摘要:

ISSN 0265-0568 NPRRDF 6(4) 311-432 (1989) Natural Product Reports A journal of current developments in bio -organic chemistry Volume 6 Number 4 CONTENTS 31 1 Enzyme Inhibitors in Medicine C. S. J. Walpole and R. Wrigglesworth Selectively reviewing the literature to the end of 1987 347 Diterpenoids J. R. Hanson Reviewing the literature published during 1987 359 Carotenoids and Polyterpenoids G. Britton Reviewing the literature published between January I986 and December 1987 393 Steroids Physical Methods D. N.Kirk Reviewing the literature mainly between mid 1985 and the end of 1987 405 P-Phenylethylamines and the Isoquinoline Alkaloids K. W. Bentley Reviewing the literature published between July 1987 and June 1988 NPR 6 Cumulative Contents of 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 I43 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 (July 1986 to June 1987) D. J. Robins 23 1 Fatty Acids and Glycerides (1986 and 1987) M. S. F. Lie Ken Jie 263 The Biosynthesis of Shikimate Metabolites (1987) P. M. Dewick 291 Limonene A. F. Thomas and Y. Bessikre Articfes that wifl appear in forthcoming issues include Triterpenoids (July I985 lo December 1987) J. D. Connolly and R. A. Hill Muscarine Oxazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1986 to June 1987) J. R. Lewis Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1987 zo June 1988) J. E. Saxton Indolizidine and Quinolizidine Alkaloids (July 1985 lo June 1987) M. F. Grundon Steroids Reactions and Partial Syntheses (November 1986 to October 1987) A. B. Turner Pyrrolidine Piperidine and Pyridine Alkaloids (July 1987 to June 1988) A. R. Pinder Recent Advances in the Use of Enzyme-catalysed Reactions in Organic Synthesis (January 1986 to June 1988) N. J. Turner Natural Sesquiterpenoids (1987) B. M. Fraga Coumarins (mid1980 to mid 1988) R. D. H. Murray Studies in Plant Cell Culture Biosynthesis and Synthesis of Indole and Bisindole Alkaloids J. P. Kutney

ISSN:0265-0568    

DOI:10.1039/NP98906FP011

出版商:RSC    

年代:1989

数据来源: RSC

摘要:

Natural Product Reports Editorial Board Professor G. Pattenden (Chairman) University of Nottingham Dr D. V. Banthorpe University College London Dr J. R. Hanson University of Sussex Dr R. B. Herbert U n iversi ty of Leeds Professor M. I. Page The Polytechnic Huddersfield Professor T. J. Simpson University of Leicester Natural Product Reports is a journal of critical reviews published bimonthly which is intended to foster progress in the study of natural products by providing reviews of the literature that has been published during well-defined periods on the topics of the general chemistry and biosynthesis of alkaloids terpenoids steroids fatty acids and 0-heterocyclic aliphatic aromatic and alicyclic natural products. Occasional reviews provide details of techniques for separation and spectroscopic identification and describe methodologies that are useful to all chemists and biologists who are actively engaged in the study of natural products.Articles in Natural Product Reports are commissioned by members of the Editorial Board or accepted by the Chairman for consideration at meetings of the Board. ~ ~~ Natural Product Reports (ISSN 0265-0568) is published bimonthly by The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge CB4 4WF England. 1989 Annual Subscription Price U.K. f169.00 Rest of World f194.00 U.S.A. $388.00. 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 11 003. US Postmaster send address changes to Natural Product Reports Publications Expediting Service Inc. 200 Meacham Avenue Elmont NY 11 003. Second-Class postage paid at Jamaica NY 11431 -9998. All other despatches outside the U.K. are by Bulk Airmail within Europe and Accelerated Surface Post outside Europe. Printed in the U.K. 0 The Royal Society of Chemistry 1989 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 1989 U.K. f169.00 Overseas f1 94.00 U.S.A. US $388.00 Subscription rates for back issues are U.K. (1984) f120.00 (1985) f125.00 (1986) f130.00 (1987) f142.00 (1988) f159.00 Overseas f1 26.00 f131.OO f143.00 f159.00 f183.00 U.S.A. US $240.00 US $242.00 US $252.00 US $280.00 US $342.00 Members of the Royal Society of Chemistrl should order th 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/NP98906FX013

出版商:RSC    

年代:1989

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP98906BX015

出版商:RSC    

年代:1989

数据来源: RSC

摘要:

Enzyme Inhibitors in Medicine C. S. J. Walpole and R. Wrigglesworth Sandoz Institute for Medical Research Gower Place London WCI E 6BN Selectively reviewing the literature to the end of 1987 1 Types of Enzyme Inhibition 1.1 Reversible Competitive In hi bi tors 1.2 Transition-State Analogues 1.3 Active-Site-Directed Irreversible Inhibition 1.4 ‘Suicide Substrates ’ 2 Examples of Enzyme Inhibition 2.1 Enzymes Related to Metabolism of Nucleosides 2.1.1 Anti-folates 2.1.2 Biosynthetic Enzymes 2.1.3 Nucleoside-Metabolism Analogues 2.2 Proteases 2.2.1 Thiol Proteases 2.2.2 Serine Proteases 2.2.3 Metalloproteases 2.2.4 Acid Proteases 2.2.5 Pro tease- Related Enzymes 2.3 Neurotransmission-Processing Enzymes 2.3.1 Acetylcholine 2.3.1.1 The carbamates 2.3.1.2 Organophosphates 2.3.2 Aromatic Amino Acid Neurotransmitters 2.3.3 y-Aminobutyric Acid (GABA) 2.4 Other Enzymes 2.4.1 Aromatase 2.4.2 H+/K+-transporting ATPase 2.4.3 Inhibition of the Biosynthesis of Icosanoids 2.4.4 Hydroxymethylglutaryl-CoA Reductase 2.4.5 Inhibition of the Biosynthesis of Vitamins 2.4.6 Miscellany 3 Future Prospects 4 References Ever since the identification of enzymes as biological catalysts scientists working with these macromolecules have unwittingly (and at first uncritically but latterly by design) been finding inhibitors.The study of the consequence of these inhibitors has had profound implications for medical practice.The introduction of such enzyme inhibitors has transformed attitudes to and expectations of the effects of many diseases and their ultimate outcome. Moreover such studies have led to a greater understanding of the aetiologies of disease. This review will implicitly attempt to cover both aspects of these advances namely the description of enzyme inhibitors which have found use in clinical practice and also the illustration of mechanistic information underlying such studies; we hope that this may act as a signpost towards future developments and targets. Any attempt to review exhaustively all of the contributions in this area (even over the past ten years) is precluded by considerations of space and indeed time and would in any event result merely in a catalogue of enzymes diseases enzyme inhibitors and their attendant clinical benefits.This would not serve the purpose which we would suggest is of greatest value in this exercise which is to identify the general principles of design of enzyme inhibitors. Accordingly this review is structured towards this end with an emphasis on molecular events rather than clinical detail. Also no account will be taken 31 1 in this review of the synthetic achievements that have been made in reaching many of the target molecules whilst always remembering that this is often the controlling factor in the whole design process. The subject matter will be split into two parts.The first section will review different types of enzyme inhibitors with examples ;these are not necessarily included for any therapeutic benefit they may impart but rather to illustrate this mechanistic classification. The second section will illustrate the best known examples of these inhibitors which have established or potential therapeutic value and will not attempt to be in any way exhaustive. These examples have been broken down into broad categories which it is hoped will assist the reader in locating a particular type of enzyme or disease. Consequently this is not a molecular-structural classification. There are also several examples which defy such classification and these have been collected together as a miscellaneous group at the end.The origins of enzyme inhibition lie in the beginnings of chemotherapy with Paul Ehrlich. As early as 1910 it was shown that amylase was inhibited by glucose,’ but the originating impetus developed from two areas of chemotherapy which occurred in the 1930s; first the discovery of the sulphonamides and the subsequent elucidation of their mode of action by Woods.2 This in turn led Fildes3 to present ideas of rational chemotherapy based on inhibition of enzymes by substrate analogues. These ideas laid the foundations for Hitchings’ classical work4 on purine and pyrimidine antimetabolites which resulted in several of the most successful clinically useful enzyme inhibitors. The second area of (seminal) importance was the chance discovery of penicillin by Fleming5 in 1928.Following the investigations of Florey and Chain6 at Oxford from which they obtained pure material the structure of penicillin G was identified in 1945. This has led to an effort over the past forty years which indeed still continues to develop more effective analogues. Over this period there have been many ‘spin-offs’ from this effort that have had important implications for the design of enzyme inhibitors. 1 Types of Enzyme Inhibition The emphasis of this review will be on inhibitors which are competitive (with the natural substrate) in their action. Consideration of other types of enzyme inhibition e.g. uncompetitive and non-competitive,’ allosteric,* and pharma- cological/physiological,gwill only be given to products that are of significant medical importance and these examples will not be discussed in mechanistic detail.The conventional representation of the productive interaction of an enzyme E with its normal substrate S to give product P is shown in Scheme 1 where K is the dissociation constant of the enzyme-substrate complex and k,, is the rate constant (first-order) for chemical catalysis (also known as the turnover number). The Michaelis constant Km which is derived from this equation (making the assumption that the binding steps are much faster than the step of chemical transformation) is only equal to K under these conditions. There are several examples where this is in fact not the case :however for the purposes of discussions of structure-activity relationships in medicinal NATURAL PRODUCT REPORTS 1989 -E+S E*S E*P E+P KS kcat ‘ef f ect ’ Scheme 1 E + S Z E*S -E-P -= E + P Km kcat E+I E-I -+* ‘effect’ Ki kcat =O Scheme 2 chemistry it is acceptable to equate the measured K with the true dissociation constant K, and this will be assumed in subsequent discussion.We can now consider the intervention of different types of inhibitor I in the process of Scheme 1. 1.1 Reversible Competitive Inhibitors This situation can be visualized as in Scheme 2 where Ki is the dissociation constant Of the enzyme-inhibitor complex’ The inhibitor ’ may ‘OmPete* with s for its binding site On to form a non-productive enzyme-inhibitor complex E-I.This serves to reduce the effective concentration of the enzyme- substrate complex E-S and thereby ultimately attenuates the ‘effect’ thus producing the observed physiological response to the inhibitor. Because of the competitive nature of such inhibition build- up of substrate in front of the block (which would be expected to occur in a multi-enzyme pathway) would ultimately overcome the effects of the inhibitor. From a consideration of Scheme 2 competitive inhibition can be represented mathematically and analysed graphically’ lo (e.g.by a Lineweaver-Burk plot or a Dixon plot). This enables Ki (the dissociation constant of the non-productive enzyme- inhibitor complex) to be calculated. Comparison of this value with that of K gives some measure of the tightness of binding of the inhibitor relative to a defined substrate (see however ref.11 p. 182). Structure-activity profiles of series of inhibitors can be built up from such data and often allow prediction of increased affinity (SAR Structure-Activity Relationships). A classical example which is often held up as exemplifying competitive inhibition is that of the sulphonamides (l) which compete with p-aminobenzoic acid (2) for its binding site on I and S do not have to bind to the same site on E; competitivity implies only that binding of S and of I is mutually exclusive. dihydrofolate synthase. However the situation is not as simple as that because under certain conditions the sulphonamides act as alternative (and dead-end) substrates.12 Another early example which is much clearer kinetically is the inhibition by malonate of succinate dehydrogenase.l3 More recently the inhibition of angiotensin-converting enzyme (ACE) [dipeptidyl carboxypeptidase I; E.C. 3.4.15.11 by the larger bradykinin- potentiating peptides (e.g. BPP, and BPP,,,) was shown to be competitive in contrast to that with the pentapeptide BPP,,.14 Many of the clinically relevant examples mentioned below were designed as and are believed to be reversible competitive inhibitors. 1.2 Transition-State Analogues An elegant extension of the design of inhibitors from consideration ofthe substrate S is the concept ofthe transition- state analogue which has developed from a consideration ofthe of into the product This idea was implicit in a far-sighted statement by Pauling15 in 1948 I believe that... the surface configuration of the enzyme is.. . complementary to the ‘ activated complex ’ for the reaction that is catalysed by the enzyme. The assumption that the enzyme has a configuration complementary to the activated complex and accordingly has the strongest power of attraction for the activated complex means that the activation energy for the reaction is less in the presence of enzyme than in its absence and accordingly that the reaction would be speeded up by the enzyme.. . . The picture even presents us with ideas as to the nature of substances which would be effective inhibitors -they shouZd resembZe the activated complex. This assertion was recognized developed and put on a semi- quantitative basis by Wolfenden in 1969.16 Figure 1 shows the free-energy profile of the reaction co- ordinate (0) for a one-substrate enzymic reaction and its non- enzymic counterpart where E -S and E Pare enzyme-substrate e and enzymeproduct complexes respectively TS is the tran- sition state of the non-enzyme-catalysed reaction and E -TS is that of the enzymic reaction.NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH I’ E TS ’\ AG E+S E+ P E*S E*P e Figure 1 E +S E + TS -KNc KTSll E-S E*TS KE* Scheme 3 The equilibria relating the transition states of a one-substrate enzymic reaction E + S E * S -+E.P $E + P and its non-enzymic counterpart S+P can be represented as a thermodynamic ‘box’ as shown in Scheme 3 where K, KTs,K,’ and K are the related dissociation constants.From this K,/KT = K,+/K,f. Since rates of reaction are related to transition-state dis- sociation constants by the Eyring equation l7 and assuming that the transmission coefficient is 1 then K,/K, = k:/k; where k is the first-order rate constant for the conversion of E-S into E-P and k is the first-order rate constant for the corresponding non-enzymic reaction. What this important equation means is that an inhibitor which resembles the substrate portion of the enzyme-transition- (3) state complex (in its binding properties) should be bound more tightly to the enzyme than is the substrate in the same ratio as the rate of the enzymic reaction exceeds the non-enzymic one.Obviously there are problems in reducing this idea to practice. It is unrealistic to make a stable analogue of an evanescent species with a lifetime of < seconds and often the model that is used seemingly without consideration is not the postulated transition state but a closely related high-energy intermediate. The Hammond postulate’*. l9 (where it is ap- plicable) which states that such metastable intermediates will be similar in structure to the transition state supports this extension of the original idea. Even with this constraint there are now many examples in the literature of the successful development of ‘transition-state analogues’. It is perhaps a moot point to question whether many of these examples actually fit the definition as outlined above.The practical end result has been the preparation and identification of very tightly binding inhibitors. An early example for the case of a one-substrate reaction as discussed theoretically above illustrates very clearly the application of the principle. This concerns inhibitors of proline racemase. 20*21 Proline racemase catalyses the reversible transformation of (3) into (4) and the observation that pyrrole-2-carboxylic acid (6) inhibits the enzyme by 50 O/O at a concentration 160-fold less than that of (3) prompted the suggestion that this compound resembles the postulated transition state (9,where C-2 assumes a planar configuration although the electronic analogy between (5) and (6) may also be important.This example is of a one-substrate case. That the principle applies to bi-substrate (and indeed multi-substrate) reactions has been established by Wolfenden22 and has been realized in practice. There is however one important difference between the one- and the two-substrate situation; an entropic factor. Formation of the bi-substrate enzyme complex from two isolated substrates involves loss of entropy from both sub- strates. If the two substrates are (artificially) joined prior to any interaction with the enzyme such as would be the case with the majority of conceivable transition-state analogues for such reactions then the entropic loss from such a ‘combined’ substrate would be less than in the normal situation.[The enzyme is precluded from collecting two isolated substrates from solution.] This difference akin to the differences in binding between monovalent and polyvalent ligands for their particular receptor has been estimated by Page and Jenck~~~ to be as large a factor as lo8mol dm-3 in terms of binding. This means that such a multi-substrate andogue would bind tightly independently of its resemblance to a transition state. Structural similarities with the latter would enhance the binding yet further. It is important from the point of view of design to make this distinction which is not always made clear in the examples in the literature. Examples of bi-substrate analogues that have therapeutic potential will be discussed below.J (4) e,u H COzH NATURAL PRODUCT REPORTS 1989 CH20H (NAG13 NHAc (NAG)3 NHAc + RO H CH20H I NAG)^ NHAC (7 1 Scheme 4 0-H L RI-C-NHR~ RI-c1%-NHR* + H2NR2 f I r GO-Enz 0-Enz 0 R'-C-H c..II -[R-rH I ] HO-Enz 0-Enz L OH J Hemiacet al It 0 II R'-C + HO-Enz I OH Scheme 5 Lysozyme hydrolyses glycosidic bonds of the polysaccharide The further examples given here serve to illustrate some component of bacterial peptidoglycan as shown in Scheme 4. general principles of design which have become important to This is an enzymic reaction involving two substrates (the medicinal chemistry as well as exemplifying the theory of second one is water) but the proposed oxycarbonium-ion-like transition-state analogues.transition state is obtained from the polysaccharide component Aldehydes that are structurally related to the acyl portion of only. This structure would exist in a half-chair conformation peptide substrates are potent inhibitors of serine protease~.~' It where C-1 and the oxygen of the ring are spz hybridized. The has been suggested that a hemiacetal is formed between the lactone (7) was designed as a transition-state analoguez4 on the active-site serine residue and the aldehyde. This tetrahedral basis that it would exist in a similar conformation and indeed intermediate is thought to resemble the transition state for it binds 40 times more tightly to the enzyme than does the hydrolysis of substrate and accounts for the tight binding substrate.25 It is interesting that the accepted mechanism is (Scheme 5).More recently direct evidence for such an called into question in a recent paperz6 on the basis of intermediate has been obtained using "F n.m.r.28 molecular-dynamics calculations but no mention is made An extension of this idea was first enunciated by Brodbeck who pointed out that trzJ7uoromethylketones therein of the enhanced binding by the transition-state analogue. and co-worker~,~~ NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH Scheme 6 would tend to exist as the tetrahedral hydrates (cf. chloral hydrate) and thus may act as transition-state analogues where it is suspected that there is sp3 hybridization in the transition state between trigonal substrates and products (e.g.in hy- drolysis of esters and amides). This was exemplified for acetylch~linesterase~~ and has since been extended to other enzymes. A similar explanation using this principle has been offered for the potent inhibition by boronic acids of chymotrypsin (see Scheme 6).30*31 Transition-state-analogue theory has become an accepted part of the medicinal chemist’s armamentarium and several extensive reviews of the subject are a~ailable.,~.~~ Several examples of designed or suspected transition-state analogues will be mentioned in the second part of this review. 1.3 Active-Site-Directed Irreversible Inhibition (ASDII) A criticism that has been directed at reversible inhibition of enzymes is that the inhibition can be overcome by increasing the amount of competing substrate.This may be expected to happen in the physiological situation where the target enzyme is likely to be a component of a multi-enzyme pathway (Scheme 7) i.e. competitive inhibitor I causes build-up of S (from S,) whicli ultimately over-rides the blocking effect of I,. This problem manifests itself either in the necessity to use large amounts of drug or to administer it frequently. B. R. Baker36 advanced the theory of active-site-directed irreversible inhibition as one way to get over this problem and his attempts to exemplify this theory over a period of ten years until his death in 1967 have had profound implications for s1 -sp -s3 medicinal chemistry.The basis of the idea is the Bridge Principle of Specificity Compared to a reversible inhibitor the active-site directed type of irreversible inhibitor can have an extra dimension of specificity; this extra specificity is dependent upon the ability of the reversibly bound inhibitor to bridge to and form a covalent bond with a nucleophilic group on the enzyme surface and upon the nucleophilicity of the enzymic group being covalently linked.36 In effect the idea is to generate potent inhibitors by combining active-site recognition features with a reactive functional group which is capable of covalently linking to the enzyme in question. The reactivity of the functional group is designed to be such that it allows a high rate of reaction at the active site (making use of the entropic advantage of the ‘local’ high concentration) whilst minimizing the rate of non-specific covalent interaction outside the target of interest.The process is illustrated in Scheme 8. Representative groups X which have been found (from experience) to work are a-haloketones diazoketones maleimides sulphonyl fluorides p-nitrophenyl esters and nitrogen mustards. These are all alkylating or acylating agents which are capable of reacting with the nucleophilic groups present in proteins. The necessary initial formation of a reversible enzyme- inhibitor complex followed by covalent modification (see Scheme 8) has kinetic consequences which are testable and diagnostic.,’ The inactivation should give a first-order time-dependent loss of catalytic activity.Analysis allows the separate evaluation of kinact (the rate constant for irreversible in- activation) and Ki(the dissociation constant of the initially formed enzyme-inhibitor complex). Another consequence is that substrate should protect from inactivation i.e. it should retard catalytic loss but because formation of the enzyme- substrate complex is reversible it should not ultimately prevent it. Establishing the irreversible nature of the complex can be achieved by dialysis or gel-filtration experiments using radiolabelled ligand when the label will remain attached to the macromolecule. Controlled degradation of the isolated complex and identification of the labelled peptide or amino acid product ~4 -’ *effect* I3 Scheme 7 Scheme 8 NATURAL PRODUCT REPORTS 1989 fPh TosNAC(0)CH2ClH 0 TPCK (81 (8a) .His-57 ASD -102 / Figure 2 TLCK (8b) may give an insight into the catalytic mechanism of the enzyme particularly when this experiment is coupled with sequence and crystallographic information about the structure of the enzyme. An early example which nicely illustrates all of these facets of ASDIIs is the work of Schoellmann and Sha~~~ on inhibitors of chymotrypsin. It was known that N-tosyl-L-phenylalanine ethyl ester (8) is a substrate for chymotrypsin ; a closely related analogue TPCK @a) incorporating a reactive chloro-ketone group was radiolabelled inhibitor identified the active-site residue that is covalently attached to the inhibitor as His-57.It was known from a wealth of experimental evidence that chymotrypsin cleaves ester and amide substrates by nucleophilic attack on the carbonyl carbon of the cleavable bond. From the experiment with TPCK it might be inferred that this active-site nucleophile would be His-57. This is in fact not the case -chymotrypsin is a serine protease the nucleophile is Ser- 195 (see Figure 2). This result illustrates the care necessary in interpreting such ex- periments where clearly small differences in orientation and reactivity of functional groups at the active site can select nucleophiles other than the one that is involved in the ‘normal’ catalytic process. TLCK (8b) is a compound that is closely related to TPCK and which was designed to inhibit tryp~in.~~ Trypsin is specific for cleaving peptides that have acyl groups containing basic side-chains (Lys and Arg).TLCK was found to inhibit trypsin irreversibly and selectively ;chymotrypsin was not affected. An inverse profile was observed for TPCK. A closely related inhibitor of chymotrypsin which was designed to (temporarily) form the acyl intermediate as a prelude to irreversible inhibition has been described.40 Although several irreversible inhibitors are used clinically (particulary against cancer) there is criticism of and scepticism about their value. It is argued that such molecules will be toxic or will be removed by scavenging sacrificial nucleophiles as a consequence of their intrinsic reactivity.That these arguments cannot be sustained is shown by the fact that these compounds are indeed drugs. Another criticism aimed at such compounds is that ir-reversible inhibition of essential enzymes is dangerous per se when the only way to replace inactivated material is by ‘expensive’ synthesis of protein. Again this argument should not be generalized as synthesis turnover rates vary dramatically. However attempts to overcome these criticisms without losing the potential of irreversible inhibition whilst gaining specificity have led to the mechanism-based irreversible inhibitors -the ‘suicide substrates’. 1.4 ‘Suicide Substrates’ A ‘suicide substrate’ is an enzyme inhibitor which although initially innocuous is activated by the target enzyme itself by using some part of the normal catalytic mechanism that is effected by the enzyme.The activated species then binds irreversibly to the enzyme and thereby destroys it. Semantic and indeed pedantic argument has followed this nomenclature and the above term has been criticized as inaccurate because the enzyme is a victim of deception whereas suicide is considered to be a voluntary act. The alternative suggestion is Trojan Horse Inhibitor ! These anthropomorphic aphorisms apart more prosaic representations of these compounds are Mechan- ism-Based Inhibitors or Latent Alkylating Agents. made as a candidate irreversible inhibitor. When incubated The general process is illustrated in Scheme 9. Comparison with the enzyme TPCK causes irreversible inhibition with of Scheme 9 with Scheme 8 which illustrates the mode of action kinetics consonant with ASDII.A subsequent experiment with of ASDIIs shows the value of the suicide-substrate approach. - E+S E-S E*P E+P Km kcat - - E t I €01 E*I*“ E + 1’ Ki kcat (11 kdiff kinac t E-I Scheme 9 NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH Ph Em -OH -)-C(0) /cp" )-C(O)N cTN =O C02-Enz H fPh N=N+ 3- fPh N=N-OH -A /PhHN-N=O Lf - PhCH; + Enz PhCH2-Enz Scheme 10 The reactive species I* is generated locally at the active site of the target enzyme and should therefore be selective for the target. This gets over one of the main criticisms of ASDII. The possibility still exists however that the activated species I* can diffuse away from the active site and not be covalently captured by it.A convenient measure of this is the partition ratio kdifl/kinael (see Scheme 9). Maximum selectivity is obtained when the partition ratio is small. This cannot be designed for however; it is dependent upon the nature of the amino-acid residues at the active site. An example which illustrates the selectivity inherent in this approach is the inhibition of chymotrypsin by N-nitroso-pep tide^.^^. 42 The L-phenylalanine-derived nitroso-amide (9) was designed as a potential suicide substrate of chymotrypsin. The proposed mode of action of (9) which is initiated by enzyme-catalysed cleavage (resulting in formation of a car- bonium ion at the active site) is illustrated in Scheme 10.However on testing ~-(9) against the enzyme there was no inhibition although ~-(9) was cleaved by the enzyme. In contrast and most surprisingly the D enantiomer of (9) efficiently and irreversibly inhibited the enzyme after initial cleavage had taken place. Because both D and L forms of (9) are cleaved the differentiation of effect must occur at the stage of formation and/or reaction of the carboniurn ion. An explanation based on studies of molecular models has been offered in which it is suggested that the carbonium ion precursor (10) is generated at the surface of the enzyme when ~-(9) is cleaved and hence is able to escape into the solvent before it has chance to bind to the enzyme.However ~-(9) is able to bind to the enzyme in a different mode (which still allows enzymic cleavage) where the unit (10) is embedded in the hydrophobic cleft of the active site and is therefore not free to escape. An attempt to illustrate this schematically is given in Figure 3. There are many other examples in the literature and some will be discussed below. Because this subject has been extensively and well reviewed over the past ten a detailed list will not be provided here but some generalizations (Ph flescape C-N-N=O 0-H '///,lg5 ///// -I L -(9) Ph 0 0 0-H /< / Figure 3 about the design of suicide substrates are worth a brief commerl t . Because the reactive groups available to (apo)enzymes are nucleophiles it follows for covalent addition of reactive species to the enzymes itself that the reactive species needs to be some form of electrophilic moiety.To date the generation of these species (using the catalytic mechanism of the enzyme) falls into a few distinct categories such as the rearrangement of acetylenes to reactive allenes (via carbanion intermediates with subsequent protonation) the generation of Michael acceptors from alkenes and haloalkyl derivatives and rearrangements subsequent to the formation of an acyl-enzyme intermediate (e.g. for many proteases). Several others are described in the literature (see particularly ref. 47). In contrast a less common mode of inactivation has been via covalent labelling of an essential co-factor and these examples have involved nucleophilic attack by the generated active species on the electrophilic cofactors (e.g.flavin and pyridoxal).Clearly there is scope both inside these two mechanistic constraints and outside (e.g. generation of free radicals) for innovative design using the mechanisms of the enzymes concerned. This completes the brief survey of the different types of enzyme inhibitors. In the second section of this review these categories will be illustrated by examples of therapeutic interest. 2 Examples of Enzyme Inhibition The design of enzyme inhibitors as drugs has followed the routes common to the development of all such entities namely (i) Random screening of miscellaneous chemicals. This relies on a high throughput of compounds (> 10000 per annum) on there being a fast and reliable screening procedure and on chance.(ii) Molecular modifications of existing active (‘ lead ’) struc-tures. These prototypes may either be natural (e.g. hormones and vitamins) or synthetic (e.g. drug structures of proven activity). (iii) Development of new indications from the observation of side effects of existing drugs. These usually arise from clinical observations and the identification of new indications is not a design process per se; however the result is often the divergence of structure-activity profiles and the identification of alternative molecular targets e.g. the original development of allopurinol as an adjunct agent for leukaemia therapy resulted after clinical trials in its introduction as an anti-gout agent and the identification of xanthine oxidase as a therapeutic target.(iv) Leads from metabolites of existing drugs e.g. the cycliza- tion of the acyclic compound proguanyl to the active antimalarial agent and inhibitor of dihydrofolate reductase cycloguanyl (Scheme 11). (v) Screening of natural products. This can be considered a specific example of (i) and is practised by many pharmaceutical companies. Examples of success are the monobactam anti- biotics and the antifungal immunosuppressant cyclospbrin. The application of this approach for the identification of enzyme inhibitors has been pioneered by Ume~awa,~O whose group has provided notable leads such as leupeptin pepstatin phosphoramidon bestatin and mevinolin among others.(vi) Developments from biochemical hypotheses. The so-called ‘logical’ approach! In fact this approach relies on astute observation of the other approaches combined with directed- hypothesis testing of the resulting clues. Many quoted examples are post hoe rationalizations. The pros and cons of these variations are discussed endlessly and value judgements are more often than not subjective. The results of each approach must speak for themselves. Suffice it to say that (i) and (ii) have been the earlier and more classical ways to drug design where chemists did not have to define a biological rationale in order to make whereas the more recent approaches [particularly (iii) (iv) and (vi)] necessitate close interaction of biologist and chemist.NATURAL PRODUCT REPORTS 1989 ‘I‘ n ProguanyI Cyclo guany l Scheme 11 The illustration of these approaches and of the types of inhibitor that were discussed in Section 1 will be broken down into four categories 1. Enzymes related to metabolism of nucleosides 2. Proteases 3. Neurotransmission-processing enzymes 4. Others. Within this crude classification individual target enzymes and clinically useful inhibitor drugs will be indicated in emboldened type. The miscellaneous category 4 above will be arranged simply in alphabetical order of enzyme target for want of any other more coherent method of grouping. 2.1 Enzymes Related to Metabolism of Nucleosides It is convenient to split this group into two biosynthetic enzymes and processing enzymes as targets.Although folate analogues are structurally unrelated to the nucleosides the ultimate therapeutic influence of anti-folates is on nucleoside metabolism so folate analogues are included here as a separate group. 2.1.1 Anti-folates Folic acid (11) is a vitamin i.e. a dietary requirement for mammals. The active cofactor forms of folate are derived from this via the key intermediate tetrahydrofolate [H,folate (14)] and function as one-carbon-transfer agents in a variety of important metabolic processes. This transfer of C units at different levels of oxidation is particularly important in the biosynthesis and metabolism of precursors of nucleic acids and for this reason it is included here.In contrast to mammalian systems which require dietary folate the majority of micro-organisms (in particular the bacteria but also some protozoa e.g. the sporozoan parasites of the genera Plasmodia Toxoplasma and Eimeria) cannot take up folate from exogenous sources and are totally dependent on this obligate material for survival. This exemplifies a general therapeutic principle for the development of anti-infective agents which is discussed more fully below. This difference between parasite and host is illustrated in Scheme 12. All of the biosynthetic enzymes leading to the production of dihydrofolate (H,folate) are potentially good therapeutic targets but the most widely exploited is dihydropteroate synthase (E2 in Scheme 12).This enzyme catalyses the nucleo- philic substitution of p-aminobenzoic acid [pAB (1 2)] into an activated (by ATP) dihydropterin to give 7,8-dihydropteroic acid (13). Sulphonamides [see structure (1) above] block this step by competing with pAB for the pterin. Comprehensive reviews on all aspects of sulphonamides are available52* 53 so no discussion is detailed here. Pyrimidines have also been described as inhibitors of this Inhibition of the previous enzyme in this pathway hydroxy-methyldihydropterin pyrophosphokinase [2-amino-4-hydroxy- NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH Parasite Host GTP Diet 0 folate (11) El [+ATPI 0 H 2folate [+L-GIuI 0 E3 1 Hzfolate (14) Cofactor forms FUNCTlON Scheme 12 6-hydroxymethyldihydropteridinepyrophosphokinase] (El in Two types of inhibitor of DHFR have been described.The Scheme 12) which activates the dihydropterin as the pyro- 'small molecule ' inhibitors are exemplified by the antibacterial phosphate by substrate analogues has been described5' but agent trimethoprim (15) and the antimalarial compound the inhibitors do not have antibacterial activity. In contrast pyrimethamine (1 6).57The 'large molecule ' substrate-analogue synergistic effects are observed when these inhibitors are used in combination with other anti-folate~.~~ In practice a more important target has been dihydrofolate reductase (DHFR) (E3 in Scheme 12). At first sight this enzyme would appear to be less attractive than the above examples because it catalyses a reaction which is common to H~N OMe parasite and host (see Scheme 12) but it is clear that this OMe H2N enzyme differs dramatically from species to species and indeed it is one of the most studied of all enzymes.(15) (16) (17) R =H (18) R =Me Table 1 Values of IC;, for inhibitors of dihydrofolate reductase in three classes of organisms IC,,/mol dm-3 x 1O-’ Inhibitor Bacteria Malaria Mammals Trimethoprim (15) 0.5 7 26000 Pyrimethamine (16) 250 0.05 70 Methotrexate (18) 0.01 0.07 0.02 (a) IC, is the concentration of an inhibitor that is required for 50% unhibition (see ref. 60). OH I @ = -P=O I OH NATURAL PRODUCT REPORTS 1989 inhibitors illustrated by aminopterin (17) and methotrexate (18) which were first made in 1947 are still important therapeutic agents in the treatment of childhood leukaemia~.~~ The remarkably broad spectrum of anti-infective activity resident in these different types of inhibitor of DHFR reflects the importance of the tetrahydrofolate cofactor in one-carbon- transfer reactions (see below) ;the latter are clearly important to all organisms.Why then are these compounds not toxic to the human host? In fact methotrexate is a very toxic agent and its use in cancer chemotherapy has to be carefully controlled and monitored to achieve the best balance of benefit from side effects. A recent development in the clinical use of methotrexate is leucovorin-salvage therapy.The potentially lethal shut-down of one-carbon folate-mediated metabolism by large doses of methotrexate can be reversed by the subsequent addition of 5-formyltetrahydrofolate (leucovorin folinic acid citrovorum factor) which provides a reservoir of one-carbon units that is able to rescue the blocked metabolism of the host.jS In contrast the ‘small molecule’ inhibitors are selective agents (see Table 1).60 These differences in potency reflect real differences in the molecular structure of the particular enzymes which the ‘small molecule’ inhibitors in contrast to metho- trexate are able to exploit. This has been established more recently by X-ray crystallography of several enzyme-inhibitor c~rnplexes.~’-~~ The observation and exploitation of these enzymic differences in DHFR culminating in the development L -ASP lE2 HA LPRPP= phosphoribosyl diphosphate 1 Scheme 13 NATURAL PRODUCT REPORTS 19894.S. J. WALPOLE AND R. WRIGGLESWORTH 32 1 PRPP +-Gin Glu I W" -E4 LGln Gl u Fum Asp I+ HCOfI u- E8 E7 E6 " N H2' I @R @R Al CAR E9 I+'C1'] J 0 Fum = fumarate IMP (20) Scheme 14 of several anti-infective agents have been uniquely productive in the history of medicinal chemistry aimed at target enzymes. The X-ray-crystallographic results of the methotrexate-NADPH-DHFR ternary complex" suggested the possibility of a stereospecific mode of transfer of hydride from the reduced cofactor to C-6 of the inhibitor (and by implication to the substrate HJolate).In fact stereochemical of the reduction of H,folate by DHFR have established that the reduction at C-6 occurs on the opposite face of the pyrazine ring to that predicted from the X-ray work. The only possible rationalization of these two sets of results is that methotrexate binds 'upside-down' relative to the substrate at the active site. This difference in binding which can be rationalized post hoc in terms of individual interactions of functional groups between molecules which are at a cursory glance so similar strikes a note of caution in the design of analogues based on the structure of the substrate. In fact methotrexate contains a 2,4-diamino-substituted pyrimidine (as does trime tho prim which binds in the same orientation to the enzyme as methotrexate) which significantly alters its chemico-physical properties relative to the 2-amino-4-oxopyrimidine-containingsubstrate dihy-drofolate.Attempts to improve on the design of trimethoprim using the binding information available from X-ray crystallography illustrate another of the pitfalls of rational drug design. An appropriately placed carboxyl group that was introduced into the trimethoprim structure interacted as predicted with a positively charged site on the bacterial enzyme (Arg-57) and PALA (21) Scheme 15 increased the tightness of the binding of the analogue to the enzyme by a factor of 15.65 That this increase in binding was not reflected in antibacterial activity is presumably because of the inability of this negatively charged molecule to penetrate the bacterium.Recently,66 suicide-substrate inhibition of DHFR by a spirocyclopropyl-pteridine has been reported. 2.1.2 Biosynthetic Enzymes A somewhat artificial distinction allows the separation of nucleotide anabolism and catabolism. For the purposes of this discussion the biosynthesis of pyrimidines (Scheme 13) is defined as the synthesis of uridylic acid (UMP) (19) and the biosynthesis of purines (Scheme 14) as the formation of inosinic acid (IMP) (20). Further transformations of the intact nucleotides are presented in Section 2.1.3. The importance of these biosynthetic pathways is obvious and in principle each of the constituent enzymes of the pathways is a good target if elements of selectivity can be achieved.The relative importance of these pathways in relation to the salvage of pre-formed nucleosides varies from organism to organism and provides some basis for this selectivity. One noteworthy difference between the two pathways is the point of insertion of the sugar phosphate moiety; i.e. early in purines late in the pyrimidines. This has implications for design because phosphorylated analogues rarely penetrate cells intact. The second enzyme in pyrimidine biosynthesis aspartate transcarbamylase (ATC'ase) (E2 in Scheme 13) has been extensively studied because of its allosteric properties and it is also an important point of inhibition of the formation of pyrimidine nucleotides.This is illustrated by N-(phosphono- acety1)- a as par tic acid (PALA) (21) a potent inhibitor of the enzyme which was rationalized as a transition-state anal~gue~~.~* (Scheme 15). Mechanistically the transition state for such a reaction would resemble the tetrahedral intermediate addition product whereas PALA has a trigonal carbon at the reacting centre. Given the fact that the Ki of PALA is N mol dm-3 it is perhaps a moot point whether PALA should be classified as a true transition-state analogue or as a 'collected-substrate ' inhibitor (see below). Although PALA clearly lowers the size of pools of pyrimidine nucleotide~,~~ resistance characterized by large compensating increases in ATC'ase activity occurs readily in murine le~kaemia,~' and clinical anti-cancer studies have been disappointing with the appearance of gastro-intestinal dermatological and neurotoxicological side effects." The fourth enzyme in Scheme 13 is dihydro-orotate de- hydrogenase; in contrast to other enzymes of the pathway this is a particulate enzyme which is linked to oxygen via the electron-transport chain.There are no good substrate-analogue inhibitors of the enzyme but hydroxynaphthoquinones which are antagonists of ubiquinones are potent inhibitors by virtue of the fact that they shut down the electron-transport chain.72 These compounds are powerful antiparasitic agents possibly because of their ability to block the biosynthesis of pyrimidines. 322 0 11H2 NC H HO HO OH S 02Nx) Me I H 6 -Mercaptopurine (23a) Atathioprine (23 b) The last two enzymes of the pathway shown in Scheme 13 i.e.orotate phosphoribosyltransferase (OPRT'ase) and orotidine monophosphate decarboxylase (ODC'ase) [orotidine-5'-phos- phate decarboxylase] are found together as a complex. An inhibitor that has powerful biological in vivo is pyrazofurin (22) which is a C-nucleoside that has been isolated from Streptomyces species. Structurally (22) has more resemblance to the intermediate AICAR of purine biosynthesis (see Scheme 14) but it has been shown that the molecule blocks the enzyme complex for the biosynthesis of pyrimidines at the decarboxylation Inhibition of this target enzyme by pyrazofurin exemplifies a general principle that is widely prevalent in nucleotide metabolism which is that the inhibitor molecule requires activation to the corresponding nucleotide prior to it being able to exert its effects.5'-Phosphorylation (by various kinases) of nucleoside analogues is very common and is crucial to the therapeutic benefits these molecules impart in that the pre- formed nucleotides are incapable (with rare exceptions) of penetrating cellular barriers and it is therefore critical that the activation process occurs once the nucleoside has penetrated the target cell. Pyrazofurin is phosphorylated by adenosine kinase an enzyme totally unconnected with the final enzyme target. This situation whereby analogues are recognized as substrates or inhibitors by widely differing enzymes makes design aimed R R Adenosine [KM= 31 x 10-6mol dm-3] NATURAL PRODUCT REPORTS 1989 at a particular target very difficult and it is still the case today that the therapeutic successes in this area of metabolism generally arise from acute observation of the effects that are produced by molecules that were originally designed for entirely different reasons.The most important inhibitor of the biosynthesis of purines is 6mercaptopurine (23a) which works by an indirect mech- anism. After activation of the base to the nucleotide by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT'ase) [hypoxan t hine p hosphori bosyl transferase] the latter acts as an allosteric inhibitor of the first enzyme of purine biosynthesis i.e.phosphoribosyl pyrophosphate amidotrans- ferase [amidophosphoribosyltransferase] (El in Scheme 14).74 6-Mercaptopurine is an effective anti-cancer agent particularly for acute childhood leukaemia. The molecule is rapidly inactivated by xanthine oxidase and various attempts to find sustained-release pro-drug forms of 6-mercaptopurine that were resistant to this degradation resulted in azathioprine (23b).75 Although this slow-release form of 6-mercaptopurine was disappointing as an anti-tumour agent the attendant immunosuppressant properties of this molecule have assumed importance; until the discovery of the peptide antibiotic cyclosporine azathioprine was the agent of choice in transplant surgery. 2.I .3 Nucleoside- Metabolism Analogues There are a large number of transformations of the biosynthetic end-product nucleotides UMP (19) and IMP (20) and many of the enzymes catalysing these transformations are important therapeutic targets.In particular there are a large number of anti-infective agents which act on one or more target enzymes in this area. It is interesting that many of these enzymes are promiscuous and it is often difficult to pin-point the biochemical site of action of these therapeutic agents. There are many reviews a~ailable~~-~* and only important selected examples will be detailed here. Adenosine deaminase (adenosine aminohydrolase) is an enzyme of purine catabolism which is important in the deactivation not only of endogenous adenosine but also of several chemotherapeutic purine analogues (e.g.ara-adeno-sine). Inhibitors of this enzyme may be useful in combination with such therapeutic agents as protectors of the latter from such metabolic inactivation thereby prolonging their period of effectiveness.'I9 The designing of inhibitors to this end has followed widely differing paths. One of the earliest examples purporting to illustrate transition-state-analogue theory was the observation 0 G,A N7 I R Inos ine = 160x 10' 6mot drn-3] R R (21d (24b) [Ki = 0.76 x lO*mol dm-31 [Ki ~12.5x 10-6mol dm-3] Scheme 16 323 NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH virat 'thymidine) ACV -8 y2 CH /I HoTl HO R H,,C6 CH(0H)Me (25) (26) EHNA 0 S# r-i 8I 1 HO OH (27) ACV that the methanol addition product (24a) to purine ribo- nucleoside but not the diastereoisomer (24b) was a potent inhibitor of adenosine deaminase (Scheme 16).These results were rationalized by suggesting the similarity of (24a) to the stereospecifically formed tetrahedral water-addition-product intermediate of the enzyme-catalysed reaction which closely resembles in structure the transition state.so Coformycin (25; R = OH) and deoxycoformycin (25; R = H) are naturally occurring antibioticsa1 which are very potent inhibitors (K,-mol dm-3) of adenosine deaminase. These structures are suggested to be good models of the above tetrahedral intermediate.34 Parenthetically similar transition-state analogues for the closely related enzyme cytidine deaminase have been designed by strict analogy with structures (24a) and (25).82* 83 Another approach to inhibitors of adenosine deaminase namely the development of erythro-hydroxynonyladenine (EHNA) (26),is a classical story in medicinal hemi is try.^^ The rational stepwise development of (26) from the structure of the normal substrate adenosine illustrates an elegant topographical exploration of the active site of the enzyme which resulted in the identification of particular binding domains (a hydrophobic region a hydroxyl-binding site and a specific methyl-binding site).EHNA is the culmination and combination of features which recognize these binding sites and is a very potent enzyme inhibitor.As an extension of these studies Schaeffer and co- workers designed a series of active-site-directed irreversible inhibitors84 which exploited the existence of the hydrophobic binding site that had been defined by the earlier work. These investigations have continued to be fruitful. Further structure- activity studies on inhibitors of adenosine deaminase led to a compound which has transformed the whole approach to the chemotherapy of viral disease. In order to evaluate quan- titatively whether putative inhibitors of adenosine deaminase would be valuable as 'adjunct agents' to prolong the metabolic half-life of existing anti-viral agents it was considered important to evaluate the intrinsic anti-viral activity of the inhibitors themselves.This situation provided the back-drop to the development of acyclovir (ACV) (27) (Zovirax) which is an anti-herpes agent of unparalleled importance. Following on from the development of EHNA the pro- gramme of synthesis had continued and truncated nucleoside structuress5 had been investigated [see (27)] which on testing showed exciting anti-viral activity against herpes in culture and ultimately clinically in Man. Carrying the trade name Zovirax (27) became the first effective treatment for genital herpes and for the serious herpetic complications commonly kinase' Host' enzyme lr ACV -@@ nost' enzyme 1' ACV -@@@ Scheme 17 5' CH2 5' -end Ude IH 0 I 0-P=O Growing DNA II Chain I O-5' CH;! 10jhy 3' -end 5' CH2 Gua ACV -@@@ -No 3' -OH for further coupling :.Chain Terminat ion Scheme 18 occurring in immunosuppressed patients. At the time of its development the mode of action of acyclovir was unknown. The unravelling of this web by Elion and c~-w~rker~~~~~~ established a principle in anti-viral chemotherapy -that there are ex$oitable differences between mammalian host enzymes and their virally induced counterparts. This has since proved useful and no doubt will be important in the future. Acyclovir is an effective non-toxic anti-viral agent because it is selectively concentrated in the virally infected cell where it is phos- phorylated to the monophosphate ACV- @ by a virally induced 'thymidine kinase'. The host's counterpart enzyme is unable to effect this change and so ACV-@ is not produced in uninfected cells.Host kinases then further phosphorylate the mono-phosphate to the triphosphate ACV- . The compound is now primed to do its damage (Scheme 17). The virally encoded DNA polymerase mistakenly recognizes ACV- as one of its natural substrates GTP. In fact the analogue acts as a substrate and also as a time-dependent inhibitor of the As a substrate it is incorporated into the growing DNA chain but because of the lack of a 3'-hydroxyl group in acyclovir it is not possible to form the next phosphodiester link and consequently chain termination occurs (Scheme 18). The R (28) R =H (29) R =NHz Me OH N3 OH (30) (31) AZT -& AZT-@ I A2 T-@@ Viral RNA DNA +-Viral Enzymes i thymidine kinase ; ii induced reverse transcriptase Scheme 19 0 NATURAL PRODUCT REPORTS 1989 small pieces of DNA so formed are non-functional and ultimately lead to inhibition of viral replication.As a consequence of this novel biochemistry but also because of the commercial success of acyclovir an enormous amount of effort has gone subsequently into developing other analogues aimed at this molecular target. The pharmacokinetic properties of acyclovir are not ideal and an elegant approach to this problem has been the development of enzyme-activated pro-drug forms (28) and (29). The compound (28) is eighteen times more soluble in water than acyclovir and is activated to it by xanthine oxidase.88.89 The amino-compound (29) is similarly activated by adenosine deaminase to acyclovir; oral dosing in dogs and rats achieved plasma concentrations of acyclovir greater than those that were observed after equivalent oral doses of the parent drug itself.90 A derivative of acyclovir called gancyclovir (30) has anti- herpes activity similar to acyclovir in vitro but unlike the parent drug is also highly active against human cytomegalo- virus.87, 91 It is interesting that (30) is stereuspeczjically phosphorylated by HSV-I thymidine kinase and that it is only the triphosphate derived from this enantiomeric monophosphate that is a potent inhibitor of the viral DNA p~lyrnerase.~~ Many other closely related compounds have been reported which show varying degrees of anti-viral activity as a consequence of this unique activation-inhibition sequence.g3 If herpes can be regarded as a ‘topical’ disease of the 1970s then most certainly AIDS is its counterpart of the 1980s.An enormous effort has gone into finding agents which inhibit the human immunodeficiency virus (HIV). Compounds which seem at present to be effective are nucleoside analogue^.^^^ 94 The mode of action of these analogues seems to exemplify the principle established by the action of acyclovir. For example 3-azido-3-deoxythymidine (31) (AZT Retrovir Zidovudine) is activated by a cytosolic thymidine kinase to the monophosphate (see Scheme 19) which is further phosphorylated to the triph~sphate.~~ This activated species AZT triphosphate is a potent inhibitor of the viral reverse transcriptase [RNA-directed DNA polymerase] causing termination of a DNA chain (cf.acyclo~ir).~~ Presumably the other effective nucleoside analogues such as 2’,3’-dideoxycytidine act in a similar manner.91 The rapid oxidative inactivation of the anti-cancer agent 6- mercaptopurine by xanthine oxidase has been mentioned above. Besides the development of azathioprine another approach to avoiding this degradation was to co-administer an inhibitor of xanthine oxidase. The inhibitor that was used was allopurinol (32). This combination showed improvement over 6-mercapto- purine alone but more importantly showed a second effect of the allopurinol. 0 0 II H xo _c (32) (36) ( XO = xanthine oxidase ) Scheme 20 NATURAL PRODUCT REPORTS 1989-C.S. J. WALPOLE AND R. WRIGGLESWORTH H fol ate 5,lO-CH2- H,folate TS Hzfolate T dUMP dTMP Enzymes TS = thymidylate synthase; DHFR = dihydrofolate re- ductase;SHMT = serine hydroxymethyltransferase Scheme 21 The physiological substrate for xanthine oxidase is in fact hypoxanthine (33) which is first oxidized at C-2 to give xanthine (34) and then at C-8 to give uric acid (33 as shown in Scheme 20. Excessive production of uric acid occurs in gout and crystallization of salts in joints causes the acute pain which is characteristic of the disease. Allopurinol (Xyloric) is effective in alleviating this by acting as a suicide substrate. Oxidation occurs at C-2 to give alloxanthine (36) but further oxidation is I’ R2 HS-Enz + H2fo(ate 0 HNyMe OAN I R2 + HS-Enz precluded by the transposition of the atoms in the five-membered ring.Alloxanthine binds tightly to the enzyme presumably by chelation to the molybdenum at the active site and thereby inactivates the The folate cofactors act as one-carbon-transfer agents in several enzymic reactions connecting the metabolism of amino acids and nucleic acids.’ One of the most important of these is the reaction catalysed by thymidylate synthase i.e. the methyla- tion of 2’-deoxyuridylic acid (dUMP) to thymidylate (dTMP) which is a key precursor in the biosynthesis of DNA. This reaction which is formally a transfer of a one-carbon unit at the oxidation level of methanol is mediated via 5,lO-rnethylene- tetrahydrofolate as the cofactor.The reductive step is achieved by concomitant oxidation of the cofactor. The cycle is illustrated in Scheme 21. This is the only tetrahydrofolate-mediatedone-carbon-unit transfer which involves the dihydrofolate oxidation level and clearly this reaction will be of importance in depleting tetrahydrofolate pools when DHFR is blocked. The mechanism of the reaction has been studied extensively100-102 and collation of this information has established that there is addition of an enzymic thiol group to the pyrimidine followed by attack of the resulting enolate anion (not shown in Scheme 22) on the methylenetetrahydrofolate cofactor. The details of the sub- sequent transfer of the ‘formaldehyde’ unit and its reduction to give the methyl group are still the subject of debate but can be represented as shown in Scheme 22.I R2 R2 L Scheme 22 I R 0 HO b; R=H CLEAVA GE 4 trate p3 p2 PfP3 Enzyme Figure 4 Figure 5 5-Fluorodeoxyuridylic acid (37a) is a potent inhibitor of thymidylate synthase by virtue of the fact that it is processed as a substrate to the point along the reaction co-ordinate where loss of a proton from C-5 would occur with the normal substrate. Because F' cannot be lost the ternary complex is stabilized and the enzyme is thereby inactivated.lo3 5-Fluorouracil (37b) is activated to the nucleotide level (37a) by various enzymes in vivo and has been extensively used as an anti- tumour agent.Other inhibitors of thymidylate synthase have been described all of which seem to function by forming non-productive ternary complexes. lo4*lo5 With the availability of an X-ray crystal structure,lo6 the designing of further inhibitors of this important therapeutic target should be forthcoming. This completes this brief summary of this area of metabolism except to indicate two particular areas where the future development of enzyme inhibitors might have important therapeutic application. S-Adenosylmethionine (SAM) acts as a cofactor in a whole variety of methyl-transfer reactions in a similar way to the various folate coenzymes which act as one- carbon-transfer agents.lo7 Indeed these two crucially important metabolic areas can be seen as intermeshed cogs relating transformations of nucleic acids and of amino acids where the NATURAL PRODUCT REPORTS 1989 connecting link is the formation of methionine from L-homocysteine using 5-methyltetrahydrofolate as the cofactor.Selective blocking of SAM-mediated methyltransferase ac- tivities which are important inter alia in the methylation of macromolecules (post-transcriptional methylation of RNA post-translational methylation of proteins) and in phospholipid and catechol metabolism must be valuable therapeutic aims.lo8 Blocking of the above-mentioned 'folate-SAM link ' en-zyme 5-methyltetrahydrofolate-homocysteinemethyltransfer-ase would have profound effects on the supply of these vital cofactors.lo9 The other area of potential exploitation is biochemically connected to SAM metabolism -this is the biosynthesis of polyamines. After a flurry of interest in designing inhibitors of the various enzymes mediating the synthesis of spermidine and spermine,l'O the area has once again become quiescent. The involvement of polyamines in the control of the synthesis of macromolecules111 and their consequent connection to cellular proliferation112,113 are potential targets for therapeutic in-terven tion. 2.2 Proteases Proteases are enzymes which catalyse the cleavage of specific peptide amide bonds within a polypeptide substrate. A common notation and clas~ification"~ of such processes is illustrated schematically in Figure 4.The detailed mechanisms of action vary from enzyme to enzyme but all are encapsulated in the representation shown in Figure 5. The identities of the nucleophile 'Nu ' and of the polarizing moiety '8' ' can be used to classify all of the known proteases into four classes 1. Thiol proteases for which Nu = SH e.g. papain. 2. Serine proteases for which Nu = OH e.g. trypsin subtilisin and chymotrypsin. 3. Metalloproteases for which 8' = M2+ where M is a metal e.g. carboxypeptidases and thermolysin. 4. Acid proteases for which 8' = CO,H e.g. pepsin and penicillopepsin. Many of the studies of inhibitors of these enzymes have been mechanistic rather than therapeutic in origin and consequently there is a large mass of literature on model studies which is aimed at enzymes of no apparent medicinal relevance; in fact there has been relatively little therapeutic 'success' at inhibiting proteases with one or two notable exceptions which will be discussed below.It is beyond the scope of this review to present all examples of inhibitors of these various classes of enzymes but in several instances particularly in the case of ACE inhibitors (see below) these studies have formed the basis from which therapeutic products have been designed. These relevant examples will therefore be presented here. Other reasons for the relative paucity of useful therapeutic inhibitors have been alluded to by Barrett in an important review of the therapeutic potential of 'proteinase ' inhibitors. '15 These are that proteases may often be performing a useful function in one location whilst causing damage at another.Conventional chemotherapeutic approaches cannot address this fundamental problem. The development of site-directed drug-delivery systems is an important direction for the future which the pharmaceutical industry is now getting to grips with. A second problem is that many protease-mediated pathological processes involve not one but several related enzymes (having different specificities) acting together. Blocking just one enzyme in these instances will be therapeutically useless. Even though the success rate has been low the importance of proteases in many physiological and pathological processes is clear. They are involved in for example digestive processes the formation and dissolution of blood clots the activation of the complement system fertilization infection inflammation and malignancy.Each of the four categories of proteases will be reviewed briefly with the emphasis on the therapeutically valuable inhibitors which at present exist. NATURAL PRODUCT REPORTS 1989-4. S. J. WALPOLE AND R. WRIGGLESWORTH Ph Ph L Enz-OH 11 ?TH R'-C -OH Enz -3 Enz -0 R'C02H t Enz-OH Scheme 23 2.2.1 ThioI Proteases The mechanism of action of thiol proteases exemplified by the plant enzyme papain is similar to that of the serine proteases which is presented in more detail below. There are no inhibitors of medicinal importance but the important observation by Wolfenden116 that aldehydes that are structurally related to the acyl portion of the substrates for thiol proteases were potent 'transition-state-like' inhibitors (see also ref.27 and Scheme 5) was seminal in generating aldehyde inhibitors of other proteases. 2.2.2 Serine Proteases An enormous amount of work has been dedicated to the investigation of the structure and catalytic mechanism of the serine proteases. Two distinct groups represented by trypsin and subtilisin exist. They have different three-dimensional geometries and low sequence homologies suggesting their descent from unrelated ancestral enzymes. The arrangement of functional groups (the 'charge-relay triad ') in the active site however is identical in the two families. It would appear that Nature has invented the same 'mechanism' twice.The details of the structure of an enzyme in relation to its catalytic function (represented by chymotrypsin which is prototypical of the group) will not be presented here but the subject has been exhaustively reviewed. 11' The reaction se-quence which has emerged from such studies is represented in Scheme 23. An identical process has been formulated for the thiol proteases with sulphur replacing oxygen as the active-site nucleophile. Many enzyme inhibitors that were designed to substantiate to test or to exploit this mechanistic hypothesis have been generated. Aldehydes1l8 and boronates3l are potent transition- state inhibitors. N-Nitro~o-amides~~. 42 (see Scheme lo) isatoic anhydride^,"^.120 halo-en01 lactones,l2l- 122 and 6-halo-2-pyr- ones123* 124 have all been proposed as mechanism-based inacti- vators of serine proteases. Although several enzymes are indeed inactivated by these molecules there is little positive evidence for the mechanisms that have been suggested. One exception is the inactivation of chymotrypsin by 5-benzyl-6-chloropyran-2-P bo-Enz-0 k381 Enz -OH Scheme 24 Ho \ IJ Enz FOR N"' 0-Enz CIA OR COOR J 0 C02R i.e. alkylated enzyme Scheme 25 by interaction of the 5-benzyl group with the hydrophobic pocket of the enzyme. The enzyme leukocyte elastase (LE) is a serine protease that is thought to be responsible for the destruction of connective tissue (elastin and collagen) in pulmonary emphysema.115 It has been suggested that the deficiency of a natural inhibitor (a1- proteinase inhibitor) may be the cause of the pathology. Exacerbation of the condition by smoking is due to further oxidative inactivation of the available inhibitor. Exogenous application of low-molecular-weight inhibitors of the enzyme would therefore be a useful therapeutic goal. Haloisocoumarins have been as suicide sub- strates for serine proteases ;in particular 3-alkoxy-7-amino-4- chlorocoumarins (39)126 are potent inhibitors of leukocyte elastase. There is some supporting evidence12' for the proposed mechanism of inactivation (Scheme 25) from X-ray studies with the model system porcine pancreatic elastase. Latent isocyanates in the form of azolides are potent irreversible inhibitors of leukocyte elastase.12* one (38) where 13CN.M.R.and X-ray-diffraction evidence123 support the mechanism shown in Scheme 24. 2.2.3 Metalloproteases Note that no enzyme-catalysed esterolysis of the pyrone Prototypical enzymes of this class are carboxypeptidase A occurs. Nucleophilic attack is directed to position 6 seemingly possibly the best studied of all enzymes and thermolysin. These NATURAL PRODUCT REPORTS 1989 H-0 directly generating tight-binding reversible inhibition. The former category is exemplified by the inhibition of carboxy- peptidase A by cyclopropane-containing peptide~l~~ (Scheme cH Enz-CO; R1C02H + Enz-COz-Scheme 26a H+ir R'-C-N-R~ __t R'C02H + H2NR2 tH + Enz-CO2H Scheme 26b are not therapeutic targets in themselves but are important because of the amount of effort aimed at them as models for related enzymes which are of great therapeutic relevance.X-ray-diffraction studies of both enzymes have been undertaken an,d the three-dimensional structure pf carboxypeptidase A to 2A129 and that of thermolysin to 2.3A130 are both known. This information has been very valuable in inhibition studies although the detailed mechanism of action (of carboxypeptidase A) remains controversial. The distinction between the so-called 'anhydride' mechanism (Scheme 26a) where Glu-270 attacks the carbonyl of the substrate to form an acyl-enzyme intermediate (an anhydride) and the 'direct' mechanism (Scheme 26b) where Glu-270 acts as a general base to activate a water molecule which directly cleaves the substrate cannot be made.l3l Various mechanistic claims have been made for particular substrates but even these have been disputed.132* 133 Combined with the observation that this enzyme can catalyse stereospecific enolization of a ketonic and a$-elimination reactions,135 it is perhaps a moot point to attempt to formulate a general mechanism for all substrates for this (and indeed for any other) enzyme.All of the inhibitors that have been described for these 'model' enzymes exploit in some way the polarizing property of the zinc cation in the active site either by activating some part of the inhibitor which then is susceptible to nucleophilic attack by an active-site residue or by linking to the zinc 27) although the proposed mechanism which is based on related studies of the inhibition of alcohol dehydrogenases and lactate dehydr~genases,'~' has not been established.The principle of using metal-co-ordinating analogues of the sub- strate as inhibitors13* will be discussed within the context of angiotensin-converting enzyme below. The metalloprotease angiotensin-converting enzyme (ACE) [dipeptidyl carboxypeptidase I] plays a crucial physiological role in controlling blood pressure. This enzyme is a component of the renin-angiotensin ~ystem,'"~which is illustrated in Figure 6. Angiotensinogen is a high-molecular-weight blood glyco- protein which is cleaved by the acid protease renin (see below) into the decapeptide angiotensin I (AI).This is cleaved to the octapeptide angiotensin I1 (AII) by ACE in blood vessels but particularly in lung tissue. This latter peptide in contrast to AI is a potent hypertensive agent which acts as shown in Figure 6. Furthermore the vasodilating peptide bradykinin is cleaved by ACE into inactive fragments; hence the overall effect of this enzyme acting on these two physiological substrates is to increase blood pressure. [Recent has shown that blocking the breakdown of bradykinin makes a significant contribution to decreasing blood pressure.] Clearly the renin-angiotensin system plays a major physio-logical role in controlling blood pressure but it is now equally clear that malfunctions of the system are pathologically important in hypertension and related diseases.Pharmacological intervention to inhibit the two important enzymes in the pathway i.e. renin and ACE which it is predicted will lower blood pressure has been successfully achieved. The important story of the development of inhibitors of the metalloprotease ACE has been described in great detail in the literat~re'~' so only an outline is presented here. ACE is a peptidyldipeptide hydrolase i.e. it cleaves dipeptide units from the carboxyl terminus of several peptides although its substrate preference is for A1 and bradykinin. Two factors were crucial in the development of the small 'non-peptide ' inhibitors of this enzyme which have subsequently proved to be so clinically valuable.These were first the structure-activity information that was available about peptidic inhibitors of the enzyme and second the recognition of the similarity of ACE with the 'model ' enzyme carboxypeptidase A. The first of these points the availability of a large amount of structure-activity information originates from the discovery that certain peptides from the venom of the Brazilian 'arrowhead ' viper (Bothrops juraracu) were potent competitive inhibitors of ACE. The most potent of these a pentapeptide BPP, (which was also a poor substrate) was extensively investigated. The results of this effort are summarized in Table 2; it was established that some activity was retained in di- and tri-peptide fragments of the original structure. Thus one of the goals which medicinal chemistry aims to achieve i.e.simpli-fication of structure with retention of activity seemed attainable. The small peptides (40)and (41) were to form the basis of all that was to follow. ..J Nu -Enz Nu-Enz Scheme 27 NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH ANGIOTENSINOGEN Vasodilation RENIN t ANGIOTENSIN I BRADYKININ ANGIOTENSINIt Breakdown products Vasoconstriction Release of aldosterone ELEVATION OF BLOOD PRESSUREI I Retention of Na'and H20 Figure 6 Table 2 Values of IC; for the competitive inhibition of peptides from the venom of Bothrops jararaca against angiotensin-converting enzyme Peptide IC5,IcGg cm-3 < Glu-Lys-Trp-Ala-Pro (BPP,,) 0.05 < Glu-Lys-Phe-Ala-Pro 0.05 Lys-Trp- Ala-Pro 1.2 Phe- Ala-Pro (40) 1.4 Ala-Pro (41) 50 (a) IC, is the concentration of inhibitor that is required to give 50% inhibition under the specified conditions (see ref.141). Although BPP5" was a potent inhibitor of the isolated enzyme it was very short-acting in vivo and was not active by oral absorption; consequently it was never a serious drug candidate and the search continued from this point for a compound without these drawbacks. From a large number of biochemical studies it was clear that ACE was a zinc-containing metalloprotease very similar to carboxypeptidase A. Its substrate specificity was similar in that it would only bind compounds with a free carboxyl terminus and several of the active-site residues of the two enzymes were identical.One fundamental point of difference however is that ACE cleaves dipeptide units from the carboxyl terminus whereas carboxypeptidase A cleaves single residues. From these considerations workers at the Squibb Institute for Medical Research were able to construct a schematic Figure 7 operational model of the active site of ACE and used it as a design template. This is illustrated in Figure 7 in which a representative tripeptide substrate [N-acylated (40)] is shown in the active site. At this time (1972) a fundamentally important contribution to the design of mechanism- based inhibitors appeared. Byers and W01fenden'~~. 143 described the potent inhibition of carboxy- peptidase A by the L-isomer of benzylsuccinic acid (42).This was rationalized as a 'bi-product ' (not 'by product '!144) inhibitor of the enzyme i.e. a compound which combines in a single molecule some of the enzyme-binding features of the NATURAL PRODUCT REPORTS 1989 Substrate OHiR-C=O + N 0 II c -0- Products (42) ki = 5 x ~ o Idrn- (cornpet i t ive) Figure 8 Table 3 Values of IC, for competitive inhibitors of angiotensin-converting enzyme Inhibitor IC,o/,umol dm-3 H02CCH2CH2-C -N "3 A Cop H Me 0 v II H02CCH2 CH-C-Pro-OH Ye H02 CCH2bH -C-Pro -OH 0 li H SCH2- C -Pro-OH 0 II HSCH CH2-C-Pro-OH 0 II HSCH,CH2CH2-C -Pro-OH Me 0 v II H SCH2CH -C -Pro -OH (captopril) Me HSCH2bH-C-Pro-OH 330 22 1480 1.1 0.20 9.3 0.02-0.08 2.4 collected products of the normal enzyme-catalysed reaction (or viewed in the reverse direction the collected substrates for formation of a peptide bond) (Figure 8).The origins of the potency of this inhibition have been attributed to the entropic advantages that are gained by the enzyme in having to 'gather' one ligand and not two from solution (see also p. 315 of this review). This is in contrast to any resemblance to the transition state of the reaction that is being catalysed. Application of these ideas using the 'stretched' (relative to carboxypeptidase A) model of the active site of ACE (see Figure 7) predicted that succinyl-amino acids could be accommodated as similar bi-product inhibitors of the enzyme.The more important developmental steps along this structure- activity 'track' are illustrated in Table 3. The dramatic increase in potency on changing the carboxyl function which is a 'weak' ligand for zinc to the stronger ligand of a sulphydryl function was a crucial advance. The stereochemistry (R) of the methyl group reflects the stereospecific requirements for an L-amino-acid analogue at this position [L-Ala in Ala-Pro (41)]. The end-point of this endeavour was the structurally simple potent competitive inhibitor of ACE called captopril (Capoten) (see Table 3). Captopril was shown to very specific for ACE. It did not inhibit a wide range of proteases at concentrations 100000-times higher than that effective for inhibition of ACE.The specific inhibitory effects were retained in an isolated muscle preparation in a variety of animals and ultimately in Man where captopril was shown to inhibit the vasoconstrictor actions of A1 and to potentiate the vasodilator effects of bradykinin. Captopril was marketed in 1980 and the clinical evidence that has been collected to date shows that it is very effective. Normalization of blood pressure of 50% of patients with essential hypertension has been observed and combined therapy with diuretics extends this profile to give benefit in 90% of patients with mild to moderate hypertension. A recent and exciting development from the clinical studies is that captopril is proving useful in treating congestive heart failure.Whether this benefit is mediated via inhibition of the renin-angiotensin system is not at present known. Although captopril is without doubt a good drug like many others it does have some side-effects -e.g. rashes and loss of taste which disappear on stopping drug treatment. These side- effects have been ascribed to the presence of the thiol group and a large effort has been put into developing an alternative to captopril which lacks the thiol group. A group of workers at Merck Sharp and D~hrne'~~ in the U.S.A. returned to the original Wolfenden 'bi-product ' analogue hypothesis and exploited a feature in the active-site model of ACE which had not been considered in the NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH Table 4 Values of IC, for various inhibitors of angiotensin- converting enzyme that were produced and tested during the discovery of enalapril and enalaprilat Inhibitor 0 II H02CCH2CH2C-Pro-OH 0 II HOZCCHZCH~CH~C-P~O -OH Me0 1 II H02CCHzNHCHC-Pro-OH Me I H02C C H-Al a-Pro-0 H HOZCCH- Ala-Pro-OH f Ph HO2 C C H-AL a-Pro-OH HO2 CC H-A Ia-P ro -0H R I HO2 CCH-Al a-Pro -OH ( S r Et 02 CCH-Ma-Pro-OH S -Nu H Figure 9 development of captopril -namely the hydrophobic site for binding of the antepenultimate aromatic residue [i.e.Phe in Phe-Ala-Pro (40)]. The path leading to the Merck compound MK421 from N-succinylproline is shown in Table 4. The postulated mode of binding of the parent compound MK422 is shown in Figure 9 (cf.Figure 7). The ester enalapril (MK42 1) is relatively inactive against the isolated enzyme but in contrast is highly active in vivo IC,,/,umol dm-' 330 70 2.4 0.09 0.0026 0.039 0.82 0.0012 l"O',fr i at ) 1.2 Enalapril 331 (43) -C(O)NHCH,CO,H (44) Ph NHC(O)CH~CO~H (45) Figure 10 HSCH2-CH R s HSCH~ -CH-R,S presumably by de-esterification to the parent diacid (enala- prilat). The former is therefore a pro-drug and has been developed because of its superior pharmacokinetic profile in Man. Clinical experience has shown that enalapril shows less adverse side-effects than ~aptopril'~~ and has a longer biological half-life. Many other inhibitors of ACE are now at various stages of development.Several are in clinical t~ia1s.l~' The majority of these are close structural analogues of captopril or enalapril but the development of the bicyclic inhibitor cilazapril (Ro 31-2848) (43) is worthy of note because of the extensive use which was made of molecular graphics in the design of this structure.14* The membrane- bound enzyme enkephalinase [membrane metallo-endopeptidase ; E.C.3.4.21.111 has been characterized as a metallo-endopeptidase that is similar in many respects to ACE.149,150The enzyme cleaves the Gly3-Phe4 bond of the enkephalins but has a fairly wide substrate specificity. A potent inhibitor of enkephalinase thiorphan151 (44),was the first of many such compounds that were designed by using the principles that had been established for ACE but which attempted to exploit fine differences between the two enzymes.These are that the Sl sub-site exhibits a specificity for an aromatic or hydrophobic residue (cf. Ala in ACE) and that the S2 site shows a preference for amino acids that have a small side-chain (e.g. Ala and Gly) (see Figure 10). The selectivity of thiorphan for enkephalinase relative to ACE was only forty-fold however and further efforts NATURAL PRODUCT REPORTS 1989 0% c Substrate Enzyme Tetrahedral NHZ Enzyme + 0 9 /\ HO -Products Figure 11 Jc CIearge NL II H 0 R' R2 N L ACH2 H R' R2 OH Figure 12 have achieved effectively complete separation of the two activities with retention of the potency of enkephalinase inhibition.152 These properties are resident in retro-thiorphan (45).Both thiorphan and retro-thiorphan show weak analgesic activity in hot-plate and writhing tests in mice but at high concentrations. 151*152 It is likely that the enkephalins are degraded by other enzymes as well (aminopeptidases) and blocking enkephalinase alone is therefore not surprisingly insufficient to protect the analgesic peptides completely against breakdown 153 and consequently may be of limited therapeutic value. 2.2.4 Acid Proteases As their name implies these enzymes catalyse the hydrolysis of a peptide bond at acidic pH. The best known of these enzymes is pepsin but others are pencillopepsin chymosin and renin.Information about the mechanism of action of this class was focussed into a proposal by the crystallographic study of pencillopepsin which was solved to 1.8A res01ution.l~~ This proposal is illustrated in Figure 11. The active-site aspartate residues activate a water molecule which then becomes the nucleophilic species which attacks the substrate. The similarity of the residues in the active site for all acid proteases gives support to this mechanistic hypothesis being a general one. The enzyme renin is an important constituent of the renin-angiotensin system (see Figure 6)and would appear to be a good therapeutic target in the control of hypertension for similar reasons as have been presented for (and realized in practice for) ACE.Accordingly an enormous effort particu- larly by the pharmaceutical industry has been expended over the past ten years in attempts to make potent orally active inhibitors of renin as potential antihypertensives. Renin cleaves the high-molecular-weight substrate angio- tensinogen at the amino-terminus into the decapeptide angio- tensin I. The angiotensinogens vary from species to species and the human N-terminal sequence is different from non-primate sequence and indeed is not cleaved by non-primate renins.lS5 The relatively recent identification of part of the N-terminal sequence (46) of human angi~tensinogen'~~ was important in directing synthetic efforts towards this structure rather than the different but previously available equine sequence.The shortest peptide fragments of angiotensinogens that have reasonable rates of cleavage and affinities for the enzyme are octapeptides around the Leulo-Val" cleavage site although shorter peptides show weak inhibition (see Figure 13). Using this information two approaches have been adopted to designing inhibitors. The first has been to make substrate analogues. Replacement of residues 10 and 11 by Phe'O-Phe" resulted in potent competitive inhibitor^,'^' but these lacked suitable solubility in water and were only weakly and transiently active in vivo. An important advance was made by Szelke and co-workers who introduced the 'reduced isostere' of the cleavable amide bond as a potential transition-state- analogue inhibitor.Their first example158 was to introduce the reduced amide isostere into the equine sequence H76 (47a) (see Figure 12). This change produced potent inhibition of canine NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH -s5-s4-s3 -s2-s1 -Sf-S2'-S3' -(Enzyme) -Plj-P4 -P3 -P2 -P -P,. -P2' -P3' -(Peptide) 6 8910 His -7Pro -Phe -His -Leu -4 ;a -Yle -;is His -Pro -Phe -H is-Leu -Val -Ile -Tyr His-Pro -Phe -His- Leu -Val-Ile-His isovaleryl -Val -Val -Sta -Ala -Sta Boc -Phe -His -Sta -Ala-Sta -OMe isobutyryl -Pro -Phe -His -Sta -Val -Phe -NH2 J,represents point of cleavage; R = reduced amide bond Figure 13 Isovaleryl -Val -Val -Sta -Ala-Sta -OH 0 0 II H 11 H H -C-N R-C-N 0 U I I 602H C02H renin; thus modification of the scissile bond increased the -D-Aia-D-Ala Penicillin potency 10000-fold over that of the analogous amide.This analogue lowered blood pressure in dogs. Extension of this idea Scheme 28 to the human sequence led to potent inhibitors [e.g.H113 (47b)l of the human enzyme.159 More recently these workers have described hydroxy RC(0)NH isosteres160 (see Figure 12). The second approach to designing inhibitors is from a natural peptide pepstatin (48). 161 This is a pentapeptide SO3H containing the unusual amino acid statine. Pepstatin is a potent inhibitor of pepsin in particular but of (52) acid proteases including renin in Structure-activity (51) studies of analogues162 and X-ray crystallography on enzyme- pepstatin complexe~'~~~ 164 suggest that the 3-hydroxyl group of the (3S)-statine resembles the putative tetrahedral intermediate proteins' (PBPs) as the targets of these drugs.More biochemical (or transition state) and also that the statine spatially represents approaches have identified these same PBPs as 'penicillin-a dipeptide unit (see Figure 13). sensitive enzymes ' (PSEs) having carboxypeptidase and trans- Modifications of pepstatin away from the central statine peptidase activities,16s which are involved in the biosynthetic residue to give analogues which more closely resemble the assembly of bacterial cell walls. sequence of the human renin and also incor- It is not clear whether the inhibition of these enzymes by the poration of statine in place of the central dipeptide unit of p-lactams causes cell death directly by blocking the trans- human renin substrate166 have both produced potent inhibitors peptidation of the peptidoglycan matrix and thereby lethally of the enzyme [(49) and (50) respectively].weakening it or indirectly by the subsequent release or Further information on these compounds (and indeed many activation of endogenous 'autolysins ' which then hydrolyse the derivative analogues reported in the literature) is not available peptidoglycan. 169-171. in which it at present because of the usual secrecy in the pharmaceutical The original model of Tipper and Str~minger,~~~ industry shrouding this stage of development but it seems was suggested that the penicillins were structural analogues of likely that the problem of retaining oral activity has not been a particular conformation of the D-Ala-D-Ala C-terminal solved that the reduction in size (and hence cost) of these portion of the nascent peptidoglycan chain is illustrated in expensive molecules has not been achieved and that no real Scheme 28.advantages of inhibition of renin have emerged over the The stereochemistry at C-6 of the penicillin is inverted from clinically established inhibitors of ACE. Given these problems the configuration that would be required for complete structural it may be more fruitful and appropriate to consider the renin overlap but the model has been accepted and extended in the system as a model and to apply the enormous amount of succeeding years. The distortion of the D-Ala-D-Ala peptide mechanistic and molecular information which has come out of bond in this conformation has been proposed as an approximate this work to other proteases.Cathepsin D is such an example; representation of transition-state geometry and hence the p-it is a protease which has been implicated in inflamrnati~n.'~~ lactams have been labelled as transition-state analogues. Further suggestions identifying the p-lactams as active-site-directed irreversible inhibitors173 (via formation of a relatively stable 2.2.5 Protease-Related Enzymes serine-linked acyl-enzyme intermediate) and as suicide sub-It seems appropriate to include the p-lactam antibiotics and strate~,~'~ have also been put forward. related antibiotics here. Although the molecular mechanism of In the past fifteen years 'non-classical' p-lactams have been e.g.(51) and the action of these potent antibacterial agents is still unclear the isolated and identified. The no~ardicins,'~~ available evidence strongly points to several ' penicillin-binding mon~bactams,~~~ e.g. (52) are monocyclic p-lactams which are NATURAL PRODUCT REPORTS 1989 OH 0gyoH-bO2H (53) (path 1)/ GyoHOH ! acyl -enzyme 1(path 2) ,Nu' Enz intermediate (path 3)\ C02H + Nu C02H Active enzyme Michael addition Er3Nu NHJJ \ 8 CO2H OH hydrolysis1 0LCHONu-Enz inactivated inactivated enzyme enzyme Scheme 29 OH CO,H (54)R=H (55) R =CH=NH powerful antibacterial agents. This biological activity thus dispels the myth that active p-lactams had to be bicyclic.The strained geometry of the amide bond in such bicyclic systems does not necessarily confer either increased reactivity or biological activity.l7' Although clavulanic acid (53) from a Streptomyces species is itself a poor antibiotic it is a powerful inhibitor of p-lactamases. These enzymes (penicillinases) are clearly closely related to the PSEs inhibition of which confers antibiotic activity. Thus combination of P-lactamase inhibitors with p-lactamase-sensitive antibiotics produces increased drug potency and indeed overcomes resistant bacteria in vivo. This important therapeutic strategy of combination is exemplified by Augmentin a clinically successful mixture of clavulanic acid with amoxycillin. The mode of action of clavulanic acid has been studied in some detail.178 It inactivates the enzyme by a complex mechanism which involves the initial formation of an acyl- enzyme intermediate (Scheme 29).Normal turnover by deacyla- tion (path l) where clavulanate acts as a substrate competes with two different fragmentation reactions (paths 2 and 3) of the intermediate both of which result in inactivated enzyme. D r? I FCH2 -C-CO2H I NH2 (56) R'=R~=H (58) (57) R' =Na,R2= 0 Me Me II H \ 0 OH (59) This suicide-substrate-like behaviour is also shown by penicillin sulphones 178 and the mechanism has been exploited in this series by modulating the ratio of turnover to inactivation (i.e.the rate of path 1 relative to paths 2 and 3) by varying the 6a-su bsti t uen t .The drug thienamycin (54),17g a broad-spectrum antibiotic with stability towards cleavage by p-lactamases has had a chequered history. The compound proved to be too unstable for development. This problem was overcome by its synthetic modification to the N-formimidoyl derivative imipenem (55),"* which was chemically stable and indeed more active than the parent compound. On testing in mammalian systems (55) was rapidly deactivated by metabolism. 181 A renal dipeptidase NATURAL PRODUCT REPORTS 198P-C. S. J. WALPOLE AND R.WRIGGLESWORTH ti*N-C~O I slow 0 -I re -activation ///// /// Scheme 30 Me (62) (611 acting as a ‘mammalian p-lactamase ’,thus precluded clinical use of this compound for infections of the urinary tract.The latest twist in this story is the development of a synthetic inhibitor of the renal dipeptidase which will be used in combination with imipenem to protect the antibiotic.ls2 There are other points along the multi-enzyme assembly path to cell-wall peptidoglycan which are useful therapeutic targets. Alanine racemase is the enzyme which converts L-into D-Ala prior to its incorporation as the C-terminal dipeptide into the uncrosslinked peptidoglycan precursor. D-Cycloserine (56) is an inhibitor of this enzyme in contrast to the L-enantiomer which is surprisingly inactive.ls3 The explanation that has been offered is that the enzyme which accepts both enantiomers of the flexible substrate/product (D-and L-Ala) is unable to bind both stereoisomers of the rigid inhibitor ;only D-cycloserine can assume the conformation common to both D-and L-Ala which allows binding tothe enzyme.A pro-drug form of cycloserine the enamine (57) of pentane-2,4-dione and cycloserine has been developed as a form of the drug which is more stable in aqueous solution. Combination of (57) with 2-deuterio-fluoro-~-alanine (DFA) (58) which is an inhibitor of the synthesis of ~-Ala,l’~ produced a highly effective anti-infective agent in animals. 185 The drug alafosfalin [L-alanyl-(R)-1-aminoethylphosphonic acid] (59) is an analogue of D-Ala-D-Ala which is a potent inhibitor of alanine racemase.186 The ‘dipeptide’ also has antibacterial activity by virtue of the fact that it is transported across the bacterial cell wall by L-amino-acid-specific peptidases and is then cleaved intracellularly by L-specific permeases to generate the actual inhibitor (R)-aminoethylphosphonate.2.3 Neurotransmission-Processing Enzymes This miscellaneous group of enzymes has been collected here because of their relative therapeutic importance. These enzymes along with uptake release and storage systems and also the pre- and post-synaptic receptors themselves are the component parts of all neurotransmission systems. Important neuro-transmitters include acetylcholine dopamine and noradren- aline y-aminobutyric acid (GABA) glutamate glycine the neuropeptides and serotonin. Further discussion of these systems is beyond the scope of this review (see ref. 188 for detail) and the information included here relates only to interference with the various enzymes that modulate the actions of these neurotransmitters and the therapeutic consequences of this interference.Brief mention has been made of the enzymes that affect the neuropeptides (specifically the enkephalins) in the section on proteases. 2.3.1 Acetylcholine The enzyme acetylcholinesteraseis present in the synaptic cleft where it is responsible for the inactivation (by ester hydrolysis) of the neurotransmitter acetylcholine. Two main types of inhibitor have been described. 2.3.1.1 The carbamates. Neostigmine (60) is representative of this group of clinically important compounds which contain a quaternary ammonium or tertiary base function along with a carbamoyl (instead of acetyl) ester.Another example is the alkaloid physostigmine (eserine). As a class these drugs bind at an anionic site of the enzyme using the basic or quaternary ammonium moiety and undergo the normal catalytic process. This involves transient acylation of an active-site serine residue (cf. serine proteases) but with the carbamoyl esters (unlike the substrate) the acyl-enzyme intermediate is slow to break down. The result is a slowly reversible inhibition of the hydrolytic function of the enzyme (Scheme 30). These compounds find effective clinical use in the treatment of myasthenia gravis where the low number of acetylcholine receptors results in a failure of neuromuscular transmission. Inhibition of acetylcholinesterase preserves the acetylcholine in the synaptic cleft making it more likely to reach the few remaining receptors.The carbamates are also used in the treatment of glaucoma and in reversing the action of neuromuscular blocking agents after anaesthesia. 2.3.1.2 Organophosphates. In contrast to the former group these compounds [e.g. dyflos (61)] are very toxic because of their ability to phosphorylate the active-site serine residue irreversibly. They have been used as war gases and as insecticides. Pralidoxime (62) is an antidote to poisoning by organophosphates by virtue of the fact that the oxime is nucleophilic enough to attack the serine phosphate intermediate and release the free enzyme. 3 36 NATURAL PRODUCT REPORTS. 1989 6.B.B * L -Tyrosine L -Tyrosine +I-OH HO H0m2:2 HO Adrenal ine L -Dopa E4 E2 t 1 OH - HO O W N H z €3 HO .Noradrena Iine Doparnine (*6.6.6. = Blood- Brain Barrier ) Enzymes E1 tyrosine hydroxylase ;E2 dopa decarboxylase ; E3 dopamine &hydroxylase ;E4 phenylethanolamine N-methyltransferase Scheme 31 HO OH HOG CHZNHNHC H ,;zoH (63) (6 4) Per iphery B.B.6 Bra in L -Dopa L -Dopa DEGRADATI0 N II EFFECT D.D. = -Dopa decarboxylase Scheme 32 NATURAL PRODUCT REPORTS 19894. S. J. WALPOLE AND R. WRIGGLESWORTH 2.3.2 Aromatic Amino Acid Neurotransmitters This group comprises dopamine noradrenaline and adrenaline (which are related biosynthetically) and serotonin (5-HT). The former group are derived from L-tyrosine which circulates in the periphery.It is able to pass the blood-brain barrier (B.B.B.) and is then metabolized to the neuro-transmitters as shown in Scheme 31. Inhibitors of the component enzymes of this pathway have been described. Tyrosine hydroxylase [tyrosine 3-mona-oxygen- ase] which is the control point for synthesis of noradrenaline is blocked by a-methyltyrosine. The resulting block in noradrenaline synthesis produces improvement in the hy-pertension caused by pheochromocytoma. lag DOPA decarboxylase [aromatic-L-amino-acid decarboxy- lase] is a non-specific enzyme which catalyses the decarboxy- lation of a variety of ‘aromatic’ L-amino acids. Mechanism- based inhibitors have been de~cribed’~~-~~~ which exploit the pyridoxal-5-phosphate-mediated decarboxylation process.This is discussed below in more detail (see Section 2.3.3). These (and other such inhibitors) have proved to be of little value as therapeutic agents on their own but they have found an important use as adjunct therapy for the treatment of Parkinson’s Disease. The tremor muscle rigidity and hypo- kinesia that are characteristic of the disease are due to low levels of dopamine in the brain (consequent upon a loss of dopaminergic neurons in the substantia nigra) and these symptoms have been alleviated by replacement therapy. Since dopamine itself does not cross the blood-brain barrier the QOH 0 (66) R Clorgyline R =H; Pargyline Form A R =Me ;Deprenyl Form 8 Figure 14 R 0 + +CH,-N /Me ‘Me biosynthetic precursor L-dopa is used.This enters the brain and is decarboxylated to dopamine which then exerts the desired effect. However L-dopa is extensively degraded by peripheral dopa decarboxylase before it enters the brain and therefore large and frequent dosing of the drug is necessary. Peripherally acting inhibitors of dopa decarboxylase (i.e. inhibitors that do not penetrate the brain) such as carbidopa (63) and benserazide (64) when administered with L-dopa allow the dose of the latter to be reduced and also lower the occurrence of peripherally mediated side-effects (Scheme 32).Ig3 Recently inhibitors of dopamine b-hydroxylase [dopamine P-mono-oxygenase] have been describedlg4 which show anti- hypertensive activity in rats.Diethyl dithiocarbamate is a major metabolite of disulfiram (65) which is used to treat alcoholism. The metabolite blocks dopamine P-hydroxylase and this may be the reason for the hypotensive effects characteristic of the disulphiram-mediated modulation of ethanol metabolism. Ig5 L-Mimosine (66) is a toxic amino acid found in legumes which inhibits dopamine P-hydroxylase. This structure contains an interesting bio-isosteric replacement for the catechol moiety but it may inhibit this copper-containing enzyme simply by chelation. lg6 The biosynthesis of serotonin follows an identical path to that of noradrenaline but in this instance starting with tryptophan instead of tyrosine. Catabolic inactivation of the aromatic amino acid neuro- transmitters occurs by methylation (e.g.by catechol methyl- transferase) or by oxidation [by monoamine oxidase (MAO)]. Inhibitors of the latter enzyme have established therapeutic importance. Monoamine oxidase [amine oxidase (flavin-containing) ;E.C. 1.4.3.41 is a flavoenzyme responsible for the deamination of a large number of monoamines such as the transmitter amines e.g. adrenaline noradrenaline serotonin and dopamine. It also deaminates potentially toxic amines in food (e.g.tyramine) and a wide variety of amines that are not found in the body (e.g. benzylamine). Inhibitors of the enzyme have achieved importance because they are effective as antidepressants and more recently particularly in combination with L-dopa they have been found to be effective against Parkinson’s Disease.lg7 One problem of using these inhibitors is the ‘cheese effect’ which is a severe hypertensive crisis (often fatal) in patients who have eaten foods (such as cheese) with a high tyramine content.Inhibition of MA0 stops the normal metabolism of this toxic amine in the intestine. Two forms ((A and B) of the enzyme have been described but differences at the molecular level are not understood. This subdivision is based on inhibition profiles ; Form A is inhibited by clorgyline and Form B is inhibited by deprenyl as shown in Figure 14. The mode of irreversible inhibition by these acetylenic amines (pargyline is another example) is not understood at the present time but model studies with 14C-labelled dimethyl- propynylamine (67) have shown that formation of a stable covalent adduct (68) with the flavin occurs198 (Scheme 33).R hp ‘Me (68) Scheme 33 NATURAL PRODUCT REPORTS 1989 ii 1 ___c Q P'" OHC I I (74) Me Me Enzymes i glutamate decarboxylase ;ii GABA transaminase (70) (71) Scheme 34 C02H I H;! N6 + CHO C02H CO;! H I I Hk< fN 'fl (77) Scheme 35 NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH CO2 H I CO;! H I + CHO CO;!H I H Scheme 36 Several cyclopropylamines are suicide substrates for MA0 ; for example (f)-tranylcyprarnine (69) is used clinically for treatment of depression. Depending upon the substituents attached to the ring the compounds bind directly to the enzyme to the flavin cofactor or to both.lg9 Further details of the inhibition of MA0 and also of other flavin-linked enzymes have been reviewed.2oo One curious twist of the potential therapeutic uses of inhibitors of monoamine oxidase involves a compound called MPTP [1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (70)]. This compound was a side-product contaminant of illicitly produced ‘synthetic heroin’. The addicts who used this contaminated material developed irreversible Parkinson-like effects and the pathological effects were soon traced to MPTP.201 It was subsequently established that MPTP is a substrate for MAO-B and is activated to MPP’ (71) via the reactive intermkdiate dihydropyridinium ion (72).These reactive species have been implicated in the destruction of the nigro-striatal neurons which would account for the symp- toms. Furthermore prevention of these neurotoxic effects has been observed with pargyline and suggests that inhibitors of MA0 may have value in the treatment of Parkinson’s disease.202 2.3.3 y-Aminobutyric Acid (GABA) GABA (73) is an inhibitory neurotransmitter found throughout the brain. Decreased levels of GABA in the brain have been implicated in epilepsy and other convulsant states and attempts to raise these levels have been made. As well as the approach of making central-nervous-system-penetrating GABA agonists effort has been put into blocking the enzymic degradation of endogenous GABA. GABA is made from L-glutamate by decarboxylation {by glutamic acid decarboxylase (GAD) [glutamate decarboxylase]) and is degraded by the enzyme GABA transaminase (GABA-T) [4-aminobutyrate amino-transferase] to succinate semialdehyde (74) (see Scheme 34).Both of these enzymes use pyridoxal 5-phosphate as a cofactor which is intimately involved in the decarboxylation (by GAD) and the transamination (by GABA-T). Mechan- ism-based inhibitors of both of the enzymes have been described and these exemplify a general principle of inhibition of pyridoxal-phosphate-dependentenzymes44 which was de- veloped and elaborated by Rando203 and by Wal~h.~~.~~ y-Vinyl-GABA (75) is a selective time-dependent inhibitor of GABA-T.204 The compound raises endogenous levels of GABA in animal models205 and shows anticonvulsant properties at the same doses.206 The proposed mechanism of action of this molecule acting as a suicide substrate is illustrated in Scheme 35.The inhibitor (75) follows the normal ‘substrate mechanism’ for transamination up to the ‘transiminated’ intermediate (76). The normal substrate would at this point suffer hydrolytic cleavage of the imine bond to generate the product i.e. succinate semialdehyde. Instead subversion of (76) occurs because of the susceptibility of the newly generated Michael- acceptor species to nucleophilic attack by the enzyme. The result compound (77) is a molecule of irreversibly inhibited enzyme. This mechanism although reasonable has not been established nor has the identity of the enzyme nucleophile been determined.Following the promise that (75) has established as a clinically useful anti-epileptic fluorine-substituted y-vinyl-GABA analogues have recently been described which are more active than the parent compound.208 Elegant though the design of a molecule such as (75) is it seems to have been upstaged by Nature. Gabaculine (78) is a natural product which is a potent mechanism-based inhibitor of GABA-T.209-211 The inactivation is based on the propensity of the intermediate iminocyclohexadiene system to undergo oxidative aromatization which results in a stable (covalent) complex of inhibitor and cofactor. Because this is tightly bound to the active site the result is an irreversible inhibition of the enzyme (see Scheme 36).(79) x=o (80)X = S 0 01 loss of HCOOH 0 Scheme 37 Isosteric replacements of the dihydroaromatic ring of gabaculine by dihydrofuran212 [in compound (79)] and by dihydr~thiophene,~~~ in (80) have resulted in inactivators of GABA-T. Inhibition of GAD has been achieved by fluoro-alkyl- substituted derivatives of glutamate [e.g. (8l)]."'"+215 Presum-ably subversion of the normal catalytic process leads to elimination of fluoride and generation of a reactive Michael acceptor which then irreversibly inhibits the enzyme. Both enantiomers of 4-aminohex-5-ynoic acid (82) [cf. y-vinyl-GABA (75)] irreversibly inhibit GAD. The principle of microscopic reversibility has been invoked to rationalize this process.216 The (9-enantiomer also inactivates GABA-T.217 NATURAL PRODUCT REPORTS 1989 C02H I (81) (82) 2.4 Other Enzymes There remain several enzyme targets of therapeutic interest which do not fall into the three categories discussed above.The more interesting of these are discussed below in simple alphabetical order. 2.4.1 Aromatase Aromatase catalyses the conversion of androgens into oestro- gens. This involves the aromatization of ring A of the steroid by a cytochrome- P-450-dependent sequence of hydroxylations of the methyl group C-19 ultimately leading to its loss as formic acid (Scheme 37). Inhibition of the biosynthesis of oestrogens induces re-gression of hormone-dependent breast cancer; blocked of the aromatase-catalysed reaction would be a way of achieving this.Consequently much effort has gone into designing inhibitors of aroma tase. The acetylenic analogues (83) and (84) were made and shown to be potent time-dependent irreversible inhibitors of aroma- ta~e.~l'" A mechanism was advanced that involved the formation of the acetylenic ketone (85) which underwent Michael addition with the enzyme. This mechanism has been debated and an alternative involving a reactive oxirene species (86) has been made (Scheme 38). Epoxy-steroids have recently been made which are potent inhibitors of aromatase,216 and the activity of these compounds adds weight to the argument that the mechanism of inhibition proceeds via the oxirene intermediate. Non-steroid inhibitors have also been described and one such aminoglutethimide (87) has been shown to cause significant tumour regression in clinical trials against breast cancer.217 9 218 2.4.2 H+/K+-transpor t ing A TPase The therapeutic value of inhibition of gastric acid secretion has been established by the unique success of the histamine H-2 antagonists for the treatment of peptic ulcers and related diseases.The mechanism of secretion of acid from the parietal cells of the gastric mucosa is shown in a simplified form in Figure 15. The final step mediating the release of acid is catalysed by H+/K+-transporting ATPase which translocates protons for potassium ions. In turn this can be controlled indirectly via the second messenger CAMP by activation or blockade of the histamine H-2 receptor; hence the effectiveness of the H-2 an tagonis ts.Another way to stop release of acid is by blocking the H+/K+-transporting ATPase directly. This is achieved by omeprazole (88). The molecule itself is not active against the enzyme but requires activation (to '0'in Figure 15). This occurs only in the parietal cell and gives omeprazole a unique selectivity. Chemical model studies have established a re-markable series of molecular events which add up to give the unique mode of action of this agent. First omeprazole is a weak base which is concentrated selectively into the acid NATURAL PRODUCT REPORTS 1989-C. S. J. WALPOLE AND R. WRIGGLESWORTH 34 1 INA CTI VAT ED ENZYME Scheme 38 H compartment of the parietal cell.The molecule is then transformed in a non-enzymic acid-catalysed rearrangement into the cationic sulphenamide (89) [compound ‘0’in Figure 151. This activated species reacts covalently with sulphydryl groups of the enzyme thereby inactivating it. Because it is a cation this active intermediate cannot diffuse out through the parietal cell membrane and cause mayhem elsewhere further augmenting its selectivity. These molecular events are illustrated Omeprarole I H-2 antagonist 1 Lcoy 1 \ Lumen H+ of stomach Histamine H-2 receptor Parietal I cell Figure 15 13 NPR 6 NATURAL PRODUCT REPORTS 1989 H‘WMe Enz -SH S7 Me H+ Me Me J Em-S \ S-Me Meo yJ ).-i-3OMe H.. Me Scheme 39 0 Me-OR (90)R = C(0)Me (92) R =H (91) R=H (93) R =Me in Scheme 39.The compound is presently undergoing clinical trials as an anti-ulcer agent and has aroused a lot of interest in the pharmaceutical industry as an alternative to existing therapy for gastric diseases.,19 2.4.3 Inhibition of the Biosynthesis of Icosanoids Since the initial reports220-222 that aspirin inhibits the bio- synthesis of prostaglandins and thereby exerts its anti-inflam- matory analgesic and antipyretic effects there has been an enormous effort directed towards chemical and pharmaco- logical intervention in this pathway. Apart from the de- velopment of further non-steroidal anti-inflammatory drugs (N.S.A.I.D.s) this effort has been unrewarded in the therapeutic sense.The biosynthetic pathway to icosanoids as presently en-branches into two different structure types the leukotrienes and the prostanoids from the common precursor arachidonic acid. The latter is liberated from membrane phospholipids by the action of phospholipase A,. Inhibitors of this enzyme which is purported to be the rate-limiting of the whole icosanoid pathway have been but these compounds are likely to be more of use as pharmacological tools than as therapeutic entities. Arachidonate is converted by the enzyme cyclo-oxygenase into prostaglandin precursors (e.g. PGH,) which are then converted enzymically into the prostaglandins into prosta- cyclin or into the thromboxanes. The other branch of the pathway involves the conversion of arachidonate by lipoxygenase into the leukotrienes which have been implicated in inflammatory disease and in asthma.Aspirin (90) exhibits time-dependent irreversible inhibition of cyclo-oxygenase by covalently labelling the amino group of the N-terminal serine residue by acetylation.226 The situation is not as simple as this however in that salicylic acid (91) which is clearly unable to do this also has potent anti-inflammatory activity in vivo. It is known that salicylic acid is formed rapidly from aspirin in vivo. Although the main action of aspirin and indeed of other N.S.A.1.D.q is by blocking cyclo-~xygenase,~~~ the detailed molecular mechanisms are at present far from clear.228 Attempts to inhibit the biosynthetic pathway to leukotrienes which are necessary to establish its physiological importance are at present under way.229 2.4.4 Hydroxymethyiglutaryl-CoA Reductase (HMG-CoA Reduc tase) Compactin (92) and mevinolin (93) are fungal metabolites which are potent inhibitors of HMG-CoA red~ctase.~~O-~~~ This enzyme catalyses the formation of mevalonate from NATURAL PRODUCT REPORTS 1989-C.S. J. WALPOLE AND R. WRIGGLESWORTH Table 5 Enzymes that might be inhibited Enzyme Disease or indication Reference Cholesterol Atherosclerosis 236 acyltransferase Aldehyde dehydrogenase Aldose reductase Alcohol abuse Peripheral neuropathies hyperlipidaemia 237 238 in diabetes Na+/K+-transporting Cardiac diseases 239 ATPase Carbonate dehydratase Diuretics epilepsy 240 Phosphodiesterase Cardiotonic 241 (CAMP)(111) Squalene Anti fungals 242 mono-oxygenase Steroid hormone Breast cancer 243 biosynthetic enzymes hypertension anti-androgenic indications contraction abortion induction of labour hydroxymethylglutarate which is an early rate-limiting step in the biosynthesis of cholesterol.The inhibitors which are competitive with hydroxymethylglutarate have been found to lower the plasma levels of cholesterol and of low-density lipoprotein (LDL) in long-term clinical studies aimed at developing therapies for atheroscler~sis.~~~ 2.4.5 Inhibition of the Biosynthesis of Vitamins Vitamins are dietary requirements for mammals. In contrast most micro-organisms (bacteria yeast and fungi) have the biosynthetic machinery to make these essential cofactors and often are unable to use exogenously available material.Exploitation of this difference by specifically blocking the biosynthesis of vitamins should lead to anti-infective agents. This idea was enunciated in the 1960s by and was investigated by him. Whilst the idea was substantiated for the cases of folic acid (see page 318) and riboflavin (vitamin B2) there has been little exploitation since. Inhibitors of folate biosynthesis do show activity in vivo;in contrast the potent inhibitors of riboflavin biosynthesis which have been made235 do not show anti-infective activity. This most probably reflects the inability of these molecules to penetrate intact cells rather than the breakdown of the principle although it is clear that some micro-organisms do have (inducible) salvage pathways.The extension of this approach which requires a detailed understanding of the constituent biosynthetic enzymes to other water-soluble vitamins [e.g. thiamin (vitamin Bl) nicotinic acid and panthothenic acid] has not been made and still looks to bk an attractive opportunity for selective therapeutic intervention. 2.4.6 Miscellany A short list of enzymes which are or appear to be good therapeutic targets where their inhibitors are known is given in Table 5. 3 Future Prospects There can be no doubt that the selective inhibition of enzymes will continue to have important implications for medicine. There are two aspects to future progress (i) The development of improved inhibitors aimed at existing targets .(ii) The definition of new target enzymes. The first of these tasks must be fulfilled by the (medicinal) chemist. Advances being made in related disciplines will impinge more and more on this process and an awareness of the significance of these is crucially important as the approach to the design of new compounds becomes more orientated towards exploiting molecular mechanisms. For example the techniques of molecular biology will answer many questions about the molecular basis of enzymic catalysis.244 Gene sequencing will be able to explain isoenzyme specificity ;active-site-directed muta- genesis identifies important amino-acid residues; cloning techniques can provide large amounts of pure enzyme for mechanistic study.Complementary with these advances in biology are develop- ments in X-ray crystallography and n.m.r. which can provide topographical and conformational details. Visualization of this information by molecular graphics coupled with the capability to do computer-assisted theoretical calculations provides another piece of a framework that surrounds the chemist who is trying to design new compounds. The design of synthetic enzyme models,245 such as the crown ethers and cyclodextrins provides important information about the origins of binding and rate enhancement in catalysis. The intervention of immunology into this process the development of ‘Abzymes’ (catalytic antibodies) is an exciting and important development.246,247 The second aspect mentioned above of defining new targets is not the domain of the medicinal chemist.Targets are identified from the results of an amalgam of biological investigations. However it is vital that the chemist keeps an open mind and is receptive to this information. The common currency of such interactions is often a small molecule (either a synthetic ‘tool ’ or a natural product that shows novel biological action). New enzyme targets will continue to appear. Examples are virally induced enzymes that have different specificities from counterparts in the host organism enzymes that are found in micro-organisms which do not occur in mammals and enzymes that perform crucial or rate-limiting transformations (e.g. ribonucleotide reductases and enzymes of S-adenosyl-methionine metabolism).The design of inhibitors to targets such as these which in the first instance may not be directed towards a particular disease has to address both the ‘Western World ’ diseases (such as heart disease ‘cancer ’ arthritis and the many manifestations of ‘mental disease 7 and ‘Developing World ’ diseases (such as malaria schistosomiasis and onchocerciasis) which have such debilitating effects on enormous numbers of people. Clearly there is still much for the medicinal chemist to do. Acknowledgements. 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ISSN:0265-0568    

DOI:10.1039/NP9890600311

出版商:RSC    

年代:1989

数据来源: RSC

摘要:

Diterpenoids J. R. Hanson School of Molecular Sciences University of Sussex Brighton BN 7 9QJ Reviewing the literature published during 1987 (Continuing the coverage of literature in Natural Product Reports 1988 Vol. 5 p. 21 1 ) 1 Introduction 2 Acyclic and Related Diterpenoids 3 Bicyclic Diterpenoids 3.1 Labdanes 3.2 Clerodanes 4 Tricyclic Diterpenoids 4. I Pimaranes 4.2 The Chemistry of the Tricyclic Diterpenoids 4.3 Naturally Occurring Abietanes 4.4 Rearranged Tricyclic Diterpenoids 5 Tetracyclic Diterpenoids 5.1 Kaurenes 5.2 Beyerenes 5.3 Atiserenes 5.4 Gibberellins 5.5 Aphidicolin and its Relatives 6 Macrocyclic Diterpenoids and their Cyclization Products 7 Miscellaneous Diterpenoids 8 References 1 Introduction This Report follows the pattern of its predecessors' and covers the literature to December 1987.The year has seen a steady increase in the number of known structures particularly clerodanes and kaurenoids. Seco-diterpenoids are being found in increasing numbers. These compounds possibly represent the early stages in the catabolism of diterpenoid natural products. mHCopH (3) 2 Acyclic and Related Diterpenoids Linear diterpenoids including geranylgeraniol eleganolone crinitol and eleganonal (l) have been found2 in the brown alga Cystoseira balearica. Furanoditerpenoids of this type are also quite common in the Compositae (= Asteraceae). Microglossic acid (2) (from Microglossa ~eylanica)~ and thymifodioic acid (3) (from Baccharis thymif~lia)~ are further examples.The cycli- zation of ambliofuran analogues to labdane structures has been ~tudied.~ 3 Bicyclic Diterpenoids 3.1 Labdanes The number of known labdanes of both enantiomeric series continues to grow. However the absolute stereochemistry that has been assigned to some of these compounds does not appear to be secure. In view of the reported co-occurrence of members of both enantiomeric series there is an increasing need for a critical survey of this area and for some well-established inter- relationships to be examined. Examination of Ericameria laricifolia (Asteraceae) a resinous shrub that grows in New Mexico has affordeda some new labdanoid esters including (4) whilst some esters of (5) have been isolated' from Piptothrix sinaloae (Compositae).Scoparic acid A (6) has been obtained* from the Paraguayan drug Typycha Kuratu [Scoparia dulcis (Scrophulariaceae)]. The ester (7) has been obtainedg from the mucus of the marine pulmonate limpet Trimusculus reticulatus in which it acts as a repellent for predatory starfish. The corresponding 2a,7a-diacetate was also isolated. New labdanes e.g. (8) (7) 347 NATURAL PRODUCT REPORTS 1989 H @ HO2C \\\ H 02H (10) (11) PAC I H' HOW, OAc (17) a; R' = OH R2 = R4 = H R3 = Me b; R'R2 = 0,R3 =Me R4 = H GlcO' (16) c; R' = OH R2 = R4 = H R3 = Bu' d; R' = OH R2 = R4 = H R3 = Bus 0go (25) (26) (20) (211 (22) continue to be detected'O in Halimium viscosum.The ent-halimane skeleton has been assigned" to some rearranged labdanes e.g. (9) from this source. Extraction of Brickellia glomerata (Compositae) afforded (10).l2 The hot-tasting dialde- hyde (1 1) is amongst the constituents of the seeds of Aframomum daniellii (Zingibera~eae),'~ which are used as a condiment. The epoxide (12) is another component. The diene (13) and the nor- labdene (14) have been ~btained'~ from Rutidosis murchisonii (Compositae). Lagerstronolide (1 5) [from Lagerstroemia lan- casteri (Lythra~eae)]'~ and phloganthoside (16) [from Phlo-gacanthus thyrsijlorus (A~anthaceae)]'~ are butenolides which have been described during the year. Plants of the genus Grindelia (Asteraceae) are characterized by an abundant resinous exudate which contains diterpenoids.A further group of grindelane diterpenoids (17) have been obtained1' from G. acutifolia whilst the havardic acids e.g. (1 8)18 are similar compounds from G. havardii. Some B-secolabdanes e.g. (19) and (20) which were ~btained'~ from Hebeclinium macro- phyllum (Compositae) may arise through a fragmentation [(21) -,(22)] that is based on the protonation of the hydroxyl group at C-8. Pine oleoresins are a rich source of diterpenoids. Cyclo- anticopalic acid (23) and its fission product (24) have been obtained20 from Pinus strobus. en?-3P-Hydroxymanoyl oxide is a further constituent of Palafoxia rosea.21 The biological activity of forskolin (25) has stimulated synthetic and chemical modifications in this area.22-2s The oxidation of 9,13-epoxy- labdanes with Fetizon's reagent has been used29 as a means of linking the furan dubiin and leonitin with the corresponding lactones.Studies have been reported on the production of the perfumery chemical ambrox (26) from sclare01~~ and from NATURAL PRODUCT REPORTS 1989-5. R. HANSON (27) (28) (29) HO' (30) HO.. (33) (32) (34) 0 (35) (36) (37) abietic acid3' and on various oxidative tran~formations~~ and cyclization reactions of labdanes. 33 The X-ray crystal structures of andrographolide and methyl trans-communate have been 35 3.2 Clerodanes Several trans-clerodanes including (27) have been isolated36 from Aristofochia brasifiensis and A.esperenzae (Aristolochi- aceae). A systematic survey of Mexican species of Salvia (Labiatae) has afforded a number of new diterpenoids including lasianthin (28) from S. l~siantha,~'kerlinic acid (29) from S. ket~fii,~~and brevifloralactone (30) from S. breviflor~.~~ Continued investigations of plants of the genus Baccharis (Compositae) have also afforded some clerodanes such as 4/3-hydroxyisobacchasmacranone (3 1) from B. macraei4' and desoxyarticulin (32) and dihydrotucumanoic acid (33) from B. pediceffata and B. rnargin~fis.~' Scuterivulactone C (34) and scuterivulactone D (35) were ~btained~~.~~from Scuteffaria rivufaris (Labiatae) which is the source of the Chinese drug Ban Zhi Lian. The X-ray crystal structures of linearolactone (36)44 and cordatin (37) [from the bark of Aparisthmiurn cordaturn (Euphorbia~eae)]~~have been reported.Rhynchosperins A B (38) and C are similar compounds that have been obtained46 AcO from Rhynchospermum verticiffatum (Compositae). Plants of the genus Teucrium (Labiatae) have been a fruitful source of Me' clerodanes. Recent isolates include 2-deoxychamaedroxide (39) (from T. div~ricatum)~'and teucretol (40) from T. ~reticurn.~~ Further studies on Ajuga charnaepitys (Labiatae) afforded (411 chamaepitin (41).49 In the same paper is reported an X-ray NATURAL PRODUCT REPORTS 1989 OAc (42) (43) 0 OH ROFOH 0 (46) R = CH3 or CH20H (471 study on auropolin (42) which established its stereochemistry. Contrary to previous suggestions n.m.r.measurements in- dicate50 a (12s) stereochemistry for teupolin I. The number of cis-clerodane lactones that are known has continued to increase. Ten lactones exemplified by (43) and (44),have been isolated5' from Gutierrezia texana (Compositae) whilst ephemeric acid (45) was from Ephemerantha comata. The cis A/B fusion in these compounds was established by n.0.e. measurements between 19-H and 10-H. Several X-ray &... -td' (50) (511 ene- 1 5p,16-diol has been reportedG5 as a constituent of Rabdosia glutinosa. 4.2 The Chemistry of the Tricyclic Diterpenoids The stereochemistry of the tetrahydroisopimaric acids has been studied,6s as has the epoxidation of various abietic acid Various methods have been exploredG9. deri~atives.~~.~~ 70 in attempts to convert the tricyclic diterpenoids into steroids.The synthesis of pisiferic acid and its analogues has attracted intere~t'l-~~ because of their antifungal activity and their effectiveness in regulating the growth of mites. The synthesis of abietatrien-l2,16-oxide (50) and its C-15 epimer has been crystal structures have also been reported in this ~eries.~~-~~ in~estigated~~ and some further studies on the modification of The pilosanones A and B (46)5sare rearranged versions of the clerodane skeleton which have been isolated from Portulaca pilosa. Synthetic endeavour in this area continues with reports having been made on the synthesis of the aj~garins~~ and methyl kola~enate.~~ 4 Tricyclic Diterpenoids 4.1 Pimaranes Both enantiomers of sandaracopimaradiene have been de- te~ted~~ in the foliage hydrocarbons of Podocarpus spicatus (the Matai tree).The carbon-13 n.m.r. spectra of pimarane diterpenes have been examined.60 7-0xoisopimara-8,15-dien-18-01 (47) has been obtainedG1 from Nepeta tuberosa subsp. reticulata (Labiatae). Sandaracopimara-8( 14) 15-diene-2~~,18- diol(48) is a minor constituent of Tetradenia riparia (= Riboza riparia) (Labiatae),62 which is used as a medicinal plant in Rwanda. The phytoalexin oryzalexin D (49) has been shown to podocarpic acid have been reported. 75 4.3 Naturally Occurring Abietanes The genus Calceolaria (Scrophulariaceae) contains a number of plants which are used in folk medicine. The dehydroabietinol (5 1) has been from C.ascendens. 18-Oxoferruginol (52) has been from the leaf oil of the conifer Torreya nucifera (Taxaceae) 1-oxohinokiol (53) is amongst the ter- penoids of Cafocedrus formosana (C~pressaceae),'~ and nimbi- diol (54) has been ~btained'~ from the root bark of the neem tree [Azadirachta indica (Meliaceae)]. The seco-abietane di- aldehyde (55) has been foundso in Chamaecyparis formosensis. Salvicanaric acid (56) and the carnosic acid derivative (57) have been iso1ateda1qa2 from Salvia canariensis (Labiatae). The monomethyl ether (58) was obtaineds3 from Salvia fanigera whilst some more highly oxidized compounds e.g. (59)s4 and (60) and (61),85 were isolated from Salvia cryptantha and Salvia interfere with cell-membrane function in Pyricufaria oryz~ze.~~ regia respectively.Tan-shen (Salvia miltiorrhiza) is one of the 14) 15-diene has been obtaineds4 most important Chinese herbal medicines. The tanshinone 12#?,18-Dihydroxypimara-8( from Chinese Pinus massoniana rosin whilst ent-isopimar-8- pigments are major constituents of the drug -new examples are NATURAL PRODUCT REPORTS 1989-5. R. HANSON 351 0 CHO OH (54) (55) (56) (57)R = Me (58) R = H HO (59) (62) (65) (68) neocryptotanshinone (62) and isotanshinone I1 (63).E6The glandular pigments obtained from plants of the genera Coleus and Plectranthus (Labiatae) continue to attract attention. Analysis of the constituents of Plectranthus edulis has affordedE7 a range of oxidized abietanes and their relatives including edulon A (64) and the 1,lO-secoabietane (65).Small amounts of seco-abietanes [e.g. (66) from Plectranthus sanguineusEE] and (67)(from Coleus barbat~s)~~ have been reported. The synthesis (69) of (1 5R)-and (1 SS)-coleon C from methoxyabietatrienols has been described. 4.4 Rearranged Tricyclic Diterpenoids Betolide (68) which was isolatedg1 from Betonica oficinalis has an unusual skeleton. An X-ray crystal structure has been reporteds2 for triptophenolide methyl ether (69). Myrocin C NATURAL PRODUCT REPORTS 1989 (73) (74) R = CH20H CHO or C02H (70) which is a tumour-inhibitory antibiotic from a species of Myrothecium has an interesting structure containing a cyclopropane ring.93 Some rosane diterpenoids such as (7 1) and (72) are constituents of Hyrnenothrix wislizenii (Com- po~itae).~~ The cleistanthane stereochemistry of auricularic acid (73) has been clarifiedg5 whilst continuing investigations on the Velloziaceae have affordedg6 the cleistanthenes (74) from Vellozia Jlavicans.Some tumour-inhibitory diterpenoids the taxa-mairins [e.g. (75)] have been isolated from Taxus mairei,97*98 which also contains some taxane diterpenoids. 5 Tetracyclic Diterpenoids 5.1 Kaurenes Some new models for the cyclization steps in the biosynthesis of tetracyclic diterpenes have been considered ;99 loo in one instance these involve cyclobutyl derivatives. The preparation of ent-kaurene from the more readily available ent-kaurane- 17,19-diol has been reported.lO' Some 15- and 18-oxygenated kaurenes such as ent-15-0x0-kaur- 16-ene- 18-oic acid have been obtained from the liverwort Porella densifolia.lo2 Simple derivatives of kaurenoic acid have been reported in Viguiera latibracteata (Compositae),lo3 Annona reticulata (Annon-aceae),lo4Solidago nemoralis (Asteraceae) lo5Helichrysam davgi (Compositae),'OG Robinsonia thurifera (Comp~sitae),'~' and Montanoa tomentosa (Compositae).lo8 The 2,17-diglycoside of ent-2a7 16p 17-trihydroxykaurane has been isolatedlog from Turbina corymbosa.The genus Sideritis (Labiatae) contains a number of species endemic to the Canary Islands and these have been a fruitful source of diterpenoids. Sidendrodiol (76) has been ObtainedllO from Sideritis dendrochahorra.ent-3P,16/? 17-Trihydroxykaurane has been obtained,"' to-gether with some beyerenes from Peteravenia malvaefolia (Composi tae). 6.H 'OMe (75) R I HO (77) R = H (85) R =OH (79) (80) R = H (81) R=OH R' 0 AcO OAc (82) R' = H R2 =OH (86) (83) R' = R2 = OAc (84) R' = R2 = H AcO OH OAc (87) (88) (891 NATURAL PRODUCT REPORTS 1989-5. R. HANSON HO Table 1 Tetracyclic diterpenoids from Rabdosia species Name Structure Source Ref. Kamebakaurin (77) Kamebakaurinin (78)) R. umbrosa var. leucantha 112 Kamebacetal (79) Inflexanin A i:!; } Inflexanin B R. injexa 113 Lushanrubescensin B Lushanrubescensin C $;} R. rubescens 114 Lushanrubescensin E (84) Rabdoserrin B (85) R.serra 115 Adenanthin (86) R. adenantha 116 Macrocalyxin E (87) R. macrocalyx 117 $;;1 Rabdosinke R. japonica 118 Rabdosinatol Coetsin A } R. coetsa 119 Coetsin B Ludongnin B (92) R. rubescens 120 Medicinal plants of the genus Rabdosia (Labiatae) par- ticularly of Chinese origin have been the source of many highly hydroxylated kaurenes several of which have tumour-in-hibitory activity. Some further examples are given in Table 1. The crystal structure of rabdoserrin A (93) has been reported.lZ1 Coestinol (94) is a similar diterpenoid which has been obtained122 from Plectranthus coetsa [ = Rabdosia coetsa] (Labiatae). The seco-kaurene derivative (95) has been reportedlZ3 as a constituent of AIepidea amatynsia (Umbelliferae).The B-seco- aldehyde anhydride fujenal (96) shows some interesting reactionslZ4 in which despite the possibility of rotation about the 9-10 bond which is observed in the enmein series there is neighbouring-group participation between C-6 and C-7 with the formation of for example lactones such as (97). There has been a renewal of interest in atractyligenin with reports on photochemical addition to the do~ble-bondl~~ and on the structure and stereochemistry of atractyliretin (98).lZ6 The total synthesis of ( & )-atra~tyligenin'~'and cafesto1128 have been reported. The coffee constituents cafestol and kahweol are inducerslZ9of the detoxifying enzyme glutathione transferase in laboratory animals. It has been suggested that the gibberellin biosynthetic system in the d dwarf mutant of maize (Zea mays) is unable to handle ent-kaur-1Sene and convert it into gibberellins.Consequently the discovery of the biotransformation of ent-kaur- 15-enes by Gibberella fijikuroi into a range of gibberell-15-enes was of interest.130 5.2 Beyerenes Phytochemical investigations of the large genus Stevia (Com- positae) have continued with a report13' being published on the isolation of the beyerene (99) from S. aristata. The rearrangement of 15,16-epoxybeyerenes that are functionalized at C-14 [e.g. (1 OO)] to kauranoids [e.g. (1 01)] has been investigated. 13 HO OH (93) (94) (p \\ CHO 'C 8 d' (95) (96) C02Me (97) (98) OH OH CO2H (99) OAc OAc (100) (1011 5.3 Atiserenes The total synthesis of atiserene has been ~ep0rted.l~~ 3-Oxoatisane- 16p 17-diol has been from Euphorbia acaulis.The microbiological transformation of some isoatisene diterpenoids into isoatisagibberellins A, and A, and into isoatisenolide has been described.135 5.4 Gibberellins Reviews have appeared on the biosynthesis and metabolism of gibber ell in^.'^^ The isolation of gibberellin A, (102) from seeds of Tropaeolum majus (nasturtium) has been described.13' Methods for the radio-immunoassay of the dimethyl esters of GA, and GA, have been deve10ped.l~~ The analysis of the MeO-C-0Me HO k02Me H02C OH OH .'C H2 0H OH H0' BzO 0 AcO PAC 1 Me Et ,CHC02-*. '0 H OAc NATURAL PRODUCT REPORTS 1989 13C-lH two-dimensional n.m.r.spectrum of gibberellic acid has been re~0rted.l~~ Papers have appeared on the cleavage of ring A of 3-oxogibberellins that occurs when they react with primary and secondary amines,140 the reductive cleavage of the lactone ring,14' and the preparation of thiogibberellins. 142 The crystal structures of 19- 10-thiogibberellin A,142 and the 19-201-isolac- tone of gibberellic acid have been re~0rted.l~~ An unusual C-7 orthoester (103) which was obtained from 6-epi-gibberellin A13 has been described.I4 The oxidation of 16( 17)-terminal epoxides to allylic aldehydes with sulphuryl chloride has been described.145 Methods for distinguishing the stereochemical consequences of reactions on ring D by using n.m.r.spec-trometry have been developed. 146 The X-ray crystal structures of gibberellin A and gibberellin A hydrochloride methyl esters were described in the same paper. The recent discovery that the antheridiogens (substances which induce the formation of antheridia in fern gametophytes) are gibberellins has introduced an interesting new dimension to gibberellin research. The biological activities of synthetic and natural antheridic acid (104) have been compared.14' The possible biosynthetic origin of these substances through the rearrangement of appropriately substituted gibberellins has led to consideration of their synthesis from the much more readily available gibberellins such as gibberellin A,. 148 5.5 Aphidicolin and its Relatives Aphidicolin (105) is of considerable interest since it is a specific inhibitor of DNA polymerase a.Chemical modifications of the structure in the course of investigations of structure-activity relationships have been reported1, whilst the late stages in the biosynthesis of aphidicolin involving the sequential hydrox- ylation of aphidicolan- 16-01s have been examined.lS0 The total syntheses of aphidic01in'~l and 2-deoxy~temodinone'~~ have been reported.The scopadulcic acids A (106) and B which have been isolated from the Paraguayan drug Typycha Kuratu (Scoparia dulcis) possess similar 6 Macrocyclic Diterpenoids and their Cyclization Products The synthesis of macrocyclic diterpenoids has continued to attract attention with reports on the use of tin compounds in cyclizations in this area.Recent isolates include agrosti- AcO-0,CCHMeEt NATURAL PRODUCT REPORTS 1989-5. R. HANSON 6 I OAc (1 18) OAc OAc C02Me (120) (1211 @ (125) Br stachin (107) from Agrostistachys h~okeri,'~~ and some cembranediol~~~~ from the soft coral Sinularia mayi. Esulone C (108) is a jatrophane diterpenoid which has been found in the roots of the leafy spurge (Euphorbia e~ula).'~~ This toxic weed poses a threat to livestock. The correct structure of euphorianin (1 09) has been e~tablished'~~ by X-ray crys-tallography. A review on the taxane constituents of Taxus species has been presented.159 Taxusin (1 10) and taiwanxan (1 11) are two further members of the series which were obtained from Taxus mairei.Their structures were established by X-ray crystal- C02H H02C& / (116) OH (1 19) AcO 0 0 lography.160,'61The 18-0x0-virg-3-ene (I 12) was obtained from a Virginia 115 cultivar of tobacco (Nicotiana tabacum).'62 7 Miscellaneous Diterpenoids The variety of diterpenoid skeleta continues to increase. Several prenylated versions of sesquiterpenoid compounds such as pachytriol (1 13) (from the brown alga Dictyota di~hotoma)'~~ and dictyotriol A (1 14) (from the alga Glossophora kuntii),lB4 have been reported. The brown alga Dilophus guineensis affordedlB5 dilophic acid (1 15). Some isoprenologues of bisabolene e.g. (1 16) have been isolatedlB6 from Eremophila foliosissima whilst E. exotrachys was the source of the viscidane diterpenoid (1 1 7).lB7 The liverworts contain a number of diterpenoids.Extraction of the oil glands of Anastrophyllum minutum afforded168 the sphenolobane diterpenoid (1 18) whilst sacculaplagin (1 19) is a hemiacetal which was obtainedlBg from Plagiochila acantho- phylla. The diterpene (120) has been obtained170 from the mollusc Aplysia dactylomela. Other new diterpenoids from marine sources include sarcodictyin A (121) from the stolonifer Sarcodictyon ro~eum,'~~ the briarene derivatives (122) from the soft coral Briareum ~teckei,'~~ and verecynarmin A (123) from the Mediterranean nudibranch Armina rnac~lata.'~~ The crystal structure of brianthein X (124) has been Nors-phaerol (125) is a bis-norditerpene which was ~btained,'~~ A AcO ' (128) (129) along with the (129- 12-hydroxybromosphaerodio1(126) from the red alga Sphaerococcus coronopifolius.176 The kalihinols e.g. (127) are a family of antibiotics which were from marine sponges of the genus Acanthella. Diterpene isocyanides e.g. (l28) have also been obtained17* from sponges of the genus Hulichondria. Chromophycadiol monoacetate (129) represents a new carbon skeleton which was from a Dictyotu species whilst dictymal (130) is a seco-fusicoccin- type diterpenoid which was isolated180 from Dictyota dichotomu. Vinigrol (1 3 1) is an unusual structure possessing antihyper- tensive and platelet-aggregation-inhibitoryactivity that has been isolated from the fungus Virguria nigru.181 Synthetic activity in this area is represented by reports on the synthesis of secotrinervitenes182+183 and laurenenes.184*185 8 References J.R. Hanson Nat. Prod. Rep. 1988 5 21 1. V. Amico P. Neri M. Piattelli and G. Ruberto Phytochemistry 1987 26 2637. A. A. L. Gunatilaka B. Dhanabalasingham L. Paredes J. Jaku- povic F. Bohlmann and N. K. B. Adikaram Phytochemistry 1987 26 2408. J. R. Saad M. J. Pestchanker and 0.S. Giordano Phytochem-istry 1987 26 3033. M. Nishizawa H. Yamada and Y. Hayashi J. Org. Chem. 1987 52 4878. J. J. Hoffmann S. D. Jolad B. N. Timmerman R. B. Bates F. A. Camou and T. J. Siahaan Phytochemistry 1987 26 2861. M. Miski D. A. Gage andT. J. Mabry Phytochemistry 1987,26 2753. M. Kawasaki T.Hayashi M. Arisawa M. Shimizu S. Horie H. Ueno H. Syogawa S. Suzuki M. Yoshizaki N. Morita Y. Tezuka T. 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Prod. 1987 50 995. 109 M. G. Nair B. A. Burke K. S. Manning and D. G. Lynn J. Chem. Res. (3, 1987 318. 110 B. M. Fraga M. G. Hernandez C. Fernandez and J. M. Arteaga Phytochemistry 1987 26 775. 111 E. Ellmauerer J. Jakupovic F. Bohlmann and R. Scott J. Nat. Prod. 1987 50 221. 112 Y. Takeda T. Ichihara Y. Takaisha T. Fujita T. Shingu and G. Kusano J. Chem. Soc. Perkin Trans. I. 1987 2403. 113 Y. Takeda T. Ichihara T. Fujita K. Kida and A. Ueno Chem. Pharm. Bull. 1987 35 3490. 114 J.C. Li C.-J. Liu H.-D. Sun and Z.-W. Lin Yunnan Zhiwu Yanjiu 1986 8 93 (Chem.Abstr. 1987 107 130873); J.-C. Li H.-D. Sun and Z.-W. Lin ibid, 1987,9,485 (Chem. Abstr. 1988 108 164 740). 115 R.-L. Jin P.-Y. Cheng and G.-Y. Xu Zhongguo Yaoke Daxue Xuebao 1987 18 172 (Chem. Abstr. 1988 108 72070). 116 Y.-L. Xu H.-D. Sun D.-Z. Wang T. Iwashita H. Komura M. Kozuka K. Naya and I. Kubo Tetrahedron Lett. 1987 28,499. 117 X.-R. Wang Z.-Q. Wang and J.-Q. Dong Zhiwu Xuebao 1986 28 415 (Chem. 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Kurogochi I. Yamaguchi M. Feyerabend N. Murofushi N. Takahashi S. Kuyama and E. W. Weiler Phytochemistry 1987 26 2895. I39 A. Preiss G. Adam D. Saman and M. Budesinsky Magn. Reson. Chem. 1987 25 239. 140 G. Adam and T. V. Sung Z. Chem. 1987 27 146. 141 B. Jiang X.-F. Pan and H. Zhao Synth.Commun. 1987 17 997. 142 A. Schierhorn G. Adam L. Kutschabsky and P. Leibnitz J. Chem. Soc. Perkin Trans. 1. 1987 21 1 1. 143 L. Kutschabsky G. Adam and B. Voight Z. Chem. 1987,27,73. 144 B. M. Fraga I. Gonzalez-Collado M. G. Hernandez F. G. Tel- lado and A.A. Perales J. Chem. Soc. Perkin Trans. l. 1987 1955. 145 C. L. Willis Tetrahedron Lett. 1987 28 2175. 146 K. M. Abouamer J. R. Hanson and P. B. Hitchcock J. Chem. Soc. Perkin Trans. 1. 1987 1991. 147 K. Takeno H. Yamane K. Nohara N. Takahashi E. J. Corey A. G. Myers and H. Schraudolf Phytochemistry 1987,26 1855. 148 M. Furber and L. N. Mander J.Am. Chem. Soc. 1987,109,6389. 149 S. Hiranuma T. Shimizu H. Yoshioka K. Ono H. Nakane and T. Takahashi Chem. Pharm. Bull. 1987 35 1641. 150 M. J. Ackland J. F. Gordon J. R. Hanson B. L. Yeoh and A. H. Ratcliffe J. Chem. SOC.,Chem. Commun. 1987 1492. 151 R. A. Holton R. M. Kennedy H. B. Kim and M. E. Krafft J. Am. Chem. Soc. 1987 109 1597. 152 J. D. White and T. C. Somers J.Am. Chem. Soc. 1987,109,4424.153 T. Hayashi M. Kishi M. Kawasaki M. Arisawa M. Shimizu S. Suzuki M. Yoshizaki N. Morita Y. Tezuka T. Kikuchi L. H. Bergansa E. Ferro and I. Basualdo. Tetrahedron Lett. 1987 28 3693. 154 J. A. Marshall B. S. Delloff and S. L. Crooks Tetrahedron Lett. 1987 28 527. 155 Y. H. Choi J. Kim J. M. Pezzuto A. D. Kinghorn N. R. Farns- worth H. Lotter and H. Wagner Tetrahedron Lett. 1986 27 5795. 156 M. Kobayashi T. Ishizaka N. Miura and H. Mitsuhashi Chem. Pharm. Bull. 1987 35 2314. 157 G. D. Manners and D. G. Davies Phytochemistry 1987 26 727. 158 J. L. Okogun C. 0.Fakunle D. E. U. Ekong H. J. Linders and G. G. Habermehl 2. Naturforsch. Teil B 1987 42 243. 159 F. Gueritte-Voegelein D. Guenard and P. Potier J. Nut. Prod. 1987 50 9.NATURAL PRODUCT REPORTS 1989 160 T. I. Ho G. H. Hsiang S. M. Peng M. K. Yeh F. C. Chen and W. L. Yang Acta Crystallogr. Sect. C 1987 43 1378. 161 T. I. Ho Y. C. Lin G. H. Lee S. M. Peng M. K. Yeh and F. C. Chen Acta Crystallogr. Sect. C 1987 43 1380. 162 R. Uegaki T. Fujimori N. Ueda and A. Ohnishi Phytochem-istry 1987 26 3029. 163 A. G. Gonzalez E. Manta J. D. Martin and C. Perez J. Nut. Prod. 1987 50 500. 164 A. P. Rivera L. A. Astudillo A. G. Gonzalez E. Manta and F. Cataldo J. Nut. Prod. 1987 50 965. 165 D. Schlenk and W. H. Gerwick Phytochemistry 1987 26 1081. 166 P. G. Forster E. L. Ghisalberti and P. R. Jefferies Tetrahedron 1987 43 2999. 167 P. G. Forster E. L. Ghisalberti and P. R. Jefferies Aust. J. Chem. 1986 39 21 11.168 J. Beyer H. Becker M. Toyota and Y. Asakawa Phytochemistry 1987 26 1085. 169 T. Hashimoto M. Tori and Y. Asakawa Tetrahedron Lett. 1987 28 6293. 170 A. G. Gonzalez F. Cataldo J. Fernandez and M. Norte J. Nut. 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ISSN:0265-0568    

DOI:10.1039/NP9890600347

出版商:RSC    

年代:1989

数据来源: RSC

摘要:

Carotenoids and Polyterpenoids G. Britton Department of Biochemistry University of Liverpool P.O. Box 147,Liverpool L69 3BX Reviewing the literature published between January 1986 and December 1987 (Continuing the coverage of literature in Natural Product Reports 1986 Vol. 3 p. 591 ) 1 Carotenoids 1.1 Introduction 1.2 New Structures and Stereochemistry 1.2.1 Carotenoids 1.2.2 New Natural Products Related to Carotenoids 1.3 Synthesis and Reactions 1.3.1 Carotenoids 1.3.2 Retinoids I .3.3 Carotenoid-like Substances 1.4 Carotenoid-Protein Complexes 1.5 Physical Methods 1.5.1 Separation and Assay 1S.2 N.M.R. and E.S.R. Spectroscopy 1.5.3 Mass Spectrometry 1.5.4 Circular Dichroism and Linear Dichroism 1S.5 Raman and Infrared Spectroscopy 1S.6 Electronic Absorption and Other Spectroscopic Methods 1S.7 X-Ray Methods 1.5.8 Miscellaneous Physical Chemistry 1.5.9 Photoreceptors 1.6 Biosynthesis and Metabolism 2 Polyterpenoids and Quinones 2.1 Polyterpenoids 2.2 Isoprenylated Quinones 3 References 1 Carotenoids 1.1 Introduction The most useful new publication in the field of carotenoids is an up-to-date ‘Key to Carotenoids’ ;’ this supersedes the first edition,2 which was published in 1976.This book catalogues all known naturally occurring carotenoids and gives compre- hensive lists of references to their occurrence distribution spectroscopic properties and synthesis.A two-volume Rom- anian monograph on carotenoids and their metabolites has been published (Volume 1 chemistry and biochemistry;3 Volume 2 :functions and utilization4). A new book on pigments in fruit5 contains extensive information on carotenoids and a general book on the biochemistry of natural pigments containing a detailed chapter on carotenoids has been published in Russian translatiomB Extensive review articles have appeared dealing with recent advances in carotenoid chemistry,’ with the application of microbial transformations in the synthesis of carotenoids and vitamins,s and on the metabolism functions and nutritional importance of caro- ten~ids.~ Other articles on the chemistry of leaf pigmentation,l0 pigments and lipids in photosynthetic bacteria,ll plant pigments as natural food colours,12 and the chemistry of isoprenoids13 include information on carotenoids.Degradation products of carotenoids as possible flavour or aroma compounds are discussed in three reviews.14-16 Methods for analysis of the carotenoids in micro-organisms1’ and for the measurement of carotenoids vitamin A and retinoidsls have been surveyed and three volumes in the series ‘Methods in Enzymology’ include articles on the purification assay etc. of derivatives of vitamin A,19 on membrane-bound retinal-proteins (especially bacteriorhodopsin),20 and on the analysis biosynthesis etc. of carotenoids in plant organelles and membranes.21 The main lectures of a symposium on retinoids have been publishedzz and several aspects of vitamin A and retinoids have been re-viewed.z3-24 1.2 New Structures and Stereochemistry I .2.1 Carotenoids The first ‘Key to Carotenoids’ published in 1976 consisted of an exhaustive list of all natural carotenoids then known together with a comprehensive collection of references.2 In a new edition of this work Pfander’ has expanded this list to include all carotenoids newly discovered up to 1986 and greatly extended the compilation of references given for the previously known compounds.The number of new natural carotenoid structures continues to increase and the stereo- chemical characterization of known substances shows that the existence of mixed enantiomers is more widespread than was previously thought especially in the animal kingdom.Carotenoid epoxides are being found in increasing variety. Although xanthophyll epoxides are among the most common naturally occurring carotenoids and carotene 5,6-epoxides have frequently been reported as minor constituents of carotenoid extracts the identification of luteochrome [(5R 6S,5’R,8‘RS)-5,6;5‘,8‘-di epoxy -5,6,5‘ 8‘-tetra h y d ro -P,p-carotene] (1) in Brazilian sweet potatoes25 is the first report of the isolation of a carotenoid with an unhydroxylated 5,6- epoxy-5,6-dihydro-P-ring in optically active form. The chirality of C-5 and C-6 is the same as in violaxanthin [(3S,5R,6S 3’S 5’R,6’S)-5,6 ;5’,6’-diepoxy-5,6,5‘ 6’-te trahydro-P,p- car0 tene- 3,3’-diol] (2). The most interesting new end-group is the 3,6-epoxy-5,6- dihydro-P or 7-oxabicyclo[2.2.llheptane group which was first identified in eutreptiellanone (3,6-epoxy-3’,4‘,7’,8’-tetra-dehydro-5,6-dihydro-P,B-caroten-4-one) (3) and which has now been found in several different carotenoids from higher plants and algae. Thus Eutreptiella gymnastica the original source of eutreptiellanone has now yielded26 the other related com-pounds a-cryptoeutreptiellanone [(3S,sR,6&3’R,6’R)-3’-hy-droxy-3,6-epoxy- 5,6-di hydro-P,e-car0 ten-4-onel (4) and the acet ylenic compound (3S,5 R,6S,3’R)-3’- hydroxy- 3,6-epoxy- 7’,8’-didehydro-5,6-dihydro-P,P-caroten-4-one (5). In higher plants cucurbitaxanthin A [(3R,3’S,5’R,6’R)-3’,6’-epoxy-5’,6’-dihydro-P,P-carotene-3,5’-diol] (6) and its 5’,6’-epoxide cucur- bitaxanthin B [(3S,5R,6S,3’S,5’R,6’R)-5,6;3’,6’-diepoxy-5,6, 5’,6’-tetrahydro-P,/l-carotene-3,5’-diol] (7) have been isolated from pumpkin (Cucurbita maxima).2‘ Cucurbitaxanthin A was also found in the fruit of red pepper (Capsicum annuum),28 together with a second 3,6-epoxide namely 5,3’-dihydroxy- 3,6-epoxy-5,6-dihydro-P,~-caroten-6’-one (8) with capsanthin 5,6-epoxide [(3S,5R,6S,3’S,5’R)-5,6-epoxy-3,3’-di h ydrox y-5,6-dihydro-P,~-caroten-6’-one] (9) and with 5,6-dihydro-P,P-car0 tene- 3,5,6,3’- tetra01 (10) which was assigned (3 S,5S,6S,3’R) chirality i.e.different from the chirality (3S,5R,6R,3’R) that had been proved for karpoxanthin (1 l) which is a carotenoid of similar constitution that had previously been isolated from 359 NATURAL PRODUCT REPORTS 1989 R2 HOJ Y HOkP f 9 h I i H0“cr k / m n (1) R’ =a R2 =b (6) R’ = h R2 = i (10) R’ =k R2 =i (2)R’ = R2 =c (7) R’ = h R2 =c (11) R’ =/ R~ =i (3)R’ = d R2 = e (8)R’ =h R2 =j (12) R’=m R2=f (4) R’ = d R2 = f (9) R’ = C R~ =j (13) R’ =m R2 =n (5) R’ =d R2 =g HOu the fruit of Rosa p~mifera.~~ Two new carotenoids related to prasinoxan thin (3,6,3’- trihydroxy- 7,8-di h ydro- y,e-caroten-8-one) (12) have been isolated from eukaryotic ultraplankton clones (Prasin~phyceae).~~ One has the novel feature of a 43- epoxy-4,5-dihydro-e end-group and both have 7,s-dihydro- structures ;this latter feature has previously only been reported in cyclic carotenoids isolated from animal sources.The compounds were characterized by spectroscopic methods including n.m.r.as (3S,3’R) 4‘,5’-epoxy-3,6,3’-trihydroxy-7,8,4’,5’,7’,8’-hexahydro-y,e-caroten-%one (13) and (3S,3’R)- 5,6-epoxy-3,3’-dihydroxy-5,6,7’,8’-tetrahydro-~,~-caroten-1l’ 19’-olide (14). Detailed n.m.r. studies leading to the rigorous assignment of stereochemistry as (3S,6R,3’R,6’R) for prasino- xanthin itself have been pre~ented.~~ 1’2’-Epoxy- 1’,2’-dihydro- e,$-carotene (15) the presence of which in the delta strain of carotenoids have been isolated from marine sponges. Aapto- purpurin from Aaptos aaptos was shown by mass spectrometry and ‘H n.m.r. to have the methoxylated structure 3-methoxy- p,x-carotene (18)35 whereas the elimination of mesylate from a 3-hydroxy-e end-group was used to characterize the minor metabolite 3,4-didehydro-y,~-carotene (19) from Microciona pr~lifera.~~ Gelliodes callista yielded the apocarotenal gelliodes- xanthin (3-hydroxy-4-oxo-2’-apo-~-caroten-2’-al) (20) together with its artefactual acetone-condensation product (21).37 Two novel apocarotenoids cochloxanthin and dihydrocochloxan- thin have been obtained from Cochlospermum tinctorum and identified by spectroscopic methods as 6-hydroxy-3-oxo-8’-apo-e-caroten-S’-oic acid (22) and 6-hydroxy-3-oxo-4,5-di-acid (23) respe~tively.~~ hydro-8’-apo-e-caroten-8’-oic The constitution and stereochemistry of the apoviolaxanthinol has been re-isolated and its chirality proved (by circular dichroism) to be (6R,2’S).33 Several new carotenoids have been isolated from animal sources.Two examples 2-hydroxy-P,P-caroten-4-one (16) and 2-hydroxy-P,P-carotene-4,4’-dione (1 7) which were obtained from the crustacean Daphnia magna contain the previously unknown 2-hydroxy-4-0x0-P end-group and were assigned (2R) chirality on the basis of their c.d. proper tie^.^^ Three new tomato (Lycopersicon esculentum) was reported previou~ly,~~ persicaxanthin [(3S,5R,6S)-5,6-epoxy-5,6-dihydro- 12’-apo-P-carotene-3,12’-diol] (24) have been established by ‘H n.m.r. and c.d. and by comparison with viola~anthin.~~ Four (di-Z)- isomers of violaxanthin itself namely the (9Z 9’Z)- the (92,13Z)- the (9Z 15Z)- and the (9Z 13’Z)-isomer have been isolated from Viola tricolor and characterized by two-dimen- sional lH n.m.r.The development of methods for the separation of carotenoid enantiomers either directly or as their simple derivatives on NATURAL PRODUCT REPORTS 198P-G. BRITTON RZ a b C d e f h I i k I m (15) R’ =a R2 =b (18) R’ =f R2 =g (21) R’ =i,R2 =k (16) R’ =c R2 =d (19) R’ = h R2 =g (22) R’ =/ R2 = C02H (17) R’ = c R2 = e (20) R’ =i R2 =j (23) R’ = m R2 = C02H (24) optically active h.p.1.c. columns has led to the discovery that the occurrence of different enantiomers or of enantiomeric mixtures is widespread in animal extracts. Thus mixtures of (3 R,3’R)- (3 R,3’S)- and (3 S,3’S)-zeaxanthin @,,&car0 tene- 3,3’-diol) (25) were present in shrimps fish and turtles although other sources e.g. higher plants shellfish and starfish contained exclusively the (3R,3’R)-i~omer.~l Comparative studies have revealed the presence in various fish of several R’ HO& HOJ3 o& isomers of lutein @,e-carotene-3,3’-diol) (26) namely the (3R,3’R,6’R)- the (3R,3’S,6’S)- the (3R,3’R,6’S)- and the (3S,3’R,6’S)-isomer which have been designated luteins A,D,F and G respecti~ely,~~.43 and of tunaxanthin (e,e-carotene-3,3’- diol) (27) namely the (3S,6S,3’S,6’S)- the (3R,6S,3’S,6’S)- the (3R,6S,3’R,6’S)- the (3R,6S,3’S,6’R)- the (3R,6R,3’R,6’R)- the (3S,6R,3’S,6’S)- the (3R,6R,3’R,6’S)- and the (3S,6R 3’S,6’R)-isomer; these have been designated tunaxanthins A B C D F G,H and J respecti~ely.~~~~~-~~ Similarly various C-6 and C-6’ epimers of 3’-hydroxy-e,e-caroten-3-one(28) [(6S 3’R,6’R) and (6R 3’R,6’R)] and of €,€-carotene- 3,3’-dione (29) [(6R,6’R) (6R,6’S) and (6S,6’S)] have all been detected in Japanese fish along with (3R,6’R)-3-hydroxy-P,ecaroten-3’-one (30).44-46 The two epimers of 3’-hydroxy-e,e-caroten-3-one (28) were also isolated from hen’s egg yolk,47 together with (6’RS)-P,e-caroten-3’-one (31).“O+ OH HO n a b C d e V f (25) R’ = R2 =a (29) R’ = R2 =c (32) R’ =a R2 =e (26) R’ =a R2 =b (30) R’ =a R2 =c (33) R’ = R2 =e (27) R’ = R2 = b (31) R’ =d R2 =c (34) R’ = R2 = f (28) R’ =c R2 =b NATURAL PRODUCT REPORTS 1989 (351 WH OH 0-Glc (38) R’ = H R2 = OH (39) R’ = OH R2 = H (40) R’ R2 = 0 @02H I 0-Glc (42) R = Me; XY = 0 (411 (43) R = Me; X = OH Y = H (44) R = CH2OH; XY = 0 An extensive and detailed n.m.r.study has allowed the carbohydrate moieties that are bound to the carotenoids myxol (3’,4’-didehydro-1’,2’-dihydro-P,+-carotene-3,1’,2’-triol) (32) and oscillol (3,4,3’,4’-tetradehydro-1,2,1’,2’-tetrahydro-+,+-carotene- 1,2,1’,2’-tetraol) (33) in Cyanobacteria to be identi- fied.48 Thus in oscillaxanthin and myxoxanthophyll from Arthrospira species the sugar present was a-linked chinovose tentatively assigned the L configuration ;this was also the sugar bound to myxol in Oscillatoria agardhii whereas in 0.bornetii J tenuis the sugar bound to myxol was 3-0-methylfucose. The fatty acid composition of astaxanthin bis-acyl ester isolated from flowers of Adonis aestivalis has been determined;49 the astaxanthin (3,3’-dihydroxy-P,P-carotene-4,4’-dione) (34) con-sisted of the (3S,3’S)- and the (3R,3’S)-isomer in the ratio 92 :5.A new tetraterpenoid hydrocarbon constituting up to 8YOof the dry weight of the green alga Botryococcus braunii has been identified as the highly saturated lycopene derivative lyco- padiene (39 and is presumably derived by biochemical condensation of two molecules of phyt01.~~ I .2.2 New Natural Products Related to Carotenoids Many of the smaller natural products contain ring structures similar to those found in carotenoids and some of these substances may be formed by degradation of natural caro-tenoids in vivo. The custom that has been established in previous Reports will be followed and the carotenoid number- (45) YcH2C02Me HO ’CH2C02CH2 0p&02H (46) (47)n = 1,m= 4 (48)n= 2,171= 3 ing scheme will be used whenever such compounds are described or discussed.The occurrence of such degraded carotenoids in tobacco and other leaves and their possible formation from carotenoids are discussed in three reviews. 14-16 A new constituent of Greek tobacco has been identified as (5R,6S,9S,7E)-megastigm- 7-ene- 5,6,9- triol (36). 51 The related compound (2S,5R,6E,8E)-2,5-epoxy-megastigma-6,8-diene (37) which is a constituent of osmanthus absolute has been found to be of low optical The structures and absolute configurations of rehmaionosides A B and C which are three new ionone glucosides from dried root of Rehmannia glutinosa have been determined as (38) (39) and (40) respecti~ely.~~ The related monoterpene glucoside rehmapicroside (4 1) was also obtained.Further possible biosynthetic precursors of abscisic acid (42) have been identified namely the 1’,4’-trans-diol [3,6- diol in the carotenoid numbering scheme] (43) in and 3-hydroxy-y-ionylideneacetic acid (45) in the fungus Cercospora ~ruenta,~’ and several metabolites of abscisic acid have been identified in leaves namely 18-hydroxy abscisic acid (44)58* and the conjugate metabolite the 17-[methy1-(3-hydroxy-3-methylglutaryl)] ester of 17-hydroxyabscisic acid (46).60 Two interesting new menaquinone derivatives (47) and (48) (see Section 2.2) have the terminal part of their isoprenoid side- chain in the form of an tz-ring.61v62 1.3 Synthesis and Reactions 1.3.1 Carotenoids A review on the synthesis of polyenes includes a discussion of the application of furan-opening methods for preparing dienic NATURAL PRODUCT REPORTS 19894.BRITTON a b k k e (49) R’ =R2 =a (50) R’ = R2 =b (51) R’ = R2 =c (52) R’ =R2 =d (57) (58) 0 (61) and trienic intermediates that are useful for synthetic work in the field of carotenoids and re ti no id^.^^ Synthetic methods in which sulphone intermediates are used and which may be useful for the synthesis of polyene systems in carotenoids have been described in several The synthesis has been reported69 of 4,5 :4,5’-diepoxy-4,5,4’,5’-tetrahydro-e,ecarotene(49) in different stereoisomeric forms from a-ionone (57) via the intermediate compound (58).The steric course of hydrolysis of (49)to give cis-or trans-4,5-diols (50) depending on conditions was also described. Optically active lycopene diepoxide (1,2 :1’,2’-diepoxy- 1,2,1 ’,2’- tetrahydro-$,$-carotene) (51) has been synthesized from epoxygeraniol and the products have been separated into the (all-@- the (5Z)- and the (7Z)-i~omer.~~ The related 1,2,1’,2’- tetraols (52; all-E) (52; 723 and (52; 72,7’2) were also prepared. Optically active (9-geranylgeraniol epoxide (59) prepared via a sulphone coupling reaction between the bromide C d f (53) R’ =e R~ =f (54) R’ = R2 =CHO (55) R’ =R2 =g (56) R’ =g R2 =e R (63)R’ =Me R2 =CHzOAc (64) R’ =R2 =CH20CH20Me (60) and the sulphone (61) was used to synthesize (5‘)-phytoene epoxide [(29-1,2-epoxy-1,2,7,8,11,12,7’,8’,11’,12’-decahydro-$,$-carotene] (62) by a Wittig reaction.71 In the series of C, and C, carotenoids a synthesis has been reported7 for (5‘)-trisanhydrobacterioruberin [(29-2,2’-bis-(3-methylbut-2-enyl)-3,4,3’,4’-tetradehydro-1,2-dihydro-$,$-caroten-1-01] (53) which was prepared in ten steps from MeC(O)CH,CO,Et Me,C=CHCH,Br MeC(O)CH=PPh, and crocetindial (54).(-)-P-Pinene was the starting point for the synthesis of the optically active end-group precursors (63) and (64) that were used to prepare the naturally occurring stereoisomers of C.p. 450 [(2R,2’R)-2,2’-bis-(4-hydroxy-3-methylbut-2-enyl)-P,P- carotene] (55) and C.p.473 [(2R,2’5‘)-3’,4’-didehydro-1’,2’-dihydro-2-(4-hydroxy-3-methylbut-2-enyl)-~,~-caroten-1’-011 74 (56).73* Carotenoid sulphates have been prepared from fourteen carotenols by reaction with a pyridine-SO complex.75 Of the products the mono- and bis-sulphates of zeaxanthin NATURAL PRODUCT REPORTS 1989 R2 R’ HO HOJYCA xp a b C d e “ O L Mead f 9 h OH . I HOw NC OH i k I m n (65) R’ = R2 =a (70)R’ = R2 = e (75)R’ = R’ =j (66) R’ = R2 = b (71) R’ =g R2 =h (76) R’ = k R2 =I (67) R’ = c R2 = d (72) R’ = R2 =g (77) R’ = R~ =m (68) R’ = R2 =c (73)R’ = f R2 =g (78) R’ =m R2 =n (69) R’ =e R2 = f (74) R’ = R~ = i (79) R’ = R2 = k (25) alloxanthin (7,8,7’,8’-tetradehydro-/?,/?-carotene-3,3’-diol) (65) capsorubin (3,3’-dihydroxy-~,~-carotene-6,6’-dione) (66) and astaxanthin (34) were stable in methanolic solution whereas the sulphates of carotenoids that contain vicinal diol phenolic or tertiary hydroxyl groups e.g.caloxanthin (p,/?-carotene-2,3,3’-triol (67) nostoxanthin (p,/?-carotene-2,3,2’,3’-tetraol) (68) 3-hydroxyisorenieratene (+,#-caroten-3-01) (69) 3,3’-dihydroxyisorenieratene(#,q5-carotene-3,3’-diol) (70) rho- dovibrin (l’-methoxy-3’,4-didehydro-1,2,1’,2’-tetrahydro-$,$- caroten- 1-01) (7 I) dihydroxylycopene (1,2,1’,2’-tetrahydro- $,$-carotene- 1 1 ’-diol) (72) and hydroxychlorobactene (1 ’,2’-dihydro-@,$-caroten-1’-01) (73) were less stable. Tertiary 5- sulphates obtained from azafrin (5,6-dihydroxy-5,6-dihydro-10’-apo-/?-caroten- 10’-oic acid) (80) and its methyl ester but secondary allylic sulphates of lutein (26) lactucaxanthin (27) isozeaxanthin (P,/?-carotene-4,4’-diol)(74) and crustaxanthin (P,/?-carotene-3,4,3’,4‘-tetraol) (75) and the primary allylic sulphate of 2’-apo-/?-caroten-2’-01 (76) were too unstable in solution for practical application.76 The carotenoid derivatives (77) and (78) have been prepared from crocetindial (54) for investigation as possible molecular-electronic device^.^' Mitsu-nobu reaction with diethyl azodicarboxylate was used to transform (3R,3’R)-zeaxanthin into the (3R,3’S)-and the (3 S,3’S)-fom. (3 R,3’R,6’R)-Lutein gave the (3 S,3’S,6’R)-iso-mer which had not been prepared before by a similar reaction.78 Photochemical reactions of /?-carotene (/?,/?-caro- tene) (79) in CC1 solution sensitized by chlorophyll or by other porphyrins have been st~died.~’ The primary reaction involved electron transfer from triplet-state carotene to CCl and the formation of radicals.The application of micro- heterogeneous photo-oxidation to various compounds in-cluding fl-carotene has been described.s0 In a study of the thermal degradation of /?-carotene at temperatures up to 800 “C many volatile products were detected by gas chromato- graphy-mass spectrometry including ten compounds related to /?-ionone (81).81 I .3.2 Retinoids Methods for the preparation of (all-E)- and (13Z)-retinal (82) and of retinal analogues have been reviewed.82 In a new synthesis of chain extension was achieved by lithiation of (2)-BrCH=CHOSiMe with Me,CLi and then successive reaction with /?-ionone (8 l) with p-ionylideneacetaldehyde (85) and with the C, ketone (86); (all-E)- (92)- (132)- and (92,13Z)-retinals were obtained.The highly stereoselective preparation of (all-E)-and (13Z)-retinol (83) has been rep~rted,~**~~ in a procedure which utilizes a double elimination reaction from the intermediate sulphone (87). Treatment of the C, ketone (88) with C1CH2CN and NaOH gave the epoxide (89; cis and trans) which was converted into retinonitrile (84) as a mixture of the (all-a- (927 (132)- and (9Z,132)- isomers.86 Efficient synthesis of 11 -cis-retinoids has been achieved by means of a highly cis-selective Wittig reaction between an oxido-allylic phosphorane (90) and an aldehyde e.g.(85).87A useful and extremely efficient method has been reported for the stereospecific synthesis of the retinoid synthon ethyl trans-3-formylbut-2-enoate(OHCCMe=CHCO,Et) by direct two-stage oxidation of ethyl 3-methylb~t-2-enoate.~~ A wide variety of species of retinal labelled with 13C and/or ,H have been prepared. Thus [5-13C]- [6-I3C]- [7-13C]- and [18-13C]-retinals were made with high chemical purity and isotopic enrichment by proceduress9 in which the I3C was introduced from simple molecules such as KI3CN [1-l3C]- and [2-13C]- NATURAL PRODUCT REPORTS 1989-G. BRITTON (80) (85) (86) (88) (92) R' = CF3 R2 = R3 = Me (95) (93) R' = R3 = Me R2 = CF3 (94) R' = R2 = Me R3 = CF3 A' (98) R' = CI,R2 = H (99) R' = Br R2 = H (100) R' = I R2 = H (101) R' = H R2 = F (102) R' = H,R2 = CI (103) R' = H R2 = Me (104) R' = H R2 = Et acetonitrile and 13CH31.Various routes were used to synthesize the deuteriated species [20,20,20-2H,]- [12,20,20,20-2H4]- [12,14,20,20,20-2H5]- [l 1,14-2H2]- [12,14-2H2]- and [20-13C 12 14-2H2]-retinals,90 and the preparation of [2,4,4,16,16,16,17 17,17,18,18-2Hl,]retinal has also been reported.g1 (all-E)-[lO,l 1- 14C2]Retinoic acid was prepared by the reaction between p-ionone and BrMgl4CZ4CH to give the C! product (91) and then Wittig chain-exten~ion.~~ Several fluorinated retinals have been synthesized. (all-E')- 18,18,18-Trifluororetinal(92) was obtained from trifluoro-,8-cyclocitral (95) by Wittig-Horner reaction.93 The (9cis)- and (1 1cis)-isomers of 18,18,18-trifluoro- retinal (92) 19,19,19-trifluororetinal(93) and 20,20,20-tri- fluororetinal(94) have been prepared by stereoselective (81) (83)R = CH20H SO2Ph (84) R = CN (87) Thp = tetrahydropyran-2-yl CN Ph36&CH20H Br-CH20H 3Ho (108) R = CH2Me (109) R = CMe3 (107) (110) R = SiMe3 The vicinal 13,14-difluoro-analogue of retinal (96) was ob- tainedg5 from the key intermediate (97) which was prepared by the reaction of Et,NSF3 with MeC(O)C=CCO,Et.Many other substituted retinals and retinal analogues have been synthesized and used as probes of the chromophore- binding site in the visual pigment rhodopsin and the bacterial photoreceptor bacteriorhodopsin.Compounds that have been prepared include 4-chloro- 4-bromo- and 4-iodo-retinal [(98)-(100)],96 12-butyl- 13-demethylretinal (1 05),96 naphthyl- retinal~,~~ 10-fluoro- 1 0-chloro- 10-methyl- and 1O-ethyl-retinal [(10 l)+ 104)],97 the acetylenic 9,l O-didehydro- 19-nor- retinal (106) and 9,10,11,12-tetradehydro-19-nor-retinal (107),98 9-ethyl- 9-t-butyl- 9-trimethylsilyl- 13-ethyl- and 13- NATURAL PRODUCT REPORTS 1989 (111) R' (112) R' (113) R' (114) R' (115) R' (116) R' = Et R2 = H = CMe3 R2 = H = R2 = H = [CH2]2Me R2 = H = H R2 = Me = R2 = Me VHO WC (122) R = H (123) R = Me CHO (126) t-butyl-retinal [(108)-( 1 12)],99 and the C- 13- and C- 14- modified compounds (1 13)-( 1 16).loo Other analogues have been prepared that have cis-configurations or s-cis-conforma- tions locked in the polyene chain e.g.the compounds (1 17)-(1 28).lo1-lo5 The arylpolyenes (129) and (1 30) which have the aldehyde group conjugated with the polyene chain through the benzene ring have been synthesizedlo6 and the spin-labelled analogue (1 3 1) has been prepared.lo7 The butenol- ide (132) was obtained from the C, aldehyde (133)lo8 and the ethoxycarbonyl compounds (1 34) and (1 35) have been pre- paredlog and converted into their dimedone derivatives (1 36) and (137). Methods for the preparation of 9-and 13-azidoretinals (138) and (139),llo and of a range of hetero- arotinoids (140)111 for possible use as pharmaceuticals have been described.Bromination of the hydrocarbons (141) and (143) by Br under heterolytic conditions proceeded regio- selectively by addition only to the triple bond to give the dibromides (142) and (144) as a mixture of stereoisomers.'12 Oxybromination of the tertiary alcohols (145) and (146) with N-bromosuccinimide gave the products (147) and (148) which with K,CO, underwent dehydrobromination to the diol (149) and the epoxy-alcohol (1 50) re~pectively."~ (all-E)-Retinyl P-D-ghcuronide (1 5 1) has been prepared by the reaction of retinol with methyl 2,3,4-tri-O-acetyl-l-bromo-1-deoxy-P-D-glucopyranuronate,followed by hydrolysis. '14 The preparation of retinyl palmitate by ester exchange between CHO (120) R = H (121) R = Me HO retinyl acetate and palmitic acid through the action of polyethylene-glycol-modified lipase has been described."j The coenzyme A thioester of retinoic acid has been prepared116 via a succinimidyl ester or an anhydride.The non-enzymic isomerization of (all-0- to (1 3Z)-retinoic acid (and vice versa) catalysed by thiols such as glutathione and mercaptoethanol has been reported."' The interaction of retinal with indoles in acidic media has been studied,l'* and the rapid decomposition of retinal and retinol by NO in CC1 has been re~0rted.l'~ I.3.3 Carotenoid-like Substances Many methods have been described for the synthesis of natural products which are structurally related to carotenoids and carotenoid end-groups. Some of these methods may be useful for the preparation of end-group intermediates as building blocks in the synthesis of carotenoids.Methods for the synthesis of ionones irones and their +-ionone precursors have been reviewed.120 The preparation of trans-methyl-+-ionone (1 52) and of trans-isomethyl-+-ionone (1 53) by aldol con-densation of citral with butan-2-one has been described.121 Under different acid conditions these products could be cyclized to methyl-a-ionone (1 54) isomethyl-a-ionone (1 55) methyl-P-ionone (1 56) or isomethyl-P-ionone (1 57).12 The synthesis of y-ionone (1 58) and dihydro-y-ionone (1 59) from a-cyclogeranic acid (1 60) has been described.lZ3 (f)cis-y-Irone NATURAL PRODUCT REPORTS 1989-G. BRITTON (129) q (132) 0 (138) R' = Ns,R2 = Me (1371 (139) R' = Me R2 = N3 1OsR (140) R = H Et or 2-phthalirnidoethyl (141) R = CZCH (143) R = C=CH (142) R = CBr=CHBr (144) R = CBr=CHBr (145) (146) (152) R' = H,R2 = Et (153) R' = R2 = Me (154) R' = H R2 = Et (155) R' = R2 = Me (156) R' = ti R2 = Et (158) (157) R' = R2 = Me NATURAL PRODUCT REPORTS I989 6CH20H (165) (1 6 1) was obtained stereoselectively from the (trimethylcyclo- hexeny1)methanol (1 62) via a P-alkoxyacrylate-olefin cycliza- tion of the ether (163) to (164) as the key Another synthesis of (+)-cis-y-ironelZ5 utilized 3,3- or 2,3-sigmatropic rearrangement of the hydroxymethyl compound (165) as the key step.Both cis- and trans-y-irones and y-ionone have been prepared126 via a 1,4-addition reaction and a chemo- and regio- selective olefination.Several methods have been reported for the synthesis of damascones and damascenones. Thus a-damascone (166) P-damascone (167) and P-damascenone (168) have been prepared from the carboxylic esters and carboxamides by use of mixed organolithium-magnesium reagent^,'^' and by P-cleavage of bis(homoally1ic) potassium alkoxides.128 a-Damascone has been synthesized by addition of allylmagnesium chloride to the ketene (169) as the key step,’,’ and y-damascone (170) by the reaction of methyl dithio- cyclogeranoate (171) with H,C=CHCH,MgBr via the sulphide (1 72). 130 a-Damascone and y-damascone have also been made by methods involving organoboranes e.g. (173),131 and a four-step preparation of P-damascone from 2,2,6-trimethylcyclo- hexanone (174) has been described.13 In a series of papers133-135 dealing with methods for the prenylation of isopentenol and geraniol conditions have been described135 under which acid- catalysed reaction of geranyl acetate (175) with Me,C(OH)CH=CH gave rise to cis- and trans-isomers of 2-prenyl-a- -P- and -7-cyclogeraniols [(176)-( 178)J.The trisporic acids are fungal hormones which induce and stimulate sporogenesis and the biosynthesis of carotene in some moulds notably BZakesZea trispora. New methods for the synthesis of trisporic acids and their derivatives are described in several papers. Trisporic acids A and B [(179) and (lSl)] their Lx&OH 0 (179) R = COzH X = H2 (180) R = CHzOH X = H2 0 (181) R = COzH X = 0 (1 83) (182) R = CH20H.X = 0 methyl esters and the corresponding trisporols A and B [(180) and (182)] have been prepared by a route involving Wittig reaction of the lactol (183).136 (+)-Trisporol B has also been synthesized by a highly stereoselective route which utilizes palladium-catalysed cross-coupling between the (9-borane (1 84) and (Z)-6-iodohept-5-en-2-one ethylene aceta113’ and also via ( & )-6- hydroxymethyl-2,6-dime thylcyclohex- 2-en- 1-one (185).138An enantioselective synthesis of methyl ( +)-trisporate B from the (+)-enedione (186) has been described.139 Several papers describe methods for the synthesis of the plant growth regulator abscisic acid (42) and related compounds. A four-step procedure from a-ionone proceeded via the acetylenic intermediates (187) and (188).140 An alternative route involved addition of the lithium salt of the acetylenic ester HCrCC(Me)=CHCO,Me to the protected ketone (189) which was prepared from isophorone.141 Isomers of the deoxy- abscisic acid (190) have been synthesized by elimination reaction NATURAL PRODUCT REPORTS 1989-G. BRITTON aoH pCHMeCHMe2)2 o& ECR o& LO (185) (187) R = H 0U0 ( 186) (188) R = C02Et (189) (184) 02Me (194) (196) 0 (200) from intermediate toluene-p-sulphonates. lg2 ( f)-2-Hydroxy-methyl-2,6-dimethylcyclohexanone (1 91) was the starting material for a synthesis of methyl (+)-phaseate (192) via the intermediate ketone (193) and ester (I 94).143 The abscisic acid analogue (195) with a geometrically rigid conjugated acid side- chain has been prepared.lg4 The reaction of the C, aldehyde (196) with rn-chloro- peroxybenzoic acid provided a highly efficient one-step syn- thesis of ( f)-aeginetolide (197) and (-t )-dihydroactinidiolide CO;! H (195) (202) (198).lg5A concise asymmetric synthesis of (SS,6S)-aeginetolide and (SS)-actinidiolide (199) has been described,146 and both enantiomers of dihydroactinidiolide were obtained in fifteen steps from (S)-3-hydroxy-2,2-dimethylcyclohexanone. Other 14' naturally occurring substances that contain structures related to carotenoid end-groups have been synthesized including manoalide (2OO),148-150 secomanoalide (201),148 obtusadienol (202),151 (-t)-pisiferin (203),152(+)-ambliol A (204),153(+)-ancistrofuran (205),154 and the marine natural dione (206).155 NATURAL PRODUCT REPORTS I989 (207) (208) (209) (210) R [R = CH2S02Ph C02Me,CH=CHC(O)Me or C(O)CH=CHMe] (211) R = H or Me (212) R = H or Me (213) R = H or Me (214) OH (218) AcOw 0 (2211 (224) (225) R = H Me or C02Me (226)R = CN (227) R = C02Et A general method for the conversion of methyl ketones into terminal alkynes was used to prepare the dienyne (207) from P-i0n0ne.l~~ Electrogenerated acid-catalysed cyclization of the acyclic isoprenoids (208) in 1,2-dichloroethane gave the isomeric monocyclic products (209) and (2IO).l5’ The acyclic (@-and (a-dienethiols (2 I 1) underwent acid-catalysed cycli- zation to give the trans-fused and cis-fused thiahydrindans (212) and (213) re~pective1y.l~~ A method has been described for preparing the propylthio-compound (2 14).159 Heating in aqueous acid produced a number of volatile C, nor-isoprenoid compounds from the megastigmenetriol (21 5) and the mega- stigmadienetriol (2 1 6).160 Direct anodic oxidation of p-ionone gave products such as (217)-(221) depending on the con- ditions that were used.lG1 a-Ionone was oxidized with difficulty to give the acetate (222).A method has been described162 for the regio- and stereo-selective reduction of the C=C double-bond of ap-unsaturated carbonyl compounds including a-and p-ionones with NaTeH. Various conditions have been evalu- ated for the continuous hydrogenation of /3-ionone on fixed supported palladium catalysts.163 The photochemical reactions of several ionone derivatives have been studied including the 9- demethylionylacetones (223) and (224),164 5,6-epoxy-5,6-di- hydro-P-ionone (219),165 and 5,6-epimino-5,6-dihydro-p-ionone (225; R=H) and its derivatives.166 Photolysis of P-ionone (81) and the p-ionyl and P-ionylidene derivatives (85) (226) and (227) in /3-cyclodextrin gave fairly stable water- soluble complexes and hence various products. 167 1.4 Carotenoid-Protein Complexes Further work on the blue carotenoprotein from the carapace of the crayfish Procambarus clarkii has been described in a series of papers. The natural complex is a 250 kDa protein of the a-crustacyanin type with h,, 635 nm and it can be dissociated into a 42 kDa ,&form which has A,, 585 nm and is composed of two different subunits.The carotenoid present is astaxanthin (34),168 as a mixture of the (3R,3’R)- (3R,3’9- and (3S,3’S)- isomers in the ratio 20:21:58. Studies on the stability and denaturation of the complex have been reported16’ and reconstitution has been achieved between astaxanthin and the isolated and separated apoprotein subunits in various combina- tion~.~~~ The individual subunits would accept astaxanthin to give rose-purple products of apoprotein size and with hmax545 nm whereas combinations of the two types of subunit gave blue products that were similar but not identical to the natural carotenoprotein.A high degree of homology was demonstrated between this carotenoprotein from P.clarkii and a-crustacyanin from the lobster Homarus arnericanus and reconstitution to give a hybrid P-crustacyanin complex was achieved with astaxanthin and mixtures of apoprotein subunits from the two animals.171A similar blue astaxanthin protein has been isolated NATURAL PRODUCT REPORTS 1989-G.BRITTON 37 1 R2 R’ a b C d e f 9 (228)R’ = R2 =a (229)R’ = 6,R2 = c (230)R’ = R2 = b (231)R’ = R2 =d from the carapace of another crayfish Astacus leptoda~tylus.’~~ This 440 kDa protein had A,, 634nm and contained the (3R,3’R)- (3R,3’S)- and (3S,3’S)-isomers of astaxanthin in the ratio 21 :41 38. Its stability appeared to be greater than that of most blue carotenoproteins.Other workers have reported h (232)R’ =d R2 =c (233)R’ = e R2 = f (234)R’ = R2 =g (235)R’ = R2 = h and serum182 of chum salmon (Oncorhynchus keta) have been characterized. A carotenoid-binding protein has been identified in the cytoplasmic membrane from a strain of the cyanobacterium Synechocy~tis,’~~ and a spectral shoulder at 560 nm has been the isolation of the blue Astacus protein in a-and y-form~’~~ attributed to a and have shown that the carotenoproteins in soft integument and hardened shell are immunochemically identical. 174 The carapace of A. leptodactylus also contains a yellow caroteno- protein (A,, 385 nm) which has twelve carotenoid molecules (astaxanthin :zeaxanthin is 1 :1) per molecule of protein (560 kDa).175Four different polypeptide subunits were resolved.Carotenoproteins that contain astaxanthin and canthaxanthin (P,P-carotene-4,4’-dione) (228) respectively have been ob- tained from the aquatic acari (Arachnoidea) Eylais hamata and Hydryphantes di~par.l’~ An ovoverdin-type carotenopro- tein has been identified in eggs of species of cladoceran~.’~~ In vivo this protein had a single absorption band (A,, approx. 650 nm). A detailed resonance-Raman-spectroscopic study of the starfish carotenoprotein asteriarubin (from Asterias rubens) reconstituted with astaxanthin or its acetylenic analogues 7,8- didehydroastaxanthin (3,3’-dihydroxy-7,8-didehydro-P,P-caro-tene-4,4’-dione) (229) 7,8,7’,8’-tetradehydroastaxanthin(3,3’-di hydroxy-7,8 ,7’ 8’- tetrade hydro-P,P-carotene-4,4’-dione) (230) and 15,15’-didehydroastaxanthin(3,3’-dihydroxy- 15,15’- lipopeptidocarotenoid complex in cells of Rhodococcus mari~.’~~ 1.5 Physical Methods 1.5.1 Separation and Assay In addition to the now extensive routine use of high-performance liquid chromatography (h.p.1.c.) for separating and purifying carotenoids some new developments have been reported.The difficult direct separation of mixtures of geometrical isomers of astaxanthin (34) astacene (3,3’-di-hydroxy-2,3,2’,3’-tetradehydro-P,P-carotene-4,4’-dione) (231) and semiastacene (3,3’-dihydroxy-2,3-didehydro-P,P-carotene-4,4’-dione) (232) in the presence of other ketocarotenoids has been achievedla5 with good resolution and much improved peak shapes on a column of silica that was coated with H3P0,.Optimal conditions for resolution have been determined for non-aqueous reversed-phase and normal-phase h.p.1.c. of geometrical isomers of canthaxanthin (228).le6 A reversed- phase procedure in which a Vydac TP201 column was used has proved useful for separating geometrical isomers of a-carotene didehydro-P,P-carotene-4,4’-dione) (236) has been re~0rted.I~~ (p,e-carotene) (233) 8-carotene (79) lycopene (@,+-carotene) The results revealed that delocalization of n-electrons is greater in the central part of the polyene chain that the methyl substituents at C-9 and C- 13 are in different environments and experience different interactions with the protein and that the methyl substituents at C-13 and C-13’ play an important part in the binding and the spectral shift.Resonance Raman spectra have been recorded of astaxanthin proteins in situ in corals.179 Further characteristics of the violet carotenoprotein from the haemolymph of the fly Rhynchosciara americana including its purification have been described.180 Carotenoid-carrying lipo- proteins containing mainly astaxanthin from the egg yolk18’ (234) and lutein (26). 187* 188 Geometrical isomers of a-carotene and 8-carotene have also been resolved by h.p.1.c. on a slurry- packed column of Ca(OH),.lS9 A comparison has been made of ten different columns for isocratic non-aqueous reversed-phase h.p.1.c. of leaf xanth~phylls,~~~ and the addition of n-decanol as a modifier to the mobile phase was shown greatly to improve both the chromatography and the stability of the column.lgl A non-aqueous reversed-phase procedure has been developed for the separation of both free carotenoids and their acyl Esters with fatty acids of different chain lengths could be separated and tentatively identified.Baseline sep- aration has been achieved of normal (protio-) from deuterio- (240) R = CH20H (241) R = CH=NOH carotenoids the latter being eluted lg4The preparation and evaluation of the C, analogue (237) of /3-carotene as an internal standard for quantitative analysis of carotenoids in vegetables have been described.lg5* lg6Further impressive direct resolutions of optical isomers of carotenoids and their simple derivatives have been reported in several papers.The mixtures that were resolved have already been discussed in Section 1.2.141-47 Most of the papers that were listed in earlier sections of this Report which describe the isolation of new natural carotenoids or new syntheses of carotenoids include h.p.1.c. procedures for purification. In addition there are a large number of publications in which h.p.1.c. procedures that have been developed for the routine qualitative and quantitative analysis of carotenoids and other substances in plant tissues and food~tuffs,~~~-~~~ and in in blood serum or pla~ma,~ll-~~~ human milk226 are described. Reversed-phase thin-layer chromatography (t.1.c.) is reported to have advantages over normal-phase t.1.c. for separating carotenoid~,~~~ and the separation of carotenes by t.1.c.on Ca(OH) has been described.228 A new volume of ‘Methods in Enzymology ’ includes several articles describing h.p.1.c. methods for the analysis of retinal and 3,4-didehydroretinal (238),229 retinol and retinyl retinol and retinoic acid (239),231 and retinyl phosphate manno~e.~~, A book on h.p.1.c. of small molecules contains a chapter on the analysis of vitamins including vitamin A (retinol) and re ti no id^.^^^ High-performance-liquid-chromato-graphic procedures have been described for the separation of a multi-component mixture containing fourteen geometrical isomers of of geometrical isomers of retinal that had been liberated from visual pigments,235 and of various geometrical isomers of 3-hydroxyretinal (240) and 3-hydroxy- retinal oxime (241),236 Both (all-E)-retinal and (I 12)-retinal have been purified in milligram amounts from complex mixtures of retinal isomers by centrifugal partition chromatography on cartridge^.^^' The relative merits of reversed-phase columns that are or are not fully end-capped have been assessed for the analysis of vitamin A and its derivatives.238 The preparation and use of anhydroretinal (242) as an internal standard for estimation of vitamin A by h.p.1.c.have been described ;239 NATURAL PRODUCT REPORTS 1989 (243) R’ = H or Me R2 = Me or CMe3 X = C104 or BF4 tritiated retinoids have also been used for internal standard- i~ation.~*O Some retinoid radicals have been detected by h.p.1.c. of their spin-trapped ad duct^.^^^ Retinyl esters with fatty acids of different chain length have been separated by non-aqueous reversed-phase h.p.l.~.~,~ Many papers report procedures for the detection and determination of retinol and other retinoids from various natural sources especially blood serum and p1asma,243-262 by h.p.1.c.A direct fluorometric assay for retinal in serum compared favourably with h.p.l.~.~~~ Problems with official methods and new techniques for analysis of vitamin A in food and feed have been The use of for- maldehyde in solvent mixtures has been recommended to permit the efficient extraction of the retinal chromophore in its original isomeric composition from visual pigments. 265 Methods for the separation purification and quantitative analysis of abscisic acid (42) and its derivatives by h.p.l.c.g.l.c. g.c.-m.s. and immunochemical methods266 -279 have been reported. The (R)and (S) enantiomers of methyl abscisate have been resolved by h.p.1.c. on a chiral column.280 Partial isotopic fractionation has been noted and the need for caution emphasized when deuterium-labelled internal standards are used in the analysis of plant hormones including abscisic acid,281 by h.p.1.c. 1.5.2 N.M.R. and E.S.R. Spectroscopy A two-dimensional lH-13C n.m.r. chemical-shift correlation has been used to assign the olefinic carbons of /3-carotene in the presence of degeneracy of proton chemical shifts.282 Two- dimensional ‘H n.m.r. studies of (7Z,9’Z)-isorenieratene ($,#-carotene) (235) have been reported and the spectra have been assigned.283 The interaction of lutein with phosphatidylcholine bilayers has been studied by n.m.r.and by other physical methods.284 Solution and solid-state 13C n.m.r. spectra of the retinal iminium salts (243) have been determined ;2e5 they provide evidence for a 6-s-trans conformation. Two-photon spec-troscopy and 13C and two-dimensional lH n.m.r. spectroscopic studies of retinyl Schiff bases protonated Schiff bases and Schiff-base salts have given evidence for a protonation-induced NATURAL PRODUCT REPORTS 1989-G. BRITTON (244) ordering reversal of nm* excited state levels.286 The n.m.r. spectra (13C and 15N) of equimolar mixtures of all-trans-retinylidene-t-butylamine (244) with substituted acetic acids in C2HC13 and C2H,02H have been studied in relation to the nature of the state of protonation of retinal Schiff bases in the presence of acids of different pK values and in solvents of different and the results have been discussed in relation to the environment of the chromophore in rhodopsin and bacteriorhodopsin.Extensive low-temperature solid-state 13C n.m.r. studies of samples of the visual pigment rhodopsin and of bacterio- rhodopsin that had been prepared from retinals that were enriched with 13C at various positions have been per-formed.2s9-294 This work has provided a great deal of information about the stereochemistry of the chromophore and about chromophore-protein interactions in these photo- receptors (see Section 1.5.9).Steric factors relevant to the conformation of p-ionone and structurally related compounds have been studied by measure- ments of dynamic 13C n.m.r. and of ‘H-lH nuclear Overhauser effects.295 Electron spin resonance studies of carotenoids that were incorporated into reaction centres of Rhodobacter (formerly Rhodopseudomonas) sphaeroides have been described296 and triplet-triplet energy transfer in light-harvesting complexes of photosynthetic bacteria has been investigated by e.~.r.,~~ 1 S.3 Mass Spectrometry (246! 1 S.5 Raman and Infrared Spectroscopy Excitation spectra for narrow Raman lines and for a broad luminescence band have been measured for p-carotene in isopentane at 177 K over a wide spectral range309b310 and compared with the results from a theoretical treatment.31’ Resonance Raman profiles of the 1005 1155 and 1525 cm-’ modes of p-carotene in CS have been determined at room temperature and at 177 K.312 Observed and calculated profile lineshapes were in good agreement.The absolute differential cross-sections of these lines in p-carotene have been measured by resonance Raman The use of near-infrared laser light for Raman measurements (to solve the problem of fluorescence) has been applied to p-carotene in CS,.314 Resonance Raman absorption and luminescence spectra of an aqueous suspension suggest that the long-wavelength-absorb- ing (540 nm) form of p-carotene is a fine crystalline form in which there is head-to-tail aggregation of the Resonance Raman spectra have been obtained from solid amorphous and crystalline ,&carotene ;,16 specific bands were identified for the crystalline sample.Saturation and non-linear electromagnetic field effects in the picosecond resonance Raman spectra of p-carotene have been studied.,” Raman and e.s.r. spectra of alkali-metal-doped p-carotene have been meas-red.^'^. 319 A Raman-spectroscopic study showed that the conformation of /?-carotene in phosphatidylcholine liposomes depends on membrane potential.320 The solvent-induced broad- ening mechanisms have been considered in calculations of the resonance Raman excitation profiles for the v1and v vibrational modes of lycopene. 321 A review on mass spectrometry of tobacco i~oprenoids~~~ includes sections dealing with degradation products of cyclic carotenoids.Mass spectrometry (electron-impact and desorp- tion chemical ionization with NH and CH as the reactant gases) has been applied to p-carotene and some of its oxidative degradation Desorption-chemical-ionization mass spectrometry has proved to be a useful aid in the structural characterization of underivatized conjugated metabolites of abscisic acid.300 I S.4 Circular Dichroism and Linear Dichroism In the pigment-protein complex of photosystem I of barley p-carotene shows strong circular dichroism indicating that it is held in a particular c~nformation.~~~ ( -)-1-Phenylethyl retino- ate shows a Cotton effect with c.d. bands at 360 245 and 2 15 nm whereas ( +)-octan-2-yl retinoate does This result has been explained in terms of the dis-symmetric environment of the polyene chain or dipole4ipole interaction between it and the phenyl group.Circular dichroism and ultraviolet spectra of eight chiral P-cyclocitral Schiff bases e.g. (245) have been determined and calculations have been made of twist conformation^.^^^ A paper304 in which the c.d. of linearly conjugated chromophores is described includes dis- cussion on configurational and conformational effects relevant to considerations of the c.d. of carotenoids and related polyenes. Circular dichroism studies of intermediates in the bacteriorhodopsin photo~ycle,~~~ of bacteriorhodopsin-lipid interactions,306 and of rhodopsin analogues that possess conformationally fixed retinylidene chr~mophores~~~ have been reported.A picosecond linear dichroism study of (1 12)- and (all-E)-retinols incorporated into polyethylene films that were ordered by stretching gave information about the molecular geometry in the excited singlet and triplet Resonance Raman excitation profiles have been measured for the vl v, and v3 vibrations of l~tein~~ and ~iolaxanthin~,~ in acetone toluene and CS,. /?-Carotene has been used as a probe in studies of the structural rearrangements that are induced in human plasma lipoprotein carotenoids by malondialdehyde. 324 Several structural popu- lations of p-carotene were indicated and its reaction with malondialdehyde caused a complex change in the carotene Raman bands. A study of resonance Raman spectra of carotenoids in vivo and in vitro suggested that in some species of photosynthetic bacteria the major fraction of carotenoids associated with the light-harvesting system is distorted from the planar all-trans c~nformation.~~~ Resonance Raman spectra have been obtained for carotenoids in vivo in and in green Spectra obtained from pigment cells of crusta- ceans allowed the detection of both free and protein-complexed car~tenoids.~~~ Resonance Raman spectra for astaxanthin and its 7,8-didehydro- 15,15’-didehydro- and 7,8,7’,8’-tetra- dehydro-derivatives [(229) (236) and (230) respectively] alone and complexed with the protein of the starfish carotenoprotein asteriarubin have been recorded and assigned.178 The spectra of the free and complexed carotenoids were compared and possible protein-carotenoid interactions discussed.Greater delocal-ization of the m-electron system in the central part of the polyene chain in the complexes and an important role of the lateral methyl substituents in these interactions were discussed. A combination of resonance Raman spectroscopy and laser microanalysis has allowed carotenoids to be detected in a single living cell.329 A review has been published on vibrational analysis of isomers of retinal. 330 Detailed analyses and assignments have been made for the resonance Raman spectra of species of retinal that were labelled with 13C at positions 5 6 7 8 9 10 11 12 13 14 or 15 or with ,H at positions 7 8 10 11 12 14 NPR 6 NATURAL PRODUCT REPORTS 1989 or 15 or the NH group and of bacteriorhodopsin and its intermediates that had been prepared from these Raman spectra have been determined for geometrical isomers of homologues of retinal including the (all-E)- (9Z)- (1 la- and (13Z)-isomers of the C, aldehyde (246) and the (all-@- (72)- and (9Z)-isomers of the C, and C, aldehydes (247) and (85).332 Key bands ascribed to an unmethylated double-bond in the 2 configuration (72 or 113 were distinguished from those due to a methylated Z double-bond (92 or 132) and key bands were revealed for the (all-E) parts of the (mono-2)- isomers.A resonance-Raman- and electronic-absorption-spec-troscopic study of retinal and of the unprotonated and protonated retinylidenehexylamine Schiff base showed that there was strong enhancement of the scattered light when the Schiff base was protonated by strong proton donors e.g.HC1.333 An investigation of absolute resonance Raman inten- sities for retinal and 5,6-dihydroretinal (248) showed that the spectral broadening that is induced by the @-ring is homo- gene~us.,~~ Resonance Raman spectra of (all-E)-retinal Schiff bases have been measured in fluoro-alcohols in which Amax is considerably red-shifted.,, In the resonance Raman spectrum of retinylidene-n-butylamine the C=N stretching frequency was shown to increase upon complexation with general Lewis Factors affecting the C=N stretching in protonated retinal Schiff bases have been studied with a series of synthetic chrom~phores.~~~ Protonation of (all-E)-retinylidene-n-butyl-amine Schiff base with protected N-acetyl-L-tyrosine ethyl ester at various molar ratios has been studied by infrared methods.338 An attenuated-total-reflection infrared study of the protonation of a retinylidene Schiff base on crystal surfaces has been The vibronic structure of (all-E)-retinal in different environments has been discussed.340 A semi-empirical cal- culation has been made of force constants of stretching vibrations in excited states of retinal and its unprotonated and protonated Schiff base.341 Resonance Raman spectral bands for the retinal chromophore of rhodopsin isorhodopsin and bathorhodopsin and of bacteriorhodopsin have been assigned by the use of an extensive series of ,H-and 13C-labelled retinal~.,~,-,~~ This work has generated much information about the geometry of the chromophore in these photoreceptor pigments and about the primary photoreactions (see Section 1.5.9).Quantum-chemical calculations of vibrational spectra of isomers of protonated retinylidene Schiff bases from previously reported resonance Raman and infrared data were in agreement with the involvement of the (13Z)-isomer in the bacteriorho- dopsin cycle.346 In bacteriorhodopsin that contained a strongly electronegative CF group at C-5 resonance Raman spec- troscopy showed that the chromophore is protonated but cannot bring about proton pumping suggesting that the CF group hinders the approach of the ring to the negatively charged protein site near the ring.347 The mechanism of formation and hydrolysis of N-retinylidene-n-butylamineas a model of the rhodopsin chromophore was investigated by a resonance Raman study of the kinetic and equilibrium properties in detergent micelle Surface-enhanced Raman spectra have been used to gain information about the position of the retinal Schiff base relative to the surface of the outer segment of the photoreceptor disc of bovine visual rods.349-351 0t her resonance- Raman-spectroscopic studies have been reported for octopus rhodopsin and its photoprodu~ts,~~~ for an ultraviolet-sensitive insect rhodop~in,~~~ on bacteriorho- dopsin and intermediates in its phot~cycle,~~~-~~~ on the halorhodopsin photocycle of Halobacterium h~lobiurn,~~~ and on purple blue and pink membranes isolated from 370 A Halobacterium h~lobiurn.~~~ review of time-resolved resonance Raman spectroscopy of photobiological and photo- chemical transients includes considerable discussion on bac- teriorh~dopsin.~~~ Raman microscopy and studies of quantum yield of the primary photochemistry of visual pigments that contain 3,4-didehydroretinal have been The use of Fourier-transform infrared (F.T.i.r.) spectroscopy to investigate the bacteriorhodopsin photocycle has been reviewed,,, and detailed F.T.i.r.studies of bacteriorhodopsin and the intermediates in its photocycle have been reported in several A survey of difference F.T.i.r. studies on rhodopsin and bacteriorhodopsin that had been prepared from dihydroretinals has led to a modified version of the external- point-charge model for bacteriorhodopsin.381 1 S.6 Electronic Absorption and Other Spectroscopic Methods Extinction coefficients have been calculated for a large number of carotenoids from their triplet-triplet absorption spectra in condensed phases.382 By using an exciting laser pulse of wavelength 510 nm and of only 4 picosecond duration the lifetimes of the lowest excited singlet states of (all-E)-P-carotene [8.4 ps] canthaxanthin (228) [5.2 ps] and 8’-apo-P- caroten-8’-al (249) [25.4 ps] have been measured Solvent effects on the intensities of nn* and ~n* absorptions of /I-carotene have been studied and calculations In spite of their different chromophores methoxyneurosporene (1-methoxy-1,2,7’,8’-tetrahydro-$,$-carotene)(250) and meth- oxyspheroidene (l,I’-dimethoxy-3,4-didehydro-1,2,1’,2’,7’,8’-hexahydro-$,$-carotene) (251) had identical singlet and triplet absorption spectra when bound in the reaction centres of Rhodobacter sphaeroides indicating the existence of strong interactions with protein.385 A lifetime of the triplet state of 4.2 ,us has been determined for spheroidene (1 -methoxy-3,4-didehydro- 1,2,7’,8’- tetrahydro- carotene) (252) in Rhodo-bacter ~phaeroides.~~~ The ultraviolet and photoelectron spectra of violerythrin (2,2’-dinor-p,/I-carotene-3,4,3’,4’-tetraone) (253) and of the smaller model compounds (254) and (255) have been discussed in order to explain the blue colour and Amax (580 nm) of violerythrin.A light-scattering spectroscopic investigation of potential-dependent changes in conformation in p-carotene has been reported.388 Picosecond time-resolved absorption spectra of the (all-E)- (723 (929 (1 la- and (132)-isomers of the C, compound retinylideneacetaldehyde (246) have been measured and the mechanism of Z-E isomerization for this homologue has been compared with that for Time-resolved absorption spectra of (all-E)- and (9Z)-retinal in n-hexane have been measured by laser photoly~is.~~~ A drastic change in the T + T absorption spectrum of the (9Z)-isomer in the time region of picoseconds to nanoseconds reflected configurational relaxation in the triplet state.Solvent effects on the spectral absorption maxima and kinetics of radical ions of (all-@-retinal have been examined by spectrophotometric pulse radiolysis at room NATURAL PRODUCT REPORTS 1989-G.BRITTON Meoh MeoL a b (250)R’ =a R2 = b (251)R’ =c R2 =a (252)R’ = c R2 = b 0 temperature in solvents of different polarities.391 Time-resolved emission studies of protonated Schiff bases of (all-@- and (1 127)-retinals suggest that the excited state relaxes within 7 The electronic structures of low-lying excited states of retinal and related model polyenes and of their Schiff bases have been Cross-sections of two-photon absorption absorption polarization ratios and polarization degrees of a two-photon-excited luminescence have been determined for solutions of retinal and retinyl The S tS absorptions of retinyl acetate and retinal aldimine have been identified in the infrared and their spectral polarization characteristics have been measured in liquid and solid solu- tion~.~~~ In the pulse radiolysis of (all-@-retinal retinoic acid and methyl retinoate fast transient absorption processes were explained in terms of the association of the radical cations with the parent polyenes to form dimer~.~~~ The spectroscopic and kinetic behaviour of radical ions of several retinal homologues of different chain-length in organic solvents and in aqueous micelles has been* examined by pulse radiolysi~.~~~ An investi- gation of the spectroscopy and photoisomerization of a series of poly(ethy1ene glyco1)peptide Schiff bases of (1 lZ)-retinal has been reported.398 Hydrogen bonding and proton transfer in model Schiff bases that are related to the chromophore of the visual pigment have been examined spectroscopically.399 A review has been published on the photochemistry of intermediates of the rhodopsin cycle as studied by low-temperature spectrophotometry and ~pectro~copy.~~~ Studies on the intermediate bathorhodopsin were described in detail.Photochemical reactions of rhodopsin analogues that had been prepared from 10-fluororetinal (101) and 12-fluororetinal (256) have been investigated by low-temperature spectro-photometrygo1 and a flash-spectroscopic photoelectric study of bacteriorhodopsins that had been prepared from retinal or its 13-ethyl (1 1I) 13-methoxy (257) and 13-demethyl (258) analogues has been described.402 The photochemical cycle of bacteriorhodopsin has been investigated by low-temperature absorption ~pe~tr~~~~py,~~~ by picosecond and nanosecond C (254)n = 1 (255)n = 3 (253) (257)R = OMe (258)R = H (259) index.407 A review of applications of two-photon spectroscopy of protein-bound chromophores includes detailed consideration of rhodopsin and bacteriorhodopsin.40s 1.5.7 X-Ray Methods The crystal structures of (9Z)-retinal and (92)- 19,19,19- trifluororetinal (93) have been determined.409 In its crystal structure (all:E)-3,4-didehydroretinal(238) has been shown to be nearly isomorphous with (all-E)-retinal.410 A trans dis-position ofthe hydroxyl groups with that at c-3 beingequatorial was determined from the crystal structure of ‘grasshopper ketone’ (259).411 X-Ray absorption studies on bacteriorho-dopsin have been described412 and the crystallographic charac- terization of a photo-active yellow protein with photochemistry similar to that of rhodopsin has been I S.8.Miscellaneous Physical Chemistry An extensive review has been published which deals with the aggregation of carotenoids in aqueous organic solvents liposomes and micelles the optical properties of carotenoid monolayers and the participation of carotenoids in energy A calorimetric and spectroscopic study has been reported415 of the miscibility of p-carotene and zeaxanthin with dipalmitoylglycerophosphocholinein multilamellar vesicles. In dimyristoylglycerophosphocholinemodel membrane systems a,w-dihydroxy-carotenoidsexert a reinforcement effect on the phospholipid bilayer similar to that of chole~tero1.~~~ The organization of zeaxanthin astaxanthin and their C, homo-logues in dimyristoylglycerophosphocholine vesicles has been examined.417 Violaxanthin in ethanol was added to liposomes and the spectral shapes of the liposomes were correlated with pigment absorption processes on the surface of the phospholipid membrane.418 The sizes and shapes of lutein aggregates in aqueous acetone solution have been examined419 by electron microscopy and spectroscopic analysis including circular by femtosecond ~pe~tr~~~~py,~~~ ~pe~tr~~~~pie~,~~~ by flash dichroism.A similar revealed a left-handed helical ~pe~tr~~~~py,~~~ and by kinetic spectroscopy of its refractive conformation of lutein aggregates that were dispersed in 14-2 NATURAL PRODUCT REPORTS 1989 HO--OH liposomes of phosphatidylcholine from egg yolk or of digalac- tosyl diglyceride from spinach leaves.Irradiation increased the permeability of phosphatidylcholine liposomes that contained p-carotene or retin01.~~~ Zeaxanthin and violaxanthin both slowed the toluidine-blue-photosensitized destruction of phos- phatidylcholine in Both retinal and retinol have been shown to be incorporated into liposomes formed from dipalmitoylglycerophosphocholine.423 Effects of micellar solu- bilization on the excited-state properties of several retinyl- polyenes have been examined primarily by nanosecond laser The flash phot~lysis.~~~photophysical and photochemical behaviour of (llZ)-retinal and its Schiff base in Triton X-100 micelles has been studied by microsecond laser flash photolysis and by steady-state techniques.425 The interactions of zeaxan- thin and astaxanthin with electrolytes at the air-water interface have been studied and compression isotherms Similar studies have been reported for monomolecular films of the diethanolamides of 15’-benzoylamino- 14-apo-P-caroten- Halide-mediated peroxidase-catalysed oxidation of p-carotene has been studied as a model for peroxidation of lipids.443 Laser flash and steady-state excitation methods together with analysis of the products by h.p.l.c.have been used to investigate the photoisomerization of the protonated and unprotonated n-butylamine Schiff bases of (all-@- (9Z)- (112)- and (132)- In another study five products were identified after (all-E)-retinal had been irradiated at 334 nm.445 Photoisomer- ization of a naphthyl analogue (263) of (all-@-retinal mediated by the protein aporetinochrome stereospecifically produced the (1 12)-isomer which was not one of the four photochemical products that were obtained in the absence of the A thousand-fold enhancement of the rate of isomerization of (1 12)-retinal to the (all-E)-form in lipid dispersions by aromatic amines was attributed to acid-base catalysis.447 Retinal retinol and retinyl palmitate in solution were not isomerized by gold light though some isomerization occurred in white light.448 The properties of the ground states of various (Z)- and (@-isomers 14’-oic acid (260) and 7’-benzoylamino-6’-apo-~-caroten-6’-oicof retinal analogues and the effect of protonation of the retinal acid (26 1).427 Surface pressure-area isotherms of pure mono- Schiff bases have been studied at ab initio SCF and correlated layers of violaxanthin or neoxanthin [(3S,SR,6R,3’S,S’R,6’9- levels.449 The differential-pulse-polarographic behaviours of 5’,6’-epoxy-6,7-didehydro-5,6,5’,6’-tetrahydro-P,p-carotene-(all-E)-retinol retinal retinoic acid and retinyl acetate have 3,5,3’-trio11 (262) and of their mixed monolayers with arachidic acid have been A general thermodynamic treatment for protolytic equilibria in an insoluble monolayer has been tested on monolayers of carotenoids that were spread at the air-water interface.429 The photo-oxidation of water that contained NAD’ at the water-octane interface in the presence of p-carotene and chlorophyll a has been 431 Large reversible bathochromic spectral shifts have been observed with increasing surface pressure for mixed monolayers of chlorophyll and ~eaxanthin,~~~ but not with chlorophyll alone nor with chlorophyll in the presence of carotenoids that did not contain two hydroxyl groups.Photovoltaic properties of mixed mono- layers of chlorophyll a and canthaxanthin have been ex-amined.433 Synthetic covalently linked carotenoid-porphyrin dyads and carotenoid-porphyrin-quinone triads have been used to study charge separation and the rapid transfer of energy between carotenoids and porphyrins as model photosynthetic 435 Studies of conformations of fluorescence life- been A range of physicochemical properties of retinyl palmitate have been For the oxidation of retinyl acetate a mechanism has been proposed in which intramolecular and intermolecular chain propagation occur with migration of the reaction centre from one end of the polyene system to the other and Kinetic parameters have been determined for the autoxidation of retinal in the solid state at 25 “C and 20 kPa in the presence of agents such as 2-t-b~tyl-4-methoxyphenol.~~~ Thermal degradation of ret- inyl palmitate in hexane at 60 “C gave a product that was not identified.454 The mechanism of fluorescence quenching of indole by retinal and its Schiff bases has been studied by luminescence and pulse fluor~rnetry.~~~ The semiconductive properties of retinoic acid have been studied as a function of the adsorption of different vapo~rs~~~ and shown to differ from those of retinol and retinyl acetate.time~,~~~ I S.9 Photoreceptors the use of pulse radiolysis and cyclic v~ltamrnetry,~~~ and a theoretical conformational have been reported. A mechanism has been proposed for electron transfer through lipid membranes with particular reference to carotenoid- or retinol-containing systems. 439 The phenomenon of conductivity enhancement that occurs when various chemical vapours are adsorbed has been used as a probe to study the adsorption and desorption processes in crystals of (1 SZ)-P-car~tene,~~~ and the compensation effect in the electrical conduction process was Derivatives of p-carotene have been proposed as candidates for components of molecular-electronic devices.442 Impressive progress has been made in elucidating the mechan- isms of chromophore-protein interactions and the structural changes that occur during photocycling in the protein retinal photoreceptors (namely rhodopsin the visual pigment and bacteriorhodopsin and related complexes from Halobacterium species) especially through n.m.r.and resonance-Raman spectroscopic studies with 13C-and 2H-labelled chromophores. Thus extensive low-temperature solid-state 13C n.m.r. studies of samples of rhodopsin that had been prepared from species of retinal that were enriched with 13Cat C-5 or C-14 have shown NATURAL PRODUCT REPORTS 1989-G.BRITTON that in the complex the chromophore adopts a 6-s-cis conformation the C=N bond is anti and the C=N linkage is protonated. 289 Further studieszg0 with samples that were enriched with 13C at positions 5 and 12 confirmed the 6-s-cis conformation and provided evidence for a negative charge interacting near C- 12. Similar work with bacteriorhodop-sin291-294 has confirmed that the chromophore in the dark- adapted form contains a mixture of (all-@- and (132)-retinal with the C=N bond anti and syn in these two components respectively and the 6-s-trans conformation in contrast to that in rhodopsin. Evidence was also obtained for the existence of a negatively charged residue on the protein near C-5 and a positive charge near C-7.Resonance Raman work with samples of visual pigment that were enriched with 2H and 13C have shown that the primary intermediate bathorhodopsin has the 10-s-trans conforma- 344. 345TheC( 14)-C( 15) bondofrhodopsinisnotperturbed by its binding to a protein but the C(14)-C(15) and C(8)-C(9) bonds of bathorhodopsin and the C(14)-C( 15) and C(l0)-C( 11) bonds of isorhodopsin are significantly altered. Resonance Raman spectral bands especially C-C stretches for the (all-@- retinal chromophore of light-adapted bacteriorhodopsin have been assigned by using isotopically labelled retinal~.~~~ This led to the demonstration that the primary step in the bacteriorho- dopsin photoreaction is a trans to cis isomerization of the C(13)-C( 14) bond and does not involve C( 14)-C( 15) s-cis- The Schiff-base mode in bovine rhodopsin and bathorhodopsin has also been studied by resonance Raman spectroscopy of samples that had been prepared from chromo- phores that were labelled with 2H or 13C at C-15 and N-H.343 Energy barriers to cis-trans isomerization in the dark in protonated all-trans- 1Santi- and 14-s-trans-retinal Schiff base in the presence and in the absence of electrostatic and nucleophilic catalysts have been calculated by the MNDO method.457 Three general processes namely bicycle-pedal hula- twist and ordinary cis-trans isomerization were considered and their possible importance in relation to the mechanism of dark-light adaptation of bacteriorhodopsin was discussed.A review on these mechanisms for reactions of confined anchored polyenes has also been and a mechanistic-model study of processes in the vertebrate and invertebrate visual cycles has been Reconstitution experiments with 5,6-dihydroretinal (248) and 7,8-dihydroretinal (264) have indicated that much of the 'opsin-shift ' in bacteriorhodopsin is due to chromophore-protein interactions near the Schiff- base end of the chromophore and have provided further evidence for a negative charge on the protein near C-5 and a positive charge near C-7.460 Evidence has been presented for a C( I3)-C( 14) cis cycle in bacteriorhodop~in~~l and for the possible involvement of isomerizations of the C( 14)-C( 15) single bond in its proton-pumping mechanism.462 Chromo- phore-protein interactions in rhodopsin and bacteriorhodopsin have been Numerous books and reviews have been published which include discussions of a wide range of aspects of the structures photochemistry and functioning of these retinal-protein photo- receptors.Amongst the topics that have been covered are general features of the structures of rhodopsin and bacterio- rhodop~in,~~~ 468 rhodopsin and the chemistry and photo-chemistry of vision,469-483 spectroscopic studies of rhodop- sin,484488 and the structure and functioning of bacterio-rhodop~in~~~-~~~ In a range of and the related halorhod~psin.~~~ spectroscopic studies the primary event in vision has been investigated by sub-picosecond time-resolved fluorescence (264) 377 by transient-absorption difference spectro- scopy (which indicated the existence of two forms of batho-rhodop~in),~~~ and by low-temperature spectroscopy of batho-rhodopsin in a transparent medium.499 A theoretical analysis of data from picosecond spectroscopic studies of the kinetics of formation of bathorhodopsin has been aimed at understanding the initial step in visual transdu~tion.~'~ A new precursor of sR,, in the photocycle of sensory rhodopsin has been The effects of chemical modification or of deproto- nation of the Schiff base of rhodopsin on the regeneration and activation of the G-protein have been ~tudied.~~~,~~~ An investigation of the photosensitivity of 10-substituted (par- ticularly 10-fluoro) visual-pigment analogues has led to the detection of a specific secondary opsin-retinal interaction.504 Energy storage in the primary photochemical events of rhodopsin and isorhodopsin has been studied.505 Spin- trapping has been used to investigate the kinetics of retinal-sensitized photo-oxidation of rhodopsin in the photoreceptor mem-b~ane.~O~ The topic of adiabatic potentials for the cis-trans isomerization of the rhodopsin chromophore has been re-viewed.507 A kinetic study of the transfer of (1 12)-retinal between rod outer-segment membranes has been The chemical environment around the chromophore of cepha- lopod retinochrome and the stereospecific transformation of (all-@- into (1 12)-retinal have been investigated and several artificial retinochromes have been prepared with analogues of The functioning of bacteriorhodopsin as a light-driven proton pump has been and the importance of lipid-protein interactions5" and conformational changes512 and the effect of cross-linkers on the photocycle513 have been studied.The position of the retinal chromophore in bacterio- rhodopsin and in the purple membrane has been investigated by fluorescence and resonance energy tran~fer,~l~-~l~ and by neutron diffra~tion.~~' Two classes of binding site for hydro- phobic molecules on bacterio-opsin have been identified.518 Samples that had been prepared from artificial analogues of retinal have been used to study several properties of bacterio-rhodop~in.~l~-~~~ Early picosecond events in the photocycle of bacteriorhodopsin have been and a theoretical treatment of the molecular dynamics of the primary photo- chemical event has been The time-course and the stoicheiometry of light-induced release and uptake of protons during the bacteriorhodopsin photocycle have been investi- gated.527 Several new transient intermediates in the bacterio- rhodopsin photocycle have been identified.528 533 Flash-spectroscopic studies of the kinetics of the halorhodop- sin photocycle have been and the quantum yield of the primary photoreaction has been determined.535 Dark- adapted halorhodopsin has been to contain (132)-and (all-@-retinal in the ratio 7:3. The mechanism of the halorhodopsin photocycle is considered to be similar to that of bacteriorhodopsin and to involve the series of isomerizations all-trans -f 13-cis 14-s-cis + 13-cis-f all-trans of the retinal Schiff-base chr~mophore.~~~ The change from 1344 14-s-cis -+ 13-cis however is not induced by deprotonation of the chromophore but by movement of C1- towards the protonated Schiff-base group.The effects of azide on this deprotonation of the chromophore have been Flash-spectrophoto-metric work has revealed the presence of a fourth protein retinal complex in Halobacterium halobium .539 This complex given the name photorhodopsin is correlated with a photo- repellant response to light of wavelength 480 nm. A water- soluble yellow photoreceptor protein similar to rhodopsin has been isolated from a halophilic bacterium,540 and photo-active retinal pigments in halo-alkaliphilic bacteria have been de- scribed.54 1.6 Biosynthesis and Metabolism Several reviews have been published on the general biosynthesis of carotenoid~,~'~ biosynthesis of carotenoids in higher plants,543.544 and the biosynthesis of chloroplast carotenoid~.~~~ NATURAL PRODUCT REPORTS 1989 R2 R' HO JPp HO b OC a HO4ic" OH e (267) R' =a R2 = b (268) R' =c R2 =b (269) R' =d R2 =e Many papers report progress with carotenogenic cell-free systems.Methods have been described for the solubilization and reconstitution of carotenogenic enzymes including phy- toene desaturase from chromoplasts of daffodil (Narcissus pseudonarcissus) with the detergent 3-[(3-~holamidopropyl)di-methylammonio]propane-1-sulphonate(CHAPS).546 The en-zymic conversion of prephytoene diphosphate (265) into carotenoids by isolated chloroplasts chromoplasts of red pepper (Capsicum annuurn) and etioplasts of wheat has been reported ;547 efficient incorporations were achieved especially into the (1 SZ)-isomer of phytoene (7,8,11 12,7',8' 1l' 12- octahydro-+,+-carotene) (266).The plastid stroma was shown to be the sole site of biosynthesis of phytoene from prephytoene diphosphate or isopentenyl diphosphate in chloroplasts etio- plasts and amylopla~ts.~~~ The incorporations were enhanced by factors such as Mg2+ Mn2+ neutral detergents ATP and glycolytic intermediate^.^^^ Partially purified phytoene synthase from chromoplasts of Capsicum annuum was inhibited by aminophenethyl diphosphate and azidophenethyl diphosphate which were competitive with respect to isopentenyl diphos- hate.^^^ Lycopene cyclase from chromoplasts of Capsicum annuum has been s~lubilized.~~~ In etioplasts and etiochloro- plasts of mustard cotyledons (Sinupis ah) the phytoene 9 h (270) R' = f R2 =g (271) R' = R2 = f (272) R' =a R2 =h jx 0 (273) X = 0 (274) X = H OH synthase complex was present in soluble form in the stroma whereas subsequent enzymes were bound in the membrane fraction.552 Membranes of a strain of the cyanobacterium Aphanocapsa have been used to demonstrate the formation of /3-cryptoxanthin (P7P-caroten-3-ol) (267) and 3-hydroxyechi- nenone (3-hydroxy-/3,/3-caroten-4-one) (268) from phyt~ene~~~ and the hydroxylation of p-carotene to P-c~yptoxanthin,~~~ the latter conversion showing characteristics of a mono-oxygenase reaction.The effects of twelve ionic and non-ionic detergents on the conversion of [14C]geranylgeranyl diphosphate into phytoene lycopene and p-carotene by membranes of a strain NATURAL PRODUCT REPORTS 1989-G. BRITTON (275) of Aphanocapsa have been tested.555 The ionic detergents were inhibitory and the best solubilization was achieved with Tween 40. Regulatory effects of a variety of chemicals on the bio- synthesis of carotenoids have been studied. Thus methyl (92)- trisporate B (273) stimulated the biosynthesis of /?-carotene under the same genetic Genetic studies with a series of carotenogenic mutants of Gibberella fujikuroi have been described.576 Biogenetic relationships between ketocarotenoids and the polymeric cell-wall material sporopollenin in green algae have been explored.577 whereas methyl (9Z)-trisporate C (274) was less effective and the (all-E)-isomers had no effect.The stimulatory effect was additive with those of light retinol dimethyl phthalate and mutation of the gene cars. Of 160 aromatic compounds that were tested on Phycomyces blakesleeanus 77 affected the biosynthesis of car~tenoids.~~~ Many stimulated the biosyn- thesis of p-carotene whereas others caused the accumulation of phytoene lycopene and other intermediates. In Blakeslea trispora biosynthesis of p-carotene was stimulated four-fold by the addition of a-ionone or p-ionone and further stimulated by a mixture of the /?-Ionone and diphenylamine blocked the desaturation of phytoene in Gibberella fujikuroi whereas other chemicals had no effect even though they were active with Phycomyces blakesleean~s.~~~ It was concluded that G.fujikuroi lacks feedback regulation of carotenogenesis like that in Phycomyces species. Biosynthesis of coloured carotenoids in fruits of Capsicum annuum was inhibited by 2-hydroxybiphenyl diphenylamine and 9-fluorenone. 560 Phytoene and phytofluene (7,8,11,12,7’,8’-hexahydro-$,$-carotene) (275) accumulated. MPTA [2-(4-methylphenoxy)triethylamine] induced the syn- in (-)-cultures of the mould Phycomyces bl~kesleeanus~~~ The metabolism of carotenoids in animals especially fish is the subject of many papers though in most cases the putative metabolic pathways have been proposed only from con-sideration of the structures of the compounds present and direct conversions have not been demonstrated.Feeding experiments with unlabelled carotenoids have however dem- onstrated the conversion of canthaxanthin into (2R)-2-hydroxy- canthaxanthin (1 7) but not into astaxanthin of p-carotene into echinenone canthaxanthin and their 2-hydroxy-derivatives and of (3R,3’R)-zeaxanthin into (3S,3’S)-astaxanthin in the crustacean Daphnia magn~.~~~ Astaxanthin that was isolated from the flesh of wild rainbow trout (Salmo gairdneri) was exclusively the (3S,3’S)-‘ isomer whereas the skin contained (3R,3’R)-zeaxanthin (3R,3’R,6’R)- lutein and (3R,3’S,6’R)-epilutein these being compounds that were assumed to be metabolites of asta~anthin.~~~ Other workers have reported the reductive metabolism of astaxanthin bisesters to isomers of zeaxanthin in trout and in other The reductive metabolism of astaxanthin to zeaxanthin in muscle of chum salmon (Oncorhynchus keta) during the spawning migration has also been The presence of 3,4,3’- tri hydroxypirardixanthin thesis of phytofluene and lycopene in lemons (Citrus lim~n).~~~ The induction required gene expression.In a range of fungi photoinduction of carotenogenesis involved an initial time-independent photochemical step fol- lowed by a series of dark metabolic reactions which involved synthesis of proteins de ~10~0.~~~ In Neurospora crassa nitrate at high concentration inhibited the photoinduction of proto- perithecia but not of carotenoid~.~~~ Effects of Ca2+ on the activity of some enzymes which play a role in regulating carotenogenesis in N.crassa have been Contrary to previous reports red light had no effect on carotenogenesis in Verticillium agaricinum although an effect of ultraviolet A was easily demonstrated. 565 Considerable progress has been made in studies of the genes for carotenoid biosynthesis especially in photosynthetic bac- teria. A review on research on genetics in photosynthetic bacteria includes a survey of some of this Several genes for the biosynthesis of carotenoids from Rhodobacter sphaeroides have been cloned567 and expressed in phylo-genetically related non-photosynthetic bacteria.568 It has been determined by complementation and by deletion mapping that the gene crtl mediates the conversion of phytoene into coloured carotenoids in Rhodobacter capsulat~s.~~~ The roles of light and 0 in regulating the expression of genes for the development of pigment-protein complexes and for biosynthesis of carotenoids in Rhodobacter capsulatus have been investigated.570 Most genes were repressed by 0 but one crtA which is responsible for the conversion of spheroidene (252) into spheroidenone (I-methoxy-3,4-didehydro- 1,2,7’,8’-tetrahydro-$,$-caroten-2- one) (269) was Aspects of the genetic regulation of biosynthesis of p-carotene and B gene specificity in tomatoes have been The genetic control of photoinduced carotenogenesis in Myxococcus x~nthus~~~ and in Mycobac-terium smegmati~~~~ has been discussed.In M. smegmatis photoinduced and constitutive synthesis of carotenoids were (5,6,5’,6’- tetra h ydro-P,P-caro- tene- 3,4,3’- triol) (270) and 3,4,3’,4’- tetrahydroxypirardixan t hin (5,6,5’,6’- tet rahydro-P,P-caro tene- 3,4,3’,4’- tetraol) (271) showed the operation of reductive metabolism of double-bonds in the prawn Pennaeus j~ponicus.~~~ As discussed earlier (see Section 1.2.1) there are reports of the identification of new enantiomeric forms of zeaxanthin lutein tunaxanthin 3’-hydroxy-e,e-caroten-3-one, etc. in fish in several pa~ers~l-~~ and pathways have been proposed for the metabolic formation of these isomers.Based on structural considerations pathways have also been proposed for the formation of the newly identified carotenoids in hen’s egg yolk namely (6R 3’R 6’R)- 3’-hydroxy-e,e-caro ten- 3-one (28) (6S 3’R 6’R)- 3’- hydroxy-e,e-car0 ten-3-one and (6’ RS)-P,s-car- oten-3’-one (3 1) from lutein zeaxanthin and P-~ryptoxanthin.~~ Several reviews deal with the conversion of carotenoids into vitamin A (retinol) and with the metabolism of vitamin A and related comp~unds.~~-~~+ 5839 584 The low-yield conversion of labelled canthaxanthin p-carotene lutein and zeaxanthin into retinol and less efficiently into vitamin A (3,4-didehydro- retinol) has been demonstrated to occur in goldfish (Carassius aurat~s).~~~ The results were considered to be inconsistent with the previously proposed pathway for the formation of vitamin A from lutein via anhydrolutein (3’,4‘-didehydro-p,P-caroten-3-01) (272).The isomerization of (all-E)- to (112)-retinol in the retina has been dem~nstrated,~~~-~~~ and the metabolism of (al1-E)- retinol and -retinoic acid to the (132)-isomers has been achieved in rabbit tracheal epithelial cells in The formation of retinoyl P-glucuronide as a biologically active metabolite of retinoic acid has been The kinetics of the enzymic esterification of retinol and 3,4-didehydroretinol by retinol ester synthase in cell membranes from frog retina pigment epithelium have been The 5,6-epoxidation of retinoic acid by haemoproteins has been NATURAL PRODUCT REPORTS 1989 (276) R = CHpOH (277) R = C02H (280) X = 0 (281) X = H OH CH20H (283) (284) R2 L J4 (286) R' = CHO R2 = C02Et (287) R' = R2 = CH2OH The metabolism of (22,4E)-y-ionylidene-ethanol(276) and (22,4E)-y-ionylideneacetic acid (277) in the abscisic-acid-synthesizing fungus Cercospora cr~enta~~~ and of a-ionylidene compounds by Cercospora rosi~ola~~~ has been demonstrated.The biosynthesis of abscisic acid in C. rosicola was inhibited by paclob~tra~o1.~~~ Deuterium-labelling studies of the synthesis of abscisic acid xanthoxin (278) and carotenoids in tomato shoots that were maintained in 70% ,H,O gave mass-spectrometric deuterium-labelling patterns which were inter- preted as showing that abscisic acid was not derived from the caroten~ids.~~~ In contrast inhibition studies with fluridone- treated barley indicated that xanthophylls are converted into a bscisic acid in dehydrating plants.597 2,7-Dimethylocta- 2,4- dienedioic acid (279) has been considered as a possible by- product of the biosynthesis of abscisic The catabolism of (f)-abscisic acid to phaseic acid (280) dihydrophaseic acid (281) and 18-hydroxy abscisic acid (44)by excised leaves of Hordeum vulgare has been The vacuolar-extravacuolar distribution of metabolites of abscisic acid in cell suspension cultures of Lycopersicon esculentum has been determined.600 Metabolites such as phaseic acid and dihydro- phaseic acid were mainly extravacuolar whereas sugar esters and other conjugates were found only in vacuoles.The reduction of aldehydes and ketones including ionones and other norcarotenoids to the corresponding alcohols by cyanobacteria has been reported.601 2 Polyterpenoids and Quinones 2.1 Polyterpenoids New IUPAC-IUB recommendations have been made for the nomenclature of preno1s.602 Configurations of double-bonds are specified as cis or trans and listed from the w end of the molecule. A scheme for the shorthand designation of structure is proposed; in this W,T,C and S are used to specify o,trans cis and saturated residues respectively. The general collective term 'prenols' is considered to include the dolichols as 2,3- dihydropolyprenols. Reviews have been published which deal with the dis- tribution biosynthesis metabolism and functions of dolichols and polypren~ls,~~~.604 the large variety of mainly-cis-poly-prenols from leaves of woody plants,605 the structures biosynthesis and physicochemical properties of the isoprenoid ether lipids of Archaebacteria,606-610 and 'H and ,H n.m.r. studies of 'H-labelled polyprenols that provide information on their organization and dynamics in membranes.611 Novel tetraether lipids from Methanobacterium thermoauto- trophicum have been identified as glucosyl and phosphoethanol- amine derivatives of the di(biphytany1) diglycerol tetraether (282) designated caldarchaeo1.612 The related ester of phospha- tidyl-myo-inositol has been obtained from Thermococcus celer and characterized by 'H and 13C n.m.r. The arrangement of isoprene units in pig liver dolichols- 18 -19 and -20 (283; n = 17 18 or 19) has been established614 by 'H and 13C n.m.r.to be WTT-[C],,-,,-S-OH. Directed aldol condensation was used as a stereoselective route for synthesizing (2)-trisubstituted 01efins.~'~ Thus in the presence of LiN(CHMe,), the aldimine (284) reacted with the (2)-aldehyde (285) to give the acrolein (286) and thence the substituted polyprenol (287). Substituted hexaprenols have been prepared by a similar route.616 Base-induced coupling of NATURAL PRODUCT REPORTS 1989-G. BRITTON 381 (288) X = OThp Y = MeC6H4S02 (289) X = CI Y = OCH2Ph CI OCH2Ph (290) OH (292) (294) (296) R' = Me R2 = H (297) R' = H R2 = Me I CH20R3 (298) R' = H R2 = R3 =a (299) R' = R2 =a R3 = H r 1 'NOH the C,,compounds (288) and (289) gave an all-cis C, building block (290) which was used to synthesize (22,62,102,142 l82,222,262,302,34E,38E)-undecaprenol (291).617 The synthe- sis of polyisoprenepolyols such as (292) and (293) containing multiple tertiary hydroxyl groups has been achieved efficiently by using hydromagnesation.618 A new approach to the synthesis of polyisoprenepolyols that possess glycerol termini e.g.(294) used the highly oxygenated C,-unit 4-( 1,2-epoxy-l-methylethyl)-2-phenyl- 1,3-diox0lane.~~~ The saturated head-to-head iso-prenoid (295) has been prepared from phyto1.620 The related diol (296) has also been synthesized and shown to be a stereoisomer of the C, diol (297) from Methanobacterium thermoautotrophicum.621The archaebacterial membrane lipid 2,3-di-O-phytanyl-sn-glycerol(298) and its 1,2-isomer (299) have been synthesized in good yield by a route involving protection of position sn-1 of 3-0-benzyl-sn-glycerol with Me3CSiPh2C1 and alkylation with (3RS,7R,11R)-phytanyl triflate.622 [U-'4C]Solanesol (300) has been prepared623 by growing tobacco on 14C02.The use of an acidified solvent has been shown to improve the efficiency of extraction of polar tetraether lipids from Methanobacterium thermoautotrophicum .624 Methods have been described for the estimation of dolichol (283) in of dolichyl phosphate in tissues,626 and of both of these compounds in soybean embryo The surface behaviour of six archaebacterial ether lipids in monolayers at the air-water interface has been investigated.628 The organization and dynamics of bipolar lipids from Sulfo-fobus solfataricus in the bulk phase and in monolayer membranes has been studieds2' and phase transitions of bipolar lipids in thermophilic archaebacteria have been inve~tigated.~~' Structure and polymorphism of tetraether lipids from S.solfataricus have been studied by crystallographic analysis as a function of water content and temperature.631 A spin-label e.s.r. and saturation-transfer e.s.r. study of archaebacterial polar lipids has been The modulation of the properties of macrovesicular lecithin membranes by a hom- ologous series of polyprenols has been The stereochemistry of the elimination of hydrogen in the biosynthesis of polyprenols in Mallotus japonicus has been Unexpectedly the same hydrogen atom was lost from the prochiral position C-4 of the isoprene units in the formation of both (Q-and (3-prenyl residues.A method for studying the stereochemical direction of formation of the C-C bond in prenyltransferase reactions with respect to the face of the double-bond of isopentenyl diphosphate has been applied to the (2)-and (E)-prenyl chain-elongation reactions that are catalysed by undecaprenyl-diphosphate synthase and hepta- prenyl-diphosphate synthase respectively in Bacillus s~btifis.~~~ In both cases formation of a C-C bond took place at the si-face of the double-bond with elimination of a hydrogen-atom from C-2 in a syn fashion.The enzymes isopentenyl-diphos- 382 8 (301)n= l,rn=3 (302) n = variable rn = 0 (303) n = 6 rn = 1 (304) n = 5 rn = 1 (305) n = 7 rn = 1 (306) n = 5 rn = 2 (307) n = 0 rn = 4 (308) n = 0 rn = 5 (309)n = 0 rn = 6 WMe R2 0 R3 (310) R' or R2 = Me R3 = polyprenyl (311) R' or R2 = Me R3 = octaprenyl R' 9 (312) R' = R2 = Me R3 = R4 = H 4 (313) R' = R2 = H R3 = R = Me phate A-isomerase farnesyl-diphosphate synthase [geranyl- transtransferase] octaprenyl-diphosphate synthase [trans-octa- prenyltranstransferase] and undecaprenyl-diphosphate syn-thase [ditrans,polycis-decaprenylcistransferase] from Escher-ichia coli have been separated and partially Dynamic interactions between the components of hexaprenyl-diphos- phate synthase [trans-pentaprenyltranstransferase]from Micro- coccus luteus have been Several studies of the biosynthesis and metabolism of dolichols in the rat have been rep~rted.~~~-~~~ a-Saturation during biosynthesis of dolichols by rat liver homogenates required NADH but saturation of dolichyl phosphate could not be achieved.641 Studies on the biosynthesis of polyisoprenoid lipids in the rat tapeworm Hymenolepsis diminut~~~~ and on the metabolism of exogenous polyisoprenols by animal cells if they were cultured in ~itr-0~~~ have been reported.A review has been presented of the pathway of assembly of isoprenoids the mechanism of formation of ether linkages etc. in the biosynthesis of archaebacterial ether lipids.644 2.2 Isoprenylated Quinones A review has been published on the isolation structure elucidation biological activity and especially chemical syn- thesis of the K vitamins i.e.phylloquinone (301) and menaquin- ones (302).645A new naturally occurring vitamin K isolated from Nocardia species has been identified as a derivative of menaquinone-8 [MK-81 with a cyclized side-chain (47).61 A similar compound (48) was later identified in Nocardia NATURAL PRODUCT REPORTS I989 Me0 L M e (314)n = 4 R = *H L -13 (315) n = 2 R = L -Jff (316) n = 6 (317) n = 5 (318) n = 4 (319) n = 3 (320) n = 6; didehydro 'H (3211 ,H 0 (322) brasiliensis.62 Several new hydrogenated menaquinones have been isolated including the terminally saturated compounds MK-7(VII-H2) and MK-6(VI-H,) [(303) and (304) respect- ively] from sulphur-reducing bacteria,64s MK-8(VIII-Hz) (305) from Halococcus morrh~ae,~~' MK-7(VI-H2 VII-H,) (306) from Thermoleophilum album,648 and a range of derivatives of MK-4 MK-5 and MK-6 namely MK-4 (8H) (307) MK-4 (6H) MK-5 (10H) (308) MK-5 (8H) MK-6 (12H) (309) and MK-6 (10H) from Thermoproteus tena~.~~' The latter organism also contained menaquinone derivatives (310) that contain an additional methyl substituent at position 5 or 8.Similar monomethylated menaquinones e.g. (3 1l) together with the $6-or 7,8-dimethylated derivatives (3 12) or (3 13) have also been identified as minor components in Natronobacterium greg~ryi.~~' Two novel ubiquinones (UQs) have been isolated from methane-utilizing bacteria and identified as structures and (314) and (315) designated 18-meth~Iene-UQ-8~~~ 11-methylene- 18,l 8-dimethyl-UQ-6,652 respectively.A new sul-phur-containing quinone has been isolated from the archae- bacterium Sulfolobus solfataricus and identified as the benzo- [b]thiophen-4,7-quinone (3 16) designated SQ-6 (1 2H).653. Several lower homologues designated SQ-5 (lOH) SQ-4 (8H) NATURAL PRODUCT REPORTS 1989-G. BRITTON 383 8 Meo%HMe0 M e bMe 0 R (323) (327)R = [CH2InMe;n = 1,4,6,8,10,12,14,or 16 (328)R = [CH2]8CH=CH2 (329)R = H Me0 Meo%HMe0 L -13 0 (330) (325)R' =a R2 = H (326)R' = H R2 = a SQ-3 (6H) SQ-6 (IOH) and the related caldariaquinone CQ-6 (3311 (10H) [i.e. compounds (3 17)-(321) respectively] were also present as minor components.The novel benzo[l,2-b;4,5- b'ldithiophen compound (322) was also identified. 654 A method for regio- and stereo-selective desulphonylation of ,Me allylic sulphones with lithium triethylhydroborate has been applied in the synthesis of ubiquinone-10 (323; n = A new procedure for the de-methoxycarbonylation of activated 0 methyl esters was used for the simultaneous removal of two methoxycarbonyl functions from a single molecule as a crucial step in another synthesis of ubiquinone- Shorter-chain (332)R' = H R2 = OH (333)R' = OH R2 = H ubiquinones e.g. UQ-2 (323; n = 2) and UQ-3 (323; n = 3) have been prepared from p-HOC6H4Me via bromination copper-catalysed polymethoxylation and copper-mediated coupling with the appropriate prenyl bromide.657 Ubiquinone- 9 (323; n = 9) has been prepared by oxidative demethylation of the ubiquinol monomethyl ether (324) with ceric ammonium nitrate.658 Photoaffinity-labelled analogues (325) and (326) of ubiquinone- 1 containing the 4-azido-2-nitroanilino-group have been prepared.659 A sample of UQ-10 that was labelled with "C has been prepared and used for distribution studies in rats.660 The plastoquinone analogues (327) and (328) have been synthesized by treating (329) with the appropriate acid.gs1 Progress in the synthesis of vitamin K has been re- viewed.645,662 In a pieparation of phylloquinone (30 l) menadiol monoacetate and isophytol were condensed to give the intermediates (330) and (33 1).663 Pristane and 1,5,9,13-tetra- methyltetradecyl acetate were treated with m-chloroperoxy- benzoic acid to give hydroxylated derivatives which were then condensed with the quinone core to give the hydroxy-phylloquinone derivatives (332) and (333).664 L (335) Several h.p.1.c.procedures have been described for the separation purification and quantitative assay of isoprenylated quinones e.g. mixtures of homologues of ubiquinone mena- quinone and demethylmenaq~inone,~~~of the ubiquinone homologues UQ-6 to UQ-10,666of UQ-9 and UQ-10,667 of ubiquinones and ubiquinol (334),668 of oxidized and reduced pla~toquinones,~~~of menaquinone and of vitamin K in plasma serum and Methods for the purification and analysis of oxidized and reduced phyllo- H quin~ne,'~~of phylloquinone and its 2,3-epoxide (335),680 and of phylloquinone chromenol (336)681 have been described and (3361 3 84 OH (337) (338) 0 (339) partition coefficients of quinones have been determined by h.p.l.c.6s2 In a negative-ion FAB mass-spectrometric study ubiquinone-6 (323; n = 6) and phylloquinone in a sulpholane matrix unexpectedly yielded abundant [MI-ions.683 A first- derivative absorption-spectroscopic assay of ubiquinone- 10 has been The location molecular interactions and mobility of isoprenoid quinones in lipid membranes and vesicles have been widely studied.Thus the behaviour of ubiquinone-10 and ubiquinol-10 (334; n = and of various ubiquinone homologues and analogueP6 in phospholipid bilayers has been investigated by fluorescence measurements with diphenylhexa- triene and n-(9-anthroyloxy)stearic acids respectively and deuterium-labelled analogues of ubiquinone- 10 dispersed in plasma membranes of E.coli or inner membranes of beetroot mitochondria have been investigated by solid-state 2Hn.m.r. spectroscopy and their orientational order at various sites determined.687 Other n.m.r. studies have investigated the interactions of UQ-10 with phospholipids in model mem-branesGs8 and the location of UQ-10 in the membrane core in phosphatidylcholine liposome~.~~~ The molecular motion of plastoquinone-9 (337) in dimyristoylglycerophosphocholine membranes has been studied by solid-state proton-enhanced 13C n.m~.~’O The molecular interactions of UQ-10 and ubiquinol- 10 with bilayers of dipalmitoylglycerophosphocho-line have been studied by F.T.i.r.and by differential scanning calorimetry and by measurements of t~rbidity.~’~ The interactions of phylloquinone with dipal- mitoylglycerophosphocholine vesicles have also been studied by fluorescence and F.T.i.r. The critical micelle concentration of short-chain ubiquinones in models system has been determined.694 Another study described the incorporation of ubiquinone into lipid vesicles and its antioxidative effect on peroxidation of lipids.695 The photolysis of plastoquinones and plastoquinols (338) in model membranes and in protic and aprotic solvents has been inve~tigated.~’~ The chronopotentiometric behaviour of UQ- 10 in carbon- paste electrodes has been examined.697 Combined e.s.r.and time-resolved CIDEP techniques have been used to identify NATURAL PRODUCT REPORTS 1989 plastoquinones and phylloquinone in the chloroplast has been shown to take place solely on the inner envelope membrane.703 In rat liver biosynthesis of ubiquinones from tyrosine and mevalonic acid has been shown to take place not only in the mitochondria but also in microsomes and in endoplasmic reticulum.704* ’05 3 References 1 ‘Key to Carotenoids’ ed. H. 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Kido J. Biochem. (Tokyo) 1986 99 63. 593 T. Kato T. Oritani and K. Yamashita Agric. Biol. Chem. 1987 51 2695. 594 S. J. Neill R. Horgan D. C. Walton andC. A. M. Mercer Phyto-chemistry 1987 26 2515. 595 S. M. Normal R. D. Bennett S. M. Poling V. P. Maier and M. D. Nelson Plant Physiol. 1986 80 122. 596 H. M. Nonhebel and B. V. Milborrow J. Exp. Bot. 1987,38,980. 597 P. E. Gamble and J. E. Mullet Eur. J. Biochem. 1986 160 117. 598 R. S. T. Linforth W. R. Bowman D. A. Griffin P. Hedden B. A. Marples and I. B. Taylor Phytochemistry 1987 26 1631. 599 A. K. Cowan and I. D. Railton Plant Physiol. 1987 84 157. 600 H. Lehmann and K.Glund Planta 1986 168 559. 601 F. Jiittner and R. Hans Appl. Microbiol. Biotechnol. 1986,25,52. 602 H. B. F. Dixon A. Cornish-Bowden and C. Liebecq Eur. J. Biochem. 1987 167 181. 603 J. W. Rip C. A. Rupar K. Ravi and K. K. Carroll Progr. Lipid Res. 1985 24 269. 604 E. L. Appelkvist and G. Dollner in ‘Peroxisomes in Biology and Medicine [Lect. Int. Symp.] 1986’ ed. H. D. Fahimi and M. Sies Springer Berlin 1987 p. 53. 605 T. Chojnacki E. Swiezewska and T. Vogtman Chem. Scr. 1987 27 209. 606 M. de Rosa A. Gambacorta and A. Gliozzi Microbiol. Rev. 1986 50 70. 607 V. Luzzati A. Gambacorta M. de Rosa and A. Gulik Annu. Rev. Biophys. Biophys. Chem. 1987 16 25. 608 .V. Luzzati and A. Gulik Syst. Appl. Microbiol. 1986 7 262. 609 T. A. Langworthy and J.L. Pond Syst. Appl. Microbiol. 1986,7 253. 391 610 D. M. Ward S. C. Brassell and G. Eglinton Nature (London) 1985 318 656. 61 1 J. S. de Ropp M. J. Knudsen and F. A. Troy Chem. Scr. 1987 27 101. 612 M. Nishihara H. Morii and Y. Koga J. Biochem. (Tokyo) 1987 101 1007. 613 M. de Rosa A. Gambacorta A. Trincone A. Basso W. Zillig and I. Holz Syst. Appl. Microbiol. 1987 9 1. 614 Y. Tanaka H. Sato A. Kageyu and T. Tomita Biochem. J. 1987 243 481. 615 N. Ya. Grigor’eva I. M. Avrutov 0.A. Pinsker 0.N. Yudina A. I. Lutsenko and A. M. Moiseenkov Zzv. Akad. Nauk SSSR Ser. Khim. 1985 1824. 616 N. Ya. Grigor’eva 0.N. Yudina and A. M. Moiseenkov Izv. Akad. Nauk SSSR Ser. Khim. 1986 2036. 617 K. Sato 0. Miyamoto S Inoue Y. Matsuhashi S.Koyama and T. Kaneko J. Chem. SOC. Chem. Commun. 1986 1761. 618 Y. Kobayashi F. Sato T. Miyakoshi Y. Fujita M. Shiono K. Kanehira and S. Suzuki Synth. Commun. 1986 16 597. 619 S. Suzuki Y. Fujita Y. Kobayashi and F. Sato Tetrahedron Lett. 1986 27 69. 620 M. E. Jung and C.-Y. Liu J. Org. Chem. 1986 51 5446. 621 C. H. Heathcock and P. A. Radel J. Org. Chem. 1986 51,4322. 622 T. Aoki and C. D. Poulter J. Org. Chem. 1985 50 5634. 623 S. B. Hassam J. Labelled Compd. Radiopharm. 1985 22 1261. 624 M. Nishihara and Y. Koga J. Biochem. (Tokyo) 1987 101 997. 625 G. N. Morris and R. K. Pullarkat Lipids 1987 22 58. 626 K. Yamada S. Abe T. Suzuki K. Katayama and T. Sato Anal. Biochem. 1986 156 380. 627 J. W. Rip and K. K. Carroll Anal. Biochem. 1987 160 350.628 R. Rolandi H. Schindler M. de Rosa and A. Gambacorta Eur. Biophys. J. 1986 14 19. 629 A. Gliozzi S. Bruno T. K. Basak M. de Rosa and A. Gamba- corta Syst. Appl. Microbiol. 1986 7 266. 630 A. Gliozzi G. Paoli D. Pisani F. Glioui M. de Rosa and A. Gambacorta Biochim. Biophys. Acta 1986 861 420. 631 A. Gulik V. Luzzati M. de Rosa and A. Gambacorta Syst. Appl. Microbiol. 1986 7 258. 632 S. Bruno S. Cannistraro A. Gliozzi M. de Rosa and A. Gambacorta Eur. Biophys. J. 1985 13 67. 633 T. Janas J. Kuczera and T. Chojnacki Stud. Biophys. 1987,121 45. 634 T. Suga T. Hirata T. Aoki and T. Kataoka J. Am. Chem. SOC. 1986 108 2366. 635 M. Ito M. Kobayashi T. Koyama and K. Ogura Biochemistry 1987 26 4745. 636 S. Fujisaki T. Nishino and H.Katsuki J. Biochem. (Tokyo) 1986 99 1327. 637 I. Yoshida T. Koyama and K. Ogura Biochemistry 1987 26 6840. 638 E.-L. Appelkvist Acta Chem. Scand. Ser. B 1987 41 73. 639 P. G. Elmberger A. Kalen E.-L. Appelkvist and G. Dallner Eur. J. Biochem. 1987 168 1. 640 R. K. Keller Trends Biochem. Sci. 1987 12 443. 641 T. J. Ekstrsm T. Chojnacki and G. Dallner J. Biol. Chem. 1987 262 4090. 642 W. J. Johnson and G. D. Cain Comp. Biochem. Physiol. B 1985 82 487. 643 I. Krajewska-Rychlik Acta Biochim. Polon. 1985 32 21 1. 644 M. de Rosa and A. Gambacorta Syst. Appl. Microbiol. 1986 7 278. 645 A. Riittimann Chimia 1986 40,290. 646 M. D. Collins and F. Widdel Syst. Appl. Microbiol. 1986 8 8. 647 B. J. Tindall and M. D. Collins FEMS Microbiol. Lett.1986,37 117. 648 M. D. Collins 0.W. Howarth and J. J. Perry FEMS Microbiol. Lett. 1986 34 167. 649 S. Thurl I. Buhrow and W. Schaefer Biol. Chem. Hoppe-Seyler 1985 366 1079. 650 M. D. Collins and B. J. Tindall FEMS Microbiol. Lett. 1987,43 307. 651 M. D. Collins and P. N. Green Biochem. Biophys. Res. Commun. 1985 133 1125. 652 M. D. Collins 0.W. Howarth and P. N. Green Arch. Micro- biol. 1986 146 263. 653 S. Thurl W. Witke I. Buhrow and W. Schaefer Biol. Chem. Hoppe-Seyler 1986 367 191. 654 V. Lanzotti A. Trincone A. Gambacorta M. de Rosa and E. Breitmaier Eur. J. Biochem. 1986 160 37. NATURAL PRODUCT REPORTS 1989 655 M. Mohri H. Kinoshita K. Inomata H. Kotake H. Takagaki and K. Yamazaki Chem. Lett. 1986 1177. 656 E.Keinan and D. Eren J. Org. Chem. 1986 51 3165. 657 E. Keinan and D. Eren J. Org. Chem. 1987 52 3872. 658 E. A. Obol’nikova I. V. Kozlova T. H. Filippova and G. I. Samokhvaiov Zh. Org. Khim. 1986 22 2258. 659 H. D. Campbell B. L. Rogers and I. G. Young Biochemistry 1986 25 172. 660 K. Ishiwata Y. Miura T. Takahashi K. Kawashima K. Yanai M. Monma and T. Ido Eur. J. Nucl. Med. 1985 11 162. 661 L.-Q. Gu B.-L. Liu and J.-L. Zhang Youji Huaxue 1986 463. 662 J.-H. Guo R.-S. Yu and D.-Q. Gu Yiyao Gongye 1986,17,468. 663 N. A. Sokolova V. N. Byzova I. K. Sarycheva and R. P. Evstig- neeva Khim.-Farm. Zh. 1985 19 995. 664 M. Tori M. Sono and Y. Asakawa Bull. Chem. SOC. Jpn. 1985 58 2669. 665 D. B. Hendrick and D. C. White J. Microbiol. Methods 1986 5 243.666 M. Ts. Yanotovskii M. P. Mogilevskaya E. A. Obol’nikova L. M. Kogan and G. I. Samokhvalov Zh. Anal. Khim. 1986,41 152. 667 J. K. Lang and L. Packer J. Chromatogr. 1987 385 109. 668 J. K. Lang K. Gohil and L. Packer Anal. Biochem. 1986 157 106. 669 Y. Isogai M. Nishimura and S. Okayama Plant Cell Physiol. 1987 28 1301. 670 T. Sakano T. Nagaoka A. Morimoto and K. Hirauchi Chem. Pharm. Bull. 1986 34 4322. 671 W. E. Lambert A. P. de Leenheer and E. J. Baert Anal. Bio- chem. 1986 158 257. 672 A. Shirahata and T. Nakamura Ketsueki To Myakkan 1985 16 395. 673 M. Lucock R. Hartley and N. J. Wild J. Liq. Chromatogr. 1987 10 191. 674 P. M. M. van Haard R. Engel and A. L. J. M. Pietersma-de Bruyn Clin. Chim. Acta 1986 157 221.675 K. Hirauchi T. Sakano and A. Morimoto Chem. Pharm. Bull. 1986 34,845. 676 Y. Haroon D. S. Bacon and J. A. Sadowski Clin. Chem. 1986 32 1925. 677 W. E. Lambert A. P. de Leenheer and M. F. Lefevere J. Chro-matogr. Sci. 1986 24 76. 678 Y. Haroon D. S. Bacon and J. A. Sadowski J. Chromatogr. 1987 384,383. 679 S. E. Hamilton I. L. Ross and B. Zerner Anal. Lett. 1986 19 177. 680 S. Cholerton and B. K. Park J. Chromatogr. 1986 375 147. 681 M. J. Fasco A. C. Wilson R. G. Briggs and J. F. Gierthy Arch. Biochem. Biophys. 1987 252 501. 682 B. S. Braun U. Benbow P. Lloyd-Williams J. M. Bruce and P. L. Dutton Methods Enzymol. 1986 125 119. 683 J. R. Lloyd and M. L. Cotter Biomed. Environ. Mass Spectrom. 1986 13 447. 684 J.-Y. Cui and G.-Z.Wang Nanjing Yaoxueyuan Xuebao 1985,16 60. 685 F. J. Aranda and J. C. Gomez-Fernandez Biochem. Int. 1986,12 137. 686 R. Fato M. Battino M. Degli Esposti G. Parenti Castelli and G. Lenaz Biochemistry 1986 25 3378. 687 B. A. Cornell M. A. Keniry A. Post R. N. Robertson L. E. Weir and P. W. Westerman Biochemistry 1987 26 7702. 688 M. Ondarroa and P. J. Quinn Eur. J. Biochem. 1986 155 353. 689 L. Michaelis and M. J. Moore Biochim. Biophys. Acta 1985,821 121. 690 A. Post R. G. Hiller and B. A. Cornell Photosynth. Res. Proc. Int. Congr. Photosynth. 7th 1986 2 509. 691 F. J. Aranda J. Villalain and J. C. Gomez-Fernandez Biochim. Biophys. Acta 1986 861 25. 692 F. J. Aranda and J. C. Gomez-Fernandez Biochim. Biophys. Acta 1985 820 19. 693 A.Ortiz J. Villalain and J. C. Gomez-Fernandez Biochim. Bio- phys. Acta 1986 863 185. 694 M. Battino T. Fahmy and G. Lenaz Biochim. Biophys. Acta 1986 851 377. 695 L. Landi L. Cabrini B. Todolini A. M. Sechi and P. Pasquali Ital. J. Biochem. 1985 34 356. 696 M. Blackwell and K. Gounaris Biochem. SOC.Trans. 1986 14 51. 697 T. Erabi K. Nishimura and M. Tanaka Tottori Daigaku Kogak- ubu Kenkyu Hokoku 1985 16,48. 698 M. T. Craw M. C. Depew and J. K. S. Wan J. Magn. Reson. 1985 65 339. 699 E. Leistner Recent Adv. Phytochem. 1986 20 243. 700 R. Bentley and R. Meganathan in ‘Escherichia coli Salmonella typhimurium ’ ed. F. C. Neidhardt American Society for Micro-biology Washington D.C. 1987 Vol. 1 p. 512. 701 R. Kolkmann and E. Leistner Z.Naturforsch. Sect. C 1987 42 542. 702 M. G. Marley R. Meganathan and R. Bentley Biochemistry 1986 25 1304. 703 E. 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ISSN:0265-0568    

DOI:10.1039/NP9890600359

出版商:RSC    

年代:1989

数据来源: RSC

摘要:

Steroids Physical Methods D. N. Kirk Department of Chemistry Queen Mary College Mile End Road London El 4NS Reviewing the literature mainly between mid 1985 and the end of 1987 (Continuing the coverage of literature in Natural Product Reports 1986 Vol. 3 p. 505) 1 Introduction 2 Structure Conformation and Receptor Binding 2.1 Receptor Binding of Steroids 3 N.M.R. Spectroscopy 3.1 'H Spectra and 'H-13C Correlated Spectra 3.2 'H-Labelled Compounds 3.3 13C Spectra 4 Other Spectroscopic Methods 5 Mass Spectrometry and Gas Chromatography-Mass Spectrometry 6 High-Performance Liquid Chromatography and Liquid Chromatography-Mass Spectrometry 7 Immunoassay and Miscellaneous Physical Methods 8 References 1 Introduction The arrangement of this Report is similar to that of the previous one,' but with more attention being given to papers concerned with the interactions of steroids with their biological receptors which is an area of rapidly increasing activity.The section dealing with n.m.r. spectroscopy emphasizes the applications of two-dimensional methods to steroids these have permitted full assignments of 'H spectra to become almost routine. Probably the other most important development has been the maturing and wide application of h.p.l.c.-(thermo- spray)-m.s. an analytical technique for the separation and study of highly polar steroids and their conjugates without the need for derivatization. A new book2 dedicated to the bile acids reviews the applications of all of the main physical methods of analysis to this important group of compounds and an~ther,~ on sterols and bile acids includes a chapter on those physico-chemical properties of the bile acids (solubility surface properties formation of micelles etc.) which are of most importance in the biological context.2 Structure Conformation and Receptor Binding It is now recognized that neither the biologically active molecular conformation nor the conformation of a molecule in a crystal need correspond exactly with the energy minimum of an isolated steroid molecule. For this reason a systematic comparison of the calculated ('molecular mechanics ' MM) and actual crystal conformations of a wide variety of steroids has been ~ndertaken.~ Disparities may partially reflect the use of inaccurate torsional parameters in the calculations but still leave room for real deviations of crystal conformations from the calculated energy minima of isolated molecules.The recent examples which follow include several in which crystal-packing forces clearly influence molecular conformations although none is as clear-cut as the difference between the conformations in solution and in the solid state of 17a-acetoxy-6a-methyl- pregn-4-ene-3,20-dione as reported a few years The geometry of progesterone calculated by the semi-empirical all-valence-electron molecular-orbital (MNDO) method corresponds very closely to the results that have been obtained by X-ray-crystallographic analysis. The only deviation that is considered possibly to be significant is in the preferred conformation of the pregnan-20-one side-chain ; the authors6 point out that they compute a gas-phase structure in which no allowance is made for any solid-state effects which may operate in the crystal.Calculated (by MM) conformations of some progesterones that are substituted at C- 16 C- 17 and/or C-2 1 agree with crystallographic data ;'molecular mechanics has also been used to predict the conformation of the 17P-acetyl side-chain in a 16a 17a-cyclohexano-steroid.* Steric energies and conformations of rings B and c have been calculated for a series of 14-methyloestra- 1,3,5( 10)-trien- 17- ones with the 8P,9a 14a- 8p,9P l4a- 801,9a 14a- and 8P,9a 14P-configurations as well as for some 1 1-oxygenated derivatives.The structures of two such compounds have been confirmed by X-ray cry~tallography.~ The butyl acetate solvate of 1 1~-[4-(dimethylamino)phenyl]-17P-hydroxy- 17a-(prop- 1-ynyl)oestra-4,9-dien-3-one (l) which is a steroid with powerful antiprogestational and antigluco- corticoid properties was found to have a rather flat steroid skeleton with the phenyl group perpendicular to the skeleton after successful co-crystallization of what had previously been an intractable compound with butyl acetate." Crystal structures for a series of esters of llp,17a,21-trihydroxypregn-4-ene-3,20-dione(cortisol ; hydrocortisone) and its 9a-fluoro-derivative show two distinct conformations of ring A corresponding respectively to hexagonal (la,2P-half-chair) and orthorhombic (inverted conformation) crystal forms." The hexagonal crystals bind solvent strongly and are readily oxidized in air to give the 1 1-oxo-derivatives whereas the orthorhombic forms are stable in air.The implications of these observations for the storage of these pharmaceutical products are obvious. Tetragonal unsolvated crystalline 901- fluorocortisol acetate has a disordered crystal structure with ring A in both the normal and the inverted conformation whereas the propanol solvate has ring A wholly in the normal la,2P-half-chair conformation.'* The difference in free energy between the two conformers must be relatively small. The iodo-substituent in 16a-iodo- 17P-oestradiol has little effect on its molecular geometry consistent with the high affinity of this steroid for the oestrogen re~eptor.'~ The 1401- 393 NATURAL PRODUCT REPORTS 1989 CH~OH (5) ? I N7 hydroxymethyloestrone derivative (3) which exists in solution as an equimolar mixture with its hemiacetal (2) crystallizes wholly as the hemiacetal which is stabilized by intermolecular hydrogen-bonding.l4 19-Nor- 17a-pregna- 1,3,5( 10)-trien-20- yne-3,17P-dioi (1 7a-ethynyloestradioI) as its hemihydrate has a crystal structure comprising bilayers of hydrogen-bonded steroid and water molecules similar to that of oestradiol hemihydrate but with staggered instead of stacked bi1a~ers.l~ 13-Ethyl- 17P-hydroxy- 1 1 -methylene- 18,19-dinor- 17a-pregn-4- en-20-yn-3-one (4) has ring A statistically disordered (1 :l) showing a normal 1 a,ZP-half-chair and an inverted lp,2a-half- chair.16 The former is calculated to be marginally the more stable conformer.The related 15-ene (9,lacking the 11-methylene group has two crystalline forms; in one of these the ethyl group at C- 13 has its normal preferred conformation anti to the C/D ring junction but in the other the ethyl group is twisted to lie above ring D." The stereochemistry of compounds [e.g.(6)] in which a steroid hormone is linked to naloxone via an azine has been examined by 13C n.m.r. and confirmed by X-ray crystallography. l9 These complexes are &selective opiate antagonists. The configuration around each end of the azine is specifically (E)[anti].The photoaddition of acetophenone and p-fluoroaceto- phenone as guests in deoxycholic acid complexes at -170 "C comes close to the maximum that is permitted by the crystal packings.2o Molecular-mechanics calculations have been used to predict the relative steric energies and preferred conforma- tions for a series of stereoisomeric hexahydrobenzo[4,5,6]-cholestanes.21 2.1 Receptor Binding of Steroids Some forty years after conformational analysis was introduced into steroid chemistry research emphasis has largely shifted from intramolecular manifestations of conformation affecting physical properties and chemical reactivity to an intensive study of the interactions between hormonal steroids and their biological receptors and other biological molecules.With the aid of conformational trends determined by X-ray crystal- lography and even more recently by the computer modelling of molecules and their 'fit' with receptors considerable progress is being made in the mapping of receptors for steroid hormones even though much remains to be done before the precise nature of the binding to the receptor and the three-dimensional structure of the binding site itself can be defined. Recent work,22 particularly on the androgen-and progesterone-receptor binding capabilities of a series of hormonally active steroids has shown that the subtle conformational changes and variations in skeletal flexibility which result from differing patterns of unsaturation and substitution in the steroid can result in a gradual shift from essentially androgenic to essentially progestational receptor binding.It seems probable that the receptors have much in common structurally differing slightly but significantly in the ligand-binding capabilities of their active sites. Present views on the structural basis of interaction between steroids and their receptors based on X-ray studies have been summarized with particular reference to synthetic hormone antagonist^.^^ Variations in binding to oestrogen receptors in response to substitution of the steroid at C-17 have been reported for both the 8P-and 8a-oestradiol series.24 17a- Methyl 17a-viny1 or 17a-ethynyl groups do not interfere significantly with binding unlike 17a-ethyl (see below). Reversal of the configuration of C-17 (to 17a-OH 17P-R) reduces the binding affinity of an oestradiol ;compounds of the 8a-series are always less effective than their natural 8P-isomers.The extent of steric shielding of the 17P-hydroxyl group of synthetic steroid oestrogens by substituents at the 17a-position correlates with their variety of abilities to elicit the oestrogenic response.25 Studies on the 0-H stretching band in their infrared spectra and of the OH signal in the lH n.m.r. spectrum in [2H6]dimethyl sulphoxide lead to the conclusion that a 17a- ethyl group in particular inhibits hydrogen-bonding inter- actions between the steroid and its receptor by blocking approach to lone-pairs of electrons on oxygen whereas 17a- ethynyl groups at the other extreme leave hydrogen- bonding unimpaired and allow high biological Molecular-mechanics calculations concerning conformations of the 17- ethyl side-chain have confirmed this conclusion.26 The abilities of the oestrogen androgen and glucocorticoid receptors to bind simpler (non-steroidal) phenols now assessed for a wide variety of compounds confirm the strong preference of the oestrogen receptor for meta-substituted phenols which are the closest in structure to the natural ~estrogens.~~ The binding requirements of the aromatase enzyme complex which is responsible for a key step in the conversion of androst- 4-ene-3,17-dione into oestrogens have been explored by means of a series of potential steroid substrates with differing patterns of substitution. Optical-difference spectra representing the change in absorbance when the steroid and enzyme interact were strongest for those steroids that are best able to inhibit the activity of the enzyme complex.Inhibitory steroids include various 6-substituted derivatives suggesting that the substituent at this position does not interfere appreciably with binding to the enzyme in contrast to substitution at C-2 C-1 I C-16 or C- 19," Molecular-mechanics calculations have been used in a new attempt to assess the energetics and conformational preferences of steroidal 4-en-3-ones that contain and do not contain the methyl group C-19.29 The 4-en-3-one is essential if a steroid is to bind to the progesterone receptor. Previous studies had indicated that binding forces depend critically on the ability of NATURAL PRODUCT REPORTS 1989-D.N. KIRK (7)R=H (8) R = Me; 11,12-didehydro ring A to adopt the abnormal Ip,2a-half-chair conformation. The new results however indicate that a 19-nor-4,9-dien-3-0ne has somewhat lower affinity for the receptor than has a 19-nor- 4-en-3-one whereas the 4,9-dienone is better able to adopt the inverted conformation. The conformation of the steroid in the steroid-receptor complex is as yet unknown. The topic is clearly still open to study. A curious correlation has been noted between 13C chemical shifts (for C-17) and the abilities of various derivatives and analogues of 17a-ethynyl- 17P-hydroxyoestr-4-en-3-0ne(' nor-ethisterone') to bind to the human endometrial proges- terone receptor.30 The receptor-binding and hormonal ac-tivities of 9P l0a-steroids have been considered in the context of steric requirements of the receptor^.^' Some des-,+steroids are bound surprisingly well by the androgen receptor.32 Compounds of the aldosterone and corticosterone type which bind to the mineralocorticoid receptor do so with increasing affinity the flatter the New X-ray analyses have added 21-hydroxy-19-norpregn-4-ene-3,20-dione (19-nor-1 1-deoxycorticosterone) (7) and 2 1 -hydroxypregna-4,11 -diene- 3,20-dione (8) to the list of those whose structures are known in detail.The 19-nor-compound (7) has the flattest structure of those studied and bonds most strongly to the receptor exceeding the affinity (though not the biological activity) of the main natural mineralocorticoid aldosterone.Electronic effects in this series although detectable by small variations in the 13C chemical shift (especially of C-5) appear to be of secondary importance in affecting affinity for the receptor.33 The long-held view that mineralocorticoid and glucocorticoid hormones are differentiated by oxygenation at C- 18 and C- 17 respectively has been ~hallenged.~~ New clinical evidence indicates that the main natural glucocorticoid cortisol (see above) is strongly bound and active at rthe mineralocorticoid receptor but is normally prevented from reaching it by 1 lp-hydroxysteroid dehydrogenase which converts cortisol into inactive cortisone. The mineralocorticoid receptor is clearly much less specific in its requirements than was previously believed.The spin-labelled spiro-doxy1 androstane derivative (9) binds at the substrate-binding site of crystalline A5-3-keto-steroid isomerase [steroid A5-isomerase; E.C. 5.3.3. l].35 The location of the steroid has been found (by X-ray crystallography and n.m.r. spectroscopy) to be in a hydrophobic cavity which is accessible from the external environment and probably corresponds closely to the docking position of androst-5-ene- 3,17-dione which is the natural substrate for the enzyme. Computer modelling of the receptor site for cardiac steroids F of Na+/K+-transporting ATPase aided by experimental data from X-ray crystallography site-specific labelling and bio- logical studies suggests that cardiac glycosides are bound mainly by interactions involving the carbonyl group in the steroid side-chain the sugar molecule that is attached directly to the steroid and possibly also any 16P-ester group.3s As with receptors the ability of an antibody to recognize and bind a steroid is thought to be enhanced if the steroid molecule has sufficient conformational mobility to allow it to adapt easily to the requirements of the antibody.Unsaturation in steroid rings by generally lowering the difference in free energy between conformers appears to increase the likelihood of cross-reactivity with antibodies3' Other X-ray analyses of steroids which have come to the Reporter's attention are collected in Table 1. The list is not necessarily exhaustive because a few X-ray analyses that are reported incidentally in papers concerned mainly with synthesis may have been overlooked.3 N.M.R. Spectroscopy 3.1 'H Spectra and 'H-13C Correlated Spectra Considerable progress has been made since the last Report' on complete assignments of the high-field 'H n.m.r. spectra of steroids. Two-dimensional (2D) techniques (known by their acronyms!) are now almost routinely applied to assist the interpretation of high-field 'H spectra. The 2D methods that have so far been used for steroids are usefully reviewed,94 with emphasis on problem solving and on those methods which can be informative with samples at or below the milligram level. If the size of a sample is not a problem a full and reliable I3C analysis can be carried out by the 13C-13C connectivity (INADEQUATE) method.The assignment of 13C spectra forms the basis for a 'H-13C 2D heteronuclear correlated (HETCOR) spectrum which provides chemical shifts for the protons that are attached to each carbon atom. When sample size is insufficient for these procedures 2D lH homonuclear shift-correlated (COSY) spectra often provide enough in- formation to enable a partial if not complete analysis of the 'H spectrum. Details including configurational assignments can be filled in by either 1D [nuclear Overhauser effect (n.0.e) or decoupling difference] or 2D (NOESY) methods. Full 'H n.m.r. (400 MHz) analyses for sixteen 5a-andro- stanes including the parent hydrocarbon and a series of derivatives that are substituted mainly at C-3 and C- 17 as well as for methyl 6a-fluoro- 1 lp-hydroxy- 16a-methyl-3-oxoan- drost-4-ene- 17P-carboxylate (10) made use of the 2D COSY and HETCOR methods complemented by n.0.e.difference spectra where necessary.95 Spectral simulation by computer was valuable for refinement of shift and spin-coupling data when signals overlapped. Spin couplings supported by molecu- lar-mechanics calculations revealed that there are conforma- tional variations especially in ring D. Substituent increments in 'H chemical shifts due to carbonyl hydroxyl and halogeno- groups were evaluated and have been interpreted in terms of a combination of anisotropy and electric-field effects.95 Accurate experimentally determined 'H-lH coupling con- stants for the protons at C-1 and C-2 in a series of steroidal 4-en-3-onesg6 and some 2-methyl-4-en-3-onesg7 have been used to establish the preferred conformations of ring A in solution.396 Table 1 Steroids that have been studied by X-ray crystallography. (Compounds with references 9-37 are mentioned also in the text). Compound Ref. Oestranes 17a-Azidomethyl- 1 7P-hydroxyoestr-4-en-3-one 38 17a-Chloromethyl-17/3-hydroxyoestr-4-en-3-one 39 17a-Cyanomethyl-17P-hydroxyoes tr-4-en-3-one 40 I OP-Hydroxyoestra- 1,4-diene-3,17-dione 41 2,CDibromo- lop 17/3-dihydroxyoestra- 1,4-dien-3-one 42 17a-Azidomethyl-17/3-hydroxyoestra-4,9-dien-3-one 43 11/3-[4-(dimethylamino)phenyl]-17P-hydroxy- 17a-(prop- 1-10 ynyl)oestra4,9-dien-3-one 11/3-[4-(dimethylamino)phenyl]-17P-hydroxy- 17a-(prop-2- 44 eny1)oest ra-4,9-dien-3-one Oestra- 1,3,5( 10)-triene 45 16a-Iodo-oestra- 1,3,5( lO)-triene-3,17P-diol 13 (+)-and (-)-3-Methoxy- 18-methyl-8a-oestra- 1,3,5( 10)-trien- 45 17-one 14-Hydroxy-3-methoxy-18-methyl-l4~-oestra-l,3,5(10)-trien-46 17-one (-t)-14-Hydroxy-3-methoxy-18-methyl-8a,9/3,14P-oestra-45 1,3,5( lO)-trien- 17-one 3-Methoxy- 14-hydroxymethyloestra- 1,3,5( 10)-trien- 17-one 14 hemiacetal (+)-3-Methoxy-14-methyl-9P-oestra-1,3,5( IO)-trien-I7/3-01 9 (&)-3-Methoxy- 14-methyloestra- 1,3,5( 10)-triene-1 la,l7/?-diol 9 16a-(l-Acetoxyethyl)-3,15a-dimethoxy-l7~-phenyl-l4,17a-47 etheno-oestra- 1,3,5( 10)-triene (+)-15a-Methyl-8-aza- 16-oxa- 13a-estra- 1,3,5( lO)-trien-17-one 48 Oestrone naloxone azine 19 17a-Ethynyl- 17P-hydroxyoestrane derivatives see 19-nor- 17a-pregn-20-ynes (below) Androstanes 4-Chloromercurioandrosta-4,6-diene-3,17-dione (acetone 49 solvate) 5,17~-Dihydroxy-~-nor-5P-androstan-3-one 17-acetate 5-(R)-50 methanesulphinate (f)-14P-Hydroxy-l~,4,8-methano-5/3,8a,9#l-androstane-7,17-51 dione 3P-Acetoxy- 17,2O-epoxy- 17-picolylandrost-5-en-7-one (1 7,20- 52 isomers) 4a-Carbomethoxy- 1 5a-cyano-4P-methyl- 5a,I 3a-androstane 53 17P-Hydroxy-4-aza-5/3-androst-1-en-3-one 54 17&Acetoxy-3-aza-~- homoandrost-4a-en-4-one 54 Methyl (4’S 16$)-3P-acetoxy- 17-oxospiro[androst-5-ene-16,3’-55 (4,5-dihydropyrazole)]-4’-carboxylate 17a-Ethynyl- 17P-hydroxyandrostane derivatives see 17a- pregn-20-ynes (below) Pregnanes 1l&Hydroxymethyl-5a-pregnane-3~,20~-diol 3,20-diacetate 56 (20R)- 19-Nor-5P 14P-pregnane- 3#?,14,20- trio1 3,20-diacetate 57 2 1-Hydroxy-19-norpregn-4-ene-3,2O-dione [19-33 nordeoxycorticosterone] 21-Hydroxypregna-4,l l-diene-3,20-dione 33 17a,21-Dihydroxypregn-4-ene-3,11,20-trione 2 I-acetate 58 [cortisone acetate (modification IVac)] Cortisone acetate (modification Vaq) 59 Esters of 1lp,l7a,21-trihydroxypregn-4-ene-3,20-dione [cortisol ; hydrocortisone] 21 -butyrate (hexagonal and orthorhombic forms) 11 21 -propionate (orthorhombic) 11 21-pentanoate (hexagonal) 11 21 -decanoate (hexagonal) 11 21-(3-~yclopentylpropionate)(hexagonal) 11 9a-Fluorocortisol 2 1 -butyrate (hexagonal) 11 9a-Fluorocortisol 2 1 -pentanoate (hexagonal) 11 9a-Fluorocortisol 2 1 -acetate 12 6a,9a-Difluoro- 1 1/3,16a 174 7a,2 l-tetrahydroxypregna- 1,4- 60 diene-3,20-dione 21 -acetate 16,17-acetonide [‘fluocinonide’] 20P-Hydroxy- 164 17a-cyclohexanopregn-4-en-3-one 61 1 1 -Methylene- 19-nor- 17a-pregn-4-en-20-yn- 17P-01 62 13-Ethyl- 17P-hydroxy- 1 l-methylene- 18,19-dinor- 17a-pregn-4- 16 en-20-yn-3-one [‘3-ketodesogestrel’] 13-Ethyl- 17P-hydroxy- 18,19-dinor- 17a-pregna-4,15-dien-20-17 yn-3-one [‘gestogene ’1 NATURAL PRODUCT REPORTS 1989 19-Nor- 17a-pregna- 1,3,5( 10)-trien-20-yne-3,17,8-diol 15 [‘ethynyloestradiol ’I 13-Ethyl- 18,19-dinor- 17a-pregn-4-en-20-yn- 17p-01 63 1la-Chloro- 13-ethyl- 18,19-dinor- 17a-pregn-4-en-20-yn- 17p-01 64 (methanol solvate) 11~-(4-Fluorophenyl)-17P-hydroxy- 19-nor- I7a-pregna-4,9- 65 dien-20-yn-3-one 11/3-(4-Fluorophenyl)- 17a-hydroxy- 19-nor- 13a-pregna-4,9- 65 dien-20-yn-3-one 1,11~,17,21-Tetrahydroxy-4-methy1-19-norpregna-1,3,5(10) 66 8(14)-tetraen-20-one 1.2 1-diacetate Sterols and miscellaneous Cholesteryl formate 67 Cholesteryl cis-dec-9-enoate 68 Cholesteryl trans-dec-9-enoate 68 Cholesteryl cis-octadec-9-enoate [oleate] 69 Cholesterokholesteryl oleate binary mixtures 70 Cholesteryl perfluoropropionate 71 Cholesteryl p-hexyloxybenzoate 72 (25S)-Cholest-5-ene-3,8-26-diol 73 5a-Cholestane-3P,6/3-diol 3-acetate 6-[3-(4- 74 iodopheny1)phenyllacetate (23R,24R)-4a,23,24-Trimethyl-5a-cholestan-3,!-01 75 3~-Acetoxy-7a-aza-~-homocholest- 5-eno[7a,7-djtetrazole 76 (1S,lo$)- and (1 R,10R)-1,5 ;5,10-diepoxy-5 I0-secocholestan- 77 3P-yl acetates 17,25-Epoxy-20-oxo-2 1,22,23,27- tetranor- 16,25-cyclocholest- 78 5-en-3P-yl acetate Calciol (vitamin D3) 5,6;7,8-diepoxide 79 Calciol (vitamin D3) 5,6;7,8;10,19-triepoxide 80 3a,7/3-Dihydroxy-5/3-cholan-24-oic acid [ursodeoxycholic 81 acid] Sodium taurodeoxycholate (monohydrate) 82 (25R)-Spirost- 5-ene-3,8,17a-diol [pennogenin ;hemihydrate] 83 Spirosta-5,25(27)-diene-1/3,3/3,11 a-trio1 (monohydrate) 84 (25R)-Spirost-5-ene-3& 12P 15a-trio1 [bahamagenin] 85 22,23-Dibromo-1O-methyl-19-noranthraergosta-5,7,9,14-86 tetraene 2/3,3a,22,23-Tetrabromo- 18-nor- 17-iso-5a-ergosta-8,11,13-86 triene 19-Nordigitoxigenin 87 14-Hydroxy-3~-(a-~-rhamnosyloxy)- 14P-bufa-4,20,22-88 trienolide [proscillaridin] 3P 1 1 a,14-Trihydroxy-SP,14P-bufa-20,22-dienolide 89 [gamabufotalin] 3/3,11a,14-Trihydroxy- 12-oxo-5/? 14P-bufa-20,22-dienolide 89 [arenobufagin] Deoxycholic acid-ethyl acetate (2 :1 complex) 90 Deoxycholic acid-ferrocene (2 :1 complex) 91 Deoxycholic acid-phenylacetylene (2 1 complex) 92 Deoxycholic acid-thiocamphenilone 93 Only one of the compounds that were investigated 2p,17p- diacetoxyandrost-4-en-3-one, has the inverted lp,2a-half-chair conformation as reported previously.The authors caution against equating signal splittings measured directly from the spectrum with coupling constants for an ABX or a more complicated spin system.Such a simple first-order analysis is likely to be unreliable even at high fields. We are advised,96 when reporting such data to state clearly that they are only estimates of ‘apparent’ J values. In the work under review computer simulation of spectra was used to refine approximate chemical shifts of the four coupled protons at C-1 and C-2 and to refine the initial estimates of J values obtained from 2D COSY spectra. Iterative analyses gave chemical shifts which are quoted to four decimal places and values of 2J and 3J to two decimal places. A two-dimensional 13C-13C connectivity experiment (IN-ADEQUATE) at natural abundance has provided all 13C n.m.r.assignments for each of the two principal tautomers (1 1 a) and (1 1 b) of aldosterone in the equilibrating mixture in solution. The results were used via two-dimensional lH-13C heteronuclear spectroscopy together with phase-sensitive double-quantum-filtered COSY 2D J-resolved lH spectra and NATURAL PRODUCT REPORTS 1989-D. N. KIRK a few selective n.0.e. measurements to determine all lH chemical shifts and most of the 'H-'H coupling constants for each taut~mer.~~ Two-dimensional 'H n.m.r. has been used to confirm the structures of the y-lactones (12) and (13) which were obtained by oxidation of 19-noraldosterone and 1lp 18,2 1-trihydroxy-19-norpregn-4-ene-3,20-dione,re-spectively with peri~date.~~ The known I3C chemical shifts for 5/3-cholan-24-oic acid and its common hydroxylated derivatives [i.e.3a-OH lithocholic acid ; 3a,7a-(OH), chenodeoxycholic acid ;3a,7P-(OH), urso- deoxycholic acid ; 3a 12a-(OH), deoxycholic acid ; and 3~~,7a,12a-(OH)~, cholic acid] have been used via two-dimensional 'H-I3C heteronuclear correlated spectroscopy to assign all of the 'H signals for these compounds.100 This is the first major series of 5P-steroids to be subjected to full 'H analyses providing valuable reference data for others of the 5p class. The increments in 'H chemical shift for individual hydroxyl substituents show good additivity within this series. Fully assigned 'H and 13C spectra of sodium cholate and sodium deoxycholate have been used to probe the effects of concentration on the association of these bile salts in solution.Two details of an earlier 'H assignment (for 6a-H and 8P-H) have been reversed.'O' The rotations of the methyl groups in micelles of sodium deoxycholate have been studied by observing the intensities of forbidden peaks in double-quantum- filtered one-dimensional spectra as a function of excitation time in combination with Tl and T datasets.lo2 The binding of Ca2+ and of Na+ by glycocholate and taurocholate ions has been studied by measuring the 'H shifts that are induced by the lanthanide ion Dy3+ as a paramagnetic isomorphous replacement for Ca2+ alone and in competition with added Ca2+ or Na+. Derived dissociation constants for the metal-bile salt complexes show that Ca2+ is more strongly bound by the carboxylate group of glycocholate than by the sulphonate group of taurocholate.In all cases the ionic terminus of the side-chain provides the main binding site for the metal The Dy3+ ion has also been used in estimating the metal- ion-induced relaxation rates of protons in glycocholate ions and thence to construct a model of the glycocholate-Dy3+ complex in submicellar concentrations in aqueous solution. The metal ion appears to associate strongly with one carboxylate oxygen atom of a glycine residue and at longer 0v F range with the peptide carbonyl oxygen and the other carboxylate oxygen but not with skeletal hydroxyl groups. lo4 Proton chemical shifts (only for protons at C-18 C-19 and C-21 and geminal to hydroxyl groups at C-3 C-7 and/or C-12) have been reported for all of the isomeric methyl 501-cholanoates that contain from one to three hydroxyl groups or from one to three 0x0-groups at positions 3 7 and 12.Additivity of substituent effects is again A complete assignment of the 'H and 13C n.m.r. spectra of 1701-ethynyl-3-methoxyoestra- 1,3,5( 10)-trien- 17p-01 (' mestran-01') made use of both homonuclear and heteronuclear two- dimensional methods including spin-echo J-correlated spectroscopy (SECSY). Spectral differences were then used to aid the structural elucidation of photo-oxidation products of mestranol modified in rings B and c.lo6Proton assignments confirmed by computer simulation have also been reported for the 3-methyl ethers of oestrone oestradiol and their 14-methyl derivative^.'^' Full assignments ('H) obtained by the COSY method have been reported for the isomeric phototrans- formation products (14) and (15) which were obtained from 17P-hydroxy-1$nor-17a-pregn-4-en-20-yn-3-one (norethister-one).lo8 High-field n.m.r.spectroscopy leading to full lH and 13Cassignments of the n.m.r. spectra of 17P-hydroxy- 19-nor- 5~,17a-pregn-2O-yn-3-one and its 5p-isomer has provided vicinal coupling constants for conformational analysis of these compounds. The conformations of the molecules in solution that were determined in this way agree well with the conformations that have been derived by X-ray-crystallographic and molecular-mechanics methods.log COSY and n.0.e. dif- ference spectra aided by computer simulation were employed in a full analysis (lH 13C and I9F) of the spectra of 6a,9- difluoro-1lP 1601,17,21-tetrahydroxypregna-1,4-diene-3,20-dione 2 1-acetate 16,17-acetonide ('fluocinonide') (1 6) which included the measurement of long-range 'H-I9F coupling constants (e.g.7a-H,6-F = 13 Hz; 7P-H,6-F = 2.5 Hz and 12a-H,9-F= 3.1 Hz).~" COSY spectra under conditions that were chosen to emphasize long-range 'H-lH couplings simplify the assignment of signals in oestrogens. The 4J and 6Jcouplings from protons at C-6 and C-9 to protons of the aromatic ring provide entry points for analysis of the COSY cross-peaks relating to protons in rings B and c with the advantage that the method is NATURAL PRODUCT REPORTS 1989 H applicable to quite small samples.llf The diastereoisomers (17) and (1 8) in which a tricarbonylchromium moiety is attached to the a-or the /?-face of the aromatic ring of an oestradiol derivative are distinguished by their high-field n.m.r.spectra (500 MHz for 'H; 125 MHz for 13C). Full assignments achieved by the two-dimensional COSY and SECSY techniques as well as 'H-13C heteronuclear-shift-correlated spectra show that there is strong deshielding of neighbouring protons on the same face as the tricarbonylchromium. The 13C spectra have been tabulated for oestradiol and fifteen of its derivatives in this series.l" The 500 MHz 'H n.m.r. spectra for the cluster complexes formed from 17a-propynyloestra- 1,3,5( 10)-triene- 3,17/?-diol and Co,(CO) or (~5-C5H,)2Mo,(CO)4 have been fully assigned by the 2D COSY method.Anisotropic effects of the propynyl group cause very large down-field shifts of signals from neighbouring protons especially those at the 1201- and 14a-positions.'13 Fourier-transform 'H n.m.r. at 300 MHz has been proved to be capable of giving a useful spectrum with as little as 5 pg of compounds of the vitamin D series by using standard n.m.r. tubes of 5 mm internal diameter fitted with glass inserts and isotopically enriched (99.96 %) CDC1,. Some re-assignments of signals have been reported.ll4 The equilibrium between the two conformations of ring A in solutions of compounds of the vitamin D series is sensitive to the polarity of the solvent. Polar solvents (methanol and DMSO) favour the conformer with an equatorial hydroxyl group.Inversion is too fast for the individual conformers to be observed by n.m.r. so the conformational preferences in a range of solvents were obtained from the variations in vicinal 'H-'H coupling constant^."^ A 'H and 13C n.m.r. study of the products of dienone-phenol rearrangement of I ,4-dien-3-ones in deuteriated acidic media showed that deuterium was incorporated only into the phenolic ring and at C-6 in proportions depending upon the reagents that were used. No deuterium was found at C-8 at C-9 or elsewhere showing that these sites are not subject to exchange during the reaction. Even C-6 is probably only involved via a reversible pre-enolization of the dienone.'16 Proton n.m.r. spectra of a series of ~-homo-Sa-androstan- 17a-one derivatives reporting chemical shifts for up to half of the ring protons in each case have been analysed to provide values of the b; R = C(0)Me b; R = C(0)Me increments in the shifts that are induced by hydroxyl groups at various positions."' Proton n.m.r.studies of solutions in sulphuric acid at various concentrations have shown that the chromogenic reaction of 17a-hydroxyprogesterone involves a rapid D-homo-annulation followed by dehydration and mi- gration of C-18 leading to coloured cationic specie^.^^*^^^^ Proton n.m.r. spectra at 500 MHz permit easy distinction between a-and P-anomers of some steroid glucuronides; the synthetic method is critical in deciding which anomer is the predominant product.120 Fully assigned 'H and 13C spectra have been reported for 3P-acetoxypregn-5-en-20-one (pregne-nolone acetate) and a series of derivatives with modified side- chains (including 20P-OH) leading to the pregn-20-ene derivative,lZ1 and 2D n.m.r.has been used to determine the structures of two oxidation products of cholesterol which include a dimeric steroid.lZ2 Proton and carbon- 13 spectroscopy including 2D experi- ments has defined the geometries of the unsaturated side- chains in a series of fluorescent analogues of cholesterol e.g. (19) that had been designed as membrane Proton and 13C spectra have been related to the configurations of the alkyl groups at C-24 in sterols from members of the Cucurbitaceae. lZ4 The 13,17-seco- 12,17-cyclo [17( 13 -+ 12) abeo] structures (21a) and (21 b) of the solvolysis products of the 12P-mesylates (20a) and (20b) have been determined by 2D n.m.r.spectroscopy.125 The lH (nearly complete) and 13C assignments have been reported for three novel bufadienolide glycosides (' orbicusides A-C ') from Cotyledon orbiculata126 and for rubellin (22) which is a novel bufadienolide glycoside.12' A combination of n.m.r. relaxation studies and MM calculations for a series of unsaturated steroids indicates that the rotational barriers of the angular methyl groups are highly sensitive to their interactions with axial hydrogens; some of these interactions are relieved by the presence of unsaturation nearby.lZ8 N.m.r. relaxation rates for protons of water have been used to probe the interaction between cholesterol and its side-chain-cleavage enzyme cytochrome P450.A qolecule of water that was initially at a distance of only ca.2.5 A from the NATURAL PRODUCT REPORTS 1989-D. N. KIRK ! (23) haem iron in the cytqchrome compatible with direct binding is displaced to ca. 4 A if cholesterol is present.lZ9 3.2 *H-LabelledCompounds Cholesteryl acetate and 3P-acetoxypregn- 5-en-20-one are among compounds that can be used to illustrate the detection of sites of deuterium labelling by means of two-dimensional lH-13C heteronuclear shift correlation with 2H decoupling. Only signals from incompletely deuteriated sites (CDH CDH or CD,H) can be observed. This method has established that catalytic reduction of cholesteryl acetate with deuterium gas exchanges the 7a-proton selectively to give the 5a,6a,7a-'H3- labelled species.13o The 2H n.m.r.spectra of specifically deuteriated cholesterol (3mZH- 7,7-,H2- and 2,2,4,4,6-2H,- labelled) and its palmitate have been used to study its organization in multilamellar dispersions of dipalmitoylglycero- phosphocholine. 131 Deuteron n.m.r. has provided evidence for the orientation of [3a-2H]cholesterol and [3p-2H]epicholesterol in dipalmitoylglycerophosphocholinelipo~omes'~~ and meas- urements of the spin-lattice relaxation of ,H were used to study the complex motions of [2,2,3,4,4,6-,H6] cholesterol in model membranes of dimyristoylglycerophosphocholine. 133 Cholest-eryl oleate selectively deuteriated in the acyl chain was the subject of ,H n.m.r.experiments to determine its orientation ordering and mobility in association with low-density lipo- protein.134 Isotopic incorporation experiments with ,H n.m.r. spectroscopy showed that the aromatic ring of the antifeedant steroid NiC-1 (23) is formed with incorporation of the angular methyl group (C-18) into ring D at the 17a-po~ition.l~~ 3.3 I3C Spectra Three pseudopolymorphic forms of testosterone (one an-hydrous and the others polymorphic monohydrates) give distinctive infrared spectra. Their 13C solid-state CP/MAS spectra also have interesting features. The a-form (anhydrous) showed two p&ks each for most of the carbon atoms corresponding to the two independent molecules which make up the asymmetric unit in the crystal.C-17 showed the largest splitting with resonance signals separated by 2.3 p.p.m. The two hydrated polymorphs each showed its own characteristic pattern of 13C chemical shifts.136 Examples of the use of 2D-INADEQUATE experiments have already been mentioned (Section 3.1) and 2D-JNAD- EQUATE experiments have also been used to determine carbon connectivities and all of the lJ(13C-13C) coupling constants for stigmasterol and androsta- 1,4-diene-3,11,17- trione. One conclusion that has been drawn from the results is that J10,19 i.e. the coupling constant to the methyl group C-19 is reduced significantly by its proximity to more unsaturation in the latter compound.137 It remains to be seen whether this effect is general.A down-field shift of the 13C signals that is seen for all carbon atoms except those that are a to carbonyl when the solvent is changed from CDCl to CDC134ioxane (1 :4) offers a simpler and more versatile alternative to or-deuterium exchange as an aid to assignment of 13C Rather few steroids of 5/3 configuration have been subjected to a full analysis of their 13C spectra (the same is true at present of high-field 'H spectra!) so the publication of full 13C assignments for a series of 23 compounds of the 5#? type is welcome. They comprise methyl 5P-cholanoates SP-pregnan- 20-ones and 5P-androstane- 17P-carboxylates ;conformational and substituent effects associated with the various side-chains are ana1y~ed.l~~ The available collections of 13C n.m.r.data are also usefully supplemented by the publication of fully analysed spectra for 247 compounds of the cardenolide and bufadienolide series. These compounds include many in which the configu- rations (SP 14P and 17a) unsaturation (Aac14) A14 and A") and substitution (e.g. 5P-OH 8/3-OH 12/3-OH 14P-OH 1501-OH 16P-OH 19-OH 19-0X0 and various epoxides) were not previously well represented among compounds for which fully assigned 13C spectra were available.140 The corresponding increments in chemical shifts will have wider application. Another group141 of workers has reported 13C spectra of thirty substituted 5P 14P-dihydroxy-steroids with particular empha- sis on the effects of 12a- and 12P-substituents. Carbon-13 n.m.r. data have also been listed for 130 steroidal sapogenins and saponin derivatives for which spectra were published up to 1983 most of the sapogenins are of the spirostan type but a few others are included.The sugar components of the saponins and their anomeric configurations can readily be distinguished by comparing 13Cdata.142 Lanthanide-induced shifts {e.g. by I\l'b(fod),]) have aided the full assignment of 13C n.m.r. spectra for a number of spirostan derivatives;14 data for sixteen (25R)- 5a-spirostanes have been tab~1ated.I~~ Other compounds for which 13C spectra have been assigned include all 26 isomeric methyl 5a-cholan-24-oates that possess one two or three hydroxyl groups at positions 3,7 and 12,145 epimeric 22,23- epoxy derivatives of over a hundred sterols and triterpene alcohols,147 a series of 3-methyl-substituted 501-cholestanes with 301- and 3P-OH -C1 -F and -0Ac (as well as discussion of carbon-halogen stretching frequencies in the infrared some bromo-derivatives of 5a-cholestan-3- one and cholest-4-en-3-0ne,l~~ 6-methylated and 3P-acetoxypregn-5-en-2O-onederivatives in which there is an additional ring fused at C-16 and C-17.15' The exact fate of hydrogen atoms originating in [2-13C ,H,]acetate in the biosynthesis of sitosterol has been studied by 13C n.m.r.152 Earlier assignments of 13C n.m.r.spectra for ecdysterone polypodiene B and pterosterone have been revised on the basis of 2D spectroscopy.153 Carbon-13 n.m.r. has been used to determine the temperature dependence of molecular motions in liquid-crystalline and isotropic liquid phases of some cholesteryl 4 Other Spectroscopic Methods Methoxymethyl-protected hydroxyl groups are characterized by three strong absorption bands in the infrared due to coupled vibrations of the H,C-O-CH,-O-C group in the region 1200-1000 Differences have been observed between the infrared spectra of some B-nor-steroids and the corresponding normal ~ter0ids.l~~ A transient triplet excited species (lifetime ca.10 ,us) has been detected from the laser flash excitation of 17P-hydroxyandrosta- 4,6-dien-3-one at 355 nm. It has been suggested that this may be the species that is involved in photocycloaddition of olefins to the dienone. 157 The fluorescence characteristics of ergosterol in hydrocarbon matrices at low temperatures15* and the fluorescence characteristics of ergosta-5,7,9(11),22-tetraen-3/?-01 and cholesta-5,7,9( 1 l)-trien-3P-ol have been Cotton effects (n+n*) for over 30 saturated and a,P-unsaturated steroidal ketones in the strongly associating solvent 1,1,1,3,3,3-hexafluoropropan-2-0l(HFIP) show differences from the spectra in heptane which roughly parallel those previously observed for 2,2,2-trifluoroethanol.Some Cotton effects are strongly enhanced some others are only weakly effected while those in a third group are reversed in sign. All show blue-shifts of the circular dichroism maximum in HFIP. The results may be partially and qualitatively rationalized in terms of differing contributions of structural features to the Cotton effect according to the strength of association with the solvent and the weaker solvation of those ketones which are subject to most steric hindrance.160 The ' 1,2-glycol' component of the side-chain of 5p-cholestane-3a,7a 12a,25,26-pentaol which is a product of impaired biosynthesis of bile acids has the (25S)-configuration.This was established from the negative sign of the Cotton effect that was induced in the circular dichroism spectrum in the presence of the lanthanide reagent [Eu(fod),] and was confirmed by comparison of the 13C chemical shifts of carbon atoms in the side-chain with those for related compounds whose structure had previously been established by X-ray crystallography. 161Measurements of rotational strength at 303 273 and 208 nm have been used to study the binding of testosterone to human serum albumin and the effects of varying pH and concentrations of urea and salt which alter the conformations of the protein.162 Circular dichroism studies have also been reported for the pentadienolide chromophore in b~fadienolides,~~~ to quantify some corticosteroids 164 and for steroidal 4-en-3-ones in drug preparations.165 Free-radical species that are generated in single crystals of cholest-4-en-3-one by irradiation with X-rays have been characterized by e.s.r. and ENDOR methods. Three distinct free radicals were recognized one being formed by hydrogen abstraction (from C-6) another by addition of hydrogen (to oxygen) and the third probably by abstraction of the methyl group C-19.166 Electron spin resonance spectroscopy of the radicals that were generated by y-irradiation of a single crystal of androst-4-ene-3,17-dione indicated that hydrogen atoms are abstracted leaving the unpaired electron in an orbital that is delocalized over C-6 C-4 and 0-3; the spectrum showed appropriate hyperfine splitting.167 y-Irradiation of crystals of testosterone monohydrate168 and of 17p-hydroxy-5a-andro-stan-3-0ne'~~ also gave radicals by loss of hydrogen atoms. An e.s.r. study of y-irradiated cholesterol and some of its derivatives confirms that the ring B allylic radical and C-25 radical are the main species formed. In the presence of oxygen the corresponding peroxy-radicals are produced ;these are the species that are believed to be implicated in the formation of stable auto-oxidation products in solid cholesterol.170 The nitroxide spin-probe 3-doxylcholestane has been incorporated into liquid crystals for e.s.r. and ENDOR study of their ordering and dynamic behaviour. 171 5 Mass Spectrometry and Gas Chromatogra phy-Mass Spectrometry An authoritative and wide-ranging review of mass spectrometry applied to steroid and peptide research discusses its use for analysis of urinary and plasma steroids including the g.c.-m.s. of derivatized steroids their characteristic fragmentations and the use of selected ion monitoring with deuteriated internal standards for q~antification."~ Gas chromatography-mass spectrometry with selected ion monitoring has been applied to the determination of androgens with 19-2H,-labelled internal A special issue of Steroids dedicated to vitamin D includes a review of methods for assay of hydroxylated metabolites in serum or plasma.Most methods so far used have depended upon saturation analysis using a binding protein which may not however be specific in its affinity. A reliable procedure based on gas chromatography-mass fragmento-graphy with deuteriated internal standards is described. Although too costly in equipment to be suitable for routine assays the g.c.-m.s. method allows accurate evaluation and NATURAL PRODUCT REPORTS 1989 parameters especially analyte concentrations. 177 FAB mass spectra of some steroidal oligoglycosides that contain two to four sugar units gave intense [M+HI' ions.Collision-activated mass spectra of these mass-selected ions yield information on the sequence of the oligoglycoside while avoiding interference from imp~rities.'~~ Negative ions that are produced by FAB from salts of bile acids show little fragmentation and hence give limited structural information. Collision-activated de-composition spectra in contrast give products of fragmen- tation remote from the charged site which can be useful in assigning detailed structures and for quantifying individual components of a mixture of bile ~a1ts.l~~ Gas chromatography-mass spectrometry with chemical ionization (by NH,) and negative-ion scanning gives good mass spectra of steryl esters allowing identification of both the sterol and the acyl components.180 The mass-spectrometric identifi- cation of saturated and unsaturated sterols has been re-viewed.lal The negative-ion mass spectra of some brassino- steroids show intense molecular ions a curious [M-4]- ion appears for such compounds if there is a vicinal diol system in the side-chain.ls2 The high-temperature (up to 350 "C) g.c.-m.s. of derivatized ecdysteroids and steryl esters has been re-viewed.la3 Other reports provide g.c.-m.s. data for trimethyl- silylated ecdysteroid~'~~ and g.c. retention data for 56 bile acids of the 5a-and the 5P-series derivatized as their methyl ester or their trimethylsilyl or ethyldimethylsilyl Positive- and negative-ion mass spectra have been obtained by laser-desorption-Fourier-transform mass spectrometry from a-solanine a-tomatine and three cardenolide glycosides.la6 Pentafluorobenzyl (PFB) esters of bile acids give good negative-ion mass spectra (g.c.-m.s.) in which the [M-1811-ion resulting from loss of the PFB group is most abundant. Further derivatization of hydroxyl groups in the PFB esters (as ethyldimethylsilyl ethers) allowed the common bile acids to be separated by gas chromatography. 3,5-Bis(trifluoromethyl)-benzoyl derivatives of steroid alcohols are detectable in electron- capture negative-ion chemical-ionization mass spectra to as little as 1 pg and have been proposed for the quantitative analysis of mixtures of steroids by g.c.-m.s. These esters formed at skeletal hydroxyl groups normally give the molecular ion as the base peak.188 (2-Cyanoethyl)dimethylsilyl (CEDMS) derivatives of hydroxyl groups are suitable for g.c.analysis with the very sensitive nitrogen-phosphorus detector. The mass-spectral fragmentation of the CEDMS derivative of cholesterol has been illustrated. Mass spectrometry has been used to characterize 16-amino-oestrogen derivative^'^^ and metabolites of the aromatase inhibitor 4-hydroxyandrost-4- ene-3,17-dione.lg1 Fragment ions (24) formed via a retro-Diels-Alder reaction were observed in the mass spectra of a series of oestra- 1,3,5( 10)-trienes. lS2 Protium-deuterium ex-changes were observed in the E.I. mass spectra of the steroidal amides (25).lS3 The translational energy that is released when a methyl group (C-18 or C-19) is lost from unsaturated steroids has been investigated.lS4The translational energy that is released during the loss of the methyl group C-19 of cholesterol and related sterols is greater if dehydration occurs first. The effect is less pronounced for C- 1 8.1S5 6 High-Performance Liquid Chromatography (H.P.L.C.) and Liquid Chromatography-Mass Spectrometry (L.C.-M.S.) A new and detailed review of analytical procedures for steroids calibration of the other assay procedures which are a~ai1able.l~~ concentrates on methods that are aimed at determining in a single assay the full range of steroids present in plasma or urine Recent developments in this field are discussed in another paper from the same group. 175 Desorption-chemical-ionization (DCI) mass spectrometry has been used to quantify vitamin D sulphate in human milk.176 The daughter-ion spectra of [M+H]+ ions derived from tandem mass spectrometry of steroid glucuronides generated by fast atom bombardment (FAB) are sensitive to experimental samples taken from patients suffering from a variety of clinical disorders and congenital hormone deficiencies.Steroid 'pro-files' that have been obtained by h.p.l.c. preferably in conjunction with m.s. are illustrated.196 The hormonal steroids and their metabolites are the subjects of another review of h.p.1.c. methods for their separation and quantitation in the NATURAL PRODUCT REPORTS 1989-D. N. KIRK 401 (24) R' = Ac HO CHO or Me0 R2 = H orMe clinical context with practical examples given to illustrate the techniques employed.lg7 The separation of isotopically labelled from unlabelled steriods by h.p.1.c. has been used as the basis for isotope- dilution analysis of various hormonal steroids and their metabolites in serum. The labelled internal standards contained six or more deuterium atoms which had been introduced by chemical exchange and were easily separated from the natural substrates; peaks were quantified by conventional immuno- assay methods.198 Ferrocenoyl azide reacts readily with steroidal alcohols and other alcohols to give urethanes which are suitable for separation by h.p.l.c. with electrochemical detection showing maximum sensitivity at +0.4 V vs a silver-silver chloride reference electrode and a detection limit of 0.5 pm01.'~~ In another h.p.1.c.application of the electrochemical detection of ferrocene 2-ferrocenylethylamine has been condensed with the carboxyl group of steroid glucuronides. The resulting ferrocenyl amides are also detectable to a limit of ca. 0.5 pmo1.200 Separations of ercalcidiol and calcidiol(25-hydroxy-vitamins D and D3)by automated h.p.1.c. use an ultraviolet-absorbing internal standard."l Increased resolution of steroid mixtures has been achieved by h.p.1.c. at low temperatures; with MeCN-MeOH as the mobile phase a reverse-phase column could be used at temperatures as low as -50 OC.,02 Optical resolution of racemic norgestrel achieved by h.p.1.c. with y-cyclodextrin as a chiral additive in the mobile phase,2o3 provides a method that is likely to find wider application.A p-cyclodextrin column has been used for separation of epimeric steroids by h.p.l.~.~O~ Equations have been developed for predicting reversed-phase h.p.1.c. retention data for Thermospray 1.c.-m.s. of steroids and of other classes of compounds of biomedical interest with mixed aqueous solvent systems that contain 0.1 mol dmP3 of ammonium acetate generally gives a positive-ion spectrum in which there is a strong (M+H)+ peak.206 In the case of highly hydroxylated steroids a series of peaks with masses (M+H -1%I)+ correspond to sequential losses of up to n molecules of water. Corticosteroids that contain the dihydroxyacetone side-chain are characterized by the (M+H -60)' ion resulting from loss of the whole side-chain as the base peak.Steroid glucuronides give negative-ion spectra comprising the (M-H)- ion with only very weak peaks for fragment ions whereas their positive- ion spectra include (M+NH,)+ peaks and show the usual losses of water molecules. Another report on thermospray negative-ion mass spectrometry of steroid glucuronides in which a salt-free aqueous solvent system was used shows that they give molecular anions only with gradient-elution h.p.1.c. and selected ion monitoring mixtures of glucuronides can be analysed with a detection limit of 100 pg.207 The thermospray method is excellent for selected ion monitoring.206 Thermospray 1.c.-m.s. with dilution with a stable isotope provides a fast quantitative assay for cortisol in serum although the reliability of the results has not yet reached (25) R' = Me 0,(CD3)2CH,or CD3C(0) R2 = D H or Me R3 = H or Me the high level that can be achieved by the more tedious g.c.-m.s.method.208 The use of thermospray 1.c.-m.s. in combination with isotope dilution for the quantitative analysis of various compounds including cortisol testosterone and 1,25-di-hydroxyvitamin D is described in a short review.2og Thermo- spray 1.c.-m.s. has also been applied effectively to the identification of aldosterone 18-hydroxycorticosterone and related metabolites from rat adrenals. Most of the mass spectra are dominated by either [M+HI+or [M-H,O +HI' ions with little other fragmentation.210 Liquid chromatography-mass spectrometry with gradient elution has been applied to the study of bile acids.211 Statistical methods have been applied to the selection of optimized solvent systems for two-dimensional t.1.c.of steroids.212 7 Immunoassay and Miscellaneous Physical Methods Applications of aminop h thal hydrazides aminonaph tho hydra- zides and acridinium esters as labels for luminescence immunoassays of steroids213 and chemiluminescence immuno- assays of steroids and peptide hormones in body fluids214 have been reviewed. N-(Fluoranthen-3-y1) maleimide and its deriva- tives can serve as fluorescent tracers in immunoassays for progesterone.215 With chelated europium(Ir1) as the label for either the antibody or the antigen time-resolved fluorescence immuno- assays have been developed for progesterone and oestradiol ; solid-phase supports were used to simplify the experimental procedure.216 Sensitivity is high.A time-resolved fluoro-immunoassay for testosterone uses antibodies that are con- jugated to isothiocyanatophenyldiethylenetriaminepenta-acetic acidmropium chelate as a marker.217 The separated (2)-and (,!?)-isomers of the 3-carboxymethyl-oxime of 1 1 -deoxycortisol gave antisera with enhanced specificity and low cross-reactivity ; the (2)-isomer was particularly effective.218 Electron diffraction has been used to study the mesomorphic behaviour of cholesteryl myri~tate.~" A theoretical and experimental study has been made of the optical properties of blue phases of cholesteryl nonanoate.220 The uses of polarizing microscopy differential scanning calorimetry 13Cn.m.r.X-ray diffraction and other techniques to study phase characteristics of cholesteryl esters have been reviewed.221 Some cardiac glycosides which adsorb strongly on Hg electrodes may be determined quantitatively at nanomole sensitivity by adsorptive stripping voltammetry.222 A novel microdetermination of mercury (as Hg2+) depends upon its ability to inhibit the oxidation of deoxycholic acid by 3a-hydroxysteroid dehydrogena~e.~~~ The natural level of 13C in cholesterol from human food tissues and serum showed little difference; no isotope fractionation was observed during its isolation and analysis. 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Gravel Acta Crys- tallogr. Sect. C 1986 42 1833. 79 W. Reischl H. Bernhard C. Kratky and E. Zbiral Monatsh. Chem. 1985 116 831. 80 H. Bernhard C. Kratky W. Reischl and E. Zbiral Monatsh. Chem. 1985 116 1221. 81 P. F. Lindley and M. C. Carey J. Crystallogr. Spectrosc. Res. 1987 17 231. 82 A. R. Campanelli S. Candeloro de Sanctis E. Giglio and L. Scaramuzza J. Lipid Res. 1987 28 483. 83 M. Soriano-Garcia I. Lopez y Celis R. A. Toscano J. M. Barba Chavez P. Enriquez R.A. Hernandez and A. Rodriguez Acta Crystallogr. Sect. C. 1987 43 1163. 84 A. Kalman G. Argay B. Ribar D. Zivanov-Stakic and S. Vladimirov Acta Crystallogr. Secr. C 1985 41 1645. 85 D. Pfeiffer L. Kutschabsky R. G. Kretschmer F. Coll and G. Adam 2. Chem. 1985 25 183. NATURAL PRODUCT REPORTS 1989-D. N. KIRK 86 G. Ferguson B. Kaitner A. F. Somogyvari V. I. Bendall W. B. Whalley C. L. Yeats R. Edmunds and J. M. Midgley J. Chem. SOC. Perkin Trans. 1 1985 1337. 87 D. Scharfenberg-Pfeiffer E. Hoehne and M. Wunderwald Cryst. Res. Technol. 1987 22 1403. 88 H. Wiedenfeld F. Knoch I. Naidis and B. Kopp Sci. Pharm. 1987 55 167. 89 G. Argay A. Kalman B. Ribar S. Vladimirov and D. Zivanov- Stakic Acta Crystallogr. Sect. C 1987 43 922.90 L. R. Nassimbeni M. L. Niven D. A. Stuart and K. J. Zemke J. Crystallogr. Spectrosc. Res. 1986 16 557. 91 K. Miki N. Kasai H. Tsutsumi M. Miyata and K. Takemoto J. Chem. Soc. Perkin Trans. I 1987 545. 92 E. Giglio F. Mazza and L. Scaramuzza J. Inclusion Phenom. 1985 3 437. 93 K. Padmanabhan V. Ramamurthy and K. Venkatesan J. In-clusion Phenom. 1987 5 315. 94 W. R. Croasmun and R. M. K. Carlson Methods Stereochem. Anal. 1987 9 (Two-dimensional NMR Spectroscopy) 387. 95 H. J. Schneider U. Buchheit N. Becker G. Schmidt and U. Siehl J. Am. Chem. Soc. 1985 107 7027. 96 K. Marat J. F. Templeton and V. P. S. Kumar Magn. Reson. Chem. 1987 25 25. 97 K. Marat J. F. Templeton R. K. Gupta and V. P. S. Kumar Magn. Reson. Chem. 1987 25 730.98 B. G. Carter D. N. Kirk and P. J. Burke J. Chem. Soc. Perkin Trans. 2 1987 1247. 99 M. Harnik Y.Kashman S. Carmely M. Cojocaru S. L. Dale M. M. Holbrook and J. C. Melby Steroids 1986 47 67. 100 D. V. Waterhous S. Barnes and D. D. Muccio J. Lipid Res. 1985 26 1068. 101 M. Campredon V. Quiroa A. Thevand A. Allouche and G. Pouzard Magn. Reson. Chem. 1986 24 624. 102 L. E. Kay and J. H. Prestegard J. Am. Chem. Soc. 1987 109 3829. 103 E. Mukidjam S. Barnes and G. A. Elgavish J. Am. Chem. Soc. 1986 108 7082. 104 E. Mukidjam G. A. Elgavish and S. Barnes Biochemistry 1987 26 6785. 105 T. Iida and T. Shinohara Nihon Daigaku Kogakubu Kiyo Bunrui A 1987 28 175. 106 A. G. J. Sedee G. M. J. Beijersbergen van Henegouwen M. E. de Vries and C.Erkelens Steroids 1985 45 101. 107 K. Bischofberger J. R. Bull and A. A. Chalmers Magn. Reson. Chem. 1987 25 780. 108 A. G. J. Sedee G. M. J. Beijersbergen van Henegouwen W. Guijt and C. A. G. Haasnoot Pharm. Weekbl. Sci. Ed. 1985 7 202. 109 A. G. J. Sedee G. M. J. Beijersbergen van Henegouwen W. Guijt and C. A. G. Haasnoot J. Org. Chem. 1985 50 4182. 110 V. J. Robinson A. D. Bain and C. A. Rodger Steroids 1986,48 267. 11 1 E. T. K. Haupt Spectrosc. Lett. 1986 19 1091. 112 S. Top G. Jaouen A. Vessieres J.-P. Abjean D. Davoust C. A. Rodger B. G. Sayer and M. J. McGlinchey Organometallics 1985 4. 2143. I13 M. Savignac 0.Jaouen C. A. Rodger R. E. Perrier B. G. Sayer and M. J. McGlinchey J. Org. Chem. 1986 51 2328. 114 N.J. Koszewski T. A. Reinhardt D. C. Beitz J. L. Napoli E. G. Baggiolini M. R. Uskokovic and R. L. Horst Anal. Biochem. 1987 162 446. 115 B. Helmer H. K. Schnoes and H. F. Deluca Arch. Biochem. Biophys. 1985 241 608. 116 A. G. Avent J. R. Hanson Y.Z. Ling and I. H. Sadler J. Chem. SOC. Perkin Trans. I 1985 2129. I17 S. A. Campbell T. A. Crabb and R. 0.Williams Magn. Reson. Chem. 1986 24 803. 118 H. Takagi T. Hayakawa T. Miura and M. Kimura Chem. Pharm. Bull. 1985 33 3224. 119 H. Takagi T. Hayakawa T. Miura and M. Kimura Chem. Pharm. Bull. 1985 33 3317. 120 P. N. Rao A. M. Rodriguez and D. W. Miller J. Steroid Bio- chem. 1986 25 417. 121 R. D. Dawe and J. L. C. Wright Can. J. Chem. 1987 65 666. 122 S. M. Koll. M. Vuilhorgne B. Dang Vu D.Ba S. Bourdon and R. Bourdon Tetrahedron Lett. 1986 27 2613. 123 J. Drew J. R. Brisson P. Morand and A. G. Szabo Can. J. Chem. 1987 65 1784. 124 T. Akihisa S. Thakur F. U. Rosenstein and T. Matsumoto Lipids 1986 21 39. 125 G. G. Habermehl and P. E. Hammann Z. Naturforsch. Teil B 1985 40 656. 126 P. S. Steyn F. R. van Heerden R. Vleggaar and L. A. P. Ander- son J. Chem. Soc. Perkin Trans. I 1986 1633. 127 P. S. Steyn R. F. van Heerden and R. Vleggaar S. Afr. J. Chem. 1986 39 143. 128 J. M. Bernassau M. Fetizon L. Hanna and J. A. Pinheiro J. Phys. Chem. 1986 90,3941. 129 R. E. Jacobs J. Singh and L. E. Vickery Biochemistry 1987 26 4541. 130 P. B. Reese L. A. Trimble and J. C. Vederas Can. J. Chem. 1986 64 1427. 131 R.S. Chana R. J. Cushley S. R. Wassall I. C. P. Smith and E. J. Dufourc Chem. Phys. Lipids 1985 37 345. 132 R. Murari M. P. Murari and W. J. Baumann Biochemistry 1986 25 1062. 133 E. J. Dufourc and I. C. P. Smith Chem. Phys. Lipids 1986 41 123. 134 W. D. Treleaven Y. I. Parmar H. Gorrissen and R. J. Cushley Biochim. Biophys. Acta 1986 877 198. 135 H. K. Gill R. W. Smith and D. A. Whiting J. Chem. SOC. Chem. Commun. 1986 1457. 136 R. A. Fletton R. K. Harris A. M. Kenwright R. W. Lancaster K. J. Packer and N. Sheppard Spectrochim. Acta Part A 1987 43 1111. 137 A. Neszmelyi W. E. Hull G. Lukacs and W. Voelter Z. Nutur-forsch. Teil B 1986 41 1178. 138 J.-C. Gramain and J. C. Quirion Magn. Reson. Chem. 1986 24 938. I39 A. M.Seldes,M. E. DeLuca,andE. G. Gros,Magn.Reson. Chem. 1986 24 185. 140 W. Robien B. Kopp D. Schabl and H. Schwarz Prog. Nucl. Magn. Reson. Spectrosc. 1987 19 131. 141 G. G. Habermehl P. E. Hammann and V. Wray Magn. Reson. Chem. 1985 23 959. 142 P. K. Agrawal D. C. Jain R. K. Gupta and R. S. Thakur Phytochemistry 1985 24 2479. 143 P. K. Agrawal and R. S. Thakur Indian J. Chem. Sect. B 1986 25 469. 144 J. A. R. Garzia and H. T. V. Castro Magn. Reson. Chem. 1987 25 831. 145 T. Iida F. C. Chang J. Goto and T. Nambara Chem Phys. Lipids 1987 45 1. 146 M. G. Sierra D. A. Bustos M. E. Zudenigo and E. A. Ruveda Tetrahedron 1986 42 755. 147 T. Akihisa and T. Matsumoto Yukagaku 1987 36 301. 148 M. van Robays R. Busson and H. Vanderhaeghe J.Chem. SOC. Perkin Trans. I 1986 251. 149 J. Romer D. Scheller and G. Grossmann Magn. Reson. Chem. 1987 25 135. 150 T. Iida T. Tamura and T. Matsumoto Magn. Reson. Chem. 1987 25 558. 151 V. S. Bogdanov E. G. Cherepanova I. S. Levina L. E. Kulikova and V. N. Ignatov Izv. Akad. Nauk SSSR Ser. Khim. 1986,2315. 152 S. Seo U. Sankawa H. Seto A. Uomori Y. Yoshimura Y. Ebizuka H. Noguchi and K. Takeda J. Chem. Soc. Chem. Commun. 1986 1139. 153 N. Nishimoto Y.Shiobara M. Fujino S. Inoue T. Takemoto F. de Oliveira G. Akisue M. K. Akisue G. Hashimoto 0. Tanaka R. Kasai and H. Matsuura Phytochemistry 1987 26 2505. 154 D. 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Reson. 1987 71 461. 167 R. Krzyminiewski A. M. Hafez A. Szyczewski and J. Pietrzak J. Mol. Struct. 1987 160 127. 168 A. Szyczewski R. Krzyminiewski and J. Pietrzak Ser. Fiz. (Uniw. im. Adama Mickiewicza Poznaniu) 1985 53 201. 169 A. M. Hafez R. Krzyminiewski and J. Pietrzak Ser. Fiz. (Uniw. im. Adama Mickiewicza Poznaniu) 1985 53 205. 170 C. L. Sevilla D. Becker and M. D. Sevilla J. Phys. Chem. 1986 90 2963. 171 B. Kirste Z. Naturforsch. Teil A 1987 42 1296. 172 C. H. L. Shackleton and W. Chai Endocrine Rev. 1985 6 441. 173 T. Yamaguchi K. Konno K. Kawabe K. Yasuda H.Mori T. Suzuki T. Yanaihara and T. Nakayama Endocrinol. Jpn. 1985 32 279. 174 R. D. Coldwell C. E. Porteous D. J. H. Trafford and H. L. J. Makin Steroids 1987 49 155. 175 C. E. Porteous R. D. Coldwell D. J. H. Trafford and H. L. J. Makin J. Steroid Biochem. 1987 28 785. 176 N. Le Boulch L. Cancela L. Miravet and C. Lange Biomed. Environ. Mass Spectrom. 1986 13 53. 177 R. B. Cole C. R. Guenat and S. J. Gaskell Anal. Chem. 1987 59 1139. 178 Y.-Z. Chen. N.-Y. Chen. H.-Q. Li F.-Z. Zhao and N. Chen Biomed. Environ. Mass Spectrom. 1987 14 9. 179 K. B. Tomer N. J. Jensen M. L. Gross and J. Whitney Biomed. Environ. Mass Spectrom. 1986 13 265. 180 R. P. Evershed and L. J. Goad Biomed. Environ. Mass Spectrom. 1987 14 131. 181 M.Dumazer M. Farines and J. Soulier Rev. Fr. Corps Gras 1986 33 151. 182 J. Schmidt H. M. Vorbrodt and G. Adam Zfl-Mitt. 1986 115 165 (Chem. Abstr. 1986 105 191 470). 183 R. P. Evershed M. C. Prescott L. J. Goad and H. H. Rees Bio-chem. Soc. Trans. 1987 15 175. 184 T. Iida T. Momose T. Mamura T. Matsumoto J. Goto T. Nambara and F. C. Chang J. Chromatogr. 1987 389 155. 185 R. P. Evershed J. G. Mercer and H. H. Rees J. Chromatogr. 1987 390 357. 186 M. L. Coates and C. L. Wilkins Biomed. Environ. Mass Spec- trom. 1986 13 199. 187 J. Goto K. Watanabe H. Miura T. Nambara and T. Iida J. Chromatogr. 1987 388 379. 188 A. Murray and D. Watson J. Steroid Biochem. 1986 25 255. 189 M. J. Bertrand S. Stefanidis and B. Sarrasin J. Chromatogr.1986 351 47. 190 W. Schade B. Schoenecker and J. Vokoun J. Prakt. Chem. 1985 327 589. 191 A. B. Foster M. Jarman J. Mann and I. B. Parr J. Steroid Biochem. 1986 24 607. 192 X.-Z. Yuan Org. Mass Spectrom. 1985 20 792. 193 P. Longevialle Org. Mass Spectrom. 1985 20 644. 194 Z. V. I. Zaretskii Z. Kustanovich E. E. Kingston J. H. Beynon NATURAL PRODUCT REPORTS 1989 R. Lauber U. P. Schlunegger and C. Djerassi Org. Mass Spec- trom. 1985 20 471. 195 Z. V. I. Zaretskii Z. Kustanovich E. E. Kinston J. H. Beynon C. Djerassi and L. Toekes Org. Mass Spectrom. 1986 21 125. 196 C. H. L. Shackleton J. Chromatogr. 1986 379 91. 197 J. W. Honour in ‘H.p.1.c. of Small Molecules’ ed. C. K. Lim IRL Press Oxford 1986 p. 117. 198 J. J. Pratt Ann.Clin. Biochem. 1986 23 251. 199 K. Shimada S. Orii M. Tanaka and T. Nambara J. Chro-matogr. 1986 352 329. 200 K. Shimada E. Nagashima S. Orii and T. Nambara J. Pharm. Biomed. Anal. 1987 5 361. 201 E. B. 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Mass Spectrom. 1986 13 21.

ISSN:0265-0568    

DOI:10.1039/NP9890600393

出版商:RSC    

年代:1989

数据来源: RSC

摘要:

/?-Phenylethylaminesand the lsoquinoline Alkaloids K. W. Bentley Department of Chemistry Loughborough University of Technology Loughborough Leicestershire LEl l 3TU Reviewing the literature published between July 1987 and June 1988 (Continuing the coverage of literature in Natural Product Reports 1988 Vol. 5 p. 265) 1 /3-Phenylethylamines 2 Isoquinolines 3 Benzylisoquinolines 4 Bisbenzylisoquinolines 5 Cularines 6 Pavines and Isopavines 7 Benzopyrrocolines 8 Berberines and Tetrahydroberberines 9 Azaberberines 10 Secoberberines 11 Protopines 12 Phthalide-isoquinolines 13 Spirobenzylisoquinolines 14 Isoindolo benzazepines 15 Rhoeadines 16 Emetine and Related Compounds 17 Benzophenanthridines 18 Aporphinoid Alkaloids 18.1 Proaporphines 18.2 Aporphines 18.3 Dimeric Aporphines 18.4 Benzylisoquinoline-Aporphine Dimers 18.5 Oxoaporphines 18.6 Dioxoaporphines 18.7 Phenanthrenes 18.8 Aristolochic Acids and Aristolactams 18.9 Azahomoaporphines 19 Morphine Alkaloids 20 Phenethylisoquinolines 21 Homoaporphinoid Alkaloids 22 Colchicine 23 Other Alkaloids 24 References I 8-PhenylethyIamines from Denmoza been isolatedfrom Opuntia hickenii, and from Candicine has rhodacantha,2 Alphonsea sclerocarpa,’ Trichocereus andagalensis, hordenine has been isolated from Trichocereus andagalensis,2 and trimethyl(p-phenylethy1)am-monium hydroxide has been isolated from Alphonsea sclero-carpa.l Absorption ~pectra,~ molecular-orbital calculation^,^ thermodynamic parameters,4and methods of determination5.of ephedrine and pseudoephedrine have been studied and ephedrine that catalyst for onto silicon has been used The heterogeneous is grafted Michael addition reactions.’ as a effects of ephedrine on muscle* and of mescaline on behaviourg have been reported. 2 lsoquinolines Thalifoline has been isolated from Abuta pahni.lo Patents have been published covering the preparation1’. l2and p~rification’~ of cotarnine. The crystal structures of salsoline and salsolidine-hydrochlorides have been determined. l4 Asymmetric syntheses of some alkaloids of this group have cyclized by treating it with a base to give an approximate 2 1 excess of the base (2) over its enantiomer and catalytic desulphurization of (2) yielded (+)-(R)-carnegine.The (2)-isomer of (1) cyclized to a 1:5 mixture of (2) and its enantiomer.I5.l6Reduction of the chiral iminium ions (3; R = Me) and (3; R = CH,Ar) with sodium borohydride yielded the bases (4)and their C-1 epimers in the ratio 7.5 1 for R = Me and 16 I for R = CH,Ar. Catalytic removal of the N-benzylic group from (4; R = Me) afforded (-)-(S)-salsolidine.” Con-version of the dihydroisoquinoline (5) into the enamide (6) followed by oxidation with osmium tetroxide yielded the hydroxy-ketone (7) which on reductive removal of the benzyloxycarbonyl group suffered reductive cyclization to (+)-calycotomine (8).’* Condensation of the aldehyde (9) with ethyl azidoacetate led to the diester (lo) which was reduced by :Izq -C02CH2Ph (5) (6) ““w$O2CH2Ph Me0 \ CH2OH CH20H (7) (8) C02Et phcH20m \ C02Et phcH20aCHo PhCH20 C02Et PhCH20 been accomplished.The (E)-vinyl sulphoxide (1) has been (9) (10) 405 15 NPR 6 NATURAL PRODUCT REPORTS 1989 C02Et phcH20Q7 PhCH20 OEt (1 1) (13) triethyl phosphite to the ester (11). Conversion of the C0,Et group of this (through CH,OH and CH,Cl) into CH, followed by treatment with boron tribromide afforded siamine (12).19 The anti-tumour effects of quinocarmycin have been stud- ied.zOA review of the chemistry of the naphthylisoquinoline alkaloids has been published.,l 3 Benzyl isoquinolines Benzylisoquinoline alkaloids have been isolated from the following plant species that marked with an asterisk being new Abuta pahni'O coclaurine Alphonsea sclerocarpa' petaline methine Cocculus laurifolius22 coclaurine N-methylcoclaurine and reticuline Corydalis bungeanaz3 juziphine and norjuziphine Duguetia ~pixiana~~~~~ codamine cis-N-oxide (13)* Manglie t ia duclouxiiz magnocurarine Oxandra majorz7 reticuline Stephania gracilenta2g papaverine Stephania venosa2' reticuline The isolation of papaverine from Stephania gracilenta is the first reported finding of this alkaloid outside the Papaveraceae.The conformation of the alkaloid in solution has been studied by n.m.r. and the spectral characteristics of several alkaloids of this group have been re~iewed.~' The oxidation of papaverine and other alkaloids of the group to N- oxides with m-chloroperoxybenzoic acid has been 6'-Nitropapaverine on oxidation with iodine yields the meso- ionic azaberberine (14) and the zwitterionic compound (1 5) ;if heated with methanolic potassium hydroxide it affords the isoxazole (1 6) which on thermolysis or photolysis gives a compound that was first assigned the structure (17)33but which has been shown by X-ray studies to have the constitution (18).34 The use of Reissert compounds in the synthesis of benzyl- isoquinoline alkaloids has been reviewed.35 The Reissert compound (19) has been converted into (20) which on treatment with sodium hydroxide and then being debenzylated yielded cristadine (2l).36The preparation of roemecarine and isoroemecarine from 1,4-trans- and 1,4-cis-4-acetoxy- 1-benzyl-"'"qN Me0 \ OMe " PhCH2O 'T N C(0)Ph CN (19) MeoqN HO R,' R' Me0 \ *.H "@NMe '0 SbMe 0 Li+ OH (24) NATURAL PRODUCT REPORTS 1989-K.W. BENTLEY OH (26) (28) (30) tetrahydroisoquinolines indicates that the former alkaloid has the structure (22; R1 = H R2= OH) epimeric with that originally assigned i.e. (22; R1= OH R2 = H) which repre- sents isoroemecarine.37 (-)-(S)-Norlaudanosine has been prepared by catalytic reduction of (4; R = 3 4-dimethoxy- benzyl).17 The seco-alkaloid cryptopleurospermine (26) has been synthesized from the aldehyde (23) and the dilithio-salt (24) which gave (25; R = C0,Et); this was reduced to (25; R = Me) which was hydrolysed and then oxidized with tri- fluoroacetic acid and sodium nitrite.3s The biological effects of papa~erine,~~-~' of 4-[4-(2-methoxy- pheny1)piperazin-1 -ylmethyl Jpapaverine 42 43 of higena-mine,44-46 and of atracuri~m~'-~~ have been studied and a method of estimating higenamine in blood53 has been described.(29) A patent covering the manufacture of atracurium has been published.54 4 Bisbenzyl isoqui no1ines Bisbenzylisoquinoline alkaloids have been isolated from the following species the twenty nine marked by an asterisk being new alkaloids. Abuta pahni'" daurisoline 2'-nordaurisoline* (27; R' = R3= Me R2= H) lindholdamine (27; R' = R2 = R3= H) 2-N-methyl-lindholdamine* (27; R' = Me R2 = R3 = H), 2'-N-methyl-lindholdamine*(27; R' = R3 = H R2= Me) and N,N-dimethyl-lindholdamine(27 ; R' = R2 = Me R3= H) Albertisia pap~ana~~ apateline 2,2'-noraromoline coscoline 0-methylcoscoline cosculine 2'-norcosculine* (28) daphnandrine daphno- line lindholdamine pangkoramine* (29) pangkorimine* (30) and 2,2'-norphaeanthine* (31) Aristolochia gigantea56 geraldoamine* (32) pampulhamine [2'-nordaurisoline] (27; R' = R3= Me R2 = H) and pedroamine [2-N-methyl-lindholdamine] (27; R1= Me R2 = R3= H) Berberis k~reana~~ aromoline berbamine obamegine and oxyacanthine Berberis pse~dambalata~~ oxyacanthine and 0-methyloxyacanthine NATURAL PRODUCT REPORTS 1989 (34) Hi OMe (40) (35) Me Cocculus pendul~s~~ kohatamine* (33 ; R = Me) 1,2-didehydrokohatamine* (34;R = Me) kohatine (33; R = H) 1,2-didehydro-kohatine* (34; R = H) 5'-hydroxyapateline* (35; R = H) 5'-hydroxytelobine* (35; R = Me) siddiquine* (36; R = H) siddiquamine* (36; R = Me) and 1,2-didehydro- 2'-nortelobine* (37) Pachygone loyaltiensis6' apateline 1,2-didehydroapateline 2,2'-noraromoline daphnandrine daphnoline 0-methylcoscoline 1,2-dide- hydrotelobine and isotrilobine Pycnarrhena ozantha61 daphnoline 2-norberbamine 2-norobamegine7 2,2'-noro- bamegine* (38; R1 = R2= R3 = H) 2-northalrugosine* (38; R1 = H R2= R3= Me) bisnorthalrugosine* (38; R' = R2 = H R3 = Me) and pycnazanthine* (39) Stephania epigeae62 cepharanthine (36) Stephan ia ven osa2 thalrugosamine Thalictrum collin um 0-methyl thalicberine Thalictrum ultrat turn^^ aromoline cultithalminine* (40) neothalibrine obaberine oxyacanthine 2'-noroxyacanthine* (41) thaligosine 2'- northaliphylline* (42) thalirugine neothalibrine 2'-a-N- oxide* (43) thalidasine 2-a-N-oxide* (44; R = H) 5-hydroxythalidasine 2-a-N-oxide* (44;R = OH thali-gosine 2-a-N-oxide* (45 ;R = H) thalrugosamine 2-a-N- oxide* (45 ; R = Me) and thaliphylline 2'-P-N-oxide* The production of alkaloids in callus cell cultures of 34 cell lines representing 33 species of Berberis has been studied.In (46). all of these cultures the principal alkaloid that was isolated was (37) the berberine analogue jatrorrhizine but the production of the NATURAL PRODUCT REPORTS 1989-K. W. BENTLEY H" OMe (43) R n Me"\ HO-q o m M e 0 (50) bisbenzylisoquinoline alkaloids aromoline berbamine berba- munine isotetrandrine and the new base 2-norberbamunine (47) was also The structures of the new alkaloids were determined by spectroscopic methods and by their conversions into known bases by reduction and by 0-and N-methylations.The co- occurrence in Cocculuspendulus of the (IS 1'8-dimers kohatine (33; R = H) and kohatamine (34; R = Me) and their (lR 1's) isomers 5'-hydroxyapateline (35 ; R = H) and 5'-hydroxy-telobine (35; R = Me) probably results from oxidation of the former pair (which have the same configuration as the common * alkaloids with three ether bridges such as trilobine isotrilobine cosculine and coscoline) to their 1,2-didehydro-compounds followed by reduction on the less hindered face of the molecule.Further oxidation of the dehydro-compounds would afford siddiquine (36 R = H) and siddiquamine (36;R = Me).59 The tertiary amines related to the N-oxides that have been isolated from Thalictrum cultraturn have been isolated from the same plant and it is possible that some of the oxides are artifacts of the extraction procedure. The known photo-oxidative cleavage of laudanosine to veratric aldehyde and a mixture of 6,7-dimethoxy-N-methyl- tetrahydroisoquinolone and 6,7-dimethoxy-N-methyl-3,4-di-hydroisoquinolinium hydroxide66*" has been developed to provide a new method of cleavage of bisbenzylisoquinoline alkaloids that is of considerable value in the determination of their structures.68 Isotetrandrine (48 ;R = Me) in methanol was irradiated in the presence of oxygen and it was cleaved to the dialdehyde (49) and a substance that showed the properties to be expected of the lactam iminium salt (50) which was reduced Me0 P M e Me0E ' Me COzMe OMe Me0 OMe (52) (53) Me Hog Me -Meo??Me0\/OH \/ Me0 (54) (55) H Me0 (56) (57) by sodium borohydride to the lactam (51).The process works equally well with the phenol berbamine (48; R =H) with its 0-acetyl ester (48; R =Ac) with phaeanthine [the (lR 1'R)-isomer of (48; R =Me)] and with a range of other alkaloids that contain two and three diphenyl ether linkages including the 'head-to-tail '-linked alkaloid cycleanine. The chemistry of bisbenzylisoquinoline alkaloids69 and of those obtained from Thalictrum species70-71 has been reviewed.The biological effects of tub~curarine,~~-~~ of cepharan- thine,76-soof tet~andrine,~~-~~ of N,N'-dimethyltetrandrine,88 of berbamine,sg*90of da~ricine,~'.~~several ethers of of berbamine and da~ricine,~~ lima~ine,~~ dimethyl-of of tril~bine,~~-~~ and of methyl-liensinineg9 have been studied. Methods for the estimation of trilobine in plasma have been described.loo* lo' 5 Cularines Alkaloids of the cularine group all of which are new bases have been isolated from the following species Corydalis claviculata'02* '03 noyaine (52) and clavizepine (53) NATURAL PRODUCT REPORTS 1989 I ,Me N 'R2 Me0 (58) MeOs:;-+ M e 0 9 Me \ OMe OMe Me0 OMe (59) (60) HO\ $e Me0 OMe Me0 R2 (611 (62) Sarcocapnos cras~ifolia'~~* lo5 enneaphylline- (54) sarcophylline (55) norsarcocapnidine (56) norsecocularidine (57),secosarcocapnine (58 ;R1= R2=Me) norsecosarcocapnine (58; R' =Me R2=H) secosarcocapnidine (58; R' =H R2=Me) and norseco- sarcocapnidine (58; R' =R2=H) Sarcocapnos enneaphyllalo4* lo5 enneaphylline sarcophylline norsecocularidine seco-sarcocapnine norsecosarcocapnine secosarcocapnidine and norsecosarcocapnidine.The structures of these alkaloids were determined by spec- troscopic methods and by their conversion into and their preparation from known alkaloids. Clavizepine (53) is the first alkaloid to be discovered that has a dibenzopyranazepine skeleton; it presumably belongs to the cularine group and a plausible origin is by oxidation of cularine to (59) rearrange-ment of this to the iminium salt (60) and subsequent reduction.Noyaine (52) represents a secocularine arising from oxidation similar to that involved in the conversion of bisbenzylisoquino-lines into secobases such as punjabine and gilgitine. Several syntheses have been achieved within this group of alkaloids. Oxidation of cularidine with lead tetra-acetate afforded a 2 3 mixture of limousamine (61) and its C-4 epimer. 0-Methylation and further oxidation with 2,3-dichloro-5,6-di- cyanobenzoquinone then afforded dioxocularine (62 ;R' =H R2=OMe).lo6 Oxidation of the diphenol (63) with potassium ferricyanide yielded a mixture of sarcocapnidine (64; R' =OH R2=H R3 =Me) and enneaphylline (64; R' =H R2 =OH R3=Me) but oxidation of the N-borane complex of (63) with NATURAL PRODUCT REPORTS 1989-K.W. BENTLEY 41 1 Me07 Me R3 Me0qOH M e MeOg Me HO Me0 Me0 R2 (65) OvO $.e H OH M:% MeogMe Me0 \ --Meo \ / Meo \ / Me0 Me0 (67) (68) vanadium oxyfluoride yielded only enneaphylline ; cularine only was obtained in the same way from the 3'-methyl ether of (63).'07 Ullmann cyclization of the bromophenols (65; R' = H, R2= R3= OMe) and (65; R1= R2= OMe R3= H)afforded cularine (64;R' = H,R2= OMe R3= Me) and sarcocapnine (64; R' = OMe R2= H R3= Me) respectively 0-Methyl- cularicine (66) was synthesized in a similar manner from (65 ; R' = H,R2R3= OCH20);10Sthe N-benzyl analogue of (65; R' = R2= OMe R3= H)was cyclized and debenzylated to norsarcocapnhe (64; R' = OMe R2= R3= H),which was selectively demethylated to norsarcocapnidine (56).lo4 Com-bination of the Ullmann approach and oxidation with lead tetra-acetate has afforded syntheses of 4-hydroxysarcocapnine (67) yagonine (62; R1= OMe R2= H),and aristoyagonine (68).lo* The trypanosomicidal activity of claviculine has been st~died.'~ 6 Pavines and lsopavines Amurensine has been isolated from subspecies album and xanthopetalum of Papaver nudica~le.'~~ The chemistry of the alkaloids of these groups has been reviewed.'1° 7 Benzopyrrocolines (-)-Cryptaustoline (69; R1= R2= Me) and (-)-cryptowoline (69; R1R2= CH,) isolated from Cryptocarya bowiei have been shown to have a cis B/C ring fusion with (7R 13s) 1-\OR' OR2 (69) (70) absolute stereochemistry."' Cryptaustoline has been synthe- sized by the cyclization of the iminium salt (70) followed by reductive debenzylation. 8 Berberines and Tetrahydroberberines Alkaloids of the berberine and tetrahydroberberine groups have been isolated from the following plant species the eight marked with asterisks being new alkaloids Alphonsea sclerocarpa' stepholidine Berberis pseudambalata5* berberine and palmatine Corydalis b~ngeana~~ cheilanthifoline and scoulerine Corydalis ochoten~is''~ cheilanthifoline Corydalis pallida114 canadine corydaline dehydrocorydaline and tetra-hydropalmatine Duguetia spixiana24- 25 spiduxine* (7l) tetrahydropalmatine and xylopinine Fumar ia indica' dihydrocoptisine* (72) Fumaria macrosepala 'l6 scoulerine and stylopine Hypeco um lep to carp um ' ' coptisine and trans-N-methylstylopinium hydroxide Hypecoum procumbens"? coptisine and trans-N-methylstylopiniumhydroxide Isopyrum thalictroides"* coptisine Meiogyne virgata' ' corydalmine dehydrocorydalmine discretamine and stepholidine Papa ver nudicaule log coptisine mecambridine palmatine and cis-N-methyl- stylopinium hydroxide Stephania hainanensis120 corydalmine isoscoulerine palmatine and tetrahydro- palmatine NATURAL PRODUCT REPORTS 1989 Stephania suberosa'21 capaurimine coreximine corytenchine discretine kike- manine pseudopalmatine oxypseudopalmatine* (73) tetrahydropalmatine tetrahydropalmatrubine stepha-bine* (74) tetrahydrostephabine* (75; R = Me) steph- abinamine* (75; R = H),stepholidine xylopinine cis- xylopinine N-oxide* and trans-xylopinine N-oxide* Step han ia ven osa kikemanine and tetrahydropalmatine Thalictrum ~ollinurn~~ berberine Thalictrum cultratum 122 berberine columbamine jatrorrhizine palmatine thali- dastine and thalifendine Thalictrum delava~il~~ berberi ne Thalictrum gland~losissimum~~~ berberine coptisine columbamine groenlandicine jatrorrhizine palmatine and thalifendine Tetrahydropalmatine has been isolated from callus cell cultures of Corydalis yanhu~uo'~~ and a patent has been published covering the isolation of berberine from rhizoma coptidis [the rhizomes of Coptis s~ecies1.l~~ The oxidation of berberine with m-chloroperoxybenzoic acid followed by photochemical rearrangement has yielded OMe OMe (73) (74) OR (75) Me0 \ MeS (79) 0 C(01CMe the 2,3,10,11,12-~ubstitutedbase (76; R = OH),which on treatment with diethyl chlorophosphate followed by reduction with sodium in liquid ammonia gave the pseudoberberine (76; R = H).The process must involve oxidative fission of the 8-8a bond and re-cyclization between C-8 and the original C-12. Similar reactions resulted in the conversion of tetrahydro- palmatine into xylopinine and of tetrahydrocoptisine into tetrahydropseudocoptisine. 12' Treatment of oxyberberine with methyllithium followed by treatment with an acid afforded the 8-methylberberinium salt which on reduction gave a mixture of 8a-and 8P-methyltetrahydroberberine.128 Several syntheses of alkaloids of this group have been achieved. The N-benzyldihydroisoquinolinium salt (77) on treament with ethoxyethene afforded the enol ether (78) which was hydrolysed to the ketone (79); cyclodehydration of this followed by reduction of the resulting iminium salt yielded (f)-~orydaline.l~~ The amide (80) (prepared from the secondary amine and methylthioacetyl chloride) was oxidized to the sulphoxide which on cyclization with toluene-p-sulphonic- acid yielded the isomeric lactams (81) and (82); further cyclization of (8 I) followed by reduction (with concomitant desulphurization) yielded tetrahydropalmatine.Production of the lactam (82)can be blocked by a suitably placed bromine atom which is easily removed prior to further cyclization. Tetrahydro- berberine stylopine and sinactine were synthesized by similar processes.130 The Grignard reagent (83) obtained from the corresponding N-pivaloyltetrahydroisoquinoline,t-butyl-lithium and mag- nesium bromide reacts readily with aldehydes with a high degree of stereospecificity to give secondary alcohols in which the alcoholic system may be inverted through the trifluoro- acetate. The product of the reaction between (83) and 3,4- dimethoxy-2-methoxycarbonylbenzaldehyde, of structure (84) was hydrolysed; the resulting amino acid was converted into the lactam (85) which gave (f)-epiophiocarpine (86; R' = OH R2 = H) on reduction to the amine.Reduction of (84) (77) (78) MeS (82) NATURAL PRODUCT REPORTS 1989-K. W. BENTLEY (94) afforded the diol (87) which was converted (by using trifluoroacetic acid and trifluoroacetic anhydride) into the ester (88) with inversion at the alcoholic carbon atom. Hydrolysis of this and cyclization (by thionyl chloride) yielded ( k)-ophio-carpine (86; R' = H R2 = OH).131 Berberastine iodide (94) has been synthesized from the isocoumarin (89) which was reduced to the diol and then oxidized to the keto-aldehyde (90). This with aminoacetal gave the imine (91) which underwent reductive cyclization to the amine (92); mild acid cyclization then yielded the alcohol (93) which was oxidized to berberastine by iodine.132 The biological effects of be~berine,'~~-'~~ of tetrahydrober- berine,l4O.lJ1 of 8-(piodobenzyl)tetrahydroberberine, 14' of pal- matine,138 of tetrahydropalmatine 142-145 of 8-benzyltetrahydro- ~almatine,'~~ of stylopine,140 of stepholidine 142 147 of jatror- rhi~ine,l~~ and of gindarinelJ8 of tetrahydr~jatrorrhizine,'~~ have been studied.A method for the estimation of palmatine has been described149 and the basic polarographic characteris- tics of jatrorrubine have been determined. I5O OH 9 Azaberberines The dihydroisoquinolinium salt (95) has been condensed with the lithium derivative (96) to give the amidine (97; X = NH). Hydrolysis of this to the lactam (97; X = 0),followed by 0-debenzylation afforded ( )-alangimaridine which was oxidized to alangimarine (98) by iodine.151 Following the successful conversion of oxyberberine into 8-methylberberine by methyl-lithium an analogue of (97 X = 0) lacking the vinyl group was synthesized and converted (by treatment with methyl-lithium) into the enamine (99) and the iminium ion (100).Reduction and debenzylation of the iminium salt afforded (k)-alamaridine (I 0I). 128 10 Secoberberines Hypecorine has been isolated from Hypecoum chinensis'" and a new secoberberine alkaloid procumbine (102) has been isolated from Hypecoicm pvocumbens.'17 414 Li' COzMe (103) CaMe R (108) clq) HO OMe \ OMe (110) CH~OAC c% Me OMe OMe OMe (1 12) Treatment of hydrastinine chloride with the lithium salt of the propylenedithiolacetal of 2-carboxymethylpiperonal (103) has afforded the ketal (104) which on reduction with lithium aluminium hydride yielded the alcohol (105).This when converted into the ketone and oxidized yielded the carbinola- mine ether hypecorinine (106). Desulphurization of (104) followed by reduction gave corydalisol (107; R = CH20H) which was oxidized to aobamine (107; R = CHO).153Treatment of the lithium derivative (103) with N-methylhydrastinine gave the lactone (108) which was desulphurized (by reduction over Raney nickel) to pe~hawarine.'~~ 11 Protopines Alkaloids of this group have been isolated from the following plant species Corydalis b~ngeana~~ corycavine and protopine Corydalis g~vanianal~~ protopine NATURAL PRODUCT REPORTS 1989 (:x$jMe n OH (113) Corydalis pallida114 allocryptopine and protopine Fumaria macrosepala116 protopine Glaucium flavum156* 15' allocryptopine and protopine Hypecoum ~hinensis'~~ allocryptopine cryptopine and protopine Hypecoum leptocarpum"' allocryptopine and protopine Hypecoum procumbens" ' allocryptopine and protopine Papaver nudicaule log allocryptopine cryptopine muramine and protopine Thalictrum delavayi122 cryptopine and pseudoprotopine A patent covering the extraction of allocryptopine from Thalictrum species has been published.158 An X-ray determination of the crystal structure of corycavine has been reported. 159 Cleavage of tetrahydroberberine and tetrahydropalmatine with cyanogen bromide under solvolytic conditions has afforded the N-cyano-compounds (109; R1R2 = CH,) and (109; R' = R2 = Me) respectively and these have been hydrolysed to the secondary bases N-methylated and oxidized to allocryptopine and muramine.160 Anhydroiso- dihydrocorycavine (1 10) has been oxidized with osmium tetroxide to the glycol which has been cleaved to the keto- aldehyde; the N-oxide of (1 10) has been converted into a mixture of (111) and (112) by acetic anhydride and acetic acid.l6 12 Pht ha Iide-isoqu ino1ines Phthalide-isoquinoline alkaloids have been isolated from the following plant species Corydalis bungeana23 bicuculline Corydalis racernosal6 adlumine bicuculline and carlumine Fumar ia indica adlumidine and bicuculline Fumaria macrosepala'16 adlumine aobamidine and bicuculline An X-ray-crystallographic study of humosine-A (1 13) has been re~0rted.l~~ The compound (84) which is an intermediate in NATURAL PRODUCT REPORTS 1989-K.W. BENTLEY 415 IH Me.% < H6 Me0 OMe (122) (122a) ( 123) the syntheses of ophiocarpine and epiophiocarpine has been hydrolysed at the amide system only to give nor-/3-hydrastine which yielded /3-hydrastine when it was N-rnethylated.l3l The biological effects of bicu~ulline,~~~ and of capn~idine,~~ of the narcotine analogue fritoq~aline'~~. have been studied 166 and a patent covering the purification of analogues of narcotine has been published. 167 'oz Me0 13 Spirobenzylisoquinolines Spirobenzylisoquinoline alkaloids have been isolated from the following species the two marked by an asterisk being new alkaloids Corydalis g~vaniana'~~ 13-epi-yenhusomine*(1 14) and ochotensine CorydaIis ochotensis' ' ochotensine and iso-ochotensine* (1 15) Fumar ia macrosepala 'l6 fumaritine and parfumine Further details of the acid-catalysed conversion of the cycloberberines (116; R = H) (116; R = Me) and (116; R = Et) into spirobenzylisoquinolines that had previously been reported have been published.'68 The trypanosomicidal activity of corpaine has been examined.g3 14 lsoindolobenzazepines The phthalimide (1 17) has been cyclized photochemically to the isoindolobenzazepine (1 18) which was dehydrated to the olefin.This olefin on oxidation successively with lead tetra- acetate and m-chloroperoxybenzoic acid gave the cyclic orthoacetate (119) which was hydrolysed to the lactam (120; R = H); this has the carbon-nitrogen skeleton of the alkaloids palmanine (120; R = OMe) and chilenine.'69 15 Rhoeadines Alpinigenine epialpinine and papaverrubines A B D and G have been isolated from Papaver nudica~le'~~ and a new alkaloid of this group zangezurine (121) has been isolated from Papaver zangezuricum. 170 This is the first rhoeadine alkaloid to be discovered that bears five oxygen substituents on the aromatic nuclei. Thermal degradation of alpinigenine N-oxide has been found to proceed principally by Meisenheimer rearrangement to the base (122) though under more extreme conditions some of the product (122a) of Cope elimination was obtained as well.Similar results were obtained with cis-alpinigenine N-oxide and with the two 0-methyl derivatives. In all of the rearrangements full retention of configuration was 0b~erved.l~~ Full details of the conversion of palmatine betaine (123; R' = OMe R2= H) through its isomer (123; R1= H R2= OMe) and (124) into (1 29 which comprises a formal synthesis of cis-alpinigenine (previously reported) have been published. 172 NATURAL PRODUCT REPORTS. 1989 Me0P h/ C a Me0 \phcH209CH2C02H\'E t 16 Emetine and Related Compounds Ankorine has been synthesized from the lactam (126)173 and 9- demethyltubulosine has been synthesized from the acid (127).174 Methods for the total synthesis of emetine have been reviewed.175 The biological effects of emetine176-178 and of dihydr~emetine'~~ have been studied.(127) 17 Benzophenanthridines Benzophenanthridine alkaloids have been isolated from the following plant species the seven marked with an asterisk being new alkaloids Coptis japon ica' sanguinarine norsanguinarine oxysanguinarine and 6- aceton yldi h ydrosanguinarine Corydalis b~ngeana~~. la0 bungeanine* (1 28) corynoline U-acetylcorynoline U- acetylisocorynoline 11-epi-corynoline I 3-epi-corynoline* L O LO (1 29) 12-hydroxycorynoline and dihydrosanguinarine Fumar ia indica ' norsanguinarine Glaucium jIav~m'~~* 157 dihydrochelerythrine dihydrochelirubine dihydrosan- guinarine and norchelidonine Hypecoum lep tocarp um chelerythrine and sanguinarine Hypecoum procum bens ' ' chelerythrine and sanguinarine Zanthoxylum leprieuriila1 chelerythrine dihydrochelerythrine dihydroavicine fag- aridine and nitidine A Zanthoxy lum spinosum la bocconoline decarine 0-acetyldecarine* chelerythrine norchelerythrine dihydrochelerythrine 6-acetonyldihydro- chelerythrine [130; R = CH,C(O)Me] 6-carboxymethyl- dihydrochelerythrine* (1 30 ;R = CH,CO,H) 6-(4-methyl- 2-oxopentyl)dihydrochelerythrine* [130; R = CH,C(O)-CH,CHMe,] chelelactam* (1 3 l) and caymandimehe* The structures (132) of the new alkaloids were determined by spectroscopic methods and that of 13-epi-corynoline was confirmed by an X-ray-crystallographic study.la0 An X-ray study has also been made of coryn01ine.l~~The structure of bocconine has been supported by further spectroscopic stud- ie~.'~~A patent covering the extraction of chelidonine from Chelidonum majus has been p~b1ished.l~~ The effect of pH on the iminium salt/carbinolamine balance in solutions of san- guinarine has been studied.la6 Full details of the conversion of berberine into oxo- chelerythrine and dihydrochelerythrine by a route previously reported have been published,la7 as have details of similar conversions of 12-methoxypalmatine into sanguilutine la*of 0- benzyldehydrodiscretine into fagaronine of pseudoberberine into nitidine,la9 and of 10-0-demethylpseudoberberine into oxyterihanine. Dihydrochelerythrine has been prepared presumably through an intermediate benzyne by treatment of the bromo- compound (133 R = Me) with sodamide.Oxidation of the product gave chelerythrine. Decarine was prepared in a similar manner from (133; R = H)lS1 Condensation of the amide (134) with the imine (135) in the presence of lithium CH-CHO with trapping of the resulting 4-lithio-3,4-dihydroisoquino-lone with ethylene oxide gave the alcohol (136; R = CH,OH). Oxidation of this to (136; R = CO,H) and cyclodehydration afforded the ketone (137) which was converted into oxonitidine by reduction dehydration and dehydrogenation. Syntheses of fagaroninelS3 and of teri- hanine and is~terihanine'~~by conventional Bischler- Napieralsky cyclizations of appropriately substituted forma- mides have been reported. The dihydrobenzophenanthridi- NATURAL PRODUCT REPORTS 1989-K.W. BENTLEY 417 p:) I 0 NMe 0 (136) Me Me0 ( 143) none system has been synthesized by condensation of benzyne with the keto-lactam (1 38).lS5 The biological effects of sanguinarine,lS6 of fagaro-nine,197.198 of fagarine,lg9 and of other alkaloids of the group200 have been studied 18 Aporphinoid Alkaloids 18.1 Proaporphines Proaporphine alkaloids have been isolated from the following plant species the nine marked with asterisks being new alkaloids Abuta pahni10 stepharine Alphonsea sclerocarpa' crotsparine and stepharine Annona hayesii201 stephari ne Meiogyne virgata' l9 stepharine (134) ( 135) 0 II op NMe It (137) (138) Me0 OMe ti0 (1411 (142) Roemeria hybrida202 mecambrine orientalinone (1 39 ; R = H) iso-oriental-inone* (140; R = I-?) roemerialinone (139; R = Me) isoroemerialinone* (140; R = Me) 1 1,12-dihydro-orient- alinone* 8,9-dihydroisoroemerialinone* a-roemehy-brine* (141) and roehybrine (142) Stephania hainanensis120 stepharine Stephania veno~a~~ stepharine N-carboxamidostepharine* (143) stepharino- sine* (144; R = H) and 0-methylstepharinosine* (144; R = Me) The structure of the previously isolated203 roehybrine (142) was elucidated for the first time.202 In addition it has been shown202 that 8,9-dihydroiso-orientalinoneis identical with an alkaloid of undetermined structure previously isolated from Papaver ~rientale.~~~ Controlled hydrogenation of stepharine has been shown to take place principally on the side of the molecule opposite to the hydrogen atom of C-6a leading mainly to 8,9- dihydrostepharine with small amounts of the 11,12-dihydro-and 8,9,11 12-tetrahydro-compounds.205 18.2 Aporphines Aporphine alkaloids have been isolated from the following plant species the twenty marked with an asterisk being new alkaloids Adonis aestivalW8 corytuberine and magnoflorine Adonis vernaliP8 corytuberine and magnoflorine Alphonsea sclerocarpa' anonaine isoboldine laurotetanine magnoflorine sparsi- florine ushinsunine and norushinsunine NATURAL PRODUCT REPORTS 1989 Me0 / MeoFH Me0 \ (1 45) (147) (1 48) (1 52) Annona hayesii2" anonaine asimilobine litsiferine nordomesticine nuci- ferine nornuciferine 3-hydroxynornuciferine and ro-emerine Aristolochia clematitis"' corytuberine and magnoflorine Caltha palustris"8 corytuberine and magnoflorine Clematis rectalla corytuberine and magnoflorine Clematis vitalba118 corytuberine and magnoflorine Consolida regalis ' corytuberine and magnoflorine Corydalis b~ngeana~~ isoboldine Corydalis g~vanianal~~ isocorydine Dactylicapnos scandens206 corydine and glaucine Discaria chacaye207 0-methylnoriso thebaine* (145) Duguetia spixiana24* 25 anonaine N-methylasimilobine duguexine* (146; R = H) duguexine N-oxide* 0-methylisopiline nornuciferine 3-hydroxynornuciferine nornuciferidine* (147 ;R = Me), oliveridine oliveridine N-oxide noroliveridine pachy- confine pachyconfine N-oxide* norpachyconfine* (147 ; R = H) roemerolidine (148) rurrebanine* (149; R = Me) rurrebanidine* (149; R = H) spixianine* (146; R = (149) OMe) spixianine N-oxide* and duguespixine (1 50 ;R' = R2 = R3= Me) Epimedium versicolor'18 corytuberine and magnoflorine Eranthis hiemalis' l8 corytuberine Fumaria macrosepala116 isoboldine Glaucium j?a~urn'~'* 208 cataline corydine isocorydine isoboldine glaucine de- hydroglaucine and didehydroglaucine Helleborus foetidus"' co rytu berine Helleborus niger' l8 Cory tube rine Helleborus viridis"' corytuberine and magnoflorine Hernandia bivalvi~~~~ hernandine Hypecoum lep tocarpum' ' corydine and isocorydine Hypecoum procum bens '" corydine and isocorydine Isopyrum thalictroides'" coptisine corytuberine and magnoflorine Meiogyne virgata"' anonaine corytuberine and norushinsunine Nelumbo nuci$era2'0 asimilobine and lirinidine Oxandra major27 anonaine and nornuciferine Phoebe valeriana2" 3-hydroxyglaucine nantenine 3-hydroxynantenine* (1 51; R1= H R2 = Me) nordelporphine* (152) phoebine* (151 ; R1= R2= Me) norphoebine* (1 51 ; R' = Me R2 = H) dehydrophoebine* (1 53) and thaliporphine Rollinia mucosa212 anonaine and N-formylanonaine Sinomenium acut um213 N-formylnordehydronuciferine*(1 54) Stephania epigeae62 dicentrine and oliveroline Stephania gracilenta2' magnoflorine 419 NATURAL PRODUCT REPORTS 1989-K.W.BENTLEY ORL OR Stephania hainanensid2' crebanine and dehydrocrebanine Stephania veno~a~~ anonaine asimilobine apoglaziovine crebanine 4-hydroxycrebanine dehydrocrebanine mecambroline nuciferoline stephadiolamine P-N-oxide* (1 55 ;R = OH) stesakine sukhodianine sukhodianine P-N-oxide* (1 56) tuduranine ushinsunine ushinsunine P-N-oxide* (1 55 ; R = H) Thalic trum aquilegifolium 21 isoboldine isocorydine and magnoflorine Thalictrum c01linum~~ glaucine isoboldine and magnoflorine Thalictrum cultratum'22 magnoflorine Thalictrum glandulosissimum'24 magnoflorine Me0 OMe CHO OMe OMe (163) (164) Thalictrum hazarica215 N-methyl-lauro tetanine Zanthoxylum leprieuriil8' magnoflorine Reviews of the chemistry of aporphines of species of Thal-ictrum7'." and of species of members of the Annonaceae216 have been published.The n.m.r. spectra of glaucine217 and of derivatives of tuduranine218 have been studied. Hofmann degradation of steporphine methiodide has been found to give only the ~henanthrene.,'~ Baicalidine,220glaucine,221* 223 N-methyl-222 thalicsimidine,222 di~entrine,~~~ laur~tetanine,~~~ and ca~sythicine,,~ have been synthesized by oxidation of the appropriately substituted benzyltetrahydroisoquinolines with thallium trifluoroace-tate,220v 221 ruthenium(1v) trifluoroacetate,222 or lead tetra-acetate.223 Pschorr cyclization of 6'-aminobenzyltetrahydro-isoquinolines has afforded syntheses of isob~ldine~~~ and of hernandine,225 and wilsonirine and nordomesticine have been prepared by acid-catalysed cyclizations of the quinone deriva- tives (157;R' = R2 = Me) and (157;R'R2 = CH,) respect- ively.226 Treatment of the Grignard reagent (83) with 2-bromobenzaldehyde afforded the N-pivaloyl derivative of (1 58; R1 = H R2= OH) which was hydrolysed to (158;R1= H R2= OH) and converted [through the 0-trifluoroacetyl ester] into (158;R' = OH,R2 = H); these epimeric alcohols were photochemically cyclized to oliveroline and ushinsunine re~pectively.'~~ Oxidative cyclization of the lactams (1 59; R = H) and (159; R = Me) has yielded the pentacyclic compounds (1 60 ;R = H) and (1 60; R = Me) in a study of the effects of conformation on ease and direction of cy~lization.~~~ The reaction of l-ethylidene-2-formyl-6,7-methylenedioxy-tetrahydroisoquinoline with benzyne has afforded the N-formyl dehydroaporphine (1 50; R1R2= CH, R3 = CHO) the struc- ture of which was confirmed by an independent photo- chemical synthesis.This proved to be different from the alkaloid trichoguattine to which that structure has been assigned.228 The dehydronorglaucine derivative (1 61) has been shown to be a very potent inhibitor of phosphodiesterase. 229 Treatment of dehydroglaucine with chloroform and 50% sodium hy- droxide yielded (162) which was hydrolysed by hot aqueous ethanol to (163; R = CHCl,) whereas the reaction in 30% sodium hydroxide afforded the aldehyde (163; R = H). 7-Chlorodehydroglaucine under similar conditions gave the tetrachloro-compound (1 64).230 NATURAL PRODUCT REPORTS 1989 (165) (166) Methods of preparing apomorphine and its analogues from alkaloids of the morphine group have been reportedz3'-232 and the biological effects of apomorphine have been 18.3 Dimeric Aprphines Five new 7,7'-dimeric dehydroaporphines have been reported.Urabaine (165; R1 = R2 = R3 = R4 = Me) N-methylurabaine and N,N'-dimethylurabaine have been isolated from Oxandra major27,248 and urabaine unonopsine (165; R1R2 = R3R4= CH,),and heteropsine (165; R1R2= CH, R3 = R4 = Me) have been isolated from Unonopsis spe~tabilis.~~~ Urabaine and N,N'-demethylurabaine have been synthesized by the oxidation of deh~dronantenine~~~ respect-and of dehydr~nuciferine,~~~ ively with mercuric nitrate ; similar oxidations of dehydro-anonaine and of dehydroxylopine have been accomplished.250 18.4 Benzylisoquinoline-Aporphine Dimers A new benzylisoquinoline-aporphine dimer thalifaboramine (I 66) has been isolated from Thalictrum fabe~i.~~' 18.5 Oxoaporphines Oxoaporphine alkaloids have been isolated from the following plant species that marked by an asterisk being new Alphonsea sclerocarpa' liriodenine Annona hayesii"' liriodenine and lysicamine (1 68) (169) (171) Duguetia ~pixiana~~. 25 lanuginosine lysicamine and U-methylmoschatoline Glaucium j?a~um'~~ corunnine and oxoglaucine Meiogyne virgatallg liriodenine Oxandra major,' liriodenine and lysicamine Phoebe valeriana211 oxophoebine* (1 67) and 0-methylmoschatoline Rollinia mucosa212 lanuginosine and liriodenine Stephania hainanensis120 oxocrebamine The chemistry of the alkaloids of Liriodendron tulip$era has been reviewed.252 An X-ray-crystallographic study of 0-acetylisomoschatoline confirming the structure of isomos- chatoline has been The antifungal properties of liriodenine liriodenine methiodide and oxoglaucine methio- dide have been 18.6 Dioxoaporphines Norcepharadione-A has been isolated from Annona hayesii.201 Cepharadione-A dioxodehydroasimilobine and the new al- kaloid aristolodione (168) have been isolated from Aristolochiu chilen~is.~~~ A new alkaloid dihydropontevedrine [represented in its carbinolamine form by the structure (169)] which is a reduced dioxoaporphine has been isolated from Glaucium fla~um.'~~ It is identical with material that had previously been produced by the irradiation of glaucine with ultraviolet light.256 18.7 Phenanthrenes Atherosperminine atherosperminine N-oxide and methoxy- atherosperminine have been isolated from Duguetia spixiana.24 The new alkaloids phoebine normethine (170; R = H)and thalihazine (170; R = Me) have been isolated from Phoebe va1eriuna"l and Thalictrum ha~arica,,'~ respectively. 18.8 Aristolochic Acids and Aristolactams Two new aristolactams F-I (171 ; R' = R3 = H,R2 = Me) and F-II(l71; R' = OMe R2 = H R3 = Me) as well as the known 42 1 NATURAL PRODUCT REPORTS 1989-K. W. BENTLEY (172) H OMe aristolactams I and A-11 have been isolated from Par-aristolochia Jlos-avi~.~~' Aristolochic acids I and I1 have been found to be metabolized in rats to aristolactams I Ia and 11 aristolochic acid Ia and 3,4-methylenedioxyphenanthrene-1-carboxylic acid and its 8-hydroxy-derivati~e.~~~ The chemistry of these compounds has been reviewed.259 18.9 Azahomoaporphines Two new azahomoaporphine alkaloids spiguetine (1 72; R = Me) and spiguetidine (172; R = H) have been isolated from Dugue tia spixiana .25 19 Morphine Alkaloids Alkaloids of the morphine group have been isolated from the following plant species the six marked by asterisks being new alkaloids Cocculus laurlfolius2' sebiferine Cocculus trilobus260 sinococculine* (1 73) Papaver nudica~le'~~ nudaurine Papaver somniferum26 somniferine* (1 74; R = H) and 0-methylsomniferine* (174; R = Me) Stephania epigeae62 sinomenine Stephan ia gracilen ?azs sinoacutine and isosinoacutine NMe2 Stephan ia su berosa2 62 delavaine nordelavaine* (1 75 ; R1R2= CH,) stephanu-bine* (175; R1= R2 = Me) and stephaphylline* (176) In addition the dibenzazonine alkaloids laurifinine and laurifonine which are analogues of neodihydrothebaine have been isolated from Cocculus laurifolius.22 The dimeric bases somniferine and U-methylsomniferine represent a new group of morphine alkaloids.O-Methylsomni- ferine is a dimer of thebaine with 14-hydroxycodeinone which has been found to occur naturally in Papaver orientale; the new base could result from the oxidation of a 7,16'-dimer of thebaine itself formed by oxidation of thebaine to the iminium salt and reaction of this with the enol ether system of unchanged thebaine.Codein~ne~~~ and 3-benzylmorphinoneZ6* have been sub- jected to reductive opening of the 4,5-oxide bridge to give thebainone-A and its 3-U-benzyl analogue and these have been converted through the toluenes-p-sulphonylhydrazones,into deoxycodeine-A and deoxymorphine-A. 264 Mixing U-rnethyl- metathebainone methine (1 77) or 0-methylsalutaridine methine (1 78) with methyltriphenylphosphonium bromide in an at-tempted Wittig reaction resulted in their rearrangement to the phenanthrenes (179; R = H) and (180; R = H). The bases (179; R = Me) and (180; R = Me) were obtained if ethyltri- phenylphosphonium bromide was 6-Demethoxytheb-aine and its 6,7-dichloro- and 6,7-dibronio-derivativeshave been oxidized to the 8,14-P-epoxide (1 8 1) and its derivatives which were hydrolysed to 8a 14P-dihydroxydeoxycodeine-C (1 82) and its halogenated analogues.266 N-Formyl-6-demethoxynorthebaineinteracts with nitro-ethene to give a mixture that contains two major and three \OSi Me But minor components of which only the product (183) of Diels-Alder addition from the rear has been identified.267 The adducts (1 84)and (1 85) have been obtained from thebaine and 2-allylphenol and 4-phenyl- 172,4-triazoline-3,5-dione, respect-ively.268 Some conformationally constrained analogues of etorphine have been prepared.In an early approach the lactone (186) which had been prepared by reduction of the Diels-Alder addition product of thebaine and dimethyl acetylenedicarboxylate was converted successively into the ether esters (187; R = Me) (187; R = H) and (187; R = CH,CO,Me) ; Dieckmann cyclization of the last of these afforded the keto-ester (1 88).269 The diol (1 89) which was prepared by the reduction of the Diels-Alder adduct of N-NATURAL PRODUCT REPORTS 1989 R OSiMe2Bu' benzoylnorthebaine and maleic anhydride was successfully converted in good yield into both the 7-and the 8-mono-t- butyldimethylsilyloxymethyl compound.Of these the 801-hydroxymethyl compound was oxidized to the 8a-aldehyde and this was epimerized to the 8P-aldehyde (190; R = CHO) which was reduced to (190; R = CH,OH).Cyclization of the mesyl ester of this alchohol gave the quaternary salt (191) which was reduced to the base (192; R = CH,OSiMe,); but this was converted {through (1 92 R = CHO) and [192; R = CH(OH)CH,]) into [192; R = C(O)CH,] which was converted into the 8P,N-methanonoretorphineby normal processes.27o The 8P,N-ethano-analogue of [192 R = C(O)CH,] was pre- pared from (1 92 ;R = CHO) via [192:R = CH(OH)CH,SiMe,] 423 NATURAL PRODUCT REPORTS 1989-K. W. BENTLEY (192; R = CH=CH,) (192; R = CH,CH,OH) and (192; R = CH,CH,OMes) and it too was converted into an analogue (193) of etorphine. The 8a,N-ethano-analogue of (193) was prepared from the 8a-t-butyldimethylsilyloxymethylanalogue of (189) via (194; R = CH,OH) (194; R = CHO) (194; R = CH=CH,) (195; R = CH,OH) (195; R = CH,CN) (195; R = CH,CHO) and (195 ;R = CH,CH,OH) followed by con- version into the secondary base and the N-benyloxycarbonyl analogue of (195; R = CH,CH,Br) which was cyclized to (196; R = CH=CH,).This was converted through (196; R = CHO) and [196; R = C(O)CH,] into the related tertiary carbinol which was demethylated to the etorphine analogue. (197) X=O All three analogues showed very greatly diminished analgesic (198) X = NMe activity compared with et~rphine.,~~ The n.m.r. spectra of a number of compounds in the morphine-thebaine group have been and an X-ray-crystallographic study of naltrexonazine has been re-Details of the preparation of the following have been published :3-O-propanoyl- and 6-O-propanoyl-m0rphine,~~~ 3-O-(2-hydroxyprop-2-yl)-morphine and -norm~rphine,~~~ N-substituted norheroin~~~' and nor morphine^,^'^ samples of "C-labelled 14-hydro~ydihydromorphinone~~~ and diprenor-phine,280 tritium-labelled 14-hydroxydihydromorphinone hy-drazone,281 oxime ethers of 14-hydroxydihydromorphinone naloxone and naltrexone,282 hydrazones of morphinone, RJ di h ydromorphinone and its 14- hydroxy-deriva tive 283 crown ethers derived from 6-(3,4-dihydroxybenzoyl)-estersand amides (199) of morphine and 6-aminodihydrodeoxymorphine 284 phenols by demethylation of codeine buprenorphine,286 14-alkyl-substituted dihydromorphines dihydrocodeines and their 6-amino-6-deoxy-analogues,2a7 heterocyclic compounds of general structures (197),288 (1 98),288 and (199) and OMe condensation products of steroidal ketones with the hydrazones of naloxone and of naltrex~ne.,~~ Full details of the synthesis of the morphinan ring system from the benzocyclobutene (200) previously reported have MeoQ been Diels-Alder addition of 1-chloro- l-cyano- ethene to the appropriate diene yielded (201) which was hydrolysed to the ketone and Beckmann transformation of the oxime of this gave (202; R = CN).With suitable protection of OMe the keto-group this was converted [through (202 ; R = CHO)] into (202; R = CH,OCH,OMe). Reduction of this to the alcohol followed by Claisen rearrangement of its ally1 ether then afforded the aldehyde (203) which was cyclodehydrated to (204; R = OCH,OMe). Conversion of this into (204; R = NHMe) followed by treatment with N-bromosuccinimide in methanol yielded (205).295 Electrolytic oxidation of (R)-laudanosine has given (9R)-O-methylfla~inantine.~~~ Me0 Methods for the detection and estimation of m~rphine,~~~-~~~ of codeine,300-303,306,307,309of her~in,~l~-~'~ of 6-O-acetylmor- ~hine,~O~ of 3-O-morpholinoethyl- of 3-O-ethylm0rphine,~~~ R of 3- and 6- and 3,6-di-O-ni~otinylmorphine,~~~ of Meo2f naltre~one,~,~ have been described.and of b~prenorphine~~l The analgesic propertie~~~~-~~~ of and pharmac~kinetics~~l-~~~ morphine have been studied as have the effects of the alkaloid on the gastro-intestinal tra~t,~~~-~~~ 0 on beha~iour,~~~-~~~ on the (203) cardiovascular ~y~tem,~~~-~~~ on on body temperat~re,~~~-~~~ on the brain,401-404 on the (202) re~piration,~~~,~~~ on the eye,405.406 liver,407.408 on the kidney,409 on reproductive endocrin~logy,~~~ on the pituitary gland,411 on the ovaries,412 on the uteru~,~'~.~~~ on neu~ones,~'~*~~~ on the immune response,417 on energy balance,418 on intracranial on the metabolism of glucose42o and of n~cleotides,~~~the plasma levels of on cortic~sterone,~~~ of cholesterol,*** of prolac-of tin,423-425 of growth of luteinizing of thyrotropin-releasing of antidiuretic and of melat~nin,~,~ levels of @-endorphin and [Met5]- on enkephalin in brain,428 on the release of ad~enalin,~,~ of and of histamine,431.5-hydro~ytryptamine,~~~ 432 and on the of amphetamine,"' of thi~pental,~~~ effects of hal~thane,~~~ and of clomipramine.436 The morphine antagonist properties of naloxone have been NATURAL PRODUCT REPORTS 1989 Me0 OMe (207) (206) (208) AC:qMe Me0 Meow Me0 Me0 (21 1) Me0 Me0 (212) ~t~died,~~~-~~~ as have the effects of this compound on behavi~ur,~~~' 3639 369.371+443-448 on the intake of 450 and of water,451-453 on the cardiovascular on body temperat~re,~~~-~~~ on the ~ystem,~~~-~~'gastro-intestinal on respira-MI? -0 OMe lowing have also been studied morphine 6-gluc~ronide,~~~~~~~ 495 497 codein~ne,~~~ codeine,400. dihydroc~deine,~~~ N-allylnormorphine 500 N-cyclopropylmethylnormor-502 phine,jO' 14-hydroxydihydromorphinone402~ and its hy-504 naloxa~one,~~~ draz~ne,~~~.naloxonazine and some of its analogues,506 naltrex~ne,~~~. naltrexone N-507-515 meth~bromide,~~~3-0-salicylylnaltrexone,516 nalbu-phine,365.402,437,438,517,518 nalmefene,418.462,519.520 p-funal trexa- mine,521-524 the pyrrole (199; R1 = R3 = H R2 = CH2C3H5 R4 = OH),525 condensation products of steroidal ketones with 14-hydroxydihydro-N-allylnormorphinone etor-hydraz~ne,~~~ ~hine,~,~ dihydroetorphine 526 buprenorphine 365. 422. 437. 529-534 dipren~rphine,~~~. 7-[di(2,chloroethyl)amino]-6,14-~~do-535-537 ethano-~ripavine,~~~ 7,8-dihydro-5',6'-dimethylcyclohex-5- sin~menine,~~~. eno[1 ',2' 8,14}~odeinone,~~~ 540 and 0-methyl- sinomenine.541 20 Phe net h yIisoquino1ines The new alkaloid isoautumnaline (206) together with the new homomorphinandienone alkaloids ( )-colchiritchine (207; R1R2= CH,) and (4-)-androcymbine (207; R' = H R2 = Me) has been isolated from Colchicurn rit~hii.~'~ Androcymbine had previously only been known as the laevorotatory enan- tiomer.In addition to isoautumnaline only three simple 1-phenethylisoquinoline alkaloids are known the others being autumnaline homolaudanosine and dysoxyline. 21 Homoaporphinoid Alkaloids An X-ray-crystallographic study of luteidine has shown this alkaloid to have the structure (208) enantiomeric with that reported Regecoline (209) has been prepared by the oxidation of the corresponding phenolic tetrahydroisoquinoline kesselringine photochemically and with iodine.544 Homoglaucine (210; R' = Me R2 = H) has been synthesized by the oxidation of homolaudanosine with ruthenium(1v) trifluoroacetate,222 0-methylkreysigine (210; R' = Me R2 = OMe) has been obtained by oxidizing 5'-methoxyhomolaud- and anosine with thallium(II1) trifluor~acetate,~~~ 0-acetyl-homothalicmidine (210; R1 = Ac R2 = H) has been prepared by the acid-catalysed cyclization of the quinone ketal (2 11)546 and its 4-acetoxy-3-methoxy-isomer has been prepared in the same way from the isomeric ketal (212).547 The homoaporphine system has also been synthesized by photochemical cyclization of the bromo-amide (213) both ortho and para to the hydroxyl group followed by Bischler-Napieralsky ring-closure and reduction.546 ti~n,~~~*~~~ on the eye,405g406 on the brain,465 on the kidney,466 on the pituitary gland,411*467 on the hypothalamus,468 on neur- ones,469 on the spinal on T-lyrnpho~ytes,~~~ on the on endotoxin ~ho~k,~~~-~~~ growth of t~mours,~~~ haemorrhagic 22 Colchicine 476,477 and hypovolaemic on levels of pr~lactin,~~~-of luteinizing hor- 480 of growth horm~ne,~~~~ m~ne,~~O of luteinizing-hormone-releasing hormone,482 of aldo- ~terone,~~~ of of 0estradio1,~~~ cat echo la mine^,^^^ and of glucose,485 and on the effects of antidepre~sants,~~~ of bromo- ript tine,^'^ of pent~barbital,~~~.and of N-methyl-D-aspartic acid.490 The pharmacological and physiological effects of the fol- Colchicine 3-P-glucoside (colchicoside) has been isolated from Colchicurn uut~rnnule~~~ and the two new alkaloids speciorit- chine (214; R1= H R2 = Me) and speciocolchine (214; R1 = Me R2= H) have been isolated from Colchicurn rit~hii.~'~ Colchicine has been reduced to two isomeric tetrahydro- colchicines and four isomeric hexahydrocolchicines and de- tailed structures have been assigned to these on the basis of their n.m.r.The circular dichroi~m~~~ and resonance NATURAL PRODUCT REPORTS 1989-K. W. BENTLEY 15 S. G. Pyne J. Chem. Soc. Chem. Commun. 1986 1686. 16 S. G. Pyne and S. L. Chapman J. Chem. SOC. Chem. Commun. MeowN MeowN Me0 \ Me0 \ 1986 1688. HQJ Meow H‘\ MeO2C-F Raman552 spectra of colchicine and its derivatives have been studied. . The biological effects of colchicine have been reviewed553* 554 and the effects of the alkaloid on the brain,555 on the liver,556*557 on the eye,558 on pulmonary epithelial cells,559 on growth-arrested cells,56o on the immune response,561 on the neuro- 17 R.P. Polniaszek and J. A. McKee Tetrahedron Lett. 1987 28 451 1. 18 G. R. Lenz Heterocycles 1986 26 721. 19 M. Kennedy C. J. Moody C. W. Rees and J. J. Vaquero J. Chem. Soc. Perkin Trans. 1 1987 1395. 20 C. D. Chiang F. Kanzawa Y. Matsushima H. Nakano K. Nakagawa H. Takahashi M. Terada S. Morinaga and R. Tsuchiya J. Pharmacobio.-Dyn. 1987 10 43 1. 21 G. Bringmann in ‘The Alkaloids’ ed. A Brossi Academic Press New York 1986 Vol. 29 p. 141. 22 S. Jain K. P. Madhusudanan and D. S. Bhakuni Indian J. Chem. Sect. B. 1987 26 308. 23 W.-G. Zeng W.-Z. Liang and G.-S. Tu Planta Med.1987 53 418. 24 D. Debourges F. Roblot R. Hocquemiller and A. Cave J. Nut. Prod. 1987 50 664 and 852. 25 S. Rasamizafy R. Hocquemiller A. Cave and A. Fournet J. Nut. Prod. 1987 50 674. 26 H.-G. Tian D.-W. Li and H.-C. Lu Zhongcaoyao 1987 18 151. 27 G. J. Arango D. Cortes B. K. Cassels A. Cave and C. Meri- enne Phytochemistry 1987 26 2093. 28 R. L. Khosa Y. Mohan and A. K. Wahi Indian J. Nut. Prod. 1987 3 8. 29 B. Charles J. Bruneton K. Pharadai B. Tantisewie H. Guinaud- eau and M. Shamma J. Nut. Prod. 1987 50 11 13. 30 I. D. Rae and P. M. Simmonds Aust. J. Chem. 1987 40,9 15. 31 H. Guinaudeau A. J. Freyer and M. Shamma Nut. Prod. Rep. 1986 3 477. muscular junction,562 on experimental encephal~myelitis,~~~ 32 and on the central nervous have been studied.23 Other Alkaloids A synthesis of aaptamine (216) has been achieved by the reduction of the nitro-compound (2 15) by triphenyl phos- and another synthesis by a route similar to that previously used has been The two unusual isoquinoline alkaloids jamtine N-oxide (217)567and cohirsine (218)568have been isolated from Cocculus hirsutus their structures being deduced from their spectra. The occurrence of these two alkaloids in the same plant and the fact that (217) is the N-oxide of a base of the same composition (C,,H,,NO,) as cohirsine suggests that they are derived from a common precursor which may well belong to the tetrahydro- berberine group. 24 References 1 D. Tadic G. P. Wannigama B. K. Cassels and A. Cave J.Nut. Prod. 1987 50 518. 2 M. Nieto An. Asoc. Quim. Argent. 1987 75 11. 3 R. H. Abu-Eittah M. M. Hamed and M. M. Abdou Int. J. 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ISSN:0265-0568    

DOI:10.1039/NP9890600405

出版商:RSC    

年代:1989

数据来源: RSC