摘要:

ISSN 0265-0568 NPRRDF 8(5) 441-526 (1991) Natural Product Reports A journal of current developments in bio -organic chemistry Volume 8 Number 5 CONTENTS 44 I Biosynthesis of C,-C, Terpenoid Compounds M. H. Beale Reviewing the literature published during 1989 455 The Lycopodium Alkaloids W. A. Ayer Reviewing the literature published between January 1986 and October 1990 465 Marine Sterols R. G. Kerr and B. J. Baker Reviewing the literature published to July 1990 499 Diterpenoid Alkaloids M. S. Yunusov Reviewing the literature published between the middle of 1985 and the end of 1989 31 NPR 8 Cumulative Contents of Volume 8 Number 1 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 lo 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 415 Withanolides and Related Ergostane-type Steroids E. Glotter Articles that will appear in forthcoming issues include A Unified Mechanistic View of Oxidative Reactions Catalysed by P-450 and Related Fe-Containing Enzymes M. Akhtar and J. N.Wright Pyrrole Pyrrolidine Piperidine Pyridine and Azepine Alkaloids (July 1989 to June 1990) A. R. Pinder Tropane Alkaloids (1990) G. Fodor and R. Dharanipragada The Biosynthesis of Polyketides (January 1986 to December 1988) T. J. Simpson Indolizine and Quinolizidine Alkaloids (July 1989 to June 1990) J. P. Michael 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

ISSN:0265-0568    

DOI:10.1039/NP99108FP011

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 Cambridge Dr J. R. Hanson University of Sussex Dr R. B. Herbert U n iversity of Leeds Professor J. Mann University of Reading Dr D. A. Whiting University of Notting ham 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 1HN 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 11 431 -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. f198.00 Overseas f228.00 U.S.A. US $467.00 Subscription rates for back issues are U.K. (1986)f 130.00 (1987)f 142.00 (1988)f 159.00 (1989)f 1 69.00 (1990)f 177.00 Overseas f 143.00 f 159.00 f 183.00 f 1 94.00 f 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/NP99108FX017

出版商:RSC    

年代:1991

数据来源: RSC

ISSN:0265-0568    

DOI:10.1039/NP99108BX019

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

Biosynthesis of C,-C, Terpenoid Compounds M. H. Beale Department of Agricultural Sciences University of Bristol AFRC Institute of Arable Crops Research Long Ashton Research Station Long Ashton Bristol BS 18 9AF UK Reviewing the literature published during 1989 (Continuing the coverage in Natural Product Reports 1990 Vol. 7 p. 387) 1 Introduction 2 Mevalonic Acid 3 Hemiterpenoids 4 Prenyltransferases 5 Monoterpenoids 6 Sesquiterpenes 7 Diterpenoids 8 Mode of Action 9 References I Introduction The format of this review is the same as that of last year’s. Over the past few years the impressive progress made in the area of mevalonate biosynthesis using molecular biological techniques has been discussed. This year we have seen the first reports of the cloning of genes encoding some of the later enzymes of terpenoid biosynthesis.It is interesting to note that of the four enzymes successfully cloned this year two of them (IPP :DMAPP isomerase and FPP synthase) were identified using oligonucleotide probes based on amino acid sequence obtained from purified enzyme whilst the other two (trichodiene synthase and juvenile hormone esterase) were acquired by screening of expression libraries with antibodies raised to enzymes. With larger amounts of purified enzymes now being made available by over expression techniques researchers are now able to apply affinity labelling and even NMR techniques to the study of the active sites and the enzyme mechanisms. It is not inconceivable that in the next few years we will see reports of the first crystal structure of a terpenoid biosynthetic enzyme.2 Mevalonic Acid New syntheses of chiral mevalonate (MVL) reported this year involve enzymic hydrolysis of esters of racemic 4,6-dihydroxy-4-methyl-hexenel or direct synthesis of both unlabelled and [2R-2H]MVL from geraniol.2 The two most widely studied enzymes of mevalonate biosynthesis are 3-hydroxy-3-methyl glutaryl-coenzyme A synthase (HMG-CoA synthase; EC 4.1.3.5) and HMG-CoA reductase (NADPH) (EC 1.1.1.34) which carry out the conversions shown in Scheme 1. These enzymes are considered to be control points in mammalian cholesterol biosynthesis and their activities are modulated at transcriptional translational and enzyme levels.This year’s papers are discussed in these terms after review of new work on enzyme structure and mechanism. The genes encoding these enzymes in mammalian and yeast systems have been well characterized. HMG-CoA reductase in hamster (871 amino acids (aa)) and yeast (1050 aa) consist of an amino-terminal region containing seven hydrophobic membrane-spanning domains and fairly homologous carboxy-terminal regions where the catalytic site is situated. Rajkovic et al.3 have now reported the cloning and sequencing of HMG-CoA reductase from the human parasite Schistosoma mansonii. Again the enzyme (948 aa) consists of the seven trans-membrane OH CH3CO-SCoA I L + “ZH CH, CH~COCH~CO-SCOA OZC\ Co2H C\H2 C02H SCoA OH YI I Enzymes i HMG-CoA synthase; ii HMG-CoA reductase Scheme 1 domains and a catalytically active carboxy terminus with ca.50 % homology with enzyme from other species. However the situation with HMG-CoA reductase from the plant Arabidopsis thaliana is different and two groups have in-dependently reported the characterization of the gene from this source. Caelles et al.4 and Learned and Fink5 report identical sequences for the gene. The deduced sequence of 592 amino acids revealed that enzyme from this source is smaller than that previously described. The truncation occurs in the amino-terminal domain while the carboxy-terminal shows high homology with known enzymes. The two groups differ in their interpretation of hydropathy profiles of the Arabidopsis enzyme with one5 reporting a single trans-membrane domain (residues 86-118) while the other4 postulates two such regions (47-69 and 83-117).However even though plant HMG-CoA re-ductase is thought to be a membrane-bound enzyme it is obvious that its interaction with the membrane is less complex than that of mammalian or yeast proteins. In contrast the sequence of HMG-CoA reductase from Pseudomonas mevalonii also reported this year,6revealed a shorter (428 aa) protein with no membrane spanning domains. Homology in the carboxy-terminal region was not high although there were some similarities with the above enzymes suggesting a related function. In an investigation of the role of thiol groups in the HMG-CoA reductase catalytic cycle Jordan-Starck and Rodwell’ carried out chemical modification of Pseudomonas mevalonii enzyme overexpressed in E.coli. They concluded that a sulphydryl group which was protected from modification by HMG-CoA may not be a catalytic residue but was involved in a conformational change. Indeed site-directed mutagenesis of cysteine residues (Cys 156 and 296) to alanine yielded* mutant enzymes which were catalytically fully active with no major changes in substrate affinity. Mutant C156A and the double mutant C156A ;C296A were not affected by sulphydryl specific reagents. However mutant C296A was inactivated to the same extent as wild type on treatment with these reagents. These results indicated that Cys 156 was not catalytic but was nevertheless important for structural stability.Roitelmann and Shechter9 have also shown in rat liver enzymes that bound HMG-CoA protects a thiol residue from reaction with iodoacetamide. They also report on the inhibition of enzyme by a series of CoA esters of mono-and dicarboxylic acids of different chain length and conclude that the thiol residue in the enzyme was protected by the CoA moiety of substrate. The inhibition kinetics observed were consistent with a hydrophobic non-catalytic site which has some conformational function. Indeed Dugan’’ has presented results indicating that the 44 1 31-2 potent inhibitor of HMG-CoA reductase mevinolin does not compete for the substrate binding site but may also exert some conformational change thereby decreasing activity.This conclusion was derived from the observation that irreversible inactivation by the active-site-directed affinity label (S)-4- bromo-2,3-dioxo-butyryl-CoA was prevented by substrate but not by mevinolin at concentrations where it should compete effectively for substrate binding site. In previous reviews efforts to understand the regulation of expression of the genes for HMG-CoA synthase and reductase in mammalian and yeast systems have been reported. Here transcription of these genes is repressed by oxysteroids by a mechanism believed to involve a protein binding to the promoter regions. Wang et reported results indicating that transcription of HMG-CoA reductase gene in Pseudomonas mevalonii is induced by mevalonate. This is perhaps not surprizing as this organism uses mevalonate as a carbon source with the enzyme operating in the mevalonate -+ HMG-CoA direction.Further evidence that the repression of HMG-CoA- reductase gene by oxysterols involves synthesis of proteins has been presented. Trzaskos et a/.12 report that 25-hydroxy-cholesterol suppresses transcription of this gene in hamster cells and that this effect is blocked (and can be reversed) by the addition of the protein synthesis inhibitor cycloheximide. Co- ordinate down-regulation of HMG-CoA synthase and re-ductase as well as farnesyl diphosphate synthase by 25-hydroxycholesterol at the transcriptional level has been demonstrated in human hepatoma HepG2 ~e1ls.I~ Molowa and Cimis14 report similar co-ordination of transcription of re-ductase and low-density-lipoprotein receptor 7 (LDL-R) genes in the same system.A co-ordinate increase in transcription of HMG-CoA reductase and LDL-R genes in phorbol ester induced macrophage differentiation has also been noted.I5 It was suggested that a negative regulatory protein was inactivated by protein kinase C thereby allowing increased gene tran- scription. In yeast two genes HMGl and HMG2 encode HMG-CoA reductase. Thorsness et a/.16have shown that in the presence of heme expression of HMGl is stimulated whereas HMG2 is repressed. This effect is not uncommon for pairs of genes which encode isozymes. The construction of hybrid genes containing promoter regions of interest fused to reporter genes whose products are easily measured and integration of them into a host genome is a common technique in the study of gene expression.In this area I have reported previously on the use of HMG-CoA reductase promoter / chloramphenicol acetyltransferase constructs. Abrams and Schimke17 have prepared constructs of HMG-CoA reductase promoter fused in both orientations to dihydofolate reductase (DHFR) genes. When transfected into hamster cells DHFR was produced regardless of the orientation of the HMG-CoA reductase promoter. However sterol induced suppression was only observed for properly orientated promoters. As no evidence for this effect could be found for endogenous HMG- CoA reductase promoters present in the cells it was concluded that the flanking regions control the direction of transcription as these are different for constructs and endogenous promoters.Post-transcriptional regulation of HMG-CoA reductase is observed mainly at the enzyme level although this year there is a publicationls reporting an interplay between glucorticoids and thyroid hormones on the stability of HMG-CoA reductase mRNA. One mechanism of modulation of enzyme activity is by phosphorylation and Parker et a/.19 have described the phosphorylation of native HMG-CoA reductase of rat. Treatment of rat hepatocytes with mevalonate resulted in an increase in phosphorylation of whole (97 kDa) enzyme. Proteolysis of the whole enzyme after phosphorylation with [32P]ATP in an in vitro system gave a labelled 56 kDa fragment and an unlabelled 52 kDa protein suggesting phosphorylation occurred at the region joining the membrane spanning and soluble catalytic domains.However von Gunten and SinenskyZ0 argue against phosphorylation as a control mech- anism but postulate the presence of an enzyme binding protein. NATURAL PRODUCT REPORTS 1991 The same group2' also have looked at the magnitude and specificity of mevalonate dependent regulation of both HMG- CoA synthase and reductase in hamster cells. The medicinal chemistry of the potent inhibitors of HMG- CoA reductase compactin and mevinolin continues and papers this year report the synthesis and activities of fluorinated analogues containing nitrogen heterocycle^^^^^^ and 4-0x0- quinoline rings.24 Rawsonol a brominated diphenylmethane from green algae has also been to be inhibitory toward this enzyme although its activity was circa one thousandth of that of mevinolin.3 Hemiterpenoids Mevalonate-5-diphosphate decarboxylase [EC 4.1 .1 .33] cata- lyses the third step in the conversion of mevalonate to isopentenyl diphosphate (IPP). Two further papers on the mechanism of this enzymic reaction have been published by the Jabalquinto and Cardemil group. In the first,26 purified enzyme from chicken liver was shown to be inactivated by methyl- methane thiosulpha te and by 5,5'-di thio bis(2-ni tro benzoa te) which are both sulphydryl specific reagents. Substrate or ATP protected the enzyme against these reagents. Further experi- ments with dithiol-specific reagents (e.g. phenylarsine oxide) led to the conclusion that the enzyme contains vicinal sulphydryl groups that are involved in substrate binding.In a second paper27 the effects of competitive inhibitors on substrate and ATP binding was examined. It was concluded that mevalonate- 5-diphosphate was the first molecule to bind to the active site and that the diphosphate group is an important functionality in that binding. Activities of this enzyme as well as those of the preceding enzymes mevalonate kinase and mevalonate phosphokinase have been examined in seedlings of the groundnut Arachis hypogaea. 28 Anderson et al.29 have reported the isolation of the gene encoding isopentenyl diphosphate dimethylallyl diphosphate isomerase the next enzyme of the terpenoid pathway.Enzyme was purified from yeast and N-terminal sequenced. This allowed the construction of oligonucleotide probes which were used to isolate the gene from a h library of yeast genomic DNA. The gene was sequenced and encodes a 288 amino acid (33.35 kDa) protein whose deduced sequenced matched the N-terminal sequence. A further paper from this examines the mechanism of irreversible inactivation of this enzyme by 3- (fluoromethyl)-3-butenyl diphosphate (1). Using IH,13C and 19FNMR techniques and 13C labelled (1) they concluded that the mechanism of inactivation of this enzyme by (1) is by S,2 displacement of fluoride ion as shown in Scheme 2. 4 Prenyltransferases The prenyl transfer reaction is central to isoprenoid bio-synthesis providing the different length prenyl-diphosphate precursors for all the terpenoid families from mono- terpenoids to natural rubber.Most prenyltransferases appear to be non- specific and will catalyse the condensation of dimethyallyl disphosphate (DMAPP) with various numbers of isopentenyl diphosphate (IPP) molecules. However last year the work of Heide who described the partial purification of a specific geranyl-diphosphate synthase which would only couple IPP with DMAPP was reported. Further details of this work have now been p~blished.~' This soluble enzyme from cell cultures Enz-X Scheme 2 NATURAL PRODUCT REPORTS 1991-M. H. BEALE of Lithospermum erythrorhizon has a molecular weight of 73 kDa and a requirement for Mg2+ or Mn2+. Geranyl diphosphate (GPP) (K 79 pM VmaX, 0.17 pmol/min/mg) was able to bind to the enzyme competitively but reacted at a much lower rate than DMAPP (K 83 pM VmaX,9.4,umol/min/mg).This was exploited3 in an enzymatic synthesis of [14C]-GPP from [14C]-IPP. Croteau and P~rkett~~ also describe this year a chain-length specific GPP synthase which separated from a mixture of prenyl transferase activities in a cell-free homogenate of sage (Salvia oficinalis). This enzyme had a molecular weight of circa 100 kDa with metal ion requirements and kinetic parameters (K IPP = 7.3 pM DMAPP = 5.6 pM) expected for a prenyl transferase and was inactivated by thiol specific reagents. It was shown to be localized in leaf epidermal glands which are the sites of monoterpene biosynthesis and thus appears to have evolved a specialist role providing GPP for monoterpene cyclases rather than for further prenyltransferase reactions.There are several papers this year describing the cloning sequence and expression of farnesyl diphosphate (FPP) synthase. Anderson et al.34 have isolated the gene for this enzyme from yeast using an oligonucleotide probe based on NH,-terminal sequence of purified enzyme. The amino acid sequence deduced from the gene shows this enzyme to be a 40 kDa protein of 342 amino acids with good homology with previous sequence data from affinity labelling and also a putative clone for rat liver enzyme. Sheares et ~71.~~ also report homologous sequence data for an almost full length cDNA encoding FPP synthase in human liver.They also demonstrated that this gene is transcriptionally regulated by MVL low density lipoproteins and 25-hydroxycholestero1 in the same way as HMG-CoA reductase. Ashby and have confirmed a previously known rat cDNA as a FPP synthase and again demonstrates regulation of its expression in co-ordination with HMG-CoA synthase and reductase genes. A group at Genetech have published a series of papers on a prenyltransferase that elongates the polyisoprene chains of rubber. In the first3’ they describe the isolation of this enzyme from latex of the rubber tree Hevea brasiliensis. This enzyme is a dimer of monomeric mass 30 kDa requires Mg2+ and will catalyse the polymerization of [1-14C]-IPP onto rubber particles containing polyisoprene chains of 720 kDa.These additions occurred to form cis-polyisoprene chains. Conversely reaction with DMAPP in the absence of rubber primers yielded GPP and FPP in the normal trans-configuration. Further investigation^^^ of this prenyltransferase were made with [14C 2S-3H]-IPP as substrate. When [3H]-DMAPP was the co-substrate tritium label was retained in FPP confirming the normal trans-addition process. However when rubber particles were the co-substrate 3H was not retained in the polymeric product indicating cis-addition. Apparently the prenyl trans- ferase has the ability to switch stereochemistry of proton removal from IPP dependent on the substrate. The authors attributed this to a protein termed rubber elongation factor (REF) tightly bound to the rubber particles used as substrate.Further papers rep~rt~~.~~ on the nature of this protein. It is solubilized by detergent treatment of rubber particles. Rubber treated in this way no longer acted as substrate for the prenyltransferase. Unfortunately the authors were unable to reinstate prenyltransfer by adding back REF to depleted particles. Thus evidence for the involvement of this protein in both turning on prenyltransferase and causing a stereochemical switch remains circumstantial. Working with extracts of guayale (Parthenium argentatum) Madhavan et ~21.~’ have investigated the properties of a polyprenyltransferase which forms rubber chains of MW lo3-lo’ with particular reference to its increased activity in cold-treated plants.Appleton and van Staden42 have also discussed the effects of temperature on this polymerization in this species. Yoshida et have demonstrated that a hexaprenyl-diphosphate synthase from Micrococcus lutens consists of two separable components. Neither component A (24 kDa) or B (27 kDa) which were separated by gel filtration had catalytic activity alone but when mixed they efficiently converted FPP and IPP into hexaprenyl chains. Further gel filtration experi- ments with components A +B in the presence of FPP and IPP yielded a single peak at 50 kDa consistent with a ternary complex of A +B +X where X is IPP FPP or even inorganic diphosphate. Thus it is the removal of substrate which causes the complex to dissociate.The same research group has been using prenyltransferases to carry out enantioselective synthetic reactions on artificial substrates and have rep~rted~~.~~ success with an undecaprenyl diphosphate synthase from Bacillus subtilis a nonaprenyldiphosphate synthase from Micrococcus lutens and a FPP synthase from rat liver. The screening of temperature-sensitive mutants of E. coli for defects in isoprenoid biosynthesis led Fujisaka et to a FPP synthase mutant. Sherman et ~1.~’ have also characterized E. coli mutants for FPP synthase as well as higher prenyl transferase and IPP isomerase activities. 5 Monoterpenoids The chemical synthesis of [4-2H]- and [5-2H2]-geranyl di- phosphates have been described.48 Croteau et al. have published further details of the monoterpene cyclases of sage.This species contains two separable enzyme activities which catalyse the cyclization of geranyl diphosphate (GPP) to (+)a-pinene (pinene cyclase I) or to (-)P-pinene (pinene cyclase 11) as shown in Scheme 3. These enzymes operate via an initial isomerization to the enantiomeric (-)(3R)- and (+)(3S)-linalyl disphosphates (LPPs) followed by the mirror image cyclizations as shown. The mechanisms call for the retention of con-figuration at C-1 of GPP and the first paper from this group experiments to test this. (1 R)-[2-14C 1 :3H]- and (1 S)-[2-l4C 1-3H]-GPP were prepared and incubated with these enzymes to give (+)a-and (-)P-pinene with no loss of tritium in all cases. The tritium location in the products was determined by chemical conversion to the corresponding enantiomers of camphor.Exchange curves for the base catalysed removal of 3H from the labelled camphor where the exolendo hydrogen exchange rates are very different (21 :l) were plotted. This revealed the positions of the 3H atoms in camphor from each incubation and the results confirmed that the configuration at C-1 of GPP is retained in the enzymatic isomerization/ cyclization reactions. Feeds of (3R)- 12-[ 1-3H]-LPP and (3s)- 1Z[l-3H]-LPP to cyclase I and 11 respectively followed by chemical degradation as before confirmed when considered with the GPP results the syn stereochemistry in the initial isomerization. Finally (I R)-[2-14C 1 -3H]-neryl diphosphate (NPP) the cis isomer of GPP was fed and the results indicated a net inversion of configuration at C-1 for this substrate.This is predicted since in this case cyclization does not require the C-2-C-3 rotation in the intermediate LPPs. Although these enzymes accept GPP NPP or LPP as substrates and convert them to the appropriate pinene (and also camphenes) they also produce verying amounts of monocyclic (limonene and ter- pinolene) and acyclic (myrcene) monoterpenes. The amounts of these products are different according to the substrate with exogenous NPP and LPP yielding much more than the natural substrate GPP. Croteau and Satterwhiteso have now addressed this problem using methods that include the resolution of limonene the only chiral product of these ‘abnormal’ incubations.They concluded that the ‘unnatural ’ substrates NPP and LPP are ionized by the enzyme before they can adopt the optimum conformation for the collapse to bicyclic products. Thus they give rise to abnormal levels of mono and acyclic products. In the case of GPP in vivo they suggest that folding of the hydrocarbon chain to the correct orientation for bicyclic products precedes ionization of the diphosphate group. Croteau et have reported the results of a thorough investigation of the mechanism of GPP :( -) endo-fenchol cyclase from fennel (Foeniculum vulgare) which produces (-)- NATURAL PRODUCT REPORTS 1991 /' GPP (-) (3R)-LPP (+) (3s)-LPP J (+) a-pinene (-) P-pinene Scheme 3 GPP (3R)-LPP J (-) (endo)-fenchol Scheme 4 NATURAL PRODUCT REPORTS 1991-M.H. BEALE GPP LPP A cis + trans sabinene hydrate Scheme 5 I 6 /\ S+ (7) endo-fenchol by the route outlined in Scheme 4. l80labelled water was used to confirm the source of the hydroxyl group while a number of substrate analogues were used to probe the initial isomerizationsyclization reactions. Results with the non-cyclizable analogue 6,7-dihydrogeranyl diphosphate (2) and the cyclopropyl analogue (3) revealed that the isomerization component of the reaction was the same as in other monoterpene cyclases. The cyclization component was examined with feeds of combinations of labelled GPP LPP and NPP while the ionization of the allylic diphosphates necessary for both steps were probed using 2-fluoro-GPP (4) and 2- fluoro-LPP (9 which are effective inhibitors but poor substrates due to the retardation of the ionization by the electron withdrawing fluorine atom.Similar experiments have been carried outs2 with partially purified GPP sabinene hydrate cyclase from sweet majoram (Majoranahortensis).This enzyme catalyses the formation of both (+)-cis and (+)-trans sabinene hydrates as shown in Scheme 5. The 1,2 hydride shift was (9) demonstrated directly using [tb3H]-GPP as substrate. The sulphonium ion analogues (6) and (7) of the carbocations involved were potent inhibitors of the enzyme especially when paired with diphosphate ion. These and other experiments involving non-cyclizable analogues and feeds of mixtures of GPP LPP and NPP confirmed that this enzyme operates in the same way as the other monoterpene cyclases.There is an overwhelming weight of evidence indicating that these mono- terpene cyclases function by initial isomerization of GPP to enzyme bound LPP. Thus a paper by Portilla and R~jas,~ describing the separation of three enzyme activities utilizing GPP NPP and LPP respectively and converting them to the same products is difficult to rationalize. Interest in the C, homoterpene (8) and its C, analogue (9) has been increased as they have recently been found to be major constituents of volatile oils of several plants. It was tenatively suggested that these compounds arise by oxidative cleavage of a C unit from corresponding C, and C, NATURAL PRODUCT REPORTS.1991 0 + 1 yc, D D3C H O (10) R = H = ['HS]-[2H2]-(8) R = H R = D = [2H~]-[2H31-(8) R= D Scheme 6 COOH gy+c-$ OH OH 0 0 0 -l&-~ (12) (13) iridodial (14) iridotrial otiC COOMe COOMe COOMe OGlc HO OGlc OGlc (21) sweroside (19) secologanin (18) loganin (15) deoxyloganin COOMe [HO*M] O q M e Ho*o OGlc HO OGlc OGlc (20) (17) cornin (16) dihydrocornin Scheme 7 compounds. Boland and Gabler54 have now synthesized [2H,]- occurred with retention of configuration the first example of nerolidol (10) and demonstrated its conversion to [2H,]- (8) this in a P450catalysed allylic hydroxylation. along with [1-2H,]-but-3en-20ne in several species as shown in This year's papers on iridoid glucosides are discussed with have examined the Scheme 6.Results with [2H,]-nerolidol confirmed the loss of reference to Scheme 7. Jensen et ~21.~~ hydrogen from C-5 of (10) as shown. biosynthesis of dihydrocornin (16) and cornin (17) in whole The 8-hydroxylation of geraniol by a microsomal cytochrome plants of Verbena oficinalis. In a comprehensive study I3C-P-450preparation from Cutharanthus roseus has been studied labelled 8-hydroxygeraniol (12) iridial (13) iridotrial (14) by Fretz et using synthetic geraniol containing 13Clabel at deoxyloganin (13 and other putative precursors were C-9 and 2H,3H-labelled chiral methyl groups (C-8)as shown in synthesized and fed. This led to the construction of the pathway (11). Their results indicated that the hydroxylation at C-8 shown in Scheme 7.The still unsolved problem of the NATURAL PRODUCT REPORTS 1991-M. H. BEALE mechanism of the conversion of loganin (1 8) into secologanin (19) has been considered by Inoue et The epimers of 6- hydroxy-loganin (20) were synthesized in labelled form but these possible intermediates were shown not to be incorporated into secologanin. In a separate experiment [6,6,7,8-2H,]-loganin yielded sweroside (21) in which the precursor aldehyde group had been reduced from the si face. Glandular trichomes are modified epidermal hairs which accumulate large quantities of monoterpenes. Gershenzon et have demonstrated that these organs are in fact the exclusive sites of carvone biosynthesis in Mentha spicata. This was accomplished by comparison of enzyme activities in cell free systems prepared from trichomes with those from leaves and also histochemically by locating carveol dihydrogenase directly to trichomes.Yamura et have demonstrated that the formation of trichomes in thyme is light-dependent. In a further paper60 the same group showed that irradiation of etiolated seedlings with red light promoted monoterpene production suggesting that phytochrome was involved in the photoregulation of terpene biosynthesis. Biotransformations of monoterpenes reported this year are of (-)-methanol by cell cultures of Eucalyptus perriniandl and of geraniol by cultures of several plant species,62 including a 4&60% conversion to geranial by Glycine max. 6 Sesquiterpenes The current situation with his work on the mechanism and stereochemistry of the enzyme trichodiene synthase from Trichothecium roseurn has been summarized by Cane.63 This enzyme which carries out the conversion of farnesyl diphosphate (FPP) to the hydrocarbon trichodiene (22) as shown in Scheme 8 was purified to homogeneity from Fusarium sporotrichioides several years ago by Hohn and co-workers.This group have now reported64 the isolation and sequence of the gene coding for this enzyme. This was achieved using antibodies to pure enzyme to screen a hgt 11 DNA library followed by the use of DNA fragments thus identified to identify phage containing the whole gene. The deduced amino acid sequence (MW = 44 kDa) was identical to previous sequences obtained from peptides arising from cyanogen bromide cleavage of pure enzyme.In order to align the deduced sequence and known fragments and to arrive at the known molecular weight a 60 nucleotide intron was postulated. This was by expressing unaltered gene in E. coli. The result was the production of inactive polypeptide with a molecular weight 2000 higher than the active enzyme. Insertion of DNA in which the postulated 60 nucleotide intron had been specifically deleted resulted in the expression of active enzyme and the production of trichodiene by E. coli. Trichodiene is the precursor of the trichothecene family of mycotoxins and thus regulation of trichodiene synthase activity may be relevant to levels of mycotoxin production. Hohn and Beremand66 have investigated levels of enzyme activity trichothecanes and immunodetectable enzyme levels in cultures of F.sporo-trichioides and Gibberella pulicaris. They concluded that enzyme activity is regulated by changes in cellular levels and demonstrated that activity increases several hours prior to tricothecane production suggesting that immunochemical analysis of enzyme levels may be a useful indicator of mycotoxin biosynthesis. Desjardins and PlattnerG7 have measured levels of diacetoxyscirpenol (23) (Scheme 8) and other tricothecanes in Gibberella pulicaris. ZarniP has updated her previous review69 on the biosynthesis of 3-acetyldeoxynivalenol (24) in Fusarium culmorum. Last year the intermediacy of the hydrocarbon bergamotene (25) in the biosynthesis of ovalicin in Pseudeuratium ovalis was reported.Cane et have now examined the formation of ~1.~~9~~ (25) from FPP in this species. Feeds of [12,13-14C]-FPP were used to confirm the presence of the sesquiterpene cyclase in a cell free system while (1 S) [1 -2H]- and (1R)[1-2H]-FPP feeds revealed that the cyclization to the six membered ring occurs FPP J H H OAc (23) diacetoxyscirpend (24) 3-acetyldeoxynivalenol Scheme 8 with net retention of configuration at C-1 of FPP (= C-6 of 25). The enzyme aristolochene synthase which catalyses the cyclization of FPP to the hydrocarbon (+)-aristolochene (26) has been purified71 to near homogeneity from Penicilliurn roqueforti. The enzyme which has a molecular weight of 37 kDa by SDS-PAGE has a K for FPP of 0.55pM and required Mg2+.Unusually for a terpene cyclase the enzyme was not inhibited by diphosphate ion. Using a crude preparation of this enzyme from Aspergillus terreus Cane et have investigated the mechanism of the cyclization reaction. As well as demonstrating the incorporation of [3H]- and [13CC,]-FPP into (26) the stereochemistry of the initial cyclization was investigated by feeding (1R) and (1 S) [l-2H]-FPP separately. 2H NMR analysis of aristolochene so produced indicated that the initial cyclization of FPP proceeds with inversion of con-figuration at C-1 of FPP (C-6 of 26). This indicates that the mechanism depicted in Scheme 9 via initial cyclization of FPP to the 10-membered germacryl ring system is operative.Last year it was suggested that 5-epi-aristolochene (27) was a precursor to the phytoalexins capsidiol (28) and debneyol (29) in cellulase-elicited cell cultures of Nicotiana tabacurn. White-head et have now confirmed their earlier suggestions by isolation of (27) from cultures in which hydroxylation is inhibited by exclusion of oxygen or NADPH. Furthermore 3H-labelled (27) was shown to be incorporated into (28) and (29) and it was suggested that the 3-hydroxylase has a role in the regulation of capsidiol(28) biosynthesis. The mechanism of the formation of (27) remains to be determined but it must be NATURAL PRODUCT REPORTS 1991 J T Scheme 9 (27) OH different than that of the 5-epimer (26) (Scheme 9).Also in the phytoalexin area Desjardins et al.74 have studied the de- toxification of lubimin by various strains of the pathogen Gibberella pulicaris. In the juvenile hormone (JH) area [3H-methyll-methionine is often used to follow the formation of methyl farnesoate (30) and its active metabolite JH-111 (31). This year Wennaur and H~ffmann’~ report on in vitro studies with isolated corpora allata of Gryllus bimaculatus (cricket) and Jones and Yin76 describe similar studies with Lymantria dispar (gipsy moth). Belles et al.” have examined the metabolism and effects on JH- (34) I11 release of fluorinated analogues such as methyl- 12,12,12- trifluorofarnesoate (32) which appears to be epoxidized at the C-6,7 double bond rather than at C-10,ll by the enzymes of corpora allata of Blatellu germanica (German cockroach).Considering its importance in genetics it is surprising that there has been little progress in juvenile hormone research in Drosophila melanogaster. Richard et al.78 have now shown that JH-111 is only a minor component of the corpora allata of flies. The major radioactive product from 3H-methionine labelling was identified as the bisepoxide (33).Bioassay experiments with synthetic (33) provided additional support for the postulate that (33) is the fruit fly juvenile hormone. Methyl farnesoate (30) also appears to have a hormonal role in crustaceans and in this context Tobe et ul.” have developed an assay of (30) levels in mandibular organs of Scylla serratu (mud crab).The carboxylesterase JH-esterase (EC 3.1 . 1 . 1) has an important role in the control of JH-111 levels and hence insect developmental processes. Hanzlik et a1.80have reported for the first time the isolation and sequencing of cDNA clones coding for this enzyme. Using antiserum to pure enzyme isolated from Heliothis virescens screening of a hgtl 1 expression library resulted in the isolation of several clones. The sequence of one of these predicted an enzyme of molecular weight 61 kDa. When aligned with sequences of other known carboxyesterases an active site serine at position 201 was predicted along with a different catalytic mechanism for this type of enzyme when compared with serine proteases. Much effort has been put into the design and synthesis of inhibitors of JH-esterase.The majority of the compounds have trifluoromethyl keto- functions which act as reversible transition state analogues. The addition of a sulphur atom ,h’ to the carbonyl leads to highly potent inhibitors such as octylthio- 1,l ,l -trifluoropropan-2-one (34). NATURAL PRODUCT REPORTS 1991-M. H. BEALE (35)e.g. R = (CH2)3-12 cF3nR (36) R = alkyfaryl m3yR 0 (37) R = alkyVaryl Szekacs et af.81have prepared and tested a series of bis (2-0x0- trifluoropropylthio) alkanes (35). The most effective inhibitor was (35 n = 8) with an of 8 x (cf. 1.8 x for 34). In this type of compound the increased potency of compounds with the ,&sulphur atom has been attributed to sulphur mimicking the double bond of the natural substrate JH-111..~~ Lindermann et ~1have tested this theory by preparing and testing a series of a$-unsaturated and a-acetylenic-trifluromethyl ketones (36) and (37). None of these new compounds were as potent as (34) indicating that the sulphur atom plays a more complex role than as a double bond analogue. Both in vivo and in vitro effects of the hormone mimic epofenonane (38) on JH-esterase levels have been studied by Newitt and Further examinations of JH-esterase activity in vitro have been described by Sparks et 121.~~ and by Meyer and Lan~rein.”,~~ Abscisic acid (ABA) (39) -biosynthesis in higher plant has been reviewed by C~eelman.~’ Over the last few years techniques such as the use of mutants inhibitors of carotenoid biosynthesis and oxygen- 18 incorporation have indicated that this sesquiter- penoid originates from the breakdown of a carotenoid (C,) precursor in higher plants (cf.direct synthesis from FPP in several species of fungi). Zeevaart et af.88~89 have now added further results with 1802 incorporation into ABA using both leaf and fruit tissue of several plant species. After incubation with 1802 ABA was isolated from the plant material and analysed by gas-chromatography-mass spectrometry. Of the labelled molecules present those containing one l80 atom were universally the most abundant. The l80 atom was located in the carboxyl group thus indicating the ABA is biosyntheized by cleavage of a larger molecule with the ring oxygen atoms already present.Willows and Milborrowgo have isolated ABA from avocado fruit incubated in 2H20. They concluded by a combination of mass spectral and lH NMR (but not 2H NMR) that the 5’-pro-S hydrogen atom was labelled and therefore introduced from the medium during cyclization of the linear .~~ precursor. Meyer et ~1 have reported on the metabolism of ABA in barley while Abrams et describe an NMR method for the measurement of (S) to (R) ratios in mixtures of enantiomers and use it to measure the rate of metabolism of racemate by cell suspension cultures. trans-/3-Farnesene (40) has been showng3 to accumulate in callus cultures of hop (Humufus lupulus) and tracer experiments with [2-14C]-MVA yielded a good incorporation. Biomimetic transformations of sesquiterpenes reported this year are of lactonesg4 and of epoxy-eudesmanesg5 under acidic conditions.The regiospecific microbial hydroxylation of caryolanol by Aspergillus niger gave the 14-hydroxy-compound in good yield.96 7 Diterpenoids Following last year’s paper in which they proposed a route for the biosynthesis of traversianal (42) Stoessl et ~1.~’ have now shown that the suggested precursor traversiadiene (4 1) is indeed present in cultures of Cercospora traversiana. The structure was determined by ‘H and I3C NMR techniques with the relative stereochemistries being established by NOE difference spectroscopy. The biosynthesis of the abietane diterpenes is usually formulated as occurring via pimaradiene (43) as shown in Scheme 10.Tomita et al. have now provided direct evidence for the stereochemistry of the 1,2 methyl shift from C-13 to C-15 in the conversion of (43) to the abietane skeleta. In their first studyg8 they fed D-[U-13C6]-glucose to cell cultures of Salvia miftiorrhiza and examined the labelling pattern in the metabolite cryptotanshinone (44). In the 13C NMR of this compound C-13 and C-17 both appeared as singlets confirming that the C- 13-C- 17 bond of pimaradiene (43) was broken in the formation of the ispropyl group of abietanes. This 1,2 Me shift was demonstrated to occur on the si face of the double bond by a method involving determination of the absolute configuration of C-15 of (44) by oxidative cleavage of the C- 15-C- 17 fragment as (S)-(+ )-2-hydroxy-methyl propionic acid.In a second paperg9 the I3C NMR of ferruginol (45) from these feeds confirmed this stereochemical assignment. Here enzymatic resolutions of 12-0-methyl- 16- hydroxyferruginol allowed assignment of the 13Csignals for the methyl groups C- 16 and C- 17 in 12-0-methylferruginol. Observation of the singlet 17-pro-R signal in the biosynthetic sample again confirmed the 1,2 si shift. Last year the work of Hanson’s group on aphidicolin (46) biosynthesis in Cephafosporium aphidicofa was reported. Here the order of hydroxylation of the precursor (47) was thought to NATURAL PRODUCT REPORTS. 1991 y GGPP I-.+ (44) Scheme 10 OH R3 4 (46) R' = R2 = R3= OH (47) R'=R2=R3=H be C-18 followed by C-3/C-17.Oikawa et al.loO have clarified the order of the second and third hydroxylations using cultures of Phoma betae grown in the presence of cytochrome P-450 inhibitors. Their results suggest the order C-18 then C-17 and finally C-3 for this organism. This year's crop from the gibberellin field are reviewed as usual with reference to the overall route shown in Scheme 11. The cyclization of GGPP to ent-kaurene (48) is thought to be a two step process involving the bicyclic intermediate copalyl diphosphate. Detailed information of the type being accrued for the mono- and sesquiterpene cyclases is lacking in this area despite the ubiquitous occurrence of kaurenoids in higher plants. One of the most studied systems is HeZianthus annuus.GafnilO' has reported that although seedlings yield cell-free extracts with good kaurene synthase activity cells of crown galls induced by Agrobacterium on these plants do not produce kaurene. Indeed evidence for the active suppression of kaurene synthase by an inhibitory substance in these tumour cells was obtained. The kaurenolides e.g. (50),are formed as a result of a significant branching of the main gibberellin pathway. In both fungi and plants this occurs via the diene-acid (49) which is converted to the 6p-7p epoxide which undergoes spontaneous intra molecular ring opening by the 19-carboxylate to form the lactone. Diaz et al.lo2have made use of the fact that 3a- hydroxykaurenes are not converted to their corresponding 19- carboxylates by the fungus Gibberella fujikuroi.They synthesized 3a-hydroxykaur-6,16-diene and showed that it was converted to the stable 6/3,7/3-epoxide by the fungus providing further evidence for this pathway of kaurenolide biosynthesis. There has been some progress in the purification of soluble dioxygenases responsible for the latter steps of GA biosynthesis in higher plants. Lange and Graebelo3 have achieved a 270-fold purification of a C-20 hydroxylase which catalyses the conversion of GA, to GA, (see Scheme 11) in pea embryos. The enzyme has a molecular mass of 44 kDa required 2-oxoglutarate Fe2+ and ascorbate and had a K for GA, of 0.7pM. Although the purified enzyme contained one major band at the above molecular weight on SDS-PAGE it was demonstrated that this was not the enzyme by isoelectric focusing where the major protein separated from enzyme activity.Albone et aZ.lo4 have made a comprehensive study of the GA and GA,,-hydroxylases of Phaseolus vulgaris. Using stereospecifically labelled substrates and partially purified enzyme extracts it was shown that the hydroxylation of GA, to GA and of GA to GA occur with retention of configuration and that GA is formed from GA, with loss of the 2p and 3p hydrogen atoms. Evidence was also presented that the conversions of GA, to GA and to GA are carried out by the same enzyme. Endo et aZ.lo5 have examined the GA content of shoots of P.vulgaris. They identified the members of the 13- hydroxy pathway GA, through GA in both tall and dwarf varieties.They suggested that as in pea rice and maize GA was the only active compound of this pathway. However the dwarf phenotype was not the result of decreased GA levels but appeared to be due to less ability to respond to active GA. NATURAL PRODUCT REPORTS 1991-M. H. BEALE 45 1 OH J + 0" Turnbull and Crozier1o6 have examined [3H]-GA4 metabolism in cell-free supernatants and seedlings of Phaseolus coccineus. Metabolites identified only on the basis of HPLC retention time included GA,. The biological significance of these results is unclear. Pisum sativum (pea) is a widely used system for the study of gibberellin control of stem elongation. This is because the endogenous GAS have been well characterized and there is a wide range of mutants available.This year Halinska et al.lo7 have published further on the endogenous GAS of the line G2 while Ross et al.loS have measured GA, 3-epi GA and GA levels in plants of the genotypic sequence le (tall) le (intermediate) and led(short) fed with the precursors [3H,13C]- GA,,. A quantitative relationship between GA level and the extent of stem elongation was established adding further evidence that GA is the active compound of the early 13- hydroxylation pathway (Scheme 11). In a further paper Ross and Reidlog have examined the expression of the le gene in dark-grown plants. They concluded that although GA levels were not increased by growth in darkness the overall metabolism of GA, was elevated under these conditions and that some aspect of sensitivity to GA was increased in darkness.The same authors also reportl'O the characterization of two further short but GA-insensitive mutants of pea. These genes lka and lkb act in young apical tissue but do not appear to involve GA-perception but influence steps in the biochemical process between perception and stem elongation. Feeds of [,HI- GA to Ika and other GA-sensitivity mutants (lk lw) were compared'l' with those to GA-biosynthesis mutants (le lh) and slender (la,cry'). Amounts of [3H]-GA8 formed were monitored by HPLC and found to be similar in all mutants indicating that GA action is independent of GA metabolism. Analysis of the GA-status in mutants of other plant species also reported this year include the dy and dx mutants of rice112 which respectively control the 3P-hydroxylation of GA, and pre-GA-skeleton formation.Rood et ~1.l~~ report on a GA- deficient rosette mutant of Brassica rapa. Triazole plant growth Gbspem lactone + OH GA20 regulators have been used to influence GA levels in two of the related Brassica napus. Photoperiodic control of GA metabolism in Salix pentandra has been investigated116 and found not to be exerted at the GA, GA, step in the pathway. GAS may also be involved in the photoperiodic control of flowering and aspects of this have been reported'l' for Silene armeria. In the Pinaceae GAS influence cone formation and Mortiz et al.l18 have examined GA metabolism in Picea sitchensis in this context. Immunological techniques were introduced into this field several years ago.This year two papers describing immuno- affinity chromatography of gibberellins have appeared. Smith and MacMillanllg report on the use of specific monoclonal antibodies to give remarkable purifications of GAS in plant extracts while Durley et al.lZ0 used polyclonal sera raised against a mixture of GA-protein antigens to separate GAS as methyl esters from plants. For kaurenoid precursors of GAS Metzger and Hazebrock,121 have used size-exclusion chromato- graphy to good effect. The dwarf rice micro-drop assay is the most widely used test for active gibberellins. Nishijuma and KatsuralZ2 describe improvements to the assay whereby dwarf varieties Tag-ginbozu and Waito-C treated with the growth retardant uniconazole were shown to respond to as little as 3.5 pg of gibberellin per plant.In fern gametophytes some gibberellin methyl esters and analogues with re-arranged C/D carbon skeleta have been identified as extremely active growth factors called anther- idiogens. Takeno et ~2l.l~~ have reported bioassay data which indicate that the principal antheridiogen of Lygodium japonicurn is the methyl ester of the recently identified GA, (51) the synthesis of which has been reviewed by Mander.124 In the species Ceratopteris the nature of the antheridiogen is unknown. However Warner and Hi~kok'~~ have published some cir- cumstantial evidence that similar compounds are involved. They show that the GA-biosynthesis inhibitors AMO- 1618 and ancymidol blocked the production of male gametophytes and NATURAL PRODUCT REPORTS 1991 that this effect was overcome by exogenously supplied filtrates from uninhibited cultures.8 Mode of Action In the insect juvenile hormone area many types of synthetic analogues have hormonal activity. Indeed many hundreds of compounds have been synthesized and tested. Structure and bio-activity of the different chemical classes of juvenoids have been reviewed by Wimmer and Romanuk.12'j This year's new reports in this area describe 5-oxa-3,8,12-trimethyl-decenoates,12' phenoxyphenoxy- and benzylphenoxy-propyl ethers,'28 isothio~yanates,~~~ linear thiocarbonates and related a thia~olylurea'~' and a new class of anti- juvenile hormones arylpyridyl thiosemicarbazones.Over the last ten years a number of juvenile hormone binding proteins have been isolated from various sources. However demonstration that these proteins are receptors requires observation of a physiological response to hormone-protein interactions. This is difficult to achieve in most systems but the Drosophila K cell line shows a number of measurable responses to juvenile hormone action. These include changes in energy metabolism as indicated by increased cytochrome oxidase activity. Thus the full characterization of hormone binding proteins in K cells is an important goal for those interested in juvenoid receptors. Wang et al.133have photo-affinity labelled and purified a binding protein to homogeneity from cytosol of K cells.This protein appeared to be a dimer with subunit mass of 24600 Da and was shown in the absence of affinity labelling to bind [3H]-juvenile hormone with unchanged kinetics through- out the purification procedure. In contrast to this high affinity low molecular mass protein much larger juvenile hormone binding proteins have been described. This year King and T~be'~~ report the partial purification of a high-affinity binding protein (K -3 nM) of molecular weight of -680000 Da from heamolymph of Diploptera punctata (cockroach). Binding assays with natural (1OR)-JH-I11 and racemic (lOR,S)-JH-I11 showed the (10R) enantiomer to have a higher affinity. Indeed Kindle et also report the higher biological activity of the (IOR) enantiomer in three different bioassays including a binding protein assay.Wisniewski13'j has described the suc- cessful identification of JH-binding proteins by probing with [3H]-JH-III ion-exchange membranes onto which proteins separated by 2-dimensional native polyacrylamide gel electro- phoresis had been electroblotted. The steroid model of hormone action has led most workers to search for soluble JH-receptors. However there are some indications of JH action at membranes and van Mellaert et al.137 have developed a glass fibre filter assay for microsomal binding sites in Sarcophaga bullata. Sevala and Da~ey'~~ have presented evidence that JH-I11 acts on membranes if cells of Rhodinius prolixus by a mechanism involving activation of protein kinase C. The cereal aleurone system where the plant hormones gibberellin (GA) and abscisic acid (ABA) play a role in regulating gene transcription was outlined in last year's review.New papers on ABA action in this area are by Jacobsen and have used [35S]-methionine labelling to show that ABA induced the synthesis of at least 25 polypeptides and that most of these were not denatured on heating to 100 "C for 10 minutes. The role of these polypeptides which contain higher glutamic acid/glutamine glycine and proline levels than average is unclear but they may function in the protection of cellular structures during seed desiccation. Napier et al. lgohave examined the effect of [Ca2+] on this ABA modulation of polypeptides in wheat aleurone tissue and showed that Ca2+ is essential for this ABA action.ABA also causes induction of proteins in other plant tissues and this year Barratt et aI.lgl describe the partial characterization of two immunologically similar proteins of MW 17200 and 18 100 induced in cultured embryos of pea (Pisum sativum) while Harada et allg2 report sequence data for a 30 kDa protein induced in Brassica napus seed. Mutants with a reduced capacity for ABA biosynthesis or response are important tools in this area and Koornneef et al.lg3have examined storage protein levels in the context of dormancy induction and seed development in mutants of Arabidopsis thaliana. Similarly Pla et al.lg4 have described the ABA-regulation of a set of phosphorylated 23-25 kDa proteins in normal and vivaparous mutants of Zea mays.Another process in which ABA is implicated is in plant responses to wounding or attack by pathogens. One example is the induction of expression of proteinase inhibitor genes throughout the plant by a local attack. Using ABA-deficient mutants of potato and tomato Pefia-CortCs et allg5report results that indicate ABA is directly involved in the induction of proteinase inhibitor I1 gene. In a similar context Roberts and Kolattuk~dy'~~ have shown that ABA induces a highly anionic peroxidase involved in the formation of suberin a mixed polymer comprizing long chain fatty acids within an aromatic matrix which is a component of wound-healed tissue. On the technical side of the study of ABA action immunoassay has become important for measurement of ABA levels.Papers published this year include production of a new monoclonal antibody,lg7 warnings of interference in use of a commercial ABA antibodylg8 and the use of immunoaffinity chromato- graphy to isolate ABA from conifer Gibberellin action on aleurone cells requires high concentra- tions of extracellular Ca2+. Bush et al.l5' report experiments using 45Ca2+ as a tracer and the Ca2+ sensitive dye Info- 1 which indicate that GA-action causes increased transport of Ca2+ in the endoplasmic reticulum. In animal cells Ca2+ is involved with phosphoinositides in signal transduction across cell membranes. There is some evidence for the presence of these messengers in plant cells. In this context Murthy et al.I5l have examined the incorporation of labelled inositol and 32Pi (inorganic phosphate) into barley aleurone phospholipids and have observed that GA enhances labelling of phosphatidyl- inositol within 30 seconds of treatment.Also in this system an endochitinase has been added152 to the list of hydrolases regulated at the transcriptional level by GA. The diterpenoid phytotoxin fusicoccin is thought to act by stimulation of the H+-ATPase in the plasma membrane. The presence of a fusicoccin-binding protein has been suspected for some time and this year we have two reports of significant progress in this area. De Boer et al.'53 have stabilized and purified a membrane protein from oat (Avena sativa) roots. Their procedure which included affinity chromatography over immobilized fusicoccin yielded two major protein bands of molecular weights of 29.7 and 31 kDa in the ratio 1:2 suggesting that the fusicoccin-binding protein in its native form is a heterotrimer with a molecular weight of 92 kDa.Working with leaves of Arabidopsis thaliana Meyer et al.'54 have solubilized a fusicoccin-binding protein of high affinity (K = 42 nM). Photoaffinity experiments with a p-azidobenzoyl derivative indicated specific labelling of a ca. 34 kDa protein on denaturing gels although gel filtration chromatography of solubilized binding proteins indicated a molecular weight of ca. 80 kDa. 9 References 1 E. Santaniello B. Canevotti R. Casati L. Ceriana F. Ferraboschi and P. Grisenti Guzz. Chim.Ital. 1989 119 55.2 T. Ohta N. Tabei and S. Nozoe Heterocycles 1989 28 425. NATURAL PRODUCT REPORTS 1991-M. H. BEALE 3 A. Rajkovic J. N. Simonsen R. E. Davis and F. M. Rottman Proc. Natl Acad. Sci. USA 1989 86 8217. 4 C. 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P. S. Steyn and R. Vlegaar 1986 Elsevier Amsterdam. 70 D. E. Cane D. B. McIlwaine and P. H. M. Harrison J. Am. Chem. SOC. 1989 111 1152. 71 T. M. Hohn and R. D. Plattner Arch. Biochem. Biophys. 1989 272 137. 72 D. E. Cane R. C. Prabhakaran E. J. Salaski P. H. M. Harrison H. Noguchi and B. J. Rawlings J. Am. Chem. SOC. 1989 111 89 14. 73 I. M. Whitehead D. R. Thelfall and D. F. Ewing Phyto- chemistry 1989 28 775. 74 A. E. Desjardins H. W. Gardner and R. D. Plattner Phyro-chemistry 1989 18 43 1. 75 R. Wennauer and K. H. Hoffman Insect Biochem. 1989 18 867. 76 G. L. Jones and C-M. Yin Comp. Biochem. Physiol. B 1989,92 9. 77 X. Belles F. Comps J. Cases B. Manchamp M-D. Picelachs and A.Messenguer Arch. Insect Biochem. Physiol. 1989 11,257. 78 D. S. Richard S. E. Applebaum T. J. Sliter F. C. Baker D. A. Schooley C. C. Reuter V. C. Henrich and L. I. Gilbert Proc. Natl Acad. Sci. USA 1989 86 1423. 79 S. S. Tobe D. A. Young and H. W. Khoo Gen. Comp. Endocrin. 1989 73 342. 80 T. N. Hanzlik U. 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Reaney G. D. Abrams T. Mazurek A. C. Shaw and L. V. Gusta Phytochemistry 1989 28 2885. 93 D. V. Banthorpe J. T. Brown and G. S. Norris Phytochemistry 1989 28 1847.94 A. G. Gonzalez A. Galindo and H. Mansilla Heterocycles 1989 28 529. 95 P. Ceccherelli M. Curini M. C. Marcotullio and 0.Rosati Tetrahedron 1989 45 3809. 96 V. Lamare A. Archelas R. Faure M. Cesario C. Pascard and R. Furstboss Tetrahedron 1989 45 3761. 97 A. Stoessl G. L. Rock and J. B. Stothers Can. J. Chem. 1989 67 1302. 98 Y. Tomita and Y. Ikeshiro J. Chem. SOC. Chem. Commun. 1987 1311. 99 Y. Tomita M. Annaka and Y. Ikeshiro J. Chem. SOC.,Chem. Cornmun. 1989 108. 100 H. Oikawa A. Ichihara and S. Sakamura Agric. Biol. Chem. 1989 53 299. 101 Y. Gafni Plant Sci. 1989 65 171. 102 C. E. Diaz B. M. Fraga and M. G. Hernandez Phytochemistry 1989 28 1053. 103 T. Lange and J. E. Graebe Planta 1989 179 211.104 K. Albone P. Gaskin J. MacMillan V. A. Smith and J. Weir Planta 1989 177 108. 105 K. Endo H. Yamane M. Nakayama I. 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ISSN:0265-0568    

DOI:10.1039/NP9910800441

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

The Lycopodium Alkaloids W. A. Ayer Department of Chemistry University of Alberta Edmonton Alberta T6G 2G2,Canada ~~ Reviewing the literature published between January 1986 and October 1990 1 Introduction 2 The C,,N Alkaloids 2.1 The Lycopodane (1) Group 2.2 The Fawcettimane (20) Fawcettidane (21) Group 2.3 The Magellanine Group 2.4 Annotinine (45)and Annotine (46) 3 The Cl,N2 Alkaloids 3.1 The Flabellidane (47) Group 3.2 The Fastigiatane (68) Group 4 References 1 Introduction The present review covers the literature for the period from the beginning of 1986 to October 1990. The Lycopodium alkaloids have not been previously reviewed in publication and were last reviewed in Specialist Periodical Reports on Alkaloids in 1983,l covering the literature to the end of 1981.Since that time an extensive review of the synthesis and biosynthesis of these alkaloids has appeared,2 and a review of all aspects of the chemistry of the Lycopodium alkaloids for the period 1973- 1985 has been p~blished.~ This review is intended to cover the developments in the field since the appearance of MacLean’s re vie^.^ The period is highlighted by the discovery in China that some of these alkaloids are potent acetylcholinesterase inhibitors. They are reported markedly to increase efficiency for learning and memory in animals4 and huperzine A shows promise in the treatment of Alzheimer’s disease and myasthenia gra~is.~ The classification of the alkaloids into the various of C,,N C16N2 and C2,N3 groups3 will be used except that the biologically important C,,N2 alkaloids are treated as members of the closely related C,,N group and the new C,,N alkaloid is placed with the biogenetically related C,,N2 group.2 The C,,N Alkaloids 2.1 The Lycopodane (1) Group Much of the pioneering work on the Lycopodium alkaloids was carried out in Canada in the late forties and early fifties by R. H. F. Manske and L. Marion. During that period the isolation and characterization of thirty-five alkaloids was reported. These were assigned numbers from Ll to L35. Most of these alkaloids have since been investigated and their structures a~signed.~ Recently many of the original samples of Manske and Marion have been re-examined by GC-MS and GC-IR and the identity of all of the L alkaloids except for L10 is now established.It turns out that several of the original L alkaloids (often characterized as the hydroperchlorates) were mixtures and only two new alkaloids 5,15-oxidolycopodane (2) a component of alkaloids L28 and L31 and acetyl-annofoline (3) a component of L 17 were uncovered. GC-MS has also been used to re-examine the alkaloids of L. clavatum var. borbonicum and L. deuterodensum .’ Flabelliformine (4) alkaloid L20 (5) and lycodine (6) were identified as constituents (4) R=OH R’= H of L. clavatum var. borbonicum. L. deuterodensum previously (5) R=H R’=OH examined by Manske in 1953 contained lycopodine (7 ca. (7)R = R’ = H 32 455 NPR 8 NATURAL PRODUCT REPORTS 1991 10 HO 94% of the total alkaloids) along with lycodine (6) anhydro- lycodoline (7 flabelliformine (4) lycodoline (7 C- 12 OH) clavolonine (7 C-8 OH) lycoflexine (8) and flabelline (9).In agreement with these results alkaloid L35 reported from this species by Manske has been shown6 to be a mixture containing lycopodine (7) flabelliformine (4),and lycodoline (7 C-12 OH). The GC-MS method was used to examine two new species L. australianum (shown to contain lycodine (6) cernuine (lo) and a new alkaloid whose structure was not established) and L. fastigiatum.' The latter species contains several known members of the lycopodane (1) group along with the new alkaloid fastigiatine (see below) and its des-N-methyl derivative.The structure of paniculine (1 1) has been confirmed by an X-ray study of the hydrobromide,s and detailed 13C NMR assignments for (11) and related compounds have been rep~rted.~ Paniculine (11) is an interesting member of the lycopodane group in that it is the only alkaloid functionalized at C- 10. In the related pentacyclic alopecurane (1 2) skeleton C-10 is the terminus of a carbon-carbon bond. A new alkaloid with the 7,8-seco-lycopodane skeleton lyconnotinol (1 3) has been isolated from L. obsc~rum.~~ The structure of (13) was confirmed by its preparation from lyconnotine (14) the only other alkaloid reported to possess this particular skeleton. An elegant new synthesis of lycopodine (7) has been reported by Kraus and Hon.11,12 5-Methylcyclohexenone was readily transformed into 2-allyl-5-methylcyclohexenone(15 Scheme 1) using a thiol addition alkylation elimination sequence. Michael addition of acetoacetate to (15) and internal aldol condensation followed by decarbomethoxylation provided (16) which was then subjected to Brown hydration. The resulting primary hydroxyl was converted to the benzenesulphonate and the bridgehead hydroxyl was transformed to the bromide (1 7). Treatment of (17) with silver triflate followed immediately by an excess of 1-amino-3-benzyloxypropane,gave the tertiary amine (18). Alternatively generation of the bridgehead enone and addition of 3-amino- 1 -propano1 provides (1 9) which also may be prepared by catalytic hydrogenolysis of (18).The (9) U (13) R=CHzOH (14) R = COOCH3 crystalline keto alcohol (19) an intermediate in Heathcock's synthesis was converted into racemic lycopodine in two steps using Heathcock's method.2 The overall yield from (15) to lycopodine was 25 %. 2.2 The Fawcettimane (20) Fawcettidane (21) Group These two skeletal groups are logically treated together since fawcettimine the prototype alkaloid exists as an equilibrium mixture of the keto amine form (22a) and the carbinolamine form (22b). Recent structural and synthetic studies by Heathcock and co-~orkers~~*'~ have provided valuable new information discussed later on this interesting keto amine-carbinolamine tautomerization. The starting material for the synthesis of fawcettimine (22) is the cyano-enone (23) also used as the starting material for the earlier syntheses of lycopodine (7) lycodine (6) and lycodoline.2 The enone (23) was subjected to Sakurai reaction with the allylsilane (24) to give alcohol (25) as a mixture of diastereo- isomers in quantitative yield (Scheme 2).Oxidation of (25) to the aldehyde Wittig reaction with [(ethoxycarbonyl)-methylenel-triphenylphosphorane,followed by treatment with sodium ethoxide gave hydrindanone (26) as a single diastereo- isomer in close to 90% yield from (25). Arndt-Eistert homologation of the acetic acid side chain provided (27) containing the additional carbon required for fawcettimine. Hydride reduction and selective tosylation gave the N,O-bis(tosy1) derivative (28).Treatment of (28) with tetra-n-butylammonium hydroxide in benzene gave the desired nine- membered heterocycle in good yield. Deprotection of the nitrogen and oxidation of the alcohol gave the aminoketone (29). The perchlorate salt of (29) was subjected to ozonolysis at -78" to give the perchlorate salt of the diketone. Careful neutralization of the salt with sodium bicarbonate and treatment of the resulting amine with aqueous HBr gave racemic fawcettimine hydrobromide identical (lH NMR and IR) with natural fawcettimine (22 = 30). The structure of the synthetic hydrobromide was verified by X-ray crystallography. This established that the hydrobromide is the salt of the NATURAL PRODUCT REPORTS 1991-W. A. AYER 457 i ii iii-v =-/& / OH /j+ ~ viii ix N-OR (18) RzPhSO2 (19) R= H Reagents i MeCOCH,CO,Et NaOMe MeOH; ii KOH heat; iii BH,-THF; iv PhS0,Cl; v PBr vi AgOTf H,N(CH,),OBn; vii H,N(CH,),OH DBU ;viii ButOK Ph,CO benzene heat ; ix H,,Pt Scheme 1 HN 0 0 H3C...q + H2cysiM*3 H3c...v0H inv CH2 -H3c-.*p OSiMe3 '-CO,Et CN CN CN v-viii i H ~ xi-xiii ix.x -CH2 CN (29) Reagents i TiCl, CH,Cl,; ii Cr0,Py; iii Ph,PCHCO,Et EtOH; iv NaOEt EtOH; v NaOH EtOH H,O; vi (COCl), C,H,; vii CH,N,; viii PhCO,Ag Et,N MeOH; ix LAH THF; x Ts,O DMAP; xi BuiN+OH- C,H,; xii Na NpH DME; xiii CrO, HOAc Scheme 2 32-2 NATURAL PRODUCT REPORTS 1991 H (30a) (R=H) (31a) (R = H) (32a) (R =Ac) (33a) (R =Ac) 1 (31b) Scheme 3 (32) R = HI R' =OH (33) R=OH R'=H (34) R= HI R'=OAc (35) R = R' =carbony1 carbinolamine form (22b) of fawcettimine and proved that the stereochemistry at C-4 of fawcettimine had been correctly assigned.Since the keto olefin (29) has the opposite stereo- chemistry at C-4 to that of fawcettimine this means that epimerization at C-4 is extremely facile. Conformational analysis in the fawcettimine-4-epifawcet-timine case is complicated by the possibility of concurrent keto amine-carbinolamine tautomerization. The recent work by Heathcock'" has done much to throw light on this interesting conformational problem. Since the 'classical ' Lycopodium structures (22a) and (22b) do not lend themselves well to this discussion fawcettimine and 4-epifawcettimine (keto amine forms) are redrawn in Scheme 3 as (30a) and (3 1a) respectively.Early work15 had suggested that fawcettimine exists mainly as the carbinolamine tautomer (30b) [IR 3585 (OH) 1730 (cyclopentanone)] but that it acetylates on nitrogen to give N-acetylfawcettimine (32a) IR 1615 (amide) 1690 (cyclohex- anone) 1730 (cyclopentanone). It was reported,16 however that the hydrochloride and perchlorate salts of fawcettimine showed carbonyl absorption at 1690 cm-' only suggesting that the cyclopentanone carbonyl is involved in carbinolamine formation. This is stereochemically possible only if fawcettimine has the 4R configuration (31a) which allows formation of the tautomer (31c). The N-acetyl derivative (33a) of (31a) was prepared during the synthesis of fawcettimine and subjected to base catalyzed equilibration.The trans epimer (32a) pre- dominates (ratio 2 1) in the equilibrium mixture. A careful study of the IR and NMR spectra of the product of ozonolysis of the hydroperchlorate of synthetic (29) indicates that neutralization affords first the diketone (3 1a) (4-epifawcet- timine) in equilibrium with keto carbinolamine (31c) [and perhaps some (31b)l. With time (31a) epimerizes to (30a) which exists in solution almost completely in the keto carbinolamine form (30b) (Scheme 3). Molecular mechanics calculation^'^ indicate that carbinolamine (30b) is the lowest energy species in Scheme 3 and that in the 4-epi series the cyclopentanone carbinolamine (31c) is preferred over the cyclohexanone carbinolamine (3 1b).The possibility remains that the hydrochloride and hydroperchlorate salts isolated many years ago16 are salts of the cyclopentanone carbinolamine (3 lc) of 4-epifawcettimine. Several new members of the fawcettimane (20) group have been isolated from L. obscururn." The structures of lobscurinol (32) epilobscurinol (33) and acetyllobscuriol (34) were determined mainly on the basis of extensive 2D COSY 'H NMR studies which allowed the complete determination of the spin system corresponding to carbons 9 to 11 and of another spin system correlating H-14 with the alkenic methyl and on through the C-8 hydrogens all the way to C-2. The a,p-unsaturated carbonyl system present in these alkaloids gives rise to absorption at 234 nm in the UV and at 1658 cm-l in the IR.The stereochemistry at C-5 was assigned on the basis of coupling constants and NOE enhancements. In particular the hydrogen at C-5 in (33) and its 0-acetyl derivative shows vicinal coupling with both C-6 H's (7 Hz 7 Hz) allylic coupling (2 Hz) with H-3 and W plan coupling with H-7. H-5 in (32) and (34) shows observable coupling to only one of the H-6's (5 Hz) smaller allylic coupling (1 Hz) and no coupling to H-7. Oxidation of lobscurinol provided the diketone (35) which was named lobscurinine (carbonyl absorption at 1722 cm-' (cyclo- pentanone) and 1663 cm-' (cyclohexenone)). Lobscurinine was not isolated among the alkaloids of L. obscuruyy~,but its interesting ammonia adduct obscurinine (36) was present.The structure of obscurinine initially believed to be a new alkaloid was determine by X-ray crystallography. l7 Since in both isolation procedure~,~~~" aqueous acidic extracts of the 459 NATURAL PRODUCT REPORTS 1991-W. A. AYER P Hg 0 H (39) (40) i-iii Reagents i 4-bromo-l-butene Mg Me,S.CuBr THF; ii LHMDS PhSeCl THF; iii H,O, py-CH,Cl,; iv PdCl, CuCl DMF H,O 0,;v NaH THF; vi HOCH,CH,OH PTSA C,H,; vii; 0,,MeOH then NaBH,; viii MsCI Et,N CH,Cl,; ix CH,NH, DMSO sealed tube; x aq. HCl THF Scheme 4 alkaloids were basified with aqueous ammonia it seems probable that obscurinine is an artefact of the isolation process. Indeed treatment of a chloroform solution of lobscurinine (35) prepared by oxidation of lobscurinol (32) with aqueous ammonia led to the formation of obscurinine (36) in virtually quantitative yield,’O presumably by internal Schiff base formation in the ammonia adduct (37) of lobscurinine.This partial synthesis of obscurinine provides confirmation of the structures of lobscurinol (32) and epilobscurinol (33). 2.3 The Magellanine group A very neat approach to the synthesis of the complex tetracyclic skeleton of magellaninone (38 Scheme 4) and related alkaloids has been described recently.18 The starting material is the pentacyclic dione (39) derived from the Diels-Alder adduct of cyclopentadiene and p-benzoquinone. This was transformed in a series of steps to the triquinanedienone (40). Conjugate addition of 4-bromomagnesium- 1-butene furnished as the major product the cis-anti-cis addition product which was transformed by phenylselenylation-selenoxide elimination to the unsaturated ketone (41).Wacker-type oxidation of the side- chain gave enedione (42). This on base-catalyzed ring closure provided the tetracyclic diketone (43 j containing the required stereochemistry at ail five ring junction stereogenic centres. The cyclopentene ring was then transformed to an N-methyl-piperidine ring by ozonolysis reduction mesylation and go *-H “H (45) treatment with methylamine to provide the tetracyclic diketo- amine (44). There remains the task of adding the methyl group at C-15 and introduction of an oxygen substitutent at C-13. 2.4 Annotinine (45) and Annotine (46) A detailed ‘H and 13C NMR study of these two structurally unique alkaloids has been reported.lg These studies confirmed the constitution of annotine (46) deduced earlier from degradation studies spectroscopic information and biogenetic considerations and allowed the assignment of stereochemistry at C-4 and C-12 in annotine (46).The conformation of the molecules determined by NMR shows good correlation with those determined by molecular mechanics calculations. NATURAL PRODUCT REPORTS 1991 A 3 2 (47) (48) R= H (50) R = AC (49) R = H (51) R=CH3 (52) (53) (54) (55) R=OH R'=H (56) R = R' = carbonyl 3 The &N Alkaloids 3.1 The Flabellidane (47) Group Huperzine A (48) and huperzine B (49) are two new alkaloids isolated from the Chinese folk medicinal plant Huperzia serrata (= Lycopodium ~erratum).~ They exhibit strong anticholin- esterase activity2' and huperzine A (48) has been shown to improve animal performance in Y-maze experiments and to be useful in treating myasthenia gravis in human^.^ Huperzine A is currently in clinical testing in China for the treatment of Alzheimer's disease.21 The structure of huperzine A was determined on the basis of extensive NMR IR and UV data dehydrogenation to give 6-methyl-a-pyridone preparation of mono- di- and tri-N-methyl derivatives and hydrogenation to dihydro and tetrahydro derivative^.^ The hydrogen on C-8 appears as a doublet (5 Hz) and is coupled to a proton at 13 3.56 (H-7).Irradiation of the C-10 methyl hydrogens causes NOE enhancement of the S 3.56 signal allowing the assignment of the stereochemistry of the exocyclic ethylidene group.The alkenic hydrogen at C-11 and the C-3 pyridone hydrogen are both shifted upfield in N-acetylhuperzine A (50) confirming the proximity of these hydrogens to the amino nitrogen. Huperzine B (49) lacks the ethylidene group signals in the NMR but this spectrum clearly shows the features of the C-8 C-15 endocyclic bond and the a-pyridone ring system. Dehydrogenation of huperzine B (Pd/C 300") furnishes 7- methylquinoline and 6-methyl-a-pyridone accounting for all of the carbon and nitrogen atoms in (49). Eschweiler-Clarke methylation gives N-methylhuperzine B (5l) the spectral characteristics of which closely resemble those of /3-obscurine (52).Catalytic hydrogenation of (5 1) gives 15-epi-/3-0bscurine characterized by the high-field chemical shift (6 0.60) of the C-15 methyl (anisotropically shielded by the pyridone ring). The fact that hydrogenation occurs exclusively from the side of C-12 suggests that the stereochemistry at C-12 is the same as in /3-obscurine. This conclusion is supported by NOE enhance- men t experiments. The structure of huperzine A (48) is similar in many respects to that reported for selagine (53),3 an alkaloid of L. selago. In an effort to determine the pharmacological activity of selagine the author of this Report and his collaborators examined the alkaloids of L. selago indigenous to northern Canada.22 The major pyridone alkaloid isolated proved to be identical with an authentic sample of huperzine A (48).Since the optical rotation (57) and melting point reported for selagineZ3 differed significantly from those of huperzine A (selagine [a],-99" mp 224-226°C ; huperzine A [a],-147" mp 214-215') it was essential to obtain a sample of the material originally isolated from L. selago and assigned structure (53). The only sample remaining in the late Professor Wiesner's collection was in the form of a KBr pellet used for the determination in 1960 of the IR spectrum. The material recovered from this pellet was identical with huperzine A. It thus appears that the structure (53) of selagine was misassigned and selagine is identical with huperzine A (48). It has also been shown that isoselagine originally assigned structure (54),24 is identical with huperzine A (48).22*25 A minor alkaloid of L.selago has been shown to be 6/3- hydroxyhuperzine A (55).22 Oxidation of (55) with Jones' reagent or manganese dioxide gives the conjugated ketone (56). N-Methylhuperzine B (51) has been reported as one of the minor alkaloids of Huperzia serrata and a new alkaloid huperzinine (57) has been described from this source.26 Phlegmariurus fordii (= Lycopodium fordii) is a tropical epiphytic species of Lycopodium belonging to the Huperzia group. The genus Lycopodium has over 400 species and the taxonomy of the genus and the family Lycopodiaceae is still in a state of Some botanists have subdivided the genus into four genera (Lycopodium Diphasiastrum Lycopodiella and Huperzia) and some have gone so far as to place Huperzia in a separate family Huperziaceae.Since all of the alkaloids reported to date are clearly biogenetically related we prefer to retain the single genus name and where necessary subdivide the genus into the four groups mentioned above as proposed genera. This has the advantage that the genus name is familiar and typifies plants that can be recognized at a glance as being closely related to one an~ther.~' A new alkaloid named phlemariuine M has been isolated from L. fordii and assigned structure (58).28 The lH and 13C NMR spectra are reported for phlemariuine M and are consistent with the proposed structure but other physical properties (m.p.IR UV) are not reported. Another paper from China also discusses the alkaloids of P. f~rdii.~'This paper reports the isolation of a compound Cl,H2,N20 which is named fordimine and assigned structure (49) (huperzine B). It is not clear whether fordimine which is claimed to be a new alkaloid is thought to be a stereoisomer of huperzine B. This paper also reports the isolation of an alkaloid C3,H,,N20, named phlegmariurine C also said to be new. The structure NATURAL PRODUCT REPORTS 1991-W. A. AYER 46 1 ?n i-vi vii viii _L (59) (60) ix x xiv-xvi J (64) (65) Reagents i pyrrolidine PhH p-TsOH ; ii acrylamide dioxane heat ; iii H,O dioxane heat ; iv. KH BnCl THF; v LDA PhSeC1 THF -78 "C; vi NaIO, Et,N MeOH heat; vii H, Pd/C; viii Ag,CO, MeI CHCI,; ix aq.HC1 Me,CO; x KH,(MeO),CO heat; xi methacrolein tetramethylguanidine CH,Cl,; xii MsCl Et,N DMAP CH,CI,; xiii NaOAc HOAc heat; xiv Ph,P=CHCH, THF ; xv PhSH AIBN heat; xvi aq. NaOH THF MeOH heat; xvii SOCl, toluene; xviii NaN, heat; xix MeOH heat; xx TMSI CHCI, heat Scheme 5 assigned to this alkaloid on the basis of limited evidence seems tenuous at best. The pronounced physiological activity of huperzine A (48) has provided the impetus for renewed interest in the synthesis of these tricyclic Lycopodium alkaloids and 1989 saw the completion of two very ~imilar~~~~' syntheses of huperzine A. In one of these,30 the a-pyridone (59) (Scheme 5) was prepared by treating the enamine derived from the monoethylene ketal of cyclohexane- lY4-dione with acrylamide.The resulting mixture of unsaturated lactams was N-benzylated and then dehydro- genated by a-selenation and oxidative elimination to give (59). The pyridone ring in (59) proved rather sensitive in subsequent steps and was thus protected by conversion to the methoxy- pyridine (60). Hydrolysis of the ketal and carbomethoxylation gave the enolized P-ketoester (61). Base-catalyzed addition of (61) to methacrolein and subsequent aldolization gave the bridged ketoalcohol (62). This was transformed via the mesylate to olefin (63). The ethylidene group of requisite stereochemistry was introduced by Wittig reaction followed by isomerization. Hydrolysis provided acid (64) which was subjected to Curtius rearrangement to give urethane (65).Treatment of (65) with trimethylsilyl iodide effected both N- and O-deprotection and afforded racemic huperzine A (48).30 It was later found that the debenzylated derivative of pyridone (59) could be prepared from the monoketal of cyclohexane- 1,4- dione in one step by treatment with methyl propiolate in methanolic ammonia under pressure.32 The synthesis of unnatural ( & )-(2)-huperzine (54) using essentially the route outlined in Scheme 5 but omitting the double bond isomeriz- ation step has also been rep~rted.,~ The (2)-isomer (54) is not as active an acetylcholinesterase inhibitor as huperzine A itself at least when the two racemic forms are compared.32 Several other analogues of huperzine A all less active than the alkaloid itself have been reported.34 The second total synthesis of huperzine A3' also proceeds through the bridged hydroxyketone (62) (Scheme 5) prepared by treating (61) with methacrolein in the presence of methanolic sodium methoxide.Mesylation and elimination gave the olefin (63). Wittig olefination gave a mixture of the 2 and E ethylidene derivatives in which the desired E isomer was the minor component. The mixture of Z/E isomers was treated with methanolic potassium hydroxide which brought about hydrolysis of the less sterically hindered E isomer to provide acid (64). Curtius rearrangement and transformation of the methoxypyridine to the pyridone provided huperzine A. A biogenetically interesting new C, alkaloid possessing the flabellidane nucleus (47) but with 3 additional carbon atoms attached at C-1 has been described.1° The new alkaloid has structure (66) and is named hydroxypropyllycodine because of NATURAL PRODUCT REPORTS 1991 Scheme 6 UCH2 COCHS & (69)R=CH3 (70)R= H its similarity to the C,,N alkaloid lycodine (6).The structure was elucidated mainly by spectroscopic methods particularly NMR and mass spectrometry. The mass spectrum shows an intense peak at m/z 199 attributed to ion (67) formed by fragmentation involving loss of the ‘bridging ’ atoms C- 14 C-15 C-8 a common fragmentation pattern in related Lycopodium alkaloids ; and McLafferty rearrangement with loss of acetaldehyde from the hydroxypropyl side chain. Hydroxypropyllycodine (66) is the first Lycopodium alkaloid encountered possessing a 19-carbon skeleton and as illustrated in Scheme 6 may be constructured biogenetically by combining two pelletierine units and one acetoacetate unit.This is consistent with the ~urrent,~,~ though still tentative biogenetic hypotheses for this group of alkaloids. 3.2 The Fastigiatane (68) Group Fastigiatine (69) and des-N-methylfastigiatine (70) were initially detected in the GC-MS examination of the crude alkaloids of L. fastigiatum a species of Lycopodium indigenous to New Zealand.’ The structure of fastigiatine was established by an X-ray analysis of the free base.35 The pentacyclic structure is related to the tetracyclic flabellidane (47) group with an extra carbon4arbon bond between C-4 and C-10 A C-4 C-10 bond is also found in the alopecurane (71) = (12) group of alkaloid^.^ The paper describing the X-ray analysis also discusses the mass spectrum and gives detailed 13C NMR assignments for fastigiatine.Des-N-methylfastigiatine (70) was correlated with (69) by N-methylation.’ It is suggested that fastigiatine may be derived biogenetically from a C-10 substituted N-methylfalbellidine as shown in (72). 4 References 1 W. A. Ayer in ‘The Alkaloids’ ed. M. F. Grundon (Specialist Periodical Reports) The Royal Society of Chemistry London 1983 Vol. 13 Chapter 12 pp. 277-280. 2 T. A. Blumenkopf and C. H. Heathcock in ‘Alkaloids Chemical and Biological Perspectives’ ed. S. W. Pelletier Vol. 3 John Wiley and Sons New York 1983 Chapter 5.3 D. B. MacLean in ‘The Alkaloids’ ed. A. Brossi Vol. 26 Academic Press New York 1985 Chapter 5. 4 J. S. Liu Y. L. Zhu C. M. Yu Y. Z. Zhou Y. Y. Han F. W. Wu and B. F. Qi Can. J. Chem. 1986 64 837. 5 Y. C. Cheng C. Z. Lu Z. L. Ying W. Y. Ni C. L. Zhang and G. W. Sang New Drugs Clin. Remed. 1986 5 2560. 6 W. A. Ayer L. M. Browne A. W. Elgersma and P. P. Singer Can. J. Chem. 1990 68 1300. 7 R. V. Gerard and D. B. MacLean Phytochemistry 1986 25 1143. 8 V. Manriquez 0. Munoz R. Quintana M. Castillo H. G. von Schnering and K. Peters Acta Crystallogr. Sect. C 1988 44 165. 9 0.M. Munoz M. Castillo and A. San Feliciano J. Nat. Prod. 1990 53 200. 10 W. A. Ayer and G. C. Kasitu Can. J. Chem. 1989 67 1077. 11 G. A.Kraus and Y. S. Hon J. Am. Chem. SOC. 1985 105,4341. 12 G. A. Kraus and Y. S. Hon Heterocycles 1987 25 377. 13 C. H. Heathcock K. M. Smith and T. A. Blumenkopf J. Am. Chem. SOC. 1986 108 5022. 14 C. H. Heathcock T. A. Blumenkopf and K. M. Smith J. Org. Chem. 1989 54 1548. 15 Y. Inubushi H. Ishii T. Harayama R. H. Burnell W. A. Ayer and B. Altenkirk Tetrahedron Lett. 1969 861. 16 R. H. Burnell C. G. Chin B. S. Mootoo and D. R. Taylor Can. J. Chem. 1963 41 3091. 17 T. Ho R. F. Chandler and A. W. Hanson Tetrahedron Lett. 1987 28 5993. 18 G. Mehta and M. S. Reddy Tetrahedron Lett. 1990 31 2039. 19 D. W. Hughes R. V. Gerard and D. B. MacLean Can. J. Chem. 1989 67 1765. 20 Y. E. Wang D. X.Yue and X. C. Tang Acta Pharmacol. Sinica 1986 7 109.21 Drugs Future 1988 13 575. 22 W. A. Ayer L. M. Browne H. Orszanska Z. Valenta and J. S. Liu Can J. Chem. 1989 67 1538. 23 Z. Valenta H. Yoshimura E. F. Rogers M. Ternbah and K. Wiesner Tetrahedron Lett. 1960 26. 24 C. H. Chen and S. S. Lee J. Taiwan Pharm. ASSOC. 1984 36 1. 25 W. A. Ayer H. Orszanska and L. K. Ho Unpublished. 26 S. Q. Yuan and T. T. Wei Acta Pharm. Sinica 1988 23 516. 27 W. J. Cody and D. M. Britton ‘Ferns and fern allies of Canada’ Canadian Government Publishing Centre Ottawa 1989 Chapter 1. NATURAL PRODUCT REPORTS 1991-W. A. AYER 28 Z. C. Miao Z. S. Yang Y. A. Lu and X. T. Liang Acta Chim. Sinica 1989 47 702. 29 B. M. Chu and J. Li Acta Pharm. Sinica 1988 23 115; J. Li Y. Y. Han and J. S. Liu Chinese Tradit.Herbal Drugs 1987 18 50. 30 Y. Xia and A. P. Kozikowski J. Am. Chem. SOC. 1989,111,4116. 31 L. Qian and R. Ji Tetrahedron Lett. 1989 30 2089. 32 A. P. Kozikowski E. R. Reddy and C. P. Miller J. Chem. SOC. Perkin Trans. I 1990 195. 33 A. P. Kozikowski F. Yamada X. C. Tang and I. Hanin Tetra-hedron Lett. 1990 31 6159. 34 A. P. Kozikowski Heterocycles 1990 27,97. 35 R. V. Gerard D. B. MacLean R. Fagianni and C. J. Lock Can. J. Chem. 1986 64 943.

ISSN:0265-0568    

DOI:10.1039/NP9910800455

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

Marine Sterols R. G. Kerra and B. J. Bakerb a Department of Chemistry Florida Atlantic University Boca Raton FL 33431 Department of Chemistry Florida Institute of Technology Melbourne FL 3290I Reviewing the literature published to July 1990 1 Introduction 2 Prokaryotic Marine Organisms 3 Marine Micro-algae 3. I Dinoflagellates 3.2 Diatoms 3.3 Miscellaneous Micro-organisms 4 Macro-algae 4.1 Red Algae 4.2 Brown Algae HOAdd 4.3 Green Algae 5 Sponges R=H,Me 6 Cnidaria 7 Bryozoans Figure 1 8 Mollusca 9 Echinoderms The material covered in this review has been limited to a 10 Annelids 11 Arthropoda discussion of mono- and polyhydroxy sterols as well as sterol 12 Tunicates sulphates from marine organisms.Steroidal saponins prevalent 13 Sediment and Sea Water in many echinoderms have recently been re~iewed~,~ and will 14 References not be dealt with here. A number of review^'-^-'^ dealing at least in part with marine sterols have appeared in the literature since Bergmann’s pioneering work in 1949.15 1 Introduction 2 Prokaryotic Marine Organisms In recent years research in the marine sterol field has progressed Sterols are ubiquitous components of eukaryotic membranes in at an impressive pace. Over two hundred new monohydroxy which they are known to improve the mechanical properties of sterols have now been isolated from marine organisms many the phospholipid bilayers. Prokaryotes are more primitive with no terrestrial counterpart. Sterols are required compounds organisms which possess a cell membrane yet lack mem-in all eukaryotes but the nature of the sterols in a particular braneous organelles.While sterols are essential for eukaryotic organism varies considerably. Sterol mixtures are generally life they are generally not required in prokaryotes. It was once more complex and unconventional structures more common in believed that sterols were absent in prokaryotes. However the less advanced eukaryotes. Prokaryotic organisms are while this is generally true the presence of sterols has normally devoid of sterols but often contain polyterpene been established in the aerobic bacterium Methalococcus alcohols which are believed to be functional equivalents of capsulatus16 as well as in some marine blue-green algae sterols.Thus the evolution of sterols seems to be associated (cyanobacteria). 173 l8 In many instances prokaryotes contain with the progression from prokaryotic to eukariotic life. The polyterpene alcohols or polar caroten~ids~~* 2o with structures basic role of sterols is the maintenance of optimal fluidity of cell comparable with their being structural and functional membranes although these compounds also serve as precursors equivalents of sterols. Ourisson and Rohmer have suggested for the production of diverse steroid classes such as the that these terpenes can be considered as ‘phyletic ancestors’ of as they are produced from similar precursors by polyhydroxylated marine sterols. ~terols~~.~~ This review is intended to provide a description of the similar biosynthetic pathways.characteristic features of sterols of the major marine phyla and There are a number of reports of the isolation of conventional highlight some of the more unusual structures. The material phytosterols (Figure 1) from blue-green algae.21-24 In many of has been organized phylogenetically and where appropriate these cases however the sterols were present in much lower the chemical ecology of sterols and the value of sterol concentrations than in eukaryotes. At these levels contamin- composition as a chemotaxonomic tool are discussed. ation cannot be ruled out especially as it has been ~ho~n~~*~~ Goad’ has suggested that there are four possible sources of in Anacystis nidulans and Azotobacter chromococcum that when sterols in marine invertebrates and that each organism must strict precautions were taken lower sterol concentrations were establish a balance between these factors.The four possible found. Nevertheless there appears to be genuine cases where contributing sources are (a) de novo biosynthesis; (b) as-sterols are present in blue-green algae in similar concentrations similation by host organism of sterols produced by symbiotic to those in eukaryotic algae. In Chlorogloeafritschii,” the yield algae or other associated bacteria or fungi; (c) assimilation of of sterols was 0.13% of the dried algal biomass; the major dietary sterols ; and (d) modification of dietary sterols. sterol was stigmasterol (Al) which was accompanied by smaller Significant progress has been made in determining the amounts of cholesterol (A2).A recent studyla of five species of biogenetic origin of sterols and the present state of knowledge blue-green algae (Anabaena cylindrica A. solitaria A. viguiere in this area will be discussed for each phylum. Techniques used Nostoc carneum and Nodularia harveyana) showed that 24-and problems encountered in biosynthetic studies of marine ethylcholesterol (A3) was the predominant sterol in all five invertebrates have recently been reviewed. species. Sterol mixtures represented ca. 0.03% of the dried 465 NATURAL PRODUCT REPORTS. 1991 side chain HO algae indicating again that they were not due to contamination. Sterol biosynthesis has not thus far been demonstrated in blue-green algae. Knowledge of the occurrence and identity of sterols in blue- green algae is important to an understanding of sterol biosynthesis in invertebrates such as sponges many of which are known to harbour considerable colonies of these symbiotic algae.3 Marine Micro-algae 3.1 Dinoflagellates Dinoflagellates are primitive unicellular eukaryotic organisms and are a major component of phytoplankton which serves as the basis of the marine food chain. The distribution of phytoplankton affects the pattern of marine life and is of enormous economic importance. Since many filter feeding invertebrates obtain sterols from their diet it is also of ecological interest to determine the primary producers of these compounds. Dinoflagellates typically contain a complex mixture of sterols many with unique structural features.8 The presence of both unconventional side chains and nuclei account for the diversity of dinoflagellate sterols.The sterol most characteristic of this group is dinos terol 4a,23,24 (R)-trimethyl- 5a-cholest- 22- en-3P-01 (B4). The unusual features of this sterol include side chain alkylation at both C-23 and C-24 and the presence of a 4a-methyl substituent in the nucleus. Retention of the 4-methyl group is considered to be a primitive feature from a biosynthetic point of view. 27 Other dinoflagellate sterols contain nuclei with an 8,14-double bond and a 4a-methyl group (C)z8which are similar to sterols of some prokaryotes. Many invertebrates particularly coelenterates and some molluscs contain dino- flagellate symbionts known as zooxanthellae which have been assigned to the single species Symbiodinium micro-adriaticum ( = Gymnodinium microadriaticum = Zooxanthella micr~adriatica).~~ Examination of zooxanthellae-containing has revealed the presence of the cyclopropyl sterols gorgosterol (A6) and 23-demethylgorgosterol (A7).Their unique structures raise intriguing questions as to their bio- synthetic origin and biological role. Considerable variation in the sterol composition of zooxanthellae isolated from taxonomically diverse host animals has been ob~erved.~~.~~ This suggests the existence of many species of zooxanthellae. From sterol analyses of zooxanthellae from eight taxonomically different hosts,33 three cohesive and distinct groups were evident.It appears therefore that sterol analysis can be a valuable tool in taxonomic studies of dinoflagellates. A significant proportion of some marine sediments is composed of dinoflagellates and characteristic sterols such as dinosterol (B4) and gorgosterol (A6) serve as valuable ‘biological markers ’ (see Section 13). There has been much discussion concerning the biosynthesis of dinosterol and g~rgosterol,~-~~-~~ yet the precise mechanism remains to be established. The biogenetic origin of gorgosterol (A6) was initially not amenable to experimental scrutiny as (A6) had only been found in zooxanthellae-containing hosts (coelenterates molluscs and algae). In a study of three Caribbean g~rgonians,~~ gorgosterol (A6) was isolated from the zooxanthellae-containing host but not found in the isolated zooxanthellae.Interestingly gorgosterol was absent in three Pacific gorgonians that were devoid of zooxanthellae suggesting that these symbionts may be involved in the production of (A6).30While dinosterol is usually present in cultured zooxanthellae numerous analyses32 revealed that the cyclopropanes gorgosterol (A6) and 23-demethylgorgosterol (A7) generally are not present. In a few cases,33 however these cyclopropanes have been isolated. It therefore appears that while some zooxanthellae are capable of synthesizing NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER Scheme 1 I (6) chain 4 0P gorgosterol the cyclopropanation of dinosterol (B4) [assuming (B4) is the precursor] is greatly facilitated when the zooxanthellae form a symbiotic association with a host invertebrate.It is not yet clear whether the dinoflagellate transfers dinosterol (B4) to the host which then converts it to gorgosterol (A6) or the dinoflagellate is responsible for the production of (A6) but this only occurs in the presence of a suitable host. Isolation of some likely precursors to gorgosterol has led to the proposal of plausible biosynthetic pathways. It has been postulated3’ that gorgosterol could be produced by the bioalkylation at C-23 of the brassicasterol side chain (8) to initially form the dinosterol side chain (4). Cyclopropanation of the C,,-C, double bond would then afford a gorgosterol side chain (6) (Scheme 1).The co-occurrence of (4,24)-dimethyl-5a- cholesta-22-en-3/3-01 (B8) dinosterol (B4) and gorgosterol (A6)33offered indirect support for this proposal. An alternative pathway has been proposed based on the isolation of 23-methyl-22-dehydrocholesterol(A9).36 The presence of this sterol suggests the operation of a fundamentally different metabolic route involving the direct bioalkylation at C-23 of the 22-dehydrocholesterol side chain (10). Formation of the dinosterol side chain from (9) could then occur by (4) Scheme 2 side chain HO (D) Scheme 3 introduction of a A24double bond followed by bioalkylation at C-24 (Scheme 1). The 4a-methyl derivative (B9) has also been isolated.39 Preliminary biosynthetic studies40 using an axenic culture of Crypthecodinium cohnii and CD,-labelled methionine demonstrated that the C-23 methyl group in dinosterol was introduced intact by transmethylation via S-adenosyl-methionine (SAM).The C-24 methyl group of dinosterol was shown to contain only two deuterium atoms implying the formation of a 24-methylene intermediate (1 1). Subsequent biosynthetic experiments using Peridinium balticum were performed with 13CH3- and CD3-labelled methionine.s Here NMR analysis showed that two deuterium atoms were present on the cyclopropyl methylene of 4a-methylgorgostanol (B6). The presence of the latter sterol (B6)41 suggests that cyclo- propanation precedes demethylation at C-4; however this remains to be verified experimentally.The above results lend support to the originally postulated scheme for the biosynthesis of gorgosterol as outlined in Scheme 2. The presence of 3-keto steroids in some dinoflagellates particularly 4a723,24-trimethylcholest-22-en-3-one[dino-sterone (D4)] has led to the of a novel biosynthetic route to the dinosterol nucleus (Scheme 3). NATURAL PRODUCT REPORTS I991 t (7) (13) Scheme 4 (15) C24R (17) c24s Scheme 5 HLOP .+ * L O P P 1 R=MeI+-,.=" (15/17) (16) Scheme 6 Shortly after the isolation of dinosterol (B4) and gorgosterol (A6) from dinoflagellates a new Al'-unsaturated sterol peridonasterol (B 12) was isolated from two cultured marine dinoflagellates Peridinium foliacium and P. baltic~m.~~ Two pathways for the biosynthesis of peridonasterol (B12) were postulated (Scheme 4).42 One possibility is the double bond migration of a Azz unsaturated side chain presumably by a biological reduction4ehydrogenation.The other invoked the ring opening of a 23-demethylgorgosterol precursor (7) to produce a A20(22)unsaturated side chain (1 3) which undergoes isomerization to peridonasterol (B12). A novel sterol 23-methylene-24-methylcholestanol(E14) has been tentatively identified from Gonyaulax moniZata by NMR and mass spectral data.43 As with the dinosterol side chain (4) the biosynthesis of (14) presumably arises from SAM bio- methylation of the brassicasterol side chain followed by loss of a proton from the C-29 methyl group. Another striking feature of the sterols of dinoflagellates is the abundance of sterols with A8(14)and A14 nuclei (C) & (F).35,38 In Scrippsiella tro~hoidea,~~ sterols with the A8(14) unsaturation account for 26% of the total sterols and in Amphidinium ~arterae~~ they represent 85% of the total free sterols.The preponderance of these compounds suggests that they have a biological function and that the unusual unsaturation is important for this. The biosynthetically intriguing 27-nor-(24R)-methylcholest-5,22-dien-3/3-01 (A15) and 24-nor-cholest-5,22-diene-3~-01 (A 16) have been isolated from the dinoflagellate Gyrnnodinium simplex,44where they comprised 35 YOof the total sterols. The former sterol (A15) has the opposite configuration at C-24 to that of occelasterol (A17) which has the same carbon skeleton and has been isolated from various invertebrates.The presence of these sterols in large quantities in G. simplex indicates that dinoflagellates are an important source of nor- sterols which are frequently found in marine invertebrates. The biosynthesis of these compounds is not known; however two different routes have been suggested. The most likely route involves demethylation of the (epi)brassicasterol side chain (8) to (epi)occelasterol (1 5/ 17) followed by a second demethylation to (16) (Scheme 5).45 De novo synthesis of sterols (15/17) and ( 16) involving nor-isoprenoid pyrophosphates and nor-squalene (Scheme 6) has been suggested44 as an alternative route. 3.2 Diatoms Diatoms comprise a significant portion of the phytoplankton NATURAL PRODUCT REPORTS 1991-R.G. KERR AND B. J. BAKER and thus as with dinoflagellates knowledge of the sterols of this group is important to an understanding of the chemical ecology of sterols. The sterols of diatoms have not received the same amount of attention as the dinoflagellates. The majority of sterol analyses of reveal that these organisms are generally composed of conventional C-27 C-28 and C-29 phytosterols (Figure 1). Nevertheless this is an important observation as many filter feeding invertebrates known to be incapable of de novo sterol biosynthesis contain the same sterols. There are reports of the rare occurrence of 22-dehydrocholesterol (A10) as the major sterol of the diatoms Biddulphia sine~sis~~ Sterol (A 10) is and Nitzschia ~y1indru.s.~~ found as a minor component of many organisms yet the biosynthesis of this simple derivative of cholesterol has not been elucidated.The diatoms B. sineusis and N. cylindrus would be good candidates for such a biosynthetic investigation. 3.3 Miscellaneous Micro-organisms Coccolithophorids (class Haptophyceae) are microscopic uni- cellular algae which are widely distributed in the oceans at times providing a major component of phytoplankton. Sterol compositions of coccolithophorids are generally very simple. In each of four species,55 Emiliania huxleyi Hymenomonas carterae Isochrysis golbana and Crystallolithus hyalinus the major sterol was brassicasterol (A8).However the unusual sterol 4a-demethyldinosterol (A4) has been isolated as a minor component in H. carterae," distinguishing this algae from the other three in this study. Hymenosulphate (G4) a sterol sulphate with the dinosterol side chain has recently been isolated56 from the coccoli-thophorid Hymenomonas sp. While many sterol sulphates have / been isolated from sponges and echinoderms this is the first documentation of the isolation of such a compound from marine micro-algae. Hymenosulphate has potent sarcoplasmic reticulum Ca-releasing a~tivity.~~ The marine unicellular chlorophyte Dunaliella tertiolecta is unusual in that it has no cell wall.57 The major sterols of this ~n~~~~~ algae were ~ h oto be the A5r7sterols ergosterol (H8) and 7-dehydrostigmasterol (H1).The minor components included A' and A8,14sterols. Apparently the absence of a cell wall does not affect the sterol composition as the sterols of D. tertiolecta are similar to those of other unicellular chlorophytes which possess a cell wall. This is an interesting observation since the biological role of sterols is the regulation of membrane fluidity where the nature of the sterol is critical.59 The biosynthesis of brassicasterol (A8) and poriferasterol (A 18) in a number of chrysophytes has been investigated using various approaches. The feeding of 3H-labelled 1401-methyl-5a- ergost-8-en-3P-01 (18) and 5a-ergost-8( 14)-en-3P-ol (58) to Ochromonas mallharnensis6O demonstrated that these com- NATURAL PRODUCT REPORTS 1991 side chain .-..&.*..A %.A .#me HO \ pounds are both precursors of the chrysophyte sterols (A8) and (A18).The hypocholesterolemic drug AY-994461 and the sterol synthesis inhibitor Triparin~l~~,~~ showed a pronounced change in the sterol composition in some chrysophytes notably a build up of sterols. This suggests the intermediacy of sterols with a nucleus and corroborates the results from the above 3H-labelling studies. The capability for de novo sterol biosynthesis in a chrysophyte was demonstrated by the incubation of cultures of Pseudoisochrysis paradoxa with 14C- and 3H-mevalonic acid.50 Cell-free extracts of Trebouxia sp. and Scenedesrnus obliquus were used to examine the presence of methyl-transferase activity in these chrysophyte~.~~ Both 3H-labelled cycloartenol (K 19) and lanosterol (L 19) (the sterol precursors of photosynthetic and nonphotosynthetic organisms respectively) were transformed to their 24-methylated deriva- tives indicating the relatively low substrate specificity of the transme thy la ting enzyme.A detailed 66 of an unidentified chrysophyte algae has revealed novel sterols as minor components including epimers of the cyclopropane 24,28-methylene-Sstigmasten-3/3-01 (A20) as well as E-24-propylidenecholesterol(A21) as the main sterol. Labelling experimentP indicate that the 24- propylidene sterol is not formed by ring opening of the cyclopropane (A20) and thus presumably arises from bio- alkylation of a 24-vinyl sterol (A22).Eighteen different sterols have been isolated6' from the marine Euglenid Eutreptia viridis. Included in this mixture was a novel A23-unsaturated sterol viz. 24-ethylcholesta-5,7,232-trien-3P-01 (H23). There are two reports68p69 of the sterols of marine occurring yeast. Sterols appear to be of some taxonomic value for this group as the A5q7 sterol ergosterol (H8) was found to be the predominant sterol (69-97 %) of all six species analyzed. 4 Macro-algae 4.1 Red Algae Sterols of macro-algae show characteristic patterns within the various divisions. As described above phytosterols generally have a structure similar to cholesterol with the exception of a methyl or ethyl group at C-24 and A2' or unsaturation NATURAL PRODUCT REPORTS 1991-R.G. KERR AND B. J. BAKER 47 1 0 (Figure 1). The sterols of red algae (Rhodophyta) are unusual as they generally lack C-24 alkylation; cholesterol (A2) with its 22-dehydro and 24-dehydro derivatives (A 10) and (A 19) are essentially the only sterols of this group. Conventional C-26 C-28 and C-29 sterols have occasionally been found in trace amounts. Rytiphlea tin~toria~~ are and Goniotrichum eleg~ns~~ exceptions as they contain campesterol (A24) and brassicasterol (A8) respectively as their major sterols. The Rhodophyta are composed of two subclasses the Bangiophycidae and Florideophycidae. 71 With a few exceptions sterol composition is consistent with this div- ision.71-76 The Florideophycidae generally contain cholesterol as the sole sterol while desmosterol (A19) and 22-dehydro- cholesterol (A1 0) are generally found in the Bangiophycidae.Within the subclass Bangiophycidae the orders Bangioles and Porphyridioles predominantly contain desmoster01~~~ 74 (A 19) and 22-dehydro~holesterol~~ (A10) respectively. The ratio of cholesterol (A2) to desmosterol (A19) varies seasonally in Rhodymenia palmata.77~ 78 Desmosterol maxima occurred in May and November which corresponds to the times of maximum total sterol content of the algae (0.019% of the dry weight). The A5nucleus is the predominant one in the sterols of red algae although cholestanol (E2) has been found7s as the major sterol of three species of the order Gelidiales and one of Cryptonemiales (subclass Florideophycidae).From the above discussion it appears that at least most red algae are incapable of side chain alkylation. However the presence or absence of methyl transferase activity using for instance tissue homogenates has yet to be established. R. palmata appears to be capable of de novo sterol biosynthesis as radioactive sterols were obtained after the feeding of 14C- acetate and 14C-mevalonic acid.80 Side chain hydroxylated sterols have been isolated from several Rhodophyta. Dihydroxy steroids (A25) and (A26) have been isolated as minor components of Asparagopsis armata,81 Rissoella verruculosa,82and Rhodymenia palmata ;83 and diol (A25) has been found in Liagora distentas4 and Scinaia fur cell at^.^^ In addition 24,25-epoxycholesterol (A27) was also isolated from A.arrnata8l and R. verruculosa.82 It has been sugge~ted~~.~~ that these steroid diols are artefacts generated in the air drying of algae. Diols (A25) and (A26) were subsequently shown to result from the autoxidation of desmosterol (A19),81q82 however these diols were also present in a freshly harvested and carefully handled sample of Asparagopsis armata.81 Two ecdysone-like metabolites pinnasterol (M28) and its 2- acetyl derivative have been isolated from the red algae Laurencia pinnata and the structures determined by X-ray crystallogra- ~hy.*~ These sterols show biological activity as moulting hormones. 4.2 Brown Algae It is well known that fucosterol (A29) is the predominant sterol of brown algae (Phaeophyta).72,86-88 It is often the sole sterol (33) (34) (35) side chain side chain present but has also been fo~nd~~~~~,~~ to co-occur with its biosynthetic precursors 24-methylene cholesterol (A 11) and desmosterol (A19). Other conventional sterols such as chol- esterol (A2) and 22-dehydrocholesterol (A 10) are occasionally encountered as minor components in this group.8s*91,s2 As with other algae seasonal variations in sterols have been observed in members of the Phaeophyta. In Padina vickersiae and Cystoseira zosteroides the yield of total sterols increased from March to June (0.01-0.07%0 dry weight of algae) corre-sponding to growth spurts in the summer. A lack of sterols with saturated side chains in brown algae may reflect an inability to reduce double bonds.Side chain-oxygenated sterols saringosterol (A30) and 24- oxocholesterol (A3 1) have been isolated87~8s~s3~ 94 from a number of brown algae. In 1970 it was recognizeds2 that both of these compounds can arise from the air oxidation of fucosterol (A29) as saringosterol (A30) and 24-oxocholesterol (A3 1) were isolated from a sample of stored milled and dried Ascophyllum nodosum but were not present in freshly harvested material. In a recent report concerning the lipids of the brown algae Hizikia the fusiformi~,~~ presence of saringosterol (A30) 24-0x0-cholesterol (A3 1 ) and 24,28-epoxyfucostero1 (A32) was noted. Only 24-oxocholesterol (A3 1) was suspected of being an artefact as when air was bubbled through a solution of fucosterol (A29) for 50 h in addition to recovered (A29) the sole product was (A31).Another novel steroid diol (A33) was isolateds5 from Desmarestia aculeata collected in the Bay of Fundy. The side chain of this unprecedented C, sterol poses an interesting biosynthetic problem. An unusual sterol 24-vinyloxycholesta-5,23-dien-3~-ol (A34) was isolated as a minor component of the sterols of the brown algae Sargassum th~mbergii.~~ Sterol (A34) is not believed to be artefactual as it was found in both fresh and dried material. The authors propose that (A34) is derived from the cleavage of the C-24,28 bond of 24,28-epoxyfucostero1 (A32). Cytosterol (A35) a sterol with a side chain cyclopropane has been found in numerous species of the genus Cy~toseira.~~ This is an interesting observation as sterol cyclopropanes are extremely rare in macro-algae.Sterols containing a 3,5-dien-7-one (N) and a 7a-hydroxy- cholesterol nucleus (0)have been found in Fucus evanescens.s4 NPR 8 NATURAL PRODUCT REPORTS 1991 f The authors suggest that (N) is an unlikely structure for a natural product and was formed during the isolation procedure from 3P-hydroxy-5-stigrnasten-7-one (P3). 4.3 Green Algae The Rhodophyta and Phaeophyta are considered to be distinct from other algae and apparently are evolutionary dead ends. The green algae (Chlorophyta) are thought to have led to the evolution of higher plants.98 Whereas red and brown algae are characterized by predominantly containing the single sterols cholesterol and fucosterol respectively green algae typically have complex mixtures of sterols.Generalizations about the sterols of green algae are difficult to make. Chlorophyta are known to contain cholesterol (A2)97,98 as is found in the Rhodophyta fucosterol (A29)99 as in the Phaeophyta isofucosterol (A36)97,99,loo as in higher plants and ergosterol (H8)97as is found in many fungi. In addition the C-25 methylene sterols codisterol (A37) and clerosterol (A38) have also been fo~nd~~~-~~~ in Chlorophyta. The total sterol content of green algae varies from one species to an~ther'~.~~~ (0.05YOof the dry weight in Hufimedu incrassuta to 0.38% in Chfamydomonus rheinhardi) as well as seasonally within a species.1oo Sterol compositions of green algae can be of value to the systematist as the occurrence of certain sterols is often characteristic of particular orders.72 979983101 For instance examination of eight Mediterranean green algaelol showed that isofucosterol (A36) is a typical component of Ulotricholes and that clerosterol (A38) is common to the genus Codium.These results are in good agreement with subsequent work which showed that clionasterol (A39) is characteristic of all species of the Siphonales not belonging to the genus Codi~m.~'More recently it was suggesteds9 that isofucosterol (A36) may not be a reliable marker for the Ulotricholes as fucosterol (A29) was found to be the predominant sterol of some members of this group. It has been SuggestedlOl that the occurrence of isofucosterol (A36) in Chlorophyta confirms their ancestral role in the evolution of higher plants.There is one report" of the occurrence of a sterol with a shortened side chain 24-nor-22-dehydrocholesterol (A 16) in the green algae Ulva rigida. Sterol (A16) has been found in red algae but only in trace quantities and it is suspected that (A16) is produced by micro-organisms present in the red algae. In the Chlorophyta U. rigida sterol (A16) represented 30% of the total free sterols however the detection of (A16) was shown'' to be related to the presence of large amounts of phytoplankton. As is frequently found in Phaeophyta saringosterol (A30) the 24-a configuration thus demonstrating agreement with the higher classification of these plants.As originally suggested lo5 assignment of C-24 configuration has both taxonomic and biosynthetic significance. 5 Sponges Sponges (Porifera) were the first invertebrates shown to contain sterols other than chole~terol.~~.~~~ Since then it has become clear that sponges have the most diverse array of novel sterols of all organisms.11-13.106,107 Generally ten to twenty monohydroxylated sterols are present in a given sponge although this number is known to vary between onelos and seventy-four.log In the latter case Axineffa canna bin^'^' was shown to contain A5 A7 A' A537.s(11), A5s7 5a-saturated and 5a-metho~y-A~,~(~~) nuclei each with different side chains. In light of the rich diversity of sponge sterols their use in chemotaxonomy has been investigated.In fact sterols were the first class of compounds to be used for chemotaxonomic studies of sponges. In the first such investigation by Bergmann,15.110 it was shown that all sponges contain sterols and that the sterol composition is different for each sponge. Some of Bergmann's findings were inaccurate due to the lack of modern analytical instruments at that time however his ideas led to future investigations. 111,112* 113 In two separate studies Bergquist and co-workers examined the sterols of 55 species of the Demospongiae'll and later 23 additional members of the Demospongiae and four belonging to the class Calcarea.l12 Sterol composition was found to be qualitatively consistent with season and location and the occurrence of particular sterols was shown to be of some chemotaxonomic value.Sponges belonging to the order Verongida have similar patterns of sterols which are characterized by a high content of aplysterol (A40) an unconventional sterol with methyl groups at C-24 and C-26. Two sponges within the order Verongida and belonging to the family Lanthellidae have very similar sterol compositions and both lack aplystane sterols thus lending support to the separation of the Lanthellidae from other members of the order Verongida. Dictyoceratid sponges are characterized by an extremely low sterol content."l-112 Amphimedon sp. (Haplosclerida) is such an example and it has been suggested that the triterpene diisocyanoadociane (41) may either replace or co-occur with sterols in cell memberane~.~'~ This is analogous to the triterpenes that are believed to be functional equivalents of sterols in many prokaryotes.19,20 A number of representative examples of unconventional sterol side chains are shown in Figure 2 which with few exceptions are unique to the phylum Porifera.Certain points has been isolated from the Chlorophyta Enteromorphu finz~.~~ The sterol analysis was performed on fresh algae and it is therefore believed" that this sterol is not an artefact from the air oxidation of fucosterol as has been suspectedg2 in certain brown algae. The configuration at C-24 of sterols from some marine Phanerogames collected in the Mediterranean has been examined.lo4 In general the sterols of algae have the 24-P configuration while higher plants primarily produce 24-a sterols.The most abundant sterols in the Phanerogames had should be noted about these structures. The most intriguing feature is the presence of numerous cyclopropanes such as petrosterol (A60) initially isolated from Petrosiu ficiformi~,'~~ hebesterol (A56) from Petrosiu hebes,'16 nicasterol (A59) from Cufyx nicaeensi~"~and 24,26-cyclocholesterol (A55) from an unidentified deep seal sponge;lls and one group of cyclo-propenes (calysterol A63) and its isomers (A64) and (A65) from Cufyxnicueensis).llg~ 120Also alkylation (biomethylation) can occur at ail carbons of the cholesterol side chain (C,,-C,,). NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B.J. BAKER (42) (45) (46) (49) R =H (50)R =Me 0 (55) R =H (56) R=Et (61) R=H (62) R =Me For example aplysterol (A40) was one of the first sterols found12' with a methyl group at C-26 ;25-methylxestosterol (A45)122 has 'extra' methyl groups at C-24 C-25 C-26 and C- 27 ;ficisterol (A48)123 has a 23-ethyl group and 22-methylene- cholesterol (A52) has been isolated124 from Halichondria panicea. Sterol side chains with three four and in one case five 'extra' methyl groups have been reported from sponges. Sutinasterol (A46) apparently the result of four bio-methylations was isolatedlo8 as the predominant sterol (96 YO of steroi mixture) of a Xestospongia sp. from the Caribbean. Its structure was determined by 'H-and 13C-NMR spectroscopy (39) (43) (44) (47) (52) R (57) R =H (58) R =Me (59) (63) and the stereochemistry of the C-24 ethyl group established by X-ray crystallography.Also present in this sponge in trace quantities is sterol (A47) which is the product of an unprecedented five biomethylations and thus represents the largest free sterol isolated to date. It is also noteworthy that a number of sterols with quaternary carbons such as mutasterol (A43),'25 durissimasterol (A44),126 and 25-methylxestosterol (A45)125 have been isolated from various sponges. Two rare sterols with doubly unsaturated side chains [(H49) and (H50)] have been isolated from Ciocalypta sp.12' collected in Hawaii and Pseudaxinella lanaecharta128 from the Senegalese coast.Also the allenic sterol (A51) has been isolated from NATURAL PRODUCT REPORTS 1991 30 Scheme 7 Axinefla austrufiensiP as the major sterol and in trace quantities from Calfyspongia dzflu~a.'~~ Sponges also contain sterols with degraded side chains such as 24-nor-22-dehydro- cholesterol (A 16) 24-methyl-27-norcholesterol (A 17) and the rare 26,27-bisnor-22-dehydrocholestero1130 (A53) found in Damariana hawaiiana. The biosynthetic origin of some of these novel sterols has been elucidated. The 24-isopropylcholesterols (A68) (A69) and (A70) in the Great Barrier Reef sponge Pseudaxinyssa sp. were one of the early biosynthetic targets addressed by Djerassi and co-w~rkers.~~~~ This sponge is of particular interest as 98% of the sterol mixture is composed of unconventional sterols; and analysis of membrane fractions of this sponge133 confirmed that these unusual sterols are almost exclusively associated with membranes and are thus likely to have a role in membrane function.The first two alkylations to (iso)fucosterol (A29) and (A36) were shown to proceed along a pathway analogous to that established in ~1ants.l~~ Biomethylation at C- 28 followed by proton migration from C-28 to C-24 and subsequent proton abstraction from an adjacent methyl group affords the C,,-isopropenyl sterol (A68). A lack of substrate specificity was observed in this key biomethylation step as both E and 2 isomers of 24-ethylidenecholesterol (A29/A36) were transformed to the three isopropylcholesterols.The regio-selectivity of proton migration was established by the feeding of [26(27)-3H] 24-methylenecholesterol. Stereoselectivity was observed in this final step as biomethylation at C-28 occurred exclusively on the a-face. The reported decreasing specific activity in sterols (A68) (A69) and (A70) for all feeding experiments is in agreement with the proposed biosynthetic scheme (Scheme 7). The biosynthesis of mutasterol (A43) in the Caribbean sponge Xestospongia muta has recently been el~cidated,'~~ representing the first such investigation of a sterol with a quaternary carbon in its side chain. X. muta is one of the large 'barrel ' sponges similar to X. testudinaria in Australia. Examination of specimens of the Australian Xestospongia showed that there were two different sterol compositions with some of the organisms possessing xestosterol (A42) as the predominant sterol while the remainder only contained con- ventional sterols.There were no obvious morphological differences between the two types except that sponges containing xestosterol (A42) had a softer (less dense) con-sistency. Three different sterol compositions were found in nine specimens of X. muta from Puerto Ri~0.l~~ Two of these were very similar to the two types of X. testudinaria and a third contained mutasterol (A43) as well as 24(28)-dehydroaplysterol (A71) and verongulasterol (A72). No differences were noted in NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER 475 I I (37) (73) 1 .-..+- I-"* / -H+ .*..A-I-.+ (74/75) I (43) (71) Scheme 8 the morphology of the specimens of X.muta. In view of the similarity in morphology and sterol contents of the Australian and Caribbean Xestospongias it has been that there may be three separate species of the Caribbean X. muta with two of these also occurring in Australia. However unambiguous demonstration of this awaits a more complete taxonomic investigation. The biosynthesis of mutasterol was to proceed via three consecutive SAM biomethylations of desmosterol (A 19) (epi)codisterol (A37/A73) and 24,26-dimethyldesmosterol (A74/A75). Considerable stereoselectivity was observed for the second step [codisterol (A37) favoured 10 times over its C-24 epimer (A73)] while there was no difference in the metabolisms of the E and 2 isomers of 24,26-dimethyldesmosterol (A74/A75) (Scheme 8).Undoubtedly the most unusual and biosynthetically in- triguing group of marine sterols are the cyclopropenes calysterol (A63) and its isomers (A64) and (A65). Naturally occurring cyclopropenes are extremely rare and except for the calysterols have been limited to sterculic acid and its congeners.136-138 Calysterol (A63) was first isolated by Fattorusso and co- worker~~~~ as the major sterol of the Mediterranean sponge Calyx nicaeensis (= C. niceaensis). Later with the aid of reversed phase HPLC Djerassi and co-workers120 also isolated 23H-isocalysterol (A65) 24H-isocalysterol (A64),139 23,24- dihydrocalysterol (A58) fucosterol (A29) 23-dehydro-24- ethylcholesterol (A76) as well as the two acetylenes (A66) and (A67) from the same sponge.The C-23 epimer of 23H-isocalysterol has also been as the major sterol of the Caribbean sponge Calyx podatypa. It appears that cyclo- propenes are characteristic of Calyx species providing ad- ditional support for the use of sterol composition as a chemotaxonomic tool in sponges. Also the two Calyx sponges are the only known source of steroidal acetylenes. Early feeding experiments141 demonstrated that fucosterol (A29) was transformed to calysterol (A63) in C. nicaeensis. It was later that 24-methylenecholesterol (A1 1) and dihydrocalysterol (A58) can also act as precursors to the three isomeric cyclopropenes (A63) (A64) and (A65) as well as to the acetylenes (A66) and (A67) (Scheme 9).Feeding of labelled calysterol (A63) and its isomers dem~nstrated'~~ the existence of an isomerization of the cyclopropenes and the lack of hydrogenation of (A63) (A64) and (A65) to dihydrocalysterol (A58). This implied that dehydrogenation of dihydrocalysterol (A58) produces a single cyclopropene which then undergoes an isomerization to the others. Using [3,28-3H,]-labelled dihydro- calysterol (A58) it was demon~trated'~~ that calysterol (A63) is not the initial dehydrogenation product but arises from isomerization of (A64) or (A65). The cyclopropane petrosterol (A60) was initially isolated as the predominant sterol of the Mediterranean sponge Petrosia Jiciformis115 and subsequently found in P.he be^,^^^ Halicondria sp.146 and very recently isolated with dihydrocalysterol (A58) from the Bahamian sponge Cribrocalina vasculum.14' Incor-poration experiments with P.fifi~iformisl~~ revealed that petro- sterol (A60) is not synthesized from epicodisterol (A73) as is known for aplysterol (A40)149 (Scheme lo) but rather from 24- methylene cholesterol (A1 1) (Scheme 11). The conversion of this latter sterol to petrosterol (A60) in C. vascul~m'~~ was shown to proceed via an unprecedented cyclopropane-cyclopropane rearrangement in which the intermediacy of a NATURAL PRODUCT REPORTS 1991 * . (63) Scheme 9 .%+y* d (73) %.+* \ fistularisAplysina I -*-A Scheme 10 / (60) Scheme 11 NATURAL PRODUCT REPORTS 1991-R.G. KERR AND B. J. BAKER side chain (R) Scheme 12 (S2) Scheme 13 protonated dihydrocalysterol was postulated. Degradation experiment^'^^ clearly demonstrated that C-28 of 24-methylene cholesterol (A1 1) gives rise to C-24 of petrosterol (A60). Due to the structural similarity of the petrosterol side chain (60) and the aplystane sterols e.g. (40) it is tempting to assume that sponges containing these sterols are closely related. Analysis of the biosynthetic routes to aplysterol (A40) and petrosterol (A60) show that in spite of their structural similarity the 'biosynthetic machinery ' used in the generation of (A40) and (A60) is quite different. Thus when using sterols as a chemotaxonomic tool it is important to consider both the structure and its biosynthesis.The aforementioned biosynthetic experiments were per-formed using five sponges. While this has provided valuable information concerning sterol metabolism in sponges the lengthy incubation periods and the logistical problems of working with sponges from remote locations or from the deep sea is limiting. The use of cell-free extracts of sponges solves these problems and has recently been des~ribed.'~~,~~~ In one study methyl transfer reactions were investigated using commercially available radiolabelled S-adenosylmethionine 150 while in the other labelled sterols were In addition to unconventional side chains sterols with unusual nuclei have also been isolated from sponges.Sterols with a contracted A-ring the 3P-(hydroxymethyl)-~-norsteranes (Q) have been found in sponges of the families Axinellidae (including Acanthella cri~ta-galli,'~~ Acanthella a~rantiaca,'~~ A. verr~cosa,'~~~ Axinella SP.,'~~ 156 Homaxinella trachys,'j7 Teichaxinella marchella,158 and Phakellia aruensi~l~~) and Hymeniacidonidae (Hymeniacidon perlevis). 160*16' Some of the first biosynthetic work performed on sponges demon-~tratedl~~~l~~ that A-nor sterols are the result of a ring contraction of A5 sterols. Feeding experiments with A. verruco~a~~~* 156 162*163 using (4-14C)-cholesterol showed that C- 4 of cholesterol gives rise to C-3 of the ring-contracted nucleus via the A4-3-keto intermediate (R) (Scheme 12). The majority of sponges containing A-nor steranes lack sterols with conventional nuclei indicating the presence of an efficient enzyme system responsible for A-ring contraction and the likelihood that A-nor steranes play a structural role in cell membranes.A-nor sterols with unusual nuclear unsaturation and un-conventional side chains have been isolated from some of the aforementioned sponges. For instance ~-norgorgostanol'~~ (Q6) was found in Stylotella agminata and the first A15 unsaturated sterol (3P-hydroxymethy1)- nor-Sol-choles t- 15-ene (S2) has been isolated from Homaxinella trachys15' and Phakellia aruen~is.'~~ Labelling experiments165 demonstrated that the latter sterol (S2) is generated by ring contraction of dietary cholesterol followed by introduction of the A15 unsaturation rather than by ring contraction of 15-dehydro- cholesterol (Scheme 13).NATURAL PRODUCT REPORTS 1991 side chain HO side chain As described above many sponges of the family Axinellidae contain sterols with the unusual A-nor nucleus. The Mediter- ranean sponge of this family Axinella polypoides,166-168 contains sterols with the extrmely rare 19-nor nucleus (T). As with many Axinellid sponges there are no conventional sterols in A. polypoides and thus as with the A-nor steranes in other Axinellidae 19-nor sterols are believed to have a structural role in the cell membrane. In a competitive feeding experiment,166* 167 it was demonstrated that A. polypoides was able to transform cholesterol (A2) but not cholestanol (E2) to 19-nor-cholestanol (T2) thus indicating that the A5 unsaturation is involved in the loss of the angular methyl group.The presence of sterols with a variety of conventional side chains and the A-nor nucleus has been reported168 in A. polypoides and it has been suggested that the 19-nor stanols with a A22 double bond (42% of the total sterols) could provide a source of starting material for certain oral contraceptives which also contain the rare 19-nor nucleus. Sterols with the very unusual 4-meth~lene-A~'~~) nucleus (U) have been isolated as the major component in Theonella conica and as a minor sterol in T. s~inhoei,'~~ both from the Red Sea. Other unconventional sponge sterol nuclei include A7s9(11) sterols (V) of Haliclona JEave~cens,~~~ and 5P-stanols (W) in Calyx nicaeensis.171 The latter class of sterols are believed to arise from bacterial metabolism of conventional A5 sterols.l'l One question which remained unanswered until recently was whether sponges are capable of de novo sterol biosynthesis.For many years results concerning the operation of this pathway were inconcl~sive.~~ 172-174 However the feeding of 3H-labelled lanosterol (L19) has dem~nstratedl~~ that most sponges are indeed capable of this process. In addition labelled cycloartenol (K 19) the sterol precursor of photosynthetic organisms was also transformed to the sterols of many sponges. It is noteworthy that lanosterol and cycloartenol were transformed into both conventional and unconventional sterols.From subsequent experiments with labelled squalene (77),176 it is evident that while sponges are able to modify cycloartenol (K19) the sole cyclization product of squalene (77) is lanosterol (L19) (Scheme 14). The observed metabolism of cycloartenol was attributed to the sponge cells and not photosynthetic symbionts such as blue-green algae as sponges that were shown to be devoid of these symbionts by electron microscopy were among those that transformed both lanosterol (L19) and cycloartenol (K 19). 175 In Xestospongia rn~ta,l~~ squalene was transformed to the unconventional sterols such as mutasterol (A43) verongulasterol (A72) and 24 (28)-dehydroaplysterol (A7I) but with the exception of cholesterol not to the side chain conventional sterols.This indicates that the latter group are obtained from the diet. The production of cholesterol in sponges by the dealkylation of 24(28)-unsaturated sterols was recently demonstrated. 177 This was a surprizing finding as certain sponges (Tethya aurantia and Microciona prolifera) are also known to be capable of de novo biosynthesis and side chain alkylation. The reason for the co-occurrence of enzymes responsible for alkylation dealkylation and de novo sterol synthesis in a single organism is unclear. However such metabolic plasticity does have obvious adaptive value. The mechanism of the degradation seems to largely parallel that described for insects,6 178 which involves the intermediacy of a 24,28-epoxide (78) (79) (Scheme 15).Whole organism feeding experiments177. 179 showed that 24-methylenecholesterol (All) and (iso)fucosterol (A29/A36) as well as their 24(28)- epoxides (A78) (A79) are converted to desmosterol (A19) and cholesterol (A2). The conversion of 24-methylenecholesterol (All) to its epoxide(s) (A79) was later demon~tratedl~' using cell-free extracts of various sponges. Unlike insects sponges seem to be incapable of dehydrogenating saturated side chains as (2-24 epimers of 24-methylcholesterol (A24) and 24-ethylcholesterol (A3) were not metab01ized.l~~ It is likely that this reflects differences in the dietary sterols to which these different organisms are exposed. It has been noted that certain molluscs arthropods and coelenterateslso are also capable of side chain dealkylation although in these cases the precise mechanism has not been elucidated.Unlike sponges these organisms are incapable of de novo biosynthesis of cholesterol (see sections 6,8 and 11). It has been suggested that the novel sterols found in sponges have a role analogous to that of cholesterol in higher animals. This seems reasonable as cholesterol is often completely replaced by unconventional sterols. Subcellular fractionation of two marine sponges has been achieved133 by the use of differential centrifugation and has provided the first direct evidence for the presence of unconventional sterols in cellular membranes. In fact the sterol compositions of membrane isolates were virtually identical to that of the whole sponges indicating that a sterol analysis of a whole sponge is a valid representation of the sterol content of sponge cell membranes.133 The determination of sterol content of different cell types has recently been addressed. Cell separations of Pseudaxynissa sp. using Ficoll gradients gave cell fractions enriched in each of the major cell types.lsl As discussed previously this sponge contains two unusual sterols 24-isopropylcholesterol (A69) NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER ? 4,4,14-demethyl sterols Scheme 14 (11) R=H (78) R = H (29136) R = Me (79) R = Me Scheme 15 (CN19) side chain HO*.. & HO HO OH and 22-dehydro-24-isopropylcholesterol(A70) that comprise 99 % of the sterol mixture.24-Isopropylcholesterol (A69) was found to predominate in Pinacocytes and Choanocytes whereas 22-dehydro-24-isopropylcholesterol(A70) was the major sterol in the larger Archeocytes.181 The question of why sponges have unconventional sterols rather than cholesterol in their cell membranes remains to be answered. However it is likely that this is related to the other membrane components notably the phospholipids. It has been determined that the phospholipid head groups and fatty acids from a number of sponges are very different from those of higher animals.182-18s They are characterized by (a) longer chain lengths (up to C30)than those of other animals (b) unusual locations and stereochemistry of unsaturation (c) uncommon branching patterns and (d) the presence of large quantities of phosphatidylethanolamines (PE) and phosphatidylserines (PS) in contrast to the predominance of phosphatidylcholines (PC) in mammalian phospholipids.Blo~h~~ has described the structural specificity of cholesterol for optimal membrane function. The structural modifications exhibited by the novel sponge sterols perhaps suggests a structural adjustment for a better 'fit' with other membrane components. In addition to the monohydroxylated sterols an increasing number of sterols with multiple oxygen functionalities have been isolated from sponge^,'^^-^^^ particularly in the genus Dysidea. Several new polyhydroxylated sterols which share the 5a-cholest-7-ene-2a,3/?,5a,6/3,9a, 1la,19-heptol nucleus (X) and possess common phytosterol side chains have been isolated from Dysidea etheriu collected in Berm~da.'~'*~~~ A series of sterols analogous to (X) but with the 5/3 skeleton (cis AB ring fusion) were later reportedlE7 from the same sponge.The trans- NATURAL PRODUCT REPORTS 1991 HO 0 side chain AB steroid was moderately cytotoxic whereas the cis-m isomer was inactive. The suggest that both the 5a,6p and 5p,6a hydroxylated sterols arise from a 5a,6a epoxide which could undergo ring opening to afford both sets of sterols. Such a 5,6-epoxide has been isolatedlS2 from Haliclona oculata from the Bay of Fundy. The polyhydroxylated 9,ll secosterol herbasterol (Y2) has been isolated from Dysidea herbacea.la8 Three samples of this sponge from different depths and locations on the Great Barrier Reef were found to have essentially identical com-positions of secondary metabolites.The 9,ll secosterol was shown to be ichthyotoxic and mildly antimicrobial. la8 The isolation of a seemingly biosynthetically related sterol with a 9,l I -epoxide (22) from an unidentified species of Dysidea collected in Guam has been reported.la9 The stereochemistry at C-6 has been questionedlS1 and through evaluation of the pyridine-induced 'H-NMR shifts of the C-4 protons it was concluded that the stereochemistry proposed initially was incorrect and the epoxide is the 6a-isomer. It is also noteworthy that herbasterol (Y2) and the 9,ll- epoxy sterol (22) contain the rare 19-hydroxy functionality.Such sterols are believed to be intermediates in the biosynthesis of 19-nor sterols. The high degree of functionality of (Y2) and (22) makes it unlikely that they play a role in cell membranes. Due to the similarity between these compounds and the crustecdysones it has been suggestedlS7 that sterols (Y2) and (22) could act as a feeding deterrent to crustacean predators. A ring B secosterol hipposterol (AA2) was isolated from Hippospongia communis;lS5 the structure was determined by MS and NMR and confirmed by synthesis. This is the first report of a ring B secosterol from a marine organism. 3,5,6-Trihydroxy sterols (AB) with various conventional side chains have been recovered from Spongionella gracilis,lS4 (AD10) Hippospongia communis lS5 Spongia oficinalis lS5 and Ircinia ~ariabi1is.l~~ Sterols with this nucleus have also been isolated from the bryozoan Myriapar trun~ata.'~~ It has been sug- gestedlg4 that these could arise from sterols.Hipposterol A5s7 (AA2) also present in H. communis could be derived from cleavage of the C-5/C-6 bond of the 3/3,5a,6/3-trihydroxy More recently the isolation of novel highly degraded sterols (AC) from Dictyonella incisa has been reported ;lS7 the authors postulated that hipposterol (AA2) and 3,5,6-trihydroxysterols (AB) are biosynthetic precursors of (AC). Extensive NMR and MS analyses revealed that this new class of sterols (AC) lacked an A-ring and the B-ring was an unsaturated y-lactone. Other sterols of this sponge included a series of A5.7sterols and 3,5,6-trihydroxy-A7-sterols, both with the same side chains as the degraded sterols (AC) and the novel steroidal ketone (AD10).lg7 A plausible biosynthesis of these highly degraded sterols (AC) has been proposed.lg7 This involves the conversion of A5s7 sterols to the 3,5,6-triols (AB) both of which are present in D.incisa.It is followed by an oxidative cleavage of the C- 5/C-6 bond to afford 5,6-secosterols analogous to hipposterol (AA2) isolated from H. communis. Subsequent cleavage of the C-9/C-I0 bond provides a route for the removal of ring A. The authors acknowledge that since methanol was used in the extraction it is possible that the (AC) nucleus is an artefact and suggest that an endoperoxide (AE) or keto acid (AF) are the actual natural products in D.incisa.In light of the number of sponges which contain different polyoxygenated sterol nuclei yet with seemingly bio-synthetically related structures it appears as though there is a common (or similar) enzyme system(s) present in these sponges. The isolation of penasterol (AG19) (in 0.02% wet weight) NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER 481 R 0-... HOOC 0 from the Okinawan sponge Penares sp.Ig8 has interesting biosynthetic implications. This is a lanosterol-derived metab- olite with a 14-carboxy group that exhibits potent antileukaemic activity. Penasterol (AG19) may be an important intermediate in the biosynthesis of In the conversion of lanosterol to A5 sterols in animals C-4 demethylation is known to involve the intermediacy of carboxylic acids.lo5 Loss of the C-14 methyl group is not so well understood although it has been demonstrated that this methyl group is expired as CO in animals,199 thus suggesting oxidation to the carboxyl stage followed by decarboxylation.Other experiments,200 however indicate that loss of the C-14 methyl occurs at the aldehyde level of oxidation. Isolation of penasterol (AG19) may well have biosynthetic implications concerning the synthesis of sterols in sponges. Some interesting sterols with side chain oxygenation have been reported from several sponges. 24-Keto-A5 and Ao sterols (A3 l) (E3 l) as well as their A25-unsaturated derivatives (A80) and (E80) were isolated201v202 as minor components of the sterol mixture of H.chilensis. In addition to 24-keto sterols (A33) and (A81) a 22,23-epoxide (A82) and a degraded aldehyde (A83) have been isolated from Hyrtius SP.'~~ These (AG19) 0 -0,SOY OH (AI84) are believed to be of biological origin and not artefactual although their biosynthesis has not been elucidated. The pregnane derivatives (A84) (A85) (A86) and (AH84) have been isolated203 from the Caribbean sponge Haliclona rubens representing the first report of such sterols in the marine environment. A 20,22-dihydroxy sterol was proposed as a precursor to these compounds. A sulphated pregnane 3P,4P-dihydroxypregn-5-en-20-one-3-sulphate (AI84) has been iso1ated2O4 from the New Zealand sponge Stylopus australis.Sulphate (AI84) was present in NATURAL PRODUCT REPORTS 1991 (AJ87) (A74/75) R u"'( -03so3R= concentrations comparable to that of the free sterols. Various other steroid sulphates have been reported from marine sponges. Two independent reports describe the isolation of halistanol sulphate (AJ87) from different species of Hali-~hondria.~~~,~~~ This is an unusual compound as it is the first trisulphate to be isolated from a sponge and the side chain is an unconventional one with a t-butyl moiety. Halistanol sulphate exhibits antimicrobial haemol ytic and ichthy otoxic activity .205 Another Halichondria sp. furnished a second trisulphated sterol sokotrasterol sulphate (AJ88) which was shown206 to possess the same nucleus as halistanol sulphate (AJ87) with a tetramethylated side chain containing a quaternary centre at C-25.The structure was determined by spectral methods and from analysis of ozonolysis products. The structures of the free sterols of this sponge are surprising as they have an un-conventional side chain [24-isopropylcholesterol (A69)] which is biosynthetically unrelated to the sterol sulphate. The species of Halichondria which contains Halistanol sulphate was also shown to contain 24-isopropyl-22-dehydrocholesterol(A70) as its sole This is the only report of single organisms possessing two biosynthetically unrelated unconventional sterols. It has been suggested206 that the biosynthesis of the free sterols and sterol sulphates involves different precursors.Trachyopsis halichondrioides contains the free sterol 24-isopropyl-cholesterol (A69) and its 22-dehydro derivative (A70) as well as the 2P,3a,6cr-sterol trisulphate (AJ) with the same side The side chain of sokotrasterol sulphate (AJ88) has a similar carbon skeleton to the previously described mutasterol (A43). It has been suggested208 that the biosynthesis of sokotrasterol sulphate involves (epi)codisterol (A37/A73) and 24,26-di-methyldesmosterol (A74/A75) as was recently dem~nstratedl~~ with labelled precursors for mutasterol. A novel type of sterol sulphate has been isolated from Toxadocia zumi collected near San Diego California.209 The nucleus of these compounds (AK) is unusual as in addition to a sulphate at C-3 the C-19 angular methyl has been oxidized to a carboxyl group.A variety of biological activities including antimicrobial antifoulant and cytotoxic properties were reported for the three steroid sulphates. The authors demonstrated that these compounds are active against many types of fouling organisms and point out that such protection against fouling is bioenergetically attractive as it involves a 'low cost ' production from sterols which are readily available from the diet. 6 Cnidaria Cnidarians of the class Anthozoa are a major source of sterols in the marine environment second only to the Porifera in absolute numbers. The most characteristic sterols of this group include gorgosterol (A6) and its 23-demethyl derivative (A7) ; their biosynthesis is discussed in section 3.1.These unusual sterols are only found in zooxanthellae-containing organisms and as described earlier (section 3.1) the precise role of the symbiont and host in gorgosterol biosynthesis is not yet understood. Of the Alcyonarian corals (class Anthozoa order Alcyonacea) the genus Sinularia is characterized by a diverse group of sterol types including and uncon-~entional~~l-~l~ side chains polyhydroxylated ~tero1~,~~~-~~~ and 9'11-seco Sterols with the unusual C-23 NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER 483 (89) (93) side chain (AL) R = H AC %.yyOH.*..p+ ....p/y -...rJI, OAc -HO R= OH -.MN R’ (AM) CIR R’ = OH (97) (AN) CIS R’ = OH (AO) CIS,R’ = H alkylation have been found from various species of Sinuluriu.From the South China Sea S. remei is reported211 to con- tain remeisterol 23,24,26-trimethylcholest-5,24(28)-dien-3/3-01 (A89). Both S. sipulosu212and S.jibrillu213contain sipalosterol A 23-ethyl-24-methylcholest-5,24(28)-dien-3/3-01 (A90) and S. rumul~sa~~~ produces 24-ethyl-23-methylcholest-5,24(28)-dien-3/3-01 (A9 1). The Chinese gorgonian Echinogorgiu pseudossapo is reported220 to contain echissaposterol (A92).221 Other Alcyonarian sterols include the biosynthetically intriguing 22( S),23( S)-methylenecholesterol (A93) from Siphonoborgiu sp.222 A sterol with a rare doubly-unsaturated side chain (AP105) R = AC cholest-5,24(28),25-trien-3/3-01 (A94) has been isolated from (AP106) R=H Sinuluriu gyrosu.210 In a series of papers detailing the sterols from Surcophyton appeared in the literature.The corresponding 5a,6-dihydro gluucum collected in Ishigaki a subtropical island near Taiwan analogue of (A96) has also been reported from S. gluucum.228 eight new sterols were described. The rare 17(20) olefin first The Pacific S. dissecta collected near Palau contains a observed in a starfish was found in sarcosterol 23,24- number of polyhydroxylated sterols characterized by the dimethylcholest-5,27(20),dien-3/3-ol(A12).224 The biosynthesis la,3/3,1 la-trihydroxy nucleus (AL) of potential utility as a of peridonasterol (B 12) the 4-methyl derivative of sarcosterol corticosteroid intermediate.214 S. numerosu collected near was discussed in section 3.1.It has been that the Guam produces similar polyhydroxylated stero1s216 including report of 23,24-dimethylcholest-5,23-dien-3/3-01 (A95) from S. numersterol A (AM 1I) whose nucleus differs from the gluucum is incorrect and this compound is actually sarcosterol Lobophytum metabolite (AN97) only by the stereochemistry at (A12). C-1 and numersterol B (A098) which bears an unusual 10- The sterol cyclopropane 22-dehydro-24,26-cyclocholesterol carbon side chain. (A96) has been isolated from S. gl~ucurn.~~~ This sterol was The much studied Surcophyton gluucum has been shown to isolated and characterized concurrently and independently by contain several polyhydroxylated sterols many with the two groups. Stereochemical assignment was ultimately achieved 1/3,3/3,5a,6p tetrahydroxy nucleus (AM2 24 105).235*236 Also by synthesis.226 There is some confusion concerning the trivial present in this soft coral are sterols with the 3P,5a,6#? nucleus name of (A96) as both papakuste~-01~~’ have (AP97 105 106).235,238 and glaucaster01~~~ Sterols (AP105) and (AP106) have also NATURAL PRODUCT REPORTS 1991 side chain HO HOJ3p OH (AR) R=CHZOH (AM107) (AS) R = CHO (AQ11) (AT) R=COZH 0- (AU99) R OH I (AX100) A' R = R' = Me (AY100) R = R' = Me (AY101) R = Me R' = H (AY102) R = H R' = Me 0 w (AZ103) been found in Lobophytum paucz~or~m.~~' The first poly- oxygenated androstane from a marine organism lp,3p,5a,6p- tetrahydroxy-5a-androstan- 17-one (AM 107) has been isolated from S.glaucum and the structure confirmed by Sterols (AP24) and (AQ11) have been reported from the Cuban Plexaurella gri~ea.~~~ Related polyhydroxylated sterols contain the rare 13-hydroxymethyl group (AR) the 13-fomyl and I3-carboxy moieties (AS) and (AT).214,215 The hippurins hippurin 1 (AU99),229 hippuristanacetal (AV99),230 and hippuristanolide (AW99)230were isolated from the gorgonian Isis hippuris from Okinawa the latter two contain unusual 18-oxygenation. Several related desacetoxy hippurins have also been de-~cribed.~~~~~~~ X-ray structure of The originally AcO" (AW99) R= 4....* .MN HO OH (BA) R' = Ac hippurin-I performed on its acetate derivative has the incorrect stereochemistry at C-22. It appears that in the course of the acetylation reaction epimerization occurred at this site.232 Isis hippuris from the Andaman Islands India was found to contain five hippurins closely related to (AU99).233 I. hippuris from both locations contain polyhydroxylated derivatives of gorgosterol. Other C- 18 oxygenated sterols include 24-methyl- cholest- 1,4,22E-trien- 16p 18,20E-triol-3-one (AX 100) and the related metabolites (AY 100)-(AY 102) from L. sarmentosa as well as the pregnane 1S-hydroxypregna- 1,4,2O-trien-3-one (AZ103) from Tellesto rii~ei.~~~ An Okinawan Xenia sp. was to contain xenia- sterol-a (BA24) xeniasterol-b (BAS) and a derivative with the gorgosterol side chain (BA6); the former two are weak inhibitors of B-16 melanoma cells. A tetrol related to xeniasterol-a has been isolated from Anthelia gla~ca.~~~ The pentahydroxy sterol monoacetate (BB108) was found in the newly described Asterospicularia randaIIi from Guam 243 while the soft coral Minabea sp.contains sterol lactones of the withanolide class typified by minabeolide 1 (BC 109).244 NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER (BB108) (BC109) (BD11) R= H (BEll) R = OH (BF11) (A1 03) (BH16) O/OR HOLfP R= 1 (110) R=Ac (112) R= H (111) R=OAc (113) R=OH (114) R= H Lobophytum viridis has been shown to contain unusual 19- oxygenated sterols which exhibit antileukaemia activity. The simplest member bears a 19-hydroxy nucleus (BD)245 and is related to the previously described 7/3,19-dihydroxy-24-methylenecholesterol (BE1 1),246 for which an X-ray analysis has been performed.247 Sterols with oxidation at C-19 are believed to be biosynthetic precursors to the rare 19-nor sterols.Another of the 19-oxygenated sterols in L. viridis containing a 5,6-epoxide (BFl 1) has also been reported from the Red Sea L. depre~sum.~~~ 5,6,-Epoxy sterols with oxygenation at C-22 and C-28 as either a lactol (BG110) or the corresponding diol (BGl 1l) and the 5,6,-olefins lobophytosterol (A1 12) and depresosterol (A1 13) have been A subsequent paper identified an analogue lacking C-28 oxygenation (BG114).249 The cold-water sea raspberry Gerseminu rubiformis from Newfoundland Canada was reported to produce pregnanes and 24-norsterols. The first in the series identified pregna-5,20-dien-3P-o1 (A1 03) along with the previously described A1g4-diene-3-one pregnanes first described from an unidentified Pacific soft Subsequently 12P-hydroxy- 24-norcholesta- 1,4,22-trien-3-0ne (BH 16) with the rare 12-hydroxy moiety was reported along with its corresponding acetate.252 253 9,ll -Seco sterols were first reported from a gorgonian.254 Two such compounds from Sinulariu sp. collected from the Great Barrier Reef (BIll) and BI24) were initially reported without stereochemistry. 217 In a subsequent paper describing five additional members of the group including one with the gorgosterol side chain (BI6) the stereochemistry of all previously reported 9,ll -seco sterols was A New Brunswick collection of the sea anemone Metridium senile (class Anthozoa order Actinaria) resulted in the isolation and characterization of (22E)-5,8-epidioxy-5a,8a-stigmasta-6,9(11),22-trien-3P-o1 (BJl),255 which presumably arises from the rare A5v7,9(11)nucleus.The Mediterranean NATURAL PRODUCT REPORTS 1991 OH (BK115) (BL116) OH (BO117) (BQl18) zoanthid Gerardia savaglia is reported to produce the highly oxygenated ecdysteroid gerardiasterone (BK 1 1 5),256 though only spectroscopic data are provided and stereochemical detail is lacking. The Mediterranean hydroid Eudendrium spp. (class Hydrozoa order Hydroidea) are prey to several nudibranches and host to eggs of the n~dibranch.~~’ Both organisms possess the polyhydroxylated sterol cholest-4-en-4,16p7 18,22R-tetrol- 3-one 16,18-diacetate (BLll6) as well as several related minor An investigation of the Mediterranean Eudendrium glomeratum identified cholest-5-en-2a,3~,7P,15p 18-pent01 2,7,15,18-tetraacetate (BM2) with the unusual 2-hydroxylation and the 3a-stereochemistry.259 The Californian gorgonian Muricea californica is the first organism reported to contain 19-norcholestenone (BN2).260 Biosynthetic considerations addressing the similarity between this coral metabolite and the 19-nor sterols found in sponges and gorgonians has been discussed. 260 R= (7) \ In addition to conventional sterols the gorgonian corals (class Anthozoa order Gorgonacia) have been shown to produce keto-sterols and pregnanes as well as side chain cyclic ethers.261 A reactive sterol cholest-4,14-dien- 15,20-diol-3,16- dione (BO 1 17) has been isolated from Leptogorgia saremtosa from the Mediterranean.262 The side chain of this sterol was readily cleaved under mild acetylation conditions.The Okinawan stolonifer Clavularia viridis (class:Anthozoa order Stolonifera) contains a series of cytotoxic keto sterols,263 stoloniferone a4 (BP7 8 11 24) highly substituted sterols bearing the unusual 2-en-1-one function in addition to the 5,6- epoxide and oxygenation at C-11. The sea pen (class Anthozoa order Pennatulacea) Virgularia sp. was reported to contain a series of previously described sterol peroxides. 264 Synthesis the un- expected 20s configuration of a series of cholanic acid derivatives (BQ118) isolated from the sea pen Ptilosarcus gurneyi which had previously been assigned on the basis of spectral evidence.266 7 Bryozoans The sterols of Bryozoans have received very little attention and in fact only two reports dealing with this subject have appeared in the literature.Five 3/3,5a,6P-trihydroxy sterols (AB) also found in a sponge,194 were reported in Myriapora truncatalg6 from the Mediterranean. Bugula neritina was shown to contain cholesterol (A2) as the predominant sterol as well as dinosterol NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER (El19) (2) = -; '"py (10) = y-y (B4) and 23S,24R-23,24-dimethylcholestanol (El 19) as minor components.267 8 Mollusca The sterols of molluscs have been extensively examined due in part to the economic importance of many shell fish.l In general terms mollusc sterols have conventional structures although there appears to be characteristic differences between phylo- genetic lo Sterol composition is simplest within the most advanced class the Cephalopods (squid cuttlefish octopus) where cholesterol (A2) generally represents ca.90 YO of the total Gastropods are somewhat less advanced than Cephalopods and this is reflected in their sterol content. Cholesterol is again the major sterol but only represents ca. 40-60 YOof the total sterol Amphineura is considered to be the most primitive molluscan class and here A7 sterols often predominate with a complex mixture of con-ventional side chains. While there are some exceptions to the above generalizations there does appear to be an evolutionary trend within the phylum Mollusca towards the single sterol cholesterol.This has been attributed to biosynthetic capabilities of the various groups as well as to selective feeding habits.268 The origin of sterols in molluscs has been considered by many research groups. While de novo sterol biosynthesis could not be demonstrated in certain molluscs it has been shown to operate in others. In one 14C-labelled acetate was transformed to the A5 sterols of the bivalves Anodanta cygnea Cardium edule Cypriva islandica and Mya arenaria but not in Mytilus edulis Atrina fragilis and Ostrea edulis. A separate investigation demonstrated270 the conversion of mevalonate to cholesterol (A2) desmosterol (A19) 24-methylenecholesterol (A 11) and 22-dehydrocholesterol (A 10) in the mussel Mytilus edulis.De novo synthesis of 22-dehydrocholesterol has not previously been reported. However this experiment27o was performed prior to the advent of HPLC and the sterols were separated by argentic TLC. Contamination cannot therefore be excluded. Members of the class Gastropoda have also been shown to synthesize sterols de novo. The whelk Buccinum ~ndatum~~' transformed 3H-lanosterol to cholesterol and also was shown to produce cholesterol from 24-methylenecholesterol (A 1 1). This is the only example of a mollusc that can generate cholesterol by de novo biosynthesis and by dealkylation of a dietary sterol. Sterols with degraded side chains are frequently found in less advanced molluscs.The scallop Placepec ten magellanicus2 contains 24-norcholesterol (A 120) and its 22-dehydro derivative (A16). Patinosterol (E17) and a lesser amount of its 24-epimer were isolated from Patinopectin yessoen~is.~~~ Also the first of the isolation of 222-dehydrocholesterol (A121) was from P. uragellonicus. The biosynthesis of these compounds in molluscs has not been discussed. The sterols of bivalves generally possess A597or A5 nuclei with conventional side chains. It has been demonstrated that the sterols of certain bivalves exert a hypocholesteolemic effect when fed to ~hi~k~.~~~,~~~ 'Killer' clams Tridacna squamosa T. noae T. crocea and Hippopus hippopus were shown by GC analysis to contain predominantly common C27 C28 and C, How-ever the Tridacnid species also contain gorgosterol (A6),277 comprising ca.6% of total sterols. Giant clams are known to contain substantial populations of symbiotic zooxanthellae. However the zooxanthellae from T. gigas were found to contain no gorgoster01.~~~ The authors did not however mention the identity of the sterols in the zooxanthellae. It would be of interest to see if dinosterol (B4) [the presumed biosynthetic precursor of gorgosterol (A6)] was present in this algae. Like many bivalves killer clams are commercially important organisms. Sterol analyses of eggs larvae and adult specimens of T. gigas from Australia have recently been performed,279with the aim of determining the sterol nutritional requirements of this clam.Gorgosterol (A6) 23-demethyl-gorgosterol (A7) and 22-dehydro-23,24-dimethylcholesterol NPR 8 NATURAL PRODUCT REPORTS 1991 side chain side chain side chain (A4) (the A5 analogue of dinosterol) were found in similar relative abundances in all samples. The eggs and larvae are devoid of the symbiotic zooxanthellae which may suggest that T. gigas has the biosynthetic machinery required for the production of gorgosterol (A6). Feeding experiments with labelled sterols are however required to confirm this. The occurrence of polyoxygenated sterols in molluscs is relatively rare. Keto steroids (P) and (BR) with conventional side chains were reported280 from the prosobranch mollusc Patingera magellanica.3-Keto sterols are known intermediates in A5 sterol biosynthesis,281 however the biological function of the 7-keto sterols in molluscs is not known. Certain oxygenated sterols including 7-keto derivatives are inhibitors of sterol synthesis282 and consequently are cytotoxic. Sterols with polyhydroxylated nuclei including ones with the rare 901-hydroxylation (AB) and (BS) have been isolated283 from the hepatopancreas of the scallop Patinopectin yessoensis. The trihydroxy sterols (AB) have also been reported from a sponge.194, Ig5 In view of the well documented predator-prey relationship of certain molluscs and sponges it is not surprizing that some sponge derived sterols and sterol derivatives have been found in molluscs.Peltodoris atromaculata was to have a similar sterol composition to the sponge Petrosia Jiciformis upon which it feeds. The unconventional sterol petrosterol (A60) was present in similar amounts in both organisms. The isolation of two steroidal ketones with C-24 carboxyl groups (BR122) from the nudibranch Aldisia songuinea has been The acids were not found in the sponge Anthoarcuata graceae upon which the nudibranch feeds although A. graceae does contain the same mixture of steroidal ketones with saturated alkyl chains that is found in the nudibranch. The acid (BR122) displayed antifeedant activity in the standard goldfish assay whereas the sponge metabolite cholestenone (BR2) was inactive. The therefore suggest that the nudibranch obtains the inactive metabolite from the sponge and modifies it to produce an active antifeedant.(AB) R=H (BS)R=OH R= (AH) R’ =R2=OH (BU) R’ =OSO,R2 =H (BV) R’=OSO~R2=OH (126) 9 Echinoderms Toxic saponins are abundant in many echinoderms ;they have recently been reviewed3 and will not be discussed in the present article. Another review addressing the secondary metabolites of echinoderms as chemotaxonomic markers has also recently appeared in the literat~re.~ Sea cucumbers (class :Holothuroidea) and starfishes (class Asteroidea) produce stanols (E) and A7 and A9(11) sterols whereas sea lilies (class Crinoidea) sea urchins (class Echinoidea) and brittle stars (class Ophiuroidea) provide predominantly conventional A5 sterols.’ There are however some exceptions to this division.All five echinoderm classes contain free sterols as well as their s~lphates.~~~~~~~ Thornasterol A (AH123) and B (AH124) as well as sterol sulphates (BU123 124 125) were originally isolated from an Okinawan collection of the crown-of-thorns starfish Acanthaster plan~i.~~~. 290 Sterols (AH 123) and (AH 124) have since been found in many other starfishe~.~~~~~~~ A r ecent report identified 24-northornasterol A (BVl26) as an aglycone from HaceZia atten~ata,~~~ along with the polyhydroxylated aglycones (BW127,128). In addition Acanthaster planci contains the A7 isomer of gorgosterol and 23-demethyl g~rgosterol.~~~.~~~ Sterol sulphates have been reported from Euretaster insignis collected in New Caled~nia.~’~ Characterized as the free sterols these metabolites are unique in containing the 3p,2 1 -dihydroxy function (A 129 13 1) and (E129-1 32).The authors report that The cytotoxic 9a,l lol-epoxycholest-7-ene-3~,5~~,6~-triol (BT2) has been isolatedzs6 from the mollusc Planaxis sulcatus. this organism lacks asterosaponins and produces mostly stanols This is very similar to the 9,ll-epoxy sterol (22) reported from (E) in contrast to all other starfish. the sponge Dysidea sp. and it is therefore suspected that (BT2) 24E-ethyl-5a-cholest-3~,6a,20S-trihydroxy-9( 1 l)-en-23-one is of dietary origin. (AH1 33) and the unusual 22,23-epoxide (BV134) have been NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER OH OH OH (BW127) R = OH,R' = H (BW128) R = H R1= OH OH HO OH OH (AH133) (BX135) 'OH R (1 37) R= I HO R1 OH (BY136) (CA) R1=OH I (CB) R' = H c (138) (BZ136) reported292 from the saponin hydrosylate of Asterina pectinifera Sphaerodiscus placenta also contains (BX136) as well as the (= Patiria pectinifera).Subsequent rep~rt~~~~,~~~ identified the corresponding 8P-hydroxy metabolite (CBl 37).301 3p,6a,8,15a,24-pentol (BX135) and 3P,4/3,6~~,7a,8,15,8,16P,26-octol (BY136) in the hydrosylate of the same organism. The Mediterranean Hacelia attenuata contains the penta-hydroxylated sterol (BZ1 36)29g and norsterols (CA137 138) and (CB138)300 as minor products. It has been suggested that brassicasterol (A8) (or its C-24 epimer) is a biosynthetic precursor to the nor sterols (CA137 138) and (CB138).300 Protoreaster nodosus from the Pacific has yielded several new moderately cytotoxic polyols (CC136)-(CF1 36),302,303 three of which (CC 136)-(CE 136) have also been isolated The from P.pe~tinifera.~~~ stereochemistry at C-25 was established by The 6-sulphate of (CD136) has been described from Patiria pectinifera306 and from Oreaster reticulat~s.~~~ Poraster superbus from New Caledonia contains NATURAL PRODUCT REPORTS I991 peso (CG140) R=OH (CH140) R = OSO; R R= (141) OH OH (AH107) (AH85) oso;I a number of polyhydroxylated sterols including (CCI 39) with the rare 29-sulphate group.3o8 The first nonol 27-nor-5a-cholestan-3P,4P,5,6a,7P,8,14,15a 24a-nonol (CG140) as well as its 6-sulphate (CH140) and related polyols (CI141 142) from the starfish Archaster ty~icus,~~~ are the most highly oxygenated sterols isolated to date.The structure of (CG140) has been confirmed by X-ray analysis ;310 and the absolute stereochemistry at C-24 of (CI142) was established by spectroscopic comparison with synthetic model compounds. The aglycone (CJ143) bearing the unusual 8,14-diene was reported from the hydrosylate of Echinaster ~epositus.~~’ A subsequent report concerning this organism identified the 22,23-epoxide (CJ 144).312 The glycone bearing the reduced progesterone side chain (asterogenol AH85) was isolated from the saponin hydrosylate of Asterias forbesi and A. vulgaris collected in New Brun~wick,~~~ along with the previously described asterone (AH 1 07).3143 315 Some controversy has surrounded asterone due to the co-occurrence of the /3-hydroxyketone (thornasterol A or B AH123,124).The presence of (AH107) may therefore be explained as a retro-aldol The isolation of asterogenol (AH85) offers the best evidence yet that asterone is indeed a natural product. Also when the crude saponin isolate was treated with NaBH, greater quantities of asterogenol resulted implying the initial presence of asterone. OH-. (CK145) (CL2) R = R’ = H (CM2) R = H R’ =Me (CN) R = R’ = Me [(19) -] Sterol sulphates of brittle stars are characterized by the presence of a C-21 sulphate group a 3a-hydroxy function and a cisA/B ring fusion. The Mediterranean ophiuroid Ophiodevrna longicaudum contains several C-2 1 sulphates including 5p-cholestan-3a,4a,l lP 12P,21-pentol3,21-disulphate (CK145).316 C-21 Sulphates have also been found in various New Caledonian317 and Okinawan318 ophiuroids.Sea cucumbers besides producing the triterpene- based holothurins are characterized by the presence of A7 and A9(11) The unusual sterol 14a-methylcholest-9( 11)-en-3p-01 (CL2) from the Pacific holothurian Cucurnaria japonica is an example of the latter.321 Several other representatives of this class were reported from a New Brunswick collection of NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER 49 1 Psolus fabri~il,~, including the 4a-methyl (CM) and 4,4-dimethyl (CN) derivatives of (CL2) which have important biosynthetic implications.Their presence suggests that holo- thurians are able to modify triterpenes to sterols and are thus capable of de novo sterol biosynthesis. This has been sub- stantiated by feeding experiments in the holothurians Bohadschia argus Holothuria mexicana H. arenicola and Stichopus californic~s.~~~,~~~ The de novo synthesis of sterols was unambiguously demonstrated by the conversion of labelled squalene (77) to the A9(11)sterol (CL2).323 Interestingly while the primary cyclization product of squalene is cycloartenol (K19) and lanosterol (L19) in photosynthetic and non-photosynthetic organisms respectively parkeol (CN 19) appears to be the only cyclization product of (77) in holothurians (Scheme 14).Sea urchins characteristically contain only conventional A report concerning a Columbian echinoid has identified the rare sterol (A146).325 10 Annelids Annelids are relatively advanced invertebrates and this is reflected in their sterol content. Cholesterol (A2) is generally the dominant sterol of this group although complex mixtures of conventional C-26 C-27 C-28 and C-29 sterols are often pre~ent.'*~~~-~~' There are no reports of new sterols from annelids in recent years. As one might expect from the preponderance of cholesterol members of this phylum do not simply obtain sterols from their diet. It is well recognized that annelids are capable of de novo biosynthesis of sterols.' However no effort has been made to separate the components of sterol mixtures and it is not known which sterols are in fact generated by this route.It seems likely that cholesterol is the sole product of de novo biosynthesis and that the other minor components are of dietary origin. 11 Arthropoda For a considerable period of time cholesterol (A2) was believed to be the sole sterol present in animals of the phylum Arthropoda. Later it was demonstrated that while cholesterol is the predominant sterol other conventional C-27 C-28 and C-29 sterols are often present. Notably desmosterol (A19) has biosynthesis it appears that arthropods produce cholesterol (A2) by a modification of dietary sterols. A of the sterol content of decapods from a wide range of depths and different geographical locations revealed that cholesterol was uniformly predominant implying a specific requirement for this sterol.In some decapod~,~~ the minor C, and C, sterols were found to be concentrated in the hepatopancreas which is believed to be responsible for much of the biosynthetic activity of these animals and presumably the site of modification of dietary sterols. The ability of crustaceans to dealkylate phytosterols has been confirmed by the production of labelled cholesterol (A2) from 14C-labelled ergosterol (H8) in Artemia ~alina~~~ as well as from and the crab Partunus trituberc~latus~~~ [4-14C]-sitosterol (A39) in the prawn Penaeus japoni~us.~~~ The mechanism for C-24 dealkylation has not been established in crustaceans although it is presumably analogous to that found in phytophageous insects.6 The presence of desmosterol (A 19) one of the intermediates in the 'insect' pathway suggests that the mechanisms may well be the same.Crustaceans seem to have some selectivity in the assimilation of dietary sterols as in a simulated feeding with Neomysis integra Scrobicularia plana and Nereis diversicolor [14C]-cholesterol (A2) was absorbed from the diet and [14C]- dinosterol (B4) was not. The spiny lobster Panulirus japonica transforms [14C]-cholesterol to various steroid hormones including progesterone and testosterone.335 While this has been well documented in vertebrates little information is available for invertebrates. Since crustaceans lack the ability to produce cholesterol de novo the steroid hormones of P.japonica presumably originate from the abundant dietary phytosterols. 12 Tunicates been found in appreciable amounts in many cru~taceans.~~~-~~~ The biogenic origin of sterols in Arthropods has been addressed by several researchers and two points seem to be clear. First de novo sterol synthesis does not seem to operate in this group; and secondly cholesterol is produced from the modification of dietary sterols -analogous to that known to operate in phytophageous insects.6 Labelled acetate and mevalonate were not transformed to the sterols of numerous crustaceans.l There is however a single which suggests that in Neomysis integra there is some capacity for de novo synthesis of sterols. This claim is based on the observation that when N.integra was fed a sterol- free diet it maintained its original sterol composition. The sterol composition of arthropods is much simpler than that of their diet and coupled with the apparent absence of de novo As with most advanced invertebrates tunicates contain predominantly conventional sterols with cholesterol (A2) generally the major component. The relative abundance of cholesterol in Styela plicata is known to be dependent on the site of co1lection.l the tunicates Styela plicata Microcosmus sulcatus Ciona intestinalis and Halocynthia papollosa transformed 14C-acetate to their free sterols demonstrating that they are capable of de novo biosynthesis of sterols.' Polyoxygenated sterols have been found in many tunicates. The tunicates Phallusia mamillata and Ciona intestinalis were reported336 to contain the 5,8-endoperoxide of several A5*7,9(11) sterols (BJ2 8 11 39) whose structures were confirmed by synthesis.Subsequently the same organisms were found to contain 24-hydroperoxy-24-vinylcholesterol (A 147) and its NATURAL PRODUCT REPORTS 1991 (11) R= (82)R = A I corresponding 24-hydroxide (A30).337 The authors suggest that (A147) probably originates from fucosterol (A29) as may be the case with (A30) in brown algae. Ascidia nigra is reported to contain endoperoxide (BJ) with conventional side chains,338 as well as several coprostanols (W10 11 82) along with new 4-methyl sterols (B7) and (B39).339 13 Sediment and Sea Water Examination of the sterols of marine sediment and of those ‘dissolved ’340 in sea water in conjunction with analyses of marine organisms provides a complete picture of the ecology of marine sterols.In addition such analysis can provide information concerning the biological origin and the state of diagenesis of sediment and thus can help reconstruct ancient depositional environments -the ultimate goal of organic geochemistry. The uniqueness of occurrence of specific compounds which is determined by the uniqueness of the biosynthetic machinery in various groups of organisms is of obvious importance. Sterols are comparatively stable and thus can have a long geological The presence of sterols in Cretaceous sediments (ca. 100 million years old) illustrates that such compounds are indeed long lived.Cretaceous sterol distri- butions are less complex than those of younger Pleistocene sediments and it has been that such differences may reflect different depositional environments. Various steroid hydrocarbons are found in sediment which are not known in living organisms. These can however be traced back to natural precursors (sterols). Diagenetic pathways of sterols have been ~tudied,~~~-~*~ and it is believed that 4-methyl steranes present in ancient sediments arose from known dinoflagellate It is evident from the preceding sections of this review that sterols from the major marine phyla can be of considerable taxonomic value. Thus sterol analyses can provide information on the natural origin and state of diagenesis of sediment.The use of sterols in geochemical investigations does however have some pitfalls.346 Oceanic environments are extremely complex ecosystems and thus in any sample of sediment there are many potential sources of organic matter.346 Furthermore only a small fraction of the organic matter produced in surface waters reaches the sea floor to be incorporated into the sediments ;and compounds are subject to considerable m~dification~~’ by heterotrophic organisms. Also various organic compounds can be produced in situ or transported by currents from distant locations. Further it has been suggested that evolutionary changes in sterol compositions of organisms may have occurred.341 4-Methyl sterols notably dinosterol (B4) and other dinoflagellate sterols are known to occur in both recent and ancient sediments.8 In fact dinosterol has been called a ‘molecular fossil ’ for its use in determining past dinoflagellate blooms.Furthermore it has been that the ratio of dinosterol to gorgosterol in sediment extracts can be correlated to the relative amount of free and symbiotically occurring dinoflagellates in the depositional environment. A determi- NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER (A21) L\5,24(28) (A147) A5 Scheme 17 nation of the earliest presence of gorgosterol and dinosterol in fossils should provide a means for determining the time when the symbiosis was established between dinoflagellates and 12 invertebrates (notably the corals).8 A-Nor-steranes (CP) and de-A-steranes (CQ) have been 13 isolated from Cretaceous black hales.^^^*^^^ A proposed 14 diagenetic pathway350 analogous to that suggested for 3-oxy- triterpenoids is shown in Scheme 16.A-Nor-steranes are 15 unknown in living organisms however as discussed in section 16 5 A-nor-sterols have been found in a number of sponges. It has been that the existence of A-nor-steranes is due to 17 a diagenetic transformation of certain sponge sterols. However it seems possible that since de-A-steranes co-occur with A-nor- steranes the latter may arise by a diagenetic ring contraction of 18 19 cholesterol. The presence of 4-demethyl C,,-steranes (24R and 248-24-11- 20 propylcholestane (CR148) in marine petroleum and sedi-mentary rock has been These were identified352 by 21 comparison of lH-and 13C-NMR spectra with those from authentic material and use of GC-MS in the metastable 22 scanning mode.The steranes are assumed to be derived 23 from 24-n-propylidene- and 24-n-propylcholesterol (A2 1) and (A147) (Scheme 17). While these sterols occur in trace amounts 24 in many organisms their production can probably be attri- 25 buted to crysophyte algae of the order Sarcinochrysidoles 352 which are known to have high concentrations of 24-n-propyl 26 ~ter01~.~~.~~~~~~~ The C,,-steranes were isolated from samples dating back to Devonian (ca. 360 million years) but were not 27 found in Cambrian and Precambrian petroleum. 352 The authors admit that their survey is far from complete but suggest that the C,,-steranes first appeared in the early Paleozoic and thus 28 the algae responsible for the biosynthesis of the precursor 29 sterols evolved between the early Ordovician and the Devonian.30 14 References 31 1 L. J. Goad in ‘Marine Natural Products’ ed. P. J. Scheuer Academic Press New York 1978 vol. 2 chapter 2. 32 2 M. J. Garson Nat. Prod. Rep. 1989 6 143. 3 D. J. Burnell and J. W. ApSimon In ‘Marine Natural Products’ 33 ed. P. J. Scheuer Academic Press New York 1983 vol. 5 chapter 6. 34 4 V. A. 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NATURAL PRODUCT REPORTS 1991-R. G. KERR AND B. J. BAKER 306 A. A. Kicha A. I. Kalinovsky and E. V. Levina Khim. Prir. Soedin 1984 20 738. 307 R. S. de Correa R. Riccio L. Minale and C. Duque J. Nat. Prod. 1985 48 751. 308 R. Riccio M. Iorizzi 0.S. Greco L. Minale D. Laurent and Y. Barbin Gazz. Chim. Ital. 1985 115 505. 309 R. Riccio 0.S. Greco and L. Minale J. Chem. SOC. Perkin Trans. I 1986 665. 310 C. A. Mattia L. Mazzarella R. Puliti R. Riccio and L. Minale Acta Cryst. Sect. C 1988 44,2170. 31 1 L. Minale R. Riccio F. de Simone A. Dini C. Pizza and E. Ramudo Tetrahedron Lett. 1978 2609. 312 L. Minale R.Riccio F. de Simone A. Dini and C. Pizza Tetrahedron Lett. 1979 645. 313 J. W. ApSimon S. Badripersaud J. A. Buccini J. Eenkhoorn and M. Gilgan Can. J. Chem. 1980 58 2703. 314 J. W. ApSimon J. A. Buccini and S. Badripersaud Can. J. Chem. 1973 51 850. 315 J. W. ApSimon and J. A. Eenkhoorn Can. J. Chem. 1974 52 41 13. 316 R. Ricio M. V. D’Auria and L. 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Org. Chem. 1981 46 3860. 339 T. B. Tam Ha W. C. M. C. Kokke and C. Djerassi Steroids 1982 40 433. 340 For a definition of ‘dissolved’ molecules in sea water see J. L. Boutry A. Salist and M. Barbier Experientia 1979 35 1541. 341 S. C. Brassell and G. Eglinton in ‘Advances in Organic Geo- chemistry’ John Wiley & Sons 1981 p. 684. 342 J. W. de Leeuw Org. Mar. Geochem. 1986 305 33. 343 C. D. Taylor S. 0. Smith and R. B. Gagosian Geochim. -Cosmochim. Acta 1981 45 2161. 344 K. M. Arima M. B. Nagasawa and G. Tamura Agric. Biol. Chem. 1969 33 1636. 345 A. S. Mackenzie S. C. Brassell G. Eglinton and J R. Maxwell Science 1982 217 491.346 J. K. Volkman Org. Geochem. 1986 9 83. 347 S. G. Wakeham and C. Lee Org. Geochem. 1989 4. 83. 348 C. Djerassi Pure Appl. Chem. 1981 53. 873. 349 G. van Graas F. de-Lange J.’W. de Leeuw and P. A. Schenck Nature 1982 296 59. 350 G. van Graas F. de Lange J. W. de Leeuw and P. A. Schenck Nature 1982 299 437. 351 J. M. Moldowan Geochim. Cosmochim. 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ISSN:0265-0568    

DOI:10.1039/NP9910800465

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

Diterpenoid Alkaloids M. S. Yunusov Institute of Chemistry Bashkirian Research Centre Ural Department of the USSR Academy of Science U.S.S.R.450054 Ufa Reviewing the literature published between the middle of 1985 and the end of 1989 (Continuing the coverage of literature in Natural Product Reports 1986 Vol. 3 p. 451 ) 1 Introduction 2 Phytochemical Studies 2.1 Alkaloids of Aconitum austroyunnanense 2.2 Alkaloids of Aconitum barbatum Pers 2.3 Alkaloids of Aconitum bullatifolium Levl. Var. Homotrichum W. T. Wang 2.4 Alkaloids of Aconitum carmichaeli Debx 2.5 Alkaloids of Aconitum coreanum Levl. Raipaies 2.6 Alkaloids of Aconitum crassicaule W. T. Wang 2.7 Alkaloids of Aconitum czekanovskyi Steinb.2.8 Alkaloids of Aconitum delavaji Franch 2.9 Alkaloids of Aconitum delphinifolium DC 2.10 Alkaloids of Aconitum falconeri Stapf. 2.1 1 Alkaloids of Aconitum jinetianum Hand-Mazz 2.12 Alkaloids of Aconitum flavum Hand-Mazz 2.13 Alkaloids of Aconitum forrestii Stapf. 2.14 Alkaloids of Aconitum geniculatum 2.15 Alkaloids of Aconitum heterophylloides Stapf. and A. paniculatum Lam. 2.16 Alkaloids of Aconitum japonicum Thunb. 2.17 Alkaloids of Aconitum karakolicum Rapaics 2.18 Alkaloids of Aconitum kojimae Ohwi Var. Lassiocarpium 2.19 Alkaloids of Aconitum leucostomum Vorosch. 2.20 Alkaloids of Aconitum longtounense T. L. Ming 2.2 1 Alkaloids of Aconitum nagarum Var.Lasiandrum W. T. Wang 2.22 Alkaloids of Aconitum napellus L. 2.23 Alkaloids of Aconitum nasutum Fish. et Rechb. 2.24 Alkaloids of Aconitum nevadense Vechtr. 2.25 Alkaloids of Aconitum orientale Mill. 2.26 Alkaloids of Aconitum palmatum Don. 2.27 Alkaloids of Aconitum paniculatum Lam. 2.28 Alkaloids of Aconitum polyschistum Hand-Mazz 2.29 Alkaloids of Aconitum pseudohuiliense Chang et Wang 2.30 Alkaloids of Aconitum sanyoense Nakai Var. Tonenze Nakai 2.3 1 Alkaloids of Aconitum scaposum Var. Vaginatum 2.32 Alkaloids of Aconitum septentrionale Koelle 2.33 Alkaloids of Aconitum sibiricum 2.34 Alkaloids of Aconitum subcuneatum Nakai 2.35 Alkaloids of Aconitum sungpanese Hand-Mazz 2.36 Alkaloids of Aconitum szechenyianum Gay 2.37 Alkaloids of Aconitum talassicum M.Pop. 2.38 Alkaloids of Aconitum yesoense Var. Macroyesoense (Nakai) Tamura 2.39 Alkaloids of Aconitum zeravschanicum Steinb. 2.40 Alkaloids of Delphinium andersonii Gray 2.4 1 Alkaloids of Delphinium anhwiense 2.42 Alkaloids of Delphinium barbeyi Huth 2.43 Alkaloids of Delphinium bicolor Nutt. 2.44 Alkaloids of Delphinium brunonianum Royle 2.45 Alkaloids of Delphinium cardiopetalum DC 2.46 Alkaloids of Delphinium confusum M. Pop. 2.47 Alkaloids of Delphinium corumbosum Regel 2.48 Alkaloids of Delphinium delavayi Franch Var.Pogonantum (H.-M.) Wang 2.49 Alkaloids of Delphinium elatum L. 2.50 Alkaloids of Delphinium geyeri 2.51 Alkaloids of Delphinium gracile DC 2.52 Alkaloids of Delphinium macrocentrum Oh. 2.53 Alkaloids of Delphinium nudicaule Torr. and Gray 2.54 Alkaloids of Delphinium nutallianum Pritz. 2.55 Alkaloids of Delphinium occidentale S Wats 2.56 Alkaloids of Delphinium paciJc Giant Mix. 2.57 Alkaloids of Delphinium pentagynum Lam. 2.58 Alkaloids of Delphinium peregrinum Var. Elongatum Boiss 2.59 Alkaloids of Delphinium pictum Willd. subsp. Pictum 2.60 Alkaloids of Delphinium staphisagria L. 2.61 Alkaloids of Delphinium taliense 2.62 Alkaloids of Delphinium tatsienense Franch.2.63 Alkaloids of Delphinium ternatum Huth 2.64 Alkaloids of Delphinium vestitum Wall 2.65 Alkaloids of Spiraea japonica L. F. Var. Fortunei Rehd 2.66 Alkaloids of Consolida species 2.67 Alkaloids of Thalictrum sessile Hayata 3 NMR Studies 4 Mass Spectrometric Studies 5 General Studies 5.1 Mechanism of Oxidation of Aconitine by Potassium Permangana te 5.2 The Kinetics of Hydrolysis of Aconitine 5.3 The Selective Removal of the Hydroxyl Group Present at C-7 in Lycoctonine 5.4 Methylenation of Clg-diterpenoid Alkaloids 5.5 Deoxygenation of Diterpenoid Alkaloids 5.6 Alkylation of Acetoxy-bearing Diterpenoid Alkaloids 5.7 Replacement of the C-8 Acetoxyl Group in Clg- di terpenoid Alkaloids 5.8 Oxidation of Cl,-diterpenoid Alkaloids with OsO 5.9 Reaction of Clg-diterpenoid Alkaloids with Sodium and Liquid Ammonia 5.10 Rearrangement of Hetisine Derivatives 5.1 1 Cleavage of the N-C-6 Bond of Alkaloids of the Hetisine Type 5.12 Derivatives of Delphisine Neoline and Aconitine for Pharmacological Studies 6 Synthetic Studies 7 Chromatographic Studies 8 References 1 Introduction This review concerns papers on the isolation determination of structures synthesis transformation and spectral studies of diterpenoid alkaloids isolated from plants of Aconitum Delphinium Spiraea Consolida and Thalictrum species.Approximately 170 reports were published during the review period. The chemical structures given in the review are predominantly those of new alkaloids but structures of known compounds are also given where needed. 499 OH 1 F F Et 'OMe dMe (2) 2 Phytochemical Studies 2.1 Alkaloids of Aconitum austroyunnanense From the roots of A. austroyunnanense a new alkaloid designated as austroconidine A( I) and seven known diter- penoid alkaloids vilmorrianine B (karakoline) isotalatizidine vilmorrianine D (sachaconitine) vilmorrianine C talatizamine vilmorrianine A and 8-deacetylyunaconitine were isolated. Austroconidine A was identified as 14-acetylsachaconitine on the basis of spectral data.' 2.2 Alkaloids of Aconitum barbaturn Pers From the aerial parts of A.barbaturn collected at the pre-bud stage (1.5 YOof total alkaloids) four known alkaloids delcosine lycoctonine songorine and senbusine A were i~olated.~' The structure of senbusine A was confirmed by correlation with the known alkaloid neoline ; methylation of both alkaloids with CH31and NaH in refluxing dioxane gave the same product i.e. 1,6,14-tri-O-methylne01ine.~~ 2.3 Alkaloids of Aconitum bullatifoliurn Levl. Var. Homotrichum W. T. Wang The structures of the diterpenoid alkaloids bullatine E (85) and bullatine F (86) isolated earlier from A. bullatifolium have been studied. On the basis of spectral data and X-ray crystallographic analysis the structure (85) was determined for bullatine E and bullatine F (86) was identified as 15 p-hydroxyneoline (nagarine).47 2.4 Alkaloids of Aconitum carrnichaeli Debx It has been reported that hokbusine A isolated from A.carrnichaeli2 and A. napellus3 and alkaloid 14 also isolated from A. carrnichaeli are the same compound and have the structure (2).5 The spectroscopic data of hokbusine A were corrected. The structure (2) has been confirmed by hetero- nuclear NOE's selective INEPT and long-range homonuclear COSY experiments.5 In a further study of tissue cultures of medicinal plants Japanese workers have described the clonal multiplication of A. carmichaeli by tip tissue culture and the subsequent restoration of adult plants.6 Quantitative analysis of aconitine-type alkaloids (mesaconitine hypaconitine and aconitine) in the dried roots was carried out.Only small differences in individual alkaloid content between roots of the clonally propagated plants and normally grown plants were observed. A review and discussion on methods especially TSC in analysis of aconitine and related alkaloids in the roots of A. carmichaeli and methods of structure determination have been published.' NATURAL PRODUCT REPORTS 1991 R20 (3)R' = R2= H R3 = AC (4) R' = H R2= R3 =Ac (5) R' = R3 = Ac R2 = H (7)R' = R2= H R3 = OCOCH(CH3)Z (8) R' = H R2= Ac R3 = OCOCH(CH& 2.5 Alkaloids of Aconitum coreanurn Levl. Raipaies Further investigation of the alkaloid fraction from A. coreanurn has furnished a new alkaloid of the hetisine type guan-fu base (acorine) (3).8,9 Its structure was deduced on the basis of spectral data.A complete assignment of NMR peaks was made with NOE and 2-D experiments which also suggested a revised structure (4) for guan-fu base A (5). The structure (5) was based on a positive periodic acid test for vicinal diols.I0 Subsequently it was assumeds that this reaction was accompanied by deacetylation or more probably an 0-13 to 0-11 acetyl migration which generates the cleavable vicinal diol (5). Atisine chlorine and isoatisine were also isolated from the aerial parts of A. coreanurn and A. rotundifoliurn Kar. et Kir. (total alkaloids 8.8Yand 0.7 YO,respectively.'l From the latter plant a small amount of the elusive 19 epimer of isoatisine (6) has also been isolated.I2 It is known that several compounds closely related to isoatisine do exist as C-19 epimers according to the doubling of several signals in their 13C NMR spectra.13 A close inspection of the 13CNMR spectra of a sample of isoatisine (6) also reveals doubling of all signals at 15-30 YOof their intensity due to a minor alkaloid.Studies of literature data and 13Cand IH-NMR spectral data showed that the 19s epimer is the major one in solution as well as in the crystal.12 The structures of the new diterpenoid alkaloids guan-fu base Z (7)14 and guan-fu base F @).I5 from A. coreanurn were determined by analysis of their spectral data. 2.6 Alkaloids of Aconitum crassicaule W. T. Wang In the course of studies of the total alkaloid content of roots of the Chinese plant A.crassicaule five new alkaloids crassicauline B (9) crassicaulidine (10),l6 crassicaudine (1 l) crassicausine (1 2) and crassicautine (13) and five known alkaloids chasmanine (14 crassicauline A (19,foresaconitine forestine (16) and yunaconitine (197) have been is01ated.l~ The structures of the new alkaloids were established on the basis of spectral data and correlation with compounds of established structures. Benzoylation of chasmanine (14) with benzoyl chloride and pyridine gave 14-benzoylchasmanine acetylation of which with Ac,O and p-toluene-sulphonic acid gave crassicaudine (1 1). Treatment of crassicaudine A (15) and yunaconitine (17) with MeOH at 140 "C gave crassicausine (12) and crassicautine (13) respectively.Heating a mixture of crassicauline A perchlorate and H,O at 140 "C for 8 hours afforded forestine (16). 2.7 Alkaloids of A conitum czekanovskyi Steinb. The alkaloids hypaconitine mesaconitine songorine and napelline were obtained from A. czek~novskyi.~~ The deter- NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV 501 R2 Et OMe O‘Me (11) R’=R4=H R2=Ac R3=Bz (18) R’ = R2= H (10) (12) R’ = H R2= Me R3 = As R4= OH (19) R’ = Ac R2= H (13) R’ = R4= OH R2= Me R3 = As (20) R’ = H R2= OAC (14) R’= R2= R3= R4= H (15) R’ = H R2 = Ac R3 =AS R4= OH (16) R’ = R2= H R3=As R4= OH (17) R’ = R4= OH R2 = Ac R3 = As (anisoyl) Et Me (21) R=AC (23) R’ = H R2 = AC (26) R’ = Me R2 = H H (22) R= H (24) R’ = R~= (27) R’ = Et R2 = OH (25)R’ = R2= AC 2.9 Alkaloids of Aconitum delphinifolium DC From the whole plants of A.delphinifolium collected while in blossom in N. America (Alberta) ten alkaloids were isolated.20*21 Flowers leaves and stems and roots of this plant contained substantial amounts of alkaloids the flowers being particularly rich but there seemed to be no qualitative differences in the distribution of individual components within the plant park2’ OMe OMe On the basis of IR mass lH and I3CNMR spectra eight of the alkaloids were identified as known C,,-diterpenoid (28) R’ = OH R2= H R3 = CO-(=$-OMe(Vr) alkaloids :14-0-acetylbrowniine 14-0-acetyltalatizamine (29) R’=OH R2=Ac R2=Vr u browniine condelphine delcosine delphinifoline isotalatizi- (30) R’ = H R2 = Ac R3 = Vr dine and virescenine.A ninth 14-0-acetylsachaconitine (21) (31) R’ = R2 = H R3= Vr has not been described before. Its structure was deduced from spectral data and by saponification to sachaconitine (22). (32) R’ = H R2 = Et R3 = Vr Comparison of spectral data of the new alkaloid gomandonine (33) R’=OH R2=R3=H 13-0-acetate with that for gomandonine (24) and yesoxine (25) (34)R’ = R2 =R3 = H allowed structure (23) to be proposed for it.21 Such an epoxide might be an intermediate in the conversion of a A16/17-~y~tem into the vic-diol of dictyzine (26)22 and macrocentrine (27).23 mination of the total alkaloids of this plant during the bud and flowering stages in leaves stems and flowers have also been reported.The major amount of alkaloids was in flowers 2.10 Alkaloids of Aconitum falconeri Stapf. (0.74%) and minor in stems at the same stage (0.16%). The Five new C,,-diterpenoid alkaloids designated as falconerine aerial parts of plants during the bud stage contained 0.44% of (28) falconerine 8-acetate (29),24falconericine (30) falconeri- the total alkaloids and during the flowering stage 0.23%. dine (31) and falconeridinine (32)25 were isolated from the roots of A. falconeri. The structures of these alkaloids were derived from their spectral data and by alkaline hydrolysis of 2.8 Alkaloids of Aconitum delavaji Franch (28) and (29) to esochasmanine (33) and (30) and (31) to From the roots of this plant new diterpenoid alkaloids chasmanine (34).25 Deacetylation of (29) at the C-8 position by delavaconitine C (18) delavaconitine D (19) delavaconitine E a gentle boiling with water gave falconerine (28)24.(20),19 delavaconine and delavaconitinel* were isolated. Their Falconeridine (32) is likely to be an artefact of the isolation structures were established by spectral data and chemical conditions. It has been synthesized by refluxing a solution of evidence. Delavaconitine showed significant analgesic activity. falconerine-%acetate in NATURAL PRODUCT REPORTS. 1991 Et-M@:F _---N OH R3 R4 OH R’ OH 6NHR2 Me Me (35) R’ = H R2 = AC (36)R’=R~=H (37) R’ =OH R2=Ac OH OH R’-’ 7 OMe OwMe OMe OMe (42) R’ = Me R2= H (51) R’ = OH R~=AC (52) R’ (45) R’ = CHO R2 = OH (54) R’ = H R2 = Et (53) R’ (46) R’ = Ac R2 = OH (55) R’ = OH R2 = Et (57) R’ = H R2= Et R3= Ac = OMe R2 = H R3 =As = OMe R2 = Ac R3= As = OH R2 = H R3 = AS (47) R’ = Ac R2 = H (48) R’ = H R2= OH 2.11 Alkaloids of Aconitum Jinetianum Hand-Mazz From the roots of A.finetianurn new diterpenoid alkaloids deoxylappaconitine (35) neofinaconitine (36) isolappaconitine (37).26 and finetianine (142) and 1-deoxy- 1-oxosongorine (143)87 were isolated. The structures of new alkaloids were elucidated on the basis of spectral and chemical data26 and confirmed for finetianine by single crystal X-ray diffraction analysis. 157 The known alkaloids songorine nominine anthranoyllycoctonine and ajacine were also isolated.26.87 2.12 Alkaloids of Aconitum fravum Hand-Mazz Continuing the search for pharmacologically active compounds Chinese researchers have reinvestigated the constituents of the roots of A.flavum and isolated ten new diterpenoid alkaloids along with six known compounds napelline lucidusculine deoxyaconitine flavaconitine benzoylaconine and neo-1ine,27-29 in addition to the previously reported aconitine and 3-acetylaconitine.The structures of new alkaloids named dehydronapelline (38) 12-acetyllucidusculine (39) 1 -epi- napelline (40) 12-epi-napelline (4 I) and 1 -demethylhypa- conitine (42);27 flavamine (43) and flavadine (44);28 flava- conidine (45) N-acetylflavaconitine (46) and flavaconijine (47).29 The latter three alkaloids are characteristic of the presence of an acyl instead of the usual alkyl group on nitrogen and their structures were determined on the basis of spec-troscopic evidence and chemical correlations.Flavaconitine (48) reacted with N-formyloxysuccinimide at room temperature (58) R’ and afforded flavaconidine (45) while in the partial acetylation with acetic anhydride at room temperature flavaconitine (48) furnished N-acetylflavaconitine (46) identical with the natural The structures of the other alkaloids were determined using spectral data and chemical transform-ation~.~~~~~ The acetylation of flavamine (43) and flavadine (44) with acetic anhydride in pyridine afforded the same unusual product N-deethyl-N-acetyl- 1,12,15- triacetylnapelline. Napel- line N-oxide was isolated in 1979 from Aconitum karukolicum Rapai~s,~~ but the absolute configuration of the chiral nitrogen atom was not determined.As the physical properties of napelline N-oxide were not in good agreement with those of flavamine (43) the authors assumed that the two compounds are epimers. 28 Pharmacological tests showed that 3-acetylaconitine has strong analgesic and antiphlogistic effects and (38) (39) (49) and (50) had the effect of lowering heart rate in Langendorffs guinea pig hearts. 12-Acetyllucidusculine (39) has the strongest effect. It had no effect on the amplitude of cardiae c~ntraction.~’ 2.13 Alkaloids of Aconiturn fovrestii Stapf. From the roots of A.forrestii five known alkaloids crassicauline A. forestine and y~naconitine,~~ plus aconosine and dola- ~onine~~ new alkaloids 34 13-dihydroxyforesaconitine and (51),35 acoforine (52) acoforesticine (53) acoforestine (54) and acoforestinine (55),33 as well as liconosine A (56)34 were isolated.The structures of the new alkaloids were determined NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV R3 R2-3 OMe OMt? (59) (60) R' = a-OMe R2 = Bz (78) R' = P-OMe R2= Ac (79) R' = P-OMe R2 = H Et OMe (63) R = OSO2CF3 (64) R = H on the basis of spectral data and correlation with alkaloids of established structure. The structure of acoforestine (54) was confirmed by an X-ray crystallographic analysis. Acoforine (52) was shown to be identical with 14-acetylcolumbidine.36 Acetylation of acoforesticine (53) afforded the known alkaloid foresaconitine (57).Acoforestine (54) and acoforestinine (55) have been obtained by heating crassicauline A (15) and yunaconitine (1 7)in ethanol at 130-1 35 0C.33 Liconosine A (56) is the first C,,-diterpenoid alkaloid with N=C double bond. 2.14 Alkaloids of Aconitum geniculatum From the roots of this plant seven diterpenoid alkaloids have been isolated six of which were identified as yunaconitine crassicauline I vilmorrianine C talatizamine chasmanine and 8-deacetyl-yunaconitine. The structure of the new alkaloid geniconitine (58) was established by spectral and chemical methods. 37 2.15 Alkaloids of Aconitum AeterophylIoides Stapf. and A. paniculatum Lam. Pelletier and co-workers have reported that heterophylloidine (59) from A.heterophylloides and panicutine from A. panicu-latum are identical. Location of keto-groups in hetero-phylloidine at C-6 and C-13 was confirmed by application of CD measurernent~.~~ 2.16 Alkaloids of Aconitum japonicum Thunb. Japanese researchers have isolated from the roots of A. japonicum (the previous name A. subcuneaturn Nakai has been OH OH OMe (70) revised to A. japonicurn) a new alkaloid 14-benzoylneoline (60) and four known alkaloids neoline karakoline 14-acetyldelcosine and pend~line.~~ The structure of 14-benzoyl- neoline was determined on the basis of spectral data and by derivation of this compound from neoline. Heating of neoline with benzoyl chloride in trifluoroacetic acid at 80 "C for 6 h gave the 1-benzoate (27Y0) 14-benzoate (21 YO) and 1,14-dibenzoate (12 YO).In order to confirm the structure of penduline (61) the latter was derived from aconitine (62) through three steps (62) -+ (63) +(64) +(61). 1rradi:tion of the 13-trifluoromethane sulphonate (63) with a 2537A lamp at room temperature for 3 hours gave a deoxygenated compound (64) hydrogenation of which afforded (61) in 53% yield.39 The structural elucidation of the new alkaloids aljesconitine A (65) and aljesaconitine B (66) isolated from A. japonicum as well as the quantitative determination of the main components aconitine (62) mesaconitine (67) and jesaconitine (68) and 8-0-methyl derivatives hokbusine A (2) and aljesaconitine A (65) have been reported.40 Hokbusine A and delcosine were found in this plant for the first time.The structures of the new alkaloids were determined by spectral data and transformation of jesaconitine (68) to (65) and (66) by treatment with methanol and ethanol respectively. The authors have concluded that (2) and (65) with an alkoxy group at C-8 were natural products since (2) and (65) were found in both the chloroform and the ether extracts by means of HPLC. It was noted that when mesaconitine N-oxide was allowed to stand in methanol at 60 "C for 7 days no transformation into the 8-0-methyl derivative occurred. This result favours a mechanism for the formation of 8-0-alkyl derivatives which involves the lone pair electrons on the nitrogen atom.40 The structures of two new alkaloids secojesaconinitine (70) and NPR 8 OAc (80)R' = H R2 = Me (82) R' =Me R2 = H (81)R' = Bz R2= Me (84) R' + R2= CH2 (83)R' = R~ = H subdesculine (71) were also determined on the basis of spectral and chemical evidence.,l Secojesaconitine (70) was the first known example of a C-19 diterpenoid with an epoxy ring between C-3 and C- 17 and its structure was confirmed by X-ray analysis.Acetylation of (70) with acetic anhydride in pyridine gave 3-acetyljesaconitine (72) and 3,15-diacetyljesaconitine(73). Treatment of (70) with acetic acid gave (68).,l Further investigation of the roots of A. japonicurn resulted in the isolation of two new diterpene alkaloids along with the eight known bases talatizamine delcosine neoline isolatizidine hypaconitine 14-O-acetyldelcosine takaosamine and kobusine., The structures of new alkaloids named as 3-epi- ignavinol (75) and 2,3-dehydrodelcosine (76) were proposed through spectroscopic analysis and confirmed by X-ray analysis for (75) and a direct correlation with 2,3-dehydrodelcosine obtained by the NaBH reduction of takaonine (77)., Two new C,,-diterpenoid alkaloids subcumine (78) and subcusine (79) together with known alkaloids ezochasma- conitine and anisoezochasmaconitine were also isolated.43 The structure of subcumine (78) was confirmed by X-ray crystal analysis.43 The LD, values of 8-O-alkyl derivatives (65) (66) (2) and (74) were also determined.In the toxicity experiment charac- teristic aconitine syndromes were observed ; the 8-O-alkyl derivatives still had high toxicities.40 2.17 Alkaloids of Aconitum karakolicum Rapaics From the aerial parts of this plant collected during the bud stage eight known diterpenoid alkaloids karakoline neoline delsoline monticamine songorine napelline acetylnapelline and napelline N-oxide; and new alkaloids that were designated as karasamine (80) I -benzoylkarasamine (8 l),, 12-epinapel-line (41)455" and dihydrosongorine (82)45b were isolated.Phenyl-P-naphthylamine and the aporphine alkaloid isoboldine were also isolated.44 The structures of new alkaloids were established on the basis of spectral data and by correlation with known alkaloids. Methylation of the known alkaloid karakoline (83) by CH,I and NaH in dioxane gave karasamine (80) as a major product.Hydrolysis of (8 1) afforded karasamine (80) and benzoylation of (80) gave I-benzoylkarasamine (8 1). Reduction of songorine (84) with NaBH gave 12-epinapelline (41). NATURAL PRODUCT REPORTS 1991 Et-OMe (76)R' = H R2 = OH (77)R' + R~=O (86)R' = H R2 = Me R3 = OH (92) R' = Me R2= R3 = H Chinese researchers have also isolated the known diterpenoid alkaloids deoxyaconitine songorine aconitine neoline and karak~line.~~ 2.18 Alkaloids of Aconitum kojimae Ohwi Var. Lassiocarpium One more C,,-diterpenoid alkaloid containing the 15,16,17- trihydroxy system named as lassiocarpine was isolated from the roots of A. kojimae. Its structure (87) was deduced on the basis of lH- and 13CNMR-spe~tra.,~ 2.19 Alkaloids of Aconitum Ieucostomum Vorosch.New alkaloids sepaconitine (88)49 50 N-acetylsepaconitine (89) and amorphous base IV (90)51 have been isolated from A. leucostomurn. Structures of new alkaloids were assigned from spectral data and by hydrolysis of base IV to lycoctonine. The base IV may be an artefact derived from lycaconitine (91) during the isolation procedure. The method of quantitative determination of the alkaloid lappaconitine in plant material (A. leuco~tomurn)~~ and a method of isolation of that alkaloid from aerial parts of A. leucostomurn using an ethanol-water (4 :1) extraction has been 2.20 Alkaloids of Aconitum Iongtounense T. L. Ming Four C- 19-di terpenoid alkaloids chasmanine yunaconitine longtouconitine A and longtouconitine B have been isolated from the roots of A.longtounense. The last two are new compounds. The structure (92) for longtouconitine B was established on the basis of spectroscopic data and X-ray analysis and by chemical transformation. 54 The known alkaloids chasmaconitine and crassicauline A and the new base 8-acetyl- 14-benzoylchasmanine (93) were also isolated from this plant., 2.21 Alkaloids of Aconitum nagarum Var. Lasiandrum W. T. Wang Seven known diterpenoid alkaloids denudatine songorine songoramine virescenine neoline 14-acetylneoline and flava- NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV Me0 Et 0 0 I c=o c=o Et-b””” I (87) (88)R= H (90) R = -NHCH&H&OOEt (89)R=Ac 0 (91) R= -N 3 0 OH 1 Et -OMe OMe OMe (93)R’ = Me R2 =Ac R3= Bz (94)R’ = H R2= Ac R3 = OH (95)R = a-H P-OH (99)R’ =Ac R2= H R3 =Ac (97)R’ = R2 = Et R3 = H conitine have been isolated from the roots of A.nagarum. Phytogenesis and morphological evolution of this plant were studied on the basis of distribution of diterpenoid alkaloids. Chemotaxonomical properties of the isolated diterpenoid alkaloids were also 2.22 Alkaloids of Aconitum napellus L. From the roots of A. napellus N-deethylaconitine (94) has been i~olated.~’ This alkaloid has not been previously found in nature but it has been prepared by oxidation of aconitine (62).58 Composition and content of the diterpenoid alkaloids in A. napellus ssp. neomontanum were studied during their ontho- genetic cycle by HPLC.59 Alkaloids aconitine N-deethyl-aconitine and two bases with M’ = 688 and 629 were detected during the vegetative cycle.It was shown that the content of aconitine in underground parts over the growing season was higher than in the aerial parts. The highest content of aconitine (1.68%) was detected in the roots at the early fruit bearing stage. The content of aconitine in aerial parts does not vary significantly (0.65% in stems and 0.50% in leaves) N-desethylaconitine was accumulated in the roots and leaves in equal amounts.59 The alkaloid content in samples of A. napellus ssp. tauricum (Wulfen) Gayer harvested during a one year period was studied to determine the influence of seasonal factors on such a content.60 In the population examined the major alkaloid present is aconitine.The highest content of aconitine appears during flowering; the richest organs are the thread like roots (0.44% d.w.) and the aerial parts (leaves 0.46% and flowers 0.47% d.w.). In the parent tuberous roots which bear the aerial stem the content of aconitine is at a maximum (0.36% d.w.) during the vegetative growth period and decreases gradually (96) R = a-H P-OAC (100) R=O during the year reaching a minimum (0.11 YO)during quiesc- ence. In the daughter tuberous roots which bear the apical bud the content of aconitine remains almost constant (about 0.2%) during the year. The content of other alkaloids is always far less remarkable in all parts of the plant and throughout the year (not above 0.04% d.w.).Diterpenoid alkaloids have been used to elucidate the taxonomical position of some minor taxa of A. napellus L. The alkaloid fraction of A. napellus ssp. tauricum (Wulfen) Gayer was investigated in eight populations growing in different localities.61 Five diterpenoid alkaloids were identified N-de-ethylaconitine aconitine mesaconitine and two other un-identified alkaloids. The alkaloid composition was shown to be quite different in the population examined and the authors could recognize at least two different diterpenoid alkaloid patterns :a pattern showing aconitine as major alkaloid and the other four compounds only in trace amounts (populations from Central and Eastern Alps); the second pattern was characterized by mesaconitine as major alkaloid and the other alkaloids were always detectable in good amount (populations growing mainly in the Eastern Alps).61 Spanish researchers have isolated from the aerial parts of A.napellus ten known alkaloids aconitine neoline 3-acetyl- aconitine chasmanine delsoline delcosine songoramine,62 cardiopetamine 15-acetylcardiopetamine and songorine ;63 and four new alkaloids 12-epidehydronapelline (95) 12-epi- acetyldehydronapelline (96)62 and 8-0-ethylbenzoylaconine (97) and 15-acetyl-13-dehydrocardiopetamine (98).63 The structures of the new bases were determined from spectroscopic data and chemical correlation. 1,14-Diacetylneoline (99) isolated from this plant has not been previously reported as a natural alkaloid.62 Reduction of songoramine (100) with LiAlH in boiling ether afforded 12-epi-napelline (41) and 12- epi-dehydronapelline (95) in 78 O/O and 6 YOyields respectively.NATURAL PRODUCT REPORTS 1991 Ob Et - ’OH O’Me OMe (101) R=H (103) R= H (102) R=OH (104) R=OH ..-Me OAc (106) R’ = OAC R2= OCOCH=CH-C6H5 (107) R’ = OH R2= OCOCH=CH-C~HS (109) R’= OAC R2= OH (110) R’ = R~=OH 2.24 Alkaloids of Aconitum nevadense Vechtr. Gonzales and co-workers have reported the isolation of five alkaloids from plants of this species growing in the Sierra Nevada Granada Spain. In addition to the known Clg-diterpenoid alkaloids neoline chasmanine and isotalatizidine two new alkaloids nevadenine (101) and nevadensine (102) were isolated.65 The structures of the new alkaloids were established by high resolution mass measurement lH and 13C- NMR spectroscopy and chemical correlations.The oxidation of isotalatizidine (103) with KMnO led to nevadenine (101) in 80 YOyield and the NaBH reduction of nevadensine (102) gave virescenine (104) in 90 YOyield. The assignments published for the 13C NMR spectrum for C-10 and C-13 in virescenine have been 2.25 Alkaloids of Aconitum orientale Mill. From the aerial parts of this plant collected during the flowering stage (0.53 Yo of total alkaloids) the known alkaloids lappaconitine N-deacetyllappaconitine and ranaconitine have been isolated.66 2.26 Alkaloids of Aconitum palmatum Don. The further investigation of the roots of A. palmaturn has led to the isolation of four known diterpenoid alkaloids :vakognavine heteratisine isoatisine and hetidine and four new alkaloids :15-deacetylvakognavine (1 09 palmadine (1 06) palmasine (107) (111) R’ =OH R~=OB~ (112) R’ =OH R~=O (113) R’ = OBZ R2 = OH 2.27 Alkaloids of Aconitum paniculatum Lam.The structure of the C,,-diterpenoid alkaloid paniculatine isolated from A. paniculatum has been revised from (1 11). A comparison of the CD spectrum of dehydropaniculatine (1 12) (negative Cotton effect) with those of the parent 1 1-(positive Cotton effect)- and 13-(negative Cotton effect)-keto derivatives showed that the previously established structure’O (1 1 1) for paniculatine has to be revised to (1 13).69 2.28 Alkaloids of Aconitum polyschistum Hand-Mazz The continued investigation of the constituents of the roots of A.polyschistum collected in China has led to the isolation of two new alkaloids polischistine D (1 14) and benzoyl-deoxyaconine (1 15) and three known alkaloids benzoyl- aconine deoxyaconitine and a~onitine.~~ 158 The structures (1 14) and (1 15) were confirmed on the basis of the spectral data and the chemical correlations of the alkaloids. Hydrolysis of 3- acetylaconitine (1 16) with dioxane-H,O (1 :1) gave polys- chistine D (1 14). Deoxyaconitine (1 17) was hydrolyzed with 10% aq. H,SO to afford (1 15).’l 2.29 Alkaloids of Aconitum pseudohuiliense Chang et Wang Three new C,,-diterpenoid alkaloids designated lepenine (1 18) lepedine (1 19) and lepetine (120) have been isolated from the roots of plants of this species as minor principles along with known bases denudatine atisinium chloride isoatisine iso- NATURAL PRODUCT REPORTS 1991-M.S. YUNUSOV OH Et Me dMe H (114) R’ = OAC R2= OH (118) R’ = R~= (121) R’ = R2= H (115) R’ = H R2 = OH (119) R’ = Me R2= H (122) R’ = Me R2 = H (1 16) R’ = R2 = OAC (120) R‘ = H R2= AC (123) R’ = H R2=Ac (117) R’ = H R2= OAC oh3 O’Me R3‘ (124) R‘ (125) R’ (126) R’ (130) R’ = R3 = H R2= Me (127) R’ = aH OH R2 = aOH H (131) R’ = OH R2 = R4 = H R3 = OH = H R2= Me R3= 2-NHAcC6H4C& (128) R’ = aH OH R2 = 0 (136) R’ = R3 = H R2 = Bz R4 = OH = H R2= Me R3 = 2-NH2C6H4CO-(129) R’ = R2= 0 = H R2 = R3 = Me Et- -N *--’ OH (133) R‘ = OMe R2= OH (134) R’ = H R2=OH (1%) R’ = R~= H vaginaline (128) and vaginadine (129)76 have been isolated.Their structures were determined from spectral and chemical data. Vaginaline (128) was transformed to vaginatine (127) and vaginadine (129) and methylation of scaconine (124) by Me1 and NaH in dioxane under reflux gave (130) which was identical to 14,18-O,O-dimethyltalatizamine. The hydrolysis of (125) and (126) afforded (124). 2.32 Alkaloids of Aconitum septentrionale Koelle From the aerial parts of A. septentrionale collected in the pre- bud stage (0.32% of total alkaloids) a known alkaloid lappaaconitine and a new alkaloid sepaconitine (88) have been isolated.77 The latter has also been isolated from A. leuco-~tomurn.~~ The isolation of a new minor alkaloid septentriosine (1 3 l) obtained in 0.015% yield from the roots of this plant has been reported.Its structure was determined from spectroscopic and X-ray data.78 2.33 Alkaloids of Aconitum sibiricum The structure (1 32) assigned previously to tuguaconitine a C18- diterpenoid alkaloid from A. sibiricum has been revised. The correct structure (1 33) for tuguaconitine was deduced from a reinterpretation of spectral data and by a comparison of the 13C-NMR spectrum of tuguac~nitine~~ with those of monti-coline (1 34) and monticamine (1 35).80 Thus there are now three diterpenoid alkaloids bearing a C,-C epoxy bridge. 2.34 Alkaloids of Aconitum subcuneatum Nakai From the roots of A. subcuneatum two new diterpenoid alkaloids named torokonine (1 36) and gomandonine (24) have been isolated together with eight known Cls-diterpenoid alkaloids mesaconitine jesaconitine 14-dehydrobrowniine neoline 1Sa-hydroxyneoline isotalatizidine senbusine A and virescenine.The structures of the new alkaloids were established atisinum chloride kobusine neoline and yunaconitine. The structures of the new alkaloids were determined on the basis of spectral data. Lepedine is the first C,,-diterpenoid alkaloid bearing a methoxyl group.72 The methoxyl group had been previously found only in bis C,,-diterpenoid alkaloids. 73 2.30 Alkaloids of Aconitum sanyoense Nakai Var. Tonenze nakai. From the aerial parts of A. sanyoense two new CIS-diterpenoid alkaloids 10-hydroxyisotalatizidine (1 2 1) and 10-hydroxy-talatizamine (122) have been isolated together with five known diterpenoid alkaloids isotalatizidine talatizamine 14-0-acetyl- talatizamine condelphine and sanyonamine ; and also two aporphine alkaloids.N-methyllaurotetanine and isoboldine. The structures of the new alkaloids were established by ‘H-and 13C NMR spectroscopy and confirmed by X-ray analysis of the diacetate (123).74 2.31 Alkaloids of Aconitum scaposum Var. Vaginatum Six new Cls-diterpenoid alkaloids scaconine (124) scaconitine (125) and N-deacetylscaconitine (126) ;75 and vaginatine (127) NATURAL PRODUCT REPORTS 1991 OH Et-HO'" OMe OMe Me (138)R = -CO-CH=CH-Ph (1 39) OR2 R3 (141)R'=OH R~=H,R~=R~=o (142)R' = OH R2= H R3= Me (144)R' = Et R2= H (147)R' = OBz R2= R4 =OH R3 = H (143)R' + R2= 0 R3= Et (145)R' = R~= H (148)R' = OVr R2= $=OH,R3 = H (146)R' = Et R2 = AC R' Me-R3 (149)R=Vr (151)R' = CHO R2= H R3 = OMe (150)R= H (158)R' = Et R2= Ac R3=p4 R4= HN-COCH2CH(Me)-COOMe or HN-COCH(Me)CH2COOMe (159)R' = Et R2= Ac R3= ~-NH~CGH~COO-coo 0 (160)R' = Et R2= H R3= &:>Me U (161)R' = Et R2= Ac R3= H (162)R' = Et R2= Ac R3= as in (160) through spectroscopic analysis using the pyridine-induced 2.37 Alkaloids of Aconitum talassicum M.Pop. solvent shift technique for gomandonine. The structure of the From the aerial parts of A. talassicum collected during the latter was confirmed by X-ray analysis.81 flowering stage (0.7% of total alkaloids of dry weight of plant material) new alkaloids actaline (140y4 and 11-dehydro-2.35 Alkaloids of Aconitum sungpanese Hand-Mazz kobusine (141),85 and seven known diterpenoid alkaloids A new Clg-diterpenoid alkaloid sungpanconitine (1 38) was monoacetyltalatizamine talatizamine talatizidine isotalatizi- isolated along with four known C?q-diterpenoid alkaloids dine talatizine kobusine and pseudokobusine have been chasmanine crassicauline A aconitine and yunaconitine isolated.This is the first report of the isolation of the two latter from the roots of this plants2 alkaloids from this plant. Actaline is unique among the known diterpenoid alkaloids as it contains a terminal methylene group at C-14 in a lycoctonine-type skeleton. It is a very interesting 2.36 Alkaloids of Aconitum szechenyianum Gay alkaloid with which to study the biosynthesis of C,,-diterpenoid A new C,,-diterpenoid alkaloid szechenyine (139) and known alkaloids ;it contains only two oxygen functions.The structure alkaloids aconitine 3-acetylaconitine and songorine as well of actaline (140) was established by X-ray analysis. 11-as p-sitosterol were isolated from the roots of plants of this Dehydrokobusine had been obtained earlier from Aconitum species.83 sanyoense and its structure was confirmed by spectral data NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV OH CH,CH,N( Me) (154) (157) N-0 Me ,OMe Me 040 -OMe R= Et (165) R' = OAc R2 = OMe R3 = H (166) R' = R3 = H R2= OMe (169) R' = R2= OAC R3 = OH (171) R'=O R2=OAc R3=OH (172) R' = OAC R2 = R3= OH and by comparison with an authentic sample obtained from kobusine.86 2.38 Alkaloids of Aconitum yesoense Var.Macroyesoense (Nakai) Tamura Continuing the study of alkaloids of roots of this plant new diterpenoid alkaloids dehydrolucidusculine (144) N-deethyl- dehydrolucidusculine (145),88 and 12-acetyldehydro-lucidusculine (146) 12-acetyllucidusculine (39) 15-benzoyl-pseudokobusine (147) 15-veratroylpseudokobusine(1 48),89990 and yesoxine (25) together with known alkaloids kobusine pseudokobusine delcosine 14-acetyldelco~ine,~~ and luciduscu- line luciculine 1-acetylluciculine 14-acetylbrowniine browniine virescenine isotalatizidine and karakolinegO have been isolated. Further investigation of A. yesoense gave new alkaloids yesoline (149)," yesonine (1 50) a-oxobrowniine (15 1),92 and the known alkaloid 14-dehydrodel~osine.~~ The structures of the new compounds were determined from spectral and chemical data.A single crystal X-ray analysis confirmed the structure of yesoxine (25) which is the first C,,-diterpenoid alkaloid containing an epoxy function at C,6-C,,.90 Treatment of pseudokobusine (152) with Ch,I in methanol gave a methiodide (1 53) which was stirred with silver oxide in 50 O/O aqueous methanol to give yesonine (150). The latter and the product derived by the hydrolysis of yesoline (149) were identical.91 2.39 Alkaloids of Aconitum zeravschanicum Steinb. Three C,,-diterpenoid alkaloids isolated earlierg3 from A. zeravschanicum were previously known nominine atisine and is~atisine.~~ Zeraconine (1 54) was one of the new alkaloids.The structure for zeraconine was established on the basis of mass- spectral and 'H NMR data and also by hydrogenolysis of it to hordenine (1 55) and the diterpenoid amine (1 56).95 Zeraconine is unique among the known diterpenoid alkaloids because of the 0-phenethylamine function of C17. A new alkaloid zeraconin N-oxide (1 57) was also isolated from this A review of structure reactions and 'H and I3C NMR spectra of napelline type C,,-diterpenoid alkaloids has been published.96 2.40 Alkaloids of Delphinium andersonii Gray From the aerial parts of this plant (0.35% of total alkaloids) known diterpenoid alkaloids delavaine delectinine takaos- amine 14-acetylbrowniine 14-acetyldelcosine browniine delcosine deltaline dictyocarpine methyllycaconitine and nudicauline ;97p98 and new alkaloids andersonine (1 58) ander-sonidine (1 59) 14-de-acetylnudicauline (1 60) and 14-acetyl- nudicaulidine (1 6 1) have been isolated.The structures of new alkaloids were deduced by spectroscopic methods and con- firmed by a correlation with nudicauline (162). The latter was stirred for two days in methanol in the presence of a large amount of alumina and this gave some andersonine (158). Thus andersonine may be an artefact formed from nudicauline (1 62) during the isolation process. 2.41 Alkaloids of Delphinium anhwiense The known alkaloid methyllycaconitine and a new anhwei-delphinine (163) have been isolated from the roots of this plant.99 2.42 Alkaloids of Delphinium barbeyi Huth Pelletier and co-workers have reported the isolation from the aerial parts of D.barbeyi and structure elucidation of new alkaloids barbeline (1 64) 6-acetyldelpheline (165) 6-deoxy- delpheline (166) ;looand 14-acetyldictyocarpine (1 69) barbinine NATURAL PRODUCT REPORTS 1991 Ro\ Et (167) R = H (168) R=Ac (173) R' (174) R' (180) R' = Ac R2= R3= H = R2 = R3 = H = Me R2= OH R3 = Bz Et- EtO' OMe (1 75) (176) R' = R2 = OH (177) R' = OMe R2= OH (178) R' = R2 = OMe (1 79) (1 70) and barbinidine (1 7 1) ; and known alkaloids deltaline delpheline dictyocarpine delcosine glaucenine browniine 14- dehydrobrowniine glaucerine methyllycaconitine 14-deacetyl- nudicauline delelatine and 6-dehydrodeltamine.lo2Barbeline is the only naturally occurring 19-diterpenoid alkaloid con-taining an azomethine function and its structure was confirmed by an X-ray crystal diffraction study. lo2Barbinidine (1 7 1) and 14-acetyldictyocarpine (1 69) were correlated with dictyo-carpine (1 72). lo2 The alkaloid (1 69) had not been isolated previously as a natural product. From a hybrid population of D. barbeyi and D. occidentale S. Wats a new alkaloid delbidine (167) has been isolated and its structure elucidated by spectroscopic methods and confirmed by hydrolysis of the known alkaloid geyeridine (168) to delbidine (167).1°' 2.43 Alkaloids of Delphinium bicolor Nutt. From whole plants of D.bicolor collected during full blossom were isolated known Clg-diterpenoid alkaloids bicoloridine and bicolorine (the alkaloids A and B isolated previously from this plant were named bicoloridine and bicolorine respectively) condelphine delcosine isotalatizidine karakoline and methyl- lycaconitine;and a new alkaloid bicolorine-6-0-acetate (1 73).The structure of new alkaloid was established on the basis of spectral data and by saponification of (173) to bicolorine (174).lo3 2.44 Alkaloids of Delphinium brunonianum Royle Four new alkaloids brunonine (175),lo4 delbrunine (176) delbruline (1 77) and delbrusine (178) ;lo5 and the known diterpenoid alkaloids dictysine ajac~nine,~~~ delcosine browniine lycoctonine and septentriodine were isolated from whole plants of this species.The structures of new alkaloids were established on the basis of spectral and chemical datalo4,105 and confirmed by X-ray crystallography for brunonine. lo4 2.45 Alkaloids of Delphinium cardiopetalum DC Further studies of plants of this species led to the isolation of the new alkaloids 16benzoylgadesine (1 79) and 14-benzoyl- dihydrogadesine (1 80) and known alkaloids karakoline dihydrogadesine and 14-acetyldihydrogadesine. lo6The struc- tures of the new compounds were determined with the aid of lH and 13C-NMR spectroscopy and chemical correlation with known alkaloids. 2.46 Alkaloids of Delphinium confusum M.Pop Three reports on a study of alkaloids of aerial parts of this species the specimens of which were collected during the flowering stage at two locations have been published.Plant material from Kirghiz SSR yielded 0.6% of total alkaloids from which the known Clg-diterpenoid alkaloids condelphine virescenine 14-acetylviresceninelo7 isotala tizidine neva-densine delcosine delsoline and the aporphine alkaloid isoboldinelo8 were isolated together with two new alkaloids 14-acetylkarakoline (18 ,)lo? and 14-U-methylisotalatizidine (182).'O8 Plant material from Tajikistan USSR yielded 0.54% of total alkaloids from which the known Clg-diterpenoid alkaloids dephatine 14-O-acetylbrowniine and new alkaloids 14-0-acetylnudicaulidine (1 6 1) and 18-deoxylycoctonine (1 84) were is01ated.l~~ The structures of the new alkaloids were established on the basis of spectral data and hydrolysis of (1 8 1) to the known alkaloid karakoline (83) and by transformation of 1-0-acetylcondelphine (1 83) and isotalatisidine (103) to (1 82).Treatment of 1-0-acetylcondelphine obtained from condelphine (185) with 5% methanolic KOH at room temperature gave 1-0-acetylisotalatizidine methylation of which by Me1 and NaH in dioxane followed by alkaline hydrolysis gave (182). Methylation of isotalatizidine (103) by CH,I and NaH in dioxane at room temperature for two hours gave (1 82) in excellent yield.'08 NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV 51 1 (181) R' (182) R' (183) R' (185) R' (190) R' (191) R' (192) R' (193) R' (194) R' (195) R' Et ? c=o = R2 = H R3 = AC (184) R' = Me R2 = R3 = H = H R2= OMe R3= Me (186) R' = R2 = R3= H = R3 = Ac R2= OMe (187) R' = H R2 + R3 = CH2 = H R2= OMe R3=Ac (188a) R = -NHCOCH(Me)CH2COOMe (188b) R = -NHCOCH2CH(Me)COOMe (189) R= -N 05Me R20>*.HO = Ac R2 = OH R3= Me R4= H = R4= Me R2= R3= H = R2= R4= H R3= Me = R2= R3= H R4= Me = R3= Ac R2 = H R4= Me (196) R' = COCH(Me)CH2Me R2 = H (198) R = COCH(Me)CH2Me = R3 = Ac R2 = CI R4= Me (197) R' = H R2 = AC Et OH Me OMe OH 2.47 Alkaloids of Delphinium corumbosum Regel From the aerial parts of this species collected during the flowering and the beginning of the fruit-bearing stage (0.4 % of total alkaloids) five known Cls-diterpenoid alkaloids del- corine methyllycaconitine deoxydelcorine dehydrodelcorine and delpheline and the new alkaloid demethylendelpheline (186) were isolated.The structure of the new alkaloid was assigned from mass-spectral and 'H NMR data and by hydrolysis of delpheline (187) with aqueous sulphuric acid to afford demethylendelpheline (1 86).llo 2.48 Alkaloids of Delphinium delavayi Franch Var. Pogonantum (H.-M.) Wang Nine known diterpenoid alkaloids :deltaline deltamine methyl- lycaconitine anthranoyllycoctonine lycoctonine delsemine hetisinone hetisine and ajaconine and two new regioisomeric alkaloids delavaine A (188a) and delavaine B (188b) have been isolated. The structures of the new alkaloids were determined on the basis of spectroscopic evidence and synthesis from methy1lycaconitine;'l' (188a) and (188b) may be artefacts formed from methyllycaconitine and methanol used in the separation procedures.2.49 Alkaloids of Delphinium elatum L. Four new alkaloids delelatine (193)113 elasine (190) iso- delpheline (191) and eladine (192) and the known alkaloids nudicauline 14-deacetylnudicauline and lycoctonine112 were isolated from the seeds of this plant. The structures of the new alkaloids were elucidated by spectro-analytical methods,l12* 113 and for delelatine by two correlation sequences (192) + 14-acetyl- 10-deoxydictyocarpine (194) and dictyocarpine (1 72) -+ 14-acetyldictyocarpine (169) -+ 10-chloro-10-deoxy-derivative (1 95) -+(1 94) -+ (1 93). '13 Methylation of eladine (1 92) afforded delpheline (l87).ll2 2.50 Alkaloids of Delphinium geyeri From the aerial parts of this plant the new alkaloids geyerine (196) geyeridine (197) and geyerinine (198); and the known Cls-diterpenoid alkaloids :dictyocarpine glaucenine delcosine browniine 14-acetyldelcosine 14-acetylbrowniine 14-dehydro- browniine and delphatine were isolated.The structures of the new alkaloids were determined by spectroscopic methods. The total alkaloid mixture was shown to have feeding-deterrent activity against the migratory grasshopper but results on the purified alkaloids were equivocal. 114 2.51 Alkaloids of Delphinium gracile DC In continuing work on D.gracile collected during the flowering period Spanish researchers have isolated a new alkaloid gracinine (199) -the first Cls-diterpenoid alkaloid bearing a C-NPR 8 NATURAL PRODUCT REPORTS 1991 AcO Et (201) R' = R2= R4 = H R3 = CH20Me (208) R1=R2=H R3=R4=Me (210) R1 = OpSMe R2 = H R3 = R4= Me S (212) R' = OMe R2= H R3 = R4= Me (213) R1 + R2= 0 R3= R4= Me Et -(200) R' (202) R' (209) R' (21 1) R' = R3 = R4= H R2 = OMe = R4 = Me R2 = R3 = H = R4= Me R2 = OMe R3= OCOCH(Me)2 = R4 = Me R2= OMe R3= OH R' (204) R' = OMe R2 = P-OH R3 = OH R4= Me R5= Ac (205) R' = OMe R2 = a-OH R3= H R4 = Me R5 = Ac (206) R1=R2=R3=H R4=R5 =Me (207) R' = OH R2 = P-OMe R3 = R4 = R5= H 12 functionality -and known bases gadesine dihydrogadesine and nudicaulidine.The structure of the new alkaloid was established by means of 'H and 13CNMR spectro~copy.~~~ 2.52 Alkaloids of Delphinium macrocentrum Oliv. Research workers from Canada and Kenya have investigated the alkaloids of the epigeal parts of this plant collected in Kenya prior to blossoming (0.3% of total alkaloids) and have isolated three new alkaloids macrocentrine (27) macro-centridine (200) and deacetyl nudicauline (160).The latter was also isolated from Delphinium ~ndersonii.~~ The structures of new alkaloids were deduced from the spectral data and by correlation with known alkaloid^"^^^'^ and for macrocentrine (27) was confirmed by X-ray crystallographic analysis.116 Macrocentrine is the second example [after dictyzine (26)22] of a C,,-diterpenoid alkaloid were the unit which is usually present as an exocyclic methylene group has been converted into a vic. diol. The authors noted that macrocentrine (27) probably arises by hydrolytic cleavage of a 16a 17-epoxy precursor and so might be an artefact of is01ation.l'~ 2.53 Alkaloids of Delphinium nudicaule Torr.and Gray Benn and Kulanthaivel have reported the results of a study of alkaloids of D. nudicaule native to N. California and 0regon.'l8 The work was carried out with whole plants collected in the wild and a horticultural strain grown in Calgary as well as the seeds. Qualitatively there was little difference in the TLC patterns of the alkaloids from the seeds or plants and fractionation of these bases yielded eleven diterpenoid alkaloids eight of which were known (hetisine 2-dehydrohetisine 6-deoxydelcorine dictyocarpine dihydrogadesine methyllyca- conitine lycoctenine and takaosamine) and three hitherto Et-d0-f: 'N _.I- Et O'%-OH Me OMe (214) R' = OH R2= BZ (215) R' = H R2 = Ac undescribed bases nudicaulamine (20 l) nudicauline (1 62) and nudicaulidine (202).The structures of the new alkaloids were determined on the basis of spectral data. 2.54 Alkaloids of Delphinium nutallianum Pritz. From D. nutallianum new alkaloids hetisine 13-0-acetate (203),'19 6-epi-pubescenine (204) 7-deoxypubescenine (205) 8- 0-methylkarasamine (206) and 6-epi-neolinine (207) ; and the known alkaloids anwheidelphine browniine browniine 14-0- acetate condelphine 14-dehydrobrowniine delbonine delcosine delcosine 14-O-acetate deltatsine deacetylnudi- cauline isotalatizidine karakoline karakoline 14-0-aceate methyllycaconitine nudicauline and subcumine120 were iso- lated.The structures of the new alkaloids were determined from spectral data and that for hetisine-13-acetate proven by X-ray crystallography of the perchlorate salt.'" 2.55 Alkaloids of Delphinium occidentale S Wats In continuing studies on alkaloids of aerial parts of D. occidentale new alkaloids occidentaline (208) occidentalidine (209) 6-acetyldelpheline (1 65),12' and delbidine (1 67)lo1 were isolated. The last two were also isolated from D.barbeyi.lOO*lO1 The structures of the new alkaloids were deduced from spectroscopic methods and chemical correlations. Thus con- version of delpheline (1 87) into the 5'-methyl dithiocarbonate (210) and subsequent reduction with tributyltin hydride produced (208).Acylation of browniine (21 1) with isobutyryl chloride gave (209). 12' Fourteen known diterpenoid alkaloids deltaline deltamine dictyocarpine delcosine delpheline 14- dehydrodelcosine browniine 14-dehydrobrowniine dictyo- carpinine glaucerine glaucenine glaucedine hetisine and hetisinone were also isolated. lZ1 NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV Me0 Me- OMe OR' OMe OMe (217) R' = R3 = Me R2= Ac (221) (222) R' = Et R2= R3= H (218) R' = Me R2=Ac R3= H (225) R' = R2= R3 = H (219) R' = H R2= Ac R3 = Me (226) R' = Et R2 = R3= AC (220) R' = H3 = Me R2= H (230) R' = HI R2= R3 = AC R2 R2 Et- OR3 OM8 R'=O R2= Et R3= R4=Ac (227) R'=R3=R4=R5=H R2=Et (232) R' = R2= H R' = OH R2 = R3 = R4 = H (228) R' = R3 = Me R2= CHO R4= Ac R5 = Bz (233) R' = Ac R2= Me R'=OH R2=H R3=R4=Ac R'=OH R2=Et R3=R4=Ac OH ,OMe Me0 dR' (234)R' = HI R2= AC (235) R' =Me R2= H 2.56 Alkaloids of Delphinium pacific Giant Mix.Two new C,,-diterpenoid alkaloids paciline (21 2) and pacinine (213) and the known alkaloid delpheline have been isolated from the seeds of D. paciJc. The structures of both new alkaloids were determined on the basis of their spectral data and chemical correlation with delpheline. The methylation of delpheline (187) with CH,I and NaH in dimethyl sulphoxide afforded paciline (2 12) ;and oxidation of (1 87) with silver oxide gave pacinine (213). The previous assignment of the chemical shifts for C-1 C-9 C-10 C-14 and C-16 for delpheline were revised on the basis of 2D NMR measurements.122 2.57 Alkaloids of Delphinium pentagynum Lam.Further studies on plants of this species have led to the isolation of two new alkaloids gadeline (214) and 14-acetylgadesine (21 5). Their structures were determined with the aid of 'H and 13C NMR spectroscopy and chemical correlation with known alkaloids.lo6 2.58 Alkaloids of Delphinium peregrinum Var. Elongatum Boiss Investigation of aerial parts of plants of this species collected during the flowering period resulted in the isolation and structure elucidation of known diterpenoid alkaloids bi-coloridine dihydrogadesine nudicaulidine and 13-acetyl-hetisinone and a new alkaloid peregrine (21 6).123 2.59 Alkaloids of Delphinium pictum Willd. subsp. Pictum Spanish researchers have isolated from seeds of this plant known C,,-diterpenoid alkaloids neoline bullatine C delphisine delphinine chasmaconitine and chasmanthinine ; and a new alkaloid pictumine (217) the structure of which was established from lH and I3C NMR 2.60 Alkaloids of Delphinium staphisagria L.Pelletier and co-workers have published a number of reports on the isolation and structure elucidation of alkaloids from seeds ofplants of this species.125,127-130 The following new diterpenoid alkaloids obtained were delstaphisine (2 18) delstaphisagrine (2 19) delstasgnine (220),125 delstaphinine (222) 1-dehydro-delphisine (223) 12' delstaphidine (226) neolinine (227) a-oxodelphinine (228),12* delstaphisine (232) 1-acetyldelphisine (233),12' delstaphigine (234) and 14-0-benzoyldelphonine (235).130 The compounds (223) (228) and (233) have not been reported before as natural products.Delstaphisine (21 8),125. is the second reported Clg-diterpenoid alkaloid containing a C- 16 hydroxyl group. The first example was delbiterine (221) isolated from D. biternaturn Huth.126 There exist now five alkaloids of this type together with delbiterine and del- staphisine the alkaloids macrocentridine (200),'17 elasine (1 go) and eladine (192).l12 The structures of the new alkaloids were deduced from their spectral data and on the basis of correlation NATURAL PRODUCT REPORTS 1991 0 ~ 3 dMe Me OR1 OR2 (236) R' = Me R2= H (238) R' = R2 = H (240) R'= Me R2= R3= R4= H (237) R' = Ac R2 = OH (239) R' + R2= CH2 (241) R' = R4 = Me R2 + R3= CH2 (242) R' = R4 = H R2 + R3 = CH2 = H R2= ""0 R3=Me(244) R' = R3= Me R2= as in (244)(245) R' (249) R' = R2= 0 = R2 = R3 = H(246) R' (251) R' = H R2 = OH = Me R2= as in (244) R3= H(247') R' (248) R' = Me R2 = R3= H Et (243) with alkaloids of established structure.Oxidation of neoline (60) with KMnO in Me,CO/H,O solution at room temperature for 24 h resulted only in dealkylation of the N-ethyl group to give N-deethylneoline (224) as the main product. Under more vigorous conditions (warming at 70-80 "C) neoline was oxidized to the dealkylated carbinolamine ether N-deethyldel- staphinine (225). Boiling of the latter with C,H,Br in acetone solution in the presence of dry K,CO for 3 h gave (222).lZ7 N- deethyldelphisine (229) and N-deethyldelstaphidine (230) two new synthetic diterpenoid alkaloids have been prepared by KMnO oxidation of delphisine (23 1).Chasmaconitine an alkaloid reported previously only from Acanitum species has also been isolated from D. staphi~agria.'~~ A review of the isolation structure elucidation physico- chemical and biological properties of di- and tetra- terpenoid alkaloids isolated from D. staphisagria has been published. 131 2.61 Alkaloids of Delphinium tafiense From the roots of D. taliense the new diterpenoid alkaloids talitine A talitine B (236) and talitine C (237) have been isolated. The structures of (236) and (237) were determined on the basis of spectral and chemical data.The known alkaloids methyllycaconitine and delsemine were also is01ated.l~~ 2.62 Alkaloids of Delphinium tatsienense Franch. The structures of the diterpenoid alkaloids tatsinine (238)133 and tatsidine (239)13 isolated from the roots of this plant have been verified by X-ray analysis (as the perchlorate and by complete 'H and 13C NMR spectral ana1~sis.l~~ It was shown that the conformation of tatsinine perchlorate is not unusual. The A ring assumes a twisted boat form with an oxygen lone pair apparently oriented towards the ammonium hydrogen. 133 A new C,,-diterpenoid alkaloid delelatine (193) was also isolated from D. tatsienense. 113 2.63 Alkaloids of Delphinium ternatum Huth From the aerial parts of D. ternatum collected at the pre-bud stage new C,,-diterpenoid alkaloids delterine (240) and terdeline (241) were is01ated.I~~The structures (240) for delte~ine',~ and (241) for te~delinel,~ were assigned from spectral data and by transformation of the known alkaloid eldelidine (242) into delterine (240) and terdeline (24 1).Methylation of (242) by CH,I and NaH in DMF under reflux gave (241) while methylation of (242) in dioxane with subsequent treatment of the methylation product with 10 % H,SO at -100 "C afforded (240). Structure (243) was proposed for oxosecodelterine the transformation product of delterine.137 Terdeline is the first Clg-diterpenoid alkaloid containing a C( 10)-methoxyl group. 2.64 Alkaloids of Delphinium vestitum Wall Two new alkaloids delvestine (244) and delvestidine (245) have been isolated from the aerial parts of this plant.Their structures were deduced from spectroscopic data and by correlation with compounds of known structure.138 Alkaline hydrolysis of delvestine (244) gave the amino-alcohol which afforded gigactonine (246) on heating with aqueous sulphuric acid. Demethylation of delvestine (245) with 3M sulphuric acid afforded anthranoyllycoctonine (247) and saponification of the latter gave lycoctonine (248). 2.65 Alkaloids of Spiraea japonica L. F. Var. Fortunei Rehd Further studies on the roots of S. japonica has led to the isolation and structural elucidation of new alkaloids designated as spirasine IV (249) spirasine IX (250) spirasine XI (251),139 spirasine V (252) spirasine VI (253),140 spirasine I (255) spirasine I1 (256) spirasine VII (257) spirasine VIII (258) 14' and spirasine I11 (259).14 The structures of these alkaloids were established on the basis of chemical and spectroscopic evidence.NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV (250) R = H (252) R'=Me R2=OH R3=H (254) R = H (259) (255) R=OH (262) R=OH (253) R' = OH R2 = Me R3 = H (256) R' + R2 = CH2 R3 = OH (257) R' = Me R2 = R3= OH (258) R' = R3= OH,R2= Me c H (263) R=Ac (264) R=Ac (265) R = H (266) R = H OCOR (271) R = bNHAc (267) R' + R2 = 0,R3 = OH R4= H (272)R'=H R2=Me R3+d=0 (268) R'+R2=0 R3=H R4=OH (269) R' = R2 = R4 = H R3 = OH (270) R' = R2 = R3 = H R4 = OH The structures of spirasine I (255),141 spirasine V (252) spirasine VI (253) and spirasine I11 (259)142 were confirmed by X-ray diffraction analysis.Dehydration of (257) and (252) gave (255)14' and (254) and some (253),140 respectively and hydration of (255) afforded (257). The compounds (252)-(259) exist in solution as C-19 epimers in a ratio of approximately 1:1. But crystals of spirasine I 111 V and VI or their salts having the carbinolamine structures are only 19(S) epimeric mixture takes place upon dissolution in CDC1 as was evidenced by 'H NMR data [S approx. 4.2 (19R) and 3.7 (195')].144 It was shown that the salt of (259) in methanol exists predominantly in the form of (260) as evidenced by the 6 = 1055 (s) of C-6. In aqueous medium the existence of (261) as a minor isomer was detected by the methine signal at 6 = 185.142 In a continuation of the study of alkaloids of S.japonica the structural elucidation of new alkaloids spirasine X (262),143 spiramine A (263) B (264) C (265) D (266),144 spirasines XI1 (267) XI11 (268) XIV (269) and XV (270)145 have been reported.The compounds (263) (264) and (265) (266) are respectively epimeric at C-19; this is the first reported case of an isolation of such an epimeric pair in pure form. Hydrolysis (273)R' = Me R2 = R3 = R4 = H (274)R' = R2 = Me R3 = H R4 = OAc of spiramine A (263) afforded an approximately 2 1 mixture of spiramine C (265) and spiramine D (266). Spiramine B (264) epimerized at C- 19 in methanol to give a pair of C-19 epimers spiramine A (263) and B (264) in an approximate ratio of 1 :1.Three-dimensional single-crystal X-ray analysis provided the total structure for spiramine A (263).Ia4 A review of the isolation and structure determination of 61 diterpenoid alkaloids isolated from Aconiturn Delphinium and Spiraea species in China between 1981 and 1985 has been published. 146 2.66 Alkaloids of Consolida Species Alkaloids were isolated from the following plant species those marked with an asterisk are the new alkaloids. Consolida ambigua (L) P. W. Ball and Heywood:14' 14- deacetylajadine (27 1)* Consolida divaricata (Flowering period 0.3 YO of total alkaloids) :las delcosine delsoline. Consolida grandulosa (Boiss et Huet). Borum :149 grandu- losine (272)* lycoctonine delcorine deoxydelcorine demethyl- Et Et OMe (275) Et (279) endeoxydelcorine delsoline (ilidine bicolorine isoboldine and delporp hine (aporphine) Consolida hohenackeri (Boiss) Gro~shl~~ hohenackerine (273)* tortumine (274)* lycoctonine delcorine dehydro- delcorine delectinine bicolorine and isoboldine (aporphine).Consolida orientale (flowering period 0.15YO of total alkaloids) :14* delcosine delsoline lycoctonine. Consolida pubescens :151 pubescenine (275)* Consolida regalis S. F. Gray ssp. paniculata (Host) So0 var. paniculata :152 regaline (276)* paniculatine (277)* paniculine (278)* corepanine (279)* lycoctonine delcorine deoxydelcorine dehydrodelcorine delcoridine and bicolorine. 2.67 Alkaloids of Thalictrum sessile Hayata Further investigations on bioactive compounds of the roots of T.sessile resulted in the isolation of seven C,,-diterpenoid alkaloids along with two aporphinoid alkaloids ( +)-thali-farazine and magnoflorine and one protoberine alkaloid berberine.153,155,156 The seven C,,-diterpenoid alkaloids are spiradine A spiredine spirasine I spirasine 11 and spirasine 111 as well as two new alkaloids thalicsessine (280)153,156 and thalicsiline (28 1). 155. 156 The structures of the new alkaloids were established from spectral data. Moreover the complete structure and relative stereochemistry of thalicsiline were defined by a single crystal X-ray analysis. This is the second report on diterpenoid alkaloids obtained from the plants of the Thalictrum genus. The diterpenoid alkaloid aconitine had been found earlier in Thalictrum minus L.var. elatum (Ja~q).’~~ 3 NMR Studies The 13C NMR spectra of the diterpenoid alkaloid episcopalidine (282) and 10 analogues have been studied. The chemical shifts which are associated with different structural features of substituents were discussed and signal assignments made for a future structural determination of diterpenoid alkaloids.159 Spanish researchers have carried out a 13CNMR study of some hetisine subtype alkaloids and their derivatives which should be of considerable value in solving structures of related compounds.16o Australian workers have used high field (5.87 T) lH and 13C NATURAL PRODUCT REPORTS 1991 Et (277) R’ = H R2 = OMe (278) R’ =OMe R2=OH 0 H homonuclear and lH 13C heteronuclear one-dimensional (1 D) and two-dimensional (2D) NMR data to interpret un-ambiguously the complete ‘H and 13C NMR spectra of anopterine (283).A comparison of the NMR data for anopterine with those of six minor alkaloids from Anopterus macleayanus has allowed the assignment of the structures of the minor alkaloids 7-P-hydroxyanopteryl 1 la 12a-ditiglate (284) 7-P-hydroxyanopteryl 1 1 -a-(E)-4’-hydroxy-2’-methylbut-2’-enoate- 1 12a-tiglate (285) anopteryl 12,a-tiglate (286) anopteryl 1 la-4’-hydroxybenzoate l2a-tiglate (287) 7P-hydroxyanopteryl 1 1 a-4’-hydroxybenzoate 12a-tiglate (288) and 7P-hydroxyanopteryl 1la-benzoate 12a-tiglate (289).161 Pelletier and co-workers have undertaken a detailed study of the 13C NMR chemical shift assignments for the well-known alkaloid aconitine (62).“j2 A variety of methods to obtain the lH and 13C NMR assignments was used with the major emphasis on the use of 2-D NMR.Chemical shift revisions have been suggested for certain carbon atoms of the Clg- diterpenoid alkaloids. 162 4 Mass Spectrometric Studies Mass spectra of the diterpenoid alkaloid episcopaladine (282) and 14 analogues have been studied for fragmentation patterns in relation to A number of articles on the mass spectra of the diterpenoid alkaloids has been published by Russian researchers. It was shown that the presence of a C-6(OCH3)-C-7(OH)-C-8(OH) grouping in Clg-diterpenoid alkaloids leads to high intensity (M -15)+ ions at the expense of the C-6(0CH3) group [using the C-6(0CD3) analogue] and considerably suppresses the competing processes of forming (M -OH)+ and (M -OCH,)’ ions from the alkaloids and the (M -56)’ ions in the anhydroxy bases.164 When the above-mentioned grouping is absent the (M -15)+ ions are formed mainly by the loss of a CH from the N-ethyl group.In the spectra of 7,8-dihydroxy bases not containing a 6- methoxy group the intensity of peaks for (M-CH,)’ ions decreases. On passing from 1 -hydroxy derivatives to 1-methoxy the contribution of the processes involving the ejection of a radical from C-1 increases and that of (M-CH,)+ ions decreases [for example a pair of compounds such as browniine NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV R20 I R'O ... .....@ Me-N H2 Et Me OH (283) R' = R2 = tigloyl R3= H (284) R' = R2 = tigloyl R3= OH (285) R' = E-HOCH2CH=C(Me)C0 R2 = tigoyl R3 = OH (286) R' (287) R' (288) R' (289) R' = H R2 = tigloyl R3 = H = ~-HOCGH~CO, R2 = tigloyl R3 = H = 4-HOC&,&O R2 = tigloyl R3= OH = PhCO R2 = tigloyl R3 = OH -@ -N Scheme 1 (21 1) and delcorine (290)l.On passing from 7,8-dihydroxy bases to the 8-mono-01s the intensity of the peaks for the (M -CH,)' ions decreases [dihydromonticoline (29 1) and dihydromonticamine (292)].164 The mass-spectral properties of 21 lycoctonine bases with 1- methoxy-7,8-methylenedioxygroups have been The fragmentation of alkaloids and their derivatives has been found to be influenced by a methylenedioxy group the nature of substituents at C-6 and C-10 and a A10(12)bond.The main processes in the spectra of AloCl2)compounds involves two competing processes the ejection of a formaldehyde molecule at the expense of the 7,8-methylenedioxy group and the ejection of an OR radical from C-6 (Scheme l).165 The parameters of the metastable defocusing spectra of the fragment ions of 21 lycoctonine alkaloids with 7,s-methylene- dioxy groups have been investigated. 166 The main parameter studied was the ratio of the intensities of the peaks of the metastable and parental ions in percentages (magnitude A). A criterion of the common nature of the mechanisms of multistage processes was the agreement of three factors the ratio of the heights of peaks in the MD spectra the positions of the maxima of all peaks and the closeness of values of A.On this basis the monotypicity of the main fragmentation processes characteristic of the individual groups of compounds has been confirmed. 166 Et-OMe (290) (M-Me)+ 100% (291) (M -Me)+ 70% (M -OH)+ 36% (M -OH)+ 100% (292) (M -Me)+ 28% (21 1) (M -Me)+ 28% (M-OH)' 100% (M -OMe)' 100% (293) R = H (294) R=C2D5 (295) An analysis of literature material on the mass spectra of hetisine alkaloids and a study of the breakdown of hetisine nominine talatisine and their derivatives with the aid of high-resolution mass-spectrometry and MD spectra have been p~b1ished.l~~ The fragmentation patterns of this type of compound are not monotypical and depend greatly on the positions of oxygen substituents.The mass spectra of hetisine alkaloids of a new type i.e. zeraconine and its N-oxide have been characterized. 167 The use of GC-MS techniques in studies of alkaloid content in the roots of Aconitum septentrionale has been reported.16* The alkaloid composition of the roots of Aconitum septen-trionale collected from different regions have been investigated. The mass spectrometric method of multipeak monitoring was used for the express control of the major alkaloid lappaconitine and seven 0the~s.l~~ The mass-spectra of eleven C,,-diterpenoid alkaloids in- cluding N-deethylmonticamine (293) d,-analogues of monti-camine (294) and A2-dihydromonticamine (295) have been considered. Using the parameters of the MD spectra the main processes of forming (M -CH,)' ions for 3,4-epoxy- and A2-dihydrobases have been prop~sed."~ The X-ray diffraction analysis of 30 Clg-diterpenoid alkaloids has been reviewed.171 NATURAL PRODUCT REPORTS 1991 >N-CH213CH3 ->N=CH-13CH3 -CH3CHO Z= CHZ=CH-OH -H-13CH0 + OH OMe (296) R = CHO (62) R = Et (297) R=Ac (69) R = H Et OH (299) t Me0 OH (248) Mechanism 2 (296) Mechanism3 Et Et Et (298) Scheme 2 5 General Studies 5.1 Mechanism of Oxidation of Aconitine by Potassium Permanganate Oxonitine (296) which is one of the permanganate oxidation products of aconitine (62) has been the subject of study for over 50 years.Recently three papers have treated the mechanism of its formation.172-174 The results reported demonstrate that an oxidation of aconitine to oxonitine with KMnO in acetone proceeds by the three mechanisms shown involving the CH and CH carbons of the N-ethyl group and the carbon of the solvent as sources of the > N-CHO group of aconitine. Mechanism 1 and 3 operate to an extent of about 84% although the proportion of oxonitine formed via each mech- anism is not kn0~n.l'~ 5.2 The Kinetics of Hydrolysis of Aconitine The hydrolysis of aconitine (62) catalyzed by OH-ion was found to be a first order reaction of two successive steps. The rate constants and activation energies of the two steps at different pH's were determined.'75 5.3 The Selective Removal of the Hydroxyl Group Present at C-7in Lycoctonine The first chemical conversion of a lycoctonine- type alkaloid [lycoctonine (248)] into an aconitine-type [7-deoxylycoctonine (298)] has been de~cribed."~ The crucial step was intramolecular C-C bond formation between the iminium carbon and the carbanion at C-7 produced by the Huang-Minlon reduction of hydroxylycoctonine (299) (Scheme 2).The latter was obtained from lycoctonine (248) by Ag,O 0xidati0n.l~~ The structure of the major product (298) was established by spectral data and was confirmed by X-ray analysis. The minor product of the Huang-Minlon reduction has the structure (300) on the basis of spectroscopic analysis. 176 5.4 Methylenation of C,,-diterpenoid Alkaloids Methylenation of a number of C,,-diterpenoid alkaloids containing a cis-diol system at C-7 and C-8 using diethoxy- methane or aqueous formaldehyde in the presence of p-toluenesulphonic acid has been reported.178 Treatment of methyllycaconitine (189) with 30 % aqueous HCHO and p- NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV Et Et GN>0 Me (302) R' = OAC R2= CI (304) R' = OAC R2= OH (305)R' = OCS(SMe) R2= OH (306)R' = OCS(SMe) R2= H (303) (301) (307)R' = H R2 = OH (308) R' = R2 = H toluenesulphonic acid in refluxing benzene afforded elatine (301). 14-Acetylbrowniine delphatine ajacine 1,14-diacetyl- delcosine and 14-dehydrobrowniine were also transformed to their methylenedioxy analogues. 178 5.5 Deoxygenation of Diterpenoid Alkaloids Pelletier and co-workers while re-investigating a reductive dehalogenation of 10-deoxy- 10-chlorodeltaline (302) obtained an unusual elimination product (303) as a minor side product.This product was formed in a yield of 60% when crude (302) was reduced with LiAlH,. Deltaline (304) was deoxygenated in almost quantitative yield to 1 0-deoxydeltaline by reduction of the intermediate (302) with Bu;SnH. Reduction of the S-methyl dithiocarbonate derivatives (305) and (306) with Bu; SnH furnished deoxy-components (307) and (308) respect- ively.178 5.6 Alkylation of Acetoxy-bearing Diterpenoid Alkaloids 1P-OH and/or 1P-OMe derivatives of delphisine (23 1) and neoline (60) have been used to study lH and 13C NMR spectral characteristics of C,,-diterpenoid alkaloids. In the course of this study the alkylation of acetoxy-bearing substrates with Me1 and NaH or KH yielded a mixture of products in which the replacement of acetoxy groups by either OH or OMe substituents occurred.However 0-alkaylation proceeded with- out cleavage of the acetoxy functionalities with trialkyloxonium salts in the presence of a strong non-nucleophilic base.180 5.7 Replacement of the C-8 Acetoxyl Group in C,,-diterpenoid Alkaloids Pelletier and co-workers have shown that the C-8 acetoxyl group can be readily replaced by a methoxy group by refluxing the appropriate alkaloid in MeOH.l8l By this reaction (312) R' = Me R2 = OAc R3= OH (313) R' = Me R2 = 0 R3 = OH (314) R' = CHZOMe R2 = 0,R3 = H hokbusine A (2) was synthesized from mesaconitine (67) and aconitine (62) delphinine 3-deoxyaconitine (1 17) and falconerine-8-0-acetate afforded the corresponding 8-deacetyl- 8-0-methyl derivatives.Artefacts bearing a methoxy or an ethoxy group at C-8 may be formed while isolating Clg- diterpenoid alkaloids. 5.8 Oxidation of C,,-diterpenoid Alkaloids with OsO The oxidation of Cl,-diterpenoid alkaloids of the aconitine type (C-6-a-OR) with OsO,. gave an N-acyl or N-deethyl derivative and of the lycoctonine type (C-6-p-OR) a lactam or carbinolamine ether derivative. This oxidation is of diagnostic value in determining the orientation of the C-6 oxygenated functional group.184 Aconitine (62) with OsO afforded N-deethylaconitine (69) (39 YO)and N-acetyl-N-deethylaconitine (17.4%) and in contrast to oxidation by permanganate,lE3 never give any detectable amounts of oxonitine (298).Reaction of deltaline (304) afforded the crystalline P-oxodeltaline (309) (77 YO),and delcosine (3 10) afforded 18-methoxygadesine (31 1).laz 5.9 Reaction of C,,-diterpenoid Alkaloids with Sodium and Liquid Ammonia The reaction of Clg-diterpenoid alkaloids with sodium and liquid ammonia in the presence and in the absence of a proton donor an alcohol has been investigated. In the absence of proton donor a hydroxy group is converted into a carbonyl group which is characteristic only for C-6 hydroxy or acetoxy derivatives. Thus eldeline (3 12) eldelidine (242) and delcorine (290) in the absence of alcohol gave 6-keto derivatives (313) and (314) respectively. The yield of 6-keto products reached 60%.In the presence of alcohol starting materials were recovered almost completely and compounds (3 13) and (3 14) have not been dete~ted.~~~,~~' NATURAL PRODUCT REPORTS 1991 Ho\ (315)R = 0 (316)R = OH (317)R=O (318)R = OH Ho\ @EH3Me 0 0 (322)R' + R2 = 0 (323)R = u.-OAS (325)R' = OH R2 = H (325)R = P-Oti 5.10 Rearrangement of Hetisine Derivatives The reaction of 2,ll -didehydrohetisine (3 1 5) and 1 1-dehydro-hetisine (3 16) with aqueous sulphuric acid led to the compounds (317) and (318) respectively. The structure of the latter was solved by a single-crystal X-ray analysis. The structure of (317) was confirmed by the oxidation of (318) with piridinium chlorochromate to afford (3 17).A plausible mechanism for this rearrangement involves hydration of the exocyclic methylene group followed by dehydration and an internal Michael addition (Scheme 3).185 Refluxing of 1l-acetyl-2,13-didehydrohetisine (321) with aqueous potassium carbonate in methanol gave a rearrange- ment product (322). Its structure was established through an X-ray crystallographic study.ls6 Treatment of (323) with methanolic potassium carbonate at 25 "C gave an isometric (1 1-epi) compound (324). The structural assignment for (324) was based on its 'H NMR spectrum. Reaction of (323) with aqueous methanolic potassium carbonate under reflux gave the rearrangement product (325). Oxidation of (325) with chro- mium trioxide-pyridine afforded the compound (322).A possible mechanism for the formation of rearrangement products (322) and (325) and the isomeric compound (324) is suggested to be as follows (i) hydrolysis of the C-11 acetyl group accompanied by a reverse aldol condensation; (ii) a Cannizzaro-like process followed by lactone formation (Scheme 4). 5.11 Cleavage of the N-C-6 bond of Alkaloids of the Hetisine Type A simple and efficient modification of the Hoffman degradation for the cleavage of the N-C-6 bond of diterpenoid alkaloids of the hetisine type has been proposed.ls7 Treatment of srirasine IX (326) with methylene bromide in ethyl acetate gave the N-bromomethyl derivative. The latter was refluxed in 10% sodium hydroxide to afford the N,6-seco-6,7-didehydro product (327) (yield 50 YO).N-chloromethylation of (326) gave the best yield of (327) but more prolonged degradation was needed.lE7 5.12 Derivatives of Delphisine Neoline and Aconitine for Pharmacological Studies In connection with a study of the pharmacological properties of diterpenoid alkaloids a series of synthetic esters of delphisine (356)-(360) and neoline (361)-(370) have been prepared.The physical and spectral data of these fifteen new compounds are given.2o4 The preparation of aconitine derivatives for examination as analgesic and anti-inflammatory agents has been described. 205,206 These compounds have the structure (371) (R1= Bz or anisoyl R2= Me or Et R3 = H or Ac R4 = H or higher fatty acid residue) Acetylation of mesaconitine (67) with Ac,O in the presence of pyridine gave 3-acetylmesaconitine which was treated with linolic acid and pyridine to afford (371) (R1 = Bz NATURAL PRODUCT REPORTS 1991-M.S. YUNUSOV 52 1 Scheme 4 @ Me Me Me Me Me Me (327) (329) (330) R'O Et OM0 OMe (356)R = BZ (361) R' = R2= BZ OH & @ R30". R2-M$of-N --"-OH Br N-CI I 0 OMe Me Me Me Me OMe R2 = Me R3 = Ac R4= linolicyl). This compound showed an sponds to a partial structure for kobusine-type alkaloids. lee ED, of 4.82 mg/kg (S.C.)205 and 8-O-methyl-14-The synthesis was carried out from the 4a-nitromethylhydro- benzoylaconine (R'= Bz. R2 = Et R3= H R4= Me) had an phenanthrone (329) which was obtained from the tetrahydro- ED, of 0.45 mg/kg (S.C.)206for an AcOH writhing test in phenanthrol(330) by means of an abnormal Reimar-Tiemann mice.reaction via the bromocyclopropane derivative (33 1). The bridged nitrogen structure was formed from the bicyclic chloramine (332) by means of the Hofmann-Loffler reaction. 6 Synthetic Studies The structure (328) was confirmed by X-ray diffraction Shibanuma and Okamoto have reported the synthesis of (+)-ana1~sis.l~~ 6,15,16-iminopodocarpane-8,11,13-triene (328) which corre-For the functionalization of the aromatic c ring of (328) the NATURAL PRODUCT REPORTS 1991 HOfloMe <@o -N N&No2 0 0 H 'SR Me Me Me Me Me Me Me (333) (334) R=OH Me02C-8(337) R = OMe 1. Me02C -N Me02C-qpo r @C02Me 0 -y qco2Me H H88 Ph 0 .~ Me Me C02Me C02Me (339) 42 goMe HO"' Me0mMe Me (343) (344) (345) Me ,OAc KOH 0*C b0 *CI 0 fYMe (347) I 0 OMe OMe 0 (346) nitration of the hydrogenation product of (329) was carried out giving the 7-nitro compound (333) (79 YOyield);189phenol (334) was obtained in two further steps (in 73.8% yield).When (334) was treated with diazomethane in ether at room temperature an unusual reaction of the angular nitromethyl group was observed i.e. an isomeric mixture of aldoximes (335) and (336) was obtained along with the desired methoxy derivative (337). The authors showed that the diazomethane promoted a reductive conversion of the nitromethyl group to an aldoxime group.18s Japanese researchers have communicated1s0 a stereo-controlled formal total synthesis of (f)-atisine (338) in which the bicyclo[2,2,2]octane system (339) is constructed by an intramolecular double Michael reaction from (340).The latter has been constructed in an optically active form from a symmetrical azabicyclo[3.3.llnonane (341) prepared by a double Mannich condensation of dimethyl cyclohexanone-2,6-dicarboxylate (342). Since (339) has been correlated with atisine by Pelletier and the present work represents a stereoselective formal total synthesis of (+)-atisine (338). A novel synthesis of Nagata's intermediate (343) for ( & )-atisine (338) using benzocyclobutene (344) has been described.lg2 A number of chemical transformations of hetidine (345) relevant to the cleavage and regeneration of the C(14)-C(20) bond have been reported.lS3The simultaneous presence of 2a-hydroxy and 13-keto groups in hetidine allows a facile conversion into a compound (346) of the atisine-type by heating in alkaline media.A reversion of (346) to (345) can be simply effected by treatment with acid.lg3 The synthesis of a number of acetyl-and dehydro-derivatives of hetisine (109) has been reported.lS4 The construction of the BCD ring system of Cls-diterpenoid alkaloids has been initiated by the development of a ring expansion reaction of the 5-membered ring ketone (347) to the 7-membered ring ketone (348).lg5From the latter the cyclic p-keto ester (349) was obtained which has a full potential of NATURAL PRODUCT REPORTS 1991-M. S. YUNUSOV 0 Me (354) 0 LO /C02C2H5 OH (355) (353) functional groups suited for the further elaboration into the A E and F rings plus substituents of a variety of Clg-diterpenoid alkaloids.lg5 The successful synthesis of the tricyclic enamine (350) as a model for the A E and F ring system of diterpenoid alkaloids has been rep~rted.'~~.~~~ Trienone (351) afforded amine (352) in four steps. Heating (352) in acetic acid in the presence of formalin at 150 "C for 10 hours provided the expected tricycle (350) in a completely regioselective manner in 97% yield.lg6 Fukumoto and co-workers utilized a method of stereo- selective construction of spirofused bicyclo[2.2.2]octane systems by an intramolecular double Michael reactionlgs to achieve a highly stereocontrolled construction of the CDF part of the lycoctonine skeleton (353) -+ (354) -+(355) in a model study ultimately directed towards a total synthesis of Aconitum alkaloids.lg7 7 Chromatographic Studies Pelletier and co-workers have demonstrated the versatility of centrifugal chromatography (Chromatotron) in the separation of complex diterpenoid alkaloid mixtures. The use of aluminium oxide containing fluorescent materials active at long (365 nm) and short (254 nm) wave lengths improves the efficiency and reduces the time required for separations. 2oo The separation of some closely related diterpenoid alkaloids using the Targett simplified vacuum liquid chromatography technique (VIC) has been reported. 201 The researchers con- cluded that VLC is an extraordinarily simple technique that consumes small amounts of solvents has reasonable resolution and requires a relatively short time to carry out a separation.The VLC method can be used for the separation of relatively large as well as small amounts of mixtures. The method also uses simple and inexpensive equipment.201 A chromatographic system involving the use of a simple mobile phase consisting of 2 mM ammonium carbonate and tetrahydrofuran for the separation of certain groups of diterpenoid alkaloids by high performance liquid chromato- graphy has been described.202 A significant separation was achieved for the permanganate oxidation products of aconitine and other Clg-diterpenoid alkaloids.202 A capillary gas chromatography method has been described for the analysis of diterpenoid alkaloids which occur in Delphinium barbeyi and D.occidentale (larskpur) to provide a rapid quantification of specific diterpenoid alkaloids occurring in larkspur and to allow the correlation of phenological ocurrence of those alkaloids with observed rangeland livestock The standard alkaloids were delpheline deltamine deltaline 14-acetyldictyocarpine dictyocarpine dictyo-carpinine and delcosine. An acetone solution of total alkaloids of a plant extract containing approximately 200 ng/pl was used for analysis. Dictyocarpine and dictyocarpinine had identical retention times in the chromatographic system. Five remaining standard alkaloids are clearly resolved. Yields of total alkaloid extract (based upon air dry plant weight) for two larkspur species varied according to the phenological growth stage of the samples (from 0.5 to 5% for D.occidentale and from 0.4 to 3% for D. barbeyi). In young leaf material of the two Delphinium species deltaline (304) was the major alkaloid (over 30% of the total alkaloid extract).203 8 References 1 Z. Jiang S. Chen and J. Zhou Acta Bot. Junnan. 1988 10 317 2 H. Hikino Y. Kuroiwa and C. Konno J. Nat. Prod. 1983 46 178. 3 H. Hikino Y. Kuroiwa and C. Konno J. Nat. Prod. 1984 47 190. 4 J. Z. Wang Master's Thesis Second Military Medical College of Shanghai Shanghai China 1982. 5 G. Y. Hang Cai J. Z. Wang and J. K. Suyder J. Nat. 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ISSN:0265-0568    

DOI:10.1039/NP9910800499

出版商:RSC    

年代:1991

数据来源: RSC