摘要:

Natural Product Reports Editorial Board Professor T. J. Simpson (Chairman) University of Bristol Dr C. Abell University of Cambridge Dr J. R. Hanson University of Sussex Dr R. B. Herbert University of Leeds Professor J. Mann University of Reading Dr D. A. Whiting University of Nottingham Natural Product Reports is a bimonthly journal of critical reviews. It aims to foster progress in the study of bioorganic chemistry by providing regular and comprehensive reviews of the relevant literature published during well-defined periods. Topics include the isolation structure biosynthesis and chemistry of the major groups of natural products-alkaloids terpenoids and steroids aliphatic aromatic and 0-heterocyclic compounds. Many reviews provide details of biological activity and wider aspects of bioorganic chemistry including developments in enzymology genetics and structural spectroscopic and chromatographic methods of general interest to all workers in the area.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge CB4 4WF England. 1993 Annual Subscription Price E.C. f242.00 Overseas f266.00 U.S.A. $532.00 Canada f279.00. Change of address and orders with payment in advance to The Royal Society of Chemistry The Distribution Centre Blackhorse Road Letchworth Herts.SG6 1 HN England. Air Freight and mailing in the U.S. by Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. US Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11003. Second-Class postage paid at Jamaica NY 11431-9998. Afl other despatches outside the U.K. are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the U.K. 0 The Royal Society of Chemistry 1993 All Rights Reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photographic recording or otherwise without the prior permission of the publishers. Printed in Great Britain by the University Press Cambridge Subscription rates for 1993 E.C. f242.00 Overseas f266.00 U.S.A. US $532.00 Subscription rates for back issues are (1988) (1989) (1 990) (1991) (1 992) U.K. €1 59.00 f169.00 f177.00 f198.00 f222.00 Overseas f 183.00 f194.00 f204.00 f228.00 f250.00 U.S.A. US $342.00 US $388.00 US $398.00 US $467.00 US$474.00 Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge CB4 4WF England

ISSN:0265-0568    

DOI:10.1039/NP99310FX021

出版商:RSC    

年代:1993

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99310BX023

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ISSN 0265-0568 NPRRDF 1O(6) 541-632 (1 993) Natural Product Reports A journal of current developments in bio -organic chemistry Volume 10 Number 6 CONTENTS 541 HMG-CoA Reductase Inhibitors A. Endo and K. Hasumi Reviewing the'literature published up to October 1992 551 A Survey of Natural Products which Abstract Hydrogen Atoms from Nucleic Acids J. A. Murphy and J. Griffiths 565 The Strobilurins Oudemansins and Myxothiazols Fungicidal Derivatives of /I-Methoxyacrylic Acid J. M. Clough 575 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites R. B. Herbert Reviewing the literature published between January and December 1991 593 Biosynthesis of Fatty Acid and Polyketide Metabolites D. O'Hagan Reviewing the literature published between mid-1991 and mid-1992 625 Cumulative Contents Volumes 1-10 NPR 10 Cumulative Contents of Volume 10 Number 1 1 Lignans Neolignans and Related Compounds (January 1989 to December 1991) R.S. Ward 29 Muscarine Oxazole Imidazole Thiazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1990 tu June 1991) J. R. Lewis 51 Indolizidine and Quinolizidine Alkaloids (July 1990 to June 1991) J. P. Michael 71 Microbial Pyran-2-ones and Dihydropyran-2-ones (up to December 1991) J. M. Dickinson Number 2 99 Quinoline Quinazoline and Acridone Alkaloids (July 1990 to June 1991) J. P. Michael 109 The Chemistry of Azadirachtin S. V. Ley A. A. Denholm and A. Wood 159 Diterpenoids (1991) J. R. Hanson 175 Chemical and Biochemical Manipulations of Nucleic Acids M.J. McPherson and J. H. Parish 199 Tropane Alkaloids (January and December 1991) G. Fodor and R.Dharanipragada Number 3 207 NMR of Proteins M. P. Williamson 233 The Biosynthesis of Shikimate Metabolites (1991) P. M. Dewick 265 Biological Variation of Microbial Metabolites by Precursor-directed Biosynthesis R. Thiericke and J. Rohr 291 Amaryllidaceae and Sceletium Alkaloids (1991) J. R. Lewis 301 Stevioside and Related Sweet Diterpenoid Glycosides (up to May 1992) J. R.Hanson and B. H. De Oliveira Number 4 31 1 Obituary David N. Kirk 1929-1992 313 Steroid Reactions and Partial Synthesis (1991) J. R. Hanson 327 Advances in Chemical Ecology (January 1988 to June 1992) J. B. Harborne 349 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1991 to June 1992) J.E. Saxton 397 Natural Sesquiterpenoids (1991) B. M. Fraga 421 Arsenic Compounds from Marine Organisms (up to October 1992) J. S. Edmonds K. A. Francesconi and R.V. Stick Number 5 429 Macrocyclic Trichothecenes (up to December 1991) J. F. Grove 449 P-Phenylethylamines and the Isoquinoline Alkaloids (July 1991 to June 1992) K. W. Bentley 471 Diterpenoid Alkaloids (December 1989 to January 1992) M. S. Yunusov 487 Pyrrolizidine Alkaloids (July 1991 to June 1992) D. J. Robins 497 Marine Natural Products (1991) D. J. Faulkner Articles that will appear in forthcoming issues include Pigments of Fungi (Macromycetes) (July 1986 to August 1992) M. Gill Plant Polyphenols E. Haslam Recent Advances in the use of Enzyme-catalysed Reactions in Organic Synthesis (July 1988 to December 1992) N.J. Turner Triterpenoids (January 1988 to December 1989) J. D. Connolly R. A. Hill and B. T. Ngadju Indolizidine and Quinolizidine Alkaloids (July 1991 to June 1992) J. P. Michael Novel Constituents of Uvaria Species (1968 to February 1993) V. S. Parmar et al. Quinaline Quinazoline and Acridone Alkaloids (July 1991 tu June 1992) J. P. Michael The Biosynthesis of Shikimate Metabolites (1992) P. M. Dewick The Fluorinated Natural Products D. B. Harper and D. O’Hagan Deoxynojirimycin Synthesis and Biological Activity (up tu December 1992) A. B. Hughes and A. J. Rudge Isoprenoid-substituted Phenolic Compounds of Moraceous Plants T. Nomura and Y. Hano Monoterpenoids (1990) D. H. Grayson Secondary Metabolism in Plant Tissue Culture Scope and Limitations (up to end of 1992) D. V. Banthorpe Mechanism of Action of Vitamin K P. Dowd. R.Hershline S. W. Ham and S. Naganathan Diterpenoids (1992) J. R. Hanson Amaryllidaceae and Sceletium Alkaloids (1992) J. R. Lewis Muscarine Oxazole Imidazole Thiazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1991 to June 1992) J. R. Lewis

ISSN:0265-0568    

DOI:10.1039/NP99310FP025

出版商:RSC    

年代:1993

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99310BP027

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

HMG-CoA Reductase Inhibitors A. Endo and K. Hasumi Department of Applied Biological Science Tokyo Noko University Fuchu Tokyo 183 Japan Selectively reviewing the literature published up to October 1992 1 Introduction 2 Biochemical and Pharmacological Mechanisms 2.1 HMG-CoA Reductase 2.2 Plasma Cholesterol 3 Mevinic Acids of Fungal Origin 3.1 Isolation 3.2 Biosynthesis 3.3 Modification 4 Synthetic Analogues 5 References 1 Introduction Extensive epidemiological studies performed in many countries have shown that increased blood cholesterol levels or more specifically increased levels of low density lipoprotein (LDL) cholesterol are a major cause of coronary heart disease. There is also substantial evidence that lowering total and LDL cholesterol levels will reduce the incidence of coronary heart disease.l In 1976 Endo et al.reported the isolation of mevastatin (formerly called ML 236B or compactin) (1) as a potent inhibitor of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase the rate-limiting enzyme in endogenous cholesterol synthesis.2. They elucidated biochemical mechanisms for the action of me~astatin~-~ and by 1980 had shown that mevastatin strikingly lowers total and LDL cholesterol in patients with hyperch~lesterolemia.~ These findings stimulated the world- wide development of mevastatin analogues in the 1980s and by 1990 three drugs lovastatin (formerly called mevinolinlO or monacolin K11,12) (2) simvastatin13 (3) and pravastatin14 (4) had been marketed in many co~ntries.~~,~~ In addition to these many other mevastatin analogues have been synthesized some of which are now under clinical development.2 Biochemical and Pharmacological Mechanisms 2.1 HMG-CoA Reductase HMG-CoA reductase a 97 kDa glycoprotein," catalyses the reductive deacylation of HMG-CoA to mevalonate in a two- step reaction (Scheme 1). The stereospecificity of HMG-CoA reductase is illustrated in Scheme 1. Only the 3sisomer of HMG-CoA is utilized in the reaction.1s-20 Each hydride transfer involves the pro-R or 'A' side of the NADPH pyridine ring,21.22 the first forming the 3S HO ,CH3 NADPH NADP+ CoA-S &02H 5R-thiohemiacetal and the second incorporating the hydrogen into the 5-pro-S position of (3S)-me~alonate.~~.23. 24 Mevastatin analogues are reversible competitive inhibitors of HMG-CoA red~ctase.~~'~ The K value for mammalian HMG- CoA reductase is -10 ,UM while the Ki value for the ring- opened acids of mevastatin (5) and lovastatin (6) are in the .~~ range of 0.2-1 n ~Thus the affinity of HMG-CoA reductase for mevastatin analogues is 10000-fold or more than its affinity for the natural substrate HMG-CoA. The 3,5-dihydroxy- heptanoic acid portion of these compounds resembles the HMG portion of HMG-CoA (Scheme 1). The 3,5-dihydroxyheptanoic acid chain of mevastatin inter- acts with the HMG binding domain of the enzyme's active site. It has been postulated that the tight binding of mevastatin is the result of its ability to simultaneously interact with both the HMG binding domain and an adjacent hydrophobic pocket which is not utilized in substrate binding.26 Kinetic studies have shown that the enzymatic reaction is consistent with the general chemical mechanism postulated for (1) R =H (3) (2) R = Me C02Na "'YOH 0 (4) (5) R = H (6) R = Me NADPH" NADP' 3s-HMG-COA 3S,5R-Mevakfyl-CoA 3R-Mevalonate thiohemiacetal Scheme 1 541 38-2 NATURAL PRODUCT REPORTS 1993 "Do"vo"U0H0qC02H OH 0 +& R'-&&&& R'-tiIN (8) R = H H 00 R.* (9) R = 0 ,.-' (12)0 ..*-\(13) "OH (7) R=H (10) R =Me (11) R = Me (14) R=Me dehydrogenase catalysis in assisting direct transfer of a hydride ion between nucleotide and The pKa of this catalytic group is dependent on whether reduced or oxidized cofactor (NADPH or NADP' respectively) is bound at the active site.Mevastatin (1) apparently owes its inhibitory activity to the ability to mimic the half-reduced substrate mevalonate hemithioacetal. Therefore by analogy the 5-hydroxy group of mevastatin must interact with the un-protonated form of this catalytic group. Mevastatin analogues inhibit cholesterol biosynthesis in a variety of mammalian cell cultures at nM concentration^.^. 6 25 In human skin fibroblast cultures sterol synthesis from [14C]acetate is inhibited by 50% at -1 nM. When mevastatin analogues are given orally to rats sterol synthesis in the liver the major organ of sterol synthesis is acutely inhibited.Mevastatin and lovastatin do not lower plasma cholesterol levels in rats and mice,28 but these agents are highly effective in reducing plasma cholesterol in dogs,29 30 rabbit^^^,^^ and h~mans.~.~~*~~,~~ 2.2 Plasma Cholesterol Cholesterol is transported in plasma in the form of lipoproteins. By means of ultracentrifugation six distinct classes of lipo- protein can be isolated from plasma. Most of the cholesterol in humans is carried by LDL. Mammalian cells from normal subjects possesses LDL receptors on the cell surface which bind LDL with a high- affinity.35 The bound LDL is incorporated into the cells and undergoes lysosomal digestion leading to hydrolysis of cholesterol esters. The free cholesterol that is released serves to control the rate of cholesterol synthesis within the cells by down-regulating HMG-CoA reductase.l' The chief role of the LDL receptor is to provide a constantly available source of cholesterol throughout the body.Mutations of the gene encoding the LDL receptor results in impaired degradation of LDL and thus cause familial hypercholesterolemia (FH).36 Endogenous cholesterol synthesis is decreased by exposing cells to LDL which facilitates delivery of exogenous cholesterol and thus down-regulates HMG-CoA red~ctase.~~ The co-ordinate regulation of LDL receptor expression and _. CH3CCOONa A CH3SCH2CH&H(NH*)COOH Scheme 2 HMG-CoA reductase activity provides a homeostatic mech- anism for ensuring an adequate supply of cholesterol to cells such as hepatocytes which metabolize large amounts of LDL each day.36 HMG-CoA reductase inhibitors typified by mevastatin (1) block endogenous cholesterol synthesis es- pecially in the liver which requires cholesterol as a substrate for bile acid synthesis4 To overcome the short-fall hepatocytes express a greater number of LDL receptors and thereby promote influx of LDL cholesterol from ~lasma.~~,~' The net result is a decrease in plasma LDL cholesterol.Lovastatin (2) and simvastatin (3) are both lactones and inactive until metabolized in the liver to the open-ring hydroxy acids. 3 Mevinic Acids of Fungal Origin 3.1 Isolation Mevastatin (l) a metabolite of Penicillium citrinum was isolated in 1973 filed for patent in 1974 and first described in the literature in 1976.2.This compound was independently isolated from P. brevicompactum as an antibiotic by Brown et al.38Subsequently lovastatin (2) was isolated from Monascus ruberll.l2 and Aspergillus terreus,1° respectively. Lovastatin is slightly more potent than mevastatin in inhibiting HMG-CoA reductase. These compounds can be easily converted to the respective open-chain dihydroxy acids (5) and (6). Along with mevastatin dihydrocompactin (7),39 ML-236A (S),2 and ML-236C (9)2 have been isolated from P. citrinum. Monacolin J monacolin L (1 l),,O dihydromonacolin L (l2),,l and 3a-hydroxy-3,5-dihydromonacolin L acid (1 3)42 are minor metabolites of M. ruber. Dihydromevinolin (14) is a product of A. terre~s.,~ The class of compounds mentioned above distinguished by a highly functionalized hexalin or octalin unit and a /3-hydroxy- 6-lactone portion linked by an ethylene bridge are collectively referred to as mevinic Dihydrocompactin (7) and dihydromevinolin (14) are com- parable to mevastatin and lovastatin respectively in inhibiting HMG-CoA reductase while other metabolites that lack the side chain ester are far less active.25 3.2 Biosynthesis [13C]Acetate [methy1-l3C]methionine and 1802 are incorporated into mevastatin and lovastatin in cultures of P.citrinum M. ruber and A. terreus and the 'H and 13C NMR spectra of the two products have been fully assigned by a combination of spectral analyses. Both compounds are formed by the head-to- tail coupling of two polyketide chains (C and C18)each derived from acetate units.The C chain has one methionine derived methyl group and a methyl group at C-6 in the bicyclic ring system of lovastatin is also derived from methionine (Scheme 2).4547 The above data suggest involvement of a biological Diels- Alder cyclization to generate the correct ring stereochemistry in a single step. Such ring forming processes have been suggested for a number of reduced polyketide metabolites. Mevinic acids isolated from M. ruber by Endo and co-workers include dihydromonacolin L (1 2) 3a-hydroxy-3,5- NATURAL PRODUCT REPORTS 1993-A. END0 AND K. HASUMI Methionine (2) (10) (3) Reagents i LiOH H,O 100"C; H,O+ toluene 110°C; ii TBSCl; iii 2,2-dimethylbutyryl chloride; Bu"NF HOAc Scheme 4 Table 1 Effects of modification of the side chain ester moiety of lovastatin.R Relative potency" 254 FA 15 w a Potency of mevastatin is assigned a value of 100. dihydromonacolin L acid (1 3) monacolin L (1 I) monacolin J (10) and monacolin K (lovastatin) (2). They have shown that dihydromonacolin L acid (1 5) is converted to 3a-hydroxy-3,5-dihydromonacolin L acid (13) by M. ruber in the presence of molecular oxygen.42 The latter can be spontaneously dehydrated to monacolin L acid (16) although conversion of exogenously added 301-hydroxy-3,5-dihydromonacolin L acid to monacolin L acid by M. ruber has not been successful. Monacolin L acid is hydroxylated to monacolin J acid (17) by the action of a mono~xygenase.~~ The end-product monacolin K acid (6) can be derived from monacolin J acid (17) by M.r~ber.~~ One possible mechanism for this conversion is the esterification of monacolin J acid with a-methylbutyryl-CoA to monacolin K acid. These results are summarized in Scheme 3. 3.3 Modification The 2s-methylbutyryl ester of mevastatin and lovastatin gave ML-236A and monacolin J respectively by hydrolysis with either alkali or carboxylesterase of the fungus Emericellu ur~guis.~O,~~ Hoffman et ul. at Merck Sharp & Dohme (MSD) synthesized a series of the side chain ester analogues of lovastatin from monacolin J (Scheme 4).13 A systematic exploration of the structure-activity relationships showed that the introduction of an additional aliphatic group on the carbon 01 to the carbonyl group increased potency (Table l).13 This observation led to the synthesis of simvastatin (3) (Scheme 4) which has about 2.5 times the intrinsic inhibitory activity of lovastatin (2).13 A process involving enolization/methylation of the 2s-methylbutyrate side chain of lovastatin also affords simvastatin highly effi~iently.~ Simvastatin has been marketed since 1988.Side chain ether analogues of lovastatin are weaker inhibitors of HMG-CoA reductase than the corresponding side chain ester analogues. Of the ether analogues prepared by Lee et al. the 4-fluorobenzyl ether analogue (1 8) proved to enhance the potency.53 "OU0 (19) R = P-OH (20) R = a-OH Modification of the hexahydronaphthalene ring 6-position in simvastatin (3) viaoxygenation and oxa replacement by Duggan et al.led to the selection of (19) and (20) for pharmacological eval~ation.~~ These two compounds proved to be orally active as hypocholesterolemic agents in cholestyramine-primed dogs and also exhibit low peripheral plasma drug activity levels which may minimize pharmacologically related side effects since the major site of cholesterol synthesis is the liver. (+)-6- Ethylmevastatin (21) is comparable to lovastatin in inhibitory PO tency.55 Pravastatin (4)14 is prepared by microbial transformati~n.~~. 57 The enzyme cytochrome P-450,, is responsible for this conversion by Streptomyces carbophil~s.~~ Pravastatin is struc- turally different from lovastatin in that it contains a hydroxyl group in the hexahydronaphthalene ring making pravastatin more hydrophilic than lovastatin.Pravastatin is comparable to mevastatin and lovastatin in inhibiting HMG-CoA reductase and in lowering plasma cholesterol. It has been on the market since 1989. The phosphorylated derivatives (22) and (23) are produced by the action of several fungal These derivatives are converted to the respective parent compounds (5)and (6) in the liver when administered to rats. Compound L-669262 (24) is derived from simvastatin (3) by microbial conversion and is 6-7 times more active than simvastatin in inhibiting HMG-CoA reductase.60 4 Synthetic Analogues Since the mid 1980s a plethora of work has been directed towards the preparation of synthetic analogues of mevastatin (1) and lovastatin (2) by many pharmaceutical companies.Of these studies initial investigations at MSD served to delineate key structure-activity relationship for mevastatin-like mimics and afforded a series of moderately effective HMG-CoA reductase inhibitors bearing a monocyclic substituent of which the ring-opened form of lactone (25) was the most potent.61.62 In general unless the hydroxy groups remain unsubstituted in an eryfhrorelationship inhibitory activity is greatly reduced. Furthermore only one enantiomer of the ring-opened form of the lactone possesses the activity displayed by the racemate.61 NATURAL PRODUCT REPORTS 1993 0 ,OqrO 0 (22) R=H (23) R =Me (27) R' = CI R2 = H (28) R'=H R2=CI These findings reveal that the chiral lactone moiety is essential for biological activity.Insertion of a bridging unit other than ethyl or (E)-ethenyl between the Scarbin01 moiety and an appropriate lipophilic moiety (e.g. 2,4-dichlorphenyl) attenuates activity.61 Further studies of a series of substituted derivatives of (25) at MSD provided a series of 7-[3,5-disubstituted( 1,l '-biphenyl)-2- yl]-3,5-dihydroxy-6-heptanoicacids of which (26) possessed 2.8 times the inhibitory activity of me~astatin.~~ X-ray crystallography studies on compound (26) showed it to possess the same chirality in the lactone ring as mevastatin. Potent inhibitory activity was not retained without concomitant sub-stitution at the 3- and 5-positions of the central phenyl ring of the biphenyl moiety with methyl or chloro The type and position of substituents on the external phenyl ring is critical.An electron-donating group (CH or CH,O) in the 4'- position is detrimental whereas a halogen (C1 or F) in this position is beneficial. Ring fusion of substituted biphenyls afforded products that were substantially less active indicating that the dihedral angle between phenyl rings must be greater than 0" to maintain a high level of inhibitory potency.64 Substituted naphthalene derivatives (27) and (28) display about the same potency as me~astatin.~~ Investigations at the Sandoz Research Institute further extended the findings obtained at MSD. The researchers chose indolyl derivatives by considering structures and molecular shapes of both coenzyme A and mevastatin.An extensive and rapid analogue program led to the choice of fluvastatin (XU62- 320) (29) (Scheme 5) as a candidate for extensive biological testing.66 As compared to the respective sodium salts of mevastatin and lovastatin fluvastatin is 22- and 10-fold more potent in inhibiting HMG-CoA reductase. However fluvastatin is comparable to mevastatin in cholesterol-lowering activity in patients.67 This drug is now under development and expected to be marketed in 1993. Many synthetic analogues of mevastatin have been prepared at Hoechst AG. Baader et al. prepared compound (30) which is comparable to lovastatin with respect to inhibition of HMG- CoA reductase.68 However the cholesterol-lowering activity of (30) in rabbits is slightly lower when compared to that of lovastatin.The same group prepared the pyridine analogue HR 780 (31) (Scheme 6),69370 which exceeded the activity of NATURAL PRODUCT REPORTS 1993-A. END0 AND K. HASUMI +k-alp F F F ___L i ii iii __c CI IA F C02Na 2 COOR v Reagents i EtOH A ii ZnC1,; iii Me,NCH=CHCHO POCl,; NaOH; iv NaH Bu"Li; v Et,B THF; NaBH, -78 "C; vi MeOH NaOH. Scheme 5 Reagents i Bu'Ph,SiCl; imidozole; ii CH,CO,But LDA; iii Et,B NaBH,; iv MeC(OMe),H H+; v Bu,NF; vi Swern oxidation; vii base; viii CF,CO,H Scheme 6 (31) NATURAL PRODUCT REPORTS 1993 C02Na ""'COH HoqoFwR i F C02Na HoqoI ONa (33)R=Pr' (34)R = pCeH4F (36) (37) (38) (35) R = S@-CeH4F) F 'w F v \/ /0 \/- coNHQ (39) (40)R = CI (41) R=OMe F F F F F .COOCH3 F6 t F H3C0 - vii vi V f- c- H3c0* H3c0&wE' viii C02Na ""-C,H N H (+)-Enantiomer (6) Reagents i neat 180 "C; ii CH,Cl, DDQ; iii THF Red-Al; iv THF CH,I; v THF LiAlH, reflux; CH2C12 PCC rt; vi THF (EtO),POCH=CHNHCy NaH rt; vii THF pentan-2,4-dione NaH BuLi 0 "C; THF BEt, NaBH, -65 "C; viii THF S-( +>-phenylglycinol 50 "C;chromatography; ix EtOH NaOH reflux.Scheme 7 END0 AND K. HASU3A1 NATURAL PRODUCT REPORTS 1993-A. is several times higher than that of lovastatin analogues enhancing the in vivo efficacy of the drug. Clinical trials with HR 780 are in progress.Compound (32) prepared by the investigators at Hoechst is 3-5 times more active than lovastatin in both in vitro and in vivo assays and is a promising candidate for development as a choles terol-lowering agent. The phenoxy- type inhibitors (3 3) (34) and (35) show more pronounced changes in their pharmacological These compounds are considerably stronger in vitro than lovastatin and highly efficacious in rabbits whilst they have only moderate activity in dogs. Lovastatin exhibits comparable activity in these animal species. The analogues having 1-N-methyltetrazol-5-y1 attached to the C-8 position of 9,9-bis(substituted aryl)-3,5-dihydroxy-6,8-nonadienoic acids have been prepared at Bristol-Meyers Squibb of which BMY 22089 (36) is a promising candidate for de~elopment.'~,~~ The open chain salt form of the 4R,6S enantiomer of (36) inhibits HMG-CoA reductase with a K value of 4.3 nM a value comparable to that for lovastatin (2).75 The hydroxyphosphinyl-containinginhibitor SQ 33 600 (37) is about as effective as pravastatin and lovastatin in inhibiting de novo cholesterol biosynthesis on intravenous ad-mini~tration.~~ SQ 33 600 also shows oral activity equivalent to that of pravastatin and lovastatin and is also effective as a hypocholesterolemic agent in rabbits dogs and monkey^.'^ It has been chosen for clinical study in humans.A series of substituted pyridines containing a hydroxy-phosphinyl functionality have been prepared at Bristol-Meyers Squibb leading to the synthesis of compound (38).77 Compound (38) exhibited acute in vivo activity in rats comparable to that of lovastatin and pravastatin.Investigators at the Warner-Lambert Company have reported a variety of substituted pyrr~le,~~,~~ quinoline,'O and pyrazolea1,82 mevalonolactones. Compound (39) is 5 times more potent than mevastatin in ~itro.~~ Two quinoline mevalonolactones (40) and (4 l) are comparable to mevastatin both in vitro and in vivo.'O The optically active form of compound (42) is 5-10 times more potent than mevastatin both in vitro and in vivo.'2 Thus the hypocholesterolemic activity of compound (42) in dogs was significant at doses of 0.1 mg/kg while lovastatin did not show a statistically significant lowering at doses lower than 1 mg/kg.82 Investigators at Rhone-Poulenc Rorer reported a series of mevastatin analogues containing a phenylcyclohexane group.Of these compounds RG 12561 (dalvastatin) (43) was slightly more potent in inhibitory potency than 10vastatin.~~ RG 12561 was evaluated in clinical trials for the treatment of hyper-cholesterolemia. The analogue (44) is 5 times more potent in vitro than l~vastatin.'~ A research group at SmithKline Beecham reported alterna- tives to the 5-hydroxymethylene group of mevastatin analogues including a,a-difluoroketonesa4 and phosphinic Highly potent HMG-CoA reductase inhibitors have recently been discovered via synthetic variation of the substitution pattern of functionalized pyridines by investigators at Bayer AG. The 5-methoxymethyl derivative BAY W 6228 (45) is the most potent compound and has been selected for further development (Scheme 7).86 In the in vitro assay BAY W 6228 is 110 times more potent than lovastatin.The absolute 3R,5S- stereochemistry in the 3,5-dihydroxy acid side chain is necessary for the inhibitory activity (Table 2). For optimal activity a branched substituent i.e.isopropyl cycloakyl or aryl at C-6 of the pyridine ring in combination with an isopropyl (or cyclopropyl) group at C-2 and a p-fluorophenyl substituent at C-4 is required. Substitution at position 5 of the pyridine ring increased the potency and the 5-methoxymethyl group of BAY W 6228 is essential for its outstanding in vitro and in vivo activity (Table 3). BAY W 6228 significantly reduces serum cholesterol levels in dogs at a daily dose of 30 ,ug/kg and is thus at least 200 times more potent than lovastatin under the same condition^.'^ BAY W 6228 is under clinical development.Table 2 HMG-CoA reductase inhibitory activities of substituted pyridines (I). Stereochemistry of the side chain. F R Relative potency" OH OH &&COONa 110 ?H ?H COONa 0.2 4-4-xc,,a 0.2 OH OH &Coma 0.03 COON a 40 0.4 a Potency of lovastatin is assigned a value of 1. lovastatin (2) in inhibiting sterol synthesis in HEP G2 cells. HR 780 effectively lowers plasma cholesterol levels in normo-lipidemic and hyperlipidemic animals (rats dogs and monkeys). Based on equipotent doses the potency of HR 780 appears to be 5-10 times that of lovastatin.The plasma half-life of HR 780 NATURAL PRODUCT REPORTS 1993 Table 3 HMG-CoA reductase inhibitory activities of substituted pyridines (11). F COONa H3C0 Substitution in position 5 Substitution in positions 2 and 6 Relative Relative R5 potency" R6 R2 potencya H 0.8 CH3 CH3 < 0.01 CH3 12 CH3 CH(CH,) 0.1 20 CH(CH3)2 CH3 0.1 CH,OH 20 CH(CH,) CH(CH3) 50 CH,OCH 50 CH(CH3)2 40 CH(CH3)2 2o 0"""2o Q, '0 10 CH(CH,) 1.6 0""" 3 CH(CH,) 0.02 '0 COOCH 30 CH(CH3)2 l6 Substitution in position 4 Linker modifications Relative Relative R4 potency" A-B potency" Q 6 CH,-CH 0.7 Q 1 CH=CH (E) 50 F 50 c=c Q a Potency of lovastatin is assigned a value of 1. 5 References 1 Lipid Research Clinics Program JAMA 1988 251 351.2 A. Endo M. Kuroda and Y.Tsujita J. Antibiot. (Japan) 1976 29 1346. 3 A. Endo M. Kuroda and K. Tanzawa FEBS Lett. 1976,72,323. 20 4 A. Endo Y. Tsujita M. Kuroda and K. Tanzawa Eur. J. Biochern. 1977 77 31. 5 I. Kaneko Y. Hazama-Shimada and A. Endo Eur. J. Biochem. 1978 87 313. 6 M. S. Brown J. R. Faust J. L. Goldstein I. Kaneko and A. Endo J. Biol. Chem. 1978 253 1121. NATURAL PRODUCT REPORTS 1993-A. END0 AND K. HASUMI 7 0. Doi and A. Endo J. Med. Sci. Biol. (Japan) 1978 31 225. 8 K. Tanzawa and A. Endo Eur. J. Biochem. 1979,98 195. 9 A. Yamamoto H. Sudo and A. Endo Atherosclerosis 1980 35 259. A. W. Alberts J. Chen G. Curon V. Hunt J. Huff C. Hoffman J. Rothrock M.Lopez H. Joshua E. Harris A. Patchett R. Monaghan S. Currie E. Stapley G. Albers-Schonberg 0. Hensens J. Hirshfield K. Hoogsteen J. Liesch and J. Springer Proc. Natl. Acad. Sci. USA 1980 77 3957. 11 A. Endo J. Antibiot. (Japan) 1979 32 852. 12 A. Endo J. Antibiot. (Japan) 1980 33 334. 13 W. F. Hoffman A. W. Alberts P. S. Anderson J. S. Chen R. L. Smith and A. K. Willard J. Med. Chem. 1986 29 849. 14 Y. Tsujita M. Kuroda Y. Shimada K. Tanzawa M. Arai I. Kaneko M. Tanaka H. Masuda C. Tarumi Y. Watanabe and S. Fujii Biochim. Biophys. Acta 1986 877 50. 15 S. M. Grundy N. Engl. J. Med. 1988 319 24. 16 D. B. Hunninghake Curr. Opin. Lipidol. 1992 3 22. 17 J. L. Goldstein and M. S. Brown J. Lipid. Res. 1984 25 1450. 18 D. Veloso W. W.Cleland and J. W. Porter Biochemistry 1981 20 887. 19 A. S. Beedle K. A. Munday and D. C. Wilton FEBS Lett. 1972 28 13. T. C. Linn J. Biol. Chem. 1967 242 984. 21 R. E. Dugan and J. W. Porter J. Biol. Chem. 1971 246 5361. 22 A. S. Beedle K. A. Munday and D. C. Wilton Eur. J. Biochem. 1972 28 151. 23 P. Blattmann and J. Retey J. Chem. Soc. Dalton Trans. 1970 1394. 24 P. Blattmann and J. Retey Hoppe-Seyler’s Z. Physiol. Chem. 1971 352 369. 25 A. Endo J. Med. Chem. 1985 28 401. 26 R. H. Abeles and H. Nakamura Biochemistry 1985 24 1364. 27 W. E. Brown and V. W. Rodwell in ‘Dehydrogenases Requiring Nicotinamide Coenzymes’ ed. J. Jeffery Birkhauser Verlag Basel 1980 p. 232. 28 A. Endo Y. Tsujita M. Kuroda and K. Tanzawa Biochim.Biophys. Acta 1979 575 266. 29 Y. Tsujita M. Kuroda K. Tanzawa N. Kitano and A. Endo Atherosclerosis 1979 32 307. P. T. Kovanen D. W. Bilheimer J. L. Goldstein J. J. Jaramillo and M. S. Brown Proc. Natl. Acad. Sci. USA 1981 78 1194. 31 M. Kuroda Y. Tsujita K. Tanzawa and A. Endo Lipids 1979 14 585. 32 Y. Watanabe T. Ito M. Saeki M. Kuroda K. Tanzawa M. Mochizuki Y. Tsujita and M. Arai Atherosclerosis 1981,38,27. 33 P. A. Kroon K. M. Hand J. W. Huff and A. W. Alberts Atherosclerosis 1982 44,41. 34 A. Endo J. Lipid Res. 1992 33 1569. 35 M. S. Brown and J. L. Goldstein Proc. Natl. Acad. Sci. USA 1979 76 3330. 36 M. S. Brown and J. L. Goldstein Science 1986 232 34. 37 M. S. Brown and J. L. Goldstein N.Engl. J. Med. 1981,305,515.38 A. G. Brown T. C. Smale T. J. King R. Hasenkamp and R. H. Thompson J. Chem. SOC. Perkin Trans. I 1976 1165. 39 Y. K. T. Lam V. P. Gullo R. T. Goegelman D. Jorn L. Huang C. De Riso R. L. Monagham and I. Putter J. Antibiot. (Japan) 1981 34 614. A. Endo K. Hasumi and S. Negishi J. Antibiot. (Japan) 1985 38 420. 41 A. Endo K. Hasumi T. Nakamura M. Kunishima and M. Masuda J. Antibiot. (Japan) 1985 38 321. 42 T. Nakamura D. Komagata S. Murakawa K. Sakai and A. Endo J. Antibiot. (Japan) 1990 43 1597. 43 G. A. Schonberg H. Joshua M. B. Lopez 0.D. Hensens J. P. Springer J. Chen S. Osterove C. H. Hoffman A. W. Alberts and A. A. Patchett J. Antibiot. (Japan) 1981 34 507. 44 T. Rosen and C. H. Heathcock Tetrahedron 1986 42 4909. 45 J.K. Chan R. N. Moor T. T. Nakashima and J. C. Vederas J. Am. Chem. SOC. 1983 105 3334. 46 R. N. Moor G. Bigam J. K. Chan et al. J. Am. Chem. SOC. 1985 107 3694. 47 A. Endo Y. Negishi T. Iwashita K. Mizukawa and M. Hirama J. Antibiot. (Japan) 1985 38 444. 48 D. Komagata H. Shimada S. Murakawa and A. Endo J. Antibiot. (Japan) 1989 42 407. 49 K. Kimura D. Komagata S. Murakawa and A. Endo. J. Antibiot. (Japan) 1990 43 780. D. Komagata H. Yamashita and A. Endo J. Antibiot. (Japan) 1986 39 1574. 51 S. Murakawa T. Nakamura D. Komagata E. Sunagawa and A. Endo Agric. Biol. Chem. 1987 51 1879. 52 D. Askin T. R. Verhoeven T. M.-H. Liu and I. Shinkai J. Org. Chem. 1991 56 4929. 53 T.-J. Lee W. J. Hotz R. L. Smith A. W. Alberts and J. L. Gilfillan J.Med. Chem. 1991 34 2474. 54 M. E. Duggan A. W. Alberts R. Bostedor Y.-S. Chao J. I. Gemershausen J. L. Gilfillan W. Halczenko G. D. Hartman V. Hunt J. S. Imagire M. S. Schwartz R. L. Smith and R. J. Stubbs J. Med. Chem. 1991 34 2489. 55 D. L. J. Clive K. S. K. Murthy R. George and M. J. Poznansky J. Chem. SOC. Perkin Trans. I 1990 2099. 56 N. Serizawa K. Nakagawa K. Hamano Y. Tsujita A. Terahard and H. Kuwano J. Antibiot. (Japan) 1983 36 604. 57 N. Serizawa S. Serizawa K. Nakagawa K. Furuya T. Okazaki and A. Terahara J. Antibiot. (Japan) 1983 36 887. 58 T. Matsuoka S. Miyakoshi K. Tanzawa K. Nakahara M. Hosobuchi and N. Serizawa Eur. J. Biochem. 1989 184 707. 59 A. Endo H. Yamashita H. Naoki T. Iwashita and Y. Mizukawa J.Antibiot. (Japan) 1985 38 328. 60 H. Joshua M. S. Schwartz and K. E. Wilson J. Antibiot. (Japan) 1991 44 366. 61 G. E. Stokker W. F. Hoffman A. W. Alberts E. J. Cragoe Jr. A. A. Deana J. L. Gilfillan J. W. Huff F. C. Novello J. D. Prugh R. L. Smith and A. K. Willard J. Med. Chem. 1985 28 347. 62 W. F. Hoffman A. W. Alberts E. J. Cragoe Jr. A. A. Deana B. E. Evans J. L. Gilfillan N. P. Gould J. W. Huff F. C. Novello J. D. Prugh K. E. Rittle R. L. Smith G. E. Stokker and A. K. Willard J. Med. Chem. 1986 29 159. 63 G. E. Stokker A. W. Alberts P. S. Anderson E. J. Cragoe Jr. A. A. Deana J. L. Gilfillan J. Hirshfield W. J. Holtz W. F. Hoffman J. W. Huff T. J. Lee F. C. Novello J. D. Prugh C. S. Rooney R. L. Smith and A. K. Willard J. Med. Chem.1986,29 170. 64 G. E. Stokker A. W. Alberts J. L. Gilfillan J. W. Huff and R. L. Smith J. Med. Chem. 1986 29 852. 65 J. D. Prugh A. W. Alberts A. A. Deana J. L. Gilfillan J. W. Huff R. L. Smith and M. Wiggins J. Med. Chem. 1990,33 758. 66 F. G. Kathawala in ‘Trends in Medicinal Chemistry ’88’ ed. H. van der Goot G. Domany L. Pallos and H. Timmerman Elsevier Amsterdam 1989 p. 709. 67 J. Yuan M. Y. Tsai J. Hegland and D. B. Hunninghake Atherosclerosis 1991 87 147. 68 E. Baader W. Bartmann G. Beck A. Bergmann H. Jendralla K. Kesseler G. Wess W. Schubert E. Granzer B. v. Kerekjarto and R. Krause Tetrahedron Lett. 1988 29 929. 69 G. Beck K. Kesseler E. Baader W. Bartmann A. Bergmann E. Granzer H. Jendralla B. v. Kerekjarto R. Krause E.Paulus W. Schubert and G. Wess J. Med. Chem. 1990 33 52. 70 G. Wess K. Kesseler E. Baader W. Bartmann G. Beck A. Bergmann H. Jendralla K. Bock G. Holzstein H. Kleine M. Schnierer Tetrahedron Lett. 1990 31 2545. 71 H. Jendralla E. Baader W. Bartmann G. Beck A. Bergmann E. Granzer B. v. Kerekjarto K. Kesseler R. Krause W. Schubert and G. Wess J. Med. Chem. 1990 33 61. 72 H. Jendralla E. Granzer B. v. Kerekjarto R. Krause U. Schacht E. Baader W. Bartmann G. Beck A. Bergmann K. Kesseler G. Wess S. Granata J. Herchen H. Kleine S. Schussler and K. Wanger J. Med. Chem. 1991 34 2962. 73 N. Balasubramanian P. J. Brown J. D. Catt W. T. Han R. A. Parker S. Y. Sit and J. J. Wright J. Med. Chem. 1989 32 2038. 74 S. Y. Sit R. A. Parker I. Motoc W.Han N. Balasubramanian J. D. Catt P. J. Brown W. E. Harte M. D. Thompson and J. J. Wright J. Med. Chem. 1990 33 2982. 75 R. A. Parker R. W. Clark S.-Y. Sit T. L. Lanier R. A. Grosso and J. J. K. Wright J. Lipid Res. 1990 31 1271. 76 D. S. Karenewsky M. C. Badia C. P. Ciosek Jr. J. A. Robl M. J. Sofia L. M. Simpkins B. DeLange T. W. Harrity S. A. Biller and E. M. Gordon J. Med. Chem. 1990 33 2952. 77 J. A. Robl L. A. Duncan J. Pluscec D. S. Karanewsky E. M. Gordon C.P. Ciosek Jr. L. C. Rich V. C. Dehmel D. A. Slusarchyk T. W. Harrity and K. A. Obrien J. Med. Chem. 199 1 34 2804. 78 B. D. Roth D. F. Ortwine M. L. Hoefle C. D. Stratton D. R. Sliskovic M. W. Wilson and R. S. Newton J. Med. Chem. 1990 33 21. 79 D. B. Roth C. J. Blankley A.W. Chucholowsky E. Ferguson M. L. Hoefle D. F. Ortwine R. S. Newton C. S. Sekerke D. R. NATURAL PRODUCT REPORTS 1993 Sliskovic C. D. Stratton and M. W. Wilson J. Med. Chem. 84 G. B. Dreyer and B. W. Metcalf Tetrahedron Lett. 1988 29 1991 34,357. 6885. 80 D. R. Sliskovic J. A. Picard W. H. Roark B. D. Roth E. 85 G.B. Dreyer C. T. Garvie B. W. Metcalf T. D. Meek and R. Ferguson B. R. Krause R. S. Newton C. Sekerke and M. K. Mayer Bioorg. Med. Chem. Lett. 1991 1 151. Shaw J. Med. Chem. 1991 34,367. 86 R. Angerbauer P. Fey W. Hubsch T. Philipps and D. Schmidt 81 D. R. Sliskovic B. D. Roth M. W. Wilson w. L. Hoefle and ‘XI International Symposium on Drugs Affecting Lipid R. S. Newton J. Med. Chem. 1990 33 31. Metabolism’ Florence Italy Abstract 1992 p.69. 82 D. R. Sliskovic C. J. Blankley B. R. Krause R. S. Newton J. A. 87 G. Thomas and R. Paglia ‘XI International Symposium on Picard W. H. Roark B. D. Roth C. Sekerke M. K. Shaw and Drugs Affecting Lipid Metabolism ’ Florence Italy Abstract R. L. Stanfield J. Med. Chem. 1992 35 2095. 1992 p. 70. 83 J. R. Regan J. G. Bruno K. Gustafson D. Amin K. Neuenschwander and M. Perrone Eur. J. Med. Chem. 1992,27 735.

ISSN:0265-0568    

DOI:10.1039/NP9931000541

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

A Survey of Natural Products which Abstract Hydrogen Atoms from Nucleic Acids J. A. Murphy* and J. Griffiths Department of Chemistry University of Nottingham University Park Nottingham NG7 2RD 1 Introduction 1 Introduction 2 Bleomycin A number of intriguing antibiotics with pronounced antitumour 2.1 Structure activity have been reported in recent years which have a 2.2 Binding to DNA common mode of action via nucleic acid modification by 2.3 Mechanism of Action hydrogen atom abstraction from either deoxyribose or ribose. 2.3.1 Base Selectivity The potency of many of these compounds has led to enormous 2.4 Reactivity with RNA interest in their mode of action. This survey unites current 3 Neocarzinostatin information on these compounds.3.1 Structure 3.2 Binding to DNA 3.3 Mechanism of Action 2 Bleomycin 4 Calicheamicins Bleomycin is used in the treatment of malignant lymphomas 4.1 Structure and squamous cell carcinomas. The clinically used antitumour 4.2 Binding to DNA agent blenoxane is a mixture of bleomycins but features 4.3 Mechanism of Action bleomycin A (1) as the principal component Figure 1. The 5 Esperamicins chemistry of bleomycin has been reviewed.' 6 Dynemicin A 7 Kedarcidin 2.1 Structure 8 References The structure of bleomycin A has been confirmed by total synthesis. Related bleomycins have also been reported. The OH OA NH2 0 0 Bleomycins X = t&B:fr t$>:er (2) Phleomycins X = A, R = (CH2)3SOCH3 D1 R = (CH2)4NH(C=NH)NH2 (1) A5 R = (CH2)3S+Me2 E R = (CH,)dNH(C=NH)NH(CH2)4NH(C=NH)NH2 (5) Demethyl A, R = (CH2)giMe AY~:R = (CH2)$lH2 Arb R = (CH2)3NH2 Azc R = (CH2)2 $ H A, R = (CH2)3NH(CH2)4NH2 Ag R = (CH~)SNH(CH~)~NH(CH~)~NH~ B1 R=H (8) B2 R = (CH2)4NH(C=NH)NH2 B4 R = (CHZ),NH(C=NH)NH(CH2)4NH(C=NH)NH2 Figure 1 55 1 NATURAL PRODUCT REPORTS 1993 OANH2 (3) H2NOC H2N.CH3 Figure 2 OY‘ :o \ 0 Figure 3 phleomycins (2) differ from the bleomycins in possessing a thiazoline-thiazole rather than a bisthiazole. Tallysomycins e.g. tallysomycin A (3) differ principally in the presence of an extra sugar ring.3 Bleomycin was first isolated as its copper(I1) comple~,~ but it is believed to exert its antitumour action as an iron complex.The structure of the iron complex which forms from Fe(11)- bleomycin A and dioxygen has been the subject of much debate.5 Figure 2 shows one coordination pattern which has been suggested for the initial oxygen complex but in recent NMR studies of a bleomycin A,-iron(n)-carbon monoxide complex evidence6 has been found that the carbamoyl group attached to mannose is coordinated to iron (Figure 3) ;this may or may not have relevance to the antitumour compound. Bleomycin A (1) is believed to effect its antitumour action by binding to and reacting with duplex DNA1 and/or RNA.’ Complexes with metals other than iron have been prepared. Cobalt manganese copper and vanadyl complexe~,~~~ as well as the iron complex cause DNA degradation under appropriate conditions.2.2 Binding to DNA The complete details of binding of bleomycin A (1) to DNA are not yet clear. It is certainly known that the molecule performs its cleavage chemistry in the minor groovelo of B- DNA and that therefore this is the site of the metal-complex. Investigations of the DNA cleavage efficiency and specificity of modified bleomycins have provided useful information on which sections of the molecule are necessary for the binding of bleomycin A (1). The thiazole end of the molecule tripeptide S (4) binds to DNA in the absence of active metal ions with a strength similar to bleomycin itself.ll The mode of binding of this portion of the bleomycin molecule is complex.There is some evidence for intercalation but minor groove binding and partial inter- calation have also been suggested for the bithiazole moiety and it seems likely that more than one mode of binding applies.’ Phleomycins (2) which contain a thiazoline-thiazole unit rather than a bithiazole cleave DNA with an efficiency and specificity very similar to that of bleomycin A (1).l2 It is very unlikely that a single thiazole would intercalate so intercalation cannot be a pre-requisite for the observed cleavage chemistry. A positive charge at this end of the molecule increases the NATURAL PRODUCT REPORTS 1993-5. A.MURPHY AND J. GRIFFITHS H2N++zN ~ N OHof))l"fNH HMe H H 0 Me HNp!Me HO Me S>'vNuMe SMe 4yNyN H HOQY) N H (9) OH strength of binding to DNA ~onsiderab1y.l~ It is to be noted however that molecules such as demethylbleomycin (5) (figure l) which are uncharged do still effect DNA ~leavage.'~ Bleomycin performs most of its chemistry at the pyrimidine of 5'-G-Py-3' sites.The cause of the specificity is not clear but it has been suggested that hydrogen-bonding occurs between the bithiazole of bleomycin and the 2-amino group of the G base.14 Recently,15 a study of the deglycobleomycin mono- thiazoles A (6) and B (7) demonstrated that these compounds were inefficient cleavers of DNA. Moreover no sequence selectivity was apparent and this was taken to reflect the importance of the bithiazole moiety in causing efficient and site-selective cleavage of DNA by bleomycin.Another study16 compared the reactivity of bleomycin A (l) bleomycin B (8) (figure I) deglycobleomycin A (9) and decarbamoylbleomycin A (10). All of these compounds show equal activity and good specificity for GC in d(CGC3TTT- AAAGCl'G). The site of action for B (8) is same as for A (1) i.e. at Cll; however the site of action is predominantly C3 for deglycobleomycin and decarbamoylbleomycin suggesting that the site of binding and cleavage is affected by the metal- binding region of these compounds. NATURAL PRODUCT REPORTS 1993 (1 1) IJ = 0,1,2,4 Scheme 1 I I I .MN I3' -y-D OpJe 0p-e o2 ___) H reduction -;y-0Lo-oJBae HO 0 3'-Glycolate Base Propenal 0- Sl -Generation of Abasic site (13) abasic site Strand scission under alkaline conditions Scheme 2 In a direct test of the role of the bithiazole unit in affecting damage sites.These results suggest that the bithiazole is not cleavage site selectivity derivatives of bleomycin (ll) which responsible for specific binding to G; it may be that added feature the bithiazole moiety separated from the metal-binding binding is needed from the bithiazole and charged terminus to centre by rigid of differing lengths were prepared. augment that of the metal-binding centre but that this need not Here the sites of lesions on DNA did not vary significantly be a very specific binding. through this series of compounds showing that the metal Although the metal-binding site usually determines the site binding centre rather than the bithiazole unit determined the of DNA cleavage modified bleomycins have now been NATURAL PRODUCT REPORTS 1993-5.A. MURPHY AND J. GRIFFITHS mn T I * 0. O ? w-T Oho-0 DNA Glycolate -0y I 0.? OVase O'r' "I'u. o-,/Base Mechanism A Scheme 3 I -HOI ?v OVase-1 Mechanism B Scheme 4 synthesized where this is not true. Attaching a slightly modified metal-binding domain to a distamycin moiety resulted in1' the distamycin dictating the site of binding and cleavage. 2.3 Mechanism of Action The mechanism has been extensively investigated. It is known that activated bleomycin A can be produced either by exposing Fe(1r)-bleomycin to oxygen and a reducing agent" or by reacting Fe(m)-bleomycin with hydrogen peroxide.[It has also been reported that activated bleomycin may be produced from the interaction of an alkyl hydroperoxide with Fe(I1)-bleomy~in,'~~.~~ but it is not currently certain if this is the same activated complex]. The activated complex is known to have two more oxidizing equivalents than F~(III)~~~.~~ and so the perferryl species (12) in Scheme 1 has been proposed as the reactive form.lsa This is formed from the dioxygen complex by electron donation and proton capture to give a hydroperoxide and then heterolytic breakdown to form the perferryl in- termedia te. This compound then abstracts a hydrogen from the C-4' of deoxyribose.l0" The removal of the C-4' hydrogen is very specific and this is proof that the reactive entity is not a diffusible hydroxyl radical but rather a bound species.The product radical can undergo decomposition in two ways giving rise either to direct strand cleavage or to generation of an abasic site (Scheme 2). In the first of these which requires the presence of dioxygen a peroxyl radicalz1 is formed and after conversion to the hydroperoxide this is proposed to undergo a Criegee-type rearrangement,22 which is thought to be either acid-catalysed or metal-ion assisted. The resulting compound reacts further giving spontaneous fragmentation to the base- propenal and a DNA strand with a modified 3'-end i.e. bearing a 3'-glycolate. Until 1992 the favoured mechanism for this fragmentation was mechanism A (Scheme 3).However this needs revision. In particular the glycolate carboxyl group bears two oxygens one of which has been shown to be the original deoxyribose oxygen and the other to be derived from a molecule of dioxygen. Kinetic studies had previously shown that it was possible to distinguish between the molecule of dioxygen required to activate bleomycin and the second molecule of dioxygen required for the production of the glycolates. Labelling studies showed that the oxygen in the carboxylate derives from this second molecule of dio~ygen,~~ and also that the oxygen in the aldehyde group of the base-propenal derives from water and not from dioxygen. Another fact which influenced the proposal of the alternative mechanism was that tritium was rapidly liberated from DNA containing deoxyriboses specifically labelled in the 2-pro-R position but not from the 2-pro-S position."' The tritium was liberated at about the same rate as DNA cleavage occurred and considerably faster than base-propenal formation.To account for these facts mechanism B was proposed (Scheme 4). NPR 10 NATURAL PRODUCT REPORTS 1993 Scheme 5 0,. 1 2-deoxyaristeromycin (14) Scheme 6 tf Figure 4 The alternative breakdown of the initial deoxyribosyl radical occurs to a greater extent at low oxygen tension.25 Here the 4'- radical undergoes electron loss to give a carbocation (Scheme 2) which is intercepted by This latter fact which was demonstrated by labelling with H,lsO was quite a surprise since it had been expected that the radical would react with the bleomycin complex to give the hydroxylated product by a 'rebound' mechanism by analogy with the reaction of the activated intermediates in cytochrome P-450 hydroxylations (Scheme 5).The only way in which the observed labelling results could be consistent with the mechanism shown in Scheme 5 would be if the intermediate iron-hydroxyl compound underwent very rapid exchange of the hydroxyl group with the solvent water. This would be very different from the behaviour of cytochrome P-450 where the rate constant for the rebound reaction is among the fastest ever quoted.26 Whichever mechanism applies the product of this second decomposition is (13). This compound is not very stable but does not lead to spontaneous cleavage of the DNA strand.Rather it may be cleaved by reaction with hydrazine or other amines.25,27 A very cunning probe of the mechanism of these reactions was devised by Saito,28 who incorporated a molecule of 2'-deoxyaristeromycin [d(Ari)] in place of a deoxyadenosine in the hexamer duplex d(GGAriAGG)-d(CCTTCC). Reaction with peplomycin a modified bleomycin [Figure 1 R = (CH,),NHCHMePh] was observed at the modified nucleoside and two products were isolated both derived from the 2'-deoxyaristeromycin. These were the alkene (14) and the alcohol (15) Scheme 6. Only this isomer of the alcohol was detected. Controls were performed to demonstrate that the two com- pounds (1 4) and (1 5)were produced directly i.e. that the alkene did not result from the alcohol during work-up and isolation.Formation of (14) was taken as additional evidence that a cationic intermediate is formed during the normal reaction of bleomycin A (1) with DNA; in this case deprotonation can dvvv-I n Base Ho.oy-J Scheme 7 occur to give the alkene. Interestingly no trace of hydro- peroxide-derived product was detected in this reaction even when the oxygen tension was drastically increased. 2.3.1 Base Selectivity Bleomycin can effect damage to either one strand or both strands of B-DNA. When damage to one strand occurs it is seen at aprimary target site. There is considerable evidence that when damage to two strands occurs the site-selectivity on the second strand is governed by the site of the initial lesion.Primary reaction is most commonly seen on the pyrim- idine29.30 in a 5'-GC-3' but sometimes on a 5'-GT-3' or less frequently 5'-GA-3'. When linked damage occurs on the second strand the site of that damage is governed by a set of selection rules as indicated in Figure 4. The primary target is represented in the top strand; the sites of damage are indicated by the arrows.3o The fact that the damage to the second strand occurs so frequently with these patterns at sites which are not primary targets for bleomycin suggests that the initial lesion potentiates the second reaction. Another interesting fact however is that if cleavage of the first strand has occurred then either cleavage or generation of the abasic site may occur on the second strand NATURAL PRODUCT REPORTS 1993-J.A. MURPHY AND J. GRIFFITHS 557 5' 3' GAAUACAAGCUUUAUCAAUAUGCUUUG "WJAAAAAUUGAAUU ,U"'A U'G-A.A U-A-C-C-C \ \I ! G \ /' 7-U-G-G-G, 4 G-C-G-G u-u/c 0 G C-G-C-A CH30aoH OK0 II ?Y GC II UOA 'Xc but if an abasic site is formed on the first strand then no 0 damage is seen to the secondary site. This suggests that damage 8, to the secondary site is mediated by a bound bleomycin qKo 01 molecule after in situ reactivation and it has been proposed that the peroxyl radical may on occasion abstract the hydrogen of the iron-hydroxyl from the reacting bleomycin Scheme 7. This would regenerate the active bleomycin for further reaction.30b The damage on the second strand involves reaction at the 4'-carbon again so this selectivity is maintained even at non-primary target sites.2.4 Reactivity with RNA Whereas the cleavage of duplex DNA has been intensively investigated studies on RNA have not progressed so easily.31 Recent studies indicate that bleomycin can cleave some RNA RO-DNA molecules selectively. Thus a Bacillus subtilis tRNAHis precursor (16) was cleaved very predominantly at one site as indi~ated.~~" 3 Neocarzinostatin The chemistry of neocarzinostatin has been the subject of recent reviews. 32 3.1 Structure This antibiotic exists as a chromophore (17) bound strongly but non-covalently to a 109 amino acid protein of molecular weight 10.7 kD.33 The chromophore can be dissociated from the pr~tein.~~.~~ Estimates of the dissociation constant of the holocompound (chromophore +protein) vary from Kd = 7 x M34 to ca.M.35bIn the absence of the protein the chromophore is unstable. The substance has been subjected to an X-ray diffraction and the secondary solution structure of the apoprotein (i.e. protein without chromophore) and holo-neocarzinostatin have been investigated. Hirayama Adjadj and Kobayashi report3' that the protein features extensive P-sheet structures and forms a deep cleft which protects the chromophore from exposure to solvent. Although the holocompound is reported to penetrate the cell wall,38 the chromophore is thought to be released from the protein prior to interacting with DNA. 3.2 Binding to DNA The chromophore binds to duplex DNA with a dissociation constant of ca.lop6 M.39 The naphthoate moiety is thought to Scheme 8 intercaloate unwinding the helix by 21" and extending its length by 3.3 A for each bound m01ecule.~~ Evidence for intercalation comes from electric dichroism measurements. The molecule extends into the minor groove; evidence for this comes from the chemistry it performs (reacting with the C-1' C-4' and C-5' hydrogens which are all found in the minor groove of B-DNA). It has also been found that major-groove binding agents do not interfere with the binding of neocarzino~tatin,~~ but minor-groove agents such as netropsin do inhibit binding. It has been proposed that the amino group of the amino sugar is protonated and electrostatically bound to phosphate on the periphery of DNA,40 but Myers suggests that the amine may have an alternative function as a local base in deprotonating a thio14' in order to initiate the cascade of reactions that occurs with this drug.3.3 Mechanism of Action The action of neocarzinostatin is thought likely to be initiated in vivo by glutathione. Reaction can be initiated by various thiols in vitro. When the thiol attacks42 at C-12 a very unstable cumulene (18) forms as shown in Scheme 8. This undergoes a rearrangement to give a diradical which then reacts by 39-2 NATURAL PRODUCT REPORTS 1993 7-OYO (20) Scheme 9 + sugar fragments OH -I 5‘ 3’ Figure 5 X and Y represent the two sugars which undergo attack.abstracting hydrogen either from a deoxyribose or from elsewhere. The indene product has been isolated; it has been shown that the hydrogens at C-2 and C-6 derive from DNA.43 Thus if the molecule is activated in the absence of DNA but in deuterated solvent then both C-2 and C-6 are deuterated but if the same experiment is repeated in the presence of DNA then protium is incorporated. More recently it has been shown that C-2 can alternatively abstract a hydrogen from a bound thiol e.g. from the CH,S of gl~tathione.~~ The molecule produces both single-stranded and double- stranded breaks in duplex DNA. For single-stranded breaks there is a strong preference for the base thymine (T > A $ C > G).45For double-stranded scission there is sequence selectivity.The two sequences which have been most studied are shown below. (The numbers refer to the positions of the deoxyribose hydrogens which are removed at the underlined sites). c-1’ c-4’ AGC AGT TCG TCA C-5’ C-5’ It is seen that in each case hydrogen atom abstraction occurs from C-5’ at a T base. This results principally in the formation of a thymine aldehyde (19) by the mechanism shown (Scheme 9).46(See below for a detailed discussion of these reactions.) The chemistry on the other strand is more varied. Detailed investigation by Kappen and Goldberg has just shown that in the AGC sequence described above the principal damage does indeed occur at C-1’ (72 YO), but that lesser damage occurs at C-4’ (1 3 %) and C-5’ (1 5 Many of the details of the chemical preferences of the radicals have emerged from specific deuterium labelling of the deoxyribose in question.The direction of stagger of the targets on the complementary strands is evidence for minor-groove binding,32a and the separation between the two cutting sites is consistent with intercalation of the naphthoate moiety as shown Figure 5. The C-5’ hydrogen is transferred to C-6 of neocarzinostatin specifically as seen from NMR.48 This is in line with molecular models based on energy minimization and molecular dynamics simulations which further predict that it is the pro-S hydrogen which is transferred. In the other double strand cleavage sequence the C-5’ hydrogen is again transferred to C-6 of neocarzinostatin.In further studies of the specificity of cleavage Frank et al. labelled one strand of a duplex of the HindIII-BamH 1 fragment of pBR322 with 32P,and examined the chemistry of cleavage at thymidine sites. They found49 that all the thymidine sites were targets for chemistry and that all suffered attack at the hydrogen at C-5’. However at four T sites in the first 44 residues hydrogen transfer from C-4’ was seen also. All these four sites were in GT steps and so this suggested that at GT NATURAL PRODUCT REPORTS 1993-J. A. MURPHY AND J. GRIFFlTHS [xxq El I3 Arrows show the relevant NH2groups Figure 6 Arrows show the relevant NH groups. Table 1 Distance (A)to Deoxyribose Hydrogen oligomer C4’ CS‘(pr0-R) CS’@ro-S) d(GCATGC) 4.47 3.12 4.68 ~(GcGTG~) 4.49 { d(CGC ACG)} 3’27 3’ t .Mnr T I I 5’ -0-02,RSH -ow HO 9 .MN T I Scheme 10 steps the binding might be slightly altered leading to a different chemistry of cleavage.[The C-4’ chemistry was isotope dependent ; i.e. on using the DNA with the C-4’ hydrogen in the target deoxyribose replaced by deuterium a substantial difference in the relative amount of C-4’/C-5’ chemistry was observed]. Saitoso in an extension of earlier studies with base substitutions51a has recently discovered that the base which is 5’ to the target T is of prime importance in determining the C-4’/C-5’ ratio. He suggests that the chemistry observed is dependent on the depth of the minor groove at this position.So if a G base is present the 2-amino group makes the minor groove shallow and causes a slightly different mode of binding than for example an A base. In using ANH,-T base pairs he finds that C-4’ chemistry is seen and in using I-C in this position it was found that C-4’ chemistry is not seen (ANH contains an offending NH group and I does not) Figure 6. He further suggests that the MeH,N+ group of the amino sugar hydrogen bonds to the C-2 carbonyl of thymine accounting for the selectivity for cleavage at thymine. When so positioned molecular models suggest that the distances to the relevant hydrogens favour C-5’ chemistry as shown in Table 1. However the relative distances appear to predict removal of the C-5’ (pro-R) hydrogen rather than the pro-S hydrogen.From the published paper it is not easy to see if geometric factors might assist selectivity for the pro-S hydrogen. The presence of a GT step in d(GCGTGC)-d(GCACGC) brings the C-4’ hydrogen a lot closer. The chemistry which occurs at the C-4’ position of deoxyribose bears close analogy with that arising from the chemistry of bleomycin (Scheme 1O).51 However whereas bleomycin degradation of DNA can occur in the absence of molecular oxygen dioxygen is needed for the neocarzinostatin- mediated decomposition of deoxyribose by C-4’ chemistry. It has been established that 3’-glycolates are formed and the most favoured mechanism for their production must be analogous to that suggested for bleomy~in.~’~ However it must be borne in mind that very recent revisions of the bleomycin mechanism have been proposed and that similar revision of the neo-carzinostatin mechanism may therefore be required.The unknown component in the products is thought likely to be the ba~e-propenal.~ The presence of a thiol has been invoked as the source of the hydroperoxyl hydrogen and also as the agent which reduces the hydroperoxide. The alternative product derived from reaction at C-4’ is an hydroxylated abasic lesions3 as in bleomycin. Here this must arise from the thiol-mediated reduction of the hydroperoxide. For chemistry occurring at the C-5’ position (Scheme 9) the initially formed radical again interacts with dioxygen. It is known from labelling studies that the C-5’-aldehyde oxygen in (19) derives from dioxygen not water.54 This product pathway is the predominant route following C-5’ activation.However as a minor pathway DNA fragments terminating in 3‘-and 5’-phosphate are found. This 3’-phosphate is thought to arise via the 3’-formyl phosphate (20) which spontaneously hydrolyses in sit^.^^ There are different suggestions on how this derivative (20) arises. Kawabatas6 has proposed a Criegee-type rearrangement of the intermediate hydroperoxide. However this would require the adjacent phosphate oxygen to assist in the electron-push. Goldberg has suggested that there is initial conversion of the hydroperoxide to an oxyl radical (21);55 this radical would certainly fragment to give the formyl phosphate. The sugar fragment would then be converted to the hemiacetal by oxidation and trapping by water prior to fragmentation.An alternative mechanism which does not seem to have been previously proposed uses the NATURAL PRODUCT REPORTS 1993 I Table 2 The Calicheamicins O-OH GoIdberg mechanism OY I T T OYO Calicheamicin Hov=e Derivative X R 3-met hoxyrhamnose Aminopentose Fo:z3R’ I .Nvv. R‘ R” 3-methoxyrhamnose Me,CH 3-methoxyrhamnose MeCH H MeCH -3-methoxyrhamnose 3-methoxyrhamnose Me,CH 3-methoxyrhamnose MeCH 3-methoxyrhamnose Me OY T OGH OYO Alternative mechanism Owase -0,. Scheme 11 1 Scheme 12 deoxyribose ring oxygen to assist the heterolytic cleavage of the peroxide as shown Scheme 11.The reaction following hydrogen abstraction from C-1’ is known to give the deoxyribonolactone (22),57Scheme 12. This product does not result in strand cleavage but cleavage does result from subsequent treatment with alkali.57 It is not currently known whether the carbonyl oxygen of the lactone originates from molecular oxygen or from water. 4 Calicheamicins 4.1 Structure The calichearnicin~~~~ (Table 2) are produced by bacteria isolated from soil samples in Texas. The first structures were Pl Br Br Aminopentose YIB‘ Br Aminopentose 4 I Aminopentose 4 IH I Aminopentose Pl‘ YI1 (23) 1 Aminopentose 4 I Aminopentose identified by and colleagues from the American Cyanamid Company. The concentrations of calicheamicins produced were miniscule (ca.0.1 mg litre-l of fermentation broth) and so attempts were made to produce higher yields by mutating the original Micromonospora echinospora ssp cal-ichensis by exposure to ultraviolet irradiation or to mutagens. However one of the most helpful aids to production was supplementation of the nutrients with sodium iodide. This led to production of new metabolites in which the normal bromine atom of calicheamicins was replaced by iodine. The yields of the major compound calicheamicin ylI (23) were increased to ca. 2 mg litre-l. The calicheamicins vary in the nature of the sugars attached to the reactive aglycone and in the halogen atom attached to the arene. The most notable features of the molecules are the enediyne unit which forms the reactive core and the trisulfide unit.The structures (Table 2) were originally determined by spectroscopic means principally NMR and mass spectrometry and by degradation and partial syntheses. Recently the structure of the aglycone has been confirmed by total synthesis of the natural enanti~mer~~ and of the racemic aglycone,60 and the oligosaccharide moiety has been synthesized by Nicolaou.61 Very recently Nicolaou has also announced the total synthesis of calicheamicin.62 4.2 Binding to DNA As yet no X-ray structure of a DNA-calicheamicin complex has been reported but from the chemistry effected on DNA it is known that binding occurs in the minor groove of B-DNA (see below). Kahne63a suggests that the oligosaccharide has an extended conformation and like other minor-groove binding molecules is pre-organized to bind in the minor groove.His results are based on NMR studies of calicheamicin e (24) which is the rearrangement product of calicheamicin ylI (23). Very few inter-sugar residue NOE effects were observed and many features of the spectrum were invariant as the solvent was changed. The solvents used were organic solvents because of the hydrophobicity of the molecule. It has been suggested that this hydr~phobicity~~ (there are only 4hydroxyl groups present) enhances the binding to DNA. Other investigations into the roles of the oligosaccharide moiety have In one of these studies,68 the aryl tetrasaccharide of calicheamicin yI1was preincubated with a duplex which contained recognition NATURAL PRODUCT REPORTS 1993-5.A. MURPHY AND J. GRIFFITHS 56 1 ‘-OR’ ’-OR’ “SSMe Bergman cyclization I Scheme 13 sites for drug cleavage. Addition of DNAse1 then showed protection by the tetrasaccharide against DNA cleavage at particular sites which were not always identical with the expected binding sites of the antibiotic. Quantitation of the relative cleaving abilities of the aglycone calicheamicinone and calicheamicin yl’ has been performed.70 The role of the sugars and the iodobenzoate was evident from the relative efficacy of DNA cutting but it was also noticeable that the natural compound was much more effective at inducing double-strand cleavage than the aglycone suggesting that the sugars not only have a recognition role for the cleavage site but also help to orient the drug for efficient cutting.4.3 Mechanism of Action The mechanism of reaction of calicheamicins with DNA has been widely studied. It has been established that both single- and double-stranded scission of duplex DNA from bacterio- phage @X 174 occur. No cutting of single-stranded DNA has been observed at low concentrations indicating a preference for binding and cleaving double-stranded DNA. Model studies in vitro show that triphenylphosphine can attack the trisulfide unit liberating a thiolate which attacks the a$-unsaturated ketone in a Michael reaction.’l This changes the geometry at the bridgehead carbon from trigonal to tetrahedral and prepares the way for the Bergman cy~lization~~ to give the diradical Scheme 13.The slow step is the initial cleavage of the trisulfide unit. It is assumed that an analogous reaction occurs in vivo where it is assumed that a thiol attacks the trisulfide. Calicheamicin shows a remarkable degree of sequence specificity cutting preferentially at tetrapyrimidine tract~~~,~’ such as 5’-PyPyPyPy-3’. The attack occurs at the sugar attached to the base highlighted in bold. When double- stranded cutting occurs the complementary oligopurine-containing strand cut occurs 3 nucleotides offset towards the 3’-end from the pyrimidine tetrad cutting site. This 3’-offset is consistent with minor-groove binding.71 5’-N N PyPyPyPy-3’ 3‘-N N PuPuPuPu-5’ The precise chemistry occurring during the hydrogen atom abstraction has been published.Thus in an elegant series of experiment^,'^ specifically labelled deoxyriboses have been prepared for incorporation into oligodeoxynucleotides. It had previously been showni1 that removal of a C-5’ hydrogen caused the major chemistry seen with calicheamicin. Thus the new radical reacts with dissolved molecular oxygen to give ultimately the hydroperoxide. Reductive cleavage by a thiol yields the C-5’ aldehyde (19) as shown in Scheme 9. This aldehyde can be reduced to the C-5’ alcohol with sodium OH R R‘ R2 n (25) Esperamicin A H A Pr’ 3 EsperamicinAlb H A Et 3 EsperamicinA, H A Me 3 EsperamicinA2 A H Pr‘ 3 EsperamicinA2b A H Et 3 EsperamicinAPc A H Me 3 EsperamicinP H A Pr‘ 4 0 $5H3 R CH3S3 Fo::cHMe2 *vIIv H3C NH B I R’ R R’ MeS9L (26) EsperamicinC B C (27) EsperamicinD B H OH (28) Esperamicin E H H C Figure 7 borohydride to assist isolation.It has now been demon~trated’~ using the oligomer 5’-d(C C C G G T Ci C T A A G)-3’ 3’-d(G G G C21 C AG G AT T C)-5’ containing a dideuteriolabelled C-5‘ methylene group at the nucleotide-dCi that C-5’ hydrogen is abstracted by the calicheamicin and transferred exclusively to C-4 of calicheamicin with 81 YOefficiency (none was transferred to C-1 ;see Scheme 13 for numbering system used here7”. No isotope was transferred from nucleotide-dCi specifically deuterated in the C-1’ or C-4’ positions. Furthermore the hydrogen abstraction is specific to the 5‘-(pro-S) hydrogen of the sugar.Similarly by labelling the sugar of nucleotide-dC” on the complementary strand it was determined that the product resulted from transfer of deuterium from the C-4’ position giving 63% of product deuteriated at the 1-carbon of ~alicheamicin.~~ This excitingly defines the exact orientation of the drug in the minor groove of this duplex. 5 Esperamicins The espe~amicins~~” bear remarkable similarity to the calicheamicins; however there are significant differences. The different esperamicins are depicted in Figure 7. These antibiotics do not show the same sequence selectivity as the calicheamicins. Esperamicin A (25) is unique in producing solely single- stranded lesions from duplex DNA.It is not yet known what the fate of the second radical in the diyl is but it has been established that the radical that does react with DNA abstracts a C-5’ hydrogen. With the esperamicins C (26) D (27) and E (28) which are obtained by chemical hydrolysis of natural esperamicins hydrogen atom abstraction from C-4’also occurs. In the light of the specific labelling results reported above for the calicheamicins where the second radical of the diyl abstracts a C-4’ hydrogen it is suggested that the diradicals from esperamicins C-E abstract C-5’ and C-4’ hydrogen^.'^ 6 Dynemicin A Dynemicin A (29) is isolated from the fermentation broth of Micromonospora cher~ina’~ and is a hybrid antibiotic containing both an anthraquinone and an enediyne moiety.Its mechanism of action has not yet been completely elucidated but initial studies provide significant information on its likely mechanism. &OH \ OCH3 / \ / OH 0 OH OH 0 OH OH OH OH OH NATURAL PRODUCT REPORTS 1993 The drug itself has been reacted in the presence of sodium hydrosulfite and d*-di~xane.’~ This led to formation of dynemicin H (30) specifically labelled with deuterium at C-24 and C-27. Other reducing agents have also been investigated. Methyl thioglycolate and NADPH led79 principally to dynemicin H also but a second product dynemicin S (3 l) was also isolated from the methyl thioglycolate reaction Scheme 14. An oxidation is required to form the quinoid ring in (31). The timing of this step is unknown.Dynemicin H also resulted from visible-light irradiation of dynemicin A in the presence of DNA. When incubated with G4 DNA from phage R199/G4ori extremely similar cutting patterns were obtained when methyl thioglycolate or NADPH were used to activate the molecule. The need for reductive activation of this drug suggests that the anthraquinone unit is reduced in either a I-electron or 2-electron reduction. (When visible light is used it is suggested that a photoreduction occurs initially.80) This would allow opening of the epoxide as shown in Scheme 14 and hence permit the enediyne unit to aromatize more easily to a benzenediyl. Abstraction of hydrogen from deoxyribose units would then lead to DNA-strand cleavage. It is interesting to note that double-strand cleavage occurs only occasionally with this drug ;single-strand cleavage is more common.If the diyl mechanism is operating graphics simulations indicate that the diyl would feature a much greater distance to one strand than to the other.81 This may account for a low efficiency of double-strand cleavage. The sequence-specificity of the drug is not high but its preference is to react at a base to the 3’-side of a purine e.g. OH OH OH 1 NucleophileRS-H*ObH-\2. PI 0 OH OH 0 &I \ / &;; \ / OH 0 OH (30) X=H (31)X = SCH2C0fle Scheme 14 NATURAL PRODUCT REPORTS 1993-J. A. MURPHY AND J. GRIFFITHS MeYMe 5‘-AG,-3’ 5’-GC-3’ 5‘-AT-3’ and of bases in such a position G is a favourite.Whether this indicates preferential intercalative interaction with a GC base-pair or preferential non-radical alkylation of the most nucleophilic of the bases G is not known.** The double-strand cleavage must of course involve radical chemistry. It is not known which hydrogens are removed by this compound but electrophoresis of the cleavage products suggests that DNA 3’-phosphates are formed; this would be consistent with abstraction of the C-5’hydrogen. Dynemicin A is thought to intercalate and to occupy the minor groove; its cleavage chemistry is affected by preincubation of DNA with minor-groove binding agents such as distamycin. It shows preferential reaction with duplex DNA rather than single- stranded DNA. 7 Kedarcidin The anti-tumour agent kedarcidins3 was recently isolated from the culture supernatant of an actinomycete strain L585-6-(ATCC 53650) found in India.Its mechanism of action has not been determined but it deserves mention here because of its structural analogy84 with other enediyne anti-tumour agents. The chromophore (32) of kedarcidin is shown. 8 References 1 (a) S. M. Hecht Acc. Chem. Res. 1986,19,383; (b) J. Stubbe and J. W. Kozarich Chem. Rev. 1987 87 1107. 2 (a) T. Takita Y. Umezawa S. Saito H. Morishima H. Naganawa H. Umezawa T. Tsuchiya T. Miyake S. Kageyama S. Umezawa Y. Muraoka M. Suzuki M. Otsuka M. Narita S. Kobayashi and M. Ohno Tetrahedron Lett. 1982,23,521; (b) Y.Aoyagi K. Katano H. Suguna J. Primeau L.-H. Chang and S. M. Hecht J. Amer.Chem. SOC. 1982 104 5537; (c) S.-I. Saito Y. Umezawa T. Yoshioka T. Takita H. Umezawa and Y. Muraoka J. Antibiot. 1983 36 92. 3 (a) H. 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Xu N. Murugesan S. M. Hecht G. A. van der Marel and J. H. van Boom Biochemistry 1988 27 58; (b) L. E. Rabow J. Stubbe J. W.Kozarich and J. A. Gerlt J. Amer. Chem. Soc. 1986 108 7130. 28 H. Sugiyama T. Sera Y. Dannoue R. Marumoto and I. Saito J. Amer. Chem. SOC. 1991 113 2290. 29 (a) A. D. D'Andrea and W. A. Haseltine Proc. Nat. Acad. Sci. U.S.A. 1978 75 3608; (b) M. Takeshita A. Grollman E. Ohtsubo and H. Ohtsubo ibid. 1978 75 5983; (c) C. K. Mirabelli A. Ting C.-H. Huang S. Mong and S. T. Crooke Cancer Res. 1982 42 2779. 30 (a) L. F. Povirk Y.-H. Han and R. J. Steighner Biochemistry 1989 28 5808; (b) R. J. Steighner and L. F. Povirk Proc. Nat. Acad. Sci. U.S.A. 1990 87 8350. 31 (a) B. J. Carter E. de Vroom E. C. Long G. A. van der Marel J. H. van Boom and S. M. Hecht Proc. Nat. Acad. Sci. U.S.A. 1990,87,9373; (b) R. S. Magliozzo J. Peisach and M.R. Ciriolo Mol. Pharmacol. 1989,35,428; (c) C. W. Haidle and J. Bearden Jr. Biochem. Biophys Res. Commun. 1975 65 815; (d) C. 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Tanaka Biochem. Biophys. Res. Commun. 1980,94,255; (e) M. A. Napier B. Holmquist D. J. Strydom and I. H. Goldberg Biochemistry 1981 20 5602. 36 L. C. Sieker L. H. Jensen and T. S. A. Samy Biochem. Biophj)s. Res. Commun. 1976 68. 358. 37 (a) H. Takashima S. Amiya and Y. Kobayashi J. Biochem. 1991 109 807; (b) E. Adjadj J. Mispelter E. Quiniou J.-L. Dimicoli V. Favaudon and J.-M.Lhoste Eur. J. Biochem. 1990 190 263; (c) E. Adjadj E. Quiniou J. Mispelter V. Favaudon and J.-M. Lhoste Eur. J. Biochem. 1992 203 505; (d) L. S. Kappen M. A. Napier andI. H. Goldberg Proc. Nat. Acad. Sci. U.S.A. 1980 77 1970; K. Hirayama T. Ando R. Takahashi and A. Murai Bull. Chem. SOC. Jpn. 1986 59 1371. 38 (a) H. Maeda S. Aikawa and A. Yamashita Cancer Research 1975 35 554; (b) J. Takeshita H. Maeda and K. Koike J. Biochem. 1990 88 1071. 39 N. Dasgupta and I. H. Goldberg Biochemistry 1985 24 6913. 40 M. A. Napier and I. H. Goldberg Mol. Pharmacol. 1983,23,500. 41 A. G. Myers P. M. Harrington and B.-M. Kwon J. Amer. Chem. SOC.,1992 114 1086. 42 (a) A. G. Myers and P. Proteau J. Amer. Chem. Soc. 1989 111 1146; (b) A.G. Myers Tetrahedron Lett. 1987 28 7212. 43 D.-H. Chin C.-H. Zeng C. E. Costello and I. H. Goldberg Biochemistry 1988 27 8 106. 44 (a) D.-H. Chin and I. H. Goldberg J. Amer. Chem. SOC. 1992 114 1914; (b) S. E. McAfee and G. W. Ashley Nucleic Acids Research 1992 20 805. 45 L. S. Kappen I. H. Goldberg and J. M. Liesch Proc. Nat. Acad. Sci. U.S.A. 1982 79 744; L. S. Kappen and I. H. Goldberg Biochemistry 1983 22 4872. 46 R. L. Charnas and I. H. Goldberg Biochem. Biophys. Res. Commun. 1984 122,642; L. S. Kappen and I. H. Goldberg NUC. Acids Res. 1985 13 1637. 47 L. S. Kappen and I. H. Goldberg Proc. Nut. Acad. Sci. U.S.A. 1992 89 6706. 48 S. M. Meschwitz and I. H. Goldberg Proc. Nat. Acad. Sci. U.S.A. 1991 88 3047. 49 B. L. Frank L.Worth Jr. D. F. Christner J. W. Kozarich J. Stubbe L. S. Kappen and I. H. Goldberg J. Amer. Chem. SOC. 1991 113,2271. 50 H. Sugiyama T. Fujiwara H. Kawabata N. Yoda N. Hirayama and I. Saito J. Amer. Chem. SOC. 1992 114 5573. 51 (a) L. S. Kappen C.-Q. Chen and I. H. Goldberg Biochemistry 1988,27,4331; (b) L. S. Kappen I. H. Goldberg B. L. Frank L. Worth Jr. D. F. Christner J. W. Kozarich and J. Stubbe Biochemistry 1991 30 2034. NATURAL PRODUCT REPORTS 1993 52 P. C. Dedon Z.-W. Jiang and I. H. Goldberg Biochemistry 1992 31 1917. 53 I. Saito H. Kuwabata T. Fujiwara H. Sugiyama and T. Matsuura J. Amer. Chem. SOC.,1989 111 8302. 54 D.-H. Chin S. A. Carr and I. H. Goldberg J. Biol. Chem. 1984 259 9975. 55 D.-H. Chin L. S. Kappen and I.H. Goldberg Proc. Nut. Acad. Sci. U.S.A. 1987 84 7070. 56 H. Kawabata H. Takeshita T. Fujiwara H. Sugiyama T. Matsuura and I. Saito Tetrahedron Lett. 1989 30 4263. 57 L. S. Kappen and I. H. Goldberg Biochemistry 1989 28 1027. 58 M. D. Lee G. A. Ellestad and D. B. Borders Ace. Chem. Res. 1991 24,235. 59 A. L. Smith C.-K. Whang E. Pitsinos G. R. Scarlato and K. C. Nicolaou J. Amer. Chem. SOC. 1992 114 3134. 60 M. P. Cabal R. S. Coleman and S. J. Danishefsky J. Amer. Chem. SOC. 1990 112 3253; J. N. Haseltine M. P. Cabal N. B. Mantlo N. Iwasawa D. S. Yamashita R. S. Coleman S. J. Danishefsky and G. K. Schulte J. Amer. Chem. SOC. 1991 113 3850. 61 K. C. Nicolaou R. D. Groneberg T. Miyazaki N. A. Stylianides T. J. Schulze and W.Stahl J. Amer. Chem. Soc. 1990,112,8193. 62 K. C. Nicolaou C. W. Hummel E. N. Pitsinos M. Nakada A. L. Smith K. Shibayama and H. Saimoto J. Amer. Chem. Soc. 1992 114 10082. 63 (a) S. Walker K. G. Valentine and D. Kahne J. Amer. Chem. SOC.,1990 112 6428; (b) S. Walker D. Yang D. Kahne and D. Gange J. Amer. Chem. SOC. 1991 113,4716. 64 W.-D. Ding and G. A. Ellestad J. Amer. Chem. Soc. 1991 113 6617. 65 R. C. Hawley L. L. Kiessling and S. L. Schreiber Proc. Nut. Acad. Sci. U.S.A. 1989 86 1105. 66 N. Zein M. Poncin R. Nilakantan and G. A. Ellestad Science 1989 244 697. 67 N. Zein M. A. Sinha W. J. McGahren and G. A. Ellestad Science 1988 240 1198. 68 K. C. Nicolaou S.-C. Tsay T. Suzuki and G. F. Joyce J. Amer. Chem. SOC. 1992 114 7555.69 J. Aiyar S. Danishefsky and D. M. Crothers J. Amer. Chem. SOC.,1992 114 7552. 70 J. Drak N. Iwasawa S. Danishefsky and D. M. Crothers Proc. Nat. Acad. Sci. U.S.A. 1991 88 7464. 71 M. D. Lee G. A. Ellestad and D. B. Borders Acc. Chem. Res. 1991 24 235. 72 (a) T. P. Lockhart P. B. Comita and R. G. Bergman J. Amer. Chem. SOC. 1981 103 4082; (b) T. P. Lockhart and R. G. Bergman ibid. 1981 103 4091 ; (c) H. N. C. Wong and F. Sondheimer Tetrahedron Lett. 1980 21 217. 73 S. Walker R. Landovitz W.-D. Ding G. A. Ellestad and D. Kahne Proc. Nat. Acad. Sci. U.S.A. 1992 89 4608. 74 J. J. De Voss C. A. Townsend W.-D. Ding G. 0.Morton G. A. Ellestad N. Zein A. B. Tabor and S. L. Schreiber J. Amer. Chem. SOC. 1990 112 9669. 75 J. J.Hangeland J. J. De Vos J. A. Heath C. A. Townsend W.-D. Ding J. A. Ashcroft and G. A. Ellestad J. Amer. Chem. SOC.,1992 114 9200. 76 D. F. 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ISSN:0265-0568    

DOI:10.1039/NP9931000551

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

The Strobi luri ns Oudemansins and Myxothiazols Fungicidal Derivatives of B-Methoxyacrylic Acid J. M. Clough Zeneca Agrochemicals Jealott’s Hill Research Station Bracknell Berkshire RG 72 6EY 1 Introduction 2 Isolation Structural Elucidation and Synthesis 2.1 The Strobilurins 2.1.1 Introduction 2.1.2 Strobilurin A (Mucidin) 2.1.3 Other Strobilurins 2.2 The Oudemansins 2.2.1 Isolation and Characterization 2.2.2 Synthesis 2.3 The Myxothiazols 3 Biosynthesis 4 Biological Activity 5 Biochemical Mode of Action 6 Synthetic Analogues as Agricultural Fungicides 7 References 1 Introduction The strobilurins oudemansins and myxothiazols are naturally- occurring derivatives of P-methoxyacrylic acid which are produced by various fungi and bacteria.The interest in these compounds stems not only from their novel structures but also from the fact that many have pronounced biological activity. For example many are able to control the growth of fungi or have insecticidal antiviral or antitumour activity. Furthermore it has been established that they have a novel mode of action -they inhibit mitochondrial respiration by binding at a specific site on cytochrome b-and this has made them valuable biochemical tools. The first member of this family of natural products to be discovered originally named mucidin but now generally known as strobilurin A (l) was isolated in Czechoslovakia in the 1960s by Musilek and his co-workers.1~2 Since that time ten additional strobilurins three oudemansins and 24 myxothiazols (only 18 in fact being derivatives of P-methoxyacrylic acid) have been characterized.I Y MeO2C (1) X = Y = H Strobilurin A (Mucidin) (1 1) Strobilurin C (10) X = MeO Y = CI Strobilurin B (5) X = HO Y = H Strobilurin F-1 (14) X = MeO Y = H Strobilurin H (4) X = H Y = MeO Strobilurin X 4 The aim of this Report is to describe the isolation structural elucidation and synthesis of these natural products and to summarize what is known of their biosynthesis biological activity and biochemical mode of action. Our interest in these compounds at Zeneca Agrochemicals is a result of their fungicidal activity and a programme of synthesis in which analogues have been prepared as potential fungicides for use in agriculture is described in the final section of this Report.This work culminated in the synthesis of a broad-spectrum systemic fungicide which is now in development. It is worth adding at the end of this introduction that other naturally-occurring derivatives of P-methoxyacrylic acid are known. Several of the corynanthe alkaloids for example incorporate a methyl P-methoxyacrylate group.3 However to my knowledge none of these inhibit mitochondrial respiration or show fungicidal properties and consequently they fall outside the scope of this-Report. 2 Isolation Structural Elucidation and Synthesis 2.1 The Strobilurins 2.1 .I Introduction Eleven strobilurins have been isolated and characterized.Each comprises an (a-methyl P-methoxyacrylate group linked at the a-position to a (substituted) phenylpentadienyl unit. Despite early confusion it has now been firmly established that the resulting triene system has the (E 2,E)-configuration in all of the strobilurins (strictly according to the Cahn-Ingold-Prelog rules hydroxystrobilurin D (2) has the (all-E)-configuration). Most of the literature from the early 1980s erroneously shows strobilurins with the (all-E)-configuration and this error still persists in a few recent publications. The members of the strobilurin family therefore differ from each other only in terms of the substituents at the 3-and 4-positions of the benzene ring with the single exception of hydroxystrobilurin D (2) which (12) X = H StrobilurinD (2)X = OH HydroxystrobilurinD (3) Strobilurin E (6) Strobilurin F-2 (13) StrobilurinG (* unknown configuration) 565 NATURAL PRODUCT REPORTS.1993 uniquely carries a hydroxy substituent on the methyl group of the phenylpentadienyl unit. They vary in complexity from strobilurin A (1) to the relatively elaborate strobilurin E (3) which incorporates complex spiroketal functionality. A few points concerning the nomenclature of the strobilurins are worth making. The first strobilurin to be discovered was originally named mucidin but is now usually referred to as strobilurin A (1) in the chemical literature (and will be so named throughout this Report) although biochemists have generally retained the term mucidin.The strobilurin termed strobilurin X (4),throughout this Report was not named when it was first described but has subsequently been termed strobilurin X (4) throughout this Report was not named when Finally two different strobilurins have been termed strobilurin F as a result of overlapping publication^.^^^^^ In this Report the terms F-1 (5) and F-2 (6) will be used priority being given to the strobilurin F which was isolated by Steglich and his co- workers because of the earlier date on which their work was submitted for publication. 2.1.2 Strobilurin A (Mucidin) Strobilurin A (1) was first isolated in the 1960s in Czechoslovakia by Musilek and his co-workers and was at that time named rnucidin.'t2q7 A fruiting body of the basidiomycete fungus Oudemansiella mucidu found growing on a beech tree was allowed to develop mycelia on an agar slant; the resulting culture when transferred to a liquid nutrient medium produced up to 600 mg I-' of strobilurin A (and improved conditions described later enabled even larger amounts -up to 1800 mg 1-' -to be obtaineds).The characteristic infrared and ultraviolet absorption spectra of the antibiotic were reported as well as its molecular weight and empirical formula but these were not sufficient to enable its structure to be determined at that time.' Long before the structure of strobilurin A (1) had been established the Czechoslovakian group had thoroughly evaluated its antimicrobial activity it controls the growth of a wide range of fungi but is inactive against ba~teria.'-~-~ By the late 1970s it had been shown during clinical trials to be effective against various fungal skin infections when applied as a topical treatment and was in use in human and veterinary medicine under the trade-name 'Mucidermin Spofa'.l0.l1 Furthermore its potential for use as an agricultural fungicide was recognized on the basis of its activity in vitro against a variety of plant pathogenic fungig For reasons described below it is worth noting that the strobilurin A (1) isolated by the Czechoslovakians was in its purest form a white crystalline solid with a melting point of about 50 0C.739.12313 Indeed crystallization was the final step in a preferred method of purification and tens of grams of pure crystalline material were obtained in this way.7 Furthermore when a deuterium-labelled sample of strobilurin A was prepared (by alkaline hydrolysis of the natural product and successive treatment of the resulting acid with deuterium oxide and diazomethane) it too was a crystalline s01id.l~ The Czechoslovakians also reported that strobilurin A is dextro- rotatory although this was later retracted.l' The Czechoslovakian group first published the structure of strobilurin A (1) in patents filed in 1974 and published in 197815 and 1979." The second of these patents shows the correct (E,Z,E)-geometry of strobilurin A and describes a non-stereocontrolled synthesis from which material identical to the natural product could be isolated.Quite independently of the work described above strobilurin A was isolated by Anke and Steglich and their co-workers in Germany in 1977,16 with a structure being assigned the following year.17 Its source in this instance was another basidiomycete Strobilurus tenacellus a fungus which grows on decaying pine cones. The fungus was maintained on agar slants and then allowed to ferment in a nutrient liquid medium to produce after purification about 3 mg 1-' of strobilurin A as an oil with no optical activity.16 Subsequently strobilurin A has been NCHO Ph \ I I /id 'i' 32% ii-iv Reagents i KOH; ii SOCl, MeOH; iii Ph,P'C-HOMe; iv hv; v H,O, Na,CO Scheme 1 (9) 26% ii v I Reagents i LiAlH,; ii MnO,; iii LiC(SMe),; iv HgCl, HgO aq.MeOH ; v Ph,P+C-HOMe Scheme 2 isolated from several other basidiomycete fungi,1s-20 as well as from the ascomycete Bolinea l~tea.~ The Czechoslovakian group had not at this time published a structure for mucidin and although Anke and Steglich recognized the similarity between strobilurin A and mucidin,17 the crystallinity and optical activity claimed for the latter appeared to preclude any possibility that the two compounds were identical.Only in 1981 did it become clear that the two compounds are in fact one and the same; claims that mucidin was optically active had apparently been made in error and were However on the basis of the evidence above it does in fact seem likely that strobilurin A when sufficiently pure is a solid at ambient temperatures.With the publication of their structure for strobilurin A in 1978 the German group introduced what was to become another source of confusion although spectroscopic and chemical methods had enabled the gross structure of the compound to be elucidated it had incorrectly been assigned the (all-E)-c~nfiguration.'~A total synthesis of strobilurin A did not at first correct this error because it involved an ambiguous photoisomerization step and intermediates whose own ge-ometry had been wrongly assigned.22 However in 1984 the authors published the same synthesis again this time with the configuration of the intermediates assigned correctly (Scheme 1);importantly the (E,E)-configuration of the key intermediate (7) had by then been established by converting it into the (E,E)-dienoate (8) a known A further stereo- controlled synthesis of strobilurin A (1) later confirmed the (E,Z,E)-geometry of the natural product.The (Z,E)-dienoate (9) which provided from the outset two of the three olefinic bonds of strobilurin A was chosen as starting material (Scheme 2).24 The unnatural (all-E)-isomer of strobilurin A has also been synthesized by several method^,^^-*^ and its single crystal X-ray structure has been recorded.25 NATURAL PRODUCT REPORTS 1993-5. M. CLOUGH i-iii ___) 41% iv v 56% + other stereoisomers 45 48 7 Reagents i ButOK; ii p-TSA aq. acetone; iii Ph,P+C-HC0,Me; iv HCO,Me NaH; v Me,SO, K,CO Scheme 3 2.1.3 Other Strobilurins Since the discovery of strobilurin A ten additional strobilurins have been isolated and characterized mainly by Anke and Steglich and their co-workers.Like strobilurin A (I) these have been isolated from a variety of fungi almost exclusively basidiomycete fungi but also from the ascomycete Bolinea lutea. Strobilurins are not present to any significant extent in the fruiting bodies of these fungi; instead they are formed when mycelia are allowed to grow as submerged or surface cultures. All of the strobilurins exhibit fungicidal activity and in addition several have other useful biological properties such as antitumour or antiviral activity (see Section 4). The strobilurins B (lo) C (11) and X (4)were discovered before the configuration of strobilurin A had been properly defined and so were originally described as (all-E)-isomers.Since there is no doubt that the strobilurins all have the same geometry it is clear that the configuration of these strobilurins must be revised together with that of strobilurin A.23*24 Strobilurin B (lo) a solid was first reported in 1977 as a congener of strobilurin A (1) in the fermentation broth of Strobilurus tenacellus,16 and it was later isolated from various other fungi as well.4~1s.20 It was shown to be a simple derivative of strobilurin A with chloro and methoxy substituents on the benzene ring ;oxidative degradation gave 4-chloro-3-methoxy- benzoic acid which established the positions of these sub~tituents.'~ The synthesis of strobilurin B (10) was reported by Sutter in 1989.27 Two successive Wittig reactions followed by a Claisen condensation were used to construct the carbon framework of strobilurin B (Scheme 3).The fact that mixtures of isomers were produced usually perceived to be a problem in synthesis was turned to advantage in this instance because it allowed various stereoisomers of strobilurin B to be isolated and their biological activities to be compared. The procedure shown in Scheme 3 was also used to prepare strobilurins A (l) C (1 1) and X (4). Strobilurin C (1 1),O and strobilurin X (4),13 further simple derivatives of strobilurin A were first described in 1983. The source of strobilurin C an oil was Xerula longipes while strobilurin X a crystalline solid was isolated from Oudemansiella mucida.The isolation of hydroxystrobilurin D (2) was also announced in 1983,28329 although five years were to pass before details were published.30 Isolated from the fungus Mycena sanguinolenta hydroxystrobilurin D is a dextrorotatory oil the first optically active strobilurin to be discovered. The compound is structur- ally unique in that the vinyl methyl group present in all the other strobilurins is oxidized to a hydroxymethyl group in hydroxystrobilurin D. Its optical activity arises from a chiral epoxide unit in one of the ring substituents which was shown OMe 1 (1 5) X = Y = H. Oudemansin A (16) X = MeO Y = CI Oudemansin B (17) X = H Y = MeO,Oudemansin X group strobilurin D (12) was first described in 1986.s,25 It is a dextrorotatory oil and was isolated from Cyphellopsis anomala a basidiomycete fungus which grows on the decaying wood of deciduous trees.Strobilurin E (3) structurally the most complex of all the strobilurins was first described in a patent application with a 1988 priority date;31 further details were published in 1990.32 It is a colourless oil obtained from the fungus Crepidotus fulvotomentosus. Four other strobilurins were described for the first time in 1990 the strobilurins F-1 (5),5 F-2 (6) G (13) and H (14).4.6 Strobilurin F-1 a waxy solid isolated with strobilurin D from Cyphellopsis anomala was shown to be a simple derivative of strobilurin A.5 The strobilurins F-2 G and H were each isolated from Bolinea lutea the only known example of an ascomycete fungus to produce strobilurins ; strobilurins A (1) and B (10) were produced at the same time.Strobilurin F-2 (6) is a colourless crystalline solid its structure being established both by spectroscopic methods and degradation studies.6 Strobilurin G (13) is a dextrorotatory oil. Although its gross structure was determined by spectroscopic methods and by degradation studies its absolute configuration remains un-known.6 Finally strobilurin H (14) also an oil was shown to be another methoxy-derivative of strobilurin A (l) a regioisomer of strobilurin X (4).6 No syntheses of the more recently isolated strobilurins have been published. However 2,2-dimethyl-3-hydroxy-3,4-di-hydro-2H- 1,5-benzodioxepin the fused ring system found in strobilurin G (13) has recently been synthesized for the first time.33 2.2 The Oudemansins 2.2.1 Isolation and Characterization Just three oudemansins are known oudemansins A (15) B (1 6) and X (1 7).Formally they are derivatives of the corresponding strobilurins in which the elements of methanol have undergone syn-addition across the central olefinic bond of the triene system. The oudemansins therefore have two contiguous chiral through degradation studies to have the (S)-config~ration.~~ centres; they are all laevorotatory with the (9S,lOS)-The corresponding strobilurin with the usual vinyl methyl configuration. Anke and Steglich and their co-workers were the first to isolate each of the oudemansins.They discovered that mycelial cultures obtained from tissue plugs or spore prints of the fruiting bodies of various basidiomycete fungi produce one or more oudemansins when allowed to ferment in suitable nutrient media. Strobilurins are often formed at the same time. The biological activity of the oudemansins closely resembles that of the strobilurins they control the growth of fungi (though not bacteria) and show antitumour activity (see Section 4). The fact that oudemansins show activity in various in vitro assays strongly suggests that they are active in their own right and that they do not simply function through elimination of methanol as biological precursors of strobilurins. The observation that low energy conformations of strobilurin A (1) and oudemansin A (15) are very similar in shape size and charge distribution is also consistent with this conclusion it is not necessary to postulate that an oudemansin must be converted into the corresponding strobilurin for the two compounds to bind at the same site.34 Oudemansin A (15) (initially known simply as oudemansin) a colourless solid was first described in 1979.35 Mycelial fermentations of Oudemansiella mucida which were already known to produce strobilurin A (l),1,2s* were now shown also to give oudemansin A (about 2.5 mg 1-l) under appropriate conditions.Later oudemansin A was also isolated from the basidiomycete fungi Mycena polygramma18 and Xerula melanotricha.20 Its structure including its relative configuraJion was established by both spectroscopic methods and single crystal X-ray analysis.35 Oudemansin B (16) was first described in 1983.20928 It is produced (about 2 mg 1-l) by submerged cultures of Xerula melanotricha (oudemansin A (15) and strobilurins A (1) and B (10) being formed at the same time). Its spectroscopic features demonstrated its close structural relationship with oudemansin A while ozonolysis gave 4-chloro-3-methoxybenzaldehyde thereby establishing the orientation of the ring substituents. From a comparison of the CD spectra of oudemansins A and B it was clear that both compounds have the same absolute configuration although this could not be assigned at the time.20 Oudemansin X (17) was first described as recently as 1990.36 Its source is yet another basidiomycete fungus Oudemansiella radicata from which it was isolated as a colourless oil (2 mg 1-1 of mycelial culture).As described below by this time it had been established through homochiral synthesis that both oudemansin A (15) and oudemansin B (16) have the (9S,lOS)- configuration. The close similarity of the CD spectrum of oudemansin X with those of oudemansin A and B therefore immediately allowed the (9S 10s)-configuration to be assigned to this new member of the family as well.36 2.2.2 Synthesis The oudemansins have attracted considerable interest as targets for synthesis and several approaches to racemic or homochiral oudemansins have been described. The most interesting challenge of course is how to construct suitable intermediates with the required syn-stereochemistry and in the case of the homochiral syntheses with the appropriate absolute configuration.Three different approaches to racemic oudemansins have been described. In the first of these the key step was the diastereoselective reduction of the a-methyl-P-ketoester (1 8) using zinc borohydride to give mainly the corresponding syn-a- methyl-P-hydroxyester (1 9). Methylation and Arndt-Eistert homologation of the ester (19) followed by formylation and further methylation then gave racemic oudemansin A (15) (Scheme 4).37 Later an alternative and more efficient method for the homologation of the ester (19) was des~ribed.~~ In the second approach a highly diastereoselective [2,3]-Wittig rearrangement of the silylated propargyl crotyl ether (20) gave the required intermediate propargyl alcohol (2 1) contaminated NATURAL PRODUCT REPORTS 1993 CO2Me (19) 91 9 38% ii-vi\ OMe OMe T .[+ 7% (7€,11 Z)-isomer] Reagents i Zn(BH,),; ii Me30+BF4-; iii LiOH; iv SOCl, py; v CH,N,; vi PhCO,Ag Et,N MeOH; vii HCO,Me LDA Scheme 4 (Z):(E)= 937 (20) 51% iiiiv I OMe OMe (22) Reagents i Bu"Li -85 "C; ii CsF; iii PhI Et,NH CuI (Ph,P),PdCI,; iv LiAIH,; v MeI NaH; vi 9-BBN then H,O, NaOH; vii 0, Pt-C NaHCO,; viii CH,N Scheme 5 with only 2% of the unwanted diastereomer. Several further steps produced the ester (22) which had previously been converted into racemic oudemansin A (15) (Scheme 5;compare Scheme 4).39 Finally in the third procedure the crotyl ester (23) was converted via a diastereoselective Ireland-Claisen re-arrangement into a 7 1 mixture of the ester (24) and its diastereomer.The ester (24) was then converted into racemic oudemansin A (1 5) by the steps depicted in Scheme 6 while the use of (4-chloro- 3 -met hoxy benzy1)dip heny lphosp hine oxide enabled racemic oudemansin B (16) to be prepared by a parallel route.40 Several syntheses of homochiral oudemansins have been published with the work of Akita and Oishi and their co- workers at the Riken Institute Japan being especially prominent. Two alternative general approaches to inter-mediates with the required absolute stereochemistry have been employed. In the first the intermediates were homochiral secondary alcohols produced by microbial reduction of the corresponding ketones while in the second the intermediates were derived from homochiral natural products.The earliest synthesis of (-)-oudemansin A (1 5) was described in 1983 by Akita and his co-workers and importantly established for the first time that the natural product has the (9S,10s)-configuration (Scheme 7).41-43 This synthesis is a modification of the method used earlier by the same group to prepare racemic oudemansin A (Scheme 4) with a microbial reduction replacing the diastereoselective zinc borohydride reduction and an alternative homologation sequence. Thus NATURAL PRODUCT REPORTS 1993-5. M. CLOUGH 0 OMe OMe (24) 7 1 OMe OMe phyC02H 60% ii vii viii (57%) ix viii x (37%) ?Me Phv &OMe Me02C Reagents i LDA Me,SiCl then aq.NH,C1; ii CH,N,; iii Ph CH,P(O)Ph, Bu"Li; iv LiBH,; v 9-BBN then H,O? NaOH; vi Jones' oxidation; vii LDA N-formylimidazole; viii Me,SO, K,CO,; ix HCO,Me 2ButLi; x H' MeOH Scheme 6 s>H Phdf Phy CO2Et COpEt (26) (25) 68% ii-iv 1 OM0 OM0 \C02Me (34) 26% x ix I OMe Ph &OMe Me02C Reagents i Candida albicans; ii LiAlH ;iii ButMe,SiC1 imidazole; iv MeI KH; v AcOH; vi TsCl py; vii NaCN; viii KOH; ix CH,N,; x HCO,Me LDA Scheme 7 the key intermediate (25) (the stereochemistry of which was established by converting it into a compound of known absolute configuration) was prepared by stereoselective re- duction of the ketone (26) using Candida albicans.A small quantity of diastereomeric material was formed at the same time but was readily separated by chromatography. A lengthy sequence of conventional steps then enabled (25) to be converted into (-)-oudemansin A (1 5) whose spectroscopic properties including optical rotation corresponded to those of the natural material (Scheme 7). An alternative synthesis of (-)-oudemansin A again by Akita and Oishi and their co-workers is depicted in Scheme 8.42-44 Once again the synthesis takes advantage of a highly stereoselective microbial reduction this time of the fury1 ketone 569 0 OH +\I C02Me 1 OMe (?Me ~ viii-x Me02C Ph7 "i 23% OSiMe2Bu' OSiMe2Bu' (29) Reagents i Saccharomyces fermentati; ii LiAlH ; iii ButMe,SiC1 imidazole; iv MeI NaH; v 0,;vi H,O,; vii CH,N,; viii DIBAL; ix Ph,P+C-HPh; x separation Scheme 8 I HO MeO -45% 84% C02Me CI (30) (+)-cawone (31) (32) >70% v J Ye OMe Ph7 mcOzMe vi-viii C02Me (34) (33) Reagents i H,O, NaOH; ii NaOEt then H,O; iii 2Me1 2NaH; iv HCl(g); v SmI,; vi O, then Me$; vii PhMgBr; viii Me,SO, DMAP Scheme 9 (27) which gave the syn-alcohol (28) in greater than 99% enantiomeric excess (together with an equal quantity of diastereomeric anti-material) on treatment with Saccharomyces fermentati.The syn-alcohol (28) was then converted into the silylated intermediate (29) which was identical to that produced by the earlier sequence shown in Scheme 7. Later the group from the Riken Institute developed two further synthetic sequences based on a microbial reduction this time providing the first total synthesis of (-)-oudemansin B (16) and confirming its (9S lOS)-config~ration.~~,~~~~~ A formal synthesis of (-)-oudemansin A (15) from (+)-carvone (30) has recently been described (Scheme 9).47 Following epoxidation (+)-carvone (30) underwent Favorskii rearrangement to give the highly substituted cyclopentane (31).Successive bis-0-methylation and hydrochlorination then gave the chloro-ester (32) which in the key step of the whole sequence underwent reductive fragmentation on treatment with samarium(I1) iodide to give the acyclic ester (33). Three conventional steps then furnished the ester (34) identical in all respects to that described in a previous synthesis of (-)-oudemansin A (15) (Scheme 7).(-)-Oudemansin X (17) has recently been prepared for the first time from (-)-quebrachitol a cyclitol obtained from the serum of the rubber tree.48 The synthesis confirmed the (9S 10s)-configuration proposed earlier for (-)-oudemansin X on the basis of the similarity of its CD spectrum with those of oudemansins A (15) and B (16).36 (35) Myxothiazol A OMe (36) Myxothiazols B to I and (37) K to 0 (R’ = H) (38) Myxothiazols Q X and Y (R’= Me) (R2defined in text) OMe I 0 0 2 m CMe0 N H 2 NH2 (39) Myxothiazol P (40) Myxothiazols R to W (R3 defined in text) Finally Meyer has prepared (+)-oudemansin A the enantiomer of the natural product as well as (+)-epioudemansin A one of the two possible epimers from homochiral precursors of known absolute configuration.It was shown that (+)-oudemansin A has the (9R 10R)-configuration thereby confirming the previous assignment of the (9S 10s)-configuration to the natural (-)-oudemansin A.49 2.3 The Myxothiazols Just one myxothiazol has been reported in the chemical literature. However I am grateful to Prof. Dr G. Hofle of the Gesellschaft fur Biotechnologische Forschung (GBF) Braunschweig Germany for a copy of an extract from the Scientific Annual Report of the GBF for 1986 which describes how he and his colleagues have isolated a total of 33 NATURAL PRODUCT REPORTS 1993 0 CH3CO2Na C 0 CH3C&Na cH3s-YC02H 0 NHp PhC02H Figure 1 myxothiazols and have established structures for 24 of them.so The compound known throughout the literature simply as myxothiazol should therefore more strictly be called myxothiazol A (35).Instead of the ‘a-linked ’ (E)-methyl P-methoxyacrylate group found in each of the strobilurins and oudemansins myxothiazol A contains an (E)-P-methoxyacrylamide group which is linked to the rest of the molecule at its ,&position. The backbone to which the acrylamide group is attached bears some resemblance to that of the oudemansins. However in place of the (substituted) benzene ring of the latter myxothiazol A possesses an interesting bisthiazole moiety substituted with a branched (E,E)-nonadienyl chain.Myxothiazol A with three chiral centres has the (7S 18S 19R)-configuration shown. Myxothiazol A (35) was first described in a patent application with a priority date in 1978,jl and full details of its isolation physical properties and gross structure were published in 1980.s2*s3 It was shown to have high levels of in vitro activity against a variety of filamentous fungi and yeasts but also proved to be toxic in all the animal systems tested (see Section 4). A particular strain of a gliding bacterium Myxococcus fulvus Mx f16 which had been found in a soil sample from Java was the original source; when allowed to ferment under optimum conditions it produced up to 5.6 mg of myxothiazol A per litre of culture medi~m.~~-~~ Subsequently another strain of the same bacterium M.fulvus Mx f85 was found to produce much larger quantities (up to 40 mg 1-’) of myxothiazol A together with the wealth of other myxothiazols referred to above.50 More recently myxothiazol A has also been isolated from other gliding bacteria such as Stigmatella aurantiaca5’ and Angiococcus dis~forrnis.~~ Myxothiazol A is a dextrorotatory solid originally isolated in the form of an amorphous powder,j2 but later crystallized and found to have a melting point of 79 0C.50Its gross structure including the geometry of the olefinic bonds was determined on the basis of spectroscopic studies and the characterization of the products of hydrolysis hydrogenation ozonolysis and Diels-Alder reaction with maleic anhydride.53 Its absolute stereochemistry was established through degra- dation studies the configurations of the two chiral fragments obtained by ozonolysis being determined by correlation with (2R,3R)-P-methylmalic acid and by X-ray ~rystallography.~’ Studies directed towards the total synthesis of myxothiazol A have been The 23 other myxothiazols to have been characterized are grouped here by structural type.Myxothiazols B to I (36) and K to 0 (37) inclusive (there is no myxothiazol J) are modifications of myxothiazol A in which the (E,E)-nonadienyl chain is truncated or oxidized. The substituent R2 represents either a I-hydroxyethyl or an acetyl group (myxothiazols N and 0respectively) or a branched 9-carbon chain incorporating olefinic hydroxyl ketone and/or epoxide functionality.The myxothiazols Q X and Y (38) are related compounds which NATURAL PRODUCT REPORTS 1993-5. M. CLOUGH A OCH3 I A CH3SqC02H CHSCO2H eo CH,CO,H Figure 2 contain an additional methyl substituent on the acrylamide unit presumably the result of the incorporation of propionate in place of acetate during biosynthesis (see Section 3); myxothiazol Q is the exact analogue of myxothiazol A. Myxothiazol P (39) is unique in that it contains just one thiazole ring. Finally myxothiazols R to W (40) inclusive contain no P-methoxyacrylamide unit the group R3 representing an oxidized 3- 5-or 6-carbon chain. The stereochemistry of the various chiral centres of the myxothiazols B to I and K to Y have not been defined,50 though it is likely that the centres which they have in common with myxothiazol A have the same configuration as in that compound.3 Biosynthesis It has been shown by feeding suitable isotopically-labelled precursors to Oudernansiella mucida that strobilurin A (1) is of mixed biosynthetic origin (Figure l).59 The aliphatic portion is mainly acetate-derived and the fact that two contiguous carbon atoms originate from C-1 of acetate suggests that some kind of rearrangement occurs during bio~ynthesis.~. 6o By contrast the benzene ring and benzylic carbon atom appear to be synthesized via the shikimate pathway since 13C- or 14C-labelled phenyl- alanine and benzoic and cinnamic acids are incorporated at these positions. Interestingly all three methyl groups are derived from methionine.Similar detailed labelling experiments with the other strobilurins have not been reported. Nevertheless there is evidence that at least some of the strobilurins are formed from strobilurin A analysis of the strobilurins present at various stages of the fermentation of Bolinea lutea suggests that strobilurin A (1) is not stable in the liquid culture but is converted into strobilurins B (lo) F-2 (6) G (13) and H (14).4 Trowitzsch-Kienast and his co-workers have used feeding experiments to study the biosynthesis of myxothiazol A (35) in Myxococcusfulvus (Figure 2).61 The non-aromatic portions are derived from one leucine two propionate and three acetate units. It is probable that leucine is converted into isovaleroyl- CoA which then serves as the starter unit for polyketide synthesis.["SI-Cysteine was also incorp~rated~l.~~ and it is likely that the bisthiazole unit is built up from this amino acid. The carbon atoms of both methoxy groups originate from methionine. It appears that the myxothiazols Q X and Y use propionate instead of acetate as the terminal building block in the polyketide synthesis and the other myxothiazols are oxidative modifications or degradation products of myxothiazol A,or are the products of incomplete biosynthesis.62 57 1 4 Biological Activity The strobilurins and oudemansins have a variety of potentially useful biological activities and indeed it was these properties which first enabled them to be detected in and then isolated from crude fermentation broths.They all have activity against a wide range of filamentous fungi and yeasts; although precise comparisons are not easy to make on the basis of published data the results of in vitro agar diffusion assays indicate that all of the strobilurins and oudemansins have broadly the same high level of a~ti~ity,~.~.~"~~.~~~~~-~~~~~~~~ except for strobilurin F-1 (5) which is distinctly ~eaker.~ They have no significant activity against bacteria. The (E,Z,E)-configuration of the strobilurins is critical for high fungicidal activity synthetic analogues with other geometries are at best only very weakly active." 27,60 It is intriguing from an ecological point of view that fungi are able to biosynthesize fungicidal compounds.It seems likely that fungi which produce strobilurins and oudemansins have an advantage over other fungi as they compete for nutrients in their natural environment. Indeed Musilek and his co-workers have reported that generally no other types of parasitic fungi appear on beech trees infected with Oudemansiella rnucida.2 Trumpower has discussed possible reasons why Oudemansiella and Strobilurus species are not themselves inhibited by the methoxyacrylates they produce. It appears that Strobilurus species at least contain a cytochrome bc complex (see Section 5) but it is not known whether this complex is inherently resistant to the methoxyacrylates or if the organism relies on an alternative toxin-insensitive oxidase pathway for growth."j Like the strobilurins and oudemansins myxothiazol A (35) is highly active in vitro against a wide range of filamentous fungi and yeasts but in addition it controls the growth of certain Gram-positive bacteria.,l< 52.64 None of the myxothiazols shows higher activity than myxothiazol A.62 Several of the strobilurins and oudemansins inhibit the growth of human tumour cells and strobilurins E (3),31.3' D (l2), and G (13)4 appear to be particularly active.Furthermore it has been reported that strobilurins D (l2) and E (3j31.32.60 have antiviral activity while myxothiazol A (35) has insecticidal properties." 65 563 Tests for acute toxicity to mice have shown that the strobilurins A (l) B (lo) C (11) and X (4) as well as oudemansins A (15) and B (16) are not particularly toxic compounds.Two different values have been reported for the acute oral LD, of strobilurin A 500mg kg-' and 825 mg kg-1,7.13 while strobilurin X is slightly more toxic with an acute oral LD, of about 420mg kg-'.13 Furthermore intraperitoneal application of strobilurins B and C and oudemansins A and B gave little or no acute toxicity at rates as high as 300 mg kg-1.34.66 Nothing has been reported for the remaining strobilurins or for oudemansin X (17). By contrast myxothiazol A (35) was found to be highly toxic in all the animal systems tested it has an acute LD, of just 2 mg kg-' in mice and is highly toxic to chicken embryo fibroblasts.,' 5 Biochemical Mode of Action The fungicidal activity of the strobilurins oudemansins and myxothiazols stems from their ability to inhibit mitochondrial respiration in fungi.The strobilurins for example typically show complete inhibition of respiration in Penicillium notutum at concentrations in the micromolar range. More specifically it is known that these compounds inhibit respiration by interfering with the function of the cytochrome bc complex an enzyme which is located in the inner mitochondrial membrane of fungi and other eukaryotes. The function of this enzyme during respiration is to catalyse the transfer of electrons from the lipid-soluble ubiquinol to the water-soluble electron-acceptor cytochrome c; as this takes place protons are translocated across the membrane in which the cytochrome bc complex is NPR I0 Me0 Me0 OH H Recent studies have turned to the question of which amino 572 NATURAL PRODUCT REPORTS 1993 Citric AFid Cycle during 75 The strobilurins oudemansins and myxothiazols are the only members of the first class while the :.213-+ 2H' naturally-occurring chromones stigmatellin A and stigmatellin B76 and the synthetic compound 3-n-undecyl-2-hydroxy- naphth~quinone'~are representatives of the other two classes. All these inhibitors bind competitively at the Q,-centre. Thus the Q,-centre is perceived to be a crevice which contains at least three distinct binding domains ; although independent steric hindrance does not allow more than one of these domains to be occupied at the same time. MeohHMeoh" Ubiquinone Ubiquinol (n= 6-10) ADP +Pi]? Inhibition :OudemansinsStrobilurins Myxot hiazols ATP Cytochrorne c1 iCytochrome c iCytochrorne a tCytochrome a3 I Figure 3 Mitochondria1 Respiration the Electron Transport Chain embedded and this establishes the proton gradient which drives the synthesis of ATP (Figure 3).63 As early as 1974 even before its structure was published strobilurin A (1) was reported to inhibit electron transport between cytochromes b and c and its fungicidal activity was linked to this effe~t.~' Soon after its isolation myxothiazol A (35) was shown to have a similar mode of a~tion.~~.~~ Furthermore in important publications by von Jagow and his co-workers strobilurins A (1) and B (lo) oudemansin A (15) and myxothiazol A (35) (the four members of this family which had by then been isolated) were compared and shown to have the same biochemical mode of action in addition to their clear structural similarities.21~69 Further studies have confirmed these results and have provided additional details about the mode of action of these fungicides.It has been established that the strobilurins oudemansins and myxothiazol A (35) bind at a specific site on cytochrome b now known as the Q,-~entre.'~*'~ Since these inhibitors can displace one another from the Q,-centre it is clear that they are reversibly bo~nd.~~,'~ In the presence of one of these inhibitors ubiquinol is still able to bind in its usual fashion at cytochrome b but it is not oxidized and it has been speculated that this is the result of a conformational distortion of cytochrome b which slightly displaces ubiquinol at its binding Interestingly the strobilurins oudemansins and myxo-thiazols are not the only compounds which have been found to bind at the Q,-centre and three distinct classes of inhibitors have been defined on the basis of spectroscopic changes which occur in various components of the cytochrome bc complex acid residues of cytochrome b make up the Q,-centre.This problem is being addressed through the use of carefully designed photoaffinity-labelled /3-methoxyacylates,i1 and by comparison of the amino acid sequences of cytochromes b from mutants of various micro-organisms which are resistant to the P-methoxyacrylates with that of cytochrome b from sensitive strains." 6 Synthetic Analogues as Agricultural Fungicides We at Zeneca Agrochemicals first became interested in the strobilurins oudemansins and myxothiazols in the early 1980s having been attracted by their fungicidal activity and potential as starting points for the synthesis of novel fungicides for use in agriculture.It was clear that the natural products themselves could not be used as agricultural fungicides because of insufficient levels of activity unsuitable physical properties and/or problems associated with their preparation on a large scale and at a suitable price either through fermentation or synthesis. Nevertheless it was believed that a knowledge of their structures and properties could provide a useful lead to synthetic fungicides having a new spectrum of biological physical and environmental proper tie^.^^.'* 79 This family of natural products was attractive for a variety of reasons. Firstly several of the strobilurins and oudemansins have rather simple structures. Furthermore the fact that several related fungicidal natural products were known indicated that within this area of chemistry there was scope for structural modification without loss of activity. In addition the novel biochemical mode of action of these fungicides implied that there would be no cross-resistance between compounds of this class and agricultural fungicides already in use. A knowledge of their mode of action was also important because it meant that an in vitro assay useful in directing a programme of synthesis could be established.Finally although inhibitors of respiration have the potential to be toxic to mammals the low acute toxicity of several strobilurins and oudemansins indicated that toxicities towards fungi and mammals are not inextricably linked in this series of com- pounds. Samples of oudemansin A and myxothiazol A were gen- erously provided by Prof. Dr T. Anke (University of Kaiserslautern Germany) and Prof. Dr H. Reichenbach (GBF Braunschweig Germany) and showed activity against several fungi growing on plants in the glasshouse when applied as foliar sprays at a concentration of 33 mg 1-'. By contrast a synthetic sample of strobilurin AZ4 showed no useful activity in similar glasshouse tests and this was shown to be due to a combination of its photochemical instability and relatively high volatility through which it is rapidly lost from a leaf A programme of synthesis was initiated; its aim was to prepare analogues of the natural products with high levels of activity and suitable physical properties for an agricultural fungicide.Synthesis of the stilbene (41) in which the (2)-olefinic bond of strobilurin A is incorporated within a benzene ring was an important first step (Scheme 10). Although the stilbene (41) has excellent activity in the glasshouse a result of its improved photostability and reduced volatility in com-parison with strobilurin A it still showed only moderate activity in the Quite independently Anke and Steglich NATURAL PRODUCT REPORTS 1993-J.M. CLOUGH -OMe *.a- Q-0D0Q CN &OMe Me02C (44) ICIA5504 Evolution of ideas Scheme 10 and their co-workers have also prepared the stilbene (41) as well as other synthetic analogues of the str~bilurins.~~.~~. Photostability was increased still further by replacing the styryl side-chain of the stilbene (41) with a phenoxy group; the resulting diphenyl ether (42) was found to control the growth of a variety of commercially-important fungi in the field. In addition it has the important property of systemic movement within plants a key attribute of modern fungicides which improves field performance through redistribution of the compound in plant tissues after appli~ation.~~ “9 79 While examining the scope for the introduction of substituents in (42) it was discovered that the tricyclic compound (43) has improved levels of activity.However (43) is not systemic because its partition coefficient is too high [log P(n-octanol/water) = 5.1 (estimate) for (43) and 3.3 (measured) for (42)]. Extensive further work showed that certain hetero- cyclic analogues of (43) retain high activity and because of their lower partition coefficients have systemic m~vement.’~ The broad-spectrum systemic fungicide ICIA5504 (44) [log P (n-octanol/water) = 2.641 now in development is the cul- mination of this work (Scheme In field trials it has shown good efficacy against a wide range of economically important fungal diseases of crops such as temperate cereals rice vines and apples.As expected on the basis of its novel mode of action it controls the growth of strains of fungi which are resistant to other classes of fungicide. BASF has also recently announced its own development fungicide of this type BAS 490 F (45).82 It is interesting that the P-methoxyacrylate toxophore of the strobilurins and oudemansins has been replaced with the isosteric methoxyiminoacetate group in this compound. The importance of P-methoxyacrylate chemistry within the agrochemical industry is demonstrated by the fact that more (45)BAS490F than ten companies are now conducting research in the area. By January 1993 these companies had published a total of more than 130 patent applications which claim synthetic analogues of the strobilurins oudemansins and myxothiazols mainly as fungicides for use in agriculture.Acknowledgements. I am grateful to Prof. Dr G. Hofle of the Gesellschaft fur Biotechnologische Forschung Braunschweig Germany for generously providing me with unpublished information about the myxothiazols. I also thank my colleagues Drs. B. C. Baldwin C. R. A. Godfrey and C. J. Urch for their helpful comments on this Report. 7 References 1 V. Musilek Czech. Pat. CS 136492 (= British Pat. GB 1163910) 1965 (Chem. Abstr, 1969 70L18900y and 1971 74 123689s). 2 V. Musilek J. Cerna V. SaSek M. Semerdiieva and M. VondraEek Folia Microbiol. (Prague) 1969 14 377. 3 ‘Dictionary of Alkaloids’ ed. I. W. Southon and J.Buckingham Chapman and Hall London and New York 1989 pp. xxxiii and 363. 4 A. Fredenhagen A. Kuhn H. H. Peter V. Cuomo and U. Giuliano J. Antibiot. 1990 43 655. 5 W. Weber T. Anke M. Bross and W. Steglich Planta Med. 1990 56 446. 6 A. Fredenhagen P. Hug and H. H. Peter J. Antibiot. 1990 43 661. 7 M. VondraEek J. Capkova J. Slechta A. Benda V. Musilek and J. Cudlin Czech. Pat. CS 136495 (= British Pat. GB 1181892) 1967 (Chem. Abstr. 1970 72,41752b and 1971 75 4029n). 8 L. Homolka,_ E. Toscaniova V. Musilek M. Vondratek V. Cechner K. Culik and P. Ettler Czech. Pat. CS 209382 1980 (Ctem. Abstr. 1983 99 193190n). 9 V. SaSek and V. Musilek Folia MicJ.obiol. (Prague) 1974 19 139. 10 M. OtEenaSek J. Kejda and J. Sich VII Internatn.Congress Infect. Parasit. Diseases Varna 2-6 Oct. 1978 3 132; J. Kejda Prakt. Lek. 1980 60 26 (Chem. Abstr. 1980 93 143792r); Z. Zouchova F. Nerud and V. Musilek Folia Microbiol. (Prague) 1982 27,35. 11 P. Sedmera V. Musilek F. Nerud and M. Vondratek J. Antibiot. 1981,_34 1069. 12 M. VondraEek J. Capkova and K. Culik Czech. Pat. CS 180775 1974 (Chem. Abstr. 1980 93 204286~). 13 M. VondraEek J. VondriEkova P. Sedmera and V. Musilek Collect. Czech. Chem. Commun. 1983 48 1508. 14 F. Nerud P. Sedmera and K. VereS Radiochem. Radioanal. Lett. 1981 46 303. 15 M. Nadrchalova and J. Capkova Czech. Pat. CS 172754 1974 (Chem. Abstr. 1979 90 85263~). 16 T. Anke F. Obenvinkler W. Steglich and G. Schramm J. Antibiot. 1977 30 806. 17 G.Schramm W. Steglich T. Anke and F. Oberwinkler Chem. Ber. 1978 111 2779. 18 J. Bauerle and T. Anke Planta Med. 1980 39 195. 19 J. Bauerle Dissertation University of Tubingen Germany 198 1. 20 T. Anke H. Besl U. Mocek and W. Steglich J. Antibiot. 1983 36 661. 21 G. von Jagow G. W. Gribble and B. L. Trumpower Bio-chemistry 1986 25,775. 22 W. Steglich G. Schramm T. Anke and F. Oberwinkler Eur. Pat. EP 44448 1980 (Chem. Abstr. 1982,96 162440~); G. Schramm Dissertation University of Bonn Germany 1980. 23 T. Anke G. Schramm B. Schwalge B. Steffan and W. Steglich Liebigs Ann. Chem. 1984 1616. 24 K. Beautement and J. M. Clough Tetrahedron Lett. 1987 28 475. 25 B. Schwalge Dissertation University of Bonn Germany 1986. 26 H. Gayer P.Gerdes and A. Klausener German Pat. DE 4012792 1990 (Chem. Abstr. 1992 116 83453n). 27 M. Sutter Tetrahedron Lett. 1989 30 5417. 28 T. Anke J. Bauerle S. Backens and W. Steglich Abstr. Ann. Meet. Am. Soc. Microbiol. 1983 244 (Biotech. Abstr. 1983 83 3872). 29 S. Backens Dissertation University of Bonn Germany 1983. 30 S. Backens W. Steglich J. Bauerle and T. Anke Liebigs Ann. Chem. 1988 405. 31 L. Daum G. Keilhauer G. Lorenz E. Ammermann T. Anke W. Weber W. Steglich B. Steffan and A. Scherer Eur. Pat. EP 342427 1988 (Chem. Abstr. 1990 113 189792a). 32 W. Weber T. Anke B. Steffan and W. Steglich J. Antibiot. 1990 43 207. 33 R. M. Williams and T. D. Cushing Tetrahedron Lett. 1990 31 6325. 34 K. Beautement J. M. Clough P. J.de Fraine and C. R. A. Godfrey Pestic. Sci. 1991 31 499. 35 T. Anke H. J. Hecht G. Schramm and W. Steglich J. Antibiot. 1979 32 1112. 36 T. Anke A. Werle M. Bross and W. Steglich J. Antibiot. 1990 43 1010. 37 T. Nakata T. Kuwabara Y. Tani and T. Oishi Tetrahedron Lett. 1982,23 1015; Jpn. Pat. JP 58/144348 1982 (Chern. Abstr. 1984 100 68084~). 38 C. J. Kowalski M. S. Haque and K. W. Fields J. Am. Chem. Soc. 1985 107 1429. 39 K. Mikami K. Azuma and T. Nakai Chem. Lett. 1983 1379; Tetrahedron 1984 40 2303. 40 J. Kallmerten and M. D. Wittman Tetrahedron Lett. 1986 27 2443; J. Org. Chem. 1987 52,4303. 41 H. Akita H. Koshiji A. Furuichi K. Horikoshi and T. Oishi Tetrahedron Lett. 1983 24 2009; Jpn. Pat. JP 59/163348 1983 (Chem.Abstr. 1985 102 1489962). 42 T. Oishi and H. Akita J. Synth. Org. Chem. Jpn. 1983 41 1031. 43 H. Akita A. Furuichi H. Koshiji K. Horikoshi and T. Oishi Chem. Pharm. Bull. 1983 31 4376. 44 H. Akita H. Koshiji A. Furuichi K. Horikoshi and T. Oishi Chem. Pharm. Bull. 1984 32 1242. 45 H. Akita H. Matsukura and T. Oishi Tetrahedron Lett. 1986 27 5397; Jpn. Pat. JP 62/205048 1986 (Chem. Abstr. 1988,109 110164h). 46 H. Akita H. Matsukura and T. Oishi Jpn. Pat. JP 62/205047 1986 (Chem. Abstr. 1988,109 110165j); H. Akita H. Matsukura H. Karashima and T. Oishi Chem. Pharm. Bull. 1992 40,2847. 47 T. Honda K. Naito S. Yamane and Y. Suzuki J. Chem. Soc. Chem. Commun. 1992 1218. 48 N. Chida K. Yamada and S. Ogawa Chem. Lett. 1992 687. 49 H.H. Meyer Liebigs Ann. Chem. 1984 791. 50 N. Bedorf B. Kunze H. Reichenbach and G. Hofle in ‘Scientific Annual Report of the Gesellschaft fur Biotechnologische Forschung mbH’ Braunschweig (Germany) 1986 p. 14. 51 H. Augustiniak K. Gerth L. Grotjahn H. Irschik T. Kemmer B. Kunze H. Reichenbach G. Reifenstahl W. Trowitzsch and V. Wray German Pat. DE 2838542 1978 (Chem. Abstr. 1980 92 179082~). 52 K. Gerth H. Irschik H. Reichenbach and W. Trowitzsch J. Antibiot. 1980 33 1474. 53 W. Trowitzsch G. Reifenstahl V. Wray and K. Gerth J. Antibiot. 1980 33 1480. 54 J. Lehmann L. Berthe-Corti W. Steven J. Nothnagel G.-W. Piehl and K. Gerth German Pat. DE 2924868 1979. 55 H. Reichenbach K. Gerth H. Irschik B. Kunze and G. Hofle Trends Biotechnol.1988 6 115. 56 W. Kohl B. Witte B. Kunze V. Wray D. Schomburg H. Reichenbach and G. Hofle Liebigs Ann. Chem. 1985 2088. 57 W. Trowitzsch G. Hofle and W. S. Sheldrick Tetrahedron Lett. 1981 22 3829. 58 I. R. Waldron Ph.D. Thesis University of Nottingham U.K. 1987 (British Library DSC D74409/87); S. Comins Ph.D. Thesis University of Nottingham U.K. 1990 (Chem. Abstr. 1991 115 289488); M. Ramsden Ph.D. Thesis University of Newcastle-upon-Tyne U.K. 1990 (Aslib Index to Theses 40 5865). 59 F. Nerud P. Sedmera Z. Zouchova V. Musilek and M. VondraEek Collect. Czech. Chem. Commun. 1982 47 1020. NATURAL PRODUCT REPORTS 1993 60 T. Anke and W. Steglich in ‘Biologically Active Molecules Identification Characterization and Synthesis’ ed. U. P. Schlunegger Springer-Verlag Berlin and Heidelberg 1989 p.9. 61 W. Trowitzsch-Kienast V. Wray K. Gerth H. Reichenbach and G. Hofle Liebigs Ann. Chem. 1986 93. 62 Prof. Dr G. Hofle Gesellschaft fur Biotechnologische Forschung mbH Braunschweig Germany personal communication August 1992. 63 B. L. Trumpower Microbiol. Rev. 1990 54 101. 64 B. Thierbach and H. Reichenbach Antimicrob. Agents Chemother. 1981 19 504. 65 G. Hofle and H. Reichenbach in ’ Scientific Annual Report of the Gesellschaft fur Biotechnologische Forschung mbH’ Braunschweig (Germany) 1990 p. 5. 66 T. Anke in ‘Cellular Recognition and Malignant Growth’ ed. S. Ebashi Japanese Scientific Society Press Tokyo/ Springer Verlag Beein 1985 p. 169. 67 J. Subik M. Behuii and ,V. Musilek Biochem Biophys.Res. Commun. 1974 57 17; J. Subik M. Behuii P. Smigaii and V. Musilek Biochim. Biophys. Acta 1974 343 363. 68 W. F. Becker G. von Jagow G. Thierbach and H. Reichenbach Hoppe-Seyler’s Z. Physiol. Chem. 1980 361 1467; G. Thierbach and H. Reichenbach Biochim. Biophys. Acta 1981 638 282; G. von Jagow and W. D. Engel FEBS Lett. 1981 136 19; S. W. Meinhardt and A. R. Crofts FEBS Lett. 1982 149 217. 69 W. F. Becker G. von Jagow T. Anke and W. Steglich FEBS Lett. 1981,132 329; G. von Jagow and W. F. Becker Bull. Mol. Biol. Med. 1982 7 1. 70 G. Thierbach and H. Reichenbach Arch. Microbiol. 1983 134 104; G. von Jagow P. 0. Ljungdahl P. Graf T. Ohnishi and B. L. Trumpower J. Biol. Chem. 1984 259 6318. 71 R. W. Mansfield and T.E. Wiggins Biochim. Biophys. Acta 1990 1015 109. 72 Y.Kamensky A. A. Konstantinov W. S. Kunz and S. Surkov. FEBS Lett. 1985 181 95. 73 U. Brandt U. Haase H. Schagger and G. von Jagow J. Biol. Chem. 1991 266 19958. 74 L. Yu and C. -A. Yu Biochemistry 1987,26,3658;U. Brandt H. Schagger and G. von Jagow Eur. J. Biochem. 1988 173 499. 75 G. von Jagow and T. A. Link Methods Enzymol. 1986,126,253 76 B. Kunze T. Kemmer G. Hofle and H. Reichenbach J. Antibiot. 1984,37,454; G. Thierbach B. Kunze H. Reichenbach and G. Hofle Biochim. Biophys. Acta 1984,765,227; G. Hofle B. Kunze C. Zorzin and H. Reichenbach Liebigs Ann. Chem. 1984 1883; G. von Jagow and T. Ohnishi FEBS Lett. 1985 185 31 1. 77 J.-P. di Rago J.-Y. Coppee and A.-M. Colson J.Biol. Chem. 1989,264 14543; F. Daldal M. K. Tokito E. Davidson and M. Faham EMBO J. 1989,8,3951; D. E. Robertson F. Daldal and P. L. Dutton Biochemistry 1990 29 11249; T. Tron M. Crimi A.-M. Colson and M. Degli Esposti Eur. J. Biochem. 1991,199 753; B. M. Geier H. Schagger U. Brandt A.-M. Colson and G. von Jagow Eur. J. Biochem. 1992 208 375; A.-M. Colson B. Edderkaoui and J.-Y. Coppee Biochim. Biophys. Acta 1992 1101 157. 78 J. M. Clough P. J. de Fraine T. E. M. Fraser and C. R. A. Godfrey in ‘Synthesis and Chemistry of Agrochemicals 111’ ed. D. R. Baker J. G. Fenyes and J. J. Steffens A. C. S. Symposium Series No. 504 American Chemical Society Washington D.C. 1992 p. 372. 79 J. M. Clough D. A. Evans P. J. de Fraine T. E. M. Fraser C.R. A. Godfrey and D. Youle in ‘Natural and Derived Pest Management Agents’ ed. P. Hedin R. Hollingworth and J. Menn American Chemical Society Washington D.C. 1993 in press. 80 T. Anke G. Schramm W. Steglich and G. von Jagow in ‘The Roots of Modern Biochemistry’ ed. H. Kleinkauf H. Von Dohren and L. Jaenicke de Gruyter Berlin and New York 1988 p. 657. 81 J. R. Godwin V. M. Anthony J. M. Clough and C. R. A. Godfrey ‘Brighton Crop Prot. Conf. Pests and Diseases -1992 Vol. 1 ’ British Crop Protection Council Farnham U.K. 1992 p. 435. 82 E. Ammermann G. Lorenz K. Schelberger B. Wenderoth H. Sauter and C. Rentzea. ‘ Brighton Crop Prot. Conf. Pests and Diseases -1992 Vol. 1 ’ British Crop Protection Council Farnham U.K. 1992 p. 403.

ISSN:0265-0568    

DOI:10.1039/NP9931000565

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites R. B. Herbert School of Chemistry University of Leeds Leeds LS2 9JT Reviewing the literature published between January and December 1991 (Continuing coverage of literature in Natural Product Reports Vol. 9,p. 507) 1 Pyrrolidine and Piperidine Alkaloids 1.1 Pyrrolidine Alkaloids 1.2 Piperidine Alkaloids 1.3 Pyrrolizidine Alkaloids 1.4 Quinolizidine Alkaloids 2 Isoquinoline Alkaloids 3 Tryptophan Metabolites 3.1 Terpenoid Indole Alkaloids 3.2 Miscellaneous Metabolites 4 Other Metabolites of the Shikimate Pathway 4.1 Dike topiperazines 4.2 Tenellin and Tropic Acid 4.3 Xanthocillin 4.4 Dhurrin 4.5 Betalains 4.6 PQQ 4.7 Hexapeptide Antibiotic L-67 1 329 4.8 Quinoline Alkaloids 4.9 Sarubicin A and Antibiotics LL-C10037 and MM 14201 4.10 Ansamycin Antibiotics 4.1 1 Aphelandrine 5 p-Lac tams 5.1 Clavulanic Acid 5.2 Penicillins and Cephalosporins 6 Miscellaneous Metabolites 6.1 Nucleoside Antibiotics 6.2 Unusual Amino Acid Derivatives 6.3 Citreamicins 6.4 Streptothricin F 6.5 Tabtoxin 7 References As always appropriate reference is made in the following discussion to earlier reports and surveys which provide background information.1-8 This Report in a change from past practice covers a calendar year (1991).In part access to the literature was obtained through the IS1 Data Service at Bath.Attention is drawn to a collection of papersg on ‘enzymo- logical and molecular genetic advances’ in the study of the biosynthesis of natural products. 1 Pyrrolidine and Piperidine Alkaloids 1.1 Pyrrolidine Alkaloids Factors which affect endogenous regulation of the biosynthesis of tropane alkaloids (cf. ref. 5 p. 507) have been carefully explored in transformed root cultures of Datura stramonium.1° The regulation is not a simple one and (i) the early enzymes of the pathway are at near rate-limiting levels (ii) there is a major limitation to flux at the level of tropine esterification and (iii) the level of free tropine may be important in determining flux into and through the tropane pathway.The relative contributions to hyoscyamine biosynthesis of the pathway from L-ornithine and the alternative from L-arginine have been examined in D.stramonium root cultures using m-a-difluoromethylarginine and m-a-difluoromethyl- ornithine as irreversible enzyme inhibitors.ll It was concluded that the two routes which yield putrescine as a common intermediate of alkaloid biosynthesis do not act in isolation from one another that arginine decarboxylase rather than ornithine decarboxylase is the more important activity for hyoscyamine formation and that the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. It has been observed that DL-a-difluoromethylornithine (but not DL- a-difluoromethylarginine) induces a phenotype in Nicotiana tabacum similar to that described for the root-inducing left- hand transferred DNA of the soil bacterium Agrobacteriurn rhizogenes.12 At the molecular level polyamine biosynthesis is being interfered with.In an examination of enzyme activities (which are associated with alkaloid biosynthesis in these root cultures) in relation to root morphology it has been shown13 that there is a notably close correlation between root morphology putrescine N-methyltransferase activity and the ability of the cultures to make alkaloids. The regulation and expression of the methyl- transferase is crucial to the overall activity of the tropane alkaloid biosynthetic pathway. Preliminary promising results in relation to alkaloid biosynthesis with somatic hybrids of Duboisia leichhardtii and N.tabacum have been reported.14 Tropinone (1) is a proven precursor for tropane alkaloids e.g. hyoscyamine (3) (cf. ref. 5 p. 508). Following earlier work on tropinone reductase a crude enzyme preparation has been obtained from roots of Datura inn0~ia.l~ Two different enzymes were present in the preparation one yielded tropine (2) (apparently irreversibly) and the other y?-tropine (5) ;NADPH was for both the preferred reducing co-enzyme. The sulphur analogue (6) of tropinone (1) has been found to be metabolized to a large degree in D. stramunium.l6 The alcohol (7) and its 3-0-acetate were among the metabolites. Tropane alkaloid biosynthesis was perturbed and it was argued that (6) may be a useful tool for studying tropane biosynthesis.@O -@-OH @OH (5) (3)R =H (4) R = OH (6) R’R2 = 0 (7)R’=OH R2=H 575 NATURAL PRODUCT REPORTS. 1993 oMeMeCO-SCoA - % C-SCOA * SCoA (9) * C02Me 'C02Me C02Me &OH G O t- &O 0 (14) Cocaine Scheme 1 (15) A='% o='~N (16) I t H (17) 1 COZH 2 Scheme 2 Hyoscyamine 6-P-hydroxylase catalyses the hydroxylation of (3) to give (4) en route to scopolamine. The occurrence and location of the enzyme within the plant has been probed with monoclonal antibodies raised against the hydroxylase in Hyoscyarnus niger.17 The enzyme was found to be localized in the pericycle of cultured plant roots. For new information on the biosynthesis of tropic acid see Section 4.2.The biosynthesis of cocaine (14) is different in important respects to that of the tropane alkaloids; the common precursor (8) suffers different fates on further reaction. In the case of cocaine it has been concluded that two sequential condens- ations with acetyl coenzyme A occur to give (lo) via (9) which has been identified as an intermediate. (cf. ref. 4 p. 185). In further experiments methyl (RS)-[1,2-13C2 1-14C]-4-( 1-methyl-2-pyrrolidiny1)-3-oxobutanoate(1 l) labelling shown (0 = 13C;* = has been found to be an intact precursor for cocaine (14) labelling shown and methyl ecgonine (13) in Erythroxylon coca.l* (Because of the coupling between the 13C enriched carbons low levels of label could simply be detected as doublets flanking natural abundance singlets in the 13C NMR spectrum of the metabolite).The proposed biosynthetic route (Scheme 1) is much strengthened by this result. Further support comes from the biornimetic synthesis of (12) by oxidation of (11) with mercuric acetate and the observation that [9-14C]-2-carbomethoxytropinone [as (12)] was incorporated into (1 3) and (14) in vivo. Simultaneous administration of the N-acetylcysteamine ester of benzoic acid with labelled (12) increased the incorporation into cocaine (14) at the expense of methyl ecgonine (1 3). 1.2 Piperidine Alkaloids The orchid alkaloid anosmine has the unique imidazole structure (1 8). Anosmine has been shown to be biosynthesized in Dendrobium parishii from two molecules of lysine DL-[~-~~C,~-~~N] lysine (15) afforded a sample of (18) the 13C NMR spectrum of which showed doublets for C-2 and C-9 i.e.intact and specific incorporation of two units of the precursor amino acid. The lysine decarboxylation product cadaverine (16) provides one of these units [ 1,5-13C2,15N,]cada~erine led to (18) labelled on C-9 (with retention of 15N) and no label on C-2 ; [ 1,5-13C,]cadaverine gave a complementary result with labelling of C-9 only. (The alternative to C-9 i.e. C-13 was not recorded as being labelled in either case it should be noted however that this labelling may not be seen in the 13C NMR spectra as C-13 is q~aternary.'~) It is likely that lysine is incorporated into the second unit by way of pipecolic acid (17); the biosynthetic pathway which the results suggest is shown in Scheme 2.Representatives of a most interesting and excitingly valuable new group of alkaloids deoxynijirimycin (21) and deoxy- mannonojirimycin (22) have received welcome biosynthetic scrutiny.20 Both metabolites derive from D-glucose in Strepto- rnyces subrutilis. The incorporation of [ 1-2H]-~-glucosewas into the 6-pro-S positions of (21) and (22) whilst [2-2H]-~-glucose labelled the 6-pro-R position in both cases. These results are consistent with the glucose isomerase catalysed conversion of glucose (1 9) into fructose (20) which becomes labelled from both precursors on C- 1 (but diastereotopically) prior to utilization in the biosynthesis of (21) and (22). The keto group in fructose serves presumably for introduction of the amino group by a transamination reaction.1.3 Pyrrolizidine Alkaloids Otonecine (28) the base portion of the alkaloid emiline (29) is biosynthesized via the well established route8 for pyrrolizidine alkaloids; retronecine (27) is a late intermediate (cf. ref. 5 p. 5 10). The incorporation into emiline in root cultures of Emilia flarnmea of samples of putrescine (23) chirally labelled with deuterium at C-1 and at C-2 has now been examined.'l The 1-pro-S hydrogen is removed and the 1-pro-R hydrogen is retained in all oxidations of amino groups yielding aldehydes (or equivalents). During reduction of the aldehyde function that leads to the alcohol that becomes C-9 of (27) hydride attack occurs on the C-re-face of the carbonyl group; the same result was observed earlier for the retronecine portion (27) of retrorsine.22 Formation of the 1,a-double bond in otonecine (28) involves loss of the 2-pro-S proton in putrescine and NATURAL PRODUCT REPORTS 1993-R.B. HERBERT S20H d20H HO,Me been shown using two species each of Senecio and Cynoglossum that this iminium salt [(25); labelled with 14C at C-41 is an efficient precursor (more efficient than tritiated putrescine serving as an internal marker) for alkaloids derived from the three skeletal types (26) (30) and (31).23 Furthermore the oGo (33) retention of the 2-pro-R proton at C-2 of emiline (29). Hydroxylation at C-7 takes place with orthodox retention of configuration.Introduction of the carbonyl group at C-8 of retronecine (27) and cleavage of the bicyclic system en route to (28) does not involve loss of deuterium at any of the sites on the pyrrolizidine ring of retronecine apart of course from the The triamine (24) is a crucial intermediate in the biosynthesis of pyrrolizidine alkaloids' and the iminium salt (25) which logically succeeds it on the biosynthetic pathway has been shown to be an efficient precursor for otonecine (28). It has now iminium salt (25) could be identified as a natural constituent of Senecio pleistocephalus since it was obtained in radioactive form after adding unlabelled carrier following a feeding experiment with [1,4-14C]putrescine. It is thus firmly located as an intermediate in pyrrolizidine alkaloid biosynthesis.On the other hand its reduction product N-(4-aminobutyl)- pyrrolidine is apparently not involved in biosynthesis. The biosynthesis of the diverse necic acids which bridge and embellish the base portions of the pyrrolizidine alkaloids has received most welcome further attention24 (for earlier work see ref. 8 p. 139). The necic acid portion of dicrotaline (33) 3- hydroxymethylglutaric acid (HMG) was not specifically derived from acetate mevalonate valine or surprisingly HMG and leucine. But threonine and isoleucine (32) were efficient precursors which labelled the HMG portion much more heavily than the base portion. Importantly ~-[4,5-'HH]isoleucine gave dicrotaline (33) in which 90.5% of the label present was located in the methyl group.This indicates that isoleucine is metabolized as shown by the dotted lines in (32) to give four of the six carbon atoms of the HMG portion of (33). NATURAL PRODUCT REPORTS 1993 0 0 0 && H5 MeN ’ (34) (35) (37) I la-H (39) (38) 1lP-H H3c013& HO -H3:s -H3;To) OH OH 2‘\ OCH3 \ OCH3 \ o (40)(S)-Reticuline (41) (S)-Scoulerine ro 0% (42) 1-<%He- ‘ ’ 1.4 Quinolizidine Alkaloids Results of experiments in Baptisia australis with samples of cadaverine chirally deuteriated on C- 1 show ingeniously that if ( +)-sparteine (34) and (-)-N-methylcytisine (35) are bio-synthesized from the same (similar) tetracyclic intermediate then ring A [as in (34)] must be degraded and ring D must be converted into a pyridone (cf.ref. 1 p. 425). This has been confirmed using deuteriated cadaverine samples with Anagyris f~etida.~~ More importantly the results obtained show that if (-)-anagyrine (36) and (+)-sparteine (34) are formed from a similar tetracyclic intermediate in this plant then it is ring D that must be converted into a pyridone. Problems remain with the reality and stereochemistry of the tetracyclic intermediates [cf. stereochemistry in (34) and (36)]. The efficient interconversionsZ6 of 14C-labelled lupanine (37) and a-isolupanine (38) in Lupinus texensis have been observed and evidence has been adduced that this occurs via an inter- mediate such as (39). Labelled lupanine (37) was incorporated into A5-dehydrolupanine and anagyrine (36).Although these results suggest a solution to the stereochemical problems above anagyrine (36) was from (R)-[ l-2H]cadaverine with deuterium still present at C-1 1 which is incompatible with the postulation of an intermediate such as (39) for epi- merization. 2 lsoquinoline Alkaloids The very profitable use of plant cell suspension cultures in the quest for the enzymes of secondary metabolism notably those for benzylisoquinoline biosynthesis has been briefly but authoritatively s~rveyed.~’ The pathway that leads from (S)-reticuline (40) to sanguinarine (44) over eight enzymically defined steps has been reviewed.28 The pivotal step in the pathway is the conversion of (S)-reticuline (40) into (S)-scoulerine (4 l) catalysed by berberine bridge enzyme (EC 1.5.3.9).The gene coding for this enzyme in Eschscholtzia californica has been sequenced; it has been expressed in catalytically active form in Saccharomyces cerevisae.28g 29 The ’I O) I O) ‘0 ‘0 OMe OMe (45) R‘ R2 = CH2 (47) (46) R’ = R2 = CH3 data obtained emphasize the role of this enzyme in plant response to pathogens. Full details have been published30 on the two P-450 enzymes that are contained in a microsomal preparation of E. californica cell cultures and which specifically catalyse one each of the steps from (5‘)-scoulerine (41) through (42) to (S)-stylopine (43) (cf. ref. 5 p. 51 1). Both enzymes were markedly induced after challenging the E. californica cell cultures with an elicitor.Tetrahydroprotoberberine alkaloids with the (R)-configur- ation at C-14 are rare in nature. In precursor feeding experiments with Cocculus laurifolius and Corydalis cava it has been shown31 that precursors such as reticuline [as (40)] cheilanthifoline [as (42)] and scoulerine [as (41)] with the (R)-configuration are not precursors for (R)-tetrahydroproto-berberine alkaloids contrary to a previous report but that (S)-precursors are incorporated. Further a specific enzyme system has been isolated from C. cava which catalyses in the presence of NADPH the reduction of berberine (49 via its 7,8-dihydro derivative into canadine (47) with (R)-configuration as shown. The reductase acts on relatives of berberine the highest turnover being observed with palmatine (46).The combined evidence indicates strongly that the reductase system is involved generally in the biogenesis of tetrahydroprotoberberine alkaloids with (R)-configuration. A plant cell bioreactor has been developed for the production of protoberberine alkaloids using immobilized Thalictruwl NATURAL PRODUCT REPORTS 1993-R. B. HERBERT OH 0 (49) HoQ OH (51) rugosum cultures.32 Berberine production in cell suspension cultures of Thalictrum minus is significantly enhanced by added spermidine.33 It is an extraordinary observation that morphine (50) and its congeners occur naturally in mammalian tissue. Demonstration of the occurrence in this tissue of enzymes able to effect the same steps as in plants would prove that these alkaloids are indeed endogenous.Important relevant evidence has now been obtained. A crucially important enzyme in the biosynthesis of morphine alkaloids in plants has been identified in a microsomal preparation of Papaver somniferum (cf. ref. 5 p. 511). It is a highly specific P-450 enzyme and catalyses the phenol oxidative coupling reaction whereby (R)-reticuline (48) is converted into salutaridine (49). Now a P-450 enzyme has been identified in a microsomal fraction of pig liver which is similarly specific it converts (R)-reticuline (48) but not the (S)-isomer into salutaridine (49).34 Other evidence relates to earlier and later stages of biosynthesis. Thebaine follows salutaridine in the biosynthetic pathway to codeine and then morphine in plants.It has been convincingly demonstrated that thebaine is converted inter alia into codeine f H (54) the precursor for over 1200 richly diverse terpenoid indole alkaloids.8 The cDNA coding for the enzyme strictosidine synthase has been cloned and its expression in various heterologous systems has been examined.28 Expression in Escherichia coli results in the quantitative production of strictosidine from tryptamine plus secologanin when they are added to the bacterial culture. The systems under development allow large scale production of the synthase. and morphine (50) in rat liver kidney and brain micro~omes.~~ There is high homology in the genes coding for strictosidine The first isoquinoline in morphine (and other benzyliso-quinoline) biosynthesis in plants is (S)-norcoclaurine (5 1) (cf.ref. 5 p. 512 Scheme 8) which is first methylated on the oxygen at C-6. Mammalian catechol methyltransferase has been to catalyse methylation of (51) in this position to the extent of 80% (20% of the 7-0-methyl isomer) in the presence of S-adenosylmethionine. Using the same enzyme system in the methylation of later potential intermediates has been examined but the results are not clear The efficient incorporation has been of norreticuline and its 1,2-didehydro derivative into papaverine in a cell free system from P. somniferum. The 2’-bromo and 2’-nitro analogues of norreticuline [as (40)] were transformed into the corresponding papaverine analogues (cf. ref. 4,p. 190). The production of emetine and cephaeline in cell suspension and excised root cultures has been The structures of three new alkaloids which were isolated from Colchicum decaisnei suggest a possible route for the catabolism of col~hicine.~~ 3 Tryptophan Metabolites 3.1 Terpenoid Indole Alkaloids A simple enzyme-catalysed Pictet-Spengler reaction of tryptamine and secologanin yields strictosidine which is then synthase in five RauwolJa species examined.2s The relationship between alkaloid content and type and the activity of the enzyme in different parts of Cinchona ledgeriana has been The effects of precursors and stimulating factors on the formation of indole alkaloids by Catharansus roseus in a biofilm reactor have been as has the optimization of growth and alkaloid production by hairy root cultures of C.roseus.43 Raucaffricine is the 0-glucosyl derivative of vomilene. The glucosyltransferase which catalyses the derivatization has been identified in cell-suspension cultures of RauwolJia serpentina as being microsomal-bound and it has been ~haracterized~~ (cf. ref. 4 p. 191). It is highly specific for vomilene and UDPG. 3.2 Miscellaneous Metabolites The catabolism of gramine which eventually leads to break-up of the aromatic system in the alkaloid has been tracked in Hordeum vulga~e.~~ Indolactam V (52) is biosynthesized in Streptoverticillium blastmyceticum from the appropriate a-amino acids via N-methyl-L-valyl-L-tryptophan01 (cf. ref. 5 p. 5 16). An analogue indolactam I (54) which was isolated as a new minor metabolite from S.blastmyceticum is biosynthesized very efficiently in vivo from N-methyl-L-isoleucyl-L-tryptophan01 (53).46 NATURAL PRODUCT REPORTS 1993 * wN=c=s H H (55) (56) u O-!L>SMe S H Ph-SMe 0 Me,*' oyOH -&NMe -dyNMe O s; MeS -OH OH OH The recently discovered compounds brassinin (57) cyclobrassinin (58) and spirobrassinin (59) are the first cruciferous phytoalexins.Their expected biosynthesis from tryptophan (55) has been confirmed by the very efficient incorporation of 4'-deuterated L-amino acid into (59) in irradiated turnip tissue. Methionine labelled the methyl groups of each metabolite. Labelled brassinin (57) was very efficiently transformed into labelled (58) and (59) but neither (58) nor (60) are precursors for (59).The results of a crucial experiment with ~~-[2-~~C]tryptophan [as (59 * = 13C3 show that rearrangement of the side chain of the amino acid occurs during biosynthesis since the label was located as shown (*) in the derived cyclobrassinin (59); the isothiocyanate (56) (cf. ref. 5 p. 519) is a very reasonable intermediate in this re-arrangement. The combined results lead to the pathway shown as Scheme 3. 4 Other Metabolites of the Shikimate Pathway The attention particularly of new readers is drawn to the excellent Reports by P. M. Dewick in this journal which are complementary to the material surveyed in this section (the last Report is given as ref. 48).Chorismate is the starting point for the elaboration of aromatic metabolites. The enzymology and roles of three chorismate utilizing enzymes in plants chorismate mutase anthranilate synthase and isochorismate synthase have been reviewed with emphasis on their importance for secondary rnetab~lism.~~ 4.1 Diketopiperazines The diketopiperazines are a group of diverse metabolites based on cyclic dipeptides which are constituted from two a-amino 3\~~ Gliotoxin (61) is biosynthetically one of the most interesting of these metabolites. It is formed via the LL-dipeptide (62); this is the only diastereomer to be incorporated. The biosynthesis of the related metabolite (63) has now been examined in Hyalondrin sp. (FSC-601) cultures.50 It is to be noted that (63) and its co-metabolite (64) are of different relevant stereochemistry to that of gliotoxin (61).Following the successful incorporation into (63) of cyclo-(L-[U-14C]Phe-~-Ser) was [as (62)] cycZo-~-[4'-~H]Phe-~-[3-~~C]Ser) tested. A very good incorporation (42 %) was observed with maintenance of the 3H:14C ratio thus demonstrating intact incorporation. A similar result was obtained for (64). The diastereoisomers of (62) were not significant precursors for (63). Thus from the results for (61) (63) and (64) sulfur is not introduced with unique stereospecificity. Further evidence for intact incorporation is that (62) with a 13C label at C-3 of the serine fragment gave (63) exclusively labelled at the corresponding position. Additional results for gliotoxin (61) in Gliocladium virens are (i) that cyclo-(~-[4'-~H]Phe-~-[3-~*C]Ser) [as (62)] is in- corporated without scrambling of either label ;(ii) the N-methyl compound (65) corresponding to (62) is not a precursor nor is it for (63); and (iii) the two linear dipeptides corresponding to (62) are not incorporated into (61) without prior substantial hydrolysis in viv~.~O 4.2 Tenellin and Tropic Acid Tenellin (66) is produced by the fungus Beauvaria hassiana.Tropic acid (67) is produced by higher plants e.g. Datura innoxia as part of the structure of e.g. scopolamine and hyoscyamine (3). Tenellin and tropic acid have biosynthetic pathways in common that involve the apparent intramolecular rearrangement of a molecule of phenylalanine.Although much work has been done in an endeavour to understand what is NATURAL PRODUCT REPORTS 1993-R. B. HERBERT 58 1 'CHO H+OH CHpOH (72) - - - Tyrosine H0-CO2H - HO*C02H NHOH oSN 0- -o":oH (73) (74) (75) NOH (78) R=H (79) R = glucose (77) (76) Scheme 4 going on in the rearrangement reactions the mechanisms remain largely It has been hypothesized that such a rearrangement occurring on phenylalanine (68) itself would afford 2-amino-3-phenyl- propionic acid (69) as an intermediate. This has been tested in B. bassiana and D.inn~xia.~' Racemic (69) failed however to label either (66) or scopolamine and hyoscyamine in carefully monitored experiments and its status as an intermediate must therefore be denied.(For other work on tropane alkaloids see Section 1.1 ). 4.3 Xanthocillin The biosynthesis of the curious isocyanide groups found in a few natural products has provided a long running puzzle with some resolution (cf. ref. 2 p. 532; ref. 3 p. 118 ref. 4 p. 206; ref. 52). The origin of the isocyanide groups in marine terpenes is now clearly established as being the cyanide ion (cf. ref. 4 p. 206). Work on the fungal isocyanide xanthocillin monomethyl ether (70) which had previously been published in preliminary form (cf. ref. 2 p. 532) is now available in a full paper.53 Having failed to identify a source in Dichotomomyces cejpii for the isocyanide groups in (70) especially in metabolism involving C units recourse was made to the use of ~-[U-~~C]glucose.Possible incorporation of label into the isocyanide groups was to be measured against the level of unexceptional label in the rest of the molecule particularly the two C PEP-derived units [see (71)]. It was hypothesized (apparently correctly) that this would provide insight into how metabolically close to glucose the isocyanide origins are. Satisfactory incorporation into (70) of D-[u-l3c U-14C]- glucose was obtained using glucose depleted cultures of D. cejpii. Most interestingly the 13C enrichments of the isocyanide carbons in derivative (71) were significantly higher than for any other carbon atoms. Therefore the isocyanide carbons are biosynthetically close to glucose (i.e. less dilution by endogenous intermediates implies a smaller number of reactions) than are either of the PEP C units [see (71)] the O-methyl group and the C unit in the aromatic ring (the labelling of this last unit was not purely intact C,; some labelling arose by cycling through the pentose phosphate pathway).A hypothetical mechanism could be drawn for the C of glyceraldehyde (72) providing the isocyanide carbons in (7 1). However DL-[ 1J3C 1 -14C]glyceraldehyde failed to label these groups. A similar result was obtained with [l-13C]glucose. The mystery remains. 4.4 Dhurrin The biosynthetic pathway to the cyanogenic glycoside dhurrin (79) in Sorghum bicolor has been delineated in fair detail (cf. ref. 5 p. 522; see Scheme 19). New results provide corroborating 55 Biosynthesis begins with tyrosine and three molecules of dioxygen are deduced to be involved in the conversion to the intermediate p-hydroxymandelonitrile (78) Scheme 4.Results5 of experiments with [''O]dioxygen demonstrate that two N-hydroxylations occur on the nitrogen atom present in tyrosine [the first oxidation yields (73)] and that these two oxygens remain enzymically distinguished through the steps of biosynthesis which involve the nitro-acid (74) and its decarboxylation product the aci-nitro-compound (75).aci-Nitro-compounds were proposed as branch points for the biosynthesis of cyanogenic glucosides and glucosinolates. Two cytochrome-P-450-dependent mono-oxygenases have been identified in S. bi~olor.~~ One of these is responsible for N-hydroxylation of tyrosine to give N-hydroxytyrosine (73) and the other in the hydroxylation which converts (77) into (78).The data support the earlier conclusion that the conversion of (76) into (77) may be by simple dehydration. 4.5 Betalains The betalains are a group of pigments found in plants of the Centrospermae order and also in some mushrooms. A key NATURAL PRODUCT REPORTS 1993 as some derivative c- HO,C 0 (86)PQQ Scheme 5 sequence of biosynthesis involves the oxidative conversion of DOPA (80) by an extradiol cleavage which leads to betalamic acid (82).6 3,4-Dihydroxyphenylalanine4,5-dioxygenase the enzyme responsible for the ring scission has now been isolated from the mushroom Amanita muscaria purified and ~haracterized.~~ Like other extradiol-cleaving enzymes it is an oligomer.The enzyme does not exhibit a strict specificity for DOPA. In other work on this Amanita enzyme good circumstantial evidence for the existence of (81) as an intermediate between (80) and (82) has been ~btained.~’ Alternative cleavage between positions 2 and 3 of (80) was also observed. This affords muscaflavin (83) via an intermediate similar to (81). Muscaflavin occurs naturally in A. muscaria. This cleavage reaction was thought to be probably due to an isozyme of the enzyme which cleaves between positions 4 and 5. 4.6 PQQ Pyrroloquinoline quinone (PQQ) (86) is one of several o-quinones which serve as prosthetic groups in some redox enzymes. Earlier studies on the biosynthesis of PQQ (cf.ref. 4 p. 206) which in part relied on the incorporation of [ l-13C]ethanol have been carried further.58 Now using [ 1-13C] and [2-13C]ethanol it has been firmly concluded that PQQ is constructed via one molecule of glutamate (85) (the conclusions derive from a comparison of the labelling patterns in the amino acids that were generated in the cultures with those in PQQ). The previously deduced role of tyrosine in PQQ biosynthesis (phenylalanine was an alternative) was established firmly in experiments with ~-[3’,5’-l~C,]- and [3-13C]tyrosine [as (84)] appropriate labelling of C-5 C-9a and C-3 was observed. Results of an experiment with L-[ l’,6’-l3C,,l5NJtyrosine es- tablished that the pyrrole nitrogen in PQQ comes intact from the amino acid [coupling between N-1 and C-la in the 13C NMR of derived (86)].A biosynthetic pathway consonant with these results has been proposed (Scheme 5).58 4.7 Hexapeptide Antibiotic L-671329 The peptide antibiotic L-671329 (87) which is produced by the fungus Zalerion arboricola contains unusual amino acids. After pilot biosynthetic studies with 14C-labelled precursors the biosynthetic origins were clearly e~tablished~~ with 13C-labelled compounds to be those shown in Scheme 6. Particularly of note are the origins of (i) the 3-hydroxy-4-methyl proline residue from leucine and not proline and (ii) the homotyrosine residue from tyrosine plus acetate (for the latter unit compare the origins in tyrosine plus glyoxylate of the similar residue in the Pseudomonas antibiotic obafluorin;60 cf.ref. 5 p. 520). 4.8 Quinoline Alkaloids The biosynthesis of furoquinoline alkaloids has been established in considerable New work on the biosynthesis of kokusaginine (92) maculosidine (93) and ptelefoline (94) in Ptelea trifoliata resulted in the incorporation of (88) and (90); the dimethoxy analogues (89) and (9 1) were much less effective precursors. Evidence was adduced that biosynthesis may involve intermediates related to derivatives of myrtopsine (95).61 N-Methylation of anthranilic acid followed by ATP-de- pendent activation of the N-methylanthranilic acid produced are the first steps in the biosynthesis of acridone alkaloids (last reported work ref. 4 p. 193 and ref. 5 p.522). Now the corresponding S-adenosyl-L-methionine anthranilic acid N- methyltransferase and activating enzyme have been found in tissue cultures of Choisya ternata which produce dihydro- furoquinoline alkaloids and in a culture of Ruta graveolens which produces dihydrofuro- and dihydropyrano-quinoline alkaloids.62 The implication is that these enzymes are involved in quinoline alkaloid biosynthesis and N-methylation of anthranilic acid is the first step in the biosynthetic pathway. 4.9 Sarubicin A and Antibiotics LL-C10037 and MM 14201 Sarubicin A (98) is biosynthesized from 6-hydroxyanthranilic acid (96) and glucose (cf. ref. 4 p. 201; ref. 1 p. 430). The amide (97) has now been identified as the next biosynthetic intermediate after the acid (96) :63 [13C015NH,]-6-Hydroxy-anthranilamide (97) was found to be an efficient and largely intact precursor (10% hydrolysis to the acid prior to NATURAL PRODUCT REPORTS 1993-R.B. HERBERT A [MeIMethionine . __ .un A -\ HO NH OH -0 0 \/ "\ Tyrosine Scheme 6 OMe OMe OMe OMe II (88) R = H (90) R = H (92) R' = R2 = OMe R3 = H (95) (89) R=OMe (91) R = OMe (93) R' = R3 = OMe R2 = H (97) O= I3C * = 15N (98) NHCOCH3 __c OH (99) 0 0 o@NH2 oQNH2 OH 0 incorporation) for sarubicin A (98) in Streptomyces helicus. from cultures of the bacteri~rn.~~ It converts (99) found now to Synthetic work relating to other potential intermediates has be its true substrate (cf. ref. 5 p. 521) into (101).Presumably been published.64 (100) is an intermediate in this reaction. Only reduction of (101) The results of a mixture of labelling and enzymic techniques is then required to give the antibiotic (102). Interestingly the has led to the clear definition of most of the stages of same found that an enzyme isolated from biosynthesis of the Streptomyces antibiotic LL-C10037 (102); Streptomyces MMp 305 1 [which produces antibiotic MM 14201 biosynthesis begins with 3-hydroxyanthranilic acid (cf. ref. 5 p. (103)] showed opposite facial selectivity yielding (104) as its 521;ref. 3 p. 121). An epoxidase has been isolated and purified product. 584 4.10 Ansamycin Antibiotics A rneta-C,N unit derived from 3-amino-5-hydroxybenzoicacid (105) is a common feature of a number of ansamycin and other antibiotics.'-* In further work it has now been shown using [carboxy-13C]-3-amino-5-hydroxybenzoic acid that (1 05) is unexceptionally also a specific precursor for the streptovaricins by incorporation into streptovaricin C (106) in Streptomyces spectabilP (the C,N unit is shown with heavy bonding).The cyclohexanecarboxylic acid moiety in ansatrienin A (109) derives from shikimic acid (107) via (108) as shown in Scheme 7 (* = I3C label from shikimic acid) (cf. ref. 3 p. 120). An NADPH-specific enoyl-CoA reductase which converts (1 10) into (1 11) has been isolated from Streptomyces collinus and partially purified. 67 The stereochemistry of double bond saturation has been determined to be that shown in Scheme 8.The stereochemistry of addition to the P-carbon of (1 10) finds analogy in the action of enoyl-CoA reductases involved in fatty acid synthesis whilst that of addition to the a-carbon is exceptional. 4.11 Aphelandrine Through the incorporation of radioactive precursors pre-liminary evidence has been obtained that the unusual alkaloid aphelandrine (1 12) is biosynthesized in plants of an Aphelandra species from cinnamic acid putrescine and spermidine.68 It is Me C02H HO' Scheme 7 NATURAL PRODUCT REPORTS 1993 uncertain if spermine and (phydroxycinnamoy1)spermidine are involved in biosynthesis because of their observed degra- dation in vivo. 5 P-Lactams 5.1 Clavulanic Acid The fascinating story of the biosynthesis of clavulanic acid (119) continues (for earlier work see ref.5 p. 524; ref. 4 p. 199; ref. 3 p. 124). The biosynthesis of (119) proceeds via proclaviminic acid (1 13) and claviminic acid (1 18). Further work concerns the identification of an intermediate between (113) and (1 18).'j9 To this end the deuteriated compound (1 14) has been used following the ingenious idea borrowed from penicillin biosynthesis of using a primary deuterium isotope effect to lead to the accumulation of an intermediate. Thus incubation of (1 14) with partially purified claviminic acid synthase afforded (1 16) [analysis by lH NMR; comparison with synthetic (1 17)]. Incubation of a pure sample of (1 16) with the synthase resulted in an efficient conversion into claviminic acid (1 18).The results show that in the conversion of (1 13) into (1 15) an oxidative cyclization/desaturation process occurs rather than the hydroxylation reactions more usually associated with the class of a-ketoglutarate dependent oxygenases of which this synthase is a member. Further it is apparent that the synthase catalyses two sequential steps of the biosynthetic pathway which is consistent with other kinetic evidence.'O An outcome of some synthetic work has been to confirm that the absolute configuration of proclaviminic acid (1 13) is L-threo as The synthesis has been reported of two peptides made through joining 3-hydroxypropionic acid with L- ornithine and DL-3-hydroxyornithine ;these are for biosynthetic studies.72 5.2 Penicillins and Cephalosporins The biosynthesis of penicillins is shown in outline in Scheme 9 ; isopenicillin N (IPN) (122) is the first penicillin to be formed the one from which all the others derive.Two manifestly important enzymes ACV synthetase and isopenicillin N synthase (IPNS) are involved sequentially in the two steps of the pathway. New work concerns almost all aspects of penicillin biosynthesis. (For earlier work see ref. 8; ref. 5 p. 524; ref. 4 196; ref. 3 p. 121). There is an enigmatic change in the stereochemistry of L- valine (120) on incorporation into the ACV tripeptide (121). Incubation of ~~-[l~O~]valine and cysteine and a-aminoadipate with partially purified ACV synthetase (from Cephalosporium acremonium) yielded ACV (121) with exclusive loss of a single oxygen The valine recovered from this experiment and from another in which the other two amino acids were omitted from the incubation showed no exchange of label.Thus activation of the valine prior to condensation is irreversible. Further the previously observed oxygen exchange in vivo (cf. ref. 5 p. 524) with labelled valine is not associated with the operation of the synthetase. The reason for the inversion of the configuration of the valine in going to ACV remains obscure. The Aspergillus nidulans gene (acvA) encoding the ACV synthetase (ACVS) has been positively identified and chara~terized.~~ The gene is transcribed in the opposite direction Scheme 8 NATURAL PRODUCT REPORTS 1993-R. B. HERBERT to ipnA (encoding IPNS) with an intergenic region of 872 nucleotides.The gene has been completely sequenced and was found to encode a protein of 3770 amino acids. The enzyme is a glycoprotein. The derived amino acid sequence contains three homologous regions of 585 amino acids each of which shows areas of similarity with adenylate-forming enzymes and several multienzyme peptide synthetases. However cysteine residues thought to be essential for a thio-template mechanism of peptide biosynthesis were not detected in the ACVS sequence. There are on the other hand putative 4’-phosphopantetheine- attachment sites and a putative thioesterase site. It is speculated that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.In independent ACVS has been isolated from C. acremonium and Streptomyces clavuligerus and has been partially characterized. Phosphopantothenic acid was shown to be associated with the enzyme from both sources. The possible involvement of pantothenate (as a ‘swinging arm’) in the formation of the ACV tripeptide (121) is suggested and is OH C02H (113) R=H (1 14) R = 2H (racemic) (115) R=H (116) R=2H (117) enantiomer of (115) C02H (121) ACV ACV Synthetase 1 L-CX-A mi no adipate + L-Cysteine + C02H (120) L-Valine supported by the two different pieces of e~idence.~~.~~ IPNS from C. acremonium contains two cysteine residues in positions 106 and 255 which are invariant in all the IPNS sequences reported so far.The functions of these residues in the C. acremonium enzyme have been examined through chemical modification and site directed m~tagenesis.~~ Three mutant enzymes were obtained. The stoichiometry of metal required for activity remains as one equivalent of Fe2+ per molecule of enzyme in the mutants as in the wild-type IPNS. Compared with wild-type enzyme Cys-255 + Ser shows a reduction of V,, by 33 % and a 1.4 fold increase in K whilst Cys-106 + Ser and Cys-106 255 + Ser had identical kinetic properties exhibiting a decrease in V,, by 63 % but a 14-fold increase in Krn. The results from chemical modification and studies with the mutants show that (i) both cysteines are free thiols; (ii) Cys-106 is more exposed than Cys-255; (iii) substrate-induced inactivation is not caused by cysteine modification ; (iv) neither cysteine is absolutely essential for bond making or breaking events during catalysis.It was concluded that Cys-106 is likely to be involved in substrate binding whilst Cys-255 has a role only in maintaining protein structure. The binding of endogenous ligands to iron in the active site of IPNS has been the subject of an NMR st~dy.’~ Electron spin echo envelope modulation spectroscopy has been used to study the active site structure of IPNS from C. acremonium with CU(II) as a spectroscopic A model system has been developed involving substrate oxidation in the presence of iron which results in ligand transfer from iron to The observations lend credence to proposed mechanisms for /I-lactam biosynthesis involving high-valent iron intermediates.The IPNS genes from C. acremonium Penicillium chrysogenum and A. nidulans have been expressed at high levels in soluble form in E. coli.80 Available evidence strongly indicates that in the cyclization of ACV (121) to isopenicillin N (122) the p-lactam ring is C02H CO2H CO2H (122) lsopenicillin N (IPN) Scheme 9 NATURAL PRODUCT REPORTS 1993 RR H **"G%AA'&L Fe2+,0; IPNS 0 I C02H C02H C02H COpH (124) R= H (125) R=F (126) R =H (127) R=F (129) AA = L-a-Aminoadipoyl ]R-F HAANxy0 H AAN-SH Z' C02H H AAN% z' i COQH (132) X=Y=Z=H (133) X=H Y =Z=D (134) X=D Y=Z=H (135) X=Y=Z=H (136) X=H Y=Z=D (137) X=D Y=Z=H ACV(121) - C02H 1 -m H Enz AAN 0rl+;y Me CO2H EnzH AAN S-te-OHD*; 0 CO2H -IPN (122) (1 38) Scheme 10 formed first followed by a further ring closure involving the sulphur atom.One of the pieces of evidence is based on the isolation of the shunt metabolite (123) from incubations of IPNS with deuteriated ACV [as (121)] (cf ref. 4,p. 198). Full details of this work have now been published.81 When the homocysteinyl tripeptide (124) was incubated with IPNS the products were the epimeric y-lactams (129). Their formation was rationalized as being via (126) and (128) thus providing evidence for p-lactam formation occurring first in the normal IPNS reactions2 (cf ref. 4,p. 198). When the fluorinated analogue (125) was similarly incubated the product of the enzymic reaction was (1 30) ;it incorporated > 90 % of one l80 atom from 180,.s3 The fluorines clearly bias the reaction in a new direction but again a cyclic intermediate i.e.(127) is deduced to be involved. The fluorines presumably stabilize (127) against collapse to an iminium ion [as (128)] and oxidation of the remaining 4-H by the attached iron-oxo species occurs with ring opening to the thiocarboxylate (130). Further evidence is provided for initial p-lactam ring formation in the normal reaction with ACV (121). Incubation of the ACV analogue (1 3 1) with IPNS leads to six penam/cepham products three arising by a desaturase pathway ( -4H) and three by a mono-oxygenase pathway ( -2H +0). Interestingly and usefully the reaction course is divertable through a primary isotope effect by the presence of a deuterium label in the allylglycine part of (131)84 (cf ref.4 p. 196). This has been exploited in a set of incubations of nine deuteriated versions of (1 3 1) with IPNS and product analysiss5 (these papers include full details of the earlier preliminary com- munications4). The results allowed a cogent set of possible mechanisms to be advanced for the two pathways. A reasonable mechanism for the formation of IPN (122) from ACV (121) is shown in Scheme 10 in which the diradical (1 38) is an intermediate. This insertion-homolysis mechanism is supported by results obtained with (132)-(134) on in-cubation with IPNS.s6 The stereochemistry of the C-S bond formed here would be dictated by competition between the rate of coupling and the rate of bond rotation in (138) (for IPN itself no bond rotation is observed).The radical nature of this bond formation was explored with the ACV analogue (132) which gave (135) as the product" (cf ref. 3 p. 122). It is known from the results of studies with deuterium- labelled substrates that rearrangement of cyclopropylcarbinyl radicals is reversible and at room temperature the label is fully scrambled (Scheme 1 1).88 Following this the compounds (1 33) and (134) have been used as 'radical-clock' substrates for IPNS.86 The products were respectively the single regioisomers (136) and (137) i.e. no scrambling of the type shown in Scheme 11. More sensitively (139) was then also examined (sensitivity is associated with seeing simple bond rotation in any radical formed as indeed was observed here).The product was (140) in which the deuteron at C-5 was present as a 1 :1 mixture of epimers i.e. randomization at the site of single deuterium labelling. These results lead to the alternative mechanisms shown in Scheme 12 (my version cf original in ref. 86) in which the formation of the P-lactam-bound iron oxene (141) is assumed. The alternative mechanisms involve either a cyclopropyl-ene reaction (path a) or a C-H insertion (path b) followed by a concerted [1,3]shift in which the pro-S cyclopropane C-C bond is opened stereospecifically. Equilibration of (142) with diradical (143) yields product (140) in which labelling at C-5 is NATURAL PRODUCT REPORTS 1993-R.B. HERBERT randomized. Since no randomization of the type illustrated in Scheme 11 is seen free radical character is only manifested at a stage after cyclopropane ring cleavage. These results then support the mechanism for IPN formation shown in Scheme 10. The tripeptides (144)-( 146) have been incubated with IPNS.89 Penam products were obtained and in the case of (146) cepham products also. A rationale for the stereochemistries observed for the penams obtained here and for other mono- substituted penams was proposed. Enz C02H -The biosynthesis of cephalosporins e.g. cephalosporin C (1SO) begins with the isomerization of isopenicillin N (1 22) to penicillin N (147). Ring expansion follows to give deacetoxy- cephalosporin C (DAOC) (148) and then a hydroxylation step which affords deacetylcephalosporin C (DAC) (149).* In- cubation of penicillin N (122) with partially purified DAOC/DAC synthase from C.acremonium has been found to give in addition to DAOC and DAC a third p-lactam (151) which is a shunt metabolite (cf ref. 3 p. 123; ref. 4 p. 199). Previously available in preliminary forrn,'O the results are now available in full.91 With the aim of understanding the mechanism of hy- droxylation whereby DAOC (148) is converted into DAC (149) in vivo the three cyclopropyl cephalosporin analogues (1 52)-( 154) have been examined as substrates for DAOC/DAC synthase from C. ~cremonium.~~ Whilst the first two compounds failed to yield products (154) afforded the alcohol (1 55).Labelling of the hydroxy group by 1802 was to an extent similar to that observed for the transformation of (148) into (149). The role of sulfur in the hydroxylation step of cephalosporin biosynthesis i.e. (148) to (149) has been probed with the carbocyclic analogue of (148) namely (1 56).93 Incubation of this compound with recombinant DAOC/DAC synthase afforded (1 57). An experiment with 1802 resulted in labelling of Enz Enz Scheme 12 AANzs* H H2:m'D>Me CO H Me o HFR0 C02H k02H (147) (148) R=H DAO (151) (149) R=OH DAC (150) R = OAc Cephalosporin C ~ H H H D-CX-AAN~~ H 0 0 0 C02H C02H (153) (154) (155) H H D-a-AAN 0 -D-a-MNU&OH 0 C02H C02H (1 56) (1 57) 41 NPR 10 588 NHQ Adenine H6 'OH Hd 'OH Hd 'OH (158) R= H (159) R= P (160) R =H (161) R=P the new hydroxy group in (157); the less than stoichiometric incorporation of label (ca.70 YO)has been observed both in the formation of (149) and of (151). A significant increase in K for the unnatural substrate (1 56) compared to (148) was observed i.e. reduced binding which it was thought might be due to conformational changes caused by the change of S for CH in the substrate. A much less marked change in V,, indicated that sulfur is not directly involved in the hydroxylation reaction. In a notable paper the 'in vitro' synthesis of forty seven different penicillins using 6-aminopenicillanic acid (6-APA) and different side-chain precursors has been described ; the conversions were effected with a coupled system of phenyl-acetyl-CoA ligase (from Pseudomonas putida) and 6-APA acyltransferase (from Penicillium chrysogenum) in the presence of ATP CoA Mg2+ and dithiothreitol at pH 8.94 The expression of the penDE gene of P.chrysogenum which encodes for isopenicillin N acyltransferase in C. acrernonium transformants has been reported to afford ben~ylpenicillin.~~ Molecular characterization and functional analysis in A. nidulans of the 5'-region of the IPNS gene from P. chrysogenum has been recorded.96 Regulatory effect regions concerned with nitrogen metabolism have been tentatively identified. Lysine 6-amino-transferase initiates p-lactam biosynthesis in Actinomycetes.Results of experiments with S. clavuligerus indicate that the gene encoding the transferase is located within the cluster of p-lactam biosynthetic genes.97 The cloning and characterization of the IPNS gene pcbC in Streptomyces griseus and the expression of the cephamycin pathway in S. clavuligerus as host has been The cephamycin biosynthetic genes pcbAB (encoding a large multidomain peptide synthetase) and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in A. nidulans and P. chrysogen~m.~~ Different intracellular locations of the enzymes of the penicillin biosynthetic pathway in P. chrysogenum have been identifiedlOO and this compartmentation has also been observed in a study in this organism with [14C]valine.101 6 Miscellaneous Metabolites 6.1 Nucleoside Antibiotics The carbocyclic ring of aristeromycin (1 62) is biosynthesized in Streptomyces citricolor from glucose by way of (158) or its phosphate (159) (cf.ref. 5 p. 526). It has now been shown by isotope trapping experiments that the amine (160) or its phosphate (161) is to be included in the biosynthetic NATURAL PRODUCT REPORTS 1993 pathway;lo2 the isolated (160) showed incorporation of label in the manner observed earlier for aristeromycin (162) with [3-3H l-'4C]-~-glucose (no loss of tritium) and (6RS)-[6-3H 6-14C]-~-ghcose (loss of approximately half the tritium). Although it has been deduced from the results of experiments with cell-free preparations of Streptomyces incarnatus that sinefungin (163) is constructed in vivo by simple combination of ATP and arginine (cf.ref. 5 p. 527) further experiments by others indicate that this is not Results of experiments with doubly labelled ATP and adenosine using cell-free preparations of Streptomyces griseolus indicate that significant degradation occurs before incorporation into sinefungin (very low incorporations were observed anyway). In support of this a 20-fold reduction in the incorporation of both adenosine and ATP was noted if 8-amino-2'-nordeoxyguanosine,which is a potent inhibitor of purine nucleoside phosphorylase was present also. On the other hand the ribose ring of adenosine was shown to be incorporated intact into (163). No loss of tritium label from C-5' of the precursor was seen which excludes the intermediacy in sinefungin biosynthesis of A9 145C (I 64) another nucleoside antibiotic.[l-14C]Ribose arginine ATP and pyridoxal phosphate when incubated together in the cell-free extract yielded sinefungin with an incorporation of radioactivity (1.1 %) far higher than in any other experiment. [8-3H]Adenine when substituted for ATP in the above gave a satisfactory incorporation (0.07 YO)of radioactivity indicating that adenine itself can be used to make sinefungin in this system. Incubation of arginine ribose and pyridoxal phosphate with the cell-free system gave a putative intermediate containing both amino acid and diol functionality. This putative in- termediate appeared to be converted into sinefungin when incubated with adenine and the cell-free extract.It certainly seems from the combined evidence that in sinefungin biosynthesis the amino acid reacts with ribose first and the product then reacts with adenine. Blasticidin S (169) is an antifungal antibiotic produced by Streptomyces griseochromogenes. It is biosynthesized from cytosine (165) D-glucose L-a-arginine (see ref. 4 p. 201 for the associated mutase reaction) and the methyl group of meth- ionine. Enzyme inhibitors have been used recently with the aim of obtaining the accumulation of intermediates in blasticidin S biosynthesis.lo4 With three compounds (a transaminase in- hibitor an inhibitor of arginine biosynthesis and a methyl- transferase inhibitor) the accumulation of two metabolites (166) and (167) was observed which was at the expense to varying degrees of the production of (169).A cell-free extract of S. griseochromogenes was obtained which catalysed the condensation of cytosine plus exclusively UDP glucuronic acid and the product was (166); this product was then shown to be a specific and intact precursor for (169) in S. griseochrornogenes cultures. The identification of a specific enzyme which catalyses the synthesis of (166) is notable. A cell-free preparation has been obtained which converts the naturally occurring (168) into blasticidin S (1 69) in the presence of S-adenosylmethionine (SAM) ; the specific methylation in the P-arginine part of (1 69) was established in an experiment with [14CH3]SAM.105 Evidence is thus provided that N-methylation is the terminal step in the biosynthesis of blasticidin S.6.2 Unusual Amino Acid Derivatives The coronamic acid (1 73) part of coronatine (1 70) is known to be derived in Pseudomonas syringae from isoleucine/ alloisoleucine (for earlier work see ref. 2 p. 537). It has now been establishedlo6 that L-alloisoleucine (1 72) is a better precursor for (173) than L-isoleucine (1 71) (both are specifically incorporated; [1-13C] labelled precursor yielded metabolite labelled on C-1'). Further experiments were carried out to probe the mechanism NATURAL PRODUCT REPORTS 1993-R. B. HERBERT (168) R= H SAM K (169) R = Me oxidative ring closure discussed in Section 5 seems likely then.3-Nitropropanoic acid (175) is known to arise from L-aspartic acid (174) in Penicillium atrovenetum (cf. ref. 4 p. 206). This acid (175) is a constituent part of one of two defensive compounds (176) and (177) found in Chrysomelid beetles and HO a similar origin from (1 74) is apparent as it is for A3-isoxazolin- OH 5-one moieties of (176) and (177) ~-[U-l~C]aspartic acid was H022C/YC02H H02C-N02 H NH2 (175) (174) (179) (176) R =H (177) R = -EmNo2 0 of cyclopropane ring formation. The C-6 methyl group of (1 72) was shown to provide C-6' of (170) and in the process a single proton was lost (13C,2H labelling with isotope shift in 13C NMR). The C-3 proton in (172) was retained into (170) so a C-3 methylene intermediate in ring formation is excluded.6- Hydroxylated isoleucine and alloisoleucine failed to label (1 70) so a hydroxylation mechanism is also excluded. The sort of found to label in adult Chrysomela tremulae not only the nitropropionate moiety of (177) but also the A3-isoxazolin-5- one moieties of both.lo7 /3-N-Oxalyl-a,P-diaminopropionic acid (ODAP) (1 80) is a neurotoxin produced by Lathyrus species. It can be formed by acylation of (179) with oxalyl CoA using enzymes from Lathyrus sativus. But (179) was not detected in plant species producing ODAP. The alanine derivative BIA (178) has been found to act as a precursor for ODAP (only) in young seedlings of L. sativus.lo8 Incongruities in the deduced biosynthesis of ODAP have been removed in experiments with callus tissue of L.sativ~s.~~~ The incorporation of radioactivity from [l4C]B1A (178) into (180) was found to be reduced in the presence of (179) and this reduction was dependent on the amount added. The production of ODAP was stimulated if oxalate was also added. The pathway follows clearly as (178) +(179) -+ ODAP (180) with (179) being a short-lived intermediate. Glycerinopyrin (182) is a simple but quite unusual naturally occurring pyrrole which is elaborated by Streptomyces violaceus. The pyrrole moiety of the metabolite turns out to have an unprecedented origin in leucine (1 81):DL-[1-13C]leucine was efficiently incorporated with labelling of C- 1' of (1 82).llo The origin of the remainder of the side chain was established to be in glycerol.Alternative possible origins for the pyrrole ring of (182) were excluded by the results of experiments with 13C- labelled acetate alanine ( = pyruvate) and methionine.'1° NATURAL PRODUCT REPORTS 1993 0 [MeIMet hionine A 02 U (183) R = CH3 (184) R = H Scheme 13 0 H2N $C02H H H H2N 2C02H I HF2 H2N CO,H Scheme 14 6.3 Citreamicins The citreamicins e.g. alpha (183) are a group of xanthone- based antibiotics produced by Micrornonospora citrea. Ex- tensive feeding experiments establishedll' the labelling pattern shown in Scheme 13. Clearly severe rearrangement centring on ring F is involved in formation of the xanthone part of (183). Also the origins of the oxygen atoms were not always as predicted and the pathway to the isobutyryl residue which forms part of ring A could not be identified.Contrary to expectation the deduced biosynthesis had turned out to be quite different to that of the superficially similar simaomycin The methylation inhibitors sinefungin and aminopterin have been found to bias citreamicin biosynthesis towards the citreamicin (184); other known methylation inhibitors were without effect . l3 6.4 Streptothricin F Streptothricin F (185) is biosynthesized in part from a molecule of arginine (186) in Streptomyces L-1689-23 (for the most recent work see ref. 4 p. 201). Earlier work had shown114 that [2-,H]- [3,3-,H,]- and [2,3,3-2H,]-arginine were incorporated in each case into (185) with loss of label. Recent published work has been concerned with confirmation of these negative results and further study of the transformation of (186) into (185).l15 ~~-[2,3,3,5,5-*H,]Arginine [as (1 86)] was incorporated into streptothricin F (185) with retention of label equally in both diastereotopic positions at C-5 in (185); the loss of label from C-2 and C-3 as seen with the other precursors was confirmed.These results are consistent with the metabolism of arginine involving hydroxylation at C-3 to give (187) then oxidation ring closure and further reactions (Scheme 14). However when tested as precursors neither DL-threo-P-hydroxyarginine[as (I 87)] nor its erythro-isomer were incorporated into (I 85) nor could they be trapped from the culture medium after feeding radioactive arginine.These are puzzling findings without present resolution as in mechanistic terms are the observed losses of deuterium from C-2 and maybe also from C-3 of labelled arginine. The DL-[~,~-'H,]- and ~-[4R-~H]-arginines ~-[4,4-~H,]-gave (185) with retention of deuterium. On the other hand L-[~S-'Hlarginine gave (185) devoid of label. Thus hydroxylation at C-4 of arginine (186) proceeds with orthodox retention of configuration en route to streptothricin F.'I5 6.5 Tabtoxin The cloning and expression of a region of the chromosome of Pseudornonas syringae BR2 that contains the biosynthetic and resistance genes required for the production of the p-lactam tabtoxin (188) has been studied116 (for earlier biosynthetic work see ref.2 p. 536). 7 References 1 R. B. Herbert Nut. Prod. Rep. 1987 4 423. 2 R. B. Herbert Nut. Prod. Rep. 1988 5 523. 3 R. B. Herbert Nut. Prod. Rep. 1990 7 105. 4 R. B. Herbert Nut. Prod. Rep. 1991 8 185. 5 R. B. Herbert Nut. Prod. Rep. 1992 9 507. 6 R. B. Herbert in 'Rodd's Chemistry of Carbon Compounds' ed. S. Coffey Elsevier Amsterdam 1980 2nd edn. Vol. IV Part L p. 291. 7 R. B. Herbert in 'Rodd's Chemistry of Carbon Compounds' ed. M. F. Ansell Elsevier Amsterdam 1988 2nd edn. Vol. IV Part L Supplement p. 155. 8 R. B. Herbert 'The Biosynthesis of Secondary Metabolites' 2nd edn. Chapman and Hall London 1989. 9 Tetrahedron 1991 47 pp. 5919-6078. 10 R. J. Robins A. J. Parr E. G. Bent and M. J. C. Rhodes Planta 1991 183 185. 11 R.J. Robins A. J. Parr and N. J. Walton Planta 1991 183 196. 12 D. Burtin J. Martin-Tanguy and D. Tepfer Plant Physiol. 1991 95 461. 13 R. J. Robins E. G. Bent and M. J. C. Rhodes Planta 1991 185 385. 14 T. Endo N. Hamaguchi T. Eriksson and Y. Yamada Planta 1991 183 505. 15 M. M. Couladis J. B. Friesen M. E. Landgrebe and E. Leete Phytochemistry 1991 30 801. 16 A. J. Parr N. J. Walton S. Bensalem P. H. McCabe and W. Routledge Phytochemistry 1991 30 2607. NATURAL PRODUCT REPORTS 1993-R. B. HERBERT 17 T. Hashimoto A. Hayashi Y. Amano J. Kohno H. Iwanari S. Usuda and Y. Yamada J. Biol. Chem. 1991 266,4648. 18 E. Leete J. A. Bjorklund M. M. Couladis and S. H. Kim J. Am. Chem. SOC. 1991 113 9286. 19 T. 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Bacteriol. 1991 173 4124.

ISSN:0265-0568    

DOI:10.1039/NP9931000575

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

Biosynthesis of Fatty Acid and Polyketide Metabolites D. O'Hagan Department of Chemistry University of Durham Science Laboratories South Road Durham DH I 3LE Reviewing the literature published between mid-1 991 and mid-I 992 (Continuing the coverage of the literature in Natural Product Reports 1992 Vol. 9 p. 447) 1 Introduction 2 Fatty Acids and Related Metabolites 2.1 From Plants 2.2 From Fungi 2.3 From Insects 3 Prostaglandins and Related Metabolites 3.1 From Plants 3.2 From Mammals 4 Polyketides from Bacteria 4.1 Aromatic Polyketides 4.1.1 Genetic Studies 4.2 Macrolides and Macrolactams 4.2.1 Macrolides 4.2.2 Macrolide Genetic Studies 4.2.3 Macrolactams 4.3 Polyethers and Related Metabolites 5 Polyketides from Fungi 6 Marine Polyketides 7 References 1 Introduction This review follows on from the previous one1 in this series and covers the literature until mid-1992.The last review' brought together a number of very exciting developments in the polyketide biosynthesis arena and on reflection the period between 1988 and 1991 is beginning to emerge as a 'golden era ' in polyketide biosynthesis. In particular a key highlight was the analysis of the erythromycin PKS genes2," from Saccharopolyspora erythraea which revealed a highly ordered Type I PKS and suggested that these higher polyketides are assembled in a modular manner. This is discussed in Section 4.2.2. This revelation came along at the time when partially assembled intermediates were being successfully introduced into the bacterial (Actinomycete) polyketides such as tylosin4 and erythr~mycin,~ again sup- porting a processive modular assembly of the polyketides.This is summarized in Section 4.2.1. The period covered by this review is very much one of consolidation. The introduction of partially assembled fragments has been demonstrated now in fungal systems and the studies of Sta~nton~-~ and Vederas'O,l' on aspyrone and dehydrocurvularin respectively demonstrate how powerful this approach can be in identifying the nature of intermediates in polyketide assembly. The ongoing genetic analysis of bacterial aromatic polyketide synthases particularly that focused on the actinorhodin PKS genes and the exploitation of these genes as hybridization probes suggests that these systems are assembled by Type 11 PKSs.The fundamental organization and structure of these systems is being unravelled. This is discussed in section 4.1.1. The next major goal must now be the isolation and characterization of bacterial enzymatic systems. A noticeable aspect of compiling this review is the increasing number of new biologically active polyketides reported from Streptomyces species particularly in The Journal qf' Antibiotics. Only a selection of these have been highlighted and then on the basis of their biosynthetic interest or structural relationship to other polyketide systems. This is 593 clearly a consequence of improving sophistication in screening technologies and in particular assay development.2 Fatty Acids and Related Metabolites 2.1 From Plants The discovery of a short chain condensing enzyme (Condensing enzyme 111) mediating the first condensation between acetyl CoA (not acetyl ACP) and malonyl ACP in plant (and bacterial) fatty acid assembly is now well established." This enzyme is differentially inhibited with respect to condensing enzymes I and 11. For example cerulenin does not inhibit condensing enzyme 111 but does inhibit condensing enzyme 11. Thiolactomycin an antibiotic from a Nocardia sp. inhibits both. A short review13 has appeared summarizing these studies and more generally discusses the inhibition of the early steps of fatty acid biosynthesis in plants by different xenobiotics.For example acetyl CoA synthase is specifically inhibited by allicin (l) an antibiotic from garlic. Allicin (1) has also been shown to inhibit this enzyme from both yeasts and bovine heart." The fatty acid synthase (FAS) enzyme enoyl reductase is the only enzyme of FASs which displays a variation in its stereochemical course in moving from one FAS to another.15 Stereochemical studies on this enzyme continue to attract interest. An investigation16 probing the stereochemical course of the enzyme from dyers thistle Carthamus tinctorius and also from the flour beetle (see also 2.3) Tribolium confusumon demonstrated remarkably that the enoyl reductases from these sources have the same stereochemical course.This was deduced to be an anti-2Re,3Re addition of the two hydrogen atoms a combination that was without precedent (Scheme 1). Now all of the possible stereochemical combinations of hydrogen addition are represented in various systems. The information was gained by incubating (E)-12-phenyl-[2-2H,]-dodec-2-enoic acid (2) as a fatty acid surrogate with the plant and insect systems. The enoyl reductases accept this substrate and reduce it to the corresponding saturated fatty acid (3). Both biological RvsEnz enoyl reductase (anti-2Re,3Re) ~ D (3) C02(-D) 4 decarboxylase H R+ D (4) Scheme 1 NATURAL PRODUCT REPORTS 1993 0 OH -3-(c H 2) 16-CO2H oleic acid (5) desat urase 1 decarboxylase -(CH2)144 -* 1 (CH2)16-C02H OH (7) Scheme 2 systems possess a decarboxylase activity that generates terminal olefinic hydrocarbons and the stereochemical course of this enzyme has been thoroughly investigated.l’.l* It proceeds with an anti-elimination of the carboxyl group and the 3-pro-S hydrogen atom.Subsequent olefin analyses using a com-bination of GC and FTIR demonstrated that in both experiments the product was (Z)-l l-phenyl-[ l-2H]-undec- 1-ene (4).The location of the label on the terminal carbon and the Z-0 fatty acyl geometry can only be rationalized if the enoyl reductase (9) m= odd 15 to 21 n=even14to20 presented hydrogen in an anti-2Re-3Re mode. The cellular location of the enoyl reductase in these in vivo studies is unclear x= 15,17,19 at present (cytosolic mitochondrial etc.?).A series of high molecular weight ether lipids (botryococcoid ethers) have been isolated from the green microalga Botryo-coccus braunii and biosynthetic experiments carried OU~.~~,~~ The major compound (7) which accounts for 26% of the dry biomass contains a C, unsaturated hydrocarbon and ‘a C, alcohol connected by an ether linkage. The efficient incor- poration of [14C]-oleic acid (5) into (7)’ and in a further study the successful incorporation of 9,l O-epoxynonacos-28-ene (8) into other lipid constituents led the authors to propose a biosynthetic pathway to (7) involving differential epoxidation (10) R1=C02H R2=H of triene (6) to generate two distinct epoxides which can then (13) R’ =H R2=OH couple as outlined in Scheme 2.Other related lipids in lesser concentrations were also identified and their structures and biosynthetic relationships to (7) are discussed. Of interest is the ability of B. braunii to generate an insoluble biopolymer (PRB) or ‘macromolecular lipid ’ which forms the chemically resistant part of the outer walls of the algae.20 The polymer appears to have ether linkages and a degree of unsaturation and oleic acid has previously been identified as a biosynthetic precursor to PRB. The authors raise the speculationz1 that the identified epoxides (9) or related species would fulfil the criteria for monomers of PRB. (11) R~=CO~H,R~= H (14) R’=H R2=OH Some insights have been gained into the biosynthetic origin of the polyunsaturated anacardic acids (lo)-( 12) and cardanols (13)-(15) from cashew nuts.22 A time dependent study on the 1 levels of unsaturated anacardic acids and cardanols over the eight week development time of the cashew nut revealed that the relative proportion of monoene which was high in the first week decreased during the following two weeks and that of the diene and triene increased over this time.By implication it was deduced that desaturase enzymes operated on the monene (12) R1=C02H R2=H to generate the di- and trienes as illustrated in Scheme 3. The (15) R’=H R2=OH alternative pathway where di- and triunsaturated fatty acids undergo elongation and aromatization was judged less likely by Scheme 3 NATURAL PRODUCT REPORTS 1993-D.O’HAGAN HH X (21)X= H OH (25)X= 0 (26)X = H OAC OH OR OH (24)R = H (27)R = AC 0 Lo-ASCoA OH OH R (22)R =CI (23)R =OH characteristic odour and arise as a consequence of fungal decomposition of the leaf litter. New polyacetylenic compounds continue to emerge from plant sources. For example a series designated the ginsenoynes A-H (2 1)-(28) were identified25,26 from roots of Panax ginseng one of the most important oriental medicinal plants. The Asteraceae (sun flower) family of plants is known to accumulate polyacetylenes which have a diverse range of interesting biological activities. Common to many of these plants are polyacetylene thiophene and 1,2- dithiin derivatives. Recent biosynthetic experiments2’ 28 on hairy root cultures have shed some light on their mode of assembly in Asteraceae.[1-13C]- [2-13C]- and [1 ,2-13C2]-acetates were successfully incorporated2’ into the two bithiophenes 5-(3- flm-v buten-1-ynyl)-2,2’-bithiophene (BBT) (29) and 5-(4-acetoxy- 1-H3C (=) 1 H3C-Z n==a-H3C++47 s-s H3C= 0 -’?- s-s (31) Scheme 4 the authors. Interestingly saturated structural analogues of the cardanol series which have been termed adipostatins A (16) and B (17) have been isolated from the bacterium Streptomyces cyaneus.23 They are powerful inhibitors of glycerol-3-phosphate dehydrogenase (Ki= 4.1 mM and 4.5 mM). Adipostatin A (16) also occurs in cashew nuts and other plants. The macrocyclic lactones I 12-decanolide (18) 1,14-tetra- decanolide (19) and 1,16-hexadecanolide (20) have been detected in the leaf litter from Pinus radiata forest They are constituents of the volatiles that give pine forests their OH 0 butynyl)-2,2’-bithiophene (BBTOAc) (30) in root cultures of Tagetes patula.The results demonstrated an essentially aceto- genic origin for the carbon chain. The peripheral carbons C-1 and C-12 are isolated and not bonded to their original acetate partners and the authors suggest that (29) and (30) are derived from at least a 14-carbon precursor. [1-13C]- [2-I3C]- and [1,2-13C2]-acetates were also incorporated into the C13 1,2- dithiin thiarubrine A (31)28 in root cultures of Ambrosi artemisiifolia. The results were consistent with the origin of thiarubrine A (31) from a C, acetogenic precursor.Six intact acetate units were apparent from I3C-l3C couplings within the enriched sample of (30) after the [1 ,2-13C,]-acetate experiment and C-1 of (31) was enriched but not significantly coupled. In addition C- 1 was enriched from [2-13C]-acetate. The bio- synthetic pathway in Scheme 4 was proposed in the light of these results and is consistent with an earlier peculation.^^ It is interesting to note that the acetylenic 1,5-diyne aglycone of the Streptomyces sp. antibiotic neocarzin~statin~~ appears also to derive from oleic acid via a C, acetylenic precursor and it has been proposed that the 1,5-diyne aglycones of the calicheamicins and esperimicins are derived from a related C, precursor. No evidence was presented for a role for oleic acid in these root metabolites but on analysis oleic acid would appear to be implicated.Hairy root or transformed root cultures of plants are emerging as powerful systems in which to carry out biosynthetic studies. For some recent reviews see references 3 1 and 32. NATURAL PRODUCT REPORTS 1993 OH OH (32) R' = H R2 = H H (33) R' =OH R2= H H (34) R' = H R2= 0 (35) R' = H R2= H OH (36) R'=OH R2=0 (37) R' =OH R2= H OH HO HO OH OH (38) R= H (39) R =OH (40) R= H (41) R=OH 0 0 t>H OH (42) (43) OR OH ,I C02H 2 OH b~ NHR~ (44) R = H R' = C(NH)NH2 (45) R = R'=H (46) R=Ac R' = H (47) R=H R'=Ac Six extremely cytotoxic compounds (32)-(37) have been isolated, from seeds of Annona muricata the parent compound 2.3 From Insects being solamin (32).This brings to eighteen the number of The stereochemical course of the enoyl reductase from the flour monotetrahydrofuranyl-y-lactone acetogenins which have been beetle Tribolium confus~mon'~has already been discussed in identified from Annonaceae. section 2.1 and Scheme 1. All-Palmitoyl CoA desaturase which converts palmitoyl-CoA (51) to (a-1 1-hexadecenoyl CoA (52) as shown in Scheme 5 is a key enzyme involved in moth 2.2 From Fungi pheromone biosynthesis. It has been characteri~ed,~ from The structures of four hydroxylated unsaturated C, fatty pheromonal glands of the adult females of Spodoptera littoralis. acids psi factors Ba,B, Ca X, (38t(41) which induce The enzyme had a preference for NADH over NADPH and premature sexual sporulation in the ascomycetous fungus activity was greatest for C, (palmitic) (100 YO),followed by C, Aspergillus nidulans have been reported after verification by (myristic) (50%) and then C, (stearic) (18%) acids.This This follows on from the previous identification of study extends the range of desaturase enzymes that have been ~ynthesis.~~ blactones (42) and (43). investigated from insect species in recent year~.~O.~l Highly modified fatty acids are emerging as potential A series of paper^^"^^ reports the characterization and therapeutic agents from screening programmes. The sphingo- biosynthesis of long chain (LMA) and very long chain (VLMA) fungins A-D (44t(47)35 display antifungal activity and have methyl branched alcohols in the developing pupae of Lepi- been isolated from Aspergillus fumigatus.doptera insect species. Typically these compounds contain The number of metabolites isolated from plants [see (32t(37)] one to three branching methyl groups and a terminal primary and fungi with an a$,-unsaturated-y-alkyl-butenolide moiety hydroxyl functionality. Lipids of the male pupae of the tobacco is increasing. The y-butenolide harzianolide (48) was isolated 36 hornworm Manduca sexta contain isobutyrate n-butyrate iso- from the fungus Trichoderma harziunum and joins a small valerate propionate and n-pentanoate esters of C,,-C, LMAs. group of fungal butenolides such as phytotoxin seiridin (49) Propionate esters of C, to > C, VLMAs were also identified.from Seiridium cardinale3' and the butenolide (50) from The variation in levels of these compounds throughout pupal The biosynthetic origins of these ring development is discussed. A biosynthetic study involving [1-Hypoxylon ~erpens.~~ systems are unknown. 13C]-propionate set out to probe the origin and relationship NATURAL PRODUCT REPORTS 1993-D. O'HAGAN 0 A" desaturase i SCoA (52) Scheme 5 OH 0 9-do- (53) I OH (54) Scheme 6 OH (55) lipoxygenase HO? OOH -I C02H C02H ? I -YCHO Scheme 7 between the LMAs and VLMAs. Analysis was carried out after feeding experiments by either converting the primary hydroxyl to chloride or reduction with LiAlD to generate a labelled hydrocarbon.These derivatized species proved more amenable to GC-MS analysis. Both the LMAs and the VLMAs in- corporate [1-13C]-propionate into methylene groups adjacent to the branching methyl group consistent with an origin from methylmalonyl CoA. Scheme 6 illustrates this for the LMA 6,lO-dimethyltetracosan-l-ol (53) and the VLMA 22,26-dimethyloctatriacontan- l-ol (54). The labelled carbons were also located between the methyl group and the terminal hydroxyl consistent with the idea that the hydroxyl end represents the chain elongation terminus. Significantly GC-MS analysis revealed that the methyl groups of the LMAs are located much closer to the hydroxyl terminus than previously deduced and a structural revision of the LMAs is suggested.For example 15,l g-dimethyltetracosan- 1-01 is now 6'10-dimethyltetracosan-1-01 (53) (the hydroxyl group has been moved to the opposite end of the molecule). This has biosynthetic implications and clearly methylmalonyl CoA is involved much later in chain assembly of the LMAs than the VLMAs where it is introduced in the early stages. Both structural classes appear to utilize acetate as a starter unit. The same research group has identified VLMAs from pupae of the Southern army worm Spodoptera eridania. The major com- ponents contain between 38 and 42 carbons in the alkyl chain and have an additional one to three branching methyl groups with 22,26- and 22,34-dimethyloctatricontanol,24,28-dimethyl-tetracontanol and 26,30-dimethyldotetracontanol(55) com- prising over 50% of the lipid and lipid esters.Radiolabelled acetate and propionate were efficiently incorporated into the lipids. Questions are raised as to the role of the VLMAs and their catabolic products in the new adults although no conclusions are drawn. It would clearly be interesting to investigate further the fatty acid synthases responsible for their production. 3 Prostaglandins and Related Metabolites 3.1 From Plants Linolenic acid (56) is acted upon by various lipoxygenases in various plants to give either the (13s)-hydroperoxide (57) (soya bean flax cotton cucumber) or the (9s)-hydroperoxide (58) (potato tomato rice embryo) and in some systems (gooseberry bakers' yeast) both.45 The resultant hydroperoxides are in general subsequently modified to short chain unsaturated aldehydes which have distinctive flavours and odours.Some of the known aldehydes which derive from linolenic acid (56) are shown in Scheme 7. Early studies in potato had identified NATURAL PRODUCT REPORTS 1993 C02H D DD D DDD C02H D D D D Scheme 8 CO2H a218 C02H D DD D D D D (61) (67) C02H C02H (65) (66) Scheme 9 colneleic (59)46 and colnelenic (60)47 acids as metabolites of the (9s)-hydroperoxide of linoleic and linolenic (56) acids re-spectively. It had previously been that the ether oxygen of these metabolites did not derive from the hydro- peroxide oxygens however the I80 used in the study was of low abundance (10%).Repetition of this experiment in potato,45 using enzymatically prepared [9-lsO2]-(9S)-hydro- peroxide (58) with 80 atom% 1802 has however established the origin of the ether oxygen of colneleic acid (59) to be from one of the hydroperoxide oxygens. Further details of the overall transformation were gleaned from the incubation of a tetradeuterated linoleic acid (61).45 The labelled linoleic acid was converted enzymatically to the corresponding (9s)-hydroperoxide (62) with retention of four deuterium atoms and then incubated with the potato homo- genate to yield colneleic acid. Subsequent analysis revealed that all of the deuteriums were retained and located as shown in structure (64). From these observations a mechanism (Scheme 8) was proposed for the generation of colneleic acid from the hydroperoxide.A key feature involved an electropositive oxygen atom which is attacked by a double bond to generate an intermediate epoxycarbonium ion (63). Elimination of the 8-pro-R hydrogen atom49 followed by C-C bond cleavage results in colneleic acid. An analogous process presumably follows for colnelenic acid (60) from linolenic acid (56). This tetra-deuterated linoleic acid (61) was also used to probe mechanistic details of the conversion of linoleic acid and linolenic acids into a-and y-ketols e.g. (65) and (66) in flax It was known that in this system the (13s)-hydroperoxide (67) is generated initially. Resultant analyses of ketols (65) and (66) established the location of the deuterium atoms as shown in Scheme 9.Analysis of the current and available data then led the authors to propose a common epoxycarbonium ion (68) leading to a common allene-epoxide intermediate (69) for the biosynthesis of both of the ketols (65) and (66). Linoleic acid (56) is known to be a precursor to 12- NATURAL PRODUCT REPORTS 1993-D. O'HAGAN oxophytodienoic acid (12-oxoPDA) (70) in addition to the hydroxyketols (71) and (72),which are formed by an analogous process to that of Scheme 9.51 12-OxoPDA (70) has similar structural characteristics to the prostaglandins although it is a C, and not a C, compound and has cis rather than trans ring substituents. The generation of 12-oxoPDA by a classic mammalian pathway would require a 12-lipoxygenase activity with loss of one deuterium atom.However this does not appear to happen. The incubation of doubly labelled [14-2H,]-linolenic acid (73) with flax seeds resulted51 in the incorporation of both of the deuterium atoms into C-14 of 12-oxoPDA (70a). This is consistent with a previously proposed mechanism5 involving the conversion of doubly labelled (1 3s)-hydroperoxylinoleic acid (74) to an epoxycarbonium ion (73 which loses the 12- proton to generate a zwitterion (76) and is then followed by cyclization to give 12-oxoPDA (70a) (Scheme lo). The intermediacy of such a zwitterion has precedence in other non- mammalian prostanoid biosyntheses particularly those from the marine molluscs such as preclavulone A and the prosta- glandins of the A and E series whose biosynthesis has been studied in some detai1.53.54 OH C02H 0 (72) (77) 0-OH PGG2 (78) cyclo-oxygenase activity PGH2 synthase 3.2 From Mammals An excellent review55 summarizes recent progress on the biosynthesis of prostaglandins and thromboxanes.A particular feature is the collation of evidence that implicates the mechanistic involvement of a tyrosyl radical in prostaglandin synthase (PGH synthase). PGH synthase is a bifunctional enzyme that utilizes arachidonic acid (77) as a substrate and mediates both the peroxidase and cyclooxygenase activities the first two steps in prostanoid biosynthesis to generate PGG (78) and PGH (79) sequentially. There would appear to be one haem per enzyme molecule which is loosely bound C02H DD C02H DD (74) H C02H DD C02H (704 Scheme 10 OH PGH2 (79) peroxidase activity prostaglandins thromboxanes prostacyclines Scheme 11 NATURAL PRODUCT REPORTS 1993 H/ HOOC ISER5301 Figure 1 Model for the cyclooxygenase and peroxidase active sites of PGH synthase 0 OAc (K = M) orientated by axial and distal histidine co-ordination to the iron.From information obtained from sequence analysis an active-site model for PGH synthase has been proposed which highlights the intimacy of tyrosine-385 the haem and the arachidonic acid binding site. This is shown in Figure 1. In sheep mouse and human PGH synthases tyrosine-385 is a conserved residue and site-directed mutagenesis replacement of tyrosine-385 by a phenylalanine results in a catalytically incompetent enzyme.56 This as well as spectroscopic evidence supports the involvement of a tryosyl radical.Compound I1 (a spectroscopically identified entity) for example has been postulated by comparison with an analogous more firmly established intermediate in the catalytic cycle of cytochrome c peroxida~e.~~ It can be rationalized that both the cyclooxygenase and peroxidase activities of PGH synthase have independent cycles which are linked by compound I1 and PGG as shown in Scheme 11. The tyrosyl radical abstracts the 13-pro-S hydrogen of arachidonic acid (AA) and the AA. radical reacts in sequence with two molecules of molecular oxygen. The resultant peroxide radical then takes back the hydrogen radical from tyrosine to generate PGG and regenerate the tyrosyl radical.The reduction of PGG to PGH primes the oxidation of ferrous haem followed by electron transfer from tyrosine to generate Compound 11. The rhizomes of Zingiber oficinale (ginger) and Alpinia oficinarurn plants have been found to contain a wide range of potent inhibitors of PGH ~ynthase.~~ The active compounds were identified as gingerols e.g. (80)-(82) (IC50between 1-2 ,UM)and diarylhepatanoids the most active compound being the diarylhepatanoid yakuchinone (83) (IC50= 0.5 p~). Struc-ture activity relationships are discussed in detail ; however for each class the presence of a phenol was essential for good inhibition. This clearly suggests that these compounds are inhibiting the catalytic cycle for the regeneration of the tyrosyl radical as discussed above and outlined in Scheme 12.Many of 0 0 OMe (83) AA h compound II Scheme 12 these compounds were also found to be good inhibitors of 5-lipoxygenase the first committed enzyme on the leukotriene biosynthesis pathway. The authors suggest that these activities may be responsible for the efficacy of ginger and related plant extracts in traditional Japanese Kampo medicine. NATURAL PRODUCT REPORTS 1993-D. O’HAGAN 60 1 OH 0 OH (84) Scheme 13 r cosx - I 0 0 0 OH OH 0 OH L (86) (85) Scheme 14 4 Polyketides from Bacteria 4.1 Aromatic Polyketides There are an increasing number of benz[a]anthraquinone antibiotics being reported in the literature.Dehydro-rabelomycin (84) typifies this class and has recently been shown to be labelled by acetate,59 in an unexceptional manner as shown in Scheme 13. Antibiotic PD 116 198 (86) produced by Streptomyces phaeochromogenes WP 3688 has a similar carbon skeleton to (84) but is apparently derived in quite a different manner.59 Incorporation of [2-13C]-acetate labelled PD 116 198 (86) consistent with the expected hypothesis however the labelling pattern from [1,2-13C2]-acetate left C-4 enriched but isolated from an adjacent label. The result was interpreted as shown in Scheme 14 where a linear tetracyclic intermediate OH 0 O H O kH3c@ H3C@ HO OH A B (87) R=R’=H (88) R=A R=B R‘ (85) derived from a decaketide is cleaved between C-lOa and C-11 followed by bond formation of C- 1 1 to C-3.Aquayamycin (87) is the most common aglycone of the angucycline (e.g. urdamycins) antibiotics and it too belongs to the banz[a]anthraquinone class although it is biosynthesized in the classic manner outlined in Scheme 13. The origin of the oxygen atoms of this aglycone have been probed60 during a biosynthetic study on urdamycin A (88) from Streptomyces fradiae. [l-13C 1802]-Acetate labelled those sites shown in Scheme 15 but most significantly the oxygen atom attached to C-4a was derived from this source. A complementary experiment carried out under an atmosphere of 1802 labelled only the oxygen atom attached to C-12b.The intermediacy of an epoxide (89) or more obviously an endoperoxide (90) is discounted therefore and the hydroxyl at C-12b would appear to be introduced by a monooxygenase acting on an intermediate such as (91). NATURAL PRODUCT REPORTS 1993 flH3 e3 \ 0 \ / OCH30 R’ OCH30 (92) R’ =OH R2= H (94) (93) R’ = H R2= OH 0 OCH3 OCH 0 (95) HO OH 0 (98) R =Me (100) R= OH R’ = R2=H (99) R = H (101) R=R’=OH R~=H (102) R = R’ = R2 = OH The structures of a number of new benz[a]anthraquinone antibiotics have been reported recently. For example the hatomarubigins A-D (92)-(95)61 were all identified from Strepto-myces sp. 2238-SVT4 and WS009 A (96) and B (97) were isolated62 from Streptomyces sp.No 89009. The latter two compounds are agonists of the vasoconstrictor endothelin. Naphterpin (98) reported in 1 990,63 is a biosynthetically intriguing Streptomyces antibiotic which is constructed from a polyketide derived naphthaquinone and a C, terpenoid moiety.64 More recently several novel Streptomyces metabolites have been identified which appear to belong to the naphterpin (98) class. 7-Demethylnaphterpin (99) was from Streptomycesprunicolor. Naphterpin (98) was also identified at low levels from this strain however (99) could not be identified from Streptomyces aeriouvifer the original naphterpin pro- ducer. The naphthgeranines A to E (99)-(102)66 from Strepto-myces sp. (Tu 35556) are closely related to naphterpin (98) and indeed naphthgeranine A and 7-demethylnaphterpin (99) are identical compounds.The origin of these antibiotics is unusual in bacteria and is much more characteristic of plant and fungal metabolites. 4.1.I Genetic Studies The analysis and utility of the actinorhodin (103) PKS genes from Streptomyces coelicolor continue to attract considerable interest and have been extremely powerful in revealing the nature and organization of aromatic PKSs of Streptomyces. Several reviews summarizing the details of actinorhodin (103) biosynthesis and the related genetic developments have ap- ~eared,~’-~O in addition to the detailed summary in the previous review of this series.’ The act PKS genes from S. coelicolor have been used as hybridization probes to identify PKS genes in other polyketide producing Streptomyces.Figure 2 illustrates the comparisons that are emerging between PKSs from different producing strains. The polyketide synthase genes are arranged in short clusters of open reading frames (ORFs) each ORF C02H 0 X=OH (96) R = H (97) R =OH coding a discrete protein/activity of the PKS as shown in Figure 2. For the first three of these tetracenomycin (104) actinorhodin (103) and granatacin (105) PKSs the activities have been deduced largely after sequence homologies to proteins with known activities. The remaining three are partially studied systems and the sequence data is not currently available but they clearly contribute to an emerging common structure. These are Type I1 PKSs where several discrete proteins are involved in the assembly of the polyketides rather than the multifunctional proteins of Type I PKSs and FASs.The top three cases in Figure 2 all possess three common proteins coded by ORFs 1-3. ORFs 1 and 2 which correlate to the act I gene appear to be translationally coupled due to overlapping 3‘ stop and 5‘ start codons and are therefore secreted together and in equal amounts. This coupled with their substantial sequence homology to each other and to the Fab B condensing enzyme of E. coli has been taken as evidence that ORF 1 and ORF 2 code a dimeric condensing enzyme. ORF 3 has homologies to Type I1 FAS acyl carrier proteins (ACP) and ORF 4 has similarities to cyclase and dehydratase enzymes and may be a bifunctional cyclase/dehydratase activity.ORF 5 is believed to code a ketoreductase activity and is found in the actinorhodin (103) and granatacin (105) systems. It is interesting and poignant that the tetracenomycin (104) PKS is apparently devoid of such an activity consistent with its biosynthesis from a polyketide such as (104a) where no such reduction is required. From these studies a clear picture of the structure and organization of the aromatic polyketide synthases is beginning to emerge. They are Type I1 PKSs and can be contrasted with the multifunctional Type I PKS which has been deduced in erythromycin biosynthesis (see Scheme 21 vide infra). There appears to be a quite different level of complexity and programming at the enzymatic level required in bacteria for the construction of these largely acetate/malonate metabolites and those that assimilate acetate propionate and butyrate such as the macrolide and polyether antibiotics.This distinction is already clear and the ongoing revelations emerging from the genetic analysis of these systems make exciting viewing. Mutant studies on Streptomyces tanashiensis which produces the benzoisochromanequinone antibiotic kalafungin (106) have identified seven phenotypic mutant classes which have been arranged in the most likely linear sequence7I consistent with the developing hypothesis for the linear arrangement of Type I1 PKS genes outlined in Figure 2. KaZI-II-III-IV-V-VI-VII-kalafungin Large (20-40 kb) chromosomal DNA fragments from S.tanashiensis have been cloned and shown to hybridize to the act1 and act111 DNA fragments.‘* These act fragments are believed to code for polyketide synthase and ketoreductase NATURAL PRODUCT REPORTS 1993-D. O'HAGAN I I 1 I I I I I 0 1 2 3 4 5 6 7kb I?>I1)12>014) S. glaucescens(tet racenomycin) S. coelicolor (actinorhodin) ,act 111 act I act VII act IV S. violaceoruber (granaticin) S. coelicolor whi E S. cinnamonensis S. rimosus (oxytetracycline) Figure 2 CH3 0 0 0 RS 0 H OH 0 " activities respectively of the actinorhodin polyketide synthase and therefore successful hybridization should identify those DNA fragments of S. tanashiensis which possess the kal PKS genes. One successfully hybridized fragment was sub-cloned to confirm this.The sub-clone was able to cosynthesize kalafungin (106) with all of the seven classes of kal mutants that were interrupted in kalafungin biosynthesis. This provides con-vincing evidence that the sub-clone contained the DNA necessary for instructing polyketide biosynthesis. Kalafungin (106) is structurally related to actinorhodin (103) and is possibly an intermediate (or shunt product) on the biosynthetic pathway. The expected homology at the genetic level between the two systems is only partially borne out however and there appear to be fundamental differences. The kal PKS genes only hybridize to four (actI 111 VA and VI) out of eight act genes and do not for example hybridize the actIV and VII genes which operate early in actinorhodin biosynthesis and would be expected to have analogous partners on the kalafungin pathway.The origin of these differences remains to be resolved. When a recombinant plasmid (pKU523). carrying the complete set of kal PKS genes was reintroduced into the wild type strain or kal mutants of S. tanashiensis then a novel metabolite tetrahydrokalafungin (107) was i~olated.'~ The role of tetrahydrokalafungin (107) on the biosynthetic pathway is -M e \/ ow 0 Me0 OMe OH unclear and it could be an intermediate a shunt product or a reduced final product. 4.2 Macrolides and Macrolactams 4.2.1 Macrolides Evidence supporting the processive assembly of the polyketide frameworks contributing the aglycones of the macrolide (and polyether) antibiotics continues.The key issue here is whether the final oxidation level (ketone alcohol double bond or methylene) is introduced by reduction of a fully assembled polyketo intermediate or whether each ketone functionality is appropriately reduced in a processive manner prior to the next malonate condensation. The processive mode of assembly gains support from three lines of evidence I partially constructed but appropriately reduced polyketide fragments have been successfully incorporated into completely assembled (see aglyc~nes~~~below and Section 5); 2 intermediate fragments of macrolide aglycones have been isolated from ty10sin~~ fermentation broths (see below) ;3, and my~inarnicin'~ isolation and analyses of the 6-deoxyerythronolide PKS genes2g3 suggest that the enzymes involved construct the molecule in a modular manner consistent with the processive hypothesis (see Scheme 21).It had previously been shown that NPR 10 NATURAL PRODUCT REPORTS 1993 I / (110) R=OH \ / (111) R=H (108a) Scheme 16 __t OH the doubly labelled reduced polyketide fragment (1 08) introduced as an N-acetylcysteamine derivative could be incorporated into the erythromycin A (110) and B (1 11)5 and nargenicin A (1 12)i6 aglycones as shown in Scheme 16. Clearly direct incorporation of the acyl moiety onto the polyketide synthase in each case is implied and supports the processive assembly where an interception has taken place after the condensation of two propionate units and a ketone reduction.However adventitious oxidation of the alcohol in (108) to a p-keto intermediate (109) would result in an intermediate on a non-processive polyketo pathway and therefore the original conclusions require confirmation. To delineate these two possibilities the same fragment (108a) labelled with both 13C and 2Hat C-3 was fed" to the erythromycin and nargenicin A producing strains as before and in each case the deuterium was retained and regiospecifically incorporated into the antibiotics as shown in Scheme 16. Clearly oxidation of the alcohol in (108a) to the /3-keto intermediate (109) could not have occurred as this would result in deuterium loss. These experiments secure the original conclusions supporting the processive assembly of such systems.The mycinamicins are 16-membered macrolides produced by Micrornonospora griseorubida. Analy~is'~ of the fermentation broth has revealed the presence of partially assembled fragments (113)-( 116) of protomycinolide IV (120) the mycin- amicin aglycone. These fragments can be rationalized as a stepwise series of intermediates in the processive assembly of the aglycone as shown in Scheme 17 where each is prematurely 605 NATURAL PRODUCT REPORTS 1993-D. O'HAGAN )+r $OH ?OH 0'0 carboxylase -OH CoAS CoAS 0 0 i Scheme 18 I released from the polyketide synthase. Also,'8 two novel macrolides (118) and (119) and a fully assembled seco acid (1 17) have been isolated from the culture filtrates of a mutant strain of M.griseorubida and they illustrate the capacity of this strain to oxidize/reduce alcohol functionalities. The authors a biosynthetic pathway to protomycinolide IV as illustrated in Scheme 17 however the intermediates (1 17) (1 18) and (119) cannot be dismissed as metabolic products rather than precursors of protomycinolide IV (120). The structure and stereochemistry of epimycinoic acid (12l) (124) which was isolated from the mutant strain of M. griseorubida was unambiguously secured by X-ray structure analysis of its p-bromophenacyl deri~ative.'~A biosynthetic role for this HoHi/&.H;Ho-OCH3 ..oJ&;3 o (125)/ D y3 intermediate is not clear. The authors raise an interesting speculation that involves coupling of the CoA ester (122) of (1 15) with the CoA ester (123) of a C-6 carboxylated derivative of epimycinoic acid (121) to afford the required structural and stereochemical fragment to complete the construction of protomycinolide IV (120) (Scheme 18).Although contrary to the hypothesis of a processive stepwise assembly for macrolide aglycones more generall~,~.~this convergent proposal is intuitively satisfying due to the economic use of synthesized fragments. However the proposal requires a novel carboxylase activity for activation of C-6 of (121). A further weakness of the proposal is the origin of (116) in Scheme 17 which is obviated as a true intermediate if this novel cyclization mode takes place. Clearly epimycinoic acid (121) could arise from adventitious oxidation/reduction of the alcohol moiety of (1 13),a hypothesis which gains some support in the light of the various oxidation y3 states of the lactones (1 18)and (1 19) and the seco acid (117) all isolated from the same mutant strain.A whole series of novel OCH3 (126) mycinamicins have been is~lated'~. as minor components from M. griseorubida. The most interesting structures from a biosynthetic point of view are mycinamicins XI1 (124) XI11 (125) and XIV (126) which indicate that the polyketide synthase shows a degree of tolerance and will substitute malonyl CoA (acetate subunit) for methylmalonyl CoA (propionate subunit) at various points (arrowed) during the assembly process. 42-2 H3CO OR* R' R2 0 It (127) Et H2N'C'O-HO 0 (128) Me H2N/"o-HO (129) Et HOh HO (130) Me HO-HO 0 (132) Et H H3C0 '%,acetate %,-propionate 2x (134) I NATURAL PRODUCT REPORTS 1993 Concanamycins A-C (1 27)-( 129) are products of Strepto-myces diastatochromogenes.Four new analogues D-G (130)-( 133) have been identified from Streptomyces sp. A1 509.82 The modification of the aglycone at C-8 after incorporation of presumably acetate propionate or butyrate subunits again illustrates how PKSs will often tolerate various subunit incorporations. Elaiophylin (1 35) is a 16-membered macrodiolide exhibiting C symmetry which has been isolated from a number of Streptomyces. The biosynthetic origin of the carbon and oxygen atoms has been studieds3 in Streptomyces sp. (DSM 4137) and the results are summarized in Scheme 19.The aglycone is assembled from acetate propionate and butyrate subunits and all of the aglycone oxygen atoms derive intact from their carboxylate precursors. The dilactone is presumably assembled from the condensation of two elaiophylinic acid moieties most probably activated as their coenzyme A esters (1 34). A novel family of milbemycin antibiotics with anthelmintic and ectoparasiticidal activities have been identified from Streptomyces hygr~scopicus.~~ These are C- 13P-acyloxomilbe- mycins which possess a unique 13P-oxygen functionality and are typified by UK 78 629 (1 36). This novel structural class can be compared to the structurally related avermectin antibiotics e.g.,avermectin Al (137) which possess a 13a-oxygen function- In alit~.~~order to probe the origin and timing of the introduction of the 1 3P-oxygen7 milbemycin LL-F28 2497 (1 38) a metabolite not secreted by the organism was incubated with S.hygroscopicus. This compound was cleanly converted to its 13/3-isobutyroxy derivative (139) as shown in Scheme 20. This biotransformation clearly suggests that the 13P-oxygen is introduced by a monooxygenase activity onto the milbemycin aglycone as a late modification. It should be noted that in the biosynthesis of the avermectins (e.g. 137) the 13a-oxygen is HHO OS OH OH [1-13C]-gluCose I OH OH (1 35) Scheme 19 NATURAL PRODUCT REPORTS 1993-D. O'HAGAN Hfi OMe I OCH3 Scheme 20 MeoY-Y introduced during aglycone assembly from the carboxylate of a propionate precursor.86 The structure of ascomycin (140) an immunosuppressant from Streptomyces hygroscopicus var.asomyceticus has been reported8' and shown to be identical with that of the previously reported FR-900 520.88 Assuming a similar stereochemistry this compound is structurally identical to the clinically significant immunosuppressant FK-50689 (141) except that an ethyl group replaces the pendant propenyl group of FK-506 (arrowed). This ethyl group presumably arises in the classic manner after incorporation of a butyrate subunit into asco- MeoYY OMe mycin (140).Extension of this analogy would require incorpora- tion of an unusual pentanoate subunit into FK-506 however this has never been probed.4.2.2 Macrolide Genetic Studies The key highlight of the last few years in macrolide biosynthesis has been the isolation2 of the polyketide synthase (PKS) genes from Saccharopolyspora erythraea which are responsible for the synthesis of the erythromycin aglycone 6-deoxyerythronolide NATURAL PRODUCT REPORTS 1993 ORF-1 module 1 module 3 module 2 [AT ACP KS AT KR ACP KS AT KR ACP module 4 4'0 '1 HO-5,H$ HO HO-ORF-3 (ORF-A) module 5 I I AT = acyltransferase ACP = acylcarrier protein KS = P-keto-ACP synthase KR = P-keto-ACP reductase DH = dehydrogenase ER = enoyl reductase TE = thioesterase (cyclase?) module 1 module 4 module 3 module5 module 2 module 6 6-MSA module 1 (starter) rat chicken Tcm L Gra Whi E Barley I -Barley Ill I -%H!{ HO-module 6 I I -HI-1-HO--..-0 Scheme 21 ORFs.The synthase is set such that after each module the developing polyketide chain is passed to a new acyl carrier protein (ACP) which chaparones it through the next module and so on. This is summarized in Scheme 21. A comprehensive computer based matching analysisg1 of the sequence of the ORFs with known sequences coding different activities from other PKSs has strengthened the assignments and sequences of the domain activities as detailed in Scheme 21. Several interesting features emerged from this study which have a bearing on the evolution of this system. (a) In the first instance there was a greater sequence homology between similar domains within the PKS than to corresponding activities from other polyketide synthases.(b) Only module 4contains a functional DH-ER activity however a stretch of about 80 amino acids from the DH-ER interdomain region of module 4 was conserved in each of the other five modules. This suggests that all six modules possess the relics of a DH-ER sequence and clearly that they are closely related and have evolved from one another. (c) There are seven ACPs coded for in the EryPKS. Six of the seven ACPs were found to be significantly homologous. The unique ACP is the first of module 1 which accepts the propionate starter unit and then releases it directly after the first methylmalonyl condensation. The remaining six ACPs in addition to the condensation mediate the transacylation of the extending chain for further processing through the system.This first ACP appears to have a modified N-terminus and is shorter than the other six by 15 aa residues at this end. The sequence homologies between various ACPs selected from a wide range of polyketide (PKS) and fatty acid (FAS) synthases were compared and their interrelatedness is illustrated in the dendogram shown in Figure 3.91 It is clear from this analysis that the multifunctional Type I PKS and FAS are closely related and that the dissociated Type I1 systems group together. The evidence does not support the evolution of PKSs of secondary metabolism from FASs of the same organism. For PKS Type1 FAS Type I PKS Type II FAS Type II Nodulation Type II Figure 3 Dendogram illustrating the relatedness of ACP genes from polyketide and fatty acid synthases after sequence homology analysisg' B (142).This was discussed in the last review' of this series and other review+ have appeared detailing these developments. In summary three open reading frames (ORF 1-3) have been identified which are deduced to code for three multifunctional proteins. Each of the multifunctional proteins mediates two of the six methylmalonyl condensation cycles (modules 1-6) required for the complete assembly of 6-deoxyerythronolide B (142). The ORFs coding each of the six modules have the appropriate activities arranged in the correct sequence along the NATURAL PRODUCT REPORTS 1993-D.O’HAGAN OH OH (143) R’ = R2 = R3 = H (147) R’ (144) R’ = R2 = H R3= CH3 (148) R’ (145) R’ = H R2 = R3 = CH3 (149) R’ (146) R’ = R2 = R3 = CH3 (150) R’ unknown stereochemistry (153) R = CH3 R’ = H R2 = OH (154) R = CH2CH3 R’ = OH R2 = H (155) R = CHZCHS R’ = H R2 = OH propionate 4 acetate I citric acid cycle Scheme 22 R-‘ = R2 = R3 = H (151) R=CH3 = R2= H R3=CH3 (152) R = CHOHCH = H R2 = R3 = CH3 = R2 = R3 = CH3 &0YR I 0 YPh HN (156) R=OH R’=H (157) R=H R’=OH a series of eight macrolactams (143)-( 150) identified from several Actinomadura strains and which possess antifungal activity. They consist of four homologous aglycones attached to one of two possible sugars.The absolute stereochemistry has been established for (149) by X-ray structure analysisg6 and is assumed for the remaining metabolites. The second group are the fluvirucins A (1 5 l) A (1 52) and B,-B (I 53)-( 157) which were isolated from five unidentified soil actinomy~etes.’~ Not unsurprisingly they were detected on the basis of their inhibitory activity against the influenza A virus. Their absolute stereo- chemistry was establishedg7 after X-ray analysis of an acetate derivative of fluvirucin A (15 1). It is clear from their structural similarity and matching aglycone stereochemistry that both of these groups are biogenetically related however it is intriguing that they have enantiomeric sugar moieties. In both cases a biosynthetic investigation has been carried out giving rise to complementary result^.^^^^^ These are summarized in Scheme 22.The aglycones are constructed largely from combinations of acetate and propionate C-3 of propionate giving rise to the pendant methyl groups of the aglycones. In each case acetate labelled the pendant ethyl groups however these presumably arise more directly from butyrate although this was not studied or discussed in either of the reports. The amide nitrogen of the macrolactams was labelledg8 from [15N]-aspartic acid (1 59) and carbons C-1 1 to C- 13 most probably derive from this source. [2-l3C]-Acetate labelled98~99 both C-12 and C-13 and C- 11 and C-12 were derived from an intact acetate unit. This is consistent with acetate processing through the citric acid cycle to an intermediate such as oxaloacetate (158) which may be used directly or be transaminated to aspartic acid (159) and incorporated either directly or after decarboxylation to p-alanine.Glycine was not incorporated into the macrolides. The involvement of citric acid cycle intermediates in polyketide biosynthesis is unusual but has precedence with the brevetoxins example S. erythraea has a Type I PKS but a distantly related Type I1 FAS. Support for this contention also comes from fungal ~ystems~~.~~ where it has been demonstrated that the stereochemistry of the enoyl reductase of the FAS is opposite to that of the polyketide synthase in the same organism which clearly suggests that the enoyl reductases of primary and secondary metabolism are not related.4.2.3 Macrolactams Two new groups of related 14-membered macrolactam anti- biotics have been independently isolated. The first group94,95 is NATURAL PRODUCT REPORTS 1993 0 t 0 HO p he ny lalan ine methionine (161a) propionate acetate and other dinoflagellate metabolites. loo.lolViridenomycin (160) n=OCH3 an antitumour antibiotic has recently been isolatedlo2 from Streptomyces gannmycicus. Although the compound had been known previously reports of its structure and relative stereo- chemistry were ambiguous but are now resolved. Virideno- mycin (160) has some similarities to hitachimycin (161) whose biosynthesis has been studiedlo3 as illustrated in (161a) and clearly viridenomycin (160) might also be expected to in- corporate a phenylanine or even a P-phenylalanine moiety.Antibiotic BE-14 106 (162) is another antitumour macrolactam isolated recently from Streptomyces spheroides.lo4 (164) R=H 4.3 Polyethers and Related Metabolites The bisglycoside polyether ionophore UK-58 852 (1 63) is Me0 Me Me hydrolysed chemically to obtain the more efficacious mono- glycoside ionophore semduramicin (164) a poultry feed additive. Mutagenesis on the producing organism Actinomadura roseorufa have given rise to a semduramicin Me (1 64) producing mutant strain. The productivity of sem-duramicin (164) was ten-fold lower than that of UK-58852 Me- C02H (165) (163) however the development is attractive and serves as a basis for culture improvement.In this respect the titre of nigericin (1 65) from Streptomyces hygroscopicus was improved dramatically by 20-fold,lo6 from 25 mg 1-' to 600 mg I-l after addition of methyl oleate to the medium. The fatty acid composition of the mycelium was also studied and iso-C-16 fatty acids were seen to increase. It is noteworthy that oleic acid 0 + H CO2H itself had no stimulatory effect and the authors suggest that exogenous methyl oleate may alter membrane permeability leading to the accumulation of the antibiotic. threomyceticus that had previously been shownlo7 to derive from two acetate units and a propionate starter unit. The Furanomycin (1 66) is an antibiotic produced by Streptomyces oxygen atom of the ether linkage is derived from molecular oxygen.1o8 A biosynthetic pathway was proposed as outlined in Scheme 23.After challenging the wild-type strain simultaneously with UV light and chemical mutagens,1og a series of 19 stable C02H:&"T&H"OH- - C02H "OH blocked mutants of S. threomyceticus were identified combina- tions of which could secrete furanomycin (166) in co-synthesis indicating that the resistance gene had been left intact. On the basis of co-synthesis studies the mutants were classified into ten studies. All of the mutants were resistant to furanomycin (166) distinct phenotypic classes. The largest group Class I was able Scheme 23 to generate furanomycin in co-synthesis with Classes 11-VII and therefore it was concluded that Class I mutants are unable NATURAL PRODUCT REPORTS 1993-D.O'HAGAN 61 1 r I 1 L Scheme 24 H O=5 HO OH 0 (171) Scheme 25 to mediate the early stages in the biosynthesis most probably at the polyketide synthase level. The identity of mutant Classes 11-VII clearly suggests that there are at least six discrete biosynthetic steps after the polyketide synthase. I-111-IV-VI-( II,V,VII)-furanomycin These mutants are being analysed further for the presence of biosynthetic intermediates. HOqoH HOF''. OH A number of cyclohexane and cyclohexene containing antibiotics have been isolated and their structures elucidated. PI-200 (167) and PI-201 (168) were identifiedl'O from a Streptomyces sp. after screening for platelet aggregation inhibition. The structure and relative stereochemistry was established as shown after X-ray analysis of a suitable crystal of PI-201 (168).The origin of these compounds can clearly be rationalized in terms of an intramolecular Diels-Alder cyclo- addition as illustrated in Scheme 24. The origin of aldecalmycin (169)y recently isolated from a Streptomyces can be similarly rationalized. Tetronothiodin (1 7 1)'12 is more intriguing from a biosynthetic point of view. The presence of a tetrahydro-thiophene ring system is unusual and carbons C-23 C-30 and C-3 1 almost certainly derive from cysteine. The cyclohexene ring system may derive via an intramolecular Diels-Alder cycloaddition of an intermediate such as (170) shown in Scheme 25 after condensation of the polyketide terminus with pyruvate.Pyruvate would then contribute carbons C-3 C-4 and C-5. All of these hypotheses remain to be verified. 5 Polyketides from Fungi It is well established that the pendant methyl groups of polyketides generally arise from the methyl group of meth- ionine rather than from the methyl group of propionate the most common process in bacteria. This is further confirmed in the following biosynthetic investigations. The three pendant methyl and one hydroxymethyl groups of Antibiotic 1233A (172) a product of Scopulariopsis sp. were significantly enriched113 by [methyl-l3C]-methionine with the backbone originating from acetate. This study allowed the direction of chain assembly to be established and clearly the terminal methyl group of the polyketide chain is oxidized to a carboxylate group.Antibiotic 1233A (172) is a potent inhibitor of HMG CoA reductase and studies with biosvnthetically prepared radiolabelled material are being conducted to assess-whether the inhibitor covalently attaches itself to the enzyme. The NATURAL PRODUCT REPORTS 1993 Scheme 26 OH OH OH OH C02H \ \ \ \ "fl^ly3yJrJy 30 HO 27 30 27 26 25 24 23 (174) NH2 0 ( 174) (175) L-v-J--A B C D E module1 module 2 module 3 module 3 module 2 module 2 module 3 module 1 KS MT KR DH ER KS MT KR DH ER module 2 KS MT KR DH KS MT KR module 3 KS MT KR DH KS KR Scheme 27 biosynthetic origin of L-67 1329 (1 73) from Zalerion arboricola metabolite ACRL toxin (1 73 was highlighted116 as illustrated has been studied114 and the results are summarized in Scheme in Scheme 27.Analysis of the structure of cubensic acid (174) 26. It is a member of the echinocandin class of antibiotics suggests that there are five discernible biosynthetic regions which are essentially amino acid derived. However the lipo- A-E which require a series of enzymatic activities such that the philic 10,12-dimethylmyristoyl side chain is of true polyketide functionality along the backbone of the molecule is app- origin again with both pendant methyl groups coming from ropriately adjusted. These regions amount to three modules met hionine. with each module possessing two condensing enzymes (KS) for Cubensic acid (174) is a metabolite of the fungus Xylaria two malonate condensations and the appropriate reduction cubensis which possesses seven pendant methyl groups and one activities.It can be seen that modules 2 and 3 are repeated in hydroxymethyl group. It was initially proposed115 on the basis cubensic acid and that fusion of modules 2 and 3 provides the of structural similarities to the polyether and macrolide right activities for the construction of the first half of ACRL antibiotics that cubensic acid (174) would be derived from a Toxin I (175). Module 1 in cubensic acid which links together combination of acetate and propionate subunits. However all three acetate groups and two methionine derived methyl of the eight pendant groups (C-23-C-30) were enriched after groups gives rise to a structural feature common to many The struc- reduced fungal polyketides.This can be seen in L-671329 (173) the incorporation of ~-[methyl-'~C]-methionine.~~~ tural similarity of cubensic acid (174) to another fungal and other examples include radiclonic acid (1 76),11' funiculosin NATURAL PRODUCT REPORTS 1993-D. O'HAGAN Ho .OH /'r*-IOH 0 Scheme 28 a (184) =170 0 = 50% scrambled I7O Scheme 29 (1 77),118 and obionin (178).l19 There exists therefore a possible biogenetic basis underlying these structural similarities and there are clear comparisons to be drawn with the modular assembly of 6-deoxyerythronolide B (142) from a bacterial Type I PKS as shown earlier in Scheme 21. The antifungal agents lanomycin (1 79) glucolanomycin (1 80) and lanomycinol (1 8 1) have been isolated120*121 from Pycnidiophora dispersa.Lanomycin (179) and glucolanomycin (1 80) inhibit the cytochrome P-450 enzyme lanosterol 14a- demethylase. A biosynthetic investigation122 has been carried out on lanomycin (179) and the results are summarized as shown in Scheme 28. Lanomycin is derived from a hexaketide with the two pendant methyl groups and the methoxyl methyl derived from methionine. Glycine contributes directly the glycyl ester moiety. A series of biosynthetic studies have been carried out on aspyrone (1 83) and its related co-metabolites asperlactone HO HO 0 OH 0 OH (186) I Scheme 30 (1 84) and isoasperlactone (1 85) from the fungus Aspergillus melleus. The use of 170for labelling in biosynthetic studies is relatively rare but was powerfully exploited6 to trace the origin of the oxygen atoms of these metabolites.170can be observed directly by NMR and as natural abundance is low (0.04%) very low levels of incorporation can be detected after comparison with natural abundance spectra. A previous with lSO2had established that all of the oxygen atoms of aspyrone (183) were derived from this source but not to the same extent. The lactone carboxylate oxygens were only labelled 50% each and it was deduced that they had been scrambled the remaining 50 YOoriginating from the aqueous medium and not acetate. Due to the small quantities produced and with the low level incorporation of lag,no biosynthetic results could be obtained for the co-metabolites asperlactone (184) and isoasperlactone (185).The sensitivity of the 1702 study did however allow incorporations into these co-metabolites to be detected. The level of isotope incorporation for each metabolite was calculated after integration of the 170 spectra and comparison with natural abundance. The dis- tribution of isotope in asperlactone (184) was similar to that found in aspyrone (183) however the distribution in iso- asperlactone (1 85) is different. These results are summarized in Scheme 29 and a previously proposed common diepoxide intermediate (182) can be rationalized to account for the observed labelling patterns. No incorporation of isotope from [l'ol-acetate could be detected in these metabolites. In view of the low natural abundance of 170 this would suggest a mechanistic impediment to such oxygen incorporation rather than reduced levels of incorporation as a consequence of exchange processes with the medium.Three further on aspyrone (183) report the results of a very elegant series of experiments where partially assembled intermediates on the biosynthetic pathway to aspyrone have been identified. In all of these cases 2H NMR was used as the analytical method. Similar to 170 the low natural abundance of 2H allows the detection of low level enrichments provided a natural abundance spectrum can be obtained. In the first phase of aspyrone biosynthesis it can be predicted that a polyketide synthase assembles a C, pentaketide such as (1 86). Subsequent processing of (1 86) following the intermediates outlined in Scheme 30 provides a rationale for the generation of the diepoxide precursor (182) the implicated intermediate as discussed above.In firm support of this hypothesis the labelled ethyl ester (187) carrying three deuterium atoms was regioselectively incorporated' into aspyrone (1 83a) as shown in Scheme 3 1. Four other putative intermediates (188)-( 19 1) were not successfully incorporated in similar experiments. Three of these (1 88)-( 190) were considered potential intermediates on an alternative pathway as shown in Scheme 32 however their failure to be incorporated into aspyrone and the successful incorporation of (187) clearly suggests that Scheme 30 more closely represents the true situation. The acyl moiety of the ester (187) represents a completely assembled product of the polyketide synthase (PKS) and has most probably intercepted the pathway beyond the PKS stage after transacylation onto the PKS.In general it has proved difficult to intercept the PKS and introduce partially assembled fragments. A number of successful cases have been reported both in fungall0*'l and bacterial T6,i7. lZ4but it is well known throughout the community that the reported successes are grossly dispro- portionate to the failed attempts! In the aspyrone study however the series of deuterated N-acylcysteamine (NAC) thioesters (192) (193) and (194) were all successfully incorporated' into aspyrone in a regiospecific manner as shown 0 OH (183a) Scheme 31 NATURAL PRODUCT REPORTS 1993 in Scheme 33.This provides clear evidence for a processive assembly of the polyketide chain by the PKS and provides a window into the system between acetate and the fully assembled PKS product (183). Finally the structure and stereochemistry of some of the very early intermediates of the PKS assembly process have been identified.g In particular the C moieties of the NAC thioesters of acetoacetate (195) and (R)-P-hydroxy- butyrate (196) were incorporated into aspyrone as shown in Scheme 34. The (S)enantiomer of (196) was not a precursor and clearly the alcohol at this stage is processed uniquely with the (R) absolute configuration. These species can be considered to be the first two products of the polyketide synthase in the processive assembly towards (187); this is clear evidence that the reduction of the carbonyl occurs immediately consequent to the first condensation of acetate and malonate.This series of papers on aspyrone illustrates the power of this approach to reveal the nature of enzyme bound intermediates on a polyketide pathway. The optimization of the incorporation of partially assembled polyketide fragments into dehydrocurvularin (1 98) from Alternaria cinerariae has been explored." It had already been shownlO that the labelled acyl moieties of (1 97) and (199) could be incorporated intact into the framework of dehydrocurvularin (197). The success of intact incorporation depends on com- petition between incorporation and the destruction of the labelled species largely by P-oxidation.Successful incor- porations into dehydrocurvularin were carried out with a mutant strain of A. cinerariae deficient in its ability to utilize fatty acids and therefore presumably suppressed in its P-oxidation activity. In the more recent study,'l these experiments were repeated with precursor (1 99) labelled at carbons C-2 and C-3 as shown in Scheme 35 such that any acetate generated by P-oxidation would only possess one isotopically enriched carbon and could not be misleadingly recycled into dehydro- curvularin (198) carrying a dual label. In addition a series of known P-oxidation inhibitors (200)-(203) were simultaneously Scheme 32 N,SWDA DC H DF DEWDD D O DC A PKS ] -[ OH DB (193) (186a) 0 I H NACS= /"rfLNf (1 94) 0 0 0 J Scheme 33 615 NATURAL PRODUCT REPORTS 1993-D.O'HAGAN (195) (197) t "Oy 0 Scheme 35 (202) R = CH,(CH,)i,- (203) R = CH,(CH,)r (204) R' =H R2=OH (205) R' =OH R2 = H (206) HO OH 0 (211) R' = R2=Ac 0 (212) R' = R2=H 6 0 (213) R' =H R2=Ac OH H0,~..C02H \ oAo$y-0 added in separate experiments and the relative intact incor- porations of (199) compared. In the case of (202) an amazing 70 % incorporation of (199) into dehydrocurvularin (198) was observed and clearly little if any of the precursor had been degraded. A further experiment in this series established the intact incorporation of the hydroxyl group at C-4 of (197) viu 180-enrichment as illustrated in Scheme 35.The decarestrictines exemplified by structures (204)-(209) are an extensive novel group of 10-membered macrolides isolated from several Penicillium sp.125-127 The structure and absolute configuration of decarestrictine B (206) was established by X-ray structure analysis of a 2-bromobenzoate derivative and the (R)configuration is assumed for the other members of the group. The structure and relative configuration of decare- strictine D (207) was also confirmed with a suitable crystalline derivative.126 The decarestrictines were isolated during a screening programme that focused on the inhibition of cholesterol biosynthesis and are potent inhibitors in this regard. They can all be considered to arise from a common pentaketide precursor with decarestrictine L (208) and M (209) as shunt products.Depudecin (210) is perhaps a related hexaketide which has been isolated128 from Alternaria brassici- cola. X-ray analysis of a bis-( 1 S)-(-)-camphanate derivative of (210) has secured the structure and absolute stereochemistry as shown. The squalestatins (21 lk(213) were is~lated~~~~'~~ from a species of Phoma on the basis of their powerful inhibitory activity on squalene synthase. They are approximately 100-fold more active than other known inhibitors and are potentially therapeutic agents for controlhg serum cholesterol levels. Their structures and absolute stereochemistry have been established as shown. The squalestatins (21 1)-(2 13) are obviously amphiphilic molecules with an interesting 2,8- dioxabicyclic [3.2.lloctane core.Another family of amphiphilic metabolites are the cinatrins exemplified by cinatrins A (214) and C (2 19 i~olated'~~.~~~ from Circinotrichum falcatisporum NATURAL PRODUCT REPORTS. 1993 CHQ 0 0 0 1 1 iqDo 0 H OGO \ / 13 Scheme 37 as a consequence of their inhibition of phospholipase A,. Their structures and absolute stereochemistry were established to be as shown using a combination of X-ray structure analysis and circular dichromism. A biosynthetic study has been carried out on the polycyclic xanthone antibiotic citreamicin-a (2 17) from Micromonospora In view of the structural similarity of citreamicin to simaomicin-a (216) at the outset of this study it was predicted that the two systems would be similarly constructed.This however proved not to be the case. Simaomicin-a (216) from Actinomadura madurae was already known134 to arise from a single polyketide chain with oxidative removal of one carbon as shown in Scheme 36. Citreamicin-a (217) on the other hand undergoes a much greater extent of oxidative modification and rearrangement. The labelling pattern from 13C and l80labelled precursors is summarized in Scheme 37. It is particularly noteworthy from the doubly labelled 13C,-acetate study that C-21 and C-22 derived from C-1 of acetate are divorced from their original C-2 partner and (2-13 which is derived from C- 2 of acetate is also isolated. This is more complex than simaomicin-a (2 16) and several possible rearrangement scenarios consistent with this labelling pattern are outlined in Scheme 37.An interesting additional feature in this study was the labelling pattern from acetate of the isobutyrate unit in ring A of citreamicin-a (217). The two methyl groups are labelled from C-2 of acetate and were isolated from their partners in the doubly labelled study. There is no contiguous assembly of acetate and routes from valine or mevalonic acid are not obvious here. Green compounds are relatively rare in the currency of secondary metabolites with the exception of the chlorophylls however hypoxyxylerone (21 8) is a bright green pigment from the fungus Hypoxylon fragif~rme.~~~ The structure was es-tablished by X-ray analysis of a suitable crystal and it was proposed that hypoxyxylerone (2 18) originates from the folding of an undecaketide as shown by pathway a in Scheme 38.It is possible that the folding pattern is a little more convoluted as illustrated by pathway b which would be more closely analogous to that e~tablished'~~ for rubrofusarin (219) a metabolite sharing a structural relationship. The purpactins A-C [(222) (220) (221) respectively] are three closely related metabolites13' of Penicillium purpurogenum. Purpactin B (220) NATURAL PRODUCT REPORTS 1993-D. O’HAGAN 0 0 SR 00 pathway\a /pathway b OH Scheme 38 ow cH30m OH OH 0 00 (219a) Scheme 39 (and C (221)) which has a carbon skeleton similar to griseofulvin (223) is believed to be a precursor to purpactin A (222) as shown in Scheme 39 and can be converted to purpactin A (222) in aqueous alcohol.A biosynthetic study13* on purpactin A (222) has revealed that the 3-methylbutyryl side chain is derived from mevalonate and the acetate ester from acetate. Methionine contributes the methoxyl methyl group. The carbons of the tricyclic system all derive from acetate as shown NATURAL PRODUCT REPORTS 1993 OH 0 OH 0 methionine A (224a) (224b) o\ 0 -OH COH I IW (220a) (222a) I (221) Scheme 40 Scheme 41 in Scheme 40. It is proposed that the purpactins derive from an original octaketide which cyclizes to an anthraquinone and undergoes decarboxylation. Following a similar pattern to griseofulvin bio~ynthesis,~~~ oxidative fission would generate benzophenones (224a) and (224b) distinguished by the ori- entation of the labels in ring A followed by ring closure to an intermediate such as (225).Appropriate modifications would generate purpactins B (220) and C (221) and rearrangement of B will generate purpactin A (222) with the observed labelling pattern. The benzophenones (224) and (225) and the corresponding aldehydes have previously been proposed as intermediates in the biosynthesis of the variecoxanthones and tajixanthone in Aspergillus variecolor. A full paper describing previously reported studies has appeared.140 Following on from a very elegant series of experiment^^^^-'^^ where chiral malonic acids were used to establish the stereospecific nature of the elimination processes involved in the generation of the aromatic ring of 6-methylsalicylic acid a similar study has been executed on orsellinic acid (227).144 Generation of the aromatic ring of orsellinic acid requires two enolizations.This is distinct from 6-methylsalicylic acid where a dehydration and an enolization are required to generate the aromatic ring. The studies were carried out using purified orsellinic acid synthase from Penicillium cyclopium. Incubation of (A)-and then (S)-[l-13C 2-2H]-malonates with succinyl CoA transferase and succinyl CoA generated paired malonyl CoA in situ. In the presence of acetyl CoA the synthase then incorporated these species into orsellinic acid.In each ex-periment a maximum of three 13C atoms and two 2Hatoms can be incorporated into orsellinic acid and an analysis of the various stereochemical possibilities gives rise to different statistical distributions of the label in the resultant metabolite. Evaluation of the molecular mass distributions of the isolated orsellinic acid from each experiment led to the conclusion that the enolizations did indeed proceed in a stereochemically defined mode and that the hydrogens eliminated from the methylene groups at C-2 and C-4 of orsellinic acid are from opposite prochiral sites in malonyl CoA. This is consistent with a single base sitting either above or below the plane of the developing aromatic ring mediating each of the elimination reactions as shown for intermediate (226) in Scheme 41.A previous study on orsellinic carried out in the same manner drew the conclusion that this process was now stereospecific. In the light of the more recent result it must be concluded that the rate of deuterium isotope exchange from the malonate had competed unfavourably with the rate of orsellinic acid synthesis during the experiment leading to racemization. NATURAL PRODUCT REPORTS 1993-D. O’HAGAN 0 0 (228) R’ = R2 = R3 = H (229)R’ = H R2 = R3 = AC (230) R’ = H R2= (CO)Et R3 = AC (231) R’ = H R2= Ac R3 = (C0)Et (232) R’ = Ac R2 = R3 = (C0)Et (238) (238a) +NH2 II Scheme 43 NH H2N KNH2 H2N NH2 I I NH t y42 NH2 I II II NH~ NH; (244) n= 9-12 (245) Scheme 44 0 0 (233)R’ = R2 = R3 = H (234)R’ = R2= H R3 = AC (235)R’ = H R2 = (CO)Et R3 = AC (236)R’ = H R2 = Ac R3 = (C0)Et (237)R’ = Ac R2 = R3 = (C0)Et (239) /Me Me (240)R’ = R2= H (241)R’ = R2 = OH (242)R’ = H R2 =OH Scheme 42 6 Marine Polyketides Marine organisms continue to contribute some of the most interesting and elaborate polyketides.One of the fastest growing class of metabolites is the polypropionates from pulmonate molluscs. Eight new polypropionates have been isolated146 from an unidentified Onchidium sp. Compounds (228)-(232) are esters of a common triol termed onchitriol I (228) and compounds (234)-(237) are esters of another triol onchitriol I1 (233). The absolute stereochemistry of the core onchitriols I (228) and I1 (233) has been established to be as The absolute stereochemistry of (238) a metabolite of Siphonariu australis has been determined14’ as (4R) (729 (8R)by total synthesis of its degradative product (239).It is proposed as shown in (238a) that all of the alkyl groups will occupy equatorial positions with the hydroxyl taking up the axial anomeric site. Ptilomycalin A (240) is a guanidine alkaloid isolated from sponges in the Caribbean (Ptlocaulis spiculifer) and Red Sea (Hemimycale sp.). The crambescidins 800 (241) and 816 (242) are structurally related sponge metabolites from the Mediter- ranean sponge Crambe crambe. Ptilocaulin (243) from Ptlocaulis spiculifer and the crambines A (244) and B (245) also from Crambe crambe are other guanidinium containing metabolites.A biosynthetic rational has been for all of these metabolites which involves Michael attack of guanidinium to appropriate enone frameworks. This is illustrated in Schemes 42 43 and 44. Scheme 43 outlines the basis of a synthetic strategy to (243) previously executed,150 and more recently an elegant synthetic approach towards crambines A (244) and B (245) employing the general strategy of Scheme 44 lends support to this general hypothesis.149 The key reaction sequence 43 NPR 10 NATURAL PRODUCT REPORTS 1993 OMe TBDMSO TBDMs0+(CH2)1 lCH3 H,N A~~ ~ Scheme 45 HoQQYBr OH 0 (246) R=OH (247)R=H (246) R=OH (247) R = H OH (249) diepoxide precursor Scheme 46 NATURAL PRODUCT REPORTS 1993-D.O'HAGAN OMe OMe (2511 OMe (252) is outlined in Scheme 45 where U-methylisourea was employed as a guanidium equivalent. Tedanolide (246) is a macrocyclic lactone isolated from the Caribbean sponge Tedania ignis.151 A C-13 dehydroxy analogue (247) has been isolated152 from the Japanese sponge Mycale adhaerens along with the brominated isocoumarin (248). The structural and stereochemical homology between the function- ality along the backbone of tedanolide (246) and now (247) with that along the backbone of the diepoxide precursor (249) of the polyether antibiotic noboritomycin A (250) a product of Actinomycete bacteria,153 has been highlighted154,155 and is shown in Scheme 46. The two asterisks indicate only two mismatches over the boxed region.It should be noted that the stereochemistry of tedanolide (246) was incorrectly drawn at C- 17 when first re~0rted.l~~ This stereochemical correlation suggests that these sponge metabolites are a product of symbiotic bacteria rather than products of the sponge. Further the structure of the isocoumarin (248) is more typical of algal plant and fungal metabolites and similar structures have been isolated from such sources as brown algae.156 This is again indicative of the symbiotic origin of these metabolites. Swinholide A (25l) a potent cytotoxic agent is a 44-membered 62 1 OMe Homo (257) dilactone which has been isolated from the Okinawan marine sponge Theonella swinhoei.15' It is one of a class of related dimeric ring compounds.An X-ray crystal structure of the di- p-bromobenzoate diketone derivative (252) of swinholide has been reported,15s establishing the absolute stereochemistry to be as shown and some conclusions are drawn regarding the preferred conformation of swinholide itself and how that relates to its biological activity. For example the oxygen atoms of (252) are directed to the interior of the molecule suggestive of ionophoric properties. The monomeric carboxylic acid of swinholide A (253) has also been isolated15' from Theonella swinhoei. Whether this is a degradative product of the parent compound or a biosynthetic intermediate remains to be established. It is interesting to note the striking structural and stereochemical similarities between the swinholide monomer (253) and for example scytophycin A (254) a metabolite of cyanobacterial origin.160 This clearly suggests a cyanobacterial origin for swinholide (25 1) and related structures.The amphidinolides are a class of macrocylic lactones isolated from laboratory cultured dinoflagellate Amphidinium sp.161 These are typified by amphidinolide A (255) and C (256) The latest of the series to be reported162 is amphidinolide F (257) NATURAL PRODUCT REPORTS 1993 HO' Na0,SO Na03sks S 0 'O-L 0 which has been identified from a new dinoflagellate strain Amphiscolops magniviridis associated with an Okinawan flat 7 References worm. Amphidinolides C (256) and F (257) are structurally 1 D. O'Hagan Nat.Prod. Rep. 1992 9 447. related with C (256) possibly being a precursor to F (257). The 2 J. Cortes S. F. Haydock G. A. Roberts D. J. Bevitt and P. F. origin of the carbon framework of the amphidinolides is not Leadlay Nature 1990 348 176. obvious. The methyl groups are not arrayed regularly which 3 S. Donadio M. J. Staver J. B. McAlpine J. B. Swanson and would tend to disqualify an origin from acetate and propionate. L. Katz Science 1991 252 675. An acetate/methionine framework remains a possibility 4 D. E. Cane and C.-C. Yang J. Am. Chem. SOC.,1987 109 1255. however the involvement of citric acid cycle intermediates 5 S. Yue J. S. Duncan Y. Yamamoto and C. R. Hutchinson would parallel more closely the biosynthesis of other dino- J. Am. Chem. SOC. 1987 109 1253.Chem. Commun. flagellate metabolites such as the brevetoxins (e.g. 259),100*101 6 J. Staunton and A. C. Sutkowski,J. Chem. SOC. 1991 1106. and could account for the erratic carbon skeleton of the 7 J. Staunton and A. C. Sutkowski,J. Chem. Soc. Chem. Commun. amp hidinolides. 1991 1108. The elaborate nature of some of the polyketides from the 8 J. Staunton and A. C. Sutkowski J. Chem. SOC. Chem. Commun. marine environment is amply illustrated by maitotoxin (MTX) 1991 1110. (258) isolated from the dinoflagellate Gambierdiscusus toxicus. 9 A. Jacobs J. Staunton and A. C. Sutkowski J. Chem. SOC. a poisoning Chem. Commun. 1991 1113. Maitotoxin (258)is a causative agent of ~iguatera,'~~ caused by ingestion of coral reef fish and in this respect it is 200 10 Y.Yoshizawa Z. Li P. B. Reese and J. C. Vederas J. Am. Chem. SOC.,1990 112 3212. times more toxic than tetrodotoxin. It has structural and 11 Z. Li F. M. Martin and J. C. Vederas J. Am. Chem. SOC. 1992, presumably biosynthetic similarities to other dinoflagellate 114 1531. metabolites such as brevetoxin A (259j1Oo- lol and yessotoxin 12 J. G. Jaworski R. C. Clough and S. R. Barnum Pfant Physiol. (260).164Maitotoxin (258) has a molecular weight of 3424 Da 1989 90 28. and to date only 30% of the structure has been reliably 13 M. Focke A. Feld and H. K. Lichtenthaler Physiol. Plant. 1991 elucidated165 after degradation studies. Periodate oxidation 81 251. followed by sodium borohydride reduction gave rise to three 14 M. Focke A. Feld and H. K. Lichtenthaler FEBS Lett.1991 fragments. The deduced structure of the two terminal fragments 261 106. is as shown (258). The central fragment which has a molecular 15 S. A. Benner A. Glasfled and J. A. Piccirilli Topics Stereochem. 1989 19 193. weight of around 2300 Da is not adequately characterized 16 C. Frossl and W. Boland J. Chem. Soc. Chem. Commun.. 1991, however NMR evidence suggests there exists between 22 and 27 1731. fused ether rings. Therefore maitotoxin (258) contains between 17 G. Gorgen and W. Boland Eur. J. Biochem. 1989 185 237. 28 and 33 ether rings and is a truly massive secondary 18 G. Gorgen C. Frossl and W. Boland Experientia 1990,46 700. metabolite. 19 P. Metzger and E. Casadevall Phytochemistry 1992 31 2341. NATURAL PRODUCT REPORTS 1993-D.O’HAGAN 20 E. Villarreal-Rosales P. Metzger and E. 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ISSN:0265-0568    

DOI:10.1039/NP9931000593

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

Cumulative Contents Volumes 1-10 Volume 1 Number 1 1 Introduction G. Pattenden 3 Rotenoids and their Biosynthesis L. Crombie 21 Indole Alkaloids and Mould Metabolites (July 1982 to June 1983) J. E. Saxton 53 Triterpenoids (October 1981 to September 1982) R. B. Boar 67 Carotenoids and Polyterpenoids (October 1981 to September 1982) G. Britton 87 Macrocyclic Microbial Metabolites (1982) R. C. F. Jones Number 2 105 Sesquiterpenoids (September 1981 to December 1982) J. S. Roberts and I. Bryson 171 Diterpenoids (August 1981 to April 1982) J. R. Hanson 181 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1982 to June 1983) R. B. Herbert 195 Quinoline Quinazoline and Acridone Alkaloids (July 1982 to June 1983) M. F. Grundon 201 Aporphinoid Alkaloids (July 1982 to June 1983) M.Shamma and H. Guinaudeau Number 3 209 Steroids Physical Aspects (September 1981 to August 1983) D. N. Kirk 219 Steroidal Alkaloids (July 1982 to June 1983) D. M. Harrison 225 Pyrrolidine Piperidine and Pyridine Alkaloids (July 1982 to June 1983) A. R. Pinder 23 1 Tropane Alkaloids (July 1982 to June 1983) G. Fodor and R. Dharanipragada 235 Pyrrolizidine Alkaloids (July 1982 to June 1983) D. J. Robins 245 Indolizidine Alkaloids (July 1982 to June 1983) J. A. Lamberton 247 Amaryllidaceae Alkaloids (July 1982 to June 1983) M. F. Grundon 25 1 Marine Natural Products Metabolites of Marine Algae and Herbivorous Marine Molluscs (1977 to October 1983) D. J. Faulkner [Corrigendum p. 5921 28 1 The Biosynthesis of Polyketides (January 1982 to June 1983) T.J. Simpson 299 Insect Pheromones and Related Natural Products (January 1982 to December 1983) R. Baker and R. H. Herbert Number 4 319 Monoterpenoids (1982) D. H. Grayson 339 Diterpenoids (April 1982 to December 1982) J. R. Hanson 349 Quinolizidine Alkaloids (July 1982 to June 1983) M. F. Grundon 355 /3-Phenylethylamines and the Isoquinoline Alkaloids (July 1982 to June 1983) K. W. Bentley 371 Erythrina and Related Alkaloids (July 1982 to June 1983) A. S. Chawla and A. H. Jackson 375 Diterpenoid Alkaloids (July 1982 to June 1983) S. W. Pelletier and S. W. Page 387 Muscarine Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1982 to June 1983) J. R. Lewis 391 Steroids Reactions and Partial Syntheses (September 1981 to December 1982) B.A. Marples Number 5 409 Prostaglandins Thromboxanes Leukotrienes and Related Arachidonic Acid Metabolites (1982) S. M. F. Lai and P. W. Manley 433 The Biosynthesis of C,-C, Terpenoid Compounds (1982 and 1983) J. R. Hanson 45 1 The Biosynthesis of Shikimate Metabolites (1982 and 1983) P. M. Dewick 47 1 Isolation of Natural Products by Droplet Counter-Current Chromatography and Related Methods (1980 to March 1984) K. Hostettmann M. Hostettmann and A. Marston 483 Fatty Acids and Glycerides (1982 and 1983) F. D. Gunstone 499 The Chemistry and Biochemistry of Simple and Complex Lipids (1982 and 1983) W. W. Christie Number 6 513 Phases of Membrane Polar Lipids in Aqueous Systems (up to end of 1983) P. J.Quinn 533 Diterpenoids (1983) J. R. Hanson 545 The Biosynthesis of Cyanogenic Glycosides and Glucosinolates (1979 to 1983) P. M. Dewick 551 Marine Natural Products Metabolites of Marine Invertebrates (1977 to July 1984) D. J. Faulkner [592] Corrigendum to Marine Natural Products Metabolites of Marine Algae and Herbivorous Marine Molluscs D. J. Faulkner 625 NATURAL PRODUCT REPORTS 1993 599 Book Review Natural Products Chemistry Volume 3 by K. Nakanishi S. Nozoe S. Ito T. Goto and S. Natori Reviewed by G. Pat ten den Volume 2 Number 1 1 Triterpenoids (October 1982 to December 1983) J. D. Connolly and R. A. Hill 19 The Biosynthesis of Porphyrins Chlorophylls and Vitamin B, (January 1978 to December 1983) F. J. Leeper 49 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1983 to June 1984) J.E. Saxton 81 /I-Phenylethylamines and the Isoquinoline Alkaloids (July 1983 to June 1984) K. W. Bentley Number 2 97 Sesquiterpenoid Synthesis (1983) J. S. Roberts 147 Natural Sesquiterpenoids (1983) B. M. Fraga 163 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1983 to June 1984) R. B. Herbert 181 Pyrrolidine Piperidine and Pyridine Alkaloids (July 1983 to June 1984) A. R. Pinder 189 Book Review Dictionary of Organic Compounds (Ftfth Edition) First and Second Supplement ed. J. Buckingham Reviewed by J. R. Hanson 189 Book Review The Chemistry of Natural Products ed. R. H. Thomson Reviewed by R. B. Herbert ~ 190 Book Review Phj’tochemical Methods A Guide to Modern Techniques of Plant Analysis (2nd edition) by J.B. Harborne Reviewed by J. R. Hanson Number 3 191 Lignans and Neolignans (1976 to December 1983) D. A. Whiting 213 Pyrrolizidine Alkaloids (July 1983 to June 1984) D. J. Robins [Erratum p. 3911 221 Tropane Alkaloids (July 1983 to June 1984) G. Fodor and R. Dharanipragada 227 Aporphinoid Alkaloids (July 1983 to June 1984) M. Shamma and H. Guinaudeau 235 Indolizidine and Quinolizidine Alkaloids (July 1983 to June 1984) M. F. Grundon 245 Muscarine Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1983 to June 1984) J. R. Lewis 249 Amaryllidaceae Alkaloids (July 1983 to June 1984) M. F. Grundon 253 Camphor A Chiral Starting Material in Natural Product Synthesis T.Money 291 Book Review Natural Products and Drug Development ed. P. Krogsgaard-Larsen S. B. Christensen and H. Kofod Reviewed by P. M. Dewick Number 4 293 Enzymology in Biosynthesis Mechanistic and Stereochemical Studies of B-Lactam Biosynthesis and the Shikimate Pathway (to December 1984) J. A. Robinson and D. Gani 321 The Biosynthesis of Polyketides (July 1983 to June 1984) T. J. Simpson 349 Carotenoids and Polyterpenoids (September 1982 to August 1984) G. Britton 389 Book Review Metabolites and Metabolism A Commentary on Secondary Metabolism by E. Haslam Reviewed by D. A. Whiting ~ 39 1 Erratum to Pyrrolizidine Alkaloids D. J. Robins (Volume 2 p. 2 13) Number 5 393 Quinoline Quinazoline and Acridone Alkaloids (July 1983 to June 1984) M.F. Grundon 401 Olefinic Microbial Metabolites excluding Macrocyclic Compounds (1982 and 1983) R. C. F. Jones 427 Phytoalexins (January I982 to July 1984) C. J. W. Brooks and D. G. Watson 461 Steroids Reactions and Partial Synthesis (1983) J. Elks Number 6 495 The Biosynthesis of Shikimate Metabolites (1984) P. M. Dewick 513 The Biosynthesis of C&, Terpenoid Compounds (1984) D. V. Banthorpe and S. A. Branch 525 The Biosynthesis of Triterpenoids and Steroids (1979 to 1983 incl.) D. M. Harrison [Errata Vol. 5 No. 1 p. 991 561 The Biosynthesis of Porphyrins Chlorophylls and Vitamin B1 (1984) F. J. Leeper Volume 3 Number 1 1 Marine Natural Products (October 1983/July 1984 to July 1985) D. J. Faulkner 35 Recent Developments in the Birch Reduction of Aromatic Compounds Applications to the Synthesis of Natural Products J.M. Hook and L. N. Mander NATURAL PRODUCT REPORTS 1993-CUMULATIVE CONTENTS Number 2 87 Avermectins and Milbemycins H. G. Davies and R. H. Green 123 Sesterterpenoids (to June 1985) J. R. Hanson 133 Biological Methods for Studying the Biosynthesis of Natural Products C. R. Hutchinson 153 /I-Phenylethylamines and Isoquinoline Alkaloids (July 1984 to June 1985) K. W. Bentley 171 Pyrrolidine Piperidine and Pyridine Alkaloids (July 1984 to June 1985) A. R. Pinder 181 Tropane Alkaloids (July 1984 to June 1985) G. Fodor and R. Dharanipragada Number 3 185 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1984 to June 1985) R. B. Herbert 205 The Biosynthesis of Carotenoids (January 1979 to December 1983) D.M. Harrison 217 Secondary Metabolism -Fact and Fiction E. Haslam 251 Monoterpenoids (1983) M. S. Carson and D. H. Grayson 273 Natural Sesquiterpenoids (1984) B. M. Fraga Number 4 297 Pyrrolizidine Alkaloids (July 1984 to June 1985) D. J. Robins 307 Diterpenoids (1984) J. R. Hanson 323 Recent Advances in Chemical Ecology (January 1982 to June 1985) J. B. Harborne 345 Aporphinoid Alkaloids (July 1984 to June 1985) M. Shamma and H. Guinaudeau 353 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1984 to June 1985) J. E. Saxton 395 The Biosynthesis of the Vitamins Thiamin Riboflavin and Folic Acid (to June 1985) D. W. Young Number 5 421 Triterpenoids (January 1984 to June 1985) J.D. Connolly and R. A. Hill 443 Steroidal Alkaloids (July 1983 to June 1985) D. M. Harrison 451 Diterpenoid Alkaloids (July 1983 to June 1985) S. W. Pelletier and S. W. Page 465 Chromanols Chromanones and Chromones (1976 to 1985) S. T. Saengchantara and T. W. Wallace 477 Spectral Characteristics of Bisbenzylisoquinoline Alkaloids H. Guinaudeau A. J. Freyer and M. Shamma 489 Recent Advances in the Use of Enzyme-Catalysed Reactions in Organic Research The Synthesis of Biologically Active Natural Products and Analogues S. Butt and S. M. Roberts 505 Steroids Physical Methods (June 1983 to June 1985) D. N. Kirk Number 6 5 15 Steroids Reactions and Partial Synthesis (1984) J. Elks 555 Erythrina and Related Alkaloids (July 1983 to June 1985) A.S. Chawla and A. H. Jackson 565 The Biosynthesis of Shikimate Metabolites (1985) P. M. Dewick 587 Muscarine Imidazole and Peptide Alkaloids and other Miscellaneous Alkaloids (July 1984 to June 1985) J. R. Lewis 591 Carotenoids and Polyterpenoids (September 1984 to December 1985) G. Britton Volume 4 Number 1 1 Centenary Tribute to Sir Robert Robinson (18861975) G. Pattenden 3 Robert Robinson (18861975) Lord Todd 13 Sir Robert Robinson -His contribution to Alkaloid Chemistry K. W. Bentley 25 Anthocyanins Brazilin and Related Compounds R. Livingstone 35 Steroids and Synthetic Oestrogens Sir John Cornforth 41 Sir Robert Robinson and the Early History of Penicillin E. P. Abraham 47 Theoretical Organic Chemistry before Robinson C. A. Russell 53 The Development of Sir Robert Robinson’s Contributions to Theoretical Organic Chemistry M.D. Saltzman 6 1 Electronic Theories of Organic Chemistry Robinson and Ingold J. Shorter 67 Chemistry in Manchester in the Twenties and some Personal Recollections W. Cocker 73 The Dyson Perrins Laboratory in Robinson’s Time M. L. Tomlinson 77 Nature’s Pathways to the Pigments of Life (The Robert Robinson Lecture) A. R. Battersby Number 2 89 Amaryllidaceae Alkaloids (July 1984 to June 1985) M. F. Grundon 95 Fatty Acids and Glycerides (1984 and 1985) F. D. Gunstone NATURAL PRODUCT REPORTS 1993 113 Simple and Complex Lipids Their Occurrence Chemistry and Biochemistry (1984 and 1985) W. W. Christie 129 Phase Behaviour of Binary Mixtures of Membrane Polar Lipids in Aqueous Systems (January 1985 to June 1986) P.J. Quinn 139 Chemical and Biochemical Manipulation of DNA and the Expression of Foreign Genes in Micro-organisms J. H. Parish and M. J. McPherson 157 The Biosynthesis of C5&20 Terpenoid Compounds (1985) D. V. Banthorpe and S. A. Branch 175 Chemical Systematics P. G. Waterman and A. I. Gray Number 3 205 Applications of Recombinant DNA in Biotechnology M. J. McPherson and J. H. Parish 225 Quinoline Quinazoline and Acridone Alkaloids (July 1984 to June 1985) M. F. Grundon [Erratum p. 4721 237 Tobacco Isoprenoids (1975 to 1984) I. Wahlberg and C. R. Enzell 277 The Use of Stable Isotopes in Biosynthetic Studies (January 1982 to December 1985) J. C. Vederas Number 4 339 The Biosynthesis of Polyketides (July 1984 to Eecember 1985) T.J. Simpson 377 Monoterpenoids (1984) D. H. Grayson 399 Diterpenoids (1985) J. R. Hanson 415 Indolizidine and Quinolizidine Alkaloids (July 1984 to June 1985) M. F. Grundon 423 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1985 to June 1986) R. B. Herbert 441 The Biosynthesis of Porphyrins Chlorophylls and Vitamin B, (1985) F. J. Leeper 471 Book Review Natural Product Chemistry ed. Atta-ur-Rahman Reviewed by E. Haslam 472 Erratum to Quinoline Quinazoline and Acridone Alkaloids M. F. Grundon (Vol. 4 No. 3 p. 225) Number 5 473 Natural Sesquiterpenoids (1985) B. M. Fraga 499 Lignans Neolignans and Related Compounds (1984 and 1985) D. A. Whiting 527 Pyrrolidine Piperidine and Pyridine Alkaloids (July 1985 to June 1986) A.R. Pinder 539 Marine Natural Products (July 1985 to September 1986) D. J. Faulkner 577 Pyrrolizidine Alkaloids (July 1985 to June 1986) D. J. Robins Number 6 591 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1985 to June 1986) J. E. Saxton 639 Benzenoid and Polycyclic Aromatic Natural Products (1984 and 1985) T. J. Simpson 677 P-Phenylethylamines and the Isoquinoline Alkaloids (Jury 1985 to June 1986) K. W. Bentley 703 Book Review Immunology in Plant Sciences ed. H. F. Linskens and J. F. Jackson Reviewed by D. V.Banthorpe Volume 5 Number 1 1 Prostaglandins Thromboxanes Leukotrienes and Related Arachidonic Acid Metabolites (1983 and 1984) T. W. Hart 47 Antibiotics with Antifungal and Antibacterial Activity Against Plant Diseases P.A. Worthington 67 Tropane Alkaloids (July 1985 to December 1986) G. Fodor and R. Dharanipragada 73 The Biosynthesis of Shikimate Metabolites (1986) P. M. Dewick 99 Errata to The Biosynthesis of Triterpenoids and Steroids D. M. Harrison (Vol. 2 No. 6 p. 525) Number 2 101 The Use of N.M.R. Spectroscopy in the Structure Determination of Natural Products One-Dimensional Methods I. H. Sadler 129 The Biosynthesis of Penicillins and Cephalosporins J. E. Baldwin and Sir Edward Abraham 147 Steroids Reactions and Partial Syntheses (1985) J. Elks 187 Non-Macrocyclic Trichothecenes (January 1970 to December 1986) J. F. Grove Number 3 21 1 Diterpenoids (1986) J. R. Hanson 229 Naturally Occurring Isocyanides M.S. Edenborough and R. B. Herbert 247 The Biosynthesis of C,&, Terpenoid Compounds (1986) M. H. Beale and J. MacMiUan 265 /3-Phenylethylamines and the Isoquinoline Alkaloids (July 1986 to June 1987) K. W. Bentley 293 Quinoline Quinazoline and Acridone Alkaloids (July 1985 to June 1987) M. F. Grundon 309 Book Review Secondary Metabolism (Second Edition) by J. Mann. Reviewed by G. W. Kirby 309 Book Review Biologically Active Natural Products ed. K. Hostettmann and P. J. Lea. Reviewed by A. Pelter NATURAL PRODUCT REPORTS 1993-CUMULATIVE CONTENTS Number 4 31 1 Steroids Reactions and Partial Syntheses (December 1985 to October 1986) A. B. Turner 351 Imidazole Oxazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1985 to June 1986) J.R. Lewis 363 Brain Chemistry and Central Nervous System Drugs R. I. Brinkworth E. J. Lloyd and P. R. Andrews 387 The Biosynthesis of Triterpenoids Steroids and Carotenoids (1984 and 1985) D. M. Harrison 417 Book Review Dictionary of Antibiotics and Related Substances ed. B. W. Bycroft. Reviewed by R. B. Herbert Number 5 419 Monoterpenoids (1985 and 1986) D. H. Grayson 465 Trends in Protease Inhibition (November 1984 to January 1987) G. Fischer 497 Natural Sesquiterpenoids (1986) B. M. Fraga 523 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1986 to June 1987) R. B. Herbert Number 6 541 Synthesis of Gibberellins and Antheridiogens (up to June 1988) L. N. Mander 581 Natural Products from Plant Tissue Culture (January 1979 to December 1986) B.E. Ellis 613 Marine Natural Products (September 1986 to December 1987) D. J. Faulkner Volume 6 Number 1 1 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1986 to June 1987) J. E. Saxton 55 Erythrina and Related Alkaloids (July 1985 to June 1987) A. S. Chawla and A. H. Jackson 67 Pyrrole Pyrrolidine Piperidine,Pyridine and Azepine Alkaloids (July 1986 to June 1987) A. R. Pinder 79 Amaryllidaceae Alkaloids (July 1985 to June 1987) M. F. Grundon 85 Recent Advances in Chemical Ecology (July 1985 to December 1987) J. B. Harborne Number 2 11 1 The Use of N.M.R. Spectroscopy in the Structure Determination of Natural Products Two-Dimensional Methods A. E. Derome 143 Biosynthetic Studies on Marine Natural Products (to April 1988) M.J. Garson 171 The Biosynthesis of Porphyrins Chlorophylls and Vitamin B, (1986 and 1987) F. J. Leeper 205 The Polyether and Macrolide Antibiotics Biogenetic Analysis and Structural Correlations D. O’Hagan Number 3 221 Pyrrolizidine Alkaloids (Jury 1986 to June 1987) D. J. Robins 231 Fatty Acids and Glycerides (1986 to 1987) M. S. F. Lie Ken Jie 263 The Biosynthesis of Shikimate Metabolites (1987) P. M. Dewick 291 Limonene A. F. Thomas and Y. Bessiere Number 4 31 1 Enzyme Inhibitors in Medicine (to December 1987) C. S. J. Walpole and R. Wrigglesworth 347 Diterpenoids (January to December 1987) J. R. Hanson 359 Carotenoids and Polyterpenoids (January 1985 to December 1987) G. Britton 393 Steroids Physical Methods (mid 1985 to December 1987) D.N. Kirk 405 8-Phenylethylamines and the Isoquinoline Alkaloids (Jury 1987 to June 1988) K. W. Bentley Number 5 433 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1987 to June 1988) J. E. Saxton 475 Triterpenoids (July 1985 to December 1987) J. D. Connolly and R. A. Hill 503 Muscarine Oxazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1986 to June 1987) J. R. Lewis 5 15 Pyrrolidine Piperidine and Pyridine Alkaloids (July 1987 to June 1988) A. R. Pinder 516 Indolizidine and Quinolizidine Alkaloids (July 1985 to June 1987) M. F. Grundon Number 6 537 Obituary Michael F. Grundon 1926-1989 539 Steroids Reactions and Partial Syntheses (November 1986 and October 1987) A.B. Turner 577 Pyrrolizidine Alkaloids (July 1987 and June 1988) D. J. Robins 591 Coumarins (mid-1980 and mid-1988) R. D. H. Murray 625 Recent Advances in the Use of Enzyme-Catalysed Reactions in Organic Synthesis (January Z986 to June 1988) N. J. Turner 645 Book Review The Dictionary of the Alkaloids ed. 1. Southon and J. Buckingham. Reviewed by R. B. Herbert NATURAL PRODUCT REPORTS 1993 Volume 7 Number 1 1 Natural Sesquiterpenoids (1987) B. M. Fraga 25 The Biosynthesis of C5<20 Terpenoid Compounds (1987) M. H. Beale 41 The Chemistry of the Gibberellins (to April 1989) J. R. Hanson 61 Recent Advances in the Chemistry and Biochemistry of Inositol Phosphates of Biological Interest B. V. L. Potter Number 2 85 Biosynthesis and Synthesis of Indole and Bisindole Alkaloids in Plant Cell Cultures A Personal Overview J.P. Kutney 105 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (July 1987 to July 1988) R. B. Herbert 131 Quinoline Quinazoline and Acridone Alkaloids (July 1987 to July 1988) M. F. Grundon 139 Steroidal Alkaloids (July 1985 to December 1987) D. M. Harrison Number 3 149 Diterpenoids (1988) J. R. Hanson 165 The Biosynthesis of Shikimate Metabolites (1988) P. M. Dewick 191 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1988 to July 1989) J. E. Saxton 245 P-Phenylethylamines and the Isoquinoline Alkaloids (July 1988 to July 1989) K. W. Bentley Number 4 269 Marine Natural Products (December 1987 to December 1988) D.J. Faulkner 31 1 Applications of Interactive Computer Graphics in Analyses of Biomolecular Structures (June 2982 to January 1988) D. J. Barlow and T. D. J. Perkins 327 Monoterpenoids (1987) D. H. Grayson 349 Lignans Neolignans and Related Compounds (January 1986 to December 1988) D. A. Whiting Number 5 365 Muscarine Oxazole Isoxazole Thiazole Imidazole and Peptide Alkaloids (July 1987 to July 1988) J. R. Lewis 377 Pyrrolizidine Alkaloids (July 1988 to June 1989) D. J. Robins 387 The Biosynthesis of C,-C, Terpenoid Compounds (1988) M. H. Beale 409 Inhibitors of Gastric Acid Secretion (to 1988) D. E. Bays and H. Finch 447 Pyrrolidine Piperidine and Pyridine Alkaloids (July 1988 to June 1989) A. R. Pinder Number 6 459 The Biosynthesis of Triterpenoids Steroids and Carotenoids (January 1986 to December 1987) D.M. Harrison 485 Indolizidine and Quinolizidine Alkaloids (July 1988 to June 1989) J. P. Michael 5 15 Natural Sesquiterpenoids (1988) B. M. Fraga 539 Tropane Alkaloids (January 1987 to December 1989) G. Fodor and R. Dharanipragada 549 Amaryllidaceae Alkaloids (July 1987 to December 1989) J. R. Lewis 557 Steroidal Alkaloids (Jul-v 1987 to December 1989) R. Schakirov and M. S. Yunusov 565 Erythrina and Related Alkaloids A. S. Chawla and A. H. Jackson Volume 8 Number 1 1 Diterpenoids (1989) J. R. Hanson 17 Steroids Reactions and Partial Synthesis (November 2987 to October 2988) A. B. Turner 53 Quinoline Quinazoline and Acridone Alkaloids (July 1988 to June 1989) J. P. Michael 69 Terpenoid Glycosides (1987 and 1988) H.Pfander and H. Stoll Number 2 97 Marine Natural Products (1989) D. J. Faulkner 149 The Biosynthesis of Shikimate Metabolites (1989) P. M. Dewick 171 Muscarine Oxazole Thiazole Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1988 to June 2989) J. R. Lewis 185 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (August 2988 to July 2989) R. B. Herbert Number 3 213 Pyrrolizidine Alkaloids (July 1989 to June 1990) D. J. Robins 223 Carotenoids and Polyterpenoids (2988) G. Britton 251 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1989 to June 1990) J. E. Saxton NATURAL PRODUCT REPORTS 1993-CUMULATIVE CONTENTS 631 309 The Occurrence and Biological Activity of Drimane Sesquiterpenoids (up to January 1990) B.J. M. Jansen and A. de Groot 319 The Synthesis of Drimane Sesquiterpenoids (up to January 1990) B. J. M. Jansen and A. de Groot Number 4 339 P-Phenylethylamines and the Isoquinoline Alkaloids (July 1989 to June 1990) K. W. Bentley 367 Terpenoid Phytoalexins (August 1984 to December 1989) C. J. W. Brooks and D. G. Watson 391 Modern Separation Methods A. Marston and K. Hostettmann 41 5 Withanolides and Related Ergostane-type Steroids E. Glotter Number 5 441 Biosynthesis of C,-C, Terpenoid Compounds (1989) M. H. Beale 455 The Lycopodium Alkaloids (January 1986 to October 1990) W. A. Ayer 465 Marine Sterols (up to July 1990) R. G. Kerr and B. J. Baker 499 Diterpenoid Alkaloids (middle of 1985 to end of 1989) M.S. Yunusov Number 6 527 A Unified Mechanistic View of Oxidative Reactions Catalysed by P-450 and Related Fe-Containing Enzymes M. Akhtar and J. N. Wright 553 Indolizidine and Quinolizidine Alkaloids (July 1989 to June 1990) J. P. Michael 573 The Biosynthesis of Polyketides (January 1986 to December 1988) T. J. Simpson 603 Tropane Alkaloids (January 1990 to December 1990) G. Fodor and R. Dharanipragada Volume 9 Number 1 1 Diterpenoids (1990) J. R. Hanson 17 Pyrrole Pyrrolidine Piperidine Pyridine and Azepine Alkaloids (July 1989 to June 1990) A. R. Pinder 25 Quinoline Quinazoline and Acridone Alkaloids (July 1989 to June 1990) J. P. Michael 37 Steroids Reactions and Partial Syntheses (1989) A. B. Turner 8 1 Muscarine Oxazole Thiazole Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1989 to June 1990) J.R. Lewis Number 2 103 Angucycline Group Antibiotics J. Rohr and R. Thiericke 139 The Microbiological Transformation of Diterpenoids (1973 to June 1991) J. R. Hanson 153 The Biosynthesis of Shikimate Metabolites (1990) P. M. Dewick 183 Amaryllidaceae and Sceletium Alkaloids (1990) J. R. Lewis Number 3 193 Natural Products and the Sesquicentenary of the Royal Society of Chemistry Some Random Comments L. Crombie 199 Intracellular Steps of Bacterial Cell Wall Peptidoglycan Biosynthesis Enzymology Antibiotics and Antibiotic Resistance T. D. H. Bugg and C. T. Walsh 217 Natural Sesquiterpenoids (1989) B. M. Fraga 243 Clerodane Diterpenoids A. T.Merritt and S. V. Ley Number 4 289 Peroxy Natural Products D. A. Casteel 3 13 Pyrrolizidine Alkaloids (July 1990 to June 1991) D. J. Robins 323 Marine Natural Products (1990) D. J. Faulkner 365 P-Phenylethylamines and the Isoquinoline Alkaloids (July 1990 to June 1991) K. W. Bentley Number 5 393 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1990 to June 1991) J. E. Saxton 447 Biosynthesis of Polyketide Metabolites (1989 to mid 1991) D. O’Hagan 481 The Sesterterpenoids (July 1985 to October 1991) J. R. Hanson 491 Azetidine Pyrrole Pyrrolidine Piperidine and Pyridine Alkaloids (July 1990 to June 1991) A. R. Pinder Number 6 505 Appreciation Edward Leete 1928-1992 507 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (August 1989 to December 1990) R.B. Herbert 531 Monoterpenoids (1988 and 1989) D. H. Grayson 557 Natural Sesquiterpenoids (1990) B. M. Fraga 58 1 Steroids Reactions and Partial Synthesis (1990) J. R. Hanson 632 NATURAL PRODUCT REPORTS 1993 Volume 10 Number 1 1 Lignans Neolignans and Related Compounds (January 1989 to December 1991) R. S. Ward 29 Muscarine Oxazole Imidazole Thiazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1990 to June 1991) J. R. Lewis 5 1 Indolizidine and Quinolizidine Alkaloids (July 1990 ro June 1991) J. P. Michael 71 Microbial Pyran-2-ones and Dihydropyran-2-ones (up to December 1991) J. M. Dickinson Number 2 99 Quinoline Quinazoline and Acridone Alkaloids (July 1990 to June 1991) J.P. Michael 109 The Chemistry of Azadirachtin S. V. Ley A. A. Denholm and A. Wood 159 Diterpenoids (1991) J. R. Hanson 175 Chemical and Biochemical Manipulations of Nucleic Acids M. J. McPherson and J. H. Parish 199 Tropane Alkaloids (January ro December 1991) G. Fodor and R. Dharanipragada Number 3 207 NMR of Proteins M. P. Williamson 233 The Biosynthesis of Shikimate Metabolites (1991) P. M. Dewick 265 Biological Variation of Microbial Metabolites by Precursor-directed Biosynthesis R. Thiericke and J. Rohr 29 1 Amaryllidaceae and Sceletium Alkaloids (1991) J. R. Lewis 301 Stevioside and Related Sweet Diterpenoid Glycosides (up to Majj 1992) J. R. Hanson and B. H. De Oliveira Number 4 31 I Obituary David N. Kirk 1929-1992 313 Steroid Reactions and Partial Synthesis (1991) J.R. Hanson 327 Advances in Chemical Ecology (January 1988 to June 1992) J. B. Harborne 349 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1991 to June 1992) J. E. Saxton 397 Natural Sesquiterpenoids (1991) B. M. Fraga 421 Arsenic Compounds from Marine Organisms (up to October 1992) J. S. Edmonds K. A. Francesconi and R. V. Stick Number 5 429 Macrocyclic Trichothecenes (up to December 1991) J. F. Grove 449 P-Phenylethylamines and the Isoquinoline Alkaloids (July 1991 to June 1992) K. W. Bentley 471 Diterpenoid Alkaloids (December 1989 to January 1992) M. S. Yunusov 487 Pyrrolizidine Alkaloids (July 1991 to June 1992) D. J. Robins 497 Marine Natural Products (1991) D. J. Faukner Number 6 541 HMG-CoA Reductase Inhibitors (up to October 1992) A. Endo and K. Hasumi 551 A Survey of Natural Products which Abstract Hydrogen Atoms from Nucleic Acids J. A. Murphy and J. Griffiths 565 The Strobilurins Oudemansins and Myxothiazols Fungicidal Derivatives of P-Methoxyacrylic Acid J. M. Clough 575 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (1991) R. B. Herbert 593 Biosynthesis of Fatty Acid and Polyketide Metabolites (mid-1991 and mid-1992) D. O’Hagan 625 Cumulative Contents Volumes 1-10

ISSN:0265-0568    

DOI:10.1039/NP9931000625

出版商:RSC    

年代:1993

数据来源: RSC