摘要:

Natural Product Reports Editorial Board Professor G. Pattenden (Chairman) University of Nottingham 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 University 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. For any individual topic successive reviews will deal with consecutive periods and while the coverage that was provided by the Specialist Periodical Reports on 'Aliphatic and Related Natural Product Chemistry' 'The Alkaloids' 'Biosynthesis' and 'Terpenoids and Steroids' will be continued this will be supplemented by occasional reviews of areas of both general and specific interest to workers in these and in other fields.Bimonthly publication allows greater flexibility than the annual or biennial publication of volumes of each of the series of Specialist Periodical Reports mentioned above in that individual reviews can be published as they become available. All articles in Natural Product Reports are commissioned by members of the Editorial Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Burlington House London W 1V OBN England. 1985 Annual Subscription Price U.K.€125.00 Rest of World €131.00 U.S.A. $242.00. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts. SG6 lHN England. Air Freight and mailing in the U.S. by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 1 1003. US. Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. Second-Class postage paid at Jamaica NY 11431 -9998. All other despatches outside the U.K. are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the U.K. 0The Royal Society of Chemistry 1985 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 Subscription rates for 1985 U.K. f125.00 Overseas f13 1 .OO U.S.A. USS242.00 Subscription rates for back issues (1984) are U.K. f120.00 Overseas f126.00 U.S.A. USS240.00 Members of the Royal Society of Chemistry should order the journal from The Membership Officer The Royal Society of Chemistry 30 Russell Square LONDON WClB 5DT England

ISSN:0265-0568    

DOI:10.1039/NP98502FX005

出版商:RSC    

年代:1985

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Biological Oxidation of Nitrogen in Organic Molecules edited by J. W. Gorrod and L. A. Damani Ellis Horwood Health Science Series 1985. 445 pages with 144 figures and 84 tables. Hardcover. DM 150.-/f 43.50. Nitrogen occurs in a wide variety of drugs natural products and environmental chemicals. In the body oxidation of these com- pdunds at the nitrogen atom is an important metabolic step. Many on the products formed in this way are unstable and undergo enzymic and/or non-enzymic conversions to other structures. This book summarizes the results of recent research in the field. It starts with three chapters about the analysis and the chemical behavior of N-oxygenated compounds. Then the metabolic mechanisms of N-oxidation are considered in detail.Here each of the nearly 40 chapters presents a case study that is of particular biological pharmacological or toxicological interest. In conclusion cytotoxic actions of N-oxidized compounds are re- viewed. To obtain this book please contact The Royal Society of Chemistry Blackhorse Road Letchworth SG6 1 HN.Phone (04626) 72555 In Customers outside the UK and Eire please contact VCH Verlagsgesellschaft P.O. Box 1260/1280 0-6940 Weinheim Federal-Republic of Germany. / I Advanced Methods in the Biohwl-Series Advanced Methods Edlted by V Neuhoff and A Maelicke in the Biological Sciences edited by V. Neuhoff and A. Maelicke Andreas Chrambach The Practice of Quantitative Gel Electrophoresis Electrophoresis is one of the most important methods for the investigation of biological materials and probably the most efficient procedure for the separa- tion and detection of proteins and other charged species.Special methods which can be summarized as gel electrophoresis include polyacrylamide and ag arose electrophoresis isot ac hop horesis and elect ro-f ocussi ng . This book presents gel electrophoretic procedures which are applicable to the identification and isolation of charged molecules. All methods have proved reliable and are used in daily laboratory work. The equipment required for gel electrophoresis is also described in detail. For items that are not commercially available the reader is given all the information needed to do the construction himself. Theoretical aspects are dealt with to the extent that they are indispen- sable for the application of a method. A comprehensive index completes the book and makes it a reliable reference work. 1985 XV 265 pages 72 figures 5 tables Hardcover ISBN 3-527-26029 0 Price €35.00 To obtain this book please contact The Royal Society of Chemistry Blackhorse Road Letchworth SG6 1 HN. Phone (04626) 72555 Ill Customers outside the UK and Eire please contact VCH Verlagsgesellschaft P.O. Box 126011280 0-6940 Weinheim Federal Republic of Germany

ISSN:0265-0568    

DOI:10.1039/NP98502BX007

出版商:RSC    

年代:1985

数据来源: RSC

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H M. F. Grundon Department of Chemistry The University of Ulster Coleraine Co. Londonderry Northern Ireland B T52 ISA Reviewing the literature published between July 1983 and June 1984 (Continuing the coverage of literature in Natural Product Reports 1984 Vol. 1 p. 195) 1 Quinoline Alkaloids 1 Quinoline Alkaloids 1.1 Occurrence 1.1 Occurrence 1.2 Non-terpenoid Quinolines 1.3 Prenylquinolinones and Hemiterpenoid Tricyclic Six new quinoline and quinolinone alkaloids that have been Alkaloids identified this year are listed together with known alkaloids 1.4 Furoquinoline A1 kaloids from new sources in Table l.l-l5 2 Quinazoline Alkaloids 3 Acridone Alkaloids 1.2 Non-terpenoid Quinolines 3.1 Occurrence The structure of swietenidin-A (9,which is an alkaloid of 3.2 Spectroscopic and Chemical Properties Chloroxyfon swietenia was originally assigned mainly on the 3.3 Synthesis basis of spectroscopic data (cf ref.16a) and has now been 3.4 Dimeric Acridones confirmed by the synthesis of its 0-methyl ether (6) (Scheme 4 References 1y7 A new alkaloid broussonetine (7) has been isolated from Broussonetia zeyfanica (M~raceae),~ which was shown pre- viously to contain 4-formyl-8-hydroxyquinoline(cf. ref. 16b). The presence of 8-hydroxyquinoline units in.the alkaloid was The emphasis this year has been on synthesis. Particular indicated by the formation of a fluorescent complex with Mg2+ advances are concerned with the use of isatoic anhydrides with and by the I3C n.m.r.spectrum; the structure of the y-strong bases to prepare 4-hydroxy-3-prenylquinolin-2-ones butyrolactone portion of the molecule was apparent from its i.r. hydroxyisopropyldihydrofuroquinolinones,and 2,2-dimethyl- absorption at 1775 cm-l by the 'H n.m.r. spectrum and by the pyrano-quinolinones and -acridones. An extensive study of the cleavage of the alkaloid under electron impact as indicated by acid-catalysed dimerization and trimerization of acridone the broken line in (7). alkaloids and the isolation of a related dimer are also of Rees and co-workersI* have reported a new seven-stage considerable interest. synthesis of the bacterial coenzyme methoxatin (10) (Scheme 2) Table 1 Isolation of quinoline alkaloids Species Alkaloid (Structure) Ajraegle paniculata 4-Methoxy-1-methylquinolin-2-one Baljourodendron riedelianum ( +)-Ribalinidine (1 8) *( +)-Riedelianine (19) Broussonetia zeylanica *Broussonetine (7) Fagara heitzii Flindersine (2; Rl = R2 = H) Fagara mayu Edulinine (3) Glycosmis citrifblia 4,8-Dimethoxy-3-(3,3-dimethylallyl)-l-methylquino~in-2-one (13) Glycosmis pen taphy lla *Glycophylone (1 2) Haplophyllum dauricum Dictamnine (1 ;RI = R2 = R3 = R4 = H) 4-Methoxy-1-methylquinolin-2-one Folimine Haplopine (1; R1 = R2 = H R3 = OH R4 = OMe) Robustine (1; RI = R2 = R3 = H R4 = OH) Robustinine (edulitine) Haplophyllum obtusiJblium Evodine [l; R' = R2 = H R3 = CH2CH(OH)CMe=CH2 R4 = OMe] *Hapaltine acetate [l; R' = R2 = H R3 = OCH,CH=C(Me)CH,OAc R4 = OMe] Haplopine Methylevoxine [I; RI = R2 = H R3 = OCH,CH(OH)C(OMe)Me, R4 = OMe] Haplophyllum suaveolens y-Fagarine (1; RI = R2 = R3 = H R4 = OMe) Flindersine *Haplophylline (16) Haplophyllum vulcanicum Kokusaginine (1; RI = R4 = H R* = R3 = OMe) Melicope leptococca Acronycidine (1 ; R1 = R3 = R4 = OMe R2 = H) Acronydine (4) Kokusaginine Monnieria trij'olia y-Fagarine Kokusaginine Skimmianine (1; R1 = R2 = H R3 = R4 = OMe) Ravenia spectabilis *Ravesilone (1 5) Zanthoxylum coco Flindersine * New alkaloids NATURAL PRODUCT REPORTS 1985 R4 Me0 \ N/ Me0 H a.\ i,ii (5) R = H (6) R = Me Reagents i HCl H,O, AcOH at 85 "C; ii NaOMe MeOH reflux; iii Zn,AcOH MeOH reflux; iv Me,SO, KOH DMF at 60-65 "C Scheme I Broussonetine (7 ) Me02C / ___) PhCHzO \ NHAc Me0 Me RO Me Glycophylone (12) R = H (14) R =Me (13) R = Me Ravesilone (15) R = H do \ I "&--(-OH Me Y Haplop hy lline (16 ) (cf ref.16c). The key indole intermediate (9) was prepared from the readily available benzaldehyde derivative (8); acid cleavage of the acetamide group was followed by annulation of the pyridine ring by a Doebner-von Miller-type reaction and then oxidation of the phenol (1 1) by a nitroxide to the required quinone system. 1.3 Prenylquinolinones and Hemiterpenoid Tricyclic Alkaloids Bhattacharyya and Chowdhury isolated the new alkaloids glycophylone (1 2) from the seeds of Glycosmispent~phylla~ and ravesilone (15) from leaves of Ravenia spectabilis. The solubility of glycophylone in aqueous alkali its cyclization with acid to compound (14),and its i.r.and 'H n.m.r. spectra indicate that the alkaloid is a 4-hydroxy-3-prenylquinolin-2-one. Evidence for the structure of ravesilone was provided by i.r. and H n.m.r. spectroscopy. The additional oxygen function in the two alkaloids was assigned to C-8 on biogenetic grounds but this argument is unreliable in view of the great variation in oxygen substitution that occurs in the homocyclic rings of quinoline alkaloids. Although not mentioned by the authors glycophylone (12) and the pyrano-derivative (14)were synthe- sized some years ago,I9 and the correspondence in melting points provides good support for the structure of the recently isolated alkaloid. Confirmation of the structures of the alkaloids for example by direct comparison of synthetic and natural samples..of glycophylone and by correlation of ravesilone (15) with compound (14) is clearly desirable. Haplophylline (16),which is a new N-functionalized alkaloid of the flindersine group has been isolated from Haplophyllum Me02C /7NH Reagents i Me0,CCH,N3 NaOMe MeOH at -10 "C; ii xylene reflux; iii MeOH HCI heat; iv MeO2CC0CH=CHCO2Me CH2C1, then H+; v H,,Pd/C MeOH; vi Bu'(C0Ph) NO' radical CH,Cl,-MeOH (9 :1); vii HC(OMe)3 MeOH H+;viii aq. K,C03 at 85 "C then acidify with HCI to pH 2.5 Scheme 2 NATURAL PRODUCT REPORTS 1985-M. F. GRUNDON suaueolens; its structure was apparent from the 'H n.m.r. and mass spectra and from the i.r.absorption at 1720cm-'.'O Jurd and Wong' have studied the alkaloid content of the heartwood of Baljourodendron riedelianum for the first time. The known alkaloid (+)-ribaline (17) was the principal alkaloid and minor constituents [(+)-ribaline and (+)-ribalinidine (18)] were also isolated; it is of some interest that the (-)-enantiomer of the latter alkaloid was obtained previously from the bark of B. riedelianum. A new alkaloid (+)-riedelianine (19) is unusual in containing no oxygen -function at C-4; its structure was determined by single-crystal X-ray diffraction. 0 (18) (+) -Riedelianine (19) (20) R' = R2= H (23) (21) R' = H R2= OMe (22) R' =OMe,R2=H (20) + & C02Et (29) li 3-Prenylquinolin-2-ones and their epoxides are key interme- diates in the synthesis of hydroxyisopropyldihydrofuro-quinoline and hydroxydimethyldihydropyranoquinolinealka-loids; a new approach to their synthesis from isatoic anhydrides (cf.ref.20a) has been described by Coppola21 (Scheme 3). The ester (23) which was readily prepared by alkylation of lithio ethyl acetate with 3,3-dimethylallyl bromide was converted into its lithium enolate; the reaction with the isatoic anhydrides (20)-(22) gave compounds (24)-(26) which were cyclized to 4-hydroxy-3-prenylquinolin-2-ones (27) in overall yields of 45-52% from the isatoic anhydrides. The prenylquinolinone (27 ; R1= OMe R2 = H) afforded the 2,2-dimethylpyranoquino-linone alkaloid oricine (28). Alternative syntheses of the alkaloids araliopsine (3 1) (cf ref.16d) and isoplatydesmine (32) were also reported (Scheme 3).** The reaction of isatoic anhydride (20) with the epoxide (29) furnished the intermediate (30) which in a neutral medium gave araliopsine (31) (33%) and in the presence of a peroxy-acid yielded a mixture of araliopsine (25%) and isoplatydesmine (32) (1 1%). Me (24)R' = R2= H (25) R' = H R2 = OMe (26) R' = OMe R2= H Ii i OH R2 Me (27) Iiii Me OH (281 (32) Reagents i LiNPri THF at -78 "C;ii PhMe reflux; iii 2,3-dichloro-5,6-dicyanobenzoquinone, PhMe at 80 "C; iv m-CIC,H,CO3H CH,Cl, at 0 "C Scheme 3 NATURAL PRODUCT REPORTS 1985 - i \ H o (33) (34) R = H (35) R = AC 1iii,iv 1i i &+-OH (36) (37) OR Reagents i AgOAc I, AcOH then HCI EtOH reflux; ii polyphosphoric acid at 100°C; iii PhC03H CHCl,; iv 4% aq.NaHC03 MeOH reflux Scheme 4 (39) (401 were reported for the alkaloid. Glycarpine clearly is not 5,7-dimethoxyisodictarnnine (41); although its structure remains in doubt it is worth pointing out that 6,8-dimethoxyisodictam- nine (isomaculosidine) (39)16 has the same melting point as glycarpine and that the * H n.m.r. spectra of the two compounds __ -are similar particularly with respect to the chemical shifts of the aromatic protons. 2 Quinazoline Alkaloids A review has been published on the occurrence physical properties mass spectra and H n.m.r. spectra of quinazoline alkaloids.17 A study of the seasonal variations of alkaloids of Adhatoda vasica also resulted in the isolation of deoxyvasicine (42; R' = H2 R? = H) which is a minor alkaloid that is new to this species (cf ref.16h).Is A convenient synthesis of glycosminine (43; R = H Ar = CHIPh) and some of its derivatives (Scheme 6) involves the reaction of anthranilic acids with imino-esters in methanol (cf ref. 16~?).~~ (38) iii iv (41) Reagents i CH,(CO,Et), NaH then CICH2COCI THF; ii Ph,O at 240-256 "C;iii NaBH,,NaOH in aq EtOH reflux; iv Me,SO, K2C03 DMF at 40 "C; v NaBH,. EtOH at room temperature; vi fused K2S0, dioxane reflux Scheme 5 Modification of previous syntheses of geibalansine (34) and platydesmine (36) (cf ref. 23) resulted in more selective preparations (e.g.Scheme 4).24 Thus the reaction of the 3- prenylquinolin-2-one (33) with iodine and silver acetate followed by acid hydrolysis gave geibalansine (34); heating geibalansine (34) or 0-acetylgeibalansine (35) with polyphos- phoric acid gave the pyranoquinoline (37) that was obtained for the first time in 1983 from a bromodihydropyranoquinoline(cf ref. 200). Epoxidation of the 3-prenylquinolinone (33) and then treatment with sodium bicarbonate gave platydesmine (36) (70%). 1.4 Furoquinoline Alkaloids Glycarpine which is an alkaloid of Glycosmis cyanocarpa was assigned structure (41) on the basis of spectroscopic evidence (cj ref. 16g). In an attempt to confirm this structure 5,7-dimethoxyisodictamnine (41 ) has been synthesized by the modified procedure of Tuppy and Bohm (Scheme 5).15 Compound (41) which was obtained either by reduction with a hydride and then methylation of the intermediate (38) or by dehydration of the alcohol (40) had the same melting point as glycarpine but differed in the spectroscopic properties that d R' I R* (421 0 d (43) Scheme 6 In an attempt to modify the biological activities of vasicine (42; R' = H,,R2 = OH) vasicinone (42; RI = 0,R2 = OH) and deoxyvasicinone (42; R' = 0,R' = H) the three alkaloids were converted into their 7-nitro-deri~atives.~~ NATURAL PRODUCT REPORTS 1985-M.F. GRUNDON Table 2 Isolation of acridone alkaloids Species Alkaloid (Structure) Ref. Acronychia baueri Des-N-methylacronycine (48) Noracronycine (66) 1,2,3-Trimethoxy-N-rnethylacridone (44) 1,3,4-Trimethoxy-N-methylacridone (45) Citrus decumana *Alkaloid (52) Alkaloid (53) Glycosmis citrijolia Atalaphyllidine (50) Citracridone-I (51) Des-N-methylacronycine (48) Des-N-methylnoracronycine(49) *Glycobismine-A (74) 5-Hydroxy-N-methylseverifoline (56) ] 5-Hydroxynoracronycine (54) 3: N-Methylseverifoline (55) Noracronycine (66) Melicope Iep tococca A c ron ycine (64) Melicopicine (46) Melicopidine (47) }I2 Teclea trichocarpa Melicopicine (46) 6-Methoxytecleanthine (58) I Tecleanthine (57) 34 * New alkaloids 3 Acridone Alkaloids 3.1 Occurrence Table 26,' 2.31-35 records known alkaloids that have been isolated from fresh sources as well as new alkaloids.A detailed account has been published6 of the seven new acridone alkaloids that had been isolated from Glycosmis citrijolia and briefly described previously (c$ ref. 16f) and eight known acridone alkaloids have now been obtained from this species. The c.d. spectrum of alkaloid (59) (now given the name glycofoline) indicates an R configuration with the prenyl group at C-13 in a pseudo-equatorial position. The structure (52) of a new alkaloid that has been isolated from Citrus decumana was indicated by spectroscopic studies and by its conversion with diazomethane into the monomethyl derivative (53) which is also a constituent of this species.33 The three acridone alkaloids that were obtained from the bark of Teclea trichocarpa were found to have antimicrobial activity; melicopicine (46) and tecleanthine (57) also showed mild antifeedant properties.34 Tissue clones from the roots of Boenninghausenia albijloru contained rutacridone isogravidonechlorine 1-hydroxy-3-methoxy-N-methylacridone,and gravidonediol glucoside but noracronycine was not found (cf ref.16i).35 3.2 Spectroscopic and Chemical Properties A study of the 3C n.m.r. spectra of acridone alkaloids that had previously been reported by Reisch and co-workers (cj ref. 16e) has now been supplemented by data for twenty-seven alkaloids including pyranoa~ridones.~~ Observations from both research groups contribute to the following conclusions. Chemical shifts of N-methyl carbon atoms in acridone alkaloids are especially sensitive to the presence of substituents at theperi-positions (C- 4 and C-5) [cf (44)]; for N-methyl-acridones that bear oxygenated substituents at both C-1 and C-3 the chemical shifts of C-1' of prenyl groups have value in placing prenyl groups at C-2 or C-4.Carbon-13 n.m.r. spectroscopy can distinguish between linear pyrano[3,2-b]acridones and angular pyrano[2,3-~]acridones in the N-methyl series; the former group shows signals for C-1 1 at ca 1 16 p.p.m. and the latter at CQ 121 p.p.m. [cj (54)]. 397 0 k3 (44) R' = R2 = OMe R3= H (48) R' = Me R2= H (45)R'=H,R2=R3=O& (49) R' = R2= H (46 1 R' = R2 = R3 = OMe (50) R' = H R2= OH (47)R'R2= OCHzO ,R3= OMe (51) R' =OH,R2= OMe (55) R = H (52) R' = OMe R2= OH (56) R = OH (53) R' = R2=OMe (54) RLH,R~= OH 0 Me0 Me (57) R = H (58) R = OMe a Glycofoline (59) (601 0 0 Me -c (62) 0 (61) fJ0OMe Me (63 1 Hydrogenation of acridone alkaloids with a platinum catalyst under acidic conditions has been studied.If rings A and/or c contained only 0-substituents these rings were reduced and the 0-substituents were eliminated ; for example compound (61) gave the reduction products (62) and (63). Ring c of pyranoacridones was ~naffected.~~ 3.3 Synthesis The synthesis of the antitumour alkaloid acronycine (64) and related acridones continues to attract attention. Watanabe has developed a regiospecific synthesis of N-methylacridones by the reaction of benzynes with the lithium salt of methyl N-methylanthranilate and applied the procedure to the prepara- tion of acronycine (64) (Scheme 7).38 As part of a study of the halogenation of acridones Reisch et al.39 synthesized noracronycine (66) from the iodo-acridone (65) (Scheme 8); at 70°C noracronycine and the ether (68) were obtained in the ratio of 3 :1 and at 100 “C in a sealed tube an improved yield of noracronycine was obtained.Copp01a~~ has applied his isatoic anhydride route to quinolinone alkaloids to the synthesis of 6-demethoxyacrony- cine (67) from the pyrano-ketone (69) (Scheme 8). 3.4 Dimeric Acridones Cordell and co-worker~~*-~~ carried out an interesting study of the reactions of acronycine (64) and noracronycine (66) with OMe li 0 Reagents i lithium N-cyclohexylisopropylamide,THF at 78 to -10 “C then at room temperature Scheme 7 0 OH OH 0 0 NATURAL PRODUCT REPORTS 1985 acid (Scheme 9).When refluxed with IOM-HCl in methanol demethylation of acronycine (64) occurs to give noracronycine (66) which then yields the yellow crystalline dimeric acridones AB-1 (71) and AB-2 (70) and the trimer AB-3 (73). The structures of the products were determined by extensive ‘H and I3C n.m.r. spectroscopic studies and in the cases of AB-1 and AB-2 by single-crystal X-ray crystallography. Since the dimer AB-1 (71) is composed of units of noracronycine and dihydronoracronycine it appears to be formed by protonation at C-2 of noracronycine followed by electrophilic aromatic substitution at C-5 of a second unit.The dimer AB-2 (70) contains a noracronycine (angular) unit and a dihydronorisoacronycine (linear) unit. Further experi- ments for example the observations that the reaction of noracronycine (66) and 2,3-dihydronoracronycine(in a ratio of 1 :10) with acid gives dihydro-AB-2 [cf:(70)] and that refluxing AB-I with acid affords AB-2 (70) and noracronycine (66) indicate that AB-1 is an intermediate in the formation of AB-2. As dihydronoracronycine is unaffected by acid rearrangement to the linear (isoacronycine) structure in dimer AB-2 occurs -. .-during its formation ;a mechanism involving intermediate (72) has been proposed.41.43 The third product i.e. AB-3 of the reaction of noracronycine with acid was shown to be the angular-angular-angular trimer (73) by spectroscopy.The reaction of noracronycine (66) with methanolic hydrochloric acid at room temperature gives AB-1 (71) AB-3 (73) and 2,3-dihydronoracronycinewhile a mixture of AB-1 and dihydronoracronycine (I lo) under the same conditions gave dihydro-AB-3 (73; 1”,2”-Hz) It thus appears that trimer AB-3 is formed from AB-I in the reaction of noracronycine with acid and alternative mechanisms were proposed.42 Further exploration of condensation-dispropor- tionation reactions in this series has been reported.j3 Immediately following the study of acid-catalysed dimeriza- tion of acronycine that is described above Furukawa et al.33 reported the isolation of a new alkaloid glycobismine-A from Glycosmiscitrijolia and showed that it was the first example of a dimeric acridone alkaloid containing C-C-linked units.Struc- ture (74) was proposed for glycobismine-A on the basis of spectroscopic studies a hydroxyl group being placed at C-3 rather than at C-2 on biogenetic grounds. The alkaloid thus appears to be derived by condensation of des-N-methylnor- acronycine (49) and glycocitrine-I1 (60) (both constituents of 0 Reagents i I? HI04 EtOH; ii Me2CCIC-CH DMF K2C03 KI at 70 OC; iii LiNPr; THF at -65 “C,then heat in PhMe; iv 2,3-dichloro- 5,6-dicyanobenzoquinone,Ph Me reflux Scheme 8 NATURAL PRODUCT REPORTS 1985-M. F. GRUNDON 0 Me (66) AB-1 (71) (72) 0 0 I AB-2 (70) 2" AB -3 (73) Scheme 9 0 4 References 1 J.Reisch and M. Mueller Pharmazie 1983 38 631. 2 L. Jurd and R. Y. Wong Aust. J. Chem. 1983 36 1615. 3 A. A. L. Gunatilaka S. Surendrakumar and R. H. Thomson 4 S. Ahmad J. Nut. Prod. 1984 47 391. 5 E. M.Assem I. A. Benages and S. M. Albonico PIanta Med. 1983 Phytochemistry 1984 23 929. 48 77. 6 T.4. Wu H. Furukawa C.4. Kuoh and K. S. Hsu J. Chem. SOC. Perkin Trans. 1 1983 1681. 7 P. Bhattacharyyaand B. K. Chowdhury Chem. Ind. (London) 1984 352. 8 I. A. Bessonova D. Batsuren and S. Yu. Yunusov Khim. Prir. 9 I. A. Bessonova D. Kurbanov and S. Y.u. Yunusov Khim. Prir. Sokdin. 1984 73 (Chem. Abstr. 1984 100 206 492). Glycobismine -A (74) Soedin. 1984 124 (Chem. Abstr. 1984 100 188 797). 10 A. Ulubelen Phytochemistry 1984 23 2123.11 T. Gozler B. Gozler A. Patra J. E. Leet A. J. Freyer and M. Shamma Tetrahedron 1984 40,1145. 12 F. Tillequin M. Koch and T. Sevenet J. Nat. Prod. 1983,46 732. 13 J. Bhattacharyya L. M. Serur and U. 0.Cheriyan J. Nat. Prod.. 16 M. F. Grundon in ‘The Alkaloids’ ed. M. F. Grundon (Specialist Periodical Reports) The Royal Society of Chemistry London (a) 1979 Vol. 9 p. 78; (b)1980 Vol. 10 p. 76; (c)1983 Vol. 13 p. 102; (41979 Vol. 9 p. 82; (e) 1980 Vol. 10 p. 82; (f) 1983; Vol. 13 p. 116;(g)1980 Vol. 10 p. 77; (h)1982 Vol. 12 p. 89; (i)1979 Vol. 9 p. 87. 17 K. S. Bhide and R. B. Mujumdar IndianJ. Chem. Sect. B 1983,22 1254. 18 A. R. MacKenzie C. J. Moody and C. W. Rees J. Chem. SOC. Chem. Commun. 1983 1372.19 E. A. Clarke and M. F. Grundon J. Chem. SOC. 1964 438 4190. 20 M. F. Grundon Nut. Prod. Rep. 1984 1 (a) p. 195; (6) p. 199. 21 G.M. Coppola J. Heterocycl. Chem. 1983 20 1217. 22 G. M. Coppola J. Heterocycl. Chem. 1983 20 1589. 23 R. M. Bowman and M. F. Grundon J. Chem. SOC. C 1966 1504. 24 M. Ramesh P. Rajamanickam and P. Shunmugam Heterocycles 1984 22 125. 25 S. C. Kuo T. P. Lin L. D. Lin H. Y. Hsu and C. H. Wu J. Nut. Prod. 1984 47 47. 26 R. Storer and D. W. Young Tetrahedron 1973 29 1217. 27 A. Prakash and S. Ghosal J. Sci. Ind. Res. 1983 42 309. 28 K. Pandita M. S. Bhatia R.K.Thappa S. G. Agarwal K. L. Dhar and C. K. Atal Planta Med. 1983 48 81. 29 I. Ganjian and I. Lalezari Synth. Commun. 1984 14 33. NATURAL PRODUCT REPORTS 1985 30 M.P. Jain C. K. Atal R. Bandhyopadhyay and B. G. Nagavi Indian J. Pharm. Sci. 1983 45 178. 31 S. Funayama and G. A. Cordell J. Nut. Prod. 1984 47 285. 32 S. C. Basa and R. N. Tripathy J. Nut. Prod. 1984 47 325. .33 H. Furukawa T.-S. Wu C.-S. Kuoh T. Sato Y.Nagai and K. Kagei Chem. Pharm. Bull. 1984 32 1647. 34 W. Lwande T. Gebreyesus A. Chapya C. Macfoy A. Hassanali and M. Okech Insect Sci. Its Appl. 1983 4 393. 35 I. N. Kuzovkina Z. Rozsa K. Szendrei and A. M. Smirnov Rastit. Resur. 1983 19 374 (Chem. Abstr. 1983 99 191 652). 36 H. Furukawa M. Yogo and T.-S. Wu Chem. Pharm. Bull. 1983 31 3084. 37 T.4. Wu; Yakugaku Zasshi 1983 103 1103 (Chem. Abstr. 1984 100 68 572). 38 M. Watanabe A. Kurosaki and S. Furukawa Chem. Pharm. Bull. 1984 32 1264. 39 J. Reisch I. Mester and S. M. El-Moghazy Liebigs Ann. Chem. 1984 31. 40 G. M. Coppola J. Heterocycl. Chem. 1984 21 913. 41 S. Funayama G. A. Cordell H. Wagner and H. L. Lotter J. Nut. Prod. 1984 47 143. 42 S. Funayama and G. A. Cordell Planta Med. 1983 48 263. 43 S. Funayama and G. A. Cordell Heterocycles 1983 20 2379.

ISSN:0265-0568    

DOI:10.1039/NP9850200393

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

Olef inic Microbial Metabolites excluding Macrocyclic Compounds R. C. F. Jones Department of Chemistry University of Nottingham University Park Nottingham NG7 2RD Reviewing the literature published during 1982 and 1983 (Continuing the coverage of literature in Aliphatic and Related Natural Product Chemistry Vol. 3 p. 288) 1 Pyran-Pyranoid Compounds 1.1 Pseudomonic Acids 1.2 Pyrone Metabolites 1.2.1 Meroterpenoids 2 Butenolide Metabolites 2.1 Tetronic Acids 2.2 Miscellaneous Butenolides 3 N-Heterocyclic Compounds 3.1 Acyltetramic acids 3.2 Other Pyrrolidines and Pyrroles 3.3 Piperidines and Pyridines 4 Other N itrogen-containing Metabolites 5 Miscellaneous Olefinic Microbial Metabolites 5.1 Methylenecyclopentanone Metabolites 5.2 Cyclopen tene Metabolites 5.3 Compactin and Mevinolin 5.4 Other Cyclohexene Metabolites 6 References This Report is the successor to the section “on-macrocyclic Olefinic Microbial Metabolites’ in the chapter entitled ‘Ole- finic Microbial Metabolites including Macrocyclic Com- pounds’ of the series of Specialist Periodical Reports on Aliphatic and Related Natural Product Chemistry.The selection and organization of material have been made on the same basis as previously and the literature surveyed is that for 1982 and 1983. 1 Pyran-Pyranoid Compounds New biosynthetic studies in Penicillium patulum using [1J3C g02]acetate and 802, have shown (using isotope shifts in the 13C n.m.r.spectra to locate l80 enrichment) that only the carbonyl oxygen atom of the antibiotic patulin (1) originates from acetate whilst the rest derive from molecular oxygen. This has been taken as support for the sequence that is summarized in Scheme 1 i.e. a mono-oxygenase mechanism of oxidative cleavage of the aromatic ring. In addition results with 12-l 3C 2-2H3]acetate indicate that the side-chain protons of the aromatic intermediates are lost during the biosynthesis. Incorporation of these same doubly labelled acetates into citrinin (2) in Aspergillus terreus produces the illustrated labelling pattern consistent with the previously determined biosynthetic pathway involving the keto-aldehyde (3);* dehy- dration of the hemi-acetal(4) would then produce the quinone- methide functionality.Full details are now available of c- incorporation studies with 3C-labelled precursors in Aspergil-lus ustus that implicate the symmetrical dialdehyde (5) as an intermediate in the biosynthesis of austdiol (6).3 New results from incubation in an atmosphere that was enriched in 1802 show that austdiol(6) is only labelled in the hydroxy-group at C- 7 again suggesting a mono-oxygenase mechanism for the oxidation of (5) or a later inte~mediate.~ 1.1 Pseudomonic Acids The structure elucidation and chemistry of pseudomonic acid C (7a) (a minor component isolated from Pseudomonas Jluores- cens) have been reported in full;s amongst the evidence are an X-ray crystal structure of the derived ethyl monate C (7b) and an efficient stereospecific preparation of (7a) from pseudo- monic acid A (8a).Pseudomonic acid D which is a further minor metabolite from the same organism has been identified as (8b) and prepared by partial synthesis from sodium monate A (8c).‘j A second total synthesis of (+)-pseudomonic acids A (8a) and C (7a) has been formally completed by Snider et al. (Scheme 2) using an interesting appr~ach.~ A Lewis-acid- catalysed ‘ene’ reaction of hexa-l,5-diene with formaldehyde afforded a dienol whose acetate (9) underwent a further ‘ene’ reaction with excess formaldehyde and ethylaluminium dich- loride and a subsequent quasi-intramolecular Diels-Alder reaction [as (lo)] in situ. Relatively straightforward modifica- tions led to an intermediate (11) that had been used in the earlier Kozikowski synthesis.Two groups have reported the enantiospecific total synthesis from carbohydrate building 8 8 Me CHO OL Scheme 1 Me Me p /CD3 I \ 13 4”” f-++-Me-C Me ‘ CHEO ‘%H OH NATURAL PRODUCT REPORTS 1985 I OH OH OH (8) a; R’ = H R2= [CH,],CO,H b; R’ = HI R2= [CH2l,CHhCH[CH2]2COqH c; R1=H,R2=Na d ; R’ = OH R2= [CH2]8C02H I OH AcOA’ \v-x 4, ‘u -b..<o ’ PhZButSiO’ ‘ (1 1) Reagents i CH,O Me,AlCl; ii Ac20 pyridine; iii CH,O (3 equivalents) EtAlCl (4.5 equivalents); iv H,O; v pyridinium chlorochromate NaOAc; vi MeMgCl; vii Ph2Bu1SiC1 Et3N 4-(dimethy1amino)pyridine ; viii pyridinium chlorochromate; ix OsO, N-methylmorpholine N-oxide; x cyclohexanone TsOH cuso Scheme 2 + FMgC‘ OSiMe2But .li OSiMe2Bu ii-vi,ii I A OH Reagents i CuI (catalytic); ii HCl (as.) dioxan; iii Me,CO H2S04 4A molecular sieves; iv TsCI Et3N 4-(dimethy1amino)pyridine;v KCN HMPT 18-crown-6; vi AIMe, Ni(acac) ; vii MeC(0Si- Me,)==NSiMe3 ; viii (EtO),P(O)CHCO,Et Na+ Scheme 3 blocks of ethyl monate C (7b) and hence by known conversions of pseudomonic acids A (8a) and C (7a).*~~ In the first route (Scheme 3) a key step is the regiospecific copper(1)- catalysed opening of the tetrahydropyran epoxide (1 2) which had been prepared from D-xylose by the Grignard reagent (1 3) which is available from D-glucose.* The second due to Fleet et ai.,(Scheme 4) is strategically dependent on two amide-acetal Claisen rearrangements to convert the dihydropyran (14) (obtained from D-arabinose) via the lactone (15) into (16).9 Further manipulation produced the aldehyde (1 7) that could be coupled to a phosphonium salt (18) which had been produced from L-arabinose ; compounds (1 7) and (1 8) are enantiomeri- cally pure forms of intermediates that were prepared and used in racemic form in the Kozikowski synthesis.The preparation from D-ribose of an intermediate (19) which is obviously potentially suitable [cf (17)] for conversion into pseudomonic acid B (8d) has also been reported.*O In a further synthetic approach to the pseudomonic acids the dihydroxytetrahydropyran(20) has been constructed from L-arabinal diacetate using a single ester enolate Claisen rearrangement and a palladium(0)-mediated allylic displace- ment to elaborate the differentiated carbon substituents;’ ] the latter tactic has also been employed to convert racemic lactone (15) which had been prepared by two alternative approaches into (21).12 1.2 Pyrone Metabolites The isolation and structure elucidation of the macommelins (22) which are newly discovered metabolites of the phytotoxic fungus Mucrophorna cornrnelinue has recently been described.NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES 403 ?H 0 o'\coNMe2 i-iii o<~Qo iv,v.i ~ Ph ButSi 0-' (14) (15) (16) vi-xiI Q +O+c**E' (7b) xi i ,xiii ++ PPh I- + OHC\ OH (18) (17) Reagents i MeC(OMe),NMe, xylene heat; ii 12 THF (aq.); iii 1,8-diazabicyclo[5.4.O]undec-7-ene; iv NaBH ;v Ph2ButSiC1 imidazole; vi MeLi at -78°C; vii OsO, N-methylmorpholine N-oxide; viii cyclohexanone TsOH CuSO,; ix (EtO),P(O)CHCO,Et Na+; x Bu4N+ F-; xi pyridinium chlorochromate molecular sieves; xii 2BuLi at -40 to 0°C; xiii AcOH (aq.) + OHC\ OfJ 'YCOpMe OH (19) (22) a ;R b;R c; R d; R Me-COzH 0 [Me] methionine = CH,CH,OH = CH(0H)Me = CH2Me = CH(OH)CH20H Scheme 4 -copMe "~ Eto2cY C02Et (20) (23) Biosynthetic studies on astepyrone (23) in a strain of Aspergillus terreus have shown that the aldehyde (24) is a precursor and the pathway has been suggested to proceed uia oxidative cleavage of the aromatic ring at the site indicated.14 Additional precursor-incorporation experiments in Diplodia macrospora [a fungal contaminant of maize (Zea mays)] have revealed a polyacetate origin as illustrated with C-12 as the starter for the mycotoxin diplosporin (25);' the carbon atoms 2 and 5 are derived by C-methylation (from methionine) and only the carbonyl oxygen atom at C-4 is derived from acetate.The simple a-pyrone sibirinone (26) which is a metabolite of (26) Hypomyces semitranslucens has been synthesized by [4+ 21 dimerization of vinylketene (obtained from pyrolysis of but-2-enoic anhydride) and treatment of the adduct with an acid.16 MeopcY C02Me (21) (24) R' H (27) a; R' = CHO RZ=OMe b ; R' = CHpOH R2 = OMe c ; R' = CHO R2= NH[CH,],OH NATURAL PRODUCT REPORTS 1985 OMe HO A A [Me)mathionine OMe OHCdo (28) 0 (29) Me Me H (30) (32) (34)a; R = [CH2I2Me (33) b; R = [CH2I3Mc Three host-specific phytotoxins i.e.solanapyrones A B and C have been isolated from Alternuriu sofani which is the causal organism of early blight disease of tomatoes and potatoes and identified as (27a) (27b) and (27c) respectively. A complete assignment of the 13C n.m.r. spectrum of citreoviridin (28) (the potent neurotoxic mycotoxin that has been isolated from Penicillium citreo-viride and Penicillium puluillorum) has been reported;18 using this as a basis incorporation experiments with 3C-labelled precursors have revealed the full pattern of labelling as shown and have confirmed the derivation of (28) from a CI8 polyketide with an acetate starter and five C units from methionine.I8 A synthesis of the related metabolite secocitreoviridin (29) which is a minor component of P.citreo-viride has been completed and confirms the structural assignment. l9 A novel sulphur- containing metabolite has been found in the same organism and has been identified as (30) and named citreothiolactone.20 Closely related to citreoviridin is the mycotoxin asteltoxin (3 l) from Aspergillusstellatus. There is evidence to suggest that the bis-tetrahydrofuran moiety is responsible for the ATPase- inhibitory properties of (31) and a recent report has outlined the synthesis of the part-structure (32) using the photo- cycloaddition of a furan to a carbonyl compound as the key step.The structure elucidation of verrucosidin (a tremorgen isolated from Penicillium uerrucosum) has shown it to be (33),22 i.e. once again structurally related to citreoviridin (28); every C2 unit of verrucosidin appears to be methylated but it is not (37) a; R = Me b; R = Et known whether the biosynthesis is based either on propionate or on acetate with polymethylation. Other new a-pyrone metabolites whose isolation and structure determination have been described recently include the myxopyronins [(34a) and (34b)] which are inhibitors of the bacterial synthesis of RNA and which are found in the gliding bacterium Myxococcus fufv~s,~~ and islandic acid (35) (an antitumour metabolite of Peniciffium i~fundicurn).~~ The isola- tion characterization and biological properties of the dihydro- pyrones CI-920 (36a) PD113,270 (36b) and PDll3,271 (36c) which are also said to be antitumour agents from Streptomyces pufveruceussubsp.fostreus have been reported ;25 the dihydro- pyrone moiety also appears in the leptomycins [(37a) and (37b)] these being newly isolated and identified antifungal antibiotics from a species of the genus Streptomyces.26 I .2.I Meroterpenoids The 13C n.m.r.spectrum of austin (38a) which is a meroterpenoid metabolite of Aspergillus ustus has been fully assigned by methods including the analysis of long-range 'H-13C couplings.27 The new meroterpenoids austinol (38b) and dehydroaustin (39) have been isolated from A. ustus and dehydroaustin has also been found in a chance mutant of an andibenin-producing strain of Aspergillus vuriecolor.27 Austinol (38b) and yet another new metabolite isoaustin (38c) have been found in Peniciffium diuer~um.~~ These various co- 405 NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES (38)a; R'= OAc,R2= H (39) (40) X = H or OH b; R'=OH,Rz=H C; R'= H,Rz=OAc 0 0 C02Et OH (41) (42) (43) OHC\/\I v-viii OH I IIW HO A-'PO I (46) (45) Reagents i NaCN EtOH (as.);ii TsOH PhH; iii LiNPr'(cyclohexyl) HMPA MeI; iv 03,then Me,S; v MeCH=CHCH=C(SiMe,)MgBr; vi Bu4N+ F-; vii MnO,; viii Me2S0 (CF,C0)20 Et,N Scheme 6 occurrences suggest that all of these new metabolites are formed by the same biosynthetic pathway as has previously been proposed for the meroterpenoid group and proceeding from .a precursor such as (40) consisting of a triprenyl moiety attached to a bis-C-methylated tetraketide.Further evidence in support of this pathway has come from the recent finding (using 2Hn.m.r. spectroscopy) that the deuterium-labelled ethyl 3,5-dimethylorsellinate (41) is incorporated specifically into the methyl group C-10' of both austin (38a) (in Aspergillus ustus) and terretonin (42) (in Aspergillus terreus).28 A new meroterpenoid paraherquonin which was isolated from Penicillium paraherquei has been shown to have structure (43) by spectroscopic methods and an X-ray crystal structure;29 it would appear to be biosynthetically related to austin etc. 2 Butenolide Metabolites 2.1 Tetronic Acids Incorporation experiments with [1-l 3C 802]acetate in Penicil-lium multicolor and examination by spin-echo F.T.3C n.m.r. spectroscopy of derivatives of the samples of labelled multicolanic acid (44a) multicolic acid (44b) and multicolosic acid (44c) that were isolated has shown that only the oxygen atom at C-4 and one of those at C-1 1are derived from acetate.30 This confirms the previously proposed biosynthetic pathway via 6-pentylresorcylate and subsequent oxidative fission as illustrated in Scheme 5; the results are also consistent with lactonization via displacement at C-1 (in a thioester?) by the oxygen at C-4. Full details including an X-ray crystal structure determina- tion have appeared of the structure elucidation of vertinolide (43 which is a new tetronic acid from Verticillium intertex- An enantioselective total synthesis of natural (-)-vertinolide has been completed (Scheme 6) from the chiral epoxide (46) itself available from geraniol by the Sharpless procedure thus unambiguously determining the absolute configuration of vertinolide to be as shown.32 This route exemplifies a potentially general approach to chiral tetronic acids and butenolides from allylic alcohols.Amongst other recent synthetic work in the tetronic acid area is the use of the silyl enol ether (47) as a C-5 nucleophile NATURAL PRODUCT REPORTS 1985 MeO MeO MeO. I ii ,iii -+ h (47) Reagents i BuLi Me3SiC1; ii RCH(OMe), ZnBr or TiC14 (catalytic); iii heat Scheme 7 0 (48) 0 ; R'R2 = CHMe R3= H b ; R' R2 = -[CH2 J5- R3= H C ;R' R2 = -[CH2I5- R3= Br Reagents i LiNPr', at -78°C; ii BuLi at -78°C; iii R4C02Me or R4COCI or (R4CO),0; iv R4CHO; v Mn02or pyridinium dichromate [if R4 = Me] Scheme 8 (50)a ; R2=Me (R' = Me or Et 1 b ; R2 = CH-CHMt xii -xiv 1 Reagents i LiNPr', at -78°C; ii MeCOCH=CHCH=CHEt; iii MeCHO; iv pyridinium dichromate; v (MeCO),O or MeC02Me; vi MeCH=CHC02Me; vii Bu'Li; viii MeCOC0,Et; ix Ac20 4-(dimethylamino)pyridine;x 2LiNPri2 Br,; xi CH,N,; xii Bu"Li; xiii MeCH=CHCHO; xiv MnOz Scheme 9 under non-basic conditions for the construction of 5-substi-tuted tetronates (e.g.Scheme 7).33 Three groups have investi- gated the directed metallation at C-3 of 4-0-methyltetronic acids.Pattenden and Clem~~~ have lithiated the 5-ethylidene- tetronate (48a) with lithium di-isopropylamide at low tempera- ture to form the corresponding vinyl anion (49a) (Scheme 8) whilst R. R. Schmidt et al.35 have deprotonated the 53-disubstituted tetronate (48b) by the same method to give (49b) and a Japanese group have also generated (49b) but by means of halogen-metal exchange between the bromotetronate (48c) and b~tyl-lithium.~~ The lithio-derivatives (49) have been shown to undergo reaction at C-3 with various electrophiles such as acylation with e~ters,3~-~~ anhydride^,^^ or acid ch10rides,~6 and addition to aldehyde^^^-^^ (Scheme 8); in some cases oxidation of the latter adducts provides a more efficient sequence for 3-C-a~ylation.~~ These methods have been used to provide short syntheses of the 3-acyltetronate structures (50a) and (50b) (Scheme 9)35-37athat had been 0 lie (51) a; R 2 Me b ; R = CH=CHMe proposed for the antibiotic metabolites gregatin B and aspertetronin A.All three groups however agree that the properties of the synthetic materials (50) do not match those of the natural products and it is suggested (on the basis of spectroscopic correlations with model compounds) that the structures of the metabolites be revised to the isomeric 5-methoxy-3(2H)-furanones (51a) and (51b) respective-ly ;35,36,37b the corresponding revision is presumably applicable to other metabolites of the gregatinlaspertetronin group. NATURAL PRODUCT REPORTS 1985 -R.C. F. JONES (52) Bu'S JA iy 0 [CH2I2Me v,viii i,ii,vi\VII Ft[CH212Me ___) H# C02Me C02 H (53) Reagents i NaH DME; ii BuLi DME; iii I[CH2],0SiMe2But; iv MeCH(OH)CO,Et AgOCOCF,; v Bu4N+ F-; vi EtI; vii (S)-MeO,CCH,CH(OH)CO,Me AgOCOCF3 ;viii NaOH (aq.) Scheme 10 Na+ OH A regiospecific y-alkylation of P-keto-thioesters has been developed and employed in the synthesis of the 3-acyltetronic acids carolic acid (52) and (S)-carlosic acid (53) (Scheme The structure and absolute configuration of tetronomycin which is a novel polyether antibiotic that has been isolated from a Streptomyces strain have been determined from spectroscopic data and an X-ray diffraction study on the silver salt of its monoacetate to be the complex 3-acyltetronic acid (54a).39 Tetronomycin is thus very similar in constitution to the antibiotic ionophore M 139603 whose structure determination (also by X-ray methods) was reported in 1981 but it apparently has the opposite absolute configuration at all comparable centres; structure (54b) therefore illustrates the enuntwmer of M139603.A study of the structures in solution and of the cation-binding properties of tetronomycin and M 139603 using high-field lH n.m.r. methods suggests that they have virtually identical geometries and that these are very reminiscent of the structures in the solid state.40 The X-ray crystal structure of the p-bromobenzoate of FR-900109 (a new antibiotic isolated from Streptomyces prunicolor) has been reported and shows that FR-900109 has the constitution (55);41 the molecule thus contains a 3-acyltetronic acid 'disguised' as an internal ketal of the 3-acyl carbonyl group.(56) a; R = Me b; R = Et (60)a ; R = CH2CH=CMe2 b;R=H 0 c ; R =CH2CL'CMe2 Details of the isolation and structure elucidation of the related thiotetronic acid antibacterial antibiotics thiolactomy- cin (56a) (from a species of the genus Nocardia that was found in a soil isolate)42 and thiotetromycin (56b) (from Streptomyces OM-674)43 have appeared recently. The newly isolated antibiotic basidalin (57) from Leucoagaricus nuucina is a simple enamine derivative of a tetronic acid,44 and as such is included in this section of the Report. 2.2 MiscellaneousButenolides Two new short syntheses have been published of lepiochlorin (58) which is a chlorinated antibiotic from a fungus of the genus Lepiot~.~~ Serpenone (a metabolite of Hypoxylon serpens) has been found to have the methoxybutenolide structure (59).46 The structures of two new butyrolactones that are related to the butyrolactone (60a) and which have been isolated from two strains of AspergiZ1u.s terreus have been reported as (60b) and (~OC).~~ Biosynthetic investigations had previously shown that (60a) is derived from phenylalanine via tyrosine whilst the present report47 includes results indicating that these amino acids are converted into 4-hydroxyphenylpyruvic acid two units of which condense together to produce first (60b) and thence after prenylation the butyrolactone (60a).408 NATURAL PRODUCT REPORTS 1985 n- 0 o*o H o e C O z H n-C6H17 hH2 OH H (61) (62) (63) ( 64) (65) (66) MeAC02H -1 Me-CO2 H I--(681 OH 0 (70) a ; R = pH b;R=O Further synthetic routes to the antifungal bis-lactones canadensolide (61)"* and avenaciolide (62)49 have been disclosed. 3 N-Heterocyclic Compounds 3.1 Acyltetramic Acids The lH and 2H n.m.r. spectra of the unique 3-acyltetramic acid malonomicin (63) have been analysed for use in biosynthetic investigations with 2H-labelled precursors. Such experiments in Streptomyces rimosus have indeed been reported and suggest that the first tetramic acid intermediate on the pathway is (64);51 the sequence to this point is not yet fully proven.New incorporation studies with [ 1,2-l 3C,]acetate have shown that the pro-2 methyl group C-22 in P-cyclopiazonic acid (65) becomes the 20P-methyl group (again C-22) upon biological conversion into a-cyclopiazonic acid (66) thus indicating a syn-addition across the carbon-carbon double- bond of the prenyl group.52 During synthetic studies directed at a-cyclopiazonic acid (66) the hexahydroisoindoloindole ring system [e.g. (67)] has been prepared apparently for the first time.53 (67) R = Hz or 0 CONHMe " O y OH 0 e O H (69) The low incorporation of acetate into streptolydigin (68) in Streptomyces Iydicus had previously caused problems with the detection of enrichments when using [ 1-l 3C]acetate as precur- sor but these problems have now been overcome by using [ 1,2-13C2]a~etate.54 The four intact acetate units that have been found to be incorporated can be combined with the previously determined incorporations of propionate to complete the labelling pattern that is illustrated for the 3-acyl side-chain and for carbon atoms 2' and 3' of the tetramic acid ring;54 derivation of C-2' and C-3' from acetate is in line with results for other 3-acyltetramic acid metabolites.A simple synthesis of rhodinose (69) which is the sugar portion of streptolydigin (68) from ethyl (S)-lactate has been c~mpleted.~~ The 3-dienoyltetramic acid metabolite tirandamycin (70a) is closely related to streptolydigin. DeShong ef al. have recently disclosed a short and efficient synthesis (in racemic form) of the alcohol (71) (Scheme 11),56 which is an intermediate in the previously reported Ireland route to tirandamycic acid (70b) (the acyl side-chain of tirandamycin).The key strategy of this new approach is the oxidative cleavage of furan-alcohol (72) to an ene-dione moiety which is found in the alcohol (71) as an acetal. The macrocyclic antiprotozoal acyltetramic acid ikarugamy- cin (73) has also been the subject of synthetic studies. A route that is based on an endo intramolecular Diels-Alder cycloaddi- tion has been developed to the tetracyclic intermediate (74) in optically active form and is outlined in Scheme 12.57 Ketone (74) has the A B and c rings of ikarugamycin with the carbon appendages to ring c in a masked form for further elaboration.A key point in the total synthesis of naturally occurring 3- acyltetramic acids is the attachment of the tetramic acid (pyrrolidine-2,4-dione) unit to the appropriate side-chain at C- 3 and there have recently been several studies of methodology in this area. Jones and Peterson have developed an approach that is based on directed metallation of the 4-0-methyltetra- mates (75) to provide a vinyl-lithium (see Scheme 13) that is acylated via its addition to aldehydes and subsequent oxidation NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES i ,ii OCHzPh j (71) Reagents i Bu'Li; ii separation of epimers at C-1' undesired epimer oxidized and then reduced [by Zn(BH,)J to (72); iii m-ClC,H,- C03H; iv HF MeCN (aq.); v Me3SiC1 NaI Scheme 11 and hydrolysi~.~~ These same authors have also reported an improvement on their earlier Lewis-acid-catalysed acylation of pyrrolidine-2,4-diones (also Scheme 13) if the boron difluoride complexes (76) are isolated,s9 whereas acylation of the quaternary ammonium enolates of tetramic acids leads to 0-acylation with bond migration [see for example (77)].60 The phosphonate-activated 3-acyltetramic acids (78) have been prepared by Boeckman and Thomas from the ketene-acetone adduct as shown in Scheme 14;61 the great potential of such intermediates for elaboration of a 3-acyl side-chain has been demonstrated by their condensation with aldehydes in a Wadsworth-Emmons fashion as illustrated.A new approach to (73) v-xii I yxvi.vi xvii -0 (74) Reagents i LiNPr', at -78 "C; ii Me3SiC1 at -78 to +25"C; iii H30+;iv CH,N,; v Bui2AIH at 0°C; vi TsCl pyridine; vii KCN; viii Bu',AIH at -20°C; ix AcOH (aq.) NaOAc; x (EtO),P(0)(5HCO2Et Na+; xi AcOH THF (as.); xii pyridinium dichromate; xiii BuLi at -50 to +25"C; xiv I,; xv toluene at 140°C for 70 hours; xvi HCl THF (aq.); xvii KOBu' Bu'OH Scheme 12 NATURAL PRODUCT REPORTS 1985 (77) (76) Reagents i Bu,N+ OH-; ii Me2S0,; iii BuLi at -78°C; iv RCHO; v MnO,; vi NaOH (aq.); vii BF3.0Et2 RCOCl; viii MeOH; ix Et,N+ OH-; x AcCl Scheme 13 0 iii,iv HN0piOEt)* ___) R2 A.+* H;e i,ii * R' 0=P(OEt )* R' (781 Reagents i LiNPrI2 at -78"C C13CCC13; ii NaH HP(O)(OEt),; iii R1CH(NH2)C02Me pyridinium tosylate; iv NaOMe MeOH PhH; v 2LiNPrI2 R*CHO sheme 14 R1 I [Rl=Mel iv -vi I t O H tO H R2 22 Reagents i R*CHXCO,Et AgBF,; ii NaHC03 (aq.) CH,Cl2; iii NaOEt EtOH; iv N-bromosuccinimide CCl,; v P(OEt),; vi EtOzCCH20S02CF Scheme 15 3-acyltetramic acids is founded on the base-mediated fragmen- is part of the evidence for the structure of the yellow metabolite tation of suitable isoxazolium salts (79) to produce P-keto- reductiline (from a variant of Streptomyces orientalis) being as amides (Scheme 15);62 this route can be modified as shown in shown in (81) and thus reminiscent of the antibiotic Scheme 15 to produce phosphonate-activated tetramic acids of reductiomycin (see later in this Report).65 the type discussed above.Cyclization of P-keto-amides is the Antibiotic X-14547A (82) which has been isolated from most frequently used method of ring-closure to form tetramic Streptomyces antibioticus and which has a variety of biological acids (for example Schemes 14 and 15) but the alternative properties (for example as an ionophore as an antibacterial sequence of bond formation that is featured in Scheme 16 has an antitumour and an antihypertensive agent and as a recently been reported.63 promoter of feed utilization in ruminants) has continued to be a target for synthetic chemists.The second total synthesis of the natural enantiomer of (82) has been communicated by Ley et al. 3.2 Other Pyrrolidines and Pyrroles and is outlined in Scheme 17.66 The racemic 'right-wing' The chemical and biological properties and structure elucida- tetrahydroindane-lactone(83) which is available as reported tion of a simple new antibiotic isohematinic acid (80) from previously by these authors (using an intramolecular Diels- A synthesis Alder cycloaddition strategy) was resolved by lactone opening Actinoplanes philippinensis have been de~cribed.~~ NATURAL PRODUCT REPORTS 1985 -R.C. F.JONES with (-)-(S)-1-phenylethylamine chromatographic separation of the amides and acid-mediated re-lactonization. Optically pure lactone (83) was converted into the sulphone (84) and this was coupled (by the sequence indicated here) to a chiral ‘left- wing’ tetrahydropyran-aldehyde (85) itself prepared from 1 ,6-anhydro-P-D-glucose to complete the formation of X-145471-2 (82).66 Another possible ‘left-wing’ precursor (86) has been prepared.67 Roush et al.have suggested the possibility of the construction of antibiotic (82) by intramolecular Diels-Alder Y COPh bOPh 0 ti: H (Y = CN ,C02R or COMe) Reagents i YCH2C02R KOBu‘ or NaH; ii NaOMe or NaOEt Scheme 16 (81 (831 (82) Li 0 41 1 reaction on a pentaene that contains all of the carbon atoms of the full skeleton and as a model for this strategy they have performed the cycloaddition of pentaene (87) to produce (88) as the major product. 68 3.3 Piperidines and Pyridines The recently reported X-ray crystal structure of an 0-acetylated dihydro-derivative has further secured the constitution (89) of streptazolin (an antibiotic that has been found in Streptomyces virido~hromogenes).~~ X-Ray methods have also been used along with the usual spectroscopic information to determine the structure (90) having a unique cyclopropyl side-chain for cyclizidine which is an indolizidine metabolite that has non-selective immunostimulatory properties and which was isolated from a species of the genus Streptomyce~;~~ the classi- fication of cyclizidine as a piperidine in this Report is obviously arbitrary.Biosynthetic studies with 3C-labelled acetate and methion- ine and with 5N-or l4C-labe1led phenylalanine as precursors in Cylindrocladium ilicicola have revealed patterns of incorpora-tion into the antifungal antibiotic ilicicolin H (91) that are consistent with the pathway that is shown in Scheme 18 from a polyketide and phenylalanine via a 3-acyltetramic acid.7 The ring-expansion of tetramic acids as a biogenesis of the pyridone ring had been proposed earlier by others in connection with tenellin (92)’ which is the pyridone metabolite of the insect- pathogenic fungi Beauveria bassiana and Beauveria tenefla and it is now speculated that it might apply to other related metabolites such as funic~losin.~~ A synthesis of (& )-tenellin (92) has recently been described.Details have been published of the isolation and biological properties of kirrothricin which is an antibiotic of the aurodox group that has been found in Streptomyces cinnam~meus,~ and of factumycin (93) which is a closely similar new antibiotic from Streptomyces la~endulae;~~ the structure of kirrothricin the elucidation of which had been reported in 1981 differs from (93) in having a 5,6-dihydro-2-pyridone terminus and the 2 geometry of the double-bond between C-14 and C-15.The stereochemistry of piericidin A ,which is an antibiotic and an inhibitor of mitochondria1 electron transport has been Reagents i,@NCH20~CH2hsiMe3; ii phsN* Bu3P; iii (PhSe), H,02; iv BuLi at -78”C THF-HMPA; v PhCOCl; vi \ 0 sodium amalgam MeOH-THF; vii Bu,N+ F-; viii NaOH MeOH (aq.) Scheme 17 NATURAL PRODUCT REPORTS 1985 0 (86 1 (87) I Scheme 18 OH (92) revised to the 9R,lOR-configuration that is shown in (94) after correlation of a degradation product with (S)-2-methylpentan- 3-01.~~ Fredericamycin A is a potent antitumour agent recently isolated from Streptomyces griseus and it has been shown to have the novel spiro structure (95).76 4 Other Nitrogen-containing Metabolites A group of homologous antibiotics have been isolated from the gliding bacterium Myxococcus xanthus and termed myxala- mides A B C and D.77aThe structures of these metabolites which are all strong inhibitors of the respiratory chain at the NADH :ubiquinone oxidoreductase have been determined to OH OH 0 OH (93) be the alaninol amides (96a-d) respectively by spectroscopic investigation initially of the major component myxalamide B (96b).776 The unsaturated fatty acid portion bears some relationship to the side-chain of the piericidins e.g.(94) (see above). Full details are now available of the isolation (from Streptomyces xanthochromogenes) and structure elucidation of the antitumour metabolite AM-6201 (97);78 this was deduced NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES R OH (96) a ; R = MeCH2MeCH b; R = Me2CH C; R = Et d;R=Me 0 0 OH If Ho* U (98) a ;R = CH,CHMe b;R= to be the same as reductiomycin which is a known metabolite of Streptomyces griseorubiginosus and a synthesis of (&)-reductiomycin has been completed.79 The 2-aminocyclopent- ane-l,3-dione amide moiety of reductiomycin has already featured in this Report as a part structure of reductiline (8 1) and is also seen in the new antibiotic U-56407 (98a) whose isolation and characterization have recently been described.80 This bright yellow crystalline metabolite of Streptomyces hagronensis is obviously related to the known asukamycin (98b) and also to U-62 162 which is another metabolite newly isolated from Streptomyces verdensis and identified as (99).8* The 2-aminocyclopentane- 1,3-dione unit that is mentioned above is also found (as an amide) in the saccharide portion of the complex antibiotics moenomycin and diumycin and hydrolysis of these antibiotics leads inter aha to the C25 alcohols moenocinol (100a) and diumycinol (100b) respectively as major fragments.Full details have now been published of the previously reported Kocienski synthesis of moenocinol,82 and the same authors have also completed a synthesis of diumy- cin01.~~ new synthesis of moenocinol (lOOaj based on A isoprenoid-type precursors has been performed.84 Pholipomy- cin which is a member of the moenomycin family of natural products has been identified as (1OOcj by chemical degrada- tion FAB m.s.techniques et~.~~ OH C02H (99) The 1,3-dipolar cycloaddition of diazoalkanes to a protected 2-aminoacrylic acid followed by thermolysis of the pyrazoline that is thus formed is the basis of a simple new synthesis of the cyclopropyl amino acid coronamic acid which is found as an amide in coronatine (101) (a phytotoxic metabolite of Pseudo-monas coronafaciens).86 An esterified amino-acid unit features in the structures that have been reported for the AK-toxins I (102a) and I1 (102b) on the basis of spectroscopic data;87 the AK-toxins are host-specific phytotoxic metabolites of Alter-naria kikuchiana which is the causal fungus of black spot disease on susceptible cultivars of Japanese pear (Pyrusserotina var.culta). The biosynthesis of the amino acid furanomycin (103) which is both an antibiotic and a competitive antagonist of L-isoleucine in Streptomyces threomyceticus has been investi- gated and an unexpected derivation (as illustrated) from propionate and two intact acetate units has been revealed.88 A full account has appeared of the transformation of D-ribose into the dihydrofuran amino acid (104);89 this is the structure that was originally proposed for furanomycin but now of course it has been found to be a diastereoisomer of the natural product.The first total synthesis of natural (+)-thermozymocidin (105) (also known as myriocin) which is an antibiotic from the thermophilic fungus Myriococcum albomyces has been com- ~leted.~O D-Fructose was the chiral starting material and some key intermediates are shown in Scheme 19. The isolation properties and structure confirmation (from an X-ray crystal structure determination) have been reported for the novel polycyclic amide antibiotic echinosporin (106) which occurs in Streptomyces echino~porus.~' A full account is now available of the synthesis that was first reported in 1977 (by Tishler et al.) of (+)-cerulenin (107);92 this is the inhibitor of fatty acid synthesis that is produced by Cephalosporium caerulens.This route (Scheme 20) is based on construction of NATURAL PRODUCT REPORTS 1985 b; R1= ,R2=H c; R1= ph> \\ ,,NHCO -Co," AcNHTq (101) (102) a ; R = Me CO,H b;R=H NHCOPh Ho\J-qoH H:if:Hz ++ H OH OAoYOH H OH OH J OH OH OH J NHCOPh PhCOO OCOPh n \0 LOTs I [CH&C$HZ]SM~ + i-iii H0zC.f;;~ HO H (105) Reagents i Bu'Li CuBr.SMez; ii MeI Me,CO (aq.); iii NaOH (aq.) Scheme 19 NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES CONH2 I OBoH z (11 1) (106) RH&o H (108) v,vi J [R= R ’ 13 (107) Reagents i LiC=CCO,Me; ii H, Lindlar catalyst; iii NaOCl pyridine; iv 65°C; v NH40H (as.); vi Cr03.2pyridine Scheme 20 A r [Me] methionine Me0 CON H2 OH (109) li N-N=NOH Rw (113) a; R = Me [CH212CHLCHCH2 b; R = Me [CH213CH=CH E OH (114) the key butenolide (108) from a C9 aldehyde and a C3 propiolate fragment and its subsequent epoxidation.Incorporation studies in Penici//ium expansum with 3C-labelled precursors have shown that the mycotoxin viridica- tumtoxin (109) has the labelling pattern shown implying a mixed polyacetate-terpenoid bi~genesis;~~ the origin of C-3 is not yet clear. The hydroxamic acid antifungal compound trichostatin A (1 10) (from Streptomyces hygroscopicus) has been the subject of total synthesis; of the two routes that have been reported by the same authors the better uses the reaction of a silyl enol ether with an acetal in the presence of a Lewis acid to establish the side-chain (Scheme 21).94 Further biosynthetic experiments on the isonitrile acid (1 1 1) with various 4C-labelled tyrosines in the fungus Trichoderma hamatum have provided support for an earlier proposal that C-3 of tyrosine is lost and that the side-chain of (1 1 1) derives from the aromatic ring with C-4 C-5 and C-6 of tyrosine providing C-1 C-2 and C-3 respectively of the isonitrile acid.95 Another nitrogenous metabolite that has an unusual functional group and which exhibits interesting activity against tubercle bacteria is the azoxy-compound elaiomycin (1 12); it has been shown to incorporate octylamine intact (into the ‘left-hand’ portion as drawn here) in Streptomyces gelaticus and the subsequent dehydrogenation to give the cis double-bond between C-5 and C-6 has been found to involve syn removal of OMe hydrogen atoms.96 Full details of the discovery (in Streptomyces I aureofaciens),characterization and structure determination of the vasodilators WS-1228A (1 13a) and WS-1228B (1 13b) have appeared and syntheses of these unique natural triazenes have Me2NmcHo also been performed.97 ii-iv ..5 Miscellaneous Olef inic Microbial Meta-0 bolites The format of this section which covers metabolites that do not fall easily into one of the previous sections follows that which was used in corresponding earlier Reports in the appropriate series of Specialist Periodical Reports. Compounds with an (1 10) acyclic ‘olefinic’ portion are reviewed before cyclopentene- and Reagents i TiCl,; ii Ph,PCHCO,Et; iii 2,3-dichloro-5,6-dicyano-cyclohexene-based structures.A newly reported metabolite that has been isolated from benzoquinone; iv NH20H Streptomyces jimbriatus and which is devoid of anti biotic Scheme 21 activity is the simple dihydroxy-ketone (1 14).98 The two 416 NATURAL PRODUCT REPORTS 1985 E (115) a ; R = H02CCH=CHCH2 (117) a; R’ = R2= H b ; R = HO~CCH~CH(OAC)CH~ (116) a; R = Me b; R = Et b ; R1 = CI R2= OMe c02Me 1 Ph”I/’C02R Q& [CH21 Me 0 (118) a; R = Me (119) (120) a; R’= R~= H (121) b;R = Et b; R1= OMe R2= CI c ; R’ = OCHzCH-CMe2 R2= H d;R’=H,R2=OMe OH Me (124) a; R = H Me ,:,.”+&$ (122) (125) (123) 0 Me (126) pigments (1 15a) and (1 15b) have been isolated from the fungus Piptoporus au~traliensis.~~ Ebelactone A (1 16a) and ebelactone B (116b) are two esterase inhibitors that have recently been identified from Streptomyces MG7-G 1 which is closely related to Streptomyces aburaviensis;’ OoO incorporation experiments with 3C-labelled precursors have shown that these p-lactones are derived biosynthetically from one acetate and either six propionate units (ebelactone A) or five propionate units and one butyrate unit (ebelactone B).toob The structure and relative configuration of oudemansin (1 17a) which is a metabolite of Oudemansiella mucida that has strong antifungal properties are known from spectroscopic and X-ray studies. A recently published total synthesis of (+)-oudemansin has used the stereoselective reduction of the p-keto-ester (1 18a) (itself available from the Reformatsky reaction of methyl 2-bromopropanoate and 3-phenylpropanal followed by oxidation) to set up the required C-9 C-10-erythro relationship.O In a subsequent publication from the same research group the keto-ester (1 18b) was reduced by the yeast Candida albicans to provide an optically active P-hydroxy-ester that was converted into natural ( -)-oudemansin (1 17a) thus defining the absolute configuration to be 9S,lOS,as drawn. lo2 The (&)-erythro-ester (1 19) which is an intermediate in the above route to (&)-(117a) has also been prepared but by a different sequence based on the erythro-selective [2,3]Wittig rearrangement of a crotyl propargyl ether.O3 Oudemansin OH 0 (128) should in fact be termed oudemansin A now that a new antifungal metabolite (1 17b) has been identified and designat- ed oudemansin B from Xerula melanotricha. lo4 This organism also produces the previously known metabolites strobilurin A (120a) and strobilurin B (120b); to this group must now be added strobilurin C (120c) newly isolated (with strobilurin B) from Xerula longipes,lo4 and a new metabolite (120d) of Oudemansiella mucida. O5 Syntheses of the antibiotics aurocitrin (121) (a pigment of Hypocrea citrina) and frustulosin (1 22) (from Stereum frustulo- sum) have been completed by elaboration from 3,6-dihydroxy- 2-iodoben~aldehyde.’~~ The enyne moiety of (122) is also seen in oxirapentyn newly isolated from the fungus Beauveria felina ; the structure and relative configuration were determined as (1 23) from spectroscopic and X-ray crystallographic evi-dence.A new synthesis has appeared of 5,7-dihydroxy-4- methylphthalide (124a),l O8 which is a key intermediate in the synthesis of mycophenolic acid (125). The biosynthesis of mycophenolic acid in Penicillium brevicompactum is known to involve oxidative degradation of the side-chain of the farnesylphthalide (124b) by at least two pathways. New incorporation and isotopic-trap experiments suggest that the alternatives i.e. either direct oxidation of the central carbon- carbon double-bond or a two-stage cleavage of the terminal group and then the central group are equally important ;lo9 this contrasts with an earlier finding.The data also suggest a NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES 0 (129) (130) Reagents i Bu'Li THF-HMPA at -78°C; ii N-chlorosuccinimide (2 equivalents); iii H,O+; iv 1,8-diazabicyclo[5.4.O]undec-7-ene; v EtMgBr; vi HC(OEt),; vii HClO (aq.) Et,O viii AcOH (aq.) Scheme 22 (132) sequence involving epoxidation of a double-bond rearrange- ment of the epoxide to a ketone and hydroxylation to form the a-hydroxy-ketone leading to cleavage of the carbon-carbon double-bonds. O9 The 2',3'-dihydro-derivative of sorbicillin (1 26) has been isolated as a new metabolite of Verticiflium intertextum along with the known compounds sorbicillin itself and bisvertoquinol (127) for which the X-ray structure is reported;"O this latter compound appears to be a Diels-Alder adduct of the quinols (128) which are related to sorbicillin and its dihydro-derivative but which as yet are not known as natural products.Syntheses have been reported of two arylated tri-isoprenoid- derived chlorinated metabolites of Ascochyta viciae namely ascochlorin (129),' which is an antiviral antibiotic and ascofuranone (1 30),' I which is an antibiotic that has hypolipidemic and antitumour properties. Both are based on lithiation and alkylation of the cyclohexadiene (1 31) as illustrated in Scheme 22. It is known that ring A of flexirubin (1 32) (the main pigment of the gliding bacterium Flexibacter elegans) is derived from tyrosine which acts as a C,C starter unit for a polyketide chain; methionine supplies the methyl substituent on ring A.Acetate provides the rest of the polyene and also ring B and its side-chain. New experiments on the origin of ring B have confirmed that all of the carbon atoms are acetate-derived that orsellinic acid (1 33a) and 3-dodecylorsellinic acid (1 33b) are intermediates and that decarboxylation occurs before an ester is formed between an alcohol that includes ring B as its aryl group and the o-arylpolyenoic acid that includes ring A as its aryl group. 5.1 Methylenecyclopentanone Metabolites Once again there has been a deluge of publications dealing with synthetic efforts in this field. There have for example been no less than seven reports of routes to sarkomycin (134) which is the antitumour agent from Streptomyces erythrochromo-genes.' l4-I 2o Three approaches that are based on conjugate (133) a ; R = H b; R = n-C12H25 addition to a 2-substituted cyclopentenone or derivative are outlined in Scheme 23.' I4-l The bicyclic lactone (135) of the route that has been described by A.B.Smith et ~1.l'~ has also been prepared by an interesting reaction between the ethyne- hexacarbonyldicobalt complex carbon monoxide and 2,5- dihydrofuran (see also below). ' Another distinctive new approach to the ethyl ester of sar.komycin employs the intramolecular 1,3-dipolar cycloaddition of a nitrile oxide to an alkene to close the cyclopentane ring (Scheme 24).'18 Other reports include a route from a-tropolone to the methyl ester of sarkomycin' I and two topologically distinct syntheses of (1 34) that use intramolecular insertion reactions of carbenes (derived from a-diazo-ketones) to bring about ring-closure.2o The organocobalt-mediated approach to 3-oxabicyclo-[3.3.0]oct-7-en-6-ones that is mentioned above [for lactone (1 35)] has also been applied to the construction of the bicyclic lactone (136) which is a known intermediate en route to methylenomycin A (1 37) (Scheme 25).' l7 The lithiated thioenol ether (138) which has previously been used in a synthesis of methylenomycin A has recently been employed in a new approach to intermediates (1 39) and (140) (Scheme 26) both of which have featured in earlier syntheses of the unstable metabolite desepoxy-4,5-didehydromethylenomycin A (141);l the lithio-derivative (1 38) has also been converted into methylenomycin B (142) (also Scheme 26).lZ2 The anthracene-methyl acrylate Diels-Alder adduct (143) has been elaborated to the spiro-cyclopentenone (144) and the a-methylene functionality was unmasked by vacuum pyrolysis to provide a novel access to methylenomycin B (142);12 two further short routes to metabolite (142) are outlined in Scheme 27.124 Finally in this group A.B.Smith et al. have published in full their synthetic endeavours that led to ( f)-xanthocidin (145) which is a metabolite of a species of the genus Strepto-myces to two of its diastereoisomers and to (i-)-desdihydroxy- 4,5-d ide hyd roxan thocid in [( f)-4,5-d ide hyd ro-4,5-d ideoxy- xanthocidin] which is a possible biogenetic precursor.25 The route to xanthocidin is shown in Scheme 28 and the other targets were also made from the key intermediate bicyclic enone (146). 418 NATURAL PRODUCT REPORTS 1985 0 i-v -% 0 (135 1 n COzMe vii -ix x ,vi ____) '+OH ____) "CN '\ COzH xi -xiii C02Me -O& (Thp = tetrahydropyran -2 -yl 1 Reagents i LiCu(CH=CH2)? at -78°C; ii O3;iii Me,S; iv 1,8-diazabicyclo[5.4.O]undec-7-ene;v pyridinium chlorochromate; vi H30+ ;vii Et,AICN; viii HO[CH2],0H H+; ix LiBH4 NaOH (aq.); x NaOH (as.); xi MeN02 Me,NC(NMe,)=NH; xii AlH,; xiii dihydropyran H+; xiv alkaline KMnO,; xv HCI (aq.) Scheme 23 Reagents i LiNPr'? Br[CH,I3Br; ii NaI Me,CO; iii AgN02; iv 4-C1C6H,NCO Et3N; v Raneynickel H2,AcOH or B(OH),; vi MsCl Et3N at 0°C Scheme 24 (136) Reagents i Co,(CO),; ii for 8 days at 85°C; iii HzOz,OH-; iv RuO Scheme 25 5.2 Cyclopentene Metabolites A group of microbial metabolites having some structural similarities to the methylenecyclopentanones that were dis- cussed above and which are found variously in Streptomyces eurythermus Streptomyces lavenduligriseus Streptomyces catt- leya and Streptoverticillium eurocidicum are the pentenomy- cins and full details of the syntheses of (-t)-pentenomycins I (147a) I1 (147b) and 111(147c) and of dehydropentenomycin I (148) have been disclosed again by A.B. Smith et al. (Scheme 29). 26 A key intermediate is the 2-hydroxymethylcyclopenten-one (149) which has been prepared by two routes from cyclopent-2-enone; the variant that is suitable for large scale is illustrated.In a further synthesis of (*)-pentenomycin I (147a) the substituents are elaborated whilst the double-bond is masked as a Diels-Alder adduct with cyclopentadiene (Scheme 30). 17 Stoodley et al. have reported along with an approach to the racemic material the preparation of natural (-)-penteno-mycin I (147a) from (-)-~,-quinic acid (Scheme 31).'2s A new synthesis of cryptosporiopsin (1 50a) has been detailed,' 29 and biosynthetic experiments on the co-metabolite cryptosporiopsinol (1 50b) in the fungus Periconia macrospinosa suggest that the chlorinated isocoumarin (1 5 1) is an advanced precursor. 30 5.3 Compactin and Mevinolin Work in the area of cyclohexene metabolites is dominated by the attention that has been given to the hypocholesterolemic NATURAL PRODUCT REPORTS 1985 -R.C. F. JONES COzMe ___) OSiMezBut PhS PhS PhS (1 38) ii iii vii ,viii ii .1 x +OzEt CH2SPh *Sph PhS PhS 0siMe,Bu *o H 0 iv,vI (140)I 0 -$Y‘CO 2 H (139) (141) Reagents 1 Bu‘Me,SiOCH,CH=CHCO,Me; ii NaCl DMSO (aq.); iii LiN(SiMe3), PhSCHJ at -76 to -40°C; iv Me,CuLi; v HF MeCN (aq.); vi HBr AcOH; vii MeLi; viii CF3CO2H PhH; ix H,C=C(CH,SPh)CO,Et at -80°C; x 4-C1C,H,CO3H (2 equivalents) at 0°C; xi NaOH DMSO Scheme 26 (143) (144) 0 0 /. VIII,III. vi ,vii Me02no, -0 Reagents i (EtCO),O pyridine; ii MeLi at -78”C THF-toluene (1 :3); iii OH- (as.); iv H+; v Pb(OAc), Cu(OAc), pyridine; vi (PhC02)2 EtCHO heat; vii NaH HCrCCHzBr or H2CrCHCH2Br; viii Hg” H,O or Wacker oxidation respectively Scheme 27 OH ___) HO HO “COz H 0 H HOI\ (146) (145) Reagents i hv PhzCO sensitizer; ii LiAlH,; iii TsCl(1 equivalent) pyridine at 0°C; iv heat; v 03,MeOH; vi PPh,; vii NaOH MeOH (aq.); viii Cr03 AcOH Ac20 then separation of regioisomers; ix; OsO, pyridine; x NaHS03 (aq.); xi MeCOC1 Me,Si[CH,],OH; xii Na,CO (as.) Scheme 28 NATURAL PRODUCT REPORTS 1985 0 (148) i -v vi -viii b (149) (147) a ; R' = R2= H b; R'=H,R2= AC c ; R' = AC R2= H Reagents i Br, CCI,; ii base; iii HO[CH2I20H TsOH; iv BuLi at -78"C H2C0 (g.); v H30+; vi ButMe2SiC1 imidazole; vii OsO,; viii NaHSO (aq.); ix Se0,; x AcOH THF (aq.); xi Ac,O pyridine; xii CrO, H2S04 Scheme 29 i-iv -% bOAc + (1470) xi xii &OM= OM OH 0 HO Reagents i e- MeOH NHf Br-; ii H2S04 (catalytic); iii cyclopentadiene; iv base; v H202 OH-; vi HBr MeOH; vii Ac20 Et3N 4-(dimethy1amino)pyridine; viii BBr, at -78 to + 20°C; ix Ac20 4-(dimethylamino)pyridine;x AgOAc AcOH; xi H+ MeOH; xii flash vacuum pyrolysis at 525°C and 0.04mmHg Scheme 30 n i-v vi-ix + $p'"" dCH2Ph OH Reagents i Pb(OAc),; ii piperidinium acetate; iii LiAIH,; iv NaH PhCH,Br; v CuO CuCl, Me,CO (aq.); vi OsO, pyridine; vii Na2S205 (as.); viii H, Pd; ix HC1 (aq.) Scheme 31 agents compactin (1 52a) (from Penicillium breuicompuctum and Penicillium citrinum) and mevinolin (1 52b) (from Monuscus ruber and Aspergillus terreus) and especially to their synthesis.Interest in hydroxylated derivatives of compactin and mevinolin prompted by the increased biological activity (as an inhibitor of the biosynthesis of cholesterol) that is shown by a minor co-metabolite that carries a hydroxy-group at C-3 has (151) led to the discovery of some micro-organisms that can selectively convert compactin (152a) either into a 30- or into a 3cl-hydroxy-derivative and that can perform various other selective transformations of (1 52a) or (1 52b). A full assignment of the 3C n.m.r. spectrum of mevinolin (1 52b) has been completed using double-quantum coherence n.m.r. after 0 incorporation of [1,2-13C2]acetate into (152b) in A.terreus;13* this in turn has permitted biosynthetic studies on (1 52b) using 3C-labelled precursors and in particular by means of a pulse- feeding technique that leads to multiple incorporation of acetate units and which allows observation of 13C-13C coupling in adjacent biosynthetic units to establish a complete R-& connectivity pattern. 32 The results are indicated in (153) and provide support for a biogenesis from a partially reduced CI8 polyketide (with the 2-methyl group as starter) and either (152) a; R = H intramolecular Diels-Alder reaction or intramolecular anionic b;R=Me condensations to generate the hexahydronaphthalene system. NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES 421 A [Me] methionine {OYOEt PhCHz? +__) PhCHzO PhCHzO 1 Scheme 32 H? H Me? H OMe H -_j w Br HO IH 0 /OS iMe2 But 0 AcO'HO d Ht) Scheme 33 Scheme 34 422 NATURAL PRODUCT REPORTS 1985 (R = SiMe2Bu') Reagents i EtAICl, at room temperature for 72 hours; ii LiAIH,; iii HF MeCN; iv Bu'Me,SiCl imidazole; v Br,; vi 1,8-diaza- bicycle[5.4.0lundec-7-ene Scheme 35 Jc.,viii OR2 0 /OH (156)a; R1 = H ,R2=CHMeOEt b; R' = Me R2=Thp pi,viii OH 0 (Thp = tetrahydropyran -2-yl ) Reagents i MeMgCl (2 equivalents) MeCH=CHCH,Br CuCl (catalytic); ii (COCl), DMSO Et,N ;iii BrMgC=C[CH,],OCH(Me)OEt; iv NaOMe-LiAlH (2 1); v KOBu' Bu'OH; vi pyridinium chlorochromate NaOAc; vii heat at 150"C PhH; viii L-Selectride@; ix BuLi (2.5 equivalents) at -30 to + 25 "C oxirane then MeCH=CHCH,Br CuCl (catalytic); x LiC-C[CH,],OThp (Thp = tetrahydropyran-2-yl); xi (S)-2-methylbutyric anhydride pyridine 4-(dimethy1amino)pyridine; xii EtOH pyridinium tosylate then separation by h.p.1.c.Scheme 36 0 x ,xi f-(157) Reagents i MeCH=CHCO,Et at 145°C; ii LiAlH4 then H,O+; iii Bu'Me,SiCI Et,N 4-(dimethy1amino)pyridine; iv (157) THF-HMPT at -78°C; v HCl MeOH (aq.); vi 2,4,6-tri-isopropylsulphonylhydrazine,MeQH HCI; vii BuLi (4 equivalents) TMEDA; viii N-chlorosuccinimide AgN03 collidine; ix LiBHBu" ; x (S)-2-methylbutyric anhydride Et,N 4-(dimethy1amino)pyridine; xi Bu,N+ F-Scheme 37 NATURAL PRODUCT REPORTS 1985 -R. C. F. JONES 423 Reagents i Br on Ag+ salt; ii 1,5-diazabicycl0[4.3.O]non-5-ene; iii LiAlH,; iv Mn02 ;v Me3SiC1 Et,N 4-(dimethylamino)pyridine; vi LiNPr' H2C=CH[CH,I2CHO at -78°C; vii 03,at -78°C; viii PPh,; ix TiC13 Zn/Cu couple DME then separation of epimers Scheme 38 (1581 1ii OR C02Me I fOCHIPh iii-viii t--- +& @ RO RO ix-xi ix xii-xiv,iii,v,iv,xv,vi,vii 1 I (152a) ix-xi,ix (152b) ( R = SiMe2But 1 Reagents i NaH at 0°C; ii chlorobenzene at reflux ;iii K-Selectride@; iv (S)-2-methylbutyric anhydride pyridine 4-(dimethylamino)pyridine; v Li liquid NH, at -78°C; vi Cr03*2pyridine; vii pyridinium dichromate DMF; viii CH2N2; ix HF MeCN (as.); x Bu'Me2SiC1 imidazole; xi SOCI pyridine; xii LiNPr', Me3SiC1 Et3N at -78 to +20"C; xiii Pd(OAc), p-benzoquinone; xiv LiCuMe,; xv NaOH EtOH (as.) Scheme 39 Full details are now available of the chiral synthesis of natural (+)-compactin that was first reported by Sih et a/.in 1981 and some of the key intermediates in their approach are illustrated in Scheme 32. 33 The butadiene-benzoquinone adduct is the starting point for a new route that has been reported by Girotra and Wendler and which is summarized in Scheme 33;134 this sequence and the route of Sih et al. (mentioned above) converge on the key alcohol (1 54) and they subsequently use similar steps to elaborate the lactone moiety. A second route from the intermediate (159 which is available (in six steps) from the butadiene-benzoquinone adduct to the alcohol (1 54) has subsequently been published by Girotra and Wendler (Scheme 34).' 35 Several groups have reported syntheses of hexahydronaph-thalene units that are related to the alcohol (154) and which could each potentially be converted into compactin (152a) by elaboration of the lactone unit.Funk and Zeller have explored the intramolecular Diels-Alder cycloaddition of 4-oxygenated NATURAL PRODUCT REPORTS 1985 "eovoMe I -v x -xiii +$ C0,Me ___) ___) (152a) c02Me SPh (161) (160) Reagents i 125"C in toluene; ii 4-ClC,H4C03H; iii P(OMe),; iv EtO,CN=NCO,Et PPh3 PhC0,H; v NaOMe MeOH; vi Ac,O Et,N 4- (dimethy1amino)pyridine;vii LiCuMe?;viii LiAlH,; ix KH heat; x (S)-2-methylbutyric anhydride Et3N 4-(dimethylamino)pyridine; xi HCl THF (as.); xii Ag2C03 PhH; xiii BBr3 at -23°C Scheme 40 ButP h2Si 0 cH20cb""v'OMe Rovo"e R i HOi PhCH2O (162) R = I or S(O) Ph (n=l or 2) (163) R (165) a ; R = 0 b; R = P-OH,H dodeca-2,8,1O-trienoatesas illustrated in Scheme 35,' 36 where-as Deutsch and Snider in a novel approach have employed a vinylallene as the dienophile in an intramolecular [4+21 cycloaddition to incorporate the required 'extra' double-bond directly into the hexahydronaphthalene (1 56a) (Scheme 36).37 The latter scheme has also been extended into the mevinolin (1 52b) series to provide the hexahydronaphthalene alcohol (156b) (also Scheme 36) that has been resolved as its (S)-2- methylbutyrate ester as illustrated. 38 Two further approaches to appropriate hexahydronaphthalenes based on different annulations of cyclohexenones are due to the groups of Heathcock (Scheme 37)139 and Clive (Scheme 38).I4O Next in this section are two distinctive convergent ap- proaches to natural (+)-compactin (152a) that include the carbon atoms of the lactone moiety at an early stage in contrast to the strategy of late elaboration [from (154) or a related intermediate] that is employed in all of the routes discussed above.From two optically active fragments Hirama and co- workers have constructed the chiral trienone (158) (which contains all of the carbon atoms that are required for the framework of compactin) as a substrate for intramolecular cycloaddition (Scheme 39); modification of the cyclo-adduct (159) then led either to (+)-compactin (152a)141 or (+)-mevinolin (1 52b),142as shown. In the synthesis due to Grieco et af.(Scheme 40) the key step is intermolecular Diels-Alder reaction of the diene (160) which had been elaborated from the commercially available tri-0-acetyl-D-glucal with the chiral dienophile (161) itself available from furan by a short sequence that includes an optical resolution. 43 The second double-bond that is needed in the hexahydronaphthalene portion is created by a Grob-type fragmentation. (164) R = H or Ac 4P CL (166) Finally the preparations have been reported of several candidates as precursors to the lactone moiety of compactin or mevinolin including (162)144 and (163)145 from carbohydrate sources and (164) from a hetero-Diels-Alder cycloaddition of the Danishefsky diene to an aldehyde.146 5.4 Other Cyclohexene Metabolites New syntheses have appeared of methyl trisporate B (165a)14' and the reduced derivative (165b),I4* which are both fungal prohormones.This latter work confirms the structure and stereochemistry of the reduced compound. 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Chem. 1983 48 3581. 121 Y. Takahashi H. Kosugi and H. Uda Chem. Lett. 1982 815. 122 Y.Takahashi H. Kosugi and U. Uda J. Chem. SOC.,Chem. Commun. 1982 496. 123 T. Siwapinyoyos and Y. Thebtaranonth J. Org. Chem. 1982,47 598. 124 G. M. Strunz and G. S. Lal Can. J. Chem. 1982,60 2528; T.-L. Ho Synth. Commun. 1983 13 435. 125 A. B. Smith 111 and D. Boschelli J. Org. Chem. 1983,48 1217. 126 A. N. Smith 111 S. J. Branca N. N. Pilla and M. A. Guaciaro J. Org. Chem. 1982 47 1855. 127 J. M. J. Verlaak A. J. H. Klunder and B. Zwanenburg Tetrahedron Lett.1982 23 5463. 128 J. D. Elliott M. Hetmanski M. N. Palfreyman N. Purcell and . R. J. Stoodley Tetrahedron Lett. 1983 24 965. 129 G. B. Henderson and R. A. Hill J. Chem. Soc. Perkin Trans. I 1983 2595. 130 G. B. Henderson and R. A. Hill J. Chem. Soc. Perkin Trans. I 1982 3037. 131 N. Serizawa K. Nakagawa K. Hamano Y. Tsujita A. Terahara and H. Kuwano J. Antibiot. 1983 36 604; N. Serizawa K. Nakagawa Y. Tsujita A. Terahara and H. Kuwano ibid. p. 608; N. Serizawa S. Serizawa K. Nakagawa K. Furuya T. Okazaki and A. Terahara ibid. p. 887; N. Serizawa K. Nakagawa Y. Tsujita A. Terahara H. Kuwano and M. Tanaka ibid. p. 918. 132 J. K. Chan R. N. Moore T. T. Nakashima and J. C. Vederas J. Am. Chem. SOC.,1983 105 3334. 133 C.-T. Hsu N.-Y.Wang L. H. Latimer and C. J. Sih J. Am. Chem. SOC.,1983 105 593. 134 N. N. Girotra and N. L. Wendler Tetrahedron Lett. 1982 23 5501. 135 N. N. Girotra and N. L. Wendler Tetrahedron Lett. 1983 24 3687. 136 R. L. Funk and W. E. Zeller J. Org. Chem. 1982 47 180. 137 E. A. Deutsch and B. B. Snider J. Org. Chem. 1982 47 2682. 138 E. A. Deutsch and B. B. Snider Tetrahedron Lett. 1983 24 3701. 139 C. H. Heathcock M. J. Taschner T. Rosen J. A. Thomas C. R. Hadley and G. Popjak Tetrahedron Lett. 1982 23 4747. 140 P. C. Anderson D. L. J. Clive and C. F. Evans Tetrahedron Lett. 1983 24 1373. 141 M. Hirama and M. Uei J. Am. Chem. Soc. 1982 104 4251. 142 M. Hirama and M. Iwashita Tetrahedron Lett. 1983 24 181 1. 143 P. A. Grieco R. E. Zelle R. Lis and J.Finn J.Am. Chem. Soc. 1983 105 1403. 144 J. D. Prugh and A. A. Deana Tetrahedron Lett. 1982 23 281. 145 Y.-L. Yang and J. R. Falck Tetrahedron Lett. 1982 23 4305. 146 S. Danishefsky J. F. Kerwin and S. Kobayashi J. Am. Chem. Soc. 1982 104 358. 147 B. M. Trost and P. L. Ornstein Tetrahedron Lett. 1983,24 2833. 148 K. Takabe and J. D. White Tetrahedron Lett. 1983 24 3709. 149 E. R. Koft and A. B. Smith 111 J. Am. Chem. Soc. 1982 104 2659.

ISSN:0265-0568    

DOI:10.1039/NP9850200401

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

Phytoalexins C. J. W. Brooks and D. G. Watson Chemistry Department University of Glasgow Glasgow Scotland G I2 800 Reviewing the literature published between January 1982 and July 1984 1 Introduction 2 Terpenoid Phytoalexins 2.1 Sweet Potato (Ipomoea batatas) 2.2 Cotton (Gossypium spp.) 2.3 Elm (Ulrnus glabra) and Lime (Tilia x europaea) 2.4 Tobacco (Nicotiana spp.) 2.5 Sweet Pepper (Capsicum annuum) 2.6 Potato (Solanurn tuberosum) 2.7 Aubergine (Solanurn melongena) 2.8 Solanurn aviculare 2.9 Coffee (Cofea arabica) 2.10 Rice (Oryza satiua) 3 Phytoalexins of Miscellaneous Structural Types 3.1 Linoleic Acid Derivatives 3.2 Acetylenes and Polyacetylenes 3.3 Bibenzyls 3.4 Stilbenes 3.5 P henan t hrenes and Di hydrop henan threnes 3.6 Benzofurans and Phenylbenzofurans 3.7 Furocoumarins 3.8 Avenalumins 3.9 Flavans 3.10 Phenylbenzofurans from Mulberry and Paper Mulberry 3.1 1 Benzoxazinones 3.12 Alkaloids 4 Isoflavonoid Phytoalexins 4.1 Introduction 4.2 Biosynthesis Enzymic Aspects 4.3 Biosynthesis Mechanistic Aspects 4.3.1The Phytoalexins of Pea (Pisum satiuum) 4.3.2The Phytoalexins of Soybean (Glycine max) 4.3.3The Phytoalexins of Phaseolus vulgaris 4.3.4The P hytoalexins of Red Clover (Trifoliumpratense) 4.4 The Total Synthesis of (t-)-Phaseollin 4.5 Newly Isolated Isoflavonoid Phytoalexins 4.6 The Analysis of Phytoalexins 4.7 Fungal Degradation of Phytoalexins 4.7.1 Studies on the Degradation of Phytoalexins by Fungi 4.7.2The Tolerance of Fungi towards Phytoalexins 4.7.3The Association between Degradation of Phytoalexins and Virulence of Pathogens 5 Elicitors of Accumulation of Phytoalexins 5.1 Introduction 5.2 Modes of Action of Elicitors 5.3 Biotic Elicitors 5.3.1 P-Glucans 5.3.2 Glucomannans 5.3.3 Chitosan and Oligomers of Glucosamine 5.3.4Glycoproteins 5.3.5 Cell-wall-degrading Enzymes from the Parasite 5.3.6Fungal Phytotoxins 5.4 The Influence of Growth Hormones on the Phytoalexin Response 5.4.1Ethylene 5.4.2Abscisic Acid and Cytokinins 5.5 Endogenous Elicitors 5.5.1 Release by Enzyme Action 5.5.2 Release by Autoclaving or by Acid Hydrolysis 5.5.3 The Characterization of Endogenous Elicitors 5.6 Abiotic Elicitors 5.6.1The Mechanism of Action of Abiotic Elicitors 5.6.2Sulphydryl Reagents 5.6.3The Release of Endogenous Elicitors by Abiotic Elicitors 5.7 Fungicides as Abiotic Elicitors 5.8 The Analysis and Characterization of Carbohydrate Elicitors of the Accumulation of Phytoalexins 5.9 The Synthesis of a Hepta-P-glucoside Elicitor 6 References 1 Introduction The term ‘phytoalexin’ was coined by Muller and Borgerl to denote defensive substances (then chemically undefined) that are produced by plants in response to an infecting organism.Muller studied the protection of susceptible cultivars of potato (Solanurn tuberosum) against infection with a virulent race of Phytophthora infestans (potato late blight) that is afforded by pre-inoculation of tuber tissue with a race of P.infestans to which the potato cultivars are resistant. This work1,* estab- lished the following typical features of the phytoalexin response (cf. refs. 3 and 4):(i) the inhibition of the development of the fungus on tubers of resistant varieties is effected by an active principle that is formed only by the host-parasite interaction; (ii) the tissue layers that are infected by the fungus suffer necrosis during which phytoalexins are produced which have fungitoxic properties; (iii) the defence reaction is limited to the tissue that has been invaded by the fungus and to neighbouring tissues ;(iv) resistant and susceptible hosts show similar basic responses but differ in the speed of formation of the phytoalexin; (v) the sensitivity of the host cell that determines the speed of reaction of the host to the challenge by the infecting organism is specific and genotypically determined.Biological and chemical studies of phytoalexins have continued to focus largely on plants that are of economic importance especially members of the Solanaceae and Legu- minosae. The production of phytoalexins by plants and also by plant tissue cultures can be stimulated not only by elicitorss that are of fungal origin but also by other natural organic materials (biotic elicitors) by a variety of abiotic elicitors such as inorganic compounds,6 or in some instances by wounding or cutting of plant tissue.These topics are discussed further in Section 5. The first phytoalexin to be identified was (+)-pisatin [(6aR 11aR)-6a-hydroxy-3-methoxy-8,9-methylenedioxypterocarpan (l)],’from seed pods of peas (Pisumsatiuum) that were infected with Monilinia fructicola. More than 200 phytoalexins are now known and certain chemical types tend to be associated with particular plant families. In this Report work on phytoalexins is classified principally on the basis of the plants studied but (1 1 NATURAL PRODUCT REPORTS 1985 the underlying chemical pattern is apparent. A general term for compounds that are formed by the plant in response to elicitory agents is 'stress metabolites' not all of which have been found to have antifungal activity in some cases it has been deemed of interest to include reference to these as well as to proven phytoalexins.It should also be mentioned that certain compounds have been encountered both as phytoalexins and as apparently normal antimicrobial constituents ('prohibitins') of plants. Excellent recent surveys of phytoalexins have appeared in a book8 and in re~iews.~*~~'O Other useful reviews cover stress metabolites of edible plants' and the role of natural products in plant-insect and plant-fungus interactions. Other books13-'5 are noted here to facilitate the citation of relevant chapters. 2 Terpenoid Phytoalexins These are reported under headings that indicate the plant species concerned.2.1 Sweet Potato (Ipomoea batatas) Rapid progress has been made in the isolation of intermediates in the biosynthesis of ipomeamarone (8) which is the most abundant phytoalexin of the sweet potato (Ipomoeu batatus Lam.). Also various side-products from the main biosynthe- tic pathway have been isolated; the biological properties of many of these have yet to be studied. Scheme 1 has been proposed to account for the numerous minor stress metabolites that are derived from farnesol (2). The following stress metabolites of the furanoterpenoid and related types had been isolated (up to 1981) from slices of the root of sweet potato that had been treated either with 0.1% HgClz or with spores of Cerutocystis jmbriutu:17~18 ipomea-marone (8) ipomeamaronol (9) 7,8-didehydroipomeamarone (7) 7-hydroxymyoporone (16) 4-hydroxymyoporone (24) 4- hydroxy-7,8-didehydromyoporone (23) 6-oxodendrolasin (5),I9v2O 6-hydroxydendrolasin (l0),l9 9-oxofarnesol (4),19 9-hydroxyfarnesol (3),' ipomeabisfuran (21),I9v2O 6-myoporol (1 l) 1-myoporol (1 4) myoporone (1 5) and 6-deoxy-6,7-p-selinenc (29) R = H 7 -Hydroxycostol (32 ) R = CHIOH 1 p -costol (30) R = CH,OH 7 -Hydroxycostal (33) R = CHO p-costal (31 ) R = CHO / I \ / r .1 / A \ 1 10 (21) R = H (22) R = OH I I I 1 q +OHI\ 0 Scheme 1 NATURAL PRODUCT REPORTS 1985 -C.J. W. BROOKS AND D. G. WATSON dihydroxymyoporone (1 2).Schneider et a/. isolated nine new stress metabolites these were 9-hydroxyfarnesoic acid (1 3) 6-oxodendrolasinolide (20) ipomeatetrahydrofuran (1 7) (42)-and (4E)-1,6-dioxoisodendrolasin[(18) and (19)] 10-hydroxy- ipomeabisfuran (22) ipomeamaronolide (27) 4-hydroxymyo- poronol (25) and 4-hydroxymyoporonol ketal (26). Carbon- 14-labelled 6-oxodendrolasin (5) and 9-hydroxyfar- nesol (3) were incorporated into ipomeamarone (8) in HgC12- treated tissue (3) was the more efficient precursor.19 In tissue that was infected with C.jimbriata [14C]-(3) was incorporated into 7,8-didehydroipomeamarone(7) ipomeamarone (8),and ipomeamaronol (9).,O [14C]Ipomeabisfuran (21) was also fed to tissue that was infected with C.jmbriata; it was incorporated into unidentified materials.2o [14C]7,8-Didehydroipomeamar-one (7) was incorporated into (8),(9) (28) and an unidentified component B,.20 The diketones (18) and (19) are possible intermediates in the biosynthesis of (8) since reduction would give the hydroxy-enone (6) the formation of which could be followed by a facile Michael addition.I8 However the incorporation of [2-2H2]mevalonate into (8) indicated that the proton at C-1 in (8) was labelled at the same time the isomerization of the 3(4) double-bond to the 4(5)-position [as in (6)] before cyclization was established.2 The individual enzymatic steps in the proposed biosynthesis of (8) have not been elucidated to a great extent.Ipomeamar- one 15-hydroxylase activity was found in the microsomal fraction from cut-injured tissue or from tissue that had been infected with C.jimbriata,and was much higher in the latter.22 The enzyme required oxygen and NADPH and its inhibition by carbon monoxide or cytochrome c suggested it to be a cytochrome-P-450-dependent mixed-function oxidase.Farne- sol dehydrogenase (which may be involved in the formation of the furan ring) was found to be present in both cut-injured tissue and tissue that had been infected with C.fimbriar~.~~ It had mol. wt 90000 consisted of two identical subunits and showed high substrate specificity. Incubation of HgC12-treated tissue with I8O2and with H2I80indicated that all three oxygen atoms in (8) are derived from atmospheric oxygen.24 The absence of the original oxygen atom of farnesol suggests that the formation of the furan ring might involve loss of the hydroxyl group of farnesol and oxidation of the proximal methyl group to an alcohol or aldehyde function.When root tissue of sweet potato was treated with HgCl, the maximum concentration of furanoterpenoids was observed ten hours after the HgCl had been added suggesting that degradation subsequently occurs when cycloheximide was administered nine hours after treatment of the tissue with HgCl, the decline in the concentration of furanoterpenoids after ten hours was arre~ted.’~ The tissue was found to metabolize T-labelled ipomeamarone initially to ipomeamar- onol(9) and then to unidentified water-soluble materials.26 The metabolism of (8) was inhibited by cycloheximide and also (ca.50%) by HgC1,. The finding of the butenolides (20) and (27)’ perhaps indicates a biochemical route for degradation of the furan ring and the structure (26) represents a further stage in one of the catabolic pathways of (8). Changes in levels of enzymes in response to elicitation up to the point of the formation of mevalonic acid have been previously revie~ed.~.~ Recent work has shown that hydroxy- met h ylglu taryl-Co A red uc tase activity which was apparently detected in the mitochondria1 fraction both from infected and from cut-damaged tissue of sweet potato was in fact hydroxymethylglutaryl-CoA lyase activity [E.C.4.1.3.4] which produced acetoacetic acid this co-chromatographed with mevalonic acid causing some initial c0nfusion.2~ The levels of mRNA in cut root tissue of sweet potato were enhancement of RNA polymerase activity whereas ethylene suppressed the activity.Wounding also increased the synthesis of bulk proteins and the formation of polysomes.28.29 These observations are consistent with the hypothesis that in response to wounding some of the enzymes that are involved in the formation of furanoterpenoids are synthesized but they do not function until HgC12 is applied.25 Tissue of sweet potato that is infected with C.Jimbriatahas been found to contain phytoalexins with the eudesmane skeleton. Two new sesquiterpenoids [7-hydroxycostol (32) and 7-hydroxycostal (33)] were isolated along with the known p-selinene (29) costol (30) and costal (31).7-Hydroxycostal was found to be more potent as an inhibitor of the germination of spores of C.jimbriata than ipomeamarone. The ketone (34) was detected in the medium after (33) had been incubated with C. jimbriata for several days and the action of (33) was postulated to involve the release of acrolein (Scheme 2).30 Conflicting evidence in the literature as to the absolute configuration of ( +)-ipomeamarone has been resolved (Scheme 3).3 The unequivocal determination of the configura- tion at C-1 as R was based on cleavage of the tetrahydrofuran ring of ipomeamarone (8) by LiNPr’ to give an enone alkoxide which was reduced in situ to isomeric diols. The corresponding diacetates (35) were converted into the homogeneous ketone (36) which proved to be antipodal to the ketone (37) that had been derived from (-)-(S)-malic acid.The (4s) configuration in (8) was established by synthesis of the lactone (42) [enantiomeric with ipomolactone (38) which was produced by oxidative ozonolysis of ipomeamarone] from ( -)-(A)-linalyl acetate (39). Epoxidation cleavage (using a periodate) and reduction (using sodium borohydride) gave the diol (40) the bis-ethoxyethyl ether of which underwent hydroboration oxidation to the aldehyde and Grignard reaction to afford the diastereoisomers (41). Jones oxidation afforded pure (42). 2.2 Cotton (Cossypium spp.) Several species of Gossypium are grown commercially to produce cotton. The principal phytoalexin that is produced by cotton plants in response to attack by Verticillium albo-atrum is hemigossypol(43).Several related cadinane-type sesquiterpen- oids also accumulate as stress metabolite^.^^ Treatment of the leaves of a resistant strain of cotton with the bacterium Xanthomonas campestris pv. malvacearum led to the accumu- lation of 2,7-dihydroxycadalene (44) and lacinilene C (45) at concentrations of up to 171 and 112 nmol per gram of fresh weight respectively. These compounds were inhibitory to the bacterium and were found to accumulate in those cells of the host that were closest to the pathogen.33 Compound (44) and its 7-methyl ether had been previously detected in the bracts of green and of field-dried A study of the spread of Verticillium dahliae through cotton tissue has shown that the fungus can grow only while it maintains an active distribution front that outruns the accumulation of terpenoid aldehydes.(33) \ found to increase rapidly after wounding reaching a maximum after 6 hours and declining rapidly by 12 hours. Chromatin- bound DNA-dependent RNA polymerase activity also in- creased reaching a maximum after 6 hours and decreasing gradually thereafter. Gibberellic acid 2,4-dichlorophenoxy- acetic acid (2,4-D) and cyclic AMP (CAMP) stimulated the Scheme 2 NATURAL PRODUCT REPORTS 1985 OAc iv v,viii viii ix -H02C (-) -(S)-Malic acid x -xi i ... HO RO (38) (42 1 Reagents i LiNPr’ (-78 to 0 “C) added to LiAIH,; ii Ac,O py; iii 03,H202; iv LiAIH,; v Me,CO,p-MeC,H,SO,H; vi Cr0,.2py; vii N-bromosuccinimide Ph3P; viii 2-methyl-2-lithio-l,3-dithian; ix N-chlorosuccinimide NaIO, H20; x rn-chloroperbenzoic acid; xi H5106;xii NaBH,; xiii EtOCH=CH, p-MeC,H,SO,H; xiv BzH6 HzOz; xv MezCHCH,MgBr; xvi KzCrz0, H2S0 Scheme 3 The permeation of infected tissue by terpenoid aldehydes precedes a physical walling-off of the infected vessels.In susceptible cotton the walling-off process is slowed down and the invasion by the pathogen is not contained.35 A histochemi-cal study of the distribution of terpenoid aldehydes in relation to an invasive mycelium of Verticillium dahliae indicated that inhibited mycelia became ‘coated’ with terpenoid aldehydes which accumulated largely in this area and in cells immediately adjacent to the infected vessels.36 2.3 Elm (Ulmus glabra) and Lime (Tilia x europaea) (-)-7-Hydroxycalamenene (46) was isolated from wood of Tilia x europaea that was infected with Ganoderma applanatum and was found to be inhibitory to this organism in ~itro.~’ Eight cadinane-type sesquiterpenoids were isolated from sapwood of Ulmus glabra that had been infected with any one of three pathogens including Ceratocystis ulmi (which is the causal agent of Dutch elm disease).Various levels of the compounds (46)-(53) were found in response to the three fungi.38 Different @ \/ R2N\ O/ R’H I (46) R = Me (48) (49) R’ = CHO R2 = H *(47)R = CHO (50) R’ =CHO R2 = OMe *absolute configuration (51) R’ = Me ,R2 = H not defined 0 0 yJyJ \/ \/ (52) (53) degrees of antifungal activity were found for these compounds with (-)-7-hydroxycalamenene having marked anti fungal properties against all three organisms.38 In sapwood of both Tilia x europaea and Ulmus glabra the compounds were isolated from a narrow band between infected and healthy tissue.All of the components have been previously observed in the heartwood of healthy NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON 43 1 2.4 Tobacco (Nicotiana spp.) Previous reviews have reported the occurrence of five stress metabolites from tobacco these are glutinosone (54) rishitin (59,solavetivone (56) phytuberin (57),and capsidiol (59). In recent work phytuberin and phytuberol(58) were isolated from tobacco leaves that had been treated with ethrel(2-chloroethyl- phosphonic acid).40 In addition to previously observed compounds 3-hydroxysolavetivone (60) and solanascone (61) were isolated from tobacco leaves that had been treated with tobacco rattle virus or with tobacco mosaic virus (TMV);41 the principal components in this case were solavetivone (1 30 pg per gram of dry weight) 3-hydroxysolavetivone (50 pg per gram of dry weight) and glutinosone (580 pg per gram of dry weight).A similar range of compounds was observed in a g.c.- m.s. analysis of an extract from tobacco leaves that had been inoculated with Pseudomonas l~chrymans.~~ In addition to the usual stress metabolites tobacco leaves that had been inoculated with TMV accumulated occidol(62) occidol acetate (63) occidol isomer 1 (64) and occidol isomer 2 (65).The amounts of (62) (63) (64) and (65) that accumulated were 28 5 5 and 1 pg per gram of fresh weight re~pectively.,~ Tobacco tissue cultures that had been treated with Phy-tophthora parasiticu were found to produce rishitin a compound that was designated as ‘epirishitin’ capsidiol and a vicinal diol that was provisionally assigned the capsidiol skeleton with hydroxyl groups at C-11 and C-12.44345 A similar diol was observed together with ‘epirishitin’ and other components in a g.c.-m.s. analysis of an extract from TMV-infected leaves of Nicotiuna rustic^.^^ Apparently the same diol was also isolated from TMV-infected leaves of Nicotiana debneyi.,’ Structure (66) has been recently verified for this diol [R. S. Burden et al.and D. G. Watson et al. Phytochemistry in press]. A review of the uses of tissue cultures in the study of host-pathogen interactions has been published with particular reference to tobacco cultures.4s Total syntheses of (5)-solavetivone and phytuberin (57) have been described in previous Reports.49 (54) (55) 2.5 Sweet Pepper (Capsicum annuurn) An antifungal mixture ‘capsicannol’ was found to accumulate in the flesh of immature capsicum fruits which had been punctured and inoculated with conidia of Glomerella cingu- luta.50,51 Capsicannol appears to be a complex mixture containing small amounts of two sesquiterpenols [1-deoxycap-sidiol (67) and capsidesmol (68)] that are closely related to capsidiol (59) which is the principal phytoalexin from ripe capsicum The alcohols (67) and (68) were found to occur at levels of ca 2 and 1 pg per gram of fresh weight respectively in fruits that had been treated with cellulase pectinase or 0.~M-CUSO,.~~.~~ Small amounts of two isomers [(69) and (70)] of capsidiol were isolated from capsicums that had been treated with CuSO, but were not present in the mixtures of stress metabolites that were produced by other treatments.52 The absolute configurations of capsidiol(59) and its 1-epimer have been determined by the formation of their dibenzoates and the determination of their circular dichroism and magnetic circular dichroism. 53 13-Hydroxycapsidiol has been found to be formed in healthy capsicums when capsidiol in solution is incubated in the cavities of halved capsicum fruits.This process was thought to be a step in the degradation of capsidiol by the plant.54 However 14C-labelled 13-hydroxycapsidiol when incubated with healthy peppers was not appreciably metabolized during 24 hours.55 The degradation of phytoalexins is of importance in plant metabolism because many of these compounds are toxic to the host tissue.56 Capsidiol was found to be more toxic towards protoplasts from resistant capsicums than towards those from susceptible fruits. This suggests that in the case of the susceptible plants capsidiol may contribute towards the rapid death of cells and towards hypersensitive response (see Section 2.6).57 It was considered that the formation of capsidiol alone could not account for the difference in resistance of stems from susceptible and resistant varieties of Capsicum annuurn to Phytophthora cupsi~i.~~ Considerable changes were observed in R oT (57) R = AC (58)R = H HO’/h13q 3 OR OH /QI& 12 (59) (62)R = H (63)R = AC (69) R’ = OH R2= H (70) R’ = H R2 = OH the levels of free sugars upon infection of stems and leaves of both resistant and susceptible capsicums.In particular the levels of sucrose were greater than normal in the infected stems of resistant plants.59 The availability of sugars for conjugation might have a bearing on several aspects of the action of phytoalexins,e.g.the production of hydrophilic phytoalexins or the detoxification of phytoalexins that are toxic to host cells.59 Capsidiol was found to reduce the secretion of pectinases by Phytophthoru capsici and partly to inhibit in uitro the action of pectinases that had been isolated from this organism.60 Pectinases are largely responsible for the maceration of tissue that occurs in diseased plants.61 A protein that was isolated from capsicum fruits was found also to inhibit pectic enzymes from Glomerellu cingulatu in vitro.62Capsidiol was found in uitro to change the composition of cell membranes from P.cupsici altering the levels of proteins phospholipids and neutral lipids therein.63 Similar action in uivo was postulated in the light of the observed inhibition by capsidiol of the D-laCtate- dependent transport of [I4C]glycine into the vesicles of Escherichiu coli a process which was not affected by inhibition of respiration.64 2.6 Potato (Solanum tuberosum) Full papers have appeared on the isolation and identification of rishitinone (71)65 and of epilubimin (72) epioxylubimin (73) and isolubimin (74),66 all of which were isolated (as minor stress metabolites) from diseased potato tubers. Acetyldehy- drorishitinol (75) was isolated from slices of potato tuber that had been treated with a cell-free extract from Phytophthoru injestans and incubated in an atmosphere of 10 pl of ethylene per litre of oxygen. The compound occurred at 40 ng per gram of fresh weight with larger amounts of the usual potato phytoale~ins.~7 The new vetispirane derivatives (76) and (77) have been isolated from potato tubers that were infected with Phomu exiguu:68u the 3-deoxy-analogue (78) of the latter compound had already been isolated from the same source.68b (72)R = H (73) R = OH (75) NATURAL PRODUCT REPORTS 1985 The 1,2-hydride shift that is involved in the biosynthesis of rishitin (55) (Scheme 4a) was confirmed by the feeding of [2- 13C 2H3]acetate to tuber tissue slices that had been treated with spores of Monilinia fructicolu.The shift of the deuterium within the acetate unit that was incorporated at positions 4 and 5 (from C-5 to C-4) was indicated by the observation that a small percentage of the 13C signal due to C-5 is shifted downfield by 0.045 p.p.m. due to the deuterium atom at the p-posit ion. 69 The proposed biosynthetic pathway to rishitin has been confirmed by feeding (~)-[8,8-zHz]solavetivone (56a) to slices of healthy tuber of a potato that was a hybrid of S.tuberosurn and S. demissum (Scheme 4b). The first incubation yielded 29% of ( +)-[8,8-*Hz]1ubimin (79a) 7% of (+)-[8,8-2Hz]oxylubimin (8Oa) and 10% of ( -)-[8,8-zHz]rishitin (55a) (+)-solavetivone was not metabolized. Re-incubation of (79a) with tissue yielded 1 H Scheme 4a (56a) (79a) [and its enantiomer] I OH (76) (77)R = OH (55a) (80a) (78)R = H Scheme 4b NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON 11% of (80a) and 10% of (55a). Optical rotation data for products suggested that the deuterium content was diluted by unlabelled material that had been elicited by ( -)-solavetivone and that this acts as an activator of the whole enzyme system.70 The metabolites rishitin solavetivone and lubimin have been found to accumulate in cell-suspension cultures of potato in response to challenge by Phytophthora infestans the compounds occurred at levels up to ca 60 pg per gram of fresh weight largely in the culture medium.71 The incorporation of [ 14C]mevalonic acid into steroids was suppressed after inocula- tion of the cultures with P.infe~tans.~~ The steroidal glyco- alkaloids (81) and (82) have antifungal action and accumulate in potatoes that are under The incorporation of [14C]mevalonic acid into these compounds was inhibited by the application of arachidonic acid (83) or icosapentaenoic acid isolated separately from P.infestans they were each found to be precipitated with the carbohydrates by potato lectins indicat- ing that they each bind to the glucans.82 Glucans from P.infestans though weakly active as elicitors on their own enhanced the eliciting activity of (83).83 The most active p-glucan fraction from the mycelial extracts suppressed the eliciting activity of both compatible and incompatible racesf of P. infestans while enhancing the activity of (83).83 Water-soluble glucans from compatible races of P. infestans sup-pressed the reducing activity of potato protoplasts that had been treated with a cell-free extract from P.infestans i.e. they suppressed the hypersensitive response.79 The action of (83)as an elicitor may be dependent on lipoxygenase which might catalyse the formation of active moieties from (83) the latter compound was rapidly metabolized by the potato tissue and (84) which are elicitors of the sesquiterpenoid phyt~alexins.~~ had disappeared 24 hours after it was applied.84 The action of Rishitin was metabolized by healthy potato tuber slices into three unidentified metabolites when the tissue was incubated in air.Incubation of rishitin with tissue in an ethylene-xygen atmosphere produced a similar pattern of metabolism but solavetivone lubimin and phytuberin (57) were found to accumulate in the tissue by synthesis de notro this suggests a role for ethylene in the regulation of the accumulation of stress metabolites (cf Section 5.4. l).74 Cell-free extracts from discs of potato tuber that had been treated with Phytophthora infestans were found to support the NADPH-dependent synthesis of lubimin from isopentenyl dip hosphate.Hypersensitive response is the rapid death of cells and the browning of tissue that is associated with attack by a pathogen and which usually results in the accumulation of phytoalexins. Hypersensitive response in resistant cultivars of potato was associated with rapid death of cells and rishitin rapidly accumulated until the levels were inhibitory to the further growth of the fungus. In susceptible cultivars the accumulation of rishitin was slower although the levels that were eventually reached were higher than in resistant cultivars. In this case rishitin accumulated in dead cells after invading hyphae had grown out of them.76*77 Hypersensitive response might be a consequence of the generation of the superoxide anion (02-).78 Potato protoplasts were found to reduce extracellular cyto- chrome c two minutes after treatment with a cell-free extract from Phytophthora injestans.The reducing ability of protoplasts was inhibited by the enzyme superoxide dismutase which catalyses the reaction 202- + 2H+ e O2 + H202.79 Arachidonic acid (83) and icosa-5,8,11,14,17-pentaenoic acid (84) were isolated from the mycelial walls of Phytophthora infestans and found to be active elicitors of the accumulation of phytoalexins in potato.80 They are released from cell walls of the mycelium of P.infestans by lipase8' and are probably present in the walls in forms that are bound to carbohydrate.81 When (83)or (84) was mixed with P-glucans that had been (81) R = -solatriosyl (82) R = -chacotriosyl (83) was blocked by salicylhydroxamic acid (which is an inhibitor of cyanide-resistant respiration and of lipoxygenase) but not by tetraethylthiuram disulphide which inhibits only cyanide-resistant re~piration.~~ The stimulation of production of phytoalexins in potato by (83)appears to be characteristic of this plant amongst several plant species that were tested only capsicums were found to respond to (83)by producing phytoalexins.86 The Solanaceous plants tomato (Lycopersicon esculentum) and tobacco also failed to respond.The activity of (83)may be dependent on the type of tissue from each plant that is used in the assay.Several plant species have been found to produce jasmonic acid (85) from linolenic acid (86) by the action of lipoxygenase it seems possible that jasmonic acid and related compounds might play a role in metabolic regulation in plants by analogy with the activities of the icosanoids in mammalian bio-chemistry.87 Total syntheses of lubimin (79) and oxylubimin (80)have been described in earlier Reports.49 The synthon (2R* 5R* 1OS*)-2-hydroxy-6,1O-dimethylspiro[4.5]dec-6-en-8-one (87),88 which had already served for the synthesis of (i-)-solavetivone and related vetispiranes was applied in a highly stereoselective synthesis of ( f)-lubiminol (88) (Scheme 5),89 which also constitutes a formal synthesis of ( k )-isolubimin.66 A stereose- lective synthesis of ( -)-[12,12-2H2]rishitin (55b) from (1 1s)- 2a-acetoxy-3-oxo-4a,5a-eudesman-6~, 1 2-olide (89) [derived from (-)-a-santonin] has been describedg0 (Scheme 6).t When they refer to races of fungi that interact with host plants the words 'compatible' and 'incompatible' have opposite meanings to those that might be expected from the description of fungicides as being compatible if they can be mixed without deleterious effect to one another or of mating types (or strains) of fungi that are compatible if they are capable of being cross-mated. The growth of a compatible fungus is not effectively restricted by its host and causes it serious damage whereas the growth of an incompatible fungus is inhibited by its host and there is only limited damage.C02H PH 2 (861 NATURAL PRODUCT REPORTS 1985 \ \ *\ \ OH bCOBu' bCOBu' OMS (87) vi i RON ix,x viii f--t OH do2Et C/02Et Iiii vi x xi A -OH OMS (R = CH20Me Reagents i Bu'COCI py; ii Red-Al@ [NaA1H2(OCH2CH20Me)2]; iii MOM-CI (MeOCH,Cl) PhNEt,; iv Se02 then NaBH,; v MeLi; vi MsCl py; vii NaCH(CO,Et),; viii Hz Raney nickel; ix NaH then Red-AD; x 3M-HCl THF; xi NaI 1,8-diazabicyclo[5.4.0]undec-7-ene Scheme 5 ,NOH i -iii iv,v Ac 0 Ac04,0 ____ d AcO\QOO AcO OH CD20Ac OH CD2OAc '0 (891 t-'o x,iii . ... ,xi-xiv "a w 0 ix ~ 11;- vi -viii 1 CN 0 0 HO HO CD2 CD2OH CD~OAC (55b) Reagents i Bu'NH .BH,; ii LiAlD,; iii Ac,O py; iv NOCl py; v hv; vi POCl, py (3 days); vii MsCl py; viii collidine; ix Na PhMe EtOH; x Ph3CCI Et,N; xi p-TsOH MeOH; xii TsCl py; xiii NaI Me,CO; xiv KOH Scheme 6 2.7 Aubergine (Soianummeiongena) when these were vacuum-infiltrated with water then incubated Aubergenone (92) has been synthesized from the readily at 25 "C for 3 days.92 available (-)-a-santonin (90) uiu a-cyperone (91) as shown in 2.9 Coffee (Coflea arabica) Scheme 7.91 Coffee leaves that had been treated with Pseudomonas syringae were found to accumulate fungitoxic compounds identified tentatively as rishitin and capsidiol :93 the latter compound was 2.8 Soianum auicuiare also formed along with two unidentified phytoalexins in an Two antifungal metabolites 5P-solasodan-3-one (93) and interaction between coffee leaves and an incompatible race of solasodenone (94) were formed in the leaves of S.auiculure the fungus Hemileiu u~statrix.~~ NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON HO OH OH OH (92) Reagents 1 HCl 73% in DMF; ii LiNPri2 at -78 "C for 1 hour then MeI; iii H2 (Ph,P),RhCl; iv hv; v HC1-saturated EtOH; vi HOAc NaOAc at 100 "C; vii perbenzoic acid; viii LiAlH,; ix H2 PtO,; x Jones reagent; xi LiNPr', at -78 "C for 1 hour then PhSeBr; xii NaHC03 NaIO Scheme 7 H I H (93) 0* (94) 2.10 Rice (Ovyza satiua) Oryzalexins A (99 B (96) and C (97) were isolated from rice that was infected with Pyricularia oryzae. The oryzalexins can be regarded as derivatives of the previously characterized diterpe,ne sandaracopimaradiene.The structures (95)-(97) were assigned on the basis of m.s. and 'H n.m.r. evidence. Absolute configurations were determined by correlation with authentic ( -)-3-hydroxysandaracopimaradiene(98). The 7a- hydroxy-3-ketone that was derived from (98) by Jones oxidation followed by allylic hydroxylation (using SeO,) was enantiomeric with oryzalexin B as indicated by the opposite Cotton effects (e.g. at 300 nm). Similar evidence confirmed that (95) and (97)also belonged to the (+)-sandaracopimara- diene series. Oryzalexins are thought to be accumulated in rice through the changes that are brought about in the biosynthetic pathway to diterpenoids via copalyl diphosphate as a result of infection with P.oryz~e.~~,~~ # 0 OH HO" (95) (96 1 HOJ$-0&@-(97) (98) 3 Phytoalexins of Miscellaneous Structural Types 3.1 Linoleic Acid Derivatives Four antifungal substances were isolated from rice leaves that had been treated with probenazole and then infected with Pyricularia oryzae. The substances inhibited the germination of conidia of P. oryzae and were inhibitory to the growth of Xunthomonas campestris pv. oryzae. Spectroscopic analysis and comparison with standards revealed the materials to be linoleic acid (92 1 1 E 15Z)-13-hydroxyoctadeca-9,11,15-trienoic acid and (1 OE,122,152)-9-hydroxyoctadeca-10,12,15trienoic acid ; the fourth material was not identified. The hydroxy-acids are thought to be produced by the action of lipoxygenase on linoleic acid in the site of the lesion on infected rice.Their antifungal action may be the basis of the effectiveness of probenazole; this compound itself is only very weakly antimicrobial but in the NATURAL PRODUCT REPORTS 1985 iii 1 x xi ,v lix vi -viii 1 0 0 II "'7II ""9 (99) C0,Me (100) C02Me Reagents; i CuBr Me2S; ii HC=CH; iii IC=CCH20Thp (Thp = tetrahydropyran-2-y]) NNN'N-tetramethylethylenediamine; iv p-MeC,H,SO,H MeOH; v MnO,; vi EtMgBr; vii HC(OEt),; viii 10% aq. HCI; ix hexamethylphosphoramide LiNPr',; x at -78 "C then at 15 "C for 1 hour; xi CH2N2 Scheme 8 MeCH=CH [CEC13 CH=CHCHCH,OH MeCH=CH [C=Cl,CH,Me or Me[CEC],CH=CHCH Me I OH (104) (106) MeCH=CH [C~Cl,CHCH,OH MeCH=CH [C=Cl,CH-or Me[CEC] CH=CHCH-CH I OH field it provides protection against infection by Xanthomonas campestris pv.oryzae (cf Section 5.7).97-99 3.2 Acetylenes and Polyacetylenes The acetylenic phytoalexins wyerone (99) and dihydrowyerone (100) of Vicia faba have been synthesized via the condensation of (a-hept-4-en-2-ynal (1 01) and hept-2-ynal (102) respect- ively with a dianion that was generated from methyl 3-(2-fury1)acrylate (103) (Scheme 8). O0 '0' Extracts from the cell walls of Phytophthora megasperma f.sp. gl'ycinea or Al'ternaria carthami elicited the accumulation of fourteen polyacetylenes in cultures of safflower (Carthamus tinctorius). The two principal compounds that were present in newly established suspension cultures were identified as safynol (104) and dehydrosafynol (105).After two months in suspension culture however the major polyacetylenes that were elicited were (106) and (107) (each representing two possible structures). Further studies indicated that (104) and (105) accumulated at different rates after an elicitor had been NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G WATSON 437 added reaching maximal levels after 4 and 7 days respectively. Eleven of the induced polyacetylenes were tested for antifungal activity and found to have it in varying degrees; (106) was found to inhibit the growth of A. carthami in culture at a concentration of 50 pg ml- I. O * 3.3 Bibenzyls Aucuparin (1 08) was induced in the cortical layer of the shoot of the loquat (Eriobotrya japonica) when the tissue was challenged with Colletotrichum lindemuthianum.O2 The compound is strongly inhibitory towards C. lindemuthianum but only temporarily inhibits the growth of Pestalotiafunerea which is a pathogen of loquat. Aucuparin had been observed previously as a constituent of the heartwood of mountain ash (Sorbus aucuparia). This is the first report of a bibenzyl phytoalexin. 3.4 Stilbenes Stilbenoid phytoalexins have previously been isolated from grapevine (Vitis vinifera) and from peanut (Arachis hypogaea). Two new stilbene-2-carboxylic acid phytoalexins [(109) and (1 lo)] have been isolated from pigeon pea (Cajanus cajan) that was challenged with Botrytis cinerea. O3 3.5 Phenanthrenes and Dihydrophenanthrenes Two of the principal phytoalexins of orchids (Orchis spp.) are the dihydrophenanthrenes hircinol (1 11) and orchinol (1 12).These are present at ca 50pg per gram of fresh weight in untreated tubers and the levels are found to rise to ca 500-900 pg per gram of fresh weight in pathogen-infected or in irradiated tubers. lo4 Tuber slices from Orchis militaris that had been irradiated with ultraviolet light (to induce the formation of phytoalexins) were found to incorporate L-[U-'4C]phenyl-alanine into orchinol. Cinnamic m-coumaric and dihydro- m-coumaric acids were also incorporated into orchinol. In similar experiments with Orchis italica and Orchis anatolica the incorporation of phenylalanine (and also of cinnamic acid) into hircinol was observed.Further examination of the feeding with [U-'sC]phenylalanine indicated that it is incorporated into m-coumaric acid and into dihydro-m-coumaric acid. Cell-free HO2C HO 2C \ \ systems from the tubers of several species of Orchis were found to condense dihydrocinnamoyl-CoA esters with malonyl-CoA units ca 20 times more effectively than when cinnamoyl-CoA esters were used the products being bibenzyls. Dihydro-m- coumaroyl-CoA was efficiently incorporated into 3,3',5-trihy- droxybibenzyl in cell-free systems and when this compound was fed to orchid tubers it was effectively converted into orchinol. The compound 3,4,5-trihydroxybibenzyl,which was produced by incubation of p-coumaroyl-CoA in the cell-free system was not incorporated into orchinol in tubers.Thus Scheme 9 may be proposed for the biosynthesis of orchinol and may be valid in general for the formation of dihydrophenanth- renes and of bibenzyls in uivo it represents the first demonstration of the meta-hydroxylation of cinnamic acid in vivo. O4 Feeding of the [*H,]phenylalanine (1 13) to tuber slices of Orchis militaris provided data which were consistent with an incorporation of deuterium at C-5 and at either C-6 or C-8 in orchinol (as well as at positions 9 and 10).lo5Incorporation at C-8 would require the biosynthesis to proceed via a spiro- compound of the type (114) which together with a dihydro- phenanthrene (1 15) occurs naturally in Cannabis sativa. The pathway that has now been discovered via meta-hydroxylation of cinnamic acid obviates the need for a rearrangement to account for the position of hydroxylation in orchinol and accords with the appearance of deuterium at positions 5 and 6 after the [2H6]phenylalanine (1 13) has been incorporated.Hircinol (1 11) and the phenanthrene isobatatasin I (1 16) occur as pre-formed antifungal compounds in the peel of the yam Dioscorea rotundata.lo6 Levels of hircinol in the yam increase ca 10 times in response to infection with Phytophthora OH m 0 OMe (109) R' = H R2= Me2C=CHCH (108) (110) R' = Me2C=CHCH2 R2=H Ho2ch HO Q HO J J 3AcSCoA n A OH / (111) OMe (112) Scheme 9 438 NATURAL PRODUCT REPORTS 1985 cannabivora. A repetition of the careful labelling studies that were conducted on Orchis militaris led to the proposal of two separate biosynthetic pathways one providing isobatatasin I via p-coumaric acid and 3,4’,5-trihydroxystilbene(Scheme lo) and the other producing hircinol (as in the orchid) via m- coumaric acid and 3,3’,5-trihydroxybibenzyl.O7 3.6 Benzofurans and Phenylbenzofurans (113) (1 4) (115) Three benzofurans a-,p- and y-pyrufurans [(1 17)-( 119)] were isolated from the dark pigmented zone between healthy and diseased tissue in the sapwood of perry pear (Pyrus communis) that was infected with Chondrostereum purpureum.The struc- tures were determined spectroscopically and by total synthesis. Bioassays (on a t.1.c. plate) showed that the compounds were the principal anti fungal components in infected wood.Concen- trations in infected tissue rose to 5 mg per gram of fresh weight OH OH in the pigmented zone. Since dibenzofuran may be oxidized by bacteria at positions 2 and 7 there was a possibility that (1 19) was a degradation product of (1 17) and (1 18) but incubation of (117) with cultures of C. purpureum did not result in the formation of (1 19). 08 O9 Cotonefuran (120) was isolated from the pigmented zone adjoining fungally infected tissue in Cotoneaster lactea and was found to be antifungal. The structure was determined by X-ray &OHMeo \ / diffraction. lo OMe OMe 3.7 Furocoumarins Scheme 10 (116) The antifungal furocoumarins psoralen (1 21) bergapten (1 22) xanthotoxin (1 23) and isopimpinellin (124) can be isolated in small amounts from healthy celery (Apium graveolens).R’ Irradiation of celery with U.V. light chilling (to -15 “C for 70 minutes) or treatment with sodium hypochlorite led to three- R3 nine- and two-fold elevation respectively of the levels of the furocoumarins as compared with those in untreated celery. R4 Copper(I1) sulphate also elicited an increase in the levels of furocoumarins in proportion to the concentration of CuSO above a threshold value of 5 x lo- mol dm-3. Psoralen and isopimpinellin occurred at much lower levels than xanthotoxin and bergapten in treated celery. Levels of psoralen which is the presumed biosynthetic precursor of bergapten xanthotoxin and isopimpinellin rose in response to cold treatment presumably due to decreased rates of transformation into the alkoxy-compounds.’ MeO OMe Cultures of parsley (Petroselinum hortense) produced furocou- marins in response to treatment with extracts from the cell walls of incompatible races of the fungi Phytophthora mega- sperma f.sp.glycinea and Alternaria carthami. Treatment with the extract from P. megasperma caused the accumulation of (121) (122) (123) and graveolone (125). In response to A. carthami compounds (1 22) and (1 24) accumulated with small amounts of (1 25). The furocoumarins are the first phytoalex- 5 i OMe ins that have been observed to be induced in parsley in response to a fungal agency. Ultraviolet light caused the accumulation of flavone glycosides in the culture. and a fungal extract had previously been shown to stimulate the enzymes of phenylpro- panoid metabolism but without causing any observed accumu- lation of phytoalexins.Ih 3.8 Avenalumins Compounds (1 26) (1 27) and (1 28) are formed in oats (Aoena spp.) in response to infection with crown rust (Puccinia coronata). Avenalumins I 11 and 111 are highly soluble in 0 water unlike most other phytoalexins. The structures (126)-(1 28) for avenalumins 1-111 respectively were confirmed &J-J+10 0 spectroscopically and by total synthesis.’ 7-1 l9 The com-pounds caused 50% inhibition of the growth of the germ tube from spores of P. coronata at 200-250 pg ml-l; the acetylated derivatives were inactive.’I9 High levels of avenalumins (125) accumulated in resistant strains of oat in response to challenge NATURAL PRODUCT REPORTS 1985 -C.J. W. BROOKS AND D. G. WATSON HO 5 4 w (129) (130) HO.1 / fiOH by P. coronata the time of most rapid accumulation coinciding with the cessation of fungal growth; susceptible lines accumu- lated very little of the compounds. Resistance was correlated with high rates of accumulation of the compounds:120 if oats were grown at high temperatures (30°C) the levels of avenalumins fell. Treatment of oat leaves with amino-oxyacetate which is an inhibitor of phenylalanine ammonia-lyase (see Section 4.2) caused up to 88% inhibition of the production of avenalu-mins. Avenalumins were found to accumulate mainly in the infected cells. Microspectrophotometric observation of the strongly fluorescent avenalumins showed that an increase in fluorescence in infected resistant cells coincided with the collapse of the cells after ca 36 hours.No fluorescent cells were observed amongst the infected cells of a susceptible line of oats. Enhanced levels of p-coumaric and ferulic acids were observed by g.c.-m.s. and by h.p.1.c. in leaves that were infected with P. coronata these compounds being direct precursors of avenalumins. Pre-formed antifungal steroids and flavonoids that were present in oat leaves remained at the same levels after infection with P. coronata.' 23 3.9 Flavans Epidermal strips from bulb scales of the daffodil (Narcissus pseudonarcissus) were inoculated with Botrytis cinerea nine antifungal fractions were obtained by gel filtration of the extract and separation by h.p.1.c.yielded twelve antifungal OH I QOH (132) fractions. Three of the antifungal compounds were identified as the flavans (129) (130) and (131) all of which had been isolated in previous work. 24 7-Hydroxyflavan (1 29) proved to be the most strongly antifungal of the substances that were isolated.' 25 3.10 Phenylbenzofurans from Mulberry and Paper Mulberry Eight 2-phenylbenzofuran phytoalexins and two stilbene-type phytoalexins have been reported to occur in the shoots of mulberry (Morus alba) that was infected with Fusarium solani f.sp. mori. 26 More recently two natural Diels-Alder adducts with antimicrobial properties have been isolated. C halcomor-acin (1 32) was isolated from leaves that were infected with the same fungus as 0.012% of the dry weight.It can be considered as a Diels-Alder adduct of morachalcone (133) and dehydro- moracin C (134) or its equivalent. The compound was optically active unlike a number of other presumed natural Diels-Alder adducts. 27 Another optically active metabolite dimoracin (1 39 was isolated from mulberry shoots that were infected with F. solani f.sp. mori as 0.0028% of the fresh weight. It can be considered as a modified Diels-Alder adduct of moracin C (136) with its dehydro-compound.' 28 Seventeen phytoalexins and prohibitins (i.e. pre-formed antimicrobial compounds) from mulberry were tested against 30 species of fungi and 24 species of bacteria. In general it was found that most of the compounds inhibited the growth of the mycelium in most of the fungi that were tested once a concentration of 56 p.p.m.was reached. In the assays of bacterial growth many species of the NATURAL PRODUCT REPORTS 1985 (143) R' = OH R2 = CH2CH=CMe2 (144) R' = R2= H 0 (145) R = H (146) R = Me (149) (150) (152) R = H (155) R = H E (153) R = OH (156) R = 02CC=CHMe genera Pseudomonas and Erwinia proved to be tolerant at concentrations of up to 224 p.p.m. of all of the compounds that were tested. * 29 In addition to the previously isolated broussonin A (1 37) and broussonin B (138) eight minor phytoalexins have been isolated from the cortical tissue of paper mulberry (Broussonetia papyrifera)that was infected with Fusarium solani f.sp.mori. Six of these [(139)-(144)] were of the broussonin type and the remaining two [(145) and (146)J were chalcones. Yields of (139)-( 146) from infected shoot tissue were 0.038 0.012 0.0032 0.033 0.014 0.0014 0.0032 and 0.0014% of the fresh weight respectively. 30 All of the compounds were found to have antimicrobial action. The chalcone (146) had been previously isolated from CuC1,-treated Pisum satiuum. l3 Two optically inactive spiro-broussonin compounds [(147) and (148)] were isolated from paper mulberry that was infected with F. solani f.sp. mori as 0.027 and 0.064% of the dry weight respectively. These are presumably derived from the oxidative coupling of broussonins. 32 The antimicrobial activity of broussonins A and B and of marmesin (149) which was also isolated from paper mulberry was tested against 30 species of fungi and 24 species of bacteria.Marmesin was only weakly inhibitory but (137) and (138) were inhibitory to many fungi and bacteria; amongst the fungi Chaetomium globosum was tolerant and many bacteria of the genus Pseudomonas were also tolerant. 33 3.11 Benzoxazinones Dianthalexin (150) was isolated from carnation (Dianthus caryophyllus) that was infected with Phytophthora parasitica. The structure of this benzoxazinone phytoalexin was con- firmed spectroscopically and also by its synthesis via thermal I Me cyclization (at 240 "C) of N-benzoyl-4-hydroxyanthranilic acid (151). This is the first instance of the isolation of a phytoalexin from the Caryophyllaceae.l 347 35 3.12 Alkaloids Growth of a culture of Ruta graveolens in the presence of fungi that are not specific to the host on which they grow led to increases in the content of rutacridone epoxide (152) and hydroxyrutacridone epoxide (1 53) of ca 50-fold.Culture filtrates from the fungi produced comparable effects on the plant cultures. The acridone epoxides were found to inhibit the growth of Bacillus subtilis. Yields of the epoxides were up to ca 2 mg per gram of fresh weight. 37 367 Leaflets of Lupinus polyphyllus when wounded (by cutting) or incubated on water were found to accumulate up to 400% of the normal amounts of alkaloids chiefly comprising tetra- hydrorhombifoline (1 54) lupanine (1 59 and 13-tigloyloxylu- panine (1 56).However there was no indication of an increase in oxosparteine synthase activity that might have been expected to be associated with the production of phyto-alexins. 38 The accumulation of lupanine and related compounds in suspension cultures of Lupinus polyphyllus could be elicited by a number of agents including papaverine coniine spermidine and CAMP which produced (respectively) increases of 75 4.5 15- and 18-fold in the alkaloid content relative to those in unelicited cultures. Levels of the enzymes that are involved in the synthesis of alkaloids such as oxosparteine synthase were unchanged in elicited as compared with unelicited cultures. It was considered that the accumulation of alkaloids was due to the inhibition by the elicitors of their normal degradation.Increased levels of lupanine and related compounds were also NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON 44 I (1 58) Reagents i phenylalanine ammonia-lyase; ii cinnamic acid 4-hydroxylase; iii 4-coumarate-CoA ligase; iv chalcone synthase; v chalcone isomerase Scheme 11 observed when cultures of plants [such as the carrot (Daucus carota)] that do not accumulate alkaloids were treated with the alkaloid-eliciting agents. Even though the plants that were tested do not accumulate alkaloids under normal circum- stances oxosparteine synthase activity could be observed in unelicited cultures. 39 The results support the view that the genetic information for versatile secondary metabolism is present in all plants but that this information is expressed in a selective manner which in many instances transcends taxo- nomic classifications.4 lsoflavonoid Phytoalexins 4.1 Introduction The biogenesis of isoflavonoid phytoalexins follows the general phenylpropanoid pathway that is well established for isoflavon- oid secondary metabolites. Indeed many of these phytoalexins are also found amongst normal secondary metabolites e.g. in roots and heartwoods. The work on the biosynthesis of isoflavonoid phytoalexins up to ca 1983 has been well re~iewed.~.~,~,~~~ Work in this area has had two themes. The first is the study of the phytoalexin response in terms of the rapid induction of the enzymes that catalyse the earlier steps in the biosynthetic pathway to isoflavonoids whereas the second is the elucidation of the later steps in the biosynthesis of isoflavonoid phytoalexins involving further elaboration of the isoflavonoid skeleton.There is some degree of overlap in the two areas of study; however the first theme has moved towards the study of molecular genetics as a basis for the elucidation of the mechanisms of response to phytoalexins. 4.2 Biosynthesis Enzymic Aspects The enzymes which have been most studied in relation to the formation of isoflavonoid phytoalexins are those occurring early in the biosynthetic pathway up to the point where an isoflavonoid precursor such as formononetin (157) is formed. The steps on the pathway to 2',4,4'-trihydroxychalcone (158) are outlined in Scheme 11 ; the principal enzymes that are involved are phenylalanine ammonia-lyase (PAL) cinnamic acid 4-hydroxylase (CA4H) [trans-cinnamate 4-mono-oxygen- ase] 4-coumarate-CoA ligase (4-CL) chalcone synthase (CHS) and chalcone isomerase (CHI).All of these enzymes have been studied to some extent but recent work has focused principally on the induction of PAL and CHS. Treatment of cell cultures of Phaseolus uulgaris with an elicitor that could be extracted into water by autoclaving the mycelium of Colletotrichum lindemuthianum was found to increase the rate of synthesis of PAL after ca 20 minutes. The levels of PAL reached a maximum after 3 hours and rapidly declined thereafter. Levels of CHS were found to increase and decrease in concert with the elicited levels of PAL.141 Similar patterns of induction of enzymes in the cultures were observed in response to a glucan elicitor that was prepared from the cell walls of Phytophthora megasperma f.sp.glycinea. 141 In these systems the levels of polysomal mRNA activity that were associated with the synthesis of PAL and CHS were found to be closely correlated with the pattern of synthesis of these enzymes in the elicited cultures.142 These observations reinforce the view that PAL and CHS are key regulatory enzymes in the biosynthesis of isoflavonoid phytoalexins and indicate that the formation of phytoalexins is stimulated by fungal elicitors as a primary defensive response resulting from a rapid sequence of events. The mRNA that is involved in the biosynthesis of PAL and CHS is transcribed de nouo rather than drawn from an intracellular pool of pre-formed mRNA.In related work cultures of soybean (Glycine max) were treated with an elicitor from P. megasperma f.sp. glycinea and levels of PAL and CHS were found to reach their maxima seven and twelve hours respectively after the elicitor had been added. The activities of mRNAs coding for PAL and CHS were found to reach maxima 5-7 hours after treatment and to decline by the tenth hour. A polygalacturonase from Aspergillus niger and a bacterial polysaccharide (xanthan) from Xuntho-monas campestris were elicitors of high activities of the mRNAs for PAL and CHS. However in contrast to the elicitor from P. megasperma the polygalacturonase elicited only very low levels of accumulation of phytoalexins while xanthan did not elicit phytoalexins.In these experiments the question of the regulation of the biosynthesis of isoflavonoids by PAL and CHS remained unres01ved.l~~ It is possible that the products of PAL and CHS activity are being rapidly broken down in those systems where they do not accumulate. Soybean hypocotyls that were infected with P. megasperma were found to show a similar pattern of induction of the mRNAs that code for PAL and CHS to that which was observed in the cultures.144 Cultures of parsley do not appear to produce isoflavonoid phytoalexins but have been used in studies of the early stages of the biosynthesis of isoflavonoids since they afford PAL 4- CL and CHS in response to irradiation with U.V.light and produce flavonoid glycosides. (The cultures also yield furocou- marin phytoalexins when treated with fungal elicitors cf Section 3.7). In experiments that employed the techniques of molecular genetics complementary DNA (cDNA) was pro- duced from mRNAs (isolated from parsley cultures) which coded for PAL 4-CL and CHS respectively. The cDNA was cloned by incorporation into a plasmid and insertion into Escherichiu coli. The use of cDNA allowed the determination of the amounts of hybridisable mRNA that code for the respective enzymes in parsley cells after irradiation with U.V. light or treatment with a fungal elicitor:145* 146 these amounts were found to correspond closely with the translatable activities of the mRNAs except in the case of PAL in parsley cultures after these had been treated with an elicitor.In this instance the levels of the mRNA that codes for PAL increased beyond the time that it attained its maximal translatable activity this NATURAL PRODUCT REPORTS. 1985 n 0 0 \ (158) ii \ Reagents i H+; ii S-adenosylmethionine Scheme 12 result implies that the mRNA is inactivated by unknown factors. The cDNA probes that have been developed in the work on parsley cultures are being applied in investigations of other isoflavonoid-synthesizing systems. 44 The induction of chalcone isomerase (CHI) in cultures of Phaseolus vulgaris by the elicitor from Colletotrichum lindemuth- ianum was examined by labelling the induced enzyme with D20.The resulting changes in the buoyant density of the protein could not be correlated with the doubling of CHI activity that was observed in response to elicitation. This supported the earlier view that CHI activity increased largely via activation of pre-existing enzyme and that little synthesis of CHI de novo took place during the initial stages in the formation of phytoalexins. However it has been noted that isoflavonoids such as kievitone (177) (see p. 444) inhibit the action of the enzyme suggesting that its regulation involves a ‘feed back’ mechanism. 14 Prenylation is required in the formation of a number of isoflavonoid phytoalexins including the glyceollins (172)-( 174) (see below) of soybean accordingly the enzymes of terpenoid biosynthesis have also been studied.The levels of hydroxy- methylglutaryl-CoA reductase (NADPH) [E.C. 1.1.1.341 in wounded soybean cotyledons increased rapidly up to 6 hours after the wounding had occurred declined between 6 and 20 hours after the event and then increased again but there was little accumulation of glyceollins. When the cotyledons were treated with elicitor from Phytophthora megasperma the HMG- CoA reductase activity showed little change until 18 hours had elapsed when a slow rise ensued however between 5 and 24 hours after elicitor was added glyceollins accumulated steadily in the tissue. A similar pattern of action of the elicitor was found in soybean cultures. In contrast the activity of a prenyltransferase was markedly elevated in response to the elicitor in both cotyledons and cultures.Wounding of cotyledons induced little change in prenyltransferase activity. It was concluded that the evidence supported a role for prenyltransferase but not for the HMG-CoA reductase in the regulation of the biosynthesis of glyce~llins.~~~~ [It may be noted that a prenyltransferase appeared to exert control over the biosynthesis of furocoumarin phytoalexins in elicitor- treated cultures of parsley. 48b] Enzymological studies of isoflavonoid phytoalexins have also been used in exploration of the distinctions between interactions between compatible and incompatible pathogens and hosts. Differences in levels of PAL between soybean seedlings that had been inoculated with compatible and incompatible races of P.megasperma were not apparent until 14 hours after inoculation the maximal activity of PAL was higher in the interaction between an incompatible pathogen and the host.The kinetics of induction of CHS and the maximal levels of activity that were attained were similar in interactions between hosts and both compatible and incompatible pathogens in this instance the similarity of the induction of the enzyme may reflect the effects of the wounding of the seedlings prior to their inoculation by insertion of fungal mycelium.149 When the spores of either compatible or incompatible races of Colletotrichum lindemuthianum were applied to the surface of hypocotyls of Phaseolus vulgaris the activity of the mRNA that codes for CHS was observed to appear 52 hours after inoculation with the incompatible fungus but not until 130 hours after inoculation with the compatible race.For the incompatible fungus the mRNA activity that codes for CHS was not found in the tissue of the host at sites that were remote from the point of inoculation whereas for the interaction with a compatible pathogen the mRNA activity was observed at such sites possibly indicating the release of larger amounts of endogenous elicitor as a result of extensive colonization of the host.’50 4.3 Biosynthesis Mechanistic Aspects The first committed step in the biosynthesis of isoflavonoids involves the rearrangement of an aryl group. The mechanism of this process is not yet known but Scheme 12 is in accord with the known facts.In a recent investigation 2’,4,4’-trihydroxy- [carbonyl,a-I3Cz]chalcone (1 58a) was fed to seedlings of Trifolium pratense that had been stimulated (by treatment with CuCl,) to produce the phytoalexins formononetin (1 57) ( -)-medicarpin (163) and (-)-maackiain (161). The I3C2 unit was found to be incorporated intact into all three metabolites indicating that aryl migration had occurred and not a rearrangement of the C3 chain of the chalcone. In addition 2‘,4,4’-trihydro~y-[P,3,5-~H,]chalcone (1 58b) was incorporated into formononetin and (-)-medicarpin with retention of all three ‘H atoms.I5’ 4.3.1 The Phytoalexins of Pea (Pisum sativum) Scheme 13 has been proposed for the biosynthesis of (+)-pisatin (l) which is the principal phytoalexin of the pea.The 4C-labelled compounds phenylalanine 2’,4,4’-trihydroxychal- cone (1 58) formononetin (1 57) 3’,7-dihydroxy-4’-methoxyiso-flavone (1 59),7-hydroxy-3’,4’-methylenedioxyisoflavone (1 60) NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON H°CI&-Jo) -0 (re-addition of H at C-3) (160) Reduction ( si-addition of H at C-3) HO MeoQpyJ;, Meo%;) (1 67) (1 1 Scheme 13 and (+)-maackiain (162) were all incorporated (in good yield) into (+)-pisatin (1) in CuC1,-treated pea pods.150 Compound (164) was found to be a poor precursor of (+)-pisatin in accord with the hypothesis that methylation occurs during the aryl shift (see Scheme 12).Compounds (165) and (166) were incorporated at low levels indicating the possibility of less (1 63) direct biogenesis. 52 (+)-Pisatin is unusual among pterocar- pans in having a (+)-(6aR 1 laR) configuration; most other pterocarpan phytoalexins are laevorotatory including ( -)-maackiain (161) which occurs in small amounts [with (+)-pisatin] in the pea. The stereochemistry of addition of hydrogen during the biosynthesis of (+)-pisatin was confirmed by feeding studies using [2-*H,]formononetin (157a). Deuteron n.m.r. of the product revealed it to be (6R)-[6-*H1]pisatin 0 which was consistent with the reduction of the double-bond (165) R = Me with re-addition of hydrogen at C-3 in formononetin.' 53 (+)-(164) (166) R = H [6,6,I 1a-2H3 JMaackiain (162a) was incorporated into pisatin NATURAL PRODUCT REPORTS.1985 (168) (169) v (172) / I Scheme 14 (174) *O% 0 HO\ OH (175) (1 761 Scheme 15 (177) with retention of all of the label thus eliminating the possible intermediacy of a pterocarp-6a-ene or -6-ene. Therefore the 6a- hydroxylation of (+)-maackiain occurs directly with retention of configuration.I 54 Further evidence of this was obtained when it was found that (+)-maackiain was incorporated into (+)-pisatin in good yield in pea pods while (-)-maackiain (161) was incorporated in low amounts into the abnormal metabolite (-)-pisatin (167).* 56 559 4.3.2 The Phytoalexins of Soybean (Glycine max) The l4C-labe1led compounds phenylalanine daidzein (1 68) 2’,4’,7-trihydroxyisoflavone(169) 3,9-dihydroxypterocarpan (170) and glycinol (171) were all found to be efficient precursors of glyceollins I (172) I1 (173) and I11 (174) in both seedlings and pods of soybean that had been treated with CuC12.This is consistent with Scheme 14 for the biosynthesis of glyceollins.Compounds (1 70) and (171) were particularly efficiently incorporated suggesting that prenylation occurred after the formation of glycinol. 57 Compound (170) (labelled with 3H) was incubated with a microsomal fraction from soybean cell cultures that had been treated with an elicitor from Phytophthora megasperma. The product was identified as glycinol (171) into which no more than 50% of the racemic substrate was converted. Optical rotatory dispersion spectra recorded for the product and for the unreacted starting material showed them to have opposite absolute configura- tions.This indicated that 6a-hydroxylation also proceeds with the retention of configuration in the biosynthetic pathway to glyceollins. 6a-Hydroxylase activity was found to be six-fold higher in elicitor-challenged cultures. 58 4.3.3 The Phytoalexins of Phaseolus vulgaris Phaseolus vulgaris produces a range of isoflavonoid phytoalex- ins these are formed via two separate pathways leading to 5-hydroxy-isoflavonoids and the corresponding isoflavonoids that are not hydroxylated at C-5. The pathways to kievitone (1 77) and (-)-phaseollin (180) which are the principal phytoalexins of P. vulgaris from genistein (175) and the chalcone (158) respectively are indicated by Schemes 15 and 16.Many of the intermediates (and related compounds) in the two pathways have been found to accumulate in wounded cotyledons or in cotyledons that have been treated with a fungal elicitor. 59 A study of the course of accumulation of isoflavon- oids in cotyledons of P. vulgaris with time indicated that 5-hydroxy-isoflavonoids such as kievitone (1 77) and 2’-hydroxy- genistein (1 76) appeared earlier in elicitor-treated tissue than the isoflavonoids such as (-)-phaseollin (180) and coumestrol (18 1). In wounded cotyledons kievitone accumulated most rapidly between 8 and 20 hours after wounding. The levels of (-)-phaseollin remained low until 60 hours had elapsed and then rose rapidly up to 96 hours reaching a value similar to that of kievitone.Treatment of wounded cotyledons with various NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON 445 Ho% -* Ho’I I % Hoe \ OH \ OH HO 0 (168) (158) c-\HoqyJ0 (1 80) (179) a (170) Scheme 16 moH &-/OH (1 58) (1 80) HO 0 I HO 1HO (1 78) (1 77a) (177b) Scheme 17 \ OH (181) concentrations of elicitor from Colletotrichurn lindemuthianum led to accumulations of kievitone that were broadly similar in time course but quantitatively dependent on the concentrations of elicitor. Major changes in levels of phytoalexins were preceded by transient increases in activities of PAL CHS and CHI. 59 The growth hormones abscisic acid and benzylamino- purine may have a role in regulating the pathways to 5-hydroxy- isoflavones and the corresponding Sunsubstituted isoflavones in P.vuZgaris (see Section 5.4). Evidence in support of the pathway to (-)-phaseollin has been obtained through the efficient incorporation of the 4C-labelled substrates 2’,4,4’-trihydroxychalcone (1 58) daidzein (1 68) 2’,4,7-trihydroxyiso- flavone (169) 3,9-dihydroxypterocarpan (170) and (-)-(6aR 1 1 aR)-phaseollidin (1 79) into (-)-phaseollin (1 80) in CuC12-treated seedlings of P. vulgaris.160 The feeding of [13C2]acetate to seedlings of P. vulgaris that had been treated with CuC12 gave incorporation of intact acetate units as indicated in Scheme 17. This result paralleled earlier results from the incorporation of [13C,]acetate into pisatin and indicated a specific folding of the polyketide chain from which ring A is derived.The lack of randomization of the label supported the intermediacy of 2’,4,4’-trihydroxychalcone (158) in the synthesis of phaseollin since the asymmetric ring A in this compound affords only one position in which a hydroxyl group can react to form the pyran ring. In contrast for kievitone there was an indication that specific folding of the polyketide chain was followed by randomization of the acetate units consistent with the intermediacy of the symmetric 2’,4,4’,6’-tetrahydroxychalcone(1 78) in its biosynthesis. There was no significant incorporation of acetate into isoprenoid units. 61 NATURAL PRODUCT REPORTS 1985 0 (158) 71 \ (182) I (157) 1 (1 63) Scheme 18 0 D 0 (1 57b) Scheme 19 (161a) 4.3.4 The Phytoalexins of Red Clover (Trifolium pratense) The metabolic grid that is involved in the biosynthesis of (-)-medicarpin (163) in T.pratense is indicated in Scheme 18. Feeding 2’,4,4’-trihydroxy[P,3,5-ZH3]cha1cone(1 58b) to CuC1,- treated seedlings yielded formononetin (1 57) and (-)-medicar- pin (163) both retaining all three deuterium atoms. (-)-Maackiain was also isolated and consisted of 50% dideuteriated and 50% trideuteriated molecules. This was due to the NIH shift that is involved in the 3’-hydroxylation of formononetin (see Scheme 19). This observation also provided evidence for the hydroxylation of a 4-methoxy-system rather than a LC‘-hydroxyisoflavone since empirical rules for the NIH shift indicate that the migration of ,Hlabel would be expected during ortho-hydroxylation of a 4-methoxy- but not of a 4-hydroxy-system.Further confirmation of these observations was obtained by using [3’,5’-*H,]formononetin (157b) as a precursor. Confirmation of the metabolic grid that is outlined in Scheme 18 was obtained by feeding (-)-7-hydroxy-4’- methoxy[2,2-2H,]isoflavanone(1 82a) to CuC1,-treated T. pra-tense. The compound was incorporated into (-)-medicarpin (163)largely with retention of both deuterium atoms i.e.(182a) was incorporated into medicarpin via a route that does not involve its conversion into formononetin with consequent loss of label.151 4.4 The Total Synthesis of (k)-Phaseoh The first syntheses of (i-)-phaseollin have been achieved via the chalcone-isoflavone route as in Scheme 20a.162 The chalcone (184) afforded a yield of 34%(based on the amount of chalcone that was converted) of the oxidative rearrangement product (i 85) via preferential reaction of the a@double-bond.The yield of (i-)-phaseollin from the acetal (185) was 30%. In order to prevent competing side-reactions of the double-bond of the chromene this was protected by regioselective radical addition163 of thiophenol [(183) -+ (188) as shown in Scheme 20b] and later regenerated by pyrolysis of the sulphoxides of (190) and (192). Extension of the synthesis to (+)-phaseollidin is feasible since phaseollidin has been obtained by reduction of natural phaseollin.6o An alternative route to phaseollin and phaseollinisoflavan has been reported in outline. 64 4.5 Newly Isolated Isoflavonoid Phytoalexins Isosativanone (193) in racemic form was isolated from fungus- inoculated leaflets of Medicago rugosa together with the known phytoalexins (-)-medicarpin (1 63) (-))-vestit01 (208) isosati- van (209) and (+)-vestitone (210).165 Diphysolone (194) was isolated from Diphysa robinioides that was challenged by Helminthosporium carbonurn with smaller amounts of its #-(or NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON v,vi $. -R VII VI OO (180) (186) R = CH20Me + (187) R = H ent -(179) ent -(180) Reagents :i Me,C(OH)CH2CH(OMe)21 py ;ii methoxymethylation reagent (unspecified); iii resacetophenone monomethoxymethyl ether OH-; iv Tl(NO,), MeOH; v NaOMe; vi H30+; vii NaBH,; viii Li NH3 Scheme 20a SPh SPh 1 ... I,II (183) + (188) R = H 0 (189) R = CH20Me (R = CH20Me) iv-vi 1 viii ix vii vi viii ix (180) + ent -(180) -(186) 0 (190) R = CH20Me (192) SPh (191) R = H Reagents:i hv PhSH (PhS)* ;ii methoxymethylation reagent (unspecified); iii resacetophenone monomethoxymethyl ether OH-; iv T1(N03)3 MeOH; v NaOMe; vi H30+; vii NaBH,; viii rn-chloroperoxybenzoic acid; ix PhMe heat Scheme 20b 2'-)methyl ether and the known isoflavonoids kievitone (177) treated with CuCl,. These included genistein (175) 2'- and ferreirin (21 1). 66 Sliced seeds of Cajanus cajan when hydroxygenistein (1 76) 2,3-didehydrokievitone (214) dalber- incubated with their own native microflora produced cajanin gioidin (215) kievitone (177) cyclokievitone (216) 5-deoxy- (212) and cajanol (213) both of which had previously been kievitone (217) and phaseollidin.5-Deoxykievitone was the isolated from C.cajan and two new isoprenylated isoflavones most fungitoxic of the compounds that were isolated. 68 (195) and (196). 67 Eight known isoflavonoid phytoalexins Sixteen isoflavonoids were isolated from seedlings of Phaseolus were isolated from seedling of Phaseolus aureus that had been mungo that had been treated with CuC1,. Thirteen of these were NATURAL PRODUCT REPORTS 1985 ‘1 OH 0 HO\ OH 0 \ OH (193) (194) (195) YI Ho% 0 HO\ OMe (196) (197) (198) (199) (2001 (204) (205 1 (206 1 R’% R2 (207) HO\ OH kOH0 HO\ How 0 \ OMe (214) R1 =OH R2= CH2CH=CMe2 (216) (218) previously known phytoalexins.Three new isoflavones were nissicarpin (201) fruticarpin (202) and nissolicarpin (203) characterized as 4’-O-methylkievitone (197) cyclokievitone were isolated from leaflets of Nissolia fruticosa which had been hydrate (198) and 5-deoxykievitone hydrate (199). 169 The treated with Helminthosporium carbonum or with CuS04. 71 pterocarpan phytoalexin desmocarpin (200) was isolated from Leaflets and pods of Lathyrus sativus have been found to Desmodium gangeticum that was infected with Helminthospor-produce (+)-pisatin (1) in response to challenge with H. ium carbonum. 70 Three new dextrorotatory pterocarpans i.e.carbonum or Botrytis cinerea; 72 lathycarpin (204) was isolated NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON from the plant if it had been inoculated with H. curbonum.173 (+)-Maackiain (162) and (+)-medicarpin (218) were isolated from Sophora juponica that had been challenged with Nectria haematococca. An isolate of N. huematococcu rapidly metabo- lized (+)-maackiain and (+)-medicarpin into unknown products; however (-)-maackiain (161) and (-)-medicarpin (1 63) were converted by the same isolate into their 6a-hydroxy- derivatives. 74 Tephrosia bidwilli that had been inoculated with H. carbonum accumulated (-)-maackiain (-)-pisatin (167) (-)-4-methoxymaackiain and the new compounds tephrocar- pin (205) and acanthocarpan (206).All of these compounds were strongly laevorotatory. This is the first instance of (-)-pisatin occurring as a natural product.' 75 The pterocarpan phytoalexin apiocarpin (207) was isolated from Apios tuberosa that had been challenged with H. carbonum. The absolute configuration of (207) was assigned as (6aR,1laR 139 using circular dichroism in comparison with data from the c.d. spectra of glyceollin I11 (174) and rotenone. 76 4.6 The Analysis of Phytoalexins Gas chromatography-mass spectrometry has been the most frequently employed technique for the quantitation and identification of sesquiterpenoid phytoalexins :recent applica- tions are to the phytoalexins from species of the genera Capsicum5 and Nicoti~nu.~~ The norsesquiterpenoid rishitin (55) and three acetylenic phytoalexins including falcarinol and falcarindiol were identified in extracts from Verticilliurn-infected tomato by capillary g.c.-m.s.of their trimethylsilyl (TMS) ethers. 77 Gas chromatography-mass spectrometry has been less frequently applied to the phenolic phytoalexins but the analysis of a number of isoflavonoid phytoalexins including dalbergioidin (21 5) and kievitone (177) after trimethylsilylation revealed that they formed either two or three derivatives. In some cases this was due to the opening of the furan ring and in others to the formation of enol trimethylsilyl derivatives from the carbonyl group. All of the mass spectra exhibited base peaks due to retro-Diels-Alder fragmentation with predominant retention of charge on the fragment that contains ring B.l 78 Gas chromatography-mass spectrometry was also used to detect the formation of small amounts of the avenalumin precursors p-coumaric acid and ferulic acid in infected oat leaves the compounds were analysed as TMS derivatives.' l High-performance liquid chromatography has been used both to separate and to determine small quantities of phytoalexins.The method has been most frequently applied to the analysis and quantitation I R HOuOMe (1 63) R=H R = OH of isoflavonoids. The phytoalexins of Phaseolus vulgaris were 1 analysed by gradient elution of a polar phase column with hexane-chloroform and chloroform-methanol mixtures en- abling the resolution of six isoflavonoids.79 The phytoalexins of soybean have been analysed by straight phase h.p.1.c. on silica ge1.180p181 The phenolic acid precursors of the avena- lumins have been analysed using reverse-phase h.p.1.c.' Radioimmunoassay has been developed in order to quantitate the soybean phytoalexin glyceollin I (172) which could be I detected by this method in the range 0.34-34 ng. The method was applied to the analysis of sections of soybean hypocotyls 15 pm thick and indicated that there is a more rapid diminution of glyceollin with increasing distance from the infected site in the interaction between a host and a compatible than an incompatible pathogen. 13 Laser microprobe analysis has also been applied to quantitate glyceollin at the cellular level in soybean.* 4.7 Fungal Degradation of Phytoalexins The metabolism of phytoalexins by fungi has been reviewed up to 1982. 183 Recent work in this area has focused principally on attempts to correlate phytoalexin-degrading ability with the virulence of an organism in uivo. 4.7.1 Studies on the Degradation of Phytoalexins by Fungi Fusarium oxysporum f.sp. lycopersici was found to degrade (-)-medicarpin (1 63) completely to non-aromatic compounds within 20 hours. The (-)-medicarpin was added to the culture medium of the fungus at a concentration of mol dm-3 :the initial step in the degradation was a reductive cleavage of the dihydrofuran ring to afford vestitol and degradation then proceeded via (219) (220) and (221) (see Scheme 21) to give uncharacterized non-aromatic compounds.It was postulated that degradation might involve hydrolysis of the lactone group in (221) decarboxylation to a stilbene and cleavage to benzoic acid derivatives. 84 Botrytis cinerea appears to have the ability to demethylate pisatin (1) (see Scheme 22) since pisatin and 3-0-demethylpisa- tin (223) were isolated from Lathyrus sativus that was infected with the fungus. Pisatin alone was isolated from the same plant when it was irradiated with U.V. light or inoculated with Helminthosporium carbonum. 72 The isoflavone phytoalexin luteone (224) was converted by AspergillusJlavus into (225) and (226) and by Botrytis cinerea into (226) (227) and (228) as shown in Scheme 23. Compounds (225) and (227) exhibited weaker antifungal activity than (224).185 Scheme 21 1 r p" (224) \ r 1 Ho% --+Ob \ OMe OH OH (229) (230) Scheme 24 OH Degradation of the pre-formed antifungal metabolite bio- chanin A (229) by twelve isolates of Nectria haematococca was found to proceed via several steps to yield (230) (Scheme 24).All of the fungal isolates showed similar ability to degrade (229) but varied in their ability to demethylate pisatin (1). This suggested that the degradation of the phytoalexin might be more specific than the degradation of pre-formed antifungal compounds. 86 NATURAL PRODUCT REPORTS 1985 (225) The most fully characterized enzyme that is concerned in the metabolism of a phytoalexin is kievitone hydratase (KHase) which catalyses the hydration of kievitone (Scheme 25) and is found in the culture fluid of Fusarium solani after its induction by the addition of kievitone to the culture medium.Kievitone hydratase is the only phytoalexin-degrading enzyme that has thus far been observed to be secreted into a culture medium and this feature has allowed a number of its properties to be determined. 87 Three isoflavonoid phytoalexins were concur- rently metabolized by F. solani f.sp. phaseoli. Phaseollidin (1 79) kievitone (1 77) and phaseollinisoflavan (232) were each added at a concentration of ca 25 pg ml-l ,to the fungal culture medium :kievitone disappeared within 8 hours phaseollin within 24 hours and phaseollinisoflavan within 30 hours. The degradation products that appeared in the medium were respectively kievitone hydrate (23 l) phaseollidin hydrate (233) and an uncharacterized metabolite from (232).[Cell-free filtrates from the same fungus also converted (177) into (231) but did not metabolize the other two isoflavonoids.] Fungal growth was inhibited during the period of detoxification of the added phytoalexins but resumed rapidly thereafter. 88 4.7.2 The Tolerance of Fungi towards Phytoalexins Certain isolates of Nectria haematococca were found to be tolerant to pisatin without effecting its demethylation. It was considered that tolerance might be associated with a modifica- tion of membrane structure. 89 Four types of micro-organism were found to develop a non-degradative tolerance to the phytoalexin 6-methoxymellein (234) from carrot (Daucus carota).In three instances pre-treatment of the organism with (234) followed by a second treatment indicated an induced tolerance as growth was less inhibited by the second treatment. Carbon-14-labelled (234) was taken up by the mycelium of two of the organisms that were studied without being degraded. 90 NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON 45 1 5.2 Modes of Action of Elicitors Several elements are present in the interaction between a host plant and a parasite. When biological extracts or abiotic elicitors are used experimentally to stimulate the phytoalexin response certain features of the conditions occurring in natural host-parasite interactions are lacking.The use of such unnatural elicitors may nevertheless give an insight into the processes occurring in Nature.2oo The main features that are involved in the initial interaction between host and parasite (232) may be summarized as follows with particular reference to the OH phytoalexin response. The binding to the cell wall of the host of components of the cell HO (233) wall of the parasite that are capable of inducing the accumulation of phytoalexins. This process is dependent on the architecture of the cell wall of the parasite; the components on the outer 0 (234) 4.7.3 The Association between Degradation of Phytoalexins and Virulence of Pathogens A number of isolates (28 altogether) of species of the genera Fusarium and Nectria were tested for their ability to produce kievitone hydratase (KHase).Only ten of the isolates metabo- lized kievitone to kievitone hydrate within 6 hours. As the enzyme is typically extracellular cell-free culture filtrates were examined for KHase activity. Only three isolates from Fusarium species which were known to be pathogenic on Phaseolus vulgaris produced extracellular KHase indicating that there is a link between the enzyme and the virulence of the fungus. 91 Several variants of Fusarium solani f.sp. phaseoli which is a pathogen of Phaseolus vulgaris were obtained by treatment with a mutagen the variants exhibited different abilities to produce KHase and their virulence was correlated with their ability to hydrate kievitone. The KHase activity was found to be of particular importance during the early stages of colonization of the plant by the fungus.192 The enzyme that is responsible for the demethylation of the pea phytoalexin pisatin in the course of its detoxification by Nectria haematococca has been found to be a cytochrome-P-450- linked mono-oxygenase which catalyses the formation of the hemiacetal (222) 93 resulting in the elimination of formalde- hyde to yield (223) (Scheme 22). Genetic analysis of phenotypes that can demethylate pisatin has indicated that a single gene confers the ability to demethylate pisatin rapidly. 94 In numerous crosses of strains of N. haematococca which produce isolates that have different demethylating abilities the most virulent progeny were always those which yielded an isolate that had high demethylating power.Accordingly it appears that the ability to degrade pisatin is an important factor in the virulence of N. haematococca on pea although it is likely that other genes for virulence are also involved.195 5 Elicitors of Accumulation of Phytoalexins 5.1 Introduction The term ‘elicitor’ was introduced to describe any substance that stimulates the biosynthesis of phytoalexins. 96 Elicitors have been divided into biotic and abiotic types to describe respectively elicitors that are of biological origin and those of chemical or physical origin;6 generally there may be little difference in the effects of biotic and abiotic elicitors.197 Another class of elicitors comprises the constitutive198 or endogenous elicitors;’ 99 these are mediators of the phytoalexin response and are produced within the cells of the elicitor- treated or microbially inoculated plant.The formation of endogenous elicitors which can be stimulated by a wide range of biotic or abiotic elicitors appears to be the first stage of a chain of events that are mediated by chemical agents within the plant cell and which lead ultimately to the transcription of mRNA to the synthesis of proteins de novo and finally to the accumulation of phytoalexins. surface of the cell wall are more likely to bind to the cell wall of the plant.201.202 Binding may be facilitated either by enzymes that are normally present within the host or by enzymes that are activated or synthesized in response to infection (e.g.chitin-ase,Io2 P-glucanases or glycosylases) which degrade the cell wall of the parasite releasing elicitor fragment~;~O~-~O~ the activity of these enzymes may be supplemented by that of enzymes that are present within the parasite itself.202*205 Ultimately the transcription of mRNA and the synthesis de novo of the enzymes that are involved in the formation of phytoalexins occur. These processes take place extremely rapidly in response to extracts from cell walls of fungi.141-143 The intercalation of components of its cell wall from the parasite into the DNA of the host. The interaction of materials from the cell wall of the parasite with the genes of the host may elicit the formation of phytoalexins. This process again could be facilitated by cell-wall-degrading enzymes in the host acting to release elicitors from the cell wall of the para~ite.~O~-~O~ Components of the cell wall of the parasite may bind to regulatory genes in the chromatin of the plant thereby directly stimulating the transcription of mRNA and the ensuing synthesis of proteins that are involved in the phytoalexin response.202 The direct release of elicitors from the cell wall of the host.Enzymes that are secreted by the parasite e.g. pectinase can directly release elicitors from the cell wall of the host. These may interact either directly or indirectly with the nucleus of the plant cell ultimately initiating the formation of phytoalexins.206v 207 The indirect release of endogenous elicitors which may be stimulated by one or more of the three previous factors.Elicitors may stimulate either the activation or the synthesis of cell-wall- degrading enzymes within the plant cell which then releases elicitor fragments from its walls. 208 209 Other elicitation processes not yet well defined. Amongst elicitors for which no clear mode of action has been established are arachidonic acid (83)80 and sucrose.210~ Experiments in uitro 21 have shown that arachidonic acid may be released from cell walls of fungi by the action of lipase.80y81 The suppression of the phytoalexin response by compounds of the cell wall of the parasite. Materials from the cell wall of the parasite may suppress the accumulation of phytoalexins. These materials may be specific to those pathogenic races of a fungus which suppress the phytoalexin response in the host facilitat- ing the process of colonization.The suppressor materials possibly act by competing with elicitors for binding sites on the cell wall of the host.211-214 The suppression of the accumulation ofphytoalexins by toxins that are produced by the parasite. lol Such toxins may contribute to the specific effects of virulent fungi which produce a hypersensitive response only when applied to resistant plants (See Section 5.3.6).215,216 5.3 Biotic Elicitors 5.3.1 P-Glucans Elicitor materials that are rich in P-glucans can be produced from a number of fungi by autoclaving their mycelia and NATURAL PRODUCT REPORTS 1985 OH -f OH I OH OH OH PH OH GIC(pl-6 ) GIC(p1-6) G IC(pl-6) G IC(p1-6) G IC 3 3-t t 1 1 Glc G Ic Glc(p1- 6)Gl~((1-6)Gl~ 3 3 t t 1 1 GIc( p1-6)GIc(pl- 6)GIc G Ic (240) OH OH GIC( pl -3 t 1 G Ic GIc(p1 -3 t 1 G lc 6 1GIC(p1-6)GIC(81-6) G IC( p1-6) G IC 3 t 1 Glc (237) 6)GI~((jl-6)Gk(~1-6)Gk(p1-6)Gl~ 3 t 1 G Ic Glc ( pl-6 1Glc (PI -6) GIC( Pl-6) Glc (p1-6) Glc 3 3 t t 1 1 G Ic GIc (242) retaining the aqueous fraction.2 7-2 l9 The best-characterized extract of this type is that from the cell walls of Phytophthora meg~sperma.~~~ This material was found to contain a P-glucan fraction of low molecular weight which was highly active in eliciting the accumulation of phytoalexins in soybean (Glycine max).Larger amounts of this material were produced by acid hydrolysis of the fungal cell The active component in this extract has been characterized as the heptaQ-glucoside (235) which will stimulate the formation of phytoalexins at 50%of the maximum (ED50 response) in soybean cotyledons at a concentration of ca 6 ng m1-1.220v221 The structure of this material is highly specific seven other heptaQ-glucosides [(236)-(242)] that were isolated from the same source were ina~tive.~~~,~~~*~~~ The structure of (235) was confirmed by synthesis (see Section 5.9)2249225 and the synthetic sample was found to have biological action identical with that of the natural material.221 The material is degraded by glycosylase I (an enzyme that has P-1,3-glucanase P-glucosidase and glucosyl transferase activity) which is sited on the outside of the cell walls of soybean and which may be responsible for the to degradation by soybean p-1,3-endoglucanase once it was bound to a receptor site on isolated soybean membranes.227 As the hepta-&glucoside is active at very low concentrations it is important to use an assay method that reduces the likelihood of degradation either by enzymes that are within the plant or by microbial contamination.P-Glucans from Phytophthora infestans have been found to have low elicitory activity in potato (Solanum tuberosum). However when the glucans were applied together with arachidonic acid they enhanced the elicitory activity.of arachidonic acid ca four-fold.82 Similar P-glucans from pathogenic (compatible) races of P. infestans have been found to contain suppressor glucans which block the accumulation of phytoalexins that occurs in response to challenge by incompati- ble races of the fungus.212*213 5.3.2 Glucomannans Elicitors that contain glucomannans were released from the cell walls of Phytophthora megasperma by a purified P-1,3-release of the heptasaccharide from larger gl~~ans.~~~~~~~*~~~ The elicitors produced an It endoglucanase from soybean.201*228 has been shown that a model /3-1,3-glucan was not susceptible EDs0 response in soybean at a concentration of 0.3 pg ml-l. NATURAL PRODUCT REPORTS 1985 -C. J. W. BROOKS AND D. G. WATSON Since the elicitors contained glucosyl residues in high propor- tions it is not possible to rule out that these only partially characterized elicitors may contain the hepta-P-glucoside (235) within their structure.The glucomannans were found to be located on the surface of the fungal mycelium and it was considered that this made them more likely elicitors than p-glucans which are overlaid by both the glucomannans and glycoproteins that together form the surface layers of the mycelium.2o 5.3.3 Chitosan and Oligomers of Glucosamine Most fungi contain the glucosamine polymer chitosan in their cell walls although often in small amounts. Its function in the fungus is perhaps related to dormancy since dormant forms of Fusarium solani contain larger amounts of chitosan than active forms.*02 The function of chitosan as an elicitor has been studied chiefly in relation to the interaction of Fusarium solani with the pea.Chitosan is a potent inducer of the accumulation of pisatin in the pea of the formation of the antifeedant proteinase inhibitor in tomato and of casbene synthetase activity in castor bean. 229 Unlike glucan elicitors chitosan has an affinity for DNA and has been shown to accumulate in the nuclei of pea cells.202 Many plants contain chitinase activity although chitin has not been reported to occur in any plant. Levels of chitinase in tomato have been found to increase in response to fungal infection,,04 and an increase in these levels was rapidly induced in cultures of parsley cells in response to an elicitor that had been prepared from the cell walls of Phytophthora mega~perma.,~~ Such chitinases within the plant may serve along with other enzymes (such as the 1,3-endoglucanases) both to degrade the cell walls of fungi and to release elicitor fragments from the chitosan in those walls.A glucosamine oligomer (DP = 4) was found to be a potent elicitor of proteinase inhibitor factor in tomato.231 A model for the types of genetic interaction that are involved in the elicitation by chitosan has been proposed.202 5.3.4 Glycoproteins Glycoproteins from various races of Cladosporium fulvum were found to be elicitors in tomato in this case the elicitor activity was dependent on both the glycosyl and the protein portions of the mole~ule.~~~.~~~ A similar type of glycoprotein obtained by autoclaving the germ tubes of Uromyces phaseoli was shown to elicit the accumulation of phytoalexins in Phaseolus vulgaris.234 The glycoprotein that could be released from the cell walls of Phytophthora megasperma by the action of PronaseR was a glucomannan conjugate and it was active at concentrations of ca 3 pgml-l as an elicitor in soybean. Glycoproteins from different races of the fungus were found to exhibit race-specific elicitation.2o ,23 An extracellular invertase glycoprotein from the culture filtrates of P. megasperma was found to have race- specific suppressor action. When the glycoprotein was applied with the P-glucan elicitor fraction from P. megasperma it suppressed the accumulation of phytoalexins in those instances where the glycoprotein was derived from a compatible race of the f~ngus.~ * 5.3.5 Cell-wall-degrading Enzymes from the Parasite A highly purified glycoprotein from the culture filtrates of Rhizopus stolonifer proved to have pectinolytic activity and to be an elicitor of casbene synthetase activity in castor bean236.237 and of pisatin in pea.229 A pectinase from Erwinia carotovora has been found to be an elicitor of the accumulation of glyceollins in soybean cotyledons.207 A glycoprotein that has pectinolytic activity and which was isolated from the larvae of the sweet potato weevil (Cyclas formicarius) was an elicitor of the formation of phytoalexins in sweet 239 Various commercial enzyme preparations have been found to be active elicitors.Cultures of carrot cells produced 6-methoxymellein (234) in response to commercial preparations of PronaseR 453 trypsin and polygalact~ronase.~~~~ 241 Commercial polygalac- turonase from Aspergillus niger was found to induce the accumulation of glyceollins in soybean tissue cultures143 and of capsidiol in capsicum fruits:242 the latter effect was also produced by commercial cellulase from Trichoderma viride. 242 5.3.6 Fungal Phy to toxins The production of acetylenic phytoalexins by cultures of safflower (Carthamus tinctorius) in response to an extract from Alternaria carthami was suppressed by a concentration of 100 pmol dm-3 of brefeldin A which is a macrolide toxin that is produced by the fungus.1o1 Such toxins might act as race- specific suppressors; Cladosporium fulvum was found to produce a host-specific toxin which caused necrosis and chlorosis in leaves of the tomato.The toxin was not produced in vitro but was produced when the fungus was grown on a plant with which it was compatible. The intercellular fluid from the tomato plant on which the fungus had been cultured caused necrosis and chlorosis if it was applied to tomato plants with which the fungus was incompatible.215v216 5.4 The Influence of Growth Hormones on the Phytoalexin Response 5.4.I Ethylene The amounts of ethylene that are released by plants are found to increase in response to infection. The production of ethylene by plants that are under stress is caused by enhancement of the activity of 1-aminocyclopropane-1-carboxylate synthase (ACC-synthase).2433 244 1-Aminocyclopropane-1-carboxylic acid is the precursor of ethylene in natural plant systems.Ethylene causes large increases in chitinase activity in a number of plants including the broad bean the tomato and the pea when they are incubated in air that contains ethylene (1 p1 ml-1).245 The addition of the elicitor extract from Phytophthora megasperma caused rapid induction of the biosynthesis of ethylene in parsley cultures. The fact that cycloheximide (which is an inhibitor of the translation of mRNA) inhibited the induction of ACC-synthase activity whereas actinomycin D (which is an inhibitor of the transcription of DNA) did not do so suggested that the mRNA that codes for ACC-synthase was pre-formed.If the production of ethylene was caused to be inhibited by the addition of aminoethoxyvinylglycine to the cultures the PAL activity that was normally induced by the elicitor from P. megasperma was also found to be inhibited. The PAL activity could not be restored by adding exogenous ethylene. These experiments suggest that ethylene is not functioning as an elicitor of PAL but that in order for PAL to be stimulated the reactions that are involved in the production of ethylene must be allowed to function.230 5.4.2 Abscisic Acid and Cytokinins The levels of phytoalexins that were elicited in fresh potato tubers by Phytophthora infestans were very low. The application of abscisic acid (ABA) to the tubers increased the accumulation of phytoalexins in response to challenging by P.infestans. Conversely ABA was found to inhibit the accumulation of phytoalexins in old tubers and to induce the susceptibility of the tissue to incompatible races of P. ' infe~tans.,~~ Induced susceptibility has also been observed in resistant lines of tobacco tissue cultures that had been treated with levels of kinetin or of benzylaminopurine (BAP)247 that are slightly above those that would normally be included in the growth medium. Abscisic acid was found to increase the levels of phaseollin in both light-and dark-grown cotyledons of Phaseolus vulgaris levels of kievitone were increased in dark- but not in light-grown cotyledons. When cotyledons were treated with HgCl, there was a large increase in the levels of both phaseollin and kievitone.Treatment with ABA elevated the levels of phaseollin further but inhibited the accumulation of kievitone. The effects of BAP on the cotyledons were found to be similar to those of ABA. It was suggested that ABA or BAP may modulate the activity of chalcone ~ynthase.~~* 5.5 Endogenous Elicitors 5.5.1 Release by Enzyme Action A polygalacturonase from Rhizopus stolonijer released a heat- stable elicitor from the cell walls of seedlings of castor bean (Ricinus communis); this elicitor stimulated the formation of casbene synthetase and was presumed to be composed of pectic fragments from the cell Material having similar activity and which was released from the cell walls of soybean by pectinase from Erwinia carotovora caused the accumulation of glyceollins in soybean.207 Carrot tissue that had been treated with PronaseB trypsin or polygalacturonase was found to yield an elicitor of 6-methoxymellein in carrot cells.241 A heat-labile elicitor was isolated from soybeans which had been frozen and then thawed.The elicitor might be presumed to be a pectinase since it released a heat-stable elicitor if it was incubated with the cell walls of soybean. This suggests that injury to a plant might activate such endogenous enzymes thereby effecting the release of cell-wall fragments which act as the next link in the chain of elicitor-mediated response.208 5.5.2 Release by Autoclaving or by Acid Hydrolysis An aqueous extract that was prepared by autoclaving soybean tissue was found to elicit the accumulation of phytoalexins in soybean cotyledons.This material was considered to be a polysaccharide containing uronic acid residues. 99 A polysac- charide composed of 67% galacturonic acid residues with arabinosyl galactosyl and rhamnosyl residues making up the NATURAL PRODUCT REPORTS 1985 castor bean. Fractions ranging from nonamers to penta-decamers had some degree of activity the most active material was shown by FAB m.s. to be trideca-a-1,4-galacturonide.A pool of the active elicitors gave an ED50 response at a concentration of 0.6 mg ml-I. Methyl esterification of the pooled elicitor reduced its activity twenty-fold and hydrolysis of the ester restored the activity.2s1 The PIIF polysaccharide from tomato has been partially characterized.The material is similar to the large (ca 200 000 dalton) rhamnogalacturonan I which has been isolated from tissue cultures of sycamore in certain aspects of its structure. Rhamnogalacturonan I has been found to have elicitor activity (in the soybean) after it has been partially hydrolysed by acid. Mild acid hydrolysis of PIIF cleaves it to a polymer (DP = 20) that consists largely of galacturonic acid residues. Further hydrolysis of this material using tomato polygalacturonase suggested that the smallest fragment that was active in eliciting the tomato proteinase inhibitor was 4-0-a-D-galaCtUrOnOSY1-D-galacturonic acid. *5 The addition of elicitor fragments from sycamore cultures to the medium in which sycamore cells were cultured resulted in a suppression of the uptake of [14C]leucine by the culture.This observation indicates that pectic fragments may be responsible for the rapid death of cells and for the hypersensitive response that is observed in infected plants.253 5.6 Abiotic Elicitors 5.6.1 The Mechanism of Action of Abiotic Elicitors Very diverse materials have been found to act as abiotic elicitors of phytoalexins. Their action may be primarily due to injury of the plant cell causing the release of endogenous elicitors but there are other factors involved for instance for This material corresponds to the 'proteinase-inhibitor-inducing factor' (PIIF) that is released when tomato plants are attacked by insects or otherwise injured.This factor has also been shown to elicit both casbene synthetase activity in castor bean and the accumulation of pisatin in the pea.229 Elicitors were released by acid hydrolysis of cell-wall material from soybean sycamore (Acer pseudoplatanus) to-bacco and wheat (Triticum spp.). The elicitor that was released by acid hydrolysis of cell walls of soybean was found to be composed largely (96%) of galacturonosyl residues. It was found that elicitor activity could also be released by the acid hydrolysis of citrus pectin.199 5.5.3 The Characterization of Endogenous Elicitors The elicitor fragments that are produced by the acid hydrolysis of citrus pectin were fractionated and the fractions assayed for their activity in eliciting the accumulation of phytoalexins in soybean.The most active purified fraction was found to be dodeca-cr-l,4-galacturonide.The elicitor that was released by acid hydrolysis of cell walls of soybean was also extensively characterized and was believed to have the same structure as the oligogalacturonide from citrus pectin.250 The soybean oligogalacturonide elicitor was active only in rather high concentrations (ca 250pgml-') so it was possible that the activity might have been due to a small amount of highly active contaminant. It is possible that the structure that is active in vivo is slightly modified and consequently more highly active,220or possibly the material binds less efficiently when it is applied to the cell surface than in vivo.When the oligogalacturonide elicitor and the hepta-P-glucoside elicitor from Phytophthora megasperma were applied together there was an enhancement of activity.220 A polygalacturonase from Rhizopus stolonfer was used to degrade polygalacturonic acid of high molecular weight yielding elicitor fragments that were fractionated and assayed for their ability to stimulate casbene synthetase activity in the potato the conditions of storage of the tubers were found to remainder was isolated from tomato leaf by aut~claving.~~~ influence their ability to respond to abiotic elicitors by accumulating phytoalexins. 2s4 When pea endocarps were treated with Fusarium solani and the proteins that were formed in response to this challenge were pulse-labelled with [35S]methionine two-dimensional electrophoresis revealed the presence of ca twenty proteins that could be associated with resistance.255 By means of translation in uitro of induced mRNA very similar patterns of induced synthesis of proteins were observed after treatment of a pea plant with the abiotic elicitors actinomycin D psoralen U.V.light or quinacrine or with the biotic elicitors Fusariumsolanior chitosan. Only CdCl produced a radically different pattern.?s5 Most of the agents that were used can intercalate with DNA and this suggests the possibility that material (such as chitosan) from a pathogen may directly affect DNA thus inducing the synthesis of phytoalexins.* 97 5.6.2 Sulphydryl Reagents Sulphydryl reagents were used to elicit the accumulation of phytoalexins in callus tissue from Trfolium repens.N-Ethylma-leimide (NEM) iodoacetamide HgCl, and p-chloromercuri- benzenesulphonic acid (PMBS) were all active as elicitors. Co- application of the elicitors with dithiothreitol (DTT) caused inhibition of the activity of the elicitor. The metabolic inhibitors KCN NaF and Na3As04 were not elicitors. It was considered that elicitor action was due to condensation with the thiol groups of proteins in the tissue. 256 N-Ethylmaleimide PMBS p-hydroxymercuribenzoate (PMB) and DTT were found to elicit the accumulation of glyceollins in soybean. The order of effectiveness was PMBS >> DTT > PMB > NEM. Co-application of PMBS PMB or NEM with DTT inhibited their action; PMBS and PMB are about equally reactive with thiol groups but PMB penetrates cells whereas PMBS does not and it was postulated that the high elicitor activity of PMBS was due to its reaction with thiol groups on the surface of the outer cell membrane.257 NATURAL PRODUCT REPORTS 1985 -C.J. W. BROOKS AND D. G. WATSON 455 o xow++o qQH HO H 0 Ph HO OH OH OTH Ph Gent iobiose [Bz = PhCO] OBz OBz OBz BZO BZO OBZ OBz Ph OBZ Ph BtO I OBz OBZ OAc OBZ ~0Bz OBzY 0 Bz BzO AcO Ph OBt OAc 0 Bz OAc Ph OBZ OBt V,VIII 3* 34 -Di -D -glucopyranosylgentiopentaose (235) Reagents i PhCHO ZnC1,; ii CF,SO,Ag; iii Ac20 pyridine; iv 80% CHC1,CO2H HOAc; v 90%CF,CO,H; vi BzC1 pyridine; vii HBr HOAc CH,CI,; viii NaOMe MeOH Scheme 26 5.6.3 The Release of Endogenous Elicitors by Abiotic Elicitors Mercury(I1) chloride was found to cause the formation of an endogenous elicitor of accumulation of phytoalexins in Phaseo-lus ~ulgaris,~~~ possibly via the binding of Hg2+ to thiol groups.Other work209 on the release of an endogenous elicitor by treatment with an abiotic elicitor has involved treatment of tissue cultures of P. uulgaris with denatured RNase A the resulting elicitor material was found to diffuse across a dialysis membrane and to stimulate the enzymes of phenylpropanoid synthesis. A crude exudate from autoclaved hypocotyls of the same bean was found to contain a diffusible component that enhanced the activity of enzymes that are involved in the synthesis of phenylpropanoids.A fungal extract from Colleto-trichum lindemuthianum did not produce transmissible elicitor activity from cells of P. ~u1gari.s.~~~ 5.7 Fungicides as Abiotic Elicitors In an increasing number of instances the activity of fungicides may be attributed to an elicitation of the natural defences of the plant. Soybean plants that had been treated with metalaxyl and then incubated with a compatible race of Phytophthora megasperma developed festricted lesions the levels of accumu- lation of glyceollins,were low and most of the restriction in fungal growth was attributed to the high concentrations of metalaxyl that were found to be in the lesions.258 In another experiment higher local concentrations of glyceollins were found in soybeans that had been treated with an incompatible race of P.megasperma than in plants that had been inoculated with a compatible race. The application of metalaxyl to plants followed by inoculation with a compatible race of P. megasperma resulted in localized accumulation of glyceollins (similar to that which was observed in the interaction with the incompatible race) and an associated local restriction of the growth of the fungus.259 A similar potentiation of the rate and amount of accumulation of phytoalexins was observed in capsicum fruits and in tobacco stems that had been treated with aluminium phosethyl [aluminium tris(ethy1 phosphonate)] prior to inoculation with Phytophthora nicotiana.In tobacco plants that had been treated with aluminium phosethyl and then inoculated with compatible P. nicotiana fungal growth was restricted to within 5 mm of the point of inoculation whereas in untreated plants the growth was not restricted. Aluminium phosethyl itself has little antifungal activity.260 Treatment of rice seeds with a number of abiotic elicitors prior to germination increased their resistance to brown spot disease (Drechslera oryzae). Ions of heavy metals were particularly effective the increased resistance in the plants persisting for 5-7 weeks. It was found that three weeks after germination extracts from plants that had grown from treated seed were not antifungal but that if diffusates were taken from such plants after they had been infected with a fungus they had antifungal activity.26 1,262 Some fungicides have been found to elicit antifungal activity directly.Treatment of the roots of capsicum tomato and eggplant (Solanum melongena) with carbendazim or benomyl led to the accumulation of fungitoxic compounds. Some of the antifungal materials were shown to contain known phytoalex- ins e.g. capsidiol in capsicum and rishitin in tomato.263 Pretreatment of seedlings of tomato and of eggplant with sublethal doses of dinitroaniline herbicides markedly increased their resistance to attack by species of the genera Fusarium and Verticillium; the herbicides were found to stimulate the production of fungitoxic materials by the plants but these were probably phenolic rather than terpenoid compounds.264 Aci-fluorfen was found to increase the levels of phytoalexins in several crops e.g. glyceollins in soybean; medicarpin and wyerone in broad bean (Vicia faba) and hemigossypol in cotton. Acifluorfen is phytotoxic and in the field its toxicity has to be counteracted by its co-application with agents such as amino-oxyacetic acid. 265 The potential for induced resistance to fungi in plants has been the subject of a recent article.266 NATURAL PRODUCT REPORTS 1985 5.8 The Analysis and Characterization of Carbohydrate Elicitors of the Accumulation of Phytoalexins Initial purification of carbohydrate-type elicitors from crude active mixtures has been carried out chiefly by gel chromato- graphy and by ion-exchange chromatography.High-perform- ance liquid chromatography has proved to be effective for the separation of small quantities of active material the hepta-& glucoside elicitor from Phytophthora megasperma was isolated by h.p.1.c. first on an aminopropylated silica column and then on a reverse-phase column with acetonitrile-water (2 :98 v/v) as the eluent (0.3ml min-I). Under these conditions the anomers of each oligosaccharide were separated but this complication was removed by reducing the terminal aldose groups -a process which did not materially alter the activity as an elicitor.222 Oligogalacturonide-type elicitors have been separated by h.p.1.c. on anion-exchange phases.250*252 Appli-cations of h.p.1.c. to separations of carbohydrates have been reviewed.267 Fast-atom-bombardment mass spectrometry (FAB m.s.) has been used to characterize oligogalacturonide elicitors that had been derived from the cell walls of plants,250.251 and the technique has been reviewed.268 The determination of the primary structure of carbohydrate- type elicitors is based on increasingly refined analyses of the compositions and sequences of glycosyl residues followed by assignments of absolute configuration and of the ring form of each glycosyl component.Typical procedures involve permeth- ylation partial hydrolysis reduction of the resultant partially methylated oligosaccharides ethylation and separation by reverse-phase h.p.1.c. The 0-methyllo-ethyl oligomers may be hydrolysed to monomeric species that are suitable (with or without prior reduction to alditols) for determination and identification by g.1.c.-m.s.The structures of the oligomers that were originally formed by partial acid hydrolysis may then be inferred. The anomeric configurations of glycosyl linkages in peralkylated oligosaccharides are determinable from H n.m.r. measurements. The methods that have been outlined here are applicable to microgram amounts of materia1.2691270 5.9 The Synthesis of a Hepta-P-glucoside Elicitor The glucan elicitor 32,34-di-(0-~-~-glucopyranosyl)gentiopen-taose (235) has been synthesized from gentiobiose as outlined in Scheme 26.2249 225 6 References 1 K. 0. Muller and H. Borger Arb. Biol. Reichsunst. Land. Forstwirtsch. Berlin-Dahlem 1940 23 189.2 K. 0. Muller Phytopathol. Z. 1956 27 237. 3 B. J. Deverall in ‘Phytoalexins’ ed. J. A. Bailey and J. W. Mansfield Blackie Glasgow and London 1982 p. 1. 4 H. Grisebach Mycol. 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Nishi Phytochemistry 1983 22 669. 241 F. Kurosaki and A. Nishi Physiol. Plant Pathol. 1984 24 169. 242 D. G. Watson and C. J. W. Brooks Physiol. Plant Pathol. 1984 24 331. 243 J. Fuhrer Plant Physiol. 1982 70 162. 244 A. M. M. de Laat and L. C.van Loon Plant Physiol. 1982,69,240. 245 T. Boller A. Gehri F. Mauch and U. Vogeli Planta 1983 157 22. 246 R. M. Bostock E. Nuckles J. W. D. M. Henfling and J. A. KuC Phytopathology 1983 73 435. 247 T. Haberlach A. D. Budde L. Sequeira and J. P. Helgeson Plant Physiol. 1978 62 522. 248 J. F. V. Goossens and J. C. Vendrig Planta 1982 154 441. 249 C. A. Ryan P. Bishop G. Pearce A. G. Darvill M. McNeil and P. Albersheim Plant Physiol. 1981 68 616. 250 E. A. Nothnagel M. McNeil P. Albersheim and A. Dell Plant Physiol. 1983 71 916. 251 D. F. Jin and C. A. West Plant Physiol. 1984 74 989. 252 P. D. Bishop G. Pearce J. E. Bryant and C. A. Ryan J. Biol. Chem. 1984 259 13 172. 253 N. Yamazaki S. C. Fry A. G. Darvill and P. Albersheim Plant Physiol.1983 72 864. 254 A. S. Cheema and N. F. Haard Physiol. Plant Pathol. 1978 13 233. 255 W. Wagoner D. C. Loschke and L. A. Hadwiger Physiol. Plant Pathol. 1982 20 99. 256 D. L. Gustine Plant Physiol. 1981 68 1323. 257 P. Stoessel Planta 1984 160 314. 258 G. Lazarovitz and E. W. B. Ward Phytopathology 1982,72,1217. 259 H. Borner G. Schatz and H. Grisebach Physiol. Plant Pathol. 1983 23 145. 260 D. 1. Guest Physiol. Plant Pathol. 1984 25 125. 261 A. K. Sinhaand G. N. Hait Trans. Br. Mycol. SOC. 1982,79,213. 262 D. N. Giri and A. K. Sinha Ann. Appl. Biol. 1983 103 229. 263 V. Emmanouil and R. K. S. Wood Physiol. Plant Pathol. 1983,22 51. 264 A. Grinstein N. Lisker J. Katan and Y. Eshel Physiol. Plant Pathol. 1984 24 347. 265 T. Komives and J.E. Casida J.Agric. Food Chem. 1983,31,751. 266 L. Sequeira Trends Biotechnol. 1984 2 25. 267 L. A. T. Verhaar and B. F. M. Kuster J. Chromatogr. 1981,220 313. 268 A. Dell and G. W. Taylor Mass S ectrom. &v. 1984 3 357. 269 M. G. McNeil A. G. Darvill P. {man L.-E. Franzen and P. Albersheim Methods Enzymol. 1982 83 3. 270 T. J. Waeghe A. G. Darvill M. McNeil and P. Albersheim Carbohydr. Res. 1983 123 281.

ISSN:0265-0568    

DOI:10.1039/NP9850200427

出版商:RSC    

年代:1985

数据来源: RSC

摘要:

Steroids Reactions and Partial Syntheses J. Elks formerly of Glaxo Group Research Ltd. Green ford Middlesex UB6 OHE Reviewing the literature published during 1983 (Continuing the coverage of literature in Natural Product Reports 1984 Vol. 1 p. 391) 1 1.1 1.1.1 1.1.2 1.1.3 1.1.4 1.2 1.2.I 1.2.2 1.3 1.3.1 1.3.2 1.3.3 1.4 1.5 1.6 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 3 Reactions Alcohols and Carboxylic Acids and their Derivatives Halides and Epoxides Oxidation Substitution and Reduction Ethers and Esters Epoxide Ring Opening Other Reactions Unsaturated Compounds Electrophilic Addition Other Reactions of Olefinic Steroids Carbonyl Compounds Reduction and Dehydrogenation Other Reactions Reactions of @Unsaturated Carbonyl Compounds and Enols or Enolic Derivatives Compounds of Nitrogen Phosphorus Sulphur and Selenium Remote Functionalization Reactions Photochemical Reactions Partial Syntheses Cholestane Derivatives and Analogues Vitamins D their Derivatives and their Metabolites Pregnanes Androstanes and Oestranes Cardenolides and Bufadienolides Heterocyclic Compounds Cyclopropano-steroids Microbiological Transformations References A review of steroids' and one on the brassinosteroids* have been published.The proceedings of the Sixth International Congress on Hormonal Steroids have appeared .3 Under the auspices of the World Health Organisation a search has been started in fourteen centres world-wide for a long-acting oral contraceptive.The first results have been published in an issue of Steroids.j 1 Reactions 1.1 Alcohols and Carboxylic Acids and their Derivatives Halides and Epoxides 1.1.1 O.uidation Substitution and Reduction Pyridinium chlorochromate in the presence of excess 33- dimethylpyrazole oxidizes steroid allylic alcohols selectively; saturated secondary hydroxy-groups are not affe~ted.~ The same oxidizing agent in the presence of calcium carbonate is used in a simplified one-step procedure for the preparation from cholesterol of cholest-5-en-3-one of very high purity.6 3p-Hydroxy-compounds react with pyridinium chlorochromate to give chromate complexes that yield the 3-ketone dimethyl acetal on treatment with methanol.' Steroids are specifically deuteriated or tritiated by reduction of the appropriate tosylate or mesylate with zinc and sodium iodide in 1,2-dimethoxyethane that contains deuterium oxide or tritium oxide; other reducible groups are unaffected i -? AcO C H2CH2F lii tiv I CHzCHZX C HzCH20TS (X = I or Br) Reagents i CIFCHCF2NEt,; ii Ph,PBr2 or (PhOCH?),P.HI; iii TsOAg; iv KF HOCH,CH,OH Scheme 1 although label may be introduced into the adjacent positions to a ketonic group.Secondary tosylates undergo some epimeriza- tion during labelling.8 3~-Acetoxy-6P-(2-fluoroethyl)-19-norcholest-5( 10)-ene (2) is prepared from the corresponding hydroxyethyl compound (1) by use of (2-chloro-l 1,2-trifluoroethyI)diethylamineor via the iodide (or bromide) and tosylate as shown in Scheme 1.9 1.1.2 Ethers and Esters 3-Ethers are prepared as mixtures of epimers (30 predominat- ing) by the action of cupric chloride and the appropriate alcohol upon the 3P-to~ylhydrazide.'~ Both methyl and methylthio- methyl ethers of 3-hydroxy-steroids are cleaved by trimethyl- silyl chloride in acetic anhydride to give the 3-acetate with retention of configuration.Another method of cleaving methylthiomethyl ethers involves the use of trityl tetrafluoro- borate; an oxidative mechanism is proposed with MeSCH- (0H)O-steroid as an intermediate. The method is appli- cable to 17~-hydroxypregnan-20-ones as well as to 3-hydroxy- compounds. A 17cr-ethynyl-17p-ol is converted into its tetra-0-acetyl-D- glucuronide as a mixture of anomers by treatment with methyl tetra-0-acetyl-P-D-glucuronate in the presence of stannic chloride.I3 Dihydrotigogenin diacetate (3) is oxidized by ozone in chloroform that contains 10%ethanol to give a 90% yield of the 16a-ethoxy-derivative (4;R = OEt) which is converted into the hemiketal (4;R = OH) on chromatography on silica.14 NATURAL PRODUCT REPORTS 1985 w CH~OAC Me0 AcO I H (3) 1 (5) J NZCHCOZJ53% 0Oh (7) Hindered hydroxy-groups including 1 1P-hydroxy-groups are benzoylated by treatment with benzoyl triflate (Bz-OSO,CF,).' Whereas a 17cr-hydroxy-20-ketone if it is unsub- stituted at C-21 cannot be converted into its pivaloate by treatment with pivalic anhydride in the presence of 4-dimethylaminopyridine the corresponding 21-acetoxy-com-pound is so converted in 70% yield ; the reaction is thought to involve a 20,21-enol pivaloate which rearranges.l6 Cholesterol on treatment with t-butyl nitrite in chloroform gives cholesteryl nitrite in quantitative yield ; a 17P-hydroxy- compound reacts similarly. Cholesteryl diazoacetate (6) is prepared by esterifying cholesterol with the tosylhydrazone of glyoxyloyl chloride and treating the product (5) with triethylamine. An improved method has been described for the preparation 1 $8"17 OH OH Me0m Me0& . OH OH of methyl esters of bile acids.' The reaction of cholic acid or deoxycholic acid with benzenesulphonyl chioride (or its 4- methyl or 2,4,6-tri-isopropyl derivative) gives the 12,24-lactone (7) as its 3-aryl~ulphonate.~~ I.I .3 Epoxide Ring Opening 5,6P-Epoxy-3P-methoxy-5f3-cholestane (8) on treatment with boron trifluoride etherate in diethyl ether gives the 5cr-fluoro- 6P-hydroxy-compound (9) together with a little of the backbone-rearranged product (1 0); the latter predominates if benzene is the solvent." The reaction of a 5a-hydroxy-3P-methoxy-6-ketone (1 1) with dimethylsulphonium methylide gives none of the 6~,6a-epoxy-6cr-methyl-compound (1 3) but instead produces a mixture of the 5a,6cr-epoxy-6f3-hydroxy-methyl-steroid (12; R = H) and its methyl ether (12; R = Me); the 6~,6a-epoxy-5cl-hydroxy-6a-methyl-steroid (13) which was made by another route equilibrates to give predominantly (12; R = H) on treatment with potassium hydroxide in boiling tetrahydrofuran.' Other examples of participation of a neighbouring group in the opening of an epoxide ring are provided by the conversion of 3cr-acetoxy-5,6P-epoxy-5P-cholestane into the 5cr-acetoxy-3cc,6~-dihydroxy-compound on storage in solution or treatment with boron trifluoride2' and by the hydrogen-bromide-catalysed conversion of 4af3,5-epoxy-3~- hydroxy-4,4-dimethyl-~-homo-5~-cholestane (14) into the 3cr,5cr-epoxy-4a~-hydroxy-compound (1 5);analogous reactions NATURAL PRODUCT REPORTS 1985 -J.ELKS occur with the corresponding 3P-hydroxy-4aa,Sa-epoxide and with the S~,6~-epo~y-3a-hydroxy-analogue.~~ The conversion in the presence of perchloric acid of (24R,25S)-30,26-dibenzoyloxy-24,25-epoxycholest-5-ene(1 6) into both (24R 25R)-3P,24-di benzoyloxy-25,26-di h ydrox yc ho- lest-5-ene (1 7) and the product of simple ring-opening has been examined with the benzoyl group labelled on the carbonyl oxygen with l80 and with H2180.The mechanism of the rearrangement is thought to involve the intermediates that are shown. An analogous reaction occurs with the (24S,25R)- analogue of (1 6). 24 2~-Halogeno-3a-hydroxy-compounds (including the iodo-compound) are prepared from the 2a,3a-epoxide by treatment with the halogen and triphenylpho~phine.~~ The reaction of androstd-ene-3,17-dione with alkaline hydrogen peroxide gives a mixture of epoxides in which the p-isomer predomi- nates.Treatment of this mixture with Olah’s reagent (HF in pyridine) gives the 5a-fluoro-4a-hydroxy-compound.26 4-Acetoxywithanolide E (18) is converted into withaperuvin C (19) by treatment with tetrakis(tripheny1phosphine)palla-(16 1 dium; other withanolide derivatives analogous to (1 8) behave in a similar fashion. The reaction appears to involve the rapidly formed q*-complex and the (n-al1yl)palladium complex which is formed relatively slowly.27 The reaction of a Sa,lOa-ep~xy-A~(~ ‘)-compound (20) with vinylmagnesium bromide and cuprous chloride gives the 5a-hydroxy-1 l~-vinyl-9(10)-olefin (21 ; R = CH=CH2); the dipyridylcopper magnesium bromides react in a similar way to give (21 ; R = 2- 3- or 4-~yridyl).*~ Potassium t-butoxide and lithium di-isopropylamide do not catalyse the opening of 43-or 5,6-epoxides presumably because of their bulk ; lithium diethylamide catalyses the conversion of the epoxides (a-or p-) into one or other of the allylic alcohols that are formed by abstraction of P-hydrogen or into a mixture of both.29 An 8a,9a-epoxy-7P-hydroxy (22) or an 8a,14a-epoxy-7P-hydroxy-cholestane (23) is converted by alcoholic hydrogen CHZOH &OH (17) ‘SHl7 4 OH (25 1 (24) AcO (18) (19) 2 464 NATURAL PRODUCT REPORTS 1985 chloride into the aromatic ring c compounds (24) (the major reaction of the acid chloride with the sodium salt of 2-product) and (25) together with the 7-oxo-8(9)-ene or 7-0x0- mercaptopyridine N-oxide gives the N-acyloxy-pyridine-2-8( 14)-ene ;the 7a-hydroxy-analogues give only these unsatu- thione which rearranges on being heated with loss of carbon rated ketones.The difference in the behaviour of the epimers dioxide to give a 2-pyridyl sulphide (26). This is reduced to the has been explained on geometrical grounds. 30 decarboxy-compound on treatment with nickel or nickel boride. Alternatively the pyridine-2-thione is converted directly into the product by treatment with tributyltin I.I .4 Other Reactions h~dride.~' The 9,ll-seco-l l-oic acid (27) is converted into the Barton et al. have described a new radical method for chloro-decarboxy-compound (28) by treatment with lead tetra- decarboxylation of side-chain carboxylic acids (Scheme 2). The acetate and trityl chloride under irradiation with visible light.32 Lorenc et al.have examined the effect of iodine and mercuric oxide upon the 19-nor-compound (29) in order to discover the influence if any of the 10-methyl group on the reaction. The products were the 5,lO-seco-compounds (30; 2 and E isomers) analogous to those that are formed from the 10-methyl compound.33 il i I .2.1 Electrophilic Addition The reaction of 5a-androst-2-ene with iodine and thiocyanogen under irradiation with ultraviolet light gives a mixture of isomers with the 2P-iodo-3a-thiocyanato- and 3a-iodo-2P-thiocyanato-compounds predominating; when the reaction occurs in the dark the 3a-iodo-2~-isothiocyanato-compound is the major product. A free-radical pathway has been suggested for the irradiated reaction.34 Whereas iodine azide (from N-(26) iodosuccinimide and hydrazoic acid) reacts with the A14-compound (31) to give the 14a-azido-1 Sa-iodo-compound (32) Reagents i 2-Mercaptopyridine N-oxide 4-dimethylaminopyridine; which is reduced by lithium aluminium hydride to the 14P 1Sg-ii heat; iii Ni or nickel boride; iv Bu,SnH epimino-compound (33) the reaction of the same olefin with N-Scheme 2 bromosuccinimide and hydrazoic acid gives the 1Sa-azido-14P-bromo-compound (34).The latter is converted (by triethyl phosphite followed by lithium aluminium hydride) into the 14a 15a-epimine (35). Similar results were obtained by using cyanamide with the N-halogeno-imides (giving after treatment of the products with potassium bicarbonate the N-cyano- epimines) or by using cyanic acid and the N-hal~geno-imides.~~ The difference has been explained in terms of an ionic mechanism for the reactions of N-iodosuccinimide and a radical one for the reactions of N-bromosuccinimide.A 3-0x0-(27) 4,6-diene on reaction with bromine azide yields the 7a-azido- 6~-bromo-compound as the major product through conjugate addition of azide ion followed by reaction of the azido-dienol with positive bromine. A 3-oxo-l,4,6-triene reacts similarly but in the presence of hydrazoic acid the reaction gives mainly the la-azido-2~-bromo-compound.The 6-bromo-7-azides react AcO with sodium azide in dimethvlformamide to give the 4-azido- OH 0 4,6-dienes. If the polarity of the 6-7 bond is reversed as in compound (36) the reaction with bromine azide gives a (30) mixture of stereoisomeric 6-azido-7-bromo-compounds (37) 082 OH ti (32) (33) OBr OH NATURAL PRODUCT REPORTS 1985 -J.ELKS CHzOAC I Ph N3 N3 (36) (37) (38) which can be dehydrobrominated to the 6-azido-A6-compound copper but very slowly by methylcopper; hence the first two (38).36 reagents are best avoided if a single isomer is required.42 Testosterone propionate reacts with allene in the presence of P. Kocovsky and his co-workers in a series of pape~s,~~.~~ silica to give the adducts (48) and (49) in roughly equal describe the effects of neighbouring oxygen-containing groups or of double-bonds on the action of hypobromous acid upon ring A or ring B olefins. They conclude that if the participating group is an alcohol or ether 5(0)" participation takes precedence over 6(0)" participation to the extent that it will if necessary force a di-equatorial ring-opening of the intermedi- ate bromonium ion.However in a competition between an alcohol or ether group and an acyloxy-group the former will take precedence even if it involves 6(0)" participation against 5(0)"." participation by the ester group. I .2.2 Other Reactions of'Olefinic Steroids The ergosterol-triazoline adduct (40) can be prepared by oxidizing the triazolidine (39) with phenylseleninic acid or its anhydride dianisyl telluroxide or diphenyl selenoxide in the presence of ergosterol. Regeneration of the 5,7-diene can be achieved with 2M potassium hydroxide in ethanol.39 A 4-allenyl-3-ketone (42) is prepared from the 3a,4a-epoxide by treatment with the lithium acetylide-ethylenediamine complex to give the ethynyl alcohol (41) followed by oxidation with a chromate.s0 The reaction of 17cr-ethynyl-17P-hydroxy-compounds (esterified with trifluoroacetic methanesulphinic or methanesulphonic acid) with phenylzinc chloride in the presence of tetrakis(tripheny1phosphine)palladium as the catalyst gives the two isomeric allenes (43) and (44) with heavy predominance of the former.The allene-palladium complex (45) has been postulated as an ir~termediate.~' The isomeric 17-allenyl compounds (46) and (47) are interconverted by dimethylcopper magnesium chloride or by lithium dimethyl- amounts. In the absence of silica compound (48) is favoured by a factor of 5 :1 at 11"C or of 9.5 :1 at -78 "C.This and some allied results has been explained by adsorption of the unhindered a-face of the enone on the silica thus leaving the p-face more available for reaction with the allene.43 Both 4P-acetoxy-3P-hydroxy-(50) and 3P-acetoxy-4P-hy-droxy-androst-Sen-17-one (5 I) undergo rearrangement in boiling acetic acid to the 3~,6~-diacetoxy-A4-compound (52) but the 3~,4~-diacetoxy-compound does not. There is evidence from labelling experiments that the acetoxylium ion (53) is involved in the rearrangement.4s The 9a-hydroxy-l5-0~0-8( 14)-ene (54) or the 8(9) 14-diene (55) on oxidation with chromic oxide in acetone at IOOC give the 8cr,9cr-epoxy-l4a-hydroxy-l5-ketone (56); further oxidation of (54) or (55) with chromic acid at room temperature gives the 11,15-dione (57).45 Ozonolysis of cholesterol has been re-examined.The reaction in non-participating solvents (carbon tetrachloride alcohol- free chloroform or methylene chloride) at -78 "C gave complex mixtures of products which on reduction yielded the seco-aldehyde (59) as the major product. If chloroform that contained an alcohol was used as the solvent the epidioxy- compound (58; R = alkyl) resulted while if the ozonolysis occurred in water with a dispersion of cholesterol or in 50% aqueous tetrahydrofuran or 50% aqueous acetic acid the corresponding alcohol (58; R = H) was obtained. The isomeric 5,6-epoxides were minor products. The conclusion has been drawn that the epidioxy-alcohol is the primary product and that this gives rise to the seco-aldehyde on reduction.Indeed while NATURAL PRODUCT REPORTS 1985 + c L2 il X-Me0(yp \ (461 (47) + "// 0a-a H2C (50) (52) (51) A (54) \ 'BHl7 +do I A c0 ;I OH --7+&0 O A c0 ;I OH AcO c AcO A A ~ NATURAL PRODUCT REPORTS 1985 -J. ELKS 7 HO OR HO2C d? (61) (60) 0&-:&A A (Me2N),P0 (62) A (63) - H H (64) 2a -Br 5a -H (65) 501-H (66) 28 -Br 5p -H (67) 58-H ___) 0GP H (68) 4a-Br 501 -H (69) 5a -H (70)48-Br,58-H (71) 58 -H (58 ;R = H) is relatively stable in anhydrous methanol or acetic acid the addition of a drop of water causes reduction to the seco-aldehyde (59) to occur within 48 A5-Steroids are converted into the 5a-hydroxy-compounds by a system consisting of oxygen chloromanganese(rI1) 5,10,15,2O-tetraphenylporphyrinate and sodium borohy-dride.47 Cholest-5-ene-3a-carboxylicacid on treatment with perchloric acid in dioxan at 80°C for 10 days gives the hitherto undescribed lactone (60).48 The hazards of ozonolysis can be avoided in the preparative- scale oxidative cleavage of 4-en-3-ones by alkaline hydrogen peroxide in the presence of AdogenB 464 as a phase-transfer catalyst.The ozonide is decomposed as it is formed with formation of the 0x0-acid ;progesterone and cholest-4-en-3-one give the respective 5-ox0-4-nor-4,5-seco-3-oic acids (61) in ca 85% yield.49 1.3 Carbonyl Compounds I .3.I Reduction and Dehydrogenation Hydrogenation of a series of A4-compounds some of which have 10-methyl groups and some of which do not has been studied.With pyridine as the solvent the angular methyl group had little effect on the rate of hydrogenation and only a very small effect in improving the stereoselectivity. In tetrahydro- furan-hydrogen bromide 19-nor-steroids are hydrogenated much faster than their 10-methyl counterparts and give almost wholly the 5P-compound. The results have been interpreted in terms of a transition state with an sp3-like conformation at C-5 in the first instance and an $-like conformation in the second. 0x0-groups at positions 11 or 17 increase the speed of hydrogenation and reduce the stereoselectivity either in acidic or in basic condition^.^^ I .3.2 Other Reactions A good procedure has been described for the conversion of 17P- t-butoxy-5a-androstan-3-one into the A2-compound (63) by reduction of the enol phosphoramidate (62) with lithium in ammonia.51 Dithioketals that are difficult to prepare by conventional methods can be made by catalysis with magne- sium or zinc trifluoromethanesulphonate ;even highly hindered ketones will undergo the reaction.52 In order to improve the regiospecificity of homologation of 3- ketones the migratory aptitudes of carbon atoms 2 and 4 were made more diverse by bromination of one of them.Thus 2a- bromo-5a-cholestan-3-one (64)and 2P-bromo-5p-cholestan-3-one (66) gave respectively ~-homo-Sa- (65) and ~-homo-SP- cholestan-3-one (67) on treatment with diazoacetic ester and boron trifluoride etherate followed by debromination with zinc hydrolysis and decarboxylation.The corresponding 4- bromo-compounds (68) and (70) yielded the isomeric A-homo- 501- and ~-homo-5~-cholestan-4-ones(69) and (71). A 2a-acetoxy-group has the same directing effect as bromine at position 2 although a Baeyer-Villiger oxidation of the 2a- acetoxy-3-ketone gives the 4-0x0-3-oxa-compound ;the conclu- sion is drawn that steric factors over-ride the electronic ones in the diazo-homologat ion.53 A hydroxy- methoxy- or acyloxy-group at position la 2a 2p 3a or 30 has the effect of increasing the proportion of the 7- oxa-6-one (73) over the 6-oxa-7-one (74) that is formed in the Baeyer-Villiger oxidation of 5a-steroid 6-ketones.The effect increases with two such groups or with strongly electron- withdrawing groups such as to~yloxy.~~ Since a 12a-acetoxy- group increases (somewhat) the proportion of 17p-methyl-17a- carboxylic acid that is formed in the Favorskii rearrangement of 17ar-brom0-20-ketones the effect of removing the 13p-methyl group was investigated. As expected rearrangement of 468 NATURAL PRODUCT REPORTS 1985 MeOA @-Q+E 0 HO H A H (83) 3P-acetoxy- 17a-bromo- 18-nor-5a-pregnan-2O-one (75) was still less stereoselective giving the 17a-methyl-l7~-carboxylic ester (76) and its epimer (77) in a ratio of ca. 5:1 along with the 170- hydroxy-18-nor-5a7 17a-pregnan-2O-one which was the major product.Both 5a-and 5P-cholestan-3-one react with potassium chlorate and 30% sulphuric acid in dioxan to give respectively the 2,2,4a-trichloro- and the 2P,4,4-trichloro-derivatives. Re-duction of the former with chromous acetate gives successive- ly the 2a,4a-di- and the 4a-mono-chloro-compound while the latter yields the 2P,4P-di- and the 2~-mono-chloro-compound ; (751 these monochloro-compounds are difficult to prepare by other methods.56 3-Ketones 17-ketones and 20-ketones react with 2-bromo- methylacrylic ester and zinc to give the a-methylene-lactones [e.g. (78)].s7 A 1w-hydroxy-17-ketone yields almost entirely the 17P-alkyl-l7a-01 on reaction with a Grignard reagent but C02Me lithium alkyls give the 17a-alkyl-l7P-01; in the absence of the 14-hydroxy-group attack by either reagent occurs from the 0-face.The effect has been explained by the formation of a cyclic complex between the 14P-OLi and the carbonyl group.s8 + POzMe (76) (77) 1.3.3 Reactions of aP-Unsaturated Carbonyl Compounds and Enols or Enolic Derivatives A one-step preparation of 17a-acetoxy-6-methylpregna-4,6-diene-3,20-dione involves the reaction of 17a-acetoxyproges- terone with methoxymethyl acetate and phosphorus oxychlor- ide in the presence of sodium acetate in chlorof~rm.~~ 16-Methylene-17-ketones (82) are prepared from the 16-hydroxy- methylene-17-ketone by the action of formaldehyde; the primary products (79) and (80) are each converted by alkali (78) into the 16a-hydroxymethyl-17-ketone(8 1) and thence into compound (82).60 2-Hydroxymethylene-5a-cholestan-3-one reacts with p-methoxyphenyl-lead triacetate to yield 24p- 0 methoxyphenyl)-5a-cholestan-3-one(83);a similar reaction has been used to prepare a 16a-(p-methoxyphenyl)-17-0ne.~A new method for the preparation of derivatives of 6-formyltes-tosterone involves the use of ethyl orthoformate in the presence of boron trifluoride etherate.62 19-Nortestosterone 17-trimeth- HoH ylsilyl ether reacts with lithio-phenylacetonitrileto give both isomers of compound (84).This is protected (as the ethylene ketal) and then treated with sodium hydroxide in dimethyl sulphoxide that contains benzyltrimethylammonium chloride 0 0 to give after deprotection the 50-benzoyl-3-ketone (85).'j3 Steroid ap-unsaturated ketones are reduced to the saturated ,,CHzOH ketones by tributyltin hydride in the presence of azobisiso- butyronitrile in high yield.A4-3-Ketones give mixtures of PCHO epimers with the SP-compound predominating except in the presence of a la-methyl (79) (80) 1lp-Aryl-4,9(10)-dien-3-ones (86) if they are subjected to a Birch-type reduction yield the 11p-ary1-q lO)-en-3-ones (87) in which the double-bond cannot be made to migrate to the 4-5 position; this has provisionally been attributed to the effect of the hydrogen atoms at the ortho-positions of the aryl ring. With 0 a benzyl group replacing the aryl group the reaction succeeded 0 but required rather drastic condition^.^^ Cyanohydrins of ring A ap-unsaturated ketones are prepared by the action of trimethylsilyl cyanide with subsequent acid hydrolysis of the trimethylsilyl ether.Mixtures of epimers are formed except from a A'-3-ketone which gives solely the 3p-trimethylsilyloxy-3~tr-cyano-compound.~~ NATURAL PRODUCT REPORTS 1985 -J. ELKS 0siMe +&+oL@ 0 0 Ph CHCN COPh CL H HH c I0-qJ ;a d cI-H OCOR OMe An 1 1 a-formylmethyl-11P-hydroxy-l,4-dien-3-one (88) reacts with lithium metal and biphenyl in tetrahydrofuran to give the epimers (89). Mesylation of the alcohols and treatment of the products with sodium acetate and acetic acid causes dehydration accompanied by migration of the double-bond and gives compound (90).67 A mixture of trimethylsilyl chloride and acetic anhydride acts as a source of acylonium ions and this mixture optionally with the addition of sodium iodide converts ketones into their enol acetates in high yield; the reaction has been applied to a saturated 3-ketone to a 3-oxo-4-ene to a 3-oxo-5-ene to a 7- one to a 17-one and to progesterone; the last was enol acetylated at both position 3 and position 20.@ Enol trifluoromethanesulphonates are prepared in good yield from the enolate anion and N-phenyldi(trifluoromethanesu1pho-ny1)imide in tetrahydrofuran or dimethoxyethane ;they can be used for the generation of olefins with known orientation of the d~uble-bond.~~ Absolute rates of bromination were measured for A2-3-enol ethers and A2-3-enol acetates.Axial substituents in the ring e.g. 5a-substituents slowed the reaction by large factors. From the effect of bromide ion the conclusion has been drawn that the intermediate is a highly unsymmetrical bromonium ion rather than a simple oxocarbenium ion.70 The reaction of a 15-en-17-one with oxygen and alumina causes its slow transformation into a mixture consisting chiefly of the 14P-hydroperoxy- and the corresponding hydroxy- compound; the reaction which does not occur on silica is thought to involve equilibration with the 14-en-17-0ne.~~ The dichloroketene adduct (91) yields the rearranged chloro- acyloxy-compound (92) on reaction with triethylammonium salts of organic acids. The reaction of these compounds with sodium methoxide or with methanol alone gives the methoxy- compound (93); the reaction is thought to involve enolization of the ketone with subsequent ionization and trapping by the nucleophile.1.4 Compounds of Nitrogen Phosphorus Sulphur and Selenium 3,6-Dinitrocholesta-3,5-dieneis prepared from 6-nitrocholes- teryl acetate by elimination of the acetoxy-group with sodium azide and the reaction of the product with nitrosyl chloride in carbon tetra~hloride.~? The hitherto undescribed 7P-amino- 17P-oestradiol has been prepared from 7a-hydroxy-l7P-oestra- diol dibenzyl ether by conversion into the tosylate and subsequent reaction first with sodium azide then with lithium aluminium hydride with final deprotection by catalytic hydrogenolysis; 7P-amino-2-hydroxy- 17P-oestradiol has been prepared similarly.73 An improved preparation of 2-amino-oestrogens involves treatment of the 2,4-dibromo-3-hydroxy- compound with sodium nitrite in acetic acid reduction of the resulting 4- bromo-2-ni tro-compound with hydrosulp hite and catalytic debromination.74 Steroidal nitro-amines are denitro- aminated thermally or with acetic anhydride-pyridine to give olefins with or without molecular rearrangement. In the presence of a vicinal axial hydroxy-group they are converted :nto the ep~xides.~~ 2P,3P-Epimino-5a-cholestanereacts with hydrogen fluoride in pyridine to give the 2~-amino-3a-fluoro-compound;the 3a- amino-2~-fluoro-compoundis obtained from the a-epimine the best method of preparation being with N-protection by carbo-t-butoxylation.The 3a-and 3~-amino-2a-fluoro-compounds result from reductive ammonolysis of the 2ar-fluoro-3-ketone with sodium cyanoborohydride in the presence of ammonium acetate.76 Nitro-compounds are converted into the corresponding aldehyde or ketone by treatment with NNN”’-tetramethy1-N”- t-butylguanidinium 3-iodoxybenzoate in the presence of guani- dine. The conditions are sufficiently mild to be used in the presence of a dithioketal group. Primary nitro-compounds gave much lower yields than secondary ones.77 Oestrone oxime is reduced by diborane or sodium borohydride to the 17p-q R~ C’ -6H II =6O-(94) HON ’ H H-NATURAL PRODUCT REPORTS. 1985 hydroxylamine which reacts with ketones or aldehydes to give the nitrones (94) in a reversible reaction.78 Sodium azide and chromium trioxide in acetic acid acting as a source of chromyl azide convert a 5,6-olefin into the 6p- azido-5a-hydroxy-compound and a 6,7-olefin largely into the 7a-azido-6~-hydroxy-compound.79 Lead tetra-acetate and tri- methylsilyl azide react with 17a-acetoxy-6,7-didehydroproges-terone to give the 6P,7a-diazido-compound as the major andthe 7a-azido-6-ketone as the minor product; the 1,4,6-triene behaves similarly.Treatment of the diazide with sodium azide or with tetramethylammonium fluoride yielded the 6-azido-4,6- diene-3-one.8o 2a-Azido-3-ketoximes undergo cleavage of a C-C bond on treatment with phosphorus oxychloride in pyridine to give the dicyanomethyl compound (95); in the same way a 21-azido-20- ketoxime gives the 17~-cyano-compound as well as the product of Beckmann rearrangement.The mechanism that is shown in Scheme 3 has been suggested.81 7-Dehydrocholic acid reacts with hydroxylamine 0-sulphate in methanolic ammonia to give the diaziridine (96) which is oxidized to the diazirine (97) by silver oxide.82 Steroid phosphates are prepared in a very pure state by the reaction of the alcohol with (1,2-dibromo-l-phenylethyl)phos-phonic acid in the presence of ethyldi-isopropylamine. There is some evidence for the intermediate formation of monomeric metaph~sphate.~~ Steroid 1,2-diacylglyceryl phosphates are prepared by the reaction of the steroid alcohol with the dioxaphosphole (98) and subsequent reaction with triethyl- amine.84 Cholestanone bis(diphenylphosphinomethy1)ethyl-ene ketal (99) is prepared as shown in Scheme 4; the catalyst that is prepared from this compound and [Rh(cod)Cl] (cod = H cyclo-octadiene) has been used for the reduction of suitably substituted olefins with high stereoselectivity.It is less effective in other catalytic processe~.~~ A t-butyl thioester has been prepared from the acid chloride and thallium(1) 2-methylpr0pane-2-thiolate.~~ 17P-Hydroxy-NCCH;! NccH2\B +‘ N;!-N=C-CH;! t-- k NCCH A 17a-vinyl-steroids cannot be inverted by classical means; however their reaction with benzenesulphenyl chloride gives the (S)-sulphoxide (loo) which is equilibrated in boiling (95 1 benzene to a mixture with the (R)-isomer (101).Treatment with Scheme 3 trimethyl phosphite then gives a mixture of starting material H0” gH z YN H H H CHzOCOR CHZOCOR I I RC0,-C H + -RCOZcCH I IrO CHzOH CH 0P”K”‘ Me 0 CH2OCOR CHZOCOR I I RC$mCH OH RC02LCH 0 CHMeCOMe II II CH20P-0-Stero id CH 0P-0 -Steroid II 11 0 0 NATURAL PRODUCT REPORTS 1985 -J. ELKS 47 1 and the required isomer (102) which is the major product.87 of the lcl-trimethylsilyloestr-4-en-3-one and its A5(10)-i~~mer Some bile acids in which selenium replaces a methylene group the corresponding lp-isorner~.~~ in the side-chain have been prepared.88 The reduction of 3-methoxyoestra-l,3,5( 10)-triene with 1it h ium in dime t hox ye t h ane that contains t r ime t h y1sily1 c hlo- 1-5 Remote Functionalization Reactions ride gives all four isomers of the 3-methoxy-1 ,Cdi(trimethylsi- A non-microbiological method (see Scheme 5) for converting ly1)-compound which is hydrolysed by acetic acid to a mixture cholesterol and related compounds into androst-4-ene-3,17- Reagents i diethyl (R,R)-tartrate; ii LiAIH,; iii TsCl; iv Ph,PNa Scheme 4 V I Iiv + 0 A c02' OCOCHZQ II 0 AcO hCOCH2 I Reagents i 02,hv,sensitizer; ii H, catalyst; iii CaH, 4-IC,H,CH2COCI Bu,N+ I-; iv SO,CI or Ph1Cl2,hv;v 1,8-diazabicyclo[5.4.0]undec-7-ene; vi 0,; vii LiOH pyridinium chlorochromate Scheme 5 412 dione involves first the conversion into the 3P-acetoxy-5a- hydroxy-compound which is then allowed to react with (4- iodopheny1)acetyl chloride in the presence of calcium hydride and a catalytic amount of tetrabutylammonium iodide.The resulting ester is then treated with sulphuryl chloride and azobisisobutyronitrileor with iodobenzene dichloride in either case under irradiation. The product is the 17a-chloro-compound which on dehydrochlorination with 1,g-diazabicy- clo[5.4.0]undec-7-ene(DBU) gives a 4 :1 mixture of the 17,20- and the 16,17-olefin. Ozonolysis of the mixture gives the 17- ketone which is converted into androst-4-ene-3,17-dioneby hydrolysis oxidation and elimination of the ester group at C-5 with lithium hydroxide. Yields are high throughout the sequence of reactions.90 Present methods for remote functionalization have the disadvantage that the chlorine-transfer group (e.g.m-iodoben- zoate) has to be covalently attached and can only be recovered by hydrolysis after the reaction is complete. The same effect can be achieved by having the transfer group in an anion or cation. Thus (5a-cholestan-3a-yl)trimethylammonium m-iodo-benzenesulphonate (1 03) on treatment with iodobenzene dichloride (under irradiation) and subsequent dehydrochlori- nation (by methanolic sodium hydroxide) gives the A9-and A14-compounds [(104) and (105) respectively] in a ratio of 3.5 :1. With the ortho-iodo-isomer (106) the preference for 9- chlorination was greater (ratio 5.7 :I) but 80%was unfunction- alized as compared with only 22% for the meta-isomer. Similarly the m-iodo-trimethylaniliniumsalt (1 07) of 3-sulpho- 5a-cholestane gave a ratio of 2.4 :1 of 9- and 14-attack with 44% unreacted,whereas the para-iodo-isomer (1 08) gave a ratio of 1 :1 with 76% unreacted.The unsubstituted trimethylanilin- ium salt underwent attack only at C-9 but 6P/ was unreacted while the tetraethylammonium salt left 100% ~nreacted.~~ 1.6 Photochemical Reactions Photocatalysed rearrangement of 5a-androst- 1-en-3-one in concentrated sulphuric acid gives in addition to the starting material its simple reduction product and the l-methyl-19- nor-compound (109) (which was also obtained in the absence of light) the 10-epimeric 5(10+1)abeo-compounds (1 lo) which form the major part of the product.92 Some AS-steroids have been oxidized to the 7-0x0-com- pounds in high yield by treatment with oxygen in the presence of mercuric bromide under irradiati~n.~~ 17a-Ethynyloestradiol is converted into the corresponding 1OP-hydroxy-3-oxo-l,4-dieneby treatment with oxygen in the presence of methylene blue or haematoporphyrin under irradiation with visible light with subsequent reduction of the 1OP-hydropero~ide.~~ The photocatalysed reaction of proges- terone with acetylene and pent-1-yne has been shown not to be very stereoselective both a-(1 11) and P-isomers (1 12) of the adducts being formed; the isomers are interconvertible by U.V.light of wavelength 300 nm.95 2~-(N-Methylanilino)-5a-cholestan-3-one,on irradiation gives the azetidino-steroid (1 13); the configurations at C-2 and C-3 are ~ncertain.~~ 2 Partial Syntheses 2.1 Cholestane Derivatives and Analogues Methods for the stereoselective synthesis of the 17-side-chain of sterols have been re~iewed.~' New methods for the stereospeci- fic preparation of the side-chain of cholesterol from a 17-ketone are shown in Scheme 6.They depend upon the addition of a lithium copper reagent to the A17-16-ketone or upon its reaction with the pivaloate of the 16P-hydro~y-17-ene.~* The first synthesis of gorgosterol(l14) has been described ;it is shown in Scheme 7.99 Crinosterol (1 16) which is a marine sterol that is the C-24 epimer of brassicasterol has been synthesized as shown in Scheme 8 ; the isomers of compound (1 15) were separated and their configurations determined by Horeau's NATURAL PRODUCT REPORTS 1985 A (109 1 (110) 0rn (111) (112) PhN44 ti HO H (1131 NATURAL PRODUCT REPORTS 1985 -J.ELKS /iii Reagents i MeCH=PPh,; ii SeO, ButO,H; iii (COCI), Me2SO; iv LiCu(CH,CH,CH,CHMe,),; v NtH4 OH-; vi LiAIH,; vii Bu'COCI; viii LiCu(CH,CH2CHzCHMe,)CN; ix H2 Pd; x PhNCO Scheme 6 CH2OH -8 ... C02Et i ,ii Ill I iv,v C02Me ix vi -viii .c---I x,xi i ii 1 CH2OH iii x,viii,x,iv,v __j viii ,XI xii ' HO (114) Reagents i EtO,CCH=PPh,; ii Bui2AIH; iii EtC(OEt),; iv 0,; v NaBH,; vi OH-; vii CH2N2; viii MsCl; ix KOBu'; x LiAIH,; xi pyridinium chlorochromate; xii H+ Scheme 7 method. loo Another marine sterol pulchrasterol (1 17) from Didehydrodesmosterol (122) which was required for conver- Aciculites pulchra provides the first example of double sion into the corresponding vitamin D has been synthesized biomethylation at C-26.1°1 A new synthesis of desmosterol from the aldehyde (121 ;n = 0) uia its homologues (121 ; n = 1) (120) from dehydroepiandrosterone is summarized in Scheme and (121 ; n = 2).'03 Both (24R)- and (249-stigmasta-5,28- 9; both C-20 epimers of compound (1 18) are formed but the dien-3P-01 have been prepared from 24-methylenecholesterol (20s)-epimer of the lactone (1 19) is epimerized by treatment via the isomers of 24-hydroxymethyI-3,5-cycIocholestan-~-ol with strong base and subsequent quenching with water.Io2 7,8-methyl ether and the corresponding aldehyde.lo4 NATURAL PRODUCT REPORTS 1985 (115) I iii vi iv,v iv f$ cll;”b.,.f-(1 16 1 Reagents i MeCrCMgBr; ii H, Lindlar’s catalyst; iii EtC(OEt),; iv LiAlH,; v MsCI; vi H, Pd Scheme 8 Since there is reason to believe that 22- and 23-methyl-sterols may occur naturally (23R)- and (23S)-23-methyl- and 23- methylene-cholesterol have been synthesized as shown in Scheme 10. The configuration of the (23R)-compound was determined by X-ray analysis.Io5 (24S)-4ol,24-Dimethyl-5ol-cholestan-3~-ol is the major sterol of some dinoflagellates and other marine organisms. Both C-24 epimers have now been synthesized from a mixture of sitosterol and campesterol. Io6 26,27-Hexadeuteriocholesterol(124) has been synthesized (117) by the action of hexadeuterioacetone upon the anion from v,vi 1, CHzOCH2Ph OH H OCH2Ph viii ix vii xiii (119) x -(120) 0 /\ Reagents i (EtO),P(O)CH,CN NaH; ii Mg MeOH; iii MeO[CH,],OCH,CI; iv CH,CHCH,OCH,Ph; v OH-; vi (Me,Si),NLi; vii LiAIH,; viii MsCl; ix LiBEt,H; x Li liquid NH,; xi (COCl), Me,SO; xii Ph,P=CMel; xiii ZnBrl Scheme 9 NATURAL PRODUCT REPORTS 1985 -J.ELKS (121) (122) CHO i-iii iv or v-vii -OMe Reagents i Me,CHCHzMgBr; ii CrO,; iii HIC=PPh,; iv (Ph,P),RhCl; v B2H6 H202 vi MsCl; vii LiAlH Scheme 10 ( Thp = tetrahydropyran -2 -yl (123) (124) CH(OH)CH=CHCHMe2 ACHO iii iv dji 6 ___j ___) i,ii 0Me (126) (125) Reagents i LiC=CCHMe2; ii H2 Lindlar's catalyst; iii MeC(OMe), 2,4,6-Me3C,H,CO,H; iv LiAlH,; v Et,NSF,; vi Hz Pd Scheme 11 24-phenylsulphonyl-3~-(tetrahydropyran-2-yloxy)chol-5-ene C-24 has been prepared (as mixed isomers) from the reduction of the product (123) with sodium amalgam and corresponding alcohols and diethylaminosulphur trifluoride ; reduction of the 24-25 double-bond with di-imide.O7 25-fluorocholesterol has been made in a similar way. The Cholesterol that is substituted with fluorine at C-20 C-22 or compounds were required as potential inhibitors of biological 476 NATURAL PRODUCT REPORTS 1985 hydroxylationof the cholesterol side-chain for the synthesis of CH2CH2F) 29-fluorositosterol (126; R1 = CH2CH2F,R2 = ecdysones but only the 24-fluoro-compounds showed relatively H) and 29-fluoroclionasterol(l26;R1= H R2 = CH2CH2F) high activity.’O* 29-Fluorostigmastero1(125;R1= CH2CH2F have been prepared as shown in Scheme 11.They all showed R2 = H) 29-fluoroporiferasterol (125; R1 = H R2 = significant impairment of the growth of the larvae of the C02H i-iii,i ___+ ThpO Ii yH ,ii ,v-I viii 1 pH I Reagents i LiAIH,; ii pyridinium chlorochromate; iii Ph,P=CMeCO,Et; ivy Bu*02H Ti(OPr’)* (-)-diethy1 Martrate; v H,C=CMeMgBr; vi pyridinium dichromate; vii LiAIH, ( -)-(S)-2,2’dihydroxy-l,l‘-binaphthyl; viii H1,Pd; ix But02H VO(MeCOCHCOMe) ;x K2C03 Scheme 12 OH OMe (127) CHO -ThpO (Thp = tetrahydropyran -2 -yl) Reagents i H2C=CMeCH2MgCI; ii PhCOCl; iii Hg(OAc)? NaBH,; ivy OH-; v H2 Pd Scheme 13 NATURAL PRODUCT REPORTS 1985 -J. ELKS tobacco hornworm (Manduca sexta) possibly because of liberation of fluoroacetate during dealkylation of the com- pounds to cholesterol.O9 A compound that was isolated from the tunicate Ciona intestinalisand which is active against murine L-1210 leukaemia proved to be 24<-hydroperoxy-24<-vinylcholesterol. It was synthesized by the photo-oxygenation of fucosterol. lo Stereo-selective introduction of hydroxyl groups at C-23 C-24 and C-25 is difficult because there are no neighbouring chiral groups to direct the entering groups into a specific configuration. In a synthesis that has been described by N. Koizumi and co- workers and which is shown in Scheme 12 chiral reagents were used to prepare (25s)- and (25R)-25,26-dihydroxycholes-terol (24R)- and (24S)-24-hydroxycholesterol and (24R)- and (24S)-24,25-dihydroxycholesterol.(Only one enantiomer of each chiral reagent is shown in the Scheme; the other enantiomer produces the epimeric form of the product.) The compounds were converted (by conventional means) (Thp = tetrahydropyran -2 -yl ) into the corresponding hydroxylated vitamins D.* Another stereoselective synthesis of the (24R)- and (24S)-epimers of 24-hydroxycholestero1 uses (S)-isobutene oxide and (R)-2- benzyloxy-3-methylbutyl iodide respectively to alkylate 6p-me thoxy -22-p henylsulp hony 1-3a,5 -c yclo -23,24-d inor -5a-cholane (1 27).The same publication describes a more convenient synthesis of the same two compounds from the (22E)-24-hydroxy-22-ene (1 28) the isomers of which are easily separable. In order to establish the configuration at C-23 of some naturally occurring sterols 23-hydroxy-25,26-didehydro-cholesterol was synthesized (Scheme 13) and the epimers were separated and converted into (23R)- and (23S)-23,25-di-hydroxycholesterol whose configurations at C-23 were deter- mined by X-ray analysis.The epimers of 23-hydroxycholestero1 were prepared from the 25,26-didehydro-c0mpounds.~ An interesting new synthesis of 25-hydroxycholestero1 is shown in Scheme 14. The final hydrogenation showed a good degree of stereoselectivity at C-20 the ratio of (20R) :(20s) epimers Iiii OH SMe OH iv t Reagents i EtCHLiMe H,C=CHCH(SMe)SiMe,; ii EtCHLiMe Me,CO; iii NiCI,; iv H2 Ni Scheme 14 H OH (129) -Reagents i Me,CCHICHMgBr; ii LiAIH,; iii HCIO,; iv bis(cyclopentadieny1)zirconium hydride Scheme 15 NATURAL PRODUCT REPORTS 1985 being around 4 :1 l4 25-Hydroxy-7,8-didehydrocholesterol of unstated configuration at can 22,25-Epoxycholest-5-en-3~-ol, be prepared from the adduct (129) as shown in Scheme 15.C-22 and C-25 has been synthesized from desmosterol(l20) as Good use is made of silicon and organometallic reagents in the shown in Scheme 18.11* synthesis (shown in Scheme 16)of some hydroxylated steroidal Because 3a,7a,12a,24~-tetrahydroxy-5~-cholestan-26-oic side-chains. acid is considered to be an intermediate in the bioconversion of A precursor to 25-hydroxy-vitamin D2(25-hydroxyercalciol) cholesterol into the bile acids all four isomers epimeric at C-24 has been prepared as shown in Scheme 17.The initial reaction and C-25 have been synthesized by the action of 2-produced the C-22 isomers in similar amount but oxidation to bromopropionic ester and zinc on the hydroxy-protected 24-the ketone and subsequent reduction with lithium aluminium aldehyde. The isomers were separated and their configurations hydride in the presence of (-)-N-methylephedrine gave the determined. For the same reason 301,701,12~,25-tetra-97 (22R)-isomer (130) in much the greater proportion. The hydroxy-5P-cholestan-24-one(131) has been prepared. A reaction of the (232)-23-ene as its phenylcarbamate with synthesis of the 22-isomers of 21,26,27-trinor-5cl-cholestane-Li2Cu,Me5 yielded the protected 25-hydroxy-derivative of 22,25-diol has been described.' 22 7,8-dihydroergosterol ; the (22S)-isomer gave the 24-epimer.24,26-CycIocholesterol which was isolated from marine Acid hydrolysis then produced 25-hydroxy-7,8-dihydroergos-sources has been shown to have the (24S,25S) terol or its 24-e~imer.I'~ configuration. PhMe2Si+\, i,ii /I MeC-CH + PhMe2SiAIEt2 w Me OH SiMezPh p"i-viii iv,v OMe Reagents 1 Pd(OAc), P(o-MeC6HJ3; ii I,; iii BuLi; iv Bu*O,H,VO(MeCOCHCOMe),; v Bu4N+ F-; vi PhCH,Cl; vii Pr',Cu(CN)Liz; viii Li liquid NH Scheme 16 OCHzOMe i ii,iii OCH2OMe ___) -(130) OMe iv,v i I PhNHCOO I OCH20Me vi p:-" H20Me Reagents i LiC=CCMe@CH,OMe; ii pyridinium dichromate; iii LiAlH, (-)-N-methylephedrine 3,5-MezC,H,0H; iv Hz Lindlar's catalyst; v PhNCO; vi LizCu3Me,; vii H+ Scheme 17 NATURAL PRODUCT REPORTS 1985 -J.ELKS 479 An improved [but still low-yielding (3%)] synthesis of brassinolide (1 32) from stigmasterol is shown in Scheme 19. 24 Castasterone which is an intermediate in the synthesis of brassinolide has been prepared from the same starting material. 25 Dolicholide (1 33) which is the 24,28-didehydro- derivative of brassinolide has been stereoselectively synthe- sized again from stigmasterol (Schemes 20 and 21),126,'27 as has the naturally occurring 28a-homodolichosterone (1 34). 28 Rings A and B bearing the functional groups of the brassino- steroids have been elaborated from 3P-bromo-6-oxo-Sa-cholestane 29 and various side-chain analogues have been synthesized.' 30-1 32 2-Deoxyecdysone which has been postulated to be biologi- cally active per se rather than having first to be 2-hydroxylated has been synthesized from ergosterol in both unlabelled form and as the 23,24-tetratritio-derivative.I33 la-Hydroxylithocholic acid which is a possible metabolite of lithocholic acid has been synthesized,] 34 as has chiogralac- tone (135).135 A new method of synthesis of the cholanic acid side-chain has been used in the preparation of (20S)-3P-hydroxy-22,23- didehydrochol-5-en-24-oic acid (1 36) which is identical with material from the sea pen Ptilosarcus gurneyi.136 5P-Ranol which is the major constituent of bull-frog bile was known to be a 27-nor-SP-cholestane-3a,7cr 12a,24,26-pentaoI.Both 24- epimers of this compound have been synthesized and SP-ran01 iv has been shown to be the (24R)-isomer (137).13' The 3a,7a,23- c-- trihydroxy-5P-cholanic acids and their 12a-hydroxy-deriva- tives have been synthesized and the isomers that are found in marine organisms have been assigned the (23R) Reagents i Tl(OAc)?; ii TsOH; iii Hg(OAc),; iv H? catalyst configuration.38 Scheme 18 0 2.2 Vitamins D their Derivatives and their Metabolites Methods of manufacture of the vitamins D their activities and their metabolites have been reviewed,139 as have the synthesis and biological activity of fluorinated analogues of the vitamins D.I4O Previtamin D [(6Z)-tacalciol] on irradiation in a viscous solvent at 92 K is converted into the cis,cis,E-rotamer of tachysterol (tacalciol). This on being allowed to warm to 100-105 K gives the trans,cis,E-and the trans,trans,E-H rotamers.The phenomena have been rationalized on the (131) principle of the non-equilibration of excited rotamers. 41 OH ~CHCH=CHPr 1 -HOJ2Y i ,ii liii HO HO OH HO, HO'. " (132) 0 Reagents i Pr'C-CLi; ii H2 Ni H,NCH2CH,NH2; iii 3-C1C,H,CO3H; iv AlMe, BuLi (catalytic); v H+ Scheme 19 NATURAL PRODUCT REPORTS 1985 f AcO- AcO" iv,v OH viii-x -t HO, HO' / (133) -Reagents i CF3C03H; ii Me,CO; iii pyridinium chlorochromate; iv S[CH,],SCHLi; v ClCH,OMe; vi HgO BF,-Et,O; vii PriC(=CH,)MgBr; viii Ac,O; ix HClO,; x OH-Scheme 20 OH HO i ii-iv -P-HO- H0°' (133) Reagents i LiAIMezBu(CH=CMePri); ii 3-C1C,H,CO3H ; iii Al(OPri)3; iv AcOH Scheme 21 0 C02H HO.HO" HO H.0 A new approach to the partial synthesis of the vitamins D transition-metal catalysts largely into the hemiketals (1 41) has produced compound (138) as both (20R)- and (20s)-which give the furan (142) on storage in chl~roform.'~~ isomers of which only the former showed some vitamin D Whereas oxidation of 25-hydroxy-vitamin D33-acetate [(3S)-3- activity. '42 O-acetyl-25-hydroxycalciol]by osmium tetroxide gives the Photo-oxygenation of vitamin D2 (ercalciol) or vitamin D3 7a,8a-dihydroxy-derivative(143) the 3,5-cyclo-ether is oxi-(calciol) gives the hydroperoxides (139) as the minor and the dized at the other double-bond to give the 105,19-diol (144). epidioxides (140) as the major products.The epidioxides The change in selectivity has been attributed to the methoxy- which are thermally stable are converted by base or by group in the 3,5-cyclo-ether shielding the 7-8 double-bond. NATURAL PRODUCT REPORTS 1985 -J. ELKS 48 1 OH CH20H Hd' OH H (1 37) HO R R R HO. OH OH OH OH AcO" AcO" $0.. (146) (147) (148) Scheme 22 Treatment of (144) with acetic acid gives the 105,19,25-trihydroxy-l0,19-dihydro-vitamin D3 acetate [(3S)-3-O-acetyl- 10~,19,25-trihydroxy-l0, 19-dihydrocalciol] with the (5Z)-form (145) as the major product.'44 Some analogues of vitamin D3 containing a carboxyalkyl substituent at C-19 were obtained from the sulphur dioxide adduct (146; R' = H). Each of the C-6 isomers of this compound was carboxyalkylated in the presence of a strong base and the products (146; R' = a-or P-[CH2],CO2Bu') were thermolysed to give the required compounds (see Scheme 22).482 NATURAL PRODUCT REPORTS 1985 -iii-v @cH20H OH HO" -0" Reagents i N-Chlorosuccinimide ; ii Ph,PLi; iii BuLi H,C=CMeCH,CI; iv Hg(OAc), NaBH,; v pyridinium dichromate; vi N-trimethylsilylimidazole vii BuLi; viii Bu,N+ F-Scheme 23 CH2OH CH~OAC I -ii &02)iii-" fi -fiNo2-fi Reagents i MeNO, H,N[CH,],NH,; ii HCHO Et,N; iii AclO; iv CrCI,; v TiCI Scheme 24 Reagents i NCCHICO,Et; ii Bu',AIH; iii AczO; iv KMnO, i li PhCH,kEt Cl-Scheme 25 The (50-isomer (147) was the major product whichever isomer was used as a starting material the desulphonylation proceed- ing preferentially in the antarafacial manner.The (5Z)-isomer (148) could be prepared by photosensitized isomerization of (147).'45 Reagents i Pb(OAc),; ii Li H2N[CHI],NH,; iii O3 la-Hydroxy-25-methyl-vitamin D3 (la-hydroxy-25-methyl-calciol) has been ~ynthesized.'~~ Since oxidation at C-23 is Scheme 26 NATURAL PRODUCT REPORTS 1985 -J. ELKS 483 considered to be involved in the catabolism of 25-hydroxy-vitamin D3 (25-hydroxycalciol) the effect of replacing this carbon with an oxygen atom has been investigated. A synthesis of the analogue (149) is shown in Scheme 23.147 The P-D-glucopyranosides of vitamins D2and D3and of the 1-hydroxy- 25-hydroxy- and 1,25-dihydroxy-derivatives of the latter compound have been described.148 Syntheses of a number of precursors of the metabolites of the vitamins D are described in Section 2.1.2.3 Pregnanes The chemistry of synthetic progestogens has been reviewed. 149 Barton and his co-workers have published details of their synthesis of corticoids from 17-ketones via 17-nitromethylene compounds (Scheme 24).l 51 Another method for this transformation has been described and is shown in Scheme 25.Is2Methods for converting bile acids into corticoids have been explored by Do-Trong and his co-workers; they employed a new way of degrading the side-chain (Scheme 26).Is3 Methods have been described for the specific labelling of progesterone with deuterium at the 15a- 15p- and 15,15- positionslS4 and for the labelling of pregnan-20-ones with I8F at C-21.155*1 56 [3,4-13C2]Progesterone has been synthesized as shown in Scheme 27.Is7 The diazoacetate of 1 1 a-hydroxyprogesterone has been prepared from the glyoxylic ester tosylhydrazone by the action of pyridine.It was required as a photo-affinity analogue of progesterone. 58 Cupric bromide in methanol that contains pyridine converts pregnenolone into its 21-bromo-derivative. This is the first direct method of brominating (2-21 that does not affect the 5-6 double-bond. Oxidation at C-3 and controlled displacement of the bromine by hydroxyl then gives deoxycorticosterone. * 59 1 7ct-Acyloxy-2 1 -chloropregna-l,4-dien-20-ones are prepared by the action of phenyl chloroformate in dimethylformamide or of N-formylmorpholine upon the 17,2l-orthoester.It is thought that the iminium salt that is formed from these reagents is involved. 6o 17a-Hydroxy-20-oxopregn-4-en-2 1 -oic acids are prepared from the 21-aldehyde by oxidation with silver oxide.l An improved route to 19-hydroxyprogesterone and to its 19,19-dideuterio-derivative has been described 62 as have Reagents i l3CH3I3COCl C,H ,NLiPr'; ii HCl AcOH routes to 19,21-dihydroxy- and 11~,19,21-trihydroxy-progester-one and 21 -hydroxy- 19-norprogesterone. 63 By a combination of chemical and microbiological methods the 3-hydroxylated saturated ring A derivatives of aldosterone and 18-hydroxycorticosterone have been prepared. They were wanted as possible metabolites of aldosterone.164The synthesis of a number of analogues of 18-hydroxy- and 18-0x0-progesterone and of 18-hydroxy- and 18-0x0-deoxycorticoster-one in which the side-chains at positions 13 and 17 are interchanged has been reported.165 In an experiment that had been designed to show whether the 13-17 or the 16-17 bond migrates during the hydrogen- chloride-catalysed D-homoannulation of 5P-pregnane-3a,20-diol disulphate (either 20-isomer) 20-l 3C-labelled material was used. The products i.e. (150; R = OH) and (1 50; R = Cl) were enriched only at C-17 showing that only the 16-17 bond had migrated.166 A mixture of 17a- and 17p-18-norpregna-4,8(9), 11,13( 14)- tetraene-3,20-dione (152) has been prepared by the action of lead tetra-acetate and cupric acetate upon compound (1 5 l) followed by treatment with boron trifluoride etherate.67 2.4 Androstanes and Oestranes Derivatives of 14a,15a-ditritio-19-nortestosterone,in which the label is pharmacologically stable have been prepared from 14a,15a-ditriti0-8,9-didehydro-oestrone methyl ether. 68 The preparation of the 3- and 17-sulphates of 5a-androstane-3a 17p- diol of 5a-androstane-3P 17P-dio1 and of the corresponding 17-ketones all of which are specifically labelled with deuterium in rings A or D has been reported.169 17a,2 1 -Dihydroxy-20-ketones are oxidized by manganese dioxide in boiling chloroform to give good yields of the 17- ketones. 70 1~,3P,5,6~-Tetrahydroxy-5a-androstan-l7-one (1 53) which has been isolated from the soft coral Sarcophyton glaucum has now been synthesized from 1P,3P-dihydroxy-pregna-5,16-dien-l7-one by Beckmann rearrangement of the diacetate oxime followed by hydrolysis then hydroxylation of the 5-6 double-bond with performic acid.171 Cathodic reduction of 17p-oestradiol 3-methyl ether at a mercury cathode using an aqueous solution containing tetrabutylam- monium hydroxide as the electrolyte gives the 1,4-dihydro- compound in greater than 90% yield; the conclusion was drawn Scheme 27 R H (150) NATURAL PRODUCT REPORTS 1985 HO OH HO& \ 0 4 i AcO@ \ 0 bH iv,v 0 0 T Reagents i CrO,; ii HSCH,CH,SH BF,.Et20; iii NaBH,; iv HCI MeOH; v HgCI, CaCO,; vi T2 Pd Scheme 28 that an amalgam of tetrabutylammonium ion is an intermediate.72* 73 A series of 7a-alkyl-testosterones has been prepared by the conjugate addition of Grignard reagents or lithium copper alkyls to 6,7-didehydrotestosteronepropionate' 74 and a new route to the anti-androgen 16P-ethyl-19-nortestosteronehas been reported. 7s The 14P-epimer of the boar-taint steroid 5a-androst- 16-en-3-one has been synthesized from 3P-hydroxy- 5a,14P-androstan-1 7-one via its hydrazone and the 17-iodo-16- ene. Its odour is reminiscent of that of the boar-taint ketone but very much less intense. 76 Methods have been described for the labelling of 17P-oestradiol or 17a-ethynyloestradiol with deuterium in ring A' 77 or ring c178 or with tritium in rings A and B17' or in ring C.'*O Oestriol has been prepared by treatment of oestrone with cupric bromide and hydrolysis of the 2,4,16a-tribromo-oestrone followed by debromination and reduction with sodium borohy- dride and palladium chloride; the 16-0-glucuronide is prepared in a similar fashion.A new and improved method for the synthesis of 2-hydroxy- 17P-oestradiol and -17P-oestrone involves a Friedel-Crafts reaction of 17P-oestradiol with acetyl chloride and oxidative cleavage of the resulting acetyl compound (154) with alkaline hydrogen peroxide. 82 Both 2- and 4-hydroxyoestrone methyl ether may be obtained (in yields of 30% and 15% respectively) by treatment of the lithio-derivative of oestrone methyl ether 17-ethylene ketal with diborane and trimethyl borate with subsequent oxidation with hydrogen peroxide.83 Methylation of 2-and 4-hydroxyoestrone and demethylation of the corresponding dimethyl ethers has been studied.lg4 2,4-Dibromo-oestradiol reacts with sodium methoxide and cuprous iodide in the presence of a crown ether to give the 2,4- dimethoxy-compound. 85 The 2- and 4-methoxy-compounds can be prepared in a similar fashion.lg6 2-Substituted oestradiol methyl ethers are prepared by the reaction of the unsubstituted compounds with mercuric acetate and sodium chloride and by the reaction of the resulting 2-chloromercuri- compound with halogens or by oxidation to give the 2- hydroxy-compound. 87 2-Nitro-oestrone free of the 4-nitro- isomer is prepared by use of (a) N-nitropyrazole or silver nitrate and boron trifluoride etherate' 88 or (b) ferric nitrate supported on a clay.189 NATURAL PRODUCT REPORTS 1985 -J.ELKS Improved yields of 6-0x0-derivatives of oestrogens have been obtained by oxidation of the 3-acetate of the oestrogen with the chromium trioxide-dimethylpyrazole complex. 90 1 O-Hydroperoxyandrosta- 1,4-dien-3-0nes have been pre-pared by photocatalysed oxygenation of the ring A phenolic compound and are reduced by iodide ion to give the lop- hydroxy-compound together with a little of the epimer. Catalytic reduction to the tetrahydro-compound followed by dehydration with perchloric acid gives the 3-0x0-19-nor-4- ene.19' In the same way a 3-oxo-5(10)-ene is converted by x,xi c-- HO H (155) 485 oxygen under irradiation with visible light into the lop-hydroperoxy-3-oxo-4-ene.* A method has been described for the tritiation of 16a-hydroxyoestrone at C-6 and C-7 (Scheme 28); the tritiated compound was needed for the radioimmunoassay of 16~-hydroxyoestrone which may play a part in systemic lupus erythematosus. 93 Some 2-and 16-alkyl derivatives of 8a,17P-oestradiol have been prepared ;the anti-uterotrophic activities were generally less than those of the unsubstituted compounds. 94 0 -H SPh viii ix EH vii f--f--Reagents i H,02 OH-; ii dihydropyran; iii Me3SiCH2C02Me BuLi; iv LiAlH,; v PhSCH,C02H dicyclohexylcarbodi-imide;vi (Ph,P),Pd ; vii HN=NH; viii 3-CIC,H,C03H; ix heat; x MeSO2C1; xi TsOH Scheme 29 (Thp = tetrahydropyran -2 -yl) Reagents i NCCH,CO,Et; ii NaBH,; iii dihydropyran; iv Bui2AlH; v NaCN HCl; vi SOCI,; vii Li2C0, LiCl; viii (Et0)2P(0)CH2C0,Et NaH; ix TsOH; x pyridinium dichromate Scheme 30 NATURAL PRODUCT REPORTS 1985 2.5 Cardenolides and Bufadienolides genin followed by deprotection with lithium aluminium 14,15-Anhydrodigitoxigenin(155) has been synthesized as hydride gives predominantly the p-anomer (158) of the shown in Scheme 29; its conversion into digitoxigenin has digitoxoside which can be oxidized to the digitoxigenin already been reported.95 The 17-ylidenecyanoacetate (156) glycoside. 97 The cr-~-arabinofuranoside~~~ and the b-D-glUC0- has been used for the synthesis of both the cardenolide and the side and p-D-galactoside'99 of digitoxigenin have been bufadienolide rings (Scheme 30).196The reaction of the prepared.digitoxose derivative (157) with the fury1 analogue of digitoxi- The configurations at C-20 of the epimers of dihydrodigoxi- MeOoC02yk + OCONHMe OH HO H 0 PhCH2O&-+ Li+ gOH ~H HO8 i ,viiI v,vi t- OH li,ii I0.I_ 0 H H Bufalin a'-Isobufalin Reagents i MsCl; ii pyridine; iii N-bromosuccinimide; iv NaBH,; v H+; vi Ag2C0, Celite; vii 1,5-diazabicyclo[4.3.0]non-5-ene Scheme 31 NATURAL PRODUCT REPORTS 1985 -J. ELKS 0 k P (159) H Reagents i PhSCH(SiMe,)CH=CH, EtMeCHLi; ii EtMeCHLi 0,; iii HBr Scheme 32 0 H OH MeOC (1 60) 0 +i ,ii (164) + (162) N+ poAc -@ iii-vi vii ,viii ,v Reagents i 3-Pyridyl-lithium; ii Ac,O 4-dimethylaminopyridine; iii xylene AcOH; iv HN=NH; v OH-; vi MsCI pyridine; vii A1(OPri) ; viii N-bromoacetamide; ix LiAlH Scheme 33 geninZo0 and dihydrodigitoxigenin201 have been established.A new synthesis of bufalin has been described; it is The synthesis has been described of analogues of digitoxi- summarized in Scheme 3 1. A slight modification of the method genin digoxigenin the corresponding isocardenolides and the has been used in the preparation of a’-isobufalin.206 The 14a-corresponding furans in which the 3P-hydroxy-group is 14-deoxy-derivative (1 59) of bufalin has been prepared using a replaced by ct-or P-azido- or by a-or ~-amino-functions.202~203novel method of synthesis of the side-chain (Scheme 32).207 Some ring-A-oxygenated derivatives of 5a-and 5P-cardeno- Several 3-esters of hellebrigenin (1 60) have been prepared and lideszo4 and some 22-alkyl derivatives of digitoxigenin digoxigenin gitoxigenin diacetate and digitoxinZo5 have been reported.The alkyl derivatives were active in the Na+/K+- transporting ATPase inhibition assay but of those that were tested only 22-( 3-oxoprop- 1-enyl)digitoxigenin 3-acetate was more active than the parent. tested for cardiotonic activity. All were highly active and the dimethylacrylate appeared to have a better therapeutic index than conventional cardiac glycosides. 208 The furan (162) and the butenolide (163) are formed by oxidation of the (222)-22-ene-20,24-diol (1 6 1) with silver carbonate on Celite.*09 The analogue (164) of a cardiac 488 NATURAL PRODUCT REPORTS 1985 C8H17 Ik HO OH OH (165) (166) H (167) I I I RC024-""' I H Reagents i NH20H; ii TsCI; iii HCI; iv LiAlH,; v 2,3-dichloro-5,6-dicyanobenzoquinone Scheme 34 II (168) 0 (174) (173) @" (170) J (171) 2.6 Heterocyclic Compounds 4-Hydroxy-3~-methyl-4-aza-5a-cholestane (1 66) has been pre- pared by reduction of the acetylenic oxime (165) with sodium (172) borohydride; the reaction is novel.21 The chromophore of the antifungal antibiotic A 25822B (167) has been synthesized by the method that is shown in Scheme 34.212Steroids (168) with aglycon with a pyridine ring replacing the butenolide ring has an oxazole ring fused on at C-6 and C-7 are produced by the been prepared as shown in Scheme 33;it is equi-active with the action of acetyl chloride and acetic anhydride in pyridine upon corresponding butenolide.2 O a 6-oxime followed by cyclization with hydrogen chloride.The NATURAL PRODUCT REPORTS 1985 -J. ELKS (176) Reagents i HC02Et NaOMe; ii AcOH; iii NaBH,; iv Ac,O Scheme 35 (178) Ii 3. Reagents; i H, Pt; ii OH-; iii H+; iv LiNPr'? PhSeBr; v LiNPr'? MeI; vi H,O Scheme 36 (179) reaction is thought to involve compound (1 69) as an intermedi- ate. A 17-aza-D-homo- 16,17a-dione (1 72) has been prepared from the 16P-azido- 17-ketone (1 70) by the action of bromine in acetic acid conversion of the resulting carboxy-nitrile (1 71) into the methyl ester amide and finally cyclization with sodium methoxide.' A 6~-(diethoxyphosphinylamino)-compound(1 73) on reac- tion with lead tetra-acetate and iodine under U.V.irradiation gives a very high yield of the 6P 19-diethoxyphosphinylimino-compound (I 74). In a similar way the (209-1 8,20-disubstitut- ed imino-compound (1 75) can be ~repared.~ Steroid pyrans (1 76) and (1 77) have been prepared as shown in Scheme 35. * 2~-Hydroxy-3~-phenylcholestanone is con-verted by treatment with chloromethyl ether into the isochroman (178); the 2a-hydroxy-3P-phenyl- and the 2a- hydroxy-3a-phenyl-isomers behave similarly to give isomers of (1 78).21 Since a-methylene-y-lactones are often cytotoxic the same group has been fused to a steroid at C-16 and C-17; the synthesis is shown in Scheme 36.218 4-Thiacholest-5-en-3-one and some related steroids have been synthesized from the corresponding 5-0x0-seco-acids (179) by treatment with phosphorus pentasulphide; the 6-thia- 7-one is prepared similarly.2l9 3-Thia-and 3-sulphinyl-derivatives of testosterone have been synthesized as shown in Scheme 37.220 Several oxathiazolidines (180) have been prepared from the l7a-methylaminomethyl-l7~-ols by treat- ment with thionyl chloride.221 The platinum and palladium complexes (18 1 ;M =Pt or Pd) and (182; M = Pt or Pd) have been made as potential antitumour agents.22 2.7 Cyclopropano-steroids 1a-Hydroxy-1~,5-cyclo-5~-cholestan-7-one (1 83) has been pre- pared from cholest-5-en-1 -one by oxidation with Collins' reagent and reduction of the 5-ene-177-dione with lithium in liquid ammonia.On reaction with acid it gives compounds (184) (185) and (186) in yields of 13% 38% and 25% respectively; with base the same compounds are formed but with the first predominating and the second reduced to traces. On treatment of (183) with a very mild base the primary (1 87) which is converted product is the 5~,7~-cyclo-compound with strong base into (184)and (186).223 Reduction of 6-nitrocholesteryl acetate with lithium di- methylcopper gives the E-oxime of 3a,5a-cyclocholestan-6-one from which the ketone is obtained by treatment with sodium bisulphite.*'j Mild reagents for the conversion of a 6-oxo-3~-tosyloxy-5c-steroid into the 3a75a-cyclo-6-one are tetramethylguanidine at 60 "C for 5 minutes or benzyltri-methylammonium hydroxide in pyridine at 60 "C for 10 minutes.225 Cholesteryl tosylate reacts with triethylaluminium to give 6E,-ethyl-3a,5a-cyclocholestane,together with a much smaller amount of 3~-ethyl~holest-5-ene.~ 26 4a,5a-Methylenecholestan-3-onereacts with bromine in acetic acid to give mainly the 2a-bromo-5-bromomethyl-5a-cholestan-3-one (188) together with the 2a-bromo- and 2,2- dibromo-derivatives of the starting ketone.Compound (1 88) is reduced by lithium tri-t-butoxyaluminium hydride to inter alia the 2a-bromo-3a,5a-epoxide (189; X = Br) which can be debrominated with tributyltin hydride to (189; X = H) (the unusual reduction of a 3-ketone to the axial hydroxy-derivative is explained by the presence of axial substituents at C-2 and C- 5); analogous reactions occur with the @,SP-methylene-cholestan-3-0ne.~ A shorter and higher-yielding route to the anti-aldosterone 6P,7P-methylene-1SP 16P-methylene-compound spirorenone (190) has been described; it is summarized in Scheme 38.228,229 The cyclopropane ring in the 20a- or 20P-hydroxy-l6a 17a- methylene-compound (191) is opened by mesyl chloride to give the 16a-mesyloxy- and 16~-chloro- derivatives [(192; R = OMS) and (192; R = Cl) respectively] of 3P-acetoxy-~- NATURAL PRODUCT REPORTS 1985 OH ii iii 1 0 ix- xi ii 1 Reagents i TsNHNH, HOAc; ii KOBu'; iii HOCH,CH,OH; iv Bu'Me,SiCl; v H,,Pd/BaSO,; vi 0,; vii NaBH,; viii PhCOSH Pri02C- N=NCOIPr' Ph,P; ix OH- 02;x Ph,P=CH,; xi H,O+; xii HIO Scheme 37 OS-Nluk @ (181) n = 2 or 3 homopregna-5,17-diene.Acetic acid similarly gives the 16a- 6p,19-Epoxycholest-4-en-3-one and 6p 19-epoxysitost-4-en-acetoxy-compound (1 92;R = OAc). Analogous reactions are 3-one on fermentation with species of the genus Mycobacter-shown by the 16P 17P-methylene-analogue (193).230 ium both give the 17-ketone in 51-64% yield; the 3p,19- dihydroxy-5-enes give high yields of 19-hydroxyandrost-4-ene-3,17-di0ne.~~~ The use of Arthrobacter simplex immobilized in 2.8 Microbiological Transformations various ways for the dehydrogenation of 4-en-3-ones to 1,4- Streptomyces roseochromo- Reviews have appeared on microbiological transformation of dien-3-ones has been st~died.~~~,~~~ steroids including an assessment of the use of immobilized genes immobilized in a cross-linked BPE-resin has been used and on the production of sex hormones and for the 16a-hydroxylation of dehydroepiandrosterone.238 spironolactone using fermentation methods.233 Hecogenin on fermentation with Cunninghamella elegans Cells of species of the genus Dioscorea cultured in a bubble- gives the lP,7P-dihydro~y-derivative;~~~ withaferin A with the column-type fermenter gave a maximum of 7.8% of diosgenin same organism gives the 12P-hydroxy- and 1SP-hydroxy-(dry weight). 234 derivatives. 240 Androst-4-ene-3,17-dione, with Mucor species NATURAL PRODUCT REPORTS 1985 -J. ELKS 491 CH,Br 6-(188) (189) 0 0 0 -@ "OH "OH HO liii,iv J. 0 (190) Bu'CO*e Reagents i Colletotrichum hi; ii H+ ; iii Bu'COCI 4-dimethylaminopyridine;iv Me2S(0)=CH2 Scheme 38 MeCHOH (191) is converted into 65,14or-dihydroxyandrost-4-en-3-one, the 17-0x0-group having been reduced to methylene; this is the first example of such a reduction by microbiological means.**l 3 References 1 G.R. Lenz In 'Kirk-Othmer Encyclopedia of Chemical Techno- logy' 3rd edn. John Wiley New York 1983,Vol. 21 pp. 645-729. 2 S. C. Chen Chem. Can. 1983 35 13. 3 J. Steroid Biochem. 1983 19 IA B C. 4 P. Crabbe in 'Progestogens in Therapy' ed. G. Benagiano P. Zulli and E. Diczfalusy Raven Press New York 1983,pp. 13-26;various authors Steroids 1983,41,Number 3. 5 E. J. Parish and A. D. Scott J. Org. Chem. 1983,48,4766. 6 E. J. Parish and S. Chitrokorn Org. Prep.Proced. Int. 1983 15 365. 7 I. Aljancic-Solaja M. Bralovic B. Solaja and M. Stefanovic Glas. Hem. Drus. Beograd. 1983,48,299. 8 F.TureEek K.VereS P. 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ISSN:0265-0568    

DOI:10.1039/NP9850200461

出版商:RSC    

年代:1985

数据来源: RSC