THE BIOSYNTHESIS OP SINOMENINE. a Thesis Submitted

THE BIOSYNTHESIS OP SINOMENINE.

A thesis submitted by

AUDREY J. RIMY

in partial fulfilment of the requirements

for the degree of

Doctor of Philosophy

The University of London.

Imperial College September 1970 London SM.7. (1963-1966) 3.

Abstract.

The biosynthesis of sinomenine has been investigated (14_, in Sinomenium acutum using uj_ and [3W-labelled precursors. Reticuline was found to be a better precursor for the alkaloid than was protosinomenine.

Reticuline was also incorporated into the dienone sinoacutine, which in turn was transformed by the plant into sinomenine. Two predicted pathways, involving isosinomenine and sinoacutinol, for this transformation were shown to be incorrect. The unusual, chlorine-containing alkaloid, acutumine, was shown not to be an artefact, and not derived biosynthetically from sinomenine, isosinomenine or sinoacutine.

A review of the biosynthesis of the morphine alkaloids is presented. 4.

Acknowledgements.

I thank Professor D.H.R.Barton, F.A.S., and Professor G.W.Kirby for their continuing advice and encouragement during the course of this work, and my husband for permission to write this Thesis during working hours. I also thank the S.R.C. for financial support. 5.

Contents. Page

Chapter I. 6 The Biosynthesis of the Morphine Alkaloids

Chapter II. 60 The Biosynthesis of Sinomenine

Chapter III. 100 Experimental

References 129 6.

Chapter I

The Biosynthesis of the Morphine Alkaloids 7.

Introduction.

Before any tracer work on the biosynthesis of the morphine alkaloids had been reported three basic proposals were made which laid the foundations for future work. The first of these 1 was made as long ago as 1910 by Winterstein and Trier who suggested that the benzylisoquinolines are built up from two eight-carbon units, derivatives of 3,4-dihydroxyphenylalanine. Two likely derivatives are 3,4-dihydroxyphenylacetaldehyde (I) and 3,4-dihydroxyphenethylamine (dopamine)(II) since these can be condensed chemically to give the benzylisoquinoline (III).

The second proposal was made in 1925 by Robinson2'3 who recognised the structural relationship between the benzylisoquinolines and the morphine alkaloids and suggested that morphine

(IV) was derived from the oxidative cyclisation of a benzylisoquinoline precursor. ire third proposal concerned the mechanism of this oxidative 4 cyclisation. In 1957, Barton and Cohen put forward possible schemes for the biosynthesis of many natural products based on the idea of the coupling of phenolate radicals. Thus a benzylisoquinoline (V), having protecting groups Ri and R2 to allow specific oxidation, could be oxidised to a diradical which could undergo intramolecular ortho-para coupling to yield the dienone

(VI). Formation of the fifth ring, as in (VII), followed by 8.

HO HO

I. dopamine

norlaudanosoline morphine 9

V. reticuline: B71=R2=Me.

0 VI. VII.

VIII. IX. tyrosine 10. reduction and dehydration would give the skeleton and level of oxidation of the morphine alkaloids, as in (VIII). Precursors. C6-C2 In the first tracer experiments58 designed to test the validity of the first proposal, tyrosine (IX) and Ohenylalanine were chosen as possible precursors, since it is known that phenylalanine can be a precursor of tyrosine9 and that tyrosine is 10 a precursor of 3,4-dihydroxyphenylalanine . Bettersby et al.5,6 fed (-)42-14Otyrosine to Papaver somniferum var. Noordster by injection into the seed capsules, and isolated morphine (IV), codeine CO, thebaine (XI) and papaverine (XII), which were all found to be radioactive. The morphine-was degraded to show that it was labelled specifically and about equally at positions 9 (2-14c and 16. ]menylalanine was also incorporated into morphine, though much less efficiently. These results were in

7,8 r agreement with those of Leete who fed (-)-L214 Chyrosine and )42).4 t Clphenylalanine to a different variety of P.somniferum via the roots of the plant, and are proof that the morphine skeleton is built up from two Cos-C2 units which can be biosynthetically 11 derived from tyrosine. It has been pointed out that decarboxylation could occur after condensation, in which case one of the two units would be C6-C2-CO2H. The fact that (-)-3,4-dihydroxyphenylalanine 12 was later shown to be a precursor for morphine led Battersby to 11.

X XI codeine thebaine

XII papaverine 12.

suggest also that tyrosine was first converted into the dihydroxy derivative before any further reaction or condensation took place.

The original proposal was for the condensation of two different C6-c2 units, one of which could be dopamine (II). 14 13 When [1- C]dopamine was fed to P.somniferum the resulting morphine was found to be radioactive, with the activity essentially confined to C-16. Once again this was confirmed by independent 14 work in which both morphine and codeine were isolated from 14 plants fed with [1- C]dopamine. In this case degradation showed a complete loss of activity from both alkaloids when the ethanamine side-chain, containing C-16, was eliminated, proving that C-9 carried no significant activity.

Thus it has been shown that 3,4-dihydroxyphenethylamine, (or some closely related compound), derived from phenylalanine via tyrosine and 3,4-dihydroxyphenylalanine, is used by the plant to build up half of the morphine skeleton, and that the second unit is different but is also derived from 3,4-dihydroxyphenylalanine and so is presumably dihydroxylated. Tyrosine and the related aromatic amino-acids are products of the well-known shikimic-prephenic acid pathway and so further 15 support for the whole scheme was obtained when it was found that [H]shikimic acid was incorporated into morphine and papaverine. 13.

16,17 Recently isolated poppy latex has also been shown to be capable of converting radioactive 3, k-dihydroxyphenylalanine into morphine. Interconversion of the Morphine Alkaloids.

The order in which morphine (TV), codeine CO and thebaine (XI) are formed in the plant is important for the consideration of the methoxylation or hydroxylation pattern of a benzylisoquinoline precursor. Two different studies of incorporation rates, and another involving the feeding of individual alkaloids have shown that thebaine is formed firstly and is further metabolised 8 into codeine and finally morphine. Battersby and Harper fed r 14 L2- CItyrosine to a large number of poppies and extracted the alkaloids from a few at a time at intervals ranging from a few hours to two weeks. The ratios of the total activities of the three alkaloids at each time interval showed a rapid incorporation of tyrosine into thebaine, followed by a drop in thebaine activity as first codeine and finally morphine became active. 19 Rapoport et al. grew poppies in an atmosphere containing rL14 Clcarbon dioxide and showed that the rate of incorporation of activity into the skeletons of the alkaloids varied in a 20 21 similar way. These workers also ' fed separate plants with labelled morphine, codeine and thebaine. They found that the activity from thebaine passed into codeine and morphine, and 14. that from codeine passed into morphine but not thebaine, and that there was no incorporation of morphine activity into codeine or thebaine. Penzylisoquinoline Precursors.

The choice of a benzylisoquinoline to test the second proposal is confined to those with a tetraoxygenation pattern if the above results of amino-acid feedings are accepted. The simplest of these is (t)-norlaudanosoline (III). Accordingly (1. )-(1-14C]norlaudanosoline was fed11 to P.somniferum plants and was found to be efficiently incorporated into morphine (IV), codeine (X), thebaine (XI) and papaverine

(XII). The morphine was degraded to show that the activity was r 14 confined to position 9 as expected. (-)-L3- CD1Orlaudanosoline 11 was also fed and again incorporation into the morphine alkaloids was achieved. In this case the derived thebaine was degraded to show that it was labelled specifically at C-I6. That the radioactive codeine had also used the precursor without scrambling of the label was shown by elimination of the ethanamine side chain with resulting complete loss of activity. Thus it is established that the morphine alkaloids are biosynthetically derived from a benzylisoquinoline.

The N- and 0-methyl groups in the three morphine alkaloids have been shown5 to be derived from the S-methyl group of 15.

methionine as would be expected, but the exact stage of the biosynthesis at which this occurs remains to be established. Since norlaudanosoline is so efficiently incorporated it seems unlikely that methylation occurs before the benzylisoquinoline stage. This has been supported by the feeding22 of 4-hydroxy- 14 3-methoxyphen[1- C]ethylamine which proved one hundred times less efficient as a precursor than dopamine.13,14

However, as the first-formed alkaloid of the three is thebaine, the most highly methylated, it does seem possible that norlaudanosoline Is itself methylated before the oxidative coupling step occurs. Methylation of the 6-and 41-hydroxyls of norlaudanosoline would not only result in the required methoxylation pattern of thebaine, but would also provide the protecting groups necessary 4 for the specific radical oxidation mentioned earlier. Reticuline

(V,R132=Me) fulfils these requirements and is also N-methylated as are the morphine alkaloids. It has the further merit of being a known natural product: (+)-reticuline was isolated from 23 Anona reticulate in 1959 (see also below). In the first experiments (t)-[1-3H, 4-methy1-14C]reticuline24 was fed to P.somniferum plants and incorporation into morphine was shown to occur. Zeisel demethylation of the morphine showed 14 that the C activity was confined to the N-methyl group as 16. expected. (t)43-14-,Uffleticuline was incorporated11'25 and with great efficiency into morphine and also into codeine and thebaine. Elimination of the ethanamine bridge from the radioactive morphine removed all the activity, which was presumably confified to C-16. In a parallel experiment (4-.)- [3-14 C]bi-nor-reticuline was also incorporated into morphine, though less efficiently, indicating that methylation of the amino group can occur as a separate step after methylation of the hydroxyls. However, since the plant is capable of demethylation of thebaine to yield successively codeine and morphine, it was particularly necessary to show that the reticuline and nor- reticuline were not being demethylated to give norlaudanosoline or laudanosoline before incorporation into the morphine alkaloids. 3H,3,N-methyl, 00'-meth ,-1 -1 - 1meticuline ' was To do this (t)-[1- C4 26 fed to P.somniferum and the resulting thebaine was shown to be radioactive. Selective Zeisel demethylations gave the activities of the three methyl groups and the activity at C-16 was obtained by difference.. The results showed that incorporation had occurred without prior degradation. This finding is supported by the 11 14 feeding of (-)-(3- Cltetrahydropapaverine (KIII) with negligible incorporation into morphine, showing that demethylation 17.

XIII XIV tetrahydropapave rine laudanosoline

XV XVI protosinomenine orientaline

XVII 18.

does not occur, and also that the oxidative coupling step is blocked as would be expected, by the methylation of the 3'- and 7-hydroxyls of the benzylisoquinoline.

22 r 3 1 14 1 In a later experiment (-)-0. - Hs,- 0,N-methyl.- Ci- laudanosoline.(XT7) was fed to P.somniferum var. Schlanstedt to give incorporation into morphine. Assuming that no N-demethylation occurs this is in apparent disagreement with the incorporation of nor-reticuline since in this case the amino group is already methylated and methylation of the hydroxyls is then necessary for the production of reticuline. It seems therefore that the plant is capable of methylating the groups in either order to give reticuline. Whether both pathways are normally used by the plant is however not clear. Indeed, the specificity of the enzyme systems of P.somniferum has yet to be explored. Thus it 27 has been established that this plant can convert dihydrocodeine into dihydromorphine with an efficiency of about half that of the corresponding conversion of codeine to morphine. If these dihydro derivatives are not natural products this is an indication that the plant enzymes are not highly selective.

The final proof of the necessary part played by reticuline in the biosynthesis was obtained by feeding its three structural 28 + 3 14 isomers suitably labelled: (-)-[1- H,3- C]protosinoMenine (XV), 19.

(t)-[1-3H]orientaline (XVI) and the unnamed (t)-[1-3R]benzylisoquinoline (XVII). In each case the incorporations were negligible. Later, in 1967, Rapoport et al.29 reported confirmation of the above results, obtained by exposure of ,14 1 plants to an atmosphere containing L Clcarbon dioxide. They first established the presence of reticuline in the plants by the isolation of (-)-reticuline from the seed capsules of

P.somniferum. None was apparently detectable in the rest of the plant, or in poppy seedlings. Budding plants and seed- 14 lings were then exposed to [ C]carbon dioxide for a brief period, and after the addition of carrier in the form of reticuline

perchlorate, reticuline and thebaine were shown to be the only major radioactive alkaloids present. In each case the specific activity of reticuline was higher than that of thebaine. The specific activities of the total N- and 0-methyl groups were

also compared. Oxidative Coupling. Having established the importance of benzylisoquinolines in morphine biosynthesis the next step in the proposed scheme can

be considered: that is the formation by phenol oxidation of a

dienone (VI) intermediate, or its tautomer (VII), and its

transformation into the morphine alkaloids. The dienone 20. predicted from the oxidative coupling of reticuline would have two methoxyl groups (XVIII). This is now known to be a natural product and has been called salutaridine. Salutartdine has 30 been isolated from Croton salutaris Croton balsamifera 1 32 and, most importantly, from Papaver orientale which also produces thebaine. However, at the start of the investigation of this stage of the biosynthesis, salutaridine was an unknown 24 compound, and a synthesis was achieved using thebaine (XI) as the starting material. It was also shown24133'34 that reticuline could be converted chemically into the dienone by phenol oxidation with potassium ferricyanide, though in very small yields detectable only by tracer work. [1-3H]Salutaridine was prepared26 as follows. Thebaine was reduced with sodium in liquid ammonia to give the dihydrothebaine (XIX). This phenol was then treated with hot, alkaline tritiated water, a process known35 to exchange specifically those protons ortho and para to a phenolic hydroxyl group. The resulting [1-3H]dihydrothebaine gave [1-3H]salutaridine after 14 successive acetylation, oxidation and hydrolysis. [16- C]Salut- aridine was prepared from biosynthetic [16-14C]thebaine, obtained ,‘ r 14 from poppies fed with t-J-1.3- C]norlaudanosoline. The 14 26 [1-3H]salutaridine and [16- C]salutaridine were then fed 21.

thebaine salutaridine

XX XIX Pummerer's ketone

XXI narwedine salutaridinols-I and -II. 22. separately to P.somniferum and in each case high incorporation into the morphine alkaloids was observed, without scrambling of the label. Also salutaridine was isolated by dilution from the phenolic alkaloids from two earlier feedings, those of (t)- [2-14_ u]tyrosine and [3-14C]norlaudanosoline, and was found to be radioactive. Thus it is clear that salutaridine must lie on the pathway from tyrosine to morphine. Salutaridinols-I and -II. The formation of thebaine from salutaridine still requires a ring closure, a reduction and dehydration. The original hypo- 4 36 thesis had been put forward in the light of the true structure of Pummerer's ketone (XX), which is formed by ortho-para coupling of the phenolate radical of p-cresol, followed by spontaneous cyclisation to form the third ring. Also the oxide ring of narwedine (XY2) was known37 to close spontaneously during the formation of this alkaloid by ferrinyanide oxidation of the 26,33 appropriate diphenol. However salutaridine showed no tendency to cyclise spontaneously in either acidic or basic solution, and it became clear that a separate biochemical step would be required. An alternative sequence of steps from the ,38,39,4o dienone to thebaine had already been proposed12 involving the reduction of salutaridine to the dienol (XXII). Then displacement of the allylic hydroxyl group, possibly as a phosphate 23% ester, by the phenolic hydroxyl group would cause ring closure to give thebaine. This sequence was proved at least partially correct by chemical and biosynthetic means. Salutaridine was reduced26'33 with sodium borohydride to give the two epimeric alcohols, salutaridinols-I and -II (XXII), both of which readily dehydrated in aqueous acidic solution to yield thebaine. Reduction of [1-3H]salutaridine gave the [1-3H]- dienols, and reduction of unlabelled salutaridine with sodium

[3Hlborohydride gave the [7-31fldienols. [1,7=-3H2]Salutaridinols-I 26 and -II were then fed separately to P.somniferum plants, and high incorporation of salutaridinol-I into thebaine was observed.

Degradation of the thebaine showed a slight (ca.31) loss of tritium from C-1, but none from C-7, indicating that the alcohol had not been oxidised to the dienone in the plant before incorporation. The salutaridinol-II was less efficiently converted into thebaine in the plant than in a control experiment designed to measure the "spontaneous" in vitro conversion which occurred in aqueous solution during the time of the feeding experiment. Confirmation of this result was obtained by feeding 3 14 , 14 [7- H,16. C]salutaridinols-I and -II. The [16- C]dienols were 14 obtained by conversion of biosirthetically derived [16- C]thebaine into [16-14C]salutaridine, followed by reduction to the two alcohols. 24.

Again salutaridinol-I was much more efficiently incorporated, the ratios for the incorporations of dienol-I to dienol-II into the three alkaloids ranging from 14.5:1 to 18.7:1. Comparison 3 14 of the ratios of H: C in the precursor and the derived alkaloids in both the feedings showed a substantial loss of tritium (14-19%) from C-7 during the conversion of thebaine (XI) to codeine (K), a point to be considered when studying the steps involved in this transformation. The absolute stereochemistry of the two alcohols was determined34 in the following way. Salutaridine was reduced with sodium [3H]borohydride to give [7-3H]salutaridinols-I and -II which were separately subjected to ozonolysis followed by catalytic reduction and alkaline hydrolysis to give glyceric acid. The yield from this degradation was very low, so inactive, carrier D-(-)-glyceric acid was added in each case and was isolated by conversion into the p-bromophenacyl ester. Chroma- tography and repeated crystallisation gave radioactive 11-bromo-

Phenacyl D-glycerate from the salutaridinol-I experiment and inactive material from the salutaridinol-II. Hence salutaridinol-I must have the configuration (XXIII) corresponding to D-glyceric acid. Bacemisation would have resulted in loss of tritium, and a further check on the results waa made by repeating the 25.

Me0 Me0

HO HO' H 5 NMe HO

Me0 Me H XXIV XXIII salutaridinol-I

HO HO IV X morphine codeine

XI thebaine 25. experiments, using L-(+)-glyceric acid as carrier. In this case salutaridinol-II produced the radioactive ester, as expected. The remainder of the proposed sequence for the conversion 2' displacement of of salutaridine into thebaine involved an SN the hydroxyl group with the closure of the fifth ring. However, this mechanism requires the opposite configuration at C-7, i.e. that of salutaridinol-II, while it has been shown above that salutaridinol-I (XXIII) is the precursor. Two alternative suggestions have been made34 to overcome this difficulty: first, direct displacement of the hydroxyl group at C-7 by an enzyme functional group with consequent inversion of configuration, followed by displacement of this function by a normal SN2' process; and second, a preliminary allylic rearrangement to give the isomer (XXIV), followed by an SN2 displacement at C-5. Thus the sequence tyrosine (IX) --.4norlaudanosoline (III)

reticuline (V, II=R=Me)--1, salutaridine salutaridinol-I thebaine codeine (X)--j morphine(IV) has been established as an important biosynthetic pathway in the opium poppy, and the main steps, from reticuline to thebainc, have been carried out in vitro to give a synthesis of

the morphine alkaloids by the biogenetic route.33 27.

Optically Active Precursors. So far in this account only racemic benzyl isoquinolines have been mentioned, but since the morphine alkaloids and later intermediates are optically active and the phenolic coupling would be expected to be stereospecific, it should be possible to predict the configuration of these precursors.

The absolute configuration of the morphine alkaloids (as in IV, X, XI) has been established by chemical41 and X-ray42 means. 23 The (+)-reticuline isolated from Anona reticulata was shown to have the absolute configuration (0ar) by methylation to (+)-laudanosine (XXVI) whose absolute configuration is known 43 from chemical correlation with the natural amino-acids.

Comparison of (00T) and (XI) therefore suggests that (-)- reticuline should be the precursor for thebaine Oa). Also W- 28 and (-)-norlaudanosoline hydrochloride were correlated with (+)- and (-)-tetrahydropapaverine respectively, whose absolute 43 configuration is well-established, with the result that in this case (+)-norlaudanosoline (XXVII) is the predicted precursor. Direct confirmation of these correlations has been obtained 34 by chemical means as follows. It has already been mentioned (page 20 ) that (t)-0Hireticuline could be converted in trace

amounts to the dienone by oxidation with potassium ferricyanide. The yield from this experiment presumably represented only half 28.

Me0

.Me0

XXV (+)-reticuline

Met) H MeO

Me 0 XXVI XI (+)-laudanosine thebaine

HO 11 0

XXVII XVIII

(+)-norlaudanosoline (.0-salutaridine 29.

the true conversion since (+)-salutaridine (XVIII) was used as a carrier to isolate the product. The experiment was repeated using (+)- and (-)-[3Hireticuline separately and in each case the product was diluted with (+)-salutaridine. The diluted material from the oxidation of (-)-reticuline (enantiomer of XXV) only retained its activity on purification and conversion to thebaine. As yet another check on these findings (0-salutaridine (XVIII) was correlated with (-)- laudanosine (enantiomer of XXVI). 0-Methyl-(1-310salutaridine 44 was reduced with sodium in liquid ammonia, a process known to cleave dienones of the proaporphine groups to their related benzylisoquinolines, and the phenolic products were methylated with diazomethane. The laudanosine obtained was shown by various means to be the (-)-form. In apparent disagreement with the above conclusions, the naturally occurring reticuline so far isolated does not seem to have a predominance of the (-)-form. Besides the (+)- reticuline isolated from Anona reticulata 23 and from Phylica 4 + rogersii. 5 ,(-)-reticuline46 and later reticuline containing 47 the (+)- and (-)-enantiomers in the ratio 3:2 was isolated 14 from opium. When (-)-(3- C)norlaudanosoline was fed to 48 P.somniferum plants and the reticuline content of the resulting alkaloids was examined by dilution analysis, it was found that 30. there was a predominance of (0-reticuline over (-)-reticuline (ratio of 6:1). As far as this result can be taken as being representative of the normal situation, then it is in agree-

ment for the findings for crude opium. However, it was also 14 , shown that poppy seedlings grown in an atmosphere of [ C]- carbon dioxide produced racemic reticuline.48 (-)-Reticuline, possibly partly racemised, has been isolated from Romneya 49 29 coulteri and from Papaver somniferum seed capsules. The following experiments with optically active precursors have clarified these observations by showing the existence of a further step in the pathway and have also helped to confirm

the finding mentioned earlier that the plant seems to be able 28 to N- and 0-methylate in either order. In the first experiments (+)- and (-)-(31kdinorlaudanosoline were prepared from the correspondingly labelled tetrahydropapaverines (XIII) after proof that the demethylation step did not involve racemisation. The two enantiomers were fed to P.somniferum with the unexpected result that (-)-norlaudanosoline (enantiomer of XXVII) was incorporated more than ten times more efficiently than the (0-isomer (XXVII) whose absolute configuration corresponds to that of the morphine alkaloids (as in XI). FUrther experiments 28 (..)41.3H,3_14c, .140,4f_o_ showed that (+)- and N-methyl methyl-14 Cjreticuline, , resolved to ca. 96% optical purity by fractional crystallisation of the 00-dibenzoyl-tartaric acid salts of the 00-dibenzyl-reticulines, were incorporated into the 14 morphine alkaloids with the same efficiency, based on C activities. The thebaine from the (-)-reticuline feeding was 14 degraded to show that there had been no scrambling of the C labelling, but it was also found that there had been a consid- erable loss of tritium in all three morphine alkaloids. This loss was least in morphine and most in thebaine. In the (-0- reticuline feeding experiment almost all the tritium was lost, 14 although the C incorporation was the same. 28 These results were explained by postulating a further intermediate, 1,2-dehydroreticuline (aVIII) which allows an oxidation-reduction. interconversion of (+)- and (-)-reticuline. Then (.0-reticuline, of the opposite absolute configuration to the morphine alkaloids, would have to pass through the dehydro- intermediate with consequent loss of tritium. The (-)-reticuline could be converted into thebaine and thence codeine and morphine directly, but would also be subjected to the oxidation-reduction process so that as time went on it would contain less and less tritium. Hence, at harvesting, the morphine would show the least loss of tritium as it would be formed from the (-)-reticuline present in the plant for the shortest time. It is assumed that 32.

Me 0

Me0

XXVIII XXIX 1,2-dehydroreticuline (+)-codamine

IV III morphine norlaudanosoline

HO Me0

XXX XXXI 1,2-dehydronorlaudanosoline 1,2-dehydronorreticuline 33.

the tritium is not lost by this process after the phenolic coupling has taken place since this would involve a violation of Bredt's rule. 1,2-Dehydroreticuline could then represent the branching of the pathways, (-)-retiouline leading to the morphine alkaloids and (t)-reticuline to (0-codamina (XXIX) and laudanosine (XXVI). 14 To test this theory [3- C]dehydroreticuline chloride was fed28 to P.somniferum with a resulting highly efficient incorporation into morphine (IV). The label was shown to be confined to the C-15, C-16 bridge of the morphine as would be expected, and the existence of the postulated oxidation-reduction system is thus established. The norlaudanosoline results can now be explained by assuming that 1,2-dehydroreticuline is an intermediate between norlaudanosoline and reticuline, and that (-)-norlaudanosoline is more easily metabolised than its enantiomer. Thus (t)-41-3H,3-14Cinorlaudanosoline

(III) was found22 to be converted into morphine by the poppy with more than 90% loss of tritium. The unequal incorporation of the 28 two norlaudanosoline enantiomers points against a rapid interconversion by an oxidation-reduction system at this point in the biosynthetic pathway, and this was borne out by the feeding of 14 [3- C1-1,2-dehydronorlaudanosoline (OCK) which showed a 34. comparatively very poor incorporation into morphine. Further experiments22 along these lines showed that 14 [3- C]-1,2-dehydronorreticuline (XXXI) was incorporated significantly into morphine, presumably either by methylation to dehydroretiouline (aVIII) or by reduction to nor-reticuline

(XXXII), previously showj1 (see above) to be a precursor for morphine. Confirmation that the latter compound was a precursor was obtained by feeding (4-. )41-3H,3-14C]nor-reticuline. Incorpor-

ation was observed but involved an 82% tritium loss. Also (t).41_3H0_14c ,N-methy1-14C]laudanosoline (XIV) was incorporated, as mentioned earlier, into morphine by poppies of the Schlanstedt variety with 88% tritium loss. These losses can be explained either by prior conversion of both precursors into reticuline

and subsequent oxidation-reduction at this point, or by similar

oxidation-reduction processes operating on nor-reticuline (XXXII) and on laudanosoline (CEIV). To make the comparison more complete (_)-(1-3H,3_14c (+)- and ,N-methy1-14C,41 -0-methy1-14C]reticuline

were fed to poppies of the Schlanstedt variety with essentially the same results as those obtained previousiy28 from NOordster

plants. One further minor possibility, that 1,9-dehydroreticuline

(XXXIII) could also be involved in the oxidation-reduction system, 22 3 14 , was ruled out by the feeding of (-0- and (-)49- H,3- Cj reticuline. The resulting morphine in both cases showed a good

XXXII MEV norreticuline laudanosoline

Me0

MeO

XXXIII 1,9-dehydroreticuline 36, incorporation of reticuline and no loss of tritium. Consideration of other routes. 22 Battersby et al. have also supplied evidence against three more controversial routes to morphine which have been suggested at various times. Before the theories of phenolic coupling, as applied to benzylisoquinolines, had been published, Cohen suggested that the imine (XXXIV) might be a precursor for morphine. Since this compound might readily be converted into a benzylisoquinoline, the corresponding secondary amine, labelled 14 with C at C.,1 was prepared instead. It was hoped that the oxidation-reduction system obviously available in the plant could generate the required base (xxxiv) in vivo. The morphine obtained from the feeding experiment was, however, virtually inactive. 51 A second possibility, that the carbon-carbon bond formation during the conversion of the benzylisoquinolines into tne morphine skeleton might occur through electrophilic substitution of the aromatic nucleus by a phenoxonium cation, rather than by radical

pairing, suggested that (-)-codamine (enantiomer of XXIX) might be 1 2 an intermediate between reticuline (V,11 =14 =Me) and salutaridine (XVIII). (+)-Codamine (xxix) is a known constituent of opium. Accordingly (.0- and (-)-codamine, labelled with tritium in the 14 aromatic ring and with C at C-3, were prepared by an unambiguous route and separately fed to P.somniferum plants. The resulting 37•

XXIX XXXIV (+)-codamine

NMe

V 1 2 reticuline:R =19 =Me XVIII salutaridine

Me0

+ HO N Me X

Me0 OMe XXVIII XXXV 1,2-dehydroreticuline sinomenine 38. incorporations into morphine were insignificant when compared with that of a parallel experiment with (-)-reticuline.

A suggestion52,53 based on the results of oxidation experiments with nitrogenous phenols in vitro, that the nitrogen atom of the benzylisoquinolines might be quaternised 22 before oxidation in vivo was similarly discounted. 14 (-)-(3- methochloride, also known as tembetarine chloride, was not significantly incorporated into morphine in P.somniferum, a negative result strengthened by comparison with the high incorporation (300 times higher) of the quaternary 1,2-dehydroreticuline (XXVIII). Further evidence against this 54 type of nitrogen protection was obtained from work on sinomenine

(XOW). Sinomenine has the opposite configuration to that of the morphine alkaloids, and (+)-tembetarine Chloride, the methochloride of (+)-reticuline, would in this case be the required compound. Natural (+)-tembetarine chloride (isolated55 from Fagara naranjillo) was labelled with tritium in the aromatic rings ortho and para to the phenolic hydroxyl groups and was fed

to Sinomenium acutum. The results showed that reticuline was over 100 times more efficient as a precursor for sinomenine 54 than was tembetarine chloride. However, it was pointed out that though these results are evidence against quaternisation

39. by methylation, they do not rule out the possibility that the precursor is covalently bonded to an enzyme at the nitrogen atom, a situation that would be more difficult to study.

To summarise the conclusions which can be drawn at this point the steps leading to morphine are shown below.

NH CO H 2 phenylalanine tyrosine 7 HO NH2 HO '`CO2H 3,4-dihydroxyphenylalanine

HO -C or C -C -CO H HO )0J112 C6 2 6 2 2 dopamine

HO HO HO norlaudanosoline 110.

norlaudanosoline

HO Me0 Me0

HO HO H HO HO HO

HO Me0 Me() laudanosoline norreticuline dehydronorreticuline

Me0 Me0 Me0

HO HO HO H HO er- HO stIMe HO Me0 Me0 Me0 (+)-reticuline dehydroreticuline ( -) -reticuline

Me0

salutaridine salutaridine

H MeO H' salutaridinol-I thebaine

Me0

H HO

codeine morphine 42.

Codeinone as an Intermediate.

The sequence thebaine (XI), codeine CO, morphine (IV) has 18-21 been established .: and two theories have been considered regarding possible intermediates between thebaine and codeine.

Either56 reduction of thebaine to give codeine methyl ether 4,12,33,57 (XXXVI), followed by demethylation, or hydrolysis of thebaine to yield neopinone isomerisation to codeinone (=VIII), followed by reduction could give codeine. 56 To test these routes Rapoport et al. prepared codeine 14 methyl ether and codeinone labelled with C in the carbon skeletons and after feeding separately to poppy plants isolated

codeine and morphine. Codeinone was shown to be a more efficient

precursor than was codeine methyl ether. Also radioactive codeinone was isolated from plants which had been exposed

to [14C]carbon dioxide, but radioactive codeine methyl ether

was not detected. Addition of inamtive codeine methyl ether as a carrier also failed to reveal any activity. These results are in agreement with those of Battersby et al.15,58

[2-31.1]14brpMne (IV) was methylated to [2-31.1]codeine CO, which was shown to be specifically labelled. Oxidation of the codeine gave [2-3 Hj1 codeinone (XXXVIII) which after purification was shown to contain a negligible amount of labelled codeine. 43.

XI thebaine

IV morphine codeine methyl ether

)(X VII xxxv. II neopinone codeinone 44.

[2,6-3 H2]Codeine (K) was prepared by reduction of the enone with sodium OH)borohydride. Feeding experiments then

showed that the activity from [2-3 N],oodeinone was incorporated efficiently and specifically into morphine and codeine. Also [2,6-3H2]codeine was incorporated into morphine without loss of label from C-6, indicating that reduction of codeine to codeinone is not a reversible

process in the plant. This then is evidence of the existence of one more

step in the biosynthesis of morphine in P.somniferum, and it seems that the picture must be very nearly complete.

It remains to be proved whether thebaine is converted directly into codeinone or via neopinone (XXXVII). If neopinone were an intermediate the small loss of tritium from C-7 observed 3 , 1 during the conversion of [7- H,lo- C]salutaridinol-I into

codeine might be explicable since the tritium would appear a to a carbonyl group. Enzymically controlled protonation of thebaine and removal of a proton from C-7 of neopinone

(XXXVII) would presumably be stereospecific and cause either complete retention or complete loss of tritium during biosynthesis. Therefore, either at least one of

the steps is not enzymically controlled, or the tritium

loss is due to some secondary, "spontaneous" process occurring in the plant. 45.

We are concerned here mainly with the biosynthesis of morphine, but it is itself subjected to changes in the plant. Labelled morphine has in fact been shown59 to be rapidly metabolised by P.somniferum to form two non- alkaloidal, polar substances which in their turn undergo further changes. Structural Relatives of Morphine. The number of known natural products with a morphine- like skeleton is rapidly growing, and some of those recently chara-xterised are outlined below. They fall naturally into four groups, and those most closely related to the morphine alkaloids will be discussed first. The isolation of sinoacutine60'61 (enantiomer of XVIII) and salutaridine -)2(XVIII) led to the recognition of a number of similar alkaloids, mainly from Croton and Papaver species. Norsinoacutine (XXXIX) was found in 62 63 Croton balsamifera and C.flavens , and 8,14-dihydrosalutaridine (XL,R=Me) and 8,14-dihydronorsalutaridine 64 (XL,R=H) were obtained from C.linearis 8,14-101hydro- salutaridine (X14R=Me) was shown to be the enantiomer of isosinomenine65) 66 (see chapter II) and thus must have the configuration shown in (XL). Flavinine (XLI, R=H) 46.

XVIII XXXIX salutaridine norsinoacutine

OMe HO

0 XL XIS flavinine: flavinantine: R=Me 0

MeO HO H XLII XLIII amurine nudaurine • 47.

and flavinantine (XLI,R=Me) were isolated from C.flavens 63,67,68 and provide examples of a different oxygenation pattern in ring A. Further examples of this kind are provided by amurine and nudaurine, constituents of

Papaver nudicaule, whose structures have been shown to be (XLII) and (XLIII) respectively. The absolute configuration of nudaurine (XLin) was determined70 in a 34 manner analogous to that used for salutaridinols-I and -II. Amurine (XLII) was reduced with lithium aluminium 0E11-- hydride to give nudaurine and its epimeric alcohol. The 17-r 3 HjnudaurineI was subjected to ozonolysis, alkaline hydrolysis and dilution with D-(-)-glyceric acid. Purification as its p-bromophenacyl ester proved the D-(-)-glyceric acid to be radioactive, hence establishing the absolute stereochemistry shown in structure (XLITI), which is the same as that of salutaridinol-I, the morphine intermediate. Finally, sinoacutine (enantiomer of XVIII) and pallidine (XLIV) have been found in Corydalis pallida.71

The probable biosynthetic pathways to these alkaloids are all fairly obvious. Thus reticuline or nor-reticuline could lead to salutaridine and norsalutaridine, which in turn could be reduced to 8,14-dihydrosalutaridine and 48.

OH Me0 HO

MeO

XLV XGIV 1 2 coclaurine: R =H,R =Me pallidine I 2 norcoclaurine :R =R =H 1 2 isococlaurine : R =Me ,R =H OH HO

Me0

XLVII XIVI crotonosine reticuline

OMe

XVI orientaline k9.

8,14-dihydronorsalutaridine. Haynes et al.72 found that labelled (+)-coclaurine (KUV,R1=H;R2=Me), (t)-norcoclaurine ocur;R1=R2.10 and (t)-isococlaurine (KLV,R=Me,112=H) were all incorporated to a small extent into 8,14-dlhydronor-.. salutaridine in Croton linearis, presUmably by prior conversion to reticuline or nor-reticuline. The remainder of this group, those carrying oxygen functions on C-2 and C-3, are more interesting since they are likely to arise from reticuline (as in XLVI) by parl,7 para phenol coupling. Thus, oxidation of (f)-reticuline would give pallidine (XLIV) directly, and oxidation of (-)-reticuline, followed by closure of the methylenedioxy ring73 would give amurine (XLII) which could be reduced to nudaurine Flavinantine R=Me) would arise from the oxidation of (-)-reticuline, followed by demethylation and remethylation or possibly by conversion to amurine (XLII) as an intermediate. Flavinine (XLI,R=H) could be derived in a similar manner from nor-reticuline, or from de-N- methylation of flavinantine. The rearrangement of hydroxyl and methoxyl groups via the methylene dioxy ring of amurine would be similar to that observed in the transformation of (0-coolaurine into crotonosine (XLVII) in vivo.74,75 5o:

76 Stuart et a1. suggested the further possibility that flavinantine is derived from orientaline (XVI) by para- para coupling to the bisdienone (XLVIII), followed by a dibenone-phenol rearrangement. However, these authors then went on to show that reticuline is approximately ten times more efficient as a precursor for flavinantine than is orientaline.

The second group of related alkaloids are the homo- morphines. Androcymbine was isolated from Androcymbium melanthoides77 and assigned the structure (XLIX, R1 R3= 2 Me,R =H). Kreysigia multiflora yielded kreysiginine, which was shown by X-ray78 and chemical79,8° analysis to have the structure (L). As has been suggested 77androcymbine is probably derived by phenol oxidation of a 1-phenethylisoquinoline base such as (LI). Kreysiginine could arise by cyclisation, reduction and methylation of androcymbine. Indirect proof that androcymbine is indeed derived from such a base was obtained during a study8/ of the biosynthesis of colchicine (LII) in Colchicum autumnale. In this

(3-0-methylA l-methylandrocymbine (XLIX,R =R2=R3 =M6) was efficiently incorporated into colchicine, as also was the

1-phenethylisoquinoline base (LI). Para-para coupling of 51.

1 OR OMe

Me0

e H Me() 8 'OH XLIX L 1 3 androcymbine: R =R =Me, kreysiginine R2* =H

OMe

LII colchicine 52

this precursor would give (XLIX,l&H,R2=R3=Me) and ortho- yara (KLIX,R1=R2=Me, R3=41), either of which could methylated to 0-me thylandrocymbine. Also rearrangement of the methoxyl and hydroxyl groups of either intermediate would lead to androcymbine. The third group of alkaloids is larger and comprises those

related to hasubanonine82'83 (LIII,R=Me) in which the ethanamine bridge is attached at C-14 instead of 0-9. Hasubanonine, homostephanoline84'85 metaphanine

86,87(LIV) and prometaphanine88'89(LV) are all constituents of Stephania japonica. Cepharamine9° (LVI) has been isolated from S.cepharantha, and aknadinine (4-demethylhasubanonine)'

(LVII) and aknadilactam (LVIII) from S.sasakii 91.In addition, 4-demethylhasubanonine and 4-demethylnorhasubanonine have been found in S.hernandifolia.92

Tracer work93 to establish ;;he biogenesis of hasubanonine 14 , gave mainly negative results. However, (-)-[2- Cjtyrosine was efficiently incorporated, and partial degradation of

the resulting hasubanonine suggested the involvement of

two C6-C2 derivatives in the usual manner. TWo possibilities were considered: either the formation of the morphine-type

skeleton, followed by C-9,N bond migration; or the coupling 53 •

Me LIII LIV hasubanonine: R=Me metaphanine homostephanoline: R=H

Me0

'‘• NMe e '•OH Me LV LVI prometaphanine cepharamine

Me0

Me OMe LVII LNIII aknadinine aknadilactam 5k. of a suitably oxygenated benzylisoquinoline precursor in such a way as to provide a driving force for the migration.

For example, ortho-pare coupling of (L1X,R?-=Me or H, 2 R =Me, R3=H) to (UX,RMe or H), followed by bond migration as indicated would leave a carbonium ion at C-14 which could lead to migration of nitrogen and the formation of hasubanonine. No evidence could be obtained in support of the former pathway. Neither reticuline, nor sinoacutine nor either of the bases (LIX,R1 =R2=H, R3=Me) or (LIX,R1=R3=Me, 2 R =H) was incorporated into hasubanonine. It was further suggested93 that hasubanonine should be the first-formed of the S.japonica alkaloids. Reduction of the keto-group to (LXI), de-O-methylation with allylic elimination of the hydroxyl group as indicated, aixd hydroxylation of C-lO would give prometaphanine WO and thence metaphanine (LW). The remaining members of the group differ only in their oxygenation patterns and fit quite easily into the scheme. The last group of alkaloids are not quite so obviously related to morphine, but are relevant to the present Thesis.

Acutumine has long been known to be a constituent of 94 Sinomenium acutum but only recently has its structure (LXII) 55•

OMe

LIX LX 56.

OMe OMe

LXII LXIII acutumine acutuminine

Me0

XXXV LXIV

sinomenine isosinomenine

Me0

LXV sinoacutine 96 been determined by X-ray95 and chemical means. Also characterised was acutumidine as N-noracutumine, and both alkaloids were found in S.acutum and Menispermum dauricum. Later97 acutuminine (LXIII) was also obtained from M. dauricum. Neither sinomenine(XXXV),isosinomenine

(LXIV) nor sinoacutine (LXY) were incorporated into 65 acutumine in S.acutum (see chapter II), so it is unlikely that acutumine is a further oxidation product of these 65 alkaloids. However, a scheme has been proposed for the biosynthesis of acutumine from a suitably oxygenated benzylisoquinoline (LXVI) precursor as follows. Para-para coupling of the phenolate radicals in the usual way would

produce the bis-dienone (LXVII). Bis-epoxidation of ring A, as in the partial structure (=III), followed by a

Favorski-type rearrangement would give the five-membered ring (LXIX). Decarboxylation with epoxide opening and subsequent oxidation would then give ring A of acutumine (LXX). The C-N bond migration is similar to that required in the hasubanonine alkaloids and could be accomplished by formation of an iminium ion as shown in the partial structure (LXXI).

Ring opening as indicated, followed by a non-concerted chloride ion attack on the carbonium ion (LXXII) would give

58. OMe HO OMe

Me0

LXVI LXVI I

Me0 OMe HO2C411L

LXVIII LXIX

Me0 OH

LXX LXXI ' Cl .H

NMe Me0 Me Me0 OMe

LXXII LXXIII 59.

(LXXIII) which could isomerise to acutumine. There is obviously plenty of scope for further work in the field of the biosynthesis of morphine and its related alkaloids. The structural variations found in these recently characterised alkaloids will make their biosynthesis particularly interesting when considered in relation to that of morphine.. 6o.

Chapter II.

The Biosynthesis of Sinomenine. 61.

Introduction. Sinomenine (XXXV), a constituent of the Japanese plant Sinomenium acutum, has an especial interest biosynthetically owing to its enantiomeric relationship with the morphine alkaloids. It is now well established that these alkaloids are derived from benzylisoquinoline precursors (see Chapter I), and similarly two benzylisoquinolines have been suggested as possible precursors for sinomenine, namely protosinomenine and reticuline. 98-100 Protosinomenine (XV) was proposed after consideration of the oxygenation pattern of sinomenine, but the suggested pathway involved ortho-meta coupling of the phenolic ring. However, the more recent theories 4 of phenol oxidation and radical coupling provide an 101 alternatIve, acceptable mechanism for the conversion. Thus, phenol oxidation of protosinomenine with intramolecular ortho-para coupling would give the dienone

(LXXIV)and then rearrangement to the carbonium ion (LXXV)and reduction would lead to sinomenine. The alternative proposal4,101,102 that (f)-reticuline (XXV) is a precursor for sinomenine followed from the biological relationship between (-)-reticuline and morphine

(IV). It has been proved that (-)-reticuline is oxidised 62.

Me0

8 OMe XXXV sinomenine

Me0

XV protosinomenine 63.

Me0

XXV IV

(-0-reticuline morphine

XVIII LXV. salutaridine sinoacutine to salutaridine (XVIII) which is further reduced and demethylated to provide morphine in Papaver somniferum

(Chapter I), and similarly (f)-reticuline could be oxidised to the enantiomeric dienone (LXV) which with reduction could give sinomenine. This dienone, now called sinoacutine, would not be predicted as an intermediate on a pathway from protosinomenine to sinomenine and so it was obvious that the feeding of suitably labelled protosinomenine, reticuline and sinoacutine would decide the correct

pathway.

Protosinomenine and Reticuline.

The first step in the investigation was the synthesis and labelling of protosinomenine, and the synthesis was carried out by a modification of the original procedure 98 .

O-Benzylisovanillin was converted into the nitrostyrene

(LXXVI) with methylamine hydroch.oride and sodium acetate 103 in an excess of nitromethane Reduction gave the

corresponding amine. A Schotten-Baumann reaction between the amine and 3-benzyloxy-4-methoxyphenylacetyl chloride104

gave the amtde (LXXVII) which was converted into the dihydro-

isoquinoline (LXXVIII) by a Bischler-Napieralski cyclisation. 65.

MeO

PhCH2O

Me0 N.HC1 PhCH 0 2 LXXVIII LXXIX laudanosine 66.

Instead of methylation with methyl iodide preceding reduction it was found that better yields were obtained by first reducing the dihydroisoquinoline to the tetrahydro-derivative, and then methylating by the Eschweiler-Clark method to 105 obtain the dibenzyl ether of protosinomenine . The methylation was carried out under nearly neutral conditions to discourage Pictet-Spengler cyclisation. Hydrogenolysis of the ether gave protosinomenine which could be methylated with diazomethane to give (-)-laudanosine (LXXIX) as expected. The shielding effect of the benzylic aromatic ring in 106 benzylisoquinolines was revealed in the n.m.r. spectra of reticuline and protosinomenine and their respective dibenzyl ethers (Table I). Thus there is a diamagnetic shift observed in the proton resonances of substituents at position 7. In protosinomenine the 7-methoxyl signal appears at 1: 6.46, a shift of 0.32 ppm from the 6-methoxyl group of reticuline, whereas in reticuline dibenzyl ether the methylene resonance of the 7-benzyloxy group appears about 0.22 ppm upfield from that of the corresponding group in protosinomenine dibenzyl ether.

67. Table Nuclear Magnetic Resonance Spectra values for protons)

Me0 4' 1 Rb R20 7

R30 6 Ph 6-0CH Ph 7-0CH Ph 41 -0Me 6-0Me 7-0Me 3'-OCH2 2 2 protosinomenine 6.17 6.46 (R?"=R3=H, R2=Me) reticuline 6.14 6.14 (n1=R2=H,R3=me) protosinomenine di- 4.96 4.98 benzyl ether or 4.98 4.96 1 3 2 (R =R =CH2Ph,R =Me) reticuline dibenzyl 4.99 5.20 ether 2 (R =R =CH2Ph,R=Me) 68.

A similar shift was noted93 in the resonances of protons in groups at C-7, among others, (LXXX, R2=CH2Ph) and 2 (LXXX,R=CH2Ph, R =Me). In (LXXX, 112=CH2Ph) the C-7 methoxyl signal appeared 0.29 ppm upfield and in (LXXX, 1 2 R =CH2Ph, R =Me) the C-7 benzyloxy methine protons were about 0.22 ppm upfield from the normal positions. (±)-Reticuline and (t )-protosinomenine were labelled with tritium ortho and para to the phenolic hydroxyl groups by base-catalysed exchange in tritiated water 35. The tritiated reticuline was degraded to establish the relative activity at each labelled position. Methylation gave laudanosine (LXXXI), which was converted into its methiodide and thence to the 107 methine (LXXXII) Oxidation gave veratric acid (LXXXIII) which was brominated in aqueous solution to give 6-bromoveratric acid 1081,purified and crystallised as its methyl ether. A control experiment using inactive veratric acid which was brominated under the same conditions but in the presence of tritiated water gave inactive (<0.2% exchange) methyl-6- bromoveratrate, confirming that there is negligible exchange of the aromatic protons during the reaction. Comparison of the relative molar activities of the laudanosine, veratric acid and methyl-6-bromoveratrate established the labelling 69.

OMe

LXXXII laudanosine

LXXXIII

Me0

0 LXXXIV LXXXV dehydrobromosinomenine 1-bromosinomeninone 70.

pattern (LMOCI ). + 14 Preliminary experiments of feeding (-)-[2- C]tyrosine (October 1963), and (t)-[8,2',6'-3H3]reticuline (July 1964 and May 1965) showed incorporations of 0.08%, 0.007% and 4. 0.001% respectively into sinomenine (see Table II). It was later shown that incorporations were always small during the summer but increased later in the year. Accord-

ingly aqueous solutions (ca. pi-16) of (t)-[5,2',6'-3H3]- protosinomenine and (t)-[8,2',6? -3H ]reticuline were fed 3 (September 1965) separately by the wick method to two similar S.acutum plants, to give incorporations of <0.003% and 0.094% respectively. The incorporation of reticuline was calculated after the biosynthetic sinomenine had been purified until the radioactivity remained constant after successive crystallisations. As a further test of radiochemical purity the alkaloid was converted into its picrate from which the free base was regenerated for further crystallisation and counting. There was insufficient material from the protosinomenine feeding however, and the level of activity was too low to allow the formation of a derivative in this case.

The sinomenine (relative molar activity: 1,0) derived from the (t)-reticuline feeding was degraded to establish Table II Feeding Experiments Precursor Year Wei- Activ- Incorporations (%) ght ity (mg.) (mCi.) sino- sino- isosino- acutumine menine acutine menine (t)-[2-14_, ujtyrosine Oct.1963 0.01 0.08 (-)-[8,2.6 3H3]- July 1964 22 0.3 0.007 0.006 reticuline It It May 1965 14 0.2 50.001 It Sept.1965 16 0.2, 0.094 40.00k (±)-[5,2',6'-3H3]- Sept.1965 14 0.2 0.003 protosinomenine [1-3H]sinoacutine Oct.1964 19 0.1 1.0 It Oct.1965. 20 0.06 0.55 50.01 <0.002 [8,8,6-methoXy-3H3]- Oct.1965 4.3 0.07 0.02 isosinomenine [1,6-methoxy-3H2]iso- Oct.1965 25 0.5 <0.01 sinomenine Oct.1966 29 0.1 0.006 <0.007 [1-3H]sinoacutinols-I and -II Oct.1965 7 0.04 <0.01 0.04 [1-3H]sinoacutinol-I oot.1966 18 0.05 0.05 [1-3H]sinoacutinol-II oot.1966 17 0.05 ‘0.01 [3H]sinomenine Nov.1966 31 0.1 0.002 72. the position and relative activity of the tritium labels.

Bromination of sinomenine had formerly been carried out with one mole of bromine in acetic acid to give varying yields of 1-bromosinothenine together with the dehydrobromosinomenine (LXXXIV). Since the formation of dehydrobromosinomenine requires two moles of bromine, unchanged sinomenine was also present. We found that one mole of bromine in chloroform, however, gave a clean and fast bromination of position 1. Thus the biosynthetic sinomenine was brominated to give 1-bromosinomenine (r.m.a: 0.42). Hydrolysis of the bromo-derivative gave essentially inactive 109 1-bromosinomeninone (LXXXV)(r.m.a: 0.04). In the acid conditions of the hydrolysis the diketone would be expected to enolise sufficiently to allow exchange of protons at positions 5 and 8, and hence the activity remaining after bromination must reside in one or other of these positions.

Treatment of sinomenine with 0.1N-deuterium chloride under the conditions used for the extraction of the alkaloids from the plant material caused exchange of only the two protons at position 5 for deuterium (see Table III, p.80 ). Thus there can be no tritium activity at position 5 of the biosynthetic sinomenine, and the tritium in 1-bromosinomenine 73. must be confined to position 8. The sinomenine from the (-)-reticuline feeding therefore contains 58% of the activity at position 1 and the remaining 42% at position 8. The values predicted from the distribution of activity in the precursor (as in (LXXXI)) would be 46% at position 1 and 545 at position 8, and hence there has been a loss of tritium (22%) from position 8 during the biological conversion of reticuline into sinomenine. The similar lcsses (14-18%) of tritium from position 7 occurring in the conversion of thebaine into codeine and morphine in 26 Papaver somniferum have already been discussed. The incorporation of (-)-reticuline into sinomenine without "scrambling" of the label, and the lack of incorporation of (-)-protosinomenine provide strong evidence in support of the idea that reticuline, presumably the (+)-enantiomer, is the true precursor of sinomenine. Further confirmation was obtained by feeding the dienone sinoacutine.

Sinoacutine. At this point we were extremely pleased to receive from Professor Chu66 '61 a sample of the hitherto unknown sinoacutine (LXV) which he had just isolated from S.acutum for the first time and characterised. Comparison with 74. salutaridine (XVIII) showed the two to be enantiomers as expected, and Professor Chu kindly sent us a good supply of sinoacutine for our work. It was then only necessary to label the alkaloid with tritium. The first exchange experiments were carried out with salutaridine, since this was available in larger quantities. Attempts to deuterate the dienone para to the phenolic hydroxyl group in the presence of triethylamine or potassium hydroxide caused degradation. Fortunately it was found that the alkaloid itself was sufficiently basic to catalyse exchange to give [1-2H]salutaridine. This is comparable with the labelling of morphine 35. An experiment with [1-3H)salutaridine (labelled by an earlier, more lengthy 26 procedure described in Chapter I) and unlabelled water determined approximately the rate of exchange, and accordingly salutaridine in dimethylformamide was treated with tritiated water for thirty hours at 100°C. The resulting [1-3H]salutaridine (9k exchange) was treated with bromine in chloroform to give essentially inactive 1-bromosalutaridine (0.1% activity). Sinoacutine was then exchanged with tritium under the same conditions. If the sinoacutine had contained traces of sinomenine then the labelled material could have been 75. contaminated with radioactive sinomenine. However, dilution of the mother liquors from the crystallisation of [1-3H]sinoacutine with inactive sinomenine, followed by purification of the sinomenine, showed the contamination to be less than 0.006%. [1-3H]sinoacutine was fed in two consecutive seasons (October 1964 and October 1965) to S.acutum, with incorporations of 1.0% and 0.55% respectively into sinomenine. In the first experiment dilution of the basic plant extract with inactive sinoacutine, re-isolation of the dienone and purification, recovered 35% of the labelled precursor. Part of the biosynthetic sinomenine was brominated to 1-bromosinomenine

(0.1% activity) and part was subjected to a base-catalysed exchange at position 1 (see later) to remove 97% of the activity. Thus sinoacutine was shown to be an efficient

precursor for sinomenine. Further support for the reticuline-sinoacutine pathway was then obtained from an earlier (Y)-reticuline feeding (0.007% incorporation into sinomenine). The phenolic bases remaining from the plant extract after removal of sinomenine were diluted with inactive sinoacutine. The dienone was then recovered, and purified by successive 76. crystallisations, conversion to sinoacutine picrate followed by recovery of the free base, and was found to be radioactive (0.006% reticuline fed). The sinoacutine was brominated to 1-bromosinoacutine (44% activity). This agrees quite closely with the result of the bromination of the sinomenine from another reticuline feeding which gave 1-bromosinomenine containing 42% of the activity (see above). However, the activities of the sinoacutine and bromosinoacutine were very low so this agreement may be largely fortuitous. Further Intermediates. We could now rule out the possibility that protosinomenine was a precursor for sinomenine. Reticuline and sinoacutine were proved to be involved in the biosynthesis, but it was obvious that at least one further intermediate was required, and a reductive step must be involved. The alternatives to consider were: reduction of the 8,14-double bond of sinoacutine to give (LXIV)(followed by isomerisation to sinomenine ); reduction of the carbonyl group to give one or other alcohol (LXXXVI); or reduction of the 5,6- double bond to give the enone (LXXXVII). If demethylation were to precede reduction the diketone (LXXXVIII) could be

77.

0 LXIV LXXXVI isosinomenine sinoacutinols

-‘, "NMe

LrOCVII LXXXVIII

MeO Me0

HO HO

Et Et

LXXXIX XC XCI 78. reduced to (LXXXIX) and then methylation and conjugation could

give sinomenine. Time allowed investigation of the first two

possibilities only.

Isosinomenine. It was very interesting to find that, in 1958 Sasaki and 110 Ueda had reported the isolation of isosinomenine (LXIV)

from S.acutum, and a paper in 19601/I gave n.m.r. evidence

in support of this structure. However, in 1963 it was 112 reported that the alkaloid did not have the structure

(LXIV) but was in fact either (XC) or (XCI). Such an

ethoxyl group would be unusual in an alkaloid, but, since

the reported isolation procedure involved keeping the plant

extract in ethanolic HC1 solution, it appeared likely that

this compound was an artefact. We decided to investigate

with a view to preparing labelled isosinomenine for

feeding purposes.

No change was observed on a thin layer chromatogram

when a solution of sinomenine in chloroform saturated with

dry hydrogen chloride was allowed to stand at room temperature

for twenty hours. However, addition of dry methanol gave an

equilibrium mixture of two components in that time. Separation

of the two components by preparative thick layer chromatography 79. gave sinomenine andisosinomenim.The latter was shown by n.m.r.

(Table i.r., u.v., and mass spectroscopy (Table IV) to have the structure (LXIV). It also proved to be identical with a sample prepared by K. Goto and kindly sent by K.Takeda (Shionogi Co.), but it was not clear whether this sample had been isolated from natural sources. The reaction was shown to be reversible by treatment of isosinomenine with methanolic hydrogen chloride, but attempts at isomerisation without the addition of methanol were unsuccessful. The stereochemistry at C-14 in isosinomenine was not defined by the spectral evidence, but proof was obtained by equilibration of [1,10,10,14-2HOsinomenine (XON) (see later) in methanolic hydrogen chloride. The resulting sinomenine and isosinomenine were both shown by mass spectroscopy (Table IV) to have retained the four deuterium atoms, and hence no change in the stereochemistry at C-l4 was involved in the transfor- 3 mation of sinomenine into isosinomenine. Okabe et al./1 have recently prepared isosinomenine by treatment of sinomeninone (XCII) with methanolic hydrogen chloride, and have shown by chemical methods that the stereochemistry is indeed as shown in (LXIV). It has also been observed/14 that the reduction of salutaridine with lithium aluminium hydride gives, besides the two dienols enantiomeric with (LXXXVI), a small

80.

Table III

Nuclear Magnetic Resonance Spectra (T values for protons. Solvent: deuterochloroform) 1 or 2 5 eq., ax. sinomenine 3.40 5.66d 7.55d 4.53d 6.21 6.53 7.54 3.42 J 15.5 cps J2 cps Asosinomenine 3.28 3.21 6.14 6.30 7.59

2 i 4.52d 6.22 [5,5- H2]sino- 3.42 6.53 7.55 menine J2 cps [5-2H]sinomenine 3.40 5.66 4.52d 6.21 6.53 7.56 3.42 J2 cps [1,10,10,14-2H4]- sinomenine 3.42 5.70d 7.61d 4.61s 6.27 6.58 7.63 J16 cps 14-episino- 3.31 5.77d 6.37d 4.24d 6.14 6.31 7.64 menine J 18 cps J2 cps 1-bromosino- 3.09 5.66d 7.54d 4.54d 6.19 6.49 7.57 menine J 16 cps J2 cps 1-bromoiso- 2.95 3.30 6.11 6.31 7.45 sinomenine 81. Table IV Mass Spectra sinomenine 33o (14%), 329 (m+68%), 328 (7%), 315 (21%), 314 (100%), 301 (18%), 286 (11%), 204 (9%), 201 (9%), 192 (37%), 190 (16%), 179 (11%), 178 (23%), 146 (8%). isosinomen- 330 (19%), 329 (m+84%), 315 (21%), 314 (100%), 286 (10%), ine 243 (26%), 192 (14%), 190 (5%), 178 (7%), 176 (5%), 146 (10%). [1,10,10,14-335 (3%), 334 (20%), 333 (78%), 332 (29%), 331 (7%), 2 H] sinom- 320 (4%), 319 (27%), 318 (100%), 317 (31%), 316 (6%), enine 306 (4%), 305 (20%), 304 (7%), 290 (9%), 193 (20%), 192 (36%), 182 (13%), 181 (27%), 149 (11%). [1,10,10, 335 (3%), 334 (19%), 333 (81%), 332 (29%), 331 (5%), 14-2H ]iso- 320 4 (5%), 319 (24%), 318 (100%), 317 (33%), 316 (5%), sinomenine 290 (10%), 247 (7%), 246 (29%), 245 (10%), 193 (19%), 181 (7%), 149 (12%). 2Hisino- 331 (14%), 330 (66%), 329 (51%), 316 (20%), 315 (100), menine 314 (72%), 302 (17%), 301 (14%), 287 (11%), 286 (9%), 193 (9%), 192 (51%), 191 (14%), 190 (14%), 180 (9%), 179 (23%), 178 (17%), 147 (7%), 146 (7%). 14-episino- 330 (17%), 329 (m+95%), 315 (22%), 314 (L00%), 286 (17%), menine 204 (11%), 192 (39%), 190 (17%). [8,8 -21i2] - 333 (5%), 332 (22%), 331 (90%), 330 (45%), 329 (9%), isosinomen- 317 (23%), 316 (loO), 315 (47%), 314 (9%), 246 (5%), ine 245 (23%), 244 (13%), 243 (5%), 194 (16%), 193 (8%), 178 (10%). 82.

sinomenine XCII XXXV sinomeninone

HO'*\"(' XCIII OMe XCIV

XCV 83. amount of an enone. This has been shown, from its spectra, to be the enantiomer of isosinomenine. Since our work was done the mass spectrum of isosinomenine has been studled115 and some interesting points arise in comparing it with the mass spectrum of sino- 116 menine '117 and of our deuterated species. Thus the ion at m/e 192 in the spectrum of sinomenine (Table IV) has been variously interpreted as having the structures 117 (XCIII) 115,(XCIV)116 and (XCV) all arising from fragmentation of the benzylic ion (XCVI). The corresponding peak in the spectrum of isosinomenine is less intense, and

Wheeler et.a1.115,giving it the structure (XCVII), have suggested that this is because the hydrogen at C-14 is less labile in the benzylic ion (XCVIII) arising from isosinomenine. However, we now find (Table IV) that this peak appears at m/e 193 in the spectrum of (1,10,10,14-2H4lisosinomenine (LXIV), and hence the deuterium at C-14 has been retained by the ion. This suggests that fragmentation of the benzylic ion (XCVIII) gives the ion (XCIX) rather than (KCVII). In the case of [1,10,10,14-2HOsinomenine two peaks appear, the one at m/e 192 being more intense than that at m/e 193, which perhaps suggests that both types of fragmentation operate in the mass spectrum of sinomenine. 84.

Me0

XCVII

XCVIII

0 LXIV isosinomenine

Me° Me XCIX C 85.

The other peak which is of particular interest appears

at m/e 178 in both sinomenine and isosinomenine and has been 115, given the structure (C) 116, corresponding to ring A and

0-9 and 0-10 of the alkaloids. In both of the tetradeuterated

species this ion appears at m/e 181, confirming the presence

of the three deuterium atoms at C-1 and C-10. A small,

unexplained peak appears at m/e 182 in the spectrum of

[1,10,1o,14-2H ]sinomenine.

It has been suggested that the ion appearing at m/e 243

in isosinomenine, which is absent in the spectrum of sino-

menine, could arise by loss of C-6, C-7 and C-8, plus their

substituents and one additional proton from the molecular ion.

It seems likely that this proton is the one from C-14 since

in the spectrum of the tetradeuterated isosinomenine this peak

occurs at m/e 246, indicating that only three of the deuterium

atoms have been retained.

The possibility that isosinomemine could be a precursor

for sinomenine received further support at this time by the 64,66 isolation of its enantiomer from Croton balsamifera.

Exchange Experiments with Sinomenine.

The next step in the investigation was the preparation

of suitably labelled isosinomenine. This could in principle 86. have been done either by labelling sinomenine with tritium, and then equilibrating in methanolic hydrogen chloride, or

by labelling isosinomenine directly. The labelling of sinomenine was attempted first. Preliminary experiments showed that with both 0.1N- deuterium chloride and 0.6N-potassium deuteroxide exchange of only the protons at C-5 for deuterium occurred, to give (5,5-2H2isinomenine. In one experiment chromatography of the (5,5-2H2jsinomenine on alumina caused preferential exchange of the 5-deuterium cis to the aryl ring for hydrogen. These

changes were reflected in the n.m.r. spectra (Table III). The cis and trans protons at position 5 of sinomenine were represented by doublets at t 5.66 and 7.55 (J=15.5 cps) respectively, the cis proton appearing at low field due to deshielding by the aryl ring. (5-2H)Sinomenine showed a broad singlet at "t 5.66, due to the proton cis to the aryl ring. In the di-deuterated species there were, of course,

no methylene signals in these positions. Sinomenine was then exchanged with deuterium oxide in dimethylformamide under the conditions used for the labelling of sinoacutine, and again the alkaloid was sufficiently basic to catalyse the exchange. Treatment with 0.6N KOH removed

the deuterium from position 5, and comparison of the mass 87. spectrum of the product with that of sinomenine (Table IV)

showed that there had been 50% exchange, presumably at

position 1. An exchange was then carried out under the

same conditions, but with diluted tritiated water, again with base treatment to remove label from position 5. Bromination of the resulting tritiated sinomenine removed

all but 1.6% of the radioactivity, confirming that exchange

occurs in position 1. However, when the exchange was

carried out in highly active (200 mCi/m1) tritiated water an anomalous result was obtained. In this case, after the

usual base treatment, bromination of the sinomenine

removed only 30% of the radioactivity. Also, approximately

10% of a by-product was present in the reaction mixture.

This was isolated, crystallised and found to be radioactive.

It was thought that the highly active tritiated water might

be causing a radiolytic breakdown of the solvent to give dimethylamine. Accordingly sinomenine was treated with water and dimethylamine under the exchange conditions to

give a mixture of sinomenine and approximately 10% of the same by-product. When sinomenine was treated with deuterium

oxide and dlalethylamine in dimethylformamide, a mixture of deuterated derivatives of sinomenine was obtained, showing 88.

molecular ions at m/e 334 and 333 (cf. sinomenine:m/e 329) in the mass spectrum. Treatment with dilute potassium hydroxide removed the deuterium atoms a to the carbonyl as usual, and the resulting tetradeutero-derivative was shown to be ]sinomenine (XXXV) by n.m.r. spectroscopy [1,10,10,14-2H4 (Table III). The protons at C-8 (olefinic) and C-9 (methine) appeared as singlets at 1,4.61 and 1.6.88 respectively, showing that the protons at C-10 and C-14 had been replaced by deuterium. The mass spectrum (Table IV) showed a molecular ion at m/e 333. The by-product was again present, and isolation, crystallisation and mass spectroscopy revealed a mixture of two deuterated species with molecular ions at We 335 and 334. The usual mild base treatment removed one deuterium to leave as the main species a pentadeutero-derivative with a molecular ion at m/e 334. These observations led to the conclusion that epimerisation occurs in the presence of dimethylamine, and that the by- product is 14-episinomenine. Removal of the proton at C-14 would give a stabilised carbanion, which could lead reversibly to the dienone (CI). The protons at C-10 would then be available fur exchange, being vinologously a to the carbonyl group, and reformation of the sinomenine system would allow 89.

CII CI thebainone

=CV isosinomenine• 90.

both epimerisation and the introduction of deuterium

at position 14. If one of the protons at position 5 of

episinomenine were much more readily exchanged than the other the presence of the pentadeuterated species would be accounted for. 14-Episinomenine was also produced by treatment of sinomenine with ethanolic sodium ethoxide at room temperature, conditions which are knownil8 to cause 14-epimerisation of thebainone (CII). The structure of 14-episinomenine followed from the above observations; the mass spectrum, which was very similar to that of sinomenine (Table IV); the analysis, and the n.m.r. spectrum (Table III). Also, treatment of 14-episinomenine with ethanolio sodium ethoxide caused approximately 90% reconversion to sinomenine. Further tritiations of sinomenine were carried out in the presence of sodium formate as a radical trapping agent,

as suggested by Dr.E.A.Evans (U.K.A.E.A. Amersham), and normal results were obtained, with, for example, 98% of the total activity of the derived sinomenine in position 1.

One later tritiation without the use of sodium formate

gave a normal result also, showing the effect to be variable. The exchange of isosinomenine in tritiated water and

dimethylformamide was also attempted. However, with or 91. without added base no radioactivity was found at position 1. This was shown by bromination to 1-bromoisosinomenine (Table III), a derivative which could also be obtained by equilibration of 1-bromosinomenine with methanolic hydrogen chloriae, thus establishing its structure. The label at C-8 of the tritiated isosinomenine, a to the carbonyl, proved to be more difficult to remove by base treatment at room temperature than in the sinomenine case. The Preparation of Labelled Isosinomenine. [1-3Wisosinomenine (LXIV) was prepared for feeding by equilibrating [13H]sinomenine with methanolic hydrogen chloride. After separation of the resulting mixture by preparative thick layer chromatography, the isosinomenine was mixed with inactive sinomenine and again recovered, to remove traces of labelled sinomenine. [6-methoxy-3WIsosinomenine was prepared by equilibrating sinomenine with [methoxy-3Himethanol in the usual way. The labelled methanol119 was obtained by hydrolysis of glutaric anhydride with tritiated water, methylation of the resulting acid with diazomethane followed by ester interchange with ethylene glycol to which had been added sodium hydride. A prior run with deuterium oxide gave deuterated methanol, whose p-nitrobenzoate salt had the expected n.m.r. spectrum.

The (6-methoxy-3Wisosinomenine was mixed with inactive 92. sinomenine and separated again as before. The sinomenine, after purification, was found to have 0.02% of the radioactivity of the isosinomenine. After crystallisation of the thus purified isosinomenine, inactive sinomeninewas added to the mother liquors, where a concentration of impurities would be expected. ,The sinomenine was purified and this time was found to have only 0.0004% of the activity of the isosinomenine. [8,8,6-methoxy-3IyIsosinomenine was obtained by treatment of the above 16-methoxy-31ilisosinomenine at 100°C with tritiated water containing an excess of potassium t-butoxide, to exchange the protons a to the carbonyl group. A parallel experiment using isosinomenine in deuterium oxide confirmed that exchange took place at C-8. The mass spectrum (Table IV) showed that the product was predominantly [8,8-2Hlisosinomenine (m/e 331), fragments at m/e 194 and 178 showing that the deuterium was confined to the lower section of the molecule. [1,6-methoxy-3H2)Isosinomenine was also purified by

"washing" with inactive sinomenine which, after separation and crystallisation had 0.001% of the activity of the isosinomenine. 93-

Thus isosinomenine variously labelled with tritium was prepared and shown to be uncontaminated ( < 0.001%) with sinomenine. This rigorous proof of the purity of the isosinomenine was particularly necessary because the separation of sinomenine and isosinomenine when both were present in approximately equal quantities was not easy.

Preparative thick layer chromatography seemed the only suitable method, and small loadings of the mixture were required to obtain any separation. Feeding Experiments with Isosinomenine. (8,8,6-methoxy-3VIsosinomenine (Oct. 1965) and (1,6-methoxy-3S2]isosinomenine (Oct. 1965 and Oct. 1966) were fed to S.acutum with incorporations of < 0.02%, < 0.005% and <0.00% respectively into sinomenine. These values are insignificant when compared with those obtained with sinoacutine (1.0% and 0.55%) Moreover, when the alkaloids remaining from the second sinoacutine feeding (0.55%) after removal of sinomenine were diluted with inactive isosinomenine, recovery and purification of the isosinomenine gave only 0.01% of the activity fed. Tritium could not have been lost from position 1 during the acidic extraction of the alkaloids from the plant in this experiment 94.

since it had previously been shown that treatment of isosinomenine with 0.1N-deuterium chloride at room temperature for two days caused no exchange of the aromatic protons. Hence it seems unlikely that isosinomenine is a precursor of sinomenine. During these feeding experiments it was noted that formation of sinomenine picrate was often accompanied by a drop in activity. This could either be due to removal of a radioactive impurity, or to some equilibrium of the sinomenine with ethanol in the presence of picric acid. Hence the [7-methoxy-3H]sinomenine from the equilibration of sinomenine in [methoxy-3H]methanol was crystallised to constant radioactivity, and then treated with picric acid to give the crystalline picrate in the usual way. Recovery of the free base and crystallisation showed no loss of radioactivity, thus proving that no equilibration occurs.

Sinoacutinols. We now had to consider the second possible pathway: that of reduction of the carbonyl group of sinoacutine to give the sinoacutinols (LXXXVI). Such a reduction occurs (Chapter I) in the biosynthesis of the morphine alkaloids 34 when salutaridine is reduced to salutaridinol-I before conversion into thebaine. 95•

LX)OCVI DOCXVII sinoacutinols

CIII LXII erythratinone acutumine 96.

(1-3H)Sinoacutine was reduced with sodium borohydride to give a mixture of the two epimeric dienols. Addition of inactive sinoacutine and separation removed any traces of radioactive dienone. In the first experiment the dienols were fed as a mixture. Inactive sinoacutine was added to the plant extract, and isolation and purification of the dienone and sinomenine showed incorporations of 0.04% and 0.01% respectively. When the dienols were fed separately the incorporations of sinoacutinol-I and -II were respectively 0.05% and 0.01% into sinomenine. Again these figures are insignificant when compared with the incorporations of sinoacutine, and the possibility that the alcohols were first being oxidised to sinoacutine in vivo could not be ruled out. Alternative Routes. Of the remaining possible pathways, reduction of the

5,6-double bond of sinoacutine give the enone (LXXXVII) is favoured by analogy with the enone erythratinone (CIII) formed during the biosynthesis of the Erythrina alkaloids 120,121. However, further work would be necessary to

distinguish between this and the alternative of the demethylation of sinoacutine preceding the reductive step. 97.

Acutumine. During the biosynthetic experiments the alkaloid acutumine was isolated from S. acutum. The alkaloid was unusually insoluble, was non-aromatic as judged by u.v. and n.m.r. spectroscopy, and was shown by mass spectroscopy 9 to contain chlorine (Table v). Earlier workers-4,122 had proposed the molecular formula C20H27NOB for acutumine, but now spectroscopy and microanalysis showed it in fact to be C1A4C1N06. Goto et al123 also proposed this revised formula. At this stage the structure of acutumine was not known, but the possibility that the chlorine had been introduced during the isolation procedure was considered. Isolation of acutumine in the presence of hydrochloric 36 acid containing C1 showed, however, that the alkaloid was not an artefact. Plant material was macerated with the radioactive hydrochloric acid and acutumine w,..s isolated in the usual way. The counting procedure was standardised with dibenzylamine hydrochloride obtained from the amine and hydrochloric acid of known activity. The counting efficiency was found, and acutumine in dimethylformamide was shown to have no quenching effect on the scintillator 98.

Table V

Spectral Properties of Acutumine m/e 397 (M 6.3%), 362 (18%, C19H24N06), 334 (10%), 318 (2%), 209 (100%, C11HNO3), 208 (23%), 194 (8%), 181 (lo%, C10H15NO2), 166 (58%,

C9H12NO2), 150 (40%, C9H12N0). m*/e 343-4 (397 —4 369, 399 --371), 180 (209 —4194), 157 (209 —4181), 152 (181 166).

t values (solvent: hexadeuterodimethylsulphoxide) 3.88d J 6 cps. 1H 4.64s 1H 5.54d J 6 cps. 1H ca.5.6m ca.1H 6.06s 3H 6.19s 3H 6.49s 7.76s t 3.88 and 5.54 are coupled together. 99.

(Nuclear Enterprises 220). The acutumine was found to be virtually inactive (less than 0.1% of the activity of the 36 H C1). Addition of dibenzylamine to the aqueous extract from the plant gave dibenzylamine hydrochloride which, after purification, was shown to have only 800 of the activity of the original hydrochloric acid. Hence there was an appreciable dilution of the H3601 by chloride ions present in the plant. Use of the radioisotope was therefore justified since the extraction of acutumine with, for example, dilute sulphuric acid, might still have introduced chlorine into the molecule. Acutumine was isolated from plants which had been fed (1,6-methoxy-3H2]isosinomenine, [1-3H]sinoacutine and OHisinomenine, but in each case the incorporation was negligible. Further work on the structure of acutumine was dis- continued when papers by Tomita et al.95, appeared giving details of their extensive chemical and X-ray studies leading to the complete structure (LXII). 100.

Chapter III.

Experimental. 101.

Melting points were taken on a Kofler hot- stage apparatus. I.r. spectra were measured with chloroform as solvent. N.m.r. spectra were run on a Varian A60 Spectrometer and mass spectra on an A.E.I. MS 9 double focussing spectrometer with an ionising potential of 70 ev, with direct probe insertion. Alumina chromatography refers to column chromatography using grade III neutral alumina unless specified otherwise. Light petroleum is the fraction boiling from 60-800. 102.

3-Benzyloxy-4-methoxy-w-nitrostyrene (LXXVI) A mixture of 0-benzylisovanillin (2g.), methylamine hydrochloride (0.24 g.) and sodium acetate (0.24 g.) in nitromethane (8.3 ml.) was shaken overnight at room temperature. The crystalline product was recovered by filtration. Dissolution in chloroform and filtration removed the salts and evaporation of the chloroform solution gave the nitrostyrene (2.3 g.), m.p. 124-128° (from ethanol) 124 (lit., 126-128°). 3-Benzyloxy-4-methoxyphenethylamine hydrochloride. The nitrostyrene (lg.) in tetrahydrofuran (12 ml.) was added slowly to a mixture of lithium aluminium hydride (1 g.) in tetrahydrofuran (12 ml.). The reaction mixture was then heated to reflux for 2 hours. Excess water (20 ml.) was added with cooling, and the precipitated aluminium salts were filtered off and extracted thoroughly with tetrahydrofuran (45 ml.). The combined filtrates were evaporated and the amine was extracted from the aqueous residue with ether. The ethereal solution was dried, and addition of dry ethereal hydrogen chloride gave the amine

m.p. (lit.,124,67 hydrochloride (0.89 g.), 155-164°. -168°). 103.

Me0

LXXVII LXXVI

LXXV1II XV protosinomenine

LXXIX XXV laudanosine reticuline 104.

N-(3-Eenzyloxy-4-methoxyphenethyl)-3-benzyloxy-4-methoxyphenyl- acetamide (LXXVII). 3-Benzyloxy-4-methoXyThenylacetic acid (1.2g.) was suspended in dry ether (40 ml.) and oxalyl chloride (2 ml.) was added. After 2 hours at room temperature the homo- geneous solution was evaporated to give the acid chloride. This chloride was dissolved in tetrahydrofuran (11 ml.) and added to a mixture of the amine hydrochloride described above (1.14 g.) in tetrahydrofuran (19 ml.) and aqueous sodium hydroxide (0.37 g. in 0.75 ml.) over 1 hour with vigorous stirring. The tetrahydrofuran was removed under reduced pressure and the alkaline residue was distributed between water (30 ml.) and chloroform (4o ml.). The organic layer was washed successively with water, 6N-hydrochloric acid, aqueous sodium bicarbonate, and finally water again, dried (Na2SO4) and evaporated. Crystallisation of the residue from ethanol gave the amide (1.74g.), m.p. 114-116°. (lit.,98 115-118°). 6-Eenzyloxy-1-(3-benzyloxy-4-methoxybenzy1)-7-methoxy-3,4- dihydroisoquinoline hydrochloride (LXXVIII). The amide (LXXVII)(0.2g.) in dry toluene (3.5 ml.) was treated with freshly distilled POC1 (2 ml.) and heated 3 under gentle reflux for 45 minutes. The solvent and excess 105. reagent were removed under reduced pressure and the oil was washed with light petroleum. The imine hydrochloride (0.17 g.) crystallised slowly from ethanol-ether; m.p. 144-149°. (lit.,98 146-148°). 6-Benzy1-1-(3-benzyloxy-4-methoxybenzy1)-7-methoxy-1,2,3,4- tetrahydroisoquinoline hydrochloride. The imine hydrochloride (8 13.) in methanol (400 ml.) was treated with sodium hydroxide (0.7 g.) at 0°C. under nitrogen. Sodium borohydride (4.6g.) was added slowly, and after 2 hours at room temperature the methanol was removed under reduced pressure. The residue was shaken with water and chloroform, and the chloroform layer was washed with 6N-hydrochloric acid, dried (Na2SO4) and evaporated. The residue (6.9 g.) gave the amine hydrochloride, m.p.197-202° (sintering from 195°.) (from ethanol-ether) (Found: C, 72.0; H, 6.3. C32H34C1N04 requires 0,72.1; H, 6.4%). Protosinomenine (XV). The amine hydrochloride (1.08g.) was suspended in water (4 ml.) containing 4N-sodium hydroxide (1 ml.). Formic acid (0.2 ml.) was added with vigorous stirring, followed by sufficient sodium hydroxide to bring the PH to ca. 5. Aqueous formaldehyde (38%, 0.2 ml.) was added and the mixture was warmed until the evolution of gas had ceased. 106.

The oily suspension was treated with an excess of sodium hydroxide and the product was extracted with chloroform.

12-Dibenzylprotosinomenine (0.81g.) gave needles, m.p. 0 93-95 (from chloroform-ether) (lit.,98 96-97Q). Hydrogenolysis over 10% palladised charcoal gave protosinomenine. Methylation of Protosinomenine. Protosinomenine (30mg.) in dry methanol (5 ml.) was treated with ethereal diazomethane. After standing at 0°C overnight the solvents were removed by evaporation and the residue was chromatographed on alumina. Elution with chloroform and ethyl acetate gave laudanosine (LXXIX), identified by m.p. and mixed m.p. 113-115° (from light petroleum). Tritiation of Protosinomenine (XV) and Reticuline (aV). A solution of (I)-reticuline (85 mg.) and potassium t-butoxide (76.5 mg.) in tritiated water (0.3 ml.) was heated to 100°C for 5 days in a t,:be sealed under nitrogen. The solution was cooled, acidified with hydrochloric acid, treated with an excess of sodium hydrogen carbonate and extracted with chloroform. Chromatography on alumina (grade V) with chloroform-ethanol (94:6) as eluent gave

(t)-[8,2',6'-3H3]reticuline (65 mg.). (1)-Protosinomenine 107. was labelled in the same way to give (t)-(5,21,6L3H3)protosinomenine. Degradation of Tritiated Reticuline. Labelled reticuline (20 mg.) in methanol (6 ml.) was treated with ethereal diazomethane. The resulting (-)- laudanosine (24 mg.) was diluted with inactive laudanosine and crystallised from light petroleum to constant activity, 107 (relative molar activity 1.0). The laudanosine was degraded to give veratric acid (r.m.a. 0.63). The veratric acid (42.5 mg.) in water (6.5 ml.) was treated dropwise, with stirring, at 90°C with bromine (0.02m1.) in water (3m1.) during 10 min. 6-Bromoveratric acid (32 mg.) crystallised from the cooled reaction mixture. The acid was methylated with diazomethane in methanol-ether to give methyl 6-bromoveratrate(20 mg.), m.p. 84-85° (from light petroleum)(lit.,125 88_8y_0. ). The methyl 6-bromoveratrate had a relative molar activity of 0.30. In a control experiment inactive veratric acid (50 mg.) in tritiated water (7.6 ml.) was treated with bromine (0.02 ml.) in tritiated water (3m1.) as before. The resulting 6-bromoveratric acid was methylated with diazomethane to give methyl 6-bromoveratrate which contained <0.2% of the tritium activity calculated for the exchange of one aromatic proton with the solvent. 108.

Sinomenine picrate. Sinomenine (13.8 mg.) was dissolved in ethanol (2 ml.) and picric acid (9.8mg.) in ethanol (1.5 ml.) was added with shaking. Sinomenine picrate crystallised as cubes, m.p. 186-188°. (Fbund: C, 54.0; H, 4.7; N, 9‘7. C25H26N4011 requires C, 53.8; H, 4.7; N, 10.0%). 1-Bromosinomenine. Sinomenine (XXXV) (50 mg.) in chloroform (4 ml.) was treated dropwise with shaking with bromine (0.0068 ml.) in chloroform (1 ml.) Evaporation of the solvent and crystallisation of the residue from water gave 1-bromosinomenine hydrobromide dihydrate (65 mg.), m.p. 209-212°. (Fbund: C,43.5; H,5.55. C1023Br2N04,2H20 requires C,43.4; H,5.2%). Chromatography on alumina and crystallisation from aqueous ethanol gave 1-bromosinomenine hemihydrate, m.p. 124-126°. (Fbund: C, 54.5; H, 5.9; N, 3.5. C19H22BrN04, 0.51120 requires C,54.7; 11,5.55; N,3.4%). 1-Hromosinomeninone (LXXXV). 1-Bromosinomenine (50 mg.) was heated at 100° for 2 hours in 2N-hydrochloric acid (3 ml.). The solution was cooled, the pH was adjusted to ca.8 and the solution was extracted with chloroform. The combined extracts were dried

109.

XXXV DOCCV

sinomenine 1-bromosinomeninone

LXV =CV sinoacutine isosinomenine

OMe

LXXXVI LXII sinoacutinols acutumine

•, - 110.

and evaporated, and the residue was crystallised (Na2SO4) from acetone to give 1-bromosinomeninone (31 mg.) as o. ait.,1262 o needles, m.p. 234-235 ) (Found: C,54.9; H, 5.2; N,3.5. Cale. for C1020BrN04: C,54.8; H,5.1; N,3.55%). Acid-catalysed Exchange of Sinomenine (XXXV). Sinomenine (50 mg.) was kept for 2 days at room temperature in deuterium oxide (1 ml.) containing 6N- hydrochloric acid (0.02m1.). The n.m.r. of the recovered sinomenine showed that both methylene (C-5) protons had been replaced by deuterium, but no further exchange had occurred. (1-3H)Sinoacutine. Sinoacutine (LXV) (51 mg.) in dimethylformamide (0.2 ml.) and tritiated water (0.13 ml.) was heated in a tube sealed under nitrogen for 30 hours at 100°C. The reaction mixture was diluted with methanol and evaporated to dryness. Chromatography of the residue on alumina with chloroform elution and crystallisation from aqueous dimethylformamide gave [1-3H]sinoacutine 26 (25 mg.). Bromination gave 1-bromosinoacutine containing 0.1% of the original tritium activity. In a second experiment the (1-3141sinoacutine was crystallised from ethyl acetate. The mother liquors from the crystallisation were mixed with inactive sinomenine, and the mixture was then crystallised three times from ether. kinally sinomenine picrate was prepared and crystallised. Recovery of the free base gave sinomenine which contained 0.000% of the tritium activity of the original sinoacutine. Isosinomenine turn. Sinomenine hydrochloride (530 mg.) in methanol (11 ml.) was added to chloroform (480 ml; freshly distilled from which had been saturated with dry hydrogen chloride. P205) The solution was kept overnight at 10°C. Shaking under reduced

pressure removed excess hydrogen chloride, and the solution was then washed with II-sodium bicarbonate. The aqueous phase was extracted with chloroform and the combined chloroform

solutions were washed with water, dried (Na2SO4) and evaporated. The residue was chromatographed on ten Merck

alumina PF254 thick layer plates developed in chloroform. The combined fast- and slow-running bands were extracted with chloroform to give, after alumina column chromatography,

respectively crude isosinomenine (247 mg.) and crude sinomenine

(238 mg.). Crystallisation from ether gave isosinomenine 3.12.

(130 mg.), m.p. 198-202°, [a]D-1- 73° (c 1.2 in EtOH), A max.(Et0H) 208, 238 and 265 nip. (t 31,600, 7,710 and 8,300), )) m .1690 and 1625 cm.-1 (Found: 0,69.3;

H,6.9. C19H23NO4 requires C, 69.3; H, 7.0%). The mother liquors from the ether crystallisation were shown by i.r. spectroscopy to contain a saturated ketone (1725 cm.-1). No change was observed in the i.r.spectrum after treatment with 5% NaOH under nitrogen for 2 hours. Isosinomenine picrate. Isosinomenine (10.5 mg.) was dissolved in ethanol (2 ml.) and picric acid (7.6 mg.) in ethanol (1 ml.) was added with shaking. Isosinomenine picrate crystallised as needles, m.p. 219-223° (Found: C, 53.8; H, 4.7; N,9.8. C25H20403.1 requires C, 53.8; H, 4.7; N, 10.0%). 1-Bromoisosinomenine. a) Isosinomenine (30mg.) was dissolved in chloroform (3 ml.) and bromine (0.0046 ml.) in chloroform (1 ml.) was added dropwise with shaking. The solution was evaporated to dryness and the residue was chromatographed on alumina. Crystallisation from ether gave 1-bromoisosinomenine (23 mg.), m.p. 204-210°

(decomp.), 9 max. 1690 and 1625 cm 1 (Found: C, 55.8; H,5.35; N,3.2. Ci ,H22BrN04 requires C, 55.8; H,5.4; N,3.4%). 113. b) 1-Bromosinomenine (125 mg.) in dry methanol (0.8 ml.) was treated with chloroform (150 ml.) which had previously been saturated with hydrogen chloride. The solution was kept overnight at room temperature and was then shaken under reduced pressure to remove excess hydrogen chloride, and washed with N-sodium bicarbonate. The aqueous phase was extracted with chloroform and the combined extracts were evaporated to dryness. Chromatography on silica thick layer plates gave 1-bromoisosinomenine. Base-catalysed Exchange of Sinomenine.

Sinomenine hydrochloride (60 mg.) was dissolved in deuterium oxide (1 ml.) under dry nitrogen, and the solution was treated with potassium t-butoxide (92 mg.). After 2 hours at room temperature the solution was treated with excess carbon dioxide and then extracted with chloroform. 2 i jsino- Evaporation of the solvent left a residue of [5,5- H2 menine which was shown before furLher purification to have an n.m.r. spectrum (Table III) identical with that of sinomenine except for the lack of signals at t 5.66 and T 7.55, due to exchange of both protons on C-5 for deuterium. The residue was then chromatographed on alumina to give [5-2H]- sinomenine (46 mg.) whose n.m.r. spectrum had a singlet signal 114. at 1: 5.66, showing that the deuterium cis to the aryl ring had been replaced by hydrogen. Deuteration of sinomenine. Sinomenine (50 mg.) was dissolved in dimethylformamide (0.2 ml.) and deuterium oxide (0.03 ml.) under nitrogen. The solution was heated in a sealed tube to 100°C for one week. The solution was then evaporated to dryness and the residue was treated with water (1 ml.) and potassium t-butoxide (70 mg.) under nitrogen. After being kept at room temperature overnight the pH of the solution was adjusted by addition of excess carbon dioxide. Extraction with chloroform followed by evaporation of the organic solvent left a residue which was chromatographed on an alumina column. Crystallisation from ether gave a 50% mixture (29 mg.) of [1-2H]sinomenine (m/e 330) and sinomenine (m/e 329). Exchange of Sinomenine with Tritiated Water of Low Specific

Activity. Sinomenine (20 mg.) in dimethylformamide ml.) was treated with tritiated water (20 mg.) in water (0.1 ml.) under nitrogen in a sealed tube at 100°C for one week. Extraction of the product in the usual way (see previous paragraph) gave crystalline [1-3H]sinomenine (12 mg.). 115.

Bromination gave 1-bromosinomenine containing 1.65 of the original tritium activity. Exchange of Sinomenine with Tritiated Water of High Specific

Activity. Sinomenine (500 mg.) was treated with dimethylformamide

(2 ml.) and tritiated water (0.3 ml.) under the conditions given above. Crystallisation of the product from ether gave (3H]sinomenine (200 mg.) which contained 985 of the activity calculated for the exchange of one proton. The mother liquors from the crystallisation were evaporated to give a residue (180 mg.) which was chromatographed on alumina. Crystallisation from ether gave further r3Hjsinomenine (50 mg.) and mother liquors containing a mixture. The mother liquors were chromatographed on silica thick layer plates with development in methanol:ether. The faster fraction (60 mg.) was crystallised from ether to give tritiated 14-episinomenine (40 mg.) (see below), m.p. 990. [4)- 44.8 (c 1.03 in CHC1 ). 3 An aliquot of the [3H]sinomenine was diluted with inactive sinomenine and treated with bromine in chloroform to give

1-bromosinomenine hydrobromide dihydrate which contained 70%. of the original tritium activity. 116.

Further treatment of the [3H)sinomenine with potassium hydroxide failed to reduce the activity. (1,10,10,14-2HOSinomenine.

Sinomenine hydrochloride (122 mg.) and dimethylamine hydrochloride (52 mg.) were dissolved in deuterium oxide (0.4 ml.) in a Carius tube flushed with nitrogen. The solution was frozen (methanol-solid carbon dioxide) and potassium t-butoxide (112 mg.) in deuterium oxide (0.46 ml.) and dimethylformamide (0.5 ml.) was added. The tube was sealed under nitrogen and was then heated to 100°C for one week. After cooling the solution was diluted with water, saturated with sodium bicarbonate, and the product was extracted with chloroform. Chromatography on alumina gave a mixture which was separated on silica thick layer plates to give deuterosinomenine (58 mg.) (m/e 334, 333) and deutero- episinomenine (11 mg.) (m/e 335, 334). Treatment of the products separately with potassium hydroxide in the usual way gave [1,10,10,14-2 4)sinomenine, identified by its n.m.r. (Table III) and mass spectra (Table Iv), and pentadeutero- episinomenine (m/e 334). 14-Episinomenine. a) Sinomenine hydrochloride (61mg. ) and dimethylamine 117. hydrochloride (25 mg.) in water were treated with potassium t-butoxide (56 mg.) in water and dimethylformamide in the manner described above. After one week at 100°C the solution was cooled, diluted with water and extracted with chloroform. Evaporation of the dried (NA2SO4) extracts gave a residue (37 mg.). Chromatography on silica thin layer plates gave crude 14-episinomenine (6 mg.), and ether crystallisation gave cubes (4 mg.), m.p. 100-102°. b) Sinomenine (1.05 g.) in ethanol (80 ml.) containing sodium ethoxide (from sodium, 2g.) was kept overnight at room temperature under nitrogen. The solution was diluted with water, treated with excess solid carbon dioxide and extracted with chloroform. The combined extracts were dried (Na2SO4) and evaporated. Crystallisations from ether gave sinomenine (868 mg.) as fine colourless needles, m.p. 152-154°, and 14-episinomenine (27 mg.) as large yellow needles, m.p. 99-101°, which were E...,parated mechanically. 14-Episinomenine crystallised from ether as colourless cubes (22.5 mg.), m.p. 105-107°, [a]D- 43° (c 0.8 in EtOH) -1 and -45° (c 1.1 in CHC1 ), ,1) 1675 and 1630 cm. 3 max. X, max(Et0H) 235 and 270 mil (E. 6,200 and 5,600), (Found: C, 69.3; H, 7.1; N, 4.1. C19H23/04 requires C, 69.3; H,7.0; 118.

N,4.3%). Treatment of 14-episinomenine with ethanolic sodium ethoxide as above gave mainly sinomenine which was isolated, crystallised and identified by m.p. and mixed m.p. 159-162°C. (1-3H)Sinomenine. Sinomenine (500 mg.) and sodium formate (102 mg.) in dimethylformamide (2.5 ml.) and tritiated water (0.5 ml.) were heated at 100°C for one week in a tube sealed under nitrogen. The solvent was removed by evaporation and the residue was treated with potassium t-butoxide (700 mg.) in water (10 ml.) under nitrogen overnight at room temperature. Excess solid carbon dioxide was added and the product was extracted with chloroform, and purified by crystallisation from ether. Bromination of an aliquot gave 1-bromosinomenine containing 2.3% of the original tritium activity. Treatment of Isosinomenine with lJeuterium Oxide or Tritiated

Water. a) Isosinomenine (50 mg.) was kept for 2 days at room temperature in deuterium oxide (1 ml.) containing 6N-hydrochloric acid (0.02 ml.). The n.m.r. spectrum showed that there had been no exchange of the protons in the aromatic ring for deuterium. 119. b) Isosinomenine (5 mg.) and potassium t-butoxide (13 mg.) were dissolved in deuterium oxide (0.25 ml.) under nitrogen, and heated at 100oC for 2 hours. The solution was cooled, neutralised and extracted with chloroform. The mass spectrum

(Table IV) of the resulting isosinomenine showed that exchange had occurred only in the methylene group (C-.8). o) Isosinomenine (22 mg.) in dimethylformamide (0.15 ml.) and tritiated water (0.05 ml.) was heated at 100°C in a tube sealed under nitrogen. After one week the solvent was removed by evaporation and the residue was treated with aqueous potassium hydroxide at room temperature in the usual way. Excess solid carbon dioxide was added and the proauct was extracted with chloroform. Bromination of an aliquot gave 1-bromoisosinomenine which had lost none of the original tritium activity. Treatment of the bromoisosinomenine with strong base in the usual way removed 25% of the activity, and further treatment of the isosinomenine with strong base removed 31% of the activity. d) Isosinomenine (40 mg.) and potassium t-butoxide (4.7 mg.) in dimethylformamide (0.2 ml.) and tritiated water (0.1 ml.) were heated tor three days at 100°C in a tube sealed under nitrogen. Solid carbon dioxide was added and the mixture 120. was evaporated to dryness. The residue was shaken with water and chloroform. Evaporation of the organic layer gave isosinomenine which was purified by alumina chromatography. Bromination gave 1-bromoisosinomenine which had lost none of the original activity, but treatment of the isosinomenine with strong base removed 72% of the tritium activity.

[1-3 Wasosinomenine.1 [1-3H]Sinomenine was treated with methanol and hydrogen chloride in chloroform as before. The mixture was separated on silica thick layer plates with methanol development. The isosinomenine fractions were eluted from the silica with wet methanol and chromatographed on alumina. Crystallisation from ether gave isosinomenine. The [1-3H]isosinomenine (62 mg.) was mixed with inactive sinomenine (30 mg.) and the separation was repeated. Crystallisation from ether gave o [1-3H]isosinomenine as needles, m.p. 182-184 . Recrystallisation from ether gave cubes, m.p. 195-2000.

[6-methoxy-3H]Isosinomenine.

Glutaric anhydride (1.27 g.) was dissolved in tritiated water (0.2 ml.) and heated to 1000C in a sealed tube overnight. The solution was cooled and diluted with ether, and ethereal diazomethane was added slowly. The solution was washed with 121. water and evaporated. The residue was dissolved in benzene and dried over magnesium sulphate. Removal of the solvent gave [methyl-3.141methyl glutarate (1.69 g.). The giutarate was then treated with sodium hydride (1.1 g., 50% disp. in oil) in ethylene glycol. The reaction mixture was heated and the [methyl-3H]methanol distillate (640 mg.) was collected. A parallel experiment with [methyl-2H]- methyl glutarate gave [methy1-2.1.1)methanol which was converted into its p-nitrobenzoate and crystallised. The n.m.r. spectrum showed almost complete exchange of one proton for deuterium. Sinomenine hydrochloride (260 mg.) was dissolved in chloroform (240 ml.) which had previously been saturated with hydrogen chloride, and the tritiated methanol was added. After being kept at room temperature overnight the solution was neutralised, the isosinomenine (110 mg.) was recovered in the usual way, and was recrystallised from ether. The [6-methoxy-3H]isosinomenine (46 mg.) was mixed with inactive sinomenine (23 mg.) and again recovered by silica thick layer chromatography. The sinomenine fractions were also eluted from the silica, chromatographed on alumina and diluted with further inactive sinomenine (55 mg.). 122.

Successive ether crystallisations reduced the activity to a constant level, 0.020 of that of the tritiated isosinomenine. The isosinomenine fractions from the thick layer chromatography were chromatographed on alumina and crystallised from ether to give a first crop (25 mg.), a second crop (3 mg.), and a residue (3 mg.). Inactive sinomenine (58 mg.) was mixed with the residue, and five successive ether crystallisations, followed by the formation of sinomenine picrate, recovery of the free base and a final ether crystallisation reduced the activity to 0.0004% of that of the labelled isosinomenine. [8,8,6-methoxy-39Isosinomenine.

[6-methoxy-3H]Isosinomenine (6 mg.) and potassium t-butoxide (15.6 mg.) were dissolved in tritiated water (0.3 ml.) and the solution was heated at 100°C for 2 hours in a tube sealed under nitrogen. After being kept at room temperature overnight the solution was diluted with an equal volume of water and excess solid carbon dioxide was added. Extraction with chloroform gave [8,8,6-methoxy-3H]isosinomenine (l.7 mg.).

(1,6-methoxy-3H2]Isosinomenine. (6-methoxy-311)Isosinomenine (14 mg.) and [1-31flisosinomenine 123.

(20 mg.) were combined and crystallised from ether. The residue (3.6 mg.) left after evaporation of the mother liquors was diluted with inactive sinomenine (21 mg.). Successive ether crystallisations and picrate formation reduced the activity of the sinomenine to 0.001% of that of the isosinomenine. [7-methoxy-3H]Sinomenine picrate.

(7,methoxy-3 HjSinomenine was crystallised from ether until its activity remained constant. The labelled sino-; menine (44 mg.) was dissolved in ethanol (8 ml.) and picric acid (32 mg.) in ethanol (1.2 ml.) was added. The sinomenine picrate (61 mg.) was collected and dried. The picrate was dissolved in methylene chloride and chromatographed on alumina with chloroform elution. The resulting sinomenine, after crystallisation from ether, was found to have lost none of its original activity. (1-3H]Sinoacutinols-I and -II. (LXXXVI) [1-3H]Sinoacutine (37 mg.) was dissolved in methanol (2.4 ml.) and sodium borohydride (102 mg.) was added at 0°C. The mixture was stirred for 2 hours at 0°C, followed by 1* hours at room temperature. The solvent was removed by 124. evaporation and the residue was shaken with water and chloroform. The organic extracts were dried and evaporated.

Thin layer chromatography showed the reaction to have gone to completion. Inactive sinoacutine (10 mg.) was added to the reaction mixture, which was then chromatographed on alumina. Elution with 50% chloroform and ethyl acetate, chloroform, and 2% methanol in chloroform gave impure sinoacutine (15 mg.), sinoacutinol-I (20 mg.) and sinoacutinol-II (20 mg.) respectively. TWo successive ethyl acetate crystallisations reduced the activity of the sinoacutine fraction to 0.8% of that of either of the dienols. The dienols were purified by further alumina chromatography. A Typical Feeding Experiment. An aqueous solution of (t)-[8,21,6?-3H ]reticuline 3 (22 mg., 2.4 x 106 counts/sec.) was fed to a small Sinomenium acutum plant by the wick method. The plant (wet weight 38 g.) was harvested after nine days and was then macerated in 0.1N-hydrochloric acid (150 ml.). Inactive sinomenine hydrochloride dihydrate (38 mg.) was added and the mixture was kept for three days at room temperature. The extract was then filtered through a small

pal of celite and the residue was washed with water. The combined filtrates were washed with benzene and basified 125. with solid sodium bicarbonate. The alkaloids were extracted with chloroform (6x80 ml.), emulsions being broken by centrifugation. The combined chloroform extracts were dried and evaporated to give the crude basic extract (132 mg., 6.14 x 105 counts/sec.). Chromatography on alumina with chloroform, followed by 6% ethanol in chloroform elution gave four main fractions. The second fraction (84 mg.) contained mainly sinomenine, as revealed by thin layer chromatography, and was crystallised from ether. Four successive crystallisations brought the activity of the sinomenine to a constant level (1.38 counts/sec./Mg.) which was not altered by formation of the picrate and recovery of the free base. The first fraction from the alumina Chromatography was combined with the mother liquors from the first two sinomenine crystallisations and with inactive sinoacutine (20 mg.). The mixture (60 mg.) was separated on a silica thick layer plate. The sinoacutine fraction was eluted from the silica with methanol, the solvent was removed by evaporation, and the residue was chromatographed on alumina to give the dienone (21 mg.).

Ethylacetate crystallisations brought the activity to a 126. constant level (4.8o counts/sec./mg.) which was diminished by formation of sinoacutine piorate and recovery of the free base. Isolation of Acutumine (LXII). In the above experiment no acutumine separated, but in other cases when the content of this alkaloid was higher, some acutumine crystallised out when the chloroform solution of the crude basic extract was concentrated. The remainder of the acutumine was recovered as the final fraction from the chromatography. Acutumine crystallised from chloroform-ether, or from ethanol, as needles, m.p. 237-238.5°, (lit. 95,238-240°), [a]D-121° (c 1.1 in pyridine) [lit., -120 094,122,and -206° 95 (both in pyridine)]. (Found: C, 57.2; H, 6.3; Cl, 8.9; N, 3.3. Cale. for Ci ,024C1N06: C, 57•x+; H, 6.1; Cl, 8.9; N, 3.5%). 36 Isolation of Acutumine in the Presence of Cl. 36 The efficiency of the counting of Cl was found by counting a solution of dibenzylamine hydrochloride (from hydrochloric acid of known specific activity) in dimethylformamide in the presence of the scintillator Nuclear Enterprises 220. The addition of acutumine and of further 127. dimethylformamide was shown to have no effect on the efficiency. One S.acutum plant was macerated with 0.1N-hydrochloric 36 acid containing Cl. After 2 days at room temperature the extract was filtered through celite. The specific activity of the hydrochloric acid was found by addition of dibenzylamine to an aliquot portion. Concentration of the solution gave a precipitate of dibenzylamine hydrochloride which was crystallised from ethanol-ether 4 and found to have a specific activity of 2.64 x 10 dis./Sec./m mole. The specific activity of the acidic plant extract was found in the same way. Addition of dibenzylamine to an aliquot portion and concentration of the solution gave a dark precipitate which was collected and washed successively with water, ethanol, benzene and ether until the washings were colourless. The residue was then triturated with ethanol and the colourless ethanolic solution was diluted with ether. Dibenzylamine 4 hydrochloride (2.15 x 10 dis./sec./m mole.) crystallised on keeping the solution at room temperature. The rt.mainder of the acidic extract was basified and extracted with chloroform in the usual way. Chromatography 128. on alumina gave four main fractions. The final fraction gave acutumine (20 mg.) which was crystallised from chloroform- ether to give needles, m.p. 231-233°, (< 30 dis./sec./m mole). Biosynthesis of Acutumine. Acutumine was isolated from plants which had been fed [1,6-methaxy-3B2]isosinomenine and [1-31-1]sinoacutine, with resulting incorporations of 0.007% and 4 0.002% respectively. These values were considered to be negligible in comparison with the incorporation of [1-3H]sinoacutine into sinomenine (0.55%) in the second feeding experiment. Sinomenine labelled with tritium mainly in the 1-, 10- and 1k-positions and in the 7-methoxyl group was also fed to S.acutum,and acutumine was isolated in the usual way. The specific activity of the acutumine was again very low (0.002%). 129•

References. 130.

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