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Contents pages |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 009-010
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摘要:
24 NPR 8 Cumulative Contents of Volume 8 Number 1 1 Diterpenoids (1989) J. R. Hanson 17 Steroids Reactions and Partial Synthesis (November 1987 to October 1988) A. B. Turner 53 Quinoline Quinazoline and Acridone Alkaloids (July 1988 to June 1989) J. P. Michael 69 Terpenoid Glycosides (1987 and 1988) H. Pfander and H. Stoll Number 2 97 Marine Natural Products (1989) D. J. Faulkner 149 The Biosynthesis of Shikimate Metabolites (1989) P. M. Dewick 171 Muscarine Oxazole Thiazole Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1988 to June 1989) J. R. Lewis 185 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (August 1988 to July 1989) R. B. Herbert Number 3 213 Pyrrolizidine Alkaloids (July 1989 to June 1990) D.J. Robins 251 Carotenoids and Polyterpenoids (1988) G. Britton 309 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1989 to June 1990) J. E. Saxton 319 The Occurrence and Biological Activity of Drimane Sesquiterpenoids (to January 1990) B. J. M. Jansen and A. de Groot Articles that will appear in forthcoming issues include Terpenoid Phytoalexins (August 1984 to December 1989) C. J. W. Brooks and D. G. Watson The Lycopodium Alkaloids (January 1986 to October 1990) W. A. Ayer A Unified Mechanistic View of Oxidative Reactions Catalysed by P-450 and Related Fe-Containing Enzymes M. Akhtar and J. N. Wright Biosynthesis of C,-C, Terpenoid Compounds (1989) M. H. Beale Pyrrole Piperidine Pyridine and Azepine Alkaloids (July 1989 to June 1990) A. R. Pinder Diterpenoid Alkaloids (July 1985 to December 1989) M. S. Yunusov Tropane Alkaloids (1990) G. Fodor and R. Dharanipragada The Biosynthesis of Polyketides (January 1986 to December 1988) T. J. Simpson
ISSN:0265-0568
DOI:10.1039/NP99108FP009
出版商:RSC
年代:1991
数据来源: RSC
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Front cover |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 013-014
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Natural Product Reports Editorial Board Professor T. J. Simpson (Chairman) University of Bristol Dr C. Abell University of Cambridge Dr J. R. Hanson University of Sussex Dr R. B. Herbert U niversity of Leeds Professor J. Mann University of Reading Dr D. A. Whiting University of Notting ham Natural Product Reports is a journal of critical reviews published bimonthly which is intended to foster progress in the study of natural products by providing reviews of the literature that has been published during well-defined periods on the topics of the general chemistry and biosynthesis of alkaloids terpenoids steroids fatty acids and 0-heterocyclic aliphatic aromatic and alicyclic natural products. Occasional reviews provide details of techniques for separation and spectroscopic identification and describe methodologies that are useful to all chemists and biologists who are actively engaged in the study of natural products.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge CB4 4WF England. 1991 Annual Subscription Price E.C. fl98.00 Overseas f228.00 U.S.A. $467.00. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. SG6 1 HN England. Air Freight and mailing in the U.S.by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. US Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 1 1003. Second-Class postage paid at Jamaica NY 11431 -9998. All other despatches outside the U.K. are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the U.K. 0 The Royal Society of Chemistry 1991 All Rights Reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photographic recwding or otherwise without the prior permission of the publishers. Printed in Great Britain by the University Press Cambridge Subscription rates for 1991 E.C. f198.00 Overseas f228.00 U.S.A. US $467.00 Subscription rates for back issues are U.K. (1986)f 130.00 (1987)€1 42.00 (1988)f 159.00 (1989)f 169.00 (1990)f 177.00 Overseas f 143.00 €1 59.00 f 183.00 f 1 94.00 f 204.00 U.S.A. US $252.00 US $280.00 US $342.00 US $388.00 US $398.00 Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge CB4 4WF England
ISSN:0265-0568
DOI:10.1039/NP99108FX013
出版商:RSC
年代:1991
数据来源: RSC
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3. |
Back cover |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 015-016
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ISSN:0265-0568
DOI:10.1039/NP99108BX015
出版商:RSC
年代:1991
数据来源: RSC
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4. |
β-Phenylethylamines and the isoquinoline alkaloids |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 339-366
K. W. Bentley,
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p-Phenylethylamines and the lsoquinoline Alkaloids K. W. Bentley Department of Chemistry Loughborough University of Technology Loughborough Leicestershire LEl 7 3TU Reviewing the literature published between July 1989 and June 1990 (Continuing the coverage of literature in Natural Product Reports 1990 Vol. 7 p. 245) now including coverage also of Erythrina Alkaloids last reviewed in Natural Product Reports 1990 Vol. 7 p. 565 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 17.1 17.2 17.3 17.4 17.5 17.6 18 19 20 21 22 23 P-Phenylethy lamines Isoquinolines Benzylisoquinolines Bisbenzylisoquinolines Cularines Pavines and Isopavines Benzopyrrocolines Berberines and Tetrahydroberberines Seco berberines Protopines Phthalide-isoquinolines Spirobenzylisoquinolines Rhoeadines Other Modified Berberines Emetine and Related Alkaloids Benzophenanthridines Aporphinoid Alkaloids Proaporphines Aporphines Oxoaporphines Dioxoaporphines Aporphine-Benzylisoquinoline Dimers Aristolochic Acids and Aristolactams Morphine Alkaloids Phenethylisoquinolines Homoaporphines Colc hicine Erythrina and Related Alkaloids References 1 /3-Phenylethylamines Four new amides of 3,4-dimenthoxycinnamic acid namely rubemamin (1 R = H) rubemamide (1 R = Me) dioxamin (2 R = H) and dioxamide (2 R = Me) have been isolated from Zanthoxylum rubescensl and hordenine has been isolated from the red marine alga Mastocarpus sellatus.Reviews of the isolation and chemistry of the P-phenylethylamines and ephedra alkaloid^,^ and of ephedrine have been published. Dialkylthiophosphate esters of ephedrine have been prepared by heating the alkaloid with dialkyl phosphites and sulphur;5 the electron-impact mass spectra of the N-trifluoroacetyl-L- prolyl derivatives of ephedrine of norephedrine and of their trimethylsilyl derivatives have been studied,6 and the effects of ephedrine on cholinergic neurotransmission have been examined.' Optically pure 1S,2S-norpseudoephedrine has been synthesized from 1 S,2S-2-amino- 1-(4-nitrophenyl)propane-1.3-dioL8 2 lsoquinolines N-methylcorydaldine has been isolated from Berberis empetri- foliag and its methylenedioxy analogue oxyhydrastinine has been isolated from Hypecoum imerbe H.pendulum and H. procumbens.lo The new alkaloid berbidine (36) obtained from Berberis brandisiana,'l though a relatively simple isoquinoline OR' 0 OH (3) (4) Me0 Me0 Me0 Me''' 0 Me0 Me0 'G-CF 'S-pTol 0 2 (7) is clearly a modified bisbenzylisoquinoline and is discussed in section 4. Thalflavine originally assigned the structure (3 R1 = Me R2R3 = CH,) has been proved to have the constitution (3 R'R2 = CH, R3 = Me) by the synthesis of both isomers.12 Highly stereospecific syntheses of 54 -)-salsolidine (6 R = 13) and 54 -)-carnegine (6 R = Me) have been achieved. Cyclizations of the chiral iminium salt (4) with base afforded the carbinolamine ether (5) reduction of which with benzylic cleavage yielded (6 R = H).13 The cyclization of the vinyl sulphoxides (7) and (8) obtained by Wittig reaction from the 339 NATURAL PRODUCT REPORTS 1991 MeNANMe MeowNMe Me0 1,) H' CH2SO-pTol Me (9) OMe I Me0 R2 OMe OMe OMe OMe C0,Me Me0 OMe OMe OMe Me (20) corresponding aldehyde was achieved by base when the 2-isomer (7) gave the tetrahydroisoquinoline (9) and the E-isomer (8) yielded the enantiomer of (9).Desulphurizing reduction of (9) yielded 5-( -)-carnegine (6 R = Me) and desulphurization of its enantiomer gave R-(+)-carnegine.I4 Mescaline and homoveratrylamine have been converted by Pictet-Spengler cyclization with the masked aldehydes (10) and (I I) respectively into anhalinine (12 R1= OMe R2= H)15 and calycotomine (12 R1= H R2= CH20H).16 The mass spectra of esters of N-2-hydroxyethylsalsoline have been studied.17 The isomeric alkaloids cherylline (14 R1= H R2= OH) and latifine (14 R' = OH R2 = H) have been synthesized by isocyanate cyclization of the azides (13 R1= H R2 = OMe) and (1 3 R1 = OMe R2 = H) followed by reduction and regio- selective demethylation by dimethylsulphide in methane-sulphonic acidla and O,O-dimethylnorcherylline has been OMe OMe OMe OMe OMe @Me Meo% \N OMe OMe Me (21) obtained together with 5-bromo-O,O-dimethylnorlatifine by the acid-catalysed cyclization of the alcohol (1 5).19 In the naphthylisoquinoline group of alkaloids bisdehydro- anicistrocladisine (1 9) has been synthesized from the aldehyde (16) via the nitrostyrene (17) which was converted via the saturated amine into the alcohol (18 R = OH) the chloride (18 R = CI) and the C-methyl compound (18 R = H).Bischler-Napieralsky ring closure of this gave dehydroan- cistrocladisine which on heating with Raney nickel was dehydrogenated to the isoquinoline (19).20In approaches to the synthesis of ancistrocladisine itself it has been found that cleavage of the lactone (20 X = 0) with sodium methoxide gave a mixture of the two rotational isomers of the hydroxy ester (21) that underwent rapid equilibration 21 whereas cleavage of the cyclic ethers (20 X = 2H) yielded a mixture of rotamers sufficiently stable to be separated.22 NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY 341 <% OCH2Ph OH (24) LI ' -,CN POPh H vOCH,Ph O W O OMe OMe 3 Benzylisoquinolines Benzylisoquinolines alkaloids have been isolated from the following plant species the three marked with asterisks being new alkaloids Annona ~herimolia~~ reticuline Berberis chilen~is~~ reticuline Cananga odor~ta~~ reticuline Cryptocarya phyllostemon2s phyllocryptine* (25) and phyllocryptonine* (24) Cryptocarya velutinosa2' velucryptine* (26) Stephania pierrii28 coclaurine N-methylcoclaurine and reticuline Thalictrum pedunculat~m~~ reticuline The structures of the new alkaloids were deduced from spectroscopic data and that of phyllocryptonine was confirmed by synthesis from (22) (obtained by Bischler-Napieralsky synthesis) by oxidation to (23) followed by reduction debenzylation and quaternization ;26 and velucryptine was obtained by oxidation of the corresponding benzyldihydro- isoq~inoline.~~ The fast atom bombardment mass spectra of ' OMe (23) MeoqN HO OMe (26) Me0 IbOMe OMe (34) benzylisoquinoline alkaloids have been shown to be very useful in structural analyses in this series giving abundant molecular ion peaks often hardly detectable in electron impact The oxidation of papaverine with vanadium pentoxide and periodic acid has been found to give papaveraldine together with 4-hydroxy-6-0-demethypapaveraldineand 4-hydroxy-4'- 0-demethylpapaverine and their 3-hydro~y-isomers.~~ N-methylcoclaurine and N-acetylnorreticuline have been oxidized to the proaporphine glaziovine and to isomeric N-acetylnora- porphines re~pectively~~ and 2,3-di(methoxycarbonyl)-nor-isoboldine has been oxidized to a mixture of derivatives of norisoboldine and n~rsalutaridine~~ (see sections 17.1 17.2 and 18).( +)-Reticuline has been synthesized from the formamidine (27) by treatment with 3- benzyloxy-4-methox ybenzylbromide followed by N-methylation and debenzylation ;34 this route was first used successfully to synthesize salsoline and la~danosine.~~ Isosevanine (28 R1= R2= H) and berbitine (28 R1 = OH R2 = Me) have been synthesized by the conventional route from the Reissert compound (29);36 cryptopleurospermine (32) has been synthesized from the aldehyde (30) by reaction with (31) followed by reduction and ~xidation,~~ following a variant of an earlier synthesis of the alkaloid38 and dihydropapaveraldine (33) has been synthesized by Bischler-Napieralsky cyclization of homoveratrylamine with the diketo-ester (34).39 342 NATURAL PRODUCT REPORTS 1991 The biological effects of papa~erine,~O-~~of 4-[4-(2-methoxy- pheny1)-piperazin-I -yl]methylpapa~erine,~~of la~danosine,~~of reti~uline,~~of higenamine,47-49 of jatrorrhi~ine~~*~~of 6'- brom~codamine,~~and of atrac~rium~~.54 have been studied.Me0 4 Bisbenzylisoquino1ines Bisbenzylisoquinoline alkaloids have been isolated from the following plant species the twenty-one marked with asterisks OH being new alkaloids Anisocycla ~ymosa~~ coscoline 1,2-dehydroapateline 1,2-dehydrotelobine and trilobine (-)-temuconine* (35) Aristolochia elegan~~~ Berberis brandisianal' berbidine* (36) chenabinol methyl ether* (37) Berberis ~hi1ensi.v~~ espinine and 12-O-demethyllauberine* (38) berbamine berbamunine and oxyacanthine Berberis vulgaris5' cordobine* (39 R' = R2= H) cordobimine* (39a) granjine* (39 R' = R2= Me) and monterine* (39 R1 = H R2= Me) Crematosperma (Species undis~losed)~~ (39) Daphnandra dielsii5'.6o diekine* (40) N-methylapateline O,N-dimethylapate-line nortenuipine nortenuipine-2P-N-oxide*(4I) repan-dine 0-methylrepandine repanduline and $-repandu- line MeNP O M OHe Me0MeoT!Me H Me0 MeN Me0 SOTNMe (36) HO (37) NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY Dehaasia triandra6I dehatrine* (42) dehatrdine* (43) and obaberine Phaeanthus vietnamensis" U,U-dimethylgrisabine* (44) Stephania pierii2" berbamunine cycleanine N-demethylcycleanine de- hydroapateline isotetrandrine 2-norisotetrandrine* (49 2'-norisotetrandrine 2-norberbamine 2'-nor-cepharanthine* (46) 2-norisocepharanthine* (47) 2-norcepharoline* (48) 2'-norobaberine* (49) stephi-baberine* (50) stepierrine* (5I) and thalrugosamine Thalictrum bu~chianum~~ thalmine Thalictrum havum64 hernandezine thalfoetidine thalidasine and U-methyl- thalic berine MeN%oMTH*' OMe (47) / OMe \ HNH*-*E \ o g M/ "H e / OH 0 \ Tiliacora racemosae5 N-methyltiliarnosine* (52) Tiliacora triandra66 tiliacorine tiliacorinine and nortiliacorinine The distribution of bisbenzylisoquinolines in Thalictrum minus plants in southern Bulgaria has been reviewed.'j7 The structures of the new alkaloids cited above were determined principally by spectroscopic studies.In addition berbidine (36) was prepared by the oxidation of chenabinol methyl ether (37);l' dehatrine (42) was reduced by sodium and liquid ammonia to a mixture of 2-0-methylarmepavine and (& )-coclaurine and was also reduced to a mixture of secondary bases that gave the diastereoisomers phaeanthine and iso- tetrandrine on N-methylation.sl (+)-and (-)-temucomine represent one of a small number of enantiomeric bisbenzyl- isoquinoline alkaloids though no racemic mixtures have so far been isolated from plants.Methods of determining the structures of alkaloids of this group have been reviewed.68 Studies of the NMR spectra of NATURAL PRODUCT REPORTS 1991 5 Cularines Cularidine oxocompostelline oxosarcocapnine and the new alkaloid norsarcocapnine (55) have been isolated from Cerato-capnos hetero~arpa.~~ Cope degradation of the N-oxides of cularine cularidine sarcocapnine and sarcocapnidine has been shown to give the stilbenoid hydroxylamines of general structure (56 R = OH) which have been reduced to the corresponding secondary amines (56 R = H).98 6 Pavines and lsopavines Norargemonine and argemonine have been isolated from Cyclea atjehensis;99 this is the first recorded occurrence of a pavine in the Menispermaceae.In approaches to the synthesis of pavine and isopavine alkaloids thermal cyclization of the azide (57) above 111 "C has been found to give the aziridine (58) which on reduction with dibutylaluminium hydride gave the pavine analogue (59 R = H) whereas (59 R = C1) and tetrandri~~e~~ (59 R = OAc) were obtained by treating the aziridine with and the mass spectra of alkaloids of the gr~up~"~~" have been reported. In approaches to the synthesis of the hydrochloric and acetic acids respectively. Cyclization of the Tiliacora alkaloids the amide (53)71and the diphenyl (54)72 azide (57)below 11 1 "C,however gave the isopavine-like imine have been prepared. Berbamunine has been obtained by the (60) which was reduced to the secondary base.loO oxidation of (+)-N-methylcoclaurine with oxygen and NADPH linked to cytochrome P450.73 The physiological and pharmacological effects of berbamine,74-76 of cephar-anthine,77-82 of da~ricine~~.~~ of of 7-O-demethylthali~berine,~~ of tetrandrine,75 88-g1 of tilia~orinine,~~ nefe~ine,~~.~~ of nortili- acorinineg2 of tubo~urarine,~~~ 94 and of metocurineg5 have been studied and a method of isolation and estimation of tubo- curarine has been described.96 OMe Me0 (55) R' OMe OH (52) \ Me Me (57) C02CH2Ph C0,Me R +-(=j OCH2Ph Me -Me (54) (59) NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY 7 Benzopyrrocolines Two new benzopyrrocolines cryptowolidine (61 R’ = H R2 = Me) and crytowolinol(62) have been isolated together with cryptowoline (61 R’ = Me R2 = H) from Cryptocarya phyl- lostemonZ6* lo’ and from Cryptocarya oubatchensis.lo’ The structures of the alkaloids were determined spectroscopically and that of cryptowolidine was confirmed by synthesis from the benzylisoquinoline (64) by displacement of chlorine by the anion of the secondary base followed by N-methylation and debenzylation.lol Hofmann degradation of cryptowolidine was found to give the styrene (63) rather than the isomeric stilbene.Syntheses of cryptaustoline have been achieved by cyclization of the lactam (65) and the indole (66) followed by reduction and N-methylation.lo2 HO Me0 H0’ n H0Y3TMe U (63) 8 Berberines and Tetrahydroberberines Alkaloids of this group have been isolated from the following plant species the four marked with asterisks being new alkaloids Anisocycla ~ymosa~~ Anisocycline* (67) and palmatine Annona cherim~lia~~ corypalmine discretamine and tetrahydropalmatine Berberis chitrialo3 dihydropalmatine N-oxide* (68) Berberis vulgaris5’ berberine berlambine lambertine and palmatine Cananga odor at^^^ coreximine Coptis quinq~efolia’~~ thalifaurine Corydalis intermedialo5 berberine canadine corydaline dehydrocorydaline coptisine palmatine tetrahydropalmatine scoulerine and stylopine Corydalis rnajorilo6 cavidine cheilanthifoline corysolidine scoulerine stylo- pine and tetrahydrocorysamine Corydalis nobilislo5 coptisine corybulbine corydalidzine corydaline de- hydrocorydaline corypalmine corysamine palmatine tetrahydropalmatine scoulerine sinactine stylopine and cis-N-methylstylopinium hydroxide Corydalis pallidalo7 capaurimine pallimamine* (69) and tetrahydro-palmatine OMe Me0 OMe PhCH20m MeO-NH~i Me0 OMe Me0 Meo%OMe he / w OMe ‘ OMe Coscinium fenestratumlo8 berberine canadine oxyberberine and oxotetra-hydroberberine* (70) Oncodostigma mono~perma'~~ discretamine and stepholidine Papaver conjine' lo berberine coptisine corysamine scoulerine sinactine and stylopine Papaver rhoeas chelidoniodes'" berberine coptisine corysamine scoulerine stylopine and trans-N-methylstylopiniumhydroxide Stephania yunanensisll corydalmine dehydrocorydalmine palmatine tetra-hydropalmatine stepharranine and stepholidine Thalictrum acutifo1ium1l2 ox yberberine Thalictrum Jlav~m~~ berberine Thalictrum purpure~cens~'~ berberine Xanthorhiza simplici~sima"~ palmatine The structure of the new alkaloids were deduced from spectroscopic data and confirmed in the case of pallimamine by an X-ray crystallographic study.lo7 Several berberine alkaloids have been converted by stages into benzophenanthridine alkaloids (see section 16).The absorption of berberine and of canadine at a mercury electrode has been studied.l15 A synthesis of dehydronorcoralydine perchlorate (72) has been achieved by the reaction of the pyrrilium salt (71) with 2,2- diethoxyethylamine under acidic conditions. 116 Thaicanine (73 R = H)ll' and 0-methylthaicanine (73 R = Me)118 have been synthesized from p-(2- benzylox y-3,4-dime thox yp heny1)- ethylamine and ,!&(2,3,4-trimethoxyphenyl)ethylamine respect-ively by heating with the isochromone (74) followed by Bischler-Napieralsky ring closure and conventional reactions.A related isochromone synthesis has been used to prepare 13- methylxylopinine (75).ll9 The deuterium labelled bases (76 R' = H R2 = Me) and (76 R' = Me R2= H) have been synthe- sized for biogenetic studies of the origins of benzo-phenanthridine alkaloids. The physiological and pharmacological properties of ber- berine,74,121-123 of dehydr~corydaline,'~~ of tetrahydro-of ~almatine,'~~ 743 ~optisine,~~ of tetrahydropalmatine 126 of benzyltetrahydropalmatine 127 and of stepholidine3'** 128 have been studied.OMe NATURAL PRODUCT REPORTS 1991 OMe OR (73) 0 OMe (74) OMe (75) NATURAL PRODUCT REPORTS 1991-K. W. BENTLEY 9 Secoberberines The absolute configuration of canadaline (77) has been confirmed by preparation of the alkaloid from S-( -)-canadine (tetrahydroberberine). 12g 10 Protopines Alkaloids of the protopine group have been isolated from the following plant species the two marked with an asterisk being new alkaloids Corydalis intermedialo5 allocryptopine and protopine Corydalis majorilo6 pro to pine Corydalis nobilislo5 allocryptopine cryptopine and protopine Corydalis pallidalo7 allocryptopine and protopine Fumaria par~iJora'~~ protopine Papaver conjinello allocryptopine cryptopine and protopine Paver cur viscapum 131 allocryptopine 1-methoxyallocryptopine* (78) 1-methoxy- I3-oxoallocryptopine* (79) and protopine Papaver dubium 110 protopine Papaver rhoeas chelidoniodesl10 allocryptopine cryptopine and protopine Oxidation of protopine with oxygen and a microsomal cytochrome P45O-NADPH-dependent enzyme has afforded 6-hydroxyprotopine (80) which underwent spontaneous cyc- lization to ~anguinarinel~~ (see section 16).The effects of protopine on platelets has been ("0 %OMe 0 ' OMe (78) 11 Phthalide-isoquinolines Phthalide-isoquinolines have been isolated from the following plant species the two marked with asterisks being new alkaloids Corydalis in termedia O bicuculline Corydalis majorilo6 bicuculline and capnoidine Corydalis nobilislo5 adlumine adlumidine bicuculline and corlumine Fumar ia indica ' papraine* (8 1) Papa ver somnifer um 35 narceine and narceinone* (82) The structure of narceinone was confirmed by preparation of the alkaloid through the oxidation of nar~eine.~~~ The electrochemical oxidation of narcotine to cotarnine and opianic acid has been studied;136 circular dichroism studies of bicuculline and its methi~didel~~ and mass spectroscopic studies of phthalide-isoquinoline alkaloids30 and a method for the isolation and estimation of narcotine13* have been reported.In approaches to the synthesis of alkaloids of this group in a process analoguous to the synthesis of dihyropapaveraldine recorded in Section 3 the 2-ethoxycarbonyl analogue of (34) was reacted with homoveratrylamine to give a keto-ester that was reduced to cordrastine (83 R1= R2 = Me) and a similar synthesis of hydrastine (83 R1R2= CH,) was achieved.39 Although the decarboxylation of potassium salts of phthalide- 3-carboxylic acids in the presence of acyclic imine methiodides normally affords acylbenzamides in the presence of 3,4-dihydroisoquinoline salts the products are phthalide-isoquino- lines; adlumine (84) has also been synthesized in this way.139 The conversion of spirobenzylisoquinoline N-oxides into dehydrophthalide-isoquinolinesis described in section 12.The physiological and pharmacological effects of bicucul- line140 141 and of hydra~tinel~~ have been studied.MeowNMe Me0 12 Spirobenzylisoquino1i nes Spirobenzylisoquinolineshave been isolated from the following plant species that marked with an asterisk being a new alkaloid Corydalis caucasialg3 corpaine fumariline fumaritine ochotensimine and si biricine Corydalis ~7abellata'~~ severzinine* (85) and sibiricine Corydalis majorPos corpaine The structures and biogenesis of alkaloids of this group have been reviewed.145 The synthetic berberine analogue (86) has been rearranged to the keto-acetal(87) which was reduced to the alcohol (88 R = H). Conversion of this into (88 R = C0,Et) followed by inversion of the alcoholic centre through the chloride yielded (89 R = C0,Et) and hydrolysis of this with subsequent treatment with formaldehyde yielded (90) which was reduced (as was 89 R = C0,Et) to (89 R = Me).Hydrolysis of the ketal (89 R = Me) gave yenhusomidine (91 R1R2 = 0) and reduction of this yielded yenhusomine (91 R1 = OH R2 = H). Hydrolyis of the ketal (88 R = Me) likewise yielded radde- anone (92 R1R2= 0),which was reduced to raddeanine (92 R1 = OH R2 = H).146 Treatment of the spirobenzylisoquinoline N-oxides (93 R1R2 = CH, R3 = R4 = Me) and (93 R' = R2 = Me R3R4 = CH,) with trifluoroacetic acid has given dehydrohydrastine (94 R1R2 = CH, R3 = R4 = Me) and dehydro-adlumine (94,R1= R2 = Me R3R4 = CH2).lg7 0 OH NATURAL PRODUCT REPORTS 1991 NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY HO '-0Me0 OMe OMe 13 Rhoeadines Rhoeadine alkaloids have been isolated from the following plant species Papaver confinello rhoeadine and papaverrubines A C D and E Papaver dubium lo rhoeadine and papaverrubines A C D and E Papaver rhoeas1l0 148 rhoeadine isorhoeadine rhoeagenine and papaverru- bines A C D and E 14 Other Modified Berberines The spirobenzazepine alkaloid turkiyenine has been isolated from Hypecoum imerbe H. pendulum and H. procumbers.1° 1-Methoxy-5-hydroxylennoxamine(96) and its dehydration pro- duct (97) have been obtained by heating dihydronarceineimide N-oxide (95) with acetic anhydride.149 15 Emetine and Related Alkaloids Two new alkaloids of the emetine group neoipecoside (98 R = H) and methylneoipecoside (98 R = Me) have been isolated from Cephaelis ipecacuanha.150 Both alkaloids give O-penta- acetates and methylation of these with diazomethane yields the same 0,O-dimethylneoipecoside penta-acetate. Spectroscopic studies led to the assignment of the positions of the hydroxyl and methoxyl groups in the alka10ids.l~~ Neoipecoside is an isomer of ipecoside which is the 6,7-dihydroxyisoquinoline analogue of (98 R = H). A highly stereoselective synthesis of ( -)-protoemetino1 has been achieved from the mixed acetal(99) which was reductively cyclized with tributyltin hydride to (100 R1 = OEt R2 = H R3 = C0,Me) and this was then further reduced at the ester group to the carbinol converted into the buytl ether and oxidized to the lactone (100 R1R2 = 0 R3 = CH,OBu).Reaction of this lactone with homoveratrylamine gave the amide (lOl) Bischler-Napieralsky cyclization of which followed by con- ventional processes gave (-)-protoemetino1 (102 R1= H R2 = CH20H).151A synthesis of hydroxyprotoemetine (102 R1 = OH R2 = CHO) has also been ~ep0rted.l~~ RO OH HO p.. 0 Br (99) \1 OBu NATURAL PRODUCT REPORTS 1991 16 Benzophenanthridines 97 Benzophenanthridine alkaloids have been isolated from the following plant species those marked with an asterisk being new alkaloids Hypecoum imerbe" nitrotyrasanguinarine* (103) and 8-methoxy-dihydro- sanguinarine 0 Me 1-Hypecoum pendulum" LO LO nitrotyrasanguinarine and 8-methoxydihydrosanguin-arine Hypecoum procumberslo nitrotyrasanguinarine and 8-methoxydihydrosanguin-R2 arine Zan thoxylum nit idum 153 6-methox y-7-0-demethyl-5,6-di h ydrochelerythrine* (104) Zanthoxylum rubescers' arnottianamide 0 The structure of the alkaloid (104) was confirmed by an X-LO (109) ray crystallographic The effect of ethylene on the production of sanguinarine in Papaver somniferum has been studied.154 Conditions for the electrochemical reduction and OR' oxidation and for the thermal demethylation of chelerythrine fagaronine and sanguinarine have been 6-Hydroxyprotopine (80) obtained by enzyme-catalysed OR2 oxidation of protopine has been found to undergo spontaneous rearrangement to sanguinarine presumably via the aldehyde Me0 (104) and the enamine (105 R = H) followed by cyclization to the chelidonine derivative (106 R = H) dehydration and oxidation.Hofmann degradation of N-methyldihydro-corysamine iodide (I 07) followed by conversion of the resulting vinyl group into CH,CHO has given (105 R = Me) cyclization of which gave cis and trans isomers of (1 06 R = Me) which on reduction afforded a mixture of corynoline (108 R' = OH R2 = H) 1 1-epicorynoline (108 R' = H R2= OH) isocorynoline (109 R' = OH R2= H) and 11-epi-isocorynoline (109 R' = H R2 = OH).'56,'5' The bases (1 10 R1R2= CH,) and (1 10 R' = R2 = Me) obtained by similar transformations of berberine alkaloids have been oxidized to the quinones (1 1 1) and these on reduction and 0-methylation have given chelilutine (1 12 R1R2= CH,) and sanguilutine (1 12 R1= R2= Me).'56.158 Oxychelirubine OR1 (113 R = H) has been converted through (1 13 R = I into oxymacarpine (1 13 R = OMe) dihydromacarpine and macar- OR2 ~ine.'~~.'~' 8-Methoxynorsanguinarine (1 14) has been prepared from luguine and its physical properties and spectra were found Me0 to differ from those of pancorine to which this structure has been assigned.160 OMe (1 12) R OH NHMe L-0 n NATURAL PRODUCT REPORTS 1991-K. W. BENTLEY Treatment of the amine (1 15) with 2-bromo-3,4-dimethyoxy- benzaldehyde and with bromodimethoxybenzoyl chloride gave the Schiff base (1 16) and the amide (1 17) respectively and both of these were cyclized photochemically (1 16) giving (1 1S) the N-metho-salt of which after removal of the isopropyl group gave tagaronine also prepared from cyclized (117) by N- methylation reduction oxidation and removal of the isopropyl group.16' Cyclization of the olefin (119) obtained from 13- hydroxy-xylopinine in acid proceeded according to Markownikov's rule to give the 11 -methyl-C-norfagaronine derivative (120).162 The physiological and pharmacological effects of cheli-d~nine'~~ of nitidine,IG4 and of of 9-metho~ychelerythrine,~~~ fagar~nine'~~ have been studied.OPri OMe OMe NH2 Me0 OMe Me0 Me0 OMe Me0 351 17 Aporphinoid Alkaloids 17.1 Proaporphines Proaporphine alkaloids have been isolated from the following plant species Annona ~herimolia~~ glaziovine Nectandra rnembranaceaels6 glaziovine Oncodostigma monospermalog stepharine Papaver conjine1l0 mecambrine Papaver dubiurn1lo mecambrine Stephania yunnanensis" stepharine Thalictrum ped~nculaturn~~ pronuci ferine The structure (121) previously assigned to roemeridine has been confirmed by an X-ray crystallographic Glaziovine (122) has been prepared by the internal oxidative coupling of N-methylcoclaurine (123 R = H) with tetraphenyl- porphyrinatomanganese (111) acetate as catalyst and sodium hypochlorite as oxidant and also by the non-oxidative coupling of the diazonium salt (123 R = +N2)in the presence of ruthenium carbonyl.168 Me0 HO Me0 OMe Me0 Me0 OMe OMe \ NMe Me0 0 Meoe Me0 \ .HO O/F M e NATURAL PRODUCT REPORTS 1991 17.2 Aporphines Thalictrum pedunc~latum~~ Aporphine alkaloids have been isolated from the following N-methyldanguyelline* (1 29) isocorydine oncovine plant species the seven marked with asterisks being new noroncovine and thalicsimidine alkaloids Tinospora malabri~al'~ Actinodaphne ~peciosa'~' magnoflorine laurotetanine and N-methyllaurotetanine The crystal structure of dehydrodicentrine has been eluci- Anisocycla ~ymosa~~ dated.175A review of the Hofmann and Cope degradations of remrefidine quaternary salts and N-oxides of aporphines has been pub- Annona ~herimolia~~ lis hed. '76 anolobine anonaine asimilobine N-methylasimilobine N-acetylnorisoboldine (130 R' = H R2= OH) and N-corydine isoboldine norsushinsunine nuciferine and acetylnorcorytuberine (130 R1 = OH R2 = H) have been xylopine Berberis empetrifolia' Me0 nantalamine* (124 R = CH20H) and nantalinine* (124 R = CHO) Cananga ~dorata~~ anonaine ushinsunine and ushinsunine N-oxide Corydalis intermedialo5 bulbocapnine isoboldine and magnoflorine Corydalis majorilo6 HO bul bocapnine Corydalis nobilislo5 isoboldine and corytuberine O\O\, Cyclea atjehensis" laurotetanine nornantenine and N-formylnornan-tenine* (125) Dehaasia triandra6' corytuberine isocorydine nantenine and xanthoplanine Diploclisia glauces~ens~~~ magno florine MHO e O F Me Hypecoum imerbe'O bulbocapnine Hypecoum pendulum" HO bulbocapnine Hypecoum procumberslo q bulbocapnine Litsea deccanensisl 'I Me0 OMe boldine norboldine corytuberine dicentrine nor-OMe OMe dicentrine isocorydine and magnoflorine Litsea gardneri16' actinodaphnine and laurolitsine Nectandra membranaceae166 apoglaziovine and isoboldine Ocotea caesia1'12 isoboldine laurelliptine pulchine zenkerine and 1-?Me hydroxy-2,9-dimethoxyaporphine*(126) OH Oncodostigma mono~perrna'~~ anonaine asimilobine corytuberine nornuciferine and norushinsunine Me0 Me0 MeoI$Me HO Papaver conJnellO corytuberine corydine isocorydine and roemerine (aporheine) Papa ver dub ium lo Me0 / corytuberine roemerine and N-met hylroemerinium OMe Me0 hydroxide Papaver rhoeasllO* 148 corytuberine isoboldine isocorydine magnoflorine and roemerine Stephan ia pierrii28 isocorydine Stephania yunuanensis"' Me0 roemerine and stephanine Thalictrum acutifo[ium112 acutifolidine* (127) HO Thalictrum buschianums3 thalicmidine Thalictrum co1linumss isoboldine Me0 Thalictrum i~hengense'~~ glaucine dehydroglaucine thaliporphine thalicsimidine and dehydrothalicsimidine* (128) NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY obtained by the internal oxidative coupling of N-acetylnor-reticuline with oxygen as the oxidant and the bis(salicy1- a1dehyde)ethylenediamine cobalt (11) complex as the catalyst.168 Photochemical cyclizations of the N-ethoxycarbonyl-6'-bromo-benzyl tetrahydroisoquinolines (131 R' = R2 = Me) (131 R' = R2 = H) (131 R' = H R2 = Me) and (131 R1= Me R2 = H) have been achieved ;and the resulting N-ethoxycarbonyl compounds have been reduced to glaucine (132 R' = R2 = Me) isoboldine (1 32 R1= R2 = H) thaliporphine (132 R' = H R2 = Me) and N-methyllaurotetanine (132 R1= Me R2 = H) ;177.178 and dehydroglaucine was prepared in a similar manner from the benzylidene analogue of (131 R' = R2 = Me).'78 The dehydroaporphines (133 R' = H R' = Me) and (133 R1= Me R2 = H) have been synthesized and the latter has been shown to be identical with goudotianine isolated from Guatteria goudotiana. 179 In this synthesis the C-methyl group was introduced into the dehydroaporphine by a Villsmeier reaction followed by reduction of the formyl group. The synthesis of 6a-13-C-labelled glaucine from 1-13-C-papaverine has also been recorded.lso In the apomorphine series the preparation of apomorphine 3-(phenyltetrazoly1)-ether by the rearrangement of 3-0-(phenyltetrazoly1)morphine has been found to be accompanied by the formation of the isomeric 4-O-(phenyltetrazolyl)-apomorphine ;Is1 the 3-0-(phenyltetrazolyl) ether has been reduced to 3-deoxyapomorphine (134 R = H) which on iodination gave (1 34 R = I) and this with sodium methoxide and copper (I) iodide yielded (134 R = OMe) which was demethylated to I-hydroxy-3-deoxyapomorphine (1 34 R = OH) the 0,O'-methylene ether of N-propylnorapomorphine has been preparedls3 and a series of N-substituted nor-apomorphines have been synthesized as potential dopamine antagonists.la4 The physiological and pharmacological effects of actino- daphninelR5 of c~ebanine,~~ 18' 18' of dehydrocrebanine,ls8 of dicentrine lHH of dehydrodicentrine 188 of glau~ine,~~~ of ~tephanine,~~ of ~ylopine,'~ of apo- of dehydr~stephanine,?~ morphine,190-207 and of N-propylnorapomorphine'08~209 have been studied.R* OR* OR2 17.3 Oxoaporphines Oxoaporphine alkaloids have been isolated from the following plant species that marked with an asterisk being a new alkaloid Alphonsea mollis210 liriodenine and oxostephanine Anisocycla racemosa55 liriodenine Annona ~herimolia~~ lanuginosine liriodenine and lysicamine Artabotrys ucinatus211 artacinatine* (1 35) atherospermidine and liriodenine Cananga odor at^'^ oxoushinsunine Dehaasia triandrd' atheroline Goniothalamus scortechnii212 liriodenine and oxostephanine Goniothalamus tapis212 liriodenine and oxostephanine Oncodostigma monos perm^^^^ liriodenine and lysicamine Xanthorhiza simpli~issima~'~ liriodenine Artacinatine (1 35) is the first representative of a new structural type of aporphinoid alkaloid being an 11-oxo-ring-c-reduced dehydro-aporphine.Its structure was deduced from its spectra and confirmed by an X-ray crystallographic study.211 The effects of oxocrebanine on cholinergic receptors have been studied. 188 17.4 Dioxoaporphines Norcepharadione-A has been isolated from Oncodostigma monospermalo' 17.5 Aporphine-Benzylisoquinoline Dimers The proaporphine-benzylisoquinoline dimers patagonine and valdivianine together with the aporphine-benzylisoquinoline dimer pakistanine have been isolated from Berberis empetri- foli~.~ It may be noted that the alkaloids natalamine (124 R = CH20H) and natalinine (124 R = CHO) obtained from the same plants are probably degraded aporphine-benzyliso- quinoline dimers.17.6 Aristolochic Acids and Aristolactams The new alkaloid piperolactam (136) has been isolated from Piper boehimerfolium and from P. 10ngum.~~~ The fluorescence characteristics and ultra-violet absorption of aristolactam N-methylaristolactam and aristolactam P-glucoside over a pH range of 1-14,214 the 13C NMR spectra of a number of aristolochic acids and ari~tolactams,'~~ and the binding characteristics of aristolochic acid216 and of aristolactam N-8-gl~coside~~~ to DNA have been studied.NPR X 354 NATURAL PRODUCT REPORTS 1991 OMe OMe OMe OMe Me0 OMe 0 OMe OMe OMe OMe Me0 0 OMe OMe (1 43) (144) Me0 R' Me0 OH R' R2 (1 47) I I OMe OMe OH Me0 0 OMe 18 Morphine Alkaloids Alkaloids of the morphine-hasubanonine group have been isolated from plants of the following species the seven marked with asterisks being new alkaloids Corydalis rnajorilo6 sinoacutine Limacia oblonga21s limalongine* (1 37 R = H) and clolimalongine* (1 37 R = C1) Papaver thebaine Sarcocapnos enne~phylla~~~ 0-methylpallidine-N-oxide* (138) Step h ania y un anensis ' sinoacutine Stephania zippeliana220 erromangine* (139) stephodeline isostephodeline tannaghe* (140) zippelhe* (141) and zippelianine* (142) Of these alkaloids clolimalongine (137 R = Cl) is an isomer of actumidine differing only in the position of the ring-A methoxyl group and 0-methylpallidine-N-oxide is the first morphinandienone N-oxide to be isolated from a plant.Since thirty free bases have been isolated from Sarocapnos enne- aphylla and no other N-oxide (138) is assumed to be a genuine constituent of the plant and not an artifact. Isostephodeline was originally assigned the structure of the C-14 epimer of stephodeline (143) but further studies have led to a revision of its structure to (144).220 6-Demethoxythebaine (145 R1= R2 = H) and its 6-chloro 6-bromo (145 R1= Br R2 = H) 7-chloro (145 R1= H R2= Cl) and 7-bromo (145 R' = H R2 = Br) derivatives have been converted into epoxides (146) and these have been further transformed into diols (147 R3= OH) azides (147 R3= N3) and amines (147 R3= NH2).221.222 14-Chloro-~-chlorocodide (148 R = Cl) and its 14-bromo analogue (148 R = Br) have NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY Me0 Me0 CIO@AMe ,A R CI / (1 49) Meon -(1 54) (155) HO Me0 HO OH "&kCHO 0 0@AMe (157) been converted into the neopine derivatives (149 R = Cl) (149 R = Br) (149 R = N3) and by rearrangement into the hasubanan derivative (1 50). 2233 224 1-Bromocodeine (1 5 1 R = Br) has been converted into the bases (1 5 1 R = CH=CH Ph) (151 R = CH=CHCO,Me) (151 R = C02Et) and (151 R = Me) by palladium-catalysed coupling reactions with the appropriate halide.225 N-formyl-6-0-methanesulphonylnorcodeine(1 52) with lit- hium bromide gave N-formylnorbromocodide (1 53) which were converted into the diene (154) by potassium t-butoxide and this was rearranged by acid to the diphenyl(l55) accessible directly from (152) by prolonged action of lithium bromide.Reduction of (153) with zinc and methanol gave N-formyl- nordeoxycodeine (1 56).226The action of potassium t-butoxide on 14-hydroxy-3,6-0-methanesulphonyl-dihydromorphine has given the structurally rigid base (157).227 N-ally1 and N-cyclopropylmethyl 14-acetoxydihydronormorphinone have been found to undergo 0to Cmigration of the acetyl group on heating with sodium hydroxide in methanol the products being the 7-acetyl compounds (1 58 R = CH2CH=CH,) and (158 R = CH2C3H,) respectively.228 Dihydromorphinone and naltrexone have been converted by the Robinson annelation reaction with methyl vinyl ketone into the bases (159 R' = H R2 = Me) and (159 R1 = OH R2 = CH2C3H5) both of which were dehydrated to equilibrium mixtures of the related ap-and py-unsaturated ketones.229 N-formyl-6-demethoxynorthebaineunderwent Diels-Alder addition of methyl vinyl ketone to give mainly the 7a-acetyl 356 Me0 io Me0 Aco@LcHo R' Me0 Me0 0 compound (160 R = COCH,) whereas addition of nitroethene gave mainly the 8P-nitro compound (161) resulting from addition on the opposite face of the diene together with smaller amounts of the isomers (162) and (160 R = Reduction of 6-demethoxythebaine with zinc and potassium hydroxide gave a 6 1 mixture of 6-demethoxy-P-dihydrothebaine (1 63 R' = R2= H) and the isomeric unconjugated diene.Addition of methyl vinyl ketone to the 0-acetyl-N-formyl compound (163 R' = Ac R2 = CHO) gave a 2:1 mixture of the adducts (164) and (165 R = COCH,) whereas the addition of nitro- ethene gave mainly (165 R = NO,).231 The addition of phenyl- sulphonylpropadiene to thebaine has also been Details of the preparation of the following have been published [1-3H]-labelled thebaine ;233 hydrazones dinitro-phenylhydrazones and semicarbazones of dihydromorphinone 14-hydroxydihydromorphinoneand their N-ally1 analogues ;234 NATURAL PRODUCT REPORTS 1991 Me0 Me0 AcogicHo HO Me0$j!hR -.-kMe HO Me 5-methyl-14-hydroxydihydromorphinone and codeinone from 5-methyl-thebaine ;235 6-N-ally1 derivatives of 6P-amino- 14-hy- droxydihydrodeoxymorphine and its N-cyclopropylmethylnor- analogue in which the acyl groups were COCH(NH,)CH,- CO,H COCH,CH,CO,H COCH=CHCO,H and COCH,- OCO,H ;236 the bases (166 R = CH(CO,Me),) (1 66 R = CH-(CN),) (166 R = CH(CN)CO,Et) and (166 R = CH(S0,Me-(CO,Et) from P-chlorocodide and the appropriate carba- nion ;237 normorphine 3 and 6-0-glucuronides ;238 N-cyclo-butylmethyl-14-methoxydihydronormorphinone;239 N-substi-tuted derivatives of 14-methoxy and 14-ethoxy-0-methyldi-hydronorthebainone ;239 240 Diels-Alder addition products of northebaine and its N-chloroacetyl and N-succinyl derivatives with nitrosobenzene ;,dl 14-hydroxydihydromorphinone oxime and its derivative^;,^ 6-S-acety1-6-thioi~omorphine;~~~ 14-N-(p-chlorocinnamoyl)-aminodihydro- N-cyclopropylmethyl-norcodeinone ;244 bases of general structure (1 67) in which R1= H or Me R2= H or OH and X = 0 or S or NH and (168) in which R1= H or Me and R2 = H or OH;245,246 binaltorphimine and some of its analogues;247 and a variety of bases of structure (1 69) in which R = CH,F (CH,),F (CH,),F CH,CHMeCH,F Et Pr and A study of the kinetics of proton transfer to thebaine has been made.249 In approaches to the synthesis of alkaloids of this group 2.3-di(methoxycarbony1)norreticuline has been oxidized to the salutaridine derivative (I 70), and salutaridine has been obtained by the oxidation of reticuline by oxygen and cytochrome P450- linked NADPH.73 NATURAL PRODUCT REPORTS 1991-K.W. BENTLEY Methods of detection and estimation of mor-Androcymbium palae~tinum~~~ phine250-'256 of codeine,250v255,256 of codeine 6-glucu-androbhe* (174 R' = H R2= Me) norandrobine* of and (174 R1= R2= H androcimine* (174 R' = R2= r~nide,~~O norc~deine,~~~ of dihydr~codeine~~~,~~~ have been described. Me) androche* (175 R1= H R2= Me) (+)-The analgesic properties of morphine have been ~t~died~~~-~~~ kreysigine* (175 R' = Me R' = H) O-methylnor-on the as have the effects of the alkaloid on behavio~r,~~~-~~~ kreysigine* (176 R = H) 0-methylkreysigine (176 R cardiovascular ~y~tem,~~~-~~~ on the gastro-intestinal = Me) and szovitsamine tract,304-300; ne~rones,~~~-~~~ the transmission of nerve imp-Merendera k~rdica~~~ u1ses,312.313 the brain 314-316 the spinal ~~rd,~~~.~~~ mono-baytopine aminergic systems,318 and temperature319 on locomotor ac- Of the new alkaloids the relationship between androbine on the norandrobine androcimine androcine kreysigine and 0-ti~ity,~~' hippocampal ce11s,322,323 oxygenation of haemogl~bin,~~~ immune methylnorkreysigine was confirmed by the preparation of 0-the nutrition,"' bronchospasm caused by histamine and by sero- methylkeysigine from all of these.44' The UV spectra of t~nin,~*~ the adrenal medulla,328 opioid patterns bechuanine kreysigine and 0-methylkreysigine have been the release of d~pamine~~l of ~leep,~:'O and of histamine,332 levels studied.447 of of the corticotrophin releasing of adenyl~ylase,~~~ A review of homoaporphine alkaloids with physical data peptides in brain,335 of luteinizing of blood has been published.449 sugar,33i,338 and of substance P in brain;339 and on the effects of chlorampheni~ol,~~~ cl~nidine,~~' of ligno~aine,~~~ mida~olam"~~ The metabolism of and of Pertussis to~in.~~~.~~~ morphine has also been The morphine-antagonist effe~t~~~~-~~~ and analgesic pro- perties35'l355 of naloxone have been studied as have the effects Me0 YOSiMe3 of this compound on behavio~r,~~~-~'~ on the cardiovascular SyStem,363-386 on the gastro-intestinal tra~t,~~~,~~~ Q-OMe on the ~~~ brain,"' on the spinal ~ ~ r on dcerebral ischaemia,3i2 ~ ~ ~ memory,373 capillary permeability,374 flash-evoked potential^,^'^ OMe regulation of the opiate re~eptor,~'~.~~' response to the cold 0 pressor test,3i8 bronchospasm induced by histamine and by the activity of GTP~S~,~'~ anaphylactic haemorhagic hock,^^'.^^^ thermal the effects of aten0101"~ and of phen~yclidine,"~ and the metabolism of serotonin in the brain.386 The binding of naloxone in the frontal and the pharmokinetics of the have also been studied.The physiological and pharmacological properties of the following have been studied her~in,~*~."~ 3053 codeine,283. 326-390-391 morphine 3-gluc~ronide,~~~ morphine 6-gluc~ronide,~~~-~~~ norm~rphine,~~~ N-allyln~rmorphine,~~'-~~~ naloxone nordihydrocodein~ne,~~~ na1trex0ne,~~~~~~~~~~~-~~~ methobr~mide,~~~ naltrexone meth-ObrOmide,356, 410 nalbuphine,272.297,407,411-413 nalmefene,414 14- hydroxydihydromorphinone methobr~mide,~~~ 14-hydroxydi-hydromorphinone hydraz~ne,~~~ 14-hydroxydihydrocodei-OMe none,417418 naloxone benzoylhydraz~ne,~~~ naloxona~one,~~~ (173) funaltre~amine,~~~-~~~ naloxone norbinalt~rphimine,~~~-~~~-~~~ 6-spi ro h ydan toin N-cycloprop ylme thyl- 6-azidodi hydrodeo- xynormorphine;428 N-cyclopropylmethyl- 14-p-methylcinnamo- ylaminonormorphine its p-chloro and p-bromocinnamoyl ana- logues and the corresponding derivatives of norcodeinone ;429 N-cyclopropylmethyl-14-p-chlorophenylacetylaminodihydron-~rmorphine,~~~,~~~ bupren~rphine,~~~.~~~-~~~ et~rphine,~~~ 7a-(2-hydroxy-5-methylhex-2-yl) -6,14-endo-ethenotetrahydro- o~ipavine,"~7a-(2,2'-dichlorodiethylaminomethyl)-6,14-endo-ethen~tetrahydro-oripavine,~~~~~~~ N-allyl-7a-(2,2'-dichloro- diethylaminomethyl)-6,l4-endo-ethenotetrahydro-oripavine,444 and N-cyclopropylmethyl-7a-(2-hydroxy-5-methy~hex-2-y1)-OH dMe 6,14-endo-ethenotetrahydronorthebaine. 433 (174) (1 75) 19 Phenethylisoquinolines A review of the chemistry of the phenethylisoquinoline alkaloids has been published.445 R-homolaudanosine (1 73) has been synthesized by trapping the acyliminium ion derived from the amide (171) by the enol ether (172) followed by catalytic reduction and N-methylation of the 20 Homoaporphi nes Homoaporphine alkaloids have been isolated from the fol- lowing plant species the six marked with asterisks being new alkaloids NATURAL PRODUCT REPORTS 1991 Me0 OH OMe (1 77) Me0 t M e O G :C0,Me Me0 H ,R NHAc Me0 Me0 'OSi Me Me Meo% OMe \ / OH But (1 87) (1 88) 21 Colchicine Alkaloids related to colchicine have been isolated from the following plant species Androcymbium pal~estinurn~~~ androbiphenyline* (177) alkaloids K3 (178 R = Me) and K4 (178 R = H) 3-demethylcolchiceine and demecolcine Merendera kurdicag4' colchicine 2-demethylcolchicine 3-demethylcolchicine N-formyl-N-deacetylcolchicine, cornigerine and deme- colcine Merendera manissadjianiig4' colchicine 2-demethylcolchicine 3-demethylcolchicine N-formyl-N-deacetylcolchicine, cornigerine and deme- colcine Merendera sob~lifera~~~ colchicine 2-demethylcolchicine 3-demethylcolchicine N-formyl-N-deacetylcolchicine cornigerine and deme- colcine An X-ray crystallographic determination of the structure of allocolchicine has been as have confirmational analyses of colchicine and isoc~lchicine.~~~ The demethylation --NHAc Meoy Me0 0-co Me Me0 HAc OMe OMe OMe n h \r) 1 0 MeoTCHo OSiSMe Q Me0 Me OMe 'But of 2-demethylcolchicine and 3-ethoxycarbonyl-3-demethyl-colchicine by sulphuric acid has been studied; the former gave 1,2-demethylcolchicine as major product with a smaller amount of the 2,3-demethyl isomer and the latter gave 2,3-demethyl- colchicine as the sole product; both demethylation products were hydrolysed to the corresponding derivatives of col-chicine.453 P-Lumicolchicine (1 79) on oxidation with m-chloroper-benzoic acid appears to suffer fission of the cyclobutene system and addition of the reagent rather than conversion into an epoxide the product having the structure (1 80) as deduced from NMR studies.With manganese dioxide P-lumicolchicine is converted into the oxazole (181) the structure of which was elucidated by X-ray crystallographic methods.454 Colchicine has been reacted with glycine in aqueous-alcoholic sodium hydroxide to give colchidicylglycine (182 R = OH) the acid chloride of which with amino-acid esters gave the glycine ester derivative (182 R = NH .CH,CO,Et) and its analogue^.^"*^^^ A patent for the preparation of derivatives of 3-demethyl-thiocolchicine has been 14C-labelled colchicine has been preparedg5' and the diester (1 83) has been converted into demethoxydeoxydihydro-colchicine (184) which constitutes a formal synthesis of the alkaloid.459 N-acetylcolchinol (1 88) a degradation product of colchicine has been synthesized from the aldehyde (1 85) and the Grignard reagent (186) via the alcohol (187 R = OH) the azide (1 87 R = N3),and the acetamide (1 87 R = NHAc).The last of these on treatment with trifluoroacetic acid trifluoro- acetic anhydride and thallium (111) trifluoroacetate gave (1 88).460 The biological effects of col~hicine,~~~-~~~ 3-demethyl-col~hicine,~~~ and methylthioether~~~~ and thioketone~~~~ of natural colchicoids have been studied.NATURAL PRODUCT REPORTS 1991-K. W. BENTLEY 359 0 0 {F OCHZPh OCH2Ph OkH,Ph 0 (1 92) (1 93) (1 94) OH 0 OCHZPh <XI(& <XI$& 0 SiMe, *: C02Me ? O=CCMe3 COCMe3 aldehyde (191). Wittig reaction of this with (MeO),P(O)CH,COCH gave the unsaturated ketone (192) 0 which was reduced with debenzylation to the saturated ketone (193) which exists in equilibrium with the semi-acetal (194). Reduction of the mixture gave the diol (195) which was oxidized by dimethylsulphoxide and phosgene to the diketone (196) and this gave (1 97) on base-catalysed aldol condensation and dehydration.467 In the cephalotaxine group both 2’S 3’s and 2’R 3’R- methylcephalotaxyltartrates have been prepared from cephalo- 22 Erythrina and Related Alkaloids taxine and (-)-and (+)-monomethyltartrates and deter- A review of the chemistry of the Erythrina alkaloids has been mination of their configurations by NMR spectroscopy has published.466 confirmed the structures (198) for harringtonine (2’S 3’s)and ( +)-3-Demethoxyerythratidinone (197) has been synthesized isoharringtonine (2’R 3’R).468 The esterifying acid of iso- from the keto-ester (189) itself obtained by the addition of harringtonine (1 99) has been synthesized in a stereochemically homoveratrylamine to the ap-unsaturated keto-ester controlled process from R,Rtartaric acid through the acetonide CH2=CHCOCOC02But followed by Bischler-Napieralsky of the dimethyl ester.469 In the synthesis of analogues of ring closure in the presence of a Lewis acid.The ketone (189) cephalotaxine the base (201) has been obtained by a photo- was reduced to the alcohol which was benzylated to (190) cyclization of the bicyclic compound (200).470The addition of which was further reduced by lithium aluminium hydride to the the nitro-compound (202) to methyl acrylate gave the ester NATURAL PRODUCT REPORTS. 1991 OH OH (206) < 0 (203) reduction of which gave the lactam-ester (204) which was reduced and cyclized to (205 R = OH) and further reduced to (204 R = Conversion of this into the diol (206) H).4713472 with osmium tetroxide followed by oxidative cleavage to the dialdehyde (207) and base-catalysed cyclization of this gave the unsaturated aldehyde (208).471 Following the successful syn- theses of chilenine and magallanesine reported in the previous review the processes used have been adapted to synthesize the tricyclic compound (216) which was an intermediate in Weinreb’s synthesis of cephalotaxine and this therefore constitutes a new formal synthesis of the alkaloid.The dihydroisoquinoline (209) and the ester-acid chloride (2 10) reacted to give a mixture of the amides (21 1) and (212) both of which were converted into the thio-acetal (213 X = 0) by heating with ethane dithio! and sodium hydride. 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ISSN:0265-0568
DOI:10.1039/NP9910800339
出版商:RSC
年代:1991
数据来源: RSC
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5. |
Terpenoid phytoalexins |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 367-389
C. J. W. Brooks,
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摘要:
Terpenoid Phytoalexins C. J. W. Brooks Chemistry Department University of Glasgow Glasgow Scotland G 72 800 D. G. Watson Department of Pharmacy University of Strathclyde Glasgo w Scotland G7 7X W Reviewing the literature published between August 1984 and December 1989 1 Introduction 2 Terpenoid Phytoalexins 2.1 Sweet potato (Ipomoea batatas) 2.2 Cotton (Gossypium spp) 2.3 Elm (Ufmusspp) 2.4 Tobacco (Nicotiana spp) 2.5 Sweet pepper (Capsicum annuurn) 2.6 Potato (Solanum tuberosum) 2.6.1 Biosynthetic Aspects 2.6.2 Aspects of Elicitation 2.6.3 Toxicity and Biotransformation 2.6.4 Synthesis 2.7 Aubergine (Solanum melongena) 2.8 Tomato (Lycopersicon esculen tum) 2.9 Coffee (Coflea spp.) 2.10 Rice (Oryza sativa) 2.1 1 Castor Bean (Ricinus communis) 2.12 Cassava (Munihot escufenta) 2.13 Lettuce (Lactuca sativa) 2.14 Lodgepole pine (Pinus contorta) 2.15 Madagascar Periwinkle (Tabernaemontana divaricata) 2.16 Periwinkle (Catharunthus roseus) 2.17 Timothy (Phfeum pratense) 3 References 1 Introduction The previous report’ covered phytoalexins of all types but the volume of work published in the period that has now elapsed has prompted the division of the material.The two major classes of phytoalexins are still the terpenoid and isoflavonoid types. This report aims to cover the principal work on the terpenoid phytoalexins but it also contains selected references to cognate researches and to some aspects of general importance.Research has been based increasingly on the elicitation and accumulation of phytoalexins in plant cell suspension cultures. Knowledge of these processes is most advanced for iso- flavonoids and related phytoalexins biosynthesized via the phenylpropanoid pathway. Studies of a variety of preparations of soybean (Glycine max) have been particularly extensive leading for example to the demonstration at the onset of phytoalexin biosynthesis of elicitor-enhanced activities of mRNA’s encoding particular enzymes of phytoalexin bio-synthesk2 Specific binding of an elicitor to membrane fractions from soybean roots has been ob~erved.~ Enzymes responsible for synthesizing phytoalexins have been isolated from other species e.g.chickpea (Cicer arietin~m).~ Reviews published since the last Report reflect the pre- ponderance of results as regards biochemistry and enzymology in the field of phenylpropanoid phytoalexins ;but they include much that is of wider significance. In addition to general surveys of the phytoalexin res~onse,~.~ some more recent reviews may be noted which feature particular themes in phytoalexin studies e.g. elicitation,?-ll metab~lism,~ genetic 367 aspects,8*11-14 role in plant resistance,? 15,16and detoxification of phytoalexins :17 also terpenoid phytoalexins,ls9 l9 their bio- synthesis,20 and their 13Cand lH NMR spectra.21 Other subjects of reviews have been phytoalexins and stress metabolites in the sapwood of trees,22 antimicrobial defence compounds in the G~aminae,~~ mycotoxins and phytoalexins in stored crops,24 and the occurrence and toxicity of phytoalexins in food derived from plants.25 Proceedings of symposia on plant-microbial interactions26 and fungal infection of plants2’ have been published. In this Report as before,l the metabolites mentioned are not necessarily all true phytoalexins but are generally antifungal compounds (or close relatives thereof) produced by the action of elicitors. 2 Terpenoid Phytoalexins 2.1 Sweet Potato (Ipomoea batatas) Sweet potato root tissue infected with Ceratocystis fimbriata (black rot fungus) produces a large group of interrelated furanoterpenoid phytoalexins (see reference 1 p.428). The enzyme that catalyses the reduction of 7,8-didehydro-ipomeamarone (1) to ipomeamarone (2) has been studied it was not present in fresh tissue but was formed within 12-24 h in response to inoculation with C. fimbriata. This enzyme required NADPH for activity and had a pH optimum of 7.5-8 the apparent K value for (1) was 45 pM.28 Strains of C. fimbriata respectively compatible and in- compatible with sweet potato have been compared markedly different growth was demonstrated in liquid media containing exudates from the infected sweet potato root tissues. Furano- terpenoid phytoalexins and umbelliferone (7-hydroxy-coumarin) (3) appeared to be the main metabolites affecting host-parasite specificity. 29 \/ (3) NATURAL PRODUCT REPORTS 1991 i ii iii ao+xo-0 iv v vi vii -0 d”rs /Po viii / Reagents i EtOH NaOEt -10 “C; ii oxalic acid H,O reflux; ill (CH,OH), TsOH PhMe heat; iv BH;THF; H,O, NaOH; v PyH+.CICrO,- CH,Cl ; vi NaH Me,SO methyltriphenylphosphonium bromide ;vii 1 M HCI silica gel CH,CI ; viii CH,=CLi-CH,OLi (3 equiv.) 0 “C; ix MnO, PhH or Cr0,-Py complex on Celite Scheme 1 O@ -@-Ho*H A A Thickened lines denote units from [l,2-13C2]acetate (6) Scheme 2 $Lo-J$k-$p-p + H * denote 14Clabels from [2-14C]or [4-14C] mevalonate J -M eOm MeOg erp -c-. HO oe H (7)-M // Me0 Me0 Me0 R2 R3 0 (11) R’ = OMe R2= Pr’ R3 = H (10) (9) (7)Y = 0 (12) R’ = OMe R2 = R3 = H (8) Y = NPh (13) R’ = R3= OAC R2= P Scheme 3 NATURAL PRODUCT REPORTS 1991-C.J. W. BROOKS AND D. G. WATSON The structures of the sweet potato phytoalexins 7-hydroxy- costol (4) and 7-hydroxycostal (5) (reference 1 p. 428) have been confirmed by synthesis of the racemic compounds (Scheme l).30 2.2 Cotton (Gossypium spp) The biochemistry of resistance to pathogens has been re-viewed.31 The biosynthesis of 2,7-dihydroxycadalene(2,7-DHC) (6) was studied by feeding [1 ,2-13C2]acetate to cotton seedlings inoculated with Xanthomonas ~ampestris.~~ The most striking feature of the 13C NMR spectrum was that the two hydroxyl- bearing carbons afforded singlets and this together with the 13C-13C couplings was consistent with the folding pattern of the farnesyl diphosphate skeleton shown in Scheme 2.A different mode of cyclization based on a cis,cis-farnesyl skeleton had been for the biosynthesis of gossypol (7) ;however compelling evidence supporting the intermediacy of cis trans-farnesyl diphosphate has emerged from two studies. Gossypol biosynthesized in the roots of cotton seedlings (Gossypium herbaeum) was 14C-labelled by the incorporation of [2-14C]mevalonate or of [4-14C]mevalonate and was purified as its dianilino derivative (8). Conversion via the bis-acetal(9) into gossic acid (10) was accompanied by loss of one-third of the molar radioactivity derived from [2-14C]mevalonate but no loss from gossypol biosynthesized in the presence of [4-14C]mevalonate. Further oxidative degradation yielding iso- butyric acid from the C-isopropyl moiety indicated the presence of one [14C]-labelled atom therein when [2-14C]mevalonate had been present in the incubation In a further experiment gossypol was biosynthesized in the presence of [5-3H]mevalonate and [2-14C]mevalonate and degraded to the compounds (1 I)-( 13).The 3H :14C atomic ratios were very close to 2 6 in (1 1) and (13) and were 0:4 in (12) consistent with the occurrence of the hydride shift indicated in Scheme 3 in the first germacrene intermediate formed by cyclization of farnesyl diph~sphate.~~ the In an independent in~estigation,~~ observed incorporation of [I ,2-13C,]acetate into gossypol (7) elicited (by chilling) in the radicles of seedlings of G. arboreum was indicative of cis,trans-farnesyl diphosphate or an equiv- alent as the main biosynthetic precursor i.e.the folding pattern was again as in Scheme 2.35 The majority of commercial cotton plants are cultivars of Gossypium hirsutum but the typical phytoalexins are also produced by other species such as G. arboreum and G. barbadense :some cultivars of the third species show particularly high resistance to wilt caused by Verticillium dahliae.36 Lesions induced in the leaves of G. hirsutum and G. arboreum by inoculation with conidia of a virulent V.dahliae mutant were in the former species reduced in surface area and in the latter completely prevented by concurrent inoculation with an avirulent mutant. The protective effect of the avirulent mutant resulted from accelerated elicitation of phytoalexins a process which was very strong in G.arb~reum.~’ A 2-3-fold increase in the content of sesquiterpenoid phytoalexins was observed in G. arboreum cell suspension cultures inoculated with a hot water extract from V. dahliae ~onidia.~* Addition of sodium [14C]acetate to the elicited cultures resulted in up to 30 YOof the label being incorporated in sesquiterpenoids. The sesquiterpenoids accumulated mainly in the cells (up to 0.27% of the dry weight) with only 20% of the cellular amounts accumulating in the medium. The phytoalexins elicited in plants or cell cultures inoculated with V. dahliae include gossypol (7) deoxyhemigossypol (14) hemigossypol (15) the corresponding 6-methyl ethers (16)-( 18) and gossypol 6,6’-dimethyl ether (19).36*38 In the work reported in reference 39 the total amounts of sesqui- terpenoids elicited were consistent between replicates but their relative amounts varied.The hot water extract from V.dahliae caused suppression of embryo production from suspended callus cultures of cotton. The culture system may have potential for the selection of resistant lines of cotton. Compounds (7) (14) R=H (15) R=H (17) R=CH3 (18) R=CH3 CHO OH OH CHO (17) R=H R’=H (16) R=CH3 R’=H (19) R=CH3 R’=CH3 (20) R = H (21) R=CH3 (14)-(16) are all fungicidal to V.dahliae but only (14) seems to have the properties that would enable it to act as a phytoalexin in cotton i.e. the phytoalexin accumulates at a fungicidal concentration in the infected stem xylem and is water-soluble up to this concentration.Histochemical studies showed that (14) accumulated in deposits on V.dahliae mycelia as early as 2 days after inoculation and was thus responsible for the death of mycelia and conidia in infected vessels in the stem stele of wilt-resistant Seabrook Sea Island (SBSI) The stems of four cotton cultivars representing G. barbadense and G. hirsutum were inoculated with conidia of mild (SS-4) and severe (T-1) strains of V.dahliae :42 Compounds (14) (1 5) (17) and (18) accumulated in the tissues of all the inoculated cultivars. After inoculation the highest concentrations of the methylated sesquiterpenoids (17) and (1 8) were present in the vascular tissues of the most resistant cultivar (SBSI); the lowest levels of (17) and (18) were present in the most susceptible cultivar.The lowest levels of the unmethylated metabolites (14) and (15) however were almost always found in the SBSI cultivar. The moderately resistant cotton cultivars contained intermediate levels of these compounds. Thus the degree of resistance to Verticillium wilt is directly related to the amounts of methylated sesquiterpenoids produced by infected tissues. The severity of the symptoms may also be related to the capacity of strains of fungi to overcome the toxicity of these compounds. Mycelial and culture fluid extracts from Aspergillus j?avus were applied to wounds on SJ-2 cotton leaves.43 Five fluorescent compounds were induced in the stems as judged by TLC and these were identified in relation to standards as lacinilene C (20) lacinilene C 7-methyl ether (21) 2,7-DHC (6) and 2,7- DHC 7-methyl ether (22).It was found that scopoletin (6- NPR 8 methoxyumbelliferone) was also elicited. Analysis of leaves of SJ-2 cotton for several days after treatment with A. flavus indicated that amounts of phytoalexins increased up to 2 days fell during the next 3 days increased again between days 5 and 6 and declined thereafter up to day The greatest concentration of compounds was found in the 3 mm zone encircling the area that had been treated with the fungal extract. Accumulation of the compounds also occurred in wounded control leaves on days 5 and 6 suggesting an effect due to constitutive elicitors which may act synergistically with fungal elicitors producing maximum accumulation at 6 days.The phytoalexin response in cotton results in the accumu- lation of the fluorescent lacinilenes in infected tissue. In order to validate fluorescence as a measure of the distribution of phytoalexins within infected cotton tissue an automatic cell sorter was used to separate highly fluorescent cells present in necrotic lesions from the less fluorescent surrounding cells.45 The fluorescent necrotic cells were found to contain ca 20 times as much lacinilene C 7-methyl ether (21) and ca40 times as much 2,7-DHC (6) as the surrounding less fluorescent cells. More than 90% of 2,7-DHC (6) and 71-100% of the other compounds were located at the infection sites.Deoxyhemi- gossypol(l4) showed the most discrimination in favour of lesion sites and 2,7-DHC 7-methyl ether (22) the least. Membrane potential-sensitive dyes were used to monitor the events involved in the early stages of the elicitation of defence response in cotton cell The cultures were equilibrated with the dyes before addition of a cell-wall extract from V.dahliae. After a lag phase of 5 minutes a rapid decrease in the fluorescence of the cells was observed in cells equilibrated with the pH-sensitive dye but the rate of decrease of fluorescence was less marked in the case of the pH-insensitive dye. Increasing the concentration of elicitor resulted in a decrease in the lag phase and also led to a decrease in the rate of fluorescence decay.These observations mirrored the fact that phytoalexins accumulate more slowly at higher elicitor concentrations in cotton cultures. Fractionation of the crude elicitor resulted in fractions in which the effect on the decay of fluorescence could be roughly correlated with their effectiveness in eliciting phytoalexins. Similar efflux of dye could be observed in tobacco and soybean cultures. These observations support the idea that elicitation involves the opening and closing of ion channels. Sodium citrate was found to inhibit the decrease in fluorescence caused by the efflux of the pH-sensitive membrane potential- sensitive dye;47 it had no effect on the efflux of a pH-insensitive dye indicating that its effect is specifically on proton fluxes.Addition of citrate immediately after the addition of elicitor had little effect on the rate of fluorescence decay. Among a number of di- and tricarboxylic acids tested the only other compound with a similar effect was sodium malate. Citrate also inhibited the accumulation of phytoalexins by the cultures. Addition of the strong Ca2+ chelator EGTA did not inhibit fluorescence decay indicating that the mode of action of citrate was not via Ca2+ chelation. Inhibition of phytoalexin ac-cumulation by citrate was also observed in cotton seedlings stimulated by biotic or abiotic elicitation. The effect of the cotton phytoalexin 2,7-DHC (6) on Cauliflower mosaic virus (CaMV) was tested. CaMV was inoculated into turnip leaves and irradiated with UV light; the irradiated virus was found to produce lesions on the leaf.48 When CaMV was irradiated in the presence of (6) the virus was not able to produce local lesions; incubation of CaMV with unirradiated DHC did not reduce the infectivity of the virus.Inoculation of the leaves with CaMV DNA did not produce lesions but the leaves became infected exhibiting chlorotic spots and vein clearing; treatment of CaMV DNA with lacinilene C (22) did not alter infectivity and electrophoretic patterns of the DNA were not altered. Similarly irradiation of CaMV DNA with UV light did not reduce infectivity or alter electrophoretic patterns. Irradiation of CaMV DNA in the presence of DHC caused extensive fragmentation of the DNA so that it was composed of relatively more linear DNA and less NATURAL PRODUCT REPORTS 1991 circular (plasmid) DNA.2,7-DHC (6) probably causes scission of DNA strands and cross-linking of DNA to the coat protein of the virus. Further investigations of the effect of UV and visible radiation on the biological activity of (22) and also on the activity of lacinilene C (20) were carried out using sunlight or ‘cool-white’ fluorescent lamps (A = 300-700 nm).49 When these compounds were irradiated they induced single strand breaks in DNA of plasmid pBR322. Irradiation at wavelengths 239 nm and 300 nm [near the absorption maxima of 2,7-DHC (6)] was more effective than irradiation at 400 500 and 600 nm in activating (6) to ‘nick’ DNA. The latter process required oxygen and was inhibited by scavengers of reactive oxygen species and of free radicals.Cultures of Xanthomonas campestris pv. maZvacearum were only partially inhibited by (6) (0.1 mM) in the dark but upon irradiation this concentration was bactericidal accordingly (6) appears to be a photosensitizer. Leaves from susceptible and resistant cotton were inoculated with X. campestris and maintained either under a light/dark cycle or in continuous darkness.50 Sesquiterpenoid phytoalexins accumulated to a much lesser extent in leaves kept in the dark compared with those kept under a light/dark cycle and although the ultrastructural reaction of the cultivars maintained in the dark resembled a hypersensitive response the growth of the bacteria was not inhibited in either susceptible or resistant plants under these conditions.The determination of four of these metabolites (14) (15) (17) and (18) in cotton stele infected with V. dahliae has been effected by HPLC.51 Gossypol (7) is chiral by reason of restricted rotation. The material isolated from cotton is racemic but interest in its activity as an oral anti-fertility agent (in males) has required studies of individual enantiomers. Res- olution has been achieved via HPLC of diastereomeric Schiff s base derivative^,^^-^^ and distinctive biological activities have been observed for (+)-and (-)-(7).54 Several techniques (CD NMR employing chiral solvating agents and chromatography on chiral stationary phases) have been applied independ- ently to the cotton phytoalexin (+)-lacinilene C (20).The R-configuration indicated by these methods was verified by X-ray diffraction and chemical correlation showed that (+)-lacinilene C methyl ether (21) itself was also the R-f~rm.~~ 2.3 Elm (Ulmus spp) The accumulation of the cadinane sesquiterpenoids mansonone A (23) and mansonones C-G(24)-(28) was studied in Ulmus americana in relation to inoculation with aggressive and non- aggressive strains of Ophiostoma ~lmi.~~ The non-aggressive strain was observed to induce higher concentrations of the phytoalexins in the upper stems of inoculated seedlings A (24) R = H (28) R=OH NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON compared with the aggressive strain. There was no difference in the levels of accumulation observed in the lower stem.Inoculation with both strains together caused accumulation of intermediate levels of the phytoalexins indicating that the aggressive strain probably suppresses the elicitation of man- sonones. A further study of mansonones E (26) and F (27) induced in Ulmus pumila seedlings after inoculation with aggressive and non-aggressive strains of 0.ulmi showed no significant differences in the amounts of these metabolites elicited by the two ~trains.~’ 2.4 Tobacco (Nicotiuna spp) Tissue cultures of Nicotiana tabacum have proved a powerful tool for exploration of the biosynthesis of sesquiterpenoid OH 1 H (31)R = H (33) (32)R = AC R’ (35)&OH R~=H (36)R’ = H R2 = OH phytoalexins.It was found that treatment of suspended callus cultures of N. tabacum with cellulase ex Trichoderma viride stimulated the formation of four major phytoalexins debneyol (29) capsidiol (30) phytuberol (3 l) and phytuberin (32).5s De novo synthesis of these compounds was confirmed by feeding of [2-14C]acetate to the cultures after they had been treated with cellulase. Debneyol had previously been isolated from leaves of Nicotiana debneyi which had been inoculated with tobacco mosaic virus (TMV).59 Callus cultures of N. tabacum inoculated with Pseudomonas spp. produced (3 1) and (32).60*61 Callus formed from different cultivars produced different amounts of (31) and (32) both in relative and absolute terms. The amounts of phytuberin produced increased with the number of sub- cultures while different strains of Pseudomonas bacteria elicited different amounts of phytoalexins.The feeding of [1,2-13C2]acetate to cultures of N. tabacum after they had been treated with cellulase ex T.viride resulted in debneyol (29) which was enriched with five intact acetate units;62 the labelling pattern provided evidence that its biosynthesis occurred via a methyl migration from C-10 to C-5 as in the course of the biosynthesis of capsidiol (30) in capsicums. The compound cyclodebneyol (33) was isolated from TMV-inoculated N. deb~zeyi.~~ Cultures of N. tabacum treated with a hot water extract from the mycelium of Phytophthora megasperma elicited (30)alone in suspension cultures of N. tabacum ;64a accumulation reached a maximum (60 pg/g fresh weight) after 24 h.In- corporation of added [I4C]acetate into capsidiol at various times after addition of elicitor reflected a transient increase in de novo synthesis. Changes in the activity of HMG-CoA reductase and the elicitor-induced accumulation of capsidiol were inhibited by adding mevinolin (69) a potent inhibitor of HMG-CoA reductase to the cell cultures. Mevinolin was also found to inhibit partially the incorporation [3H]mevalonate into ~apsidiol.~~* A number of minor diols related to debneyol were observed in cultures of N. tabacum which had been treated with cellulase (ex T. viride) when extracts from the culture medium were treated with alkane- or areneboronic acids and analysed by GC-MS.58,65 Further work led to the isolation of three further compounds from the elicited cultures 7-epi-debneyol(34) 1-hydroxydebneyol(35) and 8-hydroxydebneyol (36).66 The isolation of these further metabolites led to a proposal for a biosynthetic pathway to these compounds [Scheme 41.4-Epi-eremophila-9,ll -diene (37) was proposed as H0”-c4;-c4; J \ WH0H (35) (33) Scheme 4 NATURAL PRODUCT REPORTS 1991 y3 c=o OH qyy-w-COPP Scheme 5 (40) R = H (41) R=Ac the common precursor of both debneyol (29) and capsidiol (30). A cell-free system (cfs) prepared from unelicited cultures of N. tabacum and containing NADPH incorporated [l4CC]iso- pentenyl diphosphate (IPP) into farnesol and ~qualene.~~ A cell-free system prepared from cultures treated with cellulase and also containing NADPH incorporated IPP into four major components indicated by peaks on radio TLC due to (29) and (30) and two unknown metabolites.Incubation of [l-3H]-farnesyl diphosphate (FPP) with a cfs from elicited cultures resulted in a similar pattern of incorporation except that there was a higher degree of incorporation into the two unknown components. Unelicited cultures of N. tabacum rapidly catabolized debneyol(29) and capsidiol(30) some of the latter was converted to its 3-acetate but this was eventually converted back to capsidiol and catabolized. During the first 6 h after treatment of N. tabacum cultures with cellulase there was an inhibition of squalene synthetase activity and of the catabolism of (30) but not of (29); the cultures ceased to grow.After 6 h capsidiol and debneyol accumulated rapidly along with the phenolic compound acetosyringone (38). Both squalene synthetase activity and cellular growth remained inhibited. In the final phase levels of (30) but not (29) began to fall and at the end of this phase cellular growth resumed and squalene synthetase activity increased. 4-Epi-eremophila-9,11 -diene (37) was isolated from a large-scale incubation of [l-3H,]-FPP with a cfs prepared from cultures of N. tabacum treated with cellulase when NADPH was omitted from the incubation medium.6s [14C]-labelled (37) was rapidly and efficiently incorporated into capsidiol and debneyol in elicited cultures.The formation of (37) can be envisaged to involve two enzymes FPP carbocyclase giving germacrene A (39) and a germacrene A cyclase giving rise to a eudesmane carbocation followed by hydride and methyl migrations and proton loss. Such processes (Scheme 5) have been proposed (following Robinson's original hypothesis) on the basis of evidence from isotope labelling to occur in the biosynthesis of petasin (43) (44) capsidiol and debneyol. However the 4-epi-eremophilane skeleton present in the latter two compounds (and their congeners) implies a non-concerted process for the re-arrangement in these instances. The results of these important ~tudies~',~~ disprove -at least for the biosynthetic system in tobacco -previous proposals that oxyfunctionalization occurs prior to formation of an eremophilane structure.In a related investigation cell-wall fragments of Phytophthora parasitica were also found to elicit in cell suspension cultures of N. tabucum the rapid synthesis and secretion of sesquiter- penoid phytoalexins [(30) being the major example with unidentified congeners]. Pulse-labelling with [14C]acetate and [3H]mevalonate showed that the induction of sesquiterpenoid biosynthesis was accompanied by a sharp fall in the rate of incorporation of radioactivity into Various other elicitors were tested for their action on tobacco cultures among these sonicated Phytophthora infestans spores and mycelia arachidonic acid pectinase (ex Aspergillus niger) and copper sulphate were found to be ineffective as elicitor^.'^ Autoclaved cellulase (ex T.viride) was also inactive. The unautoclaved enzyme had earlier been found to be active5s and it was later found that the activity in this material was due to the large amounts of glucans present in the crude enzyme preparation rather than the higher molecular weight protein fraction. Methylation analysis of crude active fractions indi- cated that these were composed largely of 1,4-linked glucose units. Further purification of active fractions resulted in loss of elicitor activity (D. G. Watson and C. J. W. Brooks unpub- lished data). Stress metabolites in leaves of Nicotiana undulata inoculated with TMV were examined by GC-MS71 These analyses revealed many sesquiterpenoid stress metabolites nineteen of which were identified.Twelve of these were compounds isolated previously from tobacco leaves viz (30) (31) (32) occidol(40) occidol acetate (41) occidol isomer- 1 (42) occidol isomer-2 (43) occidenol (44) occidentalol (45) solavetivone (46) 3- NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON 0 Rqj ROH Hoqt+ 0 A (45) (46) R = H (47) R = OH R’ = H (48) R= H R’=OH OH (54) (57) R’ = OH R2 = CH3 (58) R’ = CH3 R2 = OH hydroxysolavetivone (47) and 2-keto-a-cyperone (49). Seven new stress metabolites were isolated and identified viz. 3-epi-3-hydroxysolavetivone (48) 1,2-dehydrocarissone (50) capsidiol-3-acetate (5l) aubergenone (52) 4-epiaubergenone (53) trans-dihydrocarissone (54) and 1,2-dehydro-a-cyperone (55).In-oculation of Nicotiana rustica with tobacco mosaic virus elicited the formation of (44) and (45) and a new natural sesquiterpenoid trans-occidentalol (56) [the 15-epimer of (45)].72 A protein isolated from the culture medium of Phytophthora parasitica var. nicotiana produced blackening when applied to tobacco callus and also caused the accumulation of capsidiol (30) in the The elicitor was highly active 80 ng applied to tobacco callus elicited (30) in the tissue at a concentration of (49) (52) R = CH3 R’ = H (53) R=H R’=CH3 (55) 15-20.pg/g fresh weight. The elicitor initially did not appear to be associated with any of the hydrolytic activities of the fungus although subsequently /?(I -+ 4)-endoxylanase activity was detected.Production of antibodies to the protein enabled its presence to be demonstrated in tobacco stems infected with P. parasitica by Western Blotting. The structure and activity of capsiceins apparently novel proteins derived from Phyto-phthora spp. which elicit necrosis and acquired resistance in tobacco have been studied and the complete amino-acid sequences determined. Both proteins are found to protect tobacco against invasion by the pathogen Phytophthora nicotiana (the agent of tobacco black shank) which was itself unable to produce similar elicitors.74 The hypersensitive reaction induced in cell suspension cultures or leaves of tobacco by the incompatible pathogen Pseudomonas syringae pv.pisi was not suppressed in the presence of reagents that quench singlet oxygen suggesting that such a radical has no significant role in the hypersensitive res~onse.’~ A glucan preparation obtained from the mycelial walls of Phytophthora megaspermaf. sp. glycinea and well known as an elicitor of phytoalexins in soybeans (intact plant tissues and cell suspension cultures) was reported to be a highly efficient inducer in several species and cultivars of Nicotiana of resistance to virus infection~.~~ The role of antifungal di- terpenoids notably a-and p-duvatrienediols [p-and a-cembrenediols(57,58)] that occur in healthy tobacco leaves has been further explored. Removal of most of these diols (by dipping in acetone for 1 s) increased the susceptibility of the 374 NATURAL PRODUCT REPORTS 1991 0 H&oR 0 iv - o&oH (59) I HO lo OAc viii Ho+oAcAcO-" -t(+0 0 0 Reagents i O, MeOH Me$; ii AcCl PhNEt, CHC1,; iii OsO, NaIO, aq.dioxan; iv. aq NaOH; v NaH (EtO),P ButOH DMF then 0,; vi LiCH,CO,Li HMPT (MeOCH,CH,),O then AcC1 PhNEt, CHCl,; vii OsO, py ; viii 1,8-diazabicyclo[5.4.0] undec-7-ene PhH ; ix Pb(OAc), PhH; x TsCl py then NaBH,CN HMPT; xi Li liq. NH, ButOH Scheme 6 leaves to blue mould produced by inoculation with Peronospora tabacina Adam." An interesting synthesis of phytuberin lactone (63) from elemol(59) briefly reported in 198178has been fully described79 (Scheme 6). Previous procedures for the conversion of (59) into the indenone (60) were improved and autoxidation afforded the desired cis-hydroxylation at C-4.The difficult step in the synthesis proved to be the reductive removal of the neopentylic hydroxyl group in (61) but the tosylate was formed quan- titatively and although attempted reduction caused initial cyclization to (62) this compound was readily reduced by lithium in liquid ammonia to yield (63) a known progenitor of phytuberin (32). 2.5 Sweet pepper (Capsicum annuum) Suspended callus cultures of Capsicum annuum were found to accumulate capsidiol(30) in response to treatment with various agents e.g. cellulase (ex. T. viride) a hot water extract from Gliocladium deliquescens and pectinase (ex A. niger).80 The most effective of these was the extract from G. deliquescens which elicited up to ca 3 mg of (30) per 100 ml flask of culture.Cellulase elicited up to ca 0.8 mg of (30) per flask and was an effective elicitor at concentrations down to 0.5 pglml. The rate of accumulation of (30) was greatest between 12 and 36 h but accumulation continued at a slower rate up to 72 h. Pectinase ex A. niger was a very weak elicitor of (30) in this system although in earlier work it had been found to be a powerful elicitor in capsicum fruits.81 De novo synthesis of (30) in the cultures was established by feeding them with [2-14C]acetate 12 h after they had been treated with extract from G. deliquescens. Extracts from the culture medium of C. annuum OH cultures at the stationary phase of their growth cycle were found to contain cis-9,lO-dihydrocapsenone(64).82 This com- pound may be a catabolite of (30); the fact that it was obtained from cultures in their stationary phase might suggest that stress from depletion of nutrients in the medium may have been causing some phytoalexin elicitation.Green (immature) capsicum fruits inoculated with Botrytis cinerea produced arrested and progressive lesions in approxi- mately equal amounts.83 The phytoalexin mixture capsicannol [composed largely of 1-deoxycapsidiol (65) and capsidesmol (66)81] accumulated to a greater extent in arrested lesions. NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON Capsidiol accumulated largely in dead cells whereas 'capsi- H o . . . ~ cannol' accumulated particularly in living cells surrounding arrested lesions.Fruits pretreated with a P-glucan elicitor from Glomerella cingulata 24 h prior to inoculation with B. cinerea did not develop rot during a 15-day period of incubation. The promotion of rot resistance by G. cingulata was most effective HO in wounded elicitor-treated fruits. Capsicannol appears to have a major role in the development of quiescent infections. A phospholipid fraction from Phytophthora capsici mycelium was effective in inducing resistance of sweet peppers to fungal infection.84 Incubation of P. capsici mycelium in a solution of (67) (68) R = H (70)R = OH (30) caused degradative changes similar to those seen in the intercellular hyphae in the necrotic zone formed in the leaves of a resistant line of Capsicum annuurn following inoculation with P.cap~ici.~~ The bacterial pathogen Xanthomonas campestris pv. vesicatoria overcomes genetically-defined resistance of C. annuurn by the transposon-induced mutation of a bacterial gene that provokes the resistance response of the plant.86 2.6 Potato (Solanum tuberosum) CH3 A review of phytoalexins by Kud and Rush,6 principally devoted to work on the potato summarizes the state of knowledge at the outset of the period of this report. A specific review of potato phytoalexins has stressed the uncertainties in the knowledge of their role in the resistance of potato tubers to pathogens and also makes the point that there is no evidence of any risk to health posed by the presence of the typical terpenoid phytoalexins in potatoes used as food?' 2.6.1.Biosynthetic Aspects It was found that intact potato tubers had very low levels of HMG-CoA reductase activity.88 After a lag phase of 6 h the enzyme activity in sliced tissue increased markedly reaching a maximum value at 18 h and decreasing thereafter. When slices were inoculated with incompatible Phytophthora infestans at The diff erences be tween ob served lev els of (67) and (68) may 18 h after their preparation HMG-CoA activity increased sug gest rapid c on version of lubimin to rishitin during bio- further reaching a maximum 7-8 h after fungal inoculation. synt hesi s with red uced flux through t he biosynthetic pathway separately In the case of inoculation with HgCl at 18 h the increase and cau sing depleti on of (67) or the in volvement of subsequent decrease in activity were more rapid.Application of cont rolled isoprene pools in the b iosynthesis blasticidin S completely inhibited any further increase in com pounds. HMG-CoA reductase activity stimulated by P. infestans or P otat o slices tha t were tr eated with AA and incubated in the HgCI, suggesting that such treatment promotes de novo pres ence of 180 ,/N produc ed lubimin and rishitin which were synthesis of the enzyme. labe lled with 180.90 Lubimi n (67) was l abelled only at the 2-OH Similar increases in HMG-CoA reductase activity were position the l80being lost from the observed in microsomes from both elicited and arachidonic equi libr ation w ith water; rishitin (6 8) was doubly labelled.acid (AA) treated potato slices in the period up to 12 h after Thu s th e oxyge n a toms in t hese phyto alexins are derived from wounding or wounding plus elicitati~n.~~ However by 24 h the atm osph eric ox yge n rather than from water. When aged and activity in the controls had declined markedly whereas in the une licite d potat o t uber disc s were incu bated with rishitin in an slices treated with AA activity continued to rise up to 24 h and atm osphere of "O,/N, 1 3-hydroxy rishitin (70) and did not decline to the original levels until 72 h. In an organelle- dih ydro - 13-hyd rox yrishitin (71) were produced with the side- enriched fraction from the slices there was no difference chai n hy droxyl gro ups label led with '* O. Thus (71) is produced between HMG-CoA activity in controls and in AA-treated fro m (7 0) by re duc tion of the All do uble bond in (70) rather slices.The kinetics of phytoalexin accumulation were similar than by hydroxy lat ion of ri shitin. The hydroxylation of the 13- in slices treated with either AA or an incompatible race of posi tion is pres um ably cat alysed by a microsomal NADPH- P. injestans. AA enhanced only microsomal HMG-CoA dep ende nt cyto chr ome P-4 50 mono oxygenase. This is con- reductase activity and this exceeded that in the organelle siste nt w ith the fac t that th e synthesis of lubimin in NADPH- fraction. Exposure of the tissue to 24 h of constant light reduced dep ende nt cell-f ree systems derived fr om elicited tuber discs is HMG-CoA reductase activity in the microsomal and organelle inhi bite d by me tyr apone a cytochrom e P-450 inhibitor.fractions to 10% and 50% respectively of that of tissue K enn ebec tub er discs ino culated wi th an elicitor preparation incubated in the dark. Constant light also reduced the levels of from P. infestans yielded a cell-fre e system (cfs) that phytoalexins accumulated this was largely reflected by a corp ora ted [14 C]I PP into (67) and into a decrease in the levels of lubimin (67) accumulated with rishitin unid enti fied me tabo lite^.^^ Formation of (68) was not generally (68) levels remaining unchanged. The HMG-CoA reductase obse rved in thi s s ystem. B iosynthesi s of (67) in the cfs was inhibitor mevinolin (69) reduced phytoalexin accumulation by obse rve d as ear ly as 3 h af ter elicitat ion.The biosynthesis of 60 YO; again this was largely due to a decrease in levels of (67). (67) was NADP H depende nt. Absenc e of NADPH caused an Feeding of mevalonic acid to the tissue slices did not reverse the incr ease in the am ounts of labelled unidentified components effects of mevinolin (69). Thus it was concluded that increases whi ch a re prob abl e interm ediates in the biosynthesis. Iodo- in HMG-CoA reductase activity alone were not sufficient to acet ami de comp let ely inhib ited biosy nthesis of (67) in the cfs cause phytoalexin accumulation but that reduction in its thus rul ing out the possibil ity that IP P is accumulated at the activity does as expected regulate phytoalexin accumulation. leve l of FPP synthetase. of the two C-15 aldehyde by 11,12-in-number of other NATURAL PRODUCT REPORTS 1991 Observations were made on the pattern of phytoalexin of Majestic potato was consistent with the following bio- accumulation in potato slices treated with different elicitor~.~~ synthetic sequence acetate +MVA +FPP +lubimin -P 3-Tissue treated with elicitor derived from P.infestans race 4 mycelia accumulated rishitin most rapidly in the top 0.5mm layer during the first 24 h after inoculation while increases in phytoalexin content of the subsequent 0.5 mm layers were slower. The top 0.5 mm layer 1 contained the greatest amounts of phytoalexins over the time period studied. Levels of lubimin increased more slowly than rishitin which accounted for 90 Oh of the phytoalexin content at 24 h but only for 45 % thereof at 96 h.The total phytoalexin content of the tissue at 48 h was 11.7pg/g fresh weight. Tissue inoculated with P. infestans race 4 zoospores accumulated phytoalexins mainly in layer 1 up to 24 h but the content of lower layers increased rapidly after this time while the levels in layer 1 decreased after 48 h. The phytoalexins (67) and (68) were present in approximately equal amounts at 48 h from the outset with levels of lubimin greatly exceeding those of rishitin at 96 h. The total concentration of phytoalexins at 48 h was 23 pg/g fresh weight. Tissue inoculated with zoospores of a compatible race of P. infestans (TY complex race) accumulated very low levels of phytoalexins. Tissue slices treated with arachidonic acid accumulated phytoalexins very slowly up to 24 h but thereafter levels rose linearly to a maximum of 38.1 pg/g at 96 h.The proportion of (67) gradually increased over the time period studied. Cell-free systems prepared from the differently treated tissues had biosynthetic activities which reflected the patterns of phyto- alexin accumulation in the tissues. The continued increase in phytoalexin content in the case of sodium arachidonate treatment indicates a response in which synthesis outweighs catabolism whereas treatment with race 4 zoospores produces a situation in which synthesis and catabolism are more in balance. Feeding of [2-14C]MVA to aged potato tuber discs treated with sporangia from compatible or incompatible races of P.infestans resulted initially in its incorporation into rishitin (68);93the labelled (68) was subsequently metabolized and the turnover was found to be highest in tuber discs treated with the compatible race of the fungus. There was no turnover of (68) either in discs inoculated with a sonicate prepared from P. infestans or in Kennebec cell cultures during the phase of phytoalexin accumulation. Although in previous work it has been found that aged healthy tuber discs and unelicited cell cultures transform (68) into the three metabolites (70) (71) and glutinosone (72) these compounds could not be detected in this case where discs and cultures were inoculated with sporangia. Observations of the sequential changes over a period of time of the incorporation of [2-14C]MVA or [2-14C]acetate into squalene and sterols in inoculated cell cultures (75) hydroxylubimin +rishitin.The inclusion of MVA (3.3 mM) in inoculated cultures of Majestic potato resulted in an increase in the levels of phytoalexins showing that the rate-limiting step in phytoalexin accumulation is located somewhere between acetyl CoA and MVA. Feeding of [2-14C]MVA to aged healthy tuber discs indicated that sterol biosynthesis was enhanced ; in-oculation with sporangia of P. infestans caused a rapid decrease in the rate of synthesis of squalene and sterols and a similar decrease was observed in Majestic cell cultures. Thus it was concluded that during phytoalexin biosynthesis incorporation of MVA into sterols is blocked by inhibition of squalene synthetase and this results in its diversion into sesquiterpenoid biosyn t hesis.In another study aged plugs of potato tissue were inoculated with Fusariurn roseurn and incubated for 21 h at 23 "C the plugs were then cut in half and the cut surface was incubated with (+)-[8,8-2H2]solavetivone (73) for a further 22 h.94 The [2H2]derivatives of phytuberol (31) and phytuberin (32) were formed as 0.6-0.7% of the unlabelled compounds. The 2H NMR spectra of these compounds revealed that the C-8 position was labelled indicating that these compounds are bio-synthesized from solavetivone (46). A study of the formation of (70) and (71) from rishitin in healthy potato slices was carried out (Scheme 7).95 Slices were incubated with (-)-[ 12,12-2H,]rishitin (74) yielding (-)-[1 1,l 2-2H2]- 12-hydroxy- 11,12-dihydrorishitin (75) and (-)-[ 12,12-2H,]-13-hydroxyrishitin (76) as indicated by 2H NMR spectra of the products.The formation of (75) was thought to take place via a mechanism involving stereospecific 1,2-migration of one olefinic hydrogen from C-12 to C-1 1 via an NIH-like shift; the labelling of (76) was consistent with its formation via direct oxygenation at C- 13. Incubation of [12 12-2H2]-rishitin 11,12-epoxides (77) with potato slices resulted in the formation of (78) indicating that formation of (76) via an epoxide intermediate was unlikely. The deuterium contents of (75) and (76) decreased slightly during their formation from (74) indicating the role of rishitin (68) as an activator of formation of (70) and (71) from acetate.Scheme 7 NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON 2.6.2 Aspects of Elicitation Spores of Helminthosporium carbonum and of an incompatible race of P.infestans both elicited the accumulation of rishitin and lubimin in potato tuber Autoclaving 80 YOethanol treatment or freezing destroyed the elicitor activity of H. carbonum spores but the former two treatments did not destroy the activity of P. infestans. Arachidonic and eico- sapentaenoic acids were not detected in mycelial extracts from H.c arbonum.Inoculation of potato tuber discs with compatible P. infestans spores suppressed rishitin and lubimin accumu- lation in response to subsequent inoculation (18 h later) with incompatible P.infestans spores but not in response to inoculation with H.carbonum or treatment with arachidonic acid. Neither of the latter treatments appeared to affect the growth of compatible P. infestans. There was no visible evidence for growth of H.carbonum on the tuber discs previously inoculated with a compatible race of P.infestans. Application of Ca2+ directly to AA-treated potato discs enhanced accumulation of (68) in a concentration dependent manner.97 Mg2+ Na+ and K+ were ineffective but Sr2+ had a similar effect to Ca2+. The accumulation of phytoalexins was enhanced only if Ca2+ was applied within 24 h of treatment with AA. Mg2+ inhibited rishitin accumulation by 80%. A calcium-specific ligand EGTA was found to inhibit phyto- alexin accumulation and the addition of excess Ca2+ overcame the inhibitory effect of EGTA.La3+ also was found to inhibit the accumulation of rishitin but even in the presence of La3+ the accumulation of rishitin and lubimin increased with an increase in Ca2+ concentration. An indication that Ca2+ and La3' are competing for the same ion channel is given by the fact that promotion of phytoalexin accumulation by Ca2+ was more effective if the tissue was treated with Ca2+ and La3+ together compared with addition of Ca2+ 12 h after application of La3+. Conversely La3+ was a less effective inhibitor if it was added 3 h after the application of Ca2+ to the tissue. Ca2+ may have a role in increasing pyruvate dehydrogenase activity or it may promote lipoxygenase activity producing elicitors from AA.Several factors can be considered to support a role for lipoxygenase activity in the production of elicitors from AAg8 and may be summarized as follows (i) the minimal structure for high elicitor activity is the 5,8,1l-double bond configuration in a 20-carbon acid which is also the minimal structure for the production of eicosanoids in mammals via 5-lipoxygenase activity ;(ii) several compounds which are effective inhibitors of potato lipoxygenase inhibit AA-elicited browning and sesqui- terpenoid accumulation; (iii) the activity of AA as an elicitor is enhanced by P-glucan preparations ; (iv) stimulation of p-glucan receptors in human monocytes enhances 5-lipoxygenase activity.Ca2+ enhances phytoalexin responses in potato and also the inflammatory response in humans in the latter case via participation of Ca2+ in eicosanoid production. Extensive studies have been made on the elicitation of phytoalexins in potato tubers by AA. Application of AA to plants other than potato including sweet potato broad bean French bean pea soybean pepper tobacco tomato carrot and parsnip resulted in phytoalexin accumulation only in the case of sweet pepper which accumulated capsidiol (30).99 The metabolism of AA and a number of other fatty acids by potato tuber slices was monitored by the use of radioactive sub-strates:loo the predominant metabolites observed were glycerides and no metabolites such as hydroperoxides or cyclopentanones could be detected under the incubation conditions.The elicitor activity of the fatty acids could not be correlated with their rate of metabolism. AA was found to elicit a number of other reactions in tuber tissue apart from phytoalexin accumulation. lol These included the accumulation of lignin the stimulation of peroxidase activity and the production of ethylene and ethane. Ethylene and ethane were also produced in response to the treatment of tissue with linoleic acid which does not itself elicit sesquiterpenoid production. P-glucans extracted from the mycelium of P. infestans,which alone are inactive as elicitors of phytoalexins in potato tubers,lo2 elicited lignin. The lipids from the mycelium of P. infestans were analysed and evaluated for their elicitor activity.lo3 Triglycerides were the predominant lipid class present comprising 77.8 YOof the total lipids while phospha- tidylethanolamine and ceramide aminoethylphosphonate were the most abundant phospholipids.The predominant fatty acids in all the lipids were palmitic linoleic arachidonic and eicosapentaenoic acid (EPA). All the lipids elicited phytoalexin accumulation in tuber slices but free AA elicited higher levels of phytoalexin accumulation than any lipid fraction from the fungus. Lipolytic acyl hydrolase purified from potato tuber tissue hydrolysed phosphatidylethanolamine from P. infestans but was inactive in hydrolysing triglycerides. EPA and AA were found to occur at 35 and 8 ,ug/mg respectively of dry weight in the spores of P.infestans.lo4 Bromine treatment destroyed the elicitor activity of the spores addition of mixtures of lipids from P.infestans containing (i) free and esterified EPA and AA; (ii) pure EPA and AA restored 76 '30and 55 YOrespectively of the elicitor activity present in untreated sporangia. Treatment with bromine did not remove the ability of the spores to enhance the elicitor activity of AA. Thus the eicosapolyenoic acids in the spores account for the major part of their elicitor activity provided that the enhancing activity of other com- ponents is included. Spores of Ceratocystis Jimbriata and Bipolaris carbonum elicited phytoalexin accumulation in potato slices but the effect was also abolished by treatment with bromine; the spores of these organisms did not contain eicosapolyenoic acids.Oleic linoleic linolenic acids and AA all caused cytoplasmic aggregation and cell death in potato protoplasts ;lo5 the response was morphologically identical to the response to fungal wall components from P. infestans. The most active acids in the promotion of cell death were linolenic eicosatrienoic and eicosapentaenoic acid and AA ;arachidonyl alcohol was also active with 33 % of the activity shown by AA. However only AA elicited rishitin accumulation. In addition application of a number of fatty acids including AA to potato tuber slices resulted in cell death and the only acid tested which did not cause cell death was oleic acid.lo6 AA eicosapentaenoic acid (EPA) and linoleic acid caused the highest degree of cell death but only AA EPA and the ethyl ester of AA caused appreciable accumulation of phytoalexins.Salicylhydroxamic acid was able to inhibit phytoalexin accumulation but had no effect on the degree of cell death. Thus cell death and phytoalexin accumulation are not necessarily linked. Various studies of elicitation by P. infestans reflect the complexities of observations in different experimental systems. Culture filtrates of race 4of P.infestans contained an elicitor of the hypersensitive response and of (67) and (68) in Kennebec tuber discs. The elicitor contained no fatty acids and a carbohydrate moiety appeared to be important for its ac-tivity.lo7 Elicitation by media from germinating spores of P.infestans also appeared to be due to high molecular weight constituents there being no appreciable amounts of AA present.lo8 In a study of fractions from mycelium of P.infestans it was concluded that both lipid and non-lipid components were concerned in the elicitation process. log A lipoglycoprotein complex isolated from P.infestans was found to owe its elicitor activity largely to the constituent acids AA and EPA;"' prostaglandins were also detected in the elicitor and exogenous PGE was effective in limiting penetration of the fungus into the tuber tissue. Several pathogenesis-related proteins accumulating in the intercellular space of potato leaves inoculated with P. infestans (or with fungal elicitor) are 1,3-/?-glucanases and chitinases which may play an important role in the defence response.'l' In an interesting investigation tuber discs of R,-gene-carrying potatoes were inoculated with either an incompatible or a compatible race of P.infestans. The incompatible race caused an increase in rishitin content from 4 to 96 ,ug per g of fresh weight and concomitant reduction of sterol content from 15 to 3 ,ug/g. In the response to the compatible race rishitin content remained low while sterols increased to 28 ,ug/g. [The parasite is well adapted to employ in its growth processes the major sterol (p-sitosterol) of the potato.] Similar observations were made when the glucans from an incompatible and from a compatible race of P. infestans were tested on tuber discs inoculated with an incompatible race resulting respectively in enhancement and suppression of the immune response.112 A study was made of the time courses of phytoalexin accumulation in compatible and incompatible interactions of leaves and tubers from 5 different R genotypes of p0tat0.l~~ In the incompatible non-host interaction Phytophthora mega-sperma caused a rapid accumulation of rishitin (68) up to 20 h in tuber slices [cv Datura (Rl)] followed by a slow rise up to 70 h with a decline thereafter; the maximum level of ac-cumulation was ca 25pglg. In a typical incompatible in-teraction (P. infestans Race 4)levels of (68) rose slowly up to 20 h and then rapidly to 70 h declining thereafter; the maximum level of accumulation was 40 ,ug/g. In a compatible interaction (P.infestans Race 1) levels of (68) rose slowly to 45 h and then very rapidly to a level of 60 pg/g with levels still increasing at 80 h. Arachidonic acid caused rishitin levels in tuber slices to rise steadily to a maximum (ca 40 pg/g) at 70 h declining thereafter. The same general pattern was repeated for all the cultivars. Leaves inoculated with compatible fungi died but inoculation with incompatible fungi resulted only in small brown lesions on the leaves. Neither of these inoculations caused detectable sesquiterpenoid accumulation nor did inoculation of the leaves with AA. These results suggest that the role of sesquiterpenoid phytoalexins may be largely in disease resistance in potato tubers rather than in leaves. Erw inia car0 to vora su bsp.atroseptica E. car0 to vora su bsp . carotovora and E. chrysantherni were compared for their ability to rot potatoes and to elicit the accumulation of phytoa1e~ins.l~~ NATURAL PRODUCT REPORTS 1991 (80) R = H R' = CH20H (80a) R = CH20H R' = H The tubers were found to be more susceptible to rotting at higher incubation temperatures. The extent of rotting could not be correlated with rishitin concentration in the tubers suggesting that other factors also affect their resistance. Potato tubers infected by E. carotovora or by Fusarium spp. produced phytoalexins and phenolic metabolites which were eluted from the tissue by descending paper chromatography and analysed by TLC or GLC.l15 Phytoalexins were induced in potato tissues in response to the nematodes Ditylenchus destructor and D.dipsaci. 116Rishitin (68) appeared in the discs 24 h after invasion and lubimin (67) was observed after 48 hours. D. dipsaci caused accumulation of rishitin throughout the disc whereas D.destructor caused accumulation only in the top 2 mm of disc consistent with its invasion of superficial layers. Rishitin (68) was tested for its effect on the mobility of D. dipsaci and was found to have an ED, response of 100 pg/ml. The average levels of (68) formed in the tissue were no more than 20pg/g but local concen- trations may have been much higher. Resistant potato varieties gave higher levels of (68) in response to nematodes. Rishitin produced in potato tissue treated with E. carotovora has been shown to have nematode-repellent and nematocidal activity."' 2.6.3.Toxicity and Biotransformation The toxicities of sesquiterpenoid phytoalexins from potato were tested on tissue from potato tubers. Toxicity could be related to lipophilicity with (46) (67) (68) and (32) being more toxic than the more hydrophilic 3-hydroxysolavetivone (47) oxy-lubimin (79) and (31).'18 Compounds (68) and (46) were the most toxic to P. infestans. The biotransformation of lubimin (67) and rishitin (68) by potato and soybean cultures was studied.llg Incubation of both potato and soybean cultures with rishitin produced a compound tentatively identified as glutinosone (72) without any sign of the production of (70) and (71) which are the usual metabolites of rishitin in healthy tuber tissues.Soybean cultures produced 15-dihydrolubimin (80) from lubimin but this compound was unaffected by potato cultures.11g Cell suspension cultures of two solanaceous species trans- (81) R=H R'=CH,OH (82) R = H R' = CHO (81a) R = CH20H R' = H (82a) R = CHO R' = H formed exogenous solavetivone to four hydroxylated deriva- tives associated with the response of the plants to biotic stress. It was suggested that the failure of potato cell cultures to produce these compounds under biotic stress may be due to their inability to generate adequate levels of solavetivone. 12* The biotransformation of lubimin (67) by strains of the potato pathogen Gibberella pulicaris has been studied in detai1.121v122 Fungal strain R-7715 which was tolerant of lubimin but intolerant of rishitin was initially examined.121 The major metabolites formed between 1 and 6 h were cyclo-dehydroisolubimin (84) together with three products viz.1Sdihydrolubimin (80) isolubimin (8 l) and 2-dehydrolubimin (82) accompanied by their 1 OF-epimers (80a 81 a and 82a). After 1-2 days two novel tricyclic compounds cyclolubimin (85) and 11,12-epoxycyclodehydroisolubimin(86) comprised an increasing proportion of the mixture at the expense of the early metabolites. Biotransformation of lubimin was further studied by using the two lubimin-tolerant strains (R-7715 and R-583) and one lubimin-sensitive strain (R- 110) of G. pulicaris.122Metabolism of lubimin by strain R-1 10 was slow and yielded only two major products 15-dihydrolubimin (still toxic to G.pulicaris) and isolubimin. Metabolism by strain R-77 15 yielded products (80)-(82) (84) and (85) noted above as well as cyclolubimin epoxide (87); after almost 5 days' incubation more than 15 % of the added lubimin was recovered as tricyclic metabolites. These compounds were not toxic to the lubimin-sensitive strain and their production implies that the detoxification of lubimin contributes to the virulence of G. pulicaris on potato tubers. However the mode of detoxification by strain R-583 appeared to be different as cyclodehydroisolubimin in this case was a very minor metabolite and after 48h of incubation no lubimin metabolites could be detected in chloroform-methanol extracts. NATURAL PRODUCT REPORTS 1991-C.J. W. BROOKS AND D. G. WATSON 0 (90) Reagents i Li liq. NH, FeCl, NH,Cl; ii MeMgI ether; NaOAc Ac,O Scheme 8 The results of the incubations of lubimin and of various disrupt an outer carbohydrate layer surrounding the cell an products of its transformation by G. pulicaris suggested that effect which could be observed via the binding of ferritin to likely pathways of lubimin metabolism by this organism treated cells but not to untreated controls. Such effects are would proceed via 2-dehydrolubimin (82) or 15-dihydrolubimin probably due to release of autolytic enzymes by the bacteria in (80) to isolubimin (81). It was postulated that dehydrogenation response to rishitin. could follow to yield 3,4-dehydroisolubimin (83) there was tentative evidence based on GC-MS for the presence of this compound as a transient intermediate.Cyclization would then 2.6.4. Synthesis afford cyclodehydroisolubimin (84) as the precursor of cyclo-A stereoselective synthesis of the nor-eudesmanoid 9-0x0-lubimin (85) and of the epoxides (86) and (87). The intermediacy 2P,1OP-oxymethylene- trans-decalin (88) a potential interme- of (83) is consistent with observed incorporation of a deuterium diate for the synthesis of rishitin (68) has been reported,lZ4 atom at C-3 of (84) following incubation of lubimin with starting from the known enone (89). G. pulicaris in the presence of D,O. A simple synthesis of acetylanhydrorishitinol (9 1) (reference The toxic effect of rishitin on Erwinia carotovora was 1 p. 432) has been described125 (Scheme 8) an improved Rishitin (500 pglml) was found to change the method of preparing the precursor (90) was reported.ultrastructure of the bacterial cells so that the cytoplasm Details have been reported of an interesting general gravitated towards the outer membrane. It also appeared to approach126 to the synthesis of spirovetivanes of the two major NATURAL PRODUCT REPORTS 1991 iii ~ Po Meo'Q" CN (93) 4 stereoisomers separable by chromatography two 8-anti to C(2)/C(3) iv v vi vii OMS (94) lix Reagents i LiNiPr, THF -78 "C ClCH,CN THF/HMPA (1 :1) ; ii NaH Me,SO methyltriphenylphosphonium iodide; iii CH,=CH- .COMe PhH dichloromaleic anhydride 2,6-di-t-butylcresol reflux 3 days under Ar; iv MeLi Et,O; v BuiAlH Et,O; vi NaBH, THF/H,O (2 1); vii MsCl CH,Cl, NEt,; viii 10 mol.equiv. (CO,H), Me,CO/H,O (1 :2) 85 "C 4 h; ix pyridine-modified alumina 220 "C under Ar 8 min. O Rp (96) R = C(Me)=CH2 (97) R = C(OH)Me2 (95) Scheme 9 (98) (mixture of 2-ex0 and 2-endo) reaction sequence based on cycloaddition of 4-methoxy-6- methyl-1,3-cyclohexadienylacetonitrile(95) with methyl vinyl ketone led to the synthesis of (-t)-15-norsolavetivone (96) and the (k)-1 I-hydroxy- 15-norsolavetivane (97).128 Norsolaveti- vone (96) was converted via multi-step reaction sequences into (-t)lubimin (67) and (+)oxylubimin (79).lz9 However in the synthesis of (96) the critical step of acid-catalysed cyclization of the intermediate (98) analogous to (94) (Scheme 7) but lacking the 5-methyl group proceeded with low stereoselectivity at C(7) of the cyclized products.A more efficient synthetic route to (4RS 7RS)-(97) has been devised.130 (Scheme 10). The intermediate chloro-compound (99) was accompanied by 19 % of its dehydrated (isopropylidene) analogue. The 9-step sequence afforded (97) in 44% overall yield from (100). The intermediate tertiary alcohols (102) and (103) obtained by reaction of (101) with methyllithium served as starting materials for an efficient synthesis of (+)-[8,8-2H,]-isolubimin (1O4).l3O Alternative routes to spirovetivanes have been based on the intermediates (106) and (107) derived from the spiro-enone (105) (Scheme 1 l).1319132 configurationally distinct types represented by solavetivone (46) and /3-vetivone (92) (Scheme 9).An efficient synthesisl2' of ( f)-(46) from 3-methoxy-5-methyl-2-cyclohexenone (93) via cycloaddition with methyl vinyl ketone is summarized in Scheme 9. The 8-anti isomers were preponderant among the four compounds represented by (94) and the overall yield of (+)-solavetivone (46) was 16.6%. The method was adapted for the preparation of (& )-[8,8-2H2]~~la~eti~~ne.127 An analogous NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON 381 Meoqo CN (1011 separated by chromatography iii iv v vi I Oy I ix vii CI P O H OH LOMs (97) (99) (72%) (98) (102 103) Reagents i catechylphosphotrichloride 1,8-diazabicycl0[5.4.O]undec-7-ene, PhH reflux 2 h; ii CH,=CHCOMe xylene 150 "C 7 d ; iii-vi as in Scheme 8 (iv)-(vii); vii HCO,H 20 "C 2 h; viii (CO,H), MeCOBu'/H,O (5 I) 130 "C 8 h; ix Zn-Ag MeOH/AcOH 20 "C 1 h Scheme 10 o 7qj3 i ii iii iv MoMoyJj ,2 .OCOBu' OCOBu' OCOBu' (106a) R=H (107a) R=CHO (106b) R=MOM (107b) R = CH20H MOM = CHaOCH2 Reagents i NaAlH,(OCH,CH,CH,OCH,), 0 "C; v H, PtO, EtOAc EtOH Et,O -78 "C; ii MOM-Cl PhNEt, CH,CI,; iii SeO, CaCO, xylene reflux; iv NaBH, MeOH Scheme 11 NATURAL PRODUCT REPORTS 1991 OH OH OMOM OMOM Ho.-*T Ho...% ,viii MOMO.*.($ 3 MOM0... OMS OMS (80) Reagents i MeLi; ii MsCl py 0 "C; iii NaCH(CO,Et), DME; iv H (1 atm) Raney Ni EtOH r.t.; v NaH DME; vi NaAlH,(OCH,CH,OCH,), DME reflux; vii 3 M HC1 THF 40 h r.t.; viii NaI 1,8-diazabicyclo[5.4.0]undec-7-ene, DME Scheme 12 *% Me3si0q .iv,v,vi* SQ I_ oqc" RO RO I . , Q I . OCOBu' OCOBu' OR' (110) R = ButPh2Si R' = CO~BU'; R' = H; R' = Ms vii. viii I RO (47) R' = C02Et; R' = Et R' = CH2OH; R'=H R' = CH~OAC; R' = Me Reagents i LDA (MeOCH,), Me,SiCl; ii (PhO),P 0, CH,Cl, -50 "C; Ph,P r.t. iii ButPh,SiC1 imidazole DMF; iv (CH,SH), BF;Et,O CH,Cl, 0 "C; v MeLi Me,O 0 "C; vi MsCl py 0 "C; vii NaCH(CO,Et), DME; viii KOH EtOH; ix aq. CH,O Et,N NaOAc AcOH; x (Bu'),AlH PhMe -70 "C; xi Ac,O py; xii Pd(OAc), Ph,P HCOONH, dioxan reflux; xiii Bu;N+F- THF r.t.; xiv AgNO, aq. EtOH 60 "C Scheme 13 NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON 14 (112) A3 (113) A4(14) A total synthesis of (f)-15-dihydrolubimin (80) was achieved133 (Scheme 12) from the allylic alcohol (107b in Scheme 1 1) via the introduction of a bis-(ethoxycarbony1)methyl group with inversion at C-2.The saturated alcohol [(108) in Scheme 111 proved to be an inefficient starting material for this synthesis. The first total synthesis of ( f)-3-hydroxysolavetivone (47) (Scheme 13) has been based on the trimethysilyl enol ether (109) of the spiro-enone (105)'l' (cf. Scheme 11) this was converted stereoselectively by reaction with triphenylphosphite ozonide into the C-9 alcohol (110). The remaining steps in Scheme 13 proceeded in good yield to afford (47).134 2.7 Aubergine (Solanum melongena) A of the biosynthesis of aubergenone (52) in aubergine fruits treated with Moniliniu fructicolu in the presence of [2-2H3 1-l3C]-acetate was not directly successful because of the low levels of enrichment.However the isolation of the related metabolites auberganol(ll1) and a-and /3-eudesmol (112) and (113) labelled with deuterium at C-5 (as shown by 2H NMR data) implied that these together with aubergenone are normal eudesmanes and are not produced by rearrangements analogous to those involved in the biosynthesis of rishitin (see reference 1 p. 432). Auberganol (111) is a new natural product while (112) and (1 13)have not been previously observed as aubergine stress metabolites. 2.8 Tomato (Lycopersicon esculentum) Low levels of rishitin (68) (1-3 pg g-') were recovered from stem vascular tissues of resistant (Rt.) and susceptible (Su.) isolines of tomato inoculated with both live conidia and cell- wall extracts from Verticillium ulbo-utvum.136 The activities of the cell wall extract were unaffected by deproteinization suggesting that the activity was due to carbohydrate. Higher H levels of elicitor activity were found in culture filtrates from 33-day old shake cultures and 14-day old large volume stirred cultures. In the crude form these extracts induced up to 14pg 8-l of (68) in the tissues. Larger amounts of rishitin were induced in the Su.-isoline. After dialysis the activity in culture filtrates from shake cultures was retained in the high molecular weight fraction and this fraction was again more active in the Su.-isoline.In the stirred cultures the active component was of small molecular weight (cu. 1500-1700 daltons). This elicitor induced more rishitin in the Rt.-isoline. The Su.-isoline has been shown to contain higher levels of glucosidase which would be capable of releasing elicitor fragments from higher molecular weight glucans present in the crude culture filtrates. The low molecular weight elicitor appeared to be composed of six 1 -+ 3-linked glucose units. Thus culture conditions can markedly affect elicitor activity. The non-specificity of this glucan excludes it from involvement in a mechanism for single gene resistance. Growth of pathogenic fungi may be inhibited by the presence of other microorganisms on the surface of the plant.Aureo-busidium pulluluns has been shown to be an antagonist of Alternuriu soluni a pathogen of tomato plants. Application of culture filtrates from A. pulluluns to tomato leaves prior to inoculation with A. soluni reduced the rate of infection.13' Part of the inhibitory mechanism may be due to phytoalexin induction since treatment of the leaves with A. pulluluns culture filtrates resulted in elicitation of antifungal compounds tentatively identified as (68) and falcarinol (1 14). 2.9 Coffee (Cofea spp.) Detached coffee leaves inoculated with Helminthosporium cur- bonum a non-pathogen of coffee plants showed accumulation of fungitoxic compounds (unidentified but possibly terpenoids (see reference 1 p. 434) which diffused into infection droplets on the leaf surface.13* NATURAL PRODUCT REPORTS 1991 NB trans + cis (2:l) viii ix J CHSnBu x-xii OH xiii-xv -AcO'..xvi xvii 1 aCHO xviii xix P xx-xxii xii miii TBDMSO'.. xxiv-xxvii Fiii J xxviii HO'.. OH Reagents i 2N NaOH (CH,O),SO,; ii Na C,H,OH iii CH,I NaOCH,; iv (C,H,),N*(CH,);CO.CH, CH,I KOC,H,; v KOBut CH,I; vi Pd/C H,; vii TMSCI NaI; viii Li NH,; ix CH,O.CO.CH, p-TsOH; x Raney Ni W-7 in C,H,OH; xi Jones Reagent; xii Chromatography on silica gel ;xiii HCO,C,H, NaOCH,; xiv Bu"SH p-TsOH;xv t-BDMSCI imidazole; xvi NaBH,; xvii HgCl, CdCO,; xviii KOBut CH,I; xix Bu"Li Ph,PCH,Br; xx HF in CH,CN; xxi chromatography on silica gel/AgNO,; xxii (-)camphanyl chloride; xxiii K,CO in CH,OH; xxiv acetic anhydride pyridine; xxv SeO in C,H,/aqueous acetic acid 1.5 min at 65 "C; xxvi DMSO acetic anhydride; xxvii K,CO in CH,OH; xxviii 8N CrO Scheme 14 data and optical rotation with those of the synthetic (-) isomer.The effects of (119) on P.oryzae were studied.143 The concentration for 50% inhibition of growth (ED,,) was 230 ppm. At this concentration the incorporation of [2-14C]- thymidine [2-14C]uridine ~-[U-l~C]amino acid mixture ~-[methyl-~~C]methionine, and D-[ 1-14C]glucosamine respec- tively into DNA RNA protein lipid and chitin in intact cells of P.oryzae was inhibited. These systems were not inhibited in a homogenate of the mycelium of P.oryzae by oryzalexin D at its ED, concentration; in addition the respiration of homo-2.10 Rice (Oryza sativa) genates and mitochondria was not inhibited at ED,,.However A more extensive account of the isolation and determination of ED, concentrations caused leakage of potassium from mycelial the strutures of oryzalexins A B and C (1 15)-(117) was cells and inhibited their uptake of glutamate. These findings The total synthesis of these compounds was suggest that the primary mode of action of (1 19) is interference publi~hed.~~~.~~~ carried out as outlined in Scheme 14.141 The overall yields of the with the cell membrane function in P.oryzae. compounds starting from (1 18) were 0.07 0.2 and 0.7% for The accumulation of phytoalexins by uninjured and injured oryzalexins A B and C respectively. The unnatural (-) leaves of susceptible(Su.) and resistant(Rt.) rice cultivars was enantiomers of the oryzalexins were also synthesized.Water drops incubated for 72 h on uninjured leaves Oryzalexin D (1 19) was isolated from rice leaves which were from Rt. and Su. cultivars were not toxic to P.oryzae spore infected with blast fungus Pyricularia oryzae Cavara. Its germination but the drops from the Rt. cultivar were slightly chemical structure was determined by comparison of its spectral toxic to germ tube growth. Injury of the Rt. cultivar followed NATURAL PRODUCT REPORTS 1991-C. J. W. BROOKS AND D. G. WATSON 0 by incubation with a water droplet resulted in an increase in the toxicity of the droplet to P.oryzae from 7-100 YObetween 24 and 72 h of incubation; incubation with spores of P. oryzae resulted in an increase in the toxicity of the water droplet.Injury caused little increase in the concentration of toxic substances in wound droplets from the Su. cultivar. The abiotic elicitors gibberellic acid (GA,) NaN, and penicillin induced the formation of momilactone A (120) in rice which was maximal in GAS-treated ~1ants.l~~ The induction of phytoalexins in rice leaves by heavy metal ions was 1 mM CuC1 induced the accumulation of oryzalexins and momilactones around holes punched in the leaves. Accumu- lation of momilactone A was first observed 12 h after treatment and reached a maximum at 72 h. The compounds occurred in the following relative amounts (120) > (1 16) > (1 15) > (1 17) > (1 19) =-momilactone B (121) at 72 h after inoculation.FeC1 and HgCl were respectively 38 YOand 20 YOas active as CuCl in the induction of (120) whereas Mn and Co ions showed little activity. The phytoalexins induced in rice leaves by treatment with UV-irradiation were detected by GC-MS with selected ion monitoring.14’ Accumulation of (1 15)-(117) and (1 19)-(121) was observed as well as that of an unknown antifungal substance. Large amounts of (120) were accumulated with (119) being the most abundant oryzalexin. The maximum accumulation of the phytoalexins occurred 3 days after irradiation except for (1 19) which reached its maximum after 2 days and (117) which was maximal at 6 days. The accumulation of phytoalexins was dependent on leaf ageing being much lower in the uppermost leaves. Qualitative and semi-quantitative analyses of oryzalexins A-D in rice leaves affected by blast or brown spot disease were performed by GC-MS.Concentrations were in the range 10-60 ,ug per g of lesion ; no oryzalexins were detected in healthy rice leaves.14* 2.11 Castor Bean (Ricinus communis) The regulation of the elicitation of casbene in castor bean seedlings by pectic fragments was studied. 149 Casbene H I synthetase the enzyme catalysing the formation of casbene (122) from geranylgeranyl diphosphate was purified to hom- ogeneity by dye ligand chromatography. Polyclonal anti bodies to casbene synthetase were then prepared and this permitted the determination of time-dependent changes in the levels of mRNA coding for the enzyme after elicitation of the seedlings with pectic fragments.Casbene synthetase mRNA was detected 2 h after application of the elicitor rising to a maximum at 6 h. The pattern of mRNA changes corresponded closely with the rise in casbene synthetase enzyme activity which reached a maximum at ca. 10 h after elicitation. These observations demonstrate the de novo synthesis of the enzyme. 2.12 Cassava (Manihot esculenta) Cassava roots damaged by cutting or fungal infection have been found to produce a large number of stress metabolites chiefly comprising steroids and diterpenoids. 150,151 Twenty-two of the latter have been isolated and identified most of them are new compounds comprising variously oxygenated and un-saturated structures based on four skeletal types -ent-beyerane (1 23 10 examples) ent-pimarane (1 24 9 examples) ent-atisane (125 2 examples) and ent-kaurane (126 1 example).Biological activities have not yet been reported but the isolation of these compounds greatly augments the previously small number of known diterpenoid stress metabolites. 2.13 Lettuce (Lactuca sativa) Inoculation of lettuce leaves with the bacterial pathogen Pseudomonas cichorii elicited the production of costunolide (127) (0.012% from dried leaves) and a new compound lettucenin A (128) (0.00084 Both products inhibited spore germination of Ceratocystisfimbriata -the first indication that costunolide may be classed as a phytoalexin although (127) was inhibitory at a much lower concentration. The structure (127) was assigned on the basis of spectroscopic data and represents the first guaianolide phytoalexin containing a 2H-cyclohepta[b]furan-2-one system.NPR 8 NATURAL PRODUCT REPORTS 1991 (129) R’ = C02H R2= H,R3 = R4= Me (130) R’ = C02H R2 = OH R3= R4= Me (131) R’ = C02H R2 = H R3 = CH20H R4= Me (132) R’ = Me R2= OH,R3= CH2OH R4= C02H 2.14 Lodgepole pine (Pinus contorta) Elevated levels of monoterpenes were detected in pine in phloem surrounding sites treated with the following (i) live mountain pine beetles (Dendroctonus ponderosae) ;(ii) Cerato-cystis clavigerum a fungus carried by the beetles; (iii) a pectic fragment from tomato leaves PIIF (proteinase inhibitor factor); (iv) chitosan. Chitosan was the most active elicitor stimulating a 10-fold increase in monoterpene levels.154 2.15 Madagascar Periwinkle (Tabernaemontana divaricata) Cell suspension cultures of Tabernaemontana divaricata when treated with an elicitor preparation from Candida albicans suffered rapid inhibition of both growth and of the production of the normally abundant monoterpenoid indole alkaloids. There was a rapid accumulation of at least ten pentacyclic triterpenoids. The five major metabolites of this type possibly acting as phytoalexins were ursolic acid (129) two mono- hydroxy-ursolic acids (1 30) and (1 3 l) and two dihydroxy derivatives of 3-epi-ifflaconic acid (1 32) and an isomer thereof. Restoration of cell growth and cessation of triterpenoid accumulation occurred about 36 h after elicitation and alkaloid production was resumed after a further 36 h.Cell-free preparations from elicited (but not from unelicited) cells 12 and 24 h after elicitation efficiently incorporated [l-14C]-IPP or I C2H5 C02CH3 [l-3H]-FPP into the group of triterpenoids. In elicited cultures the synthesis of phytosterols required for growth was inhibited at the level of squalene 2,3-epoxide cycloartenol cycla~e.’~~~ 2.16 Periwinkle (Catharanthus roseus) The important monoterpene indole alkaloids of Catharanthus roseus are not strictly phytoalexins being produced in healthy plants. It is however worth noting that for a number of cell lines various elicitors have been found to cause major increases in alkaloid biosynthesis in cell suspension cultures of C. roseus.In one report ajmalicine (133) and catharanthine (134) were found to be formed only in trace amounts by untreated cultures but attained levels of 400 pug 1-I and 600 pg I-l respectively within 3 days in cultures treated with cell-free culture filtrate from the growth medium of Micromucor isabellina.15’ The elicitation of tabersonine (1 39 strictosidine lactam (136) and lochnericine (1 37) has also been 0bser~ed.l~~ In one cell line of C. roseus cv. Little Delicata indole alkaloids were rapidly accumulated after treatment with a sterile preparation from Pythium aphanidermatum. Alkaloid form- ation was preceded by transient increases in extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthetase. Factors in the induction of the enzymes were exp10red.l~~ Earlier work has been briefly reviewed.160 NATURAL PRODUCT REPORTS 1991-C.J. W. BROOKS AND D. G. WATSON 387 31 A. A. Bell M. E. Mace and R. Stipanovic in ‘Natural Resistance of Plants to Pests Roles of Allelochemicals ’,ed. M. B. Green and P. A. Hedin ACS Symp. Ser. 1986 296 36. 32 M. Essenberg A. Stoessl and J. B. Stothers J. Chem. SOC. Chem. Commun. 1985 556. 33 P. Heinstein R. Widmaier P. Wegner and J. Howe Recent Adv. Phytochem. 1979 12 313. 34 R. Masciadri W. Angst and D. Arigoni J. Chem. Soc. Chem. Commun. 1985 1573. 35 R. D. Stipanovic A. Stoessl J. B. Stothers D. W. Altman A. A. Bell and P. Heinstein J. Chem. SOC. Chem. Commun. 1986 100. 2.17 Timothy (Phleurnpratense) 36 R.D. Stipanovic M. E. Mace D. W. Altman and A. A. Bell in Timothy plants infected by the phytopathogenic fungus Epichoe ‘Biologically Active Natural Products Potential Use in Agri- typhena are resistant to another pathogen Cladosporium phlei. culture’ ed. H. G. Cutler ACS Symp. Ser. 1989 380 262. When E. typhina infects a Timothy plant a peculiarly-shaped 37 L. G. Portenko I. M. Ryabova and Z. S. Azizova Zzv. Akad. stroma at the stalk is produced named choke. From this Nauk Tadzh. SSR. Otd. Biol. Nauk 1985,30 (Chem. Abstr. 1986 104 183456m). source seven fungitoxic sesquiterpenoid phytoalexins chokols lG2The 38 P. Heinstein J. Nut. Prod. 1985 48 907. A-G of general structure (138) have been isolated.lG1* simplest of these structures chokol G (139) has been 39 D.W. Altman R. D. Stipanovic D. M. Mitten and P. Heinstein In Vitro Cell Dev. Biol. 1985 21 659. synthesized. lG3 Since these metabolites are similar to other 40 M. E. Mace R. D. Stipanovic and A. A. Bell Physiol. Plant cyclopentanoids produced by fungi they are not necessarily Pathol. 1985 26 209. phytoalexins but the induced resistance suggests that exam- 41 M. E. Mace R. D. Stipanovic and A. A. Bell New Phytol. 1989 ination of the plant tissue for the presence of these or other 111 229. antifungal compounds may be of interest. 42 N. A. Garas and A. C. Waiss Phytopathology 1986 76 101 1. 43 H. J. Zeringue Jr. Phytochemistry 1987 26 975. 44 H. J. Zeringue Jr. Phytochemistry 1988 27 3429. 45 M. Pierce and M. Essenberg Physiol. Mol. Plant Pathol. 1987, 3 References 31 273.1 C. J. W. Brooks and D. G. Watson Nut. Prod. Rep. 1985,2,427. 46 P. S. Low and P. F. Heinstein Arch. Biochem. Biophys. 1986 2 H. Habereder G. Schroder and J. Ebel Planta 1989 177 58. 249 472. 3 W. E. Schmidt and J. Ebel Proc. Natl. Acad. Sci. USA 1987,84 47 I. Apostol P. S.Low P. Heinstein R. D. Stipanovic and D. W. 41 17. Altman Plant Physiol. 1987 84 1276. 4 W. Bless and W. Barz FEBS Lett. 1988 235 47. 48 T. J. Sun U. 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Fritzemeier D.Scheel and K. Hahlbrock Planta 1987 170 556. 114 G. D. Lyon Phytopathol. Z. 1984 111,236. 115 K. C. Roeber Biochem. Physiol. Pflanz. 1989 184 277. 116 S. V. Zinov’eva and L. I. Chaiova Helminthologiu 1987 24 303. 117 T. J. W. Alphey W. M. Robertson and G. D. Lyon Revue Nimatol. 1988 11 399. 118 N. Sato Y. Yoshizawa H. Miyazaki and A. Murai Ann. Phytopathol. SOC. Japan. 1985 51 494. 119 R. M. Zacharius E. B. Kalan and W. I. Kimoto Plant Cell Rep. 1985 4 1. 120 R. M. Zacharius and E. B. Kalan Plant Cell Rep. 1984 3 189. 121 H. W. Gardner A. E. Desjardins D. Weisleder and R. D. Plattner Biochim. Biophys. Acta 1988 966 347. 122 A. E. Desjardins H. W. Gardner and R. D. Plattner Phyto-chemistry 1989 28 43 1. 123 W. M. Robertson G. D.Lyon and C. E. Henry Can. J. Micro- biol. 1985 31 1108. 124 A. K. Banerjee and M. I. Pita Boente Heterocycles 1985 23 5. 125 A. Sudalai and G. S. Krishna Rao Indian J. Chem. B 1989 28 113. 126 A. Murai S. Sato and T. Masamune Bull. Chem. 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ISSN:0265-0568
DOI:10.1039/NP9910800367
出版商:RSC
年代:1991
数据来源: RSC
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6. |
Modern separation methods |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 391-413
A. Marston,
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摘要:
Modern Separation Methods A. Marston and K. Hostettmann* Institute of Pharmacognosy and Phytochemistry School of Pharmacy University of Lausanne CH-7015 La usanne Switzerland 1 Introduction 2 Planar Chromatography 2.1 Centrifugal Thin-layer Chromatography 2.2 Overpressure Layer Chromatography 3 Flash Chromatography 3.1 Normal Phase 3.2 Bonded Phases 4 Low-pressure Liquid Chromatography 5 Medium-pressure Liquid Chromatography 6 High-pressure Liquid Chromatography 6.1 Semi-preparative HPLC 6.2 Preparative and Process-scale HPLC 6.3 Biochromatography 6.4 Column Switching and Recycling 7 Countercurrent Chromatography 7.1 Droplet Countercurrent Chromatography 7.2 Centrifugal Partition Chromatography 7.2.1 Rotating Coil Instruments 7.2.2 Cartridge Instruments 8 Combination of Methods 9 Conclusions 10 References 1 Introduction The isolation of natural products from plant or animal sources poses numerous problems.The compounds being sought biologically active or otherwise may only be present in infinitesimal quantities ;when one considers for example that plants may have thousands of constituents the difficulties in separating out one particular component can be appreciated. The nature of the separation problem varies considerably from the isolation of small quantities (milligrams or less) for structure determination purposes to the isolation of very much larger amounts (hundred milligram to kilogram quantities) for comprehensive biological testing for semi-synthetic work or even for production of therapeutic agents.For these purposes a good selection of different techniques and approaches is essential. The importance of proper isolation and purification cannot be over-emphasized. Chromatographic techniques which are rapid and do not lead to decomposition material loss or artefact formation are needed. For these reasons and also with the aim of finding simpler solutions to complex separation problems the last few years have seen the development of a remarkable array of new chromatographic methods both analytical and preparative. A survey of preparative techniques used in the separation of natural products including practical aspects and applications has recently been published in an attempt to give an idea of the possibilities available.' As a product is purified during successive chromatographic steps the scale of the operation decreases.Thus the initial stages of a separation procedure involve methods with a high loading capacity and cheap stationary phases -these have traditionally been column chromatography with silica gel alumina polyamide or XAD supports although liquid-liquid methods are now finding increasing use. Subsequent steps employ techniques which require smaller samples -HPLC for example. An alternative is the use of gel filtration2 at different 39 1 stages of an isolation run. Dextran gels (Sephadex LH-20 is probably the most widely employed3) are very effective but are rather expensive for large-scale manipulations.By varying the separation mode (adsorption partition ion-exchange gel filtration etc.) and the eluent (different solvent strengths) the selectivity can be adjusted. This review describes a number of alternative techniques to classic open-column and thin-layer chromatographic methods for the preparative (as opposed to analytical) separation of mixtures. These newly introduced methods have the advantage of being rapid and often permit the isolation of previously inaccessible products. However it must be stressed that none of these provides by itself a comprehensive solution to all separation problems and the best approach is usually to employ a combination of techniques.For the description of some applications liquid-solid chromatography is treated first followed by liquid-liquid chromatography in the absence of solid stationary phases. 2 Planar Chromatography 2.1 Centrifugal Thin-layer Chromatography The technique of centrifugal thin-layer chromatography (CTLC) has been developed in order to avoid some of the problems of preparative thin-layer chromatography in which migration times are long and removal of substances from the bands on the plate is not easy or efficient. Centrifugal TLC relies on the action of a centrifugal force to accelerate mobile phase flow across a circular TLC plate. The Chromatotron manufactured by Harrison Research (Palo Alto California) consists of an inclined 24 cm diameter circular glass plate covered with a layer (1,2 or 4mm thickness) of sorbent (TLC- grade silica gel GF,, or aluminium oxide 60 GF,,4).4 The plate is rotated at 800 rpm by an electric motor sample introduction takes place at the centre and eluent is pumped across the sorbent (see Figure 1).Solvent elution produces concentric bands across the plate. These are spun off at the edges and collected for TLC analysis. The method is capable of separating 50-500 mg of a mixture on a 2 mm sorbent layer as long as the R,on an analytical TLC plate lies somewhere between 0.2 Adsorbent layer 11 ISample/Eluent 1Fraction collector Figure 1 Schematic view of the Chromatotron (model 7924). NATURAL PRODUCT REPORTS 1991 Table 1 Recent applications of CTLC with the chromatotron Sorbent Sample Sample (thickness) size Eluent Ref.Lignans Justicidin B from Silica gel -C,H,,-EtOAc (95:5) 8 Just icia pectoralis (Acan thaceae) Peltatin from a Amanoa species Silica gel -C,H,,-CHCl,-EtOH (25 :25 :3) 9 (Euphorbiaceae) (2 mm) Polyphenols Xanthones from Garcinia species Silica gel -Petrol-EtOAc (96 :4) 10 (Guttiferae) C6H CH 3-E t OAc-H0Ac (95 :5:0.5) Flavonoids from Tephrosia nubica Silica gel -CHC1,-MeOH 11 (Leguminosae) (99 1 98:2) Flavanones from Lonchocarpus Silica gel -C,H,CH,-EtOAc-HOAc 12 minimijorus (Leguminosae) (80 19 1) Prenylated flavanones from Silica gel -C,H,,-CHCl, C,H,,-Pr'OH 13 Platanus acerijblia (Platanaceae) Isoflavonoids from Erythrina Silica gel -C,H,,-EtOAc (4 1) 32 variegata (Leguminosae) (3 mm) Dichamanetin from Melodorum Silica gel -C,H,-CH,Cl,-Me,CO 14 frulicosum (Annonaceae) (1 mm) Arbutin derivatives Arbutin derivatives from Gentiana Silica gel -CHC1,-MeOH 15 pyrenaica (Gentianaceae) Quinones Naphthol-naphthoquinone dimer Silica gel -Petrol-CHC1 16 from Stypandra imbricata and Dianella revoluta (Liliaceae) Phthalides Phthalides from Meum Silica gel -CHCl, C,H,,-CHCl, C,H, 17 athamanticum (Umbelliferae) C,H,,-CHCl,-Pr'OH-MeOH Phthalide glycoside from Gentiana Silica gel -CHC1,-MeOH (9 1) 18 pedicellata (Gentianaceae) Monoterpenes Secoiridoid glucosides from Silica gel -CHC1,-MeOH 19 Gen tiana campestris (Gentianaceae) Sesqui terpenes Sesquiterpene lactones from Silica gel -C,H,,-EtOAc (90 10 75 :25) 20 Eupatorium quadrangulare (+lo% (Asteraceae) AgNO,) Diterpenes Ingenane from Mabea excelsa Silica gel -Et,O-C,H,, Et,O-Me,CO Me,CO 21 (Euphorbiaceae) (2 mm) Cucurbitacins Cucurbitacin glycosides from Silica gel -C6H ,,-CH,Cl,-EtOAc 22 Desfonta in ia spinosa (Desfontainiaceae) Alkaloids Diterpene alkaloids from Aconitum Silica gel -CHC1,-EtOAc CH,Cl,-MeOH 23 crassicaule (Ranunculaceae) (1 my) C,H,,-Et,O Et,O-MeOH Alumina Indole alkaloid from Mitragyna Silica gel 200 mg CHCl, CHC1,-MeOH 24 speciosa (Rubiaceae) (2 my) -Alkaloids from Papaver and Alumina C,H,CH,-Cyclohexane-Et,NH 25 Fumaria (1 f"m> (2:8 1) Ent-kaurene alkaloids from Silica gel -CHC1,-Me,CO-Et,NH-C,H, 26 A nop ter us glandulosus (5:4 1:6) (Escalloniaceae) EtOAc-MeOH-NH,OH (1 7 :2 1) Steroidal alkaloids from Fritillaria Silica gel -CHC1,-MeOH-H,O (1 3 :7 :2) 27 harelinii (Liliaceae) (satd.with Cyanogenic glycosides NH gas) Cyanogenic glycosides from Silica gel -EtOAc EtOAc-MeOH (19 I 9 1) 28 Merrernia dissecta (2 mm) (Convolvulaceae) Tricho thecenes Roritoxins from cultures of Silica gel -CH,Cl,-MeOH C,H,,-EtOAc 29 Myrothecium roridum (1 2 4 mm) CH,Cl,-EtOAc Baccharinoids from Baccharis Silica gel 100 mg CHC1,-MeOH (9 1) 30 megapotamica (Asteraceae) (2 mm) Miscellaneous 3-Nitropropanol glycoside from Silica gel -CHCl,-EtOH (1 4-3 :2) 31 Astragalus miser (Leguminosae) NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN and 0.5.4 One Chromatotron plate can separate the same amount of substance as three 20 x 20 cm preparative TLC plates of the same thi~kness.~ An investigation of other parameters has appeared elsewhere.s It is claimed that if the circular chromatography plates are rotated as they dry a mote uniform sorbent layer is produced.This effect is even more pronounced if drying is carried out at elevated temperatures.’ Most applications of CTLC have employed silica gel as the sorbent. An early example was the separation of the xanthones bellidifolin and desmethylbellidifolin obtained after acid hydrolysis of a methanol extract of Gentiana strictzjlora (Gentianaceae). They were separated in less than 30 minutes using chloroform with increasing amounts of methanol as solvent.The corresponding separation by polyamide open- column chromatography required at least 12 hours. Some other examples of isolation work involving the use of CTLC are shown in Table 1. In one instance a difficult separation of heliangolides sesquiterpene lactones (1) and (2) from Eupatorium quadrangulare (Asteraceae) was achieved by radial chromatography on silica gel impregnated with 10 % silver nitrate. 2o More recently aluminium oxide 60 GF,, or HF,, has been reported as an alternative s~rbent.~~. 34 Separations involving diterpene alkaloids,33 and Fumaria and Papaver alkaloid^,^ have been performed successfully on alumina plates with gradient elution in certain cases. Another commercially available instrument for CTLC is the Rotachrom (Petazon Zug Switzerland) with which the rotor speed can be regulated between 100 and 1500 rpm.It is a modular instrument which allows CTLC sequential centrifugal layer chromatography (SCLC) or centrifugal planar column chromatography (CPCC) to be performed on the same machine.35 In SCLC the mobile phase can be introduced onto the plate at any desired place and time. Difficult separations are possible on a single plate without having to recycle mixtures onto a second CTLC plate.36 By this method a prepurified fraction (150 mg) containing methoxylated coumarins from roots of Heracleum sphondyliurn (Umbelliferae) was separated after elution with various mobile phases into five components bergapten pimpinellin sphondin isobergapten and iso-pimpinelli~~.~~ A mixture (120 mg) of bipyridyl isomers was also separated by SCLC using a single mobile phase and a recycling techniq~e.~~ Me Me Mobile Phase 2.2 Overpressure Layer Chromatography Overpressure layer chromatography (OPLC) involves covering the sorbent layer of a chromatographic plate with an elastic membrane under external pressure.The plate must be tightly sealed giving a closed system (see Figure 2). By this means the vapour phase is eliminated and under forced-flow conditions of eluent sample development is 40 Two instruments are available the Chrompres 10 and the Chrompres 25 with a higher cushion pressure of 25 bar (Labor MIM Works Budapest-Esztergom Hungary). This forced-flow TLC can achieve high efficiencies due to the use of fine-particle sorbents and longer plates than those found in capillary controlled systems.As separation times are short diffusion effects are reduced. It is possible to employ mobile phases with poor solvent wetting characteristics. Both off-line36 and on-line4’ preparative separations have now been reported with OPLC apparatus but on-line applic- ations require special preparation of the plates by scraping inlet and outlet channels for the eluent and coating edges with a polymer to prevent solvent leakage.41 The method is most suitable for the purification of small (50-100 mg) amounts of partially purified samples. Natural products isolated by OPLC include the following Anthraquinones frangula-emodin from Rharnnus frangula (Rhamna~eae).~’ Alkaloids noscapine and papaverine from opium.41 Furocoumarins methoxylated furocoumarins from Hera-cleum sphondylium (Umbelliferae) Secoiridoid glycosides amaropanin amarogentin amaros- werin and gentiopicrin from Gentiana purpurea (Gentian-aceae).,’ Iridoid glycosides :shanzhizide methyl ester from Crossopteryx febrifuga (R~biaceae).,~ Cannabinoids cannabinoids from Cannabis saliva.43 Simaroubolides holacanthone and glaucaroubolone from Soulamea soulameoides (Simaroubaceae).44 Diterpenes phorbol diesters from croton oil., Cucurbitacins cucurbitacins from Hemsleya gigantha (Cucurbitaceae) .46 Steroid saponins polypodoside A from Polypodium glycyr-rhiza (P~lypodiaceae).~’ Cardiac glycosides epimeric glycosides from Streblus asper (Moraceae).42 For the separation of cucurbitacins from Hemsleya gigantha (Cucurbitaceae) an ethanol extract of the tubers was subjected to a preliminary fractionation by silica gel column chromato- graphy.Closely-related cucurbitacins with unsaturated side chains were separated from their corresponding 23,24-dihydro derivatives by OPLC using the solvent EtOAc-Et,O-CHCl,- EtOH 80 10:7:3 and 2 mm silica gel 60 F,, plates (Merck). A sample size of 50 mg was applied to each plate.46 Synthetic hernandulcin (an intensely sweet sesquiterpene) has been purified in 100 mg quantities on normal TLC plates with the mobile phase hexane+thyl acetate 10:3. Thus on-line OPLC provided a suitable method for the removal of minor reaction Pressurized Chamber Chromatographic Plate Figure 2 Preparative OPLC (Reproduced from reference 41).394 OPLC separations are usually initiated on dry plates in a non-equilibrated system. This procedure often leads to solvent demixing and the consequent formation of ill-shaped bands. However by on-plate injection the chromatographic layer can be pre-equilibrated with the solvent system thus avoiding the solvent demixing effect.42 This technique has been used for the successful separation of alkaloids cardiac glycosides and an iridoid glycoside .42 Selection of solvent systems for CTLC and OPLC can either be performed by TLC or by the ‘PRISMA’ mobile phase optimization pro~edure.~~ A description of this method with special reference to the OPLC of polar natural products (ginsenosides and flavonoid glycosides) has appeared.49 3 Flash Chromatography Flash chromatography is a preparative air-pressure-driven liquid chromatographic technique with moderate resolution first published by Still Kahn and Mitra50 in 1978 and patented in 1981 (US Patent 4293422). Its introduction was encouraged by the need to have a fast method for the purification of samples with minimal sample loss and less risk of decompo- sition so often encountered with conventional open-column chromatography. The concept of flash chromatography is exceptionally simple and preparative separations are very easy to perform using readily available and cheap laboratory glassware. A glass column of suitable length (id 10-50 mm) fitted with an exit tap is either dry-filled or slurry-filled with suitable packing material.Dry-filling gives a better packing but requires the passage of a large amount of solvent before the support is fully moistened and free of air. The attachment of a reservoir at the top of the column is optional. The sample is introduced solvent is added and the column closed with an air inlet fitted with a needle valve to control the compressed air (or nitrogen) supply. Maxirnum pressures reached at the top of the column are therefore ca. 2 bar. Separations of 0.01-10 g samples (depending on column size) can be performed in as little as 5-10 min with the eluate being collected in a fraction collector or in hand-held test tubes.Analysis of fractions is generally by TLC. Although a home-made flash chromatography apparatus is relatively simple to construct several suppliers have complete systems available :Aldrich (Milwaukee Wisconsin) J. T. Baker (Phillipsburg New Jersey) and Eyela (Tokyo Rikakikai Tokyo). A modification of the original flash chromatography apparatus to accommodate a larger solvent reservoir has also been reported.51 3.1 Normal Phase When separations are performed with silica gel typical adsorbents used are Merck LiChroprep Si 60 2540pm Merck silica gel 60,40-63 pm Merck silica gel 60,63-200 pm or Bakerbond 40 pm silica gel. With 63-200 pm silica gel flow rates of 50 ml/min are easily accessible. TLC is perfectly adequate for method development;50 a solvent is chosen that gives a good separation and moves the desired component to an R value of at least 0.35.Compounds having ARf ca.0.10 can be separated in quantities of 1 g using 50 mm id If less resolution is required these columns are capable of separating up to 10 g of sample (of crude plant extracts for example). In general the amount of sample is proportional to the cross- sectional area of the column (see Table 2). Flash chromatography has found rapid and widespread acceptance as a separation method and examples of its use are extremely abundant. One important area of application is the purification of synthetic intermediates as shown in recent work on air-sensitive compounds involved in the total synthesis of plant Flash chromatography is also an important technique for the separation of labile compounds.For example antifeedant limonoids gradually decomposed on silica gel open NATURAL PRODUCT REPORTS 1991 Table 2 Relationship between column diameter and sample size in flash chromatography (depth of adsorbent CQ. 15 cm) (Reproduced from reference 50) Column diameter Sample loading (mg) (mm) AR?2 0.2 ARf 3 0.1 10 100 40 20 400 160 30 900 360 40 1600 600 50 2500 1000 (3) columns and could only be obtained in the pure state by flash chromatography and semi-preparative HPLC.53 Flash chromatography although occasionally used as a final purification step in the separation of natural products is most often employed for the rapid preliminary fractionation of complex mixtures.It is therefore an important step in isolation strategies involving combinations of chromatographic methods (see section 8). The applications are too numerous to list here but a selection has already been documented’ and one or two pertinent examples will be described to give an idea of the possibilities of the method. A combination of flash chromatography and semi-pre-parative HPLC has been used for the isolation of neolignans with platelet activating factor binding inhibition from Piper futokadsura (Piperaceae). The crude dichloromethane extract (23 g) of the stems was fractionated on a flash column (75 x 10 cm) eluted with a hexane-ethyl acetate gradient. The three biologically active compounds (3)-(5) were purified by HPLC on a silica gel column (hexane-thy1 acetate 3 l).54 NATURAL PRODUCT REPORTS 1991-A.MARSTON AND K. HOSTETTMANN CHO (7) OH OH (9) The closely related 9-deoxy drimane sesquiterpenes (6) and (7) were isolated from Canella winterana (Canellaceae) by two flash chromatography steps. The first step employed a silica gel support and the solvent system diethyl ether-hexane 7 :3 while the pure sesquiterpenes were obtained by flash chromatography on silica gel impregnated with 10 YOsilver nitrate (diethyl ether- hexane 1 l).55 The antifungal compounds falcarindiol (8) and sarisan (9) have been isolated from the leaves of Heteromorpha trifoliata (Umbelliferae) by flash and low-pressure liquid chromato- graphy. For the first step the petroleum ether extract (4 g) was fractionated on silica gel 60 (63-200 pm) with a chloroform- methanol 99 1 -+ 96:4 gradient.Final purification was per- formed on Lobar The possibilities of separation by flash chromatography are not restricted to silica gel packings and examples of other sorbents are known. One possibility is polyamide whose use has been described for the isolation of phenolic acids.57 New quinolizidine alkaloids have been isolated from Lupinus argenteus (Fabaceae) by flash chromatography on basic alumina (Merck type T) by elution with diethyl ether diethyl ether-methanol mixtures and methan01.~~ 3.2 Bonded Phases Following the success of flash chromatography on columns packed with silica gel the use of chemically bonded phases is now assuming increasing imp~rtance.~~ With reversed-phase packing material there is increased back-pressure and this may require a shortening of the column in order to maintain adequate flow rates.Routine clean-up of crude reaction mixtures60 and separation of compounds with a values > 1.4 are easily performed. Samples may be loaded as slurries or powders in the sorbent and can for example be eluted by a step gradient starting with water moving to methanol-water mixtures and finally ending up with dichloromethane.61 It is claimed that up to 20 g of crude extracts per 100 g of support can be chromatographed in one run.61 While silica gel packings are generally discarded after use bonded sorbents are regenerated thus offsetting the problem of increased initial cost of the packing material.An alternative is to prepare the reverse-phase support from chromatographic grade silica Applications of bonded phases include the following The separation of microbiocidal methylisothiazolone deriva- tives (RP- 18; MeOH-H,0-HOAc).62 The initial fractionation of extracts from sponges ascidians and bryozoans (RP- 18; 32-63 The isolation of zwiebelanes from onion (RP-18 ; MeOH).63 The isolation of capsaicinoids (RP-1I 40 pm ; MeOH-H,0).64 Flash chromatography on chiral stationary phases holds great potential for the rapid preparative separation of enantiomers. Aminopropyl silica gel modified with dinitro- benzoylphenylglycine allows the resolution of racemic benzo- diazepiones (Valium@ analogues) on a hundred milligram scale.65 4 Low-pressure Liquid Chromatography Low-pressure liquid chromatography (LPLC) makes use of columns containing packings with a particle size of ca.4&60 pm. The relatively large particle size enables high flow rates at pressures of up to 10 bar. Because of the low pressures columns are mostly made of glass and separations can be followed visually in the case of pigmented mixtures. The best-known commercially available system is the Lobar range from E. Merck (Darmstadt W. Germany). Columns of three different standard sizes (lengths from 24 to 44 cm) can be purchased filled with LiChroprep Si60 RP-8 RP-18 or diol material. A high and uniform packing density guarantees a good separation efficiency.66 The packing material is sealed into the colunn with a glass frit and connection to a pump is provided by a metal cannula attached to a PTFE ring.The columns are re-usable although column life of the unmodified silica gel is shorter. The effect of certain parameters on separation efficiency of Lobar columns has been discussed.66 Without doubt this type of system provides one of the most versatile and simple means of isolating substances on the milligram to gram scale generally in combination with a pre- purification step (or steps). In certain cases a similar resolution can be obtained by both LPLC and analytical HPLC.66 This is of importance for the transposition of separation conditions from analytical HPLC to LPLC and is illustrated by the separation of iridoids and a phenyl glycoside from the root bark of Sesamum angolense (Pedaliaceae) a plant reputed to have anti-haemorrhagic properties in African traditional medicine.Analytical HPLC of the relevant fraction is shown in Figures 3(a) and 3(b). A NATURAL PRODUCT REPORTS. 1991 % MeOH (12) 21 10 (1 1) I 0 10 0 10 [minl (b) Figure 3 Isolation of iridoids from Sesarnurn angolense. (a) Analytical HPLC. LiChrosorb RP-8 MeOH-H,O 10:90 detection 254 nm (b) Analytical HPLC. LiChrosorb RP-8 MeOH-H,O 32 68 detection 254 nm (c) Preparative separation. Lobar RP-8 (310 x 25 mm id) sample 130 mg detection 254 nm. Table 3 Applications of LPLC in the separation of plant-derived natural products Substance class Column size Support Eluent Ref.Flavonoids B SiO Petrol-EtOAc CHC1,-MeOH 78 B DIOL CHC1,-MeOH-HOAc (950 :50 1) 79 CHC1,-MeOH (99 :1) 79 n.d. RP- 1 8 MeOH-CH,CN-THF-H,O (3 :3 10 34) 80 Flavonoid glycosides B RP-8 MeOH-H,O gradient 81 B RP-8 MeOH-H,GHCOOH (25 :73 :2) 82 B RP-8 MeOH-H,O gradient 83 n.d. RP-8 Me,CO-H,O 84 Xanthone glycosides B RP-8 MeOH-H,O gradient 81 Pterocarpans B SiO Petrol-EtOAc (5 :5 2 8) 85 Chalcone glycosides C RP-8 CH,CN-H,O (1 :9) 86 Chromenes B SiO CHCl,-C,H,,-MeOH (80 :8:0.3) 87 Phenylpropanoid glycosides -RP- 18 MeOH-H,O gradient Pr'OH-H,O gradient 88 Phloroglucinol derivatives n.d. RP-8 MeOH-H,O (2 :1) 89 Sarisan B SiO Petrol-EtOAc (10 :1) 56 Sesquiterpenes C SiO C,H,,-EtOAc (65 35) 90 Diterpenes n.d.RP- 1 8 MeOH-H,O (1 :1) 91 -RP-1 8 MeOH-H,O (1 1) 92 Limonoids -RP- 18 MeOH-H,O 93 Tri terpenes C RP-8 MeOH-H,O (4 :1) 94 Steroid glycosides B RP-8 MeOH-H,O (9 :1) 95 Iridoids C RP-8 MeOH-H,O gradient 96 Iridoid glycosides RP- 18 MeOH-H,O gradient 97 98 B RP-8 MeOH-H,O (1 :25 1 10) 99 C RP-8 MeOH-H,O 100 Alkaloids n.d. RP-8 MeOH-0.02M NH,OAc (3 :2) 101 Pol yacet ylenes B SiO Toluene-EtOAc (85 15) 56 n.d. SiO C,H,,-Et,O 102 NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN FCOOH baseline separation of phlomiol (10) and pulchelloside-I (1 1) was possible with MeOH-H,O 10:90 [see Figure 3(a)]. Verbascoside (12) was however only eluted with 32 YOMeOH [see Figure 3 (b)].These analytical conditions could be applied directly to a preparative separation on a Lobar column by performing a step-gradient elution from 10 YOto 32 %MeOH (see Figure 3~).~' In order to increase the effective column length and thus augment loading capacity and/or separating power several Lobar columns can easily be connected in series as has effectively been shown for mixtures of reaction products.68 Gradient operation is another possibility as demonstrated by the separation of flavonoid and xanthone glycosides.81 Apart from applications in the isolation of plant-derived natural products (see Table 3) LPLC has found extensive use in the purification of reaction mixtures,69 in work on the constituents of marine o~ganisms,~O-~~ in the isolation of and products in the isolation of skin alkaloids from frogs,75 and in the purification of synthetic nucleotides.76 The methyl ester of turbinaric acid (13) a cytotoxic secosqualene carboxylic acid was obtained from the brown alga Turbinaria ornata by Lobar LPLC on a size A column packed with RP-2 material and eluted with dioxane-water 10:3." 5 Medium-pressure Liquid Chromatography The concept of medium-pressure LC (MPLC) was introduced in 1979 for the separation of diastereomeric oxa~olines.~~~ A piston pump capable of producing flow-rates of 19 ml/min at 7 bar was connected via a 4-way valve (for injection of sample) to a 250 x15 mm pre-column and a main 1000 x25 mm glass chromatography column.Both columns were filled with 40-63 pm silica gel 60 (Merck) and provided separations of for example 4.4 g of (14) and (1 5) with hexane-acetone solvent combinations.Monitoring of the eluted material was performed by TLC and analytical HPLC. The technique makes use of pressures of ca. 5-40 bar and can easily accommodate much larger sample loads (100 mg- 100 g) than are generally applied in LPLC separations. Longer columns often with larger internal diameters are employed these being refillable and usually of glass. Other parameters are shown in Table 4. As far as separating power is concerned MPLC lies somewhere between LPLC and semi-preparative HPLC. A comparison with flash and open-column chromato- graphies is shown in Table 5. In this particular example open- column and flash chromatography gave the same resolution while MPLC was more efficient than the former two methods.Separations by flash and MPLC both involve a considerable gain in time. The high loading capacity of MPLC columns should be noted :a 1 :25 sample-packing material ratio could be achieved for the test mixture.lo4 For MPLC particle sizes of 15-200 pm are usually advocated and columns can be dry-packed (unmodified silica gel) or slurry-packed (reversed-phase). Verzele and Geeraertlo5 have published useful packing methods for these larger-bore columns and since the right way of filling columns is such an essential pre-requisite for good separations this item cannot be over-emphasized. Additional studies on column preparation methods have shown that dry-packing of small particle size ~ ~ ~ ~ Table 4 Comparison of preparative liquid-solid chromatographic methods Semi-prep HPLC MPLC LPLC Granulometry 5-10 pm 1540 pm 40-63 pm N/m 30000-50 000 500-10000 300-600 Column dia.8-25 mm >25mm >10mm Pressure <350 bar <40bar <6bar Table 5 Separation of 900 mg test mixture (dimethyl- diethyl- and dibutyl esters of phthalic acid 1 :1 :1) with hexane-EtOAc 4 :1 (Reproduced from reference 104). Silica gel (240 g) particle size (1Lm) Pressure (bar) Flow-rate (ml/min) Resolution (R,) Time (min) Open column 63-200 0 25 1.5 64 Flash 40-63 0.75 140 1.6 9 MPLC 2540 12 100 3.4 12 5-25 ,um silica gel is feasible and that the corresponding separations are very efficient.lo6 The influences of solvent strength flow-rate of the mobile phase capacity and dimensions of the column have been investigated for crude apolar plant extracts on silica gel.A pronounced decrease of retention time was observed on increasing flow rates from 0.9 ml/min to 4 ml/min. Still higher flow rates exerted practically no influence on retention time. With a flow rate of up to 5 ml/min no significant influence on resolution was observed. However a large increase in resolution is obtained if a separation carried out on a short column of large internal diameter is repeated on a longer column of smaller internal diameter. lo' Complete systems for MPLC separations are marketed by Buchi (Flawil Switzerland) and Labomatic (Schonenbuch Switzerland).Kusano Scientific Instrument Co. (Tokyo Japan) supply the CIG column system which comprizes a micro-pump (1-lOml/min) and a silica gel or ODS column with three internal diameter options 100 x10 mm id 100 x15 mm id or 100 x22 mm id. Separations with this system include lignan xylosides from Prunus ssiori and P. padus (Rosaceae) (RP- 18 ;20 pm). lo8 nor-Clerodane diterpenes from Croton cajucara (Euphorbi-aceae) (Silica gel 10 ,um and RP-18 20 Diterpenes from AIpinia formosana (Zingiberaceae) (60 ,um Iatrobeads spherical silica gel and RP-18 2545 Ingenol ester from Euphorbia Zathyris (Euphorbiaceae) (silica gel 10 Flavonol glycosides from Epimedium koreanum (Berberid-aceae) (RP-18 30 Chromatographs previously manufactured by Jobin-Yvon are now marketed by Axxial (see section 6.2).Although applications (one example is the isolation of bitter sesquiterpene lactones from chicory roots113) are still reported from time to time axial compression chromatography with the Jobin-Yvon NATURAL PRODUCT REPORTS 1991 4 'OH 0 OH (19) Figure 4 Analytical RP-HPLC separation of secoiridoid glycosides from Gentiunu lucteu. Hypersil RP-8 5 pm (100 x 4.6 mm id) MeOH-H,O 20 :80 30 :70 flow-rate 1.5ml/min detection 254 nm (Reproduced from reference 1). I i 60 1io [min] 1 Figure 5 Preparative MPLC separation of secoiridoid glycosides from Gentiuna lactea. RP-8 15-25 pm (460 x 25 mm id) MeOH-H,O (20 80 30 70) step gradient flow-rate 18 ml/min sample 1.5 g detection 254 nm (Reproduced from reference 1).Modulprep and related liquid chromatographs has largely been replaced by other preparative liquid chromatography tech- niques. The chromatographic conditions for MPLC are selected by analytical HPLC or by TLC. The selectivity of the eluent is first optimized and then the elution strength of the mobile phase is adjusted to suit the preparative conditions. Capacity factors (k') between 1 and 51°4 or R,values < 0.4 are appropriate. In one application of a Buchi B-680 system Schaufelberger and Hostettmann114 have described the separation of decigram amounts of secoiridoid glycosides from Gentianaceae on a LiChroprep RP-8 (Merck) support with methanol-water as eluent. Analytical HPLC was used to choose separation conditions (see Figure 4) and these were transposed directly to MPLC (see Figure 5).The separation was completed within 3 h and resolution approached that of analytical HPLC. Other MPLC applications include alkamides from plant^.^^^-'^' Tetranortriterpenoids from Azadirachta indica (Meliaceae) (separated on LiChroprep RP-18 7-10pm in glass Cucurbitacin glycosides from Picrorhiza kurrooa (Scrophul-ariaceae). 120 Sesquiterpene lactone hydroperoxides from Anthemis nobilis (Asteraceae) (separated on silica gel 63-200 pm with petrol ether-ethyl acetate).lZ1 Spiro- bicyclic triterpenes from Iris pseudacorus (Iridaceae) (methanol-water gradient elution on RP- 18 14-40 Prenylflavonol glycosides from Epimedium koreanum (Berberidaceae) (methanol-water mixtures on RP-8).lZ3 MPLC has proved very effective in the separation of saponins from molluscicidal plants.The fractionation and isolation of saponins from Phytolacca dodecandra (Phytolaccaceae) for example is shown in Figure 6. After partition of the methanolic extract of the berries between butanol and water followed by a liquid-liquid chromatographic step the bidesmosidic tri- terpene glycosides (20)-(25) were isolated by MPLC and LPLC.124 The stationary phase was of the RP-8 type and mixtures of methanol and water were employed for elution. The saponins were very soluble in the mobile phase-an important consideration in preparative chromatography. An example of MPLC separation of rather apolar compounds is given in Figure 7.The chloroform extract of the root bark of Dolichos marginata (Leguminosae) showed antifungal activity against Cladosporium cucumerinum in a TLC bioassay. A first fractionation by MPLC on silica gel was followed by a second MPLC run on silica gel (two columns in series) to obtain the antifungal pterocarpans (27) and (28). The other pterocarpans (26) and (29) were isolated by LPLC.125 As can be seen MPLC is very useful for the rapid fractionation of crude extracts provided the supports used are inexpensive enough to be discarded after the separation. Most MPLC separations have this far been performed on silica gel and various reversed-phase supports. However in one instance microcrystalline cellulose has been used for the separation of a caffeic acid glycoside ester from Plantago crassifolia (Plantaginaceae).The eluent was EtOAc-MeOH- Me(CH,),Me-H,O (90 :15:5 :11).126 NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN MeOH extract (11 g) BuOH fraction (7.5g) RLCC (2x) IAcOEtlEtOWH20 (40:20:40) laxending mode 1-11 111 Iv V VI 1101 mg 890mg 1225mg 2830mg 1195mg (20) R' (21) R' (22) R' (23) R' (24) R' (25) R' (25) 1025mg 470 mg (22) 165mg 120mg (23) (24) = CH20H R2 = OH R3 = R4= H = CH3 R2 = R3= H R4 = Gal = CH3 R2= R4= H R3= GIC = CH20H R2 = OH R3 = H R4= Gal = CH20H R2= R4= H R3 = Glc = CH3 R2 = R4= H R3 = Rha-2Glc (20) (21) 100mg 195mg (1) RP-8 MeOH-H20 (6!5:35) (2) (6535) (3)RP-8 MeOH-H20 (60:40) (4) RP-8 MeOH-H20 (60:40) (5) RP-8 MeOH-H20 (5545) Figure 6 Isolation of saponins from berries of Phytolacca dodecandra.R' 3.5 9 MPLC 920 x36 mm Silica get CHCIdeOH (10:0.8) I tpLc (26) I1 I11 IV 580 mg MPLC 920 x 36 mm Silica gel PetroVEtOAc (3:7) V lPLC (29) (26) R' = R3 = CH3 R2 = H (27) R'=R2=R3=H HO-I (28) R' = R2 = R3= H (29) R' = R2 = H R3 = CH3 Figure 7 Isolation of antifungal pterocarpans from Dolichos marginata. 6 High-pressure Liquid Chromatography High-pressure (or high-performance) liquid chromatography (HPLC) finds application in the preparative separation of samples which range from microgram to gram or even kilogram quantities. On the whole however HPLC is commonly applied as a last step in purification processes and in this respect the quantities involved tend to be at the lower end of the scale.The reason why pre-purified samples and not crude extracts are chromatographed by HPLC is that the sorbents are of small particle size and consequently very expensive. It is therefore necessary to avoid contamination and re-use the columns as often as possible to reduce costs. The preparative aspects of HPLClz7have been the subject of several international symposia and a number of books.12s*129 For all preparative HPLC techniques a judicious selection of operating parameters is required in order to achieve the desired purity and yield at an economically acceptable cost. This involves the following sequence (1) Basic choice of HPLC system.In certain cases TLC analysis of the sample can be used as a first indication of the correct operating conditions -silica gel plates for normal-phase columns and silylated silica gel plates for reversed-phase columns. When using this method it should be noted that the surface areas of silica gels employed for TLC are about twice those used in column packings and that the R,should be c 0.3. (2) Separation of a small quantity of sample on an analytical HPLC column. Once the appropriate mobile phase has been found the TLC separation can be extrapolated to an analytical NATURAL PRODUCT REPORTS 1991 Table 6 Separations of natural products by semi-preparativeHPLC Dimensions Substances separated Poly phenols Chromenes from Ageratina arsenii (Asteraceae) Column Silica gel (mm) 250 x 10 Mobile phase C,H,,-EtOAc Ref.139 Furanochromone glucosides from Ammi visnaga (Umbelliferae) Polyphenolic acids from Salvia miltiorrhiza (Labia tae) Zorbax ODS TSK-gel ODS- 120T (10 pm) 250 x 9.4 300 x 7.6 CH,CN-EtOH-H,O (1 :1 8) MeOH-H,O (4 6) +HOAc 140 141 Platanus acerifolia Kaempferol glycosides from pBondapak C, 300 x 10 MeOH-H,O (65:35 50 50) 142 (Platanaceae) Flavonoids from Senecio incanus pBondapak C, 300 x 7.8 CH ,CN-H ,O-HOAc 143 (Asteraceae) Isoflavonoids from Psorothamnus fremontii Dynamax RP- 18 250 x 21.5 MeOH-H,O (7 :3) 144 (Fabaceae) Isoflavonoids from Pueraria lobata (Leguminosae) Lignans from Forsythia intermedia Senshu-Pak ODS Aqua-Sil SN-662N Spherisorb S5 ODS2 250 x 8 150 x 8 250 x 8 CH,CH-H,O-HOAc CHC1,-MeOH-H,O (40 16 3) MeOH-H,O (9 11) 145 146 (Oleaceae) Neolignan glycosides from TSK-gel ODS-120T 300 x 21.5 CH,CN-H,O MeOH-H,O 147 Codonopsis tangshen (Campanulaceae) Ellagitannins from Quercus robur LiChrospher RP- 18 250 x 25 MeOH-H ,O-H ,PO 148 (Fagaceae) Anthocyanin from Zebrina pendula (Commelinaceae) Develosil ODS (5 Icm) n.d.CH,CN-HOAc-TFA-H ,O (6.8:5.4:0.5:87.5) 149 Sesquiterpenes from Canella winterana Sesquiterpenes pPorasil n.d. C6H1,-THF (80:20) 150 (Canellaceae) Sesquiterpene alcohols from Panax ginseng Chemcosorb 5 Si 300 x 10 (Araliaceae) Gochnatia palosanto Sesquiterpene lactones from Phenomenex R Sil 10 C-8 500 x 10 MeOH-H,O (3 :2 11 :9) 152 (Asteraceae) Diterpenes Ingol esters from Euphorbia antiquorum (Euphorbiaceae) Chemcosorb 5 Si 500x 10 C6Hl,-C,H4Cl,-EtOH (5 :2:0:2) I53 Triterpenes from Paeonia japonica callus tissue (Paeoniaceae) Saponins from quinoa Furostanol glycosides Triterpenes from Tamus communis Kusano silica gel Fuji gel R18-37 RP TSK-gel ODS- 120T pBondapak C, n.d.n.d. 300 x 21.5 300 x 7.8 C6H 14-E tOAc CH,CN-H,O (7 3) MeOH-H,O (73 :27) MeOH-H,O (62 38) 154 155 156 (Dioscoreaceae) Acylglucosyl sterols from Momordica charantia Silica gel (20 F-9 600 x 8 C6H14-THF (60 40) 157 Miscellaneous Cucurbitacin glycosides from LiChrosorb RP- 18 250x 10 CH,CN-H,O gradient 119 Picrorhiza kurrooa (Scrophulariaceae) Quassinoids from Picrasma javanica (Simaroubaceae) Limonoid from (7 lum) Capcell Pak RP-18 Phenyl 250 x 10 250 x 22.5 MeOH-H,O (2:3) CH,CH-H,O (7 13) 158 159 Azadirachta indica (Meliaceae) Cardenolides from (5 Pm) Silica gel n.d.C,H,,-Pr’OH (5 1 3 1) 160 Asclepias vestita (Asclepiadaceae) Polyacetylene from Panax ginseng (Araliaceae) Nucleosil 50-5 Senshu ODS-215 1 300 x 8 150 x 6 C,H,,-EtOAc (2 :1) MeOH-H,O (4 1) 161 Alkaloids Spermine alkaloids from Chaenorhinum minus (Scrophulariaceae) LiChrosorb NH (10 Pm) 250 x 7.8 CHC1,-MeOH-25 (191:8:1) % NH,OH 162 NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN 401 HPLC column containing the packing to be used in the preparative column. A preliminary analytical search for the right conditions saves time sample and solvent. (3) Optimization of the analytical HPLC separation aiming for small capacity factors (k’). A good analytical HPLC separation is usually a prerequisite for a successful preparative operation.Relative retention (selectivity a)is a very important parameter in determining possible sample size and it is necessary to maximize this value. An excellent discussion on how this is achieved taking into account the effects of stationary and mobile phases is given by McDonald and Bidlingmeyer,128 and also Snyder et a1.l3O (4) Scale-up to preparative HPLC apparatus.131 In many preparative HPLC examples the column is actually overloaded nonlinear adsorption isotherms are obtained and peaks are not symmetrical. Further considerations such as column geometry and sample introduction have been discussed in depth.12* Scaling-up a successful analytical separation may cause problems associated with the solubility of the sample.This is especially true of reversed-phase HPLC if the compounds under investigation do not dissolve in aqueous solvents. Diluting the sample may help but if the volume injected is too great separation efficiency If on the other hand the sample is too concentrated precipitation on the column may occur. One possible solution is to mix the crushed sample with sorbent and dry-pack into a sample column or into the chromatography column itself (‘ solid injection ’).133 Although chromatographic separations on conventional silica gel supports are very common the use of bonded phases is becoming widespread. Another trend is the move towards smaller particle higher pressure systems. A survey of stationary phases applied in preparative HPLC has appeared 134 together with a classification of reversed-phase packing^.^^^ The range of suppliers components and accessories is bewildering but clear tabulated information can be consulted in the annual Lab Guide of the journal ‘Analytical Chemistry’ and in the annual Buyers’ Guide of the magazine ‘LC-GC International ’.6.1 Semi-preparative HPLC While analytical HPLC is useful for obtaining information about sample mixtures and does not rely on their recovery the aim of semi-preparative HPLC is to isolate pure substances. In analytical HPLC emphasis is placed on resolution and analysis time but in preparative HPLC the amount of material isolated per unit time (‘throughput’) and the degree of purity are important.The use of larger particles (25-100pm) found in MPLC leads to easier packing higher permeability (requiring lower pumping pressures) larger columns and more economic equipment but there is a correspondingly lower separation efficiency. With the smaller (5-30 pm) particles used in semi- preparative HPLC the complexity and cost of the systems are greater but there is a large gain in separation efficiency. The small k’ values mean small peak volumes and higher concen- trations of the compounds in the e1~ate.l~~ An exercise has been carried out to compare the costs of separating a crude sample on 10pm and 30pm silica gel with the conclusion that the economics are in fact very ~imi1ar.l~~ ‘Semi-preparative’ is a term which has been coined for columns of 8-10 mm id often packed with 10 pm particles and useful for the separation of 1 mg-100 mg mixtures.13* Some recent representative examples are shown in Table 6 although it must be stressed that the number of applications over the last few years has been enormous.In many cases semi- preparative HPLC is used for final purification purposes. Other tabulations of HPLC separations have been given by Kingston,163 Ho~tettmann,’~~ together Verzele and De~aele,’~~ with applications in the isolation of biologically active plant constituents. Closely-related antifungal chromenes from Hypericum revo- OH OH rn 210 5nm 402 210 5nm 402 I”~.”’“”‘‘‘~ 0 10 [minl Figure 8 Separation of antifungal chromenes. (a) Analytical HPLC.LiChrosorb RP-18 (250 x 4.6 mm id) MeOH-H,O (80 20) with 5 ml/l H3P04 flow-rate 1.5 ml/min detection 254 nm. (b) Semi-preparative HPLC. pBondapak C, (300 x 7.8 mm id) MeOH-H,O (63 37) flow-rate 5 mlfmin detection 254 nm. lutum (Guttiferae) have been separated by semi-preparative HPLC. The petroleum ether extract of the leaves and twigs after flash chromatography and LPLC on silica gel gave an active fraction which appeared homogeneous on TLC. How- ever analytical HPLC on a RP- 18 column showed the presence of two homologues [see Figure 8(a)]. Since chromenes (30) and (31) degrade in the presence of acid forming the corresponding dichromenes the eluent used for the semi-preparative sep- aration did not contain acid [see Figure 8(b)]. A total of 120 mg sample was separated by repetitive injection.16’ Intermediates from organic syntheses often require purific- ation by HPLC. However some sort of sample preparation is essential in order that the crude reaction mixture does not contaminate the sorbent. Examples of separations with hexane- tert-butylmethylether pentane-acetonitrile and hexane-dichloromethane as eluents have been given with quantities of sample ranging from 5-800 rng.16 Preparative separations on analytical columns are sometimes feasible.169 For example many peptides polypeptide enzymes and hormones of current pharmaceutical interest require only microgram dosages. Worldwide demand is thus very low and chromatography of sufficient quantities can economically be performed on analytical HPLC columns.1709171 Analytical columns typically have dimensions of ca.250 x 4.6 mm id and samples of 5-100 pm can be introduced onto the column until sufficient product is obtained. For example the mass spectro- metric determination of polyamines from plants has been carried out by separating their benzamide derivatives on an analytical HPLC column containing 5 pm Ultrasphere-ODs. About 10 nmol of purified polyamine derivative was required for mass spectral analysis. 172 Polyhydroxy sterols have been isolated from the sponge Dysidea etheria by a strategy involving gel permeation (Sephadex LH-20 Bio-beads S-X4) LPLC (silica gel) semi-preparative HPLC (RP-18) and as a final step HPLC on a P-cyclodextrin analytical column (250 x 4.6 mm id) with acetonitrile-water mixtures as sol-NPR 8 NATURAL PRODUCT REPORTS 1991 Final analytical Preliminary analytical separation (1 -1 00pg) Overload separation separation (1 -1 00 pg) (0.1-25 mg) Use prep-LC column packing I I i 1 I I i Process-scale separation (0.1-10 kg) Larger cot um n Pilot plant separation (1-1OOo 9) Large column volume optimum particle size Preparative scale separation (5-5000rng) Use largediameter column I I I I 1 I Figure 9 Optimization of conditions for preparative and process-scale HPLC (Reproduced from reference 184).6.2 Preparative and Process-scale HPLC Preparative HPLC in research and development relies mainly on semi-preparative technology while commercial under-takings dictate the need for process-scale HPLC for the separation of larger amounts (reaching the kilogram scale) of compounds.While much published work is available in the field of preparative HPLC access to practical data on process- scale HPLC is much more restricted due to the confidential nature of many industrial applications. The most practical means of increasing capacity in HPLC is to enlarge the column diameter. This is not without problems especially when particle size is less than 20pm and column diameter is greater than 5 cm. True preparative and process- scale HPLC employ stainless steel columns of 5 to 200 cm id filled with silica gel-based packing materials and connected to high-pressure solvent delivery systems (ca. 70 bar) capable of delivering up to 2000 ml/min of mobile phase.These systems can accommodate 1-500 kg stationary phase.174 The packing of these columns presents a challenge especially when small a flow-rate of 210 ml/min. A 10 g charge of crude saponin fraction was resolved with an acetonitrile-water step gradient. 183 Optimization of conditions for preparative- and process- scale HPLC can be summarized as shown in Figure 9. This procedure can now to a certain extent be quantitated.lS4 When production in excess of 5-10 g of purified product is required (pilot plant stage) overlap HPLC separations can be used; the amount of solvent required per gram of purified product is substantially less than for baseline separations. 6.3 Biochromatography Biomolecules as encountered in biotechnology gene tech- nology biochemistry and molecular biology are very often ionic having both hydrophobic and hydrophilic characteristics and a high molecular weight.Special demands for their chromatography have to be met. The susceptibility of such complex samples (e.g. peptides) to change and decomposition particles with a diameter of 10-25 ,um are ~~ed.~~~,~~~ Bed means that during their separation special attention has to be compression174 was developed for this purpose achieved axially,177 radially,178 or by a combination of the two. In 1975 Waters Associates (Milford Mass. USA) intro- duced the Prep LC/System 500 preparative liquid chromato- graph. This employs radial compression of flexible plastic PrepPAK-500 cartridges with a maximum operating pressure of 35 bar,179* for multigram separations.Although numerous applications have been reported in the literature,175 this instrument shares the same characteristics as the Jobin-Yvon axial compression system :la relatively low efficiency (particle sizes are relatively large) high capacity and high solvent consumption. Millipore (Waters) market the Kiloprep 250 1000,2000 and 3000 chromatographic systems all employing radial com-pression technology. Among the applications of the Kiloprep 1000 system is the purification of the p-lactam antibiotic cefonicid on a 600 x 200 mm id C, A number of other companies manufacture equipment for large-scale preparative HPLC work including the following :174 Amicon (Danvers Mass.USA) Pharmacia (Uppsala Sweden) Separations Technology (Wakefield R.I. USA) Varex (Rockville Md. USA) and YMC (Mt. Freedom N.J USA). Axially-compressed columns are available from Axxial (continuing the activity of Jobin-Yvon ; Aulnay-Sous-Bois France) Cedi (Lannemezan France) Merck (' Prepbar ' Darmstadt Germany) Prochrom (Champigneulles France) and Rainin (Woburn MA USA). The preparative separation of the three major saponins of Bupleurum falcatum (Umbelliferae) roots saikosaponins a c and d has been performed by HPLC on a 100 x 11 cm id axially compressed column using a Kurita-LC 110 system (Kurita Tokyo Japan). The column was packed with 5 kg of ODS silica gel (20 ,urn YMC) and operated at 6&70 bar and paid to the nature of the stationary phase the mobile phase and the instrumentation.Inert metal-free systems are essential. The stationary phase pore size nature of the phase surface area of packing material and particle size are all important variables. 185 In HPLC macroporous supports are often used. The addition of salts to the mobile phase is usually necessary to minimize interactions between the silanol groups of the column and ionic groups present in the solute. In the case of proteins their sensitivity to extremes of pH contact with organic solvents high salt concentrations adsorption onto glass or hydrophobic moieties or an air-water interface means they are easily denatured. Therefore the corresponding separation conditions and equipment have to be chosen very carefully The preparative aspects and examples of HPLC for the purification of peptides and proteins have been reviewed by Hancock and Prestidge.ls6 A large number of size exclusion ion-exchange and affinity packings are available for biochromatography together with the usual silica gel normal- and bonded-phase Alternatives include hydrophobic interaction chromatography (such as Fractogel TSK supports) and hydroxyapatite columns.6.4 Column Switching and Recycling Column switchingls7 (also known as 'multidimensional chromatography'188) is useful for several purposes on-line sample clean-up. Purification of partially pure fractions from a first HPLC step. Backflushing reduction of washing times etc. There are a number of techniques for the switching of sample fractions for clean-up and re-chromatography with the analysis NATURAL PRODUCT REPORTS 1991-A.MARSTON AND K. HOSTETTMANN :Recycle I. 11...... Analytical separation -Preparative separation -Separation of recycled component Figure 10 Shave and recycle chromatography. of drugs in biological fluids providing a large area of application. la' The transfer of a fraction from one column to a second column is achieved with switching valves ;while separation is taking place on the second column the first column can be simultaneously cleaned with ~olvent.~~~ This method is very useful when overloading the column because the heart cut can immediately be passed to the next column for further purification. By this means the sweetener stevioside was directly isolated from an aqueous methanol extract of Steviu vebaudiunu (Asteraceae) leaves.las Recycling is a technique which effectively increases the length of a chromatographic bed without having to purchase longer or additional columns.It is achieved by adding a valve to permit the eluent to be directed from the column outlet back into the column inlet. An early application of recycling was reported during the total synthesis of Vitamin B, by Professor R. B. Woodward in 1973 when it was necessary to separate configurational isomers of a cobyrinic acid ester. 138 The most frequently used mode of sample recycle involves isolation of a zone that contains only two components of interest which are not completely resolved.Then during each cycle the leading and trailing edges of the common band are 'shaved ' and collected separately while the centre is recycled (see Figure Igl Isomeric labdadienes and isomeric labdatrienes have been resolved by the shave-recycle technique even though the HPLC chromatogram showed only one peak for all three isomers in each case.IS2 The Waters Prep LC/ System 500 liquid chromatograph (2 silica cartridges 300 x 57 mm) gave a 98 % recovery of starting material and since a one-component solvent system (hexane) was used the solvent could also be recycled. A model LC-09 chromatograph (Japan Analytical Industry Tokyo Japan) has been employed for the separation of sesquiterpenes (32) and (33) from Curcurnu heyneana (Zingiberaceae)IS3 and for the separation of diastereo-meric ipsenol (insect pheromone) MTPA by recycle HPLC.The same instrument was used for the isolation of cathedulins macrolide alkaloids from Cutha edulis (Khat Celastraceae). This separation was performed on a polyvinyl alcohol HPGPC column with methanol as eluent and a single 95 mg injection of sarnple.lS5 7 Countercurrent Chromatography Countercurrent chromatography is a liquid-liquid separation method which does not require a sorbent. Consequently it benefits from a number of advantages over liquid chromato- graphy total recovery of the introduced sample. No irreversible adsorption. Tailing minimized. Risk of sample denaturation minimal. Solvent consumption low. Milligram to gram amounts of mixtures can be separated and crude extracts pose no problems.Consequently the isolation of natural products is an obvious application of this technique which basically involves the partition of a solute between two immiscible solvents the relative proportions of solute passing into each of the two phases being determined by the respective partition coefficients. Descriptions of a variety of different prototype counter- current instruments have appeared largely as a result of the work of Ito.lg6 However only a limited number have actually reached the stage of commercial development. 7.1 Droplet Countercurrent Chromatography Droplet countercurrent chromatography (DCCC) relies on the continuous passage of droplets of a mobile phase through an immiscible stationary liquid phase for the continuous partition of a solute between the two A typical DCCC instrument consists of 200-600 vertical glass columns inter- connected in series by capillary Teflon tubes.These columns are first filled with the stationary phase of biphasic solvent system and the sample is injected. The mobile phase is then pumped into the first of the columns forming a stream of droplets in the immiscible stationary phase. Depending on the choice of solvents for the mobile and stationary phases these NATURAL PRODUCT REPORTS 1991 FHTl ca 300 tubes c-h-? sample sample chamber chamber i -I I Ij collector ascending mode descending mode Figure 11 The principle of DCCC (Reproduced from reference 198).Table 7 Aqueous and non-aqueous DCCC solvent systems Substances separated Glycosides tannins alkaloids antibiotics etc. A1 kaloids Basic steroid saponins Alkaloids G1 ycolipids Saponins iridoid glycosides xanthone glycosides flavonoid glycosides and anthraquinone glycosides Saponins Flavonoid glycosides Saponins A1 kaloids Saponins Anthocyanins flavonoid glycosides and peptides Naphthalide glycosides Tannins Iridoid and secoiridoid glycosides Sennosides Peptides Gibberellins and anthraquinones Diterpenoids and coumarins Essential oils Triterpenes and steroids Triterpenes steroids and depsides Triterpenes and steroids droplets are made either to ascend ('ascending mode') or descend ('descending mode ') through the columns (see Figure i1).198 The choice of a two-phase solvent system is crucial to the success of DCCC separations.Binary systems are impractical for the formation of suitable droplets because of the large difference in polarity between the two components and ternary (or quaternary) systems are required such that the addition of the third (or fourth) component miscible with the other components diminishes the difference in polarity between the two phases. The selectivity of the system is thus increased allowing the separation of closely related substances. One of the simplest means of selecting a suitable solvent system is to allow the sample to migrate on a TLC silica gel plate with the water-saturated organic layer of a two-phase Solvent system CHC1,-MeOH-H,O (7 :13:8 and other proportions) CHC1,-MeOH-Acetate buffer pH 3.6 (9 12:8) CHC1,-MeOH-1 YOaq.NH (7 :12:8) CHCl3-MeOH-5 YOHCI (5:5:3) CHCI,-MeOH-0.5 YOaq. CaC1,-Pr"0H (50 :60 :40:6) CHC1,-MeOH-H,O-Pr"0H (9 :12:8 1) CHC1,-MeOH-H,O-Pr"0H (5 :6 :4 :1) CHC1,-MeOH-H,O-Bu"0H (10 10:6 1) CHC1,-MeOH-H,O-Pr"0H-EtOH(9 :6:8 1 :8) CHC1,-MeOH-H,O-C,H,Me (5:7 :2 :5) CH,CI,-MeOH-H,O (8 :13:7) Bu"OH-HOAc-H,O (4 1 5) Bu"0H-MeOH-H,O (5:1 :5) Bu"0H-Pr"0H-H,O (2 :1:3) Bu"0H-EtOH-H,O (4 1:5) Bu"0H-Me,CO-H,O (33 10:50) Bu'OH-TFA-H,~ (1 20 1 :160) Petroleum ether-EtOH-H,O-EtOAc (5:4 1:2) C,Hl,-Et,0-Pr"OH-95 YOEtOH-H,O (4:8 3:5:4) C,H,,-EtOAc-MeN0,-MeOH (8 :2 :2:3) C,H1,-C,H,Cl,-MeOH (47 :6:72) C,H,,-Me,CO-MeOH (5 :1 :4) C,H,,-CH,Cl,-MeCN (10 3 :7) aqueous solvent system.l If the R values of the compounds to be separated are higher than about 0.5 the less polar layer is suitable for use as the mobile phase.For more polar solutes (R < 0.5),the more polar layer is used as the mobile phase. The applications of DCCC are nurnerou~~~~-~~~ and the solvent systems commonly used for certain classes of natural products are shown in Table 7. Although certain separations have been carried out with non-aqueous solventszo1 202 (see Table 7),most applications of DCCC involve polar compounds such as glycosides. Of special note are the separations of polyphenols tannins anthocyanins and saponins. Ternary CHC1,-MeOH-H20 systems of varying compositions are the most widely used.Some recent examples are as follows flavonol glycosides from Strychnos variabilis (Loganiaceae) (CHC1,-MeOH-H,O 5:6:4;descending mode).203 NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN Quercetin 3-0-glucuronide from Vaccinium myrtillus (Ericaceae) (CHCl,-MeOH-Pr OH-5 YOCH ,COOH 31.2 37.5:6.2:25; descending mode).204 Bianthrone C-glycoside from Asphodelus ramosus (Liliaceae) (CHC1,-MeOH-H,O 4 :4 :3 ; ascending mode).205 Anthocyanins from Podocarpus species (Podocarpaceae) (n-BuOH-HOAc-H,O 4 1:5; descending mode).206 Iridoid diglycosides from Premna japonica (Verbenaceae) (CHC1,-MeOH-n-PrOH-H,O 45 :60 10:40; ascending mode).207 Secoiridoid glucosides from Jasminum mesnyi (Oleaceae) (n-BuOH-EtOH-H,O 4 1 1; ascending mode).20s Hyperforin derivatives from Hypericum revolutum (Guttiferae) (petrol ether-94% EtOH-EtOAc-H,O 83 :67 33 17; ascending mode).209 Casbane diterpenes from Agrostistachys hookeri (Euphorbi- aceae) (cyclo hexane-Et ,O-i-PrOH-EtOH-H ,O 7 1 6 :6 10:8; ascending mode).210 Cucurbitacin glycoside from Persea mexicana (Lauraceae) (CHC1,-MeOH-H,O 13:7:4; ascending mode).211 Bufadienolides from Urginea pancration (Liliaceae) (CHC1,-MeOH-H,O 5 10:6 ; ascending and descending modes).212 Unsaturated alkamides from Achillea ptarmica (Asteraceae) (heptane-CHC1,-MeOH-H,O 18:25 18:3).,13 Aporphine alkaloids from Ocotea caesia (Lauraceae) (CHC1,-MeOH-H,O 5:5:3 ;descending mode).214 Chromone alkaloids from Schumanniophyton magnificum (Rubiaceae) (n-BuOH-MeOH-NH,OH 5 1 :5 ; ascending mode).215 Pyrrolizidine alkaloids have a tendency to adsorb irreversibly to solid phases such as alumina silica gel or reversed-phase silica gel.However DCCC was able to resolve the problem of separating macrocyclic pyrrolizidine alkaloids from Senecio anonymus (Asteraceae). With the solvent systems CHC1,-MeOH-H,O 13:7 :4 and CHC1,-C,H6-MeOH-H,O 5 :5:7 :2 even mixtures of cis-trans isomeric alkaloids were separated.216 During the isolation of okanin (chalcone) glycosides from Bidens pilosa (Asteraceae) a step-gradient DCCC separation was involved. After initial Sephadex LH-20 gel filtration DCCC was performed with the solvent system CHC1,-MeOH-H,O 13 7 :4 (descending mode).This was followed by CHC1,-MeOH-i-PrOH-H,O 26 :13 1 :8 and then the proportions 13:6 1:4. Final purification gave three monoglucosides. 7.2 Centrifugal Partition Chromatography Droplet countercurrent chromatography is a very versatile and straightforward technique but suffers from the disadvantage of being slow (separations require up to 2 days and longer). In addition the choice of solvent systems is limited to those which form droplets when the mobile phase is passed through the stationary phase. Centrifugal partition chromatography (CPC) on the other hand relies on a centrifugal field rather than a gravitational field for retention of the stationary phase and is consequently a much more rapid method than DCCC or rotation locular countercurrent chromatography (RLCC).CPC (or centrifugal countercurrent chromatography as it is also known) is a continuous process of non-equilibrium partition of solute between two immiscible phases contained in rotating coils or cartridges. Choice of solvent system can be guided by TLC in a similar fashion to DCCC,lg8 as exemplified by the separation of synthetic peptides.,18 For optimum separations with the cartridge method a solvent system should be chosen in which the R,values of the substances to be separated lie in the range 0.24.5 when TLC is carried out on silica gel plates with the water-saturated organic layer as mobile phase.219 For rotating coils the best R,range is 0.14.4. Alternative methods are based on the calculation of partition coefficients by analytical HPLC2,0 or by spectrophotometric measurements.221 Once the partition coefficients of the solutes are known it is theoretically possible to predict the location of eluted solute peaks and separations are highly reproducible. 222 7.2.1 Rotating Coil Instruments Instruments based on rotating coils can either involve planetary or non-planetary motion about a central axis and in fact a whole family of prototypes has been deve10ped.l~~ These include the horizontal flow-through coil-planet centrifuge the toroidal coil-planet centrifuge the countercurrent extraction coil-planet centrifuge and the high speed countercurrent chromatograph (HSCCC). Only the HSCCC has been com- mercialized to any large extent.It consists of a Teflon tube (1.6 or 2.6 mm id) wrapped as a coil around a spool. The coil is balanced by a counterweight and describes a planetary motion about a central axis (see Figure In order to prevent leakages a seal-free system has been developed for introduction of solvent into the rotating Because the coiled column assemblies can be readily interchanged both analytical- and preparative-scale separations can be performed. In one com- mercially available preparative instrument (the multilayer coil separator-extractor developed by Ito and marketed by P.C. Inc. Potomac MD USA) the coil consists of a single length of 2.6 mm id PTFE tubing with a capacity of ca. 350 ml. Rotation speeds for separations are generally of the order of 700-800 rpm.Concerning the principle of the technique the motion of the coil causes vigorous agitation of the two solvent phases. A repetitive mixing and settling process ideal for solute par- titioning occurs at over 13 times per This rapid interchange explains why efficient separations are possible in such a small volume of solvent. However the mechanism of hydrodynamic distribution of the solvent phases in the coil is not known. A suitable procedure for loading the instrument226 consists first of pumping stationary phase into the stationary coil and then introducing mobile phase after commencing rotation. When the necessary stationary phase has been displaced and mobile phase alone leaves the coil the sample (ideally in a mixture of the two phases) is introduced via a sample loop.In most instances the instrument can be connected to a UV detector for rapid indication of the progress of the separation. When the lower phase of a biphasic solvent system is the mobile phase it is introduced via the head end of the coil (i.e. near to the axis of rotation). A recently introduced modification of the pumping procedure now allows variation of phase ratios in the coil gradient outlet in'et I 1 A I Ddw \ I' I I /I Figure 12 Schematic illustration of the P.C. Inc. multilayer coil separator-extractor. NATURAL PRODUCT REPORTS 1991 R H0HO r 0ppoH HO 0 (34) (35)R=H (36)R=OH I (35) n (34) r\ (34) /I J 0 1 2 1’5 6 7 8 0 1 2 3 4 [hl [hl Figure 13 Reversed-phase operation of the multilayer coil separator-extractor.Separation of hesperetin (34) kaempferol (35) and quercetin (36). Solvent system CHC1,-MeOH-H,O (33:40 :27) flow-rate 3 ml/min rotational speed 700 rpm sample 15 mg detection 254 nm. (a) Mobile phase = upper phase. (b) Mobile phase = upper phase to 70 min then lower phase. 0 0 operation and easy reversal of mobile and stationary phases.226 This is achieved by using separate pumps for the mobile and stationary phases and simultaneous introduction of the two phases into the apparatus. To illustrate the advantages of phase reversal a separation of the flavonoids hesperetin (34) kaempferol(39 and quercetin (36) is shown in Figure 13. With the upper phase of the solvent system CHC1,-MeOH-H,O (33 :40 27) as mobile phase the chromatogram in Figure 13 (a) was obtained.In order to fill the column with exactly 50% of each phase at the beginning of the separation lower and upper phases were pumped simultaneously at the same flow-rates into the coil. For elution of the sample mobile phase alone was pumped. If after elution of the first peak quercetin (36) the phases were reversed and the lower phase was then used as the mobile phase the chromatogram shown in Figure 13(b) was obtained. Separation time was considerably reduced and the order of elution of hesperetin and kaempferol was inverted. 226 The rotating coil technique is finding extensive application in the purification of natural produ~ts.~~’~~~* One of the areas of exploitation is the preparative separation of antibiotics as shown by the isolation of siderochelin A efrotomycin COOH COOH pentalenolactone Bu 2313 B and tirandamycins A and B from crude fermentation extracts of Bacteroides fragilis and Staphy-lococcus aureus.Charges of up to 600 mg were introduced for the different Other applications are especially common in the isolation of plant-derived natural products as can be seen in Table 8. HSCCC has for example proved ideal for the separation of carotenoids. Whereas HPLC and preparative TLC were unsuitable for the isolation of these pigments from Cochlo-spermum tinctorium (Cochlospermaceae) cochloxanthin (37) and dihydrocochloxanthin (38) were obtained in one step from a methanol extract of the roots with the coil planet centrifuge.230 The two-phase solvent system used was CHC1,-MeOH-H,O 5:4 1.The extract (500 mg) was dissolved in 10 ml of a 1:1 mixture of the two solvent phases before injection. The rotational speed of the instrument was 800 rpm and the flow- rate was 4ml/min. Resolution of the mixture was achieved within 2 h with the upper phase as the mobile phase. Alkaloids are particularly suited to separation by CPC. Quaternary indole alkaloids for example are difficult to purify because of their polarity and their interaction with solid NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN Table 8 Selected applications of the multilayer coil separator-extractor in the isolation of natural products Substances separated Solvent system Ref.Flavonoids CHC1,-MeOH-H,O (4 :3:2) 233 CHC1,-MeOH-H,O (33 :40 :27) 226 Flavonol glycosides EtOAc-H,O +EtOAc-Bu'OH-H,O 234 Lignan glycosides n-C,H,,-CH,CI,-MeOH-H,O (1 :5 :4 :3) 235 Tannins Bu"0H-O. 1M NaCl (1 :1) 236 Coumarins n-C,H,,-EtOAc-MeOH-H,O 237 (18 :42 30 30) Coumarin glycosides CHC1,-MeOH-H,O (1 3 :23 :16) 238 Phenolic acids n-C,H,,-EtOAc-MeOH-H,O 237 (18 :42 :30 30) C yclohexadienone n-C,H1,-94 YOEtOH-EtOAc-H,O 239 derivatives (83:67:33 17) Alkaloids Bu"0H-Me,CO-H,O (8 :1 :10) 231 Bu"OH-O.1 M NaCl (1 1) 232 CC1,-MeOH-H,O (20 :20 :2) 240 CHC1,-0.07 M sodium phosphate (1 :1) 241 Carotenoids CC1,-MeOH-H,O (5 :4 :1) 230 Sesquiterpenes i-C,H,,-EtOAc-MeOH-H,O (7 :3 :6 4) 242 Cardiac glycosides CHC1,-MeOH-HOAc-H,O (5 :3:1:3) 243 Anthranoids n-C,H,,-CH,CN-MeOH (40 25 10) 226 Antibiotics CHC1,-MeOH-H,O (7 :13:8 1 :1 :1) 229 CCI,<HCl,-MeOH-H,O (5:5 :6 :4) 229 (4 1 :4 I) 244 Et,O-n-C,H,,-MeOH-H,O (5 1 :4 5) 245 Marine natural CHC1,-MeOH-H,O (5 10:6) 246 products CH,CI,-MeOH-H,O (5 :5:3) 247 CICH,CH,Cl-CHC1,-MeOH-H,O 248 (2:3 10:6) n-C,H,,-CH,CI,-CN (10:3 :7) 249 (5:1:4) 250 n-C,H ,,-E t OAc-MeOH-H,O 25 1 (4 7 :4 3) CHCl,-Pr;NH-MeOH-H,O (7 :1 :6 :4) 25 1 support matrices.However good results have been obtained by using centrifugal liquid-liquid techniques (see Table 8).2317 232 A new development in rotating coil centrifugal chromato- graphy is the multicoil centrifugal countercurrent instrument.This consists of twoz5 or three2, identical multilayer coils arranged symmetrically around the rotary frame of the centrifuge thus eliminating the need for balancing with a counterweight. Each coiled column undergoes synchronous planetary motion in such a way that it revolves around the central axis of the centrifuge and simultaneously rotates about its own axis at the same angular velocity. The columns are equipped with flow tubes arranged in such a fashion that they do not twist enabling seal-free operation of the instrument. Pharma-Tech Research Corp. (Baltimore Maryland USA) manufactures a range of multicoil chromatographs with different solvent capacities and rotational speeds ranging from the CCC-1000 with a capacity of 800 ml to the CCC-2000 with a capacity of 40 ml.7.2.2 Cartridge Instruments These chromatographs constructed by Sanki Engineering Ltd. (Kyoto Japan) consist of an arrangement of cartridges located at the circumference of a centrifuge rotor with their longitudinal axes parallel to the direction of the centrifugal force (see Figure 14).254Each cartridge contains the equivalent of 400 separation channels and connections between cartridges are made by narrow-bore Teflon tubes. While the rotor is spinning stationary phase is first pumped into the cartridges followed by introduction of mobile phase at the rotation speed required for the separation. Rotating seals at the upper and lower axes of the centrifuge allow the passage of solvent into the apparatus under pressure.When a steady flow of mobile 2 3 1 I II 1 g-9 t Figure 14 The Sanki centrifugal chromatograph. 1 = rotary seal joint 2 = connecting tube 3 = separation column 4 = rotor g = centri-fugal force. Table 9 Selected applications of the Sanki cartridge instrument in the separation of natural products Substances separated Flavonoids Flavonoid gl ycosides Tannins Chalcones Coumarins Phenolic acids Anthranoids Naphthoquinones C yclohexadienone derivatives Retinals Norditerpenes Saponins Tunichromes Solvent system Ref. CHC1,-MeOH-H,O (33 :40 :27) 219 EtOAc-94 Yo EtOH-H,O (2 :1 2) 219 255 EtOAc-Bu"0H-H,O (2 :I :2) 255 Bu"0H-Pr"0H-H,O (4 :I :5) 256 Bu"0H-Pr"0H-H,O (2 1 :3) 257 Bu"OH-HOAC-H,O (4 1 5) 258 CHC1,-MeOH-H,O (7 13:8) 256 259 n-C,H,,-EtOAc-MeOH-H,O 219 (I 8 :42 :30 :30) n-C,H,,-EtOAc-MeOH-H,O 219 (I 8:42 :30 30) n-C,H,,<H,CN-MeOH (40 :25 :10) 219 n-C,H,,-CH,CN-MeOH (40 :25 10) 219 n-C,H1,-94 YOEtOH-EtOAc-H,O 239 (83:67:33 17) Cyclohexane-n-C,H ,-CH,CN-MeOH 260 (500:200 :500 :1 1) CHC1,-MeOH-H,O (5:6 :4) 26 1 CHC1,-MeOH-H,O (7 13:8) 219 Am'OH-Bu"0H-Pr"0H-H,O-HCOOH 262 -t-butyl sulphide (32 :48 :40 :120 1 :4) ~~ phase alone leaves the instrument the sample is introduced and the separation is performed.The rotational speed of the instrument can be varied but although higher speeds generally lead to better resolution the pumping pressure is increased.Most separations are thus achieved at speeds of ca. 1000 rpm. Either the heavier phase or the lighter phase of a two-phase solvent system can be employed as mobile phase simply by switching the direction of flow through the instrument. A considerable number of separations with the Sanki CPC system have been reported.219* 227* 228 Both polar and relatively non-polar substances are compatible with the solvent combin- ations (see Table g) although the largest field of application has been polar substances such as tannins. Centrifugal partition chromatography is ideal for the fractionation of crude plant extracts because no solid support is present for irreversible adsorption to occur. In one example the iridoid glycoside gentiopicrin was obtained in one step from a methanol extract of the roots of Gentiana futea (Gentian-aceae).The methanol extract was first partitioned between water and petroleum ether ethyl acetate and butanol in that order. Chromatography of the butanol fraction (500 mg) by Sanki CPC NATURAL PRODUCT REPORTS 1991 0 1 2 3 4 5 [hl Figure 15 CPC separation of gentiopicrin (39) from a methanol extract of Gentiana lutea roots. Solvent system CHC1,-MeOH-H,O (9 12:8 ; mobile phase = lower phase) flow rate 2.4 ml/min rotational speed 300 rpm sample 500 mg detection 254 nm. Figure 16 mobile with CHC1,-MeOH-H,O 9 :12:8 (lower phase as mobile phase) gave 1 13 mg of pure gentiopicrin (see Figure 15).,,’ A non-aqueous solvent system has been used for CPC of the light petroleum extract of the root bark of Psorospermum febrifugum (Guttiferae) which contains a mixture of anthra-quinone anthrone and tumour cell growth-inhibitory tetra- hydroanthracene pigments.Separation of these constituents by multi-step flash and low-pressure liquid chromatographies resulted in considerable material losses owing to irreversible adsorption on the sorbents. A single CPC step however allowed the direct isolation of three pure compounds (40)-(42) and a mixture of two anthranoids (43)and a minor component (44),without loss of product (see Figure 16). A 100 mg sample of crude extract was separated within 4 h using the upper phase of the solvent system hexane-acetonitrile-methanol 40 :25 10 as mobile phase.219 Scale-up of the separation to a 500 mg loading was possible.228 The Sanki chromatograph is furthermore amenable to operation in the reversed-phase mode219 and to control of the phase ratios in the cartridges by a two-pump arrangement,263 as is the case with the rotating coil apparatus. 8 Combination of Methods No single chromatographic separation method is able to solve all separation problems and moreover it is very common to (40:25 10; find multistep chromatographic operations for the isolation of pure substances. Although it is possible to obtain pure compounds by one- or two-step procedures (some examples are given above) a combination of techniques is normally required. Of course there are hundreds of different ways of putting together all the possible separation techniques but in reality the choice of strategy is limited by a number of constraints.One of these is the nature of the substances to be separated -such as whether they are hydrophilic or hydrophobic. For example highly polar water-soluble marine natural products require special considerations and these have been dealt with by Shimi~u.,,~ An alternative strategy for marine natural products employs a combination of flash chromatography on reversed- phase supports followed by semi-preparative HPLC for final purification. For initial fractionation the extract is mixed with reversed-phase support (32-63 pm) and loaded onto the flash column as either an aqueous slurry or a powder. By this means loads of up to 20g crude extract per 1OOg of support are possible.61 Another class of substances which poses particular problems is the saponins.The approach adopted by many Japanese groups for their separation involves a preliminary fractionation on highly porous polymer (DIAION HP-20 or the like) followed by classical open-column chromatography on silica gel and/or HPLC on reversed-phase supports.265 A com-bination of liquid-liquid partition and liquid chromatography NATURAL PRODUCT REPORTS 1991-A. MARSTON AND K. HOSTETTMANN HO \ OH Hd (45) CHCIa extract of onionjuice (1) Flash chromatography RP-8 MeOH (2) Chromatotron Si02 CHC13-MeOH mixtures (3) MPLC 302 Toluene-EtOAc (10:2) 1-11 I11 Iv V (1) MPLC SiOz (1) MPLC SiO2 MPLC SiOz Pentane-acetone CHpCIpxetone n-Hexane-E tOAc (1005) (100:1) (10:3) (2) MPLC SiOz (2) MPLC SiOz I CHZClracetone CHzCIz-E tzO-acetone (200:l) (100:2:1) (52)-(57) I R'-s(o)-s-R~ / (48) R' = Me R2 = CH=CH-Me (cis) (47) (trans) \>=<:LOs-H 11 H (49) R' = Pr" R2= CH=CH-Me (cis) 0 (50) R' = Pr" R2= CH=CH-Me (trans) (52) (54) (51) R' = Pr" R2 = CH2-CH2-Me (53) diastereoisomerof (52) (55) diastereoisomerof (54) (56) (57) diastereoisomerof (56) Figure 17 Isolation of biologically-active thiosulphinates and a-sulphinyl-disulphides from Affiumcepa.268 is also very effective as shown by the isolation of molluscicidal isolated by combining silica gel flash chromatography with saponins from Hedera helix (Araliaceae) berries.A two-step petrol ether-ethyl acetate and LPLC using chloroform-procedure involving DCCC and preparative HPLC was methanol or petrol ether-ethyl acetate mixtures as el~ents.'~ sufficient to obtain four pure saponins.266 Sometimes the isolation of the required compounds (and this The liquid-liquid partition/liquid chromatography approach is frequently the case with bioactivity-guided fractionation) also enabled the separation of two new fungicidal polycyclic from a plant extract involves a very complicated series of phenols (45) and (46) from the stem bark of Cordia goetzei separation steps. In order to obtain biologically active (Boraginaceae). Although the use of DCCC for the first step thiosulphinates and a-sulphinyldisulphides from onion juice was possible,267separation was slow and a combination of CPC (Allium cepa Alliaceae) the separation sequence shown in on a Sanki instrument (cyclohexane-ethyl acetate-methanol-Figure 17 was necessary.This involved an initial reversed-water 7:8:6:6; upper phase as mobile phase) and LPLC on phase flash chromatographic step on the chloroform extract to octadecyl silica gel was preferred.263 remove triterpenes. By monitoring antiasthmatic activity the For less polar compounds recently employed separation active thiosulphinates (47)-(51) were obtained by multi-step strategies involve a preliminary flash chromatographic step centrifugal thin-layer chromatography and MPLC (Figure 17). followed by low-pressure liquid chromatography or centrifugal The disulphides were similarly separated by a final MPLC step TLC.A series of antifungal methylated flavonoids from the on silica aerial parts of Helichrysum nitens (Asteraceae) was for example In another example of a complex separation procedure 410 recourse to LPLC MPLC HPLC CTLC and DCCC was necessary for the separation of cyanogenic constituents of Xeranthemum cylindraceum (Asteraceae). The new acylated cyanogenic triglycoside xeranthin was isolated from a meth- anol extract of the fruits by an initial DCCC step (solvent chloroform-methanol-propanol-water 31 :38 :6 :25 ascending mode) followed by silica gel column chromatography (ethyl acetate-acetone-dichloromethane-water-methanol 40 :30 12 5 :8) and semi-preparative HPLC on RP- 18.269 9 Conclusions The purpose of this article is to give an idea of what is now possible with the array of modern separation techniques at the disposal of a natural products chemist or a phytochemist.The introduction of new chromatographic methods has meant not only that separation times have been reduced but that more complex mixtures can now be tackled. This is relevant for the whole range of applications-from the milligrams of a pure biomolecule which may be required for a certain therapeutical need to the kilograms of synthetic chemical or natural product which have to be separated by process-scale chromatography. 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ISSN:0265-0568
DOI:10.1039/NP9910800391
出版商:RSC
年代:1991
数据来源: RSC
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7. |
Withanolides and related ergostane-type steroids |
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Natural Product Reports,
Volume 8,
Issue 4,
1991,
Page 415-440
E. Glotter,
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摘要:
Withanolides and Related Ergostane-type Steroids E. Glotter Faculty of Agriculture The Hebrew University of Jerusalem Rehovot 76-100 Israel 1 Introduction 2 Withanolides with an Unmodified /3-Oriented Side Chain 2.1 1a,3P-Di hydroxy- 5-enes 2.2 3P-Hydroxy-5-en-1-ones 2.3 2,5-Dien-1-ones 2.4 3,5-Dien- 1-ones 2.5 4/3-Hydroxy-2,5-dien-1-ones 2.6 5/3,6P-Epoxy-4/3- hydroxy-2-en- 1 -ones 2.7 Sa-Hydroxy-2,6-dien- 1-ones 2.8 6a,7a-Epoxy-Sa-hydroxy-2-en-1 -ones 2.9 SP,6/3-Epoxy-2-en- 1 -ones and Derived Structures 2.10 18-Hydroxy -oxo and -oic acids 2.1 1 21-Hydroxywithanolides 2.12 23-Hydroxywithanolides 2.13 Epoxylactol and Epoxylactone Side Chains 3 Withanolides with an Unmodified a-Oriented Side Chain 4 Withanolides with Modified Structures 4.1 Compounds with Ring D Aromatic 4.2 13,14-Secowithanolides ;Physalins and Withaphysalin 4.3 Modifications due to the 17P-OH Group 4.4 Modifications due to Functionalization of 21 -Me 4.5 Modifications due to a 23-OH Group 4.6 Modifications due to a 28-OH Group C 5 Microbial Transformations and Cell Free Cultures 6 Biosynthesis 7 Chemotypes 8 Synthetic Achievements 9 Biological Activity 10 References 1 Introduction The withanolides are a group of naturally occurring steroids built on an ergostane skeleton in which C-22 and C-26 are appropriately oxidized in order to form a &lactone ring.This basic structure (1) was designated1" as the 'withanolide' skeleton.a b C Further investigations have led to the discovery of various biogenetically related compounds in which either the carbo- cyclic skeleton or the side chain or both are modified. Among them are 13,16seco derivatives ring D aromatic compounds bicyclic structures and epoxylactols in the side chain. All these unmodified and modified ergostanes are produced by Solan- aceae plants belonging to the genera Withania Acnistus (Dunalia) Physalis Jaborosa Datura Lycium. The names in current use for structure (1) are either withanolide or according to the Chemical Abstracts system 22-hydroxyergostan-26-oic acid 26,22-lactone. In the CAS Chemical Substances Index the compounds are catalogued as ergost...derivatives. The name ergostan-26,22-olide based on the IUPAC rules does not seem to be used. For compounds with a modified carbocyclic skeleton and/or side chain only CA names are being used along with the trivial ones. The generic name 'withasteroids ' which has been proposed,l does not seem to have gained much popularity. The progress in the chemistry of withanolides and related compounds has been reviewed; 2-5 the last review5 being from 1981. In NPR this topic was dealt with under the headings 'Steroid Reactions and Partial Syntheses ' and 'The Bio- synthesis of Triterpenoids and Steroids '. The present review is an attempt to survey this group of compounds over the last 25 years from the elucidation of the structure of withaferin A6s7 (27) to the end of 1989; ca.170 compounds have been isolated and characterized. Withania somnifera the perennial plant producing withaferin A (27) is widely distributed along the shores of the Mediter- ranean Sea as well as in India South Africa and other countries throughout the world. Various therapeutic properties have been attributed to this plant since ancient times,8-10 although they were not confirmed by studies performed at the beginning of this century." Modern research on the therapeutic properties of various withanolides is reviewed in Section 9. A common feature of all known withanolides including the related modified ergostane derivatives is oxidation at C- 1 C-22 and C-26. The primary process of oxidation at C- 1 leads to the formation of an axial la-OH (Scheme 1 structure a) which is further oxidized to a 1-one.Over 90% of these compounds are 1 -oxosteroids. With only one exception ixocarpalactone B d @ A OH HO HO e f h Major patterns of rings A and B Scheme 1 415 NATURAL PRODUCT REPORTS. 1991 (2) R’ = R2= R4 = HI R3 = OH (8) Pubescenin (3) Pubescenolide R’ = R2 = R3= H R4= OH (4) Physaldactone B R’ = Ac R2= R4= HI R3= OH (5) R’ = Ac R2= Glc R3 = OH R4= H (6) Dunawithanin B R’ = (7) Dunawithanin A R’ = (9) R = H Ac R2= Glc-Glc R3 = OH R4 = H Ac R2= Glc-Glc R3= OH R4= H I G Ic 2 Withanolides with an Unmodified P-Oriented Side Chain 2.1 la,3p-Dihydroxy-5-enes There are only three compounds in which the axial la-OH group resisted further oxidation to the 1-one.These are la,3P,2O-trihydroxywitha-5,24-dienolide(2) isolated from Withania somnifera chemotype IIIL3 (chemotypes see Section 7) pubescenolide (3),14 isolated from a variety of Physalis pubescens raised in Israel from seeds received from Walthair (India) and an additional compound pubescenin (8),15 isolated from the same source. In contrast to the last two compounds this has a S-epoxylactone instead of an unsaturated 8-lactone in the side chain. The 3/?-OH group in pubescenin is part of a glucoside. The following four compounds (4)-(7) escaped oxidation at C-1 because the la-OH is esterified (la-OAc). Compound (4) physalolactone B16 co-exists in Physalis peruuiana with the corresponding 3-monoglucoside (9.” Dunawithanin B (6) a diglucoside and dunawithanin A (7) a triglucoside both isolated from Dunalia australis are the first known glucosides with a withanolide aglycone.ls All these glucosides (5)-(7) have the same aglycone (4) named dunawithagenin by the German workers.Its structure was confirmed by X-ray analysis” of the free alcohol (2). Perulactone (167) an additional 3/?-O-glucoside has a modified side chain and is presented in Section 4.6. It is possible that many more withanolide 3P-0-glycosides exist in nature but oxidation of the free la-OH group in such compounds to 3P-0-glycosido-1-ones makes p-elimination possible thus leading to the 2,5-dien- 1-one system. 2.2 3~-Hydroxy-5-en-l-ones There are only three compounds possessing the 3P-hydroxy- 1- one system thus pointing to the ease of further transformations.Two of them have the side chain /?-oriented. 3/?,20-Dihydroxy- 1-oxowitha-5,24-dienolide (9)13 is a minor component of Physalis peruuiana growing near Varanasi (Uttar Pradesh India) where it co-exists with physalolactone B (4). 3p 14a,20,27-tetrahydroxy- 1-oxowitha-5,24-dienolide (10) is a minor component of the fruits of Withania coagulans.20 The third compound (120) isomeric with the latter has the side chain a-oriented (Section 3). 2.3 2,5-Dien-l-ones The 2,Sdien-l-one is the A and B rings system from which almost all other more elaborate patterns are derived. The (10) 14a-OHI R=OH (165) C-22 is oxidized to the hydroxyl level and its configuration is always 22P (Fieser convention12).C-26 is oxidized in most instances to the carboxylic acid level thus allowing the formation of a 26,22-lactone as shown in (1). In some of the compounds it is at the aldehyde level allowing the formation of a 26,22-lactol. In contrast to the lactone which is in most instances a/?-unsaturated the initial &/?-unsaturated lactol is never found as such but as epoxylactols or the corresponding epoxylactones. In addition to the oxidative processes at the above three centers one of the characteristic features of the plants producing withanolides is their extraordinary ability to introduce oxygen functions at almost every position of the carbocyclic skeleton and side chain.By the end of 1989 there are only one secondary (C-1 1) and two tertiary (C-8 and C-9) carbon atoms which have not been found to be oxidized. There is no direct evidence whether the major biosynthetic oxidative processes begin in ring A (C-1) or in the side chain (C-22 and C-26). However since all the withanolides have the side chain in one or other of its final forms it is reasonable to assume that the elaboration of the latter precedes or is at least concomitant with the first step in the functionalization of the carbocyclic system i.e. 1a-hydroxylation. The various compounds are divided into two major groups A.-Withanolides with an unmodified skeleton further subdivided into withanolides with a regular ,&oriented side chain (Section 2) and withanolides with an unusual a-oriented side chain (Section 3).In each subgroup the compounds are arranged as far as possible according to the substitution pattern of rings A and B as indicated in Scheme 1. B.-Withanolides with modified carbocyclic skeletons and/ or side chains (Section 4). NATURAL PRODUCT REPORTS 1991-E. GLOTTER -.-.& HO (11) DaturametelinA 14a-H R’ = H R2= OGlc (12) 7a-OH R’ = H R2= OH (13) Withanolide G 14a-OH R’ = OH R2= H (14) Withanolide H 14a-OH R’ = R2= OH (15) A16 R’ = OH R2= H (16) 17a-OH R’ = H R2= OH (17) WithanolideJ 14a-OH 17a-OH R’ = OH R2= H --. H&o OH \ simplest compound in this group is daturametelin A (1 a 27-glucoside isolated from the leaves of Datura metel.D.metel is the only known source of 21 -hydroxywithanolides (Section 2.1 l) from which several compounds with a bicyclic system in the side chain are derived (Section 4.4). 7a,27-Dihydroxy- (12) and 17a,27-dihydroxy- 1 -oxowitha-2,5,24-trienolide(1 6) are minor constituents22 of the Indian chemotype of Withania somnifera. A major hydroxylation site in the withanolides is C-14. With the exception of two compounds (36) and (62) which have a 14P-OH group most 14-hydroxywithanolides have this group a-oriented. Three of them withanolides G (13) H (14) and J (I 7) are 2,5-dien-1-ones isolated from Withania somnifera chemotype 111. These compounds and some other 1401-hydroxywithanolides were erroneously considered as A8(14) compounds.23924 Several additional 14-hydroxywithanolides have an a-oriented side chain (Section 3).A related compound la I4a-dihydroxywitha-5,24-dienolide (18)25 was isolated from Withania aristata. It was suggested that its formation is due to reduction of a 2,5-dien-l-one (19) WithanolideI R = H (20) 27-HydroxywithanolideI R = OH (21) Withanolide K 17a-OH R = H A HO (22) R’ = HI R2= OH (23) Withanolide U 14a-OH R’ = OH R2= H (24) 7P-OH R’ = OH R2= H (25) 2,3diH R’ = H R2= OH 2.4 3,5-Dien-l-ones In nature the same as under laboratory conditions the 2,5- dien- 1 -one system can be deconjugated under acidic conditions to the alternatively conjugated 3,5-dien- 1-one system which is present in compounds (1 9)-(21).The simplest 3,5-dien- 1-24 are minor components of Withania sornnifera chemotype 111 where they co-exist with the isomeric 2,5-dien-l- ones. These compounds are withanolide I (19) 27-hydroxy- withanolide I (20) and withanolide K (21) the latter possessing a 17a-OH group in addition to the 1401- and 20-hydroxy groups. Several additional compounds with the above pattern in rings A and B are described in other sections (e.g. withaphysalin D (86) and isowithametelin (1 59). 2.5 4fi-Hydroxy-2,5-dien-l-ones Withanolides possessing the 4P-hydroxy-2,5-dien- 1 -one system were considered28 as the substrate for direct epoxidation of A5 thus leading to the large group of compounds with the 5p,6p-epoxy-4P-hydroxy-2-en-l-one system in rings A and B (Scheme 1 pattern g).Recent biosynthetic investigations by Gros and co-~orkers~~ (Section 6) however support an alternative route to this system-first epoxidation of A5 and subsequent hydroxylation at C-4. and withaminimin (77)27 from P. minima. Lavie suggested26 that the A16-withanolide (1 5) might be a precursor not only for 17a-hydroxy-but also for the important group of 17p-hydroxy withanolides (Section 3). There are no biosynthetic studies so far to confirm this suggestion. 48,27-Dihydroxy- 1 -oxowitha-2,5,24-trienolide(22) along precursor. So far no other related la-hydroxywithanolide with its 2,3-dihydroderivative (25) are minor components of unsubstituted at C-3 have been isolated. the mixture of withanolides isolated from Acnistus brev~j?orus.~~ There are three A16-withanolides 20-hydroxy- 1 -oxowitha- Withanolide U (23),31 one of the 14a-hydroxywithanolides 2,5,16,24-tetraenolide (1 5),26 isolated from Withania somnifera erroneously considered as A*(14) compounds is a minor chemotype 111 withangulatin (35) from Physalis angul~ta,~~ component of the F offspring obtained by crossing W.somnifera chemotype 111with W.somnifera Indian chemotype (Section 7). 4P,7P,20-Tri hydroxy- 1 -oxowitha-2,5,24- trienolide (24)32-34 was isolated from Acnistus (Dunalia) austrah where it is accompanied by the corresponding 5/3,6/3-epoxide [7p-hydroxywithanolide D (30)]. NPR 8 2.6 5$,6~-Epoxy-4$-hydroxy-2-en-l-ones Compounds (26)-(38) are withanolides with a regular p-oriented side chain and with the above substitution pattern in rings A and B.The related 2,3-dihydro-derivatives (39) and (40) were added to this list. If we take into consideration that this pattern also exists in withanolides discussed in other sections of the review we conclude that this is the most widespread substitution pattern in this group. The structure of withaferin A (27) was elucidated by chemical degradation6 and by X-ray analysis of its 4-acetoxy- 27-p-bromobenzoate derivative. The first compounds of this 7335 type were isolated from Withania sornnifera and their investi- gation led to the discovery of chemotypes (Section 7). Chemotype 136 of W. sornnifera contains only 20-H with- anolides withaferin A (27)6 being the major constituent.It is accompanied by 27-deoxywithaferin A (26),6 14a-hydroxy-27- deoxywithaferin A (33),37 17a-hydroxy-27-deoxywithaferinA (37),22 and other related minor components. Chemotype 1136 contains only 20-hydroxywithanolides ;withanolide D (28)38 is the major constituent and is accompanied by 14a-hydroxy- (34),36 1 7a- hydroxy -(3 8),36 and 27- hydroxywithanolide D (29).36 The isomeric 7P-hydroxywithanolide D (30)32.33was isolated from Acnistus australis along with compound (24) presumably its A5-precursor. Chemotype 11123924 39 also contains only 20-OH withanolides but the major constituents have the side chain a-oriented e.g. withanolide E (107) and will be described in Section 3. Whereas the withanolides of chemotypes I and I1 have pattern g in rings A and B those of chemotype I11 have other patterns in these rings (mainly c and d Scheme 1).Two 27-0-glucosides were isolated from the roots of W. sornnifera growing in India (most probably near Varanasi)lG7 and in both compounds the aglycone is withaferin A (27). Sitoindoside IX is the unsubstituted p-glucoside and sitoindoside X is the 6'-palmitoyl ester of the latter. In addition to W. sornnifera chemotype I a major source of 20-H wi thanolides is Acnistus brevzjlorus.30 The major con- stituent is withaferin A (27) but in contrast to W. sornnifera it contains large amounts of 2,3-dihydrowithaferin A (40). Both compounds account for almost 99% of the total amount of withanolides in this plant the ratio between (27) and (40) being -2 :1.The biosynthetic process leading to the saturation of A2 is erratic in W. sornnifera but it is a major one in A. brevflorus. Dihydro-deoxywithaferin A (39) is a minor component of the latter. In nature compounds (31) and (32) have a A14 double bond presumably due to dehydration of 14-hydroxy- precursors such as compounds (33) and (34) respectively. Compound (31) was isolated from the Indian chemotype22 of W. sornnifera a producer of 20-H withanolides whereas compound (32) was found in Withania frutescens4' from the Canary Islands. Withangulatin (39 a new compound possessing a A16 double bond but with a more elaborate substitution pattern than the previously known compound (15) is a very recent addition41 to the withanolide family.It is the 14-epimer of physapubenolide (36) and is very similar to withaminimin (77) the only difference being in the pattern of rings A and B. Phy~apubenolide'~ (36) is one of the few known 14P-hydroxywithanolides. It was isolated from the seeds of a variety of Physalis pubescens growing near Walthair (India). Another variety of this plant raised from seeds received from Dijon (France) does not contain physapubenolide but another withanolide physapubescin (94) without any 14-OH group and with an epoxylactol instead of an unsaturated lactone in the side chain. There are several withanolides in which the S-lactone side chain is saturated among them compounds (41)-(43) having the same substitution pattern in rings A and B as compounds (26)-(38).Compounds (41)42and (42)43 are minor constituents of Withania somnifera of South African origin which produces 20-H along with 20-OH withanolides. In this plant the major component is withaferin A (27) which is accompanied by withanolide D (28). The ratio of (27) to (28) is -95 5 but the NATURAL PRODUCT REPORTS 1991 (26) 27-DeoxywithaferinA R' = R2 = H (27) WithaferinA R' = H R2 = OH (28) Withanolide D R' = OH R2 = H (29) 27-Hydroxywithandide D R' = R2 = OH (30) 7P-Hydroxywithanolide D 7P-OH R' = OH R2= H (31) A14 R' = R2 = H (32) A14 R' = H R2= OH (33) 14a-OH R' = R2= H (34) 14a-OH R' = OH R2= H (35) Withangulatin 14a-OH 15a-OAc A16 R' = R2= H (36)Physapubendide 14P-OH 15a-OAc R' = R2 = H (37) 17a-OH R' = R2= H (38)17a-OH R' = OH R2 = H (39) DihydrodeoxywithafennA 2,3-diH R' = R2= H (40) Dihydrowithaferin A 2,3-diH R' = H R2 = OH (41) R=H (42) R= OH (43) 2,3-diH R = H ratio between the 24,25-dihydro derivatives (41) and (42) is almost reversed (7 :93).The fully hydrogenated compound (43) is one of the minor components of Acnistus breviflor~s,~~ the same as compound (41). In all natural products with the saturated S-lactone the configurations of the newly created asymmetric centers are 24s and 25R. Two compounds isolated from Physalis viscosa have an additional oxygen in ring A Viscosalactone A (45) has a 2p,3p-epoxide and viscosalactone B (44) has a 3P-hydroxy group.44 Ring A in withanolides possessing the 5/?,6P-epoxy-4P-hydroxy-2-en- 1 -one system is in a boat conformation with C-1 and C-4 oriented upward.This conformation was inferred from NMR measurements168 on withaferin A (27) and was confirmed by crystallographic analysis.35 NATURAL PRODUCT REPORTS 1991-E. GLOTTER .%HaoH (44) Viscosalactone B 3P-OH (45) Viscosalactone A 2P,3P-epoxy H.Oh0 RO HO (52) Withacoagin R = OH (53) 17a-OH R = H Several reactions characteristic of withanolides with the above pattern in rings A and B have been reported. Under acidic conditions (H,SO in AcOH or acetone) withaferin and withanolide D (28)170undergo a pinacol type rearrangement in ring A leading to A-nor derivatives in which C-4 is lost (46).However when the reaction is carried out on 2,3-dihydro-27-deoxywithaferin A (39)169,or on 2,3-dihydrowithanolide D170 the 5P-formyl-6P-hydroxy derivatives (47) are obtained. Conjugate addition of nucleophilic reagents to the 2-en-1-one system in withanolides was first observed6 with withaferin (48) X = OMe -SC2H5 -SC6H5 -SCH2CHC02Et I aziridinyl pyrrollidinyl efc. NH2 R =Ac (f? (49) OH HA OH A (27) which was transformed under slightly acidic or basic conditions into the 3-methoxy derivative. The reaction was subsequently carried out with and with nitrogen nucleophiles 172 to give compounds with partial structure (48); they were obtained in the presence of neutral alumina at room temperature.Withanolide D (28) served as a model for several reactions with organometallic reagents. The 4-acetate was tran~formedl’~ in the presence of tetrakistriphenyl phosphine palladium Pd[P(C,H,),] into the corresponding 6P-hydroxy-2,4-dien-1-one (49). Tributyl tin hydride in the presence of a catalytic amount of the above Pd complex transformed this substrate into a (Sa)6P-hydroxy-3-en-l-onederivative (50).17,In this reaction Bu,SnH acted as a hydride donor to the n-allylic palladium complex. An analogous transfer of hydride from Bu3SnH to an activated withanolide D-palladium complex followed by concomitant protonation (H,O) resulted in the net conjugate reduction of the substrate to the 2,3-dihydro derivative (51).17 2.7 5a-Hydroxy-2,6-dien-l-ones Just as withanolides with a 4P-hydroxy-2,5-dien-1-one pattern might have been precursors of compounds in which ring B ha! a 5/3,6P-epoxide (26>-(38) withanolides with a Sa-hydroxy-2,6-dien-l-one pattern (52) and (53) might have been precursors of the large group of compounds in which ring B has a 6a,7a- NATURAL PRODUCT REPORTS 1991 (54) Withanolide B R’ = R2= H (55)R’ = H R2 = OH (56) Withanolide A R’ = OH R2 = H (57) NicandrinB 12a-OH R’ = R2= H (58) Withastramonolide 12a-OH R’ = H R2= OH (59) Withanicandrin 12-0x0 R’ = R2 = H (60) Withanone 17a-OH R’ = R2 = H (61) 14a-OH 17a-OH R’ = R2 = H (62) 14P-OH 17a-OH R’ = R2 = H (63) Withanolide T 14a-OH 17a-OH R’ = OH R2 = H epoxide (54>-(62).Withacoagin (52) has recently been iso- lated45 from the roots of Withania coagulans growing near Pondicherry (India). Along with withacoagin (52) from the roots the leaves of W. coagulans contain the corresponding 6a,7a-epoxide (56).46 This compound has also been isolated from Lycium chinense4’ (withanolide A or Lycium substance A). 5a,1701-Dihydroxy- 1 -oxowitha-2,6,24-trienolide(53) is a minor constituent of the leaves of the Indian chemotype of W.somnifera,22in which the corresponding 6a,7a-epoxide [withanone (69)]22 is the major component. The co-existence of these pairs of compounds (52) +(56) and (53) +(60) in the same plant strengthens the assumption that the 5a-hydroxy-2,6-dien- 1 -one system is the immediate precursor of the 6a,7a-epoxy-5a-hydroxy-2-en-1 -one system (Scheme 1 patterns e and g).2.8 6a,7a-Epoxy-Sa-hydroxy-2-en-l-ones The number of compounds possessing the 6a,7a-epoxy-5a- hydroxy-2-en- 1-one substitution pattern is significantly higher than the number of compounds (54)-(63) listed above. This system exists in withanolides with a saturated S-lactone an epoxylactol or an epoxylactone side chain in ring D aromatic compounds and others. The major sources of these witha- nolides are Withania somnifera Indian chemotype W. co-agulans Nicandra physaloides Lycium chinense Datura ferox and D. stramonium. Compounds of this type were first isolated and characterized22 from a W. somnifera Indian chemotype in which the major component withanone (60) is accompanied by 5a,27-dihydroxy-6a,7a-epoxy-1-oxowitha-2,24-dienolide (55) and by la,3~,5a-trihydroxy-6a,7a-epoxywith-2~nolide (64).The latter may have been biosynthesized by a slightly different pathway to those compounds which possess rings A and B with substitution pattern h (Scheme 1). It is noteworthy that neither of the compounds isolated from the Withania somnifera Indian chemotype or from W.somnifera H A 0 1\11\ (65) Functionalization of C-12 resulted in three compounds with a regular side chain two 12a-hydroxywithanolides (57) and (58) and a 12-oxowithanolide (59). Nicandrin B4s (57) was isolated from Nicandra physaloides where it is accompanied by withani~andrin~~ (59) which was isolated 13 years previously.It is noteworthy that compound (57) was isolated from the seeds whereas compound (59) was obtained from the leaves; the latter do not contain nicandrin B but several other related compounds described in Sections 2.13 and 4.1. Compound (57) was also isolated almost simultaneously from Datura ferox and given the name withaferoxolide. In view of the priority of the Indian-Japanese team the name nicandrin is retained in this review. The 27-hydroxy-analog of (57) withastra-mono1ide5’ (58) was isolated from another Datura species D. stramonium. From an F offspring whose parents are the W. somnifera chemotype I11 and W. somnifera Indian chemotype the stereoisomeric 14a-hydroxywithanone (61) and 14P-hydroxy- withan~ne~~ (62) were isolated.These compounds were not encountered in the parent plants. The structure of (62) the first known withanolide with a 14P-OH group was established by X-ray analysis. Another stereoisomer 6,8,7P-epoxy- 14a- hydroxywithanone (65) was also isolated. The unusual struc- ture assigned to this compound is based only on spectral evidence. In contrast to these compounds which do not possess a 20-OH group like all the withanolides from the parent Indian chemotype the next two compounds isolated from the above offspring are 20-OH withanolides comparable to the com-Israeli chemotype I possesses a 20-OH group. There are ponents of the parent chemotype 111. These are withanolide T however plants which are able to produce both 20-H and (63) and withanolide U53(23).20-OH compounds e.g. W. somnifera African ~hemotype~~ Some withanolides with substitution pattern h (Scheme 1) in and Lycium ~hinense.~’ The latter in addition to withanolide B rings A and B like those with pattern g have 24,25-(54) a 20-H compound produces withanolide A (56) a 20-OH dihydrocounterparts with the same configuration 24S 25R at compound previously isolated from W. ~oagulans.~~ the new asymmetric centers as compounds (41>-(43). Ixo- NATURAL PRODUCT REPORTS 1991-E. GLOTTER 421 (66)lxocarpanolide R = OH (67) Vamonolide 14a-OH R = H (68) 14a-Hydroxyixocarpandide 1 4a-OH R = OH carpan01ide~~ (66) was isolated from Physalis ixocarpa growing in a temperate region (Kharkov) of USSR along with the known ixocarpalactone A55(1 64) previously isolated from the same plant raised in Israel.14a-Hydroxyixocarpanolide56(68) was isolated from P. anguZata growing in USSR (Tashkent province) along with 24,25-epoxy-withanolide D (103). The same plant afforded a 20-H withanolide namely vamono1ide5’ (67). The structure assigned to these three compounds are based mainly on spectral analysis. 2.9 Sfl,6~-Epoxy-2-en-l-onesand Derived Structures Compounds (69)-(81) are unsubstituted at C-4 and are characterized by the presence of a 5,6-epoxide (pattern d Scheme 1) or the functional groups obtained by its opening. The genus Jaborosa is a major source of such compounds all of them being 20-H withanolides. Jaborosalactone A58* 59 (69) B58*59(73) D6* (75) and F61 (76) were isolated from J.integrifofia. Jaborosalactone L62(70) and jaborosalactone 063 (82) were isolated from J. leucotricha. The latter is the only known withanolide functionalized at C-19 (19-OH). It is quite possible that there are additional compounds of this type with C-19 at higher oxidation levels comparable to those at C-18 (Section 2.10). Withanolide Y64 (72) was isolated from the offspring obtained by cross-pollination of Withania somnifera chemotypes Indian I and Israel 111. It is the first known withanolide in which the 5,6-epoxide is a-oriented. A compound identical with jaborosalactone D (75) was isolated65 from Acnistus breviflorus and named acnistoferin. The first name is retained in this review. A. brevlJorus66 also contains 2,3- dihydrojaborosalactone A (74) along with several other known wi thanolides.17-Isowithanolide E (71) is a minor constituent of the mixture of withanolides from W.somnifera chemotype I11 ;the major component of this chemotype is the stereoisomeric withanolide E (107) the first known withanolide with an a-oriented side chain (1 7P-OH). Withaminimin6’ (77) is an additional A16 withanolide the others are compounds (15) and (35). It was suggested6’ that withaminimin might be a precursor of physalin-type com-pounds (Section 4.2). If correct this suggestion applies to withangulatin (35) as well. It is noteworthy that both Physalis angufata the source of (35) and P. minima the source of (77) are producers of physalins. Compounds (78)-(8 1) are chlorohydrins produced in nature by opening of the 5P,6P-epoxide ring in the 4-deoxywithanolides (78) and (79) and in the 4-hydroxywithanolides (80) and (81).The first two chlorohydrins jaborosalactone E6’ (78) and C (79) were isolated from Jaborosa integrifolia along with jaborosalactones A B D and F. Whereas in (78) the chlorohydrin is diaxial in (79) it is diequatorial. Under laboratory conditions opening of the 5/3,6P-epoxide in 2-en- 1- ones in the presence of HCI yields only the diaxial chlorohydrin. Chlorohydrin (80) was isolated25 from Withania frutescens growing in the Canary Islands whereas chlorohydrin (81) was isolated68 from a hybrid of W. somnifera. In both JaborosalactoneA 5P,GP-epoxy R’ = HI R2= OH Jaborosalactone L 5P,6P-epoxyI 17a-OH R’ = H R2= OH 17-lsowithanolideE 5PI6P-epoxy 17a-OH R’ = OH,R2 = H WithanolideY 5a,6a-epoxy 7a-OH 17a-OH R’ = OH R2= H Jaborosalactone B A4 6P-OH R’ = HI R2 = OH DihydrojaborosalactoneA 2,3-diH 5Pl6P,-epoxy R’= H R2= 0 Jaborosalactone D 5a-OH 6P-OH R’ = HI R2 = OH Jaborosalactone F 5a-OH 6P-OH 12a-OH R’ = H R2= 0 Withaminimin 5a-OH 6P-OH 15a-OAcI A’‘ R’ = R2 = H Jaborosalactone E 5a-CI 6P-OH R’ = H R2 = OH JaborosalactoneC 5P-OH 6a-CI R’ = H R2 = OH 4P-OH 5P-OH 6a-CI R’ = H R2 = OH 4P-OH 5P-OH 6a-CI R’ = OH R2 = H (83) Withacnisti n compounds the 5,6-chlorohydrin is diequatorial.Chlorohydrin (81) was also isolated from Acnistus brevzyorus and its structure was confirmed by X-ray analysis. It is well known that opening of the SP,GP-epoxide in a 4P-hydroxywithanolide always yields a product with a 5P-OH group.Four additional chlorohydrins 4-deoxyphysalolactone (1 lo) physalolactone (1 16) physalolactone C (1 23 and 23-hydroxy- physalolactone (127) have an a-oriented side chain (Section 3); all these chlorohydrins are diequatorial (5P-OH ; 6a-Cl). 2.10 l&Hydroxy -oxo and -oic acids Withacnistin’O (83) the first 18-functionalized withanolide was isolated from Acnistus arborescens along with withaferin A NATURAL PRODUCT REPORTS 1991 (84) Withaphysalin B (85) WithaphysalinA A2I5 (86) Withaphysalin D A3j5 (87) WithaphysalinE A2I4 w-OH (27) in the early days of research on withanolides. This compound (83) does not exist in the populations of Withania somnifera producing withaferin A.Compounds (84)-(87) designated as withaphysalins were isolated from Physalis minima. Because of the presence of a 20- hydroxyl in these compounds interaction with the 18-0x0 group led to the formation of withaphysalin B71 (84) a 18-20- y-lactol whereas the 18-oic acid gave y-lactones :withaphysalin A71 (85) withaphysalin D72(86) and withaphysalin E73*74 (87). From a structural point of view the most interesting compound of this group is withaphysalin C (154) a 13,14-seco-withanolide which is described in Section 4.2 in conjunction with the physalins. 2.11 21-Hydroxywithanolides The 21-hydroxywithanolides are one of the latest additions to the withanolide family. Functionalization of C-2 1 enabled the construction of two related bicyclic systems in the side chain (Section 4.4).In one of these the 21-OH has the role of a nucleophile whilst in the other it serves after proper transformations as a leaving group. The four compounds (88)-(91) with a free 21-OH group were isolated from Datura metel. Daturametelin B21(88) is accompanied in the plant by its 21-deoxy-analog daturametelin A (1 1). The only difference between daturametelin B and se~owithametelin~~ (89) is in the substituent at C-27 a methoxyl instead of a glucosyl group. Compound (89) could easily be obtained from withametelin (1 56) ;conversely daturametelin B (88) suspended in a mixture of CH,OH-CHCl was transformed into withametelin (1 56). Daturametelin C77 (90) the 2,3-dihydro-derivative of seco-withametelin (89) as well as its 3-sulphate ester (91) were also isolated from D.metel. (88) DaturametelinB A2 R = OGlc (89) Secowithametelin A2 R = OMe (90)DaturametelinC R = OMe (91) Daturametelin E 3P-OS03H R = OMe (92) Withanolide Q (93) Withanolide R 2.12 23-Hydroxywithanolides Withanolides Q (92) and R78 (93) are two 23-hydroxy-withanolides isolated from an artificial chemotype obtained by cross-pollination of Withania somnifera chemotypes I and 111. Self-pollination of the F offspring thus obtained afforded several F offsprings one of them remaining unchanged when re-submitted to self-pollination. The leaves of this F offspring were used in the chemical inve~tigation.~~ All the withanolides have the 22R configuration with the exception of the withanolides hydroxylated at C-23 when the designation is 22s due to a change in the order of priorities (this ambiguity can be eliminated by using Fieser’s convention;12 the designation is 22p, regardless of the substituents at C-23).The configuration at C-23 in compounds (92) and (93) is 23s. There are several additional compounds hydroxylated at C-23 but with the 23R configuration. These are ixocarpalactone A (164) with a 26,23 saturated y-lactone in a P-oriented side chain 23- hydroxyphysalactone (127) with an unsaturated 8-lactone in an a-oriented side chain and trechonolide A (166) with an unsaturated y-lactone in an a-oriented side chain.NATURAL PRODUCT REPORTS 1991-E. GLOTTER 2.13 Epoxylactol and Epoxylactone Side Chains In all the compounds with an epoxylactol side chain the 22-H 24,25-epoxide and 26-OH are on the same side of the molecule (22R 24S 25S 26R). The biogenetic conflict whether the epoxylactol side chain is formed in nature by reduction of the corresponding epoxylactone or by cyclization of an inter-mediate ap-unsaturated aldehyde and subsequent epoxidation was settled by Whiting and co-~orkers~~ in favour of the second possibility (the first possibility may constitute a minor pathway). Such a pathway was suggested4 in 1978 by the reviewer and co-workers as a reasonable biogenetic possibility. Nic-3 (95) and Ni~-7~~,~~ (96) were isolated from Nicandra physaloides (the widespread variety with blue-violet flowers) along with the unique compounds with aromatic ring D (Section 4.1).The variety N.physaloides var. albiflora produces instead of the above compounds the related nicalbin A (97) and nicalbin B (98).82 In nicalbin A (97) a compound with a 16a-oriented hydroxy group the epoxylactol side chain is as in the previous compounds. In nicalbin B (98) attack of the 16a-OH at the hemiacetal carbon leads to the closure of a new seven membered oxa-ring in a bicyclic acetal type structure. Physapubescins3 (94) is produced by a variety of Physalis pubescens raised from seeds from the Botanical Garden of Dijon (France). Another variety of P. pubescens raised from seeds from Walthair (India) does not synthesize physapubescin ..-? iu 0 ArR (94) Physapubescin 4P-OH 5P,6P-epoxy 1 5a-OAc R = a-OH,P-H (95) Nic-3 5a-OHl 6a,7a-epoxyl R = a-OH,P-H (96) Nic-7 5a-OH 6a,i'a-epoxy 12-0x0 R = a-OH,P-H (97) NicalbinA 5a-OH 6a,7a-epoxy 16a-OH R = a-OH,P-H (99) DaturalactoneD 5a-OH 6a,7a-epoxy R = 0 (100) Daturalactone B 5a-OH 6a,7a-epoxy 12a-OH R = 0 (101) Daturalactone C 5a-OHl 6a,7a-epoxy 12P-OHl R = 0 (102) Daturalactone A 5a-OH 6a17a-epoxy 12-0x0 R = 0 (103) Epoxywithanolide D 4P-OH 5P,GP-epoxy 20-OH R = 0 (104) Pubescenol 2,3-diH 4a-OH 7a-OH 20-OH R = 0 (105) Physangulide 2,3-diH 3P-OH 4P-OH SP,GP-epoxy 20-OHl R=O (98) Nicalbin B but two other withanolides the 3-glucoside pubescenin15 (8) with an epoxylactone and phy~apubenolide'~ (36) with an aP-unsaturated lactone in the side chain.In contrast to nicalbin A (97) and other withanolides with an epoxylactol side chain in which the configuration at the hemiacetal carbon is exclusively 26R physapubescin is a 4 1 mixture of 26R 26s stereoisomers. The available chemotaxonomic evidence suggests that in the genus Datura the withanolides are confined to the species D. quercifolia D. stramonium D. ferox and D. metel. The epoxylactones (99)-( 102) were isolated from D. quercifolia ; the unsaturated B-lactones nicandrin B48 (withafero~olide~~) (57) and witha~tramonolide~~ (58) were isolated from D. ferox and D. stramonium respectively. As already mentioned the 21- hydroxywithanolides and the related withametelins (Section 4.4) were found only in D.metel. Compounds (103) and (104) were isolated from plants of the genus Physalis. P. angulata afforded 24,25-epoxy-withanolide D (103) along with 14a-hydroxyix~carpanolide~~ (68). P. pubescens of unstated origin afforded pubescen01~~ (104) a compound with a quite unusual 4a-OH group in ring A. The same paper was published twice the only difference being in the order of the authors (!). Physangulide was isolated from the leaves of P. angulata. The only available informationls8 is the structural formula (105) pointing towards the 22s configuration (?).The reviewer was unable to retrieve any data in support of this assignment; there is only a reference to a 1986 symposium.206 3 Withanolides with an Unmodified a-Oriented Side Chain Compounds (106)-129) are grouped together according to their most characteristic feature an ap-unsaturated B-lactone in an a-oriented side chain with a 17P-hydroxy group.All the withanolides with unmodified or even modified a-oriented side chains invariably have this 17P-OH group. With only one exception withanolide P36 (129) a minor constituent of Withania somnifera chemotype I they also have a 20-OH group. Additional 17,8-OH,20-H withanolides are Ni~-2~~ (130) an epoxylactol isolated from Nicandra physaloides and several epoxylactols (1 30)-( 133) and epoxylactones (1 34) (1 35) isolated from Jaborosa bergii.86 Four additional 17P-OH,20-H withanolides have modified side chains Nic-11 (155) acnistin A (162) and E (163) and trechonolide A (166) (Sections 4.34.5).It is noteworthy that neither of the withanolides produced by W. somnifera chemo-type I and by plants of the Nicandra and Jaborosa genera has a 20-OH group. With very few exceptions the 17P-hydroxy- withanolides have a 14a-OH group. The exceptions are (a) A14-~~mp~~nd~ like withaperuvin FS7(1 19) withaperuvin BSs (124) and physalolactone C89 (125); (b) Nic-2 (130) which is unsubstituted at C-14 (14a-H); (c) the compounds found in J. bergii (1 3 1)-( 135) which have a 14P-OH group. The previous 14P-hydroxywithanolides (36) and (62) have a regular P-oriented side chain. The major sources of withanolides with an a-oriented side chain are Physalis peruviana and Withania somnifera chemotype 111.The compounds are presented according to the substitution pattern of rings A and B (Scheme l) the most important being the SP,6P-epoxy-2-en- 1 -one (4,the 5/3,6P-epoxy-4P-hydroxy-2-en-1-one (g) and the products resulting by modification of these patterns. The simplest compound of this series is 3P,14a,17/3,20- tetrahydroxy- 1 -oxowitha-5,24-dienolideg0 (1 20) isolated from fruits of W.coagulans in the course of a reinvestigation. It is followed by withanolide F39 (106) with a 2,5-dien-l-one in rings A and B; this is a minor component of W. somnifera chemotype 111 and may be regarded as the immediate precursor of withanolide E39,91 (107) the major component in the leaves of this chemotype. 4P-Hydroxywithanolide Eg2393 (1 13) in NATURAL PRODUCT REPORTS I991 OH tfPH PH OH HO (106) Withanolide F A5 (1 18) Withaperuvin D 14a-OH (707) Withanolide E 5P,6P-epoxy (119) Withaperuvin F A14 (108) Withanolide S 5a-OH 6P-OH (109) Withaphysanolide 4P-OH 5a-OH (1 10) 4-Deoxyphysalolactone 5P-OH 6a-CI (111) 5a-OEt 6P-OH H.Oh0 (1 12) Withaperuvin C A4 6P-OH -.(113) 4P-Hydroxywithanolide E 4P-OH !$,GP-epoxy (1 14) Withaperuvin E 4-0x0 5p,6P-epoxy (.lf-,pH (115) Withaperuvin 4P-OH 5P-OH 6a-OH OH (1 16) Physalolactone 4P-OH 5P-OH 6a-CI (117) Physanolide 2,3-diH 4-0x0 A5 (120) 3P-OH A5 (121) A3I5-diene (122) Physalactone 3-OMe 4P-OH fiP,GP-epoxy (1 23) Withaperuvin G 2P,3P-epoxy 5P,6P-epoxy OH eH &P HO OH L HO (124) Withaperuvin 8 6a-OH (126) 28-Hydroxywithaphysanolide A5 R' = H R2 = OH (1 25) Physalolactone C 6a-CI (127) 23-Hydroxyphysalactone 5P-OH 6a-CI R' = OH R2 = H ppH --.ho H I 0s eH OH HO)J \ (128) Withaperuvin H (129) Withanolide P NATURAL PRODUCT REPORTS 1991-E.GLOTTER 0 (130) Nic-2 A2 5a-OH 6aI7a-epoxy,14a-H R = a-OH,P-H (131) A2 5p,6P-epoxy 14P-OH R = a-OH,P-H (132) 5P,GP-epoxy 14P-OH R = a-OH,P-H (133) A2I4 69-OH 14P-OH R = a-OH,P-H (134) A2 5PI6P-epoxy 14P-OH R = 0 (135) 5P,GP-epoxy 14P-OH R = 0 which rings A and B have pattern h (Scheme l) is the major component of the leaves of P. peruviana. Hydrolytic opening of the 5/3,6/3-epoxide in a 4-deoxywithanolide such as withanolide E (107) leads to withanolide S39(108) a 5a,6P-diaxial diol accompanying withanolide E in the leaves of W.somnifera. It has also been isolated from the leaves of P. peruvi~na.~~ A similar hydrolytic opening of the 5/3,6P-epoxide in a 4P-hydroxywithanolide such as (1 13) occurs mainly in the roots of P. peruviana and results in several related compounds withaperuving4 (1 15) a 4P,SP,6a-trihydroxy-2-en-1-one; withaperuvin Baa (1 24) a A1*-4P,5P,6a-trihydroxy-2-en-1-one ; withaperuvin Dg6(1 l8) in which the opening of the epoxide is accompanied by conjugate addition of 6a-OH to the end of the 2-en-l-one thus leading to the formation of an oxide bridge (4P,SP-dihydroxy-3a,6a-epoxy- 1-one system) ; and witha-peruvin FS7(1 19) having in addition to the above system a A14 double bond.Withaperuvin E48(1 14) results from oxidation of the 4P-OH in 4P-hydroxy-withanolide E to give a SP,6P-epoxy- 2-en-1,4-dione. Withaperuvin GS7 (123) is the product of epoxidation of A2 in withanolide E (107) to give a 2/?,3P;5/3,6/3- diepoxy- 1-one system. In contrast to other withaperuvins which were isolated from the roots of P. peruviana and which are the result of opening of the epoxide in 4P-hydroxywithanolide E (1 13) withaperuvin CS7 (1 12) is undoubtedly obtained from withanolide E (107) in which opening of the epoxide is followed by stabilization as an allylic alcohol (6P-hydroxy-2,4-dien- 1 -one). Withaperuvin Hg7 (128) the most recent discovery within the withaperuvins from the roots of P. peruviana deserves special attention since it is characterized by the presence of an unusual sulphur containing bridge connecting C-4 with C-6.According to the authors ‘it is tempting to speculate that it is biosynthesized from 4P-hydroxywithanolide E (1 13) by condensation with a-mercaptoacetaldehyde derived from cysteine ’. Opening of the 5P,6P-epoxide in compounds having patterns d and g in rings A and B (Scheme 1) may yield not only diols as shown above but also chlorohydrins. When such an opening takes place in a 4-unsubstituted withanolide e.g. withanolide E the diol is diaxial(108) but the chlorohydrin is diequatorial e.g. 4-deoxyphysalolactoneg3(1 lo) a SP-hydroxy-6a-chloro-2- en-1-one isolated from the leaves of P. peruviana and of W. somnifera chemotype III.69 When the opening occurs in 4P-hydroxywithanolide E (1 13) the chlorohydrins as well as the diols are diequatorial (5P-OH 6a-OH and 5P-OH 6a-Cl).It is interesting that in P. peruviana the diols were isolated from the roots but the chlorohydrins were isolated from the leaves. Examples of such chlorohydrins are physalolactoneg8 (1 16) physalolactone CS8 (125) with a A14 double bond and 23- hydroxyphysalactoneg9 (127). The latter is the third known 23- hydroxywithanolide with an unmodified side chain. The HO Reagents i H,/Pd; ii LiAlH,; iii HIO,; iv CrO (Jones) Scheme 2 OH 0 A HO OH 0 previous two such compounds withanolides Q (92) and R (93) have regular P-oriented side chains and their configuration is 23S in contrast to 23R in compound (127). 28-Hydroxy~ithaphysanolide~~~ (1 26) is the only 28-hydroxywithanolide with an unmodified side chain.The two other compounds functionalized at C-28 have a modified y-lactone the first perulactone (167) in a P-oriented side chain and the second perulactone B (168) in an a-oriented side chain (Section 4.6). Compound (126) was isolated from P. viscosa along with several other 17P-hydroxywithanolides devoid of the 28-OH group the known 4P-hydroxywithanolide E (1 13) the new withaphysanolidelOl (1 09) having a 4P-Sa-dihydroxy- 2-en- 1-one system and physanolidel (1 17) with a Sene- 1,4- dione system. PhysalactoneloO* lo2(1 22) isolated from P. alkekengi growing in the Soviet Turkmenia is the product of conjugate addition of MeOH to 4P-hydroxywithanolide E (1 13).The reaction was performed in the laboratory but the compound seems to occur in nature as well. The reviewer had a frustrating experience with an artefact of this type during the work on withaferin A6 (27). In addition to other compounds already discussed two minor constituents were isolated from W.sornnifera chemotype I11 the 3,5-dien-l-one13 (121) a product of deconjugation of the 2,S-dien-1-one in withanolide F (106) and the Sa-ethoxy,6P- hydroxy-2-en-l-one13 (1 11); according to the authors the latter was isolated under conditions which excluded the possibility of it being an artefact. Two reactions of withanolide E (107) that are triggered by the 14a-hydroxy group should be mentioned. Attempted degradation of withanolide E into a pregnan-20- one derivative by cleavage of the 20-22 bond was unsuccessful since the expected pregnane derivative underwent a spon-taneous D-homo rea~rangement”~ to yield compound (1 36) (Scheme 2).Of the three tertiary hydroxy-groups in withanolide E the 14a-OH is easily eliminated under acidic conditions17’ (H2S04/acetone) to give a 14,20-oxido-bridged derivative (1 37) along with the A14-derivative. The reaction takes place with concomitant hydrolytic opening of the SP,6/3-epoxide. NPR 8 21 (138) (1 39) Nicandrenone (Nic-1 ) Nicandrenonolactone H (143) Physalin C"' (1 44) Physalin A 7a-OH H (145) Physalin B111-'13A (146) Physalin H114 A5 7P-OH (147) Physalin F719"55P,GP-epoxy (148) Physalin J7'1115 5a,6a-epoxy (149) Physalin D'16 5a-OH 6P-OH (150) Physalin Ell4 5a-OH 701-OH (151) Physalin G A4 6a-OH (152) Physalin K 4a,5a-epoxy 6a-OH (153) Physalin I 5a-OMe 6P-OH 4 Withanolides with Modified Structures 4.1 Compounds with Ring D Aromatic Natural steroids with ring D aromatic are unique compounds isolated so far from only one source Nicandra physaloides.In fact only the first two compounds (1 38) and (139) are ergostane derivatives albeit with a modified carbocyclic skeleton whereas the next three are degradation products derived from (138) by gradual cleavage of two (Nic-17 and Nic-12) and five (Nic-10) carbon atoms. The history of nicandrenone begins in 1951 when an uncharacterized bitter substance apparently a glycoside was isolated103 from N.physaloides and named nicandrin. A re- investigationlo4of this plant in 1964 resulted in the isolation of a compound with insect repellent properties which was given the name nicandrenone. Although the authors did not succeed NATURAL PRODUCT REPORTS 1991 (1 40) (141) (1 42) Nic-17 Nic-12 Nic-10) in solving its structure they pointed towards the 'probable presence of at least one aromatic ring'. The structure was determined independently and almost simultaneously in 1972 by Bateslo5 who retained the name nicandrenone and by Crombie Whiting and co-workers106 who re-named the compound Nic-1. The proposed structure is based on X-ray analysis of the related Nic- 10106*107 (142) and on spectral analy~is'~~*'~~ of Nic- 1 itself.In contrast to the 17P-hydroxywithanolides whose first representative was discovered in 197223,39 in Withania somnifera but was soon found to have many congeners in this and other Solanaceae plants the small group of ring D aromatic withanolides did not increase. The only exception is nicandreno- lactone (Nic- I-lactone) (1 39) isolated by Glotter Kirson and co-workers108 from N. physaloides raised from seeds received from Pondicherry (India). For the time being the story of such compounds seems to be closed. The biosynthetic pathway leading to the aromatization of ring D'O~ and to the epoxylactol side chain7' has been elucidated by Whiting and co-workers (Section 6). In addition to the ring D aromatic compounds N.physaloides afforded several withanolides with a normal carbocyclic skeleton withanicandring9 (59) and nicandrin B48 (57) with an @-unsaturated lactone side chain; Ni~-3~~*~~ (93 Nic-780*81 (96) and Ni~-2~~ (130) with an epoxylactol side chain; and Nic- 11 (155) with a modified side chain (Section 4.3).In the last two compounds (1 30) and (1 59 the side chain is a-oriented. 4.2 13,14-Secowithanolides;Physalins and Withaphysalin C The physalins are the most elaborate compounds of the withanolide family. They were isolated from various Physalis species (P. alkekengi P. angulata P. lanceifolia and P. minima). The structure of physalin A111,112 (144) was solved by X-ray analysis118s119of 5a-acetoxy-6/3-bromohexahydrophysalin A. The structures of physalin B (145) and C (143) were determinedlll by interrelation with physalin A whereas those assigned to physalin F (147) and J (148) were confirmed by epoxidation of physalin B.719115 In view of the a-orientation of the 6-OH group in physalin G117(1 5 1) its immediate precursor is presumably physalin J115 (148).The formation in nature of physalin K117 (152) is surprising. Under laboratory conditions steroidal 2,4-dien- 1-ones unsubstituted at C-6121 as well as those possessing a 601- hydroxy group,122 invariably give a 4P,SP-epoxide by treatment with perbenzoic or m-chloroperbenzoic acid. In the case of physalin G (1 5 l) treatment with monoperphtalic acid afforded a product identical with physalin K (152). The reviewer wonders whether physalin Ill7 (1 53) is indeed a natural product.Methanol was used for extraction of the plant material and it would not be surprizing if some physalin F115 (147) was converted into I (153). A similar reaction occurred during the processing of Withania somnife~al~~ the isolation of in withanolide E (107). NATURAL PRODUCT REPORTS 1991-E. GLOTTER (154) Withaphysalin C a b 0 0 C d e Hypothetical pathways to withaphysalin C (154) and to physalin C (143) Scheme 3 tfP HO **O (155) Nic-11 In contrast to the physalins which are withanolides modified not only in the carbocyclic skeleton but also in the side chain withaphysalin CI2*(154) is modified only in the carbocyclic moiety. It was isolated from P. minima along with witha- physalin A (85) and B (84) two withanolides with an unmodified skeleton.The structure (154) assigned to withaphysalin C was confirmed by X-ray analysis of the corresponding 18,20- lac tone. It was suggested that the biosynthetic process initiating the formation of withaphysalin C and of physalins is the oxidative cleavage of the 13,14-bond as shown in Scheme 3. In the case of withaphysalin C the immediate precursor may be a withaphysalin A like compound a but at a lower oxidation level at C-18 a 18,20-lactol instead of a 18,20-lactone. Following cleavage of the 13-14 bond by an appropriate biological oxidizing agent the transient I3P-hydroxy- 14-one in the 9 membered ring system b thus obtained is stabilized by 14P 13P-hemiketal formation (1 54).In the case of the physalins the precursor may be an unknown compound whose structure is 17a-hydroxy- 15- oxowithaphysalin A (partial structure c in Scheme 3). Oxidative cleavage of the 13,14 bond should result in a transient 13a 17a- dihydroxy-14-one d again in a 9-membered ring system which is stabilized by 14a,l7a-hemiketal formation (e).Inspection of models leads to the conclusion that this cyclic ketal is sterically favoured and easier to obtain than the alternative 14a,13a- hemiketal. Presumably the attack of the oxidizing agent was directed by the 17a-OH group the result being the a-orientation of the 13-OH in the final product. Up to this stage (e) the modifications are only in the carbocyclic framework. The presence of the 15-one activates the 16-methylene which by an intramolecular Michael type addition at the 24-25 double bond affords the final product in this case physalin C (143).4.3 Modifications due to the 17p-OH Group Nic-11 (155) is one of the components of the mixture of withanolides isolated from Nicandra phy~al0ides.l~~ The im- mediate precursor of this compound seems to be Nic-2 (130) NATURAL PRODUCT REPORTS 1991 -R = a-H X = leaving X=OH b group Formation of the modified side chains in withametelins (a)and acnistins (b) Scheme 4 (156) Withametelin (daturilin) A2 25-=CH2 (157) Datumetelin (daturametelin D) A2 25kCH20CH3 (158) Daturametelin G A2 25kCH2-OGlc (159) lsowithametelin A3 25-=CH2 (160) Daturametelin F 3P-OS03H 25-=CH2 0 H& (162) Acnistin A (163) Acnistin E 4P-OH the only component of N.physaloides with a 17P-OH group. Nucleophilic attack (in nature) of this group at the epoxidic C- 24 leads to the formation of a 17-24 oxide bridge thus closing the bicyclic dioxabicyclononane system present in the side chain of Nic- 1 1. Its structure was determined by X-ray analysis. 4.4 Modifications due to Functionalization of 21-Me Functionalization of 2 1-Me is instrumental in the modification of the @-unsaturated &lactone in two opposite albeit related directions. In a substrate like daturametelin B (88) in which the 21-Me is oxidized to the corresponding primary alcohol the hydroxy group may act as a nucleophile attacking C-24 of the unsaturated lactone thus leading to the oxabicyclic structure a (Scheme 4)which is present in compounds (156)-(160).The generic name 'withametelins ' was pr~posed"~ for withanolides with such a modified side chain. (161) Datumelin Conversely transformation of the above 21-OH into a good leaving group for instance a phosphate ester allows an opposite process in which an external nucleophile (H,O) attacks C-25 and leads to the formation of a new C-24-C-21 bond with concomitant displacement of the 21 -leaving group thus leading to the bicyclic structure b present in the two known acnistins (1 62) and (1 63). This process occurred presumably in a yet unknown substrate possessing a 17P-hydroxy group and therefore the side chain in acnistins is a-oriented. Compounds (1 56)-( 160) were isolated from Datura metel.There seems to be some confusion regarding the configurational assignments at C-20 C-22 and C-24 in this group of compounds. Treatment of ith ha met el in'^^ 125 (1 56) with MeOH in the presence of HClO afforded secowithametelin (89) showing a positive Cotton effect in the CD spectrum (AczS3+4.l); this points to the 22R configuration as in all the withanolides (except those having an oxygen function at C-23 when the designation is 22S see Section 2.12). Daturilin a compound most probably identical with withametelin (156) and several other compounds of this group126-129 were assigned 20S 22S and 24s configurations on the basis of NOE experiments done on daturilin. According to the authors these results require the 20-H 22-H and 24-Me to be on the same side of the molecule.However the 22s and 24s assignments contradict the CD measurements which indicate that 22-H and 24-Me are indeed on the same side of the molecule but on the opposite one it. 22R 24R. The configuration at C-20 is 20R. Dat~melin'~~ (161) is formed by opening the lactone ring presumably in withametelin (1 56). The configurations indicated in formulae (156)-(161) are 20R 22R and 24R. The two known acnistins,13' A (162) and E (163) were isolated from Acnistus ramiflorus and the structure of the latter was determined by X-ray analysis. The interrelation was done by conversion of acnistin E into A. NATURAL PRODUCT REPORTS 1991-E. GLOTTER HO (1 64) lxocarpalactone A (1 65) lxocarpalactone B (1 66) Trechonolide A (167) Perulactone -* -.H&oH HO (169) A2 14a-OH (170) A2 15P-OH (171) A2 12P-OH (172) 3P-OMe 12P-OH 4.5 Modifications due to a 23-OH Group There are three withanolides with modified side chains characterized by the presence of a 26,23 y-lactone ring ixocarpalactones A (164) and B (165) have a saturated lactone in a P-oriented side chain whereas in trechnonolide A (166) the side chain is a-oriented and the lactone unsaturated.The two ixocarpala~tones~~ were isolated from Physalis ixocarpa. In the major component ixocarpalactone A (1 64) the oxidation of C-23 stopped at the alcohol level resulting therefore in a lactone. In the minor component ixocarpalactone B (165) C-23 is oxidized to the carbonyl level thus allowing the formation of a spiro-lactono-acetal.The configurations of the asymmetric centers 204 22S 23R 24R and 25R were determined by 'H and 13C NMR and by CD measurements. The designations 22s and 24R instead of 22R and 24s as in other withanolides [e.g. compounds (41)-(43)] are due to changes in the order of priorities. Trechonolide A'31 (1 66) isolated from Trechonaetes laciniata is so far the only 17P-hydroxywithanolide with a 26,23-y-lactone. The same compound was isolated almost simul-taneously from Jaborosa mageZlanica and was given the name Jaborosalactone M.132 There are two independent structure (168) Perulactone B determinations by X-ray analysis (!). In view of its structure the immediate precursor of trechonolide A is presumably a 12-0x0- 178,20,22,23- tetrahydroxy-24-en-26-oic acid in which the configurations at the asymmetric centers are as in ixo- carpalactone A (164).The presence of the 12-one allowed hemiketalization with the 20-OH group. An additional com- pound (trechonolide B) isolated along with trechonolide A had the structure of the corresponding 12-methylketal ; it was shown to be an artefact formed during processing of the plant material. 4.6 Modifications due to a 28-OH Group The only 28-hydroxywithanolide with a usual unsaturated 6-lactone is 28-hydroxywithaphysanolide100 (1 26) isolated from Physalis viscosa. Its side chain is a-oriented. Two compounds related to (126) but with modified side chains are peru-la~tone'~~ (167) and perulactone B134(168) the first with a P-oriented and the latter with an a-oriented side chain.Both compounds were isolated from P. peruviana and have identical saturated side chains. The configuration at (2-25 is the same (25R) as in all withanolides with a saturated side chain either modified or unmodified. 5 Microbial Transformations and Cell Free CuItu res There are several investigations on microbiological trans-formations of withanolides. Withaferin A (27) is hydroxylated by Cunninghamella elegans to give two metabolite^'^^ one of them identified as 14a-hydroxywithaferin A (169). In a subsequent paper136 it was shown that the previous assignment is wrong and that the structure of the above compound is 15P- hydroxywithaferin A (1 70) whereas that of the second compound is 12P-hydroxywithaferin A (171).However in a patent13' of 1986 (applied for in 1981) it is claimed that fermentation of withaferin A with the same microorganism afforded 14a-hydroxywithaferin A (169). It is quite possible that the correct structure of this compound is (170) and not (1 69). 3P-Methoxy-2,3-dihydrowithaferinA was also trans-formed by C. elegans into its 12P-hydroxy derivative (172).13* NATURAL PRODUCT REPORTS 1991 0 Possible pathways for ring-D aromatization14* Scheme 5 St I St I St 'OH a b d Possible pathways to the biosynthesis of the expoxylactol side chain78 Scheme 6 Biotransformations of withaferin A were also induced by Of Arthrobacter ~imp1ex.l~~ the several derivatives thus obtained only SP,6P-epoxy-27-hydroxy- 1,4-dioxowitha-2,24- dienolide was identified.Arthrobacter itr re us"^ de-acetylated withaferin A 4,27-diacetate to the free withaferin A.lS0 From tissue cultures of W. somnifera several simple sterols such as 24-methylene- and 24-ethylidenecholesterol as well as sitosterol stigmasterol and campesterol were isolated ;139 no withanolides could be detected in the extract of these tissue cultures. Concomitantly in an investigation of W. somnifera plants,140 24-methylcholesterol 24-methylcholesta-5,24-dien-3P-01 sitosterol stigmasterol and 28-isofucosterol were iso- lated along with small amounts of 24-methylenecholesterol. This was the starting point for a study on the biosynthesis of withanolides to be discussed in Section 6.By using labeled physalin B (145) and physalin F (147) [the 5P,6P-epoxyderivative of (145)] in cell free extracts of P. minima it was that both were rapidly converted into the corresponding 546P-diol [physalin D (149)l. However callus142 derived from diploid P. minima plants synthesized only small amounts of physalin D in contrast to callus derived from triploid P. minima which synthesized physalins B D and F. 6 Biosynthesis The biosynthesis of withanolides was first investigated by Goodwin and co-worker~,'~~ by administration of [28-,H]-24- methylenecholesterol to young leaves of Withania somnifera either directly or via the stem; the second procedure gave a significantly higher degree of incorporation.The isolated withaferin A (27) and withanolide D (28) were radioactive. Similar experiments with [28-3H]-(24R,S)-24-methylcholesterol gave negative results whereas administration of [28-3H]-24- methylcholesta-5,24-dien-3P-ol led to ambiguous results ; ten years later it was unambiguously found that it is incorporated in Nic- 1 (1 38).79 We can therefore assume that both A24(28)and A24 isomers are precursors of withanolides. 144 145 Investigations of Gros and co-worker~,~~~ by ad-ministration of [2-14C]-mevalonolactone to seeds and seedlings of Acnistus brevifo~us~~~ resulted in the isolation of labelled withaferin A (27) and jaborosalactone A (69). There are indications that C-26 in these compound was directly derived from C-2 of mevalonolactone thus suggesting that the 25 pro-R methyl of cholesterol is the source of C-26 in (27) and (69).However degradation studies indicated that 98% of the radioactivity is in a C,,-hydroxyketone fragment and only 2 % in a C fragment (glyceric acid) containing carbons 25 26 and 27. The authors assume that the biosynthetic pathway involves partial cleavage of the side chain in the sterol serving as precursors. The results of the incorporation confirmed29 the ~uggestion'~~ that jaborosalactone A (69) is biosynthesized prior to withaferin A (27). The conclusion is that (69) is a plausible precursor of (27) and not as suggested by Glotter and co-worker~,'~~ that epoxidation of A5 is preceded by hy- droxylation at C-4 (the order of events in Scheme 1 should be c+d+gand not c+e+g).The biosynthesis of physalins was by experiments with radioactive acetate (l-14C and 2-14C) and mevalonic acid (2-14C) administered to Physalis minima. They were efficiently incorporated into physalin D (149). When tritiated withanolide A (56) was administered radioactive physalin D (149) was obtained; the incorporation however was low. The authors conclude that withanolides are possible precursors of physalins. Two papers by Whiting and ~o-workers~~*~~~ deal with the biosynthesis of Nic- 1 (1 38) in Nicandra physaloides the with the origin of the aromatic D-ring and the with the elaboration of the epoxylactol side chain. Two possible pathways to the enlargement and aromatization of ring D have been investigated (Scheme 5) (a) Oxidation of the angular 18-CH3 and subsequent incorporation into ring D; and (b) oxidation of 18-CH3 and its removal from the system followed by the introduction of an extraneous carbon from S-adenosylmethionine (SAM).Experiments with [3'-C2H,]- and [3'-14CH,]-mevalonic acid demonstrated that mevalonic acid was incorporated. The NATURAL PRODUCT REPORTS 1991-E. GLOTTER 43 1 R (173) and (174) R = -(1 75) trimellitic acid fragment isolated after degradation retained the activity as required by pathway A. [14CH3]-SAM is also incorporated; however the activity does not appear in ring D but is presumably located at C-28. Further experiments with 13'-C2H,]-MVA prepared from deuterioacetic acid led to the conclusion that the deuterium was located at (2-18 in the aromatic ring of Nic-1 (138).The pathway to the formation of the epoxylactol side chain in Nic- 1 was inve~tigated~~ by following the incorporation of four radioactive potential precursors [28-14C]-24-methylene-cholesterol (A) [28-14C]-24-methylcholesta-5,24-dien-3~-ol (B) [23,28-3H2]-(22R)-lactone (C) and the tritiated diol (D) obtained by LiAlH reduction of (C) (Scheme 6). The lowest absolute incorporation was that of the lactone (C) and the highest that of the diol (D). The first two compounds (A) and (B) were incorporated at levels expected in such experiments. The conclusion is that the predominant pathway from the 22,26-diol to the epoxylactol present in Nic-1 (138) is through a C-26-aldehyde in preference to the reduction of a lactone (although the latter may constitute a minor route).The outcome of administration of [3'-C2H,]-mevalonolactone re-inforces the C-26 aldehyde pathway (see also Section 2.13). Several sterols found in W. coagulansgo and P. minima,149along with withanolides were proposed as potential biogenetic precursors. These are ergosta-5,25-diene-3/3,24[-diolgO (173) isolated from the fruits of W. coagulans and two isomers of (173) named physalindicanol~,~~~ isolated from P. minima var. indica (whole plant). Physalindicanol A (1 74) is the 24-epimer of (1 73) whereas physalindicanol B (1 75) is 25- hydroxy-24-methylenecholesterol.Much as their involvement in the biosynthetic elaboration of the withanolides is reasonable such a possibility has not yet been proven.7 Chemotypes The name chemotypes denotes morphologically similar plants differing in their chemical con~tituents.'~~~~~~ The first investigations (Weizman Institute) were done on Withania somnifera growing in where three chemotypes were identified (Section 2.6). Two additional chemotypes of W. somnifera were subsequently identified. The Indian chemo- type,22 whose natural habitat is North-Western India contains only 20-H withanolides most of them with pattern h in rings A and B (Scheme l) its major component being withanone (60). The South- African ~hemotype,~ is characterized by the pro- duction of 20-H as well as 20-OH withanolides [major components (27) and (28)].It also contains small amounts of withanolides with a saturated side chain like 27-deoxy-24,25- dihydrowithaferin A (41) and 24,25-dihydrowithanolide D (42). In order to confirm that the differences between the (176) 14a-OH R = OH (177) 17a-OH R = H chemotypes have a genetic character cross-pollination* be- tween the chemotypes was performed. In the F offspring obtained by crossing W.s.1 x W.S.II,'~~ withanolide D (28) was the major component. Cross-pollination W.s.1 x W.S.III'~~ afforded an offspring again containing withanolide D (28) which did not exist in the parent chemotypes and withanolide E (107) but less than in the parent chemotype 111. In both offsprings withaferin A (27) was practically absent. As expected the F offspring from W.s.11 x W.s.111 contained withanolides D and E as the major components.Cross-pollination of W.s.111 x W.~.Indian~l.l~~ gave an off- spring in which withaferin A (27) and withanone (60) from the parent Indian chemotype disappeared being replaced by 20- OH withanolides which did not exist in the parent plants. These were withanolide D (28) 17-deoxy-20-hydroxywithanone(56) a compound previously isolated from W. co~gulans,~~ and several other compounds (61F(64) which are described in Section 2.8. The reviewer wonders whether two 2,4,6-trienes (176) and (177) isolated from the above offspring are indeed natural products. They might have resulted during processing of the plant material by dehydration of compounds with substitution patterns e or f (Scheme 1).If this assumption is correct withanolide U (23) which was isolated from this offspring might be the source of triene (176) and compound (53) which exists in the parent Indian chemotype might be the source of triene (177). The formation of the major components in the F offsprings was interpreted in genetic terms as a result of dominant versus recessive characters in the parent plants. 151 For instance hydroxylation at C-20 in chemotype I1 is considered to be induced by a dominant character in contrast to hydroxylation at C-27 in chemotype I which is induced by a recessive character. In the offspring the dominant character takes advantage over the recessive; thus the major component would contain a 20-OH group whereas 20-H:27-H and/or 20-H :27-OH withanolides would be only minor components.Such an outcome can be considered as extreme. W. somnifera African42 chemotype and Lycium chineme,' are examples of plants in which 20-OH withanolides co-exist with 20-H withanolides. The composition of the offspring obtained by cross-pollination of W.s.11 x W.s.African is as expected.205 The major components are withanolide D (28) and 24,25-dihydro-withanolide D (42). Withaferin A (27) appears only as a minor component whereas 27-deoxy-24,25-dihydrowithaferinA (41) does not appear at all. A very interesting illustration of the potential of cross-pollinations is the F offspring78 of W.s.1 x W.s.111 from which two 23-hydroxywithanolides [Q(92) and R (93)] which had never been encountered before were isolated (Section 2.12).The F hybrids50 between Datura ferox and D. stramonium or between D.quercifolia and D. stramonium produce with- astramonolide (%) not present in the parent species. It should * The terms cross-breeding and hybridization have also been used in publications on this topic; the meaning was always the same. NATURAL PRODUCT REPORTS 1991 I II 111 IV v 0 OAc OH I HO OH Reagents i N,H,; ii CrO (Jones); iii KOH; iv POC1,; v m-CPBA; vi NaOH; vii MsCl; viii OsO Scheme 7 i,ii iii iv v vi vii ___t viii ii * 0 HO ix ii i,x iv v OH lii 5p,6p + 5a,6a Reagents i LiAlH,; ii PhC0,H; iii Ac,O-py; iv CrO (Jones); v A1,0,; vi H,SO,-AcOH; vii SOCl,; viii Ba(OMe),; ix NaBH,; x,Ac,O-py-CHC1 Scheme 8 be mentioned that this work refers to a crossbreeding between unsaturated S-lactone side chain in model compounds with a two species and not between two chemotypes.Comparison simple androstane skeleton; (c) syntheses involving rings A and between W. sornnifera of West Benga1166 and W. sornnifera of B and the side chain finally leading to several natural Tamil Nadu led to the conclusion that they are chemotypes; withanolides such as withaferin A (27) 27-deoxywithaferin A the former produces withanolide D whereas the latter produces (26) withanolide D (28) jaborosalactones A (69) B (73) and withaferin A accompanied by several minor components D (79,etc. among them withanolide D in trace amounts. The first syntheses of cholestane type compounds having SP,6P-epoxy-4P-hydroxy-2-en-1-1-one and 5PY6P-epoxy-2-en- one systems due to Ikekawa and co-worker~,~~~,~~~ are based 8 Synthetic Achievements on appropriate modifications of (5a)-6P-acetoxy-la,2a-The investigations on synthesis of withanolides are divided into epoxycholestan-3-one; part of them are shown in Scheme 7.three groups :(a) methods for the synthesis of model compounds Glotter and colleague^'^'^^^^ have devised a sequence of with the most important substitution patterns of rings A and B reactions beginning with la,2a-epoxycholest-4-en-3-one, which (c d,f and g in Scheme 1); (b) methodsfor the synthesis of the are partially summarized in Scheme 8. 433 NATURAL PRODUCT REPORTS 1991-E. GLOTTER i iii iv v vi 0 a b C Reagents i LiAlH,; ii Bu'O,H/VO(acac),; iii m-CPBA; iv Ac,O-py; v CrO (Jones); vi A1,0 Scheme 9 i A HooEt COOBut HcooBut CHZOH Reagents i LDA/HMPA -78 "C; ii Steroid aldehyde -78 "C H -.,.S/CHO Scheme 10 C Scheme 11 Another sequence of reactions was devised156 for the synthesis of cholestane derivatives having the 6a,7a-epoxy-5a-hydroxy- 2-en-1-one system that is present in a large number of withanolides.The starting material was la,2a-epoxycholesta- 4,6-dien-3-one (Scheme 9). Transformation of the intermediate 4a,5a-epoxy-la-3a-dihydroxycholest-6-eneinto the desired 6a,7a-epoxy-5a-hydroxycholest-2-en- 1-one was achieved with- out purification of the four intermediate products in an overall yield of 17 %.No attempts were made to optimize the reaction conditions. The first synthetic efforts for constructing the ap-unsaturated b-lactone resulted in compounds with the unnatural 22s configuration. The synthesis of Ikekawa and co-w~rkers~~~ (Scheme 10) was based on the observation that butenoic acid esters as the corresponding lithium enolates undergo exclusive y-coupling in aldol reactions. The 3P-tetrahydropyranyl ether of 501-bisnorcholan-22-a1 served as the aldehyde component. Path A afforded a 27-deoxy- and path B a 27-hydroxy-withanolide type compound both 22s. Gonzalez and collaborators158 synthesized (Scheme 11) the unsaturated lactone by an aldol condensation of 3P-acetoxy-bisnorchol-5-en-22-al (a) with acetone leading to a 22-hydroxy-24-one (along with the 22-en-24-one).No stereo-chemical control was achieved. The P-hydroxyketone (b) was MOMO -...VCHO R = Me,Et a OH 0 *..$$ i,ii iii CH3CHBrCOBr J 0 I II Br Reagents :i Ph,P=CHCOCH,; ii H,O,/OH-; iii AI(Hg);iv P(OEt),; v NaH Scheme 13 submitted to a Reformatskii reaction with ethyl a-bromo- propionate and the resultant ester was transformed into lactone (c).According to the Cotton effect of the lactone (c)(Ae,, -2.2; personal communication 1980 from Dr J. Breton Funes) the product is most probably a mixture of 22-epirne1-s~ the major one having the 22s configuration. The lactone obtained by Ikekawa15’ had As2,,-3.72. The construction of the unsaturated B-lactone side chain by coupling lithium enolates of ap-unsaturated esters with steroidal 22-aldehydes was re-investigated by Ikekawa’s group.159 Condensation of 3~-hydroxybisnorchol-5-en-22-al (protected as the 3-THP-ether or 3-acetate) with the lithium enolate of ethyl or methyl dimethylbutenoate again afforded the corresponding 22s lactone.However (Scheme 12) when the reactions were conducted with (20R)3P-acetoxy-20-hydroxychol-5-en-22-a1 protected at C-20 [methoxymethyloxy (MOMO) derivative (a)] a mixture of the 20-MOM0 deriva- tives of the desired lactone (b)and of the corresponding 22- hydroxyester (c)was obtained. 0-NATURAL PRODUCT REPORTS 1991 + b C Upon treatment with I, not only the MOMO group was removed but concomitant lactonization of the ester took place.The lactone was directly obtained in 84% yield160 when the reaction was done with la,3~,20-tris-(methoxymethyloxy)- instead of the 3~-acetoxy-20-methoxymethyloxy-derivative (4. The protection of the 20-OH group is an essential condition for the above reaction to occur. When the unprotected 20- hydroxy-22-aldehyde was reacted with the enolate de-formylation occurred thus leading to the corresponding 20- one. The causes of the opposite behaviour of a protected 20- hydroxy-22-aldehyde (22R product) as compared to that of a 20-deoxy-22-aldehyde (22s product) are analysed in the paper. 159 Glotter’s grouplG1 succeeded in synthesizing the 22R-6-lactone side chain (Scheme 13) by using the strategy developed by McMorris for the synthesis of 23-deoxyantheridiol.162 Almost concurrently Ikekawa’s group163 solved the problem of the 22R configuration by developing a new approach for securing the correct stereochemistry (Scheme 14). The key intermediate was (22$)-22,23-epoxy- 6P-methoxy -3’5-cyclo- 24- norcholane (a). Opening of this epoxide by alkylation with the 2-methyl-l,3-dithiane anion led to the formation of a 22-hydroxydithioketal giving after treatment with HgO/BF etherate the (22R)22-hydroxy-24-one (b). Treatment with bromoacetyl bromide and continuing according to McMorris162 led to the 27-nor unsaturated lactone (c)with the correct 22R configuration. Hydrogenation and treatment with diphenyl disulphide afforded two C-25 thiophenyl stereoisomers (6). At this stage there was the choice between adding a 27-Me group or a 27-CH20H group.Treatment of d with CH,O followed by oxidation of the thiophenyl group to the corresponding sulphoxide and its thermal elimination afforded the side chain (e) present in withaferin A (27). Alternatively methylation of intermediate d with CH,I followed by the same treatment as above afforded the side chain (f) present in 27-deoxy-withaferin A (26). In subsequent investigations Ikekawa and co-workers combined the construction of the side chain with the appropriate functionalization of rings A and B thus synthesizing three 4,20- dideoxy~ithanolides,~~~ two 4P-hydroxy-20-deoxywithano-lide~,’~~ 160 and a 4P,20-dihydroxywithanolide. The starting material for the synthesis of the three jaboro- ~alactonesl~~ (Scheme 15) was 3P-hydroxybisnorchol- 5-en-22- oic acid a; which was transformed in several steps into the NATURAL PRODUCT REPORTS 1991-E.GLOTTER 435 b C A OMe vi vii a SPh viii ix x xi ix x I e d f Reagents i 2-Me-1,3-dithiane/BuLi THF -78 "C; ii HgO/BF;OEt,; iii BrCH,COBr; iv P(OEt),; v NaH; vi H,/Pd-C; vii LICHA THF -78 "C/(PhS),; viii LICHA THF -78 "C/CH,O; ix m-CPBA; x A 100 "C; xi LICHA THF/MeI LICHA = Lithium isopropyl cyclo hex ylamide Scheme 14 ....<CO*H a b iv v vi -VIII IX C OH HO OH @ @ (73) (69) e (75) Reagents i as in Scheme 14; ii aq. HCl-THF; iii TBDMS-Cl-imidazole-DMF ; iv MEM-C1; v PDC/DMF; vi AcOH-H,O-THP; vii m-CPBA; viii Al,O,-benzene; ix H,SO,-THF; x 5% aq KOH-THF TBDMS-CI = t-Butyldimethylsilyl chloride ; MEM-C1 = methoxyethoxymethyl chloride Scheme 15 NATURAL PRODUCT REPORTS 1991 ~1 R=OH QI d2 Q R=H (26) 27-Deoxywithaferin A fl * f2 gll g2 (27) Withaferin A Reagents i rn-CPBA; ii TBDMS-C1-imidazole-DMF (only for preparation of DJ; iii PDC-DMF; iv PhSH A1,0, ether; v TsOH benzene 60 "C; vi P(OMe), excess MeOH-THF Scheme 16 cf.Scheme 12 cf. Scheme 16 Withanolide D MOMO Scheme 17 (22S)-22,23-epoxide b. This intermediate was used for con- deoxywithaferin A (26),165 (Scheme 16) the side chain was struction of the side chain as outlined in Scheme 14 to give constructed as outlined in Scheme 14 the major relay being compound c (in Scheme 15).After protection of the primary compound c (in Scheme 15) with and without the 27-OH 27-OH oxidation of 1a-OH and p-elimination compound d group. was obtained. This is actually a natural product its 27- After protection of the 27-OH group in c, by transformation glucoside is daturametelin A (11). Epoxidation of d gave into t-butyldimethylsilyl ether both c and protected c1 were jaborosalactone A (69) and the 5a,6a-epoxy-stereoisomer e epoxidized to give the 5a,6a-epoxides and la-OH was oxidized subsequently converted into jaborosalactone D (75). Treatment to the 1-one (dl d,). Opening of the 5a,6a-epoxide with of (69) with base afforded jaborosalactone B (73). thiophenol gave the 6P-thiophenyl-5a-hydroxyderivative which For the stereoselective synthesis of withaferin A (27) and 27- was dehydrated to give the allylic thiophenyl ethers el and e2.NATURAL PRODUCT REPORTS 1991-E. GLOTTER OAc i ii iii AcO&0 {xb 0-N-CO,Bn a b c 14a 17a d 148,178 vi vii 0 1 {$ & [fi -xii,xiii ----viii,ix,x,xi I ' OMOM A OH OMe f e MOM0 *.,.J',CHO COCH3 xvi xvii OMOM xviii {UH h i i ' OMOM k several steps I withanolide E Reagents i TMSI (TMS),NH Et,N ClCH,CH,Cl -23 "C 45 min; ii Pd(OAc), K,CO, CH,CN 12 h; iii i-Propenyl acetate/TsOH reflux; iv Benzyl nitrosoformate; v toluene reflux; vi H,/Pd-BaSO, EtOH 3 h; vii CuCl;2H2O H,@THF 4 h; viii TsCl-py 12 h; ix TBDMSOTf Et,N CH,Cl, 0 "C 30 min; x KOAc MeOH reflux 12 h; xi Bu,NF THF 4 days; xii Ph,P=CHCH, THF; xiii MOMCl PrkNEt dioxane 80 "C sealed tube; xiv OsO, py ;xv TFAA DMSO Et,N CH,Cl, -78 "C 1.5 h; xvi CH,=CHLi THF -78 "C,1 h ;xvii O, MeOH -100 "C.Me$ 30 min; xviii Ethyl dimethylcrotonate LDA THF HMPA Scheme 18 Following oxidation to the corresponding sulphoxides these intermediates underwent the sulphoxide-sulphenate re-arrangement to give under defined conditions (absence of oxygen darkness) a mixture of the rearranged 4P-OH product ul,f,) (major component) and the unrearranged 6P-OHproduct (g! g,) (minor component). Full details are given.160 Epoxid- ation off andf gave withaferin A (27) and 27-deoxywithaferin A (26) respectively. For the synthesis of withanolide D (28) (Scheme 17) and several related 20-hydroxywithanolides 160 pregnenolone was used as starting material.After introduction of an oxygen function at C-1 (la-OH) the compound was transformed into (20R) 1 a,3~,20-tris-(MOMO)-bisnorchol-5-en-22-al (a) the ad- ditional C-22 being introduced by the dithiane procedure. The side chain was constructed in continuation as shown in Scheme 12. The functionalization of rings A and B followed the same path as used for withaferin A (Scheme 16). During this sequence in addition to the major target (28) two other natural withanolides were synthesized; physalo- lactone B (4) and (20R)3P,20-dihydroxy- 1 -oxowitha-5,24- dienolide (9). The first synthesis of a 17P-hydroxywithanolide [withanolide E (107)] due to GriecoZo4 was completed just before submitting the present review.It involves a series of steps divided according to their targets into 5 groups (Scheme 18) (a). Introduction of a 14a-OH group in the 17-oxoandrostane A (a -+ e). (b) Conversion of e into a 3,5-cycloandrostane deriva- tive (f). (c) Transformation of the androstane f into a 17P-hydroxypregnan-20-one (f+i). (d) Synthesis of the 22-aldehyde j and construction of the B-lactone moiety (j+k). (e) Functionalization of rings A and B to obtain the 5P,6P-epoxy- 2-en-l-one pattern present in withanolide E. Treatment of the dienol acetate b with benzyl nitrosoformate afforded a 2 1 mixture of the isomeric adducts c and d; the latter was isomerized into the former by brief refluxing in toluene. Treatment of the 17-ethylidene derivative g with OsO afforded a 1.4 1 mixture of 17,20-diols; the synthesis was continued with the major diol h.In view of Grieco's strategy in which the proper function- alization of rings A and B was done in the last steps of the overall synthesis most 17P-hydroxywithanolides (Section 3) become available from the intermediate (20S,22R)-la,3P 14a 17P,20-pentahydroxywith-5-enolide,whose struc-ture was confirmed by X-ray analysis.2o4 9 Biological Activity Modern investigations on withaferin A (27) before its structure elucidation led to the conclusion that the compound possesses to some extent antibacterial properties mainly against acid- fast bacilli and gram-positive microorganisms. la1-la3In a later inve~tigation'~~ on several withanolides it was found that 27- deoxywithaferin A (26) is more active than other related compounds (27) (28) (33) (39) and (40).3P-Hydroxy-2,3-dihydrowithanolide F (120) has a protective in cases of CC1 induced hepatoxicity in adult albino rats; it also produces a moderate fall of blood pressure in dogsla6 and has a significant anti-inflammatory effect in sub- acute inflammations produced by formalin. la' 24,25-Epoxywithanolide D (103) and physangulide (105) have anti- inflammatory effects Iaa when administered intra-peritoneally to mice and rats (10 mg/kg) in exudative and proliferative types of experimentally induced inflammation. The activity was comparable to that of hydrocortisone. Several daturalactones show to some extent antifertility effects on albino female rats.la9 Daturalactone B (100) is more active than daturalactone A (102) and 2,3-dihydro-daturalactone B.Withaferin A (27) and withacnistin (83) showed cytotoxicity in vitro against KB cell cultures derived from human carcinoma of the nasopharynx and in vivo activity against sarcoma 180 in mice and Walker 256 intramuscular carcino~arcoma.~~ 70 Witha-ferin A was also found to inhibit the growth of Ehrlich ascites carcinoma in mice,190 to produce mitotic arrest in the metaphase of the same tumour system,lS1 to induce vacuolization of the ~ytopla~rn,~~~ and to inhibit immunologically induced inflam- mation in rats.lS3 Withaferin A (27) and its 6a-chloro-5P- hydroxy-derivative (8 1) have cytostatic activity against HeLa 229 cells in cultures.194 Tests on several related compounds gave negative results.Withaferin A and withanolide E (107) were shown to have immunosuppressive activity on human B and T lymphocytes as well as on mice thymocyte~.'~~ 4P-Hydroxywithanolide E (113) showed a considerable life-span enhancing activity against L- 12 10 leukemia.93 Withaferin A (27) withanolide D (28) and 4P-hydroxy- withanolide E (1 13) were active against mouse leukemia L5 178Y cells,196 at a definitely higher level than synthetically prepared cholest-2-en-1-ones substituted at positions 4 5 and 6. Withangulatin (35) acts in vitro on topoisomerase I1 to induce topoisomerase 11-mediated DNA damage.Is7 Withanolide E (1 07) and 4P-hydroxywithanolide E (1 13) supplied by Kirson Glotter and Lavie were pre-clinically investigated by the National Cancer Institute (USA) on L-12 10 leukemia and B-16 melanoma; their activity was less than necessary for clinical investigations.The presence of the SP,GP-epoxide and of the 17P-hydroxy group seem to be mandatory for the antitumour activity of withanolides. Thus 17-isowithanolide E (71) is inactive as is withanolide S (108) a compound in which the 17P-hydroxy group is preserved but the 5P,6P-epoxide is converted into the 5a,6P-diol. Modified withanolides were ~ynthesized'~~ in the hope of finding more active compounds. None had even the activity of compounds (107) and (1 13). Several withanolides are insect antifeedants. Thus Nic- 1 (1 38) is a feeding deterrent for larvae of Manduca sexta L,lg9 but not for Pectinophora gossypiella Saund Heliothis virescens F and H.zea Boddie.zOO Further studieszo1 on other withanolides showed that withanolide E (107) is a potent antifeedant for larvae of Spodoptera Zittoralis Boisd. in contrast with Nic- 1 (poor activity) and other withanolides (27) (28) (30) and (145) which are inactive. 4P-Hydroxywithanolide E (1 13) is ca. 10 times less active than withanolide E whereas withanolide S (108) is inactive. Experiments with larvae of Epilachna varivestis Muls. led to the conclusion that (107) and (138) are antifeedants. However the most active compound is nicalbin A (97).202 The investigation was extendedzo3 to several other natural and synthetically modified withanolides. NATURAL PRODUCT REPORTS 1991 10 References 1 (a)D.Lavie I. Kirson and E. Glotter Zsr. J. Chem. 1968,6 671 ; (b) R. N. Tursunova V. A. Maslennikova and N. K. Abu- bakirov Khim. Prir. Soedin. 1981 17 187 (Engl. trans. Chem. Nat. Comp. 1981 17 145). 2 R. N. Tursunova V. A. Maslennikova and N. K. Abubakirov Khim. Prir. Soedin. 1977 13 147 (Engl. trans. Chem. Nat. Comp. 1977 13 131). 3 A. V. Kamernitskii I. G. Reshetova and V. A. Krivoruchko Khim. Prir. Soedin. 1977 13 156 (Engl. trans. Chem. Nat Comp. 1977 13 138). 4 E. Glotter I. Kirson D. Lavie and A. Abraham 'Bioorganic Chemistry' Vol. 11 ed. E. E. van Tamelen Academic Press New York 1978 chapter 3. 5 I. Kirson and E. Glotter J. Nat. Prod. 1981 44 633. 6 D. Lavie E. Glotter and Y. Shvo J.Chem. SOC. 1965 7517. 7 S. M. Kupchan R. W. Doskotch P. Bollinger A. T. McPhail G. A. Sim and J. A. Saenz-Renauld J. Am. Chem. SOC. 1965 87 5805. 8 W. Kopaczewski Therapie 1948 3 98. 9 K. M. Watt and M. G. Breyer-Brandwijk 'The medicinal and poisonous plants of Southern and East Africa' Livingstone Edinburgh 1962. 10 N. S. Dhalla M. S. Sastry and C. L. Malhotra J. Pharm. Science (India) 1961 50 876. 11 F. B. Power and A. H. 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Abstr. 111 166955). 189 N. Chandoke Indian J. Exp. Biol. 1978 16 419. 190 B. Shohat S. Gitter A. Abraham and D. Lavie Cancer Chemother. Rep. 1967 51 271. 191 B. Shohat S. Gitter and D. Lavie Int. J. Cancer 1970 5 244. 192 B. Shohat 2.Krebsforsch. 1973 80 97. 193 A. Fugner Arzneim. Forsch. 1973 23 932. 194 A. G. Gonzalez Y Darias D. A. Martin Herrera and M. C. Suarez Fitoterapia 1982 53 85. 195 B. Shohat I. Kirson and D. Lavie Biomedicine 1978 28 18. 196 M. Yoshida A. Hoshi K. Kuretani and M. Ishiguro J. Pharm. Dyn. 1979 2 92. 197 J. K. Juang H. W. Huang and C. M. Chen Biochem. Biophys. Res. Commun. 1989 159 1128. 198 E. Glotter S. Kumar M. Sahai A. Goldman and M. Mendelovici J. Chem. Soc. Perkin Trans. I 1991 739. 199 R. T. Yamamoto and G. S. Fraenkel Ann. Entomol. Soc. Am. 1960 53 503. 200 C. A. Elliger (personal communication to E. Glotter). 201 K. R. S. Ascher N. E. Nemny M. Eliyahu I. Kirson A. Abraham and E. Glotter Experientia 1980 36 998. 202 K. R. S. Ascher H.Schmutterer E. 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ISSN:0265-0568
DOI:10.1039/NP9910800415
出版商:RSC
年代:1991
数据来源: RSC
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