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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 ([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 ( 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
2.
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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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-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
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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.
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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 0.05 >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.
128
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
130
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.)
-
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