摘要:

ISSN 0265-0568 Coden NPRRDF Natural Product Reports A journal of current developments in bio-organic chemistry Volume3 1986 The Royal Society of Chemistry London Natural Product Reports (ISSN 0265-0568) 0The Royal Society of Chemistry 1986 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. Computer typeset by SB Datagraphics. Printed in Great Britain by Spottiswoode Ballantyne Printers Ltd ISSN 0265-0568 NPRRDF 3 1-614 1-1-1-62 (1986) Natural Product Reports A journal of current developments in bio- organic chemistry Volume 3 CONTENTS 1 Marine Natural Products D.J. Faulkner 35 Recent Developments in the Birch Reduction of Aromatic Compounds :Applications to the Synthesis of Natural Products J. M. Hook and L. N. Mander 87 Avermectins and Milbemycins H. G. Davies and R. H. Green 123 Sesterterpenoids J. R. Hanson Reviewing the literature published to June 1985 133 Biological Methods for Studying the Biosynthesis of Natural Products C. R. Hutchinson 153 P-Phenylethylamines and the Isoquinoline Alkaloids K. W. Bentley Reviewing the literature published between July 1984 and June 1985 171 Pyrrolidine Piperidine and Pyridine Alkaloids A. R. Pinder Reviewing the literature published between July 1984 and June 1985 181 Tropane Alkaloids G. Fodor and R. Dharanipragada Reviewing the literature published between July 1984 and June 1985 185 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites R.B. Herbert Reviewing the literature published between July 1984 and June 1985 205 The Biosynthesis of Carotenoids D. M. Harrison Reviewing the literature published between January 1979 and December 1983 217 Secondary Metabolism -Fact and Fiction E. Haslam 251 Monoterpenoids M. S. Carson and D. H. Grayson Reviewing the literature published during 1983 273 Natural Sesquiterpenoids B. M. Fraga Reviewing the literature published during 1984 297 Pyrrolizidine Alkaloids D. J. Robins Reviewing the literature published between July 1984 and June 1985 307 Diterpenoids J. R. Hanson Reviewing the literhture published during 1984 NATURAL PRODUCT REPORTS 1986 CONTENTS 323 Recent Advances in Chemical Ecology J.B. Harbome Reviewing the literature published between January 1982 and June 1985 345 Aporphinoid Alkaloids M. Shamma and H. Guinaudeau Reviewing the literature published between July 1984 and June 1985 353 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites J. E. Saxton Reviewing the literature published between July 1984 and June 1985 395 The Biosynthesis of the Vitamins Thiamin Riboflavin and Folk Acid D. W. Young Reviewing the literature to June 1985 421 Triterpenoids J. D. COMO~~Y and R. A. Hill Reviewing the literature published between January 1984 and June 1985 443 Steroidal Alkaloids D. M. Harrison Reviewing the literature published between July 1983 and June 1985 45 1 Diterpenoid Alkaloids S.W.Pelletier and S. W. Page Reviewing the literature published between July 1983 and June 1985 465 Chromanols Chromanones and Chromones S. T. Saengchantara and T. W. Wallace Reviewing the literature published between 1976 and December 1985 477 Spectral Characteristics of the Bisbenzylisoquinoline Alkaloids H. Guinaudeau A. J. Freyer and M. Shamma 489 Recent Advances in the use of Enzyme-catalysed Reactions in Organic Research The Synthesis of Biologically Active Natural Products and Analogues S. Butt and S. M. Roberts Reviewing the literature published between 1980 and 1985 505 Steroids Physical Methods D. N. Kirk Reviewing the literature published between mid-1983 and mid-1985 515 Steroids Reactions and Partial Synthesis J.Elks Reviewing the literature published during 1984 555 Erythrina and Related Alkaloids A. S. Chawla and A. H. Jackson Reviewing the literature published between July 1983 and June 1985 565 The Biosynthesis of Shikimate Metabolites P. M. Dewick Reviewing the literature published during 1985 587 Muscarine Imidazole and Peptide Alkaloids and other Miscellaneous Alkaloids J. R. Lewis Reviewing the literature published between July 1984 and June 1985 59 1 Carotenoids and Polyterpenoids G. Britton Reviewing the literature published between September 1984 and December 1985 Erratum to Steroids Physical Aspects D. N. Kirk (Vol. 3 No. 5 p. 505) I-1 Index of Authors Cited 1-33 Subject Index Natural Product Reports EditorialBoard Professor G.Pattenden (Chairman) U n iversi ty of Nottin g ham Dr. D. V. Banthorpe University College London Professor M. F. Grundon University of Ulster at Coleraine Professor F. D. Gunstone University of St. Andrews Dr. J. R. Hanson University of Sussex Dr. R. B. Herbert University of Leeds Dr. T. J. Simpson U n iversi ty 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. This journal includes reviews of books relating to natural products. Volumes for review should be sent to the editorial office for which the address is The Royal Society of Chemistry Burlington House London W1V OBN and marked for the attention of Mr B. J. Starkey. Contributors to Volume 3 Bentley K. W. 153 Britton G. 591 Butt S.489 Freyer A. J. 477 Grayson D. H. 251 Green R. H. 87 Kirk D. N. 505* Lewis J. R. 587 Mander L. N. 35 Carson M. S. 251 Chawla A. S. 555 Connolly J. D. 421 Davies H. G. 87 Dewick P. M. 565 Guinaudeau H. 345 477 Hanson J. R. 123 307 Harborne J. B. 323 Harrison D. M. 205 443 Haslam E. 217 Page S. W. 451 Pelletier S. W. 451 Pinder A. R. 171 Roberts S. M. 489 Robins D. J. 297 Dharanipragada R. Elks J. 515 Faulkner D. J. 1 181 Hill R. A. 421 Hook J. M. 35 Herbert R. B. 185 Saengchantara S. T. 465 Saxton J. E. 353 Shamma M. 345 477 Fodor G. 181 Hutchinson C. R. 133 Wallace T. W. 465 Fraga B. M. 273 Jackson A. H. 555 Young D. W. 395 *An erratum for this article follows this page

ISSN:0265-0568    

DOI:10.1039/NP98603FP001

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

Recent Developments in the Birch Reduction of Aromatic Compounds Applications to the Synthesis of Natural Products J. M. Hook School of Chemistry University of New South Wales P.O. Box I Kensington N.S. W. 2033 L. N. Mander Research School of Chemistry Australian National University G.P.O. Box 4 Canberra A.C. T. 2601 Reviewing the literature published to May 1985 L Introduction 3 General Considerations & 2.1 The Reduction Process 2.2 Experimental Procedures 2.3 Scope and Limitations 2.3.1 Subs t ituen t Effects 2.3.2 Secondary Reactions 3 Reduction of Alkyl- and Alkoxy-benzenes 3.1 Discussion 3.2 Intramolecular Protonation of Birch Intermediates 3.3 Synthetic Applications 3.3.1 Preparation of Cyclohex-2-enones 3.3.2 Cyclohexadienyl Anions 3.3.3 Iron Tricarbonyl Complexes 3.3.4 Carbene Addition 3.3.5 Diels- Alder Cycloaddition 4 Reduction of Aromatic Acids 4.1 Gener a1 Con side rat ions 4.1.1 Reduction 4.1.2 Reductive A1kylation 4.1.3 Re-aromatization 4.2 Alkylbenzoic Acids 4.3 Tetrahydrobenzoic Acids 4.4 Methoxybenzoic Acids 4.4.1 Reduction of 3-Methoxybenzoic Acid 4.4.2 Reduction of 2-Methoxybenzoic Acid and its Derivatives 4.5 N aphthoic Acids 4.5.1 I-Naphthoic Acids 4.5.2 2-Naphthoic Acids 4.6 Miscellaneous Aromatic Acids 4.6.1 Biphenylcarboxylic Acids 4.6.2 Furoic Acids 4.6.3 Thiophene-2-carboxylic Acids 4.7 Synthetic Applications 4.7.1 Re-aroma tization 4.7.2 Syntheses of Cyclohexenones 4.7.3 Electrophilic Additions to Dihydrobenzoic Acids 4.7.4 Nucleophilic Additions to Dihydrobenzoates 4.7.5 Derivatives of Iron Tricarbonyl 4.7.6 Ring Expansion 4.7.7 Anne1 ations 5 Reduction of Aromatic Carboxylic Esters 5.I Discussion 5.2 Synthetic Applications 5.2.1 Cyclohexenones 5.2.2 Annelations 6 Reduction of Aromatic Amides 7 Reduction of Aromatic Ketones 7.1 Discussion 7.2 Acetophenones 7.3 Tetralones 7.4 Indanones 7.5 Acetylnaphthalenes 7.5.1 1-AcetylnaphthaIenes 7.5.2 I -A1kanoyl-6-methoxynaphthalenes 7.5.3 2-Acetylnaphthalenes 7.6 Di hydrophenan threnones 8 Reduction of Aryl- and Benzyl-silanes 8.1 Arylsilanes 8.2 Benzylsilanes 9 Reductive Fission of Hetero-substituents 9.1 Hydrogenolysis of Nuclear Substituents 9.1.1 Simple Ethers 9.1.2 Deoxygenation of Phenols 9.1.3 Alkoxy-substituted Aromatic Acids and Ketones 9.2 Hydrogenolysis of Benzylic Substituents 9.2.1 Benzylic Alcohols 9.2.2 Benzylic Acetals 9.2.3 Benzylic Amines 10 Conclusion 11 References 1 Introduction The reduction of aromatic compounds by solutions of metals in liquid ammonia which was developed by Birch some four decades ago provides one of the most powerful synthetic procedures available to organic chemists.* In one simple reaction it bridges the gulf between aromatic and alicyclic molecules thereby making much of the huge arsenal of aromatic chemistry readily available for the construction of alicyclic structures.The Birch reduction also furnishes the facility to carry a synthon which is inert towards an extensive array of reagents but which is also rich in latent functionality through several stages of a protracted synthesis. The great majority of applications of the Birch reduction have been centred on aromatic ethers and simple alkyl- benzenes. Over the past decade or so however there has been increasing attention given to other derivatives. The reduction of aromatic acids has been placed on a systematic basis while new procedures have been established for substrates such as aromatic esters and ketones which have until recently been regarded as unsuitable candidates for Birch reductions.The aim of this Report is to provide a supplement to earlier reviews' -lo and an update to the scope and limitations of the reduction of aromatic substrates by metals in liquid ammonia with a view to demonstrating the extraordinary utility of these processes for the synthesis of natural products. 2 General Considerations 2.1 The Reduction Process Metals of Groups I and I1 of the Periodic Table dissolve readily in liquid ammonia. The resulting solutions of solvated electrons are powerful reducing agents that may be used to perform highly selective reactions.Our understanding of the details of the reduction process as applied to benzenoid substrates is summarized in Scheme 1.I I Electrons are added in a reversible step to form a radical anion which is usually protonated and reduced further to a pentadienyl anion.' Alternatively the radical anion may be * This review was prepared to mark the occasion of the 70th birthday of Professor Birch on 3rd August 1985. 36 NATURAL PRODUCT REPORTS 1986 HH EWG e - 7 6- c - BH ( EWG 1koH (Y = R OR ,NH2 or NR2) BH L -HH (R = H or alkyl) Ie-EWG I BH BH L 4 7 B-HH EWG e t = COz- COzR COR or Ar ) Scheme I reduced further to a dianion which is then protonated to afford the same pentadienyl anion.Protonation of this intermediate occurs predominantly at the central carbon atom15 to form the unconjugated 1,4-diene which is resistant to further reduction. With substrates that bear electron-withdrawing groups (EWG) the radical anions are formed in sufficiently high concentrations to be protonated by the ammonia or may be reduced further to dianions. Either way reduction proceeds readily (see Sections 4-7). With aromatic ethers and alkyl- benzenes however it is necessary to displace the initial equilibrium between the substrate and the radical anion through protonation by a stronger acid than ammonia (pK, CCI 35). Alcohols (pK;,16-20) are normally used for this purpose. Over-reduction may occur through initial protonation at the terminal atoms of the pentadienyl anion but occurs most frequently when the reaction medium is sufficiently basic to catalyse conjugation of the 1,4-dienes to the 1,3-isomers.The alcohol may then serve an important additional role by acting as a buffer although for substrates that bear an electron-withdrawing group the presence of an alcohol promotes over- reduction. 2.2 Experimental Procedures Procedures for metal -ammonia reductions have been de-scribed in several reviews’ lo and are outlined for specific substrates in later sections of this article. The following NATURAL PRODUCT REPORTS 1986-J. M. HOOK AND L. N. MANDER comments are therefore only a summary of the more important factors. Although many reductions are relatively insensitive to the choice of conditions the majority may be significantly improved by attention to detail.The ammonia should be carefully dried and distilled. Sodium metal is a convenient drying agent but a small amount of an iron salt should be added to catalyse the formation of sodium amide since sodium itself reacts progressively more slowly with water as the pH of the ammonia solution increases. Oxygen should be rigorously excluded and although ammonia at reflux provides quite good protection it is advisable to employ an inert atmosphere. Nitrogen is usually satisfactory but there is a potential for reaction with lithium so the use of helium or argon has been recommended for reductions with this metal.' Temperature is often an important factor and should be monitored with an internal thermometer or sensor since the assumption is rarely warranted that the temperature of the reaction mixture is the same as that of the external cooling bath.Low temperatures (<-70 "C)are crucial for the ring-reduction of aromatic esters and ketones and help to suppress unwanted side-reactions with other substrates e.g. hydrogenolysis of methoxy substituents. The choice of metal can have a profound effect on the outcome of the reduction :l lithium and sodium are usually the most satisfactory but potassium may be superior and sometimes essential for specialized applications. Calcium metal is favoured in the reduction of a-acetoxy-ketones and magnesium appeared to offer advantages in some early reductions of aromatic amides esters and polycyclic hydrocar- bons,'- but on the whole there appears to be little point to the use of metals other than the common elements of Group I.The solubilities of ionic substrates e.g. carboxylate salts and their products depend very much on the metal counter-ion. Alcohols aid solubility and it is common for insoluble substrates to dissolve as the reduction progresses. A co-solvent is usually necessary however and tetrahydrofuran (which must be dry and oxygen-free) is most commonly utilised although other ethers may be employed. Tertiary alcohols (butyl and amyl) react very slowly with the metal but methanol or ethanol protonate the intermediate radical anions more rapidly and can suppress side-reactions.Water and ammonium salts have also been used for protona- tion but may have a disastrous effect on many reactions and should be avoided for quenching purposes while any metal remains. Any excess of metal is best consumed by the addition of a simple diene (isoprene or penta-l,3-diene) since the metal will often react more rapidly with (protonated) product than with the quenching agent leading to over-reduction. Inverse quenching has been advocated for especially sensitive products. It has been noted elsewhere that the order of addition of reagents is the most important single variable to influence the distribution of products. The preferred procedure in our laboratories is based on the addition of small pieces of metal to a mixture of substrate ammonia solvent and (if appropriate) alcohol at < -70 "C until the blue colour persists for a reasonable period (approximately 10 minutes).Penta- 1,3-diene is then added followed either by ammonium chloride or an electrophile. Alkylations are best carried out on lithium derivatives so in cases where other metals have been used for reduction carefully dried lithium bromide is added before the alkyl halide. 2.3 Scope and Limitations 2.3.I Suhstituent Efl2c.t.s Elec tron-releasing groups direct reduction to unsu bsti tu ted 2,5- positions and while alkyl groups retard reduction (t-butyl > isopropyl > ethyl > methyl) amino-groups and alkoxyl groups slightly accelerate the rate of reaction.I9 Phenols are rapidly ionized and are resistant to the addition of electrons although reduction may be effected with high concentrations of In sharp contrast P-naphthols are readily re-duced furnishing the corresponding p-tetralones."- 24 Groups which allow delocalization of electrons accelerate reduction and afford 1,4-dihydro-derivatives irrespective of alkyl alkoxy and amino substituents.A major limitation on the choice of electron-withdrawing groups is the ease with which they themselves undergo reduction. Silanes and carboxylic acids present no difficulties but esters amides ketones and polycyclic aromatic compounds usually require specially selected conditions. In the absence of an electron-withdrawing group it is usually difficult to reduce benzene rings which carry bulky substituents or which do not have two unsubstituted positions in a para relationship.Compounds with 1,2,3-~ubstitution patterns for example can normally be reduced only under forcing condi- tions although there are examples where intramolecular protonation greatly accelerates reaction (see Section 3.2). 2.3.2 Secondary Reactions A variety of secondary reactions may reduce the yield of a desired product and in some cases may prevent its formation altogether. For example it is possible for both aryl and benzylic hetero substituents to undergo hydrogenolysis (see Sections 4.4 and 9). Re-aromatization may occur readily usually because oxygen is present but in some cases by loss of hydride ion; tetrahydro-products often arise as a consequence of base-catalysed conjugation of cyclohexa- 1,4-dienes or by initial protonation at C-1 or C-5 of the intermediate pentadienyl anions or during quenching procedures which do not ensure complete consumption of metal before protonation of product anions; ammonia is an excellent solvent for alkylations but may prove to be too nucleophiiic for very reactive electrophiles such as benzyl iodides.3 Reduction of Alkyl- and Alkoxy-benzenes 3.1 Discussion The Birch reduction of aromatic hydrocarbons and ethers proceeds smoothly when the substitution pattern allows the addition of hydrogen to two unsubstituted positions in a pcrru relationship thereby affording 2,5-dihydro-derivatives. The literature is replete with examples over the past four decades.The utilization of anisole moieties as precursors to cyclo- hexenones has been applied to a wide variety of synthetic aims but has been of very limited value with 1,2,3-trisubstitution patterns and more densely substituted derivatives. Compounds (1)-(5) for example have only been reduced by employing massive excesses (200-600 equivalents) of lithium metal." 29 while the aromatic rings in (6) and (7) are completely resistant to ~-eduction.~".~l 3.2 Intramolecular Protonation of Birch Intermediates A number of isolated reports in the literature indicate that neighbouring hydroxyl functions enhance the rates of reduction of substrates which would otherwise be very slow to react. It has been suggested that the accelerations of rate may stem from both complexation with the metal counter-ion that is associated with the intermediate radical anion and intramolecular protonation.It was found for example that the yield of reduction products from the I 10-hydroxy-derivative (5) was significantly enhanced (> 69q()'" when compared with the 33"/ yield from the analogue (4),2x although the reaction was still carried out with ca 600 equivalents of lithium metal. Selective reduction of ring A in the tetrahydropyran-2-yl ether (8) could be achieved satisfactorily but concomitant reduction of the 13-phenyl group was unavoidable in the alcohol (9) indicating participation by the 17P-hydro~yl.~~ Reduction of the aryl-norbornanone (10) by lithium-ammonia proceeded satisfactorily ria the eso-alcohol to give HO)/$IOMe Ho@e the normal dihydro-aromatic derivative whereas the methyl- ene analogue (1 1) gave a 1 :1 mixture of dihydro-and tetrahydro-products.33 Recent studies on the reduction of the dimethano-anthra- cenes (12)-(14) have provided some very interesting examples of intramolecular protonation of radical anions (Scheme 2).Reduction of the parent hydrocarbon (12) or the methoxy- derivative (13) proceeds very slowly (tt z 10 h) to give the expected dihydro-compounds (1 5) and (1 6) respectively. The alcohol (14) however is reduced within minutes to the tetrahydro-product (18) which arises through protonation at substituted positions on the more hindered face of the aromatic ring; the reaction is therefore clearly intramolecular.It may be assumed that the diene (17) is an intermediate and it was found that a sample that had been prepared independently was reduced under the same conditions to (1 8).-34 The reduction of the tricyclic alcohols (19) and (20) was studied during a total synthesis of the diterpenoid enmein (Scheme 3) and comparisons have been drawn with the reduction of (1). It is apparent that intramolecular protonation is involved but the systems are rather too complex for any general inferences to be made.3s 3.3 Synthetic Applications There are several important reactions in which 1,4-dihydro- benzenes and their derivatives participate. These include the formation of cyclohexenones from dihydroanisoles the forma- tion and reactions of cyclohexadienyl anions the preparation and uses of tricarbonyliron complexes and reactions with carbenes carbenoids and dienophiles.3.3.I Prepurution of‘ Cyclohex-2-enones The use of anisoles as synthetic equivalents to cyclohex-2- enones (Scheme 4) has been widespread since the original observations of Birch and there is a wealth of examples inter ah,from syntheses of steroid^,^,'.^^ terpene~,”.~~ sesquiter-penes,39 diterpenes,40 47 diterpene alkaloids,4x triterpenes,4L” and opium alkaloids.s0 s3 The reduction of silyl aryl etherss4 is a useful variation and it would seem to have considerable potential in the light of recent advances in silicon chemistry. Trimethylsilyl ethers are cleaved but both the t-butyldimethylsilyl (2 1) and the isopropyldimethylsilyl phenyl ethers (22) are stable in the reducing medium and they afford the 1,4-dihydroaryl silyl ethers (23) and (24) in 80-97% yields (Scheme 5).The dihydro- aromatics (25; R = Me) and (25; R = OMe) were used as precursors to the relatively inaccessible py-unsaturated ketones NATURAL PRODUCT REPORTS 1986 Me0 \ HO @ H (3) Me0& \ OMe (8)R = Thp (10) R = 0 (9) R = H (11) R = CH2 A (12) R = H -(15) R = H (13) R = OMe ____) (16) R = OMe (14) R = OH I (17) (18) Scheme 2 (26) through fluoride-mediated hydrolysis of the silyl enol ethers. Alternatively (25; R = Me) could be C-acylated specifically to give (28) (Scheme 6). 3.3.2 Cjrlohe.uaciienj1I Anions A variety of I -alkoxy- 1,4-dienes has been regioselectively deprotonated at the allylic orfho-position by potassium amide or alkyl-lithiums.The resulting pentadienyl anions are excel- lent nucleophiles which react selectively at C-2 and serve as valuable synthetic equivalents to cyclohex-2-enone 2-anions. In an early study by Birch the dihydroanisole (29; R = H) was converted into 2-methylcyclohex-2-enone (31 ; R = H) as outlined in Scheme 7 but the _?-methyl analogue (29; R = Me) appeared not to be deprotonated as readily under the same conditions.5s Later it was found that metallation of (29; R = Me) also failed with BunLi-HMPA-THF so ethers that were chosen for their ability to chelate more effectively with the metal centre were employed.Sh5x Excellent results were obtained if C0258and a variety of alkyl halide^^^.^' were the NATURAL PRODUC'T REPORTS 1986 -J.M. HOOK AND L. N. MANDER OMe OMe Me0 *Q0 H@o (19) Li (10 eq.) --la0/" 34'10 EtOH (50 eq.) Li (18 eq.) (19) -9"lo -64'10 EtOH (120 eq.1 Li (100 eq.) (20) -lO"10 13"lo 28 "lo EtOH (300 eq.) (1)-L i (600eq.) 8"Io 1 l"l0 EtOH (780eq.) Scheme 3 ... Me0 (29) (30) (311 1ii Reagents i KNH? liq. NH,; ii MeI; iii HCI (aq.) Scheme 7 Reagents i. metal liq. NH,3 ROH; ii (CO,H),; iii HCI Scheme 4 RMe2Si0 RMe,SiO oy (21) R =But (23) R = But (22)R = Pr' (24) R = Prl (Y = H ; o- rn- or p -Me ; or o- rn- or p -0Me) Reagents i lithium Bu'OH liq. NH3 Scheme 5 I Pr Me2Si0OR o D R + o D R '0 OMe (34) CO Me (35) Reagents i Bu"Li HMPA; ii RX; iii Bu"Li TMEDA; IV CO?;v.Met K,CO,; vi HCI (aq.) Scheme 8 [R=Me] ii (R= Me or OMe) electrophile's (Scheme 8). Very recently however the original procedure of Birch based on KNH? has been found to be highly effective for (29; R = Me) its 4-isomer and even (29; R = n-decyl).s9 The general methodology has been applied to the synthesis of oplopanone (40),h0 pregn-4-en-20-one (43),h (Z)-henicos-6-en- (28) 11-one (48) [which is the male sex attractant of the Douglas Fir Reagents i. Bu:NF H,BO, HzO THF; ii MeCOCI TiCI Tussock Moth (Orgyiu pseud~tsugata)],~~ and the alkaloid Ti(OPr'), CH2C12 at -78 "C secodaphniphylline (53),58 respectively (Schemes 9 10 1 1 and Scheme 6 12).NATURAL PRODUCT REPORTS 1986 (36) (37) (38) (39) (40) Reagents i BunLi THF at -78°C; ii isohexenyl bromide HMPA at -78°C; iii Ac20 AcOH HClO, at 0°C; iv KOH MeOH; v Me,S(O)==CH,; vi LiAlH,; vii 03,MeOH; viii Me$; ix KOBu' MeOH heat Scheme 9 . .. ... I II Ill - O-NEt 2 HO SiMe2Ph (32) (41) (42) (43) Reagents i BunLi HMPA at -70 "C; ii RBr; iii PhMe,SiLi THF; iv CF3C02H CF,CH20H at -10 "C Scheme 10 0 0 + i -iii + -iv &:."" by::l c5c10"2,&'."" ClOHZl ClOHZl 10 21 vi 0 II (48) Reagents i KNH, THF liq. NH, at -33 "C for 30 minutes; ii RBr; iii HCl H20 MeOH; iv H202 KOH MeOH; v TsNHNH, AcOH; vi H2 Pd/BaSO, EtOAc Scheme 11 C02Me II ___) 5$3 6Si Me2Bu' (49) (50) (51) iii -v HO (53) (52) Reagents i heat at 140°C for 66 hours; ii Bu~NF H,C=CHCH2Br; iii 0,; iv Zn HOAc; v Bu2NF Scheme 12 NATURAL PRODUCT REPORTS 1986 -J.M. HOOK AND L. N. MANDER 0 ... Ill Meow (55) (56) GOMe (54) (57) (58) Me0 / I OMe VI,VIII ____)Vlll,IX 0 O k (JJAq+x (y____)XI-XIII OCOPh I I nrnnL @ H ___)XIV-XIX p H 0 (611 (62) (63) Reagents i KN Hz. liq. NH,; ii ArCH,CH,Br Et,O; iii polyphosphoric acid; iv NaBH, MeOH; v 10%Pd/C at 285 'c' for 1 hour; vi. Bu'Li HMPA; vii 5-iodohex-I-ene; viii HCI Me,c'O; ix PhCOCI pyridine; x hv (450 W medium-pressure mercury-vapour lamp); xi LiN(SiMe,),; xii MeI; xiii. KOH H,O DMSO; xiv HOCH,CH,OH H+; xv LiAIH,; xvi P0Cl3 pyridine; xvii HOAc H,O THF; xviii. Ph3Pr==CH, THF; xix RhCI,.3H20 EtOH Scheme 13 Fe FeKO) I I Y Y R = HI OMe C02Me etc.Y = RzNH R3P HS03- CN- CH(C02Me)2 RLi ,R2Cd wSiMe3 Scheme 14 The C'-alkylation of cyclohexane- 1,3-diones is character- 3.3.3 Iron Tricurhotij.1 C'omp1c.w.~ istically difficult to control since dialkylation and 0-alkylation The reactivity of cyclohexudienes that are bound to iron is are serious competing reactions. The utilization of 1,3-currently 01' great interest in synthetic chemistry. The presence dimethoxycyclohexa- 1,3-diene (54) as an operational of the tricarbonyliron group ;illows the formation of stable equivalent3 5.f? 2 is accordingly a valuable synthetic procedure cations riu hydride abstraction and also determines the regio- and has been applied to syntheses of the chrysenone (56),h3 and stereo-chemical aspects of subsequent reactions of these benzo[cJphenanthrene(58),h4and ( )-1 1-cJpi-precapnelladiene cations with a wide variety of nucleophiles (Scheme 14).The (63)h5 (Scheme 13). cyclohexadiene may then be liberated from the iron complex by It is important to generate and use all of these cyclohexa- oxidative treatment. Since the dienyltricarbonyliron complexes dienyl anions at temperatures below -30 "C since there is a can be obtained in optically active form there is potential for tendency for them to behave as hydride donors and to enantioselective syntheses.h8 'O Already much of the chemis- disproportionate to the parent aromatic compound and the try of these complexes has been established and utilized in cyclohexene."" The anion that is derived from dihydroanisole syntheses of terpenes,' ~teroids,".~" alkaloids,'4 7f' trichothe-(29; R = H) functions as a hydride donor towards benzalde- canes,7' and a prostaglandin an;ilogue.7x General aspects are hyde rather than as a nu~leophile.~' covered in recent reviews"' H.3 (see also Section 4.7.5).The NATURAL PRODUC‘T REPORTS 1986 Meow ---+ C02Me (64) (65) (66) Meow Me0 iii -v f-t C02Me \OH L (69) (68) (67) (major diastereomer ) vi,vii H ‘OMe ‘OMe ‘OMe (70) Reagents i NaBH,; ii TsOH; iii SOCI?,pyridine; iv Bu>AIH;v VO(acac), Bu‘OOH; vi NaH Mel; vii Me,N -0-;viii H,30+;ix MeLi; x Cr0,.2pyridine CH2CI,; xi POC1,3,pyridine; xii Ph,P-CH,; xiii m-CIC,H,COJH ~ Scheme 15 0 Me0& Bra + BrMe0 ‘ -8 0 (73) (74) (751 Reagents i C’HBr, KOBu‘; ii AgNO, H,O MeOH Scheme 16 synthesis of the trichothecene (72),” as shown in Scheme 15 the conversions of (+)-oestrone methyl ether into 10O-methyl provides an elegant illustration of the utility of the steroids.8y.y0 In a more recent example the methodology was tricarbonyliron-diene complexes in controlling stereoche-applied to a synthesis of (-t-)-pupukeanone (94) (Scheme 18).”’ mistry and blocking unwanted reactions.The first attempt began with the Birch reduction of 2,4- dimethylanisole (83) but this led to a mixture of the olefins (86) and (87). Sequential treatment of the diene (84) using the 3.3.4 Curhene Addition Conia modification of the Simmons-Smith reaction and an The cyclopropanation of 1-methoxycyclohexa- 1,4-dienes with ethyl-diazoacetate-based cyclopropanation followed by a dihalocarbenes occurs preferentially at the enol-ether double- double fragmentation however afforded the desired inter- bond and has been exploited in syntheses of troponess5 (cf mediate enone (89).Section 4.7.9 including A-homo-steroid hormones (Scheme 16).8s Another synthesis of tropones is based on allylic oxidation of 3.3.5 Did-Alder C-vclouddition the bicyclo[4.1 .O]octenes (77) with chromium reagents (Scheme Dihydrobenzenes that have been produced by metal-ammonia 17). the 4-substituted tropones (80) are available directly and reduction of benzenoid compounds provide a rich source of the 3-substituted derivatives (81) are produced by acid-cyclohexa-l,3-dienes for [4 + 23 cycloaddition reactions.Iso-catalysed ring-expansion of the bicyclic enone (79).*” merization of the 1,4-dienes to 1,3-dienes may be achieved with y-Alkylation of cyclohex-2-enones has been achieved in a a range of catalysts,’+’ O5 although the process rarely goes to formal sense using a cyclopropanation/fragmentation method-completion. However isomerization of the 1,4-diene can ~l~gy.*~.~* Some of the first examples that were reported were usually be achieved in siru (either thermally or with a catalyst)oh NATURAL PRODUCT REPORTS. 1986-1. M. HOOK AND L. N. MANDER r 1 L J 0 (79) p (X = CI or Br) 0 (81) Reagents 1 NaOMe CI,C'C02Et; ii CHBr, KOBu'; iii CrOl.DMP (DMP = 3,5-dimethylpyrazole); iv MeSO,OH CH2C12 Scheme 17 OMe 0Me n n (82) R = H (84)R = H (83) R = Me (85)R = Me [R=H]Iiii,iv OMe V -CO;! Me MeP 0 Et02C (88) (89) (90) VII -x ,IX 1 OMc xii ,xiii -xi k, f--Me& 0 (94) (93) (92) (91) Reagents I Li.liq. Nld.< Bu'OH; ii HOCH,CH,OH TsOH; iii CH212 Ag/Zn couple; iv N,CHCO,Et copper bronze; v HCI MeOH; vi. (MeO),CH. TsOH heat for 22 hours then heat at 170 "C for 15 minutes; vii LiOH; viii LiAlH,; ix DMSO SO,.pyridine; x H2C -CHMgBr xi. PhH a1 60 'C;xii H,C-CMeLi; xiii DMSO. at 160°C; xiv H, Pt MeOH Scheme 18 in the presence of the dienophile to afford bicyclo[2.2.2]octanes As indicated in Scheme 19 the fragmentation of the Diels- directly. As expected from frontier-orbital considerations Alder adducts can be effected by a number of different electron-deficient dienophiles afford predominantly ortho-reagents9' O1 and like the cyclopropanation-based method- adducts which have been utilized in three distinct strategies ology that was described in the previous Section the overall based on fragmentations (Schemes 19 and 20) [1,2]- and [3,3]-processes may be viewed as being equivalent to y-alkylation of sigmatropic rearrangements (Schemes 2 I 26) or elimination a latent cyclohexenone system.~ of ethylene with re-aromatization (Scheme 27). This strategy of reduc t ion-cycloadd ition- fragmentation al- 44 NATURAL PRODUCT REPORTS 1986 6 (il. i,ii iii + ___) +NoHi” + / CN 0 0 OThp OH 0 Y (100) Thp = tetrahydropyran -2-yl (101) OMe 0 OH ...x ,xi XIII :$I.*-(103) vii ,xii -MeomNMe (106) CHZR (108) (107) Reagents i H,C --C(C‘I)CN; ii Na,S. EtOH; iii. NH,OH.HOAc; iv NaH. TsCI Et,O; v. MeCO,H AcOH NaOAc; vi NaOH MelSO,; vii. H,C-CHCO,Me; viii H,>O+;ix NaH; x H,C -CHC‘(O)Me; xi. MeMgI; xii RCH,MgBr; xiii IOM-HCI Scheme 19 lows a high degree of stereochemical control as illustrated by (*)-Stachenone (132) which is a member of the beyerane the recent syntheses of the spirovetivane sesquiterpenes e.g. group of diterpenes has been synthesized from the I-methoxy- ( k)-lubimin (1 17) (Scheme 2O).lo2 Other examples are found in bicyclo[2.2.2]octeno1(I 28) (Scheme 22).Io7 A detailed study of the syntheses of(+)-juvabione (I 18),lo3trichodermin (1 19),”’ the [2.2.2] + [3.2.1] rearrangement that was used in this (+)-bazzanene (120),lo1 and (+)-nootkatone (121).105 sequence has revealed that e.w-alcohols such as (128) exhibit Rearrangements of bicycle[2.2.21octane derivatives have more discrete reactivity than the isomeric endo-alcohols.lox been used to construct the bicyclo[3.2. Iloctane skeleton which In one approach to a synthesis of the gibberellins the GI YO-is a feature of a number of natural products. For example bicyclo[2.2.2]octenols (1 33) rearranged in acid (Scheme 23).”)” studiesof models for the synthesis of diterpenoid alkaloids have The alcohol (133; R = 3-oxocyclohexyl) gave the gibbanes resulted in routes to the cis-fused B/C/D ring system (127) of (1 35) directly but although the aryl derivatives (1 33; R = Ph) veatchine from the Diels Alder adduct (123) as shown in and (1 33;R = rn-MeOC,H,) rearranged to the corresponding Scheme 21.’06 bicyclo[3.2.l]octanes ( I34) they failed to undergo cyclo- NATURAL PRODUCT REPORTS 1986 -J.M. HOOK AND L. N. MANDER OMe OAc (1 09) (110) (111) (115) (116) (117) Reagents i Li liq. NH, EtOH; ii H,C-CHCO,Me dichloromaleic anhydride at 150°C for 5 days; iii (CO?H)? MeOH HzO iv Ac,O pyridine; v TsNHNH, H,O+ THF heat for 20 hours; vi MeLi; vii MeSO,CI CH2CI, at -78 "C;viii (C02H)? H,O Bu'COMe at 130 "c for 8 hours; ix. HCN Et,AI; x BH.3.NH.3 MeOH; xi BuiAIH; xii KOH MeOH Scheme 20 dehydration. Another promising approach to the gibberellins relies on Lewis-acid-catalysed rearrangement of the epoxide (1 37) (Scheme 24).' 0pco2..Yp.7 The tricyclo[5.3.1 .02."]undecane skeleton of the barbatene sesquiterpenes has been assembled using a rearrangement step similar to the previous examples (Scheme 25).' Treatment of OAc the key intermediate (142) with hydriodic acid leads to the (118) (119) synthesis of (+)-p-and (+)-a-barbatene (1 50) and (1 5 1) respectively. Alternatively exposure to rn-chloroperoxybenzoic acid and then alumina provides access to (+)-gymnornitro1 (145) and (+)-isogymnomitrol (146). The application of the oxy-Cope rearrangement to the endo-vinyl carbinols that had been derived from bicyclo[2.2.2]-octanones affords one of the best methods for the stereo-controlled synthesis of cis-octalones. 2. ' The procedure has (120) (121) been used very effectively in syntheses of the lycopodium (122) (123) (124) (1 25) (1 26) (127) Reagents i.H2C- =C'(C")OAc at 150 "C for 36 hours; ii KOH; iii HCI AcOH heat for 3.5 hours; iv Li liq. NH, EtOH; v NaIO,; vi. KOH; vii TsCI. pyridine; viii LiAIH,; ix CrO, pyridine; x Ph3P=-CH Scheme 21 46 NATURAL PRODUCT REPORTS 1986 (95) (128) (129) iii-v I (132) Reagents i EtC(OCH2CH,0)CH,CH,MgBr; ii TsOH AcOH heat; iii NaOMe MeOH; iv Li liq. NH,; v Me,SiCI; vi MeLi; vii H,C- C(SiMe,)COEt; viii Me1 Scheme 22 -%CO2 H \ COZH (133) (134) (135) ( R = 3 - oxocyclohexyl Ph ,or m -MeOC6H,) Reagents i RCH-C(OLi),; ii TsOH AcOH Scheme 23 CN -0‘ (138) Reagents i HZC=C(CI)CN; ii m-ClC,,H,C03H; iii BF,.Et,O THF Scheme 24 alkaloid luciduline (160) (Scheme 26)’ and the structure that has been proposed for cannivonine (a representative of a group of alkaloids purportedly isolated from cranberries).’ 5.1 Ih The potassium alkoxides rearrange 1O’O to lo’’ times faster than the parent alcohols leading to enhanced yields and the opportunity to trap the product enolate anion for subsequent regiocontrolled processes.’ [4 + 21 Cycloaddition reactions of dihydro-aromatics with alkyne derivatives followed by retrograde [4 + 21 cyclo- additions (the Alder-Rickett reaction) provide access to unusual substitution patterns of the aromatic ring (Scheme 27)‘’ and have been applied to the synthesis of inter uliu mycophenolic acid (161).11* Similarly the [4 + 21 adducts from quinones have been oxidized and thermolysed (Scheme 27) to afford a variety of polycyclic quinones,’ I” 33 including daunomycinone (1 62)’ 3J 3h and the benz[a]anthraquinone (163).I 3’ Much of the synthetic methodology that has been described in this Section has its roots in the earlier work of Birch clt The recent studies of Demuth and Schaffner,’ however have opened up a new area of development with exciting prospects for the synthesis of cyclopentanoid structures. The triplet- sensitized photochemically induced oxa-di-n-methane rearran- gements of bicyclo[2.2.2]octenes (1 64) have given the tricyclo- [3.3.0.0’.8]octanones (165) which are cleaved by a variety of reagents to ketones such as (166; R = H) and (167; R = H) (Scheme 28).’38 The methodology has been applied to the synthesis inter aliu of (+)-loganin aglucon 6-acetate (168)’”’’ and iridomyrmecin (169).It also has considerable potential for the preparation of polycyclopentanoid natural products e.g. coriolin (1 70).’ 38 4 Reduction of Aromatic Acids 4.1 General Considerations 4.1.1 Reduction Aromatic acids are reduced by solutions of metals in liquid ammonia very much more readily than simple alkylben~enes and anisole derivatives. In contrast to the normal requirements for the latter derivatives it is often possible to achieve reduction with close to stoicheiometric quantities of metal. The addition of aromatic carboxylic acids to liquid ammonia (or NATURAL PRODUCT REPORTS 1986 J.M. HOOK AND L. N. MANDER ~~ vii 4-++ OMe (1 40) (84) ii-iv 1 t v,vi (142) 1ix 4% + XI ,XI1 + (145) (1 46) endocyclic olefin (151) endocyclic olefin Reagents i heat toluene TsOH; ii BBr,; iii LiAIH,; iv MsCI pyridine; v Na2S HMPA; vi Li EtNH? Pr'OH; vii rn-ClC,H,CO,H; viii A120,; ix HI; x. 90,; xi MeMgI; xii P0Cl3 pyridine Scheme 25 H I Me0 I &OH H Me0 (1 52) (153 1 (154) .1 OH HI H H I vi,vii iv,v (157) (156) (155) viii -x 1 OTs Me xi 0x H (158) (160) Reagents i H,C=C(Me)MgBr; ii heat at 250 "C;iii HOCHZCH,OH H+; iv TsNHNH,; v MeLi; vi m-C1C,H,CO3H; vii PhSNa MeOH heat; viii Raney nickel EtOH; ix TsCI pyridine; x HCI Me,CO; xi MeNH, at 75 "C; xii [CH20],, Bu'OH heat Scheme 26 N4TIJRAL PRODIICT REPORTS 1986 R'CGC R3 heat R' OMe Me0 0 Me0 0 0 Scheme 27 0 0 I 0 OH HO2C (161) R W0 (164) R .li R H 0 H (165) 0 (1 67) vice ivrsa) results in the immediate precipitation of the ammonium salt.As the metal is added however the precipitate usually dissolves as reduction proceeds especially if lithium is used. If reduction is carried out in carefully dried redistilled ammonia. as little as 2.2 gram equivalents of lithium are consumed in some cases. thereby demonstrating that the substrate is reduced much more readily than the ammonium ions. which instead react with the intermediates from reduction of the substrate. However protonation by NH,+is not essential since reduction proceeds equally well on metal carboxylates (although low solubility is then often a problem).The addition of an alcohol is not necessary but it may serve as a useful buffer and can often improve solubility. The presence of an alcohol can nevertheless be deleterious. since it aliows isomerization of the initially formed I .4-dihydro-isomer to the 3.4-isomer and (in this way) the possibility of further reduction.1J0 4.1.2 Rrductiw AlkJ9lation The reduction of benzoic acids affords the dianion (17 1) which may be alkylated in situ by a variety of electrophiles. Clearly the addition of alcohol must be avoided or must be limited to small quantities. It may also be necessary to remove the ammonia before adding the electrophile.The vast majority of applications have been based on reactions with alkyl halides (see below). but additions to formaldehyde,1A' epoxides,'l' and up-unsaturated esters' l3 have also been reported (Scheme 29). 4.1.3 Rr-aromatizut ion The I .4-dihydrobenzoic acids that are derived by reductive alkylation may undergo facile re-aromatization with loss of either the carboxylic groupIAJ or the alkyl The intermediate (I 75)in the synthesis of gibberellins for example was found to be especially labile forming (176) simply on exposure to air. I l1 Oxidative decarboxylation may also be achieved with lead tetra-acetate or electrochemically (see Section 4.7.1). Lms of the I-alkyl group can be a problem when the alkyl moiety can form a reasonably stable free radical since a chain reaction may then be sustained (see Scheme 30).145Attempts to metallate C-4 with strong bases have led to aromatization in this way presumably as a consequence of the presence of traces of oxygen which allow the formation of hydroperoxides; these would then initiate the free-radical chain process.116 Reagents I.hv (1> 340 nm),PhCOMe senqitiLer. 11. Me&-Nafion". 111. Me,SiOCOCF3 Scheme 28 0 HO42 Me02CYOAc -(168) A0 H OH (169) (170) NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANIJER C02H C02H I I H02C R' Y (174) (173) /O\ Reagents I H,CO; ii R1CH=CHC02R'; Ili H2C-CHR Scheme 29 H H .R .R Y/ ~cozH Re + Scheme 30 4.2 Alkylbenzoic Acids The first studies on the reduction of aromatic acids were undertaken by Birch.These were followed spasmodically by other workers until in 1972 a comprehensive study of the Birch reduction of alkylbenzoic acids was undertaken by Van Bekkum and co-workers. 140 These authors found that 2-alkyl derivatives gave almost quantitative yields of 1,4-dihydro-products irrespective 01 the procedures that were employed. With other alkyl derivatives generally excellent yields of 1,4-dihydro-compounds were again obtained from con1 binations of Li and NH (NH4Cl quench) Li and NH and H20,or Li and NH and EtOH (NH4Cl quench). With the last combination however significant quantities of tetrahydro-products were obtained from 4-alkyl derivatives and if the quenching of the reaction (by adding N H,Cl) was omitted 4,5-dihydro-isomers became the major products.If an excess of NH,Cl was added before the lithium metal extensive reduction of the carboxyl group took place. C-Alkylation experiments were also con- ducted and invariably gave l-alkyl-l,4-dihydro-products when alkylation occurred. Not surprisingly alkylation did not occur when water had been added while variable results were obtained in the presence of ethanol. In the absence of an added proton source the yields were excellent. C 4.3 Tetrahydrobenzoic Acids The first sets of experiments that were carried out on the reduction of benzoic acids gave mixtures of dihydro- and (177) 1' C02 H CO,H C02H I 1 I (179) (180) 1181) Reagents i Li 11q.NH, then btOH; ii NH,Cl; 111 ktO-Scheme 31 w-a C02H (185) (184) VII -IX H H L IC-xiti viii 7 HOzC (186) (187) Reagents I 11,hq. NH, then LtOH; 11 LI hq. NH, then NH,CI 111 12 CHCIJ 1v (COCl)? PyllcllIle; V CH,NI; VI CU VII 11,II~. NH, BuIOH; VIII H2Cr0, ace~oiie;IX Ph,P====CH2. Bu'OH x N-bromosuccinlmlde; XI pyriolldln-I-ylacetonitrile IJMSO MI KOBu'; XIII (C02H)?,H20 Scheme 32 tetrahydro-derivatives. This is hardily surp ising 111 the Iight of the study by Van Bekkum and coIleag~es,~+~ the iesults of which are summarized in the piwiuus Section. l'he fiist systematic investigation appears to have been cai I led out un y-isopropylbenzoic acid (177) (Scheme 31),IJ7 with which it was found that reduction by lithium in liquid aniiiioiiia at -70 "C followed by the addition ot ethanol and then aniiiionium chloride at intervals of five minutes furiiished a 2 :1 mixture of trans-and cu-l,4-dihydro-derivatives(178).ln the absence of ammonium chloride the 3,4-dihydio-isorrier (1 79) was ob-tained and this could be reduced further to a 2.5 :1 mixture of' the tetrahydro-derivatives (180) (if amirionium chloride wds used to quench the reaction) or (2)-phellandric acid (181). Indane-2-carboxylic acid (182) was converted in a simildr manner (Scheme 32) into the hydrindene acid (183) and thence 50 NATURAL PRODUCT REPORTS 1986 0 L I -iii @co7H \H + ciil; \ \ CO H C02Me (188) (189) iv ii ,v vi ,vii ii I I vi,vii ii -\ \ C02Me C02Me (194) (195) (193) (1 91) viii -xiii viii -xiii I i (196) (192) Reagents i Li liq.NH, EtOH; ii CH,N,; iii KOH EtOH; iv SOCl,; v CuSO,; vi Li liq. NH3 Bu'OH; vii HZCrO,. acetone; viii. HOCH2CH20H TsOH; ix LiAlH,; x Cr03.2pyridine; xi NH,NH, KOH at 200°C; xii HC1 Me,CO; xiii Ph,P-CH Scheme 33 into (1 84) which was utilized as a precursor to the tricyclic acid (187) in an investigation into the molecular basis of the bio- activity of gibberellins. 48 A crucial step in the conversion of (-)-abietic acid into (+)-phyllocladene (192) and (+)-kaurene (196)'j9 involved the reduction of (1 88) to the mixture of epimers (189) combined with other double-bond isomers (Scheme 33). When the first reduction step is followed by alkylation at C-1 further reduction by the above routes is clearly proscribed.The diene acid can still be isomerized by strong bases however and the resulting conjugated diene can be reduced as illustrated by the sequence that is outlined in Scheme 34 leading to the analogue (204) of the plant growth regulator helminthosporic acid (205). This approach to the synthesis of cyclohexenecarboxylic acids e.g. (200) provides an attractive alternative to the [4 + 21 cycloaddition route to such compounds.lS' 4.4 Methoxybenzoic Acids Some of the synthetically most useful groups of aromatic acids are those which also bear alkoxy functions in that the reduced products are potential cyclohexenones while oxidative re-aromatization may afford useful aromatic substitution pat- terns.The electron densities of the intermediate radical anions are dominated by the carboxyl group so that 1,4-dihydro- products are formed initially although the problems of subsequent isomerization and over-reduction are similar to those that are encountered with alkylbenzoic acids. The main problem with anisoic acids is the potential for hydrogenolysis of the alkoxy function(s). Alkoxy-groups that are in a meta relationship to the acyl group present no difficulties but para-substituents are invariably hydrogenolysed' S2.1 s3 while ortho-substituents are often vulnerable to reductive cleavage.' 54.1 55 The extent of loss of ortho-alkoxy-groups depends very much on the substrate and the reaction conditions. A simple reduction of 2-methoxybenzoic acid by lithium in liquid ammonia under 'aprotic' conditions at -33 "C for example results in ca 7076 loss of the methoxy-group,' 5h whereas 2,5-dimethoxybenzoic acid under the same conditions furnishes a quantitative yield of the 1,4-dihydro-derivative.s-The problem when it occurs is most severe when lithium is employed,ISs and it appears to stem from the ammonium ions that are generated when the acid is mixed with the ammonia since pretreatment with one equivalent of base completely prevents hydrogenolysis in most cases.' s8 The reduced solubi- lity of the carboxylate salt can lead to incomp!ete reduction however although potassium salts are reasonably soluble. Alternatively hydrogenolysis can be suppressed through the use of methanol as the proton source but one-pot reductive alkylation is then impractical.4.4.1 Reduction of 3-Metho.~~~henzoic Acid Early studies on the reduction of 3-methoxybenzoic acid (206) led to a variety of products and a number of uncertainties in NATURAL PRODUCT REPORTS. 1986 -J. M. HOOK AND L. N. MANDER C02H t./i ' &I'y/ (201) XI1 -XIV IX -XI (205) (204) (203) (202) Reagents 1 LI. liq. NH,; ii Mel; iii KOH HOCH2CH,0H heat; iv Li liq. NH3 EtOH; v (COCI)?,pyridine; vi CH,N,; vil. BF.3.Et,0 MeNO,; viii H,. Pd'C; ix Ph,P=CH2 Bu'OH; x N-bromosuccinimide; xi pyrrolidin-1-ylacetonitrile,DMSO; xii KOBu'; xiii. (CO,H), H20. THF; xiv. H,CrO,, MezCO Scheme 34 (211) Reagents i Li liq. NH3 then EtOH; ii EtO-; iii Li liq.NH, then NH,Cl; iv pH 4-5 Scheme 35 structural assignments. I 52.1 53.1s9 A careful study of the reduction was therefore undertaken' 6o and revealed that when (206) was reduced by sodium-ethanol-ammonia the 1,4-dihydro-derivative (207) was formed initially but it isomerized rapidly and quantitatively to the 4,5-dihydro-compound (208) under the reaction conditions. Further reduction could then occur to give the tetrahydro-compound (209) (Scheme 35). The method of choice for the preparation of the 1,4-dihydro- compound (207) therefore entails reduction of (206) with lithium in ammonia in the absence of alcohol followed by quenching with ammonium chloride. The acid (207) is also susceptible to rearrangement under acidic conditions and the isomer (21 1) is formed at pH 4-5 (conditions which might readily prevail during the isolation procedure).3-Methoxybenzoic acid (206) undergoes reductive alkylation without difficulty and has been used extensively in synthesis (see below). 4.4.2 Reduction of 2-Metho.yybenzoic Acid and its Deriratires The reduction of 2-methoxybenzoic acid (21 2) with lithium or sodium in ammonia (with methanol as the proton source) affords the 1,4-dihydrobenzoic acid (2 13) in good yields;'5".' b'.'h2 however with more weakly acidic alcohols or in the absence of an added source of protons (conditions which are necessary for reductive alkylation) the methoxy-group suffers partial hydrogenolysis; the extent is determined by the metal that is used and the temperature at which the reaction is carried out.For example with lithium in ammonia under reflux (at -33 "C),followed by quenching with methyl iodide (204; R = Me) predominates corresponding to ca -70% hydrogenolysis of the methoxy-group. The use of sodium in place of lithium reduces the loss significantly (to <507&1s5 while reduction with potassium completely suppresses the hydrogenolysis. s8 The variable loss of the methoxy substituent is in fact a feature that is common to most derivatives of 2-methoxyben- zoic acid (Table 1). Protocols for dealing with this side-reaction have now been established however either by reduction of potassium benzoates (see Section 4.7.2) or by reduction of the benzoate esters (see Section 5. 4.5 Naphthoic Acids The reduction of naphthoic acids by a metal in liquid ammonia occurs initially in the ring that bears the carboxyl group the position of which is an important factor in governing the final outcome of the reduction.4.5.1 1-Naphthoic Acids 1-Naphthoic acids (25 1) give dihydro-acids (253) resulting from 1,4-addition to the carboxyl-bearing ring' '? (Scheme 36) if reduced by a metal in liquid ammonia. The intermediacy of the enediolate (252) is indicated since it may be trapped by a strong source of protons (such as ammonium chloride) or by an alkyl halide.' 76 If the simple 1,4-dihydro-compound (253; R = H) is required rapid quenching (by ammonium chloride) of the reaction mixture gives superior results' 77 to carrying out the reduction in the presence of an alcohol particularly ethanol; the latter conditions can lead to isomerization and/or over- reduction (CJ Section 4.4 particularly Section 4.4.2).178 NATURAL PRODUCT KEPOKTS I986 '0 Me00, Me0 \ Me0 Me0 \COzH Me09 CO;! H C02H R C02H R COzH . . C02H (212) (213) R = H (214) R = Me (220) (221) (222) R = Me (223) R = CHzPh (225) (215) R = CH,CH=CHz (224) R = CH2CH2CH=CH;! (216) R = Pr" (217) R = Pr' (218) R = n-CSHll (219) R = Et (226) (227) (228) (229) OPh (230) (231) R = CHZCH2Ph C02H H Me0H02CqD\ CO;! H M H02C e H o COZ H w (241) OCH20Me MeO\&3 Me0 C02H C02H HOZC C02H (245) (246) (247) 0CH20Me Me0 i C02H OMe (248) (249) (250) NATURAL PRODUCT REPORTS 1986 -J.M. HOOK AND L. N. MANDER Table 1 Reduction and reductive alkylation of 2-methoxybenzoic acids in liquid ammonia Substrate Reiigents" Elect roph ile Product Yield Reference (212) (212) (212) (213) (212) (213) (212) (212) (217) (217) (?I?) (113) (212) (212) (221) (221) Na. MeOH Na MeOH DME [-i7 MeOH Li. THF Li. THF LI. THF LI. THF LI. THF Li. THF Na. THF KORU' Ru'OH THF Li Nil or K at -78 "C KNH,. K at -78°C KNH,. K at -78°C KNH,. K. at -78°C Li THF KOHu'. Hu'OH THF H+ H + I4+ N H,Cl Me1 H ,C=CHCH ,C1 Pr"1 Pr'I n-C5H ,Br Me1 Me1 Me1 Et,O+ PF,-H,C=CHCH,Br Me1 Me I -looo/ 807< -IOOO; -307$ --30"p 30",',h 30°/,h 84"G 74% 79%-95"" 90:; 257gb -30%'' 4 1,/oh 80"/ 161 162 156 156 156 163 163 163 163 155 158 158 164 164 165 158 (771) (771 1 (126) (738) (230) K ;it -78 "C K.at -78 'C Li. THF 1.i. THF. at -78 Li. THF 'C PhCHzBr H ,C==CH CH ,C H Br Me1 PhOCH ,CH ,Br PhCH,CH,I 87":) 91"; >25'2 70% -60"" 158 1S8 156 166 167 (230) (210) (230) (2.15) (237) Li THF Li THF Li. THF Nit. THF Na. Ru'OH THF 3-(OMe)C H,C H ?CH ,I 3.54 0Me) C H CH,CH I ?-(OMe)C,H,CH ,Br ICH ,CH-C(Me)OSiMe H+ 63" 82:O 759 747 90" 167 168 169 170 162 (239) (241) Li. THF Nil EtZO. at -78 'C Me1 Me1 757 45" 171 172 (241) Na THF Me1 75" 172 (243) (245) Li. THF Li. THF Me1 Me1 42,; 51'; 155 155 (247) (749) KORII' Na MeOH KORu'. Na MeOH H+ H+ -loo",897 173 174 ((I) Rcactiona ucrc c,irried out at the reflux temperature of liquid ammonia (-33 C) unless otherwise indicated; (h)low yields were due to loss of 7-methoxyl 4-Methoxy-1 -naphthoic acid loses the methoxy-group com- pletely during reduction affording (253; R = H) if the reduction is by metal-ammonia solutions.Reductive methyla- tion with lithium in liquid ammonia followed by a methyl R COzH iodide quench gives a mixture of mono- and di-alkylated acids (2511 MO/\OM [(253; R = Me) and (256)].1s1 Reduction of the second ring occurs only after reduction of (252) (253) the first ring is complete. The dihydro-acids (255; R' = H R' Reagents i. metal (MI liq. NH, THF; ii. NH,Cl or RX = Me) and (255; R1 = 7-OMe R' = Me) have been reduced Scheme 36 further and the products utilized in studies of suitable models for the construction of the A-ring region of gibber ell in^,^^' while the acid (255; RI = 6-OMe R' = H) has been used as a source of the P-keto-ester (260) for conversion into the useful tricyclic enone (261) (Scheme 37).lH' 4.5.2 2-Nuphthoic Acids 2-Naphthoic acids similarly undergo preferential reduction in the carboxyl-bearing ring.If an alcohol and limited quantities of metal (cu 5 equivalents) are used the tetrahydro-derivatives are formed in excellent yield. Reduction in the second ring also The 2-methoxy-I-naphthoic acids (254; R' = H) and (254; proceeds cleanly in the expected sense when additional metal R' = OMe) have been reduced by lithium''" or sodium180 in is added and the products have been utilized in the preparation liquid ammonia with no loss of the ortho-methoxy-substituent of the potential precursors (267) and (269) for the synthesis of being reported. Thus in the presence of ethanol the 1,4-diterpenoid alkaloids'83 (Scheme 38) or of the plant growth dihydro-acids (255; R' = H) are obtained while reduction in regulator (275) (Scheme 39).8J the absence of a proton source and quenching with methyl In the absence of an alcohol the reduction may be halted at iodide. affords the alkylated acids (255; Rl = H R' = Me) the stage of the dihydro-compound but with difficulty. and (255; RI = OMe R' = Me).'" Stoicheiometric control (three equivalents of metal) gives The conditions that were used for the reductive alkylation in reasonable results,Is5 but it seems to be most effective to add the above examples (Na in NH3 at -70 "C)l7Iwould enhance small amounts of ferric chloride and to use five equivalents of the retention of the methoxy-group for reasons that were lithium.Is6 Quenching with ammonium chloride gives either mentioned earlier (Section 4.2).the 1,4- or the 1,2-dihydro-products [(280) and (279)] while the 54 NATURAL PRODUCT REPORTS 1986 I -v I ,ll,VI,lV (255) HO' I I I CO,H (258) (259) Reagents i CH,N,; ii HCI THF MeOH; iii NaBH,; iv. KOH EtOH heat; v. Na liq. NHI EtOH; vi HOCH2CH,0H TsOH; vii NaHC'O, KI.3 Scheme 37 vii -IX 1 CO2 H Me0 Reagents i Li liq. NH, Bu'OH; ii (CO,H), MeOH H,O; iii LiAIH(OBu'),; iv Ac,O NaOAc; v (COCI), pyridine; vi CH,N,; vii Cu. C,H,,; viii K,CO, MeOH H,O; ix H2Cr0,; x HCI Me2C0 HzO Scheme 38 II Ill acozH- COZH-COCHN iiv H I (cf Scheme 34) I ,V,VI -mo HOZC' (273) (275) (274) Reagents i Li liq.NH3 Bu'OH; ii (COC1)2 pyridine; iii CHIN2; iv Cu C,,H,,; v. H2Cr0,; vi H2 Pd/C MeOH Scheme 39 ~~ NATURAL PRODUCT REPORTS 1986 J. M. HOOK AND L. N. MANDER OLi (276) (278) (R=H or OMe) R\mcozH (279) (280) Scheme 40 R R Methoxy-substituents in the ring that bears the carboxyl group are prone to hydrogenolysis. A methoxy-group is invariably lost from C-1 C-3 or C-4 when reduction proceeds to the stage of the tetrahydro-compounds but retention at C-3 OMe or C-4 is possible if reduction is limited to the dihydro stage in the presence of anhydrous ferric chloride. The acids (281)- (281) R = H (283)R = H (isolated as keto -acid) (284) have been prepared thus in yields of 65-8001;.'~' (282) R = Me (284)R = Me Controlled hydrogenolysis of a methoxy-substituent has been used to advantage in an expeditious synthesis of the tetra- hydronaphthoic acid (287),' 87 which is an important starting material for the total synthesis of gibberellins.' 88 89 Thus, aoMe regioselective metallation of 1,6-dirnethoxynaphthalene(285) followed by carboxylation and then reduction furnished (287) Me0 in 71% overall yield (Scheme 41).(285) 4.6 Miscellaneous Aromatic Acids Jiit 4.6.I Biphenylcarboxylic Acids Whilst 2-phenylbenzoic acid is reduced smoothly (by Li or Na in liquid NH,; quench with NH,Cl) to the dihydro-derivative (288> the outcome from the reduction of the 4-isomer is very sensitive to reaction conditions. In one study it was found that (287) in addition to the expected products (289) and (290) (the latter Reagents i BuLi TMEDA; ii COz; iii Li liq.NH3 Bu'OH arising from isomerization of the 1,4-dihydro-isomer) a 25% Scheme 41 yield of a mixture of the alcohol (291) and the hydrocarbon (292) was formed.' 90 In a subsequent investigation in which rapid quenching procedures were employed only a 3 :1 mixture of (289) and (293) was obtained.'"' It appears therefore that (291) and (292) arose from reduction of (293) at the quenching phase of the reaction process. A precedent for such an outcome is found in Van Bekkum's earlier findings,I4O and the authors of the second study emphasize the need for care during quenching procedures. 19' They also note that superior NHZ regiocontrol of reduction was obtained by substituting the corresponding t-butyl ester as a substrate for which reduction occurs only in the more substituted ring (see Section 5).(291) R = CHzOH (294) 4.6.2 Furoic Acids (292) R = Me 2-Furoic acids are reduced (as expected) to the 2,5-dihydro- derivatives. Furanomycin (294)' c)2 has been synthesized in this (293) R = COzH way as have other derivatives that are utilized in the synthesis of derivatives of 2-alkylfurans (cf Section 4.7.11 but the reaction with methyl iodide affords excellent yields of the 2-intermediates are prone to cleavage (Scheme 42).' 93 3-Furoic methyl- 1,2-dihydro-derivatives (278) (Scheme 40). acids are equally susceptible to cleavage reactions. 94 The initial reduction of the n-bond between C-1 and C-2 in contrast to the normal 1,4-pathway that is encountered with most aromatic carbonyl compounds is presumably a conse- 4.6.3 Thiophene-2-carboxylic Acids quence of the energetic advantage in preserving aromaticity in With the propensity of carbon-sulphur bonds to undergo the second ring.Similar results have been obtained with 2- reductive cleavage it comes as no surprise that thiophene-2- ace tylnap ht halenes and 3,4-dihydrophenan thren- 1(2H >-ones carboxylic acids are readily reduced to the thiol acids (303) (Section 7). (Scheme 43).'95Reduction of the pre-formed lithium salts NATURAL PRODUCT REPORTS 1986 (295) (296) (297) R = H or alkyl iii,iv -0- $COzH (298) (299) Reagents i Li liq. NH3 at -78 NH,; iv NH,CI "C; ii NH,CI or RX; iii Li liq.[CHzI, COzEt Scheme 42 c02 Et OMe OMe (311)n = 1 (314) (312)n = 2 (313)n = 3 Iii J. FCO'" R' SH R' (303) ( 3041 (315) \i,iii Reagents i LiOH; ii Li liq. NH,; iii NH,CI; iv EtI NaOMe Scheme 43 (317) (316) (305) (306) vi 1 Meor OMe (307) Reagents i Li liq. NH,; ii C,H I Br; iii Pb(OAc), Cu(OAc) Scheme 44 under carefully controlled conditions however affords the 2,5- dihydrothiophene-2-carboxylicacids (302) in good yields (CJ Section 4.4).Iq6 The acids (303) may be utilized for the preparation of a variety of (Z)-olefinic derivatives (304) and dienes. 97 4.7 Synthetic Applications Dihydroaromatic acids have been used as a source of alicyclic compounds and as substrates for an extraordinary variety of syntheses.The procedures that have been used include re- aromatization electrophilic and nucleophilic additions to the olefinic bonds the formation of diene-tricarbonyliron com-plexes ring-expansion and a range of annelations. 4.7.I Re-aromatization The synthesis of olivetol dimethyl ether (307) [by reductive alkylation of 3,5-dimethoxybenzoic acid (305) with 1-bromo-(318) (319) Reagents i Me,NCHO POCI,; ii NaOEt; iii Na liq. NH, Bu'OH; iv (COC1)2;v CH,N,; vi CF,CO?H Scheme 45 pentane followed by oxidative decarboxylation (Scheme 44)] provides an efficient route to 5-alkylresorcinols and is a simple but elegant application of lateral logic to the design of a synthesis. 98 The more obvious alternative of homologation followed by the deletion of functional groups is just as effective with this simple example.However if the side-chain that is to be introduced is functionalized the reductive alkylation/re-aromatization strategy can offer significant advantages e.g. as in the preparation of rosefuran (308) of sesquirosefuran (3O9),lq8 and of the phenethyl alcohol (310).'6s The preparation of a-arylalkanoate ethyl esters (3 11)-(3 14) provides a further illustration of the usefulness of these procedures,' 99 and (3 13) has been utilized in the preparation of the dienone (3181 which is a key intermediate in a recent synthesis of the seco-diterpene effusin (3 19) (Scheme 45).'0° NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER Me0(33 \ -OMe COzH (322) I or II,II~,IV (323) (324) R = H (325) R = OMe Reagents i anodic oxidation (0.5 A) aq.THF N-methylmorpholine; ii Pb(OAc),. pyridine; iii BBr, at -78 "C; iv 2-methylcyclopent-ane-l,3-dione Et,N ; v L-phenylalanine Scheme 46 LiO )I R OMeCOZMe COZMe (329) (328) OMe Me0 OH OMe (330) R = H (331) R = Me Reagents i Pb(OAc), Cu(OAc), pyridine; ii KOH EtOH heat for 36 hours; iii (CF,C0)20 CF,C02H (1 :1) Scheme 47 Other examples are found in the syntheses of the steroids (324) and (325) (Scheme 46),'O' of the anthracenols (330) and (331) (Scheme 47),202 and of the cuparane sesquiterpene herbertene (337) (Scheme 48). The last example is only one of several and introduces an important variation on the alkylation step.The Ireland-Claisen [3,3] sigmatropic rearrangement provides entry to systems that are not accessible by direct alkylation and the overall result is a regiocontrolled arylation of ally1 derivatives at the more substituted terminus.203 (332) (335) (334) iV vi ,vii (336) (337) Reagents i Et,N MsCl; ii 2,3-dimethylprop-2-enol; iii lithium N- cyclohexylisopropylamide; iv Me,SiCI at O *C for 4.5 hours; v Pb(OAc), Cu(OAc),; vi CH21, Zn/Cu couple; vii H? PtOl HOAc NaOAc Scheme 48 COZH R CO2H (R = Pr',CHzCH=CH2,Bun n-C5HI1,or n-C,H,,) Reagents i metal (Li or Na) liq. NH,3;ii RX; iii HCI Scheme 49 4.7.2 Syntheses qf' Cyclohe.wnones One of the most important applications of the Birch reduction has been in the synthesis of cyclohexenones from aromatic ethers.The utilization of 2-methoxybenzoic acid (21 2) for this purpose has furnished an important variation which provides facile access to 2-alkylcyclohex-2-enones (Scheme 49). I h3.'04 In early reports the yields were poor because of partial hydrogeno- lysis of the methoxy-group during the reduction step.'55.172 2- NN-Dimethylaminobenzoic acid has been employed as an alternative substrate in an attempt to solve this problem,1sh but although hydrogenolysis is no longer observed the overall yields are barely improved. The problem has been overcome by carrying out the reductive alkylation on potassium 2-methoxy- benzoate. This method is also successful with 2-methoxy-6- methylbenzoic acid which may be viewed as a synthetic equivalent to Hagemann's ester in the preparation of 2-alkyl-3-methylcyclohex-2-enones.58 58 NATURAL PRODUCT REPORTS 1986 OMe OMe i ii iii -v -@ + HOHZC (340) (339) (341) (342) Reagents i Li liq. NH, EtOH; ii HCI; iii Ac,O pyridine; iv AcCl AlCl, PhNO,; v KI, NaOH Scheme 50 0 OMe (pop". iv-vi A A (228) (3431 (344) vii-ix .1 HO XI. xii.ix +--t 0 PHO (345) !ZBr (346) Reagents 1 Li liq. NH, at -78 "C; ii PhOCH,CH,Br; iii SM-HCl; iv H,C-CHMgBr Me,S.CuBr; v Ph,P--€H2; vi BBr,; vii AmiBH; viii HzOl NaOH; ix pyridinium chlorochromate Celite; x KOBu'; xi MeLi; xii Hg(OAc), NaBH Scheme 51 CO ZH 1-I.' -xiii vii-ix ey eo f-\ t \ I @~'x I I (351) (350) !349! Reagents i Li liq.NH3 at -78 "C; ii 6-iodo-3-methylhexa-1,3-diene; iii (CO,H), HzO; iv NaOMe MeOH; v (H,C-CMe),CuLi; vi PhMe at 170 "C for 68 hours; vii LiNPr', ZnCl, MeCHO; viii TsOH heat; ix Me,CuLi; x BuiAIH; xi S02C1, Me,NH; xii Na liq. NH3 MeOH; xiii H2 PtO? HOAc Scheme 52 Another useful example in this area has been the preparation In more complex examples reductive alkylations of 5-(Scheme 50) of podocarpenone (342) which is isomeric with isopropyl-2-methoxybenzoic acid (228) and 2-methoxybenzoic the product (340) that can be obtained by Birch reduction and acid (212) have been applied to the synthesis of (+)-hydrolysis of the parent aromatic ether (339).'OS The yields oplopanone (40) (Scheme 51)Ifi6 and of (2)-fichtelite (351) were poor as a consequence of the partial loss of the methoxy- (Scheme 52),'06 respectively.group during the reduction but could presumably be increased 5-Methoxy-2-methylbenzoic acid (352) has been converted significantly if the more recently perfected procedures were into the (-)-(S)-diol (354) (Scheme 53),207.208 which is an used.' s8 important intermediate for the synthesis of tachysterol 59 NATURAL PRODUCT REPORTS 1986-J. M. HOOK AND L. N. MANDER R2 L V I (365) (366) (367) Reagents i Br2 NaHCO,> Scheme 54 HO CH20H COC L CONHTs (354) Reagents 1. Li. liq. NH.3 EtOH then NH,CI; ii 2M-HCI; iii resolution of ( -)-quinine salt; iv NaBH,; v NaOMe MeOH; ki hTS LiAIH ‘Br Scheme 53 (368) (369) (370) Reagents i TsNH- Na+; ii Br, NaHCO Scheme 55 C02H co*Bu‘ C02Bu‘ I I (356) OH (355) OH Y-y& 0-‘ R H L\ H (374) (3751 (376) (357) (359)R = Me Reagents i Me,C=CHI H+; ii rn-CIC,H,C03H; iii N-bromosuc-(358)4 -epimer (360)R = CH2OH cinimide; iv ArCO,H; v Et,N ; vi 1,8-diazabicyclo[5.4.O]undec-7-ene; vii heat at 70°C Scheme 56 wop 0-R 4.7.3 Elect rophilic Additions to Dihydro benzoic Acids 0 Electrophiles react with the olefinic bonds in 1,4-dihydroben- H zoic acid and its derivatives with and without participation of the carboxyl group to furnish valuable intermediates for a wide (361)R = CHZOH variety of synthetic objectives.‘I6 220 Both y-and p-lactones (362)R = Me (363) [(366) and (367) respectively] are readily available (Scheme 54),‘l6.’l8 as is the p-lactam (370) (Scheme 55).’” The arene OH H oxides (373)’” and (376)223 have been formed by alternative I routes (Scheme 56).Several of these products have been employed in the synthesis of senepoxide (377) and sene01 (378),15’ of chorismic acid (379),22J of shikimic acid (380),22s and of the precursors (38 1) and (382) to synthons for a projected synthesis of maytansine.226 0 4.7.4 Nucleophilic Additions to Dihydrobenzoates (364) A short and efficient preparation (Scheme 57) of 3,4-disubstituted 3-methoxycarbonylcyclohexanones(385)is based (tacalciol) (355).”)” The carbinols (356; R = H) and (356; R = on the conjugate addition of Grignard reagents to the ester Me) which were readily obtained from 3,4,5-trimethoxyben- (383) which was derived from carefully controlled Birch zoic acid have been a valuable source of the cyclohexenones2I0 reduction of 3-methoxybenzoic acid (206) to form (208) (see that have served as key substrates in the synthesis of (2)-Scheme 35) followed by e~terification.”~ These products have ibogamine (357) and its 4-epimer (358),” (i-)-paniculides considerable potential for the synthesis of diterpenoids and A-D [(359)- (362)],”2.”3 (f)-linderalactone (363),?14 and one such application to the synthesis of gibberellins is outlined ( i-)-eriolanin (364).? in Scheme 58.228.229 60 NATURAL PRODUCT REPORTS 1986 HO OCOPh (377) (378) (389) (390) CO H C02Me C02Me I I HO' Q OH I 6H OH (379 (380) n J.ii PhCHzO C02Me CO2H Ho2c':b H0' HO I I k> (393) (394) (208) & Reagents I CH,N,; ii KNH?; iii Fe(CO),; iv H,SO, MeOH.heat / OMe for 24 hours; v resolution of PhCH(N+H,)Me salt; vi Ph.3C+ PF; MeOC vii Bu'O,CNH, Prl,NEt; viii Me,N-0-; ix NaOH MeOH. HZO; X 3M-HCI " (383) Scheme 59 Yii CH~OTS u -0 (395) (396) C02Me C02Me Reagents i HOAc NaH,PO Scheme 60 MeoWOT~ OMe \ II,llI (397) (399) Reagents i pyridine; ii Br2; iii HBr Scheme 61 4.7.5 Deriiutii-es qf Iron Tricurbotij.1 /V A recent synthesis of (-)-gabaculine (394) (Scheme 59) from + H 1,4-dihydrobenzoic acid provides an excellent illustration of the utility of Fe(CO) complexes in ~ynthesis.'~" Complexation of this type allows both regio- and stereo-chemical control in the location of substituents as well as optical resolution.23' Lz.' EtOC -O H W 4.7.6 Ring E.upuiision The solvolysis of 1,4-dihydrobenzyl tosylate (395) provides a 0 convenient synthesis of cycloheptatriene (396) (Scheme 60) but (388) substituted derivatives lead to mixtures of olefinic isomers.234 Reagents i BuqCuLi; ii LiAIH,; iii pyridinium dichromate Better control is possible in the solvolysis of methoxylated pyridinium trifluoroacetate; iv EtOC'(O)C-:CLi; v PhH heat for derivatives to afford tropone and a-,P- and y-tropolones as 160 hours exemplified by the preparation of P-tropolone (399) in Scheme Scheme 58 61.15') NATURAL PRODUCT REPORTS 1986 -J.M. HOOK AND L. N. MANDER (402) OAc I I (407) (406) (405) (404) Reagents 1 Li liq. NH,; ii Br[CH,],OCH,Ph; iii LiAlH,; iv TsCI pyridine; v LiBHEt, HMPA; vi Li liq.NH3 Bu'OH; vii N-chlorosuccinimide Me& Et,N ; viii Ac,O HClO, EtOAc; ix Ag20; x MeOH H,SO,; xi lithium 2,2,6,6-tetramethylpiperidide; xii CIC0,Et; xiii NaH; xiv MnOz; xv NaOH Ag20;xvi CH2N2;xvii MeLi Scheme 62 (410) (411) iv x xi 1 xiv iii,xii,x ,xiii '0 '0 (414) (413) (412) Reagents i Li liq. NH,; ii MeI; iii LiAIH,; iv N-chlorosuccinimide,MeZ$ Et,N; v NaH (EtO)lP(0)CH,C02Et; vi Li liq. NH3 EtOH; vii PPh3,N-bromosuccinimide;viii EtSCH,S(O)Et,BuLi; xi HClO, H,O; x Pr',NLi; xi ICH,CO,Et; xii Ag,CO, Celite; xiii MeI; xiv,OZ hv haematoporphyrin Scheme 63 4.7.7 Annelutions Total syntheses of the eudesmane sesquiterpenes ( )-occiden-talol (407) and (A)-0-santonin (414) have been based on the reductive alkylation of m-toluic acid.In the first case the carboxyl group is transformed into the angular methyl group of the target molecule (Scheme 62),23s while in the latter example the angular methyl group is introduced by alkylation and the carboxyl group is extended (Scheme 63).23hIt is important to note that because the doubly allylic C-4 is very prone to oxidation it is essential to avoid chromium(v1) reagents in the preparation of the aldehydes (403) and (409). The cis-fused 2-oxadecalin-3-one system that is found in vernolepin has been constructed in seven steps from benzoic acid (Scheme 64). The dihydro-intermediate (41 5) could be obtained directly by trapping (with chloromethyl methyl ether) the dianion that was obtained from reduction of benzoic acid with a metal in liquid ammonia.However better yields were realised from stepwise reduction with sodium and ethanol in liquid ammonia followed by alkylation (using lithium di- isopropylamide and benzyl c hloromet hyl e ther). Subsequent transformations afforded (41 7) and eventually vernolepin (41 9) and vernomenin (420).'" In another synthesis of a 2-oxadecalone derivative (Scheme 65) 3,5-dimethoxybenzoic acid in liquid ammonia was treated with 7-9 gram equivalents of sodium apparently to form the trianion (421) which was alkylated by ethyl bromide furnishing (422). Subsequent transformations led to (423) by means of an intramolecular alkylation,238 The precursor (429) to reserpine in Woodward's synthesis has been prepared from 1,4-dihydrobenzoic acid by an ingenious (but low-yielding) sequence (Scheme 66) in which the pivotal step was the intramolecular [2 + 21 cycloaddition (426) -+ (427).'39 Reductive alkylation of 2,5-dimethoxybenzoic acid with benzylic and phenethyl halides followed by cyclodehydration provides a direct entry to fluoren-2( 1H)-ones (435)' h') and phenanthren-2( 1H)-ones (433),' h7 respectively (Scheme 67).It is possible to retain the angular carboxyl group as in (436) if sufficiently mild conditions are used in the cyclization or through prior esterification. The kinetically favoured products from the cyclization of the benzyl derivatives (432; n = 1) are the bicyclononanones (434); in examples that lack the activating methoxy-group only compounds of this type were isolated.NATURAL PRODUCT REPORTS 1986 /OMe . .. I I1 iii ___) Ho2ct5-04‘ C02Me (415 1 iv- vi 0 iH OH C02Me 0 (419) (420) (418) Reagents 1 LiAIH,; 11 MeO,CCHICOCl pyridine TsN 3 Et,N; iii Cu(acac), PhMe heat; iv PhSNa DMSO; v m-CIC,H,C03H; vi (MeO),P MeOH heat Scheme 64 -o*oMe 0-I H02c+&e + 0Me OMe Jii -vi / EtOme 0 (423) Reagents i EtBr; ii LiAlH,; iii H,SO, HIO THF; iv CHIN,; v EtOCH=CHBr BF,.Et,O PhH; vi LiNPri Scheme 65 Several tricyclic intermediates that were obtained in this way have been used to prepare a diverse selection of natural products including orchinol(439) (Scheme 68),16s,z4z gibberel- lic acid (447) (Scheme 69),241 and the structure (450) that had been proposed for juncunol (Scheme 70).242 When it was found that (450) was incorrect for juncunol the correct structure (454) was synthesized from (451) as outlined in Scheme 71.24z In order to obtain a good yield of (451) however it was necessary to reduce methyl 2-methoxy-5-methylbenzoate to the 1,4-dihydro-derivative (cf:Section 5) and then alkylate the derived lithium enolate in a subsequent step. The acid (455) has been converted into (456) by reduction followed by cyclization in hot acid. A series of further transformations afforded (k)-p-vetivone (462)’j3 and a num- ber of other spirovetivanes (Scheme 72).244 A new route to bicyclo[3.3. Ilnonanes has been based on the palladium(r1)-mediated 6-endo-cyclization of substrates which are readily prepared from the reductive alkylation of aromatic acids.A selection of examples is indicated in Scheme 73.24s Alternatively this kind of substrate may be utilized for free- radical-initiated cyclizations but in these cases the preferred iiliii -Q.oMe C02H 0 (424) (425) iv.v OMe OMe (427) (426) JIV .VII AcO n Meo2i fHO Me02C OCOAr I OMe OMe (428) (429) Reagents i H,O, HC02H heat; ii heat at 183 “C for 1.5 hours; iii MeI Ag20; iv MeOH H,SO,; v 4-bromopent-3-en-4-olide Ag,O; vi hv (Pyrex-filtered) acetone; vii CF,CO,H Na,HPO,; viii 3,4,5-trimethoxybenzoyl chloride pyridine 4-dimethylamino-pyridine Scheme 66 products arise from a 5-em-process. A typical case is the preparation of the indane derivative (472) (Scheme 74) but more highly functionalized products e.g.(473)-(475) are also accessible2s6 (see Section 5.2.2 also). In a further study of free-radical carbocyclization the iodides (477) and (480) which had been prepared from the reductive alkylation of 3-methoxybenzoic acid and 3,4,5-trimethoxybenzoic acid respectively furnished the bicyclic NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER R Me0 Me0Q+ LiopOMe (230) [CH21n X (430)n = 1 or 2 ;X = Br or I OLi (431) Me0R@oMe [CH21 (436)n = 1 or 2 CO;! H (434) (435) Reagents i H,SO,; ii polyphosphoric acid; iii BF,.Et,O Scheme 67 / M&o + M&oH Me0 \ Me0 \ Br (437 (438) (439) doMe iv Me0 + Md0 Me0 C02H C02H Me0 Me0 (440) (441) Reagents i.pyridinium bromide perbromide HOAc; ii BuLi; iii H,O+; iv 75;; HISO, at 25 *C for 10 minutes; v CuBr? aq. MeOH Scheme 68 vii viii -~OCOCHICI Me0 Me0 \ Me02C 0 Me02C COCHN Me02C (442) (4431 (4 44) 'O ix ,x H I (447) (446) (445) Reagents 1 HCI NnC"; ii. HCI MeOH; iii KOH; iv (CICHICO)IO; v (COCl), DMF; vi CH,N,; vii CF,CO,H; viii K,C'O,; ix (CH,OH), TsOH x MeOCH2Cl PrINEt; xi BuLi; xii CO,; xiii H? Pd/C EtOAc Scheme 69 64 NATURAL PRODUCT REPORTS 1986 (448) (449) (450) Reagents i KCN HCI; ii POCI, pyridine; iii CuBr, MeOH; iv BuiAIH; v Ph,P==CH,; vi LiSMe HMPA Scheme 70 (4511 (452) (453) (454) Reagents i BF,.Et,O CH,CII; ii POC13 DMF; iii Ph,P=CH,; iv NaOH; v Pb(OAc), Cu(OAc),; vi LiSMe HMPA Scheme 71 Me0 C02H (4551 (456) (457) vii,iv,viri ,ix 1 (460) vi,xvii I 0 ‘CH2Br (461) (462) Reagents i Li liq.NH,3 Bu‘OH; ii (CO,H)? H,O; iii 6M-HCI; iv (CH,OH)? TsOH; v LiAIH,; vi 2M-HCI; vii Ac,O pyridine; viii KOH MeOH; ix CrO, pyridine; x NaH MeOC0,Me; xi NaBH,; xii MsCl pyridine; xiii NaOMe MeOH; xiv H2 PtO, MeOH; xv MeLi; xvi POCI, pyridine; xvii Ph.{PBr7; xviii Nal EtCOMe; ix Zn AcOH Scheme 72 ketones (478) and (481) (Scheme 75) which are promising 5 Reduction of Aromatic Carboxylic Esters intermediates for the synthesis of gibber ell in^'^' (c# Section 7.3). 5.1 Discussion Reduction of the acid (482) by lithium in liquid ammonia Early studies on the reduction of aromatic esters by metal- furnished the spiro-fused bicyclic acid (483) as the major ammonia systems afforded little if any ring-reduced com-product (Scheme 76).Equally good results were obtained with pounds but rather the products of reduction of the functional group.250.2 5 I calcium metal but lower yields were obtained with sodium and potassium.248 The isomeric acid (484) failed to give any A completely general procedure appears to be the reduction bicyclic products however.2J” of esters by sodium2s2 or potassium,’s8 at low temperatures in NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER C02Me C02Me i-iit) iv _I) OMe OMe (463) (4641 (465) C02Me C02Me i-iti iv OMe OMe C02Me “‘“Q (2 30) (468) (469) Reagents i Li liq.NH,; ii H,C-CHCH,Br; iii MeI K,CO,; iv Pd(OCOCF,)? CuCI? MeCN CHICI, 02 Scheme 73 cr 0 0 COzH 0 ‘1 (470) I Yi (4791 (480) 0 (4721 Reagents I K I :. NaHCO,; ii Bu’jSnH azobisisobutyronitrile Scheme 74 Me02C’ QoAc (481) Redgents I IO” HC‘I THF. 11 C H,N,. 111. Bu$nH mhi\isobutyr-onitrile. iv ALCI Scheme 75 \’ 0 U (473)R’= H R2= CO2But (482) (483) (474) R’= Me R2 = CO But Reagents i Li liq. NH3 THF (475) R1= OMe R2 = C02Et Scheme 76 66 NATURAL PRODUCT REPORTS 1986 (484) Table 2 Reduction of benzoate esters by metals in liquid ammoniaa Yield of OMe Me0 Substrate Metal Proton source I ,4-dihydrobenzoate -<50,; Bu'OH >95:o __ 30-509; (498)R = 3 -Me (501)R =3-Me (499) R = 4 -Me (502)R = 4 -Me Bu'OH >95% (500)R = 5 -Me (503) R = 5 -Me -20-300,; Bu'OH >95% (4 86 1 1Li Bu'OH >957; CO Me (" ._ 60-700/ (504) (505) (506) R C0,Me 0""' K Bu'OH >95% \/ (487) MeoooMe (507) R = Me 0Me (508) R =Pr' (509) R = CH2CHZOThp (512) (513) (510) R = CH2CHzCH(OSiMe,)Me (u)Reductions were conducted at 78 "C,with 2.5 equivalents of metal and I.5 equivalents of Bu'OH (if added).(511)R = Table 3 Reductive alkylation of methoxybenzoic esters Substrate Metal Cation exchange Alkyl halide Product Yield Reference (487) K -Me1 (489) 95:1 158 (487) K -Etl (490) 96:" 158 ~ (487) K Pr'I (491) 92?" 259 (487) K LII CI[CH?] ,Br (492) 67:; 259 (487) K -PhCH Br (493) 617; 259 (494) K or Na Me1 (495) 967" 158 (494) Li -Me1 (495) 737" 158 ~ (494) K or Na Pr'I (496) 94"; 158 (494) K -H ,C=CHCH ,CH ,Br (497) 00/ 158 (494) K LiBr H2C CHCH,CH,Br (497) 607 158 (498) K -Me1 (501) 499 259 (499) K Me1 (502) 80" 259 (500) K -Me1 (503) 74"; 259 (505) K Me1 (506) 96"; 158 (488) K -Me1 (507) 967; 158 (488) K Pf'I (508) 77" " 257 (488) K LiBr ThpOCH ,C H ,1" (509) 72"" 257 (488) K LiBr MeCH(OSiMe,)CH2CHII (510) 97"" 257 (488) K -~ ArCH,Br (511) 67"" 259 (512) K __ Me1 (513) 709 158 (a)Thp = tetrahydropyrdn-2-yl NATURAL PRODUCT REPORTS 1986-J.M. HOOK AND L. N. MANDER the presence of one equivalent of t-butyl alcohol. Even lithium has been shown to give excellent results and is often the preferred metal if the reduction is followed by alkylation (see below);170 lithium fails to reduce the aromatic ring of 2,6- disubstituted substrates however.The role of the proton source has been examined in one systematic study,2s3 the results of which are summarized in Table 2. It appears that methoxyl substituents promote reduction; in the case of methyl 2,6- dimethoxybenzoate such substituents obviate the need for an added proton source. Rabideau has advocated the use of water as the proton source added before the metal (sodium) followed by inverse quenching by aqueous ammonium chloride. This procedure is unsatisfactory for methyl esters but gives good results with ethyl esters and works best with t-butyl esters. However p-ethyl- p-isopropyl- and p-t-butyl-substituted t-butyl benzoates do not afford significant amounts of ring-reduced products.251 Quenching these reductions with ammonium chloride affords the 1,4-dihydrobenzoates in yields of 90-100%.2s7 On the other hand the reaction with alkyl bromides or iodides in situ generally affords the 1 -alkylated product in 70-100% yield (Table 3).These procedures have been successfully applied to more complex substrates,241 for example the gibbanes (446) and (515) (Scheme 77) (cf Scheme 69). In these cases the alkylations were completely stereoselective but in an attempt to achieve an enantioselective synthesis through reductive methylation of the (*)-menthy1 ester that had been derived from 2-methoxybenzoic acid (2 12) no diastereoselectivity was observed'5' (cf Section 6).Alkylation of the potassium enolates is not always fruitful and so the exchange of the counter-ion with lithium bromide prior to this step has been recommended.Is8 Reduction of aromatic esters (instead of acids) provides a number of potential advantages. The esters tend to be more soluble than carboxylate salts and the products are more stable. This is especially true of methoxydihydrobenzoic acids which may be prone to oxidative decarboxylation or to the hydrolysis of enol ether functions during acidic work-up procedures. Moreover hydrogenolysis of 2-alkoxy-substituents does not present the same problem as with acids (cJ Section 4.4.2). H H (515) It is of considerable interest to find that t-butyl esters may be reduced satisfactorily in contrast to aliphatic t-butyl esters which are cleaved to acids under Birch conditions.2sh Also noteworthy is the regioselective reduction of the ester (517) (Scheme 78),l9I in marked contrast to the reduction of the corresponding acid (see Section 4.6).It has also been reported that reductive methylation of naphthoate esters257*2s8 has been observed to proceed most satisfactorily without a proton source using sodium as the reductant (cJ Section 4.5). The esters (521) (522) (524) and (526) have been prepared in this way (see Scheme 79) and are envisaged as useful intermediates for the synthesis of diterpenes. The esters (521) and (522) for example were converted into the diazo-ketones (527) and (530) which underwent acid-catalysed cyclization to the tricyclic ketones (528) and (531) respectively (Scheme 80).The former compound had already been prepared by Cossey et d.2s9 by the same diazoketone-based route and had been converted into the acid (529) which is a more potent regulator of plant growth than the nor-homologue (275) (c$ Scheme 39). 5.2 Synthetic Applications Because satisfactory procedures for the nuclear reduction of aromatic esters have only recently been established there have not been many applications. The potential would appear to be even greater than that of the corresponding carboxylic acids however. 5.2.1 Cyclohexenones The reductive alkylation of t-butyl 2-methoxybenzoate (494) has been used to produce the 2-alkyl-cyclohex-2-enones (532)- (534) (Scheme 81).The conversion of the intermediate dihydro- esters into the enones is achieved in one step with hot trifluoroacetic acid,' and compares favourably with proce- dures that are described elsewhere (see Sections 3.2.2 4.7.2 and 7.1.1). H H (516) Reagents i KOBu'; ii K liq. NH,; iii Me1 Scheme 77 (517) (518) Scheme 78 NATURAL PRODUCT REPORTS 1986 (519)R = 6 -0Me (520)R = 7 -OMe (521)R=6-OMe (522) R = 7 -OMe OMe C02Me . . C02Me (525) (526) Reagents i Na liq. NH,; ii Me1 Scheme 79 0 OMe 0 (530) (531) Reagents i CF,C'O,H Scheme 80 (494) (495)R = Me (532) R = Me (496)R = Pr' (533) R = Pr' (497)R = CH2CHzCH=CH2 (534)R = CHZCH~CH-CH~ Reagent i C'F,C'O?H Scheme 81 5.2.2 Atinchriotis The application of bromination-dehydrobromination se-quences to the dihydroaromatic esters (489) (490) and (493) (Scheme 82) has furnished the cyclohexadienones (537; R = Me) (537; R = Et) and (537; R = CH,Ph).These compounds readily undergo [4 + 21 cycloadditions with iriter uliu,dimethyl acetylenedicarboxylate and gave the adduct (538) hydrogena-tion and photolysis of which led to (539)"' by means of an oxa- di-n-methane rearrangement (cf Scheme 28).I 38 The availability of this kind of cyclohexadienone has also been exploited in a new synthesis of (+)-longifolene (547) (Scheme 83). Thus the diazoalkane precursor (543) was prepared and thermolysed at 110 C to give the azo-derivative (544) photolysis of which furnished the vinylcyclopropane- based structure (545).Thermolysis of either (544) or (545) at 135 "C gave the desired tricyclic keto-ester (546) and thence the target compound (547).'h0 In a development of the approach to the secodaphniphylline alkaloid (53) that was described earlier (Scheme 12),5x the isopropyl-substituted dienophiles (551 ) have been prepared by reduction of the esters (549) (Scheme 84).'h1 The analogues (558) and (564) of the Wieland- Miescher ketone which each bear a functionalized angular substituent have been prepared by annelation of methoxydihydroaromatic NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER R R R OMe OMe OMe (489)R = Me Br (536) (490) R = Et (493)R = CH,Ph (535) (539) liv vii ,viii t vi Me02C *R __3 C02Me MeO,C Br 0 Reagents i N-bromoacetamide MeOH ; ii KOBu' Bu'OH; iii I ,5-diazabicyclo[4.3.O]non-5-ene; iv 3M-HCI MeOH ; v N-bromosuccinimide PhH heat hv; vi Me02CC-CC0,Me PhH heat for 20 hours; vii H2 Pt; viii hv (Pyrex-filtered) PhCOMe Scheme 82 Me0 C02Me C02Me i -iv V 4 "fPh 'CH (OMe) -Ph + Q (542) (543) (544) / Ivi C02Me viii,ix,v X,XI vii 4 Q GOZMe f--Reagents i N-bromoacetamide MeOH ; ii I ,5-diazabicyclo[4.3.O]non-5-ene; v PhMe heat; iii SiOz iv l-amino-rrans-2,3-diphenylaziridine; vi hv (1= 366 nm) PhH; vii xylene heat; viii H2 Pd/C EtOH; ix KOH MeOH; x LiNPr', Mel; xi Ph,P=CHI Scheme 83 (5481 (549) (550) I OSiMe2But (551)R=H or Me (552) Reagents Bu"Li TMEDA; ii CO,; iii Mel K,CO,; iv K liq.NH, Bu'OH; v IM-HC1 THF heat; vi (491,at 140°C Scheme 84 NATURAL PRODUCT REPORTS 1986 Me3Si0 i -iii + 1v.v (553) (554) (555) ivl ... Vlll -MeoP (558)R = H or Me HO (5561 Reagents i K liq. NH, Bu'OH; ii LiBr; iii RCH,CH(OSiMe,)CH,CH21; iv HOAc; v dicyclohexylcarbodi-imide,DMSO; vi Bu;NF; vii K2C0, MeOH; viii Hg(N0J2 MeCN H20 Scheme 85 1,then ii or iii iv,then -*R' Me0 \ VI or VI or VII (5591 (560)R'= H or Et (561)R' = H or Et R2= Me,Ac or MEM xi or xii or xiii IX or x R1% I 0& 0;Ix R3 R3 (564)R2= Me Ac or MEM (563)R2= Me Ac or MEM (562)R' = H or Et R3= H or Me R3= H or Me R2= Me,Ac,or MEM (MEM = MeOCH2CH20CH2) Reagents i LiNPri THF; ii H?C=CHCH,CH,Br; iii H,C=C(Et)CH,CH,I; iv LiAlH,; v NaH MeI; vi EtAc; vii MeOCH2-CH20CH2Cl Pr5NEt; viii TsOH aq.acetone; ix [R' = H] PdCl? O, CuCl DMF H,O; x 0,,MeOH Me,S; xi PhCO,H.piperidine; xii L-proline DMSO; xiii D-phenylalanjne HClO, MeCN Scheme 86 esters by two routes (Schemes 85 and 86). Both preparations this latter study apparently depends on the greater nucleophili- have relied on the use of synthetic equivalents to 3-oxobutyl city of the dihydroaromatic species which allows alkylation to and 3-oxopentyl side-chains. Of interest in the first approach is predominate over elimination. the oxidation of the hydroxyl group in the side-chain in the Reductive alkylation of aromatic esters followed by radical- presence of the cyclohexa-1,4-diene moiety and the use of the induced cyclization (initiated by tri-n-butylstannane; cf silyl ethers to permit discrimination between three masked Section 4.7.6) has been used in the preparation of a variety of ketones.262 The second approach allows enantioselective bicyclic and tricyclic molecules (Scheme 88).The formation of synthesis of the bicyclic ketones (564) from the trione a five-membered ring proceeds in good to excellent yields but precursors (563) with 70-90% enantiomeric the rates of formation of a six-membered ring are ca lo4 times Following these syntheses it has been discovered that it is slower and the transfer of hydrogen may become the dominant possible to utilize the iodides (566) as operational equivalents process. 3-Alkyl and 3-methoxyl substituents lead to enhanced for vinyl ketones and acrylic esters respectively (Scheme rates of cyclization to C-2 while alkyl groups or methoxy-87)' 70 Earlier attempts to employ similar allylic halides in groups at C-2 are inhibiting.Benzyl derivatives tend to be very alkylation reactions with enolate anions that had been derived unstable and are rapidly degraded by adventitious oxygen (c;$ from simple ketones were not encouraging. 26s The success of Section 4.1.3).26h NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER 0siMe3 Me3SiO + (565) (566) (567) R = Me Et or OMe (568) Reagents i BuYNF Scheme 87 C02Me &R2 R’ (56910; R’= H,Me,or OMe; R2= CH2CH2CH2Br A m I H (570) R’ = H,Me or OMe :O2Me I b; R’= HI R2= CH2CH=CHBr _____) CD h Reagents i Bu’jSnH Scheme 88 with esters also gave excellent results with the Z-methoxybenz- amides (573) and (574) although the successful outcome was CONEt 2 tentatively attributed to the presence of the orrho-methoxyl / R’ group.While both the amide (573) and the amide (574) gave high yields of the C-methylated derivatives (575) and (576) respectively only the less hindered compound (573) could be (573) R’= H (575) R’ = H ,R2= Me alkylated with 2-bromoethyl acetate [to give (577)]. The amide (574) R’= Me (576) R’= R2 = Me (574) was prepared by lithiation of (573) at C-6 followed by C-(577) R’ = H ,R2= CH~CH~OAC methylation and opens up the exciting prospect of combining two powerful methodologies. In- what is likely to be a most important development reductive alkylations of the benzoxazepinone (578)’60 and the 6 Reduction of Aromatic Amides benzodiazepinedione (581),I7O each of which had been derived The reduction of the ring of aromatic amides and in particular from L-proline proceed with complete diastereoselectivity benzamides was once thought not to be possible,z67 and in fact (Schemes 89 and 90 respectively).In the first example the the reduction of secondary amides by sodium in liquid methodology was applied to the preparation of the enantiomer- ammonia had been recommended as a route to benzalde- ically pure ester (580) from which (-)-longifolene (547) was hydes.”j8 It was found subsequently that under the appropri- synthesized. It was found in the second study by converting the ate conditions 1,4-dihydrobenzamides could be prepared by products into the 0x0-lactones (585) that the complementary using sodium in ammonia in the presence of an alcohol.Both chirality had been obtained in the alkylation step. primary and secondary amides could be reduced although increasing the steric bulk of the alkyl group dramatically decreased the yields of 1,4-dihydro-compound while tertiary benzamides either underwent reduction of the carbonyl group 7 Reduction of Aromatic Ketones or gave a very low yield of the 1,4-dihydro-compound.’s3~2697.1 Discussion In a very recent studyz5’ it has been found that the As with esters the prevailing view for many years was that conditions (potassium metal liquid ammonia at -78 “C,with reduction of the ring of aromatic ketones was not feasible.The one equivalent of t-butyl alcohol)’s8 that had worked so well first indication that this was incorrect was provided by the 72 NATURAL PRODUCT REPORTS 1986 (578) (579) (580) (-1 -(547) Reagents i K liq. NH, Bu'OH; ii RBr; iii MeOH HCI; iv MeOCOCl NaHCO,; v (MeO),CH MeOH HCI; vi NaOMe MeOH Scheme 89 (R = H,Me,Et H,C=CHCH ,or PhCH,) -NH2 -0 (584) (585) Reagents i K liq. NH, Bu'OH; ii RX; iii 50% H2S0, at 100°C for 6 hours; iv see ref. 271 Scheme 90 (586) (5871 Reagents i M (M = Li Na or K) liq. NH,; ii NH,Cl Scheme 91 0 I (588) (589) ,. ... II,III I (591) (592) (590 1 Reagents i metal liq. NH3 Bu'OH at -78 "C; ii LiBr; iii RX Scheme 92 reduction of the hydrophenanthrenone (586) to (587) (Scheme 91).272Further isolated examples of ring-reduction in naphthyl ketones and in molecules that contain such moieties have been reported subsequently (see below) but it is clear that these systems are much more easily reduced than simple benzenoid ones.Optimal procedures for the reduction and reductive alkylation of such systems were first established by Watanabe and Narisada for acetophenone and its methoxy-derivatives. ' 0 -7 0 (593) R' = 2 -OMe (598) R'= HI R2= Et (594) R' = 3 -OMe (599) R' = HI R2 = H2C-CHCH2 (595) R' = 4 -OMe (600) R'= H R2= CHzCOZEt (596) R' = 4 -Me (601) R'= H R2= CHZCN (597) R' = 2,s -(OMe) (602) R' = Z-OMe R2 = Me (603) R'= 2-OMe R2= Me (604) R'= 4 -Me R2= Me (605) R'= 2,5 -(OMe) ,R2= PhCH 7.2 Acetophenones A systematic study of the reductive alkylation of acetophen-onesi6revealed that the desired transformation (588) -+ (590) (Scheme 92) required a careful choice of reagents and conditions.The best results were obtained by reducing with potassium in ammonia at -78 "C with t-butyl alcohol as the proton source. Exchange of the potassium counter-ion of the enolate (589; M = K) for lithium then ensured regioselective alkylation at C-1 to give (590)in 80-90% yields. If metals other than potassium were used as the reductant undesirable side-reactions occurred [e.g.reduction of the carbonyl group to form the alcohol (591) and ethylbenzene if lithium or sodium was used] while the absence of a proton source or the presence of a strong one (e.g.water or acetic acid) during the reduction encouraged the formation of the pinacol (592). Over-alkylated and 0-alkylated products were obtained if sodium was the counter-ion in the enolate (589; M = Na). Acetophenone and several derivatives (593)-(597) have been subjected to reductive alkylation using the appropriate conditions. As might be expected from results with benzoic acids the methoxy-group is lost from 4-methoxyacetophenone NATURAL PRODUCT REPORTS 1986 -J. M.HOOK AND L. N.MANDER 0 OMe COfH C02 H (606) (607) lii lii (608) (338) Reagents i NaBH,; ii H,O+; iii KOBu‘; iv KOH Scheme 93 iii (609) R1= H I -(610) R’ = 5- OMe (611) R’ = 6,7 -(OMe);! (612) R’ = 5,6,7 -(OMeI3 @J-R’ (613) R’= H R2 = Me (614) R’= 5-OMe R2= Me (615) R1=7-OMe R2= Me (61 6) R’= 5,7 -(OMe)2 R2= Me (617) R’= 5-OMe R2= CH,C(Br)=CH Reagents i K liq.NH., Bu‘OH; ii LiBr; iii R?X Scheme 94 but the 2- and _?-derivatives (593) and (594) gave good yields of alkylated cyclohexa-2,5-dienes as did the 2,Sdimethoxy-compound (S97).Ib5 A range of products from various electrophiles was obtained [(598)-(605)] in good to excellent yield except with chloroacetonitrile. Proton transfer is expect- ed to be a problem with this reagent however. The reductive alkylation of 2-methoxyacetophenone (593) forms the basis of another route to 2-alkyl-cyclohex-2-enones and compares fdvourably with that from 2-methoxybenzoic acid (see Section 4.7.2).”’ The one important change in the general procedure that was described above involves adding the alkylating agent in aqueous THF at -78 *C in order to buffer the system further and to suppress bis-alkylation.The conversion of the 1-acetyl-1-alkyl-cyclohexa-2,S-dienes(606) into cyclohex-2-enones (338) was achieved by two distinct reaction sequences (Scheme 93) both of which were executed in high overall yields. The first involved reduction (by sodium borohydride) of the carbonyl of the acetyl group hydrolysis of the enol ether and then a base-catalysed retro-aldol reaction of (607). The second (more direct) route used acid hydrolysis of the en01 ether to give (608) followed by base-catalysed deacetylation and concomitant conjugation affording (338). 7.3 Tetralones It has been reported that 1-tetralones are reduced by sodium or lithium in liquid ammonia at -33 “C to the corresponding (619) 48 [19°/o] (620)4a [64”/0] iv-vi .1 TsO COzMe (622) \ CO 2 Me (6231 Reagents i K liq.NH, KOBu‘; ii LiBr; iii MeI aq. THF; iv CH,N,; v HOCH,CH,OH TsOH; vi H2 Pd/C EtOH; vii NaBH,; viii TsCI pyridine; x LiNPr? Scheme 95 tetralols or tetralin~,”~ but the conditions for reductive alkylation that have been applied so successfully to the nuclear reduction of acetophenone also succeed with 1 -tetralones. 1-Tetralone itself was transformed into (613) in 60% yield,I6 while modifications to the original procedure allowed the methoxylated derivatives (614)-(617) to be prepared in excellent yields (Scheme 94).27s The methoxyl group at C-6 in (61 1) and (612) is inevitably hydrogenolysed during reduction but it allows the tetralones to be formed more efficiently by cyclization of precursor phenylbutyric acids (cf Section 9.1.3).The ketone (61 7) has been converted into a tricyclic intermediate for the synthesis of gibberellins by an application of the radical-initiated cycliza- tion procedure that has been described earlier (Scheme 75).’” In a further application the keto-acid (618) has been transformed into the keto-acid (619) which is an intermediate in the synthesis of the germacrene precursor (623) (Scheme 95). It was found to be essential to neutralize the ammonium ions that had been generated from the addition of the substrate acid (618) to the liquid ammonia before the potassium metal was added.7.4 Indanones I-Indanones also undergo reductive alkylation although not as well as 1-tetralones. Good yields were obtained with methyl iodide and I-indanone or its 4- 6- or 7-methoxy-derivatives to give the dihydroindanones (624)-(627).”:” 278 These products have been used as precursors to the interesting 3a-methyl-3aH- indenes (628),276 which undergo both [4+ 21 and [8 + 21 cycl~additions~~~.~~’ and hence provide access to the new [lolannulene (629).277,280 74 NATURAL PRODUCT REPORTS 1986 0 R .ae (629) voM (630) M = Li Na ,or K (631) R = H or Me \ (632) Reagents i HA; ii Me1 Scheme 96 OYR Me0mj \ OMe OMe (6331 (634) Iii $.OYR (635) Reagents i RCOCl AlCl,; ii Na liq. NH Scheme 97 7.5 Acetylnaphthalenes The reduction and reductive alkylation of acetylnaphthalenes follows a similar course to that which has been found for naphthoic acids in that the ring that bears the acetyl group may be preferentially reduced. Simple reduction products are sufficiently stable to be isolated and regiospecific alkylation of the enolates that are generated does not appear to be as dependent on the cation as it is with acetophenones 1-tetralones. and 1-indanones. 7.5.I I-Acetylnaphthalenes 1-Acetylnaphthalene may be reduced by lithium sodium or potassium in liquid ammonia at -33 "C to the dienolate (630); this is alkylated regiospecifically at C-1by methyl iodide [to give (631 ;R = Me)] or it may be protonated at C-1 [to give (631 ;R = H)] if the reaction mixture is subjected to inverse quenching with a large excess of aqueous ammonium chloride.2s4 Traditional quenching procedures afford the 3,4- dihydro-isomer (632) presumably as a result of isomerization of (631 ;R = H) (Scheme 96).7.5.2 I-Alkanoyl-6-metho.xynaphthalenes The 1 -methoxy-group of 1,7-dimethoxynaphthalene (633) directs electrophilic attack to the para position; solutions of sodium in liquid ammonia then readily hydrogenolyse this group (cf Section 9.1.3) thereby giving ready access to 1-alkanoyl-6-methoxynaphthalenes (635) (Scheme 97) (c$ Section 9.1.3). 7.5.3 2-Acetylnaphthalenes Early studies on the reduction and reductive alkylation of 2- acetylnaphthalenes afforded mixtures of dihydro- and tetrahy- dro-derivatives.282In one case the alcohol (639) was the major product.286 A comprehensive examination of the reductive process and of quenching methods has been carried out recently however and the conditions that are required if the yields of either dihydro- or tetrahydro-products are to be optimized have been defined.283 These are summarized in Scheme 98.The mixture of (638; R = OMe) and (639; R = OMe) has been used for the synthesis of the aromatic c-ring steroid (645) (Scheme 99).287 The formation of (645) is unexpected and is not consistent with a similar reduction of the corresponding 17- alcohol [(724) + (725) as shown in Scheme 1091. 7.6 Dihydrophenanthrenones The dihydrophenanthren-4( 1H)-one (646) is readily reduced by lithium sodium or potassium in liquid ammonia at -33 "C presumably to the dienolate (647) (Scheme loo) since the reaction of this with methyl iodide affords (648).Quenching with ammonium chloride however leads to the conjugated enone (650) (Scheme 100) [c$ (631) and (632)].288 The isomeric dihydrophenanthren-l(4H)-one (651) is re-duced equally well with lithium sodium or potassium in refluxing liquid ammonia (Scheme 101). Optimal yields of the tetrahydro-ketone (653) were obtained with four molar equiv- alents of metal and catalytic amounts of ferric chloride.2ss The presence of the iron salts in this example did not limit the reduction to the dihydro stage contrary to the findings with 2- acetylnaphthalenes (Section 7.5.3) but gave slightly higher yields than reductions in their absence.The reduction of (651) with sodium or potassium in the presence of ethanol afforded a mixture of the ketone (653) and the corresponding alcohol. It has been suggested that the exclusive formation of the thermodynamically less stable cis-ring-fused compound (653) is a consequence of protonation of the enolate (652) on the sterically less hindered @-face. Reductive alkylation of the phenanthrenone (65 1) affords a variety of products depending on the metal that is used. Potassium and sodium are effective reductants but encourage bis-alkylation yielding mostly (655) with a cis fusion of rings. Alkylation of the lithium enolate (652; M = Li) is regiospecific but less stereoselec t i ve.Ring B in the tetracyclic compounds (656; R = H) and (656; R = OMe) has been reduced to the a@-unsaturated ketone i.e. (657; R = H) and (657; R = OMe) with sodium and ethanol in liquid ammonia (Scheme 102).'89 The tricyclic ketones (658) and (659) have been converted into the a@-unsaturated ketones (660) and (661) respectively (Scheme 103) by reduction with sodium and ethanol in liquid ammonia and these are regarded as potential substrates for the synthesis of diterpenes. Reductive methylation of the ketone (660) for example afforded (662).2s77.290 NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER (638) ... .. . .. III,II I,II + [l:11 R\ -&-OH (639) 0 (R = H or Me) (641) (640) Reagents i K liq.NH,; ii NH,CI; iii Li or Na liq. NH,; iv Li liq. NH,; v Mel; vi Li liq. NH, FeCI Scheme 98 I (638; R = OMe) + (639;R= OMe) + (642) (643) I..Ill -v IV -0 (645) (644) Reagents i 2,3-dichloro-5,6-dicyanobenzoquinone; ii furfuraldehyde NaOMe; iii HCI EtOH; iv KOH; v Ac,O at 165 "C; vi Li liq. NH,; vii MeI K,C0,3;viii H,O+ Scheme 99 Me0& Me0& \ \ (648) (650) Reagents i M (M = Li Na or K) liq. NH,; ii MeI; iii NH,CI Scheme 100 76 NATURAL PRODUCT REPORTS 1986 (651) (654) Reagents i M (M = Li Na or K) liq. NH,; ii NH,Cl; iii Me1 Scheme 101 n (6561 (657) Reagents i Na liq. NH, EtOH; ii NH,Cl Scheme 102 -[CH21n (658)n=2,R = H (660)n= 2 (659)n= 1 ,R = OMe (661)n = 1 ii,iiiJ[n= 21 (662) Reagents i Na liq.NH, EtOH; ii Li liq. NH,; iii Me1 Scheme 103 8 Reduction of Aryl- and Benzyl-silanes 8.1 Arylsilanes Birch reduction of trialkyl(ary1)silanes by lithium in liquid ammonia-ethanol at -70 "C has been studied systematically and a summary of results is provided in Scheme 104.'q1 The major products are usually the 1,4-dihydro-derivatives,which is in accord with e.s.r. evidence that shows that a trimethylsilyl group stabilizes aromatic radical anions.'"' A significant exception is p-trimethylsilyltoluene (669) which affords the 2,5-dihydro-product in 95% yield and is probably typical of aryl-silanes that bear a para-substituent which is electron- releasing. Cleavage of allyl(ary1)silanes by ethoxide ion is a significant problem especially at higher temperatures (>-30 "C) under which conditions further reduction to tetrahydro-and perhydro-derivatives also occurs.Electro-chemical reduction (in methylamine-lithium chloride) affords better yields of the simple dihydr~aryl-silanes.'~~ The 'activating' influence of the trimethylsilyl function is also apparent in the reduction of derivatives of naphthalene (Scheme 105). The 1,4-disilyl-derivative (68 1) is reduced exclusively in the substituted ring to give (682) while 2-trimethylsilylnaphthalene (683) affords a 4 :1 mixture of (684) and (685) respectively. 292 As would be expected the reduction of C-silylbenzoic acids and their esters is controlled by the carboxyl function.The results of an unpublished study on the reductive methylation of such compounds are indicated in Scheme 106.294 8.2 Benzylsilanes The Birch reduction of benzylsilanes generally proceeds in high yield and affords the opportunity to prepare alkylidenecyclo- hexene structures by protodesilylation of the dihydroaromatic products (Scheme 107).'"' Reduction of the parent benzyltri- methylsilane proceeds satisfactorily with lithium in liquid ammonia-ethanol but fails with some substrates. The substitu- tion of t-butyl alcohol for ethanol however avoids the generation of nucleophilic ethoxide thereby furnishing a more satisfactory procedure. The methodology also has potential for the preparation of y-hydroxysilanes e.g. (7I3) which are suitable for oxidative fragmentations (Scheme 1 08).'9h 9 Reductive Fission of Hetero-substituents 9.1 Hydrogenolysis of Nuclear Substituents 9.I.1 Simple Ethers Hydrogenolysis of simple aryl alkyl ethers is rare but there are a number of well-defined situations in which this is likely to occur. The methylenedioxybenzenes (71 5) and (716) are reduced to phenoj (71 7) and p-cresol (718) respectively,'" while the methoxyl group is lost completely from 4-methoxy- biphenyl (719).298 This loss is prevented however by the presence of a strategically located hydroxy-group (which is converted into phenoxide during the reduction) as indicated in examples (720),'9" (722),300 and (724)30' (Scheme 109). It is also significant that in these cases the olefinic bonds that are conjugate to the residual aryl ring are also preserved although excessive metal will lead to their reduction.302 The benzylic 17- hydroxyl was also retained in the reduction of (724) (see below) as it was in the reduction of (726) to (727)."13 77 NATURAL PRODUCT REPORTS 1986 -J.M. HOOK AND L. N. MANDER Si Me3 Si Me3 S iMe3 Si Me2 i 8 0+ 0+ Me3SiOEt* + (663) (664) [5 "lo] (76)* Si Me3 Si Me3 S iMe3 (682) -b Me3sim (665) (666) [60°/o] (683) Si Me3 Si Me3 -yy ii (667) (668) [70°/o] (684) (685) S i Me3 SiMe3 r80 "lo] [20O/O] Reagents i Na liq. NH, NH,CI 0 -9 Scheme 105 (669) (670) [70°/o] Si Me3 S i Me2 I C02Me C02Me (686) (687) (6881 14:11 v ii iii o!3iMe3 -S iMe3 C02Me A (674) (675) [3'/0] (673)[el O/o] C02Me Si Me2 (at -3OoC) 8 -0 * " + Me3SiOEt 6SiMe3 V,II,III) QSiMe3 SiMe3 (67'10 yield ) (676) (761* C02Me C02 Me SiMe3 SiMe3 (690) (691) Reagents i LI liq.NH, at -78 OC;ii Mel at -33 OC ~li 4 H,C iv. CHIN2;v Na liq. NH3 Bu'OH at -78°C (6771 (675) [85 "/o] Scheme 106 SiMe3 Biaryl ethers undergo reductive fission ; this reaction has been applied extensively to the elucidation of the structure of S iMe3 bisbenzylisoquinoline alkaloids. Cleavage tends to take place adjacent to a methoxy-group but is inhibited by a para-(6781 (679) [55 "lo] methoxy- or a hydroxy-group the latter as a consequence of the formation of a phenoxide. Thus oxyacanthine (728) is reduced to (729) which after O-methylation may be reduced further to (730) and (731) (Scheme 1 9.I .2 Deoxygenation of' Phenols (679) [3 O/oI (680) [ 50 "/0 ] Deoxygenation of phenols may be achieved by reduction of aryl * Detected by g.1.c.diethyl phosphates with lithium or sodium in liquid am-Scheme 104 m~nia.~~~ A recent application of the methodology is outlined 78 NATURAL PRODUCT REPORTS 1986 OMe OMe ii R* R$ R+ CH2 Si Me3 (693) (694) (R=H or Me) ... Ill + S i Me3 R (696) (697) (R = H or Me) \SiMe3 SiMe3 ... Ill ___) '0 (698) (699) (700) (701) (702) Si Me3 C02Mea,,,(703) - i,iv SiMe3 (704) vi ii Qq-+ a+ isomers SiMeg Si Me3 (709) (707) (708) Reagents i Li liq. NH3 EtOH; ii HCI THF; iii HCI THF MeOH; iv CHIN,; v 48% HI PhH; vi Li liq.NH3 Bu'OH Scheme 107 (714) Si Me3 (713) Reagents i BunLi HMPA; ii C,H,,Br; iii. Li liq. NH3 EtOH; iv H30+;V H, Pd/C; vi LiAIH,; vii (NH,) [Ce(NO,),] MeCN H,O Scheme 108 NATURAL PRODUCT REPORTS 1986-J. M. HOOK AND I,. N. MANDER 'OoH mOMe (715)R = H (717)R= H (719) (716) R = Me (718)R = Me \ HO@& I \ N Me (728) OMe 0Me OH OH HOq& \ N (724) (725) OH OH (726) (727) Me Reagents i Na liq. NH3 Bu'OH; ii Li liq. NH, Bu'OH; iii Li liq. (730) (731) NH,? EtOH Reagents i Na liq. NH,; ii CH2N> Scheme 109 Scheme 110 0 II OP(OEt) iii,iv J R MeO($OMe (R = Me,Et,or Pr') 'QOH But BU' (737 1 (7361 Reagents i (EtO),P(O)H CCI, Et,N; ii Li liq.NH, Bu'OH; iii Bu'Li THF; iv RX; v IM-HCI at 80-9O"C Scheme 111 H02C i ,ii M e O d Me0 Me0 \ HO OMe (738) iii ,iv (739) 0 (740) Reagents i 85% H2S0,; ii NaOH Me2S0,; iii Na liq. NH,; iv NH,Cl Scheme 112 0 Me0 \e~O d ~ MeO + ~M Me0 \ Me0 OMe C02H (741) (7421 1 ii (743) Reagents i polyphosphoric acid; ii Na liq. NH3 Scheme 1 13 Me0 O^OH (744) (745) (746) (7471 ,O NATURAL PRODUCT REPORTS 1986 Me0sp"' \ (754)R',R2= H,H; H,OH; Me.OH; H.CH2CH(OH)Me; H,CH2$0] ;or H,CH2C02H 0 Me OH 1 om ~ I W O M e f-f-O=c/ H02C C02H \ (758) Om (757) Reagents i Na liq. NH3 Scheme 114 in Scheme 11 1.306 The reaction works well with a variety of substituted phenols but not with dihydric phenols or naph- thols.The alternative reduction of aryl sulphonates has also been examined but the limited solubility of these derivatives can present difficulties. 9.1.3 Alkoxy-substituted Aromatic Acids and Ketones It has been noted in earlier sections (4.4) that alkoxy-groups that are in a para-relationship to a carbonyl group are invariably lost during reductions by a metal in liquid ammonia. This may be put to good advantage in obtaining patterns of aromatic substitution which would otherwise be difficult to achieve as in the preparation of 5,7-dimethoxy-1 -tetralone (740) (Scheme 112),307 in the synthesis of the acid (743) H ,NH2 0% (Scheme 1 13),308 and in the synthesis of 6-methoxy-I-naphthyl ketones (635) (Scheme 97)285 (Section 7.5.2).9.2 Hydrogenolysis of Benzylic Substituents C02H O-0 I I (748) C02 Et (7491 9.2.I Benzylic Alcohols OMe As a general rule oxygen-containing substituents are lost from benzylic positions during reductions by metals in ammonia. An (750)R'= Me,R2= H (751)R'= R2= Me R (752)R = H or Pri c H2OH (753) important set of exceptions are those compounds which possess a methoxy-group in a para-relationship to the benzylic substituent. Thus 4-methoxybenzyl alcohol (744) may be reduced in high yield to the 2,5-dihydro-derivative (745),whereas benzyl alcohol itself and its 2- and 3-methoxy-derivatives are each reduced to the dihydrot~luenes.~~)" Reduction of 4-methylbenzyl alcohol (746) gives a 4 :1 mixture of (747) with 4-ethylt0luene,~'~ confirming the influence of an electron-releasing substituent in the para-position.NATURAL PRODUCT REPORTS 1986 -J. M. HOOK AND L. N. MANDER . .. I ,ll 4 HO 1 ?H The result with 4-methoxybenzyl alcohol appears to be completely general and (745) has been used in the synthesis of anticapsin (748)31 and the intermediate (749) for the synthesis of ~ernolepin.~~* The analogous compounds (750)- Me V O M e (754)3083313-316have also been reduced with retention of the Na02C RO OR benzylic substituent. There appear to be significant advantages in deprotonation prior to red~ction,~ I ’and derivatization as a tetrahydropyran-2-yl ether may also be effe~tive.~ Reduction (759) (760) of the gibberellin intermediate (755) with sodium in liquid ammonia furnished (758) but it is possible to rationalize this result in terms of the sequence that is outlined in Scheme 114 1iii i.e.by a mechanism which is initially independent of the aryl system. Hydrolysis of (759) to (760) followed by Birch reduction however afforded (761) which could be readily re- lactonized to (762) (Scheme 115).319 9.2.2 Benzylic Acetals Metal-ammonia reduction of the acetal (763) was reported to afford the deoxygenated derivative (764) as the major product (Scheme 1 16),320 but the choice of dioxolane as the protecting group turns out to be unfortunate since it is possible to avoid (762) (7611 hydrogenolysis with other acetal functions. Reduction of the (R = Me or RR =-CH CH,-) acyclic acetals or 1,3-dioxane derivatives furnished yields of 30-75% of the dihydro-derivatives (766) (Scheme 1 1 7).321 Reagents i NaOH; ii CO,; iii Na liq.NH,; iv HOAc Scheme 115 9.2.3 Benzylic Amines Benzylic amines are resistant to hydrogenolysis under the conditions of Birch reduction unless the amine is quaternary as in the Emde reaction. A means of preserving a benzylic carbonyl group is therefore to protect it as an aminal derivative. An illustration is provided by the preparation of the aldehyde (769) which is an important constituent of the flavour of (7631 (764) cumin as outlined in Scheme 1 18.322 Reagents i Na liq. NH, MeOH Scheme 116 10 Conclusion The catalogue of examples that is outlined above provides a compelling illustration of the utility of reductions by metals in R20 OR2 R20 OR2 liquid ammonia.Applications of these processes to the reduction and alkylation of aromatic esters amides and ketones have yet to be fully developed and thus hold considerable potential for future exploitation. The scope of this article has been confined to the reduction of aromatic (765) (766) substrates by metals of Group I in liquid ammonia. Alternative reducing systems may allow greater selectivity or may lead to (R’= H or Me R2= Me or Et,or R2R2=-CH2CMe2CH2-) different outcomes. Of special note are reductions in Reagents i Na liq. NH amines323 326 and in hexamethylphosphoric t~-iarnide,~” cathodic reductions,328 and photochemically based reduc-Reductive ~ilylation~~~ Scheme I 17 tion~.~’~ 332 is also a potentially useful procedure.11 References 1 A. J. Birch Q. Rer. Chem. Soc. 1950 4 69. 2 A. J. Birch and H. Smith Q. Rec. Chem. Soc. 1958 7 17. 3 H. Smith ‘Organic Reactions in Liquid Ammonia’ Wiley-Interscience New York 1963 Vol. I Part 2. (767) 4 F. J. Kakis in ‘Steroid Reactions’ ed. C. Djerassi Holden Day San Francisco 1963 pp. 267-298. 5 R. G. Harvey Synthesis 1970 161. 6 A. J. Birch and G. S. R. Subba Rao in ‘Advances in Organic Chemistry Methods and Results’ ed. E. C. Taylor. Wiley- Interscience New York 1972 pp. I-65. 7 H. L. Dryden in ‘Organic Reactions in Steroid Chemistry’ ed. J. Fried and J. A. Edwards Van Nostrand Reinhold New York 1972 VOI. I pp. 1-60. 8 A. J. Birch J. Agric. Food Chem. 1974 22 162. 9 G. S. R. Subba Rao and K.Pramod Proc. Indian Acad. Sci.,Chem. (769) Sci. 1984 93 573. Reagents i MeNHCH2CH2NHMe at60 “C;ii. Li liq. NH, Bu‘OH; 10 A. J. Birch and J. Slobbe Heterocycles 1976 5 905. iii 2M-HCI 11 H. E. Zimmerman in ‘Molecular Rearrangements’ ed. P. de Scheme 118 Mayo Wiley-Interscience New York 1963 Vol. I pp. 347-352. 12 A. J. Birch A. L. Hinde and L. Radom J. Am. 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ISSN:0265-0568    

DOI:10.1039/NP9860300035

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

Avermectins and Milbemycins H. G. Davies and R. H. Green Medicinal Chemistry Department Glaxo Group Research Green ford Middlesex UB6 OHE 1 Introduction 2 Fermentation and Isolation 3 Structure Determination 4 Biosynthesis 5 Metabolism 6 Chemistry 6.1 Total Synthesis 6.2 Partial Synthesis 6.3 The Interconversion of the Avermectins and Mil bemycins 6.4 Protection and Acylation 6.5 Oxidations 6.6 Alkylation 6.7 Reduction 6.8 Avermectin Aglycons and their Monosaccharide Analogues 7 Structure-Activity Relationships 8 Conclusion 9 References 1 Introduction Milbemycins and avermectins are related members of the class of sixteen-membered-ring macrolides which are destined to play an increasingly important role in the treatment of parasitic diseases of animals and it is to be hoped of humans.An indication of their commercial importance can be gained from the estimate that parasitic infections of livestock cost more than $3 billion annually in the United States of America alone (see footnote in ref. 1). The structures of the naturally produced avermectins are (1)-(8). They fall into two main groups designated A and B the former possessing a methoxy-group at C-5 and the latter possessing a 5-hydroxy-group. These groups are further subdivided into the 1-series with a C-22-C-23 double-bond and a 2-series which has an axial hydroxyl group at C-23. A final subdivision into a and b series designates the presence of a s-butyl or an isopropyl group respectively at C-25.* Milbemycins while being structurally simpler than the avermectins have a greater diversity of functionalization which results in a less structured nomenclature.The only real subdivision which can be made of milbemycins is into the c1 series [(9)-(23)] members of which possess a fused tetrahydro- pyranyl ring and are true analogues of avermectins and the p series [(24)-(28)] in which the tetrahydropyranyl ring is missing. The aromatic ring of milbemycin p3(28) is a notable feature in this otherwise alicyclic series and it is of interest to note that an analogous aromatic avermectin has been described.' Comparison of structures (1)-(23) reveals that the only major difference between avermectins and milbemycins is that the latter lack the a-L-oleandrosyl-a-L-oleandrosylgroup at C- 13.Confirmation of this inter-relationship has recently been provided by the conversion of 22,23-dihydroavermectin B1 b aglycon into milbemycin D.3 This is described in a later section of this review. * Wherever avermectins are described in this Report by using only the first two identifying characters as in A2 this refers to mixtures of the a and b analogues. In these cases only the structure of the major component is indicated in the diagram. Where fully identified as in B2b this implies the use of pure compound. 2 Fermentation and Isolation In 1974 workers in the Sankyo laboratories5 described the isolation of a crude preparation of macrolides with far superior biocidal activity to that of any known insecticides and acaricides.These compounds were produced by Streptomyes strain B41-146 which was isolated from a soil sample that had been collected at Kuttian-Cho in Hokkaido Prefecture Japan and given the delightful name Streptomyces hygroscopicus subsp. aureolacrimosus from its propensity to form 'golden- yellow teardrops' of exudate on aerial mycelia. Extensive chromatography of the culture broth of this Streptomyces strain which was aided by the ready visualization of milbemycins with ultraviolet light gave thirteen milbemycins namely a1to al0and p1to p3.6Small amounts of milbemycins D to H were also produced by the parent strain.' Mutation of the original B41-146 strain by exposure to ultraviolet light gave two mutants with very different milbemycin-producing characteristic^.^ Strain Au-3 gave markedly higher yields of milbemycins D to H but did not produce milbemycins a5 to ag which are those milbemycins with a 2-methylhexanoyl group at C-23,' while strain Rf-107 gave only milbemycins J and K.* The history of the avermectins closely parallels that of the milbemycins.As part of a routine screening programme using an assay in uivo for anthelmintic activity workers at Merck Sharp & Dohme tested an actinomycete that had been isolated by workers at the Kitasato Institute of Japan.9 The source was another example of the fecund Japanese soil this time from Kawana Ito City. A concentrate of the broth in which the actinomycete had been cultured showed remarkable activity OMe ..-.A OMe Avermectin Ala (1) Avermectin Alb (2) Avermectin A2a (3) Averrnectin A2b (4) Averrnectin Bla (5) Avermectin Blb (6) Avermectin B2a (7) Averrnectin B2b (8) 23 .R' R2 x-Y Me BUS CH=CH Me Pr' CH=CH Me BUS CH*-CH(OH) Me Pr' C H2-CH (OH) H BUS CH=CH H Pr' CH=CH H BUS CHz-CH (OH) H Pr C Hz-C H(0H1 NATURAL PRODUCT REPORTS 1986 R4 ‘R2 R’ R2 Milbemycin a1 (9) OH Me Milbemycin Qz (10) OMe Me Milbemycin 43 (11) OH Et Milbemycin 44 (12) OMe Et Milbemycin 45 (13) OH Me Milbemycin 46 (14) OMe Me Milbemycin a7 (15) OH Et Milbemycin a8 (16) OMe Et Milbemycin txg (17) OH Milbemycin 410 (18) OH R’ RZ R3 Milbemycin D (19) H,p-OH Pr’ Me Milbemycin F (20) H,p-OH Pr‘ CH20C4N> 11 H 0 Milbemycin G (21) H,p-OH Pr’ Me Milbemycin J (22) 0 Me Me Mil bemycin K (23) 0 Et Me against the helminth Nematospiroides dubius in mice which is very difficult to remove with common anthelmintics (such as benzimidazoles) with no apparent toxic effect to the host over an eight-fold dosage range.9 The actinomycete that is respon- sible for producing the active agent was found to be novel and given the name and designation Streptomyces avermitilis MA-4680(NRRL8165).Extensive work on this species by causing mutation with various agents and by modification of the R3 R4 R5 Me H H Me H H Me H H Me H H Me OCOCHMeBu” OH Me OCOCHMeBu” OH Me OCOCHMeBu” OH Me OCOCH MeBu” OH H H X X R’ R2 Milbemycin (24) H,p-OMe CH20H Me Milbemycin 82 (25) H,p-OMe CH20H Et Milbemycin E (26) H,p-OMe CH20H Pr‘ Milbemycin H (27) 0 CH3 Pri OH Milbemycin 83 (28) NATURAL PRODUCT REPORTS 1986 -H.G. DAVIES AND R. H. GREEN HO \ ?H OMe (34) R = H OR (35)R = OH (29) R = H (30)R = Me OH R' 0 .. I I X OH (36) R'= H;R2= Pr'; X = H,p-OMe (31) R = BuS;X = HIp-OH (37) R1= H ;R2= Bus; X = 0 (32) R = Pr';X = H p-OH (38) R'= H ;R2= Pri ; X = 0 (33) R = Bus; X = 0 (39) R'= a-L -oleandrosyl -a-L -oleandrosyl ; R2= Bus; X=H p-OH growing medium gave more than a fifty-fold improvement in yield from the mutated strain S. avermitilis MA-4848 (ATCC 31271),y*'0 which could now produce 53 pg of avermectins.The choice of suitable methods for the isolation of the avermectins was initially guided by biological assays but as with the milbemycins a correlation which was established between their absorbance of ultraviolet light and their anthelmintic activity allowed a more rapid assay and facilitated the chromatographic purification. Solvent extrac- tion and partition of the fermentation broth gave a crude mixture of avermectins; chromatography on Sephadex allowed the separation of the avermectins A from the avermectins B. Further chromatography on Sephadex separated the A 1 components from the A2 components of the mixture. The BI component could be separated from the B2 component by crystallization from ethylene glycol or by chromatography on Sephadex followed by crystallization from benzene.The final separation of the a components from the b components in each of these mixtures was again by chromatography on Sephadex under guidance by h.p.1.c. analyses.' I Further purification of the mother liquors from the crystallization of the B components gave small amounts of the 22-hydroxyavermectins (29) and (30) of unspecified stereochemistry at C-22.' Similarly the A fractions of the fermentation broth furnished the aromatic avermectins (31) -(33) and the avermectins (34) and (35) (in which the tetrahydrofuran ring has been opened),' very reminiscent of the p series of milbemycins. Further ring-opened avermectins both substituted with the disaccharide moiety [as in (39)] and unsubstituted [as in (36)] and also further oxidized at position 5 [(37) and (38)] are produced by the mutant strain S.avermitilis MA-5218.I"t 3 Structure Determination The primary literature contains full details of the physico- chemical data of the CI and pseries of milbemycins.h The infra- red spectra of all milbemycins display absorption bands due to hydroxyl groups and the intramolecular lactone group is characterized by its absorption at 1707-1710cm-I. The aromatic ring of milbemycin p3 is also indicated in the infrared spectrum. Milbemycins as to ctg show an ester group with absorptions at 1721-1722 cm-I. The ultraviolet data on the milbemycins indicate that they contain a conjugated diene and the absorption at 266 nm of milbemycins c19 and cll0 is consistent with a pyrrole subunit.The mass spectra of these compounds are particularly informative as the molecular ion is very intense and each milbemycin gives five to seven prominent fragment ions which are clearly related to those from other milbemycins. High- resolution mass spectrometry of the prominent ions gave accurate formula weights and hence irrefutable constitutions for each ion. From the spectral data it was apparent that milbemycin PI possesses a primary hydroxyl group which was derivatized to provide a crystalline p-bromophenylurethane that was suitable t Curiously this patent also claims that derivatives of avermectins [v.g.(40) (41) and (42)] which have a [5,5]-oxaspiro ring and not a spiro-ketal group are formed.This is clearly a mistake since examination of the equivalent US. patent" indicates that the compounds that were isolated have the more familiar trienic macrocyclic ring with a spiro-ketal ring appended. NATURAL PRODUCT REPORTS 1986 for X-ray studies.] The resulting structure (24) revealed the required elements of a conjugated diene lactone and primary alcohol. It now becomes obvious that the mass-spectral fragmentation is due to a combination of retro-Diels-Alder cleavage allylic fission cleavage a to an oxygen atom and McLafferty rearrangement that leads to the elimination of the ester group (Scheme 1). With the mass-spectral fragmentation of milbemycin p in hand the solution of the structure of milbemycin pz was straightforward.Spectroscopically it is very similar to milbe- mycin PI apart from an increase of fourteen mass units in the three fragment ions (43) (44) and (45). This indicates milbemycin pzto be the 25-ethyl homologue of milbemycin p ; this conclusion is supported by the presence of a three-proton triplet at 6 0.82p.p.m. in the 'H n.m.r. spectrum. The l3Cn.m.r. spectrum of milbemycin p3 showed an ester carbonyl group twelve sp' carbon atoms and eighteen sp3 carbon atoms three of which are tertiary carbon atoms with an oxygen substituent. In combination with the infrared ultraviolet spectra an aromatic ring is indicated; the 'H n.m.r. spectrum shows a 1,2,4,5-tetrasubstitutedsystem. A combina-tion of nuclear Overhauser enhancement and double-irradia- (40)R' = a-L-oleandrosyl R2= Me (41) R' = a-L-oleandrosyl R2 = H (42)R' = a-L-oleandrosy\ -a-~-oleandrosyl R2 OMe (24) (45)[m/z 1531 tion experiments showed one aromatic proton to be ortho to the methyl group at C-4 and the other aromatic proton to be near to the olefinic methyl group.The chemical shift of the former proton further implies an ortho relationship to the aromatic carbonyl group leading to part-structures (46) and (47).Of these structures (47) was rejected on the grounds that (a)no intramolecular hydrogen-bonding between the phenolic proton and the carbonyl group is seen and (b) this would give an eighteen-membered lactone ring rather than a sixteen-mem- bered ring as is seen in other milbemycins. This was considered to be biogenetically unlikely.The similarity of milbemycin p3 (28) to other milbemycins was also apparent from the mass-spectral data. Proton n.m.r. spectra of milbemycins D E F G and H indicated the characteristic sixteen-membered lactone ring and [5.5]-spiro-ketal features and further indicated an isopropyl group at C-25 to be common to all of these milbernycins.'.l6 Comparison of the ultraviolet spectra of milbemycins D E and and G with those of milbemycins a,, PI and p2demonstrated that there is a common diene subunit while the ultraviolet spectrum of milbemycin F also showed absorbances that are character- istic of a pyrrole subunit (which was confirmed by 'H and '3C n.m.r. spectroscopyi6) as seen in milbemycin aY.The presence of an ap-unsaturated ketone in milbemycin H was suggested by the ultraviolet spectrum and by an absorption in the infrared spectrum at 1685 cm-I.Similar evidence suggested that an up-unsaturated ketone is present in milbemycins J and K.8 The fragmentation pattern that is displayed by mass spectrometry of milbemycins D F and G distinguished them as 25-isopropyl analogues of the milbemycins. I The interpre- OH OH 0 = H (46) (47) r (43) m/z 4021 t (44)[m/z 1811 (a) retro-Diels-Alder cleavage; (b) allylic cleavage; (c) a-oxygen cleavage Scheme 1 NATURAL PRODUCT REPORTS 1986 ~ H. G. DAVIES AND R. H. GREEN tation of these mass spectra was aided by comparison with the mass spectra of the samples of I3C-labelled milbemycins that had been obtained in biosynthetic studies (see below).The mass spectrum of milbemycin F allowed the placing of the pyrrole subunit in the fragment that is eliminated upon retro- Diels-Alder cleavage. Proton n.m.r. spectroscopy indicated that the substituent is on the methyl group at C-4. The mass spectra of milbemycins D and G were identical apart from the molecular ions with milbemycin G being fourteen mass units heavier. This extra mass was accommod- ated in the fragment that is eliminated by retro-Diels-Alder fragmentation. The I3C and ‘H n.m.r. spectra pointed to the presence of a methoxy-group on C-5 in milbemycin G replacing the 5-hydroxy-group of milbemycin D. Infrared and ultraviolet spectra of milbemycins J K and H demonstrated that they contain an &-unsaturated ketone grouping and the infrared spectra of milbemycins J and K also indicated the presence of a tertiary hydroxyl group but no secondary hydroxyl group.A correlation with milbemycin (x was suggested by the ‘H n.m.r. spectrum which only differed in the resonances associated with C-6 and C-3. That milbemy- cin J was merely a 5-0x0-analogue of milbemycin a,was finally proved by reduction of milbemycin J with sodium borohydride which stereospecifically produced milbemycin CI .* The mass spectrum of milbemycin J was reminiscent of those of other milbemycins apart from a major stabilized aromatic fragment which was assigned the structure (48). In a similar manner milbemycin K was assigned as a C-25 homologue of milbemycin J and hence as a 5-0x0-analogue of milbemycin r3.Milbemycin H presented a different problem in that its infrared and ultraviolet spectra were ‘essentially identical’ to those of milbemycins J and K but the molecular formula lacked one oxygen atom. Nuclear magnetic resonance spectra and mass-spectral fragmentation patterns (Scheme 2) however allowed an assignment of the structure as (27).” Although the relative configuration of milbemycins had been well established by this time the absolute configuration had not been established by the Japanese group. Workers in the Merck laboratories however had chemically correlated an avermectin aglycon with milbemycin D,3 and had further determined the absolute configuration of the avermectins. The Japanese workers then determined the absolute configuration of milbemycin D by more direct methods.Measurement of the specific rotations of milbemycin D its hcetate and the corresponding 5-epimers led by the application of Mills’ rule,’ ’ to the assignment of R configuration at (2-5. A similar conclusion was reached by studying circular dichroism spectra of milbemycin D 5-[p-(NN-dimethyiamino)benzoate] and its epimer. This assignment of the R configuration to C-5 in the milbemycins is entirely consistent with the configuration of the corresponding centre in avermectins. (Circular dichroism spectra of avermectins have corroborated this proposal’*). The previously published determination of the structure of milbemycins greatly aided the elucidation of the skeleton of the avermectins as the identity of various structural subunits in the .+ avermectins (determined by I3C and ‘H n.m.r.and mass spectra) suggested a close similarity to the milbemycins with s-butyl or isopropyl groups replacing the substituents at C-25 of the milbemycins.” The major problem lay in the identity and position of attachment of the disaccharide substituent. Analy- sis of the mass-spectral fragments that are associated with the disaccharide unit indicated that there are two isomeric or identical monomers and the ‘H n.m.r. spectrum indicated only small coupling constants for the anomeric protons at C-1’ and C-I”. Acid-catalysed methanolysis of avermectin A2a gave a single product methyl oleandroside as a 6 :1 mixture of c1 and p anomers.The optical rotation of this mixture indicated the oleandroside to be of the L series. In combination these data established the disaccharide substituent of the avermectins to be u-L-oleandrosyl-a-L-oleandrosyl. Nuclear magnetic resonance studies of avermectin aglycons were inadequate to distinguish whether hydroxylation was at C-13 or C-16 but the mass spectrum favoured C-13 as the point of attachment. This meant that the disaccharide substituent must be at C-7 or C-13 (C-5is ruled out as the members of the A series have both a disaccharide substituent and a methoxy substituent at C-5). Carbon-1 3 n.m.r. spectral data however ruled out C-7 as a point of attachment; this conclusion is supported by a deuterium-exchange experiment and by H n .m.r. spectroscopy. Final proof that C-13 is the point of attachment of the disaccharide moiety came from chemical degradation. Ozonolysis of avermectin A2a followed by reduction with sodium borohydride gave the disaccharide and spiro-ketal fragments (49) and (50),respectively (Scheme 3). Methanolysis of the disaccharide fragment (49) gave methyl cx-and p-oleandrosides and the two epimeric 2-methylpentanetriols (5 1 ) hence confirming the attachment of the disaccharide moiety to the oxygen at C-13 of the avermectins. Both avermectin Bla and the aglycon of avermectin B2a crystallized from methanol in a form that was suitable for X-ray-crystallographic ana1y~is.I~ The assignment of L configura-tion to the disaccharide moiety permitted the assignment of the absolute configuration of the macrocyclic ring.The X-ray structure of avermectin B2a aglycon reveals several subtle but important aspects of the conformation of this molecule. The overall rigidity of the structure imposes a torsional angle of 12 O upon the cyclohexenyl double-bond which in turn causes this ring to adopt a flattened chair configuration. The cis-fused tetrahydrofuran ring adopts a conformation close to the classical envelope conformation while the two six-membered rings of the spiro-ketal group adopt chair conformations. A further torsional strain of 3 O from planarity is imposed upon the transoid diene unit by the overall rigidity of the molecule. The overall conformation of avermectin B 1 a is similar but with some crucial differences.The torsional strain about the cyclohexene double-bond is reduced to 2 O which in turn results in a more distorted envelope conformation for the tetrahydropyranyl ring. This in turn leads to a reduction in torsional strain about the transoid diene to only 1 ’ from planarity. The saturated ring of the spiro-ketal still exists as a chair conformer but the unsaturated ring adopts a half-chair conformation. Both oleandrosyl groups exist as chair con-formers with the first sugar residue lying in a plane that is perpendicular to both the macrocyclic ring and the second sugar unit. Implications of extensive hydrogen-bonding networks in both of these crystal lattices can be found. The three molecules of methanol which co-crystallize with each molecule of avermectin B2a aglycon were found to link 0-7a and 0-13a intramolecularly by a network of four hydrogen-bonds while intermolecular hydrogen-bonds link 0-13a to 0-23a and 0-5a to 0-7a.A possible hydrogen-bond may intramolecularly link 0-la and 0-7a albeit the distance (3.18 A) implies that this bond is weak. A similar network of hydrogen-bonds exists in the crystal lattice of avermectin B2a. Intermolecular hydrogen- NATURAL PRODUCT REPORTS. 1986 -*+ 0 0 ___I) d -*+ -0 -I 6 Scheme 2 bonds are implied between 0-5a and 0-3”a 0-7a and 0-4”a 0-6a and HOMe (methanol a),0-7a and HOMe (methanol h),and MeOH and HOMe (methanol a and methanol h). A slightly stronger intramolecular hydrogen-bond exists between 0-1a and 0-7a in this molecule.Correlations of X-ray structures and n.m.r. data of avermec-tin A2a aglycon agree in all respects providing compelling evidence that the conformations of the avermectin skeleton in solution and in the solid state are the same. The X-ray structure also explains an apparent anomaly in the H n.m.r. spectrum which is that the axial proton at C-16 appears at lower field than its geminal counterpart. This reversal of the usual order can now be adequately explained by the proximity of the axial proton to the plane of the C-14-C-15 double-bond resulting in a deshielding of this proton. Shielding by a proximate double-bond was thought to be the reason for the distinctive high-field n.m.r.shift of ca 0.6 p.p.m. of the axial proton at C-18. However the X-ray structure did NATURAL PRODUCT REPORTS 1986 ~-H. G. DAVIES AND R. H. GREEN OH I OMe OMe i,ii I Hoh..o..x OH I HOk; (49) 7 + AoJ-o$oH OH OH (51) (50) Reagents i 03,CH,Cl,; ii NaBH,; iii 1% H2S0, MeOH Scheme 3 0 = Oxygen Figure I The structure of avermectin Bla = Oxygen not support this hypothesis. Instead it was postulated that the Figure 2 The structure of avermectin B2a aglycon anomalous high-field shift was due to shielding by the lone pairs on the adjacent oxygen atoms on the relatively rigid 4 Biosynthesis avermectin skeleton. The Au-3 strain of S.hygroscopicus affords milbemycins u2,a,, Computer-generated drawings of avermectin B 1a and the and D as major metabolites." These provide examples of 25-aglycon of avermectin B2a derived from the X-ray structures methyl (a?), 25-ethyl (a4),and 25-isopropyl (D) groups and 5-that are described above are shown in Figures 1 and 2 hydroxyl (D) and 5-methoxyl (a,,cx4) groups and thus this respectively.20 strain was selected for studies of the biosynthesis of milbemy- 94 NATURAL PRODUCT REPORTS. 1986 cins. Off-resonance proton-decoupling and selective proton- noise-decoupling techniques were used in 3C n.m.r. spectro- scopy studies to assign methine and methyl signals. Resonances in the I3C n.m.r. spectra that are due to C-4 C-8 and C-14 were assigned by long-range selective proton-decoupling techniques. Mass spectrometry was used both to check the position of incorporation of unnatural isotopes and as a check on the postulated fragmentation patterns.If [l-13C]acetate was fed to S. hygroscopicus it produced a sample of milbemycin azthat was enriched in I3C at carbons 1 5,9 15 17 19,21 and 25; weak enrichment was also observed at carbons 3 7 1 1 13 and 23. If [ 1-I 3C]propionate was fed to the bacterium there was 3C enrichment at carbons 3,7 1 1 13 and 23 of the product and 3-labelled propionate enriched carbons 26 27 28 29 and 30. It is to be noted that the weak enrichments that were observed with [ l-13CC]acetate are paralleled by the enrichments that were achieved by feeding 1-labelled propionate. This demonstrates the metabolism of acetate into propionate and the subsequent incorporation of propionate into the milbemycin.Feeding of ~-[rne?hyl-l 3C]-methionine caused enrichment of the methoxy-group at C-5. These combined results indicate that milbemycin ct2 is derived from eight acetate and five propionate units with methionine providing the methoxy-group at C-5 and acetate providing (2-25 and the associated methyl group as shown in Figure 3. A corresponding study of the biosynthesis of milbemycins 3 and D revealed an identical mode of formation for the basic skeleton from seven acetate and five propionate units with C-25 and the associated ethyl and isopropyl groups being derived from propionate and isobutyrate respectively. The incorpora- tion of isobutyrate was proved directly by incorporation of labelled [l-'TC]butyrate and its metabolic source was sug-gested by the observation that DL-[2-' 3C]valine specifically enriched C-25 as was observed from the studies with [ l-13C]butyrate.I CH3COzH CH (from methionine) I Some indication of the biosynthetic elaboration of the milbemycins was derived from the study of milbemycins J and K.8 These milbemycins are converted into milbemycins zI and a3 respectively upon incubation with the parent strain of S. hygroscopicus. The mutant strain Rf-107 that is responsible for producing milbemycins J and K must lack the appropriate dehydrogenase enzyme. Further studies suggested that milbe- mycins J and K are true precursors of milbemycins z1and ct3 in the parent strain of S.hygroscopicus. An analogous set of experiments using [1-I 3C]acetate and [1-I "Clpropionate precursors demonstrated that the carbon skeletons of avermectins Ala A2a Bla and B2a are biosynthesized in a similar fashion to the milbemycins' with the exception of the isobutyl group at C-25 which was shown to be derived from ~-isoleucine'~ (Figure 4). The use of sodium [l-14C]propionate in a fermentation process has been used as a means of preparing I4C-labelled avermectin B la with a radiopurity of >99% for further studies.' Purification of the radiolabelled avermectin B 1a from the fermentation mixture by semi-preparative h.p.1.c. has been described by Ku and co- workers.' The use of sodium [1-I 3C 80,]propionate and sodium [1-I 3C I8O2]acetate permitted the assignment of oxygen sources by observing isotopically shifted resonances in "C n.m.r.~pectra.'~ The results are shown in Figure 5 and confirm the expected pattern. They prove that the spiro-ketal oxygen that is attached to C-25 is not derived from acetate or propionate. This was taken to indicate that the spiro-ketal was generated by ketalization of a carbonyl group (C-21) with secondary hydroxyl groups at C-17 and C-25 (Figure 6). Further points of interest arose from this study. The tertiary hydroxyl group at C-7 retains the oxygen atom of its propionate precursor strongly implying that the cyclohexenediol ring is formed by simple aldol condensation either before or after lactonization. This would rule out alternative biosynthetic schemes that are based on cyclization of polyolefins.I Im OCH3 Figure 3 The biosynthetic framework of milbemycin a? OH I I I OCH3 (R = ct -L -oleandrosyl -a-L -oleandrosyl 1 Figure 4 The biosynthetic framework (carbon skeleton) of avermectin A2a NATURAL PRODUCT REPORTS 1986 -H. G. DAVIES AND R. The question of whether avermectins are derived by functionalization of milbemycins or vice versa is clearly answered by the presence of a propionate-derived oxygen at C-13 of the avermectins. This strongly implies that milbemy- cins are derived from avermectins by dehydration and reduction prior to condensation with the acetate unit that corresponds to C-9 and C-10. It is of interest that despite the change of mechanisms that is involved the methyl group at C-12 has the same relative orientation in both avermectins and milbemycins.A simplistic study of the structures of the avermectins could lead to a precursor-product relationship whereby (a) the A components were produced by methylation of the 5-hydroxy- group of the B components and (h)avermectins A 1 and B1 were produced by dehydration of avermectins A2 and B2. A study of the rate of production of the avermectins by S. avermitilis however indicates that they are produced concomitantly rather than ~erially.~ 5 Metabolism Avermectin B2a is relatively stable to sunlight and has a half- life of 2 to 5 days in non-sterile soil depending on ambient A OH A RO mm CH3COOH AA 'j CH CH,COOH HO ( R = a-L -oleandrosyl-a-L-oleandrosyl ) Figure 5 The biosynthetic framework (oxygen atoms) of avermectin B2a c Figure 6 H.GREEN conditions. Under test conditions however in sandy loam soil nematocidal efficacy and plant protection lasted for as long as two months.26 Acetone extracts of the soil were examined by h.p.1.c. and found to contain one major metabolite which was isolated by chromatography and h.p.1.c." Proton n.m.r. and mass spectrometry clearly indicated the metabolite to be 23- ketoavermectin B2a (52); confirmation of this finding was provided by reduction of the metabolite with sodium boro- hydride to give avermectin B2a. Further verification was provided by comparison of the metabolite with synthetic 23- ketoavermectin B2a.The 23-keto-derivative (52) was found to be a potent nematicide in its own right with a half-life in soil of about one month." Incubation of avermectin B2a with sterile soils revealed that less than 1% degradation occurred over thirteen days as compared with 44% degradation under non- sterile conditions. Microbiological studies have led to the isolation of three oxidizing micro-organi~ms,~~ but this work has yet to be fully described. Avermectin Bla which cannot immediately form a 23-ketoavermectin follows a different metabolic course.28 In studies of its metabolic fate in a fine sandy loam a clay and a coarse sand it produced at least thirteen metabolic products under aerobic conditions in each soil. At a dose rate of 1 p.p.m.the half-lives were 14-28 28-56 and 56 days respectively. The labelled avermectins Bla that were used in this study were [5-3H]avermectin Bla and avermectin Bla with a I3C label at positions 3 7 11 13 and 23. Analysis of the metabolites from [5-3H]avermectin gave essentially complete recovery of radioactive material and comparison with an authentic sample revealed that 5-ketoavermectin Bla which is a possible metabolite was not present. Two-dimensional t.1.c. analysis with an authentic sample demonstrated that avermectin B 1a monosaccharide was a minor metabolite; the major metabolite in all three samples purified by preparative t.1.c. and h.p.l.c. was shown to be an equilibrium mixture of 8a-hydroxyavermectin Bla (53) and the corresponding ring-opened aldehyde (54),in an approximate ratio of 1 :2.5.This structure was assigned by a 0 (52) R = a-L -oleandrosyl -a-L-oleandrosyl OHC % HO OH (53) (54) (R = U-L -oleandrosyl -a-L-oleandrosyl) combination of mass-spectrometric and H n.m.r. studies. Mass spectrometry indicated the presence of a new oxygen function which could be trimethylsilylated. The new function- ality was not on the disaccharide moiety as was confirmed by the presence of the usual fragment (m/z 329) from the silylated disaccharide while the aglycon fragment (m/z600) was 16mass units heavier than usually observed for the aglycon of avermectin Bla (m/z 584). A fragment ion of m/z 305 is observable in the mass spectra of avermectin Bla and the metabolite (and in the spectra of their trimethylsilylated derivatives); it was allocated the structure (59 corresponding to the top half of the avermectin skeleton.This ion was not observed in the mass spectrum of 24-trimethylsilylated aver- mectin Bla (see later in this Section) providing further evidence that the soil metabolite is hydroxylated in the lower part of the avermectin skeleton. Final location of the additional oxygen of the metabolite was achieved by ‘H n.m.r. spectro- scopy. The lack of the characteristic methylene signal of C-8a allowed the assignment of an 8a-hydroxy-group that is in equilibrium with a ring-opened hydroxy-aldehyde; the assign- ment was confirmed by the presence of the signal of a formyl proton at 6 10 p.p.m.in the n.m.r. spectrum and by absorptions in the infrared spectrum at 1625 1657 and 2857 cm-I. Surface residues of avermectin Bla on leaves are rapidly degraded (half-life < 1 day) and neither whole plants nor leaves of cotton seedlings and Bermuda grass take up significant quantities of avermectin BI a. This avermectin also has little leaching potential and hence is most unlikely to contaminate surface or subterranean water supplies. The metabolism of 22,23-dihydroavermectin Bla by rat or steer liver microsomes in vitrn produced two major and two minor metabolite^,'^ which were separated and purified by h.p.1.c. A study of the mass spectrum of the metabolite that was produced in greatest abundance indicated that it had under- gone mono-oxygenation in the fragment that is associated with the peripheral ring of the spiro-ketal unit.That this mono- oxygenation product was an alcohol was indicated by the further fragmentation of the ion (56) of m/z 323 to a dehydrated ion of 117/z 305. Verification that these ions arose from the metabolism of 22,23-dihydroavermectin B1 a was achieved by inspecting a mass spectrum of the corresponding metabolite from an equimolar mixture of 22,23-dihydro- and 22,23-dideuterio-avermectin B1 a. Each of the metabolite ions at mi-?21 I 305 323 and 584 was then accompanied by an equally intense ion 2 a.m.u. higher indicating that all of these ions arose from a metabolite. Mass spectra of trimethylsilylated 22,23-dihydroavermectin B1 a and the trimethylsilylated metabolite also demonstrated that the group that had been introduced was an alcohol in that the fragmentation ion of mi/; 21 1 was shifted to m/z 283.Consideration of the H n.m.r. spectrum of this metabolite allowed unequivocal localization of the new hydroxyl group on the 24-methyl group by lack of the characteristic 24-methyl absorption at 6 0.69 p.p.m. and the appearance of a new two- proton signal at 6 3.2 p.p.m. Thus this metabolite was assigned structure (57). The second major metabolite that was isolated in this study was assigned as a monosaccharide derivative (58) of the other major metabolite (57) by differences in both its ‘H n.m.r. and mass spectra these differences being characteristic of the loss of a single saccharide unit.Incubation of avermectin B 1 a and 22,23-dihydroavermectin B1b with either rat or steer liver microsomes also produced the equivalent 24-hydroxylated metabolites as the miijor polar metabolites. In all cases the metabolic profiles for both rat and steer liver microsomes were qualitatively similar with the steer liver microsomes producing metabolites (57) and (58)in slightly greater abundance. As a footnote to this paper it is of interest to note that the list of authors includes S. Avermitilis. This is either a remarkable coincidence or a generous tribute by the authors to each living organism associated with the work. NATURAL PRODUCT REPORTS. 1986 H (55) + I (56 1 OMe A0 0Me (57) R = Me .Oh. (58) R = H In contrast to the results that are described above only traces ( < 0.1573 if any of 24-hydroxylated metabolites were isolated from the incubation of 22,23-dihydroavermectins BI a and B 1 b with pig liver microsomes in zitro.30 Two major metabolites slightly more polar than the parent compounds were isolated and purified by reverse-phase and normal-phase h.p.1.c.Both of these metabolites displayed ultraviolet spectra that were similar to those of the parent avermectins thus indicating that there is no gross change in the macrocyclic system. Electron- impact mass spectrometry gives poor or non-existent molecular ions but for the present study Fast Atom Bombardment (FAB) mass spectrometry in the presence of LiCl proved to be of great utility for the obtention of molecular weights.This gave intense (A4 + Li) ions which indicated that each of the metabolites had lost 14 mass units from its parent dihydroaver- mectin. Combination of this result with the H n.m.r. results which showed only one 0-methyl group indicated that one of the two methoxy-groups of the disaccharide moiety had been demethylated. Comparison of the chemical shifts of the OCH signals that were observed in the spectra of the metabolites the parent avermectins and their monosaccharides indicated the demethylation to be on the outer sugar in the 3”-position. The two metabolites are thus 3”-0-demethyl-22,23-dihydroaver-mectin B1b (59) and its homologue 3”-0-demethyl-22,23- dihydroavermectin B I a (60). Final proof of this assignment came from partial hydrolysis of each metabolite to give the corresponding avermectin monosaccharides.These samples were identical to authentic samples of the known monosaccharides. NATURAL PRODUCT REPORTS 1986 ~-H. G. DAVIES AND R. H. GREEN As can be seen the major metabolites that were obtained from avermectins that had been incubated with liver micro- somes from pigs and from rats and steers are radically different. xl..0. It must not be forgotten however that these are results from experiments in vitro. Indeed two of the major metabolites that .Oh.* were isolated from swine liver tissue in an experiment in uiuo proved to be identical to the two metabolites from experiments with pig liver microsomes that are described above.30 An essential part of the above work is the ability to assay accurately the very low levels of avermectins that exist in tissue or plasma.Simple reversed-phase h.p.l.c. with U.V. detection of avermectins was claimed to be insufficiently sensitive3 at low levels so an alternative method was studied. Dehydration of the crude avermectin isolate produced fluorescent derivatives which could be readily detected in h.1.p.c. fractions.3' In a later paper,32 however this procedure was claimed to be 'time consuming and sensitive to minor experimental variations'. A modification of this method using 1-methylimidazole as a catalyst for acetylation decreases the inherent variability of this method,32 but a recent report by Pivnichny and co-workers (59) R = Me offers a potentially more rapid and reliable method.33 (60) R = Et Treatment of 22,23-dihydroavermectin B 1a with dilute aqueous base causes epimerization of C-2 and then isomeriza- XI.x. xiiT ix,xvi.iv,xvii.1 Milbemycin (24) (69) (70) Reagents:i ally1 bromide Mg THF; ii (MeO),CH CeC1,.7Hz0; iii foyC=& -0; iv LiAIH, EtlO; v KH; vi PhCH,I; vii MeI; viii toluene-p-sulphonic acid water; ix Li liq. NH,; x NaBH,; xi. 03,CH,CI,; xii base; xiii isopropenyl Grignard Et,O; xiv EtCOCl; XV K N(SiMe,), Me,SiCI; xvi Bu'MelSiC1; xvii Collins reagent Scheme 4 NATURAL PRODUCT REPORTS 1986 Me0 Me0 (71) v&\ Ph*P=O Ph,P=O I I Me0 Me0 (72)R = H (73)R = H (74)R = Me (75)R = Me Reagents i SOCl,; ii Me,C(NH,)CH,OH CH,CI,; iii Bu’Li; iv Ac,O; v H,C=CHMgBr; KOH HO[CH,],OH; x CH,N Scheme 5 Me0 vi H2S04; vii Li+ Ph2P-i viii 02 CHCl,; 1% iii -v J (76) OH (28) tion so that the 3-4 double-bond moves to the more stable 2-3 position (see Section 6); the resulting A2 isomer then provides an accurate internal reference which can be assessed by h.p.1.c.(with U.V. photometric detection). A simpler procedure involves extractive clean-up of a plasma sample followed by separation by normal-phase liquid chromatography using U.V. dete~tion.~~ Ivermectin has been determined by a combined h.p.1.c.-reverse isotope-dilution assay3s and by chemical-ionization tandem mass spectrometry of samples that have been purified by extraction and liquid chrornat~graphy.~~ 6 Chemistry 6.1 Total Synthesis Milbemycin P3 (28) was the first member of the large family of milbemycins and avermectins to succumb to total synthesis.Two routes to milbemycin P3 have been described in the literature to date. The first by workers at Pennsylvania Univer~ity,~’ led to racemic material whilst the later synthe- sis3* afforded optically pure milbemycin P3 that was identical in all respects to the naturally occurring compound. The earlier method involved the transformation of the lactone (61) into the aldehyde (69) by the route that is shown in Scheme 4. The isoxazoline (62) which was obtained as a 2 1 mixture of isomers was reduced to give the amino1 (63),which was shown (by 250 MHz ‘H n.m.r. spectroscopy) to be a four- component mixture.Fortuitously treatment of this mixture as shown in Scheme 4 gave a single crystalline aldehyde (64) the stereochemistry of which was confirmed by direct spectral comparison of the derived diol (65) with an authentic sample that had been obtained by chemical degradation of milbemycin p (24). Functionalization of this aldehyde (64) with isopro- penyl Grignard in diethyl ether followed by acylation gave a 2:1 mixture of the ci-and P-propionates (66) which were readily separated by flash chromatography. Claisen-Ireland rearrangement of each of the propionates (66) gave the epimeric acids (67)and (68) which were readily converted into the aldehydes (69) and (70). The aromatic part of milbemycin P3 was constructed from 3-methyl-p-anisic acid (7 1) (Scheme 5).The phosphine oxides (72) and (73) which were obtained as a 3:l mixture were Me0 (75) Reagents i NaN(SiMe3),; ii (69)[see Scheme 41; iii Bu:NF; iv KH THF; v NaSEt DMF heat Scheme 6 smoothly converted into a more favourable 1:1 mixture on treatment with strong base. Esterification of these phosphine oxides (72) and (73) afforded the methyl esters (74) and (75). Treatment of the anion of the ester (75) with the aldehyde (69) gave the diene (76) as a 7 1 mixture of trans-and cis-isomers. This diene was then readily cyclized to racemic milbemycin P3 (28) as shown in Scheme 6. NATURAL PRODUCT REPORTS 1986 -H. G. DAVIES AND R. H. GREEN 99 =o Q 0 (78) (791 Ivev + ++ fl vi ,ii 00' ix-xi 7 fie' PhC02 '-0 PhCO "0 / OH I A Y OSiPh,Bu' OH (83) (811 (80) vii viii Reagents i I?.MeCN; ii Bu,SnH; iii MeSO,H benzene; iv PhCOCl; v P(OMe), toluene; vi Bu'OCl; vii pyridinium chlorochromate; viii NaBH,; ix Ph2Bu'SiC1; x LiOH; xi (COCI)I MezSO Scheme 7 d IV-VII VIII-x XI.XII qBr ,,cBr;tLi I-Ill + d,,tl ___) / OH OH OThp CHO (841 (85) Thp = tetrahydropyran -2 -yl Reagents i LiCHBr, ii 0,;iii Zn AcOH; iv piperidine; v PhSeC1. vi LiAI(OBu'),H; vii m-chloroperoxybenzoic acid; viii MeLi. ix. Me,Al; x I? THF; xi dihydropyran; xii BuILi THF Scheme 8 Optically pure milbemycin p3 has been obtained by the allylic alcohols (86). The conversion of these alcohols into the condensation of two asymmetric moieties both of which were xanthate (87) occurred with [3,3] sigmatropic rearrangement obtained from the same chiral starting material (-)-(3S)-and this xanthate was reduced with tri-n-butyltin hydride to citronellol (77).3xIn order to synthesize the spiro-ketal moiety yield after deprotection and Swern oxidation the aldehyde this alcohol was converted into the adduct (78) which was (88).Condensation of this aldehyde with the benzoic acid (89) obtained as a mixture of two diastereoisomers (Scheme 7). gave the lactone (90) as a mixture of diastereoisomers in high Treatment of this adduct with methanesulphonic acid in wet yield. Each of these diastereoisomers on desilylation and benzene induced internal ketalization yielding the spiro-acetal treatment with potassium hydride gave a single diene acid (79) which after protection and pyrolysis gave the olefin (80).(91). Macrocyclic lactonization and deprotection then gave A chlorohydrin was readily formed and the resulting 5:l milbemycin p3(28) in optically pure form. mixture of epimers was dehalogenated with tri-n-butyltin In a recent report workers at Oxford have described the total hydride to give the epimeric alcohols (81) and (82). Inversion of synthesis of a macrocyclic model (99) for milbemycin 13 (24) the predominant axial alcohol (81) was effected by an (Scheme This was prepared by a convergent route oxidation-reduction sequence to give the desired equatorial involving a Wittig condensation between the phosphonium salt alcohol (82); after protection debenzylation and Swern (92) and the 5H-furan-2-one (93) which produced a mixture of oxidation this afforded the aldehyde (83).(-)-(3s)-Citronella1 the (Z,Z)-and (Z,Ej-dienes. Treatment of this mixture with a (84) was converted into the chiral diene (85) (Scheme 8) which trace of iodine in benzene caused the unwanted (Z,Z)-isomers added smoothly to the aldehyde (83) (Scheme 9) to give the to isomerize to the required mixture of (Z,E)-isomers((94)and 100 NATURAL PRODUCT REPORTS 1986 OHC =0 ___) ++ V' I! 3u' OT hp 0siPh Bu' Ii + **O ii -iv -OThp MeOCH,O OSi Ph,Bu ' CHO (89) I H v vi OSiPhzBu' -"o\ MeOCH20 (901 (911 (28) Reagents i CS2,THF NaH MeI; ii Bu",nH iii pyridinium toluene-p-sulphonate MeOH; iv (COCI), Me,SO; v BulNF; vi KH THF; vii /-7 ,Me 0 +N N==C=N TsO-; viii NaI Me2C0 HCl uu Scheme 9 (95)].Although 'H n.m.r. spectroscopy indicated a single isomer it was assumed that both (94) and (95) were present since the phosphonium salt (92) was racemic. Elaboration of these isomers (as shown in Scheme 10)gave a 1 1 mixture of the two carboxylic acids (96) and (97) as evidenced by 'H n.m.r. spectroscopy. Treatment of this mixture with 2-chloro-N-methylpyridinium iodide and triethylamine in dichlorometh-ane gave a single crystalline product which was shown unequivocally (by X-ray crystallography) to be the macrocyclic lactone (98) arising from cyclization of the isomer (96). This possessed the same stereochemical configuration as the milbemycins.No products deriving from the 'unnatural' isomer (97) could be isolated indicating that this compound does not cyclize under the reaction conditions. Reduction of the methyl ester with sodium bis-(2-methoxyethoxy)aluminium hydride in tetrahydrofuran completed the synthesis of the milbemycin model (99). 6.2 Partial Synthesis In view of the complex structure of the avermectins (eleven or twelve asymmetric centres depending on which series is being considered) it is perhaps not surprising that a total synthesis of an avermectin has yet to be described. Studies of model compounds by workers at Imperial College LondonAohave shown that spiro-acetals r.g.(loo) are readily available by treating tetrahydropyran-2-ones with dianions of P-diones and quenching the reaction with toluene-p-sulphonic acid (Scheme 11).Interestingly if the reaction is quenched by acetic acid the product is the 0-dione (101) which is obtained in 95% yield. In the same paper the synthesis of the subunit (105) of milbemycin P3 is also described. It is obtained by a Diels-Alder reaction between the enone (102) and the Danishefsky diene (103) followed by aromatization of the resulting cyclohexene (104) (Scheme 12). Hanessianj' has also described the synthesis of a model for the spiro-acetal unit of avermectin B 1a (Scheme 13). Starting from 2,3,4,6-tetra-0-benzyl-~-glucono-1,5-lactone (106) the chiral spiro-acetal (107) was obtained (in high yield) by a four-stage process. The spiro-acetal unit has been the subject of a number of other reports regarding synthetic approaches to avermectins and milbemycins.Workers at Southampton University'' have described the synthesis of the enantiomerically pure spiro-acetal portion (1 19)of milbemycins PI and P3 from laevogluco-san (108) (Scheme 14). Laevoglucosan (108) which is readily available from pyrolysis of corn starch,A3is transformed into NATURAL PRODUCT REPORTS 1986 -H. G. DAVIES AND R. H. GREEN OLi OLi I H + (yyl 0si Me2 But Me0 (92 1 (93) OSiMe2Bu' 2 Bu' H02C Y.- MeO Me0 (941 iii -vi1 (95) I H + Me M eO (97) Vlll M Me0 Me0 (981 (99) Reagents i LiN(SiMe ;)?; ii I? benzene; iii. 3-trimethylsilylethanol iv. CH2N2;v. F- THF. H,O; vi. HCI; vii 2-chloro-N-methylpyri- dinium iodide.€t,N viii NaAI(OCH,CH,OMe),H, THF Scheme 10 1ii OH (101) Reagents i toluene-p-sulphonic acid THF Scheme I1 0 CO,Et Me3Si0v CL 0 ii,iii + Me3SiO OH 0 (104) (105) Reagents i benzene heat; ii HCI EtOH; iii NaOEt Scheme 12 the lactone (1 15) in twelve steps. Bis-tosylation followed by reduction with lithium triethylborohydride gave a mixture of the alcohols (109) and (1 10)in a ratio of 10 :1. Allylation of this mixture afforded the requisite ether (1 1 I) which on methanoly- sis and benzylation gave the fully protected trio1 (1 12). The ally1 ether was rearranged to the corresponding vinyl ether. which on methanolysis gave the alcohol (1 13). This material was converted into the lactol (1 14); on oxidation with silver carbonate on Celite this furnished the desired lactone (1 15).Clearly this lactone could prove to be a versatile intermediate in the synthesis of any of the large number of members of the avermectin and milbemycin families. The reaction of the lactone (1 15) with the lithium acetylide (1 16) afforded the alkyne (1 17) which after methanolysis and reduction fur- nished the spiro-acetal (I 18) on treatment with ;t catalytic amount of camphorsulphonic acid. Debenzylation gii~etho requisite optically active spiro-ketal (I 19). An alternative synthesis of the spiro-acetal portion of milbemycin p3 has been described.-14 The spirocyclic ortholac- tones (122) and (123) which were obtained by the acid- catalysed reaction of the ortholactone (1 20) with the diol (I 2 1 ).102 NATURAL PRODUCT REPORTS. 1986 ?CHzPh ?CH2Ph ?CH 2Ph PhCH2O w CH2 OCH2 Ph ... Ill ___) 0 (106) H6 1iv V t PhCHZ000y~~OCH2Ph OCHzPh + OCHzPh Reagents i LiC-C[CH,],OSiMe,; ii H+ MeOH; iii H, Pd/BaSO,; iv camphorsulphonic acid; v Li liq. NH Scheme 13 were converted into the silyl enol ethers (124) and (125) (Scheme 15). Treatment of the enol ether (124) with boron trifluoride etherate gave the spiro-acetal (1 27). It is presumed that this reaction proceeds through intramolecular attack of the silyl enol ether on the dioxonium ion (126). Although this elegant process suffers from a low yield (3573 it nevertheless constitutes a valuable alternative method for the synthesis of spiro-acetals.Ley and co-workersA5 have described a synthesis of the enantiomerically pure spiro-acetal(l3 l) which is an important building block in the synthesis of avermectins and milbemy- cins. The readily available alcohol (1 09y6 was converted into the tetrafluoroborate salt (129) in high yield (Scheme 16) and this after conversion into the corresponding phosphorane reacted with the aldehyde (1 30) which had been obtained from the lactol(l28). Deprotection and cyclization (using dilute acid) afforded the spiro-acetal (131) in optically pure form. The spiro-acetal portion of avermectin B 1a (containing a secondary butyl group at C-25 of the macrolide as opposed to a methyl group in the same position in milbemycin p3)has also been synthesized in optically pure form by workers at the University of Montreal.&’ The two key intermediates for this synthesis are the lactone (132) and the alkyne (133) both obtained from D-glucose (Scheme 17).The lactone (132) was also synthesized from natural ( -)-(S)-malic acid. Condensa- tion of the lithium acetylide of (1 33) with the lactone (1 32) in the presence of boron trifluoride etherate afforded the hemiacetal (1 34). Careful partial reduction of the alkyne bond gave the Z-olefin (135) which on brief exposure to boron trifluoride etherate gave the desired enantiomerically pure spiro-acetal (1 36) as the only detectable isomer. A synthesis of the spiro-acetal sections of avermectins B 1 b and B2b has been reported.J8 The optically pure acetylene (1 37) (Scheme 18) which was obtained from isobutyraldehyde in a nine-stage process was converted into its lithio-derivative and allowed to react with the lactone (138) obtainable from laevoglucosan whereupon the hemiacetal (1 39) was obtained in 54% yield.Methanolysis followed by desilylation and partial hydrogenation gave a Z-olefin which on treatment with a catalytic amount of camphorsulphonic acid provided the spiro-acetal portion of avermectin Bl b (140). The conversion of this into the spiro-acetal moiety of avermectin B2b (141) was simply achieved by formation of a chlorohydrin followed by hydrodechlorination to give the desired spiro-acetal in high yield. In view of the large number of partial syntheses of the spiro- acetal portion of the avermectins it is perhaps surprising that the oxahydrindene component has received such little atten- tion.However Fraser-Reidj9 has recently described an elegant synthesis of this moiety which involved an intramolecular cycloaddition between a nitrile oxide and a vinyl group (Scheme 19). The nitro-alkene (142) available from diacetone glucose underwent conjugate addition with methyl-lithium to give the nitro-alkane (143). Generation of the nitrile oxide under forcing conditionsF0 furnished the tricyclic derivative (144) in high yield. This was smoothly transformed into the alcohol (143 which after protection and ring-opening gave the trio1 (146). Cyclization followed by Swern oxidation then afforded the requisite oxahydrindene moiety (147) of the avermectins.A report by workers in California” has described the preparation of a series of lactams e.g. (148) as potential intermediates for the cyclohexyl portion of the avermectins. Workers at I.C.I.s’ have also described some approaches to the synthesis of the cyclohexyl portion of milbemycin 0 (Scheme 20). Robinson annulation of the dimethoxy-p-keto- ester (149) gave the 3-hydroxycyclohexanone (1 50),which was deprotected to give the hydroxy-diketone (1 51). When reduced with sodium triacetoxyborohydride under anhydrous condi- tions this gave both stereoselectively and regioselectively the keto-diol (152) which is a model for the cyclohexyl portion of milbemycin p . NATURAL PRODUCT REPORTS.1986 -H. G. DAVIES AND R. H. GREEN 103 HO" 8-'OH OH OH OH iii -v i iii ,vii vi w t "TO"' (112) (11 1) viii,vi I .IX. x PhCHzOToH * -I__) xi PhCHzOvo + LiC+OThp I OH RO RO (116) (113) (114) (115) PhCHzOTO+OH vi xii PhCHzO@l I Y A RO RO (117) xiii xiv -HO phcHzov A RO OSi PhpBu' (R = SiPhzBu' ; Thp = tetrahydropyran -2 -yl) Reagents i toluene-p-sulphonyl chloride pyridine acetone; ii LiBEt,H ;iii NaH ; iv ally1 bromide; v chromatography;vi MeOH Amberlite resin IR 118; vii PhCH,Br; viii (PPh,),RhCl; ix ButPh2SiC1 KH; x HC0,H; xi Ag,CO,/Celite; xii H2 Pd/C for 1 hour; xiii camphorsulphonic acid; xiv H2 Pd/C for 24 hours Scheme 14 The synthesis of oleandrose (1 57) and its coupling to form the good yield.Oxidation and debenzylation gave oleandrose (1 57) protected disaccharide grouping of the avermectins has been which was then transformed (as shown in Scheme 21) into the described by WutsS3 (Scheme 21). Swern oxidation of the chiral diprotected avermectin disaccharide moiety (1 59). alcohol (153) followed by treatment with the boronate (154) Finally Nicolaou and co-workerss4 have recently reported a provided a mixture of three olefins (1 55a) (1 55b) and (1 55c) in new synthesis of oligosaccharides from phenylthio-sugars a ratio of 8.7 1.2 1.O which were inseparable on a preparative utilizing glycosyl fluorides. The usefulness of the synthesis is scale. However benzylation and hydroboration of the mixture demonstrated by the preparation of avermectin B1a (5) from its followed by chromatography provided the alcohol (1 56) in aglycon (Scheme 22).104 NATURAL PRODUCT REPORTS 1986 EtoQ EtO (120) (121) HO7-F 1I ii 1 0 Q (1 27) 0 \-Reagents i 0,; ii Me,SiCI; iii BF,.Et,O CH2C12 Scheme 15 (109) (1 29) vi -viii 1 OCH2Ph (131) + Reagents i PhCH2Br BuYNI; ii PPh,H BF;; iii HS[CH2I2SH,TiCI,; iv MeCOCI pyridine; v TI(OCOCF,),; vi BunLi; vii NaOMe; viii HCl (aq.1 Scheme 16 H But Ph 2 Si0 0 + i - iii (1 34) (1 35) (136) Reagents i BunLi THF at -70°C; ii BF,.Et,O THF; iii H+ H,O; iv H, Pd/BaSO, EtOAc pyridine; v BuYNF THF Scheme 17 NATURAL PRODUCT REPORTS. 1986 H. G. DAVIES AND R. H. GREEN ~ ?SiMezBut OH I A A But MezSiO OCHZPh OCHZPh (1 37) (1 38) (139) lii-A OCHZPh OH OH OH lvi I A OCHZPh (141) Reagents i Bu"Li; ii Amberlyst resin MeOH; iii BuflNF; iv H2 Lindlar catalyst; v camphorsulphonic acid; vi Bu'OCI; vii Bu",nH toluene Scheme 18 CH=CHNOz Me0 H Me0 Me0 iii -vi + ; '0 I BZO OBz OC Ph3 (142) (143) (144) vii ,viii 1 Me0 xii ,xiii ix -xi f----OH HO (147) (1 46) (145) Reagents i MeLi; ii PhNCO Et,N benzene heat; iii Hz nickel; iv LiAIH,; v Ph,CCl; vi MeSO2C1; vii NaOAc HMPA at 100 c';viii camphorsulphonic acid; ix Bu'COCI; x 0.5% H2S0,; xi NaBH,; xii TsCl pyridine; xiii (COCl), Me,SO Scheme 19 6.3 The Interconversion of the Averrnectins and Milbernycins into a milbemycin-like Structure it is necessary to dehydroxy-A principal difference between the avermectins and milbemy-late the avermectin selectively at C-13.This transformation has cins is the presence of the disaccharide group at C-13 in the been accomplished by Mrozik and co-w~rkers,~-~~ as shown in avermectin series the milbemycins being unsubstituted at this Scheme 23. 22,23-Dihydroavermectin B 1 aglycon [( 160a) and position. Hence in order to transform an avermectin aglycon 160b)] which is readily available as an 85 15 mixture of 25-s- 106 NATURAL PRODUCT REPORTS. 1986 butyl :25-isopropyl homologues,s6 was treated with t-butyldi- methylsilyl chloride; only the reactive 5-allylic hydroxyl group was protected. The resulting mixture was separated chromato- graphically to give the homologues (161a) and (161b).The reaction of the 13-hydroxy-group of (1 6 1a) with 2-nitrobenzene- sulphonyl chloride in the presence of 4-(dimethy1amino)pyrid-ine and ethyldi-isopropylamine afforded the 13p-c hloro- derivative (162a) presumably by displacement of the intermediate 13a-(2-nitrobenzenesulphonate) ester with the (148) Me0 OMe 0 Me? C02Me C02Me C02Me (1 49) I + '. 0 0 OH \ ji I (150) (151) (152) Reagents i NaOH MeOH; ii H' Me,CO; iii NaBH(OAc), AcOH Scheme 20 PhCH20 H +-MeOmB{:X -OH OH OH (1 54) (155a) (155b) (155c) \-iv PhCHzO\ H OMe &CH20H A OCHZPh I M e0 Me0 (156) Me? vocoMe Me0 PhCH200 X-+ Me? ... VIII ix - (158a) + Me0 I _vli LYHHO (157) (159) PhCH20 Reagents i (COC1)2 Me2SO; ii KH PhCH,CI; iii 9-borabicyclo[3.3.llnonane HzOz;iv chromatography; v pyridinium chlorochromate; vi Hz Pd(OH)?/C; vii PhCH,OH HCl; viii Et,N AczO 4-(dimethylamino)pyridine,pyridine; ix di(2-pyridyl) disulphide Bu;P CHzCI,; x Pb(C1O,)z (1 58a) Scheme 21 NATURAL PRODUCT REPORTS 1986 H. G. DAVIES AND R. H. GREEN ~~ ButMeZSiO,. OMe II OMe But Me *Si Oh But MezSi0 I + + 'SPh Oh.. A SPh Ill IV / "b.-oh.. OH (5) Reagents I AgClO, SnCI, 4A molecular sieves; ii Et2NSF, N-bromosuccinimide; iii 5-(t-butyldimethylsilyloxy)avermectin Bla aglycon; IV Bu;NF Scheme 22 HO HO I + OSi Me2But (162) a; R = Et (160) a; R = Et (161)a; R = Et b;R= Me b; R = Me b; R = Me (163) R = Et (164) (19) R = Me Reagents i.Bu 'Me S i C' 1.1 m I d;I zole i i.2-n it roben zenesu I phony1 chloride 4-(dimethylamino)pyridine Pr>NEt;iii Bu?SnH toluene; iv toluene-p-sulphonic acid. MeOtl Scheme 23 available chloride ion. Hydrodechlorination with tri-n-butyltin hydride followed by desilylation gave the desired 1 3-deoxy-compound (1 63). The isopropyl analogue (161 b) was similarly converted into the 13-deoxy-compound (19). A compound of structure (19) has been isolated from a milbemycin-producing organism and named milbemycin D.7.'h.57 An isomer (164) was also obtained in low yield from the reduction of (1 62a) by tri- n-butylin hydride but was not isolated until after desilylation had occurred at C-5.Avermectins of the 1-series (those possessing a C-22-C-23 double-bond) are also readily available from the 2-series of compounds by the reaction of the 23-hydroxy-group of the 5-protected avermectin (1 65) with 4-methylphenyl chlorothio- formate followed by pyrolytic elimination of the thiocarbonate moiety (Scheme 24).58+s9The thiocarbonate moiety can also be reductively removed from (1 66) with tri-n-butyltin hydride to give after deprotection 22,23-dihydroavermectin B 1 a (1 67). This material which is the major component of Ivermectin is also readily obtainable by catalytic reduction of avermectin B1a (9,using W ilkinson's homgeneous catalyst [tris(tri- phenylphosphine)chlororhodium].56 A small quantity (3%) of 3,4,22,23-tetrahydroavermectinB 1a (168) was also obtained in this reduction.The conversion of avermectins of the A-series into the B-series of compounds requires cleavage of a methyl ether bond. Conventional procedures for this transformation are of no use due to the presence of other ether linkages in the molecule and the instability of the avermectins to acid. However treatment of avermectins of the A-series [such as A2a (3)](Scheme 25) with mercuric acetate in toluene effects the isomerization of the ally1 ether (3) into a vinyl ether (169) which can be subjected to mild acid hydrolysis to give a high yield of the S-keto-compound (170).60 During the isomerization some of the OMe w 0 Me OR (165) R = COCH20SiMezBu' R' 0 .. NATURAL PRODUCT REPORTS. 1986 required ketone is produced but this need not be isolated at this stage.The reduction of this ketone (I 70) with sodium borohydride stereospecifically gives the 5~-hydroxy-compound (7) i.e. avermectin B2a.h1 The hydroxylation at C-13 of milbemycin derivatives offers the possibility of introducing further substituents into the skeleton. Allylic bromination of milbemycin a? (10) with N-bromosuccinimide (Scheme 26) affords the 13-bromo-deriva- tive (171) which is converted into the 13-acetoxy-compound (172); subsequent hydrolysis yields 13-hydroxymilbemycin a? (173).(" Sugar derivatives at C-13 of the milbemycins can then be readily obtained by coupling with an acetohalo-sugar followed by deacetylation with methanolic ammonia.h3 13-(D- Glucopyranosyloxy)milbemycin a? and 13-(a-~-rhamno-pyranosy1oxy)- and 13-(~-oleandrosyl-a-~-oleandrosyloxy)-mil-bemycin a have been prepared in this way.Similar alkylations of milbemycin a furnished 5-0-sugar derivatives. A variety of sugar derivatives of the avermectins have also been prepared using similar conditions.hJ 6.4 Protection and Acylation Depending on which series of the avermectins is being considered there can be up to four hydroxyl groups in the molecule. Thus from the point of view of performing chemical manipulations of any of the avermectins it is necessary to be able to distinguish between each of the hydroxyl groups. All of the avermectins possess a tertiary alcohol function at C-7 and an equatorial secondary alcohol at C-4" of the oleandrosyl moiety whilst avermectins of the 2-series also possess a more hindered axial secondary alcohol group at C-23.Additionally avermectins of the B-series possess a reactive allylic hydroxyl group at C-5. ocso 1-J iii iv ___) A A OH (1671 (168) (R' = ct -L -oleandrosyl-ct -L -oleandrosyl ) Reagents i ClC(S)O(p-MeC,H,) pyridine; ii heat at 200°C; iii toluene-p-sulphonic acid MeOH; iv NaOMe MeOH ; v Bu;SnH azobisisobutyronitrile toluene; vi (PPh,),RhCl Scheme 24 NATURAL PRODUCT REPORTS. 1986 -H. G. DAVIES AND R. H. GREEN I09 OH OH OH I I ! I RO \ RO. + , I ____) OMe OMe OH I OH (R = a-L -oleandrosyl -OL -L-oleandrosyl ) (7) Reagents i Hg(OAcIz toluene; ii (C02H12,THF. MeOH (aq.);iii NaBH Scheme 25 In order to distinguish between the reactivities of each of these hydroxyl groups an exhaustive examination of their behaviour towards acetic anhydride has been carried out.hi Mild treatment of avermectin A2 (3)* with acetic anhydride in pyridine (at 0 “C for 5 hours) afforded only the 4”-acetate (174) in high yield.However more forcing conditions (100 “C for 2 hours) gave a quantitative yield of the 4”,23-diacetate (175); the 23-monoacetate (176) was obtained by careful hydrolysis if the diacetate (175) by a base. Avermectin B1 (5)* undergoes acetylation at 0°C during 4 hours to afford approximately equal amounts of the 4”-monoacetate (177) and the 4”,5-diacetate (178); only a small quantity of the 5-monoacetate (1 79) was obtained from this reaction.Similar acetylations at C-4” of the avermectins and at C-4’ of the avermectin monosaccharides have been described in the patent literature.hh Avermectin B2 (7)* which incorporates all four of the aforementioned hydroxyl groups gives after heating for 2 hours with acetic anhydride the expected triacetate (180). Prolonged heating (for 24 hours) of the reaction mixture affords the triacetate (180) along with an approximately equal quantity of the diacetate (181). A small quantity of this aromatic derivative (181) was also observed when avermectin A2 was subjected to the same forcing conditions; the major product from this reaction was the 4”,23-diacetate. Selective protection of the hydroxyl group at C-5 of avermectin B1 (5)has been successfully achieved by treatment with t-butyldimethylsilyl chloride in NN-dimethylformamide * see footnote on page 87.to give the silyl ether (182) (7003 and the 4”,5-bis-silyl derivative (1 83) ( 1 When avermectin B2 (7) was subjected to the above treatment but for a much longer period (10 hours) the major product was the 4”,5-di-(t-butyldimethylsilyloxy)-derivative (1 84).59In both cases the protecting groups are readily removed by the action of methanolic toluene-p-sulphonic acid. In contrast t-butyldimethylsilyloxyacetyl chloride shows no selec-tivity in its action at C-4” and C-5 avermectin B2a (7) being converted in good yield into the 4”,5-bis-protected derivative (185).s8.h7This protecting group is readily removed in two stages by treatment with methanolic toluene-p-sulphonic acid and sodium methoxide.Avermectin B2a has also been similarly protected ;is its 4”,5-bis-(0-phenoxyacety1)-derivative (1 86).’7.h7In this case de-protection is effected by using ammonia in methanol. A study of 4”-monosubstituted derivatives of avermectin B1 has been made by workers at Merck.h5 Their preparation involved selective protection of the hydroxyl group at (-5 which was carried out by using t-butyldimethylsilyl chloride as described above. Acylation of the hydroxyl group at C-4” followed by deprotection with toluene-p-sulphonic acid fur-nished a range of 4”-monoacylated derivatives (I 87). Although the 4”-hydroxyl group reacted readily with acid chlorides and acetic anhydride it failed to react (under mild conditions) with succinic anhydride; thus in order to prepare a hemisuccinate it was necessary to employ trichloroethylsuccinoyl chloride.The resulting protected hemisuccinate ( 188) after desilylation with toluene-p-sulphonic acid was cleaved with zinc in acetic acid to give the requisite hemisuccinate (I 89). Similarly. potassium isocyanate could not be induced to react with the 110 NATURAL PRODUCT REPORTS. 1986 Br (171) AcO OMe (1 73) (172 1 Reagents i. N-bromosuccinimide CCl, hv; ii NaOAc AcOH; iii NaOH MeOH (aq.) Scheme 26 R’ Oh =h R’oh.. (3) R’= R2= H (5) R’= R2= H (174) R’ = OAC R2= H (177) R’= OAC R2= H (175) R’ = R2 = OAC (178) R’ = R2= OAC (176) R’= H R2= OAC (1 79) R’ = H R2 = OAC OMe R’O..A OMe OR2 I ”o’=oh. (7) R’= R2= R3 = H (180) R’ = R2= R3 = OAC NATURAL PRODUCT REPORTS 1986 H. G. DAVIES AND R. H. GREEN ~ hydroxyl function at C-4” and thus an alternative method of preparation of a carbamate was sought. The reaction of the 5-protected avermectin B 1 (182) with 4-nitrophenyl chlorofor-mate gave the 4-nitrophenyloxycarbonyl derivative (190) in high yield. This material was treated with ammonia or dimethylamine followed by desilylation to give the carba-OMe H ’‘X (5) R’= R2= (182) R’ = H R*= SiMe,Bu‘ I (183) R’= R2= SIMe2Bu’ 0R2 R’O.. OMe OMe mates (191) and (192). A wide range of other 4”-monosubstitut-ed avermectins have been prepared by similar methods.h8 Phosphoric esters of the avermectins have also been described by workers at Merck.69 Bis-(2,2,2-trichloroethyl) phosphorochloridate reacted with avermectins in the presence of ethyldi-isopropylamine and the resulting esters were deprotected using a zinc/silver reagent to furnish the desired phosphates.For example treatment of 22,23-dihydroavermec-tin B1 (167) with one equivalent of bis-(2,2,2-trichloroethyl) phosphorochloridate in the presence of 4-(dimethylamino)-pyridine afforded the 5-phosphate ester (193) in high yield. Deprotection with zinc and silver acetate then gave the required phosphate (194). If an excess of the phosphorochlorid-ate was used reaction occurred at both C-4’ and C-5 to yield the bis-phosphate ester which was deprotected to give the bis-phosphate (195).In order to obtain the 4”-phosphate (196) it was necessary first to protect the hydroxyl group at C-5 (as its t-butyldimethylsilyl derivative) before the avermectin was allowed to react with excess of the phosphorochloridate. OMe R’O.. A OMe LA OR2 (5) R’ = R2= H (188) R1 = CO[CH2I2CO2CH2CCl3,R2 = SiMe2But (189) R’ (182) R’ (190) R’ (191) R’ (1 92) R’ OMe = CO[CH2]2C02HI R2= H = HI R2= SiMe2Bu‘ = (4-N02C6H40)CO R2= SiMe2Bu‘ = HZNCO R2= H = Me2NC0,R2 = H (167) R’ = R2 = H (193) R’ = H R2= P(O)(OCH2CC13)2 ‘’ OH (194) R’ = H ,R2= P(0) (OH) (195) R’ = R2 = P(O)(OH)* (187) R = Bu‘CO Me[CH216CO or MeCONHCH2C0 (196) R’ = P(O)(OH)2 ,R2 = H Deprotection (in standard fashion) gave the 4”-phosphate (1 96).Various 5-substituted milbemycins have been described. The reaction of milbemycin D (19) with acid chlorides in pyridine gives 5-acyloxy-derivatives (197) in high yield (Scheme 27),’O whereas its reaction with a monoprotected dibasic acid or its functional equivalent (an acid chloride or anhydride) provides carboxylic acid derivatives (198).” Also treatment of milbe- mycins with either isocyanates or alkanesulphonyl chlorides provides the corresponding 5-alkylcarbamoyloxy- or 5-alkane- sulphonyloxy-derivatives.72Recently the preparation of 5-carbonates of milbemycins D a3,and us has been described.73 6.5 Oxidations With up to three secondary alcohol functions present in the avermectins it is perhaps not surprising that numerous workers have described studies on selective oxidations.I c-- NATURAL PRODUCT REPORTS 1986 Both avermectins Bla and B2a can be selectively oxidized at the hydroxyl group at C-5 with manganese dioxide to give the corresponding UP-unsaturated ketones.6’ Reduction of each of these enones with sodium borohydride proceeded stereospecifi- cally to regenerate the natural products. By this method tritiation of both avermectins Bla and B2a was achieved using sodium borotritiide as the reducing agent. The oxidation of the hydroxyl group at C-5 of the milbemycins has been carried out by using Collin’s reagent,lh and the stereospecific reduction of the resulting ketone was confirmed. The compound that is epimeric at C-5 was obtained by the route that is shown in Scheme 28.Thus milbemycin D (19) was converted into the mesylate (199),7’ which was subjected to SN2 displacement with tetraethylammonium formate; the resulting formate (200) was cleaved with sodium bicarbonate to give the 5-epimeric alcohol (201). (1 97) (19) (198) Reagents i RCOCI pyridine; ii succinic anhydride pyridine 4-(dimethy1amino)pyridine Scheme 27 .. OS02Me H’ bCH0 (200) Reagents i MeSO,CI Et,N THF; ii Et,N+ HCO?; iii NaHCO Scheme 28 NATURAL PRODUCT REPORTS 1986 H. G. DAVIES AND R. H. GREEN ~ 5-Ketomilbemycin D has been converted into its corres- ponding oxime by treatment with hydroxylamine hydro- ~hloride.’~ In order to oxidize the hydroxyl group at C-23 of avermectin B2a (7) selectively it was first necessary to protect the more reactive groups at C-5 and C-4”.Subsequent oxidation with oxalyl chloride in dimethyl sulphoxide and deprotection afforded 23-ketoavermectin B2a (202) (Scheme 29).27 OH I I A01. 0-OH (7) PhOCH OMe 2coob An alternative preparation of 23-ketoavermectin B2a was performed by initially protecting the hydroxyl groups at C-4” and C-5 with t-butyldimethylsilyloxyacetyl chloride followed by oxidation of the 23-alcohol with pyridinium dichromate. Deprotection as described earlier afforded 23-ketoavermectin B2a (202).67 This patent also contains details for the conversion of the avermectins B2 into 23-ketoavermectins B2 in the presence of soil.OH I I OCOCH20Ph OMe 0 Oh. OH (202) Reagents i PhOCH2COCI pyridine. CH,Cl,; ii (COCI)I Me,SO; iii NH, MeOH Scheme 29 NATURAL PRODUCT REPORTS 1986 Protection of the hydroxyl group at C-5 of 22,23-dihydro- avermectin B 1 (1 67) with t-butyldimethylsilyl chloride followed by Swern oxidation gave the protected 4"-ketone derivative (203). This was deprotected in the usual fashion to give the 4"-oxo-compound (204)7s (Scheme 30). Reductive OMe b \ ,ii (167) i OMe amination of the protected ketone (203) afforded a 3 :1 mixture of the epimeric amines (205) and (206) which were functiona- lized as a dimethylamine a p-chlorobenzenesulphonylamine and an acetamide prior to desilylation. Certain milbemycins (x9 and F) possess an oxygen OMe OMe OMe Reagents i Bu'Me,SiCI imidazole DMF; ii (COCI), Me,SO; iii toluene-p-sulphonic acid MeOH; iv NH,OAc NaBH,CN MeOH Scheme 30 NATURAL PRODUCT REPORTS 1986 -~H.G. DAVIES AND R. H. GREEN functionality (2-carboxypyrrole) on the allylic methyl group at C-416 whereas none of the naturally occurring avermectins has such a substituent. Various avermectins have been converted into their 4-hydroxymethyl analogues by using a catalytic amount of selenium dioxide with t-butyl hydroperoxide as the oxidant (Scheme 31).'" Various oxidations of the 8-9 and 14-15 double-bonds of the milbemycins have been reported in the patent literature. Treatment of 5-ketomilbemycin D (207) with one equivalent of rn-chloroperoxybenzoic acid gave the 14,15-epoxide (208) whilst treatment with excess oxidizing agent gave the 8,9 14,15-bis-epoxide (209)77 (Scheme 32).The 14,15-epoxide of milbemycin D was also prepared in high yield by oxidation with one equivalent of rn-chloroperoxybenzoic whereas the 3,4 :8,9 :14,15-triepoxide is formed if five to six equivalents of the oxidizing agent are used.79 Selective epoxidation of the 8,9-olefin can be achieved by using t-butyl hydroperoxide in the presence of vanadium acetylacet~nate.~~) Similar epoxidations of the avermectins have recently been reported.8 Thus treatment with one equivalent of rn-chloroperoxybenzoic acid for 24 hours at 21 "C affords the 14,15-epoxide whereas one equivalent of oxidizing agent at reduced temperatures yields the 8,9-epoxide.In each of these cases further products were obtained but not characterized. The 8,9-epoxide was also selectively obtained on treatment with vanadium acetylaceton- ate and t-butyl hydroperoxide. It has also been reporteds' that the action of osmium tetroxide on milbemycin D (1 9) furnishes the hexahydroxy- compound (210) as shown in Scheme 33. R' R' 0 I 0' + 6.6 Alkylation Very little work has been reported on the alkylation of the avermectins presumably due to the inherent instability of avermectins to strong base.33 It is reported that the avermec- tins e.g.(167) react with hydroxide ion in aqueous methanol to give two major products. Initially epimerization at C-2 occurs to give (211); as the reaction time increases a new product appears which was shown by n.m.r.spectroscopy to be the A' compound (21 2) (Scheme 34). However alkylation of the avermectins can be successfully achieved by treatment with an alkyl halide in the presence of silver oxide.83 The most reactive site for alkylation is the hydroxyl group at C-5 and thus this constitutes a method of converting avermectins of the B-series into those of the A-series. Alkylation is somewhat slower at both C-4" and C-23 and even the tertiary hydroxyl group at C-7 has been observed to react under these conditions. Interestingly however treatment of avermectins B 1 with diazomethane produces the 7-methoxy-compound as the only isolable product albeit in low yield.Alkylation of milbemycin D under similar conditions (silver oxide and methyl iodide) gave the 5-methoxy-deriva- tives in good yield.8s The hydroxyl group at C-5 of milbemycin D (19) has also been alkylated with ethyl vinyl ether in the presence of pyridinium toluene-p-sulphonate to give the derivative (21 3).85 6.7 Reduction Wilkinson's catalyst which is well known for its ability to saturate cis double-bonds selectively was employed by R'O .. R' = cc -L -oleandrosyl -a-L -oleandrosyl or H X-Y = CH2-CH2 CH=CH or CH2-CH(OH) R2= Me or H c R3= & or o^,(y h H Reagents i SeO, Bu'OOH ; ii R3C02H,diethyl azodicarboxylate PPh3 Scheme 31 .' 0 II I -(2091 (207) (208) Reagents i rn-chloroperoxybenzoic acid (1 equivalent) at 21 'C for 24 hours; ii rn-chloroperoxybenzoic acid (excess) at 21 "C for 5 hours Scheme 32 116 NATURAL PRODUCT REPORTS 1986 (19) (210) Reagents i OsO, pyridine Scheme 33 RON NaOH - + OH (1 67) (211) (21 2) (R = Q -L -oleandrosyl -a-L -oleandrosyl ) Scheme 34 H I I 0 % A OH (19) workers at Merck 8h.X7 in an attempt to reduce the C-22- C-23 double-bond of the avermectins specifically.Thus hydrogena- tion of avermectin Bla (5) in benzene provided 22,23-dihydroavermectin Bla (167) in 8506 yield (Scheme 35) along with 3% of the more fully reduced 3,4,22,23-tetrahydroaver-mectin (214). Under the same conditions avermectin Ala (1) gave only 22,23-dihydroavermectin Ala (215) in 9276 yield. A mixture of 22,23-dihydroavermectins B1 has been assigned the non-proprietary name Ivermectin.I As was mentioned earlier this selective reduction offers a method of converting avermec- tins of the I-series into milbemycin-like structures; only naturally occurring milbemycins possess the fully saturated spiro-ketal system. Reduction of avermectins B 1 with W ilkin-son's catalyst in the presence of tritium gas provides a means of producing radiolabelled Ivermectin.'" Triethylsilane with rhodium or ruthenium as a catalyst selectively reduces the 3-4 double-bond of 5-ketomilbemycin D.S8 (213) 6.8 Avermectin Aglycons and their Monosaccharide Analogues A study of the cleavage of the a-L-oleandrosyl-a-L-oleandrosyl moiety of the avermectins has been carried out by workers at Mer~k.~~~~~ Methanolysis of avermectin A2 (3) or B2 (7) with 1% sulphuric acid in methanol (Scheme 36) readily cleaved the 2-deoxy-sugar glycosides to give the aglycon (216) or (217) respectively in good yield.Substitution of the bulkier propan- 2-01 for the alcoholysis resulted in selective cleavage of the inter-saccharide bond and gave the monosaccharide analogue (218) or (219) in high yield. In contrast methanolysis of avermectins of the 1-series (those possessing a double-bond between C-22 and C-23) under similar conditions did not yield pure aglycons. The reaction proceeded to a large extent with the addition of methanol to the double-bond to yield mainly a mixture of 23a- and 230- methoxy-aglycons (220) and (221) along with a small amount of the required product (222) (Scheme 37).In order to obtain reasonable quantities of the aglycon it was found to be NATURAL PRODUCT REPORTS 1986 H. G. DAVlES ,AND R. H. GREEN OMe OR (5)R = H (1) R = Me OMe H OMe + (167)R = H (214) R = H (215) R = Me Reagents i (PPh,),RhCl H, benzene Scheme 35 necessary to employ a solution of 10% concentrated sulphuric acid in 50% aqueous tetrahydrofuran when a 2 :1 mixture of the aglycon (222) and the monosaccharide analogue (223) was obtained. 7 Structure-Act ivity Relationships Testing in tiw a laboratory assay has revealed all eight major naturally occurring avermectins to be potent anthelmintic agents but there are considerable variations in potency.56 Compounds of the B-series (possessing a 5-hydroxy-group) are generally more potent than those of the A-series (possessing a 5-methoxy-group).Variations in potency between members of the 1-series (22-23 double-bond) and the 2-series (hydroxyl group at C-23) are slight with members of the 1-series being marginally more active; differences in activity between members of the a-series (s-butyl at C-25) and the b-series (isopropyl at C-25) are infinitesimal. Studies of a range of avermectin analogues demonstrated that reduction of the 22-23 double-bond has little effect on anthelmintic activity; indeed 22,23-dihydroavermectin B 1 is fully as active as avermectin Bla in many species,9’ while additional reduction of the 3-4 double-bond has a deleterious effect on activity.Monosaccharides of the aglycons of avermectins B1 and B2 and of 22,23-dihydroavermectins B1 have only half to one-quarter the activity of the corresponding avermectins while 22,23-dihydroavermectin B 1 aglycon is only one-thirtieth as active as the corresponding disaccharide. Acetylation of the 4”-position (on the terminal disaccharide moiety) of avermectin A2 or B1 gave a compound that was equi-active with its precursor (see ref. 91 for reference to the activity of 4”-O-acetylavermectin Bl) while acetylation of avermectin A2 at C-23 or bis-acetylation at positions 4” and 23 reduced the activity.65 The triacetate of avermectin B2 is only one fortieth as active as the parent compound while avermectin B1 5-0-acetate and 4”,5-di-O-acetate are very much less active than avermectin Bla and 5-t-butyldimethyl- silylavermectin B2a is virtually inactive.The conclusion must be that a free hydroxyl group at C-4” is not essential for activity while the hydroxyl groups at C-5 and C-23 are necessary. Other 4”-substituted derivatives of avermectin B 1 were prepared in order to modify the highly lipophilic character of the 118 NATURAL PRODUCT REPORTS 1986 (3) R = Me (7) R H J (216) R = Me (218) R = Me (217) R = H (219) R = H Reagents i. 1% H,SO, MeOH; ii 1% H2S0, Pr'OH Scheme 36 avermectins and to assess the effect on biological activity. The hemisuccinate carbamate and acetylaminoacetate were as active as the parent avermectin B2a while the lipophilic octanoate and pivaloate were considerably less active.In conclusion natural avermectin B2a and its 4"-O-acetyl derivative are the most active compounds. An observation which was to assume critical importance for the commercial future of avermectins was that the avermectins B1 are more active if orally administered than avermectins B2 while the converse is true of parenteral administration. Furthermore avermectins B 1 are much less active against species of Cooperiu when dosed parenterally and avermectins B2 have generally lower activity against Huenronchs species.'" As it was assumed that this effect was correlated with changes in the conformation of the outermost spiro-ketal ring a logical target analogue was the 22,23-dihydroavermectins B1 which have the conformation of the 2-series but which lack the hydroxyl substituent at C-23 that may be responsible for the variation in bioactivity between members of the two series.65 These compounds proved to be equi-active with the avermec- tins B1 but possessed both a better safety profile and the expected efficacy upon both oral and parenteral administration -a rare case of two and two making four! On these grounds a mixture of 22,23-dihydroavermectins B la and B 1 b now given the non-proprietary name Ivermectin was selected for com- mercial development and introduced as an antiparasitic agent in 1981.This dosage form contains at least 80% of 22,23-dihydroavermectin B1 a with the residue being 22,23-dihydro- avermectin B 1b.A discussion of the mode of action of avermectins with a summary of their veterinary efficacy would be beyond the scope of this review but several reviews of this topic have been published.',"' ')') Ivermectin has gained an important and well- deserved place in the treatment of many intestinal and extra- intestinal nematodes and arthropods and is licensed in many countries for veterinary use. An important pointer to the future of Ivermectin in the human clinic lies in the report of its preliminary testing against Onchocercu Z~O/Z~O/US,'~~~' which is the causative agent of River Blindness. A single oral dose of 50 pg per kg of body weight eliminated microfilaria in 75% of lightly infected sub-jects,'O'.'O' which is a startlingly high degree of efficacy.Further work on more heavily infected subjects has confirmed Ivermectin as a potent microfilaricidal agent in Onchocerciasis is a major scourge in the Third World; until now it could only be treated effectively with diethylcarbama- zine and suramin -drugs which are chillingly described as causing 'severe side-effects including death'!'"' These are NATURAL. PRODUCT REPORTS 1986 H. G. DAVIES AND R. H. GREEN ?Me I HO.. (220) + OMe (221 1 + Reagents i. I" preliminary results but if further work confirms and extends these results this would represent a major advance in the treat men t of h u m;I n parasitic infect ions. 8 Conclusion The commercial advantage to be gained from such powerful antiparasitics as avermectin and milbemycin ensures their place in the forefront of industrial research for years to come.It can confidently be expected that this will lead to further advances in macrocyclic chemistry. OMe OH (5) I ii J. Hob.. (223) + HO .. OH (222) H2S0, MeOH; ii H2S0, THF (aq.) Scheme 37 It is to be hoped however that the greatest advantage of these antiparasitic agents will be felt in the Third World. The easing of the parasitic burden should provide both higher- yielding livestock and healthier people with a concomitant improvement in the health of poor agrarian economies. AckrzoIr,l~ci~emmts; The authors would like to thank Dr S. M. derivatives Roberts for his invaluable assistance in the preparation of this manuscript.We also gratefully acknowledge the help of Miss J. Bradshaw and Dr L. E. Clough for literature searches and Dr A. P. Tonge for the computer-generated drawings. 120 9 References 1 W. C. Campbell M. H. Fisher E. 0. Stapley. G. Albers-Schonberg and T. A. Jacob Scinicr 1983 221 823. 2 B. H. Arison R. T. Goegelman and V. P. Gullo (Merck and Co. Inc.) U.S. P. 4285963 (1981). 3 H. Mrozik J. C. Chabala P. Eskola A. Matmk F. Waksmunski M. Woods and M. H. Fisher Tmwheclroti Lett. 1983 24. 5333. 4 H. Mishima M. Kurabayashi C. Tamura S. Sato H. Kuwano. A. Saito and A. Aoki 'Structuresof Milbemycinsa, r?,a, rl a, r,, x7 'rx,x~,, a,,,and PI'. Abstract Papers 18th Symp. Chem. Natural Products Kyoto 1974.p. 309. 5 T. Okazaki M. Ono A. Aoki and R. Fukuda J. Atltihiot. 1983 36 438. 6 Y. Takiguchi H. Mishimu M. Okuda and M. Teruo J. Atitihiot. 1980. 33 1 120. 7 Y. Takiguchi. M. Ono S. Muramatsu J. Ide H. Mishimu and M. Terao J. Atirihiot. 1983. 36 502. 8 M. Ono H. Mishima Y. Takiguchi and M. Terao J. Atitihiot. 1983 36 509. 9 R. W. Burg B. M. Miller E. E. Baker J. Birnbaum S. A. Currie. R. Hartman Y.-L. Kong R. L. Monaghan. G. Olson I. Putter J. B. Tunac H. Wallick E. 0.Stapley R. Oiwii. and S. Omura Atitiniicroh. Agetits Chmiothrr. 1979 15 361. 10 G. Albers-Schonberg H. Wallick R. E. Ormond T. W. Miller and R. W. Burg (Merck and Co. Inc.) US. P. 4429042 (1984). I I T. W. Miller L. Chaiet D. J. Cole L. J. Cole J. E. Flor. R. T. Goegelman V.P. Gullo H. Joshua A. J. Kempf. W. R. Krellwiti R. L. Monaghan R. E. Ormond K. E. Wilson G. Albers-Schonberg and I. Putter. Atitimicwh. Agtvits C'hcmothcr. 1979 15 368. 12 R. F. Ormond (Merck and Co. Inc.) Eur. Pat. Appl. 73660 (1983). 13 R. T. Goegelman V. P. Gullo and L. Kaplan (Merck and Co. Inc.) Eur Pat. Appl. 58518 (1982). 14 R. T. Goegelman V. P. Gullo and L. Kaplan (Merck and Co. Inc.) US. P. 4378353 (1983). 15 H. Mishima. M. Kurabayashi C. Tamura S. Sato H. Kuwano and A. Saito Tctruheclron Lett. 1975 71 I. 16 H. Mishima J. Ide S. Muramatsu and M. Ono J. Atitihiot. 1983 36 980. 17 J. A. Mills J. Chrni. SOL'..1952 4976. 18 T. W. Miller and W. C. Randall personal communication reported in ref. 17. 19 J. P. Springer B. H.Arison J. M. Hirshfield and K. Hoogsteen. J. Am. Chem. Soc. 1981 103 4221. 20 CHEMGRAF program suite 0 E. K. Davies Chemical Crystallography Laboratory University of Oxford 1982. 21 G. Albers-Schonberg B. H. Arison J. C. Chabaln A. W. Douglas P. Eskola M. H. Fisher A. Lusi H. Mrozik J. L. Smith and R. L. Tolman J. Am. Chmi. Soc. 1981 103 4216. 22 M. Ono H. Mishima Y. Takiguchi and M. Terao J. Antihior. 1983 36 991. 23 D. E. Cane T.-C. Liang L. Kaplan M. K. Nallin M. D. Schulman 0. D. Hensens A. W. Douglas and G. Albers-Sehonberg J. Am. Chem. Soc. 1983 105 4110. 24 C. C. Ku S. C. Hwang L. Kaplan M. K. Nallin andT. A. Jacob. J. Luhelled Conipd. Rutiiophurni. 1984 22 45 I . 25 C. C. Ku S. C. Hwang and T. A. Jacob J. Liq. Chrotmtogr. 1984 7 2905.26 I. Putter J. G. MacConnell F. A. Preiser A. A. Haidri S. S.. Ristich and R. A. Dybas E.\-porietitiu 1981 37 963. 27 V. P. Gullo A. J. Kempf J. G. MacConnell H. Mrozik B. Arison and I. Putter Pesfic Sci.. 1983 14 153. 28 D. L. Bull G. W. Ivie J. G. MacConnell V. F. Gruber C. C. Ku B. H. Arison J. M. Stevenson and W. J. A. VandenHeuvel J. Agric. Food Chrni. 1984. 32 94 see also D. L. Bull Southwst. Etitonrol. 1985 Suppl. No. 7. p. 2. 29 G. T. Miwa J. S. Walsh W. J. A. VandenHeuvel B. H. Arison E. Sestokas R. Buhs A. Rosegay S. Avermitilis A. Y. H. Lu M. A. R. Walsh R. W. Walker R. Taub and T. A. Jacob Drug Merab. Dispos. 1982 10 268. 30 S.-H. L. Chiu E. Sestokas R. Taub J. L. Smith. B. Arison and A. Y. H. Lu Drug Metuh. Dispos. 1984 12 464.31 J. W. Tolan P. Eskola D. W. Fink H. Mrozik. and L. A. Zimmerman J. Chromutogr. 1980 190 367. 32 P. C. Tway J. S. Wood and G. V. Downing J. Agric. Food Chem. 1981 29 1059. 33 J. V. Pivnichny J.-S. K. Shim and L. A. Zimmerman J. Phurni. Sci. 1983 72 1447. 34 H. J. Schnitzerling and J. Nolan J. Assoc. On. Atid. Chcnl. 1985. 68. 36. NATURAL PRODUCT REPORTS. 1986 35 S.-H.L. Chiu R. P. Buhs E. Sestokas R. Taub. and T. A. Jacob. J. Agric. Food C'hwi.. 1985. 41 99. 36 P. C. Tway G.V. Downing. J. R. B. Slayback G. S. Rahn and R. K. Isensee Bioniid. Muss Spc~.trorn.,1984 I I 172. 37 A. B. Smith S. R. Schow J. D. Bloom A. S. Thompson and K. N. Winrenberg .I. Am. C'honi. SOL...1982 104 4015. 38 D. R. Williams B. A. Barner K. Nishitani and J.G. Phillips J. Am. C'hmi. Soc.. 1982. 104 4708. 39 M. J. Hughes E. J. Thomas M. D. Turnbull R. H. Jones and R. Warner J. C'hcwi. Sot.. C'hcwi. C'onit?iutr.. 1985. 755. 40 S. V. Attwood. A. Ci. M. Barrett and J.-C'. Florent J. C'hcni. Soc. C'hrv?l. ~~ot?lt?llm.. 198I. 556. 41 S. Hanessian and A. Ugolini C'urhohjdr Rc.v.. 1984. 130 261. 42 R. Baker R. H. 0.Boyes D. M. P. Broom J. A. Devlin and C. J. Swain .J. Chcwi. Soc...C'hcni. Coniniuti. 1983. 829. 43 R. B. Ward. Methods Crirhoh,dr. C'hrwi. 1963 2. 394. 44 P. Kocienski and S. D. A. Street J. C'hcwr. So(... Chcwi. Coninrun. 1984 57 I. 45 J. Godoy S. V. Ley and B. Lygo J. ('hm. So(..,C'hc~.Cbtnniutz. 1984 1381. 46 A. G. Kelly and J. S. Roberts C'urhohjdr. Rvs. 1979 77 231 47 S. Hanessian A.Ugolini and M. Therien J. Org. Chcwi. 1983,48 4427. 48 R. Baker C. J. Swain. and J. C'. Head J. Chm. SOC.,Chc~r. Coninnui. 1985 309. 49 M. Prashad and B. Fraser-Reid J. Org. Cheni. 1985 50 1564. 50 T. Mukaiyama and T. Hoshino J. An?.Chcnr.SOL'.,1960,82 5339. 51 M. E. Jung and L. J. Street J. ,401. Chm. Soc. 1984 106 8327. 52 M. D. Turnbull G. Hatter. and D. E. Ledgerwood Tetrcrhcdroti Lrtt. 1984. 25 5449. 53 P. G. M. Wuts and S. S. Bigelow. J. Org. Chmi. 1983 48 3489. 54 K. C. Nicolaou R. E. Dolle D. P. Papahatjis. and J. L. Randall J. An?.C'hwi. Soc. 1984. 106 41 89. 55 J. C. ('hahala M. H. Fisher. and H. H. MroJik (Merck and Co. Inc.) Eur. Pat. Appl. 2615 (1979). 56 J. C. Chabala H. Mroik. R. L. Tolman P. Eskola A. Lusi L. H. Peterson M.F. Woods and M. H. Fisher J. Mcti. Chem. 1980 23. 1134. 57 Y. Takiguchi. H. Mishima S. Yamamoto and M. Terao (Sankyo Co. Ltd.) Ger. Often. 3031 756 (1981 ). 58 H. Mrorik P. Eskola and M. H. Fisher. Tetruheciron Lrtt. 1982. 23. 2377. 59 H. H. Mrozik (Merck and C'o. Inc.) U.S. P. 4328335 (1982). 60 H. H. Mrorik (Merck and Co. Inc.) U.S. P 4423209 (1983). 61 J. C. Chabala A. Rosegay and M. A. R. Walsh J. Agric. Food Chrni. 1981 29 881. 62 M. H. Fisher (Merck and Co. Inc.) U.S. P. 4093629 (1978). 63 M. H. Fisher and R. L. Tolman (Merck and Co. Inc.) Br. P. I579 118 (1980). 64 M. H. Fisher and R. L. Tolman (Merck and Co. Ine.) Eur. Pat. Appl. 7812 (1980). 65 H. Mrozik P. Eskola M. H. Fisher J. R. Egerton S. Cifelli and D. A. Ostlind J.Med. Cheni. 1982 25 658. 66 Merck and Co. Inc. Jpn. Kokai Tokkyo Koho 54 61 197 (1979). 67 H. H. Mrozik J. G. MacConnell and A. J. Kempf(Merck and Co. Inc.) U.S. P 4289760 (1981). 68 H. H. Mrozik M. H. Fisher P. Kula and C. Albers-Schonberg (Merck and Co. !nc.). Eur. Pat. Appl. 1688 (1977). 69 H. H. Mroiik (Merck and Co. Inc.). Fur. Pat. Appl. 115930 ( 1984). 70 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 59 16894 (1984). 71 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 5920285 (1984). 72 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 57 139081 (1982). 73 Sankyo and Co. Ltd. Eur. Pat. Appl. 142969 (1985). 74 Sankyo and Co. Ltd.. Eur. Pat. Appl. I10667 (1984). 75 H. H. Mrozik (Merck and Co. Inc.) Eur. Pat. Appl. 89202 (1983). 76 H. H. Mrozik (Merck and C'o.Inc.). Eur. Pat. Appl. 74758 (1982). 77 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 5933288 (1984). 78 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 57 139080 (1982). 79 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 5936681 (1984). 80 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 57 139079 (1982). 81 H. H. Mrozik (Merck and Co. Inc.) Eur. Pat. Appl. 136892 ( 1985). 82 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 5936682 (1984). 83 M. H. Fisher A. Lusi and R. L. Tolman (Merck and Co. Inc.) Eur. Pat. Appl. 8184 (1980). 84 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 57 I20589 (1982). 85 Sankyo and Co. Ltd. Jpn. Kokai Tokkyo Koho 5920284 (1984). 86 J. C. Chabala and M. H. Fisher (Merck and Co. Inc.) Eur. Pat. Appl. 1689 (1979). H. G.DAVlES AND R. H. GREEN NATURAL PRODUCT REPORTS 1986 ~ 87 J. C'. C'habala and M. H. Fisher (Merck and Co. Inc.) U.S. P. 4 199339 ( 1980). 88 Sankyo and ('0. Ltd. Jpn. Kokai Tokkyo Koho 5933289 (1984). 89 H. Mro;rik P. Eskola. B. H. Arison G. Albers-Schonberg and M. H. Fisher. J. Org. Ckcni. 1982 47 489. 90 H. H. Mrorik and M. H. Fisher U.S. P. 4206205 (1982). 91 J. R. Egerton. J. Birnbaum. L. S. Blair J. C. Chabala. J. Conroy. M. H. Fisher. H. Mrozik. D. A. Ostlind C. A. Wilkins and W. C. Campbell. Br For. J.. 1980. 136 88. 92 W. c'. ('ampbell R. W. Burg M. H. Fisher and R. A. Dybas AC'S Sjwp. Svr. 1984 255 5. 93 J. K. Hotson J. S. A,fi. Vrt. Axsoc. 1982 53 87. 94 A. Misra and J. C'. Katiyar J. Sci.itid. Rcs. 1984 43 976. 95 H. Koch Phrirtn./tit. 1984. 5 55. 96 W. C'. Campbell Lind G. W. Benz J. Vc)t.Phrirniricol. T/ier(ip. 1984 7. I. 97 W.-H. Wagner. Phrirt?i. itid.. 1984. 46 507. 98 B. Robin R~JI.. Mcd. Vot. (Tou1ou.w).1983 134 495. 99 W. C. Campbell N.Z. Vct. J.. 1981 29. 174. 100 L. B. Townsend and D. S. Wise. Ph(irni. /tit . 1983. 5. 70. 101 M. A. Aziz S. Diallo I. M. Diop and M. Lxiviere. /-utw/.1984 2 171. I02 Editorial Loticet 1984. 2. I021. 103 K. Awadzi K. Y. Dadzie H. Schulz-Key. D. R. W Haddock. H. M. Gilles. and M. A. Azir Anti. Trop. Mrd. Puru.!ifol. 1985 79. 63. 104 B. M. Greene. H. R. Taylor E. W. C'upp R. P. Murph). A. T. White. M. A. A~i7,H. Schulz-Key. S. A. D'Anna.H S.Newland L. P. Goldschmidt C. Auer A. P. Hanson S. V. Frecman. F.. W. Reber.and P. N. Williams Nm. EtiKI. .I. Mod 1985. 313 133.

ISSN:0265-0568    

DOI:10.1039/NP9860300087

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

Sesterterpenoids J. R. Hanson Schoof of Mofecufar Sciences University of Sussex Brighton Sussex BN I 9QJ Reviewing the literature published up to June 1985 1 Introduction 2 Linear Sesterterpenoids 3 Mono- and Bi-carbocyclic Sesterterpenoids 4 Tricarbocyclic Sesterterpenoids of the Cheilanthane TY Pe 5 Scalarane Sesterterpenoids 6 Ceriferene Sesterterpenoids 7 Ophiobolane Sesterterpenoids 8 Miscellaneous Sesterterpenoids 8.1 Gascardic Acid 8.2 Retigeranic Acid 8.3 Heliocide H2 8.4 Stellatic Acid 9 References 1 Introduction The sesterterpenoids are a group of pentaprenyl terpenoid substances whose structures are derivable from geranylfarnesyl diphosphate. This report is a successor to that which appeared in Volume 4 of the Specialist Periodical Reports on Terpenoids and Steroids.' Several other reviews covering the sesterterpenoids have appeared.' As a number of the sesterterpenoids are of marine origin they have been described in reviews devoted to those topic^.^ Although most of the sesterterpenoids have been discovered during the past twenty years their sources are very widespread.Thus they have been isolated from fungi lichens higher plants insects and various marine organisms. Never- theless not all C, natural products are sesterterpenoids. For example there are a group of meroterpenoids such as austin which are derived from a sesquiterpenoid (C,,) and a substi- tuted tetraketide (C,(])moiety and there are also a number of degraded triterpenoids which are C15compounds.The latter have structures embodying the head-to-head prenyl linkage that is characteristic of a biosynthesis involving squalene. The carbon skeleta of the sesterterpenoids reveal examples of the two main modes of cyclization of isoprenoids namely structures arising from acid-catalysed polyene cyclizations that are reminiscent of those by which the higher terpenoids are formed and those that are based on intramolecular alkylation by the terminal pyrophosphate. The latter lead in the first instance to medium and large rings which often undergo further cyclization reactions. In some instances these lead to structures that are prenylated analogues of diterpenoid ring systems. In this review the structures will be covered in a biogenetic sequence.2 Linear Sesterterpenoids A large number of linear sesterterpenoids are known. The simple geranylfarnesol (1) and geranylnerolidol (2) have been obtained from the scale insect Ceroplustes alholineatus6 and from the fungus Cochliobolus heterostrophus,' respectively. The partially saturated analogue (ZZ,6E)-3,7,11,15,19-pentamethylicosa-2,6-dien-1-01is a constituent of the lipids of Solarium tuhero.sum.8 Marine organisms have provided a large group of C2s furans and their Czl degradation products.3 The simple furans are exemplified by furospongin-3 (3) and furospongin-4 (4) from Spongiu oficinali~,~ and by idiadione (9,from Spongia idiu.Io Ircinin-1 (6) and ircinin-2 (7)-which were obtained from the sponge Zrcinia oros contain a second furan ring.Quite a number of these compounds contain a tetronic acid moiety and show strong antibiotic activity against Staphylococcus aureus; they are exemplified by \ ariabilin* (8)'' (from Zrcinia rariabilis) its double-bond isomer strobilinin (9)' (from Zrciniu srrohilinu) and kisciculatin (10)'j (from Zrcinia jasciculura). The location of double-bonds Cti ,OH OH (2) (3) R1=Me R2= C02H (4)R' = COzH R2= Me 0 0 0. OH a 0. OH b b (8) Aa;Z (9) Ab (10) * This should not be confused with the substituted pterocarpan of the same name (also called homopisatin). 124 NATURAL PRODUCT REPORTS. 1986 in 'non-biosynthetic' positions in some of these structures is rather unusual.The co-occurrence of these compounds with a group of CZ1furanoterpenes is indicative of the origin of the latter. Thus ircinin-3 (1 1) and ircinin-4 (12) may arise by cleavage of the open forms of the tetronic acids (see Scheme A related series of compounds [(13) and (14)] and the corresponding C2 carboxylic acids occur in kink dendroides. Many of these Czl furanosesterterpenes contain a furan OH ring at both ends of the molecule as exemplified by anhydrofurospongin-1 (15)' ' and furospongin-1 (16)' * from Spongiu oficinalis. The compounds (17) and (18) have been detected in a species of Spongiu and one of Leiosellu respec-tively," whilst the y-lactone furospongolide (19)?O was ob-(18) tained from Dysideu herbucea. A C2?&lactone furodendin (20) has been reported" in a sponge of the genus 0 0 Carteriospongiu.01 / \ \ H02C (19) 0 *R 3 0 OH 0 J Scheme I 1 HO 0 CHO (22) (14) (15) OH NATURAL PRODUCT REPORTS 1986 J. R. HANSON 3 Mono- and Bi-carbocyclic Sesterterpenoids In some instances a cyclization that is reminiscent of the carotenoids has occurred. This is exemplified by a group of antimicrobial y-hydroxybutenolides which have been ob-tained from Lu@aridu ruriuhilis and include the E-and Z-neomanoalides (2 1),.. secomanoalide (22) and manoalide (23).23Degradation products of these compounds have also been observed. These include the unusual cyclic peroxide muqubilin (prianicin A) (24),'4*'5 from a sponge of the genus Prianos.A more extensively degraded compound methyl nuapapuanoate (25),'h has also been reported. Further cyclization (see Scheme 2) also occurs to give compounds with structures reminiscent of the clerodane diterpenoids. Thus the sesterterpenoid palauolide (26),?7 which is an antimicrobial constituent of sponges represents this structural type. Norsesterterpenoids containing cyclic peroxides were ob-tained from the sponge Sigmosceptrella lurt.is's and are repre- sented by sigmosceptrellin-A (27) sigmosceptrellin-B (28) and sigmosceptrellin-C (29). A diterpenoid labdane cyclization may lead to the unusual plant sesterterpenoid salvileucolide methyl ester (30) and the lactone (311. which were isolated from Salziu hypoleucu (Labia t ae) . " Some rather unusual cyclizations have been observed in the 0 R 125 structures of ircinianin (32)30 and wistarin (33).3 The struc- ture of the former was determined by an X-ray-crystallographic analysis.4 Tricarbocyclic Sesterterpenoids of the Cheilanthane Type The fern Cheilanthes jarinosu afforded two types of sesterterpenoid namely an ophiobolin cheilarinosin (see be- low) and a tricarbocyclic compound cheilanthatriol (34).32 The perhydrophenanthrene carbon skeleton was assigned to ~heilanthatriol,~'" based on dehydrogenation studies and on H n.m.r. spectral evidence. Some conventional synthetic studies directed at perhydrophenanthrenes [t..g.(3S)]that are related to this structure have been reported.33 Several marine natural products that have this carbon skeleton have been detected.These include suvanine (36),34 from a species of Zrcinia which contains a guanidinium residue. Luteone (37) may be the fragrant principle of a Canadian specimen of the dorid nudibranch Cudlinu luteowiurginata.3i Its structure which represents a degradation product of the perhydrophenanthrene skeleton was determined by an X-ray- crystallographic analysis. R I Scheme 2 0 Hoeo &c02H (27) 16S,17S (28)16R 17s (29) 16S 17R (321 CH20H 9 I0 (331 NMe2 I CHNHC-NH CH2CH2CCH3 II . OH OH 0 (34) (37) NATURAL PRODUCT REPORTS 1986 AcO. RO CHO CHO ecHo (38) (39) R = AC (42) (40) R = H 0 0 &O Ac I (43) R = OH (45) (46) (44) R = H &cH2cH21 POH &’R (48 1 (49) R = ( 50) (51) R = ’rf OAc @.[ ‘OAc -I / H (52) R = Me (54) R = Me (53) R = Et (55) R = Et 5 Scalarane Sesterterpenoids Spongiu idiu.These compounds have an ecological role in preventing predation. Furoscalarol (48) which was isolated4’ These are amongst the most common sesterterpenoids from Cocospongicl mollior possesses an unusual arrangement particularly in sponges. They form a closely related series of of the furan ring. The stereochemistry of the hydroxyl group compounds. In a number of instances these sesterterpenoids was determined by Horeau’s method. Not unexpectedly, have also been isolated from a nudibranch that is associated several condensation products of the scalaradials with amines with a sponge and hence the sesterterpenoid in the have been reported.These include the molliorins a (49) b nudibranch may have a dietary origin. Scalarin (38)3h was the (50),and c (51).4’.43 first of these compounds to be isolated from the marine Various homoscalarins that contain extra alkyl groups at sponge C’ncospongiu sccrluris. This sponge contains a number C-19 or C-24 have been obtained from sponges. Thus of dialdehydes which like their simpler sesquiterpenoid ana- extraction of Dj9sidcw herhaccw afforded scalardysin-A (52) logues possess quite distinctive biological activity e.g. as and its homologue scalardysin-B (53) and scalarherbacin-A antifeedant substances. These compounds are exemplified by (54) and its homologue scalarherbacin-B (55) together with scalaradial (39) desacetylscalaradial (40)-and heteronemin their 12-a~etates.~~.~~ A group of 24-methylscalarins [(56)-(41).-37 A number of 12-epimers.particularly 12-cy1-scalaradial (59)] were obtained from a member of the genus (42) 12-cy+scalarin (43) 12-c~~z-deoxoscalari1~ (44) and the Currrriospongia.4h Some rather similar compounds [(60) -(64)] rearranged product scalarolbutenolide (45) were isolated from were found4- in the nudibranch Chromocioris scdna which is Spongin niton.s.38 40 Some similar compounds e.g. associated with these sponges. Another group [(65)-(68)] scalarafuran (46) and scalarolide (47) were obtained from were isolated from a member of the genus Lrnu’twfcddin.48 NATURAL PRODUCT REPORTS 1986 -J.R. HANSON (58)R’ = Me ; R2= a-H ,g -OH ;@=OH (60)R = H (61)R1=H;R2=a-H,p-OH (59) R’ = CH~OAC;R2= 0 ; R3= OAC (64)R = AC (62) R1=OH;R2=a-OAc,b-H 0. (65)R = H (66)R = AC (70b) (71) R* (74)R’ = H ,R2= CH,OH OH (76)R’ = OMe R2= Me (77) Some of these compounds possess anti-inflammatory action and may be inhibitors of aggregation of blood platelets. Anti- inflammatory activity was also shown by foliaspongin (69) which was isolated49 from the sponge Phyllospongia joliascens. The prenylation of phenols and quinones is a common biosynthetic step. A number of sesterterpenoids of this type are known. These are exemplified by disidein (70a) from Dj,sideu pallescwu,sO and by toxistylide A (70b) and its double-bond isomer toxistylide B from Microciona to.uistyla.5 However the biosynthetic origin of the migrations of methyl groups that are required to generate the carbon skeleton seems rather obscure.The structure (70b) nevertheless rests on an X-ray analysis. Some biogenetically patterned cyclizations have been re-ported leading to this series. Thus further cyclization of the furan (71 ) with tin(1v) chloride afforded 12-deacetoxy-16-deoxyfuroscalarol (72).52.s3 The second major group of sesterterpenoids are represented by compounds in which the cyclization is initiated by the HO CHO 0 (70a) (73)R = CH,OH (72) (75) R = C02H CH,OH (78) pyrophosphate leaving group generating a primary carbo- cation which may then attack one of the double-bonds within the prenyl chain.6 Ceriferene Sesterterpenoids The scale insects have afforded a number of sesterterpenoids. Examination of the wax that is secreted by Ceroplustes cerijerus has led to the isolation of a series of macrocyclic sesterterpenoids containing a fourteen-membered ring. The structures of these were originally based on comparisons with the diterpenoid equivalent ~embrene.”.~~ However there was some controversy over the relative disposition of the cis and trans double-bonds in the ring.s6 As a result of synthetic endeavours in this area the structures have been revised.57 59 Ceriferol and ceriferol-1 are now formulated as (73) and (74) respectively. Ceriferic acid (75) is the acid corresponding to ceriferol. 13-Methoxycericerene (76) and cericerol-I I (77) are further members of this series.Albocerol (78) was isolatedho NATURAL PRODUCT REPORTS. 1986 128 from the Mexican species Ceroplastes albolinrutus. The secre- tion of Ceruplustes Jioridc~nsis which infests tangerines has affordedh' another group of sesterterpenoids which include floridenol (79) Sa-hydroxyfloridenol (80) flocerol (8 l) and R' floceric acid (82). The structures of these were based on an X-ray analysis of the 18-ketone that is related to floridenol. A biogenetic scheme accounting for the structures may be as shown in Scheme 3. This contains some features which are reminiscent of the biosynthesis of the triterpenes of the (79) R = H amyrin and lupane series. (80)R = OH The scale insect Cerop1uste.s ruhens which is another para- site on citrus trees contains five novel sesterterpenoids i.e.the cerorubenic acids I (83) I1 (84) and 111 (85) and the alcohols cerorubenol-I (86) and cerorubenol-I1 (87).h2 These compounds are reported to act as kairomones that are respon- sible for the ovipositional behaviour of a parasitic wasp Anicetus bentlficus towards Ceruplustrs rubens. 7 0phiobolane Sesterter penoids Several ophiobolane derivatives have been isolated from the wax of the scale insect Ceroplastes albolineatus which is found on the shrub Seneciu prarco.u. The structure of ceroplastol-I (88) was established by an X-ray analysis.h3 The structures of ceroplasteric acid (89) ceroplastol-I I (90),h' and albolic acid (91)65 were then deduced by chemical correlation.The ceralbic acids I (92) and I1 (93) from the same source, \ A represent further examples of this series."h Some aspects of the oxidative chemistry of the ceroplastanes have been inves- tigated involving the selective cleavage of the side-chain and the nuclear double-bonds."'.hx The ophiobolane structure (94) has been proposed3 Zh for I cheilarinosin which was obtained (along with cheilanthatriol) Scheme 3 from the fern Cheilanthes firrinosa. 'H ,'H / 2RLR H H \ H COZH (83) R = C02H (84)R = COZH (86) R = CHZOH (87) R = CH20H (85) R (90) R = CH20H (91) R = C02H C02H I (94) NATURAL PRODUCT REPORTS 1986 -J. R. HANSON Investigation of the metabolites of a number of phytopatho- genic fungi in clud ing Helm in thosporium leersii H.oryzae* H. turcicum H. zizaniae Cochlioholus heterostrophus and C. miyahranus* led to the isolation of the ophiobolane series of fungal metabolite^.^"^^^) The structures of these compounds were established by an extensive series of degradations and by an X-ray crystal structure. Since some of the compounds were isolated from more than one source and their identity was not immediately realized they were also known as cochliobolins or ziLanins and Cephalosporiuri caerulens yielded cephalonic acid (see Table). The ophiobolins G (100) and H (101) have been obtained recently from Aspergillus u~tus.'~ The structure of ophiobolin G was established by an X-ray analysis.Extensive biosynthetic studies have been carried out on the ophiobolins. Feeding studies,*".* using [2-' T]mevalonate established the positions of the isoprenoid units. Furthermore it was demonstrateds2 that the oxygen atom in ophiobolin A Table Alternative names for ophiobolins Ophiobolin A (9S)t''' ophiobolin,"" co~hliobolin.~' cochliobolin A,72 zizanin7% Ophiobolin B (96) ophiobolosin A.74 ritanin B," cochliobolin B72 Ophiobolin c' (97) tizanin A7*." Ophiobolin D (98) cephalonic acid7"." Ophiobolin F (99)'x (97)R = H H (100) (95) at C-14 was derived from atmospheric oxygen whereas that at C-3 must have come from the medium. The fate of the mevalonoid hydrogen atoms was investigated and this re-vealed a shift of hydrogen from C-8 to C-15 during the biosyn- thesi~.*~-~~ These results can be accommodated in the biosynthetic pathway that is shown in Scheme 4.The carbon-I3 n.m.r. spectra of the ophiobolins and their relatives have been assigned.8h Some aspects of the chemistry of ophiobolin D have been examined.s7.** Pyrolysis of dehydro-ophiobolin D (102) afforded8* a mixture of products (l03)-( 105). Treatment of (105) with potassium t-butoxide gave a tetracyclic product (l06) containing four fused five- membered rings. The structures (105) and (106) were estab- lished by X-ray analysis.89 The unusual 5 :8 :5 tricyclic ring system of the ophiobolins has resulted in a number of synthetic studies."" ('.i The synthetic challenge not only involves the construction of the angularly fused dicyclopentacyclo-octane backbone but also the control of six chiral centres on the ring system.Most of the strategies have involved a ring-enlargement sequence to generate the central eight-membered ring [e.g. (1 07) + ( 1 OS)]. 8 Miscellaneous Sesterterpenoids 8.1 Gascardic Acid The structure of gascardic acid (1 09) which was obtained from the insect Gascardia rnadqqmxzriensi.y was originally established by extensive chemical degradation; it was one of the first sesterterpenoids to be described."5."" The structure has now been confirmed by an X-ray-crystallographic analy- sis."' A synthesis of gascardic acid has been reported"8 in which the key steps involve the conversion of (1 10) into the aldehyde (1 11) and thence into the ester (1 12).The synthesis HO ' (101) '34 Scheme 4 NATURAL PRODUCT REPORTS 1986 I (102) (103) (104) 4T C02Me was complemented by cyclization to (1 13) and standard hydrindanone (1 15) formed the starting point and this was procedures for modification of the l'unctional groups. elaborated [ria the aldehydic ester (1 16)] to the acid (1 17) and thence ricr the corresponding ketene to the cyclobutanone (1 18). Modification of ring E and then ring c afforded 8.2 Retigeranic Acid retigeranic acid. Retigeranic acid (1 14) was obtained from various fiimalayan iichens such as hharia rc.tigciru. Its structure was established by an X-ray analysis."" Routes to the synthesis of the 8.3 Heliocide H2 triquinane portion of the molecule have been examined.Iieliocide Id2 (1 19) has been obtained'"' from the pigment Very recently a total synthesis has been described. I0I The glands of Go.s.si~piimihirsutuni (cotton) in which it shows NATURAL PRODUCT REPORTS 1986 -J. R. HANSON CHO (320 &cozMe (11 5) (1 16) &ycozH (11 7) (118) Hb &-HR / (120)R = C02H L- (121) Scheme 5 insecticidal activity against the cotton bollworm. It is not strictly a sesterrerpenoid in the sense that its structure is not readily derivable from geranylfarnesyl diphosphate and in- deed it appears to arise as the Diels -Alder adduct of myrcene and hemigossypolone both of which occur in cotton. 8.4 Stellatic Acid Stellatic acid (1 20) was isolated from Aspurgillzrs ste//atus and its structure obtained by an X-ray analysis.'03 It is structurally closely related to floceric acid (82) and is presumably derived from (1 21) which is the expected primary cyclization product from all-trrrns-geranylfarnesyl diphosphate if it is folded as shown in Scheme 5.9 References 1 J. R. Hanson in 'Terpenoids and Steroids' ed. K. H. Overton (Specialist Periodical Reports) The Chemical Society London 1974 Vol. 4 p. 171. 2 G. A. Cordell Phytochemistry 1974 13 2343. 3 D. J. Faulkner Tetrahedron 1977 33 1421. 4 D. J. Faulkner Nut. Prod. Rep. 1984 I 251 ; 1984 I 551 ; 1986 3 I. 5 L. Minale G. Cimino S. de Stefano and G. Sodano Fortschr. Chem. Org. 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R. Jefferies. E.vperimtia 1982 38 1444. 20 Y. Kashman and M. Zviely E.uperientiu 1980. 36 1179. 21 R. Kazlauskas P. T. Murphy and R. J. Wells E.uprricwtia 1980 36 814. 22 E. D. de Silva and P. J. Scheuer Trtruheclron Lett.. 1981 22 3147. 23 E. D. de Silva and P. J. Scheuer Tetrahedron Lett 1980 21 1611. 24 Y. Kashman and M. Rotem Tetrahedron Lett. 1979 1707. 25 S. Sokoloff S. Halevy V. Usieli A. Colorni and S. Sarel E.perientia 1982 38 337. 26 L. V. Manes G. J. Bakus and P. Crews Terrahrciron Lett.1984. 25 931. 27 B. Sullivan and D. J. Faulkner Tetraheilrun Lett. 1982 23 907. 28 M. Albericci M. Collart-Lempereur J. C. Braekman D. Daloze 9. Tursch J. P. DeClercq G. Germain and M. van Meerssche Tetrahedron Lett. 1979 2687. 29 A. Rustaiyan A. Niknejad L. Nazarians J. Jakupovic and F. Bohlmann Phj.tocheniistry 1982 21 18 12. 30 W. Hofheinz and P. Schonholzer Hek. Chim. Actrr. 1977 60 1367. 31 R. P. Gregson and D. Ouvrier J. Nut. Prod. 1982 45 419. 32 (a)H. Khan A. Zaman G. L. Chetty A. S. Gupta and S. Dev Twahedron Lett. 1971 4443; (h) R. T. Iyer K. N. N. Ayengar and S. Rangaswami Indian J. C'heni. 1972. 10. 482. 33 R. V. Venkateswaran. D. Mukherjee. and P. C. Dutta J. Chm. Soc. Perkin Trans. I 1981 1603. 34 L. V. Manes S.Naylor P. Crews and G. J. Riikus J. Org. Chem. 1985. 50 284. 35 J. Hellou R. J. Anderson S. Rafii E. Arnold and J. Clardy TL>traheciron Lett. 1981,22 4173. 36 E. Fattorusso S. Magno C. Santacroce and D. Sica. Tetra-hedron 1972 28 5993. 37 F. Yasuda and H. Tada Evperientiu 1981 37 110. 38 G. Cimino S. de Stefano and A. di Luccia. E.~pcric~/itim. 1979 35 1277. 39 G. Cimino. S. de Stefano L. Minale. and E. Trivellonc. J. Chctn. Sac. Pcrkiti Tram. I 1977 1587. 40 G. Cimino. S. de Rosa. and S. de Stefano Evpcricwtici. I98 1 37 214. 41 G. Cimino F. Cafieri L. de Napoli. and E. Fattorusso. Tetru-hetirun Lett. 1978. 2041. 42 F. Cafieri L. de Napoli E. Fattorusso C. Santacroce. and D. Sica Tt.trahrtlron Lcrt. 1977 477. 43 F. Cafieri.L. de Napoli E. Fattorusso and C. Siintacroce E.vp:prriontiu 1977 33. 994. 44 F. Cafieri L. de Napoli A. Iengo and C. Santacroce Experientia 1978 34 300. 45 Y. Kashman and M. Zviely Tetrahedron Lett. 1979 3879. 46 R. Kazlauskas P. T. Murphy R. J. Wells and J. J. Daly Auxt. J. Cheni. 1980 33 1783. 47 J. E. Hochlowski D. J. Faulkner L. S. Bass and J. Clardy J. Org. Chem. 1983 48 1738. 48 R. Kazlauskas P. T. Murphy and R. J. Wells Au,vt. J. Chmi. 1982 35 51. 49 H. Kikuchi Y. Tsukitani I. Shimizu M. Kobayashi and I. Kitagawa Chem. Pharm. Bull. 1981 29 1492; ;bid. 1983 31 552. 50 G. Cimino P. de Luca S. de Stefano and L. Minale. Tetra-hedron 1975 31 271. 51 G. Cimino S. de Stefano L. Minale R. Riccio K. Hirotsu and J.Clardy Tetrahedron Lett. 1979 36 19. 52 W. Herz and J. S. Prasad J. Org. Chmi. 1982 47 41 71. 53 W. Herz and J. S. Prasad Synth. C'ommun. 1983 13 1243. 54 F. Miyamoto H. Naoki T. Takemoto and Y. Naya. Totru-hedron 1979 35 19 13. 55 F. Miyamoto H. Naoki Y. Naya and K. Nakanishi Trtru-hedron 1980 36 348 1. 56 Y. Naya F. Miyamoto K. Nishida T. Kusumi H. Kakisawa and K. Nakanishi Chem. Lett. 1980 883. 57 J. K. Pawlak M. S. Tempesta T. Iwashita K. Nakanishi and Y. Naya Chem. Lett. 1983 1069. 58 Y. Ikeda M. Aoki T. Uyehara T. Kato and T. Yokoyama Chem. Lett 1983 1073. 59 S. Fujiwara M. Aoki T. Uyehara and T. Kato Tetruhedron Lett. 1984 25 3003. 60 R. Veloz L. Quijano J. S. Calderon and T. Rios J. Chem. Soc. Chem. Commun. 1975 191.61 Y. Naya K. Yoshihara T. Iwashita H. Komura K. Nakanishi and Y. Hata J. Am. Chem. Soc. 1981 103 7009. 62 M. S. Tempesta T. Iwashita F. Miyamoto K. Yoshihara and Y. Naya J. Chem. Soc. Cheni. Commun. 1983 1182. 63 Y. Iitaka I. Watanabe I. T. Harrison and S. Harrison J. Am. Chem. Soc. 1968 90 1092. 64 T. Rios and L. Quijano Tetruhedron Lott. 1969 I3 17. 65 T. Rios and F. Gomez G. Tetruhedron Lett. 1969 2929. 66 J. S. Calderon L. Quijano and T. Rios Cherii. Itid. (London) 1978 584. 67 T. Rios E. Hernandez L. Mocino and F. 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ISSN:0265-0568    

DOI:10.1039/NP9860300123

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

Biological Methods for Studying the Biosynthesis of Natural Products C. R. Hutchinson School of Pharmacy and Department of Bacteriology University of Wisconsin 425 N. Charter Street Madison Wl53706 USA 1 Introduction 2 Biochemical Studies with Intact Cells 2.1 Protoplasts 2.2 Inhibitors 2.3 Nutritional Regulation 2.4 A- B-and C-Factors 3 Enzymology 3.1 Aflatoxins 3.2 Alkaloids 3.3 Ant hraq ui nones 3.4 Antibiotics 3.5 Flavonoids 3.6 Terpenes 4 Genetic Methods 4.1 Mutation 4.2 Recombination 4.3 Gene-cloning Methods 5 Gene Cloning 6 Novel Antibiotics produced by Genetic Means Hybrid Antibiotics and Others 7 Summary and Acknowledgements 8 Addendum 9 References 1 Introduction The interest in pursuing studies of the biosynthesis of natural products that are formed by secondary metabolic pathways evolved from studies of primary metabolism as we began to wonder about the biochemical parallels between these two areas and the reason for the restricted production of metabolites that are often capable of having potent effects on other living organisms.The successful investigation of primary metabolism has depended heavily on the use of isotopically labelled compounds to trace the sequence of events and to define the mechanism of biochemical reactions in oioo an approach that began in 1935 with the use of deuterium to study some aspects of lipid metabolism’ and has continued unabated in part due to the development of increasingly sophisticated methods for the analysis of isotopically labelled compounds.’ This type of experiment later was overshadowed by metabolic mapping experiments using auxotrophs and by studies of the enzymology using purified proteins in vitro and of the physiology using mutants and biochemical modulators in civo. During the past decade the explosive growth in the use of recombinant DNA methods has revolutionized this field by shifting the focus of studies onto the molecular biology and genetics of primary metabolism as well as greatly facilitating the former types of experiments through an easier availability of scarce proteins and a deeper understanding of the interaction of biochemical systems.For the field of secondary metabolism the question naturally arises about its development has it grown in a parallel way and is it likely to experience an equally revolutionary growth through the application of recombinant DNA methods? The use of isotopes has proceeded in a nearly identical manner perhaps with even more sophistication,’ since it has been much harder to study the enzymology in uitro and to probe the physiology in ciro. This has been partly due to the difficulty in isolating the enzymes of secondary metabolic pathways and in establishing uniformly applicable conditions for the analysis of the complex biochemical systems that often can be viewed only through a narrow window in the organism’s lifespan. Such difficulties fortunately are waning for it is becoming increas- ingly clear that the use of recombinant DNA methods will overcome problems of this kind in much the same way as for primary metabolism.Therefore the answer to each of the questions that is posed above has to be affirmative although the study of secondary metabolism has developed like its counterpart its growth has been slower in some areas but promises to accelerate with the application of the same methodology that has characterized the other fields of modern biology since the post-1 973 revolution in molecular biology. My purpose in this review is to document the preceding statement by discussing some biological approaches that are useful for investigations of the biosynthesis of natural products. These include biochemical enzymatic and genetic methods taken from work that has been reported in the literature largely since 1980.I give pertinent examples of work from these three areas with comments on the advantages or limitations of the methodology rather than attempting to provide a comprehensive survey of the literature. Much of the terminol- ogy and general concepts the linguajranca of this scientific field were given by Brown in 1972.3As these have not changed in the intervening thirteen years I therefore refer the reader to this excellent source for background information and in the present review discuss only the newer concepts and the terminology that is necessary for an understanding of the biochemical and genetic experiments that have been described.2 Biochemical Studies with Intact Cells 2.1 Protoplasts Restrictions on the rate or extent of uptake of precursors can sometimes be a problem when using intact cells in biosynthetic experiments but the difficulty can often be overcome by using protoplasts (which are bacterial fungal or plant cells that lack most or all of their cell wall). Protoplasts can also be disrupted by osmotic shock which permits the isolation of proteins or subcellular organelles under very gentle conditions and are useful for the transformation of cells with DNA (see Section 4.3). Protoplasts are made by digesting away most of the cell wall with lysozyme (for bacteria) or with other lytic enzymes (for eukaryotes) under conditions that stabilize the resulting sacks of cellular constituents against rupture.Methods for their preparation can be found in the following reviews bacteria,j fungi,s and plants.b Examples of the use of protoplasts in studies of the biosynthesis of natural products are aflat~xins,~ actinomycin,8 and echinomycin”’() (to facilitate the uptake of precursor) and streptomycin’ and isopenicillin N (to facilitate the prepara- tion of cell-free extracts). 2.2 Inhibitors Inhibition of some step in a biosynthetic pathway by allowing an organism to grow in the presence of a compound that is known to be an enzyme inhibitor can provide circumstantial evidence for the involvement of similar enzymatic activity in cit.0 or show which kind of biochemical system has to be functioning for the biosynthesis of the natural product.This approach can be used when a more direct study of the enzyme- NH2 0 0 (1 1 HO catalysed step or biochemical system is not possible. It suffers from the inability of the experimenter to control the effects that an inhibitor may have at other points in the cellular metabolism and to control what happens to the substrate of the enzyme that is inhibited. Despite being unable to circumscribe the effects of the inhibitor useful information has been acquired in this way. Cerulenin (l) which is an antifungal metabolite of Cephafosporium caerufens is a potent inhibitor of the fatty-acid synthases in micro-organisms and in animal tissues. 3.14It inhibits 0-ketoacyl thioester synthetase activity’ by binding irreversibly to the SH of the peripheral cysteine residue in the active site of the condensing enzyme domain.l6 Since cerulenin also inhibits the biosynthesis of polyketides which are a class of natural products that are made from simple fatty acids cia poly-P-ketone intermediates this has been taken as evidence for the similarity of the enzymes that catalyse the Claisen condensation of acyl thioesters and a-carboxy-acyl thioesters in the two biochemical systems. The active sites of the polyketide- and fatty-acid-condensing enzymes are not identical however since the former is more sensitive to (1) than the 1atter.j Nevertheless cerulenin specifically blocks the formation of the polyketide-derived portion of macrolide antibiotics tetracycline,’ and candicidin‘O in uico and of naringenin- chalcone synthase’ and 6-methylsalicylate synthetase’ in vitro presumably by inhibiting the activity of condensing enzyme.This property of (1) has been exploited in the design of a screening technique for overproducing strains of Streptornyces peucetius that make daun~rubicin’~ and in the production of hybrid antibiotics (see Section 6). Fluorinated amino acids can affect the properties of eniymes by being incorporated into their primary structure,2a thus imparting abnormal properties that are akin to the result of mis-sense mutations,’5 and fluorinated analogues of normal substrates can inhibit the function of the catalytic site of an enzyme.26 These properties justify studies of the effects of some fluorinated compounds on the biosynthesis of natural products.Packter and co-workers noted that the addition of 4-fluorophenylalanine (2) to cultures of Aspergillus jurnzggarus markedly suppressed the formation of the characteristic acetate-derived phenols and resulted in the secretion of atypical shikimate-derived phenol^.^'.'^ These effects were different from those that were caused by the addition of cycloheximide 8-azaguanine or 5-methyltryptophan which are known inhibitors of the synthesis of proteins. The effects of (2) could not be explained clearly; however these and other data were used later to support the proposal that the aromatic [phenol] synthetase could have originated from a dissociated portion of the fungal fatty-acid synthase.29 The biosynthesis of the macrolide antibiotic erythromycin A (3) was blocked by the addition of 2-or 3-fluoropropionic acid or their ethyl esters to \ NATURAL PRODUCT REPORTS 1986 0 0 0 (7) cultures of Streptornyces erythrasu~.~~ The blockade of forma- tion of the antibiotic took place without a significant effect on cell growth and did not result in the accumulation of intermediates that are related to the deoxy-sugars that are characteristic of the biosynthesis of this macrolide. Presumably the fluorinated propionates blocked the formation of the macrolactone portion of (3) perhaps by inhibiting propionyl-CoA carboxylase since this enzyme can be inhibited by 3-fluoropropionyl-CoA in zitro3 and is believed to be involved in the biosynthesis of (3).32 In contrast the addition of ethyl (2S)-2-fluoropropionate to resting cell cultures of Streptornyces 1usulien.ris caused a two- to eight-fold stimulation of the production of the polyether antibiotic lasalocid A (4),33 which is formed by a polyketide pathway that utilizes acetate butyrate and propionate as precursors.jJ This result may have been due to induction of the formation of greater amounts of an enzyme that catalyses one step of the pathway in response to its depletion by the fluoropropionate. Inhibitors of mono-oxygenases or their associated electron- transport systems can block biosynthetic pathways by disrupting the hydroxylation or epoxidation of intermediates. The conversion of 6-deoxyerythronolide B (5) into erythronolide B (6) thus was inhibited by adding GEB (7) (an inhibitor of an NADPH-specific reductase in the biosynthesis of sterols) to cultures of Streptomyces erythraeu.~.~ The enzyme NATURAL PRODUCT REPORTS 1986 -C.R. HUTCHINSON 6-deoxyerythronolide-B hydroxylase which catalyses this conversion in citro contains a cytochrome P-450 prosthetic group and a two-component electron-transport system36 that presumably is inhibited in vivo by GEB. The formation of lasalocid A was found to be blocked in vivo by ethyl GEB and SK F525A33 (another inhibitor of mono-oxygenases) which supports the belief that its biosynthesis involves the epoxidation of a diene intermediate.37 Finally the most widely used inhibitor of cellular secondary metabolism is cycloheximide (8).This compound has often been used to demonstrate that the synthesis of new protein is needed for the biosynthesis of a natural product during the most active period of its formation as in the case of the phenols of Aspergillus fumigatus cited above. If cycloheximide inhibits the formation of a secondary metabolite if it is added at the beginning of production of the metabolite this usually is interpreted to mean that the enzymes of the pathway are not formed constitutively but are induced by some signal which turns on gene expression; if it does not inhibit the formation of the metabolite then constitutive formation of the enzymes prior to the production stage is presumed to have occurred.For example with the Aspergillus phenols it was concluded that orsellinic acid and 2,3,5-trihydroxytoluene were formed by constitutive enzymes and 2,3,4,5-tetrahydroxytolueneand its O-methyl derivative by inducible enzymes based on how cycloheximide affected the production of these compounds.27 2.3 Nutritional Regulation The effects of environmental and nutritional factors on the production of microbial metabolites have been studied exten- sively since these are important for their commercial production. Because these effects are more closely related to understanding the regulation of the production of metabolites than to learning how the metabolite is made I will not discuss this topic in depth. There are several recent reviews of this 135 A particularly interesting case is the stimulation of the production of macrolide antibiotics by agents that lower the concentration of ammonium ion in the growth medi~m.~~.~~ In one case the ability of ammonium ion to repress the production of antibiotic has been attributed to its suppression of the biosynthesis of protylonolide (tylactone) (9) which is the lactone portion of the macrolide tylosin Since there is evidence that in the biosynthesis of protylonolide valine can provide several of the carbon atoms of the lactone,5s it is significant that ammonium ions were found to inhibit the catabolism of valine in the micro-organism that was producing pr~tylonolide.~~ This limited coverage of the effects of nutrients on the production of microbial metabolites does not mean that it is relatively unimportant to studies of the biosynthesis of natural products.On the contrary this is an area where much knowledge is needed about the links between changes in nutrient levels and the appearance of secondary metabolites as part of the regulation of the biosynthesis of natural products. At the moment there is little insight into this subject at the molecular level but this will change soon through studies of the molecular biology using cloned genes (see Section 5) and the results will greatly illuminate our understanding of microbial physiology. 2.4 A- B- and C-Factors The onset of formation of natural products is felt to be part of the developmental biology of organisms which exhibit different stages in their life cycles.In bacteria the production of antibiotics usually does not commence until the organism's cells begin to differentiate,47 which also is true for the secondary metabolites of plant cells.4R This is a complex and poorly understood subject for any living organism yet in the case of bacteria there is a growing awareness that small molecules act as messengers in mediating the onset of production subject which the interested reader may consult ho~ever.~*-~~ The production of an antibiotic by a micro-organism that is cultured in a laboratory as a typical example does not begin until the easily utilizable carbon and nitrogen sources have been depleted below some low threshold level and the rate of growth has declined.The level of phosphate in the culture medium also affects the onset of production of antibiotic; low levels of phosphate usually favour this and high levels usually inhibit it. The work of Dekleva et al. about the effects of nutrients on the production of an anthracycline antibiotic is an example of some of these phenomena and how they can be 0 0 of secondary metabolites and other biological events,49 something like the roles of pheromones and hormones in more complex living systems. Here again is an area concerning the regulation of the biosynthesis of natural products but at the most fundamental level i.e. the molecular biology of gene expression. While the precise mechanism by which these messengers function is still unknown it is informative to review what is known for one system (antibiotic production) as an example of how such work is being pursued.The best-studied case is A-factor (11) which is an autoregulator that has been discovered in the streptomycin- producing bacterium Streptomyces griseus and which at picomolar concentrations is capable of inducing the production of aerial mycelia spores and antibiotics in strains that lack these characteristics of cellular differentiati~n."~.~~ A-Factor also causes changes in the levels of several intracellular enzymes,49 which may be part of the biochemistry of differentiation. Some structure-activity relationships have been reported for A-factor,s1*s2 and it recently has been suggesteds3 that only a single key enzyme is required for its production in four species of Streptomyces that normally do not produce it in detectable amounts (see Section 5).This result implies that the immediate precursors of A-factor are common metabolites in the genus Streptomyces. 0 136 NATURAL PRODUCT REPORTS 1986 (11) OH HO OAc (13) o\ O H D H I I (15) B-Factor (12) is the most recent autoregulator to be discovered and appears to regulate the production of the ansamacrolide antibiotic rifamycin in a species of Nocardia.54 C-Factor is a third autoregulator of the actinomycetes (but historically the first to be dis~overed~~). It is a protein with a molecular weight of approximately 34.5 kDa that stimulates cytodifferentiation of a mutant of Streptomyces griseus.The information in this section is typical of what can be learned -and what cannot -about the biosynthesis of natural products by working with intact cells. The limitations of this approach accrue from the difficulty in unravelling the complex biochemical and genetic interactions within cells of living organisms. As in most other situations like this one assumes that subdivision of the system through dissection of the cellular biochemistry and molecular biology into their individual components is the only way to a deep understanding of the phenomena that are observed. This consequently is the reason for including the next four sections in this review. 3 Enzymology Although it is safe to state that in no case have all of the enzymes that are required for the biosynthesis of any secondary metabolite been isolated and purified to homogeneity much progress has been made towards this goal in some cases.Yet this requirement has to be met (or at the least methods have to be devised for the assay of each enzyme) if one expects to achieve complete understanding of a given biosynthetic pathway. In this section I review several examples of enzymes that have been isolated from bacteria and plants to highlight what their study has contributed to the field of the biosynthesis of natural products and to reveal some of the technical problems that are associated with such work. 3.1 Aflatoxins Hsieh and co-workers have described cell-free systems for the conversion of versiconal hemiacetal acetate (1 3) into ,O OH O=P, 1 OBu" -0 0 II (14) 00 (16) versicolorin A (14)56 and of sterigmatocystin (15) into aflatoxin B1 (16),57 which are intermediate steps of the biosynthesis of aflatoxin~.~~ The conversion of (13) into (14) did not require oxygen (but the conversion rate was halved under anaerobic conditions) or NADP+ and was inhibited by cysteine and dichlorvos which these investigators had found earlier to inhibit the biosynthesis of aflatoxins in uivo and to cause the accumulation of (1 3).The activity in the cell-free preparation was soluble and did not require dithiothreitol (DTT) polyvinylpyrrolidone or glycerol for stabilization (unlike many other cases of the enzymes of secondary metabolism).In this way the authors were able to see chromatographically that several intermediates were involved in the conversion of (1 3) into (14) in uitro. 3.2 Alkaloids Knowledge about the enzymology of the biosynthesis of alkaloids has progressed rapidly during the past five years and the use of plant cell cultures has been vital to the success of this work.59 Examples of the enzymology of two well-studied systems i.e. for the biosynthesis of monoterpenoid alkaloids and of benzylisoquinoline alkaloids are reviewed here. A comprehensive review of the enzymology of alkaloid metabolism also is available.60 The formation of the monoterpenoid indole alkaloid ajmalicine (22) and its isomers from tryptamine (17) and secologanin (1 8) involves four key intermediates (Scheme 1).6* Strictosidine synthase has been purified from Catharanthus roseus about 50-fold to near homogeneity and the values of K,, and V,, for secologanin and tryptamine have been established along with the molecular weight and the pH optimum.62 The presence of 116 times more enzyme in the plant tissue culture than in the roots of the plant shows the advantage of the former.The partially purified enzyme was immobilized on CNBr-activated Sepharose which markedly increased its thermal stability and permitted the synthesis of strictosidine (19) in NATURAL PRODUCT REPORTS 1986 -C. R. HUTCHINSON H (17) (18) Me02C OH (d ialdehyde 1 (aglycon 1 Me OH Cathenamine (20) I HO (21) (22) Enzymes i strictosidine synthase ; ii strictosidine 0-glucosidases I and I1 Scheme I gram q~antities.~~ Two glucosidases were isolated from C.roseus which had strict substrate specificity for strictosidine but which differed in their values of K,, and V,,, their inhibition by substrate and by D-glucono- 1,5-lactone and their molecular weight (230 and >450kDa).’-j4 Reduction of 4,21-didehydrogeissoschizine (20) gives geissoschizine (21) which had previously been reported to play a pivotal role as the precursor of ajmalicine (22) and other types of alkaloids of C. roseus in uiuo. Yet the study of its metabolism in uitro revealed that it is a shunt product of the pathway leading to the other alkaloid^^^,^^ and enters the mainstream of events by the action of a dehydr~genase.~’ Thus the work with the alkaloid- metabolizing enzymes has clarified the understanding of this early stage of events in the complex biosynthetic pathway to the monoterpenoid alkaloids of Catharanthus roseus.The benzylisoquinoline pathway provides commercially important alkaloids e.g. berberine codeine thebaine and morphine. It is significant therefore that key enzymes of this pathway have been isolated and partially purified from plant tissue cultures by Zenk and co-workers. The first enzyme NATURAL PRODUCT REPORTS 1986 "HOV\N H 2 HO \ (23) (24) HO OH eo m HO \ NMe R R (26) (27) (28) of the pathway (S)-norlaudanosoline synthase which catalyses the conversion of dopamine (23) and of (3,4-dihydroxyphenyl)acetaldehyde (24) into 1 H-(S)-norlaudanosoline (25) has been purified about 40-fold from cell suspension cultures of Eschscholziu tenuij'olia.68 Its molecular weight pH optimum and values of K and V,, for the two substrates were established but the most interesting finding was that there are four isoenzymes differentiable by polyacryl- amide gel electrophoresis (PAGE) and their isoelectric points that catalyse the same reaction.While the reason for the presence of multiple forms of the enzyme remains unknown the results of this work invalidated an earlier claim that (S)-norlaudanosoline was formed by the condensation of dopamine and (3,4-dihydroxyphenyl)pyruvate followed by decarboxylation and reduction. Subsequent work by this group uncovered two kinds of methyltransferase (one of which acts on both enantiomers of norlaudanosoline) that are involved in the formation of reticuline (26) which is the central and branch- point intermediate of metabolism of benzylisoquinolines in plants,69 and the enzymes that catalyse the formation of (S)-tetrahydroprotoberberines (27)'O and protoberberines (28).' I Several properties of each enzyme were described and for the latter two it was reported that they were contained exclusively in an organelle that could be isolated from the cells by sucrose density-gradient centrifugation and that differed in density from mitochondria protoplastids and other known organelles of plant cells.3.3 Anthraquinones Metabolites that are derived from anthraquinones (which are made by the polyketide pathway) are widely distributed in fungi and plants.One of these metabolites is geodin (31) which is made from emodin (29). Sankawa and co-workers have purified two enzymes from Aspergillus terreus that act on intermediates of the biosynthesis of geodin. Emodin 1-0-methyltransferase was purified 89-fold in a two-step procedure using chromatography on blue Sepharose and on Sepharose GB.'* The use of the former separation method was predicated on the knowledge that S-adenosyl-L-methionine (SAM) is a co- substrate for this enzyme; thus the protein should have a site that has an affinity for the nucleotide-like ligand that is attached to the blue Sepharose. This technique by its speed and selectivity circumvented the extreme instability of the enzyme in crude preparations.Besides the molecular weight Michaelis constants and pH optimum the most significant property that was reported for this enzyme was its strict specificity for the natural substrate when compared with seven other compounds that are closely related to emodin. Dihydrogeodin oxidase [a blue copper protein which (R=OH or OMe) 0 HO @JMe\ 0 (29) 0 0*c' / Me HO M&:e\ C02Me L' C02Me (301 (31) oxidatively couples dihydrogeodin (30) to form (+)-(31)] was purified to homogeneity by a nine-step procedure including the use of chromatofocusing and was found to contain sixteen copper atoms per molecule and to consume 0.5 mole of oxygen per mole of (+)-(31) that is pr~duced.'~ This is the first example of the complete purification of an enzyme that catalyses the oxidative coupling of a phenolic natural product.3.4 Antibiotics Since many secondary metabolites have antibiotic activity there are a large number of examples that I could discuss in this section. To restrict my coverage within reasonable limits I have selected five examples of bacterial and fungal antibiotics the majority of which represent cases where an enzyme has been purified to homogeneity. Actinomycin D (33) is a well-known microbial secondary metabolite and the first antitumour antibiotic to be isolated from a micro-organism. It is biosynthesized from five amino acids and 3-hydroxy-4-methylanthranilic acid which is pro- duced by the degradation of tryptophan to 3-hydroxyanthranilic acid followed by methylation at C-4.The aromatic acid can be dimerized oxidatively to form actinosin (32)'j or it can first be coupled to a pentapeptide giving actinomycinic acid which then is dimerized to (33).75 Phenoxazinone synthase which catalyses the formation of (32) has been purified from Streptomyces untibioticus to homogene- ity by Choy and Jones.76 Affinity chromatography with 3-hydroxyanthranilic acid that was bound to Sepharose 4B was NATURAL PRODUCT REPORTS 1986 -C. R. HUTCHINSON Me Me (32) R = C02H (33) R = CONHCHC -D -Val-L -Pro -Sar -NMeCHPr ' II!! MeHC 0 c=o NH CHzOH MeIGe)l,T 0P OH OH (34) (35) (dTDP = thyrnidine -5'- diphospho ) NH H II NHCNH2 NH11 NH2 0 OH OH OH (36) OH an important purification step at an early stage in their isolation procedure.Two forms of this enzyme were found a large form with a molecular weight of 799-919 kDa and a small form of 237 kDa. Denaturing polyacrylamide gel electrophoresis showed that these forms were composed of a subunit of 97 kDa. It was not established that the same subunit was present in both forms of the enzyme (but see refs. 159 and 160 for more recent information) but antibody that had been raised to the combined forms or just to the large form cross- reacted equally with both forms suggesting that the two forms have some common antigenicity. Both forms catalyse the formation of (32) in a reaction that involves the consumption of 1.5 mole of oxygen per mole of (32) that is produced.The authors also noted a decline in the amount of the small form with an increase in the amount of the large form as the cultures aged. Streptomycin (37) is an aminocycli to1 (aminoglycoside) antibiotic and was one of the first antibiotics to be isolated from a streptomycete during the pioneering investigations of Selman Waksmann. It also is prominent as an antibiotic for which many of the 28 to 30 enzymes that comprise its biosynthetic pathuay can be assayed in cell-free preparations or as partially purified During the past six years Grisebach and co-workers have described enzymes that catalyse the second and third stages of the biosynthesis of streptomycin. These workers purified dTDP-L-dihydrostreptose synthase 50-fold from Streptomyces griseus by using buffers that contained diphenylcarbamyl chloride and phenylmethanesulphonyl flu- oride (PMSF) washing mycelial cells with 1M-KC1 and precipitating crude cell extracts with polyethyleneimine (for the inhibition and removal of protease activity).'" (The presence of proteases is a common problem with cellular extracts from species of Streptomyces.)They also circumvented the lack of convenient sources of dTDP-4-keto-6-deoxy-~-glucose and dTDP-4-keto-~-rhamnose (which are substrates for dTDP-L-dihydrostreptose synthase) by using a protein-free extract of E.coli Y-10; this mutant is blocked in rhamnose biosynthesis but has the enzymes that are needed for the formation of these two compounds.The product of the synthase i.e. dTDP-L-dihydrostreptose (34) is transferred onto streptidine 6-phosphate (35) to give 0-a-L-dihydrostreptosyl-(1-+4)-streptidine 6-phosphate (36) by an enzyme from S. griseus that these workers purified to near homogeneity by procedures including affinity chromatography on a streptidine 140 6-phosphate/Sepharose column.80 This enzyme has a molecular weight of 63 kDa (composed of two apparently identical subunits of M 35 kDa) requires Mn2+ or Mg2+ for activity and is stabilized by the presence of streptidine during its purification. Finally a cell-free system for the conversion of (36) into dihydrostreptomycin 6-phosphate which is the penultimate intracellular precursor of (37) has been described by the same group.81 The biosynthesis of antibiotics that contain a p-lactam ring as part of their structure has attracted much interest due to the medical and commercial importance of these compounds.Thus it is not surprising that the knowledge about the enzymology of the biosynthesis of p-lactam antibiotics is well advanced. So much has been accomplished in this area that I can mention only a few salient aspects; readers may consult several recent reviews for further information.82-s4 After it was shown that the tripeptide L,L,D-OI-aminoadipylcysteinylvaline was the immediate precursor of isopenicillin N (38) in cell-free systems,8s*86 the enzyme that catalyses the cyclization i.e. isopenicillin-N synthetase was purified to homogeneity from Cephalosporium acremonium Penicillium chrysogenum and Streptomyces clavuligerus by four research gro~ps.~~-~~ On e group who used rapid high-performance liquid chromatography as an essential part of their isolation scheme reported the presence of three forms of this enzyme distinguishable by h.p.1.c.elution time but not by molecular weight which they believed were either products of limited proteolysis of the principal protein (M,41 kDa) or true isoenzyme~.~~ The other research groups who used more conventional isolation methods did not report finding multiple forms of this enzyme. An interesting property of this enzyme is its ability to catalyse the formation of a wide variety of 0-lactam structure^,^^-^^ which may provide a practical bio-chemical route to useful analogues of the pencillins and cephalo~porins.~~~~~ [It is tempting to imagine that this lack of specificity will be found to be true for other enzymes of secondary metabolism yet it is more likely that it is a special property of the enzymatic mechanism for the conversion of the tripeptide into (38).] Note also that the tripeptide had been promulgated as the precursor of (38) for more than twenty years; yet this could not be proven until the cell-free system thence purified isopenicillin-N synthetase became available.Isopenicillin N is epimerized at the a-position of its aminoadipyl side-chain then ring-expanded to deacetoxycephalosporin C (39),from which cephalosporin C (40) results by hydroxylation and acetylation.The enzyme activities for ring-expansion and hydroxylation could not be separated in a nearly pure protein fraction from C. acremonium; thus it was concluded that these two reactions are catalysed by a A NATURAL PRODUCT REPORTS 1986 single bifunctional protein with a molecular weight of 31 kDa.94 In S. clavuligerus however these two activities were easily separable into two proteins with M 29 kDa and 26.2 kDa respecti~ely.~~ This is the first example of a major difference between the shared portions of a biosynthetic pathway that exists in prokaryotes and eukaryotes. The macrolide antibiotics typified by erythromycin A (3) and tylosin (lo) are biosynthesized by pathways that involve the formation of a macrolactone and deoxy- or deoxyamino- sugars which then merge to create the characteristic macrolide structure.Possibly because of the number of enzymes that is required (although the biosynthesis of streptomycin has an equivalent complexity) or the requirement for large catalytic proteins with their inherent instability (but this is also true of the oligopeptide antibiotics) the understanding of the biosyn- thesis of macrolides is not as well advanced as in the cases discussed above. Therefore I will describe only two examples of purified enzymes and direct the reader to recent reviews for additional inf~rmation.~~ loo In the biosynthesis of erythromycin A the formation of the macrolactone 6-deoxyerythronolide B (5) is succeeded by its hydroxylation at C-6 to form erythronolide B (6).A cell-free system for this oxidation has been described by Corcoran and Vyganta~.~~.~~ Their preliminary evidence shows that a protein of M,46 kDa containing a cytochrome P-448 prosthetic group and at least two other proteins for electron transport are required in vitro to couple the oxidation of substrate to the consumption of NADPH.We have examined this system further and by purifying the cytochrome-P-448-containing protein to homogeneity have found that an industrial strain of Streptomyces erythraeus that had been selected for its greater production of erythromycin A contains about five-fold more of this enzyme than a wild-type strain (A. Shafiee and C. R. Hutchinson unpublished results). Of more interest however is the presence in the same strain of two or more forms of this protein which could be separated by chromatography on hydroxyapatite {based on their ability to catalyse the hydroxylation of (9S)-[9-3H]-9-dihydro-6-deoxyerythronolide B}.We have not determined yet if these forms are true isoenzymes. Since the titre of this enzyme reaches its maximum coincident with the period of biosynthesis of antibiotic by S. erythraeus,"h and the production of erythronolide B can be increased markedly by adding propanol to the fermentation medium of a wild-type strain,'()' it will be important to determine if the synthesis of 6-deoxyerythonolide B hydroxylase is inducible in vitw by 6-deoxyerythronolide B. It has been noted that in an industrial strain of Streptomyces jradiae that produces about three-fold more tylosin than the wild-type strain the level of macrocin (41) which is the ~N.,/cH~R 0 C02 H (39)R = H (40)R = OAc OH o& OH NATURAL PRODUCT REPORTS 1986 -C.R. HUTCHINSON intermediate that is methylated in uiuo to give tylosin (lo) was much higher than in the wild-type strain even though the titre of the macrocin 0-methyltransferase was elevated at least two- fold.IO' This may be due to the ability of (41) and some other intermediates of the biosynthesis of tylosin to inhibit this enzyme in vim but the authors also suggest that the level of macrocin 0-methyltransferase is rate-limiting for the production of tylosin. Thus it was considered to be important to purify this enzyme for a detailed study of its properties.This has been accomplished recently to reveal the following things. It is an acidic protein (PI of 4.9 composed of two subunits (Mr 33 kDa and 65 kDa) and it requires a metal ion for maximal activity. The Michaelis constants of 64 and 24 pmol dm-3 for S-adenosyl-L-methionine and macrocin respectively and other kinetic data from experiments with these substrates and the inhibitor 2"'-O-demethylmacrocin are consistent with an Ordered Bi Bi mechanism for the enzymatic reaction with SAM as the first substrate to be bound and S-adenosyl-L- homocysteine as the second product that is released.Io3 The peptide antibiotics represent a diverse group of microbial secondary metabolites whose biosynthesis takes place non-ribosomally.The enzymology of the formation of these antibiotics has been studied for more than fifteen years and is particularly fascinating for what it has revealed about the properties of multicatalytic-site proteins. O4 Since this topic has been thoroughly reviewed re~ently,~~~,'~~ I will mention the highlights of gramicidin-S synthetase and tyrocidin synthetase' O7 only as an example of what is generally true for all of the peptide antibiotics. Gramicidin S (42) is assembled by gramicidin-S synthetase which is composed of two proteins; these are the light enzyme (100 kDa) and the heavy enzyme (280 kDa). The light enzyme accepts L-phenylalanine activates it by the transient formation of its adenylate via ATP (perhaps simultaneous with its epimerization to D-phenylalanine) and then uses D-phenylalanine to initiate the synthesis of a pentapeptide.The heavy enzyme accepts and activates the L isomers of proline valine ornithine and leucine and attaches them onto the amino-group of D-phenylalanine in sequo. Gramicidin S is then formed by dimerization of this pentapeptide through attachment of the amino-group of L-leucine to the carboxyl carbon of D-phenylalanine. The heavy enzyme contains one mole of pantetheine which is used successively to transfer the growing peptide (bound as a thioester via the cysteamine portion of pantetheine) to each amino acid that also is bound by a different (peripheral) thioester to the enzyme surface. The evidence that all of these transformations take place with enzyme-bound intermediates by means of multicatalytic-site proteins is very convincing.Furthermore work with tyrocidin synthetase which is composed of three large proteins (of M 100,230 and 440 kDa) has shown that the two larger proteins can be resolved into subunits (of M ca 70 kDa) that are believed to correspond to the regions (domains) that hold the individual thioester-bound amino acids. From these and other data the concept has evolved that the amino-acid sequence of peptide antibiotics is dictated by the spatial order of the amino- acid-binding sites on the enzymes with the pantetheine residue serving as the tether by which each amino acid can be picked up for addition to the growing oligopeptide. This idea is modelled on the biosynthesis of fatty acids but differs in its provision for the addition of different amino acids in each chain-growing step rather than of the same (two-carbon) unit as occurs in the formation of fatty acids.It also imagines that the size of the oligopeptide is limited by the physical properties D Phe-Pro-Val4Orn-Leu 1 of the enzyme that catalyses the assembly; i.e. that at some point the oligopeptide becomes too large for sufficient stability or fidelity and this limits the length of the peptide which can be assembled by one multicatalytic-site protein. Perhaps this is the reason that the alternative process of protein assembly cia mRNA and ribosomes evolved. 3.5 Flavonoids The flavonoids are widely distributed in plants and serve useful functions as insect attractants and natural defensive agents.O8 Because of this and their role in plant pigmentation (as anthocyanins) the study of flavonoid metabolism has attracted much interest. Using cultured cells of parsley most of the enzymes that catalyse the formation of flavonoid glycosides have been isolated and are available for study,109 but I will discuss only one of them naringenin-chalcone synthase since there is information about this enzyme at the protein mRNA and DNA levels. When dark-grown cell suspension cultures of parsley (Petroselinum hortense) are irradiated with ultraviolet light the three enzymes of general phenylpropanoid metabolism (Group I) are rapidly induced together with the enzymes (approximately thirteen) of the flavonoid glycoside pathway (Group 11).Two of the enzymes of Group I are found in all plants and the third 4-coumarate-CoA ligase plays a central role in directing intermediates into the various pathways of flavonoid metabolism of which the enzymes of Group I1 are one example. Naringenin-chalcone synthase which catalyses the formation of naringenin chalcone (44) from 4-coumaroyl- CoA (43) and three molecules of malonyl-CoA is the key enzyme in flavonoid biosynthesis and has been purified to homogeneity from parsley cells.' lo Since the ultraviolet-irradiated cells make large quantities of this enzyme,' ' its purification required only standard procedures except for the need to remove phenolic materials by treating the crude cell homogenate with an anion-exchange resin and polyethylenimine before subsequent purification steps.The most notable properties of this enzyme are its small size (two identical subunits of M 42 kDa) the lack of pantetheine in its primary structure and its ability to catalyse the formation of products that contain one or two two-carbon units less than naringenin chalcone (44)'12 or of analogues of (44)Il3 under certain conditions in vitro. This and other informationlog has resulted in the proposal that naringenin-chalcone synthase and the enzyme that is important for the synthesis of fatty acids in higher plants i.e. 3-oxoacyl-[acyl-carrier-protein]synthase have a functional relationship and a common origin perhaps by evolution of the former from the latter through gene duplication.3.6 Terpenes Progress in studying the enzymology of the biosynthesis of terpenes has been slow largely due to difficulties in isolating sufficient amounts of enzymes and to the need for considerable chemical work to prepare substrates and to identify products. Although none of the enzymes that is involved in the formation of terpenoid secondary metabolites has been purified to homo- geneity on a large enough scale for detailed study there is a growing understanding of the isolation techniques which can be used successfully and of the mechanisms of the enzymatic reactions. fioH NATURAL PRODUCT REPORTS 1986 (46) (45) One of the best examples in this area is a pair of soluble enzymes from the sage Salvia oficinalis which each catalyse the conversion of geranyl diphosphate (45) into either (+)-or (-)-a-pinene (46) and stereochemically related cyclic monoterpenes.Gambliel and Croteau reported low recoveries of activity from enzymes that had been partially purified by a four-step fractionation scheme that included the use of chromatofocusing yet they were able to show that the cyclization activities for the two enantiomerically related sets of products co-purified with the geranyl pyrophosphate :pinene cyclases I and 11. This is the most conclusive evidence to date that multiple products can be synthesized by a single monoterpene cyclase and points to the need for further study to learn how this occurs through the balance of chemical and enzymatic factors.4 Genetic Methods Since the formation of natural products is governed ultimately by the genes that encode the information for the production of appropriate enzymes and its regulation it is desirable to explore the genetic basis of the biosynthesis of natural products. In the next three Sections I discuss some genetic methods and their use in investigations of natural products primarily those made by bacteria. The intention is to show applications of these techniques rather than to review the concepts and methodology of bacterial or plant genetics and recombinant DNA work; therefore I have limited my explanation of genetics and molecular biology to only what is required for a rudimentary understanding of these matters in the context of the examples that are presented.Readers should consult the excellent monograph by Old and Primrose' l6 for a more detailed discussion of the concepts underlying genetic engineering in addition to any up-to-date introductory textbook of genetics e.g. those by Lewin"' and by Watson Tooze and Kurtz. 4.1 Mutation Besides being the experimental cornerstone of bacterial genetics the mutation of micro-organisms has been a mainstay of their industrial use principally to create high-yielding strains to direct the metabolic output towards one particular product or to create other desirable properties such as heat tolerance assimilation of a special nutrient or resistance to environmental toxins and to bacteriophages. For biosynthetic studies the main goal of creating mutations has been to produce strains that have metabolic blocks or high titres of pathway enzymes.This facilitates the identification of biosynthetic intermediates if these accumulate in the blocked mutant and are not further metabolized to shunt products and the isolation of enzymes of the biosynthetic pathway. Unfortunately knowledge about the art of mutagenesis is not widely disseminated since it often becomes proprietary information in the companies where it is most frequently practiced. Hopwood' l9 and Baltz' 2o have written useful reviews about methods of mutagenesis and there is a comprehensive account of the effects of mutagenizing antibiotic-producing bacteria. Mutagenesis protocols also can be found in standard laboratory manuals.' 2',1 23.138 Since it is difficult to find literature that exemplifies all of the facets of mutagenesis I offer the following comments as a distillation of our limited experience with the construction of blocked mutants of streptomycetes.Conditions for the growth and storage of the organism which do not result in significant loss of the ability to produce metabolites upon repeated culturing must be developed and a convenient biological or chemical assay for the production of metabolites that has the necessary sensitivity and selectivity must be devised. Growth on a solid medium and bioassay has been more practical in our hands than growth in a liquid medium and thin-layer chromatography of the metabolites.Since the blocked mutants often appear with a frequency between 0.1 and 1% it is desirable to screen batches of 1000 colonies when evaluating the result of mutagenesis. A combination of different mutagenetic treatments should be used (but not at the same time!) rather than just one kind based on experience' l9 and the chemistry of mutagenesis.' Mutants are usually picked from the 1 to 0.1% group that survives treatment with the mutagen (this depends on the type of mutagen that is used*20) to minimize the presence of strains that carry multiple mutations. After picking colonies that represent independent mutational events,lI9 it is best to allow the mutants to pass through one haploid growth cycle [to ensure that all progeny are derived from one and the same genome (i.e.are homozygous)] and to transfer mutants serially a few times to test the stability of the mutation. For organisms (e.g. the streptomycetes) that have a morphologically complex life cycle it is very important to choose blocked mutants for further study which appear to be normal in all other respects; otherwise the metabolic blockade may be due to pleiotropic effects of the mutation rather than to the lack of an enzyme for a particular pathway. Once a set of blocked mutants has been prepared they can be classified in several ways (accumulated metabolites cosynthesis etc.) as exemplified by the work of Sen0 and Baltz with mutants of the tylosin-producing bacterium Streptomyces .fi.adiae' 1s and of Weber et a/. for mutants of Streptomyces erythraeus.'6 Mutation of course is a function of what happens to the DNA of the organism. It therefore is useful to be cognizant of the and biology"' of the mutation process for an appreciation of why I have made some of the preceding generalizations. Changes in the sequence of nucleic acids of DNA ultimately cause the mutations and these result from mistakes in DNA repair which are natural (but rare) events but which may also be induced. As replication of DNA occurs with high fidelity alterations of sequence that occur during replication are not the main reason for the observed mutations. Some mutations stem instead from the erroneous pre- and post- replication repair of the nucleic acids in the DNA of an organism that have been affected because it has been treated with a mutagen.The post-replication events that are known as the SOS functions in Escherichia coli (a metaphor to the universal signal for distress) which are induced in response to damage to DNA are particularly error-prone. These biochemi- cal systems can result in transversions (i.e. substitution of a pyrimidine for a purine) or transitions (i.e. interchange of one purine or pyrimidine with another of the same type) as point mutations in the addition or deletion of one nucleic acid residue as frameshift mutations or in the deletion of segments of the DNA. These alterations to DNA then block the production of an enzyme or the activity of its regulatory element by interfering with gene expression (nonsense frameshift and polar mutations) or result in the production of a malfunctional gene product (mis-sense mutations).The single- base-change mutations are repairable at observable fre-quencies but the deletions are not which is why mutants can exhibit different stabilities. Moreover these effects vary depending on how the mutagen affects the nucleic acids,' which is why different mutagens should be used in constructing a set of blocked mutants. Finally some mutations largely those that are due to rearrangements of the DNA are easily induced and very unstable. These can be encountered if bacteria are treated with acridine or acriflavine dyes undergo regeneration of protoplasts are grown under stressful conditions or are just serially cultured.' 18 Such mutations often have pleiotropic NATURAL PRODUCT REPORTS 1986 -C.R. HUTCHINSON properties and normally are undesirable yet in a recent instance,' 29 treatment of a polyether-producing strain with ethidium bromide gave mutants that lacked aerial mycelia and which produced a low yield of the polyether antibiotic but which also produced a second antibiotic with a quite different structure. 4.2 Recombination Genetic recombination is the breakage of DNA and the reunion of two or more segments of DNA into a different arrangement than was present originally. This can occur with cellular genomes and with the smaller independent replicons of plasmid and viral DNA. The mechanisms of recombination and mutagenesis differ in many respects,' with recombination being the event by which organisms exchange information at the DNA level.The artificial recombination of DNA in i7itr-ois the keystone of the process of gene cloning and therefore the foundation of genetic engineering. Enzymes that cut DNA at specific sites (restriction endonucleases) and join the 3'-and 5'-ends of DNA (ligases) can be used to build molecules of DNA with endless structural variations. This enables the structural and functional analysis of genes their juxtaposition in different arrangements and thereby the construction of living organisms with properties that are quite different from those of their parents. In a sense this is evolution speeded up many-fold ; conversely the process promises to elucidate how present living organisms evolved.Genetic recombination is important to biosynthetic studies of natural products for two reasons. The first is its role in the development of strains where the goal is to create new organisms which have acquired the best properties of their parental strains. These properties might be growth on a certain nutrient better genetic stability or tolerance to a certain pH among the many desirable possibilities. Work towards such goals is currently proceeding empirically through the use of natural or artificial mating processes,I3O but is likely to become a rational endeavour as more information about the molecular biology of cellular biochemistry becomes available. The second reason for the importance of genetic recombination is its use in the search for new natural products which comes from the idea that random recombination of genomic DNA may result in the production of new secondary metabolites.Thus by artificially accelerating evolution it is expected that valuable metabolites will be found at a higher frequency than by screening the current population of living organisms. This seems sensible intuitively but there is not yet enough evidence for a valid judgement. An example from the field of antibiotics illustrates the use of genetic recombination in the production of a new natural product. Protoplasts can be made to fuse by treatment with polyethylene glycol under hypertonic conditions,5 and in the case of streptomycetes this results in more or less random genetic recombination at high frequency.l3'.I 32 Fusion of protoplasts from non-anti biotic-producing mutants of two 33 species of Streprotom~ws~ resulted in production of the antibiotic indolizomycin (47),'35 which was structurally unrelated to any of the known secondary metabolites of the two parental strains.The data do not prove that recombination actually took place in this instance nor can it be said that the production of (47) was caused by the recombination of genes (47) 143 from the two species as it could have resulted instead from the expression of normally silent genes (from either parent) in the fusant; I favour the latter explanation. Nonetheless this case invites further study to learn what did happen and justifies continued pursuit of the approach.(See also the case of iremycin in Section 6.) 4.3 Gene-cloning Methods The insight that led to the construction of chimeric DNA molecules (i.e. artificially made molecules that have an uncertain function) which was enabled by the discovery and isolation of site-specific restriction endonucleases three years earlier and the introduction of the chimeras into bacteria' 3s gave birth to the technology of recombinant DNA and the gene-engineering industry. Gene cloning in any context uses the same strategy. A vector i.e. a small DNA molecule (a plasmid or a viral genome) that is able to carry DNA into the host cell and which is capable of autonomous self-reproduction (replication) is chosen that has the following minimal properties.It must be easy to isolate in microgram amounts from the host organism; it must have at least one unique site for the insertion of foreign DNA (i.e.,DNA that was not originally part of the vector) that can be cut by a restriction endonuclease but which is in a region that does not interfere with the replication of the vector or with other functions that are essential for its existence; and it must carry a gene whose product signals the presence of the vector in the host cell (usually by making the cell resistant to some normally lethal substance). In the simplest case the appropriate restriction enzyme is used to cut the vector and the foreign DNA (eg. pieces of a bacterial genome) into pieces of a suitable size for insertion into the vector.The vector and the foreign DNA inserts are joined (by treatment with a DNA ligase) thus creating a population of molecules that have a random structure with respect to the size and the sequence of the DNA in the case of shotgun gene cloning. These vector-foreign DNA constructs (chimeras) are introduced into the host cell by one of several means the presence of the chimera in the cell being ascertained by selecting for expression of its gene that confers resistance to a lethal antibiotic for example and the population of host cells is then screened for a property that results from expression of a gene in the DNA that has been inserted. The process (and the result) of introducing the DNA into host cells is called transformation for circular (plasmid) or linear DNA transfection for viral genomic DNA and transduction for DNA that is carried within an intact virus.In the most general sense a cell is said to be transformed whenever it has been genetically modified by the introduction of purified DNA. A clone therefore is the genetically identical progeny of a cell into which has been introduced a unique piece of DNA containing a gene that imparts some specific property (the phenotype) to it. Any cell that contains this gene then has the corresponding genotype but this may not be evident phenotypically under all situations. Although gene cloning is not as simple in practice as is implied by the discussion above it is a powerful way to isolate genes and its success depends only on the availability of a suitable vector and in finding a way to detect the gene that is being sought amongst the many other clones that are of no interest to the investigator.Several strategies for gene cloning are reviewed in the monograph by Old and Primrose.' Ih I shall mention only those used in the streptomycetes since these are important for understanding the studies of gene cloning that are discussed in Section 5. Two reviews are available by Hopwood and his colleagues' 3h.1 about vectors and cloning strategies for streptomycetes. They have also recently produced an indispensible laboratory manual in which they describe this and related topics in the genetics of streptomycetes. 38 There are now several different kinds of vectors that can be used to introduce DNA into species of Strcptomj,c.es all of 144 which have been developed from indigenous plasmids or bacteriophages of this genus.pIJ61 is a typical plasmid vector and replicates at about 4-5 copies per bacterial genome (i.e.it is a vector that has a low copy number).I3' This vector contains genes that are capable of conferring resistance to thiostrepton and neomycin which are antibiotics that inhibit the growth of most strains of Streptomyces but it is currently known to have only a narrow host range. The gene for resistance to neomycin has a unique site (the cloning site) into which the insertion of foreign DNA results in its inactivation; hence the result is called insertional inactivation. Transformed host cells that contain the vector into which foreign DNA has been inserted can be identified as being resistant to thiostrepton but sensitive to neomycin whereas the undesired ones are resistant to both antibiotics.This property is very useful since construction of the DNA chimeras usually results in less than 100% with inserts (20-80% is typical). This strategy illustrates the most important attribute of a good vector for use with streptomycetes an ability to be screened easily for the presence of cloned DNA. Since the most interesting genes of streptomycetes encode properties that are not essential for bacterial growth (and therefore their presence cannot be selected directly) anything which reduces the laborious work of screening is a boon.pIJ702 which is a plasmid that has a high copy number (40-300 copies per host cell) and which can be inserted into a wide range of hosts represents another vector which facilitates gene cloning.'39 It carries the gene for resistance to thiostrepton (which is a valuable attribute because very few strains of Streptomyces are naturally resistant to thiostrepton) and the gene for tyrosinase in a small replicon (5.7 kb of DNA; 1 kb = 1000 nucleic acid bases). Tyrosinase is the enzyme from Streptomyces antibioticus' 39 that catalyses the oxidation of dihydroxyphenylalanine to melanin and its gene has three cloning sites which cause insertional inactivation. The result of this is readily apparent for the thiostrepton-resistant transformants with DNA inserts are not black in distinct contrast to those without inserted DNA.This result is easily visible of course only if the host strain does not normally produce melanin yet does produce it when it has been transformed with PI J 702. KC301 and KC400 are vectors that have been made from the bacteriophage $C3 1 which attacks a wide range of hosts; they are used somewhat differently than the plasmids.I3' KC301 imparts resistance to thiostrepton to its host and can accommodate about 4.8 kb of inserted DNA. When it is introduced into a host by transfection or transduction it integrates into the genome (chromosome) of the bacterial host by recombination between a specific 'attachment site' on the DNA of the phage and a corresponding site on the chromosome of the host (this is called lysogeny) resulting in only one copy of the vector per host cell.KC400 and its can accept 1.8to 9 kb of inserted DNA but in some of these vectors this can occur only by loss of the genes for resistance to antibiotic or for the repressor (which codes for a protein that is involved in maintaining lysogeny). The most important property of KC400 and its derivatives is the inability to lysogenize a host directly since they lack the attachment site that is required for recombination with the genome. Lysogeny results in expression of the gene for resistance to antibiotic; thus the only way in which these vectors can lysogenize a host and impart resistance to an antibiotic to it is by homologous recombination between their DNA insert and the genome of the host (or between the DNAs of two bacteriophages if the host is a lysogen of $C31 or one of its derivatives).The Campbell mechanism of homologous recombination' 42 results in duplication of the DNA insert and can cause inactivation of a gene if the insert is internal to the gene i.e. if it does not include the signals that commence and terminate the expression of that gene. This property of the vectors that have been derived from KC400 has been exploited in the powerful technique of mutational cloning that in principle enables the isolation of any gene which has a detectable phenotype without NATURAL PRODUCT REPORTS 1986 first having to isolate a strain that contains a mutation in the gene since mutants result directly from the gene disruption that accompanies lysogeny.143 It also provides an expedient way to obtain DNA fragments for use as probes in screening gene libraries (i.e. collections of randomly constructed clones representing an entire genome) for the genes that are of interest since the phage vector that contains the 'mutating' DNA fragment can be recovered easily from the mutated 1ysogen. Forms of each of these types of plasmid and phage vectors are available that also contain sequences of nucleic acids that permit their replication both in species of Streptomyces and in Escherichia coli; such vectors are 'shuttle vectors'. 37.138 Representative examples besides the vectors mentioned above are ones that have been constructed by researchers at Eli Lilly and Company such as ~FJ105'~~ It and ~HJL197.l~~ must be borne in mind that very few genes of Streptomyces are expressed in E.coli and shuttle vectors therefore are seldom used for the functional analysis of genes of Streptomyces in E. coli. Their main value is to facilitate manipulations of recombinant DNA and mutagenesis (including transposon mutagenesis e.g. ref. 152) using the many techniques that are available for E. coli.'46.147 Cloning strategies can take many forms depending on the phenotypic properties of the gene that is to be cloned and the information that is available about its product.' l6 With species of Streptomyces the genes for production of antibiotics have been most often sought and being non-essential (under laboratory conditions) usually have been found in one of two ways.The first approach requires through a shotgun cloning experiment the complementation of a mutation that has resulted in a specific metabolic block in the biosynthetic pathway. In this method a random collection of DNA inserts is used ideally to represent the entire genome of the wild-type strain and its success depends on one of these inserts providing the genetic information that is malfunctioning or absent in the mutant (complementation). Even when the insert is an incomplete gene or provides only a partial transcript the shotgun cloning method can work well enough because crossing-over between the DNA insert and resident DNA can restore the phenotype that is sought.It also works well even when the intermediates of the pathway are not freely diffusible and does not require prior knowledge of their structures unless the gene(s) for a particular metabolic function is desired. On the other hand since the preparation of a suitable mutant collection is empirical and laborious and all of the biosynthetic genes may not have a distinguishable phenotype shotgun gene cloning is not without its drawbacks. Nonetheless this approach has been used the most as evident by the examples in Section 5. The second approach depends on the fact that an organism must be resistant to the antibiotic that it makes if it is to continue to grow and that genes for resistance to its own antibiotic are known to be contiguous with the genes for their biosynthesis in several if not all cases.Since resistance to an antibiotic is a selectable genetic property it is comparatively easy first to clone the gene for resistance (because the cells of a normal sensitive host will not grow in the presence of the antibiotic unless they have been transformed with the gene for resistance to it) then to use it as a probe to isolate larger pieces of DNA that are contiguous with it or simply to clone large pieces of DNA in a sensitive host by direct selection for resistance to the antibiotic. While this approach seems easier than the shotgun method there are occasionally some practical problems stemming from the lack of sufficient knowledge about antibiotic-resistance properties or a convenient selection agent and less so from the instability of vectors that contain large DNA inserts.Until it is tested extensively I cannot predict if it will become the method of choice. Note however that it cannot supplant the use of blocked mutants since they are still needed to identify and to study the specific genes of a pathway. Two other cloning strategies based on the use of NATURAL PRODUCT REPORTS 1986 -C. R. HUTCHINSON messenger RNA (mRNA) to prepare cDNA clones (see Section 5 for an example) or the use of protein sequence data to make oligonucleotide DNA probes for locating specific genes in gene libraries are available in principle but have not yet been used in practice for genes of Streptomyces. This is due only to the comparative difficulty in identifying specific mRNAs of Streptomyce.s and in isolating samples of enzymes of the purity that is needed for protein microsequencing.5 Gene Cloning In this section I discuss examples of genes that govern the production of antibiotics by species of Streptomyces only for these illustrate best how typical genes for the biosynthesis of natural products can be cloned and studied. One example of a plant gene is included as a bellwether of the possibilities for cloning the genes for formation of secondary metabolites from plants. Previous reviews i37.148*159 on the subject of gene cloning in Streptomyces have been produced by Hopwood and co-worker s . Since resistance to antibiotics is directly selectable and sine qua non for the production of antibiotics such genes were the first to be cloned from streptomycetes and have had major importance in the development of cloning vectors.The gene for resistance to methylenomycin A from Streptomyces coelicolor was the first to be clonedlS0 and now there are many examples in the literature; for instance the cloning and characterization of genes that encode resistance to erythromycin neomycin thiostrepton and viomycin by Hopwood and his collaborators.' Further discussion of this subject is omitted because it is not directly important to the main topic of this section. Streptomyce.s coelicolor which is the organism that was used in the pioneering studies of the genetics of Streptomyces by Hopwood and his co-workers produces four different types of secondary metabolites which have measurable antibiotic activity two of which are also pigments.Thus it is not surprising that this bacterium was the source of the first genes for natural products to be cloned. All of the genes for the biosynthesis of actinorhodin (48) which is a pigment that has properties that make it suitable as an indicator of pH have been cloned in a low-copy-number vector (pIJ922)' '3 as one large (ca. 32 kb) segment of DNA.15J This accomplishment is a landmark for two reasons it showed that the genes for production of actinorhodin are clustered in one contiguous region of the bacterial genome which had been demonstrated at low resolution from the results of earlier studies of the genetics of its production by S.coelicolor 4y and it established that these genes could be expressed in another streptomycete following its transformation with the act DNA (gene descriptors usually are three-letter acronyms). Con- sequently the observations imply that the expression of genes for the production of antibiotics is not species-specific (this has been verified in several other cases; see below) and that genes for the biosynthesis of polyketides like those for actinorhodin are present as gene clusters (it is too soon to tell if this is true for all secondary metabolites of Streptomyces) which is consistent with current thinking about the relationship between the enzymology of the biosynthesis of polyketides and fatty acids. The work that IS reported in ref.154 furthermore highlights some technical considerations which may be general for gene co2 0 2 (48) cloning in species of Streptomyces. Vectors such as pIJ922 (containing large DNA inserts which impart a final size of almost 60 kb) appear to be stable in the strains that have been transformed with them whereas multicopy vectors such as pIJ702 which have large DNA inserts may not exhibit high stability. Stability in this context means an infrequent loss of the insert DNA such losses arising from recombination within the chimera or between the chimera and the genome of the host. Therefore it may not be absolutely necessary to use recombination-deficient streptomycete hosts which would minimize or avoid the loss of homologous DNA by analogy to the use of red mutants of E.coli when shotgun cloning DNA that had been obtained from the same species in which the blocked mutant was constructed. The biosynthetic pathway to actinorhodin has not been fully elucidated although it surely must resemble those for the formation of other isochromanequinone antibiotics. 52 Japa-nese workers have reported that they cloned a 4.3 kb segment of the DNA from S. coelicolor that encodes the production of a brown pigment whose chemical and spectral properties suggest that it is an intermediate or shunt-product of the pathway to actinorhodin. 56 Since the pigment was only produced if the DNA fragment was inserted into pIJ41 (which is the parent of pIJ61) downstream from a functional promoter sequence (i.e.a region at the 5'-end of a gene where a RNA polymerase binds to initiate transcription and which is thus vital for gene expression) the cloned segment did not apparently have its own promoter.Horinouchi and Beppu then used this fact to develop a plasmid which can be used to clone and study promoter sequences from Streptomyces. s6 This is an important development for the following reasons. Since the expression of many genes (and hence the level of their products) of prokaryotes is controlled transcriptionally the 'promoter probe' plasmid will enable semi-quantitative assay of the function of the promoter by monitoring the production of pigment. In organisms such as species of Streptomjws this control may be exerted through RNA polymerases that recognize two types of promoters vegetative ones which function throughout the growth period and developmental ones which function only during the period when cell growth has slowed greatly and cell differentiation has begun according to Westpheling et ~1.j~~ The production of a pigment or antibiotic is a typical property of the latter kind of promoter; therefore the plasmid could be useful in studies of how such pathways are regulated.Bibb and co-workers have constructed a different type of plasmid for use in the identification and semi-quantitative analysis of promoter sequences that occur in Streptomj-ces. 5x This vector contains a promoterless cat gene which encodes the production of a c hloramphen icol-i nac ti va ti ng ace tyl- transferase.Only when a DNA segment that contains a promoter sequence is inserted on the 5'-end of this gene can it be expressed and confer resistance to chloramphenicol to a host strain. This promoter-probe vector thus can be used to identify the promoter regions of cloned genes but only if the promoter functions early enough in the life cycle of the host to prevent excessive background growth of the non-transformed chloramphenicol-sensitivecells. It also can indicate the relative strength of promoters by the level of drug resistance that is imparted to the host strain. Both of these applications have been demonstrated recently by Bibb et a/. in a study of several of the genes for resistance to antibiotics in Streptomjws. 2'j Genes that encode the productionIho of A-factor (I 1) and indirectly control its production161 have been cloned from streptomycetes.In the former case a 1.1 kb fragment of DNA was found later53 to be sufficient for the production of A-factor in four different streptomycetes which do not produce (1 1) or contain DNA that is homologous with this fragment. The latter case is intriguing since strains of S. lividans and S. coelicolor which had lost the ability to produce actinorhodin A-factor and a red pigment regained all three properties when transformed with a ca. 2 kb segment of DNA that had been NATURAL PRODUCT REPORTS 1986 OH 7- 0 OH OH OH OH OWNH* (49) cloned from S. coelicolor.'6' This piece of DNA therefore must positively control the formation of all three substances in some way.Gil and Hopwood16' have cloned a gene that provides p-aminobenzoic acid (PABA) for the biosynthesis of folic acid by S.griseus and possibly also provides the p-aminoacetophenone portion of the polyene macrolide candicidin (49). They used both the insertional inactivation property of pIJ41 to locate clones that contained inserted DNA and the correspondence that exists between resistance to sulphonamides and the production of PABA which is known to cause over-production of the latter to facilitate identification of the pub clone. A 4.5 kb piece of cloned DNA complemented a pub mutation of and imparted resistance to sulphonamides to a strain of S. liciduns. It also complemented pubA and pubB mutations of E.coli but only after a portion of the Streptomyces DNA had been spontaneously deleted so that expression of the pub gene could occur from a promoter sequence of E. coli. DNA that is complementary to the naringenin-chalcone synthase mRNA of parsley has been cloned in E. coli by Hahlbrock and co-~orkers.'~~ This was possible because ultraviolet irradiation of dark-grown parsley cells causes a rapid increase in the concentration of mRNA for naringenin- chalcone synthase; hence the total mRNA from induced cells could be used for the identification of DNA that had been prepared from it in citro and cloned in E. coli. This type of cloning did not give the natural plant gene which encodes naringenin-chalcone synthase but a copy of it (cDNA) from which the introns (which are the non-translated sequences of eukaryotic DNA) were missing due to the way in which such cloning is done.Total mRNA from the induced cells was used as a substrate for reverse transcriptase which is the enzyme that can produce single-stranded DNA from an RNA template. The second complementary strands of DNA were then synthesized (by treating the DNA copies with DNA polymerase I) and ligated into pBR322 this being the most popular vector for gene cloning in E. coli. Clones in the latter host which contained cDNA that was complementary to the mRNA from induced cells but not from non-induced cells first were identified then screened for the production of a protein that reacts with naringenin-chalcone-synthase-specific antiserum after translation of their DNA in citro.The resulting cDNA clone had a 1.5kb insert that contained all of the coding sequence for naringenin-chalcone synthase. Ih4 The availability of this cDNA and others that were prepared similarlyIh5 permitted the discovery that the synthesis of secondary metabolites in parsley in response to stress conditions (irradiation with ultraviolet light or challenge by fungal elicitors) which has been implicated as a major defence response of higher plants is due to activation of genes resulting in transient increases in the rates of transcription of the associated genes. 63. 66 Two segments of DNA that complement a mutation in the biosynthesis of clavulanic acid (50) have been cloned from Streptomyces cluculigerus by the Beecham group using ~IJ702.'~' This represents the first report of cloned DNA from 0 (50) (51) the commercially important biosynthetic pathways to 0-lactam antibiotics.A group at the Eli Lilly company has established a second landmark in this field by their cloning of all of the genes for biosynthesis of erythromycin A (3). 68 This was accomplished in Streptomyces liciduns by the use of a cosmid vector which was made from plasmid and phage components and which could accept a DNA insert of cu 35 kb (cosmid vectors are described and defined in ref. 116) that could function in Streptomyces and E. coli. With such large inserts the complete Streptomjres genome is represented by only about 1000 independent clones which greatly reduces the screening effort.Colonies of E. coli that contained the cosmid with random DNA inserts from S. erythrueus [which is the species from which erythromycin A (3) was first isolated] were screened for the presence of the gene for resistance to (3),Is2 to identify clones which also might contain the genes for biosynthesis of erythromycin A. (It had already been shown by genetic mapping experiments that the ery genes are clustered.' 26) It was very rewarding to discover consequently that one of the clones that carries the gene for resistance to erythromycin A also conferred the ability to produce erythromycin A when plasmid DNA that had been isolated from it was transferred into S. liciduns. This resulted from the presence of an insert (of cu.35 kb) of the DNA of S. erythraeus in the vector; this amount of DNA is surprisingly small when one realizes that about 40 catalytic events are needed theoretically for the biosynthesis of erythromycin A. The presence of the erj' genes in this insert was confirmed by its ability and by the ability of specific DNA fragments that could be sub-cloned from it to complement three types of ery mutation when transformed into blocked mutants of S. erj.thrueus (P. Treadway R. H. Baltz and C. R. Hutchinson unpublished results). This work exemplifies the value of cosmid vectors when using the gene for resistance as the essential tool for cloning the genes for the biosynthesis of antibiotics. Mutational cloning is the keystone of the approach that has been used by Chater and Bruton to clone the genes that govern the formation of methylenomycin A (51) which is another antibiotic of Streptuntyces coelicolor.I 43 The methylenomycin A (mmj.)genes are carried by a large plasmid SCP 1 rather than being part of the chromosome of S.coelicolor and this fact was used to simplify the mutational cloning of mmj. genes. DNA from an SCP 1-containing strain of Streptomjws purtv.dlus was inserted into KC400 in ritro and the constructs were transfected into S. lividans to give plaques (i.e.the partially cleared areas on an agar culture plate that are produced by lysis of bacterial NATURAL PRODUCT REPORTS 1986 -C. R. HUTCHINSON colonies within which the phage is growing) that contained KC400 with a random assortment of DNA inserts that were <6 kb long.When S. liuidans that contained SCPl was transduced to viomycin resistance with this population of phages only those that contained insert DNA from SCPl were able to lysogenize this host because the rest either had no DNA insert or only contained inserts of the DNA of S. parvuflus which being essentially non-homologous with the DNA of S. lividuns could not recombine with the host at a significant frequency. Thus the majority if not all viomycin-resistant colonies contained DNA of SCPl and 278 independent colonies were screened for the absence of the ability to produce methylenomycin A due to insertional inactivation. Nine such clones were found and their genotype was designated !mmy! to distinguish them from mmy mutations that had been produced in the customary way.These clones in toto repre-sented about 7 kb of mmy DNA and three different types of !mmy !mutations based on their cosynthetic properties. The gene for phenoxazinone synthase (PHS) has been cloned from Streptomj-ces antibioticus by Jones and Hopwood169 in an elegant study that also uncovered two other unrelated DNA fragments that have the surprising ability to cause the production of this enzyme by S. liuidans.'70The PHS structural gene was cloned in pIJ702 by assaying transformants of S. liuidans for the production of PHS activity in uitro. Three different DNA fragments were isolated but only the one of 2.4 kb produced PHS in a coupled transcription-translation system of a streptomycete in citro,' and thereby was identified as the structural gene for PHS.The other two DNA fragments (i.8 and 4.3 kb) did not show this property (although they did encode the formation of other polypeptides) yet they conferred the ability to produce PHS on S.licidans if they were introduced into it by transformation. As the PHS that was produced from these latter two clones was indistinguishable from the purified PHS that has been described in Section 3 of this review the authors concluded that these two fragments could activate an endogenous PHS gene of S. lividuns which normally is silent or expressed at very low levels. Besides this unexpected outcome it was observed (as anticipated) that cells of S.liuidans that had been transformed with pIJ702 that contained the 2.4 kb fragment produced a higher level (about five-fold) of PHS than S.antibioticus presumably due to an increase in gene dosage from the high-copy-number vector. The first gene for biosynthesis of an antibiotic that was cloned"' was one that directs the formation of an 0-methyltransferase that is involved in the formation of the prodigiosin derivatives (52) and (53) which are the red pigments of Strepromj,ces coelicofur.' 73 In this instance the complementation by DNA that was carried in pIJ702 of a double mutant that was defective in the formation of the two pigments of S coelicolor was evident by the appearance of the characteristic red coloration that is due to (52)and (53) among essentially colourless colonies of the bacterium.(A similar ,OMe H [CH2IloMe (52) 0 0 advantage was enjoyed in the cloning of genes for actinorhodin when blue colonies were sought.) More recent work on this system174 has provided over 21 kb of the genomic DNA that complements red mutations of four classes and some informa- tion about the properties of the 0-methyltransferase. This enzyme seems to exist in two forms of M >5 MDa and 49 kDa respectively as deduced from studies of cell-free extracts that had been prepared from protoplasts and mycelia. The final example of cloning of genes for the production of an antibiotic concerns tetracenomycin C (561 which is a pigmented anthracycline metabolite of Streptomyces glaucescens' 75 that has significant antitumour activity.By complementation of blocked mutants of S. glaucrscerzs a total of 24 kb of DNA has been cloned in pIJ702 that contains all of the genes for the formation of (56) (H. Motamedi E. Wendt-Pienkowski S. Yue and C. R. Hutchinson unpublished results). When S. lividans or two other streptomycetes were transformed by the plasmid from one of the clones that contained a 12.4 kb DNA insert the red pigments (54)and (55) were produced in large quantity. The presence of pIJ702 with any of the cloned DNA in blocked mutants of S.glaucescens did not result in increased yields of (56) however which means that the normal mechanism by which the production of (56) is regulated can overcome the effect of an elevated gene dosage.6 Novel Antibiotics produced by Genetic Means Hybrid Antibiotics and Others In the previous sections of this review I have discussed examples of what can be learned about the biosynthesis of natural products by using biochemical and genetic methods in this section I present an interesting use of this information which may have commercial importance. It concerns a desire to increase the pace of discovery of new natural products by the application of methods by which the genetic constitution of organisms can be altered faster than the rate of spontaneous mutation but which are more directed than the outcome of random mutagenesis; it also concerns the basis for the specificity of biochemical interactions or the relative lack thereof in the case of pathways to secondary metabolites.Its output is hoped to be the creation of antibiotics that are structural hybrids or simple derivatives of known compounds and that have valuable biological activity. This is a commend- able goal yet will be difficult to reach until we truly are able to engineer organisms (or only proteins) to do whatever is desired. In the meantime the construction of hybrid antibiotics is being pursued empirically as illustrated by the three ways discussed below. Some of the early results look quite promising and certainly justify continuation of this approach. The pathways of secondary metabolism are not as tightly controlled and their enzymes not as fastidious as those of primary metabolism; thus it is not surprising to find more than one route in the pathway to a given natural product or more H H (53) than one type of product associated with an enzyme.These observations suggest that new ‘unnatural’ compounds might be formed if an organism is given analogues of the known intermediates of a pathway to natural products which is the foundation of mutasynthesis. Because Daum and Lemke reviewed this subject in 1979,’77 I mention only one recent example. There are three ways through which mutasynthesis can be accomplished by addition of analogues of the intermediates of a pathway to the normal producer to a blocked mutant of it or along with an inhibitor of some step of the normal pathway. The first approach is the least successful since the analogue must compete with the normal substrate of an enzyme that catalyses a reaction of a pathway and may do so poorly thus resulting in a low yield of the desired analogue of the usual natural product.The second method requires that the enzymes that catalyse stages beyond the metabolic blockade in the pathway function normally to transform the substrate analogue into the corresponding product analogue. Since the normal product is not formed by the mutant there is less competition for the substrate and therefore a better chance that the desired product will be formed. As the necessary blocked mutant may not be available this approach is complemented by the third where the biochemical inhibitor can produce a situation like a blocked mutant but without first having to make the mutant strain.The third approach cannot replace the use of mutants since there are few specific inhibitors of biosynthetic pathways that are suitable for use with intact cells (see Section 2.2). Cerulenin (l) however can be used for the mutasynthesis of antibiotics in this way if they are made by the polyketide pathway. Omura and co-workers have demonstrated its usefulness in their preparation of the chimeramycins (57) which are ‘hybrids’ of tylactone (9) and the deoxyamino-sugars that occur in spiramycin (58) and which have valuable antibiotic activity. 78 Other examples where cerulenin has been used for similar purposes are cited in their paper. A successful mating of two antibiotic-producing strains involves by definition the recombination of their genomic DNAs.Since mating is operationally a simple process this approach to creating novel antibiotics can be explored in a straightforward way but there is an important limitation. There are barriers to exchange of information at the DNA level between different species of most of the organisms that produce antibiotics (arising from low structural homology of DNAs and from the presence in the members of each species of restriction I M~,N 0- NATURAL PRODUCT REPORTS 1986 enzymes that recognize non-self DNA) that can prevent interspecific recombination. Nonetheless interspecific mating has been applied successfully in one case and resulted in the production of a new antibiotic. Non-producing genetically marked strains of Streptomyces hygroscopicus I MET JA6599 (which normally makes turimycin a macrolide) and of Streptomyces violaceus IMET JA6844 (which normally makes violamycin an anthracycline) were mated to give 90 recombinants which were identified by their growth on selective media.Three of these recombinants produced a new antibi~tic,”~ which was later identified as the anthracycline iremycin. The production of iremycin was apparently caused by some effect of the mating process but it cannot be said that this resulted from recombination of DNAs nor is iremycin truly a hybrid antibiotic since none of its structural features are present in the turimycins. Intraspecific mating of strains of Nocardia mediterranei resulted in the production of new ansamycin antibiotics that are related to the rifamycins.’81.’82 This is much more probably a result of the recombination of DNAs than the case above since the genome of strains that have been derived from the same species will be largely homologous but again these compounds were not true hybrid structures.The first example of the production of a truly hybrid antibiotic is the work of Hopwood and collaborators,’ 83 who capitalized on the close structural relationships between the three isochromanequinone antibiotics actinorhodin (48) medermycin (59) and granaticin (60) to create dihydrogranatirhodin (61) and mederrhodin A (62). Close inspection of structures (61) and (62) shows that C-3 of (61) is epimeric with C-3 of (60) but that the stereochemistry at C-3 of (61) is identical with that at C-3 of (48); (62) has an extra hydroxyl group at the position corresponding to C-8 of (59) where a hydroxyl group is also present in (48).These changes resulted from ‘cooperation’ between gene products in the pathways to actinorhodin and granaticin or to actinorhodin and medermycin when the strains that normally produce (59) or (60) were transformed with the act gene cluster or subportions thereof. Both (48)and (59) were produced when Streptomyces sp. AM-7 16 1 carried all of the act genes and (62) appeared only when a partial act clone was introduced into this host; in distinct contrast (61) was produced to the almost total exclusion of the normal antibiotics when S.viofaceoruberTu22 carried the act gene cluster. Therefore compounds (61) and (62) represent true hybrid antibiotics that have been created by genetic engineering. OH -OR OH (57)R = H or COMe I FCHO -0 MeO--OH OH NATURAL PRODUCT REPORTS 1986 -C. R. HUTCHINSON NMe2 I HO n Me H OH CO2 H (59) 2 7 Summary and Acknowledgments In this review of some biological methods that are useful for studying the biosynthesis of natural products I have inter- twined the discussion of methodology with examples of its use taken from the recent literature. By illustrating fruitful applications of these methods I have strived to highlight their utility to researchers who might wish to make use of them. It is clear from my presentation that enzymological studies are bearing much fruit and that suitable techniques of gene cloning are available and have great promise for rapidly accelerating the pace at which new knowledge will be acquired in this field.The preparation of this review would not have been possible without the stimulation of research support from the National Institutes of Health (GM 25799 GM 31925 CA 35381) and fellowships from the John Simon Guggenheim Foundation and Fulbright Program of the Council for the International Exchange of Scholars. I am particularly grateful to David A. Hopwood Keith F. Chater and Mervin J. Bibb for their advice and encouragement during a sabbatical leave in the Department of Genetics John Innes Institute Norwich England in 1983 and to David Hopwood for critical reading of the manuscript for this review.8 Addendum Two research groups have cloned genes from Streptomyces griseus that encode information that is needed for the formation of streptomycin (37).’ 84+1x5In both laboratories the researchers used assays for the enzymes of the biosynthetic pathway to streptomycin to characterize the phenotypes of blocked mutants,186*1 *’ and thereby to identify the particular str genes which complemented the str mutations. As noted for several other cases cited in Section 6 the results of this work showed that the genes for the production of this antibiotic and for self-resistance to it are clustered in the genome of Streptomyces griseus. The gene from the fungus Cephalosporium acremonium that encodes the formation of isopenidin-N synthetase has been cloned by researchers at the Eli Lilly Company.1ss Their work represents the first use in the field of natural products of an oligonucleotide whose nucleotide sequence corresponded to a portion of the N-terminal amino-acid sequence of the enzyme to probe a gene bank made in Escherichiu coli for DNA with a sequence that is homologous to the oligonucleotide probe.The clone that was identified in this way was shown to contain the desired gene based on the observation that isopenicillin-N (60) synthetase was formed when the correct DNA segment was isolated from the clone and was expressed in E. coli (after suitable genetic engineering to provide the nucleotide se-quences that are necessary for expression of the gene in this bacterium).Researchers at the Technical University in Berlin have cloned the genes for the production of the ornithine-activating fragment of gramicidin-S synthetase 2 from Bacillus brevis’ 89 and of tyrocidin synthetase 1 from the same bacterium.190 They used antibodies to gramicidin-S synthetase (which could cross-react to the two proteins) to identify Escherichiu coli clones that produced either protein by virtue of a special ‘expression vector’ which permitted the expression of the genes of B. breuis in E. coli. In this case and in the work on isopenicillin N alternative strategies of gene cloning e.g. complementation of mutations were not feasible. Finally Uchiyama and Weisblum have made an interesting speculation regarding the propensity of streptomycetes to produce a diverse number of natural products in their recent paper on the nucleotide sequence of the gene for resistance to erythromycin which was cloned from Streptomyces erythraeus.l9 They noted that the nucleotide sequences that signal the termination of gene expression have a much lower probability of occurrence in genes of Streptomyces than in those of other genera of bacteria.As one consequence they predicted that it should be easier for the streptomycetes to manufacture new proteins by small changes in the nucleotide sequence of a gene since there would be less chance that random mutation would be non-productive and would lead to sequence alter- ations which could not be expressed as functional proteins.Hence:191 “the environment of the Streptomyces genome can support a ‘molecular foundry’ in which experimental genes and their respective proteins are cast with high efficiency.” This would be just the attribute that was required for the diverse secondary metabolism of the streptomycetes to have evolved by natural processes. 9 References 1 R. Bentley Trends Biochem. Sci. 1985 10 171. 2 J. C. Vederas to be published in Natural Product Reports. 3 S. A. Brown in ‘Biosynthesis’ ed. T. A. Geisman (Specialist Periodical Reports) The Chemical Society London 1972 Vol. I p. I. 4 D. A. Hopwood Annu. Rec. Microhiol. 1981 35 237. 5 L. Ferenczy F. Kevei and M. Szegedi in ‘Microbial and Plant Protoplasts’ ed. J. F. Peberdy A. H. Rose H. J. Rogers and E. C. Cocking Academic Press London 1976 p. 177. 183 D. A. Hopwood F. Malpartida H. M. Kieser M. Ikeda J. Duncan I. Fujii B. A. M. Rudd H. G. Floss and S. Omura Nature (London) 1985 314 642. 184 J. Distler K. Mansouri and W. Piepersberg FEMS Microhiol. Lett. 1985 30 151. 185 T. Ohnuki T. Imanaka and S. Aiba J. Bacreriol. 1985 164 85. 186 J. Distler K. Klier W. Piendl 0.Werbitzky A. Bock G. Kresze and W. Piepersberg FEMS Micrubiol. Lett. 1985 30 145. 187 T. Ohnuki T. Iinanaka and S. Aiba Antimicroh. Agc~t.5 Chemother. 1985 27 367. NATURAL PRODUCT REPORTS. 1986 188 S. M. Samson R. Belagaje D. T. Blankenship J. L. Chapman D. Perry P. L. Skatrud R. M. VanFrank E. P. Abraham J. E. Baldwin S. W. Queener and T. D. Ingolia Noturc (London),1985 318 191. 89 M. Krause M. A. Marahiel H. von Dohren and H. Kleinkauf J. Bactrriol. 1985 162 1 120. 90 M. A. Marahiel M. Krause. and H.-J. Skarpeid. Moi.Gen. Gcwt. 1985 201 231. 91 H. Uchiyama and B. Weisblum Grnr) 1985 38 103.

ISSN:0265-0568    

DOI:10.1039/NP9860300133

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

P-Phenylethylamines and the lsoquinoline Alkaloids K.W. Bentley Department of Chemistry Loughborough University of Technology Loughborough Leicestershire 1E I I 3TU Reviewing the literature published between July 1984 and June 1985 (Continuing the coverage of literature in Natural Product Reports 1985 Vol. 2 p. 81) 1 [l-Phenylethylamines R' R' 2 I soquinolines 3 Benzyl isoq ii ino1ines 4 Bisbenzylisoquinolines 5 Cularines 6 Pavines and Isopavines H k5 7 Berberines and Tetrahydroberberines 8 Aza berberines 9 Protopines I 0 Ph t ha1 ide-i soqu inol ines I1 Spirobenzylisoquinolines Table 1 Isoquinolines (3) of Puchjrerrus weberr 12 lndanobenzazepines 13 Rhoeadines R' R2 R3 R4 R5 14 Spirobenzazepines Backebergine H OMe OMe Fi H 15 I soi ndoloisoq uinol i nes Isobackebergine H H OMe (>Me H 16 Isoindolobenzazepines Isosalsolidine* H OMe OMe 11 Me 17 Isoquinolinobenzazepines Isonorte huanine* OMe OMe OMe H H 18 I soindolobenmzocines Isonorweberine* OMe OMe OMe OMe ti 19 Emetine and Related Alkaloids Isopachycereine* OMe OMe OMe OMe Me 20 Morphine Alkaloids 21 Benzop hen an t hr id ines 22 Colchicine 23 Refere n ces Table 2 3,4-Di hydroisoquinolines (4) of PucRj*wreus ktdwri R' R2 R3 R4 RS Dehydroheliamine H OMe OMe H H 1 p-Pheny let hylamines Dehydrolemaireocereine H H OMe OMe H Dehydrosalsolidine H OMe OMe H Me Hordenine and [2-(4-methoxyphenyl)ethyl]methylaminehave Dehydronortehuanine* OMe OMe OMe H H been isolated from Erigonum ulcitum E.unnuum E. campunula-Dehydronorweberine* Ohle OMe OMe OMe k1 turn and E.infiaturn [2-(3,4-dimethoxyphenyl)ethyl]methyl-Dehydropachycereine OMe OMe OMe OMe Me amine and dimethyl(2-pheny1ethyl)amine have been detected in Buckohergiu milituri.s,2 and both of the amides (1) and (2) have been isolated from Critonidla ~curninata.~ The ionization constant of $-ephedrine hydrochloride has been determined5 Table 3 Tetrahydroisoquinoiines (5) of Puch ycerezis wchcrr and the. estimation of $-ephedrine in plasma by radioimmuno- assay has been described. R' R' R' R' R R" Weberidme H H OMc H I4 11 Heliamine H OMe OMe tl tl fl 2 lsoquinolines N-Methylheliarnine H OMe OMe II Me Tandem mass spectrometry employing a mass spectrometer Lemaireocereine H H OMe OMc tI 11 both to separate and to identify alkaloids has been shown to be Cdrnegine H OMe OMe tl Mc Me a valuable technique allowing very small samples of plant Nortehuanine OMe OMe OMe H H If material to be examined; in this way the twenty seven Tehuanine OMe OMe OMe H H Me 4nhalidine isoqu inoI ines dihydroisoquino1in e s and tetra h y d ro isoquino-Anhalonidinc H OMe OMe OIf H Me lines that are listed in Tables I 2 and 3 have been identified in Pellotine H OMe OMe 014 Me 11 11 OMe OMe 011 Me MC Puchyccwu.s whori those marked with an asterisk being 0-methyl pellotine FI OMe OMe OMe Me Mc new alkaloids.In addition a methoxytetrahydroisoquinoline Norweberine OMe Obfe OMc Ohle [I Df a hydroxytrimethoxy-N-methyltetrahydroisoquix~oline a tri-Weberine OMe Ohlc OMe OMe !I 44c methoxy-I -methylisoquinoline and a trimethoxy-l -methyl-3,4- Pdchycereine* OMe OMt OMe OMe Me ti N-Methylpachycereine OMe OMe OMe OMe Mc Vt.Thulictrum minus var. thaliadine and thalicthuberine rrtajuss Thalictrum thalictine and O-methylthalmine sultanahadenses Two stereoisomeric mono-N-oxides of calfatine”’ and of tetrandrineho and two isomers of tetrandrine di-N-oxideho have been prepared and their structures have been determined spectroscopically. The effects of tubocurarine,hl dauricine,h2. h3 tetran-on drine,hs-b6 and NN-dimethyltrilobine i~dide~’-~* the cardiovascular system and of cepharanthine on the aggregation of blood platelets,69 on the immune re~ponse,’~ and on the activity of adenosinetriphosphatase’ have been studied as have the neuromuscular blocking actions of dimethyltubocur- arine7’ and NN-dimethyltrilobine iodide7j and the pharmaco- kinetics of dimethylc~rine.’~ 5 Cularines Alkaloids of the cularine group have been isolated from the following sources bases marked by an asterisk being new Berbcris ruldiz.ianu’s linaresine (39)* and dihydrolinaresine (40)* Corydalis claviculine O-methylcularicine c[ar>icu/~t~’O.76.77 sarcocapnine breogan i ne (culacorine) (33; R’ = R-‘ = H R’ = Me) secocularine (34; R = Me)* and secocularidine (34; R = H)* Surcocupnos breoganine (culacorine) crussifolia7b-7 oxosarcophylline (35)* secoc ula ri ne and secocularidine Sarcocupnos celtine (33; R1 = R3 = M e c.nne~phyllu~~~ R’ = H)* celtisine (33; 78.7c) Rl = R’ = H R3 = Me)* 4-hydrox ysarcocapni ne (36)* oxosarcophylline yagonine (37)* and aristoyagonine (38)* R’ O R*O M V OR^ e (32) 0 0 Me (O0 M M Me0 Me0 Me0 NATURAL PRODUCT REPORTS 1986 Breoganine (33; R’= R3 = H R’ = Me) has been claimed to be a new alkaloid,7b but its structure is the same as that which was claimed for culacorine during the period of the previous review.80 These new bases now complete a series of cularine alkaloids that is as extensive in type as is found in the aporphine series.Yagonine has been converted into aristoya- gonine by benzylic acid tran~formation.~~ Linaresine (39) and dihydrolinaresine (40)are the first recorded alkaloids of the cularine group that have a 6,7,8-oxygenation pattern in the isoquinoline system and it seems probable that they are derived from a diphenolic derivative of dihydrorugosinone (41);75 this is a natural alkaloid occurring in Berheris durwinii,’” which is derived from berberine and may be prepared by the hydrolysis and aerial oxidation of polycarpine (42) itself preparable by the oxidation of berberine.81 4-Hydroxysarcocapnine (36)78 and limousaminego are the first cularine alkaloids that are known to contain a 4-hydroxy- group.6 Pavines and lsopavines In a study of the Knabe reaction which involves a radical chain mechanism it has been shown that the conversion of N-methyl- 1,2-dihydropapaverine into argemonine depends on the pres- ence of formic acid as a radical chain inhibitor. In the presence of inhibitors 1-benzyl-2-methyl-l,4-dihydroisoquinolines are persistent species.*’ ( )-Reframidine (45) has been synthe- sized from the aziridine (43) which was obtained from deoxypiperoin by treatment with ethyl chloroformate to give (44; R’ = CO,Et R2 = CH,Cl) conversion of this into (44; R’ = Me R’ = CHO) and cyclodehydration.83 The preparation of 13-oxothalidine (2I) from 3-oxoreticuline (20) is described in Section 3.7 Berberines and Tetrahydroberberines Alkaloids of this group have been isolated from the following species Ahutu hullatuHJ palmatine RO 0 -‘TMez Me0\/OMe Me0 Me0 (36) OH 0 MeO&O Meo0\ 6/ O H Me0 \ Me0 NATURAL PRODUCT REPORTS. 1986 -K. W. BENTLEY Berberis 13-me t hox y-8-0x0ber berine actinacanthas (46; R = OMe) O-methyl- prechilenine (47; R = Me) and prepseudopalmanine (48) Berberis aristataJs berberine and oxyberberine Berberis chochocoa6 berberine Berberis darwiniis 5.8' berberine 13-methoxy-8- oxoberberine jatrrorhizine U-methylprechilenine prepseudopalmanine and thalifendine Berberis glaucas8 berberine columbamine jatrrorhizine and palmatine Berberis trifoliolatas berberine and jatrrorhizine Berberis berberine 13-methoxy-8- ualdiuiana7 5.8 5.89 oxoberberine O-methyl- prec hilenine prepseudopalmanine and valachine (49) Cocculus laurijoliusg0 stepholidine Cordydulis bulbosa9 sinactine Corydalis cheilanthifoline and scoulerine gortschakovii' Corydalis mejoiia9' cavidine Corydalis p~ilida~~ tetrahydrocoptisine Fibraurea tinctoriag4 jatrrorhizine and palmatine Fumaria indica9 tetra hydrocop tisine Fumaria jud~ica~~ coptisine scoulerine and stylopine Gluucium berberine canadine a-and p-N-~quamigerum~' methylcanadinium iodide coptisine N-methylisocorypalminium hydroxide corysamine scoulerine stylopine a-and P-N-methylstylopinium iodide and N-methyl- tetrahydropalmatinium hydroxide Guatteriu discolor98 corypalmine discretamine discretine and 10-0-methyl- discre t ine Hypecoum erectum9 coptisine corydamine and N-methylcanadinium hydroxide Hypecoum coptisine corydamine and N-Ia~trJlorum~~ methylcanadinium hydroxide Mahonia siamensis50 berberine Pachygone ovata' coreximine and stepholidine Sinomenium acutum' O0 stepholidine Stylophorum berberine coptisine corysamine diphyllum' scoulerine stylopine and a-and P-N-methylstylopinium hydroxides Thalictrum berberine buicalense5 Thalictrum columbamine iongipedunculu tum Tinospora cupillipes' O2 columbamine dehydrodiscretine jatrrorhizine palmatine and stepharanine Berberine alkaloids have also been isolated from cell cultures of Coptis japonica'03 and Thalictrum minus.* OS Of these alkaloids the new bases 13-methoxy-8-oxoberberine(46) O-methylpre- chilenine (47; R = Me) and prepseudopalmanine (48) are the first highly oxidized berberines to be isolated that retain the carbon-nitrogen skeleton of berberine. Prechilenine of which (47; R = Me) is the methyl ether is presumed to be a precursor of the isoindolobenzazepine alkaloid chilenine into which it may be easily converted in the laboratory; and chilenine and a group of related isoindolobenzazepines occur together with (47; R = Me) in Berberis actinacuntha B.darwinii and B. (46) (47) (O0 OMe (48) (491 ( O0 W N y R (50) (51) R' OMe (52) (53 1 OMe (54) (55) ualdiviana (see Section 16). Valachine the structure of which was determined spectroscopically is a c-nor analogue of the previously known karachine and is assumed to arise from berberine acetaldehyde and acetone instead of berberine and two moles of acet0ne.8~ The berberine betaines (50; R = H) (50; R = Me) (50; R =Et) and (50; R = CH,Ph) have been photolysed to the corresponding keto-aziridines (51).Treatment of (51 ;R = H) with methyl iodide in methanol is accompanied by solvolysis of the quaternary salt to give the indanobenzazepine (52) whereas (51 ; R = Me) and (5 1 ; R = Et) suffer Hofmann elimination with methyl iodide to give (53; R' = H R' = Me) and(53;R' = R2 = Me). Withethylchloroformate,(51;R = H) gives the chloro-compound (54) but (51; R = Me) and (51; R = Et) lose hydrogen chloride in the process to give the olefins (53; R1 = H R2 = C0,Et) and (53; R' = Me R2 = CO'Et). Io5 Reductive removal of chlorine from (54) followed by hydrolysis affords the secondary amino-ketone which has been photolysed to the unnatural oxoberberine (55). The secondary alcohol (56) on treatment with ethyl chloroformate yields the cyclic urethane (57) which has been converted into NATURAL PRODUCT REPORTS.1986 the ketone (58); base-catalysed hydrolysis of this yields the unnatural betaine (59).Io6Photolysis of the betaine (50; R = ("0 OMe) in methanol has been shown to give the ketal (60).Ios The oxidation of 8-oxoberberines e.g. (46; R = H) with lead tetra-acetate has been found to give the 13-acetoxy-cornpounds % e.g. (46; R = OAc) in good yield and these may be reduced HO' OMe -successively with lithium aluminium hydride and sodium Meo \ / borohydride to 13-hydroxyberberines such as ophiocarpine (61) with the natural cis configuration. More prolonged Me0 oxidation of (46; R = H) yielded prechilenine (47; R = H) (56) (57) which had previously been prepared from the betaines (50; R = H) and (50; R = OMe).Io7 The condensation of 3,4-dihydroisoquinolineswith anions (O0 that have been derived from phthalides and 3-methylphtha- lides has been shown to give 13-hydroxy-compounds with only the cis arrangement of hydrogen and hydroxyl e.g.(62 R = H) and (62; R = Me) which can be reduced to the related tetrahydroberberines one of which has been dehydrated to (63).IosSyntheses of (-t)-corypalmine (64; R' = H R' = R3 Me0 OMe = R4 = Me),Io9 (+)-cheilanthifoline (64; R' = Me R' = H R3R4= CH2),Ilo and (+)-tetrahydrogroenlandicine(64; R' = (58) (59) H R' = Me R3R4= CH2)'Io have been achieved from suitable benzyl ethers of the general structure (65) which were prepared from P-phenylethylamines and isochromanones ; a similar synthesis of (+-)-discretine which is the 10,ll-dimethoxy-analogue of corypalmine has also been achieved.' (+-)-Tetrahydropalmatine(64; R1= R' = R3 = HO OMe R4 = Me) has been synthesized by thermal cyclization of the benzocyclobutene (66) followed by reduction.8,13-Dioxo-1 1-methoxytetrahydropalmatine has been synthesized by the OMe acid-catalysed hydrolysis of (68) itself obtained by photocycli- zation of the phthalimide (67). N-Methyltetrahydroberber-(60) (61) ine and N-methyltetrahydropalmatine salts have been obtained from the bases (69) and (70) where R'R' = CH or R' = Me0 / R' = Me by Pfitzner-Moffatt oxidation the cis salts being MeoqoMe formed from (69) and the truns salts from (70).' Transforma-tions of berberine derivatives into benzophenanthridines are \ OMe discussed in Section 21.An X-ray-crystallographic determination of the structure of dehydrocorydaldine chloride has been reported' and a study has been made of the pre-ionization degradation of quaternary (62) tetrahydroberberines in the mass spectrometer. I Ih An assay for (63) dehydrocorydaline in biological samples has been described. The effects of berberine on the content of bilirubin in bile,' of cavidine as a spasmolytic agent,"' and of stepholidine on behaviour119 have been studied and the use of tetrahydro- berberines as intercalating agents with DNA has been reviewed.' 2o 8 Azaberberines (64) (65) The three 10-azaberberine alkaloids alamarine (7 1 ;R' = Me R' = H) alangimarine (72; R1 = Me R' = H) and MeoaNH alangimaridine (73) previously isolated from Alungium lu-Me0 \ marckii have been found to be accompanied by isoalamarine (71 ;R' = H R' = Me) isoalangimarine (72; R' = H R' = Me) alamarinone (74) dihydroalamarine (75;R' = Me R' = H) and dihydroisoalamarine (75; R' = H R2 = Me) when the plant was re-examined.The structures of the new bases were elucidated by spectroscopy and by correlations by O-methyl- ations and reductions.' 21 (66) Me0 OMe (67) 9 Protopines Protopine has been isolated from Berberis durwinii,87 Coryciulis F. judai~a,~" Re pallid^,^^ Fumuria indi~a,~~ F. macrocurpa 22 R20'\o ~ N MFHzOH Glaucium oxylohum,' G. ~quamigerum,~~ Hypecoum erecturn,"" H. lactiJlorum," Stylophorum diphyllum' O1 and all Pupacer 0 species;' 23 allocryptopine has been isolated from F.judaicu,"h Me0 G. o.~yloburn,'~ G. squ~rnigerurn,~~ H. lucti-H. ere~tum,')~ Me0 Jiorum9" and S. diphyllum;'olcryptopine has been isolated from S. diphyllum. I (68) (69) NATURAL PRODUCT REPORTS 1986 -K. W. BENTLEY \N 0 OH (74) (75) (O0 -0 (78) (79) (82) (83) 10 Phthalide-isoquinolines Phthalide-isoquinoline alkaloids have been isolated from the following species Corydulis hulhosu'' bicucullinine Coryrirrlis bicuculline gortschukovii' Fumuriu indicu" adlumine narceimine and narlumidine (76) Fumuriu ,juduicu"h adlumine Fumuriu microcurpa' microcarpine (77) Pupucer somnjfev-umZx narcotine Microcarpine is a new alkaloid of the secophthalide-isoquino- line sub-group previously represented only by narlumidine.2-Narceine enol lactone has been found to be thermodyna- mically more stable than the E-isomer but in the less hindered hydrastine series which lacks a methoxyl group the stability of the isomers is reversed.'25 The photolysis of p-narcotine in methanol in the presence of eosin has been found to give 1-methoxyberberine betaine (78);' 2h in the absence of the dye and in the presence of oxygen the products of photolysis are cx-narcotine nornarceine methyl ester and cotarnine. ?' The negative-ion mass spectrum of corlumine has been found to be more structurally significant than the positive-ion spectrum. 28 The effect of bicucullinine on the consumption of oxygen in rat brain has been studied.I 2') (72) (73) (76) (77) OMe OMe (80) (81) -OH Q-0 (85) 11 Spirobenzylisoquinoli nes Fumaritine has been isolated from Fumuriu juduicu,"h 0-methylfumarophycine has been isolated from a Bulgarian herbal mixture of Fumuriu species,'3o the new and unusual alkaloid hyperectine (79) has been isolated from Hj>pucoum erecrum,' and the first known secospirobenzylisoquinoline alkaloid densiflorine (80) has been isolated from F'umuriu densfloru.' 32 The structure of hyperectine was determined by X-ray crystallography' and that of densiflorine by spectrosco- pic methods. 32 The structure of 'Base-C' which was obtained from (-)-epiophiocarpine methochloride by the action of callus cell cultures of Curjdulis species has been determined (by X-ray crystallography) as (81) and it has been shown that the N-methyl group of the quaternary salt migrates to oxygen during the transformation.' 3-3 The berberine alkaloid coptisine has been converted (riu the betaine by conventional methods) into the spiro-diol (82) in which one hydroxyl group was protected by its linkage to the nitrogen atom with formaldehyde to permit oxidation of the other to a carbonyl group; the product (83) was then reduced by sodium cyanoborohydride to ( )-corydaine (84).jJ The conversion of other berberine derivatives into spiro-com- pounds is discussed in Section 7. The photolysis of 6,7-dimethoxy-l-(2-methylbenzoyl)-3,4-dihydroisuquinolineinto a spiro-compound has been described. I 35 NATURAL PRODUCT REPORTS.1986 12 lndanobenzazepines One new indanobenzazepine alkaloid bulgaramine (85) has been isolated from 'Herba Fumaria officinalis' which is a commercial medicinal mixture of several Fumariu species that grow in Bulgaria. Although the alkaloid was previously known as a product of the pyrolysis of O-methylfumarophycine (86) (O-acetylfumaricine) which was also isolated from the same commercial preparation it has been established that it is not an artefact.]30 The production of the indanobenzazepine (52) from the berberine derivative (51 ; R = H) is discussed in Section 7. 13 Rhoeadines It has been claimed that rhoeadine is present in all Pupuver species. 23 14 Spirobenzazepines The new alkaloid ( & )-turkiyenine isolated from Hypecoum procurnbens has been assigned the novel spirobenzazepine structure (93) on the basis of detailed spectroscopic studies.It has been suggested that it is formed from coptisine (87) uia the polyberberine (88) (which was also isolated from H. procum-bens) and the rugosinone analogue (89). Conversion of this into the carbinolamine (90) followed by a rearrangement of the same type as that by which prechilenine (47; R = H) is converted into chilenine (96; R1R2 = CH,) would afford the isomeric carbinolamine (9 1). Reduction ofthis carbinolamine followed by its reaction with formaldehyde could then yield the oxonium salt (92) as an intermediate in the O-methylation of the phenolic hydroxyl and cyclization of (92) would lead to turkiyenine.36 (O0 (94) (95) 15 lsoindoloisoquinolines Nuevamine remains the only known isoindoloisoquinoline alkaloid but its structure has been revised following a study of the cyclization of the isomeric compounds (94; R1 = R2 = H R3 = R4 = OMe) and (94; R1 = R2 = OMe R3 = RJ= H) which were prepared by reduction of the corresponding phthalimides and by the condensation of homopiperonylamine with 3-bromomeconine and 3-bromo-$-meconine respectively. Trifluoroacetic acid causes (94; R' = R' = H R3 = RS = OMe) to cyclize to an isomer of nuevamine and (94; R1= R2 = OMe R3= R4 = H) to nuevamine which must therefore have the structure (95; R1 = R' = OMe R" = R5 = H) and not (95; R' = R2 = H R3 = R-l = OMe) as previously believed.The preparation of the alkaloid by the fission of chilenine (96; R'R2 = CH,) with methanolic sodium hydroxide to an acid that gives nuevamine on cyclization with loss of carbon dioxide is rationalized by postulating fission of the ion (97) to the dihydroxyisoindole (98) which can generate either of the carbinolamines (99; R1 = R' = H R3 = R-l = OMe) and (99; RI = R2 = OMe R3 = RJ = H).lsJ7 16 lsoindolobenzazepines The range of alkaloids of this group has been extended by the isolation from Berheris actinacantha B. ciarwinii and B. uuldiuiunu of 13-deoxychilenine (loo) chilenamine (1 01) pictonamine (1 02) chileninone (1 03) and palmanine (96; Rl = R' = Me). Protonation of the red base chileninone gives the phenolic salt (104). Pictonamine has been prepared from chilenine.*' In synthetic studies the compounds (105; R' = R' = Me) and (105; R1R2= CH,) have been prepared by the condensation of dimethoxy- and methylenedioxy-3,4-dihydro-isoqu inolin e w it h o-c h 1orom e t h yI benzoy1 c h 1orid e .(88) (89) HO-(96) (971 KL (98) (99) NATURAL PRODUCT REPORTS 1986 K. W. BENTLEY ~~ U OMe (106) (107) Me02C *C02Me (110) (111) Me0 Me0 “%!JMe\ (115) 17 lsoquino1inobenzazepi nes The new alkaloid saulatine (106; R1= R2 = Me) which is an analogue of puntarenine (106; R1R2= CH2),has been isolated from Abuta bullata. 85 18 Isoi ndolobenzazoci nes A new structural type which is believed to be derived (like puntarenine) from berberine has been identified in the alkaloid magallanesine (107) which was isolated from Berberis darwinii.Its structure was determined spectroscopically. 39 19 Emetine and Related Bases The new alkaloid alancine (1 08 ;R = C0,H) has been isolated from Alangium lamarckii and its structure has been confirmed by its preparation from ankorine (108; R = CH20H).140A synthesis of ( )-protoemetine has been achieved from secolo- ganin uia the triester (109) which was condensed with 2-(3- hydroxy-4-methoxypheny1)ethylamineto give after reduction the enamine (1 lo) which was cyclized to (1 11) with the unnatural stereochemistry. This could be equilibrated (in propionic acid) only before O-methylation. The resulting isomers were separated and methylated; (1 12; R = C02Me) was converted into protoemetine (112; R = CHO) by conventional methods.Condensation of the protoemetine with tryptamine yielded (5 )-deoxytubulosine. 41 The physical properties structures and sources of alkaloids of this group have been reviewed.I4’ A method of estimating emetine and cephaeline has been describedIs3 and the effects of emetine on tumour cells have been studied.’44 ‘R (109) (1 081 ‘Et HO OMe (112) (113) (114) (11 8) (119) 20 Morphine Alkaloids Morphine and hasubanan alkaloids have been isolated from the following species Cocculus laurijoliusgO 0-met hylflavinantine Papaver amurine amurinine apokrinomenon‘45 epiamurinine and di hydronudaurine Papauer bractazonine (1 13; R’= H R2 bracteatum’ 46 47 = OMe) neodihydrothebaine (113; R1= OMe R2 = H) the degraded thebaine (1 25) and thebaine N-oxides Papauer pilosum’ 48 amurine amurinine epiamurinine and di hydronudaurine Papaver somnijerum’ codeine morphine and thebaine Polyal th ia sebiferine cauliforal j9 Stephania oxostephamiersine japonica’ 52 oxoepistephamiersine oxostephasunoline (1 14) and stephadiamine (1 15) Stephania rotunda’ 53 cepharamine Thalictrum faberi5 pallidine Oxostephasunoline and stephadiamine are new alkaloids the latter being a novel c-norhasubanan whose structure was determined crystallographically Neodihydrothebaine and bractazonine are also new alkaloids presumably derived from thebaine (1 16) by alternative migrations of the ethanamine chain and the aromatic ring to give (1 17) and (1 IS) which collapse to the iminium salts (119; RI = OMe R' = H) and (119; R' = H R' = OMe) reduction of which would give the two alkaloids.42 Both have been synthesized by photocycliza-tionsoftheamides(l20;R' = OMe,R' = H)and(l20;R' = H R' = OMe) followed by reduction.Is4 Bractazonine has also been synthesized together with an isomer by a similar route from the amide (121),'4b and other isomers have also been synthesized. 55 Both of these alkaloids have been prepared from thebaine by photolysis and reduction. sh The unusual carbinolamine ether (125) which has not been given a trivial name is presumed to arise from thebaine N-oxide and has been prepared from one of the isomers of the oxide by refluxing it in chloroform.The reaction may be represented as a Cope degradation of the oxide (122) to (123) loss of water to give the imine (1 24) and hydride transfer (with nucleophilic attack of nitrogen at C-5) to give (I 25). Protona-tion of (I 25) yields the iminium salt (126). A second product of rearrangement of the N-oxide is the aminophenol (127) presumably formed from (123) by N-demethylation to the secondary base followed by nucleophilic opening of the oxide bridge. On this basis and on spectroscopic evidence the previous assignment of structures to the two thebaine N-oxides has been reversed.'47.'57 The two phenanthrene derivatives (128) and (129) which were originally identified as trace impurities in samples of heroin have been prepared by heating thebaine with acetic anhydride; they presumably arise from (130) by the alternative migrations shown.' s8 The 2,3-epoxypropyl ether of thebaol (131) has been produced by heating either thebaine or its N-propyl quaternary salt with epichlorohydrin.59 This type of degradation has only previously been reported to occur if quaternary salts are heated with acetic anhydride. 6-Demethoxythebaine (132; R = H) has been prepared by the action of sodium hydride on 6-0-mesylcodeine and on bromocodide,' 6o and 6-chloro-and 6-bromo-6-demethoxythe-baine [(132; R = C1) and (132; R = Br)] have been prepared by the action of lithium chloride and lithium bromide on 14-chloro-and 14-bromo-6-0-tosylcodeine respectively. 6-Deoxythebaine (132; R = Me) has been prepared from Meo& HO -\ Br NMe Br NMe R' PO OH R2 (120) MeN 'I Me0 Me0 Me0 (125) (126) NATURAL PRODUCT REPORTS 1986 6-methyl-6-0-methylcodeine by heating it with potassium t-butoxide.62 6-Demethoxythebaine has been subjected to Diels-Alder addition of methyl vinyl ketone and ethyl acrylate and the products have been converted into analogues of etorphine.Ih3 The addition of methyl vinyl ketone to 6-deoxythebaine has been shown to give the endo-adduct (133) and its C-7 epimer the c>.uo-adduct(134) and the 8cc-endo-ketone (135); both (133) and (135) have been converted into analogues of etorphine.IhZ 6-Demethoxy-P-dihydrothebaine adds methyl vinyl ketone to give the e-uo-adduct (136).'63 Attempted Diels-Alder addition of allenyl phenyl sulphone to thebaine gave a 2 1 mixture of E-and 2-isomers of (137); p-dihydrothebaine methyl ether afforded (138) under similar conditions.164A series of oxadiazoles of the general structure (139) in which R is alkyl alkylthio or tertiary amino has been prepared from both 7u-and 7P-adducts of thebaine and methyl acrylate.lhs N-Demethylation of bases in the morphine series has been achieved in high yield by treatment with a-chloroethyl chloroformate and heating of the products in methanol;lbh N-alkyl nor-bases in the series have been prepared from quaternary salts by selective N-demethylation with sodium thiophenoxide.67 Details of preparations of isomeric quater-nary saltsIh8 and diesters16" of morphine of 8-[di(2-chloro-ethyl)amino]~odeine,'~~ 7,7-disubstituted dihydrocodein-of ones 17 I -I73 of 14-methoxydihydro-codeinone,-morphinone and -thebainone,' 74 of 10-hydroxycodeine and 10-0x0-morphine,' 7s of 6,6-spirohydrazino-l4-hydroxydihydro-morphine,' 76 of the spiro-lactone (140),'77 of analogues of funaltrexamine,' 78 of 3-thioetorphine' and its derivatives,' and of 6-tritioethylmorphine have been published.' The pyrolysis of heroin,I8' the n.m.r.spectra of amurine pallidine and sebiferine' 83 and of cis-and trans-dihydrothebainone,' 8J and the metabolism of morphine'*5 have also been studied. trans-Dihydrocodeinone ketal (144) has been synthesized from the nitrile (141) by conversion into the ketone (142) cyclization to the olefin (143 ozonolysis and reduction.'*' Full details of the synthesis of 0-methylpallidinine that was reported in the previous review have been giveniss and a patent has been published concerning a previously reported MeN Me0 Me0 (130) M:$!p M:7ji!pMe R\ NMe -NMe M:?& Meo@ Me NMe Me .'COMe Me I COMe COMe (131) (132) (133) (1 34) (135) NATURAL PRODUCT REPORTS 1986 -K. W. BENTLEY MeO \ Meon "'"0, Me0 Me0@!Me HOV-iMe CHS02Ph CHS02Ph COMe (136) (137) HojQ MeOfi 0-OH Me pi Me 0 '-H Me0 Me0 (141) Me0 Me0 Lo iH2COMe (144) (1 45) synthesis of dihydrothebainone and its derivatives. 89 The preparation of 16-oxosalutaridine and 16-oxopallidine from 3-oxoreticuline is discussed in Section 3.Methods of detection and estimation of morphine,' 90-1 97 3-acetyl-and 6-acetyl-m0rphine,'~~heroin,192. 196-199*200 1953 codeine,l95.201 nicomorphine 20 14-hydroxydihydrocodein-nalbuphine,'O" nalmefene,Io5rearrangement products of morphine codeine and thebaine,'06 and trace elements in samples of morphine'07 have been described. The analgesic and pharmacokinetics2I 9,220 of morphine have been studied as has the use of the alkaloid in The effects of morphine on behaviour,z17. on the 218. 223 237 on the gastro-intestinal tra~t,~~*-~"~ (138) 0 'N LN/ R (139) Me0 Me0 'H MeO- MeO (142) CHO (146) (1 47) of m~rphine,~codeine,338 morphine ep~xide,~~'j dihydro-morphine,340rn~rphinone,~"' quaternary salts of 14-hydroxy-dihydromorphinone 342 nalorphine343 and its methiodide,344 naltrex~ne,~~~, 3~~.nal-345-351 naltrexone spirohydant~in,~~' bUphine,245.353-356 buprenorphine,'lB,227.229.2~O0.2~8.286.335 etorphine,?' 357 dipren~rphine,~~~ hydrazones of naloxone naltrexone and 14-hydro~ydihydromorphinone,~naloxone 5cJ a~ine,~~O funaltre~amine,~~~ derivatives of -362 363 364 7-amino-6 14-endo-ethenotetrahydro-oripavine,34s~ phen-3hh ethyldihydr~thebaine,~~~?~~~ and ~alutaridine,~~~O-methyl-fla~inantine.~~~ and 21 Benzophenanthridines brain,'j8 '55 on on the spinal ~ord~~~.~~~ cerebrospinal fluid,260on respiration,261on locomotor activ-Benzophenanthridine alkaloids have been isolated from the ity,262on the intake of and of on body tem-following perature,265on the eye,266on the placenta,267on induced con-Corydalis bulbosa9' vu1sions,268on frog muscle,269on neuronal ~esponses,~~O-~~~ Corydalis conspersa3 on membrane potential^,^^^.^^" on white blood on the levels of thyrotropin,"6 of CAMP and cGMP,'~~ of oxyto-cin,278.?79of dopamine,280,281 and of glucosezs2in the blood Corydalis ~allida~~ and on the effects of phy~ostigmine~~~ and of the~phylline**~ Fagaropsis have been studied.angolensis3 The narcotic antagonist properties of naloxone have been Fumaria indica9 studied,185 291 as have the effects of this compound on Glaucium behaviour,223.23'. 2'92 298 on forms of s~oc~,~~~-~~~ on the squamigerum9? brain,30h.307 on the treatment of on the spinal Stylophorum on the cardiovascular system,31 on the gastro-diphyllum' * intestinal tract,'J1.'47.313on dependence on al~ohol,~~"-~~~ on the intake of f~od~~~.~~~ and on coughing,320 Zanthoxylum on intractable pain,321on performance during exercise,322on rubescens3 nausea and emesis,323on experimental panperit~nitis,~~" Zanthoxylum on acupuncture-induced analgesia,325 on tumo~rs,~~~ tessmannii374 327 on induced convulsions,268on hypergly~aemia,~'~ on the levels of P-endorphir~"'~ and on the effects of and of d~pamine,~~~ ~aprotil,~~~.~~' and of LSD.334 of ~lonidine,~~~ dihydrosanguinarine corynoline 0-acetylcorynoline corynoloxine and conspermine (145) dihydrosanguinarine 6-hydroxymethyldi hydron i tid ine norsanguinarine chelerythrine chelidonine chelirubine and sanguinarine chelerythrine chelidonine chelirubine macarpine and sanguinarine 9-methoxychelerythrine and nitidine chelerythrine dihydro-chelerythrine 9-methoxy-chelerythrine nitidine and dihydronitidine The pharmacological and physiological effects of the Conspermine and 6-hydroxymethylnitidine are new alkaloids following have also been studied :her~in,~~~-~~~,~~~ other esters whose structures have been confirmed by partial synthesis from 164 U-acetylcorynoline and nitidine.The structures of arnottian- amide (146; R' = H R2 = R3 = OMe) isoarnottianamide (146; R1= R2 = OMe R3 = H) integriamide (146; R'R2 = OCH20 R3= H) and iwamide (146; R1 = H R2 = OH R3 = OMe) have been established by their preparations by Baeyer-Villiger oxidations of chelerythrine nitidine avicine and N-methyldecarine respectively.37s Bischler-Napieralsky cyclization of 0-methylarnottianamide and O-methylintegria- mide affords confirmation of the structures of chelilutine (147; R1 = R? = OMe) and chelirubine (147; RIR' = OCH,O) and this process has been extended to a total synthesis of sanguirubine by the conversion of the ketone (148) into an NATURAL PRODUCT REPORTS 1986 (155; R = CH=CH2) oxidation of which afforded (*)-corydalic acid (155; R = CHzC0,H).383In model syntheses the 13-methyl-analogue of (149) has been converted into (1 56) by the photocatalysed addition of nitrosoben~ene~~~ and the pyrylium salt (157) has been converted [through the isoquino- line (15S)l into the benzophenanthridine (159).38s Fusion of fagaronine (1 60) has been found to give a mixture ofthetertiarybases(161;R' = Me,R' = H),(161;R1 = R' = H) and (161 ;R' = R' = Me).386 The stereochemistry and c.d.spectra of benzophenanthridine alkaloids have been correlated387 and the high-resolution n.m.r. spectra388 and ionization constantsJ of some of the alkaloids have been analogue of 0-methylintegriamide followed by cy~lization.~~~ studied. The intercalating properties of A Pomeranz-Fritsch synthesis of such compounds containing an alkoxy-group at C-10 has proved unsuccessful. The berberine derivative (149) has been converted into the acetal (150) by treatment with thallium nitrate in methanol and cyclization of the acetal gives oxochelerythrine (1 5 I) which is reduced by lithium aluminium hydride to hydroxydi- hydrochelerythrine converted into chelerythrine chloride by 10% hydrochloric acid and then into dihydrochelerythrine by sodium b~rohydride.~~~ Similar syntheses of fagaronine and nitidine have been achieved from U-benzyldehydrodiscretine and pseudoberberine respectively.379 Sanguinarine cheleryth- rine and oxosanguinarine have been synthesized by the photolysisof (152; R1R2 = CH2,R3 = Me) (152; R1= R' = R3 = Me) and (153) respectively,380 and decarine has been synthesized by the cyclization of (152; R1 = H R' = Me R3 = H) by p~tassamide.~~' Dihydrosanguinarine has been oxidized to sanguinarine by dichlorodicyanobenzoquinone.38' The base (154) which was obtained by degradation of N-methyldihydrocorysamine has been converted into the base (148) R2 0 (152) Lo 0 (153) NPh NMe sanguinarine with DNA have been examined.1'0,38".3"0 22 Colchicine Colchicine 2-demethylcolchicine 3-demethylcolchicine de-mecolchine and 2-demethyldemecolchine have been isolated from Colchicum ~uturnnale.~~~ N-Methyl-N-deacetylthiocolchi- cine has been prepared from c~lchicine.~~' Colchicine has been converted (by hydrogen peroxide) into a mixture of the ring- contracted compounds (162; R1 = H R2 = OMe) and (162; R1 = C02Me R2 = H)393 and has been found to give condensation products with adenine adenosine monophos- phate and DNA by amination of the methoxyl group of the trop~lone.~"~ The n.m.r.spectrum of isocolchicine has been studied,39s and tritium-labelled colchicine has been prepared.3c)6 The neurotoxic effects of colchicine have been studied3'" as have the effects of the alkaloid on germinating ~eeds,~'"~ on leukaemi~~~~ and tumourl 4J.400 cells on ocular responses to 0 NMe (154) (155) Me Me 0 NATURAL PRODUCT REPORTS 1986 -K.W. 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ISSN:0265-0568    

DOI:10.1039/NP9860300153

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

Pyrrolidine Piperidine and Pyridine Alkaloids A. R. Pinder Department of Chemistry Clemson University Clemson South Carolina 29634-1905 U.S.A. Reviewing the literature published between July 1984 and June 1985 (Continuing the coverage of literature in Natural Product Reports 1985 Vol. 2 p. 181) 1 Pyrrole and Pyrrolidine Alkaloids 1.1 Sceletium Alkaloids 2 Piperidine Alkaloids 2.1 Spiropiperidine Alkaloids 2.2 Lactonic Piperidine Alkaloids 2.3 Bispiperidine Alkaloids 3 Pyridine Alkaloids 3. I Nicotine Alkaloids 4 References A review of chemicals occurring in the glands of ants includes a discussion and summary of pyrrole pyrrolidine piperidine and pyridine (nicotine-type) alkaloids present therein. A chapter in a new volume of a well-known alkaloid text reviews the use of spectral methods for structure determination in these groups of alkaloid^.'^ 1 Pyrrole and Pyrrolidine Alkaloids Pyrrole continues its emergence as a structural feature in the alkaloid field.Pyrrole alkaloids from marine sources have been reviewed. 2b Brunfelsamidine (l) which is a convulsant alkaloid that has been isolated from the roots and bark of Brunfelsia grandflow (an upper Amazonian tree) is pyrrole-3-carboxami- dine; it was identified by spectral investigation and by its synthesis from 3-cyanopyrrole by an established pr~cedure.~ 9,2l -Didehydroryanodine (2) is a new toxic alkaloidal constitu- ent of the botanical insecticide Ryania speciosa. Its structure has been deduced on chemical n.m.r.and mass-spectral evidence. 0 _HNH HO t-i (2) R~R*=CH~ (3) R' = Me,R2= H (4)R1= H,R2= Me r On reduction it affords the known alkaloid ryanodine (3) and its 9-epimer (4).j The buprestins (A and B) which are bitter principles of the jewel beetles (Family Buprestidae) may be formulated as 1,2,6-tri-O-(pyrrole-2-carbonyl)-P-~-glucopyran-ose (5) and 6-0-(p-hydroxybenzoy1)-1,2-di-O-(pyrrole-2-car-bony])-P-D-glucopyranose (6) respectively chiefly on spectral evidence. Several simple pyrrolidine alkaloids have been synthesized by a sulphide-contraction procedure outlined in Scheme 1.6 By means of it racemic hygrine (7; R = CH3) dehydrodarlin- ine (7; R = PhCHACH-) dehydrodarlingianine (7; R = PhCH'CHCHLCH -) and N-methylruspolinone [7; R = 3,4-(MeO)2C,H3] have been made.(2R 3S)-3-Hydroxy-2- (hydroxymethy1)pyrrolidine has been isolated from the seeds of Castanospermum australe A. Cunn. ; it was identified by n.m.r. and mass spectroscopy and by X-ray diffraction analysis.' Likewise 3,4-dihydroxy-2-(hydroxymethyl)pyrrolidine has been found in fronds of Arachniodes standishii (Moore) Ohwi. Its configuration (2,3-cis 3,4-tranLs) has been settled by n.m.r. spectral analysis of coupling constants.8 It has also been encountered in fruits of Angylocalyx boutiqueanus TOUSS.~ (+)-Codonopsinine (8) i.4. the enantiomer of the natural ( -)-alkaloid has been synthesized from L-tartaric acid (Scheme 2). The synthesis establishes the absolute configura- tion of the natural base to be 2S,3R,4R,5R.l0 2-Ethyl-5-heptylpyrrolidine (9),which is a component of the venom of the South African fire ant Solenopsis punctaticeps has been synthesized (as a mixture of cis- and trans-isomers) by application of a new route to pyrrolidine bases outlined in Scheme 3 which involves the formation of an enamidine from the cyclic base followed by metallation and alkylation.' I (5) 1 -II Br -+ iii QCHCOR 4@COR Me Me CH2COR Me Me Me WcoR L J (7) Reagents i BrCH,C'OR ii base [sometimes with a thiophile.such as PPh or P(OEt),]; iii LiAIH, under carefully controlled conditions Scheme 1 NATURAL PRODUCT REPORTS 1986 OMOM i -iv L -Tartaric acid PhCH20+OH MOMO OMOM 1 c-PhCHzO PhCH2O wAr vi MOMO OH MOMO vii 1 OMOM OMOM PhCH204 Ar ...VIII O V + HMOM0 h A r + MOMO 2 cpi mers (separable) H? OMS OMOM OH OMOM x,xi xii,xiii /rkAr YYAr f-MOMO NHCbz c-MOMO Reagents i EtOH TsOH; ii MOMCl (MOM = MeOCH,) PrlzNEt CHCl, at 60 "C for 36 hours; iii LiAIH,; iv PhCH2CI (1 equivalent) Bu",NBr; v (C0C1)2 DMSO Et,N CH,C12 at -78 "C (Swern oxidation); vi p-MeOC,H,MgBr THF warm from -10"C to room temperature over 14 hours; vii phthalimide (2.5 equivalents) diethyl azodicarboxylate PPh, THF at room temperature for 14 hours; viii Pd/C H2 MeOH; ix MeMgBr Et,O at -78 "C to room temperature; x N2H4 EtOH reflux then CbzCl (Cbz = PhCH20CO) Na,C03 CHzC12 at 0 "C; xi MsCI Et,N CH2Cl2 at 0 "C for 10 minutes; xii HCHO Pd/C Ht MeOH; xiii aq.HCI MeOH at 50 "C for 2.5 hours Scheme 2 0i_ 0 lL GSePh OR 50 ___) iv H I I I I CH-NBU' CH=NBU' CH-NBU' CH-NBU~ (9) cis and trans Reagents i Me,NCH=NBu' toluene (NH,),SO,; ii Bu'Li (PhSe)?; iii NaHC03 at room temperature; iv Bu'Li or Bu"Li n-C,HI5I; v Bu'Li EtI HMPA; vi N2H, AcOH EtOH at room temperature for 3 to 4 hours; vii LiAlH Scheme 3 NATURAL PRODUCT REPORTS 1986 -A. R. PINDER 173 Anatoxin-a (lo) which has been synthesized from a Dysidin is a unique halogenated marine alkaloidal product pyrrolidine derivative,' * is discussed in the Report on Tropane of structure (1 I) that occurs in the Indopacific sponge Dysidea Alkaloids (Natural Product Reports 1986 3 182). herbacea and which contains a trichloromethyl group.Two syntheses have been reported one (Scheme 4) leads to the racemic varietyI3 and the other (Scheme 5) to the (-)-form which is the enantiomer of the natural product.14 1.1 Scefetium A1kaloids Re-investigation of the synthesis of ( -k)-mesembranone(1 2) by dissolving-metal reduction of the cyclohexenone (13) has revealed that the by-product (14) is also formed. A pathway for Br iv 1 OMe 00 CI3C + OACI CI 0II + EtOCCHZCOzH ix OEt NPhth NPhth (Phth = phthalyl) 0 (B) Me0 0 (11) Reagents i CBrCl, azobisisobutyronitrile; ii Zn iii SOCl, DMF; iv Meldrum's acid pyridine; v HOCH,CH,SEt; vi KH HMPA Me,SO,; vii Me,O+ BF,- OH-; viii SOCl, DMF; ix Bu"Li; x KH MeOS0,F; xi N2H4; xii EtMgBr Scheme 4 C02Me COCl I I iv-vi vii viii,i> + VCOzMe -wH H H CCl3 CCI3 0 CC13 OMe CCI3 OMe (0) X (C 1 + (0) + (11 ;5R,lOR) Reagents i H2 Pd; ii NaOCEtMe, toluene; iii Me,SO, MeOH; iv SOCl,; v Meldrum's acid pyridine; vi MeOH at 65 "C; vii (MeO),CH H2S04; viii KOH EtOH then H+; ix (COCl),; x BuLi THF at 0°C Scheme 5 174 NATURAL PRODUCT REPORTS.1986 its formation has been suggested.’ (*)-Sceletium alkaloid A4 (19) and (20) of closely related structure.19 The structures of the (1 5) has been synthesized from (i-)-mesembrine by conversion latter were settled by spectral study and by correlations with of the latter into its oxime 0-ally1 ether (16) by reaction with 0-derivatives of gerrardine. allylhydroxylamine. This ether gave two products on thermal Synthetic investigations directed towards the cytochalasans rearrangement these being sceletium alkaloid AS (1 5) and its which are an important group of biologically active fungal linear isomer (17).16 Another synthesis of (15) involves an metabolites have been reported.One group has described a iminium ylide-olefin [3 + 21 cycloaddition reaction (Scheme pathway to the decenoate ester (21)’O and to the tetrahydro- 6). ’ pyrans (22) and (23) and the dioxolan (24),” all of which Gerrardine (18) has been found in the bark of Cassipourea are synthons for the construction of various subunits of the guianensis’* along with two new sulphur-containing alkaloids cytochalasan frameworks. The phosphonium salt (24) has been OMe OMe MeN Me ’IN Me H S02Ph H H HO (12) (13) (14) (1 5) (16) (17) OMe OMe OMe ...Ill iv 0”” + \ + POMe Br Nc$jOHC OMe vi -viii (15) f--Et02C Reagents i H,C-PPhs3 THF (Wittig reaction); ii,N-bromosuccinimide; iii 3-cyano-2-methylpyridine LiN(SiMe,), at -78 ”C HMPA THF; iv Bu’?AIH at 0 “C; v sarcosine ethyl ester xylene molecular sieves at 180 “C for 7 hours; vi OH-.THF MeOH; vii PhOP(O)CI, at 100 ’C for 20 minutes then H20 MeOH acidification; viii NaBH,CN Scheme 6 0 H‘ H Me H-(19) OCH,OMe 1I ThpOp ’’C02Et Br (Thp = tetrahydropyran -2 -yl) NATURAL PRODUCT REPORTS 1986-A. R. PINDER I I (23) (24) I I 'CHO HN HN 0 0 (+1 -(R)-Citronellot , 0 xi ,xii viii -x vii -t--t =&o OEt H .I I xiv,xv xvi xvii ___) -Reagents i Me,Bu'SiCI imidazole; ii 03, then Me,S; iii Ph,P=CH[CH,],CO,Et; iv Bu",NF KF.2HzO; v catalytic hydrogenation; vi Swern oxidation; vii BuLi (EtO)?P at -78 "C THF; viii OH-; ix H+; x 1,l'-carbonyldi-imidazole;xi LiN(SiMe,) THF, Y NC'OPh. at -78°C; xii LiN(SiMe,)? PhSeC1 at -78°C; xiii rn-chloroperoxybenzoic acid H202 CHCI, H,O at -50 to 0°C; GIh xiv CHCl,-toluene at 100 "C for 12 hours; xv KOH benzene MeOH; xvi LiNPr', at -78 "C PhSeC1; xvii H20, pyridine CH2CI Scheme 7 NATURAL PRODUCT REPORTS 1986 ii I I Q -LH I sOAc -C7H15 C02Me C02Me C02Me iv H CllH23 -Qc11H23 I C02 Me (29) (cis -and trans -isomers ) Reagents i Hg(OAc)? THF; ii NaBH, then C,HI ,C(0)CH=CH2; iii thioacetalization then Raney nickel; iv HCl EtOH Scheme 8 (29) Reagents I n-CgHl,C-CMgBr; ii H? Pd/C MeOH; iii Me,SiI CHCI, at 50-60 "C; iv LiAIH, EtzO at -78 to 0 "C Scheme 9 i,ii ...Ill L -Lysine + Me02C dC02bk + nH + Me0 I C02Me C02Me C02Me (+ trans -isomer 1 ix v-viii HQ -Hm JHJJ -2- Ph-C-CH2 I C02Me Ph Me Ph COzMe Ph C02Me (311 0'U C0,Me '0 +epimer ( = allosedamine)] Reagents i electrochemical oxidation; ii toluene-p-sulphonic acid MeOH at room temperature for 30 hours; iii a-trimethylsilyloxy- styrene TiCl, CH,C12 at -50 "C; iv ethylene glycol toluene-p-sulphonic acid benzene reflux for 21 hours; v NaOH MeOH (aq.) at 5 "C to room temperature for 3.5 hours; vi anodic decarboxylation MeOH; vii NH,CI toluene reflux for 2 hours; viii H, Pd/C EtOAc; ix pyridinium toluene-p-sulphonate Me,CO reflux for 3 hours; x LiAIH, Et,O reflux for 2.5 hours Scheme 10 (28) (30) 177 NATURAL PRODUCT REPORTS 1986 -A.R. PINDER pC02Me :-xiii H~;Q.J>PhCOO .. C02Me C02Me C02Me xiv -xvi 1 xvii H + _i PhCOO< W xviii w:h 0N Ph Me I Me Me HH (2 epimers) Reagents i singlet oxygen; ii EtOCH=CH, SnClz;iii ethylene glycol; iv Jones oxidation; v LiBH4 MeOH at -20 to -15 "C for 15 minutes; vi HgSO, H2S04 DME (aq.) at 0 "C for 3 minutes; vii PhCOCl pyridine at 0 "C for 5 minutes then at room temperature for 3 hours; viii LiAlH(OBu')3 THF at 0°C for 30 minutes; ix MeOCH2Cl PrI2NEt CICH2CH2Cl at 80°C for 1 hour; x K2C03 MeOH at room temperature; xi H?,Pt DME at room temperature for 1 hour; xii MeSO,CI pyridine at 0°C and then at room temperature; xiii 1,8-diazabicyclo[5.4.0]undec-7-ene, at 68-72 "C for 4days; xiv LiAIH4 THF reflux for 10 minutes; xv toluene-p-sulphonic acid (as.) acetone at room temperature for 10.5 hours; xvi PhCOCI pyridine at 0 "C for 5 minutes then at room temperature for 1 hour; xvii PhMgBr CuI.PBu, THF at 0 "C for 15 minutes; xviii KzC03 MeOH at room temperature Scheme 11 OH attached to the aldehyde (25) to generate (26) which is an 1 intermediate in the synthesis of proxiphomin (27).22 A total synthesis of (27) has been described by a route involving an intramolecular Diels-Alder addition reaction (Scheme 7).23 A similar type of approach to cytochalasin D which is another member of this group has been described; the internal Diels- Alder addition is used here to generate an eleven-membered ring.24 2 Piperidine Alkaloids A new synthesis of ( +_ )-pseudoconhydrine (trans-5-hydroxy- Piperidine alkaloids have been reviewed in a new volume of a 2-n-propylpiperidine) by application of an intramolecular monograph on alkaloids.25 An alkaloid that was isolated from amidomercuration has been described,33 and one of both Piperguineense has been assigned structure (28) on the basis of pseudoconhydrine and N-methylpseudoconhydrine,uiu anodic X-ray diffraction analysis; it is the trans,cis-isomer of wisan- oxidation of N-methoxycarbonylpiperidinesto 2,3-diacetoxy- ine.26 Hofmann's 'dimethylconiine' which was obtained over a lated products.34 4-Hydroxysedamine (33) and 4-hydroxyallo- century ago by exhaustive methylation of coniine has been re- sedamine (34) are two minor alkaloids of Sedum acre investigated using modern methods of separation and spectral formulated on spectral and chemical evidence; both have been study and found to consist of a mixture of unsaturated amines synthesized and their absolute configurations settled.35 Specta- in confirmation of earlier conclusions.27 Three new syntheses line (39 in racemic form has been synthesized (Scheme 12).36 of solenopsin A (29) which is present in the venom of fire ants The racemic forms of isoprosopinine B and desoxoprosopin- have been reported. One is by a pathway analogous to that ine (36) and (37) respectively have been synthesized (Scheme outlined in Scheme 3.' An abbreviated version of the second is 13).37 shown in Scheme 8.The final product was separated into cis- A novel glucoside 4-O-(~-D-glucopyranosyl)fagomine(38) and trans-isomers by preparative g.1.c. and configurations were which is derived from a polyhydroxylated piperidine alkaloid assigned by n.m.r. spectroscopy; the trans-product proved to be has been isolated from seeds of Xanthocercis zambesiacu. Its identical with (*)-solenopsin A.28 The third route (Scheme 9) structure has been deduced by detailed n.m.r. spectral involves a reaction between a pyridinium salt and an alkynyl analysis. Grignard reagent as the key step.29 A new synthesis of (+)-pinidine (30) has been described starting with 5-bromopentan- 2-one and 3-tosyloxyb~t-l-yne.~~ ( f)-Sedamine (31) which is 2.1 Spiropiperidine Alkaloids the enantiomer of the natural base has been synthesized from A new synthesis of perhydrohistrionic~toxin~~ and syntheses L-lysine (Scheme Sedinine (32) which is another Sedum of isonitramine (39) and sibirine (40) have been reported.40 In alkaloid has been synthesized in racemic form as outlined in the latter two endeavours an intramolecular nitrile oxide cyclo- Scheme 11.32 addition route was used.178 NATURAL PRODUCT REPORTS 1986 H2C=CH[CHzI&HzOH i+H2C=CH[CH2leCH2Br A H2C=CH[CH218CH2CHMeI iii -D H2C=CH[CH218CH2COMe bH viii ix "Om " G o -(35) (+3a-OH epimer; chromatographic separation) Reagents i PBr,; ii Mg then MeCHO; iii Jones oxidation; iv HBr hv;v Me2C=CH[CH2],C(0)CH2C02Et,NaOEt; vi Ba(OH)? H,O; vii 0,; viii EtNO, KOBu'; ix Pd/Pt/C at 30"C EtOH H2 (3 atm.) Scheme 12 OSiMe3 CHC02Me ... I ,II II + TsN C02Me (+endo -isomer ) Pi0 Ph iv TsNA-T& OH C02Me OH /vi (A) Ph . .. vii ,viii -' o, C H Oh Ts OH (36) (37) OH Reagents i benzene warm from 5 "C to room temperature over 3 hours; ii HCI THF at room temperature for 1 hour; iii MeCO,H HOAc NaOAc at 50 "C for 72 hours; iv LiAIH, EtzO at 0 "C for 1.5hours; v PhCHO toluene-p-sulphonic acid benzene at room temperature for 12 hours; vi pyridinium dichromate molecular sieve CH,CI, at room temperature for 9 hours; vii Ph3P=CH[CHJ,C(OLi)2Bu THF at 0 "C for 1 hour; viii H, Pd/C EtOH; ix ethylene glycol toluene-p-sulphonic acid benzene reflux for 16 hours; x Red-Al@ {Na[MeOCH,- CH20)2AIH2]},benzene reflux for 24 hours; xi 8M-HCl MeOH reflux for 16 hours; xii Me,C(OMe), toluene-p-sulphonic acid CH2Cl, at room temperature for 16 hours; xiii pyridinium dichromate molecular sieve CH2C12 at room temperature for 4 hours; xiv Ph,P= CH[CH,],Me THF at 0°C for 1 hour Scheme 13 NATURAL PRODUCT REPORTS 1986-A.R. PINDER OMe I OMe OMe $. 0 Me (43) (44) Me 0 OH HN10 HO OH (49) (50) (51) OH I R'. (52) R = H (54) R = OH (53)R = C02Me (55)R = H 2.2 Lactonic Piperidine Alkaloids Dumetorjne is a new alkaloid occurring in the yam Dioscoreu durnetorurn.Its structure and configuration (41) have been settled mainly by spectral study.l' 2.3 Bispiperidine Alkaloids Strictimine which was formulated as (42) largely on spectral evidence has been isolated from the roots of the Pakistani shrub Rhaz1-a strictu."' 3 Pyridine Alkaloids A new nicotinamide has been isolated from the leaves of Arnyris plurnieri; on spectral and chemical evidence it is formulated as (43)."3 A pyridine of the unusual structure (44) has been found to be a minor metabolite of the mollusc Philinopsis speciosu its structure has been elucidated chiefly by 'H n.m.r.A simple synthesis of ricinine has been devised cyclization of the dicyanide (45) affords pyridines (46) and (47) methylation of both leading to ricinine (48).jS Synthetic studies directed towards sesbanimide (49) have been rep~rted.~~.~' Three antileukaemic principles [sesbani- mide-B and sesbanimide-C] have been isolated from seeds of OH I (56) Sesbania drurnrnondii; their structures [(SO) (two diastereo- isomers) and (5 1) respectively1 have been advanced on the basis of n.m.r.spectral study and X-ray diffraction analysis.l* A new synthesis of (&)-sesbanine has been described the key step of which is the y-addition of ketene trimethylsilyl acetal to quaternized methyl nicot inate .59 The absolute configuration of venoterpine has been revised to (52) by a correlation with cantleyine (53) of which the absolute configuration is known through correlation with loganin.'O Rhexifoline is a new alkaloid that has been found in the flowers and seeds of Custilleja rhexifoliu and also in the larvae and adults of the plume moth Platjptiliu picu which feeds on the plant.Its structure (54) and relative stereo-chemistry have been settled by n.m.r. spectroscopy particu- larly n.0.e. studies. Deoxyrhexifoline (55) is also present in the Strychnovoline (56) is a new alkaloid of Strychnos dinklagei. Its structure and absolute configuration follow from spectral analysis and by its synthesis from loganin. Gentianine and cantleyine are also present.s2 3.1 Nicotine Alkaloids 4-Aminonicotine and 4-aminocotinine have been synthesized via 4-nitrocotinine N-o~ide.~~ Nornicotine has been synthe- sized by using a primary amino (protected) Grignard reagent (57) in a reaction with an N-metho~y-N-methylarnide.~~ The 180 carbon-13 and proton n.m.r.spectra of nicotine in aqueous media have been measured and discussed in some detail.55 Organolithium reagents add to (-)-nicotine regiospecifically to generate 6-substituted nicotinoids. These products are of low optical activity and evidence has been presented in support of the view that a novel racemization process caused by cleavage of the pyrrolidine ring and re-cyclization is involved.56 The alkaloid contents of 60 Nicotiana species have been analysed by g.i.c.57 Several new alkaloids have been isolated from the root bark of the tree C/eistopholis patens which grows in the Ghanaian rain forest. They are unusual azapolycyclic and naphthyridine bases.58 Full details of an earlier briefly reported synthesis of eupolauramine have been published.59 4 References 1 A.B. Attygalle and E. D. Morgan Chem. SOC. Rev. 1984,13 245. 2 (a) R. J. Highet and J. W. Wheeler in ‘The Alkaloids’ ed. A. Brossi Vol. 24 Academic Press New York 1985 Chapter 6; (b) C. Christophersen ibid. Chapter 2. 3 H. A. Lloyd H. M. Fales M. E. Goldman D. M. Jerina T. Plowman and R. E. Schultes Tetrahedron Lett. 1985 26 2623. 4 A. L. Waterhouse I. Holden and J. E. Casida J.Chem. Soc. Chem. Commun. 1984 1265. 5 W. V. Brown A. J. Jones M. J. Lacey and B. P. Moore Aust. J. Chem. 1985 38 197. 6 R. Ghirlando A. S. Howard R. B. Katz and J. P. Michael Tetrahedron 1984 40,2879. 7 R. J. Nash E. A. Bell G. W. J. Fleet R. H. Jones and J. M. Williams J.Chem. Soc. Chem Commun. 1985 738. 8 J. Furukawa S. Okuda K. Saito and S.-I. Hatanaka Phyto-chemistry 1985 24 593. 9 R. J. Nash E. A. Bell and J. M. Williams Phytochemistry 1985 24 1620. 10 H. Iida N. Yamazaki and C. Kibayashi Tetrahedron Lett. 1985 26 3255. 11 A. I. Meyers P. D. Edwards T. R. Bailey and G. E. Jagdmann Jr. J. Org. Chem. 1985 50 1019. 12 J. J. Tufariello H. Meckler and K. P. A. Senaratne J.Am. Chem. Soc. 1984 106 7979. 13 P. G. Willard and S. E. de Laszlo J. Org. Chem. 1984 49 3489. 14 H. Kohler and H. Gerlach Helv. Chim. Acta 1984 67 1783. 15 I. H. Sanchez M. I. Larraza H. J. Flores E. Diaz and K. Jankowski Heterocycles 1985 23 593. 16 I. Koyama T. Sugita K. Tagahara Y. Suzuta and H. hie Heterocycles 1984 22 1973.17 P. N. Confaione and E. M. Huie J. Am. Chem. Soc. 1984 106 7175. 18 A. Kato M. Okada and Y. Hashimoto J.Nut. Prod. 1984,47,706. 19 A. Kato M. Okada and Y.Hashimoto J.Nut. Prod. 1985,48,289. 20 I. Ackermann N. Waespe-Saracevic and C. Tamm Helu. Chim. Acta 1984 67 254. 21 D. Wallach I. G. Csendes P. E. Burckhardt T. Schmidlin and C. Tamm Helv. Chim. Acta 1984 67 1989. 22 T. Schmidlin D. Wallach and C. Tamm Helu. Chim. Acta 1984 67 1998. 23 D. J. Tapolczay E. J. Thomas and J. W. F. Whitehead J. Chem. SOC. Chem. Commun. 1985 143. NATURAL PRODUCT REPORTS. 1986 24 A. Graven D. J. Tapolczay E. J. Thomas and J. W. F. Whitehead J. Chem. SOC. Chem. Commun. 1985 145. 25 G. B. Fodor and B. Colasanti in ‘Alkaloids. Chemical and Biological Perspectives’ ed.S. W Pelletier Wiley-Interscience New York 1985 Vol. 3 p.1. 26 K. A. Woode F. L. Phillips I. Addae-Mensah J. C. J. Bart and S. Chaudhuri J. Nut. Prod. 1984 47 1024. 27 W. Cocker N. W. A. Geraghty and P. V. R. Shannon J. Chem. SOC. Perkin Trans. I 1984 2241. 28 W. Carruthers M. J. Williams and M. T. Cox J. Chem. SOC. Chem. Commun. 1984 1235. 29 Y. Nakazono R. Yamaguchi and M. Kawanisi Chem. Lett. 1984 1129. 30 S. Arseniyadis and J. Sartoretti Tetrahedron Lett. 1985 26 729. 31 K. hie K. Aoe T. Tanaka and S. Saito J. Chem. Soc. Chem. Commun. 1985 633. 32 M. Ogawa and M. Natsume Heterocycles 1985 23 831. 33 K. E. Harding and S. R. Burks J. Org. Chem. 1984 49 40. 34 T. Shono Y. Matsumura 0. Onomura T.Kanazawa and M. Habuka Chem. Lett. 1984 1101. 35 F. Halin P. Slosse and C. Hostelle Tetrahedron 1985 41 2891. 36 M. Paterne and E. Brown C. R. Acad. Sci. Ser. 2 1984,299 1183. 37 A. B. Holmes J. Thompson A. G. J. Baxter and J. Dixon J.Chem. Soc. Chem. Commun. 1985 37. 38 S. V. Evans A. R. Hayman L. E. Fellows T. K. M. Shing A. E. Derome and G. W. J. Fleet Tetrahedron Lett. 1985 26 1465. 39 A. B. Holmes K. Russell E. S. Stern M. E. Stubbs and N. E. Wellard Tetrahedron Lett. 1984 25 4163. 40 A. P. Kozikowski and P.-W. Yuen J. Chem. SOC.,Chem. Commun. 1985 847. 41 D. G. Corley M. S. Tempesta and M. M. Iwu Tetrahedron Lett. 1985 26 1615. 42 Atta-ur-Rahman and K. Zaman Heterocycles 1984 22 2023. 43 B. A. Burke and S. Phillip Heterocycles 1985 23 257.44 S. J. Coval and P. J. Scheuer J. Org. Chem. 1985 50 3024. 45 M. Mittelbach G. Kastner and H. Junek Monatsh. Chem. 1984 115 1467. 46 N. Willard M. J. Wanner G.-J. Koomen and U. K. Pandit Heterocycles 1985 23 5 1. 47 M. Shibuya Heterocycles 1985 23 61. 48 R. G. Powell C. R. Smith Jr. and D. Weisleder Phytochemistry 1984 23 2789. 49 M. Wada Y. Nishihara and K. Akiba Tetrahedron Lett. 1985,26 3267. 50 T. Ravao B. Richard M. Zeches G. Massiot and L. Le Men- Olivier Tetrahedron Lett. 1985 26 837. 51 M. R. Roby and F. R. Stermitz J. Nut. Prod. 1984 47 846. 52 S. Michel A. L. Skaltsounis F. Tillequin M. Koch and L. A. Assi J. Nat. Prod. 1985 48 86. 53 M. Shibagaki H. Matsushita and H. Kaneko Heterocycles 1985 23 1681.54 F. Z. Basha and J. F. DeBernardis Tetrahedron Lett. 1984 25 5271. 55 R. W. Slaven J. Heterocycl. Chem. 1984 21 1329. 56 J. I. Seeman C. G. Chavdarian R. A. Kornfeld and J. D. Naworal Tetrahedron 1985 41 595. 57 F. Saitoh M. Noma and N. Kawashima Phytochemistry 1985,24 477. 58 P. G. Waterman and I. Muhammad Phytochemistry 1985,24 523. 59 J. I. Levin and S. M. Weinreb J. Org. Chem. 1984 49 4325.

ISSN:0265-0568    

DOI:10.1039/NP9860300171

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

Tropane Alkaloids G. Fodor and R. Dharanipragada Department of Chemistry West Virginia University Morganto wn W V 26506- 6045 U.S.A. Reviewing the literature published between July 1984 and June 1985 (Continuing the coverage of literature in Natural Product Reports 1985 Vol. 2 p. 221) 1 Occurrence and Structure of New Alkaloids 2 Synthesis Stereochemistry and Spectroscopy of the Natural Products 3 The Chemistry of Non-natural Derivatives 4 Pharmacology 4.1 Atropine 4.2 Cocaine 4.3 Scopolamine 4.4 Miscellaneous 5 Analysis 5.1 Atropine 5.2 Cocaine 5.3 Scopolamine 5.4 Miscellaneous 6 References 1 Occurrence and Structure of New Alkaloids The isolation of six tropane alkaloids namely tropine senecioyltropine and tropane-3a,60-diol and its 3a-senecioic acid ester and its 60-angelic and tiglic acid esters from the roots of Schizanthus hookeri has been reported earlier (cf ref.la). A recent analysis* of the leaves and stem afforded the same alkaloids. The alkaloids that were produced in cultured roots of Duboisia leichardtii D. myoporoides and D. hopwoodii3 con- tained large amounts of hyoscyamine (0.53%) and scopolamine (1.16% of the dry weight). 2 Synthesis Stereochemistry and Spectros- copy of the Natural Products A synthetic route (Scheme I) which is claimed4 to be new has been described,s starting from cycloheptatriene ; this was converted (via tropone in three steps) into the cycloheptadiene derivative (1).1-Chloro-1-nitrosocyclohexanegave by a [4 + 21 cycloaddition the cyclic hydroxylamine (2) and this by catalytic hydrogenation afforded all-cis-5-amino- 1-benzoyloxy-cycloheptan-3-01 (3). The reaction of (3) with ethyl chlorofor- mate followed by treatment of the resultant acylamino-alcohol with thionyl chloride and pyridine gave 1,3-cis-5-trans-I-benzoyloxy-3-chloro-5-ethoxycarbonylaminocycloheptane(4) and the sulphite ester. The construction of the bicyclic urethane (5) was achieved by an intramolecular nucleophilic displace- ment of chlorine from (4) by the acylamino nitrogen. Lithium aluminium hydride converted (5) into pseudotropine (6). By using the chloro-amide (7) nortropacocaine was obtained after amidocyclization and hydrogenolysis; methylation then gave tropacocaine (8).The novelty of this synthesis of pseudotropine [7 steps; 1.32% overall yield (based on cycloheptatriene)] is the use of the hetero-Diels-Alder reaction to introduce the nitrogen and oxygen functionalities simultaneously. Actually the first synthesis6 of pseudotropine by Willstatter also started from cycloheptatriene and the nitrogen bridge was built from a bromocycloheptenylamine (in 8 steps ; 1.65% overall yield). The Robinson ~ynthesis,~,~ in contrast gives tropinone from succinic aldehyde (as its acetal dimethoxytetrahydrofuran) in 90%yield and the reduction of tropinone to pseudotropine was achieved by Willstatter in 90% yield (for the two steps 81% OCOPh OCOPh CI NO H (2) lii OCOPh iii iv -H3N+AO" (3) vii,iv 1 OCOPh PhCHZOCONH v ii,viii MeN&ocoph H H (6) (8) Reagents i EtOH-CCI, at -20 to -10°C; ii H, Pd/C MeOH; iii CICO,Et Na,CO, H,O-CHCI,; iv SOCI, pyridine CHCI, reflux; v KOBu' PhH HMPA; vi LiAIH, THF then 10% NaOH at 0°C; vii PhCH20COCl Na,CO, H,O-CHCI,; viii aq.HCHO H COz HI heat Scheme I overall yield). Therefore the synthesis is hardly competitive as far as practicality is concerned. As to its general use this has yet to be proven. Three years ago Pinder et al.9 showed that physoperuvine is a tautomeric mixture of 1-hydroxytropane (9) and 4-(methyl- amino)cycloheptanone (10); X-ray analysis by Ray et al.IO indicated that (+)-physoperuvine hydrochloride is (+)-(1R,5R)-l-hydroxytropanium chloride (1 1) (cf ref.1b). McPhail and Pinder" have now published X-ray data for racemic 3-(N-benzoyl-N-methylamino)cycloheptanoneand for the 4-substituted regio-isomer; resolution of synthetic (*)-4- (methy1amino)cycloheptanone to give the natural (+)-(4R)-enantiomer thus constitutes a total synthesis of the alkaloid. 182 NATURAL PRODUCT REPORTS 3986 0 QH I OH hl-0 4 NHMe l-i 0 (10) (13) OH (14) (15) (17) R = NMe2 NEt2,l-piperidyl or 1 -morpholinyl Me (21) (22) Tufariello et al.’ have extended their synthesis of tropanol; starting with a pyrroline N-oxide the new synthesis proceeds cia the bridged hydroxylamine (1 2) to anatoxin-a (1 3) which is a homotropene. The ’3C spin-lattice relaxation times of tropine and pseudotropine in [2H]chloroform have been measured as a function of concentration.The rotational diffusion coeffi- cients were calculated from the relaxation data. Reorientation of both molecules has been shown to be moderately anisotropic. The main rotational axis is parallel with a line that passes through the centre of mass of the molecule and the nitrogen atom. 3 The Chemistry of Non-natural Derivatives The quaternization of N-isopropylnorscopolaminewith methyl bromide gavels N-isopropylscopolaminium bromide (1 4) in which the methyl group is equatorial (cf. ref. 15). The abstract in Chemical Abstracts does not reveal the kind of approach that was used to discover that the methyl group is equatorial but it probably involves H n.m.r.spectroscopy. Nortropine reacts with 1 -bromo-2-fluoroethane to form a tertiary amine which has been esterified with benzilic acid to obtain a new active spasmolytic agent (1 5).] Tropine has been alkylated’ with a-chloroacetanilides to give new biologically active quaternary tropanium compounds (16). Tropine and pseudotropine have been acylated with alkoxy- and halo-benzoates18-20 to form the esters; these are used in the treatment of migraine. The interchange of methyl and other alkyl groups in alkaloids inter aha tropine has been achieved,” z’iu the N-alkyl-N-methyl quaternary salts followed by demethylation with thiophenoxide ion. 3-(Alkylamino)-N-butylnortropanes (17) have been pre-pared” by reductive amination of N-butylnortropinone.(1,2- (18) 0 J3c (23) I3CC,)Granatanine (18) has been synthesizedz3 from (2,3-I 3CC,)glutaricacid. The intermediate (2,3-’ 3C,)glutaraldehyde gave by the Robinson route (1,2-’3Cl)granatanin-3-one which was then reduced to the granatanine (18). Homograna- tanin-3-one has been convertedz4 into the spirohydantoin (19). Dynamic 13C n.m.r. studies of restricted rotation about the C-N bond in N-alkoxycarbonylgranatanin-3-ones(20) have been described.25 The free energy of activation (AG:) was estimated as 14.5- 16.6 kcal mol-I at the coalescence tempera- ture (T,). The corresponding tropane derivatives show appre-ciably lower energies of activation. The greater value ofAG,+of the granataninones rs. the value for each corresponding tropanone was ascribed to the steric compression that exists between the cw-protons at C-2 C-4 C-6 and C-7 and the alkoxycarbonyl group in the transition state where the plane of COOR is perpendicular to the plane that includes C-1 N and C-5.The barrier to rotation is increased by substituents in the granatane system if they are at y-positions to the nitrogen atom. 4 Pharmacology 4.1 Atropine f1 (1 ,4-Die t h oxy benzene)bi satropinium di bromi d e which is an analogue top-xylylenebisatropinium di bromide,2hh has been synthesized and found to be a muscle relaxant of high A new procedure has been elaborated” for administering atropine by developing a depot-form prepara- tion in which there are high-molecular-weight compounds. The pharmacological activity of Nh-isopropylatropinium bromide (21) has been reviewed28 (for a description of the Nb convention see ref.15) and its use in anticholinergic therapy of chronic obstructive pulmonary diseases was clarified. The efficiency of competition of stored and of newly made mixtures of atropine and toxogonin with the binding of 3H-QNB at the 183 DHARANIPRAGADA NATURAL PRODUCT REPORTS 1986 -G. FODOR AND R muscarinic acceptor has been ~ompared.'~ No decrease in effectiveness was observed if atropine was stored in an autoinjector for up to 15 years. 4.2 Cocaine Sex and strain differences in the response of mice to cocaine have been reported .30 Benzoylecgonine and benzoylnorecgon- ine have been used3' for the treatment of rheumatoid arthritis.Norcocaine has been found3' to be responsible for the convulsant and lethal actions of cocaine. The experiments consisted of altering the hepatic microsomal metabolism of cocaine to norcocaine via pretreatment of the experimental animals with phenobarbital. Both the EDso and the LDsOof cocaine were significantly increased. 4.3 Scopolamine The analgesic action of scopolamine has been measured.33 4.4 Miscellaneous 3-Aminotropanes have been synthesized and used for a variety of therapies. N-Benzyl-3-(quinazolin-4-ylamino)nortropane has a strong antiemetic effect.3J The analogous N-benzyl-3-[(5- dimethylaminosulphonyl-2-~uorobenzoyl)amino]nortropane is a dopamine antagonist. Similarly the di hydropyrano[2,3- blbenzoyl derivative (22) shows dopamine-blocking The ester of tropan-3a-ol with 3,5-dichlorobenzoic acid is a potent and selective antagonist at neuronal receptors for 5-hydr~xytryptamine.~' Other aroyl esters of the same type have been used in migraine therapy.'*-1° 6P-Hydroxyhyoscyamine (23) was originally found in Datura jero.9 and was ~ynthesized.~') This alkaloid has since been rediscovered in Przewalskia tangutica and named aniso-damine;aoit inhibitss' hemorrhagic shock in cats.Tropaphen (24) which is the 3-(p-acetoxyphenyl)-2-phenylpropionyl ester of tropan-2P-01 is an cx-adrenoceptor blocker which poten- tiatesJ2 the protective action of adrenaline in bronchospasm. Tropapride (25) [N-benzyl-3-(2,3-dimethoxybenzamido)nor-tropane] is localizeds3 in the pituitary gland of the rat to a lesser extent than I-sulpiride {5-aminosulphonyl-N-[(l-ethylpyrroli-din-2-yl)methyl]-2-methoxybenzamide). Therefore it has little effect on the secretion of prolactin.Troparil (2P-methoxycar- bonyl-3P-phenyltropane) showedaS central-nervous-system-stimulating activity in mice that far exceeds that of amphetamine. 5 Analysis 5.1 Atropine The stability of atropine sulphate in the presence of aluminium hydroxide gela5 and in syringesJ6 has been studied. Methods for the simultaneous determination of atropine sulphate and tropic acid by reversed-phase h.p.1.c. have been elaborated.17 The ion-pair reversed-phase h.p.1.c. of tropane alkaloids particularly of atropine of belladonine and of their products of Ph I -O-C-CHCH M~NA 0 It / Y (24) PhCH2 N,* Y I Me0 \ (251 degradation (e.g.tropic acid and atropic acid) has been studied ;48 1 % aqueous atropine sulphate shows nine peaks which were assigned to these compounds. A method for gas- chromatographic determination of atropine in tablets has been described.J9 The determination of atropine in injections that contain atropine sulphate has been achieved the technique requires basification extraction and the use of infrared spectroscopy or of t.1.c. or paper chromatography. 5.2 Cocaine The vapour pressures of cocaine heroin and amphetamine have been determi~~ed,~ by using a dynamic gas-blending system and gas-chromatographic analysis. 5.3 Scopolamine Quantitative and qualitative analyses of scopolamine atro- pine and hyoscyamine in pure tinctures of members of the Solanaceae were achieveds2 by h.p.1.c.The biphasic titrimetric determination of scopolamine butobromide (Nh-butylscopola- minium bromide = Buscopan) has been elaborated..'3 Another approach involved titration with 0.01 N mercury(rr) nitrate in the presence of diphenylcarbazide.s4 5.4 Miscellaneous Concentrations of tropacin tropaphen and their degradation products have been determined by ultraviolet spectroscopy.5s Tropaphen has also been extracted and determined by spectrophotometry. s6 6 References 1 G. Fodor and R. Dharanipragada in 'The Alkaloids' ed. M. F. Grundon (Specialist Periodical Reports) The Royal Society of Chemistry London (u) 1983 vol.13 p. 55; (h) 1983 vol. 13 p. 56. 2 V. Gambaro C. Labbe and M. Castillo Phr.tochrmisfr,,. 1983 22 1838. 3 T. Endo and Y. Yamada Phj*tochumistrji 1985 24 1233. 4 H. Iida Y. Watanabe and C. Kibayashi T~.truhcclronLctt. 1984 25 509 I. 5 H. Iida Y. Watanabe. and C. Kibayashi J. Org. Chum. 1985 50 1818. 6 R. Willstiitter Liehigs Ann. Chrm. 1901 317 204 267. 307; ihil. 1903 326 23; Chem. Ber. 1901 34 129 3163. 7 R. Robinson J. Chem. Soc. 1917 111 762. 8 G. Gil I. Simonyi and G. Tokir Actu Chim. Acrid. Sci Hring. 1955 6 365. 9 A. R. Pinder J. Org. Chrm. 1982. 47 3607. 10 A. B. Ray Y. Oshimo H. Hikino and C. Kabuto t-/cic~roc~~'c.l~~s. 1982 19 1233. 11 A. T.McPhai1 and A. R. Pinder Tc.truhedmn 1984 40. 1661.12 J. J. Tufariello H. Meckler and K. P. A. Senaratne J. Am. Chem. Soc. 1984 106 7979. 13 R. Uusvuori and M. Lounasmaa Org. Magn. Reson.,1984,22,286. 14 G. Li P. Liu J. Shan K. Zhou and P. Wang Yijm Gongw 1984 No. 9 p. 12. (Chrm. Ahstr. 1985 102 95877). 15 Suggestion by R. S. Cahn then Editor; see footnote. G. Fodor or u/. J. Chrni. Soc. 1955 3504. 16 R. Banholzer (Boehringer lngelheim K.G.) Ger. Offen. 3 320 138 (Chem. Ahstr. 1985 102 149580). 17 P. Gorecki M.Drozdzynska B. Kedzia and D. Przybylska Horhu Pol. 1983 29 135 (Chrm. Ahstr. 1985 102 6888). 18 J. R. Fozard and M. W. Gittos Eur. Pat. Appl 112276 (Chrm. Ahsfr. 1984 101. 230 152). 19 J. R.Fozard and M. W. Gittos Eur. Pat. Appl. I1 1608 (ChOm. Ahsrr. 1984 101 230 154). 20 J.R. Fozard and M. W. Gittos Br. Pat. Appl. 2131419 (Chcwi. Ahstr. 1985 102 46176). 21 T. S. Manoharan K. M. Madyastha G. P. Bhatnagar. and U. Weiss Indiun J. Chem.,Swt. B 1984 23 5. 22 J. Wolinski A. Baranowski and B. Gutowska Actu Pol. Phurni. 1984 41 25. 23 J. J. C. Barna and M. J. T. Robinson J. Luhrlld Compd. Radiophurm. 1984 21 727. 24 G. Gonzalez Trigo E. Martinez Munoz and E. Llama-Hurtado J. Hererocycl. Chem. 1984 21. 1479. 25 0.Muraoka T. Minematsu J. Tsuruzawa and T. Momose Heterocycles 1985 23 853. 26 (a)G. Chen R. Fang and Y. Zhang Yaoxue Xuebao 1984,19,21; (h)L. Gyermek and K. Nador Acta Physiol. Acad. Sci. Hung. 1952 3 183 (Chem. Abstr. 1953,47,7111a). 27 V. I. Kuleshov S. N. Golikov V. K. Kozlov Yu.A. Lyubimov V. K. Sukhankin G. A. Gur’yanov A. Ya. Bespalov and S. G. Kuznetsov USSR P. 1 113094[see Otkrytiya Izohret. 1984,34 171 (Chem. Abstr. 1985 102 50899). 28 K. L. Massey and V. P. Gotz Drug. Intell. Clin. Pharm. 1985,19,5. 29 B. Karlsson and V. Oegren Report FOA-C-40191-C3, 1984[from Govt. Rep. Announce Index (U.S.) 1984,84 601(Chem. Abstr. 1985 102 32046). 30 M. L. Thompson L. Shuster E. Casey and G. C. Kanel Biochem. Pharmacol. 1984 33 1299. 31 M. Lowell U.S. P. 4469700 (1984) (Chem. Abstr. 1984,101 21 6 442). 32 D. Huang R. Borne and M. Wilson Res. Commun. Suhst. Abuse 1984 5 201. 33 G. Hong J. Li and G. Jin Shengii Xuehao 1984,36 149(Chem. Ahstr. 1984,101,143 990). 34 F. D. King PCT Int. Appl. 84 1157 (Chem. Ahstr.1984,101 11 1240). 35 F. D. King R. T. Martin and G. Wootton Eur. Pat. Appl. 102 195 (1984)(Chem. Abstr. 1984,101,72986). 36 C. Summers PCT Int. Appl. 843281 (Chem. Abstr. 1985 102 203 957). 37 J. R. Fozard Naunyn-Schmiedeberg’s Arch. Pharmacol. 1984 326 36. 38 A. Romeike Naturwissenschqfien 1962 49 281. NATURAL PRODUCT REPORTS 1986 39 G. Fodor I. Koczor and G. Janzso Arch. Pharm. (Weinheim Ger.) 1962 295 91. 40 J.-S. Yang Y.-W. Chen and H.-C. Feng. Chung Ts’ao Yao 1981 12,No. 2,p. 4;cf. ref. Ih. 41 J. Su Zirun Zazhi 1984 7,638 (Chem. Ahstr. 1984,101,222297). 42 F. S. Zarudi Farmakol. Toksikol. (Moscow) 1984 47 81. 43 M. Strolin-Benedetti J. Dow S. Dostert D. Parisy and J. F. Rumigny J. Pharmacol. 1984 15 433. 44 V.V. Zakusov and V. I. Naumova Farmakol. Toksikol. (Moscow) 1985,48 15. 45 E. Gafitanu I. Popovici I. Matei and V. Dorneanu Farmacia (Bucharest) 1984 32 83 (Chem. Ahstr. 1984,101,116667). 46 R. S. Rhodes P. J. Rhodes and H. H. McCurdy Am. J. Hosp. Pharm. 1985 42 112. 47 A. Richard and G. Andermann Pharmazie 1984 39 866. 48 T. Jira T. Beyrich and E. Lemke Pharmazie 1984 39 351. 49 P. MajlLt Pharmazie 1984 39 325. 50 M. Cifani and 0. Modica Rass. Chim. 1983 35 413. 51 A. H. Lawrence L. Elias and M. Authier-Martin Can. J. Chem. 1984 62 1886. 52 A. H. Paphassarang J. Raynaud R. Godeau and A. M. Binsard J. Chromatogr. 1985 319 412. 53 W. Shen and X. Zhang Yaowu Fenxi Zazhi 1984 4 117 (Chem. Abstr. 1984,101,157748). 54 Nguyen Kim Can Tap Chi Duoc Hoc 1984,No. 3 pp. 24,32(Chem. Abstr. 1985 102 119719). 55 V. A. Karpenko and S. N. Stepanyuk Farmatsiya (Moscow) 1984 33,50. 56 V. G. Belikov V. A. Karpenko and S. N. Stepanyuk Farmatsiya (Moscow) 1984 33 40.

ISSN:0265-0568    

DOI:10.1039/NP9860300181

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites R. B. Herbert Department of Organic Chemistry University of Leeds Leeds LS2 9JT Reviewing the literature published between July 1984 and June 1985 (Continuing the coverage of literature in Natural Product Reports 1985 Vol. 2 p. 163) 1 Pyrrolidine and Piperidine Alkaloids 1.1 Tropane Alkaloids and Nicotine 1.2 Pyrrolizidine and Quinolizidine Alkaloids 2 Isoquinoline Alkaloids 3 Metabolites Derived from Tryptophan 3.1 Streptonigrin 3.2 Terpenoid Indole Alkaloids 3.3 Actinomycin 4 Metabolites Derived from Phenylalanine and Tyrosine 4.1 Naphthyridinomycin 4.2 Isocyanides and Tuberin 4.3 Cinodine 4.4 Lincomycins 4.5 Saframycin A 5 Other Metabolites Derived from the Shikimate Pathway 5.1 Ansamycins 6 P-Lactams 6.1 Penicillins and Cephalosporins 6.2 Nocardicins 6.3 Clavulanic Acid 6.4 Carbapenem Antibiotics 7 Miscellaneous Metabolites 7.1 Virginiamycin Antibiotics 7.2 Elaiomycin 7.3 Showdomycin 7.4 Astromicin and Streptomycin 8 References In order to provide access to useful background material appropriate reference is made in the following discussion to earlier reviews.1-5 1 Pyrrolidine and Piperidine Alkaloids Ornithine (1) and putrescine (2) are key early intermediates in the biosynthesis of pyrrolidine alkaloids4v5 and precursors for the polyamines spermine and spermidine (4).The biosynthesis of putrescine in Tetrahymena thermophila (a unicellular eukaryote) has been studJed.6 From the results it was concluded that the only major route to putrescine during the exponential phase of growth in this organism was by decarboxylation of omithine (1); the pathway via arginine and agmatine was not detected. 1.1 Tropane Alkaloids and Nicotine N-Methylputrescine (3) is an intermediate in the biosynthesis of tropane alkaloids and of nic~tine.~,~ Following work on putrescine methyltransferase from Nicotiana tabacum' (c$ ref. 1 Vol. 4 p.7) similar enzyme activity has been identified again in N. tabacum and also in whole plants and root cultures of Datura stramonium; specific activities of the enzyme and K values were determined.8 [6-'4C]Spermidine [as (4)] has been incorporatedg into nicotine (5) and nornicotine (6) in Nicotiana glutinosa with labelling of C-2' and C-5'.The results indicate that the spermidine is utilized via the symmetrical amine putrescine (2) which is a normal intermediate in the biosynthesis of (5) and (6). Tropane alkaloids e.g. hyoscyamine (7) are formed from ornithine (1) by a biosynthetic sequence which does not involve any symmetrical intermediates such as putrescine (2) and hygrine (8) is a late intermediate (results are for D.stramonium and Scopolia 1urida'O.' 1).4*5 The incorporation of [2-I4C]ornithine [as (l)] into hygrine (8) in Nicandra physaloides has been reported to give exclusive labelling of C-2.I0 The incorporation of ornithine into (8) in this plant has been re- examined,'* using DL-[5-14C]omithine.On the basis of the earlier results,Io labelling of C-5 in the hygrine (8) was expected. Instead the results of careful degradation revealed that there was equal labelling of C-2 and C-5 of the hygrine. * Thus it appears that the earlier results are in error and that hygrine is formed via the symmetrical diamine putrescine (2) in this plant. This is in accord with the findings for several other pyrrolidine alkaloids in other plant^^.^.^ (cf. ref. I Vol. 13 p. 3). Further errors in the earlier paperlo indicate that the more recent results are more reliable. These errors concern the incorporation of hygrine (8) into cuscohygrine (9) in Scopolia lurida.O They involve discrepancies in the physical properties of some of the degradation products of cuscohygrine and in the calculations of specific radioactivity.' The results that were obtained were those expected but for the wrong reasons arising from what was called the 'ESP-syndrome' * *Acronym for Eager to Satisfy the Preconceived ideas of one's research supervisor. :irI\ NHZ NHR (2) R = H (3) R = Me (5) R = Me (6) R = H (7) Me The mechanism whereby phenylalanine (10) is transformed into the ($)-tropic acid moiety (11) of tropane alkaloids e.g. hyoscyamine (7) has long been an intriguing mystery4y5 (cf:ref. 2 p. 181). One important fact is that during the rearrangement the carboxyl group undergoes an intramolecular 1,2-shift from C-a to C-0 in phenylalanine (or a derivative thereof).The fate of the hydrogen atoms at C-p during the rearrangement has now been probed in a preliminary way with a mixture of equal amounts of the four possible stereoisomers of [~arboxyl-~~c p-3H ]phenylalanine.I4 In experiments with Datura stramonium and D. innoxia the hyoscyamine (7) and scopolamine that were isolated showed high retention of tritium and both C-1 and C-2 of (1 1) were labelled. It is thus clear that the rearrangement of phenylalanine which affords tropic acid involves a 1,2-migration of hydrogen as well as of the carboxyl group. Further work has been reported on tissue cultures of Nicotiana tabacum' and Duboisia leichhardtii' and the alkaloids that they produce.1.2 Pyrrolizidine and Quinolizidine Alkaloids Aspects of the biosynthesis of pyrrolizidine and quinolizidine alkaloids have been reviewed." Full details of results from studies of the incorporation of putrescine (2) into the retronecine portion (14) of retrorsine (1 2) which had previously been published in preliminary form,18,19 have appearedZo (cf ref. 1 Vol. 12 p. 4). Additional C02H N"2 HO ,CHZOH & N NATURAL PRODUCT REPORTS 1986 results have now been reported for experiments with [l- I3C]putrescine which support earlier ones with [1,4-l 3C2]- [2,3- 3C2]- and [1-I3C l-amino-lSN]-putrescine. Evidence relating to the intermediacy of homospermidine (13) in the biosynthesis of pyrrolizidine alkaloids previously published in preliminary form,19 is now available in a full paper.21 In addition to the results reported previously with [1,9-14C2]homospermidine [as (1 3)] new complementary results that were obtained with [4,6-14C2]homospermidine [as (1 3)] have been published.Valuable information on the course of biosynthesis of the quinolizidine alkaloids lupinine (1 6) and sparteine (1 7) has been provided inter alia by the results of experiments with [l- 13C l-amino-lSN]cadaverine (15) (cf ref. 3 p. 165). Similar results have now been reported for lupanine (18);22 the combined results are summarized in Scheme 1 (the thickened bond indicates an intact I3C-lSN moiety) where the dotted lines further indicate the units of cadaverine which are used to elaborate the alkaloids.Similar results have been obtained for angustifoline (19) but appropriately where two 3C-15N doublets are seen in the 3C{ H} n.m.r. spectrum of sparteine and lupinine only one is seen in the spectrum of angustifoline.24 Useful confirmation of the correctness of results from biosynthetic experiments is provided when the results are obtained by more than one group of workers particularly if the experiments are sophisticated ones. The closely corrobor- ating results from two research groups examining the incorporation of (1R)-[l-ZHl Icadaverine (20) and (15')-[ 1-2Hl]cadaverine (21) into sparteine (24),22,23 lupanine (18),23 and angustifoline (19),23 are to be welcomed for this reason but more especially because they provide valuable insight into the way in which these alkaloids are biosynthesized.The (1 R)-[ l-ZH,]cadaverine (20) stereospecifically afforded sparteine (24) that was labelled with deuterium at C-2a C-60 C-1 la C-1501 and C-17a whilst the(lS)-[l-2Hl]cadaverine(21) gave alkaloid that was labelled at C-2p C-lOa and C-17a. Appropriately similar results were obtained for lupanine (1 8) and angustifoline (19). These results are consistent with the hypothesisz5 (cf. ref. 1 Vol. 8 p. 3) that these alkaloids are modified trimers of A'-piperideine (22) (Scheme 2). In accord [2-2H]-A1-piperideine [as (22)J gave labelling of C-60 C-1 la and C-17a.22 Sparteine that had been derived from ~-[2- 2H]lysine [as (23)] was labelled in the same way as that which had been derived from (IS)-[ l-2H,]cadaverine (21) which means that decarboxylation of lysine to give cadaverine proceeds with normal retention of configuration.22 0 Angustifoline (19) 0 Lupinine (16) Sparteine (17) Lupanine (18) Scbeme 1 NATURAL PRODUCT REPORTS 1986 -R.B. HERBERT Cadaver ine 1 Iiii H HR Sparteine (24) Processes i si attack at C-2 from the face that is si at C-3; ii loss of lO@ro-lOR)-proton;iii re attack at C-11 from the face that is si at C-9; iv re addition of H-at C-10 and C-17. Scheme 2 Enzymic evidence has been obtained that 17-oxosparteine plays a key role in the biosynthesis of tetracyclic quinolizidine alkaloids26(cf. ref. 1 Vol. 11 p. 4). Since however lupanine (18) and sparteine (24) retain deuterium from (1R)-[1-2Hl]cadaverine and from [2-2H]-A1-piperideine at C-l7a at a similar level to that at positions lla and @ 17-oxosparteine cannot be an intermediate in the formation of these alkaloids.Similar evidence excludes 10-oxosparteine or 1,2-didehydrospartenium ion as intermediates and excludes the possibility that sparteine is derived by way of lupanine. Recent results2' on the derivation of lupinine (16) from (20) and (21) have been confirmed23 (cf. ref. 3 p. 164). The presence of an S-adenosyl-L-methionine :cytisine N-methyltransferase has been detected in crude enzyme prepara- tions from LaburniLm anagyroides plants and from cell cultures of Laburnum alpinum and Cytisus canariensis. 28 The transferase specifically catalysed the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to cytisine (25) yielding N-methylcytisine (26).Other quinolizidine alkaloids e.g. angustifoline (19) and albine were only N-methylated at a much lower level. Other properties of the enzyme were determined. 2 lsoquinoline Alkaloids The proceedings of a symposium which dealt with aspects of the chemistry and biology of isoquinoline alkaloids have been published29 as has a review of the biosynthesis of aporphine alkaloids.30 Good incorporation of [2-'4C]tyrosine into Erythrina alka-loids in young expanding tissues of the shoots of Erythrina crista-galli has been reported.31 A diverse spectrum of benzylisoquinoline alkaloids has been isolated from cell suspension cultures of Fumaria ~apreolata.~ The report of the occurrence in Corydalis claviculata of the benzylisoquinoline precursor for cularine [previously published in preliminary (cf.ref. 3 p. 167)] has now been published in full.34 Detailed and valuable enzymic information is now available on the course of the biosynthesis of protoberberines(cf. ref. 3 p. 166). (S)-Reticuline (27) is a key biosynthetic intermediate which is converted via the tetrahydroprotoberberine (9-scoulerine (28) into alkaloids such as berberine (31). A new enzyme has been isolated from suspension cultures of Berberis wilsoniae var. subcaulialata and has been purified and characteri~ed.~~ The enzyme S-adenosyl-L-methionine :(9-scoulerine 09-methyltransferase is a highly stereospecific and regiospecific transmethylase.Of several closely related tetrahydroprotoberberines,only scoulerine (28) served as a substrate for the enzyme; it was specific for the (9-isomer of this alkaloid and methylation only occurred on the oxygen atom at C-9 to give (9-tetrahydrocolumbamine(29) as the product. This alkaloid and several other tetrahydroprotoberberines (with differing patterns of 0-methylation and of substitution by methylenedioxy-groups in rings A and D) can serve as substrates for the 'STOX' enzyme which catalyses their conversion into protoberberines exemplified by berberine (3 1) and columbamine (32)35 (cf ref. 3 p. 166). Like the berberine- bridge and STOX enzymes the 09-methyltransferase has a pH optimum of pH 8.9. qpR (25)R = H (27) (26) R = Me HO (311 0 0 OMe (34) (35) A methyltransferase which converts tetrahydrocolumb-amine (29) into tetrahydropalmatine (30) in the presence of SAM has been isolated from Berberis aggregata.37 It is specific to (29) as a substrate; no reaction was observed with tetrahydrojatrorrhizine tetrahydropalmatrubine columbamine jatrorrhizine or palmatrubine.The enzyme preparation also showed oxidase activity converting tetrahydroberberine tetrahydrocolumbamine and tetra-hydropalmatine into their quaternary counterparts [as (3 l)]. This oxidase activity is similar to that reported by others,36 except that here37 no oxidation of tetrahydrojatrorrhizine was observed which is in contrast to the earlier results (cf:ref.3 p. 166). An iron(@-containing enzyme which catalyses the conversion of columbamine (32) into berberine (31) has been isolated from cell cultures of Berberis st~lonifera.~~ The enzyme has been purified and characterized; it has a pH optimum of pH 8.9 like the other enzymes of the berberine pathway that were discussed above. Neither (R)-nor @)-tetra-hydrocolumbamine acted as a substrate for the enzyme. Interestingly the conversion of [3-O-methyl-3H3]columbamine [as (3211 into (31) was found to involve the loss of exactly one third of the radioactivity; i.e. there was no isotope effect. In conclusion work at the enzymic level has led to the isolation of four enzymes that are responsible for the individual steps which lead from (8-reticuline (27) to (8-scoulerine (28) to (3-tetrahydrocolumbamine (29) to columbamine (32) to berberine (31).39 The enzymes that are involved in the formation of (9-norlaudanosoline and the subsequent three methylation reactions to give reticuline (27) have also been isolated purified and chara~terized.~~ The complete pathway NATURAL PRODUCT REPORTS 1986 (28) (29)R = H (30) R = Me MeoqMe HO \ Me0@!Me \ OH (33) 0 OMe (36) to berberine is thus known in impressive detail; this constitutes a quite outstanding piece of research.A cell-free preparation has been obtained from cultures of Coptis japonica which converts (3-tetrahydroberberine into berberine (3 l).40 (R)-Tetrahydroberberine (as expected) and tetrahydropalmatine (30) failed to act as substrates for dehydrogenation the findings with the latter compound being in contrast to results referred to above.2’-Methyl[3-14C]reticuline (33) has been examined as a precursor for morphinan alkaloids in Papaver ~ornniferurn.~~ A codeine fraction was obtained on isolation and purification which by its radioactivity (5.3% incorporation) appeared to contain methylcodeine. Incubation of codeine with isolated seed capsules of P. somniferum leads to the production of morphine.42 Cofactors such as NAD+ ATP acetyl-CoA and pyridoxal phosphate were not required for the conversion. Reducing agents strongly promoted the degradation of morphine and codeine. 3 Metabolites Derived from Tryptophan 3.1 Streptonigrin Streptonigrin (34) and streptonigrone (35)43 have been isolated from an unidentified Chinese species of Streptomyces.It is probable that (35) is derived simply from (34). The biosynthesis of streptonigrin has been shown to follow an interesting course (cf. ref. 3 p. 172; ref. 1 Vol. 13 p. 18) from P-methyltryptophan in which the heterocyclic ring in the indole moiety undergoes scission; this ring is still intact in lavendamycin (36).44 A mechanism for the ring-opening reaction has been suggested,43 based on chemical precedent (Scheme 3). NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT 0 H-0 :O COZH Me H+8' HO- Ho \ Scheme 3 &OH /COH +OH +OH (37)2E (39) (411 (42) (38) 22 (0 =13C) HO I OAc \ %OH -OR (43) (44)R = H (45)R = -D -glUCOSyl OH OH (46) (47) (48) 3.2 Terpenoid Indole Alkaloids Evidence has been obtained that secologanin and vindoline are biosynthesized via 1 0-oxogeranial (37)/1O-oxoneral (38) and iridodial (39) in Catharanthus roseus and Lonicera morrowiP5 (cf ref.3 p. 170). This work has been extended to include the alkaloids ajmaline (43) and vomilenine (44) using suspension cultures of RauwoBa serpentina to which were fed a mixture of 1O-hydroxy[4-' 3C]geraniol (40) lO-hydroxy[9-I 3C]nerol (41) 9,1O-dihydro~y[2-~ 3C]geraniol (42) and [lO-*H,]iridodial [as (39)].46As before,45 the trihydroxy-compound (42) was poorly utilized for biosynthesis but the other compounds were well incorporated into the two alkaloids.46 It was concluded that the biosynthetic pathway that was deduced45 to occur in C.roseus and L. morrowii also occurred in the suspension cultures of R. serpentina. It follows that this pathway is likely to be a general one. The reported4' transformation of [2-'4C]tryptamine plus secologanin into vindoline by a crude enzyme preparation of the leaves of C. roseus (cf:ref. 2 p. 185) has been carefully re- examined.48 No evidence could be obtained for this trans- formation which requires many enzyme-catalysed steps. Convincing evidence was obtained however that the earlier positive result arose from radioactive impurities in the sample that was presumed to be pure vindoline. Raucaffricine has been found to be a major constituent of cell suspension cultures of Rauwolfia serpentina (cf:ref.3 p. 170). Its structure has recently been revised from vomilenine a-D-galactoside to vomilenine P-D-glucoside (45) by enzymatic and n.m.r. Guettardine an alkaloid which has been isolated from Guettarda heterosepala has been assigned the structure (47).50 It may represent a structural type that is intermediate between the CorynanthP (46) and cinchonamine (48) groups of alkaloids which are precursors of alkaloids such as q~inine.~.~ If so it appears that cleavage of the P-carboline system of (46) precedes the formation of the quinuclidine ring. Very interesting results concerning the biosynthesis of alkaloids and the vacuoles of plant cells have been p~blished.~' Vacuoles were isolated from different plant cell cultures.The transport mechanism for alkaloids at the tonoplast membrane as well as the compartmentation of enzymes and their products were examined. While serpentine which is the major alkaloid of Catharanthus roseus cells was clearly located inside the vacuole the two key enzymes that are involved in the biosynthesis of terpenoid indole alkaloids (strictosidine synthase and a specific glucosidase) were found to be located in the cytosol. The transport of alkaloid across the tonoplast into the vacuolar space has been characterized as involving an active energy-requiring mechanism which is sensitive to the temperature and pH of the surrounding medium. Cell cultures of Catharanthus roseus RauwoIJia serpentina Nicotiana sylvestris Datura meteloides Papauer somniferum and Daucus carota (which produces no alkaloids and into the vacuoles of which no alkaloids were transported) were examined in this investigation.The alkaloids which were examined (serpentine ajmalicine catharanthine vindoline nicotine scopolamine morphine and codeine) were taken up into the vacuoles against a concentration gradient and the system for their uptake was specific for the alkaloids that are indigenous to the plant from which the vacuoles were isolated. Serpentine was an exception it was not transported into the vacuoles of R. serpentina and C. roseus. 3.3 Actinomycin The phenoxazinone actinomycin is an important antibiotic which is produced by Streptomyces antibioticus. Several DNA sequences from this organism have been cloned leading to the expression of phenoxazinone synthase activity in Streptomyces livid an^.^^ Of three recombinant plasmids which resulted in expression of the synthase one contained the gene for phenoxazinone synthase.Evidence has been presented that the other two activate a normally 'silent' synthase gene in S. lividans.53 4 Metabolites Derived from Phenylalanine and Tyrosine 4.1 Naphthyridinomycin Further results have been reporteds4 on the biosynthesis of naphthyridinomycin (49). Labelling of C-1 and C-2 in (49) by 3 C H20H I H-;f\; HZN COzH (50) 4-&Kx +-(54) X = N=C (55) X = N=C=S (56)X = NHCHO OH NATURAL PRODUCT REPORTS 1986 [1,2-13C2]glycine had been observed previouslys5 (cf ref.2 p. 187). However [2-l 3C]glycine gave almost identical labelling of (49) but with higher enrichment which suggested that glycine was being incorporated into this C2 unit via serine. (This is a normal metabolic reaction in which one molecule of glycine condenses with C-2 of another molecule of glycine as a one- carbon unit that is linked to tetrahydrofolate). In support of this suggestion ~-[3-'~C]serine [as (50)] gave (49) in which C-2 was labelled; the I4C label from ~-[l-l~C]serine was not incorpor- ated. As expected the three methyl groups of (49) were also labelled by [3-' 3C]serine (via the C,-tetrahydrofolate pool) (cf ref. 2 p. 187). 4.2 Isocyanides and Tuberin The biosynthesis of the isocyanide (51) in Trichoderma hamatum has been studied by two groups of workers (cf ref.2 p. 188). The results that were obtained by one of these groupss6 have now been published in full.57 No positive information on the origin of the isocyanide function has been obtained. Like the isocyanide (51) hazimycin factor 5 [(R,R)+ (S,S)] and hazimycin factor 6 [(R,S)](53) have been shown to derive from tyro~ine.~~ DL-[@-'3C]Tyrosine [as (52)] was well in- corporated into the metabolites (53) in cultures of Micromonospora echinospora var. challisensis and the label was shown to be located as required at the two C-3 positions. [methyl-' 3C]Methionine was also incorporated if considerably less well than tyrosine. In the 13C n.m.r. spectrum of the metabolites enhancement of the signal for C-4 was apparent.This indicates that the carbon atoms of the isocyanide groups in (53) may originate from methionine and suggests that the derivation of (53) is uia N-methyltyrosine. However label was not incorporated into (53) from [N-methyl-' 3C]-N-methyl-~~-tyrosine. Further 4C-labelled formaldehyde was not incorpor- ated. It should be noted that results of experiments on xanthocillin monomethyl ether (61) clearly rule out the derivation of the isocyanide functions in this metabolite as being from C -tetrahydrofolate intermediates (such as formal- dehyde and methionine) (see below). H02C/ (52) fl OR2 H-' R'O \ NEC +-H-$? XI (59) R' = R2= H (57)X = NHCHO (60)R1=R2= Me (58)X = AE6 (61) R1= Me R2= H OH &NH2 'C02H HO \ Me0rNHCHO \ (62) (65) NATURAL PRODUCT REPORTS 1986 -R.B. HERBERT The isocyanides that are discussed above and below are produced by micro-organisms Isocyanides are also produced by marine sponges and the biosynthesis of one of these 2- isocyanopupukeanine (54) has been examineds9 (under conditions and in surroundings more pleasantly romantic than those possible with micro-organisms). Experiments were carried out on a species of Hymeniacidon using samples of (54) (59,and (56) that were labelled with I3C on the C1 unit. It was shown that whereas (54) was converted into (55) and (56) neither (55) nor (56) served as a precursor for (54). This important finding corroborates the observation that I4C-labelled (57) failed to yield labelled isocyanide (58) in the sponge Axinella cannabina.60 As was observed for the biosyn- thesis of xanthocillin (59)61and its monomethyl ether (61),64 formate was not incorporated into (54).s9 The structure of tuberin (62) is similar to that of xanthocillin (59).Like the latter metabolite tuberin has been shown to derive from tyro~ine~~ (cf ref. 3 p. 173). Racemic threo-p- hydroxytyrosine (63) was shown to act as a precursor for tuberin (62) in Streptomyces amakusaensis but only after it had been degraded. C-a of the precursor served as a source for the C units in the metabolite. This degradation occurs plausibly uia a retro-aldol-type reaction to give 4-hydroxybenzaldehyde and glycine with C-2 of the latter then serving as a normal C1 source via tetrahydrofolate-linked intermediates (cJ ref.3 p. 173). This hypothesis is supported by the results of further experiments.62 A cell-free preparation of S. amakusaensis was obtained which converted racemic threo-p-hydroxytyrosine (63) into 4-hydroxybenzaldehyde. Appropriately for an enzyme-catalysed reaction approximately 45% of the material was converted. Racemic erythro-p-hydroxytyrosine(63) was inert in a parallel experiment. In accord with the above observations neither racemic [a-4C 3 S2H,]-erythro-P-hydrox ytyrosine [as (63)] nor 4-hydro~y[3,5-~H,]benzaldehyde served as precursors to label (62) in intact cultures of S. amakusaensis. It was concluded that there is a normal enzymic degradation of one of the enantiomers of threo-P-hydroxytyrosine (63) to give 4- hydroxybenzaldehyde and glycine in S.amakusaensis. This may well be a general degradative pathway for tyrosine and a novel one. It is apparent from these62 and earlieF3 results that the double-bond in tuberin (62) is not introduced by way of tyrosine derivatives that are hydroxylated at C-p. Similar conclusions were made for the biosynthesis of xanthocillin dimethyl ether (60) in Aspergillus clauatus neither tyramine nor octopamine (64)was incorporated into this metabolite whereas tyrosine was a good pres~rsor.~~ From the results6* of experiments in S. amakusaensis with samples of tyrosine that were chirally deuteriated at C-p and with D-and L-tyrosine that were deuteriated at C-a it is clear that tuberin is formed from L-tyrosine (65) with retention of the proton at C-a (incomplete due to competing transamination) and retention of the P(pro-PS)-proton.The trans double-bond in (62) is formed therefore by an antiperiplanar elimination of carbon dioxide and of the P(pro-pR)-proton in tyrosine (65). This result provides a nice contrast with the introduction of double-bonds in the biosynthesis of dehydroamino-acid moieties in several secondary metabolites. In the cases that are known two hydrogen atoms are lost and in a syn stereoc hemical sense. The stereochemistry that is associated with the introduction of the cis double-bonds in the biosynthesis of xanthocillin monomethyl ether (61) has been probed in a similar way.62 Here instead of loss of the P(pro-PR)-proton in tyrosine it is the P(pro-PS)-proton which is lost.Results of further experiments have been reported on the biosynthesis from glycine of the two C units (the N-formyl group and the 0-methyl group) in tuberin (62).64 The results show that glycine (66) is.incorporated into both C units with partial non-stereospecific loss of the two protons at C-2 and also that the N-formyl group is formed as well with stereospecific loss of the 2(pro-2S)-proton. Since biosynthesis proceeds in all probability by way of C,-tetrahydrofolate intermediates (Scheme 4) the results complement those65 which show that the ll(pro-1lR)-proton in (67) is removed during its enzymatic conversion into (68). Thus overall in the conversion of C-2 of glycine (66) into C-11 of (68) the 2(pro-2S)-proton in the amino acid becomes the 11-(pro-1lR)-proton in (67).It should be noted however that if a different system (but one which also implicates C1- tetrahydrofolate intermediates) is used it is the 2(pro-2R)- proton in glycine (66) which becomes the 11(pro-1 1R)-proton in (67).(j6 Further work is clearly needed to resolve this difference. The origin of the isocyanide functions in xanthocillin monomethyl ether (6 1) that is biosynthesized in Dichotomo- myces cejpii has been investigated using glycine serine and methionine as potential donors of C units.64 In this metabolite the 0-methyl group provides an internal check on the incorporation of C1 units. As expected the 0-methyl group was labelled by methionine and also by glycine but no labelling of the carbon atoms of the isocyanide groups was apparent (from I3Cn.m.r.spectra). Although both radioactive formic acid and serine (which is a better donor of C than glycine) were incorporated into (61) again no labelling of the carbon atoms of the isocyanide groups was apparent. Thus the metabolism of C,-tetrahydrofolate units is not directly linked to the biosyn- thesis of the isocyanide functions in xanthocillin monomethyl ether (61) from this micro-organism. The different results for the biosynthesis of the isocyanide functions in the hazimycins (see above) should be noted however. (66) H (67) YNHCHO Tuberin (62) Met hionine ___) cACH-jO Scheme 4 NATURAL PRODUCT REPORTS,1986 4.3 Cinodine The origins of the antibiotic cinodine (69) which is produced by species of Nocardia have been determined using 14C- and 3C-labelled precursors.The cinnamoyl residue was shown to derive from L-tyrosine (p-coumaric acid was incorporated at a level similar to that of tyrosine; phenylalanine was also incorporated but less effi~iently).~' The three sugar residues derive directly from D-glucosamine; D-[ 1 3C]glucosamine gave a sample of (69) that was labelled to a similar extent at the anomeric centre of the hexose unit and the anomeric centres of the two pentose ~nits.6~ It has been found68 that the amino acids (arginine citrulline and ornithine and also putrescine) that are involved in the urea cycle are well incorporated into cinodine (69).Results of experiments with [guanidino-l4C]- and [U-l4C]-arginine indi- cated that both the guanidine side-chain (ring A) and the ureido-groups in the pentoses were more efficiently labelled by this amino acid than was the spermidine fragment (via ornithine and putrescine). Further more definitive results with L-kuanidin0-l3C]arginine and L-[ureido-*3C]citrulline are that these precursors label only the guanidino-group and not the ureido-functions.68 Labelled urea was a poor precursor for cinodine6' and potassium [ 3C]cyanate68 was not in-corporated. It was concluded68 that the urea cycle is not directly important in the biosynthesis of (69). The guanidino- group is derived directly from arginine (by the action of an amidinotransferase) and citrulline is probably converted into arginine before use.(69) R = ~~-[5-'~C]Ornithine gave cinodine in which C-6 was labelled;68 as expected for a precursor of ~permidine,~~ methionine was efficiently incorporated into ~inodine.~~ DL-[5,ureido-C2]Ci trulline labelled the guanidino-moiety and C-6.68 4.4 Lincomycins The lincomycins A (70) and B (71) contain respectively a trans-4-propyl-~-hygric acid moiety (73) and a truns-4-ethyl-~- hygric acid fragment (74). In this they are structurally similar to a number of other antibiotics namely anthramycin tomaymycin and sibiromycin (cf. ref. 4 and ref. 1 Vol. 11 p. 26). These units in the lincomycins are formed from tyrosine by way of dopa (72).'O The way in which the dopa molecule is modified in the course of biosynthesis of the lincomycins has been explored with ~-[3,5-~H,]tyrosine [as ~-[2,5,6-~HJdopa (72)] and ~-[rnethyl-~H,]methionine.~ * From the deuterium retentions that were observed after the tyrosine and dopa samples had been incorporated it was deduced that biosyn- thesis followed the course that is shown in Scheme 5 rather than one that had been proposed earlier.'O This deduction was supported by the results that were obtained with the labelled methionine the alkylated hygric acid moiety (73) in lincomycin A (70) but not that in lincomycin B (71) showed incorporation of label as a C-methyl group [the earlier proposal had required that both (73) and (74) should incorporate label from methionine].Scheme 5 193 NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT Further support for this pathway was obtained71 by an experiment involving D-[U-'3C]glucose. From the couplings that were observed in the 3Cn.m.r. spectrum of the hygric acid moiety in the derived lincomycin A it was possible to deduce the fate of the original tyrosine skeleton that had been formed from glucose via shikimic acid. (For other similar examples involving the use of [U-13C]glucose see ref. 1 Vol. 13 pp. 18 and 22 and Vol. 12 p. 24). These results support a previously proposed unified biosynthetic pathway72 leading from tyrosine uia dopa and involving a 2,3-extradiol cleavage (Scheme 5) to produce a common precursor of the C2-substituted and C3-substituted proline units that are found in the lincomycins anthramycin tomaymycin and sibiromycin.D-[U-I 3C]Glucose also labelled the amino-octose moiety [1 -deoxy- 1-methylthio-a-D-lincosamine (MTL)] in the lincomycins. An analysis of the very complex coupling that was observed in the 13C n.m.r. spectrum of this moiety in lincomycin A (70) in association with labelling data that had been obtained through the use of D-[l-13C]- and D-[6-13C]- glucose [1-I 3C]pyruvate and [2-' 3C]- and [1,3-l 3C2]-glycerol as precursors allowed some conclusions to be made on the course of the biosynthesis of MTL.73 It was concluded that the C8 skeleton of MTL was formed by condensation of a (C,) pentose unit with a C3 unit. The C5 unit arises by two pathways either from glucose (uia a hexose monophosphate shunt) as an intact unit or from a C3 unit (glyceraldehyde 3-phosphate) with a C2 donor [such as sedoheptulose 7-phosphate (SH7P)] via a transketolase reaction.The C3 unit which combines with the C5 fragment to form the C8 skeleton is likely to arise from a suitable donor molecule such as SH7P uia a transaldolase reaction. Dependent on the origin of the donor of the C3 unit this unit may consist either of an intact C3 moiety or of a C2 unit combined with a C1 unit. 4.5 Saframycin A Saframycin A (75) is an antibiotic that is produced by Streptomyces lauendulae. It was previously shown to be labelled by ~[U-l~CItyrosine (cf. ref. 2 p. 187). A detailed study of its biosynthetic origins which were determined by using 3C-labelled precursors has been published (Scheme 6)74 (cJ naphthyridinomycin above).The derivation of the pyruvamide side-chain from alanine has not yet been proved although derivatives of saframycin that contain an alaninyl side-chain have been isolated. Furthermore a crude enzyme preparation has been obtained from S. lauendulae which would convert the metabolite with the alaninyl side-chain into saframycin A (73.75 A OCH3 I GIyc ine 5 Other Metabolites Derived from the Shikimate Pathway 5.1 Ansamycins The biosynthesis of rifamycins has been reviewed.76 The stimulation of the production of rifamycin in Nocardia species by 'B-factor' [3'-0-(butylphosphono)adenosine] which is present in a yeast extract has been rep~rted.'~ Ansamitocins P-2 (76) P-3 (77) and P-4 (78) are produced by Actinosynnema pretiosum.The effect of various compounds on the production of ansamitocins has been examined.78 It was found in particular that compounds which could be metabo- lized to one of the three side-chains in the ansamitocins led to enhanced production of that antibiotic. Thus the production of ansamitocin P-2 (76) was stimulated by the addition of isoleucine propionate propionaldehyde or n-propyl alcohol. Incorporation of these compounds presumably occurs uia propionyl-CoA. Similar results were obtained for the other two ansamitocins. In each case there appear to be two pathways leading to the particular antibiotic ansamitocin P-2 (76) is formed either beginning with n-propyl alcohol or with isoleucine (and a-amino-n-butyric acid) P-3 (77) is formed from isobutyl alcohol or valine and P-4 (78) is formed from isoamyl alcohol or leucine.The origins of the ansa-framework of ansamitocin P-3 (77) have been explored with 14C- and 3C-labelled prec~rsors.~~ The results are illustrated in Scheme 7. They are similar to those for other an~amycins~~ (cf. ref. 2 p. 188; ref. 3 p. 174; ref. 1 Vol. 8 p. 30 Vol. 12 p. 21 and Vol. 13 p. 22). The aromatic C7N unit is known as in other antibiotics to arise via 3-amino-5-hydroxybenzoic acidg0 (cf. ref. 2 p. 189). C-9 and C- 10 were formed not from acetate but from a metabolite of glucose. C-24 was labelled by L-[carbamoyl-' 4C]citrulline but curiously not by L-[guanidino-1 4C]arginine or carbamyl phosphate.The methyl groups were labelled by [2-l 3C]glycine and by L-[methyl- 3C]methionine. The labelling by glycine is the result of normal metabolism involving tetrahydrofolic acid. 6 P-Lactams 6.1 Penicillins and Cephalosporins Aspects of the biosynthesis of cephalosporins and penicillins have been reviewed.81 Further studies with modified tripeptides that are based on the tripeptide L,L,D-ACV [N-(L-6-aminoadipyl)-L-cysteinyl-D-valine] (79) which affords the penicillins and cephalosporins in uiuo have been published (cf ref. 3 p. 174). The outcome of this work is the production of new p-lactams and additional insight into the mechanisms of normal biosynthesis. 6 HOm2C0zH \ Tyrosine JCOZH Methionine Alanine Scheme 6 194 NATURAL PRODUCT REPORTS 1986 A - ' [Me]Methionine CI A OR Me0 0 (78) R = Scheme 7 The cyclase which normally converts L,L,D-ACV into the first penicillin isopenicillin N will adipyl-L-cysteinyl-D-valine as its substrate suggesting that the amino- group of the 6-aminoadipyl moiety is not essential for cyclization.When D,L,D-ACV was incubated with a partially purified extract of Cephalosporium acremonium and the nec- essary cofactors (the extract contained cyclase and expandase activities but no penicillin epimerase) the formation of penicillin N and deacetoxycephalosporin C was detected ;8 if purified isopenicillin-N synthetase from C. acremonium was used penicillin N was formed.82 The D,L,D-ACV was a poorer substrate for the enzyme than the natural peptide L,L,D-ACV (79).Several other compounds that are based on ACV modified in the 6-aminoadipyl moiety were tested as substrates for the synthetase with negative results (there was one exception see ref. 88; cf. ref. 3 p. 175). It was concludeds2 that the minimal structural requirement for N-acyl-L-cysteinyl-D- valines to be converted into penicillin products by the isopenicillin-N synthetase is that the N-acyl group has a six-carbon (or equivalent) chain terminating in a carboxyl group; i.e. there is a binding site for the carboxyl group which is separated from the catalytically active site (which may bind to the cysteinyl sulphur atom of the substrate) by a distance equivalent to the length of one of the conformations of the adipyl side-chain.The synthetase from Streptomyces clauuligerus showed very similar substrate specificity to that for the enzyme from C. acremoni~m.~~ A change in configuration of the 6-aminoadipyl moiety from L to D or a decrease in its chain length by one or two carbon atoms was found to abolish its ability to be cyclized by the enzyme. The modified tripeptide (80) when incubated with an enzyme preparation of C. acremonium,afforded penam (84) and the cephams (87) and (88); (81) similarly afforded (89) and (90).86 These results complement ones that were obtained earlier8' (cf. ref. 3 p. 175 Scheme ll) and it was concluded that penam and cepham products arise as a consequence of the relative stability of a radical at C-3 and C-4 of (91) and also of steric factors (Scheme 8).With isopenicillin-N synthetase the tripeptide (82) has been found to be converted into six p-lactam products i.e.(85) (86), (92) (93) (94) and (95).89 The formation of these products has been rationalized as shown in Scheme 9. H 0 0 Acetate = 0 Propionate = 6 (79)R' = R2= Me (80) R' = Et ,R2= Me (81) R' = H ,R2= Et (82) R' = HI R2= CH=CH2 C02H (87)R'= R2= Me ,R3= H (88) R1=R3 =Me R2= H (89) R' = R3 = HI RZ = Me (90)R' = R2 = HI R3 = Me 195 NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT S -Enzyme v'>T$I [R= Me] ____) y'HJMey'FxMe c---[R = H orMe1 0 \ 0 R 0 R I 1 \ CO2H iO2H bO2H (91) R = H or Me Scheme 8 R' -OH ( R1 = L -8 -aminoadipyl) H S-Enzyme RNxJ-$Hz0 I C02H (851 (86) (93) Scheme 9 (96) Work relating to (96) as an isolable intermediate in the biosynthesis of penicillins (previously available in a prelimi- nary formg0) has been published in full9' (cf.ref. 2 p. 190). The intact conversion of L,L,D-ACV(98) labelled as shown with I3C and I5N into isopenicillin N (IOl) using a cell-free preparation of C. acremonium has been reported (IJCN = 4.4 Hz).~~ An attempt to detect intermediates between (98) and (101) failed (cJ ref. 1 Vol. 12 p. 25 for similar results) (101) The isopenicillin-N synthetases from C. acremonium and Penicillium chrysogenurn have been purified.93 The enzymes from these sources are similar in their values of M, their electrophoretic mobilities and their requirements for cofactors.Preliminary results indicate that the enzyme from Streptomyces clavuligerus is likewise similar (cf:ref. 2 p. 190). The isopenicillin-N synthetase from P. chrysogenum has been isolated and characterized inde~endently.~~ NATURAL PRODUCT REPORTS 1986 A synthesis of penicillins has been reported which begins cephalosporin C (105) has been probed with (3S,4S)-and with a monocyclic p-lactam and involves the oxidative (3S,4R)-[4-2H1 ,4-3HI]valines (107).101 The precursors were formation of the thiazolidine ring using Fe2+ and ascorbic incorporated into cephalosporin C (106) in cultures of C.acid which are the same cofactors as are needed in the enzymic acremoniurn.The analysis of the antibiotic was as follows the reaction.9s Further evidence that the enzymic reaction involves cephalosporin C was chemically degraded to glycolic acid (108) free radicals is thereby adduced. [C-2 =C-3’ of (106)] which was then incubated with glycolate Useful evidence is accumulating that in the conversion of oxidase to give glyoxylate (109) with known removal of the L,L,D-ACV (79) into isopenicillin N (83) the P-lactam ring is shut first to be followed by the formation of the thiazolidine ring87*96 (cf ref. 3 p. 174). Additional impressive evidence has been obtained from experiments with deuteriated ACV.97 A 1 :1 mixture of L,L,D-ACV (97) and L,L,D-A[~,~-~H,]CV (99) was incubated with crude isopenicillin-N synthetase.Analysis during the course of the reaction of recovered starting material and of derivatized penicillin showed that there was a preferential conversion of the protio-tripeptide (97) compared to the conversion of the deuterio-substrate (99); if (99) was incubated with enzyme deuteriated penicillin was formed. A similar experiment with a 1 :1 mixture of (97) and L,L,D-AC[~-*H~]V (100) was carried out. Unlike the previous experiment no isotopic discrimination was observed. Both cysteinyl- and uulinyl-deuteriated substrates show significant V,, (V,, =maximum velocity) deuterium isotope effects as measured in separate non-competitive experiments. In competitive mixed-label experiments isotopic discrimi- nation is a Vmax/Kmeffect98 (K =Michaelis constant) and reflects events only up to the first irreversible step.From the above results it is clear that only one of the bond-cleavages shows a Vmax/Km effect i.e. the cleavage of the cysteinyl C(3)-H bond and it was deduced therefore that this is the first chemical step in the reaction sequence; i.e. a P-lactam ring is the first to be formed. An energy-level diagram that is consistent with the above observations has been proposed assuming that there is a single isotopically-sensitive step for the cleavage of each C-H bond (Figure 1); the values of A1 and A2 must be comparable if the observation of V,, isotope effects at the positions 3 of the cysteine and valine residues is to be rationalized.Full details have been publishedg9 of very interesting results relating to the incorporation into cephalosporin C (106) of valine in which the methyl groups are chirally labelled (C1H2H3H)100(cf ref. 3 p. 176). The results that were obtained have led to the proposal of a mechanism for the conversion of the penicillin nucleus (102) into that of the cephalosporins (104) (Scheme 10); it is envisaged that the methylene group in the hypothetical radical intermediate (103) can undergo rotation. The stereochemistry associated with the hydroxylation of deacetoxycephalosporin C (1 04) which affords deacetyl-A Kx: \ 8 8 C02H (102) (103) 2(pro-2R)-proton of (1 08). Complementary results were obtained with the two samples of labelled valine and they show that hydroxylation is stereospecific (cJ refs.99 and loo) proceeding with normal retention of configuration. Deacetoxycephalosporin-C synthetase and deacetoxyceph- alosporin-C hydroxylase catalyse the sequential reactions in which penicillin N undergoes ring-expansion to deacetoxy- cephalosporin C (DAOC) (104) and then DAOC is hydroxyl- ated to give deacetylcephalosporin C (105). These two enzymes are intermolecular dioxygenases which require molecular oxygen iron ascorbate and 2-oxoglutarate for activity in both C. acremoniurn and S. cluvuligerus. An attempt to separate the two enzymes that are present in C. acremoniurn failed.lo2 More recently however the two enzymes from S. clauuligerus have been separated.Io3 The difference between the two organisms represents the first major difference to have been observed between the shared portions of the biosynthetic pathways to cephalosporin in prokaryotes and in eukaryotes.It has been found that glucose (and other monosaccharides) represses the formation of penicillin-synthesizing enzymes. Reaction co-ordinate Figure I (104) Scheme 10 HV? H02cxoH -Ho2cYo HS HR H(S) C02H H,““y’1”1F&OR N/ 3f H02C CH3 C02H (105) R =H (107) (106) R =AC NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT Glucose does not inhibit enzyme activity. Io4 High levels of glucose have been reported to regulate the formation of cephamycin C in Streptomyces luctumduruns by repressing the formation of deacetoxycephalosporin synthetase and by inhibiting its activity.lo5 6.2 Nocardicins The biosynthetic origins of the skeleton of nocardicin A (1 10) have been established1°6.107 (cf.ref. 1 Vol. 12 p. 27; Vol. 9 p. 33; ref. 2 p. 191). Two molecules of tyrosine provide part of the skeleton with loss of the carboxyl group from each molecule of the amino acid. Tyrosine is utilized via ~-(p-hydroxypheny1)glycine (1 11) (PHPG); PHPG that was labelled with 13Cat the carboxyl carbon gave (1 10) that was labelled at C-1' and C-10. Results of further experiments have now been published. DL-[CI-' C N]-(p-H ydrox yphen yl)gl ycine [as (1 1 l)] was incorporated into (1 10) in Nocardia uniformis. The nocardicin was enriched at C-5 and C-2' by 3C label (detected by I3C n.m.r.spectroscopy); in each case doublets (due to species that contained both 3C and 5N) were superimposed on singlets (due to I3C-containing species in which 5N label has been lost by transamination). The extent of double labelling at both sites was roughly equivalent (47 5%). It follows that the precursor was incorporated substantially intact into both PHPG units and that the nitrogen atoms that are attached to C-5 and C-2' derive from PHPG. Thus the unusual oxime function in (1 10) derives by oxidation of an amino-function. The similarity in the labelling of the two units suggests that there is some unison in the assembly of two PHPG units into an as yet unknown precursor of nocardicin A (1 10). 6.3 Clavulanic Acid Previous lo had shown that clavulanic acid (1 12) is biosynthesized from a C and a C5unit [see dotted line in (1 12)] H -NHz H C02H 10 (110) [TCA Cycle] 0 197 (cf.ref. 1 Vol. 10 p. 29 and Vol. 13 p. 36). The C3 unit is formed from a C3intermediate of glycolysis whilst the C5 unit has its origins in a C5amino acid that is directly related to 2- oxoglutarate (1 13) which is an intermediate in the TCA cycle; glutamic acid (114) was an intact source for this Cs unit.110 Further results show that amino acids [ornithine (1 15) and arginine] of the urea cycle particularly ornithine are much better [15-20 times] precursors for clavulanic acid (1 12) in Streptomyces cluuuligerus than are glutamic acid (1 14) 5-hydroxynorvaline (1 16) and proline (1 17).I There was minimal randomization of label from the ornithine precursor when it was converted into the C3 unit. The derivation of the C5 unit in clavulanic acid (1 12) appears therefore to be most directly from ornithine which may in turn be formed from 2- oxoglutarate (1 13) and glutamate (1 14) (Scheme 1 1). In an interconnected series of experiments 3H-and I4C- labelled glycerol pyruvate serine glycerate and glycine have been examined as precursors for the C3 unit in clavulanic acid (112).l** It is clear from the pattern of the results that were obtained that D-glycerate (1 18) is the most direct precursor; D-[l-14C 2-3H]glycerate [as (118)] gave (112) that was predominantly labelled by I4C in the C3 unit with tritium principally present at C-6.L-Serine (1 19) and glycine were incorporated uia glycerate; tritium at C-2 in each of these amino acids turned up at C-5 (and also C-8) in (1 12) consistent with its biosynthesis via [3-3H1]glycerate. 6.4 Catbapenem Antibiotics The biosynthetic origins of the carbapenem antibiotics are obscure. However a mutant of Streptomyces fulvoviridis has been obtained which accumulates OA-6129A (120; 6R) OA- 6129B (121; 6R) OA-6129B2 (121; 629 and OA-6129C (122; 6s) instead of PS-5 (123; 6R) epithienamycins A (124; 6R) and C (1 24; 69 and MMl7880 (1 25 ;6R) which are produced by the parent strain.l13 The OA-6129A (120; 6R) could be C02H (112) ___) -YCozH C02H H C02H (113) (114) f-H2NrCHo -"'"';.r^OH H C02H H' C02H Argi n ine [ Urea Cycle ] Scheme 11 198 NATURAL PRODUCT REPORTS 1986 SCHZCHZNHCCHZCHZNHCCH-C-CHzOHI t II0 II 0 I Me (120) R = H (123)R = H (121) R = OH (124) R = OH (122) R = OS03H (125) R = OS03H 0 (127) (128) 0 P C -0II N rC02H (134) (135) (136) R = H converted into NS-5 (1 26) plus pantothenate by using a cell-free preparation of the parent strain (but not of the mutant) and the same reaction could be achieved with amino-acid acylases.The results that were obtained led to the conclusion that the pantothenyl-containing carbapenems e.g. (1 20) were normal precursors in vivo for those with N-acetyl groups e.g. (123). The enzyme in S.fulvouiridis which catalyses this deacylation reaction has been designated A933 acylase.It has been isolated purified and characterized.' l4 A933 acylase was found to catalyse the depantothenylation of the OA-6 129 carbapenems the acyl-exchange reaction of these carbapenems with acyl- CoA's and the deacetylation of PS-5 (1 23 ;6R) and of N-acetyl- L-amino acids. Similar activities were detected in other species of Streptomyces. The properties of A933 acylase in relation to other amino-acid acylases have been further examined in some detail.115 It was further found that although the mutant that was referred to above lacked depantothenylating activity it had the same L-amino-acid acylase activity as A933 acylase. 7 Miscellaneous Metabolites A unique 2-amino-3-hydroxycyclopent-2-enone (128) moiety is present in asukamycin (129) amongst other antibiotics.Results of experiments with cultures of Streptomyces nodosus subsp. asukaensis indicate that this moiety is formed by a novel intramolecular cyclization (possibly involving pyridoxal phos- (137) R = OH phate) of 5-aminolaevulinic acid (1 27). Appropriate to the known biosynthetic origins of (127) (cf ref. 117) glycine was incorporated with loss of C-1 [C-2 of glycine becomes C-2" in (129)l; the nitrogen atom at C-2" was derived from glycine. Evidence was obtained that the remaining carbons could arise via succinate. It has been shown using cultures of Penicillium atroueneturn that 3-nitropropionic acid (13 1) derives intact from aspartic acid (130);l1*DL-[2-13C 15N]a~partic acid gave (131) the 13C n.m.r.spectrum of which showed a doublet for C-3 (arising from its coupling to I 5N).It has thus been established that the nitro-group in (1 31) arises by oxidation of the amino-group in aspartic acid. The biosynthesis of the phytotoxin coronatine (1 32) which is produced by Pseudornonas syringae pv. atropurpurea has been examined. ~-[U-~~C]isoleucine and ~-[U-l~C]threonine were incorporated specifically and efficiently into the aminocyclopropane moiety of (1 32); the incorporation of the former was better than of the latter indicating that isoleucine is the more immediate precursor. A compound that was identified as (133) has been isolated from a coronatine-producing pseudomonad. *O It is likely to be an intermediate in the biosynthesis of (132).L-[U-'4C]Valine was a good precursor for N-coronafacoylvaline (1 34) but a poor precursor for (1 32). By contrast labelled threonine and isoleucine were poor precursors for (1 34). NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT It has been well established that cyanogenic glycosides derive from the corresponding a-amino acid.' 21 It has been shown that deidaclin (136) (and its epimer tetraphyllin A) derive specifically in seedlings of Turnera ulmifolia from the unusual amino acid (cyclopent-2-eny1)glycine (1 35) [a-] 4C](cyclopent-2-enyl)glycine gave (1 36) which was labelled in the cyano-group.122 The amino acid (135) is known to occur naturally and has been isolated from T. ulmifolia.'22Previous work had shown that (135) is biosynthesized from a-ketopimelate.I 23 During the experiments with T. ulmifolia it was found that tetraphyllin B and epitetraphyllin B (137) were not labelled by radioactive (135) thus suggesting that the hydroxyl group on the cyclopentene ring is introduced at an earlier stage of the biosynthesis than (1 35). An enzyme preparation from the leaves of T. ulmifolia liberated hydrogen cyanide with notable efficiency from cyanogenic fractions that had been obtained during isolation thus suggesting the presence in T. ulmifolia of glucosidase(s) that are specific to the cyanogenic glucosides of this plant.'22 The biosynthesis and degradation of the steroidal alkaloid tomatine in tomato fruits have been studied using I4C-labelled materials. A study of the biosynthesis of fredericamycin A (138) in Streptomyces griseus has revealed chiefly using 3C-labelled acetate that the metabolite is a polyketide which includes nitrogen in the course of biosynthesis.125 C-1 is formed from the carboxyl group of acetate with loss of the methyl group.Although the pattern of labelling by acetate has been established [as shown in (139) and (140)] whether two chains are involved [as (139)] or only one chain [as (140)] is involved is unknown. The latter could involve a re-ordering of the skeleton associated with the formation of C-2 as the spiro- centre and loss of the methyl group that is attached to what becomes C-7' in (1 38). Although outside the scope of this review the interesting report' 26 that the biosynthesis of asparagusic acid (141) is from isobutyric acid via methacrylic acid 3-mercapto-2-methyl- propionic acid and S-(2-~arboxypropyl)cysteine, is to be noted.7.1 Virginiamycin Antibiotics The biosynthetic origins of virginiamycin MI have been defined with clarity12' (cf ref. 3 p. 177). Antibiotic A2315A .-(1 42) differs from virginiamycin M I in having a hydroxy-group HO 199 at C-16 instead of a carbonyl and a D-alaninet residue instead of a dehydroproline unit. The biosynthetic origins of (142) in cultures of Actinoplanes philippinensis have turned out to be similar to those of virginiamycin Most of the investigation was carried out with precursors that were labelled with stable isotopes (Scheme 12). Of particular note is the observation that like virginiamycin M the C-33 methyl group in (142) derives from the methyl group of acetate and the oxazole unit derives from serine ; 28 the valine is presumed to be incorporated via isobutyryl-CoA which serves as a starter unit for the western part of (142) as it is drawn here.Results of separate experiments with DL- L- and ~-[1- ''C]alanine samples each of which was mixed with DL-[~-~H]- alanine before it was fed to the micro-organism are that D-and L-alanine are equally good precursors for the D-alanine residue in (142).Iz8 The conversion of L-alanine into the D-isomer that exists in (142) does not involve a dehydro-amino- acid for serine was not incorporated into the alaninyl residue and ~-[3-'3C 3-ZH,]alanine was incorporated into (142) without loss of deuterium.7.2 Elaiomycin Carbon atoms 5 to 12 and the P-nitrogen atom in elaiomycin (143) are formed from n-octylamine (cf. ref. 2 p. 191). It has now been shown using cultures of Streptomyces gelaticus that C-2 C-3 and C-4 plus the a-nitrogen atom originate in serine.'29 It was adventitiously discovered in the course of checking the fatty acid origin of the n-octylamine portion of (143) that C-1 derives from C-2 of acetate. This unusual origin for a methyl group finds a parallel in the biosynthesis of the virginiamycin antibiotics (see above). The two mechanisms of biosynthesis however probably differ. [methyl-' 3C]-Methionine labelled only the 0-methyl group in (143). 7.3 Showdomycin Showdomycin (144) one of a group of C-nucleoside antibiotics is produced by Streptomyces showdoensis.Previous work had shown that the antibiotic was formed from ribose (145) and a precursor that is associated with the Krebs cycle such as 2- oxoglutarate.130 Further work has led to the firm conclusion that this precursor is glutamic acid (1 46). t The structures in ref. I28 appear as it were in a looking-glass; A23 15A is shown (in error) as having an L-alanine residue and Dalanine is drawn as the L-isomer and vice versa for i-alanine (D. G. I. Kingston personal communication; see J. Org. Chem. 1985 50 4666). NATURAL PRODUCT REPORTS 1986 H2N AtO;,H Glycine f-.. \ \ \ [Me] Methionine 0 \ I I I / / I \ A / \ NH2 \ Ho2cY \ \ \ 'I\OH \ Valine \ Serine .0 OH OH (143) (144) 5 CO;,H H02C Hoc~orOH NH2 Hs-:vqHH OH OH (145) The results of a combination of experiments using samples of 14C- I3C- and SN-labelled glutamic acid are that glutamic acid (146) is incorporated specifically into the maleimide ring of (144) with significant retention (ca. 35%) of the amino-group (lSN label was assumed to be lost by normal transamination) and loss of the carboxyl group; C-5 of glutamate labelled C-1 of (144). L-Glutamate was found to be a six-fold better precursor than the D-isomer. The role of glutamate as precursor for (144) was confirmed by the results of an experiment with [13CZ]acetate showdomycin was labelled at C-1 and C-2 by an intact acetate unit which is in accord with the metabolism of acetate via the Krebs cycle leading to [4,5-' 3CZ]glutamate (cf ref.130). L-Pyroglutamate (147) was found to be an excellent precursor for showdomycin and could be a normal intermediate.I3* However DL-[5-' 3C SN]pyr~glutamicacid was incorporated into (144) with some loss of lSN label (28% retention). For this loss to take place interconversion with glutamate must have occurred so the proof that pyroglutamate is an obligatory intermediate is currently lacking. C02H 5 0 (146) (1 47) The results of experiments with samples of glutamic acid that were chirally deuteriated at C-3 show that in the formation of the maleimide ring of showdomycin (144) the 3(pro-3R)- hydrogen of glutamate is lost and the 3(pro-3S)-hydrogen is retained.32 The results of investigating the incorporation of [I-l4C 5-ZHl]ribose have confirmed that ribose is the source of the remaining atoms in showdomycin ( 144).13* 7.4 Astromicin and Streptomycin Astromycin (149) is an aminoglycoside antibiotic that is produced by Micromonospora olicoasterospora. A detailed pathway to the antibiotic has been worked out by using mutants of M. olivoasterospora.'33 The structures of the intermediates that are produced by the mutants were estab- lished and the biotransformations of the compounds were then studied. In plotting out the course of biosynthesis radioactive methionine and glycine were used together with the various intermediates which lacked N-methyl and glycyl substituents.NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT CH2OH CH20H I I OH Ith rtn HO HO - H2N I A 0-OH scyllo -Inosose scyllo -1nosamine (148) OH OH Me Me Me I I I -1 H-C-NH;! H-C-NH;! H-C-NH~ CH2NH2 I I I 1 0 t 0-0-0 Me Me Me I I I H-C-NH2 H-C-NHz H-C-NH;! t;; 0 til 0 (-4 0 NHMe MeNCOCH2 NH; Scheme 13 The pathway to astromicin (149) which was deduced is shown in Scheme 13. It is notable that this antibiotic is formed by a pathway in which the second of two amino-groups is introduced into the cyclitol ring (ring A) after the pseudodisac- charide has been formed. In other aminocyclitol antibiotics (e.g. kanamycin gentamicin sagamicin and butirosin) by contrast a 1,3-diaminocyclitol is used for biosynthesis.The course of the biosynthesis of the important antibiotic streptomycin (I 50) is known in considerable detail. The 163 pathway to (1 50) through streptidine 6-phosphate from glucose 6-phosphate uia scyflo-inosamine (1 48) has been re-examined Me using mutants of Streptomyes griseus.' 34 The results are generally in agreement with knowledge gained hitherto. HO I 8 References R. B. Herbert in 'The Alkaloids' ed. J. E. Saxton (Specialist Periodical Reports) The Chemical Society/The Royal Society of Chemistry London 1971 Vol. I ; 1973-1975 Vols. 3-5; ibid.,ed. M.F. Grundon 1976-1983 Vols. 6-13; J. Staunton ibid. 1972 Vol. 2. OH (1 50) 2 R. B. Herbert Nut. Prod. Rep. 1984 1 181. 3 R. B.Herbert Nut. Prod. Rep. 1985 2 163. 4 R. B. Herbert in “Rodd’s Chemistry of Carbon Compounds” ed. S. Coffey Elsevier Amsterdam 1980,2nd edn. Vol. IV Part L p. 291. 5 R. B. Herbert ‘The Biosynthesis of Secondary Metabolites’ Chapman and Hall London 1981. 6 K.-M. Yao W.-F. Fong and S. F. Ng J. Biol. Chem. 1984 222 679. 7 S. Mitzusaki Y. Tanabe M. Noguchi and E. Tamaki Plant Cell Physiol. 1971 12 633; ibid. 1973 14 103. 8 F. Feth H.-A. Arfmann V. Wray and K. G. Wagner Phytochemistry 1985 24 921. 9 E. Leete Phytochemistry 1985 24 957. 10 D. G. O’Donovan and M. F. Keogh J. Chem. Soc. (C) 1969,223. 11 H. W. Liebisch K. Peisker S. Radwan and H. R. Schiitte 2. PJanzenphysiol. 1972 67 1. 12 E. Leete Phytochemistry 1985 24 953. 13 E. Leete J.Am. Chem. SOC. 1982 104 1403. 14 E. Leete J. Am. Chem. SOC. 1984 106 7271. 15 G. B. Lockwood and A. K. Essa Plant Cell Rep. 1984 3 109. 16 Y. Yamada and T. Endo Plant Cell Rep. 1984 3 186. 17 I. D. Spenser Pure Appl. Chem. 1985 57 453. 18 H. A. Khan and D. J. Robins J. Chem. SOC. Chem. Commun. 1981 146. 19 H. A. Khan and D. J. Robins J. Chem. Soc. Chem. Commun. 1981 554. 20 H.A. Khan and D. J. Robins J.Chem. SOC. Perkin Trans. I 1985 101. 21 H. A. Khan and D. J. Robins J.Chem. SOC. Perkin Trans. I 1985 819. 22 W. M. Golebiewski and I. D. Spenser J. Am. Chem. Soc. 1984 106 7925. 23 A. M. Fraser and D. J. Robins J. Chem. SOC. Chem. Commun. 1984 1477. 24 J. Rana and D. J. Robins unpublished results quoted in ref. 23.25 W. M. Golebiewski and I. D. Spenser J.Am. Chem. Soc. 1976,98 6726. 26 M. Wink T. Hartmann and H.-M. Schiebel 2.Naturforsch. Sect. C 1979,34 704; M. Wink and T. Hartmann FEBS Lett. 1979 101 343; M. Wink T. Hartmann and L. Witte 2.Naturforsch. Sect. C 1980 35 93. 27 W. M. Golebiewski and 1. D. Spenser J. Am. Chem. Soc. 1984 106,1441. 28 M. Wink Planta 1984 161 339. 29 ‘The Chemistry and Biology of Isoquinoline Alkaloids’ ed. J. D. Phillipson M. F. Roberts and M. H. Zenk Springer-Verlag Berlin 1985. 30 M. Shamma and H. Guinaudeau Tetrahedron 1984 40 4795. 31 P. G. Mantle and M. J. Coleman Phytochemistry 1984,23 1617. 32 T. Tanahashi and M. H. Zenk Plant Cell Rep. 1985 4 96. 33 G. Blaschke and G. Scriba 2.Naturforsch. Sect. C 1983,38,670.34 G. Blaschke and G. Scriba Phytochemistry 1985 24 585. 35 S. Muemmler M. Riiffer N. Nagakura and M. H. Zenk Plant Cell Rep. 1985 4 36. 36 M. Amann N. Nagakura and M. H. Zenk Tetrahedron Lett. 1984 25 953. 37 C. W. W. Beecher and W. J. Kelleher Tetrahedron Lett. 1984,25 4595. 38 M. Riiffer and M. H. Zenk Tetrahedron Lett. 1985 26 201. 39 M. H. Zenk in ref. 29 p. 240. 40 Y. Yamada and N. Okada Phytochemistry 1985 24 63. 41 T. G. Waddell and H. Rapoport Phytochemistry 1985 24 469. 42 A.-F. Hsu R. H. Liu and E. G. Piotrowski Phytochemistry 1985 24,473. 43 A. J. Herlt R. W. Rickards and J.-P. Wu J. Antibiot. 1985 38 516. 44 T. W. Doyle D. M. Balitz R. E. Grulich D. E. Nettleton S. J. Could C. Tann and A. E. Moews Tetrahedron Lett.1981 22 4595. 45 S. Uesato S. Matsuda and H. Inouye Chem. Pharm. Bull. 1984 32 1671. 46 S. Uesato S. Matsuda A. Iida H. Inouye and M. H. Zenk Chem. Pharm Bull. 1984 32 3764. 47 J. P. Kutney L. S. L. Choi T. Honda N. G. Lewis T. Sato K. L. Stuart and B. R. Worth Helv. Chim. Acra 1982 65 2088. 48 J. Stockigt H. Grundlach and B. Deus-Neumann Helv. Chim. Acta 1985 68 315. 49 H. Schiibel A. Treiber and J. Stiickigt Helu. Chim. Acta 1984 67 2078. NATURAL PRODUCT REPORTS 1986 50 M. H. Brillanceau C. Kan-Fan S. K. Kan and H.-P. Husson Tetrahedron Lett. 1984 25 2767. 51 B. Deus-Neumann and M. H. Zenk Planta 1984 162 250. 52 G. H. Jones and D. A. Hopwood J.Biol. Chem. 1984,259,14 151. 53 G. H. Jones and D. A. Hopwood J.Biol.Chem. 1984,259,14 158. 54 M. J. Zmijewski Jr. J. Antibiot. 1985 38 819. 55 M. J. Zmijewski Jr. M. Mikolajczak V. Viswanatha and V. J. Hruby J. Am. Chem. SOC. 1982 104 4969. 56 J. E. Baldwin A. E. Derome L. D. Field P. T. Gallagher A. A. Taha V. Thaller D. Brewer and A. Taylor J. Chem. Soc. Chem. Commun. 1981 1227. 57 J. E. Baldwin H. S. Bansal J. Chondrogianni L. D. Field A. A. Taha V. Thaller D. Brewer and A. Taylor Tetrahedron 1985,41 1931. 58 M. S. Puar H. Munayyer V. Hegde B. K. Lee and J. A. Waitz J. Antibiot. 1985 38 530. 59 M. R. Hagadone P. J. Scheuer and A. Holm J.Am. Chem. SOC. 1984 106 2447. 60 A. Iengo C. Santacroce and G. Sodano Experientia 1979,35 10. 61 H. Achenbach and H. Grisebach 2.Naturforsch. Ted B 1965,20 137.62 R. B. Herbert and J. Mann Tetrahedron Lett. 1984 25 4263. 63 R. B. Herbert and J. Mann J.Chem. SOC. Chem. Commun. 1983 1008. 64 R. B. Herbert and J. Mann J. Chem. Soc. Chem. Commun. 1984 1474. 65 L. J. Slieker and S. J. Benkovic J. Am. Chem. Soc. 1984 106 1833. 66 D. J. Aberhart and D. J. Russell J. Am Chem. SOC. 1984 106 4907; ibid. p. 4902. 67 S. H. L. Chiu R. Fiala R. Kennett L. Wozniak and M. W. Bullock J. Antibiot. 1984 37 1000. 68 S. H. L. Chiu R. Fiala and M. W. Bullock J.Antibiot. 1984 37 1079. 69 D. C. Billington B. T. Golding and I. K. Nassereddin J. Chem. SOC. Chem. Commun. 1980 90. 70 D. F. Witz E. J. Hessler and T. L. Miller Biochemistry 1971 10 1128; A. D. Argoudelis T. E. Eble J. A. Fox and D. J. Masan ibid.1969 8 3408. 71 N. M. Brahme J. E. Gonzalez J. P. Rolls E. J. Hessler S. Mizsak and L. H. Hurley J. Am. Chem. Soc. 1984 106 7873. 72 L. H. Hurley Acc. Chem. Res. 1980 13 263. 73 N. M. Brahme J. E. Gonzalez S. Mizsak J. R. Rolls E. J. Hessler and L. H. Hurley J. Am. Chem. SOC. 1984 106 7878. 74 Y. Mikami K. Takahashi K. Yazawa T. Arai M. Namikoshi S. Iwasaki and S. Okuda J. Biol. Chem. 1985 260 344. 75 T. Arai K. Yazawa K. Takahashi and Y. Mikami unpublished work quoted in ref. 74. 76 0.Ghisalba Chimia 1985 39 79. 77 T. Kawaguchi T. Asahi T. Satoh T. Uozumi and T. Beppu J. Antibiot. 1984 37 1587. 78 K. Hatano E. Higashide S. Akiyama and M. Yoneda Agric. Biol. Chem. 1984 48 1721. 79 K. Hatano E. Mizuta S. Akiyama E. Higashide and Y.Nakao Agric. Biol. Chem. 1985 49 327. 80 K. Hatano S. Akiyama M. Asai and R. W. Rickards J.Antibiot. 1982 35 1415. 81 E. P. Abraham Biochem. Soc. Trans. 1984 12 580. 82 J. E. Baldwin E. P. Abraham R. M. Adlington G. A. Bahadur B. Chakravarti B. P. Domayne-Hayman L. D. Field S. L. Flitsch G. S. Jayatilake A. Spakoskis H.-H. Ting N. J. Turner R. L. White and J. J. Usher J. Chem. Soc. Chem. Commun. 1984 1225. 83 J. Kupka Y.-Q. Shen S. Wolfe and A. L. Demain Can. J. Microbiol. 1983 29 488. 84 S. E. Jensen D. W. S. Westlake R. J. Bowers C. F. Ingold M. Jouany L. Lyubechansky and S. Wolfe Can. J. Chem. 1984,62 2712. 85 Y.Q. Shen S. Wolfe and A. L. Demain J. Antibiot. 1984 37 1044. 86 J. E. Baldwin R. M. Adlington N. J. Turner B.P. Domayne- Hayman H.-H. Ting A. E. Derome and J. A. Murphy J. Chem. SOC. Chem. Commun. 1984 1 167. 87 J. E. Baldwin E. P. Abraham R. M. Adlington B. Chakravarti A. E. Derome J. A. Murphy L. D. Field N. B. Green H.-H. Ting and J. J. Usher J. Chem. SOC. Chem. Commun. 1983 1317. 88 J. E. Shields C. S. Campbell S. W. Queener D. C. Duckworth and N. Neuss Helo. Chim. Acta 1984,67 870; R. J. Bowers S. E. Jensen L. Lyubechansky D. W. S. Westlake and S. Wolfe Biochem. Biophys. Res. Commun. 1984 120 607. NATURAL PRODUCT REPORTS 1986 -R. B. HERBERT 89 J. E. Baldwin R. M. Adlington A. E. Derome H.-H. Ting and N. J. Turner J. Chem. Soc. Chem. Commun. 1984 1211. 90 E. P. Abraham R. M. Adlington J. E. Baldwin M. J. Crimmin L. D. Field G.S. Jayatilake and R. L. White J. Chem. SOC. Chem. Commun. 1982 1130. 91 J. E. Baldwin E. P. Abraham R. M. Adlington M. J. Crimmin L. D. Field G. S. Jayatilake R. L. White and J. J. Usher Tetrahedron 1984 40,1907. 92 R. L. Baxter C. J. McGregor G. A. Thomson and A. I. Scott J. Chem. Soc. Perkin Trans. 1 1985 369. 93 C.-P. Pang B. Chakravarti R. M. Adlington H.-H. Ting R. L. White G. S. Jayatilake J. E. Baldwin and E. P. Abraham Biochem. J. 1984 222 789. 94 F. R. Ramos M. J. Lopez-Nieto and J. F. Martin Antimicrob. Agents Chemother. 1985 27 381. 95 J. E. Baldwin R. M. Adlington and R. Bohlmann J.Chem. Soc. Chem. Commun. 1985 357. 96 J. E. Baldwin E. P. Abraham C. G. Lovel and H.-H. Ting J. Chem. Soc. Chem. Commun. 1984 902. 97 J.E. Baldwin R. M. Adlington S. E. Moroney L. D. Field and H.-H. Ting J. Chem. SOC.,Chem. Commun. 1984 984. 98 H. Simon and D. Palm. Angew. Chem. Int. Ed. Engl. 1966,5,920. 99 C.-P. Pang R. L. White E. P. Abraham D. H. G. Crout M. Lutstorf P. J. Morgan and A. E. Derome Biochem. J.,1984,222 777. 100 E. P. Abraham C.-P. Pang R. L. White D. H. G. Crout M. Lutstorf P. J. Morgan and A. E. Derome J. Chem. SOC. Chem. Commun. 1983 723. 101 C. A. Townsend and E. B. Barrabee J. Chem. Soc. Chem. Commun. 1984 1586. 102 A. Scheidegger M. T. Kuenzi and J. Neusch J. Antibiot. 1984 37 522. 103 S. E. Jensen D. W. S. Westlake and S. Wolfe J. Antibior. 1985 38 263. 104 J. F. Martin G. Revilla M. J. Lopez-Nieto F. R. Ramos and J. M. Cantoval Biochem.Soc. Trans. 1984,12,866; G. Revilla M. J. Lopez-Nieto J. M. Luengo and J. F. Martin J. Antibiot. 1984 37 78 1. 105 J. Cortks P. Liras J. M. Castro J. Romero and J. F. Martin Biochem. SOC.Trans. 1984 12 863. 106 J. Hosoda N. Tani T. Konomi S. Ohsawa H. Aoki and H. Imanaka Agric. Biol. Chem. 1977 41 2007. 107 C. A. Townsend and A. M. Brown J.Am. Chem. SOC. 1983,105 913. 108 C. A. Townsend and G. M. Salituro J. Chem. Soc. Chem. Commun. 1 984 163 1. 109 S. W. Elson and R. S. Oliver J.Antibiot. 1978,31,586; I. Stirling and S. W. Elson ibid. 1979 32 1125; S. W. Elson in ‘Recent Advances in the Chemistry of P-Lactam Antibiotics’ ed. G. I. Gregory The Royal Society of Chemistry London 1981 p. 142. 110 S. W. Elson R. S. Oliver B. W.Bycroft and E. A. Faruk J. Antibiot. 1982 35 8 1. 11 C. A. Townsend and M.-F. Ho J. Am. Chem. Soc. 1985 107 1065. 12 C. A. Townsend and M.-F. Ho J. Am. Chem. SOC. 1985 107 1066. 13 Y.Fukagawa M. Okabe S. Azuma I. Kojima T. Ishikura and K. Kubo J. Antibiot. 1984 37 1388. 14 K. Kubo T. Ishikura and Y. Fukagawa J. Antibiot. 1984 37 1394. 15 K. Kubo T. Ishikura and Y. Fukagawa J. Antibiot. 1985 38 622. 16 A Nakagawa T.-S. Wu P. J. Keller J. P. Lee S. Omura and H. G. Floss J. Chem. Soc. Chem. Commun. 1985 519. 17 F. J. Leeper Nut. Prod. Rep. 1985 2 19. 118 R. L. Baxter E. M. Abbott S. L. Greenwood and I. J. McFarlane J. Chem. Soc. Chem. Commun. 1985 564. 119 R. E. Mitchell Phytochemistry 1985 24 247. 120 R. E. Mitchell unpublished results quoted in ref.119. 121 E. E. Conn in ‘The Biochemistryof Plants’ ed. P. K. Stumpf and E. E. Conn Academic Press New York 1981 Vol. 7 p. 479. 122 I. Tober and E. E. Conn Phytochemistry 1985 24 1215. 123 I. Tober and F. Spencer Plant Cell Rep. 1982 1 193. 124 E. A. Eltayeb and J. G. Roddick Phytochemistry 1985 24 253. 125 K. M. Byrne B. D. Hilton R. J. White R. Misra. and R. C. Pandey Biochemistry 1985 24 478. 126 R. J. Parry A. E. Mizusawa I. C. Chiu M. V. Naidu and M. Ricciardone J. Am. Chem. SOC. 1985 107 2512. 127 D. G. I. Kingston M. X.Kolpak J. W. LeFevre and I. Borup-Grochtmann J. Am. Chem. SOC. 1983 105 5106. 128 J. W. LeFevre and D. G. I. Kingston J. Org. Chern. 1984 49 2588. 129 R. J. Parry and J. V. Mueller J.Am.Chem. SOC.,1984,106,5764. 130 E. F. Elstner and R. J. Suhadolnik Biochemistry 1972 11 2578; E. F. Elstner R. J. Suhadolnik and A. Allerhand J. Bid. Chem. 1973 248 5385. 131 J. G. Buchanan M. R. Hamblin A. Kumar and R. H. Wightman J. Chem. Soc. Chem. Commun. 1984 1515. 132 J. G. Buchanan A. Kumar R. H. Wightman S. J. Field and D. W. Young J. Chem. SOC. Chem. Commun. 1984 1517. 133 S. Itoh Y. Odakura H. Kase S. Satoh K. Takahashi T. Iida K. Shirahata and K. Nakayama J. Antibiot. 1984 37 1664; Y. Odakura H. Kase S. Itoh S. Satoh S. Takasawa K. Takahashi K. Shirahata and K. Nakayama ibid. p. 1670. 134 T. Ohnuki T. Imanaka and S. Aiba Antimicrob. Agents Chemother. 1985 27 367.

ISSN:0265-0568    

DOI:10.1039/NP9860300185

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

The Biosynthesis of Carotenoids D. M. Harrison Chemistry Department University of Ulster Coleraine County Londonderry Northern Ireland B T52 7 SA _____~ Reviewing the literature published between January 1979 and December 1983 (Continuing the coverage of literature in Biosynthesis Vol. 6 p. 95) 1 Introduction 2 The Formation of Phytoene and General Aspects of the Biosynthesis of Carotenoids 3 The Biosynthesis of Carotenoids 3.1 Higher Plants and Algae 3.2 Fungi 3.3 Bacteria 3.4 Animals 4 The C30 Carotenoids 5 References I introduction This Report presents the more significant results that have been announced in the five-year period following the previous review by Mulheirn. Detailed reviews have been published on the biosynthesis of carotenoid~,~~~~~*~ on the light-induced interconversions of xanthophylls in the chlor~plast,~~ and on have also been identified.The formation of p-carotene [p,p-carotene] (7a) from carbon dioxide or acetic acid or mevalonic acid in chloroplasts of the latter species has been studied.I7 Biosynthetic studies have revealed the presence of two pools of p-carotene in chloroplasts from both the alga Chlorella pyrenoidosa and seedlings of radish (Raphanus sativus). The smaller pool of p-carotene is available for the biosynthesis of other carotenoids while the larger pool serves to protect the photosynthetic apparatus of the chloroplast from damage by light. Light-induced biosynthesis of carotenoids has been studied in seedlings of radish19 and barley (Hordeurn uulgare).20 The biosynthesis of carotenoids from [2-’4C]acetate is most rapid during the early logarithmic phase of growth in tissue cultures of the carrot (Daucus car~ta).~l A cell-free enzyme system from the blue-green alga Aphanocapsa ATCC 6714 is able to biosynthesize p-carotene and xanthophylls from geranylgeranyl diphosphate.Many herbicides function in part by inhibiting the biosynthesis of caroten~ids.’~? 23 Chromoplasts that had been Proceedings of the 5th6 and 6th7 International Symposia on Carotenoid Chemistry and Biochemistry have been published. Amongst other papers these proceedings contain short reviews on aspects of the biosynthesis of carotenoids,6a~6b including gene ti^^^,^^ and stere~chemical~~ studies.An important review on the stereochemistry of natural carotenoids contains useful biosynthetic discussion.8 The isolation structure and chemis- try of carotenoids have been re~iewed.~ 2 The Formation of Phytoene and General Aspects of the Biosynthesis of Carotenoids Early steps in the biosynthesis of carotenoids have been covered in a report on steroids and triterpenoids.1° The earliest step that is unique to the biosynthesis of carotenoids is the condensation of two molecules of geranyl diphosphate to furnish prephytoene diphosphate (la). Altman et al. have now reported full details of their total synthesis of prephytoene alcohol (1 b). I In most of the organisms that synthesize carotenoids prephytoene diphosphate is converted into cis-phytoene (2a).A series of dehydrogenation reactions then ensue with the formation of cis-phytofluene (3a) all-trans-6-carotene (4) neutosporene (9,and lycopene [$,+carotene] (6) (Scheme 1). [1-14C]Isopentenyl diphosphate was incorporated into cis- phytoene and not into lycopersene (1 5,15’-dihydrophytoene) by intact chromoplasts from fruits of sweet pepper (Capsicum annuum),12 in agreement with the prevailing view that lycopersene is not an intermediate in the biosynthesis of carotenoids. Since (-carotene contains a trans-1 5-1 5’ double-bond isomerization of the 15-1 5’ double-bond of cis-phytoene must be an early step in the formation of coloured carotenoids. It was shown that the cis-trans isomerization occurs at the phytofluene stage in C.annuum.13 The enzymes that are involved in the biosynthesis of carotenoids in chromoplasts of the same plant are compartmentalized; the biosynthesis of phytoene occurs in the stroma while the subsequent desatura- tion reactions and the formation of cyclic carotenoids occur in the membranous fra~ti0n.l~ The sites of biosynthesis of carotenoids in c hromoplasts of the daffodil (Narcissuspseudo-narcissus)’ and in chloroplasts of spinach (Spinacia oleracea)’ isolated from harcissus pseudonarcissus were able to synthesize the photo-regulation of the biosynthesis of carotenoid~.~~~~~~~ phytoene and 0-carotene from [1 -14C]isopentenyl diphosphate ; incubation of the chromoplasts with the herbicide SAN 6706 resulted in decreased formation of carotenoids and the accumulation of phytoene.24 The same herbicide and others inhibited the biosynthesis of carotenoids from [2-ISC]acetate and from [2-3H]mevalonate in Raphanus sativus,25leading to the accumulation of cis-phytoene and all-trans-lycopene.26 Other inhibitors of the biosynthesis of carotenoids have been studied.For example dibutyl phthalate inhibits the biosyn- thesis of carotenoids and leads to the accumulation of phytoene in illuminated radish plants and in Browallia speci~sa,~~ while a group of diphenyl ethers inhibits the desaturation of phytoene in algae of the genus Scenedesmus. 28 Some sec~ndary~~?~~ amines act as inducers of and tertiar~~~,~~ the biosynthesis of poly-cis-carotenoids such as prolycopene (8) in species of Citrus.The early stages of the biosynthesis of carotenoids in plants have been reviewed.31 It is known that the biosynthesis of carotenoids in Neurospora crassa is induced by light and that cis-phytoene accumulates if the fungus is grown in the dark. A cell-free enzyme system prepared from cells that were grown in the dark was able to convert [1-“T]isopentenyl diphosphate into 14C-labelled cis-phytoene; Mg2+ or Mn2+ ions were the only additional requirement for a~tivity.~’ Cultures of N. crassa that had been treated briefly with blue light furnished cell-free enzyme preparations that were more active in the biosynthesis of phyt~ene.~~ The role of light in the induction of biosynthesis of phytoene has been studied in mutants of the same species.j3 Neurospora crassa that was grown in light furnished a cell-free enzyme preparation that was active in the biosynthesis of phytoene and higher carotenoid~.~~ Centrifugation of this enzyme preparation at 1 15 000 g furnished a supernatant fraction that was able to convert [2-14C]mevalonic acid into 4C-labelled geranylgeranyl diphosphate ; the phytoene- and carotenoid-synthesizing enzymes were found in the particulate fraction.34 A cell-free enzyme system that had been prepared from a white mutant of Phycomyces blakesleeanus similarly converted [2-14C]mevalonic acid into ~is-phytoene.~~ The herbicide norflurazon blocked the formation of phytoene and of p-carotene from [2-14C]mevalonic acid in this enzyme system; since 4C-labelled geranylgeranyl diphosphate was still formed NATURAL PRODUCT REPORTS 1986 (2) a;15,15'-cis b;15,15'-trans (3)a;15,15'-cis b;15.15'-trans (61 Scheme 1 (7)a; R = H b;R=OH NATURAL PRODUCT REPORTS 1986 -D.M. HARRISON it was concluded that the herbicide inhibits the action of the A phytoene ~ynthetase.~~ solution of diphenylamine (70 pmol dm-3) completely blocked the formation of unsatu- rated carotenoids in P.blakesleeanus and led to an increase in the synthesis of phyt~ene.~~ It was shown that diphenylamine inhibits the desaturation of phytoene by a post-translation mechanism. It was shown earlier that the non-photosynthetic bacterium Halobacterium cutirubrum utilizes trans-phytoene directly in the biosynthesis of carotenoids; the exclusive formation of trans-phytoene (2b) by a colourless (i.e.non- carotenoid-forming) mutant of H. halobium has now been dem~nstrated.~~ 3 The Biosynthesis of Carotenoids 3.1 Higher Plants and Algae The biosynthesis of lutein (1 la) from mevalonic acid (9) in Calendula oficinalis has been re-in~estigated.~~ Thus (3R,5R)- [2-14C 5-3H Jmevalonic acid and the (3R,SS)-isomer each with a 3H I4C ratio of 1 :1 furnished samples of lutein in which the ratios of ‘H to I4C were 3:8 and 5 :8 respectively. These results are consistent with the hypothesis that the biosynthesis of lutein proceeds via two hydroxylation reactions acting on a precursor such as a-carotene [(6’R)-P,~-carotene] (lo) each of which occurs with retention of configuration (Scheme 2); this is contrary to the earlier result.The biosynthetic samples of lutein were each oxidized with nickel peroxide to furnish two samples of the conjugated enone (11 b); the ratios of 3H to 14C in the latter two samples demonstrated that the 3’-proton of lutein which is lost in the oxidation was derived from the S(pro-SR)-position of mevalonic acid.39 Violaxanthin (12) is the major carotenoid in the root cap of maize (Zea mays) during seed germination. Violaxanthin is not present in ungerminated seeds and is presumably formed from zeaxanthin (7b) in a reaction sequence that does not require light during the germination of maize seeds.40 The biosyn- thesis of violaxanthin from 4C-labelled zeaxanthin has been demonstrated to occur in isolated chloroplast envelopes of spinach; this transformation required NADPH and molecular oxygen.41 It has long been known that the illumination of green leaves results in a decrease in the concentration of violaxanthin and an increase in the concentrations of antheraxanthin (1 3) and zeaxanthin (7b).The reverse transformations occur when illuminated leaves are removed to the dark. This ‘xanthophyll cycle’ (or ‘violaxanthin cycle’)6d is common to all higher plants. The substrate specificity for the de-epoxidation reaction of violaxanthin in chloroplasts from lettuce (Lactuca sativa) has been studied.42 It has been shown also that the de-epoxidation of violaxanthin in chloroplasts from leaves of beech and spinach is linked to the oxidation of plastohydroquinone-9.43 Other studies have been reported on the biochemistry of the violaxanthin cycle.44 It has long been suspected that the biosynthesis of the modified carotenoids capsorubin (1 4) and capsanthin (15) may involve pinacol-like rearrangements of violaxanthin (1 2) and antheraxanthin (1 3) respectively.The time-course of the incorporation of [2-14C]acetate into each of these four compounds in fruits of sweet pepper (Capsicum annuum) was in agreement with the postulated biosynthetic relationship^.^^ Definitive results were obtained by observing the incorpora- tion of [U-14C]violaxanthin into capsorubin (5%)46 and the incorporation of [ 15,l 5’-3H2]antheraxanthin into capsanthin (4.4%) and capsorubin (0.3%)47 in chromoplast preparations from fruits of C.annuum. It was shown also that [15,15’- 3H2]zeaxanthin is a precursor of violaxanthin and anthera- an thin.^^ cis-Phytoene (2a) cis- and trans-phytofluene [(3a) and (3b)] and 6-carotene (4) each had a 3H:14C ratio of 8 :8 after their biosynthesis from (3R,4R)-[2-I4C 4-3H ,]mevalon- ate (3H:14C= 1:l) in fruits of C. annuum; in the same experiment the samples of 0-carotene zeaxanthin anthera- xanthin violaxanthin capsorubin and capsanthin that were produced each possessed a 3H:14C ratio of 6 :8 as expected.48 The absolute stereochemistry of eschscholtzxanthin (16) which is a pigment of the yellow poppy Eschscholtzja californica is the same as that of zeaxanthin (7b); thus the earlier suggestion that eschscholtzxanthin arises via dehydra- 1 H* 1 R‘ H0-(Itla;R’=oH,R~= b;R’ R2 = 0 Scheme 2 NATURAL PRODUCT REPORTS 1986 on HO -OH (15) tion of a monoepoxide of zeaxanthin remains an attractive hyp~thesis.~~ The stigmas of Crocus sativus contain the CzO dicarboxylic acid crocetin (17y0 together with glucosyl esters of the latter.The isolation of zeaxanthin (7b) from the same source and the failure to isolate plausible CzO precursors render it probable that crocetin is a degraded C40 carotenoid rather than the first member of a novel class of Cz0 caroten~ids.~~ (-Carotene (4) accumulated when Scenedesmus obliquus was grown in the dark; subsequent transfer of this green alga to a deuteriated water medium in the light led to the formation of a-carotene (lo) p-carotene (7a) lutein (1 la) and zeaxanthin (7b) each of which occurred in dideuteriated and fully deuteriated populations.The 2H2-labelled compounds must have been formed via cyclization of an existing C40 precursor; the results provide evidence for the postulate that (-carotene is the last acyclic intermediate in the biosynthesis of carotenoids in this species.51 The marine dinoflagellate Amphidinium carterae contains inter alia the allene neoxanthin (18) the acetylene diadino- xanthin (19) and as the most abundant carotenoid the C3 allene peridinin (20). Intact cells of this organism or a cell-free enzyme system furnished P-carotene (7a) zeaxanthin (7b) neoxanthin (18) and peridinin (20) each with a 3H :14C ratio of approximately 12:8 when (3R)-[2-I4C 2-3H,]mevalonic acid (3H :I4C = 2 :1) was supplied as a precursor; a sample of diadinoxanthin (19) was isolated in which the ratio of 3H to I4C was 11 :8.52 If (3R,4R)-[2-14C 4-3H1]mevalonate (3H:14C= 1 :1) was fed to the organism the peridinin that was produced had a 3H:I4C ratio of 5 :8 while the other metabolites each displayed an isotopic ratio of 6:8.The most efficient interpretation of these data is summarized in Scheme 3 and in equation (1). The data show in particular that neoxanthin could be a precursor of diadinoxanthin but that the converse is not true; furthermore the data suggest that the three carbon NATURAL PRODUCT REPORTS 1986 -D. M. HARRISON HH atoms that are eliminated in the biosynthesis of peridinin are carbons 13 14 and 20 of neoxanthin (18).52 The biosynthetic sequences were each confirmed by study of the time-course of incorporation of radioactivity from 4C-labelled zeaxanthin into the later metabolites and by the quantitative incorporation HOh O 2 H I I zeaxanthin I+ (7b) 1 neoxanthin d iad in oxan th in 7 (19) (1) (6) (18) peridinin I (20) I I $.)OH 6 (7b) (21) Scheme 4 of 4C-labelled neoxanthin into peridinin (66%) and diadino- xanthin (33%).53 The labelled precursors that were used in this study were prepared biosynthetically from racemic [2-l "C]- mevalonate. 3.2 Fungi In the Mucorales which include Blukeslea trisporu and Phycomyces blukesleeanus mating of the morphologically indistinguishable (+) and (-) types results in a dramatic increase in the production of carotenoids such as p-carotene (7a).This increase in the rate of biosynthesis of carotenoids has been attributed to the action of the hormone trisporic acid (21); the latter also stimulates the biosynthesis of carotenoids if it is applied to unmated cultures of the (-) type of fungus. It has been shown that in Blakeslea trisporu this effect can also be achieved by compounds that are structurally related to trisporic acid.54 The stimulatory effect of trisporic acid has been explained by the increased synthesis of a membrane-bound protease which inactivates a protein that acts as an inhibitor of the biosynthesis of carotenoid~.~ Carotenoid biosynthesis in mated B.trisporu is stimulated by Cu2+ ions.56 The effects of the inhibitor CPTA on the biosynthesis of carotenoids in mutant strains of P. blukesleeunus have been des~ribed.~' The mechanism of the photoregulation of the formation of carotenoids in the red yeast Rhodotorula minutu has been inve~tigated.~~ The photoreceptor system that is involved in the biosynthesis of carotenoids in Verticillium ugaricinum has also been studied. 59 3.3 Bacteria The stereochemistry of the cyclization step that occurs in the biosynthesis of zeaxanthin (7b) in a species of Flavobacterium has been elucidated by the use of 13C n.m.r. spectroscopy.60 HO I I I * 8. H,CH NATURAL PRODUCT REPORTS 1986 The organism furnished lycopene (6) that was labelled as expected when it was incubated with [2-l3C]mevalonate in the presence of nicotine.If the incubation was conducted in the absence of an inhibitor of cyclization zeaxanthin was formed with the labelling pattern shown in Scheme 4. The origins of the methyl groups at C-1 and C-1' of zeaxanthin in the Flavobacter-ium are the opposite to that reported previously for p-carotene in Blukeslea trispora. The present result,60 together with the known stereochemistry of protonation * reveals that the cyclization reaction proceeds via a trans addition to each terminal double-bond of lycopene (Scheme 4). The biosynthesis of the Cso carotenoids C.p. 450 [of revised structure (22)6'1 and sarcinaxanthin (23) in cell-free enzyme preparations from Corynebucterium poinsettiae and Micrococcus luteus respectively has been studied.62 The ratios of 3Hto I4C that were observed for each compound when (3R)-[2-14C 2-3H2]- and (3R,4R)-[2-14C 4-3H1]-mevalonic acids were sup- plied as precursors were interpreted in terms of the labelling patterns that are summarized in Scheme 5.When taken together with earlier results for decaprenoxanthin (24) these data exclude the possibilities that the ring arises by isomerization of an E ring (or vice versa) and that the y ring is formed by isomerization of either a p ring or an E ring. The simplest interpretation of the results is that the p y and E rings of the three metabolites all arise from a common intermediate carbo-cation as shown in Scheme 5.62 The acyclic C50 carotenoid bacterioruberin (25) had the same ratio of 3H to 14C as neurosporene (5) if it was biosynthesized from (3R,4R)-[2-14C 4-3H1]mevalonic acid in Halobacterium hulobium; it was concluded that the 2-proton and the 2'-proton of bacterioruberin were both derived from the 4(pro-4R>proton of me~alonate.~~ The biosynthesis of bacterioruberin in H.halobium is completely inhibited by a concentration of 25 pmol dm-3 of di~henylamine.~~ A concen- tration of 1.O mmol dm-3 of nicotine similarly blocked the .. H-CH (22) =. H,CH NATURAL PRODUCT REPORTS 1986 -D. M. HARRISON formation of bacterioruberin in H. c~tirubrum,~~ and in H. halobium Sarcina litoralis and Amoebobacter morrhuae;65 in each case lycopene (6) and bisanhydrobacterioruberin (26) accumulated.The biosynthesis of canthaxanthin (27a) in Micrococcus roseus was inhibited by nicotine and by CPTA.66In each case p-zeacarotene (28) accumulated together with other metab- olites. Since neither neurosporene (5) nor lycopene (6) could be detected in inhibited cultures it was suggested that c-carotene (4) is the substrate that undergoes cyclization in this organ- ism.66 Both cis-and trans-geranyla~etone~~ and several related compounds68 inhibited the biosynthesis of carotenoids in a OH (25) (27)a;R = H b;R = OH RO (29)a;R = H b;R = Me RO (30)a;R = H b;R = Me RO 211 species of Synechococcus; phytofluene (3) and l,-carotene accumulated if active inhibitors were present.68 The photo- induction of formation of carotenoids in Brevibacterium surfureum has been studied.69 The photosynthetic purple bacteria of the Rhodospirillaceae furnish inter alia a group of oxygenated acyclic carotenoids.Studies with mutants of Rhodopseudomonas capsulata have revealed that the hydration of neurosporene to furnish (29a) and the oxidation of (30a) to demethylspheroidenone (31a) are each controlled by a single gene. The dehydrogenation of (29a) or (29b) to furnish (30a) or (30b) is controlled by two genes (Scheme 6). It appears that a single gene controls the (31)a;R = H b;R =Me represents a genetic block ) methylation of any member of the hydroxylated series (29a)- (31a) to give the corresponding member of the methoxylated series (29b)-(31b); the gene that controls the hydration of spheroidene (30b) [to furnish the hydrate (32)] was not identified.70 Nicotine inhibited the biosynthesis of spheroi- dene (30b) in Rhodopseudomonas sphaeroides and led to the accumulation of neurosporene ;resuspension of the organism in a nicotine-free medium prepared from deuteriated water resulted in the formation of [2Hl]spheroidene (30b) in confirmation of the postulated role of neurosporene as a precursor of this caroten~id.~~ In a similar manner it was shown that 0-carotene (7a) and rhodopin (33) are both derived from lycopene in Rhodomicrobium uannielii and that ‘hydroxy- spheroidene’ (32) is derived from spheroidene (30b) in Rhodo-pseudomonas gelatin~sa.~’ The effect of light on the formation of carotenoids in Rhodopseudomonas acidophila has been studied.72 Spheroidenone (31 b) is the major carotenoid in uninhibited cultures of Erythrobactersp.OCh 114; cultures that were grown in the presence of diphenylamine accumulated phytoene (2) phytofluene (3) and 6-carotene (4).73 Me0 HO (34) HO 0 HO 0 OHC HO-HO OH NATURAL PRODUCT REPORTS 1986 The photosynthetic green sulphur bacterium Chlorobium limicola elaborates the aromatic compound chlorobactene (34) as the major carotenoid. The addition of nicotine to the bacterial culture inhibited the biosynthesis of chlorobactene and led to the accumulation of lycopene (6).74Subsequent removal of the inhibitor led to the rapid conversion of lycopene into chlorobactene even when the biosynthesis of carotenoids de novo was blocked by the addition of di~henylarnine.~~ 3.4 Animals It is generally assumed that insects do not synthesize carotenoids de novo but some species are clearly capable of metabolizing carotenoids from dietary sources.75 The stick insects Ectatosoma tiarat~m~~ each and Carausius moros~s~~ converted [ 15,l 5’-14C2]-P-carotene into a range of 14C-labelled 2-oxygenated and 2,2’-dioxygenated derivatives; the final product in each case was the diketo-carotenoid (35).76-77 The hermit crab Clibanarius erythropus incorporated radioac- OH 0 (35) OH OH (38) (39) NATURAL PRODUCT REPORTS 1986 -D.M.HARRISON tivity from [ 15,l 5’-3H2]-P-carotene into several carotenoids including canthaxanthin (27a) and astaxanthin (27b); signifi- cantly no radioactivity was incorporated into lutein (1 la) and a-doradexanthin (36) which are presumably formed from a-carotene Unlabelled zeaxanthin (7b) was reduced to the 7’,8’-dihydro-derivative (parasiloxanthin) and to the 7,8,7’,8’- tetrahydro-derivative by the Japanese catfish Parasilurus asotus; lutein (1 la) was similarly reduced to its 7,8-dihydro- deri~ative.~~ Unlabelled lutein was metabolized to or-dorade- xanthin (36) by the goldfish Carassius auratus; zeaxanthin (7b) similarly furnished P-doradexanthin (37).80 Lutein was degrad- ed to derivatives of retinol in the freshwater fishes Clarias batrachus and Ompok pabo.8’ 4 The C30 Carotenoids It has been known for some ten years that carotenoids of the non-photosynthetic bacterium Streptococcus faecium are exclu- sively C30 compounds exemplified by 4,4’-diapolycopen-4-a1 (38) or derivatives of C30 compounds such as the glucoside (39).The preparation of a cell-free enzyme extract from this organism has been described ; the crude enzyme preparation showed an absolute requirement for Mn2+ ions for the incorporation of [I-’ 4C]isopentenyl diphosphate into squalene and into C30carotenoids.8 Adenosine triphosphate was also required for the incorporation of [2-14C]mevalonic acid into C30 carotenoids. The possibility that the biosynthesis of C30 carotenoids f f OHC f (43)a; R = H b; R = -D -glucosyl I c R = HO represents a genetic block Scheme 7 214 NATURAL PRODUCT REPORTS.1986 R’ involves partial degradation of a C, precursor such as phytoene (2) has been investigated by supplying [4,8,12- 3H3]farnesyl diphosphate and [ 1-i4C]isopentenyl diphosphate to the cell-free enzyme preparation from S. fae~iurn.~~ It was argued that the condensation of tritium-labelled farnesyl dip hosp hate with ,C-la belled isopen teny 1 dip hosp ha te to furnish labelled geranylgeranyl diphosphate would lead to phytoene with a nominal 3H I4C ratio of 6 :2; subsequent removal of a C unit from each end of the phytoene molecule would furnish 4,4’-diapophytoene (40) with a nominal 3H 14C ratio of 4 2. Alternatively direct condensation of two molecules of tritium-labelled farnesyl diphosphate would give 4,4’-diapophytoene with a 3H I4C ratio of 6 :O.In the event the results of this experiment were consistent only with the latter route after allowance had been made for the biosynthesis of IT-labelled (40) de now from [ l-i4C]isopentenyl diphos- phate alone. The biosynthesis of the glucoside (39) from [U-SC]glucose and from uridinediphospho[ U-14C]glucose was also reported.83 The pigments of Staphylococcus aureus (strain S41) have been identified as C3 carotenoids; the most advanced carotenoids were the carboxylic acid (43a) and the glucosides (43b) and staphyloxanthin (43~).~ Another strain (strain 209P) of this species showed significant differences in the nature of its C30 car~tenoids.~~ On the basis of studies with mutants the route that is summarized in Scheme 7 has been proposed for the biosynthesis of carotenoids in S.aureus.86 The four genetic blocks that were identified are shown in the Scheme; it may be significant that no mutant was found that accumulated intermediates between 4,4’-diapophytoene (40) and 4,4’-diaponeurosporene (41) and it was suggested that a single dehydrogenase system is responsible for the stepwise conver- sion of the former compound into the latter.86 The carotenoids of wild-type Pseudomonas rhodos are characterized by the presence of 6-O-acyl-P-~-glucopyranosyl esters of the mono- and di-carboxylic acids (44a) and (44b) respectively. Variously coloured mutants of this bacterium could be divided into four main types type 1 mutants (white) furnished no carotenoids ; type 2 mutants (orange) furnished 4,4’-diapolycopene (44c) as the sole carotenoid; type 3 mutants (red to violet) synthesized the aldehydes (44d) and (44e) as the major carotenoids; and type 4 mutants (bright red) produced the carboxylic acids (44a) and (44b) as the major pigments8’ The immediate precursor of 4,4‘-diapophytoene (40) has not been identified though it is unlikely to be squalene.86 It may be significant that (40) is formed by the action of squalene synthetase when microsomes of yeast or of pig liver are incubated with farnesyl diphosphate in the absence of NADPH even though neither species synthesizes carotenoids.8* 5 References 1 L.J. Mulheirn in ‘Biosynthesis’ ed. J. D. Bu’Lock (Specialist Periodical Reports) The Royal Society of Chemistry London 1980 Vol. 6 p. 95. 2 (a)S. L. Spurgeon and J. W. 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ISSN:0265-0568    

DOI:10.1039/NP9860300205

出版商:RSC    

年代:1986

数据来源: RSC