摘要:

Natural Product Reports A journal of current developments in bio-organic chemistry Volume 8 Number 6 CONTENTS 527 A Unified Mechanistic View of Oxidative Reactions Catalysed by P-450 and Related Fe-Containing Enzymes M. Akhtar and J. N. Wright 553 Indolizidine and Quinolizidine Alkaloids J. P. Michael Reviewing the literature published between July 1989 and June 1990 573 The Biosynthesis of Polyketides T. J. Simpson Reviewing the literature published between January I986 and December 1988 603 Tropane Alkaloids G. Fodor and R. Dharanipragada Reviewing the literature published between January 1990 and December 1990 37 NPR 8 Cumulative Contents of Volume 8 Number 1 Diterpenoids (1989) J. R. Hanson 17 Steroids Reactions and Partial Synthesis (November 1987 to October 1988) A.B. Turner 53 Quinoline Quinazoline and Acridone Alkaloids (July 1988 to June 1989) J. P. Michael 69 Terpenoid Glycosides (1987 and 1988) H. Pfander and H. Stoll Number 2 97 Marine Natural Products (1989) D. J. Faulkner 149 The Biosynthesis of Shikimate Metabolites (1989) P. M. Dewick 171 Muscarine Oxazole Thiazole Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1988 to June 1989) J. R. Lewis 185 The Biosynthesis of Plant Alkaloids and Nitrogenous Microbial Metabolites (August 1988 to July 1989) R. B. Herbert Number 3 213 Pyrrolizidine Alkaloids (July 1989 to June 1990) D. J. Robins 223 Carotenoids and Polyterpenoids (1988) G. Britton 251 Recent Progress in the Chemistry of Indole Alkaloids and Mould Metabolites (July 1989 to June 1990) J.E. Saxton 309 The Occurrence and Biological Activity of Drimane Sesquiterpenoids (up to January 1990) B. J. M. Jansen and A. de Groot 319 The Synthesis of Drimane Sesquiterpenoids (up to January 1990) B. J. M. Jansen and A. de Groot Number 4 339 P-Phenylethylamines and the Isoquinoline Alkaloids (July 1989 to June 1990) K. W. Bentley 367 Terpenoid Phytoalexins (August 1984 to December 1989) C. J. W. Brooks and D. G. Watson 391 Modern Separation Methods A. Marston and K. Hostettmann 4 15 Withanolides and Related Ergostane-type Steroids E. Glotter Number 5 441 Biosynthesis of C,-C, Terpenoid Compounds (1989) M. H. Beale 455 The Lycopodium Alkaloids (January 1986 to October 1990) W. A. Ayer 465 Marine Sterols (up to July 1990) R.G. Kerr and B. J. Baker 499 Diterpenoid Alkaloids (middle of 1985 to end of 1989) M. S. Yunusov Articles that will appear in forthcoming issues include Pyrrole Pyrrolidine Piperidine Pyridine and Azepine Alkaloids (July 1989 to June 1990) A. R. Pinder Steroids Reactions and Partial Syntheses (1989) A. B. Turner Quinoline Quinazoline and Acridone Alkaloids (July 1989 to June 1990) J. P. Michael Muscarine Oxazole Thiazole Imidazole and Peptide Alkaloids and Other Miscellaneous Alkaloids (July 1989 to June 1990) J. R. Lewis Diterpenoids (1990) J. R. Hanson Angucycline Group Antibiotics J. Rohr and R. Thiericke The Biosynthesis of Shikimate Metabolites (1990) P. M. Dewick Amaryllidaceae and Sceletium Alkaloids (1990) J. R. Lewis The Biosynthesis of Shikimate Metabolites (1990) P. M. Dewick The Microbiological Transformation of Diterpenoids (1973 to June 1991) J. R. Hanson

ISSN:0265-0568    

DOI:10.1039/NP99108FP013

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

Natural Product Reports Editorial Board Professor T. J. Simpson (Chairman) University of Bristol Dr C. Abell University of Cam bridge Dr J. R. Hanson University of Sussex Dr R. 6. Herbert University of Leeds Professor J. Mann University of Reading Dr D. A. Whiting University of Nottingham 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. 1991 Annual Subscription Price E.C. f 198.00 Overseas f228.00 U.S.A. $467.00. 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 1991 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 1991 E.C. f 198.00 Overseas f 228.00 U.S.A. US $467.00 Subscription rates for back issues are (1986) (1987) (1988) (1989) (1990) U.K. f 130.00 f 142.00 f 159.00 f 169.00 f177.00 Overseas f 143.00 f 159.00 f 183.00 f 194.00 €204.00 U.S.A. US $252.00 US $280.00 US $342.00 US $388.00 US $398.00 Members of the Royal Society of Chemistry should order the journal from The Membership Manager The Royal Society of Chemistry Thomas Graham House Science Park Milton Road Cambridge CB4 4WF England

ISSN:0265-0568    

DOI:10.1039/NP99108FX021

出版商:RSC    

年代:1991

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99108BX023

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

A Unified Mechanistic View of Oxidative Reactions Catalysed by P-450 and Related Fe-Containing Enzymes? M.Akhtar" and J. N. Wright Department of Biochemistry University of Southampton Bassett Crescent East Southampton SO9 3TU 1 Introduction 2 P-450 Dependent Hydroxylation 2.1 Camphor Hydroxylase 2.1.1 The Structure of the 0 Complexes of P-450 2.1.2 The Iron-hydroperoxy Derivative (Fell'-OOH) 2.1.3 The 0x0-derivative (Fe = 0)and its Structure 2.2 The Mechanism of Hydroxylation 2.3 The Stereochemical Control in P-450 Dependent Hydroxylations and the Protection of the Decor of the Active Site 3 P-450 Enzymes Endowed with Multiple Activities 3.1 Aromatase and 14a-demethylase 3.2 The Conversion of Hydroxy into Carbonyl Compounds 3.3 The Mechanism for the Cleavage of Carbon-Carbon Bond Using Either Fe"'-OOH or the 0x0-derivative in a Crucial Role 3.4 The Mechanism of the Pregnene Side-chain Cleavage Reaction 4 Epoxidation of Olefins and Hydroxylation of Aromatic Compounds 4.1 Possible Mechanisms 5 Haem Containing Peroxidase and Related Enzyme Systems 5.1 Catalase 5.2 Peroxidases 5.3 Compound I and Compound I1 5.4 Peroxidases in Phenol Coupling 5.5 Chloroperoxidase 5.6 Cytochrome c Oxidase 6 Non- haem Iron Containing Oxygenases Requiring a-Oxoglutarate 7 Non-haem Iron Containing Oxygenases and Desaturases Linked to NAD(P)H 7.1 Oxygenases 7.2 Desaturases 8 Concluding Comments 9 References Synopsis (1) A large number of biological oxidation reactions use dioxygen 0,,and proceed through a step-wise cleavage of the two bonds of 0,.The reversible reaction of 0,and haemoglobin is prototypical of a widespread phenomenon in which the forward reaction depends on the reductive cleavage of one of the 0 bonds to give a superoxide anion that then forms a hexacoordinated iron complex (see below).It is argued that this feature of haemoglobin represents a fundamental reaction which operates in all iron-containing oxidases ;irrespective of whether they contain haem-iron as in P-450 enzymes or a non- haem iron as is present in prolyl and lysyl hydroxylases. + The hydroxylating P-450 enzymes are also known as monooxygenases because these transfer one oxygen atom to the organic substrate.a-Oxoglutarate- requiring hydroxylases on the other hand are classified as dioxygenases because in this case both the oxygen atoms of 0 are incorporated into organic molecules; one into the hydroxyl group and the other into succinate. From a mechanistic view point this distinction is misleading and therefore the nomenclature is avoided in the present article. 527 Haemoglobin + 0=0 Oxyhaemoglobin Nhis Nhis r Nhis (2) In P-450 dependent hydroxylases the initially formed Fe"'-O-O' species is converted into Felll-O-OH and the heterolysis of the second oxygen-oxygen bond of the latter then gives the 0x0-derivative for which a number of canonical structures are possible ; for example FeV=O t.(+') FeIV=O ++FeIV-0'.One of these FeIV-0' behaves like an alkoxy radical and participates in hydrogen abstraction from a C-H bond to produce FeIV-0H and a carbon radical. The latter is then quenched by the delivery of hydroxyl radical from FeIV-0H. The latter species may thus be regarded as a carrier of hydroxyl radical. (3) Some P-450 systems notably aromatase and 14a-demethylase catalyse not only the hydroxylation reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C-C bond cleavage process. All these reactions occur at the same active site. The conversion -CH,OH +-CHO has been explained using the 0x0-derivative Fe'"-O'. (4) The C-C bond cleavage reaction conforms to the general equation shown below and may be rationalized by two alternative mechanisms one of which uses the 0x0-derivative FeIV-0' and the other Fe"'-OOH.The merits as well as the drawbacks of the two mechanisms are considered and our own bias for a mechanism involving Fe"'0OH has been emphasized. x=o or x=c< (5) The 0x0-derivative is also involved in reactions with olefinic and aromatic double bonds and in our view these reactions are best rationalized by invoking that in these cases the electronic structure of the substrate promotes the oxo-derivative to reveal its electrophilic character [(+') FeJV=O++ Fe"'-O+]. (6) Catalase and peroxidases are also haem containing enzymes and these use a variety of peroxides as oxidants. Since in the peroxides one of the double bonds of the putative precursor 0 is already reduced these react with the FeIII form of enzyme directly to produce the Fe1''-OOH species that is converted to the 0x0-derivative (FeV=O c)FeIV-0').The latter species then participates in the decomposition of H,O, catalysed by catalase or the dehydrogenation of AH catalysed by various peroxidases via a radical mechanism involving an initial hydrogen abstraction followed by disproportionation. (7) Cytochrome c oxidase is present in all aerobic organisms and catalyses the terminal 4 electron reduction of dioxygen into H,O. This reaction may be considered as a step-wise process in which 2 electrons are first used to convert 0 into the 0x0-derivative via the usual hydroperoxide species.The remaining 2 electrons are then used for the reductive cleavage of the 0x0- derivative into water. (8) Hydroxylation reactions normally associated with P-450 types of cytochrome are also catalysed by enzymes which contain non-haem iron for example proline and lysine hydroxylases. In the resting states these enzymes contain Fe'I and use a-oxoglutarate as the reductant. The mechanism of the hydroxylation reaction catalysed by these enzymes is similar to that of P-450 enzymes. Except that the stoichiometry of electrons involved dictates that compared to the haem containing P-450 enzymes the 0x0-derivative in the non-haem enzymes may be at a higher reduction level i.e. Fe1''-O' instead of Fe'"-O'. (9) Another group of enzymes which have not yet been studied in detail are related to P-450 as well as to non-haem oxygenases.These like the former use NAD(P)H but like the latter contain non-haem iron. An oxygenase of this type could be involved in the oxidation of the 4a-methyl group of sterols to the corresponding carboxylic acid. It is hypothesized that the enzymes of this class may also catalyse the hydroxylation reaction through the participation of FeIV-0' as described in (2) except that in this case all the five ligands for the coordination of iron are provided by amino acid side chains. (10) The desaturation process -CH2-CH2-+-CH= CH- requires NAD(P)H and 0 and is also catalysed by enzymes containing non-haem FerI1 as in (9). This trans-formation may be envisaged to represent an alternative reaction course in which the neutralization of the two radical species (FeIV-0H and the carbon radical) is achieved not by an associate reaction as in hydroxylation but through dispro- portionation.(1 1) It is argued that the stereochemical control exercised by P-450 and related enzymes in which the retention of stereo- chemistry is generally observed in hydroxylations and the removal of cis-oriented hydrogen atoms in desaturations are not the consequences of the reaction mechanism itself. These are however due to the constraints on the mobility of the substrate within the Michaelis complex. It makes sense to assume that enzyme systems operating through the inter- mediacy of particularly reactive species such as free radicals have evolved to allow a minimum motion of bonds during catalysis in order to ensure that the species do not participate in random reactions and damage the decor of the active site.1 Introduction The chemical principles used by nature to produce the complicated and fascinating array of biomolecules bear a close resemblance to those used for synthesis in test-tube chemistry. In both cases molecular architecture is built up using similar laws discovered by a process of trial and error. Nature had a longer learning-period than the chemist which it has used not so much to improve on the laws but to use them with grace and subtlety. Nature's chemistry needs to be performed at or near 37 "C its reagents must be stable in water and it has available to it acids and bases which cover only a narrow pH range between 4 and 10.It could be argued that these are the constraints which necessitated that from a myriad of theo- retically possible courses for a given biological reaction the one NATURAL PRODUCT REPORTS 1991 selected is that which occurs by the lowest energy path. Energy minimization seems to be the hallmark of evolutionary biology. A large number of biological transformations like their chemical counterparts involve at some intermediary stage the cleavage of C-H or C-C bond to produce a carbanion or an equivalent species. The latter is then exploited to produce the desired target compound via condensation elimination or subs ti tution reactions. The fundamental laws which govern the cleavage of C-H and C-C bond in laboratory chemistry are now understood if not completely at least at a sufficiently advanced level to allow their description in quantitative terms.The extensive studies of Eigen' and Bell2 may be used to give us a numerical view of the ease with which a carbanion may be generated from the cleavage of a C-H bond. This process is dependent on two factors the pK value of the carbon acid (C-H) and of the base used for the deprotonation process. The reaction shown in equation I will be greatly facilitated in the forward direction if the pK of the conjugate acid of the Base B- is greater than pK of the carbon acid (for example see data in Figure 1). The classical chemistry had relied on the uses of bases with pK values between 10 (triethylamine) to around 15 (hydroxide methoxide) enabling the generation of carbanions from reagents such as ethyl acetoacetate (PK = 9.0) diethyl malonate (pK = 13.3) acetone (PK = 20) and related compounds.In recent years the introduction of stronger bases (lithium diisopro- pylamide) has extended the range of carbon acids whose carbanions may be conveniently produced and used in organic syntheses. The law underpinning the formation of carbanions from the cleavage of C-C bond also seems to be related to pK values. We know that the facility with which the C-C bond is cleaved depends on the extent to which the negative charge in the carbanion is stabilized by resonance. For example the decarboxylation of a P-keto acid occurs with relative ease (Equation 2) whereas aliphatic acids are decarboxylated only under exceptionally vigorous conditions (Equation 3).The expected reaction rates in these two cases are inversely related to the pK value of the conjugate carbon acid of the carbanion; the lower pK of the conjugate carbon acid the easier is the decarboxylation. These values are 20 and 50 respectively for the two examples cited. A cursory examination of the literature would reveal that as precursors of carbanions only those carbon acids which have pKvalues below 20 have found application in classical synthetic chemistry. Interestingly a similar situation is found in biological systems and authentic examples of reactions known to occur by carbanion intermediates are invariably those which involve precursors with pK values below 25.Organic chemists have learned from experience that non- activated carbon atoms that is those containing C-H bonds with pK values 25-50 are best manipulated not by an heterolytic cleavage mode but by the homolytic process involving free-radicals. The same is the biological experience and a free-radical mechanism appears to be preferred over an ionic process when the cleavage involves C-H or C-C bonds which would produce carbanions whose conjugate acids have pK values greater than (say) 20. A wide spread mechanism NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT 1o6 1o5 1o4 1o3 1o2 10 al CI 1 CT 10'' 1o-2 15 17.5 20 22.5 25 27.5 pK of the base Figure 1 The estimated rates of proton transfer from acetone (PK = 20) to bases of various pK values.The estimate was made using data on the rates of proton transfer to hydroxide from various carbon acids. These rates are lo-' 1.6 10 3 x lo2 and lo5s-l M-' f or the difference of pK values between hydroxide and the relevant carbon acid of -5 -4 -1.2,0.2 and 7 units respectively.'S2 In the replot above it is assumed that the rates of proton transfer from acetone to different bases are related to ApK in a proportionate manner when the rate of proton transfer from acetone to hydroxide (10-' s-' M-l) is used as a reference value. The figure above highlights the difficult nature of proton transfer from carbon acids and shows that the removal of proton from a carbon acid of pK of -20 (as in acetone) at a rate commensurate with normal enzyme catalysis (> 10' s-') will require a base with pK of around 24.In reality of course this is not necessary since the rate enhancement is achieved partly by increasing the acidity of the C-H bond through activation of the carbonyl group (viaprotonation or Schiff-base formation) and only partly by making the enzyme base behave more strongly than expected from its pK value in solution. In the present context our concern is not to delineate how this extra basicity is achieved but to emphasize that there is a limit to this process which enables enzymes to operate efficiently by a deprotonation mechanism only on carbon acids which have pK below (say) 25 R'\ RZ7C-H + O2 + 28-+ 2H' I/ Me R3 I (4) operating in biological systems for generating radical inter- mediates is the use of an 'active oxygen' species.This article lP7co; brings together existing knowledge on this class of enzymic co systems and attempts to rationalize their mechanisms in terms of a unified hypothesis. (1) Fe Protoporphyrin IX (haem) 2 P-450 Dependent Hydroxylation 2.1 Camphor Hydroxylase Prototypical of biological reactions which occur on non-activated C-H bonds is the hydroxylation process given in Equation (4) in which the hydroxyl oxygen introduced into the organic molecule is derived from 0 and the overall process requires a source of 2 electrons which usually is NADPH. The most extensively studied group of enzymes catalysing the hydroxylation reaction are cytochromes P-450.These contain Fe-protoporphyrin IX (haem b,l) as the prosthetic group and derive their name from the difference spectrum obtained by the addition of CO to the reduced form of the enzyme when a peak at 450 nm is prod~ced.~-~ A large number of P-450 enzymes have now been char- acterized and found in the case of drug metabolizing enzymes 60; to possess a relatively broad substrate specificity while those involved in biosynthesis have been found to be remarkably (2) P-450 (prosthetic group) specific for a single substrate. In all cases the protein moiety of the cytochromes contains an essential cysteine whose thiolate group serves as one of the axial ligands for the haem iron (2).6 Scheme 1 NATURAL PRODUCT REPORTS 1991 NADPH + 02 + -‘&OH (3) camphor (4) Scheme 2 I I I I I S S S s s R0.“OH k-H k-H k-H k-H The sequence of events involved in the formation of the hydroperoxy intermediate (9) during the P-450dependent hydroxylation. It is to be noted that in the resting state the enzyme contains a low-spin iron that is converted into high-spin iron upon substrate binding Scheme 3 Individual members of the P-450 family are identified by the inclusion in the subscript of the name of the substrate upon which it acts. For example the camphor hydroxylase of Pseudornonas putida catalyses the conversion of D-camphor (3) into 5-exo-5-hydroxycamphor (4) Scheme 2 and is known as cytochrome P-45OC,,,,,,. This is the first enzyme to be subjected to detailed and most elegant physico-chemical studies by Gunsalus and coworkers.’ This work has provided important information applicable to all other P-450 hydroxylases.We have therefore developed the mechanistic argument primarily using P-45OC,,,,, as an example. In the resting state cytochrome P-450 enzymes have the haem iron as FeIII (5 Scheme 3). The first step in catalysis is the compulsory binding of the substrate to the enzyme giving a binary Since this seemingly prosaic feature is retained in a wide variety of P-450 enzymes it is likely to confer some sort of advantage which is considered elsewhere in this section. 2.1.1 The Structure of 0 Complexes of P-450 Next the Fell1 in the binary complex is reduced to Fe” by a single electron transfer.12 It is the Fe’I form of the binary complex that then reacts with 0 producing a covalent adduct (8) whose spectroscopic and magnetic properties are similar to those of oxyhaemoglobin.ll The reaction (7) (8) being reminiscent of the conversion of haemoglobin into oxyhaemo- globin allows by analogy the oxygen adduct (8) to be formulated as the coordination complex of Fell1 in which one of the ligands is a superoxide anion.13u-13d The rationale for this curious formulation is best considered by reference to the haemoglobin field. During the last half a century the haemoglobin system has been subject to an extensive physico-chemical examination with the accumulation of a wealth of detailed information. Yet from the view point of bioinorganic chemistry the most outstanding contribution overshadowing any other by a long way was made by Pauling and Coryell in 1936.14a Using magnetic susceptibility measurements it was found that the iron in haemoglobin had 4 unpaired electrons an arrangement found in FeC1;6H20 however its oxygen adduct oxy-haemoglo bin was diamagnetic like ferrocyanide Fe(CN)t- and other coordination complexes of haem formed when the latter reacts with 2 molecules of a base.This observation suggested that the 0 binding to haemoglobin is attended by the conversion of a high spin iron into a low spin iron (Scheme 4). There was however a long period of indecision14b before the precise chemistry through which 0 becomes a coordination ligand for Fe” could be deduced.There is now a general consensus that oxyhaemoglobin be formulated as (12). The structure is supported by a number of physical techniques13d and may be viewed to arise from the initial interaction of Fell with 0 which causes a redox reaction producing Ferrr and the superoxide ion 0-0 the latter then acts as the sixth ligand to give the final coordination complex (12 Scheme 4).Throughout our discussion in this chapter we will assume that this mode of binding represents a general phenomenon and is applicable to a wide variety of systems where iron whether haem or non- haem is involved in the manipulation of 0,. 2.1.2 The Iron-hydroperoxy Derivative (Fe”’-OOH) We return to the P-450 system and deal with the fate of the Fell’-superoxide species (8) which following one electron reduction rapidly decomposes to produce the hydroxylated product regenerating the resting state of the P-450 (Scheme 3).The process is normally too fast to allow the identification of an intermediary species however evidence for the involvement of a hydroperoxy species in P-450 systems was provided by the demon~trationl~ that in the absence of substrate rat liver microsomes generated H,O from NADPH and 0 in a stoichiometric amount. In subsequent StudieP using purified- reconstituted enzyme the reaction was shown to be the property of P-450,,,. The formation of H,O is best rationalized by assuming the dissociation of a Fe”’-hydroperoxy species (9) and suggests that a similar intermediate may also participate during the normal catalytic turn over.2.1.3 The 0x0-derivative (Fe=O) and its Structure It was conceivable that the Fell1-peroxide species (9) is endowed with the chemical characteristic required for it to participate directly in the hydroxylation process however other obser- vations pointed to a more interesting scenario. It was found that certain artificial oxygen donors”* l8(e.g. organic peroxides iodosobenzene and NaIO,) could promote hydroxylations NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT 531 (10) Haemoglobin (high spin) t l Nhis Me o'o. Me CO co (12) Oxyhaemoglobin (low spin) The illustration highlights that the high-spin iron in haemoglobin which occupies a 'domed' configuration is forced into a planar orientation in oxyhaemoglobin Scheme 4 using a variety of P-450 enzymes without requiring either 0 or a reducing agent.The regio and stereospecificity of hydroxy- lations with these artificial oxidants were often the same as with the normal physiological system.l* Since the most effective oxygen donor iodosobenzene contains only one oxygen atom it provided the clue that it is a mono oxygen form of P-450 that is the active species involved in hydroxylation. The structure of such a species may be deduced logically by assuming the initial formation of a coordination complex (13 Scheme 5) between P-450 and iodosobenzene followed by an elimination reaction to give (14). The same intermediate may be obtained from the Fe"*-peroxide species (9) in the normal physiological reaction.That the mono oxygenated derivative of P-450 exists pre- dominantly in the double bonded structure (Fe=O) is deduced indirectly from EXAFS19"-lQd (extended X-ray absorption fine structure spectroscopy) and chemical studieslge performed on an analogous species obtained from horseradish peroxidase. The species of the type Fe=O is often referred to as the oxo- derivative. It should be noted that the 0x0-derivative (14 Scheme 5) is shown to formally contain FeV. This is because of the convention of inorganic chemistry which dictates that the electron pair forming a coordination bond is deemed to reside entirely on the ligand and not shared with the metal. Notwithstanding this formulation the 0x0-derivative can exist in a number of canonical forms shown by the structures (14a 14b 14c and 14d Scheme 6).There is however a further fact requiring comment. The results obtained from a range of physical studies (ultraviolet-visible,20 Mossbauer,21 magnetic susceptibility,22 and magnetic ~esonance,~) on the oxo-de-rivative of horseradish peroxidase have been reconciled by assuming that the species in fact contains FeIV and a porphyrin 7r radical cation. This species may be envisaged to be formed by an intramolecular redox process driven by the greater stability of Fel"=O over FeV=O. If this interpretation is accepted then the role of porphyrins in biology is not merely conveniently to provide four coordination ligands in a single structure but also to regulate the redox state of Fe through the donation of an electron.The latter property no doubt must depend on the fact that the porphyrin radical cation thus produced is stabilized by resonance over the extensive network of conjugated double bonds. The above consideration necessitates that the canonical NATURAL PRODUCT REPORTS 1991 0, \I/ I ____c /FY 1 /;\ + 0, 0 I 'OH The formation of the FeV=O intermediate (14 also see scheme 6) during the physiological (9) -* (14) and artificial (13) -+ (14) reactions Scheme 5 I I 7-l-I I -r I S S S S (14e) (149 (1 49) (14h) It should be noted that the ground state structure of the 0x0-derivative is heavily weighted in favour of (140 but in the hydroxylation process the canonical form (14d) is used Scheme 6 forms (14a) (14b) (14c) and (14d) have their 7r radical cation counterpart as shown in (14e) (140 (14g) and (14h) respectively.This is an important point and should be remembered for future reference though in the discussion that follows we will use three structures; (14b) for convenience and (14d) and (140 because these represent the reactive and resting states respectively. Since the 0x0-derivative is of central importance to the main theme of this review we take an overview of the reactions involved in its formation and the chemical characteristics which may be attributed to it. Taking the Fe" form of P-450 as the starting point the 0x0-derivative is formed by three reactions. The first reaction (7) -+(8) involving the formation of the superoxide adduct Fe"'-OO' should be reversible by analogy with the behaviour of oxyhaemoglobin.Although at present no direct information is available regarding the properties of the FeII'OOH species the fact that certain peroxides can react with the Ferrr form of P-450 to produce an oxidant which promotes hydroxylation can be readily rationalized by assuming the intermediacy of a hexacoordinated derivative whose formation utilizes the chem- istry that would be involved in the reversal of the dissociation reaction (Scheme 7). In principle there are two possible routes24 for the further conversion of the Fe"'0OH species into the oxo- derivative (Scheme 8). This may occur either by a homolytic cleavage producing a hydroxyl radical and Fe"'-O' which are then converted through an intermolecular electron transfer (t, reaction into HO-and FeIV-0' FeV=O t*etc.etc. Scheme 8). The operation of a homolytic mechanism in the cleavage of the 0-0 bond of peroxyphenyl acetic acid by a liver microsomal P-450 has indeed been demonstrated. 25 NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT 533 (5) OH4 Scheme 7 homolytic (9) heterolvtic 0 0 Scheme 8 H I The sequence shows the loss of stereospecificity when the substrate containing 2H in the 5-exo-position is used Scheme 9 Alternatively the species (14) may be directly produced by a heterolytic process. Although at present choice between these two routes is not possible in the normal reaction cycle the heterolysis process appears preferable because in this case the cleavage of the peroxide bond can be aided by an enzymic protonation process (see Reference 73).In the physiological reactions catalysed by P-450 enzymes the equilibrium of the heterolysis reaction (9)+ (14) will be expected to be in the forward direction. This is because in the physiological reaction the 0x0-derivative is only formed when the substrate is already bound to the active site and under such conditions its utilization for the hydroxylation process must be an overwhelmingly favourable process. The law of microscopic reversibility however requires that the reformation of the peroxy derivative from the two components may occur by the attack of the hydroxide ion on the oxygen of the 0x0-derivative.It thus seems reasonable to associate the canonical form represented by structure (14a) (14b) (14e) and (140 with electrophile reactions. The canonical forms (14c) and (14g) should be a base with pKa value of around 11.6 while (14d) and (14h) endow a radical character. 2.2 The Mechanism of Hydroxylation The question as to which of these forms participates in the final hydroxylation process and the detailed chemistry of the cleavage of the R-H bond remained unclear for two decades.26 The underlying cause of this was the repeated demonstration that a variety of aliphatic hydroxylation reactions occurred with overall retention of stereo~hemistry,~’ i.e. that in Equation (5). Consequently it became an obligatory requirement that any accepted mechanism of hydroxylation must also explain the retention of stereochemistry for which no precedent was known.Attention however was drawn to the fact that certain reactive carbenes (:CH,) could insert into C-H bonds to produce compounds with retention of stereochemistry. 28 It was hence deduced that the 0x0-derivative may generate ‘oxene ’ (an oxygen counterpart of carbene) that may insert into a C-H bond producing a hydroxylated product with definite retention of stereochemistry. 28 Further developments in the field however showed that although the predominant course of biological hydroxylation reactions is indeed the retention of stereochemistry there is also a distinct component of inversion the extent of which may be increased by using substrate- analogues substituted with 2H or 3H in the sensitive carbon- hydrogen bond.The first example of such a phenomenon was provided by Groves et aZ.29and the approach was then extended30 to show that P-450,,,,,, catalysed hydroxylation of D-camphor containing deuterium in the Sex0 position gave the expected 5-ex0 alcohol that still contained a substantial amount of 2H which was found to be located in the 5-endo-position7 Scheme 9. A similar isotope effect induced racemization was NATURAL PRODUCT REPORTS 1991 1 l-I I I S S S S S OH2 0' 0 ''OH JR-H JR-H k-H JR-H L t I I 1 I S S S S The postulated mechanism for the P-450dependent hydroxylation process.The scheme enlarges the partial sequence already considered in Scheme 3. The mechanisms for the conversion of (9) -+ (14) are considered in Scheme 8. It should be noted that in the above sequence the complex (15) is produced by a hydrogen abstraction reaction another possibility is that this is achieved by a four-centred cyclic process using the canonical form (14f Scheme 6). (+ *) FeIv=O (+*)Fe"-OH F~~-OH -% -11 R-H R' In our view this alternative though obviates the need to involve the participation of a reactive ferroxy radical (14d Scheme 6) the four-centred mechanism lacks generality and is not applicable to peroxidases considered in Scheme 25a Scheme 10 subsequently shown in the hydroxylation of deuterated ethyl- benzenes.31 These findings are best rationalized by assuming that the cleavage of the C-H bond at its conversion into C-OH must in fact be a step-wise process which can occur by one of three routes involving a carbanion carbonium ion or radical.The last process is most likely on chemical grounds especially when the hydrogen to be abstracted is bonded to a non-activated position (in other words the carbon acid has a pK greater than 25) as is the case in most P-450 dependent aliphatic hydroxy- lations. Groves et aZ.32should be accredited with the original suggestion that a ferroxy radical represented by the canonical form (14d) may be a suitable candidate for hydrogen abstraction. In the mechanism of Scheme 10 this species is used for hydrogen abstraction leading to the formation of a carbon radical which is neutralized (1 5) +(16) by association with a hydroxyl radical generated from (1 5).The driving force for the formation of a hydroxyl radical 'equivalent ' from (1 5) must be the energetic advantage accruing from the attendant conversion of Fe'" into Fe"'. The radical mechanism has subsequently been advocated by other~,~~,~~ but the most convincing evidence for this mechanism has come from the elegant approach of Montellano and who exploiting the broad substrate specificity of liver microsomal P-450 found that whereas the hydroxylation of methylcyclopropane (17 Scheme 11) gave only the normal product (20) the strained cyclopropane system (2 1) yielded about 15 YOof a rearranged derivative (25) whose formation may be rationalized by invoking the participation of a radical pathway in which the key event is the fragmentation reaction (22) +(23) characteristic of the behaviour of cyclopropylmethyl radicals.The contrasting profiles displayed by the two substrates (17) and (21) were further interpreted to deduce that during biological hydroxylation the collapse of the carbon radical by reaction with the hydroxyl radical (in free radical chemistry this is called the association reaction and in enzymology the process has come to be known as the oxygen rebound step) must be a fast process having a rate in excess of 1 x lo9s-l. The argument for the deduction is as follows. It is known that the cyclopropylmethyl radical (1 8) rearranges to (19) at a rate of 1 x lo8s-l since in the biological hydroxylation of (17) no rearranged material was formed meant that the rate of this process (18) -+ (19) is slower than that of the competing radical association reaction (18) -+(20).The latter process therefore must have a rate in excess of lo8s-l. A corollary of this is that in the case of the metabolism of the strained derivative (21) when a rearranged product was formed the fragmentation process (22) -+(23) must be sufficiently fast to compete with the association reaction. Subsequent studies by Ing01d~~ validated these estimates and showed that the rearrangement process (22) +(23) has a rate of 2 x lo9s-' thus highlighting that the rate constant for the oxygen rebound step must approach a value of 2 x 10" s-'.2.3 The Stereochemical Control in P-450 Dependent Hydroxylations and the Protection of the Decor of the Active Site In the light of the foregoing discussion we suggest that the retention of stereochemistry generally observed in hydroxy- lation reactions is not the consequence of the reaction mechanism itself. It is however due to the constraints on the mobility of the substrate within the Michaelis complex. It makes sense to assume that enzymes operating through the NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT 535 The formation of a rearranged product (25) during the hydroxylation of (21) Scheme 11 0 (27) Scheme 12 intermediacy of particularly reactive species such as free radicals have evolved to allow a minimum motion of bonds during catalysis in order to ensure that species do not participate in random reactions and damage the decor of the active site.In support of the above hypothesis the following observations may be cited (1) In physiological hydroxylation reactions the formation of the reactive 0x0-derivative always occurs when the substrate is already bound to the en~yrne.~-ll (2) When an artificial oxidant e.g. iodosobenzene is used in the hydroxylation reaction the formation of the 0x0-derivative will be expected to occur without the substrate being already positioned at the active site. It is known that under these conditions P-450 enzymes are rapidly inactivated. la (3) There are inhibitors known for certain P-450 enzymes which when incubated with their target enzymes in the presence of 0 and NADPH lead to the inactivation of the enzyme activity.3su Examples of this class of inhibitors are steroids of the type (26)-(28) (Scheme 12) which inactivate aromatase in the presence of 0,+ NADPH and these have been designated as suicide inhibitors of the enzyme.The assumption under- pinning this description is that these compounds are handled by the enzyme as 'pseudo-substrates ' producing reactive electrophilic species which then alkylate the enzyme. In our view some of these inhibitors for example (27) and (28) which do not contain functional groups suitable for producing electrophilic species may merely 'deputize ' for the substrate promoting the formation of the 0x0-derivative which then decomposes by reaction with active-site groups.3 P-450 Enzymes Endowed with Multiple Activities 3.1 Aromatase and 14a-demethylase During the last decade fragmentary evidence accumulating from studies on steroid biosynthesis has raised the possibility that P-450 linked enzymic systems in addition to their role in hydroxylation and related oxygen insertion reactions may also participate in the oxidation of alcohols into carbonyl com- pounds and more importantly in C-C bond cleavage reaction^.^^^^^^^ . In particular our studies on the mechanism through which the lO,&methyl group of androgen is removed in oestrogen biosynthesis (29) -+ (32)37a-h and the 14a-methyl group of lanosterol in cholesterol biosynthe~is~~ (33) -+ (36) have highlighted that in these cases the same P-450 system is involved in catalysing reactions belonging to entirely different generic types.The two conversions (Scheme 13) may be viewed as representing a general phenomenon which involves the creation of an olefinic linkage in conjugation with an existing system of double bonds. The new olefinic linkage being produced through the cleavage of a C-C and a neighbouring C-H bond. The support for the view that the two transformations are mechanistically related was provided by our extensive studies which showed that in both cases the overall process occurs in three stages each requiring the participation of 1 mole of 0 and 1 mole of NADPH.37a Furthermore in both cases hydroxyl and aldehydic derivatives are formed as intermediate^^^"^ 37e+38 and the C fragments are released as formic a~id.~~~,~~~e~~~,~~.Our experiments and those of also suggested that in both systems the three reactions are catalysed at a single active site. This conjecture was subsequently supported by the purification to homogeneity of the two enzymes (P-450a,,,atase40 and and P-45018~.demethylase41)the demonstration that these consist of single polypeptide chains and are endowed with the property of catalysing the overall conversions (29) + (32) and (33) -+ (36) respectively. Thus the work on aromatase and 14a- demethylase provided the challenge to rationalize how the same enzyme may catalyse the hydroxylation as well as the oxidation and C-C bond cleavage processes.The mechanism of the NATURAL PRODUCT REPORTS 1991 NADPH NADPH NADPH 02 0 @2 -HO OH The conversions catalysed by aromatase (29) -* (32) and I4a-demethylase (33) -+(36) may be viewed to represent the same generic process in which a double bond is produced by the cleavage of two syn-orientated substituents Scheme 13 \ FelV-O. + H-C-OH -+ [ FelV-0H ?C-OH] Patv (37) / / Fe"' + H20 +'C=O / Path c Possible mechanisms for the P-450 dependent conversion of alcohols into carbonyl compounds Scheme 14 hydroxylation reaction catalysed by these enzymes must be the same as described above and illustrated in Scheme 10. This assertion is supported for the aromatase catalysed conversion by the demonstration that the hydroxyl oxygen atom of (30) is derived from 0237e and that the reaction (29) 4(30) occurs with the retention of stereo~hemistry.~~ These are the two features which characterize most P-450 dependent hydroxylation re- actions 3.2 The Conversion of Hydroxy into Carbonyl Compounds We now deal with the oxidative reaction converting an hydroxymethyl into an aldehyde group.In the case of oestrogen biosynthesis it is known that the reaction (30) 4 (31) is attended by the loss of HR:7b*37c,43 and in the conversion the hydroxyl oxygen of (30) is retained in the aldehyde group of (31).37e The most likely possibility is that this reaction is also catalysed by the same FeIV-0' species that is involved in the initial hydroxylation process. At least three mechanisms for the oxidation may be envisaged.The first two (paths a and b Scheme 14) are modelled on the hydroxylation mechanism in which the initial abstraction of HRe produces the C-radical which is either quenched by the usual oxygen rebound process to give the gem diol(37) from which the desired aldehyde group is produced by a stereospecific dehydration. Alternatively the C-radical is neutralized by disproportionation via hydrogen abstraction from the hydroxyl group (path b Scheme 14). The retention of the hydroxyl oxygen atom in the aldehyde group shown by our lS0experiments (30)+(31) would be more consistent with the direct pathway b. Other experiment^,^^ however have shown the incorporation of lSOfrom 1802 in the 1Pcarbonyl group during the oxidation of a substrate analogue (38 Scheme 15) by aromatase.In this case a gem-diol is presumably formed which dehydrates removing the original hydroxyl group. Since the C-19 hydrogen atom of the analogue (38) removed in the oxidation has the opposite stereochemistry to that involved in the oxidation of the real substrate (Scheme 15) the experiment cannot be used to infer the mechanism of the physiological reaction. The final possibility (path c) is that in the initial step FeIV-0' abstracts a hydroxyl hydrogen to give an alkoxy radical that then loses the H, in a dispro- portionation reaction. All the three courses (paths a b and c Scheme 14) are theoretically feasible and are likely to be encountered somewhere in biological systems. However ther- mochemical data available on alkoxy if they were to be extended to ferroxy radicals suggest that paths a and b should be preferable to path c.The argument for this is as follows the initial event in paths a and b may be viewed to 537 NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT -HR* Ho+;Si IOSSof HRE * (31) NADPH+ 02 The illustration highlights that in the oxidation of the physiological substrate H, is removed however when the artificial substrate (38) is used then the C-19 hydrogen atom involved in the oxidation occupies an orientation corresponding to the HSi position Scheme 15 correspond to the reaction of Equation (6) which depending on the substitution state of the carbon radical should have a favourable AH value of about -8 kcal m01-l.~~~ The initial step in path c represented by the reaction given in Equation (7) on the other hand would be expected to have AH of zero.These considerations make path c less likely albeit only marginally. 3.3 The Mechanism for the Cleavage of Carbon-Carbon Bonds Using Either Feul-OOH or the 0x0-derivative in a Crucial Role Numerous examples of biological C-C bond cleavage re- actions are known in which the scissile bond is located between the a and p positions with respect to an existing electron withdrawing group or such an electronic arrangement is created by adduct formation between the substrate and a prosthetic group of the enzyme. The latter situation is encountered in pyridoxal phosphate and thiamine pyrophosphate linked enzymic decarboxylation reactions.In all these cases the C-C bond cleavage occurs by the more familiar heterolytic mech- anism using the retro-aldol principle shown in Equation (8). Earlier conjectures rationalizing the mechanism of C- IO-C-19 bond cleavage in oestrogen biosynthesis or C-14-C-32 bond cleavage during sterol biosynthesis were influenced by the precedent above and the mechanisms considered for the two processes conformed to the general principle underpinning the cleavage reaction of Equation (8). The observations made at Southampton that NADPH and 0 were required for the two bond-cleavage processes (31) + (32) and (35) +(36)37a,38 and that in the case of oestrogen biosynthesis an atom of oxygen from 0 was incorporated into f~rmate,~~~.highlighted the participation of a novel mechanism for the cleavage process and pointed to the possibility that an activated form of oxygen is somehow directly involved in the scission of the carbon-carbon bond. Although a large number of mechanisms are possible and indeed have been for the rationalization of the mechanism of the C-1O-C-19 bond cleavage step we have narrowed these down to the two most likely options shown in Scheme 16a. One of these uses FeIII-OOH in a crucial role. This species produced as an intermediate in P-450 linked reaction will be expected to possess strong nucleophilic properties for addition to the C- 19 carbonyl group to produce the adduct (39) that decomposes with the incorporation of one atom of oxygen from 0,,into the released formic acid.In principle the fragmentation of the adduct (39) can occur by heterolytic homolytic or a cyclic pathway and the choice between these processes is dictated by geometrical considerations together with the acidity of the R1-0' + -CH20H R1-OH + -6H-OH (6) -C Rl-0' + -CHzOH R'-OH + -CH20* (7) X = HP; H OH; or 0 C-H bond. In the aromatase catalysed reaction (31) +(32) when one of C-H bonds cleaved in the process (C-lp) will be expected to have a high pK value (-45) a cyclic mechanism (Mechanism la Scheme 16a) or one operating via a radical intermediate (Mechanism 1b) seems most appropriate. The latter process is initiated by a homolytic cleavage of the peroxy bond in (39) to furnish Fe"'-O' and an alkoxy radical.There are numerous precedents47 to suggest that a favourable course for the decomposition of such an alkoxy radical (40) is the fragmentation reaction to produce formate and a tertiary carbon radical which is quenched by a disproportionation process creating the 1,2-ene system of oestrogen. With respect to the stage at which the 2,3-double bond of the A-ring is created we have argued37h that this occurs before the removal of the lp-hydrogen since a pre-existing double bond in the position would aid the abstraction of the lp-hydrogen. (A2s3) Support for this conjecture has come from the chemical model studies of Robinson and and more importantly their experiment^^^ which showed that a 3-deo~y-A~~~-analogue of the 19-aldehyde is a substrate for aromatase and is converted into deoxyoestrogen (Scheme 17).A possible criticism of the above mechanism is that an FeIII-OOH species has previously not been implicated in the oxidation of organic compounds by P-450 types of enzymes. It should however be borne in mind that in the context under consideration the Fe'II-OOH species is used specifically for reaction with a substrate that contains a carbonyl group. Attention has already been drawn to the fact that in most P-450 reactions the formation of a binary complex between substrate and enzyme is the obligatory first step and only then does the binding of 0 to the haem iron occur. It could be argued that in such a situation when the substrate is already bound to the active site with its carbonyl group correctly positioned then Fe'''-OOH is intercepted to produce the adduct of the type (39) rather than decompose to the usual FeIV-0' (i.e.conversion 9 -+ 14 Scheme 10). The Fe"'-O' species used in Mechanism I b (Scheme 16a) has not been implicated in any of the reactions considered previously in this review. The involvement of Fe"'-O* in hydrogen abstraction from (41) thus appears inconsistent with the general principle we have attempted to develop. The inconsistency is however only superficial and may be explained as follows. In the hydroxylation and other reactions considered above two types of enzymic intermediates with radical characters have been used; the 0x0-derivative [(14); Fe"=O t) ( +')FeIV=O ++ Fe'"-O'] in the hydrogen abstraction step and FeIV-0H which may be regarded as a 'hydroxyl radical carrier' in the oxygen rebound step.The Fe"'-O' species is in fact an unprotonated form of FeIV-0H as shown in Scheme 16b. If consistency is mandatory and the roles assigned to various participants in the hydroxylation process are to be strictly adhered to then the Mechanism lb (Scheme 16a) may be redefined as shown in Scheme 16b. In this case the removal of the C- la hydrogen during the disproportionation reaction occurs following the conversion of Fe"'-O' into Fe'"-OH. The two versions (Mechanism lb Schemes 16a and 16b) differ mainly with respect to the stage at which protonation occurs. NATURAL PRODUCT REPORTS 1991 Mechanism la Mechanism lb Mechanism 2 Mechanism 3 7 FeIti-O-OH +(31) Feiv-Og +(31) FeIV-O* +(31) t Fe&0 (39) (39) homolytic cleavage 1 FeIV-OH CHO fragmentation1 FdV- OH 'CHO HOJyr (41) disproportionation &isomerization Fe"' +H20 + HO Felt'-OH O,-?H H+ HO Fe"' HCOOH HO& (32) CHO Two classes of mechanisms for the cleavage of C-C bonds using either Fe"'0OH or FeIV-0 in a crucial role.In Mechanism 1b the hydrogen abstraction in the disproportionation reaction involves Fe"'-0 *. An alternative to this is the use of the protonated form of the latter as shown in Scheme 16b Scheme 16a See legend to Scheme 16a for explanation Scheme 16b The second class of mechanisms use the oxo-derivative (FexV-O') to promote the cleavage of C-1M-19 bond either by hydrogen abstraction5O" from C-1 (Mechanism 2) or via a Scheme 17 hemiacetal radical formation (Mechanism 3).The Mechanism 2 though an attractive explanation for the cleavage process suffers from the disadvantage that it involves the creation of a reactive species (HCO) that is too small to be adequately held at the active site and may therefore undergo random de- composition rather than be directed in the desired course of the reaction. Mechanism 3 is the weakest of the alternatives NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT 539 m &-OHI I > m 0 A-4 I I 02 HO NADPH HO NADPH HO (45) The three reactions (42) -+(43) (43) -+ (44) and (42) -+ (45) catalysed by 17a-hydroxylase- 17,2O-lyase Scheme 18 :I NADPH E RC-C-XH -RC-OH + >=X (9) 02 X=O or C< considered in Scheme 16a and its main weakness lies in the difficulty of justifying why the initial reaction of FeIV-0' with the aldehydic group results in an adduct formation and not hydrogen abstraction causing further oxidation of C-19 to a carboxylic acid.It is known that cytochrome P-450 enzymes can convert aldehydes to carboxylic acids by this type of In sum the advantage of Mechanisms 2 and 3 is that these use the same iron-monooxygen species as used in the preceding hydroxylation and oxidation steps whereas the Mechanism 1 involves an iron-dioxygen species not previously thought to interact with substrates in cytochrome P-450 catalysed re-actions.We have however favoured the latter mechanism because it most directly explains the incorporation of l80into formate during the reaction (31)-+(32). Further support for this view is provided by recent work on 17a-hydroxylase-17 20- lyase. 3.4 The Mechanism of the Pregnene Side-Chain Cleavage Reaction The mechanisms considered above for the aromatase catalysed reactions (Scheme 16) can without modification be extended to several other lyase reactions involved in steroidogenesis. We have already presented evidence to show that the enzyme involved in the removal of the 14a-methyl group during sterd biosynthesis (14a-demethylase) is closely related to aromatase and therefore should have a similar mechani~m.~~~~+ 37g. 38 The cleavage of the side-chain in androgen biosynthesis (42) + (44) and the formation of the A16-steroid (42) -,(45) are the other examples of the same generic reaction (Scheme 18).All these are reducible to the formulation of Equation (9) and are also catalysed by a cytochrome P-450 en~yme.~'.~~ The strategy used to explore the mechanism of the aromatase catalysed reaction has recently been extended to 17a-hydroxy- lase- 17,2O-lyase (P-45Ol,,). The enzyme acts on the pregnene nucleus (42) and promotes first the hydroxylation at the 17a- position (42) -+ (43) and then a cleavage reaction to produce the 17-keto steroid51 (#) releasing the side-chain as acetate.53 The enzyme is believed to possess another which culminates in the formation of the A16-steroid (45).The mechanism of these two side-chain cleavage reactions were studied54 using deuterium labelled pregnenolone (42a) under 1802. The salient features of the results obtained from the experiments are illustrated in Scheme 19 which shows that in both cases (42a) -+ (44a) and (42a) -+ (45a) an atom of l80is incorporated into the side-chain released as acetate. Fur-thermore the formation of the A16-steroid is attended by the removal of the 16a-deuterium atom and the 17P side-chain thus showing that the conversion occurs by a trans-scission process. The side-chain cleavage reactions can be rationalized by the two classes of mechanism considered for aromatase which use either Fe1I1-0OH or Fe'"-O' in a crucial role. A serendipitous observation made during the course of the isotopic experiments showed the formation of a third metab- olite 17a-hydroxyandrost- 5-en-3P-01 (46) a finding that has provided a strong pointer to the fact that carbonxarbon bond cleavage reactions of Scheme 18 and 19 occur via a peroxy adduct.The 17a-hydroxyandrogen (46) was originally isolated from pig testes55 and its formation during in vitro incubation was also but the mechanistic significance of these observations was at that time not obvious. We have shown that the 17a-hydroxyl oxygen of the latter (46) is derived from 1802 and its formation from (42a) is attended by the complete retention of the C-17a and C-1601 deuterium atoms of the precursor. The most important conclusion to be drawn from these results is that the 17a-hydroxylation occurs solely at the expense of the cleavage of the C-17-C-20 bond and that the overall process corresponds to an inversion of configuration.The results forcefully highlight that the conversion must occur by a step-wise process. Although the nature of intermediates which may participate in the conversion is not directly revealed by our experiments chemical considerations dictate that these should be radical species. As far as we are aware the example under consideration provides the first experimental evidence using a natural substrate to show that the C-C bond cleaving P-450 enzymes operate via a radical mechanism. These features are most readily accommodated by extending the Mechanism lb considered above for aromatase to the pregnene side-chain cleavage reaction producing a 17a-hydroxy androgen (46) as follows.The initial event in the process is the formation of the adduct (47 Scheme 20) which decomposes by a radical mechanism producing acetate and the C- 17 radical species (48). The formation of the 17a-hydroxyandrogen from the carbon radical then occurs by an oxygen rebound process (path a) while the same carbon radical by the loss of the l6a-hydrogen atom furnishes the A16-steroid (path b). The two modes for the NATURAL PRODUCT REPORTS 1991 D 4sH* + CD3-COO-(454 The status of hydrogen and oxygen atoms during the formation of (44a) (45a) and 17a-hydroxyandrogen (46) 0 is the original C-20 oxygen of 42a whereas azand Ozdenote the oxygen used in the steps shown.D = 'H Scheme 19 (47) + OHH* + Fe"' -(44) The upper sequence shows the mechanism of the cleavage of the pregnenolone side-chain via the peroxide (47) to give the 17a-hydroxy and A16-steroids. In the lower sequence (49) -+ (44) the equivalent peroxy derivative containing a 17a-hydroxy group is shown to fragment by the conventional heterolytic process Scheme 20 neutralization of the carbon radical are in keeping with the expected chemical behaviour of such a species in non-enzymic systems. Another point to note is that in both cases the bonding events occur from the a-face of the steroid skeleton which indicates but does not prove that the two products may be produced at the same active site through the loss of stringent control by the enzyme.In our view the formation of the 17a-hydroxyandrogen as shown in Scheme 20 is at present the strongest evidence available for the participation of a peroxy adduct in a carbon<arbon bond cleavage process. This conclusion is valid whether the formation of the 17a-hydroxyandrogen (46) is catalysed by 17a-hydroxylase- 17,2O-lyase or another enzyme. Should the three cleavage reactions (Scheme 19) turn out to be the property of the same enzyme then the justification for invoking the participation of a peroxy adduct to the other two transformations (42) -+ (44) and (42) +(45) will be very strong indeed. It should be borne in mind that in the conversion of 17a-hydroxypregnenolone into the 17-ketosteroid (44) the equivalent peroxy adduct (49) could decompose by an ionic mechanism because the System now contains a readily ionizable O-H bond.In this case the necessity of invoking a radical process is not as paramount as in those fragmentations where the C-H bond to be broken has a high pK value (> 40) as in the case of the reaction (31)+(32) (35)+(36) and (42a)+ (45). The cholesterol side-chain cleavage process is an example of a related but not identical process and by the nature of the substitutents present at the termini of the scissile bond this reaction can only be rationalized by invoking the participation of an oxo-derivative species FeIV-0' as was proposed by Larroque et ~1.~' NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT 541 R R R RO H" ,Q +4 H* H* OH L (55) The sequence shows the NIH shift via an arene oxide or a carbocation Scheme 23 intermediate.In both cases a carbonyl compound may be the initial product of rearrangement. R = H or FeIr1 Scheme 21 I-------/-\ I 1 1 0 Fe"' + A I 4 (52) Possible mechanisms for the primary event in the interaction of the 0x0-derivative with olefinic and aromatic double bonds. Aromatic hydroxylation may occur through the rearrangement of the carbocation above or an equivalent species produced from an arene oxide intermediate. Mechanism 3 assumes that the 0x0-derivative in the canonical form represented by (14a Scheme 6) is an electrophilic agent Scheme 22 4 Epoxidation of Olefins and Hydroxylation of Aromatic Comnounds Besides the hydroxylation of non-activated C-H bonds one of the earlier processes known to be associated with P-450 enzymes is the oxygenation reaction involving carbon-carbon 7~ bonds.Olefinic double bonds are usually converted to the corresponding epoxides with the overall retention of stereo- chemistry.58 Aromatic compounds on the other hand give rise to phenols. The phenols arise either from the rearrangement of initially produced arene oxide species59 or perhaps through the intermediacy of a carbocation60 and their formation is invariably attended by a NIH shift61 (Scheme 21; NIH stands for National Institute of Health where the original discovery was made). The latter refers to a phenomenon in which the hydrogen bonded to the carbon undergoing hydroxylation is shifted to the neighbouring position in the product.4.1 Possible Mechanisms With both types of oxygenation reactions involving aliphatic and aromatic double bonds the problem is reduced to one of defining the mechanism through which the 0x0-derivative is used for the formation of an epoxide. In the preceding sections we have seen that the mechanism of a wide variety of reactions can be explained through the involvement of Fe'"-O' it is therefore not surprising that the same species has been inplicated in the epoxidation reaction. Even though the dominant property of the carbon-carbon 71 system is to undergo reactions with electrophilic agents there are re-spectable chemical precedents for justifying a radical addition of FerV-0' to a double bond to produce an intermediate of the type (50 Scheme 22).The conversion of the latter into an epoxide is however more problematical. The difficulty is tacitly recognized in the literature and at least two solutions for this problem are theoretically p~ssible.~~.~~,~~ Firstly that an internal redox reaction (50) -P (51) produces a cationic in- termediate which then undergoes the expected cyclization to the epoxide (52). The second is an ingenious proposal (Mechanism 2 Scheme 22) which envisages that prior to any bonding process there is a transfer of an electron from the double bond to the 0x0-derivative producing a radical cation (53) which following radical recombination gives the cationic intermediate suitable for epoxide formation (53) +(51) -+ (52).The proposal makes the unconventional assumption that an olefinic double bond is a reducing agent capable of converting FeIV into Fell1. Finally we have already emphasized that some of the reactions of the 0x0-derivative are best explained by assigning it an electrophilic character. In view of the propensity of NPR 8 Scheme 24 carbon<arbon double bonds to undergo electrophilic additions it is quite possible that this property of the C=C determines that it is the electrophilic character of the 0x0-derivative that is used in the epoxidation reaction as shown in Mechanism 3 (Scheme 22). A number of approaches have been used to shed light on the mechanism of aliphatic epoxidations as well as aromatic hydroxylations however none of these has provided conclusive evidence in support of any of the alternatives considered above.62 The only firm conclusion drawn from these is that epoxide formation does not occur by a concerted process.Interestingly however rearrangement observed during the oxidation of certain olefins has in the authors’ view provided more meaningful mechanistic pointers. For example the oxidation of norbornadiene leads to the formation of the aldehyde63u(55 Scheme 23). The reaction is best rati~nalized~~ by invoking the participation of the carbocation (54) but the question remains whether the latter is formed by the direct electrophilic addition of (+‘)Fe’”=O to the diene or through an initial radical formation.Another example involves the microsomal epoxidation of (Scheme 24) where the aldehyde (57) is formed as a side reaction similarly the oxidation of 1-phenylbutene (58)63cgives small amounts of 1-phenyl- 1-butanone and the 1-phenyl-2-butanone (60). It is known that these side products do not arise from the corresponding epoxides hence their formation is best ration- alized by invoking the participation of carbocations (56) and (59) formed as intermediates during the oxidation process. Although it is not possible to be certain whether similar carbocations are also involved in the main pathway to epoxide formation the mechanistic significance of the observations is considerable. Since philosophically at any rate once it is realized that the 0x0-derivative is capable of producing carbocations from olefinic substrates then it is legitimate to use such intermediates in other pathways so long as their involvement is justified from a theoretical view point.This is the case for epoxide formation. Circumstantial evidence for the involvement of carbocations in aromatic hydroxylation is provided by kinetic in which it was shown that the rates of hydroxylation of monosubstituted (H F C1 Br and I) benzenes correlated linearly with CT+values for these substituents. The importance of this result was however marred by the finding that a similar correlation was not found for the individual ortho or para hydroxylation rates.64 The difficulty of using the approaches of physical-organic chemistry to probe the mechanisms of enzyme catalysed reactions is that whereas in non-enzymic systems the reaction rates are governed solely by the stereo-electronic properties of the reactants in enzymic reactions these properties are further NATURAL PRODUCT REPORTS 1991 2H202 catalase -2H20 + 02 (10) AH2 + R-OOH peroxidaset A + R-OH + H20 (11) R=alkyl or H “\C-H + CI-+ R-OOH chloroperoxidase /I ‘.\c-Cl + R-OH + H20 / R=alkyl or H perturbed by the steric constraints imposed by the protein.Therefore the possibility that the correlation deduced from test tube chemistry will faithfully hold in enzymic systems is a remote one. Under these extenuating circumstances if the kinetic evidence is viewed leniently then it clearly points to a mechanism in which the initial bonding event is accompanied by the formation of a cationic species.In our view such a requirement is filled best by Mechanism 3 and then by Mechanism 1 (Scheme 22). Further support for the notion that in the biological oxidation of unsaturated carbon compounds cationic species are involved comes from the study of the hydroxylation of isotopically labelled chlor~benzenes~~ and from experiments in which it was shown that the reactions of certain acetylenic compounds with m-chloroperbenzoic acid gave products similar to those formed during biological oxidations.66 5 Haem Containing Peroxidase and Related Enzyme Systems Attention has already been drawn to the fact that the oxo- derivative may be produced ‘artificially’ from the resting state of P-450 (in the Fe’I’ form) by reaction with certain per- oxide~~~,~~ (see Scheme 5).In our view this ‘non-physiological’ pathway for the generation of the 0x0-derivative in P-450 group of enzymes is reminiscent of the mechanism through which catalase peroxidases and chloroperoxidase catalyse their respective reactions shown in equations (lo) (11) and (12). 5.1 Catalase Beef liver catalase which has been extensively studied by Chance67 was one of the first enzymes to be isolated in a high state of purity and its crystallization by Sumner and Dounce in 19376s is an important landmark in the history of biochemistry. The beef liver enzyme is composed of 4 identical subunits. Each subunit consists of a single polypeptide chain (M 60000) that is associated with one molecule of ferric protoporphyrin IX.67 The axial ligand involved in the direct binding of haem iron to the protein is now firmly established by X-ray diffraction studies to be a tyrosine residue.69 As is the case for most other haemoproteins the second axial ligand is believed to be a H,O molecule.The action of catalase is very fast each catalytic centre promotes the decomposition of about 9.5 x lo4molecules of H,0,/second67 making it one of the most active enzymes known. The crucial feature of the mechanism of the catalase catalysed reaction is the involvement of an oxo-derivati~e.~~~* lgd9 13’ Scheme 25a shows that of the two molecules of H,O consumed in each round the first is used to produce H,O and the 0x0-derivative the latter then via hydrogen abstraction and disproportionation reactions furnishes 0 and the second molecule of H,O.In our NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT Catalase hydrogen -abstraction disproportionation-H20 + O2 + Ent. 'OH co-0 Peroxidase r 1 .I. ,I ,I hydrogen d I abstraction /Iq& disproportionation-+ + H20 A Enz. \ HO H 'A0 (611 (14 Scheme 6) L Chloroperoxidase \L& /I \ (63) The literature on peroxidases and catalase is heavily centred around two enzyme-intermediates known as Compounds I and 11. The relationship of these to the intermediates proposed in the above mechanisms is defined in Scheme 25b Scheme 25a (+.) FeIV=O FeIv=O Compound I Compound I1 It is now generally accepted that horseradish peroxidase and catalase Compound I have the same structure as the 0x0-derivative involved in P-450 systems.The Compound I1 is usually formulated as above and may be regarded as the unprotonated form of (62) used in mechanisms of Scheme 25a Scheme 25b view the mechanism of Scheme 25a is the simplest explanation of the diverse observations made on the en~yme,~~,~~ though other 'non' radical mechanisms of the reaction have been considered by the previous investigat01-s.~~ 5.2 Peroxidases This group71 of enzymes catalyse the general reaction of Equation (1 1) in which the decomposition of H202or an organic peroxide is coupled to the dehydrogenation of a range of substrates such as hydroquinones ascorbate phenols and amines.Peroxidases are widely distributed but most extensive studies have been performed on enzymes isolated from horseradish (horseradish peroxidase) milk (lactoperoxidase) and fungi (chloroperoxidase). Horseradish peroxidase contains an imidazole-ligated ferric protoporphyrin IX as the prosthetic group72 and like related peroxidases from other sources displays Enz-Fe"' + R-OOH -Enz-Fe"-b + R-OH (13) Enz-Fe"-b + AH2 -Enz-Fe"' + A + H20 R = H or alkyl; AH2 = H202 or an oxidizable organic compound a wide range of overlapping activities all of which may be rationalized by invoking the participation of an oxo-deriva- tive.19 In the mechanism of Scheme 25a the latter species is shown to arise from the heterolysis of the hexa-coordinated adduct (Fe"'-OOR) produced by the association of the resting state of peroxidase with an oxidant peroxide (Scheme 25a).73 The dehydrogenation reaction then may be envisaged to involve successive hydrogen abstraction and disproportionation sequences considered above for the reaction catalysed by catalase.If this analysis is accepted then in broad chemical terms catalase and peroxidases could be viewed to catalyse identical transformations consisting of the two reactions given in Equation (1 3) and these enzymes differ only in their substrate specificity. 5.3 Compound I and Compound II The literature on peroxidases and catalase has been dominated by the study of two complexes Compound I and Compound 11 ever since their discovery nearly half a century Compound I is produced by mixing horseradish peroxidase or catalase with a peroxide in a one to one molar ratio.There is now general agreement that Compound I is the FeIV=O porphyrin radical cati~nl~-~~ (149 for which a number of canonical structures have already been considered (Scheme 6). The mechanism of the formation of Compound I and its role in a wide variety of enzymic reactions has also been dealt with in detail above. Compound I1 is usually produced by the action of reducing reagents such as K,Fe(CN) on Compound I.75976 The attendant Compound I Compound I1 reaction was envisaged by nearly 4 decades ago to involve a one-electron reduction which now may be formulated as shown in Equation 14.It has been generally assumed that horseradish peroxidase Compound I1 has a double bond and is a compulsory intermediate in the normal enzyme reaction. The participation of an equivalent species in the decomposition of H,O by catalase has also been considered but the evidence for this is not overwhelming since under the conditions of steady state kinetics this species is not produced in easily detectable amounts.67 The reader will note that a FeIV=O species is not directly involved in the mechanisms we have considered for peroxidases and catalase (Scheme 25a). In our view the formulation of Compound I1 as FeIV=O is justified when this species is produced from Compound I by reduction with single electron donors e.g.K,Fe(CN), however in the physiological reactions catalysed by peroxidases or for that matter catalase the reductants are hydrogen atom donors (AH,) which by analogy with P-450 linked reactions should participate in reduction by the transfer of a hydrogen atom. This is the consideration that has led us to deduce that in the catalytic cycles of peroxidases and catalases the reduction of Compound I ( +')FeIV=O produces Fe'"-OH. The latter species may be regarded as the protonated form of Compound I1 as elaborated in Scheme 25b. Several observations already recorded in the literature may be quoted in support of the proposition that the form of Compound 11 which is produced during catalysis has the Fe'"-OH structure. Horseradish Compound I has been subjected to extended X-ray absorption fine structure analysis by several groups and has beep found to have a short iron-oxygen bond length (about 1.6A) characteristic of a double bond ~tructure.~~~-~ There is however disagreement about the Fe-0 bond length for horseradisb peroxidase Compound I1 which owas found to be I .60-1.66A by Penner-Hahn et ~1.~~" but 1.9A by Chance.lSc It is not certain how the Compound I1 was produced by workers reporting the short Fe-0 bond length characteristic of the double bond structure however Chancelse obtained Com- pound I1 by the reaction of Compound I with ascorbic acid at low temperature.In our view ascorbic acid should reduce Compound I by a hydrogen atom transfer producing a FelV-OH species w$ich will be expected to have a Fe-0 bond length of about 2.OA as was found e~perimentally.~~~ Resonance Raman on horseradish Com-pound I1 at alkaline pH values (9 to 11.5) shows a peak at 787 cm-l attributed to a Fe=O structure.The intensity of this vibration is greatly reduced at pH 7.00. This behaviour is consistent with the acid-base equilibrium of Fe'"=O and Fe'"-OH species outlined in Scheme 25b. Interestingly however the authors reporting the aforementioned observation assigned a hydrogen-bonded rather than Fe'"-OH structure to the acid form of Compound 1177 for the following reasons. The Fe"'-OH stretching frequencies of haemoglobin and myoglobin are known to be 490-495 cm-' since resonance in this region of the spectrum was not observed for the 'acid' form of Compound I1 it was not formulated as FeIV-0H.At the risk of betraying caution we view the acid form of Compound I1 as FeIV-0H and draw attention to fact that such a formulation was implied by Critchlow and D~nford~~ in 1972. Furthermore the structural assignment (FeIV-0H) will be consistent with the elegant and careful EXAFS data of Chancelsc cited above. 5.4 Peroxidases in Phenol Coupling Peroxidases are also involved in the elaboration of the skeleton NATURAL PRODUCT REPORTS 1991 Scheme 26 of a variety of polyaromatic natural products whose biogenesis was elegantly rationalized by the 'oxidative phenol coupling ' hypothesis proposed by Barton and Cohen in 1957.7sThe key proposal in the theory is the formation of phenol radicals the various canonical forms of which can then combine in a number of ways to give a myriad of new C-C and C-0 bonds as shown in Scheme 26.The hydrogen abstraction steps in the biosynthesis must be similar to the ones involved in the removal of two hydrogen atoms from AH in the conventional peroxidase reaction (Scheme 25). Except that in the 'oxidative phenol coupling' the two hydrogen atoms are obtained from different molecules (Scheme 26). 5.5 Chloroperoxidase Chloroperoxidase from Culduriomyces fumugo is a tetramer with subunits M of 40000 and its enzymo10gy80,81 and structureaZ has been extensively studied by Hager. Each subunit contains one ferric protoporphyrin IX molecule that is ligated to a cystein thi~late.~~ A remarkable property of peroxidases is their ability to catalyse the oxidation of halide ions to produce species which participate in electrophilic substitution reactions.This reaction (Equation 12) though a dominant feature of the fungal chloroperoxidase is also catalysed by other peroxidases notably lactoperoxidase. In order to rationalize this facet of peroxidases we make the assumption that the heterolysis of the peroxide bond in the conversion (61) -+(14) Scheme 25a is a reversible process and that an halide ion instead of an alkoxide participates during the reversal process to produce an hypohalite intermediate (63) that will be expected to be a powerful reagent for electrophilic halogenation. The involvement of an hypohalite in the chloroperoxidase catalysed reaction was originally proposed by Hager and coworkersa1 to rationalize the fact that enzyme in the absence of ROOH could chlorinate dimedone using Na3T10,; and when the reaction was carried out in the presence of a large excess of non-isotopic C1- ions the chloro group incorporated into the organic compound was exclusively from Na3T10,.5.6 Cytochrome c Oxidase This ubiquitous enzyme is involved in the terminal 4 electron reduction of 0 to water and has been the subject of extensive physico-chemical studiesa4 since its original discovery by Keilin in 1925.85 The functional unit of the beef heart enzyme which has a M of around 140000 is composed of 7 to 12 polypeptides and contains two molecules of haem uand two copper atoms.86 It is known that only one of the two haem moieties is involved in direct interaction with 0 and this has historically been designated as u3 and the other as a (Scheme 27).The resting state of the enzyme in which both haem iron atoms are present as Fellr and the copper as Cu2+ may be represented as in NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT (64) A +H+ -H20 The oxygen-binding iron in the south-east corner denotes cytochrome a3and that in the north-east cytochrome a. The two copper atoms of the oxidase which are usually defined as Cu and Cu are not differentiated here. In the pasts4c it had been assumed that in the conversion (66) -f (67) the electron is donated by a copper atom; this view has now been q~estioned"~ and the new evidence suggests that the reduction may in fact involve the iron of cytochrome a as shown above.g0 The intermediacy of (68) shown above is deduced from chemical considerations and is not the feature of existing mechanisms in the literature Scheme 27 structure (64) Scheme 27.For illustrative convenience we consider the mechanism of the overall process by assuming that all the four metal atoms in the enzyme are reduced by the ferrous form of cytochrome c prior to interaction with 0,. Attempts at the identification of the intermediates parti- cipating between 0 and H,O were originally made using opti~al*~*~' and ESR techniques and the conclusions drawn from these studies have now been confirmed and extended by resonance Raman spectroscopy.It was found that the primary adduct formed from reduced cytochrome c oxidase and 0 during the first 100 micro seconds had iron4oxygen stretching at 568 cm-l characteristic of oxygen complexes of haemoglobin and myoglobin.88 Since the latter complexes have already been formulated as low-spin Fe"'-OO' species the primary oxygen- derivative of cytochrome c oxidase may be represented as (66). In similar experiments when the spectra were recorded at 800 microseconds a mode at a frequency of 790 cm-l was observed. The frequency is similar to that for Compound I1 of horseradish peroxidase lactoperoxidase and other related By analogy with the aforementioned examples the intermediate with a frequency at 790 cm-' may be formulated as the 3 electron reduced species having the structure (69) containing the Fe'"=O functionality.8s*s0It seems reasonable to assume that the latter (69) is formed via the usual oxo-derivative (68) though the currently in vogue for the cytochrome c oxidase reaction envisages the direct conversion of a hydroperoxide species of the type (67) into (69).In our view there is a cause for reflection here since the conversion (67) + (69) discounting the proton addition steps entails two reactions; rupture of the 0-0 bond and a one electron reduction-these are more attractively seen as step-wise processes rather than being packaged into a single concerted event. The reader will remember that in all the examples considered above the oxo-derivative of the type (68) was reduced by hydrogen atom transfer however its postulated reduction by an electron transfer is reminiscent of the conversion of Compound I into Compound I1 by K,Fe(CN),.The corollary from the contrasting behaviour is that the oxo-derivative (i.e. Compound I) is endowed the dual property of being reducible by a hydrogen atom or an electron to produce the protonated form of Compound I1 or Compound I1 respectively. Returning to the oxidase reaction the addition of the fourth electron (plus a proton) to (69) then gives the Fe"'-OH derivative (70). The evidences1 of the formation of such a species (70) has recently been obtained by resonance Raman spectroscopy which showed the appearance of resonance at 450 cm-l during the late phase (800-300 ps) of the oxidase reaction.By analogy with other related systems the resonance has been attributed to an Fe"'-OH stretching mode.s1 The regeneration of the resting state of the oxidase from (70) then occurs following protonation. The overwhelming picture emerging from the cumulative information available to date is that the mechanism of the oxygen reduction by cytochrome c oxidase is identical to that operating in P-450 enzymes. In both cases the first two electrons are used for the formation of the crucial oxo-intermediate. The only difference being that the oxo-derivative in the cytochrome c oxidase reaction is reduced by the transfer of two single electrons and two protons are then added separately to produce HOH while in that derived from P-450 the two electrons are provided in the form of two radical species to give ROH.6 Non-haem Iron Containing Oxygenases Requiring a-Oxog lutarate The types of reactions normally characteristic of P-450 oxygenases are also catalysed by enzymes containing non-haem iron. The active forms of this class of enzymes contain Fe" and like their P-450 counterparts have a compulsory requirement for 0 but instead of NAD(P)H these enzymes use a- NATURAL PRODUCT REPORTS 1991 + a-Ketoglutarate Ascorbate -c-H N + Succinate + C02 02 C=O C=O I I -NH -NH fNH2 + a-Ketoglutarate + Succinate + COP C=O $=O I -NH -NH (73) (74) 0 0 0 H H H H (75) Ascorbate is not consumed in the above reactions but has been suggesteds2 to be required for maintaining the iron or another sensitive centre of the enzyme in a reduced state Scheme 28 L3,**-\ /I \ L2 o\ OH (79) 0-B-H R-OH (83b) (=) The five hypothetical ligands used above (L,-L,) are the amino acid side-chains.In the text the possibility is considered that the hydrogen abstraction may in fact use an Fe'"-O. produced by a second redox centre M2eM2+. M and M are not elemental metals but metal ions Scheme 29 oxoglutarate as the reductant (Scheme 28).92 The simplest is sparse and we consider their mechanism by analogy with P-450 enzymes. If the iron bonding site of these enzymes is examples of these non-haem enzymes are prolylg3 and ly~yl~*"~~ hydroxylase which catalyse the reactions (71) +(72) and (73) +(74) respectively while thymine-5-methyl ~xygenase~~~ is a multifunctional enzyme and involved not only in the initial hydroxylation reaction (75) +(76) but also in two further oxidations to give thymine-5-carboxylic acid (78).The information regarding the detailed chemistry through which these non-haem a-oxoglutarate-requiring enzymes work modelled on P-450 oxygenases then the resting state of the enzymes will need to contain 5 strategically located side-chain groups; one to serve as the axial ligand present in P-450 enzymes and four other groups to take the place of the four pyrrole nitrogen atoms of haem (79 Scheme 29). The Fe" in such an environment should react with 0 to produce a low- spin hexa-coordinated adduct (80) that may be regarded as a NATURAL PRODUCT REPORTS 1991-M.AKHTAR AND J. N. WRIGHT [acceptor] X-B-H 0 -CH= c H -C X-7 Non-haem Iron Containing Oxygenases and Desaturases Linked to NAD(P)H 7.1 Oxygenases There is circumstantial evidence for the existence of yet another class of oxygenases which combine features of P-450 as well as a-oxoglutarate-requiring oxygenases. These contain non-haem Ferll but use NAD(P)H as reductant. A possible example of this type of oxygenase is the enzyme that catalyses the oxidation of the 4a-methyl group of sterols during cholesterol biosynthesis in animals and ergosterol biosynthesis in yeast. It was known from experiments using microsomal fractions from rat liver that the multistep oxidation of the 4a-methyl group of sterols into the corresponding carboxylic acids required NADPH and hydrogenatoms -&0 and it was assumed that a single enzyme was involved in the loss of Sp-and 7k process and it belonged to the P-450 class.Genetic experiments then revealed that mutants of yeast deficient in haem biosynthesis could oxidise the 4a-methyl group thus indicating HO the enzyme may not contain haem and therefore is not a P-450 HO&H (87) (89) Scheme 30 non-haem analogue of oxyhaemoglobin. For the resulting adduct Fe"'-00' to be able to be utilized the 'reducing equivalent ' entrapped in a-oxoglutarate it must be converted into a peroxy derivative Fe"'-OOH. We envisage that this may be achieved by the presence of another redox centre in this class of enzymes.The two states of the redox centre are symbolized as M and Mi and the formation of the peroxy derivative is shown to be achieved by the attendant conversion of M into M,+. Next the reaction of the peroxy derivative with a-oxoglutarate and the subsequent decarboxylation of the adduct (82) occurs using the chemistry for which there is a well established precedent. In accordance with the requirement of the stoichiometry of this process it is already known that during the conversions catalysed by prolyl and thymine-5- methyl hydroxylases an atom of oxygen from 0,is incorporated into s~ccinate.~ The more important aspect of the decarboxy- lation reaction is the formation of an '0x0-derivative' for which a number of canonical structures may be deduced for example (83) -,(83c).It is possible that the latter species is oxidized by the aforementioned reaction centre regenerating the M form of the enzyme and producing Fe"'-O' which is then used for the hydroxylation reaction by a sequence involving hydrogen abstraction and oxygen rebound steps. The analysis above succeeds in making the two classes of hydroxylases (P450 and a-oxoglutarate-requiring) mechan- istically similars5 but not identical since the ferroxy-radical involved in P-450 enzymes formally contains FeIV while that in non-haem a-oxoglutarate-requiring hydroxylase is Fe"'. If hydrogen atom abstraction by a ferroxy radical was the property of a unique valence state of iron then there would be required another redox centre in this class of enzymes to adjust the oxidation state of iron.For example the conversion of Fe"'-O' into FeIV-0' may be coupled to the reduction of a second redox centre (M,+ -+ M,). FeIV-0' then participates in hydroxylation following the reaction course pursued by P-450 enzymes. The catalytic cycle releases the Fell' form of the enzyme from which active enzymes containing Fe" is re-generated by the reoxidation of M to M,+. oxygenase.96 Assumings7 that the enzyme contains non-haem iron as Fell' its mechanism can be modelled on the P-450 system in which all the five ligands involved in the formation of the peroxide intermediates are provided by the protein. The FeIV-O' species formed from the peroxide then participates in hydrogen abstraction followed by oxygen rebound to produce the hydroxylation product.The conversion of the hydroxy- methyl to formyl and thence to a carboxylic group may be rationalized by the various alternatives considered in Scheme 14. The best studied examples of enzymes containing non-haem iron and using NAD(P)H are however those involved in the desaturation process and this aspect is considered next. 7.2 Desaturases In 1967 during the course of study on the formation of the 5,6-double bond in sterol biosynthesis we highlighted the operation of two types of mechanism for biological desaturation reactions.g8a The first type concerns substrates in which one of the two hydrogen atoms involved in the formation of the olefinic linkage is located adjacent to an electron withdrawing group so that it may be removed to give a resonance stabilizing carbanion.In this case the reaction occurs by a stepwise ionic process shown in Equation (15). The examples of enzymes acting by this mechanism are acyl CoA and succinate dehydrogenases. The second type of mechanism operates when the substrate contains only non-activated C-H bonds as examplified by the conversion (86 -+87 Scheme 30) in insects,99 the introduction of the 5,6-double bond in the biosynthesis of cholesterols8 and ergosterolloo(88) -+ (89) the desaturation reactions involved in the biosynthesis of unsaturated fatty acids,lo1 and the formation of the unsaturated side chains of plant With the example^^*-^^^ studied at the time it was found that these reactions occurred with an overall cis-elimination of hydrogen atoms and the enzyme system showed an obligatory re-quirement for 0 and a reducing agent (NADPH or NADH).In the light of this information it was postulateds8* loo that this class of enzymes contain a metal ion which following reduction reacts with 0 to produce a metal-peroxide species. The latter is then involved in the cis-removal of the two hydrogen atoms by a concerted mechanism. This mechanism was proposed before any information was available on the nature of the enzymes involved in the desaturation process or there was any appreciation of the theoretical principles which govern the generation of an 'active species' from 0,. It has subsequently been shown that 5-desaturase,lo3 stearyl CoA desaturase,lo4 and linoleoyl CoA desaturaselo5 contain non-haem iron.By analogy with reactions discussed above it is thus reasonable to assume that metal peroxy species proposed by us nearly a quarter of a century ago is the hexacoordinated iron compound of the type (91); which NATURAL PRODUCT REPORTS 1991 Fe"' disproportionation\ Scheme 31 Panel E Panel D Panel C Panel 6 Panel A Catalase Pe roxidase P-450 (artificial) P-450 (physiol.) Haemoglobin Nhis I I I s 1 n .'-I (R H)'1 Fe ,.a. \ \ \ iOOH 3OOH 'hIO 1. lo, RH) /I \ Nhis \I& /I \ o\ OR +H+l-HoH +H+l-RoH 37 (RH) FeIV /I \ 0' jH202 JAH2 1 02+ H20 + Enz. A + H20+ Enz. ROH + Enz.The scheme shows the common chemical features of different haem-containing proteins and emphasizes that the same species FeV=O -(+ -) Fe'"=O -Fe'"-O * ,is involved in the catalytic cycles of all the proteins Scheme 32 NATURAL PRODUCT REPORTS 1991-M. AKHTAR AND J. N. WRIGHT does not directly participate in the removal of hydrogen atoms but is converted to the usual 0x0-derivative which then produces olefinic double bond through hydrogen abstraction followed by disproportionation (Scheme 31). The desaturation process is thus closely related to the hydroxylation reaction and the two differ only in the final step ;in one case the carbon radical (94) is quenched by an oxygen rebound reaction (94) +(95) and in the other by disproportionation (94) +(96).How the choice is made between these two equally plausible courses and what determines the strict fidelity displayed by these enzymes is an interesting avenue for future research. 8 Concluding Comments We have seen above that the mechanism used in haemoglobin for the binding of oxygen to iron without any further refinement has been conserved in a wide variety of iron-containing oxygenases and related enzymes. The binding to iron is achieved at the expense of cleaving one of the bonds of 0 to produce a superoxide ‘equivalent ’. For the cleavage of the second oxygen-oxygen bond the superoxide moiety needs to be reduced to the level of a peroxide. Experimental evidence already exists to show that such a mechanism operates in P-450 types of enzymes and we have proposed that a similar process may be involved in certain non-haem iron containing oxygenases and desaturases.The cleavage of the oxygen-oxygen bond of a peroxide to give the 0x0-derivative is a crucial event under- pinning the mechanism of action of catalase peroxidases chloroperoxidases P-450 systems and several other non-haem iron containing enzymes participating in oxygenation and oxidation reactions. The resulting 0x0-derivatives may be represented by several canonical forms and one of these (14d Scheme 6) is involved in hydrogen abstraction. The latter process forms the basis of achieving a myriad of chemical transformations which may be viewed as variations on a common theme.lo6 Notwithstanding this we have now un-covered evidence for the direct participation of an Fe”’0OH species in the cleavage of acyl-carbon bonds (-CO-C).The haem containing enzymes if not from an evolutionary then certainly from a chemical viewpoint can be regarded as an hierarchical family in which the fundamental mechanism used by haemoglobin for binding 0 has been conserved and extended as displayed in Scheme 32. An important feature to note from Scheme 32 is that the cleavage of oxygen-oxygen bond to produce the 0x0-derivative is not a property that depends on the nature of the axial ligand since the process occurs as readily in horseradish peroxidase that contains histidine as the axial ligand as it does in catalase and P-450 enzymes which use tyrosine phenolate and cysteine thiolate respectively in this position.Acknowledgements. Our work in the P-450 field was supported by the Science and Engineering Research Council and Cancer Research Campaign. We thank Mrs Sue Broomfield for the preparation of the manuscript from untidy scribbles. 9 References 1 M. Eigen Angew. Chem. Int. Ed. 1964 3 1. 2 R. P. Bell ‘The Proton in Chemistry’ 1959 Methuen London. 3 M. Klingenberg Arch. Biochem. Biophys. 1958 75 376. 4 D. Garfinkel Arch. Biochem. Biophys. 1958 77,493. 5 T. Omura and R. Sato Biochim. Biophys. Acta 1963 71 224; J. Biof. Chem. 1964 239 2379 and references cited therein. 6 (a) S. D. Black and M. J. Coon in ‘Cytochrome P-450 Structure Mechanism and Biochemistry’ ed. P. R. Ortiz de Montellano 1986 Plenum Press New York and London p.161; (b) T. L. Poulos in ‘Cytochrome P-450 Structure Mechanism and Bio- chemistry’ ed. P. R. Ortiz de Montellano 1986 Plenum Press New York and London p. 505; (c) P. M. Champion B. R. Stallard I. C. Gunsalus and G. C. Wagner J. Am. Chem. SOC. 1982 104 5469. 7 I. C. Gunsalus J. R. 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ISSN:0265-0568    

DOI:10.1039/NP9910800527

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

lndolizidine and Quinolizidine Alkaloids J. P. Michael Centre for Molecular Design Department of Chemistry University of the Witwatersrand Wits 2050 South Africa Reviewing the literature published between July 1989 and June 1990 (Continuing the coverage of literature in Natural Product Reports 1990 Vol. 7 p. 485) 1 Slaframine 2 Hydroxylated Indolizidines 2.1 1-Hydroxyindolizidine and 1,2-Dihydroxyindolizidine 2.2 Swainsonine 2.3 Castanospermine and Related Alkaloids 3 Indolizomycin 4 Monornorium Alkaloids 5 Alkaloids from Amphibians 6 Elaeocarpus Alkaloids 7 Phenanthroindolizidine Alkaloids and Seco Analogues 8 Furylquinolizidines and Furylindolizidines 9 Lythraceae Alkaloids 10 Plumerinine 11 Myrtine 12 Alkaloids of the Lupinine-Cytisine-Sparteine-Matrine-Orrnosia Group 12.1 Occurrence and Detection 12.2 Structural Spectroscopic Chemical and Biological Studies 12.3 Synthesis 13 Alkaloids from Marine Sources 14 References 1 Slaframine L-Lysine was the starting material in a formal synthesis of this fungal metabolite (Scheme 1).l Anodic oxidation of the lysine derivative (1) was accompanied by migration of the terminal p-toluenesulphonylamino group to give acetal (2).However the optical activity at the stereogenic centre was lost in the process. Uncomplicated transformations provided the piperidine de- rivative (f)-(3) which has previously2 been converted into slaframine (4)[cf. Reference 3(a)]. 2 Hydroxylated lndolizidines The distribution and structure of swainsonine (9,castano-spermine (6) and related polyhydroxylated pyrrolidine pip- eridine and pyrrolizidine alkaloids have been reviewed in an article that also contains a succinct account of their biological a~tivity.~ Amongst the effects described are inhibition of glycoprotein processing the inhibition of plant and intestinal glycosidases insect antifeedant properties effects on cancer and the immune response and -of considerable current interest -antiviral activity.Swainsonine and related glycosidase inhib- itors also form the subject of a book edited by a group of prominent workers in the field.5 Very useful information on 5--OH (5) (-)-Swainsonine Rl* R3*'* R2 (6) (+)-CastanospermineR' = R3= OH R2 = H (7) 6-Epkastanospermine R' = R2 = OH R3 = H (18) 7-Deoxy-6-epi-castanospermine R' = R3 = HI R2 = OH C02Me C02Me --..---.--.-* NHCOMe 92% NHCOMe 7 I *4? TsHN NH2 NHTs Me0 OMe (82% OCOCH3 t C02Me .r,- - - - - --.iii iv 60% H2N TsHN 'COMe TsHNDCOMe (4) (k)-Slaframine (W3) Reagents i MeOH NaOMe KI Pt electrode 15 mA cm-' 12 F/mol at 0 "C then 17 F/mol at 25 "C; ii NH,Cl 130 "C/20 mmHg; iii H, PtO, HOAc; iv HCl EtOH Scheme 1 553 NATURAL PRODUCT REPORTS I991 Reagents i Hg(OAc), THF r.L; ii NaBH(OMe), H,C=CHCO Me CH,CI,; iii H, Pd(OH), MeOH; iv LiAlH Scheme 2 Ms I OH? Ms "( OMS N3 N3 (11) iv v OH o-f H viii ix vii ...0 vi --OH -60% 92% HN 80% C02But C02But 0 (5) Swainsonine (13) (12) Reagents i NaN, DMF-H,O; ii camphorsulphonic acid H,O-MeOH; iii Ba(OH), MeOH ;iv (F,CSO,),O; v LiCH,CO,But THF; vi H, Pd-C EtOH ;vii NaOMe MeOH reflux ;viii BH * Me,S ; ix CF,CO,H-H,O then ion-exchange chromatography Scheme 3 H ?H (9) 2-Epklentiginosine R1= H R2= OH (10) Lentiginosine R' = OH R* = H practical methods for the detection and isolation of alkaloidal glycosidase inhibitors including swainsonine castanospermine and 6-epi-castanospermine (7) may be found in a book dealing with the isolation of natural products.s 2.1 1-Hydrox yindolizidine and 1,2-Dihydroxyindolizidine 1,8a-cis- 1 -Hydroxyindolizidine (8) postulated as an inter-mediate in the biosynthesis of slaframine but still unknown as a natural product has been prepared in racemic form by the short reaction sequence involving intramolecular amidomercur- ation shown in Scheme 2.' A related synthesis of (8) proceeds by way of iodine-induced cyclization of a y &-unsaturated thioimidate.* Last year's review summarized reports of the occurrence of 1,8a-trans-1,2-cis-dihydroxyindolizidine(9) in nature [cf.Ref-erence 9(a)]. Leaves of Astragalus Ientiginosus var. diphysus have now been shown to contain the same optically active alkaloid [a],-32.5" (c 0.13 CHCl,) as well as the (-)-1,2- trans-diol isomer (lo) -3.3" (c 0.33 MeOH).]-O Swain- sonine is the major metabolite. The new alkaloid (10) has been named lentiginosine the previously characterized cis-diol thereby becoming 2-epi-lentiginosine.The relative stereo-chemistries of both compounds were deduced from analysis of 'H and 13CNMR spectra of the diols and their diacetates. The absolute configuration of lentiginosine has been assigned on biogenetic grounds only whereas Overman's synthesisll of (-)-2-epi-lentiginosine discussed in last year's report [cf. Reference 9(a)] effectively confirms the absolute structure given in (9). Lentiginosine found to be a reasonably good inhibitor of amyloglucosidase is in fact the first known glycosidase inhibitor from this family of alkaloids to have only two hydroxy groups. Neither it nor its 2-epimer were effective against other glycosidases. 2.2 Swainsonine (-)-Swainsonine (5) has been revisited by Fleet and co-workers who have devised a new synthesis in which the mannose-derived epoxide (1 1) plays a pivotal role (Scheme 3).12 The oxirane ring remained intact during homologation of the triflate of (1 1) with t-butyl lithioacetate but was opened in S,2 fashion when the amine group produced by reduction of azide (12) attacked it intramolecularly to give the pyrrolidine intermediate (13).This compound contains the four stereogenic centres of the target alkaloid to which it was converted by straightforward reactions. Not all steps proceeded with 100 YO conversion; but if one allows for recovery and recycling of unconverted intermediates the overall yield based on D-mannose was a respectable 15YO.This new route is important in that both pyrrolidine and pyrrolizidine congeners of swainsonine can also be obtained from the key epoxide (1 1).NATURAL PRODUCT REPORTS 1991-5. P. MICHAEL OH 0 H oH --OH --OH --OH oH~OH OH OH Bu,BuO'? O d ii - HO"' 0-" K, "f0 OH "f0 OH iv V 7% 64% HO"' (22) (23) (20) Reagents i Bu,SnO MeOH reflux; ii RCOCl NEt, r.t.; iii subtilisin dry C,H,N CH,(CH,),CO,CH,CCl, 45 "C; iv lipase CV CH,(CH,),CO,CH,CCl, THF 45 "C ;v subtilisin phosphate buffer pH 6.0 Scheme 4 Divergent synthetic routes based on reactions of the diastereomeric epoxides of the L-erythrose derivative (14) have been used to prepare (-)-8-epi-swainsonine (15) (-)-8a-epi- swainsonine (1 6) and (-)-8,8a-di-epi-swainsonine (1 7).13 Studies on the biological effects of swainsonine and its isomers continue to proliferate.A substantial body of work deals with the alkaloid's immunomodulatory activity and its potential for cancer immunotherapy. 14-25 For other biological and clinical studies the interested reader is referred to the following effects on male reproductive tissue in the rat;26 post- translational processing of rat prolactin ;,'appetite suppression and growth retardation in the rat;28 secretion of thyroid- stimulating hormone ;29 and inhibition of enzymes associated with cell walls.30 An artificial intelligence program developed for evaluating structure-activity relationships of naturally occurring plant pesticides predicted low rodent carcinogenicity for both swainsonine and castano~permine.~' Molecular orbital and molecular graphics studies of swainsonine castano-spermine and related glycosidase inhibitors have shown that their activity probably derives from their topographical equivalence to the mannopyranosyl cation a result that augurs well for the rational design of potential mannosidase inhibitors and anti-HIV 2.3 Castanospermine and Related Alkaloids Yet another biologically active new alkaloid has been isolated from the seeds of the Moreton Bay chestnut Castanospermum a~strale.~~ Preparative TLC of mother liquors obtained after crystallization of castanospermine and removal of pyrrolizidine alkaloids gave a new compound of molecular formula C,H,,NO which formed a triacetate under mild acetyl-ation conditions.The gross structure was deduced from MS 'H and I3C NMR data and the relative stereochemistry at the stereogenic centres was assigned on the basis of proton coupling constants.The new alkaloid (+)-7-deoxy-6-epi-castanospermine (1 8) is the first trihydroxyindolizidine to be isolated from C. australe. Its absolute stereochemistry was not determined but is probably as shown by analogy with other alkaloids from the same plant. Unlike its structural isomer swainsonine (18) does not inhibit a-mannosidase but is a moderate inhibitor of amyloglucosidase and yeast a-gluco- sidase. The structure of 6-epi-castanospermine (7) has been con-firmed by X-ray crystallographic analysis of its hydr~chloride.~~ This study taken in conjunction with the previously reported synthesis, from L-gulonolactone [cf.Reference 9(b)] should now lay to rest all doubts about the absolute structure. Thermospray liquid chromatography -mass spectrometry has been used as a rapid sensitive analytical technique for analysis of the alkaloids in C. australe extract^.,^ It is of interest that castanospermine transported in the phloem of C. australe is found in the honeydew excreted by the sap-sucking insect Pseudococcus longispinus (mealyb~g).~~ A recent that the anti-HIV activity of castano- spennine can be increased by as much as twenty-fold on esterification has sparked a flurry of publications describing positionally selective acylation procedures as well as several Traditional approaches involving sequential pro- tection acylation and deprotection steps have been used to make both 6- and 7-butanoyl or -benzoylcastanospermines (19 and 20 R = CH3(CH2) or Ph).40 An alternative chemical procedure for making 6-acylcastanospermines (1 9 R = CH, CH3(CH2)2 (CH3)2CHCH2Y CH3(CH2), CH3(CH2),, CH3(CH2),, Ph) using a pre-formed intermediate (21) in which the dibutyltin unit served as both protecting and activating group gave yields of between 18% and 44% (Scheme 4).41 Most interesting however are procedures in which the acylation is en~yme-catalyzed.~~-~~ Treatment of castanospermine in dry pyridine with a variety of activated NATURAL PRODUCT REPORTS 1991 i-iii ___F 88% O 0 V (26) (27) xii 30% OH I xiv xiii 43% from (29) 93% TEOC ' TEOC i TEOC ' A 1 B '1 Me3Sido (30) (29) (TEOC = Me$3iCH2CH20CO) (28) xx xxi xxv xxii-xxiv -"e-d 56% from (31) 29% TEOC ' TEOC ' $ 9 CHO TEoC 4 \ (33) (24) lndolizomycin (32) Reagents i Ph P=NCH2CH,C0 Me; ii NaBH, MeOH ; iii H,C=CHCH,SiMe, TiCI,; iv Lawesson's reagent; v 1N NaOH MeOH; vi iBuOCOC1 N-methylmorpholine THF; vii CH,N, Et,O; viii Rh,(OAc), C,H, reflux; ix W-2 Raney Ni acetone; x Me,O+Bf,-; xi NaBH,; xii Me,SiCH,CH,OCOCl C,H, r.t.; xiii 30% H,O, NaOH MeOH; xiv H,NNH, cat.AcOH MeOH; xv rn-CPBA CH,CI,; xvi TBSCl; xvii 0, CH,Cl,-MeOH then Me S; xviii Ph,P=CHOMe; xix 0, hv then PPh,; xx lithium reagent (32) THF -78 "C; xxi Ac,O; xxii 5 % Na(Hg); xxiii 1N HIO, THF; xxiv tetrapropylammonium perruthenate; xxv Bu,NF Scheme 5 esters (chloroethyl 2,2,2-trichloroethyl 2,2,2-trifluoroethyl or inhibitors that effectively reduced the glycaemic response to There are implications in vinyl esters of alkyl aryl or a-aminoalkyl carboxylic acids) in orally administered sucrose in ~ivo.~ the presence of the proteolytic enzyme subtilisin gave exclusively these results for the treatment of diabetes mellitus.The 1-acylcastanospermines (22) in yields of 23 % to 91 %. By glucosides were less effective against other disaccharidases but contrast use of porcine pancreatic lipase (PPL) gave mixtures were in general more selective than castanospermine itself. of 6-and 7-acyl castanospermines usually in poorer yields. However simple structural comparisons between castano-Further acylation of (22) in the presence of subtilisin PPL or spermine glucosides and disaccharide substrates failed to lipase from Chromobacteriurn uiscosum gave varying mixtures correlate with their ability to inhibit appropriate disacchari- of 1,6-and 1,7-diacyl castanospermines.Yields for a specific dases.47 example (R = CH,(CH,),) are shown in Scheme 4.Acylation The interested reader is referred to the following papers for could also be reversed enzymatically for example the developments relating to the biological activity of castano- action of subtilisin on 1,7-dibutanoyl castanospermine (23 spermine screening against simian immunodeficiency virus ;48 R = CH,(CH,),) produced the 7-monobutanoyl product thyroglobulin secretion in porcine thyroid cells ;49 effect on while porcine liver esterase removed the ester on position 7.synaptic membranes in the rat brain50 and on fetal cerebellar The selectivity was better than 25 :1 for both hydrolyses. explants ;51 inhibition of thioglycosidase-catalyzed gluco-Another class of castanospermine derivatives receiving sinolate hydrolysis;52 inhibition of intestinal sucrase and attention as potential therapeutic agents are the glucosides the reduction of the glycaemic response to sucrose;53 inhibition of preparation of which has been described so far only in the enzymes associated with cell walls;30 inhibition of yeast patent literature.45 Both 7-and 8-O-a-~-glucopyranosyl exoglucanases;54 and effect on the low-density lipoprotein castanospermine in particular were potent long-acting sucrase receptor.55 NATURAL PRODUCT REPORTS 1991-5.P. MICHAEL (35) H -I H3(cH2)3 (C H2)3C H=C H2 Reagents:i THF -78 "C to r.t.; ii H,NNH;H,O C,H, reflux Dean-Stark trap; iii KOH (HOCH,), 170 "C; iv H (3.4 atm) PtO, HOAc; v EtOH reflux; vi DIBAL Et,O -78 "C then HClO, r.t.; vii KCN H,O r.t.; viii H,C=CH(CH,),MgBr THF 0 "C Scheme 6 3 lndolizomycin A spectacular route to the bizarre antibiotic indolizomycin (24) by Danishefsky and co-~orkers,~~ given in detail in Scheme 5 must surely take the laurels amongst the syntheses in this year's crop. The cyclopropane ring probably the least labile feature in a target alkaloid bristling with highly sensitive functionalities was present at the start in the bicyclic anhydride (25). An early step involving the intramolecular rhodium-catalyzed conden- sation between the diazoketone and thiocarbonyl groups of (26) represents a new annulation procedure that although tailor-made for the present synthesis is clearly of greater general applicability.New too is the fragmentation of enol ether (27) induced by treatment with a chloroformate ester that also serves to protect the amine functionality. This unique construction of an azonine ring also merits further investigation as a method for preparing medium-ring heterocycles. The product (28) gave a mixture of isomers of (29) on epoxidation but the subsequent Wharton reaction afforded a single allylic alcohol (30). The sensitive conjugated triene side chain was introduced comparatively late in the sequence by treating the aldehyde (3 1) with lithiated sulphone (32) acetylating the condensation product in situ and reductively eliminating the /3-acetoxy and sulphone groups of product (33).Deprotection oxidation and spontaneous transannular ring closure by carbinolamine formation gave the alkaloid (24) in 0.46% overall yield based on (25). The synthesis did not establish the stereochemistry of the carbinolamine linkage. The product like the natural antibiotic decomposed within a matter of hours precluding direct comparison of natural and synthetic samples. However the NMR spectra of (24) agreed with those published for the natural product. Further evidence for the relative stereochemistry of substituents came from X-ray crystal- lographic analysis of suitable derivatives of (28) and the epoxide of (30).4 Monomorium Alkaloids The venom of the New Zealand ant Monomorium smithii contains a unique and distinctive mixture of alkaloids that serves to differentiate the species from another endemic specie^.^' Two of the three major components are known pyrrolidine and pyrrolizidine alkaloids while the third is a new 3,5-dialkylindolizidine7 (34). This is the first instance of the simultaneous occurrence of indolizidines and pyrrolizidines in an ant. Furthermore (34) is the first 3,5-trans-3,8a-cis-disubstituted indolizidine from an ant all previously known alkaloids of this group having the all-cis relative configuration. The structure of the new compound suggested by MS data was confirmed by two independent syntheses. Stereochemically unspecific reductive amination of the triketone (35) gave the four possible diastereomers of (34) as a mixture GC-MS analysis of which indicated that the second-eluting component correlated with the natural product.Since prior experience suggested that the second component in mixtures of 33-dialkylindolizidines should have 3,5-trans-3,8a-cis geometry a stereoselective synthesis of (34) was carried out as shown in Scheme 6. After Wolff-Kishner reduction of (36) the relative stereochemistry between position 5 and 8a was set up in a reasonably selective (9 1 cis :trans) catalytic hydrogenation of the pyrrole ring. The substituent at position 3 was introduced efficiently by intercepting an intermediate a-cyanoamine prepared in situ from (37) with 4-pentenyl Grignard reagent.The synthetic sample of (34) containing about 7 YOof both 3,5- cis isomers showed the expected GC-MS behaviour and gave 13CNMR shifts that accorded with those of known 3,5-trans- 3,8a-cis-disubstituted indolizidines. No fewer than three syntheses of the popular target (i-)-monomorine I (38) appeared during the review period. An intramolecular nitroso Diels-Alder reaction lies at the heart of Kibayashi's somewhat lengthy synthesis previously published as a communication5a [cf. Reference 3(a)] and now revealed with full experimental details.59 The last step in this synthesis reductive cyclization of ketone (39) produced monomorine I (38) and its 3-epimer (40) in yields of 71 % and 15 % respectively (Scheme 7).A formal synthesisGo commencing with 6-methylpiperidin-2-one has as its key step the quantitative catalytic hydrogenation-cyclization of the acyclic alkynone (41) which gave exclusively the 2,6-cis-disubstituted piperidine (42) (Scheme 7). The conversion of (42) into (f)-monomorine I has been described elsewhereG1 [cf. Reference 3(a)J. A short route to the alkaloid (Scheme 8) exploits rhodium(I1)-catalyzed decomposition of diazoketone (43) the bicyclic system forming as a result of intramolecular carbene insertion into the C(2)-H bond of the pyrrole ring.62 Catalytic hydrogenation was also used to ensure excellent stereocontrol at the four stereogenic centres of the alcohol product (44) which was deoxygenated to NPR 8 NATURAL PRODUCT REPORTS 1991 Reagents i H, 5 YOPd-C MeOH; ii LDA Et,O -78 "C; iii H (2 atm) 5% Pd-C EtOH r.t.Scheme 7 H' N? (43) (38) Monomorine I (44) Reagents i KOH MeCN then ethyl crotonate 0 "C to 25 "C; ii N-methylmorpholine Bu'OCOCl Et,O iii CH,N, Et,O 0 "C to r.t.; iv Rh,(OAc), CH,CI,; v H, PtO, EtOH-AcOH ;vi (CH,Cl), N,N-thiocarbonyl(diimidazole) reflux; vii Bu,SnH toluene reflux Scheme 8 the target compound (38) via the imidazolecarbothioate. The overall yield for this attractive six-step synthesis was 26 %. 5 Alkaloids from Amphibians Australian myobatrachid frogs of the genus Pseudophryne the skin alkaloids of which have been explored cursorily in the past have now been screened more thoroughly by means of GC-MS.s3 While pseudophrynamines (3a-prenylpyrrolo[2,3- blindoles) were generally the major components alkaloids of the pumiliotoxin A class were also present.Pumiliotoxin 267C (45) was a trace or minor component of Pseudophryne bibronii P. coriacea P. corroboree and P. occidentalis and a major component of P. semimarmorata. Pumiliotoxin 277 (46) perhaps an artefact formed by degradation of threo-pumiliotoxin 323A (pumiliotoxin B 47) which it always accompanied occurred in traces in P. australis and in one population each of P. coriacea and P. corroboree. Pumiliotoxin B itself was a major alkaloid of P. australis and P. corroboree and a minor or trace component of P. coriacea P. occidentalis and P. semimarmorata. By contrast the erythro isomer (48) was found in traces only in P.corroboree. Allopumiliotoxin 323B (49) was a major constituent of only one population of P. coriacea and a trace alkaloid in P. corroboree and P. semimarmorata. Pumiliotoxin 325B the structure of which is tentatively shown as (50) was present in trace amounts in all but one population of P. coriacea. It has been confirmed that threo-pumiliotoxin 323A is the sole isomer present in the following dendrobatid frogs Dendrobates auratus D. histri- onicus D. lehmanni D. pumilio D. speciosus Epipedobates tricolor and Minyobates bombetes. The intramolecular nitroso Diels-Alder route to the synthesis of indolizidine 223AB (51) by Kibayashi's group previously reported as a comm~nication~~ [cf. Reference 3(a)],has now been published in full.59 Another approach (Scheme 9) exploited cycloaddition of the methylenecyclopropane (52) to nitrone (53) which afforded a partly separable mixture of the expected four regio- and stereo-isomers (54) and (55).65 Thermolysis of (55)produced the two inseparable indolizidinones (56) and (57) in a ratio 1.4:1 as well as the vinylogous amide (58).39-2 NATURAL PRODUCT REPORTS 1991 v,vi 48%-7% SEt U (62) viii 66%-73% OSiPh2(But) $' %.* OSiPh,(But) \ \ xi xii 0 - 0 (67) (+)W) "OH X UI I un (68) (66) (65) Reagents i (COCI), C,H,N DMF; ii MeNHOMe-HCl C,H,N; iii MeMgBr THF 0 "C; iv CF,CO,H CH,Cl,; v THF Ti reagent (64) -78 "C; vi (CF,CO) 0; vii Me,O+BF,- CH,CI, then NaOH H,O-MeOH ; viii 1.5 YO(dba) Pd -CHCl, 12YOEtC(CH,O) P 10 eq.H,O THF 65 "C; ix CF,CO,H CF,CO,H; x sulphone reagent (67),5 YO(dba),Pd;CHCl, 20% dppf 10 eq. H,O THF r.t.; xi 6% Na(Hg) Na,HPO, MeOH; xii LiAlH, THF -20 "C Scheme 11 (72) 166% viii (69) (f)-ElaeokanineA (75) (74) (73) Reagents i LDA THF -78 "C; ii PrkSiCl; iii H, 5% Pd-C NaOH EtOH r.t.; iv MeOCOCl THF -23 "C; v MeCH(OEt)O(CH,),MgBr THF -23 "C to r.t.; vi 10% HCl H,O; vii NCS PPh, DMF 0 "C to 50 "C; viii AgOSO,CF, CH,(CH,),COCl CH,Cl, -78 "C to 0 "C; ix 30% HBr in AcOH CH,Cl,; x Et,SiH CF,CO,H CH,Cl, 0 "C to reflux; xi NaI MeCN 0 "C then Me,SiCl 0 "C to reflux Scheme 12 selective Pdo-catalyzed cyclization as key step (Scheme 1 l).67 The substrate (62) for this reaction prepared from the L-proline-derived ketone (63) and allyltitanium reagent (64) followed by epoxidation cyclized cleanly in the 6-endo-trig mode as dictated by stereoelectronic constraints to yield (65) as the sole isomer.Another Pdo-induced reaction between the epoxide (66) and sulphone (67) served to introduce the side chain of the target alkaloid. The sequence was completed by threo-selective reduction of ketone (68) to give (+)-allopumiliotoxin 339B (61) [aID+7" (c 0.20 MeOH) the NMR spectra of which were in agreement with those of authentic (+)-(61). This new approach to the construction of alkaloids of the pumiliotoxin class would seem to offer a general and valuable alternative to Overman's imaginative strategy [cf Reference 9(c)] and further developments are sure to be forthcoming.Overman's team incidentally has been respon- sible for preparing a variety of pumiliotoxin B analogues for biological studies aimed at demonstrating their ability to function as sodium channel agents.68 6 Elaeocarpus Alkaloids 1-Acyl- 1,2-dihydropyridines are versatile intermediates for the synthesis of alkaloids as Comins et al. continue to dem- on~trate.~' By using 3-trialkylsilyl substituents as removable blocking groups on various pyridine substrates they have now NATURAL PRODUCT REPORTS 1991-5. P. MICHAEL 561 i,ii =-iii iv ws,:* (& 92% 'To1 4:1 t I (80a) + (80b) 1:1 (81a) + (81b) 1:l (83) iii vii/ \ii vii \75%-78% 92% viii viii \ I;% IW% / (-)-Elaeokanine A (76) (-)-Elaeokanine B (+)-(86) (69) (+)-Elaeokanine A Reagents :i LDA THF -25 "C; ii (S)-(-)-~-menthyl p-toluenesulphinate THF -50 "C; iii LDA THF -78 "C; iv I(CHJZI -78 "C to r.t6; v NaBH, MeOH 15-20 "C;vi W-2 Raney Ni EtOH 25 "C ;vii THF butanal -78 "C to 25 "C ;viii NEt .toluene reflux; ix PCC 3 A molecular sieves CHzC1, r.t. Scheme 13 managed to achieve excellent or even exclusive regiocontrol in directing the addition of Grignard reagents to position 6 of the ring. The application of this refinement in the synthesis of (&)-elaeokanine A (69) is shown in Scheme 12 where the conversion of (70) to (71) exemplifies the new development. The butanoyl side chain of the target alkaloid was introduced also regiospecifically by acylation of the endocyclic dienamide (72) with the mixed anhydride of butanoic and triflic acids.The blocking group was then removed from the product (73) by protonolysis. The resulting dihydropyridine (74) was selectively reduced to (75) with triethylsilane in acidic medium after which removal of the N-acyl group and cyclization completed the synthesis of (i-)-elaeokanine A (69) in 3.6% yield based on 2-chlorop yridine. The first asymmetric syntheses of elaeokanines A (69) and B (76) (Scheme 13) are unusually interesting not only because of the methodology employed but also because they serve to establish the absolute configurations of the alkaloid^.'^ The chiral auxiliary was an optically active sulphoxide introduced by alkylating the anion of 2-methyl-A'-pyrroline (77) with (-)-(S)-L-menthyl p-toluenesulphinate.Further alkylation of the product (78) yielded the key bicyclic intermediate a-sulphinylketimine (79) borohydride reduction of which gave four separable dastereomeric indolizidines (80a,b) and (8 1a,b) in a ratio 4 4 1 :1 and a yield of 81 YO.The stereochemistry at C-8 was not determined since thermal elimination of the sulphinyl group ultimately makes the point irrelevant. However as an interesting sideline desulphurization of both major isomers gave the known (R)-( -)-indolizidine (82) and of both minor isomers gave (S)-(+)-indolizidine (83) thereby pin- pointing the absolute stereochemistry at the bridgehead carbon C-8a. Alkylation of the anions of either major isomer (80) with butanal gave in both cases a 76% combined yield of the separable alcohols (84a) and (84b) in a 2 1 ratio while similar reaction of either minor isomer (81) gave a 2 I mixture of (85a) and (85b).Single crystal X-ray analysis of alcohol (84a) provided definitive evidence for the relative stereochemistry at the four stereogenic centres. In all four products the configur- ation of C-1' in the butyl side chain was (S)and transition state models were proposed to account for the stereospecificity of the alkylation. Dehydrosulphinylation of both (84a) and (84b) in refluxing toluene furnished the natural enantiomer ( -)- elaeokanine B (76) [aID-76" (c0.4 CHCl,) in 92 % and 90 % yields respectively. The absolute configuration is therefore as shown in the diagram. Similar reactions on (85a) and (85b) gave (+)-(86) the C-1' epimer of elaeokanine B.The preparation of both (76) and (86) now allows a misconception in the literature to be clarified since the NMR spectra of these two compounds are quite different it can be confidently stated that natural (-)-elaeokanine B is a single isomer and not a 562 OMe OMe Me0 Me0 R Me0 (87) (-)-Antofine R = Me (88) Dehydroantofine (91) (-)-6-Desmethylantofine R = OH R' OMe (92) Deoxytylophorinine Noxide R' = H R2 = OMe R3 = 0 (93) Tylophorine R' = R2= OMe R3 = - (94) 6-Desmethyltylophorine R' = OMe R2 = OH R3= - mixture of C-1' epimers as was formerly hyp~thesized.~~ Finally oxidation of (76) and of (86) gave the unnatural (-) and natural (+) enantiomers of elaeokanine A respectively.The optical rotation of (+)-elaeokanine A (69) was measured as [a],+49" (c. 0.5 CHCl,) in marked contrast to the reported value of + 13" for the naturally occurring 7 Phenanthroindolizidine Alkaloids and Seco Analogues No fewer than ten new alkaloids of this group have been characterized in the period under review. The previously unexplored New Caledonian tree Cryptocarya phylZostemon (Lauraceae) has yielded new and known alkaloids of several different structural types all characterized most thoroughly by spectroscopic techniques. 73 Alkaloids of interest include the known phenanthroindolizidine (-)-antofine (87) in its first isolation from a lauraceous plant. Dehydroantofine (88) precipitated as the reineckate salt and characterized as the chloride proved to be identical to the known product of oxidation of antofine with mercuric acetate and underwent reduction to antofine with borohydride.This is the first report of the compound as a natural product though the authors comment on the possibility of its being an artefact formed during the extraction and isolation procedure. (-)-Phyllostemine (89) [a] -8" (cO.28 EtOH) and (-)-phyllosteminine (90) [a],-49" (c 0.4 EtOH) are new seco- phenanthroindolizidine alkaloids. The location of phyl-lostemine's phenolic groups at the para positions was inferred from the negative Gibbs test results. There is some uncertainty about which aromatic ring the methoxy group is attached to but the structure shown in (89) was preferred on the grounds of structural and spectroscopic analogies with known alkaloids.NATURAL PRODUCT REPORTS 1991 OMe (89) (-)-Phyllostemine (90)Phyl losteminine R4 (95) Tylophorinidine R' = R2 = H R3 = R5= OH,R4= OMe (96)R' = H R2= R5 = OH,R3= R4 = OMe (97) Tyloindicine A R' = R2= R3 = OMe R4 = R5= H (100) Tyloindicine D R' = R3 = R4 = OMe R2 = OH R5 = H (101) Tyloindicine E R' = R2 = R5 = H R3 = OH R4= OMe (102) R' = R3= R4= OMe R2= H R5 = OH (103) R' = R3= OH R2= R5 = H R4 = OMe The 4a-R absolute configuration was assigned only on the basis of the negative specific rotation. The locations of the two substituted aromatic rings in phyllosteminine were also chosen in terms of spectroscopic analogies. This compound is the first septicine-type alkaloid to be reported with a 5-hydroxy group the location of which was evident from a consideration of mass spectral breakdown patterns.(-)-Antofine (87) and (-)-6-desmethylantofine (91) identi- fied by a combination of high-resolution NMR and chiroptical methods have been isolated from a Mongolian specimen of Cynanchum hancockianum (Asclepiadaceae).74 The latter al- kaloid could be converted into the former on treatment with diazomethane. Positive Cotton effects in the ORD and CD spectra of both supported the 13a-R assignment of absolute configuration. A related species Cynanchum komarovii has been reported to contain antofine and a new alkaloid deoxytylophorinine N-oxide (92) but as this work was published in an inaccessible journal it has not been possible to confirm the details.75 Two varieties of Tylophora indica (Asclepiadaceae) a species well known as a source of phenanthroindolizidine alkaloids contain an astounding number of hitherto unknown alkaloids as well as the known alkaloids (-)-tylophorine (93) (-)-6- desmethyltylophorine (94) ( +)-tylophorinidine (99 and (+)-5-hydroxy-0-methyltylophorinidine(96).76 The full range of standard spectroscopies complemented by chemical tests and derivatization studies provided convincing evidence for the structures of the following new alkaloids (+)-tyloindicine A (97) [a],+7.2" (c 2.1 MeOH) (+)-tyloindicine B (98) [a],+ 14.3" (c 1.05 MeOH) (+)-tyloindicine C (99) [a],+ 16" (c 0.25 MeOH) (+)-tyloindicine D (loo) [a],+ 1.6" (c 0.6 MeOH) (-)-tyloindicine E (101) [a],-12" (c 0.25 MeOH) (+)-14-hydroxyisotylocrebrine (102) [aID+30"(c 0.3,MeOH) and (+)-4,6-desmethylisotylocrebrine (103) [a],+7.3" (c 1.75 NATURAL PRODUCT REPORTS 1991-5.P. MICHAEL 563 Me HO OCOCH OMe (98) Tyloindicine B (99)Tyloindicine C (104) Thionuphlutine lMe (105) Neothiobinupharidine (106) 902Et ii 86% i (qH 93% 'BOC' -/' BOC 0 31% -+ 19% iv Reagents i LDA THF -78 "C; ii hlsC1 NEt, CH,Cl, -7 "C; iii 1 eq. DBU DMSO 120 "C; iv 2 eq. DBU DMSO 120 "C; v H, 5 % Pd-C MeOH; vi LiAlH, Et,O; vii MsCl NEt, CH,Cl, 0 "C Scheme 14 MeOH). Tyloindicine B (98) the only new secophenanthro- 8 FuryIquinoIizid ines and FuryIindoIizid ines indolizidine in the group is unique amongst TyZophora alkaloids in having an acetoxy group at C-7.Both it and tyloindicine C Methylation of the isomeric alkaloids thionuphlutine (104) and (99) are unusual in bearing angular methyl groups. (+)-14-neothiobinupharidine (105) under thermodynamic conditions Hydroxyisotylocrebrine (102) is apparently a diastereomer of was in both cases accompanied by spontaneous cleavage of the Rao's alkaloid A [cf. Reference 771 reported in 1970 with a tetrahydrothiophene ring to give the quaternary species (106).82 negative specific rotation. 78 In the process the trans AB quinolizidine rings underwent a The mechanism of resistance of mammalian cells to conformational change to the cis system evidence for which tylocrebrine and cryptopleurine amongst other compounds came from NMR spectroscopy.Further treatment of (106) has been reviewed.79 In view of possible therapeutic benefits of with methyl iodide followed by Hofmann degradation was not Tylophora alkaloids the toxicity of pure alkaloid of Tylophora regioselective and gave mixtures of (107) and (108). asthmatica in the male rat has been studied and effects on A synthesis of the Nuphar indolizidine (109) by Kurihara enzyme levels and organ morphology have been evaluated.80 and co-workersg3 involves decarboxylative recyclization of a Tylophorine has antifeedant properties towards the insect tetrahydro- 1,3-0xazin-2-one to a tetrahydropyridine (Scheme Spilosoma obliqua.8' 14) a strategy previously employed by them in a synthesis of NATURAL PRODUCT REPORTS 1991 0 i\Me (1 13) Demethylvertine R' R2 = H Me (115) Plumerinine (1 14) Vertine R' = R2= Me i.ii. iii Reagents i BuLi THF -42 "C; ii I(CH,),Cl; iii oxalic acid H,O; iv BF;Et,O BF;Et,O -91 "C to -78 "C; vi CF,CO,H 0 "C; vii NaHCO, H,O r.t. THF -78 "C; v LiAlH,(OMe), CuBr.Me,S then Scheme 15 lupinine [cf. Reference 9(d)]. In the present case treatment of the focal intermediate (110) with an equivalent of DBU in dimethyl sulphoxide at 120 "C gave the hexahydroindolizine (1 11) as a single isomer. With two equivalents of base (1 11) (31 YO)and its epimer (1 12) (19 %) were isolated. The DBU- induced epimerization of (1 1 1) to the thermodynamically more stable (1 12) was also demonstrated. The stereochemistry of the two isomers was established with the aid of NOE experiments that showed the proximity of 5-H and 8a-H in (112) but not (1 11).The conversion of the latter compound to the target indolizidine was completed by standard transformations. Spectra on the final product correlated well with those reported by other workers for the synthetic alkaloid and the mass spectrum admirably matched that of the natural product the stereochemistry of which was not determined at the time of its i~olation.~ 9 Lythraceae Alkaloids The little-known South American species Heimia montana produces five known lactonic biphenylquinolizidine alkaloids (heimidine lyfoline lythridine lythrine and vertine) the new compound demethylvertine (1 13) and several unidentified minor alkaloids.85 Batches of the plant harvested in different years gave substantially different quantities of the alkaloids (seasonal variation?) and the new alkaloid was a significant component in only one batch.Apart from the problem of the OH/OMe substitution pattern in the aromatic ring the structure of (1 13) could be assigned on spectroscopic grounds and the stereochemistry of the substituents on the quinolizidine nucleus was well supported by 'H NMR data. Methylation with diazomethane gave a mixture of vertine (114) and 0-methylvertine thereby clinching the structural assignment. Insufficient amounts of the minor alkaloids designated H-17 to H-20 were isolated for anything other than mass spectrometric work. The following molecular formulae were assigned H- 17 C,,H,,NO ; H- 18 C,,H,,NO ; H-19 C,,H,,NO,; and H-20 C25H29N06' 10 Plumerinine ( + )-Plumerinine (1 15) a quinolizidine alkaloid isolated from Plumeria rubra is incorrectly described by its discoverers as a new lupin alkaloid.86 The source plant belongs to the Apocyanaceae not the Leguminosae and the structural features of the alkaloid are quite unlike those of simple lupin quinolizidines.The compound a viscous oil having [aID14.4" (c .0.31 CH,OH) gave a molecular ion in the high-resolution MS at 225.2078 corresponding to a molecular formula C,,H,,NO and fragment ions arising from simple pathways involving dehydration loss of the isopropyl group cleavage a to nitrogen and retro-Diels-Alder reactions. Exhaustive NMR data established the carbon skeleton as well as the positions and orientations of substituents while Bohlmann bands at 2820 and 2720cm-' in the IR spectrum indicated that the quinolizidine system was trans-fused.This seems to be the first report of a quinolizidine alkaloid in the subfamily Plumer- ioideae of the Apocyanaceae. 11 Myrtine A synthesis of (+)-myrtine (1 16) by the Comins team provides a further illustration of their use of dihydropyridines in alkaloid synthesis (Scheme 1 5).87 Lithiation and alkylation of dihydropyridine (1 17) prepared in three steps by addition of methyl Grignard reagent to the appropriate N-acylpyridinium salt from 4-methoxypyridine gave the cyclic enaminone (1 18). A great deal of experimentation was necessary before the right conditions were found to bring about the stereoselective 1,4- reduction to the 2,6-trans-disubstituted piperidinone (1 19).The workers hoped to capitalize on the axial orientation of the methyl group a consequence of strain between it and the substituent on N and find a reagent that would deliver hydride from the less hindered face of the molecule. The reagent of choice turned out to be a 'CuH * BF,' reductant prepared in situ. Although the isolated yield of (119) was only 56% the crude product consisting of a 95 :5 mixture of the trans-and cis-2,6-disubstituted isomers was obtained in 97 % yield. The NATURAL PRODUCT REPORTS 1991-5. P. MICHAEL 0 (120) Nuttaliine R' = R2= R3 = H R4= OH (1 23) 12-Ethoxycarbonylcytisine (121) 3g-Hydroxylupanine R' = R3 = R4= H R2= OH (132) Sessilifoline R' = R4 = H R2= OH R3= OAng (133) Lebeckianine R' = R4 = H R2= R3 = OH new records for the genus.A minor alkaloid was deduced from (138) 3a-Hydroxylupanine R' = OH R2= R3= R4 = H mass spectrometric fragmentation patterns to be an isomer of 13-hydroxylupanine possibly a-iso- 13-hydroxylupanine ;if the surmize is correct this will be a new alkaloid. &poH The alkaloidal constituents of the hybrid Laburnum watereri and their seasonal variation have been determined by various chromatographic and spectroscopic technique^.^^ The presence of epibaptifoline (one of three major alkaloids the others being cytisine and N-methylcytisine) /3-isosparteine and 14a-hydroxysparteine (122) in Laburnum is new.The authors made no special comments about the last-named compound and indeed characterized it only by mass spectrometry but it is unclear whether or not it is a new alkaloid. There appear to be no other records of it as a natural product in the literature synthesis was completed unexceptionally to give ( _+ )-myrtine (1 16) in 39 % isolated yield based on (1 17). through it is known as a synthetic cornpound.lo8 Another component has tentatively been identified as N-ethoxycarbonylcytisine (123); it is probably the same as a new alkaloid isolated from various genera of the Leguminosae in a 12 Alkaloids of the Lupinine-Cytisine-Spa rtei ne-M at i-ine-Ormosia Group 12.1 Occurrence and Detection Table 1 includes new alkaloids of this group as well as new sources of known alkaloid^.^^-^^^ The chemotaxonomic significance of alkaloids in the tribe study that will be described fully in next year's report.log Transfer of quinolizidine alkaloids from host plants to plant parasites or to insects continues to elicit interest.A group of new carbinolamide quinolizidine alkaloids (see Section 12.2) inherent to Lupinus argenteus subsp. rubricaulis is transferred by root parasitism to Castilleja sulphurea and C. flava .(Scrophulariaceae).91 The hemiparasitic herb genus Pedicularis Crotalarieae of the Leguminosae (Fabaceae) has been explored further by van Wyk and his c ~ -~ ~ r k e rOn the basis of qualitative similarities in alkaloidal composition they have postulated a close relationship between Lebeckia [cf. Reference 9(e)] and the genera Aspalathus Rafnia and Wiborgia in which trace amounts of alkaloids have been ~ .~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ (Scrophulariaceae) also takes up alkaloids from the roots of its hosts :a Lupinus argenteus hybrid ( x L. caudatus or L. alpestris) transfers lupanine and tetrahydrorhombifoline to P. racemosa while P. gruyi acquires N-methylcytisine from Thermopsis divaricarpa and P. crenulata anagyrine from Thermopsis montana.lo2The significance in the latter two cases is that while ~ ~ detected for the first time.89 The close relationship does not extend to the genus Spartidium.lo6 The co-occurrence of above-ground parts of the host contain several other quino- lizidine alkaloids only the alkaloids found in their roots are quinolizidine and macrocyclic pyrrolizidine alkaloids appears transferred to the hemiparasites.Alkaloid uptake by Pedicularis to be a unique chemotaxonomic marker for certain sections of the morphologically complex genus Lotononis and may be of value in clarifying the infrageneric cla~sification.~~ The genus semibarbata which obtains a-isolupanine from Lupinus julcratus appears not to deter oviposition by the butterfly Euphydras edithag7 The larvae of the moth Uresiphita reversalis Pearsonia is the source of several new alkaloids to be described in the next section; it like Rothia (cf. Reference 107) is unusual amongst the Crotalarieae in producing esters of quinolizidine alkaloids.lOO.lO1It has now been shown for Pearsonia that an which feed on Genista monspessulana showed similar alkaloidal profiles to the plant; while most of the alkaloid was excreted the portion sequestered by the insect was localized in the cuticle possibly conferring some protection from predator^.^^ alkaloid previously believed to be nuttalline (4p-hydroxylupanine (1 20) is actually the new compound 3p- Quinolizidine alkaloid production in cultured cells from leguminous plants appears to correlate with greening of the hydroxylupanine (121) ;lol by extension all previous claims for nuttalline as a metabolite of the Crotalarieae may have to be reviewed.The absence of a-pyridone quinolizidine alkaloids from the genus Virgilia (tribe Podalyrieae) is at variance with former reports and casts doubts on previous suggestions about generic relationships. lo4At the infrageneric level however the distribution of alkaloids appears to be sufficiently characteristic to be of taxonomic value.Several new alkaloids (including 4- hydroxylupinine and its esters) have apparently been detected in this genus and publication has been promised. The alkaloidal and botanical evidence for transferring the genus Argyrolobium from the Genisteae to the Crotalarieae has been reviewed. lo5 A study of the chemosystematics of Bulgarian Chamaecytisus (10 species 34 population^)^^ was subsequently followed by more detailed examination of polymorphic examples of C. absinthioides and C. austri~cus.~~4-Hydroxylupanine 13-angeloyloxylupanine 13-tigloyloxylupanine and the bispiper- idine alkaloid ammodendrine all detected in the former are plants implying a connection between chloroplast formation and alkaloid biosynthesis. Green callus cultures of Thermopsis lupinoides produced (+)-lupanine while white callus and adventitious roots were free of alkaloids.Similarly matrine was detected in green callus of Sophora flavescens var. angustifolia the quantities correlating well with the amounts of chlorophyll in the cells.111 At a later stage of differentiation multiple shoots produced not only matrine but also 5,6-dehydrolupanine and anagyrine. In experiments with Lupinus succulentus the rate of alkaloid production was shown to depend on the ability of plants to fix nitrogen and to increase in response to leaf damage.l12 The findings are significant in view of plant-herbivore interactions. Analytical studies published during the review period include the HPTLC densitometric analysis of some well-known alkaloids in Sophora alopecuroides ;l13 and the chromatographic and spectrophotometric determination of cytisine in Thermopsis extracts.l4 NATURAL PRODUCT REPORTS 1991-5. P. MICHAEL Table 1 Isolation and detection of alkaloids of the lupinine-cytisine-sparteine-matrine-Orrnosiu group Species Alkaloid Ref. Ammopiptanthus mongolicus Aspalathus spp.' *(+)-3a-Hydroxylupanine (138) a-Isolupanine Lupanine 88 89 Nuttalline (120) Bowdichia virgiloides Sparteine *( +)-Homo-l8-epi-ormosanine(144) (-)-Homoormosanine (142) 90 *(-)-Homopiptanthine (143) (-)-Ormosanine (140) Castilleja fiava and C. sulphurea (see text) Chamaecyt isus absin th ioides (-)-Piptanthine (141) (+)-Aphyllidine (1 27) *(-)-Argyrolobine (130) *2(R),9(R)-Dihydroxyaphyllidine (1 29) *( +)-2(S),9(R)-Dihydroxyaphyllidine (1 28) *2(S)-Hydroxyaphyllidine (1 3 1) 13-Angelo yloxylupanine 13-Benzo yloxylupanine 4-H ydrox ylupanine 13-Hydroxylupanine 91 92 13-Hydroxylupanine isomer (new?) 7-H ydroxysparteine ct-Isosparteine (+)-Lupanine 17-Oxosparteine 93 Genista monspessulana Laburnum watereri (-)-Sparteine Tiglo ylox ylupanine Aphylline Deh ydroaph ylline Virgiboidine N-Ace tylcytisine Anagyrine Cytisine 92 94 95 5,6-Dehydrolupanine Dehydrosparteine Epibaptifoline*12-Ethoxycarbonylcytisine (1 23) N-Formylcytisine 14a-Hydroxysparteine (1 22) Isolupanine P-Isosparteine Lupanine Lotononis ~pp.~ N-Methylcytisine Sparteine a-Isolupanine Lupanine Nuttalline 96 Lupinus argenteus subsp.rubricaulis Lupinus fulcratus Maackia amurensis var. buergeri Maackia tashiroi Sparteine (+)-Aphyllidine *(-)-Argyrolobine *2(R),9(R)-Dihydroxyaphyllidine *(+)-2(S),9(R)-Dihydroxyaphyllidine *2( S)-H ydroxyaphyllidine a-Isolupanine 5,6-Dehydrolupanine (-)-Rhombifoline (+)-Sparteine (-)-Anagyrine 91 97 98 99 (-)-Cytisine (+)-Epilupinine (1 25) (-)-N-Formylcytisine (-)-N-Methylcytisine Pearsonia spp." (-)-Rhombifoline *Tashiromine (124) *(-)-Cajanifoline (1 35) *(+)-Cryptanthine (1 37) *3p-Hydroxylupanine (121) a-Isolupanine Lupanine (+)-13a-(Angeloy1oxy)lupanine (1 34) *(+)-Pearsonine (1 36) 100 101 101 100 loo.101 Pedicularis crenulata (see text) Pedicularis grayi (see text) *(-)-Sessilifoline (132) Sparteine Anagyrine N-Methylcytisine 100 102 NATURAL PRODUCT REPORTS 1991-5. Table 1 continued Species Pedicularis racemosa (see text) Pedicularis semibarbata (see text) Rafnia ~pp.~ Thermopsis chinensis Thermopsis divaricarpa Virgilia spp." Wiborgia spp.' * New alkaloids P.MICHAEL 567 Alkaloid Ref. Lupanine Tetrahydrorhombi foline a-Isolupanine Lupanine Nuttalline 97 89 Sparteine *(-)-O-Acetylbaptifoline(1 39) (-)-Sparteine Thermopsine *Alkaloid 8 (structure unknown) Lupinine Oroboidine I03 102 104 Sparteine Virgiboidine Virgiline Virgiline 13-(2-pyrrolylcarboxylate) Lupanine Nuttalline 89 Sparteine a Aspalathus capitata A. carnosa A. chortophila A. cordata A. hirta A. juniperina A. linearis A. longifolia A. nivea A. perfoliata A. spinosa Lotononis adpressa L. bainesii L. calycina L. carinata L. curvicarpa L. eriantha L. hirsuta L. lanceolata L. listii L. mucronata L. platycarpa Pearsonia aristata P.cajanifolia subsp. cajanifolia P. cajanifolia subsp. cryptantha P. obovata P. sessilifolia subsp. marginata P. sessilifolia subsp. sessilifolia Rafnia angulata R. capensis R. elliptica R. opposita R. perfoliata R. racemosa Virgilia divaricata V. oroboides subsp. oroboides V. oroboides subsp. ferruginea ' Wiborgia fusca W. obcordata W. sericea CHZOH I (124) Tashiromine n= 1 (125) Epilupinine n= 2 12.2 Structural Spectroscopic Chemical and Biological Studies The new alkaloid tashiromine (124) isolated along with ammodendrine and seven common quinolizidines from Maackia tashiroi is of considerable interest as the first example of a simple indolizidine formally belonging to the lupin group of alkaloid^.^^ Previously known as a synthetic compound it bridges the structural gap between epilupinine (129 which coexists in the same plant and the pyrrolizidine alkaloid trachelanthamidine.Since other Maackia species are known to produce pyrrolidine and complex indolizidine alkaloids the implication is that they have the ability to use both ornithine and lysine in biosynthesis. The structure of tashiromine was assigned on the basis of spectroscopic data the stereochemistry being deduced from 13C NMR chemical shift comparisons with epilupinine and its cis-diastereomer lupinine. Synthetic samples of (&)-tashiromine and its diastereomer prepared by catalytic hydrogenation of the known cyclohexapyrrole (1 26) showed comparable 13C chemical shift differences to those of epilupinine and lupinine; and the synthetic and natural alkaloids were chromatographically and spectroscopically indistinguishable.The optical rotation and absolute configuration of tashiromine were not determined. An asymmetric synthesis of (-)-( 124) has recently been reported115 (see Section 12.3) and the spec- troscopic data of the natural and synthetic alkaloids are in good agreement. Four new cyclic carbinolamides isolated from Lupinus argenteus subsp. rubricaulis and its hosted root parasite Castilleja sulphurea were the subject of an extremely thorough investigation involving mass spectrometry IR UV and NMR F$ R2 R' 0 (127) (+)-Aphyllidine R' = R2 = H (128) R' = P-OH R2 = OH (129) R' = a-OH R2 = OH (130) (-)-Argyrolobine R' = a-OH R2= H (131) R' =P-OH R2= H spectroscopy optical measurements chemical derivatization and in one case X-ray crystallography.g1 The new alkaloids are C-2-mono- and C-2 C-9-bis-hydroxylated analogues of the known but uncommon alkaloid (+)-aphyllidine (127) which was a minor component of the plant extracts.The compounds were isolated as mixtures of diastereomers containing epimeric OH groups at C-2. Ready interconversion of the epimers in aqueous medium probably through an acyliminium inter- mediate made isolation of pure substances difficult but HPLC separation followed by immediate removal of solvent at low temperature (or alternatively acetylation followed by chroma- tographic separation) gave samples of alkaloids (or alkaloid acetates) that were pure enough for characterization.For the 2,9-diols a substantial range of 'H and 13C NMR techniques was used to deduce the structures of (+)-2(S),9(R)-dihydroxyaphyllidine (128) its diacetate and the diacetate of the epimer 2(R),9(R)-dihydroxyaphyllidine (1 29). The structure of (128) was ultimately corroborated by X-ray crystallography. The absolute configurations of both (128) and (129) were determined on bis(4-bromobenzoate) salts by CD methods. Consideration of coupling constant and NOE data also permitted a full conformational analysis of both alkaloids which for (128) gave results that agreed with those of the crystallographic study. The most significant finding is that the conformation of ring A in both alkaloids is such that the C-2 hydroxy group is axial and approximately perpendicular to the NATURAL PRODUCT REPORTS 1991 0 Lupanine 13a-Oangelate R' = R2= R3 = H Cajanifdine R' = OH R2= R3 = H Pearsonine R' = R3 = OH R2 = H Cryptanthine R' = R2 = HI R3= OH 0 (1 39) GAcetylbaptifoline (140) Ormosanine plane of the enamide group.The same is true for the second pair of epimers (130) and (131) NMR spectra of which were recorded on pure samples of (130) its acetate and the acetate of (1 3 1). Compound (1 30) is (-)-argyrolobine the enantiomer of a known alkaloid whose stereochemistry at C-2 was previously undetermined. Hydroxylated lupanines and their angelate [(2)-2-methy1-2- butenoate] esters appear to be the hallmark of the genus Pearsonia (see section 12.l) from which no fewer than five new alkaloids have been isolated. lol These compounds were fully characterized spectroscopically with IH and 13C NMR and various two-dimensional NMR techniques providing definitive evidence for the positions and orientations of substituents. The (E),or tiglate esters were absent. An unusual feature of these new compounds is the presence of 3P- and/or 8a-hydroxy groups the latter being unprecedented. (-)-Sessilifoline (1 32) [a],+72" (c 1.6 CHC1,) is the 3-angelate ester of the alkaloid lebeckianine (1 33) recently described by these workers116 [cf. Reference 9(e)]. The other esters all contain a 13a-angeloyloxy group and include the known (-)-lupanine 13a-angelate (1 34) and the new compounds (-)-cajanifoline (1 39 [a] -11" (c 1.4 CHCl,) (+)-pearsonine (1 36) [a] +7" (c 0.6 CHCl,) and (+)-cryptanthine (137) [a] +11 7" (c 1.4 CHCI,).Hy- drolysis of cajanifoline gave a product spectroscopically identical to the known alkaloid 3P,13a-dihydroxylupanine. The similarity in the position of the hydroxy groups in the new compounds suggested a biosynthetic relationship between them and it prompted the question whether the monohydroxy- lupanine that occurs with them in Pearsonia and previously identified as nuttalline (4/3-hydroxylupanine 120) might not in fact be the 3P-hydroxy isomer (121) instead. In support of this hypothesis the carbinol proton at 6 3;91 showed coupling constants of 1 1.45 (trans-diaxial orientation of H) and 5.62 Hz correlating in the COSY spectrum with only two other signals at about S 1.6.3P-Hydroxylupanine [a] 0" is thus a new alkaloid and its identification now leaves the structure of nuttalline open for speculation since there are no NMR data for it in the literature. By a curious coincidence at much the same time a Czech group reported the isolation of (+)-301- hydroxylupanine (1 38) [a] +73.8" from Ammopiptanthus mongo1icus.88 Their NMR data nicely complement those of the South African workers the 3P proton signal for instance occurs as a double doublet at 6 4.12 with J 5.6 and 4.5 Hz. (-)-0-Acetylbaptifoline (1 39) isolated from Thermopsis (141) Piptanthine (1 42) Homoormosanine 18a-H (143) Homopiptanthine (1 44) Homo-1 8-epi-ormosanine 18P-H chinensis is a new alkaloid only insofar as it has been isolated and fully characterized for the first time.lo3 Previous in which it was named as 13-acetoxyanagyrine contained only tentative identification based on GC-MS data.Spectroscopic studies hydrolysis to (-)-baptifoline and synthesis from (-)-baptifoline with acetic anhydride and pyridine served to confirm its structure. Of more interest are the absolute stereochemical relationships between the alkaloids isolated in this study explored to some extent by examination of Cotton effects in their CD spectra all the a-pyridone alkaloids isolated (including (1 39) (-)-anagyrine (-)-baptifoline (-)-cytisine (+)-5,6-dehydrolupanine (-)-N-formylcytisine (-)-N-methylcytisine and (-)-rhombifoline) belong to the 7R,9R 11R series while (+)-lupanine and (-)-sparteine have the 7S,9S 11S configuration.Thermopsis chin- ensis is thus exceptional in its ability to produce alkaloids of both enantiomeric series. The Colombian tree Bowdichia virgiloides is the source of five alkaloids of the Ormosia group.so Three known alkaloids (-)-ormosanine (140) (-)-piptanthine (141) and (-)-homoormosanine (1 42) were identified spectroscopically. The relative stereochemistry of the new alkaloid (-)-homopiptanthine (143) [a],-40" (c 0.32 CHCI,) was inferred from NMR data and was confirmed when (143) was shown to be identical to the homo-alkaloid formed by derivatizing piptanthine with formaldehyde. A similar relationship was established between ormosanine and homoormosanine.Piptan- thine and ormosanine epimeric at C-6 can be interconverted by hydrogenation in acidic medium; a sample of racemic ormosanine after epimerization with hydrogen and platinum dioxide in acetic acid followed by treatment with formaldehyde gave a mixture of homo-alkaloids (142) and (143). The remaining new alkaloid (+)-homo- 18-epi-ormosanine (1 44) [a] +6" (c 1.03 CHCl,) gave well-separated lH NMR signals for the aminal group indicative of a nearly planar orientation of rings A/F/E. Other NMR data provided evidence for the trans orientation of H-11 and H-16 and the proximity of hydrogens at C-8 C-11 and C-18 (NOE). The absolute configuration was not established. Structural and conformational studies on quinolizidine alkaloids and their derivatives continue to appear in profusion.Recent publications include conformational investigations of ring A and D enamines of phenylsparteines;'lg synthesis and structure of 2-phenyl-2-dehydro- 17-substituted sparteines ;120 NATURAL PRODUCT REPORTS 1991-5. P. MICHAEL COzEt I I-70% 0~'' ii 90% Cl (1 47) C02Et CCLFt /OH 200°C 4 t 76% 76% (1 25) Epilupinine (1 49) (148) (1 45) Lupinine Reagents i EtOH BF;Et,O reflux; ii NaI MeCN reflux; iii Raney Ni EtOH H (150 atm) heat; iv LiAIH, Et,O r.t. Scheme 16 Br C02Me Epilupinine (125) 49% 95% (150) (1 51 1 21% (152) 43% Reagents i NaH DMF r.t.; ii Br(CH,),Br; iii Bu,SnH AIBN C,H, reflux; iv LiAlH, THF 0 "C Scheme 17 conformational and configurational studies on 17-isopropylsparteine and 17-isopropyllupanine ;121 13C NMR studies of sparteines bearing substituents at C-2 C- 15 or C-17 ;12 CD studies of multiflorine 1 1'12-seco- 12- dehydromultiflorine and their 5-dehydro derivatives ;123 the X-ray crystal structure determination of 2-phenylsparteine ;124 and a new X-ray crystal structure analysis of (-)-~ytisine.'~~ The latter investigation was used to establish structural correlations between nicotine and cytisine which is an important nicotine agonist.13C NMR studies of quinolizidine and bis(quino1izidine) alkaloids have been reviewed by Polish workers.126 Publications dealing with biological effects of the lupin quinolizidines are numerous. Recent studies of interest report the nematicidal properties of (-)-anagyrine and (-)-N-methylcytisine towards pine wood nematodes ;127 the poor antifeedant properties of lupinidine [= (-)-sparteine] towards larvae of the tobacco hornworm Munducu sextu;12* and syntheses of lupinine esters and their interaction with choline- sterases.129 Genetic polymorphism of sparteine metabolism a condition in which the absence of the enzyme cytochrome P-450IID6 in certain individuals results in an inability to metabolize the uterotonic and antiarrhythmic drug sparteine has been the subject of three reviews that deal with mechanistic and clinical aspects of the dis~rder.'~~-l~~ The detection of the oxidation products of sparteine in mammalian urine is important for these studies and recent papers containing results of interest to chemists include the simultaneous determination of sparteine and its 2- and 5-dehydro metabolites by an HPLC method with electrochemical detection ;133 a compilation of GC-MS data on sparteine and its urinary metabolites;134 and the metabolic oxidation of sparteine and a-isosparteine to lupanine and a-isolupanine respectively in rats.135 12.3 Synthesis As usual most of the synthetic work in this area is aimed at the bicyclic quinolizidine alkaloids epilupinine (125) and lupinine (145),which are common targets when new methodology needs to be exemplified. Full details of the synthesis of (f)-lupinine by Kurihara and co-workers [cf. Reference 9(d)] have now been publi~hed.~~ Another synthesis of (f)-lupinine by Lhommet and co-workers also mentioned in last year's review [cJ Reference 9(d)] has now been published in full together with significant e~tensi0ns.l~~ The new results pertain to the reduction of the bicyclic vinylogous urethane (146) formed by alcoholysis and subsequent cyclization of the Meldrum's acid derivative (147) (Scheme 16).High-pressure hydrogenation over Raney nickel at 100 "C gave the saturated compound (148) in which the bridgehead hydrogen atom and ester group were trans to each other. At 200 "C,however the cis-isomer (149) was the sole product. Moreover (148) could be epimerized to (149) on heating at 200 "C. The stereoselection in these reactions and the structures of the isomers were determined with the aid of NMR spectroscopy.Since both saturated esters may easily be reduced with lithium aluminium hydride the synthesis provides access to both epilupinine (125)and lupinine (145)from a single key intermediate (146). Another vinylogous urethane (1 50)' prepared unexception- ally by reduction of methyl nicotinate was exploited in a radical-mediated synthesis of (+_ )-epilupinine (125) (Scheme 17).137 In the step of interest treatment of (151) with tri-n- butyltin hydride stereoselectively produced the saturated ester (1 52) in 43 % yield thereby providing an uncommon example of radical cyclization on to an enamine system. Enantioselective syntheses of both (+)-and (-)-epilupinine with 4-isopropylthiazolidinethionesas chiral auxiliaries were NATURAL PRODUCT REPORTS 1991 OfO2CF3 CI H H s (-)-Tashi romine (124) 41% 22% 0 Reagents i THF -5 "C ; ii LiAlH, THF 0 "C to reflux Scheme 18 CO2R C02Me HI i 76% (946) C.',, orii 84% (14236) Ha -+ (15%) R = ally1 (155) (153b) R = Me iii iv C02Me Hr a a ~ ~ 84% v-vii viii 63% &OH "BOC (1 45) (+)-Lupinhe (156) Reagents i allyl ester (1 53a) LiN(SiMe,), Me,SiCl THF -78 "C to 60 "C then CH,N ;ii methyl ester (153b) LiN(SiMe,), -78 "C then H,C=CHCH,Br; iii BH,.SMe,; iv OH- H,O,; v MsCl C,H,N; vi CF,CO,H; vii NaOH H,O; viii LiAlH, Et,O Scheme 19 described in last year's review [cf.Reference 9(d)]. Full descriptions of these results and related syntheses of pyr- rolizidine and indolizidine alkaloids have now been pub-lished.'15 Of interest amongst these results is the total synthesis of (-)-tashiromine (124 absolute configuration shown in Scheme 18) carried out before the compound was known to be a natural product.The observed optical rotation for the synthetic alkaloid was [a],-25.9" (c 1.16 EtOH). The first asymmetric synthesis of the unnatural (+)-enantiomer of lupinine (145) makes use of optically resolved .. :..a (S)-2-piperidinylacetic acid protected and esterified to give .... compounds (1 53).13* Ester enolate Claisen rearrangement of the allyl ester (1 53a) yielded a 94 :6 diastereomeric mixture of (157) Petrosi n (158) Petrosin A the two products (1 54) and (1 59,while allylation of the enolate of methyl ester (153b) gave reversed diastereoselectivity (14 86) (Scheme 19).Product (155) was converted by means of standard reactions to the saturated ester (156) reduced in turn with the vasodilative bis( 1-oxaquinolizidine) alkaloids known as the lithium aluminium hydride to (+)-lupinhe (145) [a],+19.5". araguspongines has now been found to produce the known The optical rotation of natural (-)-lupinhe under comparable bisquinolizidines petrosin (1 57) and petrosin A (158).140 conditions has previously been recorded as -2lo.l3' Attempts Derivatization and NMR spectroscopic studies were used to to epimerize the saturated ester (156) with sodium ethoxide show that the sample of petrosin isolated was racemic and that followed by hydride reduction gave epilupinine but with a petrosin A has a meso structure.More intriguing is the isolation lower than expected optical rotation. Epimerization might thus of a unique new hybrid quinolizidine/ 1-oxaquinolizidine occur partly through retro-Michael reaction followed by aragupetrosine A (159) [a],-18.8" (CHC1,). The new com- Michael ring closure. pound had Bohlmann bands in the IR spectrum pointing to a trans-fused quinolizidine structure. Detailed 'H and 13C NMR analysis and a range of two-dimensional techniques indicated 13 Alkaloids from Marine Sources the number and length of the methylene chains and the An Okinawan marine sponge Xestospongia sp. the source of structure and stereochemistry of the heterocyclic portions. NATURAL PRODUCT REPORTS 1991-J. P. MICHAEL (159) Aragupetrosine A Resonances indicative of a petrosin moiety and the 3a-methyl- trans-1 -oxaquinolizidine unit of one of the known aragu- spongines were also present.The absolute configuration shown in (159) was determined from NMR analysis of the esters produced by reducing the ketone group to the equatorial alcohol followed by esterification with both (+)-and (-)-a- methoxy-a-trifluorophenylacetylchloride. 14 References 1 T. Shono Y. Matsumura S. Katoh K. Takeuchi K. Sasaki T. Kamada and R. Shimizu J. Am. Chem. SOC. 1990 112 2368. 2 M. J. Schneider and T. M. Harris J. Org. Chem. 1984 49 3681. 3 M. F. Grundon Nut. Prod. Rep. (a) 1987,4,417; (b) 1989,6,527. 4 L. E. Fellows G. C. Kite R. J. Nash M. S. J. Simmonds and A. M. Scofield Recent Adv. Phytochemistry 1989 23 395.5 ‘Swainsonine and related glycosidase inhibitors’ ed. L. F. James A. D. Elbein R. J. Molyneux and C. D. Warren Iowa State University Press Ames 1989. 6 L. E. Fellows and G. W. J. 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ISSN:0265-0568    

DOI:10.1039/NP9910800553

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

The Biosynthesis of Polyketides T. J. Simpson School of Chemistry University of Bristol Cantock's Close Bristol BS8 1TS Reviewing the literature published between January 1986 and December 1988 (Continuing the coverage of literature in Natural Product Reports 1987 Vol. 4 p. 339) 1 Introduction 2 Fatty Acids and Related Compounds 3 Tetraketides 4 Pentaketides 5 Hexaketides 6 Heptaketides 7 Octaketides 8 Nonaketides 9 Decaketides 10 Dodecaketides 11 Polyether Ionophores 12 Macrolides 13 Miscellaneous Metabolites 14 References 1 Introduction This report follows the pattern of previous ones' in the series and covers the literature appearing in the period between January 1986 and December 1988. The area continues to be tremendously active and has been given fresh impetus by the increasing application of genetic analysis and gene cloning methodology which will soon occupy a dominant place in biosynthetic studies.It is clear from work in the fatty acid P-lactam shikimate and tetrapyrrole areas that the previous marked distinctions in studies of primary and secondary metabolic pathways are rapidly breaking down as the en-zymology of these secondary pathways is being increasingly understood. We can safely anticipate the extension of these methodologies to polyketide biosynthesis. Among many highlights are the genetic analysis of the actinorhodin gene cluster' and the synthesis by two groups of chiral (R)-and (S)-[1 -'3C,2-2Hl]-malonates. Both syntheses3. proceed via [1 ,4-'3C,]-acetylene dicarboxylic acid as indicated in Scheme 1.The absolute configurations were confirmed by using the chiral malonates as substrates for yeast fatty acid synthase followed by mass spectral analysis of the derived palmitic acid3 and by NMR analy~is.~ Li -C=C-Li 13702~ 111 H2P2) ~ 7 13~0,~ f f N'3C-CH2CH2-'3CN KI3CN t CICH2CH2CI 2 Fatty Acids and Related Compounds Fatty acid synthase (FAS) is multifunctional enzyme complex which shows various patterns of both structural and functional variation depending on its rigi in.^ In most prokatyotes and in chloroplasts the FAS comprises eight structurally independent and monofunctional enzymes. In vertebrates all these com- pounds are combined within a single octafunctional polypeptide chain.However in yeasts and lower fungi the FAS consists of two components. In Sacccharomyces cerevisiae the two subunits are encoded by the FAS 1 and FAS 2 genes the former encoding acetyl- malonyl- and palmityl-transferases the dehydratase and the enoyl reductase active sites and the latter the P-ketoacyl synthetase P-ketoacyl reductase and acyl carrier protein (ACP) domains. Both genes have been fully ' Similar results have been reported by another group.8s9 The complete DNA sequence of the FAS 2 gene from another fungus Penicillium patulum reveals a protein sequence with 5&70% similarity to the S. cerevisiae FAS 2." A homologue of the S. cerevisiae FAS 1 subunit has also been identified in P. patulum. The fab B gene of Escherichia coli encoding P-ketoacyl synthetase I has also been sequenced and the active site identified by tagging with [3H]-cerulenin and sequencing of the radiolabelled peptide fragments that result from proteolysis.l1 The E. coli and S. cerevisiae condensing enzymes have a common sequence of five amino acids (Ala-Cys-Ala-Thr-Ser) which are believed to contain the active site cysteine. The stereochemical course of the hydrationdehydration reaction catalysed by the E. coli P-hydroxydecanoyl thioester dehyd- ratase has been shown'' to be a syn process with protonation occurring on the si face at C-2 of the dec-2-enoyl thioester (Scheme 2). The enzyme has been sequenced and the active site identified.l3 There is growing evidence that acyl carrier proteins may play a crucial role in the overall control of both fatty acid and polyketide chain assembly.A three-dimensional structure for the E. coli ACP has been determined by NMR methods.14 This structure shows the presence of a hydrophobic cleft which has been identified as a likely site of acyl chain binding. A heat D(HI -OH KMn04 H20 pH10 J 13c0,~ (D)H... I /" C0,H (HID Scheme 1 573 NPR 8 NATURAL PRODUCT REPORTS 1991 *H-H Scheme 2 CO2H 0 -CO2H DD (4) Scheme 3 stable factor required for the synthesis of fatty acids in the erythromycin-producing organism Saccharopolyspora erythrea has been purified and identified as a discrete ACP suggesting that the FAS of S. erythrea is a dissociable complex like that of E.c01i.l~ A number of studies on the conversions of unsaturated fatty acids in plants and marine organisms have appeared. Contrary to previous reports it has been demonstrated using an enzyme preparation from potato that the oxygen inserted enzymatically between 9 and 10 of the C-18 chain of linoleic acid (1) in forming colneleic acid (4) originates from ISO via the 9- hydroperoxide group of (2).16 A mechanism is proposed in which protonation of the hydroperoxy group and loss of water leads to the epoxycarbocation (3) which undergoes cleavage of the 9,lO bond as shown in Scheme 3. In agreement with this mechanism all four deuterium atoms in [9,10,12,1 3-2H,]-linoleic acid (1) were retained being in~orporated'~ as shown in Scheme 3.This intermediate has also been used to follow the fate of olefinic deuterium labelling in formation of the 13- hydroxy-12-ketone (8) via the hydroperoxide (5) using a flax- seed derived (1 3s)-lipoxygenase and isomerase via the mech- anism shown in Scheme 4.Deuterium label was incorporated at the 9,10 and 13 positions of (8) as expected but there was none present at C-11. While control experiments suggested that the loss may be due to rapid exchange from C-1 1 the absence of deuterium can also be explained mechanistically if the C- 12 deuterium if first lost from epoxide (6) giving the allene oxide (7) which is then attacked by water with inversion at C-13 as shown." Further evidence for the involvement of allene oxide intermediates has been provided by studyls of the minor product of the isomerase enzyme the y-ketol (9).The pattern of incorporation of deuterium from the tetradeuterio-linoleic CO2H (8) Scheme 4 acid was established by 'H NMR. The lack of incorporation of deuterium label at C-11 of (9) can only be satisfactorily explained by the loss of deuterium from C- 12 of an intermediate (6) to give the allene oxide (7) followed by y-attack as shown in Scheme 5. Loss by exchange is not reasonable in this system. The enzyme system from flax-seed converts linolenic acid (10) into the a-ketol(1 I) the y-ketol(l2) and the prostaglandin- like 12-oxophytodienoic acid (1 3). experiment^'^ with [14-2H]- linolenic acid confirm the requirement for a 13-hydroperoxide for 12-oxophytodienoic acid formation.All three products (1 1)-(13) can be rationalized in a single scheme in which the main feature is the formation of a protonated allene epoxide (14) which is then deprotonated to form the allene epoxide (Scheme 6). One branch leads to the formation of the a-ketol one to the y-ketol and a third to the oxophytodienoic acid itself. It has not been possible to separate the flax enzyme preparation into an isomerase and a cyclase. Together with the present results this leads to the suggestion that a single enzyme initiates these processes. Corey has proposed an antarafacial pericyclic closure to explain the formation of preclavulone A (19) via (8R)-HPETE (16) on treatment of arachidonic acid (15) with a homogenate of the coral Clavularia viridis.,O An enzyme preparation from the unrelated coral Pseudoplexaura porosa is also capable of converting arachidonic acid to preclavulone A (1 9) indicating NATURAL PRODUCT REPORTS 1991-T.J. SIMPSON /H+ OH I (7) CO2H O D (9) Scheme 5 OOH Scheme 7 that the biosynthesis of (19) may be widespread in This has been confirmed by the demonstrated ability of homogenates from several species Plexaura homomalla Plexaura nina Plexaura jlexuosa Pseudopterogorgia americana Muriceopsis jlavida and Euricea asperula to convert arachidonic acid to (8R)-HPETE and preclavulone A.22It is suggested that (19) may be a key intermediate from which other more highly oxygenated marine prostanoids are formed.The biogenesis of (19) from arachidonic acid by lipoxygenation at C-8 migration of oxygen from C-8 to C-9 and ring closure provides an alternative pathway for the formation of marine prostanoids which contrasts sharply with the mammalian endoperoxide route. A biomimetic synthesis of analogues of (19) via allene oxide and 2-oxopentadienyl cations (cf. Scheme 7) has been NATURAL PRODUCT REPORTS 1991 R’ R1 J Scheme 8 HO’ II ,. HO OH CO2H (211 Scheme 9 R2 CO2H (23)R’ = R2= H (24) R’ = Me R2 = H (25) R’ = H R2= Me described.23 Corey has also a biomimetic synthesis of colneleic acid from (9s)-hydroperoxy-( 1OE 12Z)-octadienoic acid based on the Baeyer-Villiger-like rearrangement sum-marized in Scheme 8.Epoxy-tetraenoic acids e.g. (20) have been as the immediate biosynthetic precursors to lipoxins e.g. A (21) and B(22) as shown in Scheme 9. The synthesis of these precursors have been reported by several groups.26-28 The synthesis of 7,7-and 10 lo- and 13,13-difluoroarachidonicacids has been reported.2910,lO-Difluorarachidonic acid serves as a substrate for PGH synthase and soybean lipoxidase. Hydroxylation is observed to occur but no cyclization to prostaglandin deriv- atives was Studies on the biosynthesis of long chain branched fatty acids in marine sponges have been reported. 5,9-Hexaco- sadienoic acid (23) and the corresponding 24- and 25-methyl acids (24) and (25) were shown to be derived from the short chain precursors palmitic 12-methyl-and 13-methyl-tetradecanoic acids respectively in Jaspis stellifera so that branching does not occur after chain el~ngation.~~ In studies with Aplysina ri~tularis~~ both enantiomers of 10-methyl-hexadecanoic acid (27) were incorporated into 22-methyl-5,9- octacosadienoic acid (28) again by chain elongation.Studies with theR and S enantiomers indicated a slight preference for the R isomer. To investigate the methylation process the incorporation of [3H]-methionine was investigated. A rapid incorporation into the short branched fatty acids was observed with subsequent slower incorporation into the long chain compounds. 14C-Labelled palmiteolic acid (26) was also incorporated to suggest the biosynthetic pathway shown in Scheme 10.Incorporation of [1,2-13C,]-acetate into the botryal~,~~ a group of C52-C54 aldehydes e.g. (29) from the green alga Botryococcus braunii suggest their formation via aldol con- densation of the corresponding unsaturated aldehydes followed by dehydration of the resulting aldol intermediates. The 28-27l 2-isomers predominate. Cerulenin (30) a metabolite of Cephalo-sporium caerulens is a potent inhibitor of FAS acting specifically by blocking the condensing enzyme 2-oxoacyl- thioester synthase. It is finding widespread use as a biochemical tool to inhibit polyketide and fatty acid biosynthesis. A protocol for the preparation of cerulenin of high specific radioactivity by biosynthetic incorporation of 14Cor 3Hlabelled acetate has been described.34 3 Tetraketides Lathodoratin (3 1) is a 3-ethylchromone phytoalexin produced by cupric sulphate treatment of Lathyrus odoratus (Sweet pea).Incorporation of [13C2]-acetate gave the labelling pattern The involvement of a 2-methylbutyrate starter derived from isoleucine was confirmed by the incorporation of 14C- labelled isoleucine. The randomization of 13C-13C couplings in the phenolic ring is consistent with the involvement of a symmetrical intermediate as shown in Scheme 11. 13C-Labelled acetates were in~orporated~~ into flavipucine (32) an antibiotic metabolite of AspergilZus Javipes as shown in Scheme 12. Flavipucine co-occurs with the isomeric metabolite (33). This suggests a tetraketide origin for these metabolites with branched starter units derived from amino acid metabolism.The authors suggest biosynthesis via condensation of two diacetate units but a pathway via cleavage of a preformed carbocyclic ring would NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON I Scheme 10 CHO Me(CH,),CH =CH(CH,) ,CH =C' \ (CH2),&H=CH(CH2),Me (29) OH 0 Scheme 11 Me (32) R = -CH2CH Me Me-C0,Na -(33) R = -CHCH,Me I H Me Scheme 12 be an interesting alternative. Incorporation experiments with 13C,2H3-labelled acetate would provide information on this problem. A number of meroterpenoid metabolites are known which contain a tetraketide derived moiety substituted by a complex terpenoid-derived portion. Full details have appeared3' of biosynthetic studies on the austalides a group of mycotoxins produced by Aspergillus ustus.Incorporations of [1-l3C]-,[1,2-"C2]- and [1-13C,2H3]-acetates and [2-13C]-mevalonate into austalide D (38) confirm its derivation via 6-farnesyl-5,7- 578 NATURAL PRODUCT REPORTS 1991 \ \ OH OH L 0 d\\\ OH H Me Me OH /OMe (38) OMe (35)R=HL(36)R=OH (37) Scheme 13 - A Me=CO2Na A A {Me}-Methionine Me OH C02Et HOZH2 OH0 (39) Scheme 14 dihydroxy-4-methylphthalide (34). The mechanism proposed (Scheme 13) for the subsequent cyclization and oxidative modification of the farnesyl moiety is consistent with the relative stereochemistry of the austalides and the structures of the co-metabolites austalide J (37) K (35) and L (36) and is supported by the observed incorporation of austalide K (35) into D.Addition of ethanol to cultures of A. ustus was found to inhibit production of normal metabolites and induce formation of a new metabolite ethyl-2,4-dihydroxy-3-methyl-benzoate (39). Incorporation of 13C-labelled acetate and methionine gave the labelling pattern shown in Scheme 14 suggestive of bis-methylation of a tetraketide precursor. However no incorporation of deuterium from [1-13C,2-2H3]-acetate was observed at C-9 which indicates that C-9 may not be part of a starter unit. This result is interpreted to favour a pentaketide origin with subsequent loss of a starter unit for (39). Interestingly stellatin (40)which has the same carbon skeleton as (39) was isolated from strains of the meroterpenoid producing organism Aspergillus vuriecolor (see below) which no longer produced meroterpenoid~.~~ Carbons 9 and 10 are also derived from methionine in this metab~lite.~~ Austin (42) is a further meroterpenoid mycotoxin isolated from A.ustus. Incorporation of ethyl [13C,180]-3,5-dimethylorsellinate (41) into austin and analysis of the 13C NMR spectrum of the enriched metabolite showed isotope shifts for the C-6’ and C-8’ resonance^.^^ These clearly indicate their derivation from the 6-hydroxy and C-7 carbonyl oxygens of 3,5-dimethylorsellinate. The relative size of the isotopically shifted and non-shifted signals show that there is no significant loss of label from C-6 of (41). However approximately half the label is lost from C-7 a result consistent with formation of a free carboxylate at some stage during the biosynthesis followed by nucleophilic attack of the hydroxy group at C-5’ on C-8’ to form the y-lactone as indicated in Scheme 15.The origins of all the oxygen atoms in andilesin A (43) a meroterpenoid metabolite of Aspergillus vuriecolor have been determined by labelling studies with [1-13C,1802]-acetate,laO and ethyl [13C,180]-3,5-dimethylorsellinate.41 The C-6’ hy- droxyl is derived from C-6 of 3,5-dimethylorsellinate a result which indicates a pathway in which andilesin A is the first metabolite to be formed and that it is converted by dehydration into andilesin B which is then reduced to andilesin C. Pyncnophorin (44)is a novel antimicrobial diterpene a-It has a pyrone isolated from Macrophornu kuwats~kui.~~ similar carbon skeleton to a number of other diterpenoid se~quicillin.~~ pyrones e.g.c~lletotrichin,~~ Although the NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON I Scheme 15 Me Me HO Me 0AoJ; // Me Me Me OH / 0 Me HO "0 s" Me I OH (45) R= Et (46) R= Me Me0 studies have been carried out the aglycone appears to be derived from 3 acetates 5 propionates and 1 butyrate. It would be of great interest to see whether the orsellinate homologue present in A3 is derived via a propionate starter unit. Orsellinate moieties are found as sub-units in a number of larger structures. A particularly interesting example is the iodoorsellinate unit bound as a thioester in the calichemicins e.g.(47) a family of novel potent antitumour agents isolated from Micromonospora echinospora.46 The isolation of fusalanipyrone (48) from Fusarium solani HO OH Et pyrone moieties in these metabolites are formally derived from a triketide it is possible that the intermediate precursor is a tetraketide derived phenol which is subsequently degraded to form the pyrone. Biosynthetic studies to test this point would be worthwhile. Lipiarmycin A3 (45) and A4 (46) are novel macrolides isolated from Actinoplanes deccanen~is.~~ They differ in the structure of the orsellinate residue present in the structure. Whereas lipiarmycin A4 (46) contains a 3,5-dichloroorsellinate A3 (45) contains the ethyl homologue.Although no biosynthetic NATURAL PRODUCT REPORTS I991 0 MeCOSCoA - __Lt 0 00 SR C1 Me (48) Scheme 16 HO&HoxYco2H-‘HO ’OH Me MeOJ Me Me ‘ 0 ’OH Me (49) Scheme 17 fl-HO OH 0 HO2C OH 0 h02c 0 0 (53) R = H (54) R = Me Scheme 18 has been rep~rted.~’This compound is described as a 4 Pentaketides monoterpenoid but is almost certainly derived from a bis-C- The biosynthesis of canescin (54) a metabolite of Aspergilfus methylated tetraketide precursor as indicated in Scheme 16. mafignushas been using [rnethyl-13C,2H3]-methionine [2,3-13C,]- and [2-13C,2- Feeding with 13C labelled precursors in young plants [1 ,2-13C,]- and [1-13C,2-2H3]-a~etates of Garcinia mangostana have shown (Scheme 17) that the 2H,]-succinate as simple precursors and 2H-labelled isoco- xanthone skeleton of mangostin (50) is derived from m-umarins as potential advanced intermediates.Analysis of the hydroxybenzoic acid and three malonates via the benzophenone labelled products by NMR and mass spectrometry indicates (49). It is interesting to compare mangostin with the structurally that 6,8-dihydroxy-3,7-dimethylisocoumarin(5 1) is the first similar tajixanthone (107) see below which is in fact derived enzyme-free intermediate and that 7-formyl-6,8-dihydroxy-3,7-via an octaketide precursor. dimethylisocoumarin (52) is a later intermediate. The retention NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON 581 A -wMe.-@fMe MeCb2Na -SEnz \ \ o* o* *OH 9 9H ? (55) Scheme 19 I 0 Me OMe -0ao -M Me-C02Na SEnz e o w 0 Scheme 20 Me Me H02C WMe O OH A (58)R = ) A MeYo (59) R = HO Medo /'Me Me Me O 0 (63) (64) (60)R = CI Me of one methione derived deuterium at C-10 proves the formyl group is not oxidized to the carboxylic acid level.No deuterium from succinate is retained in the final metabolite. On the basis of these results the pathway shown in Scheme 18 is proposed. No distinction between routes (a) and (b) can be made. The isolation of demethylcanescin (53)suggests that 0-methylation is a late step. Canescin is isolated as a mixture of diasteriomers due to facile epimerization at C-10. The ready formation of an o-quinone methide is suggested to account for this.The origins of the oxygen atoms in hydroxymellein (56) have been determined50 by incorporation of 170-labelled acetate and oxygen into (56) by cultures of Aspergillus melleus followed by 170 NMR analysis of the enriched metabolites. ,H-Labelled mellein (55) was efficiently incorporated into (56). These results indicate the pathway shown in Scheme 19 in which hydroxy- mellein (56) is formed by benzylic oxidation of mellein (55). Alternative pathways via epoxide intermediates are precluded. Incorp~ration~~ of [13Cz]-acetate into kotanin (57) in As-pergillus niger confirm its formation via folding and dimer- ization of a pentaketide precursor as indicated in Scheme 20. Incorporation of [2-13C]- and [1,2-13C,]-acetates indicate that the B and c rings of the napyradiomycins (58)-(61) metabolites of the ascomycete Chaina rubra are derived via a pentaketide 1,3,6,8-tetrahydro~ynaphthalene.~~ Consistent with this the 13C-couplings in the naphthalene ring are randomized.The side chain carbons appear to be of terpenoid origin although no incorporation of label from [5-13C]-mevalonate was observed. Interestingly randomization of acetate labelling at the end of the geranyl-derived substituent was observed in (59) and (60). The incorporation of [2-2H3,1,2-13C,]-acetate into citrinin (62) in Penicillium citrinum has been reported.j3 The use of this precursor is advocated as a convenient method of analysing both u and p 2H-isotope shifts. It also allows 13C from labelled acetate to be distinguished from endogenous precursor.The structures of monochaetin a metabolite of Monochaeta compta was originally assigned54 as (63) a structure not readily rationalized biosynthetically. On the basis of NMR studies this has been revised55 to that of the azaphilone (64). The fusion of a triketide to a pentaketide moiety is reminiscent of the di ketide-octake tide fusion in ochrephilone. 56 NATURAL PRODUCT REPORTS 1991 00 CD3C02Na -0 0 11 (65) 2€ x=o (68)R’ =H R~=OH (66) 22 x = 0 (69) R’ = OH R2= H (67) 2€ X=H,OH *Ov SEnz Me -C62Na 0 * Scheme 21 5 Hexaketides Penicillium urticue normally produces the tetraketide metabolite patulin. A strain of P. urticae in which the patulin pathway is blocked between neopatulin and ascladiol produces three new macrolides patulolides A B and C (65)-(67).Incor-poration~~’ of l3C-1abelled acetates confirm a hexaketide origin for these metabolites which are closely related to cladospolides A (68) and B (69) isolated58 from Cludosporium cludosporoides. Multicolosic acid (71) and related tetronic acids from Penicillium multicolor have been the subjects of a number of previous biosynthetic studies which have demonstrated their biosynthesis via ring cleavage of 6-pentylresorcyclic acid (70) as shown in Scheme 21. The origins of a number of the oxygen atoms have previously been established by incorporation of [l- 13C,180,]-acetate. The origins of the remaining oxygens have been defined by with 1802. The C-1 carboxyl C-3 enolic and C-11 carboxy oxygens all contain the same amount of l80,whereas C-9 has much less perhaps indicative of loss by exchange at the aldehyde level.Equal amounts of oxygen at C-1 and C-1 1 suggests that aromatic cleavage proceeds by a different mechanism to that observed for patulin.,O It appears therefore that a number of different pathways for oxidative aromatic ring cleavage are available in Penicillium species. Deoxyradicinin (72) is one of a number of related metabolites produced from Alternuriu heliunthi and other plant pathogens. A number of pathways can be envisaged for the biosynthesis of the carbon skeleton of (72) by alternate condensations of preformed polyketide chains or by cleavage of a carbocyclic intermediate derived from a single polyketide chain.ResultsG1 from incorporation of 13C-labelled acetates were inconclusive. However on incorporation of [2-2H3]-acetate the levels of enrichment were such that isotope shifts could be seen at natural abundance on the 13C NMR signals due to both C-10 and C-5. In each case three isotopically shifted signals were observed due to the incorporation of up to 3 deuterium atoms on C-1 1 and C-12 respectively. This confirms the presence of two acetate starter units in deoxyradicinin. In order to distinguish between alternative pathways involving two C or C and C . precursors the incorporation of [1-13C]-3,5-dioxohexanoic acid was studied. However no incorporation was observed. Coronatine (73) is a novel phytotoxin isolated from a species of Pseudomonas syringae which infects rye-grass and soybean plants.Incorporation of [1-13C]-isoleucene resulted in en-richment at C-1 of the coronamic residue.62 [l ,2-13C2]-acetate was incorporated into the coronofacic acid (74) portion as indicated in Scheme 22. Surprisingly no label was incorporated into C-3 or C-3a. These were shown to be enriched from C-2 and C-3 or pyruvate suggesting derivation of (74) via a branched polyketide with pyruvate serving as a starter unit for one of the chains. 6 Heptaketides Full details have appeared of previously communicated studies on the biosynthesis of polivione (75),63 palitantin (76), and citromycetin (77)65. Much of this work was discussed in the previous report .l The biosynthetic interrelationships among this group has long been a subject of study and speculation.Convincing results have been obtained for a pathway to citromycetin polyvione and the related metabolites fulvic acid (78) and lapidosin (79) via ring cleavage of a carbocyclic intermediate e.g. (80) (cf. rubrofusarin itself derived from a single polyketide precursor). However this conclusion was not NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON Me-C02Na heEOC02H mc02h \ (74) Scheme 22 OH Me0 Y M e "&Me C02H Hoe.. HOC 0Me II HO". "CH20H OH 0 0 0 (75) (77) Me0 C02Me O H 00 X Me Howe HO C02H 0 OH 0 (79) -Rsv -(76) 0 + 4 H02CCH2COSCoA [HI 0 0 0 J OH OH 000 Scheme 23 readily compatible with previous evidencess-ss that citro-shown in Scheme 23 three acetate-derived units would therefore mycetin and by implication poliovione fulvic acid and differ from the remaining four.Cleavage is indicated would lapidosin incorporate two acetate starter units. To reconcile then give two apparent acetate starter units. The main drawback this apparent conflict a pathway is proposeds3 for the to this Scheme is the required 'reoxidation 'of C-2 methylene to biosynthesis of a modified precursor as shown in Scheme 23. a carbonyl which contradicts the currently accepted ideas of According to this the chain is initiated by a preformed C unit polyketide chain assembly in which the appropriate function- followed by four successive condensations with malonate to ality is generated during chain assembly.However precedents generate the necessary C,,chain. On the basis of the pathway for a preformed C starter unit and subsequent oxidation have NATURAL PRODUCT REPORTS 1991 (81) R= H (82) R = Me Scheme 24 Me 0 OH OR Scheme 25 been found in the biosynthesis of aflatoxins (see below for further discussion). One extra reductive step would generate the corresponding heptaketide precursor for palitantin (76) biosyn- as indicated in Scheme 23. Further evidence for this intriguing suggestion is awaited. Alternariol (81) has long been regarded as an archetypal example of an aromatic metabolite biosynthesized via cycliz-ation and aromatization of a simple polyketide precursor as shown in Scheme 24 and this has been tested by a number of biomimetic syntheses (for example see discussion below).However due to the co-occurrence of alternariol with other heptaketides related to lichexanthone (84) in a number of organisms it has been speculated that alternariol could be biosynthesized via a ring cleavage process as illustrated in Scheme 25 where the necessary ring fragmentation would occur either (a) by oxidative scission or (b) by a retro-aldol process. The incorporation of [1,2-13C,]-acetate into alternariol methyl ether (82) did not shown the randomization of 13C couplings which would be anticipated from the involvement of a symmetrical intermediate such as (83). However feeding norlichexanthone (84) labelled with 14C in the carbonyl group resulted in a 3.8% incorporation of label into alternariol methyl ether (82) so providing some evidence for the ring cleavage pathway.69.However this was ruled out by the reported incorporation of [1-13C,’802]-acetate into alternariol methyl ether.70 This showed high incorporation of acetate derived oxygen into all the oxygen bearing carbons including the benzopyrone carbonyl and so precludes either of the ring cleavage pathways shown in Scheme 25. In an extension of previous studies on the biomimetic synthesis of polyketides Staunton has studied the cyclization of the partially cyclized masked heptaketide analogue (85).’l On treatment with strong base this cyclized to give exclusively the biphenyl structure of alternariol methyl ether (82).On conversion of (85) to the corresponding pyrilium salt the triketo-intermediate (86) could be released by mild hydrolysis allowing its cyclization behaviour to be studied (Scheme 26). Treatment under a variety of mild conditions always resulted in formation of the biphenyl rather than the naphthalene. This suggests that only a small degree of enzymic control over the folding of the polyketide residue is required for the conversion of (86) into (82). The naphthalene (87) could be formed in high yield by carrying out the cyclization of (85) under basic conditions in non-aqueous solution which prevents hydrolysis of the pyrone. This provides support for a previous s~ggestion’~ that naphthalene ring formation in rubrofusarin could be controlled in vivo by pyrone ring formation as an early step in cyclization of the heptaketide precursor.Further work on the synthesis of polyketide intermediates and their biomimetic cyclizations have been ~eviewed.’~ Brefeldin A (88) is another much studied fungal polyketide. Previous studies have shown that one acetate-derived deuterium label is incorporated at C-14. Degradation of (88) to the lactone (89) and 2H NMR analysis has shown that both diastereotopic hydrogens at C-4 of (89) and hence C-14 of (84) are equally labelled.74 This implies that a non-stereospecific hydrogen exchange must be occurring during biosynthesis. It is suggested that this most likely occurs from a malonyl thioester in- termediate. It appears that this type of exchange must be dependent on the producing organism because equivalent positions in other polyketides show stereospecific incorporation of acetate derived hydrogen e.g.monocerin,’ averufin and cladosporin (see below). Brefeldin C (90) is the immediate precursor of brefeldin A. Stereospecifically labelled forms of (90) have been incorporated into (88) by cultures of Eupeni-cillium brefeldianum (Scheme 27) to show that hydroxylation NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON 585 -MeomMe SEt Meoy& \ OH 0 OH 0 J I M e 0\ s o H OH OH 0 (87) OH 0 Scheme 26 CD3C02Na -HO t OH (90) Scheme 27 MeCO2Na-1 MeCH2C02 Na Scheme 28 at C-7 of (90) involves stereospecific removal of the 7 pro4 hydrogen from Cyclizidine (91) and the closely related indolizomycin (92) are the only indolizidine alkaloids known to be produced by Streptomyces species.In contrast to other organisms where this class of alkaloid is known to be biosynthesized via lysine incorporations of 13Cand 2Hlabelled acetates and propionates indicate that the carbon skeleton of cyclizidine is derived via a polyketide precursor. The unusual terminal cyclopropyl ring is derived from a single propionate as shown in Scheme 28. NATURAL PRODUCT REPORTS 1991 HO 0 Meq0 0 OH OH Me 0 ‘0 (95) Scheme 29 Me o 0 Me 7 Octaketides Actinorhodin (93) a dimeric isochomanequinone antibiotic produced by Streptomyces coelicolor is a representative of a family of such antibiotics produced by a variety of Streptomyces species.It has become a focus of work on the genetics of polyketide antibiotic biosynthesis. The complete gene cluster for the biosynthesis of actinorhodin has been cloned as a contiguous stretch of DNA. 77 Co-synthetic studies in blocked mutants have identified at least six biosynthetic genes for actin~rhodin~~ and preliminary evidence suggests that the act I act 111 and act VII genes code for the necessary condensation reduction and dehydration activities associated with polyketide ~ynthases.~~ The act I11 gene has been sequenced and does indeed show homology with other oxido-reductases.80 The act I and act I11 genes have been used to probe restriction digests of genomic DNA from a large number of other strepto- mycetes.al Many of these showed the presence of DNA homologous to both act I and act 111.Interestingly the tetracenomycin (1 36) producer Streptomyces gzaucescens con-tains DNA homologous to act I but not act 111 consistent with the fact that tetracenomycin biosynthesis does not require a reduction step during polyketide chain assembly. These and many other aspects of genetic studies on polyketide assembly have been revieweda2 as have biosynthetic studies over a number of years on the isochromanequinone antibiotic^.^^ From two types of class V act mutants of S. coelicolor two monomeric precursors of actinorhodin have been isolated.83 One is the known antibiotic kalafungin (95) and the other is a yellow pigment with structure (94).On the basis of this and other work with mutant the overall pathway shown in Scheme 29 is suggested. 14C-Labelled kalafungin was efficiently incorporated (1 7.7%) into actinorhodin. Crisamycin A (96)84 and the corresponding monoepoxide crisamycin C (97)a5 are novel isochomanequinones isolated from Micromonospora purpureochromogenes. They are struc- turally closely related to actinorhodin. Incorporation of 13C- labelled acetates confirm their octaketide origin. A particularly unusual feature of their biosynthesis is the presence of a hydroxyl group at C-6 and the lack of oxygen substitution at the ‘biosynthetically predicted ’ position C-9. Presumably dimerization occurs via an oxidative coupling mechanism which would require the presence of a phenolic substituent at C-9.This implies that loss of oxygen from C-9 occurs after aromatization and after dimerization and that hydroxylation at C-6 occurs late in the pathway as has been suggested for the equivalent hydroxylation in actinorhodin biosynthesis. Deoxyfrenolicin (98) and frenolicin B (99) are nonaketide isochroanequinones isolated from Streptomyces roseofulvus. The nanaomycins (100) and (101) have been isolated as co- metabolites of frenolicins in S. roseofulvus.86This is a significant observation as it is the first finding that two types of isochromanequinone antibiotics with different configurations at C-1 and C-3 are produced by the same organism. In addition the co-occurrence of such closely related metabolites which are derived from octa- and nona-ketide precursors suggests that the polyketide synthase may be able to accommodate precursors of different chain lengths.The differing stereochemistry at C-1 and C-3 may reflect different modes of binding required for these different chain lengths. Although it is now generally accepted that many deoxy- NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON Me Me (100) R = CH20H (101) R = CO2H OH 0 OH OH 0 OH Me OH Me 0 enzyme NADPH. OH 0 OH OH 0 mH' OH ~ \ 'H* -H20 \ MemH. Me OH 0 H' 0 H H' (103) Scheme 30 genation processes in polyketide biosynthesis take place during chain assembly by reactions analogous to those catalysed by fatty acid synthases it is clear that some deoxygenations must occur at a post-aromatization stage such as in the conversion of versicolorin A to sterigmatocystin during aflatoxin biosyn- thesis.More surprising is recent evidence that the anthra- quinone chrysophanol (103) is formed via emodin (102). Preliminary worke7 showed that a crude cell-free enzyme system from Pyrenochaeta terrestris converted emodin gen- erally labelled with 3H into its 6-deoxy-derivative chryso- phanol. This has been further studied by NMR. Incubation of emodin in a cell-free medium containing 50 70D,O resulted in incorporation of deuterium into chrysophanol as indicated in Scheme 30 which suggested that reductim was occurring via a keto-tautomer of the resorcinol ring of emodin.88 Repeating the experiment with deuterated NADPH confirmed the origin of the hydride necessary for reduction.Related work on the pentaketide derived melanins in phytopathogenic fungi has been reviewed.89 A large number of fungal metabolites including benzo-phenones and xanthones are believed to be derived via oxidative metabolism of anthraquinone intermediates. Previous work NATURAL PRODUCT REPORTS 1991 OH 0 OH 0 Me Me HO Me (104) Me \ 0 Me Mehie @MZ 0 LMe \/ ‘ ’OMe Me Me OAc has shown a probable biosynthetic relationship (Scheme 3 1) between the arugosins e.g. (109 variecoxanthones e.g. (106) and tajixanthone (107) in Aspergillus variecolor. The essential intermediacy of chrysophanol in this pathway has been demon- strated by synthesis of [methyl-2H3]-chrysophanol (104) and demonstration of its specific incorporation into tajixanthone by ,H NMR.90 Related metabolites have been isolated from a number of other fungi.Arugosin E (108) and cycloisoemericellin (109) have been isolated along with known arugosins from Aspergillus silvaticus and Emericella striata respectively.g1 A novel prenylated anthranoid metabolite (1 10) has been isolated from Psorospermum glaberrirn~m.~~ It is probably derived via ring cleavage of a vismione e.g. (11 l) via a Baeyer-Villiger type of oxidation. A similar process seems likely for the formation of the well-known metabolite lambertellin (1 l 2).93 Austrocorticin (1 13) and austrocorticinic acid (1 14) novel pigments bearing a unique C side chain at C-3 in the anthraquinone nucleus have been isolated from a toadstool belonging to the genus Cortinarius.Incorporation experimentsg4 with 13C-labelled propionate indicate their origin via a propionate-triggered octaketide precursor (Scheme 32). The use of a propionate ‘starter’ is highly unusual among fungal polyketides. The origins of the oxygen atoms in the phytotoxin betaenone OH 0 B (116) have been established through a feeding experiment with [1-13C,180,]-acetate and by treatment of cultures of Phoma betae with the cytochrome P,, inhibitor ancymidol (1 16). As a result of the latter experiment the intermediate probetaenone I (1 15) accumulated which suggests a biosynthetic pathway involving an intramolecular Diels-Alder cyclization via a triene precursor at a late stage (Scheme 33).95 The lack of in-corporation of acetate derived oxygen into the terminal carbon may be ascribed to rapid exchange at the aldehyde level.However it is noteworthy that Vederas showed that the oxygens in the lactone ring of mevinolin were derived from atmospheric oxygeng6 and not from acetate as anticipated. This could be due to P-oxidation processes so it is conceivable that betaenone B could be derived from degradation of a longer polyketide precursor cf. mevinolin. Biosynthetic studies on marine polyketide-derived meta- bolites are rare. However [l-14C]-proprionate has been suc- cessfully incorporated (Scheme 34) into the dendiculatins (1 17) produced by the marine pulmonate Siphonaria dendic~lata.~’ These are presumably formed by decarboxylation of a propionate-derived octaketide.It is not possible to distinguish between the chain starter and termination units. Vermistatin a metabolite of Penicillium vermiculatin has been assignedg8 structure (1 18). This clearly indicated an NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON 59I 0 0 OH 0 CD3C02Na -HO Mel3Cl8O2Na H Scheme 37 MMeeMe MMe __c @H Me ' \ \I \ 0* -'I \ I -/I L I \'I I *' OAc 0 *' OH 0 OH OH 0 H 0 CN OH 0 CN (1 27) Scheme 38 Me Me O Me \OH 0 OH 0 HO OH (130) R= (131) R= Q H COpH C02Me C02Me 2-HMe wHMe \ 0 0 ,-+ \ / I OH 0 OH 0 OH 0 OH 0 OH 0 OH OH aklanoic acid Aclacinom ycins Py rrom ycinones Rhodomycinones Daunom ycinone Scheme 39 may represent an alternative folding of the decaketide precursor (130) and (131).The most interesting feature of these to that in kinamycin D. compounds is the tyrosine and tryptophan derived moieties Urdamycins C (130) and D (131) have been isolated from linked in quinone methide-type structures to the polyketide Streptomyces fradiae.'14 The term 'angucyclins' is proposed115 derived angucyclin backbone. A new metabolite aklaviketone to describe this group of antibiotics based on the (1 32) has been isolated from fermentations of a mutant strain benza[a]anthracene ring system. A forthcoming review in this S-383 of Streptomyces ga1ilaeus.ll6 This compound is most journal will describe the chemistry and biological activities of likely the first cyclization product on the anthracycline this growing group of metabolites in more detail.Incorporation biosynthetic pathway to possess the tetracyclic skeleton. The of 13C-labelled acetate was used to aid structural assignment of pathway shown in Scheme 39 is proposed. 41-2 NATURAL PRODUCT REPORTS 1991 Me 0 Me OH 0 OH Me OH 0 OH - EnzS 0 L 0 HO 0 HO \' 0 'OH Me OH 0 OH Ho2c\HO \ 0 OMe 0 II OH 10 OH OMe Me OH 0 OH Me0 'OMe (137)R'=R2=R3=Me 0 (138)R'=R~=H,R~=M~ (139)R' = R2 = Me R3 = H (140)R' = R3 = Me R2 = H Me0 OMe (136) Scheme 40 O O O O O O O H - 0 0 0 0 0 Me-C02Na J J C02H 0 OH OMe OMe OH 0 OH (141) Scheme 41 NATURAL PRODUCT REPORTS 1991-T.J. SIMPSON 6eCOa -+ ieCOSCoA J - -COG mutase 'eFCOSCOA Me Me dSC~A =Me '>COSCOA HOCH I Scheme 42 Tetracenomycins B (1 35) and D (1 33) have been isolated1" along with tetracenomycin D (134) from a blocked mutant of Streptomyces olivaceus TU 2353 which normally produces the ellaromycins (1 37)-(1 40).'18* 119 Tetracenomycin B (1 35) appears to be the key intermediate where the biosynthesis of the ellaromycins branches off from the pathway leading to tetracenomycin C (136). On the basis of these and other results120* 121the pathway shown in Scheme 40 is proposed. The cloning and heterologous expression of the gene cluster for the biosynthesis of tetracenomycin C in Streptomyces glaucesens has been reported. 122 10 Dodecaketides Thermorubin (141) is a polycyclic metabolite of Thermo-actinomyces vulgaris.Incorporation with 13C-labelled acetate and salicylic acid indicate a pathway in which salicylic acid acts as the starter unit for formation of a dodecaketide which cyclizes and undergoes cleavage of the terminal ring [cf. compound (1 10) above] to form thermorubin as shown in Scheme 41. 11 Polyether lonophores Full details 124 of the assignment of the 13C NMR spectrum of monensin (142) and 1802 enrichment studies have appeared. The incorporation of [2-2H2]- and (S)-[2-2Hl]-propionate into monensin in cultures of Streptomyces cinnamonensis occurs with retention of label only at C-4 and C-6 whereas during the incorporation of (R)-[2-2Hl]-propionate the deuterium label is lost.These are consistant with the formation of (5')-methyl-malonyl CoA from the labelled propionate by car-boxylation of propionyl CoA with the loss of the 2-pro-R hydrogen. The (3-methyl-malonyl CoA is subsequently inc- orporated into the antibiotic by decarboxylative condensation occurring with overall inversion. The incorporation of 2H-Me Me ?do CO-CH~ 0 II Ado 0 CO-CH~ II Scheme 43 labelled acetates provide evidence for a pathway of metabolism leading to methylmalonyl CoA that does not proceed via succinyl CoA. Instead acetyl CoA may be metabolized via butyryl CoA and isobutyryl CoA to afford (9-methyl-malonyl CoA. The results for interconversion of these precursors are summarized in Scheme 42. Incorporations of 13C- and 2H- labelled isobutyrates provided further evidence for the existence of a novel rearrangement which leads to the conversion of isobutyrate into butyrate.The rearrangement was shown126 to proceed in an intramolecular fashion by migration of the carboxy carbon of isobutyrate to the 2-pro-S methyl with a concomitant back migration of a hydrogen atom from the methyl group into the 3-pro-R position in butyrate. Normally therefore the carboxy carbon is replaced with overall retention of configuration in a vicinal interchange rearrangement. As indicated in Scheme 43 a close relationship is likely between this NATURAL PRODUCT REPORTS 1991 Et Me Me C02H __t Me MSCoAMe I OH Me - Me wIC~A Me ASCoA 0 Scheme 44 Me Me Me Scheme 45 __t Me0,C OH Me Me (149) Scheme 46 novel rearrangement and the well-known coenzyme-B, medi- ated methylamalonyl CoA-succinyl CoA mutase reaction.[1,3-13C,]-acetoacetate is incorporated via its conversion to [1,2-13C2]-propionate. A proposed pathway via isobutyryl CoA is shown in Scheme 44. Analogous results have been reported for the metabolic interrelationships of butyrate isobutyrate and propionate when these acids are used as precursors for the biosynthesis of lasalocid A (143) in Streptornyces lasaliensis. 12' Full details of the synthesis of the proposed triene precursor (144) to monensin have appeared.12* This synthesis uses the successful assembly of three key chiral synthons prepared by enzymatic and microbial techniques.An alternative synthesis based on direct aldol methodology has also be and a synthesis of the model triene (145) uses a modification of the Julia-Lythgoe olefination reaction as a key step. 130 The biosynthesis of monensin and other polyethers is believed to proceed by an epoxide-mediated cyclization cascade from the putative polyene precursors e.g. (144). The chemical feasibility of these cascades has been demonstrated by two groups,131.132 who synthesized isomeric epoxides ; e.g. (146) and demonstrated their rapid stereospecific cyclization to polyether structures e.g.(147) upon saponification followed by acidification (Scheme 45). The stereospecific synthesis of the diepoxide (148) has also been described.133 On treatment with pig-liver esterase this was converted in > 70% yield into the cyclized product.However monitoring by NMR showed that this is a stepwise process. A monocyclic lactone (149) was formed within 1 h but further conversion into (150) occurred over a 24 h period (Scheme 46). This contrasts with the rapid cascade observed in aqueous solutions at low pH and has obvious implications for the enzyme-catalysed process. The origin of the oxygen atoms in the polyether antibiotics narasin (151),134 maduramycin (152)135 and lenoremycin (1 53)136 have been established by stable isotope labelling methods. The 'ultimate' in polyether metabolites must be the brevetoxins produced by the notorious 'red tide' organism Gyrnnodiurn breve. The biosynthesis of brevetoxin B (154) has been studied by two groups137 138 who report the incorporation pattern from 13C-labelled acetate and methionine shown in Scheme 47.A surprising observation was the sequence of atoms enriched from the methyl carbon of acetate which can best be accounted for by involvement of Krebs Cycle intermediates.Therefore it is proposed that the 3-carbon fragments c d g h NATURAL PRODUCT REPORTS 1991-T. J. SIMPSON HO& Me OH 1 Me le H02C Me HO Me-Hod‘CHO U * eMe=-C02Na Methionine A Scheme 47 NATURAL PRODUCT REPORTS 1991 0 0 II 0 Scheme 48 r L SR 0 (158) Scheme 49 i k and 1 are derived from succinate either directly or indirectly and the 4-carbon fragment b from a-ketoglutarate.The remaining unusual building blocks a f and n show labelling patterns consistent with their formation via p-bydroxy-/?-methyl-glutarate.On the basis of these obser- vations a new-type of polyketide formation involving dicar- boxylic acids is suggested,13* in which a Claisen-type con- densation occurs to the a-position of the second carboxylic function with a loss of carboxyl group as shown in Scheme 48. A rather similar type of process involving condensation onto the carboxyls of succinate is proposed for the assembly of (1 55) a key intermediate in the biosynthesis of the nonactins e.g. (158) in Streptomyces griseus (Scheme 49). Full details of previous reported work in this area have appeared. 13' 12 Macrolides Results over a number of years mainly from stable isotope labelling have indicated that the necessary reductions and loss of oxygen from polyketide intermediates generally (though not always) occur concomitant with chain elongation as in fatty acid biosynthesis.Further support has come from the successful incorporation of partially elaborated chain elongation intermediates (1 59)-(161) into tylactone (1 62),141 as indicated in Scheme 50. Success was achieved by feeding the intermediates as their N-acetylcysteamine thioesters. When fed as the corresponding ethyl oxoesters incorporation of label occurred only after prior degradation. Similar results were reported for the incorporation of (163) into erythromycin NATURAL PRODUCT REPORTS 1991-T.J. SIMPSON I 00 Me,),&-NHAc . Me (159) 0 OH 0 MeAS-NHAc I Me+ s-NHAc Me Me (161) J Scheme 50 Me Me-’-NHAc OH 0 (163) 0 Me$ *..o ‘‘‘OR1 Me Me “OR Me OH Me Me (164),142 (1 56) and (1 57) as their N-caprylcysteamine thio- into nonactin (1 58) and (163) into nargenicin (165).144 Until recently there had been only one report of the isolation of partially assembled polyketide intermediates from cultures of producing However compounds (1 66)-(168) which are clearly derived from the chain assembly process 0 0 Me @OH E OH Me hie leading to the mycinamycins e.g. (169) have been isolated from mutants of Micromonospora griseorubidi~.~~~ There has been much speculation over the years concerning the biogenetic basis of the extensive structural and stereo-chemical homologies observed within each of the polyether and macrolide classes of antibiotics.These ideas have been extended NPR 8 NATURAL PRODUCT REPORTS 1991 mycinamycins I-IV 0fe \ o:.. I 0-sug OH 10 OH 0 0 OH I OH OH PPPAAPP proposed polyene precursorsto norboritomycinA R' = Me R2= H norboritomycin B R' = Et R2 = H X-14766 A R' = Me R2= CI Scheme 51 0 A Me=CO2Na Me A Methionine HOCHZCH(NH~)CO~H (170) Scheme 52 to show that these homologies can transcend both classes. For example as shown in Scheme 51 significant correlations exist between the mycinamycins and the polyethers norborito-mycins A and B and antibiotic X-14766A over a structural sequence derived from 7 biosynthetic building blocks.The analogy is further extended by comparison of a model which describes all the 16-membered macrolides with the Cane-Celmer PAPA prototype which summarizesthe stereochemistry of all the monovalent bisdispiroketal polyether antibiotics. Although the subunit constitution varies in individual cases the absolute configuration maintains its consistency at five of six asymmetric centres over the seven subunit sequence. 13C-labelled acetate methionine serine and glycine have been incorporated into the antitumour antibiotic rhizoxin (170) in Rhizopus chinensis.148 As indicated in Scheme 52 0-acetyl-L-serine appears to act as the chain starter unit prior to or after formation of the 2-methyloxazole-4-carboxylic acid unit.All three carbons of the serine-derived unit were also enriched on feeding [1,2-13C2]-glycine.The S-lactone ring is formed via a chain-branching acetate unit. This is a very unusual feature although a number of metabolites (e.g. see the aurantinins below) contain methyl branches derived from the methyl carbon of a cleaved acetate unit. Since compounds without the methoxyl and epoxy groups have been isolated,149it is assumed that 0-methylation and epoxidation occurs after formation of the macrocyclic ring. The structure of notonesomycin A an antifungal antibiotic isolated from Streptomyces aminophilus has been shown to be (171). The structure elucidation was aided by l3C-l3C couplings observed after incorporation of [13Cz]-acetate.lS0The biosyn-thesis of oligomycin (172) from acetate propionate and butyrate has been demon~tratedl~l by incorporation of the appropriate [ 1-13C]-labelled precursors in cultures of an unnamed Streptornycete. Fungichromin (173) and filipin (174) isolated from Streptomyces cellulose are typical members of the polyene family of antibiotics. Incorporations of 13C-labelled acetate propionate and octanoate demonstrated the origin of the carbon skeleton as that shown in Scheme 53. Octanoate was incorporated intact with little observable degradation.152 13 Miscellaneous Metabolites Incorporation of 13C-labelled acetate and propionate estab-lishedlS3the origins of the polyene chain in manumycin (175) as shown in Scheme 54.Surprisingly13C-acetate was in-corporated into the m-C,N unit apparently via the Krebs cycle and succinate.This suggests a novel biosynthetic pathway for the C,N moiety. In many antibiotics such C,N units have been shown to be derived from a branch of the shikimate pathway and ultimately via 3-amino-5-hydroxybenzoicacid or 3-aminobenzoic acid. However neither were incorporated into manumycin or asukamycin (176). The incorporation of acetate into carbon 4-7 of asukamycin (176) suggested their origin from succinate.154 This was confirmed by feeding [ 1,4-13Cz]- 42-2 NATURAL PRODUCT REPORTS 1991 0 0 C02HH02Em Scheme 55 R’ H Me Me Me (177) R’ = H R2= N h M * 0 (178) R1= NH2 R2= H * .Me-C0,NaMethionineA 1- AMe AMe C02H Scheme 56 succinate. [U-13C]-glycerol enriched the remaining 3 carbons of the C N unit to demonstrate it formation via intermediates of the Krebs cycle and the triose (Scheme 55). A number of manumycin analogues have been isolated as a result of feeding aminobenzoic acids as alternative C,N starter units in Streptomyces parvu1~s.l~~ Feeding 3-aminobenzoic acid suppresses manumycin biosynthesis and results in accumulation of the analogue (177) 4-aminobenzoic acid induced the production of (178) which lacks the manumycin side chain. 2-Aminobenzoic acid was not incorporated. The incorporation of 3-aminobenzoic acid with no further functionalization suggests that the natural precursor is non-aromatic. The origins of the carbon skeleton of the aurantins e.g.(179) novel carbocyclic metabolites of Bacillus aurantinus have been determined by feeding 13C-labelled precursors. 15’ They contain five C-methyl groups from methionine two from the methyl of cleaved acetate units and a carboxyl derived from a cleaved acetate unit as indicated in Scheme 56. 14 References 1 T. J. Simpson Nut. Prod. Rep. 1987 4 339; 1985 2 321; 1984 1 28. 2 D. H. Sherman F. Malpardita M. J. Bibb H. M. Kieser S. E. Hallam J. A. Robinson S. Bergh N. Uhlen T. J. Simpson and D. A. Hopwood. In ‘Proceedings of the 8th International Biotech- nology Symposium ’,ed. G. Durand L. Bobichon and J. Florent Paris 1988 SocietC Francais de Microbiologie Vol 1 pp. 123-137. 3 P. M. Jordan J.€3. Spencer and D. L. Corina J. Chem. Soc. Chem. Commun. 1986 91 1. 4 S. Huang J. M. Beale P. G. Keller and H. G. Floss J. Am. Chem. SOC.,1986 108 1100. 5 A. D. McCarthy and D. G. Hardie TIBS 1984 9 60. 6 M. Schweizer L. M. Roberts H.-J. 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Chem. 1988 53 2660.

ISSN:0265-0568    

DOI:10.1039/NP9910800573

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

Tropane Alkaloids G. Fodor and R. Dharanipragada" Department of Chemistry West Virginia University Morganto wn WV 26506-6045 USA Reviewing the literature published between January 1990 and December 1990 (Continuing the coverage of literature in Natural Product Reports 1990 Vol. 7 p. 539) 1 Occurrence and Structure of New Alkaloids 2 Synthesis and Transformations of Natural Tropanes including Spectroscopic Studies 3 Synthesis of Tropane and Homotropane Derivatives 4 Pharmacology of the Main Tropane Bases and of their New Derivatives 4.1 Atropine 4.2 Cocaine 4.2.1 The Relationship between Cocaine Receptors Monoamine Transporters and Sodium Channels 4.2.2 The Interaction of Cocaine with Enzymes and Hormones 4.2.3 The Toxicity of Cocaine 4.2.4 Prenatal Cocaine Administration 4.2.5 Antagonists of Cocaine and Attempts to Cure Addiction 4.2.6 Cocaine Self-adminis tration 4.2.7 The Discriminative Stimulus Properties of Cocaine 4.3 Scopolamine 5 Analytical 6 References 1 Occurrence and Structure of New Alkaloids A very unusual alkaloid grahamine (1) has been isolated recently' from Schizanthus grahamii.Schizanthine was isolated earlier from the same plant. It is the bis-3a,6p-dihydroxytropanyl ester of angelic acid linked to mesaconic acid.2 The structure determination of this rather complex alkaloid has been achieved by an ingenious combination of mass spectrometry and one and two-dimensional NMR spectroscopy. Fast atom bombardment (FAB) mass spec-trometry showed that the molecular mass of grahamine is 871.The lH-broadband-decoupled 13C NMR spectrum indicated 46 signals two of which belonged to a monosubstituted benzene ring therefore 48 carbon atoms were assumed to be present. The DEPT subspectra were consistent with the presence of 7 methyl 9 methylene 22 methine and 10 quaternary carbon atoms. The sum of the mass of these groups was 637 versus the total mass of 871. The difference of 234 was accounted for by assuming 3 nitrogen and 12 oxygen atoms. The empirical formula then was deduced to be C,,H,,N,O,,. One and two- dimensional NMR methods revealed that all 61 H atoms were bound to carbons therefore no hydroxyl or secondary and primary amino groups can be present.The cross signals of an H,H COSY diagram revealed the presence of three 3,6-dihydroxytropane units also a methylcyclobutane partial structure based on typical CH coupling constants in the coupled NMR spectrum. The connectivity of all six carbonyl carbons was proven in the same way. All these and other data were consistent with the structure of two tropane rings being linked as mesaconic diester and that a 2-methyl-4-phenyl-cyclobutane1,2,3-tricarboxylic acid is at the centre of the molecule. The CH COLOC diagram revealed the * Present Address Department of Chemistry University of Arizona Tucson AZ 85721. USA. 603 N I CH3 0 f? FgL: N -CH/3 \/ 0 H CH3 (1) OH presence of an angelic ester unit.Combination of all these partial structures gave the structure of grahamin as (1). It was suggested that the cyclobutane ring was formed from mesaconic and truxillic acid esters via 2+2 cycloaddition in the plant by the action of sunlight. 6-Hydroxyhyoscyamine has been found in a Duboisia myoporoides-Datura leichardtii hybrid and in Hyoscyamus alb~s.~ 2 Synthesis and Transformations of Natural Tropanes including Spectroscopic Studies Baogongteng A was described in 1987 by Chinese author^,^ as 2p,6p-dihydroxynortropane (3). A synthesis has now been reported5 starting with 6p-acetoxytropinone (2). The absolute configuration of baogongteng A has been studied6 with the [Pr(dpm),] induced CD split Cotton effect method the exciton chirality method and the Horeau method see (3).Anisodamine that was claimed earlier' to be a 6p-hydroxyhyoscyamine has been erroneously formulated in a recent chemical abstract* as scopolamine. The paper itself8 describes a new 6-step synthesis of 6~-hydroxy-3a-tropoyloxytropanefrom 6p-methoxymethoxy-3-tropanone,via reduction to the 3,6-diol monomethoxymethoxy ether acylation by transesterification with formyl-phenylacetate reduction of the aldehyde-ester NATURAL PRODUCT REPORTS 1991 COOMe COOMe Of-Ph Flashthermolysisvacuum - MeN* + PhCOOH (4) VNMe retro-ene otn COOMe with sodium borohydride to the 6-methoxymethoxy-3-tropoyloxytropane and deblocking to give the 3-tropoyloxy-6-hydroxytropane. Unfortunately there is no indication of a resolution and so the product must be 6P-hydroxyatropine while the natural product is most likely the optically active 6p-hydroxyhyoscyamine.The structure of (-)-hyoscine hydrobromide hemihydrate was studied by X-ray techniquesg 20 years ago. A recent study of anhydrous hyoscine hydrobromide has been done by X-ray crystal1ographyl0. The S( -) absolute configuration" of the tropic acid moiety was confirmed by anomalous dispersion of the bromide atom.loThe N-methyl group is axial as it is in the hemihydrateg but the conformation of the tropic acid ester part resembles that in N-butylhyoscinium bromide while the same group of the anhydrous salt has conformations similar to that in hyoscyaminium bromide. A conformational studyI2 of cocaine (4) (a,e) and its three diastereoisomers pseudococaine (e,e) allococaine (a,a) and allopseudococaine (e,a) has been carried out using molecular mechanics and quantum mechanical semi-empiricaltechniques.These studies have confirmed previous NMR evidence that the N-methyl group is more frequently equatorial in cocaine and allococaine than in the C-2 equatorial isomers that the piperidine ring is in the chair form in all 4 diastereoisomers and that the conformation of the benzoyloxy substituent on C-3 is such that a minimal interaction of the tropane and phenyl rings is achieved. A very interesting study13 of the flash vacuum thermolysis of (-)-cocaine (4) has been carried out in view of the importance of a knowledge of the thermal behaviour of this drug.The products were benzoic acid N-methylpyrrole and methyl 3-butenoate. The mechanism was interpreted as leading first to methyl 3-tropene-2-carboxylate,'ecgonidine' (5) which in turn undergoes a retro-ene reaction to N-methyl-4,5-dihydropyrrole-2-butenoicester (6) and this by a second ene reaction gives the end-products. Photo-oxidation of tropinone and scopolamine occurs at the N-methyl group. This oxidation is sensitized by electron acceptors like 9,1O-dicyanoanthracene,and gives nortropinone and nor-scopolamine respectively besides the appropriate N-formyl nor-bases. Adding a salt e.g. lithium or magnesium perchlorate resulted in the quantitative formation of the secondary amines.l4 The conversion of cocaine into pseudo-cocaine has been ~imp1ified.l~ The 'H and I3CNMR spectra of oscine (in the German literature 'scopoline') has been published.l6 Oscine is a product of rearrangement of scop-olamine (hyoscine) that was interpreted independently by three groups" in 1953 as rearward nucleophilic attack by the 3a-hydroxyl in scopine upon the C-6(7) epoxide.Further direct QMe proof for the P-configuration of the C-7 hydroxyl group was lactone salt formationlg from oscine and iodoacetic ester (1954). Hence oscine is (+)3a,6a-oxido-7P-hydroxytropane which is in harmony with the present NMR data. 3 Synthesis of Tropane and Homotropane Derivatives During this period of review three new ingenious entrieslg,21 22 into the tropane field have been published. The first asym-metric19 1,3-dipolar cycloaddition20 of 1-methyl-3-oxidopyridinium and (R)-p-tolylvinylsulphoxide led to 6-tolylsulphinyl-3-tropen-2-one(7) ; reduction to toluene-sulphenyl-3-tropen-2-one(8) followed by hydrogenation to (9) and desulphurization gave optically active 2a-hydroxytropane (10).The second entry21 used rhodium acetate-stabilized vinylcarbenoids (12) with pyrroles (1 1) to give alkoxycarbonyl-2,6-tropadienes (13). The third approach22is based upon the reaction of 1,4-dibenzenesulphonylbutane(14) the dianion of which (15) with methallyl chloride produced a substituted a-methylene cycloalkane (16) that by ozonolysis gave a 1,4-bisbenzenesulphonyl cycloalkanone (17). Amine-induced elimination leads to an azabicyclo-alkanone ;e.g. from the 1,4-dianion of bis-benzenesulphonylbutane3-tropanone (18) was obtained ; from the a,o-disulphonyl pentane derivative pseudopelletierine (19a) was produced ;from an amyl derivative N-benzyl adaline (19b) was obtained.As a further extension23 a disulphone with a hydroxyl group had been converted into a trianion (20) which was reacted with dibromoisobutene to give the 6a-and 6P-anomers of N-benzyl-6-hydroxytropinone(21) upon benzylamine induced elimination of the sulphone groups. This third method has already proved adaptable22*23 to the synthesis of natural tropanes. The two previously quoted new approaches also hold the promise to be of more general value; e.g. the asymmetric synthesis of a 2-hydroxy-tropanelg may be relevant to the synthesis of a natural tropane baogengteng A ;4s the 2,6-tropadienes2' may prove potential intermediates for hyoscine (6,7-epoxytropanes) and the cocaines.Among the new synthetic approaches one may mention a new method of manufacturing tropane bases by plant tissue cultures.24 Amines sulphur containing amino acids giberellin and/or abscisic acid have enhanced the yields of scopolamine and atropine. Some new 3-aroylamine derivatives of tropane have been synthesized and tested.25 'Decarbonylated ' cocaines e.g. 3-aryl-3-desoxyecgonines (as 22) which act as inhibitors of cocaine binding and dopamine uptake have been obtained from cycloheptatriene carboxylic acids by Michael addition of methylamine or enantiomerically from natural cocaine.26 NATURAL PRODUCT REPORTS 1991-G.FODOR AND R. DHARANIPRAGADA jPB0 0 0 Tb (9) COOMe COOMe Rh2(OAc)4 MeN@ ~ ? +N;> H H COOMe 03 S02Ph HO A R* (19a) R' = Me R2 = H (19b) R' = H R2 = C5Hll COOMe OMe 4.1Et X-J=H,CH,-OCOCHPh OMe MeNF NiPo OMe HO Analogues of the ericybe alkal~id,~? 2P,6@-dihydroxy- nortropane namely the 2-deoxy derivative with an N-methyl group and a quaternary salt (23) have been prepared from 6P-acetoxytropan-3-one (2) in several steps. New N-acyloxyalkylnortropinones (as 24) have been synthe- sized2' by the Robinson condensation of acetonedicarboxylic acid with succindialdehyde and 2-aminoethanol. The etha- nolamine hydroxyl was acylated subsequently with diphenyl- acetic acid and its homologues in the presence of DCC.The X-ray diagram showed that the piperidine ring is flattened to NATURAL PRODUCT REPORTS 1991 "\ c=o [Me3H]+Brs MeN$ accommodate an axial acyloxyalkyl substituent on the ring nitrogen. There have been relatively few papers dealing with homotro- panes e.g. anatoxin and its derivatives. The Rapoport school has elaborated28 a method for the synthesis of anatoxinal and N-methylanatoxinic acid (29) starting with (+)-t-BOC anatoxin- a (25). Dimethyl-butyl-silylation leads to enolether (26) which upon oxidation with a peroxyacid gave the ketol(27). Periodate oxidation of (27) gave t-BOC anatoxinic acid (28). Hydrolysis afforded anatoxinic acid while Eschweiler methylation lead to N-methylanatoxinic acid (29). On the other hand (28) was converted via the acid chloride into the isoxazolidide (30),and this in turn was hydrogenolized with LAH to t-BOC-anatoxinal (31).(+) Anatoxin has been obtained by classical res01ution~~ of (_+ )-N-methylazabicyclo[2.4.llnonane (33). The latter was prepared by an improved procedure (rearrangement and oxidation) of 9-methyl-9-aza-bicyclo[3.3. llnonane (32) by using trimethylammonium perbromide hydrobromide instead of pyridinium perbr~mide.~~ The concluding steps of the anatoxin synthesis followed Stjernloefs previously described synthesis.31 4 Pharmacology of the Main Tropane Bases and of their New Derivatives 4.1 Atropine Although there are a large number of publications in this area there is no main theme or focus.The effect of intravenous atropine on gastric emptying has been Presynaptic effects of atropine have been e~tablished.~~ Pre-treatment of MCPBA ____) HF i.'. COOH COOH -(+)-Anatoxin asthmatics with atropine sulphate affected adenosine-induced bronchoconstriction in humans.34 Atropine affects the p-carotene-induced cytoprotection in Liposomal ophtalmic drug delivery has been studied revealing pharmacodynamics and biodisposition of High potassium causes atropine-resistant relaxation in the iris dilator muscle of rat and pig3' Effects of atropine on visual performance has been e~tablished.~~ Atropine-induced antinociception has been stud- ied.39 The mutagenicity of atropine in Salmonella typhimurium has been explained.40 A subchronic toxicity study of inhaled aerosolized atropine sulphate formulations has been carried Cholinergic supersensitivity in the rat salivary gland caused by atropine has been ~bserved.~~,~~ A review of atropine and scopolamine-resistant subtypes of muscarinic receptors in the rabbit aorta has been published.44 Atropine-resistant transmission in partially depolarized rat urinary bladder has been described.45 Combination of arecoline with atropine affects mouse motor Atropine inhibits nicotinic acetylcholine response in isolated frog neuron^.^' Behavioural teratogenesis of neuroactive drugs has been studied in prenatal and postnatal The effect of atropine upon glucose metabolism has been e~tablished.~~ It has been observed that cold exposure decreases the effectiveness of atropine treatment in organophosphate intoxication in rats and mice.5o Atropine diazepam and physostigmine have a thermoregulatory effect in the heat-stressed rat.51 Atropine-induced cutaneous vaso-dilation decreases oesophageal temperature during exercise ;52 atropine also adjuncts to physostigmine in exercizing rats.53 Atropine has a short term effect on schedule-controlled behaviour in rats.54 The toxicology of Datura stramonium seed containing atropine has been evaluated.55 A brief review of NATURAL PRODUCT REPORTS 1991-G.FODOR AND R. DHARANIPRAGADA atropine effects on performance and physiology has been published.56 Chiral separations of atropine and homatropine on an a-1-acid glycoprotein-bonded stationary phase has been achieved.57 4.2 Cocaine This year there has been an ever-increasing interest in the pharmacology and biochemistry of cocaine.The main focus of these investigations were receptor studies interaction of cocaine with dopamine and serotonine and with hormones ;the toxicity of cocaine upon liver the cardiac system and the foetus ;effect upon self-administration ;to find new antagonists etc. We have tried to select the most important review articles and papers accordingly. First the reviews cocaine pharmacokinetics and biotrans- formation in man;5s on the respiratory effects of smoking cocaine ;5g recent findings on cocaine addiction from animal studies psychomotor versus local anaesthetic effects ;61 de-velopmental effects ;62 tight binding dopamine re-uptake inhib- itors as cocaine antagonists ;63 high affinity dopamine re-uptake inhibitors as potential cocaine antagonists ;64 mechanism of cocaine abuse and toxicity ;65 pharmacological effects of cocaine relevant to its abuse;66 on the effects of cocaine on local cerebral metabolic activity;67 modulation of cocaine receptors ;68 reinforcement pathways for cocaine ;6g neuro-behavioural pharmacology of cocaine,7o and neurological consequences of cocaine use.71' 72 The individual papers that are concerned with receptors and address mapping cocaine binding sites in human brain,73 binding to sodium channels,74* 75 labelling with 3H cocaine in v~vo,~~ determination of benzodiazepine receptor densities as modified by chronic cocaine,77 finding cocaine 78 binding sites related to drug self-admini~tration,~~ design of ligands for the cocaine receptor,80.81 cocaine inhibition of the acetylcholine receptor and characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles,82 chronic cocaine treatment influencing six protein subunits in discrete regions of the brain;83 the P-endorphin-specificE receptor could be found by the affinity of the drug U-69 593;84 chronic cocaine administration has an effect on brain-neurotransmitter receptors behavioural and biochemical evidence has been provided for cocaine rebound in rats,86 radioiodinated tropeines have been used to establish the biodistribution and hence the molecular probe for the characterization of the cocaine receptor ;87 chronic treatment with dopamine active drugs did not alter 3H-GBR- 12395 binding ;89 5-hydroxytryptamine receptor antagonists atten-uated cocaine-induced locomotion in mice,9o the cocaine sensitive catecholamine transporter has been covalently labelled.91 4.2.1 The Relationship between Cocaine Receptors Monoamine Transporters and Sodium Channels This relationship has been studied by Reith.92 Cocaine has an effect on norepinephrine stimulated phosphoinositide hydroly- sis and locomotor activity in rats.93 Acute and chronic cocaine administration has an effect on somatostatin level and binding in the rat brain.g4 Also cocaine administration influences the cholinergic enzyme levels of certain regions of the rat brain.95 The interaction of cocaine with central serotonergic neuronal systems has been ~tudied.~~-~~ In the very centre of interest is the interaction of cocaine with dopamine and the dopaminergic system100-120 since cocaine is a dopamine uptake inhibi-tor.106,107 4.2.2 The Interaction of Cocaine with Enzymes and Hormones This is illustrated by the release of prolactin in rat~,l~l-l~~ the influence on the cerebral ornithine decarboxylase and on other hormones ;124-127 testosterone influences the effect of 607 cocaine.124 The metabolism of cocaine has been studied ;129* 130 four unreported metabolites [ecgonidine (5) norecgonidine methyl ester norecgonine methylester and m-hydroxybenzoyl- ecgonine] have been found in urine.131 Rapid stereoselective hydrolysis of (+)-cocaine in plasma prevents its uptake in the brain.132 4.2.3 The Toxicity of Cocaine This was the subject of a large number of paper~.l~~-l~~ Th e clinical pharmacology and toxicity received close attention.138 Acute cocaine toxicity means antagonism by agents interacting with adrenoceptors. 134 Biodisposition plays a role in acute toxo~ity.'~~ The most fearful aspect of cocaine abuse is cardiovascular toxicity which attracted special intere~t.'~~-'~~ The hepatotoxicity of cocaine was known but many new data have confirmed previous fears. 153-158 The carcinogenic potential of cocaine has been established.159 However there is an antagonism of toxic effects on the dopamine and serotonin neurons in the brain.160.161 Cocaine-induced toxicity showed an anticonvulsant modification ;Is2 its lethal effects are reduced by the dopamine antagonist SCH 23390;163 a similar effect was attributed to cholinomimetics. 164 Cocaine tolerance165 in a gunshot wound fatality was reported. 4.2.4 Prenatal Cocaine Administration Such administration has several ill effects pregnancy increases cardiovascular toxicity.166 There are neurobehavioural and immunological effects,167 cocaine induces embryopathy in mice;ls8 the receptors in the placenta are down regulated;169 cocaine is the cause of congenital malformation^^^^ and gestation mortality and postnatal maturation are affec-ted171-173 ; uterine vascular clamping and other teratogenetic effects were 175 183 the effect on brain myelin development was demonstrated ;176* 177 cocaine prenatally affects prostacyclin neurobehavioural sexual differen- tiati~n,~~~.180 the a-receptor is blocked,lsl the developmental and neurological indices are harmed,lE2 cocaine-induced arrhytmia in the human foetus causes foetal death by affecting the myocardium;184 the cocaine binding site in the placenta was detected;Is5 locomotion and stereotype show however no long-term effects in rats;la6 genetic differences in response to cocaine were indicated. lE7Noradrenergic mechanisms are not involved in cocaine-induced lethality. 188 4.2.5 Antagonists of Cocaine and Attempts to Cure Addiction Several compounds have been tried amino acids,lsg bromo- criptine;lgO magnesium seems to ameliorate damage in the rat brain ;lg19 lg2ondansetron inhibits a behavioural consequence of withdrawing from cocaine abuse;lg3 lg4the drug SCH 23390 has an effect against toxic doses of cocaine;lg5 new potent cocaine analogues2? influence ligand binding in rat ;lg6carbama-zepine proved useful against cocaine addiction ;lg7 5-aminocarbonyl[5H]-dibenzo[a,d]cyclohepten-5,10 imines were used to treat both epilepsy and cocaine addition;lg8 similarly azapirone compounds were applied against cocaine addic- tion;lg9 nimodipine interacts with cocaine in the squirrel monkey ;200 local anaesthetics have been applied in the treatment of cocaine 202 ketotifen influences anxiety and the behavioural consequences of withdrawing from treatment with cocaine ;203 postcocaine depression has been treated.204 4.2.6 Cocaine Sew-adm in is trat ion This has been the subject of several papers :205-215 Intracranial self-admini~tration,~~~ self-administration in pigeons,2o6 reduc- tion of i.v. self-administration by dietary tryptophanzo7 and by bromocriptine ;20s 209 the failure of magnesium to maintain self- administration in cocaine-naive rats ;210 the dopamine receptor antagonist SCH 23390 increases cocaine self-administration in rats ;211 buprenorphine 212 naltrexone,213 chlordiazepoxide214 and the antiserotonin agent fluoxetine2I5 all alter cocaine self- administration. Autonomic actions216 of cocaine and its effect on neurons has been reported.Cocaine acts upon the endocrine and nervous 218 upon protein biosynthesi~,~'~ on medial bulbore- ticular neurons,22o on central monoamine neurons,221 on neurons in the thalamus,222 and on spinal projection neurons in the rat.223 4.2.7 The Discriminative Stimulus Properties of Cocaine These properties have been studied :224* 225 the directional running of mice is affected by cocaine;226 it has an effect on the limbic dynorphin has acute and chronic effects on isolation-induced aggression of mice,228 cocaine affects loco- motor activity of rats;229 it inhibits the baroreflex control of blood pressure;23o the effect of cocaine and morphine on vision has been Actions of cocaine are potentiated by caffeine232 or by A correlation was established between cocaine-induced locomotion in the brain and cocaine disposition in mice.234 Cocaine facilitates prefrontal cortex stimulation ;235 it produces cholinergically mediated analeptic arousal effects in rabbits and rats;236 orally delivered cocaine is a reinforcer for rhesus monkeys;237 the level of arousal affects vigilance task performance.238 Cocaine influences sustained and selective attention in rats ;239 causes important behavioural effects;240 food intake,241 and 243 in general are affected by cocaine morphine and heroin. Cocaine has an effect on the body temperature of rat^.^^^,^^^ A method has been devised for delivery of precise doses of smoked cocaine-base to Finally a positive effect :cocaine enhances memory storage in mice.247 4.3 Scopolamine Most of the papers in this area are devoted to the effect of scopolamine on memory artificial amnesia and some on the binding of scopolamine to muscarinic receptors.It has been reiterated that scopolamine affects memory language visuo- spatial praxis and psychomotor Scopolamine and sustained retrieval from semantic memory has been studied. 249 The effect of scopolamine on the working memory in rats was investigated by fixed-ratio size ob~ervation.~~~~ 259 Diver per- formance upon transcutaneous scopolamine has been mea- ~ured.~~~ Scopolamine differentially disrupts the behaviour of male and female Wistar Elevated plus-maze was used for evaluating memory of mice upon treatment with nootropics scopolamine and electro-convulsive shock.253 The effect of imipramine was studied in the 'learned helplessness ' model of depression in rats by using The impact of scopolamine on antipredator defence reactions in wild and laboratory rats has been Microcomputer- based performance tests were used to differentiate between scop- olamine and amphetamine.256 TRH was found to attenuate scopolamine-induced memory impairment in humans. 257 The long-range effect of transdermal scopolamine has been investi- gated.258 The effects of scopolamine on proactive interference has been established.260 Scopolamine affects the cognitive processes involved in selective object exploration.26' The drug WEB 1881 FU has been used in the three-panel runway task it improved the impairment of working memory induced by scopolamine ;262 and the same applies to mina~rine~~~ inb bur nine.^^^ The comparative study on explicit memory and repetition priming was carried out with scopolamine trimipramine and diazepam in humans.265 Scopolamine causes lesion of the nucleus basalis in the rat brain cortex.266 In addition to the memory-amnesia studies some work has been done on the behavioural effects of scopolamine.2679 268 The efficacy of physostigmin pre-treatment in combination with scopolamine against soman challenge was investigated.269 The NATURAL PRODUCT REPORTS 1991 pH dependence of the binding of scopolamine and N-methyl- scopolamine to muscarinic receptors was 271 The influence of non-radioactive ligand~~~~ on kinetics of N-methyl-3H-scopolamine binding to the muscarinic receptor has been determined.5 Analytical New techniques in two-dimensional data processing have been Fourier-transform Raman and surface enhanced Raman-spectro~copy~~~ have been applied to illicit drug testing. Double-capture immunoassay and capillary flow device have been developed276 for cocaine. The KDI Quik Test has been applied in urine Scopolamine radioimmunoassay has been used for analyzing Datura tissue cultures.278 Indirect atomic absorption spectrometric deter- mination of alkaloids has been used with an air-acetylene flame.279 HPLC alone or in combination with MS has been applied the electrospray interface was investigated. 280 Gas-chromatography- MS was used for the detection of scopolamine in rat plasma and brain.281 HPLC-EC analysis of hyoscine in urine282 has been carried out.Atropine in human plasma has been determined by HPLC.283 Reverse-phase ion-pair HPLC separation has been carried out on Chinese solanaceous Retention reproducibility of basic drugs has been achieved by HPLC on a silica column.285 Capillary column gas chromatography has been used for the identification of cocaine286 and other underivatized Alkaloidal narcotics have been determined by pyrolysis-gas chromat- ography.288 TLC has been applied in biological matrix,289 in horse plasma,29o in drug development. 291 Tropane and other alkaloids have been resolved on silica layers impregnated with metal Laboratory robotics and capillary gas chromatography were used in the analysis of cocaine.293 Gestational cocaine has been determined by hair analysis.294 Cocaine concentrations in plasma have been determined by HPLC.Graphical295 analysis of reversible radioligand binding has been applied to [N-W- methyl]-cocaine in humans.296 Microiontophoresis has been applied to cocaine in biological Cocaine and other local anaesthetics have been determined by derivative spectro- photometry.298 Stable isotope-labelled analogues of cocaine have been determined by mass spectrometry. 299 6 References I R. 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ISSN:0265-0568    

DOI:10.1039/NP9910800603

出版商:RSC    

年代:1991

数据来源: RSC