the two alkaloids. By contrast again, it was reported to originate from a boronate reduction of the corresponding 3,4dihydroisoquinoline(“8”)-a reaction from which clearly the corresponding 1,3-cis derivative must be expected (4,28-30) (cf. also Schemes 12 and 20). All these (and other) problems and inconsistencies will never be solved because an authentic sample of the natural product is no longer

2

14a

revised structure FIG.7. Postulated (8)and revised (17) structures 2 and 14a for triphyophylline (dioncophylline A). initial proposal

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

141

available, and the same applies to the authenticity of practically all the other described naphthylisoquinolinesfrom this plant, among them isotriphyophylline (“3”)(see Section II,A), triphyopeltine (“6”) (see Section III,A, l), N-methyltriphyophylline (“5”) (see Section V,A,2), and others (see also Table V, Section X). Consequently, the decision had to be taken to rename the alkaloids, so that a new beginning could be made. Consequently, 14a, the main alkaloid of T. peltatum, was renamed as dioncophylline A, after the name of the plant family, Dioncophyllaceae. Under these circumstances-the revision and renaming of the main representative of this important class of natural products-it became even more urgent to be completely certain about its new structure 2a. For this reason, the above-mentionedmethodology (NMR/total synthesis/CD)had to be expanded, all the more so as it is not always possible, or justifiable, to perform a lengthy stereoselective total synthesis of each new alkaloid. 5. The Bridge: Relative Configuration at Centers versus Axis

In addition to the elucidation of the absolute configuration at the axis by CD spectroscopy, a chemical procedure was developed (Scheme 5 ) for the unambiguous correlation of the axially chiral information to be analyzed with the centrally chiral information, which is well known, e.g., from the total synthesis. This is realized by joining the two types of chirality elements together by a chemical bridge (31).Thus, the hydroxymethyl naphthylisoquinoline 13b (i.e., still from the old (1S,3S) series) can easily be sidechain-extended by reaction with succinic anhydride. Then, after N-deprotection and acyl activation, it can be ring-closed, in practically quantitative yield. This leads to the 13-membered macrocyclic lactam 17, but specifically only for this unlike-atropisomer with a synarray of the crucial N- and 0-functionalities. The other atropisomer 13a (with the relative configuration of dioncophylline A) would be able to undergo such a ring closure only after a conformational change of the tetrahydroisoquinolinering-a change it does not undergo under the same reaction conditions. The apparent reason for this is the steric pressure exerted by its 8-substituent, which is also confirmed by semiempirical AM1 calculations (31). This analytically very useful, absolutely atropisomerdifferentiating reaction to ansa-compounds of the type 17,which has the additional element of planar chirality, is nicely applicable to the chemical elucidation of the configuration at the axis relative to the stereocenters. 6. Long-Range NMR Interactions: Relative Configuration at Centers

versus Axis As an alternative to this chemical procedure, such information about syn- or anti-relationships related to atropisomerism can also be obtained

142

GERHARD BRINGMANN A N D FRANK POKORNY

fMe0

OH

H

Me

13b

m no "monomeric"

L-2 ansacom pound

0 -0

17

SCHEME 5 . An atropisomer-differentiating reaction for the elucidation of the relative configuration at the axis (31).

spectroscopically, by nuclear Overhauser investigations (32,33).Yet, for the stereochemically relevant interactions, very large distances of about 5 A have to be covered. Nonetheless, starting with other alkaloids in which the corresponding distances are significantly smaller (see, among others, Section V,A,6), the experimental conditions were optimized such that a clear distinction could be made between dioncophylline A (14a), in which the methyl groups at C-1 and C-2' are on the same side of the molecule, and its atropisomer 14b, in which specific interactions of CH,1 with the peri-proton at C-8' are found, as well as interactions of CH3-2' with the equatorial proton at C-1 (see Fig. 8). Assisted by two-dimensional methods, e.g., by the ROESY technique (34), which avoids the general problems caused by ordinary NOE interactions that are close to zero for medium-sized molecules, this method has now become another reliable standard procedure for the correlation of the configuration at the biaryl axis with those at the stereocenters in the tetrahydroisoquinoline ring (32,33). 7. Oxidative Degradation to Amino Acids: Absolute Configuration at the Centers

For an additional confirmation of the revised absolute stereostructure 14a of dioncophyllineA ("triphyophylline"), a ruthenium-mediatedoxidative degradation was developed (35).By destruction of the aromatic rings, with additional CN-bond cleavage, 0-aminobutyric acid (18) and alanine

4.

I43

T H E NAPHTHYLISOQUINOLINE ALKALOIDS

NOE

NOE

14a (Dioncophylline A) 14b (7-epi-DioncophyllineA) FIG.8. Atropisomer-specific NOE interactions of dioncophylline A (14a) and its naturally occurring (see Section V.B.1) 7-epimer 14b.

(19)(see Scheme 6) were obtained. With just one stereocenter each, these are simple substances which thus are easy to analyze stereochemically, either as Mosher derivatives or directly, on a chiral phase. From the Dalanine (D-19)and the (R)-3-aminobutyric acid [(R)-18] detected, dioncophylline A (14a) is unambiguously confirmed to be (lR,3R)-configured, i.e., opposite to the Ancistrocfadus alkaloid hamatine (lb), with its now H 0 2 C 7 M e NH2

H

HO2C

D

v

(R)-l8

NH2

F;(e

D-19

he

L-19

Me0 14a (Dioncophylline A)

Me0

OMe

Me0

Me

1b (Hamatine)

SCHEME6. Stereoanalysis of naphthylisoquinoline alkaloids by ruthenium-mediated oxidative degradation (35).

144

GERHARD BRINGMANN A N D FRANK POKORNY

easily controllable (lS,3S)-configuration (35).This degradative analysis, which can be performed even on a submilligramscale (36),has immediately become a most valuable analytical device. It is now routinely applied to all new naphthylisoquinoline alkaloids (see also Sections 111-VI). 8 . Crystal-Structure Analysis: Confirmation of Constitution, Relative Configuration, and Conformation

One of the methods of choice should be X-ray structure analysis, which, of course, had been attempted since dioncophylline A (triphyophylline) had become available. Yet, it turned out to be very difficult to grow suitable crystals, and only recently, after finishing all of the spectroscopic, chemical, and total synthetic structural investigations mentioned above, could a crystal-structure analysis of dioncophylline A (14a) be achieved (37). For this, the presence of dichloromethane molecules in the crystal lattice (see Fig. 9) turned out to be essential. This crystal-structure analysis again fully confirmed the constitution, the relative configuration, and finally the conformation, e.g., with the typical pseudoaxial position of the 1-methylgroup, which also had become manifest from AM1 calculations (31). E. THEMETHODS:A SUMMARIZING OVERVIEW As most of the naphthylisoquinolinealkaloids do not crystallize suitably and one thus cannot rely on obtaining supporting X-ray analytical information, it turned out to be most helpful to have expanded the methodology for the structure elucidation of naphthylisoquinoline alkaloids by the previously mentioned chemical and spectroscopic procedures (degradation, bridge methodology, long-range NMR). In Fig. 10, all of these techniques

14a .CH,CI, FIG.9. Structure of 14a.CHzCI2in the crystal (37).

I

I

a

8 %

0

c

146

GERHARD BRINGMANN A N D FRANK POKORNY

are schematically summarized-the chemical procedures, as well as the spectroscopic and physical methods. All this created the basis for the application of these methods to the other alkaloids occurring in the same plants.

111. Other Alkaloids from the Dioncophyllaceae (West Africa) A. Triphyophyllum peltatum 1 . Dioncopeltine A and Dioncolactone A

Besides dioncophyllineA (14a), Triphyophyllum peltatum also produces a side-chain functionalized alkaloid named dioncopeltine A (20) (see Scheme 7) (38). This compound is very similar to the natural product “triphyopeltine” (see also Fig. 2), to which the same constitution had been attributed in the literature ( 8 ) ,although based on different spectroscopic data. Using the above-mentioned spectroscopic and chemical methods (in particular CD spectroscopy and the oxidative degradation procedure), this new alkaloid could now be assigned the complete structure 20 (38). It corresponds, in all stereochemical details, to dioncophylline A (14a). This is underlined by the transformation of both alkaloids into 0methyldioncophylline A (15a) as a joint derivative (38).Again, the structure elucidation could be further confirmed by X-ray structure analysis (38) and by total synthesis (39). For the natural product triphyopeltine (“6”), which shows a completely different optical rotation (see Scheme 7), the same constitution as that now established for dioncopeltine A (20) has been postulated ( 8 , l l ) . Whether these two alkaloids might be identical is a question that most probably will never be answered: As for isotriphyophylline (“3”) and 0methyltriphyophylline (“4”), again authentic comparison material is no longer available for “triphyopeltine”, so that dioncopeltine A (20) has to be treated (and named) as a new natural product. Moreover, a nicely blue fluorescent, nitrogen-containing compound was isolated from T. peltatum (38).The fluorescence very strongly resembled that of the pentacyclic lactone 12 (cf. Scheme 2). The structure 22 (Scheme 8) of the new natural product dioncolactone A shows that it indeed belongs to this type of axially prostereogenic (= configurationally unstable) lactone-bridged biaryls that are used (normally in an N-protected form) for the regio- and stereoselective construction of these biaryl alkaloids (cf. Sections II,D,2 and VII,C,3) (17,18).

4. T H E

NAPHTHYLISOQUINOLINE ALKALOIDS

20 (DioncopeltineA)

147

(0-MethyldioncophyllineA)

Me0

Me0

14a

(DioncophyllineA) compare.

HO

= -125°C

6 ("Triphyopeltine") (8)

SCHEME7. Complete stereostructure of dioncopeltine A (20).as evident from chemical transformations and X-ray crystallographic studies (38); comparison with the postulated (8,111 structure 6 for "triphyopeltine."

Closely analogous to the total synthesis of dioncophylline A (cf. Scheme 2), the two new alkaloids, dioncolactone A (22) and its cleavage product dioncopeltine A (20), were synthesized (see Scheme 8) from a joint protected precursor 21. In order to obtain the particular oxygenation pattern in the naphthalene moiety, i.e., with one free phenolic oxygen function specifically at C-5', the 0-isopropyl protected precursor 21 turned out to be optimal (39).

2. Dioncophyllines B and C While all three of the alkaloids 14a, 20, and 22 described above are based on a 7 , l '-position of the biaryl axis between the two molecular halves

148

GERHARD BRINGMANN A N D FRANK POKORNY

Me Me0

0

21

//

6 = 3.31 ppm

-, U

“H-

HO

THF

Me

0 )4nax em =442 nm

I

22 (Dioncolactone A)

20 (Dioncopeltine A)

axially prostereogenic

axially stereogenic

SCHEME8. Dioncolactone A (22) and dioncopeltine A (20): characteristic spectroscopic data and joint total synthesis from a protected precursor 21 (38.39).

(A-type), the new alkaloids dioncophylline B (23)and dioncophylline C (24) (see Scheme 9) exhibit completely different coupling patterns. In dioncophylline B (40),two aromatic positions (C-7 and C-6’) with limited steric demands are connected to each other, so that it is the first (nonbridged) naphthylisoquinoline alkaloid without a stable conformation at the biaryl axis. It thus does not have chromatographicallyor spectroscopically distinguishable or even isolable atropisomers. Moreover, lacking a C,-bridgehead next to the biaryl axis, it is also the first alkaloid that cannot be synthesized according to the strategy applied in Scheme 2. Nonetheless, although no total synthetic access to this interesting natural product has yet been developed, the absolute stereostructure has been unambiguously determined by application of the oxidative degradation procedure. As for all of the Dioncophyllaceae alkaloids mentioned above, the configuration at C-3 (as also at C-1) was found to be R (40). Of great interest as well is the structure of dioncophylline C (24) (41), which is reminiscent of the Ancistrocladaceae alkaloid ancistrocladine (la), yet again exhibiting an R-configuration at C-3 and lacking an oxygen function at C-6. Whereas the configurations at the stereocenters were again elucidated by the degradation method (see Scheme 9), the absolute configuration at

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

149

3 1 ppm

no stable atroplsomen

HC

c

6 = 2 43 ppm

23 (Dioncophylline B)

24 (DioncophyllineC)

RuCIflal04

RuCIflalO4 NH2 HOzC D NH2

v Me

SCHEME9. Dioncophyllines B and C (40.41):characteristic features of the elucidation of constitution and stereocenters.

the axis of dioncophylline C (24) could not be unambiguously determined, e.g., by a comparison of its CD spectrum with that of ancistrocladine (la). This uncertainty arises from the lack of an oxygen function at C-6 and its possible influence on the CD behavior of 24. For this reason, the new alkaloid and ancistrocladine (la) were transformed into constitutionally identical derivatives 25 and 26 (Scheme lo), which turned out to be enantiomers. Hence the stereochemical array in dioncophylline C (24) must be unequivocally opposite in all regards compared with ancistrocladine (la) (41). The crucial step in this reaction sequence, the removal of the 6-oxygen function, turned out to be the trickiest, owing to the difficulty of transforming this phenolic substituent into an appropriate leaving group because of its proximity to the stereochemically demanding naphthalene substituent. For this purpose, the 0-triflate group proved to be well suited (41). This procedure also allowed for the development of a strategy for the total synthesis (42) of dioncophylline C (24) (cf. Section VII,C,3), which takes advantage of the lactone methodology, despite the absence of an oxygen function next to the biaryl axis. In close analogy to the synthesis of ancistrocladine (la) (cf. Section VII,C,3), 24 can be prepared via the pentacyclic (isopropyl-protected) lactone 27, as shown in Scheme 11, where a transient oxygen function at C-6 (see empty arrow) is used for the intramolecular coupling and is reductively removed at the end (42).

150

GERHARD BRINGMANN A N D FRANK POKORNY Me0 OMe

HO OMe

-

Me

Me

24

25

- ...........'minor plane'

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

Me0

Ye0 OMe

\

Me0

-

$.,Me

HO 6/

OMe

..Me

s KH Me0

Me

Me

la

26 (= ent-25)

SCHEME10. Transformation of 24 and l a into the chiroptically comparable (since enantiomeric) derivatives 25 and 26 ( 4 / ) .

Finally, as in the case of dioncophylline A (see Fig. 8), the attribution of the relative configuration at centers versus axis by atropisomer-specific NOE interactions fully confirmed the structure 24 for dioncophylline C (41).

All four of the naphthylisoquinoline alkaloids from T . peltaturn men-

A Me

HO OMe

-

Me

-c

-

HO

H

Me

24

SCHEME 1 I . A helicene-like distorted lactone-bridged precursor 27 in the regio- and stereoselective total synthesis of ( + )-dioncophylline C (24) (42).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

151

tioned so far-dioncophylline A (14a), dioncopeltine A (22), dioncophylline B (23), and dioncophylline C (24)-exhibit high and promising biological activities in different test systems (see Sections IX,A and IX,B). 3. Dioncophylline D and Minor A-Type Alkaloids from T. peltatum A trace alkaloid 28, named dioncophylline D (39), was found in the leaves, exhibiting an unprecedented 7,8'-coupling type (compare also the structure of ancistrobrevine A (46);see Section V,A,6). In addition, a pair of atropodiastereomeric naphthylisoquinoline alkaloids related to dioncophylline A (14a), i.e., with the A-type 7,l'-coupling site, were isolated from the stem bark of the plant (43,44).Compound 30b is the first naphthylisoquinoline alkaloid from this plant that is related to dioncophylline A, but with an opposite configuration at the biaryl axis. In still smaller amounts, the normal, i.e., P-configured atropodiastereomer Ma was isolated and its structure fully elucidated (4495). Furthermore, the naphthyldihydroisoquinolinealkaloid 29, also with the rare axial array opposite to that of dioncophylline A, was isolated (39).The structures of these alkaloids isolated from T. peltatum are shown in Figure 11.

4 . Dioncophyllacines A and B Not all naphthylisoquinolinealkaloids were found to be optically active. Figure 12 shows two fully dehydrogenated representatives isolated from

Dioncophylline D (28)

5'-O-Demethyl-8-0-methyl7-epCdioncophyllidineA (29)

5'-O-Demethyl-8-Ornethyl5'-O-Dernethyl-8-0-methyldioncophylline A (ma) 7-epi-dioncophylline A (30b) FIG. 11. Structures of four minor alkaloids from T. peltaturn ( 3 9 . 4 3 4 5 ) .

152

I

GERHARD BRINGMANN A N D FRANK POKORNY

OMe

I

OMe

(*)-Dioncophyllacine A (31):

Dioncophyllacine B (32):

chiral, but an enantiomeric mixture

achiral

SCHEME12. Isoancistrocladine (35):partial synthesis from ancistrocladinine(34)(4,29,54) and degradation (53)of the natural product.

the leaves of T. peltatum; each of these has an additional oxygen function at C-4. Thus, in dioncophyllacine B (32)(46),the isomerization barrier at the axis is very low, as already noted for dioncophylline B (23) itself. This new alkaloid, which hence possesses neither stereogenic axes nor centers, is the first naphthylisoquinoline alkaloid that is achiral at room temperature. By contrast, dioncophyllacine A (31) (47,48) has a stable configuration at the axis and is thus chiral, but it does not occur in an enantiomerically pure form. It crystallizes as a racemate, as convincingly visualized by its X-ray analysis, which reveals the presence of both enantiomeric rotational isomers in the crystal (see Fig. 13). Of high biogenetic interest is the additional 4-methoxy group in 31 and 32. This oxygen function apparently does not originate directly from an acetate unit (cf. Section VIII,A), but rather seems to be an indicator of a beginning catabolism by oxidative transformation.

B. OTHERDIONCOPHYLLACEAE SPECIES The only other Dioncophyllaceae species investigated so far is Dioncophyllum tholloni, from which six different alkaloids-“triphyophylline,” “isotriphyophylline,” “N-methyltriphyophylline,” “O-methyltetradehydrotriphyophylline,” “triphyopeltine,” and “5-0-methyltriphyope1tine”were isolated (7-10). The postulated structures of these alkaloids are shown in Fig. 1 (Section II,A) and Table V (Section X). Since the last review on naphthylisoquinoline alkaloids (2), no additional work on this particular plant species has been published, but is in progress (49).

4. T H E NAPHTHYLISOQUINOLINE ALKALOIDS

153

3

V

M-31

FIG. 13. Structure of natural racemic dioncophyllacine A (P-31/M-31)in the crystal (47) (i = inversion center).

C. JOINTSTRUCTURAL PROPERTIES OF DIONCOPHYLLACEAE-TYPE ALKALOIDS Figure 14 represents a complete list of those Dioncophyllaceae alkaloids whose structures have been fully elucidated so far. Summarizing, it shows that Triphyophyllum peltatum is capable of producing alkaloids of at least four different coupling types-most of them of the A type, having the ordinary 7,1’-linkage-but also a few of the three less common types B, C, and D. Most of these alkaloids have a stable configuration at the biaryl axis and the plant produces selected representatives of both atropoisorneric series; cf. diastereomeric alkaloids like 30a/b and enantiomeric species like 31. Some of the alkaloids are not stereochemically differentiated at the axis, either because of the flattening influence of a lactone-type bridge (as in 22) or because of the limited sizes of the ortho substitutents (as in 23), so that dioncophyllacine B (24), which has no stereocenters, is even achiral at room temperature. In addition, different oxygenation, hydrogenation, and 0-methylation types also exist. Despite the obviously broad variety of structures, T. peltatum seems to synthesize its alkaloids in a directed and highly controlled way. Two strict synthetic principles are of particular interest: 0

All of these Dioncophyllaceae-typealkaloids lack an oxygen function at C-6, which, by contrast, is present in all Ancistrocladaceae-type

Dioncophylline A

(la)

5’-ODemethyl-B-O-methyl-

Dioncopeltine A (20)

Dioncolactone A (22)

dioncophylline A (ma) OMe

5’-0Demethyl-B-O- methyl7-epi-dioncophylline A (3Ob)

5-ODemethyl-8-0- methyl7-epi-dioncophyllidine A (29)

(+)-Dioncophyllacine A (31)

HO OMe

Dioncophylline B (23)

DioncophyllaaneB (32) Dioncophylline C (24) FIG. 14. Structures of known Dioncophyllaceae alkaloids from T . pelraturn.

Dioncophylline D (28)

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

Ancistrocladine (la)

Ancistrocladisine (33)

from Ancistrocladus heyneanus

I

155

Dioncophylline A (14a)

Dioncophylline B (23)

from Triphyophyllum peltatum

FIG. 15. Structural characteristics of typical Ancistrocladaceae and Dioncophyllaceae alkaloids, exemplified by selected representatives.

0

alkaloids, as isolated by Govindachari et al. (1,2), such as ancistrocladine (la) and ancistrocladisine (33) (see Fig. 15). All of the Ancistrocladaceae alkaloids have the (S)-configurationat C-3, whereas all the Dioncophyllaceae alkaloids are (3R)-configured (except for the sp*-hybridized representatives 31 and 32).

On the one hand, this rule (50,51) demonstrates the close chemotaxonomical relationship of the Dioncophyllaceae and the Ancistrocladaceae-plant families that could be classified taxonomically only with great difficulty in the past (52,52a).On the other hand, it clearly marks a distinct borderline between these two families.

I56

GERHARD BRINGMANN A N D FRANK POKORNY

IV. New Alkaloids from Asian Ancistrocladaceae Species A. Ancistrocladus heyneanus

Govindachari’s pioneering work in the early 1970s, as already reviewed in an earlier article of this series (2), led to the well-documented and reliable structure elucidation of no fewer than six naphthylisoquinoline alkaloids ( l ) ,among them ancistrocladine (la) (3), ancistrocladisine (33) (23),and ancistrocladinine (34)(28).This plant “works” extremely selectively with respect to the synthesis of the alkaloids. Whereas a great variety of different naphthylisoquinolines occur in the aerial parts of the plants, practically only ancistrocladine (la) is found in the roots. More recent work (53)revealed the presence of an additional new alkaloid, named isoancistrocladine (35). This compound had already been prepared by Govindachari by a cis-selective reduction of ancistrocladinine (34) (4,28), and thus was, for the first time, detected as a natural product (53). Indeed, it is hitherto the only naturally occurring, cis-configured naphthyltetrahydroisoquinoline alkaloid with a free, unmethylated NH function. The rare occurrence of this type probably stems from its pronounced reactivity toward oxidants, leading back to the corresponding dihydroisoquinoline (34).This instability is in stark contrast to the corresponding trans isomers or the N-methyl analogs (see below), which are far more stable. Furthermore, such cis isomers are sensitive toward acidor base-catalyzed isomerization to give the more stable trans compounds (30). Such cis-configured naphthylisoquinolines are possibly more widespread than anticipated, and would be found more frequently if isolated under milder conditions. The instability toward oxidants makes it understandable that the ruthenium-mediated degradation (cf. Scheme 6), in the case of 35 gives rise only to 3-aminobutyric acid (U), not to alanine (Scheme 12) (54), since the stereocenter C-l is rapidly destroyed to give intermediate 34 under the oxidative conditions (3533). Several naphthylisoquinoline alkaloids from A. heyneanus have been prepared by stereoselective total syntheses (see Section VII) (29,55-53, among them the new alkaloid 35 (58).

B. Ancistrocladus hamatus From A. hamatus, which is endemic to Sri Lanka, Govindachari et al. (5) had isolated hamatine (lb; see Fig. I ) , along with its predominant atropodiastereomer ancistrocladine (la). Both natural products have meanwhile been prepared by highly efficient total syntheses (29,55)(see

4. T H E

NAPHTHYLISOQUINOLINE ALKALOIDS

157

Me0 OMe

red.

.Me

Me6

Me

Ancistrocladinine (34)

ox.

Me0

Me

lsoancistrocladine (35)

from A. heyneanus (India)

SCHEME12. Isoancistrocladine (35):partial synthesis from ancistrocladinine(34)(4,29,54

and degradation (53)of the natural product.

Sections VII,C,2 and 3). No further isolation work on this plant has been published since the last review in this series (2). C. Ancistrocladus tectorius Of the various alkaloids isolated from this Southeast Asian plant (59,60), only a single new one, named ancistrotectorine (see Section X, Table 11) (61), had been fully elucidated by the time of the last review (2). In the meantime, another new alkaloid, named ancistrocline, the gross structure of which had already been established by a Chinese group (6), could be attributed the full stereostructure 36 (54,58)by spectroscopy and chemical degradation (which was, for the first time, extended to N-methyl-1,3dimethyltetrahydroisoquinolines(62)). The structure, shown in Scheme 13, was confirmed by partial (54) and total (58) synthesis. Ancistrocline (36)is thus the (more stable) N-methyl homolog of isoancistrocladine (35). Further work with A . tectorius is under investigation (63).

D. JOINTSTRUCTURAL PROPERTIES OF ASIANANCISTROCLADACEAETYPEALKALOIDS The structural affiliation of all of the naphthylisoquinoline alkaloids from these three Asian Ancistrocladus species is in complete agreement with the chemotaxonomic Ancistrocladaceae/Dioncophyllaceaerule established above. This is again underlined by the new structures 34 and 35, both of which belong to the Ancistrocladaceae type, i.e., with the (S)configuration at C-3 and an oxygen function at C-6 (cf. Fig. 15).

I58

GERHARD BRINGMANN A N D FRANK POKORNY

Me0 OMe H 0 2 C T ” ‘Me “,NxR

/

(S)-18 R = H

(S)37 R = M e

RuC13, Nal04

1 M ~ O Me

Ancistrocline (36)

H I H02C D N, W R

h;le

D-19 R = H D-38 R = M e

from A. tectorius

(South China)

SCHEME13. Ancistrocline (36).an N-methylated naphthylisoquinoline alkaloid: absolute configuration by chemical degradation (59.62).

V. Alkaloids of African Ancistrocladaceae Species Regarding the above-established chemotaxonomic rule, as based upon the alkaloids of T. peltatum (Dioncophyllaceae)and the Asian Ancistrocladaceae plants, the chemical behavior of the African Ancistrocladaceae species, exemplified by the two West African species, A . abbreviatus and A . barteri, and the (as yet) one and only East African species, A. robertsoniorum, was completely unexpected.

A. Ancistrocladus abbreviatus: A CHEMOTAXONOMIC LINKBETWEEN THE DIONCOPHYLLACEAE A N D THE ANCISTROCLADACEAE? Ancistrocladus abbreviatus (#), a West African liana, occurs in nearly the same geographic region as the Dioncophyllaceae species T. peltatum (65). From this hitherto chemically unexplored plant, a broad spectrum of predominantly unknown alkaloids was isolated.

1 . Ancistrocladine and Hamatine: Typical Ancistrocladaceae Alkaloids Completely in agreement with the above-deduced chemotaxonomic rule, A . abbreviatus was found to contain ancistrocladine (la), albeit in very small amounts ( of lyophilized plant material), in the roots and the stem bark (66), along with its atropodiastereomer hamatine (lb), which was obtained in similar amounts. The known (67) 0-methylancistrocladine

w4%

4. THE

NAPHTHYLISOQUINOLINE ALKALOIDS

159

(39a) and its naturally occurring, previously unknown atropodiastereomer

0-methylhamatine (39b)were obtained, though only in trace amounts (68). All these ancistrocladine-related alkaloids are (3s)-configured and have an oxygen function at C-6, as expected for the constitutents of an Ancistrocladus plant (see Fig. 16).

2 . Dioncophylline A and Its Analogs: Typical Dioncophyllaceae Alkaloids Astonishingly, the two main alkaloids of A . abbreviatus (0.015% of dry weight) were found to have the constitution of an N-methyl derivative of dioncophylline A (14a), and hence of a Dioncophyllaceae type product, i.e., lacking an oxygen function at C-6. Both diastereomeric compounds proved to have a relative trans array of the two methyl groups at C-1 and C-3, thus constituting a mixture of (authentic or enantiomeric) Nmethyldioncophylline A and one of the possible respective diastereomers. Of great interest at this point were the following questions about the absolute configuration at centers and axes. Would A . abbreuiatus, as do all other known Ancisfrocladus species (see above), synthesize these two alkaloids in a (3S)-configuredform and thereby follow the Ancistrocladaceae rule, at least with respect to stereochemistry? Or would the deciding factor be the Dioncophyllaceae-type constitution of the two alkaloids, so that both would be (3R)-configured, as in dioncophylline A (14a) itself? Or would both alkaloids have identical axial configurations (i.e., both M or both P ) , but different configurations at C-3 (and hence at C-l)? These questions could not be answered immediately because the separation of the two compounds turned out to be most difficult. Although diastereomeric, these compounds exhibited very similar chromatographicbehavior, as if they were enantiomers. For this reason, separation techniques were Me0 OMe

Me0

% ;N ; RO

OMe

RO ;% :N

Me0

Me

Me0

Me

laR=H

lbR=H

39a R = Me

39b R = Me

FIG. 16. Typical Ancistrocladaceae-type alkaloids from A . obbreuiarus (66.68).

I60

GERHARD BRINGMANN A N D FRANK POKORNY

used that are normally applied to racemate resolution problems-analytically by chromatography on a chiral phase, and preparatively by enhancing the weak diastereomeric character by derivatization with menthoxy acetic acid as a chiral auxiliary. By this means, diastereomer separation and subsequent cleavage gave the pure alkaloids (Scheme 14). By CD spectroscopy, oxidative degradation, and by partial synthesis of authentic samples from the corresponding N-methyl-free dioncophylline A atropoisomers 14a and 14b, the two new (69) compounds could be clearly identified as authentic N-methyldioncophylline A (40a) and its 7-epimer 40b, the first pure Dioncophyllaceae-type alkaloids isolated from an Ancistrocludus species. Dioncophylline A (14a) itself was also isolated (66) and found to be fully identical with the material previously obtained from T. peltutum (15). Interestingly, unlike the N-methyl compounds, the corresponding 7-epimer 14b could not be detected in the same plant. By contrast, the new alkaloid 4’-O-demethyldioncophyllineA (41a) is accompanied by 41b, its atropisomer (68).Alkaloids 41a and 41b are the first Dioncophyllaceae alkaloids with a free phenolic OH group on the methyl-substituted naphthalene ring. The stereostructure of 41a, for example, was again established by CD spectroscopy, oxidative degradation, and partial synthetic transformation into the known (cf. Scheme 7) 0-methyldioncophylline A (l5a). Alkaloids 41a and 41b are clearly Dioncophyllaceae-typealkaloids, but they occur in an Ancistrocladus plant. (See Fig. 17.)

-<

N-Methyldioncophylline A (40a)

or preparative

-

(ii iv) separation

Me0 Me0

N-Methyl-7-epi-dioncophylline A (40b)

SCHEME14. N-Methyldioncophylline A (Ma) and its atropodiastereomer 40b: separation by racemate resolution techniques (51). Conditions: (i) HPLC on Chiracel OD@;(ii) menthoxyacetyl chloride, DBU; (iii) CC on S O z ; (iv) KOH/MeOH.

4.

161

THE NAPHTHYLISOQUINOLINE ALKALOIDS

4laR=H

41b

FIG. 17. Three other Dioncophyllaceae alkaloids from A . abbreuiarus (68).

3. Ancistrobrevine C: A Mixed AncistrocladaceaelDioncophyllaceaeAlkaloid

Even more amazing is the structure of the new alkaloid ancistrobrevine C (42), another chemical constituent of A. abbreuiatus (36,70).It is the first dihydroisoquinoline with a free phenolic OH group at C-8, which is responsible for its yellow color and its comparatively high polarity. 0Methylation of 42 gives 43 (Scheme 151, the enantiomer of the known Ancistrocladaceae alkaloid ancistrocladisine (33). This is underlined by CD spectroscopy and the oxidative-degradationprocedure, which delivers (R)-3-aminobutyricacid [(R)-18] (36).Again, for the structural attribution, the availability of both 33 and its atropodiastereomer from the first stereoselective total synthesis (56,57) (cf. Sections VII,C,3 and 4) was most useful. From its structural properties, this new ancistrocladisine-related compound ancistrobrevine C (42) is neither a real Ancistrocladaceae alka-

CH2N2, Et20c Me0 Me0

Me0

Me

43 = ent-33

42

1

RuC13, Nal04

......................................................... Me

no alanine (19)

SCHEME IS. Structural attribution of ancistrobrevine C (42) by degradation and stereochemical correlation with ancistrocladisine (33) (36).

162

GERHARD BRINGMANN A N D FRANK POKORNY

loid nor a true Dioncophyllaceae alkaloid, but a hybrid type, since it exhibits structural landmarks of both classes of compounds. It has (R)configuration at C-3, but also has an oxygen function at C-6. 4 . Atropisomeric Dioncolines A : The Inverse Dioncophyllaceael Ancistrocladaceae Hybrid Type

More recently, another interesting pair of atropodiastereomeric alkaloids, dioncoline A (Ma) and its atropodiastereomer 44b (Fig. 18), i.e., the 3-epimers of the N-methyldioncophyllinesA 40a and 40b, were isolated (71).Their inverse-hybrid-typestructures are unprecedented, constituting, so to speak, a missing link, with the structural characteristics of both the Ancistrocladaceae type (the (S)-configuration at C-3) and of the Dioncophyllaceae type (no oxygen function at C-6) alkaloids, but in an opposite combination compared with ancistrobrevine C (42). In contrast to the latter, which seems to be atropoisomerically pure in the plant, the two atropodiastereomeric dioncolines A, 44a and 44b, were isolated in nearly equal amounts Summarizing, it becomes clear that, in contrast to T. peltatum and to the Asian Ancistrocladaceae species, neither of the two characteristic structural features is strictly constant in the alkaloids of A . abbreviatus. Not only the oxygenation at C-6, but also the configuration at any of the stereogenic centers and axes is open to variation, which makes this plant both difficult and exciting to work with.

5 . Ancistrobrevine D: An Ordinary Ancistrocladaceae-Type Alkaloid? Thus, the Ancistrocladus plant produces a stereochemically unusual pair of alkaloids, ancistrobrevine C (42) and the likewise new ancistrobrevine D ( 4 3 , as shown in Fig. 19. Again, the structure elucidation of 45 was achieved by CD spectroscopy, oxidative degradation and, total synthesis of 45 and all its possible 1,3-cis-configuredstereoisomers (72). As already described for N-methyldioncophylline A (40a) and dioncoline A (44a), except for the small difference at C-1, the two alkaloids 42 and 45 belong

FIG. 18. Dioncoline A ( M a )and its atropodiastereomer M b , the first inverse-hybrid-type alkaloids (71).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

163

Me

Me0

42 (Ancistrobrevine C)

45 (Ancistrobrevine D)

FIG. 19. Ancistrobrevines C (42) (36,70)and D (45) (72): alkaloids from a centrally, not axially diastereorneric series.

to a diastereomeric series-but, unexpectedly, not as derived atropobut as “centrodiastereomers.” This underlines that no stereochemical prediction can be made for the alkaloid output of this plant, all of the established “rules” are violated. It is of great interest at which biogenetic stage, and by which substrate properties, the stereochemistry at centers and axes is established enzymatically. Even though the confusing impression predominates that the synthetic properties of A. abbreviatus are excessively diverse, they are by no means chaotic or uncontrolled. Thus, despite the complex mixtures of alkaloids that this plant produces, all of the alkaloids that have been examined so far are enantiomerically pure. 6. New Alkaloids with Unusual Coupling Types Besides the stereochemical variations displayed by A. abbreviatus, it also seems to be a rich source of constitutionally novel naphthylisoquinoline alkaloids with hitherto unprecedented coupling sites. This is exemplified by the structure of ancistrobrevine A (a), as isolated from the stem bark, which exhibits a novel 7,8’-location (73) of the biaryl axis (50,741. Like isoancistrocladine (39, it is cis-configured at C-1 versus C-3, but stabilized by an N-methyl group, like the related alkaloids 36, 44, 45, 55, and 56 (cf. Sections IV,C; V,A,4; V,A,5; and V1,D). The configuration at the centers was established (74) to be (lR,3S) by oxidative degradation (62),and thus, together with the oxygen function present at (2-6, characterizes 46 again as an ordinary Ancistrocladaceae-type alkaloid. The configuration at the axis, by contrast, could not be elucidated unequivocally by CD experiments, due to missing comparison data-a further field of application for modified NOE experiments, in which, as in earlier cases (see also below and Sections II,D,6; VI,A; and VI,D), the axial configura-

164

GERHARD BRINGMANN A N D FRANK POKORNY

Me

Me0

46R=Me 47R=H

NOE

FIG. 20. Ancistrobrevine A (46)and its 0-demethyl analog 47 from A . ubbreuiutus; relative axial configuration as established by long-range NOE interactions (33.50,74).

tion relative to the stereocenters could be established unequivocally by NMR spectroscopy (see Fig. 20) (33). Besides 46, its monophenolic analog 6-0-demethylancistrobrevineA (47) was isolated and fully characterized (74). A likewise unprecedented coupling type is also realized in ancistrobrevine B (&)-the first naphthylisoquinoline with a 5,8’-locationof the biaryl axis (66,75),as shown in Fig. 21. Again, the configuration at the axis, as determined by CD spectroscopy, could easily be verified by specific NOE effects across the biaryl axis, due to the favorably short distances to be covered. Together with closely related work (26,76,77)on other 5,8‘coupled alkaloids (see Sections VI,A and D), this was the first example of the attribution of the relative configuration of axes and centers in this field. From a chemotaxonomic point of view, ancistrobrevines A and B (and their derivatives) can be considered as pure Ancistrocladaceae-type alkaloids.

7 . Chemotaxonomic Position Summarizing, A . abbreuiafus produces an overwhelming variety of very similar compounds, most (although not all) of them occurring as pairs of Me0

OMe OMe

OMe

Me

no

~ . . ~ e

no

, \

Me0

Me

..Me s N.”

OMe Me

Me

48 FIG.21. Ancistrobrevine B (48): stereostructure by specific NOE interactions (66).

4. T H E

NAPHTHYLISOQUINOLINE ALKALOIDS

I65

regio- and stereoisomers. Figure 22 presents all of the naphthylisoquinoline alkaloids identified so far from this productive plant. This list of structures shows that the previously strict chemotaxonomic difference between the Ancistrocladaceae and the Dioncophyllaceae has faded away. Ancistrocladrrs cibbreviutirs violates the Ancistrocladaceael Dioncophyllaceae rule by producing not only typical (expected) Ancistrocladirs alkaloids like la/b and 39a/b, but also pure Dioncophyllaceaetype alkaloids like 14a and 40a/b and, astonishingly, mixed structures like 42 as well. These hybrids belong in part to the Ancistrocladaceae alkaloids because of the oxygen function at C-6, and in part to the Dioncophyllaceae alkaloids, since they have an (R)-configuration at C-3. Even an inversetype hybrid 44,which has the opposite characteristics, has been recently isolated. (See Fig. 23.) It is currently unknown why A . (ihhrevicitrrs does not produce these alkaloids regio- and stereoselectively. Conceivably, this lack of specificity creates better chemical protection against microorganisms and herbivores (see also Section IX), which should find it more difficult to develop deactivation mechanisms against such a broad spectrum of potentially toxic compounds. In any event, A . cibhrevicitrrs is not the only species that synthesizes such a diverse variety of alkaloids. Similar behavior is also observed for A . bcirtrri. 0. Anc~istroclcidusburteri 1 . Alkciloids Isolated: Anc~istroclcidcicc~cre-, Dioncophvllaceae-, and

Hvbrid-Tvpe Nuphthvlisoqr~inolirlrs

Recently, the author’s group has embarked on an investigation of the West African shrub A . harteri, as collected in the Ivory Coast. The work on this chemically unexplored plant has just begun, but the few alkaloids isolated so far demonstrate already that this species does not adhere to the chemotaxonomic rule either. It also produces typical Ancistrocladaceae- and as Dioncophyllaceae-type alkaloids, as well as new, mixed hybrid-type naphthylisoquinolines (see Fig. 24). The only Ancistrocladaceae alkaloids isolated thus far are the well known compounds ancistrocladine ( l a ) and hamatine (lb), which occur as minor constituents (68). As in A . (ibbreuiatus,the main alkaloids are the atropodiastereomeric N-methyldioncophyllines A, 40a and 40b (78).Complementarily to A . abbreuiatus, another main alkaloid is 7-epi-dioncophylline A (14b) (68). apparently in an atropoisomerically pure form, i.e., without the normal 7-epimer dioncophylline A (14a). Alkaloid 14b had not been detected previously. Also, its 4’-O-demethylation product 41b, which is

f II II

II

fI

II

uu

u u

n o

S G

\

'Ei

b

c

FIG.22. The structures of the known alkaloids of A . ubbreuiurus-an overview.

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

167

alkaloids of Ancistrocladaceae type

Dioncophyllaceaetype

and hybrid types

H3CO

42 (Ancistrobrevine C)

H

‘‘a--

H3CO

H3CO

A

tH3

14a (DioncophyllineA) l a (Ancistrocladine) CH3

H3CO 44a (Dioncoline A)

FIG.23. Alkaloids of the Ancistrocladaceae. Dioncophyllaceae. and hybrid type. AncisAncistrocladaceae/Dioncophyllaceaerule: Is it a chemo-

rrocladus obbreuiatus violates the

taxonomic link between the two families?

accompanied by its atropodiastereomer 41a in A. abbreviatus, has now been found to occur stereochemically homogeneously in A. barteri, albeit as a minor alkaloid (68). This plant also produces a new, mixed hybridtype alkaloid, named ancistrobarterine A (49) (79),which is closely related to ancistrobrevine C (42), but with an opposite stereochemical orientation at the biaryl axis. Still, because the OH/OMe substitution pattern at C6/C-8 is also opposite in the two alkaloids, they both have the same formal descriptor P for the axial chirality (80).

2 . Chemotaxonomic Conclusions Apparently, the chemotaxonomic Ancistrocladaceae/Dioncophyllaceae rule is strictly valid only for the “chemically disciplined” Asian Ancistrocladaceae plants, which, without known exception, produce (3S-)configured and 6-oxygenated alkaloids exclusively, and for the

I 68

GERHARD BRINGMANN A N D FRANK POKORNY

Anclstrocladaceae type alkaloids Me0

OM8

(Ancistrobarterine A)

Me

4lbR=H

^.I .

.. I

N

Dioncophyllaceae type alkaloids

FIG.24. A first look at the alkaloids o f A . harrrri-another chemotaxonomic link between the two families?

West African Dioncophyllaceae species T. peltaturn, which produces only (3R)-configuredand 6-deoxygenated alkaloids. By contrast, the West African Ancistrocludus lianas obviously employ the synthetic methodologies of both families. They produce, without noticeable selectivity or stereocontrol, structural landmarks of both classes and thus apparently form chemotaxonomic and geotaxonomic links between the two families. The morphologic and genetic aspects of this botanical relationship are also being investigated by scientists from Missouri Botanical Garden (81). 3. N-Methylphylline: The First Naphthalene-Free I ,3-Dimethyltetruhydroisoquinoline

Another alkaloid 50 (see Fig. 2 5 ) found in this interesting plant might be of great relevance with respect to the presumed biosynthetic origin of the alkaloids (cf. Section VII1,A). This is the first naphthalene-devoid 1,3dimethyltetrahydroisoquinoline50 (82),which had not been found in any of the other plants previously. The occurrence of this isoquinoline moiety supports the biogenetic hypothesis, particularly with respect to the sepa-

4.

T H E N A P H T H Y L I S O Q U I N O L I N E ALKALOIDS

OH

Me

169

MeoT Me0

50 (N-Methylphylline) F I G . 2 5 . Structure of N-methylphylline (50) (82). the first natural naphthalene-free I.3dimethyltetrahydroisoquinoline. and its synthetic precursor.

rate origin of the two molecular halves. Being the precise heterocyclic half o f N-methyldioncophyllines A (40a and 40b, respectively), the new alkaloid 50 was named N-methylphylline (82). The structure of this natural product was readily determined by spectroscopy, and, ultimately, by enantioselective total synthesis starting from 1-(3,5-dirnethoxyphenyI)-2propanone (30,83). Recently, a related tetrahydroisoquinoline building block with a characteristic Ancistrocladaceae-type substitution pattern, was isolated from A . korirpensis (see Section V1.D) (84). C. Anc.istrc)c.ladrr.srohertsotiiorirm Chemical investigation of Ancistroc.ladrrs rohertsotiiorrrtii Leonard, previously the only known East African Ancistrocludrrs species, have just begun. This plant was described botanically a few years ago (85,861. When wounded (e.g., by insects), A . robertsoniorirti~ was found to produce orange crystals (871, which, according to the spectral data, seemed to be the well known (88)naphthoquinone droserone (51). shown in Fig. 26. However, these chemically pure biogenic crystals exhibited a melting point ( 1 19°C)that was far too low for droserone (181°C).Nonetheless, X-ray structure analysis performed directly on the natural crystals (87,89)confirmed the identity of this naphthoquinone, which is apparently produced as a chemical weapon. The question remains: Why does droserone have such a low melting point in its biogenic form-is it a new modification‘?In any event, after a single recrystallization of the natural material from organic solvents, the melting point was “correct.” Different from the biogenic crystals, these classically produced crystals were found to be unsuitable for X-ray structure analysis. Preliminary investigations of the nitrogen-containing constituents of this plant species show the presence of a large number of alkaloids, among

170

GERHARD BRINGMANN A N D F R A N K POKORNY

Ww Me

0

51 (Droserone)

El but:

m.p. of ’biogenic crystals” = 1 1 9°C strongly differing from lit. (m.p. = 18lOC)

FIG. 26. Ancisrrocladus rober/soniorrrrn produces crystalline droserone (51) when wounded (86J7.89).

them, ancistrocladine (la) (90),an Ancistrocladaceae-type alkaloid. Further investigations of A . robertsoniorum are in progress. D. Ancistrocladus korupensis Scientifically, the “youngest” Ancistrocladus species is A . korupensis, a Central African species recently detected in the Korup National Park in Cameroon (91). This species was found to produce an unprecedented type of biaryl alkaloids-the michellamines. Because of the structural novelty and the pharmacological importance of these intriguing new compounds, they will be treated, together with the co-occurring ordinary (i.e., monomeric) naphthylisoquinolines, in Section VI.

VI. The Michellamines: A New Class of Naturally Occurring Quateraryls and Related Compounds A. CONSTITUTION A N D RELATIVECONFIGURATION OF DIMERIC NAPHTHYLISOQUINOLINES

In connection with a broad anti-HIV-screening program, the U.S. National Cancer Institute (NCI) has tested a large number of plants, about 23,000 extracts of 7,000 plant species (92,93).One of the most promising

4.

171

T H E NAPHTHY1.ISOQUINOL.INE ALKALOIDS

candidates was an Atic~isfroc~lrrdus species from Cameroon. The extracts of this species, which initially was supposed to be the species A t i c ~ i s t r o c ~ l ~ d u s cibbrcwiutrrs, (see Section V,A) exhibited remarkable activity against the human immunodeficiency virus (HIV) (76.Y4). In the meantime, it has become evident that this discovery is exciting in many respects. First, the plant species was found to be not A . cihhrcwicitus, but a new, hit herto undescribed Anc.istroc.ladrrs species, apparently unknown even in folk medicine. It has now been described botanically and named A . konrpetisis. after the Korup National Park in Cameroon ( 9 0 . Second, the active antiviral secondary metabolites were found to be unique quateraryls, dimeric naphthylisoquinoline alkaloids 52 (see Fig. 27), named michellamines (76). They are highly polar, possessing six free phenolic groups and two amino functions and represent constitutionally symmetric dimers of a 5,8'-coupled naphthylisoquinoline. The formal monomer is very similar to ancistrobrevine B (48) from the authentic A . abbreviarrrs (66,75).Apart from its unusual constitution, its stereochemistry is also interesting. It has four stereogenic centers, two stereogenic biaryl axes, and a configurationally unstable central biaryl axis between the two constitutionally identical molecular halves. The NCI researchers found the first alkaloid of this type, michellamine A. to consist of two spectroscopically identical halves; i.e., the two monomeric naphthylisoquinolines are either homomorphous or enantiomor-

Me

OH

naphthylisoquinoline ancistrobrevine B: Me

OH

Me0

OMe

& OH

Me

52

L

48

* stereogenic axes and centers o

configurationally labile axis

FIG. 27. Constitution 52 of the michel1;imines (76) and comparison with the similarly 5.8'-coupled monomeric alkaloid ancistrobrevine B (48).iis isolated from the true species A . cihhrc~oiiitri.r( 6 6 . 7 s ) .

172

GERHARD BRINGMANN A N D FRANK POKORNY

phous to each other, the latter case (meaning a meso-type structure) being excluded by the observed optical activity of the compound. As was done independently and simultaneously for ancistrobrevine B (48) (cf. Section V,A,6, Fig. 21) (66,75), the relative configurations at centers versus axes were elucidated by NMR spectroscopy (76) (see Scheme 16).Accordingly, both molecular halves of michellamine A have a trans configuration at the two stereocenters and a relative unlike-configuration of C-3 (or C-1, respectively) versus the axis, realized by a (IS,3S,SM)-configuration (or its enantiomer). Similarly, michellamine C, which was found in only smaller quantities, was shown to have two identical naphthylisoquinoline moieties, yet both with relative like (axial versus central) configurations within the halves. By contrast, michellamine B, which has the same constitution and the same relative trans configuration at the centers, consists of two spectroscopically different and hence diastereomorphous halves, one of which exhibits an unlike-, and the other a like-configuration of centers versus axes (76). -

Relative Configuration within the Halves

trans (/ike) like

- C-1 vs. C-3: trans (like) - C-1IC-3 vs. axis: unlike

+ e.g. 1S,3S,5P

+ e.g. 1 S,3S,5M

(or enantiomer)

(or enantiomer)

Me

- Composition of the Entire "Dimer" M. Boyd eta/.,

V

V

V

2 homomorphous

2 diastereomorphous

2 homomorphous

"halves'

'halves'

'halves'

SCHEME16. Relative configuration of michellamines A, B, and C within the "halves" by NOE experiments and the stereochemical relationships between the two moieties (76); for correct absolurc configuration, see below.

4.

173

THE NAPHTHYLISOQUINOLINE ALKALOIDS

B. ELUC~DATION OF THE ABSOLUTE CONFIGURATION AT CENTERS A N D AXES

I . Stereochemical Considerations Because of the trans configuration at the centers and the presence of two diastereomorphous halves and made plausible by the fact that the first alkaloids isolated from the Ancistrocladaceae (namely, from the Asian Ancistrocladus species; cf. Section IV) all had an (S)-configuration at C-3 (1,2) michellamine B was initially proposed to have the absolute configuration 52x, in which both halves are (lS,3S)-configured at the centers and the axes are configurationally different (76).

Me OH

OH

OMe

Me

OH

I Me

OH

Me'"

mirror plane

FIG.28. Possible absolute configurations of michellamine B .

Me

174

GERHARD BRINGMANN A N D FRANK POKORNY

But michellamine B might also have been the enantiomer of 52x, also with an ( M ) - and a (P)-configuration at the axes, but now with (R)configurations at all of the centers (see Figure 28). By contrast, the two additional stereoisomers 52y and its enantiomer ent-52y, which were not taken into consideration, are also in complete agreement with the NMR data, since the diastereomorphous character of the two molecular halves does not necessarily have to result from different axial configurations. It is also conceivable that the two halves have identical axial configurations (both ( M ) or both ( P ) ) ,but different configurations at the centers (95). And following the stereochemical variability that became manifest from other African Ancisrrocladus species (see Section V), this possibility could not be excluded a priori. 2 . Degradation Experiments A simple way to distinguish between these alternatives originated from the above-mentioned(see Section II,D,7) oxidative-degradationprocedure (35).The resulting amino acids, 3-aminobutyricacid (18) and alanine (19), were formed in a high stereochemical purity, both in the (R)-configured form (95) (see Scheme 17). This is in agreement only with the stereostructure enr-52x, and excludes the initially proposed structure 52x (which would have given (S)-configuredamino acids, exclusively), as well as 52y and ent-52y (which would have given racemic amino acids). Concluding, if the molecular halves of michellamine B are diastereomorphous to each other (as evident from NMR) and the centers are homochiral (all ( R ) ) ,it must be the axes that are different; hence one axis is ( M ) - and the other is (P)-configured. Accordingly, michellamine B unequivocally has structure ent-52x (52b). It is thus a constitutionally symmetric coupling product of two atropodiastereomeric 5,8’-linked naphthylisoquinoline alkaloids. From the chemotaxonomic point of view (compare Sections III,C and V,A,7), both of these monomers have to be considered as Ancistrocladaceae/Dioncophyllaceae hybrid types, since they have the (Rhconfiguration of C-3 and an oxygen function at C-6. Also, the degradation of michellamine A (Scheme 17)gives enantiomerically pure (R)-configured amino acids 18 and 19 exclusively (77). Given the known relative unlike-configuration at axes versus centers and the now established (R) absolute configuration at the two stereocenters, michellamine A must have the structure 52a (see Fig. 29), i.e., the enantiomer of what had been assumed arbitrarily (76),both halves being (R,R,P)configured. Therefore, michellamine A is not only a constitutionally, but also a configurationally symmetric (and hence C,-symmetric) dimeric naphthylisoquinoline alkaloid.

4.

175

THE NAPHTHYLISOQUINOLINE ALKALOIDS

2x

52

RuCI~ I Nal04 *

NH2

(R)-l8

1) MeOH I HCI

2) 'Mosher-CI"

analysis

All michellamines

have 'R,R-R,R%onfiguration

SCHEME17. Determination of the absolute configuration at the stereocenters of michellamines by oxidative degradation (77,95).

Similarly, the third related alkaloid isolated, michellamine C, which was found only in smaller amounts, was established to have a C,-symmetric stereostructure 52c,also consisting of two identical, homomorphous molecular halves, both with the (R,R,M)-configuration (77). Hence, the three michellamines have homochiral isoquinoline parts; i.e. they are stereochemically identical at all the centers and thus differ only by the axial configurations. Together they represent a complete series of all the conceivable atropodiastereomers of this constitutionally symmetric dimer. 3 . CD Spectroscopy

An additional, independent confirmation of the absolute configuration at the axes was attained through an investigation of the circular dichroism of the alkaloids (26,94).Figure 30 shows the CD spectra of michellamines A, B, and C. As expected, nearly opposite CD curves were observed for michellamines A (52a) and C (52c),completely in agreement with their opposite axial configurations.Whereas the curve for michellamine B (52b), with its two heterochiral axes, was found to be far less intense. Besides these more qualitative rationalizations, an unambiguous, independent proof of the absolute configurations was difficult to achieve either empirically, because of the unprecedented structural type, or computationally, because of the drastically larger molecular size. A useful simplification resulted from the finding that the chiroptical contributions of the particular stereogenic elements were shown to be largely additive: The CD spectra of michellamines A and C add up to virtually the same spectrum as that

176

GERHARD BRINGMANN A N D FRANK POKORNY

Me

M

Me

OH

Me

bH

Me

52b (Michellamine B)

52a (Michellamine A) Me

OH

52c (Michellamine C) FIG.

29. The absolute configurations of michellamines A , B, and

c (77,94.95).

of michellamine B (with double intensities) (26). Thus, in a first approach, the measured CD spectra of michellamines could be compared, either empirically, with the experimental spectra of known structurally related monomeric alkaloids, or theoretically, with the predicted spectra of such halves. Comparison with the experimental and theoretical CD spectra of the similarly 5,8’-coupled known (66) alkaloid ancistrobre-

177

4. T H E NAPHTHYLISOQUINOLINE ALKALOIDS 52a - c

400

-

48

h

-

A ' * \ J ,

;..,

.\"h

'c;-200 .

a

-400 .

200

230

260 290 320 wavelength A (nm)

350

-600 200

230

260 290 320 wovelength A (nm)

FIG.30. Experimental CD spectra (left side) of michellamines A ( 5 2 ~()--and C (52c) (-----), and (right side) of ancistrobrevine B (48) (---

(......),

),

350

B (52b)

).

vine B (48) (see Fig. 30) fully confirmed the structural attribution elaborated above. This was further underlined by a comparison of the experimental CD spectra of michellamines with the predicted spectra of the authentic-yet at that time hypothetical-molecular halves. These monomeric naphthylisoquinolines, the korupensamines, which were detected a short time later (see Section VI,D), showed experimental CD spectra (84) that were in very good agreement with the predicted spectra (26). C. BASE-CATALYZED INTERCONVERSION OF MICHELLAMINES During the isolation work on michellamines, it was observed that the shorter and milder the isolation procedure was, the less michellamine C (52c) was isolated. In the course of the investigation of this phenomenon, the remarkable observation was made that the michellamines, although configurationally stable in neutral media, can be atropisomerized under basic conditions. 'H-NMR analysis of michellamine A (52a) in 1 ml MeOHd4 and 0.5 ml of 0.5 M NaOD/D,O showed a slow conversion of the compound to a mixture of michellamines A, B, and C (ca. 3 : 3 : 1) over a period of 7 days (77,94). Likewise, michellamine B (52b)under identical conditions was converted to a similar mixture. This reaction (Scheme 18) could also be performed preparatively, thereby making the minor OH-

52a

OH-

= 52b = 52c

SCHEME18. Base-catalyzed interconversion of michellamines A-C (77).

178

GERHARD BRINGMANN A N D FRANK POKORNY

compound michellamine C (52c) available in sufficient amounts for further testing. The observations suggest that 52c may not be a genuine natural product, but rather an artifact formed in the course of the isolation procedure. D. KORUPENSAMINES: THEMONOMERIC MICHELLAMINE HALVES Further thorough chemical investigation showed that some of the fractions devoid of HIV-inhibiting activity also contained alkaloids, namely ordinary (i.e., monomeric) naphthylisoquinolines, subsequently named korupensamines (84,96). Korupensamines A (53a) and B (53b) represent the monomeric halves of the michellamines: Michellamine A (52a) is the 6’-coupled (73) dimer of 53a, michellamine C (52c) is the constitutionally symmetric 6’-dimer of 53b, and michellamine B (52b) is the crosscoupling product of 53a and 53b. Furthermore, two related alkaloids were isolated-korupensamine C (54), the 5’Omethyl analog of 53a, and korupensamine D (59, the N-methylated 3-epimer of 53a. By this N-alkylation, the cis-array of the 1,3-methyl groups is again stabilized. (See Fig. 31) The relative configurations at centers versus axes were determined by NOE experiments (see Fig. 32), and the absolute configurations at the centers were established by oxidative degradation to the easily analyzable OH

OMe

OH

OMe

Me0

OMe

Me

Me \

R N,

HO

6%

:

Me

HO

H

53a OH

54

53b OMe

R “Me

HO

55

Me

56

FIG.3 I . Korupensamines A-D and the free tetrahydroisoquinoline 56, other new alkaloids from A . korirpensis (84.96).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

179

Mi

53a:R=H 54 : R = M e

53b

55 FIG.32. Selected NOE interactions of korupensarnines:relative configurations at centers and axes (84).

amino acids. The deduced absolute configuration at the axes could be confirmed by experimental CD spectroscopy (84,96) and, in the case of korupensamine A (53a), also by comparison with the predicted (cf. Section VI,B) CD spectrum (26).Furthermore, the absolute configuration of korupensamine A (53a) was independently elucidated by an X-ray structure analysis of this tris-p-bromobenzenesulfonate derivative (84). Furthermore, a new naphthalene-devoid 1,3-dimethyItetrahydroisoquinoline alkaloid 56 (see Fig. 31) was found in the same plant (84). Comparable to 50, a similar isoquinoline building block with a Dioncophyllaceae-type substitution pattern previously isolated from A. barteri (82) (see Fig. 25, Section V,B,3), 56 is the first free Ancistrocladaceae-type 1,3-dimethyltetrahydroisoquinolinealkaloid. As such, it is the free isoquinoline half of ancistrobrevine D (45) (see Fig. 19, Section V,A,5) and, simultaneously, the 6-0-methylated heterocyclic moiety of korupensamine D (55).

180

GERHARD BRINGMANN A N D FRANK POKORNY

E. CHEMOTAXONOMIC POSITION OF

THE

NEWSPECIES A . korupensis

The alkaloids of A . korupensis, the michellamines, as well as the korupensamines A-D, exclusively exhibit the rare 5,8’-linkage type, hinting at an unexpectedly regioselective biogenetic coupling reaction in an African Ancistrocladaceae species. The korupensamines mainly differ in their configurations at the axes and at C-3, and in their degree of 0- and Nmethylation. They all have a 6-O-function in the isoquinoline part, but only korupensamine D (55) is a typical Ancistrocladaceae-type alkaloid. All of the other korupensamines have an (R)-configuration at C-3 and must therefore be regarded as Ancistrocladaceae/Dioncophyllaceae hybrid types-not unusual for an African Ancistrocladaceae species (cf. Section V). By contrast, no typical pure Dioncophyllaceae-typenaphthylisoquinoline alkaloid has yet been isolated from A . korupensis, which clearly distinguishes this Central African Ancisfrocladus species from the West African ones. But the most obvious and hitherto unique property is the capability of producing dimeric naphthylisoquinolines (the michellamines) along with ordinary monomeric naphthylisoquinolines and the naphthalene-free isoquinoline 56.

VII. Stereocontrolled Synthesis of Mono- and Dimeric Naphthylisoquinoline Alkaloids In past years, great success has been achieved in the chemical preparation of naphthylisoquinoline alkaloids, either by partial synthesis starting from naturally occumng alkaloids or by stereoselective total synthesis from inexpensive synthetic precursors. The syntheses are of significant value as part of the previously described work of structure elucidation. Moreover, synthetic work helps to make the alkaloids and their structural analogs available for biological testing. Finally, the work of architecturing and constructing intriguing molecules like the naphthylisoquinoline alkaloids, with their central and axial elements of chirality, represents a thrilling challenge that can be satisfied only by reaching the authentic synthetic goal, not by just making a similar model compound. Moreover, the realization of synthetic targets, as dictated by the natural lead, often warrents the elaboration of novel methodologies to make the synthetic goal attainable. Thus, desire to synthesize naphthylisoquinoline alkaloids triggered the elaboration of a novel procedure for the regio- and stereoselective construction of even highly hindered biaryl axes (see below) (97).

4. T H E

181

NAPHTHYLISOQUINOLINE ALKALOIDS

A. PARTIAL SYNTHESES OF NAPHTHYLISOQUINOLINE ALKALOIDS Based on the spendid synthetic capacities of the plants themselves, a rational way to prepare natural or modified naphthylisoquinolinealkaloids is partial synthesis from one of the readily available principal alkaloids isolated from natural material. Furthermore, the transformation of a questionable natural product into a known representative, or a known joint (or enantiomeric) derivative thereof, is often useful for its structural elucidation. Typical transformation reactions are the stereoselective reduction of dihydroisoquinolines to cis- or trans-configured tetrahydroisoquinolines, N - and O-methylation, and catalytic dehydrogenation reactions. As several examples have been mentioned in connection with the structure elucidation work (cf. Schemes 4,7,10,12, and 1 3 , this aspect will not be discussed in this section.

B. TOTALSYNTHESIS OF THE ALKALOIDS: ACCESSTO MOLECULARMOIETIES

THE

Probably of greater importance is the ab initio construction of the alkaloids by total synthesis, thus allowing the preparation of authentic natural products, as well as selected regio- and stereoisomeric analogs even with “unnatural” functionalities. A prerequisite for the first total synthesis of naphthylisoquinoline alkaloids was the development of efficient procedures for the synthesis of the possible naphthalene and isoquinoline building blocks and for the directed, i.e., regio- and stereoselective, construction of highly hindered biaryl axes. 1 . Regio- and Stereoselective Synthesis of the Chiral Isoquinoline Moieties

For the elaboration of flexible total syntheses not of one naphthylisoquinoline alkaloid, but of a series of these interesting compounds, a broad spectrum of approaches was tested. These approaches, briefly sketched in Scheme 19, ultimately resulted in direct synthetic access to practically any desired regio- and stereoisomeric forms of the isoquinoline building blocks 60 of the alkaloids. In addition to the biomimetic cyclization of appropriately protected P-polycarbonyl precursors 57 (x,y = e.g. -OCH,CH,O-, pathway A) (98-100), which is especially important biomimetically (cf. Section VIII ,B), Pomerantz-Fritsch-type pathways B and C were found to be successful (2,13).The required aminoacetal 59 is synthetically available not only from the chiral pool (i.e., from the amino acid L-alanine), but, far more easily and in both enantiomeric forms, by stereoselective reductive amination of the corresponding keto-

182

GERHARD BRINGMANN A N D FRANK POKORNY M e 0 OMe

RO

Rb

Me.

58

.YP 60

Me0 OMe

"RO

RO

~

RO

61

N

Me

O H 62

63

SCHEME 19. Synthetic pathways lo all possible isomeric 6.8-dioxygenated I .3-dimethyl1.2,3.4-tetrahydroisoquinoline methyl ethers 60 (R = H or Me). By similar techniques, the (3R)-configured enantiomers can also be synthesized.

precursor 62 (101,102)-an efficient way to such useful functionalized N containing chiral building blocks. Alternatively, the first stereogenic center can be established in the form of the substituted I-arylethylamine 63 (103-106), also a representative of a useful class of efficient chiral building blocks. It may be used for the preparation of aromatic natural products with a benzylic stereocenter, and for chiral target molecules that do not possess aromatic rings. This is achieved by regioselective cleavage of the aromatic ring after a Birch reduction (107,108).In the Pomerantz-Fritsch approach, the use of an additional oxygen function for the activation of the ring-closure position and its subsequent reductive elimination is necessary. A fourth approach (pathway D ) first builds up the stereocenter at C-3 by reductive amination of arylpropanones of type 61 (109,110) to the corresponding arylisopropylamines (30). This hitherto most successful pathway will be discussed in some more detail. Scheme 20 shows, in a simplified form, this efficient approach. The stereoinformation at C-3 is introduced by asymmetric reductive amination, using l-phenylethylamines of either enantiomeric series as chiral auxiliaries. Thus, not only 64, but also its (R)-configured enantiomer (ent-64), can be prepared in high yield. One possibility for the stereocontrolled construction of the stereocenter at C-1 is the diastereoselective Pictet-Spengler reaction of 64 (e.g., with R3 = benzyl) with acetaldehyde, leading to trans products

~

4.

R'. R~ = H. Me. i+r. etc.

( R k COCH3) POC13

66

183

THE NAPHTHYLISOQUINOLINE ALKALOIDS

I

BischlerNapieralski

67

68

SCHEME20. Directed preparation of stereochemically homogeneous di- and tetrahydroisoquinolines of any desired configuration; for the preparation of the corresponding enantiomers, (R)-1-phenylethylamine is used instead (30).

65. An alternative approach, based on a Bischler-Napieralski cyclization reaction of the corresponding acetamide 64 (R3 = COCH,), delivers the dihydroisoquinoline67, which can be reduced highly selectively either to the cis-configured tetrahydroisoquinoline 66 (in this context, cf. Section II,D,4) or to the corresponding trans isomer 68. Given this useful diastereodivergent reduction of 67, the oxidative transformation of the corresponding tetrahydroisoquinolines to 67 and subsequent reduction is another possibility for the generation of the required stereoisomer. For stereoelectronic reasons, probably due to the axial position of H-1 ,cis-configured tetrahydroisoquinolines like 66 are far more readily oxidized to 67 than the corresponding trans isomers 68. Some cisconfigured representatives even undergo spontaneous oxidation and have to be handled under a N2 or Ar atmosphere (30) (cf. Section IV, A). By contrast, the oxidation of a trans isomer, especially at the level of an entire naphthyltetrahydroisoquinoline,to give the dihydro target molecule, may become a critical step, as in the first total synthesis of ancistrocladisine (33) (cf. Scheme 29, Section VII,C,3), which is attained only with strong oxidants like KMn04 (56). In all of these reactions, solutions were also obtained for problems of regioselectivity for R' # R2, as for instance in the preparation of the enantiomer of 69, the particular monophenolic/mono-0-methylatedprotected heterocyclic half of ancistrocladine (la) (30,IZI);but these solutions will not be discussed in detail here. In this way, all imaginable heterocyclic moieties of Ancistrocladaceae alkaloids are synthetically attainable. And, given the fact that these reactions can also be performed in the

184

GERHARD BRINGMANN A N D FRANK POKORNY

enantiomeric series, the hybrid-type (i.e., (3R)-configured)alkaloids are attainable as well. All of these reactions proved to be so efficient that they were even applied to the preparation of the isoquinoline moieties of the Dioncophyllaceae alkaloids, despite the necessity of subsequently removing the 6oxygen function reductively. This is done (30)e.g., via the tetrazole method (112), as exemplified in the preparation of the Dioncophyllaceae-related building block 72 (see Scheme 21). In a modified form, i.e., via the corresponding 0-tnflate, this method was applicable even to the deoxygenation of preformed naphthylisoquinolinecompounds (41)-a useful reaction not only in the course of structure elucidation, but also for the directed total synthesis of dioncophylline C, for example (24) (see Schemes 10 and I I , Section III,A,2), from which the oxygen function at C-6, used as a transient bridgehead for the coupling procedure, could subsequently be eliminated. Thus, starting from joint synthetic precursors, rational and flexible synthetic pathways are available for the preparation of any of the imaginable heterocyclic halves of the naphthylisoquinolinealkaloids. In addition, for the synthesis of the naturally occurring naphthalene-free 1,3-dimethyItetrahydroisoquinolines, these compounds constitute useful precursors. Starting from readily available precursors, the previously mentioned (see Sections V,B,3 and VI,D) alkaloids 50 (N-methylphylline) and 56 (Fig. 33) were synthesized for the first time (68,113).

2. Preparation of the Naphthalene Moieties For the preparation of the isocyclic moieties 73 of the naphthylisoquinoline alkaloids, biomimetic and nonbiomimetic strategies can be applied. Thus, by consecutive highly regioselective aldol-type cyclization of the modified poly-P-carbonyl precursor 57, the authentic (i.e., methylsubstituted) naphthalenes 73 can be obtained in good yields (98,99,114,115). Alternatively, for the synthesis of appropriately halogenated or additionally functionalized naphthalene building blocks, nonbiomimetic syntheses via precursors of the general type 74, may be preferred. /N=N PheNyN

/N=N

ph-~yh

= p.:

CI

qc-c:l-

HO

70

Me

Me0

69

Me0

Me

71

Me0

Me

72

SCHEME21. The reductive elimination of the 6-oxygen function by the tetrazole method: the pathway to Dioncophyllaceae-type tetrahydroisoquinolines (30).

4.

185

THE NAPHTHYLISOQUINOLINE ALKALOIDS

50

56

FIG. 33. Naturally occurring naphthalene-free tetrahydroisoquinolines 50 (83) and 56 (113) as prepared by total synthesis.

These can be prepared from the corresponding 2-bromo-5-alkoxybenzaldehyde. For the annulation of the second ring, the introduction of the C4unit can be achieved by a Wittig- or a Stobbe-type approach (116,117). Scheme 22 shows some representative naphthalene building blocks 73a-c used in the total synthesis of selected naphthylisoquinoiine alkaloids (17,29,55,56,58,116).For the direct preparation of target alkaloids with a specific OH/OMe substitution pattern, the use of 0-isopropyl or 0-acetyl protective groups proved to be most convenient (39,42,116,118).The final cleavage to liberate the phenolic OH group on the level of the entire naphthylisoquinoline imposed no problem (for selected examples, see Sections VII,D and VI1,E).

74

57 (Y = H)

Me0

OM0

C02H

Bl

73a

CO2H Br

73b

Me Bl

73c

SCHEME 22. Biomimetic and nonbiomimetic pathways to appropriately substituted naphthalene building blocks and selected examples 73a-c.

186

GERHARD BRINGMANN A N D FRANK POKORNY

c. DIRECTED PREPARATION OF THE AXISBY INTRAMOLECULARCOUPLING I . The Bridge Concept Axial chirality is present in a broad spectrum of natural products with many different structures, biological activities, and biosynthetic origins (119-122). These can be very simple, constitutionally symmetric compounds without further stereoelements, thus giving rise to atropoenantiomers. Or they can be complex, like the naphthylisoquinoline alkaloids, consisting of two different moieties, and they may possess additional stereocenters, so that, for example, the alkaloids ancistrocladine (la) and hamatine (lb), are rotational diastereomers. Yet, despite the increasing importance of natural and unnatural biaryl compounds-and despite enormous efforts to develop methods for the directed, i.e., regio- and stereoselective, construction of stereochemically highly hindered biaryl axes (97,123-125)-most procedures that rely on CC-bond formation with simultaneous asymmetric induction at the axis frequently suffer from low chemical and optical yields, especially the strongly sterically hindered biaryl products. Frequently, only one particular stereoisomer can be prepared, and no possibility exists for recycling the undesired atropisomer. This makes it understandable that there are essentially no procedures that are truly, applicable to the stereoselective total synthesis of functionalized natural target molecules. Given the lack of appropriate biaryl coupling procedure, for the total synthesis of naphthylisoquinoline alkaloids a novel procedure for the regio- and stereoselective construction of biaryl axes was developed (126), exemplified by the specific biaryl substitution pattern 75 (see Scheme 23). This array, with a C,-unit and an oxygen function in opposite ortho positions next to the axis, is present in ancistrocladine (la), hamatine (lb), and many other naphthylisoquinolinealkaloids (cf. 14,20, and 33), as well as in many other natural biaryl systems (127). According to this new concept, the biaryl coupling, which, in the case of l a could not be achieved intermolecularly, could be performed intramolecularly, using the CI-and 0-functionalities as bridgeheads. Such a bridge-an ether or an ester function as in 77-is easily built up from 78 and 79, allowing the intramolecular aryl coupling to form a favorable six-membered ring ether or lactone 76. Then it is finally cleaved to afford the target molecules 75. As shown in the following sections, this concept allows for high coupling yields and a reliable regioselectivity. Moreover, it can even be performed with remarkably high stereocontrol, based on a new principle in stereoselective synthesis.

4.

Me0 OMe

Me0

Me0

l b (Hamatine)

g

q$x cJo

R

R

R

Me

78

R

R

77

76

75

OMe

Me

l a (Ancistrocladine)

R

187

THE NAPHTHYLISOQUINOLINE ALKALOIDS

X = 0 or H2

R

79

SCHEME23. Retrosynthetic analysis of a novel directed, i.e., regio- and stereoselective, biaryl synthesis.

2 . Synthesis via Cyclic Ethers An early application of this principle was the synthesis of the naphthylisoquinolines 3a,b (see Scheme 24) (12-14, 128). As mentioned in Section 11, this first total synthesis of a naphthylisoquinoline alkaloid did not confirm the structure 3 published (10) for the natural product isotriphyophylline. Still, the synthetic product rac-3 obtained could be dehydrogenated and 0-methylated to give the fully aromatic naphthylisoquinoline rac-9 (12), thereby confirming the constitution of this natural product as described earlier (10).

Me0 Me0

rac-80

rac-81

rac-9

SCHEME24. An early application of the ether methodology (2,/3,/4,/28): synthesis of rac-3, the postulated (10)structure of isotriphyophylline, and rac-9 (12).

188

GERHARD BRINGMANN A N D FRANK POKORNY

Another persuasive example of the practicability of this novel concept is the first total synthesis of the alkaloids ancistrocladine (la)and hamatine (lb) (see Scheme 25) (55). The intramolecular coupling of 82, i.e., after pre-fixingthe two molecular moieties via an ether bridge, is easily achieved by Pd-catalysis, showing that the procedure works perfectly even for highly hindered target molecules, such as l a and lb. This example demonstrates that the bridge fulfills several tasks at the same time. It results in good coupling yields and reliable regioselectivity. More interestingly-and fundamental for the concept-it drastically lowers the isomerization barrier at the axis. In contrast to the final target molecules ancistrocladine (la) or hamatine (lb), the ether-bridged biaryl83 isomerizes even at room temperature; with half-lives of a few hours for the helicene-type distorted atropisomers 83a and 83b, this interconversion is slow enough for the helimeric mixture to be conveniently resolved. The desired atropodiastereomer, 83b, with the relative configuration of hamatine (lb), can be reductively transformed into the natural product. The other (in this case undesired) atropisomer 83a can be equilibrated by standing at room temperature and can thus be recycled by renewed resolution, thereby exhibiting chiral

Me0

Me0

OMe

OMe

Me0

recycling by equilibration:

Me

AT (0.g. 25OC)

"relatively slow. Me0

82

OMe

@ I

1

0

.sMe

-

1 2) ) Li.HgPd-C biphenyl

lb

Bzl Me0

Me 83b

helicene-like distorted atropisomers

SCHEME25. Total synthesis of hamatine ( l b ) or ancistrocladine (la): chiral economy with respect to rotational isomerism (55).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

189

economy with respect to axial chirality. By a similar procedure, ancistrocladine (la) can also be prepared in high overall yield (55).

3. Atropisomer-Selective Synthesis via Lactone-Bridged Biaryls a. With Thermodynamic Stereocontrol. A significantly lower rotational barrier is found for the corresponding lactones 85a/b.Although still split into spectroscopically observable helimers, 85a/b interconvert much more rapidly than the cyclic ethers 83a/b, with half-lives of slightly less than 1 minute (97,125).Due to the central chirality present in the molecule, the helimeric ratio is not 1 : 1, but nearly 5 : 1 in favor of 85a (for R = benzyl), and can even be upgraded to 100 : 0 (for R = COCF,). Out of this equilibrium, the lactone ring can be opened with retention of configuration at the axis to give the natural product ancistrocladine (la)nearly exclusively-a thermodynamically controlled induction of the axial configuration (Scheme 26). Using this strategy, the related alkaloids isoancistrocladine (33,ancistrocline (36),and ancistrocladinine (34)were also prepared with high selectivities (58). (See Fig. 34.) The methodology could successfully be applied even to the synthesis of dioncophylline C (U), despite the lack

Me0

& WN;:' OMe

0

I

0

Br

'relatively fast.

11 -

Me0 Me0

'1OO:o' for R = COCF3

OMe

Me

04

.,M~ 0 $ p R Me0

'Hamatine type', not detected for R = COCF3

Me 85b

SCHEME 26.

A thermodynamically controlled atropisomer-specific synthesis of ancis-

trocladine (la) (29,97.125,129).

190

GERHARD BRINGMANN A N D FRANK POKORNY

Me0 OMe

Me0 OMe

Me

Me *.Me

Me0

Me

HO

Me

Me0

Me

Me0

Me

35 R = H 34 24 36 R = M e Fic. 34. Further alkaloids prepared by the lactone methodology with thermodynamic stereocontrol.

of an oxygen function at C-6 as a bridgehead (42). It was, nonetheless, synthesized initially with this oxygen function, which was subsequently reductively removed via the corresponding triflate (cf. Scheme 11, Section I1 I, A ,2).

b. With Kinetic Stereocontrol: The Atropisomer-Selective Cleavage of “Axially Prostereogenic” Biaryl Lactones. The effect of the bridge, which drastically lowers the atropisomerization barrier, has remarkable consequences for less hindered biaryls such as dioncophylline A (14a), which has only three ortho substituents adjacent to the axis, but is still configurationally stable. In this case, the cyclic precursor 12, which is not split into distinct, spectroscopically observable atropisomers even at low temperature, is hence “axially prostereogenic” (17). As already mentioned in Section II,D,2 (see also Scheme 2), this conformational lability allows a completely new strategy for stereoselective biaryl synthesis, the directed twisting of a stereochemically undifferentiated axis. Assisted by the chirality already present in the molecule, i.e., by an internal asymmetric induction, the cleavage process can be controlled in such a way that, depending on the choice of the hydride transfer reagent, one obtains either of the two atropodiastereomeric alcohols, ent-Wa, leading to dioncophylline A (14a),or, optionally, its 7-epimer ent-Wb, the precursor to 14b, in high selectivity (Scheme 27) (18). In addition to its preparative value, this conceptionally new approach to stereoselective biaryl synthesis is of fundamental stereochemical interest. For the first time, the two formal goals-the CC-bond formation and asymmetric induction at the axis-are separated. This novel principle can also be extended to atropo-enantioselective biaryl syntheses, for the directed synthesis of other axially chiral natural target molecules (126,131). Such a kinetically controlled stereoselective torsion of the axis can also

4.

191

THE NAPHTHYLISOQUINOLINE ALKALOIDS

14a (Dioncophylline A)

ent-13a I

> 95% ds

Me0

Me0

0

only 1 species

ent-12

'RedAl

r w

(NMR, HPLC ....) helical, not planar but rapidly helimerizing

ent-13b

__

14b (7-epi-Dioncophylline A)

SCHEME27. Diastereodivergent synthesis of dioncophylline A (14a) and (optionally) its atropisomer 14b, by twisting the axis with kinetic stereocontrol (/7,/8,/30).

be brought about with 0-nucleophiles, as exemplified in the first total synthesis of the Ancistrocladaceae alkaloid ancistrocladisine (33) (14,56,57)(see Scheme 28). Again, the key step is a highly atropodiastereoselective ring opening of the configurationally unstable lactone-bridged biaryl precursor 86, here using the simple achiral 0-nucleophile potassium isopropoxide. Again, not even the undesired minor (P)-configured by-product is lost. It can be recovered by ester saponification and subsequent ring closure back to 86 and renewed ring opening-a recycling by recyclization-thus again exhibiting chiral economy with respect to rotational isomerism (56). This example illustrates another typical advantage of the method. It is the first stereoselective synthesis of a natural biaryl with two identical ortho substituents (2 x OMe) on one side of the axis (at C-6 and C-8)-an unsolvable difficulty for other known coupling methods (cf. Scheme 30), but not a problem for the new procedure, as it relies on completely different stereochemical principles. The only real problem in the first total synthesis of ancistrocladisine (33) was the unexpectedly difficult ultimate oxidation step of the transconfigured tetrahydroisoquinoline 88a to the target molecule 33. This problem was finally solved by oxidation with KMnO,, which gave a maximum yield of only 16%. Because this oxidation problem is due to stereoelectronic reasons (cf. Sections, IV,A and VII,B,l), the synthetic sequence

192

GERHARD BRINGMANN AND FRANK POKORNY Me

Me

1) KOiPr

Me0

-

2) Me2S04 PTC

Me0

0

Me0

86 mially prostereogenic

87 95.4% dS

I

I

2 identical ortho substituents next to the axis

(+)-Ancistrocladisine (33)

SCHEME 28. Highly atropisomer-selective cleavage with simple 0-nucleophiles: the total synthesis of ( + )-ancistrocladisine (33) (56.57).

was repeated with the corresponding cis precursors (42).Indeed, the final oxidation reaction to give 33, now performed on 88b, occurred smoothly, giving a most satisfying yield of >70% (overall yield starting from the corresponding alcohol) (42) (see Scheme 29). But, even with the initial problems in the oxidation step, this first total synthesis of ancistrocladisine (33) gave such excellent coupling yields and asymmetric inductions that it could be performed on a gram scale, even for the last steps. Ancistrocladisine (33)is an interesting example of the efficiency of the lactone methodology, because it has also been prepared by Rizzacasa and Sargent (132,133),albeit by an intermolecular strategy (see Scheme 30).

SCHEME 29. Diastereomer-differentiating behavior of trans- and cis-configured tetrahydroisoquinolines 88a and 88b toward oxidants (42,56).

4.

193

THE NAPHTHYLISOQUINOLINE ALKALOIDS

4 pMe -

7 steps

0

Me0

91 (achiral)

ACO

89

steps

Me

Me

Pd-C

Me0 Me0

, '

QMe Me

\

rac-92 ("Dehydroancistocladine")

(23%)

/

Me0

Me0

OM0 Me

\

"Ancistrocladisine" (33 and its 3 possible stereoisomers. not resolved)

SCHEME30. Preparation of a mixture of all possible stereoisomeric forms of ancistrocladisine (33) and dehydrogenation to ruc-92 (132,133).

The key step in this synthesis is the Meyers-type (97,134-137) coupling of the naphthyl oxazoline 89 with the Grignard compound 90-of course without stereoselectivity, since the resulting coupling product 91is achiral. As the subsequent introduction of the stereocenter at C-3 and the Bischler-Napieralski ring closure were not performed selectively, ancistrocladisine was finally obtained as a mixture of all four possible stereoisomeric forms, which apparently could not be resolved. For this reason, the stereocenter at C-3 was "planarized" by catalytic dehydrogenation, leading to a more homogeneous, racemic, unnatural, fully aromatic naphthylisoquinoline 92. 4 . Selected Further Examples of Stereocontrolled Total Syntheses

The applicability of the lactone methodology could be shown in directed total syntheses of a series of further related natural products and their analogs, among them dioncolactone A (22),dioncopeltine A (20),ancistrobrevine D (45) (Fig. 3 3 , and several other natural and modified analogs (39,72).

194

GERHARD BRINGMANN A N D FRANK POKORNY

HO

0

45

20

22

FIG. 35. Further selected naphthylisoquinoline alkaloids, of which 20 and 45 were prepared by kinetically controlled atropisomer-selective cleavage reaction of axially prostereogenic lactone-bridged biaryls (29,39,42,72).

D. INTERMOLECULAR COUPLING: SYNTHESIS OF KORUPENSAMINES The ether and especially the lactone methodologies-according to the extent of steric hindrance with thermodynamic or kinetic control of the asymmetric induction at the axis-proved to be the real breakthrough in the total synthesis of naphthylisoquinoline alkaloids. By contrast, initial intermolecular strategies proved to be inadequate, since intermolecular coupling reactions suffer far more from steric constraints than do the intramolecular coupling reactions in the presented lactone methodology (97), which does not even fail for systems with extremely high steric hindrance. Even a tert-butyl group next to the biaryl axis is tolerated (138). By contrast, attempts to prepare naphthylisoquinoline alkaloids such as ancistrocladine (la) by coupling the naphthalene and the isoquinoline parts in their authentic forms failed already at the level of the preparation of its isoquinoline half as a Grignard compound with magnesium in the 5-position (see Scheme 31) (139). Nonetheless, as already described for the synthesis of ruc-92 (see Scheme 30), this principle was applicable to the synthesis of O-methylancistrocladine (39a) (140) and O-methylhamatine (39b) (140), as well as for ancis:rocladinine (34) (141,142).For this purpose, first a monocyclic, and hence sterically less crowded, precursor had to be coupled. From this the absent heterocycle had to be built up secondarily, leading to quite linear syntheses, moreover without distinct stereoselectivities.

++Me

: l :NM *

no ....__coupling

: : : N* " "

Me0

rac43

)

Me0

Me

rac44

SCHEME 3 I . Attempted synthesis of simplified analogs of ancistrocladine ( l a ) (139).

4.

195

THE NAPHTHYLISOQUINOLINE ALKALOIDS

On the other hand, a disadvantage of the lactone methodology (cf. Scheme 23) is that this procedure is not applicable to some of the naphthylisoquinoline alkaloids that have been isolated, due to the lack of a C,-unit next to the axis. An example of high synthetic priority is the group of 5,8’-coupled naphthylisoquinoline alkaloids, such as ancistrobrevine B (48) and the korupensamines, primarily korupensamines A (53a) and B (53b).Especially for a scheduled first total synthesis of their highly antiviral dimers, the michellamines 92, it became clear that methods also had to be elaborated for the directed preparation of this coupling type, not attainable by the lactone methodology in its heretofore valid form (143). The first total synthesis of korupensamines A and B, as achieved very recently (f f6,144), is outlined in Scheme 32. Among the various transition metal-catalyzed, redox-neutral coupling procedures, reaction of the naphthalene 95, in a trialkylstannyl-activated form, and the isoquinoline 97, with a bromine substituent in the scheduled coupling position and benzyl groups for the protection of the subsequently free OH groups, was the

gMe gM

L

L

O

BzlO

@ +

95

OH

,

Me

\

Me

SnBua

O

BZIO

-

,

“O

\

: “Bzl

14

: 1

Br

BZIO

Me

Me

gj

HO

97

BzlO

Me 96b

NxH

Me

53a

OH

BzlO

Me

I

OH

Me

96a +

I

OMe

OMe

\

OH

Me

53b

SCHEME32. Convergent, first total synthesis of korupensamines A (53a) and B (53b) (116,144). Conditions: (i) PdC12(PPh3)2,PPh,, LiCI, Cu(1)Br; (ii) BCI,; (iii) H2, Pd/C; (iv) HPLC.

196

GERHARD BRINGMANN A N D F R A N K POKORNY

combination of choice. Thus, reaction of 95 and 97 in the presence of a PdCI,(PPh,), catalyst, gave 96 as the first 5,8’-coupled naphthylisoquinoline in an atropisomeric ratio of 1.4 : 1. As both diastereomeric korupensamines 53a and 53b occur in nature in this free form and are simultaneously building blocks, e.g., of michellamine B (52b), the stereoselectivity was not further optimized at this point. Owing to the very similar chromatographic properties of %a and %b, these atropodiastereomers were not resolved, but immediately deprotected, giving a mixture of 53a and 53b, which was separated by HPLC on an amino-bonded phase column, as attained earlier (84). This synthesis of korupensamines A and B provides the first preparative access to 5,8’-coupled naphthylisoquinolines, which are the most polar members of this class of natural products thus far synthesized. Furthermore, it provides the basic methodology for a first total synthesis of their dimeric analogs, the michellamines.

E. TOTALSYNTHESIS OF MICHELLAMINES Because of the promising anticytopathic properties of the michellamines against HIV-1 and 2 (76,77,94),a great need exists for sufficient quantities of these natural quateraryls for preclinical and clinical tests. Substance supply from the rare tropical liana Ancisfrocladuskorupensis, which grows only in some parts of Cameroon (and possibly Nigeria), has become a serious problem (77). Hence, the elaboration of a chemical total synthesis of authentic michellamines, o r structural analogs with possibly even better biological and pharmacological properties, is a challenging and urgent goal. For this reason, the National Cancer Institute published an announcement (145)urging the research community to pursue synthetic and/ or other studies aiming at the production especially of michellamine B

(52b). Very recently, synthetic strategies were elaborated for the highly convergent construction of michellamines and related compounds. The two principal approaches very nicely complement each other. One approach is the biomimetic oxidative approach, in which the outer axes are formed first and then, highly convergently, the inner, nonstereogenic axis. By contrast, the other synthetic approach first builds up the inner axis, in the form of a central binaphthalene core, and then subsequently constructs the two outer axes simultaneously-another highly convergent (but nonbiomimetic) pathway with great flexibility with respect to structural variations.

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

197

1. Biomimetic Oxidative Coupling of Korupensamine A : The First Partial (and Total) Synthesis of the Michellamines

Despite its apparent complexity, the structure of michellamine A (52a) is relatively simple. It is, as mentioned in Section VI,B,2), a C,-symmetric dimer, consisting of two constitutionally and stereochemically identical halves. In both moieties, the ordinary monomeric naphthylisoquinoline alkaloids are likely to be derived biogenetically from identical polyketide precursors (2), as evidenced by the biomimetic cyclization reactions of P-polycarbonyl compounds (98,99,114,115) and biosynthetic feeding experiments (146) (cf. Section VIII). This suggests that for a chemical synthesis, one might build up the michellamine framework biomimetically. i.e., by oxidative phenolic coupling (147,148) of korupensamine A (53a), its natural monomer. Such a first synthesis of michellamine A (52a) from 53a is shown in Scheme 33 (149,150). In order to avoid the undesired byproducts expected for a direct coupling of korupensamine A (53a), with its three phenolic oxygen functions and secondary amino group, selectivity for the coupling reaction was guaranteed on the level of a specific protection of all these functionalities except for the 5’-OH, the oxygen function next to the required coupling site. Thus, consecutive N-formylation and subsequent selective O-acetylation, specifically and in high yields, gave the partially protected derivative 98, with only the chelated OH group at C-5’ free. Treatment of this monophenolic derivative with Ag,O in CHCI, in the presence of 0.2% NEt, led to the near-quantitative formation of the binaphthylidendione 99. This violet-colored, nicely stable diquinone was subsequently, after reduction to the corresponding binaphthol, deprotected in a single step. The resulting product 52a was found to be fully identical, in all its physical, spectroscopic, and chromatographic (77,151) properties, with an authentic sample of michellamine A as isolated from natural material. This biomimetic dimerization of korupensamine A (53a) to give michellamine A (52a) is important for several reasons. Firstly, it is a rational, first partial synthesis of a representative of this novel class of quateraryls from natural precursors. Second, given the total synthesis of korupensamine A (53a) previously achieved (116) (see Section VII,D), it represents the completion of the first total synthesis of a naturally occurring michellamine. Furthermore, based on the finding that the michellamines can be interconverted by base-catalyzed atropisomerization (see Section VI,C), it constitutes the first total synthesis of all previously known michellamines. Moreover, this synthetic pathway should easily be extended to the preparation of related, natural or unnatural homo- or heterodimeric naphthyliso-

198

GERHARD BRINGMANN A N D FRANK POKORNY OH

OMe

OH

Me

OH

OMe

Me

ACO

98

53a

1 OH

52a

0

iii

iv, v

Me

99

SCHEME33. The regiospecific oxidative dimerization of korupensamine A ( 5 3 ~ via ) its specifically protected derivative 98 (149,150). Reaction conditions: (i) (CH,),CO,CHO; (ii) CH,COCI, Et,N. DMAP; (iii) Ag20, EtjN; (iv) NaBH,; (v) MeOH/HCI.

quinolines for biological evaluation. And finally, it confirms the full stereostructure of this natural product as assigned earlier (26,77). 2. Starting with the Central Axis: A Nonbiomimetic Approach

In a very fruitful collaboration of the Wurzburg group with R. Kelly et ul., an additional, nicely complementary total synthetic route to michellamines was developed (118,152).The strategy was to build up the central axis first and subsequently the outer ones. For the required binaphthalene unit 102, acetate groups were envisaged for the protection of the 0-

4.

199

THE NAPHTHYLISOQUINOLINE ALKALOIDS

functionalities and triflate groups for the activation of the scheduled coupling positions. Its synthesis, starting from 100 and 101, is shown in Scheme 34. For the crucial coupling step, the combination of the ditriflate 102 with the boronic acid derivative 103, prepared from the tetrahydroisoquinoline precursor 97, proved to be the best combination. Thus, PdII-catalyzed reaction of these appropriately prepared building blocks gave the synthetic quateraryl 104 as a mixture of atropisomers. From this, the target molecules could be liberated by the usual deprotection methodology, leading to michellamines A (52a) and B (52b) practically exclusively (see Scheme 35). The reason for the unexpected selectivity in favor of the two “truly natural” isomers of 52 is not yet known. Michellamine C (524 is formed, if at all, in trace amounts only (118). This likewise highly convergent synthetic pathway is efficiently complementary to the above-mentioned biomimetic oxidative dimerization of entire preformed naphthylisoquinolines, and again it should allow a broad series of related or completely different structural analogs to be prepared for biological evaluation.

0

OTMS

0

OMe

Me

0

0

100

101

1 OTf

iii, iv

OAc

Me OTf

OAc

102

SCHEME34. Preparation of a novel central binaphthalene building block 102 (118).Reaction conditions: (i) 0°C. THF: SiO,; (ii) Mel, A&O; (iii) DMF. Cu, 120°C: (iv) aq. Na2S20,/ CH2C12,DMAP, Ac20: (v) DBU: (vi) 2.6-lutidine. Tf,O.

200

GERHARD BRINGMANN A N D FRANK POKORNY Me

-

OBzl

i,ii

97

Me

Me

OBzl

OBzl

iii

"'"~.:: BzlO

104

Me

iv - vi

I 03

\

52a

52b

52c

SCHEME35. Building up the two outer axes: completion of the total synthesis of michellamines (118,152). Reaction conditions: (i) nBuLi, -78°C; (ii) P(OMe),, 2 N HCI; (iii) Pd(PPh3)4,Ba(OH)2, DME-H20, 80°C; (iv) H2/Pd-C, EtOH; (v) HCI/MeOH; (vi) atropisomer resolution as previously reported.

VIII. Biogenetic Origin of Naphthylisoquinoline Alkaloids

Despite the great structural variations of the 2000 (153) to 2500 (154) known isoquinoline alkaloids, all of these natural products have in common that the key step of their biosynthesis is a Pictet-Spengler-type (155) condensation reaction of 2-arylethylamineswith aldehydes or a-ketoacids (121,153,156-158),a reaction that has been successfully imitated in numerous biomimetic alkaloid syntheses (159). Still, the unusual substitution patterns of, say, ancistrocladine (la) and dioncophylline A (14a)-especially the methyl group at C-3, the oxygen function at C-8, and the apparently acetogenic naphthalene substituent-hint at a hitherto unprecedented biosynthetic origin of isoquinoline alkaloids, not from aromatic amino acids as is usual (e.g. for 105), but rather from acetate units (3,f 14) (see Scheme 36).

4.

20 1

T H E NAPHTHYLISOQUINOLINE ALKALOIDS

?

I

Me

the "Pictet-Spengler" reaction

Me0

Salsolinol (105)

Me

Ancistrocladine (la) ('Ancistrocladaceae type')

OH

Me

Dioncophylline A (148) ("Dioncophyllaceaetype')

SCHEME36. The conventional Pictet-Spengler route to isoquinolines (f2f,159)-ako valid for ancistrocladine (la) and dioncophylline A (14a)?

A. THECONCEPT OF ACETOGENIC ISOQUINOLINE ALKALOIDS The concept of acetogenic isoquinoline alkaloids (2,13,114,146) is outlined in Scheme 37 (168). According to this concept, a central joint precursor to all imaginable isocyclic and heterocyclic moieties of naphthylisoquinoline alkaloids is the labile P-pentaketone 106 (or its undecarboxylated analog), itself arising from six acetate units. A first aldol-type condensation should lead to the monocyclic bisphenolic diketone 107. This might, after incorporation of nitrogen by reductive amination ( 1 6 0 , give 108, in its O-methylated form the heterocyclic moiety of ancistrocladine (la). The analogous sequence with reduction of the central carbon atom of 106 would lead to the monophenolic diketone 109, which should then give rise to 111,the isoquinoline part, e.g., of dioncophylline A (14a). This concept convincingly rationalizes the substitution patterns in the isoquinoline parts of l a and 14a. The similarly highly probable second aldol condensation of the same monophenolic diketone 109 to give 110, the

202

GERHARD BRINGMANN A N D FRANK POKORNY

/

OH

Me

\

HopM 108

la

OH

0

&

Me

/

I1

/f

Me/C'SCoA

+ - - co2 O

5

r

r

COSCoA

M

107

Me

e

Me

106

\ 14a OH

Me

109

\

/

qM0 H

OH

Me

111

SCHEME37. Proposed highly rational biosynthesis of acetogenic isoquinoline alkaloids ( 2 , I J .14,82,99.146,160).

bisphenolic naphthalene half of all known naphthylisoquinoline alkaloids, makes this biosynthetic sequence even more plausible. The two molecular moieties, very economically originating from joint precursors, would then couple by oxidative phenolic coupling (147,148) to give the intact naphthylisoquinoline alkaloids, e.g., l a and 14a. If true, this would mean a highly rational convergent biosynthetic origin of naphthylisoquinoline alkaloids from their two halves, which, despite their structural diversity, would be formed from identical P-polycarbonyl precursors, themselves arising from six acetate units. Even more impressively, the michellamines (52) (cf. Section VI) would convergently originate from two molecules of the corresponding korupensamines (531, which, themselves, would each result from a naphthalene and an isoquinoline half, and thus ultimately from four identical precursors 106, hence twenty-four acetate units-a remarkable biosynthesis worth elucidating and profiting from biomimetically.

4.

203

THE NAPHTHYLISOQUINOLINE ALKALOIDS B.

BIOMIMETIC CYCLIZATION REACTIONS

The first clear hint of the chemical plausibility of the presented biogenetic scheme, and, at the same time, the opportunity to take advantage of such a new synthetic principle for directed alkaloid syntheses, was obtained by the biomimetic imitation of these reaction sequences, as briefly outlined in Scheme 38. Thus, preparation of the chemical analogs 57 of the assumed precursor 106 succeeded by ozonolysis of the Birch product 112 (2,13,98,114).A still shorter, one-step preparation of 57 resulted from a double condensation of diesters of type 116 with the dianion (162) of acetone (2,98,99,115). In the very same reaction step, merely by chromatography over silica gel, 57 could be cyclized to 113 (e.g., for X,Y = - O C H , C H , W ) or to 109 (for X = H and Y = OR or N R , ) . As proposed for the biosynthesis, these monocyclic precursors can very easily be transformed into the corresponding isoquinolines 114 or 117, 'biomimetic sphere'

Me

HO

112 113

OMe OMe

Me

116

109

117

SCHEME38. Rational novel isoquinoline and naphthalene biomimetic syntheses (2.98,99,/14./15).Conditions: ( i ) 03,DMS; (ii) THF, -35°C; (iii) S O z . EtzO: (iv) Me2S04, PTC; (v) NH,OAc; (vi) KH, THF; (vii) KOH. MeOH.

204

GERHARD BRINGMANN A N D FRANK POKORNY

respectively, by reaction with NH, as a nitrogen source, or into the naphthalene moiety 115, by quantitative aldol condensation with KOH/MeOH, thus giving rise to extremely short and efficient novel isoquinoline and naphthalene syntheses, moreover via joint precursors (98,99,114).These syntheses would not have been possible without the above-mentioned biogenetic considerations. Of similarly high synthetic value was the biomimetic imitation of the ultimate dimerization step of naphthylisoquinolines, particularly of korupensamine A (53a), as part of the first total synthesis of michellamine A (52a). The step succeeded by regiospecific, near-quantitative coupling to these remarkable natural quateraryls (see Scheme 33, Section VIl,E,l) (149).

C. ISOLATION OF BIOGENETIC PRECURSORS OR MODIFIEDANALOGS A further indication of the biosynthetic origin of naphthylisoquinoline alkaloids, at least with respect to the separate formation of the two molecular halves, is the identification of the free isoquinoline and naphthalene moieties or their analogs, sometimes co-occurringwith the intact naphthylisoquinolines. The isolation of the naphthalene-free tetrahydroisoquinolines 50 (82)and 56 (84)(Fig. 36) has already been mentioned (see Sections V,B,3 and V1,D). Alkaloid 50 (N-methylphylline),a trace compound from A. barferi, is the heterocyclic half of N-methyldioncophylline A (40a), whereas 56, isolated from A. korupensis, represents the isoquinoline moiety of ancistrobrevine D (45) and is related to that of korupensamine D (55).

The free naphthalene part 110, however, has not yet been detected in any of these plants, probably because of its air-sensitivity, in contrast to related quinones. Ancistrocladus heyneanus produces, when stressed (e.g., during biochemical feeding experiments; see Section VIII,F), the well-known (122,163)antibiotic plumbagin (119) ( 1 6 4 ,possibly as a phytoalexin (165). Plumbagin formation was also found in A. abbreviatus

50 (NMethylphylline)

56

FIG. 36. Naphthalene-free 1.3-Dimethyl tetrahydroisoquinoline alkaloids from African Ancisrrocladus species (82-84).

4.

205

THE NAPHTHYLISOQUINOLINE ALKALOIDS

(83,129) and Triphyophyllum peltatum (9). From early work by Zenk's group (166), this naphthoquinone is known to be formed from acetate units. Also other, oxygen-richer quinones are produced as stress metabolites, such as the long-known (88)droserone (51), which is found in insectwounded parts of A. robertsoniorum (86,87,89).It has also been identified in A. heyneanus (167), which produces even higher oxygenated compounds like ancistroquinone (168). Similarly, isoshinanolone (118; absolute configuration to be established) has been detected in A. heyneanus (169),A . barteri (82),and T . peltatum (8). All of these bicyclic compounds can be seen in close biogenetic relationship to the sensitive (99)dihydroxynaphthalene 110, which apparently is, in the plants, either oxidatively coupled with isoquinolines to give alkaloids like ancistrocladine (la) or oxygenated to give plumbagin (119),which may then be further oxidized to 51 or reduced to 118 (see Scheme 39). By contrast, indications of open-chain or monocyclic (certainly far more reactive) precursors like 106, 107, or 109 have not been obtained yet. The co-occurrence of the isocyclic or heterocyclic alkaloid halves or their analogs is in agreement with the biogenetic hypothesis, but of

Me0 OMe

106

110

1

oxygenation

-

red.

ox.

c _

Me OH

lsoshinanolone (118) from:

A. barteri

Ancistrocladine (la)

?

Me 0

Plumbagin (119)

T. peltatum A. abbreviatus A. heyneanus (when "stressed")

7

won 0

Me

Droserone (51) A. heyneanus

A. robertsoniorum (when wounded)

SCHEME39. Naphthalene derivatives from the Ancistrocladaceae and Dioncophyllaceae: apparent biogenetic relationship to naphthylisoquinoline alkaloids.

206

GERHARD BRINGMANN A N D FRANK POKORNY

course does not prove it. For appropriate investigation of the biogenetic origin of the naphthylisoquinoline alkaloids, living, alkaloidproducing Ancistrocladaceae and Dioncophyllaceae plants must be available.

D. THEPLANTS A N D THEIR BOTANICAL ENVIRONMENT As evident from the preceding sections, the Ancistrocladaceae and Dioncophyllaceae occupy a most unusual position in the plant kingdom. The production of naphthylisoquinoline alkaloids is a specific capacity hitherto found nowhere else in nature (170). These chemical properties clearly correlate with a likewise special taxonomic position. 1 . Ancistrocladaceae

The genus Ancisfrocladus consists of about 27 species of tropical lianas and shrubs that belong to the palaeotropic plant kingdom and are indigenous to the tropical rain forests of Africa and Southeast Asia (171). These intriguing plants have always been very difficult to classify taxonomically (172-174), and thus have been assigned to a monogeneric family, the Ancistrocladaceae. The further taxonomical environment of these plants, however, remains provisional (52,171) and is the subject of intensive research (81). Morphological peculiarities include the characteristic hooked branches, from which the name Ancistrocladus was derived (175) (see Plate l ) , and the remarkable fruits, monoseeded nuts with five irregularly formed “wings” (enlarged sepals) (see Plate 2). Of the 27 Ancisfrocladus species currently known, 15 occur in Asia and 12 in Africa (85,160), among them the most recently detected “new” African species A . korupensis (91) (see Sections V,D and VI,A). Morphologically, the Ancistrocladaceae show close phylogenetic relationships to the Dioncophyllaceae (1 74), a fact that can now be fully confirmed on the basis of their chemical constituents. Both plant families produce naphthylisoquinoline alkaloids and related naphthoquinones. More precisely, the strict chemotaxonomic separation of the two families, as expressed in the Ancistrocladaceae/Dioncophyllaceaerule (see Section III,C) is, as discussed above, valid only for the Asian Ancistrocladaceae plants. The African Ancisfrocladus species, however, seem to be far more closely related to the Dioncophyllaceae with respect to their chemical constituents, since they produce, besides the typical Asian Ancistrocladaceaetype alkaloids, Dioncophyllaceae-typealkaloids, as well as mixed, hybridtype alkaloids (cf. Section V). Morphologically, but also with respect to the formation of naphthoquinones like plumbagin (119)and droserone

PLATE 1. Characteristic hooked branch of Ancistrocladaceae (here A. abbreviafus). [Photo: H. Bringmann]

PLATE2. s p i c a l seed of Ancistrocladaceae (here A. heynennus). [Photo: H. BMgmann]

PLATE3. The hooked leaves of I: pelramrn (Dioncophyllaceae) [Photo: W. Barthlott]

4. Insect-trapping organ of I: pelrum. [Photo: W. Thiele]

PLATE 5. Seedling of A. heyneanus cultivated on sphagnum moss. [Photo: J. R. Jansen]

PLATE6. A flourishing plant of A. abbreviatus cultivated in a greenhouse. [Photo: B. Wiesen]

PLATE7. A plantation of hydrocultured plants of A. heyneanus. [Photo: C. Schneider]

PLATE 8. Growth-retarding activity of dioncophylliie A (14a)against S. littoralis (209) photo: A. Z;inglein]

PLATE 9. Ancisfroclodus heyneanus overgrown by the herbal parasite Cuscufa re$’exu (mother host: a Coleus species). [Photo: C. Schneider]

PLATE 10. Microscopic view of the penetration of Cuscuta rej7exu into A. heyneanus (C=Cuscuta; X=xylem; P=phloem). [Photo: B. Wiesen]

4. THE

NAPHTHYLISOQUINOLINE ALKALOIDS

207

(51), a further taxonomic relationship can be suggested for the Droseraceae and the Nepenthaceae (50,176). 2. Dioncophyllaceae

Like the Ancistrocladaceae, the Dioncophyllaceae are not yet definitively classified taxonomically (171). It was only in 1951 that the three monotypic genera Triphyophyllum, Dioncophyllum, and Habropetalum were combined into this plant family (177). Besides the close relationship to the Ancistrocladaceae, taxonomic similarities have been noted with respect to the Nepenthaceae and Droseraceae (52). Like the latter, one of the three species of Dioncophyllaceae, T. peltatum, which has typical hooked leaves (see Plate 3), has recently been found to be carnivorous as a juvenile plant (178,179);it then produces insect-trapping organs (see Plate 4) that are morphologically derived from leaves. These plants also show very characteristic seeds (64,65).Some morphologic characteristics suggest additional relationships to the Ochnaceae and Guttiferae (176).

E. CULTIVATION OF THE PLANTS Despite great efforts, the biogenetic origin of the naphthylisoquinoline alkaloids has not yet been systematically investigated. The main reason for this is the difficult cultivation of these delicate tropical plants ( 2 ) ;some of these species (e.g. A. heyneanus) could not be grown even in their countries of origin (180). 1. Soil Cultivation

After a first successful attempt in 1980 to cultivate A. heyneanus from fruits (2,181), this species could, by further optimization of the growth parameters, be cultivated with high germination rates, giving large numbers (up to 500) of vigorously growing green plants (160,182) (see Plate 5 ) . In single cases, the plants even grew to a height of more than 3 meters and developed the characteristic hooked branches (cf. Plate 1) and, at the age of 3 years, even formed blossoms (160). The cultivation conditions could also be reproduced by other botanical gardens (160,182) and were adapted to other related species such as A. barteri, A. abbreviatus, and A . robertsoniorum (see Plate 6), as well as T. peltatum (86,182). This work is not only a fundamental prerequisite of the biogenetic experiments described below, but it is also relevant to the cultivation of the new species A , korupensis in large plantations in Cameroon, aiming at a large-scale production of the michellamines (93)-work that is just beginning.

208

GERHARD BRINGMANN A N D FRANK POKORNY

2 . Hydrocultures After the early success with soil cultivation, a more recent breakthrough occurred in the cultivation of the Indian species A . heyneanus and related species (86) on hydroculture substrates (182) (see Plate 7). This breakthrough is of tremendous value for scheduled biosynthetic feeding experiments, since an administration of biogenetic precursors to the roots is possible only in the absence of the numerous soil microorganisms that may metabolize the precious labeled precursor before resorption by the plant. Owing to the fragile consistency of the Ancistrocladus roots, the soil substrate could never be washed away without severely damaging the plants. The roots of plants cultivated on hydroculture substrate, however, proved to be more flexible, although somewhat shorter, than those grown by soil cultivation and can more easily be cleaned for feeding experiments. 3 . Sterile Plants and Cell Cultures

Attempts were undertaken to grow sterile plants and to obtain alkaloidproducing tissue cultures of A . heyneanus (164). For this purpose, fruits of A. heyneanus were defatted with ethanol, then superficially sterilized with sodium hypochlorite solution, liberated from the outer layer, and exposed to germination conditions on phytohormone-free B5 medium according to Gamborg (183).Thus, starting from parts of seedlings, a formation of callus growth could be obtained. Attempts to produce cell suspension and root cultures are also under investigation.

F.

BIOSYNTHETIC

EXPERIMENTS

I . Feeding Experiments with Acetate and Malonate Given the high content of ancistrocladine (la) in the roots o f A . heyneanus, feeding experiments were performed on this part of the plant. In previous experiments (2,) sodium [ 1-l4C]acetate had been administered to the stem of A . hamatus using the wick method (156,184). Yet, most of the radioactivity migrated to the upper, green parts of the plant, whereas the roots remained practically inactive. More recent feeding experiments (146,185)were performed by administration of the precursors by a cannula (186)in a thermostated and illuminated culture box (25"C, 17.5 h/day 1600 Lux, rel. air humidity 90%). Within 7-14 days, the solutions had been taken up by the plants, which were then harvested and worked up separately for each part. As seen in Table I, low, but significant incorporation rates of both acetate and malonate into ancistrocladine (la) were observed, comparable to those into the similarly formed plumbagin (119). Given its known (166) origin from acetate, this naphthoquinone can be considered

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

209

TABLE 1 AVERAGE SPECIFIC INCORPORATION RATES ( I , ) IN TO ANCISTROCLADINE A N D PLUMBAGIN AFTER APPLICATION OF RADIOLABELED PRECURSORS TO A . heyneanirs (146.185) I , (lo-'%)

Precursor Administered (a) Sodium [I-'4C]acetate (b) Sodium [2-'4C]rnalonate (c) ~-[U-'~C]Phenylalanine

Ancistrocladine (la)

Plumbagin (119)

0.56 6.4 0.054

0.67 3.9 0.085

an acetogenic marker. As expected, phenylalanine, a possible precursor for a conventional isoquinoline alkaloid biosynthesis, showed distinctly lower specific incorporation rates. 2 . Incorporation of the More Specific Monocyclic Precursor 107 The incorporation rates thus obtained were significant, considering the usual (187,188) difficulties with higher (especially woody) plants. Still, they were too low for a localization of the labeled positions by degradation or, after administration of 13C-labeledprecursors, by NMR spectroscopy. Such general primary metabolites, which also represent precursors to many natural products, may, in addition, be rapidly catabolized by the plant or by microorganisms. For this reason, and in order to avoid such problems, feeding experiments with more specific and highly differentiated precursors, such as the monocyclic diketone 107 seemed more promising. The isotope labeling synthesis of I4C-107(185), as shown in Scheme 40, partially follows the "inactive" synthesis of 107 elaborated previously (189). The key step is the C-acetylation of the arylpropanone 120 with the mixed pivalic acetic anhydride ( 14C-121),thus avoiding a loss of half the labeled material when using acetic anhydride. The primary product of the reaction, the stable pyrylium salt I4C-l22, can be cleaved to the diketone 14C-123and deprotected to I4C-107directly before the feeding experiments. In contrast to the technique described above, the far more specialized precursor 107 was administered, in a preliminary experiment, to the roots of A. heyneanus grown on a hydroculture substrate, and was found to be incorporated into ancistrocladinine (34)(see Scheme 41) (185). Interestingly, as already noted in other feeding experiments, this naphthyldihydroisoquinoline, normally just a trace alkaloid in the root material, had, under the feeding conditions, been formed to a much higher degree than the normal main product, ancistrocladine (la). Nonetheless, la, now a

210

GERHARD BRINGMANN A N D FRANK POKORNY

Me$

CI

+

NaO

&,

14 Me

Me Me

Bzlo'Q?bMe

54% HBF4 in Et20 CH2C12

hBzl

w:c

BzlO

Bzl Me

PI

120

14c-122

OH

Me

BZIO

Me

14C-123 l4C-1O7

SCHEME 40. Isotope-labeling synthesis of the postulated biosynthetic precursor 107 (185).

Me0

Me0

OMe

OMe

HowMe Me

HO

,.Me <- - -

Me0

Me

Ancistrocladine (la)

OH

Me

14C-107

Me0

Me

Ancistrocladinine (34)

SCHEME 41. Supposed transformation of the presumed monocyclic diketo precursor 107 into ancistrocladinine (34) and the normally predominant alkaloid ancistrocladine (la) by A . hevneanus (185).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

21 1

minor alkaloid, could be isolated and was found to exhibit a significant incorporation rate of I4C-1O7 into la. 3. Conclusions These first hints at unexpectedly high incorporation rates, which fully support the biogenetic hypothesis of an acetogenic origin of the naphthylisoquinoline alkaloids (cf. Scheme 37), make the preparation and administration of the corresponding 13C-labeledprecursors promising. This work is under investigation. If verified by further feeding experiments with NMR-relevant isotope-labeled precursors, the naphthylisoquinoline alkaloids would indeed be the first acetogenic isoquinoline alkaloids.

IX. The Chemo-ecological Context of Naphthylisoquinoline Alkaloids Owing to intensive studies during the past years, a great deal of information has become available on the occurrence of the naphthylisoquinoline alkaloids and their far-reaching structural variations. Far less is known about the chemo-ecological interaction of the alkaloid-producing plants with their environment, including the question of why they produce these interesting bi- and quateraryl compounds. The purpose is certainly not just to eliminate excess nitrogen (157), at least not for the carnivorous plant T. peltatum. In an earlier review of this series (2). spasmolytic and antitumor activities had been reported for these alkaloids. More recently, the naphthylisoquinoline alkaloids have been found to exhibit several other interesting biological activities. Some of these should be advantageous for the plants, e.g., as protection against microorganisms and herbivores. A.

BIOLOGICAL ACTIVITIES AGAINST

MICROORGANISMS

Several species of the Ancistrocladaceae and the Dioncophyllaceae play a role in the folk medicine of their countries of origin. Thus, on the Malaysian peninsula, the roots of A. tectorius are used against diarrhea and malaria (61), and T. peltaturn is employed against elephantiasis (190). The leaves of Dioncophyffumthollonii are used as an aphrodisiac, whereas the root pith is administered against leprous skin diseases (11). These and other applications hint at the promising pharmacological potential of the plants and thus warrant further investigations.

212

GERHARD BRINGMANN A N D FRANK POKORNY

1 . Fungicidal Activities

Extracts of T. peltatum were shown to be active against grainpathogenic fungi such as Leptosphaeria nodorum and Pyrenophora teres (191). The extracts exhibited particular activity against Botrytis cinerea and Plasmopara viticola, important plant-pathogenic species. Among the compounds tested, dioncophylline B (23)proved to be the most active and exhibited nearly no phytotoxicity (191). Good activities were exhibited also by the related alkaloids dioncophylline A (14a), dioncopeltine A (20), and dioncophylline C (24) (see Fig. 37). The molecular mode of action of these compounds is as yet unknown. Fungicidal activity is most likely to give the plant a specific advantage, especially in the humid rain forest.

2. Antiplasmodial and Antimalarial Properties (96,192-195) It is estimated that more than 2-3 million people die of malaria each year, and many more suffer from debilitating infection (196-198). Approximately a third of the world’s population lives in malaria-endemic areas, including Central and South America, Asia, and Africa (199). Due to the rapid spread of this tropical disease, the search for new antimalarial agents has become urgent (200). Many strains, especially of Plasmodium falciparum, have become resistant to chloroquine and other widely used antimalarial drugs (201,202). Some Ancistrocladus species are used in folk

on

Me0

Dioncophylline 0

Dioncophylline A

(23)

(14a)

OH

7-ep1-DioncophyllineA ( 1 4 ~

OH

OMe

HO

OMe

Me

HO Dioncopeltine A

Dioncophylline C

Korupensamine A

(20)

(24)

(=a)

FIG. 37. Naphthylisoquinoline alkaloids with pronounced biological activities.

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

213

medicine for the treatment of malaria and might thus be a potential source of new antimalarials. For this reason, extracts of Ancistrocladaceae and Dioncophyllaceae plants and a broad series of authentic and modified naphthylisoquinolinealkaloids were tested, mainly against P. falciparum. High in uitro activities, e.g., of the root extracts of T. peltatum (CH,CIJ were found, with IC,,-values (against P. falciparum) of 0.017 and 0.053 pg/ml using the K1 (chloroquine-resistant) and the NF 54 (clone AlA9, chloroquine-sensitive) strains (192). Extracts of A . abbreuiatus (192),A . barteri (192),A . heyneanus (203),A . robertsoniorum (203),and A . korupensis exhibited remarkable activities (96). Similar results, although with slightly enhanced IC,-values, were obtained for the rodent malaria parasite P. berghei (96,193-195). Among the pure isolated naphthylisoquinolinealkaloids tested, the most active was dioncophylline C (24) with in uitro IC,-values of 0.005 and 0.015 pg/ml against P. falciparum and P . berghei, respectively (194,204). The activity of dioncopeltine A (20) is also remarkably high, with ICs0values of 0.021 and 0.038 pg/ml, respectively (193,194).Good activities were also found for dioncophylline B (23), dioncophylline A (14a), 7-epidioncophylline A (14b), and korupensamine A (53a) (96,192-194). Interestingly, the michellamines (52) exhibited practically no antimalarial activity. This behavior is completely opposite to their antiviral properties, which were found only for the dimeric naphthylisoquinoline alkaloids. Naphthylisoquinolines were also found to show distinct antimalarial activities in uiuo (204).Mice (OFl) were inoculated with P. berghei erythrocytic forms at day 0 and treated p.0. with dioncophylline C (24)(50 mg kg-' d-') in a 4-day test (205), with the first treatment 2 hours after the inoculation. Their parasitaemia was reduced to 0% at day 4 and was found to be still 0% at day 10. The treated animals were still alive and looked absolutely normal at day 85, whereas all of the control animals had died soon after the infection. Dioncopeltine A (201, dioncophylline B (23), korupensamine A (53a) and a branch bark extract (CH,CI,/NH,) of T. peltatum were also active in uiuo against P . berghei, albeit to a lesser degree (205).Attempts to find even more active structural analogs of the new antimalarial lead-by isolation from natural sources or by directed derivatization or total synthesis-are in progress.

3. Antiviral Activities (77,94) As already mentioned in Section VI, the dimeric naphthylisoquinoline alkaloids, the michellamines (52) (cf. Fig. 38), were detected in the course of a bioassay-guided search for compounds active against the human immunodeficiency virus (HIV), the principal causal factor of the acquired

214

GERHARD BRINGMANN A N D FRANK POKORNY

immunodeficiency syndrome (AIDS). The high activity of the michellamines also led to the discovery of A . korupensis, a new Ancistrocladus species from the Korup National Park rain forest in Cameroon not scientifically known nor used in folk medicine (93,206).The most potent and simultaneously most abundant member of this series of naturally occumng quateraryls, michellamine B (52b), was shown to inhibit HIV-induced cell killing and viral replication in a variety of human cell lines, as well as in cultures of human peripheral blood leukocytes and monocytes. Michellamine B was active against a series of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6 and the pyridione-resistant strain A17. Michellamine B also inhibited several strains of HIV-2 (77). The mechanism of action of the michellamines is novel and quite distinct from any other known drug class, in that it involves at least two components, including an inhibition both of the viral reverse transcriptase and of the virus-cell and cell-cell fusion processes (207).The pharmacological and toxicological behavior of the michellamines is quite favorable. Administration of nontoxic doses of michellamine B in uiuo results in sustained blood levels of the drug well in excess of its effective antiviral concentrations (252,208). The high antiviral activity of michellamines seems to be a specific property of the dimeric nature of these interesting quateraryls, since none of the normal naphthylisoquinoline alkaloids tested-including the natural (monomeric) precursors, e.g., korupensamine A (53a)-showed any remarkable activity (84).This underscores the enormous interest in developing and improving methods for the production of dimeric naphthyliso-

"aoH Me

OH

Me

Me

OH

Me

Me0

Hi)

Me

MichellamineA (52a) FIG.38. Michellamines-antiviral

Michellamine B (52b) dimeric naphthylisoquinolines (77).

4. THE

NAPHTHYLISOQUINOLINE ALKALOIDS

215

quinolines, either from natural plant material or by total synthesis. Given the fact that only the dimeric alkaloids are active and that these alkaloids have, so far, been found only in A. korupensis (which seems to grow only in the Korup National Park), efforts to cultivate the species in large plantations in Cameroon become understandable. On the other hand, the challenge of the structurally unprecedented molecular framework of these intriguing natural products and their promising pharmacological properties have also triggered successful attempts to prepare the compounds chemically, by regio- and stereocontrolled total syntheses (see Section VI1,E). B. ACTIVITIES AGAINST HERBIVORES: INSECT-GROWTH RETARDATION A N D ANTIFEEDANT ACTIVITY Stimulated by the question of why species belonging to the Ancistrocladaceae and the Dioncophyllaceaeproduce naphthylisoquinoline alkaloids, the antifeedant activity of these natural products was studied, exemplified by activity against larvae of the polyphagous herbivore Spodoptera littoralis (Noctuidae) (209). Thus, neonate larvae of this model system were reared on an artificial diet spiked with concentrations of dioncophylline A (14a) as present in the leaves of T. peltaturn. High mortality (e.g., 86% after 20 days at 1 mg g-' fr. wt.) of the larvae and growth reduction (98% under the same conditions), as well as a pronounced increase of the larval period (50 days; controls 20 days), were observed. (See Plate 8.) The deleterious effects of 14a are probably due to the strong antifeedant activity of this alkaloid, since larval weight gain and amount of food consumed were strongly reduced compared with controls when neonate larvae were kept on a diet spiked with 0.4 mg g - ' fr. wt. 14a (209). Administration of other selected naphthylisoquinoline alkaloids to neonate larvae showed that compounds with additional hydroxy functions, such as dioncopeltine A (20), were less active than 14a (210,211). Feeding the alkaloids to second instar larvae showed that antifeedant activity and growth retardation are not strictly coupled to each other, hinting at a complex mechanism of action of the alkaloids toward S. littoralis, which is currently the subject of further structure-activity investigations. WITH HERBAL PARASITES: Cuscuta C. INTERACTION

Given the stress-induced variation of alkaloid patterns in Ancistrocladus heyneanus (see Section VIII,C), stress exerted by plant parasites belonging to the genus Cuscuta might be a useful tool to investigate plant/plant

216

GERHARD BRINGMANN A N D FRANK POKORNY

interactions. Cuscuta species are known to interfere with the metabolism of their host plants (212,213). Furthermore, a study of the possible uptake of the alkaloids by the parasite might provide information about a transporting process of the products in A . heyneanus and about their activity against the parasite (see below). As Cuscuta platyloba and C. reflexa do not grow on Ancistrocladus heyneanus on their own, they were inoculated on the host plants while still connected with a Coleus species, the usual host in greenhouse experiments (214). Within several days, the lianas were overgrown (see Plate 9), the parasites forming a lot of close connections (haustoria) to the plants (see Plate 10). Yet, as soon as the parasites were disconnected from the Coleus host, the growth process of the Cuscuta species on A. heyneanus slowed down and after 6 days, approximately half of the parasite material had died. By GC-MS analysis, both the living and the dead Cuscuta parts were shown to contain ancistrocladine (la) practically exclusively (214). Possibly, the parasite quite selectively incorporates only ancistrocladine (la) out of the complex spectrum of alkaloids. A selective uptake of ancistrocladine would hint at its transport in the xylem or in the phloem of the plant. The incorporation of the alkaloids into Cuscuta, their possible toxicity toward the parasite, and their metabolism in cell cultures of Cuscuta are under investigation.

X. Tables of Known Natural Naphthylisoquinoline Alkaloids Since the appearance of the last comprehensive article on the naphthylisoquinoline alkaloids in 1986 (2), this field has undergone tremendous development with respect to the number of known representatives, structures, methods of structure elucidation, biological activities, and information on the unusual biosynthetic origin of these intriguing natural products. Table I1 represents a complete list of naphthylisoquinolinealkaloids, categorized as Dioncophyllaceae-type, hybrid-type, and Ancistrocladaceaetype representatives and according to their coupling sites. Table I11 shows the dimeric naphthylisoquinolines, the michellamines; and Table IV shows the bicyclic naphthalene and isoquinoline halves and their analogs. Those alkaloids whose structures have turned out to be incorrect or not firmly established are listed separately in Table V.

TABLE I1 NAPHTHYLISOQUINOLINE ALKALOIDS FROM ANCISTROCLADACEAE A N D DIONCOPHYLLACEAE Name, Formula (mol. wt.), and Structure

Occurrence

mp ("C)

[a]D (Solvent)

Methods for Structure Elucidation and Remarks

Dioncophybceae-Type Structures

7,1'-CoupledAlkaloids (A Type) Dioncophylline A (14a) C24H,,NO3 (377.5)

7-epi-DioncophyllineA (14b) C*,H,,NO3 (377.5)

Me0

T. pelrarum; roots, stem bark (14) A. abbreuiarus; stem bark (66)

215 (17) 214 (15)

A. barteri; roots (68) 240-242 (68)

-

14.9" (CHCIJ (15,17) General spectroscopy (15) Far-reaching NOE interactions, 14.0" (CHCII) (66)

- 14.1" (CHC13) (68)

e.g., ROESY (3233) CD (15,20) Degradation (35) X-Ray (37) Bridge analysis (31) Total synthesis (17.18) Total synthesis of e n r - l b (17) 0-Methylation to 0methyldioncophylline A (Ua)(38) Biological activity (191-1 94,209,210,215) Former name triphyophylline (see Table 111) Spectroscopy (68) CD (68) Degradation (68) Total synthesis of en?-14b(17) Biological activity (68)

14b (continues)

TABLE I1 (Continued) Name, Formula (mol. wt.), and Structure N-Methyldioncophylline A CaHBNO, (391.5)

Occurrence

(a)A. abbreuiatus;

mp ("C) 193 (51)

[ a ](Solvent) ~

+ 15.0" (CHCI,) (51)

Spectroscopy (51) CD (51) Degradation (61) Partial (and thus total) synthesis from 14s (51) Chiral HPLC (51) Biological activity (192.193.211 ) Cf. structure of Nmethyltriphyophylline; see Table V (69) and Ref. (10)

-6.0" (CHCI,) (51)

Spectroscopy (51) CD (51) Chiral HPLC (51) Partial (and thus total) synthesis of entQOb (51) Cf. structure of Nmethyltriphyophylline; see Table V (69) and Ref. (10)

stem bark (51), roots (83) A . barter;; roots (68)

Me0 00

Methods for Structure Elucidation and Remarks

Me0 N-Methyl-7-epi-dioncophyllineA (Nb) CaHBNO) (391.5)

Me0

A. abbreuintus; 230 (51) stem bark (51) roots (83) A . barteri; roots (68)

40b

5'-0-Demethyl-8-0methyldioncophylline A C24H27NO3 (377.5)

E \o

T . peltaturn; stem bark (44)

(a)

5'-O-Demethyl-8-O-methyl-7-epi-T . pc..durn; roots, stem bark (44) dioncophylline A (30b) C24H27N03 (377.5)

oil (44)

+ 37. I" (CHCI,) (44)

General spectroscopy (44) Far-reaching NOE interactions, e.g., ROESY (43,44) CD (44) Degradation (44) Partial synthetic conversion to a derivative of 14n (44)

amorphous ( . 1)

-41.5" (CHCI,) (43, 1)

General spectroscopy (44) Far-reaching NOE interactions, e.g., ROESY ( 3 3 , 4 3 4 ) CD (44) Degradation (44) Partial synthetic conversion to a derivative of 14b (44)

HO

Me0

30b (continues)

TABLE I1 (Continued) Name, Formula (mol. wt.), and Structure

4'-0-DemethyldioncophyllineA (41a)

CzjHz5NO3 (363.5)

HO N 0 N

OH

Occurrence

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

A . abbreuiatus; roots. stem bark (68)

278 (68)

- 12" (ethanol) (68)

Spectroscopy (68) Degradation (68) CD (68)

A . abbreuiatus; roots, stem bark

244 (68) 240 (68)

-22" (ethanol) (68) - 24.4"(CHCIJ (68)

Spectroscopy (68) Degradation (68) CD (68)

Me

Me0 4'-O-Demethyl-7-epidioncophylline A (41b) CzJH2sNOI (363.5)

(68) A . barteri; roots (68)

Dioncopeltine A (20) C?3H>SNO,(379.5)

HO

233-234 (38)

- 13.1" (CHC13)(38)

Spectroscopy (38) CD (38) Degradation (38) X-ray (38) Partial synthetic conversion into 1% (38) Total synthesis (39) Biological activity (192-194) Same constitution as 6 (triphyopeltine; see Table V ) , but different physical constants

T . peltrrtrrm; roots, stem bark (38)

amorphous (38)

- 64.0"(CHCI,) (38)

Spectroscopy (38) Degradation (38) Total synthesis (39)

20

Dioncolactone A (22) C23HzrNOd (375.4)

q$E:

HO Me0

T . pelrutum; roots. stem bark (38)

Me

\

0

22 (continues)

TABLE I1 (Confirrued) Name, Formula (mol. wt.), and Structure

Occurrence

5'-O-Demethyl-8-O-methyl-7-epiT . pelfarurn: roots dioncophyllidine A (29) (39) CyHzsNO, (375.5)

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

amorphous (39)

+ 13.0" (CHCI,) (39)

Spectroscopy (39) CD (39) Degradation (39)

177-179 (47.48)

0" (CHCI,) (47,48)

Spectroscopy (47,48) X-Ray of natural racemate (PIM-31) (48)

HO

Me0 N N

29

(+)-Dioncophyllacine A (31) C26H27NOd (417.5)

T . pelfaturn; leaves (47.48)

OMe

Me0 Me0

31

7,6'-Coupled Alkaloids ( B Type)

Dioncophylline B (23) CnHaN03 O.5MeOH (379.5)

OH OH Me

IrJ

T. peltaturn; root bark (39,40), leaves (39)

- 37.6" (CHCl,) (40)'

Spectroscopy (40) Degradation (40) CD (39) Biological activity (191-194)

180-182 ( l 1 3 )

(no stereogenic centers or axes)

Spectroscopy (46)

Me

23

Dioncophyllacine B (32) CzsHzsNO, (403.5)

Me

107-108 (39)

T. peltaturn; leaves (46)

32 (conrinues)

TABLE I1 (Conrinued) Name, Formula (mol. wt.), and Structure

Occurrence

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

7.8'-CoupledAlkaloids (DType) Dioncophylline D (28) C23HZsNO3 (363.5)

T . pelraturn: leaves (39)

244 (dec.) (39)

T . pelraturn: roots. stem bark (41)

246 (dec.) (41) 248 (dec.) ( 4 2 )

+ 37.6" (CHC13)(39)

Spectroscopy (39) CD (39) Degradation (39)

5,I'-Coupled Alkaloids (C Type) Dioncophylline C (24) C23H2sN0, (363.5) HO OMe

Me

HO

Me

24

- 119. I" (CHCI,) (41) after purification via the N-formyl derivative: + 19.2" (CHCI,) ( 4 1 ) synthetic product: + 16.0" (CHCI,) (42)

Spectroscopy (41) CD (41) Degradation (41) Conversion of 24 and l a into enantiomeric derivatives (41 ) Total synthesis (42) Biological activity (194)

Hybrid-Type Structures 7,1'-Coupled Alkaloids

Ancistrobrevine C (42) CzH27N04 I .5H20 (432.5)

Me0

A . abbreviatus; stem bark (36,70), roots (216)

180-183 (36)

+ 13" (CHCI,) (36)

Spectroscopy (36) CD (36) 0-Methylation to 43, the enantiomer of ancistrocladisine (33) (36) Cis-selective reduction to the (lS,3R)-diastereomer of ancistrobrevine D (45) (42) 42 is accompanied in the plant by its 3-epimer (36)

241-242 (79)

+ 88.4" ICHCI,) (79)

Spectroscopy (79) CD (79) Degradation (79) 0-Methylation to the 3-epimer of ancistrocladisine (33) (79)

42

N h) VI

Ancistrobarterine A (49) C25H27NO.4 (405.5)

A . barteri; roots (68,791

(continues)

TABLE I1 (Continued) Name, Formula (mol. wt.), and Structure h)

Occurrence

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

5,8'-Coupled Alkaloids

Korupensamine A (53a) CzjHzsN04 (379.2)

OH

OMe

A. korupensis; aerial parts (84)

amorphous (84,116)

- 75.5" (methanol) (84)

Spectroscopy (84,96) Experimental (84)and computational (26) CD investigations Degradation (84) X-Ray analysis on a sulfonate derivative of 53a (84.96) Total synthesis (116,144) Dimerization to michellamine A (524 (149,150) Biological activity (antimalarial)

(84.96) HO

Me

53a

Korupensamine B (53b) C?,H?sNO, (379.2)

OH

amorphous (84.116)

+65" (methanol) (84)

aerial parts (84)

Spectroscopy (84.96) CD (84) Degradation (84) Total synthesis (116,144) Biological activity (antimalarial) (84.96)

A . korupensis; aerial parts (84)

amorphous (84,116)

-62" (methanol) (84)

Spectroscopy (84,96) CD (84) Degradation (84) Biological activity (84,96)

A . korupensis;

OMe

Me

HO

Ho

53b

h) -4 h)

Korupensamine C (54) C24H27N04 (393.2)

Me0

OMe

54 (continues)

TABLE I1 (Continued) Name, Formula (mol. wt.), and Structure

Occurrence

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

Inverse Hybrid-Type Structures 7,/'-Coupled Alkaloids Dioncoline A (44d CZHDNOj (391.5)

A . abbreviatus; stem bark (71), roots

amorphous (217)

+ 35" (ethanol) (217)

Spectroscopy (217) CD (217) Degradation (71) Chiral HPLC (71)

A . abbreviatus; stem bark (71). roots

amorphous (217)

+26" (ethanol) (127)

Spectroscopy (217) CD (217) Degradation (71) Chiral HPLC (71)

Me t4 h)

OH

Me0 7-epi-Dioncoline A (44b) CzsH29N0, (391.5)

Me0

Me 44a

44b

Ancistrocladaceae-Type Structures 7 ,1 '-Coupled Alkaloids Ancistrobrevine D (45) C26HJIN04(435.6)

A . abbreviarus (72)

172 (72)

+24.9" (CHCI,) (72) +21.4" (CHCI,) (42)

Spectroscopy (72) CD (72) Total synthesis (72) Partial synthesis from the 3epimer of 42 (42) Chiral HPLC (72) Biological activity (192,193)

A . heyneanus; roots (22) A . harnatus; roots (67)

178-180 (23) 178 (56) 220-222 (dec., hydrochloride) (23)

+ 7.8" (CHCI3) (56)

Spectroscopy (23) CD (36) CD (exciton chirality) (218) X-Ray (219) Absolute configuration (218) Total synthesis of stereochemically pure 33 (42S 6 S 7) Preparation of a mixture of 33 and its isomers (132,133) Biological activity (192,211)

Me0

45

Me0

N \o

Ancistrocladisine (33)b C26H~N04(419.5)

Me

- 20.5" (hydrochloride, in CHCI,) (56) - 16.1"(hydrochloride, in CHCI,) (23)

(continues)

TABLE I1 (Continued) Name, Formula (mol. wt.), and Structure

Occurrence

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

7,3'-Coupled Alkaloids

Ancistrocladidine (124)b CzsH27N04 (405.5)

A . heyneanus; roots (220), leaves (216)

245-247 (dec.) (220) 243-246 (dec.) (216)

- 149.7"(CHCI,) (220) - 144.2"(CHCIJ (216)

General spectroscopy (220) Extended I3C-NMR investigations (221) CD (exciton chirality) (218) X-Ray (219) Absolute configuration (218)

Ancistrotectorine C26H,,NO, (421.5)

A . rccrorius; leaves, twigs (61.222)

134-140 (61)

0" (CHCI,) (61 )

Spectroscopy (61) CD (61) X-Ray (61) Biological activity (61)

Me

125

7,8'-Coupled Alkaloids

Ancistrobrevine A (47) CnH33NOd (435.6)

A. abbreuiatus; stem bark (74)

144-145 (74)

+ 54" (ethanol) (74)

Spectroscopy (74) Far-reaching NOE (33) interactions (72) Partial synthesis from 46 (74) CD (74) Degradation (74)

A . abbreuiutrrs;root

125-126 (74)

- 80" (ethanol) (74)

General spectroscopy (74) CD (74) Degradation (74) Conversion to 47 (74)

Me

Me

Me0

w

I\)

Me

47

6-0-DemethylancistrobrevineA (46) C26H3,NOd (421.5)

bark, stem bark ( 74)

(continues)

TABLE I1 (Continued) Name, Formula (mol. wt.), and Structure

Occurrence

mp ("C)

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

5,I'-Coupled Alkaloids W N h)

Ancistrocladine (la)b CaHBN04 (407.5)

Me9

OMe

@

Me

Me0

Me

la

A . heyneanus; roots (3,223) A . hamatus; roots

265-267 (3) 264-266 (dec.) (216) 263-265 (66) (5) A . barreri; roots (68) 263 (222) A . abbreviarus; 262-263 (224) roots, stem bark 262 (dec.) (68) 1661 261 (29) . , A . tectorius; stems, 260-263 (6) twigs (222,224) 261-262 (dec.) A. congolensis; (90) roots, stems (225) A . robertsoniorum; stems (90)

-27.4" (CHCII) (216) - 25" (CHCIJ (3) - 25" (pyridine) (224,225) - 23" (methanol) (28,291 -20.5" (CHCI,) (224) -20" (CHCI,) (20) -21" (methanol) (90)

Spectroscopy (29.68) X-Ray (4) Absolute configuration (4) Experimental (4,20,68,216)and computational (20,24,25)CD investigation Degradation (95) Total synthesis (29,55) Biological activity (192,193,210) Biogenesis (146,185)

Hamatine (lb)b C ~ H B N O(407.5) ~ Me0

A. hamatus; roots

(5) A. barteri; roots (68) A. tectorius; stems, twigs (6) A . abbreviatus; stem bark (66)

OMe

250-252 (5.6) 250 (29) 249-250 (dec.) (68)

247-248 (66)

+77.4" (CHCI,) (5) +71" (CHCI,) (68) +68" (methanol) (29) + 66. I" (CHCI,) (6) + 64" (CHCI,) (66)

Spectroscopy (5,68) Absolute configuration (226) Experimental (24,68) and computational (25) CD investigation Degradation (35,68) Total synthesis (28,29.55)

+61.8" (CHCI?)( 4 )

Spectroscopy (27.53) CD (53) Partial synthesis from ancistrocladinine (34)

lb N W

lsoancistrocladine (35) C2jHDN04 (407.5)

A. heyneanus: roots

(53)

235-236 ( 4 ) 230-232 (53)

+ 59.5" (CHCI3) (53)

(4,27.54Ib

Me0 OMe

Total synthesis (61)

HO

Me0

Me

35 (continues)

TABLE I1 (Continued) ~

~~

Name, Formula (mol. wt.), and Structure

g

h)

Ancistrocladinine (34)b CBH27N04 (405.5)

~

Occurrence A . heyneanus; roots (28.227) A . tectorius; stems (222)

mp rC) 285-288 (28) 233-235 (227) 255-258 (142)

[ a ](Solvent) ~ - 321.8" (pyridine) (28) - 326.8" (pyridine)

(227) - 148.9" (pyridine) (142)

Methods for Structure Elucidation and Remarks Spectroscopy (4,28,216) CD (216) Absolute configuration (4,28) Partial (and thus total) synthesis via 35 (141,142) Conversion to isoancistrocladine (35) (4,27,54) and ancistrocline

(36)(54) Biological activity (antimalarial) (194) Biogenesis (185) Stress-induced formation (164,185) Me0

Me

34

( - )-0-Methylancistrocladine

A . heyneanus; roots

(67) A . abbreuiarus; roots, stem bark (68) A . congolensis; roots, stems (225)

(39p)*

C26H31N04 (421.5)

Me0 OMe

200-202 (3) 198-199 (68) 1%-197 (225) 315-317 (dec., hydrochloride) (3) 271-273 (dec., hydrochloride) (140)

- 38" (ethanol) (68) - 35" (pyridine) (225) -56.1" (hydrochloride, in CHC13)(3) -40.0"(hydrochloride, in CHCI,) (140)

Spectroscopy (140) CD (68) Degradation (68) Partial synthesis from ancistrocladine (la) (3) Total synthesis (140)

Me0 %NIY

Me0

Me

39a

N W VI

( + )-0-Methylhamatine

(39b)

Me0

A . abbreuiarus;

roots, stem bark (68)

C26H3INO4 (421.5)

OMe

160-162 ( 5 ) 159-161 (68) 318-322 (dec., hydrochloride) (5 ) 211-273 (dec., hydrochloride)

+ 25" (ethanol) (68) +21.3" (THF) (140) - 20.5"(hydrochloride, in methanol) (5)' + 28.0" (hydrochloride, in methanol) (140)

Spectroscopy (5,68,140) CD (68) Degradation (68) Total synthesis (140)

(140)

39b (conrinues)

TABLE I1 (Continued) ~

Name, Formula (mol. wt.). and Structure Ancistrocline (36)' C:,H,,N04 (421.5)

Me0

W w

Occurrence A . tectorius; leaves

(6.58)

mp ("C) 227-228 ( 6 ) 223-224 (54.58)

+ 62.0" (CHCI,) (58)

122-124 (66,75)

-68" (CHCI,) (66,75)

OMe

Me0

Me

[ a ](Solvent) ~

Methods for Structure Elucidation and Remarks

Spectroscopy (6.54.58) +61.7" (CHCI,) ( 6 ) CD ( 2 2 3 +59. I" (CHCI,) (54.58) Degradation (58) Partial synthesis from ancistrocladinine (34) and isoancistrocladine (35) (54) Total synthesis (58)

36

Q.

5,8'-Coupled Alkaloids

Ancistrobrevine B (48) CxH,oNO, (407.2) Me0

OMe

A . abbreuiatus; stem

bark (66,75),roots (83)

Spectroscopy (66,75) Axial configuration by NOE (66) CD (66,75) Degradation (66,75)

Korupensamine D (55) C26H31N04 (421.5)

OH

Me6

A . korupensis; aerial parts (84.96)

OMe

Me

55

h)

-4 W

" The published sign of the optical rotation is a misprint. For further details. see former review ( 2 ) . The sign of the rotation may be a misprint (140).

amorphous (84)

+ 6" (methanol) (84,%)

Spectroscopy (84,96) CD (84.96) Degradation (84,%)

TABLE Ilk THEMICHELLAMINES: DIMERIC NAPHTHYLISOQUINOLINE ALKALOIDS FROM Ancisrrocladus korupensis Name, Formula (mol. wt.), and Structure Michellamine A (5211) CaHaN208 (756.9)

Occurrence

mp ("C)

A. korupensis; aerial parts (76,77,94)

220 (dec.) (76.94)

[aID(Solvent) -

10.5"(methanol) (769)

[a]x5 = +65.7"

Me

w oo

W

OH

(methanol) ( 7 6 9 )

Methods for Structure Elucidation and Remarks Spectroscopy ( 7 6 , 7 7 9 ) Computational and experimental CD (26.94) Degradation (77,941 Partial and total syntheses (118,144,149-152) Interconversion to 52b and 52c (77,941 HPLC (76,77,151) Biological activity ( 7 6 , 7 7 9 )

Michellamine B (52b) CaHaNZOg (756.9)

Me

A . korupensis; aerial parts (76,77,94)

OH

230 (dec.) (76,94)

- 14.8" (methanol)

(76.94) [a1365 = -23.4' (methanol) (76.94)

Spectroscopy (76.77.94) Computational and experimental CD (26,941 Degradation (94.95) Total synthesis (118,150,152) Interconversion to 52a and 52-c (77) HPLC (76,77,151) Biological activity (76,77,94)

W N

W

HA

Me 52b

(continues)

TABLE I11 (Continued) Name, Formula (mol. wt.), and Structure Michellamine C (524 C,aH@N208(756.9)

Occurrence

mp ("C)

[ale (Solvent)

A . korupensis; aerial parts (76,77,94) Possibly a nonnatural isomerization product from

-

-

52alb

Me

OH

Methods for Structure Elucidation and Remarks Spectroscopy (77,941 Computational and experimental CD (26.94) Total synthesis (118,150) Interconversion to 52a and 52b (77) HPLC (76,77) Biological activity (77,941

TABLE IV MOLECULAR HALVESOF NAPHTHYLISOQUINOLINE ALKALOIDS A N D THEIRANALOGS Name, Formula (mol. wt.), and Structure

Occurrence

mp ("C)

[a],, (Solvent)

222-225 (subl.) (168)

-

Methods for Structure Elucidation and Remarks

Naphthalene Derivatives Ancistroquinone (126) C l o 0 5 (234.2)

A . heyneanus;

Plumbagin (119) CllHB03 (188.2)

A . heyneanus (146);roots, stems (228) A . barter; (82); roots A . abbreviatus; stems (83,129) T. pletatum; roots (8) D . tholloni; roots, stem bark ( 1 1 )

@

Me

O

119

roots (168)

78 (228) 71 (231) 74 (163)

Spectroscopy (168) Total synthesis (168)

Spectroscopy (163',229,230) Total syntheses (122.231) Biomimetic synthesis of methyl ether of 119 (2) Biogenesis (166) Biological activity (122,163,164,200) Apparent stress metabolite (164) Widely distributed natural product (122)

(continues)

TABLE IV (Continued) Name, Formula (mol. wt.), and Structure Droserone (51) CIlHsO, (204.2)

Occurrence A. hevneanits; roots (167) A . robertsoniontni.

mp C'C) 181 (232)

[aID(Solvent)

-

119 (87.89)

twig bark (86.87,89

Isoshinanolone (118) Cl1H120, (192.2)

I

OH 118

A . barter;; (82)

amorphous

+ 20.0" (CHC13)b(74)

roots A . heyneanirs; roots (235)

(74) 255 (234)

- 7" (CHCI,) (234) + 200" (CHCI,) (10)

Methods for Structure Elucidation and Remarks Spectroscopy (87.233) X-Ray (87.89) Total synthesis (233) Apparent stress metabolite (86) Occurrence in other plant families (122)

Spectroscopy (74) CD (74) Total synthesis (234)

lsoquinolines N-Methylphylline (50) C,?H,,NO(191.3)

HO

Me

A . barteri (82); roots

amorphous (82)

+ 22" (CHCI,) (82)

Spectroscopy (68) Total synthesis (68) Biological activity (236) Formal heterocyclic half of N methyldioncophyllines A (40a/b)

A . korupensis: aerial parts (84)

amorphous (84)

+ 120" (methanol)

Spectroscopy (84) CD (84) Degradation (84) Total synthesis ( I 13) Formal heterocyclic half of ancistrobrevine D (45)

50

(IR,3S)-N-Methyl-l.3dimethyl-6-methox yI-hydroxytetrahydroisoquinoline (56) !i? CI3HI9NO2 (221.3) W

" For correct "C-NMR data. see Sankaram et ol. (229).

' Absolute configuration under investigation.

(84)

TABLE V NAPHTHYLISOQUINOLINE ALKALOIDS REPORTEDI N

E

Name, Formula (mol. wt.), and Postulated Structure

Occurrence

THE

EARLYLITERATURE^

[aJD(Solvent)

mp ("C)

Methods for Structure Elucidation and Remarks

Alkaloids from Dioncophyllaceae Triphyophylline (2) CuH27NO3 (377.5)

Me0

T. peltaturn: twigs ( 8 ) .stems D.tholloni; roots, stem bark ( 1 1 )

2

215 (8)

-

14.0" (CHCI3) (8)

Spectroscopy (8,10) Conversion to racemic 9 (10) Constitution and relative configuration confirmed; absolute configuration at C1/C3 revised as 1R,3R (15,17) through total synthesis Full stereostructure, including axial chirality: 14a (17) New name: dioncophylline A (15) (see Table 11)

Isotriphyophylline (3) CZdH,,NO3 (377.5)

T. peltaturn; stems, leaves

256 (10)

-22" (CHCI3) (10)

Spectroscopy (10) Conversion to racemic 9 (10) Structure disproved by total synthesis (2.12-14)

-30" (CHCI]) ( 9 )

Spectroscopy ( 9 ) Reported partial synthesis from 8 (91, different from the 0methylation product of natural or synthetic triphyophylline/ dioncophylline A (15,17): Structure not confirmed by partial or total synthesis

(10)

D . tholloni; stems, leaves (10)

0-Methyl triphyophylline (4)

CzsHDNOj (391.5)

T. peltarum; twigs ( 9 )

(continues)

TABLE V (Coniinued) Name, Formula (mol. wt.), and Postulated Structure

mp (“0

Occurrence

[ale (Solvent)

Methods for Structure Elucidation and Remarks

0-Methyl-I ,2didehy drotriphyoph y Nine (8) C,H*,NO, (389.5)

T. peliaium; twigs (9)

amorphous (9)

0” (solvent not specified) (9)

Spectroscopy (9) Reported conversion to 0methyltriphyophylline (4) Structure not confirmed (39)

O-Methyltetradehydrotriphyophylline (9) CSH25NO3 (387.5)

T. pt..aium; stems, leaves

16

0” (CHCI,) (10)

Spectroscopy (10) Prepared from 14a (“2”) Partial synthesis of optically inactive material from triphyophylline (2) (10) and isotriphyophylline (3) Constitution confirmed by partial (15) and total (12) synthesis Stereochemistry of natural material not established

(10)

D. iholloni; stems, leaves ( l o ) ,roots, stem bark ( 1 1 )

+ 59” (CHCI, (15)

Me Me Me0

N-Methyltriphyophylline (5)

C2jHDNO3 (391.5)

N 4 P

9 T . peltaturn; stems, leaves (10)

D.iholloni; twigs, leaves (10) roots, stem bark (11)

185 (10)

+70" (CHCI,) (10)

Spectroscopy (10) Reported partial synthesis from triphyophylline (2) (10). but data different from those of Nmethylation product 4Oa of triphyophylline (2)/ dioncophylline A (14a) (51) Structure not confirmedb

(continues)

TABLE V (Continued) Name, Formula (mol. wt.), and Postulated Structure

Occurrence

T. peltaium; twigs (8) D. iholloni; roots, stem bark (11)

Triphyopeltine (6) C ~ ~ H ~ S(379.5) NO~

mp ("C) 241 (8.11)

[ale (Solvent) - 95"

(pyridine) (8) (I/)

- 125" (CHCII

Methods for Structure Elucidatiop and Remarks Spectroscopy (8,11) No stereochemical investigation reported; same constitution as dioncopeltine A (20). but different physical data (cf. Table 11)

H

6

HO

5'-O-Methyltriphyopeltine (7) C24H27N04 (393.5)

D. tholloni; roots, stem bark (11)

H OH MeO

Me

7

209 ( 1 1 )

Spectroscopy (11) No stereochemical investigation reported

Alkaloids from Ancistrochdaceae Ancistine (127) CzjHBNO, (407.5)

A . ealaensis; roots, twigs

275-276 (237)

- 34" (CHC1,-methanol 1 : 1) (237)

Spectroscopy (237) Reported (237) same 0-methylation product as ancistrine (U8) Stereostructure not investigatedb

230-231 (237)

- 35" (methanol) (237)

Spectroscopy (237) Reported (237) same 0-methylation product as ancistine (l27) Stereostructure not investigatedb

(237)

H

OH

127

Me0 N

$

Me

Ancistrine (12s) CZH29N04 (407.5)

A . ealaensis;

roots, stem bark (237)

H Me0 Me0

Me

128 (continues)

TABLE V (Continued)

Name, Formula (mol. wt.), and Postulated Structure (

Occurrence

+ )-Ancistrocladine

A . congolensis; roots, stem bark (225,238)

["(+)-la"] CljHBNOI (407.5) Me?

?Me

Me0

Me

?

"(+)-1a" = enf-1a

mp ("C) 258-259 (225,238)

[ale (Solvent)

+ 27" (methanol) (225)

Methods for Structure Elucidation and Remarks Spectroscopy (225,238) Reported (225) separation from its well established enantiomer l a on an achiral phase Structure not confirmedb

Ancistrocladeine (129) CaHaNO4 (403.5) Me0

OMe

A. iectorius; roots (224,238) A . ealaensis; roots (237.238)

285-288 (28) 275-277 (224,237,238)

0” (methanol) (224,237,238) + 21.3” (pyridine) (28)

Spectroscopy (20) Partial synthesis of optically inactive U9 from ancistrocladine (la) (237,238), Preparation of optically active PI29 from ancistrocladine (la) (20,28)and of M-129from hamatine (lb) (5) Experimental (4) and computational (20) CD investigations Stereostructure of the natural material not establishedb

A. ealaensis;

84 (239,240) 176 (238)

- 26” (methanol) (239) - 32” (methanol) (238)

Spectroscopy (238-240) Structure not established*

+)c “WMe Me

/N

Me0

Me

129 N

13

Ancistroelaensine (130) C ~ ~ H B N(419.5) O ~ (239) C26H31NO4 (421.5) (238,240)

Me0

“

‘

roots, stems (238-240)

OMe

\

O

w

?

M

N,

eH

(continues)

TABLE V (Continued) Name, Formula (mol. wt.), and Postulated Structure Ancistrocladonine (131) C26HDNOd (419.5) (239) C27H33NO4 (435.6) (238,240) Me0

OMe

Me

Occurrence A . ealaensis; roots, stems (238-240)

mp ("C)

82-83 (239,240) 135 (238)

[a]"(Solvent)

+ 20" (methanol) (239.240) +45" (methanol) (238)

Methods for Structure Elucidation and Remarks Spectroscopy (238-240) Structure not establishedb

Ancistrocongolensine (l32) C24HzsN04 (391.5)

A. congolensis; roots, twigs

258 (225,238)

0" (methanol) (225,238)

Spectroscopy (225,238) Structure not established*

298-299 (225,238)

0" (methanol) (225,238)

Spectroscopy (225,238) Structure not establishedb

(225,238) Me0

OMe

Me0 wl N

132

Ancistrocongine (l33) C22H21N03 (347.4)

A. congolensis; roots, twigs

(225,238) OMe

" For further details. see former review (2). See also comments in a former review ( 2 ) .

254

GERHARD BRINGMANN A N D FRANK POKORNY

XI. Summary and Outlook In pa: years, naphthylisoquinoline alkaloids have been intensively I Id. . ied with respect to isolation,structure elucidation, biogenesis, biomimetic and nonbiomimetic partial and total synthesis, and biological activity. That research has resulted in a broad spectrum of new representatives of these naturally occurring biaryl alkaloids, many of them exhibiting promising biological activities, such as antifungal, antimalarial, and insect growth-inhibiting properties. The bioassay-guided search for new active compounds yielded the discovery of the michellamines, a new class of antiviral agents with an unprecedented quateraryl framework, produced by a previously unknown plant species, A. korupensis. The challenging molecular structures of the alkaloids triggered the development of new synthetic methods for the regio- and stereoselective construction of highly hindered biaryl axes. Initial feeding experiments showed that the naphthylisoquinoline alkaloids are also unique with respect to their biogenetic origin from acetate units-and not from aromatic amino acids. Besides the search for new related alkaloids, the investigation of their biological activities, the development of synthetic pathways to these compounds, and the detailed elucidation of their biosynthesis via the acetate pathway, additional developments can be expected for the future, e.g., the investigation of the alkaloid-producing plants and their interaction with the natural environment, i.e. with microorganisms, animals, and competing plants. Thus, in uiuo NMR experiments, using chemical-shift imaging and other techniques (241), have just begun; these are aimed at an investigation of the genuine structures of the alkaloids in the plants (e.g., conjugates, glycosides, ammonium salts, etc.), their biosynthetic site, and their transport and metabolism (215). Finally, efforts will be made to develop the discovered leads further. The hope is that such efforts will result in new drugs, especially against diseases that are endemic to the countries in which the Ancistrocladaceae and Dioncophyllaceae plants grow. This will be a high-priority, challenging task, including the chemical or biological production of improved derivatives, e.g., the synthesis of unnatural dimeric alkaloids from easily available, natural monomeric alkaloids. It can clearly be expected that this unusual class of compounds-the naphthylisoquinoline alkaloids and their recently detected dimers, the michellamines-will play an increasingly important role in the future because of their exciting structural, biosynthetic, and pharmacological properties.

4. T H E

NAPHTHYLISOQUINOLINE ALKALOIDS

255

XII. Addendum In this chapter, we report on some of the key results achieved after finishing the review, predominantly concerning the isolation of new naphthylisoquinoline alkaloids and related compounds, as well as synthetic success. A. NEW ALKALOIDS FROM AFRICAN ANCISTROCLADACEAE SPECIES More recent isolation work of the Frederick and the Wiirzburg groups mainly concentrated on the African species Ancistrocladus korupensis and A . robertsoniorum, making use inter alia of liquid-liquid chromatographic methods and HPLC/NMR coupling.

I . Ancistrocladus korupenesis a . A New Korupensamine Derivative and the First Constitutionally Unsymmetric Michellamines. The Frederick group isolated several new alkaloids from A . korupensis, such as korupensamine E (124)(242).With its IR,3R,SM-configuration, 124 is similar to korupensamine B (53b), yet with a different O-methylation pattern. In addition, for the first time, constitutionally unsymmetric bis-naphthylisoquinoline alkaloids were identified (242),such as michellamine D (12% the 8-O-methyl ether of michellamine B (52b). Even less symmetric is michellamine E (m),with trans- and (in an N-methylated form) cisconfigurated tetrahydroisoquinoline parts. Likewise unprecedented is the structure of michellamine F (l27),which comprises a trans-configurated tetrahydroisoquinoline moiety and an additionally O-methylated dihydroisoquinoline part. b . Yaoundamine B , the First Naphthylisoquinoline Glycoside, and Its Aglycone. Besides the 5,8’-coupling type that is realized in all the other mono- and dimeric naphthylisoquinoline alkaloids from A . korupensis (cf. Section VI), this plant also produces 73’-coupled alkaloids, such as yaoundamine A (128)(243). Again, the absolute configuration at the axis was elucidated by comparison of its experimental CD spectrum with the calculated one, while the configuration at the stereocenter was established by oxidative degradation. Far more remarkable and without precedent for this type of alkaloid is yaoundamine B (129), the first naphthylisoquinoline glycoside, with rhamnose as the sugar part and yaoundamine A (128)as the agylcone (243).

256

GERHARD BRINGMANN A N D FRANK POKORNY

Me

OMe

OMe OH

HO

Me

OH

Me

125 (Michellamine D)

124 (Korupensamine E)

Me OH

Me

OMe

Me Me

OH

Me

126 (Michellamine E) FIG.

AH

Me

127 (Michellamine F)

39. Further new mono- and dimeric S,I’-coupled alkaloids from A . korupensis (242).

It may be expected that several other naphthylisoquinoline alkaloids genuinely occur as glycosides, but lose their sugar parts under the isolation conditions. This remains to be investigated. c . Gentrymine B , the First Quaternary Tetrahydroisoquinolinium Salt from Ancistrocladaceae. Again most remarkably, A . korupensis was found to produce a quaternary alkaloid, a naphthalene-devoid 1,2,2,3tetramethyl-6-methoxy-8-hydroxy-tetrahydroisoquinolinium salt, named gentrymine B (244). Whereas its relative configuration at C-1 versus C-3 was deduced from NMR investigations, the absolute configuration be-

4.

257

THE NAPHTHYLISOQUINOLINE ALKALOIDS

HoMe .Me

Me0

Me0

128 (Yaoundamine A)

129 (Yaoundamine B)

FIG.40. Yaoundamine A (128) and its glycosidated analog. yaoundamine B (129) (24-7).

came evident by comparison of its predicted CD spectrum with the experimental one, thus finally leading to the absolute stereostructure 130 for this unusual new compound. Accordingly, gentrymine B is the first 1S,3Stetrahydroisoquinoline found in this plant: all other trans-configurated alkaloids produced by A. korupensis are R,R-configurated! Still, this detection is not completely unexpected, since 130 might be formed from the likewise naphthalene-devoid co-occurring N-monomethylated tetrahydroisoquinoline 56, which has more recently been named gentrymine A (244). Like other cis-configurated alkaloids from A. korupensis (see Section VI.D), 56 has the lR,3S-configuration. It may be expected that upon further enzymatic N-methylation, the resulting cis-configurated product 132 might easily isomerize to the thermodynamically more stable transcompound gentrymine B (130),by ring cleavage to the amino ketone 131, followed by renewed cyclization, with inversion of the configuration at c-1. The isolation of this first cationic Ancistrocladus alkaloid suggests that entire naphthylisoquinoline alkaloids might also occur in N , N dimethylated, i.e., quaternary forms.

Me

Me

c

130 (Gentryrnine 8)

131

132

56 (Gentryrnine A)

SCHEME 42. Gentrymine B (130) and its proposed biogenetic origin from the cisconfigurated N-monomethylated analog 56 (244).(i) enzymatic N-methylation.

258

GERHARD BRINGMANN A N D FRANK POKORNY

2. Ancis trocladus robertsoniorum More recent work on A . robertsoniorum (245)reveals, besides the occurrence of known alkaloids such as ancistrocladine (la), hamatine (lb), and ancistrobrevine B (48) the presence of a broad series of further alkaloids. A first structural analysis shows close parallels to A . korupensis in that it produces 5,8'- and 7,8'-coupled alkaloids predominantly. The distinct difference is that A . robertsoniorum alkaloids exhibit a higher degree of 0-methylation, which may explain why, in contrast to A . korupensis, no michellamine-type dimers have as yet been found. But in contrast to the constituents of all other African Ancistrocladus species (cp. Section V), the alkaloids of A. robertsoniorum that have so far been investigated are all 3s-configurated. This similarity to the Asian Ancistrocfadus plants (cp. Section IV) is a first hint that the East African species A . robertsoniorum might be a geotaxonomic link between the African and the Asian Ancistrocladaceae plants. OF DIMERIC NAPHTHYLISOQUINOLINES B. SYNTHESIS

Since the appearance of the first two total syntheses of michellamines (see section VII.E), further synthetic success has been attained in this important and rapidly expanding field. 1 . A Further Biomimetically Oriented Synthesis of Michellamines

Hoye et al. (246) recently published another synthetic path to michellamines A-C (52) (see Scheme 43), which in its fundamental conception follows the first biomimetic total synthesis described in Section VILE. 1 (cf. Scheme 33). A key step is the coupling of appropriately protected MeO-0

OMe

OH

MeO-0

Me

Me

Br

W W z

Br

134

+

135

136

Pd(ll)

I

137

SCHEME43. A third total synthesis of michellamines, as published more recently (246).

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

259

and activated naphthalene and isoquinoline moieties to korupensamine derivatives, which, after deprotection of a phenolic OH-function in the naphthalene part, are oxidatively coupled with Ag,O to give, after deprotection, a mixture of diastereomeric michellamines. While the stereocenters of the isoquinoline part 137 were built up as described in former syntheses (29,301, a distinctly different approach was elaborated for the naphthalene building block 133, which was prepared by Diels-Alder reaction of an aryne (prepared from 135) and a substituted diene (resulting from 136). For the construction of the 5,8’-axis, an iodine, not a bromine substituent was chosen as the leaving group for the isoquinoline 137 and a boronic acid group for the MOM-protected naphthalene 133. Like the other syntheses, this approach seems flexible and well applicable to the synthesis of modified michellamine analogs. 2. First Synthesis of Non-Natural Dimers of Natural Monomeric Naphthylisoquinolines

It is an interesting question why, except for A. korupensis, none of the naphthylisoquinoline-containingAncistrocladaceae or Dioncophyllaceae species have so far been found to produce dimeric alkaloids-an as yet unexploited synthetic potential of these plants! For this reason, in an attempt to rationally extend the scope of michellamine-type antiviral compounds, the author’s group, having disposed of a broad series of naturally occurring and structurally modified monomeric naphthylisoquinolines,has embarked on the coupling of these biaryls to give their hitherto unknown dimers. For this, not only oxidative, but also reductive or “redox-neutral’’ procedures can be taken into consideration. Such a dimerizationprocedure (see Scheme 44) was elaborated exemplarily for dioncophylline A (14a), the easily available main alkaloid of Triphyophyllum peltatum (see Section 11). Thus, highly regioselective bromination of 14a at C-5 using N(nBu),Br, in chloroform in the presence of NaOAc, followed by N,O-dibenzylation, gave the dioncophylline A derivative 138, with the activation group in a promising, i.e., sterically not overloaded potential coupling position. Intermolecular coupling of 138 was achieved either by lithiation and subsequent oxidation with CuCI,, or by Pd-catalyzed intermolecular coupling of the corresponding boronic acid with l38, leading to the novel quateraryl 139. The resulting jozimine A (5,5-bidioncophyllineA) (140) is remarkable in several respects: It is the first non-natural dimer of a naturally occurring (even abundant) naphthylisoquinoline alkaloid. Moreover, it constitutes a completely unprecedented “non-michellamine-type” dimer since the two halves are coupled via the isoquinoline (“I”), not the naphthalene (“N”) parts, givingan “N-I-I-N” connectivity, in contrast to the michell-

260

GERHARD BRINGMANN A N D FRANK POKORNY

Bzl

Me0

Me0

Me0

Me0

SCHEME44. Synthesis ofjozimine A (5,5-bidioncophylline A) (140)-the first non-michell~B~~, amine type dimeric naphthylisoquinoline (247).Reaction conditions: (i) N ( ~ I B ~ )NaOAc; (ii) BzlBr; (iii) nBuLi+CuCI2; (iv) H2/Pd-C.

amines, which, following the same terminology, would be characterized sequence, Another difference from the michellamines by an “I-N-N-I” is that the newly created central biaryl axis is now stereogenic. According to AMl-calculations (247), the rotational barrier at the axis should be ca. 25 kcal/mol. This prediction indeed correlates very nicely with the experimental findings. The axis is stereochemically moderately stable, with a gradual interconversion of the two atropodiastereomers at room temperature. This isomerization is slow enough for these two compounds to be separated and analyzed. Work to extend our synthetic procedure to the preparation of further hitherto unknown dimeric naphthylisoquinoline alkaloids, their directed search in nature and their pharmacological evaluation, is in progress.

4.

THE NAPHTHYLISOQUINOLINE ALKALOIDS

138

26 1

@ = BzI

FIG.41. Absolute stereostructure of the dioncophylline A derivative 138 by anomalous X-ray dispersion (248).

C. FURTHER CONFIRMATION OF THE ABSOLUTE STEREOSTRUCTURE OF DIONCOPHYLLINE A A nice additional issue of these synthetic investigations was the fact that crystals suitable for an X-ray structure analysis were obtained from the derivative 138. Due to the presence of bromine as a “heavy atom element” in the molecule, the absolute configuration of l38 and thus of dioncophylline A (14a) could now for the first time be determined directly by anomalous X-ray dispersion (see Fig. 41) (248),again perfectly confirming the previously established (see Section 1I.D) relative and absolute configuration of this important and well-known naphthylisoquinoline alkaloid. D.

CONCLUDING REMARKS

Not described in detail, although worth mentioning, is further success, e.g., in the improved cultivation of the plants, such as the first fruiting of A. heyneunus in the greenhouse (249), as well as the recent detection of antimolluscicidal activity of naphthylisoquinoline alkaloids, e.g., dioncophylline A (14a), against the snail species Biomphalariu glabrata (250), vector of the tropical infectious disease bilharzia. Summarizing, monoand dimeric naphthylisoquinoline alkaloids continue to become a more and more stimulating interdisciplinaryfield of research of increasing importance.

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Acknowledgments

For their generous financial support of this work, we thank the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 25 1 “Okologie, Physiologie und Biochemie pflanzlicher und tierischer Leistung unter Strep,” Sonderforschungsbereich 347 “Selektive Reaktionen Metall-aktivierter Molekule,” Graduiertenkolleg “Magnetische Kernresonanz in uiuo und in uitro fur die biologische und medizinische Grundlagenforschung,” and Normalverfahren), the Fonds der Chemischen Industrie, the BASF AG, and the Max-Buchner-Stiftung. Particular thanks are due to the coworkers who have, with great skill and enthusiasm, developed this field by the isolation, structure elucidation, biosynthetic, synthetic, and computational work on naphthylisoquinoline alkaloids: Dr. J. R. Jansen, Dr. H. Reuscher, Dr. M. Rubenacker, Dr. H. Busse, Dr. P. Keller, Dr. D. Lisch, Dr. T. Geuder, Dr. A. Porzel, Dr. D. Scheutzow, Dr. R. Walter, Dr. R. Weirich, R. God, R. Gotz, K.-P. Gulden, J . Holenz, Ch. Kehr, W. Koch, D. Koppler, U. Kiinkele, R. Lardy, T. Ortmann, B. Peter, Ch. Schneider, F. Teltschik, S. Gramatzki, K. Wurm, B. Wiesen, M. Wenzel, and R. Zagst. Furthermore, this work would not have been possible without the competent botanical and ethnobotanical work of our friends and scientific partners, Prof. L. AkC Assi (Ivory Coast), Prof. T. Govindachari, Prof. S. M. Ketkar, Prof. A. S. Sankara Narayanan, Prof. N. J . De Souza, M. R. Almeida (India), Dr. R. D. Haller, Dr. S. A. Robertson (Kenya), Prof. C. Zhao (People’s Republic of China), Prof. I. Addae-Mensah (Ghana), and many more scientists, especially from tropical countries. Moreover, we are particularly grateful for the collaboration in the field of biological testing to Dr. G. Franqois (Belgium), Dr. M. R. Boyd (United States), Prof. J. D. Phillipson (United Kingdom), Prof. K. Hostettmann (Switzerland), Prof. P. Proksch, and the BASF AG (Germany). Furthermore, we wish to thank Prof. F.-Ch. Czygan, Dr. P. Bachmann, Dr. A. Abou-Mandour, H. Lorenz, H. Fleischmann, M. Erhart, Prof. J . Stockigt, and Prof. M. H. Zenk (Germany) for their engaged help with plants and cell cultures, as well as Prof. 0. Lange, Prof. U. Heber, and Prof. M. Riederer for their generous hospitality in allowing us to perform experiments in the Botanical Garden of the University of Wurzburg. We are also grateful to Dr. K. Peters, Dr. Ch. Burschka, Prof. J. Fleischhauer, and Dr. B. Kramer for assistance in X-ray crystallography and CD spectroscopy. Thanks are due also to Prof. T. R. Kelly and his group (United States) for the stimulating collaboration in the total synthesis of michellamines. We wish to thank those who prepared the photographic material: H. Bringmann (Plates 1 and 2), Prof. Dr. W. Barthlott (Plate 3), W. Thiele

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(Plate 4), Dr. J. R. Jansen (Plate 3,C. Schneider (Plates 7 and 9), A. Zanglein (Plate 8), and B. Wiesen (Plates 6 and 10). Finally, we are indebted to L. Kinzinger, M. Stablein, and M. Schaffer for their technical assistance, S. Lutz, D. Leimkotter, S. Harmsen, R. Gotz, and B. Wiesen for preparing the graphics, K. P. Gulden for the CD spectra, and to M. Lehrmann and J. Wild for producing and Dr. G. Franqois, Dr. M. R. Boyd, B. Wiesen, Dr. J. Hartung, and F. Teltschik for proofreading the manuscript.

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M. A. Rizzacasa and M. V. Sargent, J . Chem. SOC., Chem. Commun., 301 (1989). M. A. Rizzacasa and M. V. Sargent, J . Chem. SOC..Perkin Trans. 1 , 844 (1991). A. I . Meyers and E. D. Mihelich, J . Am. Chem. SOC. 97,7383 (1975). A. I . Meyers, R. Gabel, and E. 0. Mihelich, J. Org. Chem. 43, 1372 (1978). A. I. Meyers, A. Meier, and D. J. Rawson, Terrahedron Lett. 33, 853 (1992). A. I . Meyers. J. R. Flisak, and R. A. Aitken, J . Am. Chem. SOC. 109, 5446 (1987). G. Bringmann, T. Hartung, L. Gobel, 0. Schupp, C. L . J. Ewers, B. Schoner, R. Zagst, K. Peters, H. G. von Schnering, and C. Burschka, Liebigs Ann. Chern., 225 (1992). 139. M. A. Rizzacasa, M. V. Sargent, B. W. Skelton, and A. H. White, Aust. J . Chem. 43, 79 (1990). 140. M. A. Rizzacasa and M. V. Sargent, J . Chem. Soc., Chem. Commun., 894 (1990); J . Chem. SOC., Perkin Trans. 1 , 845 (1991). 141. M. A. Rizzacasa and M. V. Sargent, J . Chem. SOC., Chem. Commun. 278 (1991). 142. M. A. Rizzacasa and M. V. Sargent, J . Chem. SOC., Perkin Trans. 1 , 2773 (1991). 143. At present, very promising attempts are undertaken for the extension of the lactone methodology even to this coupling type: G. Bringmann and R. Gotz, unpublished results (1994). 144. G. Bringmann, R. Gotz, and M. Boyd, U.S. Pat. Appl. 08/279,291 (1994). 145. Anonymous, J . Nut. Prod. 55, 1018 (1992). 146. G. Bringmann, F. Pokorny, M. Stablein, T. R. Govindachari, M. R. Almeida, and S. M. Ketkar, PIanta Med. 57(Suppl. 2), 98 (1991). 147. D. H. R . Barton and T. Cohen, Festschr. Prof. Dr. Arthur Stoll Siebzigsten Geburtstag, I17 (1957). 148. W. 1. Taylor and A. R. Battersby, “Oxidative Coupling of Phenols.” Arnold, London 1967. 149. G. Bringmann, S. Harmsen, J. Holenz, T. Geuder, R. Gotz, P. A. Keller, R. Walter, Y. F. Hallock, J. H. Cardellina, 11, and M. R. Boyd, Tetrahedron 50, 9643 (1994). 150. G. Bringmann, S. Harmsen, and M. Boyd, U.S. Pat. Appl. 08/279,339 (1994). 151. J. G. Supko and L . Malspeis, Anal. Biochem. 216, 52 (1994). 152. G. Bringmann, R. Gotz, T. R. Kelly, M. R. Boyd, U.S. Pat. Appl. 08/305,211 (1994). 153. J. D. Phillipson, M. F. Roberts, and M. H. Zenk, eds., “The Chemistry and Biology of Isoquinoline Alkaloids.” Springer-Verlag, Berlin, 1985. 154. T. M. Kutchnan, H. Dittrich, D. Bracher, and M. H. Zenk, Tetrahedron 47, 5945 (1991). 155. A. Pictet and T. Spengler, Chem. Ber. 44, 2030 (1911). 156. P. Manitto and P. G. Sammes, “Biosynthesis of Natural Products.” p. 43. Ellis Horwood, Chichester, 1981. 157. K. Mothes, H. R. Schiitte, and M. Luckner, eds., “Biochemistry of Alkaloids,” p. 18. VCH Verlagsgesellschaft, Weinheim, 1985. 158. M. Shamma, “The lsoquinoline Alkaloids.” Academic Press, New York, 1972. 159. D. R. Dalton, “The Alkaloids: A Biogenetic Approach.” Dekker, New York, 1979. 160. G. Bringmann, F. Pokorny, and H.-D. Zinsmeister, Palmengarten (FrankfurtlM., Ger.) 5513 13 (1991). 161. Compare the analogous reductive nitrogen incorporation in the biogenesis of the acetogenic piperidine alkaloid coniine (161a,b). 161a. M. F. Roberts, Phytochemistry 16, 1381 (1977). 161b. M. F. Roberts, Phytochemistry 10, 3057 (1971). 162. J. S. Hubbard and T . M. Harris, J . A m . Chem. SOC. 102, 2110 (1980). 132. 133. 134. 135. 136. 137. 138.

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269

I . Kubo, M. Taniguchi, A. Chapya, and K. Tsujimoto, PIanra Med., Suppl.. 185 (1980). G. Bringmann, J. R. Jansen, F. Pokorny, and H. Reuscher, unpublished results. G. T. Gujar, Fitoterapia 61, 387 (1990). R. Durand and M. H. Zenk, Tetrahedron Lerr., 3009 (1971). H. K. Desai, D. H. Gawad, T. R. Govindachari, B. S. Joshi, N. Kamat. J. D. Modi, P. C. Parthasarathy, J. Radhakrishnan, M. N. Shanbhag, A. R. Sidhaye, and N. Wiswanathan, Indian J. Chem. 11, 840 (1973). 168. T. R. Govindachari, P. C. Parthasarathy, and J. D. Modi, Indian J . Chem. 9, 1042 ( I97 1 ). 169. G. Bringmann, D. Koppler, and A. Porzel, unpublished results. 170. Only a single occurrence of a naphthylisoquinoline alkaloid outside the Ancistroclada. could not be verified (2). ceae and Dioncophyllaceae was reported ( 1 7 0 ~ )but 170a. R. Mehta, 0. P. Arora, and M. Mehta, Indian J . Chem., Sect. B UIB, 834 (1981). 171. A. Takhtyan, in “Floristic Regions of the World” (A. Cronquist, ed.), p. 197. University Press of California, London, 1986. 172. R. Hegnauer, Phytochemistry 25, 1519 (1986). 173. R. Hegnauer, Soc. Bor. Ira/. l23, 15 (1986). 174. H . Gottwald and N. Paramareswaran. Bor. Jahrb. Sysr. Pflanzengesch. Pflanzengeogr. 88,49 (1968). 175. The name Ancistrocladus, which is of Greek origin, means “hooked branch.” 176. 0. Warburg, “Die Pflanzenwelt,” p. 490. Bibliographisches Institut, Leipzig, 1921. 177. H. K . Airy Shaw, Kew Bull. 3, 327 (1951). 178. S. Green, T. L. Green, and Y. Heslop-Hamson, Bor. J. Linn. Soc. 78, 99 (1979). 179. J. E. Marburger, A m . J. Bot. 66, 404 (1979); W. Barthlott, Bonner Universitatsbl. (Bonn, Ger.) 15 (1988). 180. T. R. Govindachari, personal communication (1979). 181. G. Bringmann and T. R. Govindachari, unpublished results (1980). 182. G. Bringmann, C. Schneider, F. Pokorny, H. Lorenz, H. Fleischmann, A. S. Sankaranarayanan, M. R. Almeida, T. R. Govindachari, and L. AkC Assi, Planra Med. 59(Suppl.), 623 (1993). 183. D. L. Gamborg, Exp. Cell Res. 50, 151 (I%@. 184. C. L. Comar, “Radioisotopes in Biology and Agriculture,” p. 148. McGraw-Hill, New York, 1955. 185. G. Bringmann, F. Pokorny, and M. Stablein, unpublished results. 186. H. Hikino, T. Miyase, and T. Takemoto, Phyrochemisrry 15, 121 (1976). 187. S. A. Brown and L. R. Wetter, Prog. Phyrochem. 3, 1 (1972). 188. S. A. Brown, in “Biosynthesis” (T. A. Geissman, ed.), Vol. I , Chapter I , p. I . Chemical Society, London, 1972. 189. G. Bringmann and J. R. Jansen, Liebigs Ann. Chem., 21 16 (1985). 190. G. P. Cooper and S. J. Record, Bull.-Yale Univ.. Sch. For. 31, 27 (1931). 191. G. Bringmann, L. AkC Assi, M. Rubenacker, E. Ammermann, and G. Lorenz, D.O.S. D E Appl. 4117080 A l (1991); Eur. Pat. Appl. E P 0 515 856 A l 1992). 192. G. FranCois, G. Bringmann, J. D. Phillipson, L. AkC Assi, C. Dochez, M. Rubenacker, C. Schneider, M. Wery. D. C. Warhurst, and G. C. Kirby, Phyrochemisrry 35, 1461 (1994). 193. G. FranCois, G. Bringmann, C. Dochez, C. Schneider, G. Timperman, and L. AkC Assi, J. Erhnopharrnacol., in press (1995). 194. G. FranCois, G. Bringmann, J. D. Phillipson, M. R. Boyd. L. Ake Assi, C. Schneider, G. Timperman, and C. Dochez, U S . Pat. Appl. 08/195,547 (1994). 163. 164. 165. 166. 167.

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195. G . Franqois, G. Timperman, R. D. Haller, S. Bar, M. A. Isahakia, S . A. Robertson, C. Zhao, N . J. De Souza, L. Ake Assi, J. Holenz, and G. Bringmann, Int. J . Pharmacognosy (submitted for publication). 1%. D. L. Klayman, Nat. Hist. 10, 18 (1989). 197. J. E. Touze and G. Charmot, Cah. SantP 3, 217 (1993). 198. WHO Malaria Unit, Bull. WHO 71, 281 (1993). 199. I. Haworth, in “Malaria: Principles and Practice of Malaria“ (W. H. Wernsdorfer and I. McGregor, eds.), Vol. 2. p. 1379. Churchill-Livingstone, Edinburgh, 1988. 200. J. D. Phillipson and C. W. Wright, PIanta Med. 57(Suppl. I), 53 (1991). 201. W. Peters, “Chemotherapy and Drug Resistance in Malaria,” Vol. 2, p. 543. Academic Press, London, 1987. 202. World Health Organization, Wkly. Epidemiol. Rec. 64, 241 (1989). 203. G. Franqois and G. Bringmann, unpublished results (1993). 204. G. Franqois, G. Bringmann, J. Holenz. G. Timperman, and L. Ake Assi, Ann. Trop. Med. Parasitol. (accepted for publication). 205. G. Franqois, G. Bringmann, and G. Timperman, unpublished results (1994). 206. M. R. Boyd, personal communication (1992). 207. J. B. McMahon, M. J. Currens, R. J. Gulakowski, R. W. Buckheit, Jr., C. LackmanSmith, Y.F. Hallock, and M. R. Boyd, Antimicrob. Agents Chemother. 39,484 (1995). 208. J . G. Supko and L. Malspeis, Antimicrob. Agents Chemother. 39, 9 (1995). 209. G. Bringmann, S. Gramatzki, C. Grimm. and P. Proksch, Phyrochemistry 31, 3821 (1992). 210. C. Grimm,P. Proksch, S. Gramatzki, C. Schneider, and G. Bringmann, Planta Med. 58(Suppl. I), 630 (1992). 211. G . Bringmann, S. Gramatzki, C. Grimm,and P. Proksch, Planta Med. 59(Suppl.),624 (1993). 212. C. P. Malik and M. B. Singh, Annu. Reu. Plant Sci. 1, 67 (1979). 213. H. C. Weber, “Parasitismus von Blutenpflauzen.” Wissenschaftliche Buchgesellschaft, Darmstadt, 1993. 214. G. Bringmann, C. Schneider, B. Wiesen, and P. Proksch, unpublished results (1994). 215. G. Bringmann, S. Gramatzki, R. God, and P. Proksch, Planta Med. 58(Suppl. I), 577 (1992). 216. G. Bringmann and F. Pokorny, unpublished results. 217. G. Bringmann, R. Zagst, D. Koppler, and L. Akt Assi, unpublished results. 218. T. R. Govindachari, P. C. Parthasarathy, T. G. Rajagopalan, H. K. Desai, K. S. Ramachandran, and E. Lee, J. Chem. SOC., Perkin Trans. I , 2134 (1975). 219. P. C. Parthasarathy and G. Kartha, Indian J. Chem., Sect. B 22B, 590 (1983). 220. T. R. Govindachari, P. C. Parthasarathy, and H. K. Desai, Indian J. Chem. 11, 1190 ( 1973). 221. D. Meksuriyen, N. Ruangrungsi, P. Tantivatana, and G. A. Cordell. Phyrochemistry 29, 2750 (1990). 222. G. Bringmann and B. Peter, unpublished results. 223. T. R. Govindachari and P. C. Parthasarathy, Indian J. Chem. 8, 567 (1970). 224. J.-P. Foucher, J.-L. Pousset, A. Cave, and R. R. Paris, Plant. MPd. PhytothCr. 9,26 (1975). 225. J.-P. Foucher, J.-L. Pousset, A. Cave, A. Bouquet, and R. Paris, Plant. Mkd. Phytoth6r. 9, 87 (1975). 226. T. R. Govindachari, P. C. Parthasarathy, H. K. Desai, and M. T. Sindane, 1ndian.l. Chem., Sect. B 15B, 871 (1977).

4. T H E NAPHTHYLISOQUINOLINE ALKALOIDS

27 1

227. G . Bringmann, L. Kinzinger, F. Pokorny, and R. Weirich, unpublished results. 228. G. Bringmann, F. Pokorny, and H. Reuscher, unpublished results. 229. A. V. B. Sankaram, V. V. Narayana Reddy, and M. Marthandamurtui, Phyrochemisrry 25, 2867 (1986). 230. G . Bringmann, F. Pokorny, W. Bermel, R. Kerssebaum. and D. Scheutzow, unpublished results (1992). 231. R. H. Thomson, J . Chem. SOC., 1237 (1951). 232. R. H. Thomson, “Naturally Occurring Quinones,” 2nd ed. Academic Press, New York, 1971. 233. R. G . F. Giles and G . H. P. Roos, J . Chem. SOC.,Perkin Trans. I , 2057 (1976). 234. M. Tezuka, C. Takahashi, M. Kuroyangi, M. Satake. K. Yoshihira, and S. Natori, Phyrochemistry U,175 (1973). 235. G. Bringmann and D. Koppler, unpublished results. 236. G. Bringmann, C. Schneider, R. M. Schmidt, and S. Dobreff, unpublished results. 237. J.-P. Foucher, J.-L. Pousset, and A. Cave, Phytochemisrry 14, 2699 (1975). 238. J.-P. Foucher, Doctoral Dissertation, Universitt Paris-Sud, Paris (1975). 239. J.-P. Foucher. J.-L. Pousset, A. Cave, A. Bouquet, and R. Pais, Plant. MPd. PhyrothPr. 5, 16 (1971). 240. J.-P. Foucher, J.-L. Pousset, A. Cave, and A. Cave, Phytochemistry 13, 1253 (1974). 241. F. W. Wehrli, D. Shaw, and J. B. Kneeland, eds., “Biomedical Magnetic Resonance Imaging.” VCH Verlagsgesellschaft, Weinheim, 1988. 242. Y. F. Hallock, K. P. Mantredi, J. H. Cardellina 11, M. Schaffer, G. Bringmann, G. FranCois, and M. R. Boyd, unpublished results (1994). 243. Y. F. Hallock, J.-R. Dai, J. H. Cardellina 11, M. Schaffer. G. Bringmann, and M. R. Boyd, unpublished results (1995). 244. Y. F. Hallock, J. H. Cardellina 11, K.-P. Gulden, T. Kornek, G. Bringmann, and M. R. Boyd, Terruhedron Lerr. (submitted for publication) (1995). 245. G. Bringmann, F. Teltschik, D. Koppler, S. Bar, and R. D. Haller, unpublished results (1995). 246. T. R. Hoye, M. Cheng, L. Mi, and 0. P. Priest, Tefruhedron Lerr. 47, 8747 (1994). 247. G. Bringmann, W. Saeb, and M. Stahl, unpublished results (1995). 248. K. Peters, E.-M. Peters, H. G. von Schnering, G. Bringmann, and W. Saeb, unpublished results (1995). 249. G . Bringmann, B. Wiesen, H. Fleischmann, W. Thiele, and H. Lorenz, unpublished results (1995). 250. K. Hostettmann, J. Holenz. and G. Bringmann, unpublished results (1994).

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-CHAPTER5-

THE BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS BY PLANT TISSUE CULTURES KINUKOIWASA Kobe Pharmaceutical University, Kobe, Japan

I. Introduction . .......................................................... 273 11. The First Path ................................................... A. Conversions of Tetrahydroprotoberberines or 13 Methyltetrahydroprotoberberines into Benzophenanthridines via 277 Protopines ................................................................................... B. Interconversions between Tertiary and Quaternary Protoberberines: Application of LC/APCI-MS to Biosynthetic Studies ................ C. Formation of Benzophenanthridines from Protoberberines Using Corydalis, Chelidonium, and Macleaya Plant Species and Plant Tissue Cultures .... 314 D. Biosynthetic Studies of Protoberberines, Protopines, and Benzophenanthridines at the Enzymic Level ....... 111. The Second Pathway ........................................... A. Conversions of Tetrahydroprotoberberines into Rh Protopines: Whole Plant Studies . ...................... ............... 329 IV. The Third Pathway .............................................. A. Formation of 13-Hydroxytetrahydroprotoberberines in Whole Plants ..... 333 B. Conversions of 13-Hydroxytetrahydroprotoberberines into Spirobenzylisoquinolinesor Benzindanoazepines via 13-Oxoprotopines .. 335 C. Conversions between Tertiary and Quaternary 13-H ydroxyprotoberberines .............................. D. Biosynthetic Pathway of 13-Hydroxytetrahydropro References ................................. ......................................... 345

I. Introduction Most protoberberine alkaloids exist in nature either as tetrahydroprotoberberines or as quaternary protoberberinium salts, and some dihydroprotoberberines are also known. A number of quaternary Nmethyltetrahydroprotoberberinium salts, as well as N-oxides, have also been reported. Substituents on the aromatic nucleus are usually present 273

274

KINUKO IWASA

at C-2 and C-3, and either at C-9 and C-I0 or at C-10 and C-11. In some instances, a hydroxyl or methoxyl substituent may be present at C-I. A methyl group is sometimes found at C-8 or at C-13, while in a few cases an alcoholic hydroxyl group is located at C-13 or at (2-5. This review will cover mainly biosynthetic studies using plant tissue cultures on nonphenolic protoberberines and 13-methyl- and 13-hydroxyprotoberberines possessing substituents at C-2 and C-3, and either at C9 and C-I0 or at C-10 and C-I 1. However, some studies using whole plants will also be included. The biosynthetic conversion of the protoberberine system into other alkaloidal types, such as protopines, benzophenanthridines, rhoeadines, benzindanoazepines, and spirobenzylisoquinolines, will be discussed. The biosynthetic routes from the primary metabolite L-tyrosine to simple protoberberines have been investigated by plant feeding experiments and enzymatic studies, and the results have been reviewed ( 1 ) . It has been reported that tetrahydroxylated (S )-norlaudanosoline is the first benzylisoquinoline formed from the condensation of two c& units derived from L-tyrosine (2). Two O-methylations and one N-methylation of (S)norlaudanosolinegenerate (S)-reticuline, although the precise methylation sequence is not completely clear when working with whole plants or plant parts. It has been firmly established that the so-called “berberine bridge” carbon [C(8) of the tetrahydroprotoberberines] arises via oxidative cyclization involving the N-methyl group of reticuline (4,5). (S)- and (R)-Reticulines undergo cyclization to give (S )- and (R)-scoulerines (l), respectively. Nonphenolic protoberberines are derived through one or more methylenedioxy ring formations and/or one or two O-methylations from scoulerine. For example, the formation of a methylenedioxy group on ring D and O-methylation at the 9-OH position of (S)-scoulerine (1) generate (S)-cheilanthifoline (2) and (S)-tetrahydrocolumbamine (3), respectively, in which formation of the methylenedioxy system on ring A produces the nonphenolic protoberberines (S)-stylopine (4) and (S)canadine (9,respectively (Scheme 1). Recently, Muller and Zenk (6) demonstrated that trihydroxylated (S)norcoclaurine, but not norlaudanosoline, is formed from different c& units derived from L-tyrosine. This finding was obtained by feeding @?‘3C]tyrosine,L-[~’-’*O]DOPA, and (S)-[ l-’3C]norcoclaurineusing cell suspension cultures of Coptisjaponica (Ranunculaceae). The entire series of steps for the formation of berberine at the enzymic level is indicated in Scheme 2. (S)-Reticuline is formed from (S)-norcoclaurine through 0methylation of the C-6 hydroxyl, N-methylation, hydroxylation at C-3‘, and subsequent O-methylation at C-4. (S)-Reticuline is then bioconverted into nonphenolic protoberberines via scoulerine (1).

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

( S t Norlaudanosoline

L-Tyrosine

275

(SI-Reticuline

OHe (SI-Scoulerine ( 1 )

SCHEME 1. A biosynthetic pathway for the formation of protoberberines from the amino acid L-tyrosine.

OH-

OM. (S>Seoulerim (1)

OM. (~TconhydroEolumb.mim(3)

OM. (SkCanndine (5)

Berberim (17)

SCHEME 2. Formation of berberine via norcoclaurine from L-tyrosine at the enzymatic level.

276

KINUKO I W A S A

Three different biogenetic pathways (Scheme 3) from nonphenolic protoberberines were delineated through feeding experiments with whole plants and through studies using tissue cultures of several Cordalis species (C. incisa, C. ophiocarpa, C . ochotensis var. raddeana, C . platycarpa, and C . pallida var. tenuis) (Fumariaceae), as well as Macleaya cordata, Chelidonium majus, Papauer bracteatum, and P . rhoeas (Papaveraceae). The first pathway involves the sequence protoberberinium salts or 13-methylprotoberberinium salts + tetrahydroprotoberberines or 13methyltetrahydroprotoberberines + a-N-metho salts + protopines + benzophenanthridines. The second pathway displays the sequence tetrahydroprotoberberines + a-N-metho salts + protopines --* rhoeadines. Finally, the third pathway involves the conversion sequence tetrahydroprotoberberines + 13-hydroxytetrahydroprotoberberines + a-N-metho salts + 13-oxoprotopines+ spirobenzylisoquinolines or benzindanoazepines. These biosynthetic conversions involve N-methylation, C-methylation or oxidation at C-13, and oxidation at C-14, or alternatively, oxidation at C-6 or C-8. The protopines are key intermediates, leading from protoberberines to the other isoquinoline alkaloids, such as benzophenanthridines, rhoeadines, spirobenzylisoquinolines, and benzindanoazepines. The evidence for these pathways will be reviewed sequentially.

Protuherhcrinm

13-H ydroxyprutoherkrines

a.N-Mcthu salts

I3-Oruprutapinrs

llenrindanoarepincs

SCHEME3. The biosynthetic routes from protoberberines to other skeletal alkaloids.

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

277

XI. The First Pathway A. CONVERSIONS OF TETRAHYDROPROTOBERBERINES OR 13-METHYLTETRAHYDROPROTOBERBERINES INTO

BENZOPHENANTHRIDINES VIA PROTOPINES 1 . Whole Plant Studies

Chelidonium majus and Corydalis incisa plants were chosen as vehicles for the biosynthetic studies of protoberberines and 13-methylprotoberberines because the former produces tetrahydroprotoberberine-, protopine-, and benzophenanthridine-typealkaloids, while the latter contains mainly the same type of alkaloids, but with a C-methyl group at the position corresponding to C-13 of the tetrahydroprotoberberine skeleton (7). The protoberberines, protopines, and benzophenanthridines involved in this review are listed in Tables I-V. a. Feeding Experiments on Chelidonium majus (Papaueraceae) (i) Feeding of (R,S)- or (S)-[N-'3CHJstylopine a-and fl-N-metho salts (4a and 4b) (8). The N-methyl derivatives of tetrahydroprotoberberines, i.e., a- and P-tetrahydroprotoberberine N-metho salts (cis- and transB/C ring junctures, respectively), have been found in members of the ( R , S ) -or ( S ) - a - and -p-[N-'3CH,]StylopinemethochloPapaveraceae (7). rides (4a and 4b) were, therefore, separately administered to Chelidonium majus plants in order to obtain evidence supporting the intermediacy of these salts in the biosynthetic sequence of protoberberine alkaloids. Feeding of (R,S)-stylopine a-N-metho salt (4a) (in a mixture with 25% of 4b) resulted in incorporation into protopine (6) and sanguinarine (7) (see Scheme 4). Sanguinarine was reduced and identified as 5,6-dihydrosanguinarine (44) (Table IV). Using 'H- and I3C-NMR spectroscopy, it was found that the N-methyl groups of protopine (6) and dihydrosanguinarine (44) were enriched with I3C. The approximate enrichments of protopine and dihydrosanguinarine were determined to be 9.9 and 5.5%, respectively. By contrast, it was found that (R,S)-stylopine P-N-metho salt (4b) is not a precursor of protopine (6) and sanguinarine (7). (S)-Stylopine a-N-metho salt (4a) (in a mixture with 25% of 4b) was satisfactorily incorporated into protopine (6), sanguinarine (7), and chelidonine (8) with '3C-enrichments of 38, 31, and 29%, respectively (see Scheme 4). Significantly, the P-N-metho salt of (S)-stylopine (4b) was not incorporated into these alkaloids. The difference in enrichment between experiments with the (S)-form and the (R,S)-form of stylopine a-N-metho salt (4a) may have arisen from the fact that the (S)-form is incorporated

TABLE I THETETRAHYDROPROTOBERBERINE ALKALOIDS A N D THEIR N-METHOSALTS

N 00 4

1 Scoulerine Cheilanthifoline Tetrahydrocolurnbarnine Stylopine 4-a-N-Metho salt 4-P-N-Metho salt Canadine 5-a-N-Metho salt 5b 5-P-N-Metho salt 9 Tetrahydrocorysarnine 9a 9-a-N-Metho salt 9b 9-P-N-Metho salt 10 Mesotetrahydrocorysarnine 1Oa 10-a-N-Metho salt 14 Ophiocarpine 14a 14-a-N-Metho salt 2 3 4 4a 4b 5 5a

OH OH OH

OMe OMe OMe OCHzO OCH2O OCH2O OCHzO OCHZO OCHzO OCHzO OCH2O OCH2O OCH2O OCHzO OCHzO OCH20

OH

OMe OCH2 0 OMe OMe OCHzO OCHzO OCHzO OMe OMe OMe OMe OMe OMe OCHzO OCHzO OCHzO OCH2O OCHzO OMe OMe OMe OMe

H H H H H

H H H H H H H H H H H

H H H H H H H H H H

H H Me Me H H

H H H H H H H H H Me Me Me H H OH OH

-

-

-

...Me -Me ...Me -Me ...Me -Me

-

...Me

...Me

h)

3

15 Corycarpine 20 Mesothalictricavine 2 h 20-a-N-Metho salt 211113-Methylpseudotetrahydroberberine a-N-metho salt Un Thalictrifoline a-N-metho salt 23 Mesocorydaline 23a 23-a-N-Metho salt 29 Thalictricavine 29a 29-a-N-Metho salt 29b 29-P-N-Metho salt 30 Corydaline 3011 30-a-N-Metho salt 30b 30-P-N-Metho salt 35 6Hydroxytetrahydrocorysarnine a-N-metho salt

36 6-Hydroxymesotetrahydrocorysamine a-N-metho salt 55 Tetrahydropalmatine 55a 55-a-N-metho salt 55b 55-8-N-metho salt 61 Tetrahydrothalifendine 62 Corydalmine 71 Nandinine 80 Epiophiocarpine 8Oa 80-a-N-Metho salt 85a 1-Methoxy-epiophiocarpine a-N-metho salt

OCHzO OCHTO OCHzO OCHzO

OCHzO OMe OMe OMe OMe H OMe

H H H OMe

H Me Me Me

OH H H H

OMe OMe OMe OMe OMe OMe OCHTO OCHzO OCHzO OMe OMe OMe OMe OMe OMe OCHzO

OCHIO OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OCHTO

H H H H H H H H H H

Me Me Me H H H H H H H

H H H Me Me Me Me Me Me Me

OCHTO

OCHzO

H

Me

H

H H H H H H H H H

H H H H H H OH

H H H H H H H

OMe OMe OMe OMe OMe OMe OCHzO OMe OMe OCHTO OCHZO OCHzO OCHZO

OMe OMe OMe OMe OMe OH OMe OMe OMe

OMe OMe OMe OH OH OMe OMe OMe OMe

OH

H

OH

H

...Me

OH

280

KINUKO IWASA TABLE 11 THEPROTOBERBERINIUM SALTS

11

53 R I + R ~ = C H7,8-Dihydroberberine ~ 56 Rl=R2=Me 7.8-Dihydropalmatine R,

13 16 17 54 57 58 60

Dehydro-ophiocarpine Coptisine Berberine Palmatine 13-Methylberberine 13-Methylpalmatine Columbamine

RI CH2 CHI CH2

Me

Me

R4

R5

Me

OH H H H Me Me H

CH2 Me

CHI Me H

R3

Me Me

Me Me Me Me Me

Me Me Me Me Me

TABLE 111 THEPROTOPINE ALKALOIDS

H H H H 13-Methylpseudoallocryptopine H 13-Methylcryptopine H 13-Methylmuramine H Muramine H 13-Hydroxymuramine H H 13-Oxomuramine I-Methoxy-l3-oxoallocryptopine OMe Ochrobirine H 13-Hydroxyallocryptopine H H 13-Oxoallocryptopine

6 Protopine 11 Corycavine 18 Allocryptopine 24 Corycavidine

25 26 27 59 65 66 72 73 81 82

OCHzO OCH20 OCH20 OCHIO OCHIO OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OCH,O OCHZO OCHIO OCH,O

OCH2O OCHzO OMe OMe OMe OMe H OMe OCHIO OMe OMe OMe OMe OMe OMe OMe OMe OMe OMe OCHIO OMe OMe OMe OMe

H H H H OMe H H H H H H H H H

H H H H H H H H H

H Me H Me Me Me Me H OH 0 0 H OH H OH 0

5 . BIOTRANSFORMATION OF PROTOBERBERINE

28 1

ALKALOIDS

TABLE IV THE AROMATIZED BENZOPHENANTHRIDINE ALKALOIDS

@ORI 9 0

\

o@:)

OR2

0

4

43 R=Me 6-0-Merhoxysanguinarine 44 R=H 5.6-Dihydrosanguinarine

OR3 Rl

7 19 48 49 50 51

Sanguinarine Chelerythrine Sanguirubine Chelilutine Sanguilutine Chelirubine 52 Macarpine

R

Lo

6

R40

N. Me

R2

CH2

CH2 CH2 Me

Me

Me

Me

Me CH2

Me CH2

Me Me

Me Me CH2 CH2

CH2 CH2

R5

R6

H H OMe OMe OMe OMe OMe

H H H H H H OMe

R4

RI

TABLE V THE HYDROBENZOPHENANTHRIDINE ALKALOIDS

R20

9') '

** H 4

6

N,

0

Me

OR1

0

Me

R,

R, CH2 CH2

8 Chelidonine

U Corynoline

28 Corynoline analog Acetylcorynoline 1 I-Epicorynoline analog Acetylcorynoline analog 1 I-Epicorynoline Acetylchelidonine 47 Hornochelidonine

32 33 34 40 46

0

31 R=Me Corynoloxine 45 R=H Dehydrochelidonine

Me

Me CH2

Me Me

Me Me CH2 CH2

Me

Me

Me

41 14-Epicorynoline

R3 H Me Me Me Me Me Me H H

R4

-OH -OH -OH -0Ac ...OH -0Ac ...OH -0Ac -OH

282

KlNUKO IWASA

just as efficiently. Based upon these results, the stereospecificconversions of the a-form of (S)-stylopine a- and P-N-metho salts (4a and 4b) into protopine (6), sanguinarine (7),and chelidonine (8) were established. (ii) Feeding of [N-13CHJprotopine (6) (8). Subsequently, [N13CH3]protopine(6) hydrochloride was fed to Chelidonium majus plants. ‘H-NMR spectroscopy showed that the N-methyl groups of dihydrosanguinarine and chelidonine were enriched with 13C (42 and 38%, respectively) (Scheme 4). Protopine (6) was thus shown to be incorporated into sanguinarine (7)and chelidonine (8). In conclusion, it was confirmed that only the a-N-metho salt in the mixture of (S)-stylopine a- and p-N-methosalts (4a and 4b) is stereospecifically converted via protopine (6) into sanguinarine (7)and chelidonine (8) (Scheme 4). It is appropriate to recall that Battersby et al. (9) obtained incorporation of (R,S)-[8-3H,N-14CH,]stylopine methochloride (4a or 4b) into protopine (6) and chelidonine (8) (1.4 and 0.2%, respectively) and suggested that an N-metho salt lies on the pathway to both protopine (6) and chelidonine (8). b. Feeding Experiments on Corydalis incisa (Fumariaceae) (i) Feeding of (R,S)-[N-13CHJstylopine a- and P-N-metho salts (4a and 4b) (8). ( R , S ) - a - And p-[N-13CH3]stylopinemethochloride (4a and 4b) were fed in separate experiments to C. incisa plants. (R,S)-Stylopine

(R.S)-[N-’3CH,14a (R,S)-[N-13CH3)4b (S)-[N-”CH3]4a

-

[N-I’CHII-~(”Ccnrichment 9.9%)+ [N-”CH3]-7(”Ccnrichmenr 5.5%)

*6+1

IN-”CH3I-6 (”Ccnrichment 38%) + [N-”CH3]-7 (’3C-enrichment31%)

+ [N-”CH,I-8 (”C-enrichment 29%)

(S)-[N-”CH,]4b

6+7+8

(R.S)-[N-”CH3]-6

[N-”CH,I-7 (”Ccnrichmnt42%)+ [N-”CH3]-8 (‘3Ccnrichment38%)

fRS1-(8-3H;N-“CH3J-4a or -4b

6 (“C-iIcO~alion1.4%)+ 8 (’4C-incorporation0.2%)

SCHEME 4. Formation of sanguinarine (7) and chelidonine (8) from (S)-stylopine a-Nmetho salt (49) via protopine (6) in Chelidonium mujus plants.

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

283

a-N-metho salt (4a) (admixed with 19% of 4b) was incorporated well into protopine (6) (13C-enrichment55%) (see Scheme 5 ) . On the other hand, there was poor incorporation into protopine (I3Cenrichment 8.0%) upon administering the p-N-metho salt (4b) (admixed with 3% 4a). The stereospecific incorporation of only the a-form of the a-and p-Nmetho salts of (R,S)-stylopine into protopine (6) is in accord with the result found with Chelidonium majus. (R,S)-[N-14CH3]Stylopinemethochloride (in a mixture of a- and p-N-metho salts) was fed to C. incisa, and radioactive protopine (6) was obtained with 0.19% incorporation (10). By standard chemical degradation procedures the label was found to be located exclusively at the N-methyl group. (ii) Feeding of (R,S)-[13-13CH3]tetrahydrocorysamine and -mesotetra13,14-2H3]Tetrahyhydrocorysumine (9 and 10) (8). (R,S)-[13-I3CH3;8, drocorysamine and -mesotetrahydrocorysamine (9 and 10)hydrochlorides were administered to C. incisa plants, and both experiments showed these alkaloids to be the precursors of the 13-methylprotopine corycavine (ll), the 13C-enrichmentsbeing 9.0 and 5.0%, respectively (Scheme 5 ) . Yagi et al. administered (R,S)-[8,14a-3H2]stylopineand -tetrahydrocorysamine

~R,S)-[N-"CHII-~P

-

-

6 ("Ccnrichment 55%)

fRS)-IN-"CH]I4n and 4b (RS)-18.14-1H]4

6 ('4C-incorporation0.19%) 12 ('H-incorporation0.14%)

(R,S)-Il3-"CHl]-9

11 ("Ccnrichment 9.0%)

(R.S)-I13-"CH]I-10 (R.S)-[8,14-1H]-9 fR.S)-[N-''CHi1-98 (R,S)-[N-11CH~]-9b (R,S)-[N-'lCHl]-lOa

a

11 (']Ccnrichmcnt 5.0%) 13 ('H-incorporation 0.87%)

11 ('k-enrichment 30%) 11 11 ("C-enrichment 39%)

SCHEME5. Formationof protopine (6). corycavine (ll),and corynoline (U) from stylopine (4). tetrahydrocorysamine (9), mesotetrahydrocorysamine (lo), and their a-N-metho salts (4a, 9a. and 1oP) in Corydalis incisa plants.

284

KINUKO IWASA

(4 and 9) hydrochlorides to C . incisa plants, and obtained incorporations into corynoline (12) of 0.14 and 0.87%, respectively ( 1 1 ) . (iii) Feeding of (R,S)-[N-13CHJtetrahydrocorysamine a- and p-Nmetho salts (9a and 9b) (8). Tetrahydrocorysamine a-N-metho salt (9a) was demonstrated to be stereospecifically incorporated into corycavine (11) (I3C-enrichment30%) through feeding experiments of ( R , S ) - a - and ( R , S)-P-[N-'3CH3]tetrahydrocorysamine methochlorides (9a and 9b) to C. incisa plants (Scheme 5). Again, the p-form (9b) was not effective as a precursor of corycavine. (iv) Feeding of (R,S)-[N-13CHJmesotetrahydrocorysamine a-N-metho salt (1Oa) (8). Feeding experiments in C. incisa that made use of ( R , S ) a-[N-13CH,]mesotetrahydrocorysamine methochloride (10a) showed good incorporation into corycavine (13C-enrichment39%) (Scheme 5). Thus, these experiments define the following sequence: tetrahydrocorysamine (9) (cis relationship of the hydrogens at C-13 and C-14) its aN-metho salt (9a) + corycavine (11) + corynoline (12).It is noteworthy that mesotetrahydrocorysamine (10) (trans relationship of the protons at C-13 and C-14) is also converted via its a-N-metho salt (10a) into corycavine (11) (Scheme 5). The biosynthetic conversion of tetrahydroprotoberberines and their aN-metho salts into benzophenanthridines via protopines was demonstrated in Chelidonium majus and C. incisa plants (Scheme 6). 2. Callus Tissue Studies In many cases, the protoberberines normally found in the plants of origin were not detected in our callus tissues, although the protopines and benzophenanthridines, especially protopine (6) and sanguinarine (7)

4 Rl=R,=H 9 Rl=Me R,=H 10 Rl=H R,=Me

4a Rl=R,=H 9a R,=MeR,=H IOa R,=H R,=Me

6

R=H

I1 R=Me

+ 8 R=H 12 R=Me

L

R=HorMe

J

SCHEME 6. A biosynthetic pathway for the formation of benzophenanthridines from protopines in Chelidonium majus and Corydalis incisa plants.

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

285

were present. For example, (S)-stylopine (4), (S )-canadine (3, (S 1tetrahydrocolumbamine (3), (S )-ophiocarpine (14), (S )-corycarpine (15), coptisine (16), berberine (17), protopine (6), allocryptopine (18), sanguinarine (7), and chelerythrine (19) were isolated from the whole plants of C. ophiocarpa (12). Only protopine and sanguinarine were produced in the callus tissue derived from the same plants (12).We have found that our cell cultures have good biosynthetic capabilities for transforming exogenously supplied protoberberine alkaloids, and some information on biosynthetic processes could be obtained even with an unlabeled precursor. For example, in the callus tissues of C. ophiocarpa, nonradioactive (R,S)-canadine a-N-methochloride (5a) was bioconverted into allocryptopine (18) and chelerythrine (19) in 36 and 1% yields, respectively (12). In line with the preceding results, (R)-canadine a-N-metho salt (5a) [[aID+ 168" (CHCI,, c 0.53)], corresponding to about 50% of the amount administered, was recovered.

a. Feeding Experiments with Corydalis incisa. Intact plant studies in C. incisa show a sequence in which tetrahydroprotoberberines undergo N-methylation (B/C-cis configuration), followed by hydroxylation at C14, oxidative cleavage of the C(6)-N bond, and then intramolecular condensation between the positions corresponding to C-6 and C-13 of the protoberberine skeleton to afford benzophenanthridines (see Scheme 6). This sequence was proved by feeding experiments with cultured cells of C . incisa. (i) Feeding of (R,S)-[N-'3CH~-trans-13-methyltetrahydroprotoberberine a-N-metho salts (10a and 20a-23a) (13). The a-N-methosalts 10a and 20a-23a were prepared by treating tetraoxygenated (R,S)-trans-l3methyltetrahydroprotoberberines, which have a trans configuration of protons at C-13 and C-l4a, with I3CH3I.These 13C-labeled a-N-metho salts 10a and 20a-23a, which bear the same relative configuration at C-13 and C-14 as the only naturally occuring trans compound, thalictrifoline (22) (7), were first fed to cultured cells of C . incisa (Table V I ) .Substrates 10a and 20a-23a were bioconverted into the corresponding 13methylprotopines 11and 24-27 (Scheme 7). The 'H- and 13C-NMRspectra for 13-methylprotopinesshowed that the 13C-enrichmentof the N-methyl group of each compound was almost the same as that of the corresponding starting materials 10a and 20a-23a (Table VI). The five different a-N-metho salts were hydroxylated at C- 14 to produce the corresponding 13-methylprotopines.There was a big difference in the conversion yield between one group of a-N-metho salts, 10a, 20a, and 21a, and the other, consisting of 22a and 23a. The former, incorporating a methylenedioxy bridge at C-2 and C-3, is a more favorable precursor

.

TABLE VI ADMINISTRATION OF THE a-N-METHO SALTS OF rrans-~3-METHYLTETRAHYDROPROTOBERBERINE ALKALOIDS TO CELL STATIC CULTURES OF Corydalis incisa Products: Yield % (I3C Enrichment %Y " a ] ~ValueIh Substrates (I3C Enrichment %)" (R,S)-lOa (99) (R,S)-20a (90) (R.S)-Zla (91) ( R . S ) - U a(90) (R,S)-23a(91)

Corycavine (11)or Its Analog 11

24 25

26 27

41 (94) 16(90) 36(88) 1.5 (88) 0.9 (90)

were determined by 'H-NMRspectra Left-callus; right-medium. There is an error ( c = 0.14).

' These values

[-112"] [-124"] [-152"1 [-152"1 [-139"1 [-145"1 [-107"Y

Corynoline Analog (28)

28

0.25 (83)

Recovered Material

57 62 34 55 41

[+124"] [+Wl [+85"] [+47"] [+II"l

[+76"] [+llol [+23"] [+5.9'] [+2.2"1

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

287

of the 13-methylprotopine-type alkaloids than the latter with methoxyl groups at the same positions. Significantly, this is consistent with the fact that no 13-methylprotopine possessing methoxyl groups on ring A has yet been isolated from nature (7).The amount of 13-methylprotopine 24 is smaller than that of 11 or 25 because 24 is further converted into the benzophenanthridine 28. In this instance, the expected benzophenanthridines were not produced from 10a and 21a via 11and 25, respectively; formation of (13R)-corynoline (12)from 10a was proved in further feeding experiments (14). The 13-methylprotopines 11, 24, and 25 were levorotatory and were obtained in states of relatively high configurational purity, as can be seen values with that of (-)-corycavine (11) ([aID by comparing their [a],, - 170")(15).These 13-methylprotopinesmust have the same configuration, since ( - )-corycavine (11) has been determined by X-ray analysis to have the (13S)-configuration (16). All recovered substrates were dextrorotatory. It follows, therefore, that the (14s)-enantiomers of the a-N-metho salts 10a and 20a-23a, rather than the (R)-isomers, were the preferred precursors of the 13-methylprotopine bases 11 and 24-27, respectively (Scheme 7). Similar feeding experiments of the substrates 20a-23a were carried out using cultured cells of C. ochotensis var. raddeana and C. ophiocarpa. The a-N-metho salts 20a and 21a were converted in low yield (0.9 and 0.5%) into the corresponding 13-methylprotopines 24 and 25, respectively. However, no 13-methylprotopine was detected in the feeding experiments

(14S)-Zl.

(13s)-I

SCHEME7. Formation of (13S)-13-methylprotopinesfrom (14S)-rrans-l3-methyltetrahydroprotoberberine a-N-metho salts in cultured cells of Corydalis incisa. (Route a was also demonstrated in intact plants.)

288

KINUKO IWASA

with 22a and 23a. Therefore, the a-N-metho salts 20a and 21a are better precursors of the 13-methylprotopines than are 22a and 23a in cultured cells of C. ochotensis and C. ophiocarpa, as well as in those of C. incisa. (ii). Feeding of (R,S)-[N-'3CH~-cis-13-methyltetrahydroprotoberberine a-N-metho salts @a, 29a, and 3Oa) (13). Next, incorporation experiments of the "C-enriched cis-13-methyltetrahydroprotoberberine a-Nmethosalts 9a, 29a, and 30a, with cis protons at C-13 and C-14, were undertaken for comparison with 10a, 20a, and 23a, which bear a trans relationship for the protons in question (Table VII). Substrates 9a, 29a, and 30a were incorporated into the corresponding 13-methylprotopines 11, 24, and 27 ('3C-enrichmentsof 77, 82, and 92%, respectively), which were found to be dextrorotatory and of high optical purity (Table VII) (Scheme 8). The recovered substrates 9a and 29a were dextrorotatory. The ( 14s)-enantiomersof the a-N-metho salts, rather than the (R)-isomers,are therefore more favorable precursors of the 13-methylprotopines. Compound 30a,having the methoxyl group on ring A, is the preferred precursor for the 13-methylprotopines as well as for 9a and 29a, which have a methylenedioxy bridge on ring A. This is in contrast to the behavior displayed by trans-13-methyltetrahydroprotoberberinea-N-metho salt (Ua), which is methoxylated on ring A and is a poor substrate. Thus, the (14S)-enantiomersof the a-N-metho salts 9a,29a,and 30a were converted and 27 into (13R)-l3-methylprotopines 11, 24 [(R)-corycavidine] (Scheme 8). In conclusion, the a-N-metho salts of the (14S)-trans- and (14S)-cis13-methyltetrahydroprotoberberines are bioconverted into (1 3s)-and (13R)-l3-methylprotopines,respectively. The trans-l3-methyltetrahydroprotoberberine a-N-metho salts methoxylated on ring A are poor substrates, but the cis derivatives are not. These bioconversions were also observed in the cell cultures of C. platycarpa whose intact plants contain several 13-methylprotoberberines. Feeding experiments with unlabeled 20a and 29a indicated that they were converted into 24 in I .9 and 0.5% yields, respectively (18). (iii) Comparison of incorporations of trans- and cis-13-methyltetrahydroprotoberberine a-N-metho salts between tissue-cultured cells and intact plants (13). Four critical studies were carried out to compare the biotransformation of protoberberine alkaloids in whole plants with those in cultured cells. Feeding experiments with I3C-labeled precursors 20a, 21a, 23a, and 29a were undertaken using C. incisa plants (Table VIII). The (14S)-enantiomers of labeled precursors 20a, 21a, and 29a were incorporated into the corresponding 13-methylprotopines (13s)24, (13S)-25, and (13R)-24 (13C-enrichmentof 88, 86, and 80%, respectively), which were partial racemates (Table VIII). The corresponding

(In,

.

TABLE VII ADMINISTRATION OF THE a-N-METHOSALTS OF CiS-13-METHYLTETRAHYDROPROTOBERBERlNE, BENZOPHENANTHRIDINE, AND PROTOPINE ALKALOIDS TO CELLSTATIC CULTURES OF Corydalis incisa Products: Yield % (I3C Enrichment %)" [[ale ValueIb Substrates ("C Enrichments %)"

Benzophenanthridines

13-Methyltetrah ydroprotoberberines

(R,S)-9a (88)

11

7.5 (77)

(R,S)-29n (85)

24

6.9 (82)

[+138"]

[+87"1'

[+ 149"l

l2 31 32 28 33

34 (R.S)-* (96) (R.S)-U

27

27 (92)

[+37"1 [+92"1

58

[+80"1

[+30"]

[+40"]'

57

[+86"1

[+18"1

[+32"]'

[+159"]

(R,S)-6 (13R,14S)-8

a

2.5 (87) 4.6 (81) 0.7 (79) 5.9(83) 1.7 (79) 1.3 (84)

These values were determined using 'H-NMRspectroscopy. Left-callus; right-medium. There is an error (c = 0.15-0. I).

l2 31 32 43 45 44

9.4 6.6 34 25 1.7 47

Recovered Material

[-71"] [+62"Ir [-73"1

[-54"] [+103"1 [-61"1

290

KINUKO IWASA

SCHEME 8. Formation of (13R)- or (13S)-benzophenanthridines from (14S)-cis-13methyltetrahydroprotoberberine a-N-rnetho salts via ( 1 3R)- 13-methylprotopines in cultured cells of Corydalis incisa. (Route a was also demonstrated in intact plants.) --+ minor pathway. TABLE VIII ADMINISTRATION OF THE a-N-METHOSALTS OF trans- AND cis-13METHYLTETRAHYDROPROTOBERBERINE ALKALOIDS TO INTACTPLANTS OF Corydalis incisa Products: mg ("C Enrichment %)" [[@IDValue] Corycavine Analog

Substrates ~~~

(R,S)-U)n

(R,S)-Zla

~~

24 25

Corycavine (11)

~

7 . e (88) [- 167"]c 6.2' (86) [-33"]

( R , S) - U p

~

Corynoline

(U)

~

9

[

+ I 147

12

[+33"1 6

[+91°] (R,S)-29P Control 1 Control 2

24

7.7'(80) [+110"1

10

[+37"] 9 [+99"1 5 [+ 19"l

' These values were determined by 'H-NMR spectra. These values represent percentage yields. There is an error (c = 0.03).

~

Corynoloxine (31) ~~

20

33

[+9.9']

[+loo"] 19

31 [ +4.2") 16 [ +5.2"] 32 [+14"]

15

+

[ 2.67

trace

[+75"] 28 [+ 1167 27

[+99"1 38 [+ 11 I"] 31 [ +957

Recovered Material

~

57b [+8.0"] 71' [+ 1201 71' 736 [+14"]

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

291

13-methylprotopine-typealkaloid was not detected in the feeding experiment of 23a. In plants, as well as in cultured cells, the a-N-metho salts of the trans-13-methyltetrahydroprotoberberines with a methylenedioxy bridge on ring A are preferred as substrates over alkaloids having methoxyl groups on ring A. The values for optical purity vary among the isolated 13-methylprotopines 24 and 25. This indicates that racemization of the 13-methylprotopinealkaloids occurs in plants. The optical purities of the samples of corycavine (11) isolated from feeding experiments and parallel controls also vary greatly. In contrast, the optical purities of other alkaloids, such as corynoline (12) and corynoloxine (31), obtained from C. incisa plants do not vary largely. In conclusion, the a-N-metho salts of the trans- and cis-13-methyltetrahydroprotoberberines are bioconverted into (13s)-and (1 3R)-13methylprotopines, respectively. The a-N-metho salts of the trans-13methyltetrahydroprotoberberines with the methoxyl groups on ring A are poor substrates in comparison with alkaloids having a methylenedioxy bridge on ring A. Consequently, the biochemical processes in the conversion of the protoberberines into the benzophenanthridines occur similarly in intact plants and callus tissues. (iv) Incorporation of 13-methylprotopines into benzophenanthridines (13). The a-N-metho salts of the (14S)-trans-l3-methyltetrahydroprotoberberines, 10a and 20a-23a were transformed into the (13S)-13methylprotopines 11and 24-27, from which the formation of the benzophenanthridines was detected only with 24 (see Table VI). On the other hand, the a-N-metho salts of the (14S)-cis-13-methyltetrahydroprotoberberines 9a, 29a, and 30a were biotransformed into ( I 3R)-13-methylprotopines 11, 24, and 27, respectively. I3C-Labeled corynoline (12) (32% optical purity), corynoloxine (31) (61% optical purity), and acetylcorynoline (32) were obtained after administering 9a (Table VII) (Scheme 8). I3C-Labeledcorynoline analog 28 and I I-epicorynoline analog 33, which are dextrorotatory , and acetylcorynoline analog 34 were found after feeding 29a (Table VII) (Scheme 8). No benzophenanthridine alkaloid was detected after feeding compound 30a (Table VII). The (13R)-enantiomers, as well as the (13S)-enantiomers of the 13-methylprotopines bearing the methoxyl groups on ring A, are not significant as precursors of the benzophenanthridines. Unlabeled (R,S)-corycavine (11) (19) was proven to be converted to corynoline (12),corynoloxine (31), and acetylcorynoline (321, which are all partial racemates with an excess of the (+)-form (see Table IX). In conclusion, the (13R)-enantiomers of the 13-methylprotopines 11 and 24, possessing a methylenedioxy bridge on ring A, are bioconverted into benzophenanthridines preferentially over the (1 3S)-isomers. The 13-

292

KINUKO IWASA

methylprotopines, bearing a methylenedioxy bridge at C-2 and C-3, are more favorable precursors of the benzophenanthridines than those with methoxyl groups at the same positions. (v) Feeding of (R,S)-[8,8-2H&eis- and frans-6-hydroxyte frahydroprotofkrberine N-metho salts (35 and 36) (19). The formation of (13R)corynoline (U), with low configurational purity (32%), from (13R)corycavine (11)with high configurational specificity (8 1%) supports the intermediacy of a hypothetical achiral amino aldehyde intermediate such as A-2 (Scheme 8). The hypothetical intermediate A-2 may be formed by C(6)-N bond fission. Hydroxylation at C-6 could be involved prior to C(6)-N bond cleavage. The C-6 hydroxylated 13-methyltetrahydroprotoberberine N-metho salts (R,S)-[8,8-’H2]-35and -36were synthesized as selected hypothetical biosynthetic intermediates (19,20). The structures of these compounds were determined by X-ray analysis and ‘H-NMR studies, which indicated that 35 and 36 exist as an equilibrium mixture of the trans-B/C and cisB/C fused salts, which interconvert through the amino aldehyde forms 35b and 36b. The major isomers of [8,8-2H,]-35and -36were found to be the trans-fused salt 35c and the cis-fused salt 36a (Scheme 9). Cultured C. incisa cells were incubated with [8,8-’H2]-35and -36,respectively. Four bases were isolated from the fraction to which [8,8-’H,]35 had been fed. Their structures were confirmed to be the deuterated corynoline ([8,8-2H,]-U, corynoloxine ([8-,H]-31), the cis amino alcohol ([1,1-2H2]-37)(21), and [l-,H]-38 (21) (Table IX) (Scheme 9). Two compounds obtained from the fraction to which [8,8-2H2]-36was administered were found to be the trans amino alcohol [ 1,1-2H,]-39(21) and its dehydro derivative [1-,H]-38 (21) (Table IX) (Scheme 9). It is evident, therefore, that the 6-hydroxy-13-methyltetrahydroprotoberberine N-metho salt 35 with the cis configuration of protons at C-13 and C-14, was converted into corynoline (12) (optical purity 78%) and corynoloxine (31) (optical purity 23%), but that 36, possessing the trans configuration was not. This is in marked contrast to the situation with the 13-methyltetrahydroprotoberberine a-N-metho salts (9a and 10a), which have the same configuration at C-13 and C-14 with 35 and 36, respectively. Both of the a-N-metho salts (9a and 10a) are converted via corycavine (11)to corynoline (12)and corynoloxine (31)(see Schemes 7 and 8). The reason for this difference will be discussed later in this review. Formation of [8,8-2H2]-12from [8,8-’H,]-35 provides key evidence supporting the intermediacy of a 6-hydroxytetrahydroprotoberberineduring the conversion of the 13-methyltetrahydroprotoberberines into benzophenanthridines.

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

d-

293

0

0

Lo

[1.1-*H21-39

Lo

[1-2H]-38

t [8.8-2H21-36a

[8.8-2H21-36b

[8.8-2H2]-36c

SCHEME9. Formation of benzophenanthridines (Uand 31)from cis-6-hydroxytetrahydroprotoberberine N-metho salt (35) in cultured cells of Corydalis incisa.

(vi) Comparison of the incorporations of 14- and 6-hydroxytetrahydroprotoberberine N-metho salts (11 and 35) into benzophenanthridines (19). Both 14- and 6-hydroxytetrahydroprotoberberine N-metho salts (11 and 35) are precursors of benzophenanthridines. The hypothetical enamino aldehyde A-2 might be generated either by the elimination of water from 11 or else by a direct dehydrogenation at C-13 and C-14 of 35 followed by an oxidative C(6)-N bond cleavage. If the dehydrogenation mechanism were operating, one would expect that 35 would be a more effective precursor than 11. In order to clarify this point, experiments with 35 and 11 were carried out under identical conditions (Table IX). Corycavine (11)and [8,8-2H2]-35were fed to the cultured cells of C. incisa

TABLE IX ADMINISTRATION OF 6-HYDROXY-I3-METHYLTETRAHYDROPROTOBERBERlNEN-METHOSALTS AND B-SECOPROTOBERBERINE-TYPE ALKALOIDS TO CELLSTATIC CULTURES OF Corydalis incisa Products: Yield or Recovery % Substrates

Corycavine (11)

Corynoline (l2) Corynoloxine (31)

Value]

(R.S )-[8,8-'H2]-35 (R,S)-[8,8-2H2]-36 (R,S)-[8,8-*H2]-35 (R.S)-ll" ( R ,S )-[ 1 , I -'H2]-37

2.6

6.0" [-I47

(R,S)-[1,1,2',2'-zH,]-39 Nondeuterated compound.

[+91"]

0.7 3.5" [+28"1

5.2

38

39

12.8 [-407

2.4 trace trace

24.0

~~

[+34"]

[+90"] 10.3" [+119"]

37

Acetylcorynoline (32) ~

2.6 [-29"]

1.7

4.5" [+41"1 18.0 [-21"]

1.7 1.5

32.0

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

295

(19).Corycavine (11)was incorporated 5 and 6 times better into corynoline (12) and corynoloxine (31), respectively, than was 35. Acetylcorynoline (32), which is formed from 12, was isolated from the fraction to which 11 was fed. It is therefore unlikely that the enamino aldehyde intermediate A-2 is generated by a direct cis-dehydrogenation of 35, since 11 is a more effective precursor than is 35 (Scheme 10). It is probable, however, that the a-N-metho salts of 13-methyltetrahydroprotoberberines undergo hydroxylation at C- 14 prior to hydroxylation at C-6 in the major biosynthetic pathway to corynoline (12).But hydroxylation at C-6 prior to that at C-14 might occur as a minor pathway. (vii) Some considerations regarding the intermediates between the 13-

methyltetrahydroprotoberberine a-N-metho salts and benzophenanthridines (Z3,Z9). Hydroxylation of 11 at C-6 or hydroxylation of 35 at C14 would lead to the intermediate 6,14-dihydroxytetrahydroprotoberberine N-metho salt (Scheme 10). The a-N-metho salts (9a and 10a) of (13R,14s)-tetrahydrocorysamine (9) with the same configuration of protons at C-13 and C-14 as 35) and (13S,14s)-mesotetrahydrocorysamine (10) (with the same configuration of protons at C-13 and C-14 as 36) can be hydroxylated at C-14 to give rise to (13R)- and (13S)-corycavine (ll),respectively, which exist as an equilibrium mixture of several forms interconverting through the tenmembered ring intermediate. The a-N-metho salt of (13R,14S)-9is hydroxylated by the introduction of a hydroxyl group on the a side of C-14, which offers a smaller steric hindrance than the /3 side, to afford (13R,14R)-

r

11

t

35

/ 4

SCHEME 10. The intermediate, 6,14-dihydroxytetrahydroprotoberberineN-metho salt between a 13-methyltetrahydroprotoberberine N-metho salt and benzophenanthridine.

296

KINUKO IWASA

11, one of several forms of (13R)-corycavine. This is then hydroxylated at C-6 by the introduction of a hydroxyl group from the CY side to afford intermediate B-2 (Scheme 11). This hydroxylation at C-6 is consistent with the stereospecificloss of hydrogen from C-6 as described by Battersby et ul. (22). It will be recalled that they administered the two doubly labeled scoulerine isomers, (6R; 14R,S)-[6-'4C,6-3H,]-1and (6s; 14R,S)-1, separately to Chelidonium mujus plants and found that incorporation occurred into stylopine 4 and chelidonine 8. (R,S)-Stylopineis biosynthesized from both the [6R-3H]-and [6S-3H]-isomerswithout affecting C-6. Chelidonines (8) with tritium retentions of 100 and 5% at C-6 were obtained from [6R-3H]-and [6S-3H]-scoulerine,respectively. The results from tritium retention prove that cleavage of the N-C-6 bond of stylopine (4), which finally leads to chelidonine (8), occurs with stereospecific loss of the proS hydrogen from C-6. The 6,14-dihydroxytetrahydroprotoberberineintermediate B-2 derived through hydroxylation at C-14 of (13R,14S)-9a, followed by hydroxylation at C-6, should be the same as that formed through hydroxylation at C-14 of 6-hydroxytetrahydroprotoberberineN-metho salt 35a. An intermediate B-2 could be formed from 35a by the introduction of a hydroxyl group at C-14 from the a side in a manner similar to that for (13R,14S)-9a. The a-N-metho salt of (1 3S, 14S)-10may be hydroxylated in like fashion to ( 1 3 s 14R)-ll, one of several forms of (13S)-corycavinethat can undergo hydroxylation at C-6 to produce intermediate C. This intermediate is not converted to corynoline, because C is also obtained from 36a, which is not a precursor of corynoline (12). It follows that (13s)-corycavine [( 13S,14R)-111has to be converted via the ten-membered ring intermediate to (13S,14S)-11, which is then hydroxylated at C-6 to give the mirror image (intermediate D) of B-2. Compounds 35 and 36 and their 14-hydroxylated derivatives may not assume the ten-membered ring structure. But if they do, both 35 and 36 can be converted to corynoline (12). (13R)-Corycavine [(13R, 14R)-111obtained from (13R,14S)-tetrahydrocorysamine a-N-metho salt (9a) can be bioconverted to corynoline (12) following hydroxylation at C-6. However, (13S)-corycavine [( 13S,14R)-lll derived from (13S,14s)mesotetrahydrocorysamine a-N-metho salt (1Oa) has to be transformed into (13S,14S)-ll, which is hydroxylated at C-6 to undergo bioconversion to corynoline (12). This could explain why the a-N-metho salt of (13R,14s)-tetrahydrocorysamine (9) is a better precursor of benzophenanthridine alkaloids such as corynoline (U), corynoloxine (31), and acetylcorynoline (32) than the a-N-metho salt of (13S, 14s)-mesotetrahydrocorysamine (10).

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

(I4S)-4a R=H (13R.l4S)-9a R=Me

(14R)-6 R=H (13R,14R)-ll R=Me

( 13R)-ll

297

(14S)-6 R=H (13R.l4S)-11 R=Me

t (14S)-4 R=H (13R,14S)-9 R=Me

8-2 R=Me

Enantiomer of C

0 '

.p ( 13s. 14S)-1 1

A-1 R=H

A-2 R=Me

36a

(Enantiomer of B-2)

( 13S,14R)-ll

(13S)-ll

(13S,14S)-lO R=H (13S,14S)-lOa R=Me

SCHEME 11. Methanism for the formation of intermediates A-1 or A-2 from a IChydroxyor 6-hydroxy- 13-methyltetrahydroprotoberberineN-metho salt.

On the one hand, an intermediate, which we can assume to be B-2 or its mirror image (D), undergoes cis-elimination of water, leading to an enamino aldehyde (A-2) that is bioconverted to benzophenanthridine alka-

298

KINUKO IWASA

loids such as (13R, 14R)- and (13S, 14S)-corynoline (l2),etc. (see Scheme 12). On the other hand, the 14-hydroxylated intermediate C (or its mirror image) of 36 is not subject to trans-elimination of water and thus cannot form enamino aldehyde A-2. Enamino aldehyde A-2, formed by cis-elimination of water from B-2 or its mirror image (D), recyclizes to produce benzophenanthridines such as corynoline (12),corynoloxine (31), acetylcorynoline (32), 11-epicorynoline (a), and 14-epicorynoline (41). Corynoline (25% optical purity), corynoloxine (79% optical purity), and acetylcorynoline (55% optical purity), which are all partial racemates with an excess of the (+)-form, were obtained from (R,S)-corycavine (Table IX)(19). The difference in the amount of the (13R, 14R)- and (13S, 14S)-isomers of 12 may be explained as indicated in Scheme 12. Enamino aldehyde A2 may produce (13S, 14S)-corynoline (12)and amino aldehyde A-3 (mirror image of 36b in Scheme 9), in which the methyl group and proton at C14 are in the P-configuration, as well as (13R, 14R)-corynoline (12), in which the methyl group and the proton are in the (Y configuration (Scheme 12). Amino aldehyde A-3 may also be converted to (3R,4R)-corydalic acid methyl ester (42) (B-secoprotoberberine-type alkaloid), which is also a component of C. incisa plants. Alternatively, the enamino aldehyde A-2 may produce the 1 1-epicorynoline analog 33 and 1 1-epicorynoline (40) (Scheme 12). The formation of (13R, 14R)-corynoline (optical purity 69%) and (13R, 14S)-14-epicorynoline (41) (optical purity 100%) from ( R , S ) - [ N -

___)

(13R.IW1

A-2

33 R,=Rz=Me 40 R,+Rz=CHz

SCHEME12. Formation of (13S,14s)- and (13R, 14R)-corynoline(U) and the related alkaloids from an enamino aldehyde intermediate A-2.

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

299

'3CH3]corycavine(11)was established with conversion yields of 7.5 and 5.6%, respectively, in young cultured cells of C. incisa (14). Finally, the

enamino aldehyde A-2 may also produce the trans-B/C fused (13R,14s)14-epicorynoline(41)(Scheme 12). (viii) Feeding of unlabeled (R,S)-corynoline (12) (13). The next objective was to look into the biotransformations of benzophenanthridines such as corynoline (12) and chelidonine (8). For this purpose, unlabeled (R,S)-corynoline (12) hydrochloride was fed to cultured cells of C. incisa (Table VII). It was established that (R,S)-corynoline (12) was converted into (13S, 14S)-acetylcorynoline(32)of 81% optical purity and (13R, 14R)corynoloxine (31) of 6% optical purity, while (13s 14S)-corynoline of 47% optical purity was recovered. (13R. 14R)-Corynoline is more easily oxidized than the (13S, 14s)-form, resulting in the formation of (13R, 14R)-corynoloxine. Alternatively, (13S, 14S)-corynoline is acetylated more readily than the (13R,14R)-enantiomer to afford (13S,14s)acetylcorynoline (32)(Scheme 13). It is also interesting to note that the corynoline analog 28, bearing methoxyl groups at C-7 and C-8 on the D ring, must not be as easily oxidized as corynoline, which incorporates a methylenedioxy bridge, because corynoloxine-type alkaloids were not obtained from 29a (Scheme 8).

A-1

8-1

Me ( I 3R.I W-8

45

1

43 R.OMe 44 R=U

SCHEME13. Biosynthetic conversions from chelidonine (8) and corynoline (U).

300

K I N U K O IWASA

(h) Feeding of unlabeled protopine (6) and (13R,l4S)-chelidonine (8) (13). Chelidonine (8) has been shown to be biosynthesized from the aN-metho salt of (S)-stylopine (4) via protopine (6) (Scheme 4) (8).It was

therefore of interest to establish the relationship between chelidonine on the one hand, and the more highly oxidized sanguinarine (7) on the other. Consequently, the hydrochlorides of unlabeled protopine (6) and ( 13R,14s )-chelidonine (8) were separately administered to cultured cells of C. incisa (Table VII). Protopine (6) was found to be converted into sanguinarine (7) by the isolation of 6-methoxysanguinarine(43). Protopine (6) has been demonstrated to be converted into sanguinarine (7) and chelidonine (8) in Chelidonium rnujus (8). (13R,14S)-Chelidonine(8), for its part, was converted into dihydrosanguinarine (44)and a small amount of dehydrochelidonine (45) (Scheme 13). Acetylchelidonine (46) (Table V) was not detected. This result indicated that (14R)-chelidonine,like (14R)corynoline, is not easily acetylated. Based upon these results, the following pathways must be operating in cultured cells of C. incisu (Scheme 13): protopine (6) + (13R,14S)chelidonine (8)+ sanguinarine (7) + dihydrosanguinarine (44) and (13R,14S)-chelidonine (8) += dehydrochelidonine (45). (x) Biosynthetic routes from tetrahydroprotoberberines to benzophenanthridines. Benzophenanthridine alkaloids are biosynthesized from protoberberine alkaloids via oxidation at C-14 and then again at C-6 to furnish an intermediate 6, 14-dihydroxyprotoberberine. This reactive intermediate undergoes fission at the C(6)-N bond followed by ciselimination of water to generate an enamino aldehyde (A-1 or A-2) (see Scheme 1 1). The enamino aldehyde in turn undergoes intramolecular condensation between the positions corresponding to C-6 and C-13 of the protoberberine skeleton to supply a benzophenanthridine. Some detailed routes to the benzophenanthridines from the protoberberine alkaloids in C. incisa are summarized in Schemes 11-13. The ( - 1-( 13R,14S)-, and ( -)-( 13S,14S)-13-methyltetrahydroprotoberberines [( 13R,14S)-9 and (13S,14S)-10] and ( -)-( 14S)-tetrahydroprotoberberine [( 1 4 0 4 1 are N-methylated to afford the a-N-metho derivatives 9a, 10a, and 4a, respectively (Scheme 11). It is the S hydrogen at C-14 of the aN-metho salts that is replaced by a hydroxyl group. The ( +)-( 13R,14R)and ( - )-( 13S,14R)-13-methylprotopines[( 13R, 14R)-11and (13S,14S)-11] and ( + I-( 14R)-protopine [( 14R)-6] result from the a-N-metho salts, (13R,14S)-9a, (13S,14S)-10a, and (14S)-4a, respectively (Scheme 11). Also, the enantiomeric (+)-(13R,14R)- and (-)-( 13S,14R)-13-methylprotopines are in equilibrium through the ten-membered ring compound with the (+)-( 13R,14s)- and (-)-( 13S,14S)-13-methylprotopines. Since the stereospecific loss of the p r o 3 hydrogen atom at C-6 of the

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

301

protoberberine skeleton has been established (22), the same hydrogen of (+)-(13R, 14R)-13-methylprotopine [(13R, 14R)-ll] and (+)-(14R)protopine [( 14R)-6] could be replaced by a hydroxyl group. This would lead to intermediate 6,14-dihydroxytetrahydroprotoberberinesB- 1 and B2, respectively (Scheme 11). The (-)-(13S, 14R)-13-methylprotopine may be converted via a ten-membered ring into the diastereomeric ( - )(13S, 14S)-13-methylprotopine [( 13S, 14S)-11], whose pro-R hydrogen at C-6 could then be substituted by a hydroxyl group to afford the 6,14dihydroxy intermediate D (Scheme 11). The low conversion yield of (-)(13S, 14R)-13-methylprotopine into benzophenanthridines might be due to its required epimerization to the ( -)-( 13S, 14S)-isomer prior to its transformation into a benzophenanthridine. The ( -)-( 13R, 14S)-6-hydroxyprotoberberine a-N-metho salt [( 13R, 14S)-35] is hydroxylated at C-14 in the same manner as (13R,14S)-9a to supply the 6,14-dihydroxy intermediate B-2, while ( - )-(13S, 14S)-6hydroxyprotoberberine a-N-metho salt [( 1 3 s 14S)-36] can be hydroxylated at C-14 to afford the 6,14-dihydroxy intermediate C (Scheme 11). An enamino aldehyde intermediate (A-1 or A-2) could then be generated by cis-elimination of water from a 6,lCdihydroxy intermediate such as B- 1, B-2, or D (Scheme 11). However, it may not be possible to eliminate water in a trans manner from intermediate C. Aldehyde A-2 evidently yields much more (13R, 14R)-corynoline (12) than the (13S, 14s)-isomer, the 1 1-epicorynoline analog 33, 1 l-epicorynoline (40) and (13R, 14S)-14-epicorynoline (41) (Scheme 12). (13R, 14R)Corynoline is oxidized to give rise to (13R, 14R)-corynoloxine (31) and ( 1 3 s 14S)-corynoline is acetylated to afford ( 1 3 s 14s)-acetylcorynoline (32) (Scheme 13). Similarly, an enamino aldehyde intermediate such as A-1 could be converted to (13R, 14S)-chelidonine (8), as well as to (13R, 14S)-homochelidonine (47) (Table V), in Chelidonium majus. (13R, 14s)-Chelidonine is readily converted into sanguinarine (7) and dehydrochelidonine (45) (Scheme 13). b. Feeding Experiments with Macleaya cordata (Papaveraceae) (23). Besides sanguinarine (7) and chelerythrine (Pi)), the alkaloids sanguirubine (48), chelilutine (49), sanguilutine (50), and chelirubine (51), bearing oxygen functions at C-2, C-3, C-7, C-8, and C-10, have been isolated from several plants (Table IV) (7). Macarpine (52), bearing oxygen functions at C-2, C-3, C-7, C-8, C-10, and C-12, as well as sanguinarine (7) and chelirubine (51), have been found in Macleaya cordata plants and in cultured cells (24). With this background, protoberberine alkaloid biosynthesis, especially the biosynthetic route for the 05-and 0,-type alkaloids was examined using cultured cells of M . cordata.

302

KINUKO I W A S A

(i) Feeding of (R)-and (S)-canadine (5) (23). The biosynthetic conversion of tetrahydroprotoberberines into 0,-type alkaloids, such as sanguinarine (7)and chelerythrine (19),was investigated first. To this end, unlabeled (S )- and (R)-canadine (5) were administered to cultured cells to provide information about the enantiomeric specificity of the conversion of protoberberines into benzophenanthridines (Table X). Administration of (S)-5 resulted in the formation of berberine (17),allocryptopine (18), and chelerythrine (19)(see Scheme 14). A similar experiment with (R)-5 yielded only dehydrogenated products such as berberine (17)and 7,8dihydroberberine (53) (Table X). Thus, only the (S)-enantiomer of 5 was the precursor of species 18 and 19 (Scheme 14). This parallels the result obtained with C. incisa. Both the (R)- and (S)-enantiomers of 5 were oxidized to produce berberine (17),while dihydroberberine (53) probably falls in the pathway between tetrahydroberberine and berberine. (ii) Feeding of a- and P-N-metho salts of tetrahydroprotoberberines (4a, 4b, 5a, and 5b) (23). Feeding experiments using the a-and p-N-metho salts of tetrahydroberberines were undertaken to provide insight into the distinction between these stereoisomers. Incorporation experiments with the a-and P-N-methochlorides of (R,S)-canadine (5) showed good incorporation of the a-N-metho salt (5a) into both allocryptopine (18)and chelerythrine (19).But no similar incorporation could be detected in feeding experiments with the 6-N-metho salt of 5 (Table X) (Scheme 14). Incorporation experiments with the a-and P-N-metho salts of ( R , S ) - [ N '3CH3]stylopine(4a and 4b) confirmed that the a-N-metho salt (4a) is a good precursor of protopine (6) (enrichment 58%) and sanguinarine (7) (enrichment 42%), while the p-N-metho salt (4b) is ineffective (Table X) (Scheme 14). The substrates recovered from feeding experiments of the a-N-metho derivatives of 4 and 5 contained substantial amounts of the (R)-enantiomer(optical purity 25 and 26%, respectively) (Table X). Therefore, it was concluded that the (S)-enantiomer of the a-N-metho salts of tetrahydroprotoberberines can be stereospecifically metabolized into protopine- and benzophenanthridine-typealkaloids. This result is in agreement with the conclusions reached with regard to C. incisa. It has also been reported that [N-'4CH3]stylopinea-N-metho salt (4s) shows good incorporation into protopine (6) (0.6%) and sanguinarine (7)(7.7%) in Fumaria capreolata callus cultures (25). (iii) Feeding of protopines 6 and 18 (23). The bioconversion of protopine alkaloids into benzophenanthridine alkaloids was determined in C. i n c h (Fumariaceae). T o ensure this fact in M. cordata (Papaveraceae), feeding experiments with allocryptopine (18) hydrochloride and [N-'3CH3]proytopine(6) hydrochloride to cultured cells of M. cordata were undertaken (Table X). These experiments showed that 18 was

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

303

TABLE X ADMINISTRATION OF TETRAHYDROPROTOBERBERINE ALKALOIDS, THEIRN-METHO SALTS,PROTOPINE ALKALOIDS, A N D BENZOPHENANTHRIDINE ALKALOIDS TO Macleaya cordata Substrate 2H Enrichment %) ( I3C or

Product: Yield % (I3C or 2H Enrichment %)

Recovered Material Yield %

Optical Purity %

42

100 [(R)-enantiomer]

25

100 [(S)-enantiomerl

17

26 [(R)-enantiomer]

Suspension cultures

(R)-Canadine (5) (S)-Canadine (5)

(R,S)-5 a-N-Metho salts (5a) (R,S )-5 P-N-Metho salts (5b) (R,S)-[N-”CH3]-4 a-N-Metho salt (4a)

(90) (R.S )-[N-”CH3]-4 P-N-Metho salts (4b) [N-”CH3]Protopine (6) (90) Allocryptopine (18) [N-C2H3]Sanguinarine (7) (99)

Berberine (17) 1 1.4 7.8-Dih ydroberberine (53) 3.7 Berberine (17) 12.1 Allocryptopine (18) 1.7 Chelerythrine (19) 0.4 Allocryptopine (18) 6.2 Chelerythrine (19) I .4

-

Protopine (6) 9.6 (58) Sanguinarine (7) 2.0 (42)

29 41

25 [(R)-enantiomer]

54

Sanguinarine (7) 24.6 (40) Chelerythrine (19) 33.4 Chelirubine (51) 4.8 (44) Macarpine (52) 12.0

48 30 18

(15)

[N-C2H3]Chelirubine (51) (49) [N-CZH3]Dihydro sanguinarine (44) (99) Plants

Macarpine (52) 1.5

[N-C2H3]Allocryptopine (18) (90)

Chelerythrine (19) 180”

(45)

-

14 80

(10)

Yield (mg).

biotransformed to chelerythrine (19) in 33% conversion yield, and that 6 was converted to sanguinarine (7) (enrichment 40%) in 25% yield (Table X).

304

KINUKO I W A S A

Feeding of [N-'3CH3]allocryptopine(18) hydrochloride to intact plants of M. cordata confirmed the conversion of 18 to chelerythrine (191, and protopine (6) into sanguinarine (7)in intact plants of C. incisa and Chelidonium majus. (iv) Feeding of the fully aromatized benzophenanthridines (7 and 51) (23). The metabolism of the fully aromatized benzophenanthridines of the 0,-type, 0,-type, and 0,-type will be described. A proposal for the biogenetic conversion of sanguinarine (7)into chelirubine (51) had already been made (26,27).It involved the oxidative fission of the C(6)-N bond of the protoberberines followed by recyclization to the benzophenanthridine skeleton. Another biogenetic pathway was assumed based on the fact that sanguinarine (7)was found at the initial stage of the callus culture period and that macarpine (52), instead of sanguinarine (7),was produced at the final stage. Direct evidence for the (0,+ 0, + 0,)-type alkaloidal sequence came (7) chloride from feeding experiments (23). Whereas [N-C2H3]sanguinarine was converted into chelirubine (51) (enrichment 44%) in the callus tissue, (44) hyan analogous experiment with [N-C2H31-5,6-dihydrosanguinarine drochloride did not yield sanguinarine (7) and chelirubine (51) (Table X). Therefore, sanguinarine (7)is methoxylated at C-10 to afford 51 in good yield, and dihydrosanguinarine (44) is not effective as a precursor of 51 (Scheme 14). When [N-C2H3]chelirubine(51) chloride was administered into the callus tissues, it was metabolized into macarpine (52) (enrichment 45%) (Table X ) (Scheme 14). These experiments confirm the sequence sanguinarine (7)+ chelirubine (51) + macarpine (52) (Scheme 14). Based upon these results, it is concluded that the introduction of an oxygen function at C-10 and then at C-12 to form the 0,- and 0,-type alkaloids occurs post facto to the formation of the fully aromatized benzophenanthridine skeleton. (v) Biosynthetic routes from tetrahydroprotoberberines to benzophenanthridines in Macleaya cordata. The incorporation experiments with callus tissues of M. cordata have defined the biosynthetic pathway (S)-tetrahydroprotoberberines (e.g., 5 ) + their a-N-metho salts + protopines (e.g., 6 or 18) + fully aromatized benzophenanthridines [e.g., sanguinarine (7) and chelerythrine (19)] + C,-0-type benbenzophenanthridines zophenanthridines (e.g., 51) + C,-0-type (e.g., 52) (Scheme 14). The conversion shown in Scheme 14 may also take place in the intact plants of M. cordata, as indicated by the incorporation of allocryptopine (18) into chelerythrine (19) in the live plant.

5.

BIOTRANSFORMATION

(14S-4a R,+R,=CH,

(14.9-5

17

305

OF PROTOBERBERINE ALKALOIDS

(IdS)-Sn RI=Rz=Me

?Me

Me

0 \-0

Me \-0

52

51

7 Rl+R,=CH, 19 R,=R,=Me

44

SCHEME 14. A biosynthetic sequence for the formation of the fully aromatized benzophenanthridines from protoberberines in cultured cells of Mucleuya cordura.

B. INTERCONVERSIONS BETWEEN TERTIARY A N D QUATERNARY OF LCIAPCI-MS PROTOBERBERINES: APPLICATION TO BIOSYNTHETIC STUDIES Callus Tissue Studies (28,291

It has been demonstrated that tetrahydroprotoberberines, 13-methyltetrahydroprotoberberines, and their a-N-metho salts with the cis-B/C fused system, but not the corresponding trans-fused j3-N-metho salts, are biotransformed via the corresponding protopines into benzophenanthridines in Corydalis species (route a in Scheme 15) (8,13,19).It was somewhat surprising, however, that the a-N-metho salts had not been isolated from Corydalis plants in general, except from C. caua (7). This may be attributed to the fact that a-N-metho salts are bioconverted rapidly into protopines. Protopines, but not the a-N-metho salts, have been isolated as metabolites in incorporation experiments of tetrahydroprotoberberines using C. i n c h and M . cordata. (8,23). The formation of the a-N-metho salts of tetrahydroprotoberberines from the corresponding parent bases has been elegantly accomplished by Zenk and co-workers using (S)-tetrahydroprotoberberine-cis-Nmethyltransferase partially purified from an Eschscholtzia californica (Papaveraceae) cell suspension culture (route b in Scheme 15) (30). This

306

KINUKO IWASA

Protoberbenniumsans

Telrahydroproloberbennes

Telrahydmprolobertmne

Protopines

eoR2 fl

13-Melhylproloberbenniumsans 13-Meihyneirahydroprol~~nnes

a

Oh-

\

NMe

' \

NMe

R.0 OR3

Benzophenanlhndnes

SCHEME 15. Known and suggested routes for formation of the quaternary protoberberine alkaloids in protoberberine biosynthesis. + known pathway; unknown pathway.

is only one example of the a-N-metho salt isolated as a metabolite. The biosynthetic conversion of the 13-methyltetrahydroprotoberberines into the a-N-metho salts was not demonstrated (suggested route c in Scheme 15). The redox interconversions between tetrahydroprotoberberines or 13methyltetrahydroprotoberberines on the one hand, and protoberberinium salts or 13-methylprotoberberinium salts on the other, have not yet been fully investigated (suggested routes d-f in Scheme 15). The bioconversion of tetrahydroprotoberberines into protoberberinium salts has also been demonstrated by Zenk and co-workers using (S)-tetrahydroprotoberberine oxidase that had been purified from Berberis wifsoniue (Berberidaceae) cell cultures (route g in Scheme 15) (31). However, demonstration of the reverse process (suggested route d in Scheme 15)-namely reductionremained to be accomplished. Evidence for the C-methylation at C-13 was provided by Holland et af. (32) in a study of the biosynthesis of corydaline (30)in C. sofidu.[9-0'4CH,]Palmatine(54), but not [9-014CH3]tetrahydropalmatine (59, was incorporated into corydaline (30)(route h in Scheme 15). These results can be rationalized as follows. One carbon unit is added to a didehydroprotoberberine intermediate (56) (see Table 11), which is reduced to generate to corydaline (30). The failure to obtain incorporation of tetrahydropalmatine (55) is due to the inability of C. solidu to oxidize 55 to palmatine (54). Methylation at the C-13 site of protoberberinium salts might occur to furnish 13-methylprotoberberinium salts (suggested route i in Scheme 15). We were thus persuaded that

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

307

studies on the metabolism of these quaternary protoberberine alkaloids were essential for a comprehensive understanding of the metabolic map of the protoberberine alkaloids. LC/APCI-MS (liquid chromatography/atmospheric-pressurechemicalionization mass spectrometry) techniques were therefore applied to the metabolic studies of the quaternary alkaloids, which are highly polar and difficult to isolate. a . LCIAPCI-MS of Protoberberines and Related Alkaloids (29). Protoberberinium salts, tetrahydroprotoberberines, their a- and P-N-metho salts, protopines, and benzophenanthridines were expected as metabolites from the biogenetic pathways as currently understood. Prior to feeding experiments, authentic samples of the expected metabolites were divided into five groups (I-V) and LC/APCI-MS was undertaken by SIM (selected ion monitoring) and TIM (total ion monitoring). Group I consisted of canadine (3, its a- and P-N-metho salts (5a and 5b), berberine (17),allocryptopine (U), chelerythrine (19),and 13methylberberine (57)(see Tables I-IV), which are the expected alkaloids to be obtained from 5 or 17 incorporating a methylenedioxy group on ring A. Indeed, in our hands, LC/APCI-MS of group-I alkaloids showed a MC (mass chromatogram) having the seven peaks shown in Fig. 1 (Table XI). Each alkaloid was identified by a quasi-molecular ion [M + H]+ or a cluster ion [M + CF3]+,or else a molecular ion [MI+,and in each case, by a characteristic retention time (Table XI). Group I1 comprised the expected products, palmatine (54), tetrahydropalmatine ( 5 9 , its a- and P-N-metho salts (55aand 55b), 13-methylpalmatine (58), and muramine (59),which can be derived from 54 or 55, which possess two methoxyl groups on ring A. The LC/APCI-MS showed a MC with six peaks (Table XI) which were duly assigned to specific alkaloids. Group 111 consisted of the alkaloids [N-'3CH3]corycavidine (U), thalictricavine (29),its a- and P-N-metho salts (29a and 29b), and 13methylberberine (57),which include a methylenedioxy substituent on ring A and a methyl group at C-13, and which may be derived from either 29 (cis configuration of the protons at C- 13and C- 14)or 5% This MC displayed only four peaks because the peak due to 24 (mlz 385) at 19.5 min and that due to 57 (mlz 420) at 20.0 min overlapped (Table XI). Group IV included the expected metabolites, berberine (17),mesothalicand tricavine (20),its a-N-metho salt (20a),[N-'3CH,]corycavidine (U), 13-methylberberine(57),which include a methylenedioxy on ring A and a methyl group at C-13 (except for 17),and could be formed from 20 (trans configuration of the protons at C-13 and C-14) or 57. The MC was observed

308

KINUKO I W A S A

17 MC

10

20

30

40

LC

10 20 (min) 3 0 40 FIG.I . Mass chromatogram (TIC method: nebulizer temperature 340")and liquid chromatogram of group I.

as only three peaks because the peaks due to 20a and 17, and also those due to 24 and 57, were not resolved (Table XI). Group V involved the alkaloids mesocorydaline (231,[N-13CH3]mesocorydaline a-N-metho salt (23a), [N-'3CH3]-13-methylmuramine (27), corydaline (30),its a-and P-N-metho salts (30a and 30b),and 13-methylpalmatine (58), Each of these alkaloids includes two methoxyl groups on ring A and a methyl group at C-13, and could have been formed from 23, 30, or 58. All seven peaks in the MC were assigned to the appropriate alkaloids by observed ion ( m l z ) and retention time (Table XI). It should be noted that under the LC/APCI-MS conditions (Table XI) the tertiary alkaloids (such as tetrahydroberberines and protopines) showed quasi-molecular ions [M + HI+, the quaternary alkaloids (such as the protoberberinium salts) showed the cluster ions [M + CF3]+, and the quaternary alkaloids (such as the benzophenanthridines and the a- and P-N-metho salts of tetrahydroprotoberberines) showed molecular ions

[MI+ . b. Feeding Experiments with Corydalis pallida var. tenuis (28,29). Following confirmation that the expected metabolites were not

TABLE XI RETENTIONTIMESA N D OBSERVED IONS I N LC/APCI-MS" OF PROTOBERBERINES A N D RELATED ALKALOIDS (GROUPSI-V) Observed Ions (rnlz) Retention Time (min) Group I Canadine (5) Chelerythrine (19) 13-Methylberberine (57) Berberine (17) 5-a-N-Metho salt (5a) Allocryptopine (18) 5-P-N-Metho salt (5b)

35.0 33.5 19.9 18.7 15.0 12.8 11.3

Group I1 Tetrahydropalmatine (55) 13-Methylpalmatine(58) Palmatine (54) 55-a-N-Metho salt (55a) Muramine (59) 55-P-N-Metho salt (55b)

28.0 19.6 18.9 13.0 11.0 9.5

Group 111 Thalictricavine (29) 13-Methylberberine (57) [N-'3CH,]Corycavidine (24) 29-a-N-Metho salt (29a) 29-P-N-Metho salt (29b)

40.0 20.0b 19Sh 18.1 16.6

Group IV Mesothalictricavine (20) 13-Methylberberine (57) [N-"CHJCorycavidine (24) Berberine (17) 20-a-N-Metho salt (20a)

32.7 20.e 19.7h 18.7h 18.7h

Group V Corydaline (30) Mesocorydaline (23) 13-Methylpalmatine (58)

[N-'3CH3]-13-Methylmuramine (27) [N-"CH3]-23-a-N-Metho salt (23a) 30-a-N-Metho salt (Ma) 30-a-N-Metho salt (30b)

33.7 26.3 19.7 18.9 18. I 17.1 15.3

[MIt

[M

+ HI+

[M

+ CF,]'

340 348 420 406 354 370 354 356 436 422 370 386 370 354 420 385 368 368 354 420 385 406 368 370 370 436 40 I 385 384 384

I

LC conditions: Hitachi L-6200 Intelligent pump; Hitachi L-4OOO UV detector (280 nm): Cosmosil SC,*-AR (4.6 i.d. x 150 mm). Mobile phase: 1 M NH,OAc (0.05% TFAkMeOH (B). Linear gradient: 0-10 min 30% of B, 15 min 50% of B, 20-25 min 60% of B, 30 min 70% of B, 35 min 80% of B, 40 min 30% of B; flow rate: I ml/min. AF'CI-MS conditions: Hitachi MIOOOH: Nebulizer and vaporizer temperature -300-340 and 399°C; drift voltage: 20 V. These peaks overlapped in LC.

310

KINUKO I W A S A

present in cultured cells of C. pallida var. tenuis,feeding experiments with unlabeled protoberberines (Table XII) were carried out. The metabolites identified by LC/APCI-MS (SIM method) of the alkaloid fraction obtained from experiments 1-10 are summarized in Table XII. (i) Feeding of tenvlhydroberberine (5) and berberine (17) (28,29}. Tetrahydroberberine (S), tetrahydroberberine a-N-metho salt @a), allocryptopine (M), thalictricavine (29), and 13-methylberberine(57) were identified in experiment 1 in which berberine (17) was fed. Deuterated allocryptopine (18-'H3)and chelerythrine (19) as well as 5a, 17, and 18 were recognized in experiment 2 in which tetrahydroberberine (5) was administered. The metabolic conversions 29, 57 + 17 * 5 + Sa, 18, 19 were thus demonstrated by the results obtained from experiments 1 and 2 (see Scheme 16). Berberine (17) is reduced to dihydroberberine (53), which is methylated at C-13 to give 13-methyldihydroberberine.This is oxidized or reduced to afford 57 or 29, respectively. Reduction of 57 also affords 29. Berberine (17) is reduced via dihydroberberine to 5, which is N-methylated to give rise to the a-N-metho salt 5a. Conversion of 5a via 18 into 19 (pathways j and k) has been confirmed in C . ophiocarpa and M . cordata (12,231. (ii) Feeding of palmatine (54) and tetmhyahpalmatine (55) (28,29). Corydaline (M), tetrahydropalmatine ( 5 3 , and 13-methylpalmatine (58) were identified in experiment 3 in which palmatine (54) was fed. Tetrahydropalmatine a-N-metho salt (55a), deuterated muramine (59-*H3),and 59, as TABLE XI1 ADMINISTRATION OF TETRAHYDROPROTOBERBERINE ALKALOIDS AND PROTOBERBERINIUM SALTSTO CELLCULTURES OF Corydalis pallida VAR. tenuis Experiment

Substrate

I" 20.'

Berberine (17) Tetrahydroberberine (5)

3 4a.c

Palmatine (54) Tetrahydropalmatine (55)

5" 6" 7b 8" 96.r

13-Methylberberine (57) Thalictricavine (29) Mesothalictncavine (20) 13-Methylpalmatine (58) Corydaline (30) Mesocorydaline (23)

I Oh.'

" In cell static cultures.

* In suspension cultures. Plus L-[Me*H,lmethionine.

Metabolites (Observed Ions: m / z )

5 (340), 5a (354). 18 (370). 29 (354). 57 (420) Sa (354). 17 (406). 18 (370). 18-*Hl (373), 19 (348) 30 (370). 55 (356). 58 (436) 54 (422), 55a (370). 58 (436). 59 (386), 59*HI(389) 29 (354). 29a (368) 20 (354). 57 (420) 29 (354), 57 (420) 30 (370) 23 (370). 58 (436) 30 (370), 58 (436)

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

3I 1

-

well as 54 and 58, were detected in experiment 4 in which tetrahydropalmatine (55) was administered. The metabolic transformations 30, 58 t 54 t* 55 55a, 59 are thus demonstrated from the results derived from experiments 3 and 4 (see Scheme 16). Palmatine (54) was bioconverted to 30 and 58 and also reduced to 55, which was biotransformed to the a-N-metho salt 55a in a manner similar to 17. Conversion of 55a into 59 parallels that of 5a into 18. (iii) Feeding of mesothalictricavine (a), thalictricavine (29), and 13mthylbehine (57) (29). Thalictricavine (29) and thalictricavine a-N-metho salt (29a) were detected in experiment 5 in which 13-methylberberine(57) was fed. 13-Methylberberine(57)and mesothalictricavine (20)01-29were identified in experiments 6 and 7 to which 29 and 20 were fed, respectively. The metabolic conversions 20 57 29, 29a are thus validated from experiments 5-7 (Scheme 16). Both trans- and cis-I 3-methyltetrahydroprotoberberines, mesothalictricavine (20) and thalictricavine (29), were oxidized to afford 13-methylberberinium salt 57. 13-Methylberberinium salt 57 was reduced to 29, which undergoes N-methylation to produce the a-N-metho salt 29a. (iv) Feeding of mesocorydaline (23), corydaline (30), and 13methylpalmatine (58) (29). Corydaline (30)was identified in experiment 8 in which 13-methylpalmatine(58) was administered. 13-Methylpalmatine(58) and mesocorydaline (23) or 30 were detected in experiments 9 and 10 in which 30 and 23 were fed, respectively. The metabolic transformations 23 58 f* 30 are supported by the results obtained from experiments 8-10 (Scheme 16). Mesocorydaline (23) and corydaline (30) undergo oxidation to 13-methylpalmatine(58), which can be reduced to generate 30. The new metabolic conversions indicated by the thick arrows in Scheme 16-viz. a (17 57, 54 + 58), c (20+ 57, 23 + 58), d (57+ 29, 58- 30), e (29- 57, 3 0 4 58), g (17- 5, 54+ 55), and i (55 55a)-were all demonstrated by the results of experiments 1-10. The known metabolic conversions h (5 17, 55 -+ 54) and i (5 + 5a) were also confirmed in cultured cells of C. pallida var. tenuis.

--

-

-

c. Feeding Experiments with Corydalis incisa (29). ' Feeding experiments using unlabeled protoberberines (Table XIII, experiments 1 1-20) were carried out using cultured cells of C. incisa. The metabolites were identified by LUAPCI-MS (SIM method). (i) Feeding of tetrahydroberberines (5 and 55) and protoberberinium salts (17and 54) (29). The metabolic transformations 17 + 5 5a, 18 were indicated by experiments I 1 and 12 (Scheme 17).The conversion of 5a into 18 (pathway j) had previously been demonstrated (12,23). The metabolic

-

312

KINUKO I W A S A

I'

Jk

SCHEME16. Metabolic transformations of protoberberinium salts and tetrahydroprotoberberines in cultured cells of Corydalis pullidu var. tenuis. -+indirect evidence.

TABLE XI11 ADMINISTRATION OF TETRAHYDROPROTOBERBERINES A N D PROTOBERBERINIUM SALTS (17, 54, 57, A N D 58) TO CELL CULTURES OF Corydalis incisa Experiment

Substrate

110

16' 17b

Berberine (17) Tetrahydroberberine (5) Palmatine (54) Tetrahydropalmatine (55) 13-Methylberberine (57) Thalictricavine (29) Mesothalictricavine (20)

18 (370) 5a (354), 17 (406). 18 (370), 18-*H1(373) 58 (436) 54 (422), 59 (386). 59-*H] (389) 20 (354). 24 (384), 29 (354) 24 (384). 24-*H1(387). 29a (368). 57 (420) % (368). 24 (384). 24-*H3(387). 29 (354), 57 (420)

18" 19',' 206,'

13-Methylpalmatine(58) Corydaline (30) Mesocorydaline (23)

30 (370) 23 (370), 58 (436) 30 (370). 58 (436)

I2O.C

13 14",' 15"

" In cell static cultures.

'

In suspension cultures. ' Plus ~-[Me~H,]methionine.

Metabolites (Observed Ions: mlz)

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

3 13

20aR=-- Me 2 9 a R = 9 Me

SCHEME 17. Metabolic transformations of the protoberberinium salts and tetrahydroprotoberberines in cultured cells of Corydalis incisa. --* indirect evidence.

conversions 58 t 54 + 55,59 were substantiated by expts 13 and 14 (see Scheme 17). (ii) Feeding of trans- and cis-Z3-methyltetrahydroprotoberberines (20, 23, and 29, 30) and 13-methy~mtoberberiniumsalts (57 and 58) (29). Mesothalictricavine (20), 13-methylallocryptopine(24), and thalictricavine (29) were detected in experiment 15 (Table XIII). Deuterated 13methylallocryptopine (24-2H,), 24, thalictricavine a-N-metho salt (29a), and 57 were identified in experiment 16 (Table XIII). Mesothalictricavine a-N-metho salt (20a) was found, as well as 24, 24-*H3, 29, and 57 in experiment 17 (Table XIII). The metabolic transformations 20a, 24 + 20 f* 57 29 + 24, 29a are thus demonstrated by the results obtained from experiments 15-17 (Scheme 17). 13-Methylberberine(57) undergoes reduction to afford mesothalictricavine (20) and thalictricavine (29), These bases can be N-methylated to give rise to their a-N-metho salts (20a and 29a), respectively, or alternatively can be oxidized to regenerate 57. The transformation of 20a or 29a into 13methylallocryptopine (24) (pathway m) has previously been demonstrated

-

314

KINUKO IWASA

(13).The metabolic conversions 23 3 58 c, 30 are thus substantiated by experiments 18-20 (Scheme 17). New metabolic transformations indicated by the thick arrows in Scheme 17-viz. a (54 + 58), b (57+ 20), c (20+ 57, 23 + 58), d (57+ 29, 58 + 30),e (29+ 57,30 + 58), f (29+ 29a),and l(20 + 20a)have been demonstrated by the results of experiments 11-20. The known metabolic conversions h (5+ 17, 55 + 54) and i (5+ 5a) were also confirmed in cultured cells of C. incisa.

d. Summary. In summary, the incorporation experiments with C. pallida var. tenuis and C. incisa indicate that certain differences exist in metabolism between these cultured cell systems. In the former, the bioconversion (route k in Scheme 16) of protopine bases (e.g., 18) devoid of a C-13-methyl group into benzophenanthridines (e.g., 19)does take place, but that (route m in Scheme 17) of 13-methyltetrahydroprotoberberines (e.g., 29a)to 13-methylprotopines(e.g., 24) was not detected. In the latter system the biotransforrnations of 29a into 24 and of the 13-methylated protopines such as corycavine (11)into benzophenanthridines such as corynoline (12) do indeed occur, but the conversion of 18 to 19 is not observed. These findings show that the metabolism of protoberberines lacking a methyl group at C-13, and those possessing a methyl group at that site, proceeds preferentially in C. pallida var. tenuis and C. incisa, respectively. In other words, corycavine (11)and corynoline (12) with a C-methyl group have been isolated from C. incisa, but not from C. pallida var. tenuis (7). The metabolism of the quaternary protoberberine alkaloids in cultured cells of Corydalis species has been clarified using LC/APCI-MS. The pathways indicated in Scheme 18 were shown to apply. Interconversions of tetrahydroprotoberberines (5 and 55) or cis- and trans-13-methyltetrahydroprotoberberines (29,30and 20,23)and of their dehydro derivatives, the protoberberinium salts 17 and 54 or 13-methylprotoberberinium salts 57 and 58, did take place via redox reactions. a-N-Metho salts 5a, 55a, 29a,and 20a incorporating the B/C-cis quinolizidine system were produced from tetrahydroprotoberberines or cis- or trans-13-methyltetrahydroprotoberberines (5, 55, 29, and 20). Finally, C-methylation at C-13 of protoberberinium salts 17 and 54 was confirmed, leading to 13-methylprotoberberiniurn salts 57 and 58. C. FORMATION OF BENZOPHENANTHRIDINES FROM PROTOBERBERINES USINGCorydalis, Chelidonium, AND Macleaya PLANTSPECIES AND PLANTTISSUECULTURES Interconversion between protoberberinium salts (coptisine (16),corysamine) and their tetrahydro derivatives (stylopine (4), tetrahydrocory-

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

5 or 55

5a or 55a

29 or 30

29a

3 15

57 or 50 20 or 23 20a SCHEME18. Metabolic transformationsof quaternary protoberberine alkaloids in cultured cells of Corydalis species.

samine (9), mesotetrahydrocorysamine (10)) via redox reactions does occur (Scheme 19). The (14s)-tetrahydroprotoberberineswere N-mefhylated to produce the corresponding a-N-metho salts incorporating the B/Ccis quinolizidine system. Hydroxylation of the a-N-metho salts at C-14 generated protopines [protopine and (13R)- and (13S)-corycavinel. The protopines were hydroxylated at C-6 to form an intermediate 6,14dihydroxytetrahydroprotoberberineN-metho salt that interconverts with the amino aldehyde form. This reactive intermediate undergoes ciselimination of water to provide an enamino aldehyde. The enamino aldehyde forms B-secoprotoberberines [(3R,4R)-corydalic acid methyl ester, corydaminel, which in turn undergoes intramolecular condensation between the positions corresponding to C-6 and C-13 of the protoberberine skeleton, resulting in the formation of the benzophenanthridines [( 13S,14s)- and (13R, 14R)-corynoline, (13R, 14S)-14-epicorynoline, 11epicorynoline, and (13R, 14s)-chelidonine]. Chelidonine is converted into the fully aromatized benzophenanthridine sanguinarine, which can be reduced to dihydrosanguinarine. The introduction of an oxygen function at C-I0 and then at C-12 to form 05-and 0,-type alkaloids, such as chelirubine and macarpine, occurs following the formation of the fully aromatized benzophenanthridine, sanguinarine. Protoberberinium salts (coptisine) are reduced to 7,8-dihydro derivatives which are methylated at C-13 to form 13-methyldihydroberberines. These are oxidized to afford 13-methylprotoberberinium salts (corysamine).

SCHEME 19. Biosynthetic pathways for the formation of benzophenanthridines from protoberberines in Corydalis. Chelidonium, and Macleaya plant species and plant tissue cultures.

5 . BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

3 17

D. BIOSYNTHETIC STUDIES OF PROTOBERBERINES, PROTOPINES, AND BENZOPHENANTHRIDINES AT THE ENZYMIC LEVEL 1 . Dehydrogenation of Tetrahydroprotoberberines to Protoberberinium Salts (31,33-35)

An oxidase has been isolated and purified from Berberis wilsoniae cell cultures (31,33).This enzyme oxidizes exclusively (S)-tetrahydroprotoberberine alkaloids to the corresponding protoberberinium salts. A considerable number of tetrahydroprotoberberines with widely differing substitution patterns in rings A and D are able to serve as substrates. However, the 13,14-dehydro substrates, the N-metho salts, and compounds with a methyl group at C-13 were not dehydrogenated by the Berberis oxidase (Table XIV). In Berberis species, tetrahydrocolumbamine (3) is first oxiwhich then elabodized to produce the quaternary base columbamine (a), rates a methylenedioxy bridge to afford berberine (17) (33) (Scheme 20). This specific enzyme was found to be active toward not only tetrahydroberberines, but also benzylisoquinoline alkaloids. The following oxidation mechanism was therefore proposed. The enzyme proceeds initially with the dehydrogenation of ring C through the formation of a N-7-C-14 iminium derivative. This intermediate is further oxidized to the protoberberine system (Scheme 20). Galneder et al. reported the isolation and purification of an oxidase from Coptisjaponica cell cultures (34).This enzyme catalyzed exclusively the oxidation of (S)-canadine (5) and not of its enantiomer. Tetrahydrocolumbamine (3) and stylopine (4) were dehydrogenated as well, while scoulerine (1) and norreticuline (benzylisoquinolines) were not (Table XV). In Coptis species, the methylenedioxy bridge is first formed to generate (S)-canadine (5) from (S )-tetrahydrocolumbamine (3), and then (S)-canadine (5) is subsequently oxidized to berberine (17) (Scheme 20). Coptis oxidase differs from the Berberis oxidase in its cofactor requirement (Fe) and in its substrate specificity. Neither (S )-norretidine nor (S )-scoulerine serves as a substrate for the Coptis enzyme, although both substrates are readily oxidized by the Berberis enzyme. Some differences were observed in the substrate specificity of a crude enzyme sample (34,35).The presence of various protoberberines in this crude protein extract may have influenced the oxidase activity.

2 . N-Methylation of Tetrahydroprotoberberines to a-N-Metho Salts (25,30) An enzyme has been partially purified and characterized from cell suspension cultures of Eschscholtzia californica and C . vaginans. This enzyme specifically catalyzed the cis-N-methylation of certain (S)-

TABLE XIV SPECIFICITY OF (S)-TETRAHYDROPROTOBEREIERINE OXIDASE USINGDIFFERENTLY SUBSTITUTED TETRAHYDROPROTOBERBERINES AS SUBSTRATES" W

Substrate (S )-Scoulerine

(R,S)-Canadine (S )-Coreximine ( R , S)-Tetrahydropalmatine ( R , S)-Corypalmine

Relative Turnover Rate (%)

Substitution Pattern Knl

C-l

100

-

97 88 23 21

-

-

C-2

C-3

N-7

C-9

C-I0

C-ll

C-13

pmol/liter

OMe OCHZO OH OMe OMe OMe OMe OH

-

OH OMe

-

-

-

OMe OMe

OMe OMe OMe OMe OMe

25 13.3 4.3 0.7

OH

-

-

OH

-

-

-

(S )-Corydalmine (S )-Stylopine

: \o

(R,S)-Isocorypalmine (S )-Isoscoulerine (S )-Isocoreximine (R,S )-Stepholidine (R,S)-Discretamhe (R,S)-Capaurimine (S )-Escholidine (S )-Canadine N-Metho Salt (R,S)-Corydaline (R,S )-Tetrahydrocorysamine (R,S)-Corybulbine (R)-Corybulbine (R)-Isocoreximine (R)-Capaurimine (R)-Canadine

OMe OMe OCH2O OH OMe OMe OH OMe OH OH OMe OMe OH OMe OMe OCHZO OCHZO OMe OMe OCH20 OMe OH OMe OH OMe OH OMe OMe OCH2O

Me Me

OMe OH OCH2O OMe OMe OH OMe OMe OMe OH OMe OH OMe OH OMe OH OMe OMe OMe OMe OCH2O OMe OMe OMe OMe OMe OMe OH OMe OMe

a Data from M. Amann ef a/. (31). Relative turnover rates were determined using an optical assay containing 8-200 nmol substrate because of differing solubilities.

320

KINUKO I W A S A

(S>Scoulerine (1)

(a-Rcticuline

(StCanadine (S)

Berkrinc (17)

SCHEME20. Biosynthetic sequence from (S)-reticuline to berberine catalyzed by two common enzymes: (a) berberine bridge enzyme. (b) (S)-scoulerine-9-O-methyltransferase; and also by two enzymes specific for the Eerbcris pathway: (c) ( S )-tetrahydroprotoberberine oxidase and (d) berberine synthase. The alternate Copris pathway is characterized by (e) (Sbcanadine synthase and ( f ) (S)-canadine oxidase.

tetrahydroprotoberberine alkaloids such as (S)-stylopine (4) and (S)canadine (5) at the expense of (S)-adenosyl-L-methionine (SAM) (Table XVI) (Scheme 21) (30).(S)-Tetrahydroprotoberberine-cis-N-methyltransferase acts on tetrahydroprotoberberines of the (S)-configuration containing a methylenedioxy bridge between atoms 2 and 3 with a few exceptions. That is, this N-methyltransferase acts on the nonphenolic tetrahydroprotoberberines, such as (S)-stylopine (4) or (S)-canadine (5). The cis-N-methyltransferase has been found in several different species of isoquinoline-bearingplant cell cultures (Table XVII) (25,30).Cultures of Fumariacae and Papaveraceaes show considerable enzyme activity toward tetrahydroprotoberberine substrates, stylopine being the preferred substrate in most cases. Additionally,the tetramethoxylated alkaloid tetrahydropalmatine can be utilized as a substrate in most species investigated. These results differ from those utilizing the purified enzyme and may be due to the presence of a second N-methyltransferase activity in the crude enzyme extracts. 3. Hydroxylation at C-14 of a-N-Metho Salts of Tetrahydroprotoberberines to Afford Protopines (36) A monooxygenase has been partially purified from C. uaginans and characterized. The enzyme catalyzes the hydroxylation of the (S)-cis-Nmethyl derivatives of canadine (5) (loo%), stylopine (4) (80%), tetra-

5.

321

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

TABLE XV SUBSTRATE SPECIFICITY OF THE HIGH-A N D LOW-MOLECULAR-WEIGHT FORMS OF Coptis (S)-CANADINE OXIDASE" Enzyme Activity (%) Substrate

155 kD

58 kD

(S )-Canadine (R)-Canadine (R.S )-Stylopine (R,S)-Tetrahydrocolumbamine (R,S)-Scoulerine (R,S)-Norreticuline

100

100

0 II 5 0 0

0 26 5 0 0

Data from E. Galneder er a / . (34).

hydrothalifendine (61)(30%),tetrahydropalmatine (55) (19%), and corydalmine (6%) (62) (Table I ) (Scheme 22). The (R)-cisltruns-N-methylcanadine epimeric mixture was found to be completely inactive toward (S)-cis-Nmethyltetrahydroprotoberberine-14-hydroxylase. The enzyme that hydroxylates C- 14 of the (S)-cis-N-methyltetrahydroprotoberberines has been discovered in a number of cell cultures derived from plants of the Fumariaceae and Papaveraceae (Table XVIII). TABLE XVI RELATIVEEXTENTSOF CONVERSION OF TETRAHYDROPROTOBERBERINES TO THEIR N-METHOSALTSCATALYZED BY SAM:(S)-TETRAHYDROPROTOBERBERINECiS-N-METHYLTRANSFERASEa

Extent of Transformation (%)

Substrate ~~~~~~

R, ~

~

(S )-Canadhe

R,

CH2 (R)-Canadine CH2 (S)-Stylopine CH2 (R)-Stylopine CH, ( R , S)-Nandinine CH2 (R,S)-Tetrahydrogroenlandicine H Me (S )-Scoulerine Me H " Data from M. Rueffer er

a / . (30)

R,

R4

C . vaginans

E. californica

100 0 104

100

~

Me Me Me Me CHI CH2 H Me CH, H Me

0 90 12

0

0 109 0 94 17 0

322

KINUKO IWASA

(S)-StylOpine (4) R,+Rz=CHz (9-Canadine (5) RI=R2=Me

( S ) 4 RI+Rz=CHz

(9-hRI=R2=Me

SCHEME 21. N-Methylation catalyzed by (g) S-adenosyl-~-methionine (SAM): (S)tetrahydroprotoberberine cis-N-methyltransferase (Eschscholrzia californica or Corydalis unginans).

4 . Hydroxylation at C-6 of Protopines to Generate Dihydrobenzophenanthridines

It has been reported by Tanahashi and Zenk (37,38) that microsomal preparations from E. californica cell suspension cultures treated with a crude elicitor preparation from yeast catalyze the hydroxylation of [6-3H]protopinewith concomitant formation of [ 1l-3H]dihydrosanguinarine and H03H. In noninduced cells the enzyme possessed only ca. 10-15% of its activity in the induced cells. Based upon the belief that stereospecific removal of the p r o 4 hydrogen from C-6 occurs during conversion of scoulerine (1) into chelidonine (S), Tanahashi and Zenk assumed that protopine-6-hydroxylaseintroduces the hydroxyl group into the protopine molecule to produce (6S)-6-hydroxyprotopine, and that spontaneous rearrangement of this molecule generates dihydrosanguinarine (44) (Scheme 23). Species belonging to the Fumariaceae and Papaveraceae contained the hydroxylase which had been strongly stimulated in almost every case after the addition of elicitor (Table XIX).N o hydroxylase activity could be detected in Catharanthus roseus (Apocynaceae) and Berberis (Berberidaceae) species, which very significantly are known to contain no benzophenanthridine alkaloids (Table XIX). It is also worth pointing out that E. californica microsomal preparations have been shown to hydroxylate dihydrosanguinarine to 10-hydroxydihydrosanguinarine.This finding suggests that a second hydroxylase may be present in these membrane fractions.

5 . Dehydrogenation of Dihydrobenzophenanthridines to the Fully Aromatized Benzophenanthridines

Zenk and co-workers (39) have partially purified and characterized a novel enzyme which catalyzes the oxidation of dihydrobenzophenanthridines in the presence of oxygen to generate fully aromatized benzophenanthridine alkaloids from E. californica. This enzyme is specific with respect to the benzophenanthridine skeleton and catalyzes dihydrobenzo-

TABLE XVII

OCCURRENCE OF

DIFFERENT PLANT CELL CULTURES ALKALOIDSO ORIGINATING FROM PLANTS CONTAINING BENZYLISOQUINOLINE

SAM:(S)-TETRAHYDROPROTOBERBERINE-CiS-~-METHYLT~NSFERASE IN

Substrate

OR,

7

Cell Culture Berberidaceae Berberis beaniana B . notabilis B. wilsoniae Furnariaceae Corydalis vaginans Dicentra spectabilis Fumaria officinalis Menispermaceae Chondodendron tomentosum Tinospora cordifolia Papaveraceae Argemone plaryceras Bocconia cordata Eschscholtzia caespitosa E . californica Ranunculaceae Thalictrum glaucum T. bautrinii Data from M. Rueffer er a / . (30).

( R ,S )-Canadine RI + R, = CHZ R3 = R4 = Me 0.9 0 0

(R,S)-Stylopine RI + R2 = CH2 R, + R4 = CH2 0.9 0 0.9

(R,S)-Sinactine R, = R2 = Me R3 + R4 = CH2 (pkat g - ' dry weight)

( R ,S )-Tetrahydropalmatine R, = R2 = Me R3 = R4 = Me

1.7 0 2

0 0 1

52 28 44

80 40 42

29 12 n.d.

31 50 52

2 0

2 0

n.d. 0

4 0

41 20 77 55

41 18 107 55

57 12 30 2

49 20 14 39

0.4 0

0.4 4

0.8 0

1.1

0

324

KINUKO IWASA

It Roropine (6) R,+Rz=CHz Allocryptopine (18) Rl=Rz=Mc

SCHEME 22. Hydroxylation at C-14 catalyzed by (h) (S)-cis-N-methyltetrahydroprotoberberine 14-hydroxylase (Corydalis vaginans).

phenanthridines incorporating methylenedioxy substituents on rings A or D, such as dihydrosanguinarine (Scheme 24). Dihydrobenzophenanthridine oxidase was not found in cell suspension cultures that do not produce benzophenanthridine alkaloids, such as those originating from Rauwoljia serpentina and Catharanthus roseus (Apocynaceae).

TABLE XVIII SURVEY OF DISTRIBUTION OF HYDROXYLATION ACTIVITY I N DIFFERENT CELLCULTURES" Plant Family

Cell Culture

Fumariaceae

Corydalis ophiocarpa Corydalis vaginans Dicenrra spectabilis Furnaria capreolata Furnaria officinalis Furnaria parvijlora Bocconia cordata Eschscholtzia californica Glauciurn jlavum Papaver sornniferurn

Papaveraceae

" Data from M. Rueffer and Zenk (36).

Enzyme Activity (pkat g - ' dry weight)

52 52 79 84 260 137 I17 73 69

44

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

-

F’rotopine (6)

325

----t--t

111

0 LO Dihydrosanguinarine (44)

SCHEME 23. Reaction catalyzed by (i) protopine-6-hydroxylase (Eschscholrziu californica) and subsequent spontaneous rearrangementof 6-hydroxyprotopine to dihydrosanguinarine (44).

6 . Hydroxylation at C-10 Followed by Methylation of the Hydroxy Group at C-10 to Furnish 10-0-Substituted Dihydrobenzophenanthridines (40a,40b)

A reaction of microsomal protein obtained from E. californica cell suspension cultures with [6-3H]dihydrosanguinarinegenerated a radioactively labeled product that was methylated to supply radioactive dihydrochelirubine, thus substantiating 10-hydroxydihydrosanguinarine as the reaction product (40a). The dihydrosanguinarine-10-hydroxylase is a microsome-associated, cytochrome P-450-dependent monooxygenase, which acts specifically at the C-10 site of dihydrosanguinarine (Scheme 24). The purified methyltransferase, IO-hydroxydihydrosanguinarine10-0-methyltransferase, methylates only the hydroxyl moiety at C-10to form dihydrochelirubine. Absolutely no reaction was observed with the quaternary benzophenanthridines hydroxysanguinarine and hydroxychelerythrine, or with the closely related dihydrobenzophenanthridines(Table XX). Recently, two enzymes transforming dihydrochelirubine to macarpine (52) were also discovered using suspension cultures of Thalictrum bulgaricum and E . californica (40b): dihydrochelirubine-12-hydroxylase and 12-hydroxydihydrochelirubine-12-0-methyitransferase. These enzymes were found to have the same high substrate specificity as the two enzymes transforming dihydrosanguinarine to chelirubine (51). Zenk et al. (40b) logically concluded that the 10-methoxyl group of chelirubine is introduced at the dihydrosanguinarine level and not at the quaternary sanguinarine stage (Scheme 24). It is probable that further 12hydroxylation, followed by methylation and oxidation, would lead to the most oxidized benzophenanthridine, macarpine, in E. californica cell cultures.

TABLE XIX DISTRIBUTION OF PROTOPINE-~-HYDROXYLASE ACTIVITY I N DIFFERENT CELLCULTURES A N D EFFECT OF YEASTELICITOR ON ENZYME ACTIVITY~ Enzyme Activityb Plant Family

Cell Culture

Apoc ynaceae Berberidaceae Fumariaceae

Catharanthus roseus Eerberis stolonifera Dicentra cucullaria Fumaria pamiflora Chelidonium majus Eschscholtzia californica strain AST strain BB strain ROT Eschscholtzia lobbii

Papaveraceae

Nonelicited

Elicited

0 0 0.3 14 6

0 0 3.4 359 57

27 8 0.3 3

187 113 31 185

THE

Induction FactorC

(18.8) (13.9) (5.9) (56.6)

a Data from Tanahashi and Zenk (37). Five-day-old cultures were used for microsomal preparation. The elicited cultures received 40 pglml elicitor 20 hours before harvest. pkatlliter. Figures in parentheses indicate pkatlmg protein. Induction factor was calculated from pkatlliter. Figures in parentheses indicate induction factor calculated from pkatlmg protein.

5.

BIOTRANSFORMATION O F PROTOBERBERINE ALKALOIDS

Macarpine ( S t )

Rotopine (6)

327

Dihydromacarpine

Dihydroyguinarine (44)

IO-Hydrnxydihydrosanguinarinc

Dihydyhelirubine

Chelirubinc (51)

Sanguinarine(7)

SCHEME24. Proposed biosynthetic sequence from proptopine (6) to the benzophenanthridine macarpine (52): (i) protopine-6-hydroxylase (Eschscholtzia californica); 6)dihydrosanguinarine- 10-hydroxylase (E. californica); (k) SAM: Whydroxydihydrosanguinarine10-0-methyltransferase (E. californica); (I) dihydrochelirubine-12-hydroxylase;(m) SAM: 12-hydroxydihydrochelirubine-12-0-methyltransferase (Thalictrum bulgaricum, E. californica); (n) dihydrobenzophenanthridine oxidase (E. californica).

TABLE XX SUBSTRATE SPECIFICITY OF THE PURIFIED 10-HYDROXYDIHYDROSANGUINARINE-10-0 METHYLTRANSFERASE USINGSAM AS METHYL-GROUP DONOR" Substrate c- 7

C-8

C-I0

C-12

Relative Enzyme Activity (%)

~~

7

7

Me

10-Hydroxydihydrosanguinarine OCHZO OH H 10-Hydroxydihydrochelerythnne OMe OMe OH H 12-H ydroxydihydrochelirubine OCH2O OMe OH 10-Hydroxysanguinanne OCH2O OH 10-Hydroxychelerythrne OMe OMe OH 12-Hydroxychelirubine OCH2O OMe

Data from De-Eknarnkul el a/. ( 4 0 ~ ) .

100

0 0 0

H 0

H 0

OH

328

KINUKO I W A S A

7 . Biosynthetic Sequence from Protoberberines to Benzophenanthridines Based on Enzyme-Level Studies

The steps that lead to the formation of benzophenanthridines from protoberberines catalyzed by enzymes are shown in Scheme 25 (6,41). All enzymes involved in this transformation have been discussed (6,41). This biosynthetic pathway is the same as that obtaining in the precursorial feeding experiments using plants and plant cell cultures, excluding the relationship between dihydrobenzophenanthridines (e.g., 44) and the fully aromatized quaternary benzophenanthridines (e.g., 7, Schemes 14 and 25).

Protopine (6) Allocryptopine (18)

0 L O

Dihydrosanguinarine(44)

\

Me \-0 Chelirubine (51)

Macarpine (52)

SCHEME25. Formation of the fully aromatized benzophenanthridines from protoberberines catalyzed by enzymes.

5 . BIOTRANSFORMATION OF PROTOBERBERINE

ALKALOIDS

329

111. The Second Pathway

A. CONVERSIONS OF TETRAHYDROPROTOBERBERINES INTO RHOEADINES VIA PROTOPINES: WHOLEPLANTSTUDIES In the rhoeadines, which are tetrahydrobenzazepine alkaloids, oxygen substituents are inevitably present at positions 7, 8, 12, and 13. These substituents may be methoxyl, methylendioxy, or, in a few cases, hydroxyl groups. There are two main series, which differ in relative configuration at C-1 and C-2, so that the BID ring juncture may be trans or cis. Some biosynthetic studies have been undertaken using the trans-fused alpinigenine (63)and rhoeadine (a), which belong to the cis series. These studies have been comprehensively reviewed (42), so the results will be described only briefly in this section. 1 . Feeding Experiments with Papaver bracteatum (Papaveraceae)

a. Feeding of I4C- andlor 'H-Labeled Tetrahydropalmatine (55) and its N-Methiodide (55a and 55b) (43). Ronsch administered (R,S)-[8-14C] tetrahydropalmatine (551, (R,S )-[N-14CH3]-,and -[N-14CH3,8-14C]tetrahydropalmatine methiodide (55b,containing a little 55a) to P. bracteaturn plants. Each of these compounds was incorporated into alpinigenine (63) to the extent of 14.8, 0.56, and 0.52%, respectively (Scheme 26) (43). Administration of [8,13,14-3H3]tetrahydropalmatine(55) and [8,13,143H3,8-14C]tetrahydropalmatine methiodide (55a and 55b, as a mixture of the a-and p- isomers) to the same plants resulted in incorporation levels of 10.4 and 1.0%, respectively. A loss of 54.8% of the tritium in the precursor was observed in alpinigenine (63). This value accounts very well for the loss of two hydrogens from C-8 and C-13, and one from C14 of tetrahydropalmatine (55) and its N-metho salt. These data conform to the hypothetical involvement of intermediates A through D (see Scheme 27). Unfortunately, in all of the feeding experiments with the N-metho salt, incorporation of radioactivity was markedly lower than with the free base precursor (55).

b. Feeding of [8-'4C]Muramine (59) (44). 14C-Labeledmuramine (59) was fed to P. bracteatum. Incorporation of [8-'4C]muramine (59) into alpinigenine (63)was very high (22.6%) (Scheme 26). This result points to the fact that two bond cleavages [N-C( 14) and N-C(8)] are required for the biogenetic transformation of tetrahydroberberines to rhoeadines (see Scheme 27).

KINUKO I W A S A

330

Me0 OMe

OMe

OMe

OMe

OMe

OMe

18-'4C]-Tetrahy&opalmatine (55)

[8-'4CJ-Murarnine(59) \

J

Me0

14.8%

1

OMe

/N-14CH3]-or [N-t4CH3;8-'4C! I-55a (B/C cis) mixture (55b: major) of 55a and 55b (B/C trans)

['H1-55: 10.4%

MeOO

-

13H;'4CI-55aand 55b: 1.0% Alpinigenine (63) [8.13,14-3H;8-'4Cl-55a(B/Ccis) R=Mc mixture (55b major) of 5Sa and 55b (B/C trans) [8,13.14-'HJ-55 R=H

SCHEME26. Biosynthetic conversions of tetrahydropalmatine (55). its N-metho salts (5% and 55b). and muramine (59) to alpinigenine (63) in Papaver bracteaturn plants.

c . Feeding of the Possible Precursors (44). The possible precursors, [8-'4C]dihydropalmatinemethosulfate (56), -13-hydroxymuramine (65), - 13-oxomuramine(66),and four more alkaloids (67-70) were administered to P . bracteatum plants (Fig. 2). Two alkaloids, 56 and 67, were incorporated into alpinigenine (63) with very low efficiency, while the other alkaloids were not taken up. The data indicate a pathway consisting of an N-methylation step and subsequent hydroxylation at C- 14 of tetrahydroprotoberberine N-metho salts to provide the quaternary carbinolamine species corresponding to the protopine alkaloids. These steps are followed by hydroxylation at C8, then C(8)-N and C(14)-N bond fission, and recyclization to generate rhoeadine-type alkaloids such as alpinigenine (a),which incorporate a B/D-trans ring juncture (see Scheme 27).

2 . Feeding Experiments with Papaver rhoeas a . Feeding of (13S)-[13-3H]- and (13R)-[13-3H]Scoulerine(1) (45). Both (13s)- and (13R)-tritium-labeled scoulerine (1)(with configurational purity of 75 and 73%, respectively) were incorporated by living P . rhoeas

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

33 1

65 X=H+OH 66 X=o

z

q

NHMe

Me0

OW

\' 68

OM0

OM0 69

FIG.2

plants into rhoeadine (64) (see Scheme 28). The rhoeadine isolated from plants fed with (13S)-labeled scoulerine (1) had lost 79% of the tritium present in the precursor, whereas the (1 3R)-epimer afforded rhoeadine (a),which retained 74% of the original tritium. Bearing in mind the configurational purity of the precursors, these values constitute good evidence that stereospecific removal of the pro-S-hydrogen must have occurred from C-13 at some stage during the transformation of stylopine (4) into rhoeadine (64).

M.0 Alpiniyninc (63)

Rhocdinc (64)

SCHEME27. Biogenetic sequence for rhoeadine alkaloids.

332

KINUKO I W A S A

( I 3R)-[ 13-’H, I-Scoulerinc (1)

[2-1HI]-Rhocadinc(64)

(I3s)-I1 3-1HI]-Scoulcrinc (1)

Rhocadine (61)

( ~ . z ~ , ~ ) - [ ~ - ” c ~ ~ ~ -a-hr-metho ~ t y ~ o psalt i n e(4.)

Rhocadine (64)

I13-)H2]-Rotopinc (6)

SCHEME28. Biosynthetic formation of rhoeadine (64) from scoulenne (1). stylopine aN-metho salt (4a), and protopine (6) in Papaver rhoeas plants.

b. Feeding of (R,S)-[N-’”CH,]-a- and P-Stylopine Methochlorides (4a and 4b) (46). Tani and Tagahara administered the a- and P-N-metho salts of (R,S)-stylopine (4a and 4b) separately to P . rhoeas. This study established the exclusive utilization of the a-N-metho salt (4a) in the biotransformation into rhoeadine (64) (Scheme 28). The incorporation of I3C-labelinto rhoeadine (64) was approximately 1%. c. Feeding of [IJ-”H,]Protopine (6) (46). In a feeding experiment of [ 13-3H,]protopine(6) to P . rhoeas, incorporation (0.03%) of the 3H-label

of 6 into rhoeadine (64) was observed (Scheme 28). Degradation of the product 64 provided evidence for the localization of the 3H-label at C-2. The biosynthesis of rhoeadine-type alkaloids, such as rhoeadine (64) bearing the B/D-cis ring juncture would be accomplished by N-methylation and then hydroxylation at C-14 of tetrahydroprotoberberines to form protopine alkaloids. These would then be hydroxylated at C-8 and suffer ring opening at the N-C(8) and N-C( 14) sites. The final step would be a skeletal rearrangement (Scheme 27). 3. A Biosynthetic Scheme for Rhoeadine Alkaloids

The natural source for all rhoeadine alkaloids can be assumed to be the amino acid L-tyrosine. The pathway from L-tyrosine to the nonphenolic tetrahydroprotoberberines [e.g., stylopine (1)and canadine (5)]has been

5.

BIOTRANSFORMATION O F PROTOBERBERINE ALKALOIDS

333

amply demonstrated by several tracer experiments (42). N-Methylation of the nonphenolic tetrahydroprotoberberine alkaloids, followed by hydroxylation at C-14 of the N-metho salts, leads to the quaternary protopine species. Hydroxylation at C-8 would then give rise to the 8,14-dihydroxylated carbinolamine species A. Scission at the C(8)-N and C(14)-N bonds subsequently generates intermediate C or D, which could undergo skeletal rearrangement to furnish the nonphenolic rhoeadine-type alkaloids (Scheme 27). It should be noted that cell cultures were not used in the above studies.

IV. The Third Pathway A. FORMATION OF 13-HYDROXYTETRAHYDROPROTOBERBERINES IN WHOLEPLANTS Of the numerous protoberberines which have been isolated from natural sources, ophiocarpine (14)and corycarpine (15)(see Fig. 3), isolated from C. ophiocarpa and other Corydalis species (7), are the only 13-hydroxylated tetrahydroprotoberberine alkaloids. Clearly, the biosynthetic conversion of the tetrahydroprotoberberine ring system to these hydroxylated systems involves oxidation at C-13 at some stage. In this section, the elucidation of the stereochemical course of the hydroxylation step will be described. Ophiocarpine (14)is expected to be formed from scoulerine (1)through 0-methylation, formation of the methylenedioxy system, and oxidation at C-13. Feeding Experiments with Corydalis ophiocarpa a. Feeding of (R,S)-[l ,12-”H2]Scoulerine(l),(R,S)-[9-0’4CH3;8,14-jH2] Tetrahydroberberine(5), and (R,S)-[12--’H]Nandinine(71). Labeled ( R , S ) scoulerine (l), (R,S)-tetrahydroberberine (3,and (R,S)-nandinine (71) were fed in separate experiments to young shoots of C. ophiocarpa (47). While scoulenne (1) is an efficient precursor (0.05% incorporation) of ophiocarpine (14),nandinine (71)does not appear to be incorporated to any significant extent (0.001%) in that plant. But tetrahydroberberine (5) is incorporated efficiently (0.13% incorporation) into ophiocarpine (14). It can be concluded that scoulerine (1)is bioconverted into tetrahydrocolumbamine (3), which is transformed into ophiocarpine (14)via tetrahydroberberine (3,and that introduction of the hydroxyl at C-13 occurs at the terminal step (Scheme 29). Incorporation of (R,S)-[8,14-3H,]tetrahy-

334

KINUKO IWASA

OMe

OH

OMe

OMe

Teuahydmcolumbamine(3)

OMe Nandminc (71)

loss of ’H of 12%

OM0

0.99%

OMe

OM0 (14S)-[13-’HI]-Ophiocarpine (14)

(13S)-Teuahydrobcrbenne (5)

loss of ’H of 86% 0.22%

0 OMe OMe

(1 3R)-Teuahydrobcrbcrine (5)

OMe

(14S)-Ophiocarpine (14)

retention of ’H of 96% L

OMe ( 14R~)-[8,14-3H,]-Tc~y~obcrbcnne (5)

(14S)-[8, 14-’H2]-Ophiocarpine(14)

SCHEME29. Formation of ophiocarpine (14)from scoulerine (1)or canadine (5) in Corydalis ophiocarpa plants.

droberberine (5) occurs without loss of tritium from the C-14 position (96% retention of tritium) (Scheme 29). Consequently, it appears that direct hydroxylation of the C- 13-methylenegroup of tetrahydroberberine (5) is somehow involved in the conversion to ophiocarpine (14). A further point of importance established by this result is that biological interconversion of the (14s)- and (14R)-forms of tetrahydroberberine (5) is unlikely. Based upon the results obtained previously from the conversion of tetrahydroprotoberberines into benzophenanthridine alkaloids (Section 11), it would also be expected that only the (14S)-form of 5 would undergo conversion to (14S)-ophiocarpine (14).

5 . BIOTRANSFORMATION O F PROTOBERBERINE ALKALOIDS

335

b. Feeding of (13s)- and (13R)-[9-0'4CH3,13-3H]tetrahydroberberines [(13S)-5 and (13R)-5] (47). The doubly-labeled samples Of (13s)- and (13R)-5 were administered separately to excised cuttings of C. ophiocarpa. Both isomers (13s)-and (13R)-5 were incorporated into ophiocarpine (14) (0.99 and 0.22% incorporation levels, respectively). In ophiocarpine (14) obtained from (13S)-5 and (13R)-5, levels of tritium retention at C-13 were 88 and 14%, respectively (Scheme 29). These results show that hydroxylation of (13S)-tetrahydroberberine[(13S)-5]at C-13 to afford (14S)-ophiocarpine (14) proceeds with removal of the pro-13R hydrogen. This corresponds to an overall retention of configuration in the hydroxylation process. The fact that only 88%, rather than the expected loo%, of the p r o 4 tritium remained after conversion of the isomer (13S)-5 to (14s)ophiocarpine (14) suggests that the starting material did indeed contain a small amount of the configurational isomer (13R)-5. The corresponding loss of 86% of the pro-R tritium from the parallel experiment with isomer (13R)-5 was in full agreement with this suggestion. It has thus been established that hydroxylation of (14S)-tetrahydroberberine [( 14S)-5] to (14S)-ophiocarpine [( 14S)-14] in C. ophiocarpa proceeds with retention of configuration, and involves the removal of the pro-R hydrogen atom from the C- 13 position of the precursor (14S)-5. B. CONVERSIONS OF

13-HYDROXYTETRAHYDROPROTOBERBERlNES

BENZINDANOAZEPINES I3-OXOPROTOPINES

INTO SPlROBENZYLlSOQUINOLINES OR VIA

13-Hydroxytetrahydroprotoberberines [e.g., ophiocarpine (14) and I-methoxy-13corycarpine (15)], protopines [e.g., 13-oxomuramine (a), oxoallocryptopine (72), and ochrobirine (73)], and spirobenzylisoquinolines [e.g., sibiricine (74), corydaine (79, raddeanone (76), and yenhusomidine (77)], which possess an oxygen function at C-13, have been isolated from some plants of the genera Corydalis, Fumaria, and Papauer (Fig. 3) (7,26,48).The benzindanoazepines [e.g., O-methylfumarofine (78) and fumaritrine (79)] (Fig. 3) have also been found in the genus Fumaria (7). From a biogenetic point of view, it was of interest to study the bioconversion of 13-hydroxylated tetrahydroprotoberberines into another class of alkaloids bearing an oxygen function at C- 13-specifically, the spirobenzylisoquinolines that have been found in the same natural sources as the 13-hydroxytetrahydroprotoberberines. 1 . Callus Tissues Studies

The callus tissues of C. ophiocarpa and C. ochotensis var. raddeana were judged to be suitable for incorporation experiments of the 13-hydroxyl-

336

Ophiocarpinc (14) Rl=R2=Mc Corycarpine (15) RI+R2=CH2

KINUKO I W A S A

13-Oxomuraminc (66) RI=H R2=R3=R4=Rs=Mc X=O I-Mcthoxy-13-oxoallocryp1opinc (72) RI=OMe R2+R3=CH2 &=R,=Me X=O Ochrobirinc (73) Rl=H R2+R,=&+R5=CH2 X=OH+H

Sibiricinc (74) Rl+R2=CH2 X=H Y=OH

0-Methylfummfine (78)

Fumprimne (79)

Corydainc (75) RI+R2=CH2 X=OH Y=H Raddcanonc (76) RI=R2=Me X=H Y=OH Ycnhusomidine (77) RI=R2=Me X=OH Y=H

FIG.3

ated tetrahydroprotoberberines since, in intact plants of C. ophiocarpa, the 13-hydroxylatedtetrahydroprotoberberines are the major components, while C. ochotensis var. raddeana contains spirobenzylisoquinolines(7). (See Table XXI.) a. Feeding Experiments with Corydalis ophiocarpa. Following hydroxylation at C-13 of the tetrahydroprotoberberines to generate 13hydroxytetrahydroprotoberberines, it was thought possible that N methylation would be required for the formation of other classes of alkaloids. (i) Feeding of unlabeled (R,S)-ophiocarpine a-N-metho salt (14a) (28). A feeding experiment (experiment 1) (Table XXI) was undertaken using the quaternary salt of (R,S)-ophiocarpine(14a) bearing cis hydrogens at C-13 and C-14. Supplying the a-N-metho salt 14a, which has a cis quinolizidine system, to C. ophiocarpa indicated the incorporation of 14a into 13-oxoallocryptopine (82), whose structure was determined by comparison with an authentic sample. (R,S)-Ophiocarpinea-N-metho salt (14a) would subsequently be bioconverted via 82 into the alkaloids 83 and 84, as described in the next section. (ii) Feeding of nonlabeled (R,S) -epiophiocarpine a-N-metho salt (Ma) (18). Calli from C. ophiocarpa were grown on agar medium containing nonlabeled (R,S)-epiophiocarpine a-N-methyl chloride (Ma), which differs from (R,S)-ophiocarpine a-N-metho salt (14a) in the relative

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

337

configurations at C-13 and C-14 (experiment 2) (Table XXI). Three alkaloids were isolated, as well as the administered material. As an added check, it was determined that these alkaloids were not detected when the exogenous a-N-metho salt 80a was absent, but with conditions otherwise kept the same. The structures of two of the alkaloids were established by comparison of the spectral data with those of authentic synthetic samples as 13-hydroxyallocryptopine (81) and 13-oxoallocryptopine (82). The structure of the third alkaloid was determined by X-ray analysis (51) to be the spirobenzylisoquinoline 83, incorporating an aliphatic O-methyl group in lieu of the N-methyl group expected on biogenetic grounds (Scheme 30). The carbonyl group is positioned at C-13, and the methoxyl group at C-8 is anti to the nitrogen. The stereochemistry at C-8 is the same as in sibiricine (74), a minor alkaloid of C. sibirica,and in raddeanone (76), isolated from C. ochorensis var. raddeana. (R,S)-Epiophiocarpine a-N-metho salt (80a) was bioccnverted into the corresponding protopine-type alkaloid, 13-hydroxyallocryptopine (81), which was then converted into 13-oxoallocryptopine(82)and spirobenzylisoquinoline 83 in callus cultures of C. ophiocarpa (Scheme 30). 13-Oxoallocryptopine (82) would thus be formed by oxidation of 13-hydroxyallocryptopine (81)in callus cultures. (iii) Feeding of unlabeled 13-oxoallocryptopine (82) (18). To clarify the relationship between 13-oxoallocryptopine(82)and the spirobenzylisoquinoline alkaloid 83 in the biosynthetic pathway, the biotransformation of 82 was further examined. Feeding (experiment 3) (Table XXI) of unlabeled 13-oxoallocryptopine (82)indicated that this compound is biotransformed into the spirobenzylisoquinoline 83 and the benzindanoazepine 84, whose spectral data were identical with those of the same alkaloid prepared by Hanaoka er al. (52). One can therefore conclude that the results described above support the sequence 80a + 81 + 82 +- 83 +- 84. (iv) Feeding of (R,S)-[N-'3CH3]-epiophiocarpinea- and P-N-metho salts (80a and 8Ob) (18). In order to provide insight into the extent of discrimination between the stereochemical a- and P-N-metho salts 80a and 80b, and in an effort to confirm the transformation of the a-N-metho salt 80a into the spirobenzylisoquinoline-and benzindanoazepine-type alkaloids 83 and 84, feeding experiments with the I3C-enriched alkaloids (R,S)-[N-'3CH3]-epiophiocarpine a- and P-N-methyl chlorides 80a and 80b were carried out (experiments 4 and 5) (Table XXI). Labeled bases 81,82,83,and 84,as well as 80a, were isolated from the alkaloidal fraction of experiment 4 to which 80a was administered (Scheme 30). The 'T enrichment of each base is summarized in Table XXI. Recovered 80a contained substantial amounts of the (14R)-enantiomer.Significantly, the

TABLE XXI OF 13-HYDROXYTETRAHY DROPROTOBERBERINE N-METHOSALTS AND 13-OXOPROTOPINE-TYPE ALKALOIDS TO ADMINISTRATION Corydalis SPECIES Products: Yield % (”C Enrichment %) W

00

Experiment

Substrates

81

82

11.7

2.2 2.7

83

2.0 (87Y

5.7 (88IC

10.7 16.4

3.6 7.2

0.4 3.6

-

72

-

0.2 0.1 (90)‘ 2.2 5.0

-

3.8 C. ochotensis VBT. raddeana

96

(R,S)-[N-”CH+SOa

I .7 (83)‘

I .4 (9w

86

Recovered Material: Yield % [Optical Purity %]

0.3 3.3 0.4 (88Id

-

84

1.2

1.7 11.8 31.2 [35, (R)-enantiomer] 26.7 6.0 4.4 49.3

10

(R,S)-[N-"CH&8Oa

11 12a.b 13'

(R,S)-82 (14S)-85a ( 14s )-85.

C . pallida var. tenuis 14 (RSI-80. IS 16" C . platycarpa 17 18"

(14S)-8os (14S)-85s

(R.S)-82 ( 14s )*a

W

W

Liquid suspension cultures. Half incubation period. The N-methyl group was labeled with I3C. The 0-methyl group was labeled with "C.

2. I (90)'

0.3 (83Id 3.3

0.4

5.1

2.4

0.6

1.1

9.4

3.4

0.9

3.4

4.9

1.1 3.1

31.4 16.5 47.6

0.7

0.7

28.4 [47. (R)-enantiomer] 17.0 5.2

2.7

0.9

2.2 52.5

2.4

340

KINUKO I W A S A

OM. OM.

- pQ87c; .I

OM

83

OM.

81 R-H

L‘

OM.

84 R-H (N-Me.)

86 R=OMe

SCHEME30., Formation of ’ 13-hydroxytetrahydroprotoberberinesand their bioconversion to other types of alkaloids.

corresponding metabolites were not detected in the alkaloidal fraction of the feeding experiment (experiment 5 ) with the P-N-metho salt 80b, which has a trans quinolizidine system. It was thus demonstrated that (14S)-epiophiocarpine a-N-metho salt Ma, bearing a cis quinolizidine ring, was bioconverted into 81, 82, 83, and 84 (Scheme 30). It was also shown that the 0-methyl group at C-8 of the spirobenzylisoquinoline 83 arises from the N-methyl group of the protoberberine 80a, so that migration of the methyl group from N to 0 obtains during the ring rearrangement. 83 and 84 was estabThe biotransformation pattern 80a + 81 + 82 jlished through feeding experiments of the a-N-metho salt 80a and 13oxoallocryptopine (82). The following mechanism (Scheme 3 1) dealing with the conversions of 13-oxoprotopines (e.g., 82) into spirobenzylisoquinoline (e.g., 83) and benzindanoazepine alkaloids (e.g.. 84) can therefore be sustained. Hydroxylation of C-8 in the 13-oxoprotopinesgives rise to a carbinolamine intermediate, which loses water to generate a protoberberine phenolbetaine. Spirobenzylisoquinoline (83) and benzindanoazepine (84) would be produced from an intermediate 8,14-cycloberberine.

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

341

84

SCHEME31. Mechanism for the formation of a spirobenzylisoquinoline (83) or a benzindanoazepine (84) from 13-oxoallocryptopine (82).

(v) Feeding of (R,S)- and (14S)-[N-13CH3]-epiophiocarpine a-Nmetho salts [(R,S)-80a and (14S)-8Oa] (50). (R,S)- And (14S)-I3Clabeled a-N-metho salts (R,S)-80a and (14S)-80a were administered to cultured cells of C. ophiocarpa (experiments 6 and 7) (Table XXI)to provide information about enantiomeric specificity in the conversion of the protoberberine skeleton into a 13-hydroxyprotopine structure. Comparison of experiments 6 and 7 shows that the (14S)-isomer of the (14s)and (14R)-forms of 80a is converted efficiently into the four alkaloids 81-84, while the (14R)-isomer is, for all practical purposes, ineffective. (vi) Feeding of (14S)-l-methoxyepiophiocarpinea-N-metho salt @a) (50). In order to establish the validity and range of the conversion of the protoberberine skeleton into the spirobenzylisoquinolinesor benzindanoazepines, the transformation of a protoberberine quaternary salt incorporating a methoxyl substituent at C-1 was examined. (14S)-1Methoxyepiophiocarpine a-N-metho salt S a , which possesses a cis-fused quinolizidine structure and a trans configuration of the protons at C-13 and C-14, was fed to C. ophiocarpa. Compound 85a was biotransformed into 1-methoxy-13-oxoallocryptopine (72), which had been previously isolated from P. curuiscapum (71, as well as a I-methoxybenzindanoazepine

342

KINUKO I W A S A

86 (Scheme 30). However, the corresponding spirobenzylisoquinolineal-

kaloid was not detected in this experiment. It was thus demonstrated that tetrahydroprotoberberines bearing a C1-methoxylgroup can be biotransformed into benzindanoazepines via the 13-oxoprotopine analog. The fact that the corresponding spirobenzylisoquinoline compound was not obtained is probably due to steric interaction of the C-1 methoxyl group with the ring-C substituent. b. Feeding Experiments with Corydalis ochotensis var. raddeana (i) Feeding of (R,S)-[N-'3CHJ-epiophiocarpinea-N-metho salt (ma) (18). The I3C-labeled a-N-metho salt 80a was administered to C. ochotensis var. raddeana (experiments 9 and 10) (Table XXI). Alkaloids 81 and 82 and bases 82 and 83 were isolated from the alkaloid fraction from feeding experiments 9 and 10 (the culture period was twice that in experiment 9), respectively. Spirobenzylisoquinoline83 was formed instead of 13-hydroxyallocryptopine (81)by extending the culture period. Failure to detect benzindanoazepine 84 in the callus cultures of C. ochotensis var. raddeana might be due to a slight difference in the experimental conditions. (ii) Feeding of unlabeled 13-oxoallocryptopine (82) (18). Feeding (experiment 1 1) 13-oxoallocryptopine (82)to C. ochotensis var. raddeana resulted in incorporation of 82 into the alkaloids 83 and 84. Based upon results obtained from experiments 9-1 1, it was demonstrated that the a-N-metho salt 80a was bioconverted into the spirobenzylisoquinoline 83 and the benzindanoazepine 34 via 13-oxoallocryptopine (82)in the callus cultures of C. ochotensis var. raddeana, as well as in C. ophiocarpa (Scheme 30). (iii) Feeding of (14S)-I -methoxyepiophiocarpine a-N-metho salt (85a) (50). 1-Methoxy-13-oxoallocryptopine (72)was obtained from a feeding experiment (experiment 12) (Table XXI) of a-N-metho salt 85a to C. ochotensis var. raddeana. In this instance, the corresponding benzindanoazepine 86 was not detected by extending the culture period two fold (experiment 13). Clearly, the a-N-metho salt of 1-methoxyepiophiocarpine 85a is a precursor of 1-methoxy-13-oxoallocryptopine (72)in C. ochotensis, as well as in C. ophiocarpa.

c. Feeding Experiments with Other Corydalis Species (i) Feeding of (R,S)- and (14S)-epiophiocarpine a-N-metho salt (Ma) to Corydalis pallida var. tenuis (50). Feeding experiment (experiment 15) (Table XXI) with the (14s)-enantiomer of epiophiocarpine a-N-metho

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

343

salt 80a was undertaken in parallel with that (experiment 14) of the racemic form. The amounts of the metabolites, 13-hydroxyallocryptopine ( M ) , 13-oxoallocryptopine(82), spirobenzylisoquinoline 83, and benzindanoazepine 84 obtained from experiment 15 were 1.4- to 3.0-fold those obtained from experiment 14. Recovered 80a from experiment 14 contained substantial amounts of the (14R)-enantiomer. These results show that the (14S)-isomer of the a-N-metho salt 80a is converted efficiently into the four alkaloids, but that the (14R)-isomeris, essentially, ineffective. This conclusion is in agreement with the results obtained with C. ophiocarya (experiments 6 and 7). It therefore seems likely that (14s)-epiophiocarpine (80) is metabolized through the sequence 80a + 81 + 82 + 83 and 84 in C. pallida, as established in C. ophiocarpa. (ii) Feeding of (14S)-I -methoxyepiophiocaqine a-N-metho salt (85a) to Corydalis pallida var. tenuis (50). 1-Methoxy-13-oxoallocryptopine (72) and benzindanoazepine 86 were obtained from the alkaloid fraction to which the (14s)-isomer of the a-N-metho salt 85a had been fed. It is thus evident that the a-N-metho salt of (14S)-1-methoxyepiophiocarpine 85a would be transformed into benzindanoazepine 86 via I-methoxy-13oxoallocryptopine (72), as indicated in the transformation of 13-oxoallocryptopine (82) into the benzindanoazepine 83. (iii) Feeding of 13-oxoallocryptopine (82) and (lAS)-l-methoxyppiophiouupine a-N-metho salt (85a) to Corydalis phatyuupa (18,50). The callus cultured with 13-oxoallocryptopine (82) produced the spirobenzylisoquinoline 83 and benzindanoazepine 84 (experiment 17) (Table XXI). The N-metho salt 85a was also incorporated into l-methoxy-l3-oxoalIocryptopine (72) and benzindanoazepine 86. It is therefore very likely that the bioconversion of the a-N-metho salts 80a or 85a into benzindanoazepines 84 or 86 and/or spirobenzylisoquinoline83 via the 13-oxoprotopines 82 or 72 prevails in C. platycarpa (Scheme 30). C. CONVERSIONS BETWEEN TERTIARY AND 13-HYDROXYPROTOBERBERINES QUATERNARY 1 . Callus Tissue Studies: Feeding Experiments on Corydalis ochotensis var. raddeana

a. Feeding of Unlabeled (R,S)-Ophiocarpine (14) and (R,S)-Epiophiocarpine (80). The cis- and trans-13-hydroxytetrahydroprotoberberines 14 and 80 were administered separately to C. ochotensis var. raddeana (53).Thalictricavine (29),berberine (17) (Scheme 17),and 13-oxoallocryptopine (82) were identified through LC/APCI-MS (SIM method) in the

344

KINUKO IWASA

alkaloidal fraction from both experiments. Ophiocarpine (14) and epiophiocarpine (80) were metabolized to berberine (17) by removal of water rather than by conversion to their dehydro derivative l3. Berberine (17) is methylated at C-13 via an intermediate dihydroberberine, and subsequently 13methyldihydroberberine is reduced to thalictricavine (29), as described in Scheme 17. Formation of the metabolites 17, 29, and 82 from 14 or 80 was also observed in C. pallida var. tenuis. b. Feeding of Unlabeled Dehydro-ophiocarpine (53). Feeding of the dehydro derivative 13 of ophiocarpine (14) or epiophiocarpine (80) to C. ochotensis generated ophiocarpine (14) (Scheme 30). Reduction of dehydro-ophiocarpine (13) to ophiocarpine (14) also takes place in C. pallida var. tenuis. The oxidation of the cis- and trans- 13-hydroxytetrahydroprotoberberines, such as 14 and 80, was not specifically proved at that time, but the reduction process was demonstrated.

D. BIOSYNTHETIC PATHWAY OF 13-HYDROXYTETRAHYDROPROTOBERBERINES Some routes to the spirobenzylisoquinoline-and benzindanoazepinetype alkaloids from the 13-hydroxytetrahydroprotoberberinesare summarized in Scheme 30. Hydroxylation at C-13 of the (14s)-tetrahydroprotoberberines (e.g., 5) (providing a stereospecific loss of the pro-13R hydrogen) generates ( 14S)-cis-13-hydroxytetrahydroprotoberberines (e.g., 14). (14S)-trans-l3-Hydroxytetrahydroprotoberberines(e.g., 80), not yet isolated from nature, might be formed from the corresponding (14s)-tetrahydroprotoberberines(e.g., 5) via stereospecific removal of the pro- 13s hydrogen (Scheme 30). The cis- and trans- 13-hydroxytetrahydroprotoberberines would be N-methylated to supply the a-N-metho salts of the alkaloids, such as (14s)-ophiocarpine (14) and (14S)-epiophiocarpine (80), in which the hydrogens at C-13 and C-14 are in a cis and trans relationship, respectively. The a-N-metho salts of the cis- and trans-13hydroxytetrahydroprotoberberines (e.g., 14a and 80a) were bioconverted into 13-oxoprotopines(e.g., 82), which were transformed to spirobenzylisoquinolines (e.g., 83) and benzindanoazepines (e.g., 84) in several Corydalis species. The a-N-metho salts of (14s)-1-methoxy-13-hydroxytetrahydroprotoberberines (e.g., 85a), incorporating the cis quinolizidine system and a cis relationship between the hydrogens at C-13 and C-14, were also biotransformed into benzindanoazepines (e.g., 86) via 1-methoxy-13-oxoprotopines (e.g., 72). The finding that a spirobenzylisoquinoline alkaloid was

5.

BIOTRANSFORMATION OF PROTOBERBERINE ALKALOIDS

345

not obtained from the a-N-metho salts with a methoxyl group at C-1 is probably due to the steric interaction of the C-1 methoxyl with the substituent on ring C. The fact that conversions of the 13-hydroxytetrahydroprotoberberine a-N-metho salts into spirobenzylisoquinoline- or benzindanoazepine-type alkaloids were established in several kinds of cell cultures suggests strongly the generality of these ring rearrangements, including, in some instances, the in vivo migration of the methyl group from N to 0. Oxidation of the cis- and trans-13-hydroxytetrahydroprotoberberines (e.g., 14 and80) to the dehydro derivatives (e.g., W) was not demonstrated, but the reverse reduction process was indeed proved.

Acknowledgment The author is grateful to Professor Maurice Shamma for suggestions made during the preparation of this chapter.

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Perspectives” (S. W. Pelletier, ed.), Vol. 6,p. 297. Wiley, New York, 1988. 2. J. R. Gearad and I. D. Spenser, Can. J . Chem. 41, 783 (1%3). 3. 1. Monkovic and I. D. Spenser, Proc. Chem. Soc. 223 (1964). 4. D.H. R. Barton, R. H . Hesse, and G. W. Kirby, Proc. Chem. Soc. 267 (1%9); J . Chem. SOC.6379 (1965). 5 . A. R. Battersby, R. J. Francis, M. Hirst, and J. Staunton, Proc. Chem. Soc. 268 (l%3). 6. M. J. Muller and M. H. Zenk, Planra Med. 58, 524 (1992). 7. V. Preininger, in “The Alkaloids” (A. Brossi, ed.), Vol. 29,p. 1. Academic Press, New York, 1986. 8. N. Takao, K. Iwasa, M. Kamigauchi, and M. Sugiura, Chem. Pharm. Bull. 24, 2859 ( 1976). 9. A. R. Battersby, J. Staunton, and H. R. Wiltshire, J. Chem. Soc. 1147 (1975). 10. C. Tani and K. Tagahara, Chem. Pharm. Bull. 22, 2457 (1974). 11. A. Yagi, G. Nonaka, S. Nakayama, and I. Nishioka, Phytochemistry 16, 1197 (1977). 12. K. Iwasa and N. Takao, Phytochemisrry 21, 611 (1982). 13. K. Iwasa, M. Kamigauchi, N. Takao, and M. Cushman, J . Nar. Prod. 56, 2053 (1993). 14. M. Kamigauchi, Y. Noda, K. Iwasa, and Z. Nishijo, Helu. Chim. Acra 77,243 (1994). IS. M. Kamigauchi, Y. Noda, K. Iwasa, Z. Nishijo, and N. Takao, Arch. Pharm. (Weinheim, Ger.) 326, 501 (1993). 16. M. Kamigauchi, Y. Noda, K. Iwasa, and Z. Nishijo, Helu. Chim. Acta 77,243 (1994). 17. T. Kametani, in “The Chemistry of the lsoquinoline Alkaloids,” Vol. 2,p. 132. Sendai Inst. of Heterocyclic Chemistry, Sendai, Japan, 1974.

346

KINUKO IWASA

18. K. Iwasa, A. Tomii, N. Takao, T. Ishida, and M. Inoue, J. Chem. Res. Synop. 16 (1985); J . Chem. Res. Miniprint 301 (1985). 19. K. Iwasa, M. Kamigauchi, N. Takao, M. Cushman, J.-K. Chen, W. C. Wong, and A. McKenzie, J . Amer. Chem. Soc. 111,7925 (1989). 20. K. Iwasa, M. Kamigauchi, N. Takao, M. Cushman, W. C. Wong. and J. K. Chen, Tetrahedron Lett. 29, 6457 (1988). 21. The amino aldehyde forms (35b and 36b) of 35 and 36 are reduced to produce cis and trans alcohols (37 and 39), respectively, which are oxidized to give 38. The cis and trans amino alcohols ([1,1-2H2]-37and [ I , I ,2’,2’-2H,]-39)were biotransformed into 38. 22. A. R. Battersby, J. Staunton, and M. C. Summers, J. Chem. Soc. 45 (1979). 23. N. Takao, M. Kamigauchi, and M. Okada, Helu. Chim. Acra 66,473 (1983). 24. N. Takao, M. Kamigauchi, M. Sugiura, I. Ninomiya, 0. Miyata, andT. Naito, Heterocycles 16, 221 (1981). 25. M. Rueffer and M. H. Zenk, Tetrahedron Lett. 27, 5603 (1986). 26. M. Shamma and J. L. Moniot, “Isoquinoline Alkaloids Research: 1972-1977,” p. 287. Plenum, New York, 1978. 27. H. Ishii, Y. Murakami, and T. Ishikawa, Heterocycles 6, 1686 (1977). 28. K. Iwasa, Y. Kondoh, M. Kamigauchi. and N. Takao, Planta Med. 94, 290 (1994). 29. K. Iwasa, Y. Kondoh, and M. Kamigauchi, J. Nar. Prod. Submitted. 30. M. Rueffer, G. Zumstein, and M. H. Zenk, Phytochemistry 29, 3727 (1990). 31. M. Amann, N. Nagakura, and M. H. Zenk, Eur. J . Biochem. 175, 17 (1988). 32. H. L. Holland, P. W. Jeffs, T. M. Capps, and D. B. MacLean, Can. J . Chem. 57, 1588 (1979). 33. M. H. Zenk, in “The Chemistry and Biology of Isoquinoline Alkaloids,” (J. D. Phillipson, M. F. Roberts, and M. H. Zenk, eds.), pp. 240-256. Springer-Verlag, Berlin New York, 1985. 34. E. Galneder, M. Rueffer, G. Wanner, M. Tabata, and M. H. Zenk, Plant Cell Rept. 7, 1 (1988). 35. Y. Yamada and N. Okada, Phytochemistry 24,63 (1985). 36. M. Rueffer and M. H. Zenk, Tetrahedron Lett. 28, 5307 (1987). 37. T. Tanahashi and M. H. Zenk, Phytochemistry 29, 1113 (1990). 38. T. Tanahashi and M. H. Zenk, Tetrahedron Lett. 29, 5625 (1988). 39. M. H. Schumacher and M. H. Zenk, Plant Cell Repr. 7,43 (1988). 40a. W. De-Eknamkul, T. Tanahashi, and M. H. Zenk, Phyrochemistry 31, 2713 (1992). 40b. L. Kammerer, W. De-Eknamkul, and M.H. Zenk, Phytochemistry. 36, 1409 (1994). 41. M. H. Zenk, Pure Appl. Chem. 66, 2023 (1994). 42. H. Ronsch, in “The Alkaloids” (A. Brossi, ed.), Vol. 28, p. 66. Academic Press, New York, 1986. 43. H. R. Ronsch, Eur. J. Biochem. 28, 123 (1972). 44. H. R. Ronsch, Phytochemistry 16, 691 (1977). 45. A. R. Battersby and J. Staunton, Tetrahedron 30, 1707 (1974). 46. C. Tani and K. Tagahara, J. Pharm. SOC.Jpn. 97,93 (1977). 47. P. W. Jeffs and J. D. Scharver, J. Amer. Chem. Soc. 98,4301 (1976). 48. M. Shamma, “The Isoquinoline Alkaloids.” Academic Press, New York, 1972. 49. K. Iwasa, A. Tomii, and N. Takao, Heterocycles 22, 33 (1984). 50. K. Iwasa, M. Kamigauchi, and N. Takao, J. Nar. Prod, 51, 1232 (1988). 51. K. Iwasa, A. Tomii, N. Takao, T. Ishida, and M. Inoue, Heterocycles, 22, 1343 (1984). 52. M. Hanaoka, M. Inoue, K. Nagami. Y. Shimada, and S. Yasuda, Heterocycles 19,313 (1982). 53. K. Iwasa, Unpublished data (1994).

CUMULATIVE INDEX OF TITLES

Aconitum alkaloids, 4, 275 (1954), 7, 473 (1960), 34, 95 (1988) CI9diterpenes, 12, 2 (1970) Cz0diterpenes, 12, 136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, experimental antitumor activity of acronycine, 21, 1 (1983) N-Acyliminium ions as intermediates in alkaloid synthesis, 32, 27 1 (1988) Ajmaline-Sarpagine alkaloids, 8, 789 (1965), 11, 41 (1968) Alkaloid production, plant biotechnology of 40, 1 (1991) Alkaloid structures spectral methods, study, 24, 287 (1985) unknown structure, 5, 301 (1955), 7, 509 (1960), 10, 545 (1967), 12, 455 (1970), W, 397 (1971), 14, 507 (1973), 15, 263 (1975), 16, 511 ( 1977) X-ray diffraction, 22, 51 (1983) Alkaloids forensic chemistry of, 32, 1 (1988) histochemistry of, 39, 1 (1990) in the plant, 1, 15 (1950), 6, l(1960) Alkaloids from Amphibians, 21, 139 (1983), 43, 185 (1993) Ants and insects, 31, 193 (1987) Chinese Traditional Medicinal Plants, 32, 241 (1988) Mammals, 21, 329 (1983), 43, 119 (1993) Marine organisms, 24, 25 (1985), 41, 41 (1992) Mushrooms, 40, 189 (1991) Plants of Thailand, 41, 1 (1992) Allelochemical properties or the raison d’Ctre of alkaloids, 43, 1 (1993) A110 congeners, and tropolonic Colchicum alkaloids, 41, 125 (1992) Alstonia alkaloids, 8, 159 (1965), 12, 207 (1970), 14, 157 (1973) Amaryllidaceae alkaloids, 2, 331 (1952), 6, 289 (1960), 11, 307 (1968), 15, 83 (1975), 30, 251 (1987) Amphibian alkaloids, 21, 139 (1983), 43, 185 (1983) Analgesic alkaloids, 5, 1 (1955) 347

348

CUMULATIVE INDEX OF TITLES

Anesthetics, local, 5, 211 (1955) Anthranilic acid derived alkaloids, 17, 105 (1979), 32, 341 (1988), 39, 63 (1990) Antifungal alkaloids, 42, 117 (1992) Antimalarial alkaloids, 5, 141 (1955) Antitumor alkaloids, 25, 1 (1985) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4, 119 (1954), 9, 1 (1967), 24, 153 (1985) Aristolochia alkaloids, 31, 29 (1987) Aristotelia alkaloids, 24, 113 (1985) Aspergillus alkaloids, 29, 185 (1986) Aspidosperma alkaloids, 8, 336 (1965), 11, 205 (1968), 17, 199 (1979) Azafluoranthene alkaloids, 23, 301 (1984) Bases simple, 3, 313 (1953), 8, 1 (1965) simple indole, 10, 491 (1967) simple isoquinoline, 4, 7 (19541, 21, 255 (1983) Benzodiazepine alkaloids, 39, 63 (1990) Benzophenanthridine alkaloids, 26, 185 (1985) Benzylisoquinoline alkaloids, 4, 29 (1954), 10, 402 (1967) Betalains, 39, 1 (1990) Biosynthesis, isoquinoline alkaloids, 4, 1 (1954) pyrrolizidine alkaloids, 46, 1 (1995) quinolizidine alkaloids, 46, 1 (1995) tropane alkaloids, 44, 116 (1993) Bisbenzylisoquinoline alkaloids, 4, 199 (1954), 7, 429 (1960), 9, 133 (1967), 13, 303 (1971), 16, 249 (1977), 30, 1 (1987) synthesis, 16, 319 (1977) Bisindole alkaloids, 20, 1 (1981) Bisindole alkaloids of Catharanthus, C-20’ position as a functional hot spot in, 37, 133 (1990) isolation, structure elucidation and biosynthesis, 37, 1 (1990) medicinal chemistry of, 37, 145 (1990) pharmacology of, 37, 205 (1990) synthesis of, 37, 77 (1990) therapeutic use of, 37, 229 (1990) Bums alkaloids, steroids, 9, 305 (1967), 14, 1 (1973), 32, 79 (1988) Cactus alkaloids, 4, 23 (1954) Calabar bean alkaloids, 8, 27 (1965), 10, 383 (1967). 13, 213 (1971), 36, 225 (1989) Calabash curare alkaloids, 8, 515 (1965), 11, 189 (1968) Calycanthaceae alkaloids, 8, 581 (1965)

CUMULATIVE INDEX OF TITLES

349

Camptothecine, 21, 101 (1983) Cancentrine alkaloids, 14,407 (1973) Cannabis satiua alkaloids, 34, 77 (1989) Canthin-6-one alkaloids, 36, 135 (1989) Capsicum alkaloids, 23, 227 (1984) Carbazole alkaloids, 13, 273 (1971), 26, I (1985) chemistry and biology of, 44,257 (1993) Carboline alkaloids, 8, 47 (1965), 26, 1 (1985) P-Carboline congeners and Ipecac alkaloids, 22, I (1983) Cardioactive alkaloids, 5, 79 (1955) Celastraceae alkaloids, 16, 215 (1977) Cephalotaxus alkaloids, 23, 157 (1984) Cevane group of Veratrum alkaloids, 41, 177 (1992) Chemotaxonomy of Papaveraceae and Fumaridaceae, 29, 1 (1986) Chinese medicinal plants, alkaloids from, 32, 241 (1988) Chromone alkaloids, 31, 67 (1987) Cinchona alkaloids, 3, 1 (1953), 14, 181 (1973), 34, 332 (1989) Colchicine, 2, 261 (1952), 6, 247 (1960), 11, 407 (19681, 23, 1 (1984) Colchicum alkaloids and allo congeners, 41, 125 (1992) Configuration and conformation, elucidation by X-ray diffraction, 22, 51 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4, 249 (19541, 10, 463 (1967), 29, 287 (1986) Curare-like effects, 5, 259 (1955) Cyclic Tautomers of Tryptamine and Tryptophan, 34, 1 (1989) Cyclopeptide alkaloids, 15, 165 (1975) Daphniphyllum alkaloids, 15, 41 (l975), 29, 265 (1986) Delphinium alkaloids, 4, 275 (1954), 7, 473 (1960) C,,-diterpenes, 12, 2 (1970) C,,-diterpenes, 12, 136 (1970) Dibenzazonine alkaloids, 35, 177 (1989) Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8, 336 (1965) Diterpenoid alkaloids Aconitum, 7, 473 (1960), 12, 2 (1970), 12, 136 (19701, 34, 95 ( 1989) Delphinium, 7 , 473 (1960), 12, 2 (1970), 12, 136 (1970) Garrya, 7 , 473 (1960), 12, 2 (19601, 12, 136 (1970) chemistry, 18, 99 (1981), 42, 151 (1992) general introduction, 12, xv (1970) structure, 17, I (1970) synthesis, 17, 1 (1979)

350

CUMULATIVE INDEX OF TITLES

Eburnamine-vincaminealkaloids, 8, 250 (1963,11, 125 (1968),20, 297 (1981),42, 1 (1992) Elaeocarpus alkaloids, 6, 325 (1960) Ellipticine and related alkaloids, 39, 239 (1990) Enamide cyclizations in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in uito, 18, 323

(1981)

Ephedra alkaloids, 3, 339 (1953) Epibatidine, 46,95 (1995) Ergot alkaloids, 8, 726 (1963,15, 1 (1975),39, 329 (1990) Erythrina alkaloids, 2 , 499 (1952),7, 201 (1960),9, 483 (19671,18, 1

(1981)

Erythrophleum alkaloids, 4, 265 (19541,10, 287 (1967) Eupomatia alkaloids, 24, 1 (1985) Forensic chemistry, alkaloids, 12, 514 (1970) by chromatographic methods, 32,1 (1988) Galbulimima alkaloids, 9, 529 (l967),13, 227 (1971) Gardneria alkaloids, 36, I (1989) Garrya alkaloids, 7 , 473 (1960),12, 2 (1970),12, 136 (1970) Geissospermum alkaloids, 8, 679 (1965) Gelsemium alkaloids, 8, 93 (19651,33, 84 (1988) Glycosides, monoterpene alkaloids, 17, 545 (1979) Guatteria alkaloids, 35, 1 (1989) Haplophyton cimicidum alkaloids, 8, 673 (1965) Hasubanan alkaloids, 16, 393 (1977),33, 307 (1988) Histochemistry of alkaloids, 39, 165 (1990) Holarrhena group, steroid alkaloids, 7 , 319 (1960) Hunteria alkaloids, 8, 250 (1965) lbuga alkaloids, 8, 203 (1965),11, 79 (1968) Imidazole alkaloids, 3, 201 (1953),22, 281 (1983) Indole alkaloids, 2, 369 (19521,7, 1 (1960),26, 1 (1985) distribution in plants, 11, 1 (1968) simple, 10, 491 (1967),26, 1 (1985) Reissert synthesis of, 31, 1 (1987) Indolizidine alkaloids, 28, 183 (1986),44,189 (1993) In uitro and microbial enzymatic transformation of alkaloids, 18, 323

(1981) 2,2’-Indolylquinuclidinealkaloids, chemistry, 8, 238 (1969,11, 73 ( I 968) Ipecac alkaloids, 3, 363 (1953),7, 419 (1960),13, 189 (1971),22, 1 ( 1983)

CUMULATIVE INDEX OF TITLES

35 1

Isolation of alkaloids, 1, 1 (1950) Isoquinoline alkaloids, 7, 423 (1960) biosynthesis, 4, 1 (1954) 13C-NMRspectra, 18, 217 (1981) simple isoquinoline alkaloids, 4, 7 (1954), 21, 255 (1983) Reissert synthesis of, 31, 1 (1987) Isoquinolinequinones, from Actinomycetes and sponges, 21, 55 (1983) Khat (Catha edulis) alkaloids, 39, 139 (1990) Kopsiu alkaloids, 8, 336 (1965) Lead tetraacetate oxidation in alkaloid synthesis, 36,70 (1989) Local anesthetics, 5, 21 1 (1955) Localization in the plant, 1, 15 (1950), 6, I (1960) Lupine alkaloids, 3, 119 (1953), 7, 253 (1960), 9, 175 (1967), 31, 16 ( 1987) Lycopodium alkaloids, 5, 265 (1955), 7, 505 (1960), 10, 306 (1967), 14, 347 (1973), 26, 241 (1985), 45, 233 (1994) Lythraceae alkaloids, 18, 263 (1981), 35, 155 (1989) Mammalian alkaloids, 21, 329 (1983), 43, 119 (1993) Marine alkaloids, 24, 25 (1985), 41, 41 (1992) Maytansinoids, 23, 71 (1984) Melanins, 36, 254 (1989) Melodinus alkaloids, 11, 205 (1968) Mesembrine alkaloids, 9,467 (1967) Metabolic transformation of alkaloids, 27, 323 (1986) Microbial and in uitro enzymatic transformation of alkaloids, 18, 323 (1981) Mitragyna alkaloids, 8, 59 (1965), 10, 521 (1967), 14, 123 (1973) Monoterpene alkaloids, 16, 43 1 (1977) glycosides, 17, 545 (1979) Morphine alkaloids, 2, I (part I, 1952), 2, 161 (part 2, 1952), 6, 219 (1960), 13, I (1971), 45, 127 (1994) Muscarine alkaloids, 23, 327 (1984) Mushrooms, alkaloids from, 40, 190 (1991) Mydriatic alkaloids, 5, 243 (1955) a-Naphthophenanthridine alkaloids, 4, 253 (l954), 10, 485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (l986), 46, 127 (1995) Narcotics, 5, 1 (1955) Nuphar alkaloids, 9, 441 (1967), 16, 181 (1977), 35, 215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11, 205 (1968) Ourouparia alkaloids, 8, 59 (19651, 10, 521 (1967) Oxaporphine alkaloids, 14, 225 (1973)

352

CUMULATIVE INDEX OF TITLES

Oxazole alkaloids, 35, 259 (1989) Oxindole alkaloids, 14, 83 (1973) Papaveraceae alkaloids, 19, 467 (1967), 12, 333 (1970), 17, 385 (1979) pharmacology, 15, 207 (1975) toxicology, 15, 207 (1975) Pauridianrha alkaloids, 30, 223 (1987) Pavine and isopavine alkaloids, 31, 317 (1987) Penraceras alkaloids, 8, 250 (1965) Peptide alkaloids, 26, 299 (1985) Phenanthrene alkaloids, 39, 99 (1990) Phenanthroindolizidine alkaloids, 19, 193 (1981) Phenanthroquinolizidine alkaloids, 19, 193 (1981) P-Phenethylamines, 3, 313 (1953), 35, 77 (1989) Phenethylisoquinolinealkaloids, 14, 265 (1973), 36, 172 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7, 433 (1960), 9, 117 (1967), 24, 253 (1985) Picralima alkaloids, 8, 119 (1965), 10, 501 (1967), 14, 157 (1973) Piperidine alkaloids, 26, 89 (1985) Plant biotechnology, for alkaloid production, 40, 1 (1991) Plant systematics, 16, 1 (1977) Pleiocarpa alkaloids, 8, 336 (1965), 11, 205 (1968) Polyamine alkaloids, 22, 85 (1983) Polyamine toxins, 45, 1 (1994), 46,63 (1995) Pressor alkaloids, 5, 229 (1955) Protoberberine alkaloids, 4, 77 (1954), 9, 41 (1967), 28, 95 (1986) biotransformation of, 46,273 (1995) transformation reactions of, 33, 141 (1988) Protopine alkaloids, 4, 147 (1954), 34, 181 (1989) Pseudocinchoma alkaloids, 8, 694 (1965) Purine alkaloids, 38, 226 (1990) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960), 11, 459 (1968), 26, 89 ( 1985) Pyrrolidine alkaloids, 1, 91 (1950), 6, 31 (1960), 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6, 35 (1960), 12, 246 (1970), 26, 327 (1985) biosynthesis of, 46, I (1995) Quinazolidine alkaloids, see Indolizidine Alkaloids Quinazoline alkaloids, 3, 101 (1953), 7, 247 (1960), 29, 99 (1986) Quinazolinocarbolines, 8, 55 (1963, 21, 29 (1983) Quinoline alkaloids related to anthranilic acid, 3, 65 (19531, 7, 229 (1960), 17, 105 (1979), 32, 341 (1988)

CUMULATIVE INDEX OF TITLES

353

Quinolizidine alkaloids, 28, 183 (1985) biosynthesis of, 46, 1 (1995) Rauwolfia alkaloids, 8, 287 (1965) Reissert synthesis of isoquinoline and indole alkaloids, 31, 1 (1987) Reserpine, chemistry, 8, 287 (1965) Respiratory stimulants, 5, 109 (1955) Rhoeadine alkaloids, 28, 1 (1986) Salamandra group, steroids, 9,427 (1967) Sceletium alkaloids, 19, 1 (1981) Secoisoquinoline alkaloids, 33, 23 1 (1988) Securinega alkaloids, 14, 425 (1973) Senecio alkaloids, see Pyrrolizidine alkaloids Simple indole alkaloids, 10, 491 (1967) Simple indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) Sinomenine, 2, 219 (1952) Solanum alkaloids chemistry, 3, 247 (1953) steroids, 7, 343 (1960), 10, 1 (1967), 19, 81 (1981) Sources of alkaloids, 1, I (1950) Spectral methods, alkaloid structures, 24, 287 (1985) Spermidine and related polyamine alkaloids, 22, 85 (1983) Spermine and related polyamine alkaloids, 22, 85 (1983) Spider toxin alkaloids, 45, 1 (1994), 46,63 (1995) Spirobenzylisoquinoline alkaloids, 13, 165 (1971), 38, 157 (1990) Sponges, isoquinolinequinone alkaloids from, 21, 55 (1983) Sremona alkaloids, 9, 545 (1967) Steroid alkaloids Apocynaceae, 9, 305 (1967), 32, 79 (1988) Bums group, 9, 305 (1967), 14, 1 (l973), 32, 79 (1988) Holarrhena group, 7 , 3 I9 (1960) Salamandra group, 9, 427 (1967) Solanum group, 7 , 343 (19601, 10, 1 (1967), 19, 81 (1981) Veratrum group, 7 , 363 (1960), 10, 193 (1967), 14, I (1973), 41, 177 ( 1992) Stimulants respiratory, 5, 109 (1955) uterine, 5, 163 (1955) Structure elucidation, by X-ray diffraction, 22, 5 I (1983) Strychnos alkaloids, 1, 375 (part I, 1950), 2, 513 (part 2, 1952), 6, 179 (1960), 8, 515, 592 (1965), 11, 189 (1968), 34,211 (1989), 36, 1 ( 1989) Sulfur-containing alkaloids, 26, 53 (1989, 42, 249 (1992)

354

CUMULATIVE INDEX OF TITLES

Synthesis of alkaloids, Enamide cyclizations for, 22, 189 (1983) Lead tetraacetate oxidation in, 36, 70 (1989) Tubernuernontuna alkaloids, 27, 1 (1983) T a u s alkaloids, 10, 597 (1967), 39, 195 (1990) Thailand, alkaloids from the plants of, 41, 1 (1992) Toxicology, Papaveraceae alkaloids, 15, 207 (1975) Transformation of alkaloids, enzymatic microbial and in uirro, 18, 323 (1981) Tropane alkaloids biosynthesis of, 44, 115 (1993) chemistry, 1, 271 (19501, 6, 145 (1960), 9, 269 (1967), 13, 351 (1971), 16, 83 (1977), 33, 2 (1988), 44, 1 (1993) Tropoloisoquinoline alkaloids, 23, 301 (1984) Tropolonic Colchicum alkaloids, 23, 1 (1984), 41, 125 (1992) Tylophoru alkaloids, 9, 517 (1967) Uterine stimulants, 5, 163 (1955) Verutrurn alkaloids cevane group of, 41, 177 (1992) chemistry, 3, 247 (1952) steroids, 7 , 363 (1960), 10, 193 (1967), 14, 1 (1973) Vincu alkaloids, 8, 272 (1965), 11, 99 (1968), 20, 297 (1981) Voucungu alkaloids, 8, 203 (1965), 11, 79 (1968) Wasp toxin alkaloids, 45, 1 (19!24), 46, 63 (1995) X-ray diffraction of alkaloids, 22, 51 (1983) Yohimbe alkaloids, 8, 694 (1965), 11, 145 (1968), 27, 131 (1986)

INDEX

N-Acetylepibatidine, 115-1 16 7-Acetylepibatidine, racemic, 119-120 Agel-505, concentration-dependent inhibition of NMDNglycine-induced increase, 77, 79 Agel-489 NMDA selectivity, 77-78, 82 noncompetitive antagonism of NMDAinduced increases, 77, 81 Agelenopsis aperta, see Agel-489; Agel505

Amino acids, dioncophylline A oxidative degradation, 142-144 AMPNKA, Ca2'-permeable, 84 Anagyrine, 50, 53 cadaverine dihydrochloride incorporation, 50, 52 Ancistrine, 249 Ancistrobarterine A, 167-168, 225 Ancistrobrevine A, 231 Ancistrobrevine B, 164-165, 236 Ancistrobrevine C , 161-162, 225 Ancistrobrevine D, 162-163, 229 Ancistrocladaceae alkaloids, 155, 249-253 African species, 158-170 new alkaloids, 255-258 structures of alkaloids, 166 Asian species, 156 naphthalene derivatives, 205 naphthylisoquinoline alkaloids from, 2 16-237 plants, 206-207 Ancistrocladeine, 25 1 Ancistrocladidine, 230 Ancistrocladine, 158-159, 232 atropisomer-specific synthesis, 189 structure, 129 system, 194 total synthesis, 188 (+ bAncistrocladine, 250 355

Ancistrocladinine, 234 Ancistrocladisine, 229 total synthesis, 191-192 Ancistrocladonine, 252 Ancistrocladus abbreviatus, 158-167 alkaloids with unusual coupling types, 163-165 ancistrobrevine C, 161- 162 ancistrobrevine D, 162-163 ancistrocladine, 158- I59 atropisorneric dioncolines A, 162 chemotaxonomic position, 164-167 dioncophylline A. 159-161 hamatine, 158-159 Ancistrocladus alkaloids, joint structural properties, 157 Ancisfrocladus barter;, 165, 167- 169 chemotaxonomic conclusions, 167-168 N-methylphylline, 168-169 Ancistrocladus heyneanus, 155-157 plant cultivation, 207-208 Ancistrocladus korupensis, 170, 180 michellamines, 238-240 new alkaloids, 255-257 Ancistrocladus roberrsoniorum, 169- 170 new alkaloids, 258 Ancistrocladus rectorius, 157-158 Ancistrocline, 157, 236 Ancistrocongine, 253 Ancistrocongolensine, 253 Ancistroelaensine, 25 1 Ancistroquinone, 24 1 Ancistrotectorine, 230 Angustifoline, 48-49 (+)-Angustifoline, 46-47 Antimalarial properties, naphthylisoquinoline alkaloids, 2 12-2 13 Antiviral activities, naphthylisoquinoline alkaloids, 2 13-2 14

356

INDEX

Arg-636 effects on NMDA receptors, 84 locust muscle blocking, 88 Arginine. necine biosynthesis from, 5-7 Argiotoxine-636, see Arg-636 7-Azabicyclo[2,2, I lheptan-2-one, condensation, 1 1 1-1 13

Benzindanoazepine formation, 340-341 13-hydrox ytetrahydroprotoberberine conversion, 335-343 Benzophenanthridines aromatized, 281 Macleaya cordata, feeding experiments with, 304-305 biosynthesis from protoberberines, 328 studies at enzymic level, 317-328 from tetrahydroprotoberberines, 300-301, 304 formation, 288. 290 from protoberberines, 314-316 incorporation of 13-methylprotopines, 291-292 tetrahydroprotoberbine or 13-methyltetrah ydroprotoberberine

conversion, 277-305 Berberine feeding to Corydalis pallida, 310 formation, from L-tyrosine, 274-275 Biaryls, lactone-bridged, naphthylisoquinoline alkaloids synthesis, 189-193 5.5-Bidioncophylline A, synthesis, 259-260

Cadaverine, 36-37 [ 1 ,5-'4CzlCadaverine, incorporation into lupinine, 36, 38 I-zH]Cadaverine (R)- and (S)-[ dihydrochloride, incorporation, 40-4 I (-)-anagyrine and (+)-sparteine, 50, 52 quinolizidine alkaloids, 48-49 (+)-sparteine and (- )-N-methylcytisine, 50-5 1

[2,2,4,4-ZH,]Cadaverine dihydrochloride, incorporation into ( + )-sparteine and (-)-N-methylcytisine, 53, 55 [3.3-ZH2]Cadaverinedihydrochloride incorporation into quinolizidine alkaloids, 47 synthesis, 53-55 ['3C,'SN]Cadaverine dihydrochloride incorporation into quinolizidine alkaloids, 46-47 synthesis, 39-40 Canadine, Macleaya cordata, feeding experiments with, 302-303 Central nervous system, mammals, glutamate receptor subtypes, 72-73 Chelidonine, formation, 277, 282 Chelidonium majus, feeding experiments on, 277, 282 Circular dichroism spectroscopy dioncophylline A, 136-139 michellamines, 175-177 naphthylisoquinoline alkaloids, 175-1 77 6-Chloronicotinaldehyde, epibatidine synthesis, 104-106 6-Chloropyridine-3-carboxaldehyde, epibatidine synthesis, 106-107 Coptis, (S)-canadhe oxidase, substrate specificity, 319, 321 Corycavine, formation, 283-284 Corydaline, feeding to Corydalis pallida, 31 1 Corydalis, callus tissue studies, 285-301 6-hydroxytetrahydroprotoberberine Nmetho salts, 292-294 comparison with 14hydroxytetrahydroprotoberberine N-metho salts, 293, 295 intermediates, 295-299 a-N-methosalts, 285-288 cis- 13-methyltetrahydroprotoberberine a-N-methosalts, 288-290 tissue-cultured cells and intact plants, 288, 290-291 unlabeled (R,S)-corynoline, 299 Corydalis incisa, feeding experiments, 282-284, 31 1 Corydalis ochotensis, feeding experiments, 342-344 Corydalis ophiocarpa, feeding experiments, 333-334, 336-342

INDEX

Corydalis pallida, feeding experiments, 308, 310-311 Corynoline biosynthetic conversions from, 299 formation, 283-284, 298 Crystal-structure analysis, dioncophylline A, 144 Cuscuta species, interaction with naphthylisoquinoline alkaloids, 215-216 Cyclization, biomimetic reactions, naphthylisoquinoline alkaloids, 203-204 Cyclohexan- 1.2-dione. epibatidine synthesis, 109 Cynaustine, 19 Cynaustraline, 19 Cytisine, 36-37

357

Dihydroisoquinolines, preparation, 182-183 6,14-Dihydroxytetrahydroprotoberberine N-metho salt, 295 I ,3-Dimethyl tetrahydroisoquinoline alkaloids, naphthalene-free, 204 Dioncolactone A, 146-148, 154, 221 Dioncoline A, 162, 228 Dioncoline A, 7-epi-, 228 Dioncopeltine A, 154. 221 Dioncophyllaceae alkaloids from, 244-248 naphthalene derivatives, 205 naphthylisoquinoline alkaloids from, 2 16-237 plants, 207 Dioncophyllaceae alkaloids, 153-155 (2)-Dioncophyllacine, 222 (k )-Dioncophyllacine A, 151- 154 Dioncophyllacine B, 151-154, 223 Degradation, oxidative, dioncophylline A Dioncophylline A, 159-161, 217 to amino acids, 142-144 atropisomer-specific NOE interactions, Dehydrogenation, 142-143 dihydrobenzophenanthridines, CD spectroscopy, 136-139 aromatized benzophenanthridines, chemical bridge, 141-142 322, 324, 327 crystal-structure analysis, 144 6-0-DemethylancistrobrevineA, 23 1 diastereodivergent synthesis, 190-191 4‘-O-Demethyldioncophylline A, 220 isolation, 132 4‘-O-Demethyl-7-epi-dioncophylline A, 220 long-range NMR interactions, 141-143 5’-O-Demethyl-8-O-methyldioncophylline NMR spectroscopy, 133-134 A, 154, 219 oxidative degradation to amino acids, 5’-O-Demethyl-8-O-methyl-7-epi142-144 dioncophyllidine A, structure, 154 stereostructure, 261 5 ‘-0-Demethyl-8-O-methyl-7-epistructural elucidation methods, dioncophylline A, 219, 222 summary, 144-146 structure, 154 structure, 140-141, 154 I .4-Diaminobutane, see Putrescine Dioncophylline A, 7-epi-, 217 Dicrotalic acid, 34-35 Dioncophylline B, 147-151, 154, 223 Dicrotaline, 34-35 Dioncophylline C, 147-151, 154, 224 Diels-Alder reactions, epibatidine, 96- 102 Dioncophylline D, 151, 154, 224 Clayton and Regan, 99-101 Dioncophyllum tholloni, 152 Herman et al., 101-102 6,8-Dioxygenated 1,3-dimethyl-l,2,3,4Huang and Shen, 98-99 tetrahydroisoquinoline, methyl ethers, Scheeren et al., 100-102 18 1-182 Dihydrobenzophenanthridines Droserone, 169-170, 242 dehydrogenation, aromatized benzophenanthridines, 322, 324, 327 hydroxylation followed by methylation, Echinatine, 3-4, 19 325, 327 Ecology, polyamine toxins, 63-67

358

INDEX

Emiline, 22 Epibatidine, 95- I24 analgesic potencies, 120-122 comparison of activity with morphine, 119-120 induced antinociception, antagonism, 122- I23 NMR spectroscopy, 116-1 19 occurrence, 96 pharmacology, 119-123 structure and synthesis. 96-1 16 Diels-Alder reactions, %-I02 intramolecular nucleophilic substitution reactions, 101, 103-1 I6 structure comparison with nicotine, 12 I- 122 syntheses, 123 Epiophiocarpine (I-and P-N-metho salts, feeding studies, 336, 338 Ethers, cyclic, naphthylisoquinoline alkaloids synthesis, 187-189

Fungicidal activities, naphthylisoquinoline alkaloids, 212

Gentrymine B, 256-257 Glutamate, response antagonism, polyamine toxins effect, 70, 72 Glutamate receptor, subtypes in mammalian CNS, 72-73 Glutamatergic transmission. synthetic PhTX analog effect, 87-88

Hamatine, 233 structure, 129 total synthesis, 188 Heliosupine, esterifying acids, biosynthesis, 31-32 Heliotropium spathulatum, pulsed labeling, 20 Herbivores, activities against, naphthylisoquinoline alkaloids, 2 15

Homospermidine, 12-13 conversion into trachelanthamidine, 17-18 [ 1 ,9-'3C,]Homospermidine trihydrochloride, preparation, 14-16 [ 1 ,9-'4C,]Homospermidine trihydrochloride, preparation, 13- 14 [4,6-'4Cz]H~m~~permidine trihydrochloride, preparation, 13, 15 Hydrobenzophenanthridine alkaloids, 28 I Hydrocultures, Ancistrocladus, 208 10-Hydroxydihydrosanguinarine- 10-0methyltransferase. substrate specificity, 325, 327 H ydroxylation activity in different cell cultures, protoberberine alkaloids, 321, 324 protopines, dihydrobenzophenanthridine generation, 322, 325-326 (+)-13-Hydroxylupanine, 46-47 3-Hydroxy-3-methylglutaric acid, 34-35 I-Hydroxymethylpyrrolizidines,2 necine biosynthesis involving, 20-23 13-Hydroxyprotoberberines, conversion between tertiary and quaternary, 343-343 6-H ydroxytetrahydroprotoberberine, feeding experiments, 292-294 13-H ydroxytetrahydroprotoberberine biogenetic pathway, 344-345 conversion into spirobenzylisoquinolines or benzindanoazepines, 335-343 Corydalis ochotensis feeding studies, 342 Corydalis ophiocarpa feeding studies, 336-342 studies with other Corydalis species, 342-343 formation, in whole plants, 333-335 N-metho salts, administration, 338

Iminium ion, necine biosynthesis involving, 18-19 Indicine N-oxide, 4-5 Invertebrates, polyamine toxins, pharmacological effects, 67-72 Isoancistrocladine, 157, 233

INDEX

Isoquinoline, chiral moieties, regio- and stereoselective synthesis, 181-185 Isoquinoline alkaloids, 243 acetogenic, 20 1-202 Pictet-Spengler route, 200-201 (+)-Isoretronecanol, 19 ( 2)-[5-3H]Isoretronecanol,20-2 1 Isoshinanolone, 242 Isotriphyophylline, 245 atropodiastereomers, 132 structure, 131

Joro spider toxin competitive antagonism of non-NMDA receptors, 74 effect on synaptic response, 73-74 NMDA receptor antagonism, 79, 83 properties, 68 JSTX-3, synthetic analogs, 87

Korupensamines, 178-179, 194-196 Korupensamine A, 197-198, 226 Korupensamine B, 227 Korupensamine C. 227 Korupensamine D,237

Liquid chromatography/atmosphericpressure chemical-ionization mass spectrometry, protoberberine alkaloids, 307-309 Long-term potentiation, polyamine toxins effects, 85-86 (+)-Lupanine, 43-44, 46 Lupanine, 48-49 Lupinine biosynthesis, 36-40 enzymic process stereochemistry. 40-43 via I-piperideine, 42-43 structure, 2 Lysine, structure, 2 DL-[4,5-'3C2]Lysine,incorporation into lupinine, 37-38

359

Macarpine, biosynthetic route, 324, 327 Mucleaya corduta, feeding experiments

with, 301-305 allocryptopine, 302, 304 a- and /3-N-metho salts of tetrahydroprotoberberines, 302 aromatized benzophenanthridines, 304-305 biosynthetic routes from tetrahydroprotoberberines to benzophenanthridines, 304 canadine, 302-303 protopines, 302, 304 Mammals, central nervous system, glutamate receptor subtypes, 72-73 Matrine, 36-37, 43-44 Mesocorydaline, feeding to Corydulis pallida, 3 11 Mesothalictricavine, feeding to Corydulis pallida, 3 I 1 I -Methoxyepiophiocarpine a-N-metho salt, feeding studies, 341-343 (-)-0-Methylancistrocladine, 235 N-Methylation, tetrahydroprotoberberines, to a-N-metho salts, 317, 320-321 13-Methylberberine, feeding to Corydulis pallida, 31 1 N-Methylcytisine, cadaverine dihydrochloride incorporation, 50-5 I , 53, 55 (-)-N-Methylcytisine, 36-38 @Methyl- 1,2-didehydrotriphyophylline, 131, 246 N-Methyldioncophylline A, 165, 168, 218 N-Methyl-7-epi-dioncophylline A, 2 18 P-Methylenenorvaline, 33 7-Methylepibatidine, racemic, 119-120 (+ )-0-Methylhamatine, 235 I3-Methylpalmative, feeding to Corydulis pullida, 3 1 I N-Methylphylline, 168-169, 243 13-Methylprotoberberinium salts, feeding to Corydalis incisa, 313-314 13-Methylprotopines formation, 287 incorporation into benzophenanthridines, 291-292 0-Methyltetradehydrotriphyophylline, 246 structure, 131

360

lNDEX

trans- 13-Methyltetrah ydroprotoberberine alkaloids, a-N-metho salts, 285-286

13-Methyltetrahydroprotoberberines conversion into benzophenanthridines, 277-305 feeding to Corydalis incisa, 313-314 redox interconversions between, 306 5’-O-MethyltriphyopeItine, 248 structure, 131 N-Methyltriphyophylline, 131, 247 0-Methyltriphyophylline, 131,245 Michellamines, 170-180,238-240 antiviral properties, 214 base-catalyzed interconversion, 177-180 biomimetically oriented synthesis, 258-259 CD spectroscopy, 175-177 chemotaxonomic position, 180 constitution and relative configuration, 170-172 degradation experiments, 173-176 korupensamines, 178-179 stereochemistry, 173-174 total synthesis, 196-200 biomimetic oxidative coupling of korupensamine A, 197-198 nonbiomimetic approach, 198-200 Microorganisms, biological activities against, naphthylisoquinoline alkaloids, 21 1-215 Monocrotalic acid, biosynthesis, 34 Monocrotaline, 3-5 Morphine, comparison of activity with epibatidine, 119-120 Muramine, ‘‘C-labeled, feeding, 329-330

Naloxone, effect on epibatidine analgesic effect, 121-122 Naphthalene, moieties, preparation,

184-185

Naphthylisoquinoline alkaloids, 127-261. see also Ancistrocladaceae alkaloids, African species; Triphyophyllum peltatum from Ancistrocladaceae and Dioncoph yllaceae, 155,216-237 Ancistrocladus hamatus, 156-157

Ancistrocladus heyneanus, 155-157 Ancistrocladus tectorius, 157-158 biogenetic origin, 200-211 acetogenic isoquinoline alkaloids,

201-202 biomimetic cyclization reactions,

203-204 biosynthetic experiments, 208-211 cultivation of plants, 207-208 isolation of biogenetic precursors or modified analogs, 204-206 plants, 206-207 biological activities against microorganisms, 21 1-215 CD spectroscopy, 175-177 chemo-ecological context, 21 1-216 determination of constitution, 132-133 dimeric constitution and relative configuration,

170-172

synthesis, 258-260 Dioncophyllum tholloni, 152 directed preparation of axis by intramolecular coupling, 186-194 atropisomer-selective synthesis via lactone-bridged biaryls,

189-I93 bridge concept, 186-187 stereocontrolled total syntheses,

193-194 synthesis via cyclic ethers,

187-189

herbivore antifeedant activity, 215 interaction with herbal parasites,

215-216

korupensamines, synthesis, 194-196 michellamines, 170-180 total synthesis, 196-200 molecular halves, 241-243 new, 255-258 oxidation reaction, 192 partial syntheses, 181 plants, 206-207 cultivation, 207-208 feeding experiments with acetate and malonate, 209-210 isotope-labeling synthesis, 209-210 monocyclic precursor incorporation,

209-21 1

reported in early literature, 244-253

INDEX

stereoanalysis by ruthenium-mediated oxidative degradation, 142-143 structures, 131 total synthesis, 181-185 chiral isoquinoline moieties, 181-185 first, 131-132 naphthalene moiety preparation, 185 triphylline, isolation, 132 Necic acids, biosynthesis, 31-36 Necines, biosynthesis enzymic process stereochemistry, 23, 25-3 1 involving iminium ions, 18-19 from putrescine precursors containing stable isotopes, 7-13 from radioactive ornithine, arginine, and putrescine, 5-7 Nicotine, structure comparison with epibatidine, 12 I- 122 NMDNglycine, effects on cytosolic Ca2+ responses, 77-80 NMDA receptor, 72-73 Arg-636 effects, 84 NMDA receptor-ionphore complex, activation, 77 Non-NMDA receptors, antagonism by JSTX, 74 Nuclear magnetic resonance spectroscopy dioncophylline A, 133-134 long-range interactions, 141- I43 epibatidine, 116-1 19 Nucleophilic substitution reactions, intramolecular, epibatidine, 101, 103-1 I6 Broka, 104-106 Corey er a / . , 106-108 Daly er a / . , 109-1 1 I Fletcher er a/., 1 11-1 16 Sz h t a y er a / . , 115-1 16

Ophiocarpine, formation, 333-334 Ornithine necine biosynthesis from, 5-7 structure, 2 Otonecine, 22

36 1

Oxidative coupling, biomimetic, korupensamine A, 197-198 13-Oxoallocryptopine, feeding studies, 337, 342-343 13-Oxoprotopine-type alkaloid, administration, 337-339 17-Oxosparteine, 48

Palmatine, feeding to Corydalis pallida, 310-311 Papauer bractearurn, feeding experiments, 329 Papauer rhoeas, feeding experiments, 330-332 Parasites, herbal, interaction with naphthylisoquinoline alkaloids, 215-216 Phenylalanine, structure, 2 Philanthotoxin-343, synthetic analogs, effect on glutamatergic transmission, 87-88 PhTX-343, see also Joro spider toxin analogs, 87 photolabile analogs, 88-89 Piperideine, in lupinine biosynthesis, 42-43 Plumbagin, 208-209, 241 Polyamine toxins, 63-90 ecological aspects, 63-67 low-molecular-weight, 69 pharmacological effects in invertebrates, 67-72 background, 67-69 site and mechanism of action, 67-71 in vertebrates, 72-86 antagonism of EAA receptormediated synaptic transmission, 79, 83 comparative inhibitory potencies, 78, 82 glutamate receptor subtypes in CNS, 72-73 patch-clamp electrophysiological studies, 79-80 radiologand binding studies, 80-82 site and mechanism of action, 73-85 sources, 66

362

INDEX

structure, 65 activity relationship studies, 86-90 Protoberberine alkaloids, 273-345 benzophenanthridines from, 3 14-3 16, 328 biosynthesis routes, 276 studies, enzymic level, 317-328 from L-tyrosine, 274-275 tertiary and quaternary, interconversions between, 305-315 Corydalis incisa feeding experiments, 311-314 Corydalis pallida feeding experiments, 308-3 I I LCI/APCI-MS, 307-309 Protoberberinium salts, 280 feeding to Corydalis incisa, 3 1 I . 3 I3 tetrahydroprotoberine dehydrogenation to, 317-328 Protopines, 280 biosynthetic studies, 283-284 enzymic level, 317-328 h ydroxylation, dihydrobenzophenanthridine generation, 322, 325-326 Putrescine biosynthesis, 6-7 necine biosynthesis from, 5-7 precursors containing stable isotopes, necine biosynthesis from, 7- 13 [ I-13Cz]Putrescinedihydrochloride, preparation, 9 [ 1 ,2-13Cz]Putrescinedihydrochloride, preparation, 10-1 1 [ 1 ,4-13Cz]Putrescinedihydrochloride, preparation, 8 [2,3-”Cz]Putrescine dihydrochloride, preparation, 9-10 [‘3C.15N]Putrescinedihydrochloride, preparation, 11-12 (R)- and (S)-[ I-zH]Putrescine dihydrochloride, incorporation into rorsine and rosmarinine, 26-28 (R)-and (S)-[2-ZH]Putrescine dihydrochloride incorporation into retrorsine and rosmarine, 28-30 preparation, 28-29

[ I , I ,4,4-zH4]Putrescinedihydrochloride, preparation, 25-26 [2,2,3,3-2H4]Putrescinedihydrochloride, preparation, 23, 25 3-Pyridyl-2-cyclohexa-I ,3-diene, in epibatidine synthesis, 109-1 10 Pyrrolizidine alkaloids, 3-36 biological activity, 4-6 lupinine, biosynthesis, enzymic process stereochemistry, 40-43 necines, biosynthesis enzymic process stereochemistry, 23, 25-31 from homospermidine, 13-18 involving I-hydroxymethylpyrrolizidines, 20-23 involving iminium ions, 18-19 from putrescine precursors containing stable isotopes, 7-13 from radioactive ornithine, arginine, and putrescine, 5-7 summary, 22-24 structures, 3-4

Quinolizidine alkaloids, 36-55 lupinine, biosynthesis, 36-40 structures and biological activity, 36-37 tetracyclic, biosynthesis, 43-47 enzymic process stereochemistry, 47-55

Retronecine biosynthesis from homospermidine. 13-16 putrescine precursors, 8-12 degradation, 5 Retronecine diester. alkaloid containing otonecine from, 22-23 Retrorsine, 3-4 biosynthesis, 6 ( R ) and (S)-[2-ZH]putrescine dihydrochloride incorporation, 28-30 Rhoeadines biogenetic sequence, 330-33 I

INDEX

biosynthetic scheme, 332-333 tetrahydroprotoberberine conversion into, 329-333 Riddelline, 20, 23 Rorsine, (R)and (S)-[l-*H]putrescine dihydrochloride incorporation, 26-28 Rosmarinecine. 12-13 Rosmarinine, 3-4, 12 (R)and (S)-[ I-2H]putrescine dihydrochloride incorporation, 26-28

Sanguinarine, formation, 277, 282 Scoulerine, feeding experiments, 330-332 Senecic acid, biosynthesis, 32-33 Senecionine, biosynthesis, 6 Senkirkine, 22 Sparteine, 48-49 cadaverine dihydrochloride incorporation, 50-53, 55 (-)-Sparteine, 36-37.43-44 biosynthesis, from I-piperideine trimer,

44-45 Spectroscopy, see Circular dichroism spectroscopy; Nuclear magnetic resonance spectroscopy Spermidine, 6-7 Spermine. 6-7 Spiders, see also Joro spider toxin; Polyamine toxins abundance, 63 predatory strategies, 66 venom gland modifications, 64 Spirobenzylisoquinoline formation, 340-341 13-hydroxytetrahydroprotoberberine conversion, 335-343 Stereochemistry, necine biosynthesis, 23, 25-3 1 (+)-Supinidine, 19

Tetrahydroberberine, feeding to Corydalis pallida. 3 10 Corydalis incisa, 3 1 I , 3 I3

363

Tetrah ydroisoquinolines naphthalene-free, 184- 185 preparation, 182-183 Tetrahydropalmatine, feeding to Corydalis pallida, 3 10-3 I I Tetrahydroprotoberberine alkaloids, 278-279

(S)-Tetrahydroprotoberberine-cis-N-

methyltransferase, occurrence, 320, 323 (S)-Tetrahydroprotoberberine oxidase, specificity, 317-319 Tetrahydroprotoberberines biosynthetic routes to benzophenanthridines, 300-301, 304 conversion into benzophenanthridines, 277-305 N-metho salts, 320-321 rhoeadines, 329-333 dehydrogenation, to protoberberinium salts, 317-321 a-N-metho salts, hydroxylation, 320-324 N-methylation, to a-N-metho salts, 317, 320-321 redox conversions between, 306 Thalictricavine, feeding to Corydalis pallida, 3 I I Trachelanthamidine, homospermidine conversion into, 17-18 [5-3H]Trachelanthamidine,2 I Trachelanthamine, biosynthesis involving 1-hydroxymethylpyrrolizidines, 20-2 I Trichodesmic acid, biosynthesis, 34-35 Triphylline, see Dioncophylline A Triphyopeltine, 131, 248 Triphyophylline, 244 constitution, 132-133 structure, 131 total synthesis, 134-136 trans configuration, 133 Triphyophyllum peltatum, 146-152, see also Dioncophylline A dioncolactone A, 146-148 Dioncophyllaceae alkaloids, 153-155 dioncophyllacines A and B. 151-153 dioncophylline D, 151

364 dioncophyllines B and C, 147-151 minor alkaloids, 151 Tryptophan, structure, 2 .. . Tyrosine, structure, 2 L-Tyrosine berberine formation, 274-275 protoberberine formation, 274-275

INDEX

Vertebrates, polyamine toxins effect, see Polyamine toxins Wasps, see also Polyamine toxins Yaoundamine B, 255. 257

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