摘要:

13 NPR 5 Cumulative Contents of Volume 5 Number 1 1 Prostaglandins Thromboxanes Leukotrienes and Related Arachidonic Acid Metabolites (1983 and 1984) T. W. Hart 47 Antibiotics with Antifungal and Antibacterial Activity Against Plant Diseases P. A. Worthington 67 Tropane Alkaloids (July 1985 to December 1986) G. Fodor and R. Dharanipragada 73 The Biosynthesis of Shikimate Metabolites (1986) P. M. Dewick 99 Errata to The Biosynthesis of Triterpenoids and Steroids D. M. Harrison (Vol. 2 No. 6 p. 525) Number 2 I01 The Use of N.M.R. Spectroscopy in the Structure Determination of Natural Products One-Dimensional Methods I. H. Sadler 129 The Biosynthesis of Penicillins and Cephalosporins J. E. Baldwin and Sir Edward Abraham 147 Steroids Reactions and Partial Syntheses (1985) J.Elks 187 Non-Macrocyclic Trichothecenes (January 1970 to December 1986) J. F. Grove Number 3 21 1 Diterpenoids (1986) J. R. Hanson 229 Naturally Occurring Isocyanides M. S. Edenborough and R. B. Herbert 247 The Biosynthesis of C,&,, Terpenoid Compounds (1986) M. H. Beale and J. MacMillan 265 8-Phenylethylamines and the Isoquinoline Alkaloids (July 1986 to June 1987) K. W. Bentley 293 Quinoline Quinazoline and Acridone Alkaloids (July 1985 to June 1987) M. F. Grundon 309 Book Review Secondary Metabolism (Second Edition) by J. Mann. Reviewed by G. W. Kirby 309 Book Review Biologically Active Natural Products ed. K. Hostettmann and P. J. Lea. Reviewed by A. Pelter Articles that will appear in forthcoming issues include Monoterpenoids (1985 and 1986) D.H. Grayson Natural Sesquiterpenoids (1986) B. M. Fraga Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1986 to June 1987) J. E. Saxton Synthesis of Gibberellins and Antheridiogens (to December 1987) L. N. Mander Trends in Protease Inhibition (November 1984 to January 1987) G. Fischer The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1986 to June 1987) R. B. Herbert Natural Products from Plant Tissue Culture (January 1979 to December 1986) B. E. EUis Marine Natural Products (September 1986 to December 1987) D. J. Faulkner Erythrina and Related Alkaloids (July 1985 to June 1987) A. S. Chawla and A. H. Jackson Pyrrole Pyrrolidine Piperidine Pyridine and Azepine Alkaloids (July 1986 to June 1987) A. R. Pinder The Use of N.M.R. Spectroscopy in the Structure Determination of Natural Products Two-Dimensional Methods A. E. Derome Enzyme Inhibitors in Medicine (to December 1987) C. S. J. Walpole and R. Wrigglesworth Amaryllidaceae Alkaloids (July 1985 to June 1987) M. F. Grundon Biosynthetic Studies on Marine Natural Products (to April 1988) M. J. Garson The Biosynthesis of Porphyrins Chlorophylls and Vitamin B, (1986 and 1987) F. J. Leeper Recent Advances in Chemical Ecology (July 1985 to December 1987) J. B. Harborne The Polyether and Macrolide Antibiotics Biogenetic Analysis and Structural Correlations D. O’Hagan Pyrrolizidine Alkaloids (July 1986 to June 1987) D. J. Robins

ISSN:0265-0568    

DOI:10.1039/NP98805FP011

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

Natural Product Reports Editorial Boa rd Professor G. Pattenden (Chairman) University of Nottingham Dr D. V. Banthorpe University College London Professor M. F. Grundon University of Ulster at Coleraine Dr J. R. Hanson University of Sussex Dr R. B. Herbert University of Leeds Professor M. I. Page The Polytechnic Huddersfield Dr T. J. Simpson U niversity of Edinburgh 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 Burlington House London W1 V OBN England. 1988 Annual Subscription Price U.K. f159.00 Rest of World f183.00 U.S.A. $342.00. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts.SG6 lHN England. Air Freight and mailing in the U.S. by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11 003. US Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11 003. 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. 0The Royal Society of Chemistry 1988 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 recording or otherwise without the prior permission of the publishers. Printed in Great Britain by the University Press Cambridge Subscription rates for 1988 U.K. €159.00 Overseas f183.00 U.S.A. US $342.00 Subscription rates for back issues are (1984) (1985) (1986) (1987) U.K. f120.00 f125.00 f130.00 f142.00 Overseas f126.00 f131.OO f143.00 f159.00 U.S.A. US $240.00 US $242.00 US $252.00 US $280.00 Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry 30 Russell Square LONDON WClB 5DT England

ISSN:0265-0568    

DOI:10.1039/NP98805FX013

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

NEW BOOKS FROM THE ROYAL SOCIETY OF CHEMISTRY Amino Acids and Peptides Vol. 19 “. . its utility to the group of readers for whom it is intended is unquestionable.” -Medical Book News reviewing Vol. 18. This series was previously entitled ‘Amino Acids Peptides and -”--Proteins’. The latest volume in the series covers the literature published on the subject during 1986. @ Brief Contents Amino Acids; Peptide Synthesis; Analogue and Conformational Studies on Peptide Hormones and Other Biologically Active Peptides; Cyclic Modified and Conjugated Peptides; &Lactam Antibiotic Senior Reporter J.H. Jones Chemistry; Metal Complexes of Amino Acids and Peptides. University of Oxford Specialist Periodical Report (1987) Hardcover 345pp ISBN 0 85186 174 1 Price f69.50 ($1 36.00) Carbohydrate Chemistry VoI.19 Part I “We can wholeheartedly support this series and recommend both this volume and the series as a whole as an essential part of the library of all practical carbohydrate workers. Our laboratories would find it difficult to review the whole of this field without the aid of this series.” -British Polymer Journal reviewing Vol. 18 Part I. From Vol. 14 onwards ‘Carbohydrate Chemistry’ has been split into two parts Part I Mono Di and Tri saccharides and Their Derivatives Part II Macromolecules. Since Vol. 19 Part I has been renamed Monosaccharides Disaccharides and Specific Oligosaccharides. This volume is a review of the literature published during 1985. Brief Contents Senior Reporter N.R.Williams Free Sugars Glycosides and Disaccharides; Oligosaccharides; University of London Ethers and Anhydro-sugars; Acetals; Esters; Halogeno-sugars; Specialist Periodical Report Amino-sugars; Miscellaneous Nitrogen Derivatives; Thio- and Seleno-sugars; Deoxy-sugars; Unsaturated Derivatives; Branched- (1987) chain Sugars; Aldosuloses and Dialdoses; Sugar Acids and Lactones; Hardcover 305pp Inorganic Derivatives; Alditols and Cyclitols; Antibiotics; Nucleosides; ISBN 0 85186 222 5 N.M.R. Spectroscopy and Conformational Features; Other Physical Price f60.00 ($116.00) Methods; Separatory and Analytical Methods; Synthesis of Enantiomerically Pure Non-carbohydrate Compounds. ______~~~ ~~~~~~~ ~ To order or for further information please write to Royal Society of Chemistry Distribution Centre Blackhorse Road Letchworth Herts SG6 1 HN UK.or telephone (0462)672555 quoting your credit card details.We now accept AccessNisalMasterCard/EuroCard. RSC Members are entitled to a discount on most RSC publications and should write to The Membership Manager Royal Society of Chemistry 30 Russell Square London WC1B 5DT UK. Molecular Biology and Biotechnology 2nd Edition Completely revised and expanded. ‘I. . . the work provides an excellent introduction to the techniques of molecular biology and their industrial applications and as such will be of particular use to undergraduates and specialists in other areas.” -Biologist 1987 reviewing the 1st Edition. ‘I. . . would not hesitate in recommending it to my own biotechnology students.Very reasonably priced.” -Society for General Microbiology Quarterly 1987 reviewing the 1st Edition. The new 2nd edition of Molecular Biology and Biotechnology provides an invaluable introduction to the subject for students and scientific workers from other areas. The book requires no specialist knowledge of the biological sciences as it reviews the fundamental aspects of the field. It covers such subjects as fermentation technology; genetic engineering; product recovery; protein technology; the cloning of yeast plant and animal cells; and much more illustrating the wide ranging nature of biotechnology. Edited by J.M. Walker and E.B. Though aimed at the non-expert the book does not approach the subject Gingold The Hatfield Polytechnic in a trivial fashion. The extensive reference lists provide an excellent March 1988 opportunity for further study for all those wishing to delve deeper into particular Softcover 452pp areas. ISBN 085186 453 8 The new 2nd edition provides an up-to-date picture of developments in Price f34.95 ($69.00) this rapidly advancing field; an additional 100pages of essential information. I The Membership Manager Royal Society of Chemistry 30 Russell Square London WC1B 5DT UK. PRINTED IN GREAT BRITAIN BY THE UNIVERSITY PRESS CAMBRIDGE

ISSN:0265-0568    

DOI:10.1039/NP98805BX015

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

Steroids Reactions and Partial Syntheses A. B. Turner Chemistry Department University of Aberdeen Aberdeen AB9 2UE Scotland Reviewing the literature published during the period December 1985 to October 1986 (Continuing the coverage of literature in Natural Product Reports 1988 Vol. 5 p. 147) 1 Reactions both 501-and 5P-cholestanone under these conditions. The 1.1 Alcohols and Carboxylic Acids and their Derivatives structure (4) has been established for the dimeric anhydride Halides and Epoxides that is formed in 15% yield by treating the Butenandt acid (5) 1.1.1 Oxidation Substitution. and Reduction (which is an oxidation product of cholesterol) with base 1.1.2 Ethers and Esters followed by acetic anhydride.'l 1.1.3 Opening of Epoxide Rings The efficiencies of pyridinium chlorochromate silver carbon- 1.2 Unsaturated Compounds ate on Celite aluminium isopropoxide and chromic acid as 1.2.1 Electrophilic Addition oxidants for a range of sterols have been evaluated in connection 1.2.2 Other Reactions of Olefinic and Aromatic Steroids with base-catalysed tritium exchange-labelling of cholesterol 1.3 Carbonyl Compounds and other A5-~tero1~.12 Tritium is introduced mainly into 1.3.1 Reduction and Dehydrogenation positions 2 and 6 rather than exclusively at positions 2 and 4 1.3.2 Other Reactions as had previously been claimed.1.3.3 Reactions of ap-Unsaturated Carbonyl Compounds Selective fluorination of steroids by elemental fluorine and Enols or Enolic Derivatives (diluted with nitrogen) is controlled by deactivating oxygen 1.4 Compounds of Nitrogen Phosphorus Sulphur and f~ncti0ns.l~ Depending upon their location substitution can be other Hetero-elements 1.5 Molecular Rearrangements 1.6 Remote Functionalization Reactions 1.7 Photochemical Reactions 2 Partial Syntheses 2.1 Derivatives and Analogues of Cholestane 2.2 Vitamins D their Derivatives and their Metabolites 2.3 Cholanes Norcholanes and Dinorcholanes 2.4 Pregnanes 2.5 Androstanes and Oestranes 2.6 Cardenolides and Bufadienolides 2.7 Heterocyclic Compounds 2.8 Cyclopropano-steroids 2.9 Microbiological Transformations 3 References During the period of coverage of this Report the following topics have been reviewed topical anti-inflammatory agents,l steroid contraceptives,2 tritium labelling of steroid^,^ sterol markers for marine and terrigenous organic matter,4 and (3) reduction of cyclic ketones by alkali metals in protic solvents;5 other reviews are mentioned in the relevant sections.A new handbook covers the chromatography of steroids.6 c8 H,7° 1 Reactions 1.1 Alcohols and Carboxylic Acids and their Derivatives Halides and Epoxides 1.1.1 Oxidation Substitution and Reduction Oxidation of cholestanes by the Gif system almost always gives the corresponding 20-ketone as the major isolated product.' The cleavage of the side-chain is more selective if there are enone systems in rings A and B.The radical mechanism that has 0 been proposed for this degradation involves abstraction of a hydrogen atom at C-25 followed by oxygenation and intra- (4 1 molecular transfer of hydrogen. However the oxidation by the Gif system of dienones such as (1) and (2) lacking a proton at C-25 still yields minor amounts of the 20-ketone (3),8 indicating that a second minor route is available for degradation of the cholesterol side-chain. This is thought to involve abstraction of a hydrogen atom at the tertiary 20P-position followed by oxygenation and cleavage of the 2&22 bond.8 Platinum-catalysed oxidation of cholesterol gives the 4-en-3- one the 5-en-3-one and several 6-and 7-oxygenated product^.^ The low reactivity and the formation of a complex product mixture in this reaction contrasts sharply with similar catalytic oxidation of A4-3-01s and saturated 3-alcohols which give the corresponding ketones in good yield.1° SP-Cholestanol affords 311 13-2 directed to any of the tertiary centres C-5 C-9 C-14 C-17 or C-25 with retention of configuration.Fluorination does not occur at position 5 in 3P-sterols whereas it is frequently observed to occur at C-5 in 3a-sterols.The configuration at C- 5 controls fluorination of the electronically favourable position C-9. In the 5P-series position 9 is severely hindered by ring A owing to the A/B-CiS ring junction and no attack takes place at C-9. A commercially available robotic system has been employed to make the positron-emitting compound 16a-[18F]fluoro- 17P-oestradiol by a three-step route (Scheme 1).Each step in the synthesis as well as the purification by h.p.l.c. is entirely a~t0mated.l~ The reaction of 16-bromo- 17-ketones with potassium cyanide in aqueous pyridine gives 17P-cyano- 1601 17a-epoxides in high yield.15 On the basis of results from kinetic and isotope- labelling studies equilibration between the 1601- and 16P-bromo-compounds precedes epoxide formation and the internal S,2 displacement in the cyanohydrin intermediate involves the 16/3-bromo-substituent. The reaction of the bromopregnenol- one (6; R = Br) with sodium methoxide in refluxing methanol gave the 21-aldehyde (7) and the 21-alcohol(6; R = OH). The 21-acetate (6; R = OAc) was obtained in good yield by prolonged reaction of the bromo-compound with acetate anion.l6 The vinyl chloride (S) in combination with zinc chloride is superior to hydrogen chloride as a reagent for replacing the hydroxyl group of the epimeric tertiary alcohols (9; X = OH) by chlorine ;17 triphenylphosphine and carbon tetrachloride or phosphoryl chloride in pyridine each give mixtures of 3-methyl-5a-cholest-2-ene 3-methyl-5a-cholest-3-ene, and 3-methylene- 5a-cholestane.Fluorine is introduced at C-3 by diethylamino- sulphur trifluoride Et,NSF,. The 3a-alcohol (9; X = a-OH) gives mainly the mixture of alkenes together with a 14 YOyield of the 3~-fluoro-compound (9; X = P-F) whereas the 3P- 0 OH Reagents i Bu,N'*F; ii LiAlH,; iii HC1. NATURAL PRODUCT REPORTS 1988 alcohol (9; X =P-OH) produces 3a-fluoro-3/3-methyl-5a-cholestane in 98% yield.Stepwise displacement of bromo-substituents on the a,y-dibromo-@-enones (10) provides a convenient route to steroidal amines (Scheme 2).18,19 The initial amination takes place stereospecifically at the 2P-position with elimination of the 6P-bromide ion to produce the 4-bromo-enones (I 1). Subsequent treatment with morpholine and piperazines gives the disubstituted 4-en-3-ones (12). Each 2a,6P-diamine can also be formed directly in high yield at room temperature by using an excess of cyclic amine. Catalytic debromination of 2a,4,6P- tribromocholest-4-en-3-one by hydrogenation over palladium also occurs in stepwise fashion starting with the bromine at C- 6 then that at C-2 and finally the bromine at C-4.20 Competitive dehydration experiments with 4-methyl-stanols that were adsorbed on a shale have illuminated some aspects of the distribution of these compounds relative to their much more abundant demethyl-derivatives in immature sedirnents.,l Degradation of ring A occurs to greatly different extents in immature marine shales as judged by the occurrence of a wide variety of des-A-steroids.22 These include B-aromatic des-A- steroid hydrocarbons of both the 14a- and the 14P-series.Diagenetic isomerization of the 14a-isomers to the more stable 14P-isomers occurs at a rate comparable to that in B-aromatic anthrasteroids. Diethyl carbonate reacts with hydrocortisone and prednisol- one to give the 21-(ethyl carbonates) oestrone and dehydro- epiandrosterone give the 3-(ethyl carbonates) and oestradiol and 3P 17P-dihydroxyandrost- Sene give the corresponding bis-deri~ative.,~ Various xanthates of 5a-cholestan-3P-01 have been used in a study of the mechanism of deoxygenation of secondary alcohols by reduction of their methyl xanthates by tin hydride~.,~ One of these the isopropyl derivative (13) reacts much faster than the 0 I -P=laF OH ...Ill - Scheme 1 0 MeZC=C< CI NMe2 (7) (8) Me I; NATURAL PRODUCT REPORTS 1988-A. B. TURNER R I [77 -92'1.1 Br X' R X' X2 n OAc 5-N-0 Reagents i HX' EtOH at 0-10 "C; ii HX2 no solvent at 30-50 "C. Scheme 2 corresponding methyl xanthate in a competition experiment in which only one equivalent of tributylstannane is used but no cholestane is formed.Instead. the labile tributylstannyl xanthate (14) and propane are obtained as a result of fission of the C-S rather than the C-O bond. Treatment of the tin xanthate (14) with methyl iodide gave the methyl xanthate and reduction with tributylstannane gave cholestane. Tributyltin hydride replaces the halogen atom by hydrogen in 5a-chloro- and 5a-bromo-cholestanes that bear hydroxy- acetoxy- or benzoxy-groups in the 3p-and 4p-or in the 3p-and 6p-po~itions.~~ Derivatives of 4a-chloro-5~-cholestane-3/3,5-diol and of 3a-chloro-5a-cholestan-2/3-ol also give simple reduction products. However radical hydrogenolysis of acet- ates or benzoates of halohydrins in which the ester group is at amount of concentrated sulphuric acid.30 Methylthiomethyl ethers yield the corresponding acetoxymethyl ethers with chlorotrimethylsilane and acetic anhydride.The method is a useful alternative to that in which boron trifluoride etherate and acetic anhydride are used. Long-chain ethers of cholesterol including tritiated cholesteryl oleyl ether are conveniently prepared by the reaction of the mesylates of fatty alcohols with the sodium salt of the sterol in hot toluene containing anhydrous dimethylformamide. 31 Steroidal alcohols are selectively P-glycosylated in high C-5 results in the stereospecific 1,2-migration of the acetoxy- group or the benzoyloxy-group from the tertiary to a secondary carbon,25e.g.6p-chloro(or 6/3-bromo)-5a-cholestane-3~,5-diol diacetate (1 5) gives the 6a-acetoxy-derivative (16) rather than the 5a-acetoxy-derivative. Isotopic labelling shows that the rearrangement involves a 1,2-shift of the ether oxygen of the RS-C-acetoxy-group in the bromo-compound.26 Corticosteroid 2 1 -sulphonates can be prepared in almost quantitative yields by using sulphonyl chlorides in aprotic solvents in the presence of a base.27 Steroid alcohols react with Meldrum's acid and its alkyl and acyl derivatives to give hemimalonates alkyl hemimalonates and acetoacetates.28 Decarboxylation of the hemimalonates yields a variety of esters and hydroxyprogesterone hemimalonate and aceto-acetate undergo base-catalysed cyclodehydration to form spiro-lactones.Dihydrotestosterone and related steroid alco- hols have been acylated with acid chlorides derived from N-succinamic acids.29 1.1.2 Ethers and Esters Cholesteryl methyl and benzyl ethers are cleaved with chloro- trimethylsilane and acetic anhydride containing a catalytic ;I (13) R = CHMe2 (14)R = SnBu NATURAL PRODUCT REPORTS 1988 yields by the oximate orthoester (17) of 3,4,6-tri-O-pivaloyl- CH,OR glucopyranose in the presence of boron trifluoride ethe~ate.~~ I The bis-acetonide (18) is formed selectively from a nona-hydroxynorcholestane that has been found in Acetylation of the bis-acetonide gives the (24R)-6a,24-diacetate. Ethereal hydrogen chloride prepared simply by co-distillation of diethyl ether with concentrated hydrochloric acid is superior to methanolic hydrogen chloride as a reagent for reproducible solvolysis of mono- and di-sulphated bile acids in biological samples.34 1.1.3 Opening of Epoxide Rings The chemistry of steroidal acetoxy-epoxides and halo-epoxides OH I has been reviewed,35 including their formation from enol ace- tates and halo-alkenes their isomerization and the mechanism of rearrangement of enol epoxides and a-halo-epoxides.4-Thioandrost-4-ene-3,17-dione derivatives (1 9) which can be obtained by the addition of various thiols to 4p,5p-epoxyandrostane-3,17-dione,exhibit varying degrees of in- hibitory activity towards oestrogen ~ynthetase.~~ The allylic epoxides (20) which had previously been postulated as intermediates in the oxidation of the cholesta- dienols (21) by per acid^,^' have been isolated from the reaction of the dienols with t-butyl hydroperoxide in the presence of VO(acac) as a catalyst.38 Reductive ring-opening of these epoxycholestenols and subsequent transformations of the product are outlined in Scheme 3.Epoxidation of the cyclodecenone (22) with a peracid gives the secocholestenone epoxide (23) which undergoes stereo- specific intramolecular cyclization with an acid to give the acetal (24).39 The a-epoxide of cholesteryl acetate and the corresponding 3~-chloro-compound each react with nitrosyl chloride to afford the isomeric chlorohydrins (25) and (26) but 3a,4a-epoxy-6-nitrocholest-5-ene gives 3a-chloro-4P-nitrato-6- nitro~holest-5-ene.~~ The controlled introduction of the 15p- hydroxyl group and of side-chains onto the steroid nucleus is achieved by the palladium-catalysed reaction of I ,3-diene monoepoxides (27) with carbon nu~leophiles.~~ 1SP-Hydroxy-cholesterol and 15~-hydroxyisocholesterol were prepared from SR dehydroepiandrosterone (Scheme 4) by this route.Neighbouring hydroxyl groups participate in the alkaline (19) R = [CH2InH (#= 1-51 CH2Ph Ph or p-C,H,X saponification of 3,4a-disubstituted 5,6P-epoxy-4,4-dimethyl-~-homo-5~-cholestanederivative^.^' The 3a- and the 4aa- hydroxyl group participate in the cleavage of the epoxide ring. Involvement of the 3a-hydroxyl group facilitates the formation of transannular 3a,5a-epoxides whereas participation by the 4aa-hydroxyl group gives rise to 4aa,5a-epoxides.The 5(0)" participation by the 3a- hydroxyl group predominates over that of the 4aa-hydroxyl group. 1.2 Unsaturated Compounds I .2.1 Electrophilic Addition Anodic acetamidosulphenylation of steroidal alkenes occurs via oxidation of diary1 and dialkyl disulphides in a~etonitrile,~~ the course of addition being established by relating products to (20)R =H or OH known steroidal disulphides following reductive desulphuriza- tion. Addition of the same disulphides to the alkenes promoted by lead(Iv) manganese(m) and copper(I1) salts in trifluoro- acetic acid gives trifluoroacetoxy-sulphides. The adduct (28) from ergosterol benzoate and 4-phenyl- 1,2,4-triazoline-3,5-dione is oxidized to the glycol (29) in 52 YO yield by potassium permanganate in toluene in the presence of dicyclohexyl- 18-crown-6 ether at room temperat~re.~~ Similar oxidation of the adduct from ergosterol benzoate and 1,4- dihydronaphthalazine- 1,4-dione gives the corresponding 6a,7a-diol in only 14 YOyield.Oxidation involves the a-face of the molecule which appears to be less hindered than the p-face even in these adducts. (21) R =Hor OH NATURAL PRODUCT REPORTS 1988-A. B. TURNER -... I Ill I -HO& HOJ$? HOJ$ OH OH OH I J. I J. HO HO OH OH i iv HO" & OH J I ii v AcOd? OH -.i, I Reagents i rn-CIC,H,CO,H; ii CrO,; iii LiAlH,; iv Ac,O; v A1,0,. Scheme 3 C8Hl7 -, [as (2211 AcO AcO 0 0 (22) (23) NATURAL PRODUCT REPORTS 1988 R@ R@ Cl OH (25) (26) R\ But Me2Si0p C0,Me 1 1.( iii /,ii 11 CO Me 1 x iii iv-vtii 1 H Reagents :i CH,(CO,Me), PdJTBAA), P(OCH,),CEt ;ii NaI HMPA ;iii H, Pt ;iv EtOCH=CH,; v Bu',AlH ;vi CrO ;vii Rh(PPh,),CI ; viii TsOH ;ix Me,CHCH,C(O)CH,CO,Me Pd,(TBAA), P(OCH,),CEt ;x NH,NH, (CH,OH), OH-.Scheme 4 1.2.2 Other Reactions of OleJinic and Aromatic Steroids Depending upon the reaction conditions norethisterone and its ketal give either (a-or (2)-alkenes (30) and the (a-alkene (3 l).45Further reaction with N-bromo- or N-iodo-succinimide leads to the (a-and (2)-vinyl halides (32).Radiolabelled (2)-17a-(2-iodovinyl)nortestosterone was obtained by the reaction of the (2)-alkene (31) with labelled sodium iodide.Treatment of cholesterol with diethyl phosphate diphenylphosphine oxide or hypophosphorous acid in the presence of benzoyl peroxide or di-t-butyl peroxide and acetic acid gives products of hydrophosphorylation of the 5-6 d~uble-bond.~~ The reaction leads mainly to phosphorylation at C-5. In studies modelling aromatization and isomerization of hydrocarbons in sedimentary rocks the rate of aromatization of the c-aromatic methylnorcholestatriene (33) to the methyl- dinorcholestaheptaene (34) is found to be more dependent on temperature than is the rate of isomerization of certain NATURAL PRODUCT REPORTS 1988-A. B. TURNER 317 pristanes under conditions that favour free-radical reaction^.^' Hydrogenation of the D-homo-oestrapentaene methyl ethers (35) over palladium charcoal in ethanolic tetrahydrofuran gives 9-iso-oestrones (36) in 9-14% yield in addition to 8-iso- oes trones (37).48 1.3 Carbonyl Compounds I .3.1 Reduction and Dehydrogenation Reduction of diones and triones that are adsorbed on silica gel occurs regioselectively with borane-trimethylamine complex.49 (33) Carbonyl groups at C-3 of the steroids were attacked in preference to carbonyl groups in rings B c or D.Catalytic hydrogenation of the (22R)-unsaturated lactone (38) and its (22S)-epimer gives the (22R,24R)-lactone (39) and its (22S,24S)- 1 isomer respecti~ely.~~ The stereochemistry at C-24 of the reduced product was established by conversion into 24-ethyl- cholesterols.\/ Me0 0 &” O\ Ph (28) (35) R = Me or Et A” t as (36) O\ Ph (29) 04L-u NATURAL PRODUCT REPORTS 1988 60 YOyield) the lactol (43) which had previously been obtained 1.3.2 Other Reactions by using oxygen in the presence of potassium t-butoxide. The Details have appeared of the cleavage of a-azido-oximes to 4a-methyl-3-ketone (44)gives the 0x0-acids (45) and (46) mono- and di-cyano-compounds under Beckmann conditions. 51 which were isolated as their methyl esters in 19% and 20% 2-Azido-5a-cholest- 1 -en-3-one (41) which is a minor product yields respectively. 5P-Spirostan-3-one (47) gives the keto-ester oxime (40) probably arises (48) as the major product (45% yield). The oxidation of the from 2a-azido-5a-cholestan-3-one by a ring-opening/re-cyclization sequence (Scheme 5).51 Oxida- ketone (44)has been rationalized by the mechanism outlined in tion of 3P-acetoxycholest-5-en-7-oneoxime with perbenzoic Scheme 6.3P-acetoxy-6-oxa-The reaction of benzeneselenyl bromide with 3-keto-5a- acid gave 3~-acetoxy-7P-nitrocholest-5-ene cholest-4-en-7-one and 31-hydroxycholest- 5-en-7-one xim me.^^ steroids in ethyl acetate leads to 2a-bromo-3-ketones whereas In the oxidation of different types of 3-keto-steroids with 5P-steroid 3-ketones undergo 4P-br0mination.~~ 19-Nor-5P-potassium superoxide in the presence of 18-crown-6 the steroid 3-ketones give the 2P-bromo-derivatives. Use of products are directly related to the direction of enolization in benzeneselenyl chloride results in the formation of a-phenyl- the Thus oxidation of 5a-cholestan-3-one gives selenenyl ketones.Bromination of SP-oestrane-3,17-dione by 7-dione7 the lactol(42) in 28 YOyield and 5a-lanost-8-en-3-one gives (in bromine in acetic acid gives 2a-bromo-5P-oestrane-3,l N N Ill 111 N+ NH I I /I H H Reagent i H,O. Scheme 5 HO,C 794411 0 0 OH (42) (43) (45) Hwy44)l H0,C 0 H (46 1 (47) (48) NATURAL PRODUCT REPORTS 1988-A. B. TURNER and not the 4P-bromo-derivative that had previously been Dehydrobromination of the product yields 5P-oestr- l-ene-3,17-dione (5YO)and oestr-4-ene-3,17-dione (66 YO),the latter by a mechanism involving loss of the 5P-proton and the 2a-bromide ion from the thermodynamically favoured 3-enol.Bromination of 5a-cholestane-3,6-dione with increasing amounts of bromine in acetic acid gives cholest-4-ene-3,6-dione then its 7a-monobromo-derivative and finally 2a,7a-dibromo- cholest-4-ene- 3,6-dione .56 Steroid ketols can react with proteins both in vivo and in vitro to form covalent adducts by a non-enzymic process that involves rearrangement of the initial Schiff-base adduct. Model adducts can be prepared by the reaction of cortisol or 1601-hydroxyoestrone with lysine derivatives in the presence of sodium cyan~borohydride.~' The product (49) of the latter reaction which can be isolated as an acetylated derivative is a reduced Schiff base. A stable cortisol-lysine adduct (50) is similarly obtained but in this case addition of the cyanide ion occurs during the condensation..L 1 (441 HO '0Jp oq H Reagents i 02'; ii H,O+. I .3.3 Reactions of up-Unsaturated Carbonyl Compounds and Enols or Enolic Derivatives The initial product of the base-catalysed bromination of cholest-4-en-3-one is 4cc,5a-dibromocholestan-3-one.5s Subse-quent treatment with N-bromosuccinimide gives 2a,4,6P-tri- bromo- and 2,2,4,6~-tetrabromo-cholest-4-en-3-one.5s Aceto-xylation of 4-en-3-ones at position 2 is achieved in 71-74% yield if lead tetra-acetate is used in boiling dry benzene with a nitrogen atmosphere.'O The P-epimer predominates in the chromatographically separable product mixtures from andro- stenedione progesterone and testosterone acetate. Methoxyl- ation of 4-en-3-ones with o-iodosylbenzoic acid in methanolic potassium hydroxide occurs at C-4 and C-6P (Scheme 7).s1 Conjugate addition of lithiated acrylacetonitriles or of cyanohydrin ethers RCH(CN)OCH(Me)OCH,Me to 4,6-dien- 3-ones requires anionic activation since the yields are higher in tetrahydrofuran-hexamethylphosphoramide than in pure tetra- hydrofuran.A mixture of stereoisomers at C-7 is formed with HO4Q -0 Jp Scheme 6 0 Reagents;i o-HOIC,H,CO,H KOH MeOH. Scheme 7 Scheme 8 NATURAL PRODUCT REPORTS 1988-A. B. TURNER 32 I OH 0& 0dP (57 1 (59) 0 It X,P -LO R' Reagents LiNPr', THF at -78 "C; ii ClP(O)X, heat from -78 "C to room temperature; iii Li liq. NH, Pr'OH THF at -35 "C; iv HCIO, H,O dioxane.Scheme 9 OC(0)Ph OH Ill -[ 86*lo overall ] Me0 'P ( OEtl2 II 0 Reagents i LiNPr', THF at -78 "C;ii ClP(O)(OEt), heat from -78 "C to room temperature; iii Li liq. NH, ButOH THF at -35 "C. Scheme 10 imino-steroids which are readily hydrolysed to ketones (Scheme I 11.70 Bis-steroid derivatives of p-xylene [of the type (60)] catalyse the hydrolysis of arylpropionate esters in which there are good leaving groups.71 Acylation is markedly accelerated by hydro- phobic binding of the aryl group of the substrate to the steroids. Rate enhancements relative to imidazole of up to 5.5 x lo2 are found and the potential rate enhancements are much higher. The preparative route for the dimers (60) involves reductive amination (using a cyanoborohydride) of tereph- thalaldehyde with 3-amino-steroids.An amino-group in the 1 lp-position can be blocked by means of a formyl group which is removable by acid hydrolysis after dimerization has occurred. The trifluoroacetyl group although cleaved from simple steroids by acid hydrolysis is not suitable as a blocking group as it is only removed from the dimer under forcing conditions which cause degradation. Although the dimer hydrochloride is very soluble in water there are still difficulties caused by aggregation of the substrates and/or catalyst plus substrate leading to spuriously low rates of hydrolysis. CR H 17 NATURAL PRODUCT REPORTS. 1988 C-Terminal protecting groups that contain the cholestanol moiety are promising tools in the synthesis and conformational analysis of peptides since they enhance solubilities of peptides in organic solvents of low polarity.Studies of fully protected oligo-L-lysines with C-terminal (cholestanyloxycarbony1)benzyl ester groups show that the cholestanyl-containing peptides have a lesser tendency to self-aggregation than the benzyl ester analogue^.?^ Several peptidosteroids (61) derived from funtu- mine have been prepared from t-butoxycarbonylated amino acids via coupling with dicyclohexylcarbodi-imide.73 The reaction of cholesterol or of p-sitosterol with I ,3-butyl-ene phosphite diethylamide gives the corresponding phosphites [e.g. (62)].74 These have been converted into their phosphate and thiophosphate derivatives. Thiol esters of I7P-carboxylic acids derived from 16 I7a-disubstituted corticosteroids have been prepared by using a variety of activation procedure^.^^ These include the use of diethyl phosphate mixed anhydrides of diphenyl chlorophosphate as an activating agent and acti- vation by carbonyldi-imidazole.The I7a-acetoxy-acids react with 2-fluoro-N-methylpyridinium tosylate to give novel spiro- Me i or ii :@ ____) / NHCN ' 12 - iv 0 N\ CN Reagents i BrCN Et,O at -30 "C; ii EtOH AcOH NaOCN reflux then MeSO,Cl pyridine at 0 "C; iii Pb(OAc), cyclohexane at room temperature; iv PhH A1,0, at room temperature. Scheme 11 H Q 6 X' H (611 X = -Gly -Ala -Met -Cys-NHEt -Ala-Gly -Met-Gly,or -Cys-Gly H (60) NATURAL PRODUCT REPORTS 1988-A.B. TURNER ketals e.g. (63) whereas 17-hydroxy-acids give products of dehydration or of migration of the methyl group C-18 including the ,&lactones (64). Conjugate addition of S,S-diphenylsulphilimine to the 16- en-20-ones (65) gives the ylides (66) which can be converted into the 16a-amino-derivatives (67) by acetylation (Scheme 12).76 Attempts to achieve 21-hydroxylation of the 16a-acetamidopregnene (68) by using iodosobenzene diacetate in methanolic potassium hydroxide led only to the 17-spiro-oxetanone (69).77 (62) 1.5 Molecular Rearrangements Solvolysis of 17a-tosyloxyandrostane and of 18-norandrostane in hexafluoroisopropyl alcohol is faster than that of the 17p- isomers by a factor of > lo4 and much faster than the solvolysis of cyclopentyl tosylate or of cyclohexyl tosylate.’* Both epimers give the same products of 1,Zmethyl migration.In 97% sulphuric acid at room temperature 1 1 -deoxycorticosterone (70) forms two rearranged products (71) and (72) which have been isolated in yields of 46 YOand 32 % re~pectively.~’ After (64) I- l$,,iyiPh2 A 0 (65)R = H or Ac (661 (67) Reagents i Ph,S=NH PhMe 6 kbar at 20 “C for 24 hours; ii Ac,O pyridine. Scheme 12 C(0IMe HO&-:NHAc (681 0dP 0 0 (70) (71) (72) NATURAL PRODUCT REPORTS 1988 Y OH (74) (75) (76) (7 7) (78) Reagents i MeMgI PhMe; ii MeSO,Cl DMF collidine SO,. Scheme 13 heating the acid solution at 60 "C and then diluting it with ethanol the tetraenone (73) is obtained in 13% yield.Acetolysis of the 19-mesylate (74) leads to the spirocyclic ketone (75) via tandem migrations of two antiparallel skeletal carbon-carbon bonds,ao thereby illustrating the importance of stereoelectronic control in the Wagner-Meerwein rearrange-ment. The 501-and 5P-hydroxy-acetals (76) react with methyl- magnesium iodide in boiling toluene to give the A-homo-B- norcholestane (77) in yields of 93 YOand 63 YO,respectively.81 The rearranged product itself rearranges when it is treated with collidine and methylsulphonyl chloride in dimethylformamide that is saturated with sulphur dioxide to give the 5a-methyl-6- ketone (78) providing a convenient new method for introducing the 5a-methyl group into the steroid nucleus (Scheme 13).1.6 Remote Functionalization Reactions The nicotinate ester of 5a-cholestan-3a-01 can be chlorinated at C-9 by the di-imine of rn-iodobenzylamine and glyoxal using iodosobenzene dichloride in the presence of nickel(I1). Catalytic directed turnovers of lo9 substrate molecules per template are reported* to have been achieved.82 This can be increased to almost lo1 by using a thioxanthone that contains a metal- binding pyridine-2-carboxaldiminegroup in the presence of nickel(1r) or copper(r~).~~ With cortexolone acetate or with 3~-methyl-5a-cholestan-3a-o1 the metal complex also carried out chlorination at C-9 by binding to the hydroxyl group with 1O2 turnovers. These template-directed chlorinations occur at concentrations of the complexing agent of 10-l' to mol dm-3 but no bimolecular reactions are observed with uncom- plexed analogues even at lo-' mol dm-3.The effective molarities that are implied by this are not possible for a reaction that is promoted only by complexing. Instead both the metal and the template must be involved in bifunctional catalysis. Also the rates are so fast that chain-carrying steps cannot involve unbound free-radical intermediates. A mechanism has been proposeda4 in which the template carrying a chlorine atom on iodine or on a sulphur atom is complexed to the substrate and performs a geometrically controlled abstraction of a hydrogen * The validity of these results has been questioned and the senior author (Breslow) has indicated that refs.82 83 and 84 should be withdrawn. See Chem. Eng. News 1986 64,No. 49 pp. 2 and 6 and ibid. 1987 65 No. 15 p. 10. atom. The substrate radical is then chlorinated by copper(I1) or nickel(I1) chloride and the resulting template-metal(1) complex is finally doubly chlorinated by iodobenzene dichloride in a concerted reaction which restores the chlorine atom to the template and at the same time regenerates metal(1r) chloride. Remote chlorination can be used in two ways to convert the side-chain of cholesterol into a 17-acetyl group.a5 In one template-directed chlorination at C-20 using iodobenzene dichloride of the cholestane (79) which has an aryl ester attached to the 6P-positior1 is used to convert i-cholesterol into pregnenolone in 15% isolated yield.In the other the 17-20 double-bond is extended to a 16,20(22)-diene system by using N-bromosuccinimide (treated with osmium tetra-oxide) to give mainly the 16a,17a-diol which is converted into the acetonide and then cleaved with ruthenium chloride and sodium periodate to give the 20-oxopregnane in 43% overall yield. 1.7 Photochemical Reactions Stereospecific photoisomerization of 5a-and SP-hydroxy-6- 0x0-steroids has been ascribed to inhibition of rotation about the 9-1 0 bond.86 Their bicyclic analogues give identical mixtures of lactones owing to equilibration of intermediate acyl alkyl diradicals. Photolysis of the lactols (80) in the presence of iodosobenzene diacetate and iodine produces alkoxyl radicals which undergo P-fragmentation to form the medium-ring lactones (81) and (82) in good yield^.^' Direct irradiation (300 nm) of the ketol(83) in hexane gives the B-seco-steroid (84) and the indene (85).88 The 5a-and 5P-hydroxy-3-ketones (86) are the major and minor products respectively of the irradiation (280-320 nm) of norethisterone (87 ;R1= H R2= C =CH).sg The 5P-alcohol is also obtained by reduction of norethisterone epoxide with aluminium amalgam in isopropyl alcohol.The photochemistry of the oestrenones (87; R' = R2= H) and (87; R1= Me R2 = C=CH) is solvent-dependent giving the oestrenones (88) in ethyl acetate or the oestranones (89) in alcoholic ~~Ivents.~~ Further irradiation of the 5a-steroid 3-ketones (89) in alcoholic solvents yields the seco-steroids (90)-(92).Photolysis of 17p- acetoxyoestra-4,9-dien-3-onegives the photodimer (93) but only polymeric material is obtained from 17P-hydroxyoestra- 4,9,1 l-trien-3-0ne.~~ NATURAL PRODUCT REPORTS 1988-A. B. TURNER H OH (80)n =1 or 2 @Ho 0 c8 H1 7@;I; (83) ( 84) R’&‘HZ #,R2 CH2CH0 0 0 OH (85) (86) (87) H H H (88) (89) (90) (91) OAc 0 0 OAc (93) 0 (95) The proportions of previtamin D [(6Z)-tacalciol] tachy- sterol [ertacalciol] and lumisterol that are formed in the triplet-sensitized photoisomerization of provitamin D have been calculated as a function of irradiation ~avelength.~’ The percentage of previtamin D reached a maximum at 296.5 nm using fluorenone as sensitizer.The excited-state proton-transfer process for equilenin and dihydroequilenin depends upon pH and the concentration of proton acceptor.g2 Both oestrogens adsorb to dimyristoyl-lecithin vesicles and rates for excited- state proton transfer are greatly reduced when dihydroequilenin is adsorbed. The accessibility of the bound probe to acetate as a proton acceptor depends on the cholesterol content of the bilayer vesicles. The diazoacetate (94) and the diazirines (95; n = 1) and (95; n = 3) containing carbene-generating substituents in the side- chain give the products of C-H or 0-H insertion upon photolysis in cyclohexane or in methanol.93 Reactions of oxygen with A5-and with A5*?-3P-acetates can be photoinduced by phenylselenenyl bromide. 94 Thus 3/3- acetoxycholesta-5,7-dienegives the corresponding endoperox- ide and cholesteryl acetate gives 3~-acetoxycholest-5-en-7-one and the corresponding alcohols.Bromine radicals arising by photodissociation of the selenium reagent appear to be responsible. Prolonged irradiation of an acetone solution of diosgenin by ultraviolet light affords the 5P,6P-epoxide in I5 YO yield.95 2 Partial Syntheses Asymmetric syntheses using organometallic reagents and their application to steroid side-chains including those of brassinolide and a-ecdysone have been reviewed. g6 2.1 Derivatives and Analogues of Cholestane (25S)-26-Deuteriocholesterol (96; R = D) has been prepared from diosgenin via the hydroxy-derivative (96; R = OH) the configuration of which was established by X-ray-crystallo- graphic analy~is.’~ Tritium-labelled 7,8-didehydrocholesterol epoxide (97) of high specific activity was prepared from the 7a-bromo-compound (98).This was first oxidized to the 3-ketone which was reduced by sodium borotritiide in a special buffer-organic solvent system (to minimize side-reactions) and finally treated with a base to eliminate hydrogen bromide.98 Cholestanols (99; R = C3H,) were prepared from 5a-cholest- 1-en-3-one by alkylation with methyl iodide or its tritiated analogue followed by hydrogenation and reduction with lithium aluminium h~dride.~~ Carbon- 14-labelled p-sitosterol NATURAL PRODUCT REPORTS 1988 has been prepared from unlabelled sitosterol in six steps,loo including the reaction of ‘*C-labelled methylmagnesium iodide with the oxa-steroid (100).Three pairs of enantiomerically pure bromo-alkanes cor- responding to the side-chains of six 24-alkyl-sterols in both enantiomeric forms can be prepared from the same chiral intermediate (R)-5-acyloxy-3-(1-methylethyl)pentan-1-01 which is readily available from (+)-(R)-l‘ imonene (Scheme 14).lo1 19-Norcholesta- 1(10),5-dienyl acetate (101) is obtained in 48 YO yield from 19-hydroxycholesteryl acetate (102) by treatment with lead tetra-acetate and cupric acetate in boiling toluene.lo2 5a-Cyanocholestanone and various 3-substituted derivatives (105) were obtainedlo3 from the nitro-cyanides (103) [by reduction with zinc dust in diethyl ether-methanol that contained ammonia solution (sp.gr.0.91) to form the oximes (1 04) followed by hydrolysis with hydrogen bromide in dioxan] in overall yields of 56-64 YO(Scheme 15). Further 5a-cyanocholestanes are formed by attack of cyanide ion upon 6-nitrocholesta-3,5-diene (106) and the formation of the oxime (107) as the major product (Scheme 16) indicates that the nitro-group cannot remain unaffected during the conjugate addition of cyanide ion to a nitro-alkene in which there is ex tended conj~gation.’~~ The configuration of the 24,25-dihydroxycholesterols(108) was established by their stereocontrolled synthesis from the iodo-lactones (109). Ring-cleavage gave the epoxide (1 lo) (96) HO T (97) (98) HoJyw I H k (99) (1001 AcOJ-d-Q (101) (1 021 NATURAL PRODUCT REPORTS.1988-A. B. TURNER -( +) -( R)-L i monene .-+ ____) OH OCPh OH OH OAc 1 i ii xi xii viii HOAOR 4 (R = COPh) XI 11 ix x iii ix viii v Ac0ABr \ Br (S) Br r" HOK Br (R) Reagents i 0,;ii NaBH,; iii Ph,CCI pyridine; iv pyridinium dichromate CH,Cl,; v TsOH MeOH; vi Ac,O or PhC(O)CI pyridine; vii TsOH Ac,O; viii CBr, PPh, Et,O; ix KOH MeOH; x Wilkinson catalyst CH,Cl,; xi TsC1 pyridine; xii LiAlH,; xiii TsOH dihydropyran; xiv H,O+. Scheme 14 which by oxidation and hydrolysis led to the diols (1O8).lo5 Marine sterols of the type (113) can be prepared from the acetylene (1 11) via the selenosulphonate (1 12) as outlined in Scheme 17.1°6 Selenocuprates being more effective than other cuprates for replacement of the selenophenyl group by an alkyl group were used in the alkylation of the intermediate adduct (1 12).A route from pregnenolone to 2-deoxycrustecdysone (116) has as its key reaction the stereoselective reduction of the lactone (1 14) to the y-butyrolactone (1 15) in which there is a (20R,22R)-20,22-diol system.1o7 Several derivatives of makister- one A are also available by applying the same sequence (Scheme 18). An improved large-scale synthesis of the 14a- methylcholest-7-en- 15-one (1 17) and related ergostenone and 22-hydroxymethylpregnane derivatives involves acid-catalysed rearrangement of the vinyl epoxide (1 18) in ethanol rather than This simple change more than doubles the NATURAL PRODUCT REPORTS 1988 (103) (104) (105) Scheme 15 No2 (106) NC,* Me0 + + NC@ NC N‘OH NC 1 NO2 ‘OH (107) ._ ...I;ii II Ill @ NC @ NC 0 NC Me0 iv 1 liv Reagents i KCN MeOH; ii NH, Zn MeOH; iii HBr dioxane; iv HN, BF, PhH. Scheme 16 NATURAL PRODUCT REPORTS 1988-A. B. TURNER CIMc OH I HoJ3FH (108) I OMe (113) 0;R'= R2 = H b; R'=Me RZ = H c; R'= R2 =Me Reagents i excess LiCH,CECSiMe, THF HMPA; ii Bu,N+F- THF; iii p-MeC,H,SO,SePh AIBN PhH; iv RCu(SePh)Li THF; v sodium amalgam Na,HPO, THF MeOH; vi TsOH aq. dioxane. Scheme 17 isolated yields of the 8(14)-en-I 5-ones by avoiding by- products resulting from solvent addition. The subsequent methylation at C-14 is carried out by using the ethoxyethyl ether protecting group for the 3P-alcohol.The synthesis of the deoxyvernolepin analogue (1 19) from cholesterol makes use of a new reaction for the preparation of dimethyl acetals using pyridinium chlorochromate and methan01.'~~ Acid-sensitive functional groups such as oxirane rings are not affected under these conditions. 6-Deoxohomodolichosterone (1 20) a new plant growth promoter has been synthesized in twelve steps from stigma- sterol. The labelled crinosterol(l2 1) has been prepared from the bisnorcholanal(l22) by addition of lithium acetylide to give a 2 1 diastereoisomeric mixture of separable 22-alcohols (123) the configurations of which were determined by chemical correlation with the known methyl analogues."' Protection of the alcohol by silylation lithiation and alkylation with CD,I introduced the first CD group; partial hydrogenation with Lindlar catalyst followed by orthoester Claisen rearrangement and reduction of the resulting ester with LiAlD, introduced the second.Final treatment with an acid gave the sterol (121) having a deuterium content of 98% in an overall yield of 50 yo. The key feature of a stereoselective synthesis of brassinolide (124) is the stereoselective reduction of the tetronate (125) to control the stereochemistry of the four contiguous chiral centres of the side-chain in a single The reaction which determines the stereochemistry is carried out in the cyclic system. Addition of the dianion of the tetronate to the 20-oxopregnane gives the (2)-isomer of (125) by syn-dehydration.An aza-analogue (126) of homobrassinolide has been prepared from 5a-stigmasta-2,22-dien-6-one.ll3 Osmylation of the enol silyl ether (127) of this dienone gives the pentahydroxy-ketone (128); this by oxidation of the a-ketol group (after protection of the glycolic systems) affords a B-seco aldehyde ester which eventually allows the formation of the lactam (126) by reductive amination. Attempts to prepare this type of compound by Beckmann rearrangement of A4-6-hydroxyimino-steroids NATURAL PRODUCT REPORTS 1988 ++ I OH HO Bu Me2Si0 0 1 KO$ - IV v , I I I I Oil OH OH (115) (114) \ VI vii HO 0 (116) Reagents i 2-lithiofuran THF; ii rn-CIC,H,CO,H NaOAc CHCl,; iii CrO, pyridine; iv H, Pt,EtOAc; v aq.NaOH; vi MeMgBr THF; vii AcOH. Scheme 18 PhC(0)O 0 H H (119) (120 1 NATURAL PRODUCT REPORTS 1988-A. B. TURNER 33 1 (129) gave solely the products of vinyl migration.'14 3p- Baeyer-Villiger oxidation of 3~-bromo-5a-cholestan-Qone Hydroxy- and 3~-acetoxy-compounds undergo elimination of followed by dehydrobromination and treatment with osmium these substituents to give lactams (130). Various catalysts were tetroxide and N-methylmorpholine N-oxide. Their growth- employed the best of which was trimethylsilyl polyphosphate. promoting activity in the bean bioassay is appreciably lower B-Homo-oxacholestanones (1 3 1) and (1 32) are obtained by than that of 24-epi-bra~sinolide.''~ OH HO OMe (121) (122 1 OH OMe OSiMe3 ( 127) (128) HOP R& 0 (129) (131) The absolute configuration of C-24 of SP-ranol (133) this being a principal bile alcohol of the bullfrog which is structurally related to a major human urinary bile alcohol has been shown to be In the cholestenetetraols (134) which can be prepared from cholic acid by condensation of vinylmagnesium bromide with the 24-aldehyde the stereochemistry at C-24 was determined by means of the difference in reactivity towards the stereoselective Sharpless epoxidation.Catalytic hydrogenation of the terminal alkene (134) gives the tetraols (135) which can also be prepared from norcholestanic acid by a Kolb6 electrolytic coupling with acetic acid. Reduction of the norcholestanic acid also provided 5P-ran01 (1 33).2.2 Vitamins D their Derivatives and their Metabolites The abstracts of the 6th Vitamins D workshop have ap- peared.'l' In a general method for the synthesis of the principal calciol metabolite (1 36) the bicyclic ketone (1 37) is obtained by coupling of the iodide (1 38) with 3-(trimethylsilyl)-but-3-en-2-one via organocuprate chemistry. 11* The preparation of 25- oxocalciol which is a suitable intermediate for radiolabelling can be achieved by coupling the carbanion of the phosphine oxide (1 39) using Lythgoe's strategy. An efficient fourteen-step synthesis of 25-hydroxyvitamin D [25-hydroxyercalciol] (140) from vitamin D [ercalciol] makes use of organocuprate methylation of the allylic carbamate (141) to control the chiral centre at C-24 and a Horner-Wittig coupling to assemble the triene system (Scheme 19).llg Efficient procedures for the protection and deprotection of this triene system involve the use of the phthalhydrazido-derivative (142) from vitamin D acetate and phthalazine- ly4-dione (Scheme 2O).l2O Stereo- selective synthesis of (24R)-la,24,25-trihydroxycholesterol from la-hydroxydehydroepiandrosteroneinvolves control of the configurations at C-20 (R) and C-17 (R) by conjugate addition of lithium dimethylcuprate to the sulphonylallene (143) followed by reduction of the 17-20 double-bond (Scheme 21)? Kinetic studies suggest that there is a mechanistic similarity between the allenic and non-allenic variants of the thermal [1,5]-sigmatropic hydrogen shift in vitamin-D-type vinylallenes (144) and simpler analogues.'22 Soholytic reactions of B-thiophene-des-A-cholestanes(145) lead to the bridged ethers (146) amongst other OH NATURAL PRODUCT REPORTS 1988 OH (135) HO" a? (136) R = H Or OH 0 (137) (138) NATURAL PRODUCT REPORTS 1988-A.B. TURNER .. ... iv ___) 1 @OTs @“O OH 0C(0)Ph v -vii 1 OH OC(0)NH Ph CMe,OMOM ... VIII ix 4 I A OC(0)Ph OC(0)Ph x-xii 1 0 + O%lZ I (MOM = CH20Me) Reagents:i 0,,MeOH pyridine then NaBH ;ii TsC1 pyridine; iii PhC(O)Cl 4-dimethylaminopyridine pyridine ;iv Me,SO s-collidine ;v LiC CCMe,OCH,OMe THF ;vi pyridinium dichromate CH,Cl,; vii LiAlH, (-)-N-methylephedrine 3,5-dimethylphenol Et,O; viii H, Pd/BaSO, quinoline then resolution ;ix PhNCO pyridine 4-dimethylaminopyridine ;x Li,Cu,Me, Et,O ;xi LiAlH, Et,O ;xii pyridinium dichromate pyridinium toluene-p-sulphonate CH,Cl ;xiii BuLi THF ;xiv AG-50WX4.Scheme 19 25-Hydroxycholesterol has been prepared from lithocholic acid via condensation of 2-methyl- 1,3-dithiane with the 24- bromocholene (147). lZ4The synthesis of the 20-oxa-2 1-norcal-ciol analogue (148) in twelve steps from dehydroepiandroster- one involves alkylation of the protected androstenetriol (149).lZ5A convenient route from the norcholestene (1 50) to the calciol precursor (1 51) involves ozonolysis of the oxime acetate hydrolysis and final fragmentation with phosphenyl chloride in pyridine.126 The overall yield approaches 33 YO.Oxidation of calciol acetate with selenium dioxide and an excess of t-butyl hydroperoxide gives a mixture of products including a dimer (1 52) of (5E)-l-oxocalciol acetate.12’ The ketone (1 53) which is obtained from the ozonolysis of calciol reacts with lithiated 1,3-bis(trimethylsilyi)propyne to give mainly the (2)-enynes (154).128 The amount of this isomer was under-estimated by molecular-mechanics calculations and the discrepancy was not resolved by an investigation of possible conformers of the hydrindanone system. A simple route to Lythgoe-type dienynes is based upon palladium-catalysed coupling of the kinetic enol triflate from Grundmann’s ketone and acetylenic compounds that contain the ring A fragment.129 Selenium(1v) reagents in particular buffered selenious acid bring about 1a-hydroxyl-ation of trialkylsilyl ethers of (5E)-ercalciols leading to a con- venient synthesis of (1 s)-1-hydroxycalciol (1 s>-1-hydroxy-ercalciol and (1 s)-1,25-dihydroxy~alciol.~~~ NATURAL PRODUCT REPORTS 1988 I 0 0 (142) Ratio 6n-H 60~-H= 2:l iv (140) -Reagents:i Pb(OAc), CH,Cl,; ii NH,NH, EtOH ;iii (p-MeOC,H,),Te=O BrCl,CCCl,Br CH,Cl, aq.K,CO,; iv hv anthracene Et,N PhH. Scheme 20 2.3 Cholanes Norchoianes and Dinorcholanes The remaining three stereoisomers (155; 701 12P) (155; 78 12a) and (155; 78 128) of the 5a-series of 7,12-dihydroxy- cholanoic acids have been prepared.l3l The main reactions that were employed were selective acylation at C-7 of a 7a- dihydroxy-ester with the propanoic anhydride/4-(dimethyl- amino)pyridine system inversion of the 7a-hydroxyl group (using potassium superoxide and 18-crown-6 ether) and stereoselective reduction of the 12-ketones with sodium borohydride/palladium chloride and t-butylamine-borane complex.Syntheses of stereoisomeric 3,7-dihydroxy-5a-chola- noic acids involve similar displacements of a variety of leaving gr0~ps.l~~ The complete set of eight stereoisomers of 3,7,12- trihydroxy-5a-cholanoic acids five of which are new has been prepared by reduction of the four possible methyl 3,7-dihydroxy-12-oxo-5a-cholanoates.133 This completes the litera- ture record of the characterization of the entire set of fifty-two possible stereoisomers (twelve monohydroxy twenty-four dihydroxy and sixteen trihydroxy) of the 5P- and the 5a-series.7-Methyl-5P-cholanoic acids including 7P-methylcholic acid and a 7-methylenecholanoic acid were prepared by Grignard reaction of the bile acid oxa~olines.~~~ A general procedure for introducing 13Cinto the side-chain of bile acids involves an ene reaction between (2)-pregn- 17(20)-enes and methyl [1,2,3-13C,!propiolate.135A degradation of lithocholic acid to 24-norl~thocholic acid (1 56) involves a photochemical modi- fication of a Hunsdiecker reaction and a Kornblum oxidation of the intermediate 23-br0mide.l~~ @-Unsaturated bile acids have been prepared by conversion of bis-norcholanols followed by condensation with the Wittig reagent Ph3P= CHC0,Me.13' Inhibitors of microbial degrada- tion of sterol side-chains including the 22-yne (157) and 23- halogenated bile acids have been prepared from cholanes and norcholanes (Scheme 22).138 The titanium-tetrachloride-medi-ated alkynylation of the C-22 aldehyde (1 58) with stannylacetyl- enes occurs with high diastereoselectivity (Scheme 23).139 This selectivity also occurs in the condensations with allylstannanes and allylsilanes under similar conditions.Acetals of the aldehyde (1 58) are useful for controlling the stereochemistry in construction of ~ide-chains.'~~ Thus the acetal(l59) reacts with allyl-silanes -boranes or -stannanes in the presence of titanium tetrachloride to give the alcohol (160) as the predominant product whereas the isomeric alcohol (162) is the main product from the reaction of the acetal(l61) with allyltributylstannane.The direction of asymmetric induction depends upon the nucleophilici ty of the organometallic reagents. The evidence is that bond making and bond breaking are concerted for the high asymmetric induction that is obtained with acetal templates. Brassinosteroids (163) comprising a wide range of acid derivatives prepared by esterification or amination of the acid chloride show growth-promoting activity on a par with that of 24-epi- brassinolide. 141 NATURAL PRODUCT REPORTS 1988-A. B. TURNER I I1 _____) HO VI,I VII SiMe 0 II PhS 6 phs$ xiii xiv J OMe YO ?I OMe OAc xv iii -xx/ Reagents i TsCI pyridine; ii HOCH,CH,OH TsOH PhH; iii KOAc MeOH; iv TsOH MeOH; v MeOCH,CI EtNPr’,; vi LEECH THF; vii PhS(O)CI pyridine; viii PhCI Li,CO,; ix MeLi THF; x Me,SiCI; xi LiCuMe ; xii Bu,NF EtOH ;xiii TsOCH,CH(OH)C(OH)Me, HNPr’, THF.xiv BuLi; xv Me,C(OMe), TsOH; xvi Li EtNH,; xvii H, Pt/C EtOAc; xviii H,SO, aq. MeC(0)Me; xix H,SO, MeOH; xx Ac,O pyridine. Scheme 21 NATURAL PRODUCT REPORTS 1988 H Ac 0boTs (144) Br (147) (148) (149) Thp = tetrahydropyran -2-y C8H17 AcO&0 (152) R = (150) (1511 -CH C8H17@0 \Si Me3 1153) (1 54) H (155) NATURAL PRODUCT REPORTS 1988-A. B. TURNER 331 OMe I Br ii,iii -(64.1,) ( 73 O/O) L6c02H OMe Reagents i BuLi CO,; ii TsOH aq. dioxane; iii CH,N, Et,O. Scheme 22 p" i Bu'Me,SiO dCHO L (158 1 (R = Pr',Bu or Ph) Reagents i Bu,SnC-CR TiCl, CH,Cl,.Scheme 23 [as (16411 {so." Norcholane 23-selenocyanates are readily obtained by irradiation of mixed anhydrides of cholic acids and a 2.4 PregnanB thiohydroxamic acid with visible light in the presence of [2,3]-Wittig rearrangement of the dianion derived from the (9-triselenium cyanide.142 Irradiation of similar mixed anhydrides pregn- 17(20)-ene (1 64) constitutes an efficient approach to 22- from bisnorcholic acids in the presence of dimethyl diselenide hydroxylated steroid ~ide-chains.'~ The rearrangement of gives 20-methylselenopregnanes. (1 64) to the (20S,22S)-hydroxy-acid (165) which can be isolated as the methyl ester in 85% yield takes place within 2 hours at -56 "C.The ester is readily converted into other 22- hydroxy-steroids e.g (22R)-22,25-dihydroxycholesterol,via ad- dition of Grignard reagents to 22,23-epoxides (1 66). The same result is achieved by a silyl-radical-mediated cyclization pro- c~ss.'~~ The pregnadiene (167) gives the dinorcholene (168) in 70 YOyield after treatment with tributyltin hydride in the pres- ence of a,a'-azobisisobutyronitrile. The reaction proceeds via the 6-endo mode incorporating chirality transmission in the annulation process and results in the formation of two new stereo-centres. The method is applicable to the synthesis of 20-iso-steroid side-chains. The dinorcholene (1 68) is readily oxidized to the corresponding 16a,22-diol. 3,8-Acetoxypregna-5,16-dien-20-one can be converted into the 16a-ethyl- 19-norpregnene (169) in eleven It shows progestational activity 36 times that of progesterone.Of the many long-chain esters of the 21-hydroxyl group that can be obtained by reaction with acyl chlorides the dodecanoyl ester is active for months after a single injection in mammals. A further synthesis of the 1701-pregn-20-yne (170) involves copper-catalysed conjugate addition of methylmagnesium iodide to the androstadienone (171) to give mainly the 7a- methyl adduct which is converted into the oestrenedione (172) prior to ethynylation. 146 19-Noraldosterone has been prepared by an extension of a recent synthesis of 19-hydroxyaldosterone. 14' The 19-alcohol is oxidized with pyridinium chlorochromate to the 19-0x0-compound which is decarbonylated by heating it with alkali.A double hydroxylation reaction for the construction of the corticoid side-chain is the final step in a total synthesis of ti-)-cortis~ne.'~~ Oxidation of the 17(20)-en01 silyl ether (1 73) with rn-chloroperoxybenzoic acid in the presence of potassium hydrogen carbonate at 0 "C in dichloromethane gives cortisone (1 74) in 83 % yield whereas excess of the peracid at room CH20H I c=o NATURAL PRODUCT REPORTS 1988 temperature gives the 17-ketone. Regiospecific deoxygenation of the dihydroxyacetone side-chain occurs at C-17 with iodotrimethylsilane in acetonitrile producing 2 1 -hydroxy-20- ketones in moderate to high ~ie1ds.l~~ If chloroform was used as the solvent there was greater deoxygenation at C-21.Progesterone was obtained in good yield from 2 1 -hydroxypro- gesterone while 17a-hydroxyprogesterone was recovered un- changed. Rapid and complete conversion of 2 1 -iodoprogester- one into progesterone by this treatment with an iodosilane suggests that the deoxygenation at C-21 proceeds via the iodide. Routes to 19-oxygenated corticosterone and 19-nor- deoxycorticosterone are available from pregnenolone acetate and dehydroepiandrosterone acetate.I5O A key step in the first route is Henbest acetoxylation at C-21 of the enamine (1 75). In the second the hydroxyacetyl side-chain is introduced via base-catalysed condensation of the 17-ketone with methoxy- acetic ester. Steroids with the but-2-enoate side-chain are prepared from pregnenolone by Wittig-Horner reaction of its methoxymethyl ether with diethyl ethoxycarbonylmethylphosphonate.The 15' condensation affords exclusively the (E)-isomer (1 76). Treat- ment of pseudotigogenin diacetate (177) with sodium nitrite in aqueous acetic acid for 30 minutes at room temperature gives the oxime (1 78) in 42 YOyield.152 This yield decreases markedly in the absence of water. 2.5 Androstanes and Oestranes The oxime of dehydroepiandrosterone acetate was converted into the D-aromatic steroid (179) by incorporation of the C/D angular methyl group into the new D ring as probably occurs during the biosynthesis of Nicandra ~ter0ids.l~~ Selective aromatization of ring D was ensured by regioselective 16a- phenylselenenylation (Scheme 24).OAc (169) (1 70) 0 Pr,SiO ,Me C 0& CH20H I (172) (173) (174) 339 NATURAL PRODUCT REPORTS. 1988-A. B. TURNER C02Et I (175) (1 76 1 foAc AcO &=O% H (17'7) NOH Iiii SePh -But Me2 S i Do 0 Po M -1q0 vii viii _________) (179) Reagents i DMSO dicyclohexylcarbodi-imide,CF,CO,H; ii NaOH aq. EtOH; iii (CF,CO),O; iv ButMe,SiC1 imidazole DMF; v LiNPr', HMPA THF PhSeCI; vi BuLi 2-methyl-1,3-dithiane THF; vii H,O, pyridine CHCI,; viii HCI HgCI, THF. Scheme 24 The peroxide (1 80) gives the di-seco-derivative (1 8 1) together with the c-seco-compound (1 82) by thermolysis under acidic The structure of the latter was established by X-ray analysis of its hydrolysis product.7-Carbethoxyethynyl-4-en-3-onesin the androstane and pregnane series have been prepared by bromination of 3,3- ethylenedioxy-5-enes with N-bromosuccinimide followed by condensation with ethyl pr~pynoate.'~~ The 7a- and 7a-epimers were separated and characterized. 5p-Androstanes (1 83 ; R = Me) and (183; R = H) can be prepared efficiently from the cholanoic acid (1 84) by a sequence involving either photodegra- dation or decarboxylation with lead tetra-acetate followed by acid- or base-catalysed isomerization of the double-bond of the side-chain alkene~.~~~ Details have appeared of the synthesis of a new class of c-aromatic steroids (185) from the tricyclic hydrocarbons (1 86).15? Two bisandrostane derivatives (187; R = H) and (187; R = Me) and one monoandrostane derivative of fluorouracil were prepared for investigation as anti-cancer agents by condensing bromodihydrotestosterones with the di-or mono-sodium derivative of 5-fluoroura~il.~~~ The synthesis of 6-iodoandrost- 5-enes and of 6-iodopregn-5-enes that are labelled with iodine- 131 has been carried out via 6-chloromercuri-derivatives,which were prepared by direct mercuration of the parent steroid~.'~' The chloromercuri-compounds provide a simple high-yield method for labelling steroids with radioiodines.The chemistry and pharmacology of oestradiol have been OAc 0 NATURAL PRODUCT REPORTS 1988 reviewed.160 Oestrone mesogens of the types (188) and (189) were obtained by acylation of oestrone and condensation with aromatic amines.161.162 The fluorescent oestradiol derivative (190) has been prepared by linking fluorescein amine to the steroidal butanoic acid (19 1).163 Dicyclohexylcarbodi-imide was used to form the peptide bond. The related pyrrolinone (1 92) is formed by the reaction of 7a-(4-aminobutyl)- 17p- oestradiol (193) with fluorescein amine. The oestradiol haptens (1 9 1) and (193) were prepared from 19-nortesto~terone.~~~ Friedel-Crafts acylation of oestradiol 3-methyl ether deriva- tives with acetic anhydride/ boron trifluoride etherate gives 15-30 YOof acetylation at C-4 and up to 10 YOof 2-acetylated The catalyst was not necessary for the preparation of 2-acetylated B-noroestrogens.165 Monoglucuronides and monosulphates of 4-hydroxyoestriol have been prepared by standard methods.166 The sites of conjugation were confirmed by methylation to guaiacol oestrogens.Ring D conjugates were also prepared by protecting the phenolic hydroxyl groups as their benzyl ethers. 4-Nitro- 4-amino- and 4-fluoro-oestra- 1,3,5( 10)-trien- 17p-01s were pre- pared from 4-nitro-oestrone (Scheme 25) for use in vitro as inhibitors of sulphoconjugation of oestrogens for studies of hormone-dependent mammary cancer.167 A similar series of 2- substituted compounds was prepared from 2-nitro-oestrone. Electronegative substitution at C-4 gives the more effective inhibitors. Two types of bis-oestrone derivatives of benzene show interesting liquid-crystalline properties.168 One series OAc OAc OH (180) (181) (182) 0 H H (183) (184) (185) R NATURAL PRODUCT REPORTS 1988-A.B. TURNER 34 I R O B 0 0%-oI N/OR& (188) HO&' [CH 1311C- OH 0 (190) (193) X =CH2NH2 (1921 OH Reagents i NaBH, C,,H,,(C,H,),P+Br- PhMe H,O; ii H, Pd/C EtOH Et,N; iii NaNO, HBF,; iv m-CIC,H,CO,H CH,CI,. Scheme 25 14 NPR 5 342 NATURAL PRODUCT REPORTS 1988 X 0 RO&aN‘ (194) R = H or C(0)C6H,0R-p (195) X = 0 or N C6H40R -p 0# Ho ,&4 0 (196) (1 97) OH 24% (15a 1600 66.10 (150 168) lv OH 0 0 0 vii/ iJjs*s LJ Reagents i PhSO,CH=CH, PhH in a sealed tube; ii sodium amalgam MeOH THF Na,HPO, then Ac,O TsOH; iii KOH MeOH; iv OsO, pyridine; v NaIO, aq.EtOH; vi L-Selectride@ (LiBus3BH) THF; vii HSCH,CH,SH; viii Raney nickel EtOH. Scheme 26 NATURAL PRODUCT REPORTS. 1988-A. B. TURNER (1 94) consists of bis-Schiff-bases derived from p-phenylenedi- amine and the keto-group of oestrone and the other (195) is formed by esterification of the phenolic hydroxyl group with terephthaloyl chloride. A modified synthesis of isomeric 16,17- epoxy-17-3H-labelled derivatives of 3-hydroxy- and 3-methoxy- oestra- 1,3,5( 10)-trienes avoids incorporation of the isotope in ~~~ ring A. The norethisterone (196) is obtained from the ketone (1 97) by perhydroxylation at C-11 followed by deoxygen- ation with trimethyl phosphite and oxidation with chromium trioxide.170 14a-Methyloestrone (1 99) and its oxygenated derivatives are formed by a cycloaddition route from the diene (198).171 Introduction of a carbon residue at C-17 is also possible if this approach is used (Scheme 26).Diels-Alder reactions of the 16- en-20-ones (200) with butadienes in the presence of Lewis acids give the 16a 17a-cyclohexeno-compounds (201).172 In this case the cycloaddition involves the less hindered a-face of the steroid nucleus. Friedel-Crafts alkylation in ring A was only occasionally observed. 2.6 Cardenolides and Bufadienolides Some recent progress in the synthetic and medicinal chemistry of cardio-active glycosides has been Reduction of gomphoside (202) with sodium cyanoborohydride occurs with opening of the 2’-hemiacetal to give the epimeric 3-glycosides (203).174 Reduction of 3’-oxo-3’-deoxygomphoside with sodium borohydride gives a 3’-epimer of the 3-glycosides (203).Stereoelectronic control may be a factor in these transforma- tions. The relationship between stereochemistry of the sugar moiety and cardiac glycoside activity has been studied using various glucosides mannosides and rhamnosides of digitoxi- genin.175 Various furanosides show weak to moderate cardio- toxic activity. 176 Hemisuccinates of digitoxigenin digitoxin and digoxin were prepared by condensation with the mono- (trimethylsily1)ethyl ester of succinic acid under the influence of dicyclohexylcarbodi-imideand 4-dimethylaminopyridine. L77 The (trimethylsily1)ethyl group was removed from the mixed succinate with tetrabutylammonium fluoride.The method was not suitable for preparing oestrone hemisuccinate as it was cleaved by the fluoride to oestrone. A route for the synthesis of bufalin from deoxycholic acid (Scheme 27) involves the photochemical rearrangement of the lactone (204) to the unstable seco-aldehyde (205) which must be immediately reduced to the alcohol (206).178 Several withanolides (207) are cleaved with bis(acety1acetonato)oxo-vanadium to diketones (208) and the lactone (209).179 The oxovanadium chelate in aprotic solvents is a selective reagent for the quantitative cleavage of ditertiary glycols under mild conditions and the reaction also occurs with catalytic amounts of the complex in the presence of t-butyl hydroperoxide or m-chloroperoxybenzoic acid.2.7 Heterocyclic Compounds The Schmidt reaction of 3-oxocholest-5-en-4a-yl acetate with an equimolar amount of hydrazoic acid in benzene gives the lactone (210) and the lactam (211) whereas an excess of hydrazoic acid gives the lactam (212) together with an inseparable mixture of azido-ketones.laO 6P-Chloro-5-hydroxy- 5a-cholestan-3-one gives lactams (213) and (214) with an equimolar amount of hydrazoic acid whereas an excess leads to the tetrazole (21 5). Prolonged base-catalysed autoxidation of 4-en-3-ones in aprotic media leads to the lactols (216) in overall yields of 85-95 YO.lal Reduction of the lactols with sodium borohydride gives the 2-oxa-steroids (21 7) in isolated yields of 80-90 % (Scheme 28). Details have appeared of the formation of 1 1-oxa- 5a,17a-pregnane-3,20-dioneby cyclization of the c-seco-pregnanediol from hecogenin acetate.la2 6,9-Epithiotachystery13 acetate (21 8) is one of the solvolysis products of the sulphonate (219) which can itself be obtained (200)R’ = Me or Et R2 = H,Ac or Me R3 (201) R3= H or Me 0 Me 0 H (202) H from the rearranged cholestanone (1 50) by ozonolysis ring- closure with phosphorus pentasulphide and retro-pinacol rearrangement. la3 2.8 Cyclopropano-steroids Cleavage of the 5p,19-cyclopropane ring in the 3-keto-steroid (220) which contains the acid-sensitive 14p-hydroxyl group can be carried out under the influence of hydrochloric acid.ls4 The success of this procedure has been ascribed to the presence of the 17-keto-group.The cyclopropane ring is opened without need for the activating effect of the 3-keto-group if mercury(I1) acetate is used as the electrophile. Simmons-Smith cyclopro- panation of 1701- and 17P-hydroxy- 14-enes of the androstane and oestratriene series is controlled by the activating and syn-directing effect of the 17-hydroxyl group leading to the NATURAL PRODUCT REPORTS 1988 .ao H? C0,H 0.'Ac++# i ii >I H H 0j+Me OSiMeZBu' > 6ut V iv c--f-(204) 0 0LSMe -___) &! viii IX-XI HO (205)X = CHO2vii H (206)X =CH,OH Reagents i ClC(O)C(O)Cl DMSO CH,CI,; ii Et,N; iii ZnCI, (ButO)(Bu'Me,SiO)C=C(H)SMe MeCN; iv KF THF MeOH; v TsOH PhH ; vi hv (Pyrex-filtered) CH,C1,; vii LiAIH(OBut), THF ; viii rn-ClC,H,CO,H CH,CI,; ix Et,N MeSO,CI CH,CI ; x (CO,H), aq.MeC(0)Me; xi Amberlite CG-120 (acid form). Scheme 27 R (209) R (2081 R = AcO HO or Me (207)R = AcO HO or Me NATURAL PRODUCT REPORTS 1988-A. B. TURNER 210 11 (21011 H@ 0 OH OH (210) (211) (212) CI (213) (214) (215) -0&-R' = H; R2 = C8H,7 OH OCH2CH20Et or C(0)Me R1 =Me; R2 = OH R' =OH; R2 = C(0)Me ... Ill 1 iv 0&-0& (217) (216) Reagents i ButOK 18-crown-6 toluene at -25 "C;ii O, at -25 "C; iii BuW O, at 25 "C; iv NaBH,. Scheme 28 17 0 0 AcO' AcO" (220) (218) (219) NATURAL PRODUCT REPORTS 1988 cyclopropanes in which the 14,lSmethylene group and the 17- hydroxyl group are cis in a stereospecific reaction.la5 Oxidation of these compounds to the 17-ketones followed by reduction with diborane or complex metal hydrides gives the isomers in which the 14,lS-methylene group and the hydroxyl group at C-17 are trans.In the cyclopropanation of the epimeric 7- hydroxycholesteryl acetates the -P-alcohol likewise gives the 5,8,6P-cyclopropane and the a-alcohol gives the Sa,6P-cyclo- propane derivative. lS6Photochemically initiated cyclization of the B-homo-compound (22 1) gives the SP,6/3-cyclopropano-7- one (222) as the exclusive product. Studies on the 7-substituted SP,6,8-~yclopropanes (223) have shown them to be more prone to elimination reactions than the corresponding 7-unsubstituted ones.lS7 Methylenation of pregna-1,4,6,16-tetraene-3,20-dione gives the trimethano-derivative (224).laa The reaction of the apocholate (225) with methylene iodide in the presence of silver/zinc couple gives the 14a 1 5a-cyclopropane (226).lag 2.9 Microbiological Transformations Hydroxylation of 5a-androstane-3,17-dioneand of the A-nor- and A-homo-steroids (227) and (228) by the fungus Cun-ninghamella elegans takes place primarily at the 9a-position whereas 17P-hydroxy-5a-androstan-3-0ne undergoes mainly 7a-hydroxylation. lS0 Two of the 9a-hydroxy-compounds can be prepared by a sequence (Scheme 29) that involves 1 la-hydroxylation of the 3,17-diones by Aspergillus ochraceus. Dehydrogenation of A5-3P-acetoxy-steroids in which there is an oxygen-containing ring E e.g. (229) and (230) with Corynebacterium mediolanum produces the corresponding ketones.lS1 Production of prednisolone on the pilot-plant scale has been achieved by using calcium-alginate-immobilized Arthrobacter simplex.192 The maximum conversion of hydro-cortisone suspensions by immobilized A.simplex in a batch reactor is 80-85Y0. This is raised to 95% by feeding [as (221)l -[as (22111 Ac0a2 (2211 (222) (223) AcOO’ H H (224) (22 5) (226) OH k (227)1 0dP 0& H (227) (228) 0 I ii-v -0& A X = CH2 or [CH212 Reagents i Aspergiffus ochraceus; ii P(O)Cl, pyridine; iii rn-ClC,H,CO,H ; iv LiAlH ;v CrO,. Scheme 29 NATURAL PRODUCT REPORTS 1988-A. B. TURNER 347 0 20 J. Roemer and G. Kunz Z. Chem. 1985 25 404.21 G. A. Wolff N. A. Lamb and J. R. Maxwell Org. Geochem. 1986 10 965. 22 T. M. Peakman P. Farrimond S. C. Brassell and J. R. Maxwell Org. Geochem. 1986 10 779. 23 I. Weisz and P. Agoco Arch. Pharm. (Weinheim Ger.) 1986,319 952. 24 D. H. R. Barton D. Crich A. Loebberding and S. Z. Zard Tetrahedron 1986 42 2329. 25 S. A. Julia and R. Lorne Tetrahedron 1986 42 501 1. 26 P. Kocovsky I. Stary and F. Turecek Tetrahedron Lett. 1986 27 1513. (229) 27 A. A. Akhrem V. N. Pshenichnyi A. L. Mikhal’chuk and V. A. Khripach Vestsi Akad. Navuk B. SSR Ser. Khim. Navuk 1985 No. 6 p. 53. 28 B. M. Seletskii G. M. Segal and I. V. Torgov Croat. Chem. Acta 1985 58 699. 29 L. N. Volovel’skii I. A. Rastrepina N. V. Popova V. N. Koryu- kina and S. P.Kustova Khim. Prir. Soedin. 1985 700. 30 J. C. Sarma M. Borbaruah D. N. Sarma N. C. Barua and R. P. Sharma Tetrahedron 1986 42 3999. 31 P. K. Sripada J. Lipid Res. 1986 27 352. 32 H. Kunz and W. Pfrengle J. Chem. SOC. Chem. Commun. 1986 713. 33 R. Riccio 0. S. Greco L. Minale D. Laurent and D. Duhet J. Chem. SOC.,Perkin Trans. 1 1986 665. (230) 34 P. Dommes N. F. Breuer and H. Goebell Clin. Chim. Acta 1986 154 237. 35 P. Catsoulacos J. Heterocycl. Chem. 1986 23 321. 36 Y. J. Abul-Haj J. Med. Chem. 1986 29 582. 37 cJ J. Elks Nut. Prod. Rep. 1986 3 516. hydrocortisone suspensions continuously to either a fed-batch- 38 E. Glotter and M. Zviely J. Chem. SOC. Perkin Trans. I 1986, operated stirred tank reactor or to a continuous-flow air-lift 321.loop reactor at a controlled rate. 39 L. Lorenc M. Dabovic V. Pavlovic I. Juramic and J. Forsek Micro-organisms have been screened for their ability to Croat. Chem. Acta 1985 58 51 1. transform fenugreek steroids. 193 Five strains converted dios- 40 Shafiullah M. R. Ansari S. Husain H. Ogura and H. Taka-genin into diosgenone and some 1,2-didehydrodiosgenone yanagi Indian. J. Chem. Sect. B 1985 24 1072. whereas Mycobacterium phlei produced some androstenes 41 T. Takahashi A. Ootake J. Tsuji and H. Tachibana Tetra-when cc,a’-bipyridyl was added. Fusarium soluni produced hedron 1985 41 5747. 42 H. Velgova Collect. Czech. Chem. 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ISSN:0265-0568    

DOI:10.1039/NP9880500311

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

Imidazole Oxazole and Peptide Alkaloids and Other Miscellaneous Alkaloids J. R. Lewis Department of Chemistry University of Aberdeen Meston Walk Old Aberdeen AB9 2UE Reviewing the literature published between July 1985 and June 1986 (Continuing the coverage of literature in Natural Product Reports 1986 Vol. 3 p. 587) 1 Imidazole Oxazole Isoxazole and Thiazole Alkaloids 2 Peptide Alkaloids 3 Miscellaneous Alkaloids 4 References 1 Imidazole Oxazole Isoxazole and Thiazole Alkaloids The seeds of Lepidium sativum contain the imidazole alkaloid lepidine (1) as well as N,N’-dibenzylthiourea N,N’-dibenzyl- urea and sinapic acid ethyl ester.’ A revised structure2 for chaksine has been obtained from X-ray measurements on its iodide; its absolute configuration is reported to be as shown in structure (2).Two cytotoxic polyenylisoxazoles have been isolated from Streptomyces species. Curromycin A (3; R = OMe) was obtained from a genetically modified Streptomyces hygroscopicus3 and curro- mycin B (3; R = H) from the non-modified bacterium;4 the latter compound was produced from glycerol as its sole carbon so~rce.~ Rhizoxin (4),a new anti-tumour agent that has a chemotherapeutic effect similar to that of vincristine against leukaemias in mice has been isolated from a plant-pathogenic f~ngus.~ Marine sources continue to yield cytotoxic secondary metabolites. The sponge Discodermia calyx contains calyculin A whose structure (5) has been determined by n.m.r. and X-‘I -\ (5) R = OP(O)(OH) 351 ray measurements.' Nudibranchs produce egg-masses which are highly coloured; that they are virtually immune to pred- ation is thought to be due to the presence in them of anti- feeding or toxic substances.From Hexabranchus sanguineus the striking red egg-masses contain the tri-isoxazole macro- lides ulapualide A (6; R = 0) and ulapualide B [6; R = CHO,CCH(OMe)CH,OMe] both compounds being cyto- toxic at concentrations of 0.01 pg against mouse L1210 leukaemia.' Five antifungal macrolides that also contain the tri- isoxazole cascade have been isolated from an egg-mass of an unidentified nudibranch ;* kabiramide C has been identifiedg as (7) and the others are thought to differ only in their functional groups. The re-isolation of the flycidal alkaloid pantherine (9; R = NH,) from Amanita pantherina and its synthesis has shown it to be identical to agarinlo (a constituent of Amanita muscaria).Treatment of ethyl orthoformate with ethanolic HCl in the presence of a molecular sieve gave the chloro-ketal (8) which on reaction with hydroxylamine followed by acid hydrolysis and ring-closure gave (9; R = Cl). Pantherine (9; R = NH,) was produced by aminolysis of (9; R = Cl). The synthesis of the same compound (9; R = NH,) [now called muscimol and being described as an important constituent of Amanita muscaria] starting with dibromoformaldoxime (10) and N-dichloroacetylpropargylamine (1 2) is described in another publication." In this 'one-pot reaction ' potassium hydrogen carbonate converted (10) into (1 l) which on cycloaddition to OMe CHMe \ / ICH21,I c=o I MeCH I CHOMe I MeCH I I HO I OMe CH=CHNMeCHO (7) C=N-OHBr\ Br ' Br - (10) NATURAL PRODUCT REPORTS.1988 (12) gave (13) hydrolysis followed by methanolysis and demethylation giving (14; R = Br) and the natural product (9; R = NH,) respectively. Muscimol is thought to be capable of existing as its zwitterion (15). The non-aromatic portion of myxothiazole (16) this being a metabolite that is produced by the gliding bacterium Myxococcus fulvus Mxfl6 is derived biosynthetically from acetate propionate and leucine while the two methyl groups Me* originate from methionine;' the thiazole rings originate from cysteine. A total synthesis of the antibiotic althiomycin (17) has been achieved starting from D-cysteine toluene-p-sulphonate and 4-hydroxy-N-methylpyrroli-done; the procedure is called a stepwise elongation method commencing at the C-terminal and ending at the N-terminal position.l3 The total synthesis of patellamide B (18) which is an oxazoline-thiazoline macrolide has been achieved by elabora- tion of the thiazolecarboxylic esters (19) and (20) to (21) and (22) respectively followed by intermolecular coupling and intramolecular cy~1ization.l~This synthesis establishes the structures for patellamides A B and C as having separated oxazoline and thiazoline components and not fused ones as was originally reported for these macrolides. Consequently their structures have been re-examined and corrected.A novel feature of the macrolide ulithiacyclamide (23)15 is that its oxazoline/ thiazoline components are not only separated from each other but the seventeen-membered ring is straddled by a disulphide link. EtO ,OEt C "OLTl Et 0,C CH ''CH2C1 '0 CH2R (8) (9) 0 + II C fN-0' CIzCH CNHCHZC ECH (12) NATURAL PRODUCT REPORTS 1988-5. R. LEWIS -0 + CH2NH3 Me0 OMe* CILOH L (20) I I I JI BocN Hq-j.*. c =o / 354 2 Peptide Alkaloids The air-dried fruit hulls of Euodia hupehensis collected from the botanic gardens at Munster Pharmaceutical Institute contain the hexadeca-2,4-dienamide (24; n = 10) as well as its lower homologue (24; n = 4).16 An interesting series of poly-unsaturated amides (25) (26) (27) (28) and (29) have been isolated from Achillea ptarmica.” Shallow-water varieties of the blue-green marine alga Lyngbya majuscula contain mal- yngamide A (30) and malyngamide B (31) these chlorine- containing amides coming from its lipophilic extract.The deep- water variety of this alga contains malyngamide C (32) malyngamide D (33) and malyngamide E (34).l8 A total synthesis of kukoamine A (37) has been achieved by the oxazine method.Ig A six-step procedure was used to generate the synthon (39 which on treatment with Me,SiN gave the azide (36); the other ‘half’ of the natural product was built up in an analogous manner and the two parts were condensed to give (37). A review of the total synthesis of the potent insect poison (+)-pederin is available ; its readership will be somewhat limited as it is available only in Japanese.2o Obafluorin is an antibacterial p-lactone (38) that has been isolated from Pseudomonas Juorescens ;zl after its skeletal structure had been determined (by n.m.r.and mass spectro- scopy) its absolute configuration was obtained via an enzymic degradation of the amino-acid fragment that could be obtained from (38) by hydrolysis. Three minor alkaloids present in the bark of Peripentadenia mearsii22 are dinorperipentadenine (39) peripentamine (40; R1= Rz= OH) and anhydroperipenta- mine (40; R1R2= 0). The two unique structural units of reductiomycin (4 l) namely the cyclopentenone moiety and the dihydrofuran moiety are biosynthesized from 5-aminolaevul- inic acid and shikimic acid re~pectively.’~ z5 one Two papers,24* in Japanese,z4 describe the isolation of a highly unstable blood pigment from the heart of the black tunicate Ascidia nigra.Tunichrome B-1 was obtained by centrifugal counter-current chromatography followed by h.p.l.c. to yield the vanadium complex which on lyophilization gave the air- and water- unstable pigment (42). This compound was best processed in its reduced form at pH 7.2. A total synthesis of hexa-acetylcelena- mide A (43) has been obtained through key condensation reactionsz6 involving a-dialkylphosphorylamino-acidderiva-tives to obtain the dehydroamino acid (44)and thence (43). A novel cerebroside isolated from the cell wall of the bacterium Cystobacterfuscus has been identified as (45) ;compounds also obtained are seemingly related but they have not yet been completely characterized.27 CH [C H ,1 C H =C H C H =C H C( 0)N H C H CH Me (24) MeCEC-C ~C-C~C-CH=CHC(0)NHCH2CHMe (25) (26) Me[CH,l,CH=CHCH=CH C(0) NHCH,CH Me HCSC -C =C -CHZCH=CH-CH=CHC(O)NHCH2CHMe2 (27) HCEC-C ECICH l,CH=CH-CH=CHC(O)N 3 (28) HCEC-C EC [CH212 CH =CH -CH= CHC(0) N 3 (291 NATURAL PRODUCT REPORTS 1988 ,O Me (31) R =C (32) R = CH =gOOH (33) R = 4“. OH (34)R = HO 0 (36) NATURAL PRODUCT REPORTS. 1988-5. R. LEWIS In the year over twenty publications have appeared in which cyclopeptides are described ; in many cases they possess important antibiotic properties but their contribution to clinical medicine has yet to be realized.A review of these com-pounds28 up to 1984 is timely and will serve as a useful intro- duction to the group as more complex members are now being isolated. Sativanine D (46) sativanine E (47) and sativanine F (48) all contain a thirteen-membered ring and have been isolated from Zizyphus ~ativa.~~-~~ Discarine E which is a fourteen-membered [CH21,C(0)NH~CH,l,NH~CH21,NH ICH,l,NHC(O) [CH 1 (37) 0 6"-oij. 0cII [C H lzMe \ OH (38) (39) OH OAc H .C/N II 0 ,CO But OH fiOMe / o=c >-CHMe H HN Me 'CHO (46) (471 (48) cyclic peptide (49) was isolated from the bark extract of Discaria febrijiuga.32 The root bark of Capparis decidua contains a new spermidine alkaloid capparidisine whose structure (50) was determined by spectral The two fifteen-membered ansapeptides biphenomycin A (51 ;R = OH) and biphenomycin B (51 ; R = H) have been extracted from the culture medium of a strain of Streptomyces griseorubiginosus (No.43703) both compounds showing antibacterial activity with low toxicity.34 Members of the genus Jaspis of soft-bodied sponges contain the cyclodepsipeptide jasplakinolide (52) which possesses antifungal and antinematodal activity as well as being effective against epithelial lung Bai Jiang Yong is used in Chinese traditional medicine for its anticonvulsant and antineo- plastic properties ;it is prepared by fermenting dried Beauveria bassiana with pupae of Bombyx mori.Beauvericin (53) has been isolated from this herbal preparati~n.~~ Trienomycin A (54a) is a novel cytocidal ansamycin antibiotic that has been isolated from a Streptomyces strain (No. 86- 16) ;it is closely related to mycotrienins I and IL3’ A second paper reports38 how two other active compounds namely trienomycins B (54b) and C (54c) were obtained from the same fungus and identified. Gliding bacteria are sources of diverse types of peptide alkaloids. Corallococcus coralloides contains corallopyronins A (55; R = Me) B (55; R = Et) and C (56); all are open-chain HN I 04 OMe (50) H Ph NATURAL PRODUCT REPORTS 1988 peptides that possess antibiotic properties and are related to the myxopyr~nins.~~ Angiococcus disciformis produces the anti- biotic cyclic peptide angiolam A,40.41 which contains a nineteen-membered lactone-lactam ring and for which the structure is (57).The epimeric myxovirescins A and A are 28- membered lactone-lactams ; the configuration of C-25 in myxovirescin is R and myxovirescin A is the (25s) epimer (58).42 Five strains of Streptomyces are reported to produce naphthoquinone ansamacrolides (ansamycins) with phar-macological properties of the antibacterial or anti-tumour type. Streptomyces diastatochromogenes subsp. variabilicor produces diastovaricins I (59; R =OH) and I1 [59; R = SCH2CH(C02H)NHAc],43Streptomyces albolongus contains TAN-528A (60),44 Streptomyces 82-217 contains awamycin (6 1),45 Streptomyces Y-8340369 produces naphthomycin H (62),46 and Streptomyces S-1998 contains naphthoquinomycins A (63; R = OMe) B (63; R = SMe) and C (63; R = Azinothricin (64) is a hexadepsipeptide built up from six un- usual amino acids by a strain of Streptomyces (X-14950);48 the nineteen-membered ring also has attached to it an unusual C, side-chain.New vancomycin derivatives have been reported4g and patented ;jOthese glycopeptides which were obtained by the cultivation of Nocardia orientalis have been designated M43A H /N,C//O H2N OH V CH,CH(OH) CH N H (511 OH CH Ph NH I 0 0 (52) (53) 0 II b; R= MezHCCH2C+ c; R = MeEtCHCf II0 II II 0 0 (55) NATURAL PRODUCT REPORTS 1988-5.R. LEWIS OH OH 04 I H bH (57) (58) OH o=c ,OH ( 591 (60) c. I OH OH 0 (61) (62) Me0 358 (65a) M43B (65b) M43C (65c) and M43D (65d). Having determined the structure of aridicin A which was obtained from Kibdelosporangium aridum by using two-dimensional n.m.r. COSY and NOESY techniques the conformational geometry of its aglycon (66) was obtained by measuring its 2D NOE n.m.r. together with interactive computer-assisted molec- ular-modelling and force-field measurements to obtain in- formation on the intramolecular H ’-H coupling in the molec- ule. 51 Des(serylserylglycy1)ferrirhodin (DDF) (67) which is produced by Aspergillus ochraceus is the first fungal sidero- phore to have a linear tripeptide backbone.Its cavity can accommodate Fe3+ and Cia3+ ions.52 The total synthesis of (+)-celabenzine [68; R = C(O)Ph] has been achieved by conjugate addition of 4-aminobutanol to acrylonitrile subsequent treatment with benzoyl chloride and hydrogenation to give the amine (69). (S)-3-Amino-3-phenyl- propionic acid via its Boc derivative (70) was converted into its urethane derivative; this on condensation with the amine (69) in the presence of 2-chloro-1-methylpyridinium iodide gave the amide (71). Hydrolysis followed by oxidation of the resulting alcohol to the aldehyde and removal of the protecting t-butyloxycarbonyl grouping resulted in a cyclization to the cyclic amine which on reduction with NaBH afforded (+)-celabenzine [68; R = C(O)Ph].Dihydrocelacinnine [68; R = C(O)CH,CH,Ph] was also synthesized by a similar procedure. j3 CI NATURAL PRODUCT REPORTS 1988 (k)-Celacinnine [68 ; R = C(0)CH =CHPh] has been syn- thesized by using the novel methodology of a dihydro-oxazine e.g. (72) as a carboxamide synthon. In this way it is not necessary for a spermidine or an amino acid to be involved in a traditional condensation of an amine and an acid.54 3 Miscellaneous Alkaloids Carnosadine (73) is a novel optically active cyclopropylamino acid that has been obtained from the red alga Grateloupia cur nos^.^^ A synthesis starting from threo-3-hydroxyglutamic acid and proceeding via N-benzyl-2,3-didehydroglutamicacid and its pyrazoline derivative (74) followed by thermal or ultraviolet-induced denitrification yielded the cyclopropane (75).Elaboration of the carboxyl group in the side-chain to an amino-group and then guanidination gave the natural product thus confirming its structure as (73). The anthelmintic principle in Quisqualis indica is quisqualic acid (76). A synthesis of this from CICH,CH(NHBz)CO,Me followed by resolution of the N-benzoylated product with brucine gave the two enantiomeric products only the L-isomer being active.56 An antibiotic (77) that has been obtained from Streptomyces cavourensis S71 13 is active against Ehrlich carcinoma5’ while another metabolite of a Streptomyces species (MH 435-hF3) inhibits protein-tyrosine (65) a; R’ = H R2= C(O)NH, R3 = Glc b; R’ = Me,R2= C(O)NH, R3 = Glc C; R’ = H R2= C02H R3 =GIc CHP CH3 d; R’= H R2= CO,H R3 = Me 0II MeNACA H II +SOH 0 T‘ Ho+&$&oH CI OH CI NATURAL PRODUCT REPORTS.1988-5. R. LEWIS kinase [E.C. 2. 7. 1 . 1121; this metabolite erbstatin (78) is described as an epidermal growth factor which interacts with the kinase receptor.58 1,4,5,6-Tetrahydr0-2-methylpyrimidine-4-carboxylic acid (79) called ectoine is a novel cyclic amino acid that has been found in the cytoplasm of bacteria of the genus Ectothiorhodospira. It is thought to be an osmoregulator as it is only produced under certain growth condition^.^^ Nigerazine A (80; R1= Me R2 = H) which has been obtained from Aspergillus niger 1-639 is the isomer of nigerazine B (80; R' = H R2 = Me).60 A minor metabolite of Streptoverticillium olivoreticuli subsp.PhC=O R neoenacticus is piperazinomycin (8l) which is a novel antifungal agent that has been synthesized from the tetrabrominated tyrosyltyrosine (82)through oxidation with thallium trinitrate prior to debromination.61 Thaliporphinemethine (83) is one of fourteen alkaloids that have been isolated from Illigera pentaphylla.62 The dibenzopyranazepine alkaloid clavezepine (84) which is the first of its type to be reported has been extracted from Corydalis clavi~ulata.~~ A light yellow alkaloid called sampangine (85) co-occurs with the related alkaloid eupolauridine (86) in the stem bark of Cananga od~rata.~~ Compound (85) loses CO to give (86) in the mass spectrometer. (69) (70) OH H PhY N-OThp BocHN' -c7 YNC(0)Ph NH OC(0)Ph (73) (71) BOC = Bu'OCO (72) Thp = tetrahydropyran-2-yI H0,c aC0,H H0$ 'OZH NHBz NHBz (74) (75) yz CH,-CHC02H I MebN Ho ' H N a C02H CH=CHNHCHO (76) (78) (79) Me 0 OH OH (80) (81) (82) Me0 HOw \ Me OMe OMe OM (831 (84) (85) (86) 360 NATURAL PRODUCT REPORTS 1988 FN C I COzMe Me0 H (87 1 (88) (89) (90) A total synthesis of the bright yellow alkaloid aaptamine 25 R.C. Bruening E. M. Oltz J. Furukawa K. Nakanishi and K. (87) which is produced by the marine sponge Aaptos aaptos Kustin J. Nat.‘Prod. 1986 49 193. has been achieved by cycloaddition of methyl propargylate (88) 26 U. Schmidt and J. Wild Liebigs Ann. Chem. 1985 1882.with 2,3-dimethoxyaniline (89) to give the quinolin-4-one ;this 27 D. Dill H. Eckau and H. Budzikiewicz Z. Naturforsch. Teil B on conversion into its 4-chloro-analogue and condensation 1985 40 1738. with aminoacetaldehyde (as its dimethyl acetal) followed by 28 M. M. Joullie and R. F. Nutt in ‘Alkaloids Chemical and Biological Perspectives ’ Vol. 3 ed. S. W. Pelletier Wiley Inter- deprotection gave (87).65The structure of a novel phytoalexin science New York 1985 p. 113. exserohilone (90) which was found in the culture broth on 29 A. H. Shah V. B. Pandey G. Eckhardt and R. Tschesche Phyto-which the pathogenic fungus Exserohilum holmii (isolated from chemistry 1985 24 2765. the weed Dactyloctenium aegyptiurn) had been grown was 30 A. H. Shah V. B. Pandey G.Eckhardt. and R. Tschesche J. Nut. determined by X-ray analysis of its bis-p-bromobenzoate.66 Prod. 1985 48 555. 31 A. H. Shah V. B. Pandey G. Eckhardt and R. Tschesche J. Nut. Prod. 1985 48 2768. 32 A. Morel R. Herzog and W. Voelter Chimia 1985. 39 98 4 References (Chem. Abstr. 1985 103 142245). 1 A. Bahroun and M. Damak J. SOC. Chim. Tunis. 1985 2 15 33 V. U. Ahmad S. Arif A. Amber K. Usmanghani and G. A. (Chem. Abstr. 1986 104 65910). Miana Heterocycles 1985 23 3015 (Chem. Abstr. 1985 104 2 W. Voelter W. Winter V. U. Ahmad and M. Usmanghani 85 385). Angew. Chem. 1985 97 970 (Chem. Abstr. 1985 103 215606). 34 I. Uchida N. Shigematsu M. Ezaki M. Hashimoto H. Aoki 3 M. Ogura H. Nakayama K. Furihata A. Shimazu H. Seto and and H. Imanaka J. Antibiot 1985 38 1462 (Chem.Abstr. 1986 N. Otake J. Antibiot. 1985 38 669 (Chem. Abstr. 1985 103 104 84995). 50929). 35 P. Crews L. V. Manes and M. Boehler Tetrahedron Lett. 1986 4 M. Ogura H. Nakayama K. Furihata A. Shimazu H. Seto and 27 2797. N. Otake Agric. Biol. Chem. 1985 49 1909 (Chem. Abstr. 1985 36 H. Mao Zhongcaoyao 1985 16 293 (Chem. Abstr. 1985 103 103 101 560). 183423). 5 T. Tsuruo T. Ohhara H. Iida S. Tsukagoshi Z. Sato I. Mat- 37 S. Funayama K. Okada K. Komiyama and 1. Umezawa J. suda s. Iwasaki s.Okuda F. Shimizu et al. Cancer Res. 1986 Antibiot. 1985 38 1107 (Chem. Abstr. 1986 104 17407). 46 381 (Chem. Abstr. 1986 104 81622). 38 S. Funayama K. Okada K. Iwasaki K. Komiyama and I. 6 Y. Kato N. Fusetani S. Matsunaga K. Hashimoto S. Fujita Umezawa J. Antibiot.1985 38 1677 (Chem. Abstr. 1986 104 and T. Furuya J. Am. Chem. SOC. 1986 108 2780. 109 321). 7 J. A. Roesener and P. J. Scheuer J. Am. Chem. SOC. 1986 108 39 R. Jansen H. Irschik H. Reichenbach and G. Hofle Liebigs 846. Ann. Chem. 1985 822. 8 S. Matsunaga N. Fusetani K. Hashimoto K. Koseki and M. 40 B. Kunze W. Kohl G. Hofle and H. Reichenbach. J. Antibiot. Noma Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 27th 1985 38 1649 (Chem. Abstr. 1985 104 105989). 1985 375 (Chem. Abstr. 1986 104 183528). 41 W. Kohl B. Witte B. Kunze V. Wray D. Schomburg H. 9 S. Matsunaga N. Fusetani K. Hashimoto K. Koseki and M. Reichenbach and G. Hofle Liebigs Ann. Chem. 1985 2088. Noma J. Am. Chem. SOC. 1986 108 847. 42 W. Trowitzsch K. Borgschulte V. Wray D. Schomburg and 10 Y.Konda H. Takahashi and M. Onda Chem. Pharm. Bull. G. Hofle Liebigs Ann. Chem. 1985 1629. 1985 33 1083. 43 M. Hotta Y. Hayakawa K. Furihata A. Shimazu H. Seto and 11 D. Chiarino N. Napoletano and A. Sala Tetrahedron Lett. N. Otake J. Antibiot. 1986 39 311 (Chem. Abstr. 1986 104 1986 27 3181. 203 539). 12 W. Trowitzsch-Kienast V. Wray K. Gerth H. Reichenbach and 44 Takeda Chemical Industries Jpn. Kokai Tokkyo Koho 60-83 586 G. Hoefle Liebigs Ann. Chem. 1986 93. (Chem. Abstr. 1985 103 140297). 13 K. Inami and T. Shiba Bull. Chem. SOC. Jpn. 1985 58 352. 45 S. Funayama K. Okada H. Oka S. Tomisaka T. Miyano K. 14 U. Schmidt and H. Griesser Tetrahedron Lett. 1986 27 163. Komiyama and I. Umezawa J. Antibiot. 1985 38 1284 (Chem. 15 U. Schmidt and D. Weller Tetrahedron Lett.1986 27 3495. Abstr. 1986 104 5689). 16 J. Reisch R. A. Hussain S. K. Adesina and K. Szendrei J. Nut. 46 T. Mukhopadhyay C. M. M. Frdnco G. C. S. Reddy B. N. Prod. 1985 48 862. Ganguli and H. W. Fehlhaber J. Antibiot. 1985 38 948 (Chem. 17 G. Kuropka M. Koch and K. W. Glombitza Planta Med. 1986 Abstr. 1985 103 138 171). 244. 47 J. Mochizuki E. Kobayashi K. Furihata A. Kawaguchi H. 18 R. D. Ainslie J. J. Barchi Jr. M. Kuniyoshi R. E. Moore and Seto and N. Otake J. Antibiot. 1986 39 157 (Chem. Abstr. J. S. Mynderse J. Org. Chem. 1985 50 2859. 1986 104 145385). 19 T. Moriwake S. Saito H. Tamai H. Mitsuda and M. Inaba 48 H. Maehr C. M. Liu N. J. Palleroni J. Smallheer L. Todaro Heterocycles 1985 23 277 (Chem. Abstr. 1985 103 22828). T. H. Williams and J.F. Blount J. Antibiot. 1986,39 17 (Chem. 20 T. Nakata S. Nagao N. Mori and T. Oishi Tennen Yuki Kago- Abstr. 1986 104 164882). butsu Toronkai Koen Yoshishu 27th 1985,76 (Chem. Abstr. 1986 49 H. M. Higgins Jr. M. H. McCormick K. E. Merkel and K. H. 104 206965). Michel Eur. Pat. Appl. 159180 (Chem. Abstr. 1986 104 21 A. A. Tymiak C. A. Culver M. F. Malley and J. Z. Gougoutas 49 845). J. Org. Chem. 1985 50 5491. 50 K. E. Merkel U.S.P. 4547488 (Chem. Abstr. 1986 104 22 I. R. C. Bick Y. A. Geewananda P. Gunawardana and J. A. 87 122). Lamberton Tetrahedron 1985 41 5627. 51 P. W. Jeffs L. Mueller C. DeBrosse S. L. Heald and R. Fisher 23 J. M. Beale J. P. Lee A. Nakagawa S. Omura and H. G. Floss J. Am. Chem. Soc. 1986 108 3063. J. Am. Chem. SOC. 1986 108 331. 52 M.A. F. Jalal J. L. Galles and D. van der Helm J. Org. Chem. 24 R. C. Bruening E. M. Oltz and K. Kustin Tennen Yuki Kago- 1985 50 5642. butsu Toronkai Koen Yoshishu 27th 1985,600 (Chem. Abstr. 1986 53 H. Iida K. Fukuhara M. Machiba and T. Kikuchi Tetrahedron 104 126852). Lett. 1986 27 207. NATURAL PRODUCT REPORTS 1988-J. R. LEWIS 54 T. Moriwake S. Saito H. Tamai S. Fujita and M. Inaba Hetero-cycles 1985 23 2525 (Chem. Abstr. 1986 104 168665). 55 T. Wakamiya Y. Oda H. Fujita and T. Shiba Tetrahedron Lett. 1986 27 2143. 56 X.-Q. Gu B.-C. Pan and Y. Gao Huaxue Xuebao 1985,43,675 (Chem. Abstr. 1986 104 149363). 57 S. S. Pharmaceutical Co. Ltd. Jpn. Kokai Tokkyo Koho 60- 126093 (Chem. Abstr. 1985 103 213384). 58 H. Umezawa M. Imoto T.Sawa K. Isshiki N. Matsuda T. Uchida H. Iinuma M. Hamada and T. Takeuchi J. Antibiot. 1986 39 170 (Chem. Abstr. 1986 104 105776). 59 E. A. Galinski H.-P. Pfeiffer and H. G. Truper Eur. J.Biochem. 1985 149 135 (Chem. Abstr. 1985 103 3305). 36 1 60 T. Iwamoto A. Hirota S. Shima H. Sakai and A. Isogai Agric. Biol. Chem. 1985 49 3323 (Chem. Abstr. 1986 104 31448). 61 S. Nishiyama K. Nakamura Y. Suzuki and S. Yamamura Tetrahedron Lett. 1986 27 4481. 62 S. A. Ross R. D. Minard M. Shamma M. 0.Fagbule and G. Olatunji J. Nut. Prod. 1985 48 835. 63 J. M. Boente L. Castedo D. Dominguez and M. C. Ferro Tetra-hedron Lett. 1986 27 4077. 64 J. U. M. Rao G. S. Giri T. Hanumaiah and K. V. J. Rao J. Nat. Prod. 1986 49 346. 65 T. R. Kelly and M.P. Maguire Tetrahedron 1985 41 3033. 66 K. Sugawara F. Sugawara G. A. Strobel Y. Fu C.-H. He and J. Clardy J. Org. Chem. 1985 50 5631.

ISSN:0265-0568    

DOI:10.1039/NP9880500351

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

Brain Chemistry and Central Nervous System Drugs R. 1. Brinkworth E. J. Lloyd and P. R. Andrews School of Pharmaceutical Chemistry Victorian College of Pharmacy L td. 38 1 Royal Parade Parkville Victoria Australia 3052 1 Introduction 2 The Biological Basis of Action of CNS Drugs 2.1 Neurotransmitters 2.2 Neuropeptides and Neurotransmitters in the Brain 2.3 Coexistence of Neurotransmitters and Neuropeptides 2.4 Receptors 2.5 Receptor Mechanisms 2.6 Localization of Neuropeptides and of Neuropeptide Receptors in the Brain 2.7 Receptor Sub-types 2.8 CNS Drugs Acting at Neuroreceptors 2.9 A Common Structural Model for Compounds Active at Brain Receptors 2.10 The Evolution of Neurotransmitters 3 Discovery of CNS Drugs 3.1 Ethnopharmacology 3.2 Medicinal Plants 3.3 Toxic Substances 3.4 Analogues of Endogenous Molecules 3.5 Synthetic Compounds 3.6 Drug Design 4 Conclusion 5 References 1 Introduction Substances affecting the central nervous system (CNS i.e.the brain and spinal cord) have been known since antiquity but only within the past 150 years have the principles that are needed to understand their action been established. Underlying these principles has been the development of our knowledge the functional anatomy of the CNS; different classes of nerve cells (i.e. neurons) and the mechanisms of neurotransmission ; causal relationships between disease states and neuronal pathways ; techniques for the study of the biochemistry and pharma- cology of neurons ; techniques to extract and characterize the pure substances that produce psychotropic effects ; the chemistry of drugs and of their interaction with biological systems and macromolecules ;and the principles of genetic engineering.Consequently; much new information is-available that could be used in the rational design of new drugs with greater potency (therefore lower doses are needed) and specificity (hence less side-effects) in contrast to older methods involving the random screening of synthetic and natural products. The purpose of this review is to outline some of the biological and chemical aspects that are important for our understanding of brain function in relation to current methods for the design of CNS drugs.2 The Biological Basis of Action of CNS Drugs The brain is an exceedingly complex organ and it has only been in comparatively recent times that increasingly sophisticated techniques have enabled us to understand some of the chemical 363 processes which occur within it at more than a superficial level. Laboratory methods (such as receptor-binding studies with radiolabelled ligands autoradiography positron-emission tomography immunohistochemistry and studies on ion chan- nels) have greatly added to our knowledge of brain chemistry but at the same time have shown that there is a lot more information still to be discovered. The answer to the question of how the brain is organized depends very much on the discipline of the particular researcher of whom the question is asked it can be described on a neurochemical electrophysio- logical anatomical functional or phylogenetic basis.In this review the neurochemical organization of the brain will be emphasized although the other organizational bases will be described when appropriate. 2.1 Neurotransmitters Transmission of a nerve impulse along the axon of a neuron is in the form of a wave of depolarization. This is caused by a change in the ion permeability of the axonal membrane which results in the transfer of sodium ions into the axon from the exterior and of potassium ions out of the axon. When the impulse reaches the vicinity of the nerve-ending calcium ions move into the cell through voltage-regulated Ca2+ channels.This in turn triggers the release of a neurotransmitter into the short gap (20-50 nm) known as the synapse between the two neurons. These events are shown in Figure 1.' The subsequent binding of neurotransmitters to receptors on the adjacent neuron will be covered in a later Section. The underlying mechanisms of chemical communication whether neuronal hormonal or pheromonal are essentially the same and the distinctions between these apparently different processes chiefly relate to the distance over which the chemical signal has to act. By definition hormones are chemical transmitters which are carried by the circulatory system from the endocrine glands to target cells. Neurotransmitters on the other hand are carried only a short distance by diffusion to their target cells.2 Pheromones such as insect sex attractants are transmitted through air or water from one organism to another.2 For a substance to be classified as a bonafide neurotransmitter a number of clearly defined criteria must be met.3s4 By consensus these are as follows.The substance must be present in the presynaptic elements of nerve cells. Precursors and biosynthetic enzymes must be present in the nerve cell usually close to the site of their presumed action. Stimulation of neurons should cause release of the substance in physiologically significant amounts. Direct application of the substance should produce re-sponses that are identical to those caused by presynaptic nerve cells.There should be specific receptors in the postsynaptic region that interact with the substance. Interaction of the substance with its receptor should induce changes in the ion permeability of the postsynaptic membrane leading to excitatory or inhibitory postsynaptic potentials (increasing or decreasing the likelihood that the cell will fire). NATURAL PRODUCT REPORTS 1988 Direction of nervous impulse Figure 1 Events involving neurotransmitters at the synapse. (7) Specific inactivating mechanisms should exist by which interactions of the substance with its receptor are halted in a physiologically reasonable time. Ever since the 1920s when Sir Henry Dale demonstrated that acetylcholine is a neurotransmitter the number of substances that have been shown to be neurotransmitters has steadily increased (Table 1).Neurotransmitters may be grouped into three classes monoamines amino acids and peptides. Adenosine also appears to be a neurotransmitter whilst other substances (such as prostaglandins and steroid hormones) may eventually be included. Some pharmacologically active mono- amines such as octopamine tryptamine,and phenethylamine have not yet been shown conclusively to be neurotransmitters in mammalian nervous systems. Substances in the monoamine and amino-acid classes are known as the ‘classical ’ and ‘canonical ’ neurotransmitters and it was generally believed that any new candidate would fall into one of these classes. This situation changed in the 1970’s with the discovery of peptides that act as neurotransmitters or neuromodulators.Sub-classification of the neurotransmitter groupings can be made on a structural or a functional basis. Amino acid transmitters can be classified into inhibitory or excitatory amino acids depending on their effects on neuronal trans- mission. Glutamate which is the archetypal excitatory amino acid binds to receptors that are linked to Na’ ion channels and aspartate apparently acts in the same fashion. The influx of sodium ions causes the neuron to become depolarized; this ylation Table 1 Neurotransmitter substances in mammalian brain Monoamines Catecholamines Other Noradrenaline 5-Hydroxytryptamine (serotonin) Adrenaline Histamine Dopamine Acetylcholine” Amino acids Excitatory inhibitory Glutamate y-Aminobutyric acid (GABA) Aspartate Glycine Taurine Peptidesb Purines VIP Adenosine CCK Neurotensin Substance P Enkephalins Endorphins ACTH Oxytocin Vasopressin Somatostatin TRH Neuropeptide Y (a) Sometimes grouped by itself.(b) For a full list see Table 2. NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS process initiates the molecular events of neuronal transmission. The inhibitory amino acid GABA (4-aminobutyric acid) on the other hand has receptors linked to chloride-ion channels. The influx of chloride ions into a nerve cell makes it resistant to e~citation.~ Glycine and taurine are also inhibitory amino Classifying the monoamines noradrenaline adrenaline and dopamine as ‘catecholamines ’ not only reflects a common structural basis but also a common biosynthetic route from L-dopa.2.2 Neuropeptides and Neurotransmitters in the Brain Amongst the first neuropeptides to be discovered were substance P by Leeman and co-workers,8 and the enkephalins by Hughes Kosterlitz and other^.^ Since the mid- 1970’s there has been a marked increase in the number of neuropeptides and potential neuropeptides and it is believed by some that the total number may exceed 200.’ Several excellent reviews have been published. Neuropeptides are usually divided into classes which reflect their originally defined roles as endocrine hormones. Of more relevance to neurochemistry is a classification based on the relative concentrations of neuropeptides or neuropeptide techniques terms such as ‘neuropeptide-like ’ or ‘neuropeptide immunoreactive’ are used.Figure 2 shows a simplified representation of the human brain in longitudinal section and various transverse sections are shown in Figure 3 ;the various regions and cell groupings that are mentioned in this review are identified in these Figures. A comprehensive review by Palkovits of the localization of peptides in the central nervous system was published in 1985.16 The work of Dahlstrom and Fuxe” established an ‘ABC’ nomenclature for groups of neurons in the hind-brain and the mid-brain of the rat whose projections extend into regions of the mid-brain and forebrain (such as the basal ganglia thalamus hypothalamus and other parts of the limbic system) respectively and into the cerebral cortex.A relatively simple system in the CNS which illustrates the multiplicity of neurotransmitters in central neurons is the pain- perception (algesia)/pain-control (analgesia) system (Figure 4). Cells of the dorsal root ganglia produce substance P along with other neuropeptides and these neurons pass the pain stimulus to the dorsal horn of the spinal cord. Neurons of the lateral spinothalamic tract carry these messages to the posterolateral nucleus of the thalamus presumably using acetylcholine as a neurotransmitter with further connections to the cerebral cortex. Pain control is carried out by inhibiting the production of enkephalins and other peptides in the interneurons in the receptors in various regions of the central nervous sy~tem~~’’~ (Table 2).On this basis there are three broad classes with no two having precisely the same distribution (1) those whose highest concentrations occur in the cerebral cortex generally in small ‘interneurons ’ (VIP and CCK,) ; (2) those for which the highest concentrations are in the spinal cord medulla oblongata and pons (enkephalins neuro- tensin and substance P); (3) those whose highest concentrations exist in the hypothala- mic nuclei (most of the remainder). Some of the more recently discovered neuropeptides are discussed in Section 2.8. In general the locations of neuropeptides in individual neurons have been demonstrated by means of immunohisto- chemical techniques ; a second antibody coupled to some fluorescent compound is frequently used.Since the presence of neuropeptides is demonstrated indirectly by immunochemical Locations of neuropeptides in the mammalian Highest concentration in the cerebral cortex Vasoactive intestinal polypeptide (VIP) Cholecystokinin (CCK) Highest concentration in midbrain hindbrain and spinal cord Neurotensin (NT) Substance P (SP) Enkephalins (ENK) Dynorphins (DYN) Endorphins (END) Highest concentration in hypo thalamus Adrenocorticotropic hormone (ACTH) Oxytocin (OXT) Vasopressin (AVP) Luteinizing-hormone-releasinghormone (LHRH) Somatostatin (SST) Thyrotropin-releasing hormone (TRH) Corticotropin-releasing factor (CRF) Angiotensin I1 a-Melanocyte-stimulating hormone (a-MSH) Bradykinin Neuropeptide Y (NPY) Bombesin (BN) Galanin (GAL) Calcitonin-gene-related peptide (CGRP) Atrial natriuretic peptide (ANP) Diazepam binding inhibitor (DBI) dorsal horn and by the descending pathway which consists of 5-hydroxytryptamine (5-HT) substance P thyrotropin-releas-ing hormone (TRH) and enkephalin-producing neurons.The periaqueductal grey and the large raphe nuclei act as relay centres.15.la 2.3 Coexistence of Neurotransmitters and Neuropeptides The term “Dale’s principle” has come to mean the idea that one neuron can only produce one type of neurotransmitter. However Dale actually proposed that all synapses of a single neuron act by the same chemical transmission mechanism which could cover more than one neurotransmitter.19 Evidence has now been accumulated by Hokfelt Lundberg and others that many neurons secrete both a classical neurotransmitter and a ne~ropeptide.’~ Table 3 lists some of the known associa- tion~.~~-~~ All aspects of coexistence that were then known were reviewed at a conference in Stockholm in 1985.24 The implications of coexistence are still mostly speculative although a number of generalizations can now be made including the differential response of classical neurotransmitters and neuro- peptides to the frequency of stimulation more complex autoregulation and synergistic effects.25 The release of neuro- peptides seems to require a higher frequency of stimulation than does the release of classical neurotransmitters.At this level inhibitory processes (such as down-regulation via auto-receptors) begin to reduce the release of classical neurotrans- mitters. The mechanism of action of autoreceptors appears to involve inhibition of Ca2+-mediated neurotransmitter release implying that autoreceptor-mediated regulation will involve all coexistent neurotransmitters at a particular synapse. Synergistic effects have been demonstrated for a number of receptors of coexistent neurotransmitter pairs including VIP/acetylcholine substance P/5-HT and neuropeptide Y/noradrenaline. 25 2.4 Receptors For the purposes of this review a receptor will be defined as a membrane-bound protein or protein complex which specifically binds a neurotransmitter a drug or a hormone.Recent reviews on receptor^^^-^' have listed the requirements for a bona$de receptor as follows (1) the binding of the ligand is saturable indicating that there is a finite number of receptors; (2) the binding is reversible; (3) the binding exhibits specificity and selectivity; NATURAL PRODUCT REPORTS 1988 Stria Te,rminalis " Figure 2 Longitudinal sections of the human brain through the mid-line with the various regions projected onto the plane of the mid-line showing the following regions A cerebral cortex B olfactory bulb C forebrain and septa1 nuclei D bed nucleus of stria terminalis E basal nucleus of Meynert F hypothalamic nuclei G amygdala H hippocampus I posterior pituitary J caudate nucleus K putamen L globus pallidus M thalamus N substantia nigra 0 habenular nuclei P superior colliculus Q inferior colliculus R dorsal raphe nucleus S periaqueductal grey T cerebellum U pons V locus coeruleus W large raphe nucleus X nucleus of solitary tract Ydorsal nucleus of vagus nerve Z substantia gelatinosa.For clarity some regions are not shown on both (a) and (b). The labels (i)-(vi) in (b) refer to positions of the transverse sections in Figure 3. Based on reference 15. NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS I(i) Reticular Formation Inferior Olivary Nucleus (iv) Corpus Caiiosum \ J Red Nucleus N I (v) ~ Corms Callosum -Anterior Commisure Figure 3 Transverse sections of the human brain at the positions as indicated by (i) (ii) etc.in Figure 2b. Identifying labels A B etc. are as in Figure 2. Based on reference 18. (4) there is a correlation of binding with the activity of agonists as measured by dose-response curves; (5) there is a correlation between the distribution of its binding in a tissue (or sub-cellular sites) and the known localization (or target site) of the ligand. The term 'ligand ' here refers to a small molecule (a neurotrans- mitter a drug or a hormone) which binds to a receptor. In this context an agonist is defined as a compound which binds to a receptor and triggers a physiological response. Virtually all neurotransmitters and hormones are agonists.An antagonist on the other hand binds to a receptor but does not trigger a response. Antagonists block the action of agonists. Two mechanisms have been proposed to explain the binding of antagonists and agonists to receptors. The concerted model involves equilibrium between two conformational states these being the agonist conformation and the antagonist conforma- tion. The ligand has a preference for one of these states thereby shifting the equilibrium. 28 In the sequential or induced-fit model on the other hand the transition between the agonist and antagonist states is induced by the binding of the ligand.29 NATURAL PRODUCT REPORTS 1988 31 Cerebral Cortex Thalamus *.*.... .. ,.;..:..’*. Central Grey Region of Mesencephalon (Periaqueductal Grey) Raphi! Nucleus Nuclei Neurotensin Enkephalin Histamine LHRH Oxytoc i n Vasopressin ACTH Endorphin M-MSH ... A mygdala ACh? atin Angiotensin I I Lateral Spinothalamic Tract - .. ‘ ._ Lateral Spirio- thalamic Tract ZSpinal Cord Figure 4 Pathways of perception and control of pain. Receptor- binding studies (using radioligands) date from the early 1970’s when the binding of [1251]bungarotoxin to acetylcholine receptors in the electric organs of Ekctrophorus electricus was studied by Changeux and co-~orkers~~ and the binding of 3H-labelled naloxone to opiate receptors in rat brain was investigated by Pert and Snyder.31 Since then radioligand-binding methods have been exten- sively utilized in the study of neurotransmitters and hormones and of the way in which these substances interact with their respective receptors.For more detailed discussions of this field consult the reviews by Carman-Kr~an~~ and Williams and U’Pri~hard.~~ Besides radioligand-binding other methods for studying receptors in situ include a~toradiography~~ and posi- tron-emission tom~graphy.~~ Graphical methods such as the Scatchard Plot,36 the Hill Plot,37 and the Eadie-Hofstee Plot38 have long been available for processing results from radio- ligand-binding experiments. More recently iterative non-linear regression-analysis techniques such as LIGAND~’ and EBDA~’,~~ have become available and can provide more accurate estimates of the parameters involved.Dorsal Root Ganglion Mostly Substance P but also VIP Somatostatin CCK,. Ansiotensin II and Dynorphin 3-Pain Control Pain Perception 2.5 Receptor Mechanisms The binding of an agonist to a receptor is linked to the production of a secondary messenger in what is known as a transduction mechanism with the secondary messenger in- fluencing an effector system (usually an enzyme or an ion channel) as shown in Figure 5. This section describes these events in more detail and in molecular terms. Where the receptor is directly linked to an ion channel (Na+,K’ Ca2+ or Cl-) subsequent events occur very quickly in milliseconds whether or not the effect of the agonist is excitatory or inhibitory.On the other hand where the receptor is ultimately linked to an enzyme changes may take from minutes to days to occur. Considering the latter case in more detail there are a number of secondary messengers and primary effectors (enzymes) that have been shown to be linked to slow-acting receptors. These secondary messengers are 3’,5’-cyclic adenosine monophos- phate (CAMP) 3’,5’-cyclic guanosine monophosphate (cGMP) 1D-myo-inositol 1,4,5-trisphosphate (IPS) and diacylglycerol NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS Table 3 Coexistence of neurotransmitters in the mammalian brain24 Classical neurotransmitter Neuropeptide Brain region Animal Noradrenaline EnkephalinNeuropeptide Y Neuropeptide Y Vasopressin Locus coeruleus Locus coeruleus Medulla oblongata Locus coeruleus Cat Rat Man rat Rat Adrenaline Neurotensin Nucleus solitarius Rat CCK Nucleus solitarius Rat Neuropeptide Y Substance P Medulla oblongata Medulla oblongata Rat Rat Dopamine CCK Neurotensin Neurotensin Ventral tegmentum Ventral tegmen tum Infundibular/arcuate nuclei of hypothalamus Rat Rat Rat man 5-HT Substance P TRH CCK Enkephalin Medulla oblongata Medulla oblongata Medulla oblongata Medulla and pons (raphe nuclei) (raphe nuclei) (raphe nuclei) Rat cat Rat Rat Cat GABA Somatostatin (SST) Somatostatin (SST) CCK Neuropeptide Y Enkephalin Thalamus Hippocampus Cortex Cortex Striatal region Cat Rat cat monkey Cat monkey Cat monkey Rat Acetylcholine VIP Substance P Cortex Nucleus of dorso-lateral Rat Rat Enkephalin Galanin Superior olivary body Septa1 forebrain tegmentum (medulla) Guinea pig Rat Stimulus (Agonist binding) Recognition r Receptor Transduction Secondary Effector Mechanism Messenger System Figure 5 Transduction of a stimulus from a receptor to effector systems.(DG). The structures of these compounds are shown in Figure enzymes and other proteins as substrates for phosphorylation 6. All of these compounds are produced by membrane-bound and which are the link in the control of metabolism by enzymes that are linked to the receptor by another protein hormones and neurotransmitters alike. A review in 1982 by which is the ‘transducing element’. The receptors the trans- C~hen~~ includes a discussion of the ubiquitous nature of ducing elements and the enzymes that are responsible for the protein phosphorylation in cellular control whilst in a review generation of secondary messengers probably only come in 1984 Nestler and colleagues45 defined the role of protein together transiently.This is the ‘floating receptor’ or ‘mobile kinases in neuronal tissue. Protein kinases other than PK-A receptor ’ hypothesis and the implications are that one effector will be discussed later. system can be served by more than one receptor and that one Binding of an agonist to a PK-A-linked receptor can result in receptor type can regulate a number of membrane-bound either an increase or a decrease in adenylate cyclase activity. functions.42 Although not directly produced by a receptor-The difference occurs at the level of transduction which links linked enzyme the metalloprotein complex Ca2+-calmodulin the recognition step of agonist binding to activation of the (CaCM) is another important secondary messenger which is adenylate cyclase.Transduction of receptor-mediated events linked to the IPJDG system. occurs almost exclusively as a function of GTP-binding 3’,5’-Cyclic adenosine monophosphate which was discovered proteins known as ‘G-proteins ’ which include (i) G (or NJ by Sutherland and co-workers in the 1950’~’~~ is responsible for (ii) G (or Ni) (iii) Go,(iv) G, (v) transducin and (vi) RAS-the control of many metabolic processes. It is synthesized from protein.46 It should be noted that G-proteins are involved in all ATP by a membrane-bound enzyme adenylate cyclase and types of ligand-receptor or stimulus-receptor interactions hydrolysed to 5’-AMP by a specific phosphodiesterase.A key including the process of olfactory reception which is discussed role for CAMP is the activation of protein kinase A (PK-A). in two recent review^.^'^^^ This is one of a group of protein kinases which use cellular The binding of an antagonist to a receptor does not trigger the conformational change in that receptor that agonist binding does. Hence binding of an antagonist does not involve G-proteins. This fundamental difference was first noticed in p-adrenergic receptors by Leflcowitz and co-~orkers,~~ and discussed in a review in 1984. A working model was developed in which the agonist-receptor complex displaces GDP from the transducing element and the complex is itself displaced by GTP.A number of substances have proved to be extremely useful as tools in the study of G-proteins. Non-hydrolysable analogues of GTP (such as GppNHp and GppSp) have helped to establish the role of GTP in this system. Toxins from several bacteria namely cholera toxin and pertussis toxin have as their mode of action a specific interaction with G-proteins. Aluminium tetrafluoride anion is an inhibitor of GTP~S~,~O while the natural product forskolin from the plant Coleusforskohlii can activate adenylate cyclase in place of CAMP. The structures of GppNHp and forskolin are shown in Figure 6. The key features of G (N,) and G (Ni) have been established by workers such as Hildebrandt Gilman and others over a number of year~.~l-~~ These results are summarized in a review by Gilman.55 Both G and Gi are trimeric proteins containing a p and y subunits.The p and y subunits with molecular weights of 35000 and 10000 respectively are common to both proteins whereas the a subunits a (mol. wt = 45000) and ai (mol. wt = 41 000) are different proteins. Both G and Gi bind GTP after they have been activated by the receptor-agonist complex causing the G-protein to dissociate into subunits ; the a-GTP dimer binds to adenylate cyclase. The a,-GTP complex stimulates adenylate cyclase whilst a,-GTP inhibits it. Following this association the GTPase activity of the a subunit hydrolyses the GTP to GDP and inorganic phosphate and the a-adenylate cyclase complex dissociates.The a-induced stimula- tion or inhibition of adenylate cyclase is therefore tran~ient.~' 3 5'-cyclicAMP 3',5'-cyclic GMP 0 NATURAL PRODUCT REPORTS 1988 Cholera toxin is an enzyme which catalyses the adenosine- diphosphoribosylation of the a subunit resulting in inhibition of its GTPase activity and thereby causing a permanent switching-on of adenylate cyclase activity. Analogues of GTP such as GppNHp cannot be hydrolysed by the GTPase activity so again the activation is prolonged. Pertussis toxin also known as islet-activating protein (IAP) has a similar effect on aias does cholera toxin on a,. In this situation however the adenosine-diphosphoribosylated aiactually activates adenylate cyclase by an as yet unknown mechanism although it seems to be related to the supply of py dimers which bind to a,.54A model for the transduction mechanism of G and G is illustrated in Figure 7.32 Receptors that are linked to G include the /3,-and p,-adrenergic adenosine-2 histamine-2 dopamine-I 5-HT vasopressin-2 glucagon and ACTH.Receptors acting via G include a,-adrenergic adenosine- 1 p-opiate 8-opiate and dopamine-2.32 Recently neuropeptide Y has been added to the list.56 Only a small number of receptors have been shown to be linked to the production of cGMP by guanylate cyclase and these include the muscarinic re~eptor,~'the H,-histamine receptor,58 the CCK receptor,59 and the ANP receptor.60 Apart from adenylate cyclase and guanylate cyclase the other key membrane-bound enzyme linked to receptors is phospholipase C (PhosC) otherwise known as phosphoinositol phosphodiesterase (PIpde).Phospholipase C catalyses the hydrolysis of I-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP,) to 1,2-diacylglycerol (DG) and D-myo-inositol 1,4,5- trisphosphate (IP3). (The residues at positions 1 and 2 of DG are predominantly stearoyl and arachidonoyl respectively.) The most comprehensive review on this subject is that by A bdel- La ti f. 61 Receptors are linked to PhosC by another G-protein known PIP Hm, O=P-OQ O=P-CP -Fbrakolin Phorbol Ester Figure 6 Structures of compounds involved in transduction mechanisms. The arrow indicates where PIP is cleaved by phospholipase C. NATURAL PRODUCT REPORTS 1988-R.I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS 37 1 as G or N,.62Like G, G is sensitive to pertussis to~in.~~,~~ The precursor of the prostaglandin/thromboxane series of bioactive /3 and y subunits of G are the same as those of G and Gi but compounds. Activation of PK-C can also be carried out by a the 01 subunit is different having a molecular weight of group of compounds known as phorbol esters (see Figure 6), 39000.62As well as being activated by G,-GTP PhosC is also which are tumour-promoting.66 Phorbol esters can thus be activated by Ca2+ in a separate mechanism from Gp.65 thought of as naturally occurring analogues of DG. The complete scheme of the functions of DG and IP, which The connection of PK-C with tumorogenesis appears to both act as secondary messengers is shown in Figure 8.1,2-involve the ras oncogenes. The ras protein of yeast was the first Diacylglycerol stays in the membrane to activate another to be recognized as a G-pr~tein.~' Recent studies have linked protein kinase protein kinase C (PK-C). The ultimate fate of the phosphatidylinositol/Ca2+ system to activation of the DG is its conversion into arachidonic acid which is the oncogenes fos and Also required for PK-C activity is Agonist Agonist CAMP -dependent Protein Kinases 1 Pro te in Ph osphoryla t ion Figure 7 Transduction mechanisms involving G and Gi. Agonist Phorbol Esters Ca2+ I I GABA I Protein Phosphorylation Ca2+-CaM --Dependent Kinases Figure 8 Transduction mechanisms involving inositol phosphates and calcium.372 pho~phatidylserine.~~ PK-C has a molecular weight of about 77000,70and there are three types a,P and y. The y type is found in many tissues whilst aand P are the predominant types in brain.71 Further aspects of PK-C may be found in a review in 1984 by Nishi~uka.~~ Protein kinase C is specific for serine and threonine residues whereas PK-A is specific for tyrosine residues.44 Whilst the role of DG is in the activation of PK-C IP is involved in calcium mobilization. The Ca2+ ion is stored in the smooth endoplasmic reticulum (smooth ER) and the normal resting concentration of Ca2+ within the cell is of the order of 0.1 pmol drn-,. IP causes release of Ca2+ from the smooth ER so that its concentration rises to levels as high as 1Opmol dm-3.72 The connection between this phenomenon and the influx of Ca2+ from outside the cell was not understood until comparatively recently.Entry of Ca2+ is stimulated by myo-inositol 1,3,4,5-tetrakisphosphate(IP4) which is synthesized from IP by a specific kina~e.~~.~~ Mobilization of the Ca2+ stores in the endoplasmic reticulum is a prerequisite for this process as proposed by P~tney.~~ The most important function of Ca2+ is in the metalloprotein Ca2+-calmodulin (Ca2+-CaM).44 This complex acts as a secondary messenger but can form only if cytosolic Ca2+ levels rise to at least 1 pmol dm-,. It is an activator for several enzymes which include a group of protein kinases and adenylate kina~e.~~ The calmodulin molecule binds four Ca2+ ions at the micromolar Neurotransmitter receptors which are linked to the inositol/ Ca2+system through phospholipase C include muscarinic acetyl- choline a,-adrenergic H,-histamine 5-HT2 vasopressin- 1 angiotensin-11 bradykinin and substance P.61,70 One of the most important therapeutic agents in the treatment of manic disorders is lithium (Li+).Recent studies strongly suggest that it interferes in the inositol-salvage process in brain neurons by which IP is recycled to produce new PIP, which in turn reduces the levels of secondary messengers in the brain.77 Calcium is an activator of the membrane-bound enzyme phospholipase A, which catalyses the hydrolysis of membrane phospholipids to lyso-phospholipids and arachidonic The latter is the precursor for prostaglandins.GABA-B receptors may be linked to phospholipase A,.79 Another enzyme which is activated by calcium is the protease calpain. Lynch and co-workers have implicated calpain in the mech- anism of long-term potentiation (LTP) in rat hippocampus which is of fundamental importance in the establishment of memory.8o The functions of the GTP-binding protein Gowere unknown for a long time despite the ubiquitous nature of Go which has been suggested as occurring in the process by which opiates and opiate peptides inhibit the release of substance P this being a Ca2+-dependent process. Regulation of these neuronal calcium channels appears to occur via Go.81 The stimulus acting on a receptor need not be a chemical substance.Rhodopsin is the chemical component of the retina which is the primary recognition site for stimulus by light. This membrane-bound protein is linked to a cGMP phosphodi- esterase through another GTP-binding protein transducin.82 Recently it was discovered that rhodopsin has a strong sequence homology with the P-adrenergic receptor and thus both proteins share structural and functional such as the location of hydrophobic membrane-binding regions. Compounds of the benzodiazepine class typified by diaze- pam are extensively used in the community as anxiolytics and ~edatives.~~ Although benzodiazepines do not competitively bind to GABA receptors there is a connection. It has been found that the binding of agonists at benzodiazepine receptors is enhanced by agonists of GABA receptors and vice versa.85 The addition of a GABA agonist such as 10-5mol dmP3 muscimol can enhance benzodiazepine affinity by as much as 2.45-fold.The ratio of activities with and without the GABA agonist is called the 'GABA ratio'.86 Compounds with affinity at benzodiazepine receptors exhibit a full spectrum of pharma- NATURAL PRODUCT REPORTS 1988 cological activity from anticonvulsant to convulsant. This spectrum is reflected in the GABA ratios which range from 2.45 to 0.46.86 Both GABA receptors and benzodiazepine receptors along with barbiturate receptors are part of one large 'supercomplex ' involving C1- channelsa7 Benzodiaz- epines therefore act by potentiating the inhibitory effects of GABA on C1- channels.Antagonists of benzodiazepines such as Ro1788 simply block the binding of benzodiazepines without affecting GABA activity. Inverse agonists of benzo- diazepines (i.e. compounds for which the GABA ratio is less than 1.0) actually prevent GABA activity and as a result behave as convulsants. Opiate receptors have been shown to be linked to ion channels with the analgesic activity of opiates and opiate peptides such as enkephalins being due to an inhibitory effect of these substances on the sensory pathways that carry pain information and which use substance P as a neurotransmitter. lo The p-and &opiate receptor that are located on substance P nerve-endings as well as causing inhibition of adenylate cyclase result in an outflow of K' ions.88 Neuronal activity is thereby decreased and the release of substance P is inhibited.K-Opiate receptors on the other hand are linked to the Ca2+ channels that are involved in translating the wave of depolarization into the release of neurotransmitter^,^^ which also causes inhibition of the release of substance P and hence a diminution in the transmission of pain. Neuronal receptors share common mechanisms of action with the whole gamut of receptors including hormonal pheromonal olfactory and light recep- tors. The evolutionary implications of this commonality will be discussed in Section 2.10. 2.6 Localization of Neuropeptides and of Neuropeptide Receptors in the Brain As has already been mentioned the localization of neuropep-tides in the various regions structures and nuclei of the brain has been extensively studied by using a range of immunological techniques.16 The other major way of studying the regional Table 4 Localization of neuropeptides in the rat CNS Neuropeptide A B C I R I R I D I E F G I R I H I J I R K L I - I R - M R I N R 0 P - R QR I - R - S I I T R U V W X Y Z (a) See Figures 2 and 3.(b) See Table 2 for identification. (c) R = Receptor I = Immunoreactive substance. NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS Table 5 Drugs acting at neuroreceptors Therapeutic class Receptor Chemical type Example Neuroleptic Dopamine agonist Cholinergic DA2 DA + DA Muscarinic Nicotinic Butyrophenone Phenothiazine Benzamide Haloperidol Chlorpromazine Sulpiride Apomorphine Oxotremorine Nicotine Anticholinergic Muscarinic Nicotinic Atropine Tubocurarine Analgesic P P K Benzomorphan Non-benzomorphan Morphine Fentanyl Ti fluadom Psychotomimetic Hallucinogenic Stimulant (T 5-HT Catecholamine Ergotarnine Phencyclidine Amphetamine LSD Convulsant An tidepressant Anxiolytic Sedative uptake G1 ycine Noradrenaline 5-HT uptake Benzodiazepine Barbiturate Strychnine Imipramine Benzodiazepine (BZP) Diazepam Non-BZP Zopiclone Phenobarbital An ticonvulsan t Convulsant GABA GABA Phenytoin Bicuculline GABA Muscimol Stimulant Adenosine Caffeine Calcium-channel blocker Ca2+ channel Dihydropyridine Nitrendipine MA0 inhibitor Anticholinesterase DOPA-decarbox ylase inhibitor Depren yl Ph ysostigmine Carbidopa GABA-transaminase Gabaculine inhi bit or (a) See Figure 9.importance of neuropeptides is to investigate the localization of The distinction between receptor sub-types can be made on neuropeptide receptors. Ideally these two areas of study a number of levels including should produce complementary results in that neuropeptide The receptor being more responsive to the ' hormonal ' receptors are more likely to be found near the terminal ends of form of the transmitter than to the 'neuronal' or nerve fibres that contain a neuropeptide-immunoreactive vice versa. The differential responses of PI-and P,-adrenergic substance. The location of neurons that contain either specific receptors to adrenaline and noradrenaline is a case in neuropeptides or neuropeptide receptors is believed to be an point.49 indication of the function the neuropeptide may play in CNS-Receptor sub-types may be distinguished by the transducer regulated processes.systems to which they are linked. a (Ca2+-inositol) a2 The localization of neuropeptide-like immunoreactivity16 (Gi) p (GJ and /? (G,) noradrenergic receptors are so and the localization of neuropeptide receptorsgo in various distinguished. parts of the mammalian brain particularly rat brain have been Tissue distribution of receptor sub-types is often different extensively studied for such peptides as VIP substance P and and usually a reflection of their function. Histamine-1 neurotensin. The results of these studies are described in receptors are found on smooth-muscle fibres whereas considerable detail in major re~iews.~~~~~ There are a number of histamine-2 receptors are found on fundic mucosal cells in polypeptides which have been shown only in comparatively the stomach.118 recent times to be important in the CNS.These include The affinities of agonists and antagonists at different sub- neuropeptide Y bornbesin galanin calcitonin-gene-related types are often different sometimes markedly so. Very peptide and atrial natriuretic peptide. Table 4 summarizes the selective antagonists with high affinity are often used (in localization of these five peptides and their receptors in the rat radiolabelled form) as specific radioligands e.g. [3H]-C'S.91-116 prazosin (a,-adrenergi~)"~ and [3H]rauwolscine (a,-adren- ergic).120 Only a relatively small percentage of receptors have been 2.7 Receptor Sub-types isolated and their properties studied.The results indicate Ahlquist first recognized that a particular hormone or that receptor sub-types are actually different proteins. neurotransmitter may bind to more than one sub-type of Differences range from relatively small @,-and P,-adren- receptor.l" In this case it was the discovery of the a-and p-ergic12'*122) to extremely marked (nicotinic and muscarinic sub-types of the adrenaline (or noradrenaline) receptor. This acetylcholine123 I"). phenomenon -that there are sub-populations of receptor types It is possible that some receptor sub-types arise through in different tissues-has been shown to occur with many differences in their membrane components as for example receptors for hormones or neurotransmitters.Further sub- in p-and &opiate receptors which differ in their suscepti- division of receptor types (a1,a, pl p,) is often necessary bility to inhibition by This may be related to particularly if analysis of binding curves for radioligands the fact that &opiate receptors lack a cerebroside sulphate indicates the presence of heterogeneous populations of recep- that has been shown to be a necessary component of the tors. p-opia te receptor NPR 5 374 2.8 CNS Drugs Acting at Neuroreceptors Many classes of drugs that are active in the central nervous system have as their mode of action their affinity for receptors with some acting as agonists and others as antagonists.Table 5 lists the major classes involved with neuroreceptors. Other drugs such as prazosin (a1)and ketanserin (5-HT2) although having high affinity for their respective receptors are unable to cross the blood-brain barrier and their primary site of action is in the cardiovascular system. Table 5 also lists other CNS 1 MeN Q-4) 6 7 11 12 13 17 18 19 NATURAL PRODUCT REPORTS 1988 drugs which act at sites other than genuine neuroreceptors. The chemical structures of these drugs are shown in Figure 9. Drugs acting at CNS receptors must possess both strong affinity at the target receptor and specificity at that receptor relative to other receptors. The simple but elegant graphical technique of the receptor- binding profile as developed by Clo~se,’~’ provides a way of presenting both affinity and specificity by means of a histogram of binding affinities at different receptors on a log scale.Figure 10 shows the receptor- binding profiles of a number of drugs with varying degrees of specificity. HOqMe OH 9 10 “Me2 14 15 16 20 21 COzH 23 24 25 26 28 Figure 9 Structures of drugs acting in the CNS. Names are shown in Table 5. NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS Figure 10 Receptor-binding profiles of a number of drugs with varying degrees of specificity. These are (a) clonidine which is an a,-adrenergic partial agonist with some a1activity; (b) mianserin with a2,5HT, and H activity; (c) spiperone an antagonist with D and 5HT2 activity; (d) imipramine a tricyclic antidepressant; (e) lisuride with very broad specificity over a range of receptors ;and (0bromocriptine with a similar broad specificity.IS-? 2.9 A Common Structural Model for Compounds Active at Brain Receptors In a series of publications from this laborat~ryl~~-l~~ we have proposed the hypothesis that there is a common structural basis for compounds that act in the central nervous system whether as a drug or as a neurotransmitter. That is there is a common structural basis for all CNS-active compounds not just those from within one class. Our results led us to propose that (1) there is a common structural basis for the activity of many different classes of CNS-active drugs ; (2) the aromatic ring and the nitrogen moieties are the primary binding groups whose topographic arrangement is funda- mental to the activity of these drug classes; (3) the nature and placement of secondary binding groups known informally as 'foliage ' determine different classes of CNS drug activity.In naturally occurring neurotransmitters whether they are monoamines or neuropeptides three types of aromatic ring can be found in the 'primary aromatic binding position'. These are as follows (1) Phenyl rings including those of tyrosine and catechol- amines. For many neuropeptides the primary aromatic binding site is a tyrosine residue or a phenylalanine residue.This is often determined by studying natural peptide analogues such as morphine (see Section 3.2) or progres- sively smaller oligopeptide fragments. (2) Indole rings as in serotonin or in peptides that contain tryptophan residues. (3) Imidazole as in histamine or in peptides that contain histidine residues. Anomalous results involving clonidine and cimetidine have led to the suggestion that there are 'imidazole-binding' sites in the brain perhaps using imidazoleacetic acid as the endogenous ligand. 132 2.10 The Evolution of Neurotransmitters The major implication that can be derived from the common pharmacophore model described in the previous section is that there once existed a primaeval receptor which was the ancestor of all neurotransmitter receptors whether monamine or neuropeptide.On the basis of the common model it is tempting to speculate that the first receptor may have been specific for phenethylamine. In any case it is worth noting that the only neurotransmitters which do not fit the common model are glutamate aspartate GABA glycine taurine adenosine and TRH. When considering the evolution of receptors and neurotrans- mitters it must be realized that evolution of other components of the system such as G-proteins adenylate cyclase phospho- lipase C calmodulin calpain and the neuropeptides them- selves must have been going on simultaneously. Perhaps the first receptor had a very broad specificity for any phenethyl- amine-type compound and this led to different receptors when individual processes had to be differentiated in multicellular organisms.The earliest receptors may have been directly linked to ion channels as nicotinic acetylcholine receptors are today. 133 Still later coupling of receptors to adenylate cyclase or to phospholipase C may have resulted in further differenti- ation into receptor sub-types such as the a, a2,/I1 and P2 adrenergic receptors. An example of the ancestral connection between apparently disparate receptors is the recent discovery of the homology that exists between the P-adrenergic receptors the muscarinic acetylcholine receptor and rhodopsin (which absorbs light). 134 All three types have seven transmembrane segments as well as a number of other aspects of homology in their amino-acid sequences.Glycine receptors and GABA receptors have been similarly matched. 135 Ancestral relationships are also apparent amongst the receptor ligands in particular the neuropeptides. Well-known 'families' of neuropeptides include the VIP-secretin group NATURAL PRODUCT REPORTS 1988 which also includes PHM PHI and GIP,13' and the CCK- gastrin group which have in common a C-terminal pentapep- tide.136 This pentapeptide is also shared with a peptide that has been isolated from the skin of some amphibians namely caerulein which has a similar bioactivity to CCK.1370ther peptides with potent activity in mammalian systems such as physalaemin kassinin and eledoisin (all of which are homo- logues of substance P) bombesin and sauvagine have been isolated from amphibian Extensive studies have also been made of the bioactive peptides of sea-squirts and jawless fish and have also pointed to an evolutionary relationship between peptides of the same family.13' Furthermore several bioactive peptides are found in organisms as primitive as the unicellular Tetrahymena pyriformis. 138 This strongly supports the idea that organisms used these substances for general cellular communication and later adapted them for the more specialized role of neurotransmitters and still later as endocrine hormones.13 Endocrine glands as we know them only first appear in vertebrates whilst primitive nervous tissue is found in the simplest multicellular animals including sponges. l3 In many cases the functions of particular bioactive peptides as neurotransmitters are unrelated to their functions as endocrine hormones.For example TRH is found as a neurotransmitter in many lower species that lack TSH or a thyroid gland and appears to have been co-opted as a releasing factor for the secretion of TSH in higher ca~dates.,~~ It should be noted that no classical or peptidyl neurotransmitter has any intrinsic vascular gastro-intestinal or neuronal activity but that its effects are dependent on the range of functions carried out by its secondary messengers in a particular tissue. As well as a phylogenetic relationship existing between various neurotransmitters it has been suggested that an ontogenetic relationship also exists.The best-known example is that postulated by Pearse the so-called APUD (Amine Precursor Uptake and Decarboxylation) theory which states that neural and endocrine cells that show particular character- istics have a common embryonic origin.140 This theory is not universally accepted,141 but it does emphasize the unified nature of the neuroendocrine system These relationships between neurotransmitters directly affect drug design. The common model should enable drugs to be designed that are specific for their own particular receptors but with structural features in common. 3 Discovery of CNS Drugs The discovery of drugs is based on two approaches knowledge of the biochemistry of the disease to be targeted and the preparation of compounds that have a structural analogy with known active types.In most cases knowledge of the biochemistry of diseases has been retrospective to the discovery of active drugs particularly those that are CNS-active. The reason for this lies in the difficulty in understanding brain processes and in isolating receptor proteins; so far detailed knowledge is only available on the nicotinic acetylcholine 143 but recent results suggest that such knowledge will soon be available for GABA receptor^,'^^ glycine and noradrenaline P-recep- Much more information is available on enzymes and related diseases and this has led to several successful drugs the most notable in the CNS area being the inhibitors of the monoamine oxidases (MAO).14' The use of antipsychotics in the treatment of schizophrenia is a clear illustration of how biochemical processes have been clarified as a result of the use of CNS-active drugs.Although reserpine had been shown to control psychotic behaviour,14' only with the discovery of the antipsychotic properties of chl~rpromazinel~~ and the subsequent correlation between clinical dose and IC, values of the more potent antipsycho- was it possible to develop the antidopaminergic hypo- thesis for the action of these drugs. The fact that other neurotransmitter systems are also invo1ved12' suggests that still NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS Me I 0 MPPP MPTP Figure 11 Formation of the active metabolite MPP' of MPTP. more complex biochemistry remains to be elucidated possibly by use of other CNS-active drugs.Greater success has been obtained from optimizing the structures of compounds that have a known activity. Histori- cally these so-called lead compounds have been derived in ways that are described in the following sections. 3.1 Ethnopharmacology Undoubtedly the first drug discoveries resulted through the interaction of primitive peoples with their environment. 150 The majority of plant products were found to be suitable for food but others would have been poisonous psychotropic or medicinally useful. CNS-active plant extracts became associated with social and religious rites as a result of their ability to induce euphoria or otherwise alter the conscious state of the user.Discoveries of drug (as against nutritional) effects of plants were made in the context of a tolerant leisurely way of life where serendipity rather than a rational scientific approach by active investigation played a major role. Because these cultures developed slowly the use of psychoactive plants and their extracts became and in many cases remains an accepted part of the life of ancient peoples. Examples include the use of khat (Ethiopia) fly agaric (Siberia) opium (S.E. Asia) cannabis (Middle East) and cocaine (South America).l5l Thus the use of drugs whether natural or synthetic for recreational purposes is not a recent aberration rather the scale and impact on societies has been amplified. Indeed we might identify a new 'ethnopharmacology' as a sub-cultural aspect of modern societies but with a change in emphasis to active attempts to circumvent modern prohibitive laws.There have thus arisen in conjunction with the development of scientific knowledge sophisticated approaches to drug manufacture in the form of clandestine laboratories.1.52 Although in most cases this trend has increased the problems of controlling drug trafficking and addiction there has recently occurred a case where a toxic by-product that was obtained in such a laboratory may curiously in the long run prove benefi~ia1.l~~ Thus the presence of MPTP as a by-product of the synthesis of MPPP (Figure 11) led to the poisoning and death of heroin addicts with the victims showing the classic symptoms of Parkinson's disease.154 155 Subsequent investigations showed that the symptoms were probably due to the toxic metabolite MPP+.154Since monoamine oxidase B (MA0 B) acts on MPTP to produce MPP+,156*157 its effects may be nullified by using MA0 inhibitors such as deprenyl. An outcome of this episode is that trials have been instituted into the use of deprenyl (a MA0 inhibitor) and tocopherol in the control of the symptoms of Parkinson's disease.lss So ironically the persistent tendency of people to explore the recreational uses of drugs has produced support for the hypothesis that Parkinson's disease is the result of a toxic deprenyl Me 1 MPPf substance (but not necessarily MPP') in the en~ironment.'~~ In addition it is now possible to define a reasonable animal model for what had previously been seen as an anthropocentric disease.16* 3.2 Medicinal Plants Medicinal plants have been the source of therapeutic substances for centuries,15* but single substances (e.g.morphine salicylic acid and quinine) were not isolated until the nineteenth century. With the development of synthetic and analytical techniques chemists modified the structures of active substances in an attempt to improve potency and lessen side-effects. The classic example of this approach is morphine which has undergone considerable investigation. Thus the five fused rings have been systematically pruned,161 leading to sub-structures which retain similar analgesic properties to morphine (Figure 12); in addition the effects of various substituents have been extensively investigated162 (Figure 13).The increased potency of these sub-structures supports the hypothesis of an analgesic pharmacophore consisting primarily of a phenyl ring and a nitrogen atom with substi tuen ts providing differential activity at the various sub-types of opioid receptor. Numerous models for opioid analgesic activity that contain this pharmacophore have been However increasing the complexity of the morphine nucleus has also led to structures of increased potency. For example etorphine (Immobilon) has 8600-times the potency of morphine162 (Figure 13). The discovery and structural determination of the endo- genous opioid peptides methionine-enkephalin (met-enkepha- lin) and leucine-enkephalin (le~-enkephalin*)'~~ 164 prompted a quest for the structural correspondence between morphine and the enkephalins.This problem is still not entirely solved deduction of the active conformation being difficult due to the flexibility of these peptides. So far alternative conformations of opioid tetrapeptides superimposed on morphine and etor- phine and of leu- and met-enkephalin on PET have been pro- p0~ed.l~~ The morphine example and those of other plant products that have been successfully investigated to produce improved justifies continuing the search for active principles of plant products but in a systematic way despite the low success rate. One reason is illustrated by the structure of morphine which being an over-determined rigid analogue of the enkephalins could not have been logically deduced from the enkephalins given their prior discovery.In turn potent molecules such as etorphine would probably not have been discovered. There is also a rational basis for screening natural * To avoid confusion between the N-terminally extended compounds and the parent enkephalins these are often identified as [Met5]enkephalin and [Leu5]- enkephalin see 3AA-22.2 of IUPAC-IUB Recommendations (1983) for Nomenclature and Symbolism for Amino Acids and Peptides. NATURAL PRODUCT REPORTS 1988 Me \ Me I Me methadone (10) \ Me / \ HqN levorphanol (I) Figure 12 The effect of structural changes on morphine activity. The figures in parentheses are consensus Ki values (nmol dm-3) based on a large number of literature values.The dotted arrow shows the conformational similarity between met-enkephalin and PET. products most have similar precursors (e.g.amino acids) to those of animalslG6 and as a consequence are inherently likely to interact with the biopolymers of receptors (e.g.proteins) in an analogous way. 3.3 Toxic Substances As emphasized by Albert,lG7 toxicity is a relative concept the emphasis in drug discovery being on the search for selective toxicity. The investigation of toxic properties of synthetic molecules has been referred to in Section 3.1 in relation to the narcotic MPPP. Amongst natural products atropine mus- cimol tubocurarine and ergotamine are well-known examples whose toxicity initiated enquiry that culminated in discovery of useful drugs.168 Only the ergot alkaloids which have recently been reviewed,165 will be discussed here.The fungus Cluviceps purpureu which is a parasite on rye plants is the source of four main classes of ergot alkaloids169 (clavines lysergic acids lysergic acid amides and ergot peptide alkaloids) whose collective toxicity ‘Me pethidine (lo3) t Me \ OH morphine (2.5) Me \ K’-HO OMe OH etorphine R = Me (0.15) PET R=CH2+ Me \ HO Me Me metazocine (15) met -enkephalin (4) centuries ago includes vomiting diarrhoea thirst convulsions tachycardia confusion coma and hallucinations. 165 Modern investigations have shown that the major class the ergot peptide alkaloids consists of five structural types and their isomers ergotamine ergosine ergocristine ergocryptine and ergocornine (Figure 14).The multitude of symptoms of ergot toxicity prompted a search for structure-activity correlations that would specify particular effects devoid of toxicity. This has led to several useful (see Table 6 and Figure 15). Many ergot-derived structures have had to be rejected either through severe toxic properties or because of their chemical lability in vivo. The search for new derivatives of ergot alkaloids continues with the setting up of host-free culture systems for the fungus and the isolation of novel metabolites together with further chemical manipulation of the various ergot structures. 165 The latter process is based on the recognition of three interrelated clinical biochemical and structural facts the wide variety of biological action of the ergot alkaloids (see Table 6); the multiplicity of their binding activity as shown by the IC, values of eight different ergot alkaloids and their derivatives in first described several NATURAL PRODUCT REPORTS 1988-R.I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS N -vPh 6x1 N -V/\ antagonist reduced 6 14 bridge 3 -OMe 1OOxf diprenorp hin 14-0H lox! 14 -OAc 200x4 14 -p -Me SOOOf 8 other substitution reduces / 6 -N3 potency and/or increases Me \ --14\ toxicity f -17 /*' in 16/7 " -.(+Me / / OH OMe substituents give large etorphine increases 8600x f 70 -WPh 700x 3 -OMe 7xc OH 3 -OAc,! morphine I 5 -Me 6-OMe 10x4 6-H 10x4 6 -N3 50x4 Figure 13 The effect of substituents on morphine activity.?XCONH-9 R1 -H H HN 'J Me CH2Ph ergotamine di hy droergotamine Me i-Bu ergosine i-Pr CH2Ph ergocristine dihydroergocristine 33 % i-Pr i-Bu ergocrypthe dihydroergocryptine a :p 22:11% ergoloid-mesylate i-Pr i-Pr ergocornine dihydroergocornine 33 % Figure 14 Structural types of ergot alkaloids. NATURAL PRODUCT REPORTS 1988 Table 6 Biological activity of ergot-derived Drug Activity Ergotamine Antimigraine Dih ydroergotamine Antimigraine vascular headache orthostatic disorders Me th ylergome trine Obstetric post-partum haemorrhage Methysergide Carcinoid syndrome antimigraine Methergoline Antimigraine vascular headache Dihydroergocristine Antihypertensive venotonic Dih ydroergocornine Dih ydro-a-ergocryptine Ergoloid-mesylate antihypertensive Dihydro-/3-ergocryptine treatment of cerebral insufficiency Nicergoline a-1Adrenoceptor blocker Lysergol An ti hypertensive LSD Hallucinogenic anxiolytic antidepressant Bromocriptine Antiprolactin (galactorrhoea) antidepressant anti-Parkinson's Lisuride Antiprolactin Pergolide Dopaminergic anti-Parkinson's LY 141-865 Antihypertensive R' methylergometrine R = H methysergide R=Me I i-Bu br om ocrip t iile M6 eight receptor-binding assays12' [see profiles (e) and (f) in Figure 101; and the fact that the structures of three of the neurotransmitters whose receptors are affected (dopamine noradrenaline and serotonin) may be considered to exist within the lysergic acid p0rti0n.l~~ It is also possible to match the CCK tetrapeptide analogue Trp-Gly-Gly-Phe topographi- cally onto ergotamine in a low-energy conformation thus establishing a possible structure-activity relationship between CCK and ergotamine.131 So far structural investigations have concentrated on either the intact alkaloids or the truncated lysergic acid moiety. Modification of the tricyclic non-LSD fragment (Figure 14) for which an improved synthesis has recently been p~blished,~'~ could also be fruitful. 3.4 Analogues of Endogenous Molecules The discovery that drugs act on neurotransmitter systems initiated the idea that all or part of the structure of a neurotransmitter matches a portion of the drug.However most drugs and neurotransmitters have a large number of alternative low-energy conformations in which they may bind at their receptors. This has led to the use of the rigid-analogue technique in which a drug structure or its analogue is fixed in MeNl methergoline @ HN nicergoline ly sergol B &,-HN LSD lisuride pergolide LY 141 -865 Figure 15 Useful drugs based on ergot alkaloids. NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS 38 I 0' HNTOoH Muscimol(O.024) Imidazole -4 -ace tic acid (0.24 ) Isoguvacine (1.4) THIP (2.6) LNMe2Cl-H2N-COOH (+) -Bicuculline methochloride (7) GABA (0.34) Baclofen (-) 6 6 rs" H6 HN H HN 0-Proline (14) Isonipecotic acid (15) Nipecotic acid (>100) Homonipeco tic acid (>100) Figure 16 Rigid analogues of GABA.Figures in parentheses are IC, values (pmol dm-3) taken from ref. 172. a given conformation by incorporating a minimum number of necessary connecting atoms without substantially affecting physicochemical properties. For example the dopamine agonist 2-dipropylamino-6,7-dihydroxytetralin (ADTN)17' was devel- oped as a rigid analogue of dopamine. Similarly comprehensive efforts have been made to define the active conformations of GABA either by limiting its abundant flexibility (by using rings double-bonds or restricting groups172) or by studying natural products and their synthetic analogues (muscimol ibotenic acid and bicuculline) in which these devices are already incorp~ratedl~~ (Figure 16).This has resulted in the clinically useful compounds THIP and ba~lofen,~~~ as well as numerous experimental compounds that have advanced our understanding of the pharmacology of GABA.I6' For example it is now likely that three sub-types (A B and C) of GABA receptor exist.174 Similar attempts have been made to define the active conformations of endogenous peptides and this process has already been illustrated for the enkephalins (Section 3.2) and CCK (Section 3.3). In those cases a given natural product (morphine and ergotamine respectively) was assumed to be a rigid analogue of all or part of the peptide molecule.The reverse process in which the endogenous peptide is restricted without knowledge of a natural or synthetic product acting at the same receptor has also been carried out. The methods for introducing conformational restrictions into peptide structures include (i) introduction of cross-linking groups generally disulphides ; (ii) replacement of individual L-amino acids by corresponding D-forms ; (iii) introduction of restrictive ana- logues of the peptide bond (e.g. ethylene cyclopropane or retro-amide groups) ; (iv) replacement of flexible bonds in the peptide backbone with rigid structures (e.g. by using proline rather than existing amino acids) to limit rotation around the C(ol)-N bond. The preceding techniques can be illustrated by the develop- ment of somatostatin analogue^.'^'-^^^ Somatostatin is a peptide hormone named from its ability to inhibit the release of growth hormone but it also inhibits the release of insulin gastrin and other hormones as well as lowering glucogen 1e~els.l~~ The properties suggest several therapeutic possibilities but these are limited by two major problems the peptide is very short- acting due to its rapid metabolism and is not active orally somatostatin has a relatively non-specific activity since it inhibits the release of many different hormones simultaneously.Somatostatin consists of fourteen amino-acid residues. It has been shown by a combination of n.m.r. computer-graphic and structure-activity techniques (using the methods listed above) that this number could be contracted to just five plus a single proline residue to replace the other nine.17' The successive stages of modification are illustrated in Figure 17.The resultant Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys 1 Somatostatin I cyclo-(Aha-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser) 2 I cyclo-(Aha-Lys-C~s-Phe-Phe-D-Trp-Lys-Thr-Phe-Cys-Ser) I 3 4 c y clo -(A ha -P he-Phe-D-Trp-Ly s-Thr-P he) 4 I cydo-( Aha-Cis-Phe-D-Trp-Lys-Thr-C$s) 5 I c y cl o-(Pro-Phe-D-Trp-Lys-Thr-Ala) 6 4 cyclo-(Pro-Phe-D-Trp-Lys-Thr-Phe) 7 Figure 17 Structural modifications of somatostatin cyclic hexapeptide 7,which retains only residues 8 to 10 (8 now being a D-tryptophan residue) has a comparatively restricted conformation and is not amenable to metabolism by trypsin with the result that its activity is significantly longer-lasting than that of somatostatin.Thus 7 continues to show activity after more than five hours while somatostatin has ceased to display action within one hour. The activity of 7 relative to somatostatin is excellent inhibition of the release of growth hormone is 1.74 times greater in vitro and approximately 20 times greater in vivo and inhibition of insulin release is approximately five times more powerful. 17' The compound also displays oral activity. It is presently in clinical trial for the treatment of diabetes and may have potential in other disorders. 3.5 Synthetic Compounds Some of the earliest drug discoveries resulted from screening of synthetic compounds using random procedures with promis- ing candidates being submitted for more extensive examination.Cl~nidine,"~ and diazepam179 are examples chl~rpromazine,'~~ of drugs whose CNS properties were discovered by chance. Whereas twenty to thirty years ago one in 2000 newly synthesized chemicals was successfully marketed,165 the rate today is more difficult to estimate but is probably considerably less. Nevertheless medicinal chemists have continued to synthe- size novel structures by exploiting the combinatorial richness of carbon compounds to form rings chains and multiple bonds that lead to chirality in various forms and to include heteroatoms in order to modify electronic and steric effects.The underlying process of discovery has consisted of making minimal structural changes to lead compounds in response to feedback information from testing these compounds in bio- logical systems. However it is possible that the vast amount of information embodied in the resultant hundreds of thousands of structures could be used as follows to design CNS drugs more rationally. The previously defined common structural model (Section 2.9) based on representative CNS-active drugs not only defines the minimum structural requirements for CNS activity in general but also allows us to define the specific spatial relationships between the secondary binding groups that are important for a particular activity and the primary groups phenyl and nitrogen of the common model.The co-ordinates for these primary groups as well as the secondary binding groups for various classes have been determined. 180*181 Thus the common model leads us to the placement of these primary and secondary binding groups without stipulating the connect- ing framework which is left as a 'wild card' to be achieved by any construction which preserves the prescribed locations. Therefore a procedure for the development of structures that are potentially capable of CNS activity based on the common model would consist of three phases (1) locate the primary groups phenyl and nitrogen on some suitable framework in the topography defined by the common model ; (2) place the secondary binding groups that are necessary for a specified activity in the correct position on the framework.Clearly this step requires some forward thinking in phase 1 to make sure that appropriate atoms are available to which secondary groups can be attached; (3) judiciously remove any unwanted atoms in such a way that the locations of key groups are preserved. Because this approach is topographical carrying out these operations requires viewing the developing structures in three dimensions by using computer graphics (see Section 3.6). A related approach has been suggested by Warrener et al.,lSz whose method termed MOLRACla3 envisages the use of rigid alicyclic systems of known shape and size to space functional groups at exactly designated positions. This concept is not new having been exploited in the design of neuromuscular blocking NATURAL PRODUCT REPORTS 1988 agents,la4 transition-state analog~es,'~~ and opioid an-algesic~.'~~~'~~ However in some cases it has suffered from the limitation of being predominantly a two-dimensional tessela- tion process or else topological in approach.Suitable frameworks and building blocks for this procedure include the many structures that are known to have CNS activity as well as synthetic organic compounds and natural products. Ideally there should be a high degree of rigidity or else limited flexibility in the initial structures to ensure that minimal alteration to the locations of binding groups can occur. Amongst rigid molecules already demonstrated as fitting the common model morphine LSD strychnine mianserin diazepam apomorphine clonidine bicuculline and the ergot alkaloids provide excellent starting points.The polycyclic structure of strychnine suggests the use of cage compoundslaS such as adamantanelas as frameworks on which to graft the necessary groups. 3.6 Drug Design The term 'drug design' has a different meaning for different researchers depending on their area of research. This was emphasized at a recent conference lgowhere drug design was acknowledged to be the rational use of a collection of different methodologies leading to the discovery of a new drug. These methodologies were agreed to have two purposes lead generation and lead optimization lead generation being achieved by first identifying the relevant biochemical pathways and then synthesizing the appropriate agonists and antagonists.However there was no general acceptance of any one technique for lead optimization although QSAR methods1g1 (i.e.methods based on quantitative structure-activity relationships) rated best. Whereas the QSAR methods handle data from the level of organisms down to individual cells and receptor preparations more recent techniques which may be classed as three-dimensional QSAR have been developed which attempt to define the structural arrangement that is adopted by molecules when they bind to a receptor (the recognition process). Approaches to the design of drugs from this viewpoint depend on one or more of three requirements (1) knowledge of the structure of the biological macromolecule involved.For example X-ray structures for various enzymes (e.g. dihydrofolate reducta~e~~~) and hormones (e.g. insulin193) provide clues to the structural requirements of the molecules that bind to them; (2) knowledge of the mechanism involved. For example the involvement of coenzymes and their implied location at the enzyme-substrate reaction interface (as determined from molecular-orbital calculations) allows the transition-state geometry of the substrate to be approximated thus providing a basis for the design of transition-state an-alogues ;la5 (3) knowledge of the structures and properties of the drugs involved. CNS drugs fall into this category; whereas comparatively little is known about the three-dimensional structure of CNS receptor-binding sites there is an abundance of information about the structures of an lg5 enormous range of different CNS-active Having postulated a pharmacophore the strategy is to discover from the structures of drugs of known activity the common three-dimensional relationship between key atoms in groups of different molecules.However since many CNS- active drugs show a diversity of structure as well as several degrees of conformational freedom resulting in some cases in there being many millions of conformers to be considered,lg6 the problem then becomes one of reducing the number of possibilities. The most likely conformers are usually determined (on an energetic basis) from X-ray crystallography lS7from nuclear magnetic resonance (n.m.r.) spectroscopy lg8and from potential-energy calculations.lS9 Being derived from the solid NATURAL PRODUCT REPORTS 1988-R. I. BRINKWORTH E. J. LLOYD AND P. R. ANDREWS (X-ray) solution (n.m.r.) or isolated states (molecular-orbital and molecular-mechanics) the resultant conformers represent a highly informed guess at those adopted in what has been called the fourth or biological state.200 Further reduction in the number of possibilities is achieved by using techniques such as distance geometry201 and the active-analogue approach,lg6 whereby optimal molecular geometries are correlated with biological potency. The role of computers in drug design has been emphasized in recent books and reviews which show their use in organizing and discerning patterns from data as well as in performing large-scale calculations or for completely searching the con- formational space of a molecule.187,202-208 Other applications include mathematical modelling,209 computer-graphic analysis of macromolecule-substrate interactions,210 and searching databa~es.~l'-~l~ The major techniques of computer-assisted drug design have been ranked by Hopfinger204 in a hierarchy of their applications. Many software packages are a~ailable~~~*~'~. Ia7 for drug design and most contain programs for (1) data acquisition and model building; (2) calculation of molecular properties and conformational energies ; (3) display and manipulation of structures in an interactive computer-graphic mode ;and (4) plotting in two and in three dimensions.4 Conclusion Drug discovery has been based on the investigation of structural and mechanistic analogies between natural and synthetic molecules and endogenous counterparts in the CNS (Figure 18). This approach rests on the large body of knowledge showing that biochemical pathways and organic reactions use the same bonding interactions and mechanistic processes. Examples include the many enzyme-based reactions215 (hydro- lysis condensation transamination) coenzyme reactions216 (e.g. catalysis of carbanion reactions by thiamin diphosphate) rearrangement^'^^ (the Claisen rearrangement of chorismate to prephenate) (pheno1,c coupling) and cyclizations2ls (the Pic tet-Spengler reaction).Medicinal chemists and biochemists find the use of structural and mechanistic analogies attractive mainly because they are consistent with life processes being a continuum of universal chemical processes. Thus concomitant with biological evolu- tion of shape and function there has been evolution of chemical Evidence to support this includes the well- established fact of the evolution of proteinsZ2O (e.g.proteolytic enzymes) with its implication that receptor proteins probably evolved into sub-types to provide subtle ways of attenuating electrical signals ;221 the evolution of neurotransmitter func-tions as shown by the wide phylogenetic distribution of neurotransmitters222 and by the recent demonstration that Figure 18 Structural and mechanistic analogies between natural and synthetic molecules.The shaded area emphasizes the common biosynthetic link between organisms. NATURAL PRODUCT REPORTS 1988 biochemical agents of vertebrate endocrine and nervous systems probably orginated in unicellular organisms ;219 and the widely accepted scenario for the evolution of living cells from non- living materials i.e. chemical Two other findings support the pursuit of drug design based on the use of structural and mechanistic analogies. First there is the discovery that low-energy conformations of representatives of different classes of CNS drugs together with their rigid analogues and neurotransmitter molecules share a common topographic arrangement of drug-receptor binding groups (i.e.a common pharmacophore Section 2.9). This common structural component which has been extensively verified for 67 CNS drugs,lso*lel provides a ‘skeleton key’ to which all CNS drugs may conform in binding to their receptors. Secondly it has long been recognized that chemical reactions whether biochemical or synthetic proceed along pathways with minimum breaking and making of bonds (i.e. with a redistri- bution of a minimum number of valence electrons). This mechanistic concept has recently been quantified into a prin- ~iple~~~ -‘the principle of minimum chemical distance’ (PMCD)-and the resultant algorithm has been used to demonstrate and successfully to predict both biochemical (e.g. the isoprene rule) and synthetic (e.g.the synthesis of strychnine) pathways.224 The key roles that are played by a-amino acids such as glutamic acid and tyrosine (Figure 18) whether as building-blocks in proteins at active sites of enzymes and receptors or biotransformed into neurotransmitters become obvious.Thus there is a common structural basis on which drugs and neurotransmitters act at CNS receptors which may be further related to a common mechanistic principle by which neuro- transmitters and receptors have synergistically evolved along well-established biochemical pathways. The mechanistic prin- ciple provides a nexus between our understanding of brain biochemistry and the frequent use by medicinal chemists of structural analogies (Figure 18). 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ISSN:0265-0568    

DOI:10.1039/NP9880500363

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

The Biosynthesis of Triterpenoids Steroids and Carotenoids D. M. Harrison Chemistry Department University of Ulster Coleraine Co. Londonderry Northern Ireland B T52 ISA Reviewing the literature published between January 1984 and December 1985 (Continuing the coverage of literature in Natural Product Reports 1985 Vol. 2 p. 525 and 1986 Vol. 3 p. 205) 1 Introduction 2 Mevalonic Acid 3 The Biosynthesis of Squalene from Mevalonic Acid 4 The Biosynthesis of Triterpenoids from Squalene 5 The Formation of Sterols in Vertebrates 5.1 The Biosynthesis of Cholesterol 5.2 The Biosynthesis of Steroidal Hormones 5.3 The Biosynthesis of Bile Acids and the Metabolism of Vitamin D 6 Triterpenoids and Steroids in Higher Plants Algae and Fungi 6.1 The Biosynthesis of Sterols in Fungi 6.2 The Biosynthesis of Phytosterols 6.3 Alkylation of the Sterol Side-chain 6.4 Further Metabolism of Steroids and Triterpenoids 7 Tri terpenoids and Steroids in Invertebrates 7.1 Insects 7.2 Other Invertebrates 8 The Biosynthesis of Carotenoids 9 References 1 Introduction In earlier volumes of this journal the biosynthesis of triterpenoids and steroids' was treated separately from the biosynthesis of carotenoids.2 These two complementary strands are brought together again in this Report.The papers that are discussed were selected mainly through the perusal of Chemical Titles and of the major chemistry and biochemistry journals for the appropriate period.As before it has often been difficult to decide which publications to discuss and which to exclude. This was particularly true of Sections 2 5.2,and 5.3 for which I have endeavoured to select those papers that present new results of interest to the biosynthetic chemist from among the overwhelming number of related publications that are mainly of medical or biological interest. Studies on the isolation purification properties and inhibition of the enzymes of biosynthesis are treated in an illustrative rather than a comprehensive fashion. Two volumes of Methods in Enzymology are devoted in large HO ,Me n R CO,H (11 a; R = CH,OH b; .R = CH(OHIS-CoA c; R = C(0)S- CoA d; R=CH,O@ 0 II 5 e; Rz CH20-P-0-P-OH I 1 OH OH HOJ3Y \ (2) a; RL tyy b; R = + c; R = fl membrane- bound ;soluble enzymically active proteins of lower molecular weight are formed by adventitious proteolysis of the native enzyme during its i~olation.~ The native reductase of rat liver is a dimer,6 the monomer units of which are linked by a carbohydrate residue.' A human HMG-CoA reductase has part to the enzymes of steroid and carotenoid biosynthe~is.~ been prepared by using cloned CDNA;~ this reductase which Some individual contributions to these works and other reviews will be cited at appropriate places in the text.2 Mevalonic Acid Mevalonic acid (la) is formed via the thiohemiacetal (lb) by the stepwise reduction of (3S)-3-hydroxy-3-methylglutarylco-enzyme A (HMG-CoA) (lc) with NADPH catalysed by the enzyme hydroxymethylglutaryl-CoA reductase (NADPH) [E.C.1 . 1 . 1 . 341. The mammalian reductase remains the subject of intensive study owing to its critical role in the regulation of the biosynthesis of cholesterol (2a). The complete amino-acid sequence of the reductase of hamster has been deduced from sequence studies on the cloned cDNA.~ This reductase is a glycoprotein of mol. wt. 97092 containing 887 amino-acid residues the N-terminal domain of which remains contains 888 amino-acid residues shows close homology with the enzyme of hamster especially in the catalytic domain and in the membrane-bound region. The latter region is responsible for the sterol-regulated degradation of the enzyme.8 Antibodies that were raised to the HMG-CoA reductase of rat liver cross- reacted with the reductase of human rig in.^ It is known that mammalian HMG-CoA reductase (NADPH) is subject to rapid modulation of activity by means of a reversible phosphorylation4ephosphorylation cycle.'.'* The less active (phosphorylated) form of the enzyme is re- activated by dephosphorylation catalysed by endogenous non- specific phosphatases."In the reverse direction the more active form of the reductase is partially inactivated by ATP-dependent phosphorylation catalysed by the enzyme [hydroxymethyl- glutaryl-CoA reductase (NADPH)] kinase [E.C. 2. 7. 1 . 1091 which is itself subject to modulation of activity by reversible 387 phosphorylation ;the phosphorylated reductase kinase is the more active form.'.'O A reductase kinase of rat liver has been purified to homogeneity.12 Further evidence has been adduced for the contention that the short-term modifications of the activity of mammalian HMG-CoA reductase (NADPH) that can be achieved by altering the diet of experimental animals is due to reversible phosphorylation of the enzyme.13.l4 In particular admini- stration of mevalonolactone to the rat stomach resulted in a 38% reduction in the activity of the reductase of the liver within 20 minutes in parallel with an increase in the level of phosphorylation both of the reductase and of the reductase kinase.14 Furthermore it has been shown that the fraction of the reductase of rat liver that is in the active (unphosphorylated) form is subject to diurnal variation in vivo.15 It was suggested earlier that the regulation of the chol- esterol-utilizing enzymes cholesterol acyltransferase [E.C.2.2.3 .1 .26] and cholesterol 7a-mono-oxygenase [E.C. 1 .14. 13 .171 is linked in each case by means of a phosphorylation-dephosphorylation cycle to the regulation of HMG-CoA reductase (NADPH) and that the former two enzymes are both more active in the phosphorylated state.l Doubt has been cast on the validity of this proposal by the report that conditions which modulated the activity of the reductase by reversible phosphorylation had no effect on the activity of the acyltransferase. l6 Furthermore a cytosolic protein which stimulated the mono-oxygenase in the presence of glutathione had no effect on the reductase; moreover neither the reductase kinase nor phosphoprotein phosphatases had any effect on the activity of the mono-0~ygenase.l~ The fungal metabolite compactin (3a) (also known as ML- 236B) and related compounds are important as potential hypocholesterolemic agents that function by inhibiting HMG- CoA reductase (NADPH).18 Several mammalian cell lines are resistant to compactin owing to an extraordinary over-production of the enzyme.Such cell lines are important sources of the mammalian reductase (e.g. ref. 4). Studies have been reported on the mechanisms of over-accumulation of HMG- CoA reductase (NADPH) in compactin-resistant cells19 and on the mode of interaction of compactin with the HMG-CoA reductase of the yeast Saccharomyces cerevisiae.2o Several structural analogues of compactin that were isolated from Monascus ruber have been described.21 The growth of tissue explants of Helianthus tuberosus was inhibited by compactin (3a) or mevinolin (3b); the addition of mevalonic acid resulted in restoration of growth.22 New synthetic inhibitors of HMG- CoA reductase (NADPH) include derivatives of 5-alkylated NATURAL PRODUCT REPORTS 1988 of rat liver microsomes but both were found to be allosteric activators of the enzyme; various analogues of these coenzymes have been tested as potential activators of the reducta~e.~~ Kinetic studies have been reported on the reductase of radish.30 The sub-cellular distribution of the HMG-CoA reductase and of mevalonate kinase [E.C.2. 7. 1 .36] in leaves of Nepeta cataria has been in~estigated.~~ 3 The Biosynthesis of Squalene from Mevalonic Acid The enzyme mevalonate kina~e~~ [E.C. 2.7. 1 .361 catalyses the reaction between mevalonic acid and ATP in the presence of a divalent metal cation (usually Mg2+) to furnish 5-phosphomevalonic acid (Id) and ADP. The manner in which the metal cation is bound to the ionized triphosphate unit of ATP at the active site of porcine mevalonate kinase has been determined by kinetic studies in which a series of adenosine 5'-thiotriphosphates were utilized as analogues of the natural substrate and a range of metal ions were used including Cd2+ and Mg2+.33 Procedures have been described for the puri- fication and assay of porcine phosphomevalonate kinase [E.C.2. 7.4. 2].34 The thiodiphosphate (le) of mevalonic acid has been prepared with the aid of the latter The ATP-requiring enzyme diphosphomevalonate decar-boxylase [E.C. 4. 1 .1 ,331 has been isolated from the lemon grass Cymbopogon citratus. The enzyme has an optimum pH of 6.0 and requires Mg2+ (or Mn2+) ions for a~tivity.~~ On the basis of responses to dietary changes it has been suggested that the decarboxylase may have a role in the regulation of cholesterol biosynthesis in the The fluoromevalonate derivative (4a) inhibits the incorporation of mevalonic acid [but not of isopentenyl diphosphate (5a)l into cholesterol in a rat liver enzyme system.38 The observed inhibition is a result of the metabolic conversion of the alcohol (4a) into the diphosphate (4b) which is a potent competitive inhibitor of diphospho-mevalonate decarboxylase.The isolation of diphosphomeva- lonate decarboxylase from avian liver has been described. 39 The equilibration of isopentenyl diphosphate (5a) with dimethylallyl diphosphate (6a) is catalysed by the enzyme isopentenyl-diphosphate A-isomerase [E.C. 5.3.3.2].40 The subsequent condensations of dimethylallyl diphosphate with isopentenyl diphosphate to furnish geranyl diphosphate (7) and of the latter with isopentenyl diphosphate to give 2-trans,6- trans-farnesyl diphosphate (8) are both catalysed by the enzyme dimethylallyltranstransferase [E.C. 2. 5 .1 .11 which is often referred to as farnesyl pyrophosphate synthetase or 3,5-dihydroxypentanoic acids and the derived B-la~tones,~~ prenyltransferase.The latter term is sometimes used also to various 3-alkyl-3-hydroxypentanedioic and a non-reducible analogue of the thioacetal (I b).25 Hydroxymethylglutaryl-CoA reductase (NADPH) is in-hibited if it is incubated with a soluble protein of rat liver named fermodulin in the presence of Fe2+ ions. The inhibitory protein has been purified and characterized.26 The effects of thi01s~~ and of coenzyme A derivatives28 on the activity of the rat liver reductase have been described. Neither NAD' nor NADH is a substrate for the HMG-CoA reductase (NADPH) HO ,H A (3) a;R=H b; R= Me encompass geranylgeranyl-diphosphate synthase activity. The names that were used for these enzymes in the papers that are cited below will be retained in this Report.The fluorinated derivatives (5b) and (6b) of isopentenyl diphosphate and dimethylallyl diphosphate respectively each proved to be potent inhibitors of the isopentenyl-diphosphate A-isomerase of Claviceps SD58 apparently owing to covalent modification of the enzyme.41 Furthermore the amine (9) inhibited the isomerase of the same strain of Clavi~eps~~ and of Saccharomyces cerevisiae ;42 presumably the protonated amine acts as an analogue of the carbo-cation that is formally an intermediate in the enzyme-catalysed equilibration of (5a) and (64. The three homologues (5c) (5d) and (5e) of isopentenyl diphosphate and the two homologues (6c) and (6d) of dimethylallyl diphosphate were each converted into the same equilibrium mixture in which the homoallylic diphosphate (5d) predominated when incubated with the isomerase of pig liver.43 The reactions that took place in deuteriated water were also studied.The results were interpreted in terms of a model for the topology of the active site and the postulated involvement of two catalytic groups for proton abstraction and proton donation respectively. Isopentenyl pyrophosphate isomerase farnesyl pyrophos- NATURAL PRODUCT REPORTS 1988-D. M. HARRISON HO CH,F R24jR3 R2 R' R' RO H 13 (&)a; R= H (5) a; R =R 2=R =H ( 6) a; R1= R2=H b; R= O@ b; R1=F R2=R3=H b; R'. F R2= H c; R~=M~, H 2= H c; R1:Me R2= R3=H d; R1=R3=H R2=Me d; R':H R2z Me e; R'= R2=H R3=Me Me (7) 0 \ 3 P-CR 21 OH (10) (11 1 (13)a; R=@ b; R = @;2,3-dihydro C; R= H d; R H ;2,3-dihydro OR (14) a; R= b; R=H -11 phate synthetase and geranylgeranyl pyrophosphate synthetase have been detected in cell-free extracts of the silkworm Bombyx mori and partially purified.44 Two non-interconvertible forms I and 11 of the farnesyl pyrophosphate synthetase were identified;44 synthetase I1 showed preference for the homo- dimethylallyl diphosphate (6c) and the homoisopentenyl di- phosphate (5c) as substrates and may be involved in the biosynthesis of juvenile The enzymes isopentenyl pyrophosphate isomerase and prenyltransferase have been isolated from plastids of tomato fruit and purified.4s Both enzymes required Mg2+ or Mn2+ ions for activity ;the isomerase was totally inactivated by iodoacetamide at a concentration of 1 mmol dm-3.46 An affinity-chromatography technique has been described for the rapid and efficient one-step purification of farnesyl pyrophosphate ~ynthetase.~' The latter enzyme is regarded as requiring Mg2+ or Mn2+ cations for activity; it has now been reported that the enzyme from porcine or avian liver 16 shows higher catalytic activity in the presence of Zn2+ ions (0.2 mmol dm-3) instead.48 The unnatural substrates (10) and (11) each furnished a mixture of monocyclic diphosphates in which the cis-com- pound (1 2) predominated when incubated with avian farnesyl pyrophosphate syntheta~e.~~ The phosphonyl phosphates (13a) and (13b) were shown to be powerful inhibitors of pig liver prenyltransferase ;not surprisingly the phosphonates (13c) and (13d) were less good as inhibit01-s.~~ It was suggested earlier that the dimer (14a) of 2-trans 6-trans-farnesyl diphosphate is an intermediate in the bio- synthesis of squalene (15).The racemic diol (14b) has been synthesized with a view to testing that propo~al.~' Practical accounts have been published concerning the isolation of prenyltransferases from yeast,52 from liver,52. 53 and from pumpkin.54 The isolation of squalene syntheta~e,~~ and the aberrant formation of 12-cis- 12,13-didehydro~qualene,~~ were NPR 5 NATURAL PRODUCT REPORTS 1988 discussed also.The reactions that are catalysed by prenyl- transferase and squalene synthetase have been re~iewed.~’ The unicellular alga Botryococcus braunii synthesizes a range of hydrocarbons including the irregular triterpene (1 6) and its homologues. In a series of preliminary experiments sodium [U-14C]acetate and [U-14C]leucine were incorporated into (1 6) in 11.8% and 5.2 % yields respectively while [2-14C]mevalonate was incorporated to an extent of only 0.2 %.58 4 The Biosynthesis of Triterpenoids from Squalene The next step in the formation of sterols is the oxidation of squalene to furnish (3S)-squalene 2,3-epoxide (17). The action of the enzyme squalene epoxidase [squalene mono-oxygenase (E.C. 1 . 14.99.7)] in a washed microsomal fraction of the yeast Candida albicans was studied utilizing the conversion of [4,8,12,13,17,2 1-3H]squalene into [3H]squalene epoxide as the assay.59 The epoxidase system required molecular oxygen NADH or NADPH and FAD for activity. Unlike the epoxidase of rat liver the Candida enzyme was more active with NADH than with NADPH and its activity was destroyed if it was mixed with Triton X-100. The epoxidase system was stimulated by a soluble fraction from the cytoplasm.59 The antimycotic agents naftifine and SF 86-327 are potent inhibitors of the squalene epoxidase of fungi; the epoxidase of rat liver is much less sensitive to these compounds.6o The isolation of squalene epoxidase from rat liver microsomes has been reviewed.61 Further studies have been reported on the role of ‘super- natant protein factor’ (SPF) in the stimulation of mammalian squalene epoxidase; it has now been shown that SPF in the presence of anionic phospholipids stimulates uptake of squalene by microsomes as well as stimulating the epoxidase.62 The efficacy of SPF in stimulating squalene epoxidase is destroyed in a time- and concentration-dependent manner if it is incubated with ATP especially in the presence of Mg2+ ions.63 Further evidence has been adduced (by 13C n.m.r.) in support of the proposal that the biosynthesis of the pentacyclic triterpene ursolic acid (19) folldws the route that is summarized in Scheme 1.64 Thus [3,5-13C2]mevalonate was incorporated into ursolic acid by cell cultures of Perilla frutescens var.acuta with the labelling pattern shown in (19a).In particular the observation of coupling between 13C-labelled carbons 16 and 17 and between C-20 and C-21 demonstrated for each coupled pair of carbon atoms that the bond between them was formed by a Wagner-Meerwein rearrangement within the same mevalonate unit. Furthermore the B-isotope shift that was observed for C-30 in ursolic acid that had been labelled biosynthetically following a feeding experiment with [2-13C 4,4-2H2]mevalonate confirmed that the postulated 1,2-mi-gration of hydride ion from C-19 to C-20 (ursene numbering) had occurred as shown in Scheme l(b).64 The results provide compelling evidence for the essential features of the biosynthetic scheme that was advanced over 30 years ago by Ruzicka et aLS5 It was established earlier that protonated 2-aza-2,3-dihydro- squalene (20a) and related compounds are potent inhibitors of the enzyme-catalysed cyclization of squalene 2,3-epoxide to ( 17 I 1 A (18) 1 A I J 21 (a) o=13c (b)r=’k Scheme 1 NATURAL PRODUCT REPORTS 1988-D.M. HARRISON furnish triterpenoids. 1,Tt has been reported that the corre- sponding N-oxides especially (20b) are yet more potent as inhibitors of the conversion of squalene epoxide both into lanosterol (21 a) [catalysed by lanosterol synthase (E.C. 5 .4 .99 .7)] in rat liver homogenates and into /3-amyrin (22) in enzyme extracts of pea.s7 Presumably these inhibitors act by mimicking the formal carbo-cation intermediate that is gener- ated by protonation of squalene epoxide.In contrast the ammonium ion (23) was a poor inhibitor of the cyclization of squalene epoxide to /3-amyrin in an enzyme system from pea despite being a structural analogue of the (formal) intermediate cation (1 8).fis The decalins (24a) and (24b) which are inhibitors of lanosterol synthase in hamster cells act as non-competitive inhibitors of the synthase in rat liver systems and do not inhibit the cyclization of squalene epoxide to triterpenoids in plants. Aza-derivatives such as (24c) showed no inhibitory activity towards the cyclization of squalene epoxide in any system that was tested.69 The antimalarial drug chloroqwine blocks the biosynthesis of sterols in mammalian cells by inhibiting lanosterol ~ynthase.’~ Other inhibitors of the latter enzyme have been st~died.’~ The bicyclic triterpenoid (25) isolated from gum mastic (the resin of the shrub Pistacia lentiscus) may be regarded as the product of the trapping by water of a cationic intermediate in the cyclization of squalene epoxide to tetracyclic and pentacyclic triterpen~ids.~~ The bicyclic hydrocarbons (26a) and (26b) similarly may be considered to be products of the interruption of the proton-initiated cyclization of squalene to furnish pentacyclic triterpenoid hydrocarbon^.^^ The prokaryotes in general lack the ability to synthesize steroids at least in quantity though there are some notable exception^.'^ On the other hand triterpenoids of the hopane variety are widespread among the prokaryotes and may constitute the evolutionary forerunners of It has been shown that the biosynthetically intriguing C, bacterio-(20)a;X=H R=Me b;X=O-,R=Et (22) H HR O W ( 24)a;R= Me ,XI CH2 b ;R H,XzCH2 c ;R= H,X = NHCH2Ph 1 (21) a; R=R2=Me 1 b; R=Me,R2=H ; R’’=R~= H hopanepolyols co-occur with their 3P-methyl-derivatives (27a) (27b) and (27c) and also that 3P-methyldiplopterol (28a) is formed alongside diplopterol (28b) in Acetobacter pas-teurianus subsp.pasteurianus. The 3p-methyl-hopanoids be- come the major triterpenoids when the latter species is grown in the presence of methi~nine.~~ Other bacteria have furnished NATURAL PRODUCT REPORTS 1988 5 The Formation of Sterols in Vertebrates 5.1 The Biosynthesis of Cholesterol Studies have been reported on the conversion of [24,2S3H]- 24,25-dihydrolanosterol (29) into 4,4-dimethyl-5a-cholest-8-en-3p-01 (Scheme 2) by rat liver microsomes in which oxidative 2P-methyl-hopanoids such as 2p-methyldiplopterol (28~).~~ demethylation at C-4 was inhibited by cyanide ion (1 mmol The results of biosynthetic feeding experiments in which either label from a mixture of [Me-3H]methionine and [Me-14C]- methionine or from [Me-2H3]methionine was incorporated into 2p- and 3p-methyl-hopanoids were consistent in each case with the transfer of an intact methyl group.77 In particular 2/3-[2H3]- methyldiplopterol and 3P-[2H,]methyldiplopterol were formed when L-(Me-2H3)methionine served as precursor in Methylo- bacterium organophilum or in Acetobacter pasteurianus subsp.pasteurianus respectively. The mixed bacteriohopanepolyols (27a) (27b) and (27c) from the latter experiment were degraded to the alcohols (27d) (27e) and (270 respectively prior to their assay (by mass spectrometry of the derived acetates). In each case the mass spectrum confirmed the presence of 2H3- labelled species though the precise location of isotopic label was not determined. The attractive possibility that the 3p- methyl-hopanoids could arise from initiation of the cyclization of squalene by means of a (formal) methyl cation is not readily reconciled with the formation of 2P-methyl-hopanoids ; it was suggested that both series of triterpenoid are formed by alkylation of a A2-olefin prec~rsor.’~ (27) a; R = W2CH (0H)CHIO H)CH (0H)CYOH b; 6,7-didehydro; R = CH,CH(OH)CH(OH)CH(OH)CH20H C; R = CH(OH)CH(OH)CH(OH)CH(OH)CH20H d; R = CH2CH2OH e; 6,7-didehydro ;R=CH,CH,OH f ; R CH,OH I _____, 02 NA DPH H dm-3).78 The conditions that were used for the microsomal oxidation of the dihydrolanosterol could be manipulated to permit the accumulation of the oxygenated intermediates (30) and (31) or the conjugated diene (32) or 4,4-dimethyl-h-cholest-8-en-3P-ol (33).It is known that the oxidation of the 14a-methyl group of 24,25-dihydrolanosterol is performed by a cytochrome-P-450-based mixed-function oxidase system. The cytochrome P-450 component has now been solubilized and partially purified.7s The reductase that catalyses the NADPH-dependent re-duction of 4,4-dimethyl-5a-cholesta-8,14-dien-3~-ol (32) to the As-sterol (33) in rat liver microsomes has been studied;79 anaerobic conditions were chosen to prevent further meta- bolism of the latter sterol.It was found that the reductase was inhibited by reagents that bind to thiol groups and by the drug AY-9944 but not by cyanide ion. The enzyme displayed its maximum activity at pH 7.4 and there was no requirement for a metal ion. The membrane-bound enzyme has been partially purified ;in particular the reductase activity has been separated from the other microsomal enzymes of cholesterol biosynthesis. n - 02 NADPH NADPH O2 J Jc HC02H Scheme 2 NATURAL PRODUCT REPORTS 1988-D.M. HARRISON The purified reductase showed essentially the same properties as the membrane-bound en~yme.’~ The penultimate step in the biosynthesis of cholesterol (2a) is the oxidation of 5a-cholest-7-en-3P-01 (34) to give the conju- gated diene (35) (Scheme 3). The ‘desaturase’ enzyme has been solubilized and purified 70-fold from rat liver microsomes.80 The purified enzyme required cytochrome b and cytochrome- b reductase [E.C. 1 .6.2. 21 for activity; the reconstituted enzyme system converted (34) into (35) in the presence of NADH and oxygen. It was shown also that one mole of NADH was consumed for each mole of diene formed (but for technical reasons this stoicheiometry was determined by using an epimer a-NADH of the natural coenzyme); it follows that the enzyme system is a mixed-function oxidase.Particularly noteworthy was the removal of the 4-methylsterol oxidase activity from the desaturase during purification of the latter.s0 Thus the two activities are not associated with the same terminal oxidase contrary to an earlier suggestion. It was reported previously that the microsomal de-hydrogenation of Sol-cholest-7-en-3P-01 (34) to the diene (35) was promoted by a ‘sterol carrier protein’ (SCP) and it was suggested that SCP may be identical to ‘fatty acid binding protein’ (Z-protein). The identity of SCP with Z-protein has now been established.s’+82 It has also been claimed that the apparent binding of sterols to Z-protein is an artefact of the assays used but that ‘sterol carrier protein 2’ (SCP-2) which was implicated in the microsomal conversion of lanosterol into cholesterol and in the metabolism of the latter in the adrenal cortex is distinct from Z-protein.82 The primary structure of the SCP-2 from bovine liver has been The literature on sterol carrier proteins SCP (i.e.Z-pr~tein)~~ 86 and SCP-285* has been reviewed. The natural minor steroids (24S)-24,25-epoxycholesterols7~ 88 (2b) and 25-hydroxycholesterolss (2c) both inhibit the biosynthesis of cholesterol in mammalian cell cultures ; the main site of action in each case is HMG-CoA reductase (NADPH). A series of derivatives of 22-hydroxycholesterol has been tested both in vitro and in vivo as inhibitors of HMG- CoA reductase (NADPH); some of these compounds showed promise as hypocholesterolemic agents.89 Other oxygenated sterols inhibited the biosynthesis of cholesterol from 24,25- dihydrolanosterol in an enzyme system from liver analogues of lanosterol with lengthened side-chains were ineffective as H (31) inhibitors of the biosynthesis of cholesterol from lanosterol in the same enzyme ~ystem.~’ The biosynthesis of cholesterol from acetyl-coenzyme Ag2 and the membrane-bound enzymes which effect the biosynthesis of cholesterol from squaleneg3 have been reviewed.5.2 The Biosynthesis of Steroidal Hormones The first committed step in the biosynthesis of the steroidal hormonesg4 is the oxidative cleavage of cholesterol to furnish pregnenolone (36a) and 4-methylpentanal.This transformation requires oxygen and NADPH is catalysed by a cytochrome-P- 450-dependent mixed-function oxidase [cholesterol mono-oxygenase (side-chain-cleaving) (E.C. 1 .14. 15 .6)] and in- volves the transient formation of (22R)-22-hydroxycholesterol (37a) and (22R)-20,22-dihydroxycholesterol (37b). Powerful new evidence for the intermediacy of these two compounds was obtained by studying the kinetics of oxidation of cholesterol by a reconstituted enzyme system that included the side-chain- cleaving cytochrome P-450 [cytochrome P-450,,,] of adrenal mitochondria and the iron-sulphur protein adrenod~xin.~ In particular by utilizing anaerobic conditions for reductive steps it was possible to perform successive single re-duction-xygenation cycles on cholesterol and to observe the sequential appearance of 22-hydroxycholesterol (37a) 20,22- dihydroxycholesterol (37b) and pregnenolone (36a).95 The cytochrome P-450,, of bovine adrenal mitochondria consists of a single polypeptide chain with two independently folded domains; the N-terminal domain contains the haem- binding and the adrenodoxin-binding The complete amino-acid sequence of the cytochrome has been It has been shown that the sterol carrier protein SCP-2 stimulates the biosynthesis of pregnenolone from cholesterol by assisting the transfer of the latter steroid from the outer to the inner membrane of adrenal mitochondria.98 Several C, derivatives of cholest-4-en-3-one all of which contained a 22R- hydroxyl group were oxidized to progesterone (38a) by adrenal mitochondria ;the parent compound cholest-4-en-3-one was It not metaboli~ed.~~seems likely that these observations merely reflect the lack of specificity of cytochrome P-450,, for its normal substrate.100 Studies continue on the development of mechanism- based inhibitors of cytochrome P-450,,,.The silicon-containing c (35) Scheme 3 [as (3611 H 137)a ; R’=H,R’= OH I361 a; R= H b; R=OH b ;R1=R2= OH 1 2 C; R=H,R =NH2 (38)a; R1=R2=H b; R’= CN ,R2=H c; R1= H R2= OH 394 analogue (39a) of cholesterol caused the inactivation of cytochrome P-450,, in a time-dependent fashion perhaps owing to its oxidation by the enzyme to form a transient 22- carbo-cation followed by silylation of the enzyme [see arrows in structure (40)].'01 (22R)-22-Aminocholesterol (37c)lo2 and the analogues (39b) and (39c) with shorter side-chains,'O3 were each potent inhibitors of cytochrome P-450,,,.It is believed that in each case the inactivation arises from binding of the inhibitor to the cholesterol-binding site on the cytochrome in such a way that the amino-group co-ordinates to the haem iron. Various inhibitors of cytochrome P-450,, have been tested for their ability to inhibit the production of corticosterone by rat adrenal cells.'04 Model systems have been described for the cleavage of the triol (37b) catalysed by cytochrome P-450,,,.105 The biosynthesis of progesterone (38a) involves the oxidation of pregnenolone (36a) to give the dione (41a) [catalysed for example by the NAD+-dependent enzyme 3(or 17)/3-hy- droxysteroid dehydrogenase (E.C.1 .1 .1 .51)] followed by isomerization of the dione [catalysed by steroid A-isomerase (E.C. 5.3.3. l)]. 2a-Cyanoprogesterone (38b) is a potent inhibitor of the biosynthesis of progesterone from [7-3H]- pregnenolone in mammalian cell-free enzyme systems ;it was shown that only the dehydrogenase was inhibited in these .systems.'O6 However 201-cyanoprogesterone was shown to be an irreversible inhibitor of the A-isomerase in the bacterium Pseudomonas testo~teroni.~~' An incompletely identified enzyme that catalyses the NAD+- or NADP+-dependent oxidation of pregnenolone (36a) to furnish pregn-5-ene-3,20- dione (41 a) has been isolated from bovine adrenal microsomes and purified.This dehydrogenase also oxidized 17a-hydroxy- pregnenolone (36b) to the dione (41 b).lo8 The biosynthesis of androst-4-ene-3,20-dionefrom pro-gesterone (38a) involves two discrete steps namely (i) hydroxyl- ation of progesterone to furnish 17a-hydroxyprogesterone (38c) and (ii) cleavage of the 17-20 bond of the latter. Both activities are believed to reside at the same active site of a cytochrome-P-450-dependent hydroxylase which will also convert pregnenolone into dehydroepiandrosterone (42) via 17a-hydroxypregnenolone (36b). The components of the hydroxylase have been isolated from mammalian sources and purified.109-111 The enzymic activity (1 7a-hydroxylase/ 17,20- lyase) of the cytochrome P-450 component could be re-constituted in vitro by addition of NADPH-cytochrome-P-450 reductase ; the reconstituted enzyme system required NADPH and molecular oxygen for the conversion of progesterone into 5a-andro~t-4-ene-3,20-dione.~~~.Other workers have identified a requirement for cytochrome b, cytochrome-b reductase [E.C. 1 .6.2.2] and NADH for reconstitution of enzymic activity under conditions of low NADPH concentration."';"f."O It was confirmed that oxygen- 18 is incorporated into the 17-carbonyl group of the product i.e. androstenedione when progesterone is incubated with the reconstituted testicular enzyme system in an atmosphere of [180]oxygen.111 The latter enzyme system also oxidized tes- tosterone to androstenedione ;no oxygen- 18 was found in the androstenedione that was formed when the enzymic oxidation of testosterone was conducted under an atmosphere of [180]oxygen.l1 The A16-steroid androsta-4,16-dien-3-one (43) represents an alternative fate for progesterone in porcine testicular micro- somes.It has been claimed that the A16-steroid-forming activity arises from the 17a-hydroxylasel 17,2O-lyase activity (discussed above) and is promoted by cytochrome b,.l12 Androsta-5,16- dien-3/3-01 (44)is converted by rat liver microsomes into the a-epoxide (45) and thence into the triol (46).lI3 The biosynthesis of oestrogens from androgens which involves the placental enzyme system 'aromatase ',remains the subject of intensive mechanistic study. Earlier work had established inter aka that the conversion of androst-4-ene- 3,17-dione (47a) or testosterone (47b) into the respective oestrogens involves three discrete oxidative steps each requiring molecular oxygen and NADPH.It was shown also that the NATURAL PRODUCT REPORTS 1988 primary alcohol (48a) or (48b) and the aldehyde (49a) or (49b) respectively are intermediates and that C-19 of the androgen precursor is released as formic acid. These and other conclusions from the earlier work are summarized in Scheme 4. These general conclusions have now been extended to encompass the biosynthesis of oestriol (50c) from 16a-hydroxytestosterone (47c) by microsomes from human ~1acentae.l'~ In particular it was shown that the triol (48c) and the aldehyde (49c) are intermediates and that the conversion of the former compound into the latter proceeds with retention of the 19(pro-199-proton.Furthermore it was shown that the 19-oxygen atom of the aldehyde (49c) is retained in the formic acid that is released while the second oxygen of the latter is supplied by the third R' (39) a; R' OH R2=CH,SiMe3 1 2 b; R = H R = CH2NH2 c; R'=H R2=NH2 H (LO) vo (41)a;R= H b;RzOH OH H (46) NATURAL PRODUCT REPORTS 1988-D. M. HARRISON equivalent of molecular oxygen.114 A similar enzyme system converted [1802]andr~~t-4-ene-3, 17-dione (47a) and [3-180]-testosterone (47b) into oestrone (50a) and oestradiol (50b) respectively with retention in each case of over 90% of the oxygen- 18 label that was present in the precursor.115 This work which confirms an earlier report by another group,' demon- strates that no Schiff base is formed as an intermediate at any stage in the aromatization reaction.Until recently the available evidence suggested that the 28- hydroxy-aldehyde (5 1) is an intermediate in the aromatase- catalysed conversion of the aldehyde (49a) into oestrone (50a).' For example the hydroxy-aldehyde (51) was trapped during an aromatase-catalysed reaction and was shown to undergo spontaneous aromatization at pH 7.1 to furnish oestrone and formic acid concomitant with loss of the 18-proton. Thus the postulate that (51) is an intermediate was consistent with the established loss of both the 1P-and the 28- proton during the conversion of an androgen into an oestrogen and with the suspicion that the final step in the formation of oestrogens is a spontaneous non-enzyme-catalysed process.Supporting evidence was furnished by the report that the rate of biosynthesis of oestrogens by placental microsomes was reduced by antibodies that bind to 2P-hydroxy-androgens. 116 Notwithstanding the previous arguments it has now been concluded on the basis of the evidence that is summarized below that the 2P-hydroxy-aldehyde (51) is not an intermediate in the biosynthesis of oestrone.'17 Thus it is known that [180]formic acid is released when the 19-0x0-androgen (49a) is incubated with placental microsomes under an atmosphere of ['80]oxygen,':cJ~'14 and this observation has been confirmed.'17 If the 2P-hydroxy-compound (51) were an obligatory inter- mediate in the latter process its 2P-hydroxyl group should be o; NAI (47) (a) R1R2=0,R3=H (b) R"OH R2:R3=H (c) R1=R3= OH ,R2= H labelled with oxygen- 18 ;furthermore the same hydroxyl group would be the source of the oxygen-18 label in the formic acid that is ultimately released.However when the synthetic [19-3H,2-180]-2p-hydroxy-aldehyde (51) was allowed to aroma- tize in the presence or absence of aromatase the tritium- labelled formic acid that was formed was devoid of oxygen-18. It was concluded that the hydroxy-aldehyde (51) is not an intermediate in the biosynthesis of oestrone from the 19-0x0- androgen.'l' Further consideration must now be given to an alternative suggestion for the mechanism of the final step in the aromatase-catalysed biosynthesis of 0estr0ne.l~~ The syntheses of (19R)-and (19S)-3@-hydroxy[ 19-2H, 19-3Hl]androst-5-en- 17-one (42) have been described briefly.l18 These compounds each of which contains a 'chiral methyl' group were used earlier to determine that the initial hy- droxylation reaction catalysed by aromatase proceeds with retention of configuration.'The two components of placental aromatase i.e. the cytochrome P-450A,, and the NADPH- cytochrome-P-450 reductase have been purified by h.p.1.c. 119 Monoclonal antibodies have been raised to cytochrome P-450A,,,.120 Syntheses of the 19-azidoandrostenedione (52a) and the 19- methylthio-derivative (52b) both of which are potent inhibitors of aromatase have been described ; ultraviolet-visible differ-ence spectroscopy showed that the sulphur atom of the latter inhibitor was co-ordinated to the haem iron when the steroid was bound to aromatase.121 Other sulphur-containing inhibitors of the same type have been described.122 Several different types of inhibitor of aromatase have been prepared including the 19,19-difluoro-steroids (52c) and (52d),123 the potent inhibitor 4-hydroxyandrosta-4,6-diene-3,17-dione (52e),124 and the 6a-and the 68-hydroperoxides (53) of androst-4-ene-3,17-dione ;125 (50) Scheme 4 16 R2 (51) (52)a; R'= CH2N R2= H b; R'zCH2SMe ,R2=H c; R1.CHF ,R2= H 1 2 d; R =CHF2 R =OH 1 e; R CH ,R2=OH ; 6,7-didehydro the latter compounds may function as inhibitors of aromatase by causing oxidation of cysteine residues.Irreversible inhibitors of aromatase have been reviewed.126 Further study has been reported on the cytochrome-P-450- dependent adrenal hydroxylase that is responsible for hy- droxylation at C- 1 1 in the biosynthesis of the corticosteroids. The active 11p-hydroxylase was reconstituted from purified cytochrome P-45Oll by adding adrenodoxin adrenodoxin re- ductase [ferredoxin-NADP' reductase (E.C. 1 .18 .1 .2)] and Tween 20.12' Synthetic 18,19-dihydroxy-1 1 -deoxycortico-sterone (54a) was shown to be identical to the minor product of the hydroxylation of 18-hydroxy-11-deoxycorticosterone (54b) by a reconstituted 1 lp-hydroxylase.128 In the presence of oxygen and NADPH the same enzyme system converted [1,2 6,7-3H]corticosterone (54c) into aldosterone (55) via the 18-hydroxy-steroid (54d).129 The role of cytochrome-P-450-dependent oxidations in the biosynthesis of the steroidal hormones has been reviewed.130 5.3 The Biosynthesis of Bile Acids and the Metabolism of Vitamin D The hydroxylation of cholesterol to furnish 7a-hydroxy-cholesterol (56) catalysed by the key enzyme cholesterol 7a- mono-oxygenase [E.C. 1 .14.13.171 is normally regarded as the rate-limiting step in the biosynthesis of the bile acids. The hydroxylase is stimulated by a cytosolic protein of mol. wt. 25000 in the presence of reduced glutathione; the stimulatory protein is not affected by ATP or by Mg2+ The biosynthesis of 7a-hydroxycholesterol by liver microsomes is also stimulated by the cytosolic protein SCP-2.13 A soluble enzyme that catalyses the NADPH-dependent reduction of 7a-hydroxycholest-4-en-3-one (57) to the 5p-steroid (58) has been isolated from rat liver and purified 230- (54)a; R'= R~=OH,R*=H b; R'= R2=H R3=OH c; R'=R3=H R2=OH d; R'= H ,R2 = R3=OH H H H H 0.' NATURAL PRODUCT REPORTS 1988 fold.133 A cytochrome P-450 that is active in the 26-hydroxylation of C, steroids has been isolated from rabbit liver mit~chondria.'~~ The 26-hydroxylase activity of the purified cytochrome was restored by the addition of ferredoxin and a ferredoxin reductase.The best substrate was the triol (59a) but the diol (59b) was hydroxylated also.The terminal steps in the biosynthesis of cholic acid have been investigated; the (25R)-and the (25,s)-[7P-3Hl]-diastereoisomers (60) were both converted predominantly into the same two metabolites namely the (24a-cholest-24- enoic acid (6 1) and (24R,25S)-3a,7a 12a,24-tetrahydroxy- 5p-cholestan-26-oic acid (62) when incubated with a rat liver homogenate. 135 The biosynthesis of bile acids has been re~iewed.',~ A practical account has been published on the isolation of cytochrome-P-450-dependent hydroxylases that are involved in the biosynthesis of bile acids.13' The first step in the metabolism of vitamin D [calciol] (63a) is its oxidation to the 25-hydroxy-derivative calcidiol (63b) which is catalysed by a cytochrome-P-450-dependent mixed-function oxidase.The requisite cytochrome P-450 has been isolated from rat liver microsomes and purified ;25-hydroxylase activity could be reconstituted by supplying NADPH-cytochrome-P-450 reductase and NADPH in the presence of 0~ygen.l~~ It is known that the biosynthesis of the dihydroxy- lactone (64a) from the diol (63b) involves the intermediacy of the triol (63c) and the tetraol (63d). The lactol (65) has now been isolated from chick kidneys and shown (without the benefit of isotopic label) to be a precursor of the lactone (64a) in kidney homogenates. 139 (23,s)-1a,23,25-Trihydroxyvitamin D [(1S,23s)-1,23,25-trihydroxycalciol](63e) has been isolated from bovine kidney homogenates that were incubated with calcitriol (63f) and shown to be a precursor of the trihydroxy- lactone (64b).140 OH HO" H (58) (59)a; R= OH b; R= H OH (61) (621 NATURAL PRODUCT REPORTS 1988-D.M. HARRISON 397 HO HO (66) H 6 Triterpenoids and Steroids in Higher Plants Algae and Fungi 6.1 The Biosynthesis of Sterols in Fungi The biosynthesis of ergosterol (66) from [U-14C]acetate or from [2-14C]mevalonate was inhibited in Candida albicans by the antimycotic drug naftifineldl and in Candida albicans Candida parapsilosis Torulopsis glabrata and Trichophyton menta- grophytes by the related antimycotic agent SF88-327.1d2 The main site of action for both fungicides was squalene epoxidase [squalene mono-oxygenase (E.C. 1 .14.99.7)] as judged by the accumulation of squalene in treated fungal 142 However at very high concentrations naftifine appeared also to inhibit the transmethylation step in the biosynthesis of ergosterol by Candida a1bi~ans.l~~ The first step in the biosynthesis of ergosterol from lanosterol (21 a) in the yeast Saccharomyces cerevisiae is the oxidative removal of the 14a-methyl group by a microsomal cytochrome- P-450-based mono-oxygenase system.l Details have now been published of the isolation purification and properties of the cytochrome P-450 component (cytochrome P-4501,,,).144 The cytochrome P-45014, required NADPH-cytochrome-P-450 reductase for reconstitution of its mono-oxygenase activity; incubation of lanosterol (21a) with the reconstituted mono-oxygenase in the presence of oxygen and NADPH gave a metabolite that was identified as the triene (67).145 Yeast microsomes converted lanosterol into 4,4-dimethylzymosterol (21 b) presumably via the triene (67) when incubated with cyanide ion (to block demethylation at C-4) and NADPH.This conversion was prevented by antibodies that had been raised to cytochrome P-45014, and by known inhibitors of cytochromes Further transformations in the main route for the bio- synthesis of sterols in the yeast Saccharomyces cerevisiae include stepwise demethylation of 4,4-dimethylzymosterol (21b) to furnish zymosterol (21c) which is converted into fecosterol (68a) and thence into ergosterol. On the other hand lanosterol is the preferred substrate for the transmethylation “moH w H 5 [as (6411 OH (65) R = H (61)a;R =H b; R=OH H R1-R2 (68)a; R’= R2= R3= H b; R’= R2= R3 Me c; R’z H R2= R3= Me d; R’= R2= H R3= Me HOWR H (70)a; R= H b; R= Me reaction in Ustilago maydis and in Penicillium italicum and the first 24-substituted sterol intermediate in the biosynthesis of ergosterol by these two species is 24-methylene-24,25-dihydro-lanosterol (68b).The effect of the systemic fungicide tridemorph on the biosynthesis of ergosterol has been clarified. In Saccharomyces cerevisiae at a concentration of 10 pmol dm-3 this fungicide markedly reduced the formation of ergosterol and caused the accumulation of the normal intermediates (67) and (68a) together with abnormal 8,14-dienes such as (32) and (69) ;in Ustilago maydis that had -been similarly grown in the presence of tridemorph the main sterols were (68a) (70a) and (7 1).146 The related fungicides fenpropimorph and fenpropidin gave results with the latter two species that were qualitatively similar to those given by tridemorph but with the difference that the 8,14-diene (69) accumulated at the expense of its isomer (68a).It was concluded that the three fungicides inhibit to differing extents both the reduction of 8,14-dienes and the isomerization of A8-sterols to A7-~tero1~.146 Other workers have observed the accumulation of the 8,14,24(28)-triene (72) upon growth of Penicillium italicum in the presence of fenpropimorph ; by contrast the imidazole- based fungicide imazalil blocked the oxidative removal of the 14a-methyl group of precursors of ergosterol in the same specie^.'^' Fungicidal triazoles of the dichlorobutrazol series similarly blocked 14a-demethylation of sterols in Ustilago maydis leading to the accumulation of obtusifoliol (68c) and the other 14a-methyl-sterols (68d) and (70b).148 [14C]Lanosterol has been prepared by the growth of yeast cultures in which 14a-demethylation was blocked by buthiobate (100 pmol dm-3) in the presence of [1-14C]isopentenyl diphosphate while zymosterol (21c) was isolated from yeast cells that were adapted to aerobic growth in the presence of ethionine which blocks the transmethylation rea~ti0n.l~' The C, triene (73) and the tetraene (74) were detected by gas chromatography-mass spectrometry in yeast cultures that were grown under the latter conditions.149 Exogenously supplied cholesterol and ergosterol each inhibited the biosynthesis of ergosterol by Saccharomyces cerevisiae.150 The three isomeric ergostatrienols (75) (76) and (77) have been isolated as minor sterols from Gibberella fujikuroi. 5a-[2,4-3H]Ergosta-8,14,22-trien-3P-ol (75) and [14C]ergosta- 5,8,22-trien-3/3-01 (76) (prepared biosynthetically from [2-14C]- acetate) were incorporated into ergosterol (66) in radiochemical HO H H NATURAL PRODUCT REPORTS 1988 yields of 6.0 and 15.4% respectively in the same organism; under identical conditions 5a-[2,4-3H,]ergosta-6,8(14),22-trien-3p-01 (77) was not incorporated into ergosterol and must be regarded as an end product of biosynthe~is.~~~ On the basis of isolation studies a scheme has been proposed for the biosynthesis of cholesterol ergosterol and /3-sitosterol(78a) by the ascomycete Aureobasidium pullulans.152 The biosynthesis of ergoster01,'~~ the inhibition by fungicides of the biosynthesis of and the use of yeast mutants in the study of the biosynthesis of have been reviewed. 6.2 The Biosynthesis of Phytosterols Feeding experiments with tissue cultures of Andrographis paniculata have shown that [3-13C]leucine is incorporated into the phytosterols ,!hitosterol (78a) stigmasterol (79a) and campesterol (78b) only after its complete breakdown to [2-13C]acetate.156 Similarly [4-14C 2-3H]-P,P-dimethylacrylic acid was incorporated into the same sterols only after its extensive degradation as revealed by a substantially lower 3H:14C atomic ratio in the metab01ites.l~~ Barley seedlings (Hordeum vulgare) that were supplied with [4-14C 22,23-3H]-p-~ito~terol (78a) yielded stigmasterol (79a) with the expected 50% reduction in the ratio of 3H:14C.157 Conversely [4-14C]-/?-sitosterol was not incorporated into stigmasterol in seedlings of tobacco (Nicotiana tabacum) despite an apparent precursor-product relationship for the time-course of labelling of these two sterols when [2-14C]- mevalonate was supplied to the ~eed1ings.l~~ It was suggested that the two sterols derive from a common phytosterol precursor in tobacco.HO (75) H (77) NATURAL PRODUCT REPORTS 1988-D. M.HARRISON In cell cultures of bramble (Rubusfruticasus)that are grown under normal conditions the A5-sterols p-sitosterol (78a) isofucosterol (80a) and campesterol (78b) constitute about 96 YO of the phytosterol content and are formed from cycloartenol (81) via the route that is summarized in Scheme 5. Critical steps in this sequence are the alkylation of cycloartenol by S-adenosylmethionine (SAM) to furnish 24-methylene-cycloartanol (82) the opening of the cyclopropane ring of cycloeucalenol (83) to give obtusifoliol (68c) and the iso- merization of the A8-sterol (84) to the A7-sterol (85). It has been reported that the tertiary amine (24c) is a powerful inhibitor of the isomerization of A*-sterols to their A7-isomers in this system owing to the structural and electronic resemblance of the protonated amine to the putative carbo-cation intermediate [as (7811 (79)a; R-Me b; R:H c; R CH,F (78) a; R = Me b; R= H H [as (7811 (80)a; R1=H R2=Me b; R’.R2:H c;R’=Me R 2= H Scheme 5 1599cf.66 in the enzyme-catalysed reaction. As-Sterols such as (86a) and (87) constituted the major phytosterols (87 %) when the cell culture was incubated with the inhibitor at a concentration of 1 mg dm-3.159 At higher concentrations of the inhibitor the isomerization of cycloeucalenol(83) to obtusifoliol (68c) was retarded also and 9,19-cyclo-steroids such as cycloeucalenol and the 24-substituted pollinastanol derivatives (88) (89a) and (89b) accumulated. The sites of inhibition of sterol biosynthesis by the amine (24c) were confirmed by studies with a cell-free enzyme preparation from maize (Zea mays).59 The inhibition of biosynthesis of sterols by triarimol tridemorph and triparanol in cell cultures of tobacco (Nicotiana tabacum) carrot (Daucus carota) and soybean (Glycine max) has been studied.160 In the absence of inhibitor each of these cell cultures furnished the A5-sterols campesterol (78b) 6-sitosterol (78a) and stigmasterol (79a) as the only significant sterols. Triarimol inhibited both the 14a-demethylation of obtusifoliol and the second transmethylation reaction leading to the accumulation of obtusifoliol (68c) and 1401-methyl-5a- ergost-8-en-3P-01 (70b) while tridemorph inhibited both the conversion of cycloeucalenol into obtusifoliol and the second transmethylation reaction.In the cultures of tobacco and carrot cells treatment with triparanol led principally to the accumulation of 14a-methyl-As-sterols such as (70b) and (86b) but cycloeucalenol and 24-methylenecycloartanol were detected also. The 4a-methylsterol (84) has been isolated from horse chestnut (Aesculus hippocastanum) and may have a major role as an intermediate in the biosynthesis of phytostero1s.l6l The biosynthesis of cholesterol and phytosterols from [2-14C]acetate has been demonstrated in leaves of sorghum (Sorghum bi-color).162The incorporation of [2-14C]acetate into phytosterols NATURAL PRODUCT REPORTS. 1988 Reviews have been published on the biosynthesis of phytosterols166 and on the membrane-bound enzymes167 and the regulation168 of phytosterol biosynthesis.6.3 Alkylation of the Sterol Side-chain Poriferasterol (90a) that was labelled biosynthetically by supplying [Me-2H3]acetate to the crysophyte alga Ochromonas malhamensis acquired deuterium label in an integrated ratio of 2 3 for the 25bro-25R)- and the 25(pro-25S)-methyl group respectively; therefore the 25(pro-25R)-methyl of (90a) is derived from C-2 of me~alonate.'~~ This result is consistent with the alkylation by S-adenosylmethionine of a sterol precursor at the re face of the 24-25 double-bond as shown in Scheme 6. For the purposes of assay the deuterium-labelled poriferasterol was first degraded to (2S 3R)-2-([*H2]methyl)- [l,l 1,2-2H,]pentan-3-ol the 'H n.m.r.spectrum of which if recorded in the presence of the shift reagent Eu(dpm), per- mitted the absolute configuration of C-2 to be determined.169 The biosynthetic origins of the 25bro-25R)- and the 25(pro- 25S)-methyl groups of p-sitosterol (78a) and clionasterol (91 a) were determined earlier 170 based on the implied assumption that the biosynthetic results for isofucosterol (80a) in Pinus pineal'l were applicable also to that sterol in Physalis peruviana. The conclusions with respect to the phytosterols (78a) (80a) and (91a) in the latter species17o have now been confirmed by unambiguous assignment of the 13C n.m.r. resonances due to the diastereotopic 25-methyls of clionasteryl acetate. 172 These assignments were performed with the aid of [13C]clionasterol (91 a) that was derived chemically from [13C]poriferasterol (90a) which in turn had been labelled biosynthetically by growing Ochromonas malhamensis in the presence of sodium [13C2]acetate.by tissue cultures of Cucurbita maxima has been re~0rted.l~~ Studies of biosynthetic relevance have been published on the sterol content of seeds of the latter species.164 The metabolism of cholesterol in higher plants has been reviewed and it was suggested that cholesterol may be more widely synthesized in plants than has been recognized hither- H H\. f b; R=Me R' (09) a; R'= R2=H R3=Me b; R"R3'HJ R2=Me 3 2 c; R'=R =Me,R =H d; R' = R2*Me R3= H Sodium [13C,]acetate was incorporated into ergosterol (66) in Saccharomyces cerevisiae and the 13C n.m.r.spectrum of the derived 5a-ergost-8( 14)-en-3@-01 (92a) showed that the 25(pro- 25S)-methyl of ergosterol is derived from C-2 of mevalonate in the latter species,173 as had been shown previously for ergosterol in Claviceps pa~pali.'~~ 22,23-Dihydrobrassicasterol (9 1 b) and ..Hi-xf H /R (91) a; R=Me b;R=H (90)a; R=Me b; R=H NATURAL PRODUCT REPORTS 1988-D. M. HARRISON campesterol (78b) were isolated from suspension cultures of Physalis peruviana and Dioscorea tokoro each of which had been supplied with [13C2]acetate; the I3C n.m.r. spectra of these labelled steroids from either source and of 24-methylene-cholesterol (80b) from P. peruviana revealed that C-2 of mevalonate furnishes the 25(pro-25R)-methyl group of 22,23- dihydrobrassicasterol but the 25(pro-25S)-methyl of campe- sterol and of 24-methylene~holesterol.~~~ The result for the latter sterol is the reverse of that published previo~sly.'~~ In this study the assignments of carbon resonances for the dia-stereo topic 25-me thyls of 22,23 -di hydro brassicasterol were made by comparison with those for the A8(14)-~tero1 (92a) which has the same side-chain.Other assignments were made with the aid of a mixed sample of 13C-labelled 22,23- dihydrobrassicasterol and campesterol which was prepared by hydrogenation of the 13C-labelled 24-methylenecholesterol (80b).'73 Irrespective of the correctness or otherwise of the new assignments for (80b) it is clear from the present results that campesterol but not 22,23-dihydrobrassicasterol,could be biosynthesized by direct reduction of 24-methylenecholesterol in P.peruviana. The authors have interpreted their results in terms of the proposed biosynthetic routes (a) and (b) in Scheme 7 for the sterols (78b) and (91b) re~pective1y.l~~ Some support for this interpretation was afforded by the de-monstration that 24-methyldesmosterol (93a) incorporated 13C label stereospecifically from [13C2]acetate in cultures of P. peruviana (see Scheme 7).175 The present status of studies on the H/ -.py -'H* 1 40I biosynthetic origins of the 25-methyls of phytosterols is summarized in Table 1. Important though circumstantial evidence was discussed in the previous Report' for the view that 2401-methyl-sterols are formed in Zea mays by route (a)while 24P-methyl-sterols are biosynthesized via route (b)or (c) of Scheme 7.A study on the sterol composition of shoots of the latter species has confirmed inter alia the presence and the biosynthesis from [2-14C]mevalonate and from [Me-14C]methionine of sterols with the side-chains that are shown in Scheme 7.'76 However evidence against the operation of routes (b)or (c) was afforded by the investigation of the biosynthesis of sterols in maize seedlings that had been treated with tridem0r~h.l~~ The major sterols that were formed under the latter growth conditions were the mixed diastereoisomeric 24-methylpollinastanols (89a) and (89b) cycloeucalenol (83) the mixed dihydrocyclo- eucalenols (89c) and (89d) and significantly the 24-methyl- AZ4-stero1 (94).24-Methylenecycloartanol cycloeucalenol 24-methylenepollinastanol and the mixed 24-methylpollinastanols each retained two 2H atoms only when [Me-2H3]methionine was supplied as precursor. Furthermore when (4R)-[5-14C,4- 3Hl]mevalonic acid was supplied to the plant the 3H:14C atomic ratios in the sterols that were formed were consistent with the biosynthesis of each of the diastereoisomeric 24- methylpollinastanols via the A2%ero1 (94) as summarized in Scheme 8.177 Microsomal extracts from seedlings of maize are capable of H -Me / ( 0 indicates carbon derived from C- 2 of mevalonate 1 Scheme 6 R H (92)a; R'=H R2=Me (93)a; R = Me b; R'=Me R2=H b R-H NATURAL PRODUCT REPORTS 1988 I'd Scheme 7 S-adenosyl [Me -*HJ methionine H+ - H* ___) *- ryw (81) (82) ( 91) HO Scheme 8 NATURAL PRODUCT REPORTS 1988-D.M. HARRISON Table 1 Biosynthetic origins of the 25-methyl groups of phytosterols Sterol Labelling pattern of side-chain Species Reference (0= derived from C-2 of mevalonate) Poriferasterol (90a) Ochromonas malhamensis 169 Stigmasterol (79a) Physalis peruviana 170 Dioscorea tokoro 170 Bupleurum falcatum 170 a-Spinasterol (1 18) Bupleurum falcatum 170 Clionasterol (9 la) Bupleurum falcatum 170; cf. 172 /3-Sitosterol (78a) Physalis peruviana 170; cf. 172 Dioscorea tokoro 170; cf. 172 Isodon japonicus 170; cf. 172 Isofucosterol (80a) =&-H Physalis peruviana 170; cf.172 Pinus pinea 171 24- Met hylene- Physalis peruviana 173; cf. 170 cholesterol (80b) 22,23-Dihydro-Physalis peruviana 173 brassicasterol (9 1b) Dioscorea tokoro 173 Campesterol (78b) Physalis peruviana 173 Dioscorea tokoro 173 Ergosterol (66) Saccharomyces cerevisiae 173 Claviceps paspali 174 catalysing the alkylation of cycloartenol (8 1) by S-adenosyl- Saccharomyces cerevisiae was inhibited in vivo and in vitro by [Me-14C]methionine to furnish 24-methylenecycloartanol (82). the 25-azacholestenols (97a) and (97b).179 Increased levels of The 24-transmethylase activity was found to be strongly zymosterol (21c) and the formation of the sterols (73) and (74) inhibited by the substrate-like analogues (95a) and (95b) which were observed in yeast cultures that were incubated with these possess a basic nitrogen atom at C-25.178 The efficacy of these inhibitors.179 aza-sterols as inhibitors may be due to the structural and The biosynthesis of the unusual side-chain of 24-propyl- electronic resemblance of their conjugate acids to the C-25 idenecholesterol (98) in an unidentified marine crysophyte cation (96) which is an intermediate in the biosynthetic alga has been studied.lsO The possibility that the propylidene conversion of (81) into (82).Significantly the N-oxide (95c) substituent is formed by direct ethylation of 24-methylene-the sulphonium salt (95d) and the arsonium salt (95e) were also cholesterol (80b) or an equivalent precursor was precluded by potent inhibitors of the transmethylase activity.178 Similarly the observations that [Et-2H,]ethionine was not incorporated the A24-~tero1 methyltransferase [E.C.2. 1 . 1 -411 of the yeast into (98) in the alga while [Me-13C]methionine furnished (98) 404 NATURAL PRODUCT REPORTS 1988 H R2 H (97) a;5,6-didehydro (95) a; R1=H R2=NMe2 b; 8,9-di dehydro b; R'=Me R2= NMe2 c; R':Me R2=$Me20 d; R'= Me R2::Me2 e; R'= Me R2=AsMe + [CD,] SAM ( 98) Scheme 9 NATURAL PRODUCT REPORTS 1988-D. M. HARRISON that was labelled equally at each of the propylidene carbons. Mass spectrometry and 2H n.m.r. spectroscopy were both used to study the incorporation of label from [Me-2H3]methionine into the 24-methyl-sterols (90b) and (9 1b) the 24-ethyl-sterols (78a) (9 1a) and (79a) and 24-propylidenecholesterol ; the most plausible interpretation of the results is that the side- chains of these sterols are formed in this alga principally or exclusively by the routes that are summarized in Scheme 9.6.4 Further Metabolism of Steroids and Triterpenoids Biosynthetically labelled [14C]deoxydemethoxyviridin(99a) was incorporated in 3.9 YO radiochemical yield into the antibiotic demethoxyviridin (99b) by the fungus Nodulisporium hinnuleum; the putative role of (99a) as a precursor of demethoxyviridin was further supported by its detection in a cold-trap experiment when [l-14C]acetate was fed to the fungus. Lemon seedlings (Citrus limon) incorporated [1-14C]acetate [2-14C]mevalonate and [ 1-14C]farnesyl diphosphate into the limonoid nomilin (100) in radiochemical yields of 1.5 YO,0.5 YO and 0.15 YO respectively.lE2 Biosynthetically labelled [14C]-nomilin was metabolized in lemon seedlings to four com-pounds one of which was identified as obacunone (101).la3 [14C]Obacunone which was prepared enzymically from [14C]nomilin was further converted by the same species into the acid (102a) and limonin (103).la4 The acid (102a) was a poor precursor of limonin; however this may simply be due to permeability problems since the unnatural methyl [14C]e~ter (102b) was metabolized in 50% overall yield to a mixture of products including limor~in.’~~ The results of these studies were discussed in terms of the sequence shown in Scheme 10 for the biosynthesis of limonin.(99) a; R=H b; RzOH In earlier studies on the biosynthesis of the meliacin nimbolide (104a) the Aa-triterpenoid euphol (105a) was a more efficient precursor than were plausible A’-triterpenoids. In more recent work the 3H-labelled epoxide (106) and the 3H-labelled diepoxide (107) were incorporated into nimbolide in leaves of Azadirachta indica in 5.9 YOand 4.0 YOradiochemical yields respectively ; under the same conditions 3H-labelled euphol showed a 2.8 YOincorporation into nirnb~lide.~~~ These results were taken to mean that a 7,9( 11)-diene derived from an 801,901- epoxide was a more immediate precursor of nimbolide than euphol. The nimbolide that was isolated following the feeding of (4R)-[2-14C,4-3H,]mevalonolactone (3H:14C atomic ratio of 1 1) had a 3H: 14C atomic ratio of 3 5; this is consistent with the biosynthesis of nimbolide [labelled as (104b)l from endogenous euphol of the assumed labelling pattern (105b).lE5 A degradation that purported to demonstrate the presence of tritium label at C-3 [or C-21 of nimbolide (104b) was described.MeO-C 0‘ (104)a ; H*=’H *=12c * 3 fL b; H=H,*= C H* H* I H 0 (106) 1 0 $1 Ro2cT[ HO (103) (102) a; R= H [as (10611 b; R=Me Scheme 10 (107) [2-14C]Mevalonolactone was incorporated into the withan- olides withaferin A (108a) jaborosalactone A (108b) and jaborosalactone D (109) in leaves and seedlings of Acnistus brevzJ7orus.Upon re-feeding it to leaves of the plant the 14C- labelled jaborosalactone A was incorporated into (108a) and (1 09) in 2.0 % and 1.4 % yields respectively. lS6 Partial degradation of the l*C-labelled withaferin A that had been derived biosynthetically from [2-14C]mevalonolactone surpris- ingly showed that a total of only 2% of the 14C label resided at carbons 25 26 and 27.1S7 Further more definitive experiments are clearly desirable. The labelling patterns that were observed when [13C,]acetate was incorporated into the spirosapogenins of cell cultures of Dioscorea tokoro were reported earlier ;the full paper includes data also on the intermediate furostanol glycosides proto- neotokorin (1 1Oa) and prototokoronin (1 10b).lS8 The furo- stanol (1 11) has been isolated from suspension cultures of Dioscorea deltoidea.189 The results of pulse-chase biosynthetic experiments with [l-14C]acetate suggested that (1 1 1) is a direct precursor of diosgenin (1 12) in vivo.[1,2-14Cz]Oxaloacetate was incorporated into scilliroside (I 13) in Scilla maritima ;the results of partial degradation were interpreted to mean that C-1 (2-2 and C-3 of the precursor furnished C-24 C-23 and C-22 respectively of the metabolite (1 13).lg0 However the high degree of randomization of label that was observed in this study as might be anticipated with such a precursor renders the result equivocal. A cytochrome-P-450-dependent hydroxylase that converts the cardenolide glycoside digitoxin into its 12P-hydroxy-derivative digoxin has been isolated from cell cultures of Digitalis lan~ta.'~~ The biosynthesis of digitoxin in cultures of Digitalis purpurea has been studied.lg2 Reviews have been published on the biosynthesis of cucurbitacins and limo no id^,'^^ steroidal glyco-alkaloids,194 and sterol glycosides.lg5 H,,+OH w>(lo8)l I H1) -OH R (108) a; R=OH (1 09) b; R= H H (110) a; R1 ia-~-Ara R"0-P-D-Glc,R3=H b; R'=cc-L-Ara R2=H R3=O-(3-D-Glc NATURAL PRODUCT REPORTS 1988 (111) H H (112) OAc (1131 7 Triterpenoids and Steroids in Invertebrates 7.1 Insects Insects appear to be incapable of biosynthesizing sterols de novo; the cholesterol requirement of some phytophagous insects is satisfied by the degradation of dietary phytosterols by the route that is illustrated for /I-sitosterol(78a) and clionasterol (91a) in Scheme 11.The dehydrogenation of p-sitosterol is non- stereospecific;a mixture of fucosterol (80c) and isofucosterol (80a) is formed in the first step. The stereochemistry of the dehydrogenation of clionasterol has now been investigated by feeding a mixture of [7,7-3H,]clionasterol and [4-14C]-p-sitosterol to larvae of Tenebrio molitor in the presence of unlabelled fucosterol or isofucosterol (to act as isotopic traps) ; the ratios of 3H to in the isotopic label that was acquired by the latter compounds demonstrated that clionasterol and p-sitosterol were metabolized to fucosterol with equal efficiency while isofucosterol was formed less readily from clionasterol than from p-sitosterol.lg6Phytosterols with a C side-chain can also satisfy the sterol requirements of T. molitor. The diastereoisomeric compounds (24R)- and (24S)-24,28-epoxy- [23,23,25-3H,]ergost-5-en-3,!3-ol [(114a) and (1 15a) respectively] have now been tested as precursors of cholesterol each in competition with [4-14C]-,!3-sitosterol ; it was clear from the 3H:14C atomic ratios of the metabolites in these experiments that only the (24R)-epoxide (1 14a) was converted into cholesterol.lg7This high degree of stereospecificity is in striking contrast to the relative lack of discrimination between the two epoxides (1 14b) and (1 15b) of fucosterol that was previously reported to have been displayed by the same species.A noteworthy feature of Scheme 11 is the hydride migration from C-25 to C-24 that is observed during the conversion of p-sitosterol into desmosterol(93b) and cholesterol (2a). Indirect evidence has been furnished for the equivalent hydride NATURAL PRODUCT REPORTS 1988-D. M. HARRISON migration in the dealkylation of stigmasterol (79a) by larvae of the silkworm Bombyx mori as summarized in Scheme 12(a); in particular mass spectrometry indicated that the deuterium label was retained in the side-chain of samples of desmosterol and of cholesterol that were formed from [23-2H]-or [25-2H]-stigmasterol (79a) while unlabelled C,,-sterol metabolites were formed when [24-2Hl]stigmasterol was supplied as prec~rsor.~~~The diastereoisomeric ergostatrienols (116a) and (1 16b) were each converted into cholesterol via the cholesta-trienol (1 17) by Bombyx m~ri.”~ The unlabelled 2401-phyto-sterols p-sitosterol (78a) stigmasterol (79a) campesterol (78b) and crinosterol(79b) and their 24P-epimerseach satisfied the sterol requirement of silkworm larvae and were converted into cholesterolwith similar efficiencies.lQQ The A5-sterolsp-sito-Ll MP H ..AH ___ *- - + =AH+-VEX H* ( 80a + 80c) M~CHO’ 4 ..& H* H” -( 91a l Scheme 11 -[as (1141 1 2 (115) a;R= H (116)a; R=Me ,R= H b; R=Me b; R’* H ,R2sMe (11Ll a; R-H b; R= Me RCHO * .* H* H* C 1 (a) R= Me (b) R=CH,F (2a) (93b) Scheme 12 sterol and campesterol several A’-sterols [such as spinasterol (1 18)] and several fully saturated sterols [e.g.campestanol] supported growth of the lepidopteran Heliothis zea ;in each case dealkylation of the steroid side-chain occurred but the steroid nucleus was mainly unaltered by this insect.200 The putative fucosterol-epoxide lyase may be assayed rapidly in cell-free homogenates of the tobacco hornworm (Manduca sexta) by monitoring the release of water-soluble tritium as C3H]acetaldehyde (see Scheme 11) or its metabolic equivalent from the (24R,28R)-[24-14C,29-3H]epoxide (1 14b). Surprisingly the (24S,28S)-epoxide (1 15b) was not a substrate in this enzyme system.201 An alternative assay for the same lyase has been described.202 Triadimefon and a series of related fungicides strongly inhibited the dealkylation of ~-sitosterol (78a) and of campe- sterol (78b) by larvae of the cotton leafworm (Spodoptera littoralis); these fungicides also inhibited the conversion of a mixture of the epoxides (1 14b) and (1 15b) of [6-3H]fucosterol into cholesterol by a cell-free enzyme system from the same species.2o3 Larvae of the tobacco hornworm (Manduca sexta) converted 29-fluor0[29-~H]stigmasterol(79c) in 0.01 YOradio-chemical yield into (2R,3R)-2-fluor0[2-~H]citricacid ;the latter compound which accounts for the toxicity of the fluoro- stigmasterol is formed from the two-carbon fragment that is released during dealkylation of (79c) according to Scheme 12(b).204 Some phytophagous insects are not able to dealkylate phytosterols ;these include the cactophilic species Drosophila mettleri and D.mojaven~is.~~~ The sterol composition of the heteropteran Dysdercus fasciatus i.e. 95 %p-sitosterol and 5 % campesterol closely resembled that of its diet of cotton seed in confirmation of the observation that this insect is unable to dealkylate p-sitosterol. 206 Moulting in insects is controlled by steroidal moulting hormones the ecdysteroids. The sites of biosynthesis of ecdysone (1 19a) by Locusta migratoria have been probed with the aid of the 22,23,24,25-3H4-labelledprecursor (1 19b);the larval prothoracic gland was identified as the tissue which was the most efficient in the biosynthesis of ecdy~one.~~’ It has been reported that the introduction of the 2P-hydroxyl group occurs with retention of configuration at C-2 during the biosynthesis of ecdysone in ovaries of the desert locust (Schistocerca gregaria).208This conclusion was reached following exami- nation of the 3H :14C atomic ratios in the ecdysone (1 19a) and 2-deoxyecdysone (1 19c) that had incorporated label from a mixture of [3H]cholesterol (containing label predominantly but not exclusively at the la-and 2a-positions) and [4-14C]cholesterol.It is generally accepted that the true moulting hormone is 20- hydroxyecdysone (1 19d) which is biosynthesized from ecdy- sone in many insect tissues. The properties of ecdysone 20- mono-oxygenase [E.C 1 .14.99 .22],209 which is a cytochrome- P-450-based mixed-function oxidase and its distribution in the bodies of larvae of Schistocerca gregaria have been studied.210 Microsomal and mitochondria1 versions of the enzyme have been identified in larvae of the tobacco hornworm (Manduca sexta).211The enzyme ecdysone oxidase [E.C.1 .1 .3. 161 catalyses the oxidation of ecdysone by molecular oxygen to furnish the dehydroecdysone (1 20a) and hydrogen peroxide.212 Ecdysone oxidase and two NADPH-dependent dehydro- ecdysone reductases have been investigated in tissue extracts of the lepidopteran Pieris br~ssicae.~~~ The two reductases have complementary specificities ;the new reductase catalyses the reduction of (120a) by NADPH to furnish ecdysone while the other (known) reductase catalyses the reduction of the same substrate to give 3-epi-ecdysone (120b).213 It is possible that the enzyme activity that had previously been known as ecdysone 3-epimerase214 arises from the sequential operation of ecdysone oxidase and the latter reductase.212 High concentrations of phosphate esters of ecdysteroids such as ecdysone 22-@phosphate (1 19e) are found in newly laid eggs of Schistocerca gregaria.This phosphate can be used by the NATURAL PRODUCT REPORTS 1988 H ( 118) 3 R* R OH Hi (120) a; R’R2=OJ R3=H b ; R1=R3=H R2= OH ~ C; R’=OAc ,R2=H R3=@ ~ OH R’ ! I OH (121) a; R’= H ,R~= co2H b; R1:OH ,R2=C02H C; R’=H ,R2= CH o@ NATURAL PRODUCT REPORTS 1988-D. M. HARRISON organism as a source of ecdysone and may serve as a storage The acetyl-phosphate (120c) has been identified as an end product of the metabolism of ecdysone in eggs of the same species;216 other metabolites of [23,24-3H]ecdysone in larvae of this organism included the hormone 20-hy-droxyecdysone (I 19d) and the carboxylic acids (1 21a) and (121b).217 The latter carboxylic acids were formed as major metabolites from [23,24-3H]ecdysone and 20-hydroxyecdysone respectively by the lepidopteran Pieris brassicae by the blowfly Calliphora vicina and by Locusta migratoria.218 The meta- bolism of ecdysone and 20-hydroxyecdysone has been studied also in the flesh-fly Sarcophaga peregrina219 and in the tick Ornithodoros moubata,220while the metabolism of 20-hy-droxyecdysone in Drosophila melanogaster has been investi- gated.221 [3H]Ecdysone was converted into 20-hydroxyecdysone and other metabolites in cell cultures of Bombyx mori.222 Pupae of Manduca sexta incorporated [4-14C]cholesterol into the 26-phosphate (12 lc) of 26-hydroxyecdysone; another labelled metabolite was shown to be a glycoside of the pregn- 5-ene-3P,I 7-diol (122).223 The biosynthesis and metabolism of ecdysteroids has been reviewed.224 Reviews have been published also on the biosynthesis and metabolism of sterols with particular reference to insects.225 7.2 Other Invertebrates Incorporation studies with [I -14C]acetate have shown that the soil amoeba Acanthamoeba polyphaga is capable of bio-synthesizing sterols de novo.226 Surprisingly the major sterols of the organism were 24-alkyl-24P-sterols such as ergosterol (66) and poriferasterol (90a) which are more typical of algae; furthermore cycloartenol (8 l) 24-methylenecycloartanol (82) and cycloeucalenol (83) were present while lanosterol was not detected.This protozoon also furnished some unusual aromatic sterols.226 The I4a-demethylation of sterols by Leishmania mexicana was inhibited by treatment with ketoconazole;227 the biosynthesis of sterols from [2-14C]mevalonate was also blocked by the drug SF86-327 which led to the accumulation of [14C]squalene.228 Like insects nematodes are incapable of biosynthesizing sterols for which they have a nutritional requirement. The free- living nematode Caenorhabditis elegans not only has the ability to remove the 24-ethyl group from dietary p-sitosterol but is also capable of altering the steroid nucleus ;the most remarkable H 5 409 transformation that has been observed was the introduction of a 4a-methyl group.224.230 Thus [4-14C]-P-sitosterol was meta- bolized to cholesta-5,7-dien-3P-ol (35) as the major product together with cholesterol (2a) cholest-7-en-3P-01 (34) and 4a- methylcholest-8(14)-en-3~-ol(92b) amongst other sterols. [26-14C]Desmosterol (93b) was metabolized to a similar range of as was unlabelled stigmasterol (79a) and other phytosterol~.~~~ It was suggested that the dealkylation of the and of ~tigmasterol~~~. side-chains of ~-sito~terol~~~ 232 may occur via the routes [see Schemes I1 and 12(a) respectively] that have been established for phytophagous insects.In keeping with this suggestion p-sitosterol was metabolized principally to A24-~tero1~, such as cholesta-5,7,24-trien-3P-o1(73),when it was incubated with the nematode in the presence of the hydro- chloride of 25-azacoprostane which is a potent inhibitor of the reduction of the 2425 double-bond of desmosterol in in- sect~.~~~ NN-Dimethyldodecanamine and related nematocides also blocked the putative A24-reductase of C. elegan~.~~~. 233 Marine invertebrates continue to attract attention. Con- ventional sterols such as cholesterol (2a) epicodisterol (1 23) and codisterol (124) co-occur in the Pacific sponge Aplysina Jistularis with sterols that possess unusual side-chains exemp- lified by the main sterol 25,26-didehydroaplysterol(I 25) and the related compounds aplysterol (126) and verongulasterol (1 27).234 [26-14C]Epicodistero1 was incorporated in 11YO radiochemical yield into 25,26-didehydroaplysteroland in significant yield into aplysterol verongulasterol and the mixed 24-methyl-sterols campesterol (78b) and 22,23-dihydrobras- sicasterol (91 b); on the other hand [26-14C]codisterol was incorporated only into the mixed 24-methylcholesterols.These data are consistent with the proposed biosynthetic route shown in Scheme 13. However the source of epicodisterol remains a puzzle since this sponge is known to be incapable of bio-synthesizing sterols de novo. [28-14C]-24-Methylenecholesterol (80b) and [4-14C]cholesterol were only incorporated to a significant extent into the mixed 24-ethyl-sterols [(78a) +(91a)l and into 5a-cholestan-3P-01 respectively.234 Cell-free enzyme preparations from zooxanthellae that had been isolated from the gorgonians Pseudoplexaura porosa P.flagellosa P. wagenaari and Pseudopterogorgia americana were able to convert [14C]farnesyl diphosphate into squalene but not into It was concluded that the biosynthesis of sterols in these symbiotic systems is completed by the respective gorgonians. (127) U I The Ag(ll)-~ter~l~ (1 28a) and (1 28b) have been isolated from a sea cucumber.236 It was suggested that squalene epoxide is cyclized directly to 5a-lanosta-9( 11),24-dien-3p-ol (1 28c) in members of the Class Holothuroidea. Liver homogenates from the sea star Asterias amurensis incorporated [4-14C]cholesterol into the pregnane aglycon (129) of an asterosaponin in 0.0002 YO radiochemical yield ; other putative precursors of this aglycon gave lower incorporations.237 The metabolism of pregnenolone (36a) and progesterone (38a) by the lobster Homarus americanus has been investigated.238 The metabolism of ecdysteroids in the crab Carcinus maenas has been 8 The Biosynthesis of Carotenoids In most species that synthesize carotenoids cis-phytoene (I 30a) is converted in a series of discrete dehydrogenation steps into all-trans-lycopene [@,@-carotene] (1 3 l) which is a precursor of the typical cyclic carotenoid p-carotene [P,p-carotene] (1 32a). Further studies have been reported on the solubilization of enzymic activity for the conversion of [2-14C]mevalonate into cis-(1 30a) and trans-phytoene (1 30b) in a phytoene-accumu- lating strain of Phycomyces blakesleeanus ; a soluble enzyme extract that incorporated [2-14C-]mevalonate into both phy- toene and p-carotene was also prepared from a p-carotene- forming strain of this The biosynthesis of p-carotene from [2-14C]mevalonate or [1-14C]isopentenyl diphosphate was inhibited when the latter cell-free enzyme system was incubated with bleaching herbicides among which fluridone and difunon led to the accumulation of phytoene while amitrole and 5852 caused the accumulation of acyclic Mutants of P.blakesleeanus that are blocked in the formation of carotenoids have been reviewed.242A gene (carC) that appears to be involved in the regulation of the biosynthesis of carotenoids in P.blakesleeanus has been identified in studies of such mu- tant~.~~~ The biosynthesis of p-carotene was stimulated by high concentrations of inorganic phosphate in the related fungus Blakeslea tri~pora.~~~ Reviews have been published on the regulation of the biosynthesis of carotenoids by tertiary amines in the latter species245 and on the biosynthesis of carotenoids in R’ R2 (1281 a; R’= H R2=Me b;R’-_R2 z H c;R’= R2=Me ; 2L 25 -didehydro (130) a; 15 15’-cis b; 15 15’ -frans NATURAL PRODUCT REPORTS 1988 and members of the Order Mucorale~~~~by Neurospora crassa.247 p-Cyclocitral (133a) and the hydroxy-derivative (1 33b) are released when cells of the cyanobacterium Microcystis PCC 7806 are ruptured by freezing.248 It was shown that these aldehydes arose from oxidative degradation of endogenous p-carotene (1 32a) and zeaxanthin (I 32b) respectively catalysed by a membrane-bound oxygenase and that crocetindial(134) is also formed as shown in Scheme 14.[180,]-~-Cyclocitral and unlabelled crocetindial were isolated when cells of this organism that had grown in an atmosphere of [1802]oxygen were disrupted. 248 Bleaching herbicides and a range of 3-phenoxybenzamide derivatives blocked the incorporation of [3-14C]geranylgerany1 diphosphate into p-carotene (132a) in a cell-free enzyme system prepared from the cyanobacterium Aphanocapsa PCC 67 14. Most of the inhibitors that were tested led to the accumulation of 14C-labelled phytoene and phytofluene (1 ~OC).~~~ Neither [2-14C]mevalonate nor [ 1-14C]isopentenyl diphospate was in- corporated into carotenoids by the cell-free homogenate from the strain of Aphanocapsa while [3-14C]geranylgerany1 di- phosphate was a poor precursor of p-carotene.However the enzymes of this strain of Aphanocapsa efficiently incorporated isotopic label into p-carotene when [14C]phytoene was generated in situ from [2-14C]mevalonate by co-incubation with a cell- free enzyme preparation from a phytoene-accumulating strain of P. blakesleeanu~.~~~ A similar ‘coupled enzyme system’ was utilized to observe the biosynthesis of /3-cryptoxanthin u,/3-caroten-3-01] (135a) and the derived ketone (135b) from [14C]phytoene by a broken-cell preparation of Aphanocapsa that retained intact No incorporation of [14C]phytoene into zeaxanthin (1 32b) was observed and little into echinenone [P,p-caroten-4-one] (I 3%) and into the carotenoid glycoside myxoxanthophyll despite the fact that these were major carotenoids in intact Aphanocapsa.A chromoplast membrane preparation from fruits of Capsicum annuum incorporated [ 15,I 5’-3H]lycopene (1 3 1) into p-carotene (1 32a) without the accumulation of y-carotene [p,@-The requisite enzyme activity was tightly mem- OH (129) 12‘ c; 15 15‘-cis or frons ;ll’ 12’-didehydro NATURAL PRODUCT REPORTS 1988-D. M. HARRISON 41 1 R R (132)a; R-H b; R-OH H H + 0 (133) a; R=H b; R-OH Scheme 14 d RL (135) a; R1=OH ,R2= H2 b; R'=OH ,R2=0 c; R~=H,R*=O 9 References brane-bound and exhibited a pH optimum at 6.8.The cyclization of lycopene was inhibited by reagents that react 1 D. M. Harrison Nut. Prod Rep. 1985 2 525. 2 D. M. Harrison Nut. Prod. Rep. 1986 3 205. with thiol groups,252 and also by 2-a~a-2,3-dihydrosqualene.~~~ 3 Methods Enzymol. 1985 Volumes 110 and 11 1 ed. J. H. Law and Practical accounts have been published on the biosynthesis of carotenoids by chromoplasts of Capsicum ann~um~~~ and of daffodil255 and by chloroplasts of and on the biosynthesis of phytoene by enzyme preparations from tomato.256 The biosynthesis of phytoene and acyclic carotenoids was found to be stimulated by red light in radish plants (Raphanus sativus) that had been grown in the dark in the presence of the herbicides SAN 6706 amitrole or J852.257The biosynthesis of @-carotene was enhanced by illumination in the yeast Rhodo-sporidium diobovat~m~~~ and in Phycomyces blakesleeanus by blue light.259 Unusually the rate of biosynthesis of carotenoids by the fungus Trichophyton mentagrophytes was reduced by visible light.260 It is generally accepted that animals lack the ability to synthesize carotenoids de novo.The wide variety of carotenoids that are found in animals arise from the modification of dietary carotenoids.261 On the basis of isolation studies only pathways have been postulated for the metabolism of carotenoids in ten species of sea squirt (Tunicata),262 in the spindle shell Fusinus perplex~s,~~~ and in some marine Reviews have been published on the biosynthesis of carotenoid~,~~~ the enzymes of carotenoid biosynthesis,266 and the metabolism of carotenoids in The proceedings of the 7th international symposium on carotenoids contain inter alia papers on carotenogenic enzymes from Neurospora crussa,268a Phycomyces bZakesleeanus,268b and chromoplasts of Capsicum annuum;268cother reviews describe the metabolism of caro- tenoids in animals,268d the use of stable isotopes in biosynthetic studies,268e and the photoregulation of the biosynthesis of carotenoids.268f H.C. 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ISSN:0265-0568    

DOI:10.1039/NP9880500387

出版商:RSC    

年代:1988

数据来源: RSC

摘要:

Book Review Dictionary of Antibiotics and Related Substances ed. B. W. Bycroft; 1988;Chapman and Hall London and New York; xviii +944pp.; f295;ISBN 0-41 2-25450-6 It has been difficult for some time to gain up-to-date information on that very important and burgeoning group of naturally occurring organic compounds the antibiotics. Certainly there has been no single reliable source that could be consulted. The problem is now solved through the publication of ‘The Dictionary of Antibiotics and Related Substances ’. There is within this weighty tome (962 pages) a veritable banquet of antibiotics. Also included are those substances which by liberal definition properly belong within antibiotics in such a dic-tionary. Approximately 8000 antibiotics and related com-pounds appear within about 4000 entries in this most welcome of books.The format of the book follows the familiar pattern of ‘The Dictionary of Organic Compounds’. Indeed the data are partly compiled from this other dictionary. Data have been collected from the fifth edition and reviewed prior to inclusion in this new dictionary; very many new substances have been included and the material has been updated to the end of 1986. The entries include structures properties synonyms and key references (the references are suitably marked as relating to structure isolation synthesis and biosynthesis -the last is particularly welcome to this reviewer). There are four indexes a Name Index a Molecular Formula Index a CAS Registry Number Index and a Type of Compound Index.The last of these includes nucleoside antibiotics ansamycin antibiotics tetracyclines polyether antibiotics and so on. This last index is supported by a description of the main antibiotic types with key references which appears at the beginning of the book. The test of a book is how well it works for the reader when he searches for things he knows well. My conclusion after tracking back and forth through the dictionary is that it works very well indeed; only occasional minor errors are apparent and the references are apposite. One quibble you have to look outside this dictionary to find that the symbol D alerts the reader to possible hazards associated with a particular compound. Following this search I have spent a most enjoyable time browsing through the book looking at compounds new to me and thinking about possible biosynthetic routes; others will be more concerned with e.g. synthesis and biological properties. ‘The Dictionary of Antibiotics and Related Substances’ is inevitably expensive though as the foregoing makes clear it is a very valuable book and at something less than 4 pence per compound is very good value for money! B. W. Bycroft and contributors A. A. Higton and A. D. Roberts are to be con- gratulated on producing such an excellent dictionary. R. B. Herbert

ISSN:0265-0568    

DOI:10.1039/NP9880500417

出版商:RSC    

年代:1988

数据来源: RSC