THE BIOSYNTHESIS of SOME PHENOLIC ALKALOIDS a Thesis Submitted by GEOFFREY MELVILLE THOMAS for the Degree of DOCTOR of PHILOSOPH

THE BIOSYNTHESIS OF SOME PHENOLIC ALKALOIDS

a thesis submitted by

GEOFFREY MELVILLE THOMAS

for the degree of

DOCTOR OF PHILOSOPHY

THE UNIVERSITY OF LONDON

Imperial College, June 1963. London, S. W.7. ABSTRACT

A brief review of the biosynthesis of alkaloids, other than those of the Amaryllidaceae and morphine groups, is given. The biosynthesis of these two groups is discussed more fully with particular reference to the evidence for the Barton and Cohen concept of phenol oxidation as a biogenetic mechanism.

The incorporation of labelled phenolic precursors, derivatives of norbelladine, has been shown and by means of multiple labelled experiments incorporation as a whole, without degradation, has been proved.

Other experiments described have thrown light on the earlier stages of biogenesis.

The norlaudanosine derivative, (±) reticuline, has been shown to be incorporated into morphine, and an in vitro synthesis of thebaine from (±) reticuline using a radiochemical dilution method is de-scribed. I am deeply grateful to Professor D. H. R. Barton and Dr. G. W. Kirby for the privilege and pleasure of working under their supervision and for their great help in matters chemical and non-chemical.

To the Salters Company I would like to express my sincere thanks for the award of a scholarship and for their interest during the tenure of it.

My thanks are also due to Dr. D. W. Turner for advice on counting techniques, Mr. D. Aldrich and his staff for valuable technical assistance, Miss J. Cuckney for microanalyses, Mr. R.H. Young who grew the daffodils and poppies and to my many friends and co-workers at Imperial College. REVIEW

Alkaloid Biogenesis 1

THE AMARYLLIDACEAE ALKALOIDS

Introduction 12 Early tracer experiments 16 Synthesis of precursors 21 Feeding experiments 23 Nature of the C - C unit .....34 6 1 THE MORPHINE ALKALOIDS

Introduction 40 Feeding experiments 43 The configuration of morphine 52

EXPERIMENTAL

Amaryllidaceae alkaloids 58 Morphine alkaloids 74

REFERENCES 83 REVIEW 31.,.r,••••

ALKALOID BIOSYNTHESIS

There has been much thought and discussion about the way in which the wide variety of plant alkaloids are produced. With the advent of tracer methods in which compounds with a radioactive label have been fed to living plants, a rapidly growing amount of information on this subject is now available. 1, 2, 3 Recent authoritative reviews of this subject are now available and, for this reason, only a brief survey will be given here. One of the alkaloids from each of seven main groups has been taken, and the way in which tracer experiments have been used to investigate the mode of biogenesis, is described.

Pyridine and piperidine alkaloids Nicotine

Nicotine (I) the main alkaloid of many Nicotiana species, has been

[2.1 extensively studied by tracer methods. 4C] Ornithine (II) was fed4' 5 to N.tabacum and N. rustica plants and found to be a good precursor for the 4 5 alkaloid. Oxidative degradation with nitric acid to nicotinic acid ' (III) 6 and the nitropyrazole (IV) showed that the label was equally divided between positions 2' and 5',

NO2 CO2 H 5' 2 Me

These results indicated the formation of a symmetrical intermediate 7 which has been suggested by Leete to be putrescine (V) or the mesomeric anion (VI),

dliO )* r* ".- H N *%••CO H 2 2

1/47"

o2}1 (II)

••••••••,..3,

nicotine (I)

Support for this scheme came from the incorporation, albeit less efficiently than ornithine, of putrescine (V), proline and glutamic acid into the 7 pyrrolidine ring of nicotine . Experiments to investigate the derivation of the aromatic system have 14 been carried out. Although it was shown that [2 - C] lysine (VII) can serve as a precursor for the piperidine ring in the closely related alkaloid anabasine (VIII), it was found that it did not provide the aromatic system in either

8 anabasine or nicotine .

Nr "Z‘` H 2 z

(VII) (VIII)

Nicotine labelled in the pyridine ring was, however, isolated from plants fed with ring labelled nicotinic acid (III) whereas the carboxyl labelled 9 acid was not incorporated . Information about the derivation of the aromatic ring has come from 14 feeding experiments with labelled acetate. [2 - C] Acetate and 14 10 11 [2 - C] propionate have been shown to be incorporated into the pyridine 14 ring of the alkaloid whereas [1 - C] acetate provided only the pyrrolidine 14 ring, and [1 - C] propionate was not incorporated. [1, 3 - 14C] Glycerol provided nicotine with 57% of its activity in the ll aromatic nucleus . Tracer experiments have also shown that the N-methyl group of 12 13 nicotine can arise from choline or methionine and in the latter case that 14 14 a true transmethylation occurs . [Methyl- C] Nicotine has also been shown 14 to give rise to [methyl- C] choline in the plants so that methyl transfer from the alkaloid to acceptors can also occur. In the light of these results the biogenesis of nicotine may be summarised as follows.

CO2H Cozx Ornithine .160••••100,00 Proline Me Glutamic acid

Choline Me . .,filethlonme

Tropane alkaloids Hyoscyamine (IX) One relationship between the tropane alkaloids, based on the skeleton (X), and the pyrrolidine bases is shown by the incorporation into 14 15 hyoscyamine of [2 - CI ornithine , the precursor for the pyrrolidine portion of nicotine, Degradation proved the incorporation to be specific with the label inpositions 1 or 516'17 . The other three carbon atoms of the tropane system were shown, by the appropriate tracer experiments, to be 18 derivable from acetate . The biosynthesis of the tropic acid (XI) residue of the alkaloid was studied in three separate experiments in which phenylalanine (XII) labelled 19 in the 1-, 2- and 3-positions was fed to Datura stramoniurn . Hydrolysis of the alkaloids to tropic acid and subsequent simple degradations proved that phenylalanine is a direct precursor. The respective positions of the labels 5. were those indicated.

H °2 C

+ HO 2C Ph

CH 0H z 0

(XI)

(IX) 14 *, o and + indicate [ C] labels from different experiments)

Isoquinoline and phenethylamine alkaloids As early as 191020 before the advent of tracer experiments, it was 21 suggested that the main precursors of this large group of alkaloids are the aromatic amino acids phenylalanine (XII), tyrosine (XIII) and dihydroxyphenylalanine, Hordenine (XV) Decarboxylation, oxidation and methylation, all well known in living systems, are all that are required to convert these amino acids into hordenine. Phenylalanine (XII), tyrosine (XIII) and tyramine (XIV)22 have all been shown to be incorporated into the alkaloid in sprouting barley. The

positions of the labels were as expected and the quantitative results suggest that hordenine is formed from tyramine in a stepwise methylation process. 23 24 This is supported by experiments in which methionine (XVI) and betaine (XVII) were shown to be sources of the methyl groups in the alkaloid. The biosynthesis may then be summarised thus:

CO H 2 ed•-\\,,C0aH

NH NH HO 2 HO 2 (XIV) (XIII) (XII)

NH 2 Me CO H (XVI) z NMe HO Me V. + - (XV) 'Me N .0" CO (XVII) 3 2 CH 2

Papaverine (XVIII) 20 In 1910 it was suggested that norlaudanosoline (XIX) is formed from two moles of dihydroxyphenylalanine and it is apparent that papaverine could be derived from this by dehydrogenation of the heterocyclic system, 25 possibly by way of the N-oxide . 7.

OH OMe

OH OMe \OMe (XIX) (XVIII) (XX)

Papaverine and laudanosine (XX) occur together in the opium poppy. 26 Battersby and Harper established the biosynthesis of papaverine from two molecules of tyrosine by feeding Papaver somniferum with 14 [2- C] tyrosine and showing equal activity in the expected positions of the alkaloid (XVIII). 27 14 In further work the same group showed that [1- C] norlaudanosoline provided papaverine specifically labelled at position 1. The relationship between the benzylisoquinoline and morphine alkaloids will be discussed in detail later. Berberine (XXI)

An examination of the structure of berberine (XXI) suggests that it might also be derived from norlaudanosoline (XIX) and evidence of this came 28 from the specific incorporation of two molecules of [2-14C] tyrosine . In recent experiments in this laboratory, Mr. R. H. Hesse has isolated active berberine from Hydrastis canadensis fed with the norlaudanosoline derivative 3 (±) reticuline (XXII), labelled as shown. This also adds weight to the suggestion that the berberine bridge carbon atom (arrowed) arises from the N-methyl group but the degradation to prove this has not yet been carried out. 8.

MeO

/•?. - Me HO

NO Me OMe

(XXI) (XXII)

Colchicine (XXIII) A complete picture of the mode of biogenesis of this alkaloid, found in the autumn crocus is not yet available. 14 [3 - C] Phenylalanine (XII) was specifically incorporated into 29 colchicine in Colchicum izirantium the label being at position 5.

Me Me 0

Me0 0 (XXIII) (XII)

Independently30 [2 - 14C] tyrosine was found to be incorporated into Colchicum autumnale but position 6 was virtually inactive. Half of the alkaloid activity was, however, found in the N-acetyl group. This suggests

that degradation of the amino acids to a C - C fragment takes place prior 6 1 to incorporation. 14 30 31 L-[methyl- C] Methionine was also found to be incorporated ' but provided only the methyl groups of the alkaloid. The origin of the tropolone part of the molecule is still unknown 14 30 31 since, although [1- C] acetate gave active colchicine, all the activity was found in the N-acetyl group.

Indole alkaloids Ajmaline (XXIV) Although [2-14C] tryptophane has been shown to be the precursor of 32 the f3-carboline moiety of ajmaline , the origin of the remainder of the molecule is still not clear and has been the subject of much discussion, 33 34 Woodward and Robinson suggested derivation from dihydroxyphenyl- 35 alanine with subsequent 'Woodward' fission, Wenkert suggested derivation 36 from prephenic acid (XXV) and Thomas from a monoterpene skeleton (XXVI, numbered as would be expected in ajmaline).

OH 18

OH 14 OH H°2C 20 1`( 19 18 '4O CO2H

(XXIV) (XXV) (XXVI)

10.

37 Leete discounted these theories when he found no significant 14 incorporation on administration of [2- C] tyrosine (Woodward), 1 4 [2-14C] alanine (Wenkert) and [2- C1mevalonic acid (Thomas), He did, 14 however, find that [1- C] acetate was incorporated and this led him to put forward a biogenetic theory, independently suggested by Schittler and 38 Taylor and Battersby, stating that the carbon atoms at positions 18, 19, 20, 15, 14 and 3 were derived from acetate. C Had previously been shown 21 39 to be derived from formate . Independent work by Battersby has confirmed 40 the incorporation of acetate, but Lee te's labelling pattern was not observed .

The Cinchona alkaloids Quinine (XXVII) Quinine occurs along with cinchonine in the bark of various Cinchona species. Examination of these and other minor alkaloids, for 41 example cinchonamine (XXVIII), led to the suggestion that the quinoline moiety of the major alkaloids is formed from an indole having a two-carbon side chain at C3, by bond cleavage shown here. This type of change was 42 . established in a model system by an in vitro synthesis

CH CH OH 2 2

HO AN T NY'o, Me a„

i (XXVII) (XXVIII)

C C 43 14 As proof of this Kowanko and Leete fed [2- C] tryptophane to Cinchona plants and isolated active quinine, the whole of the activity of the alkaloid being in the expected 2! position.

Apart from this tracer work on these groups of alkaloids, a large amount of work including that of the author has been carried out on the Amaryllidaceae and morphine alkaloids, as a result of which the main features of the biogenetic pathway to these alkaloids is now largely understood. A detailed description of this work is given in the following sections. THE AMARYLLIDACEAE ALKALOIDS OH NA10

(xxx) 'WO

Y HO 12.

Introduction. The Amaryllidaceae alkaloids constitute a large group of naturally occurring compounds which have been the subject of intense investigation 44 over the last few years . They are of widely diverse functionality and structural type but can be divided into three main classes based on (a) a dibenzofuran system, for example galanthamine (XXIX), (b) a pyrrolo- phenanthridine skeleton, for example galanthine (XXX), and (c) an ethano- phenanthridine skeleton, for example haemanthamine (XXXI). In principle, the other classes of these alkaloids can be derived from these by cleavage, rearrangement and recyclisation. Oxidative cleavage of type (XXXI) at the indicated position could lead to alkaloids based on a benzpyranoindole skeleton, e.g. homolycorine (XXXII) and oxidative cleavage of the haemanthamine type, followed by internal oxidation-reduction and 45 recyclisation • can' give the tazettine (XXXIII) type . Another rearrangement with migration of the methylenedioxyphenyl ring can give rise to alkaloids 46 based on the methan.omorphanthridine skeleton, e.g. rnontanine (XXXIV) . It can be seen that all these alkaloids are closely related in that they contain a common hydroaromatic C - C 2and an aromatic G - C unit, and 6 6 1 47 biogenetic theories for their formation have been put forward by Wenkert 48 and Barton and Cohen . 13. 15.

Biogenesis of the Amaryllidaceae alkaloids (Wenkert) Biogenesis of Amaryllidaceae alkaloids (Barton and Cohen)

C°211 -,-- 0 OR ,.., ..-- RO RO (XXXV) .. I " HONf, CO 2-H -- OH HO 1 N- HO...,../..„" O

0 ''\•,,\Nr HO H RO OH RO (XLIV) RO (XLV) (XLVI) (XXXVI) (XXXVIII) • • 4 OR OH OH

0 , -\,)NN/1 I —1 1 NMe —4 ♦ NMe NMe (XXXIX) ( XLI) o 0 O HO

O O MeO (XLVII) (XLVIII) (XLIX)

‘0, OH

NMe -4.mor•em.••••• NMe ( o ) ••"\\

( o ) (0) (XL) (XXIX) ( L ) 14.

Wenkert suggested that the alkaloids arose from the pyrroles (XXXVI) and (XXXVII), derived from prephenic acid (XXXV) by amination and hydration, Condensation of the pyrrole (XXXVI) with shikimic acid (XXXVIII) would give the intermediate (XXXIX) which by Michael reaction followed by hydration-dehydration and oxidation-reduction changes could lead to the lycorine skeleton (XL). Similarly, the second pyrrole (XXXVII) would give the intermediate (XLI) which could give rise to the haemanthamine skeleton (XLII). Quaternisation of the nitrogen followed by bond 'at cleavage would lead to the dienone (XLIII) and thus to the galanthamine skeleton. Cleavage at bond 'b' would lead to the tazettine skeleton. This theory accurately locates the positions of all the oxygen atoms in the alkaloids. Barton and Cohen's scheme, involving the concept of radical pairing, was based on knowledge gained during structural work on Pummerer Is ketone. They suggested that all these alkaloids arise from oxidative coupling of a precursor of general type (XLIV) where R = H or suitable blocking groups, which could be alkyl or part of an enzyme surface), Variations of para-para- and ortho-para-coupling reactions give rise to the three main types of alkaloid, Para-para-coupling in the precursor written as (XLV) would give rise to the haemanthamine skeleton and ortho-para-coupling of (XLVI) (= XLV) the lycorine skeleton, More specifically, oxidation of (XLVII) to the diradical (XLVIII) and radical pairing would give the dienone (XLIX). Aromatisation of the bottom ring, followed by addition of the hydroxyl group across the unsaturated system (L) would lead to the oxide bridge in narwedine (LI). Galanthamine (XXIX) is obtained by reduction. 16.

Thus all the alkaloids in the family could be derived from this general type of precursor (XLIV). The fact that belladine (XLIV, R=R1=1Vie), 49 the fully methylated precursor, has been isolated from Amaryllis belladonna 50 and narwedine (LI); a postulated intermediate in the biogenetic pathway, occurs with galanthamine in Texas daffodils has added weight to this theory. However, the earliest practical evidence for the phenol oxidative coupling concept came from the verification of the predicted structure (XXIX) of 51 galanthamine by an in vitro chemical synthesis by Barton and Kirby . 14 The phenol (XLVII) labelled with [ C) in the N-methyl position was oxidised with a wide variety of agents in the presence of racemic inactive narwedine and the alkaloid was formed in yields of up to 1.4%. The isolated -) narwedine was then reduced to (±) galanthamine which was resolved in a most interesting way. It was found that recrystallisation of (±) narwedine in the preirellce of (-) galanthamine gave partly resolved (+) narwedine. Reduction of this gave (+) galanthamine which, in turn, resolved (+) narwedine to (-) narwedine. Reduction gave (-) galanthamine identical with the naturally occurring alkaloid. Early Tracer Studies The essential difference between the two biogenetic concepts outlined earlier is that Wenkert proposed the derivation of the hydroaromatic C - C unit from a hydroaromatic precursor which never becomes aromatic, 6 2 whilst Barton and Cohen suggested that the C6 - C2 unit is derived from an aromatic precursor. 52 In early tracer experiments , where plants were fed with 14 2-i C] tyrosine, radioactive galanthamine was obtained from King Alfred 17. daffodils and active lycorine (*LH) from Texas daffodils, but no degradations were carried out to prove the position of the label in the alkaloids. Independently, Battersby and Binks S3isolated lycorine (0.23% incpn.) and norpluviine ( LV) from Twink daffodils fed with 2-(14C) tyrosine. The degradation was carried out according to the following scheme.

CO2H

HO Me + HCHO

• ( LI V) 18.

Degradation of Galanthamine (Kobayashi and Uyeo)

1.00 0.96

(XXIX) (LVI) (LVII)

HCHO

0.99

0.95 (LVLTI)

[The activities of the degradation products are shown relative to galanthamine as 1.00]. 19.

Methylation of radioactive lycorine gave a mixture of a.- and f3-lycorine methiodides, which were subjected to Hofmann degradation. The resulting methine (_LIII) was hydroxylated and cleaved to give formaldehyde containing essentially all the activity. The residue was oxidised to the lactam acid (LIV) 54 which was inactive. Also, in experiments carried out by Dr, J.B. Taylor in this laboratory with radioactive galanthamine isolated from Galanthus , elwesii snowdrops fed with 2-[14C] tyrosine the position of the label was 55 proved by degradation based on the method of Kobayashi and tTyeo . Treatment of galanthamine with aqueous 46% hydrobromic acid under reflux gave apogalanthamine (LVI) which was methylated. Erode degradation of the , corresponding methochloride gave the base (LVII) which was further degraded by Hofmann elimination to give the vinyldiphenyl (LVIII). Ozonolysis under controlled conditions gave formaldehyde, isolated as its dimedone derivative, containing all the activity originally present in the alkaloid. As it is generally accepted that the conversion of prephenic acid to 56 tyrosine is irreversible , these results establish conclusively that the hydroaromatic C6 - C2 unit can be provided by tyrosine,contrary to Wenkertts theory but in accordance with the concept of Barton and Cohen. The confirmation of this theory came from work using the postulated biosynthetic precursors. 20.

Synthesis of dimethylnorbelladine

OBz

CHO

CO2H

H OBz NMe CH=NMe

(LIX)

OH BzO

Me0 NMe NMe

(XLVII)

Bz = CH C H 2 6 5 21 4

Synthesis of precursors The synthesis of N-methyl-N-(3-hydroxy-4-methoxybenzy1)-13- (-hydroxy)phenethylamine (XLVII), referred to later as dimethylnorbelladine, 14 labelled with [ C] in its N-methyl group, was carried out as in the 14 accompanying scheme. Condensation of 0-benzylisovanillin with [ C] methylamine and reduction of the resulting imine with potassium borohydride gave the amine (LIX) which was acylated with 2-benzyloxyphenylacetyl chloride to yield the amide (LX). Reduction to the amine with lithium aluminium hydride followed by catalytic debenzylation, gave the desired phenol isolated as its hydrochloride. The corresponding [N-methyl- 14C] trihydroxy compound, N methylnorbelladine, was obtained in an analogous manner from 14 [E-methyl- C]-3,4-dibenzyloxy-N-rnethylbenzylamine prepared from dibenzylprotocatechuic aldehyde. 14 For the synthesis of [0-methyl- C] precursors 3-benzyloxy-4- hydroxybenzaldehyde was required. Reimer-Tiemann formylation of O-benzylcatechol proved to be a convenient synthetic method and was 57 preferable to direct benzylation of protocatechuic aldehyde . Methylation of 3-benzyloxy-4-hydroxybenzaldehyde with [14C] methyl iodide gave 0-methyl labelled 0-benzylisovanillin. Condensation of the latter with [14C] methylamine and reduction of the imine with sodium borohydride gave the doubly labelled* benzylamine (LXI, R = CH2C6H5).

Multiple labelled compounds are mixtures of inactive and singly labelled species containing only a small number of molecules having more than one isotopic atom.

22.

CH NHMe 2 *

* (LXI)

OH 1

NMe

RO *

OBz OBz )Bz 1

II"

CHO CHzCI CH2CN

(LXII) (LXIII) 23.

Labelling in the 1-position of dimethylnorbelladine was accomplished by preparation of the appropriately labelled p-benzyloxyphenylacetic acid ( LXIII). 2-Benzyloxybenzyl chloride was converted to the labelled nitrile '(LXII) by heating with sodium [14C] cyanide in dimethylsulphoxide58, and alkaline hydrolysis of the nitrile gave the labelled acid. Feeding experiments,. The King Alfred daffodil was chosen for experiments with these precursors since it is easy to grow and produces useful amounts of galanthamine, galanthine and haemanthamine, representing the three main classes of Amaryllidaceae alkaloids. Compounds were normally injected (in aqueous solution of pH 6) into the hollow flower stalks of the mature, flowering plants. Injections appeared to have no ill effects on the daffodils? growth or alkaloid production and the plants were worked up after eight days. Throughout this work no major biosynthetic significance was attached to the magnitude of the incorporations into the alkaloids. Both dimethylnorbelladine and N-methylnorbelladine were incorporated 59 into galanthamine in King Alfreds (0.014 and 0.018% respectively) Galanthine and haemanthamine were both inactive - neither has an 1\T,-methyl group - and this indicated that no de-N-methylation had taken place with subsequent fragment incorporation. Further support for this came when it was found that all the activity in the galanthamine was, in both cases, in the N-methyl group of the alkaloid. However, the incorporations of these compounds do not discount the possibility of fragment incorporation after, in this case, breaking of bonds a or b. 24.

Dimethylnorbelladine was then prepared with a second label in the 14 > amethyl position (R = CH ) and fed to the plants in the same way. 3 The 0- and N-methyl groups were estimated by a modified Zeisel procedure in which the compound was degraded by heating with hydriodic acid. The methyl iodide so formed was collected in ethanolic triethylamine and the resultant triethylmethylammoniurn iodide is a convenient 'heavy' derivative which is easily counted. The beauty of this method is that it is possible to separate the 0-methyl derived methyl iodide from that formed from an N-methyl group and thus to estimate each label separately. The relative activities of the 0-methyl and N-methyl groups in the precursor, in galanthamine, and, for confirmation, the derived (MnO 2oxidation) narwedine were found to be the same within the limits of experimental accuracy.

Fraction of 14C in OMe NMe Precursor 0.48 0.51 Galanthamine (XXIX) 0.48 0.48 Narwedine (LI) 0.45 0.49

This not only showed that bond b had not been broken in the biosynthesis, but also that no selective demethoxylation had occurred. As final confirmation of the fact that the precursor was incorporated as a whole, the triply labelled compound was prepared, labelled on 0- and N-methyls and in the 1-position (p. 22). 25.

z QB z

•0111•1••••14

CHO CH2CI CH2CN

Bz (LXII) B CHO

Bz0"%) NNE7'/' CH CH NH 2 2 2 (LXIV) OBz OH

HO BzO HO

HO Me 0 wiv) (LX 26.

The relative activities of the three labelled positions in the precursor, 60,61 in galanthamine and the derived narwedine were identical

, 14 Fraction of C in

OMe NMe 1 remainder

Precursor 0.19 0.21 0.60 Galanthamine 0.18 0.19 0.63 Narwedine 0.18 0.18 0.63

Experiments carried out by Dr. J.B. Taylor using the corresponding phenols norbelladine (LXX) and 0-methylnorbelladine (LXVI) labelled in the 1-position gave interesting results in King Alfred daffodils. The preparation is shown in the scheme. The nitrile (LXII) was reduced to the amine (LXIV) which was condensed with the appropriate aldehyde, dibenzylprotocatechuic aldehyde or benzylisovanillin, to give the imine. Reduction and hydrogenolysis gave the precursors. Norbelladine was incorporated into galanthamine (0.014%), 59 galanthine (0.004%) and haemanthamine (0.25%) . The galanthamine was degraded as described earlier (p. 18) and, as expected, the isolated 61 formaldehyde contained essentially all the activity . 0- Methylnorbelladine was, however, incorporated into haemanthamine (0.036%) but not into galanthine nor galanthamine. These results are interesting on two counts. In the first place, although negative results in this work should be treated with caution, the different behaviour of 0-me thylnorbelladine suggests a definite order of methylation in the biosynthesis of galanthamine. 27.

Degradation of haemanthamine

--*i i••••••••••••••••••••,....4. )E Me I 7 CH NHC CO ."/ rNyr. \,-, 2 * 2 2 11 ii

P ,,,,_--1 1 (LX VII) [1.00] 11SO4 Pb(0Ac)4

HCHO [1.05] * + HCHO [0.94] + CO2 [0.0]

(LXVIII) 28.

Since this precursor is not incorporated, the order of methylation can be postulated as:- norbelladine y N-methylnorbelladine -÷ N-,0-dimethylnorbelladine galanthamine, i.e. N-methylation of OM. norbelladine must occur before 0-methylation. Secondly, one would not expect 0-methylnorbelladine to be incorporated into haemanthamine since the alkaloid has no corresponding methoxyl group. Since the cleavage of the methoxyl group and subsequent incorporation of norbelladine is unlikely in view of the multiple labelling experiments with galanthamine, the most interesting possibility is that the methoxyl group is the source of the methylenedioxy group in haemanthamine. Experiments using the same precursor with a second label in the 62 0-methyl group were then carried out by Dr. J. B. Taylor . In the first the methylene dioxy group was hydrolysed with 63 20% H 50 and the formaldehyde so formed contained all the activity 2 4 originally in the 0-methyl position. In a subsequent experiment with the doubly labelled precursor a fuller degradation was carried out proving the positions of both labels in haemanthamine. The alkaloid was converted to N-(6-phenylpiperony1)- N-methylglycine (LXVII) which was oxidised with lead tetraacetate in glacial acetic acid to yield formaldehyde, carbon dioxide and N-methy1-6- phenylpiperonylamine (LXVIII) isolated as its hydrochloride. This result not only proves that the methoxyl group can be the source of the methylenedioxy group in haemanthamine but also provides further evidence for the incorporation of the precursor as a whole. Further confirmation of the former came in investigations of the origin of the C6 - C1 fragment in galanthamine (by the present author) when

(limn)

0 H. 2HO 0 EHo --H

Ille..11100111111.1m.

zH.J HO

.6z 30. the hydrochloride of the double labelled N-methylbenzylarnine (LXI, R = H) was injected into King Alfred daffodils and the haemanthamine obtained was active. Hydrolysis with 20% H2SO4 and measurement of the activity of the formaldehyde formed showed that all the activity was in the methylenedioxy group. The hypothesis that methylenedioxy groups could be derived biogenetically by cyclisation of o-methoxyphenols was first mentioned by 63 Sribney and Kirkwood who found that methionine was a much more effective precursor than formate for the methylenedioxy groups of protopine. The present results constitute the first experimental proof of this hypothesis. Since 1, 2-dimethoxybenzenes, o-methoxyphenols and methylene- dioxybenzenes frequently occur together in closely related natural products it seems reasonable that methylenedioxy groups in general are formed in nature by a cyclisation mechanism. Clearly the intermediates involved can be either radical or cationic in character. It is attractive to speculate that an oxidation process applied to the phenolic hydroxyl of an o-methoxyphenol can give either the radical (as LXIX) or the cation (as LXX). In so far as the radical (as LXIX) is stabilised relative to the alternative radical (as LXXI) and the cation (as LXX) is destabilised relative to the oxonium ion (as LXXII), the latter process could appear, at least thermodynamically, to be more probable, However, hydroxylation of methyl groups attached to saturated carbon is commonly observed in nature (for example in the terpenoids). Consequently, hydroxylation of an o-methoxyphenol might take place without involving the phenolic hydroxyl group and the resulting hemiformal could then dehydrate to give the ion (LXXII). 31.

O Me

Degradation of haemantharnine (Battersby et al. ) OMe MeO

NR Me0 H MeO (LXXVI) (LXXVII) (LXXVIII)

0 Me Me CH2NCH* 2CO2H

ti (LXXII) (LXXX) (LXXIX)

H CH NCH CO H + 3 2 2 CH 3 (LXXIV)

PhCO H CH NCH OEt 2 3i 2 +CO2

SO C H 2 7 7 (LXXV ) HCHO 0 (LXXXII) 32.

64 Work by Battersby, Binks, Breuer, Fales and Wildman using labelled norbelladine gave good incorporations into lycorine (LII, 0.24%) and norpluvine (LV, 0.73%) in Twink daffodils. The lycorine was degraded as before. The same group found that norbelladine and tyrosine were precursors 65 of haemanthamine and crinamine (C epimer) in Twink and haemanthamine 3 was degraded in both cases according to the scheme. The alkaloid was converted to the amino acid (LXXIII) which was hydrogenolysed to sarcosine (LXXIV) isolated as its tosylate. Kolbe electrolysis gave the sulphonamide (LXXV) which on hydrolysis gave formaldehyde containing all the activity. As proof of the intact incorporation of norbelladine the precursor; double labelled, was fed to Nerine bowdenii and active lycorine (0.07%), crinamine (0.0009%) and belladine (LXXVI, 2.64%) were isolated66. The precursor was methylated to give belladine (LXXVI) which was cleaved by cyanogen bromide to give the bromide (LXXVIII, R = CH2Br) and cyanamide (LXXVII, R = CN). Conversion of these to the aldehyde (LXXVIII, R = CHO) and the urea (LXXVII, R = CONI12) established the ratio of the labels. The isolated belladine by similar procedure gave the same fragments. The aldehyde (LXXVIII, R = CHO), on oxidation and decarboxylation gave carbon dioxide, whilst the other labelled atom was isolated by Hofmann degradation of belladine, with cleavage of the resulting styrene (LXXIX). The active crinamine (LXXX) was degraded in the same way as haemanthamine, and lycorine as before to the lactam acid (LXXXI) 33. which was decarboxylated and converted to the phenanthridiniu.m derivative (LXXXII) which was oxidised to benzoic acid. The ratios of the labels in the norbelladine, belladine, crinamine and lycorine were found to be essentially the same. Interesting experiments on the ,methylation of norbelladine have 67 recently been carried out by Fales, Mann and Mudd with partially purified cell-free enzyme systems from Nerine bowdenii. It was shown that norbelladine is preferentially methylated by (-)-S-adenosyl-L-methionine, in the presence of the Nerine enzyme, to the phenol (XLVII) rather than (LXXXIII) - the ratio of para- to meta-methylation being 22 : 1. These monomethylated derivatives were not further methylated.

4,1 NH NH

Me0

HO (XLVII) (LXXXIII)

Another enxyme, which catalyses the methylation of catechols and is present in rat liver, under the same conditions gave a ratio of para- to meta- methylation. of 0.28: 1 indicating that the highly specific para-methylation was a property of the plant enzyme and the main biosynthetic route from

-iorbeii.a.dine- to lia.ernantliarnine proceeds via rather than (LXXXIII). 34.

In experiments with plant homogenates we have shown an incorporation of tyrosine into lycorine in Irene Copeland daffodil homogenates. The flowering plants were crushed under liquid nitrogen and phosphate buffer 14 of pH 7 was added followed by an aqueous solution of [2- C1 tyrosine hydrochloride. After 24 hrs. at 25°, the mixture was worked up and active lycorine (LII) (incorporation 0.020%) was isolated.

Nature of the C6 - C1 unit

That tyrosine provides the C6 - C2 unit of the major Amaryllidaceae alkaloids has been established by several groups of workers52,53,65,68,69,70,71 However, less is known about the origin of the remaining C6 - C1 unit. To investigate this, the double labelled N-methylbenzylamine (LXI, R = H) was prepared by hydrogenolysis of the corresponding benzyl ether recovered from the preparation of dimethylnorbelladine (p.20) and fed to King Alfreds. Radioactive galanthamine was isolated (0.019% incorporation), in addition to active haemanthamine mentioned earlier in connection with the biogenetic origin of methylenedioxy groups (p.29). All of the activity in the isolated galanthamine was located in the 0-methyl group, suggesting that degradation of the amine to either 3-hydroxy-4-methoxybenzylamine or, more probably, isovanillin, had preceded incorporation. Unfortunately [0-methyl-14CI isovanillin, prepared by 0-methylation of the monobenzyl aldehyde followed by hydrogenolysis, was not absorbed by the plant - most of the activity being recovered from the stem of the daffodil. However, it was found in preliminary experiments by Suhadolnik, Fischer and 35.

68 Zulalian that tritium labelled protocatechuic aldehyde was incorporated into lycorine in Narcissus incomparabilis although the distribution of radioactivity in the alkaloid remains to be determined. In an attempt to find whether tyrosine can provide the C6 - C1 fragment, generally labelled tyrosine - tyrosine with the label spread equally over each carbon atom - was fed to King Alfred daffodils. [3-14C] Tyrosine was not available for this experiment. The isolated galanthamine was converted to apo-galanthamine (LVI) as before (p.18), this was oxidised with alkaline ferricyanide and phthalic anhydride was sublimed from the reaction residue. The results from these experiments were inconsistent but it appeared that tyrosine could supply the C - C fragment although incorporation into the C - C 2unit was more 6 1 6 efficient. Further experiments in this field using [31, 51 -3H, u] tyrosine are in progress. OH 3 H

N2 2.C° 2H 70 14 Jeffs has shown that [3- C] tyrosine does not provide the C6 - C1 69 unit of haemanthamine, and Wildman, Fales and Battersby independently confirmed this for haemanthamine, haemanthidine and tazettine. 68 However, Suhadolnik, Fischer and Zulalian and Wildman, Battersby 71 and Breuer showed that phenylalanine. is incorporated into the C - C unit 6 1 but not into the C - C unit of lycorine, and the latter group showed that the 6 2 same holds true for belladine.

36.

This is consistent with the view that hydroxylation of the aromatic ring occurs only after further transformation of phenylalanine. It has been 68 71' 72 suggested that phenylserine or cinnamic acid may be intermediates 73 in this process. Recent work where trans 43-14C] cinnamate was shown to be incorporated into lycorine, supports the latter. Thus work on galantharnine and haemanthamine has established two separate routes leading from aromatic amino acids to these alkaloids. The first, involving step-wise methylation of norbelladine, has already been discussed. The second proceeds from an aromatic unit bearing a methoxyl group, for example isovanillin, and, for galantharnine biosynthesis, must not involve the intermediate formation of 0-methylnorbelladine (XLVII). One possible intermediate might be the aldimine (LXXXIV) analogous to that formed by condensation of pyridoxal phosphate and tyrosine during enzymatic decarboxylation of the amino acid.

CHO

OH Ji (LXXXIV) 37.

QBz OBz SDBz

* H* CH2COC1 CH2CONCH 3

(LXXXV)

OH OH oic

NH2 CH 3 H (LXXXVII) (LXXXVI) 38.

Methylation, decarboxylation and reduction of this aldimine would then give N-, 0-dimethylnorbelladine and hence galanthamine, while decarboxylation and reduction alone would give 0-methylnorbelladine, the precursor of haemanthamine. An equally acceptable scheme could be written using tyramine and , accordingly [3H, 14C] tyramine (LXXXVII) and doubly [14C] labelled N-methyltyramine (LXXXVI) were prepared. 3 14 [ H, C] Tyramine was most conveniently prepared by decarboxylation 14 74 of [2- C] tyrosine in tritiated diphenylamine , and N-methyltyramine, labelled in the 1-position and the N-methyl group, was prepared as in the scheme. 14 Condensation of [1- C) g-benzyloxyphenylacetyl chloride (p.20) with labelled methylamine gave the doubly labelled amide (LXXXV). Reduction with lithium aluminium hydride followed by hydrogenolysis afforded the desired doubly labelled N-methyltyramine (LXXXVI). Neither tyramine nor N-methyltyramine was found to be incorporated into galanthamine in King Alfreds. However, here again, these negative 68 results must be regarded with caution especially since tyramine is known to be an efficient precursor for lycorine. The tracer studies described so far only establish certainly that the plant is capable of carrying out certain well-defined chemical operations. A clearer view of biosynthetic sequences came from intermediate 61 trapping carried out by Dr. J. B. Taylor . If labelled tyrosine is injected together with a large amount of an inactive compound which is a true biosynthetic intermediate, two effects should be observed. 39.

First, the incorporation of tyrosine into the alkaloids should be considerably diminished since the label will become diluted as it passes through the intermediate, provided the plant cannot convert all the intermediate into the alkaloids. Second, if the intermediate can be re-isolated it should be radioactive. In experiments exactly similar to the others no incorporation of tyrosine into galanthamine in King Alfreds was observed when a large excess of the two precursors, dimethylnorbelladine and norbelladine, were present. Dimethylnorbelladine was recovered from the appropriate plant extracts and was found to be active although the incorporation was low (0.0016%). Me thylation with diazomethane gave belladine which was converted to its methiodide and this was cleaved with sodium amalgam. 0- Methylhordenine was isolated from the reaction mixture and was found to contain essentially all the original activity. THE MORPHINE ALKALOIDS

40. 4 1 . Biogenesis of the morphine alkaloids (Barton and Cohen)

RO RO

HO n HO

RIO

OH O O OR (LXXXVIII) (LXXXIX) (XC) RO RO fr Me0 • \ RO HO RO N-

RO (XCVLII)

OMe (XCVI) OR

RO (CI) (C)

(XCV) (XCII) (XCIII), R = H "XCIV. R = Me 42.

That the skeleton of morphine could be obtained from a precursor of the norlaudanosoline type, by oxidative coupling, was first suggested by

Gulland and Robinson in 1925 75E The most satisfying of the mechanisms 76, 77, 48 suggested for the coupling process is that of Barton and Cohen The norlaudanosoline derivative (LXXXVIII, where R and R' are suitable blocking groups, for example part of an enzyme surface readily added or removed from phenolic hydroxyl groups to ensure specific coupling processes) on oxidation by a one-electron transfer process would give the diradical (LXXXIX). Coupling of this diradical would give the dienone (XC) and the oxide bridge could then be formed by addition to the enone system to give (XCI). Thebaine (XCII), codeine (XCIV), morphine (XCIII) and neopine (XCV) arise then by obvious changes. If an oxide bridge is not formed, then again only simple changes account for sinomenine (XCVI). Aporphine alkaloids can also be derived from the same type of precursor by coupling across other positions. Ortho-, ortho-coupling of the norlaudanosoline skeleton (written as XCVII) would give rise to alkaloids of the corytuberine family (XCVIII) and ortho-,para-coupling would lead to the glaucine type skeleton (XCIX). Alkaloids whose biogenesis is not immediately obvious because of the position of the oxygen functions, for example crebanine (C) can be explained on the basis of a dienone-phenol rearrangement - in this case of an intermediate such as (CI). Alkaloids such as anonaine (CII, R = H) and roemerine (CII, R = Me) at first sight do not seem to be derived by phenol oxidation. To accommodate these Barton and Cohen suggested a dienol-benzene

43. rearrangement, shown in the scheme, and this type of rearrangement has since been firmly established,

HO HO

RIO R 10

(CII) Tracer experiments with the opium poppy have been extensive. 14 In early work [2. - Cl tyrosine was fed to the plants and found to be 78,79 7 incorporated into morphine , codeine? andthebaine 9. The active 8 0 morphine (XCIII) was degraded to the perhydrophenanthrene (CIII) by 44.

Degradation of morphine

+ HCHO

( CIV)

MeO

Si Ac CO2H (XCIII) •

(CV) (C VII) CO211

CO2H MeO 'MeO

MeC

CO2H NH2 6(CIX)- (CVIII) 45. methylation, quaternisation and Hofmann degradation, Oxidation gave formaldehyde and the aldehyde (CIV), isolated as its oxime, each containing half the activity of the original alkaloid so that half of the alkaloid's activity was shown to be located in the 16-position. 81 A second degradation established the position of the second labelled carbon atom. Morphine was converted to the phenanthrene (CV) which was oxidised to the diphenic acid (CVI). The required carboxyl group was then selected by decarboxylation of the derived coumarin (CVII) and vigorous alkaline hydrolysis in the presence of dimethyl sulphate gave the dimethoxy acid (CVIII). Decarboxylation with copper chromite in quinoline gave carbon dioxide, which contained half the activity of the original alkaloid, and the biphenyl residue was essentially inactive, Thus the radioactive morphine was found to be labelled equally in the 9- and 16-positions. 9 Support for this came from an independent degradation7 . The phenanthrene (CV), containing half the activity of the alkaloid was oxidised to phthalic acid and a Schmidt reaction gave anthranilic acid (CIX) containing half the activity of the phthalic acid. Because of the equivalence of the carboxyl groups of phthalic acid, these results mean that half the activity of the original morphine is located at positions 9 or 12 or is spread between them, and it is argued that the first possibility is the correct one. Both results establish that two molecules of tyrosine, or close equivalent, are incorporated into morphine. Since two molecules of tyrosine would give a laudanosoline system equally labelled at positions 1 and 3 these results lend weight to the Barton and Cohen theory, Since [2-14C) phenylalanine has been shown to be a much less 79 , 82. efficient precursor for morphine than tyrosine , it was suggested' that the 46. Preparation of (±) reticuline

BzO

Me0' H CI Me0 CN z CH2 (CXII)

BzO BzO BzO

...••••••01...111.14411. a Me0 "\ NO Me0 /"N NH MeO CHO 2 (CXIII)

MeO Me O

w••••••11•44.

BzO NCHO CO2H COCHN BzO N 2 (CXIV) (CXV)

4111(4001•1100••••••••• BzO 'f

Me0 y MeO OH OBz OBz (CXVIII) (CXVII) (CXVI) 47. main biosynthetic route may be shikimic acid prephenic acid --> phenylalanine —4 tyrosine 3, 4-dihydroxyphenylalanine —+ morphine. In our studies of morphine biogenesis the norlaudanosoline derivative (±) reticuline (CXVIII) was prepared as shown in the scheme. O-Benzylisovanillic acid (CXIV) was prepared by oxidation of the corresponding aldehyde and reaction of the acid chloride with diazomethane gave the diazoketone (CXV). In the presence of silver oxide this gave the amide (CXVI) by reaction with the phenethylamine (CXIII) prepared as shown, via the nitrostyrene, from O-benzylvanillin. Bischler-Napieralski ring closure gave the dihydroisoquinoline (CXVII), the methiodide of which was reduced with borohydride. Debenzylation gave the precursor (CXVIII). Although it has been shown that the benzyl groups can be removed from di-O-benzyl(-)reticuline by hydrogenolysis if the benzyl ether is pure, the method used in the preparation of labelled precursors was debenzylation by concentrated hydrochloric acid in ethanol. The important dienone (CXX), intermediate in the biosynthesis of the morphine alkaloids, has been synthesised by Dr. Steglich in this 83 laboratory as in the scheme . 84 Thebaine was converted to phenolic dihydrothebaine (CXIX) , which, after acetylation, was oxidised with selenium dioxide, followed by manganese dioxide, to the acetylated dienone. Deacetylation gave the dienone which was found to exist in the open phenolic form. For this reason it was accepted 85 that, as proposed by Battersby , biological conversion to thebaine might proceed through the allylic alcohol (CXXI) rather than the enone (CXXII). Chemical support for this idea came when reduction with borohydride gave a mixture of alcohols (CXXI), which gave thebaine in fair yield under the relatively mild conditions of treatment with 1N hydrochloric acid at 83 room temperature. 48. Thebaine ( XCII)

..••••=•••••••••••11114) Me Me

O ( CXIX) (.CXX) CC XXI)

( XCII)

OH OH /1N•.,...„,./ OH HON"

I- HO CONMe HO NMe

HO (C XXIII) HO (C XXIV) 49.

Although early attempts to simulate the oxidative coupling in the laboratory were unsuccessful, mild oxidation of laudanosoline giving 86 7 dihydrolaudanosoline (CXXIII) , more recently oxidation of laudanosoline methiodide with ferric chloride resulted in a high yield of (CXXIV). This material was identical with the product obtained by demethylation and quaternisation of glaucine (XCIX, R = Me). 3 Using [1- H] (-) reticuline we have completed a radiochemical synthesis of thebaine in the laboratory. The precursor was oxidised with Mn0 inactive dienone (CXX) was added to the reaction mixture, and the 2' dienone re-isolated and found to be active. Purification, to constant count, by chromatography gave material with activity equivalent to a 0.012% reaction yield, By the method of Dr. Steglich, the active dienone was converted to thebaine which had essentially the same specific activity.

Confirmation of the fact that norlaudanosoline can act as a precursor 8 for morphine came from Battersby and Binks8 , who showed a relatively large incorporation of the precursor, [14C ] labelled in the 1-position, into morphine in Papaver somniferum, Degradation of the morphine in the same way as before (p,44) showed that all the activity was in the corresponding 89 9-position of the alkaloid . We have shown that (-) reticuline is incorporated into morphine 90 14 in Papa.ver somniferum Methiodide formation with [ C] methyl iodide and reduction with sodium [In bcrchydride gave (-) reticuline (CXVIII), doubly 3 14 labelled, with a tritium label in the 1-position. The H; C ratios in morphine and in the di-O-acetyl derivative were shown to be the same, within the limits of experimental error, as that in the precursor. The incorporation 50.

14 was 0.13% whereas that of [Z- C1 tyrosine in a parallel experiment was 0.08%. Professor Battersby's group has also found that this compound is 2 incorporated into morphine . In one case it was found that glucose was more efficiently 91 incorporated into the alkaloids than tyrosine although it is generally accepted 2 that tyrosine is formed from glucose9 and therefore should be closer to the alkaloids on the biosynthetic pathway. This led to the suggestion by Rapaport that tyrosine is incorporated into the alkaloids by a minor or aberrant pathway. As a result of experiments with morphine derived from 14 93 [ C) carbon dioxide , the same author argued that the alkaloid was not derived equally from two identical molecules of tyrosine, or its biogenetic equivalent. 78, 79,81 There is, however, a great weight of evidence for the use of two equal tyrosine molecules to provide norlaudanosoline and reticuline which have been establiEhed as precursors of the morphine alkaloids. Experiments are in hand to establish the sequence of 0-methylation of the precursors and to prove intact incorporation using multiple 14 labelled (-) reticuline. This has been prepared as before (p.40) using [ C] methyl iodide for methylation of the corresponding monobenzyl aldehydes to 14 [O-14C methyl] O-benzylisovanillin and [0- C methyl] O-benzylvanillin to *W.& wawa& moms introduce the two 0-methyl labels. The label at position 3 was introduced 14 by reduction of the corresponding [ C] cyanide to the amine (see p.40), and the remaining N-methyl and tritium labels were introduced using [140] methyl. iodide and [3i-1] bc::•ohydride., Early experiments, attempting to differentiate between the methoxyl groups by Zeisel determination, proved unsuccessful and a full degradation 51.

Degradation of thebaine

Me() Me° Me()

HO Me

H Me MeO OH

(XCII) (CXXV) (CXXVI)

* Me 0

Me0 * OH 52. for the alkaloid has been worked out for assay of the labels in the isolated thebaine. A preliminary Zeisel determination for N-methyl and total 0-methyl followed by the degradation of thebaine shown in the scheme will give the ratio of the labels in the alkaloid. In the degradation thebaine (XCII) was converted to the hydrochloride of thebenine (CXXV) in boiling hydrochloric acid94.A one-stage reaction using methyl iodide and sodium hydroxide gave the vinylphenanthrene (CXXVI) which was cleaved by ozonolysis to give formaldehyde, isolated as its dimedone derivative. The dienone (CXX), postulated by Barton and Cohen as a precursor in the biosynthesis of morphine alkaloids, and the two alcohols (CXXI) have been prepared by Dr. Steglich labelled by exchange with [3H] water for feeding to the poppies.

The configuration of morphine 95 After the stereochemistry of morphine had been established , the absolute configuration of the alkaloid was elucidated, by Jeger and 96 co-workers , chemically by correlation of dihydrothebaine with the optically active acid (CXXVII) of known configuration. Their conclusion (CXXVIII) was verified by X-ray crystallographic studies 97. Apart from morphine, codeine and thebaine, other alkaloids are found in Papaver somniferum which are of the same configuration, for example (-) laudanidine (CXXIX), and papaverine is found to occur as a racemate. Two alkaloids from the plant are, however, found which have the opposite configuration to that of morphine. These are (4-) codamine (CXXX, R = H) and (+) laudanosine (CXXX, R = Me). The configuration of 98 the latter has been established chemically by degradation to the acid . (CXXXI) and shown to be opposite to that of morphine by correlation of the

(AIXXX 0) (AXXXO) (IIIXXX 0)

HO HO

(Ixxxo) 2 0H r\DZ nT OH 0 aIAI H

(ITAxxo) (IIIAXXO) H

•ES 54.

(-) form with glaucine (CXXXII) which in turn was related to .morphothebaine (CXXXIV)99. A simple explanation of the occurrence of these compounds is thatthey are methylation products of the isomer of a phenolic precursor which, because of its configuration, cannot be converted to morphine in the plant. Some anomalouG results have, however, been reported by Battersbyln who resolved [14C] norlaudanosine (CXXXV, R = H) and separately fed both isomers to Papaver somniferum. The isomer corresponding to (+) laudanosine, having a configuration opposite to that of morphine, was incorporated more efficiently than the enantiomer with the morphine configuration. It appeared, therefore, that morphine could be derived from a norlaudanosine of the opposite configuration, possibly through the corresponding imine (CXXXIII), with the form of opposite stereochemistry completing the sequence of reactions more readily. 01 Further experiments planned by the same group should clarify this point. Our interest in this stemmed from the radiochemical synthesis of thebaine when it was realised that a method of chemically relating reticuline (CXXXV, R = Me) to thebaine was now available. Optically active and racemic reticuline are accordingly being prepared with tritium labelling by exchange 3 reactions with [ H] water in the presence. The labelled reticuline so formed can be used for chemical oxidation reactions and for experiments in the plants. 55.

Summary

The evidence for the Barton and Cohen concept of phenol oxidation as a biosynthetic mechanism in the Amaryllidaceae and morphine alkaloids has been rapidly accumulating.

The incorporation of the postulated phenolic precursors, without degradation, strictly speaking only proves that the plants are capable of carrying out the appropriate chemical changes, and not that this is the actual biogenetic pathway.

However, the occurrence, in nature, of some of the postulated intermediates or their fully methylated precursors, for example belladine and laudanosoline and the intermediate trapping experiments, provide almost conclusive evidence that this is indeed the method of biogenesis used by the plant. The accepted biogenetic pathways to galanthamine and morphine may then be summarised as in the following schemes. 56.

Biogenesis of galanthamine

OH HO OH

m=4...•••••••••41 NH 2 OH H02 CO2H CO2H y0 shikimic acid prephenic acid tyrosine

OH OH OH

NMe NH

0 OH

;me

MeO MeO na rwe dine galanthamine 57.

Biogenesis of morphine

shikimic acid V••••••••••••••••••)1 prephenic acid ---4 tyrosine

MeO

Me0 OH` OH

MeO

thebaine codeine morphine EXPERIMENTAL

The Amaryllidaceae Alkaloids 58.

The radioactive precursors were prepared, assayed and injected in aqueous solution (ca. 5 ml.) of the hydrochlorides into the hollow stalks of the flowering plants and the injections were followed, after 1 day, by injections of distilled water. Seven days later the plants were worked up and the major alkaloids separated by chromatography, counted and recrystallised to constant count. Agreement between the molar activities of an alkaloid and its derivatives was taken as proof of radiochemical purity. Measurements were taken in duplicate on thin films (ca. 0.5 mg. per cm.2 ) and were not corrected for self absorption or back scattering. A gas-flow (methane) proportional counter was used giving a back-ground of ca. 15 counts per min. The main source of error lay in non-uniform films, the measured activities of duplicate samples sometimes differing by 10%. Galanthamine was crystallised in ether and purified as its hydrochloride as was galanthine, crystallised in methanol. Haemanthamine crystallised in ethyl acetate and was purified as its picrate. Incorporations were worked out on the basis of the total amount of injected precursor and on the yield of crude alkaloid obtained from the chromatography. Melting points were determined on the Koller block and petroleum ether refers to boiling range 60 - 80°. 59.

SYNTHESIS OF PRECURSORS

N-Methylnorbelladine (singly labelled) 3,4-Dibenzyloxy-N-methylbenzylamine hydrochloride To a solution of 3,4-dibenzyloxybenzaldehycle" (320 mg.) in methanol (50 ml.) was added methylamine hydrochloride (80 mg.) in methanol (10 ml.) containing 4N-sodium hydroxide (0.8 ml.). After 1 hr. at room temperature, potassium borohydride (200 mg.) was added and the suspension set aside for 2 hr. The solvent was evaporated and the residue shaken with water and ether. Evaporation of the ether layer gave the oily amine which was dissolved in methanol (1 ml.). Conc. hydrochloric acid (0.5 ml.) was added, followed by ether (5 ml.) and the mixture kept at 0° for 1 hr. The hydrochloride which separated crystallised from ethanol-ether as needles (310 mg., 83%), m.p. 169-170°. (Found: C, 71.4; H, 6.5; N, 3.5. C22H24C1NO2 requires C, 71.4; H, 6.5; N, 3.8%). Decomposition of the hydrochloride with sodium carbonate and extraction with ether gave the amine (272 mg.) which could not be crystallised. 4-Benzyloxy-E-(3,4-dibenzyloxybenzylL -N-meth xar2iethvla1 1 m in e To a suspension of 2-benzyloxyphenylacetic acid (100 mg.) in benzene (3 ml.) was added oxalyl chloride (1 ml.) and the mixture warmed until all the acid had dissolved. After 1 hr. at room temperature the solvent and excess of oxalyl chloride were evaporated and the remaining acid chloride was redissolved in benzene (2 ml.). This solution was added dropwise with stirring to 3, 4-dibenzyloxy-N-methylbenzylamine (272 mg.) in benzene (2 ml.). After 2 hr., the precipitated amine hydrochloride was removed (160 mg.) and the filtrate successively washed with N-hydrochloric acid, water, N-sodium hydrogen carbonate, and water, dried and evaporated to give the expected amide (176 mg.) which did not crystallise. This was extracted (Soxhlet) 60. into a refluxing suspension of lithium aluminium hydride (500 mg.) in ether (10 ml.). The excess of reagent was decomposed with ethyl acetate, water was added, and the ether layer separated from the pasty aqueous layer which was extracted with further quantities of ether. Evaporation of the dried (MgSO4) ethereal solutions gave the amine which crystallised under ether as prisms (149 mg., 85%), m.p. 65 - 6°. (Found: C. 81.6; H, 6.7; N, 2.5. C H NO requires C, 81.7; H, 6.8; N, 2.6%). 37 37 3 4-Hydroxy-N- (3, 4-dihydroxybenzy1)-N-methylphenethylamine hydrochloride (N-methylnorbelladine) The corresponding tribenzyl ether (149 mg.) in ethanol (5 ml.) containing conc. hydrochloric acid (0.1 ml.) was hydrogenated over 10% palladised charcoal (20 mg.). After 3 hr. the solution was filtered and evaporated to dryness. The hydrochloride crystallised from ethanol-ether as needles (76 mg., 95%), m.p. 207 - 208°. (Found: C, 60.5; H, 6.4; N, 4.3. C I6H20C1NO3 requires C, 61.0; H, 6.5; N, 4.5%). N-, 0-, Dime thylnorbelladine (triply labelled) 3-Benzyloxy-4-hydroxybenzaldehyde 103 0-Benzyl catechol (5.0 g.) in ethanol (30 ml.) and water (15 ml.) containing sodium hydroxide (20 g.) was treated with chloroform (15 g.) added dropwise with stirring at room temperature. After 2 hr. the mixture was heated under reflux for 30 min. Excess of chloroform and ethanol were removed under reduced pressure and the aqueous solution acidified with hydrochloric acid and extracted with ether. The extract was washed with water, dried (MgSO4), treated with charcoal, and evaporated. The residual dark oil was chromatographed on neutral alumina (grade V). Elution with benzene-chloroform (1 : 1) gave 3-benzyloxy-4-hydroxybenzaldehyde which crystallised from benzene as plates (490 mg., 5%), m.p. 113 - 114° 61.

104 o (lit. 113-114 ). Mr. H.P. Tiwari later improved the yield on this experiment to 19%. 0-Benzylisovanillin 14 [ C) Methyl iodide (ca. 3 mg., 0.1 mc.) was distilled in vacuo with an acetone carrier into 3-benzyloxy-4-hydroxybenzaldehyde (9 mg.) in dry acetone (5 ml.) containing anhydrous potassium carbonate (50 mg.). The reaction vessel was sealed in vacuo and heated at 60° for 72 hr. with stirring. Inactive methyl iodide (1 ml.) was then added and the mixture refluxed for a further 6 hr. to complete methylation. Inactive 0-benzyliso- 1 05 vanillin (16 mg.) was added, the mixture centrifuged and the supernatant solution evaporated. The product was chromatographed on alumina (grade III, 3 g.) and elution with benzene afforded O-benzylisovanillin (25 mg., 0.08 mc.). 3-Benzyloxy-4-methoxy-li-methylbenzylamine (XXXI) To a solution of methylamine hydrochloride (6.5 mg.) and labelled ,14 O-benzylisovanillin (25 mg.) in methanol (3 ml.) was added L CI methylamine hydrochloride (0.36 mg., 0.1 mc.) followed by sodium hydroxide solution (4N, 0.1 ml.). After 11h hr. at room temperature, sodium borohydride (15 mg.) was added and the solution allowed to stand for a further hour. Methanol was removed, water (2 ml.) added, the mixture extracted with ether (4 x 2 ml.) and the extracts evaporated to dryness after being washed with water. The oily residue was dissolved in methanol (0.2 ml.), concentrated hydrochloric acid (2 drops) was added followed by ether (3 ml.). The hydrochloride so formed was centrifuged off, washed with ether and the amine liberated in alkali and extracted with ether m.p. 61-63° from light petroleum (lit.59 61-4°). 62.

106 Benzyloxyphenylacetic acid 14 Sodium [ C] cyanide (0.98 mg., 0.1 mc. ) with inactive sodium cyanide (4.0 mg.) in dimethylsulphoxide (0.2 ml.) was heated with a solution of 2-benzyloxybenzyl chloride (30 mg.) in dimethylsulphoxide (0.5 ml.) for o 3 hr. at 100 . The solution was cooled and the crude nitrile was obtained after salting out and ether extraction (4 x 2 ml.). The crude product was heated under reflux for 18 hr. with sodium hydroxide (4N, 5 ml.) containing hydrogen peroxide (3 drops 20 vols.), cooled and extracted with chloroform (3 x 2 ml.). After acidification (HC1) the precipitated product was extracted into chloroform (4 x 3 ml.), the extracts washed with water (2 x 2 ml.) and dried. Crystallisation from benzene-petroleum ether gave 18 mg. of the acid (85% radioactive yield) m.p. 120 - 2°. N-(3-Benzyloxy-4-methoxybenzy1)-N-methy1-2-benzyloxyphenylacetamide To a suspension of 2-benzyloxyphenylacetic acid (18 mg.) in benzene (3 ml.) was added oxalyl chloride (1 ml.) and the mixture warmed until all the acid had dissolved. After 1 hr. at room temperature the solvent and excess of oxalyl chloride were removed and the remaining acid chloride was dissolved in benzene (2 ml.). This solution was added to a solution of the benzylamine (19 mg.) in benzene. After 2 hr. the precipitated amine hydrochloride was removed (9.7 mg. after recrystallisation) and the filtrate washed with N-hydrochloric acid, water, N-sodium hydrogen carbonate and water, dried and evaporated. The crude product crystallised on addition of light petroleum and recrystallisation from ether gave the amide (32 mg., 83%) 51 m.p. 91-3° (lit. 91-93°). 4-Benzyloxy-N- (3 -4-methoxybenzyl):N-methylphenethylamine The amide (LX) (32 mg.) was extracted (Soxhiet) into a refluxing suspension of lithium aluminium hydride (100 mg.) in ether (5 ml.). Excess 63. of reagent was decomposed with ethyl acetate, and water was added. The ether layer was separated and the pasty aqueous layer further extracted with ether. Evaporation of the dried (MgSO4) ethereal solutions gave the amine which crystallised from ethanol as prisms (28 mg., 907o) m.p. 73-4° (lit.51 74o).

4-Hydroxy-N,(3-hydroxy-3-methoxybenzy1)-N7methylphenethylamine hydrochloride (N-, 0-, dimethylnorbelladine) A solution of the dibenzyl ether (28 mg.) in methanol (5 ml.) containing concentrated hydrochloric acid (0.01 ml.) was hydrogenolysed over 10% palladised charcoal (10 mg.). After 2 hr., the solution was filtered, evaporated to 0.5 ml. and diluted with ether. The amine (XLVII) separated as its hydrochloride (21 mg., 83%) m.p. 230° (decomp.) (lit.51 230°).. 3-Hydroxy-4-methoxy-N-methylbenzylamine hydrochloride (LXI, R = H) The hydrochloride of the O-benzyl ether (LXI, R = CH2Ph) (la mg.) in methanol (5 ml.) was hydrogenolysed over 10% palladised charcoal (5 mg.). After 2 hr. the solution was filtered and evaporated to dryness to give the desired amine hydrochloride which crystallised in ether (7.5 mg., 95%). Recrystallisation from ethanol-ether gave plates (6 mg., 78%), m.p. 165 - 1700. (lit.59 m.p. 171-2°). N-Methyl tyramine (double labelled) Methyl-J -benz_yloxy_phenylacetamide Into a stirred solution of 2-benzyloxyphenylacetyl chloride (prepared from the acid 24 mg., p.22) was distilled methylamine (liberated from the hydrochloride 0.1 mc., 2 mg.). After 15 min. reaction was completed by the addition of an excess of methylamine and, after a further 5 min., water (4 ml.) was added and the benzene layer separated. Benzene extraction of 64, the aqueous phase (3 x 3 ml.) and evaporation of the combined, dried (MgSO4) benzene solutions gave the amide (22 mg., 95%) m.p. 138-142°. Recrystallisation of a sample from a previous unlabelled preparation from ethanol gave material m.p. 144.5 - 145.5°, (found: C, 75.1; H, 6.2; N, 5.7. C H 0 N requires C, 75.3; H, 6.7; N, 5.5%). 16 17 2 Q-Benzyl-N-methyltyramine The amide (22 mg.) was extracted (Soxhlet) into a refluxing suspension of lithium aluminium hydride (200 mg.) in ether (5 ml.) and heated under reflux for 36 hr. Decomposition with ethyl acetate followed by water and ether extraction gave the amine (19 mg., 85%). Methyltyramine hydrochloride A solution of the amine (19 mg.) in ethanol (4 ml.) containing concentrated hydrochloric acid (1 drop) was hydrogenolysed over 10% palladised charcoal. After filtering and evaporation the amine hydrochloride was 107 obtained as plates (13 mg., 89%) m.p. 146 - 147°. (lit. m.p. 148°). Tyramine (doubly labelled) 14 [2- C] tyrosine hydrochloride (ca. 1 mg., 0.05 mc.) diluted with inactive material (3 mg.) was dissolved in [ water (0.1 ml., 20 mc.) and warmed for 15 min. Sodium acetate (3 mg.) was added and the solution evaporated to dryness. The residue was heated with diphenylamine (0.5 g.), previously tritium exchanged, at 2600 for 5 min. After cooling, inactive tyramin.e was added, followed by water (1 ml.) and diphenylamine extracted with ether. The aqueous phase was evaporated to dryness and tyramine o 108 o sublimed from the residue (5 mg.) m.p. 160 - 161 (lit. m. p. 161 ). Purity ofass.....ursors AutoradionaAly The precursors were run on circular paper chromatograms in n-butanol : acetic acid : water systems (4 : 1 : 5). The dried paper was 65.

exposed to X-ray film (Kodirex) and development showed that the bands 109 were coincident with those obtained by spraying the paper with Pauli reagent.

No radioactive compounds with RF values different from those of the precursors were present as impurities. Dilution analysis Crystallisation of the precursors, diluted with inactive material showed no drop in the activity. Diluted precursors singly labelled in the 0- or N-methyl position were further subjected to Zeisel determinations for purity assay. Zeisel determinations 110 The usual microanalytical procedure for 0- and N- methyl groups was followed using samples of isolated alkaloid or diluted precursor (ca. 10 mg.). The methyl iodide evolved was collected in ethanolic . triethylarnme1 I 1 which was evaporated and the residual triethylmethyl- ammonium iodide plated in the usual way. Control experiments showed that up to 5% of the N-methyl group was liberated along with the 0-methyl group but no correction was made for this source of error. 12 Isolation of alkaloids (general procedure of Fales, Giuffrida and Wildman ) 44 King Alfred daffodils (whole plants; typically 200 g. wet weight) were extracted with 1% ethanolic tartaric acid (2 x 150 ml.). The extract was filtered, the filtrate evaporated to small bulk (50 ml.) and acidified (2N-hydrochloric acid 15 ml., water 150 ml.). The acid solution was washed with chloroform (3 x 30 ml.) and the washings back extracted with 2N-hydrochloric acid (5 x 20 ml.). The combined acid extracts were made alkaline (aqueous sodium carbonate) and extracted with chloroform (10x 30 ml.). Evaporation gave the crude basic extract (320 mg.). The crude mixture of alkaloids was chromatographed on alumina (grade III, 30 g.) using a gradient

66.

CHROMATOGRAPHY OF ALKALOIDS FROM KING ALFRED DAFFODILS

(Gradient elution off grade 3 alumina) Optical density

3 fl

1 Ld_f_

4 f o % 2% Et01-1.„, 5% EtOH 0% EtOAc 35 45 60 EtOAc 100% C H CHC13 6 6

I. Fluorescent artifact 2. Homolycorine 3. Galanthamine 4. Galanthine 5. Haemanthamine 6. Narcis samine 67. elution technique of increasing percentage of ethyl acetate in benzene, followed by chloroform and 2% ethanol-chloroform eluent. The separation of alkaloids was followed in the U. V. at 280 mil and is represented diagrammatically opposite. Fractions (1) Fluorescent artifact (2 mg.) (2) Gum containing homolycorine (10 mg.) (3) Galanthamine (61 mg. - 0.03%) (4) Galanthine (46 mg. - 0.023%) (5) Haemanthamine (72 mg. - 0.036%) Alkaloid yields were based on the wet plant weight. The procedure for isolating lycorine from Texas and Irene Copeland plants was similar except that the final chloroform extract was evaporated to small volume and set aside for the alkaloid (0.06%) to crystallise. The alkaloids were purified for counting by crystallisation of the free bases and salts (generally hydrochlorides). Degradation of galanthamine Apo-galanthamine (XXVIII) Galanthamine (60 mg.) was heated under reflux with hydrobromic acid (46% aq., 0.6 ml.) for 4 hr. under nitrogen. The reaction mixture was cooled, allowed to stand 48 hr., and the crystalline apo-galanthamine hydrobromide collected and washed with cold ethanol (35 mg., 52%) m. p. 223 - 231°. Crystallisation from ethanol gave 21 mg., m. p. 231 -232° (lit.113 232°). Phthalic anhydride Apo-galanthamine (30 mg.) was added to potassium hydroxide (1.5 g.) and potassium ferricyanide (6 g.) in water (6 ml.) and heated at 100°. Additions of the same amounts of alkali and ferricyanide were made after 68.

12 hr.. and 36 hr. and, after 60 hr., the mixture was cooled, yellow, crystalline ferrocyanide filtered off and washed with a little cold water. The filtrate was acidified (50% H2SO4) and the solution continuously extracted with ether for 36 hr. Phthalic anhydride (4 mg,) was sublimed from the residue after evaporation of the ether. Degradation of haemanthamine - from King Alfred daffodils fed with the doubly labelled benzylamine (LXI, R = H)

The active haemanthamine (12 mg ) diluted with inactive material (12 mg.) was dissolved in 20% sulphuric acid (35 ml.) and water distilled out into a solution of dimedone (200 mg.) in water (25 ml.). Water was added at the rate of distillation to keep the acid concentration constant until ca. 150 ml. had distilled over when the distillation was stopped. Formaldehyde dimedone (13 mg., 58%) crystallised from the aqueous solution as needles on standing and was counted.

The results obtained with labelled precursors are shown overleaf. 69.

N-Methylnorbelladine (20 mg.) - 0.020 mc. — — n...0•461113

Precursor (0.30 mg.), diluted with 6 inactive material (60 mg.) 1.58 x 10 c.p.m./mMole 6 recrystallised 1.54 x 10 (1) Galanthamine (71 mg., 0.027%) m.p. 115 - 9° 4 free base (cryst.) 1.69 x 10 c.p.m. /mMole hydrochloride 1.58 x 104 4 (recryst.) 1.53 x 10 (recryst.) • 1.51 x 4 1.47 x 10 Et3NMeI derived from N-Me Incorporation 0.018%. (2) Galanthine (58 mg.) m.p. 74 - 990 - inactive (3) Humanthamine (75 mg.) m.p.196-8°- inactive

N-,0-Dimethylnorbelladine(18 mg.) - 0.018 mc. (single labelled) Precursor (0.41 mg.), diluted with 6 inactive material (55 mg.) 2.31 x 10 c.p.m. /mMole 6 recrystallised - 2.30 x 10 (1) Galanthamine (63 mg., 0.024%) m.p. 116 - 121° 4 free base (cryst.) 1.48 x 10 c.p.m./mMole 4 hydrochloride • 1.32 x 10 (recryst.) ▪ 1.29 x 10 4 4 (recryst.) 1.28 x 10 4 1.24 x 10 Et3NMeI derived from N- Me Incorporation 0.014%. (2) Galanthine (49 mg.) m.p. 77 - 96° - inactive (3) Haemanthamine (65 mg.) m.p. 194 - 8° - inactive

70.

N-a 0-Dimethylnorbelladne (20 mg.) 0,041 mc. (double labelled)

Precursor (0.23 mg.), diluted with 6 inactive material (80 mg.) - 1.80 x 10 c.p.m./rnMole recrystallised - 1.91 x 106 Et NMeI derived from 0-Me .. 9.20 x 105 3 Et NMeI derived from N-Me - 1,02 x 106 3 (1) Galanthamine (82 mg., 0.022%) m.p. 115 - 8° 4 free base (cryst.) - 2.92 x 10 c. p.m./mMole 4 hydrochloride - 2.54 x 10 4 (recryst.) - 2,43 x 10 (recryst.) 2 ,43 x 104 Et NMel derived from 0-Me 1.19 x 104 3 4 from N- Me 1.19 x 10 Narwedine - 2.51 x 104 c..p.m. /mMole Et NMeI derived from 0-Me • 1.13 x 104 3 - from N-Me • 1.25 x 104

14 Fraction of C in 0-Me N- Me

Precursor 0.48 0.51 Galanthamine 0.48 0.48 Narwedine 0.45 0.49 71.

N-,0-Dimethylnorbelladine (18 mg.) 0.049 mc. (triple labelled)

Precursor (0.21 mg,) diluted with 6 inactive material (100 mg.) - 2,06 x 10 c.p.m. /mMole (recryst.) - 2.08x 106 Et3NMeI from 0- Me - 4.00 x 105 from N- Me - 4.16 x 105 6 remainder - 1.21 x 10 (1 ) Galanthamine (72 mg., 0.024%) m. p. 118 - 122° free base (cryst.) - 3.77 x 104 c.p.m. /mMole hydrochloride 3,51 x 104 - (recryst.) - 3.47 x 10 4 4 - (recryst.) 3.46 x 10 - 3 Et3NMeI from 0-Me 6.60 x 10 - from N- Me 6.81 X 103 remainder - 2.12 X 104 Narwedine 3.48 x 104 c. p. m. /mMole NMeI from 0-Me - 6.61 x 103 Et3 from N- Me - 6.63 x 103 remainder 2.16 x 104

0-Me N- Me remainder

Precursor 0.19 0.21 0.60 Galanthamine 0.18 0.19 0.63 Narwedine 0,18 0.18 0.64 --• 72.

N-Methyl-3-hydroxy-4-methoxybenzylamine (8 mg.) 0.011 me. (double labelled)

Precursor (0.20 mg.) diluted with 6 inactive material (80 mg«) ▪ 1.08 x 10 c.p.m./mMole (recryst, ) ▪ 1.05 x106 Et3NMeI from 0-Me 5.01 x 105 - from N- Me ▪ 5.03 x 105 (1) Galanthamine (60 mg., 0.020%) imp. '116 - 90 4 free base (cryst.) - 1.18 x 10 c. p.m. imMole hydrochloride - 1.09 x 104 (recryst.) - 1.06 x 104 (recryst.) - 1.08 x 10 4

Et 7C 103 3NMeI from 0-Me - 9.61 2 ▪ from N-Me - 5 x 10

I 0-Me N-Me Precursor 0.49 0.50 Galanthamine 0,91 0.05

Incorporation 0.019%

(2) Haemanthamine (64 mg., 0.022%) m. p. 196 - 7° 3 free base (cryst.) - 6.10 x 10 c.p.m. kriMole (recryst.) - 5.91 x 103 picrate (cryst.) - 5,62 x 103 (recryst.) - 5.49 x 103 formaldehyde dimedone - 5.63 x 103 Incorporation 0.010% 73.

Tyrosine (generally labelled) 0.02 mc.

(1) Galanthamine (105 mg., 0.025%) m.p. 116 - 119° 4 free base (cryst.) - 3.14 x 10 c.p.m. /mMole 4 hydrochloride - 2.31 x 10 4 (recryst.) 1.91 x 10 4 (recryst.) 1,,83 x 10 4 (rec ryst. ) 1.78 x 10 4 Phthalic anhydride 1.28 x 10 4 If incorporated into only 1.55 x 10 C6-C2 4 If into both equally 0.96 x 10 This indicates an incorporation into both fragments with a 3 : 1 preference for the C6 - C2 unit. In a second, exactly similar experiment a ratio of incorporations of 14.5 : 1 was found. Incorporation of tyrosine into lycorine in Texas daffodils [2- 14C] tyrosine (0.02 mc.) (1) Lycorine (50 mg4, 0.04%) 5 hydrochloride - 3.25 x 10 c.p.m./mMole 5 (recryst.) - 1.47 x 10 5 (recryst.) - 1.37 x 10 5 perchlorate - 1.36 x 10

Incorporation 0.44% Incorporation of 2- Cjityrosine into lycorine in homogenates of Irene Copeland daffodils

The mature flowering plants (100 g.) were crushed under liquid nitrogen and phosphate buffer of pH 7 [100 ml.; 60 ml. Na2HPO4.2H20 (11.87 g. /1. ), 40 ml. KH2PO4 (9.08 g./1.)] was added followed by an aqueous solution of tyrosine (0.003 mc.). The mixture was allowed to stand at 25° for 24 hr., with occasional shaking, before being worked up for lycorine (p. 67) 50 mg. Lycorine (crystallised from chloroform) was converted to its hydrochloride, counted and recrystallised to constant count from ethanol-ether. 4 hydrochloride - 2.43 x 10 c. p. rn./mMole 4 (recryst.) 1.75 x 10 4 (recryst.) 1.31 x 10 4 (recryst.) 1.26 x 10 4 (recryst.) 1.26 x 10

Incorporation 0.008% EXPERIMENTAL

The Morphine Alkaloids 74.

The precursors were injected in aqueous solution of the hydrochlorides (pH 6) into the pods of mature Papaver somniferum plants. After 9 days the plants were worked up and the morphine obtained was purified as its picrate and di-O-acetyl derivative to constant count. Counting of[14C]and[3H]in the presence of each other was 14 carried out in a scintillation counter, previously calibrated with[ C1 and 3 [ H]standards, allowance being made for quenching effects.

Preparation of (1\!-[14C]-methyl, 1-3H) ± Reticuline

0-Benzylisovanillic acid (CXIV) (method of Mr. M.N. Afzal) O-Benzylisovanillin (7.2 g.) in acetone (100 ml.) was stirred while potassium permanganate (3.2 g.) in water (50 ml.) was added dropwise. After 1 hr., sulphur dioxide was passed through the reaction mixture and manganese sulphate filtered off. Acetone was removed, the aqueous solution acidified (hydrochloric acid), and the precipitated acid collected and 114 crystallised from ethanol (5.1 g., 67%) m.p. 120 - 122° (lit. 123°). 3-Benzyloxy-4-methoxy-LO-diazoacetophenone (CXV) The acid (CXIV) (2.0 g.) in benzene (10 ml.) was warmed with oxalyl chloride and the resulting solution allowed to stand for 1 hr., before being evaporated to dryness. The acid chloride, which appeared as an oil and rapidly crystallised, was redissolved in benzene (10 ml.) and added slowly with shaking to a dried (MgSO4) ethereal solution of diazomethane (prepared 75. from nitrosomethylurea - 4 g. ). After i6 hr. at 0°, the diazoketone which had separated, was collected (1.7 g., 81%) m.p. 111 - 2° (lit.115114-5°). 4-Benzylou-3-methoxy-44-nitrostyrene (LXXXIII) 0-Senzylvanillin (1.2 g.) in ethanol (40 ml.) was cooled to 5° and nitromethane (1 ml.) added. A cooled solution of sodium hydroxide (0.5 g.) in ethanol (10 ml.) was added slowly with stirring. The precipitate formed was dissolved in the minimum amount of iced water (ca. 20 ml.) and the solution poured into hydrochloric acid (6 ml. concentrated, 9 ml. water) with stirring. The precipitated nitrostyrene (0.96 g., 70%) was collected m.p. 114 - 7° (lit.116 122 - 30). 4-Benzyloxy-3-methoxyphenethylamine (LXXXIV) The nitrostyrene (LXXXIII) (0.50 g.) in tetrahydrofuran. (5 ml, ) was added dropwise to a refluxing suspension of lithium aluminium hydride (0, 5 g.) in ether (50 ml.) and the mixture heated under reflux for 2 hr. After decomposition of the hydride and ether extraction, the amine (0.40 g., 83%) was obtained as an oil. This was characterised as the oxalate obtained as 17 plates from ethanol, m.p. 162 - 4° (lit 1 p 162 - 30). 3-Benzyloxy-4-methoxyphenyl-P- (41 -benzyloxy- 3 I -me thoxyphenyl ethylacetamide (LXXXVII)117 The diazoketone (LXXXVI) (24 mg.) in benzene (40 ml.) was added to the phenethylamine (LXXXIV) (liberated from the oxalate - 25 mg.) in benzene (5 ml.). Freshly prepared silver oxide118 (10 mg.) was added and the mixture vtirred and heated at 60° for 3 hr. After a further 3z hr. heating under reflux, silver oxide was removed, solvent evaporated and the product purified by boiling in ethanol with charcoal. Evaporation of the ethanol gave the amide (32 mg., 66%) m.p, 115 - 6°. 76.

1- (3- Benzyloxy- 4-methoxybenzyI)- 6- methoxy, 7-benzyloxy- 3, 4-dihydroisoquinoline hydrochloride (LXXXVIII) Phosphorus oxychloride (0. 1 ml.) was added to the amide (LXXXVII) (50 mg.) in toluene (2 ml.) and the mixture heated under reflux for 45 min. Solvent was evaporated in vacuo until solid separated, petroleum ether (10 ml.) was added and the crude hydrochloride which separated was crystallised from ethanol-ether (39 mg., 78%) m.p. 204 - 6° (decomp.) (lit.117 m.p. 198 - 200°). (11.414_, j_ methyl, 1- [3H])-1- (3-benzylay- 4-methoxybenzyI)- 7-benzyloxy- 6-methoxy-1, 2, 3, 4-tetrahydro-2-methylisosuinoline. The dihydroisoquinoline (LXXXVIII), isolated from its hydrochloride (70 mg.) by ether extraction from bicarbonate solution, was dissolved in benzene and [14C] methyl iodide was distilled into the solution. The reaction vessel was sealed under vacuum and stirred for 5 days at room temperature. The methiodide separated and the reaction was completed by the addition of a large excess of inactive methyl iodide and a further 8 hr. stirring. The precipitated labelled methiodide (49 mg.) was combined with a further amount o 117 o (5 mg.) obtained from the mother liquors, m.p. 197 - 8 (lit. 196 - 8 ). The methiodide (54 mg.) in methanol (3 ml.) was reduced with sodium [3H1 borohydride (50 mg.). After 1 hr., methanol was removed and water (2 ml.) containing 4N sodium hydroxide (2 drops) was added. Ether extraction (5 x 2 ml.) gave the required amine (32 mg.) and chromatography on alumina (grade III) and benzene elution gave the pure product (29 mg., 70%) 117 which crystallised under petrol m.p. 86 - 8° (lit 89°). Cj methyl, 1-43F11 (±) Reticuline (LXXXIX) The di-O-benzyl ether (29 mg.) in ethanol (0. 1 ml.) was heated at 100° with concentrated hydrochloride (0.2 ml.). Ethanol was evaporated and, after cooling, benzyl chloride was removed by ether extraction and the 77. mixture basified to pH 8 before (±) reticuline (12 mg.) was extracted with chloroform. Purification was effected by chromatography on alumina (grade III) and the product eluted with 2% ethanol-chloroform, but the product did not crystallise. In the preparation of (±) reticuline, [14C] labelled in the 0-methyl positions and in the 3-position in addition to the labels mentioned above, the 14 0-methyl labels were introduced by [ C] methyl iodide methylation of the corresponding monobenzylprotocatechuic aldehydes (see p.61). The chain label was introduced by preparation of the phenethylamine (CXIII) via the labelled cyanide (CXII) as shown in the scheme. 4-Benzyloxy-3-methoxybenzyl cyanide (CXII) (cf. p.62) 4-Benzyloxy-3-methoxybenzyl chloride (1.31 g.) in dimethylsulphoxide (5 ml.) was added to sodium cyanide (0.35 g.) in dimethylsulphoxide (3 ml. ) o and the solution stirred and heated at 100 for 3 hr. After cooling, the product was salted out and extracted into ether (4 x 20 ml.). The ether extracts were washed with water (5 x 15 ml.), dried and evaporated. The cyanide crystallised from petrol (0.99 g. , 68%) m, p. 67 - 8° (lit.119 m.p.67 - 8°). 4-Benzyloxy-3-methoxyphenethylamine hydrochloride (CXIII) The benzyl cyanide (0.5 g.) in tetrahydrofuran (3 ml.) was added to a refluxing suspension of lithium aluminium hydride (0.5 g.) in ether (5 ml.). After decomposition of the hydride and ether extraction the amine was obtained, did not crystallise and was characterised as the oxalate m.p. 161 - 3° 117 (lit. 162 - 3°). 78.

Isolation of alkaloids from Papaver somniferum

Morphine12° Two plants (120 g.) were macerated in a Waring blender with 1 : 1 n-butanol-benzene (500 ml.) containing 10% sodium carbonate (70 ml.). The mixture was stirred for 2 days, filtered and the plant material washed with n-butanol-benzene (120 ml.). The organic layer was washed with 0.5N sulphuric acid (6 x 70 ml.) and the extracts were made alkaline with 2% potassium hydroxide saturated with barium hydroxide. Barium sulphate was filtered off and the filtrate passed through a column of Amberlite IRA 400 in the OH form (60 g. ). The column was eluted with 50% aqueous methanol (300 ml.) followed by 0.1N hydrochloric acid, and the fraction collected when the pH of the eluate reached pH 2. 300 ml. Were collected, evaporated to 10 ml., and the concentrate was neutralised to pH 5 with 4N sodium hydroxide. Morphine (45 mg., 0.04%) separated. Thebaine Maceration of the plants with dilute hydrochloric acid and chromatography of the crude mixture of non-phenolic alkaloids is the method to be used for extraction of thebaine from Papaver somniferum or Papaver orientalis. Thebaine is the major alkaloid in the latter variety. We have already shown, in an unsuccessful attempt to isolate the dienone (CXX) from crude opium, that thebaine can be eluted with 20% ethyl acetate/benzene from Grade III alumina. Alkaloid derivatives Morphine picrate To a suspension of morphine in hot ethanol was added an ethanolic solution of picric acid. Morphine picrate crystallised on cooling and was o 121 o recrystallised from ethanol m. p. 163 - 5 (lit, , m. p. 163 - 5 ). 79.

Di-O-acetylmorphine

Morphine (20 mg.) was dissolved in acetic anhydride (0.3 ml.) and heated at 100° for 15 min. Acetic anhydride was removed in vacuo and the residue crystallised from ethyl acetate. Recrystallisation from the same o 122 m.p. solvent gave colourless cubes m.p. 172 - 3 (lit. 173°). This di-O-acetyl crystallised easily from ethyl acetate and was soluble in all ogranic solvents suitable for scintillation counting.

14 The N-[ C] methyl label in morphine isolated from plants fed with active precursors was estimated by Zeisel determination (p.65). 14 3 4- Incorporation of (.11-[ C] methyl, 1.4 113(-) reticuline into Papaver somniferum

Morphine (39 mg., 0,03%) 6 picrate 1.42 x 10 c.p.m./mMole 6 (recryst.) 1.25 x 10 6 (recryst.) 1.27 x 10 6 diacetyl derivative 1.30 x 10 6 Et NMeI from N - Me 1.19 x 10 3 Inco3Toration 0. 13% [14C]: [3H] ratios 14C1 _ 67%, [3p_] _ 6%) Setting (1) maximum [ 14C] efficiency - ([ 14 Setting (2) maximum [311] efficiency([ -Cj 20%, [3}1] - 19%)

80.

Precursor (diluted)

At setting (1) - 1350 d.p.m. /mg. At setting (2) - 510 &p.m./mg. [14C] : -3_, HJ ratio 2.0 • 1

Di-Q-acetylmorphine

At setting (1) - 656 d. p.m. /mg.

At setting (2) - 238 d.p. m. /mg. 14 f Cl [3H] ratio 2.6.:

This gives a value for the retention of the [3H] label of 78%.

14 Incor•oration of 24 C t rosine (0.01,5 mc.) into Pa aver somniferum (Parallel experiment to that with reticuline) Morphine (18 mg., 0.03%) 5 pic rate 1.72 x 10 c.p.m./mMole 5 (recryst.) 1.54 x 10 5 (recryst.) 1.47 x 10 5 di-O-acetyl derivative 1.50 x 10

Incorporatiov

81.

Radiochemical synthesis of thebaine reticuline [ 1 -3H) Di-O-benzylreticuline (8 mg., 0.02 mc.) diluted with inactive material (50 mg.) was hydrogenolysed as before and the active reticuline so formed was dissolved in chloroform (5 ml.) and shaken with Manganese dioxide (200 mg.) for 3 hr. After filtration, inactive dienone (CXX, 7 mg.) was added, the chloroform evaporated and the dienone separated by chromatography (grade III alumina, chloroform elution). 4 First count 6.22 x 10 d.p.m. imMole 4 After rechromatography 4.51 x 10 4 Third chromatography 4.43 x 10 Radiochemical yield 0.012% (6. 5 mg.) Conversion to thebaine (method of Dr. W. Steglich) The active dienone (5.7 mg.) diluted with an equal amount of inactive material was reduced with borohydride (50 mg.) at room temperature in methanol (2 ml.). Methanol was removed and chloroform extraction gave the alcohol (CXXI) as a colourless semi-solid, which was dissolved in 1N-hydrochloric acid (1 ml.) and left at room temperature for 1 hr. The solution was made strongly alkaline with 4N sodium hydroxide and the orange turbid solution was extracted with chloroform (4 x 3 ml.). Evaporation of the colourless extracts gave thebaine which was purified by chromatography (grade III alumina, 30% Et0Ac/C6H6 elution) after 1 : 1 dilution. 4 Count 1.07 x 10 d.p.m. /mMole Radiochemical yield on basis of thebaine count 0.010% 82.

Degradation of thebaine Thebenine hydrochloride (CXXV) Thebaine (1,00 g,.) was added to boiling 4N-hydrochloric acid (5 ml.) and the yellow solution was boiled for 2 min. Thebenine hydrochloride separated and was recrystallised from water (605 mg., 61%) m.p. 233 4° (lit.123 m.p. 235 ). 3, 4, 8- Trimethoxy- 5- vinylphenanthrene (CXXVI) Thebenine hydrochloride (150 mg.) in methanol (4 ml.) was heated under reflux with methyl iodide (1 ml.) and 4N-sodium hydroxide (0.2 ml.) for 2 hr. Methanol was removed in vacuo and the residue heated at 100° with 4N sodium hydroxide (3 ml.) for 1 hr. After cooling, chloroform extraction gave a brown gum which on chloroform on alumina (grade III) with benzene elution gave the vinylphenanthrene (70 mg., 47%) m.p. 119 - 122° 124 0 (lit. m.p. 122.5 ). Formaldehyde The vinyl compound (70 mg.) in ethyl chloride (3 ml.) at -40° was treated with ozone. The reaction was followed in the U. V., (starting material

Xmax. 225* 255 and 300 mil - product Xmax.210 mil only), and was complete in 25 min. Water (5 ml.) was added and ethyl chloride allowed to evaporate. Steam distillation, after the addition of zinc dust (50 mg.), into a saturated aqueous solution of dimedone gave formaldehyde as its dimedone derivative which crystallised on standing overnight as needles (5 mg., 35%). None of the expected 3, 4, 8-trimethoxy-5-aldehydrophenanthrene was isolated from the residue. 83.

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