STUDIES RELATED TO THE BIOSYNTHESIS OF

THE MORPHINE AND ERYTHRINA ALKALOIDS

A thesis presented by

RONALD JAMES

in partial fulfilment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Imperial College, S. W. 7 . November, 1966 11 .

TO MY MOTHER ABSTRACT

The stereochemistry and biosynthesis of the morphine alkaloids is reviewed, along with the structure, stereochemistry, and attendant biogenetic theories of the Erythrina alkaloids, as the essential plinth on which the present work is based. The absolute configuration at C -9 of the morphine alkaloids has been determined by direct correlation with a benzyl- isoquinoline, The stereochemistry of the epimeric salutaridinols has been defined by degrading them to optically active glyceric acids, and the method extended to obtain the stereochemistry of the alkaloid nudaurine. The structure of erythratine has been revised in the light of biogenetic theory, and evidence provided to confirm this structure and to define its stereochemistry. The structural elucidation of three other Erythrina alkaloids has been completed. Possible precursors of the Erythrina alkaloids have been fed to Erythrina plants and a modified theory for the biosynthesis of these alkaloids is suggested based on the results from these tracer studies. iv .

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to Professor D.H.R. Barton for the privilege of working under his inspiring guidance, and also to Dr. G.W. Kirby for very many helpful and friendly discussions during the course of this work. I have to thank also Dr. D.W. Turner who obtained all the 100 Mc. /sec. n.m.r. spectral data, Dr. E.S. Waight and his staff for mass spectral measurements, Mr. Young who grew the plants, and Mr. D.R. Aldrich and his staff for all kinds of technical assistance. My colleagues in the department have given me help, advice, and friendship, all of which were much appreciated. Finally I am deeply indebted to my mother, for without her sacrifices over many years this thesis would not have been written. v.

CONTENTS

page

Abstract

Acknowledgments iv•

REVIEW Stereochemistry of the morphine alkaloids 1 Biosynthesis of the opium alkaloids 14 Structure and stereochemistry of the Erythrina alkaloids 38 Biogenesis of the Erythrina alkaloids 49

DISCUSSION Absolute configuration at C-9 of the morphine alkaloids 57 Stereochemistry of the salutaridinols 62 Stereochemistry of nudaurine 72 Structures of some Erythrina alkaloids 75 Biosynthesis of the Erythrina alkaloids 89

EXPERIMENTAL 103

REFERENCES 129 1.

REVIEW

The stereochemistry of the morphine alkaloids

In 1805 the "vegetable alkali"1 morphine (1; R=H) became the first member of a large group of naturally occurring substances, 2 later grouped together as the alkaloids, to be isolated pure . The elucidation of its structure was a spur and a building block in the progress of organic chemistry from that time until the structure 3 4 proposed by Sir Robert Robinson was finally verified in 1950 . 5 The way was then clear for Stork to deduce the relative stereochemistry of morphine and codeine (1; R=Me) from the experimental evidence already available at the time. His arguments, which rest on the known steric requirements of certain reactions, are outlined below, and are followed by some more recent evidence which supports his conclusions. Morphine and codeine have five asymmetric centres but the presence of an ethanamine bridge limits the number of possible isomers to sixteen (eight racemates) instead of thirty-two. Stork first related the hydrogen at C-6 to the C-5 oxygen function. He assumed that the C-6 hydrogen was above the plane of the ring in codeine (2; R=}1). Isocodeine is then represented by (3; R=H). Of the two derived methyl ethers (2, 3; R=Me), the isocodeine ether is recovered unchanged from treatment with methoxide, whilst the 6 codeine epimer is isomerised to the phenolic enol ether (4) .

Z.

RO MeO 1VIe0

Mule

RO H (1) nn (z)

./..CDMe

HO (5) (4)

The recovery of the isocodeine ether shows that no epimerization takes place under the conditions used, and since the f3 elimination requires the existence of a trans relationship between the hydrogen at C -6 and the oxygen at C-5, the configuration of codeine can be expanded to (5). Confirmation of the cis relationship between the adjacent oxygen functions is found in the greater rate of the lead tetra-acetate cleavage of tetrahydromorphitetrol (7), compared with 7 that of tetrahydro-a-isomorphitetrol (8) . These glycols are ? prepared from dihydrocodeine (6) and dihydroisocodeine, respectively, by the sequence shown for the codeine series (6) —> (7). 3.

COOMe

e

HO HO HO (6) LiA1H 4

HO (8) HO- (7)

Ozonolysis of the substituted catechol cleaves the most oxygenated double bond and subsequent hydrogenation reduces only one of the remaining double bonds, the tetra-substituted one being stable to the conditions used. Lithium aluminium hydride reduction of the resulting ester -lactone affords the morphitetrol (7). Since cis glycols are known to be cleaved faster than trans, by lead tetra-acetate, the two oxygen functions at C-5, C-6, must be cis. Next, the ethanamine bridge was related to the C-5 hydrogen by a consideration of the relative stabilities of cis and tram fused 6,5 ring systems. Dihydrocodeinone (10) is of the hexahydro- l-indanone (9) type. If it had a trans junction of the ether ring and 4.

and the C ring, then it would be expected to isomerise in base to a v3 cis junction, which is known to be the more stable form of (9) , 9 whereas in fact dihydrocodeinone is stable in base . Further, since dihydrocodeine (6) is the sole product of catalytic reduction of both 10 dihydrocodeinone (10) and codeine (5) , these must all have the same stereochemistry at this junction.

HCr , ' (10) (6) HO (5) If the ring junction is cis, it follows that C-15 and the bridge oxygen must be trans, and this assignment is supported by a number of concerted eliminations of the ethanamine bridge with cleavage of the oxygen bridge, by nucleophilic attack at C-15. For example, when p-methyl-morphimethine (11), a Hofmann degradation product, is 11 heated with sodium ethoxide, morphenol (12) is formed .

OEt 1 + CH CH 2— 2 NMez

(12) 5 •

These eliminations are most satisfactorily explained if a trans relationship exists between C-15 and the bridge oxygen. Stork believed that he had further evidence for this trans junction in the ease of generation of 1-bromodihydrocodeinone (13) by alkali 12 treatment of 1,5-dibromodihydrothebainone (14) .

OHO

80 %

In fact, Fieser and Fieser relied heavily on this argument in their 13 earlier deduction of the morphine sterochemistry , but later work 14 by Gates has shown that the bromine is at C-7 and is equatorial, and not at C-5 and axial, and he suggests that the reaction must proceed through the enol (15).

Bile°

0 > (13)

IC

HO HO'

(Br (15) (16)

6.

Nevertheless on the other evidence the formula for codeine can be expanded to (16). The remaining centre at C-14 was assigned on the evidence from catalytic hydrogenation of thebaine (17) which gives dihydrocodeine methyl ether (18) and not 15 the isocodeine series (19) .

/vie° w MeO w MeO

H2 / , .. a NMe N/vie

MeO H (19) (17) (18)

It is argued that hydrogen must add to the diene either from the same side as the ethanarnine bridge, in which case (18) results, or from the opposite side to give (19). Hence the configuration at C-14 must be the same as that at C-6, and since the configuration at C-6 is known then the complete relative stereochemistry of morphine (20; R=H) and codeine (20; R=IvIe) is deduced.

e

HO (zo)

16,17 Recent work by Rapoport has provided additional chemical evidence for Stork's assignments, while two complete 18 19, 20 syntheses support, and X-ray studies confirm the stereo- chemistry as shown (20). 7.

Rapoport showed that exhaustive methylation of dihydro- isocodeine (21) leads to a small yield of the cyclic ether 6-codiran (22), whereas none is formed from dihydrocodeine (6) under the same 16 conditions . Me Me MeO

(6)

Me Me N 3 HO 0 (21) (21A) (22)

Formation of this cyclic ether requires a cis relationship between the C -6 hydroxyl and the ethanaxnine bridge. If this is the case in isocodeine then it must be trans in the codeine series. In another paper apoport17 challenges the evidence on which Stork assigned the C-I4 stereochemistry, on the grounds that there are several known cases where hydrogenation of the 8-14 double bond produced the unnatural epirneric configuration at C-14, and further, that if the hydrogenation of thebaine is interrupted before it is complete, neopine methyl ether (23) can be isolated, which may in turn be reduced, but not necessarily from the same side as that from which the 6-7 double bond was reduced.

Me Me

Me

Me (17) 8.

He then arrived at the same stereochemistry at C-14 in the following 17 way . Thebenone (24) is a degradation product of thebaine (17) having the same stereochemistry as morphine at C-14.

MeO Me• Me i) Hof. H2/Pd/C H2/Pd/BaSO 4 > H AcOH iii) Hof.>

(24)

The C ring was further degraded by way of the dio2dminothebenone (25) formed by condensation at both activated methylene groups. A neat double application of a variant of the Beckmann rearrangement, followed by partial hydrolysis gave the acid amide (26), which gave a cyclic imide (27) on ring closure.

MeC (24) RONO> Ts .C1 ®OBut PY•

(27)

H •

The C-14 epimeric compound prepared from epi-thebenone in the same way did not ring close under the same conditions. Models show clearly that ring closure is only possible if C-5, C-8 are cis. In 1955 two groups published their results of X-ray 19 studies on morphine hydroiodide dihydrate , and codeine hydro- 20 bromide dihydrate , and though neither group was able at that time to ascertain the absolute stereochemistry they both confirmed the relative stereochemistry as in (20). The shape of the molecule (in the crystal) was found to be as shown (28).

1 NMe

RO (28) HO 18 The synthesis by Gates and Tschudi , apart from providing the first chemical proof of the point of attachment of the ethanamine bridge at C-13, can be rationalised by the known stereochemistry, but it contributes little by way of unequivocal evidence since, in common with the synthesis by Elad and 18 Ginsberg , it relies on an isonaerisation from a trans - B, C ring junction to the natural cis junction, without independent proof for the stereochemistry involved. Absolute configuration of morphine The first attempt to determine the absolute stereo- 21 chemistry of morphine was made by Bick . Morphine (20; 11.=H). and 10.

codeine (20; R=Me) were rearranged to apornorphine (29; R=H) and apocodeine (29; R=Me) respectively, which now have only one asymmetric carbon; that corresponding to C-9 in the morphine skelefon. (See arrows).

R.p

>HO HO NMe

Me

MeG H • • • N.1\ii:e Me I Med-- (30) (29)

Comparison of shifts of specific rotation, in differing solvents, of the apo-compounds with those of L-(+)-iaudanosine (30), of known 22 absolute configuration , led him to suggest, incorrectly, that the configuration was the same in both cases. This was later shown to 23 . be incorrect when Corrodi converted L-noriaudz.•nosine (31) into N-P-carboxyethyl-L-aspz,..rtic acid (32) and N-norapocodeine (33) into N-P-carboxyethyl-D-aspartic acid (34), which he had synthesised from D-aspa.rtic acid (35).

U. ivie

1Vie0 03 MeON HOOT HOO Me0 (31) (32)

0 3 > HOOC' NH

(33) (30 24 At about the same time Bentley published some work, in which he criticized Rapoport's stereochemical assignments. He stated that the small yield (2%) of codiran (22) could involve inversion at C -6 after replacement of the trimethylammonium group by a hydroxyl group which would lead to a less strained product (36).

( 21A) 12.

JF.aer's degradation of thebaine

(15)

CHAOH

H i) 0s04 ii) LiA1H 4

i) Pb(0Ac)4 ( CH2SH) 2 iii) Raney Ni iv) H+

MeO Me0,

Me0 i) Al/Hg i) (c.,'FI2SH)2. Me2SO4/5He ii) Ni

0

Cr03

Me

COOH C 00H Me i) 03 ii)H 20y HCOOH (37) 13.

He also objected to some of Stork's work on the grounds that cis eliminations of the ethanamine bridge are known and Stork had based his configuration at C-13 and C-19 on these eliminations being trans. 14 This means that, with Gates' work on dibromothebaine, the analogy with hexahydro-l-indane is the only chemical evidence on this point 24 (vide supra). Bentley then set out to relate the asymmetric centres by independent determinations of their absolute stereochemistry, in particular at C-5 and C-13, C-14. He assigned the absolute 25 configuration at C-5 by application of Mills' rule to codeine (2; R=H) and isocodeine (3; R=1-1). By a comparison, of optical rotation, of certain derivatives with cholestane derivatives, he predicted the absolute stereochemistry at C-13, C-14 and corrected Bick's assignment at C-9, but in view of the many mistakes that have been made by using this method, any conclusions drawn from comparison of rotations should be accepted with reserve. The best evidence for the absolute stereochemistry of the 26 morphine alkaloids is due to Jeger , who degraded thebaine (17) 27 to the acid (37) of known absolute stereochemistry . This elegant degradation is shown opposite (15) —> (37) and requires no further comment. Finally Barns has been able to refine his X-ray data to enable him to distinguish between the two possible enantiomers, 28 and his results are in agreement with those of Jeger, and show that the relative and absolute stereochemistry of morphine and codeine is represented by (20). 14.

The biosynthesis of opium alkaloids

At the present time some 2,000 alkaloids have been isolated and characterised 2(-). They fall into many different classes, with a great variety of structural types which provide a challenge to chemical synthesis and which have stimulated much speculation of possible biogenetic routes. With the advent of readily available labelled organic compounds, these speculations could be put to the test experimentally, and only since that time has real progress been made. The present position in general alkaloid biosynthesis has been 30, 31 extensively reviewed . The morphine alkaloids were a natural choice for early investigation by these techniques because a vast amount is already known about their extraction and chemical transformations, and both alkaloids and plants are easily available, so that now a very complete picture has emerged. The earliest 32 theories were due to Winterstein and Trier who suggested that the aromatic amino acids phenylalanine (38), tyrosine (39), and dihydroxyphenylalanine (dopa) (40), were the precursors of simple benzylisoquinolines.

C NH2 (38)

OH

NH2

NH2 (40) (42) 15. 33 Later, Robinson suggested that all the isoquinoline alkaloids could be derived from a common hydro-aromatic unit (41) 3,34 via the intermediates shown (12)(43). In later publications he recognised that morphine was in principle derivable from the laudanosine skeleton (44). At that time no definite mechanism was 35 proposed except the suggestion that it was oxidative and possibly "homo-polar". 0 HO

NMe

(44)

To overcome the supposed difficulties inherent in the morphine oxygenation pattern, an intermediate (45) in morphine biosynthesis was proposed, with subsequent bond migration, whereas sinomenine (46) was thought to be derived directly from a 34 laudanosine type (44) .

HO w HO

—> (20) 16.

The biosynthesis of tyrosine and phenylalanine

Photosynthesis COOH 1COOH 1 C - (P. 2 = o

CH2 3 Glucose (48) ---> HO 4 —H CHO 5 J J. ••••-••••-- OH H- - OH 6 OH OH 7CH20 ® (50) CH2O (49)

COOH COOH 7 HO COOH

HO , OH OH OH OH (47) (51)

COOH COOH COOH 1 4-) 2

OH 0;) , 0 COOH

OH OH OH (52)

NH2

CH2CHCOOH CH C00001-1 CH C.',OCOOH

(38) (54) (53)

NH2

CH2CHCOOH CFI2COCOOH

OH OH (39) (55) 17.

Subsequently there have been many mechanisms advanced for the coupling reaction and these will be considered later. For the moment suffice it to say that both tyrosine (39) and nor -laudanosine (44) are in fact incorporated into morphine in Papaver somniferurn.

Tyrosine and phenylalanine

There are two major pathways for the biosynthesis of 36 aromatic compounds; the first is based upon acetic acid units , and 37 the second, the important one for aromatic amino acids , is based 38 on shikimic acid (47) , which is in turn derived from carbohydrates. Briefly the sequence (48) —> (55) (see opposite) is as follows. 39 Glucose is produced in the plant by photosynthesis and 40 this is the precursor for both phosphoenol pyruvate (48) and 41 D-erythrose-4-phosphate (49) . These compounds are believed to condense (50), then cyclise to give 5-dehydroquinic acid (51) in the manner shown. 5-Dehydroquinic acid is then converted stepwise 42 to shikimic (41), chorisrnic (52) and finally prephenic acid (53). This may aromatise to phenylpyruvic acid (54) which serves as the precursor for phenylalanine (38), or it may be oxidised prior to aromatisation (55) in which case tyrosine (39) is produced.

1-Benzylisoquinolines

Although phenylalanine and tyrosine can both arise in nature from prephenic acid as indicated, it has been shown that the 43,44, former can be converted to the latter in vivo and that dopa can 45,46. 44,45,47 be derived from tyrosine It is also known that 3, 4-dihydroxyphenylethylamine (dopamine)(42) can be formed in higher plants by decarboxylation of dopa (40). These facts lend 18. weight to Winterstein and Trier's theory, and the probable mode of biosynthesis of the benzylisoquinoline papaverine (56) is therefore shown (40) —> (56). COOH HON H HO

NH2 NH2 HO HO HO (40) (42) (43)

(56) (57)

Proof of the essential correctness of this scheme has been provided by tracer studies. Papaver somniferurn was fed with (+_)_[2_14c].. tyrosine (39) and this was incorporated specifically into papaverine (56) 48 as was shown by its degradation . Evidence for the intermediate (57) was obtained by the same group when radioactive papaverine was 49 isolated from poppies fed with [1-14C]norlaudanosine (57) . A further example is provided by the biosynthesis of narcotine 50 (58; R=OMe) again from the equivalent of two molecules of tyrosine , 14 14 since both [2- C]tyrosine and [1- C]norlaudanosine were incorporated. 19.

—> < NH HO v 2 rto

(39) Me0,,

lvie (58)

The positions of the labels were checked by degradations. The 'extras lactone carbon was shown, by the incorporation of 14 [ C]formate, to be a C-1 unit derivable from the C-1 pool of the 50 plant . Similar experiments showed that hydrastine (58; R=H) is biosynthesised in Hydrastis canadensis from two molecules of 51 tyrosine (39) , but [1-14C]doparnine (42) was incorporated only 52 14 into the 'isoquinoline' half of the alkaloid . [2- C]Phenylalanine (38) was incorporated but with much less efficiency than tyrosine 53 showing that its conversion to tyrosine is minimal in Hydrastis Of course, the incorporation of a precursor does not necessarily mean that it is the biological intermediate in the plant, but incorporation, without scrambling, indicates that, if this is not the case, then the true intermediate must be chemically closely related to, 31,54 and derivable from, the precursor in the plant

The biosynthesis of the morphine alkaloids

Following from Robinson's initial proposals for the 3,34 biosynthesis of .morphine , there were a number of attempts 55 to predict the details of the process , and especially of the 56,57,60,61. 56 mechanism of the cyclisation step Cohen suggested

20. an ionic mechanism for ring closure of the irnine (59) followed by p addition to the resulting diosphenol (60) to give morphine (20) by obvious steps.

RON

HO —> N

HO HO OH (59)

All the other mechanisms considered involved radicals. 57 Schopf based his suggestions, involving ortho-para radical coupling, 58 on the wrong structure of Pummerer's ketone (61). Barton's insight into the mechanism of oxidative phenol coupling enabled him 59 to deduce the correct structure (62) for this ketone .

(61)

(63) o 0 •-\/- (62) 21. The biosynthesis of morphine alkaloids (Barton)

0 (65) if R, Me

RO i) reduction ii) -H20 NMe `1\TMe

(68) (17) R.1 o (80) if R, Me

neopine (69) (20) 22. 60 Acting on this Bentley briefly considered coupling of the diradical (63) but favoured his earlier proposal of radical insertion starting from laudanine (64). Ivie0

HO HO —> >(2O) Me

MeO (64) Me (65) MeO Oivie 0 6.31\de (or directly) H + It is interesting to note that he mentions the possibility of salutaridine (65) (vide infra) as an intermediate. The mechanism 61 suggested by Barton and Cohen followed logically from their work on Purnmerer's ketone, and was published along with suggestions for the biosynthesis of many other groups of alkaloids and fungal metabolites, all linked by a unifying reaction mechanism involving radical pairing at ortho or para positions, or similar oxygen carbon 30, 31 coupling. Much of this theory is now supported by tracer studies The scheme for the morphine alkaloids (66) —> (69) (see opposite) envisages ortho-para coupling of radicals formed by a one electron oxidation of the norlaudanosine derivative (66). R and R' are blocking groups, methyl or part of the enzyme surface, which exert the directing influence which ensures specific coupling. f3-Addition to the dienone (65) followed by trivial steps leads to the morphine alkaloids. An important alternative sequence for the continuation of the scheme from the dienone stage (65) has been 31,62. proposed It was suggested that, unlike the Pumrnerer's ketone, 23.

Alternative route

(65) (17) if R. R' =Me

HO (20) (69) (68) 24. 63 and narwedine precursors, the dienone (65) would not cyclise spontaneously but should first be reduced to the dienol (67), which leads to thebaine (17) by allylic elimination (67) —> (17) probably 31 through a phosphorylated intermediate . Morphine and codeine follow from thebaine, as shown, via the hypothetical intermediates neopinone (68) and codeinone (69) (see opposite).

Traccr studies

The experimental work which supports these theories falls roughly into three sections; studies with small precursors, with 1-benzylisoquinolines, and the later stages. It will for convenience be surveyed in that order rather than chronologically. Experiments with generally-labelled precursors have been ignored where they have been superceded by work with specifically-labelled precursors. All experiments were carried out with Papaver somniferum. 14 The simplest precursor fed was C0 and this was 264 incorporated into thebaine, codeine, and morphine . Rate studies showed clearly that the order of formation in the plant is precursor —> thebaine —> codeine —> morphine, which is in accord with theory. The same conclusion was reached by Battersby and 65 14 Harper from their rate studies with (+)-{2- C]tyrosine. More 66 conclusive evidence still was obtained when Rapoport re-fed the generally-labelled alkaloids morphine, codeine, and thebaine 14 isolated from CO 2 -fed plants. Whereas thebaine was incorporated efficiently into both codeine and morphine, codeine was only incorporated into morphine, and morphine itself was converted into 67 neither thebaine nor codeine. These results dispose of the theory 25.

Degradation of morphine

/vie°

COOH COOH

-\* NH2 NMe NH2 (39) (38) HO

Me MeO MeO + N-CH2 CH2C L.c0 Ac Me2

NMe2 ((*

Me

COOH CH2 COOH

i) 0s04 He ii) 104 iii) H2NOH Me0

II N- OH

HCHO

•••••••• (or by ozonolysis)

MeO tvle0

MeO CO2 COOH 26. that terminal methylation. steps (morphine —> codeine —> thebaine) give deactivated products for storage in the plants. (-4 42_14_ (39)65, 68, 69 , Lityrosine and (±)-[2-C]phenyl-14 69 alanine (33) have been shown to be incorporated specifically into the morphine alkaloid3 by two groups of workers. The labels in the alkaloids were proved to be at the predicted places by alternative 68, 69 degradations by both groups . (See opposite). These degradations are typical of those used in subsequent experiments and in the sequal 'specific incorporation' implies that non-scrambling has been proved by carrying out the necessary degradations. In identical experiments tyrosine was shown to be a more . 69 efficient precursor than phenylalamne , and this presumably indicates that phenylalanine must be converted into tyrosine before 44 incorporation into morphine . However, care is needed in interpreting the results of feeding experiments since anomolous results have been known; for example, glucose was found to be a 70 more efficient precursor than tyrosine in one case . Since glucose 66 should be the precursor of tyrosine, Rapoport ,influenced by his 14 degradations which showed C0 -derived morphine was not equally 2 71 labelled in both 'halves' , suggested that tyrosine must be incorporated by an aberrent pathway. This is against the main line 54, 68, 69 of evidence , and the inconsistencies can probably be explained in terms of pool sizes and the rate at which the specific activities of the various pools of intermediates will change after 54 labelled material has been absorbed by the plant . The idea that tyrosine is not the immediate precursor of both halves of the molecule, but only a close biochemical equivalent, gains support from results 72,73 recently obtained by feeding dopamine. Two groups have fed 27.

3, 4-dihydroxyphen-[l -14C]ethylamine (dopamine) (4.2) and found it to be specifically incorporated only into the moiety associated with the ethanamine bridge. This supports the following scheme for the early stages.

CO2 > Shikimic Acid (47) —> Prephenic Acid (53)

COOH 4.

NH2

COOH

0 (55)

HO OOH

HO

(43) 28.

It is of interest to note that phenylpyruvic acid (54) and p_-hydroxyphenylpyruvic acid (55) have been isolated from P. somniferu rn during the flowering period . A number of feeding experiments designed to investigate the coupling step have been made by feeding various 1 -benzylisoquinolines . The first intermediate to be fed was (+)41-14C]norlaudanosine (57) since this is believed to be the benzylisoquinoline first formed in the plant (vide supra). This was incorporated efficiently (3.9%) into morphine and into codeine and 45 thebaine . (c. f. tyrosine, 0.6 - 1.7%). The following year 14 [3- C]n.orlaudanosine was also fed and this too was specifically incorporated into all the major alkaloids. It is believed that methylation takes place before coupling to provide the directing 61 influence required for specific coupling . To test this 14 (+)43- C]norreticuline (70) was synthesised and fed, and equally 45 good specific incorporations were obtained , (morphine 3.2-3.6%) but even better incorporations (7.3 %) were obtained when 14 0)43- C]reticuline (66) was fed. Me HO

HO 3

H

OH (70) OH (66)

The 0- and N-methyl groups of the morphine alkaloids have been shown)by incorporation of the S-methyl group of methioninej to be derived from the C-1 pool 5. 29. 76 Similar results were obtained by Barton and his co-workers who 3 h 14 fed [1- H; N-methyl-1LC]reticuline, and proved that all t e C activity resided in the N-methyl group of the morphine isolated, and 3 14 that the H : C ratio was 83% of that in the precursor. Experimental difficulties render this last value somewhat inaccurate. Incorporation (0.13%) into morphine was higher than that of 14 (±)-[2- C]tyrosine fed under the same conditions. There is a possibility that reticuline (66) is 0-demethylated to a simpler benzylisoquinoline before incorporation since 0-demethylation has been shown to occur in the terminal 64,65,66. stages of biosynthesis To eliminate this possibility a quintuply labelled (t)-reticuline was synthesised with 14C in both methoxyl groups and the N -methyl group, and 3H at C-1 and with 14 C at C-3 as a reference. The thebaine isolated from poppies fed with this precursor was fully degraded and each label separately isolated. This proved that the precursor was incorporated intact, and that it is therefore the true biological precursor. A number of differently substituted radioactive 1-benzylisoquinolines have been 65 77 synthesised, tetrahydropapaverine (71) , protosinomenine (72) , 77 orientaline (73) , and the base (74)77 , but when fed, all were 45,77 found not to be incorporated

Me• MeO HO HO

Med HO Me • Me

N/vie

MeO HO MeO

OMe Olii.e OH OMe (71) (72) (73) (74) 30.

This proves that the terminal O-demethylation steps are specific and cannot be applied to these simpler compounds. 61 At the time the coupling theory was suggested the alkaloid reticuline was unknown. It was first isolated from 78 Anona reticulata as the dextrorotatory form . The levorotatory form has the same absolute configuration as morphine, since 78 (+) -reticuline is methylated to afford (+)-laudanosine of known 22, 23 27 absolute configuration and morphine has been shown to have the opposite configuration. (-)-Reticuline should then be the biological precursor of the morphine alkaloids, and Battersby's 77 group have carried out feeding experiments to test this point , using optically active precursors. They synthesised and resolved 00- dibenzylreticuline labelled with 14C at C-3, the 4'-methoxyl and 3 N-methyl carbons, and with H at C-1. Debenzylation afforded the enantiorneric reticulines which were fed. Both were incorporated 14 to about the same extent (ca. 570 into morphine) with respect to C but loss of tritium had occurred in each case. The tritium loss was much greater for the (+)-reticuline than for the (-)-reticuline and in both cases proportionately more tritium had been retained in the morphine isolated, than in the codeine which again retained more than 77 the thebaine. To explain these facts Battersby suggests that an equilibrium exists in the plant between (+)-reticuline (75) and (-)-reticuline (76) via 1, 2-dehydroreticuline (77).

1\deO N Me

HO

N-Me e:.-) Me X "`" MeO OH (76) OH (77) OH (75) 31.

This explains the equal incorporations and the loss of tritium. Tritium transfer in the oxidation-reduction steps is proposed to account for the small amount of tritium retained (13-13%) in the alkaloids derived from (+)-reticuline. Since morphine is further along the biosynthetic pathway than codeine and thebaine, that found in the plant at any one time must have been synthesised from (-)-reticuline at an earlier time than the codeine or thebaine with which it is isolated, hence it could be expected to retain a greater proportion of its tritium. C 1,2-Dehydroreticuline chloride was fed to test the equilibrium theory and this was incorporated extremely efficiently (10%) into morphine. In earlier experiments with optically active 77 norlaudanosines the (+)-epimer (31), which corresponds to morphine with respect to absolute configuration, was incorporated to a much lesser extent (0.4%) into morphine than the (-)-epimer 77 (33) (4-6%). These results were rationalised on the basis of the above findings by assuming that 1, 2-dehydroreticuline (77) is an obligatory intermediate between (-)-norlaudanosine and (-)-reticuline

HO /vie0,.\ MeO

HO HO7 HO --,‘ • > es ------> NH NMe NMe

HO Me OH OH (33) (77) (76) 32.

It seems reasonable to conclude that (-)-reticuline is the real biogenetic intermediate and in fact the isolation of 30 (-) -reticuline from opium has been reported7'. Later work showed this to be a mixture of (+)-reticuline (60%) and (-)-reticuline (440%) and it is suggested that the "missing" proportion of the (-)-epimer has been used for morphine synthesis. This suggestion was supported 1/1 81 when (±)-[3- -C]norlauclanosine was fed to flowering plants and the derived alkaloids halved, so that the (+)- and (-)-reticuline present could be measured by the method of isotope dilution analysis. This showed that the (+) -reticuline pool carried six times the activity of the (-)-reticuline pool; however the activities were found to be the same for both reticulines when the method was applied to seedlings 14 81 fed with CO . It was suggested that a different biosynthetic 2 82 pathway was operative in seedlings , but Rapoport and his 83 co-workers isolated reticuline containing much more activity than the thebaine isolated with it, from both flowering plants and 14 seedlings which had been fed for a short time (21/2 hrs.) with CO2. 61 According to Barton and Cohen's theory reticuline should couple to give a dienone (65). This important intermediate 84 was synthesised from thebaine (17) as follows, (17) —> (65). 33.

Reduction of thebaine with sodium in liquid ammonia gave phenolic S5 dihyclrothebaine (78; lt=1-1). This phenol was acetylated (78; /1)--Ac) and oxidised successively with selenium dioxide and manganese dioxide to give the acetyl dienone.. Mild alkaline hydrolysis removed the protecting group and gave the stable crystalline dienone (65). Following this synthesis the isolation of the dienone alkaloid, 86 (+) -salutaridine from Croton salutaris was reported , which, by direct comparison, was shown to be identical with the dienone (65). Since that time (+)-salutaridine has been isolated from Papaver 87 88 39 orientalis , P. bracteaturn , and Croton balsamifera . The optical antipode, sinoacutine (79; R=Me), has been isolated 90 from Sinomeniurn acutum , and norsinoacutinc (79; 11=I-I) occurs 89 with salutaridine in C.balsamifera . The synthetic salutaridine (65) showed no tendency to undergo ring closure to the enol (80) either 76 in basic or acidic solution , so clearly the open form is thermo- dynamically the more stable, and therefore the modified pathway 1, 6; from salutaridine to thebaine via the dienol (67) looked the more probable. 34.

Reduction of the dienone (65) with borohydride gave epimeric dienols, salutaridinol-I and -II. Chemical support for the revised theory came from acid treatment of the dienols (67) which dehydrated to give thebaine (17) (30-40%). The reaction proceeded measurably at pH 5, and salutaridinol-I was dehydrated faster than salutaridino1-11 76 both at this pH and in hydrochloric acid . MeO MeO BH4C)

HO

NMe NMe NMe

Me© (65) MeO (67) MeO (17) 0 COH H+ In a series of feeding experiments by Barton and his 76 76 co-workers , and Battersby and his co-workers , salutaridine and salutadinol-I were shown to be precursors of the morphine 3 alkaloids. [1- 1-1]Salutaridine was incorporated efficiently into morphine (ca. 10%), and the radioactive thebaine isolated was reconverted into salutaridine which had retained 97% of its activity. Bromination removed all the label which had therefore not been 'scrambled'. [1, 7-3H]Salutaridinol-I and -II, in solution at pH 6, were separately fed to poppies, small samples being kept as controls. The conversion of salutaridinol-I into thebaine was 5.6 times greater in the plant than in its control, while salutaridinol-II was less effeciently converted into thebaine by the plant than in vitro. 35. Biosynthesis of morphine

CO2 shikimic acid (47) > prephenic acid (53)

, COOH COOH COON <--- - NH2. NH2

(55) (39) (38)

HOB CHO B I (42) (43) HO v NH2

HO

NH Me

1-10 OH (57) (66) 17

Me MeO

HO

N1Vie "NMe

Me (67) MeO (65) OH

RON

NMe

HO (69) (20) 36. 3 14 Feeding experiments with [7- H; 16- C]salutaridine and 3 14 [7- H; 16- C]salutaridinols-I and -II confirmed the above results. Sztlutaridine was very efficiently (15%) incorporated into the morphine alkaloids, and salutaridinol-I was converted into the morphine alkaloids (7%) 14-18 times as efficiently as salutaridino1-11 (0.4%). The obvious conclusion is that salutaridinel-I is the true biological intermediate. It was also noticed that some loss of tritium from C-7 occurred during the conversion of thebaine into codeine in the plant and this is taken as evidence for the intermediates neopinone (68), 76 and codeinone (69) in the terminal steps . Saluta.ricline has been shown to be present in small amounts in the flowering poppy. It was detected by the method of isotope dilution analysis using the total alkaloid extract from poppies 14 76 fed with either [3- C]norlaudanosine or [2-11C]tyrosine . The biosynthetic pathway for morphine alkaloids based on all the above results is shown opposite. 37.

The plrythrina alkaloids (1964)

(81) ( 8 a) (83)

Me H or or H Me 0

Me (86) (84) (85)

Me or O

Me0' ( 87) ( 89) (90)

Me or H

(88) 38.

The Structures and Stereochemistry of the 7.7thrina. Alkaloids

Erythrina alkaloids arc very wide spread in nature. There are 105 known species of Erythrina, and alkaloids occur in all the 50 which have so far been investigated 1. Interest was first aroused when it was discovered that extracts of these plants had a curare-like paralysing action92. This led Folkers to start a systematic search which resulted in eventual isolation of nine 93 alkaloids. He grouped them as free alkaloids, to which he gave the prefix erythr- (erythraline, crythramine, crythratinc, and a- and p-crythroidine), liberated alkaloids with the prefix eryso- (erysodine, erysovine, erysopine, and erysonine), and combined alkaloids with the prefix erysothio-. The combined alkaloids were shown to be esters of sulphoacetic acid, Roso CH C001-1., where c5,1. 2 2 110H is any one of the liberated alkaloids which can be obtained from the combined alkaloids by mild acid hydrolysis.

Since that time erysodine has been isolated as a glucoside95, and 96 Tomita has isolated dihydroerysodine from Cocculus laurifolus . In practice the alkaloids fall better into two groups (see opposite); the aromatic alkaloids and the cr -lactoncs, which though structurally very similar were not recognised as such for many years. Whereas all the structures of the morphine alkaloids have been known for some years, there remained 4.t the outset of this work (1964) some structural features of the Erythrina alkaloids which had not been settled. For example erythrarnine (8 2) is the dihydro- derivative of erythraline (81) but the remaining double bond could be in either of the two positions which are shown dotted. A similar uncertainty existed for erythratine (83). 39 .

Erysodine (84) and erysovine (35) are isomeric mono- phenols derived from erysopine (86) but there was no evidence for the position of the methoxyl. Erysodine (87) is related to erysodine and therefore the same uncertainty concerning the methoxyl position existed for it, and also for dihydroerysodine (38) which also has one double bond of uncertain position. a-(89) and p-(90) Erythroidines, the cf-lactones, have well established structures. Evidence is presented in this Thesis which resolves all these remaining points and for this reason the chemistry on which the known structures are based is briefly reviewed. The earliest work was carried out by Folkers and his co-worker who established the formulae and functional groups of the erythroidines. The a- and p- forms are isomeric and they suggested that the p-form, the more stable isomer, may be produced from the a-form only during the isolation of the alkaloids. The n-form has been predominantly used in the structural determination. It was shown that acid treatment of p-erythroidine resulted in three isomeric compounds; des -methoxy-, apo-, and isoapo-erythroidine, formed in that order with increasingly forcing conditions, all of which had lost the elements of methanol. On the basis of this and the results of methylation and subsequent oxidation reactions, the 97 structure (91) was proposed for p-erythroidine. 40.

Similar experiments by the same group established the formulae, functional groups, and interrelationships of the aromatic 98 alkaloids, and the general structure (92) was proposed for these, 99 e.g. erythraline was represented by (93) . Although these structures proved to be incorrect the experimental facts on which they were based remain true and the structures served as a stimulas for many of the degradative experiments which were later performed. Boekelheide and his co-workers, who investigated the erythroidines, and Prelog and his co-workers, who worked with the aromatic alkaloids, were responsible for most of this later work. 100 They deduced the correct structures in 1951 (aromatic alkaloids) and 1953 (erythroidines)101 for the two types. Most of the aromatic alkaloids have been shown to differ only in the nature of the groups attached to oxygen in ring D. The following inter-relations have been established. Erysodine (84) and erysovine (85) and erysopine (86) have all been methylated to give the same compound, called erysotrine (91), which on oxidation 102. gave 4, 5-dimethoxyphthalamide (92) That erysodine and erys ovine possess alternative arrangements of hydroxyl and methoxyl groups was proved by ethylation of each followed by oxidation which gave 4-ethoxy-5-methoxyphthalic anhydride (93) in 99 both cases . 41.

Me (84; 85) or H

MeO

Me

Et Me

(93) (91)

HO I N j HO V

Med".. (86) Me

(9 2)

Oxidation of erythraline (81) afforded N-methy1-

4,5-methylenedioxyphthalamide (94) 103 and the alkaloid has been 1 0 related to the other aromatic alkaloids in the following way0 Erythraline (81) and erysodine (84) were treated separately with acid to remove the aliphatic methoxyl group. The products were hydrogenated and the D rings converted into a dimethoxybenzanoid system. The same product (95) resulted from both. Dihydro-derivatives have been obtained by partial hydrogenation of the corresponding dienes. 42.

(94)

Me

Me

(95) (84)

The evidence for a -lactone in the erythroidines includes its 101,104, chemical properties,- i.r. spectrum and its =reduction with 101 lithium aluminium hydride The evidence for the heteroannular diene system will be considered next. Its presence was surmised from its 105 characteristic absorption maximum in the ultraviolet at ca. 235 rap., which was drastically lowered by hydrogenation to the corresponding di- and tetrahydro- derivatives. Furthermore the presence of the allylic methoxyl was deduced since mild acid hydrolysis results in a des -methoxy derivative (vide supra) lacking the elements of methanol, and showing strong absorption at 100,101 313 mil indicative of the triene (96)

N--

(96) iVie0

43.

Stronger acid converted these trienes into isomeric 100,101,104,106 compounds by what is called the apo- rearrangement By analogy with known carbonium ion rearrangements the reaction can be represented as below, (96) —> (97).

=NM

(96) (97) With the aromatic alkaloids the ether linkages in ring D arc broken during the reaction, so that the product in every case is apo-erysopine (98). Both the asymmetric centres arc destroyed by the rearrangement.

(98)

Proof of the structures of the apo-products lies in their physical „1.07 and spectral properties and in the isolation of 7-carboxyisatin,(99). and 2-aminoisophthalic acid (100) from the oxidation products of Apo- 101 13-eryth::oicline (101) , which show the points of attachment to be • at C -1, C -7 of the resulting indoline nucleus. Hofmann exhaustive methylation was also carried out and the intermediates 107,100 characterised . Isoapoerythroidine is the more stable 101 conjugated double bond isomer (102) .

14.

''•,..V.'\. 0 0 '

O' ''''`. \/ H+ [o] > / (90)

COOH 2

COOH

(100) (102)

The second characteristic type of reaction of the 109 Erythrina alkaloids again involves aromatisation of ring A . Two instructive examples arc considered below. When the 101 lithium aluminium hydride reduction product (103) of 110 p-erythr oidine was subjected to Hofmann degradation , ring A became aromatic (10,1).

HO

HO

Me -(103)(103)

4 -Methoxyphtlialic anhydride (105) was isolated from the products of oxidation of the diol (104). This provided the sole degradative evidence for the position of the aliphatic methoxyl in the Erythrina alkaloids. When the same reduction product (103) was partially reduced to its dihydro-derivative (106) before exhaustive methylation 110 ring A became aromatic (107) with the loss of the methyl group 45.

Synthesis of apoerysopine dimethyl ether

COOH

2 Cu 02 [01 02 ----> COOH

OMe OMe OMe

COOH

Ni/H2 NO2 H 0OH COOH

MeO MeO OMe OMe OMe

i) LiA1H4 ii) PB.r.5 iii) OHU

Methylate (98)

OMe

Belleau's synthesis Me

Me• Me H3PO4 > LiA1H4

(95) 6.

(107) i) H2 2 Hof. 0 3 HO (109) (108)

Hydrogenolysis of the allylic hydroxyl group followed by exhaustive Hofmann elimination of the nitrogen led to the alcohol (108). Ozonolysis of this product gave methyl ethyl ketone (109) and this is only compatible with the structure (90) for the g -lactone. To explain the two typos of reaction leading to aroma- tisation of ring A it seems not only reasonable but necessary that C-5 is a spiro carbon, the aromatisation involving cleavage of either the 5 - 9 or 5 - 13 bond. Hence the structures (t 31) - (90) were deduced. These deductions are supported by a synthesis of the 111 dig ethyl ether of apo-erysopine (see opposite) and a later 112 synthesis of apo-13-crythroidine , both of which were shown to be identical with material from natural sources, and by various 113 syntheses of degradation products with the spiran centre intact, which predominantly follow the general pattern set by the first ll4 approaches by Belleau (see opposite).

47.

Absolute stereochemistry of(3.-.ethroidine

HO' I

i) H2 Hof. ii) H3PO4 Me0 MeO (103) ii) Hof. H2

is tns

H2 • NMe2 Et OMe cis and trans

Hof. 4 cis and trans

i;t OMe

\\\0\

e HOODc) HOO cis and trans OMe 48.

The stereochemistry of the Zigraissim. alkaloids

The interconversions of all the aromatic alkaloids (except erythratine) establishes a common configuration at C -2 and the Spiro (C-5) atom, and the conversion of a- into p-erythroidine has been reported 5. The first insight into the stereochemistry of 116 these alkaloids came from X-ray studies on erythraline hydrobromide, which was shown to have the structure (110) (in the crystal) with the relative stereochemistry indicated. This was followed by the suggestion, based on molecular rotation and o.r.d. curves of certain derivatives, that the erythroidines and aromatic 117 alkaloids have the same configuration at the Spiro centre . The same authors were however unable to effect a chemical 117 correlation . The absolute stereochemistry at C-3 of the erythroidines was then established by degrading the lithium aluminium hydride product (103) to (3S)-3-n-2ethoxyadipic acid (111)18 (see opposite).

Me0

o(1n) (110) (112)

Finally, X-ray studies119 on dihydro-p-erythroidine (112) established the relative stereochemistry for the erythroidines and confirmed the aboolute stereochemistry as well, hence (110) and (112) represent the absolute stereochemistry of the two types of alkaloid. It is of interest to note that the X-ray studies enabled the double bond to be placed between C-1 and C-6 rather than C-6 and C-7. This is

the first evidence on the bond position for dihydro derivatives 49 .

The Biogenesis of the Erythrina Alkaloids

Following the publication of the correct structures for 101 the Erythrina alkaloids 100, there have been a number of 61, 101, 120,121,122, 123,124, 125,126 proposals for their biogenesis. Since there is, to date, no published evidence from feeding experiments with labelled compounds by which to judge the validity of these proposals, they will all be briefly reviewed in chronological order. 120 In 1952 Witkop and Goodwin , in a paper on the oxidation of R-alkylphenols suggested that the key steps in the formation of the Erythrina system could be the oxidation and cyclisation of the tyrosine derivative (113) as shown below, (113) —> (114). H2 Ha N— P

OH HO R.'

Further steps and the nature of the residue R were not specified. 121 Wenkert in 1953 proposed that the oxidation of the symmetrical intermediate (115), itself derivable from the equivalent of two molecules of dopa, could lead to the Erythrina alkaloids. Nitrogen-phenol oxidative coupling (116) followed by phenol-phenol coupling of unspecified mechanism would give the basic Erythrina skeleton (117). 5 0 .

1 O ff/ NH 2 C HO

07 (115) 0

In his paper formulating the correct structure of 101 p-erythroidine, Boekelheide also took the opportunity of outlining his thoughts on biogenesis. His main point was that 13-erythroidine (90) could be formed in nature from erysopine (86) by oxidative 127 fission (118) of the general type first proposed by Woodward for the biogenesis of strychnine and later applied by Robinson to . 1 28 emetme . This process has since been called Woodward fission and was thought by Boe kelheide to involve oxidation, decarboxylation and lactonisation as shown (86) —> (90)

HOO D -

(86) (118) 51.

Biogenesis of the aythrina alkaloids (Boekelheide and Prelog)

HO

[03 HO

HO OH (115)

HO

sI (86) (121) (120)

NI(

MeO 52.

Boekelheide also suggested that erysopine could be formed from two molecules of dopa, but no details were given at 101 that tirne . However, in a review on indole alkaloids, 122 Boekelheide and Prelog do give a more detailed scheme (see opposite) involving the same symmetrical intermediate (115) 121 previously- considered by IiiTenkert . Their scheme is in fact substantially the same as his but the steps are given in greater detail, (115) —> (90). The pathway from erysopine (86) to 13-erythroidine (90) in this case was assumed to pass through a 123 dialdehyde (122). Later the same year Boekelheide , in another review, published the same scheme but with the additional intermediate (123) between (119) and (120) (as below) which perhaps makes more clear the nature of the second oxidation step.

O (119) OH (123) (120) 124 At about the same time Robinson had considered the Erythrina alkaloids in his lecture series "Structural Relations of Natural Products" and noted that they could be derived from dopa and drew attention to the degradation of the aromatic ring to obtain the erythroidines. He suggested that the oxidative steps required to obtain the dienone (121) from the symmetrical phenol (115) went via the mono-orthoquinones (119) and (124) as shown, (115) --> (121). The structure (124) is of course equivalent to (120) in Boekelheiders scheme. 53.

HO HO (115) [o] >

HO -0 (119) OH (123)

HO (121) HO (124)

125 Two years later Prelog published a slightly different scheme in which two molecules of dopa combine to give the betain (125) which is oxidised to the mono orthoquinone (124) which leads to the Erythrina skeleton as before.

(:), c 00% 0 COOS ON 1 —> 0 NH3 0 Ha c

(125) 54. 61 In the same year, Barton and Cohen fitted Erythrina alkaloid biogenesis into their general scheme for phenol oxidation in plants. This requires that the tetraphenol (115) should be suitably blocked, by methyl groups or an enzyme, to direct the oxidation in the required manner. Para-para coupling (127) of the diphenol (126) followed by a further oxidative step (128) and cyclisation gives the Erythrina skeleton (129) as shown,(126) —> (131).

HO

RO

OH (126)

/0] OH

RO

(131) 55. COOH CO2 (-)

(53) 2

0 CO2 co2 Rdn 040 0' CO2 H

rb t° co2 HO H i)—H20 aromatic ii) -CO2 alkaloids

reverse aldol O

-> ethroidines For the first time some indication is given of the intermediate (130) lying between the dienone (129) and the Erythrine, alkaloids represented by (131). 126 Finally in 1959, Wenkert took a differEnt approach to alkaloid biogenesis in that he derived a number of alkaloid types from prephenic acid (53) itself,rather than from the derived amino acids. So far, every example of Wenkert's theory put to the test experimentally has been proved wrong, but his scheme for the Erythrina alkaloids is given (see opposite) for the sake of completeness.

57. DISCUSSION Absolute Configuration at C-9 in the Morphine Alkaloids

The configuration of (+)-norlaudanosine (31) has been 77 related to that of (-)-tetrahydropapaverine of known absolute 23 configuration . The absolute configuration at C-13 of morphine has 26 been determined chemically and the absolute configuration of the 28 molecule as a whole has been established by X-ray crystallography . From these results it followed that the configuration at C-1 in (+)- norlaudanosine should correspond to that at C-9 in morphine. 77 However, Battersby and his co-workers reported an in vivo incorporation (4%) into morphine of (-)-norlaudanosine which was, unexpectedly, much greater than that of the (+)-enantiomer (0.34%) It was decided thea.efore that a direct stereochemical correlation at C-$ of the morphine alkaloids with the 1-benzyliso- quinolines would be worthwhile. The opium alkaloid (+)-salutaridine 8'1 (65) is both prepared from, and the precursor of, thebaine and must therefore have the same stereochemistry at C-9 as the other morphine alkaloids. It has been related to the benzylisoquinolines by cleavage of the same carbon-carbon bond that is formed in the biosynthetic oxidative cyclisation of (-)-reticuline, and the result obtained confirms the previous configurational assignment. 129 Cava et al. have shown that dienones of the proaporphine type (132) are readily cleaved, by sodium in liquid ammonia, to the related benzylisoquinolines (133).

Me • MeO

R. IMO MeO

C (132) HO (133) 2e 58.

It seemed that this method might usefully be applied to (+)-salutaridine, but it was anticipated that the presence of the phenolic hydroxyl might interfere with the reaction and that if this phenol could be methylated the reaction would proceed more smoothly. Accordingly, some of the known methods for the preparation of 130 codeine from morphine were applied to salutaridine, but without success. The action of methyl iodide on the phenolate ion, or of 131 trimethylphenylammonium ethoxide on the free alkaloid, gave predominantly quaternary salts, while the diene system precluded the use of diazomethane. Treatment of salutaridine with methyl 132 toluene-2 -sulphonate and sodium hydride in dimethylformamide did afford 0-methyl salutaridine (134; R=H) albeit only in moderate yield. Reduction of 0-methylsalutaridine, in dioxan as co-solvent, by sodium in liquid ammonia gave a complex mixture of products, rather than the almost quantitative cleavage which Cava et al. had obtained. Methylation of either the phenolic products or the total products gave, on examination by thin layer chromatography (t.l.c.), some indication that laudanosine (135; R=1-1) was a constituent of this mixture.

-R MeO M Me0Ts Na liq.NH3 Me

NMe NMe

OH CH2N2 Me

(135) 59.

It was found that t.l. c. examination of laudanosine on silica gel G (Merck) plates developed with methanol showed in ultraviolet light a characteristic blue fluorescence after brief exposure (1-2 sec.) to iodine vapour. This test was sensitive enough to detect quite small quantities of this base in complex mixtures. Reductions were carried out under various conditions in an attempt to obtain a leas complex mixture of products, but neither the use of different co-solvents, dioxan, toluene, or tetrahydrofuran, nor the absence of a co-solvent seemed, on examination by t.l.c., to offer any advantage. The effect of different metals, sodium, potassium, lithium, and calcium, was tested in a parallel series of reductions but again there was no visible improvement. Because of the complex nature of the reaction a radio-chemical method was used to confirm the existence of laudanosine in the products. [1-311](±)-Salutaridine76 (136; R=T) 3 was methylated as before to give 0-methyl-[1- 1-I]saluta.ridine (134; R=T) and this was reduced in tetrahydrofuran by sodium in liquid ammonia, and the phenolic products methylated with diazomethane. Repeated column and thin layer chromatography gave a small amount of material inseparable from (±)-laudanosine on t.l. c. Its rotation, [a]D = -79° (in ethanol) and -54° (in o chloroform), corresponded to that of (-)-laudanosine, [a] -90 D o 133 (in ethanol) and -55 (in chloroform) , but insufficient material was available for adequate conventional characterisation. The product was, therefore, diluted with a large excess of non-radioactive (t)-laudanosine. The specific activity of the mixture remained constant on repeated recrystallisation showing that the product was 60. indeed laudanosine. Further, the specific activity of the reaction product, calculated from that of the crystalline diluted material, 3 agreed well with that of the original 0-methyl-[ 1-1]salutaridine, so that the product must have been substantially pure (-)-laudanosine. The reductive cleavage was then carried out on a larger scale with inactive materials . On this occasion the total reduction products were methylated with diazomethane, no attempt being made to separate the phenolic products . Repeated chromatography as before gave (-)-laudanosine as an oil, and it was found necessary to resolve 134 (±)-laudanosine to provide an authentic (-)-laudanosine seed with which to induce crystallisation. The crystallisation gave (-)-laudanosine, [a] = -86° (in ethanol), m.p. 88-89° undepressed D on admixture with authentic material. This work is complementary to another chemical 135 correlation recently undertaken in these laboratories . The "biogenetic type" synthesis of (t)-salutaridine and hence (±)-thebaine, 84 by manganese dioxide oxidation of (t)-reticuline has been reported . The yield on oxidation was such that radiochemical techniques had to 135 be used to identify the product. A careful study of various oxidising systems enabled a method with reproducible results to be found, and further it was shown that under the reaction conditions used, the product was oxidised faster than the starting phenol, thus explaining the low yields. A stereochemical correlation between 135 (-)-reticuline (76) and (+)-salutaridine (65) was achieved as follows . (+)- and (-)-Reticuline were separately labelled with tritium and each was oxidised, with ferricyanide in aqueous sodium hydrogen carbonate, under the same conditions, and the resulting oxidation products diluted with (+)-salutaridine. The diluted material from the 61. oxidation of (-)-reticuline retained its activity on purification and conversion to thebaine (radiochemical yield 0.0044%), while that from (+)-reticuline lost essentially all its activity on recrystallisation. Therefore (+)-salutaridine arises from the oxidation of (-)-reticuline and not from (+)-reticuline.

MeO

HO

NMe NMe H Med' OH (76) 0 (65) These complementary experiments, the one breaking and the other forming the 12-13 bond, enable the absolute stereochemistry at C-9 to be assigned in thebaine (17) and hence in the other morphine alkaloids. This result is in agreement with the original assignments. 77 Since the completion of this work Battersby et al. have published their results from feeding experiments, with optically active reticulines, using Papaver somniferurn,and have given a possible explanation for the unexpected incorporations of (-)-laudanosine. (See review).

62.

Absolute Configuration at C-7 of the Salutaridinols 84 It has been shown that reduction of the morphine precursor salutaridine (65) with sodium borohydride gives two epimeric alcohols: salutaridinols-I and -II. Salutaridino1-1, but not its epirner, was found to be a biological precursor of thebaine (17) in Papayer somniferum76, so it became desirable to know the absolute (and hence, also the relative) stereochemistry at C-7 in salutaridinol-I. In principle, ozonolysis of either salutaridinol, followed by reduction of the ozonide (138) and hydrolysis of the reaction products (139) should give optically active glyceric acid (14C). Me

COON HO 7,--1(N/ 0 [NO OH H OH NMe 1 0 CH OH Me Me Me H6 H (137) H '11 (138) (139) (140)

If the salutaridinol has the configuration shown (137) then D-glyceric acid (140) should be obtained. A key step in this reaction sequence (137) —> (140) is the reduction of the ozonide (138). The success of the method depends on the presence of the methoxyl at C-6 preventing reduction at this centre to give eventually an optically inactive product, glycerol. In order to test the possibility of obtaining the desired mode of reduction, 2, 5-dihydroanisole (141) was prepared from 136 o anisole by Birch reduction . Ozonolysis in methanol at -20 , 63.

followed by borohydride reduction, should in theory give methyl hydacrylate (142) and propane-1, 3-diol (143). Although the isolation of these water soluble products proved difficult, the infrared spectrum of the crude reaction products did show evidence for both hydroxyl and ester groups. There was also evidence for double bonds (vide infra).

i) 03 OH H Me ii) BEI4 (141) (142) (143)

The propane-diol produced in this reaction hampered possible isolation of the ester fragment, so acetyl-phenolicclihydrothebaine (144) was examined as a preferable model. Ozonolysis of this, in methanol, followed by borohydride reduction again gave a crude non-basic fraction which had bands attributable to hydroxyl, ester, and unsaturation, in its infrared spectrum, but methyl hydracrylate could not be isolated from this mixture. Experiments with 137 authentic methyl hydracrylate led to the conviction that some dehydration was taking place during the work up of the reduction products giving methyl acrylate (145). Thin layer chromatography showed this elimination to take place quantitatively on alumina or silica, and since t.l.c. on the crude reduction product also showed the presence of methyl acrylate, this was taken as good evidence for the reduction taking place at least to some extent in the required manner. 64.

MeO

Ac0

NM' 0 OH 0 > > Me0 Me0'' 1\de0."'

(140 (142) (145)

Attention was next turned to the problem of isolation of the three carbon unit from the degradation. Glyceric acid is a viscous oil, not suitable for characterisation and the possibility of isolating its methyl ester, a liquid, was only briefly considered. Experience with model reactions had impressed the need for a well characterised solid derivative. At the same time a sequence of literature reactions were carried out with a view to obtaining optically active D-glyceric acid for comparison purposes. 138 139 I, 2 5,6-Di-isopropylidine-D-mannitol (146) was cleaved by lead tetra-acetate and the resulting aldehyde (147) further 140,161 oxidised b: potassium permanganate . The potassium salt (118), which was obtained in poor yield, had [a]p +5°, whereas 141 o Keiti-Iwadare claimed [a] +23.7 and optically purity for this D compound obtained in the same manner. Clearly some racemisation could occur at the aldehyde (147) stage and so a method of avoiding this was sought.

65.

/S -0 — °X —CH Fb(OAc)4 KMn04 —> 2 0— BzH CHO COOK \ (147)

/NO— (146) (148)

--- 0 >s/

CHOH

IO )/KMn0 4 C 4

The action of periodate with catalytic amounts of 142 permanganate on 1,2- 5,6-di-isopropylidine-D-mannitol achieved this object, the potassium salt of the acid (148) now being obtained o in one step. The salt obtained in this manner had [a]p +40.8 and its optical purity was demonstrated by conversion into its brucine salt, crystallisation of this to constant rotation, and regeneration of the potassium salt. The isopropylidine derivative (150) was prepared from an authentic sample of methyl (+)-glycerate (149) with the intention of hydrolysing this with potassium hydroxide to obtain the (±)-salt (151), but any idea of using this procedure for isolation of the degradation product was quickly dispelled since the salt is both very deliquescent and the isopropylidine group is very acid labile. 66,

COOIVIe C OPiie COOK

OH

OH O (149) (150) (151)

Acid hydrolysis of pota,...qium isopropylidine-D-glycerate did, however, give optically pure D-glyceric acid which was isolated 1+3 as its calcium salt ([a] +13.6) and characterised as its D E-bromophenacyl esteracdp Next, the complete sequence (137) —> (140) was investigated. The conditions required for the hydrolysis of methyl glycerate were determined by experiments with methyl (+)-glycerate. Borohydride, which was to be used for the reduction of the ozonide, was found not to be basic enough, but 10% pota:7,sium hydroxide at room temperature for four hours was sufficient to give complete hydrolysis. The behaviour of (1.")-glyceric acid on various ion 144 exe-lange resins was studied and conditions for its isolation were determined. The preliminary experiments completed, a sample of o salutaridinol-LI was ozonised for 5 hours in methanol at -70 and the ozonide reduced with borohydride. No glyceric acid could be detected on t.l. c. at this stage even after repeated evaporation of the crude products from methanol, which would remove any borate 145 as the volatile methyl borate . The total crude product was hydrolysed but again no glyceric acid could be detected before, or after reasonable isolation procedures. It seemed that the yield of glyceric acid produced by the degradation (if any) was very small so the method of isotopic dilution analysis was applied. 67. 3 (+)-Salutaridine (65) was reduced by sodium lasorohydride to give 76 the epimeric salutaridinols, tritium labelled at C-7 . These were separately carried through the degradation as above and the products diluted with non-radioactive D-calcium glycerate in each case. The glyceric acid was re-isolated as its calcium salt, but definitive results could not be obtained because the calcium salt proved to be insoluble in both the aqueous and non-aqueous scintillation liquids used for counting. L-Bromophenacyl glycerate had been previously discounted for isolation purposes on account of its low specific rotation, but this is of no consequence if a radiochemical method is used, so this highly crystalline, easily soluble, compound now seemed ideally suitable for isolation. One further modification was made to the degradation procedure. It was considered possible that borate complex formation during the reduction could be in part responsible for the low yields obtained, so it was decided to replace the borohydride reduction by catalytic hydrogenation. The best conditions were ascertained by hydrogenation of glyceraldehyde, and of the ozonide prepared from dihydrothebaine, and were found to be reduction over Adams' catalyst in the presence of a trace of acid. Accordingly a sample of mixed radioactive dienols was ozonised at -70° in ethanol and the reaction mixture hydrogenated directly over Adams' catalyst. The total product was hydrolysed by alkali and the resulting acid esterified with k-bromophenacyl bromide. Inactive L-brornophenacyl-D-glycerate was added to the crude product and the ester isolated and purified by chromatography and crystallisation. This ester retained its activity after repeated 68. recrystallisation and must therefore have contained E-brornophenacyl- 3 D4 Hlglycerate (0.07%). In a similar repeat experiment the radioactive yield of ester was 0.00% and this was believed to be due to failure at the final esterification step. This esterification was found to be very sensitive to pH., the best results being obtained in the range 5.5-6.0 and no ester was formed if the pH was far from this range. Consequently, in subsequent degradations inactive D-glyceric acid was added after the hydrolysis step so that there would be enough ester to isolate, and so that the yield for the esterification (low and variable) could be calculated. This ester was then further diluted with inactive material and purified to constant activity. In this way degradation of salutaridinol-I was shown to yield D-glyceric acid (0.80%) while salutaridinol-II gave essentially inactive D-glyceric acid (see Table 1). Salutaridinol-I must, therefore, have the configuration shown (137). Racemisation at any stage during the isolation would result in loss of tritium and thus could not invalidate the conclusion drawn. The activity of the end product was compared with that of the starting alcohol to give the yield of glyceric acid formed in the degradation, allowance being made for loss of material in the preparation of the z-bromophenacyl derivative. To confirm these results it was desirable to carry out a corresponding pair of degradations using L-glyceric acid and ester for dilution, when the opposite results should be obtained. To do this a supply of L-glyceric acid was required. DL-Glyceric acid had been 146 prepared by a literature method from the metal ion catalysed oxidation of glycerolbya mixture of concentrated nitric and sulphuric 147 acids. Attempts to repeat the literature resolution of glyceric 69. acid using its quinine salt failed to give more than partial resolution, nor was the use of brucine, morphine, cinchonine, ephidrine, cinchonine methohydroxide, or quinine methohydroxide, any more successful. The L-acid was finally obtained by the Sni 148 diazotisation and deamination of L-serine , and was purified via its phenacyl ester. .R.epetition of the degradations, diluting with the L-acid and ester gave the expected result: radioactive L-glyceric acid being produced from salutaridino1-11 and not from its epimer.

Table 1. Degradation of the salutaridinols to glyceric acid I Salutaridinol I. II. I. II. - - Configuration of glyceric acid D D L L Yield on esterification (%)* 28 16 9 24 Yield of glyceric acid (%) + 0.80 0.06 <0.04 0.74

* Esterified with R-bromophenacyl bromide + Calculated by isotope dilution analysis

The low yields obtained in this degradation can in part be attributed to the possible modes of breakdown of the ozonide, Apart from the possibility of obtaining glycerol already noted, there is a second spontaneous process which can occur (152) —> (154) which would result in the loss of one carbon from the three carbon fragments required. There are well known analogies for this type 149 of process 70.

—> etc • CO2 +

(153)

OP.

4G-) / 0 0 CO O I O) 00 + HCOOH MeO •---7 Me° H H CHO (K3) H etc. (154)

The biological conversion of salutaridinol-I into thebaine (17) formally requires loss of the elements of water and might 31 involve a phosphorylated intermediate . U cyclisation involved a one step, SnZ displacement of phosphate then salutaridinol-11 (155) 150 and not its epimer would have the appropriate configuration at C-7. It seems likely, therefore, that an additional step must be involved in the biochemical process. Two possibilities must be considered, 71.

Me

HO

(156) (157)

MeO MeO

HO HO CD; XO Me NMe xo Me 00 H (156) (158)

First a direct displacement at C -7 in a salutaridinol-I derivative (156) by an enzyme functional group would give an enzyme bound intermediate (157) with the inverted configuration required for Sn2' ring closure. Second, a preliminary allylic (7 —> 5) rearrangement would give an isomer (158) having the correct configuration for Sn2 displacement at C-54

72.

Absolute Configuration at C-7 of Nudaurine

The degradation procedure for the salutaridinols, as finally worked out, ought to be generally applicable to dienols of the type (159) which would result in nature from reduction of the corresponding dienones formed by oxidative phenol coupling of methoxy-phenols of part structure (160).

R' Er R If R" R N .0.

tile© Me0 HO H (159) OH (160) 0 Towards the end of the work with the salutaridinols it became apparent that there was another dienol to which the method could be applied. The alkaloid nudaurine (161) was isolated from 151 Papaver aurantiacum in 1960 together with amurine (162), which 152 152 had earlier been isolated from P. amurense and P.croceum . 151, 152 Boit determined their formulae and functional groups and converted nudaurine into amurine by oxidation with active 151 1.53 manganese dioxide . There were two incorrect attempts (163) 154 (164) to deduce the structure of amurine before the correct structures of nudaurine (161) and amurine (162) were published 155 in 1965 by Dbpke and his co-workers 73.

+ >

H2 O

Me

OH OH /

OH OH (165)

74.

They based their deductions on n. m r. data and on a rearrangement (162) —> (165) which they recognised as analogous to the thebaine-thebenine rearrangement. The absolute stereochemistry at C-7 of nudaurine (161) can, therefore, be determined by application of the method used for the salutaridinols. Ozonolysis of nudaurine, followed by reduction of the ozonide and hydrolysis of the products would again give optically active glyceric acid. D-Giyceric acid (140) would be obtained if nudaurine had the configuration (166) at C-7.

CCOH i) 03 ii) Ha/Pd H OH iii) OH -

CHOH

Me ( 1 4 0 ) HO H (166)

The yields of glyceric acid from this degradation were, as in the analogous case, very small (1 %) so the method of isotopic dilution analysis was applied exactly as before, again using the 156 R-brornophenacyl ester for isolation. Amurine was reduced with 3 lithium aluminium [ H]hydride to give nudaurine and its epimer (ca. 3 : 1) (henceforth called nudaurine-I and -II respectively) ••••••••• labelled at C-7. Borohydride reduction of arn.urine resulted in four products, the two minor ones being nudaurine-I and -II. The major products were presumably the result of reduction of the diene system 151 and were not further investigated. The reported oxidation of nudaurine-I was repeated to obtain arnurine with which to supplement 75. 156 the natura_. Inaterial A series of degradations were performed and the results are tabulated (Table 2). Table 2. Degradation of the nudaurines to glyceric acid

Nudaurine I. I. I. II.

Configuration of glyceric acid D D L D Yield of glyceric acid (%) * 0.83 0.87 <0.08 0.12

* Calculated by isotope dilution analysis

Clearly D-glyceric acid results from the degradation of nudaurine-I and not from its epinzer and hence natural nudaurine (nudaurine-I) must have the configuration shown in (166). The stereochemistry of the ethanamine bridge has been assigned by 157 Dilipke from o.r.d. measurements on amurine and by comparison with (+)-salutaridine.

Structures of some Erytikrin4 Alkaloids

As a prerequisite for biosynthetic studies on the Erythrina alkaloids, it was necessary to obtain samples of these alkaloids from plant material and to examine the alkaloid content of the available plants. During the course of this work quantities of erythratine (83), erythraline (81), and erisopine (86) were accumulated. These were the major alkaloids found in E.alz.s.uca, Wind. beans 5". The extraction procedure was 159 similar to that used by Folkers ; i.e. removal of fats by 76.

extraction with petrol ether followed by extraction with methanol and separation of the alkaloids frorzl this extract, except that isolation of the separate alkaloids was carried out by chromatography on alumina columns rather than by fractional crystallisation of salts. Erysopine (86) crystallises as the free base from a concentrated ethanol solution of the crude alkaloid mixture before chromatography.

Mee (81) OH (83) (86) Extraction of the crushed beans with cold dilute hydrochloric acid or cold methanolic hydrogen chloride did not appear to have any advantages. The hydrochloric acid used during works-up seemed acidic enough to hydrolyse the combined alkaloids, and the liberated alkaloids were obtained without further acid treatment. With supplies of eryth:z.atine available it seemed worthwhile to attempt to complete its structure elucidation. The outstanding problem was the position of its double bond, and since it seems reasonable on any biogenetic scheme that e5.-ythra.tine could be derivable from a dienone (167; 11, l,:an= H or alkyl), and ought to be the precursor of erythraline (81), it was predicted that it would have the structure (168). --•-••••-> Me0 (167) (31)

The n.m.r. spectrum (CDC13 , 100 'vie. /see.) of erythratine and its benzoate, supported by the appropriate spin- decoupling studies, support this structure. The oxidation of erythratine (169; R=H) with manganese dioxide gave the corresponding ap-unsaturated ketone (170) and this was reduced with sodium borohydrid.e to give a mixture of erythratine and its C-2 epin-er; epi-erythratine (171). The n.m.r. data for these epimers and erythratine benzoate (169; Il=COPh) are recorded (Table 3).

H (a) (169) 78.

Table 3. Nuclear magnetic resonance spectra

valueb (in CDC13L

* * + ON, Proton 1 2 3 4a 4e 14 17 o,....- OMe Erythratine (169; R=H) 4.44 5.72 6.40 8.37 7.70 3.27 3.41 4.15 6.72 Er ythr atine benzoate (169;R=COPh) 4.31 4.31 6.06 8.17 7.66 3.20 3.45 4.13 6.74 Epi-erythratine (171) 4.22 5.59 6.38 8.27 - 3.55 3.44 4.15 6.66

Coupling Constants (c./sec.)

* * * * Protons ij 1,2 2,3 3,4a 3,4e 4a.,4:e

(169; R=H) 3.2 7.5 12.5 4.0 12.75 Jij in (169; R.=COPh) - 12.0 5.0 12.5 (171) - 3-4 12.0 4-5 12.0

• a and e represent axial and equatorial + identified by irradiation of benzylic methylene group • Quartet J = 1.4 c/sec.

Firstly, from the n.m.r. spectrum of erythratine, it is possible to exclude the alternative structure (172).

(172) 79.

The olefin signal at 4.4r was complex, but on irradiation at 5.72r it gave an ill defined triplet, and irradiation at 7.78'r gave a sharp doublet. While the signals could be assigned to the structure (172) if H(b) absorbed at 5.72't (next to nitrogen) and H(a) at 7.78T- , irradiation of either position should then result in the olefinic signal collapsing to a triplet, not to a doublet and a triplet. The data fit exactly for the structure (169; R=I-I) assuming J = 3.2 c. /sec. (doublet splitting). The small triplet splitting 12 would then arise from allylic (1, 7) coupling. The signal at 5.72't moves downfield to 4.31'rin the benzoate confirming that it is due to the proton on carbon carrying oxygen. Secondly the presence of the partial structure 1 2 3 4 R.R .R . C -CH -CHOMe-CHOH-CH = CR R can be inferred from 2 the n.m.r. spectrum without any prior knowledge of the structure. Irradiation of the olefinic signal picks out the proton at C-2 and irradiation of this affects the signal from the proton at C-3, and so on. By irradiating all these protons in turn the various coupling constants given in Table 3 were obtained. The mass spectra of erythratine and epi.err:,v3f4LIB, confirm the assignment of the ethylenic linkage to the 1,6 position since the base peak in. each spectrum corresponds to the loss of 58 mass units as shown, (173) —> (174).

MeO

OH (173) OII (174)

80.

This retro Diels Alder cleavage can also be seen as the major fragmentation in the a, n-unsaturated ketone, erythratinone (170), and in this case the ketone formed then loses a further 28 mass units (CO). The other major fragmentations in ,:11 Erythrina alkaloids are t72,e lor3s of 15 units (Me) and of 31 units (Me0). >

Me0 MeO (17C) 0 0 It remains to determine the stereochemistry of erythratine. The "biogenetic type" conversion of erythratine to erythraline (168) —> (81) has been achieved by treating erythratina with tosyl chloride, or better mesyl chloride, in pyridine at room temperature. The allylic F-.ulphonate is spontaneously eliminated to give the diene. The erythraline so formed is identical in every respect to the natural aPsaloid and so erythratine must have the same configuration at C-2 and C-5 as the rest of the Erythrina alkaloids. 25 Mills' rule states that the allylic alcohol (175) is rzo laevorotatory than its epirner (176).

(175) OH (176) Although this is only an erapirical rule there are no known exceptions. The specific retP,tiors of erythratine and epi-erythratine are +145°, and +280° respectively and therefore erythratine is the more laevorotatory and should have the structure (169; R=II). 81.

The infra-red spectra in dilute solution (CC14) of epi-erythratine and erythratine showed hydroxyl bands at v 3556 and 3605 cm. , respectively, confirming the above max. assignment since the strong hydrogen bonding in epi-erythratine is 160 consistent only with a cis arrangement of hydroxyl and methoxyl groups. Examination of molecular models shows that the distance involved in the equatorial-pseudoequatorial system of erythratine precludes the possibility of hydrogen bonding, whereas in the equatorial-pseudoaxial system of epi-erythratine hydrogen bonding is quite feasible. Treatment of either epimer with hydrochloric acid resulted in an equilibrium mixture of the epimers containing ca. 85-90% erythratine, as would be expected for the more stable di-equatorial system. Finally, with the aid of models, it can be seen that all the coupling constants for erythratine and its epimer given in Table 3 are in accord with the structures (169; R=H) and (171) respectively. Erythratinone (170), prepared from erythratine, was found to correspond on t.1. c, with an E. crista-,galli alkaloid 161 which we had hitherto assumed to be erythramine (82), a known constituent of this plant. Erythratinone was then isolated from the alkaloid mixture in pure form by Dr. D. Widdowson, (who was also responsible for its first synthetic preparation and its reduction to the epimeric alcohols) and it was shown to be identical with the 162 synthetic material. Erythratinone (170) is then a new alkaloid of ErZthrina crista-galli and its isolation provides support for the biogenetic steps shown (170) —> (81). 82.

(170) (168)

(81)

It will be seen from Table 3 that definite assigments have been made for the two aryl signals, In all three compounds one of the aromatic signals was shorter and broader than the other indicating some fine splitting. The proton at C-17 (169) could be split by long range coupling with the benzylic protons at C-11, and indeed irradiation in the region of the spectrum corresponding to the benzylic protons (7. 25 't) made this signal sharper and equal in height to the other aryl signal, which was of course unaffected. Since the proton at C-14 cannot be coupled the signals can be assigned with certainty. It seemed possible that this information could be used to solve another outstanding problem: the structures of erysodine (84) and erysovine (85). A sample of erysodine had earlier 163 been isolated from some beans of E. crista,-Lalli , and its n. m. r. spectrum also showed one aromatic proton to be coupled (Fig.1). 83.

H H14 H17 14

3.24 3.35 3.24 3.35 Fig. 1. Fig. 2.

This sample was then deuterated in D zO/D.M.F. and the n.m.r. spectrum of the monodeuterated derivative (m/e 300) examined (Fig.2). It showed only one aromatic signal corresponding to H14. 164 Since it is known that exchange under these conditions only takes place at positions ortho or para to the phenolic hydroxyl, then the structures of erysodine and its deutero-derivative must be as shown., (177) and (178) respectively. H HO

MeO

(177) (178) MeO 165 A sample, reported to be erysovine (85), but which by t.l. c. and by examination of its n.m.r. spectrum (Fig.3) cou1,1 be seen to be a mixture (ca. 2 : 1) of erysovine and erysodine, was also deuterated under the same conditions as were used for erysodine.

H Erysodine H H 17 14 17 OMe OMe Erysodine

3.35 3.38 15 Fig.3. 814., Synthesis of dihydroerysodine (Mondonl

HO

HN-

K [Fe(CN) ] 3 6 (129) MeO OH (126) INaBH HON HO 4

MeO H Me0 , Ho H (180)

HO

Me

7% (182) MeO 60%

Med- MeO

(186) (185) OH (184) 85.

The n. m. r . spectrum of the deuterated derivative (Fig.4) showed, in this case, complete loss of the 3.35 signal (H of erysodine) and partial loss of the 3.15 signal (H of 17 14 erysovine). It might be expected that H14 of erysovine would exchange more sr owly than H of erysodine, the former being sterically 17 hindered by the A-ring. Thus the structure for erysovine (179) is confirmed.

(179)

162 Shortly after these results were published , Iviondon and 166 Ehrhardt reported the synthesis of dihydroerysodine (186), an 96 alkaloid of Cocculus laurifolus which establishes both the position of the double bond and the aromatic substitution pattern for this compound, and also determines the structure of erysodine since dihydroerysodine is obtained from erysodine by catalytic hydrogenation. The synthesis (see opposite) starts with the "biogenetic type" phenol coupling of the diphenol (126), as predicted 61 by Barton and Cohen . Reduction of the diene (129) was reported to be stereospecific, but repetition in these laboratories resulted in a 167 mixture of epimers (ca. 4 : 1) . A novel acid rearrangement of the dienoi (180) gave three products (181), (182) and (183). The major one (183) was further reduced to an allylic alcohol (184) of unspecified configuration and the hydroxyl group replaced by chlorine by treatment with thionyl chloride in pyridine. Catalytic hydro- genation of the chloride (185) gave (±)dihydroerysodine (186), which was resolved. 86.

If mesyl chloride had been used instead of thionyl chloride, it is believed that erysodine (177) (as its mesylate) would have been obtained as in the analogous conversion of erythratine into erythraline, which would have given Iviond.on the first synthesis of an alkaloid obtained from the Erythrina species. 100 Erysonine (87) has been related to erysodine so its structure (187) is now settled. There remains one Erythrina alkaloid with a structure which is incompletely defined: erythramine (82). It was not found possible to isolate this alkaloid from either E. crista-galli 161 or E. glauca although it is reported to occur in both species However Prelog1026" reports that catalytic hydrogenation of erythraline gave a dihydro-derivative identical with natural erythramine, so this work was repeated in slightly modified form and a sample of erythramine was thus obtained. The mass spectrum of this compound (m/e_ 299) showed base peaks corresponding to loss of 59 and 58 mass units which is characteristic of all compounds with the part structure (188). i.e. R = OH; R' = H erythratine R = OCOPh; R' = H erythratine benzoate R = H; R' = OH epi-erythratine RR' = 0 erythratinone 166 R = R' = H dihydroerysodine R = R' = H erythramine

This fragmentation pattern is not seen in the mass spectra of the dienes (189) e.g. erythraline and erysodine, or in the dienones (190).

87.

MeO (189) (190)

It is concluded that erythramine has the structure shown (191).

H (191) This is supported by n.m.r. measurements (CDC13, 100 Mc. /sec.) on erythramine, with appropriate spin-decoupling studies. The olefin signal (4.45''x) was a broad singlet which on irradiation at 7.72 C gave a sharper singlet with some indication of fine splitting, and irradiation at 7, 66'1 gave a sharp doublet. The AMX system of the proton at C-3 and the two protons at C-4 can clearly be seen and the coupling constants were measured. It was also seen that the proton at C-3 was coupled to the signal at 7.66rn and this establishes the position of the double bond. The complete data for the n.m.r. spectrum of erythramine are recorded (Table 4), cf. Table 3. 88.

Table 4.

Nuclear magnetic resonance spectrum of erythramine

'values (in CDC13)

* Proton 1 2,2 3 4a 4e 14 17 OMe

4.45 7.66 6.27 8.43 7.22, 3.4 3.4 4.15 6.74

Coupling constants (c.(sec.)

* * * * Protons i,j 3, 4a l 3, 4e 4a , 4e

J i, j 11.5 I 4.0 11.5

* a and e represent axial and equatorial

The structures of the aromatic Erythrina alkaloids which have now been fully defined are shown below together with the new alkaloid, erythratinone (170). <

0 (170) (191)

MeO

HO

(179)

89.

The Biosynthesis of the Erythrina Alkaloids

A cursory inspection of the published biogenetic theories for the Erythrina alkaloids (see review) reveals that, discounting the 120 incomplete suggestion of Witkop and Goodwin , there are only two 126 fundamentally different schemes. One, due to Wenkert , based on prephenic acid, in view of the current knowledge of alkaloid biosynthesis, seems improbable. The main line of thought, to which all the other authors subscribe, has two variants. All these theories invoke a tyrosine derived bis-phenethylamine (115), or a simple derivative, as a precursor. The Erythrina alkaloids then arise via oxidative cyclisations to a dienone of the general type (129)

Erythrina alkaloids

(129)

The variations within these theories concerns the order in which the two cyclisations necessary to obtain the Erythrina skeleton take place. Most of the theories predict that nitrogen-carbon bond formation takes place first to give a dihydroindole (192), which is further oxidised to the dienone (129).

(115) > (129)

OH (192) 90. 61 Barton and Cohen are alone in suggesting an alternative in which carbon-carbon coupling of radicals, formed by oxidation of the diphenol (126) is the first step (127), followed by oxidation to the quinone (128). Addition of the amino group to the quinonoid system then gives the dienone (129; R=10=Me). These authors were the first to recognise clearly the importance of methyl blocking groups in directing the course of oxidative phenol coupling, and because of this, their scheme appears to be more attractive than the other suggestions. In particular it accounts for the methoxyl group at C-3, which is present in all of the alkaloids bar one, and the hydroxyl at C -2 in erythratine (169).

OH (126)

(128)

OH (169) (129) 91.

Chemical support for the di-phenol (126) being the precursor of the Erythrina alkaloids was obtained when its oxidation by ferricyanide to the dienone (129; R=10=Me) was reported first by Scott and his co-workers 165, and later independently by 166 Iviondon and Ehrhart as part of their synthesis of dihydroerysodine (vide supra). In spite of the stress already laid on the importance of methyl blocking groups for specific coupling of radicals, Scott's group had first tried and, not surprisingly, failed to oxidise the 168 tetraphenol (115) to an Erythrina skeleton . Objections to Barton and Cohen's scheme have been raised concerning the order in which the cyclisations occur l6c/. In particular the major objection to the route is that examination of molecular models shows the difficulty of attaching the nitrogen atom to the quinone system, the distances involved and/or rigidity of the system being too great for the nitrogen to approach closely the point of attack. For this reason nitrogen-carbon radical coupling leading to the dihydroindole (192; R=H, R'=R"=/vie) followed by normal para-para oxidative phenol coupling to yield the same dienone (129; R=R1 =Me), is 168 considered by some to be a possible alternative There is however another possible biogenetic route for the formation of Erythrina alkaloids which has not previously been considered. In the course of considering possible biogenetic routes to protostephanine, all the possible modes of coupling of diphenolic 1-benzylisoquinolines of the type (193) (with R or R' = Me or H and R" or R"' = Me or H) were examined and it became obvious that the diphenol (194; R=H) could serve as the precursor of the Erythrina 170 alkaloids 92.

OR"' (193) OH (194)

This compound (194; R=H) has the trivial name N -norprotosin.omenine 34 since (194; R=Me) was believed by Robinson to be the precursor of sinomenine (vide supra) , although recently this has been shown not to be the case171 Para-para oxidative coupling of N-norprotosinomenine leads to the dienone (195). Fission of the 1-9 bond (see arrows) followed by reduction leads to the same nine-membered ring 61 intermediate (127) that occurs in the Barton and Cohen scheme and from this point the routes are the same.

OH (127) OH 93.

The object of the present work was to seek evidence, from appropriate feeding experiments, which would either support one of these theories or differentiate between them. 14 (±)-[2- C]Tyrosine was the compound chosen for the first feeding experiment (1965 season). It was fed by the 'wick' method to the two available species of Erythrina; E. crista-galli and E. rubrinervria and the plants harvested after 7 days. The alkaloids were obtained from the plants in this and all subsequent feeding experiments by a standard isolation procedure given in detail in the experimental section. Erythraline and to a lesser extent erythratine are the major alkaloids in both species and generally these were the only ones to be isolated and purified to constant specific activity. (±)-{2_14c-- jTyrosine was incorporated into erythraline in both species but was more efficiently incorporated into E. crista-galli (0.13%) than into E.rubrinervria (0.03%). For this reason all subsequent experiments were carried out using E. crista-galli plants. This result, apart from its general usefulness in demonstrating that feeding experiments under these conditions would (given the correct precursor) result in labelled alkaloids, provides evidence for tyrosine derived intermediates in Erythrina biosynthesis and, excepting the remote possibility of degradation of tyrosine to prephenic acid in the plant, allows the rejection of Wenket's 126 prephenic acid pathway. 172 Leete has very recently announced the results of his experiments in which he fed both tyrosine and phenylalanine to E.bertervana . He found that tyrosine was efficiently incorporated 94 . Synthesis of N-norprotosinomenine

CHO CHO OOH PhCH2C1 i)S0C12 ii)CH2N2 K2CO3 > H CHPh OMe OMe Me OMe (196) (197) (198) (199) MeNO2 V Me3413C1" CH COOH N a0A c 2 PhCH 2

(202) MeO cn2Ph L.A.H. Me (200)

COC1

CHPh OMe (201)

PhCH2Q.,

HC1 Me fa) Me O PhCH PhCH2 2 (205) (204) MeO NaBH 4

Modified method of Dr. Bhakuni185 * First prepared by Mrs. A.J. Kirby, to whom we owe our initial supplies 95. into P-erythroidine. The alkaloid was degraded to show that the labels were in the expected positions. Phenylalanine was not incorporated into 13-erythroidine and this suggests that Erythrina species cannot convert phenylalanine into tyrosine. Although there is no evidence about the stage at which the aromatic ring is degraded, the incorporation of two molecules of tyrosine into Verythroidine does provide the first evidence for "Woodward fission" in alkaloid biosynthesis. It has been shown that Woodward fission 173 does not play any part in emetine biosynthesis but it cannot yet be 174 ruled out for strychnine . The only other compound fed in the 1965 season was (±)-N-norprotosinomenine. This compound was 175,176 synthesised by conventional methods (196) —> (194) (see opposite). The dibenzyl ether was first prepared in thesdaboratories (by Mrs. A.J. Kirby) in connection with work on sinomenine biosynthesis. The conditions for isotopic exchange of the hydrogens ortho and para to the phenolic hydroxyls were investigated using the free base in deuterium oxide, dimethylformamide solution, with or without additional base. In either case there was complete exchange of three aryl protons (n.m.r. spectroscopy). The tritiated derivative was obtained by either method but in each case was accompanied by severe decomposition, not observed in the deuteration, and the product was only isolated as .a pure czystAlline hydrochloride after chromatography on silica. It was fed to E. crista-galli and incorporated into bot!a erythraline (0.048%) and erythratine (0.006%). These incorporations were real but small and no firm conclusions about Erythrina biosynthesis were drawn at this time. 96.

The following season the alternative precursor bis -(3 -hydr oxy -4 -methoxyphenylethyl) amine (126) was fed. All attempts to reduce the amide (204) with lithium aluminium hydride under a variety of conditions failed. Reduction of this amide using 169 diborane has subsequently been reported . The problem was overcome in these laboratories by Dr. D.A. Widdowson who redesigned the synthesis to give a tertiary amide (209) which was easily reduced by lithium aluminium hydride. Removal of the protecting groups by catalytic hydrogenation gave the symmetrical 177 phenol (126) . PhCH2O PhCH2O PhCHO NHz > N=CHPh MeO Me0 (203) (207) L.A.H. PhCH2O

NHCH Ph IvIe0 2 (208)

PhCH 0 2 0 (208) + ,./CH Ph 11— 2 PhCH 2 MeO OMe (199) (209)

CH2Ph L.A.Hi PhCH2 CH Ph H /Pd / 2 2 He Me

(126) (210) MeO CH2Ph 97 .

The phenol (126) was triiiated in D.M.F. /TOH solution after an experiment with deuterium oxide had established the conditions for complete exchange of the four protons ortho and Para to the phenolic hydroxyl groups (n.m.r. control). 3 The symmetrical [ H]bis-phenylethylarnine (126) was fed to a second year plant early in the 1966 season. Its incorporation into erythraline (0.0012%) and erythratine (0.0055 %) was smaller than that of the benzylisoquinoline precursor (194) (ca. 40 x smaller for erythraline). However it was found that on this occasion, and in other feeding experiments with second year plants, that the yield of alkaloids was smaller, and the proportions of the different alkaloids present were different from those found for first year plants of comparable size. Thus a parallel feeding of both possible precursors to first year plants was carried out. When, later in the season, the first year plants were large enough, both precursors were fed (two plants each) at the same time, and harvested after 10 days. Identical work-up procedures resulted in similar quantities of alkaloids being isolated from each feeding (see Experimental Section), which were purified constant activities. In this way a clear cut difference was observed in the incorporations; N-norprotosinomenine being efficiently incorporated into erythraline (0.24%) and erythratine (0.02%) while the incorporations of the bis -phenylethylamine were much smaller; erythraline (0.0043%) and erythratine (0.0022%). A confirmatory experiment about one month later in which the bis -phenylethylamine was again fed to a first year plant (1 plant) produced the same result, the incorporation into erythraline being 0.0042%. It seems clear then that the benzylisoquinoline is the true precursor of the Erythrina alkaloids. 98.

Earlier, a sample of erysodienone (129), synthesised 166,168,169 by ferricyanide oxidation of the symmetrical phenol (126), was tritiated by exchange of the C-17 proton under the usual conditions. Deuteration showed (n.m.r. control) that only the 3 17 proton was exchanged. (±)-[17- H]erysodienone was fed to a second year plant which was harvested after 6 days. The incorporations, (0.085%) into erythraline and (0.15%) into erythratine, provide good evidence for the dienone being a bio- synthetic intermediate but, since it occurs on both the suggested routes, its incorporation could not help to distinguish between them. To investigate further the inter-relation of the various aromatic alkaloids, erysodine was tritiated by exchange of the 17 proton under the same conditions as were used for its deuteration for structural studies (vide supra), and fed to a first year plant. It was efficiently incorporated into erythraline (0.154%) and erysopine (0.57%) showing that erysodine can serve as a precursor for both these alkaloids. Only feeding experiments with precursors labelled in methoxyl groups will provide detailed information about these terminal stages of biosynthesis. In the experiments so far demethylation cannot be ruled out and this leaves open more than one possible route. However these incorporations do indicate that further experiments would be worthwhile. T

s '-. ; ; T 4 „ HO

HO 99,

Although the incorporation of N-noprotosinomenine contradicts the early stages proposed in Barton and Cohenis theory, it does add weight to their suggestions about the order in which the coupling reactions occur, since the nine membered ring intermediate (127) is obligatory for a scheme involving a benzyl- isoquinoline, and nitrogen carbon coupling must then occur after this. Preparation of this key intermediate (127) is at present under investigation in these laboratories and it seems that the action of chromous chloride on the dienone (129) gives the required product

OH OH (126) (127)

It has obvious use both as a precursor and for attempts to obtain the dienone (129) from it by chemical oxidation.

The conclusion that N-norprotosinomenine is the precursor of the Erythrina alkaloids demonstrates once again that a "biogenetic type" synthesis achieved in the laboratory, even if the yield is as high as 30-40%, provides no evidence for the same synthesis taking place in the plant. It would be interesting now to subject N-norprotosinomenine to chemical oxidation by one electron oxidising agents. 100. 167 Some interesting results have been obtained from 3 feeding [2- H]erythratine and its epimer. These epimers were obtained by [3H]borohydride reduction of erythratinone and were fed at first to second year plants. In a parallel feeding, epi-erythratine was incorporated ten times more efficiently into erythraline (0.43%) than was erythratine. A repeat parallel feeding to first year plants confirmed this result. This indicates that epi-erythratine is the true biological precursor of the Erythrina alkaloids. Erythratine could result from either non-stereospecific reduction of the ketone or from epimerisation either in the plant or during work up of the alkaloids. Alternatively, two different enzymes control the reduction of erythratinone. 3 Finally [2- 1-1]erysodienol (211) prepared from erysodienone (129) by [3H]borohydride reduction was fed to a first 167 year plant but was not incorporated . It appears therefore that either erysodinol is not a precursor or that the label is lost during the subsequent transformations, in which case a 1-2 hydride shift (214) —> (212) can be ruled out. The enol (213) however could still be an intermediate. 101. Biogenetic scheme for the E ythrina alkaloids

HO

OH OH

HO [0]

MeO

OH

MeCC OH

HO

_ Erythramine Erysovine? HO a and 13 Erythroidine Erysovine? 102.

The results of feeding experiments with E. crista-galli have been tabulated (Table 5) for comparison and a plausible biogenetic scheme given based on these results. Table 5. Incorporations .ed to Er hri as.....g.... rista- alli

Plant Feeding Erythraline Erythratine Year age time /0 % (years) (days) (+)42..14c1- 1965 Tyrosine 0.12 - 1 7 1965 (-1)45,2', 6' -31-1]- N-norproto- sinomenine 0.048 0.006 1 5 1966 it 0.24 0.020 1 10 1966 Bis 42,6-311]3- hydroxy-4- methoxyphenyl- ethyl)amine 0.0012 0.0055 2 6 1966 fI 0.0043 0.0022 1 10 1966 ii 0.0042 - 1 10 1966 (1)417-31-1}- Erysothenone 0.085 0.15 2 6 1966 [2-3H]Erythra- tine 0.043 - 2 6 1966 If 0.04 - 1 6 1966 [2-3I-1]Epi- erythratine 0.43 - 2 6

If 1966 0. 27 - 1 6 1966 (±)-(2-31-1)- Erysodienol 0.00 0.00 1 10 1966 [17-311]Eryso- dine 4' 0.154 - 1 10

Incorporated into Erysopine 0.57% 103.

EXPERIMENTAL

-Methyls alutaridine (+)-Salutaridine (1 g.) in dimethylformamide (10 ml.) was treated, under nitrogen, with sodium hydride (55% dispersion in mineral oil, 160 mg.) also in dimethylformamide (5 ml.). After the evolution of hydrogen had ceased (ca. 30 min.), methyl toluene-2,- sulphonate (500 mg.) in dimethylformamide (5 ml.) was added and the mixture stirred at room temperature for 4 hr. The solvent was evaporated in vacuo and the residue treated with water (10 ml.) containing 4 N-sodium hydroxide (ca. 0.5 ml.). Extraction with ethyl acetate (3 x 10 ml.) gave the desired non-phenolic product. This crude ether was chromatographed twice on silica gel (British Drug Houses Ltd.) (100 g.), elution with ethyl acetate and ethyl acetate-methanol (1 : 1) giving substantially pure material (420 mg.) contaminated with a yellow substance. Crystallisation from ethanol-ether gave 0-methylsalutaridine as prisms (300 mg.), m.p. 147-148°. The molecular formula was determined by mass- spectroscopy (Found: M, 341.16269. C201-123N04 requires .±, 341. 16475) . The n.m.r. spectrum(in CDC13) showed the following signals ("r, values): N-Me (7.51), OMe (6.20, 6.14, 6.06), H (3.67), H and H (3.14), and H (2.70). The i.r. spectrum had 8 1 2 5 vmax.(CHC13) 1625, 1645 and 1670 (dienone) cm.-l . Reductive cleavage of 0-methyl-11-3}0-a-l-salutaridine 0-Methyl41-3H]-(+)-salutaridine (420 mg., 7.0 x 10-2 mc/mmole) in tetrahydrofuran (20 ml.) and liquid ammonia (250 ml.) o (distilled from sodium) was treated with sodium at -20 until a 104. permanent blue colour was obtained. After 30 min. ethanol was added to give a clear solution which was allowed to evaporate to 50 ml. at room temperature. This solution was poured into water (500 ml.) and excess solid carbon dioxide added. The products were extracted into ethyl acetate. Evaporation gave an oil which was redissolved in ethyl acetate and separated into phenolic and non-phenolic fractions with aqueous sodinm hydroxide in the usual way. The phenolic fraction (55 mg.), in methanol, was methylated with excess ethereal diazomethane for 2 days to give an oil (48 mg.). The 'non-phenolic' fraction (110 mg.) was treated in the same way to give an oil (90 mg.). Thin layer chromatography (t.1, c.) on silica gel G (Merck) plates, developed with methanol, showed that both methylated fractions contained at least ten components. Both contained one component running alongside laudanosine and showing in ultraviolet light a characteristic blue fluorescence after brief exposure to iodine vapour, The two fractions were chromatographed separately on grade III alumina, elution with benzene then benzene-ethyl acetate (9 : 1) giving fractions enriched in laudanosine. Further purification by t.l. c. on silica gel gave a fraction (1.5 mg.) showing only one component inseparable from (±)-laudanosine. This material had [o.]D -79° (c 0.13 in Et0H) and -54° (c 0.088 in CHC13) correspond7::-..:, to (-)-laudanosine [lit. 133 [o]r) +90° (c 1.42 in EtOH) and +52° (c 1.67 in CHC13) for the enantiomer]. The (-)-laudanosine was recovered from the rotation measurement and its weight (0.66 mg. determined by comparison of the u. v. spectrum with that of pure (+)-laudanosine. This was diluted with non-radioactive (±) -laudanosine (22.4 mg.) and the mixture crystallised to constant specific activity -3 (2.2 x 10 mc/rnmole), corresponding to an activity before dilution -2 of 7.8 x 10 mc/mmole. This value agrees reasonably with that 105. -2 (7.1 x 10 mc/mmole) of the original 0-methylsalutaridine showing that the (-)-laudanosine was substantially pure.

(-)-Laudanosine from 0-methyl-(-9-salutaridine 0-Methyl-(+)-salutaridine (1 . 05 g.) was cleaved with sodium in liquid ammonia (as above). The reaction products were methylated with diazomethane without preliminary separation into phenolic and non-phenolic fractions. Repeated column and thin-layer chromatography on alumina and silica gel gave (-)-laudanosine (8 mg.) as an oil. Seeding of a light petroleum (b.p. 60-80°) 134 solution with authentic (-)-laudanosine and recrystallisation from the same s olvent gave needles (1 . 5 mg.), m.p. and mixed m.p. 88-89°, [a]p = -86° (c 0.072 in EtOH).

Ozonolysis and reduction of 2, 5-dihydroanisole 2,5 -Dihydroanisole (1.5 g . ), was dissolved in methanol (10 ml.) and cooled to -70°. Ozone was passed through the solution until a test with starch-iodide paper showed it to be in excess (3 hr.). Sodium borohydride (1 g.) was added portionwise with stirring as the o temperature was allowed to rise to -20 . After 1 hour at this temperature the reaction mixture was allowed to warm to room temperature and stirring continued a further two hours. The solvent was then evaporated to dryness and the residue taken up in water (10 ml.), basified with sodium bicarbonate solution and extracted repeatedly with chloroform. These extracts were dried (Na2SO4) and evaporated in vacuo to give an oil. The i. r. spectrum had (CHC1 ) 3450 (hydroxyl), 1720 (ester carbonyl), 1620 (w ) M aX . 3 (unsaturation) cm71. 106.

Ozonolysis and reduction of acetoxy-dihydrothebaine Acetoxy-dihydrothebaine (1.2 g.) was dissolved in methanol (10 ml.) at -70° and ozone passed through the solution for some hours until in excess. Sodium borohydride (1 g.) was added portionwise with stirring, and after two hours the temperature of the solution was allowed to rise to 25° and stirring continued for a further two hours. The solvent was removed in vacuo and the residue was dissolved in water and filtered. The filtrate was basified with sodium carbonate solution and repeatedly extracted with chloroform. These extracts were dried (Na SO ) and evaporated in vacuo to an oil. 2 4 T.l.c. of this oil on A1 0 .G. or SiO .G. developed with petrol 2 3 2 ether showed it to contain some basic compounds as well as a compound indistinguishable from methyl acrylate. The same compound was obtained from t.l. c. of methyl hydracrylate showing that the elimination takes place both on alumina and silica. The i.r. spectrum of the crude product had vm, .(CHC13) 3500 (hydroxyl), 1740 (ester carbonyl) and 1660 cm. 1. Any attempt to purify the product by chromatography decreased the ester absorption in the i.r. spectrum.

(±)-Methyl-glycerate (±)-Calcium. glycerate-dihydrate (12 g.) was treated with oxalic acid dihydrate (6 g.) in water (20 ml.) and the precipitated calcium oxalate removed by filtration. The filtrate was evaporated in vacuo and the free acid, obtained as a viscous oil, was taken up in methanol (50 ml.) and treated with excess diazomethane in ether. The excess was decomposed with a few drops of acetic acid and the solvents removed in vacuo . Distillation of the residual oil gave the 107. product (2.4 g.), b.p. 115-120°/14-19 mm. Hg (lit.178 b,p. 123-5°/ 10 mm. Hg). The i.r. spectrum had v (liquid film) 1740 (ester max. carbonyl) cm. 1.

Isopropylidine derivative of (±) -methyl glycerate (±)-Methyl glycerate (1 g.) was dissolved in acetone (50 ml.), zinc chloride (2 g.) added, and the solution stirred at room temperature for 3 hours. Quinoline (2 g.) was added and the solution left overnight. The crystalline zinc chloride-quinoline complex was removed by filtration and the filtrate evaporated in vacuo to an oil. This was dissolved in ether (25 ml.) and washed (3 x) with water, dried (MgSO4.) and the ether removed in vacuo to give an oil. It was not possible to obtain the product completely free of methyl glycerate because of the ease with which the isopropylidine group was hydrolysed. The i.r. spectrum of the product had vm . (CHC1 ) ax 3 3550 (hydroxyl), 1740 (ester carbonyl), 1080, 1150 and 1280 (isopropylidine group) cm. 1.

Calcium D-glycerate 138 1, 2, 5, 6 -Di -is opropylidene -D-marmitol (4 g ) in 142 water (1 1.) containing potassium carbonate (6 g.) was oxidised with potassium periodate (16 g.) and potassium permanganate (0.34 g.). The reaction mixture was warmed and more water (750 ml.) added tr dissolve most of the periodate. The mixture was stirred at room temperature for 18 hr. and then warmed to precipitate manganese dioxide. This was filtered off and the filtrate evaporated in vacuo to dryness. The residue was washed with ether (3 x 50 ml.) and extracted with ethanol (5 x 50 ml.). The extract was concentrated to 15 ml. then diluted with ether to give crystalline potassium 17 9 is opropylidene -D -glyce r ate (2.1 g . ) . Re crystallis ation from 108. ethanol-ether gave hygroscopic material (dried in vacuo at 1300), m.p. 235-236°, [a]ro +41° (c 0.59 in H2O). Treatment with brucine hydrochloride in ethanol gave the corresponding brucine salt which, after crystallisation from ethanol-ether, had m.p. 254-258° softening at 90-100°. Potassium isopropylidene-D-glycerate. m.p. 232-234°, [a] +35° (c 2 in H2O), was regenerated from this D brucine salt by treatment with aqueous potassium carbonate. The 180 n. m. r. spectrum confirmed the structure of the product . Potassium isopropylidene-D-glycerate (12 g.) was heated at ca. 50° in 10% aqueous acetic acid (50 ml.) for 30 min. + Ion-exchange resin (Amberlite I.R. 120, H form) was added to remove potassium ions and the suspension filtered. The filtrate was evaporated in vacuo and the oily residue dissolved in water and treated with excess calcium carbonate. The mixture was heated for 2 hr . and then filtered while hot. Concentration and refrigeration of the filtrate gave crystalline calcium D-glycerate dihydrate (3.0 g.), [a]r) +13.6° (c 4.4 in H2O) [lit, 144 [a]r, +14.4° (c 2.12 in H2O)].

Calcium DL-glycerate A mixture of 50% sulphuric acid (100 g.), glycerol (92g. copper sulphate (0.5 g.), nickel sulphate (0.5 g.) and ferric chloride 0 (0.5 g.) was warmed to 80 on a steam bath. Concentrated nitric acid (178 g.) was added dropwise to the stirred mixture maintaining o the temperature between 75 and 80 . There was an induction period before the reaction started. Addition of copper wire was useful in initiating the reaction, which became uncontrollable if more than 5-10 ml. of nitric acid was added before the reaction began. The reaction normally proceeded with vigorous evolution of nitrous fumes and external cooling was sometimes required to keep the temperature o below 80 . When the addition of nitric acid was complete, the 109. mixture was treated with an excess of calcium carbonate and the temperature maintained at 80° for one hour, then the mixture was filtered and the filtrate evaporated to ca. a quarter of its original volume. Calcium glycerate dihydrate (26 g.) separated as white prisms on standing. The i.r. spectrum (Nujol) was identical to that 146 of the optically active salt. The yield was 20% (lit. 55%).

Calcium L-glycerate L-Serine was deaminated with nitrous fumes in the 148 usual way . The crude calcium L-glycerate was purified via 14 4 p-br omophenacyl L-glycerate : chromatography on silica gel (Hopkins and Williams Ltd. , M,F.1C44.) ngavei.p. 10material, ,m[a.]pr). 114-115°,-.0 [a]D +1.5° (c 1.80 in acetone) [lit. (2. 5.62 in acetone), for the enantiomer]. This ester was hydrolysed with hot aqueous -ethanolic calcium hydroxide to give pure calcium 148 L-glycerate dihydrate, [a] -12.3° (c 1.7 in H2O) [lit. [(d -14.6° D B in H20].

Hydrolysis of methyl (.4;)-glycerate Methyl (±)-glycerate (1 g.) was dissolved in 10% potassium hydroxide solution in ethanol (10 ml.) and left at room temperature. The progress of the reaction was followed by the disappearance of the ester carbonyl band in the i.r. spectrum. The reaction was complete after four hours and the solution was then poured through a column of ion-exchange resin (Amberlite I.R. 120, H+ form) to obtain the free acid which was then isolated in the usual way as its calcium salt (0.37 g.).

Hydrogenation of glyceraldehyde Glyceraldehyde (1 g.) in water (25 ml.) was hydro- genated over Adams's catalyst. Hydrogen uptake was slow and the 110. 1 reaction was incomplete (weak 1720 cm. band in the i. r. spectrum) after 48 hours. Uptake 190 ml . Theoretical uptake 249 ml.

Hydrogenation of phenolic-dihydrothebaine ozonide 85 Phenolic-dihydrothebaine (200 mg.) in ethyl acetate- methanol (1 : 1) (10 ml.) was cooled to -70° and ozone passed into the solution for 7 hours, after which t.l, c. examination of the reaction mixture showed no starting phenol. Oxygen was passed through the solution to remove dissolved ozone, then the total product hydrogenated in the same solvent over Adams's catalyst (25 mg.). Hydrogen uptake ceased after 11/2 hours (8.5 ml.) but after adding fresh catalyst (50 mg.) and a drop of dilute hydrochloric acid a further 18.5 ml. was absorbed overnight. This was just over half the theoretical uptake assuming 100% conversion into the ozonide. No attempt was made to isolate the products.

Ozonolysis of the salutaridinols 3 (7- H) Salutaridinol-I (28 mg., 1625 counts/sec, /mg.) was ozonised in ethanol (50 ml.) at -70° for 30 min. The solution was flushed with oxygen to remove excess ozone and allowed to warm to room temperature. Adams's catalyst (25 mg.) and a trace of hydrochloric acid were added and the mixture hydrogenated overnight The solution was filtered and adjusted to ca.. pH 10 with potassium hydroxide. After 24 hr. at room temperature, non-radioactive calcium D-glycerate dihydrate (30 mg.) was added and the solution adjusted to pH 6 with hydrochloric acid. 2.-Bromophenacyl bromide (500 mg.) was added and the solution heated under reflux overnight. Most of the solvent was evaporated and the residue diluted with water (20 ml.) and extracted with methylene dichloride (3 x 30 ml.). The extract was dried (MgSO4) and evaporated and the residue chrornatographed on silica gel (Hopkins and Williams Ltd., M.F.C.). Elution with ethyl acetate-benzene (1 : 1) gave R-bromophenacyl D- glycerate (18 mg.). This was diluted with non-radioactive ester (14 mg.) and the mixture recrystallised from benzene to a constant activity of 3.2 counts/sec. /mg., corresponding to a 0.80% yield of D-glyceric acid from salutaridinol-I. Similar degradations were carried out on sa.lutaridinol-.T.1 using D-glyceric acid for dilution, and on both salutaridinols using L-glyceric acid for dilution. The results are tabulated (see Theoretical Section).

Oxidation of nudaurine -I Nuclaurine-I (50 mg.) in chloroform (3 ml.) was shaken with active manganese dioxide (500 mg.) for half an hour at room temperature. The mixture was filtered and the filtrate evaporated in vacuo. The unreacted material was removed by chromatography on grade III alumina. Elution with ethyl acetate gave amurine (32 mg.) m.p. 211-2150 (lit.152 213-15o). 3 - Nudaurines Amurine (60 mg.) in dry tetrahydrofuran (20 ml.) was 3 treated with lithium aluminium (- 11}hydride (10 mg., 25 mc.) (New England Nuclear Corp.) under reflux for 3 days. The reducing age.- was chemically inactive and only starting material could be detected by thin layer chromatography. However, excess of non-radioactive lithium aluminium hydride was added to the mixture which was kept under reflux a further half an hour. The excess was destroyed by addition of ethyl acetate and the solvents removed in vacuo. The residue was partitioned between chloroform and water and the aqueous phase further extracted with chloroform (3 x). The combined extracts were dried (MgSO4) and evaporated in vacuo to 112: give the epimeric dienols. T .1.c. on alumina (Merck) plates, developed with methanol/ethyl acetate (1 : 19), showed nudaurine-I R 0.5, and its epimer nuelaurine-II R. 0.25, in the ratio of F F ca. 3 : 1, as the only products. The crude dienols were chromato- graphed on grade III alumina, elution. with methanol/chloroform (1 : 50) afforded nudaurine-I (28 mg.), m.p. 199-200° (lit.151, 201-20e), and with methanol/chloroform (1 : 19, I : 9) nud.aurine-II (10 rn.g.) was obtained. Both the dienols were radioactive (61,000 counts/mg./sec.). Counter efficiency 13%.

Ozonolysis of nudaurine-I [7 -311]Nudaurine-I (7.5 mg., 61,000 counts/sec./mg.) was ozonised in ethanol (20 ml.) at -70° for 30 min. The solution was flushed with oxygen to remove excess ozone and allowed to warm to room temperature. Adams's catalyst (15 mg.) and a trace of hydrochloric acid was added and the mixture hydrogenated overnight. The solution was filtered and adjusted to ca.pH 10 with potassium hydroxide. After 24 hr. at room temperature non-radioactive calcium D-glycerate .dihydrate (30 mg.) was added and the solution adjusted to pH 6 with hydrochloric acid. -Bromophenacyl bromide (500 mg.) was added and the solution heated under reflux overnight. Most of the solvent was evaporated and the residue diluted with water (20 ml.) and extracted with methylene dichloride (3 x 30 ml.). The extract was dried (MgS0 ) and evaporated, and the residue chromatographed on silica gel (Hopkins and Williams Ltd., M.F. C .). Elution with ethyl acetate-benzene (1 : 1) gave z-bromophenacyl D-glycerate (10.5 mg.) This was diluted with non-radioactive D-ester (13 mg.) and the mixture recrystallised from benzene to a constant activity of 27.5 counts/sec. /mg., corresponding to a 0.86% yield of 113.

D-glyceric acid from nuclaurine -I. The above experiment was repeated, and. similar degradations were also carried out on nudaurine-I using L-glyceric acid for dilution, and on nudaurine-II using D-glyceric acid for dilution. The results are tabulated. (See Discussion).

Typical extraction of alkaloids from Erythrina beans Erythrina glauca Willd. beans (220 g.) were crushed and then powdered and extracted for 36 hr. with petrol ether (1.5 1.) using a Soxhlet extractor to remove fats, etc. This extract was discarded and the plant material further extracted twice with methanol (1.5 1.) for 36 hr . These extracts were combined and the solvent removed in vacuo. The residue was taken up in 2N hydrochloric acid (250 ml.) and washed with chloroform (250 ml.), then filtered through celite to obtain a clear solution. This solution was basified with sodium hydrogen carbonate solution and repeatedly extracted with chloroform. The combined extracts were dried (MgSO4) and evaporated in vacuo to give the crude alkaloid mixture as a dark oil (480 mg.). Erysopine (132 mg.) separated from a concentrated solution of the crude alkaloids in ethananol (5 ml.), as white prisms, m.p. 238-240° (lit. 91 241-2°). Further purification could be effected by leaching the erysopine with boiling ethyl acetate which removed any other alkaloids which may have been present. The mother-liquor from the erysopine crystallisation was evaporated in vacuo and the residue redissolved in a small quantity of ethyl acetate and applied to a column of grade III alumina. Elution with benzene, which gave a non-basic oil plus small amounts of unidentified bases, was followed by elution with ethyl acetate-benzene mixture (1 : 9) which gave fractions consisting mainly of erythraline, 114. followed by fractions which contained the new alkaloid erythratinone. Erythraline was isolated from the combined relevant fractions as its hydrobromide (90 mg.). After recrystallisation from ethanol-ether it had m.p. 240-3°d., [a]5 +219 (H20) [lit. 91 m.p. 243°d., MD +216.6 (1120)]. Ethyl acetate-benzene mixture (3 : 1) eluted fractions from which erythratine (76 mg.) was obtained. This was crystallised 25 from ethyl acetate and hail m.p. 172-4°, [a] +113° (c 9 in EtOH) [lit. 91 m.p.170-170.5°, [a.] +145.50 (EtOH)] . Elution with D chloroform gave mixed fractions containing small quantities of erysodine and erysovine as well as a little erythratine.

Erythratine benzoate Redistilled benzoyl chloride (0.5 ml.) was added drop-wise to a stirred solution of erythratine (150 rag.) in pyridine (3 ml.). The solution was refluxed for half an hour then the solvent removed in vacuo. The residue was partitioned between sodium carbonate solution (5 ml.) and chloroform (5 ml.) and the organic layer separated. The aqueous phase was further extracted with chloroform (2 x 5 ml.) and the extracts dried (MgSO4) and evaporated in vacuo. The residue was dissolved in benzene (5 ml.) and passed through a column of grade III alumina, eluting with benzene ethyl acetate (1 : 1), which removed traces of pyridine. The product (83 mg.) was obtained by removing the solvent and 98 crystallisation from ether, m.p. 127-8° (lit. 248-9°). The • i.r. spectrum had a characteristic band at 1710 (ester carbonyl) 1 cm. . The n.m.r. spectral data are given in the Discussion Section. A sample recryctallided from 95% ethanol for analysis had m.p. 127-8° (Found: C, 68.72; H, 6.04; N, 3.07. NO H 0 C25H25 5 2 requires C, 68.63; H, 6.22; N, 3.20%. Found: M, 419.17372. C 25H25N05 requires M, 419.17326). 115.

Erythratine was recovered from this benzoate as follows: the benzoate (30 mg.) in ethanol-water (1 : 1) (5 ml.) with 3 drops of 2N. sodium hydroxide solution (pH 11) was kept at room temperature overnight. Water (20.m1.) was added and the solution extracted with dichloromethane (4 x 10 ml.). The extracts were dried (MgSO4) and the solvent removed in vacuo to give erythratine. This was crystallised from ethyl acetate to give pure erythratine (18 mg.), m.p. 173-5° identical with natural material (La-. , t.l. c.).

Erythratinone Erythratine (70 mg.) in chloroform (5 ml.) was shaken for half an hour with manganese dioxide (1 g.). The mixture was filtered and the solvent evaporated. Chromatography on grade 111 alumina eluting with ethyl acetate-benzene (1 : 1) removed traces of starting material and the product was obtained as a pale oil on evaporation of solvents. Erythratinone (51 mg.) was obtained as pale yellow crystals by dissolving the oil in a drop of chloroform and adding di-isopropyl ether (2 ml.), cooling, and scratching. rn..p. 138-140°, v (CHC1 ) 1675 cm.-l . max. 3 Erythratine and Epi-erythratine Sodium borohydride (50 mg.) was added to erythratinone (45 mg.) in methanol (5 ml.) and the solution refluxed for 1 hr. The solvent was removed in vacuo and the residue dissolved in water (5 ml.) and extracted repeatedly with chloroform: The extracts were dried (MgS0z ) and evaporated in vacuo. The epirners were separated by chromatography on grade III alumina, 2% methanol in ethyl acetate eluted erythratine (14 mg.) and 10% methanol in ethyl acetate eluted epi-erythratine (28 mg.), m.p. 147-150°, [a]D +280° (c, 0.35 in .Et0H). The i.r., n.m.r., and mass spectral data for both these compounds is given in the Discussion Section. 116.

Eyimerisation of erythratine Erythratine (100 mg.) in 2N hydrochloric acid (3 ml.) was kept at 100° under nitrogen for half an hour. The solution was basified with sodium carbonate solution and repeatedly extracted with dichloromethane. These extracts were dried (Na 50 ) and the 2 4 solvent removed in vacuo to give a crystalline solid (97 mg.). This was applied to a column of grade III alumina (7 g.) and eluted with chloroform, then methanol. The chloroform fractions contained only erythratine (90 mg.) while the methanol fraction contained erythratine and its epimer. The erythratine (90 rng.) was put through this process again, the recovered erythratine being recycled four times in all. The four mixed methanol fractions were then combined and the solvent removed in vacuo. Pure epi-erythratine (16 mg.) was obtained from this after careful chromatography. The total erythratine epirn.erised (100 + 90 + 80 + 65 = 335 mg.) yielded 5% of the epimer.

Epimerisation of epi-erythratine Epi-erythratine (20 mg.) in 6N hydrochloric acid (2 ml.) was kept at 100° for 2 hr. The solution was basified with sodium carbonate solution and repeatedly extracted with chloroform. The extracts were dried (IVI.gS0A) and the solvent removed in vacuo. The residue (20 mg.) was carefully chromatographed on grade III alumina to obtain erythratine (14 mg.) and, epi-erythratine (4 mg.).

Conversion of erythratine into erythraline Methanesulphonyl chloride (2.5 ml.) in pyridine (5 ml.) was added dropwise to a cooled solution of erythratine (500 mg.) in pyridine (5 ml.) and the solution left at room temperature for 3 hr. The solution was poured into water (100 ml.), basified with sodium 117. carbonate solution and repeatedly extracted with chloroform. The combined extracts were back-washed with water (50 ml.), dried (MgS0z ) and evaporated in vacuo to give a gum (170 mg.). The aqueous phase and back-washings were combined and made acidic with hydrochloric acid. The acid solution was washed with chloroform (5 x 30 ml.) then basified and extracted as above to yield a further 35 mg. of gummy product. The gums were dissolved in benzene (5 ml.) and chromatographed on grade III alumina. Elution with ethyl acetate-benzene (1 : 9) gave fractions from which erythraline free base (35 mg.) was obtained and also erythraline isolated as its pure crystalline hydrobromide (22 mg.). Total yield of erythraline was 12%. The hydrobromide had m.p. 242-3° d. o undepressed on admixture with authentic material, [a]D +207 (H20) 91 [lit. m.p. 243°d. [a] +216.6 (H 0)]. The i. r , u.v. and mass D 2 spectra were identical with those of authentic material.

Erythramine Erythraline (50 mg.) in ethanol (10 ml.) was hydro- genated over 5% Pd/CaCO (20 mg.). When hydrogen uptake ceased 3 the Solution was filtered and the filtrate evaporated in vacuo to give a gum. Trituration with di-isopropyl ether gave the product as a white solid which was crystallised from ether (25 mg.), 102a o rn.p. 120-121° (lit. 119.5-120.5 ). The n.m.r. and mass spectral data for this compound are given in the Discussion Section.

N-Norprotosinornenine

0, 0- Dibenzyl-N-norprotosinomenine hydrochloride 176 (1 g.) was dissolved in ethanol-water : 1) (50 ml.) and concentrated hydrochloric acid (0.1 ml.) added, and hydrogenated over 10% Pd/C catalyst (250 mg.). Hydrogen uptake (75 ml.) ceased after 2 hr. (Theory 90 ml.). The solution was filtered and concentrated to 118, ca. half volume and left at 4°C. N-Norprotosinomenine hydrochlorid. separated as white crystals (580 mg.) 67%, m.p. 241-2°. The i.r. and n.m.r. spectra support the structure of this compound. (Found: M, 315.14878. C181121N04 requires M, 315.14705).

Deuterium and tritium exchanfie reactions Two methods were used. Method- (a). The phenol was dissolved in dimethyl- forma.mide-deuterium ( or tritium) oxide and potassium. tertiary butoxide (1/2 equivalent) added, and this solution was sealed, wider nitrogen, in a glass tube and left at 100° for 3 or 4 days. The solvent was then evaporated to ca. half volume and water added, and the pH brought to 8 by addition of solid carbon dioxide. The precipitated phenol was extracted into chloroform and the extracts dried (MgSO4) and evaporated inkcuo. The phenol was then isolated either as its crystalline hydrochloride or crystallised as its free base. Method (b). The phenolic base was dissolved in dimethylformamide-deuterium (or tritium) oxide, and sealed under nitrogen in a glass tube and kept at either 100° or 118° for 3 days. The solvents were then evaporated in vacuo, water added and the solution repeatedly extracted with chloroform. The phenol was isolated from these extracts as in method (a). The various exchange reactions which were carried out are recorded below (Table 6). Table 6. Exchange Reactions

Wt. of Phenol Weight D.M. F. 0;0 T 0 Temp. Butoxide Activity PHENOL Method phenol .) isolated isolated . Note (ml s •) (mils .) ( ,, . (°C) (rags mc. (rngs .) as (ms .1 N-Norprotosinomenine hydrochloride a 100 1 1 - 100 45 H. Cl'd 35 - -3 (i) II a 100 1 - 0.3 100 60 H. Clid 5 ' 1.7 x 10 (ii) -2 il a 100 1 - 0.5 100 50 H. Cl'd 8 5.1 x 10 (ii) N-Norprotosinomenine b 40 0.5 0.1 - 100 H.H Ci'd 30 - - -2 (i) N b 110 0.3 - 0.3 100 - H. Clid 12.2 7.2 x 10 (ii) -2 " b 110 0.3 - 0.3 118 - H. Gild 10 4.5x 10 (ii) Erysodine b 50 0.5 0.2 - 118 - base 35 - (iii) -2 ri b 30 0.5 - 0.2 118 - base 15 5.3x10 - Erysovine/erysodine mixture b 40 0.5 0.2 - 118 - base 40 - (iii) Bis (3 -hydroxy-4 - methoxyphenyl- b 100 - 60 -(iv) ethyl)arrxine 1 0.5 113 - H. CPd -1 ft b 100 1 - 0.3 118 - H.C1'd 58 3.9x10 - 15 1 100 - 11 -(v) Erysodienone b 0.3 - base -2 it b 10 0.6 - 0.2 100 - base 5 2.8 x 10 (vi)

(i) Loss of 3 aryl protons confirmed by n.m. r. spectroscopy Considerable decomposition took place during the exchange and the product was only isolated pure after chromatography on silica (iii) Used for structural assignments by n.m. r. spectroscopy (iv) LOSS of 4 aryl protons confirmed by n.m.r. spectroscopy (v) Loss of 1 aryl proton confirmed by n.m.r. spectroscopy (vi) May also have exchanged a or y to the carbonyl 120.

Feeding Experiments

Typical work-up of plant from a feeding experiment

The plant (tyrosine fed) (40 g. wet weight) was macerated in a blender under N/50 hydrochloric acid (600 ml.) and left in this solution for 3 days. The mixture was filtered and the filtrate basified with saturated sodium hydrogen carbonate solution, and repeatedly extracted with chloroform. The extracts were dried (IVIgS0A) and evaporated in vacuo to give the total bases, a dark oil (60 mg.). These were chromatographed on alumina as described for alkaloids extracted from E. glauca beans. Generally only erythraline and erythratine were isolated. These were diluted, if necessary, with inactive material, and crystallised until the specific activity remained constant over three successive recrystallisations. The best results were obtained on plants which were from 4-6 months old.

Counting methods Samples (0.1 - 0.8 mg.) were dissolved in dry dimethylformamide (0.2 ml.) and liquid scintillater (1.2 ml.) (Nuclear Enterprises Ltd., Type N.E. 213) was added and the solutions shaken to make sure they were homogeneous. Duplicate samples were counted in a scintillation counter (Isotope Developments Ltd., Type 601211) and are uncorrected for self absorption. The efficiency of the counter and counting method was assessed by 3 14 counting [1, 2- II]hexadecane and [ C]hexadecane standards, under the same conditions, and found to be in the range 18-22% for 14 tritium and 68-69% for C.

121.

Tyrosine feeding A. 1 Compound fed: (+)-2- Cityrosine 0.053 mg., 0.01 mc. Plant: 3.st year E.Crista Galli, fed 25.5.65 and harvested after 7 days Wet weight of plant: /-1.0 g. otal bases isolated: 60 mg. (0.206% incorporation) t/144u.hydrobromide isolated 23 rug. which was crystallised (5 x) to constant specific activity of 7.74 x 103 d. 3 2 7,74 x 10 x 23 x 10 Incorporation 7 3.7 x 10 x 0.01 x 378 = 0.127% B. Compound fed; (±)-[2-"C]tyrosine 0.058 mg., 0.01 mc. Plant: 1st year E.Rubrinervria, fed 25.5.65 and harvested after 7 days Wet weight of plant: 417 g. Total bases isolated 75 mg. (0.179% incorporation) E:rythraline hydrobrornide isolated 16 mg, which was crystallised (6 x) to constant specific activity of 2.72 x 103 d./m.mole/sec. Incorporation = .0.031%, 122.

N-norprotosinomenine feeding

3 Compound fed: (-)-[5,21,61 - 1.1]N- - norprotosinomemne 9.65 mg., 5.63:1:10 inc . 1st year E. Crista Galli, fed 5.3.65 and harvested after 5 days Wet weight of plant: 254 g. Total bases isolated 270 nag. Erythraline hydrobromide isolated 63 mg. which was crystallised (6 x) to .constant specific activity of 4.00 x 103 d./m.mole/sec. Allowing for the loss of 1/3 of the tritium 3 2 3 x 4.00 x 10 x 63 x 10 Incorporation 7 -2 2 x 3.7 x 10 x 5.63 x 10 x378 0,043%

Erythratine isolated 30 mg. which was crystallised (5 x) to constant specific activity of 8.80 x 102 d./rn.mole/sec. Allowing for the loss of 1/3 of the tritium. 2 3 x 8.80 x 10 x30 x 10 Incorporation 2 7 -2 2 7,c. 3.7 x 10 x 5.63 x 10 x 315 = 0.006% 123.

N-norprotosinomenine feeding 3 Compound fed: (I) 45,2t, 6t- 11]N- norprotosinornenine 8 mg., 3.6 x 10-2mc. Plants: Two lst year E. Crista Galli, fed 5.8.66 and harvested after 10 days Wet weight of plants: 100 g. Total bases isolated: 62 mg., to which non-radioactive erythraline (15 mg.) was added before chromatographic separation Erythraline hydrobrornide isolated 23 mg. which was crystallised (4 x) to constant specific activity of 3.50 x 104 d./m.rnole/sec. The free base from this was crystallised and had activity of 3.61 x 104 d./m.mole/sec• Allowing for the loss of 1/3 of the tritium

3 x 3.50 x 10 x 23 x 1022 Incorporation - 7 2 x 3.7 x 10 x 3.6 x 10 x373 = 0.24%, Erythratine isolated 22 mg. which was crystallised (4 x) to constant specific activity of 2.48 x 103 d./m.mole/sec. Allowing for the loss of 1/3 of the tritium 3 2 Incorporation - 3 x 2.48 x 10 x 22 x 10 7 2 x 3.7 x 10 x 3.6 x 10 x 315 = 0.02%

124.

Bis(3-hydroxy-4-methoxy phenylethyl)amine feeding

Compound fed: Bis([2,6-31-1]3 -hydroxy - -z methoxy phenylethyl) amine 11.1 mg . 7.22x 10 mc. Plant: 2nd year E.Crista Galli, fed 16.5.66 and harvested after 6 days Wet weight

of plant: 470 g. Total bases

is olated: 205 mg., to which non-radioactive erythratine (10 mg.) was added before chromatographic separation Erythraline hydrobromide isolated 35 mg., which was crystallised (3 x) to constant specific activity of 1.72 x 102 d./m..mole/sec. Allowing for the loss of half the tritium 2 z 2 x 1.72, x 10 x 35 x 10 Incorporation - 7 -2 3:7x 10 x 7.22 x 10 x 378 0.0012%

Erythratine isolated 13 mg. to which non-radioactive erythratine (10 mg.) was added and crystallised (4 x) to constant specific activity of 1.02 x 103 d./m.mole/sec. Allowing for the loss of half the tritium. 3 2 2 x 1.02 x 10 x 23 x 10 Incorporation 7 -2 3.7 x 10 x 7.22 x 10 x 315 = 0.0055%

125,

Bis (3 -hyd r olf..-y -4 -methoxy phe nylethyl)amine feeding

Compound fed: Bis[2,6-3 H]3-hydr oxy-4 -mothoxy -2 pheny-lethyl)amine 10 mg., 6.5 x 10 mc. Plants: Two 1st year E.Crista Galli, fed 5.8.66 and harvested after 10 days -Wet weight of plants: 110 g. Total bases isolated: 102 nag.

Erythraline hydrobromide isolated 46 mg., which was crystallised (4 x) to constant specific activity of 4.30 x 102 d. /m.mole/sec. Allowing for the loss of half the tritium 2 2 x 4.30 x 102 x x 10 Incorporation 7 -2 3.7x 10 x 6.5 x 10 x 378 = 0.0043%

Erythratine isolated 11.5 mg. to which was added non-radioactive erythratine (10 mg.) and crystallised (4 x) to constant specific activity of 4.03 x 102 d. /m.mole/sec. Allowing for the loss of half the tritium 2 2 2x4.03 x 10 x 21.5 x 10 Incorporation 7 2 3.7 x 10 x 6.5 x 10 x 315 = 0.0022% 126.

Bis(3-hydroxy-4-rciethoxy phenylethyllamine feeding

Compound fed: Bis [2,6-3H]3-hydroxyl-4 -rnethoxyl phenylethyl) amine 10 nig., 6.5x10-zmc, Plant: 1st year E.Crista Galli, fed 26.8.66 and harvested after 10 days Wet weight of plant: 91.6 g. Total bases isolated: 190 mg.

Erythraline hydrobromide isolated 48 mg., which was crystallised (4 x) to constant specific activity of 3,98 x 102 d./m.mole/sec.

Allowing for the loss of half the tritium 2 2 2 x 3.98 x 10 x 48 x 10 Incorporation 7 -2 3.7 x 10 x 6.5 x 10 x 378

= 0.0042%

127.

Erysodienone feeding

Compound fed: (+)417-31.1]Erysodienone 4.2 mg., 2.35 x 10 2mc 2nd year E.Crista Galli, fed 16.5.66 and harvested after 6 days Dry weight of plant: g. Total bases

isolated: 107 mg.

Erythraline hydrobromide isolated 20 mg., which was crystallised (4 x) to constant specific activity of 1.41 x 104 d./m.mole/sec. 2 1.41x 10" x 20 x 10 Incorporation 7 3.7 x 10 x 2.35 x 10 x 378 = 0.085%

Erythratine isolated 13 mg., which was crystallised (4 x) to constant specific activity of 3.12 x 106 d./m.mole/sec. 2 3.12x10 x 13 x 10 Incorporation 7 -3 3.7 x, 10 x 2.35 x 10 x 315 = 0.15%

128.

Erysodine feeding

Compound fed: [17-31-1]Erysodine 3.0 mg . , 1.04 x 10 2mc Plant: let year E.Crista Galli, fed on 5.3.66 and harvested after 10 days

Wet weight of plant: 72 g. Total bases isolated: 33 mg., to which was added non-radioactive erysopine and this was crystallised (5 x) to constant specific activity of 6.25 x 10 d./m.mole/sec.

6.25 x 10"x 10 x 10 Incorporation 7 -2 3.7 x 10 x 1.04 x 10 x 285 0.57%

From the mother liquors Erythraline hydrobromide (18 mg.) was isolated after chromatographic separation in the usual way. This was crystallised (4 x) to constant specific activity of 1.24 x 104 d. /m.mole/sec. 4 2 1.24 x 10 x 18 x 10 Incorporation 7 -2 3.7x 10 x 1.04 x 10 x 378 7- 0.154% 129.

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