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MILLENNIUM REVIEW

Do mammals make their own morphine?


Richard B. Herbert, Henrietta Venter and Sonja Pos Centre for Biomolecular Sciences, University of Leeds, Leeds, UK LS2 9JT Received (in Cambridge, UK) 2nd February 2000 Published on the Web 19th June 2000

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Introduction Pharmacology Morphine biosynthesis in poppies Morphine biosynthesis in mammals The function of endogenous morphine Conclusion Acknowledgements References We are such stuff As dreams are made on, and our little life Is rounded with a sleep. Shakespeare, The Tempest, act 4, scene 1

Introduction 15

A knife blades scoring of the unripe seed capsules of a species of poppy after the fall of its delicate mauve and white owers results in a milky exudate (Fig. 1), that may be collected and dried to give a brown gum known as opium. The use of this exudate as a medicine in the relief of pain and the generation of euphoria can be tracked back to the fourth millennium BC. Its use is found in many different societies down to the present day.

We are all too familiar with the dark side of opium and its destructive addiction: on average, 20 people a day die from drug abuse across Europe; the 1999 opium harvest in Afghanistan is estimated to have been 4581 tons.6 However, morphine 2, one of the constituents of opium along with codeine 3, stands as the essential drug for the relief of dreadful pain. To this end opium is harvested on a multi-ton scale in India which is the only legal producer of opium 3. The nitrogenous bases alkaloidsconstitute a quarter of raw opium and two of these alkaloids, morphine (ca. 17%) and codeine are used as analgesics (relievers of pain). An illustration of the way in which this opium-producing poppy is found the world over is its innocent growth as a wild ower in Britain as recorded in the exquisite water colours of W. Keble Martin (Fig. 2).7 In his book the poppys entry reads: Papaver somniferum L. Opium Poppy. A weed of cultivation, especially in fens, not native. When one of us (RBH) was a post-doc with Alan Battersby in Liverpool in the 1960s the poppies grew vigorously to a height of ve feet or more, to yield, from pioneering labelling experiments, the rst denitive information on the course of morphine biosynthesis.4 Earlier than this Robert Robinson, with the insight of genius, together with J. M. Gulland had taken the mass of confusing chemical data relating to the uncertain structure of morphine

Richard Herbert is Reader in Bioorganic Chemistry, School of Chemistry, University of Leeds; BSc (Hons.) (Cape Town); PhD and DSc, Leeds. He is the author of The Biosynthesis of Secondary Metabolites, founder, with Tom Simpson, of the Bioorganic Subject Group of The Royal Society of Chemistry, and a member of the Editorial Board of Natural Product Reports. Research: biosynthesis of nitrogenous metabolites including alkaloids; most recently, in collaboration with Peter Henderson (Leeds) and Tony Watts (Oxford), the application of isotopic labelling and NMR in solving the 3D structures of membrane transport proteins. He is married to Margaret with two children and four boisterous grandchildren.

Henrietta Venter (right) received her BSc (Hons.) from the University of the Orange Free State (South Africa). Her honours degree was, like her following MSc, awarded with distinction. The research for her masters degree was carried out while she worked as a research assistant in the Department of Food Science (UOFS, Bloemfontein) and while she also taught students from disadvantaged communities. In 1997 she won an Overseas Research Scholarship which enabled her to undertake a PhD degree at Leeds where she carries out research aimed at establishing the 3D-structure of important membrane transport proteins by biochemical and physical methods. She is married to Gerhard who works in IT. Sonja Pos (centre) obtained her Masters degree in Natural Sciences majoring in Chemistry and Biology at the Swiss Federal Institute of Technology (ETH) in Zurich, Switzerland in 1996. She is currently working as a research administrator at the Public Health Unit of the Trinity and All Saints College, University of Leeds. She is married to Martin who is carrying out research on membrane transport proteins.

DOI: 10.1039/a809409h

Nat. Prod. Rep., 2000, 17, 317322 This journal is The Royal Society of Chemistry 2000

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Fig. 1 A seed capsule of the opium poppy showing the exudate of opium resulting from wounding. Published with permission from the Science Photo Library, London.

was consuming around two litres of laudanum a week during a time of highest creativity. Diacetylmorphine (diamorphine) 4 was marketed by Bayer at the end of the nineteenth century. It was named heroin, being seen at the time as a heroic drug. Laudanum was similarly valued: the name derives from the Latin, to praise. Following its isolation, morphine 2 was introduced into medical practice and was widely employed to treat ailments as different as asthma, diarrhea, diabetes and ulcers as well as being used in the relief of pain. Diamorphine has physiological effects that are essentially the same as those of morphine except that it acts faster and has greater potency. Because heroin is rapidly hydrolysed to morphine, most of its actions are due to morphine itself. However, there are appropriate differences: heroin 4 is very lipid-soluble and readily enters the brain, whereas morphine 1 is less lipid-soluble and thus enters the brain more slowly. Codeine 3, a major alkaloid that is approximately one-sixth as effective an analgesic as morphine, is particularly active when administered orally and is a very good cough suppressant. The harrowing addiction to morphine continues to provide impetus to the quest for alternative drugs, based on the morphine skeleton, that are powerful analgesics and that do not also have the undesirable side effects. The opium alkaloids and these analogues are called opioids and all of these compounds bear a strong structural resemblance one to another rst hinting thus that there are receptors in animals complementary in structure to morphine and its family. There are three main classes for these opioid receptors namely , and . It is a reasonable hypothesis that, if there are opioid receptors, there must be endogenous opiate-like compounds. A search for endogenous analgesics rst led to the isolation of two pentapeptides, enkephalins, which bind to opioid receptors and cause analgesia. Other bio-active peptides were subsequently discovered. Together they are called endorphins. They are true

and its opium congeners (protean molecules, Robinson called them, because of their facile and frustrating rearrangements) and measured possible structures against the template of the simple benzylisoquinoline structure of the opium alkaloid papaverine 1, to propose in 1925, on biogenetic grounds, the correct structure of morphine as 2 (Scheme 1).5,8 An X-ray crystallographic analysis and other evidence allowed the absolute stereochemistry of morphine to be dened.9 In 1804 Armand Squin rst isolated the major constituent of opium. A year later Friedrich Sertrner also isolated morphine and named it morphium after Morpheus, the ancient Greek god of dreams.1,10 Appropriate to his training as an apothecary, Sertrner established, on a dog, its narcotic potency. He also recorded that morphine was alkali-like (or basic). These early observations readily make Sertrner the father of alkaloid chemistry. 2 Pharmacology 1,2,11,12

Fig. 2 Papaver somniferum, the opium poppy ower and seed capsule. Published with permission from the Robert Harding Picture Library, London.

In the sixteenth century, Paracelsus popularized the use of opium as an analgesic when he produced various preparations under the name laudanum. Much later the poet Coleridge 318 Nat. Prod. Rep., 2000, 17, 317322

neurotransmitters and bind to the various opioid receptors, eliciting a variety of effects. Curiously morphine has a very high selectivity for a single receptor, the -receptor, which appears to be responsible for analgesia, euphoria, addiction and respiratory depression.1315 From a teleological point of view it seems very unlikely that there is a receptor in mammals whose function is mainly dependent on a substance, morphine,

that originates from a plant. The existence of the highly morphine-selective -receptors could be much better explained if morphine is produced by the mammal (translated from ref. 1; see also refs. 14 and 15). At the beginning of the last century, the French scientist Mavrojannis 16 deduced that morphine was naturally present in rats: when he administered morphine to these animals, they showed the same symptoms as in a disease state of catalepsy. After a seventy-year gap, a morphine-like substance was identied in the brains of mice, rabbits and cats by use of a radioimmunoassay for morphine. The substance was isolated and found to exert a morphine-like response in pharmacological and chemical testing. It could not be a peptidic enkephalin since it was resistant to various peptidases and to acid.17 In addition this substance was found in the central nervous system, urine and cerebrospinal uid of humans and other species.18 The skins of rabbits, rats and toads were found to be a particularly good source and a sufciently large amount of compound was isolated from the toad, Bufo marinus, for it to be isolated and characterized. It was found to be identical with morphine on the basis of immunological, pharmacological and chemical criteria.19 Subsequently, six different morphine-like compounds were identied by immunoassay and four were puried from cattle brains and adrenal glands.20 The amounts obtained were sufcient for one of the compounds to be identied unambiguously as morphine from its proton NMR spectrum. Both morphine 2 and codeine 3 have been found in human cerebrospinal uid.21 The two alkaloids were present in amounts comparable to those of opioid peptides in spinal uid; the alkaloids were present mainly as conjugates. A link between morphine 2 and its opium-poppy precursor, codeine 3, is thus established within humans. Clear evidence exists then for the endogenous occurrence of morphine, and of codeine, in animal tissue. We can be assured that is not the result of medication (see, e.g., ref. 21). One immediate possibility is that endogenous morphine is dietary in origin and evidence has been obtained that morphine is ubiquitous in plants that are eaten by animals; it is present in the milk of cows and humans.22 But it might also be that morphine is synthesized in animals and by driving down the same, or similar, biosynthetic route to that in the opium poppy. What is needed is specic evidence. Before discussing such evidence we need to describe the route that has been elucidated unequivocally for morphine biosynthesis in opium poppies. The evidence for endogenous biosynthesis in mammals will turn crucially upon the key, chemically difcult step of biosynthesis, namely the oxidative conversion of (R)-reticuline 5 into salutaridine 6, a step common to both poppies and mammals. 3 Morphine biosynthesis in poppies 4,23

Scheme 2

Robert Robinsons brilliant notion that morphine is a twisted benzylisoquinoline (Scheme 1) allowed the solution of its struc-

Scheme 1

ture (see above).5,8 The detailed results that have been obtained on the biosynthesis of opium alkaloids reveal that morphine and related alkaloids are formed in P. somniferum through a series of benzylisoquinoline intermediates which culminate in

(R)-reticuline 5. 24,25 The benzylisoquinoline skeleton is elaborated from two molecules of the -amino acid -tyrosine 12 which is converted into one molecule each of dopamine 13 and 4-hydroxyphenylacetaldehyde 14. Condensation of 13 and 14 is catalysed stereospecically by (S)-norcoclaurine synthase which affords (S)-norcoclaurine 15 from which the host of benzylisoquinoline alkaloids found in plants, including morphine, have their provenance. Two enzyme-catalysed methylations and an aromatic hydroxylation lead to (S)-reticuline 16 17 (Scheme 3).2426 Curiously this compound has the opposite conguration to that found in the morphine family of alkaloids. The necessary inversion of conguration to give (R)-reticuline 5 is effected in two steps via the dehydroreticuline 18 (Scheme 4).27 The reductase that is involved is highly specic and is dependent on NADPH/NADP . Some forty years ago, Barton and Cohen hypothesized that the biosynthesis of a diverse multitude of aromatic secondary metabolites (natural products), including morphine 2, could be brought within an orderly mechanistic and predictive framework built around the simple idea of the oxidative coupling of phenols.4,28 This is exemplied in outline for morphine 2 in Scheme 5 (cf. 6). Note that only the top hydrogen-bearing ring can rearomatize after coupling. Though these ideas have long been supported circumstantially, proof has been lacking. The proof came 25,29 with the identication of the enzyme, specically in microsomal preparations of P. somniferum, that catalyses the conversion of (R)-reticuline 5 into salutaridine 6, known from earlier evidence to be a key intermediate in morphine biosynthesis.4 The enzyme is a membrane bound cytochrome P-450 protein and its catalytic action is strictly dependent on molecular oxygen and NADPH. It follows from the mechanism deduced for P-450 catalysis that the conversion of 5 into 6 is most likely to involve the formation of two phenolate radicals and their coupling. Signicantly (S)-reticuline 16 17 is not a Nat. Prod. Rep., 2000, 17, 317322 319

Scheme 3 Enzymes: i, -tyrosine decarboxylase; ii, phenolase; iii, -tyrosine transaminase; iv, p-hydroxyphenylpyruvate decarboxylase; v, (S)norcoclaurine synthase; vi, norcoclaurine-6-O-methyltransferase (6-OMT); vii, tetrahydrobenzylisoquinoline-N-methyltransferase (coclaurine methyltransferase); viii, phenolase; ix, SAM: 3 -hydroxy-N-methyl-(S)-coclaurine-4 -O-methyltransferase (4 -OMT).

pathway via its chemical interconversion with codeinone 11. A nal demethylation of codeine 3 affords morphine 2. An alternative pathway to morphine in P. somniferum involves rst demethylation of thebaine 9 to give 19 and then 20, reduction of which by codeinone reductase affords morphine (Scheme 6).32,33

Scheme 4

Scheme 6

Morphine biosynthesis in mammals

Scheme 5

substrate for the coupling enzyme. Beyond salutaridine 6 an NADPH-dependent oxidoreductase catalyses its stereospecic and substrate-specic conversion 30 into salutaridinol 7 which undergoes enzyme catalysed acetyl CoA dependent acetylation to give the acetate 8.31 This compound undergoes ring closure at slightly alkaline pH to give the opium alkaloid thebaine 9. No enzyme has been found for this conversion and it is concluded the reaction is uncatalysed and spontaneous. It is to be noted that one other biosynthetic step, namely the conversion of neopinone 10 into codeinone is similarly spontaneous.32 Also, the use of acetate (rather than, say, phosphate) as a leaving group is remarkably novel. On the other hand, the stereochemistry of the SN2 ring closure in the formation of 7 takes the orthodox stereochemical course (the epimeric alcohol is not obtained in salutaridine reduction and it is moreover inert to enzyme-catalysed acetylation). Demethylation of thebaine affords neopinone 10, which is in chemical equilibrium with codeinone 11. A specic oxidoreductase converts 11 into codeine 3.32 Neopinone 10 is not a substrate for the enzyme; it is shuttled through the biosynthetic 320 Nat. Prod. Rep., 2000, 17, 317322

It is certainly possible to examine many of the steps of morphine biosynthesis in opium poppies (Schemes 2 and 3) and conclude that they could be effected in mammals by enterprising enzymes acting non-specically. A key early step is the condensation of the amine 13 with the aldehyde 14. Such reactions have long been known to go without catalysis, giving isoquinolines in high yield under physiological conditions (aqueous solution at a pH of ca. 7) (ref. 5, p. 81; ref. 4). But the products are racemic, of course. There is one biosynthetic step that could not be spontaneous or adventitiously catalysed and that is the one whereby (R)reticuline 5 is converted oxidatively into salutaridine 6 (Scheme 2). It is on this enzymic conversion that the evidence for mammalian synthesis of morphine crucially succeeds or fails. Early work provided some evidence for a conversion of racemic reticuline (as 5) into salutaridine with rat liver homogenates.34 Recent ground-breaking experiments 35,36 with (R)-reticuline 5 led to the identication of signicant salutaridine synthase activity in a microsomal preparation of pig liver, with the product being rmly identied as salutaridine 6 from chemical, enzymatic and mass spectrometric data.35 The enzyme which is a P-450 oxidase has been isolated from pig liver and puried to homogeneity.36 It mirrors the poppy enzyme in its dependence on NADPH, in having the same pH optimum (pH 7), and in making the same exclusive stereochemical demand with a singe substrate: it catalyses the conversion of (R)-reticuline 5 into salutaridine 6, but is indifferent to the

S-isomer. The sole difference between the enzymes from the two sources lies in the temperature optima: 37 C for the mammalian enzyme and 2025 C in poppies. The pig enzyme has been partially sequenced and it will be most interesting to compare the gene sequences of the mammalian and poppy oxidases. The enzyme has been identied in the livers of other mammals including humans. It is found only in liver and kidney tissue of the pig.35 The course of morphine biosynthesis in poppies moves on from salutaridine 6 to thebaine 9 (Scheme 2). No denite evidence has yet been gleaned on the metabolic fate of salutaridine 6 in mammals but such a conversion into thebaine 9 is reasonable. In support, it has been found 37 that feeding salutaridine, thebaine and codeine to rats results in a marked increase in morphine (and codeine) in rat tissue thereafter. More signicantly, it has been clearly demonstrated that thebaine 9 is transformed into morphine 2, codeine 3 and oripavine 19 in rat liver, kidney and brain microsomes.38 These results show that both of the routes found for the conversion of thebaine 9 into morphine 2 in poppies (Schemes 2 and 6) can operate in rats. In support of the route via oripavine 19 (Scheme 6) in mammals, an NADPH-dependent morphine 6-dehydrogenase has been puried from the liver of guinea pigs, and characterized.39 The enzyme catalyses the oxidation of morphine to morphinone 20 and the reverse reductive reaction. The biosynthetic pathway to (R)-reticuline in mammals is largely obscure. It is unknown if the rst isoquinoline has the (S)-conguration (as in plants) or is the (R)-isomer which would avoid the later change of stereochemistry from 17 to 5 (Scheme 4). In plants the rst isoquinoline in the biosynthesis of morphine and other benzylisoquinoline alkaloids is (S)norcoclaurine 15 (Scheme 3) which is rst methylated on the oxygen at C-6. Mammalian catechol methyltransferase has been found to catalyse methylation of 15 in this position to the extent of 80% (20% of the 7-O-methyl isomer) in the presence of S-adenosylmethionine.40 Results of further methylation studies are not clear cut.41 In conclusion, the isolation and characterization of a mammalian P-450 enzyme that catalyses the chemically very difcult conversion of (R)-reticuline 5 into salutaridine 6 with complete specicity and that corresponds so closely with the poppy enzyme provides the very strongest evidence that mammals do indeed make their own morphine. Further evidence outlined above ts with this conclusion. 5 The function of endogenous morphine 1

for an endogenous synthesis of morphine in mammals resides in the rigorous evidence for the specic P-450 enzyme-catalysed conversion of (R)-reticuline 5 into salutaridine 6. Questions remain: pharmacologically, about the natural role of morphine and its conjugates in mammals; chemically about the necessary change of stereochemistry in the isomerization in poppies of (S)-reticuline 16 into the (R)-isomer that is the morphine precursor; also the conguration of mammalian benzylisoquinoline precursors. But the operation of the biosynthetic pathway to poppies and mammals ensures that the opium poppy is tended on a very large scale in many parts of this planet. 7 Acknowledgements

This account is based on an article by T. Amann and M. H. Zenk. It draws on the pioneering ideas of Robert Robinson and Derek Barton, the ground-breaking biosynthetic studies of Alan Battersby, Derek Barton and co-workers and the exquisite enzyme isolation and characterization of Meinhart Zenk and colleagues. 8 References

Morphine and other isoquinoline alkaloids have been the subject of an authoritative annual review by K. W. Bentley in Natural Product Reports. The biosynthesis of these alkaloids has been similarly reviewed by R. B. Herbert. The reader may obtain up-to-date information on material in this area by consulting the relevant reviews in this journal.
1 T. Amann and M. H. Zenk, Dtsch. Apoth. Ztg., 1996, 136, 519. 2 J. Mann, Murder, Magic and Medicine, Oxford University Press, Oxford, 1994. 3 National Geographic, 1985, vol. 167, no. 2. 4 R. B. Herbert, The Biosynthesis of Secondary Metabolites, 2nd ed., Chapman and Hall, 1989. 5 R. Robinson, The Structural Relations of Natural Products, Oxford University Press, Oxford, 1955. 6 The Guardian, November 23rd, 1999. 7 W. Keble Martin, The Concise British Flora in Colour, Ebury Press, London, 1965. 8 J. M. Gulland and R. Robinson, Mem. Proc. Manchester Lit. Phil. Soc., 1925, 69, 79; see also J. M. Gulland and R. Robinson, J. Chem. Soc., 1923, 980. 9 M. MacKay and D. C. Hodgkin, J. Chem. Soc., 1955, 3261; K. W. Bentley and H. M. E. Cardwell, J. Chem. Soc., 1955, 3245. 10 F. W. Sertrner, Ann. Phys., 1817, 25, 56. 11 J. G. Bovill, in Anaesthetic Physiology and Pharmacology, ed. W. McCaughey, R. S. J. Clarke, J. P. H. Fee and W. F. M. Wallace, Churchill Livingstone, Edinburgh, 1997, p. 227. 12 L. Dykstra, in Pharmacological Aspects of Drug Dependence, Springer-Verlag, Berlin, 1996, p. 197. 13 C. K. Surratt, P. S. Johnson, A. Moriwaki, B. K. Seidleck, C. J. Blaschak, J. B. Wang and G. R. Uhl, J. Biol. Chem., 1994, 269, 20 548, and refs. cited therein. 14 H. W. Kosterlitz, Nature, 1985, 317, 671. 15 H. W. Kosterlitz, Nature, 1987, 330, 606. 16 M. Mavrojannis, CR Seances Soc. Biol. Ses Fil., 1903, 55, 1092. 17 A. Gintzler, A. Levy and S. Spector, Proc. Natl. Acad. Sci. U.S.A., 1976, 73, 2132; A. Blume, J. Shorr, J. P. M. Finberg and S. Spector, Proc. Natl. Acad. Sci. U.S.A., 1977, 74, 4927; A. R. Gintzler, M. D. Gershon and S. Spector, Science, 1978, 199, 447. 18 J. Shorr, K. Foley and S. Spector, Life Sci., 1978, 23, 2057; A. K. Killian, C. R. Schuster, J. T. House, S. Sholl, M. Connors and B. H. Wainer, Life Sci., 1981, 28, 811. 19 K. Oka, J. D. Kantrowitz and S. Spector, Proc. Natl. Acad. Sci. U.S.A., 1985, 82, 1852. 20 A. Goldstein, R. W. Barrett, I. F. James, L. I. Lowney, C. J. Weitz, L. Knipmeyer and H. Rapoport, Proc. Natl. Acad. Sci. U.S.A., 1985, 82, 5203. 21 G. J. Cardinale, J. Donnerer, A. D. Fink, J. D. Kantrowitz, K. Oka and S. Spector, Life Sci., 1987, 40, 301. 22 E. Hazum, J. J. Sabatka, K.-J. Chang, D. A. Brent, J. W. A. Findlay and P. Cuatrecasas, Science, 1981, 213, 1010. 23 R. B. Herbert, Nat. Prod. Rep., 1999, 16, 199, and refs. cited therein.

Though the effect of administered morphine on humans and the preference of morphine for -receptors 14 rather than other endogenous receptors is clear, the role of endogenous morphine is not yet resolved. This morphine is apparently present in small amounts in vivo. It is likely rst that this is because of its chemical stability and receptor selectivity relative to peptidic endorphins. Secondly, conjugates of morphine as glucuronides and sulfates are found in humans and the amounts of the alkaloid itself found may be underestimates because of the existence of these conjugates (see Section 2) and because of binding to proteins. It may be noted that the analgesic effect of morphine 3 -O-sulfate is about three times higher than that of the parent base. It seems reasonable that the conjugates of morphine and not the alkaloid itself may actually be the effective forms of active opiate in mammals. 6 Conclusion

In the foregoing discussion results have been presented which provide the strongest evidence that the opiate morphine 2 is biosynthesized endogenously in mammals. The pathway when nally dened is likely to be very similar to that so beautifully established for the opium poppy. The key, persuasive evidence

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24 M. H. Zenk, M. Rueffer, T. M. Kutchan and E. Galneder, in Applications of Plant Cell and Tissue Culture, Wiley, Chichester, 1988, p. 213. 25 R. B. Herbert, Nat. Prod. Rep., 1992, 9, 511. 26 C.-K. Wat, P. Steffens and M. H. Zenk, Z. Naturforsch., Teil C, 1986, 41, 126; T. Frenzel and M. H. Zenk, Phytochemistry, 1990, 29, 3491; R. Stadler and M. H. Zenk, Liebigs Ann. Chem., 1990, 555; S. Loefer and M. H. Zenk, Phytochemistry, 1990, 29, 3499. 27 S. Loefer, R. Stadler and M. H. Zenk, Tetrahedron Lett., 1990, 31, 4853; W. De-Eknamkul and M. H. Zenk, Phytochemistry, 1992, 32, 813. 28 D. H. R. Barton and T. Cohen, in Festschrift Dr. A. Stoll, Birkhauser, Basle, 1957, p. 117. 29 M. H. Zenk, R. Gerardy and R. Stadler, J. Chem. Soc., Chem. Commun., 1989, 1725; R. Gerardy and M. H. Zenk, Phytochemistry, 1993, 32, 79. 30 R. Gerardy and M. H. Zenk, Phytochemistry, 1993, 34, 125. 31 R. Lenz and M. H. Zenk, J. Biol. Chem., 1995, 270, 31 091.

32 R. Lenz and M. H. Zenk, Eur. J. Biochem., 1995, 233, 132; R. Lenz and M. H. Zenk, Tetrahedron Lett., 1995, 36, 2449. 33 E. Brochmann-Hanssen, Planta Med., 1984, 50, 343. 34 C. J. Weitz, K. F. Faull and A. Goldstein, Nature, 1987, 330, 674. 35 T. Amann and M. H. Zenk, Tetrahedron Lett., 1991, 32, 3675. 36 T. Amann, P. H. Roos, H. Huh and M. H. Zenk, Heterocycles, 1995, 40, 425. 37 J. Donnerer, K. Oka, A. Brossi, K. C. Rice and S. Spector, Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 4566. 38 H. Kodaira and S. Spector, Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 1267. 39 S. Yamano, E. Kageura, T. Ishida and S. Toki, J. Biol. Chem., 1985, 260, 5259. 40 Y. Sekine, C. Creveling, M. Bell and A. Brossi, Helv. Chim. Acta, 1990, 73, 426. 41 X. He, D. Tadic, M. Brzostowska, A. Brossi, M. Bell and C. Creveling, Helv. Chim. Acta, 1991, 74, 1399.

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